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Studies on sperm histones in amphibia and chondrichthyes Bols, Niels Christian 1972

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STUDIES ON SPERM HISTONES IN AMPHIBIA AND CHONDRICHTHYES by NIELS CHRISTIAN BOLS B.Sc. (Hons.) Simon Fraser University 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of . Zoology Y/e accept t h i s thesis as conforming to the required standard The University of B r i t i s h Columbia August, 1972 In present ing t h i s thes is in p a r t i a l f u l f i l m e n t o f the requirements for an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I fu r ther agree that permission for extensive copying o f th is t h e s i s fo 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 representa t i ves . It is understood that copying or p u b l i c a t i o n o f th is t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my wr i t ten permiss ion . Department of on i0 d 1 / The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The basic protein composition of sperm, as w e l l as the change i n basic proteins during spermiogenesis, has been studied i n a number of organisms, using both cytochemical and biochemical tech-niques. The sperm of the seven anurans studied are divided on cytochem-i c a l c r i t e r i a into three of the f i v e classes proposed by Bloch (1969). Rana pa l u s t r i s and R^ pretiosa are of the Rana type. Xenopus l a e v i s , Hyla vers i c o l o r , and EN r e g i l l a are of the Mytilus type while Bufo  americanus and B^ boreas appear to be of the Salmon type. Electrophoresis of t e s t i c u l a r histones from representatives of these three types reveals s i g n i f i c a n t differencesc Testis s p e c i f i c components are absent i n R^ pipiens. In l a e v i s , three t e s t i s spec-i f i c bands, migrating between salmon protamine and the somatic h i s -tones, are present. A t e s t i s s p e c i f i c band migrating close to salmon protamine i s found i n B^ americanus. The basic protein changes during spermiogenesis i n the eastern red spotted newt, Diemictylus viridescens, resemble the t r a n s i t i o n s described i n the s n a i l , Helix aspera, (Bloch and Hew 1960a), the squid, Loligo opalescens (Bloch 1962) and Pleurodeles w a l t i i (Picheral 1970). I The early stages of spermiogenesis contain somatic type histones which i n l a t e r spermatids are replaced by the Mouse/grasshopper type of pro-t e i n . In turn, these proteins are replaced by the Salmon type of protein i n the spermatozoa. Electrophoresis of t e s t i c u l a r histones of the newt supports the cytochemical events outlined. Two t e s t i s s p e c i f i c bands are found. Spermiogenesis i n three cartilaginous f i s h (dogfish, skate and r a t f i s h ) i s characterized by unusual changes i n basic proteins. Early spermatids contain somatic type histones. However, l a t e spermatids contain the Salmon type of sperm histone while spermatozoa contain the Mouse/grasshopper type. Electrophoresis of t e s t i c u l a r histones indicates that protamines are present i n elasmobranch testes. However, a Mouse/grasshopper type of protein i s not revealed. TABLE OF CONTENTS PAGE I GENERAL INTRODUCTION 1 I I BASIC PROTEIN COMPOSITION OF ANURAN SPERM 8 A. INTRODUCTION 8 B. MATERIALS AND METHODS 8 ..1.. CYTOCHEMISTRY 8 a. Frogs 8 b. Feulgen reaction 9 c« Alkaline fast green reaction 9 d. Eosin Y 11 e. Deamination 11 f. Acetylation 11 g. Alkaline fast green reaction 12 h. Feulgen-alkaline fast green procedure 12 i . Sakaguchi reaction 13 j . Dinitrofluorobenzene procedure 13 k. Controls 13 2. BIOCHEMISTRY lk a. Frogs Ik b. Preparation of chromatin 15 c. Extraction of histones 15 d. Polyacrylamide disc gel electrophoresis 16 e. Alkaline fast green staining of gels 16 f e Photographing and scanning gels 17 C. RESULTS 17 1. CYTOCHEMISTRY 17 2. BIOCHEMISTRY 27 3. SALT CONCENTRATION AND THE ALKALINE FAST GREEN REACTION jk D. DISCUSSION 37 TABLE OF CONTENTS PAGE III CHANGES IN BASIC PROTEINS DURING SPERMIOGENESIS IN THE EASTERN RED SPOTTED NEWT, DIEMICTYLUS VIRIDESCENS 42 A. INTRODUCTION 42 B. METHODS AND MATERIALS 42 1. C YTOC HEMIS.TRY 42 2. BIOCHEMISTRY 42 C. RESULTS 43 1. CYTO C HEMIS TRY 43 2. BIOCHEMISTRY 50 D. DISCUSSION 53 IV CHANGES IN BASIC PROTEINS DURING SPERMIOGENESIS IN THREE CARTILAGINOUS FISH 57 A. INTRODUCTION 57 B. METHODS AND MATERIALS 57 1. CYTOCHEMISTRY 57 2. BIOCHEMISTRY 58 C. RESULTS 60 1. CYTOCHEMISTRY 60 a. Elasmobranchii 60 b. Holocephali 68 2. BIOCHEMISTRY 75 D. DISCUSSION 81 V GENERAL DISCUSSION 87 VI REFERENCES 92 LIST OF TABLES Page I. Cytochemistry of anuran sperm nuclei . . 20-21 I I . Salt concentration and AFG reaction . . . . . . . . 35 I I I . Pretreatment and the AFG reaction 36 IV. Staining with AFG made up i n 0.30 M NaCl . . . . . 36 V. Cytochemistry of newt spermiogenesis. . . . . . . . k6 VI. Cytochemistry of dogfish and skate spermiogenesis . 66 VII. Effect of hydrolysis conditions on AFG staining i n dogfish and skate spermiogenesis 67 V I I I . Cytochemistry of r a t f i s h spermiogenesis . 73 IX. Effect of hydrolysis conditions on AFG staining i n r a t f i s h spermiogenesis X. The variety of sperm histones 86 LIST OF ILLUSTRATIONS FIGURES PAGE 1-3 Sections of Rana pretiosa t e s t i s 22 4—6 Sections of Hyla versicolor t e s t i s 23 7-9 Sections of Xenopus laevis t e s t i s 24-10-13 Sections of Bufo americanus t e s t i s 25 14- A sperm nucleus of L boreas stained with a l k a l i n e fast gr.een, p i c r i c acid hydrolysis 26 15- 17 Densitometer tracings of histones from anuran testes run on 15% polyacrylamide gels for 85 mins 30 18-28 Polyacrylamide gel electrophoresis patterns of histones from anurans 31-33 29-32 Stages of spermiogenesis i n the eastern red spotted newt 4-7 33-34- Consecutive sections of cysts of stage 3 n u c l e i stained with a l k a l i n e fast green, 5% t r i c h l o r o -acetic acid hydrolysis 4-8 35 Sperm of Diemictylus viridescens stained with al k a l i n e fast green v/ithout p r i o r hydrolysis of DNA 4-9 36-37 Densitometer tracings of histones from the eastern red spotted newt run on 15% poly a c r y l -amide gels for 70 minutes and 6 t a i n e d with buffalo black 51 38 Polyacrylamide gel electrophoresis patterns of histones from the eastern red spotted newt 52 39 Testicular histones from the newt run with t e s t -i c u l a r histones from Xenopus lae v i s and from Bufo  americanus . 5 2 4-0 The zonation of the t e s t i s of Scylorhinus canicu-lus as seen i n transverse section 62b 4-1-4-7 F o l l i c l e s containing stages of dogfish spermio-genesis 63-64-4-8 Alkaline fast green staining, 5% t r i c h l o r o a c e t i c acid hydrolysis at 97 C. 64-4-9-51 F o l l i c l e s containing l a t e stages of skate spermio-genesis 65 LIST OF ILLUSTRATIONS FIGURES PAGE 52 A l k a l i n e f a s t g r e e n s t a i n i n g , 5% t r i c h l o r o a c e t i c a c i d h y d r o l y s i s a t 97 C. 65 53-59 F o l l i c l e s c o n t a i n i n g s t a g e s o f r a t f i s h s p e r m i o -g e n e s i s 71-72 60-62 D e n s i t o m e t e r t r a c i n g s o f s k a t e h i s t o n e s r u n on "15% p o l y a c r y l a m i d e g e l s for'80 m i n u t e s and s t a i n e d w i t h b u f f a l o b l a c k 77 63-65 D e n s i t o m e t e r t r a c i n g s o f d o g f i s h h i s t o n e s r u n on 15% p o l y a c r y l a m i d e g e l s f o r 80 minutes and s t a i n e d w i t h b u f f a l o b l a c k 78 66-67 D e n s i t o m e t e r t r a c i n g s o f t e s t i c u l a r h i s t o n e s from s k a t e and d o g f i s h r u n on 15% p o l y a c r y l a m i d e g e l s f o r 80 mins and s t a i n e d w i t h b u f f a l o b l a c k . The h i s t o n e s were e x t r a c t e d from crude n u c l e a r p r e -p a r a t i o n s 79 68 P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s o f h i s t o n e s from t h e s k a t e 80 ACKNOWLEDGEMENTS I would l i k e t o acknowledge Dr. H. E. K a s i n s k y f o r h i s s u p p o r t and a d v i c e , Dr. C.V. F i n n e g a n f o r use o f h i s l a b o r a t o r y f a c -i l i t i e s , my w i f e f o r a s s i s t a n c e i n p r e p a r i n g f i g u r e s , and my mother f o r t y p i n g t h e t h e s i s . I would a l s o l i k e t o thank J i m R e i d and Bob Evans f o r t h e i r h e l p i n c o l l e c t i n g f i s h . T h i s r e s e a r c h was s u p p o r t e d by NRC g r a n t #585'+'to D r . H. E. K a s i n s k y and an NRC s c h o l a r s h i p t o t h e a u t h o r . - 1 -I GENERAL INTRODUCTION W h i l e t h e h i s t o r y o f t h e b a s i c p r o t e i n s found i n t h e sperm n u c l e u s i s a l o n g one, t h e f u n c t i o n o f t h e s e p r o t e i n s , d e s p i t e many h y p o t h e s e s , r e m a i n s unknown. B l o c h (1969) approached t h e p r o b l e m o f f u n c t i o n by c a t a l o g i n g the v a r i e t y o f b a s i c p r o t e i n s found i n sperm and s e a r c h i n g f o r p o s s i b l e c o r r e l a t i o n s w i t h p r o t e i n t y p e . T h i s a p p r o a c k , a l t h o u g h p r o m i s i n g , i s f r u s t r a t e d by the l a c k o f i n f o r -m a t i o n on the b a s i c p r o t e i n c o m p o s i t i o n o f most sperm, by t h e d i f -f i c u l t y i n i n t e r p r e t a t i n g o l d e r work i n t h e l i g h t o f r e c e n t b i o -c h e m i c a l t e c h n i q u e s ( V e n d r e l y and V e n d r e l y 1966), and by t h e pr o b l e m o f comparing c o n c l u s i o n s from b i o c h e m i c a l work w i t h t h o s e drawn from a c y t o c h e m i c a l a p p r o a c h . T h e r e f o r e , t h e b a s i c p r o t e i n c o m p o s i t i o n o f sperm, as w e l l as t h e changes i n b a s i c p r o t e i n s d u r i n g s p e r m i o g e n e s i s , has been s t u d i e d i n a number o f o r g a n i s m s , u s i n g b o t h c y t o c h e m i c a l and b i o c h e m i c a l t e c h n i q u e s . The h i s t o r y o f t h e b a s i c p r o t e i n s i n t h e c e l l n u c l e u s began i n 1874- when M i e s c h e r i s o l a t e d from salmon sperm a n i t r o g e n o u s base w h i c h he termed p r o t a m i n e ( f r o m F e l i x I960). From t h e immature t e s t e s , he i s o l a t e d a p r o t e i n but he was u n a b l e t o f i n d p r o t a m i n e s . L a t e r K o s s e l d emonstrated t h a t p r o t a m i n e s were made up o f amino a c i d s and t h e r e f o r e were p r o t e i n s . K o s s e l a l s o showed t h a t t h e b a s i c p r o t e i n from t h e u n r i p e t e s t e s b e l o n g e d t o t h e c l a s s o f b a s i c p r o -t e i n s t o which K o s s e l had g i v e n t h e name h i s t o n e (from L u c k 1964-). However, t h e d i s t i n c t i o n between t h e h i s t o n e s and t h e p r o t a m i n e s was not always a p p a r e n t . P r e s e n t l y , t h e most common d e f i n i t i o n o f h i s t o n e s i s one g i v e n by M urray (1964); " h i s t o n e s a r e b a s i c p r o t e i n s t h a t a t some time a r e a s s o c i a t e d w i t h DNA." T h i s d e f i n i t i o n i n c l u d e s p r o t a m i n e s and de-l i b e r a t e l y makes no d i s t i n c t i o n between t h e two. S i n c e i n many r e s p e c t s sperm n u c l e i a r e u n i q u e , t h e term p r o t a m i n e might be used f o r t h e b a s i c p r o t e i n s a s s o c i a t e d w i t h t h e DNA o f sperm ( J o h n s 1971)• Even t h i s d e f i n i t i o n causes d i f f i c u l t i e s s i n c e p r o t a m i n e s a r e g e n e r a l -l y thought o f o n l y i n c o n n e c t i o n w i t h t h e b a s i c p r o t e i n s f o u n d i n f i s h - 2 sperm. The most e x p l i c i t term for the basic proteins associated with sperm DNA i s sperm histone. (Bloch 1 9 6 9 ) » and, therefore, the term w i l l be used i n th i s study. Protamines w i l l refer to the very basic proteins found i n salmon sperm and the proteins that behave s i m i l a r l y to salmon sperm histones. The early work on sperm histones was lar g e l y confined to teleost f i s h . Kossel and his co-workers ( 1928) studied protamines from many species of f i s h and the work was.continued by F e l i x and his students ( i 9 6 0 ) . From these studies f i s h sperm histones were shown to contain very few kinds of amino acids, to contain about two basic amino acids to every non-basic amino acid, and to have arginine con-s t i t u t e about two-thirds of a l l amino acids. Based on these f a c t s , the sperm histones were classed by Kossel as either monoprotamines, diprotamines, or triprotamines. Monoprotamines contained only one basic amino acid:arginine. The diprotamines contained arginine and either lysine or histidine,while triprotamines contained a l l three basic amino acids. Recent work on the sperm histones of f i s h has been confined l a r g e l y to a few laboratories. Ando and Swada ( I 9 6 I ) separated the protamine from P a c i f i c herrings into two main frac t i o n s , Y and Z, and then further resolved Y into Y l and Y2. The complete amino acid se-quence of these three fractions was determined (Ando and Suzuki. 1 9 6 ? ) and with recent work by Ando and Watanabe ( 1969) the sequences of seven protamines are now known. A fundamental pentapeptide module was present i n a l l protamine sequences elucidated. This subunit may be designated as XR^, where X i s alanine, glycine, isoleucine, proline, serine, threonine or valine and R represents arginine. Ling, Trevithick, and Dixon ( 1 9 6 9 ) demonstrated that the protamine of Salmo g a i r d n e r i i was synthesized i n the cytoplasm of early sperm-atids and rapidly transferred to the nucleus. By use of various i n -h i b i t o r s , these workers were able to show that protamine was synthe-sized on ribosomes l i k e other proteins and that the protamine mRNA was very stable. Ingles and Dixon ( 1 9 6 ? ) reported extensive phosphory-l a t i o n of protamine from Sc_ g a i r d n e r i i . A l l the serine residues - 3 -were phosphorylates i n vivo. The reason for t h i s phosphorylation i s unknown. Work on the basic proteins found i n the sperm of other organ-isms proceeded much slower than the work with f i s h sperm. Perhaps the echinoderms received the most attention. The work of Kossel (1928), Hamer (1955)* and Vendrely and Vendrely (1966) indicated that the basic proteins i n the sperm of echinoderms were very s i m i l a r to those found i n somatic c e l l n u c l e i . Daly, Mirsky, and Pis (1951) studied the basic proteins from rooster sperm and found them r i c h i n arginine. Since h i s t i d i n e was present but ly s i n e ab-sent, these proteins would be classed as diprotamines. Information on the basic protein composition i n the sperm of other organisms l i k e l y proceeded slowly for two reasons. Sperm histones are most e a s i l y studied i f sperm may be obtained free of other c e l l types. "The best s t a r t i n g material for the preparation of protamines i s freshly drawn mil t from f i s h that are ready to spawn, since i t contains only one type of c e l l s , the spermatozoa" (Felix I960). With echinoderms, sperm also could be collected free from other c e l l types. The second requirement was the a v a i l a b i l i t y of large quantities of sperm i n order to do biochemical analysis. Once again f i s h s a t i s f i e d t h i s requirement. The development of cytochemical techniques to demonstrate basic proteins and to distinguish between histones and protamines was a major advance i n attempts to study the protein types i n a wide variety of organisms. A l f e r t and Geschwind (1953) used an anionic dye, fast green, at a basic pH (8.1) to s e l e c t i v e l y s t a i n proteins having high i s o e l e c t r i c points. Histones and protamines have much higher i s o e l e c t r i c points than most proteins i n the c e l l . This method does appear to be s p e c i f i c for histones and protamines since the selective staining of the nucleus i s usually achieved and the removal of DNA i s required for staining. Since DNA must be removed for staining to occur, the actual, dye binding s i t e s are l i k e l y the basic - k -groups o f h i s t o n e s and p r o t a m i n e s w h i c h a r e n o r m a l l y bound e l e c t r o -s t a t i c a l l y w i t h t h e phosphate groups o f DNA. The r e m o v a l o f DNA w i t h h o t 5% TCA r e s u l t e d i n a techn5.que t o d i s t i n g u i s h p r o t a m i n e s from h i s t o n e s . A l f e r t (1956) found t h a t w h i l e t h e e a r l y s t a g e s o f s p e r m i o g e n e s i s i n Chinook salmon s t a i n e d w i t h f a s t g r e e n a f t e r t h e e x t r a c t i o n o f DNA w i t h hot t r i c h l o r o a c e t i c a c i d , t h e mature sperm d i d n o t . S i n c e model e x p e r i m e n t s on f i l t e r paper had i n d i c a t e d t h a t h ot TCA removed p r o t a m i n e s and r e t a i n e d h i s t o n e s ( A l f e r t and Gesch-wind 1953)» t h e l a c k Of s t a i n i n g s u g g e s t e d t h e p r e s e n c e o f p r o t a -mines . A subsequent b i o c h e m i c a l s t u d y ( C a l l a n a n , C a r r o l l , and M i t c h e l l 1957) c o n f i r m e d t h a t p r o t a m i n e s were i n d e e d p r e s e n t i n t h e sperm o f t h i s s p e c i e s * Thus p r o t a m i n e s can be e x t r a c t e d f r o m f o r m a l i n f i x e d s e c t i o n s w h i l e h i s t o n e s c a n n o t . The use o f c y t o c h e m i s t r y t o d e t e r m i n e t h e b a s i c p r o t e i n compo-s i t i o n o f sperm was f u r t h e r r e f i n e d by B l o c h and Hew ( 1 9 6 0 a ) . They were a b l e t o s t a i n p r o t a m i n e s by removing DNA w i t h p i c r i c a c i d . P i c -r i c a c i d i s a s t r o n g p r e c i p i t a n t o f p r o t a m i n e s ( F e l i x I 9 6 0 ) and r e t a i n s p r o t a m i n e s i n t h e s e c t i o n s . D e a m i n a t i o n o r a c e t y l a t i o n o f l y s i n e r e s i d u e s was a l s o c a r r i e d o u t i n o r d e r t o " p e r m i t t h e s p e c -i f i c s t a i n i n g o f h i s t o n e s w h i c h have a v e r y h i g h r a t i o o f a r g i n i n e t o l y s i n e " ( B l o c h 1 9 6 6 ) . Under t h e s e c o n d i t i o n s p r o t a m i n e s d i d s t a i n w h i l e t h e b a s i c p r o t e i n s f o u n d i n t h e s o m a t i c c e l l s d i d n o t . The p r o t e i n s t h a t do s t a i n have been termed the v e r y a r g i n i n e - r i c h h i s t o n e s t o d i s t i n g u i s h them f r o m the a r g i n i n e - r i c h h i s t o n e s o f s o m a t i c c e l l s ( P i p k i n 1 969). Through the use o f t h e s e t e c h n i q u e s a number o f v e r y i n t e r e s t -i n g o b s e r v a t i o n s were made on a wide range o f o r g a n i s m s . B l o c h and Hew (1960a) found t h a t t h e mature sperm o f t h e s n a i l , H e l i x a s p e r s a , c o n t a i n e d p r o t a m i n e s . However, i n t h e t r a n s i t i o n f rom t h e n o r m a l h i s t o n e complement t o the p r o t a m i n e , s p e r m a t i d s went t h r o u g h a s t a g e where t h e y were n o t e x t r a c t a b l e w i t h h o t 5% TCA b u t y e t con-t a i n e d p r o t e i n s v e r y r i c h i n a r g i n i n e . The p r o t e i n s f o r t h i s s t a g e have been termed t h e " s t a b l e p r o t a m i n e s " B l o c h ( 1 9 6 9 ) . Subsequent s t u d i e s showed t h a t t h i s t r a n s i t i o n was common to s q u i d ( B l o c h 1962) - 5 -and P I e u r o d e l e s w a l t i i ( P i c h e r a l 1970). I n o t h e r o r g a n i s m s t h e t r a n -s i t i o n p r oceeded o n l y as f a r a s t h e " s t a b l e p r o t a m i n e . " T h i s was t h e case i n D r o s o p h i l a m e l a n o g a s t e r (Das, Gay, and Kaufmann 1964), t h e gr a s s h o p p e r ( B l o c h and B r a c k 1964) the c o c c i d ( B e r l o w i t z 1965)» the c r i c k e t (Kaye and McMaster-Kaye 1966), the mouse (Monesi 1964), and the r a t (Vaughn 1966). I n o t h e r organisms the sperm d i d n o t s t a i n a t a l l a f t e r d e a m i n a t i o n o r a c e t y l a t i o n . T h i s was the ca s e w i t h Kana  p i p i e n s sperm w h i c h were t h e r e f o r e n o t r i c h i n a r g i n i n e . P e r h a p s t h e most e x c i t i n g f i n d i n g was t h a t t h e sperm o f many c r a b s had no c y t o -c h e m i c a l l y d e m o n s t r a b l e b a s i c p r o t e i n s ( B l o c h 1966, C h e v a i l l i e r 1967* L a n g r e t h 1969« Vaughn 1968). T h i s c o n c l u s i o n was a l s o s u p p o r t e d by some b i o c h e m i c a l e v i d e n c e (Vaughn, C h a i t o f f , D e l e e n , and G a r l a n d 1969t Vaughn and H i n s c h 1970). B l o c h (1969) a t t e m p t e d t o b r i n g o r d e r t o t h i s a r r a y o f i n f o r -m a t i o n by p u t t i n g sperm i n t o c l a s s e s " a c c o r d i n g t o the n a t u r e and t h e d i s p o s i t i o n o f t h e i r h i s t o n e s . " S i n c e the c h a r a c t e r i z a t i o n o f t h e s e p r o t e i n s was l a r g e l y i n c o m p l e t e , the c l a s s e s were d e s i g n a t e d by s p e c i e s t h a t s e r v e d as t y p i c a l examples o f t h e p r o t e i n t y p e . I n o r d e r o f d e c r e a s i n g b a s i c i t y e t h e s e a r e the Salmon t y p e , t h e Mouse/ g r a s s h o p p e r t y p e , t h e M y t i l u s t y p e , t h e Kana type and t h e Crab t y p e . Salmon and organisms o f t h i s t y p e have sperm w h i c h c o n t a i n t h e monoprotamines o f K o s s e l . 