Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1972_A6_7 B64_7.pdf [ 11.21MB ]
Metadata
JSON: 831-1.0101642.json
JSON-LD: 831-1.0101642-ld.json
RDF/XML (Pretty): 831-1.0101642-rdf.xml
RDF/JSON: 831-1.0101642-rdf.json
Turtle: 831-1.0101642-turtle.txt
N-Triples: 831-1.0101642-rdf-ntriples.txt
Original Record: 831-1.0101642-source.json
Full Text
831-1.0101642-fulltext.txt
Citation
831-1.0101642.ris

Full Text

STUDIES ON SPERM HISTONES IN AMPHIBIA AND CHONDRICHTHYES  by  NIELS CHRISTIAN BOLS B.Sc. (Hons.) Simon Fraser U n i v e r s i t y 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 t h e s i s as conforming to the required standard  The U n i v e r s i t y of B r i t i s h Columbia August, 1972  In p r e s e n t i n g t h i s  thesis  an advanced degree at  further  agree  fulfilment  of  the  requirements  the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree  the L i b r a r y s h a l l make I  in p a r t i a l  it  freely  available  for  this  thesis  f o r s c h o l a r l y purposes may be granted by the Head o f my Department  of  this  thesis for  It  financial  of  on  i0 d  or  i s understood that c o p y i n g o r p u b l i c a t i o n g a i n s h a l l not  written permission.  Department  that  r e f e r e n c e and s t u d y .  that permission for extensive copying o f  by h i s r e p r e s e n t a t i v e s .  for  1/  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  be allowed without my  ABSTRACT The basic p r o t e i n 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 techniques. The sperm of the seven anurans studied are d i v i d e d on cytochemi c a l c r i t e r i a i n t o three of the f i v e classes proposed by Bloch (1969). Rana p a l u s t r i s and R^ p r e t i o s a are of the Rana type. Xenopus l a e v i s , Hyla v e r s i c o l o r , and EN r e g i l l a are of the M y t i l u s 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 T e s t i s s p e c i f i c components are absent i n R^ p i p i e n s . In l a e v i s , three t e s t i s speci 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 c l o s e to salmon protamine i s found i n B^ americanus. The basic p r o t e i n changes during spermiogenesis i n the eastern red spotted newt, Diemictylus v i r i d e s c e n s , resemble the t r a n s i t i o n s described i n the s n a i l , H e l i x aspera, (Bloch and Hew 1960a), the squid, L o l i g o opalescens (Bloch 1962) and Pleurodeles w a l t i i ( P i c h e r a l 1970). I The e a r l y stages of spermiogenesis contain somatic type histones which i n l a t e r spermatids are replaced by the Mouse/grasshopper type of prot e i n . In t u r n , these proteins are replaced by the Salmon type of p r o t e i n i n the spermatozoa. E l e c t r o p h o r e s i s of t e s t i c u l a r histones of the newt supports the cytochemical events o u t l i n e d . Two t e s t i s s p e c i f i c bands are found. Spermiogenesis i n three c a r t i l a g i n o u s f i s h ( d o g f i s h , skate and r a t f i s h ) i s characterized by unusual changes i n basic p r o t e i n s . E a r l y 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 i n d i c a t e s that protamines are present i n elasmobranch t e s t e s . However, a Mouse/grasshopper type of p r o t e i n i s not revealed.  TABLE OF CONTENTS PAGE I II  GENERAL INTRODUCTION BASIC PROTEIN COMPOSITION OF ANURAN SPERM A. INTRODUCTION B. MATERIALS AND METHODS ..1.. CYTOCHEMISTRY  D.  8 8 8  a.  Frogs  8  b. c«  Feulgen r e a c t i o n A l k a l i n e f a s t green r e a c t i o n  9 9  d.  Eosin Y  11  e.  Deamination  11  f. g. h.  Acetylation A l k a l i n e f a s t green r e a c t i o n Feulgen-alkaline f a s t green procedure  11 12 12  i. Sakaguchi r e a c t i o n j . Dinitrofluorobenzene procedure k. Controls 2. BIOCHEMISTRY a. Frogs b. Preparation o f chromatin c. E x t r a c t i o n o f histones d. Polyacrylamide d i s c g e l e l e c t r o p h o r e s i s e. A l k a l i n e f a s t green s t a i n i n g of g e l s f Photographing and scanning gels RESULTS 1. CYTOCHEMISTRY 2. BIOCHEMISTRY 3. SALT CONCENTRATION AND THE ALKALINE FAST GREEN REACTION DISCUSSION e  C.  1 8  13 13 13 lk Ik 15 15 16 16 17 17 17 27 jk 37  TABLE OF CONTENTS PAGE III  CHANGES IN BASIC PROTEINS DURING SPERMIOGENESIS IN THE EASTERN RED SPOTTED NEWT, DIEMICTYLUS VIRIDESCENS A. INTRODUCTION B. METHODS AND MATERIALS 1. C YTOC HEMIS.TRY 2. BIOCHEMISTRY C.  RESULTS 1.  CYTO C HEMIS TRY  2. BIOCHEMISTRY D. DISCUSSION IV  V VI  42 42 42 42 42 43 43 50 53  CHANGES IN BASIC PROTEINS DURING SPERMIOGENESIS IN THREE CARTILAGINOUS FISH A. INTRODUCTION B. METHODS AND MATERIALS 1. CYTOCHEMISTRY 2. BIOCHEMISTRY C. RESULTS 1. CYTOCHEMISTRY  57 57 57 57 58 60 60  a. Elasmobranchii b. Holocephali 2. BIOCHEMISTRY D. DISCUSSION GENERAL DISCUSSION REFERENCES  60 68 75 81 87 92  LIST OF TABLES  Page  I.  Cytochemistry of anuran sperm n u c l e i  ..  II.  S a l t concentration and AFG r e a c t i o n . . . . . . . .  35  III.  Pretreatment and the AFG reaction  36  IV.  Staining with AFG made up i n 0.30 M NaCl  V.  Cytochemistry of newt spermiogenesis. . . . . . . .  k6  VI.  Cytochemistry of dogfish and skate spermiogenesis .  66  VII.  E f f e c t of h y d r o l y s i s conditions on AFG s t a i n i n g i n  .....  36  dogfish and skate spermiogenesis VIII.  Cytochemistry of r a t f i s h spermiogenesis  IX.  E f f e c t of hydrolysis conditions on AFG s t a i n i n g i n r a t f i s h spermiogenesis  X.  The v a r i e t y of sperm histones  20-21  67 .  73  86  LIST OF ILLUSTRATIONS FIGURES  PAGE  1-3  Sections of Rana p r e t i o s a t e s t i s  22  4—6  Sections of Hyla v e r s i c o l o r t e s t i s  23  7-9  Sections of Xenopus l a e v i s 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 w i t h a l k a l i n e f a s t gr.een, p i c r i c a c i d h y d r o l y s i s  26  15- 17  Densitometer t r a c i n g s of histones from anuran t e s t e s run on 15% polyacrylamide gels f o r 85 mins  30  18-28  Polyacrylamide g e l electrophoresis patterns o f histones from anurans  29-32  Stages of spermiogenesis spotted newt  33-34-  Consecutive s e c t i o n s o f cysts of stage 3 n u c l e i s t a i n e d with a l k a l i n e f a s t green, 5% t r i c h l o r o acetic acid hydrolysis  i n the eastern red  31-33 4-7  4-8  35  Sperm of Diemictylus v i r i d e s c e n s stained w i t h a l k a l i n e f a s t green v/ithout p r i o r h y d r o l y s i s of DNA 4-9  36-37  Densitometer t r a c i n g s of histones from the eastern red spotted newt run on 15% poly a c r y l amide gels f o r 70 minutes and 6 t a i n e d w i t h b u f f a l o black  51  38  Polyacrylamide g e l e l e c t r o p h o r e s i s patterns o f histones from the eastern red spotted newt  52  39  T e s t i c u l a r histones from the newt run w i t h t e s t i c u l a r histones from Xenopus l a e v i s and from Bufo americanus . 5  2  4-0  The zonation of the t e s t i s of Scylorhinus canicul u s as seen i n transverse s e c t i o n  62b  4-1-4-7  F o l l i c l e s containing stages of dogfish spermiogenesis  4-8  A l k a l i n e f a s t green 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 at 97 C.  64-  F o l l i c l e s c o n t a i n i n g l a t e stages o f skate spermiogenesis  65  4-9-51  63-64-  L I S T OF ILLUSTRATIONS  FIGURES 52 53-59 60-62  63-65  66-67  68  PAGE 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. F o l l i c l e s containing stages o f r a t f i s h genesis  65  spermio-  Densitometer t r a c i n g s o f skate histones run on "15% p o l y a c r y l a m i d e g e l s f o r ' 8 0 m i n u t e s a n d stained with buffalo black  71-72  77  Densitometer 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 m i n u t e s a n d stained with buffalo black  78  Densitometer 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 a n d d o g f i s h r u n o n 15% p o l y a c r y l a m i d e g e l s f o r 80 m i n s a n d 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 r u d e n u c l e a r p r e parations  79  Polyacrylamide g e l electrophoresis patterns o f h i s t o n e s from the skate  80  ACKNOWLEDGEMENTS  I would l i k e  t o a c k n o w l e d g e D r . H. E. K a s i n s k y  a n d a d v i c e , D r . C.V. ilities, for  f o r h i s support  Finnegan f o r use o f h i s l a b o r a t o r y f a c -  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 , a n d my m o t h e r  typing the thesis.  I would a l s o l i k e  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 T h i s r e s e a r c h was s u p p o r t e d  t o t h a n k J i m R e i d a n d Bob  fish.  b y NRC g r a n t #585'+'to D r . H. E .  K a s i n s k y a n d a n 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  While 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 o n e , 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.  Bloch  (1969) a p p r o a c h e d 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 t h e 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  searching  f o r possible c o r r e l a t i o n s with protein type.  approack, although  promising,  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 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 most sperm, b y 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 chemical  techniques  (Vendrely  of comparing c o n c l u s i o n s a cytochemical  approach.  This  bio-  a n d V e n d r e l y 1966), a n d b y t h e p r o b l e m  from biochemical Therefore,  work w i t h t h o s e drawn from  the basic protein composition  of  sperm, a s w e l l a s 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  spermiogenesis,  has  b e e n 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  cytochemical  and  biochemical The  techniques.  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  w h i c h h e t e r m e d p r o t a m i n e ( f r o m F e l i x I960).  From t h e i m m a t u r e  t e s t e s , he i s o l a t e d a p r o t e i n b u t h e was u n a b l e t o f i n d Later Kossel and  base  protamines.  d e m o n s t r a t e d t h a t p r o t a m i n e s w e r e made u p o f a m i n o  t h e r e f o r e were p r o t e i n s .  p r o t e i n from t h e u n r i p e  acids  K o s s e l a l s o showed t h a t t h e b a s i c  t e s t e s belonged to the c l a s s o f b a s i c  t e i n s t o which K o s s e l had given  t h e name h i s t o n e  pro-  ( f r o m L u c k 1964-).  H o w e v e r , t h e d i s t i n c t i o n b e t w e e n t h e h i s t o n e s a n d t h e p r o t a m i n e s was not  always apparent. 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  b y M u r r a y (1964); " h i s t o n e s a s s o c i a t e d w i t h DNA."  a r e b a s i c p r o t e i n s t h a t a t some t i m e a r e  This d e f i n i t i o n i n c l u d e s protamines 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 b e t w e e n t h e t w o . respects for  i s one g i v e n  Since  sperm n u c l e i a r e u n i q u e , t h e t e r m p r o t a m i n e  i n many  m i g h t be u s e d  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 s p e r m ( 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 connection  with the basic proteins found i n f i s h  - 2 sperm. The most e x p l i c i t term f o r the basic proteins a s s o c i a t e d with sperm DNA i s sperm histone. (Bloch 1 9 6 9 ) » and, t h e r e f o r e , the term w i l l be used i n t h i s study. Protamines w i l l r e f e r t o 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 e a r l y work on sperm histones was l a r g e l y confined to t e l e o s t f i s h . Kossel and h i s co-workers ( 1 9 2 8 ) studied protamines from many species o f f i s h and the work was.continued by F e l i x and h i s students ( i 9 6 0 ) . From these s t u d i e s f i s h sperm histones were shown to contain very few kinds of amino a c i d s , to contain about two basic amino acids to every non-basic amino a c i d , and to have a r g i n i n e cons t i t u t e about two-thirds of a l l amino a c i d s . Based on these f a c t s , the sperm histones were classed by Kossel as e i t h e r monoprotamines, diprotamines, or triprotamines. Monoprotamines contained only one basic amino a c i d : a r g i n i n e . The diprotamines contained a r g i n i n e and e i t h e r l y s i n e or h i s t i d i n e , w h i l e triprotamines contained a l l three basic amino a c i d s . Recent work on the sperm histones of f i s h has been confined l a r g e l y to a few l a b o r a t o r i e s . Ando and Swada ( I 9 6 I ) separated the protamine from P a c i f i c herrings i n t o two main f r a c t i o n s , Y and Z, and then f u r t h e r resolved Y i n t o Y l and Y2. The complete amino a c i d sequence o f these three f r a c t i o n s was determined (Ando and Suzuki. 1 9 6 ? ) and with recent work by Ando and Watanabe ( 1 9 6 9 ) the sequences o f 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 a l a n i n e , g l y c i n e , i s o l e u c i n e , p r o l i n e , s e r i n e , threonine o r v a l i n e and R represents a r g i n i n e . L i n g , T r e v i t h i c k , 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 o f e a r l y sperma t i d s and r a p i d l y t r a n s f e r r e d to the nucleus. By use o f various i n h i b i t o r s , these workers were able to show that protamine was synthes i z e d on ribosomes l i k e other proteins and that the protamine mRNA was very s t a b l e . Ingles and Dixon ( 1 9 6 ? ) reported extensive phosphoryl 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 v i v o . i s unknown.  The reason f o r t h i s  phosphorylation  Work on the basic proteins found i n the sperm of other organisms proceeded much slower than the work with f i s h sperm. Perhaps the echinoderms received the most a t t e n t i o n . The work o f Kossel (1928), Hamer (1955)* and Vendrely and Vendrely (1966) i n d i c a t e d 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 P i s (1951) studied the basic proteins from rooster sperm and found them r i c h i n a r g i n i n e . Since h i s t i d i n e was present but l y s i n e absent, these proteins would be classed as diprotamines. Information on the basic p r o t e i n composition i n the sperm o f other organisms l i k e l y proceeded slowly f o r two reasons. Sperm histones are most e a s i l y studied i f sperm may be obtained f r e e of other c e l l types. "The best s t a r t i n g m a t e r i a l f o r the preparation of protamines i s f r e s h l y drawn m i l t from f i s h that a r e ready to spawn, since i t contains only one type of c e l l s , the spermatozoa" ( F e l i x I960). With echinoderms, sperm also could be c o l l e c t e d 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 l a r g e q u a n t i t i e s of sperm i n order to do biochemical a n a l y s i s . 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 d i s t i n g u i s h between histones and protamines was a major advance i n attempts to study the p r o t e i n types i n a wide v a r i e t y o f organisms. A l f e r t and Geschwind (1953) used an anionic dye, f a s t green, a t 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 p o i n t s . 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 f o r histones and protamines since the s e l e c t i v e s t a i n i n g o f the nucleus i s u s u a l l y achieved and the removal of DNA i s required f o r s t a i n i n g . Since DNA must be removed f o r s t a i n i n g to occur, the a c t u a l , 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 s t a t i c a l l y w i t h the phosphate  g r o u p s o f DNA.  The r e m o v a l o f  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  from h i s t o n e s .  Alfert  did not. h o t TCA  DNA  protamines  (1956) found t h a t w h i l e the e a r l y s t a g e s o f  spermiogenesis i n Chinook salmon s t a i n e d w i t h e x t r a c t i o n o f DNA  electro-  f a s t green a f t e r  w i t h hot t r i c h l o r o a c e t i c a c i d , the mature  the  sperm  S i n c e model e x p e r i m e n t s on f i l t e r p a p e r had i n d i c a t e d 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  that  Gesch-  w i n d 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  prota-  mines .  and  A subsequent  M i t c h e l l 1957)  biochemical study (Callanan, C a r r o l l ,  c o n f i r m e d t h a t protamines were i n d e e d p r e s e n t i n the  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 c a n be e x t r a c t e d f r o m  fixed sections while histones  cannot.  The u s e 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 s i t i o n o f s p e r m was  f u r t h e r r e f i n e d b y B l o c h a n d Hew  w e r e a b l e t o s t a i n p r o t a m i n e s b y r e m o v i n g DNA ric  r e t a i n s protamines i n the s e c t i o n s . ific  compo-  (1960a).  with picric  They  acid.  a c i d i s a strong p r e c i p i t a n t of protamines ( F e l i x I960)  l y s i n e r e s i d u e s was  formalin  Pic-  and  Deamination or a c e t y l a t i o n  of  a l s o c a r r i e d out i n order to "permit the spec-  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  to l y s i n e "  (Bloch 1966).  Under these c o n d i t i o n s protamines d i d  s t a i n while the b a s i c p r o t e i n s found i n the somatic 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 h a v e b e e n t e r m e d 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 t o d i s t i n g u i s h them f r o m t h e a r g i n i n e - r i c h h i s t o n e s somatic c e l l s Through  (Pipkin  1969).  t h e u s e 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 w e r e made on a w i d e r a n g e o f o r g a n i s m s . Hew  of  (1960a) found t h a t t h e mature  contained protamines.  B l o c h and  sperm o f t h e s n a i l , H e l i x a s p e r s a ,  However, i n t h e t r a n s i t i o n f r o m t h e  normal  h i s t o n e complement t o t h e 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 w h e r e t h e y w e r e n o t e x t r a c t a b l e w i t h h o t 5% TCA tained proteins very rich i n arginine.  but y e t con-  The p r o t e i n s f o r t h i s  stage  have been termed the " s t a b l e p r o t a m i n e s " B l o c h (1969).  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  (Bloch  common t o s q u i d  1962)  - 5 and  PIeurodeles  waltii  ( P i c h e r a l 1970).  I n other  organisms the t r a n -  s i t i o n proceeded o n l y as f a r a s t h e "stable protamine." case i nD r o s o p h i l a  T h i s was t h e  m e l a n o g a s t e r ( D a s , G a y , a n d K a u f m a n n 1964), t h e  g r a s s h o p p e r ( B l o c h a n d B r a c k 1964) t h e c o c c i d ( B e r l o w i t z 1965)» t h e cricket  ( K a y e a n d M c M a s t e r - K a y e 1966), t h e mouse ( M o n e s i 1964), a n d  t h e r a t (Vaughn 1966).  In other  organisms the sperm d i d n o t s t a i n a t  a l l a f t e r deamination o r a c e t y l a t i o n .  T h i s was t h e c a s e w i t h  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 .  Kana  Perhaps 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 s p e r m o f many c r a b s h a d n o c y t o chemically demonstrable b a s i c p r o t e i n s L a n g r e t h 1969« V a u g h n 1968). some b i o c h e m i c a l 1969  t  ( B l o c h 1966, C h e v a i l l i e r 1967*  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 b y  evidence (Vaughn, C h a i t o f f , D e l e e n , a n d G a r l a n d  Vaughn a n d H i n s c h 1970). Bloch  (1969) a t t e m p t e d t o b r i n g o r d e r  to this array o f infor-  m a t i o n b y 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 nature and t h e  disposition of their histones."  Since  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 ,  t h e c l a s s e s were d e s i g n a t e d  species order  that served  o f decreasing  grasshopper type,  the c h a r a c t e r i z a t i o n o f these by  a s t y p i c a l examples o f t h e p r o t e i n t y p e . basicity  e  t h e s e a r e t h e Salmon t y p e ,  In  t h e Mouse/  t h e M y t i l u s t y p e , t h e Kana type and t h e Crab  type.  Salmon and o r g a n i s m s 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 .  Arginine constitutes the sole basic  a c i d a n d makes up t o t w o - t h i r d s chemically, hot  o f t h e amino a c i d r e s i d u e s .  these proteins a r e c h a r a c t e r i z e d by being  amino  Cyto-  extracted  with  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 b y p i c r i c a c i d h y d r o -  l y s i s , and give 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 Sakaguchi reagent f o r arginine„ The very  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  r i c h i n a r g i n i n e b u t a r e much more c o m p l e x t h a n t h e m o n o p r o t -  amines."  Although not e x p l i c i t i n Bloch's a r t i c l e ,  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 the  "stable protamines."  critera.  