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Variability of sperm histones in Anura contrasts with relative constancy in Urodela, Squamata and Aves Mann, Mairi Elizabeth 1981

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VARIABILITY OF SPERM HISTONES IN ANURA CONTRASTS WITH RELATIVE CONSTANCY IN URODELA, SQUAMATA AND AVES. By MAIRI ELIZABETH MANN B.SC. UNIVERSITY OF B.C. 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of ZOOLOGY) 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 3 JULY 1981 c MAIRI ELIZABETH MANN, 1981 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 requirements 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 Columbia, I agree 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 agree 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 copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date ABSTRACT Sperm h i s t o n e d i v e r s i t y i n the v e r t e b r a t e s has been examined i n order to d e l i n e a t e p o s s i b l e macroevolutionary t r e n d s . Sperm b a s i c p r o t e i n s have been c h a r a c t e r i z e d c y t o c h e m i c a l l y i n t e s t i s s e c t i o n s and by e l e c t r o p h o r e s i s i n p o l y a c r y l a m i d e and s t a r c h g e l s . R e p r e s e n t a t i v e s p e c i e s of Anura ( f r o g s and t o a d s ) , Urodela (newts and salamanders), Squamata ( l i z a r d s and snakes) and Aves (birds) have been compared. The t e s t i s - s p e c i f i c b a s i c p r o t e i n s of Anura demonstrated a high degree of d i v e r s i t y . C y t o c h e m i c a l l y , sperm h i s t o n e s of the genus Rana resembled those of somatic c e l l s while those of the genera Hyla, Bufo and Scaphiopus appeared to be of the more b a s i c " i n t e r m e d i a t e type". E l e c t r o p h o r e s i s r e v e a l e d d i f f e r e n c e s between the t e s t i s - s p e c i f i c b a s i c p r o t e i n s of these genera, and i n the genus Xenopus, sperm nuclear p r o t e i n s v a r i e d at the s p e c i e s and even sub-species l e v e l s . X. t r o p i c a l i s and X. sjo. n. I l l ( Z a i r e ) , d i s t a n t l y r e l a t e d to the r e s t of the genus, were found to have s o m a t i c - l i k e sperm h i s t o n e s as opposed to the i n t e r m e d i a t e type p r o t e i n s d i s p l a y e d by other Xenopus s p e c i e s . The a r r a y of b a s i c p r o t e i n s found in the sperm of u r o d e l e s , r e p t i l e s and b i r d s appeared more c o n s e r v a t i v e than i n anurans. A l l the urodeles s t u d i e d showed a c y t o c h e m i c a l l y d e t e c t a b l e t r a n s i s t i o n from somatic h i s t o n e s to s t a b l e protamines to protamines d u r i n g the course of spermiogenesis. The p r o t a m i n e - l i k e p r o t e i n appeared to be e l e c t r o p h o r e t i c a l l y s i m i l a r i n a l l urodele s p e c i e s . L i z a r d s and snakes a l s o demonstrated t e s t i s - and sperm-spec i f i c b a s i c p r o t e i n s with s i m i l a r e l e c t r o p h o r e t i c m o b i l i t i e s , although c y t o c h e m i s t r y suggested that the l i z a r d sperm h i s t o n e s had a higher content of a r g i n i n e than the p r o t e i n s found i n snake sperm. C y t o c h e m i c a l l y , the sperm nuc l e a r p r o t e i n s of seven sp e c i e s of b i r d s , r e p r e s e n t i n g four a v i a n o r d e r s , were found to be of the protamine type. I n t a c t protamines were d i f f i c u l t to i s o l a t e from avian t e s t e s but s i m i l a r i t i e s were noted between the major t e s t i s - s p e c i f i c e l e c t r o p h o r e t i c bands of a l l the b i r d s s t u d i e d . O v e r a l l , there appeared to be a trend i n v e r t e b r a t e phylogeny from sperm h i s t o n e d i v e r s i t y i n t e l e o s t f i s h and anuran amphibians to r e l a t i v e constancy i n Urodela, R e p t i l i a , Aves and Mammalia. T h i s e v o l u t i o n a r y t r e n d was d i s c u s s e d i n terms of the p o s s i b l e , f u n c t i o n s of the b a s i c nuclear p r o t e i n s of sperm and the appearance of i n t e r n a l f e r t i l i z a t i o n and chromosomally-based sex-determination i n the v e r t e b r a t e s . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES . v i i LIST OF FIGURES , v i i i LIST OF ABBREVIATIONS x i i ACKNOWLEDGEMENTS x i i i I. GENERAL INTRODUCTION . 1 I I . DIVERSITY OF SPERM HISTONES IN THE GENUS XENOPUS AND OTHER ANURA 20 A. INTRODUCTION 20 B. MATERIALS AND METHODS 23 1. Experimental Animals 23 2. Cytochemistry 26 a. F i x a t i o n and embedding 26 b. Feulgen r e a c t i o n 27 c. A l k a l i n e f a s t green r e a c t i o n 27 d. E o s i n Y 29 e. Deamination 29 f. A c e t y l a t i o n 30 g. A l k a l i n e f a s t green r e a c t i o n without h y d r o l y s i s 30 h. F e u l g e n - a l k a l i n e f a s t green procedure 31 i . Sakaguchi r e a c t i o n 31 j . D i n i t r o f l u o r o b e n z e n e procedure 32 k. C o n t r o l s 33 3. B i o c h e m i s t r y 34 V a. Micromethod 34 b. Spermatid/sperm method 35 c. Whole t e s t i s method 37 d. C o n t r o l s 38 e. E l e c t r o p h o r e s i s 39 C. RESULTS . 42 1 . Cytochemistry 42 a. C o n t r o l s 42 b. Xenopus 47 c. Other Anura 63 d. S a l t c o n c e n t r a t i o n and the a l k a l i n e f a s t green r e a c t i o n 77 2. E l e c t r o p h o r e s i s 80 a. Xenopus l a e v i s l a e v i s c o n t r o l s 80 b. Xenopus l a e v i s subspecies 95 c. Xenopus s p e c i e s 106 d. Other Anura 126 D. DISCUSSION 132 I I I . RELATIVE CONSTANCY OF SPERM HISTONES IN URODELA, SQUAMATA AND AVES 140 A. INTRODUCTI ON 140 B. MATERIALS AND METHODS 141 1. Experimental Animals 141 2. Cytochemistry 143 3. Biochemistry 143 a. P r e p a r a t i o n of t i s s u e and i s o l a t i o n of b a s i c p r o t e i n s 143 v i b. E l e c t r o p h o r e s i s 144 C. RESULTS 145 1. Urodela 145 a. Cytochemistry 145 b. E l e c t r o p h o r e s i s 150 2 . Sguamata 155 a. Cytochemistry 155 b. E l e c t r o p h o r e s i s 169 3. Aves 180 a. Cytochemistry 180 b. E l e c t r o p h o r e s i s 195 D. DISCUSSION 205 IV. GENERAL DISCUSSION 208 V. REFERENCES 221 LIST OF TABLES v i i T a ble I. O r i g i n - of Experimental Animals from the Genus Xenopus 26 Table I I . Cytochemistry of Sperm N u c l e i of C o n t r o l Animals 46 Table I I I . Cytochemistry of Sperm N u c l e i i n the Genus Xenopus 62 Table IV. Cytochemistry of Sperm i n Anura 76 Table V. S a l t C o n c e n t r a t i o n and the A l k a l i n e . Fast Green Reaction 78 Table VI. C o n d i t i o n of B a s i c Nuclear P r o t e i n s I s o l a t e d by D i f f e r e n t Methods 81 Table V I I . Cytochemistry of Elongate Spermatids and Sperm of Drodeles ........150 Table V I I I . Cytochemistry of R e p t i l i a n Sperm N u c l e i 168 Table IX. E f f e c t o f F i x a t i o n Time on AFG S t a i n i n g f o r Protamine .184 Table X. Cytochemistry of Avian Sperm 195 Table XI. The V a r i e t y of Sperm Histones i n the V e r t e b r a t e s 209 Table X I I . Occurence of I n t e r n a l F e r t i l i z a t i o n , Sex Chromosomes and Sperm Histone D i v e r s i t y i n the V e r t e b r a t e s 218 v i i i LIST OF FIGURES F i g u r e 1. S t a i n i n g of t e s t i s s e c t i o n s from c o n t r o l animals 43 F i g u r e 2. Feulgen s t a i n i n g of s e c t i o n s of Xenopus t e s t e s . 48 F i g u r e 3. Feulgen s t a i n i n g of s e c t i o n s of Xenopus t e s t e s . 50 F i g u r e 4. High m a g n i f i c a t i o n of Feulgen s t a i n e d Xenopus sperm 52 F i g u r e 5. A l k a l i n e f a s t green s t a i n i n g of Xenopus t e s t i s s e c t i o n s 55 Fi g u r e 6. A l k a l i n e f a s t green s t a i n i n g of Xenopus t e s t i s s e c t i o n s 57 F i g u r e 7. Sakaguchi s t a i n i n g of Xenopus t e s t i s s e c t i o n s .. 60 F i g u r e 8. S t a i n i n g of t e s t i s s e c t i o n s from Rana ca t e s b e i a n a 64 F i g u r e 9. S t a i n i n g of t e s t i s s e c t i o n s from Bufo 66 F i g u r e 10. S t a i n i n g of t e s t i s s e c t i o n s from Bufo 69 Fi g u r e 11. S t a i n i n g of t e s t i s s e c t i o n s from Hyla 71 F i g u r e 12. S t a i n i n g of t e s t i s s e c t i o n s from Scaphiopus ... 73 F i g u r e 13. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s and sperm b a s i c p r o t e i n s from X. 1_. l a e v i s 83 F i g u r e 14. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s and sperm b a s i c p r o t e i n s from X. 1. l a e v i s ..... 85 F i g u r e 15. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s and sperm b a s i c p r o t e i n s from X. 1. l a e v i s 90 F i g u r e 16. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s b a s i c p r o t e i n s from f i v e Xenopus l a e v i s subspecies 96 F i g u r e 17. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s of X. _1. l a e v i s , and X. 2^ . p e t e r s i 98 F i g u r e 18. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s of X. 1_. ssp. n. (Malawi), X. 1_. v i c t o r i a n u s and X. 1. bunyoniensis 101 F i g u r e 19. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s and spermatid/sperm b a s i c n u c l e a r p r o t e i n s from the genus Xenopus 103 F i g u r e 20. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s b a s i c p r o t e i n s from f i v e Xenopus sp e c i e s 107 F i g u r e 21. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from f i v e Xenopus s p e c i e s 109 F i g u r e 22. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s b a s i c p r o t e i n s from nine Xenopus s p e c i e s 111 F i g u r e 23. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from X. 1^ . l a e v i s , X. b o r e a l i s and X. m u e l l e r i 113 F i g u r e 24. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from X. c l i v i i , X. f r a s e r i and X. t r o p i c a l i s 115 F i g u r e 25. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from X. v e s t i t u s , X. w i t t e i and X. r u w e n z o r i e n s i s 117 F i g u r e 26. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from the genus Xenopus 119 F i g u r e 27. S t a r c h g e l electrophoretograms of t e s t i s b a s i s p r o t e i n s from congeneric s p e c i e s of anurans 127 F i g u r e 28. E l e c t r o p h o r e t i c p r o f i l e s of b a s i c p r o t e i n s from anuran t e s t e s 129 X F i g u r e 29. S t a i n i n g of t e s t i s s e c t i o n s from u r o d e l e s 146 F i g u r e 30. S t a r c h g e l electrophoretograms of t e s t i s b a s i c p r o t e i n s from urodeles 151 F i g u r e 31. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from i n d i v i d u a l urodeles 154 F i g u r e 32. S t a i n i n g of ductus deferens s e c t i o n s from Thamnophis 157 F i g u r e 33. S t a i n i n g of ductus deferens and t e s t i s s e c t i o n s of Thamnophis and Elaphe 159 F i g u r e 34. S t a i n i n g of t e s t i s and ductus deferens s e c t i o n s from A n o l i s c a r o l i n e n s i s 161 F i g u r e 35. S t a i n i n g of t e s t i s and ductus deferens s e c t i o n s from Crotaphytus c o l l a r i s 163 F i g u r e 36. S t a i n i n g of t e s t i s and ductus deferens s e c t i o n s from Sceloporus magister 165 F i g u r e 37. S t a r c h g e l electrophoretograms of t e s t i s and ductus deferens b a s i c p r o t e i n s from Squamata 171 F i g u r e 38. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from A n o l i s 173 F i g u r e 39. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s and ductus deferens b a s i c p r o t e i n s from Squamata 176 F i g u r e 40. Absorbance p r o f i l e s of t e s t i s and ductus deferens p r o t e i n s from Squamata 178 F i g u r e 41. Feulgen s t a i n i n g of a v i a n t e s t i s and ductus deferens s e c t i o n s 181 F i g u r e 42. A l k a l i n e f a s t green s t a i n i n g of ductus deferens s e c t i o n s from G a l l u s domesticus 184 F i g u r e 43. A l k a l i n e f a s t green s t a i n i n g of avian t e s t i s and ductus deferens s e c t i o n s 186 F i g u r e 44. S t a i n i n g of ductus deferens s e c t i o n s from G a l l u s domesticus 189 F i g u r e 45. S t a i n i n g of avian t e s t i s and ductus deferens sect ions 193 F i g u r e 46. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from G a l l u s domesticus 196 F i g u r e 47. E l e c t r o p h o r e t i c p r o f i l e s of a v i a n t e s t i s and ductus deferens b a s i c p r o t e i n s 199 F i g u r e 48. S t a r c h g e l electrophoretograms of a v i a n t e s t i s and ductus deferens b a s i c p r o t e i n s 202 F i g u r e 49. Composite e l e c t r o p h o r e t i c p r o f i l e s of v e r t e b r a t e sperm h i s t o n e s 211 LIST OF ABBREVIATIONS x i i DNA= d e o x y r i b o n u c l e i c a c i d RNA= r i b o n u c l e i c a c i d PMSF= ph e n y l m e t h y l s u l f o n y l f l u o r i d e TLCK= N - o t - p - t o s y l - L - l y s i n e chloromethyl ketone TPCK= L - l - t o s y l a m i d e - 2 - p h e n y l e t h y l chloromethyl ketone HC1= h y d r o c h l o r i c a c i d TCA= t r i c h l o r o a c e t i c a c i d AFG= A l k a l i n e f a s t green DNFB= d i n i t r o f l u o r o b e n z e n e PBS= phosphate b u f f e r e d s a l i n e NBF= n e u t r a l b u f f e r e d f o r m a l i n EDTA= eth y l e n e d i a m i n e - t e t r a - a c e t a t e T r i s = tris(hydroxymethyl)-amino methane TEMED= N,N,N)N'-tetramethyl-ethylenediamine SSC= s a l i n e sodium c i t r a t e x i i i ACKNOWLEDGEMENTS I would l i k e to express my deep a p p r e c i a t i o n to Dr. H.E. Kasinsky f o r h i s support, encouragement and advic e dur i n g the pr o d u c t i o n of t h i s t h e s i s . Thanks are a l s o given to Drs. N.C. B o i s , M.S. R i s l e y , R.A. Eckhardt, O.H.J. Destree and E.W. Byrd f o r t h e i r t e c h n i c a l . a s s i s t a n c e and h e l p f u l d i s c u s s i o n s . S p e c i a l a p p r e c i a t i o n i s extended to Dr. M. F i s c h b e r g f o r h i s generous c o o p e r a t i o n i n p r o v i d i n g the animals from the genus Xenopus, and to the s t a f f of the S t a t i o n de Zoologie Experimentale de Geneve f o r t h e i r g e n e r o s i t y and kindness. I would a l s o l i k e t o thank Ms. J . T a i t , Dr. D. M c l n t y r e and Dr. J . B a i l e y f o r t h e i r h elp i n p r o v i d i n g and i d e n t i f y i n g the animals used i n t h i s study. And s p e c i a l thanks are due to D. D e M i l l e , B. Yee, S. Kwauk and P. C s e r j e s i f o r t h e i r technic-al a s s i s t a n c e and moral support. Thanks a l s o to the f a c u l t y , students and s t a f f of the Department of Zoology, U.B.C, and to my f a m i l y and f r i e n d s , e s p e c i a l l y Mum, f o r t h e i r constant encouragement, understanding and p a t i e n c e . T h i s r e s e a r c h was supported by NRC grant #5854 to Dr. H.E. Kasinsky. 1 I. GENERAL INTRODUCTION In v i r t u a l l y a l l animal groups, d i f f e r e n t i a t i o n of the male gamete, or spermiogenesis, i s c h a r a c t e r i z e d by s e v e r a l m o r p h o l o g i c a l and bioche m i c a l f e a t u r e s . With the completion of m e i o s i s , the spermatid l o s e s much of i t s cytoplasm and develops i n t o a f l a g e l l a t e d spermatozoon. During t h i s p e r i o d of t r a n s f o r m a t i o n , the nucleus becomes elongated and reduced i n volume, and the chromatin condenses i n t o an amorphous, electron-opaque mass with l i t t l e or no d i s c e r n a b l e i n t e r n a l s t r u c t u r e . In a l l organisms s t u d i e d to date, chromatin condensation i s accompanied by replacement of some or a l l of the DNA-associated b a s i c p r o t e i n s , or h i s t o n e s , normally found i n somatic c e l l s by p r o t e i n s which are t y p i c a l of the sperm (Subirana, 1975; Bloch, 1969, 1976). A. Somatic h i s t o n e s Somatic c e l l s g e n e r a l l y c o n t a i n f i v e c l a s s e s of h i s t o n e s : H3 and H4, which are a r g i n i n e - r i c h p r o t e i n s ; H2A and H2B, termed the " s l i g h t l y l y s i n e - r i c h h i s t o n e s " ; and the H1 h i s t o n e s , which are r i c h i n l y s i n e . The t e r m i n a l p o r t i o n s of a l l h i s t o n e s c o n t a i n the g r e a t e s t d e n s i t y of b a s i c amino a c i d s . H1 has i t s b a s i c r e s i d u e s c l u s t e r e d i n the N- and C-terminal p o r t i o n s of the molecule while the c l u s t e r i n g of b a s i c amino a c i d s i n the other h i s t o n e s occurs p r i n c i p a l l y i n the N-t e r m i n a l r e g i o n . Hydrophobic and a c i d i c r e s i d u e s tend to dominate the c e n t r a l r e g i ons ( E l g i n and Weintraub, 1975; Isenberg, -1979). 2 Because of t h e i r a b i l i t y , i n high c o n c e n t r a t i o n s , to completely i n h i b i t t r a n s c r i p t i o n , i t was at one time thought t h a t the somatic h i s t o n e s acted as s p e c i f i c gene r e p r e s s o r s (Stedman and Stedman, 1950). However, t h i s view seems u n l i k e l y because of the l i m i t e d v a r i e t y of h i s t o n e types and, with the exception of H1, t h e i r l a c k of t i s s u e s p e c i f i c i t y . With few exceptions H2A, H2B, H3 and H4 occur i n the same molar r a t i o , of approximately one, i n a l l e u k a r y o t i c n u c l e i . Furthermore, the primary s t r u c t u r e s of these p r o t e i n s have been remarkably w e l l conserved through e v o l u t i o n . For example, there are only two c o n s e r v a t i v e amino a c i d d i f f e r e n c e s between the sequences of H4 i s o l a t e d from pea bud chromatin and from that of c a l f thymus (DeLange et a l . , 1969). It i s now thought that the somatic h i s t o n e s are l a r g e l y s t r u c t u r a l r a t h e r than r e g u l a t o r y p r o t e i n s and that they are r e s p o n s i b l e f o r the secondary c o i l i n g of DNA i n t o " b e a d - l i k e " s t r u c t u r e s known as "nucleosomes" ( O l i n s and O l i n s , 1974; Romberg, 1974; Van Holde et a l . , 1974). Each nucleosome c o n s i s t s of a "core p a r t i c l e " , c o n t a i n i n g two molecules each of h i s t o n e s H2A, H2B, H3 and H4, around which 145 base p a i r s of DNA i s wound i n approximately two s u p e r h e l i c a l t u r n s . The c o n s e r v a t i o n of amino a c i d sequences of the four core h i s t o n e s i s thought to r e f l e c t the very s p e c i f i c requirements of the h i s t o n e - h i s t o n e and histone-DNA i n t e r a c t i o n s which determine t h i s fundamental s t r u c t u r e of chromatin i n somatic c e l l s . Nucleosome core p a r t i c l e s are l i n k e d by a s t r e t c h of DNA which can vary i n l e n g t h from f i v e to about n i n e t y base p a i r s 3 (Romberg, 1977). Histone H1 appears to be a s s o c i a t e d with t h i s "spacer" or " l i n k e r " region (Varshavsky et a_l., 1976; Witlock and Simpson, 1976), and i s thought to p l a y a r o l e i n s t a b i l i z i n g the nucleosome and m a i n t a i n i n g the higher order c o i l i n g of the chromatin f i b r e ( F i n c h and Klug, 1976; Thoma et a l . , 1979). Histone H1 has been l e s s s t r i n g e n t l y conserved through e v o l u t i o n than the core h i s t o n e s . Not only do the H1's vary from s p e c i e s to s p e c i e s ( B u s t i n and Cole, 1968; Panyim et a l . , 1971), but even w i t h i n one t i s s u e from a s i n g l e animal there may be at l e a s t four H1 subtypes (Welch and Cole, 1979; Smerdon and Isenberg, 1976a; Seyedin and K i s t l e r , 1979a, 1979b). S t a g e - s p e c i f i c H1 v a r i a n t s a l s o appear dur i n g the embryonic development of sea u r c h i n s ( A r c e c i and Gross, 1980a), D r o s o p h i l a (Newrock et al.,1978) and Xenopus (Roster e_t a l . , 1 9 7 9 ) . However v a r i a t i o n s i n primary sequences of the H1 subtypes appear to be r e s t r i c t e d to the b a s i c t e r m i n a l regions of the molecules, with the c e n t r a l g l o b u l a r r e g i o n being more h i g h l y conserved (Isenberg, 1979). Pa t t e r n s of sequence homology i n d i c a t e that h i s t o n e H5, found i n the n u c l e a t e d e r y t h r o c y t e s of b i r d s , may a l s o be c o n s i d e r e d an extreme v a r i a n t of H1 r a t h e r than a d i s t i n c t h i s t o n e type (Yaguchi et a l . , 1977). The p r e c i s e f u n c t i o n of these H1 v a r i a n t s i s s t i l l unknown, but they may p l a y a r o l e i n the coarse c o n t r o l of t r a n s c r i p t i o n by modulating the compaction of chromatin ( A r c e c i and Gross, 1980a) or by s p e c i f i c b i n d i n g of non-histone "high m o b i l i t y group" chromosomal p r o t e i n s 4 (Smerdon and Isenberg, 1976a; Mardian et a_l. , 1 980). S e v e r a l e x t e n s i v e reviews on nucleosome s t r u c t u r e and the f u n c t i o n of the somatic h i s t o n e s have been r e c e n t l y p u b l i s h e d and cover many other f e a t u r e s not d i s c u s s e d here (McGhee and F e l s e n f e l d , 1980; L i l l e y and Pardon, 1979; Romberg, 1977). B. Sperm h i s t o n e s The b a s i c p r o t e i n s found i n the n u c l e i of sperm are unique in many r e s p e c t s . F i r s t of a l l , they represent one of the few examples of t i s s u e s p e c i f i c i t y demonstrated by the h i s t o n e s . A l s o , i n c o n t r a s t to the somatic h i s t o n e s , sperm h i s t o n e s i n d i f f e r e n t groups of animals d i s p l a y a remarkable v a r i a b i l i t y i n s i z e , amino a c i d composition and degree of b a s i c i t y . On the b a s i s of both cytochemical and biochemical evidence, Bloch (1969, 1976) has c l a s s i f i e d these p r o t e i n s i n t o f i v e broad c a t e g o r i e s , designated by the organisms which serve as t y p i c a l examples of each p r o t e i n type. In order of de c r e a s i n g b a s i c i t y , these a r e : the "salmon type", the "mouse-grasshopper type", the "M y t i l u s (mussel) type", the "Rana (frog) type", and the "crab type". 5 1. The "salmon type" or monoprotamines: F i r s t i s o l a t e d from the Rhine salmon by Meischer i n 1874, and l a t e r c h a r a c t e r i z e d by K o s s e l (1928), t h i s c l a s s of b a s i c p r o t e i n s , commonly known as protamines, has been the most e x t e n s i v e l y s t u d i e d group of sperm h i s t o n e s ( F e l i x , 1960; Ando et a_l. , 1 973). From the work of Ando and Suzuki ( 1967), Ando and Watanabe (1969) and B r e t z e l (1973), the amino a c i d sequences of s e v e r a l protamines from salmonoid f i s h are known. They are small p r o t e i n s , 32-33 amino a c i d r e s i d u e s i n l e n g t h , and c o n t a i n very few kinds of amino a c i d s . A r g i n i n e c o n s t i t u t e s the s o l e b a s i c amino a c i d and makes up two-thirds of the t o t a l amino a c i d composition. In any one s p e c i e s , two or three c l o s e l y r e l a t e d components, which d i f f e r s l i g h t l y i n amino a c i d sequence, may be present (Ando et a_l., 1973; Dixon, 1974). A protamine, " g a l l i n e " , has a l s o been found in the mature sperm of the domestic r o o s t e r , G a l l u s domesticus (Nakano et a l . , 1976a). T h i s p r o t e i n i s l a r g e r than the f i s h protamines, having 65 amino a c i d r e s i d u e s , 38 of which are a r g i n i n e . 2. The "mouse-grasshopper type" or " s t a b l e protamines": These are c h a r a c t e r i z e d as being very b a s i c "protamine-l i k e " p r o t e i n s which are more d i f f i c u l t to e x t r a c t from the sperm head than are the f i s h protamines (Bloch, 1969,1976). Mammalian sperm h i s t o n e s f a l l i n t o t h i s category and are known to c o n t a i n a high percentage of c y s t e i n e r e s i d u e s as w e l l as a r g i n i n e (Coelingh et a l . , 1972; Monfoort et a_l. , 1973; 6 K i s t l e r et a_l. , 1976; B e l l v e and Carraway, 1978; Kolk and Samuel, 1975; T o b i t a et a l . , 1979). These p r o t e i n s are " s t a b i l i z e d " i n the mature sperm nucleus by i n t e r m o l e c u l a r d i s u l f i d e bonds and a t h i o l - r e d u c i n g agent i s r e q u i r e d f o r t h e i r removal . They have thus been termed the " s t a b l e protamines" (Bedford and C a l v i n , 1974; B e l l v ^ et a l . , 1975; C a l v i n , 1976). In a d d i t i o n to'mammals, s t a b l e protamines have a l s o been observed i n the mature sperm of c a r t i l a g i n o u s f i s h (Bois and Kasinsky, 1974, 1976; Bois et a l . , 1980; Gusse and C h e v a i l l i e r , 1978,1980) and many i n s e c t s (Kaye and McMaster-Kaye, 1966; Das et a l . , 1964; Bloch and Brack, 1964). The spermatids of the newts Notophthalmus v i r i d e s c e n s (Bois e_t a l . , 1976) and P l e u r o d e l e s w a l t l ( P i c h e r a l , 1970) a l s o c o n t a i n s t a b l e protamines, although these are l a t e r r e p l a c e d by protamines of the salmon-type. A s i m i l a r t r a n s i t i o n from s t a b l e protamine to protamine has a l s o been observed d u r i n g spermiogenesis i n the s n a i l H e l i x aspersa (Bloch and Hew, 1960a) and the squid L o l i g o opalescens (Bloch, 1962). 3. The " M y t i l u s " or " i n t e r m e d i a t e type": T h i s i s a broad c l a s s of b a s i c p r o t e i n s which share the p r o p e r t i e s of being e a s i l y e x t r a c t e d from the sperm nucleus and intermediate i n composition between the somatic h i s t o n e s and the protamines (Bloch, 1969,1976). These i n c l u d e the p r o t e i n s which were termed d i - and t r i - p r o t a m i n e s by K o s s e l (1928); that 7 i s , b a s i c p r o t e i n s which c o n t a i n other b a s i c amino a c i d s , s p e c i f i c a l l y l y s i n e or h i s t i d i n e or both, as w e l l as a r g i n i n e . E l e c t r o p h o r e t i c a l l y , the i n t e r m e d i a t e - t y p e sperm h i s t o n e s are a d i v e r s e group of p r o t e i n s and have been found in a wide v a r i e t y of organisms, i n c l u d i n g m o l l u s c s (Colom and Subirana, 1979; Subirana et a l . , 1973), f i s h ( K o s s e l , 1928; F e l i x , 1960; Y u l i k o v a et a l . , 1979; Kennedy and Davies, 1980) and amphibians (Bois and Kasinsky, 1972; Huang et a l . , 1978). The sperm of the mussel M y t i l u s c o n t a i n two such intermediate p r o t e i n s , one of which i s very r i c h i n l y s i n e and the other c o n t a i n i n g more a r g i n i n e (Bloch, 1966; Subirana e_t a l . , 1973). 4. The "Rana" or "somatic type": The sperm of s e v e r a l s p e c i e s of echinoderms (Easton and C h a l k l e y , 1972; Subirana and Paulau, 1968), of the f r o g Rana (Alder and Gorovsky, 1975; Z i r k i n , 1970) and of the c a r p and g o l d f i s h ( Z i r k i n , 1971b; Nandi et a_l. , 1979) c o n t a i n h i s t o n e s which are almost i d e n t i c a l to the somatic h i s t o n e s . Subirana e_t a l . ( 1975) r e p o r t e d that i n these animals, the somatic h i s t o n e s H2A, H3 and H4 are always r e t a i n e d i n the sperm nucleus, whereas H1 and H2B may be present or absent. In a l l cases a d d i t i o n a l components, o f t e n r e l a t e d to H1 or H2B, may a l s o be found (Subirana e_t a l . , 1975; A l d e r and Gorovsky, 1975; de P e t r o c e l l i s et a l . , 1980). 8 5. The "crab type": The sperm n u c l e i of crabs appear to c o n t a i n no c y t o c h e m i c a l l y d e t e c t a b l e b a s i c p r o t e i n s ( C h e v a i l l i e r , 1967; Vaughn, 1968; Langreth, 1969), although b a s i c p r o t e i n s may be present i n the cytoplasm (Bloch, 1966). Vaughn and Hinsch (1972) r e p o r t e d the presence of a h i g h l y a c i d i c p r o t e i n a s s o c i a t e d with the sperm chromatin of the s p i d e r crab, L i b i n i a emarginata. In view of the d i v e r s i t y of sperm h i s t o n e types found in v a r i o u s groups of organisms, two fundamental q u e s t i o n s a r i s e : (1) what purpose do these b a s i c p r o t e i n s serve i n the sperm nucleus and why do some sperm (crabs) have no h i s t o n e s ; and (2) what e v o l u t i o n a r y p r e s s u r e s l e d to such a v a r i e t y of sperm h i s t o n e s when the somatic h i s t o n e s have been so r i g i d l y conserved? D e s p i t e many hypotheses, the p r e c i s e f u n c t i o n and b i o l o g i c a l s i g n i f i c a n c e of the s h i f t from somatic to sperm-s p e c i f i c h i s t o n e s remains unknown. Se v e r a l experimental approaches have been taken i n order to i n c r e a s e our understanding of t h i s phenomenon. One has been to study the molecular mechanisms by which s p e r m - s p e c i f i c b a s i c p r o t e i n s r e p l a c e somatic h i s t o n e s d u r i n g spermiogenesis (Dixon, 1974; M e i s t r i c h et a l . , 1976; L o i r and Lanneau, 1978b), and to examine the changing subunit s t r u c t u r e of chromatin duri n g t h i s replacement (Honda e_t a_l. , 1974; Gusse and C h e v a i l l i e r , 1980; McMaster-Kaye and Kaye, 1980; 9 Spadafora et a l . , 1976; Kierszenbaum and T r e s , 1975,1978). Another has attempted to c o r r e l a t e b i o c h e m i c a l changes with morphological changes d u r i n g sperm maturation, such as chromatin packing or the shaping of the sperm nucleus (Fawcett et a l . , 1971; Walker, 1971; Gusse and C h e v a i l l i e r , 1978). A t h i r d has been to explore the range of d i v e r s i t y of sperm h i s t o n e s i n v a r i o u s animal groups to see i f any p h y l o g e n e t i c r e l a t i o n s h i p s emerge. T h i s c o u l d shed l i g h t on the p o s s i b l e s e l e c t i v e p r e s s u r e s which might have l e d to the v a r i e t y of h i s t o n e t r a n s i t i o n s which occur d u r i n g spermiogenesis i n d i f f e r e n t organisms (Bloch, 1969,1976; Kasinsky et a_l., 1978). These experimental approaches have generated s e v e r a l hypotheses concerning the f u n c t i o n of sperm h i s t o n e s and have y i e l d e d some i n t e r e s t i n g r e s u l t s . The rainbow t r o u t , Salmo g a i r d n e r i i , has been used as a model system f o r the study of protamine s y n t h e s i s and replacement of somatic h i s t o n e s d u r i n g spermiogenesis (Dixon, 1974; Davies et. a l . , 1976). A f a m i l y of three or four protamine v a r i a n t s are s y n t h e s i z e d i n l a t e spermatids (Ling et a l . , 1971) from a heterogeneous p o p u l a t i o n of s t a b l e mRNA's (Gedamu and Dixon, 1979; J e n k i n s , 1979). Binding of protamine to DNA i s accompanied by p h o s p h o r y l a t i o n and dephosphorylation of s e r i n e r e s i d u e s i n the p r o t e i n , a process which may serve i n the correct,, alignment of the protamine molecules i n the sperm chromatin (Louie and Dixon, 1972). Somatic h i s t o n e s are thought to be d i s p l a c e d from chromatin by a combination of a c e t y l a t i o n and, perhaps, s p e c i f i c p r o t e o l y s i s 10 ra t h e r than by d i r e c t displacement by protamine (Marushige and Dixon, 1971; Marushige et a l . , 1976). The f i n a l r e s u l t i s a h i g h l y condensed form of chromatin which i s i n a c t i v e as a template f o r RNA polymerase (Marushige and Dixon, 1969) and shows no sign s of nucleosomal s t r u c t u r e when d i g e s t e d with nucleases (Honda et a l . , 1974,1975). The DNA of the condensed sperm nucleus i s a p p a r e n t l y covered by protamine molecules and has no exposed r e g i o n s s u s c e p t i b l e to nuclease a t t a c k . Inoue and Fuke (1970) used e l e c t r o n microscopy to show that n a t i v e and r e c o n s t i t u t e d DNA-protamine complexes form an extended network in which DNA f i b r e s appear to be l i n k e d side by side through protamine b r i d g e s . These r e s u l t s are c o n s i s t e n t with the nucleoprotamine model proposed by Warrant and Kim (1978), i n which protamine a l p h a - h e l i c a l segments wrap around the major groove of e i t h e r a s i n g l e s t r a n d or two adjacent strands of DNA and a r g i n i n e s i d e - c h a i n s form c o n t a c t s between neighbouring DNA double h e l i c e s . An a l t e r n a t e model i s that the protamine molecule may act as a core around which the DNA double h e l i x i s wound, one s u p e r c o i l turn per protamine molecule (Bazett-Jones and Ottensmeyer, 1979). In g e n e r a l , the mature sperm of e u t h e r i a n mammals c o n t a i n a s i n g l e stable-protamine s p e c i e s ( B e l l v e , 1979), although two protamines are known to e x i s t i n the spermatozoa of humans (Kolk and Samuel, 1975) and mice ( B e l l v e et a l . , 1975). S y n t h e s i s of these protamines occurs i n the t e r m i n a l stages of spermiogenesis ( P l a t z et al_., 1975; Grimes e_t a l . , 1977) and 11 t h e i r b i n d i n g to DNA may be modulated by p h o s p h o r y l a t i o n and d e phosphorylation of s e r i n e r e s i d u e s , as seen i n the t r o u t (Marushige and Marushige, 1975,1978). The primary s t r u c t u r e of b u l l protamine, and probably of other mammalian protamines, i s such that the a r g i n i n e r e s i d u e s are l o c a t e d towards the middle of the molecule while the c y s t e i n e r e s i d u e s are grouped at e i t h e r end (Coelingh and R o z i j n , 1975). Conceivably, the very b a s i c c e n t r a l p o r t i o n of t h i s molecule would i n t e r a c t with DNA while the ends would allow f o r i n t e r m o l e c u l a r c r o s s - l i n k s . The formation of i n t e r m o l e c