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 to t w o - t h i r d s o f the amino a c i d r e s i d u e s . C y t o -c h e m i c a l l y , t h e s e p r o t e i n s a r e c h a r a c t e r i z e d by b e i n g e x t r a c t e d w i t h h o t 5% t r i c h l o r o a c e t i c a c i d , y e t a r e r e t a i n e d by p i c r i c a c i d h y d r o -l y s i s , and g i v e a b r i g h t r e d r e a c t i o n w i t h t h e S a k a g u c h i r e a g e n t f o r arginine„ The Mouse/grasshopper t y p e o f sperm c o n t a i n p r o t e i n s t h a t " a r e v e r y r i c h i n a r g i n i n e but a r e much more complex t h a n t h e monoprot-amines." A l t h o u g h n ot e x p l i c i t i n B l o c h ' s a r t i c l e , t h i s t y p e i s c h a r a c t e r i z e d s t r i c t l y on c y t o c h e m i c a l c r i t e r a . These sperm c o n t a i n the " s t a b l e p r o t a m i n e s . " These p r o t e i n s a r e not e x t r a c t e d by 3% t r i c h l o r o a c e t i c a c i d and t h e y a r e not a f f e c t e d by t h e b l o c k i n g o f l y s i n e amino g r o u p s , i n d i c a t i n g t h a t t h e y a r e r i c h i n a r g i n i n e . - 6 -This i s confirmed by the bright red reaction these n u c l e i give af t e r the Sakaguchi test for arginine. Sperm of the Mytilus type contain proteins that were c a l l e d diprotamines or triprotamines by Kossel. These histones may be cal l e d intermediate "because they f a l l between the true histones and the monoprotamines*' (Bloch 1969). Cy to chemically, Bloch did not define t h i s type. However, he gave many examples of t h i s type, c i t i n g unpublished cytochemical data as a reference, -- Bloch (1966) reported the sperm of the P a c i f i c coast mussel,'JMytilus  edulis, did stain with fast green after t r i c h l o r o a c e t i c a c i d hydro-l y s i s and continued to sta i n despite deamination of l y s i n e residues. However, t h i s staining was not as bright as that obtained with sperm of the Mouse/grasshopper type. Therefore, t h i s w i l l be used as the cytochemical d e f i n i t i o n of t h i s type. The Rana type of sperm contain histones that are s i m i l a r to those of somatic c e l l s . They do not stain after the amino groups have been blocked by deamination or acetylation. This i s how somatic histones behave. The amino acid composition of these sperm i s s i m i l a r to that obtained with somatic n u c l e i (Vendrely 1957). Sperm of the Crab type contain no cytochemically detectable basic proteins. Perhaps proteins of low b a s i c i t y s i m i l a r to the "cleavage histones" i n the developing s n a i l embryo (Bloch and Hew 1960b) are present. The most commonly held view on the function of the basic pro-teins i n the sperm nucleus i s that they are somehow involved i n the complete repression of the sperm genome* The exact o r i g i n of t h i s view i s unknown. However, t h i s view most l i k e l y stems from the hypothesis of Stedman and Stedman (1950) that the function of the basic proteins i n the c e l l nucleus " i s to act as gene repressors." Although considerable evidence has accumulated (Georgiev 1969) that histones do i n h i b i t DNA-dependent RNA synthesis and thus gene action, the almost t o t a l lack of histone tissue s p e c i f i c i t y argues against histones being s p e c i f i c gene regulators. The discovery that the - 7 -a r g i n i n e - r i c h h i s t o n e (f2al) i s r e m a r k a b l y s i m i l a r i n c a l f and pea (De Lange e t a l . 1969) a l s o a r g u e s a g a i n s t s p e c i f i c i t y i n t h e r e p r e s s i o n o f t h e genome by h i s t o n e s . However, t h e b a s i c p r o t e i n s f o u n d i n t h e sperm n u c l e u s a r e o f c o n s i d e r a b l e i n t e r e s t s i n c e t h e y a r e one o f the few examples o f h i s t o n e t i s s u e s p e c i f i c i t y , as w e l l as d i s p l a y i n g c o n s i d e r a b l e s p e c i e s s p e c i f i c i t y . Thus the b a s i c p r o t e i n s found i n sperm n u c l e i a r e unique and might prove u s e f u l i n e l u c i d a t i n g t h e f u n c t i o n o f h i s t o n e s i n g e n e r a l . A n o t h e r p o p u l a r h y p o t h e s i s i s t h a t p r o t a m i n e s a r e n e c e s s a r y f o r t h e p r o p e r " p a c k a g i n g " o f t h e sperm DNA. Thus the p a t t e r n o f n u c l e a r c o n d e n s a t i o n , the shape o f t h e n u c l e u s , and the amount o f DNA p r e s e n t i n the n u c l e u s a r e o f i n t e r e s t i n o r d e r t o t e s t t h i s h y p o t h e s i s . Sperm h i s t o n e s may have a " p r o t e c t i v e " r o l e ( B l o c h 1969). Neb-u l o u s as t h i s t erm i s , p o s s i b l e c o r r e l a t i o n s might e x i s t between sperm h i s t o n e type and e x t e r n a l o r i n t e r n a l f e r t i l i z a t i o n . A c o r -r e l a t i o n might e x i s t between s h o r t o r l o n g - l i v e d sperm and t h e h i s t o n e t y p e . O l i n s , O l i n s , and Yon H i p p e l (1968) have s u g g e s t e d t h a t the f u n c t i o n o f the p r o t a m i n e s i s t o e r a s e the de v e l o p m e n t a l h i s t o r y o f the c e l l i n o r d e r t o r e s t o r e t o t i p o t e n c y . A n o t h e r p o s s i b i l i t y i s t h a t the type o f sperm h i s t o n e p r e s e n t i s r e l a t e d to the b e g i n n i n g o f RNA s y n t h e s i s i n t h e embryo o r t o t h e c l e a v a g e p a t t e r n ( B l o c h 1969). On t h e o t h e r hand, t h e sperm h i s t o n e s might have no f u n c t i o n and the " v a r i a b i l i t y o f t h e p r o t e i n r e f l e c t s an e v o l u t i o n a r y i n d i f f e r e n c e t o a r e l a t i v e l y unimportant- p r o t e i n i n a n i n e r t n u c l e u s " ( B l o c h 19690* - 8 -I I . BASIC PROTEIN COMPOSITION OF ANURAN SPERM A. INTRODUCTION W h i l e a Rana c l a s s has been d e s i g n a t e d , work on a nuran sperm h i s t o n e s has been m i n i m a l . R e f e r r i n g t o u n p u b l i s h e d c y t o c h e m i c a l d a t a , B l o c h (1969) c l a s s i f i e d t h e sperm h i s t o n e s o f Bufo v u l g a r i s and Xenopus l a e v i s as the M y t i l u s t y p e . O n l y the sperm h i s t o n e s o f Rana p i p i e n s have been s t u d i e d b i o c h e m i c a l l y ( V e n d r e l y 1957; B l o c h 1962) and c y t o c h e m i c a l l y ( B l o c h 1962; Z i r k i n 1970). The h i s t o n e s o f t h e s e sperm were fo u n d t o be s i m i l a r t o t h o s e o f s o m a t i c c e l l s . A l t h o u g h l i t t l e i n f o r m a t i o n i s a v a i l a b l e on anuran sperm h i s -t o n e s , B l o c h (1969) used examples from anurans t o t e s t many hypo-t h e s e s on sperm h i s t o n e f u n c t i o n . I n o r d e r t o p r o p e r l y t e s t t h e s e h y p o t h e s e s i n the f u t u r e , t h e v a r i e t y o f a n u r a n sperm h i s t o n e s has been f u r t h e r examined. B. MATERIAL AND METHODS 1. CYTOCHEMISTRY a. F r o g s S e x u a l l y mature Rana p a l u s t r i s and H y l a v e r s i c o l o r were o b t a i n e d from t h e C o n n e c t i c u t V a l l e y B i o l o g i c a l S u p p l y Co. (Southampton, Mas-s a c h u s e t t s ) and s e x u a l l y mature Bufo americanus from the S t e i n h i l b e r Co. (Oshkosh, W i s c o n s i n ) i n May 1971 • Xenopus l a e v i s were p u r c h a s e d from the South A f r i c a n Snake Farm (Cape P r o v i n c e , S outh A f r i c a ) i n O c t o b e r , 1970, and were m a i n t a i n e d w i t h a l a b o r a t o r y s t o c k . Rana  p r e t i o s a , H y l a r e g i l l a , and Bufo boreas were c o l l e c t e d i n t h e l o w e r F r a s e r V a l l e y , B r i t i s h C o l u m b i a , d u r i n g t h e i r b r e e d i n g seasons i n 1971. The a n i m a l s vrere e t h e r i z e d and t h e t e s t e s q u i c k l y removed and f i x e d i n 1056 n e u t r a l b u f f e r e d f o r m a l i n ( P e a r s e 1968, page 601) o r i n a b s o l u t e e t h a n o l - g l a c i a l a c e t i c a c i d (3:l)« F o r m a l i n . f i x a t i o n was f o r 4—6 h a t room t e m p e r a t u r e f o l l o w e d by a 14—17 h wash i n r u n n i n g t a p w a ter* T i s s u e s were then d e h y d r a t e d i n a g r a d e d s e r i e s o f e t h -a n o l , c l e a r e d i n benzene, and embedded i n P a r a p l a s t o r P a r a p l a s t P l u s ( F i s h e r S c i e n t i f i c C o . ) . F i x a t i o n i n a b s o l u t e e t h a n o l - g l a c i a l a c e t i c a c i d was f o r 2 h a t room t e m p e r a t u r e f o l l o w e d by 2 r i n s e s (1 h each) i n a b s o l u t e e t h a n o l . T i s s u e s were t h e n c l e a r e d i n benzene and embed-ded i n P a r a p l a s t . S e c t i o n s (6-10u)were c u t on a Spencer "820" m i c r o -tome. W i t h t h e e x c e p t i o n o f L l a e v i s , r e s u l t s were r e c o r d e d f o r t h e sperm f o u n d i n the t e s t e s o f s e x u a l l y mature f r o g s i n , o r j u s t b e f o r e , t h e i r b r e e d i n g season (see Wright and W r i g h t 19^9» f o r b r e e d -i n g seasons and a t a b l e o f b r e e d i n g s i z e s ) . F o r X^ l a e v i s , t e s t e s were f i x e d 2k h a f t e r t h e f r o g s had r e c e i v e d i n j e c t i o n s o f c h o r i o n i c g o n a d o t r o p h i n . T h i s i s a s t a n d a r d method f o r o b t a i n i n g f e r t i l e X. l a e v i s males (Gurdon 196?)• Thus, mature sperm were p r e s e n t i n a l l f r o g s . b. F e u l g e n r e a c t i o n D e x o y r i b o n u c l e i c a c i d (DNA) was demons t r a t e d by t h e F e u l g e n r e -. o a c t i o n . S e c t i o n s were h y d r o l y z e d f o r 11 m i n u t e s i n 1 N HC1 a t 60 C. A f t e r two r i n s e s (5 m i n u t e s each) i n d i s t i l l e d H 20, 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 r e a g e n t f o r 30 m i n u t e s . T h i s was f o l l o w e d by many s h o r t r i n s e s i n d i s t i l l e d E^O, a f i v e minute r i n s e i n a c i d -b i s u l f i t e wash, and t h e n two r i n s e s (5 m i n u t e s ) i n d i s t i l l e d E\>0 a g a i n . S e c t i o n s were d e h y d r a t e d , c l e a r e d , and mounted. DNA was a l s o demonstrated by the F e u l g e n p r o c e d u r e a f t e r B l o c h and Godman (1935) and by b a s i c f u c h s i n i n a c i d a l c o h o l ( H o r o b i n and K e v i l l -Davie6 1971)» The t h r e e t e c h n i q u e s 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 i o n The p r e s e n c e o f h i s t o n e s was demonstrated by t h e A l f e r t and Geschwind (1953) p r o c e d u r e e D e o x y r i b o n u c l e i c a c i d was removed f r o m s e c t i o n s w i t h hot (85-89°C) 5% t r i c h l o r o a c e t i c a c i d (TCA), b e f o r e s t a i n i n g f o r 30 minutes w i t h 0.1% (w/v) a l k a l i n e f a s t g r e e n (AFG) a t pH' 8.1-8.3. The c o r r e c t pH was o b t a i n e d by t i t r a t i n g w i t h 0.1N NaOH. The AFG s o l u t i o n was used i m m e d i a t e l y a f t e r p r e p a r a t i o n . H y d r o l y s i s w i t h 5% TCA was c a r r i e d out w i t h f r e s h r e a g e n t on each o c c a s i o n . Re-p e a t e d use o f the same TCA s o l u t i o n r e s u l t e d i n the i n c o m p l e t e r e -- 10 -moval o f DNA w h i c h i n t u r n l e a d t o re d u c e d s t a i n i n g . S i n c e t h e sperm o f Rj_ p r e t i o s a t B. r e g i l l a , and H«_ v e r s i c o l o r appeared t o be de s -t r o y e d a t 9k-100°C and t h e i r morphology a l t e r e d between 90-94-°C, DNA was removed a t l o w e r t e m p e r a t u r e s (8f5-89°C) w h i c h l e f t t h e s e sperm n u c l e i i n t a c t . The r e m o v a l o f DNA was checked by t h e F e u l g e n p r o c e d u r e f o r DNA. A f t e r h y d r o l y s i s t h e s e c t i o n s were immersed i n t h r e e changes o f 70% e t h a n o l (10 m i n u t e s each) to remove TCA. S t a i n -i n g was "followed by a f i v e m i nute r i n s e i n d i s t i l l e d H^O, d e h y d r a t i o n , and mounting. D e o x y r i b o n u c l e i c a c i d was a l s o removed w i t h I N t r i -c h l o r o a c e t i c a c i d f o r J h a t 6o°C ( B l o c h 1966) and t h e same s t a i n i n g p r o c e d u r e f o l l o w e d . The two methods gave i d e n t i c a l r e s u l t s . How-e v e r , w i t h t h i s method, t h e d i f f e r e n c e between p r o t a m i n e and non-pr o t a m i n e c o n t a i n i n g c e l l s i s l e s s d i s t i n c t . Treatment o f s e c t i o n s w i t h hot 5% TCA ca u s e s t h e comp l e t e removal o f p r o t a m i n e . B e i n g a s t r o n g e r p r e c i p i t a n t t h a n 5% TCA, I N TCA, w h i l e removing most p r o t -amine, causes t h e r e t e n t i o n o f a s m a l l amount o f p r o t a m i n e . The IN TCA - t r e a t m e n t a l s o a l t e r s n u c l e a r morphology, p a r t i c u l a r l y t h e sperm m u c l e i o f R._ p r e t i o s a , H. v e r s i c o l o r and H«_ r e g i l l a . S i n c e p r o t a m i n e s a r e washed o u t by t r i c h l o r o a c e t i c a c i d t r e a t -ment, DNA was a l s o removed by t r e a t m e n t w i t h s a t u r a t e d p i c r i c a c i d f o r 6 h a t 60°C ( B l o c h and Hew I960). S e c t i o n s were t h e n s t a i n e d w i t h a l k a l i n e f a s t g r e e n . The p i c r i c a c i d s o l u t i o n was made j u s t b e f o r e us e . As w i t h t h e TCA method, the h y d r o l y s i s r e a g e n t gave poor r e s u l t s i f r e u s e d . C y t o p l a s m i c s t a i n i n g was o b s e r v e d u n l e s s measures were t a k e n t o remove p i c r i c a c i d from t h e s e c t i o n s b e f o r e s t a i n i n g . Two methods were t r i e d . As recoamended by P i p k i n (1968), p i c r i c a c i d was removed by r i n s i n g s e c t i o n s b r i e f l y (5 seconds) i n a c e t o n e con-t a i n i n g 1% c o n c e n t r a t e d h y d r o c h l o r i c a c i d ( v / v ) . However, t h i s p r o -c e d u re r e s u l t e d i n re d u c e d s t a i n i n g which i s perhaps due t o the ex-t r a c t i o n o f b a s i c p r o t e i n s . A l s o , many sperm n u c l e i a p p e a r e d d i s -t o r t e d as a r e s u l t o f t h i s s t e p . However, p i c r i c a c i d c o u l d be r e -moved from s e c t i o n s w i t h o u t t h e s e c o m p l i c a t i o n s by l o n g r i n s e s i n d i s t i l l e d H 20 (3-2*f h ) . The AFG r e a c t i o n was per f o r m e d o n l y w i t h m a t e r i a l f i x e d i n f o r m a l i n . - 11 -d. E o s i n Y E o s i 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 ( B l o c h and Hew 1960a) t o c o r r o b o r a t e t h e 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 the r i n s i n g a f t e r the p i c r i c a c i d h y d r o l y s i s , were s t a i n e d w i t h 0.1% (w/v) e o s i n Y b u f f e r e d a t pH 8.1-8.3 w i t h 0.01M t r i s - H C l 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 h, r i n s e d i n d i s t i l l e d H^O f o r 5 m i n u t e s , d e h y d r a t e d , and mounted. I n some c a s e s , i n o r d e r t o improve s p e c i -f i c i t y , t h e d i s t i l l e d H 20 was bro u g h t t o pH 8.3. O n l y f o r m a l i n f i x e d m a t e r i a l was used f o r t h i s p r o c e d u r e . e. D e a m i n a t i o n To d e t e r m i n e whether v e r y a r g i n i n e - r i c h h i s t o n e s were p r e s e n t , d e a m i n a t i o n o f l y s i n e r e s i d u e s ( B l o c h and Hew 1960a) 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 t o s t a i n i n g w i t h AFG . Two changes o f n i t r o u s a c i d , p r e p a r e d j u s t b e f o r e use by c o m b i n i n g e q u a l volumes o f (10% w/v) t r i c h l o r o a c e t i c a c i d and 10% (w/v) sodium n i t r i t e , were used f o r 15 m i n u t e s each. The s t o c k s o l u t i o n s o f t r i c h l o r o a c e t i c a c i d and sodium n i t r i t e were n e v e r k e p t f o r more t h a n one week. 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 p r o c e d u r e o f P i p k i n (1968) was a l s o t r i e d . T h i s c o n s i s t e d o f t h r e e s u c c e s s i v e 15 minute changes a t 4 C i n f r e s h -l y p r e p a r e d n i t r o u s a c i d s o l u t i o n . However, d e a m i n a t i o n was jud g e d i n c o m p l e t e i n the c o n t r o l Re. p i p i e n s t e s t i s . The d e a m i n a t i o n p r o -cedure 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 gave i n c o n s i s t e n t r e s u l t s . T h i s was due t o the i n c o m p l e t e r e m o v a l o f p i c r i c a c i d , w h i c h a p p e a r e d t o i n h i b i t t h e d e a m i n a t i o n r e a c t i o n . f . A c e t y l a t i o n A l s o , t o de t e r m i n e whether v e r y a r g i n i n e - r i c h h i s t o n e s were p r e s e n t , a c e t y l a t i o n o f l y s i n e r e s i d u e s ( B l o c h and Hew 1960a) was per f o r m e d 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 b e f o r e s t a i n i n g w i t h AFG o r e o s i n Y. A f t e r h y d r o l y s i s , the e l i d e s were r i n s e d i n t h r e e changes o f d i s t i l l e d E^O (5 m i n u t e s e a c h ) , d e h y d r a t e d i n e t h a n o l , and t r a n s f e r r e d t o 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 a n h y d r i d e . Time and t e m p e r a t u r e s were v a r i e d i n o r d e r t o f i n d a c e t y l a t i o n c o n d i t i o n s w h i c h gave t h e most c o n s i s t e n t r e s u l t s . S l i d e s were l e f t i n the a c e t y l a t i o n r e a g e n t f o r 1, 2, 3» ^» 8, 12, and - 12 -2k h at 60 C and at room t e m p e r a t u r e . A c e t y l a t i o n at room temper-a t u r e f o r 2k h was fou n d t o be b e s t . However, a c e t y l a t i o n f o r 1 h at 60°C gave adequate r e s u l t s i f p i c r i c a c i d was c o m p l e t e l y removed from t h e s e c t i o n s p r i o r t o a c e t y l a t i o n . S i n c e a c e t y l a t i o n a t room te m p e r a t u r e f o r 2k h was used f i r s t and d i d g i v e c o n s i s t e n t r e s u l t s , t h i s p r o c e d u r e was adopted f o r t h e r e s t o f t h e s t u d y . g. A l k a l i n e f a s t green w i t h o u t 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 , a l k a l i n e "fast g r e e n s t a i n i n g was used w i t h o u t p r i o r h y d r o l y s i s f o r t h e removal o f DNA. S e c t i o n s were brought t o w a t e r , s t a i n e d f o r 30 minutes i n AFG, r i n s e d i n d i s -t i l l e d H^O f o r 5 m i n u t e s , d e h y d r a t e d , and mounted. I n i t i a l l y , t h i s t e c h n i q u e was c a r r i e d o u t w i t h f a s t g r e e n i n c i t r a t e - p h o s p h a t e b u f f e r a t p H S . l as recommended by Chayen e t a l . (1969) f o r g e n e r a l 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 u n u s u a l r e s u l t s . T h e r e f o r e , 0.01M t r i s - H C l b u f f e r was t r i e d ; y e t the same r e s u l t s were a c h i e v e d . To determine whether t h e s e o b s e r v a t i o n s were due t o s a l t c o n c e n t r a -t i o n , s e c t i o n s were s t a i n e d i n AFG made up i n i n c r e a s i n g l y h i g h e r c o n c e n t r a t i o n s o f N a C l . A range from no NaCl t o Z'.kM N a C l was t r i e d . From t h e s e e x p e r i m e n t s ^ the g e n e r a l p r a c t i c e o f u s i n g AFG a d j u s t e d t o pH 8.1-8.3 w i t h 0 .1N NaOH was adopted. F o r t h i s t e s t , m a t e r i a l f i x e d i n 10% NBF and e t h a n o l - g l a c i a l a c e t i c a c i d (3:1) was u s e d . h. F e u l g e n - a l k a l i n e f a s t green p r o c e d u r e The method o f Vaughn (I966) was f o l l o w e d t o demonstrate non-DN A - a s s o c i a t e d b a s i c p r o t e i n s . S l i d e s were brought t o w a t e r and then h y d r o l y z e d f o r 25 minutes i n I N t r i c h l o r o a c e t i c a c i d a t 60°C. S t a i n i n g i n t r i c h l o r o a c e t i c a c i d - S c h i f f ' s r e a g e n t (made up a c c o r d i n g t o P i p k i n 1968) f o r k5 minutes a t room t e m p e r a t u r e f o l l o w e d . N e x t , t h r e e 5 minute r i n s e s i n s u l f i t e b l e a c h ( p r e p a r e d w i t h t r i c h l o r o a c e t -i c a c i d ) and then t h r e e 10 minute r i n s e s i n 70% e t h a n o l were c a r r i e d o u t . S e c t i o n s were then s t a i n e d w i t h a l k a l i n e f a s t g r e e n i n t h e norma l manner. A f t e r s t a i n i n g t h e y were d i f f e r e n t i a t e d f o r 5 min-u t e s i n two r i n s e s o f a b s o l u t e m e t h a n o l , c l e a r e d i n x y l e n e , and mounted. T h i s p r o c e d u r e was c a r r i e d o ut on f o r m a l i n f i x e d m a t e r i a l o n l y . - 13 -1. S a k a g u c h i r e a c t i o n A r g i n i n e was demonstrated by t h e S a k a g u c h i r e a c t i o n as mod-i f i e d by D e i t c h (1961). S t o c k s o l u t i o n s o f 4% b a r i u m h y d r o x i d e (w/v) and 1,5% 2 , 4 - d i c h l o r o - n a p h t h o l (w/v) i n t e r t i a r y b u t a n o l were made up i n advance and used o v e r a p e r i o d o f a month. J u s t b e f o r e u s e , 1% sodium h y p o c h l o r i t e (v/v) was p r e p a r e d . I n s t e a d o f " c l o r o x , " " w h i te magic b l e a c h " (Safeway L t d . ) was used as a s o u r c e o f 5% NaOCl. S l i d e s were brought t o water and p l a c e d i n an empty C o p l i n j a r . I m m e d i a t e l y the s t a i n i n g r e a g e n t was p r e p a r e d (5 p a r t s b a r i u m h y d r o x i d e , 1 p a r t h y p o - c h l o r i t e , and then 1 p a r t d i c h l o r o n a p h t h o l ) and poured on the s l i d e s . S t a i n i n g t o o k p l a c e a t room t e m p e r a t u r e f o r 10 m i n u t e s . S l i d e s were t h e n t r a n s f e r r e d t h r o u g h 3 changes o f t e r t i a r y b u t a n o l c o n t a i n i n g 3% ( v / v ) o f 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 s e e s and the n e x t two f o r 30 sees each. S l i d e s were c l e a r e d i n two changes (30 s e e s each) o f x y l e n e 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 and mounted i n Permount c o n t a i n i n g 10% ( v / v ) t r i - N -b u t y l a m i n e . The Sa k a g u c h i r e a c t i o n was perf o r m e d on m a t e r i a l f i x e d i n f o r m a l i n as w e l l as m a t e r i a l f i x e d i n a b s o l u t e e t h a n o l - g l a c i a 3 L a c e t i c a c i d ( 3 s i ) . j . D i n i t r o f l u o r o b e n z e n e p r o c e d u r e P r o t e i n - b o u n d 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 -zene (DNFB) p r o c e d u r e as o u t l i n e d by P i p k i n (1968). 