t h i s type i s  These sperm  contain  T h e s e p r o t e i n s a r e n o t e x t r a c t e d b y 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 n o t a f f e c t e d b y 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 b r i g h t red r e a c t i o n these n u c l e i give a f t e r the Sakaguchi t e s t f o r a r g i n i n e . Sperm of the M y t i l u s type contain proteins that were c a l l e d diprotamines or triprotamines by K o s s e l . These histones may be c a l l e d intermediate "because they f a l l between the true histones and the monoprotamines*' (Bloch 1969). Cy to chemically, Bloch d i d not define t h i s type. However, he gave many examples o f 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 e d u l i s , d i d s t a i n with f a s t green a f t e r t r i c h l o r o a c e t i c a c i d hydrol y s i s and continued to s t a i n despite deamination of l y s i n e residues. However, t h i s s t a i n i n g was not as b r i g h t as that obtained w i t h 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 s t a i n a f t e r the amino groups have been blocked by deamination or a c e t y l a t i o n . This i s how somatic histones behave. The amino a c i d composition o f 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 p r o t e i n s . Perhaps p r o t e i n s of low b a s i c i t y s i m i l a r to the "cleavage h i s t o n e s " i n the developing s n a i l embryo (Bloch and Hew 1960b) are present. The most commonly held view on the f u n c t i o n o f the b a s i c prot e i n s i n the sperm nucleus i s that they are somehow i n v o l v e d 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 o f Stedman and Stedman (1950) that the f u n c t i o n o f the basic proteins i n the c e l l nucleus " i s to act as gene r e p r e s s o r s . " Although considerable evidence has accumulated (Georgiev 1969) that histones do i n h i b i t DNA-dependent RNA synthesis and thus gene a c t i o n , the almost t o t a l l a c k of histone t i s s u e s p e c i f i c i t y argues against histones being s p e c i f i c gene r e g u l a t o r s . The discovery that the  - 7arginine-rich histone  (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 a n d p e a  (De L a n g e 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 b y h i s t o n e s . f o u n d i n t h e sperm n u c l e u s  However, t h e b a s i c  proteins  are o f considerable interest since  they  a r e one o f t h e 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 , a s 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  species s p e c i f i c i t y .  Thus t h e 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 elucidating the function of histones i n general. Another popular  hypothesis  the proper "packaging" condensation, in  the nucleus  i s that protamines a r e necessary f o r  o f t h e s p e r m DNA.  Thus t h e p a t t e r n o f n u c l e a r  t h e s h a p e o f t h e n u c l e u s , a n d t h e amount o f DNA are of interest  i n order  to test this  Sperm h i s t o n e s may h a v e a " p r o t e c t i v e " r o l e  present  hypothesis.  ( B l o c h 1969).  ulous as t h i s term 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  Neb-  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 cor-  r e l a t i o n m i g h t e x i s t between s h o r t o r l o n g - l i v e d sperm a n d t h e h i s t o n e type. O l i n s , O l i n s , and Yon H i p p e l  (1968) h a v e s u g g e s t e d t h a t t h e  f u n c t i o n o f the protamines i s to erase the c e l l i n order  the developmental h i s t o r y o f  to restore totipotency.  Another p o s s i b i l i t y i s t h a t t h e type r e l a t e d to the beginning cleavage  pattern  o f sperm h i s t o n e p r e s e n t i s  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  ( B l o c h 1969).  On t h e o t h e r h a n d , t h e s p e r m h i s t o n e s m i g h t h a v e no f u n c t i o n a n d 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  to 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*  -  II.  8  -  BASIC PROTEIN COMPOSITION OF ANURAN SPERM A.  INTRODUCTION  W h i l e a R a n a c l a s s h a s b e e n d e s i g n a t e d , w o r k on a n u r a n s p e r m 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 to unpublished  cytochemical  d a t a , B l o c h (1969) c l a s s i f i e d t h e s p e r m h i s t o n e s o f B u f o and Xenopus l a e v i s as t h e M y t i l u s t y p e .  vulgaris  Only the sperm h i s t o n e s o f  R a n a p i p i e n s h a v e b e e n 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;  1962)  a n d c y t o c h e m i c a l l y ( B l o c h 1962;  t h e s e sperm were f o u n d Although l i t t l e tones, Bloch  Z i r k i n 1970).  The  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  Bloch  histones of cells.  i n f o r m a t i o n i s a v a i l a b l e on a n u r a n s p e r m  his-  (1969) u s e d e x a m p l e s f r o m a n u r a n s t o t e s t many h y p o -  t h e s e s on s p e r m h i s t o n e f u n c t i o n .  In order to properly t e s t  these  hypotheses i n the f u t u r e , the v a r i e t y o f anuran sperm h i s t o n e s been f u r t h e r  has  examined.  B.  MATERIAL AND  METHODS  1.  CYTOCHEMISTRY  a.  Frogs  S e x u a l l y m a t u r e Rana p a l u s t r i s a n d H y l a v e r s i c o l o r w e r e 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.  s a c h u s e t t s ) and Co. from  ( S o u t h a m p t o n , Mas-  s e x u a l l y mature Bufo americanus from the  (Oshkosh, W i s c o n s i n )  i n May  1971  •  Steinhilber  Xenopus l a e v i s were  purchased  t h e S o u t h A f r i c a n S n a k e F a r m (Cape P r o v i n c e , S o u t h A f r i c a ) i n  October,  1970,  and w e r e 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 .  p r e t i o s a , H y l a r e g i l l a , and  Rana  Bufo boreas were c o l l e c t e d i n t h e  F r a s e r V a l l e y , B r i t i s h Columbia,  lower  during t h e i r breeding seasons i n  1971. The  a n i m a l s vrere e t h e r i z e d a n d  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,  absolute ethanol-glacial acetic acid f o r 4—6  the t e s t e s q u i c k l y removed  h a t room t e m p e r a t u r e  (3:l)«  p a g e 601)  Formalin.fixation  f o l l o w e d b y a 14—17  and or i n was  h wash i n r u n n i n g  tap  water*  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,  a n d embedded i n P a r a p l a s t o r P a r a p l a s t  (Fisher S c i e n t i f i c Co.).  Fixation i n absolute ethanol-glacial  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 b y 2 r i n s e s i n absolute ethanol. ded i n P a r a p l a s t .  Plus  acetic  (1 h e a c h )  T i s s u e s w e r e t h e n c l e a r e d i n b e n z e n e a n d embed-  S e c t i o n s (6-10u)were c u t o n a S p e n c e r  "820" m i c r o -  tome. With the exception 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 t h e 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 breeding season ing  ( s e e W r i g h t a n d W r i g h t 19^9»  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 breed-  F o r X^ l a e v i s , t e s t e s  w e r e f i x e d 2k h a f t e r t h e f r o g s h a d 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 gonadotrophin.  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  X. l a e v i s m a l e s  ( G u r d o n 196?)•  all  fertile  Thus, mature sperm were p r e s e n t i n  frogs. b.  Feulgen  reaction  Dexoyribonucleic acid action.  (DNA) was d e m o n s t r a t e d b y t h e F e u l g e n r e . o  S e c t i o n s w e r e 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 6 0 C.  A f t e r two r i n s e s  (5 m i n u t e s e a c h ) i n d i s t i l l e d H 0, 2  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 . many s h o r t r i n s e s i n d i s t i l l e d bisulfite again.  s e c t i o n s were  T h i s was f o l l o w e d b y  E^O, a f i v e m i n u t e r i n s e i n a c i d -  w a s h , a n d 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  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.  E\>0  DNA was  a l s o d e m o n s t r a t e d b y t h e 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 a n d Godman (1935) a n d b y 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 Davie6 1971)» c.  (Horobin and K e v i l l -  The t h r e e t e c h n i q u e s g a v e c o m p a r a b l e  A l k a l i n e f a s t green  results.  reaction  The p r e s e n c e o f h i s t o n e s was d e m o n s t r a t e d b y t h e A l f e r t a n d 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 r e m o v e d  s e c t i o n s w i t h h o t (85-89°C) 5% t r i c h l o r o a c e t i c a c i d s t a i n i n g f o r 30 m i n u t e s w i t h 0.1% pH' 8.1-8.3.  (TCA), b e f o r e  (w/v) a l k a l i n e f a s t g r e e n  The c o r r e c t pH was o b t a i n e d b y t i t r a t i n g  from  (AFG) a t  w i t h 0.1N NaOH.  The AFG s o l u t i o n was u s e d 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 .  Hydrolysis  w i t h 5% TCA was c a r r i e d o u t w i t h f r e s h r e a g e n t o n e a c h o c c a s i o n .  Re-  p e a t e d u s e o f t h e same TCA s o l u t i o n r e s u l t e d i n t h e i n c o m p l e t e r e -  - 10 m o v a l o f DNA w h i c h i n t u r n l e a d o f Rj_ p r e t i o s a troyed  t o reduced s t a i n i n g .  S i n c e t h e sperm  B. r e g i l l a , a n d H«_ v e r s i c o l o r a p p e a r e d t o b e d e s -  t  a t 9k-100°C  a n d t h e i r m o r p h o l o g y a l t e r e d b e t w e e n 90-94-°C,  DNA was r e m o v e d 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 sperm n u c l e i i n t a c t . p r o c e d u r e f o r DNA.  these  The r e m o v a l o f DNA was c h e c k e d b y t h e F e u l g e n After hydrolysis  the sections  were immersed i n  t h r e e c h a n g e s o f 70% e t h a n o l (10 m i n u t e s e a c h ) t o r e m o v e TCA. i n g was " f o l l o w e d and  mounting.  chloroacetic  b y a f i v e m i n u t e r i n s e i n d i s t i l l e d H^O,  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 a c i d f o r J h a t 6o°C ( B l o c h  procedure followed. ever, with  protamine containing with  dehydration,  removed w i t h  IN t r i -  1966) a n d t h e same s t a i n i n g  The two m e t h o d s g a v e i d e n t i c a l r e s u l t s .  t h i s method, t h e d i f f e r e n c e  How-  between p r o t a m i n e a n d non-  cells i s less distinct.  Treatment o f s e c t i o n s  h o t 5% TCA c a u s e s t h e c o m p l e t e r e m o v a l o f p r o t a m i n e .  Being 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 r e m o v i n g m o s t amine, causes t h e r e t e n t i o n  Stain-  o f a s m a l l amount o f p r o t a m i n e .  protThe  I N 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 m o r p h o l o g y , p a r t i c u l a r l y t h e s p e r m 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 a n d 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 m e n t , DNA was a l s o  removed by t r e a t m e n t w i t h  f o r 6 h a t 60°C ( B l o c h with  a n d Hew I960).  a l k a l i n e f a s t green.  b e f o r e use.  As w i t h  r e s u l t s i f reused.  saturated  S e c t i o n s were t h e n  t h e TCA m e t h o d , t h e h y d r o l y s i s  acid  stained  The p i c r i c a c i d s o l u t i o n was made  just  r e a g e n t gave p o o r  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 m e a s u r e s  w e r e t a k e n t o remove p i c r i c a c i d f r o m t h e s e c t i o n s Two m e t h o d s w e r e t r i e d . was  picric  treat-  As recoamended b y P i p k i n  removed b y r i n s i n g s e c t i o n s  briefly  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  before  staining.  (1968), p i c r i c  acid  (5 s e c o n d s ) i n a c e t o n e c o n -  acid  (v/v).  However, t h i s p r o -  c e d u r e r e s u l t e d i n r e d u c e d s t a i n i n g w h i c h i s p e r h a p s due t o t h e e x traction of basic  proteins.  A l s o , many s p e r m 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 step. moved f r o m s e c t i o n s distilled H0 2  material  However, p i c r i c a c i d c o u l d  without these complications by long  (3-2*f h ) .  rinses i n  The AFG r e a c t i o n was p e r f o r m e d o n l y  fixed i n formalin.  be r e with  - 11 d.  Eosin Y  E o s i n Y was u s 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 1960a) t o c o r r o b o r a t e t h e r i n s i n g a f t e r the (w/v)  eosin  sections  AFG s t a i n i n g .  were s t a i n e d  a t pH 8.1-8.3 w i t h  material e. To  the  w/v)  2  was u s e d f o r t h i s  fixed  procedure.  determine 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 , (Bloch  a n d Hew 1960a) w a s c a r r i e d  a n d p r i o r t o s t a i n i n g w i t h AFG  .  out  Two c h a n g e s o f  a c i d , prepared j u s t before use by combining equal volumes o f t r i c h l o r o a c e t i c a c i d a n d 10% (w/v) The s t o c k s o l u t i o n s  gave e r r a t i c r e s u l t s . This consisted  sodium n i t r i t e ,  i n c o m p l e t e i n the  o f t r i c h l o r o a c e t i c a c i d and  The p r o c e d u r e o f P i p k i n  acid solution.  i n h i b i t the f.  Old  solutions  (1968) w a s a l s o  tried.  control  However, d e a m i n a t i o n was j u d g e d  Re. p i p i e n s  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 due t o the  were used  o f t h r e e s u c c e s s i v e 15 m i n u t e c h a n g e s a t 4 C i n f r e s h -  l y prepared nitrous  testis.  gave i n c o n s i s t e n t  incomplete removal o f p i c r i c  deamination  The d e a m i n a t i o n results.  proThis  a c i d , which appeared t o  reaction.  Acetylation  Also,  t o d e t e r m i n e w h e t h e r 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  present, acetylation o f lysine residues performed 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 eosin  speci-  Only formalin  s o d i u m n i t r i t e w e r e n e v e r k e p t f o r more t h a n o n e week.  or  The  Deamination  f o r 15 m i n u t e s e a c h .  was  0.1%  0.01M t r i s - H C l b u f f e r .  d i s t i l l e d H 0 was b r o u g h t t o pH 8.3.  a f t e r TCA h y d r o l y s i s (10%  with  I n some c a s e s , i n o r d e r t o i m p r o v e  deamination o f l y s i n e residues nitrous  were s t a i n e d  the  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 , a n d mounted. ficity,  a n d Hew  The s l i d e s , f o l l o w i n g  picric acid hydrolysis,  Y buffered  (Bloch  Y.  After hydrolysis,  the  (Bloch  a n d Hew 1960a) w a s  a n d b e f o r e s t a i n i n g w i t h AFG  e l i d e s were r i n s e d  in  three  c h a n g e s 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 , a n d transferred  to a solution containing  pure a c e t i c anhydride. i n the  glacial acetic  Time a n d t e m p e r a t u r e s w e r e v a r i e d  find acetylation conditions S l i d e s were l e f t  1% ( v / v )  w h i c h gave the  most c o n s i s t e n t  acid i n  i n order t o results.  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, a n d  - 12 2k h at 60 C a n d at room t e m p e r a t u r e . a t u r e f o r 2k h was f o u n d t o b e b e s t .  A c e t y l a t i o n a t room  temper-  However, a c e t y l a t i o n f o r 1 h  at 60°C g a v e a d e q u a t e 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 r e m o v e d 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 . t e m p e r a t u r e f o r 2k h was u s e d f i r s t  S i n c e a c e t y l a t i o n a t room  and d i d g i v e c o n s i s t e n t  results,  t h i s p r o c e d u r e was a d o p t e d 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 was u s e d 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 r e m o v a l o f DNA.  staining Sections  w e r e b r o u g h t t o w a t e r , s t a i n e d f o r 30 m i n u t e s i n A F G , r i n s e d i n d i s tilled  H^O f o r 5 m i n u t e s , d e h y d r a t e d , a n d m o u n t e d .  Initially,  this  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 at  p H S . l a s recommended  f a s t green s t a i n i n g . 0.01M  buffer  b y C h a y e n e t a l . (1969) f o r g e n e r a l a l k a l i n e  T h i s method gave u n u s u a l r e s u l t s .  Therefore,  t r i s - H C l b u f f e r was t r i e d ; y e t t h e same r e s u l t s w e r e a c h i e v e d .  To d e t e r m i n e w h e t h e r t h e s e o b s e r v a t i o n s w e r e 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 w e r e s t a i n e d i n AFG made u p i n i n c r e a s i n g l y h i g h e r concentrations o f NaCl.  A r a n g e f r o m no N a C l t o Z'.kM N a C l was t r i e d .  F r o m t h e s e e x p e r i m e n t s ^ t h e 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 to  pH 8.1-8.3 w i t h 0 . 1 N NaOH was a d o p t e d .  For this test, material  f i x e d i n 10% NBF a n d e t h a n o l - g l a c i a l a c e t i c a c i d h.  (3:1) was u s e d .  Feulgen - a l k a l i n e f a s t green procedure  The m e t h o d o f Vaughn  (I966) was f o l l o w e d t o d e m o n s t r a t e n o n -  DNA-associated basic p r o t e i n s .  S l i d e s were brought t o w a t e r and  t h e n h y d r o l y z e d f o r 25 m i n u t e s 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 u p a c c o r d i n g to  P i p k i n 1968) f o r k5 m i n u t e s a t room t e m p e r a t u r e f o l l o w e d .  three 5 minute r i n s e s i n s u l f i t e b l e a c h (prepared w i t h ic  trichloroacet-  a c i d ) a n d t h e n t h r e e 10 m i n u t e r i n s e s i n 70% e t h a n o l w e r e  out.  Next, carried  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  n o r m a 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 , a n d mounted. only.  T h i s p r o c e d u r e was c a r r i e d o u t o n f o r m a l i n f i x e d  material  - 13 1.  Sakaguchi r e a c t i o n  A r g i n i n e was d e m o n s t r a t e d b y t h e S a k a g u c h i r e a c t i o n i f i e d by Deitch  (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) a n d 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 w e r e made u p i n a d v a n c e  5% N a O C l . jar.  bleach"  (v/v) was p r e p a r e d .  S l i d e s were b r o u g h t t o w a t e r a n d p l a c e d  Instead o f  i n a n empty C o p l i n  I m m e d i a t e l y t h e 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  and p o u r e d on t h e s l i d e s . 10 m i n u t e s .  t e r t i a r y butanol  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  S l i d e s were t h e n t r a n s f e r r e d through 3 changes o f 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 .  c l e a r e d i n two c h a n g e s tri-N-butylamine butylamine.  j.  The f i r s t S l i d e s were  (30 s e e s e a c h ) o f x y l e n e c o n t a i n i n g 5% ( v / v )  a n d m o u n t e d i n P e r m o u n t c o n t a i n i n g 10% ( v / v ) t r i - N -  T h e S a k a g u c h i r e a c t i o n was p e r f o r m e d o n m a t e r i a l  formalin as w e l l as material f i x e d i n absolute  acetic acid  barium  dichloronaphthol)  c h a n g e was f o r 5 s e e s a n d t h e n e x t two f o r 30 s e e s e a c h .  in  Just  ( S a f e w a y L t d . ) was u s e d a s a s o u r c e o f  hydroxide, 1 p a r t h y p o - c h l o r i t e , and then 1 p a r t for  butanol  a n d used o v e r a p e r i o d o f a month.  b e f o r e u s e , 1% s o d i u m h y p o c h l o r i t e " c l o r o x , " " w h i t e magic  a s mod-  fixed  ethanol-glacia3L  (3si).  Dinitrofluorobenzene  procedure  P r o t e i n - b o u n d l y s i n e was d e m o n s t r a t e d b y t h e d i n i t r o f l u o r o b e n zene in  (DNFB) p r o c e d u r e a s o u t l i n e d b y P i p k i n (1968).  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  Material  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 .  was  removed b y i m m e r s i n g 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  I n some c a s e s ,  This step insured the r e t e n t i o n o f a l l basic proteins. 10 m i n u t e c h a n g e s solution  r i n s e d i n s e v e r a l changes  k.  minutes.  After  (0.60ml DNFB i n 26 m l o f e t h a n o l , 4.0ml I M s o d i u m  three  bicarbon-  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 .  r e a g e n t was u s e d i m m e d i a t e l y o n p r e p a r a t i o n .  xylene,  formaldehyde  o f 70% e t h a n o l , t h e s e c t i o n s w e r e s t a i n e d i n DNFB  a t e , a n d 20.ml o f d i s t i l l e d The  fixed  The s e c t i o n s were  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  a n d mounted. Controls  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 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  very l i g h t  or  0.1N  absent.  I n a l l s t a i n i n g p r o c e d u r e s , s p e r m f r o m a number o f d i f f e r e n t s p e c i e s w e r e s t a i n e d a t t h e same t i m e 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 . number o f o r g a n i s m s s p e r m was  i n which  the basic p r o t e i n composition of  known w e r e u s e d a s c o n t r o l s f o r m o s t r e a c t i o n s .  l o w i n g organisms  were u s e d :  contain protamines  A  the  The  fol-  S a l m o g a i r d n e r i i , whose s p e r r a c e l l 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  h i s t o n e s i n the sperm s i m i l a r t o those o f s o m a t i c c e l l s  contains  (Block  Z i r k i n 1970); a n d C a r a s s i u s a u r a t u s , whose s p e r m a r e s i m i l a r o f R«_ p i p i e n s ( Z i r k i n 1971)»  1962;  to  those  S i n c e the s t a i n i n g p a t t e r n observed  w i t h B ^ b o r e a s a n d B^ a m e r i c a n u s  appeared  unusual, a l k a l i n e fast  green  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 repeated w i t h material f i x e d i n absolute e t h a n o l - g l a c i a l a c e t i c A l s o , AFG  s t a i n i n g was  w e r e t a k e n on a Z e i s s  2.  BIOCHEMISTRY  a.  Frogs  Re  p i p i e n s w e r e o b t a i n e d f r o m E. G.  W i s c o n s i n ) i n December 1971 same company i n May ized. until  The  acid.  t r i e d f o r different lengths of time, 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 A l l photographs  was  1972.  temperatures.  photomicroscope.  S t e i n h i l b e r & Co.  w h i l e B_j_ a m e r i c a n u s  (Oshkosh,  were o b t a i n e d from  the  W i t h R^ p i p i e n s , t h e a n i m a l s w e r e e t h e r -  t e s t e s a n d l i v e r s w e r e q u i c k l y r e m o v e d a n d s t o r e d a t -20°C  the time o f use.  I n D e c e m b e r , t h e m o s t common s t a g e o f  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 m a t u r e s p e r m (Rugh 1959)• t o a d s , B_._ a m e r i c a n u s , w e r e p i t h e d ; t h e t e s t e s a n d l i v e r s w e r e removed and used a l m o s t i m m e d i a t e l y .  