u l a r d i s u l f i d e bonds occurs d u r i n g the passage of sperm through the epididymis and i s accompanied by p r o g r e s s i v e s t a b i l i z a t i o n of the nucleus (Marushige and Marushige, 1975b; M e i s t r i c h et a l . , 1976; L o i r and Lanneau, 1978b). In mammalian spermiogenesis, somatic h i s t o n e s do not appear to be d i r e c t l y r e p l a c e d by protamines. Instead, an unusual and heterogeneous group of b a s i c p r o t e i n s are t r a n s i e n t l y a s s o c i a t e d with the condensing spermatid nucleus (Grimes et a l . , 1977; K i s t l e r et a l . , 1973; L o i r and Lanneau, 1978a). The f i r s t of these p r o t e i n s , TP ( t e s t i s p r o t e i n ) , i s o l a t e d from mouse t e s t i s , was c h a r a c t e r i z e d as a low molecular weight p o l y p e p t i d e c o n t a i n i n g an e q u a l l y h i g h content of l y s i n e and a r g i n i n e (Lam and Bruce, 1971). Subsequent s t u d i e s v e r i f i e d the e x i s t e n c e of s i m i l a r p r o t e i n s i n the t e s t e s of s e v e r a l other mammalian sp e c i e s ( K i s t l e r et a l . , 1973,1975) and showed that they d i f f e r from the s t a b l e protamines of mature sperm in t h e i r lower a r g i n i n e 12 and higher l y s i n e contents and t h e i r l a c k of c y s t e i n e ( K i s t l e r e_t a l . , 1974). Another group of spermatid b a s i c p r o t e i n s , more c l o s e l y r e l a t e d to the somatic h i s t o n e s , have been i d e n t i f i e d i n the r a t (Grimes et a l . , 1977) and the ram ( L o i r and Lanneau, 1978a). T h i s group of b a s i c p r o t e i n s i s t r a n s i e n t l y a s s o c i a t e d with the spermatid nucleus d u r i n g the t r a n s i t i o n of chromatin from a nucleosome-like s t r u c t u r e to a smooth branching f i b r i l (Kierszenbaum and T r e s , 1975,1978; L o i r and Courtens, 1979). TP appears to be a s s o c i a t e d with the condensing chromatin of e l o n g a t i n g l a t e spermatids (Marushige and Marushige, 1975b) and i t s appearance has been l i n k e d to the i n c r e a s e d r e s i s t a n c e of chromatin to mechanical d i s r u p t i o n ( M e i s t r i c h et a l . , 1976; L o i r and Lanneau, 1978b). It i s presumed that t h i s complex s e r i e s of nuclear p r o t e i n changes d u r i n g mammalian spermiogenesis has a r o l e i n the ordered condensation of the spermatid chromatin ( B e l l v e , 1979). Changes i n nuclear b a s i c p r o t e i n s have been l i n k e d to sperm chromatin condensation i n other organisms as w e l l . High r e s o l u t i o n s t u d i e s of spermiogenesis i n the newt P l e u r o d e l e s w a l t l have r e v e a l e d that very a r g i n i n e - r i c h p r o t e i n s are a s s o c i a t e d with the condensed c e n t r a l p o r t i o n of the e l o n g a t i n g spermatid nucleus, while s o m a t i c - l i k e h i s t o n e s are s t i l l found i n the uncondensed chromatin network which forms a "crown" around the p e r i p h e r y of the nucleus ( P i c h e r a l and Bassez, I971a,b). During sperm nuclear e l o n g a t i o n in the house c r i c k e t , Acheta domestica, there appears to be a random l o s s of nucleosomes from chromatin f i b r e s . They 13 disappear completely by the onset of l a t e spermiogenesis when un u s u a l l y s t r a i g h t , t h i c k chromatin s t r u c t u r e s appear (McMaster-Kaye and Kaye, 1980). Late e l o n g a t i n g spermatids show a c y t o c h e m i c a l l y d e t e c t a b l e increase i n a r g i n i n e (Kaye and McMaster-Kaye, 1975) and a t r a n s i t i o n i n p r o t e i n s has been r e v e a l e d by e l e c t r o p h o r e s i s (Kaye et a l . , 1978). Sperm chromatin condensation i s not always accompanied by l o s s of nucleosomes. Gusse and C h e v a i l l i e r (1980) have noted the p e r s i s t e n c e of nucleosome-like g l o b u l a r s t r u c t u r e s i n the chromatin of d o g f i s h sperm, even though spermiogenesis in t h i s animal i n v o l v e s a s e r i e s of b a s i c p r o t e i n t r a n s i t i o n s , from somatic h i s t o n e s to p r o t a m i n e - l i k e p r o t e i n s and f i n a l l y to s t a b l e protamines. Nuclease d i g e s t i o n of sperm chromatin of T r i t u r u s v u l g a r i s (newt) and Bufo bufo (toad), both of which c o n t a i n p r o t a m i n e - l i k e sperm h i s t o n e s as w e l l as some somatic h i s t o n e s , a l s o produced d i s c r e t e fragments i n d i c a t i v e of a nucleosome-like s u b s t r u c t u r e (Kharchencko and N a l i v a e v a , 1979). M i c r o c o c c a l nuclease d i g e s t s , combined with e l e c t r o n microscopy, have shown that nucleosomal s u b s t r u c t u r e i s r e t a i n e d i n the sperm of sea u r c h i n s , ' which c o n t a i n a complement of s o m a t i c - l i k e h i s t o n e s (Spadafora et a l . , 1976). However, a novel c u t t i n g p e r i o d i c i t y of 500 base p a i r s has been found when sperm chromatin i s d i g e s t e d with DNase I ( A r c e c i and Gross, 1980b). DNase I d i g e s t i o n of Rana sperm chromatin a l s o r e v e a l e d a nucleosomal. s u b s t r u c t u r e with a longer repeat length than that of l i v e r chromatin (Kharchencko and N a l i v a e v a , 1979). Both sea u r c h i n s and Rana c o n t a i n sperm-14 s p e c i f i c H1 v a r i a n t s . The former i s both l a r g e r and more b a s i c than somatic H1 v a r i a n t s (Brandt et. a l . , 1979). I t may be r e s p o n s i b l e f o r the compaction of nucleosomes i n such a way that they are more r e s i s t a n t to DNase I d i g e s t i o n and may a l s o be, i n p a r t , r e s p o n s i b l e f o r the condensation of sperm chromatin. X-ray d i f f r a c t i o n s t u d i e s i n d i c a t e that h i s t o n e c r o s s - l i n k s are present i n sperm which r e t a i n a s o m a t i c - l i k e complement of h i s t o n e s , although chromatin appears to have a much l o o s e r conformation than i n sperm which c o n t a i n more b a s i c p r o t e i n s (Subirana et a l . , 1975). Most of the evidence presented here s t r o n g l y i m p l i e s that s p e r m - s p e c i f i c b a s i c p r o t e i n s are i n t i m a t e l y i n v o l v e d i n the condensation of chromatin. In f a c t , the n u c l e i of crab sperm, which do not c o n t a i n b a s i c p r o t e i n s , f a i l to condense (Vaughn and Hinsch, 1972). The q u e s t i o n that remains, however, i s why such a d i v e r s i t y of sperm h i s t o n e types has evolved i f t h e i r only f u n c t i o n i s to maintain the chromatin of sperm i n a condensed s t a t e u n t i l a f t e r f e r t i l i z a t i o n . One suggestion i s that the type of b a s i c p r o t e i n c o u l d be r e l a t e d to the general p a t t e r n of chromatin condensation and, u l t i m a t e l y , to the shape of the sperm nucleus (Fawcett et a_l. , 1971). However there appears to be l i t t l e c o r r e l a t i o n between the p a t t e r n of chromatin condensation i n the sperm nucleus and the type of sperm h i s t o n e p r e s e n t . Walker (1971), i n a comprehensive s e r i e s of s t u d i e s on the f i n e s t r u c t u r e of sperm chromatin condensation i n s e v e r a l organisms, found that there were three b a s i c p a t t e r n s . In the f i r s t type, 15 the " f i b r o u s " p a t t e r n , f i b r e s or f i l a m e n t s occur i n the e a r l y spermatid nucleus and g r a d u a l l y become t h i c k e r , f u s i n g to produce the mature sperm head. The second or " l a m e l l a r " type of condensation i n v o l v e s chromatin f i b r e s which fuse i n t o sheets or l a m e l l a e as spermiogenesis proceeds. In the " g r a n u l a r " type the spermatid nucleus undergoes a l l a l t e r a t i o n s i n shape before the onset of chromatin condensation. Chromatin i n i t i a l l y has a f i n e l y g r a n u l a r appearance, but a f t e r n uclear e l o n g a t i o n i s complete i t clumps i n t o c o a r s e r granules which e v e n t u a l l y c o a l e s c e to form the compact n u c l e o p r o t e i n of the mature nucleus. Sperm of the f r o g Xenopus and the r o o s t e r G a l l u s domesticus both demonstrate the g r a n u l a r form of chromatin condensation (Walker and MacGregor, 1976), but Xenopus has an intermediate type of sperm h i s t o n e (Bois and Kasinsky, 1972, 1973), while r o o s t e r sperm c o n t a i n a protamine (Nakano et a l . , 1976a). Sperm of the welk N u c e l l a c o n t a i n a protamine-type of h i s t o n e but d i s p l a y the l a m e l l a r -p a t t e r n of chromatin condensation (Walker, 1971). Likewise, no c o r r e l a t i o n appears to e x i s t between the f i n a l shape of the sperm nucleus and the type of sperm h i s t o n e . Bloch (1969) notes that sperm heads bearing s o m a t i c - l i k e h i s t o n e s may be s p h e r i c a l ( t o a d f i s h ) , elongate (Rana) or filamentous ( N i t e l l a , a p l a n t ) . Sperm c o n t a i n i n g protamines or h i g h l y a r g i n i n e - r i c h p r o t e i n s may be ovate ( b u l l ) , f l a t t e n e d (guinea p i g ) , elongate (Notophthalmus, a newt) or filamentous (grasshopper). T i g h t packing of sperm chromatin c o u l d be r e l a t e d to i n h i b i t i o n of t r a n s c r i p t i o n i n the maturing sperm nucleus or to 16 p r o t e c t i o n of sperm DNA from g e n e t i c damage. A l l mature sperm n u c l e i are u s u a l l y t r a n s c r i p t i o n a l l y i n e r t . In mammalian spermiogenesis, the c e s s a t i o n of RNA t r a n s c r i p t i o n and the condensation of chromatin occur at approximately the same stage in spermatid development (Kierszenbaum and T r e s , 1978; L o i r and Courtens, 1979). However, in i n the marine worm, U r e c h i s caupo, t r a n s c r i p t i o n ceases as e a r l y as the spermatocyte stage while the appearance of protamine occurs much l a t e r , i n the spermatid stage (Das et al_., 1967). In Xenopus l a e v i s , t r a n s c i p t i o n c o n t i n u e s w e l l i n t o the spermatid stage while s p e r m - s p e c i f i c b a s i c p r o t e i n s begin to make t h e i r appearance w e l l before t h i s stage ( K a l t , 1979; R i s l e y , 1977). In a d d i t i o n , many other c e l l types, such as avian e r y t h r o c y t e s , have n u c l e i which are t r a n s c r i p t i o n a l l y i n a c t i v e . Histone v a r i a n t s (e.g. H5) are found i n these n u c l e i , but gene i n a c t i v a t i o n per se does not seem to depend on the presence of h i g h l y b a s i c p r o t e i n s such as those found i n many sperm n u c l e i . F a i l u r e to f i n d a simple c o r r e l a t i o n between sperm h i s t o n e type and the e f f e c t s of chromatin condensation may be a s s o c i a t e d with the f a c t that packaging and p r o t e c t i o n of sperm DNA may be r a t h e r u n s p e c i f i c r o l e s . I f so, then why has packing of the sperm nucleus not been achieved simply by i n c r e a s i n g the r e l a t i v e amounts of somatic h i s t o n e s ? One p o s s i b l e e x p l a n a t i o n i s t h a t i f t h i s mechanism was employed, the egg would r e q u i r e a method of removing excess h i s t o n e s from the male genome, thereby exposing i t s own h i s t o n e s to the danger o f . d e g r a d a t i o n . Since sperm n u c l e i c o n t a i n p e c u l i a r p r o t e i n s , s p e c i f i c enzymes 17 f o r t h e i r removal may have been developed which would pose no t h r e a t to the s t r u c t u r e of the egg chromatin (Marushige and Marushige, 1975a). Another p o s s i b i l i t y i s that i t i s necessary to r e p l a c e the p r e - e x i s t i n g DNA-bound p r o t e i n s of t e s t i s c e l l s with s p e r m - s p e c i f i c p r o t e i n s so t h a t , when they are removed, the male DNA becomes a " t a b u l a r a s a " upon which the p a t t e r n of h i s t o n e s and non-histone p r o t e i n s can be r e -e s t a b l i s h e d under the cytoplasmic c o n t r o l of the f e r t i l i z e d ovum ( O l i n s et a l . , 1968). It would appear, t h e r e f o r e , that replacement of at l e a s t one of the somatic h i s t o n e s by a s p e r m - s p e c i f i c p r o t e i n may be necessary d u r i n g spermiogenesis. Yet none of the hypotheses o u t l i n e d above have been ab l e to e x p l a i n the s e l e c t i v e p r e s s u r e s which l e d to the remarkable d i v e r s i t y of sperm h i s t o n e types found i n nature. In an attempt to d i s c o v e r the e v o l u t i o n a r y s i g n i f i c a n c e of t h i s d i v e r s i t y , Bloch (1969, 1976) compiled a systematic catalogue of a l l the organisms whose sperm h i s t o n e s had been c h a r a c t e r i z e d . From the i n f o r m a t i o n a v a i l a b l e , he concluded that there was no r e l a t i o n s h i p between sperm h i s t o n e type and the l e n g t h of sperm storage time, the mode of f e r t i l i z a t i o n ( e x t e r n a l vs i n t e r n a l ) or the f e r t i l i z a t i o n medium ( f r e s h water vs s a l t water). He a l s o concluded that "even though a p h y l o g e n e t i c r e l a t i o n s h i p i s o f t e n apparent w i t h i n t i g h t l y d e f i n e d taxonomic groups (e.g. c l u p e i d s and e u t h e r i a n s ) , there seems to be no e v o l u t i o n a r y t r e n d . Most of the c l a s s e s of sperm h i s t o n e s are represented i n most of the broad tax a " . 18 Bloch's (1969) statement addresses i t s e l f to the o v e r a l l e v o l u t i o n of sperm h i s t o n e s i n both p l a n t s and animals. However comparative a n a l y s i s of sperm h i s t o n e d i v e r s i t y between c l o s e l y r e l a t e d taxonomic groups was f a r from complete when he s t a t e d that no p h y l o g e n e t i c r e l a t i o n s h i p s were apparent. The e x i s t i n g l i t e r a t u r e d e a l i n g with sperm h i s t o n e s i n the v e r t e b r a t e s , where e v o l u t i o n a r y h i s t o r y has been reasonably w e l l documented, focuses almost e x c l u s i v e l y upon t e l e o s t f i s h and e u t h e r i a n mammals, to the v i r t u a l n e g l e c t of other c l a s s e s i n t h i s phylum. Boi s (1972) and Kasinsky et a l . (1978) have s t a r t e d to expand the catalogue of sperm h i s t o n e s i n the v e r t e b r a t e s , c o n c e n t r a t i n g on c a r t i l a g i n o u s f i s h , amphibians and r e p t i l e s . The r e s u l t s have i n d i c a t e d that an i n t e r e s t i n g e v o l u t i o n a r y t r e n d may be apparent i n the v e r t e b r a t e s . Among the amphibians, as i n the t e l e o s t f i s h , a v a r i e t y of sperm h i s t o n e types are d i s p l a y e d , while the sperm of v a r i o u s r e p t i l e s appear to c o n t a i n only a p r o t a m i n e - l i k e p r o t e i n . Sperm h i s t o n e s of e u t h e r i a n mammals a l s o f a l l i n t o a s i n g l e c l a s s , the s t a b l e protamines. Therefore, i n c o n t r a s t to Bloch's (1969) statement, i t would appear that i n v e r t e b r a t e e v o l u t i o n there has been a t r e n d away from sperm h i s t o n e d i v e r s i t y toward a more narrow range of p r o t e i n types. In the present study, I have endeavored to expand the catalogue of sperm h i s t o n e s , using both cy t o c h e m i c a l and b i o c h e m i c a l techniques. S p e c i a l a t t e n t i o n has been given here to the v e r t e b r a t e groups whose sperm h i s t o n e s have been l e s s w e l l s t u d i e d i n the p a s t , namely the amphibians (anurans and 19 u r o d e l e s ) , r e p t i l e s (snakes and l i z a r d s ) and b i r d s . I have a l s o attempted to c o r r e l a t e the appearance of sperm h i s t o n e d i v e r s i t y i n the t e l e o s t f i s h and amphibians, and t h e i r r e l a t i v e constancy i n the "higher" v e r t e b r a t e groups, with phenomena such as chromatin condensation and shaping of the sperm nucleus, and the occurance of heteromorphic sex chromosomes and i n t e r n a l f e r t i l i z a t i o n i n v e r t e b r a t e phylogeny. 20 I I . DIVERSITY OF SPERM HISTONES IN THE GENUS XENOPUS AND OTHER ANURA A. INTRODUCTION T e s t i s - s p e c i f i c b a s i c p r o t e i n s of v a r i o u s f r o g genera demonstrate a great deal of d i v e r s i t y . The sperm of Rana c o n t a i n only a s o m a t i c - l i k e complement of h i s t o n e s (Alder and Gorovsky, 1975; Z i r k i n , 1970, 1971a; Kharchencko and N a l i v a e v a , 1979), while, c y t o c h e m i c a l l y , members of the genera Hyla, Bufo and Xenopus show int e r m e d i a t e types of sperm h i s t o n e s (Bois and Kasinsky, 1972). The e l e c t r o p h o r e t i c p r o f i l e s of t e s t i s - s p e c i f i c b a s i c p r o t e i n s of Hyla, Bufo and Xenopus are, however, q u i t e d i f f e r e n t from each other (Bois and Kasinsky, 1973) and s t a r c h g e l e l e c t r o p h o r e s i s has even r e v e a l e d d i f f e r e n c e s between congeneric s p e c i e s of Scaphiopus and Xenopus (Kasinsky et a l . , 1978). In l i g h t of these o b s e r v a t i o n s , i t was of i n t e r e s t to examine s e v e r a l s p e c i e s from a s i n g l e genus to determine i f t e s t i s - and sperm-s p e c i f i c b a s i c p r o t e i n s c o u l d be used as p o s s i b l e molecular markers for i n d i v i d u a l s p e c i e s of Anura. Of the sperm- and t e s t i s - s p e c i f i c h i s t o n e s found i n Anura, those of Xenopus l a e v i s l a e v i s have been the most e x t e n s i v e l y c h a r a c t e r i z e d b i o c h e m i c a l l y . R i s l e y (1977) and R i s l e y and Eckhardt, (1975, 1981) have found s i x e l e c t r o p h o r e t i c a l l y d i s t i n c t b a s i c chromosomal p r o t e i n s s p e c i f i c to the l a t e r stages of spermiogenesis. Four of these, SP's 3,4,5, and 6, had e l e c t r o p h o r e t i c m o b i l i t i e s g r e a t e r than 21 those of the somatic h i s t o n e s i n a c i d / u r e a p o l y a c r y l a m i d e g e l s and were a l s o r e s o l v e d i n the phosphorylated forms. Bois and Kasinsky (1973) r e p o r t e d three bands m i g r a t i n g f a s t e r than h i s t o n e H4 on a c i d / u r e a g e l s : X1 (not found by R i s l e y ) , X2 (a doublet which corresponds to SP's 3-5) and X3 (corresponding to SP6). X2 and X3 continued to s t a i n with a l k a l i n e f a s t green a f t e r deamination of the g e l s , i n d i c a t i n g t h a t they were a r g i n i n e - r i c h p r o t e i n s . These two bands have a l s o been sub j e c t e d to amino a c i d a n a l y s i s (Huang, 1977; Huang et a l . , 1978) and have been found to c o n t a i n a high percentage of a r g i n i n e as w e l l as some l y s i n e and h i s t i d i n e . T h e r e f o r e they can be c a t e g o r i z e d as intermediate type sperm h i s t o n e s , i n Bloch's (1969) M y t i l u s c l a s s . SP1 and SP2 were s p e r m - s p e c i f i c b a s i c p r o t e i n s which migrated w i t h i n the somatic h i s t o n e region i n a c i d / u r e a g e l s ( R i s l e y , 1977), but f a s t e r than the h i s t o n e s i n polyacrylamide g e l s c o n t a i n i n g 0.375% T r i t o n X-100 and 2.5 M urea. Two v a r i a n t s of SP2 have been observed. I n d i v i d u a l f r o g s appeared to have e i t h e r SP2a or SP2b, or a 1:1 r a t i o of both. T h i s i s p o s s i b l y an example of a l l e l i c v a r i a t i o n w i t h i n the sperm chromatin b a s i c p r o t e i n s of Xenopus l a e v i s l a e v i s ( R i s l e y and Eckhardt, 1979c). Through the generous c o o p e r a t i o n of Dr. M. F i s c h b e r g of Geneva, eleven s p e c i e s of Xenopus and f i v e subspecies of Xenopus l a e v i s have been made a v a i l a b l e f o r t h i s study. T h i s i s a p o l y p l o i d genus with chromosome numbers ranging from 20 to 108. Many of these s p e c i e s and subspecies can be d i s t i n g u i s h e d 22 from one another on the b a s i s of the e l e c t r o p h o r e t i c m o b i l i t i e s of t h e i r serum albumins (Bisbee et a l . , 1977), l a c t a t e dehydrogenase p r o f i l e s (Vonwyl and F i s c h b e r g , 1980), mating c a l l s (Vigny, 1979), DNA content (Thiebaud and F i s c h b e r g , 1977) and karyotypes (Tymowska, 1976, 1977; Tymowska and F i s c h b e r g , 1973). X. t r o p i c a l i s and X. sp_. n. I l l ( Z a i r e ) are c l o s e l y r e l a t e d to each other (M. F i s c h b e r g , p e r s o n a l communication) but are p h y l o g e n e t i c a l l y more d i s t a n t to the other members of the genus (Bisbee et a l . , 1977; Tymowska, 1973). The present study i s an a n a l y s i s of i n t e r - and i n t r a -s p e c i f i c v a r i a b i l i t y of t e s t i s - and s p e r m - s p e c i f i c b a s i c p r o t e i n s i n the genus Xenopus and other anuran genera, u s i n g c y t o c h e m i s t r y as w e l l as p o l y a c r y l a m i d e and s t a r c h g e l e l e c t r o p h o r e s i s . The p r o t e i n s f o r e l e c t r o p h o r e s i s were i s o l a t e d by a micromethod which allows f o r examination of a s i n g l e t e s t i s from a s i n g l e i n d i v i d u a l , and from spermatid/sperm p r e p a r a t i o n s using pooled t e s t e s from two animals. P r e l i m i n a r y f i n d i n g s have been presented (Kasinsky et a l . , 1979; Kasinsky et a l . , 1980; Mann et a l . , 1980). 23 B. MATERIALS AND METHODS J_ . Experimental Animals S e x u a l l y mature males of the toad Bufo marinus and the t r u e frogs Rana c a t e s b e i a n a and Rana p i p i e n s were obtained from Mogul-Ed, Oshkosh, Wisconsin. The f o l l o w i n g animals were obtained from Camosun Aqua r i a , Vancouver, B.C., through the c o o p e r a t i o n of Ms. J . T a i t : the f r o g s Rana p r e t i o s a and Rana  c l a m i t a n s ; the toads Bufo a l v a r i u s , Bufo boreas, Bufo bufo  h a l o p h i l u s and Bufo punctatus; the t r e e f r o g s Hyla r e g i l l a and Hyla g r a t i o s a ; and the spadefoot toads Scaphiopus couchi and Scaphiopus bombifrons. I d e n t i t i e s were checked from standard sources (Conant, 1975; S t e b b i n s , 1966). Animals, obtained i n l a t e s p r i n g or e a r l y summer, were maintained i n the l a b o r a t o r y f o r no longer than one week. They were e t h e r i z e d and d i s s e c t e d immediately. Organs used f o r e l e c t r o p h o r e s i s were frozen on dry i c e and s t o r e d at -70°C, or f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n f o r c y t o c h e m i s t r y . S e x u a l l y mature Xenopus l a e v i s l a e v i s males were o r i g i n a l l y o b t ained from the South A f r i c a n Snake Farm, F i s h Hoek, Cape, South A f r i c a . These animals have been maintained i n c a p t i v i t y f o r s e v e r a l years i n accordance with the procedures of Care of Experimental Animals- A Guide f o r Canada (Canadian C o u n c i l of Animal Care, Ottawa, O n t a r i o ) . Animals were e t h e r i z e d and d i s s e c t e d promptly. The e x c i s e d organs were frozen immediately on dry i c e and s t o r e d at -70°C. R e p r e s e n t a t i v e s of each of eleven s p e c i e s of the genus 24 Xenopus and f i v e subspecies of Xenopus l a e v i s were generously donated by Dr. M. F i s c h b e r g , from h i s amphibian colony i n Geneva. The animals were d i v i d e d i n t o three sets as f o l l o w s : Sets #J_ and #2: Two s e x u a l l y mature males r e p r e s e n t i n g each of four Xenopus l a e v i s subspecies and e i g h t Xenopus s p e c i e s were d i s s e c t e d i n Geneva. Test e s were e x c i s e d from animals a n a e s t h e t i z e d i n 0.5% MS222. Both t e s t e s from the animals of set #1 and one t e s t i s from each of set #2 were immediately froz e n i n l i q u i d a i r and hand t r a n s p o r t e d , on dry i c e , from Geneva to Vancouver, i n the passenger compartment of an a i r p l a n e . They were s t o r e d at -70°C f o r f u t u r e e l e c t r o p h o r e t i c a n a l y s i s . The second set of t e s t e s from animal set #2 was f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n f o r cytochemical i n v e s t i g a t i o n . Set #3: Two male r e p r e s e n t a t i v e s of each of f i v e Xenopus l a e v i s subspecies and eleven Xenopus s p e c i e s were t r a n s p o r t e d a l i v e i n the passenger compartment of an a i r p l a n e , from Geneva to New York C i t y . A l l s u r v i v e d except f o r s i n g l e specimens of X. _1. bunyoniensis, X. .1. sudanensis, X. v e s t i t u s and X. t r o p i c a l i s . Frogs were kept f o r a few days i n d e i o n i z e d water at C o r n e l l M e d i c a l C o l l e g e , i n the l a b o r a t o r y of Dr. M. R i s l e y , and were fed minced c a l f l i v e r . Animals were s a c r i f i c e d by d e c a p i t a t i o n and t e s t e s were 25 d i s s e c t e d out and immediately s u b j e c t e d to c o l l a g e n a s e d i s s o c i a t i o n as descibed below. Blood was a l s o c o l l e c t e d f o r a n a l y s i s of e r y t h r o c y t e h i s t o n e s . The o r i g i n s of Xenopus specimens from s e t s #1,#2 and #3 are shown i n Table I. Taxonomic r e l a t i o n s h i p s of a l l anuran s p e c i e s used i n t h i s study are i n d i c a t e d i n Table XI. C o n t r o l s f o r the cytochemical experiments i n c l u d e d mature t e s t e s from the rainbow t r o u t , Salmo g a i r d n e r i i , a g i f t of Dr. J . B a i l e y , the mussels M y t i l u s e d u l i s , c o l l e c t e d from Brocton P o i n t , Vancouver, B.C., and M y t i l u s c a l i f o r n i c u s , c o l l e c t e d near Bamfield, B.C., the spiny d o g f i s h , Squalus a c a n t h i u s , a g i f t of Dr. P. Ford, and the g o l d f i s h , C a r a s s i u s auratus, a g i f t of Dr. R. L i l e y . 2. Cytochemistry Cytochemical procedures f o l l o w e d those of Bois (1972). a. F i x a t i o n and embedding Test e s were f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n (Pearse, 1968, page 601) f o r 7-14 days. T i s s u e s were washed i n running tap water f o r 12-18 hours, dehydrated i n a graded s e r i e s of ethanols (2-3 hours i n each), c l e a r e d i n xylene and embedded i n T i s s u e P r e p ( F i s h e r S c i e n t i f i c Co.). S e c t i o n s (6-10 um) were cut on a Spencer "820" microtome. TABLE I . O r i q i n o f E x p e r i m e n t a l A n i m a l s f r o m t h e G e n u s X e n o p u s Number o f S p e c i e s Chromosomes S e t O r i g i n a " G e n e r a t i o n * 5 , ( m i t o s i s ) XENOPUS L A E V I S S U B S P E C I E S X. 1. l a e v i s ( D a u d i n ) 36 F i s h H o e k , C a p e , P - S o u t h A f r i c a  X. 1. p e t e r s i B o c a g e 36 #1S2 Z a m b i a F1 #3 • " ' F3 X. 1. v i c t o r i a n u s A h l 36 #1 K a m p a l a , U g a n d a ( E ) F1 #2 K i s i i z i , U g a n d a (W) F1 #3 Mbyue, Rwanda : F1 X. 1. b u n y o n i e n s i s L o v e r i d g e 36 #1&2 L a k e L u h o n d a , Rwanda P #3 L a k e B u l e r a , Rwanda F1 X. 1. s s p . n . d - 3 6 h * #1,2&3 M a l a w a i F l X. 1. s u d a n e n s i s P e r r e t 36 #3 G a l i m , C a m e r o o n F1 XENOPUS S P E C I E S X. c l i v i i P e r a c c a 36 #1&2 A d d i s A b b a b a , F l c -#3 E t h o o i a F2 X. rouelleri ( P e t e r s ) 36 #1&3 M a l a w i F l #2 I f a k a r a , T a n z a n i a F l X. b o r e a l i s ( P a r k e r ) 36 #1 M a r s a b i t , K e n y a P #2S3 M a r a l a l , K e n y a P & F1 X. f r a s e r l B o u l e n g e r 36 #1 Y a o u n d e , C a m e r o o n P #2S3 F o u l a s s i , C a m e r o o n F l X. s p . n . I I 3 6 h ' #3 E t h i o p i a F1 X. v e s t i t u s L a u r e n t 72 #1S3 L a k e M u t a n d a , U g a n d a F l . #2 Mbyue, Rwanda . F l X. w i t t e i e ' 72 #1&2 L a k e B u n y o n i , F l #3 U g a n d a P X. a m i e t i ^ * 72 #3 G a l i m , C a m e r o o n P X. r u w e n z o r i e n s i s 9 - 108 #1,2&3 S e m l i k i V a l l e y , Uganda F l X. t r o p i c a l i s ( G r a y ) 20 #1S2 I v o r y C o a s t F l #3 F2 X. s p . n . I l l 4 0 h - #3 Z a i r e F l b. P d e n o t e s a n i m a l s t a k e n f r c n t h e w i l d . e. T i n s e l y e t a l . , 1979 F l d e n o t e s f i r s t g e n e r a t i o n b r e d i n f . K o b e l e t ~ a l T 7 1990 c a p t i v i t y . g. F i s c h b e T g - S K o b e l , ( 1 9 7 8 ) c. O f f s p r i n g o f 2 c o u p l e s f r o m t h e same h. M. F i s c h b e r g , p e r s o n a l c o m m u n i c a t i o n p o p u l a t i o n . 27 b. Feulgen r e a c t i o n D e o x y r i b o n u c l e i c a c i d (DNA) was demonstrated by the Feulgen r e a c t i o n (Pearse, 1968, page 822). S e c t i o n s were hy d r o l y s e d f o r 11 min i n 1 N HC1 at 60°C. A f t e r two 5-min r i n s e s i n d i s t i l l e d water, s e c t i o n s were s t a i n e d i n S c h i f f ' s reagent f o r 30 min. T h i s was fo l l o w e d by many short r i n s e s i n d i s t i l l e d water, a 5-min r i n s e i n a c i d - b i s u l f i t e wash and two 5-min r i n s e s i n d i s t i l l e d water. S e c t i o n s were dehydrated i n a graded s e r i e s of e t h a n o l s , c l e a r e d i n xylene and mounted with Permount ( F i s h e r S c i e n t i f i c Co.). DNA was a l s o demonstrated by the Feulgen procedure a f t e r Bloch and Godman (1955) using 1 N TCA i n p l a c e of HC1. The two techniques gave comparable r e s u l t s . c. A l k a l i n e f a s t green r e a c t ion The presence of h i s t o n e s was demonstrated by the A l f e r t and Geschwind (1953) procedure. DNA was removed from s e c t i o n s by h y d r o l y s i s i n hot (85-90°C) 5% TCA f o r 15-20 min. A f r e s h reagent was used on each o c c a s i o n s i n c e repeated use of the same TCA s o l u t i o n r e s u l t e d i n incomplete removal of DNA, which i n turn l e d to reduced s t a i n i n g . Higher h y d r o l y s i s temperatures ( 9 0 - 1 0 0 ° 0 caused i r r e g u l a r i t i e s i n the s t a i n i n g p a t t e r n s of amphibian sperm ( B o i s , 1972). A l t e r a t i o n s i n nuc l e a r morphology and, to some extent, d e s t r u c t i o n of sperm n u c l e i , e s p e c i a l l y towards the edges of the s e c t i o n s , were observed i n t h i s higher temperature range. T h e r e f o r e , DNA was 28 removed at lower temperatures which l e f t these n u c l e i i n t a c t . The removal of DNA was checked by the Feulgen procedure. A f t e r h y d r o l y s i s the s e c t i o n s were immersed in three changes of 70% ethanol (10 min each) to remove TCA, r i n s e d i n d i s t i l l e d water and s t a i n e d f o r 30 min at room temperature i n 0.1% (w/v) Fast Green FCF ( F i s h e r S c i e n t i f i c Co.) made up i n 0.1 M HCl-borate b u f f e r , pH 8.2 (Prento and Lyon, 1977). T h i s a l k a l i n e f a s t green (AFG) s o l u t i o n was used immediately a f t e r p r e p a r a t i o n and the pH was measured both before and a f t e r s t a i n i n g to ensure minimum f l u c t u a t i o n . The pH remained constant at approximately 8.25. In .some cases s t a i n i n g was performed i n 0.1% aqueous f a s t green brought to pH 8.1-8.3 by t i t r a t i o n with 0.1 N NaOH. T h i s procedure produced more v a r i a b i l i t y i n pH (8.0-8.5), but l e s s u n s p e c i f i c s t a i n i n g of cytoplasm, than d i d the s t a i n made up i n borate b u f f e r . S t a i n i n g was f o l l o w e d by a 5-min r i n s e i n d i s t i l l e d water, dehydration and mounting as above. Since protamines were washed out by TCA treatment, DNA was a l s o removed with s a t u r a t e d p i c r i c a c i d f o r 6 hours at 60°C (Bloch and Hew, 1960a). The p i c r i c a c i d s o l u t i o n was prepared immediately before use. As with the TCA method, the h y d r o l y s i s reagent gave poor r e s u l t s i f reused. Two methods were t r i e d f o r removing p i c r i c a c i d from s e c t i o n s before s t a i n i n g . As recommended by Bois (1972), s e c t i o n s were r i n s e d 4-24 hours i n d i s t i l l e d water. However t h i s procedure r e s u l t e d i n reduced s t a i n i n g , perhaps due to the e x t r a c t i o n of b a s i c p r o t e i n s . 