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Biology of the histones and protamines in trout testis Louie, Andrew James 1972

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BIOLOGY OF THE HISTONES AND PROTAMINES IN TROUT TESTIS by ANDREW J. LOUIE B.Sc, University of Alberta, 1968 A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry Faculty of Medicine We accept t h i s thesis as conforming to the required standard September 1972 University of B r i t i s h Columbia In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copy ing or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gai'Pi s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Department o f 3(•/"/£,t<4n T&^j The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date ABSTRACT Trout testes maturation i s characterized by the complete replacement of the histones by the protamines. A study of the d i f f e r e n t c e l l types involved i n spermatogenesis was i n i t i a t e d . C e l l s from testes at d i f f e r e n t stages of develop-ment were resolved according to t h e i r s i z e by sedimentation on serum albumin gradients. The temporal order of appearance of d i f f e r e n t c e l l types was noted. DNA determinations and analysis of basic proteins indicated that (a) large c e l l s (spermatogonia and early primary spermatocytes), (b) early , middle, and l a t e spermatids, and (c) mature sperm were re-solvable . The rates of DNA synthesis, histone synthesis, and h i s -tone phosphorylation bear a 1:1:1 r e l a t i o n s h i p to each other i n the large c e l l s . Protamine synthesis begins i n middle spermatids, while histones are progressively l o s t during the t r a n s i t i o n from middle to late spermatids. Very l i t t l e histone phosphorylation was found i n spermatids, i n d i c a t i n g that t h i s process i s not involved i n the removal of histones during the replacement process. The duration of each of the e a r l y , middle, and late spermatid stages was about 1 week, a t o t a l of 3 weeks being required for spermiogenesis i n trout. During development the t e s t i s weight increases expo-n e n t i a l l y 500 to 1000 f o l d then decreases as spermatozoa are l o s t . About 10 to 11 successive spermatogonial c e l l divisions (12 to 13 t o t a l c e l l divisions) occur during development. A k i n e t i c model for the appearance and disappearance of the d i f f e r e n t c e l l types was derived from the chronological and geometric rel a t i o n s h i p of the d i f f e r e n t c e l l types. The protamines undergo a series of phosphorylations and dephosphorylations during spermatid development. At least 6 modified bands of unsubstituted protamine were resolved by gel electrophoresis. The temporal r e l a t i o n s h i p of the bands was elucidated by following the fate of newly synthesized prot-amine labeled with [3H] arginine. Label was not found i n an unsubstituted protamine u n t i l 5 to 10 days a f t e r synthesis. The phosphorylation and dephosphorylation of protamine appear to be obligatory and u n i d i r e c t i o n a l . A large number of pools (at l e a s t 6) i s postulated to account f o r the long lapse between synthesis of protamine and appearance of l a b e l i n un-modified protamine c h a r a c t e r i s t i c of mature spermatozoa. The controlled phosphorylation of protamine may be important i n the correct binding of protamine to DNA and removal of his tones, while dephosphorylation of protamine may be involved i n the pro-gressive condensation of spermatid chromatin. Phosphorylation of protamine i s not involved i n the removal of protamine from ribosomes or i t s transport i n t o the nucleus . Substantial quantities of highly phosphorylated prot-amines suitable f o r i n v i t r o studies of the i n t e r a c t i o n of phosphoprotamine with DNA were prepared. There are at least 2 series of phosphoprotamines, each with 0, 1, 2, and 3 phosphates per molecule, and d i f f e r i n g i n the number of s e r y l residues. The metabolism of the histones was examined. The 5 major histone fractions were separated by Bio-Gel P-10 chromato-graphy; gel electrophoresis of each f r a c t i o n resolved phos-phorylated from unphosphorylated species. The lev e l s of phospho-histone were low (^5%) i n histone I I b 2 and I I I , and high i n histone IV (^30%) and I (40 to 50%) . These d i f f e r e n t l e v e l s suggest that phosphorylation may have d i f f e r e n t functions i n each histone. Labeling studies showed that newly synthesized histone IV undergoes an obligatory and sequential series of acetylations and deacetylations, which may be involved i n the correct binding of newly synthesized histone IV to DNA. After an i n i t i a l lag, newly synthesized histone I i s sequentially phosphorylated and dephosphorylated. This cycle of phosphorylation and dephosphorylation i s re-peated i n the next c e l l cycle. On the other hand, histone I l b i i s rapidly phosphorylated shortly af t e r synthesis and then dephosphorylated. After a short lag (correct binding to DNA) histone IV i s phosphorylated and then slowly dephos-phorylated. Previously formed ("old") histones I l b i and IV do not appear to be appreciably phosphorylated i n the suc-ceeding c e l l cycle. These data suggest that phosphorylation and dephosphor-y l a t i o n may have a r o l e i n the correct binding of newly syn-thesized histone I l b i to DNA. On the other hand, phosphor-y l a t i o n and dephosphorylation of "new" and "old" histone I may have an active role i n regulating the compactness of chromosomes. A scheme f o r the regulation of chromosome c o i l i n g during the c e l l cycle was proposed, i n which molecules of histone I i n extended chromosomes are highly phosphorylated while those i n condensed (super coiled) chromosomes are un-phosphorylated. The phosphorylation of "old" histone I may serve to uncoil the chromosomal f i b r e during telophase and about the DNA r e p l i c a t i o n fork during S phase; the phos-phorylation of newly synthesized histones I and IV may be necessary to maintain the d i f f u s e state of euchromatin. The dephosphorylation of I could be part of a mechanism involved i n the condensation of interphase chromosomes into metaphase chromosomes during mitosis and meiosis. V. TABLE OF CONTENTS Page ABSTRACT it TABLE OF CONTENTS V LIST OF TABLES x LIST OF FIGURES xi ACKNOWLEDGEMENT XV DEDICATION xvi INTRODUCTION 1 The Chemistry and Biology of the Histones 4 The Chemistry and Biology of the Protamines 13 The Process of Spermatogenesis 26 PART I: TROUT TESTIS CELLS EXPERIMENTAL PROCEDURES 27 I. Materials and Abbreviations 27 (a) Materials 27 (b) Source of Testes 27 (c) Abbreviations 28 I I . Characterization by DNA and Protein Analysis of C e l l s Separated by Velocity Sedimentation . . . 39 (a) Preparation of C e l l Suspensions 39 (b) Incubation of C e l l s 40 (c) C e l l Separations 40 (d) Radioactivity Analysis 42 (e) DNA and RNA Determinations 43 (f) D i s t r i b u t i o n of Histones and Protamines and t h e i r Synthesis i n the Different C e l l Types 45 I I I . Synthesis and Phosphorylation of Histones and Protamines i n the Different C e l l Types 47 (a) Incubation and Separation of C e l l s 47 (b) Starch Gel Electrophoresis 47 (c) Analysis of Radioactivity i n Starch Gels . . . 47 vi. Page RESULTS 49 I. Characterization by DNA and Protein Analysis of C e l l s Separated by V e l o c i t y Sedimentation . . . 49 (a) C e l l Separation P r o f i l e s and I d e n t i f i c a t i o n of C e l l s Synthesizing DNA 49 (b) DNA and RNA Contents of Different C e l l Types . 50 (c) Incorporation of Arginine and Lysine i n t o the Different C e l l Types 55 (d) C e l l u l a r D i s t r i b u t i o n of the Histones and Protamines 58 (e) Approximate Rate of Protamine Biosynthesis i n vivo 64 I I . Synthesis and Phosphorylation of Histones and Protamines i n the Different C e l l Types 66 (a) Rapid Extraction and Separation of Basic Proteins of the C e l l 66 (b) Separation of C e l l s from Preprotamine Stage Te s t i s 69 (c) Histone Metabolism i n Preprotamine Testis . . . 71 (d) Separation of C e l l s from Protamine Stage Te s t i s 77 (e) Histone Metabolism i n Protamine Stage Test i s . 78 (f) Protamine Metabolism 87 I I I . D i s t r i b u t i o n of C e l l s at Dif f e r e n t Stages of Spermatogenesis and a Ki n e t i c Model of Trout Te s t i s Development 89 (a) Growth of the Testis 89 (b) Proportion of C e l l s at Dif f e r e n t Stages of Development 91 (c) Spermatogonia and The j u r a t i o n of C e l l Cycle . 93 (d) A Model f o r the Development of the Testi s . . . 96 DISCUSSION 107 Nucleic Acid and Protein Analysis 110 DNA and RNA Synthesis I l l Synthesis and Phosphorylation of Histones and Prot-amines 113 Chronology of Spermatogenesis 115 Volumes of the Di f f e r e n t C e l l Types 116 Maintenance of the Germ Line and Spermatogonial P r o l i f e r a t i o n 120 Ef f e c t s of Gonadotrophins on Spermatogenesis 123 Development of Naturally Maturing Testes 125 v i i . Page PART I I : ENZYMATIC MODIFICATIONS OF THE PROTAMINES EXPERIMENTAL PROCEDURES 129 I. Material and Abbreviations 129 (a) Materials 129 (b) Abbreviations 129 I I . Overall Kinetics of Enzymatic Modification . . . .129 (a) C e l l Incubations 129 (b) In Vivo Labeling 130 (c) Starch Gel Electrophoresis 131 I I I . I n t r a c e l l u l a r Kinetics of Enzymatic Modifications .131 (a) Analysis of Protamine from Ribosomes 131 (b) P u r i f i c a t i o n of Protamine from Nuclei 133 (c) Microscale Separation of Cytoplasm from Nuclei 133 (d) Microscale Separation of Nucleohistone from Nucleoprotamine 134 (e) Extraction of Proteins and Starch Gel E l e c t r o -phoresis 135 IV. Separation and Characterization of Phosphorylated Species of Protamine from Trout Testes 135 (a) Trout Testes 135 (b) C e l l Incubations 136 (c) Extraction and P u r i f i c a t i o n of Protamines . . .137 (d) CM-Cellulose Chromatography of Protamines . . .138 (e) Amino acid Analysis 139 (f) Phosphate Determinations .139 (g) Alkaline Phosphatase Treatment 140 RESULTS 141 I. Overall Kinetics of Enzymatic Modification of the Protamines 141 (a) Nomenclature of Protamine Bands 141 (b) Incorporation o f 1 t 3H]arginine and l 3 2 P ] phosphate i n t o Protamine and the E f f e c t of Metabolic Inhibitors 143 (c) Incorporation of Labeled Methionine and Arginine i n t o Protamine as a Function of Time 146 (d) In Vivo Incorporation of [ 3H]arginine i n t o Protamine 149 (e) Overall Kinetics of Methionine Removal and Phosphorylation and Dephosphorylation of Protamine 151 (f) A Proposal for the Function of Protamine Phosphorylation 155 v i i i . Page I I . I n t r a c e l l u l a r Kinetics of Enzymatic Modification of the Protamines 160 (a) Comparison of Basic Proteins i n Various C e l l Fractions 161 (b) Presence of Labeled Protamine on Ribosomes . .164 (c) Synthesis of Protamine and t h e i r Transport from the Cytoplasm into the Nucleus 169 (d) Metabolism of Protamine i n Nucleohistone and Nucleoprotamine Fractions 172 I I I . Separation and Characterization of Phosphorylated Species of Protamine 178 (a) Chromatography of Protamines from Naturally Maturing and Hormonally Induced Testes . . . .178 (b) Amino acid and Phosphate Analysis of Prot-amines 180 (c) Methionine and 3 2P-Labeled Protamines 184 (d) P o s i t i v e I d e n t i f i c a t i o n of Phosphoprotamines on Starch G<als 189 (e) Alkaline Phosphatase Treatment of Phospho-protamines 190 DISCUSSION 194 Separation and Characterization of Protamine 196 I n t r a c e l l u l a r Transport of Protamine 199 Cytoplasmic Versus Nuclear Phosphorylation 201 Protamine Kinases . . . . .203 Nucleohistone and Nucleoprotamine 205 The Replacement Process 20 8 PART I I I : ENZYMATIC MODIFICATION OF THE HISTONES DURING SPERMATOGENESIS IN TROUT EXPERIMENTAL PROCEDURES 213 (a) Materials 213 (b) C e l l Incubations 213 (c) Labeling of Histones i n Intact F i s h 214 (d) Preparation of Histones 215 (e) Fractionation of Histones 215 (f) T r y p t i c Phosphopeptides 217 RESULTS AND DISCUSSION 218 I. I d e n t i f i c a t i o n and Levels of Phosphorylated Species of Histone i n the Major Fractions 218 (a) Bio-Gel P-10 Chromatography of 3H and 3 2P-Labeled Histones 219 ix. Page (b) I d e n t i f i c a t i o n of Phosphorylated Species of Histones 221 (c) Phosphopeptides i n the Major Histone Frac-tions . .226 (d) Proportions of Phosphorylated Histone Species .228 I I . Synthesis, Acetylation, and Phosphorylation of Histone IV 231 (a) Fate of Newly Synthesized Histone IV 231 (b) B i o l o g i c a l Function of Acetylation and Deacetylation of Histone IV 235 I I I . Overall K i n e t i c s of Phosphorylation and De-phosphorylation of Histones and Their Possible Role i n Determining Chromosomal Structure 242 (a) Histone I 243 (b) Histone I l b i ,. . .245 (c) Comparison of Histones I, I l b i , and IV . . . .245 (d) B i o l o g i c a l Role of Histone Phosphorylation . .249 CONCLUDING REMARKS 255 Acetylation of Histones 255 Phosphorylation of Histones 259 Ultrastructure of Chromosomes 262 Annealing of Histones to DNA and P a r t i a l Nucleo-histones 265 Interaction of Histones with DNA 267 Phosphorylation of Histone I as a Determinant of Chromosomal Structure 273 Chromosomal C o i l i n g During the C e l l Cycle 283 BIBLIOGRAPHY 290 X LIST OF TABLES Table Page I Chemical composition of various chromatins , 2 II P r i n c i p a l components of c a l f thymus histone and the two commonly used systems for t h e i r nomen-clature 5 III Variations i n germ c e l l nuclear volume, expressed i n y 3 30 IV Comparison of data on the duration of spermato-genesis i n animals 32 V DNA and RNA contents and RNA:DNA ratios of c e l l s from d i f f e r e n t regions of serum albumin gradients . 53 VI Relative rates of DNA synthesis i n c e l l s from d i f f e r e n t regions of serum albumin gradients . . . . 54 VII Relative rates of protein synthesis i n c e l l s from d i f f e r e n t regions of serum albumin gradients . . . . 59 VIII Synthesis and phosphorylation of histones i n c e l l s of preprotamine t e s t i s 74 IX Synthesis and phosphorylation of histone and prot-amine i n c e l l s of protamine stage t e s t i s 82 X DNA and RNA contents of immature t e s t i s 9 5 XI Possible mode of spermatogonial p r o l i f e r a t i o n i n trout t e s t i s 10 2 XII Volumes of trout t e s t i s c e l l s 103 XIII Duration of the S phases of DNA synthesis and c e l l u l a r cycles i n d i f f e r e n t categories of sperm-atogonia . . . 117 XIV Number of spermatogonial d i v i s i o n s 122 XV Amino acid analysis of protamine fractions 182 XVI Phosphate analysis of protamine fractions 183 XVII Chromosomal f i b r e dimensions and average phosphate content per mole of histone I 281 xi . LIST OF FIGURES Figure Page 1. Comparison of the NH2-terminal regions of trout t e s t i s histones and the s i t e s of enzymatic mod-i f i c a t i o n .11 2. Amino acid sequences of 3 components of clupeine and i r i d i n e 18 3. Relationship of the d i f f e r e n t c e l l types involved i n spermatogenesis 27 4. Time course of incorporation of [ 3H]arginine into c e l l s 41 5. C e l l separation and [ 3H]thymidine p r o f i l e s of c e l l s from d i f f e r e n t stages of hormonally induced testes. 51 6. C e l l separation, [ 3H]arginine, and [^C]lysine p r o f i l e s of c e l l s from testes at d i f f e r e n t stages of hormonal induction 57 7. Extended c e l l separation, gel electrophoresis, and autoradiography of [ x"*C]arginine labeled c e l l s from protamine stage testes 61 8. Separation of histones and protamines on starch gels 67 9. C e l l separation and r a d i o a c t i v i t y p r o f i l e s of c e l l s from a t e s t i s at the preprotamine stage of develop-ment 70 10. Separation and analysis of r a d i o a c t i v i t y i n h i s -tones from c e l l s of preprotamine stage t e s t i s . . . 72 11. Extended sedimentation of c e l l s from a t e s t i s at the protamine stage of development 79 12. Separation and analysis of r a d i o a c t i v i t y i n h i s -tone and protamines of c e l l s from t e s t i s at the protamine stage of development 80 13. Turnover of 3 2 P - l a b e l e d phosphoryl groups i n h i s -tones from trout t e s t i s c e l l s 84 14. Comparison of the growth of hormonally induced and naturally maturing trout testes 90 x i i . Figure Page 15. Sedimentation of c e l l s from testes at d i f f e r e n t stages of development 92 16. Hypothetical d i s t r i b u t i o n of each c e l l type as a function of the developmental stage of the t e s t i s 97 17. Pulse-chase of [ 3H]guanosine la b e l i n c e l l s from naturally maturing testes . .100 18. Estimated t o t a l c e l l number and weight of the t e s t i s at d i f f e r e n t stages of development 105 19. Nomenclature of protamine bands resolved by starch gel electrophoresis 142 20. Incorporation of [ 3H]arginine and t 3 2 P] phosphate into protamine and the e f f e c t of an i n h i b i t o r of protein synthesis and an uncoupler of oxida-t i v e phosphorylation . . . . . 144 21. Incorporation of [ 3H]arginine and [3 5S ]methionine into protamine as a function of time 147 22. In vivo incorporation of [ 3H]arginine into prot-amine 150 23. S p e c i f i c a c t i v i t y of 3 5S-protamine from the i n -cubation of F i g . 21 153 24. Proportion of [ 3H]arginine l a b e l i n the d i f f e r e n t species of labeled protamine at various times afte r the s t a r t of l a b e l i n g 154 25. A schematic representation of the k i n e t i c r e l a t i o n -ship of the d i f f e r e n t protamine species 156 26. Resolution of acid-soluble proteins from d i f f e r e n t fractions of the c e l l 162 27. Nucleohistone and nucleoprotamine fractions from testes at the middle protamine stage of develop-ment 165 28. (A), sucrose density gradient centrifugation of polysomes from trout testes. (B), starch gel electrophoresis and analysis of r a d i o a c t i v i t y of acid-soluble proteins from the diribosome region and protamine prepared from nuclei ., 167 x i i i . Figure Page 29. Starch gel electrophoresis and analysis of radio-a c t i v i t y i n acid-soluble proteins from the cyto-plasm and nucleus 170 30. Transport of protamine from the cytoplasm in t o the nucleus 171 31. Comparison of the metabolism of protamine from the nucleohistone and nucleoprotamine fractions of c e l l s 175 32. Comparison of the s p e c i f i c a c t i v i t y of [ 3^meth-ionine i n protamine from whole c e l l s , nucleo-histone and nucleoprotamine fractions 177 33. Resolution of protamines from a r t i f i c i a l l y induced and naturally maturing testes by chromatography on CM-cellulose , 179 ^ 34. P r o f i l e of [ 3 5S]methionine labeled protamine re-solved by CM-cellulose chromatography 186 35. Chromatography of 3 2 P - l a b e l e d protamine followed by starch gel electrophoresis 187 36. Starch gel electrophoresis of protamine resolved by chromatography on CM-cellulose 191 37. Alkaline phosphatase treatment of protamine . . . .192 38. Time dependence of [ 3 2P]phosphate incorporation into histones 220 39. Starch gel electrophoresis of pooled 3H and 3 2P labeled histone fractions from F i g . 38 222 40. Dephosphorylation of phospho-IIbi by a l k a l i n e phosphatase 225 41. Tryptic phosphopeptides from the major histone fractions 227 42. Incorporation of radioactive isotope into histone IV as a function of time 232 43. The fate of i n vivo labeled histone IV 234 xiv . Figure Page 44. A scheme for the possible binding of the basic NH 2-terminal region of newly synthesized histone IV i n the major groove of DNA 241 45. Starch gel electrophoresis and analysis of radio-a c t i v i t y i n histone 1 = 244 46. Starch gel electrophoresis and analysis of radio-a c t i v i t y i n histone I l b i 246 47. Overall k i n e t i c s of phosphorylation and dephos-phorylation of histone I, I l b i , and IV i n trout t e s t i s 248 48. The NH 2-terminal region of histone I l b i written i n the form of a h e l i c a l wheel and the binding of th i s region to DNA 251 49. Comparison of the possible mechanism of binding newly synthesized histone I l b i and IV to DNA . . .252 50. Possible i n t e r a c t i o n of histones with DNA 272 51. Phosphorylation and dephosphorylation of histone I as a determinant of chromosomal c o i l i n g 275 52. A scheme for the possible regulation of chromosome structure during the c e l l cycle 284 ACKNOWLEDGEMENTS I wish to thank my supervisor, Dr. Gordon H. Dixon for his encouragement, comments, and enthusiastic i n t e r e s t during the course of t h i s work. My associations with the members of t h i s laboratory have been most h e l p f u l and I wish to acknowledge the debt I owe to them. In p a r t i c u l a r , I wish to thank Drs. Michael Sung, Peter Candido, and Vi c t o r Ling for t h e i r valuable suggestions and discussion of r e s u l t s , Dr. Dominic Lam who introduced the c e l l sedimentation tech-nique to the lab, and Josef Durgo, our instruments tech-n i c i a n , whose ingenuity and hard work has been a boon to the lab. I wish to acknowledge the 4 years of f i n a n c i a l support from the National Research Council of Canada, from whom I received a Centennial Science Fellowship. The research was supported by grants to Dr. G.H. Dixon from the Medical Research Council, National Research Council, and National Cancer I n s t i t u t e of Canada. xvi . DEDICATION to My wife, Carol My son, Christopher Robert and ifly parents 1. INTRODUCTION The histones (1,2) are a class of basic proteins assoc-iated with DNA i n the nuclei of most somatic c e l l s . The i s o l a t e d DNA-protein complex from c e l l s i n the interphase stage of the c e l l cycle i s c a l l e d chromatin (3) while i n c e l l s at the metaphase stage of mitosis t h i s material i s condensed into chromosomes. Native nucleohistone i s the soluble portion of chromatin obtained af t e r shearing chromatin and consists (3) primarily of DNA, histones, a variable proportion of non-histone chromosomal proteins and i n some cases, small amounts of RNA (Table I ) . The r a t i o of the weight of histones to DNA i s approximately 1:1 i n most somatic tissues (4). However, during spermatogenesis i n many species, the histones are replaced by new species of sperm-specific basic proteins (5,6). In the salmonid f i s h , these new proteins are c a l l e d protamines (6,7). The protamines were f i r s t i s o l a t e d almost a century ago by Miescher (8,9) who was interested i n the chemistry of the c e l l nucleus. In 1868, he described the i s o l a t i o n of DNA from pus c e l l s ; t h i s was followed i n 1874 by the characterization of a "nitrogen-rich base" associated with DNA extracted from m i l t of the A t l a n t i c salmon (Salmo s a l a r ) . He thought t h i s com-pound to be quite simple i n nature (C 9H2iNs0 3) and named i t "protamine". Evidence for the sperm s p e c i f i c i t y of protamine came from immature testes, from which he i s o l a t e d a protein TABLE I Chemical compositions of various chromatins [from Bonner et a l . (4) ] Content, relative to DNA, of Tnmni-iin Source of • . . J c m P ! a t 0 chromatin u . , Nonhistone D v r A , « , a C r « l X A\ DNA Histone nrnlrin R N A (% of DNA) Pea embryonic axis 1.00 1.03 0.29 0.26 12 Pea vegetative bud 1.00 1.30 .10 .11 6 Pea growing cotyledon 1.00 0.76 .36 .13 32 Rat liver 1.00 1.00 0.67 .043 20 Rat ascites tumor ' 1.00 1.16 ' 1.00 .13 10 Human HeLa cells 1.00 1.02 0.71 .09 10 Cow thymus 1.00 1.14 .33 .007 15 Sea urchin blastula 1.00 1.04 0.48 .039 10 Sea urchin pluteus 1.00 0.86 1.04 .078 20 3. but was unable to extract any protamine. In the footsteps of Miescher, Kossel (1) showed that protamine consisted of amino acids and was therefore a protein; the protein i s o l a t e d from immature testes he c a l l e d "histone". Since then, histones have been i s o l a t e d (2,4) from many phylogenetically distant species and tissues within a species, while the d i s t r i b u t i o n (5,6) of the protamines or analogous sperm-specific proteins has been extended to a large number of f i s h , insects, amphibians, molluscs, and mammals. Early workers studying the histones and protamines were hampered by the apparent heterogeneity (2,4) of these proteins (some of which was due to impurities and aggregration), and inadequacy of methods for separating chemically similar pro-teins and t h e i r characterization. The advent of modern bio-chemical techniques of chromatography, electrophoresis, and physi c a l , chemical, and enzymatic methods for probing the molecular structures of molecules together with a sounder understanding of the molecular basis of biology led to an upsurge i n investigations of many aspects of the structure and function of these proteins. Only the s a l i e n t features w i l l be discussed here. For recent reviews on the histones, t h e i r modification* and t h e i r interactions with DNA, consult r e f s . 10 to 14; for reviews on the protamines, the early work i s covered by F e l i x (15), while l a t e r studies have been sum-marized by Bloch (5), Dixon and Smith (7), and Dixon et a l . (16). A b r i e f discussion of spermatogenesis and the e f f e c t 4. of gonadotropic hormones i s presented; for a comprehensive description of spermatogenesis, Roosen-Runge (17) and Courot et a l . (18) may be consulted while the endocrine aspects of f i s h spermatogenesis are summarized by Hoar (19). Chemistry and Biology of the Histones: Only recently have the histones from most organisms been reproducibly separated into 5 d i s t i n c t and major f r a c t i o n s . The two major systems of histone nomenclature (20,21) and some of the properties of the histone fractions are given i n TABLE I I . In t h i s t h e s i s , the nomenclature of Rasmussen et a l . (20) i s followed. The major histone fractions (I, I l b i , I I b 2 , III and IV) can be separated by ion-exchange chromatography (20,21), gel exclusion chromatography (22,23), or sel e c t i v e extractions with acid and acidified-ethanol solutions followed by d i f f e r e n t i a l p r e c i p i t a t i o n (24). Each f r a c t i o n constitutes about 20% of the t o t a l histones i n the c e l l (25). The amino acid sequences of histones I I b 2 (26), III (27,28), and IV (29) have been published while sequences of histone I l b i are almost complete (30,31). The sequence of histone I has posed some problems because of the high content (^30%) of l y s y l residues and the larger size of the molecule (30). However, i t seems clear that the complete sequences of representative species of a l l 5 histones w i l l be known i n the next year or two. Comparison (29) of the amino acid sequences of histone IV from c a l f thymus and pea seedling revealed several i n t e r -TABLE II Pr i n c i p a l Components of Calf Thymus Histone and the Two Commonly Used Systems for Their Nomenclature [from E l g i n et a l . (10)] Subclass; nomenclature of : Lys/Arg Moles/100 moles Class Rasmussen et al: Johns and Butler ratio Molecular weight total histone' N-terminal C-terminal Lysine-rich la fl 22 21,000- • 6.7 Blocked Lysine lb fl 22 21,000= 6.6 Blocked Lysine Slightly Ilbi f2a2 ~2.5 13,000-15,000= 20.0 Blocked Lysine lysine-rich IIb2 f2b 2.5 13,774- 24.6 Proline Lysine Arginine-rich III f3 0.8y 13,000-15,000 18.3 Alanine Alanine IV f2al 0.1 11,282-' 23.8 Acetylserine Glycine 6. esting points: f i r s t , the segregation of basic amino acid residues i n the NH2-terminal half of the molecules; and second, the almost i d e n t i c a l sequences of the 2 histone IV*s despite the long span of evolutionary development (600 m i l l i o n years) since the evolutionary precursors of the two species diverged. That histone IV has the slowest evolutionary mutation rate of any protein so far studied (29,32) suggests that the i n t e r a c t i o n of histone IV with DNA and other chromosomal constituents i s highly s p e c i f i c and non-random, since most mutations must be l e t h a l or suppressed. The sequences of the other histones have also revealed a segregation (11,30) of basic amino acid residues i n the molecules: a concentration of basic residues i n the NH2-terminal region of the arginine or s l i g h t l y l y s i n e - r i c h histones ( I l b i , I I b 2 , and III) and a concentration i n the COOH-terminal region i n the l y s i n e - r i c h histone I. Comparison of sequences of histone Ilbx (30,31) and III from c a l f thymus (27), trout (33), and chicken erythrocytes (28) reveal only minor amino acid replace-ments i n d i c a t i n g a slow rate of evolutionary change for these proteins; the larger v a r i a t i o n i n size of histone I shown by gel electrophoretic studies of histones from many species (34) suggests that histone I may evolve s l i g h t l y faster than the other histones implying that i t s interactions with DNA and other s t r u c t u r a l components of chromatin may be less precise. I t has been proposed (11,26,27,29,30) from the segre-gation of basic amino acid residues i n these proteins that 7. the basic regions of the histones are l i k e l y DNA binding s i t e s while the r e l a t i v e l y neutral or hydrophobic portions of the molecules might be free to i n t e r a c t with other histones or non-histone proteins. The histones are t i g h t l y associated with DNA. High s a l t concentrations (3) or a c i d i c (pH 0.2 to 2.0) conditions are required to dissociate histones from DNA. Lysine-rich histone I i s eluted from chromatin between 0.4 and 0.5 M NaCl, s l i g h t l y l y s i n e - r i c h histones I l b i and I I b 2 between 0.8 and 1.2 M NaCl, while the arginine-rich histones III and IV are eluted between 0.8 and 1.6 M NaCl. The interactions of histones with DNA are not e n t i r e l y i o n i c since urea, which disrupts hydrophobic bonds, considerably decreases the s a l t concentration required to elute histones from chromatin (36). The order of e l u t i o n of histones from chromatin with s a l t i s i n contrast to the p r e c i p i t a b i l i t y of DNA by histones i n v i t r o . The amount of histone required to p r e c i p i t a t e a given amount of DNA i s lowest for histone I and highest for the arginine-rich histones (37,190). These data seem to suggest that i n vivo, the arginine and s l i g h t l y l y s i n e - r i c h histones are more intimately associated with DNA while the l y s i n e - r i c h histone I i s more exposed on the surface of the nucleohistone complex (10,38). The histones appear to be stable s t r u c t u r a l components of chromatin as judged by t h e i r slow turnover rates. When h i s -tones and DNA are labeled with separate radioisotopes followed by 8. measurement of the s p e c i f i c a c t i v i t y of the histone and DNA at various times during the growth of the c e l l s or tissue s , the h a l f - l i v e s of both histones and DNA are very s i m i l a r (39, 40). These results suggest that the DNA-histone complex i s metabolically quite stable (39,40). Complexes of histone with DNA are considerably less extended than free DNA (41,42). The histones may c o i l (41) and intramolecularly c r o s s - l i n k (43) DNA with a r e s u l t that the packing r a t i o of a given length of DNA i n chromosomes may be as high as 100 to 1 (44). Spectroscopic studies have indicated that i n d i l u t e s a l t solutions, free histones have low levels of ordered structure while an increase of the s a l t concentration induces formation of ( A - h e l i c a l regions (45,46). In combination with DNA the histones are rather evenly d i s t r i b u t e d over the DNA double helix (41,42,47). Not a l l DNA phosphates are neutralized by the c a t i o n i c side chains of the histones since as many as 50% of the DNA phosphates of chromatin are free to bind poly-cations (48,49). As much as 50 to 65% of the proteins com-plexed with DNA are i n the o(-helical conformation (41) and i t has been proposed that the histones i n t h i s conformation l i e i n the major groove of DNA (22,37,41). Phleomycin binding (50) and studies with "reporter" molecules (51) also indicate that the major groove of DNA i s occupied by protein while the minor groove i s r e l a t i v e l y free. I t should be noted that recent 9. NMR spectroscopic studies indicate that histone I annealed to DNA i s i n the extended conformation with some 8 structure and very l i t t l e o( h e l i x (52) . In most c e l l s [cells from c a l f thymus appear to be an exception (53)] the histones are synthesized i n the cytoplasm (54) on small polyribosomes and transported into the nucleus where they bind to DNA. Most of the histones are synthesized i n the S phase (phase of DNA synthesis) of the c e l l cycle (55-57). Histone and DNA synthesis are believed to be coupled, since i n h i b i t i o n of DNA synthesis by hydroxy-urea leads to a cessation of histone synthesis and disappearance of small polyribosomes and messenger RNA believed to be involved with histone synthesis i n the cytoplasm (54,58). The close equivalence of the amount of histone to the amount of DNA i n many species and tissues within a species led to the proposal by Stedman and Stedman (59) that the h i s -tones might function as general gene repressors. Studies of the template a c t i v i t y of appropriately i s o l a t e d chromatin (Table I) and histone-free chromatin renewed i n t e r e s t i n the idea that histones might repress the genetic a c t i v i t y of DNA (3,60) . If histones are repressors of genetic a c t i v i t y , then the small number of histone species and the i n h i b i t o r y e f f e c t s of histone on t r a n s c r i p t i o n of DNA by RNA polymerase raise, ques-tions regarding the mechanism of s e l e c t i v e gene derepression necessary for control of gene expression and hence c e l l d i f f e r -10. e n t i a t i o n . The discoveries that histones i n vivo could be acetylated at the <X-NH2-terminus (61) and e-NH2 p o s i t i o n of l y s y l residues (29,62,63), and phosphorylated (64,65) on the hydroxyl group of s e r y l residues suggested that these mod-i f i c a t i o n s might modulate the in t e r a c t i o n of histones with DNA and hence regulate the expression of genetic a c t i v i t y by decreasing the binding of histones to DNA. Post-synthetic methylation (66) of the e-NH2 group of s p e c i f i c l y s y l residues and the guanidinium group of arginyl residues has also been reported (67). Since methylation of histones occurs r e l a t i v e l y l a t e i n the c e l l cycle p r i o r to c e l l d i v i s i o n , i t has been proposed that methylation may be involved i n chromosome condensation (68). Candido and Dixon (23,33,38,69). have determined the mul-t i p l e s i t e s of e-NH 2-acetylation of histone I l b i , I I b 2 , I I I , and IV i n trout t e s t i s while Sung and Dixon (22,70,71) have determined the s i t e s of phosphorylation of histone I, I l b i , I I b 2 , and IV. The s i t e s of acetylation i n histone I l b i , I I b 2 , I I I , and IV a l l occur i n the highly basic NH 2-terminal regions of these molecules as do the s i t e s of phosphorylation of histone I l b i , I I b 2 , and IV (22,70,71); these are shown i n F i g . 1. As mentioned previously, the basic regions of the histones are the l i k e l y s i t e s of attachment of histone to DNA. That a l l these modifications decrease the net p o s i t i v e charge of the basic region of the molecules by ne u t r a l i z a t i o n of the e-NH2 group 11. Comparison of N-Terminal Regions of Histones of Trout Testis . . -• ; j - - , , 1 Histone IV Histone lib. PO. Ac\ i r Ac-Sar-Oly-Arg-Gly-Lya^ -1 0 I r, Ae-Ser-eiy-Arg-eiy-Lys-Thr. r « " f S i r ~1 1 Ac 1 1 ! l l ' Iciy-Cly-Lya- Gly-Lau-Gly i 1 i Lya-1 | 1 1 ' 0 1 1 1 1 1 1 -Gly-Gly-Lys •Ale-Arg-Ala . 1 | 1 10 IIS | 20 I I I I —(Ala-LySjThr-Arg-(Argt-S»r-l _ _ I 15 Ae Ac Ac Ac I I / 1 / i / 1 Histone III 4 l a - A r B - 7 7 > r - t y « ^ / n ^ r - 4 l a - ^ r 9 - t y » ^ « f - r h r - 0 / ) ' ^ / y - t ) f » - 4 t o ^ r o ^ » » W . y » - 0 / u W . » o ^ t o - ^ / i f - l r » - * / » - i 4 r 9 -I 1 I 1 I B 10 15 20 28 Ac j*Oil. Ac Ac Ac HlStOne lib Pro<1ln-Pro-Ala<y\-Sar-Ala-Pm-Lya-Jya^ly^9r-Lya-Ly»^-Ala-Val-Thr.Lya-7^ 2 I I 10 is 20 F i g . 1. Comparison of the NH 2-terminal regions of trout t e s t i s histones and the s i t e s of enzymatic modification (from Candido and Dixon, 38 ). 12. (acetylation) or by introduction of negative charges (phos-phorylation) would seem to indicate that these post-synthetic modifications of the histones may modulate the binding of h i s -tones to DNA and perhaps allow expression of genetic a c t i v i t y . Thus, when lymphocytes are stimulated to divide and d i f f e r -entiate by phytohemaglutinin, the increase i n RNA synthesis i s preceded by an increase i n histone acetylation not assoc-iated with histone synthesis (72). Administration of the hormones glucagon or i n s u l i n to rats leads to the increased synthesis of c e r t a i n enzymes (73) ; Langan has shown that phos-phorylation of a s p e c i f i c s i t e of histone I i n rat l i v e r increases 15 to 25 f o l d following administration of these hormones (74,75). In addition, the template a c t i v i t y of chromatin increases with the degree of phosphorylation of histone I (76). Chemical acetylation of histones i n v i t r o to degrees which did not a f f e c t t h e i r a b i l i t y to combine with DNA lowered the i n h i b i t i o n of RNA polymerase a c t i v i t y i n c a l f thymus DNA by 21 to 66% (66). These results support the idea that gene a c t i v i t y may be derepressed by acetylation or phosphorylation of histones or both. However, a d i r e c t cause-and-effeet r e l a t i o n s h i p i n which histone acetylation or phosphorylation or both increase the template a c t i v i t y of DNA has not yet been demonstrated. Puffing of the giant polytene chromosomes i n some insect tissues i s a well documented case of l o c a l i z e d genetic a c t i v i t y ; intense RNA synthesis occurs at s p e c i f i c s i t e s along the 13. chromosome (77). Cytochemical staining procedures (78) re-vealed that there i s as much histone i n regions of puffing as i n inactive regions thus implying that physical removal of histones i s not required for chromosome puffing and hence gene a c t i v i t y . In these studies, an increase i n non-histones ( i . e . , a c i d i c - p r o t e i n staining) was indicated i n the puffing regions. However, autoradiography of [ 3H]acetate-labeled giant s a l i v a r y chromosomes did not reveal a concentration of counts over regions of chromosome puffing, suggesting that active acetylation of histones i s unlike l y to be involved i n t h i s example of gene derepression (80). Evidence w i l l be presented i n t h i s thesis which suggests that acetylation and phosphorylation of c e r t a i n histones (IV and I l b i , respectively) may be part of a mechanism involved i n binding newly synthesized histone to DNA, while the phosphoryl-ation and dephosphorylation of histone I may regulate the c o i l i n g of chromosomes during the c e l l cycle. The Chemistry and Biology of the Protamines; As mentioned above, the protamines were f i r s t i s o l a t e d from salmon sperm-atozoa almost a century ago by Miescher (8). About 20 years l a t e r , Kossel (81) showed that the protamines were made up of amino acids and therefore were proteins. Much of the sub-sequent work dealt with methods for t h e i r i s o l a t i o n , chemical characterization, and the d i s t r i b u t i o n of protamines or s i m i l a r proteins i n the nuclei of spermatozoa of d i f f e r e n t species (1,5,6,15). The d i s t r i b u t i o n of sperm-specific basic proteins i n evo l u t i o n a r i l y divergent species has been determined primarily by cytochemical staining techniques and, i n a minority of cases, by d i r e c t chemical i s o l a t i o n and characterization (5,6) . In only a few species have the sperm-specific proteins been adequately characterized by accurate amino acid analysis, s i z e , and amino acid sequence determination. Not a l l sperm-specific basic proteins are similar to the protamines f i r s t i s o l a t e d by Miescher. Bloch (6) has made a useful c l a s s i f i c a t i o n of the sperm-specific proteins into 5 groups by selecting those i n certain species as t y p i c a l examples of the protein type rather than by characterizing the proteins themselves. In order of decreasing b a s i c i t y , these are: (a) the salmon type with up to 2/3 of the amino acid residues as arginine and a molecular weight close to 5000; (b) the mouse and grasshopper type which are very r i c h i n arginine but more complex i n t h e i r amino acid composition and which are d i f f i c u l t to extract from c e l l s because of th e i r "keratinoid" nature; (c) the Mytilus (mussel) type whose proteins are intermediate i n nature between the histones and protamines (5); (d) the Rana (frog) type s i m i l a r to the histones i n somatic c e l l s ; and (e) the crab type which either do not possess a t y p i c a l histone (82) or have a histone of very low b a s i c i t y (5). In the f i r s t 4 types, the sperm nuclei are highly condensed; i n the l a s t (crab) type the n u c l e i are r e l a t i v e l y uncondensed and the proteins associated with the DNA are d i f f i c u l t to v i s u a l i z e by the normal staining procedures. In view of the v a r i a b i l i t y of basic proteins from sperm of d i f f e r e n t species, we s h a l l r e s t r i c t our discussion to the proteins (protamines) of the f i r s t 2 classes which are very arginine-rich and which seem to replace almost a l l of the t y p i c a l somatic histones during spermatogenesis. The protamines are sometimes generically named from t h e i r species of o r i g i n ; thus salmine comes from salmon, clupeine from herring (Clupea), sturnine from sturgeon, i r i d i n e from rainbow trout (Salmo irideus) and g a l l i n from domestic fowl (Gallus domesticus). The amino acid compositions (15) of the protamines are most unusual. In the salmonids, close to 2/3 of the amino acid residues are arginine; the remainder consists primarily of serine, p r o l i n e , glycine and alanine with lesser amounts of other amino acids. Estimates of the molecular weight of prot-amine from various species vary; most are between 4000 to 8000 with a mean close to 5000 (15). That the protamines were not minor components of sperm was shown by Miescher (8). Careful analysis showed that about 80% of the dry weight of the salmon sperm was nucleo-protamine (8,83); of t h i s , 2/3 was DNA by weight and another 1/3 was protamine (15). The protamines are bound to DNA through s a l t linkages. P o l l i s t e r and Mirsky (83) showed 16. that nucleoprotamine could be completely dissolved by 1 M NaCl and the protamine removed completely from DNA by d i a l y s i s . The DNA solution retained i n the d i a l y s i s sac was as viscous as the o r i g i n a l solution. These experiments indicated the high molecular weight of DNA, the small s i z e of protamine, and the i o n i c nature of t h e i r i n t e r a c t i o n s . There i s a near but not exact equivalence of the number of arginyl residues and DNA phosphoryl groups i n salmon sperm (15), i n d i c a t i n g that a probable function of protamine i s the n e u t r a l i z a t i o n of the negative charges of DNA which would allow the complex to pack t i g h t l y . From the DNA content (2.5 x 10~ 1 2g) of trout sperm (84), about 70 cm of DNA must be packed i n a nucleus of volume 10.4 cubic microns (83). The e a r l i e s t workers i n the f i e l d recognized that the protamines were not homogeneous proteins (15). Development of modern chromatographic techniques led to resolution of homo-geneous species of protamine i n quantities adequate for chemical characterization and sequence studies (85,86). Clupeine was resolved into 3 components, Y^ V J J » and Z , which were very s i m i l a r i n amino acid composition and s i z e . Iso-l a t i o n of a l l 3 components from an i n d i v i d u a l f i s h indicated that t h i s heterogeneity was not due to genetic polymorphism i n a population of f i s h (87). A greater degree of heterogeneity i s indicated i n the domestic fowl: at least 8 to 10 components of g a l l i n have been resolved which have sim i l a r arginine con-tents but d i f f e r i n the percentages of the lesser amino acids and the size of the molecules (88). Early attempts to determine the amino acid sequences of the protamines were hindered (a) by t h e i r heterogeneity and (b) t h e i r high arginine contents which suggested that tracts of arginyl residues might e x i s t . Before the f i r s t sequences of the protamines were known, the idea was put forward there might be a regular, repeating structure i n which blocks of 4 arg i n y l residues were separated by pairs of non-basic amino acids (89). Such t r a c t s of arg i n y l residues would allow the f u l l y extended polypeptide chain of protamine to l i e i n the minor groove and wrap around the DNA double h e l i x with the maximum number of arginyl residues i n t e r a c t i n g i o n i c a l l y with the negative charges of the DNA phosphates. Pairs of neutral residues would "loop out" between the blocks of 4 arginines (89). However, I s h i i et a l . (90) l a t e r showed that mono, d i , t r i , and t e t r a a r g i n y l sequences existed i n protamine. Ando's group at the University of Tokyo has completed the sequences (Fig. 2) of 3 components of clupeine (91-93) and 3 components of i r i d i n e (86). Of the 32 to 33 amino acid residues i n the 3 components of i r i d i n e , 21 to 22 are arginyl-and 4 are s e r y l residues. A fourth component of i r i d i n e was also re-solved but i t was heterogeneous and not sequenced (86). This component has 3 s e r y l residues. The i r r e g u l a r d i s t r i b u t i o n 18. Clupeine Y-I H-Alo A A A A Ser Ser SenA -Pro -1 la • A- A A-A P r o - A A A-Thr Thr- A-A A -A -A la Gly A A A-A -OH Clupaino Y-II H-Pro A A A - - T h r A A-Ala Ser A Pro Vol A A A A P r o • A A - • Vol Ser A-A A A Ala A A A A -OH Clupeine Z H-Ala A A A - A - S e r -A- A-Ala • Ser A Pro- Vol • A • A A A - P r o • A - A - - Va lSer A-A-A-A-Ala A A - A - A - O H B Iridine lb H - P r o A A A A A A S e r S e r S e r A P r o • I la A A A - A - Pro A A V a l S e r A A A A • A- - Gly • Gly A A A A - OH Iridine II H-Pro A - A A A Ser Ser Ser APr o Val A A A • A - - A l a A A V o l - S e r A A • A A A • A Gly Sly • A • A A A -OH la rb II Arg 22 22 21 Ser 4 4 4 Pro 3 3 2 Ala 1 Gly 2 2 2 Vol 2 1 2 l ie 1 Total 33 33 32 F i g . 2. Amino acid sequences of 3 components of clupeine (91-93) and i r i d i n e (86). The sequences are aligned so as to obtain maximum homology between the components (112). The compositions of the i r i d i n e components are shown i n C. of a r g i n y l residues within the protamines would seem to i n -dicate that the interactions of protamine with DNA are not as simple as o r i g i n a l l y conceived. A r t i f i c i a l complexes of DNA with varying proportions of protamine have been formed by annealing at high s a l t concen-t r a t i o n (94,95). The nature of the complexes was followed by monitoring the increase i n hyperchromicity of DNA upon gradual increase of the temperature of the sol u t i o n . B i -phasic melting p r o f i l e s were found i n which the lower T m t r a n s i t i o n was si m i l a r to free DNA and the higher t r a n s i t i o n was very close to that for DNA-polyarginine complexes (94, 95). In addition, the complexes could be separated by cen-t r i f u g a t i o n into a p e l l e t i n which the DNA phosphates are completely neutralized and a supernatant containing essen-t i a l l y uncomplexed DNA (94,95). These data indicate that the binding of protamine to free DNA i s highly cooperative with formation of completely complexed DNA by p r e f e r e n t i a l bind-ing of protamine to s i t e s adjacent to those already occupied by the peptide (94,95). Miescher was unable to i s o l a t e protamine from immature testes which contain large proportions of stem c e l l s and sperm-atocytes (8). In 1956, A l f e r t (96) used cytochemical s t a i n -ing procedures to show that the protamines appeared at a middle stage of spermiogenesis (maturation of the spermatid) and associated with t h e i r appearance was the loss of material with the staining properties of histone. The l a s t histones removed appeared to be arginine-rich by these procedures. Early workers could not est a b l i s h whether the protamines were newly synthesized from amino acid precursors or i n some way were the breakdown products of histones leaving an arginine-r i c h core, protamine (15). However, by autoradiography Bloch and Brack (97) studying spermiogenesis i n the grasshopper, and Monesi (98) studying the same process i n the mouse, showed that i n the middle-stage spermatids, there was the rapid synthesis of an arg i n i n e - r i c h protein, presumably a protamine. Ingles et a l . (99) studied spermatogenesis i n trout testes a r t i f i c i a l l y induced to mature by i n j e c t i o n of salmon p i t u i t a r y extracts. They showed that protamine bio-synthesis was i n h i b i t e d by i n h i b i t o r s of protein synthesis such as cycloheximide and puromycin. Ling et a l . (100) i n -cubated trout testes c e l l s with labeled arginine and showed that protamine was synthesized i n the cytoplasm on small ribosomes (disomes) and rapidly transported into the nucleus. These data indicated that protamines were synthesized de novo by the usual mechanisms involving messenger RNA, transfer RNA, and ribosomes. An i n t e r e s t i n g finding by Wigle and Dixon (101) was the incorporation of a methionyl residue at the NH2~terminus of nascent protamine and i t s l a t e r removal, thus accounting for i t s absence from protamine i s o l a t e d from mature spermatozoa. This also indicated that methionine 21. might have a role i n the i n i t i a t i o n of protein biosynthesis i n eucaryotic organisms (101). Additional evidence that protamine i s synthesized i n the cytoplasm was presented by Gilmour and Dixon (102). Using a trout l i v e r (which does not synthesize protamine i n vivo) c e l l - f r e e system, they showed that an exogenous low-molecular-weight RNA f r a c t i o n from trout t e s t i s polyribosomes can stimulate the incorporation of [* ^ C] arginine into prot-amine. Methionine was also incorporated at the NH2-terminus of the i n v i t r o labeled protamine i n d i c a t i n g that methionine was involved i n both the i n vivo and i n v i t r o i n i t i a t i o n of protamine biosynthesis. The presence of small amounts of phosphoserine i n com-mercial preparations of protamine was f i r s t noted by Murray (103). Later, Ingles and Dixon (104) found that i n the early protamine stage of trout testes development, approximately 75% of the s e r y l residues of protamine were phosphorylated, whereas i n mature spermatozoa (milt) only 5 to 6% of the s e r y l residues were modified. Moreover, a l l the s e r y l residues newly incorporated into protamine were phosphorylated (104). These discoveries were confirmed and extended by Marushige et a l . (105) who found that newly synthesized protamine eluted with phosphoprotamines from carboxymethyl-cellulose columns. These data pointed towards a cycle of enzymatic phosphoryl-ations and dephosphorylations (10.4,105) . Marushige et a l . 