Since  americanus  spermatoThe quickly  breeds i n  a n d J u n e ( W r i g h t a n d W r i g h t 19^9), t h e t e s t i s a t t h i s t i m e o f t h e s h o u l d c o n t a i n many m a t u r e s p e r m .  A l i g h t microscope  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 S e x u a l l y mature  inspection  cell  May  year of  type present.  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 from  the  S o u t h A f r i c a n S n a k e Farm and w e r e 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  years.  During t h i s time, they p e r i o d i c a l l y received  - 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  196?).  o b t a i n i n g f e r t i l e X^ l a e v i s m a l e s ( G u r d o n were p i t h e d .  The  The X ^ l a e v i s m a l e s  t e s t e s and l i v e r s were q u i c k l y removed and u s e d a l -  most i m m e d i a t e l y . b.  Preparation of chromatin  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  (1966).  u r e o f Marushige and Bonner o r on i c e .  i s o l a t e d by the p r o c e d -  T e s t e s and l i v e r s w e r e h o m o g e n i s e d i n a S o r v a l l Omni M i x e r  with saline-EDTA  (0.075  8.0)  M N a C l , 0.024- M EDTA, pH  a t a speed  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 w a s h e d c l o t h , a n d c e n t r i f u g e d a t 1,500 e r a l d r o p s o f 2p e l l e t was  o c t a n o l was  g f o r 15  minutes.  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  C h r o m a t i n was  sedimented a t  w i t h t h e same b u f f e r .  i n 0.01  10,000g  c h r o m a t i n was  o f t h e t u b e was  for  15  M Tris buffer  25ml  of  C h r o m a t i n was  1.7  homogen-  (pH  8.0).  m i n u t e s and washed once  M sucrose.  mixed g e n t l y and c e n t r i f u g e d a t  further  purified  homogenized i n  w i t h a Dounce h o m o g e n i z e r .  layered over  (on V o r t e x -  a d d i t i o n a l wash-  I n a f e w c a s e s , c h r o m a t i n was  by c e n t r i f u g a t i o n through s u c r o s e . M T r i s b u f f e r (pH 8.0)  The n u c l e a r  The n u c l e a r p e l l e t was  i z e d w i t h a Dounce h a n d h o m o g e n i z e r  cheese-  S i n c e the n u c l e a r p e l l e t s  from 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, i n g s were employed f o r t h i s t i s s u e .  elopment  (1969)  0.01  S i x ml o f  this  The  one-third  22,000  upper rpm  for  t h e SW-27 r o t o r o f a S p i n d o 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 . i c a t i o n t h r o u g h s u c r o s e was  set-  I n seme c a s e s , s e v -  added to prevent f r o t h i n g .  G e n i e 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 .  et a l .  4-°C  A l l s t e p s were p e r f o r m e d a t  o m i t t e d i n most c a s e s s i n c e  2  h in Purif-  Marushige  found t h a t w i t h t r o u t t e s t e s i n the l a t e s t a g e s o f dev-  t h e p u r i t y o f t h e 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  this step. c.  Extraction of histones  C h r o m a t i n was  h o m o g e n i z e d i n 0.2  M ^SO^  w i t h a Dounce homogen-  i z e r a n d s t i r r e d i n t h e same s o l u t i o n f o r 1 h a t 4-°C. lowed by c e n t r i f u g a t i o n a t n a t a n t , 3-4was  10,000  v o l u m e s o f c o l d 100%  g for  20  minutes.  e t h a n o l was  T h i s was To  the  fol-  super-  added and t h e m i x t u r e  p l a c e d i n t h e f r e e z e r (-20°C) w h e r e 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 w i t h i n 2k h.  The p r e c i p i t a t e was  r e c o v e r e d by c e n t r i f u g a t i o n , washed  once w i t h e t h a n o l , and d r i e d i n vacuo. p e l l e t was d.  e x t r a c t e d a second  In most c a s e s , the  time by the above  chromatin  procedure.  Polyacrylamide disc g e l e l e c t r o p h o r e s i s  H i s t o n e s were f r a c t i o n a t e d on 15% p o l y a c r y l a m i d e d i s c g e l s by the method o f Bonner ejt  a l . (1968).  i n 8 M urea a t 1 mg/ml.  P r o t e i n samples were d i s s o l v e d  T h i s c o n c e n t r a t i o n was  where o n l y a s m a l l amount o f p r o t e i n was (DTT) was  added (0.05  approximated  available.  i n cases  Dithiothreitol  ml o f 1 M DTT/l) to the samples which were then  i n c u b a t e d a t 57° C f o r 50 minutes. ml o f sample and run a t 4—5  Each g e l was  milliamperes.  l o a d e d w i t h 0.02-0.06  While g e l s from 5.0  - 7.4-  cm  i n l e n g t h were t r i e d , the l o n g g e l s (7.4- cm) were most s u i t a b l e s i n c e these g e l s c o u l d be run f o r 80-90 minutes, a t i o n of the h i s t o n e s , and s t i l l bands.  which a l l o w e d f o r the s e p a r -  r e t a i n the f a s t moving  Gels were s t a i n e d i n 1% B u f f a l o B l a c k NBR  protamine  i n 7% a c e t i c a c i d f o r  a t l e a s t two hours and d e s t a i n e d i n 7% a c e t i c a c i d f o r a p p r o x i m a t e l y 4-0 h.  A number of known h i s t o n e s were run as markers i n o r d e r to g i v e  p o s s i b l e i d e n t i t i e s to the bands o b t a i n e d . o b t a i n e d from Worthington 1) from Sigma Chemical  C a l f thymus h i s t o n e  B i o c h e m i c a l Co. and salmon protamine  Co.  T r o u t protamine  study was migrated e.  The  obtained  from  l a e v i s e r y t h r o c y t e h i s t o n e used i n t h i s  prepared by Dr. H. K a s i n s k y .  Trout and salmon  protamine  together. A l k a l i n e f a s t green s t a i n i n g o f g e l s  G e l s were a l s o s t a i n e d w i t h 0.1% pH 8.0  (grade  and h i s t o n e T were ob-  t a i n e d from Dr. G. Dixon w h i l e h i s t o n e IV from pea was Dr. D. Fambrough.  was  with 0.07  M Tris-HCl.  T h i s was  f a s t green performed  (w/v)  buffered at  on the Bonner et a l .  (1968) g e l system ( d e s c r i b e d above) e s s e n t i a l l y as o u t l i n e d by l o w i t z e t aJU  (1970) f o r the Johns (1967) g e l system.  After  Ber-  electro-  p h o r e s i s , the g e l s were removed from the tubes and p l a c e d i n 1 N a c e t i c a c i d f o r 2 h. and i n 0.07  G e l s were then washed i n d i s t i l l e d H 0 2  M T r i s - H C l a t pH 8.0  then s t a i n e d f o r 16 h. nitrous acid.  f o r 4-5 minutes.  The remainder  f o r 15  minutes  Some o f the g e l s were  o f the g e l s were deaminated w i t h  Deamination c o n s i s t e d of immersing the g e l s 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 v o l u m e s o f 10% 10%  trichloroacetic acid  (w/v).  sodium n i t r i t e  T h i s s t e p was  (w/v)  c a r r i e d out  for  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  ution.  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 ,  The  15  fusion  m i n u t e w a s h e s , and destaining  ""f.  was  stained  c a r r i e d out  " P h o t o g r a p h i n g and  16  r a t e o f 1 cm/min and  i n 0.07  scanning  M Tris-ECl  a c h a r t s p e e d o f 0.5  RESULTS  1.  CYTOCHEMISTRY  The  s p e r m n u c l e i o f R^_ p r e t i o s a  t h o s e o f Ro_ uniform. oacetic  palustris.  Alkaline  u n i f o r m and a f t e r IN  are  These n u c l e i a l s o  l e a r morphology i s s e r i o u s l y a l t e r e d  the  s p e r m o f R»_ p r e t i o s a  This indicates  R a n a s p e r m n u c l e i do gesting  not  and  forces  i f acetylation the  with a  R^  i s intense  s t a i n i n g and  conclusion that  a t 60°C.  palustris.  the  somatic type.  that  trichlorsimilarly green nuc-  With t r i c h l o r o a c e t i c  The  not  nuclei  in  the sug-  stain  hydrolysis This  rein-  arginine-rich histones  these sperm c o n t a i n  n u c l e i show i n t e n s e  present  ( F i g . 3)»  before s t a i n i n g .  histones  are of  dinitrofluorobenzene  s t a i n i n g f o r l y s i n e but  s t a i n only l i g h t l y a f t e r the  reaction  P r o t a m i n e s and  for arginine.  and  After deamination  absent.  p r o t a m i n e s and  p r e s e n t i n t h e s e s p e r m and  are  However, the  eosin Y after p i c r i c acid  not  The  is  as  stain with alkaline fast  that protamines are  i s c a r r i e d out  scan  c o n t r o l Salmo g a i r d n e r i i s p e r m  that a r g i n i n e - r i c h histones are  not  nm  s t a i n or s t a i n very s l i g h t l y  w i t h a l k a l i n e f a s t g r e e n and but  a t 660  ( F i g . 2).  protamines i n the  washed o u t .  Dif-  A Gilford  r o d - s h a p e d ( F i g . 1)  p a r a l l e l s t h e DNA  are  for  min/inch.  trichloroacetic acid hydrolysis the  8.0)  8.0).  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%  intense.  acid hydrolysis  (pH  (pH  camera.  F e u l g e n s t a i n i n g f o r DNA  acid hydrolysis  were  gels  u s e d to scan g e l s  C.  M Tris-HCl  sol-  h i n a l k a l i n e fast green.  G e l s were photographed w i t h a P o l a r o i d S p e c t r o p h o t o m e t e r 2400 was  45  minutes with a f r e s h  w a s h e d 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 two  and  Sakaguchi  a r g i n i n e - r i c h h i s t o n e s of  the  - 18 mouse a n d 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 guchi test  ( B l o c h 1969).  red reaction  W i t h no h y d r o l y s i s ,  s t a i n i n g i s a b s e n t ( T a b l e 1), s h o w i n g t h a t iated basic  proteins  are missing.  w i t h t h e Saka-  alkaline fast  green  non-nucleic acid  Alkaline  assoc-  f a s t green a f t e r  Feulgen  s t a i n i n g r e s u l t s i n a uniform purple s t a i n ; thus non-DNA-associated basic  proteins The  are  are also  a b s e n t from t h e Rana sperm.  sperm n u c l e i o f  versicolor  rod-shaped but s l i g h t l y bent.  uniform.  Alkaline  hydrolysis tern.  ( F i g . k) a s w e l l  as  Feulgen s t a i n i n g i s i n t e n s e and  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  i s intense  This indicates  ( F i g . 5) a n d f o l l o w s  histones are present.  The  ( F i g . 6), w h i c h  that  arginine-rich  line  f a s t green o r eosin 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  addition,  suggests  i n both ( T a b l e 1); In  The weak S a k a g u c h i  reaction  t h e s p e r m h i s t o n e s a r e n o t o f t h e s a l m o n , o r mouse  grasshopper type.  Non-nucleic acid-associated  non-DNA-associated basic  proteins  basic  proteins  s p e r m n u c l e i o f X ^ l a e v i s a r e w a v y ( F i g . 7)»  orates the Feulgen 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  ( F i g . 8).  Alkaline  Feulgen s t a i n -  Alkaline  This  presence o f a r g i n i n e - r i c h  histones.  f a s t green and e o s i n Y a f t e r p i c r i c  staining  demonstrates  t h e y a r e washed o u t b y s u c h  ( F i g . 9)» w h i c h i n d i c a t e s t h e The n u c l e i s t a i n w i t h acid hydrolysis  alkaline  and even  which i s another i n d i c a t i o n that  histones are present.  corrob-  f a s t green s t a i n i n g , although reduced, 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  prior acetylation,  f a s t green  ( T a b l e 1 a n d F i g . 8).  protamines a r e not present, since  treatment.  and  are absent.  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  that  alka-  these 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 but respond  demonstrates that  The  staining  arginine-rich histones are present.  moderately t o the Sakaguchi r e a c t i o n . and  pat-  The n u c l e i s t a i n w i t h  s p e c i e s and c o n t i n u e t o 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 also, suggests that  acid  the Feulgen s t a i n i n g  that protamines are not present.  i s reduced only s l i g h t l y a f t e r deamination  this,  Regilla  with  arginine-rich  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 a n d  g i v e a moderate r e a c t i o n  w i t h the Sakaguchi t e s t .  Thus, l i k e t h e  H y l a sperm, t h e 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  basic  - 19 t h a n t h e Rana t y p e ;  but they a r e not as b a s i c as those o f  Salmon, o r Mouse/grasshopper reaction.  types which g i v e a s t r o n g  the  Sakaguchi  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. A s i n R a n a , t h e s p e r m n u c l e i o f B_j_ b o r e a s a n d Bj_ a m e r i c a n u s a l s o rod-shaped The  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 ) .  n u c l e i do 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 TCA  lysis  ( F i g . 11 a n d T a b l e 1 ) .  hydro-  T h e y 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  or 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  instance  ( F i g . 12), which s t r o n g l y suggests that protamines are  present.  H o w e v e r , t h e n u c l e i do n o t s t a i n u n d e r a n y o f t h e c o n d i t i o n s acetylation  ( F i g . 13) which i n d i c a t e s t h a t p r o t a m i n e s and  r i c h histones are absent. and  T h i s paradox  of  arginine-  p e r s i s t s with both  e v e n 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  lengths of time,  fixatives  for different  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  ferent temperatures.  The n u c l e i s t a i n w i t h  dif-  dinitrofluorobenzene  but o n l y moderately a f t e r the Sakaguchi r e a c t i o n .  H o w e v e r , when  t h e 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 nuclei stain strongly. Pleurodeles 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 the Sakaguchi  were p r e s e n t i n t h i s u r o d e l e . i n another respect.  type.  The  P e r h a p s , Bufo sperm a l s o The  ( F i g . 14).  salmon  contain  n u c l e i o f Bufo sperm a r e u n u s u a l  s t a i n i n g observed a f t e r p i c r i c a c i d  i s n o t u n i f o r m ; i n s t e a d , t h e n u c l e i show c l u m p s o f  staining  Bloch  a n d Hew  that  test.  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 h i s t o n e s o f the salmon  (1960a) o b s e r v e d a  hydro-  intense  coagulation  e f f e c t w i t h t h e 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 treatment.  P e r h a p s a s i m i l a r phenomenon i s b e i n g  the Bufo sperm.  these  P i c h e r a l (1970) found t h a t t h e sperm o f  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  lysis  are  observed  Non-nucleic acid-associated basic proteins  absent i n Bufo sperm as a r e non-DNA-associated  basic  acid  with are  proteins.  20  TABLE 1 Cytochemistry  o f anuran sperm n u c l e i  Results Staining and p r e treatment  Reactive material  Feulgen  DNA  AFG-TCA 86°C  Basic proteins other than p r o t amines  AFG-TCA 86°C, deamination  Basic proteins rich in arginine  I  Rana R a n a B u f o palu- p r e t - ameris t r i s i o s a canus  AFG-picric Basic p r o acid teins i n cluding protamines AFG-picric Basic p r o acid, ac- teins rich etylation i n arginine Eosin-I picric acid  Basic prot e i n s except "cleavage2 histones"  Eosin-Y Basic propicric teins rich acid, ac- i n arginine etylation AFG  Non-nucleic acid-associated basic proteins  using  Bufo H y l a Hyla, boreas reg- v e r s i i l i a color  Xenopua jaevis  - 21 -  TABLE 1  (continued) R e s u l t s U6ing  Staining and p r e treatment  Reactive material  AFG - a f t e r Feulgen  Non-DNA-  Sakaguchi  Proteinbound arginine  Dinitrofluorobenzene  Proteinbound lysine  Rana R a n a B u f o Bufo Hyla Hyla p a l u - pret° a m e r i - b o r e a s r e g - v e r s i s t r i s ioea canus i l i a color  Xenopus laevis  associated basic proteins  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 w e r e o b t a i n e d w i t h AFG- 1 N TCA, 60°C. ^ B l o c h a n d Hew (1960b).  - 22 -  Figures 1 - 3»  Sections of Rana pretiosa t e s t i s .  Scale i n F i g . 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 t r i c h l o r o a c e t i c  acid hydrolysis at 60°C.  Sperm nuclear morphology has been ser-  i o u s l y altered.  Figure 3»  Staining with a l k a l i n e fast green, IN t r i c h l o r o a c e t i c  acid hydrolysis, a f t e r deamination.  No staining can be seen.  3  - 23 -  F i g u r e s k - 6. Fig  e  k d e n o t e s lO/i a n d a p p l i e s  F i g u r e k» sperm  Sections o f Hyla versicolor  Feulgen s t a i n i n g  testis.  Scale i n  t o F i g s , 5 and 6 as w e l l .  demonstrating  clusters  o f curved  nuclei.  F i g u r e 5»  Staining  acid hydrolysis  F i g u r e 6.  f a s t green,  IN trichloroacetic  f a s t green,  IN t r i c h l o r o a c e t i c  a t 60°C.  Staining  acid hydrolysis  with alkaline  with alkaline  a t 60°C, a f t e r d e a m i n a t i o n .  to s t a i n although  s t a i n i n g i s reduced.  Sperm n u c l e i  continue  - Zk -  'Figure's '7 -"9. d e n o t e s IG71  S e c t i o n s o f Xenopus l a e v i s t e s t i s .  and a p p l i e s  to Figs.  8 and 9 a l s o .  F i g u r e 7-  Feulgen s t a i n i n g showing c l u s t e r s  F i g u r e 8.  Staining  acid hydrolysis  F i g u r e 9»  o f wavy sperm n u c l e i .  with a l k a l i n e fast green, IN t r i c h l o r o a c e t i c  a t 60°C.  Staining  acid hydrolysis  Scale i n F i g . 7  with a l k a l i n e f a s t green, IN t r i c h l o r o a c e t i c  a t 60°C, a f t e r  deamination.  - 25 -  F i g u r e s 10 - 13.  S e c t i o n s o f Bufo americanus  F i g . 1 0 d e n o t e s 10/x  F i g u r e 10. are  and a p p l i e s  -Feulgen s t a i n i n g .  to Figs.  Clusters  testis.  Scale i n  1 1 , 12 and 13 a s w e l l .  o f rod-shaped  sperm  nuclei  evident.  Figure 11.  Staining  acid hydrolysis  F i g u r e 12. lysis.  a t 90°C.  Staining  Sperm n u c l e i  F i g u r e 13*  with alkaline  Staining  Sperm n u c l e i  with alkaline stain  trichloroacetic  do n o t s t a i n .  fast green, p i c r i c a c i d  hydro-  unevenly.  with alkaline  l y s i s , a f t e r deamination.  f a s t g r e e n , 5%  Staining  f a s t green, p i c r i c i s absent.  acid  hydro-  - 26 -  Figure  14.  A s p e r m nucleus o f B. b o r e a e s t a i n e d  f a s t green, p i c r i c d e n o t e s 10 ja .  acid hydrolysis.  with  alkaline  S t a i n i n g i s uneven.  Scale  14  - 27 2. An  BIOCHEMISTRY e l e c t r o p h o r e t i c comparison 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 , a n d 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 acrylamide  t  americanus i n d i c a t e s s i g -  17 a n d 18).  The r e g i o n o n p o l y -  g e l s bound b y t h e f a s t e s t a n d s l o w e s t moving bands o b -  t a i n e d w i t h h i s t o n e s f r o m Rj_ p i p i e n s t e s t e s i s d e f i n e d a s 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 . b i o c h e m i c a l . 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 were s i m i l a r undescribed  o f f r o g sperm and s o m a t i c  tissues  ( V e n d r e l y 1957) a n d t h e 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 a n system) o f h i s t o n e s from f r o g t e s t e s and somatic  vrere a l s o s i m i l a r  ( B l o c h 1962).  tissue  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 ,  Ilb.^ I l l ,  2  a n d I V ( J o h n s 1971).  Fambrough a n d B o n n e r (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 p e r f o r m e d in this section) w i l l bands.  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  The f a s t e s t m o v i n g b a n d c o n t a i n s h i s t o n e I V w h i l e h i s t o n e I  makes u p t h e s l o w e s t m o v i n g b a n d . histones I l b ^ , I I b  2  and I I I .  The t h i r d b a n d i s c o m p o s e d o f  Bands m i g r a t i n g s l o w e r  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 gregates  three  of histones.  contaminating  than  the H  proteins o r ag-  I n t h i s study t h e f a s t e s t moving band i n t h e  histone region co-electrophoresed  with histone IV from pea.  h i s t o n e T ( W i g l e a n d D i x o n 1971)» p r o t a m i n e  Only  (Marushige and Dixon  1969)j a n d p r o t a m i n e - l i k e p r o t e i n s (Lam a n d B r u c e 1971) a r e r e p o r ted t o migrate  f a s t e r than 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  b a n d s ( X ^ , X , X^) w e r e f o u n d t o m i g r a t e 2  three  f a s t e r than histone IV while  w i t h B o _ a m e r i c a n u s one b a n d ( B ) h a s 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 a n d 17). A comparison on p o l y a c r y l a m i d e of t e s t i s and l i v e r  from  gels o f thehistone  pipiens supports  the contention that the  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 those o f somatic ( F i g . 19).  Four bands a r e p r e s e n t  amounts v a r y . contamination  i n both  components tissues  tissues, although the  The f a s t e s t m o v i n g b a n d i s v e r y f a i n t i n l i v e r . o f l i v e r chromatin  The  w i t h p i g m e n t m i g h t a c c o u n t f o r some  of 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 these  f r o g h i s t o n e s r e v e a l s o n e more  band t h a n does t h e e l e c t r o p h o r e s i s o f c o m m e r c i a l c a l f thymus or trout t e s t i s histone. catesbiana l i v e r band.  histone  P e r h a p s , h i s t o n e V, w h i c h i s p r e s e n t  (Nelson and Yunis  Four bands a r e l e s s e v i d e n t  1969)  f  accounts f o r the a d d i t i o n a l  i n Rj_ p i p i e n s t e s t i s .  Here  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 o n t h e m a i n b a n d ( F i g . 15).  •be p r e s e n t  when t h e 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 However,  t h e two s l o w e s t  the g e l s a r e deaminated p r i o r that these  three  present  a shoulder 25 a n d 26).  i n R.  F o u r components a l s o a p p e a r t o (Figs.  m o v i n g b a n d s do n o t s t a i n when  t o s t a i n i n g ( F i g . 26).  This indicates  bands a r e n o t 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 c o m p a r i s o n o f t h e h i s t o n e s f r o m t e s t i s , and  liver,  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 main bands a r e p r e s e n t  to the testis  20).  ( F i g s . 16 a n d 20).  Three  i n t h e 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 erythrocyte histone region ( F i g .  The e r y t h r o c y t e s o f f r o g s h a v e b e e n r e p o r t e d t o c o n t a i n  V (Nelson and Yunis in  as  histone  1969) a n d 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 b a n d  the erythrocyte H region.  region co-electrophoreses  The f a s t e s t m o v i n g b a n d i n t h e h i s t o n e  w i t h h i s t o n e IV from pea.  ( F i g . 20).  When  t h e 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 , t h e same p a t t e r n i s revealed  ( F i g s . 25 a n d 27)« e x c e p t X I a p p e a r s i n a m o u n t s  be d e t e c t e d . continue  too small to  T h r e e b a n d s i n t h e h i s t o n e r e g i o n a n d b a n d s X 2 a n d X3  t o s t a i n even i f t h e 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 in arginine.  ( F i g . 27).  Therefore,  None o f t h e t e s t i s s p e c i f i c  these  bands a r e r i c h  bands c o - e l e c t r o p h o r e s e  protamine 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 migrates r e g i o n o f t h e X2 d o u b l e t  ( F i g . 22).  However,  with  i n the  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 a s d o e s X2 w h i c h i n d i c a t e s t h a t X 2 i s more r i c h  i n a r g i n i n e than the l y s i n e - r i c h  h i s t o n e T ( F i g . 28). • A c o m p a r i s o n o f t h e h i s t o n e components o f t e s t i s a n d l i v e r  from  B. a m e r i c a n u s d i s c l o s e s t h a t t h e f a s t m o v i n g b a n d , B, i s s p e c i f i c t o the  testis  ( F i g s . 17 a n d 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 are present bands a l s o 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  i n the t e s t i s histone r e g i o n .  band i n the h i s t o n e r e g i o n f r o m pea  ( F i g . 21).  (H)  A similar  co-electrophoreses  ine.  with histone  ( F i g . 25)•  of the g e l s p r i o r to s t a i n i n g w i t h a l k a l i n e f a s t  does n o t p r e v e n t and  f a s t e s t moving  t h e two  b a n d B 'from s t a i n i n g . Band B m i g r a t e s  These bands a r e  region  therefore rich i n argin-  i n the protamine r e g i o n  ( F i g . 23)»  although  quite achieved.  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 h i s t o n e s o f B^ a m e r i c a n u s i n d i c a t e s t h a t b a n d B m i g r a t e s 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 f r o m X.  De-  green  f a s t e s t moving bands i n the h i s t o n e  e l e c t r o p h o r e s i s w i t h salmon protamine i s not  the  IV  electrophoretic pattern i s revealed  when t h e 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 amination  The  minor  laevis  co-  Electestis  f a s t e r than  ( F i g . 24).  - 30 -  F i g u r e s 15 - 17.  Densitometer t r a c i n g s o f h i s t o n e s from anuran  t e s t e s run on 15%  polyacrylamide  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 d i c a t e s somatic h i s t o n e  g e l s f o r 85 minutes.  w i t h h i s t o n e IV from pea.  region.  F i g u r e 15.  Rana p i p i e n s  F i g u r e 16.  Xenopus l a e v i s  F i g u r e 3.7.  Bufo americanus  Arrow i n d i c -  testis.  testis.  testis.  H in-  - 31 -  F i g u r e s 18 - 28.  Polyacrylamide g e l electrophoresis patterns of  h i s t o n e s from anurans.  G e l s w e r e r u n f o r 85 m i n u t e s a n d s t a i n e d  with buffalo black unless indicated otherwise.  Gel patterns i n  each f i g u r e were o b t a i n e d  H i n d i c a t e s somatic  histone region.  d u r i n g t h e same r u 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 f r o m t h e a n o d e  (top o f g e l ) towards the cathode F i g u r e 18.  (bottom o f g e l ) .  A.  Pana p i p i e n s  testis.  B.  Bufo americanus  C.  Xenopus l a e v i s  D.  Same a s 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  testis. testis.  was p l a c e d on t h e g e l .  F i g u r e 19.  A. B.  F i g u r e 20.  pipiens  testis.  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 run w i t h h i s t o n e IV from pea.  A.  laevis  testis.  laevis  liver.  B  *  C.  —  Xj_ l a e v i s  erythrocyte.  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 I V f r o m p e a . F i g u r e 21.  A.  Bj_ a m e r i c a n u s  testis.  B.  Bj_ a m e r i c a n u s  liver.  C.  Bj_ a m e r i c a n u s l i v e r r u n w i t h h i s t o n e I V f r o m p e a .  19  18 A  B  C  I  A B C  D  1  — X1  •  — X2 — X3 - B  21  20 A  B  C  7"A  D  B C  —  1 •  .  H — X1 — X2 — X3  —B  A  - 32  F i g u r e 22,  Figure  23 •  F i g u r e 24-.  -  H i s t o n e s from Xenopus l a e v i s  testis.  A.  Testicular histones  alone-  B.  T e s t i c u l a r h i s t o n e s p l u s salmon protamine ( P ) .  C.  Testicular histones plus trout histone  H i s t o n e s from Bufo americanus  T.  testis.  A.  Testicular histones  alone.  B.  T e s t i c u l a r h i s t o n e s p l u s salmon protamine ( P ) .  T e s t i c u l a r h i s t o n e s f r o m X._ l a e v i s a n d Bj_ a m e r i c a n u s run  together.  — XI  —X2  — X3 —P  24  1]  —XI  - 33 -  F i g u r e 25.  H i s t o n e s from anuran t e s t e s s t a i n e d w i t h e i t h e r b u f f a l o  black or a l k a l i n e fast  green.  A, C, and E.  Stained with buffalo black.  B  S t a i n e d w i t h a l k a l i n e f a s t green.  D, and F.  s  A and B.  Rana p i p i e n s  C and D.  Bufo  E and F.  Xenopus l a e v i s  F i g u r e 26.  americanus  T e s t i c u l a r h i s t o n e s from R^ p i p i e n s s t a i n e d w i t h  alkaline fast  green.  A.  Without deamination o f g e l s .  B.  With deamination of g e l s .  F i g u r e 27.  T e s t i c u l a r h i s t o n e s from Xj_ l a e v i s s t a i n e d w i t h  a l k a l i n e f a s t green. A,  Without deamination o f g e l s .  B.  With deamination o f g e l s .  F i g u r e 28.  Trout h i s t o n e T and salmon protamine  w i t h a l k a l i n e f a s t green. A.  Without deamination o f g e l s .  B.  With deamination o f g e l s .  (P) s t a i n e d  - 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 i s unusual since the protamines of trout sperm are not free basic proteins but instead are electrostatically bound to the phosphate groups of DNA (Frederieq 1971). If the experiment i s repeated using 0.01M tris-HCl buffer in place of the citrate-phosphate buffer, the same results are achieved. However, when the experiment i s repeated 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 i s the cause of these unusual results. Since the possibility of developing a new technique to distinguish between protamine and non-protamine containing cells existed, the phenomenon was studied further. Salt concentration i s indeed necessary for staining without prior hydrolysis. As can be seen in Table II, staining only occurs at high salt concentrations. Staining i s dependent on the mixture of salt and dye (see Table III). Staining i s absent i f sections are treated with a salt solution prior to staining with AFG made up i n d i s t i l l e d 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 i s used on a variety of sperm (Table IV) at an intermediate salt concentration. As has been shown in the previous sections, 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 testing for free basic proteins.  - 35 -  TABLE I I S a l t C o n c e n t r a t i o n and the AFG R e a c t i o n *  Stain-  R e s u l t s 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 p i p i e n s  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  -  -  -  -  -  -  -  -  0.0375 M NaCl  +  -  0.0750 M NaCl  +  alight  0.1500 M NaCl  +  +  -  0.3000 M NaCl  ++  +  mm  0.6000 M NaCl  ++  +  1.2000 H NaCl  +-!-  +  2.4000  ++  +  0.0188  *  M  M  NaCl  NaCl  slight  These r e s u l t s a r e o b t a i n e d without h y d r o l y s i s  -  -  - 36 TABLE I I I  Pretreatment and the AFG Reaction * AFG made up i n  Pretreatment  Trout sperm  distilled H 0  none  d i s t i l l e d ILpO  30 minutes i n 0.300 M NaCl  2  0.30 M NaCl  none  0.30 M NaCl *  +  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 i n Arginine  Xenopus l a e v i s  +  +  Bufo boreas  +  +  B. americanus  +  +  Rana pipiens R. pretiosa R. p a l u s t r i s *  These r e s u l t s were obtained without hydrolysis.  - 37 D.  DISCUSSION  On the b a s i s o f c y t o c h e m i c a l c r i t e r i a , the sperm o f the anurans s t u d i e d may histones of  be c l a s s i f i e d i n t o t h r e e groups.  seven  The sperm  l a e v i s are o f the M y t i l u s type, which i s i n agree-  ment w i t h the data o f B l o c h (1969).  These sperm 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 xuader a l l c o n d i t i o n s o f h y d r o l y s i s , but  they  s t a i n s l i g h t l y l e s s i n t e n s e l y a f t e r a c e t y l a t i o n o r deamination.  This  d i s t i n g u i s h e s them from the Mouse and grasshopper type o f sperm h i s tone which shows no r e d u c t i o n i n s t a i n i n g i n t e n s i t y a f t e r deamina t i o n or a c e t y l a t i o n . demonstrate  The sperm o f H«_ r e g i l l a and  versicolor  a s t a i n i n g p a t t e r n s i m i l a r to that o f  t h e r e f o r e a l s o belong to the M y t i l u s t y p e .  laevis  and  The sperm o f R^ p a l u s -  t r i s and R^ p r e t i o s a s t a i n l i k e the sperm o f R^ p i p i e n s ( B l o c h 1962;  Z i r k i n 1970)  and thus a r e o f the Rana type.  These sperm do  not s t a i n or s t a i n o n l y f a i n t l y with 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 deamination.  The b a s i c p r o t e i n s i n the sperm o f B^  ameri-  canus and B_o_ boreas are unusual from a c y t o c h e m i c a l p o i n t o f view. These sperm s a t i s f y three c r i t e r i a f o r the Salmon t y p e .  They do not  s t a i n a f t e r TCA h y d r o l y s i s , 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 f o r m a l i n f i x e d m a t e r i a l g i v e a s t r o n g 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 h y d r o l y s i s i f the s e c t i o n s are a c e t y l a t e d , which suggests t h a t protamines absent.  are  T h e r e f o r e , on the b a s i s o f t h i s c y t o c h e m i c a l d a t a these  sperm are c l a s s e d as the Salmon type w i t h r e s e r v a t i o n .  B l o c h (1969),  on the o t h e r hand, c l a s s i f i e d the sperm h i s t o n e s o f Bj_ v u l g a r i s as the M y t i l u s type on the b a s i s o f 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 . 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 R^  t e s t i s and l i v e r r e v e a l s no q u a l i t a t i v e d i f f e r e n c e s .  pipiens  This i s i n  agreement w i t h the s t u d i e s o f Vendrely (1957) and B l o c h (1962). V e n d r e l y found t h a t the amino a c i d compositions o f h i s t o n e s e x t r a c t e d from f r o g sperm and from somatic t i s s u e s were s i m i l a r w h i l e B l o c h found no e l e c t r o p h o r e t i c d i f f e r e n c e s between h i s t o n e s from t e s t e s and h i s t o n e s from somatic t i s s u e s .  The  t e s t e s o f Rj_ p r e t i o s a and  R^  - 38 p a l u s t r i s would be expected  to g i v e e l e c t r o p h o r e t i c p a t t e r n s very-  s i m i l a r to those obtained w i t h R. p i p i e n s s i n c e they s t a i n  similarly.  U n l i k e R^ p i p i e n s , e l e c t r o p h o r e s i s o f h i s t o n e s from Xj_ l a e v i s r e v e a l s three f a s t moving bands s p e c i f i c t o the t e s t i s .  These bands  a r e not l i k e l y t o be d e g r a d a t i o n products s i n c e s i m i l a r bands were not found i n o t h e r t i s s u e s o f Xenopus nor i n the o t h e r anurans examined by the same procedure.  S i n c e the sperm n u c l e i were the o n l y  n u c l e i i n the Xenopus t e s t i s t h a t d i d not show s t a i n i n g p r o p e r t i e s s i m i l a r to Rana type sperm n u c l e i o r somatic are l i k e l y s p e c i f i c  to sperm.  c e l l n u c l e i , these bands  T h i s i s a l s o supported by the obser-  v a t i o n t h a t bands X2 and X3 do s t a i n a f t e r deamination,  indicating,  t h a t l i k e the sperm n u c l e i , they are r i c h i n a r g i n i n e .  On the g e l  system employed i n t h i s study bands XI, X2, and X3 demonstrate m o b i l i t i e s i n t e r m e d i a t e between protamines and somatic  histones.  Thus  M y t i l u s type o f sperm h i s t o n e does appear to be an " i n t e r m e d i a t e " type. These o b s e r v a t i o n s w i t h Xj_ l a e v i s a r e v e r y s i m i l a r to those obt a i n e d by Bloch (1966) with M y t i l u s e d u l i s .  He r e p o r t e d t h a t e l e c t r o -  p h o r e s i s o f t e s t i c u l a r h i s t o n e s showed t h r e e bands ( ^ Y ,  <5 ) whose  m o b i l i t i e s were f a s t e r than those o f somatic  h i s t o n e s (although  somatic  t i s s u e s were not s t u d i e d i n t h i s organism).  How these t h r e e bands a r e  r e l a t e d to the bands (XI, X2, and X3) o b t a i n e d i n t h i s study i s unknown s i n c e the e l e c t r o p h o r e t i c system was not d e s c r i b e d by B l o c h . Bloch d i d determine the amino a c i d composition i n g bands.  However,  o f the two f a s t e s t mov-  They c o n t a i n e d a l l three o f the b a s i c amino a c i d s :  ine, lysine,and h i s t i d i n e .  Although  argin-  the amount o f h i s t i d i n e was s m a l l ,  these p r o t e i n s appear to be s i m i l a r to the t r i p r o t a m i n e s o f K o s s e l . S i n c e techniques are being developed  to do amino a c i d a n a l y s i s o f  bands o b t a i n e d on p o l y a c r y l a m i d e g e l s (Houston 1971), the amino composition o f bands XI, X2, and X3 can a l s o be How  acid  determined.  these bands are r e l a t e d to the p r o c e s s of spermiogenesis  and  how, they are r e l a t e d to each o t h e r i s unknown. As a r e s u l t o f l a b e l Idl i n g p a t t e r n s with C -amino a c i d s , Bloch (1966) b e l i e v e s t h a t the three p r o t e i n s i n Mj_ e d u l i s a r e s y n t h e s i z e d a t the same time i n the  - 39 early spermatid; however, as development proceeds they are successively 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 present along with the sperm specific basic proteins? These questions remain to be answered. One fast moving band, B, i s specific to the testis of B^ americanus. 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 protamine containing cells are sperm. Therefore, this band i s most l i k e ly specific to sperm nuclei. Only amino acid analysis of this band w i l l determine how closely related band B i s to salmon protamine. However, the present data support the contention that sperm histones of B^ americanus are of the Salmon type. Why these nuclei do not stain after acetylation i s 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 i s low in arginine. Also, the absence of band B i n R._ pipiens and X^ laevis testis would be d i f f i c u l t 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 a l . (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 -  S i m i l a r proteins i n the sperm of frogs were not found.  Since the  appearance of non-DNA-associated basic proteins has been c o r r e l a t e d with the removal of somatic h i s t o n e s , such proteins would not be expected i n an organism with the somatic type of sperm h i s t o n e .  How-  ever, such proteins might be expected i n sperm with the M y t i l u s or Salmon type of sperm histone. An explanation f o r the e f f e c t of s a l t concentration on the r e a c t i o n i s d i f f i c u l t to e n v i s i o n .  AFG  S p e c i f i c s t a i n i n g of sperm r i c h  i n a r g i n i n e i s achieved without the removal of DNA at high s a l t concentrations.  Since i n c r e a s i n g 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 a r g i n i n e are s e l e c t i v e l y exposed by high s a l t concentrations. i a t i o n of nucleohistone and nucleoprotamine l y s i n e - r i c h histones are removed from DNA  In v i t r o d i s s o c -  by s a l t i n d i c a t e s that at low s a l t concentrations;  the a r g i n i n e - r i c h histones at much higher concentrations; and the protamines (very r i c h i n a r g i n i n e ) at intermediate concentrations (Marushige and Dixon 1971).  Thus, the s e l e c t i v e 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, s i n c e 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  s t a i n i n g would be expected due to the exposure of a r g i n i n e - r i c h h i s tones.  In f a c t , somatic c e l l s u s u a l l y do not s t a i n under any con-  ditions. A d i r e c t comparison between sectioned m a t e r i a l and chromatin  may  be misleading since i n sectioned m a t e r i a l the f i x a t i v e must be considered.  The experiments i n t h i s study were performed on m a t e r i a l  f i x e d i n formaldehyde, which i s thought to f u n c t i o n as a f i x a t i v e by forming methylene bridges between amino groups of neighboring prot e i n s (Bowes and Carter 1965)o  However, besides p r o t e i n - p r o t e i n  b i n d i n g , formaldehyde l i n k s histone molecules  to DNA.  (1969) found that the histones of formaldehyde-treated were not d i s s o c i a b l e from DNA  by s a l t .  B r u t l a g et a l . nucleohistone  The protein-DNA i n t e r a c t i o n  was no longer mainly i o n i c as i n native nucleohistone.  Instead,  - kl -  m e t h y l e n e b r i d g e s were f o r m e d b e t w e e n t h e amino g r o u p s o f h i s t o n e s and  t h e amino g r o u p s o f DNA.  are not stained despite high ever, few  salt  c o n t a i n few, i f a n y l y s i n e  f r e e amino g r o u p s .  DNA a n d m i g h t  still  the  staining  selective  at high s a l t  capable  residues  of forming  rich  linkage.  for staining dissociation  occurs  a l l the s t a i n i n g  salt while  P o s s i b l y , the protamines a r e not  f o r the s a l t  i n the presence  negative  occurs.  and t h e dye t o be  o f the dye, the s t a i n  ions f o r p o s i t i v e s i t e s  When t h i s  dissociation  s i t e s a r e blocked  mechanism.  on t h e  occurs  by n e g a t i v e  prior to  i o n s and  does n o t o c c u r . mechanism i s t e s t a b l e .  should  be o b s e r v e d  I f cross l i n k i n g  o f formaldehyde,  nucleoprotamine to a very  of forming then  then  the e f -  i n formaldehyde.  If  m e t h y l e n e b r i d g e s w i t h DNA i n t h e  the protamine o f  s h o u l d be d i s s o c i a b l e selective  due t o f o r m a l d e h y d e  exposure o f protamine,  only i n material fixed  protamine i s not capable  lead  of prot-  c a n a l s o be e x p l a i n e d b y t h i s  the basis o f the d i f f e r e n t i a l  presence  s p e r m h i s t o n e s w i t h AFG  c o n c e n t r a t i o n s u s e d due t o some p r o t -  to occur  protamine and s t a i n i n g  fect  basis,  h i s t o n e s a r e n o t e x p o s e d due t o e x -  The r e q u i r e m e n t  competes s u c c e s s i v e l y w i t h  is  with  .On t h i s  due t o t h e l a c k o f p r o t a m i n e - D N A c r o s s l i n k i n g  removed b y t h e s a l t  amine-protein  The  salt.  i n a r g i n i n e a r e exposed by h i g h  t e n s i v e histone-DNA c r o s s l i n k i n g .  staining  methylene b r i d g e s  s e c t i o n s , t h e dye b i n d i n g s i t e s  dye b i n d i n g s i t e s o f s o m a t i c  staining,  how-  c o n c e n t r a t i o n s m i g h t be e x p l a i n e d .  concentration  When s a l t  Protamines,  i n formaldehyde t r e a t e d m a t e r i a l ,  o f very a r g i n i n e - r i c h  amines and p r o t e i n s v e r y  together  cells  ( L i n g e_t a l . 1971) a n d t h u s  be diss.o.ciated .from .DNA w i t h  In f o r m a l d e h y d e - t r e a t e d  completely  e x p l a i n why s o m a t i c  concentrations.  Therefore,  p r o t a m i n e s would be l e s s  the  T h i s might  fox^maldehyde-treated  f r o m DNA b y s a l t .  method f o r i s o l a t i n g  protamine.  This  could  - hz -  III.  CHANGES IN BASIC PROTEINS DURING SPERMIOGENESIS IN THE EASTERN RED SPOTTED NEWT, DIEMICTYLUS A.  VIRISDESCENS  INTRODUCTION  The histone t r a n s i t i o n s observed during spermiogenesis i n the urodele, PIeurodeles w a l t i i , ( P i c h e r a l 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 t r a n s i t i o n only as f a r as the "stable protamine" o r the Mouse/grasshopper type o f sperm histone.  To check the d i v e r s i t y  of sperm histone types i n the urodeles, the basic p r o t e i n changes during spermiogenesis i n the eastern red spotted newt have been r e examined. B.  METHODS AND MATERIALS  1. CYTOCHEMISTRY Sexually mature Diemictylus ( T r i t u r u s ) v i r i d e s c e n s , the eastern red spotted newt, were obtained from E. G. S t e i n h i l b e r & Co.  Animals,  maintained i n the l a b o r a t o r y 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 t e s t e s were r e -  moved and f i x e d i n 10% n e u t r a l buffered formalin o r absolute ethanolg l a c i a l a c e t i c a c i d (3:1) as o u t l i n e d i n s e c t i o n I I . A l l s t a i n i n g procedures employed on t h i s m a t e r i a l have been described p r e v i o u s l y . 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 e t h e r i z e d and the t e s t e s ,  l i v e r s , hearts, and spleens were removed and stored a t -20°C u n t i l use.' With one exception a l l biochemical procedures were c a r r i e d out as described i n s e c t i o n I I , In order to c o l l e c t enough m a t e r i a l f o r the e x t r a c t i o n o f histones, somatic t i s s u e s were combined and then  - k-3 extracted. 