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 h y d r o l y z e d f o r 15 m i n u t e s i n 5% TCA a t 86-89 C t o remove f o r m a l d e h y d e . I n some c a s e s , formaldehyde was removed by immersing s e c t i o n s i n b o i l i n g w a t e r f o r f i v e m i n u t e s . T h i s s t e p i n s u r e d the r e t e n t i o n o f a l l b a s i c p r o t e i n s . A f t e r t h r e e 10 minute changes o f 70% e t h a n o l , t h e 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 (0.60ml DNFB i n 26 ml o f e t h a n o l , 4.0ml IM sodium b i c a r b o n -a t e , and 20.ml o f d i s t i l l e d w a t e r ) f o r 30 m i n u t e s a t room t e m p e r a t u r e . The r e a g e n t was used i m m e d i a t e l y on 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 o f 70% e t h a n o l , d e h y d r a t e d , c l e a r e d i n x y l e n e , 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 s u b j e c t e d t o - Ik -t r i c h l o r o a c e t i c o r p i c r i c a c i d h y d r o l y s i s and t h e n t r e a t e d w i t h 0.1N HC1 f o r 15 h r t o remove h i s t o n e s . S t a i n i n g was v e r y l i g h t o r a b s e n t . I n a l l s t a i n i n g p r o c e d u r e s , sperm from a number o f d i f f e r e n t s p e c i e s were s t a i n e d a t t h e same time i n o r d e r t o f a c i l i t a t e c o m p a r i s o n s . A number o f organisms i n which t h e b a s i c p r o t e i n c o m p o s i t i o n o f the sperm was 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 . The f o l -l o w i n g organisms were u s e d : Salmo g a i r d n e r i i , whose sperra c e l l s c o n t a i n p r o t a m i n e s ( I n g l e s e t a l . 1966); 'Hj. p i p i e n s , w h i c h c o n t a i n s h i s t o n e s i n the sperm s i m i l a r t o t h o s e o f s o m a t i c c e l l s ( B l o c k 1962; Z i r k i n 1970); and C a r a s s i u s a u r a t u s , whose sperm a r e s i m i l a r t o t h o s e o f R«_ p i p i e n s ( Z i r k i n 1971)» S i n c e the s t a i n i n g p a t t e r n o b s e r v e d w i t h B^ b o r e a s and B^ americanus appeared u n u s u a l , a l k a l i n e f a s t g r e e n s t a i n i n g w i t h b o t h t r i c h l o r o a c e t i c a c i d and p i c r i c a c i d h y d r o l y s i s was r e p e a t e d w i t h m a t e r i a l f i x e d i n a b s o l u t e e t h a n o l - g l a c i a l a c e t i c a c i d . A l s o , AFG s t a i n i n g was t r i e d f o r d i f f e r e n t l e n g t h s o f t i m e , f o r d i f -f e r e n t p e r i o d s o f h y d r o l y s i s , and a t d i f f e r e n t t e m p e r a t u r e s . A l l p h o t o g r a p h s were t a k e n on a Z e i s s p h o t o m i c r o s c o p e . 2. BIOCHEMISTRY a. F r o g s Re p i p i e n s were o b t a i n e d from E. G. S t e i n h i l b e r & Co. (Oshkosh, W i s c o n s i n ) i n December 1971 w h i l e B_j_ americanus were o b t a i n e d from t h e same company i n May 1972. W i t h R^ p i p i e n s , t h e a n i m a l s were e t h e r -i z e d . The t e s t e s and l i v e r s were q u i c k l y removed and s t o r e d a t -20°C u n t i l the t i m e o f use. I n December, the most common s t a g e o f spermato-g e n e s i s i n t h e Ro_ p i p i e n s t e s t i s i s mature sperm (Rugh 1959)• The t o a d s , B_._ a m e r i c a n u s , were p i t h e d ; t h e t e s t e s and l i v e r s were q u i c k l y removed and used a l m o s t i m m e d i a t e l y . S i n c e americanus b r e e d s i n May and June ( W r i g h t and W r i g h t 19^9), the t e s t i s a t t h i s t i m e o f the y e a r s h o u l d c o n t a i n many mature sperm. A l i g h t m i c r o s c o p e i n s p e c t i o n o f t h e t e s t i s c o n f i r m s t h a t sperm a r e the predominant c e l l t y p e p r e s e n t . S e x u a l l y mature l a e v i s males were o b t a i n e d o r i g i n a l l y f r o m t h e South A f r i c a n Snake Farm and were m a i n t a i n e d w i t h a l a b o r a t o r y s t o c k f o r a t l e a s t two y e a r s . D u r i n g t h i s t i m e , t h e y p e r i o d i c a l l y r e c e i v e d - 15 -i n j e c t i o n s o f c h o r i o n i c g o n a d o t r o p h i n w h i c h i s a s t a n d a r d method f o r o b t a i n i n g f e r t i l e X^ l a e v i s males (Gurdon 196?). The X^ l a e v i s males were p i t h e d . The t e s t e s and l i v e r s were q u i c k l y removed and used a l -most i m m e d i a t e l y . b. P r e p a r a t i o n o f c h r o m a t i n W i t h s l i g h t m o d i f i c a t i o n s , c h r o m a t i n was i s o l a t e d by t h e p r o c e d -u r e o f Marushige and Bonner (1966). A l l s t e p s were p e r f o r m e d a t 4-°C o r on i c e . T e s t e s and l i v e r s were homogenised i n a S o r v a l l Omni M i x e r w i t h s a l i n e - E D T A (0.075 M N a C l , 0.024- M EDTA, pH 8.0) a t a speed s e t -t i n g o f 5 f o r 2 m i n u t e s , f i l t e r e d t h r o u g h 4- l a y e r s o f washed cheese-c l o t h , and c e n t r i f u g e d a t 1,500 g f o r 15 m i n u t e s . I n seme c a s e s , s e v -e r a l d r o p s o f 2- o c t a n o l was added t o p r e v e n t f r o t h i n g . The n u c l e a r p e l l e t was washed t w i c e by r e s u s p e n d i n g i n s a l i n e - E D T A (on V o r t e x -Genie f o r 1 m i n u t e ) and c e n t r i f u g a t i o n . S i n c e the n u c l e a r p e l l e t s f r om l i v e r s were h e a v i l y c o n t a m i n a t e d w i t h pigment, a d d i t i o n a l wash-i n g s were employed f o r t h i s t i s s u e . The n u c l e a r p e l l e t was homogen-i z e d w i t h a Dounce hand homogenizer i n 0.01 M T r i s b u f f e r (pH 8.0). C h r o m a t i n was sedimented a t 10,000g f o r 15 minutes and washed once w i t h the same b u f f e r . I n a few c a s e s , c h r o m a t i n was f u r t h e r p u r i f i e d by c e n t r i f u g a t i o n t h r o u g h s u c r o s e . Chromatin was homogenized i n 0.01 M T r i s b u f f e r (pH 8.0) w i t h a Dounce homogenizer. S i x ml o f t h i s c h r o m a t i n was l a y e r e d o v e r 25ml o f 1.7 M s u c r o s e . The upper o n e - t h i r d o f t h e tube was mixed g e n t l y and c e n t r i f u g e d a t 22,000 rpm f o r 2 h i n the SW-27 r o t o r o f a Spindo L2-65 p r e p a r a t i v e u l t r a c e n t r i f u g e . P u r i f -i c a t i o n t h r o u g h s u c r o s e was o m i t t e d i n most c a s e s s i n c e M a r u s h i g e e t a l . (1969) f o u n d t h a t w i t h t r o u t t e s t e s i n t h e l a t e s t a g e s o f dev-elopment the p u r i t y o f the c h r o m a t i n was not s i g n i f i c a n t l y a l t e r e d by t h i s s t e p . c. E x t r a c t i o n o f h i s t o n e s Chromatin was homogenized i n 0.2 M ^ S O ^ w i t h a Dounce homogen-i z e r and s t i r r e d i n the same s o l u t i o n f o r 1 h a t 4-°C. T h i s was f o l -l o wed by c e n t r i f u g a t i o n a t 10,000 g f o r 20 m i n u t e s . To the s u p e r -n a t a n t , 3-4- volumes o f c o l d 100% e t h a n o l was added and t h e m i x t u r e was p l a c e d i n the f r e e z e r (-20°C) where p r e c i p i t a t i o n u s u a l l y o c c u r r e d - 16 -within 2k h. The p r e c i p i t a t e was recovered by centrifugation, washed once with ethanol, and dried i n vacuo. In most cases, the chromatin p e l l e t was extracted a second time by the above procedure. d. Polyacrylamide disc gel electrophoresis Histones were fractionated on 15% polyacrylamide disc gels by the method of Bonner ejt a l . (1968). Protein samples were dissolved i n 8 M urea at 1 mg/ml. This concentration was approximated i n cases where only a small amount of protein was a v a i l a b l e . D i t h i o t h r e i t o l (DTT) was added (0.05 ml of 1 M DTT/l) to the samples which were then incubated at 57° C for 50 minutes. Each gel was loaded with 0.02-0.06 ml of sample and run at 4—5 milliamperes. While gels from 5.0 - 7.4- cm i n length were t r i e d , the long gels (7.4- cm) were most s u i t a b l e since these gels could be run for 80-90 minutes, which allowed f o r the separ-ation of the histones, and s t i l l r e t a i n the f a s t moving protamine bands. Gels were stained i n 1% Buffalo Black NBR i n 7% a c e t i c a c i d for at l e a s t two hours and destained i n 7% a c e t i c acid f o r approximately 4-0 h. A number of known histones were run as markers i n order to give possible i d e n t i t i e s to the bands obtained. Calf thymus histone was obtained from Worthington Biochemical Co. and salmon protamine (grade 1) from Sigma Chemical Co. Trout protamine and histone T were ob-tained from Dr. G. Dixon while histone IV from pea was obtained from Dr. D. Fambrough. The l a e v i s erythrocyte histone used i n t h i s study was prepared by Dr. H. Kasinsky. Trout and salmon protamine migrated together. e. Alkaline fast green s t a i n i n g of gels Gels were also stained with 0.1% fast green (w/v) buffered at pH 8.0 with 0.07 M Tris-HCl. This was performed on the Bonner et a l . (1968) gel system (described above) e s s e n t i a l l y as outlined by Ber-lowitz et aJU (1970) for the Johns (1967) ge l system. After e l e c t r o -phoresis, the gels were removed from the tubes and placed i n 1 N acetic a c i d for 2 h. Gels were then washed i n d i s t i l l e d H20 f o r 15 minutes and i n 0.07 M Tris-HCl at pH 8.0 f o r 4-5 minutes. Some of the gels were then stained for 16 h. The remainder of the gels were deaminated with nitrous acid. Deamination consisted of immersing the gels i n a f r e s h l y - 17 -p r e p a r e d s o l u t i o n o f e q u a l volumes o f 10% sodium n i t r i t e (w/v) and 10% t r i c h l o r o a c e t i c a c i d (w/v). T h i s s t e p was c a r r i e d o u t f o r 45 m i n u t e s and t h e n r e p e a t e d f o r a n o t h e r 45 minutes w i t h a f r e s h s o l -u t i o n . The deaminated g e l s , w h i c h were e x c e e d i n g l y f r a g i l e , were washed i n w a t e r f o r 5 m i n u t e s , p l a c e d i n 0.07 M T r i s - H C l (pH 8.0) f o r two 15 minute washes, and s t a i n e d 16 h i n a l k a l i n e f a s t g r e e n . D i f -f u s i o n d e s t a i n i n g was c a r r i e d out i n 0.07 M T r i s - E C l (pH 8.0). " " f . "Photographing and s c a n n i n g g e l s G e l s were photographed w i t h a P o l a r o i d camera. A G i l f o r d S p e c t r o p h o t o m e t e r 2400 was u s e d to s c a n g e l s a t 660 nm w i t h a s c a n r a t e o f 1 cm/min and a c h a r t speed o f 0.5 m i n / i n c h . C. RESULTS 1. CYTOCHEMISTRY The sperm n u c l e i o f R^ _ p r e t i o s a a r e rod-shaped ( F i g . 1) as a r e t h o s e o f Ro_ p a l u s t r i s . F e u l g e n s t a i n i n g f o r DNA i s i n t e n s e and u n i f o r m . A l k a l i n e f a s t g r e e n s t a i n i n g f o r h i s t o n e s w i t h 5% t r i c h l o r -o a c e t i c a c i d h y d r o l y s i s p a r a l l e l s t h e DNA s t a i n i n g and i s s i m i l a r l y u n i f o r m and i n t e n s e . These n u c l e i a l s o s t a i n w i t h a l k a l i n e f a s t g r e e n a f t e r I N t r i c h l o r o a c e t i c a c i d h y d r o l y s i s a t 60°C. However, the nuc-l e a r morphology i s s e r i o u s l y a l t e r e d ( F i g . 2). W i t h t r i c h l o r o a c e t i c a c i d h y d r o l y s i s t h e p r o t a m i n e s i n t h e c o n t r o l Salmo g a i r d n e r i i sperm a r e washed o u t . T h i s i n d i c a t e s t h a t p r o t a m i n e s a r e n o t p r e s e n t i n the sperm o f R»_ p r e t i o s a and R^ p a l u s t r i s . A f t e r d e a m i n a t i o n the Rana sperm n u c l e i do not s t a i n o r s t a i n v e r y s l i g h t l y ( F i g . 3)» s u g -g e s t i n g t h a t a r g i n i n e - r i c h h i s t o n e s a r e a b s e n t . The n u c l e i s t a i n w i t h a l k a l i n e f a s t green 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 not i f a c e t y l a t i o n i s c a r r i e d out b e f o r e s t a i n i n g . T h i s r e i n -f o r c e s the c o n c l u s i o n t h a t p r o t a m i n e s and a r g i n i n e - r i c h h i s t o n e s a r e n o t p r e s e n t i n t h e s e sperm and t h a t t h e s e sperm c o n t a i n h i s t o n e s o f t h e s o m a t i c t y p e . The n u c l e i show i n t e n s e d i n i t r o f l u o r o b e n z e n e s t a i n i n g f o r l y s i n e but s t a i n o n l y l i g h t l y a f t e r t h e S a k a g u c h i r e a c t i o n f o r a r g i n i n e . P r o t a m i n e s and a r g i n i n e - r i c h h i s t o n e s o f t h e - 18 -mouse and g r a s s h o p p e r t y p e g i v e a b r i g h t r e d r e a c t i o n w i t h t h e Saka-g u c h i t e s t ( B l o c h 1969). W i t h no h y d r o l y s i s , a l k a l i n e f a s t green s t a i n i n g i s absent ( T a b l e 1), showing t h a t 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 a r e m i s s i n g . A l k a l i n e f a s t g r e e n a f t e r F e u l g e n s t a i n i n g r e s u l t s i n a u n i f o r m p u r p l e s t a i n ; t h u s n o n - D N A - a s s o c i a t e d b a s i c p r o t e i n s a r e a l s o a b s e n t from t he Rana sperm. The sperm n u c l e i o f v e r s i c o l o r ( F i g . k) as w e l l as R e g i l l a a r e rod-shaped but s l i g h t l y b e n t . F e u l g e n s t a i n i n g i s i n t e n s e and u n i f o r m . A l k a l i n e f a s t green s t a i n i n g a f t e r t r i c h l o r o a c e t i c a c i d h y d r o l y s i s i s i n t e n s e ( F i g . 5) and f o l l o w s the F e u l g e n s t a i n i n g p a t -t e r n . T h i s i n d i c a t e s t h a t p r o t a m i n e s a r e not p r e s e n t . The s t a i n i n g i s r e d u c e d o n l y s l i g h t l y a f t e r d e a m i n a t i o n ( F i g . 6), w h i c h s u g g e s t s t h a t a r g i n i n e - r i c h h i s t o n e s a r e p r e s e n t . The n u c l e i s t a i n w i t h a l k a -l i n e f a s t green o r 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 i n b o t h s p e c i e s and c o n t i n u e to s t a i n even w i t h p r i o r a c e t y l a t i o n ( T a b l e 1); t h i s , a l s o , s u g g e s t s t h a t a r g i n i n e - r i c h h i s t o n e s a r e p r e s e n t . I n a d d i t i o n , t h e s e n u c l e i s t a i n w i t h d i n i t r o f l u o r o b e n z e n e b u t re s p o n d m o d e r a t e l y t o the S a k a g u c h i r e a c t i o n . The weak S a k a g u c h i r e a c t i o n d e m onstrates t h a t t h e sperm h i s t o n e s a r e n o t o f the salmon, o r mouse and g r a s s h o p p e r t y p 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 and non-DNA-associated b a s i c p r o t e i n s a r e a b s e n t . The sperm n u c l e i o f X^ l a e v i s a r e wavy ( F i g . 7)» F e u l g e n s t a i n -i n g i s i n t e n s e and u n i f o r m ; t h e a l k a l i n e f a s t green s t a i n i n g c o r r o b -o r a t e s the F e u l g e n s t a i n i n g ( F i g . 8). A l k a l i n e f a s t g r e e n s t a i n i n g o c c u r s w i t h TCA h y d r o l y s i s ( T a b l e 1 and F i g . 8). T h i s d e m o n s t r a t e s t h a t p r o t a m i n e s a r e n o t p r e s e n t , s i n c e t h e y a r e washed o u t by su c h t r e a t m e n t . A l k a l i n e f a s t g r e e n s t a i n i n g , a l t h o u g h r e d u c e d , i s s t i l l e v i d e n t even w i t h p r i o r d e a m i n a t i o n ( F i g . 9)» which i n d i c a t e s t h e p r e s e n c e o f a r g i n i n e - r i c h h i s t o n e s . The n u c l e i s t a i n w i t h a l k a l i n e f a s t g r e e n 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 even w i t h p r i o r a c e t y l a t i o n , which i s a n o t h e r i n d i c a t i o n t h a t a r g i n i n e - r i c h h i s t o n e s a r e p r e s e n t . The n u c l e i s t a i n w i t h d i n i t r o f l u o r o b e n z e n e and g i v e a moderate r e a c t i o n w i t h the S a k a g u c h i t e s t . Thus, l i k e t h e H y l a sperm, t he X^ l a e v i s sperm c o n t a i n h i s t o n e s t h a t a r e more b a s i c - 19 -than t h e Rana t y p e ; but t h e y a r e not as b a s i c as t h o s e o f t h e Salmon, o r Mouse/grasshopper t y p e s which g i v e a s t r o n g S a k a g u c h i r e a c t i o n . 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 and 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 a r e absent from Xenopus sperm. As i n Rana, the sperm n u c l e i o f B_j_ b o r e a s and Bj_ a m e r i c a n u s a r e a l s o rod-shaped and F e u l g e n s t a i n i n g i s i n t e n s e and u n i f o r m ( F i g . 1 0 ) . The n u c l e i do not s t a i n w i t h a l k a l i n e f a s t green a f t e r TCA h y d r o -l y s i s ( F i g . 11 and T a b l e 1 ) . They do s t a i n w i t h a l k a l i n e f a s t g r e e n o r w i t h 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 i n each i n s t a n c e ( F i g . 1 2 ) , w h i c h s t r o n g l y s u g g e s t s t h a t p r o t a m i n e s a r e p r e s e n t . However, the n u c l e i do not s t a i n under any o f t h e c o n d i t i o n s o f a c e t y l a t i o n ( F i g . 13) w h i c h i n d i c a t e s t h a t p r o t a m i n e s and a r g i n i n e -r i c h h i s t o n e s a r e a b s e n t . T h i s paradox p e r s i s t s w i t h b o t h f i x a t i v e s and even when a l k a l i n e f a s t g r e e n s t a i n i n g i s t r i e d f o r d i f f e r e n t l e n g t h s o f t i m e , f o r d i f f e r e n t p e r i o d s o f h y d r o l y s i s , and a t d i f -f e r e n t t e m p e r a t u r e s . The n u c l e i s t a i n w i t h d i n i t r o f l u o r o b e n z e n e b u t o n l y m o d e r a t e l y a f t e r t he S a k a g u c h i r e a c t i o n . However, when the S a k a g u c h i r e a c t i o n i s p e r f o r m e d on f o r m a l i n f i x e d m a t e r i a l t h e s e n u c l e i s t a i n s t r o n g l y . P i c h e r a l (1970) found t h a t t h e sperm o f P l e u r o d e l e s w a l t l i i showed a s i m i l a r s t a i n i n g p a t t e r n , e x c e p t t h a t t h e sperm gave an i n t e n s e r e d r e a c t i o n w i t h the S a k a g u c h i t e s t . He t h e r e f o r e c o n c l u d e d t h a t p r o t a m i n e s s i m i l a r t o t h o s e i n salmon were p r e s e n t i n t h i s u r o d e l e . P e r h a p s , Bufo sperm a l s o c o n t a i n h i s t o n e s o f the salmon t y p e . The n u c l e i o f Bufo sperm a r e u n u s u a l i n a n o t h e r r e s p e c t . The s t a i n i n g o b s e r v e d 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 i s n o t u n i f o r m ; i n s t e a d , the n u c l e i show clumps o f i n t e n s e s t a i n i n g ( F i g . 