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Variability of testis-specific proteins in Gasterosteus aculeatus L. and related species Lemke, Michael J. 1985

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VARIABILITY OF TESTIS-SPECIFIC PROTEINS IN GASTEROSTEUS ACULEATUS L. AND RELATED SPECIES by MICHAEL J . LEMKE B.Sc., U n i v e r s i t y of W i s c o n s i n - S t e v e n s P o i n t , W i s c o n s i n , 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA 12 F e b r u a r y 1985 © M i c h a e l J . Lemke, 1985 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. M i c h a e l J . Lemke Department of Z o o l o g y  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 26 March 1985 DE-6 (3/81) i i ABSTRACT T e s t i s - s p e c i f i c p r o t e i n (TSP) v a r i a b i l i t y has been examined i n the three-spined s t i c k l e b a c k , Gasterosteus aculeatus and r e l a t e d species i n c l u d i n g Gasterosteus wheatlandi, P u n q i t i u s  p u n g i t i u s , and Aulorhynchus f l a v i d u s , i n order to determine i f such p r o t e i n s can act as molecular markers f o r d i f f e r e n t species of Gasterosteiformes and for d i f f e r e n t p opulations of a s i n g l e species. Cytochemistry of the sperm h i s t o n e s of Gasterosteus  aculeatus revealed that these basic p r o t e i n s can be c l a s s i f i e d as intermediate sperm histones according t o Bloch's (1969 and 1976) c a t e g o r i e s . E l e c t r o n microscopy i n d i c a t e s that the chromatin i n the nucleus condenses i n a granular pattern as the somatic histones of the spermatogonia are r e p l a c e d by the sperm histones i n the spermatid during spermiogenesis i n t h i s t e l e o s t . The t e s t i s s p e c i f i c p r o t e i n s (TSP's) of G. aculeatus and r e l a t e d species were c h a r a c t e r i z e d by e l e c t r o p h o r e s i s on polyacrylamide g e l s and by h y d r o l y s i s of the amidoblack s t a i n e d bands from the g e l and subsequent amino a c i d a n a l y s i s . The compositional a n a l y s i s revealed that a l l the TSP's of the f i s h i n the order Gasterosteiformes were intermediate type c o n t a i n i n g h i s t i d i n e , l y s i n e , and a r g i n i n e amino a c i d r e s i d u e s . However, the TSP's of d i f f e r e n t species could be d i s t i n g u i s h e d by t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s on polyacrylamide g e l s and by d i f f e r e n c e s i n the amino a c i d composition. Apparently TSP's can act as molecular markers to d i s t i n g u i s h these p a r t i c u l a r t e l e o s t s . To e s t a b l i s h the e l e c t r o p h o r e t i c p a t t e r n f o r the TSP's of mature, breeding G. aculeatus, the developmental p r o f i l e was i n v e s t i g a t e d over the course of a season f o r a population of these f i s h i n J e r i c h o Pond, Vancouver, B.C. As the t e s t i s matures, somatic histones are replaced g r a d u a l l y by one or s e v e r a l r a p i d l y moving TSP's. Some p r o t e i n bands that are present i n f i s h with immature t e s t e s are removed by the time the e l e c t r o p h o r e t i c pattern t y p i c a l for mature males occurs during the breeding season. This p a t t e r n shows only r a p i d l y moving TSP's and very low l e v e l s of somatic h i s t o n e s . Experiments using a l k a l i n e phosphatase i n d i c a t e d that the e l e c t r o p h o r e t i c p r o f i l e of the m u l t i p l i c i t y of TSP'.s of mature f i s h was not due to d i f f e r e n c e s i n charge because of phosphorylation of serine side chains i n TSP bands. In a d d i t i o n to a c t i n g as molecular markers fo r d i f f e r e n t s p e c i e s , the TSP's showed an e l e c t r o p h o r e t i c p r o f i l e i n anadromous G. aculeatus that d i f f e r e d from the p r o f i l e i n freshwater f i s h with respect to band morphology and a l s o the number of bands seen on long g e l s . Here, too, the s i m i l a r i t y of the freshwater and anadromous TSP p r o f i l e s was r e i n f o r c e d by s i m i l a r d i g e s t i o n patterns with cyanogen bromide, confirming the presence of methionine i n these p r o t e i n s . However, d i f f e r e n t populations of anadromous G. aculeatus e i t h e r from B r i t i s h Columbia or from Quebec were i n d i s t i n g u i s h a b l e by i v e l e c t r o p h o r e t i c a n a l y s i s . Moreover th e e l e c t r o p h o r e t i c and amino a c i d a n a l y s i s c o u l d not d i s t i n g u i s h between G. a c u l e a t u s from d i f f e r e n t f r e s h w a t e r p o p u l a t i o n s . There was no d i s c e r n a b l e t r e n d f o r the m u l t i p l i c i t y of TSP's from such p o p u l a t i o n s . However, i n t h e f i s h from two f r e s h w a t e r l a k e s t h e r e was an i n d i c a t i o n t h a t t h e b e n t h i c forms (bottom d w e l l e r s ) might have reached s e x u a l m a t u r i t y l a t e r than the l i m n e t i c forms ( t o p d w e l l e r s ) as the l i m n e t i c a n i m a l s l o s t t h e i r somatic h i s t o n e s b e f o r e the b e n t h i c s d i d . From t h e s e a n a l y s e s , TSP's a p p a r e n t l y can a c t as m o l e c u l a r markers between d i f f e r e n t s p e c i e s of G a s t e r o s t e i f o r m e s , and t o a l e s s e r e x t e n t between anadromous and f r e s h w a t e r forms of G. a c u l e a t u s . T h i s agrees w i t h the f i n d i n g s of Mann et. a l . ( 1982) t h a t the s p e r m a t i d / s p e r m - s p e c i f i c p r o t e i n s of the f r o g genus Xenopus can d i s t i n g u i s h between d i f f e r e n t s p e c i e s of the genus and somewhat between d i f f e r e n t s u b s p e c i e s of Xenopus l a e v i s . B o th the G a s t e r o s t e i f o r m e s and anuran TSP's a r e of the i n t e r m e d i a t e t y p e . In s e v e r a l i n s t a n c e s , p a r t i c u l a r s p e c i e s of f i s h gave anomalous r e s u l t s . For example, an anadromous G. a c u l e a t u s showed the p r e sence of a band t y p i c a l f o r t h e TSP of G. w h e a t l a n d i on p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . Perhaps t h i s i s due t o h y b r i d i z a t i o n between G a s t e r o s t e i d a e of d i f f e r e n t s p e c i e s . F i n a l l y , i n c u b a t i o n of TSP p r e p a r a t i o n s a t 37 C i n d i c a t e d the presence of endogenous p r o t e a s e a t n e u t r a l pH. V Such a p r o t e a s e was not a c t i v e a t a c i d pH and t h e r e f o r e d i d not i n t e r f e r e w i t h the e l e c t r o p h o r e t i c a n a l y s i s . v i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES i x LIST OF FIGURES x ACKNOWLEDGEMENTS x i i i I n t r o d u c t i o n 1 M a t e r i a l s and Methods ...13 A. E x p e r i m e n t a l A n i m a l s 13 B. Morphology 17 C. C y t o c h e m i s t r y 18 1. F i x a t i v e and embedding 18 2. Feulgen r e a c t i o n 18 3. A l k a l i n e f a s t r e a c t i o n 19 4. A c e t y l a t i o n 20 5. Sakaguchi r e a c t i o n 20 6. C o n t r o l s 21 7. Photography 21 D. B i o c h e m i s t r y 22 1. P r e p a r a t i o n of T e s t i s - S p e c i f i c P r o t e i n s (TSP) 22 2. E l e c t r o p h o r e s i s .. 23 3. S t a i n i n g of p o l y a c r y l a m i d e g e l s ..25 4. Photography of g e l s 25 5. Amino a c i d a n a l y s i s 25 a. Sample p r e p a r a t i o n and h y d r o l y s i s 26 b. Sample a n a l y s i s 26 v i i 6. C h a r a c t e r i z a t i o n of the TSP p r o t e i n s ; 27 a. T r y p t i c d i g e s t i o n 27 b. Cleavage w i t h cyanogen bromide ...28 c. A l k a l i n e phosphatase 28 d. C o n t r o l s 29 R e s u l t s 30 A. Morphology 30 1. L i g h t m i c r o s c o p y 30 2. E l e c t r o n m i c r o s c o p y 33 B. C y t o c h e m i s t r y 38 1. C o n t r o l s ...38 2. _G. a c u l e a t u s - l e i u r u s 43 C. B i o c h e m i s t r y 46 1 . C o n t r o l s •. .46 2. G e l s i z e .....53 3. S p e c i e s d i f f e r e n c e s 54 4. Developmental changes .63 5. Comparison of anadromous and f r e s h w a t e r G. a c u l e a t u s 67 6. B e n t h i c / L i m n e t i c comparison 81 7. P r o t e i n c h a r a c t e r i z a t i o n 92 a. T r y p t i c d i g e s t i o n 93 b. Cyanogen bromide r e a c t i o n 93 c. A l k a l i n e phosphatase and p o s s i b l e endogenous p r o t e a s e 99 8. Anomalies 107 D i s c u s s i o n 117 R e f e r e n c e s C i t e d LIST OF TABLES Tab l e 1. C y t o c h e m i s t r y of sperm n u c l e i i n G a s t e r o s t e u s a c u l e a t u s - l e i u r u s 41 Ta b l e 2. Amino a c i d c o m p o s i t i o n of Xenopus h i s t o n e H4 from p o l y a c r y l a m i d e g e l s 58 Ta b l e 3. Amino a c i d c o m p o s i t i o n of t e s t i s - s p e c i f i c p r o t e i n s from p o l y a c r y l a m i d e g e l s from G. a c u l e a t u s and r e l a t e d s p e c i e s .. . . 60 Ta b l e 4. M u l t i p l e TSP bands from G. a c u l e a t u s . Composite data from v a r y i n g s i z e s of p o l y a c r y l a m i d e g e l s 74 Ta b l e 5. Amino a c i d c o m p o s i t i o n of t e s t i s - s p e c i f i c p r o t e i n s from p o l y a c r y l a m i d e g e l s from G. a c u l e a t u s - t r a c h u r u s ....75 Ta b l e 6. Amino a c i d c o m p o s i t i o n of t e s t i s - s p e c i f i c p r o t e i n s from p o l y a c r y l a m i d e g e l s from G. a c u l e a t u s - l e i u r u s 79 Ta b l e 7. Amino a c i d c o m p o s i t i o n of t e s t i s - s p e c i f i c p r o t e i n s from G. a c u l e a t u s l e i u r u s and t r a c h u r u s forms ...80 X LIST OF FIGURES F i g u r e 1. N o r t h American d i s t r i b u t i o n of G. a c u l e a t u s 8 F i g u r e 2. Photographs of t h r e e - s p i n e s t i c k l e b a c k s ( G a s t e r o s t e u s a c u l e a t u s ) and r e l a t e d s p e c i e s 14 F i g u r e 3. L i g h t m i c r o g r a p h s of c r o s s - s e c t i o n s through t u b u l e s of 2 d i f f e r e n t G. a c u l e a t u s - l e i u r u s t e s t e s 31 F i g u r e 4. P r o c e s s of s p e r m i o g e n e s i s i n d e v e l o p i n g s p e r m a t i d s i n G. a c u l e a t u s - l e i u r u s 34 F i g u r e 5. L i n e drawing of p r o b a b l e t h r e e - d i m e n s i o n a l c o n f i g u r a t i o n of G. a c u l e a t u s sperm 39 F i g u r e 6. C y t o c h e m i s t r y done on G. a c u l e a t u s t e s t e s 44 F i g u r e 7. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s - s p e c i f i c p r o t e i n s (TSPs) from Anura and Squamata 47 F i g u r e 8. E l e c t r o p h o r e t i c p r o f i l e s of b a s i c p r o t e i n s from t e s t i s of X. l a e v i s and G. a c u l e a t u s on d i f f e r e n t s i z e s of p o l y a c r y l a m i d e g e l s 50 F i g u r e 9. Comparison of e l e c t r o p h o r e t i c p r o f i l e s of TSPs from G. a c u l e a t u s and r e l a t e d t e l e o s t s 56 F i g u r e 10. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from t e s t i s of G. a c u l e a t u s t aken from J e r i c h o Pond, Vancouver, B.C. ...64 F i g u r e 11. E l e c t r o p h o r e t i c comparison of TSPs from b r e e d i n g and n o n - b r e e d i n g G. a c u l e a t u s - l e i u r u s from M a r p o l e Pond, Vancouver, B.C 68 F i g u r e 12. E l e c t r o p h o r e t i c comparisons of TSPs from the t e s t i s of the anadromous form of G. a c u l e a t u s from d i f f e r e n t p o p u l a t i o n s i n B r i t i s h Columbia and Quebec. ...71 F i g u r e 13. E l e c t r o p h o r e t i c comparison of TSPs from f r e s h w a t e r and anadromous G. a c u l e a t u s t e s t i s 76 F i g u r e 14. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from t e s t e s of b e n t h i c and l i m n e t i c forms of G. a c u l e a t u s from Enos Lake, B.C 83 F i g u r e 15. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from t e s t e s of b e n t h i c and l i m n e t i c G. a c u l e a t u s - l e i u r u s from Paxton Lake, B.C. 87 F i g u r e 16. E l e c t r o p h o r e t i c p r o f i l e s of b a s i c p r o t e i n s from t e s t i s of b e n t h i c and l i m n e t i c forms of G. a c u l e a t u s from Paxton Lake, B.C. R e l a t i v e amounts of somatic h i s t o n e s a re compared 89 F i g u r e 17. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from one p a i r of t e s t e s from G. a c u l e a t u s - t r a c h u r u s from R a i l r o a d D i t c h , B.C. d i g e s t e d w i t h cyanogen bromide or w i t h t r y p s i n 94 F i g u r e 18. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from f r e s h w a t e r and anadromous G. a c u l e a t u s d i g e s t e d w i t h cyanogen bromide. 96 F i g u r e 19. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from G. a c u l e a t u s - l e i u r u s from J e r i c h o Pond, Vancouver, B.C. t r e a t e d w i t h a l k a l i n e p hosphatase. . ... 101 F i g u r e 20. E l e c t r o p h o r e t i c d e t e c t i o n of d e g r a d a t i o n p r o d u c t s i n G. a c u l e a t u s and P. p u n g i t i u s TSPs upon h e a t i n g a t s l i g h t l y a l k a l i n e pH 104 F i g u r e 21. E f f e c t s of ammonium s u l f a t e on endogenous x i i p r o t e a s e of G. a c u l e a t u s TSPs .. 108 F i g u r e 22. E l e c t r o p h o r e t i c p r o f i l e s of anomalous bands i n TSPs of G. a c u l e a t u s - t r a c h u r u s from Quebec. 111 F i g u r e 23. E l e c t r o p h o r e t i c p r o f i l e s of anomalous bands i n TSPs of G. a c u l e a t u s - t r a c h u r u s from B.C 114 F i g u r e 24. P l a t e morphs of G. a c u l e a t u s 122 x i i i ACKNOWLEDGEMENTS My deep a p p r e c i a t i o n to Dr. H. E. Kasinsky, f o r h i s advice and f r i e n d s h i p . I have found Dr. Kasinsky's d e d i c a t i o n to science, through h i s c o n s c i e n t i o u s r e f l e c t i o n of the l i f e around him, to be a true i n s p i r a t i o n . I am g r a t e f u l a l s o f o r the ass i s t a n c e of the f o l l o w i n g people: Dr. J . D. McPhail, R. Carveth and collegues f o r the c o l l e c t i o n of b i o l o g i c a l specimens and advice, Dr. W. Vogl for l i g h t and e l e c t r o n microscopy, M. Mann for cytochemistry, Dr. H. Guderley f o r b i o l o g i c a l specimens, Dr. J . Gosline for h i s i n s i g h t f u l c ounseling, M. Tsui for photography, H. Sabourni and U. Lee f o r t e c h n i c a l a s s i s t a n c e . Thanks a l s o for the help and support from Dr. D. Holme, M. E. DeMont, B. Young, the ' f a m i l y ' , and D. F i e l d s . I would l i k e to acknowledge the Vancouver Regional T r a n s i t System-bus route no. 41 (be t t e r luck next time boys, I'm s t i l l w a l king). This research was supported by grant no. 675854 to Dr. H. E. Kasinsky. 1 INTRODUCTION The male gametes of most s p e c i e s c o n t a i n s p e c i a l DNA a s s o c i a t e d , b a s i c p r o t e i n s ( s p e r m - h i s t o n e s ) , which are u s u a l l y d i f f e r e n t i n s t r u c t u r e and c o m p o s i t i o n than those found i n somatic c e l l s (Tsanev, 1980). I n t e r e s t i n g l y , the sperm h i s t o n e s from d i f f e r e n t organisms show marked d i f f e r e n c e s i n s t r u c t u r e and c o m p o s i t i o n . T h i s i s i n c o n t r a s t t o the extreme l a c k of v a r i a t i o n i n somatic h i s t o n e s , which a r e amongst the most s t r u c t u r a l l y c o n s e r v a t i v e p r o t e i n s known. The v a r i a b i l i t y of b a s i c sperm p r o t e i n s has been s t u d i e d i n a v a r i e t y of t a x a and has been found t o be g r e a t e s t i n the c l a s s Amphibia ( K a s i n s k y e t a l . , 1978; Mann, 1981) and i n some t e l e o s t s ( c l a s s O s t e i c h t h y e s ) ( B l o c h , 1 96.9,. K a s i n s k y et a_l. , 1984b). Sperm h i s t o n e s from the genus Xenopus ( c l a s s Amphibia) show s u f f i c i e n t d i v e r s i t y t h a t they can be used t o r e l i a b l y d i s t i n g u i s h i n d i v i d u a l s p e c i e s and i n some ca s e s s u b s p e c i e s . T h e r e f o r e , sperm h i s t o n e s produce u s e f u l m o l e c u l a r markers. S i n c e g r e a t d i v e r s i t y i n sperm h i s t o n e s e x i s t among t e l e o s t s , i t would be i n t e r e s t i n g t o know i f the b a s i c p r o t e i n s i n sperm can be used as m o l e c u l a r markers i n f i s h , as they had i n Xenopus. I f so, a r e these m o l e c u l a r markers s e n s i t i v e enough t o d i s t i n g u i s h between s u b s p e c i e s and even d i f f e r e n t p o p u l a t i o n s ? I f enough i s known about the taxonomy and e c o l o g y of the f i s h groups, then t h e s e m o l e c u l a r markers may p r o v i d e i n s i g h t i n t o the s p e c i a t i o n of the group. F u r t h e r , i t i s p o s s i b l e t h a t s t u d i e s of the v a r i a b i l i t y of sperm h i s t o n e s may u l t i m a t e l y l e a d t o a b e t t e r g e n e r a l — u n d e r s t a n d i n g 2 of the r o l e of sperm p r o t e i n s i n the c e l l . T h i s study s e t s out t o d e t e r m i n e i f t e s t i s s p e c i f i c p r o t e i n s (TSP's) can a c t as m o l e c u l a r markers f o r s e v e r a l d i f f e r e n t s p e c i e s of t e l e o s t s i n the o r d e r G a s t e r o s t e i f o r m e s and t o see i f TSP's can be used t o d i s t i n g u i s h between d i f f e r e n t p o p u l a t i o n s of the same s p e c i e s ( G a s t e r o s t e u s a c u l e a t u s ) . D u r i n g s p e r m i o g e n e s i s , the male gamete of G a s t e r o s t e u s  a c u l e a t u s l o s e s c y t o p l a s m , most of i t s o r g a n e l l e s , and reduces i t s n u c l e a r volume. Accompanying t h i s p r o c e s s i s a replacement of the 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 the DNA of the gamete. H i s t o n e s c h a r a c t e r i s t i c of somatic t i s s u e s a r e s u b s t i t u t e d by more b a s i c p r o t e i n s i n the s p e r m a t i d and sperm. The c h r o m a t i n f i b r i l s c o a l e s c e i n t o a g g r e g a t e s f o r m i n g a dense, s e m i c r y s t a l l i n e mass composed of DNA and the b a s i c sperm p r o t e i n s . H i s t o n e s a r e s m a l l b a s i c p r o t e i n s of about 102-135 amino a c i d s t h a t have a s t r o n g p o s i t i v e charge a t n e u t r a l pH. These are c h a r a c t e r i s t i c s s h a r ed by few o t h e r p r o t e i n s . In somatic c e l l s , an octamer of two of each h i s t o n e s H2A, H2B, H3 and H4 form as DNA of 146 n u c l e o t i d e s i n l e n g t h wrap around 1 1/2 times t o c r e a t e the nucleosome. Between each nucleosome are the H1 h i s t o n e s a s s o c i a t e d w i t h about 60 DNA n u c l e o t i d e s . The h i g h amount of l y s i n e and a r g i n i n e c r e a t e s the p o s i t i v e charge of the h i s t o n e t h a t e l e c t r o s t a t i c a l l y l i n k s the n e g a t i v e DNA phosphates (Wolfe, 1983). C o n s e r v a t i o n of the 4 n u c leosomal h i s t o n e s has been n o t e d , e s p e c i a l l y i n h i s t o n e H4 i n which o n l y 2 of the 102 amino a c i d s d i f f e r between the c a l f and the garden pea. The a r g i n i n e - r i c h H3 and H4 a r e very s i m i l a r i n a l l a n i m a l s p e c i e s whereas the s l i g h t l y l y s i n e - r i c h h i s t o n e p a i r H2A and H2B has r e l a t i v e l y l a r g e r s p e c i e s d i f f e r e n c e s . I s o h i s t o n e s (non-a l l e l i c v a r i a n t s ) of H2A, H2B and H3 e x i s t ( Z w e i d l e r , 1978). L y s i n e - r i c h h i s t o n e H1 i s t h e most v a r i a b l e showing s p e c i e s -s p e c i f i c h e t e r o g e n e i t y . Due t o t h i s h i g h degree of c o n s e r v a t i o n , i t can be assumed t h a t a l l p a r t s of the c h r o m a t i n s t r u c t u r e and f u n c t i o n a r e v i t a l and even s m a l l changes would have a l e t h a l e f f e c t ( E l g i n and W e i n t r a u b , 1975). D u r i n g s p e r m i o g e n e s i s the m o r p h o l o g i c a l changes i n the n u c l e u s a r e u s u a l l y accompanied by replacement of the h i s t o n e s ( B o l s , et a l . , 1976) by more b a s i c t e s t i s - and sperm- s p e c i f i c p r o t a m i n e s (Wolfe, 1981; S u b i r a n a , 1982). For example, replacement i n t r o u t sperm i s thought t o o c c u r by a c e t y l a t i o n of h i s t o n e s H1, H2A, H2B, H3 and H4 which reduces the b a s i c i t y of the p r o t e i n and causes the u n b i n d i n g of the DNA ( D i x o n , 1974). A r g i n i n e - r i c h p r o t e i n s a r e s y n t h e s i z e d i n the c y t o p l a s m , p h o s p h o r y l a t e d , and then e n t e r the n u c l e u s . These p r o t e i n s a r e c a l l e d p r o t a m i n e s and s u b s e q u e n t l y r e p l a c e the h i s t o n e s t h r o u g h c o m p e t i t i o n . A f t e r r e p l a c e m e n t , the n u c l e u s condenses as the p r o t a m i n e s a r e d e p h o s p h o r y l a t e d . RNA s y n t h e s i s i s i n a c t i v e b e f o r e the end of replacement (Wo l f e , 1981). The p r o t a m i n e s a r e not o n l y more b a s i c , but a r e s m a l l e r (50-78 amino a c i d s ) than 4 h i s t o n e s ( W o l f e , 1981; S u b i r a n a , 1982). Thus, DNA-protamine d e c r e a s e s the n u c l e a r volume more so than does the DNA-histone complex. (Wolfe, 1981). Many a n i m a l s have sperm p r o t e i n s t h a t can be r e l a t e d i n c o m p o s i t i o n t o e i t h e r p r o t a m i n e s or somatic h i s t o n e s ( S u b i r a n a , 1982). B l o c h ( 1969) c l a s s i f i e d sperm head p r o t e i n s i vnto the f o l l o w i n g 5 c a t e g o r i e s : Type 1: Salmon - up t o 2/3 of the amino a c i d r e s i d u e s a r e a r g i n i n e and t h i s c o n s t i t u t e s the s o l e b a s i c amino a c i d . A l s o r e f e r r e d t o as monoprotamine. Type 2: Mammalian - a r g i n i n e r i c h , h i g h l y b a s i c , c o n t a i n i n g -SK groups or a s s o c i a t e d w i t h -SH c o n t a i n i n g p r o t e i n s . Type 3: M y t i l u s - i n t e r m e d i a t e between monoprotamines and somatic h i s t o n e s . They c o n t a i n 2 or 3 b a s i c amino a c i d s . Type 4: Rana - no apparent d i f f e r e n c e between sperm and somatic h i s t o n e s . Type 5 Crab - no b a s i c p r o t e i n i n the n u c l e u s . The f u n c t i o n of sperm p r o t e i n s i s unknown. One approach t o t h i s problem was a t t e m p t e d w i t h the use of x -ray d i f f r a c t i o n t e c h n i q u e s by S u b i r a n a (1982). E x t e n s i v e e x p e r i m e n t s proved t h a t the DNA a r r a n g e d i t s e l f i n a p a r a l l e l f a s h i o n w i t h the p r o t a m i n e s n e u t r a l i z i n g t h e phosphate groups. I t was not d e t e r m i n e d whether the DNA wraps around protamine groups i n a 'screw' e f f e c t or i f the p o l y a r g i n i n e groups f e l l i n t o the l a r g e groove of the DNA h e l i x , making an o r t h o r h o m b i c system. I t i s 5 a l s o p o s s i b l e t h a t the protamine f a l l s i n the s m a l l groove of the DNA h e l i x . Another approach i s t o l o o k f o r p o s s i b l e p h y l o g e n e t i c t r e n d s which might g i v