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The control of cyclical changes in the testicular activity of the lake chub Couesius plumbeus (Agassiz) Ahsan, Syed Nazar 1964

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THE CONTROL OF CYCLICAL CHANGES I N THE TESTICULAR ACTIVITY.OF THE LAKE CHUB COUESIUS PLUMBEUS (AGASSIZ) by SYED NAZAR AHSAN B.Sc. (Hons.) P a t n a U n i v e r s i t y , 1953. M.Sc. P a t n a U n i v e r s i t y , 1955 A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS OF'., THE: DEGREE OF D o c t o r o f P h i l o s o p h y i n t h e D e p a r t m e n t o f Z o o l o g y 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 t h e r e q u i r e d s t a n d a r d The U n i v e r s i t y o f B r i t i s h . C o l u m b i a J u n e , 1964. In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r pub 1 f - . c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f . The U n i v e r s i t y o f B r i t i s h C o l u m b i a Vancouver 8 # 1 Canada ' * ' • Date ABSTRACT The a n n u a l t e s t i c u l a r c y c l e o f a t e l e o s t t h e l a k e chub ( C o u e s i u s p l u m b e u s ) f r o m a n o r t h t e m p e r a t e l a t i t u d e (51°N) hae be e n s t u d i e d by h i s t o l o g i c a l and h i s t o c h e m i c a l methods w i t h a p l a n i m e t r i c e v a l u a t i o n o f t h e d i f f e r e n t s p e r m a t o g e n e t i c s t a g e s * The a n n u a l c y c l e i s d i v i d e d i n t o f i v e d i f f e r e n t s t a g e s and t h e c y c l i c a l t e s t i c u l a r c h a n g e s have been c o r r e l a t e d w i t h c h a n g i n g e n v i r o n m e n t a l c o n d i t i o n s . L o b u l e b o u n d a r y c e l l s , c o n s i d e r e d t o be homologue o f t h e L e y d i g c e l l s o f h i g h e r v e r t e b r a t e s , have b e e n i d e n t i f i e d , and t h e c h a n g e s i n t h e i r s e c r e t o r y a c t i v i t y h a ve b e e n r e p o r t e d . T e m p e r a t u r e i s t h e m a j o r e n v i r o n m e n t a l f a c t o r c o n t r o l l i n g t h e t e s t i c u l a r c y c l e . The p i t u i t a r y g l a n d , t h r o u g h i t s g o n a d o t r o p i n ( s ) , m e d i a t e s b e t w e e n e n v i r o n m e n t a l c h a n g e s and d e v e l o p m e n t s i n t h e t e s t e s . H i g h e r t e m p e r a t u r e s (16°-22°C) •.>;,-• p r o m o t e s p e r m i o g e n e s i s and s p e r m i a t i o n , w h e r e a s l o w t e m p e r a t u r e s (5°-12°C)a,re more c o n d u c i v e t o g o n i a l p r o l i f e r a t i o n and t h e i n i t i a l p h a s e o f s p e r m a t o g e n e s i s . H y p o p h y s e c t o m y a f f e c t s t h e m i t o t i c a b i l i t y o f t h e s p e r m a t o g o n i a , c o m p l e t e l y b l o c k s t h e i r t r a n s f o r m a t i o n i n t o s p e r m a t o c y t e s and s u p p r e s s e s t h e s e c r e t o r y a c t i v i t y o f t h e l o b u l e b o u n d a r y c e l l s . R e p l a c e m e n t t h e r a p y w i t h f i s h g o n a d o t r o p i n and mammalian LH r e s t o r e s s p e r m a t o g e n e s i s t o a l a r g e e x t e n t and maximum r e s p o n s e i s e l i c i t e d w i t h w h o l e f i s h p i t u i t a r y e x t r a c t . I t i s p r o p o s e d t h a t t h e f i s h p i t u i t a r y g o n a d o t r o p i n i s s i m i l a r t o mammalian LH and i n t h i s s p e c i e s mammalian FSH i s ( i i i ) p h y s i o l o g i c a l l y i n a c t i v e i n t h e r e s t o r a t i o n o f t e s t i c u l a r a c t i v i t y . S i n c e w h o l e f i s h p i t u i t a r y p r o d u c e s a maximum r e s p o n s e i t i s s u g g e s t e d t h a t f a c t o r s s u c h as TSH and STH have a p r o b a b l e s y n e r g i s t i c r o l e i n t h e t e s t i c u l a r m a t u r a t i o n i n C o u e s i u s p l u m b e u s . E v i d e n c e i s s p r e s e n t e d t h a t a weak e n d o g e n o u s r h y t h m o f a c t i v i t y i s p a r t l y r e s p o n s i b l e f o r t h e t i m i n g o f v a r i o u s t e s t i c u l a r c h a n g e s . TABLE OF CONTENTS PAGE INTRODUCTION 1 MATERIALS AND METHODS 5 1. H y p o p h y s e c t o m y . 5 2. P r o c e d u r e o f h y p o p h y s e c t o m y 6 3. I n j e c t i o n s 10 4. P r e p a r a t i o n o f s a l m o n p i t u i t a r y e x t r a c t s , . . 14 5. P h o t o p e r i o d e x p e r i m e n t . ... . . . .• . . . . . 15 6. A u t o p s y 17 7. E v a l u a t i o n o f t e s t i c u l a r m a t u r a t i o n . . . . . 18 8. M e t e o r o l o g i c a l d a t a 18 9. G o n o s o m a t i c i n d e x 19 RESULTS I . ANNUAL TESTICULAR CYCLE 20 1., G e n e r a l s t r u c t u r e o f t h e t e s t i s 20 2. S e a s o n a l c h a n g e s i n t h e t e s t i s . . . . . . . 26 3. H i s t p l p g i c a l f e a t u r e s p f p is':- spawned . . . . t e s t i s . . . . . . . . . 32 4. R e l a t i o n o f t h e a n n u a l c y c l e t o e n v i r o n m e n t a l c h a n g e s . . . . . . . 33 5. L o b u l e b o u n d a r y c e l l s 35 6. C h o l e s t e r o l t e s t 35 I I . EFFECT OF HYPOPHYSECTOMY 35 1. H i s t o l o g i c a l c h a n g e s . ... 37 2. C h p l e s . t e r b l i and: l i p i d 39 ( v ) TABLE OF CONTENTS ( c o n t i n u e d ) PAGE I I I . REPLACEMENT THERAPY 40 1. E f f e c t o f mammalian f o l l i c l e - s t i m u l a t i n g hormone (FSH) . 40 2. E f f e c t o f mammalian l u t e i n i z i n g hormone (LH) ' 43 3. E f f e c t o f f r a c t i o n a t e d s a l m o n p i t u i t a r y g o n a d o t r o p i n 49 4. E f f e c t o f c r u d e s a l m o n p i t u i t a r y e x t r a c t . , 5 4 5. E f f e c t o f e x p e r i m e n t a l c o n d i t i o n s on sham o p e r a t e d u n t r e a t e d c o n t r o l s 59 6. G e n e r a l summary o f t h e r e s u l t s o f r e p l a c e -ment t h e r a p y . 60 I V . ENVIRONMENTAL CONTROL OF TESTICULAR CYCLE: . . . . 63 PHOTOPERIOD TEMPERATURE EXPERIMENTS 63 1. E x p l o r a t o r y e x p e r i m e n t . . . . 63 2. P h o t o p e r i o d - t e m p e r a t u r e e x p e r i m e n t w i t h p r e s p a w n i n g s t o c k 66 3. P h o t o p e r i o d - t e m p e r a t u r e e x p e r i m e n t s w i t h p o s t s p a w n i n g s t o c k . 77 4. P h o t o p e r i o d - t e m p e r a t u r e e x p e r i m e n t w i t h t h e ' p r e ^ f r e e z e up' s t o c k 87 5. G e n e r a l summary o f p h o t o p e r i o d - t e m p e r a t u r e e x p e r i m e n t s 93 DISCUSSION . 98 1. S e a s o n a l t e s t i c u l a r c y c l e 98 2. S e a s o n a l f a c t o r s a f f e c t i n g t e s t i c u l a r c y c l e . 99 3. R o l e o f t e m p e r a t u r e and l i g h t . 100 4. End o g e n o u s r h y t h m 103 5. M e c h a n i s m o f c o n t r o l o f t e s t i c u l a r c y c l e . . 104 6. R o l e o f g o n a d o t r o p i n ( s ) i n t h e c o n t r o l o f t h e t e s t i c u l a r c y c l e . 107 ( v i ) TABLE OF CONTENTS ( c o n t i n u e d ) PAGE SUMMARY AND CONCLUSIONS 113 LITERATURE C I T E D . . 115 APPENDIX 122 LISt OF FIGURES FIGURE 1. Camera lucida diagram of the topography of the Pituitary Gland (P) of Couesius plumbeus. Only the mucus membrane covering the roof of the buccal cavity is removed. 1. Internal carotid ar,tery; 2. Branch from the internal; carotid artery; 3. Circulus cephalicus; 4. Maxillary nerve; 5. Optic nerve; 6. First g i l l cleft - c;:i >•;• ( ] 7. Parasphenoid. (X '50) . ." . . . . . . . . . . . 2A. A general view of the set-up for hypophysectomy . . B. Some of the instruments used in the operation . , . 3A. Sagittal section of head through the pituitary gland and adjacent brain tissue of C.plumbeus (X 750) B. Sagittal section of the pituitary region of the brain after hypophysectomy (X700) . . . , 4. A general view of the aquaria used to keep experimental fish and controls . . . . 5. Seasonal variation in the GSI of Couesius plumbeus as related to changes in water temperature and daylength. »• » =GSI of the 1962-63 cycle; 0 0-GSI "of the 1963 cycle (May to November) A—--A= Water temperature. The curved line represents the seasonal changes in daylength at 51% 6. Seasonal changes in the spermatogenetic composition of normal Couesius . 0- 0 = spermatocytes;• § = (sperms j = t . * 7 f Tgpermat©gp]iia:;X»&-XorCpycn9!tio L nests. 7. Cyclical changes in the activity of the testes in normal fish. 0 0 = Dividing stages; f 1 = Resting stages (sperms excluded) . . 8. Comparison of the GSI of hypophysectomised fish with the sham operated controls, and normals (at the time of the completion of each experiment) . . 9. Compar|son of the GSI of FSH-, arid LH- treated fish with untreated hypophysectomjsed and sham operated controls. •—-—• = untreated hypophy-sectomised controls; 0 0 = FSH-treated fish; • •= LH-treated fish; 0 0 = sham operated controls ( v i i i ) FIGURE PAGE 10. C y c l i c a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f u n t r e a t e d h y p o p h y s e c t o m i s e d f i s h . ^ £ = P r i m a r y germ c e l l s ; \~~~\-P r i m a r y s p e r m a t o g o n i a ; r ~ ~ | = R e s t i n g s e c o n d a r y s p e r m a t o g o n i a ; • = D i v i d i n g s e c o n d a r y s p e r m a t o g o n i a ( r a r e ) . . . . . 44 11. S e a s o n a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f FSH t r e a t e d f i s h . (See F i g . 1 0 f o r e x p l a n a t i o n s ) 4 5 12. S e a s o n a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f mammalian L H - t r e a t e d f i s h . The r i g h t b a r i n June 1963 and s u b s e q u e n t e x p e r i m e n t s i n d i c a t e c o n d i t i o n a t h i g h t e m p e r a t u r e ; t h e l e f t t h a t f o u n d a t l o w t e m p e r a t u r e . W//M - S e c o n d a r y s p e r m a t o g o n i a ( d i v i d i n g ) = P r i m a r y s p e r m a t o c y t e s ( r e s t i n g and d i v i d i n g ) ; o t h e r e x p l a n a t i o n s as i n F i g u r e 10 4 7 13. S e a s o n a l c h a n g e s i n t h e GSI o f f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n (FSG) t r e a t e d f i s h , as co m p a r e d t o t h o s e w i t h mammalian'LH. GSI o f u n t r e a t e d h y p o p h y s e c t o m i s e d and sham o p e r a t e d c o n t r o l s i n c l u d e d f o r c o m p a r i s o n . 