1 4 ) . B l o c h and Hew (1960a) o b s e r v e d a c o a g u l a t i o n e f f e c t w i t h t he sperm o f the s n a i l H e l i x a s p e r s a a f t e r p i c r i c a c i d t r e a t m e n t . Perhaps a s i m i l a r phenomenon i s b e i n g o b s e r v e d w i t h the Bufo sperm. 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 a r e absent i n Bufo sperm as a r e non-DNA-associated b a s i c p r o t e i n s . 20 TABLE 1 C y t o c h e m i s t r y o f anuran sperm n u c l e i S t a i n i n g R e a c t i v e and p r e - m a t e r i a l t r e a t m e n t R e s u l t s u s i n g Rana Rana Bufo Bufo H y l a Hyla, Xenopua  p a l u - p r e t - a m e r i - b o r e a s r e g - v e r s i - j a e v i s s t r i s i o s a canus i l i a c o l o r F e u l g e n AFG-TCA 86°C I DNA B a s i c p r o -t e i n s o t h e r t h a n p r o t -amines AFG-TCA 86°C, deamin-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 A F G - p i c r i c B a s i c p r o -a c i d t e i n s i n -c l u d i n g p r o t a m i n e s A F G - p i c r i c B a s i c p r o -a c i d , a c - t e i n s r i c h e t y l a t i o n i n a r g i n i n e E o s i n - I p i c r i c a c i d B a s i c p r o -t e i n s e x c e p t " c l e a v a g e 2 h i s t o n e s " E o s i n - Y p i c r i c B a s i c p r o -t e i n s r i c h a c i d , a c - i n a r g i n i n e e t y l a t i o n AFG 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 - 21 -TABLE 1 ( c o n t i n u e d ) R e s u l t s U6ing S t a i n i n g R e a c t i v e Rana Rana Bufo Bufo H y l a H y l a Xenopus and p r e - m a t e r i a l p a l u - pret° a m e r i - b o r e a s r e g - v e r s i - l a e v i s t r e a t m e n t s t r i s i o e a canus i l i a c o l o r AFG - a f t e r F e u l g e n 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 S a k a g u c h i P r o t e i n -bound a r g i n i n e D i n i t r o -f l u o r o -benzene P r o t e i n -bound l y s i n e NOTE: AFG = a l k a l i n e f a s t g r e e n ; TCA = t r i c h l o r o a c e t i c a c i d . - I d e n t i c a l r e s u l t s were o b t a i n e d w i t h AFG- 1 N TCA, 60°C. ^ B l o c h and Hew (1960b). - 22 -Figures 1 - 3» Sections of Rana pretiosa testis. Scale in Fig. 1 denotes 10/1 and applies to Figs. 2 and 3 as well. Figure 1. Feulgen staining showing clusters of rod-shaped sperm nuclei. Figure 2. Staining with alkaline fast green, IN trichloroacetic acid hydrolysis at 60°C. Sperm nuclear morphology has been ser-iously altered. Figure 3» Staining with alkaline fast green, IN trichloroacetic acid hydrolysis, after deamination. No staining can be seen. 3 - 23 -F i g u r e s k - 6. S e c t i o n s o f H y l a v e r s i c o l o r t e s t i s . S c a l e i n F i g e k denotes lO/i and a p p l i e s t o F i g s , 5 and 6 as w e l l . F i g u r e k» F e u l g e n s t a i n i n g d e m o n s t r a t i n g c l u s t e r s o f c u r v e d sperm n u c l e i . F i g u r e 5» S t a i n i n g w i t h a l k a l i n e f a s t g r e e n , I N t r i c h l o r o a c e t i c a c i d h y d r o l y s i s a t 60°C. F i g u r e 6. S t a i n i n g w i t h a l k a l i n e f a s t g r e e n , IN t r i c h l o r o a c e t i c a c i d h y d r o l y s i s a t 60°C, a f t e r d e a m i n a t i o n . Sperm n u c l e i c o n t i n u e t o s t a i n a l t h o u g h s t a i n i n g i s r e d u c e d . - Zk -'Figure's '7 -"9. S e c t i o n s o f Xenopus l a e v i s t e s t i s . S c a l e i n F i g . 7 denotes IG71 and a p p l i e s t o F i g s . 8 and 9 a l s o . F i g u r e 7- F e u l g e n s t a i n i n g showing c l u s t e r s o f wavy sperm n u c l e i . F i g u r e 8. S t a i n i n g w i t h a l k a l i n e f a s t g r e e n , I N t r i c h l o r o a c e t i c a c i d h y d r o l y s i s a t 60°C. F i g u r e 9» S t a i n i n g w i t h a l k a l i n e f a s t g r e e n , I N t r i c h l o r o a c e t i c a c i d h y d r o l y s i s a t 60°C, a f t e r d e a m i n a t i o n . - 25 -F i g u r e s 10 - 13. S e c t i o n s o f Bufo americanus t e s t i s . S c a l e i n F i g . 10 denotes 10/x and a p p l i e s t o F i g s . 1 1 , 12 and 13 as w e l l . F i g u r e 10. -Feulgen s t a i n i n g . C l u s t e r s o f rod-shaped sperm n u c l e i a r e e v i d e n t . F i g u r e 1 1 . S t a i n i n g w i t h a l k a l i n e f a s t g r e e n , 5% t r i c h l o r o a c e t i c a c i d h y d r o l y s i s a t 90°C. Sperm n u c l e i do n o t s t a i n . F i g u r e 12. S t a i n i n g w i t h a l k a l i n e f a s t g r e e n , p i c r i c a c i d h y d r o -l y s i s . Sperm n u c l e i s t a i n u n e v e n l y . F i g u r e 13* S t a i n i n g w i t h a l k a l i n e f a s t g r e e n , p i c r i c a c i d h y d r o -l y s i s , a f t e r d e a m i n a t i o n . S t a i n i n g i s a b s e n t . - 26 -F i g u r e 14. A sperm nucleus o f B. boreae s t a i n e d w i t h a l k a l i n e f a s t g r e e n , p i c r i c a c i d h y d r o l y s i s . S t a i n i n g i s uneven. S c a l e d e n o t e s 10 ja . 14 - 27 -2. BIOCHEMISTRY An e l e c t r o p h o r e t i c c o m parison o f t h e b a s i c p r o t e i n s from t h e t e s t e s o f R^ p i p i e n s , X._ l a e v i s , and americanus i n d i c a t e s s i g -n i f i c a n t d i f f e r e n c e s ( F i g s . 159 l 6 t 17 and 18). The r e g i o n on p o l y -a c r y l a m i d e g e l s bound by the f a s t e s t and s l o w e s t moving bands ob-t a i n e d w i t h h i s t o n e s from Rj_ p i p i e n s t e s t e s i s d e f i n e d as t h e s o m a t i c h i s t o n e r e g i o n .(H.) s i n c e p r e v i o u s .biochemical . s t u d i e s ..had i n d i c a t e d t h a t t h e amino a c i d c o m p o s i t i o n s o f f r o g sperm and s o m a t i c t i s s u e s were s i m i l a r ( V e n d r e l y 1957) and the 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 an u n d e s c r i b e d system) o f h i s t o n e s from f r o g t e s t e s and s o m a t i c t i s s u e vrere a l s o s i m i l a r ( B l o c h 1962). The H r e g i o n s h o u l d c o n t a i n t h e f i v e main h i s t o n e f r a c t i o n s - I , I I b 2 , I l b . ^ I l l , and IV (Johns 1971). Fambrough and Bonner (1966) r e p o r t t h a t e l e c t r o p h o r e s i s ( a s pe r f o r m e d i n t h i s s e c t i o n ) w i l l s e p a r a t e whole c a l f thymus h i s t o n e i n t o t h r e e bands. The f a s t e s t moving band c o n t a i n s h i s t o n e IV w h i l e h i s t o n e I makes up t h e s l o w e s t moving band. The t h i r d band i s composed o f h i s t o n e s I l b ^ , I I b 2 and I I I . Bands m i g r a t i n g s l o w e r t h a n t h e H r e g i o n a r e l i k e l y t o be n o n - h i s t o n e c o n t a m i n a t i n g p r o t e i n s o r a g-g r e g a t e s o f h i s t o n e s . I n t h i s s t u d y t h e f a s t e s t moving band i n t h e h i s t o n e r e g i o n c o - e l e c t r o p h o r e s e d w i t h h i s t o n e IV from pea. O n l y h i s t o n e T (W i g l e and D i x o n 1971)» pro t a m i n e (Marushige and D i x o n 1969)j and p r o t a m i n e - l i k e p r o t e i n s (Lam and Bruce 1971) a r e r e p o r -t e d t o m i g r a t e f a s t e r t h a n h i s t o n e I V. W i t h X^ l a e v i s t e s t e s t h r e e bands (X^, X 2 , X^) were found t o m i g r a t e f a s t e r t h a n h i s t o n e IV w h i l e w i t h B o _ americanus one band (B) has a g r e a t e r m o b i l i t y t h a n I V. ( F i g s . 16 and 17). A comparison on p o l y a c r y l a m i d e g e l s o f the h i s t o n e components o f t e s t i s and l i v e r from p i p i e n s s u p p o r t s t h e c o n t e n t i o n t h a t t h e b a s i c p r o t e i n s o f t e s t e s a r e s i m i l a r t o th o s e o f s o m a t i c t i s s u e s ( F i g . 19). Four bands a r e p r e s e n t i n both t i s s u e s , a l t h o u g h t h e amounts v a r y . The f a s t e s t moving band i s v e r y f a i n t i n l i v e r . The c o n t a m i n a t i o n o f l i v e r c h r o m a t i n w i t h pigment might a c c o u n t f o r some o f t h i s v a r i a t i o n s i n c e c e r t a i n h i s t o n e s might be s e l e c t i v e l y bound - 28 -by pigment. E l e c t r o p h o r e s i s o f t h e s e f r o g h i s t o n e s r e v e a l s one more band than does t he e l e c t r o p h o r e s i s o f comme r c i a l c a l f thymus h i s t o n e o r t r o u t t e s t i s h i s t o n e . P e r h a p s , h i s t o n e V, which i s p r e s e n t i n R.  c a t e s b i a n a l i v e r ( N e l s o n and Y u n i s 1969)f a c c o u n t s f o r t h e a d d i t i o n a l band. Four bands a r e l e s s e v i d e n t i n Rj_ p i p i e n s t e s t i s . Here t h r e e bands a r e c l e a r l y v i s i b l e w i t h p o s s i b l y a f o u r t h band b e i n g p r e s e n t a s a s h o u l d e r on the main band ( F i g . 15). F o u r components a l s o appear t o •be p r e s e n t when the g e l s a r e s t a i n e d w i t h a l k a l i n e f a s t g r e e n ( F i g s . 25 and 26). However, the two s l o w e s t moving bands do n o t s t a i n when the g e l s a r e deaminated p r i o r to s t a i n i n g ( F i g . 26). T h i s i n d i c a t e s t h a t t h e s e bands a r e not r i c h i n a r g i n i n e . An e l e c t r o p h o r e t i c comparison o f the h i s t o n e s from t e s t i s , l i v e r , and e r y t h r o c y t e o f X^ l a e v i s r e v e a l s t h a t t h e t h r e e f a s t moving bands ( X I , X2, X3) a r e s p e c i f i c t o t h e t e s t i s ( F i g s . 16 and 20). Three main bands a r e p r e s e n t i n the t e s t i s and l i v e r h i s t o n e r e g i o n (H) w h i l e f o u r bands a r e p r e s e n t i n the e r y t h r o c y t e h i s t o n e r e g i o n ( F i g . 20). The e r y t h r o c y t e s o f f r o g s have been r e p o r t e d t o c o n t a i n h i s t o n e V ( N e l s o n and Y u n i s 1969) and t h i s c o u l d a c c o u n t f o r t h e e x t r a band i n t h e e r y t h r o c y t e H r e g i o n . The f a s t e s t moving band i n t h e h i s t o n e r e g i o n c o - e l e c t r o p h o r e s e s w i t h h i s t o n e IV from pea. ( F i g . 20). When the g e l s a r e s t a i n e d w i t h a l k a l i n e f a s t g r e e n , the same p a t t e r n i s r e v e a l e d ( F i g s . 25 and 27)« e x c e p t X I appears i n amounts t o o s m a l l t o be d e t e c t e d . Three bands i n t h e h i s t o n e r e g i o n and bands X2 and X3 c o n t i n u e to s t a i n even i f the g e l s a r e deaminated p r i o r t o s t a i n i n g w i t h a l k a l i n e f a s t green ( F i g . 27). T h e r e f o r e , t h e s e bands a r e r i c h i n a r g i n i n e . None o f the t e s t i s s p e c i f i c bands c o - e l e c t r o p h o r e s e w i t h p r o t a m i n e from t r o u t o r salmon b u t t r o u t h i s t o n e T m i g r a t e s i n the r e g i o n o f the X2 d o u b l e t ( F i g . 22). However, t r o u t h i s t o n e T does n o t s t a i n w i t h a l k a l i n e f a s t g r e e n a f t e r d e a m i n a t i o n as does X2 w h i c h i n d i c a t e s t h a t X2 i s more r i c h i n a r g i n i n e t h a n t h e l y s i n e - r i c h h i s t o n e T ( F i g . 28). • A comparison o f t h e h i s t o n e components o f t e s t i s and l i v e r from B. americanus d i s c l o s e s t h a t t h e f a s t moving band, B, i s s p e c i f i c t o t h e t e s t i s ( F i g s . 17 and 2 1 ) . I n t h e h i s t o n e r e g i o n (H) a t l e a s t two - 29 -major bands a r e p r e s e n t i n b o t h t i s s u e s w i t h p o s s i b l y two m i n o r bands a l s o p r e s e n t i n t h e t e s t i s h i s t o n e r e g i o n . The f a s t e s t moving band i n t h e h i s t o n e r e g i o n (H) c o - e l e c t r o p h o r e s e s w i t h h i s t o n e IV from pea ( F i g . 2 1 ) . A s i m i l a r e l e c t r o p h o r e t i c p a t t e r n i s r e v e a l e d when the g e l s a r e s t a i n e d w i t h a l k a l i n e f a s t green ( F i g . 2 5 ) • De-a m i n a t i o n o f the g e l s p r i o r t o s t a i n i n g w i t h a l k a l i n e f a s t g r e e n does n o t p r e v e n t t h e two f a s t e s t moving bands i n t h e h i s t o n e r e g i o n and band B 'from s t a i n i n g . These bands a r e t h e r e f o r e r i c h i n a r g i n -i n e . Band B m i g r a t e s i n the p r o t a m i n e r e g i o n ( F i g . 23)» a l t h o u g h c o -e l e c t r o p h o r e s i s w i t h salmon p r o t a m i n e i s not q u i t e a c h i e v e d . E l e c -t r o p h o r e s i s o f Xj_ l a e v i s t e s t i s h i s t o n e s t o g e t h e r w i t h t h e t e s t i s h i s t o n e s o f B^ americanus i n d i c a t e s t h a t band B m i g r a t e s f a s t e r t h a n the t h r e e t e s t i s s p e c i f i c h i s t o n e s from X. l a e v i s ( F i g . 2 4 ) . - 30 -Figures 15 - 17. Densitometer tracings of histones from anuran testes run on 15% polyacrylamide gels f o r 85 minutes. Arrow i n d i c -ates band that co-electrophoresed with histone IV from pea. H i n -dicates somatic histone region. Figure 15. Rana pipiens t e s t i s . Figure 16. Xenopus l a e v i s t e s t i s . Figure 3.7. Bufo americanus t e s t i s . - 31 -F i g u r e s 18 - 28. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s o f h i s t o n e s from a n u r a n s . G e l s were r u n f o r 85 minutes and s t a i n e d w i t h b u f f a l o b l a c k u n l e s s i n d i c a t e d o t h e r w i s e . G e l p a t t e r n s i n each f i g u r e were o b t a i n e d d u r i n g t h e same r u n . H i n d i c a t e s s o m a t i c h i s t o n e r e g i o n . The d i r e c t i o n o f e l e c t r o p h o r e s i s i s from t he anode ( t o p o f g e l ) towards the cathode (bottom o f g e l ) . F i g u r e 18. A. Pana p i p i e n s t e s t i s . Bufo a m e r i c a n u s t e s t i s . B. C. D. Xenopus l a e v i s t e s t i s . Same as C, e x c e p t a g r e a t e r q u a n t i t y o f p r o t e i n was p l a c e d on the g e l . F i g u r e 19. A. p i p i e n s t e s t i s . B. IL_ p i p i e n s l i v e r . C. p i p i e n s l i v e r r u n w i t h h i s t o n e IV from p e a . F i g u r e 20. A. l a e v i s t e s t i s . B * — l a e v i s l i v e r . C. Xj_ l a e v i s e r y t h r o c y t e . 2Li l a e v i s e r y t h r o c y t e r u n w i t h h i s t o n e IV f r o m pea. F i g u r e 21. A. Bj_ americanus t e s t i s . B. Bj_ americanus l i v e r . C. Bj_ americanus l i v e r r u n w i t h h i s t o n e IV from pea. 18 19 A B C D A B C I 1 — X1 — X2 • — X3 - B 20 A B C D — 1 . — X1 • — X2 — X3 21 7" A B C H — B A - 32 -F i g u r e 22, H i s t o n e s from Xenopus l a e v i s t e s t i s . A. T e s t i c u l a r h i s t o n e s a l o n e -B. T e s t i c u l a r h i s t o n e s p l u s salmon p r o t a m i n e ( P ) . C. T e s t i c u l a r h i s t o n e s p l u s t r o u t h i s t o n e T. F i g u r e 23 • H i s t o n e s from Bufo americanus t e s t i s . A. T e s t i c u l a r h i s t o n e s a l o n e . B. T e s t i c u l a r h i s t o n e s p l u s salmon p r o t a m i n e ( P ) . F i g u r e 24-. T e s t i c u l a r h i s t o n e s from X._ l a e v i s and Bj_ a m e r i c a n u s r u n t o g e t h e r . — XI — X 2 — X3 — P 24 1] —XI - 33 -Figure 25. Histones from anuran testes stained with ei t h e r buffalo black or al k a l i n e f a s t green. A, C, and E. Stained with buffalo black. B s D, and F. Stained with a l k a l i n e f a s t green. A and B. Rana pipiens C and D. Bufo americanus E and F. Xenopus l a e v i s Figure 26. T e s t i c u l a r histones from R^ pipiens stained with a l k a l i n e fast green. A. Without deamination of gels. B. With deamination of gels. Figure 27. T e s t i c u l a r histones from Xj_ l a e v i s stained with a l k a l i n e fast green. A, Without deamination of gels. B. With deamination of gels. Figure 28. Trout histone T and salmon protamine (P) stained with a l k a l i n e f a s t green. A. Without deamination of gels. B. With deamination of gels. - yh -3. SALT CONCENTRATION AND THE ALKALINE FAST GREEN REACTION Alkaline fast green, made up in citrate-phosphate buffer, gives unusual results when used without prior hydrolysis. The sperm of trout, Salmo gairdnerii, stain while the sperm of Rj_ pipiens do not. This is unusual since the protamines of trout sperm are not free basic proteins but instead are electrostatically bound to the phos-phate groups of DNA (Frederieq 1971). If the experiment is repeated using 0.01M tris-HCl buffer in place of the citrate-phosphate buffer, the same results are achieved. However, when the experiment is re-peated using AFG brought to the correct pH by titrating with 0.1N NaOH, trout sperm, as well as the sperm of R*_ pipiens, do not stain. This suggests that salt concentration is the cause of these unusual results. Since the possibility of developing a new technique to dis-tinguish between protamine and non-protamine containing cells existed, the phenomenon was studied further. Salt concentration is indeed necessary for staining without prior hydrolysis. As can be seen in Table II, staining only occurs at high salt concentrations. Staining is dependent on the mixture of salt and dye (see Table III). Staining is absent i f sections are treated with a salt solution prior to staining with AFG made up in distilled water. Staining appears to be confined to very arginine-rich sperm nuclei (Table IV). Deamination of trout sperm does not prevent staining and thus protamines are responsible for staining in this case. Since at high salt concentrations nuclei become distorted, the technique is used on a variety of sperm (Table IV) at an inter-mediate salt concentration. As has been shown in the previous sec-tions, the sperm that give a positive reaction are rich in arginine. Despite this i n i t i a l success, the technique was abandoned since a lot* level of staining (cytoplasmic as well as nuclear) always existed. However, this study does point out a possible source of error in test-ing for free basic proteins. - 35 -TABLE II Salt Concentration and the AFG Reaction * Stain- Results with sperm n u c l e i from AFG made Salmo g a i r d n e r i i Xenopus l a e v i s Rana pipiens up i n d i s t i l l e d E^O - -OoOC-12 M NaCl - - -0.0024 M NaCl - - -0.0047 M NaCl - - -0.0094 M NaCl - -0.0188 M NaCl s l i g h t - -0.0375 M NaCl + - -0.0750 M NaCl + a l i g h t -0.1500 M NaCl + + -0.3000 M NaCl ++ + mm 0.6000 M NaCl ++ + -1.2000 H NaCl +-!