e c l u e s about the f u n c t i o n of the sperm p r o t e i n s . K a s i n s k y , e_t a l . (1978) proposed t h a t a m a c r o e v o l u t i o n a r y t r e n d e x i s t s i n the sperm h i s t o n e s of v e r t e b r a t e s i n which the d i v e r s i t y of sperm h i s t o n e s i n f i s h and f r o g s g i v e s way t o a r e l a t i v e c o n s t a n c y i n b i r d s and e u t h e r i a n mammals. The v a r i a b i l i t y of the sperm p r o t e i n s i n anurans a l l o w e d t h e s e p r o t e i n s t o be used as m o l e c u l a r markers f o r s p e c i e s and perhaps even s u b s p e c i e s (Mann e t a_l. , 1 982). S t u d i e s on Xenopus spp. i l l u s t r a t e s t h i s v a r i a b i l i t y . Only 3 peopl e i n the w o r l d can d i s t i n g u i s h t hese s p e c i e s and s u b s p e c i e s by m o r p h o l o g i c a l f e a t u r e s ( K a s i n s k y , p e r s . comm.). There a r e 2 p o l y p l o i d l i n e s of Xenopus. The sperm p r o t e i n s p r o f i l e s of the 20 and 40 d i p l o i d l i n e s of a n i m a l s can e a s i l y be d i s t i n g u i s h e d from the sperm p r o t e i n s p r o f i l e s of the 36, 72 and 108 d i p l o i d a n i m a l s . I t had been d e t e r m i n e d by serum albumin i m m u n o l o g i c a l a n a l y s i s t h a t X. l a e v i s and X. b o r e a l i s , both w i t h a d i p l o i d number of 36, were s e p a r a t e d by an e s t i m a t e d 25 t o 30 m i l l i o n y e a r s from t h e i r common a n c e s t o r ( B i s b e e et a l . , -1977). Yet sperm p r o t e i n e l e c t r o p h o r e s i s r e v e a l e d c h a r a c t e r i s t i c bands t h a t c o u l d i d e n t i f y the 2 s p e c i e s . In the case of s u b s p e c i e s , m o r p h o l o g i c a l l y v e r y s i m i l a r X. 1. v i c t o r i a n u s and X. 1^ . b u n y o n i e n s i s a l s o had i d e n t i c a l l a c t a t e dehydrogenase isozymes. However, X. 1. b u n y o n i e n s i s c o u l d be i d e n t i f i e d by the presence 6 of s e v e r a l e x t r a sperm p r o t e i n bands on p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (Mann et a l . , 1982). A c q u i s i t i o n of d i f f e r e n t Xenopus s p e c i e s i s d i f f i c u l t , c o s t l y and the e c o l o g y of the s p e c i e s are not w e l l d e t e r m i n e d . S i n c e t e l e o s t s e x h i b i t a s i m i l a r d i v e r s i t y of sperm p r o t e i n t y p e s t o the anurans ( B l o c h , 1969, 1976), i t might be u s e f u l t o l o o k a t t e l e o s t s . The s t i c k l e b a c k s ( G a s t e r o s t e i d a e ) form a s m a l l f a m i l y of the p h y l o g e n e t i c a l l y d i v e r s e bony f i s h e s , the T e l e o s t e i . The t h r e e - s p i n e s t i c k l e b a c k ( G a s t e r o s t e u s a c u l e a t u s ) i s abundant i n c o a s t a l B.C. and has been s t u d i e d e x t e n s i v e l y . F u r t h e r m o r e , p r e l i m i n a r y o b s e r v a t i o n s show the presence of i n t e r m e d i a t e - t y p e sperm i n the t e s t i s ( K a s i n s k y e_t al_. , 1979). C o n s e q u e n t l y , G. a c u l e a t u s seems t o be an a p p r o p r i a t e t e l e o s t t o examine r e g a r d i n g sperm p r o t e i n v a r i a b i l i t y . A l t h o u g h the b r e e d i n g b e h a v i o r and o t h e r c h a r a c t e r i s t i c s of G. a c u l e a t u s are w e l l s t u d i e d , the taxonomy of the s p e c i e s i s b e w i l d e r i n g and t h e r e are a l a r g e number of m o r p h o l o g i c a l l y d i f f e r e n t p o p u l a t i o n s . B e l l (1976) su g g e s t s t h a t the t h r e e - s p i n e s t i c k l e b a c k i s a s u p e r s p e c i e s composed of s p e c i e s and s u b s p e c i e s , some of which are p o l y t y p i c and p o l y m o r p h i c but a l l of which form a c o h e s i v e taxonomic u n i t . T h i s s u p e r s p e c i e s c o n s i s t s of a c o l l e c t i o n of dynamic p o p u l a t i o n s each of which responds r a p i d l y t o changing l o c a l c o n d i t i o n s (Moyle and Cech, 7 1982). G a s t e r o s t e u s a c u l e a t u s has a c i r c u m p o l a r d i s t r i b u t i o n except on the A r c t i c - S i b e r i a n c o a s t . On the N o r t h American west c o a s t i t e x t e n d s south t o B a j a , C a l i f o r n i a and i n Europe, s o u t h t o the M e d i t e r r a n i a n Sea (Hagen, 1 9 6 7 ) ( F i g . 1 ) . A major d i v i s i o n i n G. a c u l e a t u s i s i n t o the t r a c h u r u s and l e i u r u s forms. T r a c h u r u s i s the anadromous form. I t spends most of i t s l i f e i n the marine environment but breeds i n f r e s h w a t e r . The l e i u r u s form completes i t s t o t a l l i f e c y c l e i n f r e s h w a t e r . R e p r o d u c t i v e b e h a v i o r i n G. a c u l e a t u s i s s y n c h r o n i z e d w i t h the e n v i r o n m e n t a l c o n d i t i o n s ( L i l e y , 1968). A 2 p a r t r e p r o d u c t i v e c y c l e r o u g h l y i n c l u d e s the non-breeding season from August t o A p r i l and a 2-3 month b r e e d i n g season i n the r e m a i n i n g months (Wootton, 1976). The t e s t i s of the m a t u r i n g G. a c u l e a t u s . i s d i f f e r e n t i a t e d when the a n i m a l i s 25 mm. l o n g and i s f u n c t i o n a l l y mature when i t i s 40 mm. i n l e n g t h (Swarup, 1959). P a i r e d t e s t e s of a mature, b r e e d i n g G. " a c u l e a t u s measure 1.5 x 0.9 mm. T h i s i s about 1% of the a d u l t body w e i g h t . L o c a t e d v e n t r o - l a t e r a l l y i n the body c a v i t y , t e s t e s a r e c o v e r e d by v i s c e r a l p e r i t o n e u m . Melanophores a r e p r e s e n t i n t h i s p e r i t o n e u m and make i t much d a r k e r than the o v a r i e s of the female. T h e ^ t e s t i s c o n s i s t s _ o f 150-200 t u b u l e s t h a t u n i t e a t the vas d e f e r e n s . Tubules a r e composed of e p i t h e l i a l c e l l s , c o n n e c t i v e t i s s u e , a few spermatogonia germ c e l l s as w e l l as the i n t e r s t i t i a l c e l l s between t u b u l e s (Wootton, 1976). In mature s t i c k l e b a c k s the 8 i F i g u r e 1. N o r t h American d i s t r i b u t i o n of G. a c u l e a t u s . (Wootton, 1976). 10 i n t e r s t i t i a l space becomes g l a n d u l a r and prominent, as opposed t o narrow, n o n - g l a n d u l a r i n t e r t u b u l a r space of the immature males ( C r a i g - B e n n e t t , 1931). A l t h o u g h the p r i m a r y f u n c t i o n of the t e s t i s i s sperm p r o d u c t i o n , they a l s o s e r v e as e n d o c r i n e organs t h a t produce s t e r o i d hormones and androgens (Wootton, 1976). The t e s t i s a l s o c o n t r o l s the development of the secondary s e x u a l c h a r a c t e r i s t i c s (Hoar e_t a l . , 1951). The i n t e r s t i t i a l c e l l s of L e y d i g i n the t e s t i s a r e the s i t e f o r the p r o d u c t i o n of the hormones t h a t induce t h e s e c h a r a c t e r i s t i c s . The development and m a t u r a t i o n of the t e s t i s i s dependent on hormonal response from the p i t u i t a r y g l a n d of the hypothalmus and the i n t e r s t i t i a l t i s s u e of the t e s t i s . Upon r e a c h i n g s e x u a l m a t u r i t y , t h e . a d u l t a n i m a l goes through a s e a s o n a l development c o o r d i n a t e d by hormones of t h e s e organs which t r i g g e r s s p e r m a t o g e n e s i s , mating b e h a v i o r and development of secondary s e x u a l c h a r a c t e r i s t i c s . What changes occur as s p e r m a t o g e n e s i s p r o g r e s s e s i n G. a c u l e a t u s ? A d e s c r i p t i o n of the s e a s o n a l development of s p e r m a t o g e n e s i s i n the t e s t i s of G. a c u l e a t u s has been done by C r a i g - B e n n e t t (1931). D i v i s i o n of the spermatogonia s t a r t s a t the end of J u l y and extends t o the m i d d l e of September, a t which p o i n t d i v i s i o n s t o p s . The c e l l s t r a n s f o r m i n t o p r i m a r y s p e r m a t o c y t e s which a r e c o n t a i n e d i n c y s t s t h a t e n t i r e l y f i l l the lumen of the t u b u l e s . Throughout the year spermatagonia a r e p r e s e n t i n the l i n i n g of the vas d e f e r e n s w i t h i n the t e s t i s and 11 / m i g r a t e toward the p e r i p h e r y of the t u b u l e s d u r i n g the w i n t e r . The d i v i s i o n of these c e l l s are r a r e l y seen i n the a d u l t f i s h . M a t u r a t i o n of the male germ c e l l s s t a r t s w i t h the appearance of the p r i m a r y spermatocytes i n the t e s t i s between l a t e J u l y t o e a r l y September and i s completed i n 2-3 months. The p r i m a r y spermatocyte i s about t w i c e as l a r g e as the spermatogonium and c h a r a c t e r i s t i c a l l y has a l a r g e n u c l e o l u s and g r a n u l a t e d c y t o p l a s m . The maximum s i z e of the t e s t i s i s reached when the s m a l l e r secondary s p e r m a t o c y t e s a r i s e and the i n t e r s t i t i a l t i s s u e b e g i n s to d e v e l o p i n January t h r o u g h March. Though minute, the chromosomes a r e e v i d e n t a t t h i s s t a g e . Upon f o r m a t i o n of the s p e r m a t i d s the c y s t s d i s a p p e a r . Chromatin of t h e s p e r m a t i d s i s dense and o c c a s i o n a l l y cups t o one s i d e of. the n u c l e a r membrane. The spermatozoan f i n a l l y d e v e l o p s i n l a t e March t o A p r i l . I t has an o v a l head 1.5 um l o n g w i t h a 15 urn t a i l (Ahsan and Hoar, 1963). E i g h t y p e r c e n t of s p e r m a t o g e n e s i s i s i n p r o g r e s s i n January and o n l y 20% i n June when i t i s marked by the development of the s p e r m a t o c y t e s . C o n d e n s a t i o n of the n u c l e u s d u r i n g spermatogenesis o c c u r s i n the p o s t e r i o r p a r t of the t e s t i s f i r s t . D i v i s i o n of spermatogonia t h a t w i l l produce next y e a r ' s spermatozoa s t a r t s a f t e r the p r e v i o u s spermatozoan a r e d i s c h a r g e d . In t h i s t h e s i s I propose t o examine the f o l l o w i n g q u e s t i o n s r e g a r d i n g sperm h i s t o n e v a r i a b i l i t y i n G. a c u l e a t u s and r e l a t e d spec i e s . 1 2 1. Can t e s t i s s p e c i f i c p r o t e i n s (TSP's) a c t as m o l e c u l a r markers f o r p a r t i c u l a r s p e c i e s of t e l e o s t s i n the o r d e r G a s t e r o s t e i f o r m e s ? 2. Are the TSP's i n d i f f e r e n t p o p u l a t i o n s of the same s p e c i e s s i m i l a r or d i f f e r e n t ? For example, can the TSP's s e r v e t o d i s t i n g u i s h between l e i u r u s and t r a c h u r u s forms of G. a c u l e a t u s ? Can the TSP's be used t o d i s t i n g u i s h d i f f e r e n t f r e s h w a t e r p o p u l a t i o n s from each o t h e r ? Answers t o t h e s e q u e s t i o n s w i l l a l l o w us t o d e t e r m i n e t o what e x t e n t TSP's can a c t as m o l e c u l a r markers between s p e c i e s i n the G a s t e r o s t e i f o r m e s and between p o p u l a t i o n s of G. a c u l e a t u s . 13 MATERIALS AND METHODS A. E x p e r i m e n t a l A n i m a l s T h r e e - s p i n e s t i c k l e b a c k s were p r o v i d e d by Dr. J . D. M c P h a i l and h i s c o l l e a g u e s from R a i l r o a d D i t c h , the L i t t l e Campbell R i v e r and Gr-anby, P a x t o n , Enos, P r i e s t and Goose l a k e s , B r i t i s h Columbia. Minnow t r a p s (The Gee Minnow Trap, Cuba S p e c i a l i t y Mfg. Co., Houghton, N.Y.) and d i p n e t s were used t o c a p t u r e the f i s h . I sampled p o p u l a t i o n s of the same s p e c i e s i n Marpole and J e r i c h o Ponds, B.C. An anadromous t h r e e - s p i n e s t i c k l e b a c k and A l o r h y n c h u s f l a v i d u s were c a p t u r e d w i t h a 10 meter s e i n e net a t S t a n l e y Park. Captured f i s h were kept a l i v e u n t i l f r o z e n or were im m e d i a t e l y put on d r y i c e and s t o r e d a t -70*C (*C = < ^ C ) . T e s t i c u l a r p r o t e i n s d i d not show s i g n s of d e g r a d a t i o n when a n i m a l s were c a p t u r e d and s t o r e d f o r e l e c t r o p h o r e t i c a n a l y s i s i n t h i s f a s h i o n . Anadromous G. a c u l e a t u s , P u n q i t i u s p u n q i t i u s and G. w h e a t l a n d i were p r o v i d e d by Dr. H. G u d e r l e y , U n i v e r s i t y of L a v a l , from R i v i e r e des Vase, Quebec. Upon c a p t u r e , the specimens were f r o z e n on d r y i c e and f l o w n t o Vancouver. Here they were t r a n s f e r r e d t o a -70*C f r e e z e r . A complete l i s t and map of the c o l l e c t i o n s i t e s , d a t e s of s a m p l i n g and l o n g i t u d i n a l and l a t i t u d i n a l c o - o r d i n a t e s a r e g i v e n i n Appendix IA and IB. R e p r e s e n t a t i v e p i c t u r e s of f i s h used i n t h i s s tudy a r e shown i n F i g . 2. 1 4 F i g u r e 2. Photographs of t h r e e - s p i n e s t i c k l e b a c k s ( G a s t e r o s t e u s a c u l e a t u s ) and r e l a t e d s p e c i e s . A. G. a c u l e a t u s - t r a c h u r u s (anadromous) male i n non-b r e e d i n g c o l o r s * B. G. a c u l e a t u s - l e i u r u s ( f r e s h w a t e r ) male e x h i b i t i n g b r e e d i n g c o l o r a t i o n * C. G. a c u l e a t u s - l e i u r u s , l i m n e t i c male and female from Enos Lake, B.C. (male on t o p ) * D. G. a c u l e a t u s - l e i u r u s , b e n t h i c male and female from Enos Lake, B.C. (male on bottom)* E. B l a c k - s p o t t e d s t i c k l e b a c k (G. w h e a t l a n d i ) . F. N i n e - s p i n e s t i c k l e b a c k ( P u n g i t i u s p u n g i t i u s ) G. Tube snout ( A u l o r h y n c h u s f l a v i d u s ) B l a c k l i n e i n l o w e r , r i g h t c o r n e r by each photograph = 0.5 cm. * Photographs taken by P. Bentzen. 1 5 • * — -17 X. l a e v i s was o b t a i n e d from a p o p u l a t i o n m a i n t a i n e d i n the l a b o r a t o r y . Bufo and A n o l i s were purchased from Camosun A q u a r i a , Vancouver, B.C. through the c o u r t e s y of J . - T a i t . B. Morphology T e s t e s from t h r e e - s p i n e s t i c k l e b a c k s were p r e p a r e d f o r l i g h t and e l e c t r o n m i c r o s c o p y by the f o l l o w i n g method. T e s t e s were e x c i s e d from 6 f r e s h l y caught male s t i c k l e b a c k s i n d i f f e r e n t s t a g e s of r e p r o d u c t i v e a c t i v i t y . One t e s t i s was c u t i n t o s m a l l s e c t i o n s w h i l e the e t h e r was f i x e d i n t a c t . The t i s s u e was f i x e d f o r 2.5 hours i n the 1.5% paraformaldehyde/1.5% g l u t a r a l d e h y d e / O . 1 M sodium c a c o d y l a t e , pH 7.3, made up the p r e v i o u s day. The t i s s u e was washed 3 t i m e s i n 0.1 sodium c a c o d y l a t e f o r 10 min. f o r the f i r s t 2 washes and l e f t o v e r n i g h t i n the l a s t wash. The next day the t e s t e s were put i n t o 1.0% OsO4/0.1 c a c o d y l a t e , pH 7.3, f o r 1.5 hours a t room temperature, then washed 3 ti m e s i n d i s t i l l e d water f o r 10 min. each. The samples were p l a c e d i n aqueous 1.0% u r a n y l a c e t a t e f o r 1 hour, then washed f o r 3 t i m e s f o r 10 min. i n d i s t i l l e d water. Graded e t h a n o l washes f o r 10 min. each a t 30, 50, 70, 95 % and 3 washes i n 100% e t h a n o l were done and the specimens were p l a c e d i n 100% p r o p y l e n e o x i d e f o r two, 15 min. washes. The samples were l e f t i n a 1:1 p r o p y l e n e o x i d e / p o l y - b e d 812 s o l u t i o n o v e r n i g h t . On the t h i r d day they were embedded i n p o l y - b e d 812 and l e f t o v e r n i g h t a t 60*C. The b l o c k s were s e c t i o n e d f o r u l t r a s t r u c t u r e and l i g h t 18 m i c r o s c o p y (LM). LM s e c t i o n s were 1 urn i n t h i c k n e s s and were s t a i n e d w i t h t o l u i d i n e b l u e . EM s e c t i o n s were s t a i n e d w i t h b oth u r a n y l a c e t a t e and l e a d n i t r a t e . Photographs f o r LM were taken on a Z e i s s - I I I m i c r o s c o p e and f o r EM on a P h i l l i p s 300 used a t 60 kv. C. C y t o c h e m i s t r y C y t o c h e m i c a l p r o c e d u r e s f o l l o w e d those of B o l s (1972) and Mann (1981). J_. F i x a t i v e and embedding T e s t e s were f i x e d in. 10% 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, pg. 601) f o r 2-3 months. T i s s u e s were then d e h y d r a t e d i n a graded s e r i e s of e t h a n o l (2-3 hours e a c h ) , c l e a r e d i n x y l e n e and embedded i n T i s s u e Prep ( F i s h e r S c i e n t i f i c Co., F a i r l a w n , N . J . ) . S e c t i o n s (5 urn) were c u t on a Spencer '820' microtome. 2. F e u l g e n r e a c t i o n The Fe u l g e n r e a c t i o n was used t o d e t e c t the presence of DNA ( P e a r s e , 1968, pg. 822). S e c t i o n s were h y d r o l y z e d f o r 11 min. i n 1 N HC1 a t 60*C and r i n s e d t w i c e i n d i s t i l l e d water f o r 5 min. The s e c t i o n s were s t a i n e d i n S c h i f f ' s reagent f o r 30 min. T h i s was f o l l o w e d by many s h o r t r i n s e s i n d i s t i l l e d w a t e r , a 5 min. r i n s e i n a c i d - b i s u l f i t e wash and two, 5 min. r i n s e s i n d i s t i l l e d 19 water. S e c t i o n s were d e h y d r a t e d i n a graded s e r i e s of e t h a n o l s , c l e a r e d i n x y l e n e and mounted w i t h Permount ( F i s h e r S c i e n t i f i c Co. ) . 3. A l k a l i n e f a s t r e a c t i o n The A l f e r t and Geschwind (1953) p r o c e d u r e was used t o demonstrate the presence of h i s t o n e s . H y d r o l y s i s i n hot (85*-90*C) 5% TCA f o r 15-20 min. removed DNA from the s e c t i o n s . To i n s u r e complete DNA removal and complete s t a i n i n g , f r e s h reagent was used on each o c c a s i o n . DNA removal a t t h i s t e m p e r a t u r e l e f t the n u c l e i i n t a c t . Absence of DNA was checked by the F e u l g e n p r o c e d u r e . A l t e r n a t i v e l y , DNA was removed from s e c t i o n s by h y d r o l y s i s i n a s a t u r a t e d p i c r i c a c i d s o l u t i o n f o r 6 h o u r s a t 60*C ( B l o c h and Hew, 1960a). T h i s p r o c e d u r e removes DNA from a l l b a s i c n u c l e a r p r o t e i n s , i n c l u d i n g protamine-bound DNA t h a t was not e x t r a c t e d by h y d r o l y s i s i n 5% TCA (Das et. a_l. , 1967). A f t e r h y d r o l y s i s the s e c t i o n s were immersed i n 70% e t h a n o l f o r t h r e e , 10 min. changes t o remove TCA or p i c r i c a c i d , r i n s e d i n d i s t i l l e d water and s t a i n e d f o r 30 min. a t room tem p e r a t u r e i n 0.1% (w/v) f a s t green FCF ( F i s h e r S c i e n t i f i c Co., F a i r l a w n , N . J . ) , pH 8.1-8.3.. ( A l f e r t and Geschwind, 1953). The a l k a l i n e f a s t green (AFG) s o l u t i o n was used i m m e d i a t e l y w i t h the pH b e i n g measured b e f o r e and a f t e r s t a i n i n g t o ensure minimum f l u c t u a t i o n . The pH remained c o n s t a n t a t a p p r o x i m a t e l y 8.23. S t a i n i n g was f o l l o w e d by a 5 min. r i n s e i n d i s t i l l e d "water, 20 d e h y d r a t i o n and mounting as above. 4. A c e t y l a t i o n To d e t e r m i n e whether v e r y a r g i n i n e - r i c h h i s t o n e s were p r e s e n t , a c e t y l a t i o n of l y s i n e r e s i d u e s ( B l o c h and Hew, 1960b) was 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 and b e f o r e AFG s t a i n i n g . A f t e r h y d r o l y s i s the s l i d e s were r i n s e d i n 3 changes of 70% e t h a n o l , 10 min. each, d e h y d r a t e d i n a b s o l u t e e t h a n o l , and put i n t o 1% (v/v) g l a c i a l a c e t i c a c i d i n pure a c e t i c -a n h y d r i d e . A f t e r a l l o w i n g a c e t y l a t i o n t o oc c u r f o r 1 hour a t 60*C, the s l i d e s were r i n s e d t w i c e w i t h a b s o l u t e e t h a n o l f o r 15 min. each, brought t o water, and s t a i n e d f o r 30 min. w i t h 0.1% a l k a l i n e f a s t green (pH 8.1-8.3). 5. Sakaquchi r e a c t i o n The Sakaguchi r e a c t i o n , as m o d i f i e d by D i e t c h (1961), was used t o demonstrate p r o t e i n bound a r g i n i n e . S t ock s o l u t i o n s of 4% (w/v) h y d r o x i d e and 1.5% (w/v) . 2 , 4 - d i c h l o r o n a p t h o l i n t e r t i a r y b u t a n o l were made i n advance. A f r e s h s o l u t i o n of 1% sodium h y p o c h l o r i t e was made by d i l u t i n g 'AquaA' h y p o c h l o r i t e ( M i r o c l e a n P r o d u c t s , Richmond, B.C.) by 1:10. The s l i d e s were brought t o wa t e r , b l o t t e d and p l a c e d i n an empty C o p l i n j a r . The s t a i n i n g r e agent was p r e p a r e d i m m e d i a t e l y by m i x i n g 5 p a r t s barium h y d r o x i d e , 1 p a r t h y p o c h l o r i t e , and then 1 p a r t d i c h l o r o n a p t h o l and poured over the s l i d e s . S t a i n i n g was done a t 21 room / temperature f o r 10-20 min. Next, the s l i d e s were put t h r o u g h 3 changes of t e r t i a r y b u t a n o l w i t h 5% (v/v) t r i - N -b u t y l a m i n e f o r 30 s e c . each, and c l e a r e d i n two changes of x y l e n e c o n t a i n i n g 5% (v/v) t r i - N - b u t y l a m i n e . They were mounted i n Permount c o n t a i n i n g 10% (v/v) t r i - N - b u t y l a m i n e . 6. C o n t r o l s To d e t e r m i n e the s t a i n i n g p a t t e r n s of sperm of o t h e r organisms c o n t a i n i n g i n t e r m e d i a t e - t y p e sperm s p e c i f i c p r o t e i n s , s e c t i o n s of t e s t e s from Xenopus ( t y p e 3A) and M y t i l u s (type 3B) were used ( K a s i n s k y , e t a_l. , 1984). T e s t e s of the t r o u t (Salmo) and the l e o p a r d f r o g (Rana) were used t o show the s t a i n i n g p a t t e r n s of pro t a m i n e s (type 1) and s o m a t i c - l i k e sperm h i s t o n e s ( t y p e 4) r e s p e c t i v e l y . H y d r o l a q u s c o l l e i , the r a t f i s h , was used t o show the k e r a t i n o u s p r o t e i n s of the t y p e 2 ( B o l s and K a s i n s k y , 1976). 7. Photography Photographs f o r c y t o c h e m i s t r y were t a k e n on a L e i t z Orthomat-W photomicroscope u s i n g i n t e r f e r e n c e - c o n t r a s t o p t i c s . 