52 14. S e a s o n a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n t r e a t e d f i s h . L e f t b a r ) i n May 1963, and s u b s e q u e n t e x p e r i m e n t s i n d i c a t e c o n d i t i o n a t l o w t e m p e r a t u r e , r i g h t d e n o t e s t h a t a t h i g h . E x p l a n a t i o n s same as i n F i g u r e 12 56 15. E f f e c t o f l o w and h i g h t e m p e r a t u r e s on t h e f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n and mammalian, L H - t r e a t e d f i s h i n May 1963 and s u b s e q u e n t e x p e r i m e n t s . ! • = Mammalian L H - t r e a t e d ( h i g h t e m p e r a t u r e ) , 0 0 = Mammalian L H - t r e a t e d ( l o w t e m p e r a t u r e ) ; t • = F r a c t i o n a t e d s a l m o n g o n a d o t r o p i n ( h i g h t e m p e r a t u r e ) and X X = F r a c t i o n a t e d s a l m o n g o n a d o t r o p i n ( l o w t e m p e r a t u r e ) . 57 16A. P h o t o m i c r o g r a p h o f a t e s t i s f r o m h y p o p h y s e c t o m i s e d f i s h s h o w i n g .abundant f i b r o b l a s t s s i m u l a t i n g f i b r o t i c d e g e n e r a t i o n . P r i m a r y germ c e l l s a r e most p r e d o m i n a n t . Some p r i m a r y s p e r m a t o g o n i a a r e a l s o s e e n (X 700) 58 B. P h o t o m i c r o g r a p h o f an FSH t r e a t e d f i s h t e s t i s s h o w i n g m o s t l y p r i m a r y germ c e l l s , and p r i m a r y s p e r m a t o g o n i a . A s m a l l n e s t o f o f s e c o n d a r y s p e r m a t o g o n i a on t h e l e f t c e n t r e c a n be s e e n (X 700) 58 ( i x ) FIGURE 17A. P h o t o m i c r o g r a p h o f an LH t r e a t e d t e s t i s s h o w i n g t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f a t y p i c a l LH t r e a t e d f i s h (X700) B. P h o t o m i c r o g r a p h o f a f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n t r e a t e d f i s h s h o w i n g s p e r m a t o g e n e t i c c o n d i t i o n o f a t y p i c a l t r e a t e d f i s h (X 7 0 0 ) . . . . 18. S e a s o n a l c h a n g e s i n t h e GSI o f c r u d e w h o l e s a l m o n p i t u i t a r y e x t r a c t (CSE) t r e a t e d f i s h as compared t o t h o s e t r e a t e d w i t h f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n ( F S G ) . GSI o f u n t r e a t e d h y p o p h y s e c t -o m i s e d (HPX ;) and sham o p e r a t e d c o n t r o l s (SHAM) are i n c l u d e d f o r c o m p a r i s o n 19. S e a s o n a l c h a n g e s i n t h e GSI o f f r a c t i o n a t e d s a l m o n g o n a d a t r o p i n (FSG) t r e a t e d and FSH t r e a t e d f i s h . GSI o f (.untreated h y p o p h y s e c t o m i s e d (HPX) and sham o p e r a t e d c o n t r o l s (SHAM) a r e i n c l u d e d f o r c o m p a r i s o n 20. The s p e r m a t o g e n e t i c c o m p o s i t i o n o f c r u d e w h o l e s a l m o n p i t u i t a r y e x t r a c t t r e a t e d f i s h . fflfflTTO = P y c n o t i c n e s t s ; = S e c o n d a r y s p e r m a t o c y t e s and s p e r m a t i d s ; l U i = D i v i d i n g p r i m a r y spermatocytes; O t h e r e x p l a n a t i o n s as i n F i g u r e 10. 65 21. GSI o f ' p r e s p a w n i n g f i s h ' u n d e r f o u r d i f f e r e n t p h o t o p e r i o d - t e m p e r a t u r e c o m b i n a t i o n s , a t t h e t i m e o f t h e i n d i c a t e d s a m p l i n g s compared w i t h t h o s e :Of t h e n o r m a l . The p o i n t o f c o n v e r g e n c e a t l e f t i n d i c a t e s t h e GSI o f t h e n o r m a l f i s h a t t h e s t a r t o f t h e e x p e r i m e n t . • • = S h o r t p h o t o p e r i o d (8 h r ) and h i g h t e m p e r a t u r e (19°±1.5 C ) ; • • = S h o r t p h o t o p e r i o d (8 h r ) and l o w t e m p e r a t u r e (9°-1.5 C ) ; 0 0 = Long p h o t o p e r i o d (16 h r ) and. h i g h t e m p e r a t u r e (19°±1.5 C ) ; 0 0 = Long p h o t o p e r i o d (16 h r ) and l o w t e m p e r a t u r e (9°+1.5 C ) ; X X = N o r m a l . . . . 67 22. S p e r m a t o g e n e t i c c o m p o s i t i o n o f ' p r e s p a w n i n g f i s h ' u n d e r f o u r c o m b i n a t i o n s o f p h o t o p e r i o d - t e m p e r a t u r e a t t h e t i m e o f t h e v a r i o u s s a m p l i n g s ( i n d i c a t e d by Roman n u m e r a l s ) . I.N = I n i t i a l n o r m a l ; L-L°= L o n g p h o t o p e r i o d , l o w t e m p e r a t u r e ; S-L°= S h o r t p h o t o p e r i o d , l o w t e m p e r a t u r e ; L-H°= Long p h o t o p e r i o d , h i g h t e m p e r a t u r e ; S-H° = S h o r t p h o t o p e r i o d , h i g h temperature; N = N o r m a l . M M = P r i m a r y germ c e l l s ; \' J = p r i m a r y sperm-a t o g o n i a ; liiiHfl = S e c o n d a r y s p e r m a t o g o n i a ; ~ P r i m a r y s p e r m a t o c y t e s ; = S e c o n d a r y spermatocytes; t H 3 = S p e r m a t i d s and s p e r m s ; HUGH = P y c n o t i c n e s t s . The l o w e r b l o c k i n e a c h r e p r e s e n t s r e s t i n g n e s t s , t h e u p p e r d e n o t e s t h o s e d i v i d i n g . 69 PAGE 61 61 62 64 ( x ) FIGURE 23. GSI o f 'pp'st s p a w n i n g f i s h u n d e r f o u r d i f f e r e n t p h o t o p e r i / o d - t e m p e r a t u r e c o m b i n a t i o n s a t t h e t i m e s o f 'the i n d i c a t e d s a m p l i n g s c o m p a r e d w i t h t h o s e o f \ t h e n o r m a l s . F o r e x p l a n a t i o n s see F i g u r e 21°. 24A. P h o t o m i c r o g r a p h o f a S t a g e V o r e a r l y S t a g e I t e s t i s s h o w i n g l o b u l e s c o n s i s t i n g m o s t l y o f p r i m a r y germ c e l l s . M i g r a t i n g c e l l s a r e shown i n t h e i n t e r l o b u l a r s p a c e , some o f w h i c h have e n t e r e d t h e l o b u l e s w h i c h l a c k s a lumen (X 7 0 0 ) . . B. P h o t o m i c r o g r a p h o f a s e c t i o n o f t e s t i s f r o m p h o t o p e r i o d - t e m p e r a t u r e f i s h s h o w i n g p r e m a t u r e f o r m a t i o n o f p r i m a r y s p e r m a t o c y t e s and t h e s t o p p i n g o f any f u r t h e r s p e r m a t o g e n e t i c p r o g r e s s a t l o w e r t e m p e r a t u r e s (X 700) 25A. P h o t o m i c r o g r a p h o f a p o r t i o n o f an LS o f a n o r m a l s p a w n i n g t e s t i s s h o w i n g d i v i d i n g n e s t s o f s p e r m -a t o g o n i a and s p e r m a t o c y t e s l i n i n g t h e l o b u l e f i l l e d w i t h sperms (X 7 0 0 ) B. P h o t o m i c r o g r a p h o f a p o r t i o n o f an LS o f a t e s t i s o f s i m i l a r f i s h u n d e r l o w t e m p e r a t u r e s ( e i t h e r p h o t o p e r i o d ) s h o w i n g s u p p r e s s i o n o f s p e r m a t o g e n e t i c a c t i v i t y (X700).- . 26A. P h o t o m i c r o g r a p h o f a p o r t i o n o f t e s t i s s h o w i n g l o b u l e b o u n d a r y c e l l s (X 1260) B. P h o t o m i c r o g r a p h o f a t e s t i s o f C. p l u m b e u s s t a i n e d w i t h Sudan B l a c k B s h o w i n g l i p o i d a l l o b u l e b o u n d a r y c e l l a r e a s , and a b s e n c e o f any i n t r a l o b u l a r l i p i d a c c u m u l a t i o n (X 135) 27. S p e r m a t o g e n e t i c c o m p o s i t i o n o f ' p o s t s p a w n i n g f i s h ' u n d e r f o u r c o m b i n a t i o n s o f p h o t o p e r i o d -t e m p e r a t u r e a t t h e t i m e o f t h e o v a r i o u s s a m p l i n g s i n d i c a t e d by Roman n u m e r a l s . O t h e r e x p l a n a t i o n s as i n F i g u r e 22 28. GSI o f ' p r e - f r e e z e up' f i s h u n d e r f o u r d i f f e r e n t p h o t o p e r i o d - t e m p e r a t u r e c o m b i n a t i o n s a t t h e t i m e o f t h e i n d i c a t e d s a m p l i n g s . The c o n v e r g i n g p o i n t a t l e f t i n d i c a t e s t h e GSI o f t h e n o r m a l s a t t h e s t a r t o f t h e e x p e r i m e n t . • • = N o r m a l s ; o t h e r e x p l a n a t i o n s same as i n F i g u r e 21 29. S p . e r m a t o g e n e t i c c o m p o s i t i o n o f ' p r e - f r e e z e up' • f i s h u n d e r f o u r c o m b i n a t i o n s o f p h o t o p e r i o d and t e m p e r a t u r e a t t h e t i m e o f t h e v a r i o u s s a m p l i n g s i n d i c a t e d by Roman n u m e r a l s . E x p l a n a t i o n s same as i n F i g u r e 22 (xi) FIGURE PAGE 30. Summary of the replacement therapy experiments showing seasonal effect. The smooth curve indicates the seasonal change in the activity of the normal testes. Roman numerals indicate the five stages of the annual testicular cycle. Signs at the top of the figure indicate the timing of the various experiments with reference to the annual cycle, and their effects. A (-) sign denotes regression; and a (+) sign stimulation. Increases in stimulatory and regressive effect are indicated by increase in number of these signs. 94 31. Summary of the photoperiod-temperature experiments. Symbols above the curve indicate the condition o f fish at later samplings at higher temperature, the bnesbelow the curve denote those at low temperature. The spots on the curve where these symbols are placed indicate the level of stimulation or regression exhibited by the fish at the fin a l sampling. Other explanations same as in Figure 30. 95 32. Photomicrograph showing cells lining the distal spermatic ducts of spawning fish. Note three cells in the centre with elongated nuclei in their basal half and granule f i l l e d lumen-halft adjacent cells on l e f t are spent (X 1260). . . . . 96 33A. Photomicrograph of a portion of an LS of testis of fish treated with crude whole salmon- pituitary extract. Note the lobule on l e f t upper corner almost f u l l of primary spermatocytes. Some secondary spermatocytes are seen dividing at right upper corner (X 700). . , , 97 B. Photomicrograph of a portion of an LS of a normal Srage II testis showing pycnotic nests among healthy spermatogenetic stages (X 700) 97 LIST OF TABLES TABLE PAGE I. Summary of l i p i d stain and cholesterol test results on normal Couesius testis . . . . . . . . . . 122 II. Effect of hypophysectomy on the testes in C. plumbeus. Figures under spermatogenetic condition are the percentage composition of various spermatogenetic stages from planimetry. SPGR = primary germ ce l l s ; SPG' = primary spermatogonia; SPG" = resting secondary sperm-atogonia; SPG-x = dividing secondary spermatogonia; SPC = resting primary spermatocytes; SPC'x -dividing primary spermatocytes; SPC" = secondary spermatocytes; SPD = spermatids; S = sperms; d = pychotic nests 123 I I I . Effect of hypophysectomy on the testes in C. plumbeus as measured by gonosomatic indices TGSI) and cholesterol test 124 IV. Summary of effect of replacement therapy with mammalian purified f o l l i c l e stimulating hormone (FSH). Symbols as in Table II. . 125 V. Summary of the effect of replacement therapy with mammalian purified luteinizing hormone (LH). Symbols :as in Table II. . . . . . . . . . . . . . . 