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  turn, are organized into  seminiferous tubules which, i n  c l u s t e r s of c e l l s or cysts.  A l l cysts with-  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 t h e same s t a g e spermatogenesis. e s i s may  I n September and October  a l l stages of  of  spermiogen-  be f o u n d i n t h e n e w t t e s t i s w h i l e i n December m a t u r e s p e r m  predominate  (Baker 1966).  As a r e s u l t , m o s t o b s e r v a t i o n s w e r e made  on m a t e r i a l f i x e d i n O c t o b e r . Spermiogenesis  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 .  29-32  shown i n F i g s .  summarized i n Table  and  These s t a g e s  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  and  are  V.  Stage 1 n u c l e i , the e a r l i e s t s p e r m a t i d s observed, a r e ( F i g . 29)  are  demonstrate  u n e v e n s t a i n i n g f o r DNA.  histones p a r a l l e l s the F e u l g e n - s t a i n i n g p a t t e r n .  AFG  round  staining for  Treatment of  these  n u c l e i with deamination or a c e t y l a t i o n reagents a b o l i s h e s s t a i n i n g for basic proteins, indicating tones.  the absence o f v e r y a r g i n i n e - r i c h  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  arginine.  Spermatids s t a g e 2.  reaction for  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 which  t h i s stage are s i m i l a r to those of somatic  cell  occur i n  nuclei.  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  T h e s e 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).  For  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 allel  each o t h e r .  T h e s e n u c l e i do n o t s t a i n w i t h AFG  a r e deaminated  or a c e t y l a b e d and  light  to the Sakaguchi  response  l i k e stage 1 n u c l e i , reaction.  somatic type are present i n these Stage  his-  3 n u c l e i a r e n a r r o w and  pleted nuclear elongation.  The  as  stage par-  i f the s e c t i o n s they g i v e a  Therefore, histones of  the  nuclei. c y l i n d r i c a l and have a l m o s t  com-  arrangement o f these n u c l e i i n c y s t s  i s more p r e c i s e t h a n i n s t a g e s 1 a n d  2 ( F i g . 30).  Within a cyst,  n u c l e i l i e approximately p a r a l l e l w i t h the apices p o i n t i n g  towards  the  - 44 a Sertoli cell  ( 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  uniform.  As w i t h s t a g e s 1 a n d  AFG-staining of t h i s stage occurs a f t e r t r i c h l o r o a c e t i c a c i d lysis  (Fig.33)t  nuclei.  i n d i c a t i n g t h a t protamines  W h i l e AFG  are absent  from  2,  hydro-  these  s t a i n i n g i n the a p i c a l r e g i o n of stage 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 gradient i s a c t u a l l y observed.  I n some c y s t s  o n l y t h e m o s t 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 posterior  h a l v e s o f t h e n u c l e i s t a i n ; and  i n some, t h e n u c l e i  completely s t a i n e d except f o r the a p i c e s .  are  These s t a i n i n g p a t t e r n s  suggest t h a t the very 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 the somatic h i s t o n e s from the base to the apex o f the n u c l e u s .  Within  a c y s t , a l l n u c l e i show t h e 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 synchronous.  However, the c y s t s w i t h i n  erous tubule are not q u i t e synchronous  w i t h one  another  a seminif-  ( 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 present.  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  guchi reaction,  than the a p i c a l r e g i o n s .  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 , which  cysts are s l i g h t l y curved. cysts  ( F i g . 31)  than stage 3 n u c l e i .  i n t e n s e and u n i f o r m .  packed  Feulgen s t a i n i n g i s very i n -  S i n c e s t a i n i n g w i t h AFG  acetic acid hydrolysis,  i n some  T h e s e n u c l e i o c c u r i n more t i g h t l y  t e n s e a n d 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 t h e AFG  staining i s similarly  occurs a f t e r  trichloro-  s t a g e 4 n u c l e i do n o t c o n t a i n p r o t a m i n e s .  or e o s i n T s t a i n i n g o f these n u c l e i remains  i n t e n s e and u n i f o r m  s p i t e deamination or a c e t y l a t i o n of these c e l l s . s t a i n very s t r o n g l y a f t e r the Sakaguchi  These n u c l e i  reaction.  Therefore,  c o n t a i n very a r g i n i n e - r i c h h i s t o n e s of the Mouse/grasshopper ("stable  Saka-  yet the b a s a l p o r t i o n o f stage 3 n u c l e i appear to  s t a i n more i n t e n s e l y Stage  to f o l l o w w i t h the  are  AFG de-  also they  type  protamine").  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 t h e most  a d v a n c e d s t a g e o b s e r v e d and w e r e f o u n d i n O c t o b e r testes.  Thus, t h e y a r e p r o b a b l y mature sperm.  and  February  These n u c l e i  de-  - 45 monstrate  a v e r y i n t e n s e , uniform s t a i n i n g f o r DNA.  However, these  n u c l e i do not s t a i n w i t h 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 h y d r o l y s i s , which suggests t h a t protamines  are present.  They do s t a i n w i t h AFG  and e o s i n Y a f t e r p i c r i c a c i d h y d r o l y s i s and c o n t i n u e t o s t a i n s t r o n g l y even when a c e t y l a t i o n i s c a r r i e d out p r i o r t o s t a i n i n g . Stage 5 n u c l e i s t a i n i n t e n s e l y a f t e r the Sakaguchi r e a c t i o n .  These  r e s u l t s i n d i c a t e t h a t protamines a r e p r e s e n t i n mature sperm.  Un-  l i k e the p r e v i o u s change, the t r a n s i t i o n from the Mouse/grasshopper type o f sperm h i s t o n e 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 s t e p s a r e observed. With f o r m a l i n f i x e d m a t e r i a l , AFG s t a i n i n g without p r i o r l y s i s i s absent i n a l l stages o f s p e r m i o g e n e s i s . that free basic p r o t e i n s a r e not present. PIeurodeles w a l t i i , P i c h e r a l  (1970)  hydro-  This indicates  However, w i t h the u r o d e l e ,  observed f r e e b a s i c p r o t e i n s i n  the neck and t a i l r e g i o n s o f spermatozoa.  The appearance o f f r e e  b a s i c p r o t e i n s i n these r e g i o n s was c o r r e l a t e d w i t h 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 h i s t o n e s and then to the protamines.  There-  f o r e , i n the p r e s e n t study the s t a i n i n g procedure was r e p e a t e d on material f i x e d i n absolute e t h a n o l - g l a c i a l acetic a c i d  (3:1).  Under  these c o n d i t i o n s the t a i l s o f mature sperm do s t a i n w i t h AFG without h y d r o l y s i s ( F i g . 35).  T h i s suggested t h a t f o r m a l i n was h i d i n g  free  b a s i c p r o t e i n s by b i n d i n g w i t h the amino groups o f l y s i n e , the primary dye b i n d i n g s i t e s o f l y s i n e - r i c h h i s t o n e s .  However, when f o r m a l i n  f i x e d m a t e r i a l was t r e a t e d w i t h b o i l i n g water t o remove AFG  formaldehyde,  s t a i n i n g was s t i l l absent i n a l l stages o f s p e r m i o g e n e s i s .  The  t a i l s o f mature sperm do s t a i n a f t e r the Sakaguchi r e a c t i o n f o r a r g i n ine.  Thus f r e e b a s i c p r o t e i n s that a r e o n l y r e t a i n e d by C l a r k e * s f i x -  ative  (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 ) appear t o be p r e s e n t i n  the t a i l s o f mature spermatozoa.  - 46 TABLE V Cytochemistry o f newt spermiogenesis S t a i n i n g and pretreatment  Reactive material  1  2  Stages 3  a b  AFG-TCA 86°C  Basic proteins other than protamines  AFG-TCA 86°C, deamination  Basic p r o t e i n s rich i n arginine  AFG-picric acid  Basic proteins including protamines  + +  AFG-picric acid, acetylation  Basic proteins rich i n arginine  - +  Eosin-Y p i c r i c acid  Basic proteins except "cleavage histones"  + +  1  Eosin-Y p i c r i c acid, acetylation  Basic p r o t e i n s rich 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 a r e f o r f o r m a l i n f i x e d m a t e r i a l .  a = a p i c a l region, b = basal region.  ++  ++  -  Figures 2 9 - 3 2 . newt.  Stages of spermiogenesis i n the eastern red spotted  Feulgen stained.  Figure 2 9 .  47 -  Scale denots 50 ;u .  C y s t s 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 n u c l e i .  nucleus.  Figure 31»  Cysts of stage 4 n u c l e i .  Figure 3 2 .  Cysts of stage 5 n u c l e i .  Arrow indicates S e r t o l i c e l l  - 48 -  F i g u r e s 33-34.  Consecutive s e c t i o n s o f c y s t s o f stage 3 n u c l e i  s t a i n e d w i t h 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 lysis.  S c a l e denotes 50 /x  F i g u r e 33*  Without  F i g u r e 34.  With  .  deamination.  deamination.  - 49 -  F i g u r e 35«  Sperm of D i e m i c t y l u s v i r i d e s c e n s s t a i n e d w i t h  f a s t green without  p r i o r h y d r o l y s i s o f DNA.  The m a t e r i a l *ra.s 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 (3:1). s t a i n while the n u c l e i do n o t .  alkaline  The t a i l  The s c a l e denots 50 ja •  regions  - 50 2.  BIOCHEMISTRY  The  h i s t o n e c o m p o n e n t s o f newt t e s t i s a r e c o m p a r e d  p h o r e t i c a l l y w i t h those h e a r t , and  spleen  d e f i n e d by  the s l o w e s t  somatic  pipiens testes. r e g i o n and 38).  f r o m a combined p r e p a r a t i o n o f newt  ( F i g s . 56,  t i s s u e s and  and  37  and  38).  The  i n both the  the h i s t o n e r e g i o n from a combination  runs as a d o u b l e t  m i n u t e s ( F i g . 37).  g r a t i n g slower represent  i n the  which migrates  as a d o u b l e t ,  than NI, are s p e c i f i c to the a p p e a r s i n f r o n t o f N2  and  and  N2,  with histone  electro-  very  IV from  i n the  f a s t moving bands,  NI,  slightly  I f B o _ a m e r i c a n u s 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 similar.  faster band with  E l e c t r o p h o r e s i s o f newt  t o g e t h e r r e v e a l s t h a t NI  ( F i g . 39)»  miand  Occasionally, a faint  ( F i g . 37).  pea,  testis  and  g r a t e f a s t e r t h a n t h e t e s t i s s p e c i f i c b a n d s f r o m X^ l a e v i s w i t h N2  fast-  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  laevis t e s t i s histones  band B m i g r a t e s  histone  A number o f b a n d s  which migrates  testis.  protamine from salmon or t r o u t and  Two  the  of t i s s u e s ( F i g .  than the h i s t o n e r e g i o n are present products.  is  R.  In the H r e g i o n the  t e s t i s preparation.  degradation  (H)  with  testis  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 s t moving band, which c o - e l e c t r o p h o r e s e s  may  histone region  i s s i m i l a r to the p a t t e r n o b t a i n e d  p h o r e s i s f o r o n l y 70  liver,  f a s t e s t moving bands o b t a i n e d w i t h  Four bands a r e present  H o w e v e r , two  electro-  N2  mi-  (Fig.  39).  preparation,  strongly suggesting  that they  are  - 51 -  Figures  36  and  37•  ern r e d s p o t t e d and  newt r u n  stained with  F i g u r e 36. l i v e r and Top:  ates  15%  polyacrylamide  run  37*  f o r 70  from a combination o f somatic  east-  minutes  H i n d i c a t e s somatic histone  region.  tissues —  heart,  alone. run  with  histone  IV  Testicular histones.  band t h a t c o - e l e c t r o p h o r e s e d the  faint  co-electrophoresed Top:  gels  from the  spleen.  arrow d e s i g n a t e s which  on  buffalo black.  Histones  Bottom:  Figure  Densitometer tracings of histones  run  Bottom:  from  pea.  Arrow i n the  histone  with  IV  histone  from pea.  indicSecond  band which i s o c c a s i o n a l l y p r e s e n t with  protamine  alone. run with  region  salmon  protamine.  from salmon o r  trout.  and  - 52 -  F i g u r e 58.  Polyacrylamide  g e l electrophoresis patterns  from the e a s t e r n r e d s p o t t e d newt. s t a i n e d with b u f f a l o b l a c k .  Gels were r u n f o r 85 minutes and  H designates  somatic h i s t o n e  A.  " t e s t i c u l a r "histones.  B.  t e s t i c u l a r h i s t o n e s p l u s salmon protamine ( P ) .  C.  liver  D.  l i v e r h i s t o n e s p l u s h i s t o n e IV from pea.  F i g u r e 39.  of.histones  region.  histones.  Testicular  h i s t o n e s from the newt run with  h i s t o n e s from Xenopus l a e v i s  testicular  (A) and from Bufo americanus ( B ) .  38 -A  B  *  f  C JB.  TT • 8 D  H  N1N2— P-  39 A  B  li X2-  X3-  N1N2—  -N2+ B  - 53 D.  DISCUSSION  The b a s i c p r o t e i n changes d u r i n g spermiogenesis  i n the e a s t e r n  r e d s p o t t e d newt resemble the t r a n s i t i o n s d e s c r i b e d i n the s n a i l , H e l i x aspera, (Bloch and Hew 1960a), the s q u i d , L o l i g o  opalescens,  (Bloch 1962), and P l e u r o d e l e s w a l t i i  The e a r l y  stages o f spermiogenesis  ( P i c h e r a l 1970).  c o n t a i n somatic type h i s t o n e s which i n l a t e r  spermatids a r e r e p l a c e d by b a s i c 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 but not-extrac-table w i t h hot t r i c h l o r o a c e t i c acid-. t e i n s a r e r e p l a c e d by protamines,  In -turn, t h e s e  pro-  proteins that are s o l u b l e i n 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 a r g i n i n e . In the e a s t e r n r e d s p o t t e d nev/t the t r a n s i t i o n from the somatic type o f h i s t o n e to the " s t a b l e protamine"  i s p r o g r e s s i v e and v e r y  s i m i l a r to the c o n v e r s i o n observed i n P^ w a l t i i  ( P i c h e r a l 1970)*  Besides m o n i t o r i n g t h i s t r a n s i t i o n by s t a i n i n g techniques, 3 observed  the s e q u e n t i a l i n c o r p o r a t i o n o f H - a r g i n i n e i n t o  undergoing  t h i s h i s t o n e change.  Picheral nuclei  T h e r e f o r e , the g r a d u a l change i n  n u c l e a r s t a i n i n g does not r e p r e s e n t an unmasking o f 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 but a p r o g r e s s i v e accumulation ized proteins.  As w i t h t h e n u c l e a r b a s i c p r o t e i n s found i n the sperm  of the grasshopper 1969), P i c h e r a l  o f newly s y n t h e s -  (Bloch and Brack 1964) and t r o u t  ( L i n g e_t a l .  (1970) found t h a t i n P._ w a l t i i both the Mouse/grass-  hopper type o f p r o t e i n ( " s t a b l e protamine") and protamines  were  s y n t h e s i z e d i n the cytoplasm o f s p e r m a t i d s . The change i n D_. v i r i d e s c e n s from the Mouse/grasshopper type o f p r o t e i n to protamines served.  Picheral  i s sudden and no t r a n s i t i o n a l s t a g e s a r e ob-  (1970) found a s i m i l a r s i t u a t i o n i n  waltii.  In  3 t h i s s p e c i e s even the H - a r g i n i n e i n c o r p o r a t i o n showed no t r a n s i t i o n . The Salmon type o f sperm h i s t o n e might be c h a r a c t e r i s t i c o f Urodeles.  Bloch (I969) i n d i c a t e d t h a t the sperm o f Amphiuma were o f  the Salmon type w h i l e P i c h e r a l f o r the sperm o f P^_ w a l t i i .  (1970) reached the same c o n c l u s i o n  While  Bloch (1969) ( c i t i n g  c y t o c h e m i c a l data) c l a s s i f i e d the sperm o f  unpublished  v i r i d e s c e n s as the  Mouse/grasshopper type, the present study i n d i c a t e s t h a t these sperm a r e a c t u a l l y o f the Salmon t y p e .  Bloch might have sampled t e s t e s i n  - 54  -  which the most advanced stage of spermiogenesis was l a t e spermatid with the Mouse/grasshopper type of p r o t e i n . This could account f o r his erroneous c o n c l u s i o n . P i c h e r a l (1971) was also able to c o r r e l a t e these basic p r o t e i n changes during spermiogenesis with u l t r a s t r u e t u r a l observations. In the e a r l y spermatid chromatin f i b e r s were organized i n a loose network 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 t o wards the c e n t r a l 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 s t r u c t u r e was observed on the t r a n s i t i o n to protamines. Perhaps the most i n t e r e s t i n g observation made by P i c h e r a l (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, f r e e basic proteins appeared i n the neck region of the spermatid. Subsequently, when the "stable protamine" was replaced by protamines, f r e e basic p r o t e i n s appeared i n the t a i l region. These r e s u l t s s t r o n g l y imply that the free basic p r o t e i n s r e present sloughed o f f nuclear h i s t o n e s . In the present study, f r e e basic proteins were not observed at any stage i n formalin f i x e d materi a l . P i c h e r a l employed a s i m i l a r f i x a t i v e ; however, h i s 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 v i r i d e s c e n s . Unfortunately, not a l l stages of spermiogenesis were f i x e 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 c l e a r . However, Vaughn (as reported by Bloch 1966) found that some basic proteins displaced from the nucleus during r a t spermiogenesis were r e t a i n e d 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 f o r m a l i n . The e l e c t r o p h o r e t i c comparison of t e s t i c u l a r histones from D. v i r i d e s c e n s with somatic histones i n d i c a t e s that two t e s t i s s p e c i f i c  - 55 bands, NI and N2, a r e p r e s e n t . protamine,  S i n c e N2 migrates c l o s e s t t o salmon  t h i s band i s l i k e l y c o n f i n e d to mature sperm which c y t o -  c h e m i c a l l y have been shown to behave l i k e salmon sperm.  In a d d i t i o n ,  N2 migrates w i t h band B from Bufo americanus t e s t e s , which a l s o t a i n c e l l s t h a t s t a i n s i m i l a r to the mature sperm o f  con-  viridescens.  The NI d o u b l e t , then, i s l i k e l y s p e c i f i c t o stage 3 and k n u c l e i and r e p r e s e n t s the " s t a b l e protamine."  The i n t e r p r e t a t i o n o f these r e -  s u l t s i s s i m i l a r to the one g i v e n by "Bloch (1962) to r e s u l t s o b t a i n e d with the s q u i d , which show a s t a i n i n g p a t t e r n s i m i l a r to the e a s t e r n red  s p o t t e d newt.  S t a r c h g e l e l e c t r o p h o r e s i s showed f o u r major  groups o f h i s t o n e s from s q u i d t e s t e s . corresponded  The two slowest moving bands  to h i s t o n e s from somatic t i s s u e s while the two  fastest  moving bands were c o n f i n e d t o l a t e spermatid and sperm n u c l e i  (these  stages were s e p a r a t e d by c e n t r i f u g a t i o n i n a sucrose g r a d i e n t ) .  The  f a s t e s t moving band was very s i m i l a r i n amino a c i d composition and m o l e c u l a r weight to t y p i c a l protamines  and was a l s o found i n a p r e -  p a r a t i o n c o n s i s t i n g e n t i r e l y o f mature sperm.  Therefore, Bloch i n -  d i c a t e d t h a t t h i s p r o t e i n was present i n c e l l s t h a t were cytochemi c a l l y e x t r a c t a b l e w i t h hot t r i c h l o r o a c e t i c a c i d and v e r y r i c h i n a r ginine.  The s l i g h t l y slower moving band was thought  to o c c u r i n the  l a t e spermatids and to r e p r e s e n t the " s t a b l e protamine."  This protein  c o n t a i n e d a l l the amino a c i d s of t y p i c a l h i s t o n e s but had an a r g i n i n e to  l y s i n e r a t i o o f about s i x .  contain cysteine.  T h i s v e r y a r g i n i n e - r i c h h i s t o n e d i d not  P i c h e r a l (19?0), on the o t h e r hand, r e p o r t e d t h a t  -*- Hi. ' a l t i i the n u c l e i c o n t a i n i n g t h e " s t a b l e protamine" n  v  i n c y s t e i n e w h i l e the mature sperm were n o t .  were  rich  Whether the l a t e sperm-  a t i d s o f the e a s t e r n r e d s p o t t e d newt c o n t a i n c y s t e i n e i s unknown. Two arguments may be advanced a g a i n s t 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 h i s t o n e bands i n  viridescens.  F i r s t , one would  expect band N2 to occur i n g r e a t e r amounts s i n c e a t the time the t e s t e s were e x t r a c t e d mature sperm should have been the predominant c e l l type  (Baker 1966).  However, spermiogenesis  might have been  delayed o r i n t e r r u p t e d i f the animals were i m p r o p e r l y maintained the commercial d e a l e r .  by  Secondly, s i n c e the sperm o f D. v i r i d e s c e n s  - 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  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 . s l i g h t l y s l o w e r than salmon protamine. protamine" i s present that d i f f e r s  expect the  Y e t , b a n d N2  P e r h a p s , an  mobility.  migrates  "amphibian  j u s t s l i g h t l y from salmon  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 in  prot-  prot-  difference  - 57 IV.  CHANGES IN BASIC PROTEINS DURING SPERMIOGENESIS I N THREE CARTILAGINOUS FISH A.  INTRODUCTION  I n o r d e r t o 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 t h e M y t i l u s type o f sperm h i s t o n e l a r g e q u a n t i t i e s o f s t a r t i n g m a t e r i a l a r e r e q u i r e d . An i d e a l o r g a n i s m f o r such a s t u d y must have l a r g e t e s t e s , be e a s i l y obtained,  and o f c o u r s e , have t h e M y t i l u s t y p e o f sperm h i s t o n e .  C i t i n g Kossel, Bloch  (1969) c l a s s e d t h e s h a r k , C e n t r o p h o r u s g r a n u -  l o s a , a s c o n t a i n i n g t h e M y t i l u s t y p e o f sperm h i s t o n e .  Bloch  also  c l a s s e d the r a y , D a s y a t i s s a b i n a , a s t h e M y t i l u s t y p e on t h e b a s i s of unpublished cytochemical  data.  