29 Three changes of 70% ethanol (10 min each), f o l l o w e d by a b r i e f r i n s e i n d i s t i l l e d water, appeared to remove p i c r i c a c i d and r e t a i n the s t a i n a b i l i t y of n u c l e i with AFG. S t a i n i n g was c a r r i e d out f o r 30 min as o u t l i n e d above. d. E o s i n Y Eosi n Y was used a f t e r p i c r i c a c i d h y d r o l y s i s (Bloch and Hew, 1960b) to c o r r o b o r a t e the AFG s t a i n i n g . The s l i d e s , f o l l o w i n g r i n s i n g a f t e r h y d r o l y s i s , were s t a i n e d with 0.1% (w/v) e o s i n Y ( F i s h e r S c i e n t i f i c Co.) b u f f e r e d at pH 8.1-8.3 with 0.1 M HCl-borate b u f f e r . The s e c t i o n s were s t a i n e d f o r 3 hours, r i n s e d i n d i s t i l l e d water f o r 5 min, dehydrated and mounted. e. Deamination To determine i f very a r g i n i n e - r i c h h i s t o n e s were present, deamination of l y s i n e r e s i d u e s was c a r r i e d out a f t e r TCA h y d r o l y s i s and p r i o r to s t a i n i n g with AFG (Bloch and Hew, 1960b). Two changes of n i t r o u s a c i d , prepared j u s t before use by combining equal volumes of 10% (w/v) TCA and 10% (w/v) sodium n i t r i t e , were used f o r 20 min each at room temperature. The stock s o l u t i o n s were made f r e s h on each oc c a s i o n as o l d s o l u t i o n s gave e r r a t i c r e s u l t s . The procedure o u t l i n e d by P i p k i n (1968), c o n s i s t i n g of two s u c c e s s i v e 15 min changes of f r e s h l y prepared n i t r o u s a c i d s o l u t i o n at 4°C, was a l s o t r i e d . However deamination was judged incomplete i n the 30 c o n t r o l Rana p i p i e n s s e c t i o n s . The deamination procedure gave i n c o n s i s t e n t r e s u l t s when used a f t e r p i c r i c a c i d h y d r o l y s i s , perhaps due to the incomplete removal of p i c r i c a c i d which appeared to i n h i b i t the r e a c t i o n . f_. A c e t y l a t ion A l s o , to determine whether very a r g i n i n e - r i c h h i s t o n e s were present, a c e t y l a t i o n of l y s i n e r e s i d u e s (Bloch and Hew, 1960b) was performed a f t e r p i c r i c a c i d or TCA h y d r o l y s i s and before s t a i n i n g with AFG or e o s i n Y. A f t e r h y d r o l y s i s , the s l i d e s were r i n s e d i n three changes of 70% e t hanol (10 min each), dehydrated in a b s o l u t e ethanol and t r a n s f e r r e d to a s o l u t i o n c o n t a i n i n g 1% (v/v) g l a c i a l a c e t i c a c i d i n pure a c e t i c anhydride. A c e t y l a t i o n was c a r r i e d out f o r 1 hour at 60°C. S l i d e s were r i n s e d i n two changes of a b s o l u t e ethanol (15 min each) and brought to water. To improve s p e c i f i c i t y , the s l i d e s were t r a n s f e r r e d to 0.1 M HCl-borate b u f f e r f o r 10 min before s t a i n i n g with AFG or e o s i n Y as above. g_. A l k a l i n e f a s t green r e a c t i o n without h y d r o l y s i s To t e s t f o r f r e e b a s i c p r o t e i n s , AFG s t a i n i n g was used without p r i o r h y d r o l y s i s to remove DNA. S e c t i o n s were brought to water, s t a i n e d f o r 30 min i n 0.1% aqueous AFG at pH 8.1-8.3, r i n s e d i n d i s t i l l e d water f o r 5 min, dehydrated and mounted. T h i s technique was a l s o c a r r i e d out using 0.1% f a s t green i n 0.1 M HCl-borate b u f f e r at pH 8.2, as recommended by 31 Prento and Lyon (1977) f o r general a l k a l i n e f a s t green s t a i n i n g . T h i s method gave unusual r e s u l t s , as noted by Bois (1972) with AFG made up i n other b u f f e r s . To determine whether these o b s e r v a t i o n s were due to s a l t c o n c e n t r a t i o n , and to see i f any d i f f e r e n c e s i n s t a i n a b i l i t y c o u l d be observed among the v a r i o u s anuran s p e c i e s , s e c t i o n s were s t a i n e d in AFG made up i n i n c r e a s i n g l y higher c o n c e n t r a t i o n s of NaCl. A range from no NaCl to 1.0 M NaCl was t r i e d . h. F e u l g e n - a l k a l i n e f a s t green procedure The method of Vaughn (1966) was fo l l o w e d to demonstrate non-DNA-associated b a s i c p r o t e i n s . S l i d e s were brought to water and h y d r o l y s e d f o r 25 min i n 1 N TCA at 60°C. S t a i n i n g i n TCA-S c h i f f ' s reagent (made up a c c o r d i n g to P i p k i n , 1968), f o r 45 min at room temperature, f o l l o w e d h y d r o l y s i s . Next, three 5-min r i n s e s i n s u l f i t e b l e a c h (prepared with TCA) and then three 10-min r i n s e s i n 70% eth a n o l were c a r r i e d out. S e c t i o n s were then s t a i n e d with a l k a l i n e f a s t green i n the normal manner. A f t e r s t a i n i n g the s l i d e s were d i f f e r e n t i a t e d f o r 5 min i n two r i n s e s of a b s o l u t e methanol, c l e a r e d i n xylene and mounted. i _ . Sakaquchi r e a c t i o n Protein-bound a r g i n i n e was demonstrated by the Sakaguchi r e a c t i o n as modified by D e i t c h (1961). Stock s o l u t i o n s of 4% (w/v) barium hydroxide and 1.5% (w/v) 2,4-dichloronaphthol i n t e r t i a r y butanol were made i n advance and used over a p e r i o d 32 of a month. Just before use, 1% sodium h y p o c h l o r i t e was prepared, using "AquA" h y p o c h l o r i t e ( M i r a c l e a n Products, Richmond, B.C.) as the source of 10% NaOCl. S l i d e s were brought to water, b l o t t e d and pl a c e d i n an empty C o p l i n j a r . Immediately the s t a i n i n g reagent was prepared (5 p a r t s barium hydroxide, 1 part h y p o c h l o r i t e , and then 1 pa r t d i c h l o r o n a p h t h o l ) and poured over the s l i d e s . S t a i n i n g took p l a c e at room temperature f o r 10-20 min. S l i d e s were then t r a n s f e r r e d through three changes of t e r t i a r y butanol c o n t a i n i n g 5% (v/v) t r i - N - b u t y l a m i n e . The f i r s t change was f o r 5 sec and the next two f o r 30 sec each. S l i d e s were c l e a r e d i n two changes (30 sec each) of xylene c o n t a i n i n g 5% (v/v) t r i - N -butylamine and mounted i n Permount c o n t a i n i n g 10% (v/v) t r i - N -butylamine. j . D i n i t r o f l u o r o b e n z e n e procedure Protein-bound l y s i n e was demonstrated by the d i n i t r o f l u o r o b e n z e n e (DNFB) procedure as o u t l i n e d by P i p k i n (1968). To remove formaldehyde, m a t e r i a l f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n was hyd r o l y s e d f o r 15 min i n 5% TCA at 86-89°C, or immersed i n b o i l i n g water f o r 5 min to ensure the r e t e n t i o n of a l l b a s i c p r o t e i n s . A f t e r three 10-min r i n s e s i n 70% e t h a n o l , the s e c t i o n s were s t a i n e d i n DNFB s o l u t i o n (0.6 ml DNFB i n 26-ml a b s o l u t e e t h a n o l , 4.0 ml 1 M sodium bi c a r b o n a t e , and 20 ml d i s t i l l e d water) f o r 30 min at room temperature. The reagent was used immediately a f t e r p r e p a r a t i o n . The s e c t i o n s were r i n s e d i n s e v e r a l changes 33 of 70% e t h a n o l , dehydrated, c l e a r e d i n xylene and mounted, k. C o n t r o l s As a c o n t r o l f o r h i s t o n e s t a i n i n g , s e c t i o n s were sub j e c t e d to TCA or p i c r i c a c i d h y d r o l y s i s and then t r e a t e d with 0.1 N HC1 f o r 15 hours to remove h i s t o n e s . S t a i n i n g with AFG was absent or very l i g h t . In a l l procedures, sperm from a number of d i f f e r e n t s p e c i e s were s t a i n e d at the same time to f a c i l i t a t e comparison. The f o l l o w i n g organisms, i n which the b a s i c p r o t e i n composition of the sperm i s known, were used as c o n t r o l s f o r most r e a c t i o n s : Salmo g a i r d n e r i i ( t r o u t ) , whose sperm c o n t a i n protamines ( I n g l e s et a l . , 1966); Rana p i p i e n s ( f r o g ) , which has sperm h i s t o n e s s i m i l a r to those found i n somatic c e l l s (Bloch, 1969; Z i r k i n , 1970); C a r a s s i u s auratus ( g o l d f i s h ) , whose sperm h i s t o n e s are s i m i l a r to those of R. p i p i e n s ( Z i r k i n , 1971b); and Squalus a c a n t h i u s ( d o g f i s h ) , whose sperm c o n t a i n " s t a b l e protamines" (Bois and Kasinsky, 1974). Sperm of Xenopus l a e v i s l a e v i s , which c o n t a i n h i s t o n e s of the intermediate type (Bois and Kasinsky, 1972), as w e l l as those of M y t i l u s e d u l i s and M y t i l u s c a l i f o r n i c u s (Subirana et a l . , 1973; Bloch, 1966), were a l s o used as c o n t r o l s . A l l photographs were taken on a L e i t z Orthomat-W photomicroscope, u s i n g an i n t e r f e r e n c e - c o n t r a s t p o l a r i z e r . 34 3_. Biochemistry a. Micromethod Histones from set #1 and #2 Xenopus specimens and other anurans were obtained from a c i d e x t r a c t s of t e s t i s c e l l suspensions as d e s c r i b e d i n Kasinsky et a l . (1978). S i n g l e t e s t e s , f r e s h or st o r e d at -70°C, were homogenized by hand using a Kontes D u a l l ground g l a s s homogenizer, with four volumes of i c e c o l d phosphate b u f f e r e d s a l i n e (PBS) (Dulbecco and Vogt, 1954). The c e l l suspension was checked by l i g h t microscopy to a s c e r t a i n the presence of sperm, which, i n a l l cases, made up the m a j o r i t y of c e l l types i n the homogenate. T h i s suspension was then f i l t e r e d with s u c t i o n onto a g l a s s f i b r e f i l t e r paper and r i n s e d with 70% eth a n o l which, a c c o r d i n g to Louie and Dixon (1972), f i x e s the c e l l s and s t r i p s them of t h e i r cytoplasm. Excess f i l t e r paper was trimmed away and the remainder was i n s e r t e d i n t o 200 u l of 0.4 N s u l f u r i c a c i d . A f t e r e x t r a c t i o n on i c e f o r 30 min, the e x t r a c t was removed with a flamed Pasteur p i p e t t e and 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 o v e r n i g h t at -20°C with four volumes of 95% e t h a n o l . The p r e c i p i t a t e was spun down i n an Eppendorf m i c r o c e n t r i f u g e (15,000 rpm, 10 min) and the ethanol removed. The samples were a i r d r i e d , r e d i s s o l v e d i n 0.9 N a c e t i c a c i d and s a t u r a t e d with urea f o r e l e c t r o p h o r e s i s . 35 b. Spermatid/sperm method i . D i s s o c i a t i o n of t e s t e s and s e p a r a t i o n of t e s t i s c e l l s : Four t e s t e s from each p a i r of set #3 Xenopus specimens were pooled and d i s s o c i a t e d a c c o r d i n g to the method of R i s l e y and Eckhardt (1979a). T e s t e s were cut i n t o small p i e c e s and s t r i r r e d g e n t l y at 30°C i n 15 ml of 0R2 s a l t s o l u t i o n (Wallace et a l . , 1973) c o n t a i n i n g 0.2% c o l l a g e n a s e (190-195 U/mg; Sigma Chemical Co.). D i s s o c i a t i o n time was 1-1.5 hours, u n t i l most t i s s u e fragments were d i s s o l v e d . Remaining fragments were removed with a Pasteur p i p e t t e and the c e l l suspension was then c e n t r i f u g e d at lOOOxg f o r 10 min at 4°C, using a S o r v a l l HB-4 r o t o r . C e l l p e l l e t s were resuspended i n 15 ml 15% Metrizamide (Nyegaard & Co. A/S, Oslo, Norway) i n OR2 ( c o n t a i n i n g 30 mM NaCl to compensate f o r the osmotic e f f e c t of Metrizamide) and o v e r l a y e r e d with 2 ml of OR2. E l o n g a t i n g spermatids and mature sperm were separated from somatic and other spermatogenic c e l l s by c e n t r i f u g a t i o n at 4000xg f o r 15 min. i i . I s o l a t i o n of n u c l e i and p r e p a r a t i o n of chromatin: F o l l o w i n g the method of R i s l e y and Eckhardt (1981), a l l op e r a t i o n s were performed at 4°C, u s i n g a S o r v a l l HB-4 r o t o r f o r a l l c e n t r i f u g a t i o n s . To i n h i b i t p r o t e o l y s i s , a l l s o l u t i o n s c o n tained 1 mM PMSF ( p h e n y l m e t h y l s u l f o n y l f l u o r i d e , f r e s h l y added from a 50 mM stock s o l u t i o n i n i s o p r o p a n o l ) , 0.1 mM TPCK 36 (L-1-tosylamide-2-phenylethyl chloromethyl ketone) and 0.1 mM TLCK (N-**--p-tosyl-L-lysine chloromethyl ketone; f r e s h l y added to warm s o l u t i o n s from 10 mM stock s prepared i n i s o p r o p a n o l and water, r e s p e c t i v e l y ) and 50 mM Na b i s u l f i t e (added j u s t p r i o r to use, without pH adjustment). The a d d i t i o n of Na b i s u l f i t e to s o l u t i o n s c o n t a i n i n g 15 mM T r i s lowers the pH to 6.4 ( R i s l e y and Eckhardt, 1981). 5% t h i o d i g l y c o l was added to suppress o x i d a t i o n of methionine r e s i d u e s ( A j i r o et a l . , 1978). Spermatid/sperm p r e p a r a t i o n s and e r y t h r o c y t e s were homogenized i n 0.25 M sucrose/3 mM magnesium c h l o r i d e / 1 5 mM T r i s using a T e f l o n - g l a s s homogenizer (5-6 s t r o k e s , 1500 rpm). N u c l e i were c o l l e c t e d by c e n t r i f u g a t i o n at I000xg f o r 10 min, resuspended i n 0.25 M sucrose/3 mM magnesium c h l o r i d e / 1 5 mM T r i s c o n t a i n i n g 1.0% T r i t o n X-100, and homogenized with a Dounce hand homogenizer with 5-6 s t r o k e s of the loose f i t t i n g p e s t l e . N u c l e i were c o l l e c t e d by c e n t r i f u g a t i o n as above and washed again i n the T r i t o n X-100 wash s o l u t i o n . I s o l a t e d n u c l e i were suspended i n 0.2 M NaCl/15 mM T r i s and a small sample was examined by phase-contrast microscopy to check f o r the presence of spermatid/sperm or e r y t h r o c y t e n u c l e i . Contamination by cytoplasm and n u c l e i of other c e l l types was minimal i n a l l cases. N u c l e i were washed twice i n the NaCl s o l u t i o n and once i n 10 mM EDTA/10 mM T r i s (pH 8.0) and c o l l e c t e d by c e n t r i f u g a t i o n at lOOOxg a f t e r the NaCl washes and at 4000xg a f t e r the EDTA wash. 37 Chromatin i n the f i n a l p e l l e t was swollen f o r 10-15 min i n 10% t h i o d i g l y c o l ( l a c k i n g protease i n h i b i t o r s ) and d i s p e r s e d by ge n t l e p i p e t t i n g with a Pasteur p i p e t t e . i i i . I s o l a t i o n of h i s t o n e s : Histones and sperm b a s i c p r o t e i n s were e x t r a c t e d by adding 4 N s u l f u r i c a c i d to chromatin suspensions to a f i n a l c o n c e n t r a t i o n of 0.4 N. A f t e r 1 hour of e x t r a c t i o n at 4°C, the a c i d i n s o l u b l e m a t e r i a l was p e l l e t e d by c e n t r i f u g a t i o n at 4000xg f o r 15 min. Supernatants were withdrawn and the p e l l e t s r e - e x t r a c t e d with 0.4 N s u l f u r i c a c i d f o r an a d d i t i o n a l hour. The a c i d e x t r a c t s were pooled and p r o t e i n was p r e c i p i t a t e d by the a d d i t i o n of 100% (w/v) t r i c h l o r o a c e t i c a c i d (TCA) to a f i n a l c o n c e n t r a t i o n of 20% (w/v). A f t e r 15 min at room temperature, the p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f u g a t i o n at 4000xg, 15 min, and s e q u e n t i a l l y washed i n acetone-HCl (0.33 ml c o n c e n t r a t e d HC1/200 ml acetone) and acetone, both c o n t a i n i n g 1% t h i o d i g l y c o l , and d r i e d ir\ vacuo. c. Whole t e s t i s method In the case of X. 1. bunyoniensis, X. 1. sudanensis, X. v e s t i t u s and X. t r o p i c a l i s , h i s t o n e s were i s o l a t e d from n u c l e i prepared from whole t e s t e s , without c o l l a g e n a s e d i s s o c i a t i o n and s e p a r a t i o n of the spermatid/sperm f r a c t i o n i n Metrizamide. Two t e s t e s (one p a i r from each animal) were homogenized d i r e c t l y i n 0.25 M sucrose/3 mM magnesium 38 c h l o r i d e / 1 5 mM T r i s , u s i n g a T e f l o n - g l a s s homogenizer. I s o l a t i o n of n u c l e i and p r e p a r a t i o n of chromatin and h i s t o n e s were performed as o u t l i n e d i n the spermatid/sperm method above, with a l l s o l u t i o n s c o n t a i n i n g p r o t e o l y t i c i n h i b i t o r s . d. C o n t r o l s T e s t i s - and s p e r m - s p e c i f i c h i s t o n e s of X. 1. l a e v i s were i s o l a t e d by the three methods o u t l i n e d above (micromethod, spermatid/sperm method and whole t e s t i s method), using e i t h e r f r e s h m a t e r i a l or m a t e r i a l s t o r e d at -70°C. R e s u l t s are o u t l i n e d i n Table VI. In a d d i t i o n to e x t r a c t i o n with 0.4 N s u l f u r i c a c i d , X. 1_. l a e v i s sperm b a s i c p r o t e i n s were a l s o e x t r a c t e d from chromatin with 5% (w/v) TCA f o r 20 min. TCA i n s o l u b l e m a t e r i a l 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 i n an Eppendorf m i c r o c e n t r i f u g e (15,000 rpm, 10 min) and the p e l l e t was r e - e x t r a c t e d with 0.4 N s u l f u r i c a c i d f o r 1 hour. P r o t e i n s were p r e c i p i t a t e d and d r i e d as above. E r y t h r o c y t e h i s t o n e s of set #3 Xenopus specimens were i s o l a t e d from chromatin by the method of R i s l e y and Eckhardt (1981). Frogs were b l e d i n t o c o l d (4°C) SSC (0.15 M NaCl, 15 mM Na c i t r a t e , pH 7.0) c o n t a i n i n g 1 mM PMSF, 0.1 mM TPCK, 50 mM Na b i s u l f i t e and 5% t h i o d i g l y c o l , and e r y t h r o c y t e s were c o l l e c t e d by c e n t r i f u g a t i o n at 2,000 rpm, 5 min, 4°C. I s o l a t i o n of n u c l e i and p r e p a r a t i o n of chromatin and h i s t o n e s were perfomed as o u t l i n e d i n the spermatid/sperm method d e s c r i b e d above. 39 X. 1. l a e v i s e r y t h r o c y t e h i s t o n e s , used as e l e c t r o p h o r e t i c markers, were the g i f t of Dr. M. R i s l e y . H e r r i n g protamine s u l f a t e (Sigma Chemical Co.; 1 mg/ml) was a l s o used as an e l e c t r o p h o r e t i c marker. e. E l e c t r o p h o r e s i s i . A c i d / u r e a p o l y a c r y l a m i d e g e l s : 15% p o l y a c r y l a m i d e s l a b g e l s c o n t a i n i n g 6.25 M urea were prepared a c c o r d i n g to the method of Panyim and C h a l k l e y (1969). Each g e l measured 20x15x0.15 cm and con t a i n e d 10 s l o t s . Pre-e l e c t r o p h o r e s i s was performed f o r 18-24 hours at 150 V cons t a n t , at room temperature, to remove p e r s u l f a t e and TEMED. P r o t e i n samples (75-100 u l ) , d i s s o l v e d i n 5% a c e t i c a c i d s a t u r a t e d with urea, were e l e c t r o p h o r e s e d at 150-200 V constant (8-10 mA) f o r 8-12 hours. Power was s u p p l i e d by a Heathkit Regulated H.V. power supply. i i . A c i d / u r e a / T r i t o n X-100 po l y a c r y l a m i d e g e l s : 12% p o l y a c r y l a m i d e g e l s c o n t a i n i n g 0.375% T r i t o n X-100 and 2.5 M urea were o v e r l a y e r e d with 0.375% T r i t o n X-100/2.5 M urea/5% a c e t i c a c i d , p r e - e l e c t r o p h o r e s e d overnight at 11 mA/slab to constant v o l t a g e and scavenged with 1 M cysteamine-HCl/5% a c e t i c a c i d f o r 1 hour at 200 V (Alfageme et al.,1974; Zweidler, 1978). P r o t e i n samples (25-60 ug) were d i s s o l v e d (1 mg/ml) i n 8 M urea/5% 2-40 mercaptoethanol and e l e c t r o p h o r e s e d at 190 V constant f o r 12 hours, u s i n g 5% a c e t i c a c i d as the running b u f f e r . i i i . S t a i n i n g of p o l y a c r y l a m i d e g e l s : Gels were s t a i n e d f o r 5 hours with 0.1% Coomassie Blue R-250 (Bio-Rad L a b o r a t o r i e s , Ca.) i n 25% methanol/7% a c e t i c a c i d (prepared from a 0.4% stock s o l u t i o n of the dye made up in absolute methanol). D e s t a i n i n g was by d i f f u s i o n i n 25% methanol/7% a c e t i c a c i d . Gels were a l s o s t a i n e d i n 0.2% Amido Black (A n a l i n e Blue Black; Matheson, Coleman and B e l l , Ohio) i n 30% methanol/10% a c e t i c a c i d , 5 hours to o v e r n i g h t . They were de s t a i n e d i n s e v e r a l changes of 30% methanol/10% a c e t i c a c i d and photographed, then r e s t a i n e d 2-5 hours i n 0.2% Amido Black. Restained g e l s were then d e s t a i n e d by the s e n s i t i v e method of Wray and S t u b b l e f i e l d (1970), i n 1 M s u l f u r i c a c i d / 3 M urea, which enhances s t a i n i n g approximately t e n - f o l d . Densitometer t r a c i n g s of each channel were taken on a Quickscan J u n i o r Densitometer (Helena I n d u s t r i e s , Beaumont, Texas) at 610 nm. i v . S t a r c h g e l s : P r e p a r a t i o n of t i s s u e s and e l e c t r o p h o r e s i s of t e s t i c u l a r b a s i c p r o t e i n s i n s t a r c h g e l s i s o u t l i n e d i n Kasinsky et a l . (1978), f o l l o w i n g the micromethod of Louie . and Dixon (1972). A s i n g l e t e s t i s from a s i n g l e animal was hand homogenized i n phosphate b u f f e r e d s a l i n e (PBS) and f i l t e r e d 41 onto a g l a s s f i b r e f i l t e r paper over an area the s i z e of a s t a r c h g e l sample s l o t (5X5 mm). The c e l l s were f i x e d with 70% ethanol which, a c c o r d i n g to Louie and Dixon (1972), s t r i p s most of them of t h e i r cytoplasm. F i l t e r papers were then p l a c e d i n t o sample s l o t s f i l l e d with 0.4 N s u l f u r i c a c i d and allowed to s i t in the a c i d f o r 15-30 min f o r e x t r a c t i o n of b a s i c p r o t e i n s . The f i l t e r papers remained i n the s l o t s f o r the remainder of the run, as c o n t r o l experiments i n d i c a t e d that t h e i r presence d i d not a l t e r the m o b i l i t i e s of the p r o t e i n s . S t a r c h g e l s c o n t a i n i n g 4 M urea were prepared a c c o r d i n g to the method of Sung and Smithies (1969) (0.5X12X25 cm, pH 3.4). Basic p r o t e i n s , . e x t r a c t e d as d e s c r i b e d above, were e l e c t r o p h o r e s e d at 250 v o l t s f o r 15 hours, at 4°C. At the completion of the run, the g e l s were b i s s e c t e d h o r i z o n t a l l y and s t a i n e d by the s e n s i t i v e c o b a l t Amido Black technique of Sung and Smithies (1969) (0.125% Amido Black i n 1% a c e t i c a c i d c o n t a i n i n g 0.6 mM c o b a l t n i t r a t e ) . D e s t a i n i n g was performed i n d i l u t e s u l f u r i c a c i d . According to these authors, t h i s technique i s over 100-fold more s e n s i t i v e than c o n v e n t i o n a l use of Amido Black. Pea s e e d l i n g and c a l f thymus h i s t o n e H4 ( g i f t s of Drs. D. Fambrough and J . Bonner) and t r o u t t e s t i s protamine and high m o b i l i t y group p r o t e i n T ( g i f t s of Dr. G. Dixon) were used as e l e c t r o p h o r e t i c markers. 42 C. RESULTS J_. Cytochemistry a. C o n t r o l s Sperm of the rainbow t r o u t , Salmo g a i r d n e r i i , c o n t a i n a r g i n i n e - r i c h protamines ( I n g l e s et a l . , 1966). These c e l l s s t a i n e d i n t e n s e l y with Feulgen ( F i g . l A ) . However, a f t e r h y d r o l y s i s of DNA i n 5% TCA, somatic c e l l s s t a i n e d i n t e n s e l y with a l k a l i n e f a s t green (AFG) while sperm d i d not s t a i n ( F i g . l B ) . T h i s i s c h a r a c t e r i s t i c of p r o t a m i n e - c o n t a i n i n g sperm ( A l f e r t , 1956). Protamines were r e t a i n e d when s e c t i o n s were hy d r o l y s e d i n p i c r i c a c i d ( F i g . l C ) , and sperm continued to s t a i n with AFG a f t e r a c e t y l a t i o n while somatic c e l l s d i d not. Trout sperm gave a very strong r e a c t i o n with the Sakaguchi t e s t f o r a r g i n i n e ( F i g . I D ) , but d i d not s t a i n with DNFB f o r l y s i n e . Sperm of the d o g f i s h , Squalus a c a n t h i u s , c o n t a i n s t a b l e protamines l i k e those found i n mammalian sperm (Bois and Kasinsky, 1974). Sperm n u c l e i s t a i n e d with AFG a f t e r TCA and p i c r i c a c i d h y d r o l y s i s , and continued to s t a i n a f t e r deamination or a c e t y l a t i o n of the s e c t i o n s (data not shown). They r e a c t e d s t r o n g l y with the Sakaguchi t e s t , but f a i n t l y with DNFB, i n d i c a t i n g that a r g i n i n e - r i c h p r o t e i n s were present i n these n u c l e i . The sperm h i s t o n e s of M y t i l u s e d u l i s and M y t i l u s  c a l i f o r n i c u s are of the i n t e r m e d i a t e type, c o n t a i n i n g both l y s i n e and a r g i n i n e (Subirana et a l . , 1973; Bloch, 1966). While 43 F i g u r e 1. S t a i n i n g of t e s t i s s e c t i o n s from c o n t r o l animals. A-D. Salmo q a i r d n e r i i : A. Feulgen. B. AFG a f t e r h y d r o l y s i s 'in 5% TCA, 85-90°C, 15 min. C. AFG a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 h r . D. Sakaguchi. E-H. M y t i l u s e d u l i s : E. Feulgen. F. AFG a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. G. AFG a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1 h r ) . H. Sakaguchi. I-L. Rana p i p i e n s : I. Feulgen. J . AFG a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. K. AFG a f t e r TCA h y d r o l y s i s and deamination. L. Sakaguchi. AFG= TCA= a l k a l i n e f a s t green t r i c h l o r o a c e t i c a c i d 44 45 sperm s t a i n e d i n t e n s e l y with Feulgen ( F i g . l E ) , these n u c l e i d i d not s t a i n with AFG a f t e r h y d r o l y s i s of s e c t i o n s i n hot TCA ( F i g . l F ) . They d i d s t a i n with AFG a f t e r p i c r i c a c i d h y d r o l y s i s , but only f a i n t l y i f the s e c t i o n s had been a c e t y l a t e d ( F i g . l G ) , i n d i c a t i n g the presence of l y s i n e - r i c h r a t h e r than a r g i n i n e -r i c h p r o t e i n s . T h i s was c o r r o b o r a t e d by the r e a c t i o n of these sperm n u c l e i with DNFB, and by t h e i r f a i n t r e a c t i o n with the Sakaguchi t e s t ( F i g . l H ) . C y t o c h e m i c a l l y , the sperm of Rana p i p i e n s behaved l i k e somatic c e l l s ( Z i r k i n , 1970). They s t a i n e d i n t e n s e l y with Feulgen (Fig.11) and with AFG a f t e r h y d r o l y s i s i n hot TCA ( F i g . U ) or p i c r i c a c i d . However, when h y d r o l y s e d s e c t i o n s were deaminated (Fig.IK) or a c e t y l a t e d , sperm d i d not s t a i n with AFG. They d i d s t a i n with DNFB, but re a c t e d only f a i n t l y i n the Sakaguchi t e s t ( F i g . 1 L ) . S i m i l a r r e s u l t s were ob t a i n e d with sperm of the g o l d f i s h , C a r a s s i u s auratus (data not shown), a l s o known to c o n t a i n s o m a t i c - l i k e h i s t o n e s ( Z i r k i n , 1971b). Cytochemical data f o r the sperm of the c o n t r o l animals i s summarized i n Table I I . Pro t a m i n e - c o n t a i n i n g sperm; as seen i n Salmo g a i r d n e r i i , were c l a s s i f i e d as type 1. Sperm c o n t a i n i n g s t a b l e protamines (Squalus a c a n t h i u s ) were termed type 2. Intermediate-type sperm ( M y t i l u s ) were type 3, while sperm which behaved l i k e somatic c e l l s (Rana) were c l a s s i f i e d as type 4. Table I I . Cytochemistry of Sperm Nuclei of C o n t r o l Animals Staining & Pretreatment Reactive Material Salmo n a i r d n c r l i Sqnalus acanth.Lus Organism Used M y t i l u s cduU.n M y t i l u s c a l i f o r n i c u n Rana pi r i i f.nn Ca r r a s i u s Feulgen AFG-TCA, 85°-90°C, AFG-TCA, deamination AFG, p i c r i c a c i d , 60°C. AFG, p i c r i c a c i d , 60°C, acetylat ion Eosin Y, p i c r i c a c i d Eosin Y, p i c r i c a c i d , acetylation Sakaguchi AFG, no hydrolysis AFG after Feulgen DNA Basic proteins other than protamines Basic proteins r i c h In arginine Basic proteins including protamines Basic proteins r i c h in arginine Basic proteins including protamines Basic proteins r i c h in arginine Protein-bound arginine Protein-bound lysine Non-nucleic a c i d associated basic proteins Non-DNA-associated basic proteins Sperm histone type Reference A l f e r t , 1956; Drance et a l . , 1975 Bois & Kasinsky, 1974 Subirana et a l . , -T977 Bloch, 1966 Z i r k i n , 1970 Z i r k i n , 19 71b O N 47 b. X e n o p u s The t e s t e s of a l l s p e c i e s and subspecies of the genus Xenopus were s i m i l a r i n s t r u c t u r e . As i n X. 1. l a e v i s , they were o r g a n i z e d i n t o s e miniferous t u b u l e s c o n t a i n i n g c y s t s of synchronously developing spermatogenic c e l l s (Reed and S t a n l e y , 1972; K a l t , 1976; Vigny, 1977). E l o n g a t i n g spermatids appeared to be arranged i n bundles around the p e r i p h e r y of the t u b u l e s while the more mature sperm were f r e e i n the c e n t r a l c a v i t y . Feulgen s t a i n i n g of these n u c l e i was intense and uniform ( F i g s . 2 and 3). The sperm n u c l e i of a l l s p e c i e s and subspecies were h e l i c a l i n shape. A l l s p e c i e s with h a p l o i d number n=l8 chromosomes had sperm which appeared i d e n t i c a l i n s i z e and shape to those of X. L^. l a e v i s ( F i g . 4 B ) , with n u c l e i d e s c r i b i n g 1.5 gyres of a h e l i x (Reed and S t a n l e y , 1972). The sperm n u c l e i of X. t r o p i c a l i s (n=1u) resembled those of X. 1. l a e v i s (n=l8) i n shape but appeared smaller i n s i z e (Fig.4A), while those of X. v e s t i t u s (n = 36) and X. w i t t e i (n=36), which a l s o had 1.5 gyres, appeared somewhat l a r g e r ( F i g . 4 C ) . Sperm n u c l e i of X. ruwenzoriensis (n=54) were l a r g e r than those of X. 1. l a e v i s and appeared to d e s c r i b e a h e l i x of two f u l l gyres (Fig.4D). T h i s was i n keeping with the h a p l o i d DNA content and chromosome numbers of these s p e c i e s (Table I ) . X. v e s t i t u s and X. w i t t e i are t e t r a p l o i d with respect to X. 1. l a e v i s , while X. ruwenzoriensis i s h e x a p l o i d , and X. t r o p i c a l i s has approximately one-half the DNA content of X. 1. l a e v i s (Thiebaud and F i s c h b e r g , 1977). 48 F i g u r e 2. Feulgen s t a i n i n g of s e c t i o n s of Xenopus t e s t e s . A. X. 1. bunyoniensis B. X. 1. v i c t o r i a n u s C. X. 1. ssp. n. (Malawi) D. X. 1. p e t e r s i E. X. c l i v i i F. X. m u e l l e r i 4?. Fig.2 10um 50 F i g u r e 3. Feulgen s t a i n i n g of s e c t i o n s of Xenopus t e s t e s A. X. 1^ . l a e v i s B. X. b o r e a l i s C. X. w i t t e i D. X. v e s t i t u s E. X. ruwenzoriensis F. X. t r o p i c a l i s 51. • «•-• * , • At > W..JI-1 Fig.3 lOum 52 F i g u r e 4. High m a g n i f i c a t i o n of Feulgen s t a i n e d Xenopus sperm A. X. t r o p i c a l i s (n=10) B. X. 1. l a e v i s (n=l8) C. X. v e s t i t u s (n=36) D. X. ruwenzoriensis (n=54) S3 54 A l k a l i n e f a s t green s t a i n i n g occured a f t e r h y d r o l y s i s of s e c t i o n s i n 5% TCA at 85-90°C, or with p i c r i c a c i d at 60°C, and p a r a l l e l l e d the DNA s t a i n i n g i n a l l s p e c i e s and subspecies (Fig.5A and C, and Fig.6A and C) T h i s i n d i c a t e s that t r o u t - l i k e protamines were not present i n Xenopus sperm n u c l e i , s i n c e they would have been washed out by t h i s treatment. However, as noted by Bois and Kasinsky (1973), sperm s t r u c t u r e was s e r i o u s l y a l t e r e d and AFG s t a i n i n g was reduced i f the s e c t i o n s were h y d r o l y s e d i n TCA at higher temperatures (90-100 0C). Sperm of the d o g f i s h , Squalus a c a n t h i u s , which c o n t a i n a s t a b l e protamine l i k e that found i n the mouse (Bois and Kasinsky, 1974), were a b l e to withstand t h i s treatment. Thus i t would appear that s t a b l e protamines are not present i n Xenopus sperm. With the exc e p t i o n of X. t r o p i c a l i s , sperm n u c l e i of a l l sp e c i e s c ontinued to s t a i n with AFG a f t e r deamination or a c e t y l a t i o n of the s e c t i o n s , although the s t a i n i n g was reduced (Fig.5B and D). Sperm of X. t r o p i c a l i s f a i l e d to s t a i n a f t e r deamination or a c e t y l a t i o n . T h i s was a l s o the case when AFG or eo s i n Y s t a i n i n g f o l l o wed p i c r i c a c i d h y d r o l y s i s and a c e t y l a t i o n ; s t a i n i n g was absent i n X. t r o p i c a l i s (Fig.6D), but only s l i g h t l y reduced i n a l l other s p e c i e s examined ( F i g . 6 B ) . These r e s u l t s i n d i c a t e d the presence of a r g i n i n e / l y s i n e -r i c h protein's i n the sperm of a l l s p e c i e s except X. t r o p i c a l i s . T h i s was c o r r o b o r a t e d by the Sakaguchi r e a c t i o n . Sperm of a l l s p e c i e s gave a moderate r e a c t i o n with the Sakaguchi t e s t , with the e x c e p t i o n of X. t r o p i c a l i s which gave only a f a i n t r e a c t i o n 55 F i g u r e 5. A l k a l i n e f a s t green s t a i n i n g of Xenopus t e s t i s s e c t i o n s . S e c t i o n s of t e s t e s are from X. 1_. p e t e r s i (A and B) and X. f r a s e r i (C and D). A. & C. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. B. AFG s t a i n i n g a f t e r TCA h y d r o l y s i s and deamination. D. AFG s t a i n i n g a f t e r TCA h y d r o l y s i s and a c e t y l a t i o n . 5& 57 F i g u r e 6. A l k a l i n e f a s t green s t a i n i n g of Xenopus t e s t i s s e c t i o n s . S e c t i o n s of t e s t e s are from X. 1. ssp. n. (Malawi) (A and B) and X. t r o p i c a l i s (C and D). A. & C. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 hr. B. & D. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1hr). sz 59 s i m i l a r to somatic c e l l s ( F i g . 7 ) . The presence of l y s i n e i n the sperm of a l l species was i n d i c a t e d by p o s i t i v e s t a i n i n g with DNFB. Non-nucleic a c i d - a s s o c i a t e d b a s i c p r o t e i n s were absent from a l l n u c l e i , as i n d i c a t e d by f a i l u r e to s t a i n with aqueous f a s t green without p r i o r h y d r o l y s i s and negative (pink) s t a i n i n g with AFG a f t e r Feulgen. On the b a s i s of these data, i t appears that sperm h i s t o n e s of a l l s p e c i e s of the genus Xenopus used i n t h i s study, except f o r X. t r o p i c a l i s , can be c l a s s i f i e d as the intermediate type (type 3), which c o n t a i n s both a r g i n i n e and l y s i n e . T h i s agrees with the r e s u l t s of amino a c i d a n a l y s i s of the s p e r m - s p e c i f i c p r o t e i n s of X. 1^ . l a e v i s (Huang, 1977; Huang et a l . , 1978). However, the type 3 sperm h i s t o n e s of Xenopus were u n l i k e those of M y t i l u s i n that they were not e x t r a c t a b l e from f o r m a l i n f i x e d n u c l e i with 5% TCA at 85-90°C. More d r a s t i c treatment of s e c t i o n s , such as h y d r o l y s i s at 95-100°C, was necessary to e x t r a c t these p r o t e i n s . Thus, i t no longer seems a p p r o p r i a t e to c h a r a c t e r i z e the intermediate c l a s s of anuran sperm nuclear p r o t e i n s as the " M y t i l u s - t y p e " . The h i s t o n e s of X. t r o p i c a l i s sperm appeared to be of the somatic or Rana type (type 4), i n agreement with the e l e c t r o p h o r e t i c data ( F i g . l 9 B ) . Cytochemical r e s u l t s f o r the genus Xenopus are summarized i n Table I I I . 