22. (105) also showed that i n v i t r o , a cytoplasmic f r a c t i o n of testes c e l l s could synthesize and phosphorylate protamine. Moreover, J e r g i l and Dixon (106) is o l a t e d a protein kinase, protamine kinase, from the high-speed supernatant f r a c t i o n of testes homogenates. In 0.3 M NaCl, t h i s enzyme phos-phorylated protamine more rapidly than histones and was also stimulated by c y c l i c 3',5*-AMP (106). This evidence for the cytoplasmic l o c a l i z a t i o n of phos-phorylation of protamine led to several proposals for the possible function of phosphorylation (16). I t was suggested that protamine phosphorylation might be required either for the release of the highly basic protamine from the ribo-some by decreasing the net p o s i t i v e charge of protamine, or for i t s transport into the nucleus. Dephosphorylation of protamine was a slow process since newly synthesized protamine found on is o l a t e d chromatin was phosphorylated (105). Ingles and Dixon (104) characterized some of the s i t e s of phosphorylation of protamine from trout testes while Sanders and Dixon (107) continued these studies. I s o l a t i o n of phosphopeptides aft e r t r y p t i c digestion of 3 2 P - l a b e l e d protamine indicated that a l l 4 s e r y l residues of protamine could be phosphorylated i n vivo. In addition to the Val-Ser (P)-Arg sequence common to most of the trout testes protamines, 2 other highly phosphorylated peptides were characterized: a triphosphorylated peptide Ser(P)-Ser(P)-Ser(P)-Arg-Pro-Val-Arg presumably from the protamine component with 4 serines 23. and a diphosphorylated peptide, Ser(P)-Ser(P)-Arg-Pro-Val-Arg presumably from the protamine component with 3 serines (107). During the replacement process, the template a c t i v i t y of testes chromatin decreases d r a s t i c a l l y (108). Marushige and Dixon (108) analyzed the histones from trout testes at d i f f e r -ent stages of development and found that the ar g i n i n e - r i c h histones were removed f i r s t while the l y s i n e - r i c h histone I was removed l a s t . This was i n contrast to the i o n i c d i s -placement of histones from chromatin with increasing con-centrations of s a l t mentioned previously. Attempts to d i s -place histones from chromatin with added protamine led to the removal of histone I f i r s t (109,110); thus i t would seem that i o n i c mechanisms are not e n t i r e l y responsible for the sele c t i v e removal of histones from spermatids i n vivo. Phosphorylation of histones occurs i n trout testes at a stage when the t e s t i s i s a c t i v e l y synthesizing protamine (22,104,105). Phosphorylation of a l l 5 histone f r a c t i o n s , I, I l b i , IIb2, III, and IV was described by Sung and Dixon (22). Candido and Dixon (23) also found extensive acetyl-ation of 4 of the 5 histone fractions ( I l b i / I I b 2 , III; and IV) from trout testes. With the exception of phosphorylation of histone I a l l these modifications occur i n the highly basic NH 2-terminal region of these histones (38). These results seemed to indicate that phosphorylation and perhaps acetylation of histones might be important i n the removal process (22). Construction of a CPK molecular model of the DNA double h e l i x and the NH 2-terminal region of histone IV i n the cx- h e l i c a l conformation showed that t h i s region of histone IV could f i t into the major groove of DNA with the appropriate interactions of arginyl and l y s y l residues with the negative phosphates of DNA (22). Since acetylation of s p e c i f i c l y s y l residues and phosphorylation occur i n t h i s highly basic region, i t was postulated that the combined modification of t h i s region would s u f f i c i e n t l y loosen i t s binding to DNA and e f f e c t the removal, or loosen the binding so that proteo-l y s i s could occur (22,109). In t h i s connection, a histonase (protease s p e c i f i c for histones) was found i n trout testes by Sanders and Dixon (111). An i n t e r e s t i n g example of histone removal has been found i n the decapod crabs (Emerita analoga). In t h i s species (5,82) the entire complement of histones appear to be t o t a l l y removed from the DNA and the histones are found apparently undegraded i n a cytoplasmic capsule. I t would seem, then, that i n some species, there are mechanisms for histone removal which do not involve p r o t e o l y s i s . From evidence presented i n t h i s t h e s i s , i t would appear that the mechanisms for the removal of histones from DNA and the binding of protamine are far from understood. The possible functions of these sperm-specific proteins have been considered by Bloch (6) who has t r i e d to correlate 25. protein type and possible function with phylogenetic r e l a t i o n -ships of the numerous species examined. Several possible functions were examined. These include the p o s s i b i l i t i e s that the highly basic proteins (a) cause the condensation of chromatin, shrinkage of the nucleus, and consequent stream-l i n i n g of the c e l l , (b) i n h i b i t gene t r a n s c r i p t i o n , (c) erase the developmental history of the c e l l thus providing a t o t i -potent nucleus, and (d) protect the chromosome from adverse ef f e c t s of the environment. Black and Dixon (112) have suggested that the protamines evolved through a series of p a r t i a l gene duplications and mutational events from an archetypal pentapeptide, Ala-Arg-Arg-Arg-Arg. In view of the s t r i c t conservation i n amino acid sequence of the arginine-rich histones from species d i s t a n t l y removed on the evolutionary scale, the variations i n amino acid sequence of the protamines and heterogeneity i n type of sperm-specific basic proteins suggest that there are few selective pressures for d i r e c t i n g the evolution of these proteins beyond the necessity of s u f f i c i e n t b a s i c i t y to neutralize the negative charges of the DNA phosphates. Bloch (6) has proposed "that the v a r i a b i l i t y (non-conservation) of the protein r e f l e c t s an evolutionary indifference to a r e l -a t i v e l y unimportant protein i n an i n e r t nucleus. The gen-e r a l l y high arginine contents of these proteins may be a t t r i b -uted to the fact that there are more t r i p l e t s that code for arginine than for lysine and h i s t i d i n e . Given genetic d r i f t , arginine may act as a s t a t i s t i c a l trap". The Process of Spermatogenesis: The immature t e s t i s consists of connective tissue and spermatogonia (stem c e l l s ) of which several types (A, intermediate, and B) can be distinguished c y t o l o g i c a l l y (17,18). Spermatogonia undergo a series of mitotic c e l l d i v i s i o n s which f i n a l l y give r i s e to primary spermatocytes. The primary spermatocytes undergo a process c a l l e d meiosis (two rapid c e l l d i v i s i o n s without an i n t e r -vening period of DNA synthesis) giving r i s e to four sperm-atids with half the somatic number of chromosomes and a haploid complement of DNA. The spermatids no longer divide but d i f f e r e n t i a t e to form the spermatozoan or mature sperm. The entire process from stem c e l l to sperm i s c a l l e d sperm-atogenesis while the d i f f e r e n t i a t i o n of the spermatid i s c a l l e d spermiogenesis (17,18). The r e l a t i o n s h i p of the d i f f e r e n t c e l l types involved i n spermatogenesis i s shown i n F i g . 3. The early stages of spermatogenesis are l i t t l e under-stood (17,18,113). The lack of obvious morphological changes and hence d i f f i c u l t y i n i d e n t i f y i n g spermatogonia at d i f f e r e n t stages of development has only recently been circumvented by use of radiotracers such as [ 3H]thymidine to l a b e l c e l l s syn-thesizing DNA and following the lineage of the labeled c e l l s (18,114). Microscopically, at least 3 types of spermatogonia J L [^Primary Spermatocyte ••B i*S econ dory Spermatocyte l ^ 2 0 _ r » Spermatid >•» Spermatid » Sperm Mitosis . Meiosis * Spermiogenesis Spermatogenesis F i g . 3. Relationship of the d i f f e r e n t c e l l types involved i n spermatogenesis. can be distinguished by c a r e f u l staining and noting of the r e l a t i v e positions of the c e l l s . These are the A, i n t e r -mediate, and B type spermatogonia (17,18,113). The A c e l l s are r e l a t i v e l y undifferentiated (predefinitive) while the B c e l l s are committed to divide and give r i s e to 2 daughter primary spermatocytes. In t h i s sense, the B spermatogonia are "committed" and t h e i r d i v i s i o n i s a d e f i n i t i v e one. Continuous m u l t i p l i c a t i o n of the A spermatogonia re-plenishes the stock of c e l l s which have become committed to the spermatogenetic pathway. The number of p r e d e f i n i t i v e c e l l d i v i s i o n s which occur i n a l i n e of c e l l s before a def-i n i t i v e d i v i s i o n occurs and the possible mechanisms or modes of stem-cell renewal h)awbeen reviewed by Hanna-Alava (113) and Courot et a l . (18). The l a t e r stages of spermatogenesis are marked by the transformation of the spermatid into sperm. This trans-formation involves the loss of a considerable proportion of the cytoplasm, the condensation of the chromatin into a very condensed state often accompanied by elongation of the nucleus and development of a propulsive organelle, the flagellum (17,18). In mammalian species, maturation of the spermatid occurs i n close association with the S e r t o l i or nurse c e l l s (17). However, i n many f i s h there i s no evidence for nurse c e l l s or t h e i r analogues (115) . The loss of cytoplasm i s associated with a d r a s t i c de-crease i n the RNA content of the spermatid (116,117). In mature sperm, the content of RNA i s very low (117). RNA synthesis, as measured by incorporation of t 3H]uridine f o l -lowed by autoradiography, f a l l s as the spermatid matures (98). By the time protamine synthesis begins, no RNA syn-thesis i s detectable (98). This raises questions as to the time of synthesis of the messenger RNA which codes for the sperm-specific basic proteins. I t i s possible that the periods (98) of enhanced RNA synthesis i n primary spermato-cytes at the prophase stage of meiosis may code for masked messengers which are activated i n the spermatid. The condensation of the spermatid nucleus i s associated with the loss of histone and appearance of the sperm-specific protein (96). The mechanism of the replacement, the v a r i a -tions i n packing of chromatin, and elongation of the nucleus are not understood. In mammalian, but not salmonid sperm-ati d s , there i s the formation of a nuclear cap c a l l e d the acrosome (17). Clermont and Leblond have described 19 stages of spermiogenesis i n rat testes based upon the staining and appearance of the developing acrosome (118). S t r i k i n g changes occur i n the volume of c e l l s under-going spermatogenesis, e s p e c i a l l y during meiosis and the l a t e stages when the spermatid nucleus condenses (Table I I I ) . Techniques have been developed for the separation of c e l l s TABLE III Variations i n the Germ C e l l Nuclear Volume, Expressed i n y 3 [from Courot et a l . (18)] Species Rat Ram Bull Spermatogonia A, 212 268 332 A i 212 229 555 A, 212 477 A, 180 In 151 195 356 B, 137 150 278 B, 144 212 Spermatocytes Preleptotene • 74 • 87 203 Leptotene 195 119 222 Zygotene •— 144 230 Pachytene 1 — 171 268 Pachytene 2 — 288 325 Diplotene 624 321 445 II Spermatocytes 299 258 292 Spermatids 1 Spermatids (st. 4 and 5) 92 67 90 2 Spermatids (6-8) 151 97 120 V. diplotene spermatocyte 2.9 1.2 1.3 V. A i spermatogonium V. diplotene spermatocyte 3.2 2.7 2.2 V. leptotene spermatocyte V. diplotene spermatocyte 6.7 4.8. 4.9 V. spermatid (1) 31. based upon volume differences (119). Application (120) of these techniques to mouse testes c e l l s labeled with [3H] thymidine have shown that a f t e r the f i n a l period of DNA synthesis, the volume of the primary spermatocyte rapidly increases as the c e l l s enter meiotic prophase (120). After several days the primary spermatocyte undergoes 2 rapid d i v i s i o n s i n sequence producing 4 spermatids. This i s assoc-iated with a fo u r - f o l d decrease i n volume of the labeled c e l l s . Subsequent developments considerably decrease the volume of the early spermatid (120). The chronology of spermatogenesis, that i s , the d e f i n i t i o n of, the temporal rel a t i o n s h i p of the various c e l l types i n terms of the times of c e l l d i v i s i o n and morphological changes, has been described i n d e t a i l for only a few species (Table IV). In mammals i t takes about 30 to 40 days to go from the late primary spermatocyte to mature sperm while estimates of the entire process are s l i g h t l y longer, 40 to 60 days. There i s as yet l i t t l e information on the temporal r e l a t i o n -ship of the d i f f e r e n t c e l l types involved i n spermatogenesis i n the salmonids or related species. To some extent, t h i s lack of precise information has hindered our understanding and a b i l i t y to correlate the morphological with biochemical events. Most fishes undergo an annual cycle of testes growth and decline p r i o r to mating. Much of the research i n trout TABLE IV Comparison of Data on the Duration of Spermatogenesis i n Animals [from Roosen-Rtinge (142) ] Total Species- Spermatogonia Spermatocytes Spermatids days Jellyfish (Roosen-Runge, unpublished) ( l ? ) 8 Approx. 1 Approx. 1 3 Crustacean ' 7-8 7-8 5-6 20 Silk moth ' ' 4-5 10-12 ' 5-6 20 Grasshopper - 8-9 9-10 10 ' ' ' • 28 Drosophila ? . 4 5 10? Medaka ? at25C 5 at IS C 12 7 8 ? ? Mouse . ^ Rat 7 13-14 14-15 34.5 8-9 18-19 20-21 48 Ram (8-9?) 16-17 13-14 41.6 Man (24-26?) 27-28 22-23 75 ° The duration of the spermatogonial stage is often dubious, and depends greatly on the definition of its beginning, and whether a "stem cell cycle" is included or not. as opposed to salmon i s due to the a v a i l a b i l i t y of f i s h of defined stock and varying stages of testes maturity at d i f f e r -ent times of the year. I t i s possible to induce spermato-genesis i n sexually immature trout by i n j e c t i o n s (121,122) of salmon p i t u i t a r y extracts and thus obtain testes for study at a l l times of the year. Spermatogenesis i n f i s h i s profoundly influenced by gonadotropins elaborated by the p i t u i t a r y gland (19). Two separate gonadotropins, f o l l i c l e - s t i m u l a t i n g hormone (FSH) and l u t e i n i z i n g hormone (LH) can be detected i n most ver-tebrates . However, evidence for the presence of the two hormones i n tel e o s t fishes i s not conclusive (19). FSH a c t i v i t y seems to be very low while the a c t i v i t y of LH can account for most of the e f f e c t s of crude p i t u i t a r y extracts. Robertson and Rinfret (121) were the f i r s t to show that i n j e c t i o n s of salmon p i t u i t a r y extracts into sexually immature rainbow trout lead to a dramatic (500 to 1000 fold) increase i n testes wet weight over a two month period. This response of the immature t e s t i s was developed into an assay for gonadotropic a c t i v i t y of salmon p i t u i t a r y glands by Schmidt et a l . (122). P i t u i t a r i e s from sexually mature salmon con-tained more gonadotropic a c t i v i t y than those from immature f i s h (122). The response of the immature testes to the extracts was a logarithmic function of the dose over a two week period (122). The system of hormonal injections developed by Schmidt et a l . (122) was used by Ingles (123) to stimulate immature trout testes to grow to f u l l maturity. Such testes were a convenient source of material during the o f f season. Ingles et a l . (99), and l a t e r Marushige and Dixon (108) monitored the testes for appearance of protamine and loss of histone. Protamine appeared 45 to 55 days aft e r beginning the twice-weekly series of i n j e c t i o n s . More recently, Donaldson et a l . (124) have shown that spermatozoa produced from a 10 to 12 week series of i n j e c t i o n s of p u r i f i e d salmon gonadotropin i n t o immature salmon were as e f f e c t i v e i n f e r t i l i z i n g eggs as spermatozoa from naturally maturing f i s h . Thus, i t appears that the products of spermatogenesis induced by these i n j e c -tions are normal i n a l l respects. The target c e l l s and mechanism of action of the p i t u i -tary gonadotropins are as yet l i t t l e understood (19). In f i s h , i t appears that the early stages of spermatogenesis, those of stem c e l l p r o l i f e r a t i o n and d i f f e r e n t i a t i o n which give r i s e to primary spermatocytes, are the most susceptible to the gonadotropins, while spermatid maturation can continue i n the absence of these hormones (19,145). In t h i s t h e s i s , attention has been focused on the syn-t h e s i s , appearance, and enzymatic modifications of the prot-amines i n the d i f f e r e n t c e l l types of trout t e s t i s i n an attempt to understand more f u l l y the biochemical events at 35. the terminal stages of spermatogenesis when the histones are replaced by the protamines. The c e l l sedimentation method developed by M i l l e r and P h i l l i p s (119) was used to separate testes c e l l s at d i f f e r e n t stages of development. The c e l l s were characterized by t h e i r DNA and RNA contents and by the complement of basic proteins i n the nucleus. The spermatid stages synthesizing and phosphorylating protamine were iden-t i f i e d and correlated with the loss of histone and decrease i n c e l l volume. The large number (6 to 8) of protamine species observed i n spermatids which had j u s t begun to synthesize protamine compared to the 2 main species found i n mature sperm led to an i n v e s t i g a t i o n of the k i n e t i c s of enzymatic modifications order of the protaminesAto explain the relationships of the d i f f e r -ent species. The r e s u l t s of these experiments led to a new view of the significance of protamine phosphorylation and dephosphorylation. Thus, i t was previously postulated that histone phosphorylation was involved i n the replacement of histones by protamines (49). To prove or disprove t h i s hypothesis, the c e l l types synthesizing and phosphorylating histones were investigated. The results were contrary to the hypothesis and prompted an examination of the biology of the histones i n trout t e s t i s i n an attempt to understand the functions of the observed modifications (phosphoryla-t i o n and acetylation) of the histones. The conclusions 36. reached from studying the k i n e t i c s of enzymatic modification of the histones appear to have a more general significance than previously r e a l i z e d . PART I: TROUT TESTIS CELLS 37. EXPERIMENTAL PROCEDURES I. Materials, Abbreviations, and Definitions (a) Materials: A l l chemicals obtained commercially were of the highest purity or reagent grade. [ 3H]thymidine (spec. act. 5 Ci/mmole, DL-[ 3H]arginine (spec. act. 12 Ci/mmole), [**C]arginine (spec. act. 312 mCi/ mmole) , [ l l*C]lysine (spec. act. 312 mCi/mmole) , [ 3H]lysine (spec. act. 12 Ci/mmole) and NCS S o l u b i l i z e r were obtained from Amersham-Searle; c a r r i e r - f r e e inorganic [ 3 2PJphosphate from Atomic Energy of Canada, Ltd.; p e n i c i l l i n and strep-tomycin from Baltimore B i o l o g i c a l s ; bovine serum albumin, Fraction V, Grade B from Calbiochem and Sigma; Nonidet P-40 from S h e l l O i l ; Electrostarch from Otto H i l l e r , Madison, W i s e , U.S.A.; Connaught Starch from Connaught Laboratories, Toronto, Canada; glass f i b r e f i l t e r s (Ap-200-2500 and Ap-200-4700) from M i l l i p o r e F i l t e r Corp., Bedford, Mass., U.S.A.; petrolatum j e l l y from E l i L i l l y & Co.; the pl e x i g l a s s c e l l sedimentation chamber as described i n (119), the water-cooled starch gel tray and variable-thickness gel s l i c e r , from Richmond S c i e n t i f i c Co., Richmond, B.C., Canada. (b) Source of Testes; Sexually immature rainbow trout (S. G a i r d n e r i i ) ^ weighing approximately 100 g (age about 1 year) * Salmo iri d e u s and S. g a i r d n e r i i are considered to be iden-t i c a l species, the former nomenclature being more commonly used i n Europe and Japan, and the l a t t e r i n North America. to 200 g (age about lh year) were obtained from Sun Valley Trout Farm, Mission, B.C. The husbandry of these f i s h has previously been described i n d e t a i l (123). The f i s h were housed i n aquaria at the Department of Biochemistry, Univ-e r s i t y of B r i t i s h Columbia i n fresh running water (2 to 4 l i t e r s per min) at constant temperature (12 to 13°) . These f i s h were subjected to twice-weekly i n j e c t i o n s of a standard p i t u i t a r y extract (122) prepared from p i t u i t a r i e s of spawn-ing chinook salmon (Oncorhynchustshawytscha) obtained from Green River Salmon Hatchery, Auburn, Washington. P i t u i t a r y extracts were prepared by homogenizing 1 volume of tissue with 3 volumes of 0.15 M NaCl i n a Waring Blendor and cen-t r i f u g i n g twice at 15,000 x g for 10 min. The 15,000 x g supernatant was frozen i n 5 ml aliquots and thawed as re-quired for the i n j e c t i o n s . Testes at d i f f e r e n t stages of development were obtained at d i f f e r e n t times aft e r i n i t i a t i o n of the i n j e c t i o n s . These w i l l be referred to as " a r t i f i c i a l l y induced" or "hormonally induced" testes as opposed to those maturing natur a l l y . (c) Abbreviations: For the sake of c l a r i t y , abbrevi-ations have been kept to a minimum. When used, t h e i r mean-ing i s explained i n the body of the text where they are f i r s t encountered. TMKS:- an i s o t o n i c medium containing Tris-HCl (50 mM, pH 7.4) MgCl 2 (1 mM), KC1 (25 mM) and sucrose (0.25 M) . TMKS-0.1% glucose:- a medium containing the same ingredients as TMKS with glucose added. TMK:- a buffer containing magnesium and potassium i d e n t i c a l with TMKS except that i t lacks sucrose. Phosphate-buffered s a l i n e : - an isotoni c buffered medium (pH 7.2) containing 0.14 M NaCl, 2.7 mM KC1, 8 mM Na 2 HPOi» 1.5 mM KH2PCH, 0.9 mM CaCl 2 , 0.5 mM MgCl 2. BSA:- Bovine serum albumin. PCA:- Perchloric acid. TCA:- T r i c h l o r o a c e t i c acid. PPO:- 2,5-diphenyloxazole. POPOP:- 1,4,-Bis-(5-phenyloxazolyl)-benzene. Sv:- Sedimentation ve l o c i t y constant i n mm per hr of c e l l s sedimenting i n serum albumin gradients at one gravity. I I . Characterization by DNA and Protein Analysis of C e l l s Separated by Velocit y Sedimentation (a) Preparation of C e l l Suspensions: Excised testes were minced with scissors i n 3 volumes of TMKS-0.1% glucose and c e l l suspensions were prepared by gentle hand homogen-i z a t i o n (3 strokes up-and-down) i n a Potter-Elvehjem hom-ogenizer with a Teflon pestle. The c e l l suspensions were f i l t e r e d through four layers of cheese c l o t h . Because of the large proportion of connective tissue i n testes at early stages (3 to 5 weeks), the y i e l d of testes c e l l s i s low (10 to 30%) at these times; the yi e l d s rose s i g n i f i c a n t l y (to greater than 90%) as the t e s t i s continued to hypertrophy and the number of c e l l s undergoing spermatogenesis increased. (b) Incubation of C e l l s ; The incubation mixtures (1 ml t o t a l volume) consisted of 3 to 5 x 10 8 c e l l s , labeled pre-cursor (one or two of the following: 30 to 50 uCi of [3H] thymidine, 40 to 100 uCi of [ 3H]arginine, 15 to 20 uCi of [l **C]arginine, 100 yCi of [3H] l y s i n e , 7.5 uCi of t 1 "C] l y s i n e , phenol red, and TMKS-0.1% glucose to 1.0 ml. Included i n incubations longer than 2 hours were 100 units of p e n i c i l l i n and streptomycin and 0.1 ml^Waymouth1s medium (125) with 10 mM Tris-HCl buffer (pH 7.2) instead of phosphate buffer. In-cubations were carr i e d out at 15 to 16° on a gyratory water bath and were stopped by d i l u t i n g with 20 ml of cold phosphate-buffered saline and centrifuging at 1500 x g for 10 minutes. The incorporation of [ 3H]arginine was l i n e a r up to 20 hr i n the c e l l suspensions (Fig. 4). After r i n s i n g the walls of the centrifuge tube and c e l l p e l l e t with fresh phosphate-buffered s a l i n e , the c e l l s were resuspended i n 10 ml of phosphate-buffered saline with 15 to 20 gentle strokes i n the Potter-Elvehjem homogenizer to disperse any clumps of c e l l s . (c) C e l l Separations: C e l l separation by v e l o c i t y sedimentation at unit gravity was carr i e d out as described by Lam et a l . (120) except that the bovine serum albumin (BSA) i n phosphate-buffered saline solution was t i t r a t e d to ..-PBS 30 10 O x oTMKS I ^•Waymouth's ^Hank's to 10 4 8 12  16 HOURS 20 24 F i g . 4. Time course of incorporation of [ 3H]arginine into c e l l s . A concentrated c e l l suspension was prepared from testes at the early protamine stage of development. An aliquot (0.15 ml) of the c e l l suspension (in TMKS) was d i -luted by the addition (0.30 ml) of one of the following solutions: phosphate-buffered saline (PBS), Waymouth's med-ium with Tris-HCl (pH 7.4) instead of phosphate buffer, TMKS, or Hank's balanced s a l t s o l u t i o n . The incubation was i n i t -i a ted by the addition of 50 y l of [ 3H]arginine. The f i n a l concentration of c e l l s and la b e l was 7.5 x 10 8 c e l l s per ml and 66 yCi of [ 3H]arginine per ml. At various times 20 y l (1.5 x 10 7 c e l l s ) were removed and di l u t e d i n 1 ml of TMKS. Ce l l s were preci p i t a t e d with 3 to 4 volumes of 95% ethanol, coll e c t e d on f i l t e r s , and washed with 5% t r i c h l o r o a c e t i c acid-0.25% tungstate (126) followed by 95% ethanol. Toluene s c i n t i l l a t i o n f l u i d (2 ml) was added to the dried f i l t e r s and the f i l t e r s were analyzed for r a d i o a c t i v i t y . Addition of glucose to a f i n a l concentration of 0.1% or Eagle's vitamin mixture did not af f e c t the incorporation of [3H] arginine i n the presence of PBS, TMKS, or Hank's balanced s a l t solutions. The reason for the enhanced incorporation of 3H i n the presence of PBS over that i n TMKS or Hank's balanced s a l t solution i s not known. The lower incorporation of 3H i n Waymouth's medium i s due to the presence of unlabeled arginine present i n the formulation (125). pH 7.2 with 1 M NaOH and f i l t e r e d through a glass f i b r e f i l t e r (AP-200-4700). The gradient was loaded with 20 ml of phosphate-buffered saline followed by 6 to 10 x 10 7 c e l l s i n 10 to 15 ml of 0.5% BSA i n phosphate-buffered s a l i n e . A 5 ml buffer-layer of 0.75% BSA (119) separated the sample layer from the 600 ml l i n e a r gradient (1 to 3%) of BSA. After sedimenting for 260 min or 560 min at 4°, the lower 100 ml was run out and discarded. Seventy-five 7 ml fractions (1 fr a c t i o n per 30 sec) were then c o l l e c t e d . The l a s t f r a c t i o n collected corresponds to the top of the gradient and was lab-eled Fraction 1. C e l l s were counted on a hemocytometer or al t e r n a t i v e l y / on a Coulter Model B counter. (d) Radioactivity Analysis; For studies of [3H] thymidine incorporation into nucleic acids, t o t a l acid-insoluble radio-a c t i v i t y incorporated into c e l l s was determined by f i l t e r i n g an aliquot of the formaldehyde-fixed BSA c e l l suspension (see section "e") through a glass f i b e r f i l t e r ( Millipore AP-200-2500). The c e l l s c o l l e c t e d on the f i l t e r were washed with phos-phate-buffered saline (containing 3.7% formaldehyde), 5% t r i -chloroacetic acid, and 95% ethanol. The f i l t e r s were dried at 60° for 30 min before addition of 1 to 2 ml of toluene s c i n t i l l a t i o n f l u i d (0.1 g of POPOP, 4 g of PPO per l i t r e of toluene). In some early studies, c e l l s were not fixed with formaldehyde, and form-aldehyde was omitted from the phosphate-buffered saline wash. For determination of labeled arginine or lysi n e incorporated i n t o n u c l e i , an aliquot of the BSA c e l l suspension (unfixed) was passed through a f i l t e r . The c e l l s c o l l e c t e d were washed with phosphate buffered s a l i n e , p r e c i p i t a t e d with 5% t r i c h l o r o a c e t i c acid -0.25% sodium tungstate adjusted to pH 2.0 (126) , and washed with 95% ethanol. The f i l t e r s were dried as above for 30 minutes before addition of 1 to 2 ml of toluene s c i n t i l l a t i o n f l u i d . Control studies showed that labeled DNA, histone and protamine were pre c i p i t a t e d by these methods. (e) DNA and RNA Determinations; For determination of the DNA and RNA contents of the d i f f e r e n t c e l l types sep-arated by v e l o c i t y sedimentation, one-tenth volume (0.7 ml) of 37% formaldehyde was added to each c e l l f r a c t i o n from the gradient and quickly mixed on a Vortex Mixer. After removal of an aliquot (0.5 to 2 ml) for measurement of [ 3H]thymidine incorporation (where th i s was done) the desired fractions were pooled, the c e l l s per ml counted, and the t o t a l volume recorded. The pooled fractions were f i l t e r e d onto a glass f i b r e f i l t e r ( Millipore AP-200-2500) and the c e l l s on the f i l t e r were then washed (to remove most of the BSA) with 3 x 5 ml of phosphate-buffered saline containing 3.7% form-aldehyde, 3 x 10 ml of 10% t r i c h l o r o a c e t i c acid, and 3 x 5 ml of 95% ethanol. A modification (127) of the Schmidt-Thannhauser tech-nique (128) was used for the determination of the DNA and RNA contents of the d i f f e r e n t c e l l types. KOH (1.5 ml of a 0.5 M solution) was added to the s l i g h t l y damp (from ethanol) f i l t e r s containing the c e l l s i n a s c i n t i l l a t i o n v i a l . The capped v i a l was incubated at 37° for 1 to 2 hours (127) to hydrolyze RNA. At the end of the incubation, 0.75 ml of 1.6 M perchloric acid was added and the contents cooled i n ice for 30 minutes. The f i l t e r was removed, placed over another glass f i b e r f i l t e r , and the contents of the v i a l were f i l t e r e d through these two f i l t e r s . The v i a l and the f i l t e r s were rinsed twice with 0.37 ml of 0.25 M perc h l o r i c acid. The absorption spectrum of the f i l t r a t e ( t o t a l volume 3 ml) was read against the f i l t r a t e of a g l a s s - f i b e r - f i l t e r blank treated under i d e n t i c a l conditions. The RNA content per c e l l was determined by c a l c u l a t i n g the amount of RNA (25 A26O units = 1 mg hydrolyzed RNA) and di v i d i n g by the number of c e l l s on the f i l t e r . After removal of the RNA by alkaline hydrolysis as above, the two f i l t e r s containing the cold p e r c h l o r i c acid-insoluble material were incubated with 2 ml of 1 M perchloric acid at 80 to 85° for 15 min i n the same capped s c i n t i l l a t i o n v i a l and the contents cooled on i c e for 1 hour. The two f i l t e r s were removed, and placed over a clean glass f i b e r f i l t e r . The contents of the v i a l were f i l t e r e d through these three f i l t e r s . The absorption spectrum of the f i l t e r e d hydrolysate ( t o t a l volume ^ 2 ml) was determined against a g l a s s - f i b e r - f i l t e r blank treated i d e n t i c a l l y . The DNA content was determined by assuming that 27 A 2 6 0 units of hydrolyzed DNA equal 1 mg of DNA and dividing by the number of c e l l s on the f i l t e r to obtain the DNA content per c e l l . Control studies showed that i f the c e l l s were not fixed with formaldehyde beforehand, no RNA was detectable i n the f i l t e r e d c e l l s . Thus f i l t r a t i o n stripped unfixed c e l l s of t h e i r cytoplasm. DNA determinations on the hot p e r c h l o r i c acid-soluble material by the Dische diphenylamine reaction (129) gave i d e n t i c a l results to those determined by t h e i r absorption spectrum. (f) D i s t r i b u t i o n of Histones and Protamines and Their  Synthesis i n the Different C e l l Types: C e l l suspensions from testes at the protamine stage of development were incu-bated with' i1 kC]arginine and the labeled c e l l s separated by an extended sedimentation (10 hours) to resolve the spermatids of d i f f e r e n t s i z e s . Fractions (7 ml) were c o l -lected and an aliquot was removed for the determination of the t o t a l r a d i o a c t i v i t y incorporated into n u c l e i . The con-tent of nuclear basic proteins was determined i n the re-mainder of the f r a c t i o n . C e l l s were f i l t e r e d onto a glass f i b e r f i l t e r over an area the size of the starch gel sample s l o t (5 mm x 5 mm) by using a Teflon template, washed with phosphate-buffered s a l i n e , and fixed with ethanol. The f i l t r a t i o n stripped most of the c e l l s of t h e i r cytoplasm. 46 g of Electrostarch and 46 g of Connaught Starch were mixed and starch gels were prepared and poured in t o a gel tray f i t t e d for water cooling as previously described (130). The surface of the gel around the s l o t s was lined with petro-latum j e l l y and the slot s were f i l l e d with 0.4 M HC1. The c e l l s on f i l t e r s s l i g h t l y dampened with ethanol were inserted into the s l o t and a f t e r extraction for 15 to 30 min, the slots were f i l l e d with 0.4 M HCl to replace that absorbed into the gel. The contents were mixed by moving the f i l t e r up-and-down i n the s l o t before sealing with petrolatum j e l l y . Electrophoresis was conducted at 6 V per cm for 12 hours with water cooling at 6°. After electrophoresis, the gels were s l i c e d h o r i z o n t a l l y into 3 slabs. The middle slab was stained by the sensitive cobalt-Amido Black 10B procedure and destained with d i l u t e s u l f u r i c acid (130). Control studies showed that more than 90% of the histones are extracted, since removal and extraction of the f i l t e r s for a second time led to the appearance of l i t t l e or no detectable histones on starch gels. For autoradiography, the gels destained with s u l f u r i c acid were steeped i n 2% acetic acid to remove most of the s u l f u r i c acid. The gel was then washed with fresh 2% acetic acid but not to the stage where the protein bands are l o s t , and dried as described by Candido and Dixon (69) before exposure to Kodak Blue Brand Medical X-ray f i l m for 5 months at -20°. 47. I I I . Synthesis and Phosphorylation of Histones and Protamines i n the Different C e l l Types (a) Incubation and Separation of C e l l s ; The incubation mixtures consisted of 0.2 ml of Waymouth's medium (125) with 10 mM Tris-HCl buffer (pH 7.2) instead of phosphate buffer, 100 uCi of DL- [3H] arginine or 100 uCi of DL-[ 3H] l y s i n e , 300 yCi of c a r r i e r - f r e e inorganic [ 3 2P]phosphate, phenol red, 100 units of p e n i c i l l i n and streptomycin, 2 to 4 x 10 8 c e l l s , and TMKS-0.1% glucose to a f i n a l volume of 1 ml. After i n -cubating at 15 to 16° for 5 to 6 hours, the c e l l s were washed and 6 to 10 x 10 7 c e l l s were separated by v e l o c i t y sedimentation i n a 1 to 3% bovine serum albumin gradient at unit gravity for 5 or 10 hours. At the end of the sep-aration, fractions were c o l l e c t e d and the c e l l s per ml and r a d i o a c t i v i t y i n each of the fractions were determined. (b) Starch Gel Electrophoresis; C e l l s were co l l e c t e d onto glass f i b e r f i l t e r s , acid-soluble proteins were extracted from the c e l l s on the f i l t e r , and starch gel electrophoresis was conducted as described i n I I f to separate the extracted proteins. (c) Analysis of Radioactivity i n Starch Gels; For analysis of the r a d i o a c t i v i t y incorporated into the various histone and protamine f r a c t i o n s , the middle slab of the gel was cut into 2 mm s l i c e s . Each s l i c e was then incubated with 48. 0.5 ml of NCS S o l u b i l i z e r for 8 to 12 hr at room temperature. By the end of th i s period, the opaque gel had become trans-parent. Toluene s c i n t i l l a t i o n f l u i d (5 ml) was then added and the capped v i a l s incubated for a further 3 hr at 45° before counting. For autoradiography, the gels destained i n s u l f u r i c acid were steeped i n 2% acetic acid, washed, dried, and exposed to X-ray f i l m as before (Section I l f ) . RESULTS I. Characterization By DNA and Protein Analysis of C e l l s Separated by Velocity Sedimentation ( a) C e l l Separation P r o f i l e s and I d e n t i f i c a t i o n of  C e l l s Synthesizing DNA: According to the sedimentation v e l o c i t y equation (119) larger p a r t i c l e s sediment faster than smaller ones and i n the method developed by M i l l e r and P h i l l i p s (119), c e l l s of various sizes are separated by f l o a t i n g a t h i n band of c e l l s on top of a gradient of serum albumin (to prevent convection) and allowing the c e l l s to sediment at unit gravity for several hours. At the end of t h i s time, fractions are c o l l e c t e d from the bottom of the gradient and the c e l l s are counted eit h e r v i s u a l l y on a hemocytometer or e l e c t r o n i c a l l y . C e l l s are labeled according to t h e i r sedimentation v e l o c i t y constant, Sv, i n units of mm per hr. Thus, larger c e l l s have a larger Sv value and smaller c e l l s have a smaller Sv value. Because of the d i v e r s i t y of c e l l types involved i n sperm-atogenesis (Fig. 3) and the a v a i l a b i l i t y of trout testes at d i f f e r e n t stages of development from immature trout injected with p i t u i t a r y gonadotrophins (122,123), i t was of i n t e r e s t to examine the c e l l separation p r o f i l e s of testes at d i f f e r e n t stages of development and to determine which c e l l s were ac t i v e l y synthesizing DNA. Accordingly, c e l l suspensions from trout testes were incubated with [ 3H]thymidine to l a b e l c e l l s synthesizing DNA and sedimented on serum albumin gradients for 5 hr. F i g . 5A,B, and C shows the c e l l sep-aration and corresponding [ 3H]thymidine incorporation pro-f i l e s of c e l l s from testes at d i f f e r e n t stages of hormonal induction. In early stage testes (Fig. 5A) most of the c e l l s sed-iment i n a broad band between 2.5 and 4.5 mm per hr, while i n middle stage testes (Fig. 5B) another peak of c e l l s (1.5 Sv) appears. At a s t i l l l a t e r stage (Fig. 5C), the large peak of small c e l l s has a shoulder of even smaller c e l l s (1.0 Sv). The [ 3H]thymidine incorporation p r o f i l e s indicate that the large c e l l s (2.5 to 4.5 Sv) are a c t i v e l y synthesizing DNA while the small c e l l s (1.5 Sv and 1.0 Sv) synthesize very l i t t l e DNA. Thus the larger c e l l peak (2.5 to 4.5 Sv) prob-ably includes A and B stem c e l l s and primary spermatocytes while the peak of smaller c e l l s (1.5 Sv) i s composed of spermatids. The higher proportion of these small c e l l s (1.5 and 1.0 Sv) at l a t e r stages of maturation, suggests that these c e l l s are spermatids. (b) DNA and RNA Contents of Different C e l l Types; After a f i n a l DNA synthesis, the primary spermatocyte undergoes meiosis producing four spermatids. The DNA content per c e l l of the A and B stem c e l l s and primary spermatocyte p r i o r to DNA synthesis should be twice that of the haploid spermatid. I t was therefore of i n t e r e s t to determine the DNA and RNA S v (mm/hr) 7.8 6.4 5.0 3.6 2.6 12 300-600-E Z o. o 400-200-1 600-1 O x 400H z a. ° 200H 0 A. 35 days 60 | 9-5 , p15_> 3.5 . 2.8 - 60 - 40 - 20 Thymidine-*, C. 57 days 60 40 Fraction No TOP Fig. 5. C e l l separation and [ 3H]thymidine p r o f i l e s of c e l l s from d i f f e r e n t stages of hormonally induced testes. C e l l suspensions were incubated with [ 3H]thymidine and sedimented at unit gravity on serum albumin gradients for 5.0 hours. At the end of the separation, 7.0 ml frac t i o n s , each equal to 0.57 mm of gradient, were coll e c t e d and fixed with formaldehyde. An aliquot was removed and analyzed for r a d i o a c t i v i t y . Fractions were pooled as indicated and used for DNA and RNA determinations. Sedimentation i s from r i g h t to l e f t ; larger c e l l s sediment faster than smaller c e l l s ; sedimentation v e l o c i t y was determined from the middle of the sample layer. (A) 35 days i n j e c t i o n , stem c e l l stage; (B) 44 days i n j e c t i o n , early spermatid (preprotamine) stage; (C) 57 days i n j e c t i o n , early protamine stage. (—O-r— O— ) c e l l per ml; (—• •—) [3H] thymidine. contents of the d i f f e r e n t c e l l types and thus show unequiv-oc a l l y that the small 1.5 Sv and 1.0 Sv c e l l s were indeed spermatids. Table V shows the DNA and RNA contents of d i f f e r e n t trout testes c e l l s separated by v e l o c i t y sedimentation. Pooled fractions were fixed with formaldehyde and c o l l e c t e d on f i l t e r s ; nucleic acids were extracted from the c o l l e c t e d c e l l s and quantitated spectrophotometrically. Sperm from m i l t of naturally maturing trout, and f i s h red blood c e l l s which are nucleated, are included as controls for the haploid and d i -p l o i d DNA contents respectively. The value obtained for mature sperm by our procedure agrees very well with that determined by other workers (84,131). From Table V, the 1.5 Sv c e l l s have a haploid DNA content and are indeed spermatids, while the 2.8 and 3.5 Sv c e l l s have a d i p l o i d DNA content and are probably the stem c e l l s and primary spermatocytes. The larger 4.5 to 7.0 Sv c e l l s have close to a t e t r a p l o i d DNA content and may be c e l l s undergoing mitosis or at the prophase stage of meiosis. The larger c e l l s are also synthesizing DNA (as measured by [ 3H]thymidine incorporation, Table VI) faster than the smaller c e l l s . Relative to the c e l l s i n the 3.5 Sv region, the 1.5 Sv c e l l s incorporate 4%, the 2.8 Sv c e l l s ^ 40% and and the 6.0 Sv c e l l s ^ 180% as much l a b e l . I t w i l l be shown l a t e r that the rates of histone synthesis and phosphorylation TABLE V DNA and RNA Contents and RNA:DNA Ratios of Cel l s from Different Regions of Serum Albumin Gradients Testis cell suspensions were separated on serum albumin gradients. Fractions fixed in formaldehyde were pooled and total DNA and RNA was determined as described under "Experimental Procedure." The superscript numbers in the DNA columns represent the number of cells X 10~* used in that determination. Each value represents one determination. Stage" Cell size 7.0 Sv 6.0 Sv 4.5 to 5.0 Sv 3.5 Sv 2.8 Sv 1.5 Sv6 1.5 Sv« 1.0 Sv Mature sperm 0.6 Sv Trout red blood ceil DNA* 1 2 3 4 5 10.4*' 9.9" 11.5 8 ° 10.1«-' 13.7»-« 7.78.J 8.6U 9.8'» 5.9»-» 5.3"-« 5.4" 6.7»» 5.210 5.08-' 4.5" 5.16'-' 2.55"' 2.45" 2.48* 2.5 ± 0.2 4.9 ± 0.2 RNA* 1 2 3 4 5 4.1 5.4 4.7 4.6 2.9 3.6 4.4 3.1 1.8 2.1 2.0 1.7 1.4 1.6 1.4 1.55 0.38 0.23" 0.10 0.48 RNA: DNA 1 2 3 4 5 0.42 0.46 0.47 0.34 0.38 0.47 0.52 0.31 0.30 0.39 0.37 0.25 0.27 0.32 0.32 0730 0.15 0.10 0.04 0.10 • Stages 1, 2, 3, 4, and 5 correspond to 26, 35, 40, 44, and 60 days b 1.5 Sv spermatids which have not begun to synthesize prota. of hormonal induction, respectively. Stages 1, 2, and 3 are stem mine (preprotamine spermatids). cell stage testis, Stage 4 is early spermatid (preprotamine) stage • Mixture of preprotamine and protamine stage 1.5 Sv sperma-testis, and Stage 5 is early protamine testis. tids. * Picograms per cell. TABLE VI Relative Rates of DNA Synthesis i n C e l l s from Different Regions of Serum Albumin Gradients Testis cell suspensions were incubated with ['H]thymidine and separated on serum albumin gradients. An aliquot of each frac-tion was analyzed for radioactivity as under "Experimental Pro-cedure." Specific incorporation of ['H]thymidine (counts per min per 10* cells) was determined for each fraction. A mean value averaged over three to five fractions and expressed relative to 3.5 Sv cells is indicated for each cell size. Days of hormonal induction Testis stage Cell size 6.0 Sv 4.5 Sv 3.5 Sv 2.8 Sv 1.5 Sv 35 Stem cell 1.4 1.0 0.40 35» Stem cell 1.7 1.3 1.0 0.45 44* Preprotamine spermatid 2.0 1.4 1.0 0.40 0.02 57" Early protamine 1.0 0.50 0.006 • " Same as Fig. %A, B, and C, respectively. p a r a l l e l these rates of DNA synthesis. The RNA to DNA r a t i o s of d i f f e r e n t c e l l types were com-pared. Typical r a t i o s for d i f f e r e n t tissues (132) are as follows: l i v e r , 2 to 5; pancreas, 2 to 8; kidney, brain and spleen, 0.8 to 1.5; and thymus from various species, 0.4 to 0.6. In comparison to other t i s s u e s , the lower rat i o s (Table V) of RNA to DNA i n testes c e l l s suggest that a large proportion of the volume of these c e l l s i s occupied by 2 n u c l e i while the cytoplasm i s scanty. I t should be noted that i n the early 1.5 Sv spermatid there i s s t i l l a sub-s t a n t i a l amount of RNA r e l a t i v e to the larger c e l l s (Table V) and during maturation of the spermatid, the RNA content de-creases presumably as the cytoplasm i s gradually shed. (c) Incorporation of Arginine and Lysine Into the D i f f e r - ent C e l l Types: Histone synthesis i s usually cl o s e l y associated with DNA synthesis (54-57) but during spermatogenesis, the protamines, a group of highly basic, a r g i n i n e - r i c h proteins are synthesized at a l a t e stage of t e s t i s development (99, 100). Following binding of protamine to chromatin i n sperm-a t i d nuclei there i s a progressive replacement of the normal complement of histones by protamine (108,109). A l l histones contain substantial amounts of lysine while the arginine 2 Trout red blood c e l l s are an exception; the RNA:DNA r a t i o i s low (0.10) yet there i s a considerable amount of cytoplasm which i s , however, d e f i c i e n t i n ribosomes. 56. contents vary from low i n histone I to high i n histones III and IV. In contrast, the protamines are extremely r i c h i n arginine and contain at most traces of lysine (133). Thus, i n c e l l s a c t i v e l y synthesizing protamine, an increase i n the r a t i o of arginine to l y s i n e incorporation into t o t a l protein would be expected. A s i m i l a r rationale was used by Ling et a l . (100) to determine the stage of naturally maturing trout testgs. The r a t i o of arginine to lysine incorporation was high (30 to 150) i n testes a c t i v e l y synthesizing protamine while i n "preprotamine" stage testes, the r a t i o was less than 5 (100). F i g . 6A,B and C shows the c e l l separation p r o f i l e s (A and B) and r a d i o a c t i v i t y p r o f i l e s (A and C) of c e l l sus-pensions incubated with [3H] arginine and [x ^ C] l y s i n e . In early spermatid stage testes (Fig. 6A) with a large proportion of 2.8 to 3.5 Sv c e l l s , both arginine and lysine are incorporated into the early spermatids (1.5 Sv). At a l a t e r stage (Fig. 6B) there i s a shoulder of smaller c e l l s (1.0 Sv) presumably sperm-atids at a l a t e r stage of development which are also incor-porating arginine and lysine (Fig. 6C). Before protamine synthesis and histone replacement has begun, the early spermatids can be d i f f e r e n t i a t e d v i s u a l l y i n the microscope from l a t e r stage spermatids. This i s indicated by the "non-r e f r a c t i l e " c e l l counts i n F i g . 6B (lower curve). Late spermatids and sperm have a very compact, dense nucleus and thus r e f r a c t l i g h t intensely. 57. Sv (mm/hr) 7.8 6.4 5;0 3.6 Z5 12 Fraction No. Fig. 6. C e l l separation, [3H] arginine, and [^C] lysi n e p r o f i l e s of c e l l s from testes at d i f f e r e n t stages of hor-monal induction. C e l l s were incubated with [ 3H]arginine and t ^ C ] l y s i n e and analyzed as i n Fig. 5 except that formaldehyde f i x a t i o n was omitted. (A) 49 days i n j e c t i o n , preprotamine stage; (B) 61 days i n j e c t i o n , early protamine stage (the upper curve represents t o t a l c e l l counts while the lower curve represents "non r e f r a c t i l e " c e l l counts on a hemocytometer); (C) r a d i o a c t i v i t y p r o f i l e of (B).( ) c e l l per ml; (—O O-) [3H] arginine; (—• •-) [l **C] l y s i n e . 58. The r a t i o of [3H] arginine to I 1 ''C] lysine incorporation into n uclei changes as the c e l l s progress through spermato-genesis (Table VII). In 2.8 and 3.5 Sv c e l l s , the normalized r a t i o of arginine to l y s i n e incorporation i s close to 1.0 while i n early 1.5 Sv spermatids, the r a t i o i s 2.9. This increases i n middle 1.5 Sv spermatids to 5.7 and reaches 10 to 15 i n 1.0 Sv spermatids. On the other hand, the r e l a -t i v e incorporation of [1I,C] lysine (presumably r e f l e c t i n g the biosynthesis of histones and other proteins but not protamine) decreases d r a s t i c a l l y with completion of meiosis: the early spermatids incorporate only 1 to 2% as much lysi n e as the larger c e l l s while the l a t e r spermatids incorporate even l e s s . Thus the large c e l l s which a c t i v e l y synthesize DNA also a c t i v e l y incorporate l y s i n e , while the spermatids which have v i r t u a l l y ceased DNA synthesis incorporate very l i t t l e l y s i n e . However, i n spermatids, there i s an increased incorporation of arginine r e l a t i v e to l y s i n e , and t h i s incor-poration of arginine increases as spermiogenesis proceeds. Presumably, t h i s i s due to the onset of protamine synthesis i n 1.5 Sv c e l l s and continued synthesis i n 1.0 Sv spermatids. (d) C e l l u l a r D i s t r i b u t i o n of the Histones and Protamines: To determine whether labeled arginine i s indeed incorporated into histones i n the large c e l l s and into protamine i n the spermatids, methods were developed to c o l l e c t small quantities of c e l l s , from which the basic proteins could be rapidly TABLE VII Relative Rates of Protein Synthesis i n C e l l s from Different Regions of Serum Albumin Gradients Testis cell suspensions were doubly labeled with ['HJarginine and [HC]lysine and separated on serum albumin gradients. An aliquot of each fraction was analyzed for radioactivity as under "Experimental Procedure" and *H counts corrected for 30% 1 4 C overlap into the lower channel. Specific incorporation in counts per min per 10s cells was determined over three to five fractions and expressed relative to the 3.5 Sv cells for the indicated cell sizes. Days of hormonal induction Testis stage Cell size 3.5 Sv 2.8 Sv " 1.5 Sv° U Sv* 1.0 Sv 35 Stem cell Arginine 1.0 0.62 Lysine 1.0 0.47 Ratio Arg:Lys 1.0 1.3 49 Preprotamine spermatid Arginine' 1.0 0.95 0.024 Lysine" 1.0 1.1 0.013 Ratio ArglLys' 1.0 0.85 2.9 61 Early protamine Arginine1* 1.0 1.45 0.063 0.056 Lysine1* 1.0 1.45 0.012 0.006 . Ratio Arg:Lys 1.0 -1.0 5.7 10.5 67 Mid protamine Arginine 1.0 1.45 0.93 0.43 Lysine 1.0 1.51 0.09 0.03 Ratio Arg:Lys 1.0 0.95 10 15 ° 1.5 Sv spermatids which have not begun to synthesize protamine. * Mixture of preprotamine and protamine stage 1.5 Sv spermatids. * Calculated from Fig. 6 A and C, respectively. extracted, separated, and analyzed for r a d i o a c t i v i t y . Testes c e l l s at the protamine stage were incubated with [lhC]arginine and the c e l l s of various size separated by sedimentation. To further resolve the spermatid region, the c e l l s were sedimented for 10 instead of 5 hr. C e l l s from fractions across the spermatid peak were c o l l e c t e d on glass f i b e r f i l t e r s and t h e i r proteins were extracted with acid d i r e c t l y i n the sample s l o t of the starch g e l . These proteins were separated by electrophoresis (130) and stained with the sensi t i v e Amido Black s t a i n . [This v a r i a t i o n i n technique (130) appears s p e c i f i c for arg i n y l guanidinium groups and i s close to 100 times as sensi t i v e as the normal Amido Black stain.] In addition, the gel was dried and exposed to X-ray f i l m for autoradiography. F i g . 7A and B shows the re s u l t s of two of these experiments, one conducted on "early protamine" stage testes (A), and the other on "mid protamine" stage testes (B). Three classes of spermatids are resolved by extended sedimentation of c e l l s from mid-protamine testes (Fig. 7B): 1.5 Sv, 1.0 Sv, and 0.6 Sv. The 0.6 Sv class consists of mature sperm and these are the only c e l l s found i n m i l t . The order of appearance of these c e l l s i s 1.5 (Fig. 5B), 1.0 (Fig. 7A) and f i n a l l y 0.6 Sv c e l l s (Fig. 7B). Starch gel electrophoresis to resolve the histones and protamines followed by autoradiography indicate that the complete complement of histones i s found i n the large c e l l s 61 Fraction No. T Q p Fraction No. F i g . 