The elasmobranchs then a p p e a r t o  be s u i t a b l e organisms f o r s t u d y i n g t h e M y t i l u s sperm h i s t o n e Therefore,  a s t u d y o f t h e b a s i c p r o t e i n changes d u r i n g  genesis i n the d o g f i s h skate  (Squalus  -suckleyi  (Raja rhj,na) was u n d e r t a k e n .  type.  spermio-  ) and i n t h e l o n g  nose  L a t e r , when t h e M y t i l u s t y p e  o f sperm h i s t o n e was n o t f o u n d i n these e l a s m o b r a n c h s , t h e s t u d y 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 B.  METHODS AND MATERIALS  1.  CYTOCHEMISTRY  Squalus s u c k l e y i  "';• t h e d o g f i s h , R a j a r h i n a , t h e  l o n g nose s k a t e , and H y d r o l a g u s c o l l i e i , off  colliei).  t h e r a t f i s h , were c o l l e c t e d  Conox, B r i t i s h C o l u m b i a , i n May and November.  C o l l e c t i o n s were  made by means o f a t r a w l o p e r a t e d from the Canadian F i s h e r i e s R e s e a r c h 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 d r a g o f a p p r o x i m a t e l y 15  m i n u t e s , the n e t was emptied onto t h e deck where m a t u r e - l o o k i n g s p e c imens o f each s p e c 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  e x c i s e d and t h i n s e c t i o n s were c u t a t r i g h t a n g l e s t o t h e l o n g a x i s o f t h e 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 o r 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).  not be p r o m p t l y r e t u r n e d to 32 h.  Since m a t e r i a l  to the l a b o r a t o r y , f i x a t i o n occurred  However, w i t h t h e r a t f i s h , an o c c a s i o n  could f o r 2h  a r o s e v/here m a t e r i a l  - 58 was  f i x e d f o r 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 compens a t e f o r the i n c r e a s e d time spent i n f o r m a l i n . sues were dehydrated and  embedded as d e s c r i b e d  Otherwise, the  tis-  i n the anuran s e c t i o n .  A l l s t a i n i n g procedures performed on t h i s m a t e r i a l have been described  previously.  2.  BIOCHEMISTRY  For b i o c h e m i c a l manner, were used.  work, the same organisms, c o l l e c t e d i n the same The  t e s t e s and  p o s s i b l e , under c o n d i t i o n s  l i v e r s were removed as q u i c k l y  t h a t were sometimes d i f f i c u l t , and  as  stored  on dry i c e i n Thermos b o t t l e s f o r 24-32 h, although some m a t e r i a l was  s t o r e d i n t h i s manner f o r o n l y a 6-10  a t o r y t h i s m a t e r i a l was u n t i l use.  Since  h period.  s t o r e d a t -20°C and and  cytochemical  formed on m a t e r i a l from the same t e s t i s . i n August and  labor-  i n some cases a t -70°C  j u s t a s m a l l p o r t i o n of a t e s t i s was  chemical s t u d i e s , b i o c h e m i c a l were obtained  In the  work was  used f o r c y t o often per-  Also, for dogfish,  testes  September as w e l l as i n November and  May.  I n i t i a l l y , attempts were made to e x t r a c t b a s i c p r o t e i n s from  the  d o g f i s h t e s t i s by the procedure used by I n g l e s et a l . (1966) to show the presence of protamine i n t r o u t t e s t e s .  The  sample o f t e s t i s  homogenized w i t h a Dounce homogenizer i n the presence o f 0.2 c e n t r i f u g e d a t 12,000g f o r 30 minutes. against obtained  d i s t i l l e d water f o r 24 h and  The  then l y o p h i l i z e d .  were examined by p o l y a c r y l a m i d e  iously outlined.  supernatant was  was  N HC1  and  dialyzed  The  proteins  d i s c e l e c t r o p h o r e s i s as p r e v -  T h i s procedure \vas t r i e d three times and  then aban-  doned. T e s t e s were a l s o e x t r a c t e d i n the manner used to demonstrate protamine i n the mouse t e s t i s (2 grams), obtained the t e s t i s , was  (Lam  and  Bruce 1970).  A tissue slice  by c u t t i n g a t r i g h t a n g l e s to the l o n g a x i s of  homogenized g e n t l y w i t h a Dounce homogenizer i n d i s -  t i l l e d water and  l e f t to l y s e f o r 30 minutes.  c e n t r i f u g e d at 10,000g f o r 10 homogenized again  minutes and  i n d i s t i l l e d water.  The  homogenate  the p e l l e t o b t a i n e d  T h i s homogenate was  was was  centrifuged  - 59 -  at 12,000g f o r 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 a t room temperature f o r 2 h. The suspension vras centrifuged at 12,000g f o r 20 minutes and the supernatant l y o p h i l i z e d .  The p r o t e i n s i n the l y p h o l i z e d e x t r a c t were  examined by polyacrylamide d i s c e l e c t r o p h o r e s i s .  This same proced-  ure was followed w i t h l i v e r s as w e l l as with t e s t e s . Testes were also extracted with a c i d 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 c e n t r i f u g a t i o n as  described above were homogenized with a Dounce homogenizer a t room temperature i n 5% P -mercaptoethanol (v/v) and 8 M urea i n 0.1M T r i s at pH 8.5.  The suspension was incubated a t 37° C f o r 2 h i n a  Dubnoff metabolic shaking incubator and then brought to a f i n a l concentration of 0.5 N HC1 by the a d d i t i o n o f concentrated HC1.  The  homogenate was l e f t f o r 1 h at room temperature and then c e n t r i f u g e d at 15«000g f o r 20 minutes. the H  +  The supernatant was brought to pH 5.0 by  a d d i t i o n of NaOH and a p p l i e d to a CM30-cellulose column (1x10 cm, form).  The column was f i r s t washed w i t h 200 ml o f d i s t i l l e d H.,0  and then the basic proteins were eluted w i t h 0.1N HC1.  The HC1 f r a c -  t i o n was l y o p h i l i z e d and examined by g e l e l e c t r o p h o r e s i s . Basic proteins were also extracted from a crude nuclear preparation.  E s s e n t i a l l y , the f i r s t four steps used t o obtain chromatin  (described p r e v i o u s l y ) 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 l a y e r s of washed cheesecloth, centrifuged a t l,500g f o r 15 minutes, resuspended i n saline-EDTA, and centrifuged again a t l,500g f o r 15 minutes. scope.  The nuclear p e l l e t was checked under a l i g h t micro-  To each gram of n u c l e i , 5 w l of 0.2 M HgSO^ was added (as  recommended f o r t r o u t t e s t i s n u c l e i , Ling ejt a l . 1971).  A f t e r being  homogenized and l e f t at room temperature f o r 20 minutes, the mixture was centrifuged a t 12,000g f o r 20 minutes.  Cold 100% ethanol (3-4  volumes) was added to the supernatant which was then stored a t -20°C f o r approximately 24 h«  The p r e c i p i t a t e which formed i n t h i s time  was recovered by c e n t r i f u g a t i o n (12,000g f o r 20 minutes), washed once  - 60 w i t h e t h a n o l , and d r i e d i n vacuo* Chromatin was  prepared and e x t r a c t e d w i t h a c i d as d e s c r i b e d  i n the anuran s e c t i o n .  C.  RESULTS  1.  CYTOCHEMISTRY  a.  Elasmobranchii The  t e s t e s of elasmobranch f i s h e s a r e composed o f s p h e r i c a l  f o l l i c l e s or ampullae ( S t a n l e y 1966), a l s o termed t u b u l e s by some authors the g e m  (Simpson and Wardle 1967).  Within a s p h e r i c a l f o l l i c l e a l l  elements are a t the same stage o f d i f f e r e n t i a t i o n and  o r g a n i z e d i n t o a number of spermatocysts  or c y s t s .  The  are  follicles  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 a s pect o f the t e s t i s and move s t e a d i l y away as they develop, f o l l o w e d c l o s e l y by s u c c e s s i v e l y younger s t a g e s ( S t a n l e y 1966).  F o l l i c l e s that  have reached the v e n t r a l and v e n t r o - m e d i a l area of the t e s t i s c o n t a i n the l a t e stages of spermiogenesis i o g e n e s i s i s completed,  and mature spermatozoa.  When sperm-  the f o l l i c l e s open to the c o l l e c t i n g d u c t u l e  system and the spermatozoa a r e r e l e a s e d .  Thus i n a s e c t i o n 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 i s a l l stages of spermatog e n e s i s are present  ( F i g . 40).  G e n e r a l l y , the elasmobranch t e s t i s c o n t a i n s a l l s t a g e s o f spermatogenesis  throughout  the year  (Simpson and Wardle 1967).  an annual c y c l e i n the d o g f i s h t e s t i s has been observed  While  (Simpson and  Wardle I967), o n l y the abundance of v a r i o u s stages changed.  Mature  sperm were most frequent in. t e s t e s caught between J u l y and December and were l e a s t common i n t e s t e s caught i n February and May.  No i n -  f o r m a t i o n i s a v a i l a b l e on the presence or absence o f a s e a s o n a l c y c l e in  the skate In  testis.  the present study spermiogenesis  i n the d o g f i s h and  skate  has been d i v i d e d i n t o e i g h t s t a g e s on the b a s i s o f n u c l e a r morphology and b a s i c p r o t e i n c o m p o s i t i o n .  The stages a r e i l l u s t r a t e d i n  - 61 f i g u r e s 4-1-52 and the c y t o c h e m i c a l r e s u l t s f o r these stages a r e summarized i n Tables VI and V l l . The  process o f n u c l e a r e l o n g a t i o n has been d i v i d e d  ( F i g s . 41-44).  into 4 stages  Stage 1 n u c l e i f o r both s p e c i e s a r e round and l o o s e l y  o r g a n i z e d i n the f o l l i c l e s .  Stage 2 n u c l e i a r e shaped l i k e  drops w h i l e stage 3 n u c l e i a r e r o d l i k e .  tear  Stage 4 n u c l e i a r e t h i n and  c y l i n d r i c a l and a r e o r g a n i z e d i n t o d e f i n i t e c y s t s , p a r t i c u l a r l y i n the case o f s k a t e s . and  The stage 4 n u c l e i o f the s k a t e appear l o n g e r  s t r a i g h t e r than those o f d o g f i s h . S p i r a l i z a t i o n has been d i v i d e d i n t o two s t a g e s .  Stage 5 n u c l e i  are s p i r a l i z e d a t the base and r o d - l i k e a t the apexes. i s more n o t i c e a b l e  This  with s k a t e s ( F i g . 49) than with d o g f i s h  process  ( F i g . 45).  Stage 6 n u c l e i a r e completely s p i r a l i z e d ( F i g s . 46 and 50). Stage 7 and 8 n u c l e i a r e m o r p h o l o g i c a l l y i n d i s t i n u i s h a b l e each other ( F i g s . 47 and 51); y e t d i f f e r c y t o c h e m i c a l l y . distinguished  from  They a r e  from stage 6 n u c l e i by b e i n g o r g a n i z e d i n t o v e r y dense-  l y packed c y s t s  ( F i g s . 47, 48, 51 and 52).  Stages 1, 2, and 3 i n both skate and d o g f i s h s i m i l a r (Tables V I and V I I ) . types o f TCA h y d r o l y s i s ,  are cytochemically  These n u c l e i s t a i n with AFG a f t e r a l l  i n d i c a t i n g the absence o f protamines.  How-  ever, these n u c l e i do not s t a i n w i t h AFG i f they have been deaminated o r a c e t y l a t e d tones.  i n d i c a t i n g the absence o f very a r g i n i n e - r i c h h i s -  These n u c l e i s t a i n weakly a f t e r t h e Sakaguchi r e a c t i o n f o r  arginine.  T h e r e f o r e , these n u c l e i c o n t a i n  o f somatic  cells.  h i s t o n e s s i m i l a r t o those  Stage 4 n u c l e i s t a i n w i t h AFG a f t e r IN TCA h y d r o l y s i s 5% TCA h y d r o l y s i s  a t 89°C Table V I I ) .  absent a f t e r 5% TCA h y d r o l y s i s the presence o f a protamine. a c i d despite  and a f t e r  However, s t a i n i n g i s l i g h t o r  a t 97°C (Table V I I ) . These n u c l e i s t a i n w i t h  This  suggests  AFG-picric  a c e t y l a t i o n i n d i c a t i n g the presence o f v e r y  arginine-  r i c h h i s t o n e s and s t a i n moderately a f t e r the Sakaguchi r e a c t i o n f o r arginine. Stage 5 and 6 n u c l e i show i d e n t i c a l s t a i n i n g p r o p e r t i e s VI and V I I ) .  (Tables  They both s t a i n weakly with AFG and IN TCA h y d r o l y s i s  - 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 a f t e r p i c r i c acid  and continue to do so even a f t e r acetylation. presence of very arginine-rich histones.  hydrolysis  This indicates the  This conclusion  i s rein-  froced by the intense staining a f t e r the Sakaguchi reaction. staining i s absent after TCA hydrolysis at 97°C  9  Since  these r e s u l t s sug-  gest that protamines are present i n these n u c l e i . Stage 7 nuclei stain well with AFG a f t e r IN TCA hydrolysis at 60°C" and s t a i n moderately well a f t e r 5% TCA hydrolysis at 89°C (Table VI and VII).  However they do not s t a i n with AFG when the  hydrolysis temperature i s raised to 97°C.  These nuclei are very  arginine-rich since they s t a i n with AFG - p i c r i c acid despite acetylation.  The intense red staining observed after the Sakaguchi r e -  action supports the contention that the very arginine-rich histones are present.  These n u c l e i , l i k e stages 5 and 6, appear to contain  protamines. Stage 8 nuclei stain well with AFG under a l l conditions hydrolysis  (Figs. 48 and 52).  Thus protamines are absent.  of TCA These  n u c l e i continue to stain intensely despite deamination or acetyl a t i o n 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 r e 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 i n 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 s e c t i o n . 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 i n d i c a t e areas i n which f o l l i c l e s of p r o g r e s s i v e l y l a t e r development 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  4l~47.  Figures genesis*  Follicles  Follicles  F i g u r e 42.  Tear  of a  containing stages of dogfish  Feulgen s t a i n i n g .  F i g u r e 4l.  -  S c a l e denotes  50/x  c o n t a i n i n g round stage 1  drop-shaped  stage 2 n u c l e i .  spermio-  .  nuclei.  L d e s i g n a t e s lumen  follicle.  F i g u r e 43.  Follicles  of rod l i k e stage 3  nuclei.  F i g u r e 44.  Follicles  of stage 4 n u c l e i .  The o r g a n i z a t i o n o f  nuclei  cysts i s noticeable.  into  - 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 n u c l e i occur i n densely-  packed cysts.  Figure 48.  A l k a l i n e f a s t green 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 at 97°C.  N u c l e i 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 spermiogenesis.  Feulgen s t a i n i n g .  Scale denotes 50J » 1  Figure 4-9. F o l l i c l e containing stage 5 n u c l e i which have begun spiralization.  Figure 50.  L i n d i c a t e s lumen of the f o l l i c l e .  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 n u c l e i occur i n densely-  packed c y s t s .  Figure 52.  A l k a l i n e f a s t green 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 at 97°C  N u c l e i 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 V I  Cytochemistry o f d o g f i s h and skate  S t a i n i n g and pretreatment  Reactive material  AFG-TCA  Basic p r o t e i n s o t h e r than protamines  97°C AFG-TCA  97°C  deamination AFG-picric acid  spermiogenesis  Stages  3  +  +  +  +  +  +  5  Basic proteins rich i n arginine Basic proteins including protamines  AFG-picric a c i d , Basic p r o t e i n s acetylation rich 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  -  67 -  TABLE V I I  E f f e c t o f h y d r o l y s i s c o n d i t i o n s on AFG s t a i n i n g i n d o g f i s h and s k a t e spermiogenesis  Hydrolysis conditions  1  P i c r i c acid f o r 6 h a t 60°C  +  IN TCA a t 60°C for J h  2  3  4  +  +  +  +  +  +  +  +  +  +  +  +  Stages 5  6  7  8  +  +  +  +  +  +  +  +  +  +  -  -  +  .+  -  +  5% TCA a t 89°C f o r 15 mins 5% TCA a t 97°C f o r 15 mins NOTE:  TCA = t r i c h l o r o a c e t i c  acid.  - 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 s p h e r i c a l ampullae and w i t h i n each ampullae germ elements are at the same stage of development (Stanley 1963). A l l stages of spermatogenesis are seen i n s e c t i o n s cut a t r i g h t angles to the long a x i s 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 d i v i d e d i n t o seven stages on the b a s i s of nuclear morphology and b a s i c p r o t e i n composition. The stages are i l l u s t r a t e d i n f i g u r e s 53-59 and the cytochemical r e s u l t s are summarized i n Tables V I I I and IX. In a d d i t i o n , cytochemical r e s u l t s are recorded f o r sperm i n the e p i didymis. Stages 1-4 of spermiogenesis represent steps i n the process of nuclear elongation. Stage 1 n u c l e i are round and l o o s e l y organized i n the f o l l i c l e s ( F i g . 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 ( F i g . 54). The rod shaped n u c l e i aligned at the periphery of f o l l i c l e s have been designated as stage 3 ( F i g . 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 i n t o c y s t s are stage 4 ( F i g . 56). Each cyst of stage 4 n u c l e i points toward a S e r t o l i nucleus, which i s not always apparent due to the d i f f u s e chromatin s t a i n i n g , while the t a i l s of these spermatozoa protrude i n t o the c e n t r a l lumen of the f o l l i c l e . A f t e r 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 a n t e r i o r t i p and n u c l e i demonstrating t h i s t r a n s i t i o n have been termed stage 5 ( F i g . 57 )• Stage 6 n u c l e i have completed s p i r a l i z a t i o n ( F i g . 58) while stage 7 n u c l e i are organized i n t o much more t i g h t l y packed cysts than stage 6 ( F i g . 59). The f o l l i c l e s as a whole appear to change i n s i z e as spermiogenesis proceeds. F o l l i c l e s containing e a r l y stages of spermiogenesis are considerably l a r g e r than those containing l a t e r stages. Stanley (1966) observed a s i m i l a r phenomenon i n S c y l i o r h i n u s and Torpedo. Stages 1, 2, and 3 give s i m i l a r cytochemical r e s u l t s (Tables V I I I  - 69 and  IX.  These n u c l e i s t a i n w i t h AFG a f t e r TCA h y d r o l y s i s ,  indic-  a t i n g the absence o f protamines; do not s t a i n with AFG i f s e c t i o n s are p r e v i o u s l y  deaminated o r a c e t y l a t e d ,  very a r g i n i n e - r i c h h i s t o n e s ; reaction  for arginine.  c e l l s t a i n i n g pattern  i n d i c a t i n g the absence o f  and s t a i n l i g h t l y a f t e r the Sakaguchi  These n u c l e i e x h i b i t e d and t h e r e f o r e  contain  the t y p i c a l somatic  the somatic type o f  histones. Stage k n u c l e i g i v e unusual s t a i n i n g r e s u l t s (Tables V I I I and I X ) . . S t a i n i n g i s dependent on the temperature a t which TCA hydrol y s i s i s carried out. hydrolysis  Stage 4 n u c l e i s t a i n with AFG a f t e r IN TCA  a t 60°C o r a t 89°C; and s t a i n f a i n t l y or.not a t a l l a f t e r  TCA h y d r o l y s i s acid hydrolysis  a t 97°C.  These n u c l e i s t a i n w i t h AFG a f t e r  picric  and continue to s t a i n even i f a c e t y l a t e d .  g e s t s they c o n t a i n  very a r g i n i n e - r i c h h i s t o n e s .  n u c l e i g i v e o n l y a moderate Sakaguchi  reaction. results  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 a t 60°C and do not s t a i n a t a l l a f t e r 5% TCA h y d r o l y s i s or 97°C. and  sug-  However, these  Stages 5 and 6 n u c l e i a l s o g i v e unusual s t a i n i n g (Tables V I I I and I X ) .  This  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  continue to do so d e s p i t e  hydrolysis  a c e t y l a t i o n , i n d i c a t i n g the presence  of v e r y a r g i n i n e - r i c h h i s t o n e s . a f t e r the Sakaguchi r e a c t i o n  a t 89°C  These n u c l e i a l s o s t a i n  f o r arginine.  strongly  This s t a i n i n g  pattern  suggests the presence o f protamines. Stage 7 n u c l e i and n u c l e i found i n the epididymis g i v e i c a l cytochemical r e s u l t s . o f TCA h y d r o l y s i s  These n u c l e i s t a i n under a l l  conditions  and continue to do so even i f the s e c t i o n s  aminated p r i o r to s t a i n i n g .  S t a i n i n g w i t h AFG a f t e r p i c r i c  hydrolysis  acetylation.  continues d e s p i t e  identa r e deacid  An i n t e n s e r e d r e a c t i o n  i s given by these n u c l e i a f t e r treatment with the Sakaguchi reagent. These r e s u l t s i n d i c a t e the presence o f 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 b a s i c p r o t e i n s  were not observed i n any o f the stages o f  spermiogenesis. The  e f f e c t o f f i x a t i o n on AFG s t a i n i n g and deamination was  - 70 studied on nuclei found i n the epididymis. 6-32 h did not affect the r e s u l t s .  Fixation times between  - 71 -  F i g u r e s 53-59. genesis.  F i g u r e 53.  F o l l i c l e s c o n t a i n i n g stages o f r a t f i s h  Feulgen s t a i n i n g .  S c a l e denotes 10}X  Round stage 1 n u c l e i .  spermio-  .  L i n d i c a t e s lumen o f the  follicle.  F i g u r e 5^.  Tear, drop-shaped stage 2 n u c l e i .  F i g u r e 55.  Rod-shaped stage 3 n u c l e i .  F i g u r e 56.  Stage h n u c l e i o r g a n i z e d  into  cysts.  ,.  • .  • • • .••»  ••••••  • • • •*  •• .  • * • •"•V-;-.  •  -V  .it^v^W.  • « •  •  1  «• ..  \t». —*? '  V a •  *  • •  • •  4  • •  «A  •  5*  •V *  1*  •  ^  55  ^  ^  ^  •  \ —  -  F i g u r e 57«  Stage 5 n u c l e i ,  72  -  These n u c l e i a r e undergoing n u c l e a r  spiralization.  Figure 58.  Stage 6 n u c l e i .  F i g u r e 59 •  Stage 7 n u c l e i .  S p i r a l i z a t i o n has been completed.  - 73 TABLE VIII Cytochemistry o f r a t f i s h  S t a i n i n g and pretreatnent  Reactive material  AFG-TCA 97°C  Basic p r o t e i n s o t h e r than protamines  AFG-TCA 97°C, deamination  Basic proteins rich i n arginine  AFG-picric acid  Basic proteins including protamines  AFG-picric acid, acetylation  Non-nucleic acidaesociated basic proteins  Sakaguchi  Protein-bound arginine  *  4  Stages  5  6  +  +  +  +  +  +  +  +  +  +  ++  ++  Basic p r o t e i n s rich i n arginine  AFG  NOTE:  spermiogenesis  +  ++  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 .  