22 D. B i o c h e m i s t r y J_. P r e p a r a t i o n of T e s t i s - S p e c i f i c P r o t e i n s (TSP) Somatic h i s t o n e s and TSP's were o b t a i n e d from a c i d e x t r a c t s of t e s t i s c e l l s u s p e n s i o n s as d e s c r i b e d by K a s i n s k y , e t a l . (1978). T e s t e s used were e i t h e r f r e s h or f r o z e n at -70*C and homogenized by hand u s i n g a Kontes D u a l l ground g l a s s homogenizer w i t h 4 volumes of i c e c o l d phosphate b u f f e r e d s a l i n e (PBS) (Dulbecco and Vogt, 1954). L i g h t m i c r oscopy was used t o check the p r e s ence of sperm at 400x m a g n i f i c a t i o n . In the mature, b r e e d i n g s t i c k l e b a c k , sperm appeared as the major c e l l t ype i n the t e s t i s homogenate. The s u s p e n s i o n was put d r o p - w i s e onto a g l a s s f i b r e f i l t e r paper under s u c t i o n and r.insed w i t h 2 ml. of i c e c o l d 70% (v/v) e t h a n o l . A c c o r d i n g t o L o u i e and Dixon (1972), t h i s s t e p f i x e s the c e l l s and removes the c y t o p l a s m . The e xcess f i l t e r paper was trimmed away; the remainder was p l a c e d i n t o a m i c r o - c e n t r i f u g e tube w i t h 200 u l of 0.4 N HC1 t o e x t r a c t the b a s i c p r o t e i n s . U s i n g 0.4 N s u l f u r i c a c i d gave l i t t l e d i f f e r e n c e i n the r e s u l t s except t h a t HC1 seemed t o y i e l d d a r k e r bands upon e l e c t r o p h o r e s i s and s t a i n i n g of the g e l . A f t e r e x t r a c t i o n on i c e f o r 20-30 min., the e x t r a c t was removed w i t h a flamed P a s t e u r p i p e t t e and the a c i d s o l u b l e p r o t e i n s were p r e c i p i t a t e d by the a d d i t i o n of 44 u l of 100% (w/v) t r i c h l o r o a c e t i c a c i d (TCA) t o a f i n a l c o n c e n t r a t i o n of 20% (w/v) f o r 20-30 min. a t room t e m p e r a t u r e . The p r e c i p i t a t e s were spun down i n an Eppendorf m i c r o c e n t r i f u g e (15,000 rpm f o r 10 min.) 23 and the s u p e r n a t a n t removed by s u c t i o n . The p e l l e t s were resuspended i n a c i d acetone (0.33 ml. c o n c e n t r a t e d HC1/200 ml. acetone) t o wash out the TCA and c e n t r i f u g e d as above. T h i s s t e p was then r e p e a t e d . A stream of n i t r o g e n gas was used t o dry the p e l l e t , which was then d i s s o l v e d i n 8 M urea/5% 2-m e r c a p t o e t h a n o l f o r e l e c t r o p h o r e s i s (Mann et, a l . , 1982). T h i s method of i s o l a t i n g TSP's was more r a p i d than i s o l a t i n g such b a s i c p r o t e i n s from c h r o m a t i n p r e p a r a t i o n s of p u r i f i e d c e l l t y p e s (Mann e t a l . , 1982). T h e r e f o r e , d u r i n g the i s o l a t i o n p r o c e d u r e , t h e r e was a reduced chance of p r o t e o l y s i s of the p r o t e i n s ( K a s i n s k y e t a_l. , 1978). Heart was run as a somatic t i s s u e t o check the f i t n e s s of the specimen by i n s p e c t i o n of the som a t i c h i s t o n e p r o f i l e on the p o l y a c r y l a m i d e g e l . Commercial h e r r i n g ( C l u p e i n a ) protamine "and c a l f thymus h i s t o n e (Sigma Chem. Co., S t . L o u i s , M i s s o u r i ) were a l s o run as e l e c t r o p h o r e t i c m arkers. 2. E l e c t r o p h o r e s i s P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) was based on the method of Panyim and C h a l k l e y (1969) t o make 15% p o l y a c r y l a m i d e s l a b g e l s c o n t a i n i n g 6.25 M urea t o a f i n a l pH of about 2.5 ( a f t e r p r e - e l e c t r o p h o r e s i s ) . The f o l l o w i n g s o l u t i o n s were used t o make the s e g e l s : S o l u t i o n A: 60% a c r y l a m i d e (w/v) (Bio-Rad Chem. Co., Richmond, CA) and 0.4% N , N ' - b i s a c r y l a m i d e (w/v) (Eastman Kodak Co., R o c h e s t e r , N.Y.) i n d i s t i l l e d w ater; S o l u t i o n B: 43.3% of g l a c i a l a c e t i c a c i d (v/v) and 4.0% 24 N,N,N',N'-tetramethyl e t h y l e n e d i a m i n e (TEMED) (Eastman Kodak, R o c h e s t e r , N.Y.) i n d i s t i l l e d w ater; S o l u t i o n C: 0.2% ammonium s u l f a t e (w/v) ( F i s h e r Chem. Co., F a i r l a w n , N.J.) i n 10 M u l t r a -pure urea ( A l r i c h Chem. Co., Milwaukee, WI). S o l u t i o n s A and B were s t o r e d a t 0*C. S o l u t i o n C was f r e s h l y p r e p a r e d b e f o r e use. The s o l u t i o n s were mixed i n the r a t i o s of 2 p a r t s A, 1 p a r t B and 5 p a r t s C a t room t e m p e r a t u r e , poured i n t o the g e l mold, and a l l o w e d t o p o l y m e r i z e f o r 30-60 min. b e f o r e p r e - e l e c t r o p h o r e s i s . The g e l molds were 0.5 cm. sheet g l a s s t o form g e l s i z e s i n the d i m e n s i o n s d e s c r i b e d i n Appendix I I . The g e l s were run a t room t e m p e r a t u r e , except, the 87 cm. g e l i n which p r e - e l e c t r o p h o r e s i s was done i n a c o l d room a t 4*C. The t r a y b u f f e r was 0.9 N a c e t i c a c i d . P r e - e l e c t r o p h o r e s i s was c a r r i e d out by a l l o w i n g 10 u l of p y r o n i n - Y (Eastman Kodak Co., R o c h e s t e r , N.Y.) dye p l a c e d i n one of the end l a n e s t o run the complete l e n g t h of the g e l i n t o the bottom b u f f e r t r a y . T h i s a l l o w e d e q u i l i b r a t i o n of the g e l and removal of the p e r s u l f a t e . The t r a y b u f f e r was r e p l a c e d w i t h f r e s h s o l u t i o n and the p r o t e i n samples (60-100 u l . ) were l o a d e d . The dye was a l s o a p p l i e d t o one l a n e of the g e l as a marker f o r the r e l a t i v e m i g r a t i o n of the p r o t e i n s . A f t e r s e v e r a l g e l s had been r u n , the dye m i g r a t i o n c o u l d be used as a r e l i a b l e method of c a l c u l a t i n g the m i g r a t i o n of the o t h e r b a s i c p r o t e i n s . S i n c e the TSP's m i g r a t e d s l o w e r than the dye, an a p p l i c a t i o n of a second dye marker was o f t e n used when the f i r s t had moved o f f the g e l . C o n s t a n t power (DC) was s u p p l i e d by a H e a t h k i t ( M i s s i s s a u g a , O n t a r i o ) R e g u l a t e d H.V. power s u p p l y or a Beckman Model R-120 ( F u l l e r t o n , CA) power s u p p l y . 25 3. S t a i n i n g of p o l y a c r y l a m i d e g e l s G e l s were s t a i n e d o v e r n i g h t w i t h 0.2% amidoblack ( A n a l i n e B l u e - 3 l a c k ) ( A l d r i c h Chem. Co., M i l w . , WI) i n 7% a c e t i c a c i d / 3 0 % methanol and d e s t a i n e d by d i f f u s i o n w i t h many changes of 7% a c e t i c a c i d / 3 0 % methanol u n t i l the g e l was c l e a r . Some g e l s were then r e s t a i n e d w i t h 0.2% amidoblack o v e r n i g h t and d e s t a i n e d by the s e n s i t i v e method of Wray and S t u b b l e f i e l d . (1970) i n 1 M s u l f u r i c a c i d / 3 M urea t o enhance s t a i n i n g of the p r o t e i n s by a p p r o x i m a t e l y 10 f o l d . . 4. Photography of g e l s A P o l a r o i d camera"using type 665 p o s i t i v e / n e g a t i v e l a n d f i l m a t f/11 and 1/60 s e c . was used f o r an immediate r e c o r d of the p r o t e i n bands on the g e l s . A Nikon 35 mm SLR camera w i t h a 28 mm l e n s e a t f/8 f o r 1/15 s e c . w i t h a y e l l o w f i l t e r was used t o enhance band c o n t r a s t . Kodak T e c h n i c a l Pan 35mm f i l m , a . s . a . 25, was used and the p i c t u r e s were taken w i t h the g e l on a l i g h t box i l l u m i n a t e d from below. 5. Amino a c i d a n a l y s i s 26 a. Sample p r e p a r a t i o n and h y d r o l y s i s P r o t e i n s from amidoblack s t a i n e d bands a f t e r PAGE were h y d r o l y z e d f o r amino a c i d a n a l y s i s a c c o r d i n g t o the methods of Houston (1971) and P a l l o t t a and T e s s i e r (1976) as d e s c r i b e d by Huang (1977). Bands were s l i c e d from the g e l w i t h a c l e a n r a z o r b l a d e , p l a c e d i n c l e a n t e s t tubes t h a t had been flamed r e d hot t o burn o f f any r e s i d u e and then c o o l e d . The s l i c e s were washed 3 times w i t h s e v e r a l ml. of g l a s s d i s t i l l e d water f o r 20-30 min. each. Upon removal of the w a t e r , 1-2 m l . of 6 N HCl/0.05% 2-m e r c a p t o e t h a n o l were added and the tubes s e a l e d under vacuum. H y d r o l y s i s took p l a c e a t 110*C f o r 24 h o u r s . The s o l u t i o n was removed, l e a v i n g the b u l k of the a c r y l a m i d e b e h i n d , p l a c e d i n c o o l e d , flamed t e s t tubes and d r i e d w i t h n i t r o g e n gas at 50*C. The r e s i d u e was r e d i s s o l v e d i n 300 u l Beckman sodium c i t r a t e d i l u t i o n b u f f e r , pH 2.2 f o r 5 min.. and f i l t e r e d t hrough a 45 urn M i l l i p o r e f i l t e r ( c a t . no.. HAWP 01300, B e d f o r d , MA). An a d d i t i o n a l 150 u l of d i l u t i o n b u f f e r washed t h r o u g h the f i l t e r h e l p e d t o c o l l e c t the r e m a i n i n g sample. Samples were f r o z e n a t -20*C u n t i l used. b. Sample a n a l y s i s A l l samples were run on a Beckman Amino A c i d A n a l y z e r Model 118C. The 31.5 x 0.6 cm. column was packed w i t h Beckman Type W-2 r e s i n and was run u s i n g Beckman sodium c i t r a t e b u f f e r s . A n a l y s i s was done a c c o r d i n g t o the Beckman A p p l i c a t i o n Notes 119C-AN-00 27 ( 1 975) w i t h the f o l l o w i n g e x c e p t i o n s : 1) column temperature^ was 52*C; 2) b u f f e r changes c o n s i s t e d of B u f f e r A, 52 min.;; B u f f e r B, 14 min.; B u f f e r C, 82 min.; NaOH, 52 min.; B u f f e r A, 35 min. The r e s u l t s of the amino a c i d a n a l y s i s were e x p r e s s e d as mole p e r c e n t . The s p e c t r o p h o t o m e t r i c peaks were i n t e g r a t e d by t r a c i n g the o u t l i n e of the peaks and c a l c u l a t i n g the a r e a u s i n g a g r a p h i c s t a b l e on an Apple I I computer. These v a l u e s were t r a n s f o r m e d by t a k i n g the a r c s i n of the square r o o t of the mole % so t h a t the r e s u l t s c o u l d be t e s t e d f o r s i g n i f i c a n t d i f f e r e n c e s t h r o u g h an a n a l y s i s of v a r i a n c e (ANOVA) t e s t . The GLIM computer package was used t o c a l c u l a t e the s t a t i s t i c s . The amino a c i d a n a l y s e s of p r o t e i n samples were run a t the h i g h e s t s e n s i t i v i t y of the amino a c i d a n a l y z e r . Thus, even minor c o n t a m i n a t i o n of the sample from the p o l y a c r y l a m i d e g e l and the c h e m i c a l s used t o p r o c e s s i t c o u l d a ccount f o r the v a r i a b i l i t y seen between samples i n each a n i m a l group. 6. C h a r a c t e r i z a t i o n of the TSP p r o t e i n s a. T r y p t i c d i g e s t i o n - T r y p t i c d i g e s t i o n of the p r o t e i n p r e p a r a t i o n s was adapted from E l d e r e t a l . (1977). The d r i e d sample was d i s s o l v e d i n 10 u l of 50 ug/ml t r y p s i n (10800 BAEE units/mg s o l i d ) ( S i g m a Chem. Co., S t . L o u i s , M i s s o u r i ) i n 0.05 M ammonium b i c a r b o n a t e b u f f e r 28 (pH 8.0) and i n c u b a t e d a t 37*C o v e r n i g h t . The r e a c t i o n was stopped w i t h the a d d i t i o n of 50 u l of 8 M u r e a / 5 % 2-m e r c a p t o e t h a n o l . b. Cleavage w i t h cyanogen bromide T e s t i c u l a r p r o t e i n s o b t a i n e d through a c i d e x t r a c t i o n as d e s c r i b e d above were c l e a v e d a t the m e t h i o n i n e r e s i d u e s (Gross and Witkop, 1962) by the cyanogen bromide r e a c t i o n as adapted from the work of DeLange et a l . (1968) on h i s t o n e H4. The d r i e d p r o t e i n p e l l e t (about 100 ug) p r e p a r e d from the t e s t i s homogenate was t r e a t e d w i t h an 80 f o l d molar e x c e s s of cyanogen bromide ( A l d r i c h Chem. Co., M i l w . , WI) a t room temperature and a l s o a t 37*C f o r 16 hours i n 10 u l 70% f o r m i c a c i d (v/v) a t a p r o t e i n c o n c e n t r a t i o n of 10 u g / u l . From amino a c i d a n a l y s i s , i t was e s t i m a t e d t h a t t h e r e was about 100 ug of p r o t e i n i n a sample, w i t h a m o l e c u l a r weight of l e s s than 10,000 ug/umole. T h e r e f o r e 85 ug of cyanogen bromide i n 10 u l 70% f o r m i c a c i d was added t o the p r o t e i n sample. The r e a c t i o n was stopped by the a d d i t i o n of 50 u l of 8 M u r e a / 5 % 2-mercaptoethanol and a p p l i e d t o the g e l . c. A l k a l i n e phosphatase A l k a l i n e phosphatase was used i n an attempt t o d e p h o s p h o r y l a t e the s e r i n e s i d e c h a i n s of TSP a c c o r d i n g t o the method of R i s l e y (1977). A l k a l i n e phosphatase (BAPC, 11 mg/ml, 29 33 unints/mg; W o r t h i n g t o n D i a g n o s t i c Systems I n c . , M i s s i s s a u g a , Ont.) was added a t a c o n c e n t r a t i o n of 0.0363 u n i t s / 1 0 0 ug of p r o t e i n i n 10 u l of 0.4 N t r i s / H C l b u f f e r (pH 7.8). D i g e s t i o n s were done f o r 3 and 16 hours a t 37*C i n a c o n s t a n t temperature water b a t h . / [ P r o t e o l y t i c i n h i b i t o r s were used i n some of thes e e x p e r i m e n t s . The s o l u t i o n s would then c o n t a i n 1 mM PMSF ( p h e n y l m e t h y l s u l f o n y l f l u o r i d e , f r e s h l y added from a 50 mM s t o c k s o l u t i o n i n i s o p r o p a n o l ) , 0.1 mM TPCK ( L - 1 - t o s y l a m i d e - 2-p h e n y l e t h y l c h l o r o m e t h y l ketone) and 0.1 mM TLCK ( N - o - p - t o s y l - L -l y s i n e c h l o r o m e t h y l ketone; f r e s h l y added t o warm s o l u t i o n s from 10 mM s t o c k s p r e p a r e d i n i s o p r o p a n o l and water, r e s p e c t i v e l y ) and 50. mM sodium b i s u l f i t e (added j u s t p r i o r t o use, w i t h o u t pH ad j u s t m e n t ) ( R i s l e y and E c k h a r d t , 1981). d. C o n t r o l s C o n t r o l e x p e r i m e n t s were done on TSP p r e p a r a t i o n s s u b m i t t e d t o the same c o n d i t i o n s as d e s c r i b e d above but i n the b u f f e r o n l y , w i t h o u t added r e a g e n t s (CNBr or enzymes)_,_ C o n t r o l s were a l i q u o t s of the same p r e p a r a t i o n s p l i t i n t o e q u a l amounts f o r the assay and c o n t r o l . A f r a c t i o n of the p r o t e i n p r e p a r a t i o n from the same a n i m a l was f r o z e n i m m e d i a t e l y a t -70*C and thawed s h o r t l y b e f o r e g e l a p p l i c a t i o n f o r PAGE. 30 RESULTS A. Morphology K L i g h t m i c r o s c o p y L i g h t m i c r o s c o p y of the G. a c u l e a t u s - l e i u r u s from J e r i c h o Pond, Vancouver, B.C. showed the morphology of the t e s t i s as tak e n from 2 a n i m a l s a t d i f f e r e n t s t a g e s of s e x u a l m a t u r i t y . T e s t e s from both specimens were t a k e n from the s i t e e a r l y i n the b r e e d i n g season (5 May) so t h a t a n i m a l s a t d i f f e r e n t s t a g e s of m a t u r i t y c o u l d be o b t a i n e d . A c r o s s s e c t i o n of the t e s t i s of a s t i c k l e b a c k not y e t i n b r e e d i n g c o n d i t i o n i s seen i n F i g . 3A. The male f i s h d i d not have t h e c h a r a c t e r i s t i c r e d - t h r o a t and b l u e eyes seen i n the b r e e d i n g a n i m a l . F u r t h e r m o r e , t h e t e s t i s was p r e d o m i n a n t l y w h i t e w i t h an i n c o m p l e t e s u r f a c e c o v e r i n g of m e l a n o c y t e s . C e l l t y p e s were a t v a r i o u s s t a g e s i n s p e r m a t o g e n e s i s , i n c l u d i n g p r i m a r y and secondary s p e r m a t o c y t e s . I t was d i f f i c u l t t o d i s t i n g u i s h s p e r m a t i d s from sperm i n the l i g h t m i c r o s c o p e s e c t i o n s . The t e s t i s of a s e x u a l l y mature G. a c u l e a t u s i n f u l l b r e e d i n g c o l o r s has a much d i f f e r e n t morphology. The t e s t i s i s c o m p l e t e l y b l a c k . The t u b u l e c r o s s - s e c t i o n shows m a i n l y s p e r m a t i d s and spermatozoa ( F i g . 3B). F i n e , s a c - l i k e s t r u c t u r e s a re seen i n the open a r e a of the t u b u l e . There was a l s o a 31 F i g u r e 3. L i g h t m i c r o g r a p h s of c r o s s - s e c t i o n s t h r o u g h t u b u l e s of 2 d i f f e r e n t G. a c u l e a t u s - l e i u r u s t e s t e s from J e r i c h o Pond, Vancouver, B.C. c a p t u r e d 10 May 1984. S e c t i o n s were s t a i n e d w i t h t o l u i d i n e b l u e . A. C r o s s - s e c t i o n of a f u l l y matured, b r e e d i n g t e s t i s w i t h many d e v e l o p i n g c e l l - t y p e s i n the p r o c e s s of s p e r m a t o g e n e s i s . B. C r o s s - s e c t i o n of a t u b u l e from a f u l l y matured, p o t e n t i a l l y b r e e d i n g a n i m a l . Only spermatids/sperm a r e p r e s e n t . (spc) = s p e r m a t o c y t e ; ( s p t ) = s p e r m a t i d ; ( I ) = i n t e r s t i t i u m ; (BV) = b l o o d v e s s e l ; ( s p t / s p ) = spermatid/sperm; (SC) = S e r t o l i c e l l ; (M) = m e l a n o c y t e s ; (TW) = t u b u l e w a l l . Bar = 100 urn. 32 33 'ragged' edge a l o n g the p e r i m e t e r of the t u b u l e s . T h i s was b e l i e v e d t o be S e r t o l i c e l l s which h e l d the d e v e l o p i n g gamete as t h e y reduced i n volume and then were r e l e a s e d the spermatozoa i n t o the lumen of the t u b u l e (Dr. W. V o g l , p e r s . comm.). The S e r t o l i c e l l s s u r r o u n d the d e v e l o p i n g sperm and a r e i n v o l v e d i n c o n t r o l l i n g the r e l e a s e of mature sperm i n t o the lumen of the s e m i n i f e r o u s t u b u l e ( P h i l l i p s , 1974a). The t e s t i s i n the e a r l i e r s t a g e s of spermatogenesis ( F i g . 3A) has a much narrower or reduced i n t e r s t i t i a l t i s s u e than the t e s t i s i n the more advanced s t a g e ( F i g . 3B). The i n c r e a s e i n i n t e r s t i t i a l t i s s u e t h i c k n e s s a l s o i n d i c a t e s the s e x u a l l y a c t i v e G. a c u l e a t u s ( C r a i g - B e n n e t t , 1931). 2. E l e c t r o n m i c r o s c o p y T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y was used t o study the u l t r a s t r u c t u r e of the development of G. a c u l e a t u s sperm. The s p e r m a t i d a f t e r m e i o s i s I I i s shown i n F i g . 4A. The c y t o p l a s m i s moving t o the c a u d a l p o l e of the c e l l . The m i t o c h o n d r i a p o s i t i o n t h e m s e l v e s a n t e r i o r l y and l a t e r a l a t the base of the f l a g e l l u m . The c h r o m a t i n i s b e g i n n i n g t o condense i n the n u c l e u s . In F i g . 4B, the m i t o c h o n d r i a have reached the f i n a l p o s i t i o n i n the m i d d l e p i e c e of the s p e r m a t i d . R e s i d u a l c y t o p l a s m i s seen i n the upper c e n t e r of the s e c t i o n . The c h r o m a t i n becomes much more condensed and a r r a n g e s i t s e l f i n t o a g l o b u l a r - l i k e s t r u c t u r e ( F i g . 4B'). A s i s t e r c e l l i s seen t o the r i g h t ( F i g . 4B). Sperm p r o t e i n replacement has a l r e a d y taken p l a c e by t h e s e l a s t 2 34 F i g u r e 4. P r o c e s s of s p e r m i o g e n e s i s i n d e v e l o p i n g s p e r m a t i d s i n G. a c u l e a t u s - l e i u r u s . A. M i g r a t i o n of c y t o p l a s m t o c a u d a l p o l e of s p e r m a t i d , n u c l e a r c o n d e n s a t i o n and assembly of the f l a g e l l u m . (Mi) = m i t o c h o n d r i a ; (F) = f l a g e l l u m ; (pC) = p r o x i m a l c e n t r i o l e ; (dC) = d i s t a l c e n t r i o l e ; (N) = n u c l e u s ; (pM) = plasma membrane; (nE) = n u c l e a r e n v e l o p e . Arrows o u t l i n e c e l l and n u c l e a r b o r d e r s . Bar = 1 urn. B. The a l i g n m e n t of the s p e r m a t i d i n the t u b u l e and g r e a t e r n u c l e a r c o n d e n s a t i o n . (CY) = c y t o p l a s m ( r e s i d u a l body); (F) = f l a g e l l u m ; (Mi) = m i t o c h o n d r i a ; (N) = n u c l e u s . Arrows o u t l i n e c e l l b o r d e r . Bar = 2 urn. B'. S p e r m a t i d n u c l e u s showing g r a n u l a r c h r o m a t i n c o n d e n s a t i o n . Bar = 1 urn. C. Mature spermatozoa w i t h f u l l y condensed c h r o m a t i n . M i t o c h o n d r i a a r e not i n c o n t a c t w i t h the f l a g e l l u m but ar e i n the c y t o p l a s m i c r i n g s u r r o u n d i n g the f l a g e l l a r base. (pM) = plasma membrane ( o u t e r l a y e r ) ; (N) = n u c l e u s ; (nE) = n u c l e a r envelope ( i n n e r 2 l a y e r s ) ; (pC) = p r o x i m a l c e n t r i o l e ; (C) = c e n t r i o l e , ( a d j u n c t ) ; (Mi) = m i t o c h o n d r i a ; (F) = f l a g e l l u m ; (BP) = b a s a l p l a t e . Bar = 0.5 um. 36 38 s t a g e s of s p e r m i o g e n e s i s . The mature sperm i n F i g . 4C i s seen when the e x c e s s c y t o p l a s m has been d i s p e n s e d w i t h , and the n u c l e u s has become f u l l y condensed i n t o the s e m i - c r y s t a l l i n e s t r u c t u r e . The g l o b u l a r p a t t e r n can s t i l l f a i n t l y be seen as i t has become t i g h t l y f u s e d i n t o a condensed n u c l e u s . The space seen between the p o r t i o n of the c e l l t h a t c o n t a i n s the m i t o c h o n d r i a and the base of the f l a g e l l u m e x i s t s due t o the unique shape of t h i s c e l l . A diagram d e p i c t i n g the t h r e e d i m e n s i o n a l shape of t h i s c e l l has been drawn ( F i g . 