- + -2.4000 M NaCl ++ + -* These r e s u l t s are obtained without hydrolysis - 36 -TABLE I I I Pretreatment and the AFG Reaction * AFG made up in Pretreatment Trout sperm d i s t i l l e d H20 d i s t i l l e d ILpO 0.30 M NaCl 0.30 M NaCl none 30 minutes in 0.300 M NaCl none deamination + + * These results were obtained without hydrolysis. TABLE IV Staining with AFG made up i n 0.30 M NaCl * Species Results with Sperm Nuclei Sperm Rich in Arginine Xenopus laevis  Bufo boreas  B. americanus  Rana pipiens  R. pretiosa  R. palustris + + + + + + * These results were obtained without hydrolysis. - 37 -D. DISCUSSION On the basis of cytochemical c r i t e r i a , the sperm of the seven anurans studied may be c l a s s i f i e d into three groups. The sperm histones of l a e v i s are of the Mytilus type, which i s i n agree-ment with the data of Bloch (1969). These sperm n u c l e i s t a i n with a l k a l i n e fast green xuader a l l conditions of hydrolysis, but they s t a i n s l i g h t l y l e s s intensely a f t e r a c e t y l a t i o n or deamination. This distinguishes them from the Mouse and grasshopper type of sperm h i s -tone which shows no reduction i n st a i n i n g i n t e n s i t y a f t e r deamin-ation or a c e t y l a t i o n . The sperm of H«_ r e g i l l a and v e r s i c o l o r demonstrate a staining pattern s i m i l a r to that of l a e v i s and therefore also belong to the Mytilus type. The sperm of R^ palus- t r i s and R^ pretiosa s t a i n l i k e the sperm of R^ pipiens (Bloch 1962; Z i r k i n 1970) and thus are of the Rana type. These sperm do not s t a i n or s t a i n only f a i n t l y with a l k a l i n e f a s t green or eosin Y a f t e r deamination. The basic proteins i n the sperm of B^ ameri- canus and B_o_ boreas are unusual from a cytochemical point of view. These sperm s a t i s f y three c r i t e r i a for the Salmon type. They do not stai n a f t e r TCA hydrolysis, do s t a i n 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 i n formalin fixed material give a strong Sakaguchi reaction. However, these sperm do not s t a i n a f t e r p i c r i c a c i d hydrolysis i f the sections are acetylated, which suggests that protamines are absent. Therefore, on the basis of t h i s cytochemical data these sperm are classed as the Salmon type with reservation. Bloch (1969), on the other hand, c l a s s i f i e d the sperm histones of Bj_ v u l g a r i s as the Mytilus type on the basis of unpublished cytochemical data. An electrophoretic comparison of the histones from R^ pipiens t e s t i s and l i v e r reveals no q u a l i t a t i v e differences. This i s i n agreement with the studies of Vendrely (1957) and Bloch (1962). Vendrely found that the amino acid compositions of histones extrac-ted from frog sperm and from somatic tissues were s i m i l a r while Bloch found no electrophoretic differences between histones from testes and histones from somatic tis s u e s . The testes of Rj_ pretiosa and R^ - 38 -p a l u s t r i s would be expected to give electrophoretic patterns very-s i m i l a r to those obtained with R. pipiens since they s t a i n s i m i l a r l y . Unlike R^ pipiens, electrophoresis of histones from Xj_ l a e v i s reveals three f a s t moving bands s p e c i f i c to the t e s t i s . These bands are not l i k e l y to be degradation products since s i m i l a r bands were not found i n other tissues of Xenopus nor i n the other anurans ex-amined by the same procedure. Since the sperm nu c l e i were the only n u c l e i i n the Xenopus t e s t i s that did not show s t a i n i n g properties s i m i l a r to Rana type sperm n u c l e i or somatic c e l l n u c l e i , these bands are l i k e l y s p e c i f i c to sperm. This i s also supported by the obser-vation that bands X2 and X3 do s t a i n a f t e r deamination, i n d i c a t i n g , that l i k e the sperm n u c l e i , they are r i c h i n arginine. On the gel system employed i n t h i s study bands XI, X2, and X3 demonstrate mo b i l i t i e s intermediate between protamines and somatic histones. Thus Mytilus type of sperm histone does appear to be an "intermediate" type. These observations with Xj_ l a e v i s are very s i m i l a r to those ob-tained by Bloch (1966) with Mytilus edulis. He reported that e l e c t r o -phoresis of t e s t i c u l a r histones showed three bands (^Y, <5 ) whose mo b i l i t i e s were fas t e r than those of somatic histones (although somatic tissues were not studied i n t h i s organism). How these three bands are related to the bands (XI, X2, and X3) obtained i n t h i s study i s unknown since the electrophoretic system was not described by Bloch. However, Bloch did determine the amino acid composition of the two f a s t e s t mov-ing bands. They contained a l l three of the basic amino a c i d s : argin-ine, lysine,and h i s t i d i n e . Although the amount of h i s t i d i n e was small, these proteins appear to be s i m i l a r to the triprotamines of Kossel. Since techniques are being developed to do amino aci d analysis of bands obtained on polyacrylamide gels (Houston 1971), the amino aci d composition of bands XI, X2, and X3 can also be determined. How these bands are r e l a t e d to the process of spermiogenesis and how, they are related to each other i s unknown. As a r e s u l t o f l a b e l -Id-l i n g patterns with C -amino acids, Bloch (1966) believes that the three proteins i n Mj_ edulis are synthesized at the same time i n the - 39 -early spermatid; however, as development proceeds they are succes-sively incorporated into the sperm chromatin. Bloch's data could also be interpreted to indicate a precursor-product relationship. Thus band p could be converted to <T and this in turn to T • This raises the question as to whether bands XI, X2, and X3 are specific to various stages of Xenopus spermiogenesis or whether a l l three are found in mature sperm? Are the normal somatic histones completely removed during spermiogenesis or are small amounts pres-ent along with the sperm specific basic proteins? These questions remain to be answered. One fast moving band, B, is specific to the testis of B^ amer- icanus. This band migrates similarly to salmon protamine and, like salmon protamine, continues to stain despite deamination of the gels. The only cells in the testis that behave cytochemically like prota-mine containing cells are sperm. Therefore, this band is most like-ly specific to sperm nuclei. Only amino acid analysis of this band will determine how closely related band B is to salmon protamine. However, the present data support the contention that sperm his-tones of B^ americanus are of the Salmon type. Why these nuclei do not stain after acetylation is s t i l l unexplained. The argument might be advanced that band B could be a meiotic protein, perhaps similar to the protein obtained by Sheridan and Stern (1967) from the meiotic cells in the anthers of l i l y and tulip. However, the latter protein is low in arginine. Also, the absence of band B in R._ pipiens and X^ laevis testis would be difficult to explain. Basic proteins free from DNA have been observed in the sperm of a number of animals. Vaughn ejb al. (1969) found such proteins in the capsule of the sperm cells of Emerita analoga. Das <et al. (1967) demonstrated basic proteins in the acrosomes of the sperm of Urechis caupo. In PIeurodel waitIii, Picheral (1970) saw free basic proteins in the neck and t a i l of the sperm. In the present study, free basic proteins were not observed in the sperm cells of the seven anurans examined. Vaughn (1966) observed basic proteins associated with RNA in the "sphere chroraatophile" of the rat sperm. - ifO -Similar proteins i n the sperm of frogs were not found. Since the appearance of non-DNA-associated basic proteins has been correlated with the removal of somatic histones, such proteins would not be ex-pected i n an organism with the somatic type of sperm histone. How-ever, such proteins might be expected i n sperm with the Mytilus or Salmon type of sperm histone. An explanation for the effect of s a l t concentration on the AFG reaction i s d i f f i c u l t to envision. Specific staining of sperm r i c h i n arginine i s achieved without the removal of DNA at high s a l t con-centrations. Since increasing s a l t concentrations are known to s e l -e c t i v e l y remove histones (Fambrough and Bonner 1968), one p o s s i b i l i t y i s that the dye binding s i t e s of histones very r i c h i n arginine are s e l e c t i v e l y exposed by high s a l t concentrations. In v i t r o dissoc-i a t i o n of nucleohistone and nucleoprotamine by s a l t indicates that l y s i n e - r i c h histones are removed from DNA at low s a l t concentrations; the arginine-rich histones at much higher concentrations; and the protamines (very r i c h i n arginine) at intermediate concentrations (Marushige and Dixon 1971). Thus, the selective exposure of h i s -tones very r i c h i n arginine appears an u n l i k e l y mechanism, since at low s a l t concentrations somatic c e l l s would be expected to s t a i n due to the exposure of l y s i n e - r i c h histones. At high s a l t concentrations staining would be expected due to the exposure of arginine-rich h i s -tones. In fact, somatic c e l l s usually do not stain under any con-d i t i o n s . A direct comparison between sectioned material and chromatin may be misleading since i n sectioned material the f i x a t i v e must be con-sidered. The experiments i n t h i s study were performed on material fix e d i n formaldehyde, which i s thought to function as a f i x a t i v e by forming methylene bridges between amino groups of neighboring pro-teins (Bowes and Carter 1965)o However, besides protein-protein binding, formaldehyde l i n k s histone molecules to DNA. Brutlag et a l . (1969) found that the histones of formaldehyde-treated nucleohistone were not dissociable from DNA by s a l t . The protein-DNA interaction was no longer mainly ionic as i n native nucleohistone. Instead, - kl -methylene b r i d g e s were formed between the amino groups o f h i s t o n e s and the amino groups o f DNA. T h i s might e x p l a i n why somatic c e l l s a r e not s t a i n e d d e s p i t e high s a l t c o n c e n t r a t i o n s . Protamines, how-ever, c o n t a i n few, i f any l y s i n e r e s i d u e s ( L i n g e_t a l . 1971) and thus few f r e e amino groups. T h e r e f o r e , i n formaldehyde t r e a t e d m a t e r i a l , protamines would be l e s s capable of forming methylene b r i d g e s w i t h DNA and might s t i l l be diss.o.ciated .from .DNA w i t h s a l t . .On t h i s b a s i s , the s e l e c t i v e s t a i n i n g o f ve r y a r g i n i n e - r i c h sperm h i s t o n e s w i t h AFG a t h i g h s a l t c o n c e n t r a t i o n s might be e x p l a i n e d . In formaldehyde-treated s e c t i o n s , the dye b i n d i n g s i t e s o f p r o t -amines and p r o t e i n s very r i c h i n a r g i n i n e a r e exposed by h i g h s a l t c o n c e n t r a t i o n due to the l a c k o f protamine-DNA c r o s s l i n k i n g w h i l e the dye b i n d i n g s i t e s o f somatic h i s t o n e s a r e not exposed due to ex-t e n s i v e histone-DNA c r o s s l i n k i n g . P o s s i b l y , the protamines a r e not completely removed by the s a l t c o n c e n t r a t i o n s used due to some p r o t -amine-protein l i n k a g e . The requirement f o r the s a l t and the dye to be tog e t h e r f o r s t a i n i n g to occur can a l s o be e x p l a i n e d by t h i s mechanism. When s a l t d i s s o c i a t i o n o c c u r s i n the presence o f the dye, the s t a i n competes s u c c e s s i v e l y w i t h n e g a t i v e i o n s f o r p o s i t i v e s i t e s on the protamine and s t a i n i n g o c c u r s . When t h i s d i s s o c i a t i o n o c c u r s p r i o r to s t a i n i n g , a l l the s t a i n i n g s i t e s a r e b l o c k e d by n e g a t i v e i o n s and s t a i n i n g does not occur . The mechanism i s t e s t a b l e . I f c r o s s l i n k i n g due to formaldehyde i s the b a s i s o f the d i f f e r e n t i a l exposure o f protamine, then the e f -f e c t s h o u l d be observed o n l y i n m a t e r i a l f i x e d i n formaldehyde. I f protamine i s not capable of forming methylene b r i d g e s w i t h DNA i n the presence of formaldehyde, then the protamine o f fox^maldehyde-treated nucleoprotamine s h o u l d be d i s s o c i a b l e from DNA by s a l t . T h i s c o u l d l e a d to a very s e l e c t i v e method f o r i s o l a t i n g protamine. - hz -I I I . CHANGES IN BASIC PROTEINS DURING SPERMIOGENESIS IN THE EASTERN RED SPOTTED NEWT, DIEMICTYLUS VIRISDESCENS A. INTRODUCTION The histone transitions observed during spermiogenesis i n the uro-dele, PIeurodeles w a l t i i , (Picheral 1970) are very s i m i l a r to the changes found i n the s n a i l (Bloch and Hew 1960a) and the squid (Bloch 1966)o In these organisms the mature sperm are of the Salmon type. However, Bloch (1969) reported that the urodele, Diemictylus v i r i d e s -cens, showed a tr a n s i t i o n only as far as the "stable protamine" or the Mouse/grasshopper type of sperm histone. To check the d i v e r s i t y of sperm histone types i n the urodeles, the basic protein changes during spermiogenesis i n the eastern red spotted newt have been re-examined. B. METHODS AND MATERIALS 1. CYTOCHEMISTRY Sexually mature Diemictylus (Triturus) viridescens, the eastern red spotted newt, were obtained from E. G. Steinhilber & Co. Animals, maintained i n the laboratory on beef heart and X«_ laevi3 tadpoles, were s a c r i f i c e d . i n February, May, and October. The testes were re-moved and fixed i n 10% neutral buffered formalin or absolute ethanol-g l a c i a l acetic acid (3:1) as outlined i n section I I . A l l s t a i n i n g procedures employed on th i s material have been described previously. 2. BIOCHEMISTRY Newts were obtained from the same company i n the f i r s t week of January 1972. On a r r i v a l , the animals were etherized and the testes, l i v e r s , hearts, and spleens were removed and stored at -20°C u n t i l use.' With one exception a l l biochemical procedures were carried out as described i n section I I , In order to c o l l e c t enough material for the extraction of histones, somatic tissues were combined and then - k-3 -e x t r a c t e d . C. RESULTS 1. CYTOCHEMISTRY The newt t e s t i s i s 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 t u b u l e s w h i c h , i n t u r n , a r e o r g a n i z e d i n t o c l u s t e r s o f c e l l s o r c y s t s . A l l c y s t s w i t h -i n a s e m i n i f e r o u s t u b u l e a r e a p p r o x i m a t e l y a t the same s t a g e o f s p e r m a t o g e n e s i s . I n September and October a l l s t a g e s o f s p e r m i o g e n -e s i s may be f o u n d i n the newt t e s t i s w h i l e i n December mature sperm predominate (Baker 1966). As a r e s u l t , most o b s e r v a t i o n s were made on m a t e r i a l f i x e d i n O c t o b e r . S p e r m i o g e n e s i s has been d i v i d e d i n t o f i v e s t a g e s on t h e b a s i s o f n u c l e a r shape and b a s i c p r o t e i n c o m p o s i t i o n . These s t a g e s a r e shown i n F i g s . 29-32 and the c y t o c h e m i c a l r e s u l t s f o r each s t a g e a r e summarized i n T a b l e V. Stage 1 n u c l e i , t he e a r l i e s t s p e r m a t i d s o b s e r v e d , a r e r o u n d ( F i g . 29) and demonstrate uneven s t a i n i n g f o r DNA. AFG s t a i n i n g f o r h i s t o n e s p a r a l l e l s t h e F e u l g e n - s t a i n i n g p a t t e r n . Treatment o f t h e s e n u c l e i w i t h d e a m i n a t i o n o r a c e t y l a t i o n r e a g e n t s a b o l i s h e s s t a i n i n g f o r b a s i c p r o t e i n s , i n d i c a t i n g t he absence o f v e r y a r g i n i n e - r i c h h i s -t o n e s . These n u c l e i s t a i n l i g h t l y a f t e r t h e S a k a g u c h i r e a c t i o n f o r a r g i n i n e . These r e s u l t s i n d i c a t e t h a t the h i s t o n e s w h i c h o c c u r i n t h i s s t a g e a r e s i m i l a r t o t h o s e o f s o m a t i c c e l l n u c l e i . S p e r m a t i d s b e g i n n i n g n u c l e a r e l o n g a t i o n have been d e s i g n a t e d as s t a g e 2. These n u c l e i a r e o v a l t o c i g a r - s h a p e d ( F i g . 29). F o r s t a g e 2 n u c l e i , the F e u l g e n and A F G - s t a i n i n g p a t t e r n s a r e uneven and p a r -a l l e l each o t h e r . These n u c l e i do not s t a i n w i t h AFG i f the s e c t i o n s a r e deaminated o r a c e t y l a b e d and l i k e s t a g e 1 n u c l e i , t h e y g i v e a l i g h t r e s p o n s e to the S a k a g u c h i r e a c t i o n . T h e r e f o r e , h i s t o n e s o f t h e s o m a t i c t y p e a r e p r e s e n t i n t h e s e n u c l e i . Stage 3 n u c l e i a r e narrow and c y l i n d r i c a l and have a l m o s t com-p l e t e d n u c l e a r e l o n g a t i o n . The arrangement o f t h e s e n u c l e i i n c y s t s i s more p r e c i s e than i n s t a g e s 1 and 2 ( F i g . 30). W i t h i n a c y s t , t h e n u c l e i l i e a p p r o x i m a t e l y p a r a l l e l w i t h t h e a p i c e s p o i n t i n g towards - 44 -a S e r t o l i c e l l ( F i g . 30). F e u l g e n - s t a i n i n g as w e l l as A F G - s t a i n i n g o f s t a g e 3 n u c l e i i s i n t e n s e and u n i f o r m . As w i t h s t a g e s 1 and 2, A F G - s t a i n i n g o f t h i s s t a g e o c c u r s a f t e r t r i c h l o r o a c e t i c a c i d h y d r o -l y s i s (Fig.33) t i n d i c a t i n g t h a t p r o t a m i n e s a r e a b s e n t from t h e s e n u c l e i . W h i l e AFG s t a i n i n g i n t h e a p i c a l r e g i o n o f s t a g e 3 n u c l e i i s a b o l i s h e d by d e a m i n a t i o n o r a c e t y l a t i o n , t h e b a s a l r e g i o n c o n t i n u e s t o s t a i n ( F i g . 34). A g r a d i e n t i s a c t u a l l y o b s e r v e d . I n some c y s t s o n l y the most b a s a l r e g i o n s o f "the n u c l e i s t a i n ; i n o t h e r s , t h e p o s t e r i o r h a l v e s o f the n u c l e i s t a i n ; and i n some, the n u c l e i a r e c o m p l e t e l y s t a i n e d except f o r the a p i c e s . These s t a i n i n g p a t t e r n s s u g g e s t t h a t t h e v e r y a r g i n i n e - r i c h h i s t o n e s g r a d u a l l y r e p l a c e t h e s o m a t i c h i s t o n e s from the base t o the apex o f the n u c l e u s . W i t h i n a c y s t , a l l n u c l e i show the same s t a i n i n g p a t t e r n , s u g g e s t i n g t h a t t h i s t r a n s i t i o n i s s y n c h r o n o u s . However, the c y s t s w i t h i n a s e m i n i f -erous t u b u l e a r e n o t q u i t e s ynchronous w i t h one a n o t h e r ( F i g . 3*0 and d i f f e r e n t s t e p s i n the t r a n s i t i o n t o the v e r y a r g i n i n e - r i c h form a r e p r e s e n t . T h i s t r a n s i t i o n i s more d i f f i c u l t t o f o l l o w w i t h the Saka-g u c h i r e a c t i o n , y e t the b a s a l p o r t i o n o f s t a g e 3 n u c l e i a p p e a r t o s t a i n more i n t e n s e l y than the a p i c a l r e g i o n s . Stage 4 n u c l e i appear as v e r y t h i n c y l i n d e r s , w h i c h i n some c y s t s a r e s l i g h t l y c u r v e d . These n u c l e i o c c u r i n more t i g h t l y packed c y s t s ( F i g . 31) t h a n s t a g e 3 n u c l e i . F e u l g e n s t a i n i n g i s v e r y i n -t e n s e and u n i f o r m i n t h e s e n u c l e i w h i l e the AFG s t a i n i n g i s s i m i l a r l y i n t e n s e and u n i f o r m . S i n c e s t a i n i n g w i t h AFG o c c u r s a f t e r t r i c h l o r o -a c e t i c a c i d h y d r o l y s i s , s t a g e 4 n u c l e i do not c o n t a i n p r o t a m i n e s . AFG o r e o s i n T s t a i n i n g o f t h e s e n u c l e i remains i n t e n s e and u n i f o r m de-s p i t e d e a m i n a t i o n o r a c e t y l a t i o n o f t h e s e c e l l s . These n u c l e i a l s o s t a i n v e r y s t r o n g l y a f t e r t h e S a k a g u c h i r e a c t i o n . T h e r e f o r e , t h e y c o n t a i n v e r y a r g i n i n e - r i c h h i s t o n e s of t h e Mouse/grasshopper typ e ( " s t a b l e p r o t a m i n e " ) . Stage 5 n u c l e i appear a s t h i n , c u r v e d r o d s and o c c u r i n v e r y t i g h t l y packed c y s t s ( F i g . 32). These n u c l e i r e p r e s e n t the most advanced s t a g e o b s e r v e d and were fo u n d i n O c t o b e r and F e b r u a r y t e s t e s . Thus, t h e y a r e p r o b a b l y mature sperm. These n u c l e i de-- 45 -monstrate a very intense, uniform staining for DNA. However, these n u c l e i do not s t a i n with AFG a f t e r t r i c h l o r o a c e t i c a c i d hydrolysis, which suggests that protamines are present. They do s t a i n with AFG and eosin Y a f t e r p i c r i c a c i d hydrolysis and continue to s t a i n strongly even when acetylation i s carried out p r i o r to s t a i n i n g . Stage 5 n u c l e i s t a i n intensely a f t e r the Sakaguchi reaction. These re s u l t s indicate that protamines are present i n mature sperm. Un-l i k e the previous change, the t r a n s i t i o n from the Mouse/grasshopper type of sperm histone i n stage "k n u c l e i to the protamines i n mature sperm i s very sudden and no t r a n s i t i o n steps are observed. With formalin f i x e d material, AFG stai n i n g without p r i o r hydro-l y s i s i s absent i n a l l stages of spermiogenesis. This i n d i c a t e s that free basic proteins are not present. However, with the urodele, PIeurodeles w a l t i i , Picheral (1970) observed free basic proteins i n the neck and t a i l regions of spermatozoa. The appearance of free basic proteins i n these regions was correlated with the t r a n s i t i o n to the very a r g i n i n e - r i c h histones and then to the protamines. There-fore, i n the present study the st a i n i n g procedure was repeated on material f i x e d i n absolute et h a n o l - g l a c i a l acetic a c i d (3:1). Under these conditions the t a i l s of mature sperm do s t a i n with AFG without hydrolysis (Fig. 35). This suggested that formalin was hiding free basic proteins by binding with the amino groups of l y s i n e , the primary dye binding s i t e s of l y s i n e - r i c h histones. However, when formalin fixed material was treated with b o i l i n g water to remove formaldehyde, AFG st a i n i n g was s t i l l absent i n a l l stages of spermiogenesis. The t a i l s of mature sperm do s t a i n a f t e r the Sakaguchi reaction for argin-ine. Thus free basic proteins that are only retained by Clarke*s f i x -a t i ve (absolute ethanol-glacial acetic acid) appear to be present i n the t a i l s of mature spermatozoa. - 46 -TABLE V Cytochemistry of newt spermiogenesis Staining and Reactive Stages pretreatment material 1 2 3 a b AFG-TCA 86°C1 Basic proteins other than protamines AFG-TCA 86°C, deamination Basic proteins r i c h i n arginine - + AFG-picric ac i d Basic proteins including protamines + + AFG-picric acid, acetylation Basic proteins r i c h i n arginine - + Eosin-Y p i c r i c a c i d Basic proteins except "cleavage histones" + + Eosin-Y p i c r i c a c i d , acetylation Basic proteins r i c h i n arginine - + AFG Non-nucleic a c i d -associated basic proteins Sakaguchi Protein-bound arginine + + ++ ++ NOTE: AFG = a l k a l i n e f a s t green; TCA = t r i c h l o r o a c e t i c a c i d , p l o c h and Hew (1960b). These r e s u l t s are for formalin f i x e d material. a = b = a p i c a l region, basal region. - 47 -Figures 2 9 - 3 2 . Stages of spermiogenesis in the eastern red spotted newt. Feulgen stained. Scale denots 50 ;u . Figure 2 9 . Cys ts of round stage 1 nuclei to the l e f t and cysts of • cigar—shaped -stage 2 n u c l e i to -the -ri-ght-0 Figure 3 0 . Cysts of stage 3 nucle i . Arrow indicates Ser to l i c e l l nucleus. Figure 31» Cysts of stage 4 nucle i . Figure 3 2 . Cysts of stage 5 nucle i . - 48 -Figures 33-34. Consecutive sections of cysts of stage 3 n u c l e i stained with a l k a l i n e f a s t green, 5% t r i c h l o r o a c e t i c a c i d hydro l y s i s . Scale denotes 50 /x . Figure 33* Without deamination. Figure 34. With deamination. - 49 -Figure 35« Sperm of Diemictylus viridescens stained with a l k a l i n e f a s t green without p r i o r hydrolysis of DNA. The material *ra.s f i x e d i n absolute ethanol: g l a c i a l acetic acid (3:1). The t a i l regions s t a i n while the n u c l e i do not. The scale denots 50 ja • - 50 -2. BIOCHEMISTRY The h i s t o n e components o f newt t e s t i s a r e compared e l e c t r o -p h o r e t i c a l l y w i t h t h o s e from a combined p r e p a r a t i o n o f newt l i v e r , h e a r t , and s p l e e n ( F i g s . 