Sperm found i n t h e e p i d i d y m i s gave i d e n t i c a l r e s u l t s to s t a g e 7»  - 7k -  TABLE IX  E f f e c t of h y d r o l y s i s conditions on AFG s t a i n i n g i n r a t f i s h spermiogenesis  Hydrolysis conditions  1  2  3  4-  Stages 5 6  P i c r i c acid for 6 h a t 60 C  +  +  +  +  +  +  +  3h  +  +  +  +  +  +  +  5% TCA at 8 9 ° C f o r 15 mine  +  +  +  +  % TCA a t 97°C for 15 mins  +  +  +  7  INTOA a t 60°C for  NOTE:  TCA = t r i c h l o r o a c e t i c a c i d .  - 75 -  in  2.  BIOCHEMISTRY  The  histone  components o f  60-62.  figures  phoresis  of l i v e r  b o u n d by  these  Two  histones  histone  IV  histone  region are  and  The testis two  faster  the  dogfish histone  very  fast  the  region  fastest  slower  The  his-  moving  IV  i s specific  to  ( F i g . 62).  IV  region are  This  to  Band  testis  to  S  and band  the  testis  and  dogfish  protamine.  bands w i l l  histone  the  Therefore,  Liver histones  m o v i n g band  i s specific  ( F i g . 65).  the  from d o g f i s h l i v e r  63-65.  than  with  proteins.  be  in this  migrate  defined  bands a r e a l s o p r e s e n t  histone  (D)  electro-  i n the region  from pea.  testis  and  the  testis from  The  both  bands  contaminating  the  as  as  mi-  proteins. migrates  p r o t e i n appears i d e n t i c a l  A  with  with  band  skate. use  of other  e x t r a c t i o n methods r e i n f o r c e s t h e  o n l y 1 p r o t a m i n e band i s p r e s e n t  Figures after  Two  fastest  the  f a s t moving band  The that  The  contaminating  these  l i v e r s migrates with  slower than  compared  region.  of protamine - s p e c i f i c i t y  bound by  the  the  g e l s and histone  are  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  in figures  area  r e g i o n and  after  bands.  salmon protamine  region.  salmon protamine S of  two  e l e c t r o p h o r e s i s of histones  t e s t e s and  the  electrophoretic mobility with  b a n d s and  grating  contains  than h i s t o n e  with  i s illustrated  histone  d e f i n e d as  livers  A number o f bands m i g r a t i n g  characteristics  identical  evident  polyacrylamide  l i k e l y non-histone  co-electrophoreses two  be  region  from pea.  which migrates  S has  on  testes also  band i n the h i s t o n e  t e s t e s and  major bands a r e  bands w i l l  tone r e g i o n of  skate  66  the  and  67  illustrate  In the  t r a c t e d by  the  two  mouse p r o t a m i n e . protamine region d i d not  yield  skate  methods u s e d by  with  Band D i s p r e s e n t  obtained  dogfish  testis,  b a s i c p r o t e i n s were  Lam  Bruce  and  one  ex-  (1971) t o e x t r a c t major band i n  Extraction of l i v e r s  a protamine-like  and  testes.  salmon p r o t a m i n e i s  B o t h methods y i e l d e d o n l y one ( F i g . 68).  dogfish  In both skate  co-electrophoreses  case o f the  and  electrophoretic patterns  a c i d e x t r a c t i o n of n u c l e i .  moving band t h a t  present.  the  i n skate  conclusion  by  these  the  methods  protein.  i n t e s t e s c a u g h t i n May,  A u g u s t , and  November.  - 76 A l t h o u g h no a t t e m p t was made t o q u a n t i t a t e t h e r e s u l t s , b a n d D a p p e a r s t o b e m o s t e a s i l y e x t r a c t e d f r o m t e s t e s c a u g h t i n May. Since the stages o f spermatogenesis i n the elasmobranch are  arranged i n l a y e r s from t h e l a t e r a l  ley  1966), a t t e m p t s w e r e made t o i s o l a t e c e l l  t e s t i s into long strips.  to the ventral areas  testis (Stan-  types by c u t t i n g the  However, t h i s method p r o v e d  unsuccessful  s i n c e t h e 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  difficult.  The a p p l i c a t i o n o f t h e 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 u s e d b y Lam and Bruce useful.  (1970) t o s e p a r a t e mouse s p e r m a t o g e n i c c e l l s m i g h t  prove  - 77 -  F i g u r e s 60-62. 15%  Densitometer t r a c i n g s o f skate h i s t o n e s run  polyacrylamide  black.  g e l s f o r 80 minutes and  stained with b u f f a l o  The h i s t o n e s were e x t r a c t e d from chromatin.  somatic h i s t o n e r e g i o n .  on  H indicates  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 -  phoreses w i t h h i s t o n e IV from  pea.  F i g u r e 60.  Liver histones.  Figure 6 l .  Testicular histones.  F i g u r e 62.  T e s t i c u l a r h i s t o n e s run with salmon protamine.  774  - 78 -  F i g u r e s 63-65.  Densitometer t r a c i n g s of dogfish histones run  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 m i n u t e s a n d s t a i n e d w i t h b u f f a l o black.  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 .  somatic histone region.  Arrow  phoreses w i t h h i s t o n e IV from  H indicates  i n d i c a t e s t h a t the band c o - e l e c t r o pea.  F i g u r e 63.  Liver histones  F i g u r e 64.  Testicular histones.  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 run w i t h salmon  protamine.  - 79 -  Figures 66 and 67.  Densitometer tracings of t e s t i c u l a r histones  from skate and dogfish run on 15% polyacrylamide gels for 80 minutes and stained with buffalo black. ted from crude nuclear preparations. region.  The histones were extrac-  H indicates somatic histone  Arrow indicates that the band cco-electrophoreses with  histone IV from pea.  Figure 66. Top:  Skate t e s t i c u l a r histones. electrophoresed alone.  Bottom: electrophoresed with salmon protamine.  Figure 67. Top:  Dogfish t e s t i c u l a r histones. electrophoresed alone.  Bottom:  electrophoresed with salmon protamine.  - 8o -  F i g u r e 68.  Polyacrylamide gel electrophoresis patterns  h i s t o n e s from the s k a t e . A.  G e l s r u n a s i n F i g u r e s 60-67o  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  nuclear  preparation. 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  of  Lam and B r u c e (1971). D.  Testicular histones  e x t r a c t e d from  chromatin.  of  - 81 D.  DISCUSSION  The b a s i c p r o t e i n changes d u r i n g spermiogenesis i n the t h r e e c a r t i l a g i n o u s f i s h s t u d i e d a r e remarkably s i m i l a r and v e r y u n u s u a l . In a l l three s p e c i e ; the t r a n s i t i o n to a v e r y a r g i n i n e - r i c h h i s t o n e begins a t the ends o f n u c l e a r e l o n g a t i o n and the b e g i n n i n g o f nuclear spiralization.  However, i n s t e a d of the appearance  of  "stable  protamines" o r the Mouse/grasshopper type o f sperm h i s t o n e f o l l o w e d 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 Hew  ( B l o c h and  1960a), P l e u r o d e l e w a l t i i . ( P i c h e r a l 1970), the s q u i d ( B l o c h  and the e a s t e r n r e d s p o t t e d newt (part I I I o f t h i s t h e s i s ) , appear  f i r s t f o l l o w e d by s t a b l e  1962)  protamines  protamines.  Three l i n e s o f evidence suggest that t h i s i s the c o r r e c t sequence o f events and not j u s t an e r r o r i n s t a g i n g .  I f s e c t i o n s cut a t r i g h t  a n g l e s to the l o n g a x i s o f the t e s t i s a r e scanned from the g e r m i n a l zone to the o p p o s i t e edge o f the t e s t i s , a l a y e r of protamine t a i n i n g c e l l s appears f i r s t , protamine  f o l l o w e d by the appearance  con-  of s t a b l e  c o n t a i n i n g sperm a t the v e r y o u t e r edge o f the t e s t i s .  The  d i r e c t i o n o f t h i s scan i s from the most immature s t a g e o f spermatog e n e s i s to the most mature ( S t a n l e y 1966).  In the case o f the dog-  f i s h , sperm o f the Mouse/grasshopper type a r e found i n c o l l e c t i n g d u c t u l e s w h i l e i n both d o g f i s h and skate sperm of the Mouse/grasshopper type are observed i n the lumen o f f o l l i c l e s , i n d i c a t i n g they are mature and are being r e l e a s e d ( S t a n l e y 1966).  that  Finally,  sperm i n the epididymal r e g i o n o f the r a t f i s h are found to be  the  the  Mouse/grasshopper type while the t e s t i s d i s p l a y s e a r l i e r s t a g e s o f spermiogenesis where protamines are p r e s e n t .  Thus i n c a r t i l a g i n o u s  f i s h the b a s i c p r o t e i n changes d u r i n g spermiogenesis appear r e v e r s e o f those r e p o r t e d i n o t h e r organisms  (histone  to be  the  protamine  s t a b l e protamine). The p r i n c i p a l argument a g a i n s t t h i s unusual t r a n s i t i o n i s the l a c k o f s u p p o r t i n g b i o c h e m i c a l evidence.Using the same g e l system i n the present study, Lam  and Bruce  as  (1971) r e p o r t e d t h a t the b a s i c  p r o t e i n 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 expected 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. Perhaps, 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 protamine 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) w i l l 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 d i f f i c u l t to extract Mouse/grasshopper type of protein. An alternate possibility i s that the appearance of stable protamines i s 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 i s d i f f i c u l t 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 i s obtained from these sperm similar in amino acid composi t i o n and electrophoretic mobility to the testis specific bands, the  - 83 unusual sequence of events i s l i k e l y an a r t i f a c t .  However, i f from  these sperm a protein i s obtained which i s different from the t e s t i s s p e c i f i c protein, the sequence of events outlined above i s l i k e l y to be the correct one. A t h i r d p o s s i b i l i t y i s that the appearance of protamines i s an artifact.  Some stages of spermiogenesis might be sensitive to  tri-  chloroacetic acid hydrolysis for reasons other than the presence of protamines.  The reorganization of nuclear material i n the process of  s p i r a l i z a t i o n might be the cause of t h i s .  However, i f the appearance  of protamine i s an a r t i f a c t , the co-electrophoresis  of t e s t i s spec-  i f i c bands with salmon protamine must be regarded as merely f o r t u i t ous.  This i s an unlikely p o s s i b i l i t y . Therefore, on the basis of the cytochemical r e s u l t s ,  of the dogfish, long nose skate, and the r a t f i s h w i l l be classed as the Mouse/grasshopper type.  the sperm tentatively  Future studies should c l a r i f y  the discrepancies between this conclusion and the biochemical r e s u l t s . The conclusion of this study i s not i n agreement with the  little  available information on the basic protein composition of sperm from other cartilaginous f i s h .  On the basis of Kossel's (1928) work,  Bloch (1969) c l a s s i f i e d 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  isolation  techniques u t i l i z e d i n his day were harsh and a r t i f a c t s i n amino acid composition were possible.  On the basis of unpublished cytochemical  data, Bloch (1969) c l a s s i f i e d the ray, Dasyatis sabina, as the Mytilus type.  Comment on unpublished results i s d i f f i c u l t .  However, i n t h i s  study no variation i n sperm histone type was observed i n a wide range of cartilaginous f i s h .  Therefore, differences between this study and  Bloch s results are not l i k e l y to be explained on the basis that d i f f  ferent organisms have been studied. The observation that protamines are present i n elasmobranchs might lead to some fascinating comparative studies. of elasmobranchs be fractionated,  Can the protamines  Protamines from teleost f i s h have  been fractionated by a number of workers (Ando and Sawada 1961, Ando  - 84 and Suzuki 1966, protamines  from elasmobranch  a fundamental far  L i n g et al„ 1 9 7 D «  The amino a c i d sequence o f  f i s h would make an e x c i t i n g s t u d y s i n c e  pentapeptide has been found i n a l l the protamines  elucidated.  T h i s s u b - u n i t o c c u r s some t h r e e o r f o u r times per  m o l e c u l e , w h i l e o t h e r p i e c e s a r e fragments from p a r t i a l d u p l i c a t i o n o f the c i s t r o n Protamines have been sequenced n e r i i ) , the P a c i f i c h e r r i n g (Oncorhynchus  so  of the u n i t  resulting  (Black and Dixon 1967).  from the rainbow t r o u t  (Salmo g a i r d -  (Clupea p a l l a s i i ) , and Chum salmon  k e t a ) ( P h i l l i p s 1971).  These organisms a r e bony f i s h .  The b a s i c p r o t e i n changes o f t h i s study may  be r e l a t e d to  r e c e n t u l t r a s t r u c t u r e o b s e r v a t i o n s on d o g f i s h spermiogenesis ( S t a n l e y 1971a, 1971b). t i d s was  S t a n l e y observed t h a t the chromatin o f e a r l y sperma-  f i n e l y g r a n u l a r and e v e n l y d i s p e r s e d .  f i b e r s were i n i t i a l l y  randomly o r i e n t e d , f i b e r s a t a l a t e r stage were  aligned anteroposteriorly. around 19 M to  While n u c l e o p r o t e i n  When the nucleus reached a l e n g t h o f  the i n t r a n u c l e a r f i b e r s were j o i n e d t o g e t h e r l a t e r a l l y  form a maze of s h e e t l i k e c o n f i g u r a t i o n s .  These f i b r o u s s h e e t s  f o l l o w e d a h e l i c a l course a l o n g the l e n g t h o f the n u c l e u s .  Later  these sheets were compacted i n t o a s o l i d column o f c h r o m a t i n . t h i s p o i n t the nucleus was  At  almost completely elongated and the nuc-  l e a r volume had decreased from about 1 8 0 ^  •  to a p p r o x i m a t e l y 10ya  The t r a n s i t i o n to 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 b e g i n s a t about t h i s stage which i n d i c a t e s t h a t these p r o t e i n s are not in  e a r l y n u c l e a r condensation.  Next, S t a n l e y observed the  involved  spiraliz-  a t i o n of chromatin, b e g i n n i n g a t the p o s t e r i o r end o f the n u c l e u s and p r o c e e d i n g to the a n t e r i o r t i p . Although the appearance  of  protamines  i s c o r r e l a t e d w i t h the process o f n u c l e a r 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 , s i n c e the s p i r a l i z a t i o n i s f o r e shadowed much e a r l i e r by the h e l i c a l alignment o f f i b r i l l a r n u c l e a r sheets.  The chromatin m a t e r i a l o f mature sperm was  h i g h l y compacted,  but t h i n s e c t i o n s s t i l l gave evidence o f l o n g i t u d i n a l f i b r i l s .  The  c e n t e r o f the sperm nucleus appeared more e l e c t r o n dense than the p e r i p h e r y and S t a n l e y suggested t h a t the p e r i p h e r a l m a t e r i a l may residual nuclear protein.  Such p r o t e i n might be the protamines  be that  t h i s study suggests are r e p l a c e d i n the l a t e s t a g e s o f spermiogenesis  - 85 by the Mouse/grasshopper type o f p r o t e i n .  - 86 TABLE X  The V a r i e t y of Sperm Histones Organism  Sperm Histone Type  CLASS CHONDRICHTHYES Subclass Elasmobranchii Squalus s u c k l e y i (or acanthias)  Mouse/grasshopper  Raja r h i n a  Mouse/grasshopper  Subclass Holocephali Hydrolagus c o l l i e i  Mouse/grasshopper  CLASS AMPHIBIA Subclass Lepospondyl: Diemictylus v i r i d e s c e n s Subclass Apsidospondyli Xenopus l a e v i s Rana p r e t i o s a Rana p a l u s t r i s Bufo boreas Bufo americanus Hyla r e g i l l a Hyla v e r s i c o l o r  *  C l a s s i f i c a t i o n i s from Romer (1967).  Salmon Mytilus Rana Rana Salmon Salmon Mytilus Mytilus  - 8? V.  GENERAL DISCUSSION  The v a r i e t y 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 w i t h respect to hypotheses on the f u n c t i o n of sperm histones. While the r e s u l t s i n Table X y i e l d no information on the p o s t u l ated repressor r o l e of sperm h i s t o n e s , the data can be used i n the future to c r i t i c a l l y t e s t t h i s hypothesis.  I f the f u n c t i o n 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 t r a n s ition.  Indeed, RNA synthesis i s not observed i n sperm of the M y t i l u s  type (Das et a l . 1965)» the Mouse/grasshopper type (Bloch and Brach 1964) and the Salmon type (Marushige and Dixon 1969).  RNA s y n t h e s i s  i s also absent i n sperm of the crab type t-zhich are f r e e of basic proteins (Vaughn and Thomson 1972). from the somatic histone type occurs. type no t r a n s i t i o n i s observed.  In a l l these cases a t r a n s i t i o n On the other hand, i n the Rana  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 pred i 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 d i f f e r e n t packaging r e q u i r e ments f o r sperm DNA.  This hypothesis may be discussed i n terms of  nuclear shape, nuclear condensation p a t t e r n , and the amount of DNA per nucleus. No c o r r e l a t i o n appears to e x i s t between sperm histone type and nuclear shape.  G o l d f i s h ( Z i r k i n 1971b) and frogs of the genus Rana  have sperm histones s i m i l a r to somatic c e l l s .  Yet, the sperm n u c l e i  of g o l d f i s h are round while those of the frogs are rod-shaped. sperm of trout contain protamines, as do the sperm of Bufo and Bj_ boreas.  The  americanus  Despite t h i s s i m i l a r i t y i n p r o t e i n type, t r o u t sperm  n u c l e i are round while Bufo sperm n u c l e i are rod-shaped. A c o r r e l a t i o n between nuclear condensation p a t t e r n and sperm histone type might e x i s t .  As noted i n the s e c t i o n s on the eastern red  - 88 s p o t t e d newt and the c a r t i l a g i n o u s f i s h , b a s i c p r o t e i n t r a n s i t i o n s are accompanied by marked changes i n n u c l e a r f i n e s t r u c t u r e .  A  s i m i l a r o b s e r v a t i o n was made by Bloch and Hew (1960a) f o r the s n a i l . Zirkin  (1971a, 1971b) has s t u d i e d t h e n u c l e a r f i n e s t r u c t u r e i n  sperm that c o n t a i n the Rana type o f h i s t o n e .  U n l i k e t h e above organ-  isms, h i g h l y o r i e n t e d f i b e r s and sheets were not e v i d e n t .  Instead  randomly o r i e n t e d f i b e r s with diameters  s l i g h t l y s m a l l e r than  from somatic  In crab sperm the l o s s o f  c e l l n u c l e i were p r e s e n t .  those  b a s i c n u c l e a r p r o t e i n s was c o r r e l a t e d with changes i n chromatin (Langreth 1969). and  The chromatin  changed from homogenous f i n e  granules  f i b r i l s o f f a i r l y low e l e c t r o n o p a c i t y to clumps o f f i b e r s i n an  e l e c t r o n - t r a n s l u c e n t nucleoplasm.  Thus changes i n n u c l e a r f i n e s t r u c -  t u r e do appear to be c o r r e l a t e d w i t h h i s t o n e type. suggests  This  correlation  t h e DNA i s packed i n t o the sperm head i n a manner dependent  on the sperm h i s t o n e  type.  Walker (1971) b e l i e v e s t h r e e b a s i c p a t t e r n s o f n u c l e a r condens a t i o n a r e evident i n spermiogenesis. " f i b r o u s " type.  The f i r s t p a t t e r n i s the  F i b e r s o r f i l a m e n t s occur i n the e a r l y spermatid and  become t h i c k e r and e v e n t u a l l y f u s e to g i v e the mature sperm head. the second p a t t e r n , the " l a m e l l a r " type, the f i b e r s present e a r l y spermatid eeds.  i n the  f u s e i n t o sheets o r l a m e l l a e as spermiogenesis  The t h i r d p a t t e r n i s the " g r a n u l a r " type.  In  proc-  I n 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 t o the s t a r t o f any condensation  of the nucleoprotein.  between these condensation  Walker found l i t t l e  p a t t e r n s and sperm h i s t o n e  correlation  type.  A r e l a t i o n s h i p between sperm h i s t o n e type and the amount o f DNA per nucleus i s n o t e v i d e n t . the d e n s i t y o f packaging  O l i n s e t a l . (1968) have c a l c u l a t e d t h a t  i n t r o u t sperm i s 0.7g DNA/crn^.  Since i n  the sperm head t h e r a t i o o f protamine arginine/DNA phosphate was about one,  they suggested  t h a t protamines may f a c i l i t a t e the packing o f DNA  by r e d u c i n g phosphate-phosphate e l e c t r o s t a t i c r e p u l s i o n s . formation o f aggregated s t r u c t u r e s would be f a v o r e d . the n u c l e a r volume given by Z i r k i n content  Thus the  However, u s i n g  (1970) and c a l c u l a t i n g the DNA  from the r e s u l t s o f Bachman (1970), one may compute the d e n s i t y  - 89 o f p a c k a g i n g i n Rj_ p i p i e n s sperm.  I n t h e s e sperm w i t h the  somatic  h i s t o n e type the d e n s i t y o f p a c k a g i n g i s 0.66g DNA/cm^ and  very  s i m i l a r to t h a t i n t r o u t sperm which c o n t a i n p r o t a m i n e . Bloch  ( 1 9 6 9 ) p o s t u l a t e d t h a t the v a r i e t y o f sperm h i s t o n e  t y p e s 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 o r g a n i s m s  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 type.  histone  A l t e r n a t i v e l y , organisms w i t h i n t e r n a l f e r t i l i z a t i o n  r e q u i r e a unique h i s t o n e .  might  Perhaps, sperm which are s t o r e d i n the  female f o r l o n g p e r i o d s b e f o r e  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 h i s t o n e type and  or i n t e r n a l f e r t i l i z a t i o n .  The  external  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 o f h i s t o n e t y p e s . e a s t e r n r e d s p o t t e d newt and have i n t e r n a l f e r t i l i z a t i o n .  