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Biosynthesis of protamine in trout Salmo gairdnerii testis Ling, Victor 1969

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The U n i v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of VICTOR LING B„Sc. (Hon), (Biochemistry) U n i v e r s i t y of Toronto, 1966 FRIDAY, AUGUST 29, 1969, AT 10:00 A.M. IN ROOM 241,'GRADUATE STUDIES NEW ADMINISTRATION BUILDING COMMITTEE IN CHARGE Chairman: j > P > Kutney G.H. Dixon M. Smith G.M. Tener R.A.J.. Warren J.J.R. Campbell E x t e r n a l Examiner: R.D. Cole Department of Biochemistry U n i v e r s i t y of C a l i f o r n i a , Berkeley, C a l i f o r n i a Research Supervisor: G.H. Dixon THE BIOSYNTHESIS OF PROTAMINE IN TROUT (SALMO GAIRDNERII) TESTIS ABSTRACT At a Late stage of spermatogenesis a sperm-s p e c i f i c p r o t e i n , protamine, i s synthesized i n the t e s t i s of salmonid f i s h and p r o g r e s s i v e l y replaces histones i n combination w i t h DNA. Protamine has a molecular weight near 5,000 and contains 2/3 of i t s t o t a l amino a c i d residues as a r g i n i n e . Studies on the b i o s y n t h e s i s of protamine have been made on the rainbow t r o u t (Salmo g a i r d n e r i i ) t e s t i s . Successive column chromatography on B i o -Gel P-10 and CM- c e l l u l o s e has been employed to i s o l a t e and c h a r a c t e r i z e newly synthesized l a b e l l e d protamine. Newly synthesized protamine i s phos-phorylated and i s e l u t e d e a r l i e r from the CM-c e l l u l o s e column than mature protamine. However, the two forms of protamine chromatograph c o i n -c i d e n t a l l y when newly synthesized protamine i s f i r s t t r e a t e d w i t h a l k a l i n e phosphatase. Protamine i s separated i n t o three components on CM-cellulose and the amino a c i d compositions of the components are very s i m i l a r . The r e l a t i v e amounts of the components present i n the t e s t i s n u c l e i are d i f f e r e n t at d i f f e r e n t stages of spermatogenesis and the synthesis of each component appears to be independently c o n t r o l l e d . This suggests that the components, while c h e m i c a l l y very s i m i l a r , are the products of separate s t r u c t u r a l genes and may have d i f f e r e n t f u n c t i o n s . By pulse l a b e l l i n g t e s t i s c e l l suspensions f o r d i f f e r e n t lengths of time and a n a l y z i n g the amount of ^C- protamine found i n the cytoplasm and i n the nucleus, the s i t e of protamine syn t h e s i s can be shown to be i n the cytoplasm. F u r t h e r , a c e l l - f r e e , i s o l a t e d p o s t - m i t o c h o n d r i a l cytoplasmic f r a c t i o n can incorporate l ^ C - a r g i n i n e i n t o whole protamine molecules, while both an i s o l a t e d nuclear f r a c t i o n and high-speed supernatant were r e l a t i v e l y i n a c t i v e . This i n d i c a t e s that protamine synthesis occurs on cytoplasmic microsomes. Sedimentation analys i s of p u l s e - l a b e l l e d t e s t i s r ibososes indicates that protamine is syn-thes ized on a c la s s of small polysomes, the disomes, sedimenting at 120S . While dimeric ribosomes inves t igated in various t i ssues have been shown to be inac t ive ar te fac t s formed during i s o l a t i o n , the disomes in trout t e s t i s have been demonstrated to be a func t iona l c lass of polysomes. They are not d i s soc iab le at 1 mM Mg"1-*" ion concentrat ion , are not the breakdown product of larger polysomes, nor are they produced by i n t e r a c t i o n with free protamine. These disomes conta in the major quant i ty of nascent protamine and increase in number in the t e s t i s c e l l s during the act ive protamine synthes iz ing stage of development. the packaging of DNA into the sperm head. The phosphorylat ion of protamine and the protamine components may serve to regulate th i s packaging process . The p r o b a b l ^ f unct ion of protamine i s for GRADUATE STUDIES F i e l d of Study: Biochemistry Biochemical Methods Structure and Funct ion of Prote Biochemistry of Nucle ic Acids S t a f f (Biochemistry) s G . H . Dixon G.M. Tener J . F . Richards Biochemistry of Amino Ac ids and Prote ins Biochemistry of Carboyhdrates S . H . Zbarsky W . J . Polglase P . D . Bragg G . I . Drummond Biochemistry of L i p i d s Biochemistry of S tero id C . T . Beer G . I . Drummond J . F . Richards V . J . O'Donnel l M. Darrach A . F . Burton T h e o r e t i c a l Organic Chemistry R. Stewart R . E . Pincock D irec ted Studies in Genetics D . T . Suzuki PUBLICATIONS V. L i n g and R.A. .Anwar. On the Presence of Two D i s t i n c t P r o t e o l y t i c Components i n P a n c r e a t i c C r y s t a l l i n e E l a s t a s e , Biochem. Biophys. Res. Comm. 24, 593, 1966. K. Marushige, V. L i n g , B. J e r g i l , and G.H. Dixon. Synthesis and Phosphorylation of Protamine i n Trout T e s t i s , Fed. Proc. 27 336, 1968. V. L i n g & G.H. Dixon. B i o s y n t h e s i s of Protamine i n v i t r o , 51st Annual Conference of the Chemical I n s t i t u t e of Canada, a b s t r a c t , 1968. G.H. Dixon, C.J. I n g l e s , B. J e r g i l , V. L i n g , & K. MaruShig P r o t e i n transformations during d i f f e r e n t i a t i o n of t r o u t t e s t i s , Proceedings of the Eighth Canadian Cancer Conference at Honey Harbour, O n t a r i o , 1968 (Pergamon Press, Canada). V. L i n g , J.R. T r e v i t h i c k , & G.H. Dixon. The b i o s y n t h e s i s of protamine i n t r o u t t e s t i s . I . I n t r a c e l l u l a r s i t e of s y n t h e s i s . Can. J . Biochem. 47, 51, 1969. V. L i n g , G.H. Dixon. P r o t e i n Synthesis by T e s t i s Polysomes Proc. Can. Fed. B i o l . Soc. 12, 83, 1969. K. Marushige, V. L i n g , G.H. Dixon. Phosphorylation of Chromosomal Basic P r o t e i n s i n Maturing Trout T e s t i s . J . B i o l . Chem. In press, 1969. G.H. Dixon, K. Marushige, V. L i n g , M. Sung, & D.T. Wigle. Phosphorylation of Histones i n Maturing Trout T e s t i s . Fed. Proc. 28, 599, 1969. BIOSYNTHESIS OF PROTAMINE IN TROUT (Salmo gairdnerii) TESTIS by VICTOR LING B . S c , U n i v e r s i t y of Toronto, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o lumbia, I a g r e e t h a t 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 r e f e r e n c e and Study. I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g 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 g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r 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 g a i n s h a l l not be a l l o w e d w i t h o u t my wr i t t e n permi ss i on. Department o f *B/Q C M r & M i C f/t Y 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 JUfij- '2r/ /?6f i ABSTRACT The B i o s y n t h e s i s of Protamine i n Trout (Salmo gairdnerii) T e s t i s At a l a t e stage of spermatogenesis a sperm-specific pro-t e i n , protamine, i s synthesized i n the t e s t i s of salmonid f i s h and p r o g r e s s i v e l y r e p l a c e s h i s t o n e s i n combination w i t h DNA. Protamine has a molecular weight near 5,000 and contains 2/3 of i t s t o t a l amino a c i d r e s i d u e s as a r g i n i n e . Studies on the bi o s y n t h e s i s of protamine have been made on the rainbow t r o u t {Salmo gairdnerii) t e s t i s . Successive column chromatography on Bio-Gel P-10 and CM-c e l l u l o s e has been employed t o i s o l a t e and c h a r a c t e r i z e newly synthesized l a b e l l e d protamine. Newly synthesized protamine i s phosphorylated and i s e l u t e d e a r l i e r from the CM-cellulose column than mature protamine. However, the two forms of pro-tamine chromatograph c o i n c i d e n t a l l y when newly synthesized protamine i s f i r s t t r e a t e d w i t h a l k a l i n e phosphatase. Protamine i s separated i n t o three components on CM-cellulose and the amino a c i d compositions of the components are very s i m i -l a r . The r e l a t i v e amounts of the components present i n the t e s t i s n u c l e i are d i f f e r e n t a t d i f f e r e n t stages of spermatogenesis and the s y n t h e s i s of each component appears to be independently c o n t r o l l e d . This suggests t h a t the components, w h i l e c h e m i c a l l y very s i m i l a r , are the products of separate s t r u c t u r a l genes and may have d i f f e r e n t f u n c t i o n s . By p u l s e l a b e l l i n g t e s t i s c e l l suspensions f o r d i f f e r e n t lengths of time and a n a l y z i n g the amount of 1 "^C-protamine found i n the cytoplasm and i n the nucleus, the s i t e of protamine i i s y n t h e s i s can be shown to be i n the cytoplasm. F u r t h e r , a c e l l - f r e e , i s o l a t e d p o s t - m i t o c h o n d r i a l cytoplasmic f r a c t i o n can i n c o r p o r a t e 1 1*C-arginine i n t o whole protamine molecules, w h i l e both an i s o l a t e d nuclear f r a c t i o n and high-speed super-natant were r e l a t i v e l y i n a c t i v e . This i n d i c a t e s t h a t protamine sy n t h e s i s occurs on cytoplasmic microsomes. Sedimentation a n a l y s i s of p u l s e - l a b e l l e d t e s t i s ribosomes i n d i c a t e s t h a t protamine i s synthesized on a c l a s s of small polysomes, the disomes, sedimenting a t 120S. While di m e r i c ribosomes i n v e s t i g a t e d i n v a r i o u s t i s s u e s have been shown to be i n a c t i v e a r t e f a c t s formed duri n g i s o l a t i o n , the disomes i n t r o u t t e s t i s have been demonstrated to be a f u n c t i o n a l c l a s s of polysomes. They are not d i s s o c i a b l e at 1 mM Mg + + i o n concentra-t i o n , are not the breakdown product of l a r g e r polysomes, nor are they produced by i n t e r a c t i o n w i t h f r e e protamine. These disomes c o n t a i n the major q u a n t i t y of nascent protamine and i n c r e a s e i n number i n the t e s t i s c e l l s d u r i n g the a c t i v e pro-tamine s y n t h e s i z i n g stage of development. The probable f u n c t i o n of protamine i s f o r the packaging of DNA i n t o the sperm head. The phosphorylation o f protamine and the protamine components may serve t o r e g u l a t e t h i s packaging process. i i i TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENT i x INTRODUCTION 1 The Process of Spermatogenesis 1 The Chemistry and Biology of Protamine 13 P r o t e i n B i o s y n t h e s i s 24 MATERIALS AND METHODS 35 I. Chemicals and Abb r e v i a t i o n s 35 (a) Chemicals 35 (b) Abbrevi a t i o n s 35 I I . I s o l a t i o n and C h a r a c t e r i z a t i o n of Protamine and Radi o a c t i v e Protamine 36 (a) Source of Protamine 36 (b) C e l l Incubation 38 (c) A c i d E x t r a c t i o n of T e s t i s Basic P r o t e i n s 39 (i) U n l a b e l l e d b a s i c p r o t e i n s 39 ( i i ) L a b e l l e d b a s i c p r o t e i n s 40 (d) Bio-Gel P-10 Chromatography 41 (e) CM-Cellulose Chromatography 42 (f) Amino A c i d A n a l y s i s of F r a c t i o n a t e d Protamine Components 42 (g) Measurement of R a d i o a c t i v i t y 43 (h) D i g e s t i o n of Rad i o a c t i v e Protamine w i t h A l k a l i n e Phosphatase 44 i v I I I . I n t r a c e l l u l a r S i t e o f P r o t a m i n e S y n t h e s i s 44 (a) S e d i m e n t a t i o n A n a l y s i s o f T e s t i s C y t o p l a s m i c and N u c l e a r M i c r o s o m e s 1 44 (b) P u l s e - C h a s e K i n e t i c s o f 1 ^ C - P r o t a m i n e 46 (c) Time C o u r s e o f I n t r a c e l l u l a r D i s t r i b u t i o n o f 1 ^ C - P r o t a m i n e 46 (d) S y n t h e s i s o f P r o t a m i n e i n a C e l l - F r e e S y s t e m 47 ( i ) I n c o r p o r a t i o n o f 1 ^ C - a r g i n i n e i n t o P r o t a m i n e by an i s o l a t e d c y t o p l a s m i c f r a c t i o n 47 ( i i ) D i f f e r e n t i a l i n c o r p o r a t i o n o f l a b e l l e d a r g i -n i n e i n t o p r o t a m i n e by i s o l a t e d c e l l f r a c t i o n s 47 ( i i i ) I n c o r p o r a t i o n o f 3 2 P - p h o s p h a t e and 3H-a r g i n i n e by a c y t o p l a s m i c c e l l - f r e e s y s t e m 48 IV. C h a r a c t e r i z a t i o n o f T r o u t T e s t i s Ribosomes 49 (a) P r e p a r a t i o n and P u r i f i c a t i o n o f Ribosomes 49 (b) S u c r o s e D e n s i t y G r a d i e n t A n a l y s i s o f Ribosomes 50 (c) The T e s t i s P o l y s o m e s A s s o c i a t e d w i t h t h e S y n -t h e s i s o f P r o t a m i n e 51 ( i ) C h a r a c t e r i z a t i o n o f t h e n a s c e n t p r o t e i n s a s s o c i a t e d w i t h t e s t i s p o l y s o m e s 51 ( i i ) N o n - s p e c i f i c b i n d i n g o f 1 ^ C - p r o t a m i n e t o t e s t i s r i b o s o m e s 51 ( i i i ) T i m e - c o u r s e d i s t r i b u t i o n o f 1 ^ C - a r g i n i n e l a b e l l e d p r o t e i n s a s s o c i a t e d w i t h t e s t i s p o l y s o m e s 52 ( i v ) A n a l y s i s o f t e s t i s r i b o s o m e s o b t a i n e d f r o m t h e p o s t - n u c l e a r s u p e r n a t a n t and t h e p o s t -mi t o c h o n d r i a l s u p e r n a t a n t 52 (v) E f f e c t o f low M g + + i o n c o n c e n t r a t i o n 53 ( v i ) E f f e c t o f r i b o n u c l e a s e d i g e s t 54 ( v i i ) D e t e r m i n a t i o n o f t h e s e d i m e n t a t i o n c o e f f i c -i e n t (S) o f t e s t i s r i b o s o m e s 55 (d) D e v e l o p m e n t a l Changes o f t h e T e s t i s P o l y s o m e P o p u l a t i o n 55 V RESULTS 57 I. I s o l a t i o n and C h a r a c t e r i z a t i o n of Protamine and Ra d i o a c t i v e Protamine 57 (a) Bio-Gel P-10 Chromatography of T e s t i s Nuclear B a s i c P r o t e i n s 57 (b) S u l f u r i c A c i d E x t r a c t i o n of T e s t i s Nuclear P r o t e i n s 60 (c) Time-course I n c o r p o r a t i o n of 1 ^ C - A r g i n i n e i n t o Protamine by a T e s t i s C e l l Suspension 63 c(d) Determination of the Stage of T e s t i s Development and the Bio-Gel P-10 Chromatography of Radio-a c t i v e Protamine 63 (e) CM-Cellulose Chromatography of Protamine 68 (f) Changes i n the Chromatographic P r o f i l e s of Protamine During Development 8 2 I I . I n t r a c e l l u l a r S i t e of Protamine Synthesis 87 (a) Sedimentation A n a l y s i s of Cytoplasmic and Nuclear Microsomes 87 (b) Pulse-Chase K i n e t i c s of 1 ^ - P r o t a m i n e 92 (c) Time-Course of I n t r a c e l l u l a r D i s t r i b u t i o n of 1^C-Protamine 94 (d) Protamine Synthesis i n a C e l l - F r e e System 97 I I I . C h a r a c t e r i z a t i o n of Trout T e s t i s Ribosomes 106 (a) The Polysomes Associated w i t h the Synthesis of Protamine 107 (b) Developmental Changes of the T e s t i s Polysome P o p u l a t i o n 120 DISCUSSION 133 FOOTNOTES 155 BIBLIOGRAPHY 157 v i LIST OF TABLES Table Page I E x t r a c t i o n of t e s t i s nuclear p r o t e i n s w i t h 0.2 M E2SOk 62 I I Amino a c i d composition of protamine and protamine components i s o l a t e d from a CM-cellulose column 73 I I I R e l a t i v e i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o pro-tamine by d i f f e r e n t i s o l a t e d c e l l f r a c t i o n s 101 IV C h a r a c t e r i z a t i o n of the nascent peptides a s s o c i a -ted w i t h d i f f e r e n t s i z e s of polysomes 111 V Stages of t e s t i s development i n n a t u r a l l y maturing t r o u t (Salmo gairdnerii) 122 v i i LIST OF FIGURES Figure Page 1. Comparison of amino a c i d sequences of protamines from Clupea pallasii ( P a c i f i c herring) 17 2. Bio-Gel P-10 chromatography of b a s i c p r o t e i n s e x t r a c -ted from t e s t i s n u c l e i a£ d i f f e r e n t stages of spermatogenesis 58 3. E f f i c i e n c y of e x t r a c t i o n f o r protamine by 0.2 M s u l -f u r i c a c i d 61 4. Time-course i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o a c i d -s o l u b l e p r o t e i n s by a t e s t i s c e l l suspension 64 5. Transformation from his t o n e synthesis to protamine sy n t h e s i s i n t r o u t t e s t i s . 66 6. Bio-Gel P-10 chromatography of a mixture of lhC-a r g i n i n e and u n l a b e l l e d t e s t i s a c i d - s o l u b l e p r o t e i n s 69 7. Chromatography of a mixture of 1 ^ C - a r g i n i n e and un-l a b e l l e d protamine on a CM-cellulose column (1 x 30 cm) 70 8. CM-cellulose chromatography of rainbow t r o u t t e s t i s protamine 71 9. Chromatography on CM-cellulose of newly synthesized protamine obtained from a c e l l suspension of n a t u r a l l y maturing t r o u t t e s t i s 76 10. Chromatography on CM-52 of newly synthesized phosphory-l a t e d and e n z y m a t i c a l l y dephosphorylated protamine 79 11. Rate of dephosphorylation of newly synthesized pro-tamine i n t e s t i s n u c l e i 81 12. Changes i n the chromatographic p r o f i l e s of protamine during t e s t i s development 84 13. P r e p a r a t i o n of t e s t i s cytoplasmic and nuclear micro-somes 8 9 14. Sedimentation a n a l y s i s of t e s t i s cytoplasmic and Nuclear microsomes 90 15. Pulse-chase k i n e t i c s of 1 ^ C - a r g i n i n e - l a b e l l e d pro-tamine 93 v i i i 16. Time-course of i n t r a c e l l u l a r d i s t r i b u t i o n of ll*C-a r g i n i n e - l a b e l l e d protamine 95 17. I n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o protamine by an i s o l a t e d cytoplasmic f r a c t i o n 99 18. Procedure f o r the i s o l a t i o n of d i f f e r e n t c e l l f r a c -t i o n s from t r o u t t e s t i s 100 19. C h a r a c t e r i z a t i o n by CM-cellulose chromatography of the m a t e r i a l l a b e l l e d w i t h 1 ^ C - a r g i n i n e by an i s o -l a t e d cytoplasmic f r a c t i o n from t r o u t t e s t i s 103 20. Synthesis and phosphorylation of protamine by an i s o l a t e d cytoplasmic f r a c t i o n 104 21. Sedimentation a n a l y s i s of p u r i f i e d ribosomes ob-t a i n e d from t e s t i s c e l l s pulsed f o r 1 min w i t h 1 ^ C - a r g i n i n e 108 22. Determination of the sedimentation c o e f f i c i e n t (S) of t e s t i s ribosomes 110 23. N o n - s p e c i f i c b i n d i n g of 1 hC-protamine to t e s t i s ribosomes 113 24. Time-course of 1 ^ C - a r g i n i n e i n c o r p o r a t i o n by nascent peptides a s s o c i a t e d w i t h t e s t i s ribosomes 115 25. Sucrose d e n s i t y g r a d i e n t a n a l y s i s of ribosomes obtained from d i f f e r e n t c e l l f r a c t i o n s 117 26. E f f e c t of low Mg + + i o n co n c e n t r a t i o n on t e s t i s disomes 119 27. E f f e c t of RNase on t e s t i s polysomes 121 28. P r o f i l e s of t r o u t t e s t i s polysomes at d i f f e r e n t stages of development 125 29-32. Developmental changes of t e s t i s polysomes 127 29. September t e s t i s 128 30. October t e s t i s 129 31. November t e s t i s 130 32. December t e s t i s 131 33. I n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o protamine i n the presence of actinomycin D 14 4 i x ACKNOWLEDGEMENTS I w i s h t o t h a n k my r e s e a r c h s u p e r v i s o r Dr. G o r d o n H. D i x o n f o r e n t h u s i a s t i c s u p p o r t , v a l u a b l e s u g g e s t i o n s and d i s c u s s i o n s d u r i n g t h e c o u r s e o f t h i s work. My a s s o c i a t i o n s w i t h t h e members o f t h i s l a b o r a t o r y have b e e n most h e l p f u l and I w i s h t o a c k n o w l e d g e t h e d e b t I owe t o them. I n p a r t i c u l a r , I w i s h t o t h a n k D r s . K e i j i M a r u s h i g e , B e n g t J e r g i l and J o h n T r e v i t h i c k whom I c o l l a b o r a t e d w i t h i n a number o f e x p e r i m e n t s . Thanks a r e a l s o due t o Mr. and M r s . Hans Lehmann f o r t h e i r g e n e r o u s d o n a t i o n s o f r a i n b o w t r o u t t e s t e s and t o E f o r h e r h e l p d u r i n g t h e p r e p a r a t i o n o f t h e m a n u s c r i p t f o r t h i s t h e s i s . The f i n a n c i a l s u p p o r t o f t h e N a t i o n a l R e s e a r c h C o u n c i l n o f Canada and t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada i s a p p r e c i a t e d . The r e s e a r c h was s u p p o r t e d by g r a n t s t o Dr. G.H. D i x o n f r o m t h e M e d i c a l R e s e a r c h C o u n c i l o f Canada and t h e F i s h e r i e s R e s e a r c h B o a r d o f Canada. - 1 -INTRODUCTION The Process of Spermatogenesis : The process by which the germ c e l l s (spermatogonia) of the t e s t i s p r o l i f e r a t e and transform i n t o f r e e m o t i l e c e l l s , the spermatozoa, i s known as spermatogenesis. Although there are great d i f f e r e n c e s i n the s i z e , s t r u c t u r e and development of the spermatozoa of higher organisms, (74) spermatozoa are c h a r a c t e r -i z e d by being h a p l o i d , h i g h l y m o t i l e w i t h one or more Ijlagella, and extremely compact c o n t a i n i n g h i g h l y condensed nuclear mater-i a l . Since the time of Antoni van Leeuwenhoek (75) who reported i n 1677 to the Royal Society h i s observations on sperm motion, i n v e s t i g a t o r s have been f a s c i n a t e d by the uniqueness of the spermatozoon. With the a i d of i n c r e a s i n g s o p h i s t i c a t i o n i n the l i g h t microscope and l a t e r the e l e c t r o n microscope, the s t r u c -t u r a l d e t a i l s of the spermatozoa as w e l l as c e l l s a s s o c i a t e d w i t h spermatozoan d i f f e r e n t i a t i o n have been e l u c i d a t e d f o r a wide range of animal (76-78). Once the morphological features of c e l l s i n spermatogenesis were c h a r a c t e r i z e d , i t was p o s s i b l e to c o n s t r u c t the events of spermatogenesis according to the appearance of c e r t a i n c e l l types. In the mammal at l e a s t , (79) the process of spermatogenesis has been d i v i d e d i n t o more than twenty stages c h a r a c t e r i z e d by recognizable c e l l types. However, sin c e the morphological d e t a i l s of these c e l l s vary widely w i t h d i f f e r e n t s p e c i e s , only the general scheme of spermatogenesis as represented by the f o l l o w i n g diagram w i l l be considered. - 2 -m e x o s i s m i t o s i s . s t . . . 1 m e i o t x c SPERMATOGONIA ->PRIMARY > SECONDARY (2n) SPERMATOCYTES (2n) d i v i s i o n SPERMATOCYTES ' 1 2nd m e i o t i c d i v i s i o n s p e r m i o g e n e s i s SPERMATOZOA <-(n) SPERMATIDS (n) The v a s t number o f s p e r m a t o z o a f o r m e d as t h e e n d - p r o d u c t o f s p e r m a t o g e n e s i s a ^ e s t h e r e s u l t o f r e p e a t e d m i t o s e s by t h e sperma-t o g o n i a f o r m i n g a l a r g e number o f p r i m a r y s p e r m a t o c y t e s ; e a c h p r i m a r y s p e r m a t o c y t e t h e n u n d e r g o e s m e i o s i s w h i c h l e a d s v i a two d i p -l o i d (2n) s e c o n d a r y s p e r m a t o c y t e s t o t h e f o r m a t i o n o f f o u r h a p-l o i d (n) s p e r m a t i d s , t h e number o f chromosomes i n e a c h b e i n g r e -d u c e d by h a l f . The s p e r m a t i d c e l l i s t r a n s f o r m e d w i t h o u t f u r t h e r c e l l d i v i s i o n i n t o t h e m a t u r e sperm c e l l by t h e p r o c e s s o f s p e r m i o g e n e s i s . T h i s p r o c e s s i s c h a r a c t e r i z e d by d r a m a t i c c h a n g e s i n c e l l m o r p h o l o g y (80 , 8 1 ) ; f o r example, i n t h e mammal, t h e i d i o s o m e o r G o l g i a p p a r a t u s d e v e l o p s i n t o t h e a c r o s o m e , t h e n u c l e u s c h a n g e s s h a p e , and t o g e t h e r w i t h t h e acrosome f o r m s t h e he a d o f t h e sperm c e l l . The m i t o c h o n d r i a and c e n t r i o l e s b o t h p a r t i c i p a t e i n c o n s t r u c t i n g t h e t a i l . D u r i n g t h i s m a t u r a t i o n p r o c e s s , g r o u p s o f s p e r m a t i d s a r e c o n n e c t e d by b r i d g e s o f c y t o -p l a s m and t h e s p e r m a t i d s t h e m s e l v e s a r e embedded i n l a r g e S e r -t o l i c e l l s c o n s i d e r e d t o be n u t r i t i v e c e l l s ( 8 2 ) . The mode o f a t t a c h m e n t o f t h e s p e r m a t i d c e l l t o t h e S e r t o l i c e l l , t h e mech-a n i s m o f r e l e a s e o f t h e mat u r e s p e r m a t o z o a , and t h e r o l e o f t h e S e r t o l i c e l l a r e n o t s a t i s f a c t o r i l y u n d e r s t o o d . A n o t h e r s t r i k i n g f e a t u r e o f s p e r m i o g e n e s i s i s t h e marked - 3 -r e d u c t i o n i n the amount of cytoplasm i n the d i f f e r e n t i a t i n g sper-matids. This was noted by Caspersson i n 1939 (83) who measured the p r o g r e s s i v e disappearance of r i b o n u c l e i c a c i d from the develop-i n g sperm c e l l by cytochemical and spectrophotometric means. Daoust and Clermont (84) have a l s o shown th a t i n the r a t , the RNA of the spermatid i s c o l l e c t e d i n t o l a r g e r and l a r g e r granules u n t i l they are s e t f r e e as ' r e s i d u a l bodies' s h o r t l y before the r e l e a s e of mature spermatozoa. Lacy (85), studying i n greater d e t a i l these r e s i d u a l bodies i n various stages of t h e i r develop-ment, has noted t h a t i n a d d i t i o n to the l i p i d d r o p l e t s and a mass c o n t a i n i n g numerous RNA p a r t i c l e s , these bodies i n c l u d e some mitochondria which tend to fuse w i t h each other to form membran-ous bodies. The r e s i d u a l bodies a l s o c o n tain G o l g i membranes and v e s i c l e s , and a c o n c e n t r a t i o n of cytoplasmic membranes. C l e a r l y a mechanism e x i s t s f o r the removal of the cytoplasm from the maturing sperm c e l l . The process of spermatogenesis i n the f i s h , i n p a r t i c u l a r the salmon f a m i l y (Salmonidae) has been s t u d i e d by v a r i o u s i n v e s -t i g a t o r s (9, 86-89) and i n d i c a t i o n s are t h a t i t f o l l o w s the gen-e r a l scheme of events as diagramed above. U n l i k e the mammal, however, where spermatozoa are u s u a l l y formed continuously, sper-matozoa i n the f i s h are produced and r e l e a s e d at a p a r t i c u l a r time during the year as the r e s u l t of one c y c l e of spermatogenesis. This c y c l e normally takes place over a p e r i o d of s e v e r a l months during which time the t e s t i s develops and increases i n s i z e . The mature t e s t i s can represent as much as 5% of the body weight (90,91). At any time during the development of the t e s t i s , there are u s u a l l y present a number of d i f f e r e n t c e l l types (primary and - 4 -secondary spermatocytes, spermatids and spermatozoa) (92); one type u s u a l l y predominates and i s i n d i c a t i v e of the p a r t i c u l a r stage of t e s t i s development. I t i s i n t e r e s t i n g to note t h a t i n one species of Salmonid at l e a s t , the spermatid c e l l s appear to be l y i n g f r e e i n the lumina of the t e s t i s unassociated w i t h any other c e l l ; no n u t r i t i v e c e l l s comparable to the S e r t o l i c e l l s have been observed (92). I t i s p o s s i b l e t h a t the S e r t o l i c e l l s observed i n other v e r t e b r a t e s have fu n c t i o n s other than those d i r e c t l y r e l a t e d to the maturation of the spermatid c e l l . L i t t l e i s known about the chemical nature and mode of a c t i o n of the developmental f a c t o r s which c o n t r o l the successive stages of c e l l u l a r t r a nsformation during spermatogenesis. That these stages are w e l l r e g u l a t e d were noted by Roosen-Runge (93) who has found t h a t i n the mammal, w i t h the p o s s i b l e exception of man, spermatozoa development occurs i n synchronous c y c l e s . F u r t h e r , i f a generation of c e l l s were delayed at some p o i n t , i t d i d not begin i t s development again at a random p o i n t of spermatogenesis but took i t s place among the generations i n the same c o r r e l a t i o n as before. L i k e l y candidates f o r the i n i t i a t i o n and c o n t r o l of spermatogenesis are the gonadotrophic hormones ( l u t e i n i z i n g hor-mone and f o l l i c l e s t i m u l a t i n g hormone) (94) of the adenohypophy-s i s of the p i t u i t a r y gland; however, the a c t u a l mode of a c t i o n of these hormones i s not understood. The r o l e of gonadotrophic hormones i n the c o n t r o l of spermatogenesis i n the f i s h has been reviewed by s e v e r a l authors (5, 95, 96). I t has been shown th a t t e s t e s of the immature f i s h respond r e a d i l y to treatment w i t h e x t r a c t s of p i t u i t a r y glands obtained from spawning salmon (97, 98). Immature rainbow t r o u t kept i n aquaria i n our l a b o r a -t o r y f o r example become s e x u a l l y mature i n about two months a f t e r - 5 -treatment w i t h the p i t u i t a r y e x t r a c t s . Whether the hormones of the f i s h p i t u i t a r y are i d e n t i c a l w i t h those of the mammal i s s t i l l i n doubt (95) but i t appears t h a t as i n the mammal, spermatogen-e s i s i n f i s h i s a hormonally c o n t r o l l e d c e l l u l a r d i f f e r e n t i a t i o n . A major e x t e r n a l stimulus of sexual maturation i n salmonid f i s h i s the photoperiod which acts through the hypothalamus c o n t r o l l i n g gonadotrophin s e c r e t i o n . Spermatogenesis i s ac c e l e r a t e d by ex-posing the f i s h to an appropriate number of hours of d a y l i g h t per day (99). The biochemical changes accompanying spermatogenesis have not been e x t e n s i v e l y i n v e s t i g a t e d and i t i s only r e c e n t l y t h a t e f f o r t s are being made i n t h i s area. I t i s u s e f u l a t t h i s p o i n t to review some of the macromolecular processes a s s o c i a t e d w i t h spermatogenesis. Most of the in f o r m a t i o n p r e s e n t l y a v a i l a b l e are the r e s u l t of s t u d i e s w i t h the orthopteran t e s t i s (the l o c u s t and the grasshopper) (100-102), the anther of p l a n t s , u s u a l l y the l i l y (103,104), and the developing t e s t i s of salmonid f i s h (3, 105). The reason f o r the choice of these organisms f o r study m a t e r i a l are t h a t : a) the orthopteran chromosomes are large and e a s i l y recognized; hence c e l l s at d i f f e r e n t stages of develop-ment could be i d e n t i f i e d and st u d i e d by h i s t o l o g i c a l methods, b) the maturing c e l l s of the l i l y anther are extremely w e l l syn-chronized a l l o w i n g f o r developmental s t u d i e s of the process of meio s i s , and c) the f i s h t e s t i s provides l a r g e amounts of r e l a -t i v e l y synchronized c e l l s at d i f f e r e n t stages of spermatogenesis and these can be subjected to biochemical s t u d i e s . The mammalian t e s t i s has been l i t t l e used f o r biochemical s t u d i e s s i n c e there i s present at any one time a great complexity of c e l l s a t d i f f e r e n t - 6 -s t a g e s o f s p e r m a t o g e n e s i s . R e c e n t l y Lam e't at. (106) h a v e de-v i s e d an i n g e n i o u s method o f s e p a r a t i n g and i s o l a t i n g c e l l s a t d i f f e r e n t s t a g e s o f s p e r m a t o g e n e s i s f r o m t h e mouse t e s t i s . T h i s t e c h n i q u e s h o u l d p r o v i d e t h e n e c e s s a r y i m p e t u s f o r s t u d y i n g t h e b i o c h e m i s t r y o f mammalian s p e r m a t o g e n e s i s . The s y n t h e s i s o f DNA d u r i n g s p e r m a t o g e n e s i s was i n v e s t i g a t e d by a u t o r a d i o g r a p h y and m e a s u r i n g t h e amount o f t r i t i a t e d t h y m i d i n e i n c o r p o r a t e d i n t o t h e n u c l e i o f t h e male germ c e l l a t d i f f e r e n t s t a g e s o f d e v e l o p m e n t . S t u d i e s i n t h e g r a s s h o p p e r and t h e l o c u s t (100, 101) h a v e shown t h a t DNA s y n t h e s i s o c c u r s o n l y d u r i n g p r e -o r e a r l y - m e i o s i s a s s o c i a t e d w i t h t h e s p e r m a t o g o n i a and t h e p r i -mary s p e r m a t o c y t e s , t h e f o r m e r i n p r e p a r a t i o n f o r m i t o s i s and t h e l a t t e r f o r m e i o s i s . RNA s y n t h e s i s , s t u d i e d by t h e i n c o r p o r a -t i o n o f 3 H - u r i d i n e , o c c u r s on a l l autosomes (the s o m a t i c chromo-somes) t h r o u g h o u t t h e f i r s t and s e c o n d m e i o t i c p r o p h a s e o f t h e p r i m a r y and s e c o n d a r y s p e r m a t o c y t e s r e s p e c t i v e l y . No RNA s y n -t h e s i s o c c u r s a t t h e c o n t r a c t e d s t a g e s o f f i r s t o r s e c o n d meta-p h a s e , o r f i r s t o r s e c o n d a n a p h a s e . RNA i s a g a i n s y n t h e s i z e d by t h e y o u ng s p e r m a t i d c e l l b u t s u c h s y n t h e s i s c e a s e s b e f o r e any m o r p h o l o g i c a l c h a n g es i n t h e s p e r m a t i d c o u l d be d e t e c t e d . No l a b e l was d e t e c t e d i n t h e n u c l e i o f t h e d i f f e r e n t i a t i n g s p e r m a t i d e v e n a f t e r 8 h o u r s i n c u b a t i o n w i t h 3 H - u r i d i n e . H e n d e r s o n (100) has f u r t h e r shown t h a t t h e l a b e l i n c o r p o r a t e d i n t h e n u c l e u s i s l a t e r f o u n d i n t h e c y t o p l a s m c o n s i s t e n t w i t h t h e n u c l e a r s y n t h e s i s o f RNA and i t s l a t e r t r a n s p o r t t o t h e c y t o p l a s m . No i n d e p e n d e n t RNA s y n t h e s i s i n t h e c y t o p l a s m c o u l d be d e t e c t e d . He has t e n t a -t i v e l y s u g g e s t e d t h a t t h e RNA s y n t h e s i z e d i s o f t h e m e s s e n g e r t y p e r a t h e r t h a n r i b o s o m a l RNA i n t h a t no d i s t i n c t n u c l e o l u s , t h e - 7 -p r e s e n c e o f w h i c h i s u s u a l l y a s s o c i a t e d w i t h a c t i v e r i b o s o m a l RNA s y n t h e s i s , c o u l d be d e t e c t e d . Das et al. (101) h a v e measured t h e r a t e o f p r o t e i n s y n t h e s i s i n m e i o t i c c e l l s by d e t e r m i n i n g t h e amount o f 3 H - a r g i n i n e i n c o r p o r a t e d f o r a g i v e n l e n g t h o f t i m e . They have f o u n d t h a t p r o t e i n s y n t h e s i s c o n t i n u e s t h r o u g h o u t t h e w h o l e m e i o t i c c y c l e as w e l l as d u r i n g s p e r m i o g e n e s i s . I n g e n e r a l , t h e r a t e o f p r o t e i n s y n t h e s i s , i s d e c r e a s e d d u r i n g t h e s t a g e s o f m e i o t i c c e l l d e v e l o p m e n t when t h e r e i s l i t t l e RNA s y n t h e s i s . I n t h e s p e r m a t i d c e l l , where t h e r e i s no RNA s y n t h e s i s , p r o t e i n s y n -t h e s i s c o n t i n u e s t h r o u g h o u t i t s d e v e l o p m e n t a l t h o u g h a t a much r e d u c e d r a t e . T h i s s u g g e s t s t h a t t h e RNA p r o d u c e d e a r l i e r , p o s -s i b l y d u r i n g t h e v e r y e a r l y s p e r m a t i d s t a g e , s u s t a i n s t h e p r o -t e i n s y n t h e s i s d u r i n g s p e r m a t i d c e l l d e v e l o p m e n t . B l o c h and B r a c k (107) h a v e f u r t h e r shown t h a t t h e e l o n g a t i n g s p e r m a t i d c e l l o f t h e g r a s s h o p p e r i n c o r p o r a t e d 3 H - a r g i n i n e f i r s t i n t o t h e c y t o -p l a s m and t h i s l a b e l was l a t e r t r a n s p o r t e d i n t o t h e n u c l e u s ; t h i s s u g g e s t s t h a t a t t h i s l a t e s t a g e o f s p e r m a t o g e n e s i s , a n u c l e a r p r o t e i n i s s y n t h e s i z e d by t h e c y t o p l a s m . T a y l o r ( 1 0 3 ) , and S t e r n and c o w o r k e r s (104, 108) have c o n -f i r m e d many o f t h e f i n d i n g s c i t e d above i n t h e i r s t u d y o f m e i o s i s on p l a n t m a t e r i a l s p a r t i c u l a r l y on t h e a n t h e r o f t h e l i l y . Be-c a u s e t h e y were w o r k i n g w i t h h i g h l y s y n c h r o n o u s s y s t e m s , t h e y were a b l e t o examine t h e d e v e l o p m e n t a l c o n t r o l o f p o l l e n f o r m a -t i o n . F o r e x ample, H o t t a eb al. (109) have shown t h a t a t d i f f e r -e n t s t a g e s o f m e i o s i s i n t h e a n t h e r o f t h e l i l y d i f f e r e n t p r o -t e i n s a r e s y n t h e s i z e d as j u d g e d by t h e amount o f 3 H - l e u c i n e i n -c o r p o r a t e d i n t o t h e p r o t e i n s o f t h e c e l l n u c l e i e x t r a c t e d w i t h pH 8 p h o s p h a t e b u f f e r (pH 8 n u c l e a r p r o t e i n s ) and s e p a r a t e d by - 8 -c h r o m a t o g r a p h y on D E A E - c e l l u l o s e . They have a l s o f o u n d t h a t a l t h o u g h g e n e r a l p r o t e i n s y n t h e s i s i n t h e a n t h e r i s o n l y p a r -t i a l l y i n h i b i t e d i n t h e p r e s e n c e o f a low c o n c e n t r a t i o n o f c y c l o -h e x i m i d e (0.2 u g / m l ) , t h e i n c o r p o r a t i o n o f 3 H - l e u c i n e i n t o t h e pH 8 n u c l e a r p r o t e i n s i s m a r k e d l y s u p p r e s s e d . F u r t h e r , DNA s y n -t h e s i s i s a l s o m a r k e d l y s u p p r e s s e d i n t h e p r e s e n c e o f cyclo-2". h e x i m i d e s u g g e s t i n g t h a t DNA s y n t h e s i s d u r i n g m e i o t i c p r o p h a s e i s d e p e n d e n t on t h e p r e s e n c e o f c e r t a i n newly s y n t h e s i z e d p r o -t e i n s , p o s s i b l y t h e pH 8 n u c l e a r p r o t e i n s . The c h a r a c t e r i z a t i o n and i d e n t i f i c a t i o n o f t h e s e p r o t e i n s s h o u l d be o f g r e a t i n t e r e s t . P r o g r e s s i n t h e b i o c h e m i s t r y o f chromosomal b a s i c p r o t e i n s o f s p e r m a t o g e n e s i s h a s b e e n f o c u s s e d m a i n l y on t h e f i s h t e s t i s . M e i s c h e r (1) o b s e r v i n g t h e d r a m a t i c m o r p h o l o g i c a l c h a n g es i n t h e s a l m o n t e s t i s d u r i n g t h e s p a w n i n g p e r i o d f i r s t d e t e r m i n e d t h a t t h e t e s