56, 37 and 38). The h i s t o n e r e g i o n (H) i s d e f i n e d by the s l o w e s t and f a s t e s t moving bands o b t a i n e d w i t h t h e s o m a t i c t i s s u e s and i s s i m i l a r t o the p a t t e r n o b t a i n e d w i t h R. p i p i e n s t e s t e s . F o u r bands a r e p r e s e n t i n b o t h t h e t e s t i s h i s t o n e r e g i o n and the h i s t o n e r e g i o n from a c o m b i n a t i o n o f t i s s u e s ( F i g . 38). However, two main h i s t o n e bands a r e v i s i b l e a f t e r e l e c t r o -p h o r e s i s f o r o n l y 70 minutes ( F i g . 37). I n the H r e g i o n the f a s t -e s t moving band, which c o - e l e c t r o p h o r e s e s w i t h h i s t o n e IV f r o m pea, r u n s as a d o u b l e t i n the t e s t i s p r e p a r a t i o n . A number o f bands m i -g r a t i n g s l o w e r t h a n t h e h i s t o n e r e g i o n a r e p r e s e n t i n t h e t e s t i s and may r e p r e s e n t d e g r a d a t i o n p r o d u c t s . Two v e r y f a s t moving bands, N I , w h i ch m i g r a t e s as a d o u b l e t , and N2, w h i c h m i g r a t e s s l i g h t l y f a s t e r t h a n N I , a r e s p e c i f i c to t h e t e s t i s . O c c a s i o n a l l y , a f a i n t band a p p e a r s i n f r o n t o f N2 and o n l y t h i s band c o - e l e c t r o p h o r e s e s w i t h p r o t a m i n e from salmon o r t r o u t ( F i g . 37). E l e c t r o p h o r e s i s o f newt and l a e v i s t e s t i s h i s t o n e s t o g e t h e r r e v e a l s t h a t NI and N2 m i -g r a t e f a s t e r t h a n the t e s t i s s p e c i f i c bands from X^ l a e v i s ( F i g . 39). I f B o _ americanus h i s t o n e s a r e r u n w i t h a newt t e s t i s p r e p a r a t i o n , band B m i g r a t e s w i t h N2 ( F i g . 39)» s t r o n g l y s u g g e s t i n g t h a t t h e y a r e s i m i l a r . - 51 -F i g u r e s 36 and 37• Densitometer t r a c i n g s o f h i s t o n e s from the e a s t -ern r e d s p o t t e d newt run on 15% p o l y a c r y l a m i d e g e l s f o r 70 minutes and s t a i n e d w i t h b u f f a l o b l a c k . H i n d i c a t e s somatic h i s t o n e r e g i o n . F i g u r e 36. H i s t o n e s from a combination o f somatic t i s s u e s — h e a r t , l i v e r and s p l e e n . Top: run a l o n e . Bottom: run w i t h h i s t o n e IV from pea. F i g u r e 37* T e s t i c u l a r h i s t o n e s . Arrow i n the h i s t o n e r e g i o n i n d i c -a t e s band t h a t c o - e l e c t r o p h o r e s e d w i t h h i s t o n e IV from pea. Second arrow d e s i g n a t e s the f a i n t band which i s o c c a s i o n a l l y p r e s e n t and which c o - e l e c t r o p h o r e s e d w i t h protamine from salmon or t r o u t . Top: run a l o n e . Bottom: run w i t h salmon protamine. - 52 -Figure 58. Polyacrylamide gel electrophoresis patterns of.histones from the eastern red spotted newt. Gels were run f o r 85 minutes and stained with buffalo black. H designates somatic histone region. A. "testicular "histones. B. t e s t i c u l a r histones plus salmon protamine (P). C. l i v e r histones. D. l i v e r histones plus histone IV from pea. Figure 39. T e s t i c u l a r histones from the newt run with t e s t i c u l a r histones from Xenopus l a e v i s (A) and from Bufo americanus (B). 38 -A B C JB. * f TT • 8 DH N 1 -N 2 — P -39 A B X 2 -X3-N 1 -N 2 — l i - N 2 + B - 5 3 -D. DISCUSSION The basic protein changes during spermiogenesis i n the eastern red spotted newt resemble the tra n s i t i o n s described i n the s n a i l , Helix aspera, (Bloch and Hew 1960a), the squid, Loligo opalescens, (Bloch 1962), and Pleurodeles w a l t i i (Picheral 1970). The earl y stages of spermiogenesis contain somatic type histones which i n l a t e r spermatids are replaced by basic proteins very r i c h i n arginine but not-extrac-table with hot t r i c h l o r o a c e t i c acid-. In -turn, these pro-teins are replaced by protamines, proteins that are soluble i n t r i -chloroacetic a c i d and very r i c h i n arginine. In the eastern red spotted nev/t the t r a n s i t i o n from the somatic type of histone to the "stable protamine" i s progressive and very s i m i l a r to the conversion observed i n P^ w a l t i i (Picheral 1970)* Besides monitoring t h i s t r a n s i t i o n by s t a i n i n g techniques, P i c h e r a l 3 observed the sequential incorporation of H -arginine into n u c l e i undergoing t h i s histone change. Therefore, the gradual change i n nuclear s t a i n i n g does not represent an unmasking of the very argin-i n e - r i c h histones but a progressive accumulation of newly synthes-ized proteins. As with the nuclear basic proteins found i n the sperm of the grasshopper (Bloch and Brack 1964) and trout (Ling e_t a l . 1969), Picheral (1970) found that i n P._ w a l t i i both the Mouse/grass-hopper type of protein ("stable protamine") and protamines were synthesized i n the cytoplasm of spermatids. The change i n D_. viridescens from the Mouse/grasshopper type of protein to protamines i s sudden and no t r a n s i t i o n a l stages are ob-served. Picheral (1970) found a s i m i l a r s i t u a t i o n i n w a l t i i . In 3 t h i s species even the H -arginine incorporation showed no t r a n s i t i o n . The Salmon type of sperm histone might be c h a r a c t e r i s t i c of Urodeles. Bloch (I969) indicated that the sperm of Amphiuma were of the Salmon type while Picheral (1970) reached the same conclusion for the sperm of P^_ w a l t i i . While Bloch (1969) ( c i t i n g unpublished cytochemical data) c l a s s i f i e d the sperm of viridescens as the Mouse/grasshopper type, the present study indicates that these sperm are a c t u a l l y of the Salmon type. Bloch might have sampled testes i n - 54 -which the most advanced stage of spermiogenesis was l a t e spermatid with the Mouse/grasshopper type of protein. This could account for his erroneous conclusion. Picheral (1971) was also able to correlate these basic protein changes during spermiogenesis with u l t r a s t r u e t u r a l observations. In the early spermatid chromatin f i b e r s were organized i n a loose net-work while as the gradual t r a n s i t i o n to the "stable protamine" began dense granules appeared at the base of the nucleus and moved up to-wards the central and a p i c a l part of the nucleus. At the same time a dense matrix was seen to spread between the granules from the t i p to the basal part of the nucleus. Ko change i n nuclear f i n e structure was observed on the t r a n s i t i o n to protamines. Perhaps the most inte r e s t i n g observation made by Picheral (1970) was the presence of free basic proteins i n the neck and t a i l regions of sperm. As the t r a n s i t i o n from the somatic type of histone to the "stable protamine" occured, free basic proteins appeared i n the neck region of the spermatid. Subsequently, when the "stable protamine" was replaced by protamines, free basic proteins appeared i n the t a i l region. These results strongly imply that the free basic proteins re-present sloughed o f f nuclear histones. In the present study, free basic proteins were not observed at any stage i n formalin f i x e d mater-i a l . Picheral employed a s i m i l a r f i x a t i v e ; however, his f i x a t i o n time was considerably longer than the one used i n t h i s study. On the other hand, with Clarke's f i x a t i v e , free basic proteins were present i n the t a i l region of stage 5 spermatozoa i n viridescens. Unfortunately, not a l l stages of spermiogenesis were fixe d i n Clarke's so whether free basic proteins are present i n other stages of spermiogenesis remains unknown. Why these proteins are evident only a f t e r Clarke's f i x a t i v e i s not clear. However, Vaughn (as reported by Bloch 1966) found that some basic proteins displaced from the nucleus during rat spermio-genesis were retained by Carnoy's f i x a t i v e (very s i m i l a r to Clarke's) and not by formalin. The electrophoretic comparison of t e s t i c u l a r histones from D.  viridescens with somatic histones indicates that two t e s t i s s p e c i f i c - 55 -bands, NI and N2, are present. Since N2 migrates closest to salmon protamine, t h i s band i s l i k e l y confined to mature sperm which cyto-chemically have been shown to behave l i k e salmon sperm. In addition, N2 migrates with band B from Bufo americanus testes, which also con-t a i n c e l l s that s t a i n s i m i l a r to the mature sperm of viridescens. The NI doublet, then, i s l i k e l y s p e c i f i c to stage 3 and k n u c l e i and represents the "stable protamine." The i n t e r p r e t a t i o n of these r e -s u l t s i s s i m i l a r to the one given by "Bloch (1962) to r e s u l t s obtained with the squid, which show a s t a i n i n g pattern s i m i l a r to the eastern red spotted newt. Starch gel electrophoresis showed four major groups of histones from squid testes. The two slowest moving bands corresponded to histones from somatic tissues while the two fastest moving bands were confined to l a t e spermatid and sperm n u c l e i (these stages were separated by centrifugation i n a sucrose gradient). The fastest moving band was very s i m i l a r i n amino acid composition and molecular weight to t y p i c a l protamines and was also found i n a pre-paration consisting e n t i r e l y of mature sperm. Therefore, Bloch i n -dicated that t h i s protein was present i n c e l l s that were cytochem-i c a l l y extractable with hot t r i c h l o r o a c e t i c a c i d and very r i c h i n ar-ginine. The s l i g h t l y slower moving band was thought to occur i n the l a t e spermatids and to represent the "stable protamine." This protein contained a l l the amino acids of t y p i c a l histones but had an arginine to l y s i n e r a t i o of about s i x . This very a r g i n i n e - r i c h histone did not contain cysteine. Picheral (19?0), on the other hand, reported that -*-n Hi. v ' a l t i i the n u c l e i containing the "stable protamine" were r i c h i n cysteine while the mature sperm were not. Whether the l a t e sperm-atids of the eastern red spotted newt contain cysteine i s unknown. Two arguments may be advanced against the above i n t e r p r e t a t i o n of the t e s t i c u l a r histone bands i n viridescens. F i r s t , one would expect band N2 to occur i n greater amounts since at the time the testes were extracted mature sperm should have been the predominant c e l l type (Baker 1966). However, spermiogenesis might have been delayed or interrupted i f the animals were improperly maintained by the commercial dealer. Secondly, since the sperm of D. viridescens - 56 -and salmon a r e c y t o c h e m i c a l l y s i m i l a r s one w o u l d e x p e c t t h e p r o t -amines o f t h e s e c e l l s t o m i g r a t e t o g e t h e r . Y e t , band N2 m i g r a t e s s l i g h t l y s l o w e r t h a n salmon p r o t a m i n e . P e r h a p s , an "amphibian p r o t a m i n e " i s p r e s e n t t h a t d i f f e r s j u s t s l i g h t l y from salmon p r o t -amine i n charge and m o l e c u l a r w e i g h t c a u s i n g the s l i g h t d i f f e r e n c e i n m o b i l i t y . - 5 7 -IV. CHANGES IN BASIC PROTEINS DURING SPERMIOGENESIS IN THREE  CARTILAGINOUS FISH A. INTRODUCTION In order to b i o c h e m i c a l l y c h a r a c t e r i z e the M y t i l u s type of sperm histone l a r g e q u a n t i t i e s of s t a r t i n g m a t e r i a l are r e q u i r e d . An i d e a l organism f o r such a study must have l a r g e t e s t e s , be e a s i l y obtained, and of course, have the M y t i l u s type o f sperm h i s t o n e . C i t i n g K o s s e l , Bloch (1969) c l a s s e d the shark, Centrophorus granu- l o s a , as c o n t a i n i n g the M y t i l u s type of sperm h i s t o n e . Bloch a l s o c l a s s e d the ray, Dasyatis s a b i n a , as the M y t i l u s type on the b a s i s of unpublished cytochemical data. The elasmobranchs then appear to be s u i t a b l e organisms f o r s t u d y i n g the M y t i l u s sperm h i s t o n e type. Therefore, a study of the b a s i c p r o t e i n changes during spermio-genesis i n the d o g f i s h (Squalus - s u c k l e y i ) and i n the l o n g nose skate (Raja rhj,na) was undertaken. L a t e r , when the M y t i l u s type of sperm histone was not found i n these elasmobranchs, the study i n c l u d e d another c a r t i l a g i n o u s f i s h , the r a t f i s h (Hydrolagus c o l l i e i ) . B. METHODS AND MATERIALS 1. CYTOCHEMISTRY Squalus s u c k l e y i "';• the d o g f i s h , Raja r h i n a , the long nose skate, and Hydrolagus c o l l i e i , the r a t f i s h , were c o l l e c t e d o f f Conox, B r i t i s h Columbia, i n May and November. C o l l e c t i o n s were made by means of a t r a w l operated from the Canadian F i s h e r i e s Research v e s s e l , the " I n v e s t i g a t o r No. 1." A f t e r a drag of approximately 15 minutes, the net was emptied onto the deck where mature-looking spec-imens of each spec i e s were chosen f o r d i s s e c t i o n . The t e s t e s were exc i s e d and t h i n s e c t i o n s were cut a t r i g h t angles to the l o n g a x i s o f the t e s t e s f o r f i x a t i o n i n e i t h e r 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 i n absolute e t h a n o l - g l a c i a l a c e t i c a c i d (3:1). Since m a t e r i a l c o u l d not be promptly returned to the l a b o r a t o r y , f i x a t i o n occurred f o r 2h to 32 h. However, wit h the r a t f i s h , an occasion arose v/here m a t e r i a l - 5 8 -was f i x e d for 6, 8, 10, 12, 16, and 32 h. Generally, the tissues were washed i n running tap water f o r 24 to 32 h i n order to compen-sate f o r the increased time spent i n formalin. Otherwise, the t i s -sues were dehydrated and embedded as described i n the anuran section. A l l s t aining procedures performed on t h i s material have been des-cribed previously. 2. BIOCHEMISTRY For biochemical work, the same organisms, c o l l e c t e d i n the same manner, were used. The testes and l i v e r s were removed as quickly as possible, under conditions that were sometimes d i f f i c u l t , and stored on dry i c e i n Thermos bottles f o r 24-32 h, although some material was stored i n t h i s manner for only a 6-10 h period. In the labor-atory t h i s material was stored at -20°C and i n some cases at -70°C u n t i l use. Since just a small portion of a t e s t i s was used for cyto-chemical studies, biochemical and cytochemical work was often per-formed on material from the same t e s t i s . Also, for dogfish, testes were obtained i n August and September as well as i n November and May. I n i t i a l l y , attempts were made to extract basic proteins from the dogfish t e s t i s by the procedure used by Ingles et a l . (1966) to show the presence of protamine i n trout testes. The sample of t e s t i s was homogenized with a Dounce homogenizer i n the presence of 0.2 N HC1 and centrifuged at 12,000g for 30 minutes. The supernatant was dialyzed against d i s t i l l e d water for 24 h and then l y o p h i l i z e d . The proteins obtained were examined by polyacrylamide disc electrophoresis as prev-i o u s l y outlined. This procedure \vas t r i e d three times and then aban-doned. Testes were also extracted i n the manner used to demonstrate protamine i n the mouse t e s t i s (Lam and Bruce 1970). A tissue s l i c e (2 grams), obtained by cutting at r i g h t angles to the long axis of the t e s t i s , was homogenized gently with a Dounce homogenizer i n d i s -t i l l e d water and l e f t to lyse for 30 minutes. The homogenate was centrifuged at 10,000g for 10 minutes and the p e l l e t obtained was homogenized again i n d i s t i l l e d water. This homogenate was centrifuged - 59 -at 12,000g for 20 minutes. The p e l l e t was homogenized i n 35 ml of 0,25 N HOT and l e f t to stand at room temperature for 2 h. The sus-pension vras centrifuged at 12,000g for 20 minutes and the super-natant l y o p h i l i z e d . The proteins i n the lypholized extract were examined by polyacrylamide disc electrophoresis. This same proced-ure was followed with l i v e r s as well as with testes. Testes were also extracted with acid a f t e r the c e l l s had been f i r s t disrupted with P> -mercaptoethanol and urea (Lam and Bruce 1970). P e l l e t s obtained from the f i r s t 12,000g centrifugation as described above were homogenized with a Dounce homogenizer at room temperature i n 5% P -mercaptoethanol (v/v) and 8 M urea i n 0.1M Tri s at pH 8.5. The suspension was incubated at 37° C for 2 h i n a Dubnoff metabolic shaking incubator and then brought to a f i n a l con-centration of 0.5 N HC1 by the addition of concentrated HC1. The homogenate was l e f t for 1 h at room temperature and then centrifuged at 15«000g for 20 minutes. The supernatant was brought to pH 5.0 by the addition of NaOH and applied to a CM30-cellulose column (1x10 cm, H+ form). The column was f i r s t washed with 200 ml of d i s t i l l e d H.,0 and then the basic proteins were eluted with 0.1N HC1. The HC1 f r a c -tion was l y o p h i l i z e d and examined by gel electrophoresis. Basic proteins were also extracted from a crude nuclear prepar-ation. E s s e n t i a l l y , the f i r s t four steps used to obtain chromatin (described previously) were followed i n order to get n u c l e i . The t i s -sue was homogenized on the S o r v a l l i n saline-EDTA (pH 8.0), f i l t e r e d through 4 layers of washed cheesecloth, centrifuged at l ,500g for 15 minutes, resuspended i n saline-EDTA, and centrifuged again at l ,500g for 15 minutes. The nuclear p e l l e t was checked under a l i g h t micro-scope. To each gram of n u c l e i , 5 wl of 0.2 M HgSO^ was added (as recommended for trout t e s t i s n u c l e i , Ling ejt a l . 1971). After being homogenized and l e f t at room temperature for 20 minutes, the mixture was centrifuged at 12,000g for 20 minutes. Cold 100% ethanol (3-4 volumes) was added to the supernatant which was then stored at -20°C for approximately 24 h« The precipitate which formed i n t h i s time was recovered by centrifugation (12,000g for 20 minutes), washed once - 60 -with ethanol, and dried i n vacuo* Chromatin was prepared and extracted with ac i d as described i n the anuran sect i o n . C. RESULTS 1. CYTOCHEMISTRY a. Elasmobranchii The testes of elasmobranch fishes are composed of spherical f o l l i c l e s or ampullae (Stanley 1966), also termed tubules by some authors (Simpson and Wardle 1967). Within a spherical f o l l i c l e a l l the gem elements are at the same stage of d i f f e r e n t i a t i o n and are organized into a number of spermatocysts or cysts. The f o l l i c l e s a r i s e from f i x e d germinal s i t e s on the l a t e r a l or d o r s o - l a t e r a l as-pect of the t e s t i s and move s t e a d i l y away as they develop, followed c l o s e l y by successively younger stages (Stanley 1966). F o l l i c l e s that have reached the ventral and ventro-medial area of the t e s t i s contain the l a t e stages of spermiogenesis and mature spermatozoa. When sperm-iogenesis i s completed, the f o l l i c l e s open to the c o l l e c t i n g ductule system and the spermatozoa are released. Thus i n a section cut at r i g h t angles to the long axis of the t e s t i s a l l stages of spermato-genesis are present ( F i g . 