the t h r e e c a r t i l a g i n o u s f i s h The  The  studied  Salmon type o f sperm h i s t o n e i s  found i n the newt w h i l e the c a r t i l a g i n o u s f i s h have the Mouse/grasshopper t y p e .  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  c o n t a i n s the Rana type o f sperm h i s t o n e  (Bloch  and  yet,  1969).  L o n g - l i v e d sperm do not appear t o 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  r e p o r t e d t o remain v i a b l e f o r upwards o f a y e a r (Taber and  Blum i 9 6 0 ) .  Snakes have been r e p o r t e d to c o n t i n u e b e a r i n g o f f s p r i n g more than f o u r y e a r s a f t e r mating (Wright and Wright 1 9 5 7 ) • been r e p o r t e d i n the female d o g f i s h  Sperm s t o r a g e  (Metten 1939) and might be  g e n e r a l o c c u r r e n c e i n elasmobranchs (Grover 1 9 7 0 ) .  Bloch  of  Yet bees have the  Rana type o f h i s t o n e , snakes the Salmon type ( B l o c h 1969)» and d o g f i s h the Mouse/grasshopper  has  the  type.  ( 1 9 6 9 ) , among o t h e r s  ( O l i n s et a l . 1 9 6 8 ) , has  t h a t the sperm h i s t o n e t r a n s i t i o n may  suggested  erase the d e 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 , t h e r e b y 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 cell.  T h i s view r e q u i r e s t h a t " s p e r m a t o g o n i a l c e l l s and  c y t e s be c o n s i d e r e d ( B l o c h 1969)»  spermato-  d i f f e r e n t i a t e d i n the same sense as s o m a t i c c e l l s  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  dino and H o f f n e r  Berar-  ( 1 9 7 0 ) demonstrated t h a t the n u c l e i o f s p e r m a t o g o n i a l  "  - 90 c e l l s from Rana.pipiens underwent developmental r e s t r i c t i o n s d u r i n g 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 . nuclei.  They behaved l i k e  somatic  However, i n Rj_ p i p i e n s no sperm h i s t o n e t r a n s i t i o n o c c u r s .  T h e r e f o r e , the h i s t o n e t r a n s i t i o n does not appear to be n e c e s s a r y to erase the developmental  restrictions.  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 as to what happens t o sperm h i s tones i n e a r l y development.  Work on r a t sperm suggests t h a t they are  l o s t very e a r l y i n p r o n u c l e a r formation (Kopecny T970). c o r r e l a t i o n might  However, a  e x i s t between c h a r a c t e r i s t i c events o f e a r l y  develop-  ment and sperm h i s t o n e type. Perhaps of the egg.  the sperm h i s t o n e type i s r e l a t e d to the c l e a v a g e p a t t e r n However, among the amphibians,  Rana, cleavage i s h o l o b l a s t i c .  D i e m i c t y l u s , Xenopus, and  Yet the sperm o f these organisms  are  o f the Salmon, M y t i l u s , and Rana t y p e s , r e s p e c t i v e l y .  The  cartilag-  inous f i s h have  The  cartilag-  raeroblastic  eggs as do most t e l e o s t s .  inous f i s h have the Mouse/grasshopper type o f sperm h i s t o n e \tfhile the t e l e o s t s demonstrate i a n s do.  the same range i n sperm h i s t o n e s as the amphib-  Thus no r e l a t i o n appears to e x i s t between sperm h i s t o n e type  and cleavage p a t t e r n . The beginnings o f RNA  s y n t h e s i s d u r i n g e a r l y embryonic  develop-  ment appears to be q u i t e v a r i a b l e and might be c o r r e l a t e d w i t h sperm h i s t o n e type (Bloch 1969). s y n t h e s i s i n the organisms  However, due to the l a c k o f data on  RNA  o f the present study a c o r r e l a t i o n i s not  possible. B l o c h (1969) noted that sperm h i s t o n e types d i d not show an utionary trend. represented. tones was  evol-  W i t h i n most o f the broad taxa s t u d i e d a l l types were  Indeed, i n the p r e s e n t study a wide range of sperm h i s -  observed i n the amphibians.  s e r v e d i n the c a r t i l a g i n o u s f i s h . cannot be advanced.  However, no v a r i a t i o n was  ob-  An e x p l a n a t i o n f o r these r e s u l t s  Bloch a l s o observed l i t t l e v a r i a t i o n i n sperm  h i s t o n e s w i t h i n t i g h t l y d e f i n e d taxomic groups. p r e s e n t study v a r i a t i o n i n sperm h i s t o n e type was f r o g s of the same genus.  S i m i l a r l y , i n the not found among  - 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 b e t w e e n h i s t o n e t y p e a n d a n y 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 (1969) t h a t t h e v a r i a b i l i t y o f s p e r m 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 unimportant p r o t e i n i n an nucleus." be  Bloch  inert  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  basicity.  - 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 spermatogenesis i n the salmon. J . Biophys. B i o c h e m . C y t o l . 2: 109-114 A l f e r t , M., a n d I . G e s h w i n d , 1953 A s e l e c t i v e m e t h o d f o r t h e b a s i c proteins of c e l l nuclei. P r o c . N a t . A c a d . S c i . U.S.A.  39:  992-999  A n d o , T., a n d F . Sawada, 1961 S t u d i e s o n p r o t a m i n e s . a t i o n o f c l u p e i n e . J . B i o c h e m . 49: 252-259«  IX. Fraction-  A n d o , T., a n d K. S u z u k i , 196? The amino a c i d s e q u e n c e - o f t h e t h i r d component o f c l u p e i n e . B i o c h e m . B i o p h y s . A c t a . 1 4 0 : 375-377 A n d o , T. a n d S. W a t a n a b e , 1969 A new m e t h o d f o r f r a c t i o n a t i o n o f p r o t a m i n e s a n d t h e amino a c i d s e q u e n c e s o f o n e c o m p o n e n t o f s a l m i n e and t h r e e components o f i r i d i n e . Int. J .Protein R e s e a r c h 1: 221-224 Bachman, K., 1970 S p e c i f i c n u c l e a r DNA a m o u n t s i n t o a d s o f t h e g e n u s B u f o . Chromosoma 29: 365-374 B a k e r , C. L . 1966 S p e r m a t o z o a a n d s p e r m a t e l e o s i s i n t h e S a l a m a n d r i d a e w i t h e l e c t r o n microscopy o f D i e m i c t y l u s . J . Tenn. Acad. S c i . 4 1 : 2-25 5  B e r l o w i t z , L . , 1965 A n a l y s i s o f h i s t o n e i n s i t u i n d e v e l o p m e n t a l l y i n a c t i v a t e d chromatin. P r o c . N a t . A c a d . S c i . U.S.A. 54: 476-480 B e r l o w i t z , L . , D. P a l l o t t a , a n d P. P a w l o w s k i , 1970 I s o l a t e d h i s t o n e f r a c t i o n s and the a l k a l i n e f a s t green r e a c t i o n . J . Histochem. C y t o c h e m . 18: 334-339 B l a c k , S. A., a n d G. H. D i x o n , 1967 E v o l u t i o n o f p r o t a m i n e : A f u r t h e r e x a m p l e o f p a r t i a l gene d u p l i c a t i o n . N a t u r e 216:  152-154.  B l o c h , D. P., 1962 S y n t h e t i c p r o c e s s e s i n t h e c e l l n u c l e u s . I . H i s t o n e s y n t h e s i s i n n o n - r e p l i c a t i n g chromosomes. J . H i s t o c h e m . C y t o c h e m . 10: 137-144 1966 C y t o c h e m i s t r y , o f t h e h i s t o n e s . 1969 A c a t a l o g o f s p e r m h i s t o n e s .  Protoplasmatologia Genetics  (Suppl.)  5: 1-56  61: 93-111  B l o c h , D. P. a n d G. C. Godman 155 A m i c r o p h o t o m e t r i c s t u d y o f t h e synthesis o f desoxyribonucleic a c i d and nuclear h i s t o n e s . J . Biophys. B i o c h e m . C y t o l . 1: 17-24 B l o c h , D. P., a n d H. T. C. Hew, 1960a S c h e d u l e o f s p e r m a t o g e n e s i s i n the pulmonate s n a i l H e l i x a s p e r s a , w i t h s p e c i a l r e f e r e n c e t o histone t r a n s i t i o n . J . B i o p h y s . " B i o c h e m . C y t o l . 7: 515-532  - 93 ________ 1960b Changes i n n u c l e a r h i s t o n e s d u r i n g f e r t i l i z a t i o n and e a r l y embryonic development i n t h e pulmonate s n a i l H e l i x a s p e r s a . J. Biophys. Biochem. C y t o l . 8: 69-81 B l o c h , D. P., and S. D. B r a c k , 1964 E v i d e n c e f o r t h e c y t o p l a s m i c s y n t h e s i s o f n u c l e a r h i s t o n e during spermiogenesis i n the grasshopper Chortophaga v i r i d i f a s c i a t a (De G e e r ) . J . C e l l B i o l 22: 327-340 0  Bonner, J . , G. P. C h a l k l e y , M. Dahmus, D. Fambrough, F. F u j i m u r a , R. C. Huang, J . Huberman, R. Jensen., K. M a r u s h i g e , H. O h l e n b u s c h , B. O l i v e r a , and J . Widholm, 1968 I s o l a t i o n and c h a r a c t e r i z a t i o n o f chromosomal n u c l e o p r o t e i n s . I n Methods i n Enzymology, V o l . X I I P a r t B E d i t e d by L. Grossman and K. Moldave. Academic P r e s s , pp. 3-65 Bowes, J . A., and C. W. Water, 1965 C r o s s l i n k i n g o f c o l l a g e n . J . A p p l . Chem. 15: 296-297 B r u t l a g , D., C. S c h l e h u b e r , and J , Bonner, 1969 P r o p e r t i e s o f f o r m a l dehyde-treated n u c l e o h i s t o n e . Biochem. 8: 32l4— 3218 C a l l a n a n , M. J . , W. R. C a r r o l l , a n d E. R. M i t c h e l l , 1957 P h y s i c a l and c h e m i c a l p r o p e r t i e s o f p r o t a m i n e from the sperm o f salmon (Oncorhyncus t s c h a w y t s c h a ) . J . B i o l . Chem. 229: 279-287 Chayen, J . , L. B i t e n s k y , R. G. B u t c h e r , and L. W. P o u l t e r , 1969 A g u i d e to p r a c t i c a l h i s t o c h e m i s t r y . O l i v e r and Boyd L t d . C h e v a i l l i e r , P., 1967 M i s e en evidence e t etude c y t o c h i m i q u e d'une p r o t e i n e b a s i q u e e x t r a n u c l e a i r e dans l e s s p e r m a t o z o i d e s des c r u s t a c e s decapodes. J . C e l l B i o l . 52: 547-556 D a l y , M. N., A. E. M i r s k y , and H. R i s , 1951 The amino a c i d c o m p o s i t i o n and some p r o p e r t i e s o f h i s t o n e s . J . Gen. P h y s i o l . 34: 439-450 Das,  C. C , H. Gay, and B. P. Kaufman, 1964 H i s t o n e - p r o t e i n t r a n s i t i o n i n D r o s o p h i l a m e l a n o g a s t e r . I Changes d u r i n g s p e r m a t o g e n e s i s . Exp. C e l l Res. 3 5 : 507 51^ =  Das,  N. K., E. P. S i e g e l , and M. A l f e r t , 19&5 S y n t h e t i c a c t i v i t i e s d u r i n g s p e r m a t o g e n e s i s ' i n t h e l o c u s t . J . C e l l B i o l . 25: 387-395  Das,  N. K., J.•Micou-Eastwood, and M. A l f e r t , 1967 C y t o c h e m i c a l and b i o c h e m i c a l p r o p e r t i e s o f b a s i c p r o t e i n s o f U r e c h i s acrosomes. J . C e l l B i o l . 35: 455-458 ™~~  D e i t c h , A. D., 196l An improved Sakaguchi r e a c t i o n f o r m i c r o s p e c t r o p h o t o m e t r i c use. J . Histochem. Cytochem. 9: 477-483  - 94 -  De Lange, R. J * , D. M. Fambrough, E. L. Smith, and J . Bonner, 1969 I I I Complete amino a c i d sequence of pea seedling IV. Comparison with the homologous c a l f thymus histone. J . B i o l . Chem. 244: 5669-5679  D i Bernardino, M. A. and N. Hoffner, 1970 Development and chromosomal c o n s t i t u t i o n of nuclear-transplants derived from male germ c e l l s . J . Exp. Zool. 176: 61-72 Fambrough, D., and J . Bonner, 1966 On the s i m i l a r i t y o f p l a n t and animal histones. Biochem. 5: 2565-2569 1968 S e l e c t i v e d i s s o c i a t i o n o f pea bud Biochim. Biophys. Acta. 154: 601-603  nucleohistone.  F e l i x , K., i960 Protamines. In advances i n protein chemistry. Edited by C. B. Anfinsen, M. L. Anson, K. B a i l e y , and J . T. Eds a l l . Academic Press pp 1-56 Fredericq, E., 1971 The chemical and p h y s i c a l properties o f nucleohistones. In Histones and Nucleohistones. Edited by D. M. P. P h i l l i p s . Plenum P u b l i s h i n g Company L t d . pp 135-186 Georgiev, G. P., 1969 Histones and the c o n t r o l of gene a c t i o n . Annu. Rev. Genet. 3: 115-180 Grover, C. A., 1970 The egg-cases of the s w e l l shark, Cephaloscyllium ventriosum: formation, f u n c t i o n , and population d i f f e r e n c e s . Masters Thesis, U n i v e r s i t y o f B r i t i s h Columbia Gurdon, J. B. 1967 A f r i c a n clawed frogs. In Methods i n developmental biology. Edited by F. H. W i l t and N. K. Wessells. Thomas Y.  Crowell Co. pp 75-84  Hamer, D., 1955 The composition o f the basic proteins o f echinoderm sperm. B i o l . B u l l . 108: 35-39 Horobin, R. W. and I . M. K e v i l l - D a v i e s 1971 Basic Fuchsin i n a c i d a l c o h o l : a s i m p l i f i e d a l t e r n a t i v e to S h i f f reagent. S t a i n Technol. 46: 53-58 c  Houston, L. L., 1971 Amino a c i d a n a l y s i s of stained bands from polyacrylamide g e l s . Anal. Biochem. 44: 81-88. Ingles, C. J . , J . R. T r e v i t h i c k , M. Smith, and G. H. Dixon, 1966 Biosynthesis o f protamine during spermatogenesis i n salmonoid f i s h . Biochem. Biophys. Res. Commun. 22: 627-634 Johns, E. W., 1967 The electrophoresis o f histones i n polyacrylamide g e l and t h e i r q u a n t i t a t i v e determination. Biochem. J . 104: 78-83  - 95 1971 The p r e p a r a t i o n a n d c h a r a c t e r i z a t i o n o f h i s t o n e s . I n H i s t o n e s and N u c l e o h i s t o n e s . E d i t e d b y D.M.P. P h i l l i p s , P l e n u m P u b l i s h i n g Company L t d , p p 1-4-5 K a y e , J . S., a n d R. M c M a s t e r - K a y , 1966 The f i n e s t r u c t u r e a n d c h e m i c a l composition o f n u c l e i during spermiogenesis i n the louse c r i c k e t , J . C e l l B i o l . 31: 159-179 14 K o p e n c y , V., 1970 L a b e l l i n g o f mouse s p e r m a t o z o a w i t h L - a r g i n i n e (U) m o n o h y d r o c h l o r i d e . Z. Z e l l f o r s c h . 109: 4 l 4 - 4 l 9 K o s s e l , A., 1928 The P r o t a m i n e s a n d H i s t o n e s . London  C  Longmans G r e e n a n d C o . ,  Lam, D. M. K., R. F u r r e r , a n d W. R. B r u c e , 1970 The s e p a r a t i o n , p h y s i c a l c h a r a c t e r i z a t i o n and d i f f e r e n t i a t i o n k i n e t i c s o f spermatog o n i a l c e l l s o f t h e mouse. P r o c . N a t . A c a d . S c i . 65: 192-199 Lam, D. M. K., a n d W. R. B r u c e , 1971 The b i o s y n t h e s i s o f p r o t a m i n e d u r i n g s p e r m a t o g e n e s i s o f t h e mouse: e x t r a c t i o n , p a r t i a l c h a r a c t e r i z a t i o n , and s i t e o f s y n t h e s i s . J . C e l l . P h y s i o l . 78: 13-24 L a n g r e t h , S. G. 1969 S p e r m i o g e n e s i s i n C a n c e r c r a b s . h3j_ 575-603  J. Cell  Biol.  L i n g , V., J . E . T r e v i t h i c k a n d G. H. D i x o n , 1969 The b i o s y n t h e s i s o f protamine i n t r o u t t e s t i s . I . Intracellular site of synthesis. Can. J . B i o c h e m . __7_: 51-60 L i n g , V., B. J e r g i l , a n d G. H. D i x o n , 1971 The b i o s y n t h e s i s o f p r o t a m i n e i n t r o u t t e s t i s I I I . C h a r a c t e r i z a t i o n o f p r o t a m i n e components and t h e i r s y n t h e s i s d u r i n g t e s t i s development. J . B i o l . Chem. 246: H 6 8 - I I 7 6 L u c k , J . M., 1964 H i s t o n e c h e m i s t r y : t h e p i o n e e r s . I n The n u c l e o histones. E d i t e d b y J . B o n n e r a n d P. 0. P. T s ' o . H o l d e n - D a y , I n c . p p 3-12 M a r u s h i g e , K., a n d J . B o n n e r , 1966 T e m p l a t e p r o p e r t i e s o f l i v e r chromatin. J . M o l . B i o l . 15: 160-174 M a r u s h i g e , K., a n d G. H. D i x o n , 1969 D e v e l o p m e n t a l c h a n g e s i n c h r o m o somal c o m p o s i t i o n and t e m p l a t e a c t i v i t y d u r i n g s p e r m a t o g e n e s i s in trout testis. D e v e l o p . B i o l . 19: 397-414 1971 T r a n s f o r m a t i o n Chem. 2 4 6 : 5799-5805  o f trout t e s t i s chromatin.  J. Biol.  M a r u s h i g e , K., V. L i n g a n d G. H. D i x o n , 1969 P h o s p h o r y l a t i o n o f chromosomal b a s i c p r o t e i n s i n m a t u r i n g t r o u t t e s t i s . J . Biol. Chem. 244: 5953-5958  - 96 Metten, H., 1939 Studies on the reproduction of the d o g f i s h . Trans. Roy. Soc. London 230: 217-238  Phil.  Monesi, V., 1965 Synthetic a c t i v i t i e s during spermatogenesis i n the mouse. RNA and p r o t e i n . Exp. C e l l Res. 39: 197-224 Murray, K., 1964 Histone nomenclature. In The nucleohistones. Edited by J . Bonner and P. 0. P. Ts'o. Holden-Day, Inc. pp 15-20 Nelson, R.D., and J . J . Yunis, 1969 Species and t i s s u e s p e c i f i c i t y o f very L y s i n e - r i c h -and- s e r i n e — r i c h h i s t o n e s . -Ex-p. C e l l - R e s . 57: 311-318  O l i n s , D. E., A. L. O l i n s , and P. H. Von H i p p e l , 1968 On the s t r u c t u r e and s t a b i l i t y of DNA-protamine and DNA-polypeptide complexes. J. Mol. B i o l . 33: 265-281 Pearse, A. G. E., 1968 Histochemistry: A. C h u r c h i l l L t d .  t h e o r e t i c a l and a p p l i e d .  J. &  P i c h e r a l , E., 1970 Nature et e v o l u t i o n des proteines basiques au cours de l a spermiogenese chez PIeurodeles w a l t i i Michah., amphibien urodele. Histochemie, 23: 189-206 1971 U l t r a s t r u c t u r e du noyau en rapport avec I n v o l u t i o n des proteins basiques n u c l e a i r e s au cours de l a spermiogenese du t r i t o n Pleurodeles w a l t i i Michah J. Microsc. ( P a r i s ) 12: 107-132 P i p k i n , J . L. 1968 C y t o l o g i c a l and cytochemical methodology of h i s t o n e s . In Methods i n c e l l physiology, III. Edited by D. M. P r e s c o t t . Academic Press, pp 307-346 P h i l l i p s , D. M. P., 1971 The primary s t r u c t u r e of histones and p r o t amines. In Histones and Nucleohistones. Edited by D. M. P. P h i l l i p s . Plenum P u b l i s h i n g Company L t d . pp 47-83 Romer, A. S., 1967 The vertebrate body.  W. B. Saunders Company  Rugh, R., 1939 The reproductive processes of the f r o g , R«_ p i p i e n s . J . Exp. Zool. 80: 81-105 Sheridan, W. F. and H. Stern, 1967 Histones o f meiosis. Res.  45: 323-335  Exp. C e l l  Simpson, T. H., and C. S. Wardle, 1967 A seasonal cycle i n the t e s t i s of the spurdog, Squalus acanthias, and the s i t e s o f 3 hydroxys t e r o i d dehydrogenase a c t i v i t y . J. Mar. B i o l . Ass. U. K. 47: 699-708  Stanley, H. P., 1963 Urogenital morphology i n the Chimaeroid Hydrolagus c o l l i e i . J. Morphology 112: 99-127  ~~  fish  - 97 1966 The s t r u c t u r e a n d d e v e l o p m e n t o f t h e s e m i n i f e r o u s f o l l i c l e i n S c y l i o r h i n u s c a n i c u l u s and Torpedo marmorata (Elasmobranchi). Z. Z e l l f o r s c h . 75: 4-53-468 1971a Fine s t r u c t u r e o f spermiogenesis i n the elasmobranch f i s h Squalus s u c k l e y i . I . Acrosome f o r m a t i o n , n u c l e a r e l o n g a t i o n and d i f f e r e n t i a t i o n o f the m i d p i e c e a x i s . J . Ultrastruct.  Res. 36: 86-102  1971b Fine s t r u c t u r e o f spermiogenesis i n the elasmobranch f i s h Squalus s u c k l e y i . I I . Late stages o f d i f f e r e n t i a t i o n and s t r u c t u r e o f the mature spermatozoan. J .Ultrastruct. R e s . 36:  103-118  Stedman, E . , and E . Stedman, 1950 C e l l N a t u r e 166: 78O-78I  specificity  o f histones.  T a b e r , S., a n d M. S. Blum, I960 P r e s e r v a t i o n o f h o n e y b e e s e r u m . S c i e n c e 131: 1734-1735 V a u g h n , J . C., I966 The r e l a t i o n s h i p o f t h e " s p h e r e chromatophile" to t h e f a t e o f d i s p l a c e d h i s t o n e f o l l o w i n g t h e h i s t o n e t r a n s i t i o n i nr a t spermiogenesis. J . C e l l B i o l . 31: 257-278  I.  1968 Changing n u c l e a r h i s t o n e p a t t e r n s during development. F e r t i l i z a t i o n and e a r l y cleavage i n t h e crab E m e r i t a a n a l o g a .  J . Histochem.  C y t o c h e m . 16: 4-73-4-79  V a u g h n , J . C , J . C h a i t o f f , R. D e l e e n , a n d C. G a r l a n d , 1969o Changing n u c l e a r h i s t o n e p a t t e r n s d u r i n g development. I I . I s o l a t i o n and p a r t i a l c h a r a c t e r i z a t i o n o f " d e c a p o d i n e " from sperm c e l l s o f t h e crab Emerita analoga. E x p . C e l l R e s . 54-: 362-366 V a u g h n , J . C , a n d G . V/. H i n s c h , 1970 F r a c t i o n a t i o n a n d c h a r a c t e r i z a t i o n o f n u c l e a r a n d a c r o s o m a l components o f t h e sperm o f t h e s p i d e r crab, L i b i n i a emarginata. Amer. Z o o l . 10: 523-525 V a u g h n , J . C., a n d L . A. Thomson, 1972 A k i n e t i c s t u d y o f DNA a n d b a s i c p r o t e i n metabolism d u r i n g spermatogenesis i n t h e sand crab, Emerita analoga. J . C e l l B i o l . 52: 322-337 V e n d r e l y , R., 1957 Donnees r e c e n t e s s u r l a c h i m i e de l'ADN e t d e s desoxyribonucleoproteines. A r c h i v der J u l i u s K l a u s - S t i f f . V e r e r b u n g s f o r s c h . , S o z i a l a n t h r o p o l . u . R a s s e n h y g . 32: 538-553 V e n d r e l y , R., a n d C. V e n d r e l y , I966 B i o c h e m i s t r y protamines. P r o t o p l a s m a t o l o g i a 5: 1-88  o f h i s t o n e s and  W a l k e r , M. H., 1971 S t u d i e s o n t h e a r r a n g e m e n t o f n u c l e o p r o t e i n i n e l o n g a t e sperm h e a d s . Chromosoma 34: 340-354  - 98 -  Wigle, D. T., and G. H. Dixon, 1971 A new histone from t r o u t t e s t i s . J . B i o l . Chem. 246; 5636-5644 Wright, A. F., and A. A. Wright, 1949 Handbook of frogs and toads of the United States and Canada. Comstock P u b l i s h i n g Company, Inc. 1957 Handbook of snakes o f the United States and C nada. Comstock Publ. Assoc. a  Z i r k i n , B. R., 1970 The p r o t e i n composition o f n u c l e i during spermiogenesis i n the leopard f r o g , Rana p i p i e n s . Chromosoma 31: 213-240 1971a The f i n e s t r u c t u r e of n u c l e i during spermiogenesis i n the leopard f r o g , Rana p i p i e n s . J . U l t r a s t r u c t . Res. 34: 159-174 ________ 1971b The f i n e s t r u c t u r e o f n u c l e i i n mature sperm I . A p p l i c a t i o n of the Longmuir t r o u g h - c r i t i c a l point method to histonecontaining sperm n u c l e i . J . U l t r a s t r u c t . Res. 36: 237-248  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101642/manifest

Comment

Related Items