7. Extended c e l l separation, gel electrophoresis, and autoradiography of [*"c]arginine labeled c e l l s from prot-amine stage testes. C e l l suspensions were incubated with 20 uCi of [^C] arginine for 7.0 hr and separated by sedimentation at unit gravity for 10.0 hours. Fractions (7.0 ml) were collected and c e l l s were counted e l e c t r o n i c a l l y . Nuclei from the indicated fractions were coll e c t e d on f i l t e r s and acid-soluble proteins were separated by starch gel electrophoresis i n the presence of urea. The stained gel was dried by vacuum suction and exposed to X-ray f i l m for 5 months to obtain the autoradiogram. The reference holes i n the gel correspond to the black dots on the autoradiogram. (2.8 and 3.5 Sv) as well as early spermatids. During the t r a n s i t i o n from 1.5 to 1.0 Sv spermatids, most of the h i s -tones are l o s t (Fig. 7A and B) while the residual histones are l o s t during the t r a n s i t i o n from late spermatid (1.0 Sv) to mature sperm (0.6 Sv) (Fig. 7B). The protamines are found i n spermatids (1.5 and 1.0 Sv) and sperm (0.6 Sv), but not i n the larger c e l l s (2.8 and 3.5 Sv). Several bands of protamine are resolved on the starch gels: the fas t e s t i s unmodified protamine, while i t w i l l be shown l a t e r that the slower ones are various species of phos-phorylated protamine. Phosphorylation decreases the net p o s i t i v e charge of protamine and at least 3 phosphorylated bands may be seen migrating behind the fastest (unmodified) band. During the t r a n s i t i o n from the l a t e spermatid (1.0 Sv) to the mature sperm (0.6 Sv), the residual phosphorylated protamines are progressively dephosphorylated u n t i l i n mature sperm, only unmodified protamine i s found (Fig. 7B). Thus, there appears to be a strong c o r r e l a t i o n between the dephos-phorylation of phospho-protamine and the controlled con-densation of the spermatid nucleus from 1.5 to 1.0 Sv c e l l s (Fig. 7A) and 1.0 Sv spermatids to 0.6 Sv sperm (Fig. 7B). The histones are synthesized i n the 2.8 and 3.5 Sv c e l l s (left-hand side of the autoradiograms of lkC i n F i g . 7A and B). L i t t l e or no histone synthesis occurs i n sperm-at i d s . Shortly a f t e r synthesis, most of the t 1 !*C]arg-inine label incorporated into the protamines appears i n the phosphorylated bands (Fig. 7A, autoradiogram). Thus, protamines must be phosphorylated during or shortly a f t e r t h e i r synthesis. Since I 1^C]arginine label i s seen i n the unsub-s t i t u t e d band of protamine i n the autoradiogram of F i g . 7B but not i n the corresponding p o s i t i o n i n F i g . 7A, i t i s possible that the phosphorylation of protamine may be more rapid i n the early spermatid (1.5 Sv) or the dephosphorylation of protamine may be more rapid i n the late spermatid (1.0 Sv) perhaps due to an increased a c t i v i t y of protamine phosphatases. A l t e r n a t i v e l y , i f the newly synthesized protamine pool i s small i n the early spermatid (1.5 Sv), phosphorylation would rapidly deplete t h i s pool and very l i t t l e l a b e l would be seen i n "unmodified" protamine. In l a t e r spermatids (1.0 Sv) t h i s pool may be larger (faster protamine biosynthesis) and may be depleted to a lesser extent by phosphorylation and t h i s would account for the s i g n i f i c a n t label found i n unmodified protamine i n F i g . 7B. Fig . 7B also indicates that the late spermatids (1.0 Sv) have a tendency to "dimerize": c e l l s with unmodified protamine but very l i t t l e histone are found sedimenting i n the 1.4 to 2.0 Sv region of the gradient. From the sedimentation v e l o c i t y equation (119) M i l l e r and P h i l l i p s showed that i f the volume of a c e l l increases or decreases two-fold, the sedimentation v e l o c i t y constant, Sv, changes 1.6 f o l d . Thus the volume of the spermatid c e l l decreases approximately two-fold during the t r a n s i t i o n from 1.5 to 1.0 Sv and another two-fold during the t r a n s i t i o n from 1.0 to 0.6 Sv, the o v e r a l l change being a four - f o l d decrease i n volume. The volume of a mature trout sperm calculated from the sedimentation v e l o c i t y constant and the approxima-r 2 t i o n of the sedimentation v e l o c i t y equation, Sv = -g— (ref. 119, i n which Sv i s i n units of mm per hr and r i n microns), i s 15.5 x 10~ 1 2 cm3. This agrees very well with studies of cross sections from electron microscopy by P o l l i s t e r and Mirsky (83). These workers calculated that the t o t a l volume of the sperm c e l l was 15.7 x 10 1 2 cm3. The loss of cytoplasm, histones, and e f f e c t i v e packaging of DNA i s such that of the dry weight of the mature sperm (5.0 x 10~ 1 2 g per c e l l ) , 50% i s DNA (2.5 x 10~ 1 2 g per c e l l ) , and 30% i s contributed by protamine (1.5 x 10 1 2 g per c e l l ) . (e) Approximate Rate of Protamine Biosynthesis i n vivo; I t takes close to 1 week for the 1.5 Sv spermatids which have begun to synthesize protamine to condense into 1.0 Sv sperm-a t i d s . [In Fig. 6A (49 Days) only 1.5 Sv spermatids are found while i n F i g . 7A (55 Days) 1.0 Sv spermatids have appeared.] I f most of the protamine (1.8 x 10 8 molecules per sperm) i s synthesized during t h i s period, then an average of 1.8 x 10* molecules w i l l be synthesized per minute per spermatid. From the RNA content of the spermatids synthesiz-ing protamine (0.25 x 10~ 1 2 g per spermatid) there are approximately 60,000 ribosomes per spermatid assuming that 80% of the RNA i s ribosoraal RNA (t o t a l MW ^ 2 x 10 6 per ribosome). I f a l l the ribosomes are synthesizing protamine during t h i s period, then 1 molecule of protamine w i l l be syn-thesized every 3 min per ribosome. However, not a l l ribosomes w i l l be synthesizing protamine. For example, the t a i l pro-teins and enzymes involved i n egg penetration must also be synthesized. In addition, the RNA ( i . e . ribosome) content decreases as spermiogenesis proceeds. Thus, the average rate of protamine synthesis w i l l be somewhat faster, perhaps closer to 1 molecule per min per ribosome. I I . Synthesis and Phosphorylation of Histones and Protamines i n Different C e l l Types During spermatogenesis i n trout t e s t i s , the somatic histones are replaced by the protamines and extensive phos-phorylation of both the histones and the protamines takes place. Phosphorylation has been shown to occur i n histones I, I l b i , I I b 2 , III, and IV (22), and there i s also extensive e-amino acetylation of l y s y l residues i n I l b i , I I b 2 , III, and IV (23) i n testes active i n the replacement process. Since early data indicated that phosphorylation and acetylation occurred on "old" ( i . e . preformed) histones (65,74,105), a process of histone phosphorylation and acetylation was sug-gested as a possible mechanism for the removal of histones from chromatin during spermiogenesis (22). To obtain evidence for or against t h i s hypothesis the c e l l u l a r s p e c i f i c i t y of the synthesis and metabolism of the histones and protamines was examined. (a) Rapid Extraction and Separation of Basic Proteins  of the C e l l : Histones modified by phosphorylation or ace t y l -ation migrate more slowly toward the cathode than the corres-ponding unmodified forms during starch gel electrophoresis i n the presence of urea (22,23,134). The mo b i l i t i e s of the various histone fractions i n these starch gels have previously been characterized i n rat l i v e r (130,134), and more recently i n trout t e s t i s (22,23). F i g . 8A summarizes the positions on 67. + amines F i g . 8. Separation of histones and protamines on starch gels. (A) A mixture of trout testes histones and protamines (50 ug) prepared by conventional acid extraction of nuclei and CM-cellulose chromatography to remove most of the aci d i c proteins (100) was separated by electrophoresis, stained with Amido Black, and destained with s u l f u r i c acid as des-cribed i n the "Experimental Procedures". The histones are lab-eled according to Rasmussen et a l . (20). Most of the h i s -tone III i s i n the oxidized form (III dimer). (B) Protein staining pattern and corresponding autoradiogram of acid-soluble proteins rapidly extracted and separated on starch gels. C e l l s were incubated with 100 yCi per ml of inorganic [ 3 2P]phosphate for 8 hr. At the end of the incubation, 3 x 10 7 c e l l s were fixed with 95% ethanol, co l l e c t e d on a glass f i b e r f i l t e r , and extracted with 0.4 N HCl i n a starch gel s l o t . After electrophoresis, the gel was t r i s e c t e d hor-i z o n t a l l y and the bottom slab was stained. The destained gel was equilibrated with acetic acid, dried, and then ex-posed to X-ray f i l m for 10 days before development. The phosphorylated histones and protamines both have a slower mobility than unmodified species. Most of the histone III i s i n the reduced monomer state. 68. starch gels of the various histones and protamines prepared by conventional acid-extraction of nuclei from trout t e s t i s and adsorption onto and e l u t i o n from CM-cellulose to remove most of the a c i d i c proteins (100,105). The bands between histone T (135) and unsubstituted protamine are various forms of protamine modified by phosphorylation of the hydroxy1 group of s e r y l residues (104,107) or by the presence of a methionyl group at the NH 2-terminus (101). Most of the histone III prepared i n t h i s manner i s i n the oxidized dimer form (Fig. 8A). In contrast, i f histones and protamines are extracted and separated rapidly (Fig. 8B), most of the histone III i s monomeric with the sulfhydryl group i n the reduced state. This supports the observation by Marushige and Dixon (108) that rapid extraction and separation of histones by e l e c t r o -phoresis considerably shortens the time during which oxidation can occur. Thus, most of the t e s t i s histone III i n vivo i s i n the free s u l f h y d r y l , monomer state. Besides the starch gel electrophoretic pattern, F i g . 8B also shows the corresponding autoradiogram of acid-soluble proteins of c e l l s labeled with [ 3 2P]phosphate from "early protamine" stage trout testes. Most of the phosphate incor-poration into acid-soluble proteins from testes c e l l s fixed with ethanol i s , at t h i s stage, into the histones. The three minor phosphorylated bands between histone IV and T are not found i n nuclei and may be cytoplasmic or e x t r a - c e l l u l a r proteins p r e c i p i t a t e d by the ethanol. Several phosphorylated bands migrate as a series with decreasing m o b i l i t i e s behind unmodified protamine. At least four modified protamines may be distinguished and these appear to correspond to mono, d i , t r i , and t e t r a phosphorylation of the protamine s e r y l residues and are accordingly labeled P i , P 2, P 3 and P^. In f a c t , Sanders and Dixon (107) have shown recently that one of the phosphopeptides i s o l a t e d from a t r y p t i c digest of mixed phosphoprotamines has the composition Ser (P)-Ser (P)-Ser (P) -Arg-Pro-Val-Arg and probably comes from a t r i - or t e t r a -phosphorylated species of protamine. Further evidence that these four species are indeed phosphorylated protamines i s provided by t h e i r conversion to dephospho (unmodified) prot-amine by treatment with alkaline phosphatase (PART II of thi s Thesis). (b) Separation of C e l l s from Preprotamine Stage Testis : To determine which c e l l types contain histone or protamine or both, and which are involved i n the synthesis or phos-phorylation of these proteins, or both, testes c e l l s were incubated with [3H] lysine or l 3H] arginine and inorganic [ 3 2P]phosphate and separated by sedimentation at unit gravity on serum albumin gradients. F i g . 9 shows the results of sedimentation of c e l l s from a preprotamine stage t e s t i s . Forty-two days afte r the st a r t of salmon p i t u i t a r y extract i n j e c t i o n s , a t e s t i s c e l l suspension was prepared and i n -Sj(mm/hr) Fraction No F i g . 9. C e l l separation and r a d i o a c t i v i t y p r o f i l e s of c e l l s from testes at the preprotamine stage of development. C e l l s from a hormonally induced t e s t i s (42 days) were incubated with 300 yCi per ml of inorganic [ 3 2P]phosphate and 100 UCi per ml of [ 3H]lysine for 5 hr. C e l l s (8.4 x 10 7) were sedimented at unit gravity for 260 min before beginning c o l l e c t i o n of 7.0 ml fr a c t i o n s . One f r a c t i o n was col l e c t e d every 30 sec and each f r a c t i o n corresponds to 0.57 mm of gradient. Aliquots were removed for determination of radio-a c t i v i t y on glass f i b e r f i l t e r s . 71. cubated with [3H] lysine and [3 2P]phosphate for 5 hr and then separated by v e l o c i t y sedimentation for 5 hr at one gravity. Four major c e l l fractions which sediment at 1.5, 2.8, 3.5, and 5.0 Sv are found i n preprotamine c e l l suspensions (Fig. 9). The 1.5 Sv c e l l s have been i d e n t i f i e d biochemically as spermatids by t h e i r haploid complement of DNA and by t h e i r lack of DNA synthesis (Table V). The 2.8 and 3.5 Sv c e l l s both have a d i p l o i d complement of DNA and i n addition incor-porate [ 3H]thymidine into DNA. The i d e n t i t y of the 5.0 Sv c e l l s i s not clear but they appear to have close to a t e t r a -p l o i d DNA content (Table V) . [ 3H]lysine and 3 2P are incorporated into a l l c e l l f r ac-tions of F i g . 9. The c e l l s i n the 2.8 and 3.5 Sv region are incorporating more lysine and 3 2P than the 1.5 Sv c e l l s . Some of the l y s i n e may be incorporated into non-histone pro-t e i n and i n c e l l s active i n DNA and RNA synthesis, much of the phosphate may be incorporated into DNA and RNA. To determine whether any 3H or 3 2P had been incorporated into the histones or protamines, c e l l s i n the 1.5, 2.8, 3.5, and 5.0 Sv regions were c o l l e c t e d into separate pools and the acid-soluble basic proteins extracted from them and sep-arated by starch gel electrophoresis. (c) Histone Metabolism i n Preprotamine T e s t i s : F i g . 10 shows the d i s t r i b u t i o n of [3H] lysine and [ 3 2P]phosphate la b e l i n the histone region of the 1.5, 2.8, 3.5 and 5.0 Sv H DISTANCE (cm) » » -F i g . 10. Separation and analysis of r a d i o a c t i v i t y i n histones from preprotamine stage t e s t i s . C e l l s were pooled from the c e l l separation i l l u s t r a t e d i n F i g . 9 and c o l -lected on f i l t e r s . Acid-soluble proteins were extracted i n the starch gel s l o t s . After electrophoresis, the gel was t r i s e c t e d and the bottom slab was stained. The middle slab was s l i c e d at 2 mm interv a l s and each s l i c e s o l u b i l i z e d and counted for r a d i o a c t i v i t y . (A) 2.1 x 10 7 c e l l s not subjected to c e l l separation but fixed with ethanol and collected on a f i l t e r at the beginning of c e l l separation; (B) 2.0 x 10 7 1.5 Sv c e l l s ; (C) 1.0 x 10 7 2.8 Sv c e l l s ; (D) 1.0 x 10 7 3.5 Sv c e l l s ; (E) 0.5 x 10 7 5.0 Sv c e l l s . regions a f t e r separation on starch gels. No protamine was found i n these 1.5 Sv c e l l s ; these represent "early" spermatids i n which only histones are found. The histone staining patterns are sim i l a r i n a l l c e l l types: a l l contain histone T and most of the histone III exists as the monomer and not the oxidized dimer. Furthermore, the patterns of histone synthesis ( [ 3H]lysine incorporation) and phos-phorylation ( [ 3 2P]phosphate incorporation) are a l l s i m i l a r . In a l l c e l l types, the slowest 3 2P-histone peak corresponds to monophospho-IIbi (134) and i s the phosphorylated histone present i n the highest amount. The faster running broader 3 2 P peak contains phospho-IIb 2, phospho-III monomer, and phospho-IV (PART III of t h i s t h e s i s ) . I t would appear that histone synthesis and phosphorylation are not r e s t r i c t e d to the stem c e l l s and primary spermatocytes, which are synthesizing DNA, but also take place both i n early, preprotamine sperm-atids and i n the large c e l l s i n the 5.0 Sv region. However, the various c e l l types d i f f e r widely i n the rates of histone synthesis and phosphorylation that they support. Table VIII shows the s p e c i f i c r a d i o a c t i v i t y i n cpm per 10 7 c e l l s of [3H] lysine and [ 3 2P]phosphate incorporated into histones by the d i f f e r e n t c e l l types as well as incor-poration calculated r e l a t i v e to the 3.5 Sv c e l l s . The radio-a c t i v i t y incorporated into c e l l s which had not been subjected to c e l l separation and whose metabolism had been stopped by f i x a t i o n with ethanol at the beginning of the c e l l separation TABLE VIII Synthesis and Phosphorylation of Histone i n C e l l s of Preprotamine T e s t i s Radioactivity Cell typa Unfrac-tiooated" 1.S Sv* 2.8 Sv* 3.5 Sv» 3.0 Sv» 2,250 185 850 2,450 2,450 cpm *H/107 cells' 6,700 430 4,480 11,350 11,100 »»P:»H. 0.34 0.43 0.19 , 0.22 0.22 Relative " P : ' H . 1.5 2.0 0.88 1.0 1.0 " Cell metabolism stopped by fixation with ethanol at the be* ginning of cell separation. * Cell metabolism continued for a further 9 hours. • "P and *H summations were made in the histone region (7.4 to 10.4 cm) of Fig. 10 and divided by the number of cells on each filter. i s included as a control i n case any phosphate turnover (65) occurred during the lengthy c e l l sedimentation. The rates of synthesis and phosphorylation of histone, as measured by [3H] ly s i n e and [ 3 2P]phosphate incorporation, are quite d i f f e r e n t for the d i f f e r e n t c e l l types (Table VIII). In the 1.5 Sv preprotamine spermatids there i s only a small amount of histone synthesis and phosphorylation. However, the spermatids have a haploid complement of DNA. When t h i s i s considered, the rates of synthesis and phosphorylation of histone double from 3.8 and 7.5% respectively to 7.6 and 15% r e l a t i v e to the 3.5 Sv c e l l s on a DNA basis. I t appears then, that i n preprotamine (early) spermatids which are at a r e l a t i v e l y late stage of spermatogenesis, s i g n i f i c a n t histone synthesis and phosphorylation are s t i l l occurring. In the 2.8 Sv c e l l s the rates of histone synthesis and phos-phorylation are both about 40% of that i n the 3.5 Sv c e l l s . Thus, the 3.5 Sv c e l l s are synthesizing and phosphorylating histones 2.5 times faster than the 2.8 Sv c e l l s . However, i f the r a t i o between [3H] lysine and [ 3 2P]phosphate incor-poration i s computed for each c e l l type, there i s a s t r i k i n g one-to-one re l a t i o n s h i p between these processes, although t h e i r magnitude changes from one c e l l type to another. When the rates of histone synthesis and phosphorylation are compared to the rates of DNA synthesis i n the 2.8 and 3.5 Sv c e l l types, there i s a clear r e l a t i o n s h i p between DNA synthesis on the one hand and histone synthesis and phos-phorylation on the other. For example, the r e l a t i v e rates of [ 3H]thymidine incorporation into DNA i n the 2.8 and 3.5 Sv c e l l s were determined previously (Table VI) to be 0.4 and 1.0 respectively while i n the present case, histone syn-thesis i s i n the r a t i o 0.39 to 1.0 i n the two c e l l types and histone phosphorylation i s 0.35 i n the 2.8 Sv c e l l s and 1.0 i n the 3.5 Sv c e l l s . I t seems clear that these processes occur simultaneously but that t h e i r absolute rates change as the c e l l s d i f f e r e n t i a t e . F i g . 10A, the [ 3H]lysine and [ 3 2P]phosphate pattern of incor-poration from unseparated c e l l s , shows that the pattern of histone phosphorylation has not changed appreciably from the beginning of the c e l l separation to the end (about 9 hours l a t e r when the pooled peaks were c o l l e c t e d on f i l t e r s ) . However, a higher r a t i o of 3 2 P to 3H i s observed i n the un-fractionated c e l l s (1.5:1.0) as compared to the r a t i o (1.0: 1.0) i n the 3.5 Sv c e l l s . Since histones are degraded or replaced only slowly on chromatin (39,40), t h i s r e s u l t would seem to indicate turnover or removal of the histone phosphate during the lengthy sedimentation process. Thus about one-t h i r d of the histone phosphate had turned over during the 9 hr i n t e r v a l between s t a r t i n g the c e l l separation and c o l l e c t i n g the separated c e l l s on f i l t e r s . This indicates an o v e r a l l h a l f - l i f e of 13 to 15 hours for phosphoryl groups bound to histone. As the c e l l separation was ca r r i e d out at 4 to 6°, the turnover at 15° i s probably 2 to 3 f o l d f a s t e r . The conditions of the c e l l separation method are very mild and i n other cases i t has been shown that c e l l s are s t i l l v iable a f t e r separation (136,137). I t appears that the slow removal of phosphate i s a s i g n i f i c a n t physiblogical process and i s not associated with any damage to the c e l l s during separation. (d) Separation of C e l l s from Protamine Stage T e s t i s : I t was previously proposed by Sung and Dixon (22) that en-zymatic modifications of the NH 2-terminal region of histone IV by O-phosphorylation of s e r y l 1 and e-amino acetylation of l y s y l s 5,8,12, and 16 could a l t e r the binding of t h i s basic region to DNA and thus provide a mechanism for the ob-served removal of histones from the chromatin of trout t e s t i s c e l l s at the spermatid stage. The obvious question then was "In c e l l s that have begun protamine biosynthesis and histone replacement, does extensive histone phosphorylation occur?" Testes c e l l s from a f i s h injected with p i t u i t a r y extracts for 67 days were incubated with [ 3H]arginine and inorganic [ 3 2P]phosphate for 6 hours and subjected to sedimentation for 10 hours. I t was previously shown (Fig. 7) that i n protamine stage testes, sedimentation of c e l l s for an extended period of time resolves three classes of spermatids sedimenting at 78. 1.5,-1.0, and 0.6 mm per hr i n which histones are progressively l o s t from the 1.5 Sv to 1.0 Sv c e l l types and are absent i n the 0.6 Sv mature sperm. F i g . 11 shows the p r o f i l e of these c e l l s a f t e r 10 hours of sedimentation, and i t may be seen that more than 90% of the c e l l s are spermatids. Mixed c e l l s from the end of the 6 hour incubation and pooled c e l l s from the 1.0, 1.5, and 3.0 to 3.5 Sv regions were extracted with acid and the soluble proteins were separated on starch gels as before. (e) Histone Metabolism i n Protamine Stage T e s t i s ; While the large c e l l s contain only histones, the 1.0 and 1.5 Sv c e l l s contain both histones and protamine (Fig. 12). The 1.5 Sv c e l l s which contain both histone and protamine are "middle" spermatids and the 1.0 Sv c e l l s are "late" spermatids. The major loss of histones takes place during the 1.5 to 1.0 Sv t r a n s i t i o n . As expected (Fig. 7), very l i t t l e synthesis of histone takes place i n the protamine stage spermatids; but i t was surprising to f i n d very l i t t l e phosphorylation of histone. This suggests that most of the phosphorylation of histones previously seen i n unseparated c e l l suspensions from la t e t e s t i s (22,105) was due to a small f r a c t i o n of large c e l l s a c t i v e l y synthesizing histone. The differences between the rates of histone synthesis i n the large (3.5 Sv) and small (1.0 and 1.5 Sv) c e l l s became more apparent when the t o t a l r a d i o a c t i v i t y incorporated into F i g . 11. Extended sedimentation of c e l l s from a t e s t i s at the protamine stage of development. C e l l s from a hor-monally induced t e s t i s (67 days) were incubated with 300 yCi per ml of inorganic [ 3 2P]phosphate and 100 yCi per ml of [3H] arginine for 6 hr. 10 x 10 7 c e l l s were loaded over a l i n e a r gradient of BSA i n phosphate-buffered sa l i n e and processed as i n Fi g . 9 except that t o t a l sedimentation time was 10.0 hr including 40 min for the c o l l e c t i o n of fr a c t i o n s . 80. 20 10" o E E 4- 2-<v CM 2-| i 1 o X 1 0. CSi "1 (cpm 1 20-1 10-rone 10- 5-in I 10' 5-5-25--I 1 1 I I I L. 3 2P bockground-<" Histones l,5S / 3.5 Sv 7 9 M 13 15 17 19 2T 20 10 0. CvJ 20 100 50 20 o » E E CM — 01 l 0 2 2 I X X X E o a I 0 01 10 I 0 0 » c 50 I o +-o t_ 0. D I S T A N C E (cm) F i g . 12. Separation and analysis of r a d i o a c t i v i t y i n histones and protamines of c e l l s from a t e s t i s at the prot-amine stage of development. C e l l s were pooled from the c e l l separation i l l u s t r a t e d i n F i g . 11 and processed as i n Fi g . 10. (A) 3.7 x 10 7 c e l l s not subjected to c e l l sep-aration but fixed with ethanol and co l l e c t e d on a f i l t e r at the beginning of c e l l separation; (B) 1.25 x 10 7 1.0 Sv c e l l s ; (C) 2.0 x 10 7 1.5 Sv c e l l s ; (D) 1.0 x 10 6 3.0 to 3. 5 Sv c e l l s . histone by each c e l l type was determined by integrating the i n d i v i d u a l r a d i o a c t i v i t i e s i n each gel s l i c e i n the histone region of F i g . 12. These values are presented i n Table IX and indicate that the rate of histone synthesis (per 10 7 c e l l s ) i n both the 1.0 (late) and 1.5 Sv (middle) spermatids i s less than 1% of the l e v e l i n the 3.5 Sv c e l l s . Since histone synthesis and phosphorylation appear to take place simultaneously, the r a t i o of 3 2P to 3H r e l a t i v e to the unfractionated c e l l s was examined. During the 14 hours of c e l l separation the 3 2P to 3H arginine r a t i o had 3 declined to about k of the o r i g i n a l value of 0.30, i n d i -cating an average h a l f - l i f e for histone phosphate of 7.5 to 8 hours i n the 3.0 to 3.5 Sv c e l l s (Table IX). On the other hand, i n the 1.0 and 1.5 Sv protamine/)spermatids, there was very l i t t l e histone phosphate turnover (the 3 2 P to 3H r a t i o s are 0.29 and 0.28 respectively compared to the control unfractionated c e l l s ) . To examine the turnover of the histone phosphate more cl o s e l y , c e l l s were incubated with [ 3H]arginine and inorganic [ 3 2P]phosphate for 45 minutes, and chased i n a medium with unlabeled phosphate and arginine. At various times, c e l l s were c o l l e c t e d and t h e i r basic proteins were extracted with 3" That t h i s assumption i s v a l i d i s indicated by the fact that even though less than 10% of the c e l l s are active i n h i s -tone synthesis, more than 90% of the histone synthesis and phosphorylation i s due to t h e i r a c t i v i t y . TABLE IX Synthesis and phosphorylation of histone and protamine i n c e l l s of protamine stage t e s t i s Radioactivity Cell type Unfrac-tionated* 1.0 Sv* 1.5 Sv* 3.5 Sv* Histone „ "P cpm/107 cells' 1,230 60 . 155 4,740 •H cpm/107 cells' 4,080 210 650 68,800 "P:»H 0.301 0.289 0.282 0.080 Protamine "P cpm/107 cells'.... 456 163 423 *H cpm/107 cells' 33,200 14,800 31,700 "P:»H 0.0138 0.011 0.0134 " Cell metabolism stopped by fixation with ethanol at the be-ginning of cell separation. ' Cell metabolism continued for 14 more hours. • "P and *H counts were integrated oyer the histone (7.4 to 10.6 cm) and protamine (14.6 to 20.2 cm) regions of Fig.(Sand divided by the number of cells on each filter sample. 83. acid and separated by electrophoresis. The 3H and 3 2P counts i n the histone regions were measured at various times. The resu l t s are shown i n F i g . 13: the phosphate counts i n the histones are decreasing while the arginine counts are constant. The calculated h a l f - l i f e for turnover of phosphate i s 6 to 8 hours, confirming the value obtained from the c e l l separation studies above. These turnover times for histone phosphate are v a l i d only for trout t e s t i s c e l l s at 15°. For mammalian c e l l s growing exponentially i n tissue culture, (generation time about one day) the absolute rates of DNA synthesis, histone synthesis and phosphorylation, and histone dephos-phorylation are probably much faster, although one would predict that these rates would p a r a l l e l one another. Phosphorylation of histones has been noted i n diverse phy s i o l o g i c a l s i t u a t i o n s . Thus, phosphorylation of a l l 5 major histone fractions, I, I l b j , IIb2 , III, and IV has been observed i n trout t e s t i s active i n the replacement process (22,105). A 15 to 20 f o l d increase i n phosphorylation of histone I was observed i n rat l i v e r following i n j e c t i o n of hormones which induce synthesis of new enzymes (74) . Phos-phorylation of d i f f e r e n t histones was observed as follows at d i f f e r e n t times i n regenerating rat l i v e r : phosphorylation of histone I at early Gj (1 or 2 hours) (138); phosphoryla-t i o n of histone I l b i at 15 hr (134); and phosphorylation of both histone I and III during the "S period" of the f i r s t J 1 1 I • ' ' ^ P / ' H - 0 . 5 H O U R S o f C H A S E F i g . 13. Turnover of [ 3 2P]labeled phosphoryl groups i n histones from trout t e s t i s cells.'.CCells (5 x 10 8) from an early protamine stage t e s t i s were incubated with 10 0 uCi per ml of [ 3H]arginine and 300 uCi per ml of inorganic [ 3 2P] phosphate i n 1 ml of TMKS-0.1% glucose at 15° on a gyratory water bath. After 45 min of labeling, the c e l l s were d i -luted with 25 ml of cold phosphate-buffered s a l i n e contain-ing unlabeled arginine and centrifuged at 1000 x g for 10 min. The chase consisted of resuspending the c e l l s i n 10 ml of an equal mixture of Waymouth's medium (125) and phosphate-buffered s a l i n e containing 1 mM unlabeled arginine and further incubating at 15°. At various times 0.5 ml (2.5 x 10 7 c e l l s ) was removed and d i l u t e d with cold phos-phate-buffered s a l i n e ; the c e l l s were f i l t e r e d with the aid of a Teflon template onto a glass f i b e r f i l t e r over an area the siz e of the starch gel s l o t . The c e l l s were washed with TMKS (5 ml), TMKS-1% Nonidet P-40 (5 ml) to s t r i p the c e l l s of cytoplasm (148), and fixed with ethanol. Acid-soluble nuclear proteins were extracted and separated by starch gel electrophoresis. The histone and protamine regions of the middle slab were s l i c e d into 2 mm s l i c e s and counted for r a d i o a c t i v i t y . The 3 2P and 3H counts over the histone region were integrated and the 3 2P: 3H r a t i o c a l -culated. Tritium counts i n the protamine region indicated that phosphorylation of labeled protamine continued nor-mally during the chase. c e l l cycle, 22 hr after p a r t i a l hepatectomy (139). In a l l these instances, phosphorylation seemed to occur on "old" preformed histone molecules rather than newly synthesized histone molecules. Several important points should be noted on the met-abolism of histones i n trout t e s t i s . F i r s t , the extensive phosphorylation of histones i n the large c e l l s which p a r a l l e l s the rates of histone and DNA synthesis, and lack of phos-phorylation i n spermatids active i n the replacement process suggest (a) that phosphorylation of histones does not play a s i g n i f i c a n t r o l e i n the replacement process, and (b) that histone phosphorylation plays an important role i n the metabolism of the c e l l . Second, [ 3H]arginine and ly s i n e l a b e l are found i n the phospho-IIbi region of the gels i n Fig. 10 and 12, implying that histone I l b i i s phosphorylated during or shortly a f t e r i t s synthesis. This suggests, then, that some of the histone phosphorylation associated with DNA and histone synthesis may be related to the "correct" binding of newly synthesized histone (e.g. histone I l b i ) to DNA. Third, histone IV i s the f a s t e s t migrating major histone component and i s c l e a r l y resolved as 2 bands i n approximately equal proportions from the other major histone fractions on both acrylamide (25) and starch gels (22,69). The faster band i s unmodified histone IV, while the slower band i s histone IV which i s monoacetylated on any one of several possible l y s y l residues (69). However, i n our studies, 5 hours aft e r the s t a r t of incorporation of [ 3H]lysine or arginine, very l i t t l e l a b e l i s found i n the unmodified and monoacetylated histone IV region (arrow, F i g . 10A). Even 14 hr af t e r the s t a r t ( i . e . after completion of a 5 hr c e l l separation) only a small amount of l a b e l appears (Fig. 10C,D and E). However, at the end of the extended c e l l separation, 19 hr af t e r the s t a r t of l a b e l i n g , a s i g n i f i c a n t peak i n t h i s region i s detected (Fig. 12D). This anomaly i n the labeling k i n e t i c s of histone IV cannot be explained by d i f f e r e n t i a l synthesis of histones, since i n rapidly d i v i d i n g c e l l s , a l l histones are synthesized simultaneously at s i m i l a r rates (55,57). The other pos-s i b i l i t y i s that histone IV i s enzymatically modified by phosphorylation or multiple acetylation or both shortly aft e r synthesis. Both modifications r e s u l t i n a s i g n i f i c a n t decrease i n mobility of histone IV on starch gels (22,69) and i f the amino acid l a b e l were largely i n these modified species, i t would explain the lack of l a b e l i n the major unsubstituted band of histone IV. This has prompted us to examine (PART III of t h i s thesis) the metabolism of histone IV further and our r e s u l t s indeed confirm that at early times, labeled amino acids incorporated into histone IV are found exclusively i n the modified, slower migrating species of histone IV. (f) Protamine Metabolism: As previously shown (Fig. 7) the "mature" protamines are the two fastest running bands on starch gels. The slower bands represent protamines that are presumably phosphorylated (Fig. 8) on one to four . s e r y l residues ( P i , P 2, P 3 , and P i » ) . In F i g . 12, a l l forms of modified protamine are found i n the 1.5 and 1.0 Sv c e l l s ; data i n Table IX indicate that the 1.5 Sv c e l l s are synthesizing and phosphorylating protamine twice as fas t as the 1.0 Sv c e l l s . I t should be mentioned that the 1.5 Sv c e l l peak i s heavily contaminated with 1.0 Sv c e l l s , since about 50% of the c e l l s i n the 1.5 Sv peak can be discrim-inated as 1.0 Sv on a Coulter Counter. Thus the true rate of protamine synthesis i n the 1.5 Sv c e l l s i s probably even higher. The characterization of c e l l s from testes at d i f f e r e n t stages of hormonal induction indicates that three stages of spermatid d i f f e r e n t i a t i o n can be defined: (a) early spermatids, 1.5 Sv, which have not yet begun to make protamine; (b) middle spermatids, 1.5 Sv, which have begun to syn-thesize protamine and lose histone; and (c) late spermatids, 1.0 Sv, which have synthesized most of t h e i r protamine and l o s t most of t h e i r histone. To t h i s l i s t we may add a fourth type: (d) mature sperm (0.6 Sv) which has completed protamine syn-t h e s i s , phosphorylation, and dephosphorylation and has no detectable histone (Fig. 7) . I I I . D i s t r i b u t i o n of C e l l s at Different Stages of Sperm-atogenesis and a Ki n e t i c Model of Trout Testis Develop-ment (a) Growth of the T e s t i s ; The size and shape of an, organ and i t s rate of growth are determined by the rate of c e l l d i v i s i o n and the chemical and b i o l o g i c a l properties of i t s constituent c e l l s . In the rainbow trout an annual period of spermatogenetic a c t i v i t y alternates with a period of i n v o l u t i o n a f t e r which there i s a reorganization of the t e s t i s and some spermatogonial p r o l i f e r a t i o n (115,121,140). Spermatogenetic a c t i v i t y i s influenced by p i t u i t a r y gonado-tropins (19,121,122). Accordingly, both intramuscular im-plantation (141) of p i t u i t a r i e s from sexually mature r a i n -bow trout and i n j e c t i o n (99,121,122) of salmon p i t u i t a r y extracts into sexually immature rainbow trout cause the immature testes to grow and d i f f e r e n t i a t e . In both naturally maturing and a r t i f i c i a l l y induced t e s t i s , a 500 to 1000 f o l d increase i n t e s t i s wet weight can occur (141). F i g . 14 compares the increases i n weight of testes a r t i f i c i a l l y induced to mature by hormonal i n j e c t i o n s , to testes maturing natur a l l y . In both cases, the weight i n -creases exponentially from about 10 or 20 mg to approxi-mately 10 g. When the maximum weight i s achieved, the weight decreases, also exponentially. However, the slopes of r i s e and f a l l are d i f f e r e n t for the a r t i f i c i a l l y induced F i g . 14. Comparison of the growth of hormonally induced and naturally maturing trout testes. The average weight of a p a i r of hormonally induced testes was plotted as a function of the number of days following the i n i t i a t i o n of a twice weekly series of inje c t i o n s of crude salmon p i t -uitary extract. The data for naturally maturing testes was obtained from Ling and Dixon (79). I n f a n t i l e testes can be induced to mature at any period of the year by i n j e c -t i o n of p i t u i t a r y extracts. The maximum weight varies depending upon the size of the f i s h ; small f i s h ('v-lOO g) y i e l d 3 to 4 g testes while larger f i s h (200 to 250 g) y i e l d 8 to 10 g testes. Note that the increase and decrease i n weight of the t e s t i s i s exponential with doubling or halv-ing times close to 1 week i n hormonally induced testes, and 2 weeks i n naturally maturing testes. 91. and naturally maturing t e s t i s . The a r t i f i c i a l l y induced t e s t i s doubles i t s weight every week (7 days) while the doubling time for the naturally maturing t e s t i s i s about two weeks (14 days). When the weight of the t e s t i s begins to f a l l , the rate of decline also p a r a l l e l s i t s rate of i n -crease: the a r t i f i c i a l l y induced t e s t i s halves i t s weight every week, while the naturally maturing t e s t i s halves i t s weight every 2 weeks. (b) Proportion of C e l l s at Different Stages of Testis  Development: The 500 to 1000 f o l d increase i n t e s t i s weight implies a 500 to 1000 f o l d increase i n c e l l number. The large number of c e l l s arises from the rapid d i v i s i o n of a small number of spermatogonial stem c e l l s which give r i s e to primary spermatocytes and ultimately spermatids. The r e l a t i o n -ship of the d i f f e r e n t c e l l s involved i n spermatogenesis has been depicted schematically i n F i g . 3. Because of the c y c l i c pattern of spermatogenesis i n salmonids, the population of c e l l s changes r a d i c a l l y from the immature to the mature t e s t i s . C e l l s undergoing spermatogenesis go through large changes i n volume (18) and are separable by the technique of v e l o c i t y sedimentation (120). F i g . 15A to G summarizes the p r o f i l e s of c e l l number versus c e l l size i n suspensions prepared from testes at d i f f e r e n t stages of hormonal induc-t i o n . At early stages (30 to 35 days) most of the c e l l s sedi-VELOCITY of SEDIMENTATION ( S v ,mm/hr) 7.8 6.4 5.0 3.6 ZJS 1.2 0 6.4 8.0 3.6 2.S 1.2 92. 20 SO FRACTION NUMBER F i g . 15. Sedimentation of c e l l s from testes at d i f f e r e n t stages of development. C e l l suspensions from hormonally induced testes at d i f f e r e n t stages of development were pre-pared and sedimented for a t o t a l period of 5.0 hr including 40 min for the c o l l e c t i o n of 7.0 ml fr a c t i o n s . Early sperm-atids are "non-refractile" under the microscope and can be di f f e r e n t i a t e d from spermatids at l a t e r stages of development which are highly condensed and r e f r a c t l i g h t intensely; t h i s i s indicated by the lower curve i n F (non-refractile c e l l s ) . In C,D, and E, v i r t u a l l y a l l the spermatids are early spermatids (1.5 Sv) which have not yet begun to synthesize protamine, while i n F, as many as 50% of the spermatids are early, the remainder being middle (1.5 to 1.0 Sv) and late (1.2 to 1.0 Sv) spermatids. Note that only a small pro-portion of c e l l s i n completely mature testes (G, naturally maturing testes from f i s h ready to spawn i n mid-January) are mature spermatozoa which sediment at 0.6 mm per hr (see H). 93. ment at 2.5 to 4.0 mm per hr. Presumably, these are sperm-atogonia and primary spermatocytes. At intermediate stages (42,49, and 54 days) a s i g n i f i c a n t peak of smaller c e l l s (1.5 Sv) i s found. Their sudden appearance, small s i z e , and haploid complement of DNA (Table V) indicate that they are spermatids. With increasing maturity (61 days) the proportion of large c e l l s i s considerably decreased. In addition, the peak of small c e l l s has a shoulder of s t i l l smaller c e l l s which sediment at 1.0 to 1.2 mm per hr. These are late spermatids. In f u l l y mature testes from naturally maturing f i s h , a single population of c e l l s which sediment at 1.2 mm per hr i s found while the mature sperm (milt) sediment at 0.6 mm per hr. (c) Spermatogonia and the Duration of the C e l l Cycle; A d e f i n i t e progression of c e l l types from spermatogonia to l a t e spermatids takes place i n a r t i f i c i a l l y induced testes. The 500 to 1000 f o l d increase i n t e s t i s weight can be accounted for by a geometric m u l t i p l i c a t i o n of c e l l s . Nine (2 9 = 512) to ten ( 2 1 0 = 1024) c e l l d i v i s i o n s could account for the observed 500 to 1000 f o l d increase i n weight. By c e l l counts of m i l t from 2 year old naturally matur-ing trout, i t appears that about 10" (1000 x 108)1 sperm are produced by each t e s t i s . These could be derived from 1 to 2 x 10 8 o r i g i n a l stem c e l l s i n the immature t e s t i s assum-ing 9 or 10 consecutive c e l l d i v i s i o n s . In f a c t , from the approximate volume of the stem c e l l s , i t i s possible to show that the maximum possible number of c e l l s i n the immature t e s t i s (10 to 20 mg weight) i s about 1 x 10 8 c e l l s . However, t h i s estimate assumes that there i s no contribution i n volume from connective tissues. A more r e l i a b l e estimate of the maximum number of stem c e l l s i n the immature t e s t i s can be obtained from the DNA content of each t e s t i s . Table X shows the DNA content of i n d i v i d u a l testes and the corresponding maximum number of c e l l s i n the t e s t i s . The number of c e l l s can be as low as 0.2 x 10 8 and as high as 0.4 x 10 8 depending upon the o r i g i n a l weight of the organ. I t would appear then, that more than 9 or 10 con-secutive c e l l d i v i s i o n s must be involved. Not a l l of the c e l l s i n the completely immature t e s t i s are spermatogonia; there i s some contribution from c e l l s constituting connective t i s s u e . A reasonable estimate of the o r i g i n a l number of spermatogonia would l i e between 0.1 to 0.2 x 10 8 c e l l s , which would point to 12 or 13 c e l l d i v i s i o n s i n the t e s t i s . I f each c e l l d i v i s i o n required 1 week, the geometric m u l t i p l i c a t i o n of c e l l s would not account for the observed weights of the t e s t i s ; the maximum t e s t i s weight would be reached 12 to 13 weeks afte r i n i t i a t i o n of i n j e c t i o n s . Some of the c e l l s must divide considerably f a s t e r . Examination of the DNA content per t e s t i s at various weeks afte r hormonal induction (108) reveals that at early times, the number of TABLE X DNA and RNA Contents of Immature Testi s Testes were removed from sexually immature rainbow trout (age *>1 yr, length ^20 to 22 cm, weight <v90 to lOOg) . The l e f t and r i g h t t e s t i s of each f i s h were blotted to remove excess f l u i d before weighing. As noted by Robertson (141), the l e f t t e s t i s i s consistently larger than the r i g h t t e s t i s . Each t e s t i s was minced with a razor blade and homogenized vigorously with 1 ml of TMKS i n a motor driven Potter-Elvehjem homogenizer. 1 ml of 20% t r i c h l o r o a c e t i c acid was added and the mixture was cooled on i c e for 15 min to p r e c i p i t a t e proteins and nucleic acids. The insoluble material was c o l -lected on a glass f i b r e f i l t e r , washed with 10% t r i c h l o r o -acetic acid and 9 5% ethanol. RNA and DNA contents per t e s t i s were determined spectrophotometrically as i n Section l i e of the Experimental Procedures. Note that the r a t i o of the RNA to DNA contents (1.15 ± 0.15) of immature testes i s much higher than that (0.4 + 0.1) i n the 2.8 to 5.0 Sv c e l l s i n Table V. This higher r a t i o probably r e f l e c t s the contribution of RNA from c e l l s c o nstituting connective t i s s u e . Fish Number Testis Wet Wt. (mg) RNA (yg) DNA (yg) c e l l s per t e s t i s (xlO - 7) 1 L 12 99 93 1.85 R 11 91 87 1.75 2 L 20 173 161 3.2 R 12 98 102 2.05 3 L 27 256 190 3.85 R 22 240 165 3.3 Average 17.3 160 135 2.7 a Based on the d i p l o i d DNA content (5.0 x 10~ 1 2g) of trout c e l l s (Table V) . c e l l s i n the t e s t i s doubles every 0.5 week (3 to 3.5 days), suggesting that t h i s i s the time required for spermatogonia to complete the c e l l cycle. (d) A Model for the Development of the T e s t i s ; I t i s possible to derive a model for the development of the t e s t i s , based upon the rates of c e l l d i v i s i o n and the observed c e l l sedimentation p r o f i l e s . To simplify the ca l c u l a t i o n s , we s h a l l use 960 x 10 8 as the number of sperm produced by a t e s t i s . I f we follow the c e l l types backwards, 960 x 10 8 sperm must have come from 960 x 10 8 late spermatids, 960 x 10 8 middle spermatids, 960 x 10 8 early spermatids, 240 x 10 8 primary spermatocytes, 120 x 10 8 B stem c e l l s , and 60 x 10 8 A ' c e l l s (stem c e l l s committed to divide into B stem c e l l s ) . The observed asynchrony of t e s t i s c e l l develop-ment (Fig. 15) indicates that most of the c e l l s pass through a given stage during a 3 to 5 week period. I f these c e l l types are d i s t r i b u t e d normally through time and i f we know the approximate duration i t takes to pass from one c e l l type into another, we can p l o t the number of c e l l s of each type i n the t e s t i s as a function of time. Fig. 16 shows the number and the temporal d i s t r i b u t i o n of each c e l l type, assuming that a l l the c e l l s of each type are d i s t r i b u t e d i n the proportions 5, 20, 50, 20, and 5% at each succeeding week of a 5 week span. The i n t e r v a l between A and B, and B and primary spermatocytes, has been set at F i g . 16. Hypothetical d i s t r i b u t i o n of each c e l l type as a function of the developmental stage of the t e s t i s . The number of spermatogonial c e l l d i v i sions and i n t e r v a l between each c e l l type (chronology of spermatogenesis) i s discussed i n the text. 0.5 week. The i n t e r v a l between early primary spermatocytes and early spermatids i s 2 weeks; the i n t e r v a l between sperm-atids of d i f f e r e n t stages i s 1 week. I t i s assumed that development of one c e l l into another c e l l type i s tempor-a l l y fixed (18); that i s , i n succeeding generations of c e l l s a r i s i n g from the A' spermatogonia, c e l l s at the leading portion of the A' d i s t r i b u t i o n occupy corresponding positions of succeeding d i s t r i b u t i o n s . Evidence for the i n t e r v a l between c e l l peaks ( i . e . , the chronology of spermatogenesis i n trout) follows. As mentioned above, the average time for the early (immature) t e s t i s to double i t s DNA content i s about 0.5 week. Pre-sumably, then, i t takes about 0.5 week for each of the A, A', and B c e l l cycles. I t takes about 2 weeks for trout t e s t i s primary spermatocytes to undergo DNA synthesis and the two meiotic d i v i s i o n s to produce 4 spermatids. We have followed testes c e l l s from naturally maturing f i s h pulse-. 4 labeled with [ 3H]guanosine which labels c e l l s synthesizing DNA. At appropriate i n t e r v a l s a f t e r i n j e c t i o n of label into f i s h , c e l l s were separated by sedimentation and analyzed _ -[ H]Guanosine labels both RNA and DNA; however, since (a) more DNA i s synthesized i n testes c e l l s than RNA, and (b) DNA i s metabolically stable while RNA turns over and i s gradually l o s t during spermiogenesis, [ 3H]guanosine can be used to follow c e l l s labeled i n t h e i r DNA. In f a c t , the p r o f i l e s of guanosine and thymidine la b e l i n g are very sim-i l a r (Fig. 17a). for r a d i o a c t i v i t y (Fig. 17); l a b e l was found i n early sperm-atids (17 days) and l a t e spermatids (31 days), i n d i c a t i n g that i t takes about 2 weeks to pass through the primary spermatocyte stage, and about 2 weeks to go from early spermatid to late spermatid. In vertebrate systems which have been studied, the duration of the primary spermatocyte stage i s also about 2 weeks (Table IV). Some in d i c a t i o n of the duration of each of the spermatid stages i s also found from the p r o f i l e s of c e l l sedimentation of c e l l s prepared from testes at d i f f e r e n t stages (Fig. 15) and from the acid-soluble proteins (histones and protamines) resolved by starch gel electrophoresis from each of the d i f f e r e n t c e l l types (Fig. 7). Early spermatids are found aft e r 42 to 49 days (Fig. 6A and 9), middle spermatids after 55 days, late spermatids aft e r 57 to 60 days (Fig. 5 and 6), and mature sperm afte r 67 days (Fig. 7B). Thus, the time spent by the spermatid i n the early, middle, and l a t e stages i s close to 1 week each. The 960 x 10 8 spermatozoa must have arisen from 60 x 10 8 A" stem c e l l s through four c e l l d i v i s i o n s . I f we assume the immature t e s t i s (10 to 20 mg wet wt.) contains between 0.1 to 0.2 x 10 8 o r i g i n a l stem c e l l (gonocytes), these c e l l s must have undergone 8 to 9 consecutive c e l l d i v i s i o n s to produce close to 60 x 10 8 A 1 stem c e l l s . A possible scheme for the generation of 60 x 10 8 A' stem c e l l s normally VELOCITY of SEDIMENTATION (S v,inmm/hr) FRACTION NUMBER F i g . 17. Pulse-chase of [ 3H]guanosine i n c e l l s from nat-u r a l l y maturing t e s t i s . (A), a c e l l suspension from a hormonally induced t e s t i s (early protamine stage) was labeled with 30 yCi per ml of [ H]thymidine and 3 yCi per ml of I 1 1 1C]guanosine for 0.5 hr. C e l l s of various sizes were resolved by sedimentation on serum albumin gradients for a t o t a l of 5.0 hr including 40 min for the c o l l e c t i o n of 7.0 ml fractions (8.0 ml fractions i n C). After determining the number of c e l l s per ml with the aid of a hemocytometer, c e l l s were fixed with formaldehyde and coll e c t e d on glass f i b r e f i l t e r for analysis of radio-a c t i v i t y . Note that the p r o f i l e s of [3H] thymidine (—• •-and t 1 ^ C]guanosine (—o O-) labeling are very s i m i l a r , suggesting that most of the lab e l i s incorporated into DNA. B,C,D, and F, are sedimentation p r o f i l e s of c e l l s from naturally maturing testes 1.5, 3.5, 5, 17, and 31 days, respectively a f t e r i n j e c t i o n of 500 yCi of [ 3H]guanosine int o the peritoneal cavity of naturally maturing (Oct. to Nov.) f i s h . ( ), t o t a l c e l l counts x 10"1*; ( ), non r e f r a c t i l e c e l l counts x 10"*3; (-O—O-) [ 3H]guanosine l a b e l . Note the difference i n scales between counts of t o t a l and non r e f r a c t i l e c e l l s ; less than 10% of the sperm-atids are early spermatids (1.5 Sv) i n naturally maturing testes between October and November. 101. d i s t r i b u t e d over a 5 week period by the geometric d i v i s i o n of 0.125 x 10 8 o r i g i n a l spermatogonia i s shown i n Table XI. In F i g . 