126 VI. Summary of the effect of replacement therapy with fractionated salmon pituitary gonadotropin* Symbols as in Table II. In May 1963 and subsequent experiments the upper row denoted by H indicates condition of treated fish at high temperature, the lower row^L^that at low temperature. . . . . . 127 VII. Effect of replacement therapy: Crude whole salmon pituitary extract (CSE). Symbols as in Table II. . 128 VIII. Exploratory photoperiod experiment. Fish in experimental condition, January 1, 1963.. . . . . . 129 IX. Photoperiod-temperature experiment: Effect of photoperiod-temperature combination on prespawning stock. Fish in experimental condition, April 30, 1963, L-L 0^ long photoperiod, low temperature; S-L°= short photoperiod, low temperature; L-H°= long photoperiod, high temperature; S-H°- short photoperiod, high temperature. 130 ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Dr. W.S. Hoar for his constant encouragement, enthusiastic guidance, and c r i t i c a l supervision during the entire phase of this work. thanks are due to Drs. P.A. Dehnel, P. Ford, H.D, Fisher, M, Smith and N.J. Wilimovsky for their helpful suggestions during the preparation of the manuscript. The author also wishes to thank Dr. P. Ford for the use of his equipment for histochemical and photographic work. The author wishes to express his thanks to a l l persons who have helped him in the successful completion of this work, especially; Messrs. P.E. Ellickson, W.L. Woodall and M. Teraguchi for help in collection of the fish; Mr. J.A. McCabe for recording lake temperatures; Miss Hilary Short and Mr. W.L. Woodall for some histological preparations; Miss Joanne Mathias for assistance in the greater part of the planimetric work; Miss Barbara Findley and Mr. P.J. Schmidt of the Technological Laboratory, Vancouver, B.C. (Fisheries Research Board of Canada) for the supply of the salmon pituitary extracts and salmon fractionated gonadotropin; Mrs. Joan Glass for her meticulous typing of the manuscript. The author also wishes to thank sincerely his wife for having patiently borne a l l hardships during the period of this work, and his parents for their keen interest in his education. The study and stay in Canada of the author was provided for by the award of a Commonwealth Scholarship by the Canadian Universities Foundation, Ottawa. Their assistance is gratefully acknowledged. INTRODUCTION R e p r o d u c t i v e a c t i v i t i e s i n most a n i m a l s u n d e r g o c y c l i c a l c h a n g e s w h i c h a r e c h a r a c t e r i s t i c o f e a c h s p e c i e s ( B u l l o u g h 1 9 6 1 ) . These c y c l i c a l c h a n g e s have been c a r e f u l l y s t u d i e d , i n b o t h v e r t e b r a t e s and i n v e r t e b r a t e s , and a r e g o v e r n e d by a number o f e n v i r o n m e n t a l and n e u r o e n d o c r i n e f a c t o r s . I n s e v e r a l f i s h e s t h e g o n a d a l c o n t r o l s have now been w o r k e d o u t i n c o n s i d e r a b l e d e t a i l . I t i s w e l l e s t a b l i s h e d t h a t t h e p i t u i t a r y g l a n d p l a y s an e s s e n t i a l p a r t ( V i v i e n , 1938, 1 9 4 1 P 1939; M a t t h e w s , 1939; B u r g e r , 1 9 4 1 ; P i c k f o r d , 1953; T a v o l g a , 1955; B a r r , 1 9 6 3 ; and r e v i e w s by H o a r , 1955, 1957, 1962; B a l l , 1 960; M a r s h a l l , 1960; and Dodd, 1 9 6 0 ) . C o r r e l a t i v e c y c l i c a l c h a n g e s i n t h e h i s t o l o g i c a l a p p e a r a n c e s o f t h e p i t u i t a r y g l a n d and g o n a d s have f r e q u e n t l y been d e m o n s t r a t e d ( V e r h o e v e n and Van O o r d t , 1 9 5 5 ; S o k o l , 1 9 5 5 ) . Among e n v i r o n m e n t a l f a c t o r s l i g h t and t e m p e r a t u r e a r e p a r t i c u l a r l y most s i g n i f i c a n t i n n o r t h t e m p e r a t e l a t i t u d e s (Van de E e c k h o u d t , 1947; H a r r i n g t o n , 195Q» 1956, 1957, 1959; Baggerman, 1957; H o a r , 1961; H e n d e r s e n , 1 9 6 3 ) . H a r r i n g t o n ( 1 9 5 9 b ) and A t z ( 1 9 5 7 ) have c r i t i c a l l y r e v i e w e d t h e l i t e r a t u r e . I n v e s t i g a t i o n s o f t h e s e c o n t r o l s i n f i s h e s have c o n c e n t r a t e d on t h e f e m a l e c h i e f l y b e c a u s e t h e i n f l u e n c e s o f t h e c o n t r o l l i n g f a c t o r s on t h e gonads i s r e p o r t e d t o be r e f l e c t e d w i t h g r e a t e r d e p e n d a b i l i t y and s p e c i f i c i t y i n t h e o v a r i e s t h a n i n t h e t e s t e s . O o g e n e t i c s t a g e s i n f i s h e s a r e e a s y t o i d e n t i f y , d e m a r c a t e , and q u a n t i f y ( H a r r i n g t o n , 1 9 5 9 a ) . I n t h e few s t u d i e s - 2 -involving males, the changes noted were of a gross nature and usually.did not include detailed histological examination (Medlen, 1 9 5 1 ; Baggerman, 1957; Harrington, 1956; Hendersen, 1 9 6 3 ) . Subtle cyclical changes in the testes paralleling those in the ovaries may occur but have escaped notice because of a lack of accurate methods of quantifying the level of maturation of the testes. The endocrinological studies of the controlling mechanism also have been rather inconclusive because (a) fish have usually been treated with hormones without previous hypophysectomy to eliminate the effects of endogenous gonadotropins and (b) only mammalian gonadotropins or crude fish pituitary extracts have been used in attempts to demonstrate the pituitary control. There are indications that in fishes the /gonadotropin responsible for gonadal maturation is physiologically similar in action to the mammalian Luteinizing hormone (LH). There have also been reports of a factor in fishes similar to mammalian follicle-stimulating hormone (FSH) (Kirshenblat, 1949: Witschi, 1955; Otsuka, 1 9 5 6 ) . Witschi ( 1 9 5 5 ) reported a preponderence of an LH-like factor over an FSH-like factor in fishes on the basis of standard assay methods. Ramaswami ( 1 9 6 2 ) pointed to the presence of LH in the Indian catfish, Heteropnuestes^ f o s s i l i s . but made no specific tests for the presence of FSH. Ahsan and Hoar ( 1 9 6 3 ) showed that mammalian FSH failed to produce any substantial stimulation in Gasterosteus aculeatus. The literature has been reviewed (Hoar, 1964b) and uncertainties, s t i l l exist concerning the number of gonadotropins and their physiological - 3 -action. There are several unresolved problems associated with the reproductive physiology of the male teleoit. the environmental regulation of gonadal activity varies in the different telebsts. It might be light controlled;, temperature controlled, synergistic control by light and temperature or by an endogenous rhythm modified by these factors (Bullough, 1940). Differences have been found in fishes belonging to different families and even in closely related species living in widely different latitudes (Harrington, 1959a). Moreover, there are few long term studies of the pituitary control of the fish testes. C r i t i c a l investigation of pituitary function requires hypophysectomised fish and replacement therapy with purified fish gonadatropin (preferably from the same or closely related fish). With these points in mind, male Couesius plumbeus (Agassiz) (=. Hvbopsis plumbea of Bailey) were selected for a comprehensive analysis of control of the fish testicular cycle. This fish is particularly suitable because of i t s abundance, hardiness, size, and suitability for hypophysectomy. The investigations were conducted along four different but interrelated lines: i ) Study of the annual testicular cycle of C.plumbeus with reference to the environmental temperature and photoperiod. i i ) S t u d y of the effect of hypophysectomy on the testes and the seasonal variations in the response to pituitary ablation. - 4 -i i i ) Study of the effect of replacement therapy on the testes of hypophysectomised fish using: a) Fractionated salmon pituitary gonadotropin. b) Crude salmon pituitary extract. c) Purified mammalian FSH. d) Purified mammalian LH. iv) Experimental analysis of the effect of photoperiod and temperature on stocks of wild fish captured in different phases of their annual cycle. MATERIALS AND METHODS Samples of male Couesius plumbeus (Aqassiz) (Family: Cyprinidae) were captured each month, usually in the f i r s t week, from "No f i s h " Lake and the adjacent 3-mile Lake near Clinton, British Columbia, at approximately 51°8'N and 121°40'w. They Were held under standard aquarial conditions. Collections were usually made during the f i r s t week of the month, beginning in June 1962 and ending in November 1962. Monthly collections were started again in late April 1963, the last collection being in the month of November. Surplus fish from 1962 collections were kept outdoors in troughs of running water under as near natural conditions as possible and sampled monthly from January to April in 1963. Each set of experiments were identified by the month and the year in which the fish were hypophysectomised. Regular replacement therapy began with fish hypophysectomised in July 1962. Each experiment lasted about 50 - 60 days. A batch of 20 - 30 fish was killed immediately after arrival at the laboratory for the study of the annual testicular cycle. The remaining fish were used for pituitary removal procedures and for photoperiod and temperature experiments. H v D Q D h v s e c t o m v Fish were acclimated to laboratory water and temperature conditions for at least 36 hours before hypophysectomy. The pituitary gland of Couesius plumbeus is oval anteroposteriorily and rounded dorsventrally in cross section. It is situated in the cranial floor immediately above the paresphenoid. Seen from the roof of the buccal cavity after the removal of the buccal epithelium i t appears as a white oval body about 1 mm in i t s longest diameter, situated at the level of the f i r s t g i l l c l e f t . The general topography of the pituitary gland is shown in Figure 1. The pituitary is connected to the hypothalamus by a narrow vertical stalk. Like a l l the fishes which possess a stalk, the hypophysial recess in £. plumbeus is almost obliterated (Kerr, 1942). The relationship of the pituitary to the brain i s shown in Figure 3A. It is the presence of a narrow stalk, i t s vertical disposition and the generally anteriad position of the gland i t s e l f which make C. plumbeus an ideal fish for hypophysectomy. . Procedure of hvpophvsectomv. Fish ranging from about 57 mm to 70 mm forklength were hypophysectomized by a modification of the opercular approach described by Abramowitz (1937). They were anesthetised with 112000 MS 222 (Tricaine Methane Sulfonate -Sandoz) dissolved in qquarrium water, by placing them in the anesthetic solution until swimming movements ceased and the opercular movements were appreciably slowed down. The fish was then placed belly upwards in a groove made in a soft sponge fitted into a small rectangular plastic tray (18 x 12 x 3 c)m), which constituted the 'operation table 1. The tray had an opening at either end, the opening farther from the operator serving as ah inlet for fish physiological saline (Young, 1933). This was led into the groove through a 2 mm diameter polyethylene tube projecting about 5 - 8 mm into the groove. The other opening served as an outlet for waste saline and blood. - 7 -F i g . l . Camera l u c i d a d i a g r a m o f t h e t o p o g r a p h y o f t h e P i t u i t a r y G l a n d (P) o f C o u e s i u s p l u m b e u s . O n l y t h e mucus membrane c o v e r i n g t h e r o o f o f t h e b u c c a l c a v i t y i s r e m o v e d . 