t i s t r a n s f o r m a t i o n was a c c o m p a n i e d by a t r a n s f o r m a t i o n o f t h e n u c l e a r p r o t e i n s f r o m h i s t o n e s t o p r o t a m i n e . A l f e r t (9) u s i n g c y t o c h e m i c a l t e c h n i q u e s has shown t h a t s o m a t i c h i s t o n e s a r e t r a n s f o r m e d i n t o p r o t a m i n e a t t h e s p e r m a t i d s t a g e o f d e v e l o p -ment i n s a l m o n t e s t i s . T h i s f i n d i n g has b e e n c o n f i r m e d f o r a number o f f i s h e s (5, 10, 11) by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and amino a c i d a n a l y s i s o f t h e a c i d e x t r a c t a b l e n u c l e a r p r o t e i n s o f t h e t e s t i s o b t a i n e d a t d i f f e r e n t s t a g e s d u r i n g s p e r m a t o g e n e s i s . I n g l e s et al. (11) have shown by s t u d i e s w i t h i n h i b i t o r s ( c h l o r -a m p h e n i c o l , c y c l o h e x i m i d e , p u r o m y c i n and a c t i n o m y c i n D) t h a t p r o -t a m i n e i n t h e t e s t i s n u c l e u s i s t h e r e s u l t o f de novo s y n t h e s i s p r o b a b l y v i a t h e ribosome-messenger-RNA complex and i s n o t t h e r e s u l t o f some c o n v e r s i o n o f t h e h i s t o n e s a l r e a d y p r e s e n t . T h i s - 9 -s u g g e s t s t h a t t h e t r a n s f o r m a t i o n o f h i s t o n e s t o p r o t a m i n e i n t h e s p e r m a t i d c e l l s must i n v o l v e a p r o c e s s o f r e p l a c e m e n t i n w h i c h t h e h i s t o n e s on t h e DNA a r e r e p l a c e d by newly s y n t h e s i z e d p r o t a m i n e . M a r u s h i g e and D i x o n ( 1 2 ) , s t u d y i n g t h e p r o p e r t i e s o f c h r o m a t i n f r o m d e v e l o p i n g s t e e l h e a d t r o u t t e s t i s , have n o t e d t h a t i n t h i s r e p l a c e m e n t p r o c e s s a g r e a t e r p r o p o r t i o n o f t h e a r g i n i n e - r i c h h i s t o n e s ( H i s t o n e IV) a r e r e p l a c e d f i r s t and t h e l y s i n e - r i c h h i s t o n e s ( H i s t o n e I) a r e r e p l a c e d l a t e r i n d e v e l o p -ment. T h i s s u g g e s t s t h a t t h e r e p l a c e m e n t o f h i s t o n e s by p r o -t a m i n e o c c u r s i n t e m p o r a l s e q u e n c e and i s a w e l l r e g u l a t e d p r o -c e s s . W i g l e and D i x o n (110) have r e c e n t l y i s o l a t e d a new h i s t o n e , H i s t o n e T, i n t h e r a i n b o w t r o u t t e s t i s . They have shown t h a t t h i s h i s t o n e i s n o t t h e d e g r a d a t i o n p r o d u c t o f some o t h e r h i s -t o n e s b u t i s u n i q u e w i t h a m o l e c u l a r w e i g h t o f a b o u t 7,500 and c o n t a i n i n g l a r g e amounts o f l y s i n e and a l a n i n e r e s i d u e s . Whether t h i s h i s t o n e has a s p e c i a l r o l e i n s p e r m a t o g e n e s i s i s n o t known a t p r e s e n t . I t i s i n t e r e s t i n g t o n o t e t h a t S h e r i d a n and S t e r n (111) h a v e o b s e r v e d t h e a p p e a r a n c e o f a u n i q u e h i s t o n e i n t h e d e v e l o p i n g a n t h e r s o f Lilium and Tulipa. T h i s h i s t o n e i s a b s e n t i n t h e s o m a t i c c e l l s o f t h e s e p l a n t s . They have s u g g e s t e d t h a t d u r i n g t h e f o r m a t i o n o f t h e male gamete, t h e r e i s a s h i f t f r o m t h e p r e - e x i s t i n g common h i s t o n e s t o t h i s " m e i o t i c h i s t o n e " . I t - i s s t i l l an open q u e s t i o n as t o w h e t h e r t h e ^ r e p l a c e m e n t o f s o m a t i c h i s t o n e s by a n o t h e r b a s i c p r o t e i n ( u s u a l l y r i c h i n a r g i n i n e ) s o d r a m a t i c a l l y o b s e r v e d i n t h e s a l m o n i d t e s t i s i s a g e n e r a l r e q u i r e m e n t f o r t h e p r o c e s s o f s p e r m a t o z o a f o r m a t i o n . However, t h e t r a n s f o r m a t i o n o f s o m a t i c h i s t o n e s i n t e s t i s c e l l s - 10 -a t a l a t e s t a g e o f s p e r m a t o g e n e s i s h a s b e e n o b s e r v e d i n a v a r i e t y o f o r g a n i s m s . B l o c h (13) f o r example has o b s e r v e d by h i s t o c h e m i c a l t e c h n i q u e s t h a t s o m a t i c h i s t o n e s were r e p l a c e d by p r o t a m i n e s i n t h e t e s t i s o f t h e s n a i l {Helix aspersa) and o f t h e s q u i d (Loligo opalescens). F u r t h e r , M o n e s i (112) has o b s e r v e d t h i s e f f e c t i n mouse. The p r e s e n c e o f a h i g h a r g i n i n e c o n t e n t i n mammalian s p e r m a t o z o a has b e e n n o t e d (2) b u t t h e p r e s e n c e o f " p r o t a m i n e - l i k e " b a s i c p r o t e i n s has y e t t o be d e m o n s t r a t e d . I n v a r i o u s o r g a n i s m s (2) i t has b e e n o b s e r v e d by c y t o c h e m i c a l s t u d -i e s t h a t t h e r e p l a c e m e n t o f s o m a t i c h i s t o n e s d u r i n g s p e r m a t o -g e n e s i s i s a two s t e p p r o c e s s : f i r s t , r e p l a c e m e n t by an a r g i n i n e -r i c h h i s t o n e , t h e n by p r o t a m i n e . I n some o r g a n i s m s o n l y t h e f i r s t s t e p i s o b s e r v e d and i n o t h e r s no r e p l a c e m e n t o c c u r s a t a l l ( 2 ) . As a l r e a d y m e n t i o n e d , i t i s c h a r a c t e r i s t i c o f s p e r m a t o -g e n e s i s t h a t RNA d i s a p p e a r s f r o m t h e c y t o p l a s m o f t h e m a t u r i n g s p e r m a t i d c e l l . C r e e l m a n and T o m l i n s o n (113) have o b s e r v e d a p r o g r e s s i v e d e c r e a s e i n RNA c o n t e n t i n t h e m a t u r i n g s a l m o n t e s -t i s and, Ando and H a s h i m o t o (10) have shown t h a t RNA i s p r e s e n t I n t h e n u c l e i o f m a t u r i n g t r o u t t e s t i s a t an i n t e r m e d i a t e s t a g e o f d e v e l o p m e n t b u t t h e amount o f RNA i n t h e n u c l e i d e c r e a s e s t o z e r o by t h e s t a g e when p r o t a m i n e i s p r o d u c e d . T h e s e o b s e r v a t i o n s a r e c o n s i s t e n t w i t h t h e e l i m i n a t i o n o f RNA and t h e l a c k o f RNA s y n t h e s i s i n t h e m a t u r i n g s p e r m a t i d c e l l s o f v a r i o u s o r g a n i s m s a l r e a d y m e n t i o n e d above. I t i s n o t e w o r t h y ifchat i n one s p e c i e s o f s a l m o n Oncorhynahus nerka, t h e d e c r e a s e i n RNA was a c c o m p a n i e d by a change i n b a s e c o m p o s i t i o n ; t h e RNA p r e s e n t i n m a t u r e t e s t i s was much r i c h e r i n g u a n o s i n e t h a n i n t h e immature t e s t i s ( 3 ) . T h i s may i n d i c a t e t h a t t h e e l i m i n a t i o n o f RNA i s a r a t h e r s p e c i f i c - 11 -p r o c e s s . The a b i l i t y o f t h e t r o u t t e s t i s c h r o m a t i n a t v a r i o u s s t a g e s o f d e v e l o p m e n t t o a c t as a t e m p l a t e f o r t h e s y n t h e s i s o f RNA by a p u r i f i e d b a c t e r i a l p o l y m e r a s e was examined by M a r u s h i g e and D i x o n ( 1 2 ) . B onner et al. (114) have shown t h a t t h e i s o l a t e d c h r o m a t i n when p r o p e r l y p r e p a r e d r e t a i n s t h e t e m p l a t e a c t i v i t y f o r RNA s y n t h e s i s c h a r a c t e r i s t i c o f t h e c e l l s f r o m w h i c h i t was o b t a i n e d . I n t h e t r o u t t e s t i s ( 1 2 ) , i t was f o u n d t h a t t h e chromosomal tem-p l a t e a c t i v i t y g e n e r a l l y d e c r e a s e d d u r i n g t h e c o u r s e o f s p e r m a t o -g e n e s i s . F u r t h e r , i t was d e m o n s t r a t e d t h a t t h e c h a n g e s i n t h e t e m p l a t e a c t i v i t y r e f l e c t e d t h e c h e m i c a l c o m p o s i t i o n o f t h e i s o -l a t e d c h r o m a t i n . F o r example, p r i o r t o t h e a p p e a r a n c e o f p r o t a -mine on t h e c h r o m a t i n , t h e r e was a s h a r p d e c r e a s e i n t e m p l a t e a c t i v i t y a s s o c i a t e d w i t h an i n c r e a s e i n h i s t o n e c o n t e n t and a d e c r e a s e i n n o n - h i s t o n e p r o t e i n c o n t e n t . T h i s s u p p o r t s t h e h y p o -t h e s i s t h a t h i s t o n e s a c t as r e p r e s s o r s o f gene e x p r e s s i o n and t h e n o n - h i s t o n e p r o t e i n s as a c t i v a t o r s (114, 1 1 5 ) . A f t e r t h i s s h a r p d e c r e a s e i n t e m p l a t e a c t i v i t y and d u r i n g t h e c o u r s e o f r e p l a c e -ment o f h i s t o n e s by p r o t a m i n e , t h e t e m p l a t e a c t i v i t y p r o g r e s s i v e l y d e c r e a s e d u n t i l by t h e n u c l e o p r o t a m i n e s t a g e , no t e m p l a t e a c t i v i t y was o b s e r v e d . A number o f enzymes have b e e n i d e n t i f i e d i n t h e d e v e l o p i n g s a l m o n i d t e s t i s . Some a t t e m p t s have been made t o d e t e r m i n e t h e f u n c t i o n s o f t h e s e enzymes i n t h e p r o c e s s o f s p e r m a t o g e n e s i s . J e r g i l and D i x o n (35) have i s o l a t e d f r o m t h e h i g h s p e e d s u p e r -n a t a n t o f t h e r a i n b o w t r o u t t e s t i s a p h o s p h o k i n a s e ( p r o t a m i n e k i n a s e ) t h a t i s a b l e t o p h o s p h o r y l a t e t h e s e r y l r e s i d u e s o f p r o -t a m i n e and t o a l e s s e r e x t e n t o f h i s t o n e s . A p h o s p h a t a s e c a p a b l e - 12 -of removing 3 2P-phosphate from 3 2 P - l a b e l l e d protamine a l s o has been observed (115). . Since during the replacement of histones by protamine i n the - t r o u t t e s t i s no f r e e i n t a c t h i s t o n e molecules can be detected i n the nucleoplasm or the cytoplasm (117) suggest-ing t h a t the replaced histones are degraded, an attempt to i s o l -ate an enzyme "histonase" has been i n i t i a t e d i n our l a b o r a t o r y . Presumably the s p e c i f i c i t y of such an enzyme would allow i t t o degrade histones but not protamine. Tarr and Gardner (118) r e -c e n t l y demonstrated RNA and DNA polymerase a c t i v i t i e s i n c e l l -f r e e e x t r a c t s of immature salmon t e s t i s . McDonald (119) has shown t h a t salmon t e s t i s i s very r i c h i n DNase IT. Other enzymes degrading n u c l e i c acids ( d i e s t e r a s e s , phosphatasesjg^dv^lb'ojg^qpreas^) have been observed (3). I t has been suggested t h a t enzymes i n the salmon t e s t i s capable of degrading RNA are appropriate to the l a t e r stages of spermatogenesis when the amount of RNA i n the t i s s u e d e c l i n e s but t h a t the r o l e of such an enzyme as DNase I I , s p e c i f i c f o r degradation of DNA, i s not immediately apparent (3). From the preceeding i t i s obvious t h a t the process of sperma-togenesis i s an example of i n v o l v e d and complex c e l l u l a r d i f f e r -e n t i a t i o n . As p a r t of the attempt (105) to d e f i n e b i o c h e m i c a l l y a s m a l l p o r t i o n of t h i s complex process, a study of the biosyn-t h e s i s of protamine was i n i t i a t e d and the r e s u l t s are presented i n t h i s t h e s i s . As w i l l be d i s c u s s e d , protamine i s a small pro-t e i n w i t h unusual amino a c i d composition. This i n v i t e s a d e t a i l -ed study of i t s mechanism of b i o s y n t h e s i s , p a r t i c u l a r l y since i t s appearance occurs at a l a t e stage of sperm d i f f e r e n t i a t i o n when the nuclear genes are i n a c t i v e . The f a c t that protamine i t s e l f - 13 -i s a chromosomal p r o t e i n and t h a t i t replaces histones i n a r e g u l a t e d manner adds considerable i n t e r e s t to the problem. The Chemistry and Biology of Protamine; I s o l a t i o n of protamine from salmon sperm was f i r s t described by Miescher i n 1874 (1) and soon a f t e r , the s t u d i e s of Kossel (120) e s t a b l i s h e d the presence of protamines i n the spermatozoa of sev-e r a l d i f f e r e n t species of f i s h . Since the time of K o s s e l , the search f o r the occurrence of protamine i n a wide v a r i e t y of org-anisms has been pursued by various i n v e s t i g a t o r s (1, 2). To date, as already mentioned, the presence of protamine i n the. spermatozoa of various organisms has been i d e n t i f i e d only by cytochemical methods and the a c t u a l i s o l a t i o n of protamines has been r e s t r i c t e d to the spermatozoa of a number of f i s h e s (1) and of the r o o s t e r (1). However, a consequence of such a search may be the establishment of an u n i v e r s a l b i o l o g i c a l s i g n i f i c a n c e f o r protamine. The chemistry and b i o l o g y of - protamine have been r e -viewed i n recent years by F e l i x (1), Murray (121), Vendrely and Vendrely (2), H n i l i c a (122), Dixon and Smith (3), and Dixon et al. (105). Protamine and histones are p r o t e i n s a s s o c i a t e d w i t h DNA i n the c e l l nucleus and are g e n e r a l l y e x t r a c t a b l e w i t h d i l u t e min-e r a l a c i d s . The d i s t i n c t i o n between these two c l a s s e s of pro-t e i n s was e s t a b l i s h e d by Kossel (120); however, i t i s sometimes d i f f i c u l t to d i s t i n g u i s h e x a c t l y between them (121). The " c l a s s i -c a l " protamines of f i s h spermatozoa a t any r a t e are e a s i l y d i s -t i n g u i s h e d from other b a s i c nuclear p r o t e i n s such as histones or b a s i c p r o t e i n s of ribosomal o r i g i n . Thus, almost a l l the i n f o r -- 14 -mation p r e s e n t l y a v a i l a b l e on the chemical nature of protamine has been obtained from s t u d i e s on f i s h protamines, p a r t i c u l a r l y those from h e r r i n g and salmonid f i s h . The i s o l a t i o n of l a r g e q u a n t i t i e s of protamine from f i s h spermatozoa u s u a l l y i n v o l v e s e x t r a c t i o n w i t h d i l u t e m ineral acids or high s a l t , ^ s e v e r a l p r e c i p i t a t i o n steps w i t h organic s o l v e n t or a c i d s , and i n some cases formation of s a l t s such as p i c r a t e s s or metal complexes (1, 33, 123). When spermatozoa or sperm n u c l e i are used as s t a r t i n g m a t e r i a l , these methods y i e l d p r eparations of r e l a t i v e l y pure protamine since nucleoprotamine comprises over 90% by weight of the sperm n u c l e i (124). However, these methods are not able to e x t r a c t protamine s p e c i f i c a l l y from a mixture of b a s i c p r o t e i n s (mainly histones) present i n the c e l l n u c l e i of the maturing t e s t i s . To overcome t h i s problem, Ingles and Dixon (7) have r e c e n t l y introduced a method i n which protamine i s separated from other nuclear b a s i c p r o t e i n s by chromatography on a Bio-Gel P-10 column using 0.2 M a c e t i c a c i d as eluant. The protamine f r a c t i o n was uncontaminated by any other p r o t e i n as judged by i t s s i n g l e amino-terminal residue (pro-l i n e ) and i t s m i g r a t i o n as a s i n g l e band on polyacrylamide g e l e l e c t r o p h o r e s i s at various pH's. A t a b l e of the amino a c i d composition of protamine from v a r -ious sources has been compiled by F e l i x (1). The outstanding feat u r e of a l l protamines i s t h e i r remarkably high a r g i n i n e con-t e n t ; t h i s amino a c i d c o n s t i t u t e s about two t h i r d s of the p r o t e i n . The r e s t of the protamine molecule i s mainly composed of a very l i m i t e d range of 5-7 n e u t r a l amino a c i d s , namely : a l a n i n e , g l y c i n e , - 15 -i s o l e u c i n e , p r o l i n e , s e r i n e , and t h r e o n i n e . The most a b u n d a n t a m i n o - t e r m i n a l amino a c i d s a r e a l a n i n e and p r o l i n e , a l t h o u g h s m a l l q u a n t i t i e s o f o t h e r s have b e e n f o u n d (121) . A r g i n i n e , p r e s e n t as a b l o c k o f s e v e r a l r e s i d u e s , o c c u p i e s t h e c a r b o x y l -t e r m i n a l o f a l l p r o t a m i n e s e x a m i n e d ( 1 ) . The m o l e c u l a r w e i g h t s o f v a r i o u s p r o t a m i n e s have b e e n d e t e r m i n e d by a number o f chem-i c a l methods (125-127) and i t a p p e a r s t h a t p r o t a m i n e s a r e v e r y s m a l l p r o t e i n s w i t h m o l e c u l a r w e i g h t s c l o s e t o 5,000 c o r r e s p o n -d i n g t o a c h a i n l e n g t h o f 31-34 amino a c i d r e s i d u e s . As a r e s u l t o f t h e s e f e a t u r e s , namely t h e l a r g e number o f a r g i n y l r e s i d u e s and low m o l e c u l a r w e i g h t , p r o t a m i n e p o s s e s s e s h i g h l y u n u s u a l p r o p e r t i e s . The h e t e r o g e n e i t y o f p r o t a m i n e was assumed by t h e e a r l y i n -v e s t i g a t o r s (1, 1 2 0 ) , however, t h e s t a r t i n g m a t e r i a l and f r a c -t i o n a t i o n methods a f f e c t e d t h e i r r e s u l t s c o n s i d e r a b l y and t h e a c t u a l n a t u r e o f h e t e r o g e n e i t y was n o t t h o r o u g h l y i n v e s t i g a t e d . I n r e c e n t y e a r s , t h e u s e o f more modern f r a c t i o n a t i o n t e c h n i q u e s s u c h as c o u n t e r c u r r e n t d i s t r i b u t i o n and c h r o m a t o g r a p h y on p a p e r , i o n - e x c h a n g e r s , and a l u m i n a (1, 3) h a v e r e p r o d u c i b l y i n d i c a t e d t h e p r e s e n c e o f more t h a n one component i n e a c h p r o t a m i n e examined. Due t o t h e r e m a r k a b l e s i m i l a r i t y i n p r o p e r t i e s o f t h e components, and i n d e e d o f d i f f e r e n t p r o t a m i n e s , t h e i s o l a t i o n o f a s i n g l e homogeneous p r o t e i n a p p e a r s n o t e a s i l y a c h i e v e d . I n s p i t e o f t h i s , Ando's g r o u p i n Tokyo (128-130) has i s o l a t e d and p u r i f i e d by a l u m i n a and c a r b o x y m e t h y l c e l l u l o s e c h r o m a t o g r a p h y t h r e e com-p o n e n t s ( Y l , Y I I , and Z) o f p r o t a m i n e ( c l u p e i n e ) f r o m t h e P a c i f i c h e r r i n g Clupea pallasii. T h e s e components a r e p r e s e n t i n t h e : - 16 -r a t i o of 1.0: 1.1: 0.7 r e s p e c t i v e l y . They have a l s o demonstrated that.the observed heterogeneity does not appear to be the r e s u l t of genetic polymorphism s i n c e the same components could be de-t e c t e d i n the t e s t i s of a s i n g l e h e r r i n g (31); f u r t h e r , the same species of h e r r i n g obtained from d i f f e r e n t geographical l o c a -t i o n s a l s o c o n t a i n the same three components of protamine (131). Recently the complete amino a c i d sequences f o r the three components of clupeine. were determined by Ando and co-workers (128-130) and are shown i n F i g . 1. P a r t i a l sequences of rainbow t r o u t (15) and steelhead t r o u t (5) protamines already obtained i n d i c a t e t h a t protamines from h e r r i n g and t r o u t are c l o s e l y r e -l a t e d . From the sequence data of the clupeine components, Black and Dixon (132) have proposed an e v o l u t i o n a r y pathway whereby the present-day sequences could have been deri v e d during the course of e v o l u t i o n from an a r c h e t y p a l pentapeptide, ala-arg-arg-arg-arg, by the m u t a t i o n a l processes of repeated gene d u p l i c a t i o n (133) t o -gether w i t h s i n g l e base s u b s t i t u t i o n s i n amino a c i d codons. A v a i l a b i l i t y of sequences f o r other protamines should allow f o r a c r i t i c a l t e s t of t h i s i n t e r e s t i n g hypothesis. Stedman and Stedman (134), having observed t h a t the a r g i n i n e content of the nuclear b a s i c p r o t e i n s e x t r a c t e d from salmon sperm, fowl e r y t h r o c y t e and l i v e r were d i f f e r e n t , f i r s t proposed t h a t these d i f f e r e n t b a s i c p r o t e i n s ( i n c l u d i n g protamines and histones) could o f f e r a means of r e g u l a t i n g gene expression by t h e i r spec-i f i c a s s o c i a t i o n w i t h DNA. Subsequent s t u d i e s have i n d i c a t e d t h a t histones are r e p r e s s o r s of gene a c t i v i t y and t h i s has been discussed i n s e v e r a l reviews (121, 122, 135, 137). In a few Clupeine Y I Ala Arg Arg Arg Arg Ser Ser Ser Arg Pro I le Arg Arg Arg Arg Pro Arg Arg Arg Thr Thr Arg Arg Arg Arg Ala Gly Arg Arg Arg Arg Clupeine Y 11 p r 0 Arg Arg Arg — Thr Arg Arg Ala Ser Arg Pro Val Arg Arg Arg Arg Pro Arg Arg — Val Ser Arg Arg Arg Arg Ala — Arg Arg Arg Arg Clupeine Z Ala Arg Arg Arg Arg Ser Arg Arg Ala Ser Arg Pro Val Arg Arg Arg Arg Pro Arg Arg — Val Ser Arg Arg Arg Arg Ala — Arg Arg Arg Arg F i g . 1. Comparison of amino a c i d sequences of protamines from Clupea pallasii ( P a c i f i c herring)(129-131) The components are a l i g n e d so as t o o b t a i n maximum homology i n amino a c i d sequences between the three-components (132). - 18 -cases t i s s u e - s p e c i f i c nuclear b a s i c p r o t e i n s have been observed, notab l y , the protamines of f i s h spermatozoa (1,2), the histones of avian e r y t h r o c y t e s (136) and a s p e c i f i c h i s t o n e i n p i g b r a i n (138). However, i n g e n e r a l , histones from a wide v a r i e t y of organisms appear to be s t r i k i n g l y s i m i l a r and are composed of only a l i m i t e d range of major components p o s s i b l y Mess than 10 (122, 141, 142). For example, histones i s o l a t e d from organisms as d i f f e r e n t as pea embryo and c a l f are very s i m i l a r w i t h r e -spect to amino a c i d composition, amino-terminal amino a c i d s , and e l e c t r o p h o r e t i c m o b i l i t y (135). Recently the complete amino a c i d sequence of the a r g i n i n e - r i c h h i s t o n e , histone TV i s o l a t e d from c a l f thymus has been determined (139). A unique sequence con-s i s t e n t w i t h a s i n g l e homogeneous p r o t e i n was. obtained, and, ex-cept f o r the presence or absence of the a c e t y l group on the l y s i n e at p o s i t i o n 16, no other evidence was observed f o r v a r i a n t s t r u c -tures i n the p r o t e i n . Amino a c i d composition, peptide mapping, and sequence s t u d i e s on the 18 residue ( t h i s represents about 1/5 of the t o t a l molecule) cyanogen bromide peptide obtained from the carboxyl-terminus of histone IV of pea embryo i n d i c a t e that the h i s t o n e IV of pea and c a l f are v i r t u a l l y i d e n t i c a l (140). Thus i t would appear d i f f i c u l t to e x p l a i n the genetic r e g u l a t i o n of a wide d i v e r s i t y of c e l l types by a l i m i t e d number of h i s t o n e f r a c t i o n s . However, a l s o present i n the chromosome are RNA and non-histone p r o t e i n s (114) . That these components may by them-selv e s or i n conjunction w i t h histones provide s p e c i f i c i t y f o r gene r e g u l a t i o n has been suggested (114, 115) 143, 144). The discovery t h a t histones are modified i n d i f f e r e n t ways - 19 -by a s e r i e s of enzymatic r e a c t i o n s , namely : a c e t y l a t i o n (145), methylation (145), and phosphorylation (21-24), has prompted spec u l a t i o n s t h a t these m o d i f i c a t i o n s could provide a means whereby the a s s o c i a t i o n of histones w i t h DNA are s e l e c t i v e l y a l t e r e d to enable s p e c i f i c c o n t r o l of gene expression. A l l f r e y et al. (145) and Stevely and Stocken (24) have shown th a t in vitro, DNA complexed w i t h modified histones d i s p l a y e d greater template a c t i v i t y w i t h added RNA polymerase than DNA complexed w i t h unmodified h i s t o n e s . F u r t h e r , there have been observed in vivo s e v e r a l instances i n which an increase i n RNA synthesis was preceeded by an increase i n the m o d i f i c a t i o n of histones (23, 24, 145). As y e t , however, i t i s not known whether these modi-f i c a t i o n s r e g u l a t e gene expression s p e c i f i c a l l y . That protamine i s phosphorylated was f i r s t noted by Murray (25) who demonstrated the presence of 0-phospho-serine i n the a c i d hydrolysate of a commercial p r e p a r a t i o n of protamine. Sub-sequently, Ingles and Dixon (7) have i s o l a t e d 3 2 P - l a b e l l e d pro-tamine from a c e l l suspension of steelhead t r o u t t e s t i s incubated w i t h i n o r g a n i c 3 2P-phosphate. They i s o l a t e d and c h a r a c t e r i z e d s e v e r a l 0-3 2P-phosphoseryl peptides from the t r y p t i c d i g e s t of t h i s protamine. From the f a c t t h a t a number of d i f f e r e n t phospho-peptides were observed, i t seemed probable t h a t a l l the s e r i n e residues i n the protamine could be phosphorylated. Moreover, by an i n c o r p o r a t i o n experiment using 1 ^'C-serine, i t was observed t h a t a l l the t r y p t i c peptides of newly synthesized protamine con-t a i n i n g l l f C - s e r i n e were phosphorylated as judged by the p o s i t i o n s of these peptides on high voltage paper e l e c t r o p h o r e s i s before and - 20 -a f t e r treatment w i t h E. coli a l k a l i n e phosphatase (7). Ingles (5) has a l s o noted t h a t the i n c o r p o r a t i o n of 3 2P-phosphate i n t o protamine was not i n h i b i t e d i n a t e s t i s c e l l suspension incubated i n the presence of 0.1 mM puromycin, a concentration which i n -h i b i t e d the i n c o r p o r a t i o n of 3 H - a r g i n i n e i n t o protamine by 80%. This suggests t h a t t h i s phosphorylation process i s independent of the i n c o r p o r a t i o n of amino acids and must occur at the nascent peptide or completed p r o t e i n l e v e l . As already mentioned, J e r g i l and Dixon (35) have i s o l a t e d a protamine kinase from t r o u t t e s t i s and c h a r a c t e r i z a t i o n of t h i s enzyme i n d i c a t e s t h a t i t i s r e l a t i v e l y s p e c i f i c f o r protamine t r a n s f e r r i n g the gamma-phosphate of ATP to the s e r i n e residues i n protamine y i e l d i n g O-phosphoseryl residues (54). Langan and Smith (146) have a l s o i s o l a t e d a s p e c i f i c h i s -tone kinase from r a t l i v e r . At p r o g r e s s i v e stages of t r o u t t e s t i s development, the r a t i o of t o t a l phosphate t o t t o t a l s e r i n e i n protamine decreased (7). This was i n t e r p r e t e d to mean tha t at an e a r l y stage of spermio-genesis when protamine s y n t h e s i s i s j u s t i n i t i a t e d , the greater p r o p o r t i o n of the protamine present i n the nucleus i s phosphory-l a t e d and as the t e s t i s matures, there i s a progressive decrease i n the p r o p o r t i o n of phosphorylated protamine. This probably r e f l e c t s the f a c t t h a t at the e a r l i e r stages of t e s t i s develop-ment there i s present a greater p r o p o r t i o n of newly synthesized phosphorylated protamine when protamine s y n t h e s i s had j u s t been "turned-on" but at l a t e r stages, t h i s p r o p o r t i o n i s decreased due to the accumulation of i n c r e a s i n g amounts of. dephosphorylated protamine. At any r a t e , a turnover of phosphate occurs i n the protamine molecule and probably f o l l o w s the f o l l o w i n g sequence : - 21 -f i r s t , protamine i s phosphorylated s h o r t l y a f t e r i t s synthesis (5,7), then the newly synthesized protamine binds to DNA without any appreciable dephosphorylation (48) and f i n a l l y , protamine loses i t s phosphates during sperm maturation (7). Several p o s s i b l e f u n c t i o n s f o r the phosphorylation of pro-tamine have been suggested (3); f o r example, the i n t r o d u c t i o n of 6-8 negative charges i n t o the h i g h l y b a s i c protamine molecule by the i n c o r p o r a t i o n of 3-4 phosphates may be necessary p r e r e q u i s i t e f o r the r e l e a s e of protamine from i t s s i t e of synthesis on the ribosome complex, f o r i t s t r a n s p o r t to the DNA, or f o r a modu-l a t e d i n t e r a c t i o n w i t h DNA to r e g u l a t e gene expression. Recently, a f u r t h e r p o s s i b i l i t y f o r the f u n c t i o n of phosphorylation of protamine has been proposed (33) from the observation t h a t during the replacement of histones already on the DNA by newly synthes-i z e d phosphorylated protamine i n t r o u t t e s t i s , the histones are a l s o phosphorylated. I t i s suggested t h a t the simultaneous phosphorylation of both histones and protamine, the former pre-formed and the l a t t e r newly synthesized, may be s i g n i f i c a n t i n the replacement process. Dixon et al. (49) have shown th a t i n the case of h i s t o n e I I and h i s t o n e IV of t r o u t t e s t i s , a major s i t e of phosphorylation i s at the N-terminus of the molecule as judged by the recovery of a s i n g l e phosphopeptide, N-acetyl-O-p h o s p h o - s e r y l - g l y c y l - a r g i n i n e from t r y p t i c d i g e s t s of . l 2 P - h i s t o n e s . I t i s p o s s i b l e then t h a t i n the case of h i s t o n e I I and IV at l e a s t , phosphorylation of t h i s s t r a t e g i c a l l y l o c a t e d N - a c e t y l -s e r y l - r e s i d u e could i n i t i a t e d i s s o c i a t i o n of these histones (33). The s t r u c t u r e s of nucleoprotamine (the complex of protamine - 22 -w i t h DNA) has been st u d i e d as p a r t of the e f f o r t to di s c o v e r the f u n c t i o n of protamine and as a simple model of the a s s o c i a -t i o n of b a s i c p r o t e i n s w i t h DNA. X-ray d i f f r a c t i o n s t u d i e s both on n a t i v e (147) and r e c o n s t i t u t e d (148) nucleoprotamine as w e l l as i n t a c t sperm n u c l e i (149) i n d i c a t e t h a t nucleoprotamines are very r e g u l a r s t r u c t u r e s of a c r y s t a l l i n e nature. Since chemical analyses showed t h a t the molar r a t i o of a r g i n i n e residues of protamine to phosphate groups of DNA i s c l o s e to u n i t y (1), i t has been proposed by Feughelman et al. (150) th a t the f u l l y extended protamine molecule i s wrapped around the shallow groove of the DNA h e l i x w i t h the a r g i n y l residues i n t e r a c t i n g w i t h the phosphate residues of DNA. I t has a l s o been proposed th a t non-b a s i c amino acids i n protamine "loop-out" of the nucleoprotamine complex si n c e they do not i n t e r a c t w i t h phosphates of the DNA (150). However, the sequence data of clupeine (see F i g . 1) i n -d i c a t e t h a t the a r g i n i n e s i n protamine are arranged i n blocks of d i - , t r i - and t e t r a - a r g i n y l residues separated by one to three non-basic amino a c i d s . Since at l e a s t two non-basic amino acids i n sequence are r e q u i r e d to form a "loop", the hypothesis of Feughelman et at. (150) needs to be modified. Recently O l i n s et al. (151) have st u d i e d the i n t e r a c t i o n of e a t i o n i c homopoly-peptides and protamines w i t h DNA obtained from various sources and have observed t h a t t h i s i n t e r a c t i o n i s not pu r e l y e l e c t r o -s t a t i c but n o n - e l e c t r o s t a t i c forces s e n s i t i v e to 50% methanol were a l s o i n v o l v e d . This i n d i c a t e s t h a t the non-basic amino acids i n protamine may have a r o l e i n the formation of the nucleopro-tamine complex. The#e are at l e a s t two probable f u n c t i o n s of protamine which - 23 -a r e p e r h a p s n o t m u t u a l l y e x c l u s i v e t o e a c h o t h e r . T h e s e a r e (a) p a c k i n g o f DNA i n t o t h e sperm h e a d ; and (b) t h e r e g u l a t i o n o f gene e x p r e s s i o n . The f a c t t h a t t h e r e a r e enough a r g i n i n e s i n n u c l e o p r o t a m i n e t o n e u t r a l i z e a l m o s t a l l t h e p h o s p h a t e s o f DNA has a l r e a d y b e e n m e n t i o n e d . P r e s u m a b l y p r o t a m i n e f a c i l i t a t e s t h e f o l d i n g o f t h e DNA m o l e c u l e by n e u t r a l i z i n g t h e e l e c t r o s t a t i c r e p u l s i o n o f t h e DNA p h o s p h a t e s . O l i n s et al. (151) have f u r t h e r p r o p o s e d t h a t p r o l i n e r e s i d u e s may be s t r a t e g i c a l l y l o c a t e d i n t h e p r o t a m i n e m o l e c u l e s s i n c e p r o l i n e , as a r e s u l t o f i t s s t e r e o -c h e m i s t r y , i s a b l e t o bend a p o l y p e p t i d e c h a i n a l m o s t 90? o u t o f t h e p l a n e o f t h e p e p t i d e bond. The p r e s e n c e o f p r o l i n e may c o n t r i b u t e s m a l l b e n d i n g f o r c e s t o t h e DNAmmolecule w h i c h when m u l t i p l i e d , a l o n g w i t h t h e n e u t r a l i z i n g e f f e c t o f a r g i n i n e , p r o -d u c e t h e h i g h - o r d e r f o l d i n g o f sperm head chromosome ( 1 4 9 ) . I t i s e s t i m a t e d t h a t a b o u t 2 m e t e r s o f DNA m o l e c u l e s a r e p a c k e d i n t o a volume o f 10.4 u 3 (124) i n t h e t r o u t sperm h e a d . T h a t n u c l e o p r o t a m i n e i s i n a c t i v e as t e m p l a t e f o r RNA s y n t h e -s i s has a l r e a d y b e e n m e n t i o n e d (12) and i t has b e e n s u g g e s t e d t h a t t h e t o t a l i n a c t i v a t i o n o f t h e n u c l e a r genome, w h i c h i s a p p r o -p r i a t e i n t h e t e r m i n a l l y d i f f e r e n t i a t e d sperm c e l l , may o n l y be t h e i n c i d e n t a l c o n s e q u e n c e o f t h e p a c k a g i n g o f DNA by p r o t a m i n e ( 1 2 ) . However, O l i n s et al. (151) have shown t h a t b e c a u s e t h e p r o t a m i n e m o l e c u l e i s composed o f b l o c k s o f a r g i n y l r e s i d u e s s e p -a r a t e d by n o n - b a s i c amino a c i d s , p r o t a m i n e e x h i b i t s a s h o r t e r " e f f e c t i v e p o l y c a t i o n l e n g t h " w i t h r e s p e c t t o s t a b i l i z a t i o n o f DNA t h a n m i g h t be e x p e c t e d o f an o l i g o a r g i n i n e c o n t a i n i n g t h e same number o f a r g i n i n e ( a b o u t 20) i n u n b r o k e n s e q u e n c e . T h i s s u g g e s t s t h a t p r o t a m i n e d o e s n o t f u n c t i o n m e r e l y as a p o l y c a t i o n t o - 24 -n e u t r a l i z e t h e DNA d u r i n g t h e p a c k a g i n g p r o c e s s . S i n c e p r o = t a m i n e d i s a p p e a r s s h o r t l y a f t e r f e r t i l i z a t i o n ( 1 5 3 ) , t h e r o l e o f t h i s b a s i c p r o t e i n may be t o a l l o w f o r r a p i d and s p e c i f i c p o s t - f e r t i l i z a t i o n n u c l e a r c h a n g e s f o r t h e s u b s e q u e n t gene e x p r e s s i o n d u r i n g d e v e l o p m e n t . P r o t e i n B i o s y n t h e s i s : I t i s g e n e r a l l y c o n s i d e r e d t h a t t h e c h a r a c t e r i s t i c f e a t u r e s o f a d i f f e r e n t i a t e d c e l l a r e d e t e r m i n e d by i t s p a r t i c u l a r com-p l e m e n t o f p r o t e i n s , and t h e d i v e r s i t y o f b o t h f o r m and f u n c t i o n d i s p l a y e d by d i f f e r e n t c e l l t y p e s i s t h e r e s u l t o f c y t o - d i f f e r e n -t i a t i o n , t h e c o n t r o l l e d p r o c e s s by w h i c h d i f f e r e n t s p e c i f i c p r o -t e i n s a r e s y n t h e s i z e d . Hence, t h e u n d e r s t a n d i n g o f t h e mechanism o f p r o t e i n s y n t h e s i s has become a c e n t r a l p r o b l e m i n modern b i o l -ogy. The m a i n f e a t u r e s o f t h e c u r r e n t t h e o r y o f p r o t e i n s y n t h e s i s a r e o u t l i n e d as f o l l o w s : m e s s e n g e r RNA (mRNA) i s s y n t h e s i z e d by RNA p o l y m e r a s e as a s i n g l e - s t r a n d e d m o l e c u l e homologous t o a p o r t i o n o f one s t r a n d o f t h e DNA. T h i s p r o c e s s i s known as t r a n s -c r i p t i o n . Ribosomes a s s o c i a t e w i t h t h e mRNA i n s e q u e n c e s t a r t i n g a t an i n i t i a t i n g s i t e and t r a v e l l i n g a l o n g t h e m e s s e n g e r i n a 5 1 t o 3 1 d i r e c t i o n . A t any one t i m e , s e v e r a l r i b o s o m e s may be a s s o c i a t e d w i t h t h e m e s s e n g e r f o r m i n g a p o l y r i b o s o m e complex. T h e s e c o m p l e x e s a r e t h e a c t i v e p r o t e i n s y n t h e s i z i n g u n i t s i n t h e c e l l . I t i s on t h e s e s t r u c t u r e s t h a t t h e g e n e t i c i n f o r m a t i o n c a r r i e d i n t h e f o r m o f b a s e t r i p l e t c o d o n s i n t h e mRNA i s t r a n s -l a t e d i n t o t h e p r i m a r y s t r u c t u r e o f newly s y n t h e s i z e d d p r o t e i n . The t r a n s l a t i o n p r o c e s s i s m e d i a t e d by a c l a s s o f low m o l e c u l a r - 25 -weight RNA, the t r a n s f e r RNA (tRNA). D i f f e r e n t tRNA molecules.; are s p e c i f i c f o r each of the twenty amino acids and i n the presence of s p e c i f i c aminoacyl-tRNA synthetases, ATP and amino a c i d s , form aminoacyl-tRNA 1s. In t h i s form, the amino a c i d i s c h e mically a c t i v a t e d , being attached c o v a l e n t l y by an e s t e r bond to the 3'-0Hor 2V-0H of the tRNA. Each tRNA contains an anti-codon r e g i o n i n the form of a base t r i p l e t s p e c i f i c f o r the amino a c i d which i t c a r r i e s . This anti-codon r e g i o n presumably i n t e r a c t s s p e c i f i c a l l y w i t h the codon of the mRNA during the t r a n s l a t i o n process. In E. ooli, at l e a s t , the i n i t i a t i o n of p r o t e i n s y n t h e s i s i s coded f o r on the mRNA (AUG) and re q u i r e s N-formylmethionyl-tRNA as the i n i t i a t i n g tRNA. A f t e r i n i t i a t i o n , the stepwise a d d i t i o n of amino acid s to the growing polypeptide chain i s accomplished by a n u c l e o p h i l i c replacement r e a c t i o n . The amino group of the newly s e l e c t e d aminoacyl-tRNA attac k s the es t e r bond of the peptidyl-tRNA and d i s p l a c e s the tRNA from the