16, the t h e o r e t i c a l numbers of c e l l s of each type are given at any p a r t i c u l a r time based upon the rate of c e l l d i v i s i o n and inter v a l s between the d i f f e r e n t c e l l types as discussed above. I f the wet weight of each c e l l type i s known, i t should be possible to calculate the weight of the t e s t i s at any time aft e r i n i t i a t i o n of hormonal i n -je c t i o n s . The approximate sedimentation v e l o c i t y constant of each of the c e l l types i s known (Fig. 15). From the approx-r 2 xmation of the sedimentation v e l o c i t y equation, Sv = ^— (ref. 119), the radius and hence the volume of a spherical c e l l can be estimated. Since the density of most c e l l s i s very close to one, the wet weight of a c e l l can be approximated by i t s volume. Table XII shows the calculated spherical volume for each of the c e l l types involved i n spermatogenesis i n trout t e s t i s . In other systems for which quantitative data are a v a i l -able on the sizes of the d i f f e r e n t c e l l types undergoing spermatogenesis (Table I I I ) , the spermatogonia are s i g n i f -i c a n t l y larger than the early primary spermatocytes. As discussed above, spermatogonia i n trout testes have a gen-eration time close to 0.5 week while the primary spermato-cytes have a much longer l i f e ('v 2 weeks including a very long prophase). In both spermatogonia and spermatocyte, 102. TABLE XI A Possible Mode of P r o l i f e r a t i o n of Spermatogonia i n Trout Testes Consecutive c e l l d i v i s i o n s of o r i g i n a l spermatogonia (gonocytes) at f i r s t slowly (1 d i v i s i o n per week) then rapidly (2 d i v i s i o n s per week) generate a large number of predef-i n i t i v e (A) spermatogonia. Under the influence of gonado-tropins, some of these are transformed into i n d e f i n i t i v e (A 1) spermatogonia which divide to y i e l d the d e f i n i t i v e (B) spermatogonia. The scheme i s arranged so that close to 60 x 10 8 A' spermatogonia are approximately normally d i s t r i b u t e d over a 5 week period. For comparison, a 5:20:50:20:5 d i s -t r i b u t i o n of 60 x 10 8 spermatogonia i s shown. A l l values are x 10" 8. Days Weeks 0 0 7 1 14 2 21 3 28 4 35 5 42 6 49 7 56 8 A spermatogonia A' spermatogonia 0.125 0.25 0.5 1 2 4 7 1 11 14 3 8 14 7 14 21 2 1 12 3 2 Total A' per week 5:20:50 :20 :5 Di s t r i b u t i o n 1 3 11 12 35 30 15 12 2 3 103. TABLE XII Volumes of Trout Testis C e l l s The volumes of trout t e s t i s c e l l s were estimated from the approximation (119) of the sedimentation v e l o c i t y equation, Sv = , i n which Sv has units of mm per hr and r microns. Thus, r = 2/sv and V = 4.18r 3 for spherical c e l l s . C e l l Type Sv (mm per hr) Radius (y) Diameter (y) Volume (y 3) Mature Sperm Late Spermatid Middle Spermatid Early Spermatid Spermatogonia and Early Primary Spermatocyte Mi t o t i c c e l l s and Meiotic c e l l s i n prophase 0.6 1.0 1.0 to 1.5 1 1.5 2.8 3. 5 4.5 to 5.0 1.55 2.0 2.0 to 2.45 2.45 3.3 3.7 4.2 to 4.5 3.2 4.0 4.0 to 4.9 4.9 6.6 7.4 8.4 to 9.0 15.6 33 33 to 61 61 160 220 315 to 375 104. the same amount of DNA must be synthesized per c e l l cycle. The shorter generation time for spermatogonia implies a considerably higher rate of DNA synthesis. The finding (Table VI) that the 3.5 Sv c e l l s synthesize DNA 2.5 times faster than the 2.8 Sv c e l l s i s consistent with the 3.5 Sv c e l l s being spermatogonia and the 2.8 Sv c e l l s primary spermatocytes. Thus, i n c a l c u l a t i n g the wet weight of the t e s t i s at various stages of development, a value of 215 x 10 9 mg per c e l l (3.5 Sv) has been used for the A and B spermatogonia, 160 x 10~ 9 mg per c e l l (2.8 Sv) for the early (pre-DNA synthesis) primary spermatocyte, and 350 x 10 9 mg per c e l l for the larger late primary spermatocyte (there i s a f a i r proportion of c e l l s which have a t e t r a p l o i d complement of DNA sedimenting between 4 to 5 mm per hr at early times i n F i g . 15A to E). F i g . 18 shows the t o t a l c e l l number and expected t e s t i s wet weight calculated from F i g . 16, Table XI and Table XII. Since spermatozoa leave the t e s t i s and f i l l the sperm sac, the number of sperm and the t o t a l weight of sperm c e l l s are plotted apart from the t e s t i s c e l l s . Also shown i n Fig. 18 are experimental points for the actual growth of the t e s t i s . At early times (1 to 2 weeks) the observed and c a l -culated t e s t i s ~ weights are quite d i f f e r e n t . This difference i s due to connective tissue which has not been considered i n the c a l c u l a t i o n s . In the ca l c u l a t i o n s , half of the DNA 10$. F i g . 18. Estimated t o t a l c e l l number and weight of the t e s t i s at d i f f e r e n t stages of development. The number of c e l l s and weight of the t e s t i s was calculated from (a) the p r o l i f e r a t i o n of spermatogonia (Table XI), (b) the number and d i s t r i b u t i o n of the d i f f e r e n t c e l l types as a function of time (Fig. 16) , and (c) the estimated wet weights of each c e l l type (Table XII) . The experimental weights of testes (open c i r c l e s , dotted line) are shown for comparison with the calculated weights of the t e s t i s ( s o l i d c i r c l e s , s o l i d l i n e s ) . The difference i n the actual and calculated weight of the t e s t i s at early times (1 to 28 days) i s attributed to connective tis s u e , blood c e l l s , and i n t e r s t i t i a l f l u i d . Mature sperm leave the t e s t i s ; con-sequently, the number and weight of the spermatozoa are plotted apart from those of the t e s t i s . content of the uninduced t e s t i s has been ascribed to sperm-atogonia (gonocytes); together with the estimated weight of each spermatogonia, the spermatogonia make up about 10 to 15% (2.6 mg) of the t o t a l weight (^  20 mg) of the immature t e s t i s . The other 85 to 90% i s presumably due to connective tissue, i n -t e r s t i t i a l f l u i d , and blood c e l l s . However, at l a t e r times (2 to 4 weeks) the observed and calculated weights converge (decreasing proportion of connective tissue) and reach a maximum after 7 to 9 weeks of hormonal i n j e c t i o n s . The weight of the t e s t i s peaks and begins to decline before the t o t a l c e l l number peaks. This i s attributed to the loss of cytoplasm and decrease i n c e l l mass during spermiogenesis. The maximum weight of sperm achieved (^1.6 g) i s very close to that observed i n vivo (5 ml mil t = ^1.6 g packed sperm c e l l s ) . DISCUSSION The completely immature t e s t i s i s a th i n translucent cord l y i n g dorsally i n the peritoneal cavity. A series of injections of p i t u i t a r y extracts into sexually immature f i s h results i n a rapid increase i n the weight of the t e s t i s . At early stages, the t e s t i s i s pinkish. When the maximum weight of the t e s t i s i s achieved i t s color begins to change from pink to cream and i t s weight begins to decline. The f u l l y mature t e s t i s has a milky-white appear-ance. The transformation i n colour i s associated with the maturation of the spermatid to the spermatozoan, while the decline i n weight i s associated with the passage of sperm-atozoa from the ducts of the t e s t i s into the sperm sac; t h i s process i s c a l l e d "spermiation" and i s influenced by gonadotropins (143). H i s t o l o g i c a l studies (115,141,144) of salmonid sperm-atogenesis have shown that the completely immature t e s t i s i s structured into lobules bounded by connective t i s s u e . Each lobule i s composed of 2 or 3 to half a dozen or more cysts of spermatogonia. With the onset of spermatogenesis, the sperm-atogonia divide repeatedly within each i n d i v i d u a l cyst. The large number of c e l l s undergoing c e l l d i v i s i o n cause the cysts and hence the lobules and t e s t i s to rapidly en-large. At some stage, primary spermatocytes are formed. After a f i n a l period of DNA synthesis, the primary sperm-atocytes undergo meiosis producing four spermatids with a haploid complement of DNA. The spermatids no longer divide but d i f f e r e n t i a t e through a process c a l l e d "spermiogenesis" into spermatozoa. C e l l s within each cyst are at si m i l a r stages of development. C e l l s i n d i f f e r e n t cysts, however, may be at widely d i f f e r i n g stages and i n large testes, some cysts consist of mature sperm, others of primary spermato-cytes, and others yet of spermatogonia (115,141,144). A 500 to 1000 f o l d increase i n DNA content and t e s t i s weight (implying a s i m i l a r increase i n c e l l number) had previously been observed (10 8,121) i n trout t e s t i s . An e s s e n t i a l prerequisite to c e l l d i v i s i o n i s DNA synthesis and associated with DNA synthesis i s the synthesis of h i s -tones, a group of basic proteins associated with the genetic material. The exact functions of the histones are s t i l l unclear (10,11). They are believed to play either a struc-t u r a l role by n e u t r a l i z i n g the negative charges of the DNA phosphates, thus allowing chromosomes to e x i s t i n a "compact" state, and/or a regulatory role i n repressing genetic a c t i v i t y by vir t u e of t h e i r close association with DNA. However, la t e i n salmonid spermatogenesis, the histones are replaced by the protamines, a series of small proteins (M.W. 5000) which are very r i c h i n arginine (7,16,140). Using cytochemical techniques, A l f e r t (96) was the f i r s t to show that the protamines appeared i n middle-stage sperma-t i d s and associated with t h e i r appearance was the loss of material with the staining properties of histone. I t could not then be established, however, whether the protamines were newly synthesized or whether i n some way they were products of histone degradation (15,140). However, auto-radiography of h i s t o l o g i c a l sections indicated that i n middle-stage spermatids of mouse (98) and grasshoppers (97) an arg i n i n e - r i c h protein i s rapidly synthesized. More recently i t has been shown (99,100) that protamine i s syn-thesized i n the cytoplasm and rapidly transported into the nucleus. In addition, Ingles and Dixon (104) showed that i n trout t e s t i s that had just begun to synthesize protamine, the protamines were extensively modified by the phosphorylation of s e r y l residues, whereas those extracted from mature spermatozoa (milt) were largely unphosphorylated. In .this part of the t h e s i s , the technique of c e l l sed-imentation (119,120) at unit gravity i n gradients s t a b i l i z e d by serum albumin has been used to separate c e l l s from d i f f e r -ent stages of testes induced to mature by hormones i n salmon p i t u i t a r y extracts. Different populations of c e l l s were found i n testes at d i f f e r e n t stages of development (Fig. 15). Large c e l l s (spermatogonia and primary spermatocytes) pre-dominated i n early testes (up to 35 days of hormonal i n -jection) , while small c e l l s (spermatids at varying stages of development) predominated at l a t e r stages (50 days and over). By selecting testes at appropriate stages, i t was possible to obtain s u f f i c i e n t numbers of the various c e l l types—spermatogonia and spermatocytes (2.8 to 3.5 Sv) , early spermatids (1.5 Sv), mixtures of early and middle spermatids (1.5 to 1.0 Sv), late spermatids (1.0 Sv), and mature sperm (0.6 S v ) — f o r biochemical characterization. The c e l l s were characterized by t h e i r s i z e , t h e i r time of appearance during t e s t i s development, by t h e i r DNA and RNA contents, by t h e i r complement of nuclear proteins ( i . e . histones and protamines), and by t h e i r r e l a t i v e rates of DNA, histone, and protamine biosynthesis. Nucleic Acid and Protein Analysis: By DNA analysis of c e l l s separated by sedimentation (Table V), c e l l s sedimenting at 5.0 to 9.0 mm per hr have close to a t e t r a p l o i d DNA content and are presumably mitotic c e l l s or l a t e primary spermatocytes. The 2.5 to 4.0 mm per hr c e l l s have a d i p l o i d DNA content (A and B stem c e l l s , and early primary spermatocytes) while the 1.5 to 0.6 Sv c e l l s have a haploid DNA content (spermatids and spermatozoa, r e s p e c t i v e l y ) . The complement of nuclear proteins was also characterized i n the d i f f e r e n t c e l l types by starch gel electrophoresis (Fig. 7). Large c e l l s (2.5 to 9.0 Sv), and spermatids from 42 to 49 day testes (early spermatid stage) contain only histones. Spermatids from 55 to 67 day testes were resolved by sedimentation for an extended period into a mixture of early (1.5 Sv) and middle (1.5 to 1.0 Sv) spermatids, and late (1.2 to 1.0 Sv) spermatids which contain both histones and protamines. Onset of protamine biosynthesis and histone replacement marks the beginning of the middle spermatid stage. The l a t e spermatids have synthesized almost the entire com-plement of protamines and l o s t most of t h e i r histones. During the t r a n s i t i o n from late spermatids to spermatozoa, the residual histones are l o s t (Fig. 7). The spermatozoan nucleus consists almost e n t i r e l y of nucleoprotamine (83). DNA and RNA synthesis; As judged by incorporation of [3H] thymidine, a s p e c i f i c l a b e l for DNA, most of the DNA i s synthesized by 2.8 to 3.5 Sv c e l l s and packaged into a more compact form during the transformation of spermatids into mature sperm. As expected, very l i t t l e DNA i s synthesized i n the spermatids (Table VI). However, a s i g n i f i c a n t i n -corporation of [ 3H]thymidine ('v 2 x that of 3.5 Sv c e l l s ) was found i n the large c e l l s sedimenting between 4.5 to 7.0 Sv (Table VI). As judged by microscopic examination, these large c e l l s are not dimers of smaller (2.8 to 3.5 Sv) c e l l s . Moreover, the r a t i o of RNA:DNA i s s i g n i f i c a n t l y higher i n the larger c e l l s (0.45 ± .05) than i n the smaller c e l l s (0.3 ± .05). At present, the si g n i f i c a n c e of t h i s incorporation i s not understood, although i t could be related to the synthesis of a small amount of late r e p l i c a t i n g DNA observed during the pachytene stage of meiosis i n L i l i u m (204) and mouse (205). 112. The generally larger s i z e of spermatogonia (Table III) and more rapid rate of DNA synthesis ( i . e . shorter generation time), suggest, that c e l l s sedimenting i n the 3.5 Sv region are spermatogonia, while the smaller size and slower rate of DNA synthesis (longer c e l l cycle) suggest that c e l l s i n the 2.8 Sv region are early (preleptotene) primary spermatocytes. Determinations of the RNA content of the d i f f e r e n t c e l l types (Table V) indicate that the early spermatids contain at l e a s t half as much RNA (on a DNA basis) as the large 2.8 to 3.5 Sv c e l l s . Thus, there i s a s i g n i f i c a n t amount of RNA (and hence ribosomes) i n the early spermatids for carrying on protein synthesis. The content of RNA decreases as the spermatids mature, presumably due to a sloughing of cytoplasm (18). Autoradiographic studies have indicated that RNA syn-thesis increases rapidly i n la t e meiotic prophase, ceases during the 2 meiotic d i v i s i o n s , and continues at a high l e v e l i n early spermatids (18). RNA synthesis gradually decreases, and ceases before nuclear condensation and elongation (18). However, arginine incorporation ( i . e . protamine biosynthesis) increases i n the elongating spermatids (18). The time of synthesis of the messenger RNA for protamine, i s not known. Presumably i t i s i n the early spermatids since l i t t l e or no RNA synthesis i s seen i n middle spermatids when protamine i s actually synthesized. 113. Synthesis and Phosphorylation of Histones and Protamines: Histones and DNA are synthesized simultaneously i n the 2.8 and 3.5 Sv c e l l s ; l i t t l e or no synthesis of either was seen i n spermatids (Table XIII, IX and X). Histones are the only basic nuclear proteins found i n large c e l l s (2.8 to 3.5 Sv) and early spermatids (1.5 Sv). However, they gradually disappear during the t r a n s i t i o n from middle to l a t e sperm-at i d s , and when spermiogenesis i s complete (mature sperm), no histones remain (Fig. 7). I t was previously postulated (22,105) that the exten-sive phosphorylation of histones observed i n trout testes might be important i n the removal of histones from chromatin during spermiogenesis (maturation of the spermatid). The studies here have shown that q u a n t i t a t i v e l y , histone phos-phorylation occurs i n the larger c e l l s synthesizing both histones and DNA. Thus, phosphorylation of histones does not appear to play a s i g n i f i c a n t role i n the replacement process. Similar studies on the acetylation of histones have been conducted by Candido and Dixon (146) . Acetylation was found i n a l l c e l l types; however, s i g n i f i c a n t acetylation not connected with histone synthesis was found i n spermatids, in d i c a t i n g that acetylation of histones may have a s i g -n i f i c a n t role i n the replacement process (146). The rates of DNA synthesis, histone synthesis, and histone phosphorylation bear a 1:1:1 r e l a t i o n to each other, 114. suggesting that phosphorylation of histone may have an im-portant role i n the metabolism of the c e l l . The si g n i f i c a n c e of histone phosphorylation and acetylation i n the large c e l l s i s the topic of PART III of this Thesis. Protamines are f i r s t seen i n middle spermatids and are rapidly synthesized i n these c e l l s as well as the smaller l a t e spermatids (Fig. 7). Seven hours after i n i t i a t i o n of I1 **C] arginine l a b e l i n g (Fig. 7A) , the labeled protamines are present as a series of phosphorylated derivatives which migrate more slowly than the protamines of mature sperm. As the middle spermatids develop into the smaller, late spermatids and eventually into mature sperm, these phospho-protamines are progressively dephosphorylated and converted to the single major band of dephospho-protamine seen i n sperm (Fig. 7B). This cycle of enzymatic phosphorylation and dephosphorylation of protamine had been indicated previously by Ingles and Dixon (104) who showed that i n the early protamine stage of t e s t i s development, approximately 75% of the s e r y l residues were phosphorylated, whereas i n sperm, only 5 to 6% of the s e r y l residues were phosphorylated. The dephosphorylation of protamine appears to be related to the gradual condensation of the spermatid chromatin (Fig. 7). A study of the k i n e t i c s of enzymatic modification of the protamines i s the subject of PART II of t h i s Thesis. 115. Chronology of Spermatogenesis; In most vertebrate species studied, the duration of the primary spermatocyte stage i s close to 2 weeks; the larger part of t h i s period i s spent i n meiotic prophase (18) . The time required for maturation of the spermatid i s generally between 2 to 3 weeks (Table IV). There i s l i t t l e concrete data on the duration of spermato-genesis i n f i s h . In the common "black mollie" ( P o e c i l i a  spenops), an oviviparous t e l e o s t f i s h , the duration of the primary spermatocyte and spermatid stages was estimated by [ 3H]thymidine lab e l i n g , and the frequency of the d i f f e r -ent c e l l types (147). Label was found i n sperm about 21 days a f t e r p u l s e - l a b e l l i n g with (?Hlthymidine. T h e duration of the primary spermatocyte stage was 7 days, while spermiogenesis required 14 days. The present rough estimates of the duration of the d i f f e r e n t stages of spermiogenesis i n trout t e s t i s (1 week for early spermatid, 1 week for middle spermatid, and 1 week for late spermatid stages) were obtained from the progressive changes i n the population of the d i f f e r e n t c e l l types at d i f f e r e n t periods a f t e r i n i t i a t i o n of hormonal i n -jections (Fig. 15). Isotopic labeling of c e l l s a c t i v e l y synthesizing DNA (Fig. 17) indicated that 2 weeks pass before l a b e l i s found i n early spermatids (1.5 Sv) and 4 weeks before l a b e l i s found i n late spermatids (1.0 Sv) i n naturally maturing trout. Volumes of the Different C e l l Types: Large changes i n volume occur i n c e l l s undergoing spermatogenesis (Table I I I ) . The volumes of c e l l s at s i m i l a r stages of development vary considerably from species to species (Table I I I ) . For example, the volume of A spermatogonia i n rats i s close to 200u 3, i n rams 250u 3, and i n b u l l s 400 ± 100y 3. These v a r i -ations are not due to widely d i f f e r i n g contents of DNA since the d i p l o i d content of DNA i n c e l l s of the ram and the b u l l i s almost i d e n t i c a l (18). In general, the volume of suc-ceeding generations of spermatogonia decreases progres-s i v e l y (Table I I I ) , while the duration of the S phase i n -creases (Table XIII). The increase i n duration of the S phas i s attributed to increased heterochromatization of chromatin (and hence slower DNA synthesis) i n late spermatogonia (18). The duration of the spermatogonial c e l l cycles i n d i f f e r e n t mammalian species i s shown i n Table XIII. In mouse, i t l a s t s about 30 hours, i n rats 40 hours, and i n b u l l s 50 to 70 hours. I t would appear that no generaliza-tions can be made regarding the duration of the sperm-atogonial c e l l cycles i n d i f f e r e n t species. There i s l i t t l e information on the duration of these c e l l cycles i n f i s h . Our estimates of the duration of the cycle (3 to 3.5 days) and the number of spermatogonial d i v i s i o n s (10 to 11) i n trout are based upon the geometric increase i n DNA content and weight of t e s t i c u l a r t i s s u e . 117. TABLE XIII Duration of the S Phases of DNA Synthesis and C e l l u l a r Cycles i n D i f f e r e n t Categories of Spermatogonia [from Courot et a l (18)]. Mouse Rat Bull (Monesi, 1962) (Hilschcr, 19C4) (Hochereau, 1967) Spermatogonia S phase Life span S phase Life span S phase Life span A, 7 hr 5 days 19. 5 hr 8 days 11.9 hr 9.5 days A 2 7. ,5hr 29 hr 20 . 5 hr 39 hr 11.4 hr 45 hr A 3 8 hr 28 hr 21 hr 42 hr 13.5 hr 60 hr At 13 hr 30 hr 23 hr 42 hr In 14 hr 27 hr 24 hr 42 hr 17.3 hr 60 hr B, 18 hr 29 hr . 25 ,5hr 42 hr 20.9 hr 72 hr B, — — — — 19.6 hr 48 hr . Spermatogonial divisions 12 days 20 days 24 days In comparison to the duration i n mammalian testes (Table XIII) our average value of 3 to 3.5 days i s not unduly long, con-sid e r i n g that rainbow trout normally metabolise at 10 to 15° versus 37° i n mammals. In view of the large number of spermatogonial d i v i s i o n s , i n trout t e s t i s , i t i s unlikely that the volume of succeed-ing generations of spermatogonia gradually decreases. The paucity of such information on spermatogenesis i n f i s h makes comparisons very tenuous. Since we know of no informa-t i o n regarding the duration of the S phase i n the d i f f e r e n t f i s h c e l l types, the estimates of the rate of DNA synthesis and assignment of 3.5 Sv c e l l s as spermatogonia and 2.8 Sv c e l l s as early primary spermatocytes are tentative and sub-j e c t to r e v i s i o n as the c e l l s become more accurately charac-t e r i z e d . During prophase, the volume of the primary spermato-cyte increases greatly (Table I I I ) . In mammals, the maxi-mum volume reached varies i n d i f f e r e n t species: i n mouse (120), i t approaches lOOOy3 (8 to 10 Sv); i n rams and b u l l s , 300 to 400u 3. Few data on the volume of spermatocytes i n f i s h are available. I have not been able to observe any s i g n i f i c a n t peak of c e l l s larger than 5 Sv i n trout t e s t i s . C e l l s with a t e t r a p l o i d complement of DNA sediment between 4.5 to 7.0 mm per hr (Table V). However, they have not been p o s i t i v e l y i d e n t i f i e d as primary spermatocytes i n meiotic prophase although the t e t r a p l o i d complement of DNA i s highly suggestive. These c e l l s are unlikely to be sperm-atogonia undergoing mitosis which i s a r e l a t i v e l y rapid process Spermatids are much smaller than spermatogonia and spermatocytes. Thus, they are e a s i l y resolved from the larger c e l l s by sedimentation. Estimates of the volume of spermatids from the approximation of the sedimentation v e l o c i t y equation (119) y i e l d values of 61y 3 for early spermatids, 33y 3 for late spermatids, and 15.5y3 for mature sperm (Table XII). Thus, an o v e r a l l 4 f o l d decrease i n volume occurs during spermiogenesis i n trout. Similar changes occur i n mouse sperm-atids (120). I t should be noted here that only a small proportion of the c e l l s i n f u l l y mature testes i s spermatozoa (Fig. 15G); most of the spermatozoa f i n d t h e i r way int o the sperm sac where they are stored and concentrated p r i o r to ejacula-t i o n . In e a r l i e r c y t o l o g i c a l observations of trout and salmon t e s t i s (115,141,144) the c e l l s labeled as mature spermatozoa and present i n s i g n i f i c a n t proportions may, i n fa c t , be late spermatids (1.0 Sv). Late spermatids (a) have a t a i l , (b) are highly condensed, (c) have almost completed synthesis of the entir e complement of protamine and (d) have l o s t most of t h e i r histones; they may be mistaken for mature sperm, since the diameters (Table XII) of late sperm-atids (4y) and mature sperm (3.2y) are not much d i f f e r e n t . Dry-weight analysis of c e l l s (late spermatids) from mature testes and mature sperm (from milt) y i e l d a value of 8 x 10 mg per late spermatid and 5.0 x 10 9 mg per mature sperm. For comparison, the average dry weight of c e l l s from early testes i s about 40 x 10 9 mg per c e l l . Maintenance of the Germ-Cell Line and Spermatogonial Pro-l i f e r a t i o n : The maintenance of the germ-cell l i n e , and the p r o l i f e r a t i o n and d i f f e r e n t i a t i o n of spermatogonia are poorly understood (17,18,112). Spermatogonial p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i n one species may be d i f f e r e n t from that i n phylogenetically remote species (112,142). Hannah-Alava (112) describes several possible schemes of stem c e l l renewal and spermatogonial m u l t i p l i c a t i o n . E s s e n t i a l l y , these f a l l i n t o 2 major categories. In one, the primordial stem c e l l s (gonocytes) undergo a series of dichotomous d i v i s i o n s (2 equivalent daughter c e l l s at each division) generating a large pool of " p r e d e f i n i t i v e " spermatogonia (A stem c e l l s ) . "In-d e f i n i t i v e " spermatogonia (A' stem c e l l s ) are selected almost at random from the large supply of p r e d e f i n i t i v e spermato-gonia to divide into " d e f i n i t i v e " B spermatogonia. In the other category, a quasidichotomous or "stem-cell" d i v i s i o n (two unequal daughter c e l l s , one p r e d e f i n i t i v e and maintaining the germ-cell l i n e , the other d e f i n i t i v e ) makes possible sperm-atogonial renewal and m u l t i p l i c a t i o n . I t i s possible to discriminate between these two mechanisms and variations within them by counts of the number of c e l l s i n cysts or genetic analysis of progeny. There i s some evidence (112) for the stem-cell method of spermatogonial renewal and mul-t i p l i c a t i o n i n the r a t , the mouse, Drosophila, and the s i l k worm (Bombyx mori), while dichotomous d i v i s i o n s have been reported i n b u l l s , rams, man, and rats (18). There i s l i t t l e information on the m u l t i p l i c a t i o n of spermatogonia i n the salmonid fi s h e s . Most workers have focused t h e i r attention on the f i n a l stages of spermato-genesis, the transformation of spermatids into spermatozoa. Table XIV shows some estimates of the number of spermatogonial d i v i s i o n s i n d i f f e r e n t species. In most mammals, 5 to 6 di v i s i o n s of spermatogonia (32 to 64 progeny) occur; i n f i s h , much larger numbers have been recorded. 10 to 14 spermatogonial c e l l d i v i s i o n s would y i e l d 1000 to 16,000 progeny. Our estimate of 10 to 11 spermatogonial di v i s i o n s based upon the increase i n DNA content of the t e s t i s i s comparable to that recorded i n other fishes. I t should be noted that the number of spermatogonial di v i s i o n s estimated by Henderson (112) i n brook trout (S. f o n t i n a l i s ) i s compari-t i v e l y low. In view of the lack of concrete evidence for the stem-c e l l or random se l e c t i o n mode of spermatogonial m u l t i p l i c a t i o n , the scheme depicted i n Table XI i s purely deductive. The scheme i s a gross approximation based on the necessity of generating 60 x 10 8 A' c e l l s from fewer than 0.2 x 10 8 spermatogonia i n the short period of a few weeks. As i t TABLE XIV Number of Spermatogonial Divisions [from Courot et a l . (18) ] No. of Species Method of analysis generations References Fish Guppy No. of spermatogonia/cyst 10-12 Geiser (1924) Guppy No. of spermatogonia/cyst 14 Billard (1968) Char No. of spermatogonia/cyst 6 Henderson (1962) Spurdog No. of spermatogonia/cyst 12 Mellinger (1965) Dogfish No. of spermatogonia/cyst 13 Holstein (1969) Birds Cock Nuclear variations. 1 Lake (1956) Cock Labeling index; nuclear variations 2-3 De Reviers (1968a) Duck Mitotic index 3 Clermont (1958a) Quail Nuclear variations 3 Yamamoto et al. (1967) Mammals Rat No. of spermatogonia; nuclear variations 5 Roosen-Rungeand Giesel (1950) No. of spermatogonia; mitotic index 5 Leblond and Clermont (1952b) Nuclear variations 6 Clermont and Bustos (1966) Labeled mitosis index 6 Hilscher (1964) Labeled mitosis index 6 or 7 Hilscher and Makoski (1968) Mouse Labeled mitosis index 6 Monesi (1962) Nuclear variations 5 Widmaier (1963) Hamster No. of spermatogonia; nuclear variations 5 Clermont (1954) Guinea pig Nuclear variations 4 Cleland (1951) Rabbit No. of spermatogonia 5 Swierstra and Foote (1963) Ram No. of spermatogonia; nuclear variations 5 Ortavant (1956) Bull No. of spermatogonia; nuclear variations S Ortavant (1959) No. of spermatogonia; nuclear variations 5 Kramer (I960) No. of spermatogonia; nuclear variations 5 Amann (1962) Mitotic index; labeling index 6 Hochereau (1967) Monkey Nuclear variations; mitotic index; labeling index S Clermont and Leblond (1959) Man No. of spermatogonia; nuclear variations 4 Clermont (1966) stands, the scheme f a l l s into the category "random selection of d e f i n i t i v e spermatogonia"; i t i s assumed that there i s no death or degeneration of stem c e l l s . In naturally maturing testes, h i s t o l o g i c a l studies by Henderson (115) have shown that spermatogonial p r o l i f e r a t i o n continues throughout the entire period of growth and develop-ment of the t e s t i s . Spermatogonial divisions cease only when the t e s t i s i s completely mature (December to February). There appears to be a stock of reserve spermatogonia (gono-cytes) present i n the t e s t i s at a l l times (115). E f f e c t of Gonadotropins on Spermatogenesis: Salmonid sperm-atogenesis i s influenced by gonadotropins elaborated by the p i t u i t a r i e s i n the breeding season. Robertson and Rinfret (121) noted the s t r i k i n g stimulation (500 f o l d increase i n t e s t i s weight over an eight week period) of growth of infan-t i l e testes i n rainbow trout by extracts of salmon p i t u i -t a r i e s . More gonadotropin a c t i v i t y was found i n p i t u i t a r i e s from mature than immature salmon (122). The greater the dosage of p i t u i t a r y extract injected into immature f i s h , the greater the rate of hypertrophy of the immature t e s t i s (122) . More recently, Donaldson et a l . (12) have shown that sperm-atozoa can be produced from a 10 to 12 week series of i n j e c -tions of p u r i f i e d p i t u i t a r y gonadotropins into immature salmon. Spermatozoa produced i n th i s manner were as e f f e c t i v e i n f e r t i l i z i n g eggs as those from naturally maturing f i s h . 124. There are several possible effects of the gonadotropins: (a) they may be mitogenic and be required for c e l l d i v i s i o n ; (b) they may shorten the generation time ( i . e . increase the rate of c e l l d i v i s i o n i n the t e s t i s ) ; (c) they may be re-quired for the d i f f e r e n t i a t i o n of p r e d e f i n i t i v e into d e f i n i -t i v e spermatogonia; and (d) they may be required for main-tenance of spermiogenesis. I t i s conceivable that the accumulation of a "determining" amount of gonadotropin over a period of time may cause the p r e d e f i n i t i v e A stem c e l l to be transformed into the i n d e f i n i t i v e A' c e l l which divides to form 2 B stem c e l l s . At early stages (1 to 3 weeks injection) only a small proportion of c e l l s i s committed, while at l a t e r stages (3 to 6 weeks) an increasingly greater proportion i s committed (Table XI). Possibly because of a greater accumu-l a t i o n of " h i t s " of gonadotropin, the pro b a b i l i t y of commit-ment increases with the duration of i n j e c t i o n of gonadotropins. In f i s h , the LH l i k e gonadotropins seem to be required for the early c e l l d i v i s i o n s (19,145). Thus, i n j e c t i o n of immature rainbow trout with salmon p i t u i t a r y extracts for 42 days (early spermatid stage) leads to t e s t i s hypertrophy; ces-sation of in j e c t i o n s stops further growth of the t e s t i s and the early c e l l types degenerate or are l o s t . C e l l sedimentation at 49 days (1 week af t e r the l a s t injection) indicated that most of the c e l l s are spermatids (peak 1.2 Sv), which have matured normally (or s l i g h t l y f a s t e r ) . Very few large c e l l s are found. The spermatids appear to be developing normally as judged by protamine biosynthesis and decrease i n spermatid c e l l volumes. Development of Naturally Maturing Testes: The trout t e s t i s increases dramatically i n size and then undergoes a period, of decrease i n weight during which spermatids mature and sperm leave the t e s t i s to f i l l the sperm sac. Whereas naturally maturing trout testes reach a maximum weight i n 4 to 5 months and complete maturity i n 6 to 8 months, growth of the testes i n immature trout injected with salmon p i t u i t a r y extracts i s s u b s t a n t i a l l y accelerated with the maximum weight being reached i n 2 months and complete maturity achieved i n 3 months In both cases, the testes reach the same maximum weight so that the same number of c e l l s must be involved. I t appears, therefore, that following hormonal induction of spermatogenesi the c e l l s pass through the various stages i n considerable synchrony and i t i s possible to predict the d i s t r i b u t i o n of c e l l types at any time during the maturation process (Fig. 16) The much slower doubling time (2 weeks) of naturally maturing testes (Fig. 14) may be due to a lower amount of p i t u i t a r y gonadotropins secreted naturally compared to ar t -i f i c i a l l y induced f i s h which receive large doses of gonado-tropins. The e f f e c t of this lower l e v e l of gonadotropins could be either (a) a slower rate of c e l l d i v i s i o n or (b) the same rate of c e l l d i v i s i o n as i n a r t i f i c i a l l y induced f i s h but a much lower proportion of c e l l s are " h i t " and induced to develop or divide at one time. Evidence points to the l a t t e r i n t e r p r e t a t i o n since the period between s t a r t of t e s t i s hypertrophy (June) and f i r s t detection of protamine biosynthesis (late August) i s about 8 weeks which suggests that the chron-ologies of spermatogenesis i n naturally maturing f i s h and a r t -i f i c i a l l y induced f i s h are not very d i f f e r e n t . Between late August and November, the sedimentation p r o f i l e s of c e l l s from naturally maturing testes change very l i t t l e : a l l have a high proportion of spermatids sedimenting between 1.0 to 1.6 mm per hr. During these months, the incorporation of [3H] thymidine per t e s t i s i s about the same, suggesting that a r e l a t i v e l y constant but small number of c e l l s are undergoing DNA synthesis and c e l l d i v i s i o n . These data support the argument that the d i s t r i b u t i o n of developing c e l l s of a given type i s spread over a wider period of time, and naturally maturing testes are, for that reason, less synchronized than a r t i f i c i a l l y induced testes. Obviously, some points such as (a) the mode and mechanism of spermatogonial renewal, m u l t i p l i c a t i o n and d i f f e r e n t i a t i o n , (b) the target c e l l s and mechanism of action of gonadotropins, (c) the exact chronology of salmonid spermatogenesis, (d) the sequence of events leading to unmasking and expression of the protamine genes, and (e) the mechanism of removal of the t i g h t l y bound histones from chromatin remain to be c l a r i f i e d . The descriptions and model of testes development i n terms of the changing proportions and passing of the d i f f e r e n t c e l l types should be of use i n c l a r i f y i n g these points. PART II ENZYMATIC MODIFICATIONS THE PROTAMINES By separating trout testes c e l l s on serum albumin gradients and analyzing t h e i r basic nuclear proteins on starch gels, we have shown (PART I) that protamine i s syn-thesized and phosphorylated i n middle and late spermatids. Associated with the appearance of protamine was the loss of histone (Fig. 7). On urea starch gels, at least s i x bands of protamine were observed (Fig 7,12); two were unmod-i f i e d "mature" protamines as found i n spermatozoa while the others were phosphorylated. This prompted an examination of the k i n e t i c s of incorporation of [ 3H]arginine into the various protamine bands i n order to characterize them and to explore the possible b i o l o g i c a l function(s) of protamine phosphoryla-t i o n and dephosphorylation. Positive i d e n t i f i c a t i o n of each of the bands observed on starch gels as protamine and t h e i r order of phosphorylation, was achieved by i s o l a t i o n and chemical characterization of these proteins. Hormonally induced (12 2) rather than naturally maturing trout testes were used because the accelerated development of hormonally induced testes makes i t possible to obtain testes with a larger proportion of modified protamine. EXPERIMENTAL PROCEDURES I. Materials and Abbreviations The materials and abbreviations not l i s t e d i n PART I are as follows: (a) Materials: [ 3 5S]methionine (spec. act. 430-575 C i per mmole) was obtained from Amersham-Searle; cycloheximide from Calbiochem; sucrose, density gradient grade (ribo-nuclease f r e e ) , from Mann Research Laboratories; carboxy-methylcellulose and phosphocellulose from Bio-Rad; b a c t e r i a l a l kaline phosphatase (BAP-SF 21 units per mg) from Worthington. (b) Abbreviations: CHX:- cycloheximide 2,4-DNP:- 2,4-dinitrophenol CM-cellulose:- carboxymethylcellulose I I . Overall Kinetics of Enzymatic Modification (a) C e l l Incubations: C e l l suspensions were prepared from testes at the protamine stage of development as des-cribed i n PART I. The incubation mixtures (1 ml t o t a l volume) consisted of 3 to 4 x 10 8 c e l l s , r a d i o a c t i v i t y (one or two of the follow-ing: 25 to 200 uCi of [3H] arginine, 200 yCi of [3 SS]methionine, or 300 yCi of inorganic [ 3 2P]phosphate adjusted to pH 7.2 with NaOH and T r i s b u f f e r ) , 0.2 ml of Waymouth's medium (125) (with 10 mM T r i s buffer pH 7.2 instead of phosphate buffer, and minus arginine or methionine as required), phenol red, 100 units of p e n i c i l l i n and streptomycin, and TMKS-0.1% glucose to a f i n a l volume of 1.0 ml. A l l incubations were carried out at 15 to 16° on a gyratory water bath with occasional mixing to resuspend sedimented c e l l s . In studies using i n h i b i t o r s either of protein synthesis or of energy metabolism, c e l l s were incubated with label as above for 10 min before addition of cycloheximide (2 x 10-1,M f i n a l con-centration) or 2,4-dinitrophenol (2 x 10-I*M fi n a l ) . At various times, 100 y l (3 to 4 x 10 7 c e l l s ) were re-moved and di l u t e d with 1 ml of cold TMKS. Cell s were immediately fix e d with 3 to 4 volumes of 95% ethanol and f i l t e r e d onto glass f i b e r f i l t e r s over an area s l i g h t l y smaller than the size of the starch gel sample s l o t (6 x 10 mm) with the aid of a Teflon template. The fixed c e l l s were washed with 95% ethanol and the f i l t e r s were dried. (b) In Vivo Labeling: Rainbow trout (approximately 100 g weight) i n early protamine stage (55 to 60 days after i n i t i a t i o n of hormonal induction) were injected intraper-i t o n e a l l y with 300 yCi of [ 3HJarginine i n 0.4 ml of s a l i n e . At various times, f i s h were s a c r i f i c e d . The testes were excised and c e l l suspensions were prepared e s s e n t i a l l y as above. 2.5 x 10 8 c e l l s were f i l t e r e d onto glass f i b e r discs, washed twice with 5 ml of TMKS, twice with 5 ml of 0.5% Nonidet P-40 i n TMKS, and fixed with 95% ethanol and dried. The Nonidet P-40 wash stripped the c e l l s of any cytoplasm and hence ribosomes (PART I ) . Control studies showed that nuclear histones and protamines were not removed by t h i s wash. For electrophoresis, a 6 x 10 mm2 rectangle (con-taining 'M x 10 7 c e l l s ) was cut from the f i l t e r . (c) Starch Gel Electrophoresis: Starch gels were pre-pared, basic proteins were extracted, and gel electrophoresis was conducted as i n PART I. After electrophoresis, the gels were t r i s e c t e d h o r i z o n t a l l y . The bottom slab was stained by the sensi t i v e cobalt-Amido Black procedure and destained with s u l f u r i c acid (130). The middle slab was s l i c e d into 2 mm s l i c e s which were then s o l u b i l i z e d and analyzed for r a d i o a c t i v i t y . Tritium counts were corrected for 30% 3 5S-counts i n the t r i t i u m channel. I I I . I n t r a c e l l u l a r Kinetics of Enzymatic Modifications (a) Analysis of Protamine from Ribosomes: A concen-trated c e l l suspension (1 volume of tissue to 1 volume of TMKS-0.1% glucose) was prepared from 10 g of testes at the early protamine stage of development and incubated with 15 uCi per ml of [ 3H]arginine and 15 uCi per ml of [ 3 5S]methionine for 20 min. C e l l s were sedimented at 1500 x g to remove excess label and resuspended i n 10 ml of TMKS with 2 x lO^M cycloheximide to i n h i b i t protein synthesis (99). An equal 132. volume of a 1% solution of the norwLonic detergent Nonidet P-40 i n cold TMKS was added to disrupt c e l l s ; nuclei remain in t a c t after t h i s treatment (148) . A l l further operations were performed at 4° i n the presence of 10 **M cyclohex-imide. Sedimentation at 3000 x g for 10 min removed nu c l e i ; the post-nuclear supernatant was centrifuged at 15,000 x g for 15 min. The 15,000 x g supernatant was layered over 2 ml of 1.0 M sucrose i n TMK made 10_,*M i n cycloheximide and centrifuged for 2 hr at 60,000 rpm (Spinco L2-65, 65 r o t o r ) . The ribosomal p e l l e t was washed gently with TMK and resuspended i n 4 ml of TMK. The suspension was centrifuged at 5,000 x g for 10 min to remove insoluble material. Approximately 3 mg of ribosomes i n 1 ml of TMK were c a r e f u l l y layered over the top of a 35 ml lin e a r sucrose density gradient (10 to 30% w/v i n TMK-10~14M CHX) generated by a Beckmann Density Gradient Former (DGF-IM-3) and centrifuged at 4° i n a SW-27 swinging bucket rotor (Spinco) at 24,000 rpm for 3 hr. The rotor was allowed to stop without braking, each centrifuge tube was punctured at the bottom with a needle and 35 1-ml fractions were c o l -lected. Absorbance was read at 260 nm. The monosome, d i -some, and polysome regions were pooled i n separate fractions and p r e c i p i t a t e d with 4 volumes of cold ethanol overnight. The p r e c i p i t a t e from each was f i l t e r e d onto glass f i b e r f i l t e r s over an area the size of the starch gel sample s l o t (6 ram x 10 mm) by using a Teflon template, washed with ethanol, and dried. (b) P u r i f i c a t i o n of Protamine from Nuclei: Histones and protamines were extracted twice from the above nuclei with 5 volumes of 0.4 N s u l f u r i c acid, precipitated with ethanol, and adsorbed onto a CM-cellulose column as des-cribed i n Section IV of these Procedures. Histones were eluted with 0.4 M L i C l , 3 M urea buffered with 10 mM sodium acetate, pH 5.5. The column was washed with 10 mM NH^ HCG-3 and protamines were eluted with 0.5 N HC1. The protamine solution was l y o p h i l i z e d and redissolved i n a small volume of water. (c) Microscale Separation of Cytoplasm from Nuclei: A c e l l suspension was prepared from an early protamine stage t e s t i s . The incubation mixture consisted of 5 x 10® c e l l s , 50 yCi of [ 3H]arginine, 100 yCi of [ 3 5S]methionine and TMKS-0.1% glucose to a volume of 0.5 ml. The suspension was i n -cubated at 15 to 16°. At various times aft e r i n i t i a t i o n of l a b e l i n g , 100 y l (^ 1 x 10 8 c e l l s ) were removed and added to 0.5 ml of TMKS-10_,,M CHX. A solution of 1% Nonidet P-40 i n TMKS (0.5 ml) was added to lyse the c e l l s and nuclei were c o l l e c t e d on glass f i b r e f i l t e r s by pressure f i l t r a -t i o n . The nuclei were washed twice with 0.5 ml of 1% Nonidet P-40, fixed with 95% ethanol, and dried. The aqueous f i l t r a t e (2 ml total) passed through the f i l t e r d i r e c t l y into 8 ml of cold ethanol. The solution was mixed, kept at -20° overnight, and the p r e c i p i t a t e was co l l e c t e d on glass f i b r e f i l t e r s over an area the size of the starch gel s l o t . The pr e c i p i t a t e (cytoplasmic proteins and ribosomes) col l e c t e d on the f i l t e r s was washed with 95% ethanol and dried. (d) Microscale Separation of Nucleohistone from Nucleo-protamine : The incubation mixtures consisted of 9 x 10 8 c e l l s , 200 yCi of [3H] arginine, 200 yCi of [3 5S ] methionine, 100 units of p e n i c i l l i n and streptomycin, phenol red, 0.1 ml of Waymouth's medium (125) minus arginine and methionine, and TMKS-0.1% glucose to a t o t a l volume of 1.0 ml. At various times aft e r the i n i t i a t i o n of the incubation with labeled precursors, 100 y l (^ 9 x 10 7 c e l l s ) were removed and added to 100 y l of 1% Nonidet P-40 i n TMKS. 1 ml of 10 mM Tris-HCl (pH 8.0) was added to lyse the nuclei and chromatin was sonicated at 75 to 80 watts for 90 sec using the micro-t i p probe of a Bronson s o n i f i e r . Cooling was with a mixture of ethanol, i c e , and water. I f needed, Dow Corning A n t i -foam A was added to control excess foaming. The nucleo-histone portion of chromatin i s s o l u b i l i z e d under these conditions. Nucleoprotamine remains insoluble and was f i l t e r e d under pressure onto glass f i b r e f i l t e r s , washed twice with 0.5 ml of 10 mM Tris-HCl (pH 8.0), fixed with ethanol, and dried. The f i l t r a t e ( t o t a l volume ^ 2.2 ml) consisting of s o l u b i l i z e d nucleohistone passed d i r e c t l y into 4 volumes of cold ethanol. The solution was mixed and allowed to stand at -20° overnight. The precipitated nucleohistone was co l l e c t e d on glass f i b e r f i l t e r s , washed with ethanol and dried. I t should be noted that with t h i s procedure, the cytoplasmic proteins are coll e c t e d with the nucleo-histone. However, the cytoplasm from testes c e l l s i s scanty and the contamination with cytoplasmic protamine i s not great. (e) Extraction of Proteins and Starch Gel Electrophoresis P r e c i p i t a t e d fractions (ribosomes, cytoplasmic proteins, n u c l e i , nucleohistone, and nucleoprotamine) c o l l e c t e d on f i l t e r s were extracted as before (PART I) for acid-soluble proteins and subjected to starch gel electrophoresis. IV. Separation and Characterization of Phosphorylated Species of Protamine from Trout Testes (a) Trout Testes: The stage of t e s t i s development, and whether the testes were from naturally maturing f i s h or f i s h induced to mature by inj e c t i o n s of salmon p i t u i t a r y extracts (122,123) were important i n obtaining protamine with a high proportion of phosphorylated species. A r t i f i c -i a l l y induced rainbow trout were the most favourable source. The completely immature t e s t i s i s a thin cord weighing 10 to 20 mg. After 50 to 60 days of hormonal i n j e c t i o n s , the t e s t i s wet weight has increased 500 to 1000 f o l d (PART I) to a maximum of 8 to 10 g. As described i n PART I, testes at t h i s stage are cream i n color and contain a large pro-portion of early spermatids. However, the presence of early spermatids does not necessarily indicate protamine biosynthesis since the duration of the early spermatid stage i s about 1 week. Onset of protamine biosynthesis and re-placement of histones marks the beginning of the middle spermatid stage. The duration of t h i s stage i s also about 1 week. When the colour of the t e s t i s begins to change from cream to white, a s i g n i f i c a n t proportion (^ 5 to 10%) of the nuclear basic proteins are protamines. Although the yi e l d s of protamine are r e l a t i v e l y low (10 to 20 mg per 10 g of te s t i s ) at thi s stage, approximately equal proportions of the major species of unmodified protamine and phosphorylated protamine are found (Fig. 33A). I f naturally maturing testes or hormonally induced testes at a l a t e r stage are used, the y i e l d s of protamine are much greater. However, the proportion of phosphorylated species of protamine i s much diminished (Fig. 33B). The y i e l d of phosphoprotamines i s low i n naturally maturing testes because only a small proportion of the spermatids are active i n synthesizing protamine (Fig. 17). (b) C e l l Incubations: Radioactive precursors (1 or 2 of the following: 15 to 50 yCi per ml of [3H] arginine, 15 to 50 yCi per ml of [ 3 5S]methionine, or 100 to 300 yCi per ml of [ 3 2P]phosphate) made up i n TMKS were added to give the indicated f i n a l concentrations per ml of c e l l sus-pension. The incubations were carried out at 15 to 16° on a gyratory water bath. (c) Extraction and P u r i f i c a t i o n of Protamines: At the end of the incubation, c e l l s were sedimented at 1500 x g to remove excess la b e l and resuspended i n 3 volumes of TMKS. An equal volume of a 1% solution of the nonionic detergent Nonidet P-40 i n cold TMKS was added to disrupt c e l l s . Nuclei were c o l l e c t e d by centrifugation at 3000 x g for 10 min. Histones and protamines were extracted twice with 5 volumes of 0.4 N s u l f u r i c acid, precipitated with 3 volumes of ethanol, and col l e c t e d by centrifugation (15,000 x g for 15 min). The sediment was washed with ethanol and dissolved i n water. The r e s u l t i n g solution was centrifuged to remove insoluble material, t i t r a t e d to pH 5.5 with 1.0 M Tris-HCl (pH 8.0), and adsorbed onto a 2.5 x 10 cm column of CM-cellulose The column was washed with 0.2 M L i C l , 10 mM lithium acetate (pH 5.5) and eluted with 0.4 M L i C l i n 3 M urea to remove the histones. Protamines remained on the column. The c o l -umn was then washed with 10 mM ammonium acetate (pH 7) to remove excess s a l t s and the protamines were eluted with 0.2 M HC1. The eluate was l y o p h i l i z e d . The protamines were dissolved i n a small volume of water. The solution was t i t r a t e d to pH 7.5 with 1.0 M Tris-HCl (pH 7.5) and s o l i d urea was added to make a 6 M urea solution. (d) CM-Cellulose Chromatography of Protamines: Long CM-cellulose columns (1.0 x 40 and 1.5 x 55 cm) were prepared and equilibrated with 0.1 M L i C l , 10 mM Tris-HCl (pH 7.5), i n 6 M urea. To remove insoluble p a r t i c l e s , a l l solutions were passed through glass f i b e r f i l t e r s beforehand. The prot-amine solution i n 6 M urea was applied to the column and the column was washed with 50 ml of 0.1 or 0.2 M L i C l i n 6 M urea buffered with 10 mM Tris-HCl (pH 7.5). Protamines were eluted with a l i n e a r 0.45 to 0.85 M L i C l gradient made 6 M i n urea and buffered with 10 mM Tris-HCl (pH 7.5). The flow rate was regulated at 30 to 35 ml per hr with an Auto Technicon Pump and 5 to 7.5 ml fractions were c o l l e c t e d . Absorbance was read at 230 nm on a Beckman DB-G spectro-photometer against a 6 M urea blank; r a d i o a c t i v i t y was analyzed i n every second f r a c t i o n by counting an aliquot (50 to 100 yl) i n 2 to 5 ml of Bray's s c i n t i l l a t i o n f l u i d (149); s a l t concentration was measured with a conductivity meter (Radiometer) and reference to standard solutions of L i C l i n 6 M urea; protamines i n various fractions were mon-ito r e d by removing 30 y l aliquots and subjecting them to starch gel electrophoresis. Appropriate fractions were c o l -lected into separate pools and protamines were adsorbed d i r e c t l y onto small ( 1 x 5 cm) phosphocellulose columns at pH 7.5. The columns were washed with 10 mM ammonium acetate to remove excess s a l t s and urea, followed by 30 to 50 ml of 0.05 M HC1 to remove remaining s a l t s . Protamines were then eluted with 75 to 100 ml of 0.5 M HC1. The 0.5 M HC1 eluate was l y o p h i l i z e d and the protamines were redissolved i n a small volume of water. (e) Amino Acid Analysis; An aliquot (20 to 40 nmoles) of each f r a c t i o n of desalted protamine was l y o p h i l i z e d i n a small t e s t tube and hydrolyzed under vacuum with 6 N HCl at 110° for 16 hr. The hydrolysates were dried i n a heated (50 to 60°) vacuum desicator and redissolved i n 400 y l of pH 4.25 c i t r a t e buffer. Half of this (200 m i c r o l i t r e s corresponding to 10 to 20 nmoles of protamine) was analyzed on a Technicon automatic amino acid analyzer using a single (long) column system. Serine analyses were corrected for 15 to 20% loss during hydrolysis (151) . (f) Phosphate Determinations: Phosphate was determined by the method of Ames (150). Protamines (10 to 20 nmoles) were ashed with 30 m i c r o l i t r e s of magnesium n i t r a t e s o l -ution over a strong flame. HCl (0.3 ml of a 0.5 N solution) was added to the ashed samples and heated i n a b o i l i n g water bath for 15 min. The ascorbic acid - molybdate solution (0.7 ml) was added and the mixture was incubated i n a water bath at 45° for 20 min. Absorbance at 820 nm was read on a Unicam SP 800 Spectrophotometer. The phosphate standard (K2HPO1J was treated i n an i d e n t i c a l manner. (g) Alkaline Phosphatase Treatment: Trout testes c e l l s were incubated with 25 yCi per ml of [H3 ] arginine and 100 yCi per ml of [ 3 2P'phosphate for 7.5 hr to label the protamines. Protamines were extracted, p u r i f i e d , and a f r a c t i o n enriched i n phosphoprotamines was treated with b a c t e r i a l a l k a l i n e phosphatase. Protamine (150 yg) was incubated at 37° with 60 yg of a l k a l i n e phosphatase i n a t o t a l volume of 200 y l of 0.4 M T r i s - H C l , pH 7.5. At 30 min, 1.5 hr, and 5.5 hr, two aliquots, one 60 y l and the other 5 y l , were removed. The 60 y l aliquot was frozen at -20° immediately while 50 y l of 0.2 M HC1 were added to the 5 y l aliquot before freezing. At the end of the incubation, the samples were applied i n separate starch gel s l o t s and the protamines were resolved by electrophoresis. After electrophoresis, the gels were t r i s e c t e d h o r i z o n t a l l y . The bottom slab was stained by the sen s i t i v e cobalt-Amido Black procedure (130) . The middle slab was s l i c e d into 2.0 mm s l i c e s which were then s o l u b i l i z e d and analyzed for r a d i o a c t i v i t y . Because of the extreme s e n s i t i v i t y of the protein s t a i n for arginine-rich proteins (130) the 5 y l sample was used to indicate the p o s i t i o n of the protamine bands while the 60 y l samples were counted. 