60 F i g u r e 7. Sakaguchi s t a i n i n g of Xenopus t e s t i s s e c t i o n s . A. X. 1_. v i c t o r i a n u s B. X. b o r e a l i s C. X. m u e l l e r i D. X. t r o p i c a l i s Chromosome. Number ( H a p l o i d ) Table I I I . Cytochemistry of Sperm Nuclei In the Genua Xenopus Organism Used Xenopua laevis Subspecies Xenopua Species Controls laevis p e t e r s l y l c t o r l - bunyon- asp.n. c l l v l l muellerl borealla f r a B e r l v e s t l t u a w l t t e l ruwenzor- t r o p l - Rana Salmo 18 18 lanus lensis (Malawi) 18 18 18 18 18 18 18 36 36 lens i s c a l l s pipiens q a l r d n e r l l 54 10 S t a i n i n g and p r e t r e a t m e n t F e u l g e n AFG-TCA 85-90°C AFG-TCA, 95-100°C AFG-TCA, 85-90"C deaminat i o n A F G - p i c r i c a c i d , 60* A F G - p i c r i c a c i d , 60«, a c e t y l a t i o n E o s i n Y-p l c r i c a c i d E o s i n Y-p ^ . c r i c a c i d a c e t y l a t i o n S a k a g u c h i DNFB AFG-no h y d r o l y s i AFG a f t e r F e u l g e n R e a c t i v e M a t e r i a l DNA + B a s i c p r o t e i n s o t h e r than + pro t a m i n e s S t a b l e p r otamines B a s i c p r o t e i n s r i c h i n + a r g i n i n e B a s i c p r o t e i n s i n c l u d i n g + pr o t a m i n e s B a s i c p r o t e i n s r i c h i n + a r g i n i n e B a s i c p r o t e i n s i n c l u d i n g + pro t a m i n e s B a s i c p r o t e i n s r i c h i n + a r g i n i n e P r o t e i n - b o u n d a r g i n i n e + P r o t e i n - b o u n d l y s i n e • N o n - n u c l e i c ! a c i d - a s s o c i a t e d -b a s i c p r o t e i n s Non-DNA-a s s o c i a t e d -b a s i c p r o t e i n s + + + + • • + • + + + • • + • + + • + • • -f + Sperm histone type Abbreviatlonsi HC1 AFG hydrochloric acid; TCA alkaline fast green; DNFB tr i c h l o r o a c e t i c acid; 2 t4 dlnitrofluorobenzene. ft 63 c. Other Anura The sperm of Rana ca t e s b e i a n a were rod-shaped l i k e those of Rana p i p i e n s . Feulgen s t a i n i n g was intense and uniform and AFG s t a i n i n g f o l l o w i n g TCA h y d r o l y s i s p a r a l l e l l e d the DNA s t a i n i n g ( F i g . 8 A ) . A f t e r deamination, the sperm d i d not s t a i n , or s t a i n e d only f a i n t l y (Fig.8B), suggesting that a r g i n i n e - r i c h p r o t e i n s were not prese n t . The n u c l e i s t a i n e d with AFG or eo s i n Y a f t e r h y d r o l y s i s i n p i c r i c a c i d , but not i f a c e t y l a t i o n was c a r r i e d out before s t a i n i n g ( F i g . 8 C ) . T h i s r e i n f o r c e d the c o n c l u s i o n that a r g i n i n e - r i c h p r o t e i n s were absent. The n u c l e i showed inte n s e DNFB s t a i n i n g f o r l y s i n e , but s t a i n e d only l i g h t l y with the Sakaguchi t e s t f o r a r g i n i n e (Fig.8D). With no h y d r o l y s i s , AFG s t a i n i n g was absent, showing that non-nucleic a c i d - a s s o c i a t e d b a s i c p r o t e i n s were absent. AFG a f t e r Feulgen s t a i n i n g r e s u l t e d in a uniform p u r p l e s t a i n ; thus non-DNA-a s s o c i a t e d b a s i c p r o t e i n s were a l s o absent from these n u c l e i . In a l l of the t e s t s used, the s t a i n i n g p a t t e r n s of Rana  ca t e s b e i a n a sperm were s i m i l a r to those observed i n the sperm of Rana p i p i e n s and i n somatic c e l l s . Thus, i n agreement with the e l e c t r o p h o e t i c data (Figs.27 and 28), i t can be concluded that Rana ca t e s b e i a n a has sperm h i s t o n e s of the somatic type, or type 4. As i n Rana, the sperm of Bufo marinus and Bufo punctatus were rod-shaped and Feulgen s t a i n i n g was intense and uniform (Fig.9A and B). The sperm n u c l e i , l i k e those of M y t i l u s and Salmo g a i r d n e r i i , d i d not s t a i n with AFG a f t e r 5% TCA h y d r o l y s i s (Fig.9C and D). N u c l e i s t a i n e d i n t e n s e l y with AFG 64 F i g u r e 8. S t a i n i n g of t e s t i s s e c t i o n s from Rana c a t e s b e i a n a . A. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. B. AFG s t a i n i n g a f t e r TCA h y d r o l y s i s and deamination. C. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1 h r ) . D. Sakaguchi s t a i n i n g . 66 F i g u r e 9. S t a i n i n g of t e s t i s s e c t i o n s from Bufo. S e c t i o n s of t e s t e s are from Bufo marinus (A and C) and Bufo punctatus (B and D). A. & B. Feulgen s t a i n i n g . C. & D. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. 6 7 68 and e o s i n Y a f t e r p i c r i c a c i d h y d r o l y s i s (Fig.10A and B), but s t a i n i n g was reduced a f t e r a c e t y l a t i o n ( F i g . l O C and D). The sperm n u c l e i of both Bufo s p e c i e s s t a i n e d with DNFB f o r l y s i n e and gave a moderate r e a c t i o n with the Sakaguchi t e s t f o r a r g i n i n e ( F i g . l O E and F ) . Thus a r g i n i n e - r i c h h i s t o n e s appeared to be present, and the presence of some l y s i n e was a l s o e v i d e n t . Non-nucleic a c i d - a s s o c i a t e d b a s i c p r o t e i n s appeared to be absent from these n u c l e i . The sperm n u c l e i of Bufo marinus and Bufo punctatus showed the same s t a i n i n g p a t t e r n s as those of M y t i l u s , with the e x c e p t i o n of a s l i g h t l y darker r e a c t i o n with the Sakaguchi t e s t . T h e r e f o r e , the sperm h i s t o n e s of these Bufo s p e c i e s can be p l a c e d i n the intermediate category (type 3). T h i s a l s o agrees with the amino a c i d a n a l y s i s of the s p e r m - s p e c i f i c h i s t o n e s of Bufo marinus (Huang, 1977; Huang et a l . , 1978). The sperm n u c l e i of Hyla r e g i l l a and Hyla g r a t i o s a were rod-shaped and s l i g h t l y bent (Fig.11A and B), while the sperm of Scaphiopus couchi were smal l and wavy (Fig.12A). Feulgen s t a i n i n g was intense and uniform and AFG s t a i n i n g , a f t e r TCA h y d r o l y s i s at 85-90°C, followed the Feulgen p a t t e r n (Fig.11C and 12B). However, when the temperature of h y d r o l y s i s was r a i s e d to 95-100°C, the sperm n u c l e a r s t r u c t u r e was broken down (Fig.11D), as was the case with Xenopus l a e v i s sperm. S t a i n i n g with AFG was only s l i g h t l y reduced a f t e r deamination of l y s i n e r e s i d u e s ( F i g . l I E and F, and 12C), suggesting that a r g i n i n e -r i c h p r o t e i n s were pres e n t . N u c l e i s t a i n e d with AFG and e o s i n Y a f t e r p i c r i c a c i d h y d r o l y s i s and continued to s t a i n even with 69 F i g u r e 10. S t a i n i n g of t e s t i s s e c t i o n s from Bufo. S e c t i o n s of t e s t e s are from Bufo marinus (A,C and E) and Bufo punctatus (B,D and F ) . A. & B. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 hr. C. & D. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1hr). E. & F. Sakaguchi s t a i n i n g . e l l C E » . . • > - .-Fig.lO lOym 71 F i g u r e 11. S t a i n i n g of t e s t i s s e c t i o n s from Hyla. S e c t i o n s of t e s t e s are from Hyla r e g i l l a (A and E) and Hyla q r a t i o s a (B,C,D and F ) . A. & B. Feulgen s t a i n i n g . C. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. D. AFG s t a i n i n g a f t e r TCA h y d r o l y s i s , 95-100 oC, 15 min. E. & F. AFG s t a i n i n g a f t e r TCA h y d r o l y s i s (85-90°C, 15 min) and deamination. 7 £ 73 F i g u r e 12. S t a i n i n g of t e s t i s s e c t i o n s from Scaphiopus. A. Feulgen s t a i n i n g . B. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, min. C. AFG s t a i n i n g a f t e r TCA h y d r o l y s i s and deamination. 75 p r i o r a c e t y l a t i o n . T h i s a l s o suggested that a r g i n i n e - r i c h p r o t e i n s were pres e n t . N u c l e i s t a i n e d with DNFB f o r l y s i n e , and responded moderately to the Sakaguchi t e s t f o r a r g i n i n e . The moderate r e a c t i o n with Sakaguchi i n d i c a t e d t h a t these sperm h i s t o n e s were not of the salmon or s t a b l e protamine type. The sperm h i s t o n e s of Hyla and Scaphiopus, l i k e those of most Xenopus s p e c i e s , appeared to be of the intermediate type. They were l e s s e a s i l y e x t r a c t e d fom n u c l e i than those of Bufo or M y t i l u s , but d i d not r e s i s t h i g h temperature h y d r o l y s i s i n TCA as d i d the sperm n u c l e i of mammals and the d o g f i s h . The p o s i t i v e r e a c t i o n with DNFB and the moderate s t a i n i n g with the Sakaguchi r e a c t i o n i n d i c a t e d that these p r o t e i n s contained both l y s i n e and a r g i n i n e , and thus may be c l a s s i f i e d as type 3 sperm h i s t o n e s . Bois and Kasinsky (1972) a l s o c l a s s i f i e d the sperm h i s t o n e s of Hyla r e g i l l a and Hyla v e r s i c o l o r as type 3 on the b a s i s of c y t o c h e m i s t r y . R e s u l t s of the cyto c h e m i c a l t e s t s i n the v a r i o u s s p e c i e s of Anura are summarized i n Table IV. The inter m e d i a t e - t y p e of anuran sperm h i s t o n e s has been d i v i d e d i n t o two subtypes. Type 3A sperm h i s t o n e s , found i n Xenopus, Hyla and Scaphiopus, were not e x t r a c t a b l e from n u c l e i with 5% TCA ( 8 5 - 9 0 ° C ) , while type 3B sperm p r o t e i n s , such as those of Bufo and M y t i l u s , were washed out of n u c l e i under these c o n d i t i o n s . TABLE IV. Cytochemistry of Sperm i n Anura Organism used Controls Staining s Reactive Rana Rana Hy_la Hyjja Scaphiopus Bufo Bufo Mytilus Xenopus Squalus Salmc. Pretreatment Material pTpTens c^esbeiana ^ t i o s a r e g i l l a couchi marinus punctatus edulis l a e v i s _ acanthius ^ . r d n e r u Feulgen AFG-TCA, B5-90°C. Basic proteins otlier than protamines AFG-TCA, 95-100°C. Stable protamines AFG-TCA, 85-90°C., deamination AFG, p i c r i c a c i d , 60°C. AFG, p i c r i c acid, acetylation Eosin Y - p i c r i c acid, 60°C Eosin Y - p i c r i c acid, acetylation Basic proteins r i c h i n arginine Basic proteins + including protamines Sakaguchi Basic proteins r i c h i n arginine Basic proteins including protamines Basic proteins r i c h i n arginine Protein-bound arginine DNFB AFG, no hydrolysis Protein-bound lysine Non-nucleic acid assoc. basic proteins AFG after F<julgcn Non-DNA-assoc. basic proteins Sperm histone type 3A 3A 3A 3B 3B 3B 3A 3A. = Intermediate-type, not extractable with 5% TCA (85-90°C). 3B. = Intermediate-type, extractable with 5% TCA (85-90°C). -^1 77 d. S a l t c o n c e n t r a t i o n and the a l k a l i n e f a s t green r e a c t i o n As noted by Bois (1972), sperm n u c l e i that contained a r g i n i n e - r i c h p r o t e i n s s t a i n e d with 0.1% AFG without p r i o r h y d r o l y s i s when the s t a i n was made up i n NaCl s o l u t i o n s . Somatic h i s t o n e s and Rana type sperm h i s t o n e s f a i l e d to s t a i n . Sperm n u c l e i of Salmo g a i r d n e r i i s t a i n e d w i t h i n the s a l t c o n c e n t r a t i o n range of 2.4-0.02 M NaCl, while the sperm of X. 1. l a e v i s s t a i n e d between 2.4-0.05 M NaCl. Sperm n u c l e i of Bufo americanus and Bufo boreas were a l s o s t a i n a b l e at 0.3 M NaCl. Bois (1972) suggested that AFG s t a i n i n g without h y d r o l y s i s of DNA c o u l d p o s s i b l y be due to d i s s o c i a t i o n of a r g i n i n e - r i c h p r o t e i n s from DNA i n the NaCl s o l u t i o n . When s a l t d i s s o c i a t i o n occured i n the presence of a l k a l i n e f a s t green, the s t a i n may have competed s u c c e s s f u l l y with other negative ions f o r p o s i t i v e s i t e s on the protamine or intermediate-type sperm h i s t o n e s and thus s t a i n i n g occured. As c o n t r o l s f o r t h i s experiment, s e c t i o n s of t e s t e s of Salmo g a i r d n e r i i (type 1), M y t i l u s e d u l i s (type 3) and Rana  p i p i e n s (type 4) were used. R e s u l t s are shown i n Table V. Salmo g a i r d n e r i i sperm n u c l e i s t a i n e d w i t h i n the s a l t c o n c e n t r a t i o n range of 0.9 M-0.025 M, while those of Rana d i d not s t a i n at a l l , nor d i d the sperm n u c l e i of M y t i l u s . F i x a t i o n of p r o t e i n s with formaldehyde causes the formation of methylene b r i d g e s between amino groups of neighbouring p r o t e i n s (Bowes and C a r t e r , 1965). Formaldehyde a l s o l i n k s the l y s i n e r e s i d u e s of h i s t o n e molecules to DNA i n such a way that they are not d i s s o c i a b l e by s a l t Table V. Salt Concentration and the Alkaline Fast Green Reaction i n Sperm Nuclei of the Genus Xenopus a*k Xenopus laevis Subspecies  laevis peters! v i c t o r ! - bunyon- ssp.n. lanus • iensis Chromosome No: 18 (Haploid) 18 18 18 18 Organism Used Xenopus Species Controls c l i v i l muelleri borealis f r a s e r i vestitus w i t t e i ruwenzor- t r o p i - Rana Bufo Salmo Mytilus i e n s i s c a l l s plplens marinus q a l r d n e r i i edulis and 18 18 18 18 36 36 54 10 Bufo punctatus Sperm Histone Type: Stain 0.U AFG C Made up i n : •9M NaCl ,6M NaCl .3M NaCl .15M NaCl .075M NaCl •050M NaCl • 025H H a d .010M NaCl .005H NaCl .001M NaCl d i s t i l l e d II,0 s l i g h t slight s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t s l i g h t aAnimal set #2, one. ^Results obtained without hydrolysis A b b r e v i a t i o n : AFG » Alkaline Fast Green -^1 OO 79 ( B r u t l a g et a l . , 1969). T h i s might e x p l a i n why the somatic h i s t o n e s and the sperm h i s t o n e s of M y t i l u s , both r e l a t i v e l y r i c h i n l y s i n e , were not s t a i n e d even at high s a l t c o n c e n t r a t i o n s . Formalin f i x a t i o n may cause blockage of most of the p o s i t i v e dye-binding s i t e s of these p r o t e i n s by l i n k i n g the l y s i n e r e s i d u e s to DNA. On the other hand, the h i g h l y a r g i n i n e -r i c h protamines of Salmo g a i r d n e r i i , which c o n t a i n no l y s i n e (Ling et a l . , 1971) may be l e s s capable of forming methylene bridges with DNA and were t h e r e f o r e d i s s o c i a b l e and s t a i n a b l e with AFG i n the presence of s a l t . Sperm n u c l e i of Bufo marinus and Bufo punctatus s t a i n e d at hig h s a l t c o n c e n t r a t i o n s (0.9-0.3 M) and showed s l i g h t s t a i n i n g at 0.075 M NaCl. The sperm nuclear b a s i c p r o t e i n of Bufo marinus i s a r g i n i n e - r i c h (45 mole%) with some l y s i n e (4.8 mole%). Thus i t would appear t h a t , with f o r m a l i n f i x e d m a t e r i a l , AFG s t a i n i n g i n the presence of s a l t , without h y d r o l y s i s of DNA, i s c o n f i n e d to the a r g i n i n e -r i c h sperm h i s t o n e s of types 1 and 3. In agreement with the other c y t o c h e m i c a l data, the sperm n u c l e i of X. t r o p i c a l i s d i d not s t a i n with AFG made up i n NaCl s o l u t i o n s , suggesting once again that the sperm h i s t o n e s of t h i s s p e c i e s are not r i c h i n a r g i n i n e . Sperm n u c l e i of the other s p e c i e s and subspecies of Xenopus d i d s t a i n at high s a l t c o n c e n t r a t i o n s (>0.3 M NaCl). A s l i g h t d i f f e r e n c e was noted between the sperm of X. v e s t i t u s and X. w i t t e i and sperm of the other s p e c i e s . The former d i d not s t a i n at c o n c e n t r a t i o n s of <0.30 M NaCl whereas the other s p e c i e s continued to s t a i n f a i n t l y at 0.15 M NaCl. These r e s u l t are summarized i n Table V. 80 2. E l e c t r o p h o r e s i s a. X e n o p u s l a e v i s l a e v i s c o n t r o l s T e s t i s - and s p e r m - s p e c i f i c h i s t o n e s of X. 1. l a e v i s were i s o l a t e d by the micromethod, the spermatid/sperm method or the whole t e s t i s method, using e i t h e r f r e s h t i s s u e or m a t e r i a l s t o r e d at -70°C. R e s u l t s are summarized i n Table VI. i . I d e n t i f i c a t i o n of bands: Figs.13,14 and 15 show p r o f i l e s of X. 1. l a e v i s t e s t i s -and s p e r m - s p e c i f i c b a s i c p r o t e i n s i s o l a t e d by d i f f e r e n t methods and separated i n p o l y a c r y l a m i d e g e l s c o n t a i n i n g 6.25 M urea (Figs.13 and 15A) or 0.4% T r i t o n X-100/2.5 M urea (Figs.14 and 15B). Histone nomenclature f o l l o w s that of R i s l e y and Eckhardt (1981). The p a t t e r n s of h i s t o n e m i g r a t i o n i n these g e l s were s i m i l a r to those determined by R i s l e y and Eckhardt (1981) f o r 6.25 M urea g e l s , and by Koster et a l . (1979) f o r 0.375% T r i t o n X-100/2.5 M urea g e l s . In the a c i d / u r e a g e l shown i n Fig.13, the f i r s t f a i n t band near the top should be h i s t o n e H3 i n the dimer form (H3D). The H1 h i s t o n e s were a l s o c l e a r l y evident i n t h i s r e g ion of the g e l . H1A migrated j u s t ahead of H3D and was l e d by H1B. H1C and H1D c o u l d be seen as a f a i n t doublet ahead of the sperm-s p e c i f i c p r o t e i n SP1 ( R i s l e y and Eckhardt, 1981). The H3 monomer and H2A comigrated on 6.25 M urea g e l s , as d i d H2B and SP2. These two bands were very c l o s e to each other and c o u l d be seen as a broad band m i g r a t i n g j u s t ahead of the H1's. The next 81 T a b l e V I . C o n d i t i o n of y . i . l a e v i s B a s i c Mnclp^r p Method Tissue Micromethod3- Testis-fresh Testis-frozen Whole Tissue Testis-fresh Method13 • Testis-frozen Erythrocytes Spermatid/ spera Method6, Testis-fresh Testis-frozen a*Kasinsky et al., 1978 Risley and Eckhardt, 1981 Risley and Eckhardt, 1979a, rotoip.s Isolated bv n i f f 0 ^ . Methods Condition of R a t i o HI Histones SP3-5/SP6 Intact Intact Intact Intact Intact Intact Degraded <1 <1 >1 >1 >1 <1 Other Observations "X1" present. all histones and SP's intact "XI" present, all histones and SP's intact (Figs.13 and 14) "X1" not present all histones and SP's intact (Figs.13 and 14) "XI" not present, all histones and SP's intact (Figs.13 and 14) No bands migrating faster than H4 (acid/urea gels), or HID (Triton gels) (Fig.l9B) "XI" not present, Hi's, H2A and H23 reduced, f3int bands leading SP3-5 (Fig. 19 A.) Bands leading H4, SP2, SP3-5 and S?5 (Figs.13, 14 and 15) 1981 82 d a r k l y s t a i n i n g band c o n t a i n e d h i s t o n e H4, the f a s t e s t of the somatic h i s t o n e s i n t h i s g e l system. Bands moving f a s t e r than H4 represented the s p e r m - s p e c i f i c p r o t e i n s SP3-5, seen as a doublet, and SP6, a s i n g l e band with the f a s t e s t m o b i l i t y of a l l . In the T r i t o n g e l ( F i g . 1 4 ) , the order of m o b i l i t y of the nucleosomal h i s t o n e s , from slowest to f a s t e s t , was H2A, H3, H2B and H4. These c o u l d be seen as d a r k l y s t a i n i n g bands at the top of the g e l . The next group of bands represented the H1 h i s t o n e s : H1A (very f a i n t i n t h i s g e l ) , H1B, H1C and H1D ( c a l l e d H5 by Koster e_t a l . , 1979). The s p e r m - s p e c i f i c p r o t e i n SP1 migrated between H1B and H1C and the dark band l e a d i n g the H1's was SP2. The f a s t e s t moving components rep r e s e n t e d SP3-5 and SP6 (M. R i s l e y , i n p r e p a r a t i o n ) . i i . H i s t o n e s i s o l a t e d from e r y t h r o c y t e n u c l e i : H i stones i s o l a t e d from X. 1. l a e v i s e r y t h r o c y t e n u c l e i and separated i n a T r i t o n g e l are shown in F i g . l 9 A (channel 1). The four nucleosomal h i s t o n e s were present i n approximately equal p r o p o r t i o n s at the top of the g e l . The H1 h i s t o n e s migrated ahead of H4; from slowest to f a s t e s t these were H1A, H1B, H1C ( f a i n t ) and H1D. U n l i k e the e l e c t r o p h o r e t i c p r o f i l e s of t e s t i s or spermatid/sperm b a s i c p r o t e i n s , e r y t h r o c y t e n u c l e i of X. _1. l a e v i s c o ntained no b a s i c p r o t e i n s m i g r a t i n g f a s t e r than H1D i n T r i t o n g e l s , or H4 i n a c i d / u r e a g e l s . The h i s t o n e s i s o l a t e d from e r y t h r o c y t e n u c l e i of the set #3 Xenopus sp e c i e s a l s o showed no bands moving f a s t e r than H4 83 F i g u r e 13. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s and sperm b a s i c p r o t e i n s from X. 1_. l a e v i s . E l e c t r o p h o r e s i s was performed i n a 25 cm p o l y a c r y l a m i d e s l a b g e l (15% acrylamide, 6.25 M urea) f o r 12 hr at 200 V. S t a i n i n g was performed i n 0.2% Amido Black and d e s t a i n i n g was i n 30% methanol/10% ace'tic a c i d . Basic p r o t e i n s were e x t r a c t e d from: 1. Whole t e s t i s chromatin prepared from f r e s h t i s s u e . 2. Whole t e s t i s chromatin prepared from t i s s u e s t o r e d at -70°C. 3. Spermatid/sperm chromatin prepared from t i s s u e s t o r e d at -70°C. 4. Whole t e s t e s , s t o r e d at -70°C, homogenized i n PBS. Arrows i n d i c a t e products of d e g r a d a t i o n . SP= spermatid/sperm-spec i f i c b a s i c p r o t e i n s of X. 1. l a e v i s . X1= p o s i t i o n of contaminant e x t r a c t e d from t e s t i s homogenates of X. 1_. l a e v i s . 85 F i g u r e 14. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s and sperm b a s i c p r o t e i n s from X. 1. l a e v i s . E l e c t r o p h o r e s i s was performed i n a 20 cm p o l y a c r y l a m i d e s l a b g e l (12% acrylamide, 2.5 M urea, 0.4% T r i t o n X-100) fo r 6 hr at 200 V. S t a i n i n g was i n 0.2% Amido Black and d e s t a i n i n g i n 30% methanol/10% a c e t i c a c i d . Basic p r o t e i n s were e x t r a c t e d from: 1. Whole t e s t i s chromatin prepared from f r e s h t i s s u e . 2. Whole t e s t i s chromatin prepared from t i s s u e s t o r e d at -70°C. 3. Spermatid/sperm chromatin prepared from t i s s u e s t o r e d at -70°C. 4. Whole t e s t e s , s t o r e d at -70°C, homogenized i n PBS. Arrows i n d i c a t e products of d e g r a d a t i o n . 87 i n a c i d / u r e a p o l y a c r y l a m i d e g e l s , or f a s t e r than H1D i n T r i t o n g e l s (data not shown). T h i s i s evidence that the fast-moving bands obtained by the same method from the t e s t e s and spermatid/sperm p r e p a r a t i o n s of the v a r i o u s Xenopus s p e c i e s ( F i g . l 9 A and B) were i n f a c t sperm- or t e s t i s - s p e c i f i c p r o t e i n s rather than a r t i f a c t s of the i s o l a t i o n procedure. i i i . H i stones i s o l a t e d from f r e s h and froz e n whole t e s t e s : Figs.13 and 14 compare t e s t i s - s p e c i f i c h i s t o n e s from f r e s h t i s s u e (channel 1) and from t i s s u e s t o r e d at -70°C (channel 2), i s o l a t e d from the nuclear chromatin of whole t e s t e s by the method of R i s l e y and Eckhardt (1981). A l l working s o l u t i o n s c o n t a i n e d p r o t e o l y t i c i n h i b i t o r s to prevent the breakdown of chromosomal p r o t e i n s . R i s l e y (1977) noted that the use of Na b i s u l f i t e alone, as recommended by other authors (Panyim e_t §_1., 1971), was not s u f f i c i e n t to prevent p r o t e o l y t i c degradation 'of Xenopus h i s t o n e s . T h i s degradation was apparent i n the disappearance of the H1 h i s t o n e s and as e x t r a bands l e a d i n g H2B and H4 on a c i d / u r e a p o l y a c r y l a m i d e g e l s . A d d i t i o n of PMSF, TPCK and TLCK appeared to i n h i b i t p r o t e o l y s i s e f f e c t i v e l y and whole t e s t i s h i s t o n e s i s o l a t e d from f r e s h t i s s u e (Figs.13 and 14, channel 1 and t r a c e 1) showed none of these s i g n s of de g r a d a t i o n , nor d i d the p r o t e i n s i s o l a t e d from f r o z e n t e s t e s (Figs.13 and 14, channel 2, t r a c e 2). 88 i v . H i s tones i s o l a t e d by the micromethod: Channel 4 (Figs.13 and 14) shows a p r o f i l e of b a s i c p r o t e i n s e x t r a c t e d from whole t e s t e s s t o r e d at -70°C, using the micromethod of Kasinsky ejt a_l. ( 1978). These p r o t e i n s , prepared by simple a c i d e x t r a c t i o n of a t e s t i s homogenate without the use of p r o t e o l y t i c i n h i b i t o r s , showed few apparent s i g n s of d e g r a d a t i o n . The H1 h i s t o n e s were present and i n t a c t and there were no bands l e a d i n g H2B, H4, SP2, SP3-5 or SP6. A very f a i n t band was evident m i g r a t i n g approximately half-way between H4 and SP3 ( F i g . 1 3 ) , although i t was not r e s o l v e d i n the t r a c e . A s i m i l a r band was obtained from s u l f u r i c a c i d e x t r a c t s of homogenates made from f r e s h t e s t e s (data not shown). T h i s band may correspond to "X1" r e p o r t e d by Bois and Kasinsky (1973), but i t was not apparent when h i s t o n e s were prepared from chromatin by R i s l e y and Eckhardt's (1981) method. I t may represent a minor cytoplasmic contaminant. In a l l other r e s p e c t s , the micromethod appeared to produce an e l e c t r o p h o r e t i c p r o f i l e t h a t was q u a l i t a t i v e l y comparable to that obtained by the chromatin method. However the q u a n t i t a t i v e p r o p o r t i o n of SP3-5, i s o l a t e d by the micromethod, appeared somewhat reduced r e l a t i v e to H4, while the p r o p o r t i o n of SP6 was somewhat grea t e r than seen when h i s t o n e s were i s o l a t e d by the chromatin method (Fig.13 and Table V ) . 89 v. H i s t o n e s i s o l a t e d from spermatid/sperm chromatin of f r e s h and fr o z e n t e s t e s : B a s i c p r o t e i n s obtained from nuclear chromatin of the spermatid/sperm f r a c t i o n of f r e s h t e s t e s d i s s o c i a t e d with c o l l a g e n a s e showed some s i g n s of degradation (Fig.15A and B, channels 1 and 2; F i g . l 9 B , channel 1). The a c i d / u r e a g e l (Fig.15A) r e v e a l e d that the H1 h i s t o n e s were present i n the 5% TCA-soluble f r a c t i o n (channel 1), although they were reduced i n c o n c e n t r a t i o n compared to whole t e s t i s p r e p a r a t i o n s ( F i g . 1 3 ) . [5% TCA i s known to p r e f e r e n t i a l l y e x t r a c t the H1 h i s t o n e s (DeNooij and Westinbrink, 1962)]. There were no bands evident l e a d i n g H2B or H4, but there was a f a i n t i n d i c a t i o n of banding between SP3-5 and SP6 (channel 2). SP3-5 were present i n much higher p r o p o r t i o n to SP6 i n the f r e s h t i s s u e p r e p a r a t i o n (channel 2) than i n the spermatid/sperm f r a c t i o n of frozen t i s s u e (channel 3), even though s i m i l a r amounts of p r o t e i n were a p p l i e d to each of these channels. The p r o f i l e s of b a s i c p r o t e i n s i s o l a t e d from spermatid/sperm chromatin of f r e s h t e s t e s and separated i n T r i t o n g e l s may be seen i n Figs.15B and 19B. The H1 h i s t o n e s were e x t r a c t e d from chromatin with e i t h e r 5% TCA ( F i g . l 5 B , channel 1), or with 0.4 N s u l f u r i c a c i d ( F i g . l 9 B , channel 1). As noted i n the a c i d / u r e a g e l (Fig.15A), and by R i s l e y (1977), the H1 h i s t o n e s , as w e l l as H2A and H2B, were much reduced i n p r o p o r t i o n to H3, H4 and the s p e r m - s p e c i f i c p r o t e i n s . F a i n t bands c o u l d be seen l e a d i n g SP2 and SP6, and between SP3-5 and SP6, e s p e c i a l l y in F i g . l 9 B . These bands roughly corresponded to 90 Fi g u r e 15. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s and sperm b a s i c p r o t e i n s from X. 1. l a e v i s . A. E l e c t r o p h o r e s i s was performed i n a 25 cm poly a c r y l a m i d e s l a b g e l (15% acrylamide, 6.25 M urea) f o r 12 hr at 200 V. S t a i n i n g was i n 0.2% Amido Black and d e s t a i n i n g i n 30% methanol/10% a c e t i c a c i d . B. E l e c t r o p h o r e s i s was performed i n a 25 cm poly a c r y l a m i d e s l a b g e l (12% acrylamide, 2.5 M urea, 0.4% T r i t o n X-100) f o r 7.5 hr at 220 V. S t a i n i n g was in 0.1% Coomassie Blue and d e s t a i n i n g i n 30% methanol/10% a c e t i c a c i d . 1. Basic p r o t e i n s e x t r a c t e d with 5% TCA (20 min) from spermatid/sperm chromatin prepared from f r e s h t i s s u e . 2. Basic p r o t e i n s e x t r a c t e d with 0.4 N s u l f u r i c a c i d from spermatid/sperm chromatin (prepared from f r e s h t i s s u e ) a f t e r i n i t i a l e x t r a c t i o n with 5% TCA. 3. Basic p r o t e i n s e x t r a c t e d with 0.4 N s u l f u r i c a c i d from spermatid/sperm chromatin prepared from t i s s u e s t o r e d at -70°C. Arrows i n d i c a t e products of d e g r a d a t i o n . 91 1 2 3 92 those seen i n the spermatid/sperm f r a c t i o n s of frozen t e s t e s , although t h e i r r e l a t i v e p r o p o r t i o n s were much lower. A f a i n t band m i g r a t i n g between SP3-5 and SP6 c o u l d a l s o be seen i n the chromatin p r e p a r a t i o n from whole f r e s h t e s t e s , shown i n F i g . l 9 B , channel 2. S e v e r a l o b s e r v a t i o n s suggest that the sub-bands seen between SP3-5 and SP6 may be n a t u r a l l y o c c u r i n g components of X. 1. l a e v i s sperm r a t h e r than a r t i f a c t s of the i s o l a t i o n procedures. F i r s t of a l l , spermatids and sperm prepared from f r e s h t i s s u e by c o l l a g e n a s e d i s s o c i a t i o n and c e n t r i f u g a t i o n through Metrizamide were h i g h l y v i a b l e as judged by Trypan blue e x c l u s i o n and i n c o r p o r a t i o n of r a d i o a c t i v e amino a c i d s and n u c l e o t i d e s i n c u l t u r e ( R i s l e y and Eckhardt, 1979b). Secondly, these bands were only prominent i n p r e p a r a t i o n s c o n t a i n i n g a very high percentage of mature sperm (M. R i s l e y , p e r s o n a l communication). Somatic h i s t o n e s are known to be t o t a l l y or p a r t i a l l y r e p l a c e d by protamines, or other b a s i c p r o t e i n s , i n many organisms. In the case of Salmo g a i r d n e r i i , the removal of these p r o t e i n s i s thought to be accomplished by a combination of a c e t y l a t i o n and p h o s p h o r y l a t i o n of h i s t o n e s and t h e i r subsequent p r o t e o l y s i s (Marushige and Dixon, 1971; Marushige et a l . , 1976). A s i m i l a r s i t u a t i o n may be the case i n X. 1. l a e v i s , with H1, H2A and H2B being p r e f e r e n t i a l l y d i s p l a c e d and p o s s i b l y degraded i n the spermatid or mature sperm. The sub-bands seen between SP3-5 and SP6 i n p r e p a r a t i o n s of l a t e spermatids and mature sperm may represent the n a t u r a l l y . o c c u r i n g breakdown products of these p r o t e i n s . 93 On the other hand, b a s i c p r o t e i n s i s o l a t e d from spermatid/sperm chromatin of t e s t e s s t o r e d at -70°C and then d i s s o c i a t e d with c o l l a g e n a s e (Figs.13, 14, and 15, channel 3) appeared to be much more degraded than those obtained from f r e s h t i s s u e ( F i g . l 5 A and B, channels 1 and 2). In a c i d / u r e a g e l s (Figs.13 and 15A), the H1 h i s t o n e s were v i r t u a l l y absent and minor bands c o u l d be seen l e a d i n g H2B and H4. There were a l s o at l e a s t two bands evident between SP3-5 and SP6, as w e l l as s e v e r a l f a i n t bands l e a d i n g SP6. The q u a n t i t a t i v e p r o p o r t i o n of SP3-5 was g r e a t l y reduced r e l a t i v e to SP6, compared with that seen i n whole t e s t i s p r e p a r a t i o n s and i n spermatid/sperm f r a c t i o n s prepared from f r e s h t i s s u e . In T r i t o n g e l s (Figs.14 and 15B), bands c o u l d be seen l e a d i n g SP2 and SP6, and between SP3-5 and SP6. Again, the H1 h i s t o n e s and SP3-5 appeared much reduced i n c o n c e n t r a t i o n r e l a t i v e to whole t e s t i s p r e p a r a t i o n s . Degradation of spermatid/sperm nu c l e a r b a s i c p r o t e i n s prepared from fro z e n t e s t e s was g r e a t e r than that seen when f r e s h t i s s u e i s used. However there were a few s i m i l a r i t i e s . In both cases, the H1's, H2A and H2B were g r e a t l y reduced and bands appeared between SP3-5 and SP6, perhaps the r e s u l t of n a t u r a l p r o t e o l y s i s . However, when t i s s u e had been f r o z e n , the s p e r m - s p e c i f i c p r o t e i n s SP3-5 and SP6 appeared degraded as w e l l . Destree et a l . (1975) have shown that h i s t o n e degradation i n X. 1^ . l a e v i s l i v e r was due to a c y t o p l a s m i c protease a c t i v i t y r a t h e r than a nu c l e a r enzyme. F r e e z i n g and thawing of t i s s u e i n the experiments performed here was probably r e s p o n s i b l e f o r breakage of c e l l s and leakage of p r o t e o l y t i c 94 enzymes i n t o the n u c l e i . The 60-90 min spent at 30°C dur i n g c o l l a g e n a s e d i s s o c i a t i o n would p r o v i d e ample o p p o r t u n i t y f o r p r o t e o l y s i s of h i s t o n e s and sperm n u c l e a r p r o t e i n s . However, h i s t o n e s prepared from f r o z e n whole t e s t e s appeared to be i n t a c t , suggesting that f r e e z i n g i t s e l f was not r e s p o n s i b l e f o r t h i s d e g r a d a t i o n . v i . H i s t o n e s e x t r a c t e d from chromatin with 5% TCA: Fig.15A and B (channel 1) a l s o shows t h a t , i n a d d i t i o n to the H1 h i s t o n e s , a l l the sperm s p e c i f i c p r o t e i n s of X. 1. l a e v i s were e x t r a c t a b l e from chromatin with 5% TCA, although they appeared to be l e s s s o l u b l e than the H1's. A l a r g e p r o p o r t i o n of the SP's remained i n the chromatin p e l l e t a f t e r 20 minutes of e x t r a c t i o n with 5% TCA, while most of the H1 was s o l u b i l i z e d d u r i n g t h i s time. The nucleosomal h i s t o n e s were v i r t u a l l y i n s o l u b l e i n 5% TCA, a c h a r a c t e r i s t i c which d i s t i n g u i s h e s them from the H1's (De Nooij and Westinbrink, 1962). T h i s property a l s o appeared to d i s t i n g u i s h the SP's of X. 1. l a e v i s from the protamines of salmonoid f i s h , which are h i g h l y s o l u b l e i n 5% TCA ( A l f e r t , 1956; F e l i x , 1960), and may e x p l a i n why the sperm of X. 1. l a e v i s and other Xenopus s p e c i e s , continued to s t a i n with AFG a f t e r h y d r o l y s i s i n 5% TCA while the protamines of t r o u t were e x t r a c t e d . 