40). Generally, the elasmobranch t e s t i s contains a l l stages of spermatogenesis throughout the year (Simpson and Wardle 1967). While an annual cycle i n the dogfish t e s t i s has been observed (Simpson and Wardle I967), only the abundance of various stages changed. Mature sperm were most frequent in. testes caught between July and December and were least common i n testes caught i n February and May. No i n -formation i s available on the presence or absence of a seasonal cycle i n the skate t e s t i s . In the present study spermiogenesis i n the dogfish and skate has been divided into eight stages on the basis of nuclear morphology and basic protein composition. The stages are i l l u s t r a t e d i n - 61 -figures 4-1-52 and the cytochemical r e s u l t s f o r these stages are sum-marized i n Tables VI and V l l . The process of nuclear elongation has been divided into 4 stages (Figs. 41-44). Stage 1 nuc l e i for both species are round and lo o s e l y organized i n the f o l l i c l e s . Stage 2 nu c l e i are shaped l i k e tear drops while stage 3 nuc l e i are rod l i k e . Stage 4 n u c l e i are thin and c y l i n d r i c a l and are organized into d e f i n i t e cysts, p a r t i c u l a r l y i n the case of skates. The stage 4 nuc l e i of the skate appear longer and s t r a i g h t e r than those of dogfish. S p i r a l i z a t i o n has been divided into two stages. Stage 5 n u c l e i are s p i r a l i z e d at the base and rod-like at the apexes. This process i s more noticeable with skates ( F i g . 49) than with dogfish ( F i g . 45). Stage 6 n u c l e i are completely s p i r a l i z e d (Figs. 46 and 50). Stage 7 and 8 nuc l e i are morphologically i n d i s t i n u i s h a b l e from each other (Figs. 47 and 51); yet d i f f e r cytochemically. They are distinguished from stage 6 n u c l e i by being organized into very dense-l y packed cysts (Figs. 47, 48, 51 and 52). Stages 1, 2, and 3 i n both skate and dogfish are cytochemically s i m i l a r (Tables VI and VII). These nu c l e i s t a i n with AFG a f t e r a l l types of TCA hydrolysis, i n d i c a t i n g the absence of protamines. How-ever, these n u c l e i do not s t a i n with AFG i f they have been deamin-ated or acetylated i n d i c a t i n g the absence of very a r g i n i n e - r i c h h i s -tones. These n u c l e i s t a i n weakly a f t e r the Sakaguchi reaction for arginine. Therefore, these n u c l e i contain histones s i m i l a r to those of somatic c e l l s . Stage 4 nuc l e i s t a i n with AFG af t e r IN TCA hydrolysis and a f t e r 5% TCA hydrolysis at 89°C Table VII). However, stai n i n g i s l i g h t or absent a f t e r 5% TCA hydrolysis at 97°C (Table VII). This suggests the presence of a protamine. These nu c l e i s t a i n with AFG-picric a c i d despite acetylation i n d i c a t i n g the presence of very arginine-r i c h histones and s t a i n moderately a f t e r the Sakaguchi reaction for arginine. Stage 5 and 6 nuc l e i show i d e n t i c a l s t a i n i n g properties (Tables VI and VII). They both s t a i n weakly with AFG and IN TCA hydrolysis - 62 -at 60°C. With 5% TCA hydrolysis at 89°C and at 97°C they do not stain at a l l . They stain with AFG after picric acid hydrolysis and continue to do so even after acetylation. This indicates the presence of very arginine-rich histones. This conclusion i s rein-froced by the intense staining after the Sakaguchi reaction. Since staining is absent after TCA hydrolysis at 97°C9 these results sug-gest that protamines are present in these nuclei. Stage 7 nuclei stain well with AFG after IN TCA hydrolysis at 60°C" and stain moderately well after 5% TCA hydrolysis at 89°C (Table VI and VII). However they do not stain with AFG when the hydrolysis temperature i s raised to 97°C. These nuclei are very arginine-rich since they stain with AFG - picric acid despite acety-lation. The intense red staining observed after the Sakaguchi re-action supports the contention that the very arginine-rich histones are present. These nuclei, l i k e stages 5 and 6, appear to contain protamines. Stage 8 nuclei stain well with AFG under a l l conditions of TCA hydrolysis (Figs. 48 and 52). Thus protamines are absent. These nuclei continue to stain intensely despite deamination or acety-lation of sections prior to staining. This indicates the presence of very arginine-rich histones and the conclusion i s reinforced by the intense red reaction these nuclei give after the Sakaguchi re-agent for arginine. These results suggest that the mature sperm of dogfish and skate contain the mouse and grasshopper type of histone. Free basic proteins were absent in a l l stages of both dogfish and skate spermiogenesis. This was true both for formalin and ab-solute ethanol-glacial acetic acid fixed material. -62 b -Figure 40. From Stanley (1966). The zonation of the t e s t i s of Scylorhinus canicuius as seen i n transverse section. At the l e f t the l a t e r a l area of the t e s t i s contains the germinal zone (GZ) from which seminiferous f o l l i c l e s are continuously formed. Zones 1-5 indicate areas i n which f o l l i c l e s of progressively l a t e r develop-ment are found. 1. spermatogonia; 2. primary spermatocytes; 3. secondary spermatocytes; 4. spermatids; 5« zone of sperm release and f o l l i c u l a r degeneration. E efferent ductule. - 63 -F i g u r e s 4l~47. F o l l i c l e s c o n t a i n i n g s t a g e s o f d o g f i s h s p e r m i o -g e n e s i s * F e u l g e n s t a i n i n g . S c a l e denotes 50/x . F i g u r e 4l. F o l l i c l e s c o n t a i n i n g round s t a g e 1 n u c l e i . F i g u r e 42. Tear drop-shaped s t a g e 2 n u c l e i . L d e s i g n a t e s lumen o f a f o l l i c l e . F i g u r e 43. F o l l i c l e s o f r o d l i k e s t a g e 3 n u c l e i . F i g u r e 44. F o l l i c l e s o f s t a g e 4 n u c l e i . The o r g a n i z a t i o n o f n u c l e i i n t o c y s t s i s n o t i c e a b l e . - 64 -Figure 45. Stage 5 n u c l e i . Figure 46. Stage 6 n u c l e i . Figure 47. Stage 7 and 8 n u c l e i . These nuc l e i occur i n densely-packed cysts. Figure 48. Alkaline fast green staining, 5% t r i c h l o r o a c e t i c acid hydrolysis at 97°C. Nuclei that s t a i n are stage 8 while those that do not (see arrow) are stage 7« Scale denotes 50 JU . - 65 -Figures 4-9-51. F o l l i c l e s containing l a t e stages of skate sperm-iogenesis. Feulgen st a i n i n g . Scale denotes 50J1 » Figure 4-9. F o l l i c l e containing stage 5 nuclei which have begun s p i r a l i z a t i o n . L indicates lumen of the f o l l i c l e . Figure 50. Stage 6 n u c l e i . S p i r a l i z a t i o n has been completed. Figure 51. Stage 7 and 8 n u c l e i . These nuc l e i occur i n densely-packed cysts. Figure 52. Alkaline fast green staining, 5% t r i c h l o r o a c e t i c acid hydrolysis at 97°C Nuclei that s t a i n are stage 8 while those that do not (see arrow) are stage 7» Scale denotes 50 ja « 51 4 y * — - 66 -TABLE VI Cytochemistry of dogfish and skate spermiogenesis Staining and pretreatment Reactive material Stages 3 ^ 5 AFG-TCA 97°C AFG-TCA 97°C deamination AFG-picric a c i d Basic proteins other than protamines Basic proteins r i c h i n ar-ginine Basic proteins including protamines AFG-picric acid, Basic proteins a c e t y l a t i o n r i c h i n a r -ginine + + + + + + + + + + + + + + + +• AFG Sakaguchi Non-nucleic acid-associated basic proteins Protein-bound arginine + + + + ++ ++ ++ ++ NOTE: AFG = a l k a l i n e f a s t green; TCA = t r i c h l o r o a c e t i c acid - 67 -TABLE VII E f f e c t of hydrolysis conditions on AFG s t a i n i n g i n dogfish and skate spermiogenesis Hydrolysis Stages conditions 1 2 3 4 5 6 7 8 P i c r i c acid f o r 6 h at 60°C + + + + + + + + IN TCA at 60°C for J h + + + + + + + + 5% TCA at 89°C for 15 mins + + + + - - + . + 5% TCA at 97°C for 15 mins + + + - + NOTE: TCA = t r i c h l o r o a c e t i c a c i d . - 68 -b. Holocephali Like the elasmobranch t e s t i s , the r a t f i s h t e s t i s i s composed of spherical ampullae and within each ampullae germ elements are at the same stage of development (Stanley 1963). A l l stages of spermatogenesis are seen i n sections cut at right angles to the long axis of the t e s t i s . A seasonal cycle has not been reported i n the r a t f i s h t e s t i s . In t h i s study spermiogenesis i n the r a t f i s h has been divided into seven stages on the basis of nuclear morphology and basic protein composition. The stages are i l l u s t r a t e d i n figures 53-59 and the cytochemical results are summarized i n Tables VIII and IX. In addition, cytochemical results are recorded for sperm i n the epi-didymis. Stages 1-4 of spermiogenesis represent steps i n the process of nuclear elongation. Stage 1 nuclei are round and loosely organized i n the f o l l i c l e s (Fig. 53) while stage 2 n u c l e i are tear drop shaped and have begun to migrate to the periphery of the f o l l i c l e s (Fig. 54). The rod shaped nuclei aligned at the periphery of f o l l i c l e s have been designated as stage 3 (Fig. 55) while the t h i n , c y l i n d r i c a l n u c l e i organized into cysts are stage 4 (Fig. 56). Each cyst of stage 4 nuclei points toward a S e r t o l i nucleus, which i s not always apparent due to the diffuse chromatin staining, while the t a i l s of these sperm-atozoa protrude into the central lumen of the f o l l i c l e . After nuclear elongation, spermatids go through a process of s p i r a l i z a t i o n . S p i r a l i z a t i o n begins at the ba6e of the nucleus and proceeds to the anterior t i p and nuclei demonstrating t h i s t r a n s i t i o n have been termed stage 5 (Fig. 57 )• Stage 6 nuclei have completed s p i r a l i z a t i o n (Fig. 58) while stage 7 nuclei are organized into much more t i g h t l y packed cysts than stage 6 (Fig. 59). The f o l l i c l e s as a whole appear to change i n size as spermio-genesis proceeds. F o l l i c l e s containing early stages of spermiogenesis are considerably larger than those containing l a t e r stages. Stanley (1966) observed a s i m i l a r phenomenon i n Scyliorhinus and Torpedo. Stages 1, 2, and 3 give s i m i l a r cytochemical re s u l t s (Tables VIII - 69 -and IX. These nu c l e i s t a i n with AFG a f t e r TCA hydrolysis, i n d i c -ating the absence of protamines; do not s t a i n with AFG i f sections are previously deaminated or acetylated, i n d i c a t i n g the absence of very ar g i n i n e - r i c h histones; and s t a i n l i g h t l y a f t e r the Sakaguchi reaction for arginine. These n u c l e i exhibited the t y p i c a l somatic c e l l s t a i n i n g pattern and therefore contain the somatic type of histones. Stage k n u c l e i give unusual staining r e s u l t s (Tables VIII and IX). .Staining i s dependent on the temperature at which TCA hydro-l y s i s i s carried out. Stage 4 n u c l e i s t a i n with AFG a f t e r IN TCA hydrolysis at 60°C or at 89°C; and s t a i n f a i n t l y or.not at a l l a f t e r TCA hydrolysis at 97°C. These n u c l e i s t a i n with AFG a f t e r p i c r i c a c i d hydrolysis and continue to s t a i n even i f acetylated. This sug-gests they contain very a r g i n i n e - r i c h histones. However, these n u c l e i give only a moderate Sakaguchi reaction. Stages 5 and 6 nuclei also give unusual staining r e s u l t s (Tables VIII and IX). These n u c l e i s t a i n weakly a f t e r IN TCA hydro-l y s i s at 60°C and do not s t a i n at a l l a f t e r 5% TCA hydrolysis at 89°C or 97°C. These nuclei s t a i n with AFG a f t e r p i c r i c acid hydrolysis and continue to do so despite acetylation, i n d i c a t i n g the presence of very ar g i n i n e - r i c h histones. These nu c l e i also s t a i n strongly a f t e r the Sakaguchi reaction for arginine. This s t a i n i n g pattern suggests the presence of protamines. Stage 7 nu c l e i and n u c l e i found i n the epididymis give ident-i c a l cytochemical r e s u l t s . These n u c l e i s t a i n under a l l conditions of TCA hydrolysis and continue to do so even i f the sections are de-aminated p r i o r to st a i n i n g . Staining with AFG a f t e r p i c r i c acid hydrolysis continues despite a c e t y l a t i o n . An intense red reaction i s given by these n u c l e i a f t e r treatment with the Sakaguchi reagent. These results indicate the presence of a very a r g i n i n e - r i c h histone s i m i l a r to the mouse and grasshopper type. Free basic proteins were not observed i n any of the stages of spermiogenesis. The effect of f i x a t i o n on AFG staining and deamination was - 70 -studied on nuclei found in the epididymis. Fixation times between 6-32 h did not affect the results. - 71 -Figures 53-59. F o l l i c l e s containing stages of r a t f i s h spermio-genesis. Feulgen staining. Scale denotes 10}X . Figure 53. Round stage 1 n u c l e i . L indicates lumen of the f o l l i c l e . Figure 5^. Tear, drop-shaped stage 2 n u c l e i . Figure 55. Rod-shaped stage 3 n u c l e i . Figure 56. Stage h n u c l e i organized into cysts. 1 • • « • —*? ' V a • * • • • • 4 • • • ,. • . •••••• «• .. • • • .••» • • • •* • • . • * • -V •"•V-;-. • . i t^v ^ W . \ t » . « A 5* • ^ ^ ^ ^ • 55 \ — •V * 1 * - 72 -Figure 57« Stage 5 n u c l e i , s p i r a l i z a t i o n . Figure 5 8 . Stage 6 n u c l e i . Figure 59 • Stage 7 n u c l e i . These nu c l e i are undergoing nuclear S p i r a l i z a t i o n has been completed. - 73 -TABLE VIII Cytochemistry of r a t f i s h spermiogenesis Staining and pretreatnent Reactive material Stages 4 5 6 AFG-TCA 97°C AFG-TCA 97°C, deamination AFG-picric a c i d AFG-picric acid, ac e t y l a t i o n AFG Sakaguchi Basic proteins other than protamines Basic proteins r i c h i n arginine Basic proteins including protamines Basic proteins r i c h i n arginine Non-nucleic acid-aesociated basic proteins Protein-bound arginine + + + + + + + + + + + ++ ++ ++ NOTE: AFG = a l k a l i n e fast green; TCA = t r i c h l o r o a c e t i c a c i d . * Sperm found i n the epididymis gave i d e n t i c a l r e s u l t s to stage 7» - 7k -TABLE IX Effect of hydrolysis conditions on AFG staining i n r a t f i s h spermiogenesis Hydrolysis Stages conditions 1 2 3 4- 5 6 7 P i c r i c acid for 6 h at 60 C + + + + + + + INTOA at 60°C for 3 h + + + + + + + 5% TCA at 8 9 ° C for 15 mine + + + + % TCA at 97°C for 15 mins + + + NOTE: TCA = tri c h l o r o a c e t i c acid. - 75 -2. BIOCHEMISTRY The h i s t o n e components o f skate t e s t e s and l i v e r s a r e compared i n f i g u r e s 60-62. Two major bands are e v i d e n t a f t e r the e l e c t r o -p h o r e s i s o f l i v e r h i s t o n e s on p o l y a c r y l a m i d e g e l s and the r e g i o n bound by these bands w i l l be d e f i n e d as the h i s t o n e r e g i o n . The h i s -tone r e g i o n o f t e s t e s a l s o c o n t a i n s two bands. The f a s t e s t moving band i n the h i s t o n e r e g i o n from both t i s s u e s c o - e l e c t r o p h o r e s e s w i t h h i s t o n e IV from pea. A number o f bands m i g r a t i n g slower than the h i s t o n e r e g i o n a r e l i k e l y non-histone contaminating p r o t e i n s . Band S which migrates f a s t e r than h i s t o n e IV i s s p e c i f i c to the t e s t i s and c o - e l e c t r o p h o r e s e s w i t h salmon protamine ( F i g . 62). T h e r e f o r e , band S has two c h a r a c t e r i s t i c s o f protamine - s p e c i f i c i t y to the t e s t i s and 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 y w i t h protamine. The e l e c t r o p h o r e s i s of h i s t o n e s from d o g f i s h l i v e r and d o g f i s h t e s t i s i s i l l u s t r a t e d i n f i g u r e s 63-65. L i v e r h i s t o n e s m i g r a t e as two bands and the a r e a bound by these bands w i l l be d e f i n e d as the d o g f i s h h i s t o n e r e g i o n . Two bands ar e a l s o p r e s e n t i n the t e s t i s h i s t o n e r e g i o n and the f a s t e s t moving band i n t h i s r e g i o n from both t e s t e s and l i v e r s m i grates w i t h h i s t o n e IV from pea. The bands mi-g r a t i n g slower than the h i s t o n e r e g i o n a r e c o n t a m i n a t i n g p r o t e i n s . A v e r y f a s t moving band (D) i s s p e c i f i c to the t e s t i s and m i g r a t e s w i t h salmon protamine ( F i g . 65). T h i s p r o t e i n appears i d e n t i c a l w i t h band S o f s k a t e . The use o f o t h e r e x t r a c t i o n methods r e i n f o r c e s the c o n c l u s i o n t h a t o n l y 1 protamine band i s present i n skate and d o g f i s h t e s t e s . F i g u r e s 66 and 67 i l l u s t r a t e the e l e c t r o p h o r e t i c p a t t e r n s o b t a i n e d a f t e r the a c i d e x t r a c t i o n of n u c l e i . In both skate and d o g f i s h one f a s t moving band t h a t c o - e l e c t r o p h o r e s e s w i t h salmon protamine i s p r e s e n t . In the case o f the s k a t e t e s t i s , b a s i c p r o t e i n s were ex-t r a c t e d by the two methods used by Lam and Bruce (1971) to e x t r a c t mouse protamine. Both methods y i e l d e d o n l y one major band i n the protamine r e g i o n ( F i g . 68). E x t r a c t i o n o f l i v e r s by these methods d i d not y i e l d a p r o t a m i n e - l i k e p r o t e i n . Band D i s present i n t e s t e s caught i n May, August, and November. - 76 -A l t h o u g h no attempt was made t o q u a n t i t a t e t h e r e s u l t s , band D ap-p e a r s t o be most e a s i l y e x t r a c t e d from t e s t e s caught i n May. S i n c e the s t a g e s o f s p e r m a t o g e n e s i s i n the elasmobranch t e s t i s a r e a r r a n g e d i n l a y e r s from t h e l a t e r a l t o the v e n t r a l a r e a s ( S t a n -l e y 1966), a t t e m p t s were made t o i s o l a t e c e l l t y p e s by c u t t i n g t h e t e s t i s i n t o l o n g s t r i p s . However, t h i s method p r o v e d u n s u c c e s s f u l s i n c e the c o r r e c t o r i e n t a t i o n o f f r o z e n t e s t e s was v e r y d i f f i c u l t . The a p p l i c a t i o n o f the s e d i m e n t a t i o n v e l o c i t y t e c h n i q u e used by Lam and Bruce (1970) t o s e p a r a t e mouse spermatogenic c e l l s m i g h t p r o v e u s e f u l . - 77 -Figures 60-62. Densitometer tracings of skate histones run on 15% polyacrylamide gels for 80 minutes and stained with buffalo black. The histones were extracted from chromatin. H in d i c a t e s somatic histone region. Arrow indicates that the band co-electro-phoreses with histone IV from pea. Figure 60. Liver histones. Figure 6 l . T e s t i c u l a r histones. Figure 62. T e s t i c u l a r histones run with salmon protamine. 774 - 7 8 -F i g u r e s 63-65. D e n s i t o m e t e r t r a c i n g s o f d o g f i s h h i s t o n e s r u n on 1 5 % p o l y a c r y l a m i d e g e l s f o r 80 minutes and s t a i n e d w i t h b u f f a l o b l a c k . The h i s t o n e s were e x t r a c t e d from c h r o m a t i n . H i n d i c a t e s s o m a t i c h i s t o n e r e g i o n . Arrow i n d i c a t e s t h a t the band c o - e l e c t r o -p h o r e s e s w i t h h i s t o n e IV from pea. F i g u r e 63. L i v e r h i s t o n e s F i g u r e 64. T e s t i c u l a r h i s t o n e s . F i g u r e 65. T e s t i c u l a r h i s t o n e s r u n w i t h salmon p r o t a m i n e . - 79 -Figures 66 and 67. Densitometer tracings of testicular histones from skate and dogfish run on 15% polyacrylamide gels for 80 minutes and stained with buffalo black. The histones were extrac-ted from crude nuclear preparations. H indicates somatic histone region. Arrow indicates that the band cco-electrophoreses with histone IV from pea. Figure 66. Skate testicular histones. Top: electrophoresed alone. Bottom: electrophoresed with salmon protamine. Figure 67. Dogfish testicular histones. Top: electrophoresed alone. Bottom: electrophoresed with salmon protamine. - 8o -F i g u r e 68. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s p a t t e r n s o f h i s t o n e s from the s k a t e . G e l s r u n as i n F i g u r e s 60-67o A. L i v e r h i s t o n e s e x t r a c t e d from a crude n u c l e a r p r e p a r a t i o n . B. T e s t i c u l a r h i s t o n e s e x t r a c t e d as i n A. C. T e s t i c u l a r h i s t o n e s e x t r a c t e d by the method o f Lam and Bruce (1971). D. T e s t i c u l a r h i s t o n e s e x t r a c t e d from c h r o m a t i n . - 81 -D. DISCUSSION The basic protein changes during spermiogenesis i n the three cartilaginous f i s h studied are remarkably s i m i l a r and very unusual. In a l l three specie ; the t r a n s i t i o n to a very a r g i n i n e - r i c h histone begins at the ends of nuclear elongation and the beginning of nu-clear s p i r a l i z a t i o n . However, instead of the appearance of "stable protamines" or the Mouse/grasshopper type of sperm histone followed by a t r a n s i t i o n to protamines, as i s the case i n the s n a i l (Bloch and Hew 1960a), Pleurodele w a l t i i . (Picheral 1970), the squid (Bloch 1962) and the eastern red spotted newt (part I I I of this t h e s i s ) , protamines appear f i r s t followed by stable protamines. Three l i n e s of evidence suggest that t h i s i s the correct sequence of events and not just an error i n staging. I f sections cut at right angles to the long axis of the t e s t i s are scanned from the germinal zone to the opposite edge of the t e s t i s , a layer of protamine con-taining c e l l s appears f i r s t , followed by the appearance of stable protamine containing sperm at the very outer edge of the t e s t i s . The di r e c t i o n of this scan i s from the most immature stage of spermato-genesis to the most mature (Stanley 1966). In the case of the dog-f i s h , sperm of the Mouse/grasshopper type are found i n c o l l e c t i n g ductules while i n both dogfish and skate sperm of the Mouse/grass-hopper type are observed i n the lumen of f o l l i c l e s , i n d i c a t i n g that they are mature and are being released (Stanley 1966). F i n a l l y , the sperm i n the epididymal region of the r a t f i s h are found to be the Mouse/grasshopper type while the t e s t i s displays e a r l i e r stages of spermiogenesis where protamines are present. Thus i n cartilaginous f i s h the basic protein changes during spermiogenesis appear to be the reverse of those reported i n other organisms (histone protamine stable protamine). The p r i n c i p a l argument against t h i s unusual t r a n s i t i o n i s the lack of supporting biochemical evidence.Using the same gel system as i n the present study, Lam and Bruce (1971) reported that the basic protein i s o l a t e d from mouse sperm migrated s l i g h t l y slower than salmon - 82 -protamine. Since sperm were found in dogfish and skate testes that stained exactly like mouse sperm, a similar protein would be ex-pected to be found in these testes. No such band was obtained, a l -though a hint of such a band was obtained with one skate preparation. This protein might have been missed for at least two reasons. Per-haps, the cells containing the Mouse/grasshopper type of protein were absent or present in only small numbers in the tissues extracted. However, in some cases cytochemistry was performed on a small portion of the testis to be extracted and the presence of the stable prot-amine was confirmed. This protein might have been missed for another reason. In some cases, basic proteins from mammalian sperm, the Mouse/grasshopper type, can only be isolated under extremely acid conditions and even the methods of Lam and Bruce (1971) will f a i l (Ti Wing Wu, personal communications). A similar situation might exist for the mature sperm of cartilaginous fish. The observation that protamines were most easily extracted from testes obtained in May would be consistent with this explanation. The testes at this time would be expected to contain a predominance of cells in the middle of spermiogenesis, which would be easy to extract, while testes in November would contain an abundance of mature sperm (Simpson and Wardle 1967) which would contain the difficult to extract Mouse/grass-hopper type of protein. An alternate possibility is that the appearance of stable protamines is an artifact. Perhaps, as the sperm nuclei become very condensed they resist trichloroacetic extraction for reasons other than the presence of the Mouse/grasshopper type of protein. This would explain why despite numerous extraction methods only a protamine band was obtained with skate and dogfish testes. However, why these nuclei would be resistant to extraction is difficult to explain. These possibilities may be tested by the extraction of basic proteins from mature sperm only. Sufficient mature sperm might be collected from the epididymal region of the ductus deferens. If a protein is obtained from these sperm similar in amino acid compos-ition and electrophoretic mobility to the testis specific bands, the - 83 -unusual sequence of events i s l i k e l y an ar t i fac t . However, i f from these sperm a protein is obtained which i s different from the testis specific protein, the sequence of events outlined above is l i k e l y to be the correct one. A third poss ibi l i ty is that the appearance of protamines i s an ar t i fac t . Some stages of spermiogenesis might be sensitive to t r i -chloroacetic acid hydrolysis for reasons other than the presence of protamines. The reorganization of nuclear material in the process of spiral izat ion might be the cause of th is . However, i f the appearance of protamine is an ar t i fac t , the co-electrophoresis of testis spec-i f i c bands with salmon protamine must be regarded as merely for tu i t -ous. This is an unlikely poss ib i l i ty . Therefore, on the basis of the cytochemical results , the sperm of the dogfish, long nose skate, and the ratf ish w i l l be tentatively classed as the Mouse/grasshopper type. Future studies should c lar i fy the discrepancies between this conclusion and the biochemical results . The conclusion of this study i s not in agreement with the l i t t l e available information on the basic protein composition of sperm from other cartilaginous f i sh . On the basis of Kossel's (1928) work, Bloch (1969) c lass i f ied the shark, Centrophorus granulosus, as the Mytilus type. However, Vendrely and Vendrely (1966) have pointed out that much of Kossel's work should be repeated since the isolat ion techniques u t i l i zed in his day were harsh and art i facts in amino acid composition were possible. On the basis of unpublished cytochemical data, Bloch (1969) c lass i f ied the ray, Dasyatis sabina, as the Mytilus type. Comment on unpublished results i s d i f f i c u l t . However, in this study no variation in sperm histone type was observed in a wide range of cartilaginous f i s h . Therefore, differences between this study and Bloch fs results are not l ike ly to be explained on the basis that d i f -ferent organisms have been studied. The observation that protamines are present in elasmobranchs might lead to some fascinating comparative studies. Can the protamines of elasmobranchs be fractionated, Protamines from teleost f ish have been fractionated by a number of workers (Ando and Sawada 1961, Ando - 84 -and Suzuki 1966, Ling et al„ 197D« The amino aci d sequence of protamines from elasmobranch f i s h would make an e x c i t i n g study since a fundamental pentapeptide has been found i n a l l the protamines so fa r elucidated. This sub-unit occurs some three or four times per molecule, while other pieces are fragments of the unit r e s u l t i n g from p a r t i a l duplication of the c i s t r o n (Black and Dixon 1967). Protamines have been sequenced from the rainbow trout (Salmo g a i r d -n e r i i ) , the P a c i f i c herring (Clupea p a l l a s i i ) , and Chum salmon (Oncorhynchus keta) ( P h i l l i p s 1971). These organisms are bony f i s h . The basic protein changes of t h i s study may be r e l a t e d to recent ultr a s t r u c t u r e observations on dogfish spermiogenesis (Stanley 1971a, 1971b). Stanley observed that the chromatin of ea r l y sperma-t i d s was f i n e l y granular and evenly dispersed. While nucleoprotein f i b e r s were i n i t i a l l y randomly oriented, f i b e r s at a l a t e r stage were aligned anteroposteriorly. When the nucleus reached a length of around 19 M the i n t r a nuclear f i b e r s were joined together l a t e r a l l y to form a maze of sheetlike configurations. These fibrous sheets followed a h e l i c a l course along the length of the nucleus. Later these sheets were compacted into a s o l i d column of chromatin. At t h i s point the nucleus was almost completely elongated and the nuc-l e a r volume had decreased from about 180^ to approximately 10ya • The t r a n s i t i o n to the very a r g i n i n e - r i c h histones begins at about t h i s stage which indicates that these proteins are not involved i n early nuclear condensation. Next, Stanley observed the s p i r a l i z -ation of chromatin, beginning at the posterior end of the nucleus and proceeding to the anterior t i p . Although the appearance of protamines i s correlated with the process of nuclear s p i r a l i z a t i o n , a cause and e f f e c t r e l a t i o n s h i p i s u n l i k e l y , since the s p i r a l i z a t i o n i s fore-shadowed much e a r l i e r by the h e l i c a l alignment of f i b r i l l a r nuclear sheets. The chromatin material of mature sperm was highly compacted, but thin sections s t i l l gave evidence of l o n g i t u d i n a l f i b r i l s . The center of the sperm nucleus appeared more electron dense than the periphery and Stanley suggested that the peripheral material may be residual nuclear protein. Such protein might be the protamines that t h i s study suggests are replaced i n the l a t e stages of spermiogenesis - 85 -by the Mouse/grasshopper ty p e o f p r o t e i n . - 86 -TABLE X The Variety of Sperm Histones Organism Sperm Histone Type CLASS CHONDRICHTHYES Subclass Elasmobranchii Squalus suckleyi (or acanthias) Raja rhina Subclass Holocephali Hydrolagus c o l l i e i Mouse/grasshopper Mouse/grasshopper Mouse/grasshopper CLASS AMPHIBIA Subclass Lepospondyl: Diemictylus viridescens Salmon Subclass Apsidospondyli Xenopus l a e v i s Mytilus Rana pretiosa Rana Rana p a l u s t r i s Rana Bufo boreas Salmon Bufo americanus Salmon Hyla r e g i l l a Mytilus Hyla versicolor Mytilus * C l a s s i f i c a t i o n i s from Romer (1967). - 8? -V. GENERAL DISCUSSION The variety of sperm histone types which have been demonstrated i n the present study are summarized i n Table X. These r e s u l t s are d i s -cussed below with respect to hypotheses on the function of sperm histones. While the results i n Table X y i e l d no information on the postul-ated repressor role of sperm histones, the data can be used i n the future to c r i t i c a l l y test t h i s hypothesis. I f the function of the h i s -tone t r a n s i t i o n i s to repress the sperm genome, one would expect the sperm genome to be repressed only i n organisms which showed a trans-i t i o n . Indeed, RNA synthesis i s not observed i n sperm of the Mytilus type (Das et a l . 1965)» the Mouse/grasshopper type (Bloch and Brach 1964) and the Salmon type (Marushige and Dixon 1969). RNA synthesis i s also absent i n sperm of the crab type t-zhich are free of basic proteins (Vaughn and Thomson 1972). In a l l these cases a t r a n s i t i o n from the somatic histone type occurs. On the other hand, i n the Rana type no t r a n s i t i o n i s observed. Whether the sperm genome i s complete-l y repressed i n t h i s type remains to be seen. However, i f the sperm histone t r a n s i t i o n serves to repress the sperm genome, one would pre-di c t that the sperm genome i s not repressed i n the case of the Rana type. The sperm histone types might r e f l e c t different packaging require-ments for sperm DNA. This hypothesis may be discussed i n terms of nuclear shape, nuclear condensation pattern, and the amount of DNA per nucleus. No correlation appears to exist between sperm histone type and nuclear shape. Goldfish ( Z i r k i n 1971b) and frogs of the genus Rana have sperm histones similar to somatic c e l l s . Yet, the sperm nuclei of goldfish are round while those of the frogs are rod-shaped. The sperm of trout contain protamines, as do the sperm of Bufo americanus and Bj_ boreas. Despite this s i m i l a r i t y i n protein type, trout sperm nuclei are round while Bufo sperm nuclei are rod-shaped. A correlation between nuclear condensation pattern and sperm histone type might e x i s t . As noted i n the sections on the eastern red - 88 -spotted newt and the cartilaginous f i s h , basic protein t r a n s i t i o n s are accompanied by marked changes i n nuclear f i n e structure. A si m i l a r observation was made by Bloch and Hew (1960a) for the s n a i l . Z i r k i n (1971a, 1971b) has studied the nuclear f i n e structure i n sperm that contain the Rana type of histone. Unlike the above organ-isms, highly oriented f i b e r s and sheets were not evident. Instead randomly oriented f i b e r s with diameters s l i g h t l y smaller than those from somatic c e l l n u c lei were present. In crab sperm the l o s s of basic nuclear proteins was correlated with changes i n chromatin (Langreth 1969). The chromatin changed from homogenous f i n e granules and f i b r i l s of f a i r l y low electron opacity to clumps of f i b e r s i n an electron-translucent nucleoplasm. Thus changes i n nuclear f i n e s t r u c -ture do appear to be correlated with histone type. This c o r r e l a t i o n suggests the DNA i s packed into the sperm head i n a manner dependent on the sperm histone type. Walker (1971) believes three basic patterns of nuclear conden-sation are evident i n spermiogenesis. The f i r s t pattern i s the "fibrous" type. Fibers or filaments occur i n the earl y spermatid and become thicker and eventually fuse to give the mature sperm head. In the second pattern, the "lamellar" type, the f i b e r s present i n the early spermatid fuse into sheets or lamellae as spermiogenesis proc-eeds. The t h i r d pattern i s the "granular" type. In t h i s type the spermatid undergoes a l l a l t e r a t i o n i n shape p r i o r to the s t a r t of any condensation of the nucleoprotein. Walker found l i t t l e c o r r e l a t i o n between these condensation patterns and sperm histone type. A re l a t i o n s h i p between sperm histone type and the amount of DNA per nucleus i s not evident. Olins et a l . (1968) have cal c u l a t e d that the density of packaging i n trout sperm i s 0.7g DNA/crn^. Since i n the sperm head the r a t i o of protamine arginine/DNA phosphate was about one, they suggested that protamines may f a c i l i t a t e the packing of DNA by reducing phosphate-phosphate e l e c t r o s t a t i c repulsions. Thus the formation of aggregated structures would be favored. However, using the nuclear volume given by Z i r k i n (1970) and c a l c u l a t i n g the DNA content from the r e s u l t s of Bachman (1970), one may compute the density - 89 -of packaging i n Rj_ p i p i e n s sperm. In these sperm with the somatic his t o n e type the density of packaging i s 0.66g DNA/cm^ and very s i m i l a r to that i n t r o u t sperm which contain protamine. Bloch (1969) p o s t u l a t e d t h a t the v a r i e t y of sperm histone types might r e f l e c t d i f f e r e n t p r o t e c t i v e r o l e s . Thus organisms which have e x t e r n a l f e r t i l i z a t i o n might r e q u i r e a s p e c i f i c h i s t o n e type. A l t e r n a t i v e l y , organisms wi t h i n t e r n a l f e r t i l i z a t i o n might r e q u i r e a unique histo n e . Perhaps, sperm which are s t o r e d i n the female f o r long periods before f e r t i l i z a t i o n need a s p e c i a l sperm histone . No c o r r e l a t i o n e x i s t s between sperm histone type and e x t e r n a l or i n t e r n a l f e r t i l i z a t i o n . The anurans s t u d i e d a l l have e x t e r n a l f e r t i l i z a t i o n ; y e t , they d i s p l a y a wide range of histone types. The eastern red spotted newt and the three c a r t i l a g i n o u s f i s h s t u d i e d have i n t e r n a l f e r t i l i z a t i o n . The Salmon type of sperm his t o n e i s found i n the newt while the c a r t i l a g i n o u s f i s h have the Mouse/grass-hopper type. The honey bee a l s o has i n t e r n a l f e r t i l i z a t i o n and y e t , contains the Rana type of sperm histone (Bloch 1969). Long-lived sperm do not appear to r e q u i r e a s p e c i a l sperm h i s -tone. Sperm i n the spermatheca of the queen honey bee have been reported to remain v i a b l e f o r upwards of a year (Taber and Blum i 9 6 0 ) . Snakes have been reported to continue bearing o f f s p r i n g more than four years a f t e r mating (Wright and Wright 1957)• Sperm storage has been reported i n the female dogfish (Metten 1939) and might be of general occurrence i n elasmobranchs (Grover 1970). Yet bees have the Rana type of h i s t o n e , snakes the Salmon type (Bloch 1969)» and the d o g f i s h the Mouse/grasshopper type. Bloch (1969), among others ( O l i n s et a l . 1 9 6 8 ) , has suggested that the sperm histone t r a n s i t i o n may erase the developmental h i s t o r y of the c e l l , thereby r e s t o r i n g t o t i p o t e n c y to a h i g h l y s p e c i a l i z e d c e l l . This view r e q u i r e s that "spermatogonial c e l l s and spermato-cytes be considered d i f f e r e n t i a t e d i n the same sense as somatic c e l l s " (Bloch 1969)» In a s e r i e s of n u c l e a r - t r a n s p l a n t experiments D i Berar-dino and Hoffner (1970) demonstrated that the n u c l e i of spermatogonial - 90 -c e l l s from Rana.pipiens underwent developmental r e s t r i c t i o n s during t h e i r process of c e l l d i f f e r e n t i a t i o n . They behaved l i k e somatic n u c l e i . However, i n Rj_ pipiens no sperm histone t r a n s i t i o n occurs. Therefore, the histone t r a n s i t i o n does not appear to be necessary to erase the developmental r e s t r i c t i o n s . L i t t l e information i s av a i l a b l e as to what happens to sperm h i s -tones i n early development. Work on rat sperm suggests that they are l o s t very early i n pronuclear formation (Kopecny T970). However, a co r r e l a t i o n might exist between c h a r a c t e r i s t i c events of ea r l y develop-ment and sperm histone type. Perhaps the sperm histone type i s r e l a t e d to the cleavage pattern of the egg. However, among the amphibians, Diemictylus , Xenopus, and Rana, cleavage i s h o l o b l a s t i c . Yet the sperm of these organisms are of the Salmon, Mytilus, and Rana types, respectively. The c a r t i l a g -inous f i s h have raeroblastic eggs as do most t e l e o s t s . The c a r t i l a g -inous f i s h have the Mouse/grasshopper type of sperm histone \tfhile the teleosts demonstrate the same range i n sperm histones as the amphib-ians do. Thus no r e l a t i o n appears to exist between sperm histone type and cleavage pattern. The beginnings of RNA synthesis during early embryonic develop-ment appears to be quite variable and might be correlated with sperm histone type (Bloch 1969). However, due to the lack of data on RNA synthesis i n the organisms of the present study a c o r r e l a t i o n i s not possible. Bloch (1969) noted that sperm histone types did not show an evol-utionary trend. Within most of the broad taxa studied a l l types were represented. Indeed, i n the present study a wide range of sperm h i s -tones was observed i n the amphibians. However, no v a r i a t i o n was ob-served i n the cartilaginous f i s h . An explanation f o r these r e s u l t s cannot be advanced. Bloch also observed l i t t l e v a r i a t i o n i n sperm histones within t i g h t l y defined taxomic groups. S i m i l a r l y , i n the present study v a r i a t i o n i n sperm histone type was not found among frogs of the same genus. - 91 -S i n c e no c o r r e l a t i o n p r e s e n t l y e x i s t s between h i s t o n e t y p e and any one a s p e c t o f f u n c t i o n , one i s f o r c e d t o c o n c l u d e w i t h B l o c h (1969) t h a t t he v a r i a b i l i t y o f sperm h i s t o n e s " r e f l e c t s an e v o l u t i o n -a r y i n d i f f e r e n c e t o a r e l a t i v e l y u n i m p o r t a n t p r o t e i n i n an i n e r t n u c l e u s . " The o n l y g e n e r a l r e q u i r e m e n t o f t h e s e p r o t e i n s a p p e a r s t o be b a s i c i t y . - 92 -REFERENCES A l f e r t , M., 1956 C h e m i c a l d i f f e r e n t i a t i o n o f n u c l e a r p r o t e i n s d u r i n g s p e r m a t o g e n e s i s i n the salmon. J . B i o p h y s . Biochem. C y t o l . 2: 109-114 A l f e r t , M., and I . 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