141. RESULTS I. Overall Kinetics of Enzymatic Modification of the Protamines (a) Nomenclature of Protamine Bands: To f a c i l i t a t e discussion of the r e s u l t s , F i g . 19 shows the starch gel electrophoresis pattern and incorporation p r o f i l e s of i n -organic [ 3 2P]phosphate and [ 3 5S]methionine into protamine. The various bands are labeled according to the 3 2 P and 3 SS-incorporation. The two fa s t e s t bands (P QA and P QB) rep-resent the 2 major species of unphosphorylated protamine, while the slower bands P i , P 2 , and P 3 represent protamines with 1,2, and 3 serine phosphates respectively (see Section III of these RESULTS). Wigle and Dixon (101) showed that methionine was incorporated at the NH 2-terminus of newly synthesized protamine and that the NH 2-terminus of the methionyl residue was not blocked. When 3 S S - l a b e l e d prot-amines are separated on starch gels, the 3 5S-pattern i s consistent neither with protein staining nor with 3 2 p -incorporation. For example, a [ 3 5S]methionine labeled prot-amine species migrates s l i g h t l y behind unmodified protamine pP Q]; t h i s we i n t e r p r e t as newly synthesized protamine with a labeled methionyl residue at i t s NH 2-terminus ( i . e . P QM). I t i s not clear why the presence of the methionyl residue de-creases the mobility although methionyl-protamine would have molecular weight approximately 3% greater than P . Since 142. F i g . 19. Nomenclature of protamine bands-resolved by starch gel electrophoresis. A sample of whole protamine labeled with inorganic [ 3 2P]phosphate (250 yCi per ml, 14 hr) and another labeled with [ 3 5S]methionine (50 yCi per ml, 8 hr) were separated on starch gels. The gel was t r i -sected horizontally and the bottom slab was stained (photograph). The protamine region of the middle slab was s l i c e d into 2 mm s l i c e s and each s l i c e was s o l u b i l i z e d and counted for r a d i o a c t i v i t y . The 3 2P and 3 5S counts from the two samples are superimposed. Electrophoresis i s from l e f t to rig h t . The more phosphorylated components migrate more slowly than unmodified protamine. The various protamine bands are labeled as follows: "P" represents the protamine peptide chain; the subscript numerals rep-resent the number of s e r y l residues e s t e r i f i e d by phosphate and the s u f f i x "M" indicates the presence or absence of NH2~terminal methionine. P Q i s unmodified protamine and i s the major form of protamine found i n mature sperm. Two components of P Q (P GA and P QB) are p a r t i a l l y resolved by starch gel electrophoresis. the NH 2-terminus i s not blocked, the net charge i s the same and the increase i n molecular weight would reduce the mo-b i l i t y somewhat. There are corresponding species, PiM and P 2M which migrate s l i g h t l y slower than monophospho-(Pi) and diphospho-(P 2) protamine. Triphospho-protamine (Pj) i s the slowest migrating species and there appears to be no more than a trace of t h i s species with a methionyl res-idue at i t s NH 2-terminus. (b) Incorporation of [3H] arginine and [ 3 2P]phosphate  i n t o protamine and the e f f e c t of metabolic i n h i b i t o r s : C e l l s were incubated with [3H] arginine and [ 3 2P]phosphate. After 10 min, the incubation mixture was divided into three portions, cycloheximide was added to one, 2,4-dinitrophenol (2,4-DNP) to another, and the t h i r d served as control. Incubation was continued i n each case for another 5.5 hr. C e l l s were re-moved and processed as described i n the EXPERIMENTAL PROCEDURES. Fi g . 20 shows the protein s t a i n and r a d i o a c t i v i t y i n the protamine region from these c e l l s . The fastest peak of [3H] arginine not associated with any protein band i s free arginine. After 10 min of labeling (Fig. 20A) three sharp peaks of [3H] arginine which do not correspond exactly to the protein 144. I 6 B CD O I 6 0 £ E 8 0 ™ CM I o x e a. \0** 5? - 16 - 8 17 ~~19 2 I -+ DISTANCE > ~ Fig. 20. Incorporation of [3H] arginine and [3 2P] phosphate into protamine and the e f f e c t of an i n h i b i t i o n of protein syn-thesis and an uncoupler of oxidative phosphorylation. Cells were incubated with 200 yCi per ml of [ 3H]arginine and 250 yCi per ml of inorganic [3 P]phosphate. After 10 min, the mixture was divided into three and cycloheximide (2 x 10-I*M f i n a l concentration) was added to one, 2,4-dinitrophenol (2 x 10_1,M fi n a l ) was added to another, while the t h i r d served as control. At 10 min and 5.5 hr of incubation, 3 x 10 7 c e l l s were removed, fixed with ethanol, and co l l e c t e d on glass f i b e r f i l t e r s . Acid-soluble proteins were extracted and separated i n starch gels. After electrophoresis, the gel was t r i s e c t e d hor-i z o n t a l l y , the bottom slab was stained (only the protamine region of the gel i s shown i n the photograph), the middle slab was s l i c e d into 2 mm s l i c e s , and each s l i c e was s o l -u b i l i z e d and analyzed for r a d i o a c t i v i t y . (A) 10 min of labeling; (B) 5.5 hr control; (C) 5.5 hr cycloheximide in h i b i t e d ; (D) 5.5 hr 2,4-dinitrophenol i n h i b i t e d . bands i n the protamine region are seen. These three peaks of 3H label are probably methionyl derivatives of P , P i , and P 2 protamine. Very l i t t l e [ 3 2P]phosphate was incor-porated after t h i s short time. However, the incorporation of [ 3 2P]phosphate into whole protamine continues i n a l i n e a r fashion and a f t e r 5.5 hr (Fig. 20B) there i s s i g n i f i c a n t incorporation of labeled phosphate. In addition, the pro-f i l e of 3H label has changed and shows a broad central region of incorporation with small peaks of arginine l a b e l i n the slowest ( P 3 ) and fastest (P Q) regions of protamine. I t would appear from F i g . 20A and B that the k i n e t i c r e l a t i o n s h i p of the d i f f e r e n t protamine species i s rather complex. Fi g . 20C shows the e f f e c t of cycloheximide on the patterns of [ 3H]arginine and phosphate incorporation. The i n h i b i t o r has d r a s t i c a l l y decreased the synthesis of prot-amine ( v i z . the difference i n ordinate scales between F i g . 20B and C) yet phosphorylation of the reduced amount of [ 3H]arginine-labeled protamine has continued as judged by the appearance of slower running peaks of l a b e l and the r e l a t i v e l y unchanged incorporation of [ 3 2P]phosphate. These results confirm the previous observation of Ingles and Dixon (104) that phosphorylation of protamine occurs i n -dependently of protein synthesis. I t was calculated i n PART I of t h i s Thesis that the approximate rate of protamine biosynthesis i s 1 molecule per min per ribosome. Because of the rapid transport of protamine into the nucleus (half-time 1 to 2 min, ref . 10 0) most of the phosphorylation of protamine must take place inside the nucleus and not i n the cytoplasm as previously suggested (10 5). The e f f e c t of an uncoupler of oxidative phosphorylation, 2,4-DNP, i s shown i n F i g . 20D. The [ 3H]arginine p r o f i l e changes only s l i g h t l y a f t e r the addition of i n h i b i t o r , there being no formation of P3 and much less P2• In l i n e with t h i s , no more [ 3 2P]phosphate i s incorporated into protamine. The observed increase of Pi at the expense of P Q i n F i g . 20D probably represents phosphorylation that had taken place before 2,4-DNP caused the exhaustion of the ATP pool. Thus 2,4-DNP has abolished both protein synthesis and the con-version of labeled protamine into phosphorylated derivatives. (c) Incorporation of labeled methionine and arginine  into protamine as a function of time; To study the k i n e t i c r e l a t i o n s h i p of the d i f f e r e n t protamine bands, c e l l s were doubly labeled with [ 3 5S]methionine and [ 3H]arginine. At various times, aliquots were removed and acid-soluble pro-teins were separated on starch gels. F i g . 21 shows the staining pattern obtained and the r a d i o a c t i v i t y p r o f i l e s of the protamine region a f t e r starch gel electrophoresis. The separation i s s l i g h t l y d i f f e r e n t from that i n F i g . 20 because of variations i n starch gels prepared from d i f f e r e n t lots 147. ' H ^S S 5 S "^S^H TT. 7n r n — i i 1 1 1 — 1 1 1 1 r-14 16 18 20 |4 16 18 20 14 16 16 20 + DISTANCE (cm) along GEL > -F i g . 21. Incorporation of [ 3H]arginine and [ 3 5S]methionine into protamine as a function of time. A t e s t i s c e l l sus-pension was incubated with 50 uCi per ml of [ 3H]arginine and 200 \iCi per ml of [ 3 5S]methionine. At various times, 3.2 x 10 7 c e l l s were removed and processed as i n F i g . 20. (A) 3 min of labeling; (B) 10 min; (C) 30 min; (D) 1.5 hr; (E) 3.0 hr; (F) 5.5 hr; (G) 9.0 hr; (H) 14.0 hr; (I) 24.0 hr. ( ), 3H counts; ( ), 3 5S counts. The i n -tensely stained protein band at the far l e f t of the photo-graph corresponds to histone T, which has been characterized by Wigle and Dixon (135) . 148. of starch. The fastest peak of 3 SS-counts not associated with protein staining i s free methionine which migrates s l i g h t l y slower than free arginine. The p r o f i l e s of * 3H]arginine after d i f f e r e n t periods of la b e l i n g (Fig. 21A to I) indicate that the band which i s f i r s t labeled with [3H" arginine migrates i n the p o s i t i o n of PQM (Figs. 19 and 21A) and represents newly synthesized prot-amine with a methionyl residue at i t s NH 2-terminus. There-a f t e r , the pattern of arginine labeling changes rapidly and a f t e r 10 min, l a b e l appears i n two other bands, PiM and P2M. Thus phosphorylation occurs very shortly a f t e r protamine syn-th e s i s . At 30 min, the 3 5S and 3H p r o f i l e s of labeled prot-amine coincide; however afte r 1.5 to 3 hours (Fig. 21D and E) some peaks have separated; the arginine l a b e l formerly i n PiM and P 2M i s migrating s l i g h t l y f aster i n the Pi and P 2 regions, respectively. This must be due to the removal of methionine from the NH 2-terminus of PiM and P 2M i n the nucleus to form Pi and P 2 respectively. There i s a small proportion of [ 3 5S]methionine la b e l i n the P 3 region of F i g . 21B to G and t h i s may represent P 3 M . The p r o f i l e s of labeled arginine i n protamines resolved by gel electrophoresis change most dramatically during the f i r s t 30 min of labeling (Fig. 21A to C) and thereafter the changes are less pronounced (Fig. 21D to I ) . Only af t e r 3 hr does a s i g n i f i c a n t proportion of arginine label appear i n the P 3 band of protamine. With increasing time, the proportion of arginine label increases i n P 3, P 2 , and Pi with a corresponding decrease i n P2M and PiM. After 5.5 hr, the proportion of arginine l a b e l i n Pi and P 2 i s about equal (Fig. 21F). However, t h i s too changes, with the pro-portion of 3H label i n Pi decreasing and P 2 increasing with time (Fig. 21G to I ) . The d i s t r i b u t i o n of [3 SS] methionine does not change appreciably with time (although the t o t a l 3 5S content increases) and serves as a useful reference for the p o s i t i o n of the arginine la b e l i n the various protamine bands. After the i n i t i a l 30 min of l a b e l i n g , and even afte r 24 hr of l a b e l i n g , only a small proportion of the labeled arginine i s found i n the unmodified protamine region. This implies that the phosphorylation of protamine i s u n i d i r e c t i o n a l and follows a d e f i n i t e sequence. Thus Pi labeled with arginine i s phosphorylated to P 2 and not dephosphorylated to P Q (Fig. 211). Two questions were then posed: (a) "How long does i t take newly synthesized protamine to be enzy-matically phosphorylated and completely dephosphorylated?"; and (b) "What i s the b i o l o g i c a l s ignificance of phosphoryla-t i o n and dephosphorylation of protamine?" (d) In Vivo Incorporation of [ 3H]arginine i n t o Protamine: In order to observe the passage of labeled arginine i n t o com-p l e t e l y unmodified protamine, l i v e trout were labeled with [ 3HJarginine i n vivo. F i g . 22A to D shows the labeled 150. F i g . 22. In vivo incorporation of [ 3H]arginine into prot-amine. Rainbow trout at the early protamine stage (55 days of hormonal induction) were injected i n t r a p e r i t o n e a l l y with 350 yCi of [ 3H]arginine i n 0.4 ml of sal i n e . At the i n -dicated times, a f i s h was s a c r i f i c e d , the testes were excised, and a c e l l suspension was prepared. 25 x 10 7 c e l l s were f i l t e r e d onto a glass f i b e r f i l t e r , washed with TMKS, and then TMKS-0.5% Nonidet P-40 to s t r i p the c e l l s of cyto-plasm and hence remove ribosomes. The nuclei were then fixed with ethanol. Acid-soluble proteins were extracted from 4 x 10 7 n u c l e i , separated on starch gels, and processed as i n Fi g . 20. (A) 1.0 days post-injection; (B) 4.0 days; (C) 7.0 days; (D) 10.0 days. 151. arginine p r o f i l e s of the protamine region from these i n vivo experiments. After one or two days of incubation, no additional arginine labeled protamine i s synthesized (no s i g n i f i c a n t labeling i n the PQM, PiM, and P2M regions). This indicates that the injected labeled arginine was rapidly u t i l i z e d or completely d i l u t e d by endogenous unlabeled arginine. Even a f t e r 1 and 4 days of labeling (Fig. 22), essen-t i a l l y no t 3H]arginine i s found i n the unmodified (P Q) protamine, while a f t e r 7 and 10 days of incubation, a s i g -n i f i c a n t proportion of la b e l i s found i n P . The proportion of l a b e l i n Pi decreases from days 1 to 4 and increases from days 4 to 7 and 10. Presumably these changing levels r e f l e c t the continued phosphorylation of Pi protamine to P 2 and P 3 on one hand and the dephosphorylation of P 3 and P 2 to Pi on the other hand. Thus i t takes about 5 to 10 days f o r newly synthesized protamine to go through the series of phosphorylations and dephosphorylations. (e) Overall Kinetics of Methionine Removal and Phos- phorylation and Dephosphorylation of Protamine: Wigle and Dixon (101) showed that methionine was removed from the NH2-terminus of newly synthesized protamine but i n t h e i r 2 hour chase, only 50% of the la b e l had been removed. In a s i t u a -t i o n i n which one l a b e l (methionine) i s being removed at a constant rate from molecules while the other (arginine) i s stable, i t would be expected that the r a t i o of [ 3 5S]methionine to [3H] arginine would decrease l i n e a r l y with time. F i g . 23 shows the r a t i o of 3 5S/ 3H for the PQM region and the whole protamine region calculated at the various times from F i g . 21A to I. The [ 3 SS]methionine la b e l i s being removed i n whole protamine but not at a constant rate (Fig. 23): there i s a rapid removal within the f i r s t 1*5 hr and then a decline to a much slower rate. This suggests that there may be two pools of t 3 5S]methionyl-protamine, one of which i s r e l a t i v e l y accessible to the methionine removing enzyme(s), and the other less so. A curious observation i s that the 3 5S/ 3H r a t i o for P QM increases with time. Protamine i s very r i c h i n arginine and the pool of added [ 3H]arginine l a b e l may be depleted much more quickly than the pool of [3 5S ]methionine l a b e l so that there would be a more rapid decrease of s p e c i f i c a c t i v i t y i n the arginine pool than the methionine pool. Knowing the r a t i o of [ 3H]arginine la b e l to [ 3 5S]methionine label as indicated i n PQM at the various times of la b e l i n g , i t was possible to estimate the approximate proportion of arginine label i n the d i f f e r e n t protamines with time. F i g . 24 shows these results calculated from F i g . 21 and using the 3H to 3 5S r a t i o i n P M at the d i f f e r e n t times as a factor o for the conversion of 3 5S counts to equivalent 3H counts i n PQM, PiM, and P2M. E s s e n t i a l l y a l l the arginine counts are i n P QM afte r 3 min of incubation. PiM and P 2M reach a peak within 30 min and then decline. Pi (from PiM) increases, F i g . 23. S p e c i f i c a c t i v i t y of 3 sS-protamine from the i n -cubation of F i g . 21. 3 5S and 3H counts were integrated over the PQM and whole protamine regions of F i g . 21A to H, The r a t i o of 3 5S to 3H was plotted at the various times. (—• •-) whole protamine; (-O O-) ~ P M. o 154. DAYS (in vivo) F i g . 24. Proportion of t 3H]arginine l a b e l i n the d i f f e r e n t species of labeled protamine at various times a f t e r the s t a r t of labeling. Labeled arginine i n the d i f f e r e n t mod-i f i e d species of protamine was integrated at the various times from F i g . 21 and F i g . 22. Counts of arginine i n a given protamine species were divided by the t o t a l arginine counts i n whole protamine to obtain the proportion of l a b e l i n that p a r t i c u l a r species. For P0M, PiM and P2M, values were calculated from the [ 3H]arginine la b e l at early times and from the [ 3 5S]methionine l a b e l at l a t e r times. The r a t i o of 3H to 3 5S of P QM (see F i g . 23) was used as a factor to convert 3 5S counts to 3H counts i n the methionine con-tai n i n g species of protamine. For Pi the t o t a l 3H counts i n the combined Pi and PiM regions were determined and cor-rected for 3H counts (from the 3 5S content) i n PiM. Similar calculations were employed to determine the proportion of counts i n P 2 . The proportion of arginine l a b e l i n Pi and P 2 at day 1 of the i n vivo incubation was corrected for counts i n PiM and P 2M respectively. peaks at 3 hr, and slowly declines (phosphorylation to P2 and P 3 ) . Labeled P q (protamine which has been phosphorylated and dephosphorylated and i s termed "unmodified" or "mature" protamine) does not appear u n t i l 4 to 5 days afte r the s t a r t of arginine l a b e l i n g , and increases rapidly thereafter. These data suggest the existence of "pools" of the various enzymatically modified species of protamine. For example, arginine l a b e l enters a molecule of protamine (PQM) and becomes a part ( i . e . i s d i l u t e d in) of a larger pool of unlabeled P M; labeled P M i s removed from the P M pool and 0 0 o transferred to the PiM pool through the a c t i v i t y of phospho-kinases acting randomly on the molecules of the PQM pool. Thus the arginine-labeled protamine o r i g i n a l l y d i l u t e d i n the PQM pool, i s d i l u t e d again i n the PiM pool. I t would, therefore, take a long time for arginine-labeled protamine to pass through a l l the pools and appear i n unmodified mature protamine. I f we count the number of pools involved (PiM and Pi being counted as one and Pi on the phosphoryla-t i o n pathway being d i f f e r e n t from Pi on the dephosphoryla-t i o n pathway) newly synthesized protamine must pass through at l e a s t 6 pools of modified protamines (Fig. 25). (f) A Proposal for the Function of Protamine Phos- phorylation: The sequence and u n i d i r e c t i o n a l i t y of these events together with the d i f f e r i n g metabolic behaviour of the d i f f e r e n t protamine pools, suggest that the sequential M e t A r g - * P 0 M [P0M] ^ [P,M]-*[P2M]->[P3M] C>topla_8m_ Nucleus / \ Po 5-10 hours 5-10 davs ^ F i g . 25. A schematic representation of the k i n e t i c r e l a t i o n -ship of the d i f f e r e n t protamine species. Arginine and meth-ionine are incorporated into nascent protamine (PQM) i n the cytoplasm. Protamine i s rapidly transported into nucleus where i t binds to DNA. The PGM from the cytoplasm becomes part of a larger P QM pool i n the nucleus. Protamine phospho-kinases phosphorylate PQM to PiM and P2M (and perhaps to P 3 M ) Simultaneously, methionine i s removed from PiM and P2M to formPi and P 2. Phosphorylation of Pi and P 2.continues to form P 3. Sequential dephosphorylation of P 3 eventually gives r i s e to P Q, the unmodified or "mature" protamine character-i s t i c of mature sperm. The square brackets indicate "pools" of the various protamine species and the arrows represent enzymatic removal of a molecule from one pool to another. The pool sizes of the nascent and methionine containing species of protamine are small compared to the protamine from which methionine has already been cleaved. Because of the siz e of the pools and the number of the protamine species, i t takes about 5 to 10 min for labeled arginine to reach PiM and P2M, 5 to 10 hr to reach P 2 and P 3, and 5 to 10 days for labeled protamine to be completely phosphorylated and dephosphorylated to P Q. phosphorylation of newly synthesized protamine i s involved i n some way i n i t s proper binding to chromatin. The proposal that enzymatic modifications might be i n -volved i n the correct binding of newly synthesized basic proteins to nucleic acids i s new. A p r i o r i , the p r o b a b i l i t y of a highly p o s i t i v e l y charged molecule such as protamine entering the nucleus and binding cor r e c t l y through i o n i c linkages to the negatively charged molecule of DNA would seem to be s l i g h t . Hence, some mechanism for the regulation of the binding of such highly charged polycationic molecules would seem to be required, assuming that the f i n a l , correct binding i s highly s p e c i f i c and ordered. I f phosphorylation i s involved i n the binding of newly synthesized protamine to DNA, we can envisage the following sequence of events. Nascent protamine i s synthesized i n the cytoplasm and rapidly transported into the nucleus where i t binds i n a non-specific fashion to DNA through i o n i c l i n k -ages. Protamine phosphokinases (106) which are bound loosely to chromatin might then act as "editing" enzymes, which recognize and phosphorylate the improperly bound protamine. Phosphorylation (by the same or d i f f e r e n t protamine kinases) continues u n t i l the phosphorylated protamine assumes the correct conformation with respect to DNA. Phosphoryla-t i o n of protamine would decrease i t s net po s i t i v e charge, thus decreasing the strength of i t s i n t e r a c t i o n with DNA 158. and perhaps allowing the protamine more freedom to f i n d i t s correct conformation. Simultaneously, histone i s removed i n some as yet unknown manner although i t i s possible that phos-phorylation of protamine may have some d i r e c t role i n the removal of histone. F i n a l l y , protamine phosphatases, which also act as "editing" enzymes recognize those phosphorylated protamine molecules bound i n the correct conformation and remove the negative phosphory1 groups thus increasing the net p o s i t i v e charge on protamine and "locking" i t to DNA. The progressive removal of phosphoryl groups from prot-amine would gradually contract the spermatid chromatin into a highly compact state c h a r a c t e r i s t i c of mature sperm. I t i s worth noting here that i n the 1.5 Sv spermatids which have j u s t begun to synthesize protamine, the prot-amines present are exclusively i n the phosphorylated state (Fig. 7A). Associated with the t r a n s i t i o n of the 1.5 Sv spermatid to the smaller l a t e spermatid (1.0 Sv) i s the appearance of unmodified "mature" protamine. I t takes about 1 week for 1.5 Sv spermatids to be transformed in t o the smaller late spermatids (PART I ) . This time i n t e r v a l cor-relates rather well with the 5 to 10 days required for newly synthesized protamine to be completely phosphorylated and dephosphorylated. S i m i l a r l y , the t r a n s i t i o n of l a t e sperm-atids to mature sperm (0.6 Sv) i s associated with the de-phosphorylation of residual phosphorylated protamine (Fig. 7B). These data suggest that i t i s the dephosphorylation of phosphorylated protamine that may cause the contraction and condensation of the spermatid chromatin. I I . I n t r a c e l l u l a r Kinetics of Enzymatic Modification of the Protamines Ling, T r e v i t h i c k and Dixon (100) previously showed that protamine labeled by a 30 sec pulse of [* l*C]arginine and chased with a large excess of cold arginine was rapidly transported from the microsomal f r a c t i o n into the nucleus with a half-time of 1 to 2 min. In addition, Marushige et a l . (105) incubated the post-mitochondrial supernatant f r a c t i o n from trout testes with [ 3H]arginine and $-[ 3 2P]ATP and showed that phosphorylation of newly synthesized prot-amine could occur i n the cytoplasm. J e r g i l and Dixon (106) also found a protein kinase i n the post-ribosomal super-natant f r a c t i o n which phosphorylated protamine more rapidly than histones at high i o n i c strengths. These findings led to the idea that newly synthesized protamine was phos-phorylated i n the cytoplasm perhaps as a mechanism for the removal of protamine from the ribosome or transport of prot-amine into the nucleus (16,105). However, because of the rapid transport of protamine into the nucleus and the r e l -a t i v e l y slow pace of enzymatic modification of protamine, i t was suggested i n the previous section that most of the phosphorylation and dephosphorylation of protamine occurred i n the nucleus (Fig. 25). Using small-scale incubations and procedures for rapidly separating c e l l fractions and stopping enzymatic reactions, the transport of newly synthesized protamine from the cyto-161. plasm to the nucleus and the i n t r a c e l l u l a r s i t e of phos-phorylation and dephosphorylation of protamine have been re-investigated. (a) Comparison of Basic Proteins i n Various C e l l Fractions: Ribosomes, whole cytoplasm, n u c l e i , nucleohistone and nucleo-protamine were prepared from c e l l s and c o l l e c t e d on f i l t e r s . The acid-soluble fractions from these were resolved by e l e c t r o -phoresis i n starch gels as described by Sung and Smithies (130). After electrophoresis, the protein bands were detected by staining with Amido-Black and destaining with d i l u t e s u l f u r i c a c id, a procedure which s e l e c t i v e l y stains arginyl residues and i s about 100 x more sensi t i v e than the normal Amido Black method (130). The l y s i n e - r i c h histones I and IIb2, contain a low proportion of arginyl residues and s t a i n poorly with t h i s method (130). However, microgram quan-t i t i e s of arginine-rich proteins are detectable (130) and F i g . 26 shows these proteins from the d i f f e r e n t fractions of the c e l l . For comparison acid-soluble proteins from 1 M NHi»Cl washed E. c o l i ribosomes and trout t e s t i s ribosomes ( g i f t s from Dr. R.S. Gilmour) are also shown. Examination of Fig. 26a (washed E. c o l i ribosomes), b (washed t e s t i s ribosomes), and c (unwashed t e s t i s ribosomes p u r i f i e d by sucrose density gradient centrifugation) shows that the ribosomal proteins of E. c o l i are d i f f e r e n t from those of trout testes. The unwashed ribosomes from testes m F i g . 26. Resolution of acid-soluble proteins from d i f f e r e n t fractions of the c e l l . (a), E. c o l i ribosomes and (b), trout testes ribosomes washed with 1 M NHUC1; (c and c'), di f f e r e n t preparations of trout testes ribosomes p u r i f i e d by sucrose density gradient centrifugation; (d), super-natant (top fractions) of a sucrose density gradient of ribosomes; (e) cytoplasm from ^10 x 10 7 c e l l s and (f) nuclei from ^2.5 x 10 7 c e l l s prepared by the Nonidet P-40 method; (g) nucleoprotamine and (h) nucleohistone from ^2.5 x 10 7 c e l l s prepared by sonication. 1 M NH^Cl washed E. c o l i and trout testes ribosomes were g i f t s from Dr. R.S. Gilmour. at the protamine stage of development contain a s i g n i f i c a n t amount of bound unmodified protamine ( P Q ) . Washing testes ribosomes with 1 M NH i,Cl removes bound protamine and a number of proteins associated with ribosomes. Presumably, these include i n i t i a t i o n , translocation and release factors. A f r a c t i o n from the top of a sucrose density gradient af t e r the ribosomes had been sedimented (Fig. 26d) shows that the acid-soluble proteins from the post-ribosomal supernatant are very heterogeneous and are seen as a broad smear. F i g . 26e and f shows the acid-soluble proteins from whole cyto-plasm and nuclei prepared by using the non:-ionic detergent Nonidet P-40 which lyses c e l l s and leaves n u c l e i i n t a c t (148) Most of the p r e c i p i t a t e d cytoplasmic proteins are of ribosomal o r i g i n (Fig. 26c and e). No observable histones are found i n the cytoplasm and few i f any ribosomal protein bands are seen i n the nuclear f r a c t i o n (Fig. 26e and f). We can con-clude that the cytoplasm and nuclei produced and separated by the method used here are r e l a t i v e l y clean and do not cross-contaminate each other. F i g . 26£ and h, shows the basic proteins from the nucleo histone and nucleoprotamine f r a c t i o n s . The proteins i n t h i s nucleoprotamine f r a c t i o n (g) consist mostly of phospho-protamines and very l i t t l e histone. The nucleohistone frac-t i o n contains most of the histone and about the same amount of protamine as the nucleoprotamine f r a c t i o n . The testes samples used here were at the very early protamine stage of development. I f more mature testes are used a larger propor-t i o n of the protamine i s i n the dephospho (unsubstituted) form and i s found i n the nucleoprotamine f r a c t i o n while very l i t t l e protamine i s found i n the nucleohistone f r a c t i o n (Fig. 27). This i s probably a r e f l e c t i o n of the fact that i n more mature testes only a small proportion of the sperm-atids are at the middle protamine stage; most of the other spermatids have already synthesized, phosphorylated, and dephosphorylated t h e i r complement of protamine (Fig. 7 and 16) . (b) Presence of Labeled Protamine on Ribosomes: Because of the excellent separation of unmodified and phosphorylated protamines from each other and from ribosomal proteins and histones, i t was of i n t e r e s t to see (a), i f more nascent protamine could be detected on diribosomes than on poly-ribosomes and monosomes as Ling et a l . (100) found; and (b) i f phosphorylated protamine could be found on ribosomes. Trout testes c e l l s were labeled for 20 min with [3H] arginine and [ 3 SS] methionine. Protein synthesis was stopped with cycloheximide. Ribosomes were prepared taking care to use solutions containing 1 x IO-1* M cyclohexmide to prevent run-off of ribosomes from messenger RNA. F i g . 28a shows a t y p i c a l polyribosome p r o f i l e from trout testes; separate fractions of the monosome, disome, and higher polysomes origin Histones 10cm Protamines 20cm 0 Fig. 27. Nucleohistone and nucleoprotamine fractions from testes at the middle protamine stage of development. Nucleo-histone (a) and nucleoprotamine (b) fractions were separated by sonication of 1 x 10 7 c e l l s and the compact nucleoprotamine was coll e c t e d on glass f i b r e f i l t e r s . The soluble nucleo-histone f r a c t i o n was preci p i t a t e d with ethanol and collected. Compared to early protamine testes (Fig. 26g and h) most of the protamine i n testes of l a t e r stages i s found i n the compact nucleoprotamine f r a c t i o n . Note the higher propor-t i o n of P Q protamine i n thi s preparation compared to that i n F i g . 26g and h. were pooled as indicated, p r e c i p i t a t e d with ethanol, and co l l e c t e d on f i l t e r s . F i g . 28B(a) shows the [ 3 H] arginine and [3 5S]methionine counts of the protamines extracted from the di-ribosomes and separated by electrophoresis i n starch gels. The corres-ponding p r o f i l e of protamine from the nuclear f r a c t i o n of the same incubation i s shown i n F i g . 28B(b). The two fastest intensely stained bands i n the di-ribosome f r a c t i o n have very l i t t l e [ 3 5S]methionine label and are unmodified (P Q) and monophospho (Pi) protamine. The 3 H p r o f i l e s of incorporation indicate that arginine-labeled protamine can be found i n both P Q and Pi from di-ribosomes. In the nucleus the 2 major peaks of [3H] arginine l a b e l [Fig. 28B(b)] both contain s i g n i f i c a n t 3 5S l a b e l . The presence of a methionyl residue at the NH 2-terminus of protamine decreases the mobility of protamine somewhat. Therefore the faster migrating peak of lab e l i s PQM while the slower peak of l a b e l i s PiM. The small shoulder of lab e l t r a i l i n g PiM i s P2M. The 3 H and 3 5S p r o f i l e of counts i n protamine from the mono and higher polysome fractions (not shown) i s very s i m i l a r to that i n F i g . 28B(a). In f a c t , the s p e c i f i c a c t i v i t y of t 3 H ]arginine i n protamine per A 2 6 o of ribosomes i s approx-imately the same for a l l three f r a c t i o n s . The data can be interpreted i n several ways. F i r s t , the quantitative and q u a l i t a t i v e differences i n 3 H and 3 5S labeled protamine from F i g . 28. (A), sucrose density gradient centrifugation of polysomes from trout t e s t i s . Trout t e s t i s c e l l s were i n -cubated for 20 min with [3H] arginine and [3 5S]methionine. Ribosomes were prepared by d i f f e r e n t i a l centrifugation and p e l l e t i n g through 1.0 M sucrose. Ribosomes were f r a c t i o n -ated by v e l o c i t y sedimentation on a 10 to 30% sucrose den-s i t y gradient. Separate fractions were pooled as indicated and precipitated with ethanol. (B), starch gel electro-phoresis and analysis of r a d i o a c t i v i t y of acid-soluble proteins from the diribosome region and protamine pre-pared from nuclei. (a) I O A 2 6 0 of ribosomes from the d i -ribosome region of the sucrose density gradient were ex-tracted with acid i n the starch gel s l o t . After electro-phoresis, the gel was t r i s e c t e d h o r i z o n t a l l y . The bottom slab was stained (photograph) while the middle slab was s l i c e d and analyzed for r a d i o a c t i v i t y . Only the protamine region of the gel i s shown. (b) protamine from nuclei of the above incubation. ( ), [ 3H]arginine; ( ), [3 5 S ] methionine. the ribosomes and the nuclei and lack of differences between the ribosome fractions may be attributed to the rapid ces-sation of metabolism i n nuclei with the addition of acid. The extended period of time required to prepare and separate ribosomes before f i x a t i o n with ethanol may be s u f f i c i e n t for nascent protamine chains to be completed, released and NH2-terminal methionine to be removed by methionyl-aminopeptidases from the protamine peptide chain bound to the ribosome even i n the presence of cycloheximide. Second, the majority of the protamine pulse-labeled by Ling et a l . (10 0) w i l l even-t u a l l y reach the nucleus but a small f r a c t i o n could remain bound no n - s p e c i f i c a l l y to i s o l a t e d ribosomes. In the present case (20 min labeling) most of the protamine also reaches the nucleus (Fig. 28B(b)), while the same proportion of com-pleted protamine chains remain bound no n - s p e c i f i c a l l y to ribosomes. But the amount of label i n no n - s p e c i f i c a l l y bound protamine i s very s i g n i f i c a n t , since a 20 min labeling period may allow s u f f i c i e n t accumulation of labeled prot-amine no n - s p e c i f i c a l l y bound to ribosomes. This would at f i r s t sight appear to contradict the results (protamine bio-synthesis on di-ribosomes) by Ling et a l . (10 0) . These results do point to the necessity of using i n h i b i t o r s of both protein synthesis and energy requiring processes i n order to prepare polyribosomes v i a lengthy procedures with nascent peptides attached. (c) Synthesis of Protamine and t h e i r Transport from the  Cytoplasm i n t o the Nucleus: Testes c e l l s were incubated with [ 3H]arginine and [ 3 SS] methionine for varying periods. Aliquots were removed and rapidly lysed; nuclei and preci p i t a t e d cytoplasm were co l l e c t e d on separate f i l t e r s . Starch gel electrophoresis resolved the acid-soluble proteins. F i g . 29 shows the 3H and 3 5S p r o f i l e s of the protamines from the cytoplasmic and nuclear f r a c t i o n s . The f a s t migrating peak of 3 5S counts i n the cytoplasm i s free methionine; these counts are not found i n p u r i f i e d ribosomes (Fig. 28B) or the nuclear f r a c t i o n i n F i g . 29. After 3 min of labeling (Fig. 29a and a'), the only labeled protamine f r a c t i o n found i n both the cytoplasm and the nucleus i s PQM. After 10 min, l a b e l i n cytoplasmic protamine i s s t i l l e s s e n t i a l l y a l l i n P oM while i n the nucleus, a 1:1 r a t i o of arginine lab e l i s found i n PQM and PiM (Fig. 29b and b'). After 30 min (Fig. 29c and c ' ) , some arginine l a b e l i n PjM i s found i n the cytoplasm while the proportion of arginine la b e l i n PiM has increased i n the nucleus. Fig . 30 summarizes the results of F i g . 29. The r a t i o of [ 3H]arginine i n cytoplasmic protamine to nuclear prot-amine i s plotted with time for two d i f f e r e n t incubations. The data support the results of Ling et a l . (100) that protamine i s synthesized i n the cytoplasm and rapidly trans-ported into the nucleus. However, our data c l e a r l y show that 170. + DISTANCE (cm) along GEL > F i g . 29. Starch gel electrophoresis and analysis of radio-a c t i v i t y i n acid-soluble proteins from the cytoplasm and nucleus. 10 x 10 7 c e l l s labeled with [ 3H]arginine and [ 3 SS] methionine for various times were treated with Nonidet P-40. Nuclei were c o l l e c t e d on f i l t e r s . The cytoplasm ( f i l t r a t e ) was precipitated with ethanol and also c o l l e c t e d on f i l t e r s . Acid-soluble proteins from these were separated by ele c t r o -phoresis, and analyzed for r a d i o a c t i v i t y as i n Fig . 20. (a), (b), and (c) are the cytoplasmic fractions from 3, 10, and 30 min of labeling respectively; (a'), ( b 1 ) , and (c*) are the corresponding n u c l e i . The cytoplasm was from an equivalent of ^ 10 x 10 7 c e l l s while nuclei came from ^2.5 x 10 7 c e l l s . The f a i n t bands at the far l e f t of the photo-graphs of a, b, and c are ribosomal proteins while the i n -tensely staining band at the far l e f t of the photographs of the nuclei (a", b*, and c 1) i s histone T. 171. T I M E ( m i n ) F i g . 30. Transport of protamine from the cytoplasm into the nucleus. pH]arginine counts were integrated i n the protamine region of the starch gels of acid-soluble pro-teins from the cytoplasm and nuclei. The rat i o s of cytoplasmic to nuclear counts from an equivalent number of c e l l s were plotted at the d i f f e r e n t times of incubation. The upper curve (a) was obtained from the incubation of F i g . 29 and shows the transport of newly synthesized prot-amine from the cytoplasm into the nucleus i n testes which had just begun to synthesize protamine (photograph "a"). The lower curve (b) i s from a d i f f e r e n t incubation under the same conditions as (a) except that the c e l l s were prepared from a testes of s l i g h t l y l a t e r stage (photograph "b"). Both photographs.shows protamine from approximately the same member of c e l l s (^2.5 x 10 7) and indicates that "b" has a greater amount of protamine and a higher propor-t i o n of P Q ( i . e . s l i g h t l y l a t e r developmental stage of t e s t i s ) . The explanation for, and the sig n i f i c a n c e of the differences i n rate of protamine transport between (a) and (b) a r e not known. phosphorylation occurs rapidly i n the nucleus but only slowly i n the cytoplasm. The p o s s i b i l i t y exists that de-phosphorylation of protamine occurs faster i n the cytoplasm; however, since most of the p h y s i o l o g i c a l l y important phos-phorylation of protamine occurs i n the nucleus, we can ignore the non-specific phosphorylation and dephosphorylation of protamine i n the cytoplasm. Moreover, newly synthesized unphosphorylated protamine (PQM) i s found i n the nucleus (Fig 29a) i n d i c a t i n g that, contrary to previous proposals (16,10 5) phosphorylation of protamine i s not required for (a) the removal of protamine from the ribosome or (b) the transport of protamine from the cytoplasm into the nucleus. The differences i n the r a t i o of arginine-labeled cyto-plasmic protamine:nuclear protamine i n the two curves (Fig. 30a and b) may be due to differences i n t e s t i s development. The upper curve with the slower rate of protamine transport i s found i n very early protamine stage t e s t i s , while the lower curve i n d i c a t i n g very rapid protamine transport i s found i n ^ t e s t i s of s l i g h t l y l a t e r stage. The reasons for the differences are not known. (d) Metabolism of Protamine i n Nucleohistone and  Nucleoprotamine Fractions: Marushige and Dixon (108) or-i g i n a l l y separated testes nucleohistone from nucleoprotamine by shearing whole chromatin i n a Waring blendor followed by d i f f e r e n t i a l centrifugation. They found a small amount of histone (mostly histone I) i n the compact nucleoprotamine portion of chromatin from mature testes, but were unable to detect any protamine i n the soluble nucleohistone portion (108). In our studies, we have used testes at the early protamine stage of t e s t i s development when there are large proportions of early spermatids which have not yet begun protamine biosynthesis and middle spermatids which have begun synthesis of protamine and the replacement process but not undergone s i g n i f i c a n t nuclear condensation (Fig. 7). From such testes, we f i n d (Fig. 26g_ and h) a portion of chromatin (nucleohistone) which has bound protamine but not yet condensed and hence can be sheared. The s e n s i t i v i t y of the s t a i n for arginine-rich proteins gives the impression that the amounts of protamine and histone i n t h i s preparation of nucleoprotamine are about equal; i n fact, only 5 to 10 percent of the proteins are protamine. I t was of i n t e r e s t to examine whether newly synthesized protamine bound p r e f e r e n t i a l l y to nucleohistone or to nucleo-protamine and to see i f any differences i n rates of protamine metabolism e x i s t between these two f r a c t i o n s . We have previously shown (Fig. 23) that the k i n e t i c s of methionine removal was biphasic from protamine labeled with t 3 5S]methionine and [ 3H]arginine; methionine removal was at f i r s t rapid (half-time 'v 1 to 1% hr) and then declined to a much slower rate. To determine whether these differences could be due to differences i n the rates of removal of the methionyl residue i n protamine bound to the nucleohistone and nucleoprotamine f r a c t i o n s , testes c e l l s were labeled with [3H] arginine and [3 SS]methionine. At various times aliquots were removed and nucleohistone and nucleoprotamine were prepared by sonication. The two fractions were c o l -lected on separate f i l t e r s . Acid-soluble proteins were ex-tracted and separated by gel electrophoresis. F i g . 31 shows the protein s t a i n and corresponding r a d i o a c t i v i t y p r o f i l e s of labeled protamine from the nucleohistone and nucleo-protamine f r a c t i o n s . The r a d i o a c t i v i t y p r o f i l e s appear rather s i m i l a r i n both the nucleohistone and nucleoprotamine f r a c t i o n s . After 45 min (Fig. 31a and a'), most of the 3H-lab i s i n PiM with s l i g h t l y less i n PQM and P2M. After 9 hr (Fig. 31c and c') , more lab e l i s found i n P 2 and a s i g n i f -icant proportion of l a b e l i s found i n P 3. These changing p r o f i l e s indicate the sequential phosphorylation of newly synthesized protamine and removal of NH 2-terminal methionine (Fig. 25). On closer scrutiny, the proportion o f ! t 3 5S] methionine la b e l i n the protamine of nucleohistone at 9 hr i s much less than that i n protamine from the corresponding nucleoprotamine. In addition (arrows), there i s a deeper valley between Pi and P 2 i n F i g . 31c than i n F i g . 31c'. PiM electrophoresis between Pi and P 2 (Fig. 19). The deeper valley indicates that more PiM protamine has had NH 2-terminal 175. 3 95 H. Nucleohistone Nucleoprotamine ± J. + DISTANCE (cm) along GEL > F i g . 31. Comparison of the metabolism of protamine from the nucleohistone and nucleoprotamine fractions of c e l l s . A c e l l suspension was incubated with [ 3H]arginine and [ 3 SS] methionine. At various times, nucleohistone and nucleo-protamine fractions were prepared by sonication of an aliquot (^ 10 x 10 7 c e l l s ) of the c e l l suspension. Approximately h of the aliquot was extracted with acid, and subjected to starch gel electrophoresis. (a), (b), and (c) are the protamine regions from the nucleohistone fractions of c e l l s incubated for 45 min, 3 hr, and 9 hr respectively; (a'), (b'), and (c 1) are the corresponding regions from the nucleo-protamine fract i o n s . Note that at 45 min of incubation (a'), the [3H] arginine and [3 5S]methionine p r o f i l e s coincide while a f t e r 9 hr, ( c 1 ) , the two p r o f i l e s are no longer coincident. methionine removed to y i e l d Pi i n the nucleohistone f r a c t i o n than i n the nucleoprotamine f r a c t i o n . F i g . 32 shows the s p e c i f i c a c t i v i t y ( 3 5S/ 3H) of protamine from the nucleo-histone and nucleoprotamine fractions with time, and con-firms that methionine i s removed r e l a t i v e l y rapidly at a constant rate i n the nucleohistone f r a c t i o n but only slowly i n the nucleoprotamine f r a c t i o n . Included i n F i g . 32 for comparison are the curves for P QM and whole protamine pre-viously obtained from i n t a c t c e l l s (Fig. 23). Examination of these curves indicates that the rapid rate of methionine removal i n the nucleohistone f r a c t i o n and the slow rate i n the nucleoprotamine f r a c t i o n explains the biphasic curve obtained previously for protamine from whole c e l l s . As noted before, the increase i n s p e c i f i c a c t i v i t y ( 3 5S/ 3H) of PQM with time i s probably due to rapid depletion of the endogenous arginine pool as a r e s u l t of incorporation into protamine. PQM i s the protamine species closest to free amino acids and as such r e f l e c t s most rapidly the changes i n s p e c i f i c a c t i v i t y of the two amino acid pools. 8 1 0 H O U R S 1 2 1 4 1 6 F i g . 32. Comparison of the s p e c i f i c a c t i v i t y of [ 3 5S] methionine i n protamine from whole c e l l s , nucleohistone, and nucleoprotamine fr a c t i o n s . 3 5 S and 3H counts were integrated i n the PgM and whole protamine region of Fig. 31. The r a t i o of 3*S to 3H was plotted at the various times. The data for protamine from the i n t a c t cells were obtained i n a s i m i l a r incubation (Fig. 21) and were prev-iously described (Fig. 23). (a')/ (b'), and (c') are the s p e c i f i c a c t i v i t y of P QM from the whole c e l l , nucleohistone and nucleoportamine fractions respectively; (a), (b), and (c) are the corresponding s p e c i f i c a c t i v i t i e s of whole protamine. Note that the biphasic curve (a) of whole protamine from the i n t a c t c e l l i s explained by two d i f f e r -ent rates of methionine removal: a r e l a t i v e l y rapid removal from nucleohistone (b), and a r e l a t i v e l y slow removal from nucleoprotamine (c). 178. I I I . Separation and Characterization of Phosphorylated Species of Protamine In part, t h i s section of the Thesis was prompted by the necessity to i d e n t i f y p o s i t i v e l y the series of bands resolved by starch gel electrophoresis of acid-soluble pro-teins from middle-stage spermatids (Fig. 7), and l a t e r to es t a b l i s h t h e i r degree of phosphorylation. In PART I, Fi g . 8, the labeling of the protamine bands i s erroneous. At that time, the experiments reported i n thi s Section had not been completed. In addition, the complex metabolism of the protamines and the b i o l o g i c a l s ignificance of the replacement process suggest that a supply of these phosphorylated species of protamine would greatly aid i n studying i n v i t r o the exact function of phosphorylation and i t s possible role i n the correct binding of protamine to chromatin and the removal of histones. (a) Chromatography of Protamines from Naturally Maturing  and Hormonally Induced Testes: A c e l l suspension was pre-pared from hormonally induced testes at the early protamine stage and incubated with [ 3H]arginine for 1 hr. Protamines were extracted, p u r i f i e d , and resolved on CM-cellulose c o l -umns by eluting with a gradient of L i C l i n 6 M urea. F i g . 