95 b. X e n o p u s l a e v i s subspecies Fig.16 shows the a c i d / u r e a g e l e l e c t r o p h o r e t i c p r o f i l e s of s u l f u r i c a c i d e x t r a c t s obtained from whole t e s t e s of f i v e Xenopus l a e v i s subspecies (animal set #1) prepared by the micromethod. The g e l i n Fig.16A was s t a i n e d with Amido Black and d e s t a i n e d with 35% methanol/10% a c e t i c a c i d . Only the p r o t e i n s of X. 1. l a e v i s and X. 1^ . p e t e r s i were r e s o l v e d and t r a c e s of these two channels are shown in Fig.17. The somatic h i s t o n e s appeared to be i n t a c t and bands with the same m o b i l i t i e s as SP3-5 and SP6 c o u l d be seen i n X. _1. p e t e r s i . These p r o t e i n s had m o b i l i t i e s i n t e r mediate to h i s t o n e H4 and h e r r i n g protamine. At the top of the g e l , the H1 h i s t o n e s appeared r e s o l v e d i n both X. 1^ . l a e v i s and X. .1. p e t e r s i . H1B was not apparent i n X. 1_. p e t e r s i , but a band with the same m o b i l i t y as SP1 was present, as were bands corresponding to H1A, H1C and H1D. Fig.16B shows the same g e l r e s t a i n e d with Amido Black and d e s t a i n e d i n 1 M s u l f u r i c a cid/3 M urea. S t a i n i n g was enhanced approximately t e n - f o l d (Wray and S t u b b l e f i e l d , 1970). The h i s t o n e s of X. _1. bunyoniensis, X. _1. v i c t o r ianus and X. 1. ssp. n. (Malawi) were made evident by t h i s procedure and t r a c e s of these channels can be seen i n Fig.18. The fast-moving bands of X. 1.. ssp. n. (Malawi) were s i m i l a r i n m o b i l i t y to SP3-5 and SP6 of X. 1. l a e v i s , as were those of X. 1. v i c t o r i a n u s . However the p r o t e i n s of X. 1. bunyoniensis showed s l i g h t d i f f e r e n c e s . Four to f i v e bands were r e s o l v e d i n the SP3-5 region and two bands c o u l d be seen i n the region of 96 F i g u r e 16. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s b a s i c p r o t e i n s from f i v e Xenopus l a e v i s subspecies . Testes ( s t o r e d at -70°C) were obtained from animal set #1 and b a s i c p r o t e i n s were e x t r a c t e d d i r e c t l y with 0.4 N s u l f u r i c a c i d from whole t i s s u e homogenized i n PBS. E l e c t r o p h o r e s i s was performed i n a 20 cm p o l y a c r y l a m i d e s l a b g e l (15% acrylamide, 6.25 M urea) f o r 12 hr at 150 V. A. Gel s t a i n e d with 0.2% Amido Black and d e s t a i n e d i n 30% methanol/10% a c e t i c a c i d . B. Same g e l r e s t a i n e d with 0.2% Amido Black and d e s t a i n e d i n 1 N s u l f u r i c a cid/3 M urea. 1. X. 1. l a e v i s 2. X. 1. bunyoniensis 3. X. 1. v i c t o r i a n u s 4. X. 1. p e t e r s i 5. X. 1. ssp. n. (Mala 6. H e r r i n g protamine Labels to the l e f t of each f i g u r e i n d i c a t e h i s t o n e s and s p e r m - s p e c i f i c b a s i c p r o t e i n s of X. 1_. l a e v i s . P= h e r r i n g protamine. 1 2 3 4 5 6 7 1 2 3 4 5 6 7 H4-H3D H1A-. H1B H3D-H1A-. SPK I H4-XI-rlii S P 6 _ w SP6 P- P-B Fig. 16 98 F i g u r e 17. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s of X. 1_. l a e v i s , and X. 1. p e t e r s i . Basic p r o t e i n s were e x t r a c t e d from whole t e s t i s homogenates and e l e c t r o p h o r e s e d as shown i n Fig.16A (channels 1 & 4). Absorbance readings were taken at 610 nm. SH= somatic h i s t o n e s TSP= t e s t i s - s p e c i f i c p r o t e i n s Fig.17 100 SP6. The H1 h i s t o n e s were not as w e l l enhanced in F i g . l 6 B as were the more a r g i n i n e - r i c h p r o t e i n s . However bands moving with the same m o b i l i t i e s as H1A and SP1 were apparent i n a l l subspecies. Fig.26 shows the absorbance p r o f i l e s of p r o t e i n s i s o l a t e d by the micromethod, from t e s t e s of animal set #2, and e l e c t r o p h o r e s e d i n a c i d / u r e a g e l s . X. 1_. p e t e r s i , X. 1^ . v i c t o r ianus and X. 1_. ssp. n. (Malawi) had h i s t o n e p r o f i l e s s i m i l a r to X. 1. l a e v i s , but X. 1_. bunyoniensis once again had an e x t r a band i n the SP3-5 r e g i o n . F i g . l 9 B shows the p r o f i l e s of spermatid/sperm and t e s t i c u l a r h i s t o n e s from animal set #3, i s o l a t e d from nuclear chromatin by the method of R i s l e y and Eckhardt (1981) and separated on a 0.375% T r i t o n X-100/2.5 M urea p o l y a c r y l a m i d e g e l . The somatic h i s t o n e s appeared i n t a c t and w e l l r e s o l v e d . Comparison of spermatid/sperm b a s i c nuclear p r o t e i n s with those i s o l a t e d from the e r y t h r o c y t e s of the same animals showed that bands moving f a s t e r than H1D i n the T r i t o n g e l system were s p e c i f i c to the sperm of each subspecies (data not shown). Each of the s i x subspecies c o u l d be d i s t i n g u i s h e d from the others on the b a s i s of the m o b i l i t i e s of t h e i r s p e r m - s p e c i f i c b a s i c p r o t e i n s . The SP2's of X. 1. p e t e r s i , X. 1. v i c t o r i a n u s , X. 1. bunyoniensis and X. 1_. sudanensis a l l had f a s t e r m o b i l i t i e s than those of X. 1. l a e v i s and X. 1. ssp. n. (Malawi). X. 1. bunyoniensis and X. 1. sudanensis showed four bands i n the SP3-5 region r a t h e r than the three 101 F i g u r e 18. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s of X. 1_. ssp. n. (Malawi), X. 1_. v i c t o r i a n u s and X. 1. b u n y o n i e n s i s . B a s i c p r o t e i n s were e x t r a c t e d from whole t e s t i s homogenates and e l e c t r o p h o r e s e d as shown i n Fig.16B (channels 2, 3 & 5). Absorbance readings were taken at 610 nm. Arrows i n d i c a t e b a s i c p r o t e i n s s p e c i f i c to the t e s t e s of X. 1. bun y o n i e n s i s . SH= somatic h i s t o n e s TSP= t e s t i s - s p e c i f i c p r o t e i n s X.l .v ictor ianus Fig.18 103 F i g u r e 19. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s and spermatid/sperm b a s i c nuclear p r o t e i n s from the genus Xenopus. Basic p r o t e i n s were e x t r a c t e d from chromatin prepared from e r y t h r o c y t e s , whole t e s t e s and spermatid/sperm f r a c t i o n s of t e s t e s from animal set #3 ( f r e s h t i s s u e ) . E l e c t r o p h o r e s i s was performed i n 28 cm pol y a c r y l a m i d e s l a b g e l s (12% acrylamide, 2.5 M urea, 0.375% T r i t o n X-100) f o r 12 hr at 190 V. S t a i n i n g was i n 0.1% Coomassie Blue and d e s t a i n i n g i n 25% methanol/7% a c e t i c a c i d . A. 1.X. 1. l a e v i s sperm 2. X. 1. l a e v i s t e s t e s 3. X. b o r e a l i s sperm 4. X. m u e l l e r i sperm 5. X. t r o p i c a l i s t e s t e s 6. X. sjo. n. I l l ( Z a i r e ) t e s t e s 7. X. ruwenzoriensis sperm 8. X. v e s t i t u s t e s t e s 9. X. a m i e t i sperm 10. X. w i t t e i sperm B. 1.X. 1. l a e v i s e r y t h r o c y t e s 2. X. 1. l a e v i s t e s t e s 3. X. 1. p e t e r s i sperm 4. X. 1_. v i c t o r ianus sperm 5. X. 1. bunyoniensis t e s t e s 6. X. 1_. sudanensis t e s t e s 7. X. 1. ssp. ni (Malawi) sperm 8. X. c l i v i i sperm 9. X. SJD. n. I l l ( Z a i r e ) sperm 10. X. f r a s e r i sperm La b e l s to the l e f t of each f i g u r e i n d i c a t e h i s t o n e s and sperm b a s i c nuclear p r o t e i n s of X. _1. l a e v i s . TSP= t e s t i s - s p e c i f i c p r o t e i n s (seen only i n e x t r a c t s of whole t e s t e s ) . o o o CO K o i n CO a! CN CO N o to I CO CN | I Q_ I/) I III I I I I I I III III I I I III Itttf I I I ' 1 I 1 t IJ, i N t 1 ( N ^ X X I X a. u a. to co I X i IV* I III I III I l i t l i l t I I I I I I I t • i i i i 11 i in i i i i i • $ \ \ h \ I t • • • n a. It! I f CN co CN ^ r i S - a. X X X X X X X t o i co Da CO CO o a. io I 105 bands r e s o l v e d i n X. 1. l a e v i s , X. ^L. p e t e r s i and X. 1. v i c t o r i a n u s . X. 1. ssp. n. (Malawi) showed three bands i n t h i s r e g i o n , but the f a s t e s t band had a m o b i l i t y s l i g h t l y g r e a t e r than SP5 of X. 1. l a e v i s . F u r t h e r s u b s p e c i e s - s p e c i f i c d i f f e r e n c e s were seen i n the SP6 r e g i o n . In X. 1. p e t e r s i , SP6 was r e s o l v e d as a t r i p l e t as opposed to the dou b l e t s seen i n X. 1_. l a e v i s , X. 1_. v i c t o r ianus, X. 1. bunyoniensis and X. 1. ssp. n. (Malawi). X. 1. sudanensis showed a strong doublet i n t h i s r e g i o n , r a t h e r than the strong and weak components seen i n the other subspecies, and a weak band moving ahead of SP6 c o u l d a l s o be seen. I t was a l s o i n t e r e s t i n g to note that i n the spermatid/sperm p r e p a r a t i o n s of X. 1_. p e t e r s i , X. 1_. v i c t o r ianus and X. 1. ssp. n. (Malawi) the somatic h i s t o n e s H2A and H2B were present i n amounts equal t o , or only s l i g h t l y reduced from the amounts of H3 and H4. These two hi s t o n e s were g r e a t l y reduced i n the sperm prepared from X. 1. l a e v i s ( F i g . l 9 A , channel 1 and R i s l e y , 1977). Bands which comigrated with H1D from X. 1. l a e v i s e r y t h r o c y t e s , were present i n the spermatid/sperm f r a c t i o n s of X. 1. p e t e r s i , X. 1. v i c t o r i a n u s and X. 1. ssp. n. (Malawi), and were e s p e c i a l l y e vident i n the whole t e s t i s p r e p a r a t i o n s of X. 1_. bunyoniensis and X. 1. sudanensis. However, s l i g h t d i f f e r e n c e s were evident i n the other H1 h i s t o n e s of the va r i o u s subspecies. H1A and H1B were much reduced compared with those seen i n e r y t h r o c y t e s , but three f a i n t bands c o u l d be seen mi g r a t i n g i n the region of H1B and SP1. These may represent 1 06 s p e c i e s - s p e c i f i c v a r i a n t s of H1A, H1B and SP1, s p e r m - s p e c i f i c v a r i a n t s of H1B, or v a r i a n t s of SP1 . c. X e n o p u s s p e c i e s P r o f i l e s of b a s i c p r o t e i n s i s o l a t e d from nuclear chromatin of spermatid/sperm f r a c t i o n s and whole t e s t e s , or from t e s t i s homogenates of v a r i o u s Xenopus s p e c i e s are shown i n Fig.19 (animal set #3), Figs.20-25 (animal set #1), and Fig.26 (animal set #2). Each s p e c i e s c o u l d be d i s t i n g u i s h e d from the others by the m o b i l i t i e s of these p r o t e i n s i n T r i t o n and a c i d / u r e a g e l s , and more dramatic d i f f e r e n c e s were seen between the v a r i o u s s p e c i e s than were seen between the subspecies of Xenopus  l a e v i s . E r y t h r o c y t e h i s t o n e s from each s p e c i e s , i s o l a t e d from chromatin by the method of R i s l e y and Eckhardt (1981), showed t y p i c a l somatic p r o f i l e s ( F i g . l 9 A , channel 1), with no bands m i g r a t i n g f a s t e r than H1D i n T r i t o n g e l s , or H4 i n a c i d / u r e a g e l s . Of the s p e c i e s with 2n=36 chromosomes, the s p e r m - s p e c i f i c h i s t o n e s of X. f r a s e r i were the most l i k e those of X. 1_. l a e v i s . In the T r i t o n g e l shown i n F i g . l 9 B (channel 10), a strong doublet was r e s o l v e d having the same m o b i l i t y as SP3-5, as was a s i n g l e band with the same m o b i l i t y as SP6. However, a band resembling SP2 was not p r e s e n t . In the H1 r e g i o n , there was a band with the m o b i l i t y of H1D and one resembling H1A. The l a t t e r may a l s o have been a s p e c i e s - s p e c i f i c v a r i a n t of SP1. Fig.22B (channel 5) and Figs.24 and 26 show the a c i d / u r e a g e l 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 with s u l f u r i c a c i d from 107 F i g u r e 20. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s b a s i c p r o t e i n s from f i v e Xenopus sp e c i e s . B a s i c p r o t e i n s were e x t r a c t e d d i r e c t l y from whole t e s t e s ( s t o r e d at -70°C) homogenized in PBS. T i s s u e was obtained from animal set #1. E l e c t r o p h o r e s i s was performed i n a 20 cm polyacrylamide s l a b g e l (12% acrylamide, 2.5 M urea, 0.4% T r i t o n X-100) f o r 6 hr at 220 V. A. Gel s t a i n e d with 0.2% Amido Black and d e s t a i n e d i n 30% methanol/10% a c e t i c a c i d . B. Same g e l r e s t a i n e d with 0.2% Amido Black and d e s t a i n e d i n 1M s u l f u r i c acid/3 M urea. 1 . X. 1^ . l a e v i s 2. X. b o r e a l i s 3. X. m u e l l e r i 4. X. c l i v i i 5. X. w i t t e i Labels to the l e f t of each f i g u r e i n d i c a t e h i s t o n e s and s p e r m - s p e c i f i c b a s i c nuclear p r o t e i n s (SP's) of X. 1. l a e v i s . Fig. 20 109 F i g u r e 21. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from f i v e Xenopus s p e c i e s . Basic p r o t e i n s were e x t r a c t e d from whole t e s t i s homogenates and e l e c t r o p h o r e s e d as shown i n Fig.20B. Absorbance readings were taken at 610 nm. X.I.laevis SP2 H4 A SPI U\J\ i X.borealis X.muelleri X.clivii X.wittei L i 21 111 F i g u r e 22. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s b a s i c p r o t e i n s from nine Xenopus s p e c i e s . Testes ( s t o r e d at -70°C) were obtained from animal set #1 and b a s i c p r o t e i n s were e x t r a c t e d d i r e c t l y from whole t i s s u e homogenates. E l e c t r o p h o r e s i s was performed in 15% p o l y a c r y l a m i d e s l a b g e l s c o n t a i n i n g 6.25 M urea and s t a i n i n g was i n 0.2% Amido Black. A. Gel (20 cm) e l e c t r o p h o r e s e d f o r 8.5 hr at 200 V and d e s t a i n e d i n 30% methanol/10% a c e t i c a c i d . B. Same g e l r e s t a i n e d i n 0.2% Amido Black and d e s t a i n e d i n 1 M s u l f u r i c acid/3 M urea. C. Gel (15 cm) e l e c t r o p h o r e s e d f o r 6 hr at 220 V and d e s t a i n e d i n 30% methanol/10% a c e t i c a c i d . A. & B. 1. X. 1. l a e v i s 2. X. b o r e a l i s 3. X. m u e l l e r i 4. X. c l i v i i 5. X. f r a s e r i 6. X. t r o p i c a l i s C. 1.X. ruwenzoriensis 2. X. t r o p i c a l i s 3. X. w i t t e i 4. X. v e s t i t u s 5. X. c l i v i i 6. X. 1. l a e v i s L a b e l s i n d i c a t e somatic h i s t o n e s and s p e r m - s p e c i f i c b a s i c n u c l e a r p r o t e i n s (SP's) of X. 1. l a e v i s . X1= p o s i t i o n of contaminant e x t r a c t e d from t e s t i s homogenates of X. 1. l a e v i s . Fig . 2 2 1 1 3 Figu r e 23. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from X. 1. l a e v i s , X. b o r e a l i s and X. m u e l l e r i . Basic p r o t e i n s were e x t r a c t e d from whole t e s t i s homogenates and e l e c t r o p h o r e s e d as shown i n Fig.22A (channels 1, 2 & 3). Absorbance readings were taken at 610 nm. SH= somatic h i s t o n e s TSP= t e s t i s - s p e c i f i c p r o t e i n s X. I.laevis X.borealis X.muelleri Fig.23 115 F i g u r e 24. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from X. c l i v i i , X. f r a s e r i and X. t r o p i c a l i s . B a s i c p r o t e i n s were e x t r a c t e d from whole t e s t i s homogenates and e l e c t r o p h o r e s e d as shown i n Fig.22B (channels 4, 5 & 6). Absorbance readings were taken at 610 nm. SH= somatic h i s t o n e s TSP= t e s t i s - s p e c i f i c p r o t e i n s * i n d i c a t e s t e s t i s - s p e c i f i c p r o t e i n m i g r a t i n g i n the somatic h i s t o n e r e g i o n . 'X1' i n d i c a t e s p o s i t i o n of contaminant e x t r a c t e d from t e s t i s homogenates of X. 1. l a e v i s . Fig . 24 1 17 F i g u r e 25. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from X. v e s t i t u s , X. w i t t e i and X. ru w e n z o r i e n s i s . Basic p r o t e i n s were'extracted from whole t e s t i s homogenates and e l e c t r o p h o r e s e d as shown i n Fig.22C (channels 1, 3 & 5). Absorbance readings were taken at 610 nm. SH= somatic h i s t o n e s TSP= t e s t i s - s p e c i f i c p r o t e i n s * i n d i c a t e s t e s t i s - s p e c i f i c b a s i c p r o t e i n s m i g r a t i n g i n the somatic h i s t o n e r e g i o n . SH |- T 5 P X.vest i tus .25 119 F i g u r e 26. Absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from the genus Xenopus. Testes ( s t o r e d at -70°C) were obtained from animal set #2 and b a s i c p r o t e i n s were e x t r a c t e d d i r e c t l y from whole t i s s u e homogenates. E l e c t r o p h o r e s i s was performed i n 25 cm p o l y a c r y l a m i d e s l a b g e l s c o n t a i n i n g 6.25 M urea, f o r 28 hr at 130 V. S t a i n i n g was i n 0.2% Amido Black and d e s t a i n i n g i n 30% methanol/10% a c e t i c a c i d . Absorbance readings were taken at 610 nm. SH= somatic h i s t o n e s TSP= t e s t i s - s p e c i f i c p r o t e i n s Arrows i n d i c a t e the p o s i t i o n of h i s t o n e H4. * i n d i c a t e s t e s t i s - s p e c i f i c b a s i c p r o t e i n s m i g r a t i n g i n the somatic h i s t o n e r e g i o n . - s m * T S P SP3-5 H4 Y SP6 X . I . I a e v i s © — X . b o r e a l i s X . m u e l l e r i L A X . I. l a e v i s X . I. v i c t o r i a n u s X . I. b u n y o n i e n s i s X . f r a s e n •TSP' SP3-5 © - 0 X . v e s t i t u s X . I. l a e v i s X . r u w e n i o r i c n s i s Fig.26 121 t e s t e s homogenized i n PBS. The fast-moving bands of X. f r a s e r i appeared very f a i n t i n the g e l shown i n Fig.22B and they were s l i g h t l y r e t a r d e d with r e s p e c t to SP3-5 and SP6 of X. 1. l a e v i s , but the same general tendency was apparent. The sperm of X. sp. n. II ( E t h i o p i a ) ( F i g . l 9 B , channel 9) con t a i n e d b a s i c p r o t e i n s which were s i m i l a r to those of X. 1.. sudanensis i n the T r i t o n g e l system. In the SP3-5 r e g i o n , three bands were seen, the darkest of which migrated c l o s e to SP5. A dark band with a m o b i l i t y c l o s e to that of SP6, and a f a s t e r moving component s i m i l a r to that seen i n X. 1. sudanensis, were a l s o r e s o l v e d . A very dark band, which may be a do u b l e t , comigrated with SP2. H1D was a l s o apparent, as was a component m i g r a t i n g with a m o b i l i t y s i m i l a r to SP1. From the sperm and t e s t e s of X. c l i v i i ( F i g . l 9 B , channel 8; Fig.20, channel 4; F i g . 2 1 ) , a p r o t e i n with a m o b i l i t y s i m i l a r to SP2 was r e s o l v e d i n T r i t o n g e l s , as was a band co m i g r a t i n g with SP3. A band m i g r a t i n g between SP2 and SP3, which had no counte r p a r t i n the Xenopus l a e v i s subspecies, was a l s o seen. Two bands were evident with m o b i l i t i e s s l i g h t l y f a s t e r than SP5 and a t h i r d band migrated s l i g h t l y slower than SP6. H1D and SP1 or H1B c o u l d a l s o be seen i n F i g . l 9 A . The m o b i l i t i e s of bands moving f a s t e r than H4 were s i m i l a r i n a c i d / u r e a g e l s (Fig.22A and B, channel 4; Figs.24 and 26) to the s p e r m - s p e c i f i c bands seen i n the T r i t o n g e l s . X. b o r e a l i s and X. m u e l l e r i sperm and t e s t e s c o n t a i n e d p r o t e i n s which were q u i t e d i f f e r e n t from the other Xenopus s p e c i e s . In the case of X. b o r e a l i s , a l l the s p e r m - s p e c i f i c 122 components migrated i n the r e g i o n between SP2 and SP3-5 i n T r i t o n g e l s . In F i g . l 9 A (channel 3), a s e r i e s of approximately twenty-one bands c o u l d be seen i n t h i s r e g i o n , the most prominent of which moved about half-way between SP2 and SP3 of X. 1. l a e v i s . A t r i p l e t was apparent moving ahead of SP2 and a very f a i n t doublet migrated' i n the SP3-5 r e g i o n . There were no bands evident i n the region of SP6 and there was only a f a i n t component with a m o b i l i t y s l i g h t l y slower than SP2. A s i m i l a r p a t t e r n was obtained using p r o t e i n s e x t r a c t e d from a t e s t i s homogenate (Fig.20A and B, channel 3; F i g . 2 1 ) , although there was l e s s r e s o l u t i o n of minor bands. Three major bands migrated between SP2 and SP3-5, no bands migrated f a s t e r than SP3-5, and a f a i n t doublet was seen m i g r a t i n g slower than SP2. The t e s t i s - s p e c i f i c b a s i c p r o t e i n s from X. b o r e a l i s overlapped the somatic h i s t o n e s i n a c i d / u r e a g e l s c o n t a i n i n g 6.25 M urea (Fig.22A and B, channel 2; Figs.23 and 26). A doublet c o u l d be seen l e a d i n g the H2A/H2B/H3 peak and a s i n g l e band ran j u s t behind H4. Two bands, one very dark, the other f a i n t , l e d H4. The f a s t e s t moving bands migrated midway between H4 and SP3-5. In the case of X. m u e l l e r i ( F i g . l 9 A , channel 4), a group of s i x bands c o u l d be seen i n T r i t o n g e l s , m i g r a t i n g between the f i r s t group of X. b o r e a l i s p r o t e i n s and SP3-5 of X. 1. l a e v i s • Of the two most prominent components, the slower was s i m i l a r i n m o b i l i t y to the i n t e r m e d i a t e band of X. c l i v i i . A f a i n t doublet migrated between SP3-5 and SP6. As i n X. b o r e a l i s , a f a i n t doublet was seen moving s l i g h t l y slower 123 than SP2, and no bands were apparent i n the SP6 r e g i o n . A s i m i l a r p a t t e r n was seen with the whole t e s t i s homogenate i n the T r i t o n g e l shown i n Fig.20 (channel 4) and Fig.21, and i n the a c i d / u r e a g e l s shown i n Fig.22 (channel 3) and Figs.23 and 26. Both X. m u e l l e r i and X. b o r e a l i s appeared to c o n t a i n H1D and had components which comigrated with SP1 or H1B. Sperm- and t e s t i s - s p e c i f i c h i s t o n e p r o f i l e s of the p o l y p l o i d s p e c i e s X. w i t t e i , X. v e s t i t u s and X. a m i e t i (2n=72), and X. ruwenzoriensis (2n=l08) are shown i n the T r i t o n g e l i n F i g . l 9 A (channels 7-10) and in the a c i d / u r e a g e l s i n Fig.22C and Figs.25 and 26. These p r o t e i n s had m o b i l i t i e s s i m i l a r to those of X. 1. l a e v i s although s l i g h t d i f f e r e n c e s c o u l d be seen among them a l l . Sperm of X. w i t t e i ( F i g . l 9 A , channel 10) contained a component which appeared to comigrate with, or move s l i g h t l y f a s t e r than SP2 of X. 1. l a e v i s . A doublet was seen i n the SP3-5 r e g i o n , as were two f a i n t bands moving s l i g h t l y f a s t e r than SP5. A strong band comigrated with SP6, while a l e s s prominent band moved j u s t ahead of t h i s . A very f a i n t band was a l s o apparent about half-way between SP2 and SP3, i n the region of the prominent band of X. b o r e a l i s . The p r o f i l e was s i m i l a r , a lthough not as w e l l r e s o l v e d , when a c i d e x t r a c t s from t e s t i s homogenates were used i n a c i d / u r e a g e l s (Fig.22C, channel 8; Figs.25 and 26). X. a m i e t i sperm ( F i g . l 9 A , channel 9) l a c k e d bands in the SP2 r e g i o n . A dark band comigrated with SP5 while a d i s t i n c t 124 t r i p l e t migrated s l i g h t l y f a s t e r than SP6. F a i n t bands c o u l d be seen moving j u s t behind SP3 and between SP5 and SP6. Basic p r o t e i n s prepared from the t e s t e s of X. v e s t i t u s are seen i n F i g . l 9 A (channel 8 ) . A dark doublet moved s l i g h t l y f a s t e r than SP2, the slower of which appeared to comigrate with the slowest s p e r m - s p e c i f i c band of X. w i t t e i . Only a f a i n t band c o u l d be seen i n the SP3-5 r e g i o n . A band comigrated with SP6 and a s l i g h t l y f a s t e r component was a l s o p r e s e n t . In a c i d / u r e a g e l s (Fig.22C, channel 7; Figs.25 and 26), X. v e s t i t u s showed fast-moving bands s i m i l a r i n m o b i l i t y to SP3-5 and SP6, and an e x t r a band, running between H2B and H4, which may correspond to the unique SP2 doublet seen i n Fig.1.9A. As i n X. a m i e t i , the sperm of X. ruwenzoriensis ( F i g . l 9 A , channel 7) l a c k e d SP2 or a s i m i l a r component i n T r i t o n g e l s . A band comigrated with SP5 and two bands were seen moving s l i g h t l y slower than SP3. A l s o present was a s e r i e s of f i v e bands moving i n the region of SP6'. Three of these moved f a s t e r than SP6 while the other two were s l i g h t l y slower i n m o b i l i t y . In a c i d / u r e a g e l s (Fig.22C, channel 9; Figs.25 and 26), three bands were seen near SP3-5 and two or three near SP6. As i n the case of the other s p e c i e s and subspecies examined, bands corresponding to H1D and SP1 were apparent i n the four p o l y p l o i d s p e c i e s . A l s o i n t e r e s t i n g to note was the f a c t t h a t , i n c o n t r a s t to X. 1_. l a e v i s , spermatid/sperm f r a c t i o n s prepared from the v a r i o u s Xenopus s p e c i e s a l l appeared to c o n t a i n s i g n i f i c a n t amounts of somatic h i s t o n e s H2A and H2B as w e l l as H3 and H4. 125 X. t r o p i c a l i s and X. sp_. n. I l l ( Z a i r e ) were not as c l o s e l y r e l a t e d , p h y l o g e n e t i c a l l y , to X. 1. l a e v i s as were the other s p e c i e s examined (Tymowska, 1973; M. F i s c h b e r g , p e r s o n a l communication). T r i t o n g e l p r o f i l e s of t h e i r sperm- and t e s t i s -s p e c i f i c h i s t o n e s are shown i n F i g . l 9 A (channels 5 and 6). These p r o f i l e s were d r a m a t i c a l l y d i f f e r e n t from those found i n the r e s t of the genus. There were no components moving i n the region f a s t e r than'the H1 h i s t o n e s . In both s p e c i e s , a s i n g l e dark, s p e r m - s p e c i f i c band c o u l d be seen m i g r a t i n g s l i g h t l y slower than H1D. X. sp_. n. I l l ( Z a i r e ) had an a d d i t i o n a l component m i g r a t i n g s l i g h t l y slower than SP2. Both s p e c i e s had bands m i g r a t i n g i n the region of SP1, although a s l i g h t l y slower v a r i a n t was a l s o seen i n X. sp. n. I l l ( Z a i r e ) . The nucleosomal h i s t o n e s were a l l present i n roughly equal amounts. The a c i d / u r e a g e l p r o f i l e of t e s t i c u l a r b a s i c p r o t e i n s of X. t r o p i c a l i s , i s o l a t e d by the micromethod, i s seen i n Fig.22B (channel 6) and Fig.24. A s i n g l e f a i n t fast-moving component comigrated with "X1", a band o b t a i n e d by the micromethod from X. 1^ . l a e v i s t e s t e s . The appearance of t h i s band, which may be a cytoplasmic contaminant, was one of the l i m i t a t i o n s of the micromethod, as i t was not seen when h i s t o n e s were prepared from n u c l e a r chromatin by the method of R i s l e y and Eckhardt (1981). However. Rana t e s t e s , which a l s o have s o m a t i c - l i k e h i s t o n e s , do not show 'X1' when b a s i c p r o t e i n s are i s o l a t e d by the micromethod (Fig.28B). Thus 'X1' appears to be a Xenopus s p e c i f i c p r o t e i n contaminant. An a d d i t i o n a l band c o u l d be seen j u s t behind the 1 26 H2A/H2B/H3 peak i n the somatic h i s t o n e r e g i o n . T h i s band may correspond to the s p e r m - s p e c i f i c band seen i n the H1 region of the T r i t o n g e l ( F i g . l 9 A ) . d. Other Anura Figs.27 and 28 show the s t a r c h g e l and a c i d / u r e a p o l y a c r y l a m i d e g e l p r o f i l e s of b a s i c p r o t e i n s i s o l a t e d from whole t e s t i s homogenates of v a r i o u s s p e c i e s of Anura. The s t a r c h g e l electrophoretograms confirmed the d i f f e r e n c e s i n m o b i l i t y of t e s t i s - s p e c i f i c h i s t o n e s from X. 1. l a e v i s and X. b o r e a l i s (Fig.27B) and r e v e a l e d s p e c i e s - s p e c i f i c d i f f e r e n c e s in the genus Scaphiopus, but not i n Rana, Bufo or Hyla. Testes of Scaphiopus bombifrons (Fig.27A, channel 2) produced s e v e r a l bands that moved more r a p i d l y than those of Scaphiopus couchi (channel 1). The former resembled the e l e c t r o p h o r e t i c p r o f i l e of Bufo (channels 3 and 4). The three congeneric s p e c i e s of Bufo shown i n Fig.27 d i s p l a y e d s i m i l a r e l e c t r o p h o r e t i c p r o f i l e s i n s t a r c h g e l s . Bufo  boreas (Fig.27C, channel 2), Bufo bufo h a l o p h i l u s (Fig.27A, channel 3) and Bufo a l v a r i u s (Fig.27A, channel 4) a l l had one band which migrated i n the r e g i o n of t r o u t protamine and, o c c a s i o n a l l y , two or three slower moving bands which may represent phosphorylated d e r i v a t i v e s of the main band, as seen in the t r o u t (Louie and Dixon, 1972; Dixon, 1974). In a c i d / u r e a p o l y a c r y l a m i d e g e l s ( F i g . 2 8 ) , the t e s t i s - s p e c i f i c h i s t o n e p r o f i l e s of Bufo marinus and Bufo punctatus d i s p l a y e d a s i n g l e fast-moving component with a m o b i l i t y s l i g h t l y slower than 127 Fi g u r e 27. S t a r c h g e l electrophoretograms of t e s t i s b a s i s p r o t e i n s from congeneric s p e c i e s of anurans. P r o t e i n s were e x t r a c t e d d i r e c t l y from homogenates of t e s t e s from i n d i v i d u a l animals. A. 1. Scaphiopus couchi (S.c.) 2. Scaphiopus bombifrons (S.b.) 3. Bufo bufo h a l o p h i l u s (B) 4. Bufo a l v a r i u s (B) 5. C a l f thymus H4 B. 1. Trout t e s t i s b a s i c p r o t e i n s (T & P) 2. X. 1. l a e v i s ( X . l . ) 3. C a l f thymus H4 4. X. b o r e a l i s C. 1.& 7. C a l f thymus H4 2. Bufo boreas (B) 3. Hyla regi11a l i v e r 4. Hyla g r a t i o s a (H.g.) 5. X. 1. l a e v i s (X) 6. Hyla r e q i l l a (H.r.) D. 1. C a l f thymus H4 2.& 3. Rana p r e t i o s a (R) 4. Rana c a t e s b e i a n a (R) 5. Rana p i p i e n s (R) 6. Rana c l a m i t a n s (R) T= high m o b i l i t y group p r o t e i n "T" from Salmo g a i r d n e r i i ( t r o u t ) t e s t i s P= Salmo g a i r d n e r i i protamine 129 F i g u r e 28. E l e c t r o p h o r e t i c p r o f i l e s of b a s i c p r o t e i n s from anuran t e s t e s . P r o t e i n s were e x t r a c t e d with 0.4 N s u l f u r i c a c i d from whole t e s t e s homogenized i n PBS. E l e c t r o p h o r e s i s was performed i n 15% p o l y a c r y l a m i d e g e l s c o n t a i n i n g 6.25 M urea. G e l s were s t a i n e d with 0.2% Amido Black and d e s t a i n e d i n 30% methanol/10% a c e t i c a c i d . A. Gel (15 cm) e l e c t r o p h o r e s e d f o r 2 hr at 200 V. 1. & 3. X. 1. l a e v i s 2. H e r r i n g protamine (P) 4. Bufo marinus (B) B. Gel (15 cm) e l e c t r o p h o r e s e d f o r 3 hr at 150 V. 1. Bufo mar inus (B) 2. Bufo punctatus (B) 3. X. 1. l a e v i s (heart) 4. Rana ca t e s b e i a n a 5. X. 1. l a e v i s ( t e s t e s ) C. Gel (25 cm) e l e c t r o p h o r e s e d f o r 23 hr at 150 V. 1. Hyla r e q i l l a (Hr) 2. Bufo punctatus (B) SP3-5 and SP6 i n d i c a t e the p o s i t i o n s of the sperm-s p e c i f i c b a s i c nuclear p r o t e i n s of X. .1. l a e v i s . SH= somatic h i s t o n e s I 3 O 131 t r o u t protamine. T h i s was a l s o the case with Bufo americanus (Bois and Kasinsky, 1973; Kasinsky e t . a l . , 1978). Testes of Hyla g r a t i o s a and Hyla r e g i l l a gave s i m i l a r b a s i c p r o t e i n p r o f i l e s i n s t a r c h g e l s (Fig.27C, channels 4 and 6), with three or four bands moving s l i g h t l y ahead of H4. The p r o t e i n s of Hyla r e g i l l a t e s t e s were f u r t h e r r e s o l v e d i n a c i d / u r e a p o l y a c r y l a m i d e g e l s (Fig.28C) i n t o e i g h t components. Three of these migrated s l i g h t l y f a s t e r than H4, while the other f i v e bands had m o b i l i t i e s i n the region of SP3-5 of X. 1^ . l a e v i s . The four s p e c i e s of Rana shown i n Fig.27D had t e s t i s -s p e c i f i c h i s t o n e p r o f i l e s s i m i l a r to somatic c e l l s , with no bands m i g r a t i n g f a s t e r than H4 i n the s t a r c h g e l system. T h i s was confirmed, i n the case of Rana c a t e s b e i a n a t e s t e s , by e l e c t r o p h o r e s i s i n a c i d / u r e a p o l y a c r y l a m i d e g e l s (Fig.28B). 