1. I n t e r n a l c a r o t i d a r t e r y 2. B r a n c h f r o m t h e i n t e r n a l c a r o t i d a r t e r y 3. C i r c u l u s c e p h a l i c u s 4. M a x i l l a r y n e r v e 5. O p t i c n e r v e .6. F i r s t g i l l c l e f t (X c i r c a 30) 7. P a r a s p h e n o i d - 8 -The fish was held in position by passing over the body a pair of rubber bands held on either side of the groove by pins. A beam of light from a 100-watt lamp cooled through a round-bottomc*. l i t r e flask of cold water was directed towards the inlet half of the groove. The fish was slightly t i l t e d to i t s right on i t s back. The saline inlet tube was then passed into the buccal cavity of the anesthetised fish through the mouth. The operculum was raised and the cleft between the branchiostegal membrane and the gular region of the lower jaw was extended by a forwardly directed incision with a fine pair of scissors. This provided a much larger opening of the g i l l cleft into the roof of the buccal cavity. The operculum was l i f t e d and kept parted by a retractor fashioned from a bent pin. The mucus membrane covering the palate was cut by a slightly oblique transverse incision with an iridectomy scissor. The cut membrane was flushed with saline and when necessary excess fl u i d was removed with a fine tube attached to a suction tap. Usually there was l i t t l e bleeding up to this point in the operation. The parasphenoid was now bared, thus exposing the oval whitish pituitary (Fig. l ) . It was cut obliquely immediately behind i t s widest part by an iridectomy scissor. A Morse Scaler was then inserted above the bone and the pituitary was loosened. The flow of saline which was kept at a minimum to this point and even stopped occasionally, was now increased. Blood was flushed and the pituitary was sucked out with the fine tube referred to above. The flow of saline was gradually decreased over a period of 2 minutes, and the fish was allowed to recover (on the operation table) for 5 - 1 0 minutes with constant saline flow - 9 -F i g . 2 A . A g e n e r a l v i e w o f t h e s e t up f o r h y p o p h y s e c t o m y B. Some o f t h e i n s t r u m e n t s u s e d i n t h e o p e r a t i o n - 10 -over i t s g i l l s before being transferred to a 0.2% bath of equal . parts of sodium and calcium chloride prepared in aquarium water (Chavin, 1956). The fish were left in this postoperative bath for about 6 hours. They were then transferred to 5'4 or 10 gallon aquaria in which they were kept until the autopsy. A head magnifier (about 6 times) was used during the operation. The general set-up used for hypophysectomy is shown in Figure: 2. The survival rate in the operative procedure was about 50$. This low survival was due to the caie taken to see that the pituitary was completely removed. Causes of casualty were either excessive bleeding or damage to the brain. The hypophysectomised fish were kept for 30 - 40 days before any replacement therapy was initiated. During this period they fed well and appeared to be healthy; feeding was not started until two days after hypophysectomy. They were kept in lots of about 6 in aerated aquaria, fitt e d with "outside f i l t e r s " (Fig. 4 ) ; water was usually changed once a week. The temperature ranged from 18°C to 21°C. The fish were fed daily with frozen brine shrimp (Wardley's, Saskatchewan). Feeding was curtailed to alternate days in experimental fish during the subsequent fortnight of injections. Injections Both mammalian and fish pituitary hormones were used as follows:, 1. Fractionated salmon pituitary gonadatropin. 2. Crude salmon pituitary extract. 3. Pure mammalian FSH (Armour Porcine Lot #216-175-6 and #216-177-6) - 11 -Fig. 3A. Sagittal section of head through the pituitary gland and adjacent brain tissue of C. plumbeus (X 750) B. Sagittal section of the pituitary region of the brain after hypophysectomy (X 700) - 12 -4. P u r e mammalian LH (Armour O v i n e L o t #216-176-5 and #216-178-5) I n r e p l a c e m e n t t h e r a p y a d m i n i s t e r e d d u r i n g May 1963 - O c t o b e r 1963, f i s h t h a t w e re i n j e c t e d w i t h f r a c t i o n a t e d f i s h g o n a d o t r o p i n ( s ) and mammalian LH were k e p t d u r i n g t h e d u r a t i o n 6 f t h e i n j e c t i o n a t two t e m p e r a t u r e c o n d i t i o n s , v i z : one b a t c h a t 9°C - 1.5°and t h e o t h e r a t t h e u s u a l 19.0 - 1.5°C. E a c h h y p o p h y s e c t o m i s e d f i s h t o be t r e a t e d w i t h f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n s r e c e i v e d a t o t a l o f 0.6 ml o f t h e f r a c t i o n a t e d p r e p a r a t i o n . T h i s t o t a l amount was g i v e n i n 6 e q u a l i n j e c t i o n s o f 0.1 m l / f i s h a d m i n i s t e r e d on a l t e r n a t e d a y s s p r e a d o v e r two weeks. F i s h t r e a t e d w i t h c r u d e s a l m o n p i t u i t a r y e x t r a c t r e c e i v e d a . t o t a l o f 0.3 ml o f t h e p r e p a r a t i o n a l s o d i v i d e d i n t o 6 e q u a l d o s e s . F i s h r e c e i v i n g mammalian FSH o r LH e a c h r e c e i v e d 0.3 mgm - 0.35 mgm o f p u r i f i e d powder d i s s o l v e d i n f i s h s a l i n e . E a c h i n j e c t i o n c a r r i e d 0.06 mgm o f t h e hormone i h 0.05 ml o f s o l u t i o n . N o r m a l c o n t r o l s as w e l l as h y p o p h y s e c t o m i s e d c o n t r o l s were injected w i t h a t o t a l o f 0.6 ml o f s a l i n e p e r f i s h . F i s h were l i g h t l y a n e s t h e t i s e d and i n j e c t e d i n t r a p e r i t o n e a l l y w i t h a 0.25 ml s y r i n g e and a gauge #30 n e e d l e so t h a t t h e n e e d l e p i e r c e d t h e b e l l y a h e a d o f t h e a n a l o p e n i n g . Some t i m e was a l l o w e d b e f o r e t h e n e e d l e was w i t h d r a w n frofr) t h e body. T h i s p r e c a u t i o n , c o u p l e d w i t h l i g h t a n e s t h e s i a , e n s u r e d t h a t l i t t l e o r no hormone was l o s t f r o m t h e f i s h a f t e r t h e i n j e c t i o n s . A l l t h e above h o r m o n a l t r e a t m e n t s were s t a r t e d on t h e same d a t e and f i s h were a u t o p s i e d one o r two d a y s a f t e r t h e l a s t i n j e c t i o n . - 13 -F i g . 4. A g e n e r a l v i e w o f t h e a q u a r i a u s e d t o keep e x p e r i m e n t a l f i s h and c o n t r o l s -14-Preparation of Salmon Pituitary Extracts The salmon extracts were prepared from Spring Salmon (Qncorhvnchus tshawvtscha) and Coho Salmon (Qncorhvnchus kisutch) pituitaries by Miss Barbara Findley and Mr. Peter Schmidt at the Technological Research Laboratory of the Fisheries Research Board of Canada, Vancouver, B.C. Separation of the gonadotropin was by starch gel ion exchange electrophoresis. The following outline of the procedure of fractionation has been provided by Dr. M. Smith. The crude pituitary extract was prepared by grinding 1 gram of frozen salmon glands, obtained from mature spawning fish of either sex in equal numbers, with 1.5 volumes of 1.25% NaCl solution, using a glass homogenizer. After centrifuging, the extract was dialysed in cold glycine buffer for 6 hours. Then, 0.2 ml of this extract was applied tb each of the 4 slots of the starch gel. The starch solution consisted of 11% hydrolysed starch in 0.004 M glycine solution at pH 9.5. The electrophoresis was carried out for 16 hours at 300 volts using 0.3 M borate buffer in 10% NaCl solution. On completion of the electrophoresis the gels were cut on the positive side 1.2 cm and 5.0 cm from the origin. This strip 3.8 cm long contained the desired gonadotropic fraction as evidenced by spermatokinetic tests on frogs and earlier experiments on steelhead trout. It was cut into small pieces and put into a dialysing bag f i l l e d with glycine puffer and suspended in a beaker also containing glycine buffer. Two strips of f i l t e r paper were run from the borate buffer and the NaCl solution Qf the electrophoresis apparatus - 15 -into the beaker. A three hundred volts DC was applied for 16 hours after which the glycine solution in the bag was removed, centrifuged and evaporated to approximately 0.25 ml by freeze drying. The residue was then made up to volume of 1 ml with saline solution;. The crude whole salmon pituitary extract was prepared by grinding 1 gm of frozen spring salmon gland with 2 volumes of 1.25$ NaCl. A l l these hormone preparations were stored under refrigeration. The total amount of salmon extracts injected intb each fish amounted to an equivalent of 2.5 fresh fish glands weighing a total of 200 mgm. Controls were of two kinds. Sham operated fish were kept in conditions identical to those of the operated ones and were called "sham operated controls". Some hypophysectomised fish were also kept under similar conditions. They constituted the "operated controls'*. Both these control groups were injected with equivalent amounts of fish saline and autopsied at the same time as the experimental' fish. The batch of fish autopsied at the beginning of each experiment composed the normals or i n i t i a l controls. They provide the picture of the annual testicular cycle and also give an idea of the state of the testis in the fish at the start of each experiment. Sham operation included extension of the opercular opening and the incision of the mucus epithelium of the buccal roof in a manner similar to that employed for complete pituitary ablation. Photoperiod, Experiment In January 1963 fish from the outdoor tanks were put - 16 -u n d e r c o n t r o l l e d p h o t o p e r i o d s o f 16 h o u r s l i g h t a l t e r n a t i n g w i t h 8 h o u r s d a r k n e s s and 8 h o u r s l i g h t a l t e r n a t i n g w i t h 16 h o u r s o f d a r k n e s s . The t a n k s were p r o v i d e d w i t h l i g h t - p r o o f t o p s c a r r y i n g a l i g h t s o u r c e o f 8 w a t t s e q u i v a l e n t t o 27 f t . c . T e m p e r a t u r e s r a n g e d f r o m 5°C i n J a n u a r y t o 6.5°C i n A p r i l . F i s h were s a m p l e d a f t e r 45 and 75 d a y s . L a t e r i t was d e c i d e d t o s u b j e c t f i s h e s b r o u g h t d i r e c t l y f r o m t h e l a k e a t v a r i o u s p e r i o d s o f t h e i r a n n u a l c y c l e t o a c o m b i n a t i o n o f l i g h t and t e m p e r a t u r e c o n t r o l . On A p r i l 3 0 , 1 9 6 3 , f i s h c o n s t i t u t i n g t h e p r e s p a w n i n g s t o c k were p l a c e d i n e q u a l numbers u n d e r e a c h o f t h e f o l l o w i n g r e g i m e s : 1. Long p h o t o p e r i o d (16 h r l i g h t ) and l o w t e m p e r a t u r e (8°C ± 2°C). 