33A shows the p r o f i l e s of absorbance and r a d i o a c t i v i t y of protamine eluted from the column. Eight f r a c t i o n s , labeled F i g . 33. Resolution of protamines from a r t i f i c i a l l y i n -duced (A) and naturally maturing testes (B) by chromato-graphy on CM-cellulose.(A) Protamine (approximately 60 mg from hormonally induced testes labeled for 1 hr with [ 3H]arginine) was applied to a 1.5 x 55 cm CM-cellulose column and eluted with a 900 ml gradient of L i C l (0.45 to 0.85M) i n 6 M urea and buffered with 10 mM Tris-HCl (pH 7.5). ( ) absorbance measured at 230 nM against a 6 M urea blank; (-©-•-) r a d i o a c t i v i t y of 30 y l aliquots i n 2 ml of Bray's s c i n t i l l a t i o n f l u i d (149); (-A-A-) concentration of L i C l as measured with a conduc-t i v i t y meter and reference to standard solutions of L i C l i n 6 M urea. Fractions were pooled as indicated for amino acid analysis and phosphate determinations. (B) Approx-imately 60 mg of protamine(from naturally maturing testes c o l l e c t e d on Sept. 26, 1969 at Sun Valley Trout Farm, Mission, B. C.) were applied to a 1 x 40 cm CM-cellulose column, The protamine was eluted with a 700 ml gradient of L i C l (0.45 to 0.85 M) i n 6 M urea. Most of the prot-amine i s found i n 2 fr a c t i o n s , I and I I ; only a small pro-portion of the t o t a l protamine i s found i n Fractions III to VIII i n natur a l l y maturing testes. I to VIII are p a r t i a l l y resolved. Evidence w i l l be presented l a t e r that i f protamine i s prepared from testes labeled with [ 3 2P]phosphate, Fractions I and II are not labeled while Fractions I I I to VIII are labeled, i n d i c a t i n g that the l a t t e r are phosphoproteins. F i g . 33B shows the separation of prot-amines prepared from naturally maturing testes c o l l e c t e d during mid-September. By t h i s time, the t e s t i s has reached close to i t s maximal weight (Fig. 14) and more than 80% of t h e , c e l l s are spermatids; active protamine synthesis i s occurring i n the testes (100,79 ). Only two major peaks, I and I I , are found; a much smaller proportion of phospho-proteins elutes e a r l i e r . Fractions were pooled from F i g . 33A as indicated and the proteins were adsorbed onto and eluted from small phospho-c e l l u l o s e columns. The eluate was l y o p h i l i z e d and the pro-teins were dissolved i n a small volume of water. An aliquot of each f r a c t i o n was removed for amino acid analysis and phosphate determinations. (b) Amino Acid and Phosphate Analysis of Protamines; The amino acid sequences of three components of protamine, l a , l b , and I I , from rainbow trout have been determined by Ando and Watanabe (86), and are shown i n F i g . 2B. The corresponding number of amino acid residues per molecule of protamine i s shown i n F i g . 2C. These workers also re-solved a fourth component with 3 s e r y l residues per molecule of protamine on CM-Sephadex columns. However, i t was heter-ogeneous and not sequenced. Table XV shows the amino acid composition expressed i n residues of amino acid per 32 residues of the fractions pooled from F i g . 33A. Comparison of these with the com-positions i n F i g . 2C indicates that a l l of the 8 fractions are indeed protamines. The odd f r a c t i o n s , I, I I I , V, and VII have close to 3 s e r y l residues per molecule of protamine while the even numbered fractions have close to 4 s e r y l residues per molecule. Table XVI shows the phosphate analysis of each of the 8 f r a c t i o n s . Examination of the number of phosphate res-idues per molecule of protamine indicates that Fractions I and II are unmodified protamines; Fractions III and IV have close to 1 phosphate, Fractions V and VI have close to 2 phosphates, and Fractions VII and VIII have close to 3 phosphates per molecule of protamine. Correlation of the phosphate analyses with the amino acid compositions of each f r a c t i o n indicates that two series of protamines each with 0, 1, 2, and 3 phosphates per molecule of protamine are re-solved by gradient e l u t i o n of protamine from CM-cellulose. One series (I, I I I , V, and VII) has 3 s e r y l residues per molecule of protamine while the other (II, IV, VI, and VIII) has 4 s e r y l residues per molecule. The protamines i n each series are not homogeneous as indicated by the f r a c t i o n a l 182. TABLE XV Amino Acid Analysis of Protamine Fractions Fractions were hydrolyzed i n vacuum with 6 N HCl at 110°, for 16 hr and applied to the long column of Technicon Amino Acid Analyser. Results are expressed as residues of amino acid per mole of protamine, assuming that 1 mole of protamine has 32 amino acid residues. Serine analyses were corrected for 15 to 20% loss during hydrolysis. A trace of methionine was detected i n Fraction IV; however, control studies indicated that as much as hydrolysis. 75% of methionine i s destroyed upon acid Fraction VIII VII VI V IV I I I II I nmoles of Prot-amine 16 .4 13.0 24.4 18.8 13.8 17.1 16 .7 23 Arg 21.4 23.0 22.4 23.1 21.9 21.2 22.6 22.5 Ser 3.55 3.0 3.33 2.55 3.5 2.8 3.5 2.7 Pro 2.38 2.60 2.42 2.43 2.5 2.12 2.67 2.44 Ala 0.39 0.34 0.46 0.58 0.56 0.72 0.53 0.65 Gly 2.08 2.04 2.16 2.02 2.2 1.98 1.93 2.0 Val 1.80 1.73 2.08 1.73 1.85 1.62 1.86 1.75 H e 0.33 0.19 0.27 0.31 0.27 0.28 0.27 0.28 183. TABLE XVI Phosphate Analysis of Protamine Fractions Each f r a c t i o n of protamine (20 yl) was ashed with magnesium n i t r a t e to convert organic phosphate to inorganic phosphate. Phosphate was analyzed by the method of Ames (150). The amount of protamine i n each sample was deter-mined by amino acid analysis (Table XV). Note that the most phosphorylated species of protamine (Fractions VII and VIII) contain 3 phosphates per molecule of protamine. Fraction VIII VII VI V IV II I II I Phosphate (nmoles) 50 41 51 38 14 8.6 5.3 2.3 Protamine (nmoles) 16.4 13.0 24.4 18.8 13.8 17.1 16.7 23 Phosphate: Protamine 3.10 3.15 2.07 2.02 1.0 0.50 0.32 0 .10 184 residues of p r o l i n e , alanine, and isole u c i n e . The trout protamines sequenced by Ando and Watanabe (86) a l l have 4 se r y l residues. The sequence of the l b component d i f f e r s from the sequence of l a p r i n c i p a l l y i n the substitution of an i s o l e u c y l for a v a l y l residue, while the sequence of component II d i f f e r s from l a i n the substitution of an alanyl for a p r o l y l residue. In Table XV, the sum of the p r o l y l residues (^2.5) and alanyl residues (^0.5) i s 3; the sum of the v a l y l residues (^1.75) and i s o l e u c y l residues (^0.25) i s 2. These compositions would be i n accord with three subspecies i n each of the protamines with 3 and 4 s e r y l res-idues . These three subspecies would be i n the proportions 0.25 ( l a ) , 0.25 (lb) and 0.5 ( I I ) . (c) Methionine and 3 2P Labeled Protamines: Wigle and Dixon (101) showed that a methionyl residue i s incorporated at the NH 2-terminus of nascent protamine and l a t e r removed. When the k i n e t i c s of enzymatic modification of the protamines was followed (Fig. 21 and 25) i t was found that newly synthesized protamine (PQM) was rapidly phosphorylated. Shortly a f t e r , methionine was removed. At least three peaks of methionyl l a b e l (PQM, P I M , and P2M) were observed upon starch gel electrophoresis (Fig. 19). I t was therefore of i n t e r e s t to determine where methionyl-protamine eluted from these columns. 185. A c e l l suspension was labeled with [ 3 5S"methionine. Protamines were extracted and p u r i f i e d . C a r r i e r [ 3H]arginine-labeled protamine was added. F i g . 34 shows the p r o f i l e of absorbance at 230 nm and r a d i o a c t i v i t y . Four peaks of methionine incorporation are found. These are labeled PQM, PiM, P 2 M , and P 3 M ; the subscript numerals indicate the pre-sumed degree of phosphorylation of these species of prot-amine. The 3 5S counts elute i n peaks s l i g h t l y ahead of Fractions I I , IV, VI, and VIII. To examine the p r o f i l e of [ 3 2P]phosphate incorporation, [3H] arginine and t 3 2P]phosphate doubly-labeled protamine was prepared from a 19 hr incubation of testes c e l l s . F i g . 35A shows the absorbance and r a d i o a c t i v i t y p r o f i l e s of the eluted protamines. The 6 peaks of 3 2 P label coincide with the peaks of Fractions III to VIII. I t was demonstrated previously that the p r o f i l e s of r a d i o a c t i v i t y of protamine labeled with [ 3H]arginine for varying times and resolved by starch gel electrophoresis are a function of the duration of lab e l i n g (Fig. 21A to I ) . Protamine labeled for a very short time (3 min) showed only 1 peak of r a d i o a c t i v i t y i n the unphosphorylated methionine containing species of protamine (P QM), while at s l i g h t l y l a t e r times (30 min to 1 h r ) , three methionyl peaks (PQM, PiM, and P 2 M ) and one peak i n which methionine had been cleaved (Pi) were observed. In F i g . 33A the p r o f i l e of 186. E f f l u e n t V o l u m e ( m l ) F i g . 34. P r o f i l e of [ 3 5S]methionine-labeled protamine resolved by CM-cellulose chromatography. A c e l l sus-pension from an a r t i f i c i a l l y induced t e s t i s was incubated with 15 yCi per ml of [ 3 5S]methionine f o r 7.5 hr. Prot-amine was extracted, p u r i f i e d , and mixed with a small amount of protamine labeled for 1 hr with [ 3H]arginine. 10 mg of protamine were applied onto a 1.2 x 40 cm CM-ce l l u l o s e column and eluted with a 700 ml l i n e a r gradient of L i C l (0.40 to 0.85 M) i n 6 M urea. ( ), absorb-ance at 230 nm; (-0-0-) , 3H counts;(-A-A-), 3 SS-counts; (-A-A-), L i C l concentration. 187. Effluent Volume (ml) 100 200 300 400 500 600 700 - i i 1 i , — - — . j — n • — F i g . 35. Chromatography of 3 2 P - l a b e l e d protamine followed by starch gel electrophoresis. (A), P r o f i l e of 3 2 P - l a b e l e d protamine. A testes c e l l suspension was incubated with 10 uCi per ml of [ 3H]arginine and 100 yCi per ml of [ 3 2P] phosphate for 19 hr. Protamines were extracted and p u r i f i e d ; 25 mg were applied to a 1.2 x 40 cm CM-cellulose column and eluted with a li n e a r gradient of L i C l (0.40 to 0.85 M) i n 6 M urea. (B), Starch gel electrophoresis of aliquots of fractions c o l l e c t e d from the gradient i n (A). 30 y l aliquots were applied to separate starch gel sl o t s as i n -dicated. In addition, a sample of unseparated protamine was applied at the side and centre s l o t s to serve as r e f -erences. After electrophoresis for 12 hr, the gel was t r i -sected horizontally; the middle slab shown i n the photo-graph was stained with a d i l u t e solution (0.125%) of Amido Black and destained with s u l f u r i c acid (130). 188. 3H l a b e l represents such a case. In order of e l u t i o n , the radioactive peaks are P 3 M , P2M, PiM, P i , and PQM. In protamine labeled for 19 hr with [3H] arginine (Fig. 35A) , the 6 peaks of 3H l a b e l coincide with the peaks of 3 2 P l a b e l ; i n addition there i s a shoulder of 3H counts e l u t i n g j u s t ahead of Fraction I I . As mentioned above, the prot-amines are resolved by chromatography on CM-cellulose i n the presence of urea into two s e r i e s , one of protamines containing 3 serines and the other with 4. I t i s not immediately apparent why only 4 peaks of [ 3 SS]methionine la b e l are found. One pos-s i b i l i t y i s that only one series of protamine has methionine incorporated at the NH2-terminus. One would then expect another series of 3H labeled protamine with no methionine. However, the 3H label i n F i g . 33A (protamine labeled for 1 hr i n vivo) a l l seems to correspond to methionyl-protamine except for Pi formed by the cleavage of methionine from PiM. The 6 peaks of 3H l a b e l observed a f t e r 19 hr which do not correspond to methionine labeling indicate that the protamines with 3 and 4 s e r y l residues are synthesized simultaneously ( i . e . there are no marked differences i n rates of synthesis of the d i f f e r e n t protamine f r a c t i o n s ) . This would seem to imply that the two series of methionyl-protamine are eluted together instead of being resolved into the even-numbered and odd-numbered methionyl-protamine s e r i e s . Wigle and Dixon (101) observed 6 peaks of [ 3 SS]methionine i n protamine eluted from CM-cellulose by a L i C l gradient s i m i l a r to that used by Marushige et a l . (105). Wigle and Dixon's source of protamine was also hormonally induced testes at the early protamine stage. However, the absence of urea i n the e l u t i o n gradient and the lack of chemical or gel electrophoretic data makes i t d i f f i c u l t to assess the sig n i f i c a n c e of t h e i r peaks. (d) P o s i t i v e I d e n t i f i c a t i o n of Phosphoprotamines on  Starch Gels: The 8 bands of "protamine" previously observed on starch gels (Fig. 19) had the properties of protamine. That i s , they were not found i n early spermatids but appeared at a middle stage of spermiogenesis (Fig. 7); they could be highly labeled with [ 3H]arginine (Fig. 12); some of the bands were phosphorylated (Fig. 8 and 20); and [ 3H]arginine label progressed i n a defined way from one band to another (Fig. 21 and 22) in d i c a t i n g that they were not separate proteins syn-thesized simultaneously. However, conclusive chemical e v i -dence that they were protamines was not presented at that time. To demonstrate that the "protamine" bands observed on starch gels were indeed protamines or phosphoprotamines with d i f f e r e n t l e v e l s of phosphorylation, aliquots were re-moved from the fractions c o l l e c t e d from the column i n F i g . 35A and subjected to electrophoresis on starch gels. F i g . 35B shows the protein s t a i n of the g e l . A sample of un-fractionated protamine served as a marker and was applied at e i t h e r side and i n the centre of the row of sample s l o t s . The fas t e s t migrating broad band, Po [consisting of 2 com-ponents PoA (=Fraction I) and PoB (=Fraction II)] i s the major band of protamine found i n late spermatids and mature sperm. I t i s , therefore, unmodified protamine. The 3 slower moving broad bands P i , P 2, and P 3 are c l e a r l y phospho-protamines with 1,2, and 3 phosphates respectively. The sensi t i v e staining procedure developed by Sung and Smithies (130) makes these starch gels se n s i t i v e monitors for the separation of the protamine components. F i g . 36 shows another separation of protamines monitored by these starch gels. Because of s l i g h t differences i n lots of starch and gel buffer, the resolution of PoA and PoB i n t h i s instance i s clearer than that i n F i g . 35B. (e) Alkaline Phosphatase Treatment of Phosphoprotamines: The phosphoprotamines can be converted by al k a l i n e phos-phatase treatment to unsubstituted protamine. A sample of protamine labeled with 3 2 P and [ 3H]arginine and enriched i n P 2 and P 3 species was incubated with alkaline phosphatase. At various times, aliquots of the incubation mixture were removed and subjected to starch gel electrophoresis. The photographs of the starch gel i n F i g . 37A to D show that a l k a l i n e phosphatase treatment progressively cleaves phos-phate from phosphoprotamines to form the unsubstituted 191. VIII VII VI V IV III II I 1 2 -e F i g . 36. Starch gel electrophoresis of protamines resolved by chromatography on CM-cellulose. The elu t i o n of protamines from a gradient (0.4 to 1.0 M L i C l i n 6 M urea) was monitored by subjecting 30 y l aliquots of various fractions to e l e c t r o -phoresis i n starch gels as i n F i g . 35B. At l e a s t 9 bands of protamine are resolved by the combination of column chrom-atography and gel electrophoresis. The two fastest bands (P QA and P DB) are the two unmodified protamines normally found i n mature sperm. The resolution of protamines on starch gels i n t h i s figure i s s l i g h t l y d i f f e r e n t from that i n F i g . 35B because of s l i g h t differences i n l o t s of starch and gel buffer. Note that Fractions VII and VIII d i f f e r s l i g h t l y i n mobility and are p a r t i a l l y resolved i n t h i s gel. 4800 • 2400-ice 4800 to E E 2400 CM E Q . o X 10 4000 2000-200 - 100 8000 4000 P,5.5htV T , T I VT^ P 3 P2 * 192. 200 g "co 100 | OJ \ E tx u 200 0_ M lO i 100 200 00 13 15 17 19 + Distance (cm)—> -F i g . 37. Alkaline phosphatase treatment of protamine. A sample of protamine (150 yg) enriched i n P 2 and P 3 species and labeled i n vivo with [ 3H]arginine and [ 3 2P]phosphate, was incubated with b a c t e r i a l alkaline phosphatase (60 yg). At various times, aliquots were removed, frozen, and l a t e r subjected to starch gel electrophoresis. After electrophor-e s i s , the gel was t r i s e c t e d horizontally; the bottom slab was stained (photographs), while the middle slab was s l i c e d into 2 mm s l i c e s . Each s l i c e was s o l u b i l i z e d and analyzed for r a d i o a c t i v i t y . (A) control incubated for 5.5 hr at 37°C without enzyme; (B), (C), and (D) are samples of protamine incubated with alkaline phosphatase for 30 min, 1.5 hr, and 5.5 hr respectively. (dephospho) protamine. Analysis of r a d i o a c t i v i t y i n the protamine region of the starch gel indicates that the 3 2 P lab e l i s being removed while the [ 3H]arginine la b e l i s pro-gressively concentrated i n the dephosphoprotamine band (P Q i n F i g . 37D) . DISCUSSION The mechanisms involved i n the condensation of chromatin and removal of histones during spermatogenesis are l i t t l e understood. Other than the observation that a sperm-specific basic protein has been found i n the highly condensed sperm of many organisms (5,6,15), and that this protein seems to appear i n the middle stages (96,97,98) of spermiogenesis (maturation of the spermatid), l i t t l e i s known about the chemistry or biology of these proteins. To a large extent chemical and b i o l o g i c a l studies of the f i s h (herring and salmonid) protamines have served as model studies i n attempts to elucidate the mechanism of the condensation process. The ready a v a i l a b i l i t y of testes at d i f f e r e n t stages of develop-ment, the ease of extraction of t h e i r protamines, and the high proportion of c e l l s (especially i n hormonally-induced testes) undergoing the replacement process, have made salmonid testes a favourable system for study. Condensation of the nucleus i n the spermatids of salmonid fishes i s characterized by the complete replacement (96, 108, 140) of the histones i n the nucleus by the protamines, Protamine i s synthesized i n the cytoplasm (100) by the usual mechanisms of protein biosynthesis (99,100) and rapidly transported into the nucleus (100, F i g . 29 and 30). Methionine i s incorporated at the NH 2-terminus of nascent protamine (101,102), and l a t e r removed (101, F i g . 32) thus accounting for i t s absence i n protamine i s o l a t e d from spermatozoa. These findings suggested that methionine might have a role i n the i n i t i a t i o n of eucaryotic protein bio-synthesis (101). In addition, protamine appears to under-go a series of enzymatic phosphorylations and dephosphoryla-tions (104,105, F i g . 25). However, the exact function(s) of the protamines and the b i o l o g i c a l s i g n i f i c a n c e of the observed phosphorylation and dephosphorylation are not understood. The conversion of nucleohistone to nucleoprotamine may be related to the necessity of packaging large amounts of DNA i n a very com-pact form. The replacement process occurs i n the spermatids. This was conclusively shown from histochemical studies by A l f e r t (96). From the morphological and staining charac-t e r i s t i c s of salmon t e s t i s c e l l s , A l f e r t recognized 4 species of spermatids at progressive stages of condensation and histone removal. Our analyses of the population of c e l l s i n testes at progressive stages of development and the complement of histones and protamines i n the d i f f e r e n t developmental stages have confirmed A l f e r t ' s findings; i n addition, they have yielded information on the time of appearance of protamine, the loss of histone, and the rate of protamine biosynthesis and condensation of the sperm-a t i d . C e l l s at four stages of the replacement process were resolved by c e l l sedimentation (Fig. 7): (a) early sperm-atids (1.5 Sv) which have not yet begun to synthesize prot-amine; (b) middle spermatids (1.5 to 1.0 Sv) i n which prot-amine synthesis and histone replacement has begun; (c) l a t e spermatids i n which synthesis of protamine and loss of histone i s almost complete (1.0 Sv); and (d) mature sperm (0.6 Sv), which the c e l l nucleus contains almost e n t i r e l y nucleoprotamine. The i n t e r v a l between each of the stages i s about 1 week (Fig. 16) so that about 3 weeks are required for spermiogenesis i n trout t e s t i s . Separation and Characterization of Protamine; In the middle spermatids, we i n i t i a l l y observed at least 4 and l a t e r t h i s was extended to 6 phosphorylated species of protamine resolvable by starch gel electrophoresis. In order of increasing degree of modification these are meth-io n y l (PQM) , phosphoryl ( P i , P 2, and P 3) and phospho-methionyl (PiM and P2M) species of unmodified (Po) protamine. The amino acid analyses (Table XV), phosphate determinations (Table XVI) and alkaline phosphatase treatment (Fig. 37) of p u r i f i e d protamines unequivocally demonstrate that the various bands observed on starch gels and peaks eluted from CM-cellulose are protamines of d i f f e r e n t degrees (P o, P i , P 2, and P 3) of phosphorylation. The separation of phosphoprotamines from unmodified protamines on both starch gels (pH 3.2 to 3.5) and CM-c e l l u l o s e columns (pH 7.5) i s based upon the stepwise de-crease i n net p o s i t i v e charge of the protamine molecule [from +22 i n Po to +19 i n P 3 at pH 3.5 and +16 at pH 7.5] contributed by the increasing number of phosphoryl groups each with minus -1 charge at pH 3.5 or -2 charge at pH 7.5. CM-cellulose chromatography had been used previously (100,105,133) to resolve the protamines. Marushige et a l . (105) showed that 3 2 P - l a b e l e d protamine eluted ahead of unmodified protamines. Ling et a l . (133) treated protamine with alkaline phosphatase and resolved 3 components, C^, C j j , and C J J J , eluted with increasing concentrations of L i C l from CM-cellulose columns. From amino acid analysis (133 and Table XV) C^-j. (3 s e r y l residues) and (4 s e r y l residues) appear to correspond to Fractions I and I I , respec-t i v e l y resolved by chromatography with L i C l i n the presence of 6 M urea (Fig. 35). The Cj component may be a discrete but minor component of protamine (133), or a l t e r n a t i v e l y , phosphoprotamines which have not been completely dephos-phorylated. The fractions eluted l a s t by CM-cellulose chromatography i n the presence of urea are labeled I and II since (a) these are the major components found i n mature sperm and (b) Fractions III to VTII appear to be modifications of Fractions I and I I . Comparison of protamines eluted from CM-cellulose to protamines resolved by starch gel e l e c t r o -phoresis (Fig. 19,35, and 36) indicates that Fractions I and II from CM-cellulose correspond to PoA and PoB respectively, III and IV to P i , V and VI to P 2, and VII and VIII to P 3. In the absence of urea, the unmodified components of protamine elute at a s a l t concentration of 0.9 to 0.95 M L i C l (133), while i n the presence of 6 M urea, these species elute at 0.7 to 0.75 M L i C l (Fig. 33). Thus, the i n t e r -actions of protamine with CM-cellulose are not e n t i r e l y i o n i c but must involve some hydrogen bonding and hydrophobic in t e r a c t i o n s . A s i m i l a r e f f e c t of urea was observed upon the e l u t i o n of histones from CM-cellulose (PART III of this Thesis). A l l of the histones are eluted at 0.40 M L i C l i n the presence of 6 M urea. Urea also sharpens the peaks of eluted material and considerably reduces t a i l i n g . I f urea i s omitted from the gradient a detectable amount of t r i -phosphoprotamine (P 3 or Fraction VIII) t r a i l s into the region of unmodified protamine as judged by monitoring the e l u t i o n on starch gels. From rooster sperm, Pulleyblank (88) resolved at least 8 to 10 species of the sperm-specific basic protein of fowl, g a l l i n (152), by gradient e l u t i o n from CM-cellulose columns. Amino acid analyses indicated that about 60% of the residues i n each of these species are arginine. However, the pro-portions of the lesser amino acids varied considerably. In addition, the species of g a l l i n eluted f i r s t migrated the fas t e s t upon starch gel electrophoresis, while the species eluted l a s t migrated the slowest. This i s i n contrast to the trout protamines; where the species eluted f i r s t ( P 3 ) migrates the slowest and that eluted l a s t (Po) migrates the fas t e s t . I t would appear, then, that the multiple species of g a l l i n observed by Pulleybank (88) are not phosphorylated derivatives of unmodified species of g a l l i n . I f a series of phosphorylations and dephosphorylations of g a l l i n s i m i l a r to that observed i n trout protamine occurred, i t would not be surprising that the species i s o l a t e d from mature sperm-atozoa are unphosphorylated (88). C I n t r a c e l l u l a r Transport of Protamine; The k i n e t i c studies reported here (Fig. 21,25,29, and 30), using labeled arginine and methionine indicate that newly synthesized protamine i s not phosphorylated and has methionine at i t s NH2-terminus (PoM) . Newly synthesized protamine i s rapidly transported from the cytoplasm into the nucleus i n the form of P M. o Thus phosphorylation of protamine i s not required for the removal of nascent protamine from the ribosome or the trans-port of protamine into the nucleus. The mechanism of t h i s rapid transport i s not known. Although extrapolation of results obtained from other systems i s hazardous, some recent studies should be mentioned on the k i n e t i c s and mechanism of i n t r a c e l l u l a r protein trans-port i n other systems (153-155). The pancreatic exocrine proteins are synthesized on rough endoplasmic reticulum, packaged i n zymogen granules, and secreted from the acinar c e l l (153). Using electron microscopic and autoradiographic techniques, Jamieson and Palade (153) followed the i n t r a -c e l l u l a r location of pulse-labeled nascent proteins i n pancreatic c e l l s . After several minutes, the rough endo-plasmic reticulum was highly labeled. Progressively, the lab e l was found i n smooth endoplasmic reticulum (7 to 10 min), go l g i v e s i c l e s (10 to 40 min) and zymogen granules (1 to 2 hr). Redman (154) showed that transport of nascent peptides across rat l i v e r microsomal membranes was depen-dent only upon t h e i r release from membrane bound ribosomes. This release did not require ATP and could occur i n the cold (154). Many of the soluble enzymes i n mitochondria are synthesized outside the mitochondria on microsomes. Kaden-bach (155) used a c e l l - f r e e system to study the microsomal synthesis of some mitochondrial enzymes and showed that labeled proteins could be transported from the microsomes into the mitochondria. Both the transport of polypeptides i n the microsome and into the mitochondria seemed to be dependent upon ATP. Thus the report (16) that the transport of protamine i n t o the nucleus depends upon ATP ( i n h i b i t i o n of transport by 2,4-dinitrophenol) supports the p o s s i b i l i t y that most of the protamine may be synthesized on rough endoplasmic 201. reticulum and enters the cisternae of the endoplasmic re-ticulum where i t i s transported into the nucleus. The rapid appearance of labeled protamine i n the nucleus would be explained by the scanty cytoplasm of spermatids and hence the short distance that newly synthesized protamine must traverse i n order to reach the nucleus. Cytoplasmic Versus Nuclear Phosphorylation; Ling et a l (100) showed that exogenous t 1 **C] arginine-labeled protamine binds no n - s p e c i f i c a l l y to ribosomes. We have shown here that af t e r l a b e l i n g with [3H] arginine, both unphosphorylated (P Q) and phosphorylated (P x) protamines can be found on i s o l a t e d ribosomes. From the post-ribosomal supernatant J e r g i l and Dixon (106) have i s o l a t e d a kinase which phosphorylates free protamine. In vivo, phosphorylation of protamine can occur i n the cytoplasm (105) but i t proceeds at a much slower rate and i s small i n comparison to that occurring i n the nucleus (Fig. 29,30). Phosphorylation of protamine i n the cytoplasm i s not obligatory. On ribosomes, methionine can be removed from newly synthesized protamine (PQM) to form P Q (Fig. 28) while i n the nucleus, PQM i s rapidly phosphorylated (Fig. 29). Newly synthesized protamine (PQM) i s rapidly trans-ported into the nucleus (100 and F i g . 29) where i t i s phos-phorylated (within 5 to 10 min) to PXM and P 2M (Fig. 21). Methionine i s then removed to form Pi and P 2 . Phosphoryla-t i o n of Pi and P 2 continues and labeled arginine i s found i n 202. P 3 after 5 to 10 hr. Very l i t t l e [ 3H]arginine i s found i n unmodified protamine at early times. However, 5 to 10 days af t e r i n j e c t i o n of [ 3H]arginine, l a b e l i s found i n unsub-s t i t u t e d protamine, Po, derived from the sequential dephos-phorylation of P 3 (Fig. 25). The long time (Fig. 25) required to completely phosphorylate and dephosphorylate protamine i n the nucleus compares fav-orably with the time (^1 week) observed for condensation of middle spermatids, which have just begun to synthesize protamine, into l a t e spermatids (Fig. 7). The short period required to pass through the i n i t i a l stages and the long period required to completely phosphorylate and dephosphorylate the protamines make i t unlikely that a protamine phosphokinase sequentially phosphorylates a l l si t e s on a protamine molecule before leaving to f i n d a new substrate. Rather, separate and d i s t i n c t pools of the various modified species of protamine e x i s t (Fig. 25); molecules within a given pool are modified at random by the appropriate enzyme and become part of another pool, where the process of random sel e c t i o n by phosphokinases (or phosphatases) i s repeated. The obligatory phosphorylation and dephosphorylation of newly synthesized protamine appears to be u n i d i r e c t i o n a l , since species i n the phosphorylation pathway did not behave i n exactly the same way as s i m i l a r species on the dephosphorylation pathway. This implies that protamine phosphokinases and phosphatases recognize s p e c i f i c conformations or the l o c a l environment of protamine bound to chromatin. Protamine Kinases; Ingles and Dixon (104) characterized some of the si t e s of phosphorylation of protamine, while Sanders and Dixon (107) continued these studies. I s o l a t i o n of phosphopeptides aft e r t r y p t i c digestion of 3 2 P - l a b e l e d protamines indicated that a l l 4 s e r y l residues i n protamine could be phosphorylated i n vivo (107). In addition to the Val-Ser(P)-Arg sequence common to most of the protamines, two other highly phosphorylated peptides (107) were of note: a triphosphorylated peptide Ser(P)-Ser(P)-Ser(P)-Arg-Pro-Val-Arg presumably from the protamine components with 4 s e r y l residues, and a diphosphorylated peptide Ser(P)-Ser(P)-Arg-Pro-Val-Arg presumably from the protamine components with 3 s e r y l res-idues. The most phosphorylated species reported here appears to have 3 phosphoryl groups per molecule of protamine (Table XVI). I t would appear, then, that there are mechanisms i n -volved i n regulating the degree of protamine phosphorylation i n vivo. J e r g i l and Dixon (106) have i s o l a t e d a "protamine" kinase and phosphatase from the post-ribosomal supernatant of trout testes c e l l s . This kinase i s s p e c i f i c for prot-amine and i s stimulated by c y c l i c 3',5J-AMP. Incubation of unmodified protamines (less than 5% of the s e r y l residues i n a mixture of protamines with about 10% of the se r y l res-phosphorylated) with protamine kinase and 204. idues phosphorylated. Starch gel electrophoresis revealed the appearance of a single phosphorylated band migrating s l i g h t l y slower than unmodified protamine (106). From the chemical characterizations of the i n vivo phosphorylated protamines (Fig. 33,35, Table XV, and XVI), the phosphorylated band observed by J e r g i l and Dixon (106) i s i d e n t i f i e d as mono-phosphoprotamine. That only the mono-phosphoprotamine was observed during the reaction i n v i t r o would seem to i n -dicate either that the conformation of protamine i s important i n vivo or that there are several protamine kinases which phosphorylate d i f f e r e n t s i t e s on the molecule. J e r g i l and Dixon's data indicated that t h e i r protamine kinase could phos-phorylate several s i t e s of protamine. The protamine kinase i s o l a t e d from the cytoplasmic f r a c t i o n i s d i s t i n c t (156) from the ribosomal protein kinase (157). However t h i s does not exclude the p o s s i b i l i t y that the phosphorylation of protamine on ribosomes i s due to a ribosomal kinase. . , That the protamine phosphokinases and phosphatases might recognize s p e c i f i c conformations or the l o c a l environment of protamines bound to chromatin suggests that perhaps the lack of success i n i s o l a t i o n of protamine kinase(s) from nuclei may be due to the use of free protamine as a sub-strate i n the enzyme assays. If so, the protein kinases so far i s o l a t e d should be re-examined with dephospho-protamine bound to nucleohistone as a substrate. 205. Nucleohistone and Nucleoprotamine; Examination by electron microscopy of th i n sections of trout spermatids fixed with osmium tetroxide (158) revealed that the chromatin of early spermatids i s d i f f u s e and stains poorly. At l a t e r stages (middle spermatids) loose f o c i of intensely staining material are seen. The f o c i enlarge to f i l l the nucleus and gradually become condensed (late spermatids). The nucleus of the mature sperm i s very condensed and intensely stained. In v i t r o , the annealing of protamine to DNA by d i r e c t mixing or by g r a d i e n t - d i a l y s i s r e s u l t s i n the formation of r i g i d aggregates of DNA-protamine which are separable from r e l a t i v e l y protamine-free DNA by centrifugation (94,95). This indicates that the binding of protamine to DNA i s highly cooperative with the p r e f e r e n t i a l binding of protamine to s i t e s adjacent to those already occupied by the polypeptide (94,95) . Protamine i s synthesized i n the cytoplasm and gradually accumulates i n the nucleus. The binding of protamine i n vivo to chromatin (nucleohistone) may also be cooperative with the formation of scattered f o c i of intensely staining material (nucleoprotamine) i n the nucleus of spermatids active i n the replacement process. Marushige and Dixon (108) f i r s t noted that there appeared to be a c e r t a i n temporal sequence i n the disappearance of the histone fractions during the replacement process. Chromatin from testes i n which the replacement process i s active showed a higher content of histone I. The nucleo-protamine portion was separated from the nucleohistone por-t i o n of chromatin by shearing i n a Waring Blendor followed by centifugation. The nucleohistones were s o l u b i l i z e d and were found i n the supernatant while the nucleoprotamine was recovered i n the sediment. No protamine was found i n the nucleohistone f r a c t i o n while the nucleoprotamine f r a c t i o n always contained a small amount of histone. The histone i n the nucleoprotamine f r a c t i o n was highly enriched i n histone I and d e f i c i e n t i n the arginine and s l i g h t l y - l y s i n e -r i c h histones. Thus, i t appears that the arginine-rich histones are the f i r s t and the l y s i n e - r i c h histones (his-tone I) are the l a s t to be removed during the process of displacement (108,109). Compared to mature testes, testes at the early prot-amine stage of development contain a much higher proportion of middle spermatids which have begun protamine biosynthesis and the replacement process but have not undergone s i g -n i f i c a n t nuclear condensation (Fig. 15,17). From such testes, we have found a f r a c t i o n of chromatin (nucleohistone) which has protamine bound to i t but i s not yet condensed and hence can be sheared (Fig. 26g and h, and F i g . 27). The protamines found i n th i s portion are highly phosphorylated. That the protamines i n the s o l u b i l i z e d nucleohistone f r a c -t i o n are d i f f e r e n t from that i n the compact nucleoprotamine f r a c t i o n i s shown i n F i g . 30 and 31. The NH2-terminal methionyl residue of newly synthesized protamine i s rapidly removed from protamine i n the nucleohistone f r a c t i o n , but only slowly i n the nucleoprotamine f r a c t i o n . The reasons for these differences are not clear although they could be attributed to a reduced a c c e s s i b i l i t y of the methionyl amino-peptidases to methionyl-protamine i n the nucleoprotamine f r a c t i o n . The summation of these two rates accounts for the biphasic removal of the NH 2-terminal methionyl residue ob-served i n whole c e l l s (Fig. 32). The resistance of nucleoprotamine to shearing may be due to i t s compactness. When separated by electrophoresis i n starch gels, the acid-soluble proteins from middle sperm-atids (Fig. 12) which have begun to synthesize and phosphorylate protamine resemble those from nucleohistone prepared by son-i c a t i n g chromatin from testes at the early-protamine stage (Fig. 26g). In contrast, the p r o f i l e of acid-soluble proteins from late spermatids (Fig. 12) which have l o s t most of t h e i r histone resembles the nucleoprotamine f r a c t i o n (Fig. 26h and 27) of sonicated testes chromatin. These data suggest that the protamine found i n the nucleohistone f r a c t i o n i s not associated with large tracts of condensed chromatin. Presum-ably, histone i s a c t i v e l y removed from those stretches of nucleohistone on which protamines are bound. 208. The Replacement Process: Histone I was found to be the l a s t histone replaced by protamine during spermiogenesis (108,109). The arginine-rich histones III and IV are removed f i r s t i n the i n vivo replacement (108). This i s i n contrast to e l u t i o n of histones from chromatin with increasing concentrations of sodium chloride when histone I i s the f i r s t removed (109,110). In v i t r o d i s s o c i a t i o n of histone from chromatin by added protamine (109,110) also resulted i n displacement of histone I f i r s t , suggesting that processes other than d i r e c t displace-ment or increasing i o n i c strength are important i n the bio-l o g i c a l replacement. Decreasing the i o n i c interactions of histones to DNA by phosphorylation of histones i s unlikely to be important i n the removal process since most of the phosphorylation occurs i n spermatogonia and primary spermatocytes active i n DNA and histone synthesis (Table VI and VIII). However, acetyl-ation of histones may be important since s i g n i f i c a n t a c e t y l -ation has been detected i n spermatids (146). Proteases s p e c i f i c for histones have also been suggested as important i n the b i o l o g i c a l removal during spermatogenesis (109). Marushige and Dixon (109) noted that there was a heterogeneous mixture of small proteins or peptides i n the nucleoprotamine portion of chromatin. They suggested that these could be p r o t e o l y t i c products of histones (109). In fact, Dr. M.M. Sanders has i s o l a t e d and p a r t i a l l y characterized a histonase from trout testes. However, the presence and a c t i v i t y of these enzymes and the i d e n t i t y of the observed peptides have yet to be established i n spermatids at the replacement stage. Some i n t e r e s t i n g observations on spermiogenesis i n other systems appear to be pertinent. The nuclei of crab spermatozoa (5,82) are r e l a t i v e l y uncondensed and do not contain a histone or protamine-like basic protein; however, a cytoplasmic capsule contains s i g n i f i c a n t amounts of basic proteins which appear to have s i m i l a r m o b i l i t i e s on a c r y l -amide gels as c a l f thymus histone fractions (82). In rats, the "sphere chromatophile" i n the residual body which i s sloughed from spermatids late i n the maturation process contains a histone-like protein r i c h i n lysine (159). This protein i s not of ribosomal o r i g i n and appears to be pro-t e i n synthesized i n the preceding c e l l cycle (primary spermatocytes) about the time of the l a s t (premeiotic) DNA (and histone) synthesis (159). These observations would seem to indicate that during spermiogenesis, the complement of histones may be removed apparently i n t a c t from chromo-somes, and that extensive proteolysis (to small peptides or amino acids) may not necessarily occur during the replace-ment process. I t should be mentioned that the studies (109,110) on the displacement of histones from chromatin have been con-ducted using protamines which consist largely of dephospho 210. (P Q) protamine from late stage testes or mature sperm. The present data suggest that the controlled phosphorylation of protamine might play an important role i n the correct bind-ing of protamine to DNA and perhaps simultaneous loss of histone, while dephosphorylation of protamine seems to be related to the controlled condensation of spermatid chromatin. I t i s conceivable that only i n c o r r e c t l y bound protamines, or those i n close association with histones ( i . e . recently-synthesized protamines) can be phosphorylated, while only phosphoprotamines i n the correct DNA-binding conformation and about which the surrounding histones have been removed can be dephosphorylated. That i s , the presence or absence of histones complexed to DNA and associated with protamine, may be an important c o n t r o l l i n g element determining the substrate s p e c i f i c i t i e s of the phosphokinase(s) and phos-phatase (s) s p e c i f i c for protamine. Conversely, the presence of protamine on nucleohistone may provide a signal (to d i r e c t the enzymes) involved i n the removal of histones. Investigating the i n v i t r o displacement of histones using highly phosphorylated protamines should determine whether phosphorylation of protamine i s alone s u f f i c i e n t for the b i o l o g i c a l displacement or whether other processes are required simultaneously. The sequences of the 3 herring protamines, clupeine Y I ' Y I I ' a n d Z ' r e P o r t e d bY Ando et a l . (91,92,93), are shown i n F i g . 2A. Two of the components of clupeine contain threonyl residues. The Y^ component has 3 s e r y l residues i n sequence and one Thr-Thr sequence; the Y.^ component has 2 s e r y l res-idues and 1 threonyl residue a l l occurring singly; the Z component has 3 i s o l a t e d s e r y l residues and 1 threonyl res-idue. The maximum degree of phosphorylation of protamine observed during the present work with trout i s 3 phosphates per molecule of protamine. I f an obligatory sequence of phosphorylations and dephosphorylations i s required for the binding of clupeine to DNA, and since the component has only 2 s e r y l residues, i t would be of i n t e r e s t to determine whether the threonyl residues i n Y^ or can be phos-phorylated i n vivo. Because phosphorylation of sperm-specific proteins com-parable to the protamines i n the salmonids has not.been described i n any other system, the general s i g n i f i c a n c e of the phosphorylation-dephosphorylation process cannot be established with any degree of confidence. However, i t i s encouraging that most amino acid analyses indicate that the protamines of every species contain s i g n i f i c a n t amounts of serine (5,15). PART III ENZYMATIC MODIFICATIONS OF THE HISTONES DURING SPERMATOGENESIS IN TROUT 212. In examining the rel a t i o n s h i p of histone and protamine synthesis to t h e i r phosphorylation i n d i f f e r e n t c e l l types of trout t e s t i s (PART I ) , we were struck by the fact that the amino acid l a b e l ( [3H] arginine and t3H] lysine) , did not appear i n the two major ( i . e . unmodified, and monoacetylated) bands of histone IV, which were resolved from the other histones by starch gel electrophoresis, u n t i l 19 hours a f t e r the s t a r t of the incorporation (Fig. 10 and F i g . 12). This discrepancy could not be accounted for by d i f f e r e n t i a l histone synthesis since a l l histones are synthesized simultaneously (55). How-ever, the starch gels used are capable of resolving histone IV into 10 bands (22,69) of which 9 are modified by phos-phorylation or acetylation, or both; thus an alte r n a t i v e explanation was that the la b e l i n newly synthesized histone IV was migrating i n the region of modified histone IV and did not appear i n the unsubstituted band u n t i l much l a t e r . In addition, amino acid l a b e l was found i n the phos-phorylated band of histone I l b i , implying that a s i g n i f i c a n t proportion of histone I l b i i s phosphorylated during or shortly a f t e r i t s synthesis. This was i n contrast to the results of Sung et a l . (134), and experiments which suggested that only "old" histones were phosphorylated (65,74,105). Experiments were then devised to examine the k i n e t i c s of lab e l i n g of the various modified species of the histones i n order to gain some insight i n t o the possible b i o l o g i c a l s i g -n i f i c a n c e of these modifications. 213. EXPERIMENTAL PROCEDURES (a) Materials: Additional materials not l i s t e d i n PARTS I and II of the Thesis are the following: d i t h i o t h r e i t o l was obtained from Calbiochem; porcine t r y p s i n from Novo Indus t r i , Denmark. (b) C e l l Incubations: C e l l suspensions were prepared by mincing 6 to 8 g of tissue with scissors i n 3 to 4 volumes of TMKS-0.1% glucose and gently hand homogenizing (3 strokes up-and-down) i n a Potter-Elvehjem homogenizer with a Teflon pest l e . The c e l l suspension was f i l t e r e d through 4 layers of cheesecloth. For short term (10 min to 4 hr) incubations, the c e l l s were centrifuged at 1000 x g f o r 10 min to remove excess amino acids and phosphate, and resuspended i n 2.5 volumes of TMKS-0.1% glucose. The suspension (approximately 20 ml) was pre-incubated for 10 min at 15 to 16° on a gyratory water bath before addition of radioactive precursors (100 uCi per ml of [3H] arginine, 100 yCi per ml of [3H] l y s i n e , and 300 yCi per ml of inorganic [ 3 2P]phosphate), phenol red, and 100 units per ml of p e n i c i l l i n and streptomycin. At 10 min, 1 hr, and 4 hr a f t e r addition of the radioactive l a b e l s , one-t h i r d of the c e l l suspension was removed and cycloheximide (2 x 10_,fM f i n a l concentration) and 2,4-dinitrophenol (2 x lO'^M f i n a l concentration) were added to i n h i b i t protein 214. synthesis and deplete the ATP pool by uncoupling oxidative phosphorylation. C e l l s were washed with phosphate-buffered saline containing cycloheximide and 2,4-dinitrophenol to remove excess radioactive l a b e l . For long-term incubations (to 12 hours) the o r i g i n a l c e l l suspension (approximately 25 ml) was supplemented with 2.5 ml of Waymouth's medium (125) (containing 10 mM T r i s -HCl buffer, pH 7.2, instead of phosphate b u f f e r ) , phenol red, and 100 units per ml of p e n i c i l l i n and streptomycin. Radioactive precursors (20 yCi per ml of [3H] arginine and 200 yCi per ml of inorganic [ 3 2P]phosphate) were added and the incubation was ca r r i e d out at 15 to 16° on a gyratory water bath. Control studies showed that incorporation of [ 3H]arginine and 3 2P^ was l i n e a r f o r at least 12 hours under these conditions. After the incubation, the c e l l s were di l u t e d and washed with phosphate-buffered s a l i n e . (c) Labeling of Histones i n Intact F i s h : Hormonally i n -duced rainbow trout (approximately 150 to 200 g weight) at the pre-protamine or early protamine stage (45 to 55 days a f t e r the s t a r t of hormonal induction) were injected i n t r a -p e r i t o n e a l l y with 500 yCi of [ 3H]arginine and 500 yCi of [ 3H]lysine i n 0.4 ml of s a l i n e . At various times, f i s h were s a c r i f i c e d and c e l l suspensions were prepared e s s e n t i a l l y as above. (d) Preparation of Histones: Washed c e l l s were broken (100) i n a Potter-Elvehjem homogenizer with a motor-driven pestle (5000 rpm, 30 sec). Nuclei were sedimented at 2000 x g for 10 min and then resuspended i n 15 ml of TMKS. A 1% Nonidet P-40 solution i n TMKS (15 ml) was added to s t r i p any perinuclear cytoplasmic fragments and the nuclei were sedimented again. Nuclear histones and protamines were ex-tracted twice with 5 volumes of 0.4 N s u l f u r i c acid (133) and p r e c i p i t a t e d with 3 volumes of 95% ethanol. The pre-c i p i t a t e was co l l e c t e d by centrifugation at 15,000 x g for 10 min, washed with ethanol, and dissolved i n 0.1 M L i C l . The r e s u l t i n g solution was t i t r a t e d to pH 5.5 with 1 M Tris-HCl, pH 8, and adsorbed onto a 2.5 x 10 cm carboxymethyl-c e l l u l o s e column (100). The column was washed with 0.2 M LiCl-lOmM lithium acetate, pH 5.5, and eluted with 0.4 M L i C l , '3 M urea. The eluate was passed through a phospho-cellulose column ( 3 x 3 cm) and washed with 0.05 M HC1. Histones were eluted with 0.4 M HC1 and l y o p h i l i z e d . A l t e r n a t i v e l y , t o t a l histones and protamines were eluted with 0.2 N HC1 from the carboxymethyl-cellulose column and l y o p h i l i z e d . Histones were separated from protamines by passage through a 2 x 50 cm Bio-Gel P-10 column eluted with 0.01 N HC1 (104). (e) Fractionation of Histones: The f i v e major histone fractions (I, I l b i , I I b 2 , III monomer, and IV) were resolved by chromatography on long (3 x 320 cm) Bio-Gel P-10 columns as described by Candido and Dixon (23). To reduce the d i -s u l f i d e - linked dimers of histone I I I , the histones (30 to 100 mg) were incubated for 1 hr with 20 mM d i t h i o t h r e i t o l i n a 6 M deionized-urea solution buffered with 0.1 M sodium borate, pH 9.0. The histones were then alkylated by the addition of r e c r y s t a l l i z e d iodoacetamide to a concentration of 60 mM and incubation for a further 45 to 60 min (23). In early experiments, the sample was then applied to the column; subsequently, the solutions were a c i d i f i e d with HCl before application to the column. The column was eluted with 0.01 N HCl at a flow rate of 30 to 40 ml per hr. The void volume (650 ml) was discarded and 6 ml fractions were co l l e c t e d thereafter. The e l u t i o n of histones was monitored at 220 or 230 nm. Radioactivity i n 0.2 ml aliquots of every alternate f r a c t i o n was analyzed by the addition of 4 ml of Bray's s c i n t i l l a t i o n f l u i d (149) and counting i n a s c i n -t i l l a t i o n counter. The major histone fractions were pooled, l y o p h i l i z e d , and redissolved at a concentration of 10 mg per ml of 0.01 N HCl. The histone fractions (0.3 to 0.75 mg) were further separated by starch gel electrophoresis i n the presence of 4 M urea as described i n PART I. After electrophoresis, the gels were t r i s e c t e d h o r i z o n t a l l y . The bottom slab was stained by the se n s i t i v e cobalt-Amido Black procedure (0.125% Amido Black) and destained with s u l f u r i c acid (130). The bottom slab was further destained for 217. photography by extensive washing with 2% acetic acid. In l a t e r experiments, Histone I was detected by st a i n i n g the bottom slab by the normal procedure (1% Amido Black i n 2% ac e t i c acid) and destaining by extensive washing with 2% acetic acid. For analysis of r a d i o a c t i v i t y incorporated into the h i s -tones, the middle slab of the gel was cut into 1.5 mm s l i c e s which were then incubated with 0.4 ml of NCS S o l u b i l i z e r for 8 to 12 hr at room temperature. By the end of th i s period, the opaque gel had become transparent. Toluene s c i n t i l l a t i o n f l u i d (4 ml) was then added and the capped v i a l s incubated for a further 3 hr at 45° before counting on a Nuclear Chicago Unilux II Counter. (f) T r y p t i c Phosphopeptides; Phosphopeptides i n each of the histone fractions were examined by the procedure of Sung and Dixon (22). An aliquot of each major histone frac-t i o n (0.5 to 1.0 mg) labeled with 3 2 P i n vivo for 4 hr was digested with a 1:50 r a t i o (w/w) of dialyzed porcine tr y p s i n i n 0.1 M N H i»HCO3, pH 8.0, at 38° for 3 hr. The reaction was stopped by l y o p h i l i z a t i o n . The digests were redissolved i n a small volume of water and applied to Whatman 3MM paper. Electrophoresis was conducted at pH 6.5 i n a toluene-cooled tank for 60 min. The voltage gradient was between 50 to 60 V per cm. The pH 6.5 buffer consisted of pyridine/acetic acid/ water, 100:4:900. After electrophoresis, the paper was dried at 80° and exposed to X-ray f i l m (Kodak Blue Brand X) for 3 days to v i s u a l i z e the phosphopeptides. 