5 ) . The f l a g e l l u m i s surrounded by a space. T h i s appears t o be a u n i q u e l y shaped sperm w i t h no acrosome p r e s e n t . These c h a r a c t e r i s t i c s a re t y p i c a l of t e l e o s t sperm ( P h i l l i p s , 1974b). F i s h have a m i c r o p y l e i n the egg i n t o which the sperm p e n e t r a t e s f o r f e r t i l i z a t i o n . B. C y t o c h e m i s t r y J_. C o n t r o l s C y t o c h e m i c a l s t a i n i n g of sperm n u c l e i from a n i m a l s of each of B l o c h ' s (1969 and 1976) c a t e g o r i e s of sperm p r o t e i n s was done as c o n t r o l s as i s summarized i n T a b l e 1. Sperm of the rainbow t r o u t (Salmo g a i r d n e r i i ) c o n t a i n s a r g i n i n e - r i c h p r o t a m i n e s ( I n g l e s et. a_l. , 1966) and i s d e s i g n a t e d type 1. These c e l l s s t a i n e d i n t e n s i v e l y w i t h F e u l g e n . P r o t a m i n e s were r e s t a i n e d when s e c t i o n s were h y d r o l y z e d i n p i c r i c a c i d , and 39 F i g u r e 5. L i n e drawing of p r o b a b l e t h r e e - d i m e n s i o n a l c o n f i g u r a t i o n of G. a c u l e a t u s sperm. T a b l e 1. C y t o c h e m i s t r y o f sperm n u c l e i i n G a s t e r o s t e u s a c u l e a t u s - l e i u r u s . G. a c u l e a t u s - l e i u r u s S t a i n i n g and p r e a t r e a t m e n t R e a c t i v e m a t e r i a l • m •U 00 i A ^oo Ctj CTi I B C D E F G i H C o n t r o l s •r-( CO M bC CO co r-4 CD « d co CO o O •rH P. i-i ••-( C 0 M rH o > •rt r-l cu •r< -d l-l c CD 4J a a ctj CO o <u rd >> ctj •H co. M X o x: -1 SI F e u l g e n AFG-TCA 80-85°C AFG, p i c r i c a c i d , 60°C AFG, p i c r i c a c i d , 60 C, a c e t y l a t i o n Sakaguchi DNA B a s i c p r o t e i n s ( o t h e r than protamines & type 3 B ) B a s i c p r o t e i n s ( i n c l u d i n g protamines) B a s i c p r o t e i n s ( r i c h i n a r g i n i n e ) P r o t e i n bound a r g i n i n e + + + + + + + + 1 1 1 + + 4- + + + + + + + + + + + + + + + + + ++ + + + Sperm h i s t o n e type 3 B 3 B 3 B 3 B 3 B 3 B 3 B 3 B 3 A 3 B 4 A-G = G. a c u l e a t u s from J e r i c h o Pond, B.C. H = G. a c u l e a t u s from Marpole Pond, B.C. + = s t a i n s + = s t a i n s moderately: -= does not s t a i n 1 = spermatids s t a i n 42 sperm c o n t i n u e d t o s t a i n w i t h a l k a l i n e f a s t green (AFG) a f t e r a c e t y l a t i o n . The t r o u t sperm gave a v e r y s t r o n g r e a c t i o n " w i t h the Sakaguchi t e s t f o r a r g i n i n e . Sperm of the r a t f i s h , H y d rolagus c o l l e i , c o n t a i n v e r y a r g i n i n e - r i c h h i s t o n e s l i k e t h o s e found i n mammalian sperm ( B o l s and K a s i n s k y , 1974). The k e r a t i n o u s p rotamine of t h e s e sperm n u c l e i s t a i n e d w i t h AFG a f t e r TCA and p i c r i c a c i d h y d r o l y s i s , and c o n t i n u e d t o s t a i n a f t e r a c e t y l a t i o n of the s e c t i o n . They a l s o r e a c t e d s t r o n g l y w i t h Sakaguchi s t a i n . The sperm h i s t o n e s of M y t i l u s e d u l i s and Xenopus l a e v i s a r e of the i n t e r m e d i a t e - t y p e 3 c o n t a i n i n g both l y s i n e and a r g i n i n e ( S u b i r a n a e_t a l . , 19.73; B l o c h , 1969). The sperm s t a i n e d i n t e n s i v e l y w i t h F e u l g e n . The n u c l e i of M. e d u l i s d i d not s t a i n w i t h AFG a f t e r h y d r o l y s i s of s e c t i o n s i n 5% (w/v) TCA a t 80-85*C whereas X. l a e v i s sperm d i d . The d i f f e r e n c e i n the te m p e r a t u r e of e x t r a c t i o n of the b a s i c p r o t e i n s from the sperm n u c l e i d e f i n e s the c a t e g o r y 3A f o r X. l a e v i s (95-100*C) and type 3B f o r M. e d u l i s ( 8 0 - 8 5 * C ) ( K a s i n s k y , e t a l . , 1984a). They d i d s t a i n w i t h AFG a f t e r p i c r i c a c i d h y d r o l y s i s , but o n l y f a i n t l y i f the s e c t i o n s had been a c e t y l a t e d , i n d i c a t i n g the presence of l y s i n e -r i c h r a t h e r than a r g i n i n e - r i c h p r o t e i n s a f t e r h y d r o l y s i s . C y t o c h e m i c a l l y , sperm of Rana p i p i e n s which behaved l i k e s omatic c e l l s ( Z i r k i n , 1970) were d e s i g n a t e d as type 4. The n u c l e i s t a i n e d i n t e n s i v e l y w i t h Feulgen and w i t h AFG a f t e r 43 / h y d r o l y s i s i n hot TCA or p i c r i c a c i d . However, when h y d r o l y z e d s e c t i o n s were a c e t y l a t e d , sperm d i d not s t a i n w i t h AFG. 2. G. a c u l e a t u s - l e i u r u s C y t o c h e m i c a l s t a i n i n g of sperm n u c l e i from male G. a c u l e a t u s - l e i u r u s from J e r i c h o Pond, Vancouver, B.C. was done from specimens c o l l e c t e d 29 October 1983 and 11 May 1984 and on a specimen c o l l e c t e d from Marpole Pond, Vancouver, B.C. on 8 May 1983. Feulgen s t a i n was p o s i t i v e i n a l l a n i m a l s showing the presence of DNA ( F i g . 6A). B a s i c p r o t e i n s o t h e r than p r o t a m i n e s and type 3B p r o t e i n s were not p r e s e n t i n specimens B ( F i g . 6C), E, F, G and H (Table 1) as was shown t h r o u g h a n e g a t i v e AFG s t a i n w i t h TCA h y d r o l y s i s a t 80-85*C. TCA h y d r o l y s i s a t 80-85*C e x t r a c t s the p r o t a m i n e s from the n u c l e u s . Spermatids d i d s t a i n i n specimens A ( F i g . 6D), C and D. These t e s t e s were i n an e a r l i e r s t age of development than the n o n - s t a i n i n g ones and a l s o had d i f f e r e n t t y p e s of sperm p r o t e i n s p r e s e n t than i n mature sperm. A p o s i t i v e AFG w i t h p i c r i c a c i d h y d r o l y s i s a t 60*C proved b a s i c p r o t e i n s were p r e s e n t i n a l l the specimens ( F i g . 6E). Whereas TCA h y d r o l y s i s removed the p r o t a m i n e s , p i c r i c a c i d r e t a i n s t h e s e and o t h e r b a s i c p r o t e i n s . AFG w i t h p i c r i c a c i d a t 60*C w i t h a c e t y l a t i o n gave a more l i g h t l y s t a i n e d n u c l e i r i c h i n a r g i n i n e and i n d i c a t i n g the presence of l y s i n e . A c e t y l a t i o n d e c r e a s e s the p r o t a m i n e s t a i n i n g t o h e l p show the presence of l y s i n e . A l l the specimens s t a i n e d p o s i t i v e f o r the Sakaguchi s t a i n i n d i c a t i n g p r o t e i n - b o u n d a r g i n i n e i n the n u c l e i . 44 F i g u r e 6. C y t o c h e m i s t r y done on G. a c u l e a t u s t e s t e s w i t h A, F e u l g e n s t a i n ; B, Sakaguchi s t a i n ; C and D, AFG, TCA, 80-85*C; E, AFG, p i c r i c a c i d , 60*C and F, AFG, p i c r i c a c i d , 60*C, a c e t y l a t i o n . A l l p a n e l s r e p r e s e n t c y t o c h e m i s t r y f o r a n i m a l B ( T a b l e 1) c a p t u r e d from J e r i c h o Pond, 11 May 1.984 except f o r p a n e l D which i s a n i m a l A, c a p t u r e d from J e r i c h o Pond, 29 October 1983. Dark l i n e i n p a n e l E r e p r e s e n t s s c a l e of 10 urn f o r a l l p a n e l s . 45 46 From the cyt o c h e n n i c a l a n a l y s i s of G. a c u l e a t u s , i t can be d e t e r m i n e d t h a t the sperm h i s t o n e s f a l l i n t o B l o c h ' s i n t e r m e d i a t e - t y p e c a t e g o r y . In p a r t i c u l a r , the b a s i c p r o t e i n s a r e s i m i l a r t o M y t i l u s e d u l i s ; t ype 3B. A l s o , i t can be seen t h a t d u r i n g s p e r m a t o g e n e s i s , somatic h i s t o n e s a r e r e p l a c e d by ty p e 2 s t a b l e p r o t a m i n e s or type 4 s o m a t i c - l i k e h i s t o n e s , which a r e then r e p l a c e d by the type 3B i n t e r m e d i a t e p r o t e i n s . T h i s can be seen from specimens A, C and D (Table 1) where s p e r m a t i d s s t a i n e d p o s i t i v e f o r AFG h y d r o l y z e d w i t h TCA a t 80-85*C as i n the c o n t r o l , H y d r o l a g u s c o l l e i . C. B i o c h e m i s t r y J_. C o n t r o l s E l e c t r o p h o r e t i c s e p a r a t i o n was done on t e s t i s - s p e c i f i c p r o t e i n s (TSP) from a n i m a l s known t o have the i n t e r m e d i a t e - t y p e . F i g . 7 shows a c o n t r o l g e l w i t h the b a s i c p r o t e i n s from Bufo, Xenopus and A n o l i s . S i n c e h i s t o n e 4 (H4) i s the most e v o l u t i o n a r i l y c o n s e r v a t i v e p r o t e i n known, i t s e r v e s as a u s e f u l marker f o r p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (PAGE) because of i t s f i x e d m i g r a t i o n p a t t e r n . Two bands a r e o f t e n seen due t o an u n a c e t y l a t e d form of H4 t h a t moves more r a p i d l y than the a c e t y l a t e d form. A c e t y l a t i o n i s p r o b a b l y on the E p s i l o n - a m i n o groups of l y s i n e r e s i d u e s ( I s e n b e r g , 1979). X. l a e v i s t e s t i s b a s i c p r o t e i n s ( l a n e 3) e x h i b i t e d b oth somatic h i s t o n e s and l a t e F i g u r e 7. E l e c t r o p h o r e t i c p r o f i l e s of t e s t i s - s p e c i f i c p r o t e i n s (TSPs) from Anura and Squamata. on a 42 cm. p o l y a c r y l a m i d e g e l . Lane 1, Bufo americanus; l a n e 2, Bufo m a r i n u s ; l a n e 3, Xenopus l a e v i s ; l a n e 4, A n o l i s s a q r e i ; l a n e 5, Xenopus l a e v i s h e a r t showing somatic h i s t o n e s ; l a n e 6, h e r r i n g p r o t a m i n e (P) (Sigma Chem. Co.) L a t e s p ermatid/sperm p r o t e i n s (SP) are i d e n t i f i e d f o r X. l a e v i s . E v o l u t i o n a r i l y c o n s e r v a t i v e h i s t o n e H4 i s i n d i c a t e d as a marker, (cm.) = c e n t i m e t e r s Arrow denotes d i r e c t i o n of m i g r a t i o n s . See m a t e r i a l s and methods f o r d e t a i l s of the e l e c t r o p h o r e t i c p r o c e d u r e . 48 1 2 3 4 5 6 =H4 20-• ZSP 3-5 =SP6 40" 49 spermatid/sperm b a s i c p r o t e i n s and t h e r e f o r e s e r v e s as a u s e f u l marker f o r PAGE. D a r k l y s t a i n e d p r o t e i n s moving more s l o w l y than h i s t o n e H4 were somatic h i s t o n e s . P r o t e i n s m i g r a t i n g more r a p i d l y than h i s t o n e H4 were TSP's. The sperm p r o t e i n s (SP) of X. l a e v i s t e s t i s and somatic h i s t o n e s of X. l a e v i s h e a r t have been s t u d i e d b e f o r e (Mann et a l . , 1982) and s e r v e as markers f o r m i g r a t i o n of the i n t e r m e d i a t e - t y p e b a s i c p r o t e i n s of o t h e r a n i m a l s . The 3 bands of SP 3, SP 4 and SP 5 were found moving f a s t e r than the somatic h i s t o n e s and s l o w e r than the SP 6 p r o t e i n s . The TSP from G. a c u l e a t u s ( F i g . 8, l a n e 1) was seen m i g r a t i n g s l i g h t l y f a s t e r than SP 6 of Xenopus. P r o t e i n samples a p p l i e d t o the g e l s were p r e p a r e d from t e s t i s c e l l s u s p e n s i o n s by the micromethod of L o u i e and Dixon-(1972) as d e s c r i b e d i n M a t e r i a l s and Methods. T h i s was a r e l a t i v e l y f a s t p r e p a r a t i o n t h a t a l s o d e c r e a s e d the chance of p r o t e o l y s i s . P o s s i b l e p r o t e o l y s i s c o u l d be d e t e c t e d by PAGE i f e x t r a bands o c c u r r e d i n n o n - g e r m i n a l organs l i k e h e a r t . A d i s a d v a n t a g e of the p r e p a r a t i o n was t h a t a l l c e l l t y p e s p r e s e n t i n the t e s t i s a r e p r o c e s s e d t o g e t h e r y i e l d i n g b o th n u c l e o s o m a l h i s t o n e s and spermatid/sperm p r o t e i n s . Mature G. a c u l e a t u s t e s t e s , however, c o n s i s t e n t l y had 1 t o 3 p r o t e i n bands w i t h no or few f a i n t s l o w e r , n u c leosomal h i s t o n e s . S i n c e the b r e e d i n g season of the t h r e e - s p i n e s t i c k l e b a c k i s w e l l d e f i n e d ( C r a i g -B e n n e t t , 1931), and i n l i g h t of d e v e l o p m e n t a l TSP s t u d i e s t o be d i s c u s s e d l a t e r i n t h i s t h e s i s , one can assume t h a t the p r o t e i n s o b s e r v e d by PAGE come m a i n l y (>90%) from l a t e s p e r m a t i d s and 50 F i g u r e 8. E l e c t r o p h o r e t i c p r o f i l e s of b a s i c p r o t e i n s from t e s t i s of X. l a e v i s and G. a c u l e a t u s on d i f f e r e n t s i z e s of p o l y a c r y l a m i d e g e l s . H i s t o n e H4 has been i n d i c a t e d as a n u c l e o s o m a l h i s t o n e marker. F i g . 8A. I . 25 cm. g e l w i t h Lane 1, G. a c u l e a t u s - l e i u r u s TSP; l a n e 2, X. l a e v i s t e s t i s ; l a n e 3, X. l a e v i s h e a r t . I I . 34 cm. g e l ; l a n e 1, G. a c u l e a t u s - l e i u r u s ; l a n e s 2 and 3, X. l a e v i s t e s t i s ; l a n e 4, X. l a e v i s h e a r t . I I I . 45 cm. g e l , l a n e 1, X. l a e v i s h e a r t ; l a n e 2, X. l a e v i s t e s t i s , l a n e 3, G. a c u l e a t u s TSP. F i g . 8B. IV. 61 cm. g e l , . l a n e 1, X. l a e v i s h e a r t ; l a n e 2, X. l a e v i s t e s t i s ; l a n e 3, G. a c u l e a t u s - t r a c h u r u s Tanadromous form) TSP. V. 87 cm. g e i , l a n e 1, X. l a e v i s h e a r t ; l a n e 2, X. l a e v i s t e s t i s ; l a n e 3, G. a c u l e a t u s - t r a c h u r u s TSP. 53 sperm. In G. a c u l e a t u s t h e s e c e l l s had o n l y a few b a s i c p r o t e i n s . P r e v i o u s work on the e l e c t r o p h o r e t i c s e p a r a t i o n of TSP had been done on g e l s w i t h 25-34 cm. l e n g t h s (Mann e t a l . , 1982). Experiments w i t h l o n g e r g e l s i z e s gave s u p e r i o r s e p a r a t i o n of t e s t i c u l a r b a s i c p r o t e i n s ( F i g s . 7 and 8 ) . 2. G e l s i z e The p r o t e i n s a n a l y z e d were o f h i g h b a s i c i t y and s m a l l s i z e , which made the use of a c i d urea p o l y a c r y l a m i d e d e n a t u r i n g g e l of Panyim and C h a l k l e y (1969) the most p r a c t i c a l method f o r i n v e s t i g a t i o n . Samples were d i s s o l v e d i n 8 M u r e a / 5 % 2-m e r c a p t o e t h a n o l t o d i s s o c i a t e the p r o t e i n s and t o e l i m i n a t e the chance of p o s s i b l e d e g r a d a t i o n by d e n a t u r i n g any endogenous p r o t e a s e s p r e s e n t . D i f f e r e n t s i z e s of p o l y a c r y l a m i d e g e l s were t e s t e d as a parameter f o r e l e c t r o p h o r e t i c a n a l y s i s . G e l s i z e s ranged from 25 cm t o 87 cm. These were measurements of g e l s i n the mold b e f o r e s w e l l i n g caused by d e s t a i n i n g . I n v e s t i g a t i o n of t h e TSP was f i r s t done on s h o r t e r g e l s of about 25 cm. To make sure t h e r e were no o t h e r bands moving w i t h the f a s t m i g r a t i n g p r o t e i n s , g e l s of i n c r e a s e d l e n g t h were used. The maximum g e l l e n g t h , 87 cm ( F i g . 8B-IV) (100 cm when s w o l l e n a f t e r d e s t a i n i n g ) , r e v e a l e d no f u r t h e r s e p a r a t i o n of TSP i n G. a c u l e a t u s or X. l a e v i s t e s t i s 54 and h e a r t c o n t r o l s . Longer p o l y a c r y l a m i d e g e l s improved the s e p a r a t i o n of the f a s t e r p r o t e i n bands w i t h o u t l o s s of band r e s o l u t i o n . G e l s of 61 cm l e n g t h s gave the b e s t r e s u l t s ( F i g . 8 B - I I I ) . T h i s was demonstrated by the s e p a r a t i o n of X. l a e v i s sperm p r o t e i n s . W ith s h o r t e r g e l s ( F i g . 8 A - I ) , SP 3-5 o f t e n m i g r a t e d t o g e t h e r and SP 6 appeared as o n l y 1 band. S e p a r a t i o n of X. l a e v i s SP 3, SP 4 and SP 5 as w e l l as SP 6 i n t o 2 bands were seen on l o n g e r g e l s ( F i g . 8B-1V and V ) . No f u r t h e r bands o c c u r r e d i n e i t h e r X. l a e v i s t e s t i s or G. a c u l e a t u s t e s t i s on the 87 cm g e l than on the 61 cm g e l . The l a r g e r s i z e was awkward t o work w i t h i n f r e q u e n t e x p e r i m e n t a l a s s a y s . 3. S p e c i e s d i f f e r e n c e s Once the optimum method of b a s i c p r o t e i n i s o l a t i o n and s e p a r a t i o n had been worked o u t , the f a s t m i g r a t i n g TSP's of d i f f e r e n t , r e l a t e d t e l e o s t s were compared by PAGE. Sperm p r o t e i n s have been shown t o be d i v e r s e i n amphibians (Mann et a l . , 1982; K a s i n s k y e t a_l. , 1978) and i n some t e l e o s t s ( B l o c h , 1976). I t was of i n t e r e s t t o d i s c o v e r whether the p a t t e r n of s p e c i e s - s p e c i f i c sperm p r o t e i n s found i n amphibians (Mann et~ a l . , 1982; K a s i n s k y et a l . , 1978) c o u l d a l s o be seen i n t e l e o s t s . F o c u s i n g on the e a s i l y a c c e s s i b l e and w e l l s t u d i e d G. a c u l e a t u s and t a x o n o m i c a l l y c l o s e l y r e l a t e d t e l e o s t s , e l e c t r o p h o r e t i c p r o f i l e s of t e s t i s s p e c i f i c p r o t e i n s were 55 examined ( F i g . 9 ) . Specimens of anadromous and f r e s h w a t e r G. a c u l e a t u s , and the A t l a n t i c s i s t e r s p e c i e s , G. w h e a t l a n d i ; a c l o s e l y r e l a t e d genus ( P u n g i t i u s p u n g i t i u s ) , and Au l o r h y n c h u s  f l a v i d u s , a r e l a t e d f a m i l y , were examined. G. a c u l e a t u s had the s l o w e s t m i g r a t i n g TSP ( F i g . 9, l a n e 2-4), w i t h G. w h e a t l a n d i ( l a n e 5 ) , P. p u n g i t i u s ( l a n e 6) and A. f l a v i d u s ( l a n e 7) i n t u r n h a v i n g d i f f e r e n t f a s t e r m i g r a t i n g TSP's. S i n c e the bands were s e p a r a t e d by a c o n s i d e r a b l e d i s t a n c e i t was u n l i k e l y t h a t the d i f f e r e n c e s were do t o p h o s p h o r y l a t e d s i d e - c h a i n m o d i f i c a t i o n of the p r o t e i n ( L o u i e and D i x o n , 1972). I d e n t i f i c a t i o n of the s p e c i e s was then p o s s i b l e by i n s p e c t i n g the band p a t t e r n of TSP's and t h e i r d i s t a n c e of m i g r a t i o n on the g e l . These ex p e r i m e n t s showed t h a t b a s i c p r o t e i n s i n the t e s t i s from c l o s e l y r e l a t e d s p e c i e s o f t e l e o s t s were d i f f e r e n t i n e l e c t r o p h o r e t i c m o b i l i t y and t h a t the m o b i l i t i e s of t h e s e p r o t e i n bands were c o n s i s t e n t enough t o be m o l e c u l a r markers f o r t h e s e s p e c i e s . The p r o t e i n d i f f e r e n c e s were not l i m i t e d t o e l e c t r o p h o r e t i c m o b i l i t y . Amino a c i d a n a l y s i s of the f a s t m i g r a t i n g TSP's of t h e s e s p e c i e s was done by h y d r o l y z i n g the a m i d o b l a c k - s t a i n e d bands a c c o r d i n g t o the method of Houston (1971) and P a l l o t t a and T e s s i e r (1976). X. l a e v i s H4 bands c u t from p o l y a c r y l a m i d e g e l s were used as c o n t r o l s t o s t a n d a r d i z e the amino a c i d methodology (Ta b l e 2 ) . Xenopus h e a r t was much l a r g e r than G. a c u l e a t u s h e a r t which gave more m a t e r i a l t o work w i t h . Heart was used because the t i s s u e d i d not degrade d u r i n g p r e p a r a t i o n f o r 56 F i g u r e 9. Comparison of e l e c t r o p h o r e t i c p r o f i l e s of TSPs from G. a c u l e a t u s and r e l a t e d t e l e o s t s on a 61 cm. g e l . Lane 1, X. l a e v i s h e a r t ; l a n e 2, G a s t e r o s t e u s a c u l e a t u s -t r a c h u r u s ( G . a . ) , R a i l r o a d D i t c h , B.C.; l a n e 3, G. a c u l e a t u s - t r a c h u r u s , R i v i e r e des Vase, Quebec; l a n e 4, G. a c u l e a t u s - l e i u r u s , J e r i c h o Pond, Vancouver, B.C.; l a n e 5, G a s t e r o s t e u s w h e a t l a n d i (G.w.), R i v i e r e des Vase, Quebec; l a n e 6, P u n g i t i u s p u n q i t i u s ( P . p . ) , R i v i e r e des Vase, Quebec; l a n e 7, A u l o r h y n c h u s f l a v i d u s ( A . f . ) , S t a n l e y Park, Vancouver, B.C.; l a n e 8, X. l a e v i s t e s t i s , ' t ' below g e l = t r a c h u r u s ; '1' below g e l = l e i u r u s . 5 8 T a b l e 2. Amino a c i d c o m p o s i t i o n o f Xenopus h i s t o n e H4 from p o l y a c r y l a m i d e g e l s . ^ ( V a l u e s i n mole p e r c e n t ) Xenopus l a e v i s C a l f H e a r t H4 l i v e r H 4 ^ b ) thymus H 4 ^ ( g e l ) (powder) (powder)  No. o f a n i m a l s z No. o f a n a l y s e s 4 13 .5+2 .5 ( d ) mole 7» Lys 13.4 11.4 H i s 2.5+0.5 2.2 2.2 A r g 12.4+2.1 .13.2 12.8 Asp 6.5+1.0 6.2 5.2 Thr 5.6+0.4 4.7 6.3 Ser 4.7+1.6 2.7 2.2 G l u 9.2+2.8 6.4 6.9 Pro 1.2+0.6 2.6 1.5 G l y 14.8+1.3 12.0 14.9 A l a 8.7+3.4 7.1 7.7 V a l 6.7+0.9 6.5 8.2 Met 0.2+0.1 1.2 1.0 H e 3.7+0.6 5.9 5.7 Leu 7.2+0.7 8.6 8.2 Tyr 0.9+0.4 3.9 3.8 Phe 2.2+0.6 3.6 2.1 L y s / A r g 1.1 1.0 0.9 ( e ) B a s i c / A c i d i c 1.8 2.3 2.2 (a) 157o a c i d / u r e a p o l y a c r y l a m i d e s l a b g e l , a m i d o b l a c k s t a i n i n g (Panyim and C h a l k l e y , 1969) (b) B y r d and K a s i n s k y , 1973 (c) Johns, 1971 (d) Mean+standard d e v i a t i o n (e) B a s i c / A c i d i c = L y s + H i s + A r g / A s p + G l u 59 e l e c t r o p h o r e s i s ( K a s i n s k y e t §_1. , 1978). The r e s u l t s of amino a c i d a n a l y s i s were v e r y c l o s e t o tho s e f o r commercial powder ' h i s t o n e H4 i n d i c a t i n g good methodology. Amino a c i d c o m p o s i t i o n of TSP's from G. a c u l e a t u s and r e l a t e d s p e c i e s i s seen i n Table 3. From t h e s e a n a l y s e s i t can be seen t h a t a l l these b a s i c p r o t e i n s may be c l a s s i f i e d as i n t e r m e d i a t e - t y p e s ( B l o c h 1969 and 1976) as they c o n t a i n l y s i n e and h i s t i d i n e as w e l l as a r g i n i n e , v e r i f y i n g the r e s u l t s of the c y t o c h e m i s t r y d e s c r i b e d e a r l i e r . A l t h o u g h c o n c l u s i o n s from the amino a c i d a n a l y s i s was l i m i t e d due t o the s m a l l number of samples, a n a l y s i s of v a r i a n c e (ANOVA) r e v e a l e d s i g n i f i c a n t d i f f e r e n c e s between a n i m a l groups (p<0.05). The d a t a were d i v i d e d i n t o 4 d i f f e r e n t c a t e g o r i e s so t h a t d i f f e r e n t p r o p e r t i e s of the p r o t e i n c o u l d be d e t e c t e d . Test 1. A l l of the 16 amino a c i d s were compared between the groups i . e . s p e c i e s 1 t o s p e c i e s 2 or f r e s h w a t e r p o p u l a t i o n t o anadromous p o p u l a t i o n and so on.. T h i s t e s t would g i v e an i d e a of how s i m i l a r the p r o t e i n s were i n r e l a t i v e c o m p o s i t i o n . T e s t 2. The sum of the b a s i c amino a c i d s were compared. Because the TSP's were run a t the low pH of about 2.5, a l l the a c i d i c amino a c i d s were e s s e n t i a l l y uncharged and o n l y the p o s i t i v e c h a r g e s of the b a s i c amino a c i d s would be p r e s e n t . T h i s t e s t would h e l p e x p l a i n the r e l a t i v e m i g r a t i o n of the TSP on a g e l due t o the p o s i t i v e charge of the p r o t e i n a t pH 2.5. Test 3. L y s i n e / a r g i n i n e r a t i o g i v e s an i d e a about the a r g i n i n e r i c h n e s s of the s e p r o t e i n s . T h i s t e s t becomes more r e l e v a n t when comparing t h e s e p r o t e i n s t o o t h e r sperm p r o t e i n s T a b l e 3. Amino a c i d c o m p o s i t i o n o f t e s t i s - s p e c i f i c p r o t e i n s from p o l y a c r y l a m i d e g e l s from G. a c u l e a t u s and r e l a t e d s p e c i e s . (Values i n mole p e r c e n t ) G a s t e r o s t e u s G a s t e r o s t e u s  a c u l e a t u s w h e a t l a n d i trachurus-RRD P u n g i t i u s A u l o r h y n c h u s  p u n g i t i u s f l a v i d u s No. o f anim a l s 4 2 3 2 1 No. o f a n a l y s e s 2 3 3 (a) x ( b ) Lys 0.4+0.2 ( c ) 3.4+0.6 5.1+1.9 0.8+0.8 1.6 Hys 0.6+0.3 2.8+0.3 0.8+0.4 1.5+1.4 1.6 Arg 36.8+7.9 39.1+2.0 42.1T4.4 40.2+4.2 30.6 Asp 0.7+0.6 3.4+0.3 1.8+0.6 1.1+0.8 t F ( d ) 2.5 Thr 6.7+0.9 7.1+0.8 6.7+1.6 2.5 Ser 5.3+0.3 6.8+1.3 9.7+0.8 12.0+0.2 19.1 G l u 5.7+0.8 3.8+2.0 2.1+2.3 2.1+2.3 3.0 Pro 6.8+1.0 3.4T0.3 7.0+~4.0 9 .5+"3 .7 4.9 G l y 8.2+0.6 6.9+1.3 2 .4+"2.1 4.9+"0.9 7.9 A l a 8.0+0.8 6.6+2.5 5.5+2.0 12.3+3.7 11.5 V a l 6.6+1.2 10.5+"2.0 5.7+0.3 4. 3+"0. 7 3.3 Met 1.5+1.2 t r 0.5+0.3 4.3+1.2 5.2 H e 2.9+0.6 1.2+0.1 5.6+0.3 t r 1.4 Leu 5.5+0.9 4.0+"0.2 2 .6+1.6 t r 1.1 Tyr 1.8+1.6 t r 0.5+0.2 3.9+0.9 1.4 Phe 2.7+0.5 0.8+0.2 3.0+1.1 3.5+0.7 0.8 T o t a l B a s i c s ^ e ) 37.8+7.7 45.4+1.6 48.1+4.4 42.5+6.3 33.8 L y s / A r g m B a s i c / A c i d i c w 0.011+0.006 0.087+0.016 0.121+0.047 0.020+0.020 0.052 5.9+1.8 . 