132 D. DISCUSSION In agreement with the o b s e r v a t i o n s of Bois and Kasinsky (1973, 1974) and Kasinsky et a l . (1978), the present study r e v e a l e d that sperm- and t e s t i s - s p e c i f i c b a s i c nuclear p r o t e i n s d i s p l a y remarkable d i v e r s i t y i n the order Anura. Sperm h i s t o n e s of the genera Bufo, Hyla, Rana and Xenopus had cytochemical and e l e c t r o p h o r e t i c p r o p e r t i e s which were d i s t i n c t i v e f o r each genus, and i n the genus Xenopus, the d i v e r s i t y of sperm h i s t o n e s c o u l d be used to d i s t i n g u i s h subspecies, s p e c i e s , and perhaps higher l e v e l s of c l a s s i f i c a t i o n . C y t o c h e m i c a l l y , the genus Xenopus can be d i v i d e d i n t o two groups. X. t r o p i c a l i s c o n t a i n e d s o m a t i c - l i k e sperm h i s t o n e s while a l l other species and subspecies had more b a s i c nuclear p r o t e i n s of the intermediate type. E l e c t r o p h o r e s i s confirmed t h i s o b s e r v a t i o n and showed that s o m a t i c - l i k e sperm h i s t o n e s were a l s o found i n X. sp_. n. I l l ( Z a i r e ) , a p o l y p l o i d s p e c i e s c l o s e l y r e l a t e d to X. t r o p i c a l i s (M. F i s c h b e r g , p e r s o n a l communication). The chromosome number and karyotype of X. t r o p i c a l i s d i f f e r s t r i k i n g l y from other Xenopus sp e c i e s (Tymowska, 1973; Tymowska and F i s c h b e r g , 1973), and s t u d i e s with serum albumins r e v e a l e d a great immunological d i s t a n c e between X. t r o p i c a l i s and the r e s t of the genus (Bisbee et a l . , 1977). Sperm h i s t o n e a n a l y s i s supported the c l o s e r e l a t i o n s h i p of X. t r o p i c a l i s to X. sp_. n. I l l ( Z a i r e ) , as w e l l as the p h y l o g e n e t i c d i s t a n c e of these s p e c i e s from the other Xenopus taxa. The c l o s e r e l a t i o n s h i p of the subspecies of Xenopus l a e v i s 133 was r e f l e c t e d i n the o v e r a l l s i m i l a r i t y of t h e i r sperm h i s t o n e p r o f i l e s . However minor d i f f e r e n c e s enable one to d i s t i n g u i s h each of the subspecies from the o t h e r s . T h i s i s e s p e c i a l l y important with regard to X. 1. bunyoniensis, whose l a c t a t e dehydrogenase p r o f i l e i s i d e n t i c a l to that of X. 1. v i c t o r i a n u s (Vonwyl and F i s c h b e r g , 1980). The extent to which the minor e l e c t r o p h o r e t i c bands seen here r e f l e c t a l l e l i c v a r i a t i o n or s i d e - c h a i n m o d i f i c a t i o n s of p r o t e i n s i s as yet unknown. Both f a c t o r s are known to i n f l u e n c e the sperm h i s t o n e p r o f i l e of X. _1. l a e v i s . A l l e l i c v a r i a t i o n has been noted i n SP2 ( R i s l e y and Eckhardt, 1979c), and p h o s p h o r y l a t i o n occurs i n SP3-5 and SP6 ( R i s l e y , 1977). However s i d e - c h a i n m o d i f i c a t i o n s of t h i s l a t t e r type r e t a r d the m o b i l i t i e s of b a s i c p r o t e i n s i n p o l y a c r y l a m i d e g e l s , while X. 1. bunyoniensis c o n s i s t e n t l y showed a band which migrates s l i g h t l y f a s t e r than the SP3-5-l i k e p r o t e i n s of the other Xenopus l a e v i s subspecies. Of the s p e c i e s with 2n=36 chromosomes, the b a s i c p r o t e i n s of X. b o r e a l i s , X. m u e l l e r i and X. c l i v i i sperm n u c l e i d i s p l a y e d e l e c t r o p h o r e t i c m o b i l i t i e s which were d i s t i n c t l y d i f f e r e n t from those of Xenopus l a e v i s . The p o s i t i o n s of the n u c l e o l a r o r g a n i z e r s i n these s p e c i e s l e d Tymowska and F i s c h b e r g (1973) to suggest that X. m u e l l e r i and X. b o r e a l i s may be more c l o s e l y r e l a t e d to each other, and p o s s i b l y to X. c l i v i i , than to the Xenopus l a e v i s group. The c l o s e a f f i n i t y of X. m u e l l e r i and X. b o r e a l i s i s a l s o supported by a study of b i v a l e n t formation d u r i n g m e i o t i c d i p l o t e n e of h y b r i d oocytes ( M u l l e r , 1977). S i m i l a r r e l a t i o n s h i p s are suggested when we 1 34 compare the sperm h i s t o n e p r o f i l e s of these s p e c i e s ( F i g . 1 9 ) . In g e n e r a l , the m o b i l i t i e s of these p r o t e i n s are slower than those found i n the Xenopus l a e v i s group and there i s some resemblance between the p r o f i l e s of X. m u e l l e r i and X. c l i v i i . X. sp_. n. II ( E t h i o p i a ) had a sperm h i s t o n e p r o f i l e which s t r o n g l y resembled that of the Xenopus l a e v i s group, i n c l u d i n g the presence of SP2. The taxonomic r e l a t i o n s h i p of t h i s s p e c i e s to the r e s t of the genus has yet" to be worked out by h y b r i d i z a t i o n and other b i o c h e m i c a l s t u d i e s . However the e l e c t r o p h o r e t i c m o b i l i t i e s of i t s sperm h i s t o n e s p o i n t s to a c l o s e r a f f i n i t y to the Xenopus l a e v i s group than to other s p e c i e s i n the genus. X. f r a s e r i possesses a unique LDH-A gene (Vonwyl and F i s c h b e r g , 1980) and a p a r t i c u l a r type of n u c l e o l a r o r g a n i z e r (Tymowska and F i s c h b e r g , 1973). H y b r i d i z a t i o n s t u d i e s a l s o suggest that t h i s s p e c i e s may be rather d i s t a n t to the other Xenopus taxa. While i t s sperm h i s t o n e p r o f i l e resembled that of Xenopus l a e v i s , l a c k of SP2 p o i n t s to at l e a s t some divergence of these two groups. The p o l y p l o i d s p e c i e s X. w i t t e i , X. v e s t i t u s and X. am i e t i (2n=72), as w e l l as X. ruwenzoriensis (2n=l08), d i s p l a y e d sperm h i s t o n e p r o f i l e s which were s i m i l a r to the Xenopus l a e v i s group. K a r y o l o g i c a l s t u d i e s have r e v e a l e d s t r o n g s i m i l a r i t i e s between X. w i t t e i and X. v e s t i t u s (Tymowska and F i s c h b e r g , 1980), r e f l e c t e d here, i n the presence of a s i m i l a r S P 2 - l i k e component i n the sperm of both s p e c i e s . These s p e c i e s are b e l i e v e d to be a l l o t e t r a p l o i d s d e r i v e d from d i p l o i d i z a t i o n 1 35 of a h y b r i d genome, and f e a t u r e s of t h e i r karyotypes suggest that they may p o s s i l b l y share a common ancestor from the Xenopus l a e v i s group (Tymowska and F i s c h b e r g , 1980; Tymowska et a l . , 1978). Sperm h i s t o n e p r o f i l e s , which resembled those of the Xenopus l a e v i s subspecies, support t h i s c o n t e n t i o n and p o i n t to the p o s s i b i l i t y of a Xenopus l a e v i s - l i k e ancestor i n the case of X. a m i e t i and X. ruwenzoriensis as w e l l . On the b a s i s of m o r p h o l o g i c a l s i m i l a r i t i e s , a r e l a t i o n s h i p between X. f r a s e r i , X. a m i e t i and X. ruwenzoriensis has a l s o been suggested (Kobel et a l . , 1980). S i m i l a r i t i e s i n the SP3-5 and S P 6 - l i k e sperm p r o t e i n s and l a c k of an S P 2 - l i k e component in a l l three s p e c i e s may r e f l e c t t h i s r e l a t i o n s h i p . C o n s i d e r a b l e divergence of the p o l y p l o i d Xenopus s p e c i e s i s r e f l e c t e d i n the r e d u c t i o n of SP3-5 i n X. v e s t i t u s , the apparent l a c k of SP2 i n X. a m i e t i and X. ruwenzoriensis and i n the m u l t i p l e banding p a t t e r n s seen i n the SP3-5 and SP6 region of each s p e c i e s . I f these s p e c i e s are i n f a c t the products of a l l o p o l y p l o i d y , such h e t e r o g e n e i t y of p r o t e i n s would be expected i f genes from both a n c e s t r a l parent s p e c i e s have d i v e r g e d and are expressed. A s i m i l a r s i t u a t i o n has been noted with the d i v e r g e n t oC- and/^-globin genes of X. 1_. l a e v i s and X. t r o p i c a l i s . The X. 1. l a e v i s genes appear to have a r i s e n by d u p l i c a t i o n and divergence of a s i n g l e gene p a i r , observed i n X. t r o p i c a l i s , p o s s i b l y by t e t r a p l o i d i z a t i o n of a X. t r o p i c a l i s - l i k e ancestor ( J e f f r e y s et a l . , 1980). The minor e l e c t r o p h o r e t i c bands seen i n the sperm h i s t o n e p r o f i l e s of the v a r i o u s Xenopus s p e c i e s may r e f l e c t , at l e a s t 136 i n p a r t , a l l e l i c v a r i a t i o n , s i d e - c h a i n m o d i f i c a t i o n s of p r o t e i n s and perhaps even n a t u r a l degradation products of other chromosomal p r o t e i n s which are d i s p l a c e d d u r i n g the course of spermiogenesis. S t u d i e s such as those performed by R i s l e y (1977) and R i s l e y and Eckhardt (1979a) on X. 1. l a e v i s , using s e p a r a t i o n of t e s t i s c e l l s i n t o v a r i o u s spermatogenic stages and i n c o r p o r a t i o n of r a d i o a c t i v e amino a c i d s i n t o sperm s p e c i f i c h i s t o n e s , should h e l p to d i s t i n g u i s h between these p o s s i b i l i t i e s . However i t i s apparent from the data presented here that sperm h i s t o n e s may be u s e f u l markers f o r i d e n t i f y i n g p a r t i c u l a r s p e c i e s and even subspecies i n the genus Xenopus. In the genus Rana, only s o m a t i c - l i k e h i s t o n e s have been observed i n t e s t e s and sperm. In Rana p i p i e n s , a major H1 f r a c t i o n has been found i n the t e s t i s which i s not present i n l i v e r (Alder and Gorovsky, 1975). Perhaps s p e c i e s - s p e c i f i c d i f f e r e n c e s i n H1 or other h i s t o n e m o b i l i t i e s may be noted i f Rana t e s t i c u l a r h i s t o n e s were to be separated on longer p o l y a c r y l a m i d e g e l s c o n t a i n i n g SDS. T h i s system has r e v e l e d d i f f e r e n c e s i n the sperm H1's and H2B's of v a r i o u s sea u r c h i n s p e c i e s (de P e t r o c e l l i s et a l . , 1980). Cytochemistry showed that sperm h i s t o n e s of Bufo, Hyla and Scaphiopus, l i k e those of most Xenopus s p e c i e s , were of the intermediate type, c o n t a i n i n g both a r g i n i n e and l y s i n e . However e l e c t r o p h o r e s i s r e v e a l e d great d i f f e r e n c e s between the t e s t i s -s p e c i f i c h i s t o n e s of these genera (Figs.27 and 28). L i k e the e l e c t r o p h o r e t i c d i f f e r e n c e s noted among Xenopus s p e c i e s , the two s p e c i e s of Scaphiopus used in t h i s study d i s p l a y e d 1 37 d i f f e r e n t e l e c t r o p h o r e t i c p r o f i l e s i n s t a r c h g e l s . Polyacrylamide g e l e l e c t r o p h o r e s i s may provide b e t t e r r e s o l u t i o n of these p r o t e i n s . A l s o , although the t e s t i s -s p e c i f i c h i s t o n e p r o f i l e s of Hyla r e g i l l a and Hyla g r a t i o s a appeared s i m i l a r i n s t a r c h g e l s , g r e a t e r r e s o l u t i o n of these p r o t e i n s appears to be p o s s i b l e on long p o l y a c r y l a m i d e g e l s and perhaps s p e c i e s - s p e c i f i c d i f f e r e n c e s i n sperm h i s t o n e s may be found in t h i s genus as w e l l . T e s t i s - s p e c i f i c b a s i c p r o t e i n s of Bufo resembled the protamines of f i s h i n t h e i r r a p i d e l e c t r o p h o r e t i c m o b i l i t y (Bois and Kasinsky, 1972; Kharchencko and N a l i v a e v a , 1979), h e t e r o g e n e i t y i n s t a r c h g e l s and high content of a r g i n i n e (45 mole%; Huang ej: a l . , 1978). They were a l s o washed out of s e c t i o n s h y d r o l y s e d i n 5% TCA. However the presence of l y s i n e , as i n d i c a t e d by c y t o c h e m i s t r y and amino a c i d a n a l y s i s (Huang, 1977), d i s t i n g u i s h e s Bufo protamines from those of salmonoid f i s h (Ando e_t a_l. , 1973) and p l a c e s them i n the i n t e r m e d i a t e - t y p e sperm h i s t o n e c a t egory. Although s p e c i e s -s p e c i f i c d i f f e r e n c e s i n the sperm h i s t o n e p r o f i l e s of v a r i o u s Bufo s p e c i e s have not been noted e l e c t r o p h o r e t i c a l l y , these p r o t e i n s may represent a f a m i l y of c l o s e l y r e l a t e d p r o t e i n s , separable by chromatography, s i m i l a r to the s i t u a t i o n seen i n salmon, h e r r i n g and tuna (Ando et a_l. , 1 973). F u r t h e r f r a c t i o n a t i o n and amino a c i d a n a l y s i s may r e v e a l s p e c i e s -s p e c i f i c d i f f e r e n c e s not r e s o l v e d i n t h i s study. The intermediate sperm h i s t o n e type i n c l u d e s p r o t e i n s of widely d i f f e r e n t e l e c t r o p h o r e t i c m o b i l i t i e s . C y t o c h e m i c a l l y , 138 these p r o t e i n s may be d i v i d e d i n t o two c l a s s e s on the b a s i s of t h e i r e x t r a c t a b i l i t y from f o r m a l i n f i x e d n u c l e i with hot 5% TCA. The sperm nuclear p r o t e i n s of Bufo, l i k e those of M y t i l u s , were e a s i l y e x t r a c t e d at 85-90°C (type 3B), while those of Xenopus, Hyla and Scaphiopus (type 3A) r e q u i r e d more r i g o r o u s e x t r a c t i o n c o n d i t i o n s ( 9 0 - 1 0 0 0 C ) . On the b a s i s of amino a c i d composition, Huang (1977) a l s o d i v i d e d the i n t e r m e d i a t e - t y p e sperm h i s t o n e s i n t o two groups, one having l e s s than 40 mole% a r g i n i n e content (type 3A, e.g. Xenopus  l a e v i s ) , and the other with g r e a t e r than 40 mole% a r g i n i n e content (type 3B, e.g. Bufo marinus). However, the major b a s i c p r o t e i n of M y t i l u s e d u l i s sperm n u c l e i , with an a r g i n i n e content of 30 mole% (Phelan et §_1. , 1974), would f a l l i n t o Huang's type 3A category w h i l e , c y t o c h e m i c a l l y , M y t i l u s sperm f a l l i n t o the type 3B c l a s s . Many f a c t o r s , such as molecular weight or the r a t i o of b a s i c to a c i d i c amino a c i d s , may i n f l u e n c e the s o l u b i l i t y of these p r o t e i n s i n 5% TCA. E x t r a c t i o n may a l s o be a f f e c t e d by p r o t e i n - p r o t e i n and p r o t e i n -DNA i n t e r a c t i o n s which determine the s t r u c t u r e of chromatin i n sperm n u c l e i and the e f f e c t s of f o r m a l i n f i x a t i o n on these i n t e r a c t i o n s . I t i s c l e a r that the i n t e r m e d i a t e - t y p e c l a s s i f i c a t i o n of sperm h i s t o n e s i s a very l o o s e one and i n c l u d e s a number of very d i f f e r e n t b a s i c p r o t e i n s which c o n t a i n l y s i n e and/or h i s t i d i n e as w e l l as a r g i n i n e . T h i s study has shown that the sperm h i s t o n e s of Anura are a very d i v e r s e group of p r o t e i n s . T h i s d i v e r s i t y i s c l e a r l y seen at the genus l e v e l and, i n at l e a s t one genus, Xenopus, at 139 the s p e c i e s and even subspecies l e v e l s . T h i s i s i n t r i g u i n g i n many r e s p e c t s , e s p e c i a l l y when viewed a g a i n s t the extreme conservatism of primary sequence i n the nucleosomal h i s t o n e s and the r e l a t i v e constancy of sperm h i s t o n e types seen i n higher v e r t e b r a t e o r d e r s , as shown i n the next chapter. Some ideas r e g a r d i n g the f u n c t i o n of these- p r o t e i n s and t h e i r r a p i d e v o l u t i o n i n Anura w i l l be d i s c u s s e d i n the f i n a l chapter of t h i s t h e s i s . 140 I I I . RELATIVE CONSTANCY OF SPERM HISTONES IN URODELA, SQUAMATA AND AVES A. INTRODUCTION In c o n t r a s t to the d i v e r s i t y of sperm h i s t o n e types seen i n t e l e o s t s (Bloch, 1969, 1976) and anurans (Chapter I I ) , the b a s i c sperm p r o t e i n s observed i n higher v e r t e b r a t e orders appear to be r e l a t i v e l y c o n s e r v a t i v e . Mature sperm of e u t h e r i a n mammals d i s p l a y only the " s t a b l e protamine" type of nuclear p r o t e i n ( B e l l v e et a l . , 1975; C a l v i n , 1975, 1976; C o e l i n g h and R o z i j n , 1975). Sperm- and t e s t i s - s p e c i f i c b a s i c p r o t e i n s from s e v e r a l l i z a r d s and snakes (Squamata) resemble each other when e l e c t r o p h o r e s e d i n s t a r c h g e l s (Kasinsky et a_l. , 1978), and those of s e v e r a l urodeles have s i m i l a r e l e c t r o p h o r e t i c m o b i l i t i e s i n p o l y a c r y l a m i d e g e l s ( P i c h e r a l , 1979). Chemical a n a l y s i s of t e s t i s - s p e c i f i c b a s i c p r o t e i n s from two snakes, Elaphe and Thamnophis, and a l i z a r d , A n o l i s , showed that a l l three had s i m i l a r amino a c i d compostions with 25-30 mole% of a r g i n i n e and 5-19 mole% of l y s i n e (Huang, 1977; Huang et a l . , 1978). The sperm h i s t o n e s of only one b i r d have been examined. Sperm of the domestic r o o s t e r , G a l l u s domesticus, c o n t a i n a monoprotamine, g a l l i n e , composed of 65 amino a c i d s , 38 of which are a r g i n i n e (Nakano et a l . , 1976a). Kasinsky et a_l. (1978) suggested that i n v e r t e b r a t e phylogeny there may be a t r e n d away from sperm h i s t o n e d i v e r s i t y i n f i s h and amphibians toward a r e l a t i v e constancy of 141 sperm h i s t o n e types i n Squamata and E u t h e r i a . T h i s study attempts to determine whether the apparent trend i s upheld i n the orders Urodela and Squamata and i n the c l a s s Aves. Cytochemistry and e l e c t r o p h o r e s i s were used to compare sperm-and t e s t i s - s p e c i f i c b a s i c nuclear p r o t e i n s from s e v e r a l u r o d e l e s , l i z a r d s and snakes. In a d d i t i o n , sperm h i s t o n e s from s e v e r a l s p e c i e s of b i r d s , from s e v e r a l Avian or d e r s , were a l s o compared. B. MATERIALS AND METHODS Experimental Animals The f o l l o w i n g urodeles were obtained i n June from Camosun Aq u a r i a , Vancouver, B.C., with the c o o p e r a t i o n of Ms. J . T a i t : Plethodon vehiculum, T a r i c h a t o r o s a , Cynops py r r h o g a s t e r and Notophthalmus v i r i d e s c e n s v i r i d e s c e n s . The l i z a r d s A n o l i s  c a r o l i n e n s i s c a r o l i n e n s i s , A n o l i s s a g r e i , Crotaphytus c o l l a r i s , Sceloporus magister, Sceloporus o l i v a c e u s and Gerrhonatus  c o e r u l e u s , and the snakes Thamnophi s s i r t a l i s , Thamnophis  c o u c h i , Thamnophis o r d i n o i d e s and Thamnophis eques, were a l s o o b tained from Camosun Aquaria dur i n g the summer months. Testes of the green ratsnake, Elaphe o b s o l e t a o b s o l e t a X o b s o l e t a  q u a d r i v i t t a t a , were a g i f t of Dr. D. M c l n t y r e and t e s t e s as w e l l as semen of Elaphe g u t t a t a g u t t a t a and Elaphe o b s o l e t a  q u a d r i v i t t a t a were donated by Dr. G. Slemmer. I d e n t i t i e s of a l l r e p t i l e s and newts were checked from standard sources (Conant, 1975; Stebbins 1966). Animals were kept i n the l a b f o r 142 no longer than one week and were s a c r i f i c e d by e t h e r i z a t i o n . T estes and sperm ducts were froz e n on dry i c e or f i x e d i n n e u t r a l b u f f e r e d f o r m a l i n or C l a r k e ' s f i x a t i v e ( a c e t i c a c i d / e t h a n o l , 3:1). S e x u a l l y mature White-leghorn c o c k e r e l s ( G a l l u s domesticus) and Japanese q u a i l (Coturnix c o t u r n i x japonica) were a c q u i r e d from the U n i v e r s i t y of B.C. p o u l t r y farm, with the a s s i s t a n c e of Dr. B. March and the l a t e Dr. CW. Roberts. Breeding budgies ( M e l o p s i t t a c u s undulatus) and c a n a r i e s (Serinus c a n a r i u s ) were obtained from Camosun Aqu a r i a . Mature Mallard/Rouen drakes (Anas p l a t y r h y n c o s ) were purchased from Dr. R. Fitzsimmons and were kept f o r s e v e r a l days on a 24 hour photoperiod to ensure maximum t e s t i c u l a r development (Romanoff, 1960, pp.53-57). Peking M a l l a r d (Anas p l a t y r h y n c o s ) t e s t e s were a g i f t of Dr. D. Jones. A male brown-headed c.owbird (Molothrus a t e r ) , captured on Vancouver I s l a n d , was a g i f t of Dr. J . Smith, and a domestic gander (Anser anser) was purchased from Willow Hobby Farms, Aldergrove, B.C. A l l b i r d s were s a c r i f i c e d by e t h e r i z a t i o n or d e c a p i t a t i o n . With the exception of the ducks, s a c r i f i c e was w i t h i n 48 hours of s e p a r a t i o n from t h e i r mates. T e s t e s and sperm ducts were d i s s e c t e d out as r a p i d l y as p o s s i b l e and were immediately p l a c e d on i c e and used d i r e c t l y or t r a n s f e r r e d to -70°C. T e s t e s used f o r c y t o c h e m i s t r y were f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n or C l a r k e ' s f i x a t i v e . T estes of a l l b i r d s , except Anser anser, were l a r g e and the d u c t i d e f e r e n t i a w e l l 143 developed, i n d i c a t i n g that these animals were i n breeding c o n d i t i o n (Romanoff, 1960, pp.52-53). Taxonomic r e l a t i o n s h i p s of animals used i n t h i s study are o u t l i n e d i n Table XI. 2. Cytochemistry Testes were f i x e d i n 10% n e u t r a l b u f f e r e d f o r m a l i n or C l a r k e ' s f i x a t i v e ( e t h a n o l / a c e t i c a c i d , 3:1) f o r p e r i o d s of one day to one month. In the case of the r o o s t e r , duck and q u a i l , some of the t i s s u e remained i n f i x a t i v e f o r up to one year before embedding. T i s s u e was washed overnight i n running tap water, dehydrated in ethanol and embedded i n Tis s u e P r e p p a r a f i n as d e s c r i b e d p r e v i o u s l y . S e c t i o n s (5-10 um) were subjected to the s t a i n i n g procedures as o u t l i n e d i n the pr e v i o u s chapter of t h i s t h e s i s . 3_. Biochemistry a. P r e p a r a t i o n of t i s s u e and i s o l a t i o n of b a s i c p r o t e i n s Basic p r o t e i n s were i s o l a t e d from whole t e s t e s or sperm ducts homogenized i n PBS or 1% c i t r i c a c i d by the micromethod of Kasinsky et a l . (1978), u s i n g a s i n g l e t e s t i s from a s i n g l e animal as d e s c r i b e d p r e v i o u s l y . In a d d i t i o n , when s u f f i c i e n t m a t e r i a l was a v a i l a b l e , as i n the case of Coturnix c o t u r n i x j a p o n i c a and Notophthalmus v i r i d e s c e n s , h i s t o n e s were i s o l a t e d from chromatin using the procedure of 144 Marushige and Bonner (1966) as m o d i f i e d by Bois et a l . (1976). Testes and h e a r t s were homogenized at 4°C i n a S o r v a l l Omni Mixer with saline-EDTA (0.075 M NaCl, 0.024 M EDTA, pH 8.0) at a speed s e t t i n g of 5 f o r 2 min, f i l t e r e d through four l a y e r s of washed c h e e s e c l o t h and c e n t r i f u g e d at 1500 x g f o r 15 min. The nu c l e a r p e l l e t was washed twice by resuspending i n saline-EDTA on a Vortex Genie mixer, c e n t r i f u g i n g as b e f o r e . The f i n a l p e l l e t was homogenized with a Dounce hand homogenizer in 0.01 M T r i s b u f f e r (pH 8.0), and chromatin was sedimented at 10,000 x g f o r 15 min and washed once with the same b u f f e r . For e x t r a c t i o n of h i s t o n e s , chromatin was homogenized i n 0.4 N s u l f u r i c a c i d with a Dounce homogenizer and s t i r r e d i n the same s o l u t i o n f o r one hour at 4°C. T h i s s o l u t i o n was c e n t r i f u g e d at 10,000 x g f o r 20 min and the supernatant was used d i r e c t l y i n s t a r c h g e l e l e c t r o p h o r e s i s or s t o r e d at -20°C. b. E l e c t r o p h o r e s i s In a d d i t i o n to the s t a r c h and p o l y a c r y l a m i d e s l a b g e l procedures o u t l i n e d i n the p r e v i o u s s e c t i o n , b a s i c p r o t e i n s were e l e c t r o p h o r e s e d i n 8 cm 10% p o l y a c r y l a m i d e d i s c g e l s c o n t a i n i n g 5 M urea (pH 4.5), a c c o r d i n g to the method d e s c r i b e d i n Nakano et a l . (1973). 145 C. RESULTS J_. Urodela a. Cytochemi s t r y The c y t o c h e m i s t r y of the spermatogenic n u c l e i of two newts, P l e u r o d e l e s waIt1 ( P i c h e r a l , 1970) and Notophthalmus  v i r i d e s c e n s v i r i d e s c e n s (Bois et a l . , 1976), has been d e s c r i b e d . The t e s t e s of these animals are o r g a n i z e d i n t o s e m i n i f e r o u s tubules c o n t a i n i n g c y s t s of synchronously de v e l o p i n g spermatids. Spermiogenesis can be d i v i d e d i n t o f i v e stages on the b a s i s of nuclear shape and b a s i c p r o t e i n composition. Stage 1 and 2 spermatid n u c l e i are round or c i g a r shaped r e s p e c t i v e l y , and c o n t a i n s o m a t i c - l i k e h i s t o n e s . Stage 3 n u c l e i are elongate and show a t r a n s i t i o n of h i s t o n e type from s o m a t i c - l i k e i n the a p i c a l p o r t i o n of the nucleus to a r g i n i n e -r i c h s t a b l e protamines in the b a s a l r e g i o n . N u c l e i of stage 4 appear as very t h i n , curved c y l i n d e r s and c o n t a i n s t a b l e protamines ( a r g i n i n e - r i c h ; not e x t r a c t a b l e with hot 5% TCA) throughout t h e i r e n t i r e l e n g t h . Stage 5 n u c l e i are the most advanced and c o n t a i n 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 which can be e x t r a c t e d with hot 5% TCA. A l l f i v e spermiogenic stages c o u l d be observed in the t e s t e s of the newts Cynops p y r r h o q a s t e r , Plethodon vehiculum and T a r i c h a t o r o s a (Fig.29A and B). A l l stages s t a i n e d i n t e n s e l y with Feulgen. 146 F i g u r e 29. S t a i n i n g of t e s t i s s e c t i o n s from u r o d e l e s . S e c t i o n s of t e s t e s are from T a r i c h a t o r o s a (A-D), Cynops  py r r h o g a s t e r (E) and Plethodon vehiculum ( F ) . A. & B. Feulgen s t a i n i n g . C. Sc D. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. E. Sc F. Sakaguchi s t a i n i n g . Numbers 1-5 i n d i c a t e stages of spermatid development as d e s c r i b e d i n the t e x t . W7 B p # r 20gm Fig. 29 10um 1 48 A f t e r h y d r o l y s i s i n 5% TCA at 85-90°C, stage 5 n u c l e i of a l l s p e c i e s d i d not s t a i n with AFG, i n d i c a t i n g that protamines were present i n the mature sperm n u c l e i (Fig.29D). N u c l e i of stages 1-4 s t a i n e d with AFG a f t e r TCA h y d r o l y s i s (Fig.29C). Stage 4 n u c l e i continued to s t a i n a f t e r deamination while e a r l i e r stages d i d not. In stage 3 n u c l e i there was a gr a d i e n t of d a r k l y s t a i n i n g m a t e r i a l i n the b a s a l regions to pale s t a i n i n g a n t e r i o r l y when the s e c t i o n s had been deaminated. A l l elongate stages s t a i n e d with AFG or e o s i n Y a f t e r p i c r i c a c i d h y d r o l y s i s and continued to s t a i n a f t e r a c e t y l a t i o n . A strong r e a c t i o n with Sakaguchi (Fig.29E and F) and a f a i n t r e a c t i o n with DNFB i n d i c a t e d that stage 3-5 n u c l e i of a l l three s p e c i e s of newts c o n t a i n e d a r g i n i n e - r i c h p r o t e i n s . AFG s t a i n i n g a f t e r Feulgen r e v e a l e d that non-DNA-a s s o c i a t e d b a s i c p r o t e i n s were present at the bases of elongate spermatid n u c l e i . T h i s was a l s o noted by P i c h e r a l (1970) i n P l e u r o d e l e s w a l t l sperm and by Bois et_ a l . ( 1976) i n Notophthalmus v i r i d e s c e n s sperm. Cytochemical data f o r the newts i s summarized i n Table V I I . L i k e P l e u r o d e l e s w a l t l and Notophthalmus  v i r i d e s c e n s , spermiogenesis i n Cynops, T a r i c h a and Plethodon appeared to i n v o l v e a s e r i e s of t r a n s i t i o n s i n nucl e a r b a s i c p r o t e i n s , from somatic type to s t a b l e protamines i n e l o n g a t i n g n u c l e i , and f i n a l l y to protamine i n the mature sperm. Free b a s i c p r o t e i n s i n the bas a l r e g i o n s of elongate n u c l e i may be c o r r e l a t e d to the t r a n s i t i o n from s t a b l e protamine to protamine and may represent p r o t e i n s which are sloughed o f f durin g t h i s 149 process ( P i c h e r a l , 1970). A s i m i l a r t r a n s i t i o n i n p r o t e i n types has been observed in the s n a i l H e l i x aspersa (Bloch and Hew, 1960a) and i n the squid L o l i g o opalescens (Bloch, 1966). The l a t t e r has been c o r r e l a t e d to an a c t u a l change in e l e c t r o p h o r e t i c a l l y d e t e c t a b l e b a s i c p r o t e i n f r a c t i o n s from t e s t i c u l a r spermatids and mature sperm of the spermatophore (Bloch, 1966). b. E l e c t r o p h o r e s i s As noted by Bois et a l . (1976), b a s i c p r o t e i n s prepared from Notophthalmus v i r i d e s c e n s v i r i d e s c e n s t e s t i c u l a r chromatin i n c l u d e d s e v e r a l fast-moving p r o t e i n s along with a complement of slower moving somatic h i s t o n e s (Fig.30A, channel 2). The t e s t i s - s p e c i f i c p r o t e i n s had an e l e c t r o p h o r e t i c m o b i l i t y i n s t a r c h g e l s c l o s e to slowest t e s t i s - s p e c i f i c component of Bufo  americanus (Fig.30A, channel 1). S i m i l a r r e l a t i v e m o b i l i t i e s of t e s t i s - s p e c i f i c b a s i c p r o t e i n s were noted i n the newt T r i t u r u s  v u l g a r i s and the toad Bufo bufo (Kharchencko and N a l i v a e v a , 1979). T e s t i s - s p e c i f i c p r o t e i n s of Notophthalmus v i r i d e s c e n s  v i r i d e s c e n s and Cynops pyr r h o g a s t e r had s i m i l a r e l e c t r o p h o r e t i c m o b i l i t i e s i n s t a r c h g e l s (Fig.30B) and t h i s s i m i l a r i t y extended to T a r i c h a t o r o s a (Fig.30C). Lack of c l e a r r e s o l u t i o n of the f a s t e r m i g r a t i n g newt p r o t e i n s e x t r a c t e d from t e s t i s homogenates allowed only an o v e r a l l comparison of the p a t t e r n s i n s t a r c h g e l s . However s p e r m - s p e c i f i c b a s i c p r o t e i n s of a l l newts appeared to be s i m i l a r to each o t h e r . 150 Table VII• Cytochemistry of Elongate Spermatids and Sperm of urodeles Organism Used i n 9 s Reactive Taricha Plethodon Cynops Notophthalmus PleurodeL atraent Material torosa vehiculum pyrrhogaster viridescens*' w a l t l p -Feulgen DNA +c. + d. +c. +d. + c . + d . +<=• +d. + c. + d. AFG,TCA, 85-90"C Basic proteins other than protamines + - + - + - + - + -AFG,TCA, 85-90°C deamination Arginine-rich basic proteins + - + - + - + - + -AFG, p i c r i c acid, 60°C. Basic proteins including protamines + + + + + + + + + + AFG, p i c r i c acid, 60°C. acetylation Arginine-rich basic proteins + + + + + + + + + Eosin Y, p i c r i c acid 60" Basic proteins including protamines + + + + + + + + + Eosin Y, p i c r i c acid, 60° acetylation Arginine-rich basic proteins + + + + + + + + + + Sakaguchi Protein-bound arginine + + + + + + + + + + . DNFB Protein-bound lysine - - - - - - - - - -AFG, after Feulgen Non-DIIA-associated basic proteins + e. + e. Sperm histone type 2 —> 1 2 -> 1 2 —> 1 2 - i 1 2 ^ 1 a. Bois et a l . , 1976 d. Late elongate stage 5 and mature sperm. b. Picheral, 1970 e. Basal region of nucleus c. Early elongate stages 3 and 4 151 F i g u r e 30. S t a r c h g e l electrophoretograms of t e s t i s b a s i c p r o t e i n s from u r o d e l e s . B a s i c p r o t e i n s shown in A were i s o l a t e d from chromatin prepared from pooled t e s t e s of s e v e r a l i n d i v i d u a l s , while those i n B and C were e x t r a c t e d d i r e c t l y from homogenates of t e s t e s from s i n g l e i n d i v i d u a l s . A. 1. Bufo americanus (B) 2. Notophthalmus v i r i d e s c e n s v i r i d e s c e n s (N.v.) B. 1. C a l f thymus H4 2. Cynops pyrrhogaster (C.p.) 3. Notophthalmus v i r i d e s c e n s v i r i d e s c e n s (N.v.) C. 1. & 6. T a r i c h a t o r o s a ( T . t . ) 2. & 7. Cynops pyrrhogaster (C.p.) 3. H e r r i n g protamine (P) 4. Bufo boreas (B) 5. X. 1. l a e v i s (X) 8. C a l f thymus H4 152. > z T » T T A ^ A A A O CO + • l cb • — 153 In short p o l y a c r y l a m i d e d i s c g e l s ( F i g . 3 l A ) , the t e s t i s -s p e c i f i c p r o t e i n s of T a r i c h a , Plethodon and Notophthalmus migrated as s i n g l e bands moving f a s t e r than the somatic h i s t o n e (SH) r e g i o n . However comparison of m o b i l i t i e s was d i f f i c u l t here because of the d i f f e r e n t r e l a t i v e m o b i l i t i e s of h i s t o n e s i n each d i s c g e l . In p o l y a c r y l a m i d e s l a b g e l s comparison was much e a s i e r and Fig.31B and C shows that the fast-moving t e s t i s - s p e c i f i c p r o t e i n s of T a r i c h a and Plethodon had e l e c t r o p h o r e t i c m o b i l i t i e s which were s i m i l a r to each other. These p r o t e i n s migrated s l i g h l t y slower than SP6 of Xenopus  l a e v i s , as d i d the p r o t e i n s of Notophthalmus v i r i d e s c e n s  v i r i d e s c e n s i n the same g e l system (Bois ert §_1. , 1976). P i c h e r a l (1979) a l s o noted that the sperm- and t e s t i s -s p e c i f i c b a s i c p r o t e i n s of P l e u r o d e l e s w a l t l , Ambystoma, Salamandra and Euproctes had v i r t u a l l y i d e n t i c a l e l e c t r o p h o r e t i c m o b i l i t i e s i n p o l y a c r y l a m i d e g e l s . 2. Squamata a. Cytochemistry Sperm n u c l e i of the snakes Thamnophis (three s p e c i e s ) and Elaphe (one s p e c i e s ) resembled those of Bufo c y t o c h e m i c a l l y . When hy d r o l y s e d i n 5% TCA at 85-90°C, sperm of the t e s t i s and ductus deferens d i d not s t a i n with AFG, i n d i c a t i n g that t r o u t -l i k e protamines may be present (Fig.32C; Fig.33B and F ) . AFG s t a i n i n g was intense a f t e r p i c r i c a c i d h y d r o l y s i s (Fig.32E; 1 54 Fig u r e 31. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from i n d i v i d u a l u r o d e l e s . Basic p r o t e i n s were e x t r a c t e d with 0.4 N s u l f u r i c a c i d from whole t e s t i s homogenates. A. E l e c t r o p h o r e s i s was performed i n 8 cm po l y a c r y l a m i d e d i s c g e l s (10% acrylamide, 5 M urea) f o r 45 min at 40 mA. 1. Bufo marinus 2. Plethodon vehiculum 3. Plethodon vehiculum (heart) 4. Notophthalmus v i r i d e s c e n s v i r i d e s c e n s 5. & 6. T a r i c h a torosa 7. HERRING PROTAMINE (P) B. E l e c t r o p h o r e s i s was performed i n a 15 cm pol y a c r y l a m i d e s l a b g e l (15% acrylamide, 6.25 M urea) f o r 3 hr at 150 V. 1. Bufo marinus (B) 2. T a r i c h a t o r o s a 3. Plethodon vehiculum 4. X. 1. l a e v i s C. Absorbance p r o f i l e s (610 nm) of t e s t i s b a s i c p r o t e i n s e l e c t r o p h o r e s e d as shown i n B. Arrows i n d i c a t e p o s i t i o n s of t e s t i s - s p e c i f i c b a s i c p r o t e i n s of Plethodon, T a r i c h a and Notophthalmus. SH= somatic h i s t o n e s SP= s p e r m - s p e c i f i c b a s i c nuclear p r o t e i n s of X. 1. l a e v i s 1 2 3 4 5 6 7 + > -156 Fig.33C), but much reduced i f the s e c t i o n s were a c e t y l a t e d to block l y s i n e r e s i d u e s (Fig.32F; Fig.33D). However mature sperm of the ductus deferens gave a strong r e a c t i o n with the Sakaguchi t e s t f o r a r g i n i n e . Sud (1961) a l s o noted that the sperm of the snake N a t r i x n a t r i x gave a strong r e a c t i o n with the Sakaguchi t e s t and concluded that a r g i n i n e - r i c h p r o t e i n s were pres e n t . On the b a s i s of t h i s s i n g l e o b s e r v a t i o n , Bloch (1969) c l a s s e d N a t r i x n a t r i x sperm h i s t o n e s as type 1. However, the r e s u l t s presented here i n d i c a t e that both a r g i n i n e and l y s i n e must be present i n the nuclear p r o t e i n s of Thamnophis and Elaphe sperm and t h e r e f o r e they should be c l a s s i f i e d as type 3, or intermediate-type sperm h i s t o n e s . T h i s i s i n agreement with the amino a c i d a n a l y ses of Huang (1977), which demonstrated the presence of both a r g i n i n e (23-30 mole%) and l y s i n e (4-7 mole%) i n the sperm s p e c i f i c b a s i c p r o t e i n s from Thamnophis s i r t a l i s and Elaphe g u t t a t a g u t t a t a . Sperm of the l i z a r d s A n o l i s c a r o l i n e n s i s c a r o l i n e n s i s ( F i g . 3 4 ) , Crotaphytus c o l l a r i s (Fig.35) and Sceloporus magister (Fig.36) showed the same s t a i n i n g p r o p e r t i e s as those of the snakes, with one e x c e p t i o n . Mature sperm of the ductus deferens continued to s t a i n s t r o n g l y with AFG a f t e r p i c r i c a c i d h y d r o l y s i s and a c e t y l a t i o n (Fig.34F; Fig.35E and F ) . T h i s would i n d i c a t e that the sperm of these l i z a r d s c o n t a i n a r g i n i n e - r i c h protamines with l i t t l e l y s i n e (type 1). Bloch (unpublished data c i t e d i n Bloch, 1969) a l s o found a type 1 protamine i n the sperm of the l i z a r d H o l brookia texana. Amino a c i d a n a l y s i s of the t e s t i s - s p e c i f i c b a s i c p r o t e i n s 157 F i g u r e 32. S t a i n i n g of ductus deferens s e c t i o n s from Thamnophis. S e c t i o n s of ductus deferens are from Thamnophis o r d i n o i d e s (A,D.E and F) and Thamnophis s i r t a l i s (B and cV. A. & B. Feulgen s t a i n i n g . C. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. D. Sakaguchi s t a i n i n g . E. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 hr. F. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1 h r ) . 1 5 8 Fig. 32 159 F i g u r e 33. S t a i n i n g of ductus deferens and t e s t i s s e c t i o n s of Thamnophis and Elaphe. S e c t i o n s are from the ductus deferens of Thamnophis  o r d i n o i d e s (A-D) and the t e s t e s of Elaphe o b s o l e t a  o b s o l e t a X o b s o l e t a q u a d r i v i t t a t a (E and F"T^ A. & E. Feulgen s t a i n i n g . B. & F. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C. 15 min. C. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 hr. D. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1 h r ) . lOum 161 F i g u r e 34. S t a i n i n g of t e s t i s and ductus deferens s e c t i o n s from A n o l i s c a r o l i n e n s i s . S e c t i o n s are from A n o l i s c a r o l i n e n s i s t e s t i s (A,C and E) and ductus deferens (B,D and F ) . A. & B. Feulgen s t a i n i n g . C. & D. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. E. AFG s t a i n i n g a f t e r TCA h y d r o l y s i s and deamination. F. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1 h r ) . I 6 £ lOum 163 Fi g u r e 35. S t a i n i n g of t e s t i s and ductus deferens s e c t i o n s from Crotaphytus c o l l a r i s . S e c t i o n s are of Crotaphytus c o l l a r i s t e s t i s (A,C and E) and ductus deferens (B,D and F ) . A. & B. Feulgen s t a i n i n g . C. & D. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. E. & F. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1 h r ) . Fig.35 lOum 165 F i g u r e 36. S t a i n i n g of t e s t i s and ductus deferens s e c t i o n s from Sceloporus magister. S e c t i o n s are of Sceloporus magister t e s t i s (A,B and D) and ductus deferens (C). A. Feulgen s t a i n i n g . B. & C. AFG s t a i n i n g a f t e r h y d r o l y s i s i n 5% TCA, 85-90°C, 15 min. D. AFG s t a i n i n g a f t e r TCA h y d r o l y s i s and deamination. TZSS&Qf : jagg- ; v • • C j ? 3 tip; D Fig.36 lOum 167 from A n o l i s c a r o l i n e n s i s showed a s i g n i f i c a n t amount of l y s i n e (9.8 mole%), more than i n e i t h e r of the snakes s t u d i e d (Huang, 1977). P r o t e i n s used f o r t h i s a n a l y s i s were taken from a s i n g l e fast-moving e l e c t r o p h o r e t i c component from short p o l y a c r y l a m i d e g e l s (Fig.38A). T h i s band may have represented a mixture of b a s i c p r o t e i n s from the t e s t i s , as e l e c t r o p h o r e s i s on longer g e l s r e v e a l e d the presence of two or three bands from t e s t i c u l a r p r e p a r a t i o n s of A n o l i s and other l i z a r d s (Figs.38B and 39). On the b a s i s of the c y t o c h e m i c a l r e s u l t s , we would expect to f i n d that l y s i n e i s present i n one of the t e s t i s -s p e c i f i c bands and that a more a r g i n i n e - r i c h p r o t e i n i s s p e c i f i c to the sperm. There i s some evidence f o r the presence of a l y s i n e - r i c h p r o t e i n i n the spermatids of A n o l i s . Figs.34C and E show that while l a t e spermatids of the t e s t i s s t a i n e d with AFG a f t e r TCA h y d r o l y s i s , t h i s s t a i n i n g was somewhat reduced a f t e r deamination. Thus there may be a t r a n s i t i o n from somatic-type h i s t o n e s to a l y s i n e / a r g i n i n e - r i c h p r o t e i n i n the e l o n g a t i n g spermatids, and f i n a l l y to an a r g i n i n e - r i c h protamine i n the mature sperm of A n o l i s. A s i m i l a r t r a n s i t i o n from a l y s i n e / a r g i n i n e - r i c h p r o t e i n , to a s t a b l e protamine, has been observed, u s i n g biochemical techniques, i n the d e v e l o p i n g sperm of mammals ( K i s t l e r e_t a l . , 1973; Kumaroo et a_l. , 1975). Cytochemical r e s u l t s f o r the sperm of Squamata are summarized i n Table V I I I . TABLE VIII. Cytochemistry of Reptilian Sperm Nuclei Organism Used L a c e r t i l i a Serpentes Controls S t a i n i n g s R e a c t i v e Pretreatment M a t e r i a l Anolis Sceloporus Crotaphytus Thamnophis Thamnophis Thamnophis Elaphe Salmo Soualus Mytilus ^p^s c a T o T T n l n s i s magister collarL s i r t a l i s ordinoides acanthius edulis lae^ Feulgen AFG-TCA 85-90°C. B a s i c p r o t e i n s o t h e r than protamines AFG-TCA 85-90°C., deamination Basic proteins rich in arginine A F G - p i c n c a c i d , 60°C. B a s i c p r o t e i n s i n c l u d i n g protamines A F G - p i c r i c a c i d , 60°C. a c e t y l a t i o n B a s i c p r o t e i n s r i c h i n a r g i n i n e Sakaguchi P r o t e i n - b o u n d a r g i n i n e ArG, without h y d r o l y s i s N o n - n u c l e i c a c i d a ssoc. b a s i c p r o t e i n s Sperm h i s t o n e type a. Late spermatids c o n t i n u e to s t a i n . b - o . o b s o l e t a X o . q u a d r i v i t t a t a 3B 3B 3B 3B 3B 3A 00 169 b. E l e c t r o p h o r e s i s S t a r c h g e l e l e c t r o p h o r e s i s (Fig.37) r e v e a l e d that t e s t i s -and ductus deferens- s p e c i f i c b a s i c p r o t e i n s , e x t r a c t e d from whole organ homogenates from i n d i v i d u a l snakes and l i z a r d s , gave p a t t e r n s which s t r o n g l y resembled each o t h e r . P r e p a r a t i o n s of Thamnophis and Elaphe ductus deferens, as w e l l as A n o l i s t e s t i s and ductus deferens, showed the same two-banded p a t t e r n of p r i n c i p a l p r o t e i n s , one band moving c l o s e to SP6 of Xenopus  l a e v i s and the other c l o s e to t r o u t protamine. In a d d i t i o n , both t e s t i s and ductus deferens samples f r e q u e n t l y showed the presence of a band moving only s l i g h t l y f a s t e r than H4. T h i s might represent a p r o t e i n from an e a r l i e r c e l l type than spermatozoa, s i n c e t h i s was the only band present e a r l y i n the s p r i n g and l a t e i n the summer breeding season when snake t e s t e s are aspermatogenic (Kasinsky ejt a l . , 1978). No obvious d i f f e r e n c e s were observed between the s t a r c h g e l e l e c t r o p h o r e t i c p r o f i l e s of sperm h i s t o n e s from congeneric s p e c i e s of Elaphe (Fig.37A) or A n o l i s (Fig.37C). Fig.37A (channels 3 and 4) re v e a l e d s i g n s of p r o t e o l y s i s i n Elaphe ductus deferens basic p r o t e i n s when homogenates were s t o r e d i n PBS f o r s e v e r a l days at -70°C, but not when the e x c i s e d organ was s t o r e d i n t a c t (channels 5 and 6). The banding p a t t e r n obtained with A n o l i s t e s t i s homogenates from s i n g l e animals (Fig.37B) was the same as that seen f o r p r o t e i n s i s o l a t e d from chromatin of pooled t e s t e s from s e v e r a l animals (Kasinsky et a_l. , 1978). The s t a r c h g e l e l e c t r o p h o r e t i c p r o f i l e s of b a s i c p r o t e i n s o b t a i n e d from Gecko ( l i z a r d ) and 170 Coluber (snake) t e s t e s were a l s o s i m i l a r to the p a t t e r n s observed here f o r Thamnophi s, Elaphe and A n o l i s (Kasinsky et a l . , 1978). In short p o l y a c r y l a m i d e g e l s (Fig.38A) the t e s t i s - and ductus d e f e r e n s - s p e c i f i c p r o t e i n s of A n o l i s c a r o l i n e n s i s appeared as one band. However i n long g e l s (Fig.38B and C), t e s t i s - s p e c i f i c p r o t e i n s of A n o l i s were r e s o l v e d i n t o four bands. The major component ("1") had a m o b i l i t y c l o s e to h e r r i n g protamine. Two f a i n t e r bands ("2") migrated s l i g h t l y ahead of SP6 of Xenopus l a e v i s and another very f a i n t component ("3") migrated i n the region between H4 and SP3-5 of Xenopus  l a e v i s . S i m i l a r 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 t i c p r o f i l e s were seen f o r the l i z a r d s Gerrhonatus c o e r u l e u s , Sceloporus  o l i v a c e u s and Crotaphytus c o l l a r i s , and f o r the snakes Thamnophis s i r t a l i s , Thamnophis o r d i n o i d e s and Elaphe g u t t a t a  g u t t a t a (Figs.39 and 40). In the case of Crotaphytus and Sceloporus t e s t e s (Fig.39A), o n l y components "1" and "3" were e v i d e n t , while the p r e p a r a t i o n of t e s t i s and ductus deferens from Gerrhonatus showed a s i n g l e component "2" band as w e l l as components "1" and "3" (Fig.39B and C). Mixed t e s t i s and ductus deferens samples from Thamnophis s i r t a l i s a l s o showed these three bands (Fig.39B and C). Component "3" was not apparent in the samples prepared from the ductus deferens of Thamnophis  o r d i n o i d e s nor from the semen of Elaphe g u t t a t a g u t t a t a , and component "2" was f a i n t e r than i n t e s t i s p r e p a r a t i o n s , as were the somatic h i s t o n e s (Fig.39B and C). The major p r o t e i n 171 F i g u r e 37. S t a r c h g e l electrophoretograms of t e s t i s and ductus deferens b a s i c p r o t e i n s from Squamata. P r o t e i n s were e x t r a c t e d from t e s t i s or ductus deferens homogenates from i n d i v i d u a l animals. A l l t i s s u e s had been s t o r e d at -70°C. Where i n d i c a t e d , t i s s u e homogenates were a l s o s t o r e d at -70°C. A. 1. H e r r i n g protamine (P) 2. X. 1. l a e v i s t e s t i s (X) 3. Elaphe o b s o l e t a q u a d r i v i t t a t a ductus deferens (homogenate stored) (E.o.q.) 4. Elaphe g u t t a t a g u t t a t a ductus deferens (homogenate stored) (E.g.g.) 5. Elaphe o b s o l e t a q u a d r i v i t t a t a ductus deferens (homogenate used d i r e c t l y ) 6. Elaphe g u t t a t a g u t t a t a ductus deferens (homogenate used d i r e c t l y ) 7. C a l f thymus H4 Broken arrow i n d i c a t e s degradation of s t o r e d homogenates. B. 1. Pea s e e d l i n g H4 2. C a l f thymus H4 3. A n o l i s c a r o l i n e n s i s l i v e r 4. A n o l i s c a r o l i n e n s i s heart 5. A n o l i s c a r o l i n e n s i s t e s t i s 6. A n o l i s c a r o l i n e n s i s ductus deferens 7. A n o l i s s a g r e i t e s t i s 8. A n o l i s s a g r e i ductus deferens C. 1. X. 1_. l a e v i s t e s t i s (X) 2. Thamnophis eques ductus deferens 3. Pea s e e d l i n g H4 173 F i g u r e 38. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from A n o l i s . P r o t e i n s were e x t r a c t e d with 0.4 N s u l f u r i c a c i d from whole t i s s u e homogenized i n PBS. Where i n d i c a t e d , homogenates were prepared from f r e s h t i s s u e . A. E l e c t r o p h o r e s i s was performed i n 8 cm pol y a c r y l a m i d e d i s c g e l s (10% acrylamide, 5 M urea) f o r 45 min at 40 mA. 1. H e r r i n g protamine (P) 2. A n o l i s c a r o l i n e n s i s heart ( f r e s h ) 3. A n o l i s c a r o l i n e n s i s ductus deferens ( f r e s h ) 4. A n o l i s c a r o l i n e n s i s t e s t e s ( f r e s h ) B. E l e c t r o p h o r e s i s was perfomed i n a 20 cm pol y a c r y l a m i d e s l a b g e l (15% acrylamide, 6.25 M urea) f o r 20 hr at 120 V. 1. & 4. H e r r i n g protamine (P) 2. D i e r o c h e l y s r e t i c u l a r i s ( t u r t l e ) ductus deferens (Dr) 3. A n o l i s c a r o l i n e n s i s t e s t e s (Ac) 5. X. 1. l a e v i s t e s t e s C. Absorbance p r o f i l e s (610 nm) of t e s t i s b a s i c p r o t e i n s e l e c t r o p h o r e s e d as shown i n B. SH= somatic h i s t o n e s SP= s p e r m - s p e c i f i c b a s i c nuclear p r o t e i n s of X. 1. l a e v i s ' Arrows (1, 2 & 3) i n d i c a t e t e s t i s - s p e c i f i c p r o t e i n s of A n o l i s c a r o l i n e n s i s , as d e s c r i b e d i n the t e x t . 174 1 2 3 4 Fig. 3 8 175 e x t r a c t e d from the d u c t i d e f e r e n t i a of these snakes appeared to be component "1". Fast moving t e s t i s - s p e c i f i c bands from Thamnophis proximus were not observed i n the g e l shown in Fig.39C (channel 5) but a high r e s o l u t i o n absorbance p r o f i l e of t h i s channel r e v e a l e d the presence of low amounts of components "1" and "2" ( F i g . 40). The o v e r a l l p i c t u r e which can be gathered from these e l e c t r o p h o e r t i c data i s that the sperm- and t e s t i s - s p e c i f i c h i s t o n e s of l i z a r d s and snakes appear to be s i m i l a r to each o t h e r . The f a s t e s t moving p r o t e i n ("1"), with a m o b i l i t y c l o s e to that of t r o u t protamine, appeared to be the major component of the mature sperm of the ductus deferens and semen. Component "2" appeared to be present i n greater c o n c e n t r a t i o n s i n the t e s t i s and may represent a s p e r m a t i d - s p e c i f i c p r o t e i n , as has been suggested by the c y t o c h e m i s t r y . Component "3" appeared only i n some of the p r e p a r a t i o n s from t e s t i s homogenates. I t may be another t e s t i s - s p e c i f i c p r o t e i n , perhaps homologous with the slow moving band seen i n s t a r c h g e l s . However the p o s s i b i l i t y that component "3" i s a non-histone contaminant cannot be completely r u l e d out, e s p e c i a l l y s i n c e i t s m o b i l i t y i s s i m i l a r to "X1", a band o b t a i n e d under s i m i l a r c o n d i t i o n s from Xenopus l a e v i s t e s t e s . E x t r a c t i o n of b a s i c p r o t e i n s from t e s t i s chromatin should be h e l p f u l i n r e s o l v i n g t h i s i s s u e . B a s i c p r o t e i n s e x t r a c t e d from a ductus deferens homogenate of the t u r t l e D i e r o c h e l y s r e t i c u l a r i s (order Chelonia) were r e s o l v e d i n t o two bands i n p o l y a c r y l a m i d e g e l s (Fig.38B). The f a s t e s t moving component migrated c o n s i d e r a b l y slower than that 176 F i g u r e 39. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s and ductus deferens b a s i c p r o t e i n s from Squamata. P r o t e i n s were e x t r a c t e d with 0.4 N s u l f u r i c a c i d from t i s s u e homogenized in PBS. E l e c t r o p h o r e s i s was performed i n 25 cm p o l y a c r y l a m i d e s l a b g e l s (15% acrylamide, 6.25 M urea) f o r 23 hr at 150 V. S t a i n i n g was i n 0.2% Amido Black (A & C) or 0.1% Coomassie Blue (B) and d e s t a i n i n g was i n 30% methanol/10% a c e t i c a c i d . A. 1. Bufo punctatus t e s t i s (B) 2. Crotaphytus c o l l a r i s t e s t e s 3. Sceloporus o l i v a c e u s t e s t e s and ductus deferens B. 1. Gerrhonatus coeruleus t e s t e s and ductus deferens 2. Elaphe g u t t a t a g u t t a t a ductus deferens 3. Thamnophis o r d i n o i d e s ductus deferens 4. Thamnophis s i r t a l i s t e s t e s and ductus deferens C. 1. Gerrhonatus co e r u l e u s t e s t e s 2. Elaphe g u t t a t a g u t t a t a ductus deferens 3. Thamnophis o r d i n o i d e s ductus deferens 4. Thamnophis s i r t a l i s t e s t e s and ductus deferens 5. Thamnophis proximus t e s t e s 6. X. 1. l a e v i s t e s t e s Arrows (1, 2 & 3) i n d i c a t e t e s t i s - and ductus def e r e n s -s p e c i f i c p r o t e i n s of l i z a r d s and snakes, as d e s c r i b e d i n the t e x t . SH= somatic h i s t o n e s SP= s p e r m - s p e c i f i c b a s i c n u c l e a r p r o t e i n s of X. 1. l a e v i s . m F i g u r e 40. Absorbance p r o f i l e s of t e s t i s and ductus deferens p r o t e i n s from Squamata. Basic p r o t e i n s were obtained from t e s t e s and ductus deferens homogenates and e l e c t r o p h o r e s e d as shown in Fig.39A and C. " Absorbance was measured at 6l0nm. The absorbance p r o f i l e s of X. 1. l a e v i s and Thamnophis  proximus have been enlarged to ehance the t e s t i s -s p e c i f i c peaks of Thamnophis proximus. Arrows i n d i c a t e the p o s i t i o n of somatic h i s t o n e H4. Numbers 1, 2 and 3 r e f e r to t e s t i s - and ductus d e f e r e n s -s p e c i f i c p r o t e i n s of l i z a r d s and snakes as shown i n Fig.39 and d e s c r i b e d i n the t e x t . SH= somatic h i s t o n e s TSP= T e s t i s - s p e c i f i c p r o t e i n s 180 of A n o l i s , while a slower f a i n t band migrated i n the region of component "2". S i m i l a r e l e c t r o p h o r e t i c p r o f i l e s have been observed f o r the h i s t o n e s e x t r a c t e d from t e s t i s and ductus deferens chromatin of other s p e c i e s of t u r t l e s (E.W. Byrd and L. Fabre, u n p u b l i s h e d ) . I t would seem, t h e r e f o r e , that sperm h i s t o n e s are constant i n the r e p t i l i a n order C h e l o n i a as w e l l as i n Squamata. 3. Aves a. Cytochemistry The spermatogenic c e l l s of the r o o s t e r ( G a l l u s domesticus) were organized i n t o roughly three zones in the seminiferous t u b u l e s of the t e s t i s (Fig.41A). Spermatogonia and spermatocytes, which have round n u c l e i , were l o c a t e d towards the perimeter of the t u b u l e s . Tear-drop shaped e a r l y spermatids occupied the next zone, while the more mature e l o n g a t i n g spermatids were arranged i n loose bundles, surrounded by S e r t o l i c e l l cytoplasm, i n the innermost l a y e r (Romanoff, 1960, pp. 31-43). Mature sperm c o u l d be seen f r e e i n the i n t e r i o r c a v i t y of the t u b u l e . The n u c l e i of the sperm were long, s l e n d e r rods and were s l i g h t l y bent ( Z l o t n i k , 1947). A l l spermatid stages s t a i n e d i n t e n s e l y and u n i f o r m l y with Feulgen, as d i d the mature sperm of the ductus deferens (Fig.41A and B). The mature sperm of the r o o s t e r are known to c o n t a i n a protamine (Nakano et a l . , 1976a). L i k e the protamine found i n the sperm n u c l e i of the t r o u t (Salmo g a i r d n e r i i ) , t h i s p r o t e i n 181 F i g u r e 41. Feulgen s t a i n i n g of avian t e s t i s and ductus deferens s e c t i o n s . A. G a l l u s domesticus t e s t i s . B. G a l l u s domesticus ductus d e f e r e n s . C. Anas p l a t y r h y n c o s t e s t i s . D. C o t u r n i x c o t u r n i x j a p o n i c a t e s t i s . E. M e l o p s i t t a c u s undulatus t e s t i s . F. Molothrus a t e r t e s t i s . Inset i n F shows high m a g n i f i c a t i o n of e l o n g a t i n g spermatids of Molothrus a t e r showing the onset of n u c l e a r s p i r a l i z a t i o n . Scale= 0.25um. 10ym 183 c o u l d be e x t r a c t e d by h y d r o l y s i s i n hot 5% TCA. When m a t e r i a l f i x e d i n n e u t r a l b u f f e r d f o r m a l i n f o r short p e r i o d s of time (three hours to one month) was h y d r o l y s e d f o r 20 min at 85-95°C and s t a i n e d with AFG, the n u c l e i of mature sperm d i d not s t a i n , while the n u c l e i of l a t e e l o n g a t i n g spermatids s t a i n e d very weakly. However prolonged f i x a t i o n i n f o r m a l i n ( s e v e r a l months) appeared to have a s t a b i l i z i n g e f f e c t on these n u c l e i (Table IX). When hydrolysed at temperatures of 75-95°C for 20 min, a l l n u c l e i s t a i n e d s t r o n g l y with AFG. The f o r m a l i n -s t a b i l i z e d p r o t e i n was e x t r a c t a b l e , however, when the temperature of h y d r o l y s i s was r a i s e d to 95-100 oC ( F i g . 4 2 ) . T h i s was the case f o r both mature sperm of the ductus deferens and the t e s t i s . Somatic n u c l e i and e a r l y spermatid stages continued to s t a i n , even at e l e v a t e d temperatures, while the l a t e spermatid stage s t a i n e d only f a i n t l y (Fig.43A). M a t e r i a l f i x e d i n C l a r k e ' s f i x a t i v e showed a s i m i l a r s t a i n i n g p a t t e r n . T h i s i s i n agreement with the f i n d i n g s of Mezquita and Teng (1977) that the t r a n s i t i o n from somatic h i s t o n e s to protamine takes p l a c e i n the l a t e spermatid stage. S e c t i o n s t r e a t e d with AFG without p r i o r h y d r o l y s i s d i d not s t a i n , i n d i c a t i n g that f r e e b a s i c p r o t e i n s were not p r e s e n t . When s e c t i o n s f i x e d i n f o r m a l i n f o r prolonged p e r i o d s were subj e c t e d to deamination a f t e r h y d r o l y s i s i n 5% TCA at lower temperatures, mature sperm and l a t e spermatids continued to s t a i n with AFG while somatic n u c l e i and immature spermatids d i d not. A l l stages s t a i n e d s t r o n g l y with AFG and e o s i n Y a f t e r p i c r i c a c i d h y d r o l y s i s , but only the sperm and l a t e spermatids 184 Figu r e 42. A l k a l i n e f a s t green s t a i n i n g of ductus deferens s e c t i o n s from G a l l u s domesticus. A. T i s s u e was f i x e d i n NBF f o r 1 month. H y d r o l y s i s was i n 5% TCA at 80-85°C, 20 min. B-D. T i s s u e was f i x e d i n NBF f o r 12 months. B. H y d r o l y s i s i n 5% TCA at 80-85°C, 20 min. C. H y d r o l y s i s i n 5% TCA at 85-90°C, 20 min. D. H y d r o l y s i s i n 5% TCA at 95-100 oC, 20 min. TCA= t r i c h l o r o a c e t i c a c i d NBF= n e u t r a l b u f f e r e d f o r m a l i n Table IX. E f f e c t of F i x a t i o n Time on AFG Stai n i n g f o r Protamine Organism F i x a t i o n Time Hydrolysis Temperature 3• i n NBF 75-85" 85-95° 95-100° Rooster 1 year + + Rooster -* 1 month _b. - -Duck 1 year + + -Duck 2 months - - -a. Hydrolysis i n 5% TCA ( t r i c h l o r o a c e t i c acid) b. Presence of protamine indi c a t e d by absence of AFG s t a i n i n g A « -• c D Fig.42 186 F i g u r e 43. A l k a l i n e f a s t green s t a i n i n g of avian t e s t i s and ductus deferens s e c t i o n s . H y d r o l y s i s was i n 5% TCA, 95~100 0C, 20 min. A. G a l l u s domesticus t e s t i s . B. Anas p l a t y r h y n c o s t e s t i s . C. Coturnix c o t u r n i x j a p o n i c a t e s t i s . D. Coturnix c o t u r n i x j a p o n i c a ductus d e f e r e n s . E. Molothrus a t e r t e s t i s . F. M e l o p s i t t a c u s undulatus ductus d e f e r e n s . IS7 [, \ / • •  .• \ • • »* v* ' .... ; » • • « ^ • *! • • '«v • ; . •. . * * * " - ' . ' • . . * •'• •• . . . ; . . B 1 A • < c ; 1HS • D v . * • ' '•; , ***V ' . " . • .,• . J * r - > • - v - h i K * If g , E • • • . . . F lOum 188 s t a i n e d a f t e r a c e t y l a t i o n ( F i g . 4 4 ) . These r e s u l t s may be taken as an i n d i c a t i o n that the more mature n u c l e i c o n t a i n e d only a r g i n i n e - r i c h p r o t e i n s and l i t t l e or no l y s i n e . T h i s was co r r o b o r a t e d by the f a c t that mature sperm and spermatids s t a i n e d i n t e n s e l y with the Sakagughi t e s t f o r a r g i n i n e but f a i l e d to s t a i n with DNFB f o r l y s i n e . The g e n e r a l o r g a n i z a t i o n of the semi n i f e r o u s t u b u l e s i n the t e s t e s of the q u a i l (Coturnix c o t u r n i x j a p o n i c a ) , the duck (Anas p l a t y r h y n c o s ) , the budgie ( M e l o p s i t t a c u s undulatus) and the cowbird (Molothrus a t e r ) was s i m i l a r t o that of the r o o s t e r (Fig.41).- Late spermatids appeared to be arranged i n loose bundles and occupied the innermost l a y e r of spermatogenic t i s s u e , while more mature sperm c o u l d be seen f r e e i n the lumen of the t u b u l e s . The mature sperm of Coturnix c o t u r n i x j a p o n i c a , Anas p l a t y r h y n c o s and M e l o p s i t t a c u s undulatus had long, s l e n d e r , rod-shaped n u c l e i , while those of Molothrus a t e r underwent the s p i r a l i z a t i o n t y p i c a l of the Pa s s e r i n e b i r d s (Romanoff, 1960, p.36; Henley et a l . , 1978). A l l spermatids and sperm n u c l e i s t a i n e d i n t e n s e l y with Feulgen (Fig.41B-E) and mature sperm c o u l d be seen f i l l i n g the ductus deferens i n a l l cases. The a l k a l i n e f a s t green r e a c t i o n i n these b i r d s f o l l o w e d a p a t t e r n s i m i l a r to that seen i n the r o o s t e r . Prolonged f i x a t i o n i n f o r m a l i n appeared to s t a b i l i z e the n u c l e i of the mature stages, which s t a i n e d s t r o n g l y with AFG a f t e r h y d r o l y s i s i n 5% TCA at 75-95°C. When the h y d r o l y s i s temperature was r a i s e d to 95-100 o C, mature sperm n u c l e i were e x t r a c t e d , i n d i c a t i n g 189 F i g u r e 44. S t a i n i n g of ductus deferens s e c t i o n s from G a l l u s domesticus. A. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 hr. B. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1 h r ) . C. Eo s i n Y s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 hr. D. E o s i n Y s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s and a c e t y l a t i o n . Wo 191 that protamines were present (Fig.43B-F). Somatic and immature spermatid n u c l e i continued to s t a i n under these c o n d i t i o n s . With m a t e r i a l f i x e d f o r short p e r i o d s , the protamine c o u l d be e x t r a c t e d at lower temperatures, which was a l s o the case f o r m a t e r i a l f i x e d i n C l a r k e ' s f i x a t i v e . Deamination had no e f f e c t on the f o r m a l i n - s t a b i l i z e d mature stages when i t followed low temperature h y d r o l y s i s . These stages continued to s t a i n s t r o n g l y with AFG, i n d i c a t i n g that a r g i n i n e - r i c h rather than l y s i n e - r i c h p r o t e i n s were pres e n t . S t a i n i n g was absent i n a l l stages when there had been no p r i o r h y d r o l y s i s , showing that f r e e b a s i c p r o t e i n s were absent. A l l stages s t a i n e d s t r o n g l y with AFG or e o s i n Y a f t e r p i c r i c a c i d h y d r o l y s i s . A c e t y l a t i o n p r i o r to s t a i n i n g appeared to have l i t t l e or no e f f e c t on the mature sperm ( F i g . 4 5 ) . Mature sperm and l a t e spermatids of a l l the b i r d s s t u d i e d s t a i n e d s t r o n g l y with the Sakaguchi r e a c t i o n but s t a i n e d only f a i n t l y or not at a l l with DNFB. T h i s p o i n t e d to the presence of h i g h l y a r g i n i n e - r i c h b a s i c p r o t e i n s i n the sperm n u c l e i of these animals. In a l l cases the t r a n s i t i o n from somatic h i s t o n e s to a r g i n i n e - r i c h protamines appeared to occur i n the l a t e spermatid stage. I t i s at t h i s stage that the n u c l e i became r e s i s t a n t to a c e t y l a t i o n and deamination and showed a strong r e a c t i o n with the Sakaguchi t e s t . I t was a l s o the l a t e spermatids which became e x t r a c t a b l e with hot 5% TCA. In the case of the cowbird, the t r a n s i t i o n from somatic-l i k e b a s i c p r o t e i n s to protamine appeared to take place concurrent with, or j u s t p r i o r to s p i r a l i z a t i o n of the 192 spermatid nucleus. Cytochemical r e s u l t s are summarized i n Table X. b. E l e c t r o p h o r e s i s Fig.46 (channel 2) shows the e l e c t r o p h o r e t i c p r o f i l e of b a s i c p r o t e i n s e x t r a c t e d from f r e s h r o o s t e r t e s t i s by the micromethod of Kasinsky et a l . (1978), separated i n an a c i d / u r e a p o l y a c r y l a m i d e s l a b g e l , pH 3.4 (Panyim and C h a l k l e y , 1969). A l l the somatic h i s t o n e s appeared to be r e s o l v e d , with H2A and H2B appearing as a dark doublet and H4 having the f a s t e s t m o b i l i t y i n the somatic h i s t o n e (SH) r e g i o n . Only one band (G) with a m o b i l i t y f a s t e r than H4 c o u l d be seen. T h i s band migrated to a p o s i t i o n approximately half-way between H4 and SP3-5 of Xenopus l a e v i s t e s t e s . When r o o s t e r t e s t i c u l a r or ductus deferens b a s i c p r o t e i n s were e l e c t r o p h o r e s e d i n the short g e l system of Nakano et a l . (1973), pH 4.5, a s i n g l e prominent band was seen m i g r a t i n g between the somatic h i s t o n e region (SH) and h e r r i n g protamine (P) (Fig.47A). T h i s band was obtained from t i s s u e homogenized i n e i t h e r PBS (channels 1 and 3) or 1% c i t r i c a c i d (channels 2 and 4), and a s i m i l a r band was obtained from the ductus deferens of Anas p l a t y r h y n c o s (channel 5). However f a i n t bands were a l s o e vident m i g r a t i n g more slo w l y than "G" (channels land 5) and i n one case, a band moving s l i g h t l y f a s t e r than "G" was apparent (channel 4). Basic p r o t e i n s , i s o l a t e d by the micromethod from t e s t e s and d u c t i d e f e r e n t i a of the r o o s t e r ( G a l l u s domesticus), the 193 Fig u r e 45. S t a i n i n g of avian t e s t i s and ductus deferens s e c t i o n s . S e c t i o n s were from the t e s t e s of Anas p l a t y r h y n c o s (A and B) and Molothrus a t e r (E and F ) , and the ductus deferens of Coturnix c o t u r n i x j a p o n i c a (C and D). A. & E. Eosin Y s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 hr. B. & F. Eosin Y s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s (60°C, 6 hr) and a c e t y l a t i o n (60°C, 1 h r ) . C. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s , 60°C, 6 hr. D. AFG s t a i n i n g a f t e r p i c r i c a c i d h y d r o l y s i s and a c e t y l a t i o n . TABLE X. C y t o c h e m i s t r y of A v i a n Sperm S t a i n i n g 8 Reactive Organisms Used C o n t r o l s P r e t r e a t m e n t M a t e r i a l G a l l u s C o t u r n i x Anas M e l o p s i t t a c u s M o l o t h r u s Salrno Squalus Xenopus Ran domesticus c o t u r n i x p l a t y r h y n - u ndulatus a t e r g a i r d n e r i a c a n t h i u s 1 a e v i s - pTpT j a p o n i c a chos Feulgen AFG-TCA, 95-100°C AFG-TCA, 95-100°C deamination AFG, p i c r i c a c i d , 60."C AFG, p i c r i c a c i d a c e t y l a t i o n E o s i n Y-p i c r i c a c i d E o s i n Y-p i c r i c a c i d , a c e t y l a t i o n AFG without h y d r o l y s i s Sakaguchi DNA Ba s i c p r o t e i n s o t h e r than protamines• B a s i c p r o t e i n s r i c h i n a r g i n i n e B a s i c p r o t e i n s i n c l u d i n g protamines B a s i c p r o t e i n s r i c h i n a r g i n i n e 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 r i c h i n a r g i n i n e Non-nucleic a c i d a s s o c i a t e d b a s i c p r o t e i n s P r o t e i n bound a r g i n i n e Protein-bound l y s i n e Sperm h i s t o n e type 196 F i g u r e 46. E l e c t r o p h o r e t i c and absorbance p r o f i l e s of t e s t i s b a s i c p r o t e i n s from G a l l u s domesticus. P r o t e i n s were obtained from f r e s h t i s s u e homogenized i n PBS and e x t r a c t e d with 0.4 N s u l f u r i c a c i d . E l e c t r o p h o r e s i s was performed i n a 25 cm polyacrylamide s l a b g e l (15% acrylamide, 6.25 M urea) f o r 12 hr at 150 V. Absorbance p r o f i l e s (610 nm) were taken from the g e l s t a i n e d i n 0.2% Amido Black and d e s t a i n e d i n 30% methanol/10% a c e t i c a c i d . A f t e r r e s t a i n i n g i n 0.2% Amido Black and d e s t a i n i n g i n 1 M s u l f u r i c a cid/3 M urea, the g e l was photographed as shown. 