2. S h o r t p h o t o p e r i o d (8 hr l i g h t ) and l o w t e m p e r a t u r e (8°C ± 2 ° C ) . 3. L o n g p h o t o p e r i o d (16 h r l i g h t ) and h i g h t e m p e r a t u r e (19°C ± 1.5°C). 4. S h o r t p h o t o p e r i o d (8 h r l i g h t ) and h i g h t e m p e r a t u r e (19°C + 1:5°C). The f i s h i n t h i s t e m p e r a t u r e - p h o t o p e r i o d e x p e r i m e n t w e r e h o u s e d i n f o u r s e p e r a t e f i b r e g l a s s t a n k s m e a s u r i n g 63 x 46 x 36 cm e a c h o f w h i c h was p r o v i d e d w i t h a l i g h t - p r o o f t o p c o n t a i n i n g lamps w h i c h gave 27 f t . c a t w a t e r s u r f a c e . The l o w e r t e m p e r a t u r e s were t h o s e i n t h e g e n t l y f l o w i n g t a p w a t e r ; h i g h e r t e m p e r a t u r e s were a c h i e v e d by t h e r m o s t a t i c a l l y c o n t r o l l e d h e a t e r s . P o s t p a w n i n g f i s h b r o u g h t f r o m t h e l a k e on A u g u s t 9, - 17 -1963 were l i k e w i s e p u t under' t h e above p h o t o p e r i o d - t e m p e r a t u r e c o m b i n a t i o n . The f i n a l s t o c k i n g o f f i s h u n d e r t h e s e c o n d i t i o n s was made w i t h t h e " P r e f r e e z e up" b a t c h c o l l e c t e d on November 1, 1963. C a r e and f e e d i n g o f t h e s e f i s h were s i m i l a r t o t h o s e u s e d f o r r e p l a c e m e n t t h e r a p y s t u d y . These t h r e e s t o c k s o f f i s h w ere m a r k e d d i s t i n c t i v e l y and s a m p l e d a t r e g u l a r i n t e r v a l s . A u t o p s y F i s h w e r e k i l l e d by o v e r a n e s t h e t i s i n g . The body w e i g h t and t h e f o r k l e n g t h o f e a c h f i s h were r e c o r d e d . The abdomen was o p e n e d and b o t h t e s t e s were d i s s e c t e d o u t , f r e e d f r o m a d h e r i n g a d i p o s e t i s s u e and w e i g h e d t o t h e n e a r e s t o f 2.5 m i l l i g r a m s ( on a M e t t l e r Type H3 B a l a n c e ) . U s u a l l y t h e r i g h t t e s t i s was f i x e d i n B o u i n ' s f l u i d f o r r o u t i n e h i s t o l o g y and t h e l e f t f i x e d i n B a k e r ' s F o r m o l - C a l c i u m s o l u t i o n f o r t h e c h o l e s t e r o l t e s t and l i p i d s t a i n i n g . C h o l e s t e r o l and l i p i d s t a i n i n g was r e s t r i c t e d t o t h e t e s t e s b e l o n g i n g t o t h e o p e r a t e d f i s h . The B o u i n f i x e d t e s t e s were p r o c e s s e d i n the' u s u a l way, embedded i n p a r a f f i n wax (M.P. 56-58) and r e p r e s e n t a t i v e s e r i a l l o n g i t u d i n a l s e c t i o n s c u t a t 6 / t i . a n d u s u a l l y s t a i n e d ' W i t h E h r l l c h ' s H e m a t o x y l i n and E o s i n . The f o r m a l - c a l c i u m f i x e d t e s t i s was c u t i n t o two p . e i c e s . One h a l f was f r o z e n s e c t i o n e d w i t h an I n t e r n a t i o n a l H a r r i s C r y o s t a t . S e c t i o n s were made a t 25 - 30^u and t e s t e d by S c h u l t z ' s p r o c e s s f o r c h o l e s t e r o l and i t s e s t e r s . Adam's ( 1 9 6 1 ) method was t r i e d b u t was d i s c a r d e d i n f a v o u r o f S c h u l t z ' s . The o t h e r h a l f o f t h e t e s t i s was embedded i n g e l a t i n ( G u r r , 1 9 5 3 ) , s e c t i o n e d a t 8 a n d s t a i n e d w i t h Sudan B l a c k B f o l l o w i n g t h e p r o c e d u r e g i v e n by Humason ( 1 9 6 2 ) . The s e c t i o n s - 18 -were sometimes counterstained with Carmalum. In some instances thick (25^ /u) gelatine-embedded testis sections also were subjected to Schultz test for cholesterol. Testes of the photoperiod experiment were subjected to usual histology only. Evaluation of Testicular Maturation Each testis was carefully examined under the microscope and gross observations were noted using the high power and o i l immersion lenses. Ten lobules from each testis were selected at random, and with a Spencer Camera lucida attached to a Leitz-Wetzlar microscope with monocular attachment, the outlines of the selected lobules were charted on a white sheet of paper. The spermatogenetic nests inside each cyst were identified and their outlines made within the lobule walls (Magnification C.X/. 270). The names of the stages.in a cyst and the state of activity, viz., resting or dividing were a l l noted on the projection. The areas of the various c e l l nests were then measured with an Aristo Planimeter (Dennert 8. Pape, Hamburg) in planimetric units and tabulated. The same planimetric settings were used for a l l measurements. The percentage composition in terms of the relative abundance of the various types of spermatogenetic stages was thus obtained. This process also gave an indication of the relative activity of a fish testis by giving the ratiq of the dividing and resting stages in a cyst. The condition of the spermatic ducts, i t s contents (if any) and i t s cellular lining were studied and records maintained. Ma.tfeoroloaical data Temperatures of the lake from which most of the fish - 19 -were obtained were regularly recorded by Mr. J.A. McCabe and are reliable within the limits of f i e l d data. The daylight period at 51°N latitude was calculated from the tables of Sunrise and Sunset times in 'The Observer's Handbook' published by the Royal Astronomical Society of Canada, Ottawa. Gonosomatic Index The gonosomatic index for each fish was calculated in the usual manner: Gonosomatic index ( G S I ) = weight, of, gonitis x 1 0 0 weight of body To find whether the pituitary had been successfully removed, the head was dissected by cutting the healed incision in the mucus membrane and removing the parasphenoid bone. The pituitary area was.checked for the gland. In cases of doubt the hypothalamic area was checked histologically. - 20 -RESULTS ANNUAL:.JESTI.CUL.AR. CYCLE .. General Structure of the Testis The testes in Couesius plumbeus Agassiz are a pair of elongated structures lying above the intestine laterodorsally on either side of the swimbladder. They extend from the genital opening to almost the anterior end of the abdominal cavity. At the posterior end of each testis, a vas deferens arises and opens outside by a single genital opening after meeting with i t s opposite number a very short distance from the genital pore. The testis is slightly triangular in cross section with the genital artery and vein lying on i t s ventral face. Each testis is enveloped in a very thin coelomic epithelium overlying the delicate tunica albuginea. The tunica albuginea remains uniform in i t s thickness during the entire seasonal cycle. The testis is composed of distinct seminiferous lobules of varying shapes and sizes with definite but extremely thin fibrous lobular boundaries (Fig. 17). These lobules are distinguishable with some di f f i c u l t y in very immature fish. In sexually active animals, the lobules enlarge, .and branch extensively, giving rise to a testicular structure composed of lobules in an amazing variety of shapes and sizes. Except for the lobules close to the posterior end of the testis, the identity of the lobules is evident even in highly active fish. Blood capillaries and elongate fibroblasts are found between adjacent and therefore adhering lobular walls (Fig. 16). Besides these fibroblasts, e l l i p t i c a l lobule boundary - 21 -c e l l s a l s o o c c u r c l o s e t o t h e l o b u l e w a l l . Some o f t h e s e l o b u l e b o u n d a r y c e l l s ( c o n s i d e r e d h o m o l o g u e s o f t h e L e y d i g c e l l s o f h i g h e r v e r t e b r a t e s ) l i e d e c i d e d l y w i t h i n t h e l o b u l e ( F i g . 26k). They v a r y i n shape f r o m e l l i p t i c a l o r o v a l t o a l m o s t r e c t a n g u l a r , or e v e n c u b o i d a l and c a n be d i s t i n g u i s h e d f r o m t h e r e s t i n g germ cells by the absence of any prominent n u c l e o l u s and by t h e i r less chromatic nature, .-.their shape and disposition. The l o b u l e boundary cells exhibit seasonal changes in abundance and histochemical reactions. Since only these testicular cells besides the blood capillaries give a positive test for cholesterol and are f a i r l y densely lipoidal, they a p p e a r to be similar to the l o b u l e b o u n d a r y c e l l s o f Esox lucius ( M a r s h a l l and Lofts, 1956; and L o f t s and M a r s h a l l , 1957) and p r o b a b l y c o n s t i t u t e t h e e n d o c r i n e component o f t h e t e s t i s . The s p e r m a t o g e n e t i c c omponents o f t h e t e s t i s l i e i n s i d e t h e l o b u l e i n more o r l e s s d i s c r e t e c e l l u l a r n e s t s . U s u a l l y c e l l s i n one n e s t b e l o n g t o one s p e r m a t o g e n e t i c s t a g e , a n d , e x c e p t f q r /the e a r l y d i v i s i o n s o f t h e p r i m a r y s p e r m a t o g o n i a , t h e e l e m e n t s i n a n e s t u n d e r g o c e l l d i v i s i o n i n a l l - o r - n o n e f a s h i o n . S p e r m a t o g e n e s i s p e r se has n o t been t h e m a i n p u r p o s e o f t h e p r e s e n t s t u d y . However, t h e v a r i o u s s t a g e s have been i d e n t i f i e d and d e l i m i t e d as f a r as p r a c t i c a b l e . N u c l e a r d i a m e t e r and c h r o m a t i c n a t u r e o f t h e n u c l e u s has been u s e d t o i d e n t i f y t h e s p e r m a t o g e n e t i c s t a g e s . B u l l o u g h (1939) has s u c c e s s f u l l y d i s t i n g u i s h e d t h e s p e r m a t o g e n e t i c s t a g e s i n t h e c y p r i n i d P h o x i n u s l a e v i s by u s i n g t h e n u c l e a r d i a m e t e r as t h e s o l e c r i t e r i o n . I n t h i s s t u d y n u c l e a r d i a m e t e r as w e l l as t h e a f f i n i t y o f t h e n u c l e u s f o r b a s i c d y e s s u c h as H a e m a t o x y l i n has - 22 -F i g . 