growing end of the polypeptide chain. One or more enzymes (trans -ferases) may be in v o l v e d i n t h i s r e a c t i o n . At l e a s t two s i t e s i n the ribosome are i n v o l v e d i n the t r a n s f e r r e a c t i o n (1) the de-coding s i t e which s e l e c t s the aaminoacy1-tRNA s p e c i f i e d by the mRNA and (2) the condensing s i t e which keeps the peptidyl-tRNA p r o p e r l y a l i g n e d f o r peptide bond formation. As the peptide bond i s formed, the nascent peptide chain i s t r a n s f e r r e d from the tRNA i n the condensing s i t e to the incoming aminoacyl-tRNA lodged i n the decoding s i t e . To repeat the process of chain extension, the decoding s i t e i s c l e a r e d i n a t r a n s l o c a t i o n step a s s o c i a t e d w i t h the h y d r o l y s i s of GTP. In t h i s step, the mRNA advances by one - 26 -t r i p l e t codon and the peptidyl-tRNA i s returned to the conden-s i n g s i t e . The decoding s i t e remains vacant u n t i l f i l l e d w i t h the aminoacyl-tRNA s p e c i f i e d by the newly exposed codon, thus s t a r t i n g a new c y c l e . A f t e r a s e r i e s of t r a n s l o c a t i o n s , when the ribosome has. completed i t s t r a n s i t of the mRNA molecule, the completed polypeptide chain i s rel e a s e d and the ribosome d i s -s o c i a t e s from the mRNA. I t has been suggested t h a t s p e c i f i c t e r m i n a t i n g codons e.g. amber (UAG) and ochre (UAA), and pol y -peptide chain r e l e a s i n g f a c t o r s (R f a c t o r s ) are r e q u i r e d f o r t h i s f i n a l step. The mechanism of p r o t e i n synthesis only b r i e f l y d e s cribed here has been the f o c a l p o i n t of molecular b i o l o g y i n the l a s t decade, and has been e x t e n s i v e l y reviewed (60, 155-162). I t i s q u i t e apparent t h a t the ribosome plays a c e n t r a l r o l e i n p r o t e i n b i o s y n t h e s i s . I t i s a l s o now recognized t h a t the r i b o -some i s of u n i v e r s a l occurrence (63) being found i n a l l l i v i n g c e l l s whether from micro-organisms, p l a n t s or animals; the gener-a l b a s i c s t r u c t u r e of the ribosome i s the same i r r e s p e c t i v e of source. From e l e c t r o n microscopic s t u d i e s of p u r i f i e d ribosomes i t i s seen t h a t they are l a r g e , approximately s p h e r i c a l macro-molecules w i t h diameters of about 200-250 A (165). Molecular weights v a r y i n g from 2.6 x 10§ f o r E. ooli ribosomes (163) to 3.5-5.0 x 10 B f o r mammalian ribosomes (63, 164) have been o b t a i n -ed, and the sedimentation c o e f f i c i e n t s reported f o r complete s i n g l e ribosomes i s o l a t e d from a wide v a r i e t y of organisms range between 68S and 83S (63). In gen e r a l , the ribosomes from animals are found i n the top range and those from b a c t e r i a i n the bottom range; these are g e n e r a l l y c l a s s i f i e d as "SOS" and "70S" ribosomes r e s p e c t i v e l y . A n a l y s i s shows t h a t the only c o n s t i t u e n t s of - 27 -ribosomes are p r o t e i n and RNA, and i n animals, the proportions of p r o t e i n s to RNA are c l o s e t o 1:1 (166, 167). Ribosomes are composed of two r e v e r s i b l y d i s s o c i a b l e sub-units which are depen-dent on magnesium i o n c o n c e n t r a t i o n and i o n i c s t r e n g t h (159). One of the subunits i s about h a l f the s i z e of the other (159); i n animal c e l l s these subunits have sedimentation c o e f f i c i e n t s of about 6 0S and 4OS r e s p e c t i v e l y (63). The l a r g e r subunit con-t a i n s a 28S RNA molecule and the smaller subunit an 18S RNA (168); i n a d d i t i o n , RNA species of 5S (159) and p o s s i b l y a l s o a 7S (168) are a s s o c i a t e d w i t h the l a r g e r subunit. Ribosomal s i t e s f o r mRNA (160,169) and aminoacyl-tRNA (170,171) are l o c a t e d i n the s m a l l e r subunit w h i l e the l a r g e r subunit contains the b i n d i n g s i t e f o r the peptidyl-tRNA (170). Recently by a s e r i e s of e x p e r i -ments (172, 173, 183) on the r e c o n s t i t u t i o n of the p r o t e i n s and RNA of ribosomes, some l i g h t has been thrown on the r e l a t i o n s h i p s between the s t r u c t u r e and f u n c t i o n of ribosome components. Data have accumulated which i n d i c a t e that the a c t i v e c e l l u l a r component i n p r o t e i n s y n t h e s i s c o n s i s t s of ribosomal c l u s t e r s held together by mRNA, the polyribosomes (60, 155). I t i s a l s o c l e a r from work on a wide v a r i e t y of c e l l s t h a t l i k e ribosomes, p o l y -somes are of u n i v e r s a l occurrence (60, 155). While i t i s not easy to f u r n i s h convincing evidence t h a t the ribosomes of the p o l y -somes are l i n k e d through mRNA there i s p l e n t y of c i r c u m s t a n t i a l evidence (156) and no r e a l evidence against i t . Moreover, recen-t l y Laycock and Hunt (174) have i s o l a t e d from the polysomes of r a b b i t r e t i c u l o c y t e s a mRNA which i n the presence of an E. coli c e l l - f r e e system of ribosomes d i r e c t e d synthesis of the g l o b i n of r a b b i t hemoglobin. As a measure of the s t a b i l i t y of mRNAsy• - 28 -t h e r a t e o f p o l y s o m e d e g r a d a t i o n h a s b e e n examined i n c e l l s i n c u b a t e d w i t h a c t i n o m y c i n D t o i n h i b i t RNA s y n t h e s i s . By t h i s and o t h e r t e c h n i q u e s i n v o l v i n g p u l s e - l a b e l l i n g , i t has be e n e s t i m a t e d t h a t t h e h a l f - l i f e o f a b a c t e r i a l mRNA i s o f t h e o r d e r o f 2-3 m i n , (175, 176) w h i l e t h a t o f a n i m a l e.g. i n r a t l i v e r , i s much l o n g e r up t o 80 h o u r s (177). I t has be e n s u g g e s t e d t h a t t h e number o f r i b o s o m e s on a p o l y s o m e i s r e l a t e d t o t h e s i z e o f t h e mRNA c h a i n (156) and t h a t t h e s i z e o f a p o l y s o m e s y n t h e s i z i n g a p a r t i c u l a r p r o t e i n c a n be p r e d i c t e d i f t h e s i z e o f t h e p r o t e i n i s known (50). E x a m i n a t i o n o f f r a c t i o n a t e d c e l l o r g a n e l l e s h as r e v e a l e d t h a t b e s i d e b e i n g i n t h e c y t o p l a s m , r i b o s o m e s a r e a l s o p r e s e n t i n t h e n u c l e u s (37-40) and m i t o c h o n d r i a (154) o f a n i m a l c e l l s . B o t h DNA and RNA have b e e n o b s e r v e d i n t h e c e n t r i o l e s by c y t o -c h e m i c a l methods (178) b u t no r i b o n u c l e o p r o t e i n p a r t i c l e has y e t be e n i s o l a t e d . V a r i o u s i n v e s t i g a t i o n s (37-40) have shown t h e p r e s e n c e o f r i b o s o m e s f u l l y a c t i v e i n p r o t e i n s y n t h e s i s i n t h e n u c l e i o f ' a n i m a l c e l l s . M c C a r t y et al. (39) have i s o l a t e d r i b o s o m e s f r o m p u r i f i e d r a t l i v e r n u c l e i and have f o u n d t h a t t h e s e d i m e n t a t i o n v e l o c i t i e s a s w e l l as t h e a b i l i t y t o s y n t h e s i z e p r o t e i n in vitro were s i m i l a r t o c y t o p l a s m i c r i b o s o m e s . F u r t h e r , s i n c e b o t h t h e n u c l e a r r i b o s o m e s and t h e c y t o p l a s m i c r i b o s o m e s have s i m i l a r b a s e r a t i o s f o r t h e i r r i b o s o m a l RNA, i t was s u g g e s t e d t h a t t h e s e r i b o s o m e s a r e d e r i v e d f r o m t h e same g e n e t i c l o c i . T h e r e have a l s o b e e n s u g g e s t i o n s t h a t DNA may a c t as t e m p l a t e f o r p r o t e i n s y n t h e s i s by n u c l e a r r i b o s o m e s (41, 42). - 29 -Mitochondria have been shown to be rath e r autonomous organ-e l l e s , and m i t o c h o n d r i a l DNA (179, 180) ribosomes (154) and tENA (181) have been i s o l a t e d . O'Brien and K a l f (68) have obtained ribosomal p a r t i c l e s sedimenting at 55S from c a r e f u l l y p u r i f i e d r a t l i v e r mitochondria. They have shown t h a t only the 55S r i b o -somes became l a b e l l e d when mitochondria were incubated in vitro w i t h r a d i o a c t i v e l e u c i n e , and th a t the diameter of 145 A f o r the i s o l a t e d ribosomes corresponded w e l l w i t h reported values from e l e c t r o n micrographs of i n t r a m i t o c h o n d r i a l ribosomes (100-150 A). I t i s i n t e r e s t i n g to note t h a t s i m i l a r S. values have been repor-ted f o r c h l o r o p l a s t ribosomes (182) and thus suggest t h a t a l l p l a s t i d s may have ribosomes even smaller than the 70S b a c t e r i a l ribosomes. While the synthesis of a s p e c i f i c p r o t e i n by mito-c h o n d r i a l ribosomeshas not yet been observed, i s o l a t e d mitochon-d r i a are able to incor p o r a t e r a d i o a c t i v e amino acids i n t o pro-t e i n (154). F u r t h e r , as i n b a c t e r i a , t h i s i n c o r p o r a t i o n i s very s e n s i t i v e to i n h i b i t i o n by chloramphenicol but r e s i s t a n t to c y c l o -heximide (154) and i n t h i s respect d i f f e r s s i g n i f i c a n t l y from the microsomal system. Recently, i t has been suggested from i n h i b i t o r s t u d i e s t h a t i n Neurospora, the p r o t e i n s of the mito-c h o n d r i a l ribosomes are synthesized by cytoplasmic ribosomes (184). This suggests t h a t p r o t e i n s y n t h e s i s by mitochondria and cy t o -plasmic microsomes may be c l o s e l y coupled. In the b a c t e r i a l system, the r e g u l a t i o n of p r o t e i n synthesis appears to be adequately explained by the concept of the "operon" (185) and the recent i s o l a t i o n of repressor p r o t e i n s (186-187) from E. coli has g r e a t l y supported t h i s hypothesis. Since i n the - 30 -operon concept, p r o t e i n synthesis i s regulated at the t r a n s -c r i p t i o n a l l e v e l , a necessary fe a t u r e of the concept r e q u i r e s t h a t mRNAs are s h o r t - l i v e d and r a p i d l y t u r n i n g over; t h i s has been demonstrated i n various b a c t e r i a l systems (60, 158). How-ever, as already mentioned, u n l i k e b a c t e r i a , animal c e l l s con-t a i n mRNAs wi t h r e l a t i v e l y long h a l f - l i v e s ; hence, the c o n t r o l of p r o t e i n s y n t h e s i s may be on the t r a n s l a t i o n a l l e v e l . As a p o s s i b l e example of t r a n s l a t i o n a l c o n t r o l , i t has been shown that u n f e r t i l i z e d eggs of various organisms do not synthesize p r o t e i n (188) but a f t e r f e r t i l i z a t i o n and i n the absence of RNA synthesis ( i n h i b i t e d by actinomycin D) p r o t e i n i s synthesized, and the eggs are capable of developing normally to the b l a s t u l a stage (189). This suggests t h a t the synthesis of s p e c i f i c p r o t e i n s necessary f o r p o s t - f e r t i l i z a t i o n development i s r e g u l a t e d at the t r a n s l a -t i o n a l l e v e l . A l s o , i t has been shown by Monroy et at. (190) that ribosomes i s o l a t e d from u n f e r t i l i z e d eggs are i n a c t i v e f o r in vitro p r o t e i n s y n t h e s i s , but a f t e r a treatment w i t h t r y p s i n , the f o r -merly i n a c t i v e p r e p a r a t i o n now a c t i v e l y incorporates l a b e l l e d amino acid s even i n the absence of added exogenous mRNA. Hence, i t i s p o s s i b l e that masked mRNAs may be stored i n the c e l l c y t o -plasm i n d i s c r e t e p a r t i c l e s of r i b o n u c l e o p r o t e i n e.g. informosomes (188) or a s s o c i a t e d w i t h ribosomes i n the form of i n a c t i v e p o l y -somes (191). C o n t r o l of p r o t e i n synthesis t h e r e f o r e may i n v o l v e the d i f f e r e n t i a l a c t i v a t i o n of s p e c i f i c mRNAs or polysomes already present i n the cytoplasm. That mRNA may a l s o be r e v e r s i b l y i n -a c t i v a t e d by a masking process has been suggested (192). In syn-chronized Chinese hamster c e l l s (193) i t has been shown tha t dur-i n g metaphase when there i s no p r o t e i n s y n t h e s i s , polysomes are - 31 -d i s s o c i a t e d b u t r e f o r m a g a i n a f t e r m i t o s i s e v e n i n t h e p r e s e n c e o f a c t i n o m y c i n D. T h e s e p i e c e s o f e v i d e n c e s u g g e s t t h a t mRNA i n a n i m a l c e l l s i s s t a b l e and may e x i s t i n a v a r i e t y o f fo r m s d e p e n -d i n g on t h e s t a g e o f d e v e l o p m e n t w i t h i n t h e c e l l ; t h e r e g u l a t o r y mechanisms w h i c h g o v e r n t h e a c t i v i t y o f mRNA c o n s t i t u t e s r e g u l a -t i o n a t t h e l e v e l o f t r a n s l a t i o n . The s a l m o n i d t e s t i s p r o v i d e s an i d e a l s y s t e m f o r s t u d y i n g v a r i o u s a s p e c t s o f p r o t e i n b i o s y n t h e s i s . F o r example, f r o m t h e s e q u e n c e s o f c l u p e i n e ( F i g . 1 ) , i t may be s e e n t h a t a r g i n i n e o c c u r s i n b l o c k s o f 2-4 r e s i d u e s . S i n c e i t h as be e n shown t h a t t h e RNA codons f o r t h e 20 amino a c i d s a r e i n many c a s e s d e g e n e r a t e (19 4 ) , e.g. 6 d i f f e r e n t c o d o n s h as b e e n a s s i g n e d t o t h e amino a c i d a r g i -n i n e ( 1 9 5 ) , and a s i m i l a r m u l t i p l i c i t y o f tRNAs has be e n o b s e r v e d ( 1 9 6 ) , i t w o u l d be o f i n t e r e s t t o d i s c o v e r w h e t h e r a l l t h e a r g i n -i n e i n p r o t a m i n e u s e s t h e same t r i p l e t c o d o n . I n t h e s y n t h e s i s o f r a b b i t h e m o g l o b i n f o r example, i t h as been o b s e r v e d t h a t t h e t r a n s -f e r o f a r g i n i n e i n t o d i f f e r e n t p o s i t i o n s i n t h e p r o t e i n i s accom-p l i s h e d by d i f f e r e n t tRNAs (197) . The s i g n i f i c a n c e o f u t i l i z a t i o n o f v a r i o u s m u l t i p l e c o d o n s i n p r o t e i n s y n t h e s i s i s n o t known a l -t h o u g h A n d e r s o n (198) h as s u g g e s t e d t h a t a p o s s i b l e r o l e o f some o f t h e c o d o n s may be f o r t h e c o n t r o l o f t h e r a t e o f p r o t e i n s y n -t h e s i s and he has named them r e g u l a t o r y c o d o n s . I n t h i s c o n t e x t , he h as shown t h a t in vitro,, t h e r a t e o f p r o t e i n s y n t h e s i s u s i n g s y n t h e t i c mRNA i s a f u n c t i o n o f t h e c o n c e n t r a t i o n o f tRNA and t h i s s u p p o r t s t h e p o s t u l a t e o f many w o r k e r s t h a t tRNA i s i n v o l v e d i n t h e t r a n s l a t i o n a l c o n t r o l o f p r o t e i n s y n t h e s i s (194, 199, 2 0 0 ) . S i n c e i n t h e f i s h t e s t i s , t h e s y n t h e s i s o f a s p e c i f i c p r o t e i n , p r o t a m i n e , i s " t u r n e d - o n " a t a s t a g e o f d i f f e r e n t i a t i o n when t h e - 3 2 -general r a t e of p r o t e i n s y n t h e s i s i s decreasing and the nuclear genome i s i n a c t i v e , i t provides an i d e a l system to examine the p o s s i b l e r e g u l a t i o n by tRNA of the s y n t h e s i s of a s p e c i f i c pro= t e i n . Studies on the tRNA of salmon t e s t i s have already, been i n i t i a t e d (201). I t has been shown tha t the p r o f i l e s of tRNA separated on a BD-cellulose. column (213) are d i f f e r e n t between the young and the more mature t e s t i s . C o r r e l a t i o n between these observations and the p r o t e i n s y n t h e s i z i n g a c t i v i t y of these t e s t e s should prove i n t e r e s t i n g . Progress towards a f u l l understanding of the mechanism and c o n t r o l of p r o t e i n synthesis demands a d e t a i l e d knowledge of the nature of the product synthesized, and f o r t h i s reason s t u d i e s i n p r o t e i n b i o s y n t h e s i s must u l t i m a t e l y be concerned w i t h s i n g l e , w e l l - d e f i n e d p r o t e i n s . The in vitro synthesis of s p e c i f i c pro-t e i n s i n animals have been s t u d i e d i n r e l a t i v e l y few systems (202). The main area of i n v e s t i g a t i o n has centered on the synthesis of hemoglobin (155). One of the major problems encountered i n these in vitvo s t u d i e s has been the d i f f i c u l t y of p o s i t i v e l y i d e n t i f y i n g and c h a r a c t e r i z i n g the newly synthesized product. Ingles et al. (11) have already demonstrated t h a t protamine i s synthesized by the usual route of p r o t e i n synthesis i n v o l v i n g mRNA, ribosomes, and tRNA. The low molecular weight and h i g h l y unusual amino a c i d composition of protamine bestow unusual p r o p e r t i e s on t h i s p r o t e i n which all o w i t to be. separated and i d e n t i f i e d . The s i t e of synth e s i s of nuclear b a s i c p r o t e i n s have been examined i n a v a r i e t y of systems and the r e s u l t s are not completely uniform. In the c a l f thymus system, f o r example, Reid and Cole - 33 -(44) have shown t h a t a w e l l c h a r a c t e r i z e d l y s i n e - r i c h h i s t o n e i s synthesized by i s o l a t e d n u c l e i and hence suggest a nuclear s i t e of s y n t h e s i s f o r h i s t o n e s . These observations are i n agreement w i t h the autoradiographic s t u d i e s of A l l f r e y et al. (43). On the other hand, Robbins and Borun (45) and more r e c e n t l y , G a l l w i t z and M u e l l e r (203) have shown i n synchronized Hela c e l l s , t hat histones are synthesized by small cytoplasmic polysomes and are subsequently t r a n s p o r t e d i n t o the nucleus. Further,. Bloch and Brack (107) have shown by autoradiography t h a t i n the spermatids of grasshopper, the a r g i n i n e - r i c h h i s t o n e i s synthesized i n the cytoplasm and l a t e r move i n t o the nucleus. At present i t i s not known what s i g n i f i c a n c e i s a s s o c i a t e d w i t h the nuclear or cyto-plasmic s i t e of histo n e s y n t h e s i s . I t i s t h e r e f o r e of i n t e r e s t to d i s c o v e r the i n t r a c e l l u l a r s i t e of protamine s y n t h e s i s . The mRNA of protamine should a l s o be an i d e a l candidate f o r i n v e s t i g a t i o n . As already mentioned, protamine appears to be synthesized on a s t a b l e mRNA template (11) and may be amenable f o r s t u d i e s on t r a n s l a t i o n a l c o n t r o l of p r o t e i n s y n t h e s i s . Fur-t h e r , the mRNA, i f monocistronic, would be composed of only about 100 n u c l e o t i d e s and the sequencing of a n a t u r a l mRNA of t h i s l ength may be f e a s i b l e . With the high content of a r g i n i n e i n the protamine molecule (see F i g . 1) the protamine mRNA should possess rep e a t i n g sequences of codons f o r a r g i n i n e . The work to be reported i n t h i s t h e s i s was undertaken to study the b i o s y n t h e s i s of protamine. Towards t h i s end, a method employing chromatography on Bio-Gel P-10 and CM-cellulose has been developed to i d e n t i f y and c h a r a c t e r i z e newly synthesized - 34 -protamine. I t was found t h a t the nascent protamine i s more h i g h l y phosphorylated than preformed protamine already present i n the t e s t i s c e l l and the two behave d i f f e r e n t l y on CM-cellulose. C e r t a i n aspects concerning the phosphorylation of newly synthes-i z e d protamine and i t s subsequent dephosphorylation have been examined and w i l l be described. Since the s i t e of synthesis of a nuclear p r o t e i n i s a subject of some d i s p u t e , a major p o r t i o n of t h i s study has been devoted towards the discovery of the i n t r a -c e l l u l a r s i t e of protamine s y n t h e s i s . Several l i n e s of evidence are presented to show tha t protamine i s synthesized i n the cyto-plasmic microsomes of spermatid c e l l s . C h a r a c t e r i z a t i o n of the t e s t i s polysomes shows that protamine i s synthesized on a s p e c i a l c l a s s of polysomes, the di-ribosomes (disomes). The p r o p e r t i e s of these disomes are c h a r a c t e r i z e d . C e r t a i n developmental changes a s s o c i a t e d w i t h the t e s t i s polysomes and the b i o s y n t h e s i s of d i f f e r e n t protamine components have a l s o been examined. - 35 -MATERIALS AND METHODS I. Chemicals and Abbreviations a) Chemicals Common chemicals obtained commercially were of the highest p u r i t y or reagent grade. Radioactive amino a c i d s : 1 ^ C - a r g i n i n e and lkC-lysine (uniformly l a b e l l e d , 226-243 mC/mM) and 3 H - a r g i n i n e (1.1 C/mM) were obtained from New England Nuclear Corporation; r a d i o a c t i v e 3 2P-phosphate, c a r r i e d f r e e , from Atomic Energy of Canada, L t d . ; adenosine 5 1 - t r i p h o s p h a t e (disodium) (ATP) and guanosine 5 1 - t r i p h o s p h a t e (trisodium) (GTP), from Calbiochem; 2-mercaptoethanol, from Eastman Organic Chemicals; cycloheximide, from Calbiochem; Hanks' balanced s a l t s o l u t i o n , from Baltimore B i o l o g i c a l Laboratory; Bio-Gel P-10 and CM-cellulose, from B i o -Rad L a b o r a t o r i e s ; CM-52, from Whatman; T r i t o n X-100, from Rohm and Haas; and sucrose, d e n s i t y - g r a d i e n t grade ( r i b o n u c l e a s e - f r e e ) , from Mann Research L a b o r a t o r i e s , Inc. b) A b b r e v i a t i o n s The use of common a b b r e v i a t i o n s , terminology and symbols i n t h i s t h e s i s g e n e r a l l y conforms to the usage of the Journa l of B i o l o g i c a l Chemistry ( J . B i o l . Chem. 24 3, 1, 1968). TCA :- t r i c h l o r o a c e t i c a c i d TCA-tungstate :- a p r e c i p i t a n t f o r p r o t e i n described by Gardner et al. (16) c o n t a i n i n g 5% t r i c h l o r o a c e t i c a c i d and 0.25% sodium tungstate at pH 2. TMKS :- an i s o t o n i c medium s i m i l a r to Medium A of L i t t l e f i e l d and K e l l e r (6) c o n t a i n i n g T r i s - H C l 50 mM (pH 7.6), magnesium acetate 5 mM, potassium c h l o r i d e 25 mM, and sucrose 0.25 M. - 36 -TMK :- a b u f f e r c o n t a i n i n g magnesium and p o t a s s i u m i d e n t i c a l w i t h TMKS e x c e p t t h a t i t c o n t a i n s no s u c r o s e . TMC :- a h y p o t o n i c T r i s b u f f e r u s e d i n c o n j u n c t i o n w i t h v i g o r o u s h o m o g e n i z a t i o n t o r u p t u r e t e s t i s c e l l n u c l e i ; t h i s b u f f e r c o n t a i n s T r i s - H C l 10 mM (pH 7.8).magnesium a c e t a t e 0.5 mM, and c y c l o h e x i m i d e 50 yM. PMS :- p o s t - m i t o c h o n d r i a l s u p e r n a t a n t , u s u a l l y t h e s u p e r n a t a n t o b t a i n e d f r o m t h e c e n t r i f u g a t i o n o f a c e l l homogenate a t 15,000 x g f o r 15 m i n . PNS :- p o s t - n u c l e a r s u p e r n a t a n t , t h e s u p e r n a t a n t o b t a i n e d f r o m c e n t r i f u g a t i o n o f a c e l l homogenate a t 1,000 x g f o r 10 m i n . C M - c e l l u l o s e :- c a r b o x y - m e t h y l - c e l l u l o s e i o n exchange r e s i n . CM-52 :- a m i c r o - g r a n u l a r f o r m o f C M - c e l l u l o s e m a n u f a c t u r e d by Whatman. T r i - R t i s s u e h o m o g e n i z e r : - ( T r i - R I n s t r u m e n t s , New Y o r k ) , a l l h o m o g e n i z a t i o n s were p e r f o r m e d w i t h t h i s h o m o g e n i z e r ; i t c o n s i s t s o f a g l a s s - r e i n f o r c e d t e f l o n p e s t l e and a 30 m l c a p a c i t y g l a s s h o m o g e n i z i n g t u b e . The p e s t l e t o t u b e c l e a r a n c e i s between 0.006 - 0.009 i n c h . I I . I s o l a t i o n and C h a r a c t e r i z a t i o n o f P r o t a m i n e and R a d i o a c t i v e P r o t a m i n e a) S o u r c e o f P r o t a m i n e P r o t a m i n e may be i s o l a t e d f r o m t h e t e s t i s o f s a l m o n i d and r e l a t e d f i s h a t a l a t e s t a g e o f s p e r m a t o g e n e s i s ( 1 , 2 , 3 ) . I n t h i s s t u d y , t h e r a i n b o w t r o u t (Salmo gairdnerii) was t h e main s u b j e c t o f i n v e s t i g a t i o n a l t h o u g h on o c c a s i o n t h e t e s t i s o f t h e s t e e l h e a d t r o u t , an anadromous s t r a i n o f Salmo gairdnerii was a l s o employed. - 37 -Testes at v a r i o u s stages of spermatogenesis were obtained during the months of September to December from n a t u r a l l y maturing rainbow t r o u t (13-18 months old) r a i s e d at the Sun V a l l e y Trout Farm, Coquitlam, B r i t i s h Columbia. Testes were removed from the t r o u t , immediately placed on i c e and transported to the l a b o r a -t o r y by automobile; the whole process took about two hours. The t e s t e s were then b r i e f l y r i n s e d w i t h c o l d running tap water and excess l i q u i d removed by p a t t i n g the t e s t e s on absorbent paper towels. Testes not immediately r e q u i r e d were frozen on dry i c e and stored at -80°. Spermatogenesis was induced i n aquarium-kept t r o u t by i n j e c -t i o n s of salmon p i t u i t a r y e x t r a c t s (4). The sources and husbandry of these t r o u t have p r e v i o u s l y been described i n d e t a i l (5). In essence, s e x u a l l y immature rainbow t r o u t and steelhead t r o u t were kept i n aquaria (at the Department of Biochemistry,. U n i v e r s i t y of B r i t i s h Columbia) i n f r e s h running water (2-4 l i t e r s per minute) and constant temperature (12°-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 e x t r a c t obtained from spawning salmon (5). Protamine synthesis began s i x to e i g h t weeks a f t e r the i n i t i a l i n j e c t i o n and t e s t e s obtained during t h i s p e r i o d were s u i t a b l e f o r experimentation. Since protamine i s a chromosomal p r o t e i n l o c a t e d i n the n u c l e i of t e s t i s c e l l s at the appropriate stage of development, o f t e n i t was u s e f u l to prepare n u c l e i of t e s t i s c e l l s as a purer s t a r t i n g m a t e r i a l f o r the i s o l a t i o n of protamine. Testes were minced by s c i s s o r s i n two volumes of c o l d TMKS pH 7.6 and homogenized w i t h the T r i - R t i s s u e homogenizer at 5,000 rpm f o r 20 sec. The - 38 -homogenate was c e n t r i f u g e d a t 1,000 x g f o r 10 m i n , and t h e s e d i m e n t c o n t a i n i n g t h e n u c l e i was c o l l e c t e d and washed o n c e more i n TMKS by c e n t r i f u g a t i o n . T h i s washed n u c l e a r p e l l e t was t h e n s u s p e n d e d i n 0.2 M H 2SOM. f o r e x t r a c t i o n o f n u c l e a r b a s i c p r o t e i n s . b) C e l l I n c u b a t i o n T e s t e s f o r i n c o r p o r a t i o n o f r a d i o a c t i v e amino a c i d s were o b t a i n e d a t a s t a g e o f s p e r m a t o g e n e s i s when p r o t a m i n e was a c t i v e l y s y n t h e s i z e d i n e i t h e r n a t u r a l l y m a t u r i n g t r o u t o r h o r m o n a l l y i n -d u c e d t r o u t a s a l r e a d y d e s c r i b e d . Ifctwas f o u n d t h a t f r e s h l y ex-c i s e d t e s t e s c o u l d be k e p t on i c e a t 0° f o r a t l e a s t 4 h o u r s w i t h o u t any s i g n i f i c a n t l o s s o f i n c o r p o r a t i n g a b i l i t y o f l a b e l l e d amino a c i d s i n t o p r o t a m i n e . A l l o p e r a t i o n s p r i o r t o i n c u b a t i o n were p e r f o r m e d a t 0 ° - 4 ° . T e s t i s t i s s u e m i n c e d u s i n g s c i s s o r s i n 3 volumes o f Hanks' b a l a n c e d s a l t s o l u t i o n o r i n TMKS. The m i n c e d t i s s u e s u s p e n s i o n was h o m o g e n i z e d by hand u s i n g two o r t h r e e up and down s t r o k e s w i t h t h e T r i - R t i s s u e h o m o g e n i z e r t o s e p a r a t e t h e c e l l s . T h i s homogenate was s t r a i n e d t h r o u g h 4 l a y e r s o f c h e e s e - c l o t h t o remove c o n n e c t i v e t i s s u e and any l a r g e p i e c e s o f i n t a c t t i s s u e . The s t r a i n e d homogenate c o n t a i n e d e s s e n t i a l l y o n l y s i n g l e c e l l s i n s u s p e n s i o n ( 2 7 ) . The c e l l s were p e l l e t e d by c e n t r i f u g a t i o n (1,000 x g f o r 10 min) and r e s u s p e n d e d i n a b o u t 2 volumes o f e i t h e r Hanks' s o l u t i o n o r TMKS f o r i n c u b a t i o n . I n c u b a t i o n s were c a r r i e d o u t a t 20° i n a g y r a t o r y w a t e r b a t h (New B r u n s w i c k ) and were s t a r t e d by t h e a d d i t i o n o f a l a b e l l e d amino a c i d a t a f i n a l c o n c e n t r a t i o n o f 0.1-1.0 uC/ml o f i n c u b a t i o n - 39 -m i x t u r e as a p p r o p r i a t e . I n some e x p e r i m e n t s , t h e i n c u b a t i o n medium was s u p p l e m e n t e d w i t h u n l a b e l l e d amino a c i d s , b u t i t was f o u n d t h a t f o r t h e p e r i o d o f i n c u b a t i o n o f 1 o r 2 h o u r s , t h e a d d i t i o n o f u n l a b e l l e d amino a c i d s d i d n o t s t i m u l a t e t h e i n c o r -p o r a t i o n o f t h e l a b e l l e d amino a c i d . T h i s s u g g e s t e d t h a t t h e amino a c i d p o o l s i n t h e c e l l s were n o t l i m i t i n g f o r p r o t e i n s y n -t h e s i s d u r i n g t h e t i m e p e r i o d o f i n c u b a t i o n . I n c u b a t i o n s were t e r m i n a t e d by t h e a d d i t i o n o f a c o n c e n t r a t e d s o l u t i o n o f c y c l o -h e x i m i d e t o a f i n a l c o n c e n t r a t i o n o f 0.6 mM. C y c l o h e x i m i d e a t t h i s c o n c e n t r a t i o n has p r e v i o u s l y b e e n shown t o be a p o t e n t i n -h i b i t o r o f a r g i n i n e i n c o r p o r a t i o n i n t o p r o t a m i n e by t e s t i s c e l l s (11). I n some e x p e r i m e n t s , i n c u b a t i o n s were t e r m i n a t e d by c o o l i n g t h e i n c u b a t i o n m i x t u r e t o 0° o r by f r e e z i n g t h e m i x t u r e on d r y i c e . c) A c i d E x t r a c t i o n o f T e s t i s B a s i c P r o t e i n s ( i ) U n l a b e l l e d b a s i c p r o t e i n s B a s i c p r o t e i n s i n c l u d i n g p r o t a m i n e were e x t r a c t e d f r o m t e s t i s by a method m o d i f i e d f r o m K o s s e l (1). T e s t i s c e l l s o r c e l l f r a c -t i o n s were s u s p e n d e d i n f i v e v o l u m e s o f 0.2 M H 2S0if, b r i e f l y h o m o g e n i z e d by t h e T r i - R h o m o g e n i z e r , and a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r t w e n t y m i n u t e s . The a c i d e x t r a c t was c o l l e c t e d by c e n t r i f u g a t i o n and t h e s e d i m e n t was r e - e x t r a c t e d by t h e same p r o -c e d u r e . The two a c i d e x t r a c t s were p o o l e d and p r o t e i n p r e c i p i t a t e d by t h e a d d i t i o n o f t h r e e v o l u m e s o f e t h a n o l and l e f t a t - 2 0 ° f o r s e v e r a l h o u r s . The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n , washed w i t h e t h a n o l , d i s s o l v e d i n H 2 O , t h e pH a d j u s t e d t o n e a r n e u t r a l i t y as i n d i c a t e d by pH p a p e r , and t h e s o l u t i o n a p p l i e d t o a - 40 -s m a l l C M - c e l l u l o s e column (1 x 7 cm, H f o r m ) . F o l l o w i n g e x t e n -s i v e w a s h i n g w i t h H 2 0 (200-500 mis ) t h e b a s i c p r o t e i n s were e l u t e d f r o m t h e column w i t h 0.2 M HC1. The e l u a t e was l y o p h i l i z e d i n p r e p a r a t i o n f o r f r a c t i o n a t i o n by c h r o m a t o g r a p h y , ( i i ) L a b e l l e d b a s i c p r o t e i n s R a d i o a c t i v e b a s i c p r o t e i n s were e x t r a c t e d f r o m t h e t e s t i s c e l l s w i t h a c i d as above a f t e r an a p p r o p r i a t e p e r i o d o f i n c o r p o r a -t i o n o f a r a d i o a c t i v e amino a c i d . However, i n o r d e r t o e n s u r e c o m p l e t e r e m o v a l o f t h e c o n t a m i n a t i n g f r e e l a b e l l e d amino a c i d s n o t i n c o r p o r a t e d i n t o p r o t e i n , t h e p r o t e i n s u l f a t e p r e c i p i t a t e d by e t h a n o l was s u b j e c t e d t o a s e c o n d p r e c i p i t a t i o n by r e - d i s s o l v i n g t h e p r e c i p i t a t e i n 0.2 M H 2 S O 4 and r e p r e c i p i t a t i n g w i t h ^ e t h a n o l a t - 2 0 ° . T h i s was f o l l o w e d by an e t h a n o l wash o f t h e p r e c i p i t a t e b e f o r e t r e a t m e n t f o r c h r o m a t o g r a p h y o f t h e p r o t e i n . D u r i n g t h e e a r l y p a r t o f t h i s s t u d y , some a c i d e x t r a c t i o n s f o r n e w l y s y n t h e s i z e d p r o t a m i n e were p e r f o r m e d a t 90° f o r 20 m i n u t e s i n an a t t e m p t t o i n c r e a s e t h e e f f i c i e n c y o f e x t r a c t i o n ; however, i n c r e a s e d t e m p e r a t u r e o f e x t r a c t i o n d i d n o t impjfgive t h e y i e l d and s i n c e t h e r e was a l s o t h e d a n g e r o f p e p t i d e bond c l e a v a g e a t t h i s h i g h e r t e m p e r a t u r e , t h i s method o f e x t r a c t i o n was n o t p u r s u e d f u r t h e r . I n c e r t a i n c a s e s when t h e amount o f endogenous p r o t a m i n e i n t h e t e s t i s was low e.g. d u r i n g t h e e a r l y s t a g e s o f s p e r m a t o g e n e s i s , n o n - r a d i o a c t i v e c a r r i e r p r o t a m i n e (3-4 mg) was a dded p r i o r t o a c i d e x t r a c t i o n o f t h e t i s s u e f o r l a b e l l e d p r o t a m i n e . S i n c e i t was f o u n d t h a t a t t h e l a t e s t a g e o f s p e r m a t o g e n e s i s , p r o t a m i n e i s t h e m a j o r a c i d e x t r a c t a b l e t e s t i s b a s i c p r o t e i n i n t o w h i c h l a b e l l e d a r g i n i n e i s i n c o r p o r a t e d , (see F i g . 