6.3+2.0 12.3+9.3 13.3+13.0 6.2 (a) f a s t e r , major TSP band a n a l y s e d (b) s l o w e r , minor TSP band a n a l y z e d (c) mean + s t a n d a r d d e v i a t i o n (d) t r = t r a c e (e) T o t a l B a s i c s - Lys+His+Arg ( f ) B a s i c / A c i d i c = Lys+His+Arg/Asp+Glu 61 of B l o c h ' s c a t e g o r i e s and the amount of a r g i n i n e i n these p r o t e i n s . Test 4. The b a s i c / a c i d i c amino a c i d s ( h i s t i d i n e + l y s i n e + a r g i n i n e / a s p a r t i c a c i d + g l u t a m i c a c i d ) r a t i o i s c a l c u l a t e d so t h a t an i d e a of the net charge of the p r o t e i n a t p h y s i o l o g i c a l pH of the a n i m a l may be c a l c u l a t e d . T h i s r a t i o was not e x t r e m e l y a c c u r a t e s i n c e i t was not known how much of the a c i d i c amino a c i d s were p r e s e n t as a s p a r a g i n e or g l u t a m i n e . Comparison of a l l the amino a c i d s ( t e s t 1) showed no s i g n i f i c a n t d i f f e r e n c e s between G. a c u l e a t u s (Ga), G. w h e a t l a n d i (Gw), P. p u n q i t i u s (Pp) and A. f l a v i d u s ( A f ) i n d i c a t i n g the o v e r a l l s i m i l a r i t y of the c o m p o s i t i o n of the TSP's. S i g n i f i c a n t d i f f e r e n c e s between the sum of the b a s i c amino a c i d s ( t e s t 2) were found between the f o u r s p e c i e s . A Tukey's t e s t f o r m u l t i p l e comparisons r e v e a l e d t h a t the TSP's from P. p u n q i t i u s and G. w h e a t l a n d i c o n t a i n s i g n i f i c a n t l y more p o s i t i v e l y c h a r g e d amino a c i d s than TSP's from G. a c u l e a t u s and A. f l a v i d u s . T h e - p o s i t i v e c harges of the TSP's from G. a c u l e a t u s and A. f l a v i d u s were found t o be s i m i l a r as were the TSP's from P. p u n g i t i u s and G. w h e a t l a n d i . The ranked means (mole %) from l e s s t o more b a s i c were 37.8 (Ga) < 42.5 ( A f ) < 45.4 (Gw) < 48.1 ( P p ) . C o r r e l a t i n g t h e s e d a t a w i t h the e l e c t r o p h o r e t i c m i g r a t i o n of the TSP's ( F i g . 9) e x p l a i n s the f a s t e r m i g r a t i o n of (Gw) and (Pp) over (Ga)-. I t does not e x p l a i n the f a s t e r m i g r a t i o n of (Pp) over (Gw) or ( A f ) over a l l t h r e e s p e c i e s . PAGE s e p a r a t e s 62 d i s s o c i a t e d p r o t e i n s by both s i z e and c h a r g e . T h e r e f o r e , the f a s t e r m i g r a t i o n of the p r o t e i n s on the g e l t h a t was not due t o d i f f e r e n c e s i n charge may be e x p l a i n e d by a s m a l l e r m o l e c u l a r weight of the TSP. For example, even though the A. f l a v i d u s TSP had a r e l a t i v e l y lower p o s i t i v e charge than t h e TSP's from G. w h e a t l a n d i and P. p u n g i t i u s , the A. f l a v i d u s TSP m i g r a t e d \ f u r t h e r i n PAGE. Thus, the TSP^from A. f l a v i d u s must have a lower m o l e c u l a r weight than the o t h e r 2 s p e c i e s . Comparing the l y s i n e / a r g i n i n e r a t i o ( t e s t 3) a l s o showed a s i g n i f i c a n t d i f f e r e n c e i n the ANOVA. The ranked mole % means were 0.011 (Ga) < 0.020 ( A f ) < 0.087 (Gw) < 0.121 ( P p ) . A r e l a t i o n s h i p of (Ga) = (Af) =/ (Gw) = (Pp) from the Tukey's t e s t y i e l d s r e s u l t s s i m i l a r t o the r e l a t i o n s h i p of t e s t 2 where (=) and (=/) i n d i c a t e s i m i l a r i t y and s i g n i f i c a n t d i f f e r e n c e of the v a l u e s r e s p e c t i v e l y . (Ga) was more s i m i l a r t o ( A f ) and both were l e s s a r g i n i n e - r i c h than (Gw) and ( P p ) . A r e a l d i f f e r e n c e between the TSP's p r o b a b l y e x i s t s between the f o u r s p e c i e s because t e s t s 2 and 3 show the same r e l a t i o n s h i p . These t e s t show i m p o r t a n t d i f f e r e n c e s between the TSP's t h a t p a r t i a l l y e x p l a i n the PAGE TSP m o b i l i t y as w e l l as g i v e an i n s i g h t i n t o the a r g i n i n e r i c h n e s s of the s e p r o t e i n s . The b a s i c / a c i d i c amino a c i d r a t i o showed s i g n i f i c a n t d i f f e r e n c e s between the s p e c i e s i n t e s t 4. The ranked mole % means were 5.9 (Ga) < 6.3 (Gw) < 12.3 (Pp) < 13.3 ( A f ) . The Tukey's t e s t showed the r e l a t i o n s h i p of (Ga) = (Gw) =/ (Pp) 63 ( A f ) . I t i s i n t e r e s t i n g t o note t h a t ( A f ) and (Ga) were more s i m i l a r i n t e s t s 2 and 3 w h i l e (Ga) and (Gw) were more s i m i l a r i n t h i s t e s t . T h i s index f u r t h e r p o i n t s out t h a t t h e r e a r e r e a l d i f f e r e n c e s between the TSP's from these f o u r s p e c i e s . 4. Developmental changes E l e c t r o p h o r e t i c p a t t e r n s of TSP's were compared from sample specimens of f r e s h w a t e r male t h r e e - s p i n e s t i c k l e b a c k s taken from J e r i c h o Pond, B.C. from October 1983 t o August 1984 i n o r d e r t o determine the e l e c t r o p h o r e t i c p r o f i l e of TSP's from mature G. a c u l e a t u s . In the p r e v i o u s s e c t i o n , the c y t o l o g y of the t e s t e s b e f o r e , d u r i n g and a f t e r m a t u r a t i o n was d i s c u s s e d . I n the non-b r e e d i n g p e r i o d from August t o March, many s t a g e s of the d e v e l o p i n g germ c e l l may be pr e s e n t a t any one time d u r i n g s p e r m a t o g e n e s i s i n the d e v e l o p i n g t e s t i s ( C r a i g - B e n n e t t , 1931). Both somat i c h i s t o n e s and many TSP's can be seen i n the p o s t - b r e e d i n g p e r i o d of G. a c u l e a t u s i n October t o March on PAGE ( F i g . 10). The d i s a p p e a r a n c e of the slow, s o m a t i c h i s t o n e bands and r e d u c t i o n i n numbers of the TSP bands o c c u r as the b r e e d i n g season s t a r t s a f t e r March.-In October ( F i g . 10A, l a n e s 1 and 2) as many as 5 bands were seen w i t h the f a s t e s t , most c h a r a c t e r i s t i c band of the mature t e s t i s a p p e a r i n g as o n l y a f a i n t band. In November ( F i g . 10A, l a n e s 3 and 4) 2 dark bands were seen, 1 of which was the f a s t e r , c h a r a c t e r i s t i c band of G. a c u l e a t u s mature t e s t i s . Three f a i n t e r bands were a l s o p r e s e n t , 64 F i g u r e 10. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from t e s t i s of G. a c u l e a t u s taken from J e r i c h o Pond, Vancouver, B.C. from October 1983 t o May 1984, 61 cm. g e l . A. Lane 1, G. a c u l e a t u s - t r a c h u r u s , R a i l r o a d D i t c h , B.C. b r e e d i n g male; a l l o t h e r TSP's seen i n t h e s e g e l s a r e from a n i m a l s c o l l e c t e d from J e r i c h o Pond, B.C. w i t h time of c o l l e c t i o n as f o l l o w s ; l a n e s 2 and 3, O c t o b e r ( 0 ) ; l a n e s 4 and 5, November (N); l a n e 6, December (D); l a n e s 7 and 8, January ( J ) . B. Lane 1, January ( J ) ; l a n e 2, F e b r u a r y ( F ) ; l a n e 3, March (M); l a n e 4, A p r i l ( A ) ; l a n e 5, May (My). B'. Ten f o l d more s e n s i t i v e r e s t a i n i n g of t h e t o p and bottom p o r t i o n s of g e l 'B' a c c o r d i n g t o the method of Wray and S t u b b l e f i e l d (1970). A p o r t i o n of G. a c u l e a t u s TSP's were c u t out f o r amino a c i d " a n a l y s i s b e f o r e s e n s i t i v e r e s t a i n i n g of the g e l . They a r e seen as the open r e c t a n g u l a r r e g i o n i n l a n e s 11' and 12'. The center-p o r t i o n of the g e l fragmented i n the r e s t a i n i n g s o l u t i o n . However, no a d d i t i o n a l bands were seen. 65 66 2 s l o w e r and 1 f a i n t band between the d a r k e r bands. December had a v e r y s i m i l a r p a t t e r n t o the November a n i m a l s ( F i g . 10A, l a n e 5 ) . In January ( F i g . 10A, l a n e s 6 and 7 and F i g . 10B, l a n e 8) t h e r e was a d e c r e a s e i n band number, a d i s a p p e a r a n c e of the s l o w e r , f a i n t bands and the d i m i n i s h i n g of the s l o w e r dark band. The s l o w e r dark band decrea s e d more i n F e b r u a r y ( F i g . 10B, l a n e 9 ) . The d a r k e r band w i t h an o c c a s i o n a l l i g h t e r , s l i g h t l y s l o w e r band r e p r e s e n t i n g the mature, b r e e d i n g male t h r e e - s p i n e s t i c k l e b a c k was seen i n March ( F i g . 10B, l a n e 10) and subsequent months ( F i g . 10B, l a n e s 11 and 12) i n t o the b r e e d i n g season. T h e r e f o r e , the mature f i s h show o n l y f a s t TSP bands, a d i s a p p e a r a n c e of the slower TSP, and g r e a t e r than 90% d e c l i n e i n the somatic h i s t o n e bands. S e n s i t i v e d e s t a i n i n g , u s i n g the method of Wray and S t u b b l e f i e l d (1970) c o u l d d e t e c t p r o t e i n bands on PAGE about ten times g r e a t e r than the amidoblack s t a i n i n F i g . 10B'. Somatic h i s t o n e s c o u l d be seen i n the p r e p a r a t i o n s from October t o November i n F i g . 10B, and w i t h s e n s i t i v e s t a i n , s omatic h i s t o n e s c o u l d e a s i l y be seen from J a n u a r y t o March, and t o a much l e s s e r e x t e n t i n the specimens c o l l e c t e d i n A p r i l and May. No f u r t h e r TSP's were seen w i t h the s e n s i t i v e s t a i n . C o n t a m i n a t i o n or h a n d l i n g o f t e n darkens the background of the s e n s i t i v e r e s t a i n e d g e l s . The u r e a / s u l f u r i c a c i d s o l u t i o n a l s o seems t o prevoke c r a c k i n g of the g e l . G. a c u l e a t u s - l e i u r u s was d e t e r m i n e d t o be i n b r e e d i n g c o n d i t i o n when the male secondary s e x u a l c h a r a c t e r i s t i c s of r e d -t h r o a t and b l u e eyes were observed (Wootton, 1976). The t e s t e s 67 of t h e s e a n i m a l s were b l a c k . The non-breeding a n i m a l s d i d not d i s p l a y such s e x u a l c o l o r a t i o n . T e s t e s from t h e s e a n i m a l s were w h i t e i n the e a r l y p o s t - b r e e d i n g p e r i o d and g r a d u a l l y became s p e c k l e d w i t h melanocytes as the year p r o g r e s s e d u n t i l the b l a c k , b r e e d i n g t e s t i s had d e v e l o p e d . In Marpole Pond, Vancouver, B.C., an e a r l y s p r i n g sample of G. a c u l e a t u s - l e i u r u s gave a m i x t u r e of s e x u a l l y d e v e l o p i n g f i s h . Some specimens had w h i t e t e s t e s w h i l e o t h e r f i s h had b l a c k ones. F i g . 11 shows the b l a c k b r e e d i n g t e s t i s TSP's ( l a n e 1) and the n o n - b r e e d i n g , w h i t e t e s t i s TSP p r o f i l e ( l a n e 2 ) . Non-breeders showed more TSP's than b r e e d e r s and a l s o had somatic h i s t o n e s i n the t e s t i s p r e p a r a t i o n . T h i s d e v e l o p m e n t a l p r o f i l e was t h e r e f o r e o b s e r v e d i n a n o ther p o p u l a t i o n independent of the J e r i c h o Pond p o p u l a t i o n . 5. Comparison of anadromous and f r e s h w a t e r G . a c u l e a t u s In the p r e v i o u s s e c t i o n s of t h i s t h e s i s we d e t e r m i n e d t h a t the i n t e r m e d i a t e - t y p e TSP's of G a s t e r o s t e u s and r e l a t e d s p e c i e s were s p e c i e s - s p e c i f i c m o l e c u l a r markers. I t was shown a l s o t h a t b r e e d i n g males o f f e r e d a c o n s i s t e n t p r o t e i n p a t t e r n f o r co m p a r i s o n . Next,-~ the p o s s i b l e j 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 the TSP's of the anadromous and f r e s h w a t e r forms as w e l l as i n t e r - and i n t r a - p o p u l a t i o n comparisons were a n a l y z e d . E l e c t r o p h o r e s i s of G. a c u l e a t u s - t r a c h u r u s TSP's was 68 F i g u r e 11. E l e c t r o p h o r e t i c comparison of TSP's from Marpole Pond, Vancouver, B.C. b r e e d i n g ( l a n e 1) and non-breeding ( l a n e 2) G. a c u l e a t u s - l e i u r u s . T r i a n g l e s i n d i c a t e p r e s e n c e of somatic h i s t o n e s . 69 70 performed on s e v e r a l West Coast p o p u l a t i o n s and a Quebec p o p u l a t i o n ( F i g . 12). Most i n d i v i d u a l s produced a v e r y dark band t h a t m i g r a t e d the f a s t e s t of the G. a c u l e a t u s TSP's. A t y p i c a l TSP p a t t e r n was o b s e r v e d from a l l anadromous forms on p o l y a c r y l a m i d e g e l s . A 'comet-shaped' band w i t h a t y p i c a l l y f u z z y l e a d i n g edge and a c u r v e d t r a i l i n g edge was o b s e r v e d . I mmediately above t h i s band was o f t e n 1 ( F i g . 12A, l a n e 1 and 4; F i g . 12B, l a n e 4) or l e s s o f t e n 2 ( F i g . 12B, l a n e 3) s m a l l , t h i n bands. E x a c t m i g r a t i o n of the p r o t e i n bands was dependent on band d a r k n e s s ( p r o t e i n c o n c e n t r a t i o n ) . P r o t e i n s of h i g h e r c o n c e n t r a t i o n would m i g r a t e a l i t t l e s lower than l i g h t e r bands of the same o r i g i n and presumably the same c o m p o s i t i o n . O f t e n bands were compared by l i n i n g up the 'comet-shaped' dark band, and comparing i t w i t h TSP p a t t e r n from o t h e r specimens. The number of G. a c u l e a t u s w i t h m u l t i p l e bands (Table 4) were compared u s i n g the ' G ' - d i s t r i b u t i o n t e s t . G. a c u l e a t u s - t r a c h u r u s from R a i l r o a d D i t c h , B.C. and R i v i e r e des Vase, Quebec had s u f f i c i e n t . number t o w a r r a n t s t a t i s t i c a l a n a l y s i s . These p o p u l a t i o n s showed no s i g n i f i c a n t d i f f e r e n c e s (p>0.5) between the number of TSP m u l t i p l e bands. Amino a c i d a n a l y s i s r e v e a l e d s i m i l a r r e s u l t s f o r the two anadromous p o p u l a t i o n s from R a i l r o a d D i t c h , B.C. and R i v i e r e des Vase, Quebec (Table 5 ) . There were no s i g n i f i c a n t d i f f e r e n c e s i n any of the 4 ANOVA t e s t s (p. 59) done on the amino a c i d s r e s u l t s from th e s e p o p u l a t i o n s . The s i m i l a r i t y of t h e s e amino a c i d r e s u l t s i s c o n s i s t a n t w i t h the s i m i l a r TSP m i g r a t i o n and 71 F i g u r e 12. E l e c t r o p h o r e t i c comparisons of TSPs from the t e s t i s of the anadromous form of G. a c u l e a t u s from d i f f e r e n t p o p u l a t i o n s i n B r i t i s h Columbia and Quebec. A. 34 cm. g e l , l a n e 1, G. a c u l e a t u s - t r a c h u r u s from R i v i e r e des Vase, Quebec; l a n e 2, L i t t l e Campbell R i v e r , B. C.; l a n e s 3 and 4, R a i l r o a d D i t c h , B.C. B. 61 cm. g e l , l a n e 1, X. l a e v i s h e a r t ; l a n e 2, X. l a e v i s t e s t i s ; l a n e 3, G. a c u l e a t u s - t r a c h u r u s from R a i l r o a d D i t c h , B.C.; l a n e 4, R i v i e r e des Vase, Quebec. 73 / appearance observed on PAGE ( F i g . 12). F i g u r e 13 i l l u s t r a t e s some of the v a r i a b i l i t y seen w i t h i n and between l a k e s . The TSP bands of f r e s h w a t e r p o p u l a t i o n s were more 'block-shaped' than bands of anadromous s t i c k l e b a c k s . The p r o t e i n s m i g r a t e d s i m i l a r l y t o the f a s t , 'comet-shaped' anadromous TSP's ( F i g . 13A and 13B). I f a second or more r a r e l y , a t h i r d band was seen i n the TSP p r o f i l e s from b r e e d i n g l e i u r u s males, i t was ' b l o c k - s h a p e d ' , m i g r a t i n g c l o s e l y t o the f i r s t and was of even appearance t o the f i r s t ( F i g . 13A, l a n e s 2, 3 and 6, and F i g . 13B, l a n e 1). T h i s compares t o the v e r y t h i n , m u l t i p l e bands seen i n the anadromous form (Ex. F i g . 12A, l a n e s 1 and 3 ) . As was found i n most of the TSP's from anadromous and f r e s h w a t e r G. a c u l e a t u s , t h e r e was not a c o n s i s t e n t number of bands produced t h a t c h a r a c t e r i s t i c a l l y marked the p o p u l a t i o n . R a t h e r , t h e r e were p o p u l a t i o n s t h a t had i n d i v i d u a l s showing v a r i a b i l i t y of the TSP p r o f i l e s , some specimens h a v i n g m u l t i p l e bands, o t h e r s h a v i n g a s i n g l e band. T a b l e 4 shows the composite r e s u l t s of the e l e c t r o p h o r e t i c s t u d i e s on t h r e e - s p i n e s t i c k l e b a c k p o p u l a t i o n s . A l t h o u g h i t may appear t h a t t h e r e were d i f f e r e n c e s i n the number of TSP m u l t i p l e bands among f r e s h w a t e r p o p u l a t i o n s from Cranby Lake, Goose Lake, J e r i c h o Pond and Marpole Pond, t h e r e was not a s u f f i c i e n t sample s i z e t o a l l o w s t a t i s t i c a l a n a l y s i s . Lakes w i t h the b e n t h i c / l i m n e t i c t y p e s must be a n a l y z e d 7 4 T a b l e 4. M u l t i p l e TSP bands from G. a c u l e a t u s . Composite d a t a from v a r y i n g s i z e s o f p o l y a c r y l a m i d e g e l s . T o t a l number Number o f specimens w i t h : L o c a t i o n o f specimens 1 band 2 bands 3 bands F r e s h w a t e r P o p u l a t i o n s Enos Lake L i m n e t i c B e n t h i c 6 8 4 2 2 6 0 0 Paxton Lake L i m n e t i c B e n t h i c U n s p e c i f i e d 11 8 1 10 7 0 1 1 1 0 0 0 Cranby Lake 11 8 3 0 Goose Lake 9 7 1 1 J e r i c h o Pond 3 1 2 0 M a r p o l e Pond 1 1 0 0 ** P r i e s t Lake 2 Anadromous 2 P o p u l a t i o n s 0 0 R a i l r o a d D i t c h 41 18 20 3 R i v i e r e des Vase 18 11 7 0 L. Campbell R i v e r 4 4 0 0 S t a n l e y P a r k 1 0 1 0 * M u l t i p l e TSP bands = d o u b l e t o r t r i p l e t TSP's from PAGE ** The b e n t h i c / l i m n e t i c t y p e s e x i s t e d i n t h i s l a k e , b u t c o l l e c t e d samples d i d n o t s p e c i f y w h i c h t y p e . 75 T a b l e 5. Amino a c i d c o m p o s i t i o n o f t e s t i s - s p e c i f i c p r o t e i n s from p o l y a c r y l a m i d e g e l s from G. a c u l e a t u s - t r a c h u r u s . ( V a l u e s i n mole p e r c e n t ) R a i l r o a d D i t c h R i v i e r e des Vase No. o f a n i m a l s 4 2 1 No. o f a n a l y s e s 2 (a) T_(b) Lys 0.4+0.2 ( c ) 0.5+0.1 2.6 H i s 0.6+0.3 0.7+0.1 1.7 A r g 36.8+7.9 36.3+0.1 37.4 Asp 0.7+0.6 0.9+0.2 1.3 Thr 6.7+0.9 7.4+0.6 3.5 Ser 5.8+0.3 5.4+0.4 3.5 G l u 5.7+0.8 6.2+1.1 5.2 Pro 6.8+1.0 6.2+2.3 4.3 G l y 8.2+0.6 8.4+0.1 12.2 A l a 8.0+0.8 8.3+1.0 7.8 V a l 6.6+1.2 6.0+0.5 3.5 Met 1.5+1.2 0.7+0.1 0.4 H e 2.9+0.6 3.2+0.6 3.0 Leu 5.5+0.9 4.8+0.6 6.1 Tyr 1.8+1.6 2.0+1.7 5.7 Phe 2.7+0.5 2.8+0.3 1.7 (d) T o t a l B a s i c s 37.8+7.7 37.6+0.2 41.7 L y s / A r g 0.011+0.006 0.014+0.003 0.070 B a s i c / A c i d i c 5.9+1.8 5.3+1.0 6.4 (a) f a s t e r , major m i g r a t i n g band o f a d o u b l e t a n a l y z e d (b) s l o w e r , minor m i g r a t i n g band o f a d o u b l e t a n a l y z e d (c) mean + s t a n d a r d d e v i a t i o n (d) T o t a l B a s i c s = Lys+His+Arg (e) B a s i c / A c i d i c = Lys+His+Arg/Asp+Glu 76 F i g u r e 13. E l e c t r o p h o r e t i c comparison of TSPs from f r e s h w a t e r and anadromous G. a c u l e a t u s t e s t i s . A. V a r i a b i l i t y of TSP bands from i n d i v i d u a l specimens of G. a c u l e a t u s - l e i u r u s from Cranby Lake, B.C. on a 34 cm. g e l . B. Lane 1, TSP bands from G. a c u l e a t u s from f r e s h w a t e r Goose Lake, B.C.; l a n e 2, TSP's from anadromous G. a c u l e a t u s - t r a c h u r u s from L i t t l e Campbell R i v e r , B.C. and l a n e 3, R a i l r o a d D i t c h , B.C. on a 61 cm. g e l . C. 34 cm. g e l , l a n e 1, X. l a e v i s t e s t i s ; l a n e 2, b e n t h i c G. a c u l e a t u s - l e i u r u s from Enos Lake, B.C.; l a n e 3, l i m n e t i c G. a c u l e a t u s from Enos Lake; l a n e 4, G. a c u l e a t u s from Paxton Lake; l a n e s 5 and 6, G. a c u l e a t u s - t r a c h u r u s from R i v i e r e des Vase, Quebec; l a n e 7, R a i l r o a d D i t c h , B.C.; l a n e 8, G. a c u l e a t u s -t r a c h u r u s h e a r t from R a i l r o a d D i t c h , B.C.. was run as a c o n t r o l . N o t a t i o n s below the g e l read as f o l l o w s : B = b e n t h i c ; L = l i m n e t i c ; 1 = l e i u r u s ; t = t r a c h u r u s . 78 s e p a r a t e l y because i t has s t i l l not been d e t e r m i n e d i f e i t h e r of ty p e s a re the same as t h e G. a c u l e a t u s - l e i u r u s found i n l a k e s t h a t do not have t h e s e two t y p e s p r e s e n t . The sum of the t o t a l number of TSP m u l t i p l e bands from the B.C. anadromous p o p u l a t i o n s ( R a i l r o a d D i t c h + L i t t l e Campbell R i v e r + S t a n l e y P a r k ) was s i g n i f i c a n t l y h i g h e r than the sum of the TSP m u l t i p l e bands from the B.C. f r e s h w a t e r p o p u l a t i o n s t h a t d i d not show the b e n t h i c / l i m n e t i c t y p e s (Cranby + Goose + J e r i c h o + Marpole) ( p < 0 . 0 l ) . The amino a c i d d a t a from the d i f f e r e n t p o p u l a t i o n s of G. a c u l e a t u s - l e i u r u s ( T a b l e 6) were a n a l y z e d by an ANOVA t e s t f o r each of the 4 c a t e g o r i e s d e s c r i b e d p r e v i o u s l y (p. 59) . No s i g n i f i c a n t d i f f e r e n c e was found i n any of thes e t e s t s i n d i c a t i n g the o v e r a l l s i m i l a r i t y of the TSP's. These r e s u l t s agree w i t h the s i m i l a r appearance and m i g r a g t i o n of the TSP's i n PAGE from d i f f e r e n t f r e s h w a t e r p o p u l a t i o n s of G. a c u l e a t u s . The amino a c i d c o m p o s i t i o n i s s i m i l a r i n both f r e s h w a t e r and anadromous G. a c u l e a t u s (Table 7 ) . Of the 4 d i f f e r e n t ANOVA t e s t s done on the amino a c i d d a t a , o n l y one s i g n i f i c a n t d i f f e r e n c e , i n t e s t 3 ( l y s i n e / a r g i n i n e r a t i o ) , was found. T h i s s u g g e s t s a more a r g i n i n e - r i c h TSP i n anadromous p o p u l a t i o n s , which may account f o r t h e s l i g h t d i f f e r e n c e i n appearance of the band shapes between t h e s e two groups. T a b l e 6. Amino a c i d c o m p o s i t i o n o f t e s t i s - s p e c i f i c p r o t e i n s from p o l y a c r y l a m i d e g e l s from G. a c u l e a t u s - l e i u r u s . (Values i n mole p e r c e n t ) Goose Lake Paxton Lake J e r i c h o Pond E n o s ( a ) Lake-l i m n e t i c E n o s ( b ) Lake-l i m n e t i c E n o s ( a ) Lake-benthic No. o f anim a l s 1 No. o f a n a l y s e s 1 Lys 0.7 His 2.5 Arg 36.2 Asp 0.9 Thr 5.7 Ser 8.9 G l u 4.6 Pro 4.6 G l y 8.9 A l a 6.9 V a l 5.7 Met 2.1 H e 3.4 Leu 4.6 Tyr 1.8 Phe 2.5 T o t a l B a s i c s ^ 39.4 L y s / A r g ( e ) 0.019 B a s i c / A c i d i c k } 7.2 1 . 3 + 0 . 