5 . S e a s o n a l v a r i a t i o n i n t h e GSI o f C o u e s i u s p l u m b e u s as r e l a t e d t o c h a n g e s i n w a t e r t e m p e r a t u r e and d a y l e n g t h t • GSX o f t h e 1962-63 c y c l e ; 0 0 G S I o f t h e 1963 c y c l e (May t o November) j> A> W a t e r t e m p e r a t u r e The c u r v e d l i n e r e p r e s e n t s t h e s e a s o n a l c h a n g e s i n d a y l e n g t h a t 51°N - 23 -been u s e d t o d i s t i n g u i s h t h e s t a g e s . The e a s i l y i d e n t i f i a b l e s t a g e s a r e : I . P r i m a r y Germ C e l l These a r e f o u n d t h r o u g h o u t t h e y e a r b u t a r e comparatively r a r e i n m a t u r e t e s t e s . A c l e a r mass o f c y t o p l a s m c a n be d i s t i n g u i s h e d s u r r r o u n d i n g t h e l i g h t l y s t a i n i n g n u c l e u s . The n u c l e u s a v e r a g e s 7.2/u i n d i a m e t e r , and c o n t a i n s a s i n g l e l a r g e e x c e n t r i c n u c l e o l u s . The n u c l e o p l a s m i s o t h e r w i s e c l e a r and c o n t a i n s l i t t l e c h r o m a t i n i c m a t e r i a l . I I . P r i m a r y S p e r m a t o g o n i a These c e l l s have r e l a t i v e l y l e s s c y t o p l a s m t h a n t h e p r i m a r y germ c e l l s . The n u c l e u s a v e r a g e s 5.2/u i n d i a m e t e r . The n u c l e o l u s i s s m a l l , c e n t r a l and b a s o p h i l i c ; t h e n u c l e o p l a s m i s more c h r o m a t i c . The p r i m a r y s p e r m a t o g o n i a u n d e r g o a. number o f d i v i s i o n s t o p r o d u c e t h e s e c o n d a r y s p e r m a t o g o n i a . I I I . S e c o n d a r y S p e r m a t o g o n i a T h i s st.a/ge can be d i s t i n g u i s h e d f r o m t h e p r e v i o u s by t h e s l i g h t d e c r e a s e i n n u c l e a r d i a m e t e r ( a v e r a g e 4.5^/J) and by a marked i n c r e a s e i n b a s o p h i l i a o f t h e . n u c l e u s . I t i s n o t e a s y t o d i s t i n g u i s h b e t w e e n l a t e r g e n e r a t i o n s o f p r i m a r y s p e r m a t o g o n i a and t h e new s e c o n d a r y o n e s . I V . P r i m a r y S p e r m a t o c y t e I n t h i s s t a g e t h e t h e n u c l e u s has u n d e r g o n e f u r t h e r r e d u c t i o n i n s i z e and a v e r a g e s 3.57^/J i n d i a m e t e r . T h e r e i s no d i s t i n c t n u c l e o l u s and c h r o m a t i c m a t e r i a l i s a b u n d a n t . V. S e c o n d a r y S p e r m a t o c y t e s T h i s i s a t r a n s i e n t s t a g e i n t h e s p e r m a t o g e n e s i s o f f i s h e s vand i s r a r e l y s e e n i n r e s t i n g p h a s e . The a v e r a g e - 24 -d i a m e t e r o f t h e s e n u c l e i i s 2.67^/u. I t i s d i f f i c u l t t o d i s t i n g u i s h t h e c y t o p l a s m i n s p e r m a t o g e n e t i c s t a g e s f o l l o w i n g t h e p r i m a r y s p e r m a t o c y t e , V I . S p e r m a t i d s These a r e s p h e r i c a l m asses c o n s i s t i n g a l m o s t e n t i r e l y o f b a s o p h i l i c m a t e r i a l c a u s e d by a s t r i k i n g c o n d e n s a t i o n o f n u c l e a r c h r o m a t i n . They measure 1.8/u i n a v e r a g e d i a m e t e r . S p e r m a t i d s a r e t h e l a t e s t s t a g e o f s p e r m a t o g e n e s i s c o n t a i n e d i n n e s t s . A f t e r s p e r m a t i d s a r e f o r m e d t h e n e s t membrane r u p t u r e s l i b e r a t i n g t h e s p e r m a t i d s i n t o t h e lumen o f t h e l o b u l e s where t h e y t r a n s f o r m i n t o s p e r m s . V I I . ! S p e r m a t o z o a These a r e t h e f i n a l p r o d u c t o f s p e r m a t o g e n e t i c p r o c e s s e s . I n C. p l u m b e u s t r a n s f o r m a t i o n o f s p e r m a t i d s i n t o s perms t a k e s p l a c e i n t h e l o b u l e s so t h a t t h e l o b u l e c o n t a i n s m a t u r e s p e r m s . P r i m a r y germ c e l l d i v i d e s t o f o r m p r i m a r y spermatogonia. The s t o c k o f p r i m a r y s p e r m a t o g o n i a i s d e r i v e d a l s o f r o m c e r t a i n m i g r a t i n g c e l l s . These m i g r a t i n g c e l l s a r e a b u n d a n t - d u r i n g S e p t e m b e r and O c t o b e r ( S t a g e I o f t h e a n n u a l c y c l e ) . They a r i s e f r o m m i t o t i c d i v i s i o n o f c e r t a i n c e l l g r o u p s s i t u a t e d i n two o r t h r e e l o c i o f t h e i n t e r l o b u l a r t i s s u e . These b a s o p h i l i c c e l l s a p p e a r t o m i g r a t e a l o n g t h e i n t e r l o b u l a r p a t h and e n t e r t h e l o b u l e s t o become t r a n s f o r m e d i n t o p r i m a r y s p e r m a t o g o n i a (Fig.24A). B o t h t h e p r i m a r y and t h e s e c o n d a r y s p e r m a t o g o n i a u n d e r g o a.number o f g e n e r a t i o n s o f m i t o t i c d i v i s i o n b e f o r e t r a n s f o r m i n g r e s p e c t i v e l y i n t o s e c o n d a r y s p e r m a t o g o n i a o r p r i m a r y s p e r m a t o c y t e s . C o n s e q u e n t l y n e s t s o f t h e s e t h r e e s t a g e s grow t o - 25 -f a i r l y l a r g e s i z e s . The d i v i s i o n i n t h e p r i m a r y s p e r m a t o g o n i a l s t a g e i s u s u a l l y s l o w . I n n e s t s o f e a r l y g e n e r a t i o n s o f p r i m a r y s p e r m a t o g o n i a some : d i v i d i n g n u c l e i a r e f r e q u e n t l y s e e n amongst n o n - d i v i d i n g o n e s . B r a n c h i n g e f f e r e n t d u c t s a r e s i t u a t e d on t h e i n n e r m a r g i n o f e a c h t e s t i s and t h e a p i c e s o f m a t u r e l o b u l e s open i n t o t hem., These d u c t s a r e more p r o m i n e n t i n t h e m i d d l e and p o s t e r i o r t h i r d o f t h e t e s t i s . I n m a t u r e t e s t e s t h e y a r e d i s t e n d e d w i t h sperms and t h e a d j a c e n t l o b u l e b o u n d a r y seems t o b r e a k up and t h e i r c o n t e n t s become f l u s h w i t h .those a l r e a d y i n t h e d u c t . I n p o s t spawned t e s t e s t h e s e d u c t s c o l l a p s e and t h e i r c o l l a p s e d w a l l s g i v e t h e a p p e a r a n c e o f p a r a l l e l c o r d s o f c e l l s l y i n g on t h e . i n n e r s u r f a c e o f e a c h t e s t i s . The c e l l s l i n i n g t h e s e d u c t s and t h e v a s d e f e r e n s seem t o be s e c r e t o r y i n n a t u r e and d u r i n g t h e s p a w n i n g s e a s o n show: ( i ) an i n c r e a s e i n s i z e , ( i i ) a d i s p l a c e m e n t o f t h e n u c l e u s t o w a r d t h e b a s a l h a l f o f t h e c e l l , and ( i i i ) a c c u m u l a t i o n o f g r a n u l a r s u b s t a n c e s . I n spawned f i s h t h e y a r e r e d u c e d i n s i z e and a p p e a r v a c u o l a t e d . ( F i g . 3 2 ) . H e n d e r s e n (1962) f o u n d t h a t t h e s e c e l l s i n S a l v e l i n u s f o n t i n a l i s p o s s e s s l o n g p r o t o p l a s m i c p r o c e s s e s on t h e i r f r e e s u r f a c e s . I n C. p l u m b e u s t h e i r f r e e m a r g i n s show an i r r e g u l a r o u t l i n e r e m i n i s c e n t o f mucus s e c r e t i n g c e l l s w h i c h have d i s c h a r g e d t h e i r m u c o i d a l c o n t e n t . I n p o s t spawned t e s t i s t h e y become f l a t . I t i s p o s s i b l e t h a t t h e p r o c e s s e s f o u n d i n t h e b r o o k t r o u t a r e c h a n g e s b r o u g h t a b o u t i n ' t h e c e l l a f t e r t h e end' o f i t s s e c r e t o r y a c t i v i t y . I n C. p l u m b e u s a c o r t i c a l and m e d u l l a r y zone c a n n o t be d i s t i n g u i s h e d . L o b u l e s a d j a c e n t t o t h e d u e t s n e a r t h e - 26 -p o s t e r i o r end o f t h e t e s t i s i n m a t u r i n g f i s h seem t o be s p e r m a t o g e n e t i c a l l y s l i g h t l y more a d v a n c e d t h a n t h e r e s t b u t t h e r e i s no o t h e r i n d i c a t i o n o f any a n t e r o p o s t e r i o r s e r i a t i o n . S e a s o n a l C h anges i n t h e T e s t i s I t i s e s s e n t i a l t o know t h e n o r m a l a n n u a l c y c l e o f t h e t e s t i c u l a r c h a n g e s b e f o r e p l a n n i n g any l o n g t e r m i n v e s t i g a t i o n o f t h e c o n t r o l l i n g mechanism. The male gonad o f C o u e s i u s  p l u m b e u s u n d e r g o e s s e a s o n a l l y a r e g u l a r c y c l i c a l change i n g r o s s a p p e a r a n c e , w e i g h t , and s p e r m a t o g e n e t i c a c t i v i t y ( F i g . 