5c) a r a p i d p r o c e d u r e f o r t h e measurement o f p r o t a m i n e s y n t h e s i s was d e v e l o p e d . - 41 -C e l l s o r c e l l f r a c t i o n s o b t a i n e d a f t e r t h e i n c o r p o r a t i o n o f r a d i o a c t i v e a r g i n i n e were e x t r a c t e d w i t h a t l e a s t 10 v o l u m e s o f 0.5 M HC1 p l u s 5% TCA f o r 20 m i n u t e s a t 0 ° . The a d d i t i o n o f 5% TCA p r o v i d e s some s p e c i f i c i t y f o r t h e e x t r a c t i o n o f p r o t a m i n e i n t h a t a number o f b a s i c p r o t e i n s e.g. some r i b o s o m a l p r o t e i n s and a r g i n i n e - r i c h h i s t o n e s , a r e s o l u b l e i n 0.5 M HC1 b u t i n s o l u b l e i n t h e m i x e d e x t r a c t a n t . The a c i d e x t r a c t e d p r o t e i n s were r e -c o v e r e d by c e n t r i f u g a t i o n , n e u t r a l i z e d w i t h NaOH, r a d i o a c t i v e p r o t a m i n e p r e c i p i t a t e d by pH 2 T C A - t u n g s t a t e a f t e r t h e a d d i t i o n o f c a r r i e r p r o t a m i n e and t h e p r e c i p i t a t e washed 4-5 t i m e s w i t h T C A - t u n g s t a t e c o n t a i n i n g 1 mM 1 2 C - a r g i n i n e t o remove f r e e l h C -a r g i n i n e . D u r i n g t h e f i n a l wash, t h e m i x t u r e was h e a t e d i n a b o i l i n g w a t e r b a t h f o r 2 m i n u t e s , t h e p r e c i p i t a t e washed o n c e more w i t h e t h a n o l , r e s u s p e n d e d i n a s m a l l volume o f w a t e r and t r a n s f e r r e d t o a g l a s s s c i n t i l l a t i o n v i a l f o r a s s a y o f r a d i o a c t i v -i t y . T h i s more s p e c i f i c a c i d e x t r a c t i o n p r o c e d u r e a l o n g w i t h t h e p r e c i p i t a t i o n s t e p a l l o w s a c o n v e n i e n t and r a p i d measure o f l h C -a r g i n i n e i n c o r p o r a t i o n i n t o p r o t a m i n e , and h e n c e o f p r o t a m i n e s y n t h e s i s . d) B i o - G e l P-10 C h r o m a t o g r a p h y The s e p a r a t i o n o f t h e n u c l e a r b a s i c p r o t e i n s o f t r o u t t e s t i s i n t o h i s t o n e s and p r o t a m i n e by B i o - G e l P - 1 0 . c h r o m a t o g r a p h y has b e e n d e s c r i b e d by I n g l e s and D i x o n (7) and I n g l e s ( 5 ) . Samples o f a c i d e x t r a c t e d t e s t i s p r o t e i n up t o 50 mg were d i s s o l v e d i n s m a l l v o l u m e s ( l e s s t h a n 2 ml) o f 0.2 M a c e t i c a c i d and a p p l i e d t o a B i o - G e l P-10 c olumn (2 x 30 cm) p r e v i o u s l y e q u i l i b r a t e d w i t h 0.2 M a c e t i c a c i d . T h i s c o l u m n was e l u t e d w i t h 0.2 M a c e t i c a c i d - 42 -and f r a c t i o n s c o l l e c t e d were monitored at 230 nm. This wave-length was used as there are no aromatic chromophores i n protamine and hence no absorbance at 280 nm. e) CM-Cellulose Chromatography The protamine i s o l a t e d from the Bib-Gel P-10 column may be f u r t h e r c h a r a c t e r i z e d by chromatography on CM-cellulose prepared according to Schlossman et al. (8). The f r a c t i o n s from the P-10 column comprising the protamine region were l y o p h i l i z e d , d i s s o l v e d i n a small volume of water and a p p l i e d to a CM-cellulose column (1 x 30 cm) e q u i l i b r a t e d w i t h 0.01 M l i t h i u m a c e t a t e - a c e t i c a c i d b u f f e r , pH 5. A l i n e a r g r a d i e n t of l i t h i u m c h l o r i d e prepared i n the same b u f f e r was formed by connecting two i d e n t i c a l c y l i n d e r s c o n t a i n i n g , r e s p e c t i v e l y , 350 ml of 0.2 M and 350 ml of 2.0 M l i t h i u m c h l o r i d e i n s e r i e s . The chamber c o n t a i n i n g the lower s a l t c o n c e n t r a t i o n was s t i r r e d m a g n e t i c a l l y and the e f f l u e n t from i t was used to e l u t e the column. Column e f f l u e n t s were monitored at 230 nm and the s a l t c o n c e n t r a t i o n of the e f f l u e n t s measured by a c o n d u c t i v i t y meter (Radiometer, Copenhagen). In order t o achieve b e t t e r separation of protamine components, shallower l i t h i u m c h l o r i d e gradients of 0.8 to 1.2 M were used together w i t h a micro-granular form of CM-cellulose CM-52. (Whatman). f) Amino A c i d A n a l y s i s of F r a c t i o n a t e d Protamine Components Appropriate f r a c t i o n s corresponding to peaks of protamine separated by CM-cellulose chromatography were pooled and d i l u t e d w i t h 5 volumes of d i s t i l l e d water. The d i l u t e d s o l u t i o n s were passed through a small CM-cellulose column (1 x 7 cm, H form) and the column washed w i t h a l a r g e volume of water (300-500 ml) to - 43 -remove l i t h i u m c h l o r i d e . The adsorbed protamine was e l u t e d w i t h 0.2 M HC1 and the e l u a t e l y o p h i l i z e d . I f a s i g n i f i c a n t amount of s a l t was observed to be s t i l l present at t h i s stage, protamine was d i s s o l v e d i n a small volume of water and then p r e c i p i t a t e d w i t h s e v e r a l volumes of acetone l e a v i n g the s a l t i n s o l u t i o n . The protamine p r e c i p i t a t e was then c o l l e c t e d by c e n t r i f u g a t i o n . Each protamine component was hydrolyzed i n a small volume of 6 N HC1 f o r 16 hours at 105° i n an ampule sealed under vacuum. The hydrolysate was d r i e d in vacuo, r e c o n s t i t u t e d to a known volume w i t h 0.2 M sodium c i t r a t e b u f f e r pH 2.2 and the amino a c i d composition of the hydrolysate determined on a Beckman model 120 C amino a c i d analyzer, g) Measurement of R a d i o a c t i v i t y A l i q u o t s of up to 1.0 ml were d i s s o l v e d d i r e c t l y i n 10 ml of Bray's s c i n t i l l a t i o n f l u i d (18) f o r the assay of r a d i o a c t i v i t y . When i t was necessary to concentrate samples of p r o t e i n contained i n l a r g e volumes, the pH of the sample was adjusted to near n e u t r a l i t y and p r o t e i n p r e c i p i t a t e d w i t h pH 2 TCA-tungstate. The p r e c i p i t a t e was washed once w i t h ethanol by c e n t r i f u g a t i o n , sus-pended i n a small volume of water, t r a n s f e r r e d by Pasteur p i p e t t e to a g l a s s s c i n t i l l a t i o n v i a l , and i t s r a d i o a c t i v i t y assayed i n Bray's f l u i d . A U n i l u x l i q u i d - s c i n t i l l a t i o n counter was used f o r the assay of r a d i o a c t i v i t y and, when necessary, quench c o r r e c t i o n s were performed by the a d d i t i o n of an i n t e r n a l standard to the samples. For a convenient assay of r a d i o a c t i v i t y i n f r a c t i o n s obtained during P-10 chromatography, a l i q u o t s were d r i e d on metal planchets and counted i n a gas-flow system (Nuclear-Chicago). - 44 -h) Digest of Radioactive Protamine w i t h A l k a l i n e Phosphatase About 5 mg of l a b e l l e d protamine was t r e a t e d w i t h 1 mg of E. coli a l k a l i n e phosphatase (BAP-SF 21 u/mg) (Worthington) i n 2 ml of 0.5 M T r i s - H C l pH 7.6 at 30° f o r 3 hours. At the end of the treatment, the s o l u t i o n was d i l u t e d w i t h 4 ml of d i s t i l l e d water and a p p l i e d d i r e c t l y onto a CM-cellulose column f o r chroma-tography w i t h the L i C l system. I I I . I n t r a c e l l u l a r S i t e of Protamine Synthesis a) Sedimentation A n a l y s i s of T e s t i s Cytoplasmic and Nuclear Microsomes 1 ( i ) Pulse-chase i n c o r p o r a t i o n of 1 1*C-arginine A 6 ml c e l l suspension was prepared from 3 g of hormonally induced t r o u t t e s t i s i n TMKS and d i v i d e d i n t o two equal p o r t i o n s , c e l l suspension A and B. The c e l l suspensions were allowed to inc o r p o r a t e 1 1*C-arginine (1 yC/ml) at 20° f o r 0.5 minutes a f t e r which u n l a b e l l e d 1 2 C - a r g i n i n e was added to a f i n a l c o ncentration of 1 mM as a chase. I n c o r p o r a t i o n i n c e l l suspension A was t e r -minated w i t h 0.4 mM cycloheximide a f t e r a 0.5 min chase, and B was s i m i l a r l y terminated a f t e r a 10 min chase. The c e l l s were recovered by c e n t r i f u g a t i o n (1,000 x g, 10 min) and resuspended i n two volumes of TMKS c o n t a i n i n g cycloheximide f o r p r e p a r a t i o n of microsomes. ( i i ) P r e p a r a t i o n of Microsomes The c e l l suspensions obtained a f t e r the pulse-chase i n c u b a t i o n were subjected to homogenization (5,000 rpm, 20 sec) and the homogenate c e n t r i f u g e d at 1,000 x g f o r 10 min to y i e l d a crude nuclear sediment and a post-nuclear supernatant (PNS). The PNS - 45 -was f u r t h e r c e n t r i f u g e d at 15,000 x g f o r 15 min to sediment mitochondria and y i e l d a post-rmitochondrial supernatant (PMS) . The crude nuclear f r a c t i o n was resuspended i n 10 ml of the hypertonic T r i s b u f f e r (TMC) pH 7.8 and homogenized v i g o r o u s l y at 5,000 rpm f o r 2 min to rupture the n u c l e i . That the n u c l e i were s u c c e s s f u l l y ruptured was i n d i c a t e d by a marked increase i n the v i s c o s i t y of the homogenized nuclear suspension probably as a r e s u l t of chromatin being r e l e a s e d i n t o the s o l u t i o n . This homo-genate was c e n t r i f u g e d at 30,000 x g f o r 10 min to sediment the nuclear r e s i d u e . The nuclear supernatant and the PMS were c e n t r i -fuged at 65,000 rpm (.65 r o t o r , Spinco L2-65) f o r 60 min to p e l l e t "nuclear microsomes" and "cytoplasmic microsomes" r e s p e c t i v e l y . A diagram of the steps i n v o l v e d i n t h i s p r e p a r a t i o n i s presented i n F i g . 13. ( i i i ) Sucrose d e n s i t y gradient a n a l y s i s of the microsomes The microsomal p e l l e t s were d i s s o l v e d i n 0.2 ml TMK b u f f e r and l a y e r e d onto a 15-30% l i n e a r sucrose gradient formed i n a SW-39 c e l l u l o s e n i t r a t e c e n t r i f u g e tube. C e n t r i f u g a t i o n was per-formed at 35,000 rpm f o r 1.5 hours i n a SW-39 swinging-bucket r o t o r (Spinco) which was allowed to stop without braking. Approxi-mately equal f r a c t i o n s of 18 drops each were c o l l e c t e d a f t e r punc-t u r i n g the bottom of the c e n t r i f u g e tube; the f r a c t i o n s were a p p r o p r i a t e l y d i l u t e d w i t h water and monitored f o r absorption at 260 nm; c a r r i e r p r o t e i n s (1 mg bovine serum albumin and 0.5 mg protamine) were added to each f r a c t i o n which was then a c i d e x t r a c -ted f o r protamine w i t h 0.5 M HC1 plus 5% TCA as already described. The a c i d e x t r a c t e d protamine was n e u t r a l i z e d , p r e c i p i t a t e d w i t h - 46 -pH 2-TCA-tungstate. The p r e c i p i t a t e was washed and r a d i o a c t i v i t y -assayed i n Bray's s c i n t i l l a t i o n f l u i d as before. b) Pulse-Chase K i n e t i c s of 1''C-Protamine A 15 ml t e s t i s c e l l suspension was prepared i n Hanks!i medium w i t h 5 g of t e s t i s obtained from hormonally induced t r o u t . Two mis of c e l l suspension were used f o r each i n c u b a t i o n and the c e l l s i n each i n c u b a t i o n mixture were exposed t o 1.0 yC of 1 ^ C - a r g i n i n e f o r 30 sec, and then t r e a t e d w i t h 1 2 C - a r g i n i n e ( f i n a l concentra-t i o n 1 mM) f o r v a r y i n g lengths of time as chase. P r o t e i n synthesis was terminated i n the in c u b a t i o n mixtures by adding cycloheximide to a f i n a l c o n c e n t r a t i o n of 0.4 mM. The c e l l s were ruptured by vigorous homogenization (5,000 rpm, 2 min) i n the hypotonic T r i s -b u f f e r (TMC). The nuclear residue was c o l l e c t e d by c e n t r i f u g a t i o n of the homogenate at 30,000 x g f o r 10 min and microsomes were c o l l e c t e d by f u r t h e r c e n t r i f u g a t i o n of the 30,000 x g supernatant at 65,000 rpm f o r 60 min i n a Spinco 65 r o t o r . C a r r i e r protamine (0.5 mg) was added to each f r a c t i o n and b a s i c p r o t e i n s were ex-t r a c t e d by the more s p e c i f i c mixed e x t r a c t a n t (0.5 M HC1 plus 5% TCA) as already described. The b a s i c p r o t e i n s were p r e c i p i t a t e d , washed, and t h e i r r a d i o a c t i v i t y assayed as described above. c) Time Course of I n t r a c e l l u l a r D i s t r i b u t i o n of 1*C-Protamine Two i d e n t i c a l t e s t i s c e l l suspensions i n TMKS (5 ml each) were prepared w i t h 4 g of t e s t e s obtained from hormonally induced t r o u t . A f t e r a pr e i n c u b a t i o n at 20° f o r 5 min, 2.5 yC of 1 1 1C-a r g i n i n e was added to each c e l l suspension. Incubations were t e r -minated at 0.5 min and 15 min, r e s p e c t i v e l y , by the a d d i t i o n of an equal volume of i c e c o l d TMKS c o n t a i n i n g 1 mM 1 2 C - a r g i n i n e and - 47 -1 mM cycloheximide. The suspensions were homogenized (5,000 rpm fo r 20 sec) and c e n t r i f u g e d at 15,000 x g f o r 15 min. The p e l l e t s were f u r t h e r washed by resuspension i n c o l d TMKS and c e n t r i f u g e d at 1,000 x g f o r 10 min. The r e s u l t i n g p e l l e t s were designated the nuclear f r a c t i o n s ; the r e s p e c t i v e supernatants from the f i r s t and second c e n t r i f u g a t i o n were combined and designated the cyto-plasmic f r a c t i o n s . Each f r a c t i o n was e x t r a c t e d w i t h 0.2 M s u l f u r i c a c i d f o r s e v e r a l hours, and the a c i d - e x t r a c t a b l e p r o t e i n s were processed f o r Bio-Gel P-10 chromatography. d) Synthesis of Protamine i n a C e l l - F r e e System (i) I n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o protamine by an i s o l a t e d cytoplasmic f r a c t i o n Testes obtained from n a t u r a l l y maturing t r o u t (40 g) were homogenized w i t h 10 ml of TMKS at 5,000 rpm f o r 20 sec. A post-mi t o c h o n d r i a l cytoplasmic f r a c t i o n was obtained as a supernatant f o l l o w i n g c e n t r i f u g a t i o n at 20,000 x g f o r 10 min. This f r a c t i o n , a f t e r c e n t r i f u g a t i o n twice more under the same c o n d i t i o n s to r u l e out c e l l contamination, was incubated w i t h 1 1*C-arginine f o r 60 min at 20°. The i n c u b a t i o n mixture (10 ml) f o r i n c o r p o r a t i o n had the f o l l o w i n g f i n a l composition: 1 ^ C - a r g i n i n e 0.3 uC/ml, ATP 2 mM, 2-mercaptoethanol 6 mM, GTP 0.2 mM, and 8.4 ml of the cytoplasmic f r a c t i o n . A f t e r i n c u b a t i o n , the mixture was ex t r a c t e d w i t h 0.2 M hot s u l f u r i c a c i d , 5 mg c a r r i e r protamine were added, and the e x t r a c t was f r a c t i o n a t e d by chromatography on the Bio-Gel P-10 column (2 x 22 cm). ( i i ) D i f f e r e n t i a l i n c o r p o r a t i o n of l a b e l l e d a r g i n i n e i n t o pro-tamine by i s o l a t e d c e l l f r a c t i o n s - 48 -A concentrated t e s t i s c e l l suspension was prepared w i t h 11 g te s t e s from n a t u r a l l y maturing t r o u t by adding an equal volume of TMKS to the t i s s u e . This suspension was homogenized (5,000 rpm fo r 20 sec) and c e n t r i f u g e d at 1,000 x g f o r 10 min. A nuclear f r a c t i o n was obtained from the sediment of t h i s c e n t r i f u g a t i o n and was washed twice more w i t h c o l d TMKS. A cytoplasmic f r a c t i o n f r e e of mitochondria was obtained by s u b j e c t i n g the supernatant f r a c -t i o n of the 1,000 x g f o r 10 min c e n t r i f u g a t i o n to 15,000 x g c e n t r i f u g a t i o n f o r 15 min. A high speed supernatant (60,000 rpm f o r 2 hours i n the Spinco L2-65, 65 r o t o r ) was prepared from the cytoplasmic f r a c t i o n of a second i d e n t i c a l c e l l suspension. Each f r a c t i o n was d i l u t e d to 8.0 ml w i t h c o l d TMKS and incubated at 20° w i t h GTP 1 mM, ATP 5 mM, 1 h C - a r g i n i n e 0.25 yC/ml f o r 90 min. At the end of the i n c u b a t i o n , a c i d e x t r a c t s (0.2 M HC1) were prepared from each f r a c t i o n , c a r r i e r protamine (10 mg) was added, and the e x t r a c t s were processed f o r Bio-Gel p-10 chromatography. The f r a c t i o n s corresponding to the protamine peak were c o l l e c t e d from the P-10 column, l y o p h i l i z e d , and r e d i s s o l v e d i n 1.0 ml of water. A small a l i q u o t was removed f o r determination of r a d i o -a c t i v i t y ; the remainder was a p p l i e d to the CM-cellulose column and e l u t e d w i t h a 0.2-2.0 M l i t h i u m c h l o r i d e gradient. ( i i i ) I n c o r p o r a t i o n of 3 2P-phosphate and 3 H - a r g i n i n e by a cytoplasmic c e l l - f r e e system Approximately 3 0 ml of packed t e s t i s c e l l s obtained from n a t u r a l l y maturing t r o u t were homogenized at 5,000 rpm f o r 20 sec i n TMKS and the homogenate c e n t r i f u g e d twice at 15,000 x g f o r 15 min; the po s t - m i t o c h o n d r i a l supernatant (15 ml) was incubated w i t h - 49 -2 mM GTP, 6 mM beta-mercaptoethanol, 100 yC of 3 H - a r g i n i n e ( s p e c i f i c a c t i v i t y 1.1 C/mM) and. 2 mM ATP co n t a i n i n g about 3 x 10 6 cpm gamma-32P-ATP at 20° f o r 90 min. Basic p r o t e i n s were a c i d e x t r a c t e d w i t h 0.2 M s u l f u r i c a c i d , c a r r i e r protamine (3 mg) was added and the a c i d e x t r a c t e d p r o t e i n s prepared f o r chroma-tography on the Bio-Gel P-10 column. IV. C h a r a c t e r i z a t i o n of Trout T e s t i s Ribosomes a) P r e p a r a t i o n and P u r i f i c a t i o n of Ribosomes Ribosomes were prepared from t r o u t t e s t i s and l i v e r by a method s i m i l a r to tha t described by Wettst e i n et al. (30). A l l operations were performed at 0°-4°. Frozen or f r e s h t i s s u e was minced w i t h s c i s s o r s i n 1-2 volumes of TMKS and homogenized at 5,000 rpm f o r 20 sec. The homogenate was c e n t r i f u g e d at 15,000 x g f o r 15 min to o b t a i n a post-mitochondrial supernatant (PMS). On occasion, t h i s c e n t r i f u g a t i o n was repeated, or two c e n t r i f u -g a t i o n s , the f i r s t at 1,000 1x g f o r 10 min (post-nuclear super-natant obtained) and the second, 15,000 x g f o r 15 min, were per-formed. The PMS was t r e a t e d w i t h the non-ionic detergent T r i t o n X-100 to a f i n a l c o n c e n t r a t i o n of 2% and r e - c e n t r i f u g e d at 15,000 x g f o r 15 min. The detergent t r e a t e d PMS was layered over 3 ml of dense sucrose ( u s u a l l y 40% w/v) made up i n TMK i n a 13 ml polyallomer high speed c e n t r i f u g e tube. C e n t r i f u g a t i o n was per-formed at 60,000 rpm f o r 2 h r s , the supernatant above the dense sucrose l a y e r was c a r e f u l l y a s p i r a t e d o f f , and then the dense sucrose l a y e r i t s e l f was s i m i l a r l y removed l e a v i n g a c l e a r r i b o -somal p e l l e t . The i n s i d e w a l l of the c e n t r i f u g e tube was wiped w i t h absorbent paper t i s s u e and the ribosomal p e l l e t b r i e f l y - 50 -r i n s e d w i t h a small volume of c o l d TMK. The p e l l e t was e a s i l y d i s s o l v e d i n TMK g i v i n g an opalescent s o l u t i o n . In some cases, e s p e c i a l l y when i s o l a t i n g ribosomes from t e s t i s , the ribosomal p e l l e t when r e - d i s s o l v e d i n TMK gave a cloudy s o l u t i o n p o s s i b l y due to the presence of aggregates and membranes. This s o l u t i o n was c l a r i f i e d by c e n t r i f u g a t i o n at 15,000 x g f o r 15 min 2. b) Sucrose Density Gradient A n a l y s i s of Ribosomes Sucrose s o l u t i o n s of appropriate concentrations were prepared i n TMK w i t h r i b o n u c l e a s e - f r e e d e n s i t y gradient grade sucrose ob-t a i n e d from Mann Research L a b o r a t o r i e s . Linear sucrose gradients were generated by a Beckman d e n s i t y - g r a d i e n t former (DGF-IM-3) i n c e l l u l o s e n i t r a t e tubes at room temperature. The gradients were then cooled by a l l o w i n g them to stand i n the c o l d room (4°) f o r 2-3 hiDs before use. Appropriate concentrations of ribosome s o l u -t i o n s were made up i n TMK and small volumes of 0.2-2 ml as appropriate were layered onto the sucrose g r a d i e n t s . C e n t r i f u g a -t i o n was performed at 4° i n a swinging-bucket r o t o r (SW-39) SW-25, or SW-27, Beckman) which was allowed to stop without braking. At the end of the c e n t r i f u g a t i o n , the c e n t r i f u g e tubes were punctured at the bottom w i t h a needle and approximately equal f r a c t i o n s were c o l l e c t e d f o r a n a l y s i s of absorbance at 260 nm and of r a d i o -a c t i v i t y when r e q u i r e d . For a more s e n s i t i v e a n a l y s i s of the ribosome p r o f i l e , the e f f l u e n t obtained from the sucrose gradient a f t e r puncturing the c e n t r i f u g e tube was continuously monitored and recorded at 254 nm by an Isco u l t r a v i o l e t analyser equipped w i t h a 10 mm flow c e l l . The flow r a t e of the e f f l u e n t was kept constant by means of an Accu-flow pump (Beckman); the chart - 51 -speed of the recorder was set at 12 inches per hour. c) The T e s t i s Polysomes A s s o c i a t e d w i t h the Synthesis of Protamine (i) C h a r a c t e r i z a t i o n of the nascent p r o t e i n s a s s o c i a t e d w i t h t e s t i s polysomes A 20 g c e l l suspension i n TMKS prepared w i t h t e s t i s from n a t u r a l l y maturing t r o u t at the protamine stage was incubated w i t h 1 h C - a r g i n i n e (0.5 uC/ml) i n c u b a t i o n mixture) f o r 1 min and the i n c o r p o r a t i o n terminated by the a d d i t i o n of cycloheximide. Deter-gent t r e a t e d ribosomes were p u r i f i e d by sedimentation through 2 ml of 1.0 M sucrose s o l u t i o n i n a 13 ml c e n t r i f u g e tube, the ribosomal p e l l e t was r e - d i s s o l v e d i n 2 ml of TMK b u f f e r and a p p l i e d to the top of a 28 ml l i n e a r sucrose gradient (10-30% w./v) . Cen-t r i f u g a t i o n was performed at 24,000 rpm f o r 2.5 hrs i n a SW-25 swinging-bucket r o t o r . Twenty-five equal f r a c t i o n s were c o l l e c t e d from the gra d i e n t and absorbance at 260 nm and r a d i o a c t i v i t y determined f o r each f r a c t i o n . Appropriate f r a c t i o n s corresponding to monosomes, disomes and l a r g e r polysomes (areas A, B, and C r e s p e c t i v e l y of F i g . 21) were pooled, e x t r a c t e d w i t h hot 0.2 M s u l f u r i c a c i d , 5 mg c a r r i e r protamine added, and the e x t r a c t s f r a c t i o n a t e d by P-10 chromatography. The t o t a l r a d i o a c t i v i t y a s s o c i a t e d w i t h P I , and P I I of the P-10 column was determined i n -d i v i d u a l l y f o r each e x t r a c t . ( i i ) N o n - s p e c i f i c b i n d i n g of 1^C-protamine to t e s t i s ribosomes U n l a b e l l e d t e s t i s ribosomes were prepared by sedimentation through 1.5 M dense sucrose s o l u t i o n ; t h i s ribosome p r e p a r a t i o n was incubated w i t h 1^C-protamine prepared b i o s y n t h e t i c a l l y (85 ug, 15,050 cpm) at 0° f o r 20 min, and the ribosomes r e - i s o l a t e d - 52 -by sedimenting through 1.5 M dense sucrose as before. The r i b o -somal p e l l e t was r e - d i s s o l v e d i n 0.2 ml TMK and layered onto a 4.8 ml l i n e a r sucrose gradient 10-30% prepared i n a SW-39 c e l l u -l o s e n i t r a t e tube. C e n t r i f u g a t i o n was at 37,000 rpm f o r 60 min i n a SW-39 swinging-bucket r o t o r and f r a c t i o n s c o l l e c t e d as described above. Each f r a c t i o n was assayed f o r absorbance at 260 nm and f o r r a d i o a c t i v i t y . ( i i i ) Time-course d i s t r i b u t i o n of. 1 ^ C - a r g i n i n e l a b e l l e d p r o t e i n s a s s o c i a t e d w i t h t e s t i s polysomes Two i d e n t i c a l t e s t i s c e l l suspensions were prepared i n TMKS from 15 g t i s s u e obtained from n a t u r a l l y .maturiSBg t r o u t . Each c e l l suspension was exposed to 0.5 yC/ml of 1 h C - a r g i n i n e at 20°; i n one case f o r 2 min and i n the other f o r 40 min. I n c o r p o r a t i o n was terminated i n each case by the a d d i t i o n of cycloheximide to a f i n a l c o n c e n t r a t i o n of 0.4 mM. Ribosomes were prepared from each c e l l suspension by t r e a t i n g the PMS w i t h T r i t o n X-100 and p e l l e t -i n g the ribosomes through a l a y e r of 40% w/v dense sucrose. The ribosomal p e l l e t s were r e d i s s o l v e d i n I ml of TMK and layered onto a 35 ml l i n e a r sucrose gradient (10-34% w/v); c e n t r i f u g a t i o n was at 26,000 rpm f o r 2.5 hr i n a SW-27 swinging-bucket r o t o r . At the end of the c e n t r i f u g a t i o n , 30 equal f r a c t i o n s were c o l l e c t e d from each tube, and each f r a c t i o n analyzed f o r absorbance at 260 nm and f o r r a d i o a c t i v i t y . (iv) A n a l y s i s of t e s t i s ribosomes obtained from the post-nuclear supernatant and the post-mitochondrial supernatant Two i d e n t i c a l c e l l suspensions i n TMKS (15 g each) were pre-pared from hormonally induced t r o u t . Each suspension was exposed to 1.5 yC/ml of 1 ^ C - a r g i n i n e f o r 2 min at 20° and i n c o r p o r a t i o n - 53 -was terminated w i t h the a d d i t i o n of 4 mM cycloheximide. The c e l l s were washed by c e n t r i f u g a t i o n , resuspended i n TMKS con-t a i n i n g cycloheximide, homogenized at 5,000 rpm f o r 20 sec and spun at 1,000 x g f o r 10 min to o b t a i n a post-nuclear supernatant (PNS). The PNS from one c e l l suspension was t r e a t e d w i t h 2% T r i t o n X-100 at 0° f o r 20 min, the other PNS was f u r t h e r f r a c -t i o n a t e d by c e n t r i f u g a t i o n at 15,000 x g f o r 15 min to y i e l d a PMS and a crude m i t o c h o n d r i a l f r a c t i o n as the sediment. The PMS and the crude m i t o c h o n d r i a l f r a c t i o n were a l s o t r e a t e d w i t h 2% T r i t o n X-100. Ribosomes were then i s o l a t e d from each detergent t r e a t e d f r a c t i o n by sedimentation through 3 ml of 40% sucrose s o l u t i o n . The p u r i f i e d ribosomes were layered oyer 35 ml l i n e a r , 10-34% sucrose-gradients and c e n t r i f u g e d i n a SW-27 swinging-bucket r o t o r at 26,000 rpm f o r 2.5 hr. F r a c t i o n s c o l l e c t e d from the gradients at the end of the c e n t r i f u g a t i o n were analyzed f o r absorption at 260 nm and f o r r a d i o a c t i v i t y . ++ (v) E f f e c t of low Mg i o n co n c e n t r a t i o n Frozen t e s t i s c o l l e c t e d from n a t u r a l l y maturing t r o u t at the protamine stage of spermatogenesis were allowed to thaw at room temperature u n t i l the t i s s u e was j u s t s o f t enough to be minced w i t h s c i s s o r s y i e l d i n g a frozen paste. This was d i v i d e d i n t o two equal p o r t i o n s ; one p o r t i o n was suspended i n TMKS and the other i n a medium i d e n t i c a l to TMKS except that the concent r a t i o n of magnesium acetate was 1 mM in s t e a d of the usual 5 mM. The sus-pensions were washed by c e n t r i f u g a t i o n and resuspended i n t h e i r r e s p e c t i v e b u f f e r s . Throughout each step of the i s o l a t i o n pro-cedure, the two preparations were t r e a t e d i d e n t i c a l l y except that one of the preparations was exposed to b u f f e r s c o n t a i n i n g 1 mM - 54 -magnesium and the other. 5 mM magnesium. Ribosomes from each pr e p a r a t i o n were i s o l a t e d by sedimentation through 3 ml of 40% w/v sucrose s o l u t i o n and were then layered onto 35 ml l i n e a r sucrose-gradients (10-34% w/v) made up wit h appropriate magnesium concentrations. C e n t r i f u g a t i o n was at 26,000 rpm on a SW-27 swinging-bucket r o t o r f o r 2.5 hrs. Polysome p r o f i l e s were analyzed by the Isco u l t r a v i o l e t analyzer, (vi) E f f e c t of ribonuclease d i g e s t A c e l l suspension i n TMKS was prepared from 25 gm t e s t e s obtained from n a t u r a l l y maturing t r o u t at a stage of protamine s y n t h e s i s . The suspension was exposed to 0.5 yC/ml i n c u b a t i o n mixture of 1 ^ C - a r g i n i n e f o r 10 min at 20° a f t e r which 1 mM of cycloheximide was added to terminate i n c o r p o r a t i o n . The c e l l s were sedimented by c e n t r i f u g a t i o n , resuspended i n 10 ml of TMKS plus cycloheximide and homogenized at 5,000 rpm f o r 20 sec. A post - m i t o c h o n d r i a l supernatant (PMS) was prepared by c o l l e c t i n g the top 10 ml of the supernatant obtained from 15,000 x g 15 min c e n t r i f u g a t i o n . This PMS was t r e a t e d w i t h 1% T r i t o n X-100 and d i v i d e d i n t o three equal p o r t i o n s : the f i r s t p o r t i o n was kept as c o n t r o l , the second p o r t i o n was t r e a t e d f i r s t w i t h bentonite to a f i n a l c o n c e n t r a t i o n of 2 mg/ml then w i t h 0.1 ug pa n c r e a t i c »ibo-nuclease (Worthington), and the t h i r d p o r t i o n was subjected to 0.1 yg ribonuclease treatment alone. The three p o r t i o n s were then c e n t r i f u g e d at 4° at 15,000 x g f o r 15 min to c l a r i f y the s o l u t i o n , a f t e r which, two ml from each p o r t i o n was layered onto a 28 ml sucrose-gradient 10-30% and c e n t r i f u g e d at 24,000 rpm at 3° f o r 2.5 hrs i n a SW-25 swinging-bucket r o t o r . Each gradient - 55 -was t h e n f r a c t i o n a t e d i n t o 30 e q u a l f r a c t i o n s o f a b o u t 1 ml e a c h ; a b s o r b a n c e a t 260 nm and r a d i o a c t i v i t y were a s s a y e d f o r e a c h f r a c t i o n . ( v i i ) D e t e r m i n a t i o n o f t h e s e d i m e n t a t i o n c o e f f i c i e n t (S) o f t e s t i s r i b o s o m e s P u r i f i e d r i b o s o m e s f r o m t e s t i s a t t h e p r o t a m i n e s t a g e ob-t a i n e d f r o m n a t u r a l l y m a t u r i n g t r o u t were p r e p a r e d by t r e a t m e n t w i t h 2% T r i t o n X-10 0 and s e d i m e n t a t i o n t h r o u g h d e n s e s u c r o s e as a l r e a d y d e s c r i b e d . I n o r d e r t o wash t h e r i b o s o m e s f r e e o f s u c r o s e , t h e r i b o s o m a l p e l l e t was r i n s e d w i t h TMK, r e d i s s o l v e d , and f u r t h e r r e s e d i m e n t e d by u l t r a c e n t r i f u g a t i o n . The r i b o s o m e s were t h e n r e -d i s s o l v e d i n TMK t o a c o n c e n t r a t i o n o f 8-9 mg/ml. The s e d i m e n -t a t i o n c o e f f i c i e n t o f t h e r i b o s o m e s was m e a s u r e d a c c o r d i n g t o t h e method d e s c r i b e d by Schachman (28) on a M o d e l E a n a l y t i c a l u l t r a -c e n t r i f uge (Beckman). The s p e e d o f t h e c e n t r i f u g a t i o n was a t 20,000 rpm, t h e s e d i m e n t a t i o n p r o f i l e was m o n i t o r e d by t h e S c h l i e r e n o p t i c a l s y s t e m w i t h t h e b a r a n g l e s e t a t 30 d e g r e e , t e m p e r a t u r e was a t 23.2° and p h o t o g r a p h s o f t h e s e d i m e n t a t i o n p r o f i l e s were t a k e n a t 2 m i n i n t e r v a l s a f t e r r e a c h i n g t o p s p e e d . A N i k o n p r o -f i l e p r o j e c t o r m o d e l 6C e q u i p p e d w i t h a m i c r o - c o m p a r a t o r ( A n g l o -p h o t o L t d . , T o r o n t o ) was u s e d t o measure t h e d i s t a n c e between t h e r i b o s o m e p e a k s and -the r e f e r e n c e l i n e i n e a c h p h o t o g r a p h . The s e d i m e n t a t i o n c o e f f i c i e n t s ( S 2 0 ) f o r t h e monoribosomes and t h e d i - r i b o s o m e s were c a l c u l a t e d a f t e r m a k i n g t h e a p p r o p r i a t e c o r r e c -t i o n s t o n o r m a l i z e t h e S v a l u e s o b t a i n e d a t 23.2° f o r 20° ( 2 8 ) . d) D e v e l o p m e n t a l Changes o f t h e T e s t i s P o l y s ome P o p u l a t i o n From t h e months o f A u g u s t t o December, t e s t e s were o b t a i n e d - 56 -at p r o g r e s s i v e stages of development from n a t u r a l l y maturing t r o u t . A random sample of 20 t e s t e s or more was c o l l e c t e d at the middle of each month, minced w i t h s c i s s o r s and a p o r t i o n of t h i s mince, 10.-20 g, was removed f o r p r e p a r a t i o n of a c e l l suspension. The c e l l suspension was d i v i d e d i n t o two equal p a r t s and i n c u b a t i o n s , one w i t h 1 ^C-arginine and the other 1'*C-lysine (0.5 uC/ml i n c u b a t i o n m i x t u r e ) , were performed at 20° f o r 30 min. Incubations were terminated w i t h the a d d i t i o n of cycloheximide and the. c e l l s homogenized and d i f f e r e n t i a l l y c e n t r i f u g e d to o b t a i n nuclear p e l l e t s and post-mitochondrial cytoplasmic f r a c -t i o n s as already described. The nuclear p e l l e t s obtained from the c e l l s allowed to inco r p o r a t e 1 ^ C - a r g i n i n e were e x t r a c t e d w i t h 0.2 M H 2 S O 4 , the b a s i c p r o t e i n s were f r a c t i o n a t e d on a Bio-Gel P-10 column as before and the t o t a l r a d i o a c t i v i t y contained i n protamine was assayed. Ribosomes were p u r i f i e d from the c y t o p l a s -mic f r a c t i o n s a f t e r treatment w i t h T r i t o n X-100, a small sample was removed f o r determination of ribosome content per gram t e s t i s by assaying f o r the amount of ribosomal RNA us i n g the o r c i n o l method (29) w i t h yeast RNA as standard, the r e s t of the ribosomes were layered onto a 35 ml 10-34% l i n e a r sucrose gradients and c e n t r i f u g e d at 26,000 rpm i n a SW-27 swinging-bucket r o t o r f o r 2.5 hrs. The gradients were then analyzed f o r absorbance at 260 nm and f o r r a d i o a c t i v i t y . - 57 -RESULTS I . I s o l a t i o n and C h a r a c t e r i z a t i o n o f P r o t a m i n e and R a d i o a c t i v e  P r o t a m i n e To be a b l e t o s t u d y t h e b i o s y n t h e s i s o f a s p e c i f i c p r o -t e i n , a d e t a i l e d knowledge o f t h e n a t u r e o f t h e p r o t e i n i s r e -q u i r e d s o as t o a l l o w a c o n f i d e n t i d e n t i f i c a t i o n o f any newly s y n t h e s i z e d p r o d u c t as t h a t p r o t e i n . T h i s s e c t i o n d e a l s w i t h t h e i s o l a t i o n and t h e c h a r a c t e r i z a t i o n o f p r o t a m i n e and i n p a r t i -c u l a r n e w l y s y n t h e s i z e d p r o t a m i n e l a b e l l e d w i t h r a d i o a c t i v e a r g i -n i n e . The u n u s u a l c h e m i c a l n a t u r e o f p r o t a m i n e as a p r o t e i n has a l r e a d y b e e n d e s c r i b e d i n " I n t r o d u c t i o n 1 ^ . As a r e s u l t o f i t s c h a r a c t e r i s t i c p r o p e r t i e s , p r o t a m i n e b e h a v e s q u i t e d i f f e r e n t l y on B i o - G e l P-10 and on C M - c e l l u l o s e f r o m o t h e r chromosomal b a s i c p r o t e i n s and c o u l d t h u s be e a s i l y s e p a r a t e d . T h i s t h e n a l l o w s p r o t a m i n e t o be u n i q u e l y i d e n t i f i e d . a) B i o - G e l P-10 C h r o m a t o g r a p h y o f T e s t i s N u c l e a r B a s i c P r o t e i n s F i g . 2 A t o D shows t h e B i o - G e l P-10 c h r o m a t o g r a p h y o f a c i d e x t r a c t a b l e p r o t e i n s f r o m t r o u t t e s t i s n u c l e i o b t a i n e d d u r i n g months o f September t o December f r o m n a t u r a l l y m a t u r i n g t r o u t . -The p r o t e i n s a r e e l u t e d f r o m t h e column as two m a j o r p e a k s , PI and P I I ; t h e s e p e a k s have p r e v i o u s l y b e e n c h a r a c t e r i z e d by I n g l e s (5) and I n g l e s and D i x o n (7) who have shown by p o l y a c r y l a -mide g e l e l e c t r o p h o r e s i s t h a t PI c o n t a i n s m a i n l y h i s t o n e s and o t h e r l a r g e m o l e c u l a r w e i g h t ( g r e a t e r t h a n 10,000) b a s i c p r o t e i n s w h i l e P I I c o n t a i n s o n l y p r o t a m i n e . I t may be. s e e n t h a t d u r i n g t h e e a r l y s t a g e s o f s p e r m a t o -g e n e s i s i n September t h e t e s t i s n u c l e i c o n t a i n m a i n l y h i s t o n e s , - 58 -T 1 1 r r 0 10 2 0 0 10 20 FRACTION NO. ( 5 ml) F i g . 2. P r o f i l e s of b a s i c p r o t e i n s e x t r a c t e d from t e s t i s n u c l e i a t d i f f e r e n t stages of spermatogenesis. B a s i c p r o t e i n s were chromatographed on a Bio- G e l P-10 column (2 x 30 cm) e l u t e d w i t h 0.2 M a c e t i c a c i d . Histone was e l u t e d at PI and protamine at P I I from t h i s column. Testes were obtained from n a t u r a l l y maturing" t r o u t at the middle of each month: (A) September, (B) October, (C) November, and (D) December. - 59 -PI o f F i g . 2 A, b u t by t h e end o f s p e r m a t o g e n e s i s i n December, t h e m a j o r n u c l e a r p r o t e i n i s p r o t a m i n e P I I o f F i g . 2 D. The s e d a t a s u g g e s t t h a t a ' t r a n s f o r m a t i o n ' , i . e . a r e p l a c e m e n t o f h i s t o n e by p r o t a m i n e , has o c c u r r e d i n t h e t e s t i s c e l l n u c l e i d u r i n g s p e r m a t o g e n e s i s and s u p p o r t t h e o b s e r v a t i o n s o f v a r i o u s i n v e s t i g a t o r s who have examined t h e b a s i c p r o t e i n s f r o m t h e n u c l e i o f d e v e l o p i n g t e s t i s i n s a l m o n o i d f i s h ( 1 , 2 , 3 ) . A l f e r t (9) by h i s t o c h e m i c a l s t u d i e s o f m a t u r i n g s a l m o n t e s t i s showed t h a t a t r a n s f o r m a t i o n o c c u r r e d a t t h e s p e r m a t i d s t a g e o f t e s t i s c e l l d e v e l o p m e n t i n t h a t a " h i s t o n e " i n t h e sperm-a t i d n u c l e i , s t a i n e d by f a s t g r e e n (an a c i d i c dye) and n o t e x t r a c -t e d by h o t 5% TCA, was c o n v e r t e d t o a " p r o t a m i n e " w h i c h a l s o s t a i n e d w i t h f a s t g r e e n b u t was a c i d s o l u b l e . Ando and H a s h i m o t o (10) showed by a n a l y s i s o f t h e amino a c i d c o m p o s i t i o n o f b a s i c p r o t e i n s o b t a i n e d f r o m t h e n u c l e i o f t e s t i s c e l l s i n n a t u r a l l y m a t u r i n g r a i n b o w t r o u t Salmo ivideus* t h a t t h e r e was a s p e c i f i c c hange i n t h e c o m p o s i t i o n d u r i n g a l a t e s t a g e o f s p e r m a t o g e n e s i s ( f r o m O c t o b e r t o December). A t t h a t s t a g e , t h e r e was a p r o g r e s s -i v e d e c r e a s e i n n o n - p r o t a m i n e amino a c i d s e.g. Phe, T y r , T h r , H i s , L y s , G l u , A s p , and C y s , and a l a r g e i n c r e a s e i n A r g , t h e m a j o r p r o t a m i n e amino a c i d , u n t i l t h e f i n a l amino a c i d c o m p o s i t i o n o f b a s i c p r o t e i n s o b t a i n e d f r o m sperm n u c l e i was t h a t o f p r o t a m i n e . I n g l e s e t al. (11) and I n g l e s (5) have c h a r a c t e r i z e d t h e a c i d -s o l u b l e p r o t e i n o f t h e d e v e l o p i n g t e s t i s o f h o r m o n a l l y i n d u c e d Salmo gaivdnevii by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s and have shown t h a t t h e s o m a t i c h i s t o n e s p r e s e n t up t o t h e f i r s t a p p e a r a n c e o f p r o t a m i n e were c o m p l e t e l y r e p l a c e d by p r o t a m i n e t o w a r d t h e end o f s p e r m a t o g e n e s i s . M a r u s h i g e and D i x o n (12) d e m o n s t r a t e d by - 60 -p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f b a s i c p r o t e i n s a c i d - e x t r a c t e d f r o m p u r i f i e d c h r o m a t i n p r e p a r e d f r o m t r o u t t e s t i s a t d i f f e r e n t s t a g e s o f s p e r m a t o g e n e s i s t h a t t h e r e may be a t e m p o r a l s e q u e n c e i n t h i s r e p l a c e m e n t o f h i s t o n e s by p r o t a m i n e i n t h a t t h e l y s i n e -r i c h h i s t o n e s ( h i s t o n e I) a p p e a r e d t o be t h e l a s t h i s t o n e f r a c t i o n t o be d i s s o c i a t e d f r o m DNA d u r i n g t h e r e p l a c e m e n t p r o c e s s . B l o c h (13) u s i n g h i s t o c h e m i c a l t e c h n i q u e s has o b s e r v e d s i m i l a r t r a n s -f o r m a t i o n s i n n u c l e a r p r o t e i n s d u r i n g s p e r m a t o g e n e s i s i n b o t h s n a i l {Helix aspersa) and s q u i d (Loligo opalesoens) t e s t i s . b) S u l f u r i c A c i d E x t r a c t i o n o f T e s t i s N u c l e a r P r o t e i n s I t was n o t e d t h a t t h e r a t i o o f t h e v o l u m e o f 0X2 M H 2SOit t o t e s t i s w e i g h t and t h e number o f t i m e s o f a c i d e x t r a c t i o n was i m p o r t a n t f o r t h e t o t a l e x t r a c t i o n o f p r o t a m i n e . As shown i n F i g . 3 and i n T a b l e I , a low r a t i o o f a c i d t o t e s t i s t i s s u e (1:1) e x t r a c t e d m a i n l y h i s t o n e s , P I o f F i g . 2A, w h i l e a h i g h e r r a t i o (5:1) e x t r a c t e d a b o u t 30% o f t h e p r o t a m i n e and a r e p e a t e d e x t r a c -t i o n a t t h e h i g h e r r a t i o e x t r a c t e d a l l t h e p r o t a m i n e P I I o f F i g . 1 B. From T a b l e I , i t i s s e e n t h a t a d d i t i o n a l e x t r a c t i o n s a t t h e 5:1 r a t i o a f t e r t h e s e c o n d e x t r a c t i o n d i d n o t im p r o v e t h e y i e l d o f p r o t a m i n e . I t a p p e a r s t h a t h i s t o n e s a r e more e a s i l y e x t r a c t e d f r o m t e s t i s n u c l e i t h a n p r o t a m i n e i n t h a t e v e n a low r a t i o (1:1) o f e x t r a c t a n t t o t i s s u e e x t r a c t e d q u a n t i t a t i v e l y a l m o s t a l l t h e h i s t o n e s w h i l e u n d e r t h e same c o n d i t i o n s v e r y l i t t l e p r o t a m i n e i s e x t r a c t e d . T h i s may r e f l e c t t h e f a c t t h a t p r o t a m i n e , b e i n g a h i g h l y b a s i c p r o t e i n (2/3 o f i t s r e s i d u e s a r e a r g i n i n e ) , b i n d s more s t r o n g l y t o DNA. EXTRACTION I « I B EXTRACTION 2x5=1 Q 20 30 0 F R A C T I O N NO I ood— 30 F i g . 