0 ^ 1.8+1.2 30.2+4.6 2.6+0.4 6.5+"0.6 4.8*3.2 5.6+0.6 7.2+0.5 11.9+1.3 7.8+0.6 5.4+0.4 2.2+0.2 2.8+0.3 4.2+0.5 2.3+0.4 3.0+0.4 33.3+3.4 0.043+0.007 4.0+0.7 1 1 1.1 1.9 28.0 1.5 6.7 6.3 6.7 •7.1 8.2 9.3 7.5 1.9 3.0 4.5 3.0 3.4 31.0 0.039 3.4 1 2 1.2+0.5 1.9+1.8 30.3+0.7 2.0+0.6 7.3+1.3 7.6+0.9 5.1+2.5 3.1+0.1 10.2+0.9 7.2+1.1 6.8+*0.6 2.4+0.4 3.3+1.7 5.2+0.1 3.2+1.6 3.2+0.7 33.0+2.3 0.040+0.017 4.7+2.1 1 1 0.9 1.2 37.3 1.8 8.4 6.6 9.6 4.2 8.7 6.0 6.0 3.0 1.8 3.6 0.6 0.6 39.4 0.024 3.5 1 1 1.5 2.4 39.8 1.5 4.9 4.9 3.9 2.9 9.7 4.4 8.3 2.4 2.9 4.9 3.9 1.9 43.7 0.038 8.1 (a) f a s t e r , major m i g r a t i n g band o f a d o u b l e t a n a l y z e d (b) s l o w e r , minor m i g r a t i n g band o f a d o u b l e t a n a l y z e d (c) mean + s t a n d a r d d e v i a t i o n (d) Total""fcasics » Lys+His+Arg (e) B a s i c / A c i d i c - Lys+His+Arg/Asp+Glu 80 T a b l e 7. Amino a c i d c o m p o s i t i o n o f t e s t i s - s p e c i f i c p r o t e i n s from G. a c u l e a t u s l e i u r u s and t r a c h u r u s forms. ( V a l u e s i n mole p e r c e n t ) l e i u r u s t r a c h u r u s L y s 1.1+0.3 0.4+0.2 H i s 1.7+1.6 0.6+0.3 A r g 33.4+5.9 36.7+6.1 Asp 2.0+0.7 1.5+1.8 Thr 6.6+1.4 6.9+0.8 Ser 6.9+2.0 5.8+0.8 G l u 5.7+2.1 5.8+0.8 Pr o ' 5.6+2.1 6.6+1.3 G l y 10.0+1.5 8.2+0.5 A l a 6.9+1.8 8.1+0.8 V a l 6.0+1.0 6.4+1.0 Met 2.1+0.6 1.2+1.0 l i e 2.9+0.8 3.0+0.5 Leu 4.4+0.6 5.2+0.8 T y r 2.4+1.1 1.8+1.5 Phe 2.7+1.0 2.7+0.4 T o t a l B a s i c s ( a ) 35.8+4.7 36.0+5.2 L y s / A r g 0.033+0.011 0.011+0.003 B a s i c / A c i d i c ^ 4.7+2.0 5.2+2.1 * Average o f 8 a n a l y s e s from 6 a n i m a l s ( T a b l e 6) ** Average o f 6 a n a l y s e s from 6 a n i m a l s ( T a b l e 4 ) . Only f a s t e r , major m i g r a t i n g band o f d o u b l e t used f o r t h i s c a l c u l a t i o n . (a) T o t a l B a s i c s * Lys+His+Arg (b) B a s i c / A c i d i c = Lys+His+Arg/Asp+Glu 81 In G. a c u l e a t u s , no r e l i a b l e d i f f e r e n c e s c o u l d be seen between d i f f e r e n t l e i u r u s p o p u l a t i o n s or between d i f f e r e n t t r a c h u r u s p o p u l a t i o n s . However, s i g n i f i c a n t d i f f e r e n c e s between l e i u r u s and t r a c h u r u s p o p u l a t i o n s have been d e t e r m i n e d i n the l y s i n e / a r g i n i n e r a t i o from the amino a c i d d a t a , i n the number of i n d i v i d u a l s h a v i n g m u l t i p l e TSP bands and i n the shape d i f f e r e n c e s of the TSP bands. T h e r e f o r e , TSP's may be c o n s i d e r e d as m o l e c u l a r markers between anadromous and f r e s h w a t e r p o p u l a t i o n s , but not between d i f f e r e n t anadromous or d i f f e r e n t f r e s h w a t e r p o p u l a t i o n s . 6. B e n t h i c / L i m n e t i c comparison In s e v e r a l l a k e s on Vancouver I s l a n d and s u r r o u n d i n g i s l a n d s , 2 forms of G. a c u l e a t u s - l e i u r u s e x i s t s y m p a t r i c a l l y . These a r e an upper d w e l l i n g , l i m n e t i c type and a bottom d w e l l i n g b e n t h i c t y p e . L i m n e t i c and b e n t h i c p o p u l a t i o n s from Enos Lake on Vancouver I s l a n d and Paxton Lake on Texada I s l a n d , B.C. were s t u d i e d . B i o l o g i c a l and m o r p h o l o g i c a l d i f f e r e n c e s i n t h e Enos Lake p a i r have been s t u d i e d i n g r e a t e r d e t a i l than has been done f o r Paxton Lake ( M c P h a i l , 1984a; Bentzen and M c P h a i l , 1984; Ridgway and M c P h a i l , 1984). I t i s i m p o r t a n t t o note t h a t the l i m n e t i c t ype i n one l a k e may or may not be the same type i n another l a k e . The same h o l d s f o r the b e n t h i c t y p e s . I t i s not known i f t h e i r o r i g i n s a r e the same. At t h i s t ime e v i d e n c e l e a d s t o the c o n c l u s i o n t h a t the l i m n e t i c / b e n t h i c p a i r of Enos Lake i s not the same as the p a i r i n Paxton Lake ( M c P h a i l , 1984b). In the 82 p r e s e n t study we wanted t o see i f the e l e c t r o p h o r e t i c method used t o a n a l y z e TSP's c o u l d d i s t i n g u i s h between the b e n t h i c and l i m n e t i c t y p e s . A l t h o u g h t h e r e i s much c o n t r o v e r s y as t o the taxonomic and b i o l o g i c a l s t a t u s of the s e p o p u l a t i o n s , M c P h a i l and h i s c o l l e a g u e s ( M c P h a i l , 1984a; Bentzen and M c P h a i l , 1984; Ridgway and M c P h a i l , 1984) have d e t e r m i n e d t h a t the l i m n e t i c and b e n t h i c p o p u l a t i o n s i n the s e l a k e s a r e d i f f e r e n t s p e c i e s . The TSP's of b e n t h i c and l i m n e t i c forms of G. a c u l e a t u s from Enos Lake m i g r a t e d the same d i s t a n c e on PAGE as the TSP's from o t h e r p o p u l a t i o n s of G a s t e r o s t e u s ( F i g . 13C, l a n e s 2 and 3) i n d i c a t i n g t h a t the p r o t e i n s were s i m i l a r . On 2 s e p a r a t e 34 cm. g e l r u n s , 2 d i f f e r e n t b e n t h i c i n d i v i d u a l s from Enos Lake produced 2 bands w h i l e the 2 d i f f e r e n t l i m n e t i c a n i m a l s produced a s i n g l e band. A p i c t u r e of one of these g e l s i s shown ( F i g . 14A-I). On the 61 cm. g e l ( F i g . 1 4 A - I I ) , both the b e n t h i c and l i m n e t i c a n i m a l s TSP's from Enos Lake showed double-banded p a t t e r n s . However, the s e p a r a t i o n appears t o be l e s s i n the l i m n e t i c bands of l a n e 5 than w i t h the b e n t h i c bands of l a n e s 1 and 2. T h i s s u g g e s t s t h a t l o n g e r g e l s a r e n e c e s s a r y t o r e s o l v e the l i m n e t i c bands as these TSP's appear t o have s i m i l a r e l e c t r o p h o r e t i c m o b i l i t y on the g e l s . In an e l e c t r o p h o r e t i c g e l , done by B. C o y l e i n Dr. H. K a s i n s k y ' s l a b o r a t o r y on a n i m a l s c a p t u r e d on the same day, i t was n o t i c e d t h a t i n Enos. Lake, the b e n t h i c a n i m a l s o f t e n showed t r a c e s of somatic h i s t o n e s i n the upper p o r t i o n of the g e l . 83 F i g u r e 14. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from t e s t e s of b e n t h i c and l i m n e t i c forms of G. a c u l e a t u s from Enos Lake, B.C. A. I and I I . Animals were c a p t u r e d 29 June 1983. B = b e n t h i c ( g e l I , l a n e 2; g e l I I , l a n e s 1-3); L = l i m n e t i c ( g e l I , l a n e 1; g e l I I , l a n e s 4 and 5) Lane 6 i s X. l a e v i s h e a r t , shown as a marker. B. S u l f u r i c a c i d / u r e a s e n s i t i v e r e s t a i n i n g of the upper p o r t i o n of g e l s I and I I of F i g . 14A. 84 85 8 6 R e s t a i n i n g of the g e l s i n F i g . 14A enhanced the bands ten f o l d and c o n f i r m e d t h a t the b e n t h i c a n i m a l s ( F i g . 14B, I : l a n e 2' and I I : l a n e s 1'-3') had more somati c h i s t o n e s p r e s e n t than the l i m n e t i c specimens ( F i g . 14B, I : l a n e 1'and I I : l a n e s 5' and 6'). I t was u n u s u a l t o see the somatic h i s t o n e bands when the c h a r a c t e r i s t i c 1 or 2 TSP bands of the b r e e d i n g mature male were seen w i t h amidoblack s t a i n i n g . T h e i r appearance upon s e n s i t i v e r e s t a i n i n g s u g g e s t s t h a t r e s i d u a l somatic h i s t o n e s were p r e s e n t o n l y t o the e x t e n t of s e v e r a l p e r c e n t (<10%) of t o t a l b a s i c p r o t e i n s i n the mature t e s t i s of t h e s e b r e e d i n g specimens. There was not a marked d i f f e r e n c e between the TSP's of l i m n e t i c and b e n t h i c t y p e s of G. a c u l e a t u s i n Paxton Lake, B.C. ( F i g . 15). The presence of so m a t i c h i s t o n e s i n the b e n t h i c specimens was noted ( F i g . 16A, l a n e s 4 and 5; F i g . 16B, l a n e 2) but l e s s so i n the l i m n e t i c specimens from Paxton Lake ( F i g . 16A, l a n e s 1 and 2; F i g . 16B, l a n e 1 ) . The f i s h p a i r s were c a p t u r e d on the same day. E l e c t r o p h o r e t i c a n a l y s i s of t e s t i s p r e p a r a t i o n s c o u l d d i s t i n g u i s h between b e n t h i c and l i m n e t i c forms of G. a c u l e a t u s by o b s e r v i n g the r e l a t i v e amounts of s o m a t i c h i s t o n e s p r e s e n t . The number of b e n t h i c and l i m n e t i c i n d i v i d u a l s t h a t had TSP m u l t i p l e bands ( T a b l e 4) were a n a l y z e d by the ' G - t e s t ' f o r d i f f e r e n c e s . No s i g n i f i c a n t d i f f e r e n c e i n the number of m u l t i p l e bands between the two t y p e s was found i n e i t h e r Enos or Paxton L a k e s . Enos Lake b e n t h i c s , however, were found t o have 87 F i g u r e 15. E l e c t r o p h o r e t i c p r o f i l e s of TSPs from t e s t e s of b e n t h i c and l i m n e t i c G. a c u l e a t u s - l e i u r u s from Paxton Lake, B.C. c a p t u r e d 5 J u l y 1979 on a 34 cm. g e l . Lanes 1-3, l i m n e t i c ; l a n e s 4-6, b e n t h i c ; l a n e 7, X. l a e v i s . 88 89 F i g u r e 16. E l e c t r o p h o r e t i c p r o f i l e s of b a s i c p r o t e i n s from t e s t i s of b e n t h i c and l i m n e t i c forms of G. a c u l e a t u s from Paxton Lake, B.G. R e l a t i v e amounts of somatic h i s t o n e s are compared. A. Top p o r t i o n of a 25 cm. g e l a f t e r s e n s i t i v e s t a i n i n g showing l a n e 1, G. a c u l e a t u s - l e i u r u s h e a r t ; l a n e s 2 and 3, G. a c u l e a t u s - l e i u r u s ' , l i m n e t i c ( L ) ; l a n e s 4 and 5, G. a c u l e a t u s - l e i u r u s , b e n t h i c ( B ) . B. 8 cm. g e l , l a n e 1, G. a c u l e a t u s - l e i u r u s l i m n e t i c ; l a n e 2, b e n t h i c , from f i s h c a p t u r e d 29 June 1982. 91 s i g n i f i c a n t l y h i g h e r number of TSP m u l t i p l e bands over the Paxton Lake b e n t h i c s (p<0.0l) w h i l e Enos Lake and Paxton Lake l i m n e t i c s were not found t o have s i g n i f i c a n t d i f f e r e n c e s (p>0.05) i n the number of TSP m u l t i p l e bands seen on PAGE. T h i s s u g g e s t s t h a t Enos Lake b e n t h i c s a r e not the same as Paxton Lake b e n t h i c s . A l t h o u g h i t i s thought t h a t the r e s p e c t i v e l i m n e t i c t y p e s are a l s o d i f f e r e n t ( M c P h a i l , 1984b), t h e s e d a t a does not s u p p o r t or d i s p r o v e t h i s t h e o r y . Because of the s i m i l a r i t y seen i n the TSP's of the b e n t h i c and l i m n e t i c t y p e s , i t i s p r o b a b l e t h a t d i f f e r e n t , y e t c l o s e l y r e l a t e d p o p u l a t i o n s p r o b a b l y do c o e x i s t i n Enos and Paxton L a k e s . A l a r g e r sample s i z e would be needed t o v e r i f y these f i n d i n g s . I f the TSP p a t t e r n i s i n d e e d s p e c i f i c f o r r e l a t e d s p e c i e s of t e l e o s t s , as shown p r e v i o u s l y , i t would seem t h a t t h e s e p r o t e i n s a r e not s u f f i c i e n t l y d i s s i m i l a r i n e l e c t r o p h o r e t i c c h a r a c t e r i s t i c s t o d i s t i n g u i s h t h e s e G. a c u l e a t u s forms as d i s t i n c t s p e c i e s . I f , by o t h e r s t a n d a r d s , b e n t h i c and l i m n e t i c G. a c u l e a t u s of Enos Lake a r e c o n s i d e r e d s e p a r a t e s p e c i e s ( M c P h a i l , 1984a), t h e i r d i v e r g e n c e from a common a n c e s t o r would be r e l a t i v e l y r e c e n t . The g r e a t e r amounts of som a t i c h i s t o n e s seen i n b e n t h i c than i n l i m n e t i c t e s t i s s u g g e s t s t h a t t h e r e may be a d i f f e r e n c e i n b r e e d i n g time between t h e s e 2 f i s h t y p e s . As was seen i n the development s e c t i o n , the p r e s e n c e of somatic h i s t o n e s was a s s o c i a t e d w i t h n o n - b r e e d i n g t e s t i s c o n t a i n i n g germ c e l l s w i t h v a r i o u s e a r l y s t a g e s i n s p e r m a t o g e n e s i s . T h i s l e a d s t o two h y p o t h e s e s . The f i r s t s u g g e s t s a b i o l o g i c a l d i f f e r e n c e by 92 a s e p a r a t i o n i n b r e e d i n g t i m e s . The l i m n e t i c breeds e a r l y w h i l e the b e n t h i c form breeds l a t e . The b e n t h i c has somatic h i s t o n e s a l o n g w i t h TSP's whereas l i m n e t i c has o n l y TSP's ( F i g . 14B), as i t has a l r e a d y l o s t i t s somatic h i s t o n e s . Both forms a r e p o t e n t i a l l y i n the same a r e a a t the same t i m e , y e t i n t e r b r e e d i n g i s not p o s s i b l e because of d i f f e r e n c e s i n s e x u a l m a t u r a t i o n . H y p o t h e s i s two suggests t h a t i n d i f f e r e n t r e l a t e d s p e c i e s , somatic h i s t o n e s might be l o s t t o a d i f f e r e n t e x t e n t i n mature sperm. For example, X. b o r e a l i s r e t a i n s a l l f o u r nucleosomal h i s t o n e s i n sperm whereas X. l a e v i s r e t a i n s m o s t l y h i s t o n e s H2A and H2B a l o n g w i t h the TSP's (Mann e t a l . , 1982). In t h i s i n s t a n c e , M c P h a i l (1984a) sees G. a c u l e a t u s l i m n e t i c s and b e n t h i c forms as d i f f e r e n t s p e c i e s . Perhaps one r e t a i n s more somatic h i s t o n e s than the o t h e r a l o n g w i t h TSP's i n mature sperm. T h e r e f o r e , the mature sperm of the b e n t h i c and l i m n e t i c forms may not be the same and c o u l d a c t as a b a r r i e r t o i n t e r b r e e d i n g . We have a l r e a d y seen t h a t somatic h i s t o n e s a r e p r e s e n t i n the Enos Lake b e n t h i c form t o the e x t e n t of a few p e r c e n t by s e n s i t i v e r e s t a i n i n g . The b e n t h i c form might merely have a h i g h e r p e r c e n t of the somatic h i s t o n e complements. For example X. l a e v i s l o s e s H2B i n mature sperm but not H3 and H4, w h i l e o t h e r Xenopus s p e c i e s r e t a i n H2A, H2B and H4. 1_. P r o t e i n c h a r a c t e r i z a t i o n TSP's from G. a c u l e a t u s were c h a r a c t e r i z e d , by p r o t e i n c l e a v a g e u s i n g the s p e c i f i c f r a g m e n t i n g a gents of t r y p s i n and 93 cyanogen bromide (CNBr). a. T r y p t i c d i g e s t i o n A c i d s o l u b l e p r o t e i n s from G. a c u l e a t u s t e s t i s were d i g e s t e d w i t h t r y p s i n (Sigma Chem. Co.) f o r 16 hours a t pH 8.0 a t 37*C. There was a h i g h p e r c e n t a g e of a r g i n i n e i n the s e TSP's (T a b l e 7 ) , so t h a t d i g e s t i o n w i t h t r y p s i n , which c l e a v e s a t a r g i n i n e and l y s i n e r e s i d u e s , c r e a t e d many s m a l l p e p t i d e s w i t h such r a p i d m o b i l i t y t h a t they were not d e t e c t e d on PAGE by amidoblack s t a i n ( F i g . 17A, l a n e s 7 and 8) or by s e n s i t i v e r e s t a i n i n g ( F i g . 17B, l a n e s 7' and 8'). The p e p t i d e s were e i t h e r t o o low i n c o n c e n t r a t i o n t o be d e t e c t e d or m i g r a t e d so f a s t t h a t they ran o f f the g e l . b. Cyanogen bromide r e a c t i o n Amino a c i d a n a l y s i s showed t h a t m e t h i o n i n e was p r e s e n t a t low l e v e l s i n G. a c u l e a t u s TSP's ( T a b l e s 3,5,6 and 7 ) . T h i s was c o n f i r m e d by cyanogen bromide c l e a v a g e a t the m e t h i o n i n e r e s i d u e s i n these p r o t e i n s . The cyanogen bromide (CNBr) r e a c t i o n was done a t room temperature and a t 37*C f o r 16 hours i n 70% f o r m i c a c i d . G. a c u l e a t u s TSP's i n c u b a t e d i n 70% f o r m i c a c i d w i t h o u t CNBr a c t e d as c o n t r o l s ( F i g . 17-1:-lanes 2, 4 and 6 and F i g . 18-1, l a n e 3) and were run a l o n g s i d e the CNBr d i g e s t e d sample. No d i g e s t i o n 94 F i g u r e 17. E l e c t r o p h o r e t i c p r o f i l e s of TSP's from one p a i r of t e s t e s from G. a c u l e a t u s - t r a c h u r u s from R a i l r o a d D i t c h , B.C. d i g e s t e d w i t h cyanogen bromide (CNBr) or w i t h t r y p s i n . I . Lanes 1, 3 and 5 r e p r e s e n t samples c l e a v e d w i t h CNBr and were run on t h i s 61 cm. g e l . TSP's were a l l o w e d t o r e a c t w i t h CNBr f o r 3, 2 and 1 days r e s p e c t i v e l y a t room te m p e r a t u r e . Lane 7 shows r e s u l t s of t r y p t i c d i g e s t i o n a t 37*C f o r 1 day. The TSP's from the o t h e r t e s t i s of the same r e s p e c t i v e a n i m a l s were d i s s o l v e d i n 8 M u r e a / 5 % 2-mercaptoethanol as c o n t r o l s and then f r o z e n u n t i l used i n l a n e s 2, 4, 6 and 8. Arrows i n d i c a t e l i g h t e r bands from CNBr d i g e s t i o n . I I . S e n s i t i v e r e s t a i n i n g of t r y p t i c d i g e s t i o n , l a n e 7' and u n d i g e s t e d c o n t r o l , l a n e 8'. 95 96 F i g u r e 18. E l e c t r o p h o r e t i c p r o f i l e s of TSP's from f r e s h w a t e r and anadromous G. a c u l e a t u s were d i g e s t e d w i t h cyanogen bromide and ran on t h i s 61 cm. g e l . I . TSP's e x t r a c t e d from 2 t e s t e s of a s i n g l e G. a c u l e a t u s - t r a c h u r u s were d i v i d e d i n t o l a n e 2, f r o z e n c o n t r o l ; l a n e 3, c o n t r o l i n 70% f o r m i c a c i d i n c u b a t e d f o r 16 hours a t 37*C; and l a n e 4, CNBr d i g e s t i o n i n 70% f o r m i c a c i d f o r 16 hours a t 37*C. G. a c u l e a t u s - l e i u r u s TSP's were d i v i d e d s i m i l a r l y w i t h l a n e 5, f r o z e n c o n t r o l ? l a n e 6, f o r m i c a c i d c o n t r o l and l a n e 7, CNBr r e a c t i o n . Lane 1, h e r r i n g protamine ( P ) ; l a n e 8, TSP's from P. p u n g i t i u s (P.p.) and l a n e 9, G. w h e a t l a n d i (G.w.T a r e shown as markers. I I . S e n s i t i v e r e s t a i n i n g of G. a c u l e a t u s - t r a c h u r u s TSP's; l a n e 2' f r o z e n c o n t r o l ; l a n e 3', f o r m i c a c i d c o n t r o l ; l a n e 4', CNBr r e a c t i o n ; l a n e 5', and G. a c u l e a t u s - l e i u r u s f r o z e n c o n t r o l ; l a n e 6, f o r m i c a c i d c o n t r o l ; l a n e 7', CNBr r e a c t i o n . Arrows denote p r i n c i p a l bands a f t e r CNBr d i g e s t i o n . 98 was d e t e c t e d i n the c o n t r o l s even w i t h s e n s i t i v e r e s t a i n i n g . T h i s experiment was done f o l l o w i n g two d i f f e r e n t p r o t o c o l s . In the f i r s t method, we used one, t e s t i s from the f i s h as a c o n t r o l . T h i s t e s t i s was homogenized, the b a s i c p r o t e i n s e x t r a c t e d , d i s s o l v e d i n 8 M urea/5% 2-mercaptoethanol and f r o z e n a t -70*C u n t i l PAGE ( F i g . 17-1, l a n e s 2, 4, 6 and 8 ) . The o t h e r t e s t i s from the same f i s h was p r e p a r e d i n the^-same way ex c e p t t h a t the CNBr r e a c t i o n was a l l o w e d t o oc c u r ( F i g . 17-1, l a n e s 1, 3 and 5) . In the second method, the 2 t e s t e s from the same a n i m a l were homogenized t o g e t h e r and s p l i t i n t o even a l i g u o t s . B a s i c p r o t e i n s were e x t r a c t e d and the CNBr r e a c t i o n was a l l o w e d t o take p l a c e on TSP's i n one p o r t i o n of the sample, ( F i g . 18-1, l a n e s 4 and 7 ) . The second TSP p o r t i o n was s u b j e c t e d t o the same c o n d i t i o n s as the f i r s t o n l y w i t h o u t the p r e s e n c e of CNBr ( F i g . 18-1, l a n e s 3 and 6 ) . The l a s t TSP p o r t i o n was d i s s o l v e d i n 8 M urea/5% 2-mercaptoethanol and f r o z e n a t -70*C u n t i l used ( F i g . 18-1, l a n e s 2 and 5 ) . No d e g r a d a t i o n of c o n t r o l s was d e t e c t e d even when the g e l was s e n s i t i v e l y r e s t a i n e d ( F i g . 18-11, l a n e s 2', 3', 5' and 6'). Cyanogen bromide, under the above c o n d i t i o n s , was a l l o w e d t o r e a c t w i t h the TSP p r e p a r a t i o n a t an 80 f o l d molar excess over the e s t i m a t e d p r o t e i n c o n c e n t r a t i o n . When 2 o r i g i n a l TSP bands were p r e s e n t , as seen i n the c o n t r o l ( F i g . 17, l a n e 2) 4 f a s t e r bands were produced ( F i g . 17, l a n e 1 ) . T h i s s u g g e s t s t h a t each TSP i n the d o u b l e t c o n t a i n e d a m e t h i o n i n e r e s i d u e and was c l e a v e d by CNBr. One TSP band i n the c o n t r o l ( F i g . 17, l a n e s 4 99 and 6) produced 2 f a s t bands ( F i g . 17, l a n e s 3 and 5) a f t e r CNBr r e a c t i o n . A r e s i d u a l band remained p r o b a b l y of TSP's t h a t d i d not r e a c t or m i g r a t e d s i m i l a r i l y t o the c o n t r o l TSP's. T h i s band c o u l d a l s o r e p r e s e n t a m e t h i o n i n e - f r e e b a s i c p r o t e i n t h a t co-m i g r a t e d w i t h the p r i n c i p a l TSP band of G. a c u l e a t u s . The TSP's of the t r a c h u r u s and l e i u r u s forms of G. a c u l e a t u s were not s i g n i f i c a n t l y d i f f e r e n t . T h i s was c o n f i r m e d by CNBr. No d i f f e r e n c e was seen between the f r e s h w a t e r and anadromous G. a c u l e a t u s CNBr c l e a v a g e p r o d u c t s on PAGE. Two bands were seen w i t h amidoblack s t a i n i n g i n both the f r e s h w a t e r ( F i g . 18-1, l a n e 4) and anadromous ( F i g . 18-1, l a n e 7) forms. A t h i r d , f a s t e r m i g r a t i n g band was d e t e c t e d w i t h s e n s i t i v e s t a i n i n both G. a c u l e a t u s forms ( F i g . 18-11, l a n e s 4' and 7'). c. A l k a l i n e phosphatase and p o s s i b l e endogenous p r o t e a s e I t may have been t h a t the o c c u r r e n c e of the double bands i n some of t h e e l e c t r o p h o r e t i c p r o f i l e s of G. a c u l e a t u s TSP's was due t o p h o s p h o r y l a t i o n of the s e r i n e s i d e c h a i n s y i e l d i n g p r o t e i n s w i t h 2 d i f f e r e n t m o b i l i t i e s on PAGE f o r the p h o s p h o r y l a t e d and the u n p h o s p h o r y l a t e d forms. T h i s has been observed f o r protamine d u r i n g t r o u t s p e r m i o g e n e s i s ( L o u i e and D i x o n , 1972). To t e s t t h i s h y p o t h e s i s , the TSP's of G. a c u l e a t u s t r a c h u r u s and l e i u r u s were s u b m i t t e d t o a l k a l i n e phosphatase (AP) t r e a t m e n t t o see i f d i f f e r e n c e s i n band m i g r a t i o n would o c c u r . TSP p r e p a r a t i o n s were d i v i d e d i n t o e q u a l a l i q u o t s of the 100 t e s t i s homogenate from one a n i m a l and used e i t h e r as a c o n t r o l or as AP t r e a t e d samples i n a s i m i l a r f a s h i o n as d e s c r i b e d p r e v i o u s l y f o r the CNBr r e a c t i o n . For optimum AP c o n d i t i o n s , the TSP's were suspended i n t r i s / H C l b u f f e r , pH 7.8, f o r 3 hours a t 37*C ( F i g . 19-1, l a n e s 2 and 4 ) . The r e a c t i o n was stopped w i t h 8 M u r e a / 5 % 2-mercaptoethanol. In b o t h the f r e s h w a t e r . ( F i g . 19, l a n e 2) and anadromous TSP's ( l a n e 4 ) , no apparent d i f f e r e n c e i n m o b i l i t y was seen when compared t o the TSP u n i n c u b a t e d c o n t r o l i n 8M u r e a / 5 % 2-m e r c a p t o e t h a n o l ( l a n e 6 ) . Both bands were s t i l l seen i n b oth the anadromous sample t r e a t e d w i t h AP ( l a n e 2) and the u n i n c u b a t e d c o n t r o l ( l a n e 6 ) . No a d d i t i o n a l bands were seen even w i t h s e n s i t i v e r e s t a i n i n g ( F i g . 19-11, l a n e s 2' and 4' compared w i t h 6'). T h i s s u g g e s t s t h a t the TSP's a r e not l i k e l y t o c o n t a i n p h o s p h o r y l a t e d or u n p h o s p h o r y l a t e d forms of t h e same p r o t e i n . However, i t i s s t i l l p o s s i b l e t h a t 2 d i f f e r e n t p r o t e i n s c o u l d e x i s t i n the TSP's of the b r e e d i n g males, or t h a t the enzyme d i d not r e a c t e f f e c t i v e l y w i t h the TSP's. The l a t e r p o s s i b i l i t y seems u n l i k e l y s i n c e optimum AP c o n d i t i o n s were p r e s e n t . v. C o n t r o l s from G. a c u l e a t u s TSP's i n c u b a t e d i n t r i s b u f f e r , pH 7.8, a t 37*C f o r 3 hours ( F i g . 