7 ) . T h e r e i s l i t t l e c hange i n t h e l i p i d c o n t e n t o f t h e t e s t i s ; t h i s i s n e v e r p r e s e n t i n q u a n t i t i e s s u f f i c i e n t t o be u s e f u l as a d i s c r i m i n a t o r y h i s t o c h e m i c a l t e s t . A s l i g h t s e a s o n a l v a r i a t i o n was o b s e r v e d i n t h e c h o l e s t e r o l c o n t e n t o f t h e l o b u l e b o u n d a r y c e l l s ( T a b l e I ) . S p e r m a t o g e n e t i c c y c l e . . A l t h o u g h c y c l i c a l c h a n g e s i n f i s h t e s t e s h a v e ' o f t e n been s t u d i e d ( T u r n e r , 1919; C r a i g - B e n n e t t , 1931; M a t t h e w s , 1938; B u r g e r , 1939; B u l l o u g h , 1939; J o n e s , 1940; F r e d e r i c k , 1 9 4 1; W e i s e l , 1943; James, 1946; H e n d e r s e n , 1962; B a r r , 1963; H o f f m a n , 1 9 6 3 ) , t h e methods have r a r e l y been q u a n t i t a t i v e . The t e s t i s o f C o u e s i u s p l u m beus l e n d s i t s e l f a d m i r a b l y t o a d i s c r i m i n a t o r y q u a n t i t a t i v e s t u d y due t o t h e c o m p l e t e l a c k o f z o n a t i o n and o f a n t e r o p o s t e r i o r s e r i a t i o n , and a l s o t h e f a c t t h a t t h e o r g a n i s composed o f r a t h e r d i s c r e t e l o b u l e s i n w h i c h v a r i o u s s p e r m a t o g e n e t i c n e s t s and t h e i r s t a t e o f a c t i v i t y can be e a s i l y c h a r t e d f o r t h e e s t i m a t i o n o f r e l a t i v e a b u n d a n c e . The a n n u a l c y c l e c a n be c o n v e n i e n t l y d i v i d e d i n t o f i v e c l e a r l y d i s t i n g u i s h a b l e s t a g e s as f o l l o w s : - 27 -S t a g e I . - E a r l y i m m a t u r e t e s t i s - c o n s i s t i n g l a r g e l y o f r e s t i n g p r i m a r y s p e r m a t o g o n i a and p r i m a r y germ c e l l s . The b e g i n n i n g o f s p e r m a t o g e n e s i s i s e v i d e n c e d by a • few d i v i d i n g p r i m a r y s p e r m a t o g o n i a and p r e s e n c e o f some s e c o n d a r y s p e r m a t o g o n i a b u t s p e r m a t o c y t e s i f p r e s e n t a r e n o t d i v i d i n g . R e s t i n g s t a g e s c o n s t i t u t e 6 9 - 7 7 % o f t h e mass o f t h e t e s t e s . M i g r a t o r y c e l l s w h i c h a p p e a r t o c o n t r i b u t e t o t h e new s t o c k o f > s p e r m a t o g o n i a a r e o b v i o u s i n t h e e a r l i e r p h a s e o f t h i s s t a g e . C o n d i t i o n o f t e s t i s i n f i s h u n d e r n a t u r a l c o n d i t i o n . d u r i n g S e p t e m b e r and O c t o b e r c o n f o r m s t o t h i s s t a g e . The g o n o s o m a t i c i n d e x ( G S I ) d u r i n g t h i s s t a g e r a n g e s f r o m 0.712 - 0.929 on t h e a v e r a g e . C h o l e s t e r o l t e s t s a r e . f a i n t l y p o s i t i v e t o p o s i t i v e . W i d e s p r e a d p y c n o t i c d e g e n e r a t i o n o f l a t e r g e n e r a t i o n s o f s e c o n d a r y s p e r m a t o g o n i a and p r i m a r y s p e r m a t o c y t e i s common. I n p y c n o t i c n e s t s v a r y i n g d e g r e e s o f c e l l u l a r d e g e n e r a t i o n i s s e e n . Some c e l l s a r e a l m o s t n o r m a l s'o t h a t :the s t a g e o f m a t u r a t i o n c a n be i d e n t i f i e d ; o t h e r s show i n c r e a s e d n u c l e a r b a s o p h i l i a , and f i n a l l y t h e p y c n o s i s o f t h e e n t i r e c e l l ( F i g . 3 3 B ) . S t a g e I I . - .Late i m m a t u r e t e s t i s - c o n s i s t s o f a ph a s e o f s l o w s u s t a i n e d m i t o t i c a c t i v i t y as w e l l as a b e g i n n i n g o f m e i o t i c d i v i s i o n . . S p e r m a t o g o n i a c o n t i n u e t o d i v i d e b u i l d i n g up a s t o c k o f c e l l s f o r t r a n s f o r m a t i o n i n t o s p e r m a t o c y t a l s t a g e s . P r i m a r y s p e r m a t o c y t a l n e s t s i n c r e a s e i n ' e x t e n t . P y c n o t i c d e g e n e r a t i o n s l o w s down and f i n a l l y a l m o s t c e a s e s . P r i m a r y s p e r m a t o c y t e s ^ ^ ' .' \, ..I : • - 27 - . S t a g e I . - E a r l y i m m a t u r e t e s t i s - c o n s i s t i n g l a r g e l y o f r e s t i n g p r i m a r y s p e r m a t o g o n i a and ' p r i m a r y germ c e l l s . The b e g i n n i n g o f s p e r m a t o g e n e s i s i s e v i d e n c e d by a few d i v i d i n g p r i m a r y s p e r m a t o g o n i a and p r e s e n c e o f some s e c o n d a r y s p e r m a t o g o n i a b u t s p e r m a t o c y t e s i f p r e s e n t a r e n o t d i v i d i n g . R e s t i n g s t a g e s c o n s t i t u t e 6 9 - 7 7 % o f t h e mass o f t h e t e s t e s . M i g r a t o r y c e l l s w h i c h a p p e a r t o c o n t r i b u t e t o t h e new s t o c k o f s p e r m a t o g o n i a a r e o b v i o u s i n the. e a r l i e r p h a s e o f t h i s s t a g e . C o n d i t i o n o f t e s t i s i n f i s h u n d e r n a t u r a l c o n d i t i o n ' . d u r i n g S e p t e m b e r and O c t o b e r c o n f o r m s t o t h i s s t a g e . The g o n o s o m a t i c i n d e x ( G S I ) d u r i n g t h i s s t a g e r a n g e s f r o m 0.712 - 0.929 on t h e a v e r a g e . C h o l e s t e r o l t e s t s a r e f a i n t l y p o s i t i v e t o p o s i t i v e . W i d e s p r e a d p y c n o t i c d e g e n e r a t i o n o f l a t e r g e n e r a t i o n s o f s e c o n d a r y s p e r m a t o g o n i a and p r i m a r y s p e r m a t o c y t e i s common. I n p y c n o t i c n e s t s v a r y i n g d e g r e e s o f c e l l u l a r d e g e n e r a t i o n i s s e e n . Some c e l l s a r e a l m o s t n o r m a l s'o t h a t :tthe s t a g e o f m a t u r a t i o n c a n be i d e n t i f i e d ; o t h e r s show i n c r e a s e d n u c l e a r b a s o p h i l i a , and f i n a l l y t h e p y c n o s i s o f t h e e n t i r e c e l l ( F i g . 3 3 B ) . S t a g e I I . - .Late i m m a t u r e t e s t i s - c o n s i s t s o f a ph a s e o f s l o w : s u s t a i n e d m i t o t i c a c t i v i t y as w e l l as a b e g i n n i n g o f m e i o t i c d i v i s i o n . - S p e r m a t o g o n i a c o n t i n u e t o d i v i d e b u i l d i n g up a s t o c k o f c e l l s f o r t r a n s f o r m a t i o n i n t o s p e r m a t o c y t a l s t a g e s . P r i m a r y s p e r m a t o c y t a l n e s t s i n c r e a s e i n e x t e n t . P y c n o t i c d e g e n e r a t i o n s l o w s down and f i n a l l y a l m o s t c e a s e s . P r i m a r y s p e r m a t o c y t e s - 28 -d i v i d e t o p r o d u c e a.-few- s e c o n d a r y s p e r m a t o c y t a l n e s t s ; t h e c h o l e s t e r o l t e s t i s p o s i t i v e . The c o n d i t i o n o f t e s t i s i n t h e f i s h i n n a t u r e d u r i n g November t o M a r c h c o n f o r m t o t h i s p hase o f a n n u a l c y c l e . The GSI a v e r a g e s 1.355 i n November t o .1.384 i n M a r c h . R e s t i n g s t a g e s c o n s t i t u t e a b o u t 7 5 % i n November t o 4 1 % i n M a r c h . S t a g e I I I . - M a t u r i n g t e s t i s - shows b r i s k s p e r m a t o g e n e t i c a c t i v i t y and t h e b e g i n n i n g o f s p e r m i o g e n e s i s . . S e c o n d a r y s p e r m a t o g o n i a and s p e r m a t o c y t e s c o n t i n u e t o d i v i d e f o l l o w e d by t r a n s f o r m a t i o n o f s e c o n d a r y s p e r m a t o c y t e s i n t o s p e r m a t i d s and s p e r m s . C h o l e s t e r o l t e s t s a r e n e g a t i v e . The GSI r a n g e s f r o m 1.505 i n A p r i l t o 2.069 i n May. T h i s s i t u a t i o n i s e x h i b i t e d i n n a t u r e by f i s h d u r i n g A p r i l and May. S t a g e I V . - M a t u r e t e s t i s - t h i s s t a g e e x h i b i t s c o n t i n u e d s p e r m i o g e n e t i c a c t i v i t y w h i l e t h e s p e r m a t o g e n e t i c a c t i v i t y a l s o c o n t i n u e s . R e s t i n g s t a g e s now c o n s t i t u t e o n l y 3-4% o f t h e t o t a l mass o f t h e t e s t i s . Sperms f i l l t h e d i s t e n d e d e f f e r e n t d u c t s . C h o l e s t e r o l t e s t s a r e n e g a t i v e . The s p a w n i n g t e s t i s i s f o u n d i n n a t u r e d u r i n g J u n e and J u l y . U n l i k e most o t h e r s p e c i e s ( n o n - c y p r i n i d ) o f f i s h , .the s p a w n i n g t e s t i s i n C o u e s i u s p l u m b eus does n o t show any s p e r m a t o g e n e t i c a r r e s t and s l o w s p e r m a t o g e n e t i c a c t i v i t y seems t o c o n t i n u e . The GSI a v e r a g e s 2.137 i n June t o 1.806 i n J u l y . - 29 -S t a g e V. - The s p e n t t e s t i s ^ shows c o m p l e t i o n o f s p e r m i a t i o n . M i t o t i c a c t i v i t y i s r a r e and r e s t i n g s t a g e s c o n s t i t u t e 94% o f t h e t o t a l mass o f t h e g o n a d . T h i s i s t h e o n l y q u i e s c e n t p h a s e i n t h e a n n u a l c y c l e o f C o u e s i u s testes and c a n be c a l l e d a p h a s e o f s p e r m a t o g e n e t i c r e c e s s . P r i m a r y g e r m - c e l l s and s p e r m a t o g o n i a p r e d o m i n a t e . Some r e s i d u a l sperms may be p r e s e n t . T h i s s i t u a t i o n i n n a t u r e o c c u r s i n f i s h d u r i n g A u g u s t . C h o l e s t e r o l t e s t s a r e n e g a t i v e . The GSI averag.es 0. 5 3 . These f i v e s t a g e s i n t h e a n n u a l c y c l e a r e i n t i m a t e l y r e l a t e d t o t h e c h a n g e s i n t h e e n v i r o n m e n t . The t e s t i c u l a r c y c l e i n C o u e s i u s p l u m b e u s i s c o n t i n u o u s , e x c e p t f o r a s h o r t , q u i e s c e n t p e r i o d i n l a t e J u l y and August-. From S e p t e m b e r up t o M a r c h t h e r e i s a g r a d u a l b u i l d - u p o f s p e r m a t o g o n i a ! s t a g e s w h i c h s t a r t p r o d u c i n g s p e r m a t o c y t i a i s t a g e s i n e a r l y November ( F i g . 6 ) . The n u c l e a r d i v i s i o n i s f - ' m i t o t i c t i l l t h e f o r m a t i o n o f p r i m a r y s p e r m a t o c y t e s up t o M a r c h . The f i r s t m a t u r a t i o n d i v i s i o n o c c u r s a t t h e p r i m a r y s p e r m a t o c y t e s t a g e and t h i s i s h e l d o v e r u n t i l t h e b e g i n n i n g o f s p r i n g . I n l a t e s p r i n g t h e r e i s a b u r s t o f m e i o t i c a c t i v i t y and t h e f i n a l s t a g e i n t h e m a t u r a t i o n o f t h e t e s t i s i s a t t a i n e d . I n summary t h e c y c l e i s as f o l l o w s : m i t o t i c m u l t i p l i c a t i o n o f s p e r m a t o g o n i a b u i l d - u p o f p r i m a r y s p e r m a t o c y t e ^ m e i o t i c d i v i s i o n p r o d u c i n g s e c o n d a r y s p e r m a t o c y t e s and s p e r m a t i d s • c o m p l e t i o n o f spermio gene s i s f o l l o w e d by s p e r m i a t i o n . As e v i d e n c e d f r o m t h e h i s t o l o g y o f t h e t e s t e s and t h e - 30 -F i g . 6 . S e a s o n a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f n o r m a l C o u e s i u s • • S p e r m a t o g o n i a , 0 0 S p e r m a t o c y t e s § f Sperms X X P y c n o t i c n e s t s - 31 -F i g . 7 . C y c l i c a l c h a n g e s i n t h e a c t i v i t y o f t h e t e s t e s i n n o r m a l f i s h ; 0 0 D i v i d i n g s t a g e s • f R e s t i n g s t a g e s ( s p e r m s e x c l u d e d ) - 32 -a p p e a r a n c e o f e a r l y f r y i n t h e l a k e (Geen, 1955) s p a w n i n g o c c u r s f r o m m i d June t o m i d J u l y w i t h a p o s s i b l e week's e x t e n s i o n a t e i t h e r end. F i s h s t a r t s p a w n i n g b e f o r e t h e l o b u l e s a r e c o m p l e t e l y e n g o r g e d w i t h s p e r m , and some r e m n a n t spermsmay be f o u n d i n a few f i s h as l o n g as one o r two months a f t e r s p a w n i n g . The r a n g e i n g o n o s o m a t i c i n d i c e s i n C. p l u m b e u s ( 0 . 