1. X. 1. l a e v i s t e s t e s 2. G a l l u s domesticus t e s t i s (G) 3. G a l l u s domesticus heart 4. Bufo marinus t e s t i s (B) Labels to the l e f t of the photograph i n d i c a t e somatic h i s t o n e s and s p e r m - s p e c i f i c b a s i c p r o t e i n s (SP's) of X. 1. l a e v i s . Fig. 46 198 q u a i l (Coturnix c o t u r n i x j a p o n i c a ) and two v a r i e t i e s of M a l l a r d duck (Anas p l a t y r h y n c o s ) are shown i n Fig.47B and C. The t i s s u e used i n t h i s experiment had been s t o r e d at -70°C. Using c o n v e n t i o n a l Amido Black s t a i n i n g (Fig.47C), a l l s p e c i e s showed a component which comigrated with band "G" of the r o o s t e r i n the Panyim and C h a l k l e y g e l system. However the s e n s i t i v e d e s t a i n i n g procedure of Wray and S t u b b l e f i e l d (1970) r e v e a l e d other f a s t e r moving components as w e l l (Fig.47B), e s p e c i a l l y evident i n the sample taken from the ductus deferens of Coturnix c o t u r n i x j a p o n i c a . When the t i s s u e s were homogenized i n PBS c o n t a i n i n g 50 mM Na b i s u l f i t e , no s i g n i f i c a n t d i f f e r e n c e s i n the banding p a t t e r n s c o u l d be observed, nor when the samples were homogenized in 1% c i t r i c a c i d at pH 2.7. S t a r c h g e l e l e c t r o p h o r e s i s of b a s i c p r o t e i n s from t e s t e s and d u c t i d e f e r e n t i a of G a l l u s domesticus, Coturnix c o t u r n i x j a p o n i c a , S e r i n u s c a n a r i u s , M e l o p s i t t a c u s undulatus and Anser anser showed a prominent f a s t moving band which comigrated with Xenopus SP3-5 i n t h i s g e l system ( F i g . 4 8 ) . Bands which migrated f a s t e r than t h i s were e s p e c i a l l y apparent i n the samples taken from the d u c t i d e f e r e n t i a . These may represent a r t i f a c t s of p r o t e o l y s i s . In experiments using b a s i c p r o t e i n s i s o l a t e d from Coturnix c o t u r n i x j a p o n i c a t e s t i s chromatin by the method of Marushige and Bonner (1966), there was a l s o evidence of p r o t e o l y t i c breakdown (Fig.48A-C). A s e r i e s of up to s i x bands moving f a s t e r than H4 c o u l d be r e a d i l y seen, with the most prominent band having the slowest m o b i l i t y (Fig.48B, channel 199 F i g u r e 47. E l e c t r o p h o r e t i c p r o f i l e s of avian t e s t i s and ductus deferens b a s i c p r o t e i n s . P r o t e i n s were e x t r a c t e d from whole t i s s u e ( s t o r e d at -70°C) homogenized i n PBS or 1% c i t r i c a c i d (where i n d i c a t e d ) . A. E l e c t r o p h o r e s i s was performed i n 8 cm p o l y a c r y l a m i d e d i s c g e l s (10% acrylamide, 5 M urea) for 45 min at 40 mA. S t a i n i n g was i n 0.2% Amido Black and d e s t a i n i n g i n 30% methanol/10% a c e t i c a c i d . 1. G a l l u s domesticus t e s t i s (PBS) 2. G a l l u s domesticus t e s t i s ( c i t r i c a c i d ) 3. G a l l u s domesticus ductus deferens (PBS) 4. G a l l u s domesticus ductus deferens ( c i t r i c a c i d ) 5. Anas p l a t y r h y n c o s ( M a l l a r d / Rouen) ductus deferens (PBSl 6. Anas p l a t y r h y n c o s ( M a l l a r d / Rouen) heart (PBS) 7. H e r r i n g protamine (P) B. E l e c t r o p h o r e s i s was performed i n a 20 cm p o l y a c r y l a m i d e s l a b g e l (15% acrylamide, 6.25 M urea) f o r 7.5 hr at 200 V. The g e l was s t a i n e d i n 0.2% Amido Black and d e s t a i n e d i n 1 M s u l f u r i c acid/3 M urea. C. Gel shown in B a f t e r f u r t h e r d e s t a i n i n g i n 30% methanol/10% a c e t i c a c i d . 1. & 2. X. 1_. l a e v i s t e s t i s 3. C o t u r n i x c o t u r n i x j a p o n i c a ductus deferens 4. C o t u r n i x c o t u r n i x j a p o n i c a t e s t i s 5. G a l l u s domesticus ductus deferens 6. & 7. G a l l u s domest i c u s t e s t i s 8. Anas p l a t y r h y n c o s (Peking) t e s t i s 9. Anas p l a t y r h y n c o s ( M a l l a r d / Rouen) t e s t i s G= t e s t i s - s p e c i f i c p r o t e i n of G a l l u s domesticus ( g a l l i n e ) SH= somatic h i s t o n e s P= h e r r i n g protamine Labels to the r i g h t of B S. C i n d i c a t e somatic h i s t o n e s and s p e r m - s p e c i f i c b a s i c p r o t e i n s (SP's) of X. 1. l a e v i s . 201 9). The degree of r e s o l u t i o n of these bands v a r i e d from g e l to g e l , but i t appeared that the number of secondary bands inc r e a s e d with l e n g t h of time the chromatin p r e p a r a t i o n was s t o r e d , even at -70°C. Sperm of the r o o s t e r c o n t a i n a t r y p s i n - l i k e enzyme, e i t h e r i n the acrosome or a s s o c i a t e d with the chromatin (Howarth and Digby, 1973; Nakano e_t a l . , 1976b), which was r e s p o n s i b l e f o r the breakdown of g a l l i n e i n t o at l e a s t e i g h t components' during the i s o l a t i o n procedures f i r s t used by Nakano et a l . (1973). Using p r o t e i n s i s o l a t e d from f r o z e n semen, these workers found that the f r a c t i o n with the l a r g e s t molecular weight, G V I I I , was the most r e t a r d e d of the f a s t moving bands in polyacrylamide g e l s , with the smaller f r a c t i o n s having f a s t e r m o b i l i t i e s . I t was subsequently d i s c o v e r e d that these smaller f r a c t i o n were products of p r o t e o l y t i c degradation of GVIII (Nakano et a l . , 1976a, 1976b). Perhaps t h i s was a l s o the case with the t e s t i c u l a r b a s i c p r o t e i n s i s o l a t e d by the methods used in t h i s study. The t r y p s i n - l i k e enzyme r e p o r t e d by Nakano et a l . (1976b) i s known to be a c i d s t a b l e , e x t r a c t a b l e from sperm with d i l u t e a c i d and o p t i m a l l y a c t i v e at n e u t r a l pH. The micromethod procedure employed in t h i s study probably r e t a i n e d t h i s enzyme in the a c t i v e s t a t e . The enzyme may have been e x t r a c t e d along with the b a s i c nuclear p r o t e i n s i n 0.4 N s u l f u r i c a c i d and may have acted l a t e r , e i t h e r i n the e t h a n o l p r e c i p i t a t i o n stage or during storage i n d i l u t e a c i d / u r e a . Homogenization i n 1% c i t r i c a c i d , pH 2.7, a procedure known to remove the acrosome and 202 F i g u r e 48. Starch g e l electrophoretograms of avian t e s t i s and ductus deferens b a s i c p r o t e i n s . T i s s u e s were s t o r e d at -70°C and homogenized in PBS. Basic p r o t e i n s were e x t r a c t e d d i r e c t l y from homogenates made from organs of a s i n g l e i n d i v i d u a l , or from chromatin prepared from pooled organs of s e v e r a l i n d i v i d u a l s (where i n d i c a t e d ) . Chromatin p r e p a r a t i o n s were store d at -70°C. A. 1.X. 1. l a e v i s t e s t i s 2. G a l l u s domesticus t e s t i s (G) 3. Serinus c a n a r i u s ductus deferens (Sc) 4. Serinus c a n a r i u s t e s t i s (Sc) 5. Anser anser t e s t i s (Aa) 6. Coturnix c o t u r n i x j a p o n i c a ductus deferens (Ccj) 7. Coturnix c o t u r n i x j a p o n i c a t e s t i s (Ccj) 8. Coturnix c o t u r n i x j a p o n i c a t e s t i s chromatin (Ccj) 9. C a l f thymus H4 B. 1.X. 1_. l a e v i s t e s t i s 2. G a l l u s domesticus heart 3. G a l l u s domesticus (#1) t e s t i s (G) 4. G a l l u s domesticus (#1) ductus deferens (G) 5. G a l l u s domesticus (#2) t e s t i s (G) 6. G a l l u s domesticus (#2) ductus deferens (G) 7. G a l l u s domesticus (#3) t e s t i s (G) 8. G a l l u s domesticus (#3) ductus deferens (G) 9. Coturnix c o t u r n i x j a p o n i c a t e s t i s chromatin C. 1.X. 1_. l a e v i s t e s t i s 2. Coturnix c o t u r n i x j a p o n i c a t e s t i s (Ccj) 3. Coturnix c o t u r n i x j a p o n i c a t e s t i s chromatin 4. & 5. M e l o p s i t t a c u s undulatus t e s t i s (Mu) SH= somatic h i s t o n e s SP= s p e r m - s p e c i f i c b a s i c p r o t e i n s of X. 1. l a e v i s 204 i n a c t i v a t e the enzyme (Nakano et a l . , 1976b; Mezquita and Teng, 1977), was e v i d e n t l y not s u f f i c i e n t to prevent degradation of g a l l i n e and other avian sperm h i s t o n e s when these p r o t e i n s were e x t r a c t e d d i r e c t l y from t i s s u e homogenates by the micromethod. T h i s c o u l d have been because no p r e c a u t i o n s were taken i n the micromethod to remove contaminating c y t o p l a s m i c elements from the p r e p a r a t i o n s . However i f f r e s h r a t h e r than f r o z e n t i s s u e was used, p r o t e o l y s i s seemed to be l e s s of a problem, suggesting that some degradation of p r o t e i n s was o c c u r r i n g due to damage of t i s s u e d u r i n g f r e e z i n g and thawing. I s o l a t i o n of Coturnix c o t u r n i x j a p o n i c a t e s t i c u l a r chromatin at n e u t r a l pH, by the method of Marushige and Bonner (1966) was a l s o not e f f e c t i v e i n i n a c t i v a t i n g the p r o t e o l y t i c a c t i v i t y a s s o c i a t e d with t h i s t i s s u e . Despite the problems with p r o t e o l y s i s of avian sperm h i s t o n e s , some general comments can be made regarding the e l e c t r o p h o r e t i c data presented here. F i r s t of a l l , i n both s t a r c h and a c i d / u r e a p o l y a c r y l a m i d e g e l s , there was one prominent band moving f a s t e r than H4, which was s p e c i f i c to the t e s t i s and ductus deferens and seemed to be common to a l l the avian s p e c i e s examined. A l s o , p a t t e r n s of p r o t e o l y t i c breakdown appeared to be s i m i l a r to a l a r g e e x t e n t . T e s t i c u l a r p r e p a r a t i o n s showed between two and f i v e e x t r a fast-moving bands which had s i m i l a r m o b i l i t i e s i n each of the s p e c i e s examined, although the amounts of each band v a r i e d i n each p r e p a r a t i o n . T h i s c o u l d be an i n d i c a t i o n that a v i a n protamines have s i m i l a r s i t e s f o r p r o t e o l y t i c a t t a c k . 205 D. DISCUSSION The o v e r a l l c o n c l u s i o n which can be made from the study of sperm h i s t o n e s i n Urodela, Squamata and Aves i s that there appears to be a r e l a t i v e constancy of p r o t e i n types w i t h i n each of these v e r t e b r a t e groups. T h i s i s i n marked c o n t r a s t to the c y t o c h e m i c a l and e l e c t r o p h o r e t i c d i v e r s i t y of b a s i c p r o t e i n s found i n anuran sperm (Chapter I I ) . The t h r e e urodeles examined i n t h i s study, Cynops, Plethodon and T a r i c h a , a l l had a r g i n i n e - r i c h protamines i n the mature sperm and appeared to undergo a t r a n s i s t i o n of nuclear p r o t e i n types d u r i n g spermiogenesis, from somatic h i s t o n e s to s t a b l e protamines to protamines. E l e c t r o p h o r e s i s of t e s t i c u l a r b a s i c p r o t e i n s r e v e a l e d a s i m i l a r i t y i n m o b i l i t i e s of t e s t i s -s p e c i f i c h i s t o n e s from a l l three newts. Combined with the data of P i c h e r a l (1970) on the newt P l e u r o d e l e s w a l t l , and that of B o i s e_t a l . ( 1976) on Notophthalmus v i r i d e s c e n s , i t would appear that the t r a n s i t i o n of n u c l e a r p r o t e i n s to the f i n a l e l e c t r o p h o r e t i c a l l y constant protamine may be the g e n e r a l r u l e among members of the urodele f a m i l i e s Salamandridae and P l e t h o d o n t i d a e . In a d d i t i o n , b a s i c p r o t e i n s of the sperm from the f a m i l i e s Ambystomidae and the p r i m i t i v e Hynobidae are a l s o s i m i l a r ( P i c h e r a l , 1979). R e p r e s e n t a t i v e s of the r e p t i l i a n order Squamata a l s o d i s p l a y e d a constancy of sperm h i s t o n e types w i t h i n the sub-or d e r s L a c e r t a l i a ( l i z a r d s ) and Serpentes (snakes). E l e c t r o p h o r e t i c a l l y , the t e s t i s - s p e c i f i c b a s i c p r o t e i n s from the eleven l i z a r d s and snakes used i n t h i s study appeared 206 s i m i l a r to each other, d i s p l a y i n g two or three bands with m o b i l i t i e s f a s t e r than H4 i n long p o l y a c r y l a m i d e and s t a r c h g e l s . At l e a s t one of these bands may represent a spermatid-s p e c i f i c p r o t e i n , while the f a s t e s t moving band was apparently s p e c i f i c to the sperm as i t was the only component obtained from Elaphe semen. Chemical a n a l y s i s of the s i n g l e band obtained by short polyacrylamide g e l e l e c t r o p h o r e s i s showed a c l o s e resemblance in amino a c i d composition between t e s t i s -s p e c i f i c p r o t e i n s of both l i z a r d s and snakes (Huang, 1977; Huang et a_l. , 1978). The c y t o c h e m i c a l r e s u l t s o b tained i n t h i s study suggested that l i z a r d sperm b a s i c p r o t e i n s had a higher a r g i n i n e and lower l y s i n e content than those of the snakes, but w i t h i n each suborder members resembled each other. Examination of seven s p e c i e s of b i r d s , r e p r e s e n t i n g four a v i a n o r d e r s , showed that c y t o c h e m i c a l l y , avian sperm h i s t o n e s may be c l a s s i f i e d as protamines. However i s o l a t i o n of i n t a c t protamines from the t e s t e s of b i r d s has proven to be more d i f f i c u l t than from the other animals employed i n t h i s study. T h i s may be due to the presence of an extremely a c t i v e and a c i d s t a b l e p r o t e o l y t i c enzyme, such as that found i n the acrosome of the r o o s t e r (Nakano et a l . , 1976b). Even so, the major t e s t i s - s p e c i f i c p r o t e i n f r a c t i o n s r e s o l v e d by p o l y a c r y l a m i d e and s t a r c h g e l e l e c t r o p h o r e s i s appeared to have a s i m i l a r m o b i l i t i e s i n a l l avian s p e c i e s s t u d i e d . T h i s evidence supports the hypothesis (Kasinsky et a l . , 1978) that r e l a t i v e constancy of sperm h i s t o n e s types i s c h a r a c t e r i s t i c of v e r t e b r a t e o r d e r s other 207 than t e l e o s t f i s h and anuran amphibians. 208 IV. GENERAL DISCUSSION Comparative Aspects: The main c o n c l u s i o n which can be drawn from the r e s u l t s of t h i s t h e s i s i s that there appears to be a great deal of v a r i a b i l i t y among the sperm h i s t o n e s of Anura, while there i s much l e s s d i v e r s i t y i n the orders Urodela and Squamata and i n the c l a s s Aves. C y t o c h e m i c a l l y , anuran sperm h i s t o n e s f a l l i n t o three c a t e g o r i e s : the somatic type (e.g. Rana and X. t r o p i c a l i s ) , and two i n t e r m e d i a t e sub-types, 3A (Xenopus and Hyla) and 3B (Bufo) (Table X I ) . E l e c t r o p h o r e t i c a l l y , each genus s t u d i e d appears to have a c h a r a c t e r i s t i c t e s t i s - and s p e r m - s p e c i f i c b a s i c p r o t e i n p r o f i l e (Fig.49) In the genus Xenopus, the b a s i c n u c l e a r p r o t e i n s of spermatids and sperm appear to be s p e c i e s - and even sub-s p e c i e s - s p e c i f i c . V a r i a t i o n i n the e l e c t r o p h o r e t i c m o b i l i t i e s of sperm h i s t o n e s i n the genus Xenopus seem to r e f l e c t the e v o l u t i o n a r y d i s t a n c e s between s p e c i e s i n f e r r e d by h y b r i d i z a t i o n s t u d i e s ( M u l l e r , 1977; Vigny, 1977), karyotype analyses (Tymowska, 1976, 1977; Tymowska and F i s c h b e r g , 1973), and the examination of the changes i n serum albumins (Bisbee et a_l. , 1977) and l a c t a t e dehydrogenase isozymes (Vonwyl and F i s c h b e r g , 1980). In the present study, the most s t r i k i n g d i s c o v e r y was the absence of intermediate-type sperm h i s t o n e s i n X. t r o p i c a l i s and i t s r e l a t i v e X. sp_. n. I l l ( Z a i r e ) , f u r t h e r evidence that these s p e c i e s are 209 Table X I . The V a r i e t y of Sperm Histories i n the Ve r t e b r a t e s C l a s s i f i c a t i o n Snerm H: Type* ' Superorder Amphibia**' • Order Anura Family P i p i d a e Xenopus 3 & 4 Pelobatidae Scaphiopus couchi 3 S.' bombifrons H y l i d a e Hyla r e g i l l a 3 H. g r a t i o s a 3 Bufonidae Bufo marinus 3 B. punctatus 3 B. americanus 3 ( l ? ) c -B. boreas 3 ( l ? ) c * B. a l v a r i u s B. b . h a l o p h i l u s Ranidae Rana p i p i e n s 4 R. catesbeiana 4 R. p r e t i o s a 4 C ' R. clamitans Order Urodela Family Plethodontidae Salamandridae Plethodon vehiculum 2+1 Notophthalmus v . v i r i d e s c e n s 2+1 T a r i c h a t o r o s a  Cynops pyrrhogaster 2+1 2+1 Superorder R e p t i l i a Order Squamata Suborder L a c e r t i l i a ( l i z a r d s ) Family Iguanidae Family Anguidae A n o l i s c . c a r o l i n e n s i s  A. s . s a g r e i  Sceloporus magister S. o l i v a c e u s  Crotaphytus c o l l a r i s Gerrhonatus coerulus 1 1 1 210 Table X I . cont, C l a s s i f i c a t i o n Suborder Serpentes (snakes) Family Colubridae C l a s s A v e s e ' Subclass Neornithes Order Anseriformes Suborder Anseres Family Anatidae Order G a l l i f o r m e s F a m i l y Phasianidae Order P s i t t a c i f o r m e s Family P s i t t a c i d a e Order Passeriformes Family F r i n g i l l i d a e I c t e r i d a e Thamnophis s i r t a l i s  T. ordinoides  T. couchi  T. proximus  T. eques Elaphe g.guttata E. o.obsoleta X o . q u a d r i v i t t a t a E. o b s o l e t a q u a d r i v i t t a t a Sperm Histone  Type a-3 3 3 Anas platyrhynchos  Anser anser G a l l u s domesticus  Coturnix c . j a p o n i c a M e l o p s i t t a c u s undulatus Serinus canarius Molothrus a t e r d. 1 * 1 1 1 a. Cytochemical c l a s s i f i c a t i o n , where a v a i l a b l e . * i n d i c a t e s e l e c t r o p h o r e t i c data only a v a i l a b l e . " C l a s s i f i c a t i o n from Conant,1975 and Stebbins,1966 Bois and Kasinsky,1972 Huang et al.,1978 e. C l a s s i f i c a t i o n from Farner and King,1973 21 1 F i g u r e 49. Composite e l e c t r o p h o r e t i c p r o f i l e s of v e r t e b r a t e sperm h i s t o n e s . P= h e r r i n g protamine R= Rana H4= somatic h i s t o n e H4 Hy= Hyla X= Xenopus S= Scaphiopus B= Bufo 212 Sperm Histone Type 2 Sperm Histone Types 1,3,4 / R H4 Hy X X S B — — Somatic Histones XI EE) Chondr-ichthyes Oste-ichthyes Anura Uro-d e l a 2 1 R e p t i l i a Aves Mam-mali a Fig. 49 213 only d i s t a n t l y r e l a t e d to the r e s t of the genus Xenopus. The sperm h i s t o n e s of v a r i o u s u r o d e l e s , r e p r e s e n t i n g s e v e r a l genera, may be p l a c e d i n a s i n g l e cytochemical category (type 1), and a l l the s p e c i e s examined appear to undergo the same type of b a s i c p r o t e i n t r a n s i s t i o n d u r i n g spermiogenesis (Table X I ) . E l e c t r o p h o r e s i s has a l s o r e v e a l e d a strong s i m i l a r i t y among the t e s t i s - s p e c i f i c b a s i c p r o t e i n s of t h i s order (see a l s o P i c h e r a l , 1979). L i k e w i s e , the b a s i c p r o t e i n s from snake and l i z a r d t e s t e s (order Squamata) have extremely s i m i l a r e l e c t r o p h o r e t i c p r o f i l e s , although there appears to be a cytochemical d i s t i n c t i o n between the sperm nuc l e a r p r o t e i n s of these two groups (Table X I ) . The sperm h i s t o n e s of seven sp e c i e s of b i r d s , r e p r e s e n t i n g four avian o r d e r s , a l s o appear to f a l l i n t o a s i n g l e cytochemical c l a s s (type 1) (Table XI) and have s i m i l a r e l e c t r o p h o r e t i c p r o f i l e s . Examination of the l i t e r a t u r e shows that sperm h i s t o n e v a r i a b i l i t y i s seen among t e l e o s t f i s h . S e v e r a l orders (e.g. Clueiformes and Perciformes) have protamines i n t h e i r sperm, while others (e.g. C y p r i n i f o r m e s ) r e t a i n s o m a t i c - l i k e h i s t o n e s (Bloch, 1966, 1976; Ando et a l . , 1973; Nandi et a_l. , 1979). D a i s l e y and Davies (1979) r e p o r t e d that d i f f e r e n c e s i n amino a c i d composition were s i g n i f i c a n t between protamines of v a r i o u s f a m i l i e s of the same t e l e o s t suborder. On the other hand, the sperm nuclear p r o t e i n s of c a r t i l a g i n o u s f i s h appear to be s i m i l a r to each other (Bois et a l . , 1980), as do those of e u t h e r i a n mammals (Coelingh and R o s i j n , 1975; B e l l v e et a l . , 1975; C a l v i n , 1976). 214 It would appear t h e r e f o r e that i n v e r t e b r a t e e v o l u t i o n there has been a trend away from sperm h i s t o n e d i v e r s i t y i n the t e l e o s t s and anurans, toward a r e l a t i v e constancy of sperm p r o t e i n types i n u r o d e l e s , r e p t i l e s and mammals ( F i g . 4 9 ) . What does t h i s trend s i g n i f y ? F u n c t i o n a l Aspects; Shaping of the sperm head: As mentioned i n the General I n t r o d u c t i o n , there appears to be l i t t l e or no c o r r e l a t i o n between the shape of the sperm nucleus and the type of sperm p r o t e i n p r e s e n t . Although.the. s i m i l a r i t y of sperm h i s t o n e s i n Squamata and Urodela may be r e f l e c t e d i n an o v e r a l l s i m i l a r i t y of sperm n u c l e a r morphology and chromatin condensation p a t t e r n s i n r e p r e s e n t a t i v e s of these orders ( F u r r i e r i , 1970; P i c h e r a l , 1979), t h i s does not seem to be the general r u l e . The sperm n u c l e i of of a l l the Xenopus s p e c i e s examined i n t h i s study were a l s o m o r p h o l o g i c a l l y s i m i l a r , and yet the sperm of X. t r o p i c a l i s c o n tained only s o m a t i c - l i k e h i s t o n e s while the other s p e c i e s had e l e c t r o p h o r e t i c a l l y d i s t i n c t p r o t e i n s of the intermediate type. Furthermore, Xenopus sperm n u c l e i decondensed with 2.0 M NaCl r e t a i n e d t h e i r o r i g i n a l shape to a c o n s i d e r a b l e degree, d e s p i t e the f a c t that the b a s i c protein-DNA i n t e r a c t i o n s must have been s e r i o u s l y d i s r u p t e d ( R i s l e y et a_l. , i n p r e p a r a t i o n ) . On the other hand, while a l l the avian s p e c i e s s t u d i e d appeared to have s i m i l a r sperm nuclear p r o t e i n s , nuclear shape 215 v a r i e d from a simple curved c y l i n d e r , as seen i n G a l l u s domesticus, to the complex s p i r a l shape of Serinus c a n a r i u s and Molothrus a t e r . Sperm n u c l e i of s e v e r a l c a r t i l a g i n o u s f i s h are a l s o s p i r a l i n shape, but these n u c l e i c o n t a i n s t a b l e protamines (Bois and Kasinsky, 1974; Bois et a l . , 1980; Stanley, 1971; Gusse and C h e v a i l l i e r , 1978). F e r t i l i z a t i o n b a r r i e r s : Another p o s s i b l e e x p l a n a t i o n f o r the d i v e r s i t y of sperm h i s t o n e s i s that they may p r o v i d e a kind of f e r t i l i z a t i o n b a r r i e r which prevents c r o s s - h y b r i d i z a t i o n of s p e c i e s . However, at l e a s t w i t h i n the genus Xenopus, v i a b l e h ybrids have been produced e x p e r i m e n t a l l y between many s p e c i e s , i n c l u d i n g X. 1_. l a e v i s , X. b o r e a l i s and X. m u e l l e r i ( M u l l e r , 1977; Vigny, 1977) These s p e c i e s have sperm h i s t o n e s which are d i s t i n c t l y d i f f e r e n t from each other ( F i g . 1 9 ) . F e r t i l i z a t i o n medium: Nandi et a l . (1979) have suggested that there may be a c o r r e l a t i o n between the presence of protamines i n f i s h sperm and the s a l i n i t y of the water i n which they reproduce. Somatic-l i k e h i s t o n e s were found o n l y i n the sperm of f r e s h water s p e c i e s (e.g. C y p r i n i f o r m e s ) while protamines were c h a r a c t e r i s t i c of the sperm of marine and e s t u a r i n e s p e c i e s . In 0.6 M NaCl (the approximate s a l i n i t y of sea water), h i s t o n e -type sperm were l y s e d while sperm c o n t a i n i n g protamines remained s t a b l e . However there are marine organisms, such as 216 sea u r c h i n s , which have s o m a t i c - l i k e sperm h i s t o n e s but s t i l l reproduce s u c c e s s f u l l y i n sea water. Moreover, many s p e c i e s which reproduce in f r e s h water (salmon, newts and toads) have sperm which c o n t a i n protamines or 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 . S a l i n i t y of the f e r t i l i z a t i o n medium may have some i n f l u e n c e on the e v o l u t i o n of sperm b a s i c p r o t e i n s , but i t does not seem to be the only s e l e c t i v e pressure l e a d i n g to the appearance of the h i g h l y b a s i c sperm p r o t e i n s i n e v o l u t i o n . E v o l u t i o n a r y Aspects; As p o i n t e d out i n the General I n t r o d u c t i o n , i t would appear that at l e a s t one of the somatic h i s t o n e s must be r e p l a c e d by a s p e r m - s p e c i f i c p r o t e i n d u r i n g the course of spermiogenesis. The kind of s p e r m - s p e c i f i c p r o t e i n c o u l d be anything from a h i s t o n e H1 v a r i a n t to a h i g h l y a r g i n i n e - r i c h protamine. Bloch (1969, 1976) and Subirana (1975) have suggested that the s i g n i f i c a n c e of these s p e r m - s p e c i f i c p r o t e i n s may l i e simply i n t h e i r a b i l i t y to condense the sperm chromatin and provide p r o t e c t i o n f o r the male genome, r o l e s which may be r a t h e r u n s p e c i f i c . The only requirement of the sperm h i s t o n e s appears to be t h e i r b a s i c i t y , a l l o w i n g f o r the wide range of sperm nuc l e a r p r o t e i n types found i n nature. I t appears however, that there has been a t r e n d away from sperm h i s t o n e d i v e r s i t y i n t e l e o s t f i h and anuran amphibians, toward a more narrow range of a r g i n i n e - r i c h sperm p r o t e i n s i n urodeles, r e p t i l e s , b i r d s and mammals. T h i s t r e n d appears to c o r r e l a t e with the occurence of i n t e r n a l f e r t i l i z a t i o n and the 217 appearance of heteromorphic sex chromosomes i n v e r t e b r a t e e v o l u t i o n . A study of p o s t - t e s t i c u l a r sperm maturation has shown that in t e l e o s t s and anurans, optimal sperm m o t i l i t y i s a t t a i n e d i n the t e s t i s . The sperm of elasmobranchs, u r o d e l e s , r e p t i l e s , b i r d s and mammals r e q u i r e a p e r i o d of maturation in the excurrent ducts before becoming o p t i m a l l y m o t i l e (Bedford, 1979) (see Table X I I ) . The n e c e s s i t y for post-t e s t i c u l a r maturation appears to c o i n c i d e with the s i t u a t i o n i n which i n t e r n a l f e r t i l i z a t i o n has been adopted by a l l members of the l a r g e r group: namely, elasmobranchs, u r o d e l e s , r e p t i l e s , b i r d s and mammals. These are p r e c i s e l y the v e r t e b r a t e groups which show r e l a t i v e constancy of sperm h i s t o n e type. Thus, i n c o n t r a s t to Bloch's (1969) statement, there does appear to be a c o r r e l a t i o n between the r e l a t i v e constancy of sperm h i s t o n e s and animal groups where i n t e r n a l f e r t i l i z a t i o n i s the general r u l e . In t e l e o s t s and anurans, where f e r t i l i z a t i o n i s g e n e r a l l y e x t e r n a l , sperm h i s t o n e s may have been allowed to d i v e r g e , p o s s i b l y i n response to evironmental f a c t o r s such as s a l i n i t y , and perhaps p a r t l y i n response to the e f f e c t s of genetic d r i f t on p r o t e i n s with an u n s p e c i f i c f u n c t i o n . By c o n t r a s t , the i n t e r n a l environment of the female t r a c t may have p l a c e d c e r t a i n s e l e c t i v e c o n s t r a i n t s on the e v o l u t i o n of the sperm h i s t o n e s i n animals with i n t e r n a l f e r t i l i z a t i o n . In order to e x p l a i n the v a r i a b i l i t y of h i g h l y a r g i n i n e -r i c h sperm nuclear p r o t e i n s , Bloch (1969) proposed that i n organisms with chromosomally-based sex-determination, some of T a b l e X I I . O c c u r r e n c e o f I n t e r n a l F e r t i l i z a t i o n , Sperm H i s t o n e D i v e r s i t y i Sex Chromosomes and n the V e r t e b r a t e s C h o n d r i c h t h y e s O s t e i c h t h y es A n u r a U r o d e l a R e p t i l i a . Aves Mammalia Sperm H i s t o n e D i v e r s i t y - + + - - - -I n t e r n a l F e r t i l i z a t i o n 3 ' + r a r e v e r y t a r e + + Appearance o f Optimum Sperm •'. M o t i l i t y 3 * e x c u r r e n t d u c t t e s t i s t e s t i s d u c t d u c t d u c t d u c t P o l y p l o i d y ^ • ? + c' r a r e r a r e - -H e t e r o m o r p h i c Sex Chromosomes ' ? r a r e - r a r e + + + a , f r o m B e d f o r d , 1979. b ' f r o m M o r e s c a l c h i , 1 9 7 3 and 1976. c"Ohno e t a l . , 1968 . d , B o g a r t , 1980. rO r- 1 OO 219 the h i s t o n e genes might have been f r e e d from the s e l e c t i v e r e s t r a i n t s which o r d i n a r i l y cause h i s t o n e s to be e v o l u t i o n a r i l y c o n s e r v a t i v e . By becoming l o c a l i z e d on the heteromorphic regions of the sex chromosomes, these genes would be p r o t e c t e d from c r o s s i n g over and point-mutations might accumulate. Since the only requirement of sperm h i s t o n e s appears to be t h e i r b a s i c i t y , and s i n c e there are more codons f o r a r g i n i n e than f o r l y s i n e or h i s t i d i n e , the g e n e t i c code might p r o v i d e a " s t a t i s t i c a l t r a p " , and genetic d r i f t would be the m o t i v a t i n g f o r c e behind the accumulation of a r g i n i n e i n these p r o t e i n s . Kasinsky et a l (1978) proposed that the trend toward r e l a t i v e constancy of sperm h i s t o n e s might be e x p l a i n e d by a m o d i f i c a t i o n of Bloch's o r i g i n a l h y p o t h e s i s . They suggested that the amount of sperm h i s t o n e v a r i a b i l i t y i n a given c l a s s might be r e l a t e d to the degree to which sex-determination i s chromosomally- based. As Ohno (1969) has p o i n t e d out, i t i s with the r e p t i l e s that nature ceased i t s experimentation with p o l y p l o i d i z a t i o n , because of the appearance of heteromorphic sex chromosomes that determine s e x - d i f f e r e n t i a t i o n . Heteromorphic sex chromosomes are known to be present i n c e r t a i n s p e c i e s of l i z a r d s , and then appear more r e g u l a r l y i n the snakes, b i r d s and mammals (Becak et a l . , 1975). Rare cases of sex chromosomes have a l s o been noted among the urodeles ( M o r e s c a l c h i , 1976). With some e x c e p t i o n s , f i s h and anurans do not g e n e r a l l y d i s p l a y heteromorphic sex chromosomes, and sex r e v e r s a l i s s t i l l p o s s i b l e by hormone treatment (Rei n b l o t h , 1975). Numerous cases of p o l y p l o i d y are a l s o 220 observed i n f i s h and frogs (Ohno, 1968; M o r e s c a l c h i , 1973, 1976; Bogart, 1980). (See Table X I I ) . I t may not be simply a c o i n c i d e n c e that i n organisms where sex-determination i s l e s s chromosomally-based ( f i s h and anurans), we see d i v e r s i t y of sperm h i s t o n e types, whereas in r e p t i l e s , b i r d s and mammals, with t h e i r chromosomal sex-determination, we see only the h i g h l y a r g i n i n e - r i c h types of sperm h i s t o n e s . According to Bloch's (1969) hy p o t h e s i s , one would expect to see an accumulation of a r g i n i n e i f the genes coding f o r sperm h i s t o n e s had become l o c a t e d on the sex chromosomes. The appearance of c y s t e i n e i n the mammalian protamines c o u l d be a t t r i b u t e d to a simple p o i n t mutation from an a r g i n i n e codon, and i t s s i g n i f i c a n c e might be r e l a t e d to the f a c t that the mammalian sperm head must be h i g h l y s t a b i l i z e d i n order to get through the very t h i c k zona p e l l u c i d a of the mammalian ovum (Bedford and C a l v i n , 1974). T h i s h y p o t h e s i s would be t e s t a b l e by i s o l a t i o n of protamine mRNA from r e p t i l e s , b i r d s or mammals and l o c a l i z a t i o n of the sperm h i s t o n e genes on the sex chromosomes by _in s i t u h y b r i d i z a t i o n . In summary, Table XII i n d i c a t e s that c o r r e l a t i o n s do e x i s t between sperm hi s t o n e d i v e r s i t y in the v e r t e b r a t e s and i n t e r n a l f e r t i l i z a t i o n , appearance of optimum sperm m o t i l i t y , p o l y p l o i d y and heteromorphic sex chromosomes. 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