3. E f f i c i e n c y of e x t r a c t i o n f o r protamine (PII) by 0.2 M s u l f u r i c a c i d . The c o n d i t i o n s of e x t r a c t i o n are described i n " M a t e r i a l s and Methods". P r o t e i n s e x t r a c t e d by a c i d were f r a c t i o n a t e d on a Bio-Gel P-10 column. (A) t e s t i s n u c l e i were e x t r a c t e d once w i t h 1 volume of 0.2 M H 2 S C K , and (B) twice w i t h 5 volumes of 0.2 M H 2 S C u . • TABLE I E x t r a c t i o n of t e s t i s nuclear p r o t e i n s w i t h 0.2 M H2S0it. A c i d - s o b l u b l e p r o t e i n s were separated by a Bio - G e l P-10 column as described i n M a t e r i a l s and Methods. EXPERIMENT EXTRACTION RATIO volume (ml) /weight (g) 0.2M H2SOif / t i s s u e TOTAL BASIC * PROTEIN EXTRACTED (mg) HISTONES ** PROTAMINE * P j O f P-10 P j O f P-10 column(mg) column (mg) NOVEMBER TESTIS (2 g of n u c l e i ) 1:1 2:1 5:1 2 x 5:1 8.5 8.4 11.8 20.0 8.2 8.4 8.5 8.6 0.3 <0.1 3.3 11.4 I I 2 x 5:1 DECEMBER TESTIS 3 x 5:1 (1 g of n u c l e i ) 4 x 5:1 7.3 6.5 6.8 1.4 1.4 1.3 5.9 5.1 5.5 * sum of P and P J J . ** determined by Lowry method (214) u s i n g serum albumin as standard - 63 -c) Time-course I n c o r p o r a t i o n of 1 h C - a r g i n i n e i n t o Protamine by a T e s t i s C e l l Suspension F i g . 4 shows the i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o a c i d s o l u b l e p r o t e i n s by a suspension of t e s t i s c e l l s from n a t u r a l l y maturing t r o u t at the protamine s y n t h e s i z i n g stage. I t may be noted t h a t the i n c o r p o r a t i o n proceeds i n a l i n e a r manner f o r up to 9 hours at 20°. The i n c o r p o r a t i o n of l a b e l l e d a r g i n i n e i n t o protamine by a suspension of Chinook salmon [Oncorhynchus tshawyteha) t e s t i s c e l l s has been stud i e d by Ingles et al. (11) who have followed the i n c o r p o r a t i o n f o r 4 hours at 20°. They have f u r t h e r s t u d i e d the e f f e c t s of various i n h i b i t o r s on t h i s i n c o r -p o r a t i o n f i n d i n g t h a t there was strong i n h i b i t i o n by c y c l o h e x i -mide and puromycin but no i n h i b i t i o n by actinomycin D and only s l i g h t i n h i b i t i o n by chloramphenicol. From these r e s u l t s , they have suggested t h a t d e s p i t e i t s small s i z e and unusual amino a c i d composition, protamine i s synthesized by the route i n v o l v i n g messenger RNA, ribosomes and s o l u b l e RNA. While F i g . 4 shows only the i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o a c i d - e x t r a c t a b l e p r o t e i n s , the m a j o r i t y of the counts incorporated i s l i k e l y to be i n pro-tamine i n t h a t a t t h i s stage of spermatogenesis, protamine i s the major a c i d - s o l u b l e p r o t e i n being synthesized (see F i g . 5 C). d) Determination of the Stage of T e s t i s Development and the B i o -Gel P-10 Chromatography of Radioactive Protamine V i s u a l i n s p e c t i o n of the developing t r o u t t e s t i s enables a rough e s t i m a t i o n to be made of the stage of spermatogenesis. A f t e r the major enlargement of the t e s t i s , a pink, somewhat t r a n s -l u c e n t , appearance i n d i c a t e s t h a t the spermatid stage has not;yet - 64 -2 4 6 8 10 INCUBATION TIME (HOURS) F i g . 4. Time course i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o a c i d -s o l u b l e p r o t e i n s by a t e s t i s c e l l suspension. A 12 ml c e l l sus-pension was prepared i n Hanks' s o l u t i o n c o n t a i n i n g 7 5 mg a r g i n i n e / l i t e r and was incubated w i t h 5 uC of 1 4 C - a r g i n i n e a t 20°. At va r i o u s times 0.5 ml of the c e l l suspension was removed and the c e l l s e x t r a c t e d w i t h 0.2 M HCl. The a c i d - s o l u b l e p r o t e i n s were p r e c i p i t a t e d . w i t h pH 2 TCA-tungstate, washed, and t h e i r r a d i o -a c t i v i t y assayed. - 65 -b e e n r e a c h e d and p r o t a m i n e s y n t h e s i s has n o t y e t commenced. As s o o n as t h e t e s t i s becomes w h i t e and opaque, a l a r g e number o f s p e r m a t i d c e l l s a r e p r e s e n t and t h e t i s s u e i s u s u a l l y a c t i v e i n p r o t a m i n e b i o s y n t h e s i s . A c o n v e n i e n t and r e l i a b l e b i o c h e m i c a l t e s t t o a s c e r t a i n w h e t h e r t h e t e s t i s i s a t t h e p r o t a m i n e o r p r e -p r o t a m i n e s t a g e i s t o d e t e r m i n e t h e r e l a t i v e i n c o r p o r a t i o n o f 1 ^ C - a r g i n i n e and lhC-lysine i n t o , a c i d - e x t r a c t a b l e p r o t e i n s p r e -c i p i t a t e d by pH 2 T C A ^ t u n g s t a t e . A h i g h r a t i o o f a r g i n i n e t o l y s i n e i n c o r p o r a t e d d u r i n g a 30 m i n i n c u b a t i o n a t 20$ r e f l e c t s a c t i v e s y n t h e s i s o f p r o t a m i n e . F o r example, t h e p r o t a m i n e s t a g e t e s t i s shows a r g i n i n e / l y s i n e i n c o r p o r a t i o n r a t i o s of.' 30-150, whereas b e f o r e p r o t a m i n e s y n t h e s i s s t a r t s ( p r e - p r o t a m i n e s t a g e ) , t h e r a t i o i s l e s s t h a n 5. T h a t t h i s t e s t i s v a l i d i s i n d i c a t e d i n F i g . 5 A, B, C. As a l r e a d y m e n t i o n e d , t h e f i r s t peak (PI) t o be e l u t e d f r o m t h e P-10 column i s composed m a i n l y o f h i s t o n e s w h i l e t h e s e c o n d peak (P I I ) c o n t a i n s o n l y p r o t a m i n e . A t t h e p r e -p r o t a m i n e s t a g e o f s p e r m a t o g e n e s i s ( F i g . 5 A ) , i n c o r p o r a t i o n o f l a b e l l e d a r g i n i n e i n t o a c i d - e x t r a c t a b l e p r o t e i n s i s f o u n d o n l y i n P I , i n d i c a t i n g a c t i v e s y n t h e s i s o f h i s t o n e s . S i n c e t h e t o t a l h i s t o n e f r a c t i o n c o n t a i n s a r g i n i n e and l y s i n e r e s i d u e s i n a p p r o x i -m a t e l y e q u a l amounts, t h e a r g i n i n e / l y s i n e r a t i o i n s u c h a t e s t i s w o u l d be n e a r u n i t y . However, by t h e e a r l y p r o t a m i n e s t a g e ( F i g . 5 B ) , i n c o r p o r a t i o n i n t o PI has d e c r e a s e d and most o f t h e i n c o r -p o r a t e d 1 * * C - a r g i n i n e a p p e a r s i n P I I , i . e . p r o t a m i n e . As shown i n F i g . 5 C, by t h e m i d - p r o t a m i n e s t a g e a l m o s t a l l t h e 1 " ^ C - a r g i n i n e i n c o r p o r a t e d i n t o a c i d - e x t r a c t a b l e p r o t e i n s i s now f o u n d i n p r o -t a m i n e . S i n c e p r o t a m i n e c o n t a i n s no l y s i n e r e s i d u e s 4 and o v e r - 66 -F i g . 5. Transformation from h i s t o n e s y n t h e s i s t o protamine syn-t h e s i s i n t r o u t t e s t i s . T e s t i s c e l l suspensions were prepared w i t h 1 g of t i s s u e from hormonally induced t r o u t and were i n c u -bated at 20° w i t h 1 l tC-arginine (A,B, and C) or 1 ^ C - l y s i n e (D) (0.5 uC l a b e l l e d amino a c i d per ml i n c u b a t i o n m i x t u r e ) , f o r 60 min. A c i d - s o l u b l e p r o t e i n s were e x t r a c t e d w i t h 0.2 M s u l f u r i c a c i d and f r a c t i o n a t e d by Bio-Gel P-10 chromatography as d e s c r i b e d i n " M a t e r i a l s and Methods". (A) A c i d - s o l u b l e p r o t e i n s from t e s t e s a t the pre-protamine stage plus 3 mg of c a r r i e r protamine; (B) and (D), from t e s t e s at the early-protamine stage; and (C) from t e s t e s at the mid-protamine stage. two-thirds of i t s amino a c i d residues are a r g i n i n e , the a r g i n i n e / l y s i n e i n c o r p o r a t i o n r a t i o s reach very high values at t h i s time when protamine synthesis i s maximal and histone synthesis has l a r g e l y ceased. The replacement of histones by protamine i n com-b i n a t i o n w i t h DNA i n t e s t i s c e l l s of salmonid f i s h has already been discussed. I t i s seen here t h a t the i n c o r p o r a t i o n data, ( F i g . 5 ) , i . e . decreased i n c o r p o r a t i o n i n t o PI and increased i n -c o r p o r a t i o n i n t o P I I of the Bio-Gel P-10 column a l s o r e f l e c t s t h i s t r a nsformation i n that during the stage of development i n the t e s t i s c e l l s when histones are being replaced by protamine, there i s a marked decrease i n the synt h e s i s of histones w h i l e at the same time, protamine i s being a c t i v e l y synthesized. This i m p l i e s t h a t during the p e r i o d of tr a n s f o r m a t i o n , there may be a c o n t r o l mechanism which " s h u t s - o f f " the synthesis of histones when the synt h e s i s of protamine i s "turned-on". I t has already been demonstrated t h a t protamine i s e l u t e d from the P-10 column as a c h a r a c t e r i s t i c second peak, P I I . F i g . 5 A shows th a t i n the pre-protamine stage t e s t i s , no b a s i c pro-t e i n s i n c o r p o r a t i n g 1 1*C-arginine chromatograph on the P-10 column i n the protamine re g i o n w h i l e during the protamine stage (Fig. 5 B,C) the major peak of incorporated 1 ^ C - a r g i n i n e i s i n the p r o t a -mine r e g i o n . As already mentioned, protamine does not co n t a i n any l y s i n e residues'* and F i g . 5 D shows th a t i n an i n c o r p o r a t i o n experiment using the same t e s t i s t i s s u e employed f o r F i g . 5 B, and using 1'*C-lysine as the l a b e l l e d amino a c i d , no l a b e l i s i n c o r -porated i n t o P I I of the P-10 column. This experiment provides an e x c e l l e n t i n t e r n a l c o n t r o l which demonstrates t h a t no b a s i c , l y s i n e - c o n t a i n i n g p r o t e i n chromatographs i n the protamine region. - 68 -Chromatography on Bio-Gel P-10 normally i n v o l v e s a process of g e l e x c l u s i o n w i t h an e x c l u s i o n l i m i t of 10,000 molecular weight, however protamine e l u t e s from the column even l a t e r than f r e e a r g i n i n e ( F i g. 6). This suggests t h a t there i s some i n t e r a c t i o n , probably ion-exchange between the h i g h l y b a s i c protamine molecule and a small number of c a r b o x y l groups produced by deamidation of the acrylamide matrix. This observation i s i n agreement w i t h those of Schwartz and Zabin (19) who have observed t h a t a r g i n i n e , a b a s i c amino a c i d , i s retarded and i s e l u t e d l a t e r than the n e u t r a l amino acids on a Bio-Gel P-2 column using 0.1 M a c e t i c a c i d as the e l u t a n t . e) CM-Cellulose Chromatography of Protamine The chromatography of a mixture of f r e e 1 ^ C - a r g i n i n e and u n l a b e l l e d protamine on CM-cellulose e l u t e d w i t h a 0.2-2.0 M l i t h i u m c h l o r i d e s a l t g r a dient shows th a t f r e e 1 ^ C - a r g i n i n e i s e l u t e d i n the v o i d volume and i s c l e a r l y separated from c a r r i e r protamine which i s e l u t e d at 0.9-1.0 M l i t h i u m c h l o r i d e (Fig. 7). Yaron et al. (20) have shown th a t t h i s method of chromatography can d i f f e r e n t i a t e between l y s i n e o l i g o p e p t i d e s d i f f e r i n g by s i n g l e l y s i n e residues and they have used t h i s technique to prepare o l i g o - l y s i n e s of known chain length. I t may be seen t h a t the c a r r i e r protamine i s r e s o l v e d as two p a r t i a l l y separated compon-ents, C^-j. and C n i ; f u r t h e r , when a shallower gradient of L i C l (0.8-122 M) i s used to e l u t e the column, (Fig. 8) the r e s o l u t i o n of the two components i s much improved and a t h i r d component C i s revealed. Thus, due to i t s high r e s o l u t i o n , CM-cellulose chroma-tography provides a powerful means both f o r determining whether - 69 -0 10 20 30 FRACTION NUMBER ( 5 m l ) F i g . 6. Bio - G e l P-10 chromatography of a mixture of 1 "*C-a r g i n i n e and u n l a b e l l e d t e s t i s a c i d - s o l u b l e nuclear p r o t e i n s . A c i d - s o l u b l e p r o t e i n s were prepared from 2 g of t e s t i s n u c l e i and were mixed w i t h 1 mg of c a r r i e r a r g i n i n e c o n t a i n i n g 0.1 yC of 1 4 C - a r g i n i n e . ' This mixture was a p p l i e d to a Bio-Gel P-10 column (2 x 30 cm) and e l u t e d w i t h 0.2 M a c e t i c a c i d . Histones chromatograph at PI and Protamine at P I I on t h i s column. - 70 -V I I — I E F F L U E N T VOLUME . ml F i g . 7. Chromatography of a mixture of 1 ^ C - a r g i n i n e and un-l a b e l l e d protamine on a CM-cellulose column (1 x 30 cm). A sample of protamine (10 mg) obtained from t r o u t t e s t i s was p u r i f i e d by Bio- G e l P-10 chromatography and mixed w i t h 2 mg of c a r r i e r a r g i n i n e c o n t a i n i n g 0.2 u C of C-argi n i n e . This mix-t u r e was a p p l i e d to the CM-cellulose column (1 x 30 cm) and e l u t e d w i t h a 700 ml l i t h i u m c h l o r i d e g r a d i e n t (0.2 M - 2.0 M) as d e s c r i b e d i n " M a t e r i a l s and Methods". F i g . 8. CM-cellulose chromatography of rainbow t r o u t t e s t i s protamine. About 15 mg of protamine was p u r i f i e d by Bi o - G e l P-10 chromatography and a p p l i e d to a CM-cellulose column (1.1 x 48 cm). The column was e l u t e d w i t h a 700 ml l i n e a r g r a d i e n t of l i t h i u m c h l o r i d e (0.8 - 1.2 M) b u f f e r e d w i t h 0.01 M l i t h i u m a c e t a t e - a c e t i c a c i d , pH 5.0. The three peaks of protamine are l a b e l l e d as components C , C_ , and C . i - 72 -newly synthesized m a t e r i a l represents complete protamine mole-cules and r u l i n g out p o s s i b l e contamination by f r e e 1 ^ C - a r g i n i n e . In view of the p o s s i b i l i t y t h a t there might be genetic v a r i a t i o n i n the s t r u c t u r e of protamine and t h a t each i n d i v i d u a l f i s h might produce a c h a r a c t e r i s t i c p a t t e r n of protamines, the presence of the observed three components of protamine C , C J J ' and C-T-JJ* ( F i g . 8) could be due to the f a c t t h a t protamine was e x t r a c t e d from a pool of t e s t e s from s e v e r a l f i s h . To t e s t t h i s p o s s i b i l i t y , protamine from a s i n g l e t r o u t was examined on the CM-cellulose column. The r e s u l t showed th a t the protamine o b t a i n -ed from a s i n g l e f i s h a l s o contained the three peaks C^, C j j r and C J J J , as before, and the p r o f i l e of the peaks was s i m i l a r to t h a t obtained i n F i g . 8. This suggests t h a t the maturing t e s t i s of each i n d i v i d u a l f i s h synthesizes the three components of pro-tamine. These r e s u l t s are c o n s i s t e n t w i t h those of. Ando and Sawada (31) who have i s o l a t e d protamines from i n d i v i d u a l h e r r i n g s and rainbow t r o u t and examined t h e i r chromatography p r o f i l e s on an alumina column according to the methods of Scanes and Tozer (32). They observed that the protamines from i n d i v i d u a l f i s h were hetero-geneous and t h a t they chromatograph i n a manner s i m i l a r to pro-tamines prepared from a l a r g e number of f i s h suggesting t h a t there must be s e v e r a l d i f f e r e n t s t r u c t u r a l genes f o r protamine i n the f i s h genome. I t was of i n t e r e s t then t o determine whether the separated components of protamine as observed i n F i g . 8 were homogeneous components. Table I I shows the r e s u l t s of amino a c i d analyses on a p p r o p r i a t e l y pooled f r a c t i o n s of and C^-j. of F i g . 8. i s present here ( F i g . 8) only i n a small amount and could not be - 73 -TABLE I I A m i n o a c i d c o m p o s i t i o n o f p r o t a m i n e a n d p r o t a m i n e c o m p o n e n t s i s o -l a t e d f r o m a C M - c e l l u l o s e c o l u m n ( F i g . 8 ) . A m i n o C C T T T T o t a l A c i d s P r o t a m i n e (5) m o l e % no. o f m o l e % n o . o f no. o f r e s i d u e s r e s i d u e s . r e s i d u e s s e r i n e 12.3 4.1 9.5 3.1 3.5 p r o l i n e 8.6 2.8 8.0 2.6 2.7 g l y c i n e 5.6 1.9 6.1 2.0 2.2 a l a n i n e 0.5 0.2 1.4 0.5 0.5 v a l i n e 4.9 1.6 4.7 1.6 1.8 i s o l e u c i n e 0.7 0.3 0.2 0.1 0.2 a r g i n i n e 67.2 22.1 70.0 23.1 22.1 T o t a l 99.8 33.0 99.9 33.0 33.0 - 74 -e a s i l y a n a l y z e d 5 . I t may be noted i n t h i s t a b l e t h a t a s i z e of 33 amino acids has been assumed f o r the rainbow t r o u t protamine molecule. This assumption i s based on the molecular weight data of Ando and Hashimoto (14) who have found the molecule weight of rainbow t r o u t protamine to be about 5,000. For example, i f the molecular weight of u n f r a c t i o n a t e d protamine were c a l c u l a t e d from the amino a c i d composition data (Table I I ) and assuming 33 residues f o r the protamine molecule, i t would give a molecular weight of 4,975. F u r t h e r , Ando and co-workers (15) have separated rainbow t r o u t protamine i n t o a l e a s t two components, one con-t a i n i n g 33-34 residues and the other 32-33 re s i d u e s . I t i s seen from Table I I t h a t peak i s d i f f e r e n t from CJJ-J- from the d i f f e r e n c e s i n the r e l a t i v e amounts of s e r i n e , a l a n i n e , i s o l e u c i n e and a r g i n i n e present i n each peak; a l s o , the amino a c i d composi-t i o n of each peak i s d i f f e r e n t from t h a t of whole u n f r a c t i o n a t e d protamine. However, each peak C J J , and C^^r i s not homogeneous i n that amino acids such as i s o l e u c i n e and alan i n e are present i n l e s s than s i n g l e amino a c i d residue amount. The separation of protamine components by C M - c e l l u l o s e i s based on ion-exchange and the f a c t t h a t peak C J J J i s e l u t e d at a higher s a l t c oncentration than C.J.J i s c o n s i s t e n t w i t h the observation that C J J J contains one more a r g i n i n e residue than C ^ ^ and hence i s more b a s i c . This i s o l a t i o n and se p a r a t i o n of components of protamine d i f f e r i n g by t h e i r a r g i n i n e content and other n e u t r a l amino acids i s c o n s i s t e n t w i t h the work of Ando and h i s group (15) who have found that one component of rainbow t r o u t protamine contains 22-23 a r g i n i n e residues and another 21-23 a r g i n i n e s . - 75 -F i g . 9 shows the chromatography on CM-cellulose of newly synthesized protamine obtained by i n c u b a t i o n of l a b e l l e d a r g i n i n e w i t h a t e s t i s c e l l suspension; c a r r i e r protamine was added p r i o r to chromatography and the 230 nm absorbance p r o f i l e mainly r e -f l e c t s the c a r r i e r protamine. I t i s observed t h a t the newly synthesized protamine chromatographs i n the same reg i o n as c a r r i e r protamine but i s not e x a c t l y c o i n c i d e n t w i t h the c a r r i e r > being e l u t e d s l i g h t l y e a r l i e r . F u r t h e r , 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 the two p a r t i a l l y separated peaks are a l s o d i f f e r e n t i n t h a t the r a t i o of the f i r s t protamine peak to be e l u t e d over the second peak i s much higher i n the r a d i o a c t i v i t y p r o f i l e than i n the absorbance p r o f i l e . Since CM-cellulose chromatography i n v o l v e s ion-exchange and separates molecules on the b a s i s of charge d i f f e r e n c e s , the p o s s i b i l i t y of a charge d i f f e r e n c e between the newly synthesized protamine and preformed " o l d " protamine was examined. The m o d i f i c a t i o n of nucleoproteins by phosphorylation has been w e l l documented (21-24). The presence of phosphorylated pro-tamine was detected by Murray (25,26) who noted the presence of O-phospho-serine i n the p a r t i a l a c i d hydrolysates of a commercially a v a i l a b l e p r e p a r a t i o n of protamine (salmine) . A l s o , Ingles and Dixon (7) have shown th a t 3 2P-phosphate i s incorporated i n t o pro-tamine as 0- 3 2P-phospho-serine by suspensions of t r o u t t e s t i s c e l l s . That phosphorylation of the prbtamiheemolecule could sub-s t a n t i a l l y a l t e r the net charge of protamine may be demonstrated by c a l c u l a t i n g the d i f f e r e n c e i n charge between a non-phosphory-l a t e d protamine molecule and a f u l l y phosphorylated molecule. 6 0.2 . o ro CM fe LU < C D C C O if) GO < 0.0 ro-o^o^>-o--o-^  3.0 2.0 1.0 J 0 Lul Q r r o _i X - J LL. O >-h; cr < o E F F L U E N T V O L U M E , ml F i g . 9. Chromatography on CM-cellulose of newly synthesized protamine obtained from a c e l l suspension of n a t u r a l l y maturing t r o u t t e s t i s . A c e l l suspension was prepared and incubated w i t h 1 ^ C - a r g i n i n e at 20° f o r 60 min. R a d i o a c t i v e protamine was e x t r a c t e d w i t h a c i d and p u r i f i e d by Bio-G e l P-10 chromatography. This l a b e l l e d m a t e r i a l was mixed w i t h 10 mg c a r r i e r protamine and chromatographed on the CM-cellulose L i C l system as i n F i g . 7. - 77u-For example, from the amino:acid composition of protamine, (Table I I ) i t i s seen t h a t a non-phosphcrylated protamine mole-cul e would have a net p o s i t i v e charge of 22 at pH 5 (the chroma-tographic c o n d i t i o n on CM-cellulose) due to the presence of 22 a r g i n i n e residues plus one Imino-terminal group of p r o l i n e and one c a r b o x y l - t e r m i n a l group of a r g i n i n e w h i l e a f u l l y phosphory-l a t e d protamine molecule would have a net p o s i t i v e charge of 18-19 due to the p h o s p h o r y l a t i o n of the 3-4 s e r i n e residues present. Ingles and Dixon (7) have demonstrated t h a t newly syn-t h e s i z e d protamine was phosphorylated and t h a t the r e l a t i v e amount of phosphorylated protamine present i n the t e s t i s decreas-ed as the t e s t i s matured. This suggests t h a t there must be some mechanism f o r removing phosphate from newly synthesized protamine. F i g . 10 A shows the chromatography on CM-52 of protamine e x t r a c t e d A from a t e s t i s c e l l suspension incubated f o r 60 min w i t h 3H-a r g i n i n e and 3 2P-phosphate. I t i s seen t h a t both the t r i t i u m l a b e l l e d and the 3 2 P l a b e l l e d protamine are e l u t e d e a r l i e r and do not chromatograph c o i n c i d e n t l y w i t h the preformed u n l a b e l l e d protamine monitored at 230 nm. F u r t h e r , the peak of t r i t i u m l a b e l l e d protamine e l u t e d l a s t from the column has l i t t l e 3 2 P - l a b e l a s s o c i a t e d w i t h i t . This suggests t h a t the newly synthesized protamine i s phosphorylated and that the more h i g h l y phosphory-l a t e d components are e l u t e d e a r l i e r on t h i s column. In support of t h i s o b s e r v a t i o n , Marushige et al. (33) have shown t h a t pro-tamine may be p r o g r e s s i v e l y e x t r a c t e d from t r o u t t e s t i s chromatin by i n c r e a s i n g the NaCl conce n t r a t i o n u n t i l by 1.2 M, a l l the pro-tamine was e x t r a c t e d . A more h i g h l y phosphorylated protamine f r a c t i o n (as judged by the greater number of cpm 3 2 P incorporated per absorbance at 230 nm) was obtained from protamine e x t r a c t e d at 0.6-0.8 M NaCl. The chromatography on the CM-cellulose - L i C l system showed t h a t t h i s protamine was g r e a t l y enriched i n the components of protamine t h a t e l u t e d e a r l i e r i n t h i s column. Con-v e r s e l y i t was shown,=that the protamine f r a c t i o n e x t r a c t e d at 0.9-1.2 M NaCl, was l e s s phosphorylated and was enriched i n the components of protamine t h a t e l u t e d l a t e r from the CM-cellulose column. J e r g i l (34) has a l s o shown tha t f o l l o w i n g treatment of c a r r i e r protamine w i t h a t r o u t t e s t i s phosphokinase (35, 54) which t r a n s f e r s 3 2P from gamma-.3 2P-ATP to the s e r y l residues of protamine, peaks l a b e l l e d w i t h 3 2P chromatograph on the CM-cellulose system ahead of untreated c a r r i e r protamine i n a p o s i t i o n very s i m i l a r to t h a t of the newly synthesized 3 H - a r g i n i n e - l a b e l l e d protamine. When the protamine from F i g . 10 A was c o l l e c t e d , d e s a l t e d , t r e a t e d w i t h E. eoli a l k a l i n e phosphatase to remove phosphate, and r e -chromatographed on the same CM-52-LiCl system, (see F i g . 10 B), the 3H-protamine p r o f i l e i s now more c o i n c i d e n t w i t h the absorb-ance p r o f i l e although i t s t i l l e l u t e s s l i g h t l y e a r l i e r . I t should a l s o be noted t h a t the absorbance p r o f i l e s of the preformed p r o t a -mine d i d not change (compare F i g . 10 (A) and .(B)) a f t e r treatment w i t h the a l k a l i n e phosphatase. This i n d i c a t e s t h a t newly syn-t h e s i z e d protamine i s more h i g h l y phosphorylated than preformed protamine and i s e l u t e d from the CM-cellulose column e a r l i e r due to the greater degree of phosphorylation. Since from F i g . 4 i t i s seen t h a t a t e s t i s c e l l suspension i s able to incor p o r a t e 1 ''C-arginine i n t o a c i d - e x t r a c t a b l e p r o t e i n s - 79 -T 1 1 1 I A FRAC HON NO.--1, ml F i g . 10. Chromatography on CM-52 of newly synthesized phosphory-l a t e d and e n z y m a t i c a l l y dephosphorylated protamine. A c e l l suspension was prepared from 10 g of t e s t i s and incubated w i t h 500 uC of 3 H - a r g i n i n e and 2 mC of 3 2P-phosphate at 20° f o r 60 min. Protamine was extracted' w i t h a c i d and i s o l a t e d by chroma-tography on a Bi o - G e l P-10 column. Protamine thus obtained was a p p l i e d to a CM-52- column (1.1 x 40 cm) and e l u t e d w i t h a 700 ml g r a d i e n t of 0.75-1.3 M L i C l (A). F r a c t i o n s designated by arrows i n (A) were pooled, d e s a l t e d , l y o p h i l i z e d , and incubated w i t h E. coli a l k a l i n e phosphatase as described i n " M a t e r i a l s and Methods". A f t e r i n c u b a t i o n , the s o l u t i o n was d i l u t e d 3 times w i t h water and subjected to chromatography on the CM-52 column as before (B). - 80 -at a constant r a t e f o r 9 hours or more at 20?, i t was p o s s i b l e , i n conjunction w i t h the h i g h l y r e s o l v i n g C M - c e l l u l o s e - L i C l system, to determine whether the newly synthesized protamine was dephos-phorylated s h o r t l y a f t e r t r a n s p o r t to the nucleus. F i g . 11 shows the r e s u l t of an experiment i n which two i d e n t i c a l t e s t i s c e l l suspensions were allowed to i n c o r p o r a t e 1 4 C - a r g i n i n e f o r 60 min at 20°. Protamine was e x t r a c t e d immediately w i t h a c i d from one suspension w h i l e the other was washed once w i t h Hanks.;' s o l u t i o n to remove 1 ^ C - a r g i n i n e and then chased f o r 7 hours w i t h 1 mM 1 Z C -a r g i n i n e before a c i d e x t r a c t i o n . I t may be seen from F i g . 11 t h a t when chromatographed on the CM-cellulose column, the r a d i o -a c t i v e protamine obtained a f t e r 7 hours chase at 20° s t i l l e l u t e d s i g n i f i c a n t l y ahead of the endogenous protamine at a p o s i t i o n s i m i l a r to t h a t of the r a d i o a c t i v e protamine obtained a f t e r no chase. This suggests t h a t dephosphorylation of newly synthesized protamine i s not a r a p i d process and that no s i g n i f i c a n t amount of newly synthesized protamine i s dephosphorylated w i t h i n 7 hours at 20°. While i t i s c l e a r t h a t phosphorylation plays a major r o l e i n the e a r l i e r e l u t i o n of newly synthesized protamine, a second p o s s i -b i l i t y e x i s t s f o r the non-coincidence between the r a d i o a c t i v i t y p r o f i l e and absorbance p r o f i l e on CM-cellulose chromatography. AS already mentioned, the peaks of protamine resolved on CM-cellulose are not homogeneous as judged by amino a c i d a n a l y s i s of the peaks and hence each peak may be composed of s e v e r a l protamine compon-ents. I t i s e n t i r e l y p o s s i b l e t h a t the synthesis of each compon-ent of protamiheecould occur independently at d i f f e r e n t stages of - 81 -F i g . 11. Rate of dephosphorylation of newly synthesized protamine i n t e s t i s n u c l e i . Two i d e n t i c a l c e l l suspensions (15 g t e s t i s each) were prepared i n Hanks' s o l u t i o n and incubated w i t h 2.5 yC 1 4 C - a r g i n i n e at 20° f o r 1 hr. (A) protamine was e x t r a c t e d w i t h a c i d from c e l l n u c l e i at the end of the 1 hr i n c o r p o r a t i o n ; (B) c e l l s were washed w i t h Hanks' s o l u t i o n and f u r t h e r chased f o r 7 hrs w i t h 1 mM 1 2 C - a r g i n i n e before e x t r a c t i o n f o r protamine as i n (A). Protamine was p u r i f i e d by Bi o - G e l P-10 chromatography and' a p p l i e d to a CM-52 column (1.1 x 40 cm). Conditions of chromatography were as i n F i g . 10. - 82 -spermatogenesis. Thus at any i n s t a n t , one component could be synthesized at a more r a p i d r a t e than the others le a d i n g to an asymmetrical d i s t r i b u t i o n of l a b e l through a peak c o n t a i n i n g p a r t i a l l y separated components. f) Changes i n the Chromatographic P r o f i l e s of Protamine During Development Since i t has already been demonstrated that the protamine of each t r o u t i s heterogeneous (Table I I ) and could be separated i n t o three major components by CM-cellulose chromatography (see F i g . 8), i t was of i n t e r e s t then to determine whether the com-ponents of protamine are present i n a constant r a t i o throughout t e s t i s development. A p r e l i m i n a r y examination of the chromato-graphic p r o f i l e s on CM-cellulose of protamine obtained from t e s t i s of n a t u r a l l y maturing t r o u t during the e a r l y and l a t e stages of spermatogenesis, i n September and i n December respec-t i v e l y , showed t h a t there were marked d i f f e r e n c e s i n the r e l a t i v e amounts of the three components of protamine present C ,.C , and (as c l a s s i f i e d i n F i g . 8). One s t r i k i n g d i f f e r e n c e was t h a t C j , the f i r s t protamine peak to be e l u t e d from the CM-cellulose column c o n s t i t u t e d a s i g n i f i c a n t p r o p o r t i o n of the t o t a l protamine present i n September but by December, C , was present i n almost n e g l i g i b l e amounts. This suggested that the protamine components may be synthesized at d i f f e r e n t r e l a t i v e r a t e s and/or at d i f f e r e n t times during spermatogenesis. To examine t h i s p o s s i b i l i t y , t r o u t t e s t e s c o l l e c t e d from n a t u r a l l y maturing t r o u t during the months of September to December were incubated w i t h 0.5 uC/ml of 1^C-a r g i n i n e f o r 30 min at 20° a f t e r which protamine was e x t r a c t e d and - 83 -p u r i f i e d from the t e s t i s n u c l e i by chromatography on the Bio-Gel P-10 column. The i s o l a t e d protamine was t r e a t e d w i t h E. coli a l k a l i n e phosphatase as described i n " M a t e r i a l s and Methods" i n order to b e t t e r determine w i t h which component of protamine the newly synthesized protamine was a s s o c i a t e d . The r a t i o n a l e f o r t h i s treatment was t h a t as already demonstrated i n F i g . 10 A and B, newly synthesized protamine i n c o r p o r a t i n g l a b e l l e d a r g i n i n e does not chromatograph at the same p o s i t i o n as the preformed pro-tamine; a l s o , the p r o f i l e of incorporated r a d i o a c t i v i t y i s q u i t e d i f f e r e n t from the absorbance p r o f i l e , Hence, i t was d i f f i c u l t to determine the correspondence between the r a d i o a c t i v e components and the preformed components monitored at 230 nm by absorbance. A f t e r treatment w i t h a l k a l i n e phosphatase, however,, the r a d i o a c t i v e p r o f i l e i s more c o i n c i d e n t w i t h absorbance p r o f i l e (see F i g . 10 A and B) and i t i s now p o s s i b l e to a s s o c i a t e the r a d i o a c t i v e com-ponents w i t h the absorbance components; the amount of r a d i o a c t i -v i t y a s s o c i a t e d w i t h a p a r t i c u l a r component would then give a measure of the amount of syn t h e s i s of t h a t component. F i g . 12 A to D shows the CM-cellulose chromatography of protamine of natur-a l l y maturing t r o u t t e s t i s obtained from September to December. Protamine of t r o u t sperm, F i g . 12 E was obtained i n January during the spawning p e r i o d by expressing m i l t from a number of t r o u t , c o l l e c t i n g the sperm c e l l s by c e n t r i f u g a t i o n and e x t r a c t i n g the c e l l s w i t h 0.2 M s u l f u r i c a c i d as before. In F i g . 12, the f i r s t absorbance peak to be e l u t e d from the column s h o r t l y a f t e r the v o i d volume i s the a l k a l i n e phosphatase present i n the i n c u -b a t i o n mixtures which were a p p l i e d d i r e c t l y onto the CM-cellulose - 84 -FRACTION NO. - 4ml 10 JO 30 40 V J . 60 70 Fraction No.-4ml F i g . 12. Changes i n the chromatographic p r o f i l e s of protamine d u r i n g t e s t i s development. Testes were c o l l e c t e d from n a t u r a l l y maturing t r o u t during the middle of each month and c e l l suspensions were prepared w i t h 10-15 g of t e s t i s . The c e l l suspensions were incubated w i t h 0.5 uC/ml i n c u b a t i o n mixture of 1 I*C-arginine at 20° f o r 30 min and were then e x t r a c t e d f o r protamine. Protamine p u r i f i e d on Bi o - G e l P-10 was t r e a t e d w i t h E. coli a l k a l i n e phosphatase as de s c r i b e d i n " M a t e r i a l s and Methods" and a p p l i e d to a CM-52 column (1.1 x 40 cm). Conditions of chromatography were as i n F i g . 10. Protamine was i s o l a t e d from t e s t e s c o l l e c t e d i n (A) September (B) October (C) November (D) December and (E) from mature spermatazoa expressed from t r o u t i n January. C^ ., C j j r and CJJ-J- are the components of protamine as c l a s s i f i e d i n Fxg.8. - 85 -column. At about 0.9 M L i C l , peaks of protamine are e l u t e d o f f the column and i t may be seen that there are changes i n the chromatographic p r o f i l e s of the protamine components as the t e s t i s matures. C-j., the f i r s t protamine peak decreases as the t e s t i s matures w h i l e the t h i r d protamine peak C^ ..^  increases u n t i l i n the December t e s t i s , i t i s the major protamine peak becoming s l i g h t l y l a r g e r than C^. The sperm protamine contains e s s e n t i a l l y only components and C^-j. and bears a strong s i m i l a r i t y to the protamine found i n the December t e s t i s . This suggests then t h a t the developing t e s t i s contains a changing complement of protamine components at d i f f e r e n t stages of sperma-togenesis u n t i l f i n a l l y by the sperm c e l l stage of development t h i s complement becomes f i x e d . That s p e c i f i c changes could.occur i n the popu l a t i o n of chromosomal p r o t e i n s have been shown by Stellwagen and Cole (36). They have demonstrated t h a t the very l y s i n e - r i c h h i s t o n e s , histone F l , obtained from r a t and r a b b i t mammary glands could be separated i n t o 4 components by chromatography on an Amberlite IRC-50 column e l u t e d by a shallow gradient of 7-14% guanidinum hy d r o c h l o r i d e . They observed t h a t the r a t i o s of the d i f f e r e n t components of the F l h i s t o n e change w i t h the d i f f e r e n t p h y s i o l o g i c a l s t a t e s of the mammary gland obtained at d i f f e r e n t stages of development and l a c t a t i o n . Marushige and Dixon (12) have a l s o shown th a t at d i f f e r e n t stages of t r o u t t e s t i s development, the histone popu-l a t i o n of the i s o l a t e d chromatin i s d i f f e r e n t . a s judged by p o l y -acrylamide g e l e l e c t r o p h o r e s i s ; f u r t h e r , they have shown that i n the process of replacement of histones by protamine, c e r t a i n - 86 -histones (histone IV) may.be replaced before others (histone I ) , suggesting t h a t the replacement process occurs i n a d e f i n i t e temporal sequence. I t i s not known at present whether the d i f -f e r e n t components of protamine have d i f f e r e n t s p e c i f i c i t i e s i n the replacement of hi s t o n e s . From F i g . 