218. RESULTS AND DISCUSSION I. I d e n t i f i c a t i o n and Levels of Phosphorylated Species of Histones i n the Major Fractions Extensive phosphorylation on s e r y l hydroxyl residues and e-NH 2-acetylation of histones have been observed (22,23, 33,69,105) during spermatogenesis i n trout testes. Sung and Dixon (22) found that the s i t e of phosphorylation i n both histone I l b i and IV was at the NH 2-terminus; the t r y p t i c digests yielded a single phosphopeptide, N-acetyl-Ser(P)-Gly-Arg. In addition, Candido and Dixon (23,33,69) have characterized the multiple s i t e s of e-NH 2-acetylation i n histone I l b i , I I b 2 , III and IV: a l l are i n the NH 2-terminal region (22,38,69). I t seems probable that the phosphorylation and acetylation of t h i s region may modify i t s i n t e r a c t i o n with DNA. Phosphorylation of histones takes place i n stem c e l l s and primary spermatocytes undergoing rapid DNA and histone synthesis (PART I of t h i s Thesis) rather than i n spermatids where the histones are removed and replaced by the protamines i n chromatin. C e l l s which synthesize histones also phos-phorylate histones at proportionate rates (PART I ) . This suggests that histone phosphorylation i s not important i n the removal of histones during spermiogenesis, but i s an important factor i n modulating either histone binding to DNA or the gross physical structure of chromatin. Since each histone may modulate the structure of chromatin d i f f e r -ently, studying t h e i r enzymatic modifications may y i e l d clues to t h e i r possible function. (a) Bio-Gel P-10 Chromatography of 3H and 3 2P-Labeled  Histones: Testes at the early protamine stage were excised. C e l l suspensions were prepared and incubated with 3H amino acids and inorganic [ 3 2P]phosphate for 1 and 12 hours. His-tones were extracted with acid, p r e c i p i t a t e d with ethanol, and a c i d i c proteins removed by adsorption onto and e l u t i o n from CM-cellulose. Histones were eluted with acid and l y -o p h i l i z e d . The histones were reduced and alkylated to prevent dimer formation between molecules of histone III and chrom-atographed on long (3 x 320 cm) Bio-Gel P-10 columns to sep-arate the major histone fractions (23). Fi g . 38 shows the absorbance and r a d i o a c t i v i t y p r o f i l e s of histones labeled with 3H and 3 2 P i n vivo for 1 hour (A) and 12 hours (B). The f i v e major histone f r a c t i o n s , I, I l b i , IIb2, I II monomer, and IV, are well resolved. 3 2 P la b e l i s found i n a l l f i v e fractions (Fig. 38A). However, the 3 2P l a b e l i n I l b i , IIb2, III monomer and IV, elutes s l i g h t l y ahead of the absorbance peaks. This i s probably due to the presence of a small number of anionic groups i n the Bio-Gel matrix which confer upon i t weak cation-exchange properties and the phosphorylated histones, being less basic, are eluted 20 40 60 80 100 120 140 160 180 I Fraction Number F i g . 38. Time dependence of [ 3 2P]phosphate incorporation i n t o histones. (A) A trout t e s t i s c e l l suspension was centrifuged to remove excess amino acids and phosphate and resuspended i n TMKS-0.1% glucose. The washed c e l l suspension was incubated for 1 hr with 100 yCi per ml of [ 3H]arginine, 100 yCi per ml of [ 3H]lysine, and 300 yCi per ml of [ 3 2P]phosphate. Histones were extracted and p u r i f i e d . After reduction and a l k y l a t i o n to convert h i s -tone III dimer to the monomer form, 100 mg of histones were applied to a 3 x 320 cm Bio-Gel P-10 column;. The column was eluted with 0.01 N HCl. The f i r s t 650 ml were discarded and 6 ml fractions were co l l e c t e d thereafter. Bray's s c i n t i l l a t i o n f l u i d i(4 sml)was added to 0.2 ml of every alternate f r a c t i o n which was then analyzed for radio-a c t i v i t y on a s c i n t i l l a t i o n counter. ( ) absorbance at 220 run; (-O—O-) 3 2P; (-A A - ) t r i t i u m counts. (B) Same as A except an unwashed c e l l suspension was incubated 12 hr with 20 yCi per ml of [ 3H]arginine and 200 yCi per ml of t 3 2P]phosphate. No radioactive lysine was added to the suspension and hence the t r i t i u m counts i n l y s i n e - r i c h h i s -tone I are low. 221. e a r l i e r . Candido and Dixon (23) have shown that the ac e t y l -ated histones ( [l "C] acetate p r o f i l e of incorporation) also elute i n a s i m i l a r manner on these columns. In contrast, except for histone I I I , which i s s l i g h t l y retarded, the i n -corporation of 3H coincides with the absorption p r o f i l e . At longer times (several days), the 3H p r o f i l e for histone III coincided with the absorbance p r o f i l e . The proportion of 3 2 P counts i n each of the histone fractions v a r i e s : histone I, I l b i , I I b 2 and IV have a high proportion while histone III has a low proportion of 3 2P counts a f t e r 1 hour of incorporation. However, af t e r 12 hours (Fig. 38B), the proportion of 3 2 P i n I I b 2 and III r e l a t i v e to histone I l b i i s much diminished, suggesting that the pools of phospho-IIb 2 and III are small i n com-parison to that of phospho-IIbi. These small pools e q u i l -ibrate rapidly ( 3 2P incorporation by phosphokinases equal to 3 2 P removal by phosphatases), while the pools of phospho-I, I l b i , and IV continue to increase during the incubation and more 3 2P i s incorporated than removed. (b) I d e n t i f i c a t i o n of Phosphorylated Species of His- tones : Fractions were pooled as indicated i n F i g . 38A, l y o p h i l i z e d , and redissolved i n 0.01 N HCl at a concentra-t i o n of 10 mg per ml. The histone fractions were further separated by starch gel electrophoresis i n the presence of 4 M urea. F i g . 39A to E shows the resolution of each of the histone f r a c t i o n s . 222. Fi g . 39. Starch gel electrophoresis of pooled 3H-and 3 2P-labeled histone fractions from F i g . 38. An aliquot (0.3 to 0.5 mg) of each histone f r a c t i o n was separated by e l e c t r o -phoresis i n starch gels at 7 V per cm for 16 hr i n a water-cooled gel tray. After electrophoresis, the gel was t r i -sected horizontally. The bottom slab was stained with Amido Black 10B and destained with acetic apid. The middle slab was s l i c e d into 1.5 mm s l i c e s , s o l u b i l i z e d , and analyzed for r a d i o a c t i v i t y . (A), histone I; (B), histone I l b i ; (C), histone I I b 2 ; (D), histone III monomer; (E), histone IV. A sample of unreduced whole histone i s shown below (E) to show the r e l a t i v e positions of the various bands. Note that the phosphorylated species migrate much slower than the corres-ponding unmodified histone. For example, phosphohistone IV i s found i n a p o s i t i o n corresponding to unmodified histone I l b i . From the stained gel and 3 2 P counts (Fig. 39A), four phosphorylated species of histone I are found i n trout t e s t i s . Sherod et a l . (160) have also observed a s i m i l a r series of four bands resolved by electrophoresis i n acrylamide gels for extended periods i n l y s i n e - r i c h histone of Ascites c e l l s . Most of the 3H counts are found i n the unmodified histone I band a f t e r 1 hour of labeling (Fig. 39A). This suggests that histone I i s not phosphorylated immediately following i t s syn-thesis . The phosphorylation of histone I l b i has previously been reported by Sung et a l . (134) and the acetylated bands lab-eled Ai and A 2 i n F i g . 39B were described by Candido and Dixon (23) . About 20% of the 3H l a b e l i s found i n the phos-phorylated species of histone I l b i a f t e r a 1 hour labeling period. In PART I of t h i s Thesis (Fig. 10 and 12) the d i s -t r i b u t i o n of H 3 amino acid and 3 2 P l a b e l was examined i n histones extracted from c e l l s on f i l t e r s with acid and sep-arated by starch gel electrophoresis. A peak of 3H l a b e l was found i n a band corresponding to phospho-IIbi. In the studies with unfractionated histone, i t was not possible to determine the proportion of 3H counts that were i n the phosphorylated band of histone I I b a . The results here support the idea that newly-synthesized histone I l b i i s phosphorylated shortly a f t e r i t s synthesis. As yet, t h i s has not been described i n any other system. Alkaline phos-phatase treatment (Fig. 40) of 3H and 3 2 P - l a b e l e d histone l i b a followed by gel electrophoresis and analysis of radio-a c t i v i t y i n the gel shows that the phosphorylated I l b i band, together with a l l the 3H and 3 2 P l a b e l , disappears i n d i c a t -ing that the 3H l a b e l i s indeed i n phospho-IIb a. The two phosphorylated bands i n the histone I I b 2 prep-aration (Fig. 39C) have not been reported previously. The f i r s t band, P i , i s probably phospho-IIb 2; however, i t i s not c l e a r whether the second band, P 2, i s a bona fide phosphoryl-ated component of I I b 2 since phospho-III overlaps i n t o the I I b 2 region following chromatography on Bio-Gel P-10. In histone III (Fig. 39D) there i s a series of phosphorylated bands (PAo, PAi, and PA 2) corresponding to the acetylated series described by Candido and Dixon (23). The small peak of t r i t i u m l a b e l i n the region of PA2 i n F i g . 39D comprises a s i g n i f i c a n t proportion (^25%) of the l a b e l i n the histone III f r a c t i o n at 10 min and 1 hr, but a f t e r longer periods of incubation, i t i s much diminished or absent. At present, i t s nature and r e l a t i o n s h i p to histone III are unknown. The phosphorylated and acetylated species of histone IV have been described by Sung and Dixon (22) and Candido and Dixon (69). In F i g . 39E, most of the t r i t i u m l a b e l i s i n the d l a c e t y l histone IV band (A 2) and not i n the unmodified histone IV. This finding p a r t i a l l y explains the anomaly i n l a b e l i n g of histone IV which we observed i n PART I (Fig. 10 225. 1 5 0 0 -1 0 0 0 n 5 0 0 E e E Q. O OL IOOO-1 5 0 0 5 0 0 -II II ( A ) - phospho-IIbi 1 • (B). r~h—an ^rrT L - 1 5 0 0 •1000 • o 5 0 0 = E E in \ E Q. O X IO 2 4 0 0 1 6 0 0 - 8 0 0 8 .5 10.0 11.5 13.0 14.5 + DISTANCE (cm) F i g . 40. Dephosphorylation of phospho-IIbi by alkaline phosphatase. Histone I l b i was prepared from testes lab-eled i n vivo for 4 hr with [ 3H]arginine and lysine and t 3 2P]phosphate. A sample (^250 yg) of histone I l b i was incubated with 25 yg of E. c o l i a l k a line phosphatase for 3 hr at 37° i n 0.4 M Tris-HCl buffer, pH 8.0. (A) control, no enzyme; (B) alk a l i n e phosphatase treated. Note that a l l of the 3H and 3 2 P counts and protein staining disappear from the phospho-IIbi region. and 12) : [ 3H],arginine and [3H] lysine counts were not found i n the unmodified and monoacetylated histone IV band at 5 and 9 hours a f t e r beginning the c e l l incubations; however, after 19 hr, a s i g n i f i c a n t proportion of counts was found i n the two major bands of histone IV. I t was speculated that the l a b e l might be i n the phosphorylated or acetylated bands of histone IV. These data suggest that newly synthesized histone IV undergoes an obligatory series of acetylations and deacetylations. (c) Phosphopeptides of the Major Histone Fractions: To examine the phosphopeptides i n each of the d i f f e r e n t histone f r a c t i o n s , 0.5 to 1.0 mg of each pooled f r a c t i o n was digested with dialyzed porcine t r y p s i n and separated by high voltage electrophoresis at pH 6.5. The t r y p t i c phosphopeptides were located by autoradiography and F i g . 41 shows the r e s u l t s . As described by Sung and Dixon (22), one major spot, labeled T i , i s observed i n both histone I l b i and IV, and 2 major spots, Ti and T 2, i n histone I and I I b 2 . In addition to the 2 major spots I ( T i ) j i s Ser(P)-Pro-Lys while (T2)^ i s Lys-Ser(P)-Pro-Lys] i n trout t e s t i s histone I (22,70) , several other radioactive bands are observed; they may represent the other s i t e s of phosphorylation i n histone I, but these phosphopeptides have not yet been characterized. In r a t l i v e r histone I, Langan (74,75) has observed phos-phorylation at two other s i t e s , at s e r y l residue 38 by a 32p,* e F i g . 41. Tryptic phosphopeptides from the major histone fractions. Histones labeled for 4 hr i n vivo with 3 2 P i were fractionated by Bio-Gel P-10 chromatography. A pattern si m i l a r to that i n F i g . 1A was obtained and the major histone fractions were pooled and l y o p h i l i z e d . A portion (0.5 to 1 mg) of each f r a c t i o n was digested with a 1:50 r a t i o (w/w) of porcine trypsin for 2 hr at 38°. Tryptic phospho-peptides were located by autoradiography a f t e r high v o l t -age electrophoresis at pH 6.5 according to the method of Sung and Dixon (22). Lane (A), histone 1; (B) histone I l b i ; (C) histone I I b 2 ; (D) histone I I I ; (E) histone IV. PHENOL RED 228. c y c l i c AMP-dependent histone kinase I and at s e r y l residue 106 by a second histone kinase II which i s unaffected by c y c l i c AMP. In F i g . 41, two major t r y p t i c phosphopeptides labeled ( T O J J J and ^ " ^ m a r e f ° u n d i n histone I I I . Cross-contamination of phosphopeptides i s evident i n histone I l b i and IIb2, and I I I . As mentioned above, because of the s l i g h t cation exchange e f f e c t of Bio-Gel, phospho-IIb2 elutes be-tween I l b i and I I b 2 while phospho-III elutes between IIb2 and I I I . In addition, histone III dimer (unreduced) chrom-atographs i n the I l b i and IIb2 region. These factors make i t d i f f i c u l t to p u r i f y phospho-IIb 2 and III by Bio-Gel P-10 chromatography alone. Thus Ti and T 2 from phospho-IIb2 are found i n the histone I l b j and III samples, while Ti and T 2 from phospho-III cross-contaminate the I l b i and IIb2 samples. Cross-contamination can be eliminated largely by pooling a smaller number of fractions from the column. (d) Proportions of Phosphorylated Histone Species: I t should be noted from the stained gels of the various histone fractions that i n hormonally induced trout t e s t i s at a stage active i n histone synthesis, the enzymatically modified species comprise a s i g n i f i c a n t proportion of each f r a c t i o n . As des-cribed i n PART I, spermatogenesis induced by salmon p i t u i t a r y extracts i s completed i n approximately 2% months while sperm-atogenesis i n naturally maturing f i s h takes about 6 to 7 months (Fig. 14). In both cases, the testes reach the same maximal s i z e (about 10 gm) and produce the same number of sperm (approximately 10 1 1 sperm). This means that spermatogenesis i s s u b s t a n t i a l l y accelerated and we observe consistently that the levels of phosphorylated histone are much higher i n hor-monally induced testes. Thus, i n F i g . 2 of reference 23, (resolution on starch gels of histones from naturally matur-ing t e s t e s ) , the level s of phosphorylated histone I l b i , I I b 2 , III monomer, and IV are sub s t a n t i a l l y lower than those levels reported here i n hormonally induced testes. As judged by the i n t e n s i t y of protein staining i n F i g . 39A to E, the phos-phorylated species of histones constitute about 40 to 50% of histone I, 15 to 20% of I l b i , 5% of IIb 2 and I I I , and 30 to 40% of histone IV. These differences i n levels of phos-phorylation may r e s u l t from (a) differences i n rates of phosphorylation and dephosphorylation, (b) differences i n the period that a p a r t i c u l a r histone species would remain phosphorylated ("transit time"), and (c) the metabolic state of the ti s s u e , that i s , the proportion of c e l l s i n the tissue active i n synthesis and phosphorylation of histones. This t h i r d factor i s probably the major one i n determining the differences i n l e v e l of phosphorylation between hormonally induced and naturally maturing trout t e s t i s since the hor-monally induced tissue has a higher proportion of c e l l s active i n histone synthesis and phosphorylation (PART I ) . The d i f f e r e n t l e v e l s of phosphohistone species i n rapidly 230. di v i d i n g c e l l s have not been reported previously; they indicate that phosphorylation of each histone f r a c t i o n may have separate functions other than, or i n addition to, the usually postulated one of d i r e c t gene derepression. There i s l i t t l e evidence of obligatory histone phosphorylation a f t e r synthesis, but the p a r a l l e l rates of histone synthesis and phosphorylation (Table VIII) together with the high levels of phosphorylated h i s -tone species i n trout t e s t i s suggest that for histones I, I l b i , and IV, newly synthesized molecules are phosphorylated and dephosphorylated at le a s t once during the c e l l cycle. Neither histone I (Fig. 39A) nor histone IV (Fig. 39E) are phosphorylated shortly a f t e r synthesis. The phosphorylation of newly synthesized histone I l b i may be involved i n i t s binding to chromatin. 231. I I . Synthesis, Acetylation, and Phosphorylation of Histone IV. In the previous section, we noted that after a 1 hr l a b e l -ing period, most of the 3H amino acid l a b e l i n histone IV was found i n the d i a c e t y l (A 2) species. In this section, the fate of newly synthesized histone IV i s investigated further. (a) Fate of Newly Synthesized Histone IV; C e l l suspen-sions were prepared and incubated with a mixture of [ 3H]arginine and ly s i n e and inorganic [ 3 2P]phosphate for various times. At the end of each incubation, histones were extracted with acid and p u r i f i e d . Histone IV was resolved by chromatography on long Bio-Gel P-10 columns as before. A sample of p u r i f i e d histone IV from each incubation was subjected to gel electro-phoresis. The gel was stained and analyzed for r a d i o a c t i v i t y as i n the previous section. F i g . 42A to D shows the 3H and 3 2P l a b e l i n histone IV resolved by gel electrophoresis and the corresponding stained starch g e l . In the i n i t i a l 4 hr, (Fig. 42A,B, and C) most of the 3H l a b e l i n histone IV appears i n the d i a c e t y l derivative (A 2) with less i n the mono (Ai) and t r i ( A 3 ) acetyl derivatives; very l i t t l e r a d i o a c t i v i t y i s found i n the unsubstituted h i s -tone IV band (Ao). After 12 hr of la b e l i n g (Fig. 42D) a larger proportion of arginine label i s found i n A 3 and Ai» than i n the i n i t i a l 4 hr. In addition, the proportion of l a b e l i n A.i, one of the two major bands of histone IV (as judged by protein s t a i n i n g ) , has increased s i g n i f i c a n t l y . 232. A 8 Aj F i g . 42. Incorporation of radioactive isotope into histone IV as a function of time. Trout t e s t i s c e l l s were incubated with [3H]amino acids and inorganic [ 3 2P]phosphate. At various times, the incubations were stopped, histones were extracted and histone IV was p u r i f i e d on Bio-Gel P-10 columns as i n F i g . 38 and resolved by gel electrophoresis as i n F i g . 39. (A), (B), and (C) are respectively 10 min, 1 hr, and 4 hr labeling with 100 yCi per ml of [ 3H]arginine, 100 yCi per ml of [3H] lysine, and 300 yCi per ml of [ 3 2P] phosphate; (D), 12 hr labeling with 20 yCi per ml of [3H] arginine and 100 yCi per ml of [ 3 2P]phosphate. The blurred region s l i g h t l y ahead of unsubstituted histone IV (Ao) i n (A) i s degraded histone IV. Degradation as judged by the presence of t h i s faster running material, sometimes occurs during repeated column chromatography or during prolonged storage at -20° of frozen aqueous samples. The enzymatic modifications of histone IV appear to occur at a r e l a t i v e l y slow rate (Fig. 42). Therefore the patterns of labeling were examined a f t e r extended periods of time. A group of rainbow trout at a stage of development when the testes are active i n histone synthesis was labeled i n vivo by a single i n t r a p e r i t o n e a l i n j e c t i o n of [3H] arginine and [3H] lysine i n t o each f i s h . At various times a f t e r i n j e c t i o n , a f i s h was s a c r i f i c e d , the testes were excised, and histone IV was p u r i f i e d . The results of the i n vivo labeling are shown i n F i g . 4 3 . After 16 hr (2/3 day) (Fig. 4 3 A ) there i s an appreciable pro-portion of H 3 l a b e l i n A* and A 0. In the ensuing days, the proportion of l a b e l i n A<», A 3 , and A 2 decreases while that i n Ai and A 0 increases (Fig. 43B,C,D and E). In F i g . 42, very l i t t l e amino acid l a b e l was found i n the phosphorylated bands of histone IV. However, 16 hr af t e r the s t a r t of l a b e l i n g (Fig. 4 3 A ) about 20% of the 3H l a b e l - i s i n the 2 major phosphorylated histone IV bands, A 0 P 1 and A 1 P 1 . The proportion i s higher (about 25 to 35%) i n the 1 , 3 , and 8 day samples and gradually decreases (20% a f t e r 12 days and 14% a f t e r 16 days) . From these experiments i t appears that very shortly af t e r histone IV i s synthesized, i t i s rapidly and o b l i g a t o r i l y acetyl-ated (to A 2 ) . Following t h i s i n i t i a l rapid acetylation, acetyl-ation continues (at a slower rate) to A 3 and A*. Acetylated F i g . 43. Injection of lab e l into trout and the fate of i n vivo labeled histone IV. Rainbow trout were injected i n t r a p e r i t o n e a l l y with a mixture of 500 yCi of [ 3H]arginine and 500 yCi of r H ] l y s i n e . At various times, a f i s h was s a c r i f i c e d and a c e l l suspension prepared from the excised testes. In some cases, c e l l suspensions were also incubated for 3 hours with 300 yCi per ml of inorganic [ 3 2P]phosphate to l a b e l phosphorylated histones. Histones were extracted and histone IV was separated and analyzed as i n F i g . 39. (A) , -ff day labeling; (B) , 1 day; (C) , 3 days; (D) , 8 days; (E), 12 days; and (F), 16 days. 235. histone IV i s then slowly and progressively deacetylated to Ai and A Q . The acetylation and deacetylation of a newly syn-thesized histone IV molecule take about a day. These data are consistent with a model i n which newly synthesized histone IV molecules pass through, i n sequence, various acetylated "pools" of histone IV i n which molecules i n the acetylation pathway are recognizably d i f f e r e n t from molecules i n the de-acetylation pathway. I t appears that the cycles of acetylation and deacetylation, and phosphorylation and dephosphorylation of histone IV take place simultaneously but not necessarily oh the same molecule. I f s i g n i f i c a n t phosphorylation and acetyl-ation occurred simultaneously on the same molecule, the pattern of 3H label i n the phosphorylated series of histone IV (A^Piito A 0 P 1 ) would be s i m i l a r to that i n the unphosphorylated series (A^ to Ao) for labe l i n g periods of less than one day (Fig. 42, and F i g . 4 3 A and B). In fa c t , the two patterns are d i f f e r e n t for these early times suggesting that phosphorylation does not occur on most histone IV molecules u n t i l they have gone through the series of acetylations (to Ai») and deacetylation to Ai or Ao . (b) B i o l o g i c a l Function of Acetylation and Deacetylation of  Histone IV: When DNA i s repl i c a t e d i n the S phase, the t o t a l amount of the histones must also be doubled. Since histone synthesis takes place i n the cytoplasm (54), the question arises of how the newly synthesized histones become bound to DNA. The studies described i n PART I on trout t e s t i s c e l l s separated by 236. sedimentation have shown that there i s a p o s i t i v e correla-t i o n of histone synthesis and phosphorylation with DNA syn-th e s i s ; c e l l s which synthesize DNA at d i f f e r e n t rates also synthesize and phosphorylate histones at proportional rates. In addition, studies of the c e l l cycle i n Chinese hamster c e l l s i n culture (161) indicate that histone acetylation occurs i n the S phase during the period of histone synthesis and that histone deacetylation takes place l a t e i n the same phase before mitosis. I t i s proposed here that those modifications occurring shortly a f t e r histone synthesis are involved i n the correct binding of some, of the histones to DNA i n chromatin. The evidence i s not strong that p a r t i c u l a r histones bind s p e c i f i c -a l l y to selected regions of DNA, but the work of Itzhaki (48) and Clark and Felsenfeld (49) on the binding of polylysine and the e f f e c t of deoxyribonucleases on chromatin indicates that although as much as 50% of the DNA phosphates are "free", few extensive tracts of free DNA e x i s t . Various workers (162,163) have shown that chromatin i s not uniformly covered by histones or other chromosomal proteins. These data imply some spec-i f i c i t y i n binding of histones to DNA. In the case of histone IV, precise s p e c i f i c i t y i n function of i t s various regions i s also implied i n the extreme conservation of i t s amino acid sequence during evolution (29,32). The basic regions of most of the histones are asymmetrically d i s t r i b u t e d , with c l u s t e r i n g of basic residues and hydrophobic 237. and a c i d i c residues i n d i f f e r e n t parts of the molecules (26-29). In histone IV, the NH 2-terminal region i s r i c h i n arginyl and l y s y l residues (29). The s i t e s of enzymatic acetylation and phosphorylation are also l o c a l i z e d i n thi s region (22,69). When t h i s evidence i s combined with the observations here of the obligatory passage of newly synthesized histone IV mol-ecules through pools of acetylated intermediates there i s a clear i n d i c a t i o n that post-synthetic modification of histone IV by acetylation and deacetylation may be involved i n some phase of the formation of new DNA-histone complexes. Since the binding of the polycationic histone to poly-anionic DNA i s very strong (3,14,46), any incorrect i n t e r -actions once established might be very d i f f i c u l t to reverse. Therefore, the decrease i n the p o s i t i v e charge density at the NH 2-terminus of histone IV due to the acetylation of the e-amino groups of l y s y l residues 5,8,12 and 16 (ref. 69) might serve an important function i n the formation of the correct complex with DNA. F i r s t , any l y s y l residues i n thi s region that had formed inco r r e c t interactions could be acetylated and inc o r r e c t i n t e r -actions eliminated. Thus, the chromatin-bound histone acetyl-ases (164,165) and deacetylases (166) would be acting as "editing" enzymes for detecting and reversing i n c o r r e c t i o n i c i n t e r a c t i o n s . For t h i s function i t i s unnecessary to postulate a p a r t i c u l a r conformation of the NH 2-terminal region of h i s -238. tone IV (or of the DNA binding s i t e s of the histones), merely that the i n t e r a c t i o n between each histone and DNA i s a d e f i n i t e , non-random one. However, there have been more s p e c i f i c pro-posals that histones bind i n the major groove of DNA (51) by i': t h e i r basic regions through i o n i c interactions (22,30,37,41) with the negatively charged DNA phosphates. In no case, how-ever, i s the conformation of a histone, or i t s complex with DNA, known. A possible model for the binding of histone IV has been proposed (22,37) i n which the f i r s t 18 residues from the NH2-terminus are i n an o(-helical conformation i n the major groove of DNA and the p o s i t i v e charges of the four l y s y i residues, 5,8,12*and 16, bind to a series of four phosphates on one strand of DNA. Combined acetylation of s p e c i f i c e-amino-l y s y l groups and phosphorylation of the NH 2-terminal s e r y l hydroxyl group i n the C<-helical portion was suggested (22) as a mechanism for loosening t h i s region, thus providing a mech-anism for the modulation of histone binding to DNA and perhaps removal of histones during spermiogenesis when they are re-placed by the protamines (22,109). I f the basic NH 2-terminal region of histone IV i n an o( - h e l i c a l conformation were involved i n DNA binding (22,37), i t seems l i k e l y that the negative charges of DNA would strongly s t a b i l i z e t h i s conformation once the complex had formed. How-ever, Boublik et a l . (45) estimated the p r o b a b i l i t y of OV-helical 239. formation i n various regions of histone IV i n solution by arranging the known sequences according to the h e l i c a l wheel conformation of S c h i f f e r and Edmundson (167). Using Prothero's rule (168), they came to the conclusion that the hydrophobic portion of the molecule (residues 55 to 72) had the highest p o t e n t i a l for h e l i x formation while the most basic regions (residues 1 to 36 and 91 to 102) had the lowest due to charge repulsion between the p o s i t i v e l y charged l y s y l and a r g i n y l residues. When histone IV was induced to become more cV-helical by an increase i n the s a l t concentration, high resolution NMR spectroscopy indicated, i n f a c t , that the prediction of l i t t l e cX-helix formation i n the basic regions of free histone IV was j u s t i f i e d (45). Thus, the spontaneous formation of a s i g n i f i c a n t f r a c t i o n of h e l i x i n the NH 2-terminal region of free, unmodified histone IV seems un l i k e l y . However, the enzymatic acetylation of l y s y l residues 5,8, 12 and 16 would greatly increase the p r o b a b i l i t y of h e l i x formation i n t h i s region since the main obstacle to h e l i x formation, namely, charge repulsion of the c a t i o n i c l y s y l residues, would disappear. Thus, the observation i n F i g . 42 and 43 that newly synthesized histone IV must pass through a series of acetylated forms i s c e r t a i n l y consistent with the idea that these modifications are necessary to enable a par-t i c u l a r conformation, s p e c i f i c a l l y i n t h i s case an cX h e l i x , to form p r i o r to i t s correct binding to the major groove of DNA. Sequential deacetylation could then "lock" histone IV in t o place by regenerating the p o s i t i v e charges of lysines 5,8,12, and 16 and allowing i o n i c interactions with four DNA phosphates. This i s outlined schematically i n F i g . 44. I t takes about 1 week for trout t e s t i s c e l l s to complete the c e l l cycle (chromosome r e p l i c a t i o n and c e l l d i v i s i o n ) . Thus the long period observed here for acetylation, phos-phorylation and removal of these modifying groups from histone IV i s not disproportionate to the length of the trout t e s t i s c e l l c y c l e . In c e l l s with a rapid generation time ('vl day) , such as mammalian c e l l s i n tissue culture, one would predict that the cycles of acetylation and deacetylation, and phos-phorylation and dephosphorylation would be considerably accel-erated. CYTOPLASM FIG. 44- A s c h e m e f o r t h e p o s s i b l e b i n d i n g of t h e b a s i c N H 2 -t e r m i n a l r e g i o n of n e w l y s y n t h e s i z e d h i s t o n e I V i n t h e m a j o r g r o o v e o f D N A . F o r c o n v e n i e n c e , o n l y t h e N H ^ - t e r m i n a l p o r t i o n of h i s t o n e I V i n v o l v e d w i t h D N A b i n d i n g £22,37)is s h o w n . H o w t h e r e m a i n d e r o f t h e m o l e c u l e i n t e r a c t s w i t h D N A a n d o t h e r m o l e c u l e s , s u c h as a c i d i c c h r o m o s o m a l p r o t e i n s a n d o t h e r h i s -t o n e s , is n o t k n o w n . D N A s y n t h e s i s (a) i n t h e n u c l e u s is a c c o m -p a n i e d b y h i s t o n e s y n t h e s i s (b) i n t h e c y t o p l a s m . A l t h o u g h l i t t l e is k n o w n o f t h e p a r t i t i o n of h i s t o n e s d u r i n g D N A s y n t h e s i s , i t i s a s s u m e d h e r e t h a t " o l d " h i s t o n e s , s t i l l c o r r e c t l y b o u n d t o D N A , s e g r e g a t e r a n d o m l y (a) t o e a c h d a u g h t e r h e l i x . A n e w l y s y n -t h e s i z e d h i s t o n e I V c h a i n e n t e r s (c) t h e n u c l e u s a n d b i n d s ( d ) i n a r a n d o m f a s h i o n t h r o u g h i o n i c l i n k a g e s t o D N A p h o s p h a t e s o f t h e b a r e s e c t i o n o f D N A . A c e t y l a s e s , w h i c h a c t as " e d i t i n g " e n -z y m e s , d e t e c t t h e i n c o r r e c t l y b o u n d h i s t o n e a n d a e e t y l a t e (e) t h e e - N H 2 g r o u p s o f s p e c i f i c l y s y l r e s i d u e s i n t h e N H r t e r m i n a l r e g i o n of h i s t o n e I V . T h i s d e c r e a s e s t h e i r i o n i c i n t e r a c t i o n s w i t h t h e D N A p h o s p h a t e s a n d a l l o w s t h e n e u t r a l i z e d N E b - t e r m i n a l r e g i o n t o a s s u m e t h e c o r r e c t c o n f o r m a t i o n ( i n t h i s c a s e a n a - h e l i c a l o n e ) a n d fit i n t o t h e m a j o r g r o o v e of D N A . D e a c e t y l a s e s , w h i c h a l s o f u n c t i o n as " e d i t i n g " e n z y m e s , r e m o v e (/) t h e a c e t y l g r o u p s t o u n m a s k t h e p o s i t i v e l y c h a r g e d e - a m i n o - l y s y l g r o u p s . T h i s " l o c k s " t h e N H r t e r m i n a l r e g i o n o f h i s t o n e I V i n i t s c o r r e c t D N A - b i n d i n g c o n f o r m a t i o n t h r o u g h i o n i c i n t e r a c t i o n s w i t h D N A p h o s p h a t e s . A t a l a t e r s t a g e , p h o s p h o r y l a t i o n (g) a n d d e p h o s p h o r y l a t i o n (h) o f h i s t o n e I V o c c u r s . T h e f u n c t i o n s o f p h o s p h o r y l a t i o n a n d d e -p h o s p h o r y l a t i o n a r e n o t k n o w n , a l t h o u g h t h e y m a y b e r e l a t e d t o t h e e x p a n s i o n a n d c o n t r a c t i o n of c h r o m a t i n d u r i n g m i t o s i s a n d m e i o s i s . 242. I I I . Overall Kinetics of Phosphorylation and Dephosphorylation of Histones and Their Possible Role i n Determining Chromosomal Structure When the phosphorylated histones i n each of the major fractions were resolved from unphosphorylated species by starch gel electrophoresis (Fig. 39), about 40 to 50% of histone I, 15 to 20% of histone I l b i ; 5% of histones I I b 2 and I I I , and 30 to 35% of histone IV were found i n the phos-phorylated state. Such extensive phosphorylation of histones suggests that there i s some role for phosphorylation other than, or i n addition to, the usually postulated one of d i r e c t gene dere-pression (65,74). I t was previously suggested (22,109) that the phosphorylation of histones observed i n trout t e s t i s was involved i n the loss of histones i n spermatids and t h e i r replacement by protamines. However, most of the phosphoryla-t i o n occurs i n spermatogonia and primary spermatocytes under-going rapid DNA synthesis; c e l l s which synthesized histones also appeared to phosphorylate histones at proportionate rates (PART I) . In t h i s section, the phosphorylation and dephosphorylation of histone I, l i b i , and IV are compared and the possible roles that these modifications may have i n modulating the binding of histones to DNA and the structure of chromosomes are discussed. 243. (a) Histone I; F i g . 45 shows the resolution of histone I on the starch gels and the p r o f i l e s of 3H incorporation at various times aft e r i n i t i a t i o n of labeling with amino acids. The four phosphorylated bands labeled P i , P 2 , P a , and Pi» (Fig. 45C), are due presumably to the presence of 1 to 4 phos-phoryl groups on the same molecule which has the e f f e c t of decreasing i t s net p o s i t i v e charge and retarding i t s mobility i n four steps, as compared to unmodified histone I. These bands decrease and the 3 2 P l a b e l disappears a f t e r treatment with a l k a l i n e phosphatase. From the 3H p r o f i l e s of F i g . 45, i t i s apparent that at the early times (10 min to 4 hr) newly synthesized histone I i s e s s e n t i a l l y a l l i n the unmodified form, but by 16 hr (2/3 day), a s i g n i f i c a n t f r a c t i o n has been phos-phorylated. The proportion of 3H l a b e l i n Pa and Pi, increases (1 and 3 days) then decreases dramatically (8 days). However, at 12 and 16 days, the proportion of 3H l a b e l i n the phos-phorylated species i s again very high. These changing patterns of 3H l a b e l following incorporation of [ 3H]arginine and lysine i n t o histone I show that a series of sequential phosphorylations (1 to 3 days) and dephosphorylations (5 to 8 days) take place i n trout t e s t i s and that t h i s sequence of events i s repeated (8 to 16 days). The l a b e l i n g following a s i n g l e i n t r a p e r i t o n e a l i n j e c -t i o n of 3H amino acids i s e s s e n t i a l l y that of a "pulse" of durational day (PART I) during which most of the injected 244. H DISTANCE (cm) along GEL F i g . 45. Starch gel electrophoresis and analysis of radio-a c t i v i t y i n histone I. Histone I was prepared from i n t a c t c e l l s labeled for various times i n suspension or i n vivo following i n j e c t i o n of [ 3H]arginine and lys i n e . 0.2 to 0.3 mg of histone was separated by electrophoresis, stained, and analyzed for r a d i o a c t i v i t y as i n F i g . 39. (A) and (B) are histone I preparations labeled for 10 min and 4 hr re-spectively i n c e l l suspensions with 100 yCi per ml each of [ H]arginine and [ 3H]lysine, and 300 yCi per ml of [ 3 2P] phosphate; (C) -§ day, (D) 1 day, (E) 3 days, (F) 5 days, (G) 8 days, (H) 12 days, and (I) 16 days afte r i n j e c t i o n of 500 yCi each of [3H] arginine and [3H] lysine into f i s h . In (C), (E), and (H), the c e l l suspensions prepared from i n vivo labeled testes were further incubated with 300 yCi per ml of 3 2 P^ for 3 hr to la b e l phosphorylated histones. In frame (C), P i , P 2, P 3 , and Pu are the four phosphorylated species of histone I. Gels i n (A) and (B) were stained with 0.125% Amido Black i n 1% acetic acid containing cobalt n i t r a t e , destained with 0.5 N HaSO* and then further destained with 1% acetic acid. Gels i n (C to I) were stained with a 1% solution of Amido Black i n acetic acid and destained with 1% acetic acid. The phosphorylated histone I bands stain less intensely with the d i l u t e (0.125%) Amido Black solu-t i o n . The unsubstituted band of histone I migrated about 14 cm i n a l l cases. However, the phosphorylated bands are more cl e a r l y resolved i n (C to I) than i n (A) and (B). These differences i n resolution are due to d i f f e r e n t preparations of starch gel buffer. Degradation of histones as judged by the diffuse staining material migrating s l i g h t l y faster than the unsubstituted band of histone I sometimes occurs during prolonged storage of repeated column chromatography. 245. l a b e l i s incorporated into protein or otherwise metabolised. In addition, the average generation time for trout testes c e l l s i s about 1 week (PART I ) . Thus, the 3H l a b e l i n the phosphorylated species of histone I at days 12 and 16 indicates that histone I synthesized i n the parent c e l l and par t i t i o n e d to the daughter c e l l s i s phosphorylated once more. These data show unequivocally that both "old" and "new" histone I under-go extensive phosphorylation and dephosphorylation during the c e l l c y c l e . (b) Histone I l b i ; I t was thus of i n t e r e s t to see whether other histones i n the same tissue undergo a s i m i l a r cycle of phosphorylation and dephosphorylation which i s repeated i n the succeeding c e l l cycle. F i g . 46A to F, shows the resolution of histone I l b i on starch gels and the p r o f i l e of incorporated 3H at various times aft e r i n i t i a t i o n of labeling with [3H] arginine and l y s i n e . In contrast to histone I, histone I l b i i s phosphorylated very shortly a f t e r i t s synthesis. At 10 min, 1 hr, and 4 hours, 5,18, and 25% respectively of the t o t a l 3H incorporated into histone Ilb . i i s i n the phosphorylated species of Ilb . i (Fig. 46A to C) . After longer periods of i n -cubation, the proportion of 3H i n phospho-IIbi decreases (Fig. 46E and F). (c) Comparison of Histone I, I l b i , and IV: The other histone f r a c t i o n which undergoes extensive phosphorylation i n 10.0 11.5 13.0 145 10.0 H\5 I3X> 145 + DISTANCE (cm) along GEL *> F i g . 46. Starch gel electrophoresis and analysis of radio-a c t i v i t y i n histone I l b i . Histone I l b i (0.3 to 0.5 mg) was separated by starch gel electrophoresis. (A), (B), and (C) are 10 min, 1 hr, and 4 hr labeling respectively with 100 yCi per ml each of [ 3H]arginine and [3H] l y s i n e , and 300 yCi D e r ml of 3 2P, . (D) , (E) , and (F) are preparations of I l b i 3 -, 8-, and 16 days after the i n j e c t i o n of [ 3H]arginine and lysin e into f i s h . (D) has also been labeled with 3 2P as described i n Fig . 45. The differences i n resolution of phospho-IIbi from unsubstituted I l b i i n (A to C) and (D to F) are due to d i f f e r e n t preparations of gel buffer. After 16 days, appreciable 3H la b e l i s found i n the mono-acetylated I l b i region as judged by the shoulder of 3H la b e l i n (F). This may be due to acetylation of "old" histone I l b i i n spermatids (146) since i t takes about 2 weeks for primary spermatocytes to undergo meiosis giving r i s e to spermatids (PART I) I t i s d i f f i c u l t to say whether acetylation of histone I l b i occurs shortly a f t e r synthesis as the resolution of acetylated from unsubstituted I l b i i s poorer i n (A to C). 247. trout testes c e l l s i s histone IV. The k i n e t i c s of enzymatic modification of histone IV have been described (Fig. 42 and 43), but no conclusion was possible regarding the function of i t s phosphorylation and dephosphorylation. In F i g . 47 a com-parison i s made of the f r a c t i o n of 3H la b e l i n the phos-phorylated species of histone I, I l b i , and IV with time. The levels of phosphohistone at each time point were determined from histones prepared from the same testes. Several conclusions can be drawn from F i g . 47: (a) the d i s t r i b u t i o n of 3H l a b e l i n each of the phosphorylated species of histone i s time dependent; (b) as much as 30 to 50% of the 3H l a b e l can be found i n the phosphorylated species; and (c) the k i n e t i c s of phosphate turnover are d i f f e r e n t for each of the three histones. The r i s i n g slopes represent the rates of phosphorylation of the p a r t i c u l a r histone and are equated to the formation of a 3H-labeled phosphohistone pool. The peak or plateau represents either (a) an equilibrium between con-tinued phosphorylation and dephosphorylation of labeled histone or (b) phosphorylation followed by an i n t e r v a l (equal to the breadth of the plateau) before dephosphorylation occurs. The time during which phosphorylated species remains i n that state due to (a), (b), or both i s defined here as i t s phosphorylation " t r a n s i t time". The f a l l i n g slopes would then represent greater dephosphorylation of the 3H-labeled phosphohistone pool than phosphorylation of labeled dephosphohistones. 248. C O C o o. CO o to CO u w CO 0. TIME (days) F i g . 47. Overall k i n e t i c s of phosphorylation and dephos-phorylation of histone I, I l b i , and IV i n trout t e s t i s . *H-label i n the phosphorylated and unmodified species of histones was integrated from the p r o f i l e s of r a d i o a c t i v i t y i n F i g . 42, 43, 45, and 46, and the percentage of 3H-label i n the phosphorylated histone species was plotted with time, The values at each time were obtained from histones pre-pared from the same p a i r of labeled testes or c e l l sus-pension, except for the two points for histone I at 3 days which were obtained from two d i f f e r e n t incubations. 249. Histone I and IV resemble each other i n having a lag period before phosphorylation begins as well as a long t r a n s i t time. After several days, the 3H la b e l i n the phosphohistone IV pool reaches a maximum and slowly declines. Unlike histone I, however, the dephosphorylation of histone IV proceeds at a r e l a t i v e l y slower rate and the cycle of phosphorylation and dephosphorylation i s not repeated i n the next generation of c e l l s . The NH 2-terminal regions of histone I l b i and IV show extensive sequence homology, a high proportion of basic res-idues, and a common s i t e for phosphorylation (Fig. 1): thus, t r y p t i c digests of histone I l b i and IV yielded a single phospho-peptide, N-acetyl-Ser(P)-Gly-Arg (22). Despite t h e i r sequence homology and i d e n t i c a l s i t e s of phosphorylation, histone I l b i , but not IV, i s phosphorylated almost immediately a f t e r i t s synthesis. The 3H-labeled phosphohistone I l b i pool builds up r a p i d l y , reaches an equilibrium l e v e l of 30% between phos-phorylation and dephosphorylation, and declines at f i r s t rapidly and then more slowly to a l e v e l of ^10%. (d) B i o l o g i c a l Role of Histone Phosphorylation: The present data suggest that the phosphorylation and dephos-phorylation of these three histones may serve quite d i f f e r e n t functions. For histone I l b i , the rapid phosphorylation shortly a f t e r synthesis, the short t r a n s i t time, and r e l a t i v e l y rapid rate of dephosphorylation suggest that phosphorylation may be important i n the binding of histone I l b i v i a i t s basic NH2-terminal region to DNA. I have suggested (Fig. 44) that the obligatory and se-quential acetylation of the NH 2-terminal region of histone IV might allow t h i s region to form and h e l i x and f i t into the major groove of DNA. Subsequent deacetylation by histone deacetylases, which would recognize the NH 2-terminal region of histone IV i n the correct conformation, would expose the p o s i t i v e charges of the l y s y l residues and lock t h i s region i n the correct conformation to DNA by i o n i c linkages. The extensive sequence homology i n the NH 2-terminal region of histone I l b i and IV suggests that t h i s region of histone I l b i may also form an ©< h e l i x and f i t into the major groove of DNA. Construction of a CPK molecular model of the Non-terminal segment of I l b i i n an c X - h e l i c a l conformation showed that i t could indeed f i t into the major groove with l y s y l residues 5,9,13, and arginyl residue 17 binding to a series of DNA phosphates on one side and a r g i n y l residues 3,11,17, and l y s y l residue 15 binding to a series of DNA phosphates on the other side of the major groove of the DNA double h e l i x (Fig. 48). A possible mechanism of binding of the NH2-terminal region of histone I l b i to DNA i s compared with that for histone IV i n F i g . 49. These two histones which may bind i n a s i m i l a r conformation ( o<s helix) to DNA may a t t a i n i t by apparently d i f f e r e n t mechanisms. 251. Fi g . 48. The NH 2-terminal sequence of histone I l b i written i n the form of a h e l i c a l wheel and the binding of th i s region to DNA. (A), the l i n e a r sequence of the NH 2-terminal region of histone I l b i from trout t e s t i s (Fig. 1) was plotted i n the form of a h e l i c a l wheel, a convenient method proposed by S c h i f f e r and Edmundson (167) for representing the r e l a t i o n -ship of the side chains of the amino acid residues i n the <=< - h e l i c a l conformation of a protein. On the outer per-iphery of the wheel, the positions of the known enzymatic modifications of t h i s portion of the sequence of I l b i are shown: phosphorylation of s e r y l residue 1 reported by Sung and Dixon (22) and acetylation of l y s y l residue 5 by Candido and Dixon(33),A molecular model of the NH 2-terminal region i n an ^ - h e l i c a l conformation i s shown i n (B), while one turn of the DNA double h e l i x i n the B conformation i s shown i n (C). (D) shows the proposed binding of the NH 2-terminal region of I l b i i n the ^ - h e l i c a l conformation through i o n i c interactions with the phosphates of DNA: l y s y l residues 5,9,13, and arginyl residue 7 are on one side of the major groove of DNA while arginyl 3,11, l y s y l 15, and ar g i n y l 20 are on the other side of the major groove. 252. • Acetyloted Lys F i g . 49. Comparison of the possible mechanism of binding of newly synthesized histone I l b i and IV to DNA. DNA syn-thesis (a) with the formation of two daughter helices i n the nucleus i s accompanied by histone synthesis (b) i n the cytoplasm. Newly synthesized chains of histone I l b i and IV enter (c) the nucleus and bind (d) i n a random manner through i o n i c linkages to the naked stretches of DNA double h e l i x . For convenience, only the NH 2-terminal regions of the proteins are shown. In histone IV, acetylation of l y s y l residues 5, 8,12, and 16 removes i o n i c interactions and allows the form-ation of an o( h e l i x which can f i t (e) int o the major groove of DNA. Deacetylases (f) remove the acetyl groups from h i s -tone IV i n the correct conformation thus exposing the posi -t i v e charges of the l y s y l residues and "locking" the Non-terminal region to DNA. Phosphorylation (g) of histone IV can then occur. In the case of histone I l b i , phosphorylation of the hydroxy1 group of NH 2-terminal N-acetyl serine (and perhaps acetylation of l y s y l residue 5) may i n i t i a t e the form-ation of 2% to 3 turns of o< h e l i x and p u l l the rest of the NH 2-terminal region into the correct conformation ( e 1 ) . Phos-phorylases (and deacetylases) could then remove (fj) the mod-i f y i n g groups. I t should be noted that although the Non-terminal region of histone I l b i (or IV for the matter) i n an ocT-helical conformation can f i t p r e c i s e l y into the major-groove of DNA with the appropriate i o n i c i n t e r a c t i o n s , i t does not prove that t h i s conformation exists on DNA i n vivo. The important point i s that, whatever the conformation, a s p e c i f i c one i s recognized by the phosphokinases (acetylases) and phos-phatases (deacetylases) involved, such-that newly synthesized histone i n c o r r e c t l y bound to chromatin i s d i f f e r e n t i a t e d from "old" histone c o r r e c t l y bound. 253. In contrast to histone l i b * , histone IV i s phosphorylated aft e r i t s correct binding to DNA has been achieved. The i n i t i a l k i n e t i c s of phosphorylation of both histone I and IV are very s i m i l a r : both are phosphorylated a f t e r an i n i t i a l l a g, both have a long t r a n s i t time, and the levels of phosphorylation are high. As much as 40 to 50% of newly synthesized histone I and 35% of histone IV can be found i n the phosphorylated state (Fig. 47). In f a c t , because phosphorylation and dephos-phorylation are competing yet continuous processes, these l e v e l s represent minimal estimates and suggest that more than 50% of the newly synthesized histone I and IV molecules under-go phosphorylation at some stage of the c e l l cycle. In addition, the c e l l suspensions used are not synchronous c u l -tures. I f phosphorylation and dephosphorylation of I and IV occurred at s p e c i f i c times of the c e l l cycle, the lack of synchrony would lower the percentage of 3H i n the phosphoryl-ated species and cause the plateau to have a rounded rather than square or trapezoidal top. Thus, the true extent of phosphorylation and dephosphorylation of histone I and IV would be rather higher than that shown i n F i g . 