19-1, l a n e s 3 and 5) y i e l d e d unexpected r e s u l t s . TSP's i n t h i s s l i g h t l y a l k a l i n e b u f f e r produced as many as 11 d i s c r e t e bands ( l a n e s 5 and 5') i n G. a c u l e a t u s - t r a c h u r u s and r o u g h l y 8 bands i n G. a c u l e a t u s - l e i u r u s ( l a n e s 3 and 3'). S i n c e no bands had been seen when a heated 101 F i g u r e 19. E l e c t r o p h o r e t i c p r o f i l e s of TSP's from G. a c u l e a t u s - l e i u r u s from J e r i c h o Pond, Vancouver, B.C. t r e a t e d w i t h a l k a l i n e phosphatase and run on a 61 cm. g e l . I . Lane 2, TSP's were heated a t 37*C f o r 3 hours i n t r i s / H C l b u f f e r , pH 7.8 w i t h a l k a l i n e phosphatase (AP) ( W o r t h i n g t o n D i a g n o s t i c Chem.). Lane 3, a p o r t i o n of the same sample under the same c o n d i t i o n s w i t h o u t AP; l a n e 4, e q u a l p o r t i o n s of G. a c u l e a t u s - t r a c h u r u s , R a i l r o a d D i t c h , B.C. TSP's were s i m i l a r l y t r e a t e d w i t h AP; lane 5, c o n t r o l , i n c u b a t e d i n b u f f e r o n l y ; l a n e 6, f r o z e n TSP's i n 8 M u r e a / 5 % 2 - m e r c a p t o e t h a n o l ; l a n e 7, X. l a e v i s TSP's t r e a t e d w i t h AP; l a n e 8, X. l a e v i s TSP c o n t r o l i n c u b a t e d o n l y i n b u f f e r ; l a n e 1, h e r r i n g p rotamine (P) and AP i n u r e a / 2 - m e r c a p t o e t h a n o l f o r e l e c t r o p h o r e s i s . I I . Lanes 2-8 of the g e l r e p r e s e n t e d i n F i g . 19-1 were s e n s i t i v e l y r e s t a i n e d t o enhance f a i n t bands. Arrows i n d i c a t e p r e s e n c e of f a i n t bands i n G. a c u l e a t u s TSP. T r i a n g l e s , i n d i c a t e the major d e g r a d a t i o n bands seen i n X. l a e v i s t e s t i c u l a r p r o t e i n s . 1 0 2 103 c o n t r o l a t a v e r y low pH of 1.8 was examined i n the CNBr exp e r i m e n t s ( F i g . 18, l a n e 2 ) , i t became apparent t h a t a p o s s i b l e endogenous p r o t e a s e was p r e s e n t i n the TSP p r e p a r a t i o n t h a t had become a c t i v e when heated a t s l i g h t l y a l k a l i n e pH. S e v e r a l e x p e r i m e n t s were performed s u b s e q u e n t l y t o v e r i f y the presence of an endogenous p r o t e a s e w i t h the TSP's. Endogenous p r o t e a s e s a l s o were d e t e c t e d i n X. l a e v i s t e s t i s p r e p a r a t i o n s , as c o u l d be seen i n F i g . 19. D e g r a d a t i o n of the TSP's was seen w i t h the h e a t e d , n e u t r a l pH sample ( l a n e s 8 and 8') where none was seen i n the AP sample ( l a n e s 7 and 7'). However, the d e g r a d a t i o n was c o n s i d e r a b l y l e s s than i n G. a c u l e a t u s TSP's. An experiment was d e s i g n e d t o i n h i b i t a c t i v i t y of any endogenous p r o t e a s e t h a t might be p r e s e n t by u s i n g the known i n h i b i t o r s TPCK ( L - 1 - t o s y l a m i d e - 2 - p h e n y l e t h y l c h l o r o m e t h y l k e t o n e ) , TLCK ( N - a - p - t o s y l - L - l y s i n e c h l o r o m e t h y l k e t o n e ) , PMSF ( p h e n y l m e t h y l s u l f o n y l f l u o r i d e ) and sodium b i s u l f i t e (Mann, et a l . , 1982; R i s l e y and E c k h a r d t , 1981). A g a i n , t e s t e s from the same a n i m a l were homogenized and d i v i d e d i n t o t h i r d s . F i g . 20-1, l a n e 2 shows the u n i n c u b a t e d c o n t r o l . I t underwent o n l y s l i g h t d e g r a d a t i o n as was seen tn the t e n f o l d enhanced s e n s i t i v e r e s t a i n ( F i g . 20-11, l a n e 2 ' ) . Lanes 3 and 3' show the absence of any bands moving f a s t e r than G. a c u l e a t u s TSP. Only a s m a l l amount of p r o t e i n i n the TSP p o s i t i o n was v i s i b l e i n the s e n s i t i v e l y r e s t a i n e d g e l ( F i g . 20-11, l a n e 3'). T h i s was 104 F i g u r e 20. E l e c t r o p h o r e t i c d e t e c t i o n of d e g r a d a t i o n p r o d u c t s i n G. a c u l e a t u s and P. p u n q i t i u s TSPs upon h e a t i n g a t s l i g h t l y a l k a l i n e pH. 61 cm. g e l of G. a c u l e a t u s -t r a c h u r u s from R a i l r o a d D i t c h , B.C. and P. p u n g i t i u s from R i v i e r e des Vase, Quebec. I . Lane 1, h e r r i n g protamine marker ( P ) ; l a n e .2, TSP's i n u n i n c u b a t e d G. a c u l e a t u s c o n t r o l ; l a n e 3, G. a c u l e a t u s TSP's heated i n t r i s / H C l b u f f e r , pH 7.8 f o r 3 hours a t 37*C; l a n e 4, G. a c u l e a t u s TSP's he a t e d i n the same b u f f e r i n the presence of the p r o t e o l y t i c i n h i b i t o r s TPCK, TLCK, PMSF and NaHS03. Lanes 2-4 r e p r e s e n t e q u a l a l i q u o t e s of the TSP's from 2 t e s t e s of a s i n g l e G. a c u l e a t u s . Lane 5, a d i f f e r e n t specimen of G. a c u l e a t u s - t r a c h u r u s i n 0.4 N HC1, pH 1.8, he a t e d f o r 3 hours; l a n e 6, 0.4 N HC1 heated f o r 3 hours w i t h p r o t e o l y t i c i n h i b i t o r s , f i n a l pH of 2.1; l a n e 7, unheated . c o n t r o l of P. p u n g i t i u s TSP's; l a n e 8, P. p u n g i t i u s TSP's heated i n b u f f e r ; l a n e 9, P. p u n g i t i u s TSP's heated i n b u f f e r w i t h i n h i b i t o r s . Lanes 7-9 r e p r e s e n t e q u a l a l i q u o t e s of the TSP's form 2 t e s t e s of a s i n g l e P. p u n q i t i u s . B l a c k arrows i n d i c a t e bands absent i n amidoblack s t a i n e d g e l ( I ) but p r e s e n t a f t e r t e n f o l d more s e n s i t i v e r e s t a i n i n g w i t h u r e a / s u l f u r i c a c i d ( I I - s e e w h i t e a r r o w s ) . I I . S e n s i t i v e r e s t a i n i n g of l a n e s 2-8 t o enhance i n v i s i b l e or f a i n t bands of g e l " seen i n F i g . .20-1. Arrows i n d i c a t e f a i n t bands. 105 1 06 p r o b a b l y due t o the almost complete d e g r a d a t i o n of the TSP's from a sample h e a t e d f o r 3 hours i n pH 7.8 b u f f e r ( f i n a l pH 9.4 w i t h i n h i b i t o r s ) . Some of the TSP t h a t remains ( F i g . 20-11, l a n e 3') might be due t o the s h o r t r e a c t i o n time of o n l y 3 hou r s . The a d d i t i o n o f p r o t e a s e i n h i b i t o r s d e c r e a s e d the d e g r a d a t i v e a c t i v i t y ( F i g . 20-1, l a n e 4, and F i g . 20-11, l a n e 4') s i n c e the TSP was o n l y degraded t o the p o i n t t h a t 11 f a s t e r bands were seen, r a t h e r than complete d i g e s t i o n t o no bands a t a l l . S i m i l a r r e s u l t s were seen w i t h P. p u n g i t i u s u n i n c u b a t e d c o n t r o l e x h i b i t i n g one TSP band ( F i g . 20-1, l a n e 7 and F i g . 20-11, l a n e 7') and h e a t e d TSP's i n b u f f e r showing almost complete d e g r a d a t i o n (no f a s t - m o v i n g bands; F i g . 20-1, l a n e 8 and F i g . 20-11, l a n e 8 ' ) . TSP's heated i n the presence of p r o t e o l y t i c i n h i b i t o r s ( F i g . 20-1, l a n e 9 and Fig.. 20-11, l a n e 9') gave s i m i l a r r e s u l t s under these c o n d i t i o n s as G. a c u l e a t u s TSP's, namely l e s s complete d e g r a d a t i o n . In c o n t r a s t , TSP's heated f o r the same t i m e but i n 0.4 N HC1, pH 1.8 f o r t h e c o n t r o l ( F i g . 20-1, l a n e 5 and F i g . 20-1I, l a n e 5') and 0.4 N HCl p l u s the above mentioned p r o t e o l y t i c i n h i b i t o r s , f i n a l pH of 2.'1 ( l a n e 1-6 and I I - 6 ' ) showed n e g l i g i b l e d e g r a d a t i o n . Because the p r o t e a s e i n h i b i t o r s m i n i m i z e t o some e x t e n t the d e g r a d a t i o n of the TSP's from G. a c u l e a t u s and P. p u n g i t i u s , we c o n c l u d e t h a t an endogenous p r o t e a s e i s l i k e l y t o be p r e s e n t i n the TSP p r e p a r a t i o n s . T h i s p r o t e a s e i s a c t i v e a t s l i g h t l y a l k a l i n e pH but not a t a c i d i c pH. Granted' the presence of a p r o t e a s e i n the TSP's, why then 1 07 / d i d a l k a l i n e phosphatase i n h i b i t / i t s a c t i v i t y ( F i g . 19, l a n e s 2 and 4) a t pH 7.8, i n c u b a t e d a t 37*C f o r 3 hours? One p o s s i b i l i t y i s t h a t AP may have d e p h o s p h o r y l a t e d the p r o t e a s e t o i n a c t i v a t e i t . However, the AP enzyme t h a t we used ( W o r t h i n g t o n D i a g n o s t i c Chem.) was s h i p p e d i n 2.6 M ammonium s u l f a t e t o p r e s e r v e i t s a c t i v i t y . Ammonium s u l f a t e i s known t o p r e c i p i t a t e p r o t e i n s . T h e r e f o r e , another p o s s i b i l i t y was t h a t the ammonium s u l f a t e i n the AP p r e p a r a t i o n was i n h i b i t i n g the endogenous p r o t e a s e . An experiment was d e s i g n e d t o t e s t t h i s h y p o t h e s i s u s i n g heated samples of e q u a l a l i q u o t s of a TSP p r e p a r a t i o n i n t r i s / H C l b u f f e r , pH 7.8 i n 2.6 M ammonium s u l f a t e w i t h and w i t h o u t AP. The c o n t r o l c o n s i s t e d of an u n t r e a t e d sample. F i g . 21 shows t h a t 0.36 u n i t s of AP i n 0.26 M ammonium s u l f a t e i n the 10 u l TSP sample ( l a n e s 2 and 2') had about the same, amount of d e g r a d a t i o n a f t e r i n c u b a t i o n as d i d TSP's i n c u b a t e d w i t h 0.26 M ammonium s u l f a t e a l o n e ( l a n e s 3 and 3'). I n c u b a t i o n w i t h 10 f o l d d i l u t i o n of the ammonium s u l f a t e sample ( l a n e s 4 and 4') showed more d e g r a d a t i o n of the TSP's. The u n t r e a t e d c o n t r o l ( l a n e s 5 and 5') showed no breakdown. I t can be c o n c l u d e d t h a t the presence of ammonium s u l f a t e i n the commercial a l k a l i n e phosphatase p r e p a r a t i o n c o n t r i b u t e d t o the i n a c t i v a t i o n of the endogenous p r o t e a s e by an unknown mechanism. 8. An o m a l i e s In the co u r s e of the s e i n v e s t i g a t i o n s a few unusual examples of G. a c u l e a t u s TSP's were uncovered. In one t e s t i s 108 / F i g u r e 21. E f f e c t s of ammonium s u l f a t e on endogenous p r o t e a s e of G. a c u l e a t u s TSP's on a 61 cm. g e l . I . Lane 1, h e r r i n g protamine ( P ) ; l a n e 2, G. a c u l e a t u s -t r a c h u r u s , R a i l r o a d D i t c h , B.C., TSP's i n c u b a t e d i n t r i s / H C l b u f f e r w i t h 0.26 M ammonium s u l f a t e , pH 7.8, 37*C f o r 3 hours w i t h a l k a l i n e phosphatase (AP); l a n e 3, TSP's i n c u b a t e d i n b u f f e r and 0.26 M ammonium s u l f a t e ; l a n e 4, TSP's i n c u b a t e d i n b u f f e r and a 1:10 d i l u t i o n of ammonium s u l f a t e (0.026 M); l a n e 5, TSP c o n t r o l i n 8 M urea/5% 2 - m e r c a p t o e t h a n o l . Lanes 2-4 r e p r e s e n t e q u a l a l i q u o t e s of the TSP's from 4 t e s t e s of two specimens of G. a c u l e a t u s . AP = a l k a l i n e p h o s p h a t a s e . I I . S e n s i t i v e r e s t a i n i n g of F i g . 21-1. Arrows i n d i c a t e f a i n t bands, moving between h i s t o n e H4 and G. a c u l e a t u s spp. 109 1 10 from G. a c u l e a t u s - t r a c h u r u s from R i v i e r e des Vase, Quebec, the TSP's showed a band m i g r a t i n g f a s t e r than the t y p i c a l bands t h a t were n o r m a l l y d e t e c t e d ( F i g . 22-1, l a n e 3 ) . The o t h e r t e s t i s was used t o r e p e a t the r e s u l t s t o see i f the band l i n e d up w i t h the one from G. w h e a t l a n d i . F i g . 22-1, l a n e 4 shows the p r i n c i p a l band of the TSP's from G. a c u l e a t u s l i n i n g up w i t h the p r i n c i p a l band of G. a c u l e a t u s - t r a c h u r u s from o t h e r l o c a t i o n s . The l i g h t e r , f a s t e r moving band m i g r a t e d j u s t about the same d i s t a n c e on the g e l as the TSP's from G. w h e a t l a n d i . When the g e l was s e n s i t i v e l y r e s t a i n e d , a d d i t i o n a l bands not d e t e c t e d by amidoblack s t a i n i n g were seen. G. w h e a t l a n d i ( F i g . 22-11, l a n e s 4' and 5') had a s l o w e r band t h a t m i g r a t e d s i m i l a r i l y t o G. a c u l e a t u s ( l a n e s 1' and 2') and a f a s t e r f a i n t band t h a t j u s t about matched w i t h a f a i n t , slow band of P. p u n q i t i u s - ( l a n e 6'). A d d i t i o n a l l y , P. p u n g i t i u s had a sl o w , f a i n t band t h a t m i g r a t e d w i t h G. a c u l e a t u s and the slow, f a i n t band of G. w h e a t l a n d i . T h e r e f o r e , the bands of G. w h e a t l a n d i and P. p u n g i t i u s t h a t m i g r a t e d s i m i l a r i l y w i t h each o t h e r were not the p r i m a r y p h e n o t y p i c e x p r e s s i o n of the TSP genome, but r e p r e s e n t e d l e v e l s of TSP's t e n f o l d l o w e r . N e v e r t h e l e s s , the co-m i g r a t i o n of th e s e bands su g g e s t s t h a t p a r t of the genomes may be common i n the s e r e l a t e d G a s t e r o s t e i f o r m e s . The un u s u a l G. a c u l e a t u s TSP p a t t e r n o c c u r r e d i n o n l y one of many a n i m a l s s h i p p e d t o Vancouver from a random c o l l e c t i o n of a n i m a l s c a p t u r e d i n Quebec. P o s s i b l e p a r e n t a l c r o s s e s a r e 111 F i g u r e 22. E l e c t r o p h o r e t i c p r o f i l e s of anomalous bands i n TSP's of G. a c u l e a t u s - t r a c h u r u s form Quebec. 42 cm. g e l w i t h G. a c u l e a t u s - t r a c h u r u s h a v i n g TSP bands t y p i c a l of t h a t s p e c i e s a l o n g w i t h t h o s e seen i n G. w h e a t l a n d i ( l a n e 3 ) . I. . Lane 1 , G. a c u l e a t u s - t r a c h u r u s from R a i l r o a d D i t c h , B.C.; l a n e 2 and 3, G. a c u l e a t u s from R i v i e r e des Vase, Quebec; l a n e s 4 and 5, G. w h e a t l a n d i ; l a n e 6, P. p u n g i t i u s from R i v i e r e des Vase, Quebec; l a n e 7, X. l a e v i s t e s t i s shown as a marker. B l a c k arrows show f a i n t bands. I I . S e n s i t i v e r e s t a i n i n g of g e l seen i n F i g . 22-1. White .arrows show bands v i s u a l i z e d w i t h the s e n s i t i v e r e s t a i n i n g p r o c e d u r e . The w h i t e r e c t a n g l e i n l a n e 6' shows where the amidoblack s t a i n e d band had been c u t out f o r amino a c i d a n a l y s i s b e f o r e r e s t a i n i n g i n u r e a / s u l f u r i c a c i d . \ \ \ 112 1 13 unknown. Subsequent i n v e s t i g a t i o n of G. a c u l e a t u s - t r a c h u r u s from Quebec and o t h e r l o c a t i o n s d i d not r e v e a l a band t h a t m i g r a t e d l i k e the one shown i n F i g . 22, l a n e 3. Because G. w h e a t l a n d i was caught i n the same b r a c k i s h water t i d e p o o l s of Quebec as the b r e e d i n g G. a c u l e a t u s , i t i s p o s s i b l e t h a t F i g . 22, l a n e 3 might r e p r e s e n t e l e c t r o p h o r e t i c d e t e c t i o n of TSP's from a h y b r i d between G. a c u l e a t u s - t r a c h u r u s and G. w h e a t l a n d i . M clnerney (1969) r e p o r t s t h a t G. a c u l e a t u s may breed w i t h G. w h e a t l a n d i when G. a c u l e a t u s m i g r a t e s t h r o u g h the e s t u r i n e a r e a s where G. w h e a t l a n d i i s b r e e d i n g . However, i t i s b e l i e v e d t h a t d i f f e r e n c e s i n the n a t u r a l environment, b r e e d i n g h a b i t a t and b r e e d i n g b e h a v i o r most l i k e l y m a i n t a i n r e p r o d u c t i v e i s o l a t i o n . The ' h y b r i d ' specimen was i n s p e c t e d by f i s h b i o l o g i s t R. C a r v e t h , c u r a t o r of the U.B.C. Museum of I c h t h i o l o g y . The f i s h was f o u n d t o be G. a c u l e a t u s - t r a c h u r u s w i t h no apparent major m o r p h o l o g i c a l d i f f e r e n c e s t o o t h e r specimens of the same s p e c i e s . Another anomaly can be seen i n F i g . 23. Here a G. a c u l e a t u s - t r a c h u r u s from R a i l r o a d D i t c h , B.C. was found t o have a band m i g r a t i n g v e r y f a s t i n f r o n t of the c h a r a c t e r i s t i c TSP f o r t h i s a n i m a l ( F i g . 23, l a n e s 3 and 3'). T h i s r e s u l t was c o n f i r m e d u s i n g the o t h e r t e s t i s of t h i s a n i m a l ( d a t a not shown). The p o s s i b i l i t y t h a t u n p h o s p h o r y l a t e d s e r i n e r e s i d u e s i n the p r o t e i n c o u l d cause the band t o m i g r a t e so f a r from the o r i g i n a l form i s u n l i k e l y . C o n t r o l s f o r t h e s e e x p e r i m e n t s were done under a c i d pH c o n d i t i o n s and show no d e g r a d a t i o n , as would 1 1 4 F i g u r e 23. E l e c t r o p h o r e t i c p r o f i l e s of anomalous bands i n TSP's of G. a c u l e a t u s - t r a c h u r u s from B.C. I . Lane 1, G. a c u l e a t u s - t r a c h u r u s h e a r t ; and l a n e s 2 and 3, TSP; l a n e 4, c a l f thymus h i s t o n e . Arrow denotes f a i n t r a p i d l y m i g r a t i n g band. I I . S e n s i t i v e r e s t a i n i n g of g e l i n F i g . 23-1 t o b e t t e r i l l u s t r a t e the presence of the u n u s u a l f a s t band i n l a n e 3' i s i n d i c a t e d by the arrow. 116 be i n d i c a t e d by a d d i t i o n a l f a s t moving bands, even w i t h s e n s i t i v e r e s t a i n i n g . T h e r e f o r e t h i s band was u n l i k e l y t o have been produced by d e g r a d a t i o n due t o an endogenous p r o t e a s e . 1 17 DISCUSSION T h i s t h e s i s r e p r e s e n t s the f i r s t u l t r a s t r u c t u r a l e x a m i n a t i o n of s p e r m i o g e n e s i s i n G. a c u l e a t u s . L i g h t microscopy c r o s s - s e c t i o n s of G. a c u l e a t u s t e s t i s showed many c e l l - t y p e s i n the t e s t i s t u b u l e s from non-b r e e d i n g a n i m a l s . These c e l l t y p e s i n c l u d e d p r i m a r y and secondary s p e r m a t o c y t e s and s p e r m a t i d s . The t e s t i s i s a p p a r e n t l y u n d e r g o i n g s p e r m a t o g e n e s i s as d e s c r i b e d by Wootton (1976) and Baggerman (1957). M a i n l y s p e r m a t i d s and spermatozoa were p r e s e n t i n the t u b u l e s from b r e e d i n g a n i m a l s . A l i g h t and e l e c t r o n m i c r o s c o p i c s t u d y of the brook s t i c k l e b a c k ( C u l a e a i n c o n s t a n s ) t e s t i s was done by Ruby and M c M i l l a n (1975) and showed s i m i l a r r e s u l t s . Chromatin c o n d e n s a t i o n i n t o g r a n u l e s i n both.C. i n c o n s t a n s and G. a c u l e a t u s i s of p a r t i c u l a r i n t e r e s t s i n c e no s e t p a t t e r n t o sperm c h r o m a t i n c o n d e n s a t i o n has y e t been found. C y t o c h e m i s t r y of G. a c u l e a t u s t e s t e s ( T a b l e 1) showed t h a t 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 the c h r o m a t i n f e l l i n t o B l o c h ' s (1969, 1976) i n t e r m e d i a t e p r o t e i n c a t e g o r y . As they a re e x t r a c t e d w i t h AFG/TCA a t 80-85*C, they a r e i n t e r m e d i a t e type 3B ( K a s i n s k y et a l . , 1984a). T h i s c o n c l u s i o n i s support e d by the s i m i l a r m o b i l i t y of TSP's from X. l a e v i s and from the b r e e d i n g s t a g e of G. a c u l e a t u s on a c i d / u r e a p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s ( F i g . 8 ) . Amino a c i d a n a l y s i s shows a h i g h mole p e r c e n t of a r g i n i n e , a l o n g w i t h the presence of h i s t i d i n e and l y s i n e ( T a ble 7 ) . T h i s c o n f i r m s an i n t e r m e d i a t e - t y p e of sperm 1 18 h i s t o n e . What i s the v a r i a b i l i t y of t h i s i n t e r m e d i a t e sperm h i s t o n e i n G. a c u l e a t u s and r e l a t e d s p e c i e s of G a s t e r o s t e i f o r m e s ? D i v e r s i t y of the TSP p r o f i l e on PAGE i n G. a c u l e a t u s , G. w h e a t l a n d i , P. p u n g i t i u s and A. f l a v i d u s was observed ( F i g . 9 ) . The TSP's of G. w h e a t l a n d i , P. p u n g i t i u s and A. f l a v i d u s were a l s o c o n f i r m e d t o be the i n t e r m e d i a t e - t y p e sperm p r o t e i n s by amino a c i d a n a l y s i s (Table 3 ) . D i f f e r e n c e s i n the amino a c i d c o m p o s i t i o n of the TSP's was c l e a r l y seen between t h e s e s p e c i e s . A c o r r e l a t i o n was found between e l e c t r o p h o r e t i c m o b i l i t y , the b a s i c / a c i d i c amino a c i d r a t i o and the sum of a l l the b a s i c amino a c i d s f o r the p r i n c i p a l TSP's of each s p e c i e s . Amino a c i d d a t a showed g r e a t e r p o s i t i v e charge i n the G. w h e a t l a n d i and P. p u n g i t i u s TSP's than i n the TSP of G. a c u l e a t u s , which e x p l a i n e d the f a s t e r e l e c t r o p h o r e t i c m i g r a t i o n of the former over the l a t t e r ( F i g . 9 and T a b l e 3 ) . I co n c l u d e t h a t the TSP's can be re g a r d e d as m o l e c u l a r markers f o r thes e s p e c i e s of G a s t e r o s t e i f o r m e s , as was the case f o r the i n d i v i d u a l s p e c i e s of the f r o g genus Xenopus (Mann et. a l . , 1982). The d i v e r s i t y of i n t e r m e d i a t e type sperm h i s t o n e s and t h e i r ^ s p e c i f i c i t y i n s p e c i e s of the genus Xenopus and s u b s p e c i e s of Xenopus l a e v i s has been s t u d i e d by Mann e t a l . (1982). The i n t e r m e d i a t e - t y p e sperm p r o t e i n s were d e t e r m i n e d t o be r e l i a b l e m o l e c u l a r markers f o r thes e a n i m a l s down t o the s u b s p e c i e s l e v e l . However, o n l y about 6 a n i m a l s o f — e a c h s u b s p e c i e s were 119 l o o k e d a t i n these s t u d i e s . Another e l e c t r o p h o r e t i c s t u d y by d e P e t r o c e l l i s et a l . (1980) on 3 s p e c i e s of sea u r c h i n d i s c o v e r e d d i s t i n c t i v e v a r i a n t s i n sperm h i s t o n e H2B s p e c i f i c enough t o d e f i n e each s p e c i e s . T h i s s t u d y l o o k e d a t type 4 s o m a t i c - l i k e sperm h i s t o n e s r a t h e r than type 3 i n t e r m e d i a t e sperm p r o t e i n s . The p r e s e n t study of G. a c u l e a t u s TSP's has compared the i n t e r m e d i a t e type 3 TSP's of 124 i n d i v i d u a l s and r e p r e s e n t s the f i r s t d e t a i l e d study of p o s s i b l e v a r i a t i o n s of TSP's w i t h i n d i f f e r i n g p o p u l a t i o n s of a s i n g l e s p e c i e s . F i s c h b e r g and c o l l e a g u e s (Vonwyl and F i s c h b e r g , 1980; B u r k i e t al., 1984) have a l s o found d i f f e r e n c e s i n the e l e c t r o p h o r e t i c m o b i l i t i e s of hemoglobin and LDH isozymes i n the genus Xenopus t h a t are s p e c i e s - s p e c i f i c . Thus, the s p e c i e s - s p e c i f i c TSP e l e c t r o p h o r e t i c p r o f i l e s might be c h a r a c t e r i s t i c of a l a r g e r number of p r o t e i n s t h a t can a c t as m o l e c u l a r markers f o r d i s t i n c t s p e c i e s i n f r o g s and f i s h . D i f f e r e n t c e l l t y p e s were seen i n the d e v e l o p i n g t e s t e s of G. a c u l e a t u s from J e r i c h o Pond. T h i s might account f o r d i f f e r e n c e s i n the b a s i c p r o t e i n e l e c t r o p h o r e t i c p r o f i l e o b s e r v e d . I f t h e r e i s a d i f f e r e n c e , how would i t a f f e c t the i n t r a - and i n t e r - p o p u l a t i o n a l comparisons of TSP's? L o o k i n g a t a d e v e l o p m e n t a l p r o f i l e of the J e r i c h o Pond p o p u l a t i o n a d e q u a t e l y answered th e s e q u e s t i o n s . The e l e c t r o p h o r e t i c p r o f i l e of TSP's of G. a c u l e a t u s from 120 J e r i c h o Pond, Vancouver, B.C. r e v e a l e d both somatic "and TSP bands ( F i g . 