12, i t i s seen t h a t r a d i o a c t i v i t y i s incorporated i n t o the three components of protamine throughout t e s t i s develop-ment, however, the r e l a t i v e amounts of i n c o r p o r a t i o n i s n e i t h e r the same nor i n constant p r o p o r t i o n s . This suggests t h a t once protamine sy n t h e s i s begins, a l l the components of protamine continue to be synthesized throughout the l a t e r stages of t e s t i s development but, the r a t e of synthesis of the components are d i f f e r e n t . I f the components were synthesized at the same r a t e , then the peaks of incorporated a r g i n i n e f o r the three components would be of the same s i z e . F u r t her, i t may be seen that the r a t e of synt h e s i s of any one component r e l a t i v e to the other two i s d i f f e r e n t at d i f f e r e n t times during t e s t i s development suggest-i n g t h a t any one component i s not synthesized at the same r a t e throughout spermatogenesis. In general, i t appears that there i s a r e l a t i v e decrease i n the synt h e s i s of C^. and a r e l a t i v e i n -crease i n tha t of C as development of the t e s t i s proceeds. Since t h i s i n c o r p o r a t i o n data appears to r e f l e c t the absorbance data, i t i s l i k e l y then t h a t the changes i n the r e l a t i v e amounts of the components of protamine present i n the t e s t i s n u c l e i at d i f f e r e n t stages of spermatogenesis are the r e s u l t of the d i f f e r -e n t i a l s ynthesis of the components. - 87 -I I . I n t r a c e l l u l a r S i t e of Protamine Synthesis This s e c t i o n i s concerned w i t h the i n t r a c e l l u l a r s i t e of synthesis of protamine. Ingles et al. (7) have proposed from t h e i r s t u d i e s of various i n h i b i t o r s on protamine sy n t h e s i s i n the t e s t i s c e l l suspensions, t h a t protamine i s synthesized upon a s t a b l e messenger RNA template a s s o c i a t e d w i t h ribosomes. In euka r y o t i c c e l l s , the m a j o r i t y of ribosomes are lo c a t e d i n the cytoplasm but ribosomes f u l l y a c t i v e i n p r o t e i n synthesis have been i s o l a t e d from animal n u c l e i (37-40), and i t has been suggest-ed by P a t e l and Wang (41) and Naora (42) t h a t DNA i t s e l f can under some c o n d i t i o n s act d i r e c t l y as a template f o r s y n t h e s i s . The nuclear s y n t h e s i s of histones i n thymocytes has been i m p l i c a t e d by the s t u d i e s of A l l f r e y et al. (43) and Reid and Cole (44) wh i l e Robbins and Borun (45) have shown t h a t histones are syn-t h e s i z e d i n the cytoplasm of Hela c e l l s . Since protamines, l i k e h i s t o n e s , are a l s o chromosomal p r o t e i n s , the question a r i s e s of whether they are synthesized i n the nucleus i t s e l f or at some other l o c a t i o n i n the c e l l and subsequently transported to the nucleus. F u r t h e r , because of the unusual nature of the protamine molecule, i t s r o l e as the replacement p r o t e i n f o r histones on DNA, and i t s r e s t r i c t i o n to the t e s t i s , knowledge of the c e l l u l a r locus of protamine synthesis may help to e l u c i d a t e the r o l e played by protamine i n c e l l u l a r d i f f e r e n t i a t i o n during sperm maturation. a) Sedimentation A n a l y s i s of Cytoplasmic and Nuclear Microsomes Since as al r e d y mentioned, the i n h i b i t i o n s t u d i e s of Ingles et al. (11) s t r o n g l y suggested t h a t protamine was synthesized by ribosomes, . '. experiment was performed to determine the amount - 88 -o f n a s c e n t p r o t a m i n e a s s o c i a t e d w i t h n u c l e a r m i c r o s o m e s and c y t o -p l a s m i c m i c r o s o m e s t h e r e b y d e t e r m i n i n g t h e s i t e o f p r o t a m i n e s y n t h e s i s . As m e n t i o n e d i n t h e " M a t e r i a l s and M ethods", t h e t e r m " m i c r o s o m e s " u s e d i n t h i s t h e s i s d e n o t e s t h e s e d i m e n t ob-t a i n e d e i t h e r f r o m t h e p o s t - n u c l e a r s u p e r n a t a n t o r t h e p o s t -m i t o c h o n d r i a l s u p e r n a t a n t a f t e r h i g h s p e e d c e n t r i f u g a t i o n . The p r o c e d u r e s f o r t h e i s o l a t i o n o f c y t o p l a s m i c and n u c l e a r m i c r o s o m e s a r e o u t l i n e d i n F i g . 13. F i g . 14 shows t h e d i s t r i b u t i o n o f 1 ^ C -a r g i n i n e l a b e l l e d a c i d - e x t r a c t a b l e p r o t e i n s a s s o c i a t e d w i t h n u c -l e a r and c y t o p l a s m i c m i c r o s o m e s f r a c t i o n a t e d on s u c r o s e d e n s i t y g r a d i e n t s . The method o f a c i d e x t r a c t i o n employed (0.5 M HC1 p l u s 5% TCA) was one f o u n d t o be more s p e c i f i c f o r p r o t a m i n e ( s e e " M a t e r i a l s and Methods") and a l s o , as a l r e a d y m e n t i o n e d , a t t h i s s t a g e o f s p e r m a t o g e n e s i s , p r o t a m i n e i s t h e m a j o r a c i d - e x t r a c t -a b l e p r o t e i n b e i n g s y n t h e s i z e d . T h u s , t h e peak o f 1 ^ C - a r g i n i n e l a b e l l e d p r o t e i n p r e s e n t i n F i g . 14 A i s l i k e l y t o be p r o t a m i n e . By c o m p a r i n g F i g . 14 A and B, i t may be o b s e r v e d t h a t t h e s p e c i f i c a c t i v i t y o f l a b e l p e r a b s o r b a n c e a t 260 nm i s s u b s t a n t i a l l y h i g h e r i n t h e c y t o p l a s m i c m i c r o s o m e s t h a n i n t h e n u c l e a r m i c r o s o m e s s u g g e s t i n g t h a t p r o t a m i n e i s s y n t h e s i z e d i n t h e c y t o p l a s m and t h e n t r a n s p o r t e d i n t o t h e n u c l e u s . The c o u n t s p r e s e n t i n t h e n u c l e a r m i c r o s o m e s c o u l d v e r y w e l l be due t o c o n t a m i n a t i o n by c y t o p l a s m i c m i c r o s o m e s s i n c e t h e n u c l e a r m i c r o s o m e s were o b t a i n e d f r o m a r e l a t i v e l y c r u d e n u c l e a r f r a c t i o n w h i c h was n o t washed f r e e o f c y t o p l a s m . F u r t h e r t h e c e n t r i f u g a t i o n s t e p employed t o s e d i m e n t t h e n u c l e a r f r a c t i o n w o u l d a l s o s e d i m e n t w h o l e c e l l s w h i c h were n o t r u p t u r e d d u r i n g t h e b r i e f h o m o g e n a t i o n s t e p employed - 8<J -no:: 30 sec C L L L ;:.: n:..,:;: : •clo!'.--xirido ar.ci 1 'C-arg inine 1 "C-arc ir.ino 3C ere CELL E'JSPE; tli) I | 1 'C-arc i ni ne 1 *C-arginine cvclohexircidG CELL. FRACTIONATION FOR PPVP'-.RATIOX OF MICROSOMES CELL SUSPENSIONS A and B homogenised, 5,000 rpm 20 sec i n Tl'.KS, centrifugation 1,000 x g for 10 min -CYTOPLASM (PNS) suspended in TMC hotsogemxation 5,000 rpm 2 min, centrifugation: 30,000 x g for 10 min 65,000 rpm for 1 hour 15,000 x g for IS min PFLLF.T (NUCLEAR RESIDUE, DIFCAHnED) PELLET N'UCLEAR MICROSOMES SUPERNATANT (DISCARDED) PELLET MITOCHONDRIA PELLET . CYTOPLASMIC MICROSOMES SUPERNATANT (PMS) 65,000 rpm for 1 hour SUPERNA-TANT (DISCARDED) F i g . 13. P r e p a r a t i o n of t e s t i s c y t o p l a s m i c and n u c l e a r m i c r o -^ t T 141 f a ^ s l s .£y sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n ( F i g . 14) as d e s c r i b e d m " M a t e r i a l s and Methods". - 90 -CYTOPLASMIC MICROSOMES NUCLEAR MICROSOMES - j -j-* | p— r— 1 BOTTOM TOP BOTTOM FRACTION NUMBER F i g . 14. Sedimentation a n a l y s i s of t e s t i s cytoplasmic and nuclear microsomes. C e l l suspensions were pulsed w i t h C-arginine and chased w i t h 1 2 C - a r g i n i n e as i l l u s t r a t e d i n F i g . 13. C e l l s were f r a c t i o n a t e d and cytoplasmic microsomes and nuclear microsomes were obtained ( F i g . 13). The microsomes (0.2 ml) were layered onto 15-30% (w/v) l i n e a r sucrose g r a d i e n t s and c e n t r i f u g e d at 35,000 rpm f o r 1.5 hr i n a SW-39 swinging bucket r o t o r which was allowed to stop without b r a k i n g . F r a c t i o n s from the grad i e n t s were c o l l e c t e d and analyzed f o r absorbance at 260 nm and r a d i o -a c t i v i t y as de s c r i b e d i n " M a t e r i a l s and Methods". (A) and (C) are cytoplasmic microsomes; (B) and (D) nuclear microsomes. (A) and (B) are microsomes i s o l a t e d a f t e r a 0.5 min pulse of ^ C - a r g i n i n e ; (C) and (D) microsomes i s o l a t e d a f t e r a 0.5 min pulse of ^ C - a r g i n i n e and a 10 min chase w i t h 1 2 C - a r g i n i n e . - 91 -f o r the i s o l a t i o n of cytoplasm. In an attempt to e l i m i n a t e the p o s s i b i l i t y of cytoplasmic contamination, a s i m i l a r experiment was performed i n which the crude nuclear f r a c t i o n was p u r i f i e d by c e n t r i f u g a t i o n through s e v e r a l l a y e r s of sucrose s o l u t i o n s of i n -c r e a s i n g d e n s i t y , a method developed by T r e v i t h i c k et al. (46) i n which whole c e l l s remained at an i n t e r f a c e f l o a t i n g on 2.8 M sucrose and the n u c l e i passed through to form a p e l l e t . The microsomes obtained from t h i s p u r i f i e d nuclear f r a c t i o n now con-t a i n e d almost no a c i d - e x t r a c t a b l e counts. This then supports the cytoplasmic microsomes as the s i t e of protamine s y n t h e s i s . I f one assumes th a t the peak of absorbance at 260 nm (Fig. 14 A to be a s s o c i a t e d w i t h the s i n g l e ribosomes, then the l a b e l l e d m a t e r i a l i s l i k e l y to be a s s o c i a t e d w i t h small polyribosomes. Since protamine i s a h i g h l y b a s i c p r o t e i n , there i s the p o s s i b i l i t y t h a t the observed peak of r a d i o a c t i v i t y i n F i g . 14 A i s not a s s o c i a t e d w i t h nascent protamine growing from polyribosomes but a c t u a l l y represents some n o n - s p e c i f i c i o n i c i n t e r a c t i o n between f r e e r a d i o a c t i v e protamine and the n u c l e i c acids of the ribosomes. I f the peak of r a d i o a c t i v i t y were due to n o n - s p e c i f i c binding of 1^C-protamine to ribosomes then the r a d i o a c t i v e p r o f i l e would be very s i m i l a r to the absorbance p r o f i l e (see F i g . 23). That t h i s i s not the case may be v e r i f i e d by the f a c t t h a t , as seen i n F i g . 14 A, the r a d i o a c t i v i t y p r o f i l e i s not c o i n c i d e n t w i t h the absorbance p r o f i l e . F u r t h e r , as i l l u s t r a t e d i n F i g . 14 C and D, i t i s seen that i n a 10 min chase w i t h u n l a b e l l e d l 2 C - a r g i n i n e there i s a l o s s of l a b e l l e d m a t e r i a l from the microsomes suggest-i n g t h a t l i t t l e n o n - s p e c i f i c b i n d i n g occurs and that newly - 92 -s y n t h e s i z e d p r o t a m i n e i s f r e e l y r e l e a s e d f r o m t h e m i c r o s o m e s i n s p i t e o f i t s h i g h l y b a s i c n a t u r e . b) P u l s e - c h a s e K i n e t i c s o f 1 ^ C - P r o t a m i n e The p a s s a g e o f 1 ^ C - p r o t a m i n e f r o m m i c r o s o m e s t o n u c l e u s c o u l d be e xamined by p u l s e l a b e l l i n g t h e p r o t a m i n e i n wh o l e c e l l s and s u b s e q u e n t l y c h a s i n g w i t h 1 2 C - a r g i n i n e . Fiona t h e m i c r o s o m a l s t u d i e s j u s t m e n t i o n e d , t h e r e i s v e r y l i t t l e a c i d - e x t r a c t a b l e r a d i o a c t i v i t y a s s o c i a t e d w i t h n u c l e a r m i c r o s o m e s , and hence i n t h i s e x p e r i m e n t no a t t e m p t was made t o s e p a r a t e t h e n u c l e a r m i c r o -somes f r o m t h e c y t o p l a s m i c m i c r o s o m e s . The d a t a p r e s e n t e d i n F i g . 15 show t h a t i n i t i a l l y t h e p e r c e n t a g e o f l a b e i n t h e m i c r o -somes was s l i g h t l y g r e a t e r t h a n t h a t i n t h e n u c l e a r r e s i d u e . As t h e c h a s e w i t h 1 2 C - a r g i n i n e c o n t i n u e d , t h e p e r c e n t a g e o f l a b e l l e d p r o t a m i n e on t h e m i c r o s o m e s d e c r e a s e d w h i l e t h a t i n t h e n u c l e a r f r a c t i o n i n c r e a s e d , c o n s i s t e n t w i t h a p r e c u r s o r - p r o d u c t r e l a t i o n -s h i p . F u r t h e r , i n e a r l i e r e x p e r i m e n t s , (47) T r e v i t h i c k had o b s e r v e d t h a t t h e l e v e l s o f l a b e l l e d p r o t a m i n e f r e e i n t h e s u p e r -n a t a n t were v e r y s m a l l by c o m p a r i s o n w i t h t h a t on t h e m i c r o s o m e s . T h u s , newly s y n t h e s i z e d p r o t a m i n e p a s s e s f r o m t h e c y t o p l a s m i c m i c r o s o m e s i n t o t h e n u c l e u s by some t r a n s p o r t mechanism w h i c h i s as y e t u n d e f i n e d . P r e l i m i n a r y e x p e r i m e n t s c a r r i e d o u t by M a r u s h i g e et al. (48) showed t h a t w i t h 2 , 4 - d i n i t r o p h e n o l as an i n h i b i t o r o f ATP f o r m a t i o n i n w h o l e s p e r m a t i d c e l l s t h e r e i s some i n t e r u p t i o n o f movement o f p r o t a m i n e f r o m c y t o p l a s m t o n u c l e u s . T h i s s u g g e s t s t h e p o s s i b i l i t y o f ATP b e i n g t h e e n e r g y s o u r c e f o r t r a n s p o r t . - 93 -ui . z i I O O or < i o u. o z o cc o o. cc o o z < o UJ o cc UJ \ N U C L E A R R E S I D U E M I C R O S O M E S i i_ 12. 8 10 M I N U T E S A F T E R C H A S E O F C - A R G I N I N E 14, C-ARG L P U L S E C H A S E T E R M I N A T E D BY C Y C L O H E X I M I D E A T T I M E S I N D I C A T E D F i g . 15. Pulse-chase k i n e t i c s As d e s c r i b e d i n " M a t e r i a l s and sion s were pulsed f o r 30 sec wi chase of 1 z C - a r g i n i n e (1 mM). cycloheximide at the times i n d i i n t o n uclear r e s i d u e s and micro was s p e c i f i c a l l y e x t r a c t e d f o r Protamine was p r e c i p i t a t e d w i t h a c t i v i t y i n the p r e c i p i t a t e s wa of 1 ''C-arginine l a b e l l e d protamine. Methods", i d e n t i c a l c e l l suspen-t h 1 4 C - a r g i n i n e f o l l o w e d by a The chase was terminated w i t h cated. The c e l l s were f r a c t i o n a t e d somal f r a c t i o n s and each f r a c t i o n protamine (0.5 M HCl and 5% TCA). pH 2 TCA-tungstate and r a d i o -s assayed. - 94 -c) Time Course of I n t r a c e l l u l a r D i s t r i b u t i o n of 1^C-Protamine Having e s t a b l i s h e d t h a t a r a p i d l y l a b e l l e d a c i d - e x t r a c t a b l e p r o t e i n , probably protamine, i s synthesized on the microsomes of the cytoplasm, and t h a t t h i s p r o t e i n i s then transported i n t o the nucleus, i t i s necessary to i d e n t i f y the l a b e l l e d p r o t e i n c l e a r l y as protamine. The d i s t r i b u t i o n of the l a b e l l e d protamine a f t e r i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e f o r a short time and a longer time could then be s t u d i e d . From F i g . 15, i t appears that the t r a n s p o r t of newly synthesized protamine i s r a p i d , w i t h an upper l i m i t to the h a l f - t i m e of 1-2 min at 20°. Therefore, i t may be p r e d i c t e d t h a t i f the t e s t i s c e l l s are allowed to incorporate l a b e l l e d a r g i n i n e i n t o protamine f o r a short enough time, most of the l a b e l l e d protamine would be present i n the cytoplasm since only a l i m i t e d amount of t r a n s p o r t would have occurred; at longer times of i n c o r p o r a t i o n , the nucleus would have accumulated the greater amount of l a b e l l e d protamine,being transported there from the cytoplasm. F i g . 16 shows the r e s u l t of such an experiment which confirms the p r e d i c t i o n . I t may be seen i n F i g . 16 A and B t h a t a f t e r short i n c o r p o r a t i o n p e r i o d of 0.5 min, the r a d i o a c t i v e protamine (PII of the P-10 column) i s present i n greater amount i n the t e s t i s c e l l cytoplasm than i n the nucleus. This suggests t h a t s i n c e r a d i o a c t i v e protamine appears f i r s t i n the cytoplasm, i t must be synthesized there and that the r a d i o a c t i v e protamine present i n the nucleus i s the r e s u l t of t r a n s p o r t from the c y t o -plasm. A f t e r a longer i n c u b a t i o n of 15 min, the nuclear f r a c t i o n ( F i g . 16 D) now contains s u b s t a n t i a l l y more 1^C-protamine than the cytoplasmic f r a c t i o n ( F i g . 16 C) a r e s u l t c o n s i s t e n t w i t h the continued t r a n s p o r t of protamine i n t o the nucleus. - 95 -F i g . 16. Time course of i n t r a c e l l u l a r d i s t r i b u t i o n of 1 lfC-a r g i n i n e - l a b e l l e d protamine. As de s c r i b e d i n " M a t e r i a l s and Methods", c e l l suspensions from hormonally induced t r o u t t e s t e s were pulsed w i t h C-arginine f o r 0.5 and 15 min and i n c o r p o r a -t i o n was terminated w i t h cycloheximide. The c e l l s were homo-genized and separated i n t o cytoplasmic and nuclear f r a c t i o n s by d i f f e r e n t i a l c e n t r i f u g a t i o n . The f r a c t i o n s were e x t r a c t e d w i t h 0.2 M s u l f u r i c a c i d and 1^C-protamine was i d e n t i f i e d by B i o -Gel P-10 chromatography (PII)'. (A) and (C) are the cytoplasmic f r a c t i o n s ; (B) and (D), the nuclear f r a c t i o n s . - 96 -I n any b i o c h e m i c a l s t u d i e s c o n c e r n e d w i t h t h e d e t e r m i n a t i o n o f t h e c e l l u l a r s i t e o f p r o t e i n s y n t h e s i s , t h e f r a c t i o n a t i o n o f t h e c e l l s i n t o t h e i r components i s e s s e n t i a l t o t h e f i n a l e l u c i -d a t i o n o f t h e s i t e o f s y n t h e s i s . However, one i s a l w a y s c o n c e r n e d w i t h t h e p o s s i b l e c o n t a m i n a t i o n o f one component by a n o t h e r d u r i n g t h e c o u r s e o f f r a c t i o n a t i o n . I n t h e s p e r m a t i d c e l l , t h e m a j o r c e l l t y p e p r e s e n t i n t h e t e s t i s d u r i n g a c t i v e p r o t a m i n e s y n t h e s i s ( 9 ) , t h e r e i s o n l y a s c a n t amount o f c y t o p l a s m compared t o t h e mass o f t h e n u c l e u s (9) and h e n c e , a s m a l l p e r c e n t a g e o f n u c l e a r c o n t a m i n a n t a p p e a r i n g i n t h e c y t o p l a s m d u r i n g f r a c t i o n a t i o n may r e p r e s e n t a s i g n i f i c a n t p r o p o r t i o n o f t h e c y t o p l a s m . T h i s p o s s i -b i l i t y o f n u c l e a r c o n t a m i n a t i o n i s o f s p e c i a l c o n c e r n i n t h e p r o b -lem o f t r y i n g t o d e t e r m i n e t h e s i t e o f p r o t a m i n e s y n t h e s i s s i n c e t h e n u c l e u s i s t h e f i n a l l o c a t i o n o f n ewly s y n t h e s i z e d p r o t a m i n e and t h e r e f o r e e v e n a f t e r a s h o r t i n c o r p o r a t i o n p e r i o d o f r a d i o -a c t i v e amino a c i d s by t h e c e l l s , t h e n u c l e u s a c c u m u l a t e s a l a r g e amount o f l a b e l l e d p r o t a m i n e . T h u s , t h e p r e s e n c e o f a s m a l l amount o f n e w l y s y n t h e s i z e d n u c l e a r p r o t a m i n e i n t h e c y t o p l a s m c o u l d be m i s t a k e n f o r t h e p r e s e n c e o f n ewly s y n t h e s i z e d c y t o -p l a s m i c p r o t a m i n e . To overcome t h e s e d i f f i c u l t i e s , o n l y a b r i e f and m i l d h o m o g e n i z a t i o n was employed d u r i n g t h e b r e a k a g e o f c e l l s i n o r d e r t o e n s u r e t h e l e a s t p o s s i b l e damage t o t h e n u c l e i . A l s o , u s e o f t h e p u l s e - c h a s e t e c h n i q u e was employed t o e s t a b l i s h p r e c u r s o r - p r o d u c t r e l a t i o n s h i p s . T h a t t h e c o n d i t i o n o f h o m o g e n i z a -t i o n was i d e a l i s i n d i c a t e d by t h e a b s e n c e o f a b s o r b a n c e a t 230 nm i n P I I , t h e p r o t a m i n e r e g i o n , ( F i g . 16 A and C ) , when t h e a c i d -e x t r a c t a b l e p r o t e i n s o f t h e c y t o p l a s m were c h r o m a t o g r a p h e d on t h e B i o - G e l P-10 c olumn. F o r had n u c l e a r m a t e r i a l b e e n p r e s e n t , t h e n - 97 -some evidence of the protamine present i n the a c i d e x t r a c t s of n u c l e i , P I I of F i g . 16 B and D would have been seen. I t should a l s o be noted here that the use of 1 ^ C - a r g i n i n e to study i n c o r -p o r a t i o n i n t o protamine was o b l i g a t o r y f o r the s u c c e s s f u l demon-s t r a t i o n of spermatid cytoplasm as the s i t e of protamine syn-t h e s i s . As shown i n F i g . 16 A and C, t e s t i s c e l l cytoplasm con-t a i n s no chemically detectable amounts of protamine as shown by the absence of 230 nm absorbance at P I I , however, d e f i n i t e peaks of r a d i o a c t i v e protamine could be observed i n the P I I reg i o n due to the greater s e n s i t i v i t y of d e t e c t i o n f o r r a d i o a c t i v e m a t e r i a l . The unusual nature of the protamine molecule i t s e l f has c o n t r i -buted to the ease of i d e n t i f i c a t i o n of r a d i o a c t i v e m a t e r i a l i n the cytoplasm as newly synthesized protamine. F i r s t , because of i t s h i g h l y b a s i c nature and small s i z e e , protamine could be c l e a r l y separated from other b a s i c p r o t e i n s by means of Bio-Gel P-10 chromatography and the h i g h l y r e s o l v i n g C M - c e l l u l o s e - L i C l system; t h i s allows a confide n t i d e n t i f i c a t i o n ofnnewly synthesized protamine. Second, because of the f a c t t h a t the high b a s i c i t y of protamine i s due e n t i r e l y to high content a r g i n i n e residues which comprise 2/3 of the protamine molecule, the s e n s i t i v i t y of d e t e c t i n g protamine by 1 ^ C - a r g i n i n e l a b e l l i n g i s g r e a t l y increased. d) Protamine Synthesis i n a C e l l - F r e e System E a r l y during the course of t h i s work, i t was noted t h a t a crude broken c e l l homogenate obtained from t e s t i s could i n c o r p o r -ate 1 ^ C - a r g i n i n e i n t o a c i d e x t r a c t a b l e p r o t e i n s . I n i t i a l l y i t was f e l t t hat a p o s s i b l e e x p l a n a t i o n of t h i s apparent ruptured-c e l l s y n t h e s i s of p r o t e i n was due to the presence i n the homogenate - 98 -of a small p o p u l a t i o n of i n t a c t c e l l s not ruptured by homogeniza-t i o n . However, Dr. K e i j i Marushige of t h i s l a b o r a t o r y noted that a p o s t - m i t o c h o n d r i a l cytoplasmic f r a c t i o n i s o l a t e d from t e s t i s c e l l s during the protamine stage of spermatogenesis was able to inc o r p o r a t e 1 ^ C - a r g i n i n e i n t o protamine when supplemented w i t h appropriate c o f a c t o r s . F i g . 17 i l l u s t r a t e s the chromatography on the Bio-Gel P-10 of newly synthesized protamine i s o l a t e d from a c e l l - f r e e cytoplasmic system. I t was of i n t e r e s t then to examine the r e l a t i v e i n c o r p o r a t i o n of 1 '*C-arginine i n t o protamine by v a r i o u s t e s t i s c e l l f r a c t i o n s . F i g . 18 i l l u s t r a t e s the steps i n v o l v e d i n i s o l a t i n g a nuclear f r a c t i o n , a pos t - m i t o c h o n d r i a l cytoplasmic f r a c t i o n , and a high speed supernatant f r a c t i o n , from n a t u r a l l y maturing t r o u t t e s t i s . These i s o l a t e d f r a c t i o n s were i n d i v i d u a l l y incubated w i t h 1 I tC-arginine i n in vitro p r o t e i n syn-t h e s i z i n g systems. Table I I I shows the r e l a t i v e i n c o r p o r a t i o n by each i s o l a t e d c e l l f r a c t i o n of 1 4 C - a r g i n i n e i n t o protamine i s o l a t e d as P I I of the Bio-Gel P-10 column. The cytoplasm f r a c -t i o n (comprising microsomes plus supernatant f a c t o r s ) was very a c t i v e i n i n c o r p o r a t i n g 1 ^ C - a r g i n i n e i n t o protamine while the nuclear f r a c t i o n and the high speed supernatant were r e l a t i v e l y i n a c t i v e , again confirming the cytoplasmic synthesis of protamine. When the 1 ^ C - a r g i n i n e l a b e l l e d product synthesized by the i s o -l a t e d cytoplasmic f r a c t i o n and c h a r a c t e r i z e d as P I I of the P-10 column was f u r t h e r chromatographed on the h i g h l y r e s o l v i n g CM-c e l l u l o s e L i C l system, the l a b e l was e l u t e d i n the same reg i o n as c a r r i e r protamine. But again, l i k e newly synthesized protamine obtained from whole c e l l incubations as already discussed, p r e c i s e - 99 -T 1 1 1 r E F F L U E N T V O L U M E , ml F i g . 17. I n c o r p o r a t i o n of 1 4 C - a r g i n i n e i n t o protamine by an i s o -l a t e d cytoplasmic f r a c t i o n . As described i n " M a t e r i a l s and Methods", a c e l l f r e e cytoplasmic f r a c t i o n was obtained from 40 g of t e s t i s a f t e r homogenization and d i f f e r e n t i a l c e n t r i f u g a t i o n . The i n c u b a t i o n mixture (10 ml) had the f o l l o w i n g composition: ATP 2 mM, 2-mercaptoethanol 6 mM, GTP 0.2 mM, 1 ^ C - a r g i n i n e 0.3 yC/ml, and 8.4 ml of the cytoplasmic f r a c t i o n . A f t e r i n c u b a t i o n , the mixture was e x t r a c t e d w i t h hot s u l f u r i c a c i d , 5 mg c a r r i e r protamine was added, and the e x t r a c t f r a c t i o n a t e d by chromato-graphy on a Bi o - G e l P-10 column. - 100 CYTOPLASMIC SYNTHESIS OF PROTAMINE I s o l a t i o n of D i f f e r e n t C e l l F r a c t i o n s . CELL SUSPENSION IN TMKS HOMOGENIZATION, 5,00 0 RPM, 20 Sec CENTRIFUGATION, 15,000 x g f o r 15 min —CYTOPLASM (POST-MITOCHONDRIAL SUPERNATANT) PELLET 2 x 1,000 x g 10 min\ 60,000 RPM WASH (SUPERNATANT DISCARDED) 2 HOURS •MICROSOMES (PELLET) WASHED NUCLEI HIGH SPEED SUPERNATANT F i g . 18. Procedure f o r the i s o l a t i o n of d i f f e r e n t c e l l f r a c t i o n s (cytoplasm, n u c l e i , and high speed supernatant) from t r o u t t e s t i s . Each i s o l a t e d f r a c t i o n was assayed f o r protamine s y n t h e s i z i n g a c t i v i t y i n a c e l l - f r e e system (Table I I I ) . - 101 -TABLE I I I R e l a t i v e i n c o r p o r a t i o n of 1 "*C-arginine i n t o protamine by d i f f e r e n t i s o l a t e d c e l l f r a c t i o n s (see F i g . 18). C e l l f r a c t i o n CPM i n t o of 1 "*C-arginine i n c o r p o r a t e d protamine per 90; min.. . Nucleus (1,000 x g 10 min 114 sediment Cytoplasm(15,000 x g 15 min 4,000 supernatant High-speed supernatant 236 (60,000 rpm 2 hour) Note: Protamine was i s o l a t e d by chromatography on Bio- G e l P-10 as d e s c r i b e d i n M a t e r i a l s and Methods. - 102 -coincidence i s not seen between r a d i o a c t i v i t y and absorbance at 230 nm (F i g . 19). As already mentioned, a major f a c t o r con-t r i b u t i n g to the e a r l i e r e l u t i o n of newly synthesized protamine was the greater degree of phosphorylation of the newly synthesized protamine. The p o s s i b i l i t y t h a t protamine newly synthesized in vitro was a l s o phosphorylated was examined. A cytoplasmic c e l l f r e e system from t e s t i s was prepared as before w i t h post-mitochon-d r i a l supernatant and incubated w i t h gamma-32P-ATP and 3 H - a r g i n i n e . As shown i n F i g . 20 both l a b e l s can be found i n P I I , the protamine reg i o n of the P-10 column i n d i c a t i n g t h a t t h i s protamine synthes-i z e d in vitro was a l s o phosphorylated and t h a t phosphorylation could account f o r the e a r l i e r e l u t i o n of t h i s protamine on the CM-cellulose system. These data are c o n s i s t e n t w i t h t h a t of Marushige et al. (48,33) who were able to i s o l a t e 3 2 P - l a b e l l e d protamine from the cytoplasm of t e s t i s c e l l s a f t e r a l l o w i n g whole c e l l s to in c o r p o r a t e 3 2P-phosphate. J e r g i l and Dixon (35) have a l s o found a protamine kinase i n t e s t i s c e l l cytoplasm t h a t w i l l phosphorylate protamine using gamma-32P-ATP as substrate and y i e l d s O-.3 2P-phospho-serine as a product when the phosphorylated protamine was p a r t i a l l y hydrolyzed by a c i d and the products analyzed by high voltage e l e c t r o p h o r e s i s . F u r t h e r , Marushige et al. (48) have noted t h a t using a s i m i l a r c e l l - f r e e system as above, but w i t h 3 2P-phosphate as s u b s t r a t e , no i n c o r p o r a t i o n of l a b e l i n t o protamine could be detected. Since the phosphates of protamine appear to be d i r e c t l y d e r i v e d from ATP, t h i s suggests t h a t the cytoplasmic enzyme of J e r g i l and Dixon (35) which a l s o r e q u i r e s ATP to phosphorylate protamine i s l i k e l y to be the enzyme th a t phosphorylates newly synthesizedclprotamine i n the c e l l . I t - 103 -F i g . 19. C h a r a c t e r i z a t i o n by CM-cellulose chromatography of the m a t e r i a l l a b e l l e d w i t h C-arginine by an i s o l a t e d c y t o -plasmic f r a c t i o n from t r o u t t e s t i s . The l a b e l l e d b a s i c pro-t e i n s were e x t r a c t e d i n 0.2 M s u l f u r i c a c i d , f r a c t i o n a t e d by Bi o - G e l P-10 and chromatographed on a CM-cellulose column (1 x 30 cm) as i n F i g . 7. The l a b e l l e d m a t e r i a l was th a t d e s c r i b e d i n Table I I I as protamine synthesized by the cytoplasmic f r a c t i o n . - 104 -F i g . 20. Synthesis and phosp h o r y l a t i o n of protamine by an i s o -l a t e d bytoplasmic f r a c t i o n . As des c r i b e d i n " M a t e r i a l s and Methods", a cytoplasmic f r a c t i o n (15 ml) was prepared from 30 ml packed t e s t i s c e l l s and incubated w i t h c o - f a c t o r s (as i n F i g . ' 17) i n c l u d i n g 100 uC of 3 H - a r g i n i n e and 2 mM ATP c o n t a i n i n g about 3 x 10 6 cpm gamma-32P-ATP. Basic p r o t e i n s were e x t r a c t e d w i t h 0.2 M s u l f u r i c a c i d , c a r r i e r protamine was added, and the ex-t r a c t chromatographed on a Bio-Gel P-10 column as before ( F i g . 17) . - 105 -may be noted t h a t l a r g e amounts of 3 2P and 3H a r g i n i n e were a l s o incorporated i n t o PI of the P-10 column i n B i g . 20. As already mentioned, histones and the other b a s i c cytoplasmic p r o t e i n s are el u t e d i n t h i s r e g i o n from the P-10 column. Dixon et al. (49) have employed a method of i d e n t i f i c a t i o n of 3 2P-phospho-peptides of t r o u t t e s t i s histones a f t e r d i g e s t i o n of 3 2 P - l a b e l l e d histones by t r y p s i n , s e p a r a t i o n of the phospho-peptides by high voltage paper e l e c t r o p h o r e s i s and d e t e c t i o n of the peptides by radioauto-graphy. When a t r y p s i n d i g e s t of the PI m a t e r i a l of the c e l l f r e e system was analyzed by the same method there was a broad d i f f u s e r e g i o n of r a d i o a c t i v i t y not corresponding t o the s e r i e s of phospho-histone peptides obtained by Dixon et al. This i n d i -cates t h a t the 3 2 P - l a b e l incorporated i n t o the PI reg i o n by the in vitro cytoplasmic f r a c t i o n was not i n t o histones but i n t o some other a c i d - e x t r a c t a b l e p r o t e i n s not p r e s e n t l y i d e n t i f i e d . Ingles et al. (11) have suggested from stud i e s w i t h various i n h i b i t o r s t h a t protamine i s probably synthesized on a s t a b l e messenger RNA sin c e they observed no i n h i b i t i o n of protamine syn-t h e s i s i n i n t a c t spermatid c e l l s w i t h actinomycin D at concentra-t i o n s which i n h i b i t e d RNA s y n t h e s i s . The data presented here and based on in vitro s t u d i e s of the synthesis of protamine support t h e i r hypothesis. F i r s t , the microsomal f r a c t i o n i s r e q u i r e d f o r protamine s y n t h e s i s ; the supernatant f r a c t i o n along was r e l a t i v e l y i n a c t i v e (Table I I I ) . Second, the f a c t t h a t an i s o l a t e d c y t o -plasmic f r a c t i o n was able to support p r o t e i n synthesis i n the absence of the nucleus suggests the presence of a s t a b l e endogen-ous messenger RNA i n the cytoplasm. Further, the messenger RNA f o r - 106 -p r o t a m i n e must be p r e s e n t as a c o m p l e t e m o l e c u l e and t h e i n t e g r i t y o f t h e p r o t e i n s y n t h e s i n g complex must be p r e s e r v e d i n t h i s in v i t r o s y s t e m s i n c e t h e p r o t a m i n e s y n t h e s i z e d a p p e a r s t o c o n s i s t o f c o m p l e t e d m o l e c u l e s as d e d u c e d f r o m t h e f a c t t h a t t h e l a b e l l e d m a t e r i a l s c h r o m a t o g r a p h s i n t h e same r e g i o n as c a r r i e r p r o t a m i n e on C M - c e l l u l o s e . Y a r o n et al. ( 2 0 ) , have shown t h a t t h i s method o f c h r o m a t o g r a p h y i s c a p a b l e o f s e p a r a t i n g a m i x t u r e o f o l i g o -l y s i n e s i n t o i n d i v i d u a l components, and i t has b e e n d e m o n s t r a t e d t h a t e v e n p r o t a m i n e was s e p a r a t e d i n t o d i f f e r e n t components by t h i s s y s t e m . T h u s , i f t h e p r o t a m i n e m o l e c u l e s s y n t h e s i z e d in v i t r o by t h i s s y s t e m were i n c o m p l e t e , t h e y w o u l d n o t c h r o m a t o g r a p h i n t h e same r e g i o n as c a r r i e r p r o t a m i n e b u t w o u l d be e l u t e d s u b s t a n t i a l l y e a r l i e r i n t h i s h i g h l y r e s o l v i n g s y s t e m . I I I . C h a r a c t e r i z a t i o n o f T r o u t T e s t i s Ribosomes H a v i n g e s t a b l i s h e d t h a t t h e s i t e o f s y n t h e s i s o f p r o t a m i n e was on c y t o p l a s m i c m i c r o s o m e s , a s e r i e s o f e x p e r i m e n t s was p e r -f o r m e d t o e s t a b l i s h t h e s i z e o f t h e p o l y s o m e upon w h i c h p r o t a m i n e i s s y n t h e s i z e d . R i c h and c o w o r k e r s (50-53) have shown f r o m t h e i r s t u d i e s o f t h e p o l y s o m e s i n v o l v e d i n t h e s y n t h e s i s o f v a r i o u s p r o t e i n s t h a t t h e r e was a r e l a t i o n s h i p b etween t h e s i z e o f t h e p o l y p e p t i d e c h a i n b e i n g s y n t h e s i z e d and t h e s i z e o f t h e p o l y s o m e c o m p l e x i n v o l v e d i n t h e s y n t h e s i s . S i n c e t h e m o l e c u l a r w e i g h t o f p r o t a m i n e i s known ( 1 4 ) , i t was o f i n t e r e s t t o e s t a b l i s h t h e s i z e o f t h e p o l y s o m e s a s s o c i a t e d w i t h t h e s y n t h e s i s o f p r o t a m i n e . Numerous i n v e s t i g a t o r s s t u d y i n g t h e m o l e c u l a r e v e n t s a s s o c i a t e d w i t h t h e d e v e l o p m e n t o f a n i m a l embryos (55-57) have n o t e d t h e c h a n g i n g p o p u l a t i o n and a c t i v i t y o f p o l y s o m e s a t d i f f e r e n t s t a g e s - 107 -of development suggesting t h a t a p a r t i c u l a r complement of pro-t e i n s i s synthesized at each s p e c i f i c stage of d i f f e r e n t i a t i o n . Since the t r o u t t e s t i s o f f e r s a case of d i f f e r e n t i a t i o n i n which at a l a t e stage of development a larg e q u a n t i t y of protamine i s synthesized, the polysomes of the t e s t i s may be analyzed f o r s p e c i f i c changes as s o c i a t e d w i t h the development of the t e s t i s c e l l . a) The Polysomes As s o c i a t e d w i t h the Synthesis of Protamine Since i t has already been demonstrated t h a t 1 4 C - a r g i n i n e -l a b e l l e d nascent protamine appeared on the cytoplasmic microsomes a f t e r a short i n c o r p o r a t i o n p e r i o d of 0.5 min at 20° (see F i g . 15), ribosomes were prepared from the cytoplasm of t e s t i s c e l l s which had been incubated w i t h 1 ^ C - a r g i n i n e f o r 60 sec. The i s o -l a t e d ribosomes were analyzed on a sucrose gradient as seen i n F i g . 21. I t i s evident t h a t the r a d i o a c t i v i t y p r o f i l e i s d i f f e r -ent from the absorbance p r o f i l e measured at 260 nm i n tha t the major peak of r a d i o a c t i v i t y i s found i n the di-ribosome (disome) r e g i o n c o l l e c t e d a t the 12th-13th f r a c t i o n of the sucrose g r a d i e n t , i n c o n t r a s t there i s l i t t l e r a d i o a c t i v i t y i n the monosome r e g i o n , the major absorbance peak, c o l l e c t e d at the 17th f r a c t i o n . The lac k of r a d i o a c t i v i t y i n the monosome reg i o n i s c o n s i s t e n t w i t h the observations of v a r i o u s i n v e s t i g a t o r s (58-60). The usual e x p l a n a t i o n f o r t h i s observation i s tha t the monoribosomes l a c k messenger RNA and are i n a c t i v e i n p r o t e i n s y n t h e s i s . The s e d i -mentation c o e f f i c i e n t of the ribosomes obtained from f r a c t i o n 17 of F i g . 21 determined on the model E a n a l y t i c a l u l t c a c e n t r i f u g e was found to be 77S. M a r t i n and Ames (61) have shown th a t - 108 -F i g . 21. Sedimentation a n a l y s i s of p u r i f i e d ribosomes obtained from t e s t i s c e l l s pulsed f o r 1 min w i t h 1 4 C - a r g i n i n e . The r i b o -somes were t r e a t e d w i t h T r i t o n X-100 and sedimented through dense sucrose before a n a l y s i s as described i n " M a t e r i a l s and Methods". The l i n e a r g r a d i e n t was 10-30% (w/v) of sucrose w i t h c e n t r i f u g a t i o n a t 24,000 rpm f o r 2.5 hrs i n a SW-25 swinging-bucket r o t o r . - 109 -macromolecules sediment i n l i n e a r d e n s i t y gradients at constant r a t e s and the r a t e of sedimentation i s dependent on the s i z e of the macromolecule. Hence, the p o s i t i o n of a macromolecule i n a l i n e a r gradient i s d i r e c t l y p r o p o r t i o n a l to i t s sedimentation c o e f f i c i e n t . F u r t h e r , when the sedimentation c o e f f i c i e n t of one macromolecule i s known and used as a reference p o i n t , the sedimentation c o e f f i c i e n t of another i n the same gradient may be c a l c u l a t e d by i t s r e l a t i v e p o s i t i o n to the reference molecule. Using the monosome peak w i t h a.sedimentation c o e f f i c i e n t of 77S as the reference p o i n t , the sedimentation c o e f f i c i e n t of the r a d i o a c t i v e peak of F i g . 21 may be c a l c u l a t e d to be near 120S. When t o t a l u n f r a c t i o n a t e d t e s t i s ribosomes were analyzed by the model E a n a l y t i c a l u l t r a c e n t r i f u g e , (see F i g . 22) and the s e d i -mentation c o e f f i c i e n t s of the monosome and disome peaks c a l c u l a t e d , the monosome peak was seen t o sediment at 77S and the disome peak at 121S at 20° i n TMK b u f f e r . These values are c o n s i s t e n t w i t h those reported (62) f o r monosomes and disomes i s o l a t e d from a n i -mal c e l l s ; i n a d d i t i o n , t h i s v e r i f i e s t h a t f i s h t e s t i s ribosomes are of the "80S" type c h a r a c t e r i s t i c of animal ribosomes ra t h e r than the "70S" b a c t e r i a l type (63). To c h a r a c t e r i z e f u r t h e r the nascent p r o t e i n s a s s o c i a t e d w i t h t e s t i s polysomes, appropriate f r a c t i o n s from the polysome gradient were pooled (areas A, B, and C as i n d i c a t e d i n F i g . 