47. From pulse-chase studies the o v e r a l l h a l f - l i f e of 3 2 p -labeled histone phosphate i s 6 to 8 hr i n trout testes (Fig. 13). Thus, the maintenance of the high l e v e l of phosphorylated species of histone I and IV for long periods implies that phosphorylation and dephosphorylation of these histones i s a continuous balanced process rather than an i n i t i a l rapid phosphorylation followed by a long stay i n the phosphorylated state before dephosphorylation occurs. These data suggest that phosphorylation and dephosphorylation may have an active and a major role i n determining the structure of chromosomes i n the c e l l . CONCLUDING REMARKS Modifications of histones by acetylation of s p e c i f i c l y s y l residues and phosphorylation of s p e c i f i c s e r y l residues have been reported i n various t i s s u e s . Clues to the possible b i o l o g i c a l function of these modifications have been gleaned by correlating the acetylation and phosphorylation of h i s -tones with the metabolic state of the c e l l or ti s s u e , and with the u l t r a s t r u c t u r a l morphology of the nucleus. Thin sections (169,170) of interphase nuclei reveal chromatin i n two states: d i f f u s e euchromatin with chromosomal o f i b r e s 'vlOO A i n diameter, and condensed heterochromatin with o chromosomal fibres 200 to 300 A i n diameter. As much as 80% of the chromatin i n lymphocytes i s aggregated in t o hetero-chromatin (169). The dif f u s e euchromatin, which i s the most active i n RNA (169) and DNA (169,171) synthesis, i s also the most active i n histone acetylation (172) , while the condensed chromatin i s r e l a t i v e l y i n a c t i v e i n a l l these respects. I t has been reported that much of the acetylation occurs i n the boundary of condensed and dif f u s e chromatin i n c a l f thymus nuc l e i (173). Acetylation of Histones: Post-synthetic acetylation of s l i g h t l y - l y s i n e - r i c h ( I l b i and Ilbz) and arginine-rich (III and IV) histones occurs on the e-NH2 group of s p e c i f i c l y s y l residues (23,27,28,33,69). These modifications are indepen-dent of protein synthesis as judged by continued acetylation of histones i n the presence of i n h i b i t o r s of protein syn-thesis (66,69). This i s i n contrast to the acetylation of the NH 2-terminal of histone I, I l b i , and IV which i s connected with the synthesis of these histones on ribosomes (66,174). I t has been postulated that the acetylation of "old" histones i s somehow connected with the ac t i v a t i o n of the genome and expression of genetic a c t i v i t y (175,176). This hypothesis i s supported by the following observations. The increase i n RNA synthesis i n cultured lymphocytes aft e r addition of mitogenic agents such as phytohemagglutinin i s p a r a l l e l e d by increased acetylation of the arginine-rich h i s -tones (72). On the other hand, a decrease i n RNA synthesis i s observed i n polymorphonuclear leucocytes following adminis-t r a t i o n of phytohemagglutinin, and t h i s i s p a r a l l e l e d by a decrease i n acetylation of the arginine-rich histones (72). Administration of cortisone to rats stimulates new RNA syn-thesis i n the l i v e r and also increases the acetylation of histones (175). There i s also an increase i n acetylation of histones i n rat l i v e r shortly a f t e r p a r t i a l hepatectomy, the peak i n histone acetylation (4 hr) preceding a peak i n RNA synthesis (6 h r ) . The increased acetylation i s accompanied by a decrease i n rate of deacetylation of histones as com-pared to control sham-operated rats (176). In general, the flow of genetic information i s from DNA to RNA to protein. The acetylation of old histone s l i g h t l y 257. before or during RNA synthesis suggests, but by no means proves, that acetylation may play a role i n events leading to the t r a n s c r i p t i o n a l processes (176). In fact, i n a well documented instance of l o c a l i z e d expression of genetic a c t i v i t y , puffing of polytene chromosomes, appreciable acetylation of histones, as measured by 3H-acetate l a b e l -ing, was not observed (80), although i t could be argued that acetylation of histones was already complete before puffing. Candido and Dixon (23,33,69) have shown that post-synthetic acetylations of histone occur i n developing trout testes. Acetylation occurs i n both large c e l l s synthesizing histones and DNA, and spermatids which have ceased DNA and histone synthesis (146). These workers have elegantly characterized the molecular s i t e s of acetylation i n histones I l b i , n b 2 , I I I , and IV. In a l l 4 histones, multiple s i t e s of acetylation occur i n the highly basic NH 2-terminal portions of the mol-ecules. The results i n PART III of t h i s Thesis have shown conclusively for the f i r s t time that shortly af t e r histone IV i s synthesized, i t undergoes an obligatory and sequential series of acetylations and deacetylations which may allow newly synthesized histone IV to assume the correct conforma-t i o n for binding to DNA. Since acetylation of histones continues i n the presence of i n h i b i t o r s of protein synthesis (66,72), i t appears that at least 2 pools of histone IV which can be acetylated e x i s t i n trout t e s t i s . One of these comprises newly syn-thesized histone IV i n c e l l s synthesizing histone and DNA, the other "old" histone IV i n spermatids (146). Candido and Dixon have suggested that removal of old histones i n sperm-atids by acetylation may mirror the binding of newly syn-thesized histone to DNA (146). Data supporting the idea that acetylation of histones may be involved i n the binding of newly synthesized histones to chromatin come' from studies of synchronized Chinese hamster c e l l s i n culture. Shepherd et a l . (161) have shown that the acetate contents of the arginine- and s l i g h t l y -l y s i n e - r i c h histones rose rapidly to a maximum late i n S phase coincident with the termination of DNA (and histone) synthesis and declined rapidly thereafter. However, the question was not resolved whether acetylation occurred on "new" histones synthesized i n the current c e l l cycle or "old" histones synthesized i n previous c e l l cycles. The data on the fate of newly synthesized histone IV i n trout t e s t i s are the f i r s t to show that acetylation can occur on new histone. We do not know how long after synthesis the other histones i n trout t e s t i s are acetylated and deacetylated. I f as i n histone IV, the acetylations and deacetylations of these other histones occurred . shortly a f t e r t h e i r synthesis, i t would be conceivable that these modifications might be involved i n the correct binding of these histones to DNA. I t seems clear, though, that acetylation and deacetyla-t i o n of s p e c i f i c e-NH2 groups of l y s y l residues may occur at d i f f e r e n t times (implying d i f f e r e n t functions) during the " l i f e " of a p a r t i c u l a r histone molecule. These can be l i s t e d as follows. (A) acetylation shortly a f t e r histone synthesis may be required for the proper binding of new histone to DNA; (B) acetylation during spermiogenesis may be required for the removal of histones from DNA; and (C) acetylation of old histones may occur i n non-dividing c e l l s stimulated by hormones or other agents. The functions of acetylation i n the l a t t e r case are not precisely known but i t may be involved i n the events leading to unmasking of the genome and hence synthesis of new RNA species (176). Phosphorylation of Histones: Like acetylation, i t appears that phosphorylation of the hydroxyl group of s p e c i f i c s e r y l residues may occur at various times i n the " l i f e " of a par-t i c u l a r histone molecule or species. Increased phosphoryla-t i o n of histone I has been reported during the S phase of the c e l l cycle (177,178), following stimulation of lympho-cytes with phytohemagglutinin (179), and at various times af t e r p a r t i a l hepatectomy i n rat l i v e r (178). In regener-ating rat l i v e r , increased phosphorylation of histones I, I l b i , I I b 2 , and IV occurs i n the f i r s t 6 hours aft e r p a r t i a l hepatectomy before appreciably synthesis of histones and DNA takes place (178). However, a l l histones appear to be phos-phorylated during the S phase (16 to 25 hr) i n regenerating rat l i v e r (178). Extensive phosphorylation i n trout testes of a l l 5 major histone species, I, I l b i , Ilbz , I I I , and IV was reported by Sung and Dixon (22). The view had been expressed that t h i s phosphorylation may be involved i n the removal of histones during spermiogenesis (16,22,105). In PART I of this Thesis, data were presented i n d i c a t i n g that s i g n i f i c a n t phosphorylation of histones was not involved i n the loss of histones from chromatin i n spermatids. Rather, phosphorylation occurred i n large c e l l s synthesizing both histones and DNA. Phosphorylation of histones occurs after completion of the newly synthesized protein chain as judged by undiminished phosphorylation i n the presence of i n h i b i t o r s of protein synthesis (72) . During induction of synthesis of new enzymes i n rat l i v e r by the hormones glucagon and i n s u l i n , the phosphorylation of a s p e c i f i c s e r y l residue increases 15 to 25 f o l d over control values (74,75). Since phosphohistones have a decreased a f f i n i t y for DNA (134) , and phosphorylation i s often accom-panied by increased RNA synthesis (75), Langan has proposed that this process may be important i n gene derepression (74,75). 261. The s i t e s of enzymatic phosphorylation of the d i f f e r e n t histone species have been determined by Langan (74,75), Sung (71) , and Sung and Dixon (22,70) . A single s i t e of phos-phorylation has been l o c a l i z e d i n the NH 2-terminal region of histones I l b i , I I b 2 f and IV (22), while preliminary data characterizing the phosphopeptides i n histone III (not presented i n t h i s thesis) suggest that i n trout t e s t i s , s e r y l residue 10 may be the s i t e of phosphorylation. Thus, l i k e acetylation, phosphorylation of the arginine- and s l i g h t l y - l y s i n e - r i c h histones occurs i n the highly basic NH 2-terminal region. Four phosphorylated species of h i s -tone I have been found i n trout testes (PART III) and Ascites tumor c e l l s i n culture (160). Thus, there appears to be at least 4 s i t e s of phosphorylation i n histone I. Langan (75) has reported that s e r y l residues 38 and 106 are the s i t e s of phosphorylation of histone I i n rat l i v e r . Although several phosphopeptides have been characterized (22), the s i t e s of phosphorylation of histone I i n trout t e s t i s are s t i l l unknown. The d i f f e r e n t levels of phospho-histones i n each of the major histone f r a c t i o n s , the phosphorylation of histone I l b i , shortly after i t s synthesis, the lag i n phosphorylation of recently synthesized histone I and IV, and the phosphorylation of both "old" and "new" histone I have not been reported pre-viously. I have suggested that the r e l a t i v e l y rapid phos-phorylation and dephosphorylation of histone I l b i may be involved i n the proper binding of t h i s histone to DNA. A possible "function" of phosphorylation of histone I and IV w i l l be described l a t e r . Although high levels of 3H amino acid label were ob-served i n the phosphorylated species of histone I, I l b i and IV at various times af t e r the labeling of the proteins, I have not been able to observe appreciable 3H-label i n phospho-IIb2 or I I I . These two histones have a low o v e r a l l l e v e l of phosphorylation (about 5%) i n contrast to the r e l a t i v e l y high levels i n histone I, I l b i , and IV. As mentioned above, the evidence suggests that the s i t e s of phosphorylation of histone IIb2 and III are i n the highly basic NH2-terminal region [seryl residue 5 i n I I b 2 (71) and s e r y l residue 10 i n I I I ] , and i t i s conceivable that phosphorylation and de-phosphorylation of these two histones may occur very shortly a f t e r t h e i r synthesis. If so, these processes could be related to the binding of these histones to DNA. Ultrastructure of Chromosomes; The molecular structure of chromosomes has been the object of intense study i n recent years and the f i e l d has been reviewed extensively (180-182). Although d i f f e r e n t workers in t e r p r e t the folding of chromo-somes from electron microscopic data i n various ways (180), the o v e r a l l concensus i s that the basic deoxyribonucleohis-o tone (DNH) f i b r e i n interphase c e l l s i s ^10 0 A i n diameter and results from the f i r s t - o r d e r c o i l i n g of a thinner f i b r e o ° (30 to 40 A diameter), the extended form of DNH. The 100 A f i b r e i n turn can be c o i l e d by a process of second-order c o i l -o ing into a 200 to 300 A f i b r e . This folding or c o i l i n g i s necessary f o r packaging a single long molecule of double h e l i c a l DNA i n a compact form and may represent a form of coarse control of genetic a c t i v i t y . Quantitative electron microscopic data have been obtained for the d i f f e r e n t chromosomal fibres (182,183). DuPraw and Bahr (44) interpret the data as follows. Chromosomal fibres e x i s t i n two forms, A and B. The A f i b r e has an average o diameter of 100 A and i s super c o i l e d , with a DNA packing r a t i o (DNA length/fibre length) of 5 to 10:1, while the B fi b r e s are c o i l e d super c o i l s with an average diameter o of 230 A and DNA packing r a t i o of 50 to 100:1. The smaller A fibres appear to be denser than the larger B f i b r e s ( 44) , and the higher order of c o i l i n g i n the B fibres may be associated with the addition of proteins or other r e l a t i v e l y l i g h t molecules to the B f i b r e s . There i s a v a r i a t i o n i n the diameter of the two f i b r e forms, and the exact degree of DNA packing d i f f e r s from f i b r e to f i b r e , from interphase to metaphase, from metaphase to metaphase, and from chromosome to chromosome within the same metaphase plate (44). I t would appear that chromosomes are not well defined molecular e n t i t i e s with reproducible compositions and properties. The 100 A fibres are c o i l s of a single t h i n thread of deoxyribonucleoprotein as judged by digestion of chromosomal threads with proteases (183,184) and electron microscopic observations of stretched chromosomal fibres which show thi n o o ^30 A threads merging into thicker 100 A fibres (185). During mitosis, the interphase chromosomal fibres con-dense into metaphase chromosomes, with a very high DNA pack-ing r a t i o , sometimes exceeding 100:1 (44). The condensed metaphase chromosomes i n addition, have a much larger com-plement of non-histone proteins than interphase chromosomes (180,181). X-ray d i f f r a c t i o n studies of i s o l a t e d nucleohistones (186-188) and i n t a c t n u c l e i (189) reveal a r e l a t i v e l y d i f f u s e d i f f r a c t i o n pattern with a series of low-angle rings corres-o ponding to r e f l e c t i o n s at 110, 55, 37, and 27 A which are not seen i n d i f f r a c t i o n patterns of DNA. Fourier transforms have been calculated for a h e l i c a l DNH complex; these suggest o o a "super c o i l " with diameter 100 A and pitch 120 A (187,188). A simple c a l c u l a t i o n shows that a super c o i l of such dimen-sions would have a DNA packing r a t i o of 3:1. Bram and Ris (185) interpreted t h e i r electron microscopic and low-angle X-ray scatter results as due to a t h i n f i b r e c o i l e d or folded o o to make a thicker f i b e r of diameter 100 A and p i t c h 45 A. Again, a simple c a l c u l a t i o n shows that the DNA packing r a t i o o of a f i b r e of diameter 30 A super c o i l e d into a f i b r e of diameter 100 A and pitch 45 A would have a DNA packing r a t i o of 7:1. I t seems clear then, that a variable DNA packing r a t i o can be obtained by varying the p i t c h of the superc o i l . The factors involved i n determining the diameter of the super c o i l e d f i b r e , the p i t c h , and hence DNA packing r a t i o of the f i b r e are unknown. Annealing of Histones to DNA and P a r t i a l Nucleohistones: The highly conserved primary structure of the histones and t h e i r presence on DNA i n stoichiometric amounts suggest that the histones may have an important s t r u c t u r a l role i n chromosomes (12,14,30) . Attempts have been made to elucidate the s t r u c t u r a l requirements for super c o i l i n g and the role of each i n d i v i d -ual histone i n the structure of chromosomes. P a r t i a l nucleo-histones are prepared by se l e c t i v e extraction of histones from native nucleohistone with d i l u t e acid solutions or solutions of increasing concentration of s a l t (14). Histone-nucleates can be prepared by d i r e c t mixing of histones with DNA i n phy s i o l o g i c a l saline (.15 M NaCl) or "annealing" histones to DNA by mixing the two at high s a l t concentrations (2 M NaCl) and di a l y z i n g to form the DNA-histone complex (14) The annealing of histones to DNA i s a cooperative proces with p r e f e r e n t i a l binding of histones to s i t e s adjacent to other histones (94) . This has been shown by "melting" the complexes by gradually increasing the temperature of the s o l -ution and following the hyperchromicity of the unfolding DNA double h e l i x . Biphasic melting p r o f i l e s with a lower T?M c h a r a c t e r i s t i c of free DNA and a higher T M c h a r a c t e r i s t i c of completely complexed DNA were obtained. In addition, the p r e c i p i t a t e d histone-nucleate can be separated from uncomplexed DNA by d i f f e r e n t i a l centrifugation. These re s u l t s indicate the cooperative nature of histone binding to DNA (94) The a f f i n i t i e s of the d i f f e r e n t histones for free DNA are not i d e n t i c a l (190). Addition of DNA to an excess of whole histones results i n p r e f e r e n t i a l binding of the arginine and s l i g h t l y - l y s i n e - r i c h histones followed by histone I (190). That i s , DNA does not combine with an excess of histones i n t h e i r "natural" proportions, and caution must be used i n i n t e r preting experiments on "chromatin" r e c o n s t i t u t i o n . The histones are believed to be rather evenly d i s t r i b -uted over chromatin with few stretches of "free DNA" (42, 48,163). Histone I l b i , I I b 2 , I I I , and IV are believed to be more intimately bound to DNA than histone I which i s e a s i l y removed at low s a l t concentrations (3). As judged by i n f r a red, c i r c u l a r dichroism, and deuterium exchange studies (12). 40 to 50% of the conformation of proteins bound to chromatin i s cA h e l i c a l . However, i n free solution or a r t i f i c i a l l y complexed with DNA (52), histone I has l i t t l e tendency to form £X - h e l i c e s . The evidence suggests that i t i s on the surface of the deoxynucleohistone complex i n an extended 1 or random c o i l conformation (12). o As judged by the 110, 55, 37, and 27 A r e f l e c t i o n s attributed to the super c o i l of chromatin, X-ray d i f f r a c t i o n studies have shown that (a) histone I can be removed by extraction with d i l u t e perchloric acid or 1 M NaCl without loss of super c o i l i n g (191); (b) between 60 and 88% of the histone has to be removed before the super structure breaks down (12,191); (c) histone-nucleates prepared by mixing DNA and whole histones under conditions of no s a l t , low s a l t , or high s a l t followed by d i a l y s i s to 0.25 M NaCl possess the "super co i l e d " configuration (188); and (d) the simplest complex which possesses the super c o i l e d configuration i s that of histones I l b i and III with DNA; complexes of histone I with DNA do not possess the super c o i l e d configuration. From the X-ray d i f f r a c t i o n studies described above, i t would appear that (a) histone I i s not involved i n super-c o i l i n g , and (b) the order of the histones along the DNA double h e l i x i n a r t i f i c i a l l y prepared histone-nucleates i s r e l a t i v e l y unimportant i n the c o i l e d super structure. This i s not to say, however, that the order, the proportions, or the conformation of histones on native chromatin are unim-portant for b i o l o g i c a l a c t i v i t y . Interactions of Histones with DNA; The o v e r a l l charge of the nucleohistone f i b r e i s about half that of free DNA (14). As many as 50% of the DNA phosphoryl groups are "free" and about 38% of the free phosphoryl groups are present i n f a i r l y long stretches (47). These stretches are "covered" and not t r u l y free zones of DNA (48,162,163). Electrometric and spectrophotometry-titration studies of histone and nucleohistone (192) indicate that a l l the carboxyl, imidazole, and t y r o s y l groups of nucleohistone are available for t i t r a t i o n , while 80% of the l y s y l and arginyl residues are masked or complexed with the phosphoryl groups of DNA. Not a l l of the phosphoryl groups of DNA are neutralized by the basic residues of the histone moieties. This i s e v i -dent from the stoichiometry of histone binding to DNA (30). The 5 major histone fractions are present i n nucleohistone i n approximately equal proportions. The r a t i o of histone to DNA i n chromatin i s as high as 1.25 to 1.3 to 1. We know the molecular weights of a l l the major histone f r a c t i o n s , and the dimensions of the DNA double h e l i x . I f the "inner" h i s -tones, I l b i , I I b 2 , I I I , and IV were evenly d i s t r i b u t e d over the DNA double h e l i x , the histones would be closely packed with each histone occupying 20 to 25 base pairs of DNA double h e l i x , or approximately 2 to 2h dyads of the double h e l i x (30). There would be 40 to 50 DNA phosphoryl groups for each inner histone. Since there are 25 to 30 ar g i n y l and l y s y l residues per "inner" histone, and 80% of these groups in t e r a c t i o n i c a l l y with the DNA phosphates (19 2), 20 to 25 (about half) of the DNA phosphoryl groups would be "free" and neutralized by small counter ions. In f a c t , for every 100 phosphoryl groups from nucleohistone i s o l a t e d i n the presence of Na +, there are 30 to 40 sodium ions (193j,194) . Since the minor groove of DNA i s free (50,51), i t i s pre-sumed that the "inner" histones occupy the major groove of DNA. The extended length of histone I l b i , H b 2 , I I I , and IV O O would be about 350 to 500 A (3.4 A per amino acid residue). Assuming no overlapping of the inner histones along the nucleohistone complex, the inner histones must be i n a com-pact conformation. The length of the major groove i n a segment of DNA double h e l i x consisting of 20 to 25 base pairs o o i s 140 to 175 A (7 A between DNA phosphates). I f histones I l b i , I I b 2 , III and IV were completely CX h e l i c a l , t h e i r o o h e l i c a l length would be between 150 to 225 A (1.5 A per amino acid residue i n the o<helix) (30). Although the exact percentage of ^ - h e l i c a l conformation i n each of the histone species on chromatin i s not known, i t would seem that close packing of the histones along DNA with minimal overlapping would be favoured by high contents of h e l i x . As mentioned i n PART III of t h i s Thesis, NMR studies indicate that i n free solution, the basic regions of the h i s -tone have the lowest tendency to form ordered structures while the hydrophobic regions have a high p o t e n t i a l for o{ h e l i x formation (12,45). However, construction of molecular models 270. of the highly basic NH 2-terminal region of histone IV, ' [Sung and Dixon (22) and Shih and Bonner (37)], I l b i (this Thesis), and I I I (Louie, A.J., not shown), i n the o(- h e l i c a l conformation indicates that these regions can a l l f i t i n the major groove of DNA with the p o s i t i v e charges of a r g i n y l and l y s y l residues opposed to the DNA phosphoryl groups. In histone I I I , p r o l y l residue 16 joins two s t r a i g h t sections of o( h e l i x (residues 1 to 15 and 17 to 26) i n the major groove O f DNA. Evidence was presented i n t h i s Thesis, which suggests that the sequential acetylation of histone IV, and phos-phorylation of histone I l b i may be important i n binding of the NH 2-terminal region of these histones i n proper confor-mation ( s p e c i f i c a l l y an o( helix) to DNA. F i g . 50 shows how the inner histones, I l b i , IIb2, H I , and IV might i n t e r a c t with DNA. I t may be s i g n i f i c a n t that the sum of the lengths of DNA double h e l i x occupied by each of these histones i n sequence i s close to 100 base pairs or 10 dyads of the double h e l i x . This length i s approximately equal to the length of 1 turn of the postulated super c o i l . Provided that the p i t c h i s s u f f i c i e n t l y small, one may envisage a s i t u a t i o n i n which the non-polar regions of the inner histones of one c o i l i n t e r -act with the non-polar regions of the histones i n the next higher (or lower) c o i l . For example, one could speculate that the histone III molecules may be stacked one above the 271. F i g . 50. Possible-interaction of histones with DNA. (A), supercoiled nucleohistone f i b r e of diameter 100 A and v a r i -able p i t c h . A p i t c h of 120 A as suggested by Pardon et a l . (187) would r e s u l t i n a r e l a t i v e l y open structure with a DNA packing r a t i o of 3:1, while a 45 A p i t c h as suggested by Bram and Ris (185) would give a more compact structure with a DNA packing r a t i o of 7:1. The arginine (III and IV) and s l i g h t l y - l y s i n e ( I l b i and IIb 2) r i c h histones are shown i n the major groove of DNA. Four of these c l o s e l y packed span about 100 base pairs or approximatelylcoil of the super c o i l . The l y s i n e - r i c h histone I shown i n the top c o i l o v e r l i e s the other histones and s p i r a l s along the 30 A f i b r e . (B), the major groove of DNA i s shown unfolded. The s l i g h t l y - l y s i n e -r i c h and ar g i n i n e - r i c h histones are shown i n the major .groove. The NH 2-terminal regions of these histones are i n an c ? < -helical conformation. The regions with a larger proportion of a-polar side chains may also be i n an o^-helical conformation, and be eith e r i n the major groove or looped out. Histone I i s shown i n the extended state. I t should be emphasized that (a) the arrangement of histones along the double h e l i x , (b) the regions of histones which are o< h e l i c a l , (c) the arrange-? ment and ro l e of the non-histone chromosomal proteins, and many other aspects of chromosomal structure are completely unknown. 272. other and during c e l l d i v i s i o n , when maximum oxidation of the SH group of histone III has been observed (179), formation of d i s u l f i d e bridges may decrease the pit c h of the super c o i l . Other models suggesting how histones might i n t e r a c t with DNA have been described (30,19 5). The scheme shown for the binding of histones to the major groove of DNA i s s i m i l a r i n proportions to that suggested by P h i l l i p s (30) except that i n F i g . 50, the NH 2-terminal region iscX h e l i c a l rather than an extended peptide chain. In F i g . 50, histone I i s shown overlying four of the other histones. The completely extended length of histone I (^220 amino acid residues) i s about 700 to 800 A. I t may be coin-c i d e n t a l , but the length of the major groove occupied by the o four of the inner histones i s about 700 A. Free histone I has very l i t t l e tendency to form ordered secondary struc-tures such as ( Xhelices (12) , and i t i s shown s p i r a l l i n g i n an extended conformation along the surface of the nucleohistone f i b r i l . Although the sequence of the carboxyl half of h i s -tone I from residues 107 to 216 has not been determined, i t s composition i s such that 80% of the residues are Lys, Pro, and Ala, and t h i s i s the same as the segment from 1 to 40 (52). The centre segment of histone I from residues 40 to 107 contain a large proportion of apolar residues. In the model shown i n F i g . 50, the e-NH2 groups of the l y s y l res-idues i n the NH2- and carboxyl-terminal regions anchor the molecule through i o n i c interactions with DNA phosphate*?. I t should be emphasized that the exact conformation and sequence of histones along the DNA double h e l i x and the i n t e r -actions of the d i f f e r e n t components of chromatin (DNA, h i s -tones, and non-histone chromosomal proteins) are completely unknown. The figure i s an aid i n v i s u a l i z i n g the molecular dimensions of the complex. We do not know to what extent the histone molecules overlap each other, whether the peptide backbone of the histones l i e s e n t i r e l y i n the major groove of DNA, whether histone I binds i n the major or minor groove of DNA, or the length of DNA double h e l i x covered by histone I on chromatin. In spite of the large number of researchers and the d i f f e r e n t experimental approaches taken i n attempts to understand the structure and function of the components of chromosomes, we are very ignorant of the molecular structure of the deoxyribonucleoprotein complex. At a s l i g h t l y higher l e v e l of structure, we are also s t i l l at a loss for a unifying hypothesis which explains (a) the reversible t r a n s i t i o n of chromatin from an active to an i n a c t i v e state, and (b) the condensation of interphase to metaphase chromosomes and expansion of condensed chromosomes during telophase. Phosphorylation of Histone I as a Determinant of Chromosomal  Structure: I t i s proposed here that the d i f f e r e n t physical states of chromatin may be modulated by the phosphorylation 274. and dephosphorylation of certa i n histones. S p e c i f i c a l l y , the extent of phosphorylation of the histone I molecules i n a p a r t i c u l a r stretch of deoxyribonucleoprotein i s proposed to be a major factor i n determining the degree of super c o i l i n g of the chromosomal f i b r i l (Fig. 51). F i b r i l s i n which histone I i s i n the completely phosphorylated state (average 3 to 4 phosphates per molecule of histone I) would be i n the extended form. An intermediate degree of phosphorylation (average 1 to 2 phosphates per molefcule) would cause the extended form to o collapse into the 100 A chromosomal f i b r e s , while a lower l e v e l of phosphorylation (average 0 to 0.5 phosphates per molecule) would allow the formation of a higher order of c o i l i n g , the 200 to 300 A fibres . Histone IIb2 and III show only low levels of phosphoryl-ation (Fig. 39) while, as discussed above, i t appears that the phosphorylation of I l b i i s primarily involved i n the bind-ing of newly synthesized histone l i b * to DNA ( f i g . 49). of the other histones which show high lev e l s of phosphorylated i n t e r -mediates (I and IV), histone I has four possible s i t e s of phosphorylation while histone IV has one. I f the l e v e l of phosphorylation of histones i s important, t h i s suggests that the phosphorylation of histone I might have the greatest e f f e c t i n determining the o v e r a l l structure of chromosomes. How might phosphorylation and dephosphorylation of h i s -tones a f f e c t the structure of chromosomes? The obligatory HISTONE I PHOSPHOKINASES coiled coil 200-300 A fibre 0-0.5 POj/mole coiled 80-100 A fibre 1-2 PO^/mole uncoiled 30-40 A fibre 3-4 PO4 /mole PHOSPHATASES F i g . 5.J,.. Phosphorylation and dephosphorylation of histone I as a determinant of chromosomal c o i l i n g . Chromosomal f i b r e s i n which histone I has a low overall, l e v e l of phosphoryla-t i o n (0 to 0.5 P O i , per mole of histone I) i s 200 to 300 A i n diameter. Phosphorylation of histone I on the surface of the f i b r e s by histone I phosphokinases increases the net negative charge on the surface o f o t h e f i b r e and because of charge repulsion, the 200 to 30g A f i b r e unfolds into a much longer but narrower (80 to 100 A) f i b r e . I f e s s e n t i a l l y a l l the histone I molecules i n the chromosomal f i b r e are exten-s i v e l y phosphorylated (3 £o 4 phosphates per mole of histone I) the extended 30 to 40 A f i b r e i s found. > Removal of phos-phates from histone I by phosphatases reduces the charge repulsion and allows the f i b r e to f o l d or c o i l . Note that c o i l i n g and uncoiling i s reversible depending upon the a c t i v i t y and location o f ohistone I phosphokinases and phos-phatases. The 30 to 40 A f i b r e i s c h a r a c t e r i s t i c of lamp-brush chromosomes and polytene chromosomal puffs; the 80 to 100 A f i b r e i s c h a r a c t e r i s t i c of euchromatin while the 200 to 300 A f i b r e i s c h a r a c t e r i s t i c of heterochromatin and meta-phase chromosomes. and sequential phosphorylation and dephosphorylation of prot-amine i n the spermatid nucleus i s associated with the conden-sation of spermatid chromatin (PART I and II of t h i s Thesis). The appearance of unsubstituted protamine from the dephos-phorylation of phosphoprotamine i n the spermatid i s associated with the appearance of spermatids with half the o r i g i n a l sperm-a t i d volume while the dephosphorylation of the complete com-plement of protamine results i n the condensation of the late spermatid in t o mature spermatozoa with one-quarter the volume of the o r i g i n a l spermatid. The approximate numerical equiva-lence of the arginines i n protamine and the phosphates of DNA (7) suggests that the presence of 1 to 4 phosphates (each with minus 1.7 charge) on protamine i s s u f f i c i e n t to prevent the t i g h t packing of nucleoprotamine. The events leading to nuclear condensation during spermiogenesis i n trout t e s t i s can be interpreted as follows. Chromatin i n early spermatids i s e n t i r e l y nucleohistone. Protamine, synthesized i n the cytoplasm and transported into the nucleus, binds to chromatin. Phosphorylation allows t h i s newly synthesized protamine to bind corr e c t l y to DNA. Histones are removed at t h i s stage. Upon dephosphorylation, the repulsive e f f e c t of the negative charges due to protamine phosphoryl groups d i s -appears, allowing the nucleoprotamine to condense (PART I I ) . In a s i m i l a r way, the introduction into histone I of up to four phosphoryl groups each with a minus 1.7 charge could i n h i b i t the super c o i l i n g or packing of chromosomes. Re-duction of the number of phosphoryl groups i n histone I might then allow the chromosomal f i b r i l to super c o i l (Fig. 51). The diameter of the extended deoxynucleohistone f i b r i l o i s 30 to 40 A (180-182), while the diameter of the DNA double o h e l i x i s about 20 A. Thus the DNA double h e l i x i s covered o by a 5 to 10 A layer of proteins. The DNA phosphoryl groups i n the i n t e r i o r of the nucleoprotein complex would be far from the surface and would not prevent super c o i l i n g by charge repulsion. On the other hand, when histone I i s phosphorylated, the primary and secondary ionizations of the s e r y l phosphate groups would be "fixed" on the surface of histone I and could exert t h e i r f u l l repulsive e f f e c t and prevent the formation of super c o i l s . I t i s also possible that the multiple s i t e s of phosphorylation of histone I could exh i b i t cooperative e f f e c t s s i m i l a r to those observed i n regulatory enzymes. These e f f e c t s could account for the " a l l or none" tr a n s i t i o n s i n chromosomal c o i l i n g . For ex-ample, the dephosphorylation of a number of histone I mol-ecules i n extended chromosomes would lead to a point where there would be a very small energy b a r r i e r to super c o i l -ing and the removal of a few str a t e g i c phosphoryl groups would allow the extended fi b r e s to super c o i l . Since the super c o i l i n g would reduce the surface area of the f i b r i l , further dephosphorylation might be prevented since some 278. phosphorylation s i t e s of histone I molecules would become buried. Several lines of evidence indicate that the external charge of the DNH f i b r i l can regulate the c o i l i n g of chromo-somes. Chelating agents such as EDTA and c i t r a t e cause the 200 to 300 A f i b r i l s to unfold into 100 A f i b r i l s (180). M i l l e r et a l . (196) have shown that a polyanion, polystyrene sulfonate (MW 18,000), decondenses the paternal hetero-chromatic set of mealy bug chromosomes and that t h i s decon-densation increases the a b i l i t y of the paternal chromosomes to incorporate labeled RNA precursors. These observations reinforced e a r l i e r evidence that polyanions may function as derepressors of chromatin (197). An alt e r n a t i v e i n t e r p r e t a t i o n i s that the polyanions might bind to histones on chromatin and the repulsive negative charges on the surface could d i s -rupt the super c o i l i n g of chromosomes leading to increased a c c e s s i b i l i t y i n both decondensed eu- and heterochromatin to RNA polymerase. Whereas anionic agents such as EDTA and c i t r a t e cause condensed chromosomes to expand, divalent cations such as ++ ++ Mg and Ca cause chromosomes to condense and aggregate (169,189,198). Conceivably, the divalent cations might com-plex e f f e c t i v e l y with the exposed phosphoryl groups of histones (histone I i n particular) and any other negative charges on the surface and allow the chromosomes to condense or undergo further c o i l i n g . I t could be argued that since histone I can be removed from nucleohistone without a f f e c t i n g the super c o i l e d struc-ture of the complex, histone I i s not important i n the c o i l -ing of chromosomes. A regular superhelical form may be generated by a system of r e s t r a i n t s arranged both around the DNA molecule and displaced from one another i n a regular manner along the molecule (12). However, the de l i c a t e balance between the extended, super c o i l e d , and c o i l e d super c o i l e d states could be modulated by charge-charge i n t e r a c t i o n s , and the fixed-negative charges of phospho-histone I along the o surface of the 30 A nucleohistone f i b r e could force the balance towards the less folded states. Viewed i n t h i s manner, removal of unmodified histone I [or 70 to 80% of the t o t a l histones (12)] may not s i g n i f i c a n t l y change the charge on o the surface of the c o i l e d 30 A nucleohistone cylinder, and the f i b r e could remain super c o i l e d . Histone I then, i s important i n the c o i l i n g of chromosomes by v i r t u e of i t s capacity to be reversibly charged and discharged by phospho-kinases and phosphatases. The phosphorylation of histone IV may reinforce the e f f e c t of phosphorylation of histone I. Stevely and Stocken (76) have shown that histone I from euchromatin has an average of 1.8 phosphates per mole of histone I (MW ^20,000) while that from heterochromatin has an average of 0.2 phosphates per mole. Thus, differences i n phosphohistone I content do e x i s t between eu- and hetero-chromatin. The hypothesis that the state of phosphorylation of histone I on the surface of the DNA f i b r e might control the order of c o i l i n g implies that the s i t e s of phosphorylation which are buried i n the i n t e r i o r are i n the dephospho form while those on the exterior are phosphorylated. Table XVII shows the r e l a t i v e lengths and c y l i n d r i c a l surface areas of a given amount of (feoxyribonucleohistone i n various orders of c o i l i n g . I f histones are randomly d i s t r i b u t e d over the DNA o double h e l i x , then i n the extended 35 A f i b r i l , a l l the phos-phorylation s i t e s of histone I are on the surface of the ex-tended cylinder. With c o i l i n g and packing of chromosomes, the proportion of the s i t e s on the surface decreases. In the 100 0 o A f i b r i l , approximately 30 to 40% and i n the 200 to 300 A f i b r i l approximately 10 to 15% of the t o t a l s i t e s of the h i s -tone I molecules would be exposed on the surface. I f we assume that 75% of the phosphorylation s i t e s on the surface of the cylinder are phosphorylated (that i s , 3 phosphoryl groups per histone I molecule), then an average molar phos-phate to histone I r a t i o of 3:1, 1.0 to 1.5:1, and 0.3 to o o o 0.4:1 may be calculated for 35 A, 100 A, and 200 to 300 A f i b r i l s , r espectively. These calculations agree reasonably well with the experimentally determined leve l s of phosphoryla-t i o n of histone I eu- and heterochromatin (76). Thus, the high levels (35 to 50%) of phosphorylated species of histone 1 and IV reported here for trout testes c e l l s active i n DNA 281. TABLE XVII Chromosomal f i b r e dimensions and average phosphate content per mole of histone I. Fibre Diameter (A) DNA packing r a t i o Relative f i b r e Dimensions 3 Phosphate content of histone I*3 Volume Length Surface Area 75% 50% 25% Extended 35 1:1 1 1 1 3 2 Coiled 80 5.3:1 1 0.19 0.43 1.3 0.86 100 8:1 1 0.12 0.34 1 0.67 Coiled 230 44:1 1 0.023 0.15 0.45 0.30 0.15 C o i l 300 75:1 1 0.013 0.113 0.33 0.22 0.11 a Assuming that the f i b r e i s a long cylinder. Assuming that only those s i t e s on the surface of the cylinder are phosphorylated. Numbers are computed for 25, 50 and 75% phosphorylation of the s i t e s on the surface. Since there are four s i t e s of phosphorylation i n histone I, the o v e r a l l phosphate content, assuming that 75% of the s i t e s on the surface are phosphorylated, can be obtained simply by mul-t i p l y i n g the surface area by 3. 282. and histone synthesis may r e f l e c t more the a b i l i t y of phospho-histone I to maintain the d i f f u s e state of chromatin rather than derepression of DNA dependent RNA synthesis (75,76). The phosphorylation of histone IV may reinforce the e f f e c t of phosphorylation of histone I. Phosphorylation of the ex-posed NH 2-terminus of histone IV may hinder f i r s t - o r d e r super c o i l i n g . The predicted phosphorylation of histone I on the sur-face of chromosomes may prevent side by side or transverse aggregration of chromosomal f i b r e s . Thus, the aggregration of polytene chromosomal strands (199) may be due to dephos-phorylation of histone I s i t e s on the surface of the i n d i v i d -ual f i b r i l s and the maintenance of polytene chromosomal puffs would be explained by extensive phosphorylation of histone I and possibly IV. The continuous phosphorylation and dephosphorylation of histones I and IV involved i n the long t r a n s i t times mentioned previously suggest that the maintenance of the euchromatic state i s an active metabolic process. Johns has proposed that the extended form of the chromosome i s accessible to the large and bulky RNA polymerase molecule, while the c o i l e d , condensed form i s inaccessible (200). In t h i s respect, reg-ulation of the order of c o i l i n g by phosphokinases and phos-phatases might constitute a coarse control of t r a n s c r i p t i o n , the unfolding of chromosomes being a prerequisite for fine control of t r a n s c r i p t i o n a l a c t i v i t y . Thus f a l t e r i n g the 283. amount or a c t i v i t y of phosphokinases [for example, by c y c l i c AMP (75)] and phosphatases may activate or repress the a c t i v i t y of large or small regions of chromosomes depending upon the presence and a c t i v i t y of these enzymes (75). Chromosomal C o i l i n g During the C e l l Cycle: The postulated role of phosphorylation and dephosphorylation of certa i n histones i n determining the supramolecular structure of chrom-osomes may shed some l i g h t on the regulation of chromosomal structure during the c e l l cycle. I s h a l l f i r s t present a scheme (Fig. 52) and then show that i t i s consistent with experimental evidence. Following c e l l d i v i s i o n , the metaphase chromosomes regroup during telophase and lose a large amount of non-histone protein. Simultaneously a histone I phosphokinase i s activated and phosphorylates histone 1(a). This causes o o the 200 to 300 A f i b r i l s to unfold to 100 A f i b r i l s . Some chromosomes may not be phosphorylated and these would remain heterochromatic throughout much of the c e l l cycle. During Gi, further phosphorylation at selected s i t e s could lead o to the appearance of stretches of extended (30 to 40 A) DNH. These extended regions can then be transcribed and the messenger RNA transported to the cytoplasm where new proteins are synthesized. During S phase, phosphokinase o a c t i v i t y increases (b) and both the 200 to 300 A f i b r i l s o and the 100 A f i b r i l s are converted by phosphorylation of o histone I to 35 A f i b r i l s about the DNA r e p l i c a t i o n fork. ANA-PHASE^ TELO-PHASE PRO-PHASE META-PHASE ANAPHASE CHROMOSOMES c)^^^ proteins COILED COIL COILED COIL COILED COIL p r o t * ' n 8 METAPHASE CHROMOSOMES UNCOILED—i UNCOILED —1 R N A S Y » N -T H E S I S p R 0 T E I N • S Y N T H E S I S F i g . 5%. A scheme for the possible regulation of chromo-somal structure during the c e l l cycle. See text for explanation. The c o i l i n g and uncoiling of chromosomes i s important i n DNA synthesis (chromosomal re p l i c a t i o n ) and RNA synthesis (expression of genetic a c t i v i t y , and i s reg-ulated by the a c t i v i t y , concentration, and location of histone I phosphokinases and phosphatases which act at s p e c i f i c times or l o c i during the c e l l cycle. This phosphorylation may be obligatory for the r e p l i c a t i o n fork to move along the chromosome. DNA i s synthesized (c); newly synthesized histones from the cytoplasm enter the nucleus and bind randomly to the naked stretches of the DNA double h e l i x (d). When the correct conformations of the i n t e r i o r histones ( I l b i , IIb2, III and IV) are achieved (d), histone I can bind on the surface. I f the chromosome i s to remain inactive (heterochromatic chromosomes), phosphorylation of histone I need not occur at t h i s stage. However, i f the newly synthesized portion of the double h e l i x i s part of active chromatin (euchromatin) phosphorylation of newly synthesized histone I would occur (e). To ensure that t h i s euchromatin remains active, i t may be necessary that the newly synthesized histone IV be phosphorylated to reinforce the e f f e c t of that from histone I. Dephosphorylation would cause the extended region to supercoil ( f ) . During prophase, histone phosphorylation decreases. Continued dephosphoryla-t i o n of histone I and IV would then allow extended chromo-o somes to condense, 100 A fibres being converted into 20 0 to o 300 A f i b r i l s (g). Addition of non-histone proteins and removal of residual phosphoryl groups on the exterior of the o 200 to 300 A f i b r i l would lead to the formation of metaphase chromosomes (h). Some evidence for the above scheme follows. Lake (201) has reported that the a c t i v i t y of a histone I phosphokinase i n metaphase chromosomes from cultured mammalian c e l l s i s 3 to 7 f o l d greater than that i n interphase chromatin, and that l a b e l from [ ^ 3 2PJATP could be incorporated i n t o histone I of metaphase chromosomes. Our i n t e r p r e t a t i o n i s that the rapid release of metaphase chromosomes from the t i g h t l y folded conformation may be dependent upon the action of t h i s phospho-kinase which begins phosphorylating histone I while s t i l l i n late metaphase. Removal of non-histone protein during telophase and continued phosphorylation of histone I would release the chromosome from i t s folded state and allow i t to expand and f i l l the nucleus. Ki n e t i c studies have indicated that the phosphorylation of histones not connected with histone synthesis i n regenera-ting rat l i v e r precedes the peak of DNA synthesis (178). In synchronized mammalian c e l l s i n culture, histone I phos-phorylation increases with DNA synthesis i n the S phase (177). Pawse et a l (202) have also shown that the amount and a c t i v i t y of histone I phosphokinase increases 6 to 7 f o l d while the phosphatase a c t i v i t y remains constant during the period of DNA synthesis i n regenerating rat l i v e r . The increase i n phosphatase a c t i v i t y i s due to new enzyme synthesis (202). Evidence has been presented i n t h i s Thesis suggesting that histone I synthesized and phosphorylated i n the preceding c e l l cycle i s again extensively phosphorylated i n the succeed-ing c e l l cycle. In terms of the model, the increase i n "old" histone I phosphorylation p r i o r to DNA synthesis indicates the necessity of uncoiling super c o i l e d regions of chromo-somes i n a d i f f e r e n t i a t e d t i s s u e . In rapidly d i v i d i n g (un-differentiated) c e l l s i n culture, the Gi chromatin would o already be d i f f u s e (10 0 A f i b r i l s ) . During the S phase old histone I phosphorylation i s then necessary to uncoil chromosomes about the DNA r e p l i c a t i o n s i t e while phosphoryla-t i o n of newly synthesized histone I would take place i n order o to maintain the d i f f u s e state (100 A f i b r i l s ) of interphase chromatin. The lag i n the k i n e t i c s of phosphorylation of newly syn-thesized histone I and IV reported here (Fig. 44) suggests that phosphorylation of I and IV does not begin u n t i l these histones are properly bound to chromatin. The correct bind-ing of newly synthesized histone I l b i (by phosphorylation and dephosphorylation) and IV (by acetylation and deacetylation) takes place over a period of 12-24 hours i n trout t e s t i s . As previously mentioned, t h i s period i s not disproportionate to the average length (^ 1 week) of the trout t e s t i s c e l l c ycle. Towards the end of S phase, histone I phosphorylation decreases (177). This presumably r e f l e c t s a decrease i n the l e v e l of a c t i v i t y of the histone I phosphokinase rather than increased a c t i v i t y of phosphatase. Balhorn et a l . (203) have shown that i n regenerating rat l i v e r the l e v e l of phos-phorylated histone I increased with the f i r s t wave of DNA synthesis, decreased, then increased again with the second wave of DNA synthesis. Their results are s i m i l a r to those reported here with trout testes. The decrease i n l e v e l of phosphorylated histone I due to decreased a c t i v i t y of phospho-kinases between periods of DNA synthesis may therefore be related to the condensation of interphase into metaphase chromosomes. The a t t r a c t i v e features of t h i s hypothesis for regulat-ing the c o i l i n g of chromosomes are several. (A), i t i s not necessary to postulate any new or unusual elements for cross-l i n k i n g chromosomal strands which might otherwise be d i f f i c u l t to reverse. (B), the scheme (Fig. 52) sheds l i g h t on hither-to l i t t l e understood phenomena and assigns a role to the puzzling nature of phosphorylation and dephosphorylation of histone I and IV. (C) , the enzymes [phosphokinases (75, 201,202), and phosphatases (75,202)] for phosphorylating and dephosphorylating histones e x i s t and the times at which they are active correlate with the necessary changes i n chromosomal structure. 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