10). Somatic bands c o n f i r m the presence of c e l l s t h a t have not y e t undergone m e i o s i s and subsequent replacement of the n u c l e o s o m a l p r o t e i n s w i t h TSP's. The presence of many TSP bands seem t o show s p e c i f i c b a s i c p r o t e i n s a r e a s s o c i a t e d w i t h the d i f f e r e n t g e r m - c e l l t y p e s . The g r e a t e s t number of both s o m a t i c h i s t o n e s and TSP bands a r e seen i n the e a r l y p o s t -b r e e d i n g p e r i o d (October-November) when most germ c e l l s a re u n d e r g o i n g s p e r m a t o g e n e s i s . Both the number of b a s i c p r o t e i n s and the p r o c e s s of s p e r m a t o g e n e s i s d e c l i n e u n t i l the b r e e d i n g season ( A p r i l - M a y ) . At t h i s time the slow TSP bands c o m p l e t e l y d i s a p p e a r and the somatic h i s t o n e bands decrease about 90%, as seen m a i n l y by s e n s i t i v e r e s t a i n i n g of g e l s ( F i g . 10, l a n e s 11' and 12'). The f a s t TSP bands remain p r e s e n t . They are t y p i c a l of the mature spermatozoa. E x p e r i m e n t s w i t h a l k a l i n e phosphatase i n d i c a t e t h a t the m u l t i p l e banded TSP p r o f i l e s i n mature f i s h a r e not due t o p h o s p h o r y l a t i o n of s e r i n e r e s i d u e s y i e l d i n g p r o t e i n s of d i f f e r e n t c h a r g e . However, t h i s does not r u l e out the p o s s i b i l i t y of p h o s p h o r y l a t i o n of TSP's d u r i n g development, as seen by Dixon (1974) i n t r o u t t e s t i s . Tsanev (1980) found t h a t t r o u t t e s t i s has a few p e r c e n t of s o m a t i c h i s t o n e s p r e s e n t i n the mature sperm. What has been d e t e r m i n e d by these d a t a i s t h a t the c h a n g i n g e l e c t r o p h o r e t i c p r o f i l e of b a s i c p r o t e i n s d u r i n g development of the t e s t i s does not i n v a l i d a t e i n t e r - or i n t r a - s p e c i f i c comparisons i f TSP's a r e taken from males i n b r e e d i n g c o n d i t i o n . Knowing something about the d e v e l o p m e n t a l p r o f i l e of TSP's and the v a r i a b i l i t y of TSP's from r e l a t e d 121 s p e c i e s , i t i s now p o s s i b l e t o ta k e a c l o s e r l o o k a t the complex s p e c i e s s i t u a t i o n of G. a c u l e a t u s TSP's by f i r s t l o o k i n g at the d i f f e r e n c e s between f r e s h w a t e r and anadromous forms. Bony l a t e r a l p l a t e s and h a b i t a t h e l p t o d i s t i n g u i s h l e i u r u s from t r a c h u r u s . The p l a t e s a r e not f u l l y d e v e l o p e d u n t i l the j u v e n i l e reaches about 32 mm. (Hagen and Moodie, 1982). T r a c h u r u s i s a marine f i s h but u s u a l l y m i g r a t e s i n t o e s t u a r i e s or f r e s h w a t e r t o breed i n s p r i n g (Hagen and Moodie, 1982). In autumn the a d u l t and young r e t u r n t o the marine environment. I t i s monomorphic f o r a complete s e r i e s of bony p l a t e s ( F i g . 24A) a l o n g the s i d e s of the body and i s a b r i l l i a n t s i l v e r c o l o r . T r a c h u r u s i s l a r g e r i n s i z e than l e i u r u s (Hagen, 1 9 6 7 ) ( F i g . 2A and 2B). L e i u r u s permanently i n h a b i t s f r e s h w a t e r . I t has a m o t t l e d c o l o r w i t h d i s t i n c t b a r s . In a d d i t i o n , i t has a v a r i a b l e p a t t e r n of p l a t e s , r a n g i n g from low p l a t i n g , which i n c l u d e s o n l y a n t e r i o r p l a t e s and c a u d a l k e e l p l a t e ( F i g . 24B), t o h i g h p l a t e morphs h a v i n g a c o n t i n u o u s s e r i e s of l a t e r a l p l a t e s ( F i g . 24A) (Hagen and Moodie, 1982; A v i s e , 1967). P a r t i a l p l a t e d morphs a l s o e x i s t w i t h the low and h i g h p l a t e d morphs ( F i g . 24C). The low p l a t e d l e i u r u s may c o - e x i s t i n c o a s t a l streams w i t h t r a c h u r u s (Hagen, 1967). What does the TSP p r o f i l e of t h e s e d i f f e r e n t m o r p h o l o g i c a l forms of G. a c u l e a t u s l o o k l i k e ? We have a l r e a d y seen t h a t the TSP bands from PAGE of d i f f e r e n t s p e c i e s of f i s h examined i n t h i s s tudy have been d e t e r m i n e d t o be r e l i a b l e m o l e c u l a r markers. Are they a l s o 1 22 F i g u r e 24. P l a t e morphs of G. a c u l e a t u s ( p l a t e s a r e shown w i t h s t i p p l i n g ) (adapted from Hagen and G i l b e r t s o n , 1973). c Completely plated morpfc Low plated morpti c Partially plated morph 124 m o l e c u l a r markers f o r d i f f e r e n t p o p u l a t i o n s w i t h i n a s i n g l e s p e c i e s ? TSP bands of the 2 forms of G. a c u l e a t u s were d i s c r e t e enough t o d i s t i n g u i s h the forms. T r a c h u r u s had a c h a r a c t e r i s t i c 'comet-shaped' TSP band whereas the l e i u r u s form had ' b l o c k -shaped' bands ( F i g . 13). T h i s i s not t o say t h a t the d i f f e r e n t f r e s h w a t e r p o p u l a t i o n s were o v e r a l l as homogeneous as the anadromous p o p u l a t i o n s , but r a t h e r t h a t a t t h i s l e v e l of a n a l y s i s , d i f f e r e n c e s c o u l d be seen between the two G. a c u l e a t u s t y p e s . Another d i f f e r e n c e was found i n the l y s i n e / a r g i n i n e r a t i o from the amino a c i d d a t a . A s i g n i f i c a n t l y h i g h e r amount of a r g i n i n e over l y s i n e was found i n the TSP's from anadromous p o p u l a t i o n s than was found i n the f r e s h w a t e r p o p u l a t i o n s . T h i s may account f o r the d i f f e r e n t band appearance between the two t y p e s i n PAGE. The t r a c h u r u s TSP e l e c t r o p h o r e t i c p r o f i l e more o f t e n had 2-3 bands whereas l e i u r u s had o n l y one (Table 4 ) . T h i s c o i n c i d e s w i t h B e l l ' s (1976) p o s t u l a t e s and W i t h l e r ' s (1980) work t h a t e l e c t r o p h o r e t i c g e n e t i c v a r i a t i o n i s g r e a t e r i n t r a c h u r u s than i n l e i u r u s due t o g r e a t e r age and l e s s v u l n e r a b i l i t y t o g e n e t i c v a r i a b i l i t y t h r ough founder e f f e c t s - a n d g e n e t i c d r i f t . Hagen (1967) found d i f f e r e n c e s i n muscle p r o t e i n s between t r a c h u r u s and l e i u r u s . D i a g n o s t i c , bands f o r each form were seen w i t h h y b r i d s h a v i n g b o t h bands of the p a r e n t s . A v i s e (1976) assayed 15 g e n e t i c l o c i encoding 9 enzymes and 4 g e n e r a l p r o t e i n s and c o n c l u d e d t h a t d e s p i t e the d i f f e r e n c e i n a l l e l i c 125 f r e q u e n c y a t p o l y m o r p h i c l o c i , t r a c h u r u s and l e i u r u s e x h i b i t e d a h i g h o v e r a l l b i o c h e m i c a l s i m i l a r i t y . A l t h o u g h v i s u a l i n s p e c t i o n of the t r a c h u r u s / l e i u r u s TSP bands i n d i c a t e d t h a t they were d i f f e r e n t , amino a c i d a n a l y s i s r e v e a l e d t h a t the d i f f e r e n c e i n r e l a t i v e c o m p o s i t i o n of the p r o t e i n s was n e g l i g i b l e (Table 7 ) . Between d i f f e r e n t f r e s h w a t e r p o p u l a t i o n s and between d i f f e r e n t anadromous p o p u l a t i o n s , the TSP p r o f i l e s were not c h a r a c t e r i s t i c f o r a s p e c i f i c p o p u l a t i o n , and t h e r e f o r e , were no l o n g e r r e l i a b l e as m o l e c u l a r markers. W i t h l e r (1980) found t h a t f r e s h w a t e r p o p u l a t i o n s were no more s i m i l a r t o each o t h e r than t h e y were t o the anadromous p o p u l a t i o n s r e g a r d i n g a n a l y s i s of isoz y m e s . B i o c h e m i c a l l y , the e l e c t r o p h o r e t i c a n a l y s i s found s i m i l a r l y m i g r a t i n g TSP bands and i n d i s t i n g u i s h a b l e amino a c i d c o m p o s i t i o n between d i f f e r e n t t r a c h u r u s and d i f f e r e n t l e i u r u s p o p u l a t i o n s . Cyanogen bromide c l e a v a g e of the TSP's from l e i u r u s ( J e r i c h o Pond) and t r a c h u r u s ( R a i l r o a d D i t c h ) gave 2 bands each t h a t m i g r a t e d s i m i l a r d i s t a n c e s ( F i g . 18). T h e r e f o r e , i f t h e r e i s a d i f f e r e n c e i n the p r i m a r y s t r u c t u r e of the p r o t e i n s i n the TSP bands, i t i s not d e t e c t a b l e on PAGE. These r e s u l t s a r e c o n s i s t e n t w i t h s t u d i e s on the v a r i a b i l i t y of o t h e r p r o t e i n s i n G. a c u l e a t u s . A c o m p a r a t i v e s t u d y of hemoglobins i n p o p u l a t i o n s of t r a c h u r u s and l e i u r u s forms of t h r e e - s p i n e s t i c k l e b a c k from s o u t h e r n B r i t i s h Columbia showed they were so s i m i l a r as t o c o n s i d e r both of t h e s e forms t o be p a r t of a s i n g l e s p e c i e s ( M i c h i e l , 1977). A l s o , 126 c h a r a c t e r i z a t i o n of LDH isozymes (Vonwyl and F i s c h b e r g , 1980) and hemoglobins ( B u r k i et a l . , 1984) i n d i f f e r e n t s u b s p e c i e s of Xenopus l a e v i s c o u l d not d i s t i n g u i s h the a n i m a l s of each s u b s p e c i e s , whereas sperm p r o t e i n p r o f i l e s on PAGE found d i f f e r e n c e s marked enough t o c o n s i s t e n t l y d e f i n e the s u b s p e c i e s . I t appears t h a t a n a l y s i s of TSP's by PAGE i s a r e l i a b l e way of d e t e c t i n g d i f f e r e n c e s i n the 2 forms of G. a c u l e a t u s , not d e t e c t a b l e by hemoglobin a n a l y s i s , and i s s i m p l e r t o a c c o m p l i s h than isozyme e x p e r i m e n t s . These f i n d i n g s of i n t e r m e d i a t e p r o t e i n t y p e as w e l l as d i f f e r e n c e s i n the anadromous and f r e s h w a t e r t y p e s of G. a c u l e a t u s c o n t r a d i c t the h y p o t h e s i s of Nandi e t a l . (1979) t h a t marine and e s t u a r i n e f i s h have . p r o t a m i n e s i n sperm w h i l e f r e s h w a t e r f i s h c o n t a i n s o m a t i c - l i k e sperm h i s t o n e s . These a u t h o r s proposed t h a t h i s t o n e - t y p e . f r e s h w a t e r f i s h e s o r i g i n a t e d f i r s t and the protamine type e v o l v e d l a t e r as an a d a p t a t i o n t o s a l i n e h a b i t a t s . However, b o t h the anadromous and f r e s h w a t e r G. a c u l e a t u s i n t h i s s tudy breed i n f r e s h w a t e r and n e i t h e r had s o m a t i c - l i k e h i s t o n e s but i n t e r m e d i a t e type sperm p r o t e i n s . A l s o , A. f l a v i d u s spends i t s e n t i r e l i f e i n t h e marine h a b i t a t and i n s t e a d of h a v i n g p r o t a m i n e s i n i t s TSP's as Nandi et a l . would have p r e d i c t e d , i t too has i n t e r m e d i a t e t y p e TSP's (Table 3 ) . T h i s i n f o r m a t i o n f u r t h e r d i s p r o v e s the above h y p o t h e s i s . A model of t h r e e - s p i n e s t i c k l e b a c k d i s p e r s a l i n southwest Canada and northwest U n i t e d S t a t e s has been proposed t o e x p l a i n 1 27 the r e l a t i o n s h i p of the d i f f e r e n t forms. F r e s h w a t e r p o p u l a t i o n s seem t o have o r i g i n a t e d as a r e s u l t of p o s t g l a c i a l p h y l o g e n e t i c e v o l u t i o n from t r a c h u r u s and have e x i s t e d s i n c e the L a t e Miocene ( B e l l , 1976). F o s s i l e v i d e n c e r e v e a l s t h a t G a s t e r o s t e u s  a c u l e a t u s has been on the P a c i f i c Coast f o r 10 m i l l i o n y e a r s ( B e l l , 1976; Moyle and Cech, 1982). G e o l o g i c e v e n t s such as g l a c i a l movements th r o u g h t h i s a r e a have p r o v i d e d the o p p o r t u n i t y f o r r e p e a t e d i n v a s i o n s i n t o l a k e s and c o a s t a l streams, perhaps a l l o w i n g i s o l a t i o n and d i v e r g e n t e v o l u t i o n of s m a l l p o p u l a t i o n s t o occur ( B e l l , 1976). The l a s t major g l a c i a l r e t r e a t , about 13,000 y e a r s ago, a l l o w e d l a n d l e v e l s t o r i s e as much as 200 meters (Mathews et a l . , 1970), e x p o s i n g l a r g e l a k e s and low l y i n g streams i n the F r a s e r V a l l e y and on Vancouver I s l a n d . P o p u l a t i o n s of t r a c h u r u s p r o b a b l y were t r a p p e d i n these water b o d i e s . To f u r t h e r c o m p l i c a t e m a t t e r s , about 11,000 y e a r s ago a l e s s severe l a n d submergence o c c u r r e d , a l l o w i n g a secondary marine i n v a s i o n of low l y i n g l a k e s . I t i s assumed t h a t t h e s e p o p u l a t i o n s of f i s h have had about 9000 y e a r s of s t a b l e g e o l o g i c c o n d i t i o n s i n i s o l a t i o n i n which t o d i f f e r e n t i a t e " . Gene f l o w between i s o l a t e d waters o c c u r s o n l y a l o n g r i v e r s and by f l o o d i n g of the low l y i n g a r e a s ( W i t h l e r , 1980). The above model may account f o r the f a c t t h a t some l a k e s i n the g l a c i a t e d r e g i o n s of southwest Canada and nor t h w e s t U n i t e d S t a t e s have s y m p a t r i c v a r i a t i o n s of the f r e s h w a t e r s t i c k l e b a c k . These i n c l u d e a s m a l l e r , p l a n k t o n - e a t i n g l i m n e t i c form and a l a r g e r , bottom f o r a g i n g b e n t h i c type ( M c P h a i l , 1984a). Paxton 1 28 Lake on Texada I s l a n d , B.C. and Enos Lakes / /on Vancouver I s l a n d , B.C. each c o n t a i n 2 e c o l o g i c a l l y and m o r p h o l o g i c a l l y d i s t i n c t t h r e e - s p i n e s t i c k l e b a c k p o p u l a t i o n s . In Enos Lake, the c o m b i n a t i o n of e c o l o g i c a l , m o r p h o l o g i c a l and b e h a v i o r a l d i f f e r e n c e s between the 2 s t i c k l e b a c k forms produces almost t o t a l r e p r o d u c t i v e i s o l a t i o n , thus f o r m i n g a b i o l o g i c a l b a r r i e r . T h e r e f o r e , two b i o l o g i c a l s p e c i e s have been determined t o e x i s t s y m p a t r i c a l l y due t o s e p a r a t e genomes, h i g h l y d i f f e r e n t t r o p h i c a d a p t a t i o n s and d i s s i m i l a r morphology. A l t h o u g h the b e n t h i c form resembles o t h e r s m a l l - l a k e s t i c k l e b a c k s of Vancouver I s l a n d , i t i s not c l e a r i f e i t h e r form i s G a s t e r o s t e u s a c u l e a t u s ( M c P h a i l , 1984a) . I t appears t h a t a r e l a t i o n s h i p e x i s t s between .the presence of somatic h i s t o n e s and TSP bands w i t h the b r e e d i n g development of G. a c u l e a t u s i n Enos Lake. Somatic h i s t o n e bands were i n d i c a t i v e of e a r l y s t a g e s of s p e r m a t o g e n e s i s . D i f f e r e n c e s i n the number and the r e l a t i v e m i g r a t i o n of TSP bands were seen between the b e n t h i c and l i m n e t i c t y p e s i n Enos Lake. In a d d i t i o n , the b e n t h i c a n i m a l s of b oth Enos and Paxton Lakes had a r e l a t i v e l y g r e a t e r number of somatic h i s t o n e bands than the l i m n e t i c s . T h i s was demonstrated most c l e a r l y w i t h s e n s i t i v e r e s t a i n i n g of p o l y a c r y l a m i d e g e l s ( F i g s . 14B and 16). More TSP bands a r e seen when somatic h i s t o n e bands a r e p r e s e n t . Y e t , each sample of the b e n t h i c / l i m n e t i c a n i m a l p a i r s of these l a k e s was c o l l e c t e d on the same date and w e l l i n t o t h e i r d e s c r i b e d b r e e d i n g p e r i o d s . The m i g r a t i o n of the TSP's o f • t h e l i m n e t i c and 1.29 b e n t h i c forms were s i m i l a r enough t o c o n s i d e r t h e s e a n i m a l s v e r y c l o s e l y r e l a t e d . A s i g n i f i c a n t l y g r e a t e r number of m u l t i p l e bands were observed i n the TSP p r o f i l e s of b e n t h i c a n i m a l s from Enos Lake than i n b e n t h i c s from Paxton Lake s u g g e s t i n g a d i f f e r e n c e between the b e n t h i c t y p e s i n the s e two l a k e s ( T a b l e 4 ) . At t h i s p o i n t , l e t us r e i t e r a t e the major f i n d i n g s r e g a r d i n g the v a r i a b i l i t y of TSP's i n the t h r e e - s p i n e s t i c k l e b a c k s : 1) TSP's a r e r e l i a b l e m o l e c u l a r markers between r e l a t e d s p e c i e s i n the o r d e r G a s t e r o s t e i f o r m e s . 2) G a s t e r o s t e u s a c u l e a t u s TSP's may s e r v e as m o l e c u l a r markers i n i d e n t i f y i n g anadromous ( t r a c h u r u s ) and f r e s h w a t e r ( l e i u r u s ) p o p u l a t i o n s . 3) TSP's of G. a c u l e a t u s cannot be used t o i d e n t i f y d i f f e r e n t anadromous p o p u l a t i o n s nor can they be used t o i d e n t i f y d i f f e r e n t f r e s h w a t e r p o p u l a t i o n s . 4) The b e n t h i c and l i m n e t i c t y p e s of G. a c u l e a t u s i n Enos Lake may be d i s t i n g u i s h e d by t h e i r somatic h i s t o n e and TSP e l e c t r o p h o r e t i c p r o f i l e s . In l i g h t of these f i n d i n g s , i t i s i n t e r e s t i n g t o note t h a t two anomalous s i t u a t i o n s emerged i n the s e s t u d i e s . F i r s t , one G. a c u l e a t u s specimen e x h i b i t e d a band m i g r a t i n g s i m i l a r l y t o a G. w h e a t l a n d i band. I t may be p o s s i b l e t o e x p l a i n the TSP p r o f i l e i n F i g . 22 by p r o p o s i n g a h y b r i d between G. a c u l e a t u s and G. 1 30 w h e a t l a n d i . However, r e s t a i n i n g a l s o showed G. a c u l e a t u s , G. w h e a t l a n d i and P. p u n g i t i u s a l l had TSP's t y p i c a l of the o t h e r s p e c i e s i n amounts l e s s than ten f o l d the amount of the f a i n t e s t a m idoblack s t a i n e d band. Perhaps t h i s r e f l e c t s the e v o l u t i o n of the/TSP's from a common a n c e s t o r of t h i s o r d e r (Wootton, 1976). \ \ \ - S e c o n d l y , G. a c u l e a t u s from R a i l r o a d D i t c h , B.C. had a p a r t i c u l a r l y f a s t m i g r a t i n g band. Perhaps t h e r e a r e a d d i t i o n a l genes f o r TSP's p r e s e n t i n G. a c u l e a t u s which are u s u a l l y not e x p r e s s e d . F u r t h e r hypotheses a w a i t s t u d i e s a t the DNA l e v e l f o r i s o l a t i o n and c h a r a c t e r i z a t i o n of the TSP genes. F i n a l l y , i n c u b a t i o n s t u d i e s suggested the p o s s i b i l i t y of an endogenous p r o t e a s e i n the c h r o m a t i n of G. a c u l e a t u s t h a t degrades the TSP's a t n e a r l y n e u t r a l pH a t 37*C ( F i g . 19). What might be the o r i g i n of t h i s p r o t e a s e ? Sperm of r o o s t e r c o n t a i n a t r y p s i n - l i k e enzyme, e i t h e r i n the acrosome or a s s o c i a t e d w i t h c h r o m a t i n (Howarth and Digby, 1973; Nakano e_t a l . , 1976). T h i s enzyme i s known t o be a c i d s t a b l e , e x t r a c t a b l e from sperm j w i t h d i l u t e a c i d and o p t i m a l l y a c t i v e a t n e u t r a l pH, a l l c h a r a c t e r i s t i c s of the endogenous p r o t e a s e of G. a c u l e a t u s . A method t h a t i s known t o remove the acrosome and i n a c t i v a t e the enzyme (Nakano e_t a l . , 1 976; M e z q u i t a and Teng, 1977), was not s u f f i c i e n t t o p r e v e n t d e g r a d a t i o n of g a l l i n e and o t h e r a v i a n sperm h i s t o n e s . I t i s t h e r e f o r e l i k e l y t o be c h r o m a t i n bound. 131 S e r t o l i c e l l s a r e known t o p h a g o c y t i z e the s p e r m a t i d r e s i d u a l body i n t e l e o s t s ( G r i e r et a_l. , 1980). Because t e s t i c u l a r homogenates were used i n t h i s s tudy t o i s o l a t e TSP, and S e r t o l i c e l l s are known t o be p r e s e n t ( F i g . 3B), i t i s p o s s i b l e t h a t o t h e r c e l l s c o n t a i n i n g p r o t e o l y t i c agents may be a b l e t o degrade the TSP's under proper c o n d i t i o n s . Thus the o r i g i n of the endogenous p r o t e a s e of G a s t e r o s t e i f o r m e s remains u n c e r t a i n . P o s s i b l e r e s e a r c h t o answer the many q u e s t i o n s uncovered by t h i s i n i t i a l l o o k a t TSP's i n the G a s t e r o s t e i f o r m e s can be summarized as f o l l o w s : 1. M o l e c u l a r l e v e l : I s o l a t e TSP genes t o study t h e i r m u l t i p l i c i t y and o r g a n i z a t i o n on the genome. 2. F u n c t i o n a l l e v e l : I n t e r a c t i o n of DNA w i t h the i n t e r m e d i a t e sperm h i s t o n e s can be examined by b i o p h y s i c a l methods such as x-ray d i f f r a c t i o n t o r e v e a l p o s s i b l e n u c l e o s o m a l - l i k e p a c k i n g of c h r o m a t i n , as was h i n t e d a t by the morphology of the c o n d e n s i n g sperm n u c l e u s ( F i g . 4B'). I t i s s t i l l u n c l e a r as t o whether the p r o t e i n s pack i n the major or minor groove of the DNA or i f the DNA wraps around the p r o t e i n s . 3. C e l l u l a r l e v e l : P u r i f y c e l l t y p e s t o c h a r a c t e r i z e b a s i c p r o t e i n s more a c c u r a t e l y and s p e c i f i c a l l y t o the c e l l t y p e of i t s o r i g i n . 4. Organism l e v e l : H y b r i d e x periments between the d i f f e r e n t s p e c i e s and subtypes of G. a c u l e a t u s would see i f d i a g n o s t i c 132 bands of both t y p e s a r e produced. 5. 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J e r i c h o Pond, Vancouver, B.C. October 1983 - June 1984, 49° 1 2 3 6 6. M a r p o l e Pond, Vancouver, B.C. 8 May 1983. 49° 123° 7. P r i e s t L a k e , Texada I s l a n d , B.C. 5 J u l y 1979. 49° 44' 124° 34' Anadromous P o p u l a t i o n s : 8. R a i l r o a d D i t c h , e s t u a r y , F o r t L a n g l e y , B.C. 5 May 1983. 49° 10' 122° 36' 9. L i t t l e Campbell R i v e r , e s t u a r y , W h i t e r o c k , B.C. 6 June 1983. 49° 01' 122° 46' 10. S t a n l e y P a r k , ocean, Vancouver, B.C. 15 May 1984. 49° 18' 123° 08' 11. R i v i e r e des Vase, t i d e p o o l , Quebec. 14 and 28 June 1983, 48° 00' 69° 26' See Appendix IB f o r g e o g r a p h i c l o c a t i o n s o f p o p u l a t i o n s . 142 Appendix IB. Geographic l o c a t i o n s o f G. a c u l e a t u s p o p u l a t i o n s sampled i n Canada. Appendix II. PAGE conditions for different gel sizes. length (cm.) D.C. volts mA hours/run 25 150 6 26 34 200 300 5 9 45 24 45 350 7 24 61 400 7-8 47-97 87 850 7 70 pH = 2.4-2.6 * a l l gels were Q.20 cm in width 

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