21) and a c i d e x t r a c t e d p r i o r to chromatography on the Bio-Gel P-10 column. The t o t a l amount of r a d i o a c t i v i t y i n P I , the f i r s t peak and P I I , the protamine peak of the P-10 column was determined. Table IV shows th a t the major p o r t i o n of the l a b e l i n f r a c t i o n B (the d i -some area) chromatographs i n the P I I reg i o n of the P-10 column Time a f t e r reaching speed (min) 12 14 16 18 20 Speed:- 20,000 rpm Bar angle :- 30 degree Temperature:- 23.2° Solvent:- TMK, pH 7.6 D i r e c t i o n of sedimentation:- > F i g . 22. Determination of the sedimentation c o e f f i c i e n t (S) of t e s t i s ribosomes. As des c r i b e d i n " M a t e r i a l s and Methods" a sample of p u r i f i e d t e s t i s ribosomes (8 mg/ml) i n TMK b u f f e r was analyzed on the Model E a n a l y t i c a l u l t r a c e n t r i f u g e monitored by the S c h l i e r e n o p t i c a l system. The c o n d i t i o n s of c e n t r i f u g a t i o n are as i n d i c a t e d . Sedimen-t a t i o n i s from l e f t t o r i g h t . - I l l -TABLE IV C h a r a c t e r i z a t i o n of the nascent peptides a s s o c i a t e d w i t h d i f f e r e n t s i z e s of polysomes. Region of Polysome g r a d i e n t ( F i g . 20) CPM 1 ^ C - a r g i n i n e i n P j CPM l l*C-arginine i n P I . I . A 190 352 B 188 762 C 122 286 Note: Chromatography of a c i d e x t r a c t s of pooled f r a c t i o n s from the sucrose d e n s i t y - g r a d i e n t a n a l y s i s of F i g . 21 on a Bi o - G e l P-10 column. The t a b l e shows the t o t a l r a d i o a c t i v i t y present i n the P^, and P^^ (protamine) areas of the chroma-togram. - 112 -and i s t h e r e f o r e protamine. The smaller amounts of P I I r a d i o -a c t i v i t y i n f r a c t i o n s A and C, the monosome and l a r g e r polysome areas r e s p e c t i v e l y , probably represent overlap of the disome peak w i t h areas A and C. The r a d i o a c t i v i t y a s s o c i a t e d w i t h PI of f r a c t i o n s A, B, and C could not be i d e n t i f i e d but appears to be present i n approximately equal amount throughout the gradient. These data i n d i c a t e that a f t e r a short pulse of 1 ^ C - a r g i n i n e , a peak of r a d i o a c t i v i t y i s r a p i d l y incorporated i n t o the disome re g i o n of the t e s t i s polysomes and t h a t t h i s disome re g i o n con-t a i n s most of the nascent protamine. Hence the disomes are i m p l i -cated as the polysomes i n v o l v e d i n the synthesis of protamine. Protamine i s a h i g h l y b a s i c p r o t e i n known to complex r e a d i l y w i t h n u c l e i c a c i d s ; thus, there i s a p o s s i b i l i t y t h a t the peak of r a d i o a c t i v i t y a s s o c i a t e d w i t h disomes might be due to aggregation of monosomes i n t o disomes as a r e s u l t of some i n t e r a c t i o n of the monosomes w i t h f r e e r a d i o a c t i v e protamine. That t h i s i s not l i k e l y to be the case was demonstrated i n an experiment i n which a small amount of i s o l a t e d 1 ^ C - a r g i n i n e l a b e l l e d protamine was mixed w i t h a p r e p a r a t i o n of ribosomes and t h i s mixture f r a c t i o n -ated by sucrose d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n . As may be seen (Fi g . 23), the r a d i o a c t i v i t y p r o f i l e corresponds almost e x a c t l y to the o p t i c a l d e n s i t y p r o f i l e and no r a d i o a c t i v e peak i n the d i -some re g i o n i s observed. Some r a p i d l y sedimenting m a t e r i a l i s present at the bottom of the g r a d i e n t ; t h i s probably represents aggregates of ribosomes formed i n the presence of the added pro-tamine s i n c e a c o n t r o l experiment without protamine showed no m a t e r i a l of t h i s k i n d . o 3 Oi o 3 3 OJ cn ^ CD co s ; cn i £ OJ O P o cn (D • cn P-iQ P-I cr c o (D Ct P O rt O P P (D <: p-o cn i—1 OJ ^ cn Tj tr P (D fl> Hi^d O 0) PJ tr P-o pj cn pj 3 P- rt ^ t r N fD fD rt n o p -fD O 3 tr OJ hi p j P- O *< Hi fD • f t P i-3 p- tr Kl fD £ • pj r t P-O 0 pj p. truq fD • M M to fD OJ CL . 0 a P. o P- 3 Hi I p- cn fD TO a fD O H P-P- Hi t r P o cn O 3 fD cn o tr p-Oi P-Hi 3 P. LQ O 3 O Hi rt CD ~ cn -F r t n p- I cn V P 0) rt P-O K OJ cn o rt OJ 3 p-fD OJ rt i-i p-t r o cn O 3 fD P-a o c tr OJ rt fD rt DJ fD cn * rt p- p-rt O H cn rt cn OJ PJ s: P fD o cn P; •ptr o C fD o 0 o O l cn P P *T3 O P O fD P 3 '0 W 1 O fD 3 fD P- rt cn cn PJ « Hi<X> O 3 O P, P- p-hi PJ P) 3 > CL. r t fD <Ti P- fD cn O fD Ch OJ oo 3 3 r t rt p- P-3 — p •C fD • h-1 O o £ - 0 1 Hi OJ t r P' o Ul C dp - 3 O 1 s: o < ABSORBANCE AT 260 NM c » « - « « « - o COUNTS PER MINUTE PER FRACTION - ETT -- 114 -To determine whether the disomes r e s u l t from degradation of l a r g e r polysomes i n t o disomes during the i s o l a t i o n procedure, ribosomes were prepared from two i d e n t i c a l t e s t i s c e l l suspensions a f t e r they have been allowed to incorporate 1 4 C - a r g i n i n e at 20° f o r 2 min and 40 min r e s p e c t i v e l y ; i n c o r p o r a t i o n was terminated by the a d d i t i o n of 0.4 mM cycloheximide. The polysome p r o f i l e of each i n c u b a t i o n was examined by sucrose gradient c e n t r i f u g a -t i o n on a SW-27 r o t o r ( F i g . 24 A and B). I t may be seen t h a t a f t e r a 2 min i n c o r p o r a t i o n p e r i o d , the major peak of r a d i o a c t i v i t y i s a s s o c i a t e d w i t h the disomes sedimenting at 120S w h i l e the l a r g e r polysomes and the monosomes (77S) co n t a i n l i t t l e r a d i o -a c t i v i t y . A f t e r a 40 min i n c o r p o r a t i o n p e r i o d , the amount of r a d i o a c t i v i t y a s s o c i a t e d w i t h the disomes and monosomes remained e s s e n t i a l l y the same as that observed i n the 2 min i n c o r p o r a t i o n sample but now there i s more r a d i o a c t i v i t y a s s o c i a t e d w i t h the l a r g e r polysomes. Since the r a d i o a c t i v i t y appears f i r s t on the disomes and only a f t e r a longer i n c u b a t i o n p e r i o d does more r a d i o a c t i v i t y appear i n the l a r g e r polysomes, the disomes could not be the breakdown product of l a r g e r polysomes. Further, since nuclease u s u a l l y degrades polysomes i n t o monosomes (58, 64, 65) and since there i s no a d d i t i o n a l increase of r a d i o a c t i v i t y i n the monosome region w i t h the longer i n c u b a t i o n p e r i o d , t h i s suggests t h a t the t e s t i s polysomes are not degraded by nuclease during i n c u b a t i o n or during the i s o l a t i o n procedure. In the treatment of the post-mitochondrial cytoplasmic f r a c t i o n w i t h T r i t o n X-100 during the pre p a r a t i o n of ribosomes, there was the p o s s i b i l i t y t h a t the small number of mitochondria s t i l l present a f t e r d i f f e r e n t i a l c e n t r i f u g a t i o n would be lysed - 115 -1 1 r A UOS 77S FRACTION NO. F i g . 24. Time course of 1 ^ C - a r g i n i n e i n c o r p o r a t i o n by nascent peptides a s s o c i a t e d w i t h t e s t i s ribosomes. Two i d e n t i c a l c e l l suspensions prepared w i t h 159 t e s t i s each to i n c o r p o r a t e *kC-a r g i n i n e (15 yC) f o r 2 min (A) and 40 min (B) r e s p e c t i v e l y , i n c o r p o r a t i o n was terminated by the a d d i t i o n of 0.4 mM cyclohex mide. Ribosomes were p u r i f i e d and analyzed by sucrose g r a d i e n t c e n t r i f u g a t i o n (10-34% w/v) on a SW-27 swinging-bucket r o t o r as p r e v i o u s l y d e s c r i b e d i n " M a t e r i a l s and Methods".-- 116 -(66) and the ribosomes of the mitochondria (67,68) would then be r e l e a s e d i n t o the cytoplasm. As a r e s u l t , i t i s p o s s i b l e t h a t the protamine peak of r a d i o a c t i v i t y appearing i n the disome re g i o n of t e s t i s polysomes ( F i g . 21) was a c t u a l l y due to the presence of the m i t o c h o n d r i a l ribosomes. To check the p o s s i b i l -i t y of a m i t o c h o n d r i a l s i t e of synthesis f o r protamine, the ribosomes of the post-nuclear supernatant (PNS) (cytoplasm plus mitochondria) and those of the post-mitochondrial supernatant (PMS) were analyzed a f t e r t e s t i s c e l l s were incubated w i t h 1hC-a r g i n i n e f o r 2 min at 20°. The r e s u l t s are shown i n F i g . 25 A, B and C. The m i t o c h o n d r i a l ribosomes of F i g . 25 C were prepared from the 15,000 x g - 15 min sediment of F i g . 25 B. I t i s obser-ved t h a t the absorbance p r o f i l e s of the ribosomes prepared from, the PNS and PMS are e s s e n t i a l l y the same; the r a d i o a c t i v i t y p r o f i l e s are a l s o s i m i l a r w i t h the major peak of r a d i o a c t i v i t y being i n the disome r e g i o n . While the t o t a l amount of r a d i o -a c t i v i t y i n c orporated i n t o the disome region i s s i m i l a r f o r the PNS and PMS ribosomes, i t appears that the s p e c i f i c a c t i v i t y i . e . cpm 1 4 C - a r g i n i n e per absorbance at 260 nm at the disome re g i o n i s higher i n the PMS ribosomes than i n the PNS ribosomes. This suggests t h a t the a d d i t i o n a l m i t o c h o n d r i a l ribosomes d e l i b e r a t e l y r e l e a s e d by detergent treatment of the PNS are not a s s o c i a t e d w i t h the synthesis of protamine; otherwise, the s p e c i f i c a c t i v i t y as w e l l as the t o t a l i n c o r p o r a t i o n i n t o the disome re g i o n of the PNS ribosomes would be higher than the PMS r i b o -somes. Fu r t h e r , i t i s seen t h a t the ribosomes prepared from a crude m i t o c h o n d r i a l f r a c t i o n contains l i t t l e r a d i o a c t i v i t y w i t h low s p e c i f i c a c t i v i t y i n the disome r e g i o n (Fig. 25 C). - 117 -F i g . 25. Sucrose d e n s i t y g r a d i e n t a n a l y s i s of ribosomes ob-t a i n e d from d i f f e r e n t c e l l f r a c t i o n s . As described i n " M a t e r i a l s and Methods", a post-nuclear supernatant, a p o s t - m i t o c h o n d r i a l supernatant and a m i t o c h o n d r i a l f r a c t i o n were prepared from i d e n t i c a l c e l l suspensions of t r o u t t e s t i s (15 g) p r e v i o u s l y allowed to i n c o r p o r a t e 1 ^ C - a r g i n i n e f o r 2 min. Ribosomes were p u r i f i e d from each of the i s o l a t e d f r a c t i o n s and were analyzed by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n on a SW-27 r o t o r as p r e v i o u s l y d e s c r i b e d . (A) p r o f i l e of ribosomes from the p o s t - n u c l e a r supernatant, (B) from the p o s t - m i t o c h o n d r i a l super-natant, (C) from the m i t o c h o n d r i a l f r a c t i o n . - 118 -V a r i o u s i n v e s t i g a t o r s (69-71) have n o t e d t h a t d i m e r i c r i b o s o m e s s e d i m e n t i n g a t 110-120S a r e a r t e f a c t s f o r m e d as t h e r e s u l t o f an a g g r e g a t i o n o f r i b o s o m e s a t h i g h magnesium i o n c o n c e n t r a t i o n s . S i n c e t h e s e d i m e r i c r i b o s o m e s u s u a l l y d i s s o c i a t e i n t o monosomes a t low magnesium i o n c o n c e n t r a t i o n s e.g. 1 mM o r l e s s , an e x p e r i m e n t was p e r f o r m e d t o s e e i f t h e t e s t i s d i -somes were t h e r e s u l t o f magnesium d e p e n d e n t a g g r e g a t i o n . Two p a r a l l e l e x t r a c t i o n s were p e r f o r m e d on t h e t e s t e s o f n a t u r a l l y m a t u r i n g t r o u t o b t a i n e d a t t h e p r o t a m i n e s t a g e o f s p e r m a t o g e n e s i s ; one e x t r a c t i o n was w i t h t h e u s u a l TMK b u f f e r c o n t a i n i n g 5 mM magnesium a c e t a t e and t h e o t h e r w i t h t h e same b u f f e r b u t w i t h 1 mM magnesium a c e t a t e . The p o l y s o m e p r o f i l e s were c o n t i n u o u s l y a n a l y z e d w i t h t h e I s c o u l t r a v i o l e t a n a l y z e r as s e e n i n F i g . 26. I t may be s e e n h e r e t h a t a t t h e p r o t a m i n e s t a g e o f s p e r m a t o -g e n e s i s , t h e r e i s a l a r g e peak o f d i s o m e s s e d i m e n t i n g a t 120S (see F i g . 26 A) i n t h e t e s t i s p o l y s o m e s , and t h i s peak r e m a i n s e s s e n -t i a l l y i n t a c t a t 1 mM magnesium a c e t a t e c o n c e n t r a t i o n , F i g . 26 B. T h i s s u g g e s t s t h a t a t t h e p r o t a m i n e s t a g e o f s p e r m a t o g e n e s i s , a g r e a t number o f d i s o m e s a r e p r e s e n t i n t h e t e s t i s and t h e m a j o r i t y o f t h e s e d i s o m e s a r e n o t t h e r e s u l t o f magnesium d e p e n d e n t a g g r e -g a t i o n . I t i s s e e n t h a t t h e t e s t i s p o l y s o m e s p r e p a r e d i n 1 mM magnesium a c e t a t e c o n t a i n a s u b s t a n t i a l p r o p o r t i o n o f monosomes and a d e c r e a s e d p r o p o r t i o n o f l a r g e r p o l y s o m e s when compared w i t h t h e p o l y s o m e s p r e p a r e d i n 5 mM magnesium a c e t a t e (compare F i g . 26 B and A ) . I t i s p o s s i b l e t h a t some o f t h e l a r g e r p o l y -somes have d i s s o c i a t e d i n t o monosomes a t t h e l o w e r magnesium c o n c e n t r a t i o n ; a n o t h e r p o s s i b i l i t y i s t h a t a t t h e l o w e r magnesium c o n c e n t r a t i o n , l a t e n t n u c l e a s e a c t i v i t y i s a c t i v a t e d and some - 119 -1 r 120 S EFFLUENT VOLUME - ml F i g . 26. E f f e c t of low Mg i o n c o n c e n t r a t i o n on t e s t i s disomes. As d e s c r i b e d i n " M a t e r i a l s and Methods", ribosomes were i s o l a t e d from f r o z e n t e s t i s i n TMKS b u f f e r (A) c o n t a i n i n g the normal 5 mM magnesium ac e t a t e and (B) 1 mM magnesium acetate. The r i b o -somes were analyzed i n sucrose g r a d i e n t s c o n t a i n i n g the approp-r i a t e magnesium c o n c e n t r a t i o n on a SW-27 r o t o r . The p r o f i l e s of the t e s t i s polysomes were c o n t i n u o u s l y monitored by an Isco UV anal y z e r as p r e v i o u s l y d e s c r i b e d . - 120 -l a r g e r polysomes are degraded to monosomes. I f t h i s l a t t e r p o s s i b i l i t y i s indeed the case, i t would appear th a t the disomes are not a f f e c t e d by m i l d nuclease a c t i v i t y . The e f f e c t of a m i l d treatment of t e s t i s ribosomes w i t h (pancreatic RNase (Worthington) 0.03 ug/ml) w a s examined. The ribosomes were p u r i f i e d from t e s t i s c e l l s p r e v i o u s l y incubated w i t h 1 "*C-arginine and hence contained l a b e l l e d nascent p r o t e i n s a s s o c i a t e d w i t h them. F i g . 27 A shows that the t e s t i s polysomes co n t a i n two peaks of absorbance at 260 nm, P k l and P k l l c o r r e s -ponding to the monosomes and disomes r e s p e c t i v e l y . The major peak of r a d i o a c t i v i t y i s a s s o c i a t e d w i t h P k l l (the disomes) and r a d i o a c t i v i t y i s a l s o found i n the l a r g e r polysomes. With the RNase treatment, ( F i g . 27 C), there i s a decrease i n the amount of absorbance at 260 nm i n the l a r g e r polysome reg i o n and an increase i n monosomes ( P k l ) . R a d i o a c t i v i t y associated w i t h the l a r g e r polysomes i s a l s o removed by the RNase treatment. These observations are i n agreement w i t h those of various i n v e s t i g a t o r s (59, 65, 73) who have studied the e f f e c t of m i l d RNase t r e a t -ment on b a c t e r i a l and animal polysomes. As may be seen from F i g . 27 C, the disomes are r e l a t i v e l y i n s e n s i t i v e to the a c t i o n of the m i l d RNase treatment. This suggests that the disomes are d i f f e r e n t from the l a r g e r polysomes i n some way and are r e s i s t a n t to RNase a c t i o n . F i g . 27 B shows th a t the degradation of the l a r g e r polysomes by RNase may be prevented to some extent by the a d d i t i o n of bentonite to a f i n a l c o n c e n t r a t i o n of 2 mg/ml. b) Developmental Changes of the T e s t i s Polysome Population Table V shows the development of the t r o u t t e s t i s c o l l e c t e d - 121 -0 10 20 30 FRACTION F i g . 27. E f f e c t of RNase on t e s t i s polysomes. As de s c r i b e d i n " M a t e r i a l s and Methods", ribosomes were i s o l a t e d from a c e l l suspension prepared from 25 g of t r o u t t e s t i s which had p r e v i o u s l y been incubated w i t h 1 ^ C - a r g i n i n e f o r 10 min. The ribosomes were t r e a t e d w i t h RNase (0.03 ug/ml), be n t o n i t e (2 mg/ml) p l u s RNase, or l e f t untreated as i n d i c a t e d and were analyzed on a SW-25 swinging-bucket r o t o r as before. TABLE V Stages of t e s t i s development i n n a t u r a l l y maturing rainbow t r o u t (Salmo gairdnerii) MONTH STAGE Average wt. per t e s t i s (20) i n grams Mg Ribosomal RNA/g t e s t i s CPM X I*C-Arg** In c o r p o r a t e d i n t o protamine /g t e s t i s / 3 0 min AUGUST PRE-PROTAMINE . 1.6* 1.95 -SEPTEMBER EARLY-PROTAMINE 7.1 1.54 2,422 OCTOBER PROTAMINE 10.0 0.71 11.302 •NOVEMBER PROTAMINE 9.3 0.29 9,380 DECEMBER LATE-PROTAMINE 4.0 0.03 604 LIVER 5.6 Average wt. of 6 t e s t e s . Protamine was i s o l a t e d on a B i o - G e l P-10 column - 123 -from n a t u r a l l y maturing 1 1/2 to 2 year o l d t r o u t during the months of August to December; these f i s h normally spawn during the months of January and February. From the t a b l e , i t may be seen t h a t during the pre-protamine stage of development there i s a major enlargement i n the t e s t i s as demonstrated by the increase i n t e s t i s weight from 1.6 gm to 7.1 gm during the months of August to September. At t h i s time, the spermatogonial-stem c e l l s i n the t e s t i s are presumably undergoing repeated mitoses to form a l a r g e number of c e l l s which then undergo two m e i o t i c d i v i s i o n s and subsequently develop i n t o sperm c e l l s . As already demonstrated i n F i g . 5, during the time of protamine s y n t h e s i s , there i s l i t t l e h i s t o n e synthesis and t h i s t a b l e shows from the i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o protamine t h a t protamine i s most r a p i d l y synthesized, during the months of October and November when there i s no f u r t h e r increase i n t e s t i s weight. By December, however, the late-protamine stage, there i s a marked decrease i n t e s t i s weight w i t h a s i m i l a r decrease i n protamine s y n t h e s i s . At t h i s December stage, the replacement of histones by protamine i s e s s e n t i a l l y complete and the decreased weight of the t e s t i s i s probably the r e s u l t of some r e l e a s e of mature spermatozoa. When the t o t a l amount of ribosomes present i n the t e s t i s was determined as a f u n c t i o n of time, i t i s observed t h a t from Septem-ber on, a f t e r protamine synthesis had begun, there i s a progressive decrease i n t o t a l ribosomes per gram of t e s t i s u n t i l December when there i s an even sharper decrease i n ribosomal content. Since i n the maturation of the sperm c e l l there i s a progressive decrease i n the amount of cytoplasm as detected by c y t o l o g i c a l methods, - 124 -the decrease i n the amount of ribosomes may be the r e s u l t of the general decrease i n the cytoplasm. From the t a b l e i t may a l s o be observed t h a t the t o t a l amount of ribosomes present at a p a r t i c u l a r stage of development does not r e f l e c t the r a t e of protamine sy n t h e s i s since i n the early-protamine stage i n Septem-ber where there i s l i t t l e protamine s y n t h e s i s , there are more ribosomes per gram t e s t i s than during the protamine stage i n October or November when protamine i s r a p i d l y synthesized. In December, however, protamine synthesis i s g r e a t l y decreased and t h i s corresponds to the sharp decrease i n ribosomal content. In view of the f a c t t h a t protamine synthesis increased during a time when t o t a l ribosomal content decreased, i t was of i n t e r e s t then to see i f there were any changes i n the ribosome po p u l a t i o n during t e s t i s development, and i n p a r t i c u l a r , i f these changes r e f l e c t e d the changing r a t e s of protamine s y n t h e s i s . F i g . 28 A to D shows the polysome p r o f i l e s of t e s t i s r i b o -somes obtained from the months of September to December a l l pre-pared under i d e n t i c a l c o n d i t i o n s . I t i s evident t h a t the t e s t i s polysome p r o f i l e changes from month to month and each p r o f i l e i s d i f f e r e n t from t h a t observed i n t r o u t l i v e r F i g . 28 E. The t r o u t l i v e r polysome p r o f i l e s obtained during d i f f e r e n t months were e s s e n t i a l l y the same. Of p a r t i c u l a r i n t e r e s t and s i g n i f i c a n c e is. the presence of l a r g e peaks of disomes sedimenting at 120S during the months of October and November at the time when pro-tamine i s synthesized most r a p i d l y as i n d i c a t e d i n Table V. Since we have already e s t a b l i s h e d t h a t the disomes are associated w i t h protamine s y n t h e s i s , the increase i n the number of disomes - 125 -TROUT TESTIS POLYSOMES 1 0 " S E P T E M B E R EFFLUENT VOLUME -ml F i g . 28. P r o f i l e s o f t r o u t t e s t i s polysomes a t d i f f e r e n t s t a g e s of development. Ribosomes were i s o l a t e d from t e s t i s and l i v e r o b t a i n e d from n a t u r a l l y maturing t r o u t a t the middle o f each month and were a n a l y z e d by suc r o s e d e n s i t y g r a d i e n t c e n t r i f u g a -t i o n on a SW-2 7 swinging-bucket r o t o r . The polysome p r o f i l e s were monitored on the Isco u l t r a v i o l e t a n a l y z e r as p r e v i o u s l y d e s c r i b e d . - 126 -c o i n c i d i n g w i t h the increase i n protamine synthesis i m p l i e s t h a t the c o n t r o l of the r a t e of protamine synthesis i s a f u n c t i o n of the number of polysomes, the disomes, s y n t h e s i z i n g protamine. Since there appear to be s p e c i f i c changes i n the polysome popu-l a t i o n during t e s t i s development, the decrease i n ribosomes (as seen i n Table V) i s probably not s t r i c t l y the r e s u l t of the gen-e r a l decrease i n the c e l l cytoplasm but may be the r e s u l t of s p e c i f i c degradation of c e r t a i n ribosomes or polysomes not r e -q u i r e d f o r f u r t h e r p r o t e i n synthesis a f t e r a p a r t i c u l a r stage of t e s t i s maturation. The p r o t e i n s y n t h e s i z i n g a b i l i t y of t e s t i s ribosomes during t e s t i s maturation was examined at each stage of development by the d i s t r i b u t i o n of incorporated 1 "*C-arginine and 1 ^ C - l y s i n e i n the polysome gra d i e n t . I t i s seen i n F i g s . 29 to 32 t h a t the d i s t r i b u t i o n of l a b e l of 1 "*C-arginine and 1 ^'C-lysine are d i f f e r -ent. In a d d i t i o n , the d i s t r i b u t i o n p a t t e r n p a r t i c u l a r l y of lhC-a r g i n i n e changes as development proceeds. This i s c o n s i s t e n t w i t h the assumption t h a t d i f f e r e n t p r o t e i n s are synthesizeddby the polysomes at d i f f e r e n t stages of development and t h a t d i f f e r -ent polysomes synthesize d i f f e r e n t p r o t e i n s . I t may be observed t h a t throughout the t e s t i s development from September to December, the disomes f r a c t i o n sedimenting at 120S i s the only c l a s s of polysomes which contains c o n s i s t e n t l y a peak of 1 4 C - a r g i n i n e a s s o c i a t e d w i t h i t but which incorporates very l i t t l e "-^C-lysine at any stage. This observation i s c o n s i s t e n t w i t h the disome synthesis of protamine s i n c e the protamine molecule contains over 2/3 of i t s amino a c i d residues as a r g i n i n e and no l y s i n e r e s i d u e s . - 127 -F i g s . 29-32. Developmental changes of t e s t i s polysomes. As d e s c r i b e d i n " M a t e r i a l s and Methods", c e l l suspensions were prepared from t e s t i s of n a t u r a l l y maturing t r o u t at the middle of each month. The c e l l suspensions were incubated w i t h 1 ^C-arginine and X I * C - l y s i n e f o r 30 min and ribosomes were p u r i f i e d from these c e l l suspensions as before. The polysome p r o f i l e and the r a d i o a c t i v i t y a s s o c i a t e d w i t h the polysomes were analyzed a f t e r sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n on a SW-27 r o t o r . F i g . (29) polysomes from t e s t e s obtained i n September, (30) October, (31) November, (32) December. F i g s . 29-32 (A) polysome p r o f i l e monitored by Isco u l t r a v i o l e t a n a l y z e r (B) polysomes from c e l l s incubated w i t h 1 ^ C - a r g i n i n e , and (C) w i t h C - l y s i n e . - 128 -SEPTEMBER TESTIS POLYSOMES FRACTION NO. F i g . 29. - 129 -OCTOBER TESTIS POLYSOMES A I20S I (O.D.I 3) Q FRACTION NO. F i g . 30. - 130 -NOVEMBER TESTIS POLYSOMES A 120S BOTTOM TOP FRACTION NO. F i g . 31. - 131 _ DECEMBER TESTIS POLYSOMES FRACTION NO. F i g . 32. - 1 3 2 -This l a c k of l y s i n e l a b e l i n the disomes suggests that the t e s t i s disomes may be e x c l u s i v e l y engaged i n the synthesis of protamine. The i n c o r p o r a t i o n of l a b e l i n t o the l a r g e r polysome area a l s o changes w i t h d i f f e r e n t stages of development. I t i s seen tha t i n September there i s l i t t l e i n c o r p o r a t i o n i n t o the l a r g e r polysomes but by November, there are lar g e peaks of l a b e l p a r t i -c u l a r l y of 1 ^ C - a r g i n i n e found i n th a t r e g i o n . At the present time, the l a b e l incorporated i n t o the l a r g e r polysome region has not been c h a r a c t e r i z e d but i t i s not l i k e l y to be associated w i t h nascent histones since there i s only n e g l i g i b l e histone s y n t h e s i s at the protamine stage of development. I t i s p o s s i b l e t h a t the l a b e l i s as s o c i a t e d at t h i s l a t e stage of spermatogenesis w i t h the syn t h e s i s of p r o t e i n s necessary f o r the transformation of spermatid c e l l s i n t o sperm c e l l s . - 133 -DISCUSSION This study of the b i o s y n t h e s i s of protamine has progressed through three stages of i n v e s t i g a t i o n : a) i s o l a t i o n and charac-t e r i z a t i o n of newly synthesized protamine, b) e l u c i d a t i o n of the i n t r a c e l l u l a r s i t e of protamine s y n t h e s i s , and c) i d e n t i f i c a t i o n of the c l a s s of polysomes a s s o c i a t e d w i t h protamine s y n t h e s i s . In a d d i t i o n , c e r t a i n aspects of the phosphorylation of protamine together w i t h developmental changes i n t r o u t t e s t i s have been examined. In any study of the b i o s y n t h e s i s of a s p e c i f i c p r o t e i n , a p o s i t i v e i d e n t i f i c a t i o n of the p r o t e i n i s r e q u i r e d ; f u r t h e r , i t i s advantageous to work w i t h a d i f f e r e n t i a t e d system engaged e x c l u s i v e l y i n the synthesis of t h a t p r o t e i n . In t h i s r e s p e c t , the t r o u t t e s t i s o f f e r s s e v e r a l advantages f o r the study of the b i o s y n t h e s i s of protamine. F i r s t , because of the unusual chemical nature of protamine, i t can be r e a d i l y separated from other t e s t i s b a s i c p r o t e i n s . From F i g s . 5 and 7, i t i s seen t h a t by successive chromatography on Bio-Gel P-10 and CM-cellulose, protamine can be i s o l a t e d completely f r e e from contamination by other b a s i c p r o t e i n s or by f r e e l a b e l l e d a r g i n i n e . I t i s a l s o apparent t h a t protamine i s separated i n t o at l e a s t 3 major components by chromatography on CM-cellulose ( F i g . 8) and newly synthesized protamine modified by phosphorylation (7) i s a l s o r e s o l v e d from c a r r i e r protamine ( F i g . 10 A and B). Hence, t h i s i s a h i g h l y r e s o l v i n g method which allows f o r the confident iden-t i f i c a t i o n of protamine. Second, as can be seen i n F i g . 5, during the time of protamine synthesis i n t r o u t t e s t i s , protamine - 134 -i s the major a c i d e x t r a c t a b l e p r o t e i n being synthesized as judged by the i n c o r p o r a t i o n of 1 1*C-arginine. Very l i t t l e l a b e l i s found i n histones or other b a s i c p r o t e i n s (Fig. 5 C) . To take advantage of t h i s o b s e r v a t i o n , a more s p e c i f i c e x t r a c t i o n (0.5 M HC1 plus 5% TCA) f o r protamine has been developed which when coupled w i t h a p r e c i p i t a t i o n step, allowed r a p i d deter-mination of the i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o protamine-as a measure of protamine s y n t h e s i s . Having e s t a b l i s h e d a s u i t a b l e method f o r unequivocally i d e n t i f y i n g newly synthesized protamine, i t was then p o s s i b l e to examine the i n t r a c e l l u l a r s i t e of protamine s y n t h e s i s . Several l i n e s of evidence c o n t r i b u t e to the c o n c l u s i o n t h a t the synthesis of protamine takes place i n the cytoplasm of t r o u t t e s t i s c e l l s . F i r s t , from the i n h i b i t i o n s t u d i e s of Ingles et al. (11) a microsomal mode of protamine synthesis was i n d i c a t e d , and an examination of the t e s t i s microsomes ( F i g . 14) showed th a t l a b e l -l e d nascent protamine i s almost e n t i r e l y a s s o c i a t e d w i t h cyto-plasmic microsomes; as already mentioned, the l a b e l a a s s o c i a t e d w i t h the nuclear microsomes i s l i k e l y to be the r e s u l t of cyto-plasmic contamination. Second, a more d e t a i l e d a n a l y s i s of the k i n e t i c s of l a b e l l i n g by the pulse-chase technique confirmed a precursor-product r e l a t i o n s h i p between the microsomes and the nucleus ( F i g . 15) i n tha t the p r o p o r t i o n of 1 1 * C - a r g i n i n e - l a b e l l e d protamine was i n i t i a l l y higher on the microsomes and when chased w i t h u n l a b e l l e d a r g i n i n e , the l a b e l l e d protamine was then r a p i d l y transported i n t o the nucleus. T h i r d , as summarized i n F i g . 16, protamine l a b e l l e d i n whole c e l l s by a short pulse of 1''C-arginine i s l o c a t e d mainly i n the cytoplasm, w h i l e a f t e r a longer p u l s e , - 135 -the major p o r t i o n of the newly synthesized protamine has moved to i t s f i n a l l o c a t i o n i n the nucleus. The f a c t t h a t h i g h l y l a b e l l e d protamine appears f i r s t i n the cytoplasm a f t e r a short pulse of 1 ^ C - a r g i n i n e p o i n t s s t r o n g l y to cytoplasm as the s i t e of s y n t h e s i s . The f o u r t h l i n e of evidence i s independent of the o t h e r s , being the r e s u l t of in vitro i n c o r p o r a t i o n s t u d i e s w i t h the i s o l a t e d c e l l f r a c t i o n s (Table I I I ) . Only the cyto-plasmic f r a c t i o n composed of microsomes plus supernatant can i n c o r p o r a t e a p p r e c i a b l e amounts of 1 ^ C - a r g i n i n e i n t o protamine. Both i s o l a t e d n u c l e i and supernatant alone are e s s e n t i a l l y i n a c t i v e . Thus the microsomal f r a c t i o n i s c l e a r l y i m p l i c a t e d as the s i t e of s y n t h e s i s i n the cytoplasm. This cytoplasmic s i t e of s y n t h e s i s of a b a s i c nuclear pro-t e i n i s i n agreement w i t h the conclusions of Bloch and Brack (107) who have shown by autoradiography of grasshopper (Chortophaga viridifasaiata) spermatid c e l l s t h a t incorporated^: 3 H - a r g i n i n e i s i n i t i a l l y l o c a t e d i n the cytoplasm and l a t e r moves to the nucleus. The s t u d i e s of Robbins and Borun (45) and a l s o of G a l l w i t z and M u e l l e r (203) have a l s o shown the cytoplasmic syn-t h e s i s of histones i n synchronized Hela c e l l s . However, as already mentioned, there i s a l s o evidence p o i n t i n g to the nuclear s y n t h e s i s of histones (43) and Reid and Cole (44) have demonstra-ted the s y n t h e s i s of a l y s i n e - r i c h histone i n i s o l a t e d c a l f thymus n u c l e i . The b i o l o g i c a l s i g n i f i c a n c e of d i f f e r e n t s i t e s of s y n t h e s i s f o r a chromosomal p r o t e i n , or indeed, f o r any p r o t e i n i s not c l e a r a t the moment. However, a p o i n t of p o s s i b l e s i g n i -f i c a n c e i s t h a t ribosomes might not e a s i l y be accommodated i n the - 136 -e x t r e m e l y d e n s e and compact n u c l e i c h a r a c t e r i s t i c o f s p e r m a t i d c e l l s . The p o s i t i o n o f t h e peak o f 1 ^ C - a r g i n i n e - l a b e l l e d p r o t a m i n e a s s o c i a t e d w i t h c y t o p l a s m i c m i c r o s o m e s f r a c t i o n a t e d on a s u c r o s e d e n s i t y - g r a d i e n t ( F i g . 14 A) f i r s t i n d i c a t e d t h a t p r o t a m i n e was p r o b a b l y s y n t h e s i z e d on s m a l l p o l y s o m e s . S u b s e q u e n t e x a m i n a t i o n o f p u r i f i e d d e t e r g e n t - t r e a t e d p o l y s o m e s showed t h a t t h e m a j o r peak o f n ewly s y n t h e s i z e d p r o t a m i n e was f o u n d a s s o c i a t e d w i t h a c l a s s o f p o l y s o m e s , t h e d i - r i b o s o m e s (disomes) s e d i m e n t i n g a t 120S ( F i g . 21, T a b l e I V ) . I n v a r i o u s i n v e s t i g a t i o n s i t has b e e n n o t e d t h a t t h e a p p e a r a n c e o f d i m e r i c r i b o s o m e s s e d i m e n t i n g a t 100-200S i s a r f e e f a c t u a l a n d o o f t e n r e s u l t s f r o m an a g g r e g a t i o n o f r i b o s o m e s a t h i g h magnesium c o n c e n t r a t i o n s (63, 6 9 - 7 1 ) . The p o s s i b i l i t y t h a t t h e d i s o m e s o b s e r v e d i n t r o u t t e s t i s were a l s o a r e t f a c t s was e x a m i n e d b u t v a r i o u s l i n e s o f i n v e s t i g a t i o n have shown, as f o l l o w s , t h a t t h e d i s o m e s a r e a t r u e c l a s s o f s m a l l p o l y s o m e s . 1) The d i m e r i c r i b o s o m e s n o t e d as a r t e f a c t s by v a r i o u s i n v e s t i g a t o r s (70, 71) have b e e n shown t o be r e l a t i v e l y i n a c t i v e i n p r o t e i n s y n t h e s i s , w h e r e a s t h e d i s o m e s i n t h e p r e s e n t e x p e r i m e n t s a r e h i g h l y a c t i v e ( F i g . 21) and c o n t a i n t h e m a j o r i t y o f t h e n a s c e n t p r o t a m i n e m o l e c u l e s ( T a b l e I V ) . 2) No s p e c i f i c p eak o f r a d i o a c t i v i t y was g e n e r a t e d i n t h e d isome a r e a when l a b e l l e d p r o t a m i n e was added t o a p r e p a r a t i o n o f r i b o s o m e s ( F i g . 23) and f u r t h e r , n o n - s p e c i f i c b i n d i n g t o t h e r i b o s o m e s o c c u r r e d w i t h u n i f o r m s p e c i f i c . a c t i v i t y t h r o u g h o u t t h e e n t i r e r e g i o n o f t h e g r a d i e n t . 3) T h r o u g h o u t t e s t i s d e v e l o p m e n t ( f r o m S e p t . - D e c ) , d i s o m e s a r e t h e o n l y c l a s s o f p o l y s o m e w h i c h i n c o r p o r a t e s ll*C-- 137 -a r g i n i n e but l i t t l e 1'''C-lysine, a f i n d i n g c o n s i s t e n t w i t h the f a c t t h a t the protamine molecule contains no l y s i n e r e s i d u e h . 4) The disomes are not magnesium dependent aggregates since the magnesium con c e n t r a t i o n used i n these experiments (5 mM) i s i n the normal range and cannot be c l a s s i f i e d as a "high magnesium conc e n t r a t i o n " . Further as seen i n F i g . 26, the l a r g e peak of disomes i n t r o u t t e s t i s does not appear to d i s s o c i a t e when i s o l a t e d i n 1 mM magnesium i o n c o n c e n t r a t i o n . A r t e f a c t u a l magnesium-dependent dimeric ribosomes u s u a l l y d i s s o c i a t e at t h i s lower magnesium con c e n t r a t i o n (71) and are not present i n pre-p a r a t i o n s of ribosomes e x t r a c t e d i n 1 mM Mg (70, 204). The decrease i n the amount of l a r g e r polysomes observed i n ribosomes e x t r a c t e d i n 1 mM Mg + +, ( F i g . 26 B) i s l i k e l y to be the r e s u l t of degradation of these polysomes by nuclease. I t i s p o s s i b l e t h a t bound nuclease i s r e l e a s e d i n the presence of lower magnesium i o n concentrations during the i s o l a t i o n procedure. The increase i n the number of monosomes (F i g . 26 B) supports t h i s conjecture s i n c e nuclease u s u a l l y degrades polysomes i n t o monosomes. 5) That the disomes are not the r e s u l t of degradation of l a r g e r polysomes during the i s o l a t i o n procedure i s suggested i n F i g . 24 s i n c e a f t e r a very short pulse of 1 ^ C - a r g i n i n e the major peak of r a d i o -a c t i v i t y i s a s s o c i a t e d w i t h the disomes and only a f t e r a longer pulse do the l a r g e r polysomes become l a b e l l e d w i t h no a d d i t i o n a l increase i n l a b e l l i n g of the disomes or monosomes. Since the disomes are l a b e l l e d f i r s t , i t could not be the breakdown product of l a r g e r polysomes. 6) S p e c i f i c f u n c t i o n a l polysomes are thought to form at d i f f e r e n t stages of c e l l development when p a r t i c u l a r p r o t e i n s are synthesized. In synchronized Hela c e l l s - 138 -f o r i n s t a n c e , prominent peaks of small cytoplasmic polysomes are observed when ribosomes, obtained during the S phase of c e l l development, were f r a c t i o n a t e d on a sucrose gradient (45). These polysomes are not present at other stages of c e l l development and they have been shown to be a s s o c i a t e d s p e c i f i c a l l y w i t h the synthesis of h i s t o n e during DNA s y n t h e s i s . In the t r o u t t e s t i s , the r e l a t i v e p r o p o r t i o n of disomes i n the t r o u t t e s t i s increases at the time when protamine i s most r a p i d l y synthesized during Octt.and Nov. (see Table V and F i g . 28) and the disome i s the major c l a s s of polysome present ( F i g . 28 Oct. and Nov.). Trout l i v e r polysomes i s o l a t e d under the same c o n d i t i o n s do not show these changes. Hence, the disomes are a c l a s s of f u n c t i o n a l polysomes formed s p e c i f i c a l l y f o r the synthesis of protamine at a l a t e stage of spermatogenesis. Since the amino a c i d composition (5) and the molecular weight (14) 6 of protamine from rainbow t r o u t are s i m i l a r to the w e l l - c h a r a c t e r i z e d protamines of h e r r i n g (•c3iupe'ihes;). (129, 130), the number of amino a c i d residues comprising the rainbow t r o u t protamine molecule would a l s o be s i m i l a r to t h a t found i n the c l u p e i n e s , namely 30-31 r e s i d u e s . A messenger RNA f o r such a molecule would r e q u i r e only 96-99 n u c l e o t i d e s (30-31 codons f o r the amino acids p l u s i n i t i a t i o n and t e r m i n a t i o n codons) i f i t were monocistronic. A r e l a t i o n s h i p appears to e x i s t between the number of residues of the polypeptide chain and the number of ribosomes i n the polysome complex (50); f o r example, hemoglobin chains of average length 143.5 residues (mean of 141 f o r a-chains and 146 f o r 3-chains) appear to be synthesized on p o l y -somes which c o n t a i n an average of f i v e ribosomes. Using t h i s - 139 -r e l a t i o n s h i p a monocistronic protamine messenger would r e q u i r e 1.04-1.08 ribosomes per polysome. Since ribosomes are "quantum" p a r t i c l e s , two modeOis are p o s s i b l e . The f i r s t would accommodate two ribosomes on a monocistronic messenger but packed somewhat more t i g h t l y than i n the hemoglobin example, and the second would p o s t u l a t e a d i c i s t r o n i c messenger RNA w i t h an average of two ribosomes ( t h e o r e t i c a l 2.08-2.16) spaced at the more usual i n t e r v a l . At the moment i t i s not p o s s i b l e to choose between these a l t e r n a t i v e s , although i t should be noted that under c e r t a i n c o n d i t i o n s , ' t i g h t l y packed 1 ribosomes can occur on a messenger RNA template. For example, H o r i and Rab i n o v i t z (205) have shown th a t when r a b b i t r e t i c u l o c y t e s are t r e a t e d w i t h O-methylthreonine (an i s o l e u c i n e a n t a g o n i s t ) , a new c l a s s of heavy polysomes con-t a i n i n g about 12 monomers appears i n these c e l l s w h i l e the usual c l a s s of polyribosomes a s s o c i a t e d w i t h the synthesis of hemo-g l o b i n and c o n t a i n i n g 5-6 monomers i s diminished. I t i s suggested th a t the heavy polyribosomes may have been formed by maximal packing of monomers on an extended messenger RNA strand as a r e s u l t of a block at an i s o l e u c y l residue near the C-terminal end of the B - c h a i n of hemoglobin. I t i s g e n e r a l l y assumed th a t the breakdown of polysomes to monosomes f o l l o w i n g a m i l d treatment w i t h RNase i s the r e s u l t of enzymatic degradation of the messenger RNA connecting ribosomes i n the polysome complex. (58, 59, 73). From F i g . 27 i t i s seen t h a t i n t r o u t t e s t i s the disomes d i f f e r from other c l a s s e s of l a r g e r polysomes i n t h a t they are r e s i s t a n t to m i l d RNase di g e s -t i o n . This r e s i s t a n c e however does not preclude the existence of - 140 -a m e s s e n g e r RNA i n t h e d i s o m e . A r e c e n t s t u d y by F e n w i c k (206) on t h e e f f e c t o f RNase on t h e p o l y s o m e s o f E. ooli and H e l a c e l l s has i n d i c a t e d t h a t t h e m i l d e s t f o r m o f RNase t r e a t m e n t t h a t d i s a g g r e g a t e s p o l y s o m e s a l s o a t t a c k s r i b o s o m a l RNA. He t h u s c o n c l u d e s t h a t m i l d RNase t r e a t m e n t i s n o t s p e c i f i c f o r t h e d e g r a d a t i o n o f m e s s e n g e r RNA and t h e a c t u a l mechanism o f polysome, d i s a g g r e g a t i o n by t h i s enzyme i s n o t u n d e r s t o o d . I t i s n o t known what c o n t r i b u t e s t o t h e r e s i s t a n c e o f t h e d i s o m e t o RNase b u t two p o s s i b i l i t i e s e x i s t . 1) A more t i g h t l y p a c k e d s t r u c t u r e as p r o p o s e d i n t h e m odel o f t h e d i s o m e c o n t a i n i n g a m o n o c i s t r o n i c messenger.RNA may be a f a c t o r c o n t r i b u t i n g t o t h e r e s i s t a n c e o f t h e d i s o m e t o RNase. Whether a t i g h t e r p a c k i n g o f r i b o s o m e s c o u l d c o n t r i b u t e t o t h e RNase r e s i s t a n c e o f p o l y -somes i s n o t p r e s e n t l y known b u t t h i s c o u l d be t e s t e d i n t h e r e t i c u l o c y t e s y s t e m o f H o r i and R a b i n o v i t z (205) w h i c h c o n t a i n s t h e i n h i b i t o r - i n d u c e d " t i g h t l y p a c k e d " p o l y s o m e s o f h e m o g l o b i n . 2) S i n c e p r o t a m i n e a p p e a r s t o be s y n t h e s i z e d on a s t a b l e messen-g e r RNA t e m p l a t e ( 1 1 ) , i t i s p o s s i b l e t h a t t h i s o b s e r v e d s t a b i -l i t y i s t h e r e s u l t o f some p r o t e c t i o n o f t h e d i s o m e i n t h e c e l l a g a i n s t t h e a c t i o n o f n u c l e a s e s . T h i s p r o t e c t i o n w o u l d m a n i f e s t i t s e l f a s t h e r e s i s t a n c e o f t h e d i s o m e t o m i l d RNase t r e a t m e n t . The n e e d f o r a p r o t e c t i o n mechanism c a n be a p p r e c i a t e d when i t i s o b s e r v e d t h a t t h e s y n t h e s i s o f p r o t a m i n e i s o c c u r r i n g a t a t i m e when t h e t o t a l c o n t e n t o f r i b o s o m e s i n t h e c e l l c y t o p l a s m i s d e c r e a s i n g ( T a b l e V ) . F o r example, f r o m t h e month o f O c t . t o Nov., t h e r a t e o f p r o t a m i n e s y n t h e s i s i n t h e t e s t i s i s e s s e n t i a l l y t h e same w h i l e t h e t o t a l r i b o s o m a l c o n t e n t i s d e c r e a s e d by more t h a n h a l f . - 141 -As a l r e a d y m e n t i o n e d i n t h e " I n t r o d u c t i o n " , t h e r e i s i n c r e a s i n g e v i d e n c e t h a t t h e m e s s e n g e r RNAs i n a n i m a l s a r e s t a b l e and c a n e x i s t i n a w i d e v a r i e t y o f f o r m s . The s t u d y o f d e v e l o p -m e n t a l c h a n g e s i n t h e p o l y s o m e s o f t r o u t t e s t i s ( T a b l e V, F i g s . 28-32) was i n i t i a t e d p a r t l y f r o m an i n t e r e s t c o n c e r n i n g t h e p r o p e r t i e s o f t h e p r o t a m i n e m e s s e n g e r RNA and t h e r e g u l a t i o n o f t h e l a t e " t u r n i n g - o n " o f p r o t a m i n e s y n t h e s i s d u r i n g s p e r m a t o g e n e s i s . S i n c e p r o t a m i n e s y n t h e s i s o c c u r s on a s t a b l e m e s s e n g e r RNA and i s n o t i n h i b i t e d by a c t i n o m y c i n D up t o a c o n c e n t r a t i o n o f 1 mM ( 1 1 ) , t h r e e s i m p l e m o d e l s a r e p o s t u l a t e d f o r t h e e v e n t s c o n c e r n e d w i t h t h e s y n t h e s i s o f p r o t a m i n e . (a) The m e s s e n g e r RNA f o r p r o t a m i n e i s s y n t h e s i z e d a t a t i m e e a r l y i n s p e r m a t o g e n e s i s and i s s t o r e d i n an i n a c t i v e f o r m e.g. as an i n f o r m o s o m e . A t t h e a p p r o p r i a t e s t a g e o f d e v e l o p m e n t t h i s i n a c t i v e m e s s e n g e r i s a c t i -v a t e d and a s s o c i a t e s w i t h r i b o s o m e s f o r p r o t a m i n e s y n t h e s i s . (b) T h i s i s l i k e m o d e l (a) e x c e p t t h a t t h e m e s s e n g e r RNA when s y n t h e s -i z e d i s a s s o c i a t e d w i t h r i b o s o m e s t o f o r m an i n a c t i v e d isome com-p l e x . T h e s e d i s o m e s c o u l d t h e n be a c t i v a t e d a t t h e a p p r o p r i a t e t i m e . (c) The m e s s e n g e r RNA o f p r o t a m i n e i s s y n t h e s i z e d a t a t i m e when p r o t a m i n e s y n t h e s i s i s t o be " t u r n e d - o n " ; t h e newly s y n t h e s i z e d m e s s e n g e r i m m e d i a t e l y a s s o c i a t e s w i t h r i b o s o m e s t o s y n t h e s i z e p r o t a m i n e . I n a l l t h r e e m o d e l s , i t i s p o s t u l a t e d t h a t s y n t h e s i s o f p r o t a m i n e i s c a r r i e d o u t on s t a b l e m e s s e n g e r RNA-r i b o s o m e c o m p l e x e s .•• , M o d e l (b) p r e d i c t s t h e p r e s e n c e o f a l a r g e number o f i n a c t i v e d i s o m e s i n t h e t e s t i s e v e n a t a p r e - p r o t a m i n e s t a g e o f t e s t i s d e v e l o p m e n t . I t i s s e e n i n F i g . 28 t h a t a t t h e e a r l y - p r o t a m i n e s t a g e o f d e v e l o p m e n t i n S e p t . , t h e t r o u t t e s t i s c o n t a i n s r e l a t i v e l y few d i s o m e s . T h i s o b s e r v a t i o n e l i m i n a t e s t h e - 142 -p o s s i b i l i t y o f m o d e l (b) a l t h o u g h i t i s s t i l l v a l i d i f t h e i n -a c t i v e m e s s e n g e r RNA-ribosome complex i s s t o r e d i n a f o r m o t h e r t h a n t h e di s o m e and i s c o n v e r t e d t o t h e di s o m e d u r i n g i n i t i a t i o n o f p r o t a m i n e s y n t h e s i s . M o d e l s (a) and (c) p r e d i c t t h a t t h e f o r m a t i o n o f d i s o m e s o c c u r s i n d i r e c t p r o p o r t i o n t o t h e r a t e o f p r o t a m i n e s y n t h e s i s s i n c e t h e s y n t h e s i s w o u l d b e g i n on t h e a s s o -c i a t i o n o f t h e m e s s e n g e r RNA w i t h r i b o s o m e s t o f o r m d i s o m e s . T h i p r e d i c t i o n i s o b s e r v e d i n F i g . 2.8 where t h e a p p e a r a n c e o f prom-i n e n t p e a k s o f d i s o m e s s e e n i n t h e O c t . and Nov. t e s t i s c o r r e l a t e w i t h t h e r a p i d r a t e o f p r o t a m i n e s y n t h e s i s o b s e r v e d d u r i n g t h e s e two months ( T a b l e V ) . I n m o d e l s (a) and ( b ) , p r o t a m i n e s y n t h e s i s c o u l d n o t be i n h i b i t e d by a c t i n o m y c i n D a t any t i m e d u r i n g t h e p r o t a m i n e s t a g e o f t e s t i s d e v e l o p m e n t s i n c e t h e m e s s e n g e r RNA i s p r e f o r m e d . I n m o d e l (C) however, t h e s y n t h e s i s o f p r o t a m i n e c o u l d be i n h i b i t e d by a c t i n o m y c i n D a t an e a r l y s t a g e o f p r o t a m i n e s y n t h e s i s when m e s s e n g e r RNA i s s t i l l b e i n g f o r m e d b u t a t l a t e r s t a g e s , no i n h i b i t i o n w o u l d be o b s e r v e d a f t e r t h e f o r m a t i o n o f m e s s e n g e r RNA has c e a s e d . Hence, t h e o b s e r v a t i o n o f I n g l e s et al. (11) t h a t a c t i n o m y c i n D d o e s n o t i n h i b i t t h e i n c o r p o r a t i o n o f l a b e l l e d a r g i n i n e i n t o p r o t a m i n e i n s a l m o n and t r o u t t e s t e s when p r o t a m i n e i s a c t i v e l y s y n t h e s i z e d , i s c o n s i s t e n t w i t h a l l t h r e e m o d e l s . To t e s t f o r t h e p o s s i b i l i t y o f model (c) , t h e i n c o r p o r a t i o n o f 1 h C — a r g i n i n e i n t o p r o t a m i n e -5 i n t h e p r e s e n c e o f a c t i n o m y c i n D (10 M) was examined i n a number o f t e s t e s c o l l e c t e d f r o m n a t u r a l l y m a t u r i n g r a i n b o w t r o u t a t a s t a g e when p r o t a m i n e s y n t h e s i s had j u s t begun. I t was f o u n d t h a t a t t h i s s t a g e o f t e s t i s m a t u r a t i o n , t h e i n c o r p o r a t i o n - 143 -of 1 ^ C - a r g i n i n e i n t o protamine was i n h i b i t e d by about 30% ( F i g . 33). This suggests t h a t the protamine messenger RNA i s synthes-i z e d during e a r l y protamine sy n t h e s i s and i m p l i c a t e s model (c) as the most s u i t a b l e model which at present f i t s a l l the observa-t i o n s . The a v a i l a b i l i t y of a method f o r the i d e n t i f i c a t i o n of the protamine messenger RNA should a l l o w f o r the l o c a l i z a t i o n of the time of protamine messenger synthesis and f o r the c r i t i c a l t e s t i n g of these models. I t i s p o s s i b l e , f o r example, th a t the observation of i n h i b i t i o n of protamine synthesis by actinomycin D i s not the r e s u l t of i n h i b i t i o n of the formation of protamine messenger RNA but of another r e g u l a t o r y RNA r e q u i r e d f o r the i n i t i a t i o n of protamine s y n t h e s i s . I f t h i s i s the case, model (a) may be a more s u i t a b l e model. The observation t h a t newly synthesized protamine i s phos-phorylated (7), transported i n t o the nucleus, binds w i t h DNA (48), and subsequently dephosphorylated (7) has prompted specu-l a t i o n as to the b i o l o g i c a l f u n c t i o n s served by the phosphoryla-t i o n of s e r y l residues i n protamine. I t has been suggested that the phosphorylation of protamine i n t r o d u c i n g 6-8 negative charges i n t o t h i s s m a ll h i g h l y b a s i c molecule, may be necessary f o r (a) i t s r e l e a s e from the cytoplasmic disome complex, (b) i t s t r a n s p o r t i n t o the nucleus, and (c) a modulated bi n d i n g of protamine to DNA (3). In view of the f a c t t h a t protamine i s synthesized i n the cytoplasm, a p o s s i b l e cytoplasmic s i t e of phosphorylation was examined and i n F i g . 20 i t may be seen t h a t i n a crude cytoplasmic c e l l - f r e e system, newly synthesized protamine i s a l s o phosphory-l a t e d . This observation i s c o n s i s t e n t w i t h t h a t of Marushige FRACTION NO. (6ml) F i g . 33. I n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o protamine i n the presence of actinomycin D. Two t e s t e s (3 g each) were obtained from a n a t u r a l l y maturing t r o u t a t the e a r l y -protamine stage of spermatogenesis. A c e l l suspension prepared from the t e s t e s were d i v i d e d i n t o two equal p o r t i o n s , one as c o n t r o l and other c o n t a i n i n g 10 5 M actinomycin D. A f t e r a p r e i n c u b a t i o n at 20° f o r 30 min, 1 uC of 1 ^ C - a r g i n i n e was added to each suspension and the i n c u b a t i o n continued f o r 60 min a f t e r which cycloheximide was added to a f i n a l c o n c e n t r a t i o n 0.4 mM. B a s i c p r o t e i n s were e x t r a c t e d from the t e s t i s n u c l e i as p r e v i o u s l y d e s c r i b e d , and the p r o t e i n s were analyzed on a B i o - G e l P-10 column as before. (A) c o n t r o l , (B) contains 10 5 M actinomycin D. - 145 -et al. (48) who have i s o l a t e d 3 2 P - l a b e l l e d phospho-profeamine f r o m t h e c y t o p l a s m o f t e s t i s c e l l s i n c u b a t e d w i t h 3 2 P - p h o s p h a t e . J e r g i l and D i x o n (35) have a l s o f o u n d a p r o t a m i n e k i n a s e i n t h e h i g h s p e e d s u p e r n a t a n t o f t e s t i s c e l l s . Some p r e l i m i n a r y s t u d i e s were made t h e n t o answer t h e q u e s t i o n o f w h e t h e r n a s c e n t p r o -t a m i n e s t i l l a t t a c h e d t o r i b o s o m e s i s p h o s p h o r y l a t e d . T e s t i s c e l l s u s p e n s i o n s were a l l o w e d t o i n c o r p o r a t e 3 2 P - p h o s p h a t e and 3 H - a r g i n i n e and r i b o s o m e s were p r e p a r e d as d e s c r i b e d i n " M a t e r i a l s and M e t h o d s " . T h e s e p u r i f i e d r i b o s o m e s were e x t r a c t e d f o r p r o t a m i n e and t h e e x t r a c t c h a r a c t e r i z e d by c h r o m a t o g r a p h y on B i o - G e l P-10. The r e s u l t s o f s e v e r a l e x p e r i m e n t s i n d i c a t e t h a t n a s c e n t p r o t a m i n e a s s o c i a t e d w i t h r i b o s o m e s i s p h o s p h o r y l a t e d . Whether t h i s p h o s p h o r y l a t i o n i s s p e c i f i c a l l y r e q u i r e d f o r t h e r e l e a s e o f p r o t a m i n e f r o m r i b o s o m e i s n o t known b u t p e r h a p s t h i s q u e s t i o n may be ans w e r e d by e m p l o y i n g a d e f i n e d c e l l - f r e e s y s t e m . F o r example, p r o t a m i n e s y n t h e s i s and t h e r e l e a s e o f p r o t a m i n e f r o m r i b o s o m e s c o u l d be examined i n t h e p r e s e n c e and a b s e n c e o f p u r i f i e d p r o t a m i n e k i n a s e ( 3 5 ) , t h e p h o s p h o r y l a t i n g enzyme f o r p r o t a m i n e . The c y t o p l a s m i c s y n t h e s i s o f p r o t a m i n e and i t s f i n a l l o c a -t i o n i n t h e n u c l e u s r a i s e s t h e p r o b l e m o f t h e i n t r a c e l l u l a r t r a n s -p o r t mechanism. From t h e p u l s e - c h a s e e x p e r i m e n t ( F i g . 15) i t a p p e a r s t h a t t h e t r a n s p o r t o f newly s y n t h e s i z e d p r o t a m i n e i s r a p i d w i t h an u p p e r l i m i t t o t h e h a l f - t i m e o f 1-2 m i n a t 2 0 ° . The p o s s i b i l i t y t h a t p h o s p h o r y l a t i o n p l a y s a p a r t i n t h e t r a n s p o r t o f p r o t a m i n e has b e e n examined i n a p r e l i m i n a r y way. M a r u s h i g e et al. (48) have shown t h a t t h e i n c o r p o r a t i o n o f 1 4 C - a r g i n i n e - 146 -i n t o protamine by t e s t i s c e l l s i s i n h i b i t e d i n the presence of va r i o u s l e v e l s of 2:4 d i n i t r o p h e n o l (an uncoupler of o x i d a t i v e p h o s p h o r y l a t i o n ) . However, the d i s t r i b u t i o n of newly synthesized protamine i s changed from a r a t i o of cytoplasm/nucleus of 0.18 i n the c o n t r o l s to 0.76 i n the presence of 2:4 d i n i t r o p h e n o l . This suggests t h a t when the i n t r a c e l l u l a r supply of ATP i s i n -h i b i t e d , there i s some i n t e r r u p t i o n of the t r a n s p o r t of protamine from cytoplasm to nucleus. I t i s not known at present whether t h i s i n t e r r u p t i o n i s the r e s u l t of a r e d u c t i o n i n the phosphory-l a t i o n of protamine or of some depression of an ATP dependent t r a n s p o r t mechanism f o r protamine. The c o n c l u s i o n t h a t the newly synthesized and phosphory-l a t e d protamine must a c t u a l l y be dephosphorylated in vivo stems from two p r i n c i p a l observations. F i r s t , Ingles and Dixon (7) have noted a p r o g r e s s i v e decrease i n the phosphate content of protamine obtained from t e s t e s at advancing stages of maturation; the protamine obtained from sperm c e l l s i s almost completely dephosphorylated. Second, i t i s seen t h a t the chromatographic behaviour of l a b e l l e d newly synthesized protamine and mature sperm protamine are d i f f e r e n t ( F i g . 10 A) i n that the newly synthesized protamine i s e l u t e d s u b s t a n t i a l l y e a r l i e r than mature protamine on a CM-cellulose column. As judged by the r e s u l t of treatment w i t h E. ooli a l k a l i n e phosphatase, t h i s d i f f e r e n c e i s due to a higher degree of phosphorylation on the newly synthesizeddpro-tamine ( F i g . 10 B). There must then be a mechanism f o r the r e -moval of phosphate from newly synthesized protamine as i t becomes converted i n t o mature protamine. In an attempt to d i s c o v e r the ( - 147 -r a t e o f r e m o v a l o f p h o s p h a t e f r o m p r o t a m i n e , an e x p e r i m e n t as summarized by F i g . 11 was p e r f o r m e d . From t h i s e x p e r i m e n t i t i s s e e n t h a t newly- s y n t h e s i z e d p r o t a m i n e p r e s e n t i n t h e c e l l n u c l e u s f o r s e v e n h o u r s has n o t b e e n s i g n i f i c a n t l y d e p h o s p h o r y -l a t e d as j u d g e d by t h e s i m i l a r i t y i n t h e c h r o m a t o g r a p h i c b e h a v i o u r between t h i s and newly s y n t h e s i z e d p r o t a m i n e e x t r a c t e d i m m e d i a t e l y a f t e r i n c o r p o r a t i o n (compare F i g . . 11 A and B ) . T h i s i n d i c a t e s t h a t p h o s p h o r y l a t e d p r o t a m i n e i s n o t d e p h o s p h o r y l a t e d s h o r t l y a f t e r t r a n s p o r t t o t h e n u c l e u s b u t may be p r e s e n t on t h e c h r o m a t i n f o r some t i m e b e f o r e d e p h o s p h o r y l a t i o n o c c u r s . T h a t t h e d e p h o s -p h o r y l a t i o n . p r o c e s s i s n o t a : r a p i d one h a s a l s o b e e n s u g g e s t e d by M a r u s h i g e et al. (48) who were a b l e t o i s o l a t e 3 2 P - l a b e l l e d p r o t a m i n e f r o m t h e c h r o m a t i n o f t r o u t t e s t i s . The p o s s i b i l i t y t h a t t h e f u n c t i o n o f newly s y n t h e s i z e d p h o s p h o - p r o t a m i n e p r e s e n t i n c h r o m a t i n i s d i f f e r e n t f r o m t h e ma-t u r e d e p h o s p h o - p r o t a m i n e i s s u g g e s t e d by t h e d i f f e r e n c e i n t h e i r b i n d i n g a b i l i t y t o DNA. M a r u s h i g e et al. (33) have shown by s a l t - g r a d i e n t d i a l y s i s o f p u r i f i e d c h r o m a t i n t h a t 3 2 P - l a b e l l e d -p h o s p h o - p r o t a m i n e i s e l u t e d f r o m t h e c h r o m a t i n b e f o r e t h e un-l a b e l l e d m a t u r e p r o t a m i n e . T h i s s u g g e s t s t h a t t h e newly s y n t h e -s i z e d p h o s p h o - p r o t a m i n e b i n d s l e s s s t r o n g l y t o DNA t h a n t h e m a t u r e p r o t a m i n e . I t i s p o s s i b l e t h e n t h a t t h e p h o s p h o - p r o t a m i n e p r o -v i d e s some m o d u l a t e d r e p r e s s i o n o f DNA t e m p l a t e a c t i v i t y and a t t h e a p p r o p r i a t e t i m e , a d e p h o s p h o r y l a t i o n p r o c e s s o c c u r s w h i c h v a l -lows f o r t h e t i g h t e r b i n d i n g o f p r o t a m i n e t o DNA. T h i s d e p h o s -p h o r y l a t i o n p r o c e s s , i t s mechanism as y e t unknown, may be r e q u i r e d b o t h f o r t h e p a c k a g i n g o f DNA as w e l l as t h e c o m p l e t e r e p r e s s i o n - 148 -of genome a c t i v i t y . Marushige et al. (33) have demonstrated t h a t p r e - e x i s t i n g histones l o c a t e d on the DNA i s phosphorylated during the replacement process. Since the phosphorylation of protamine occurs i n the cytoplasm, and since the high speed supernatant phospho-kinase of J e r g i l and Dixon (35) appears s p e c i f i c f o r protamine, the phosphorylation of histones l o c a t e d i n the nucleus might be mediated by another.mechanism. I n v e s t i -gations of the phosphorylated histones have revealed s e v e r a l i n t e r e s t i n g f e a t u r e s . Dixon et al., (49) have shown t h a t i n the case of h i s t o n e I I and histone IV, the major s i t e of phosphory-l a t i o n i s at the N-terminus of the molecule as judged by the recovery of a s i n g l e major phosphopeptide (N-acetyl-O-phospho-s e r y l - g l y c y l - a r g i n i n e ) from the t r y p t i c d i g e s t of 3 2 P - h i s t o n e . In a d d i t i o n , a s i n g l e t r y p t i c peptide which contains most of the l a b e l has r e c e n t l y been i s o l a t e d from 3 2 p - l a b e l l e d - h i s t o n e I of t r o u t t e s t i s (207). This peptide i s very s i m i l a r to the major phospho-peptide obtained by Langan and coworker (208) from a t r y p -t i c d i g e s t of histone I phosphorylated in vitro by a c a l f l i v e r h i s t o n e kinase. This provides c o n c l u s i v e evidence both in vivo and in vitro t h a t the phosphorylating mechanism f o r histones act only on a l i m i t e d number of s p e c i f i c s e r i n e r e s i d u e s . The phos-p h o r y l a t i o n of histones i n the d i f f e r e n t i a t i n g t r o u t t e s t i s at l e a s t , i s l i k e l y to be i n v o l v e d w i t h the d i s s o c i a t i o n of histones from DNA, during the replacementpprocess. To lend support to t h i s hypothesis, Marushige et al. (33) have r e c e n t l y shown that during the protamine stage of development i n t r o u t t e s t i s only the p r e - e x i s t i n g histones on DNA are phosphorylated; the small - 149 -amount of newly synthesized h i s t o n e s , l a b e l l e d w i t h 1 ^ C - a r g i n i n e or l y s i n e , observed at t h i s stage are not phosphorylated. This suggests t h a t phosphorylation of h i s t o n e i n general may be r e -q u i r e d f o r the d i s s o c i a t i o n of histones from DNA. The phosphorylation of histones i n c a l f l i v e r i s probably mediated by h i s t o n e kinase (141). The phosphorylation of h i s t o n e i n t r o u t t e s t i s may a l s o be mediated by the same mechanism since in vivo, the s i t e of phosphorylation on the h i s t o n e I molecule (207) appears to be i d e n t i c a l t o the s i t e of phosphorylation in vitro by the h i s t o n e kinase of Langan (208). However, the p o s s i -b i l i t y of a phospho-transferase f o r the t r a n s f e r of phosphate from the newly synthesized protamine to the p r e - e x i s t i n g histone during the replacement process cannot be r u l e d out. The existence of such an enzyme would i n d i c a t e a r e g u l a t o r y r o l e f o r the phos-p h o r y l a t i o n of protamine s i n c e the a r r i v a l of newly synthesized phospho-protamine i n the nucleus would s i g n a l the phosphorylation of c e r t a i n histonesmolecules. The attachment iof phosphate at s p e c i f i c s i t e s on these histones could i n i t i a t e a s e r i e s of events cu l m i n a t i n g i n the d i s s o c i a t i o n of the phosphorylated histones from DNA and the replacement of these histones by protamine. Such a mechanism would all o w the a s s o c i a t i o n of newly synthesized protamine to DNA and the d i s s o c i a t i o n of histones from DNA to be c l o s e l y coupled. Experiments have been i n i t i a t e d i n t h i s l a b o r a -t o r y to examine t h i s p o s s i b i l i t y . I t i s seen i n F i g . 5 A> and C, t h a t the i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e i n t o h i s t o n e and protamine r e f l e c t s the process of replacement of h i s t o n e by protamine i n t h a t when protamine - 150 -synthesis begins, histone s y n t h e s i s decreases ( F i g . 5 B) and l a t e r ceases almost completely ( F i g . 5 C). The p o s s i b i l i t y t h a t the c e s s a t i o n of histone synthesis may i n v o l v e a s p e c i a l mechanism of r e g u l a t i o n has been r a i s e d . However, since by the protamine stage of t e s t i s development DNA synthesis was already ceased (10), the c e s s a t i o n of h i s t o n e synthesis may be a r e f l e c -t i o n of c e s s a t i o n of DNA s y n t h e s i s . Histone synthesis i s u s u a l l y considered to be coupled to DNA synthesis i n that various i n v e s -t i g a t o r s have shown i n regenerating l i v e r the r a p i d synthesis of h i s t o n e j u s t p r i o r to the s y n t h e s i s of DNA (.209, 210). F u r t h e r , i n synchronized Hela c e l l s , Robbins and Borun (45) have shown t h a t there i s a p r e c i s e c o r r e l a t i o n between the synthesis of h i s t o n e and DNA. They observed t h a t histone synthesis occurs only d u r i n g DNA synthesis (during the S phase of the c e l l c y c l e) and t h a t when DNA synthesis stops, e i t h e r when the c e l l s progress i n t o the G 2 phase or as a r e s u l t of s p e c i f i c a l l y i n h i b i t i n g DNA synthesis w i t h c y t o s i n e a r a b i n o s i d e , histone synthesis a l s o ceases. This c o u p l i n g of histone s y n t h e s i s w i t h DNA synthesis appears to be mediated v i a small cytoplasmic polysomes s p e c i f i c f o r h i s t o n e s y n t h e s i s . The i n i t i a t i o n of DNA synthesis i s accompanied by the a c t i v a t i o n of these small polysomes wh i l e the a r r e s t of DNA s y n t h e s i s causes t h e i r d i s r u p t i o n . The C M - c e l l u l o s e - L i C l chromatography of rainbow t r o u t pro-tamine demonstrates the presence of at l e a s t three major compon-ents C I, CIjr and C11T ( F i g . 8). Component Cj i s present i n s u b s t a n t i a l l y smaller amounts than the other two. The presence of these components i s not the r e s u l t of genetic polymorphism - 151 -sinc e the same components could be obtained from the t e s t i s of a s i n g l e f i s h . Ando 1s group has a l s o demonstrated the presence of d i f f e r e n t components i n the protamine of h e r r i n g (130) as w e l l as i n other f i s h (15, 31). Hence these observations support the general consensus t h a t protamines are heterogeneous (1). The sequence data f o r clupeine ( F i g . 1) show th a t the three components of t h i s P a c i f i c h e r r i n g protamine are s t r i k i n g l y s i m i l a r , so much so, t h a t e v o l u t i o n of these three components from a common ances-t o r could be explained by a l i m i t e d number of mutational events (132). The components of t r o u t protamine are a l s o very s i m i l a r as judged by the f a c t t h a t they are not separable by e l e c t r o p h o r -e s i s on polyacrylamide g e l or chromatography on Bio-Gel P-10, Sephadex G-25, or on alumina (5). They are separable only on the h i g h l y r e s o l v i n g CM-cellulose system using a shallow l i t h i u m c h l o r i d e gradient as eluant ( F i g . 8). The amino-terminal (proline) and the c a r b o x y l - t e r m i n a l (arginine) of a l l three components are the same as judged by the absence of other amino acids when the N-terminal amino a c i d and the C-terminal amino a c i d of u n f r a c t i o n - ; ated protamine was determined (5). Further, the amino a c i d com-p o s i t i o n f o r a l l three components have been determined and they are a l s o very s i m i l a r (Table I I and Footnote 5). Since the com-ponents are so s i m i l a r , one p o s s i b i l i t y i s th a t they a l l have i d e n t i c a l f u n c t i o n s . The appearance of these components then could be the r e s u l t of mutations of the conservative type (133) which do not a f f e c t the f u n c t i o n of the protamine molecule and hence the mutations can p e r s i s t i n the genome. Since the C M - c e l l u l o s e - L i C l system i s able to r e s o l v e the - 152 -protamine components, i t i s p o s s i b l e to examine the b i o s y n t h e s i s of each of the components sepa r a t e l y . F i g . 12 A to D summarizes the changes i n the proportions of d i f f e r e n t protamine components present i n the t e s t i s c e l l n u c l e i during maturation. The r e l a t i v e amount of component C^ . decreases wh i l e t h a t of C J J J increases u n t i l at the mature sperm stage of development, C-J-JJ becomes the major component being present i n ' s l i g h t l y greater amounts than C^. The changes observed i n the r e l a t i v e amounts of the pro-tamine components present i n the nucleus must be the r e s u l t of d i f f e r e n t i a l s y n t h e s i s of these components as judged by the d i f f e r e n c e s i n the rates of i n c o r p o r a t i o n of 1 ^ C - a r g i n i n e ( F i g . 12). The d i f f e r e n t i a l s ynthesis of histone f r a c t i o n s has a l s o been observed i n r a t l i v e r by H n i l i c a et at. (212). In a d d i t i o n , Chalkley and Maurer .0211) i n a survey of a number of d i f f e r e n t c e l l types have suggested t h a t the synthesis of the l y s i n e - r i c h h i stones I and I I i s c l o s e l y coupled w i t h DNA synthesis whereas the a r g i n i n e - r i c h histones I I I and IV t u r n over continuously. The s i g n i f i c a n c e of the d i f f e r e n t i a l synthesis of histone f r a c -t i o n s or of the protamine components i s not known. However, since the s y n t h e s i s of the protamine components appears to be regulated i n r e l a t i o n s h i p one to another, i t i s p o s s i b l e t h a t the d i f f e r -ent components of protamine i n s p i t e of t h e i r s i m i l a r i t y , may have d i f f e r e n t f u n c t i o n s . Two probable f u n c t i o n s can be assigned to protamine, (a) packing of DNA i n t o the sperm head, and (b) r e p r e s s i o n of gene expression; i t i s p o s s i b l e that (a) i s the primary f u n c t i o n and (b) i s the d i r e c t consequence of (a) (12) although s p e c i a l - 153 -mechanism f o r gene r e p r e s s i o n may a l s o be i n v o l v e d . However, w h i l e the mechanism of f o l d i n g of sperm head chromosome i s not understood nor the exact r o l e of protamine i n t h i s complex pro-cess, i t i s c l e a r t h a t protamine does not serve merely to neutra-l i z e DNA e l e c t r o s t a t i c a l l y . I t appears t h a t d e s p i t e a small s i z e and a preponderance of a r g i n i n e , the s t r u c t u r e of protamine has been p r e c i s e l y s e l e c t e d through e v o l u t i o n f o r the r o l e i t p l a y s . Several l i n e s of evidence c o n t r i b u t e to t h i s c o n c l u s i o n . F i r s t , O l i n s e t al. wA151) have demonstrated t h a t the bin d i n g of protamine to DNA i s not pu r e l y e l e c t r o s t a t i c and t h a t n o n - e l e c t r o s t a t i c f o r c e s are i n v o l v e d . Second, as a r e s u l t of the a r g i n i n e residues on protamine being separated by one, two, or three n e u t r a l amino acids ( F i g . 1 ) , protamine binds l e s s s t r o n g l y to DNA than a pol y -c a t i o n c o n t a i n i n g about twenty a r g i n i n e residues i n sequence (151). T h i r d , i f the f u n c t i o n of protamine were only f o r the e l e c t r o s t a t i c n e u t r a l i z a t i o n of DNA, then one would expect t h a t during the course of e v o l u t i o n conservative s i n g l e base mutations could occur r e s u l t i n g i n the change of c e r t a i n a r g i n i n e residues to l y s i n e and these changes would be preserved. Yet, the remark-able constancy of a r g i n i n e both among d i f f e r e n t species of f i s h protamines and among the components of the same protamine i n d i -cates t h a t c e r t a i n p r e c i s e f u n c t i o n not yet understood must be served by a r g i n i n e i n protamine. Fourth, since protamine occurs on the chromatin both i n the phosphorylated and de-phopshorylated form, there must be some modulation of the bin d i n g of protamine to DNA. F i f t h , the f a c t t h a t d i f f e r e n t histone f r a c t i o n s are replaced p r e f e r e n t i a l l y by protamine during t e s t i s maturation - 154 -(12) suggests the p o s s i b i l i t y of d i f f e r e n t s p e c i f i c protamine components p a r t i c i p a t i n g i n the c o n t r o l of t h i s process. F i n a l l y , w h i l e the protamine components are chemically very s i m i l a r , they are synthesized independently and a l s o , t h e i r r a t e s of syn t h e s i s vary during t e s t i s maturation. This suggests t h a t the protamine components have d i f f e r e n t s p e c i f i c f u n c t i o n s . - 155 -FOOTNOTES 1. I t should be noted t h a t the term "microsomes" used i n t h i s t h e s i s i s defined i n s t r i c t l y p r a c t i c a l terms and represents the sediment of the high speed c e n t r i f u g a t i o n of a post-mi t o c h o n d r i a l supernatant which had not p r e v i o u s l y been t r e a t e d w i t h detergent to d i s s o l v e membranes. While t h i s sediment would c o n t a i n mainly ribosomes and membranes, other macro-molecules are a l s o a s s o c i a t e d w i t h these components. 2. An opaque p e l l e t was found as the sediment of t h i s c e n t r i f u g a -t i o n . This p e l l e t was discarded and not analyzed f u r t h e r s i n c e i t was found to c o n t a i n l e s s than 5% of the t o t a l r a d i o -a c t i v i t y a s s o c i a t e d w i t h t e s t i s ribosomes when the ribosomes were p u r i f i e d from c e l l s which had been allowed to inco r p o r a t e 1 ^ C - a r g i n i n e f o r 60 min. 3. The species of rainbow t r o u t used by Ando and coworkers was Salmo ivideus which i s g e n e r a l l y regarded as being completely i d e n t i c a l w i t h Salmo gaivdnerii, the species used i n these experiments. 4. No l y s i n e residue was observed when the amino a c i d composition of t o t a l u n f r a c t i o n a t e d t r o u t protamine was analyzed (5). However, a very small number of l y s i n e residues not detected by the a n a l y s i s could be present (see Footnote 5). 5. Recently J e r g i l (215) has prepared component i n s u f f i c i e n t q u a n t i t y for'amino a c i d a n a l y s i s . The amino a c i d composition was ( i n mole %) : arg 64.2, ser 11.9, pro 8.7, g l y 6.1, v a l 5.9, - 156 -a l a 0.8, i l e 0.4, l y s 2.1. Except f o r the presence of l y s i n e , the amino a c i d composition was very s i m i l a r to and CJJJ (Table I I ) . 6. The molecular weight of protamine from Salmo irideus was de t e r -mined (see Footnote 5). - 157 -BIBLIOGRAPHY 1. F e l i x , K. : Advances i n P r o t e i n Chemistry 1_5, 1, 1960. 2. Vendrely, R. , and C. Vendrely: Protoplasmatologia 5_, 3C, 1966 (Springer-Verlag, Vienna). 3. Dixon, G.H., and M. Smith: Prog, i n N u c l e i c A c i d Res. and Molecular Biocl. 8_, 9, 1968 (J.N. Davidson and W.E. Cohn, eds. Academic Press, New York). 4. Schmidt, P.J., B.S. M i t c h e l l , M. Smith,and H. Tsuyuki: Gen. Comp. E n d o c r i n o l . 5_, 197, 1965. 5. I n g l e s , C.J.: Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1968. 6. L i t t l e f i e l d , J.W., and E.B. K e l l e r : J . B i o l . 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