5 3 0 9 t o 2.1028) i s much l e s s t h a n f o u n d f o r most o t h e r t e l e o s t . I n F u n d u l u s h e t e r o c l i t u s P i c k f o r d (1953) f o u n d a r e g r e s s e d f i s h w i t h GSI a v e r a g e 0.72 w h i c h r o s e t o 5.84 d u r i n g t h e peak o f s p e r m a t o g e n e t i c a c t i v i t y . S i m i l a r e x t r e m e s o f r a n g e s o f t e s t i c u l a r s i z i $x\d w e i g h t have been r e p o r t e d by H a r r i n g t o n (1959) i n E n n e a c a n t h u s o b e s a u s ^ W e i s e l ( 1 9 4 3 ) i n O n c o r h v n c h u s n e r k a . and by B a r r ( 1 9 6 3 c ) ' i n P l e u r o n e c t e s p l a t e s s a . I n M v l o c h e i l u s c a u r i n u m . S c o t t ( 1 9 5 2 ) f o u n d a f i v e - ^ f o l d i n c r e a s e i n t e s t e s w e i g h t d u r i n g the. a c t i v e p h a s e . S e a s o n a l v a r i a t i o n i n t h e g o n o s o m a t i c i n d i c e s o f t h e male C y p r i n i d s a p p e a r t o be o f a l e s s e r o r d e r t h a n t h a t of i o t h e r t e l e o s t s . H i s t o l o g i c a l F e a t u r e s o f P o s t - S p a w n e d T e s t i s The spawned t e s t e s o f Q. p l u m b e u s h^tve s e v e r a l d i s t i n c t i v e f e a t u r e s w h i c h w i l l be r e f e r r e d t o i n l a t e r e x p e r i m e n t a l w o r k. C o n s e q u e n t l y some m e n t i o n i s made a t t h i s s t a g e . The, f i r s t d i s t i n c t i v e c h a r a c t e r i s t i c o f s u c h t e s t e s i s th e p r e s e n c e o f i n c r e a s i n g numbers o f m i g r a t i n g c e l l s w h i c h a r e s t r u c t u r a l l y and t i n c t o r i a l l y d i s t i n c t f r o m a l l o t h e r c e l l t y p e s . The o t h e r f e a t u r e i s t h e b e g i n n i n g o f p y c n o t i c d e g e n e r a t i o n o f r e m n a n t p r i m a r y s p e r m a t o c y t e s and l a t e r g e n e r a t i o n o f s e c o n d a r y s p e r m a t o g o n i a . I n Q. p l u m b e u s p y c n o t i c d e g e n e r a t i o n o f l a t e s e c o n d a r y s p e r m a t o g o n i a ! n e s t s i s a r e g u l a r f e a t u r e o f t h e a n n u a l - 3 3 . -c y c l e . I t a p p e a r s i n s m a l l m e asure i n some A u g u s t f i s h and g r a d u a l l y i n c r e a s e s t o a maximum o f an a v e r a g e o f 16% o f t h e t e s t i s mass i n O c t o b e r . A f t e r t h i s p e a k , p y c n o t i c d e g e n e r a t i o n s h a r p l y d r o p s t o 8% i n O c t o b e r and g r a d u a l l y c e a s e s by M a r c h . P y c n o t i c d e g e n e r a t i o n n e v e r o c c u r s i n f i s h w h i c h have n o t y e t c o m p l e t e d t h e i r c y c l e . F i s h w h i c h a r e a b o u t 57 mm f o r k l e n g t h o r l e s s do n o t show p y c n o t i c s t a g e s and c o n s e q u e n t l y t h i s i s a good i n d i c a t i o n o f w h e t h e r t h e f i s h has r e a c h e d m a t u r i t y o r n o t . Remnant s p e r m s , c o l l a p s e d s e m i n i f e r o u s l o b u l e s and s p e r m a t i c d u c t s a r e o t h e r f e a t u r e s common t o spawned f i s h . I n C. plumbeus unspawned sperms a r e c a s t o u t f a i r l y s oon a f t e r s p a w n i n g . A r e g u l a r p y c n o t i c d e g e n e r a t i o n o f ' s p e r m a t o g e n e t i c n e s t s o f t h i s m a g n i t u d e i s e v i d e n t l y o f a r a r e o c c u r r e n c e i n o t h e r s p e c i e s o f f i s h , and h a s n o t been h i t h e r t o d e s c r i b e d . S u c h d e g e n e r a t i o n has been n o t e d i n f r o g s by B a s u ( p e r s o n a l c o m m u n i c a t i o n ) , and by R o b e r t s o n & W e x l e r (1960) i n P a c i f i c s a l m o n . R e l a t i o n o f t h e A n n u a l C y c l e t o E n v i r o n m e n t a l Changes T e m p e r a t u r e o f t h e w a t e r o f t h e l a k e and ,the amount o f d a y l i g h t a v a i l a b l e a t t h e l a t i t u d e o f t h e l a k e were t h e two e n v i r o n m e n t a l f a c t o r s i n v e s t i g a t e d . F i g u r e 5 shows t h e g o n o s o m a t i c i n d i c e s o f f i s h o b t a i n e d f r o m t h e i r n a t u r a l e n v i r o n m e n t a t d i f f e r e n t p a r t s o f t h e i r y e a r l y c y c l e . The l a k e i s c o v e r e d , w i t h i c e f r o m l a t e November t o e a r l y A p r i l . From l a t e A p r i l , t h e t e m p e r a t u r e o f t h e w a t e r r i s e s r a p i d l y and t h e f i n a l s t a g e s o f t h e m a t u r a t i o n o c c u r d u r i n g t h i s i n c r e a s i n g t e m p e r a t u r e p e r i o d . The peak i n w a t e r t e m p e r a t u r e i s r e a c h e d i n A u g u s t . T h i s i s t h e p o s t - s p a w n i n g p e r i o d when t h e gonads a r e l e a s t a c t i v e .  *"S. L. Basu (19&3) P e r s o n a l C o m m u n i c a t i o n . - 3 4 -The f i n a l p h a s e o f m a t u r a t i o n i s a l s o c o i n c i d e n t w i t h a m a r k e d i n c r e a s e i n amount o f d a y l e n g t h . The peak gonad w e i g h t i s o b t a i n e d i n J u n e when t h e d a y l e n g t h i s maximum. From t h e p r e s e n t h i s t o l o g i c a l s t u d i e s as w e l l as t h e e a r l i e r f i e l d s t u d i e s ( G e e n , 1955) i t i s e v i d e n t t h a t s p a w n i n g commences a b o u t t h i s t i m e and s p e r m i a t i o n i s c o m p l e t e d i n J u l y . The t e m p e r a t u r e o f t h e w a t e r d e c l i n e s r a p i d l y a f t e r A u g u s t and t h e r e i s a s i m u l t a n e o u s r a p i d d e c r e a s e i n d a y l e n g t h . The r e c o n s t i t u t i o n o f the. t e s t i s , w h i c h i s a s l o w p r o c e s s , o c c u r s . , d u r i n g t h i s p e r i o d o f f a l l i n g t e m p e r a t u r e s and d e c r e a s i n g d a y l e n g t h . The s p e e d o f r e c o n s t i t u t i o n i s s l o w a t f i r s t b u t more r a p i d i n O c t o b e r . From November %o M a r c h . a n a l m o s t . s t e a d y s t a t e i s m a i n t a i n e d as i s e v i d e n t i n t h e s l o w b u i l d - u p o f b o t h t h e l a t e r s p e r m a t o g e n e t i c s t a g e s and t h e w e i g h t o f t h e gonad. " • \ I t i s " o b v i o u s t h a t w a t e r t e m p e r a t u r e r e m a i n s a l m o s t c o n s t a n t f r o m l a t e November t o e a r l y A p r i l ( Geen, 1955) a l t h o u g h d a y l e n g t h i n c r e a s e s s t e a d i l y f r o m l a t e December o n w a r d s . From b e g i n n i n g S e p t e m b e r t h r o u g h M a r c h , a l a r g e s t o r e o f s e c o n d a r y s p e r m a t o g o n i a (SPG") and p r i m a r y s p e r m a t o c y t e s ( S P C ) i s b u i l t up b u t s p e r m a t o g e n e s i s seems t o s t o p a t t h e p r i m a r y s p e r m a t o c y t e l e v e l and t h e f i s h p a s s t h e e n t i r e w i n t e r i n t h i s c o n d i t i o n . S c o t t ( 1 9 5 2 ) t h o u g h t t h a t i n a l o c a l c y p r i n i d M y l o c h e i l u s c a u r i n u m t h e q u i e s c e n t w i n t e r c o n d i t i o n i s p a s s e d i n • t h e s e c o n d a r y s p e r m a t o c y t e s t a g e , i . e . i n t h e s e c o n d m a t u r a t i o n d i v i s i o n . I t i s . u n i v e r s a l l y a g r e e d t h a t t h e s e c o n d a r y s p e r m a t o c y t e i s a t r a n s i e n t p h a s e i n s p e r m a t o g e n e t i c c y c l e o f f i s h e s ( F o l e y , 1926; V a u p e l , 1929; B e n n i n g t o n , 1936; W e i s e l , 1943; B u l l o u g h , ,1939) and c o n s e q u e n t l y S c o t t ' s s e c o n d a r y - 35 -s p e r m a t o c y t e may. have a c t u a l l y been t h e p r i m a r y s p e r m a t o c y t e s t a g e . P o s t s p e r m a t o c y t a l d e v e l o p m e n t i n C. p l u m b e u s o c c u r s i n A p r i l and May when t h e t e m p e r a t u r e o f t h e w a t e r s t a r t s i n c r e a s i n g a t a r a p i d r a t e . L o b u l e B o u n d a r y C e l l s These c e l l s a l s o a p p e a r t o e x h i b i t a c y c l i c a l change c o m p a r a b l e t o t h e s p e r m a t o g e n e t i c s t a g e s w i t h an i n c r e a s e i n number d u r i n g S t a g e s I and I I and an i n t e n s i f i c a t i o n i n t h e c h o l e s t e r o l t e s t , w h i c h l a t e r d e c l i n e s . I n t r a l o b u l a r L i p i d s T e s t e s f r o m f i s h o b t a i n e d t h r o u g h t h e v a r i o u s p h a s e s o f t h e a n n u a l c y c l e were s t a i n e d w i t h Sudan B l a c k B f o r t h e d e m o n s t r a t i o n l i p i d s . I n g e n e r a l t h e i n t r a l o b u l a r l i p i d i s v e r y l o w . P r i m a r y germ c e l l s and. t h e l o b u l e b o u n d a r y a r e t h e o n l y c e l l u l a r elements w h i c h show l i p o i d a l n a t u r e . U n l i k e b i r d s , some a m p h i b i a n s ( L o f t s , 1961) and some f i s h , e.g. E s o x l u c i u s ( L o f t s and M a r s h a l l , 1957) C o u e s i u s t e s t e s do n o t u n d e r g o any p o s t s p a w n i n g s t e a t o g e n e s i s . C h o l e s t e r o l T e s t The r e s u l t s o f l i p i d and c h o l e s t e r o l s t a i n i n g f o r n o r m a l t e s t e s a r e g i v e n i n T a b l e I . •< ' • EFFECT OF HYPOPHYSECTOMY I n t h e p r e s e n t s t u d y h y p o p h y s e c t o m i s e d f i s h were m a i n t a i n e d f o r p e r i o d s v a r y i n g f r o m 46 t o 63 d a y s . I n 14 e x p e r i m e n t s c o n d u c t e d f r o m J u l y 1962 t o M a r c h 1963 and f r o m May t o O c t o b e r , 1 9 6 3 , a t o t a l o f 374 h y p o p h y s e c t o m i s e d f i s h were k e p t s u c c e s s f u l l y u n t i l a u t o p s y . Of t h e s e 306 f i s h r e c e i v e d - 36 -C o m p a r i s o n o f t h e GSI o f h y p o p h y s e c t o m i s e d f i s h w i t h t h e sham o p e r a t e d c o n t r o l s , and n o r m a l s ( a t t h e t i m e o f t h e c o m p l e t i o n o f e a c h e x p e r i m e n t ) s z / \ /"N. NORMAL i J A S O N J F M M J J A S O EXPERIMENTS - 37 -r e p l a c m e n t t h e r a p y and t h e r e s t were c o n t r o l s . T h e r e i s a m a r k e d d e c r e a s e i n t h e g o n o s o m a t i c i n d e x ( G S I ) o f t h e h y p o p h y s e c t o m i s e d f i s h i n a l l e x p e r i m e n t s . T h i s d e c r e a s e i n t e s t i c u l a r w e i g h t i s most m a r k e d i n e x p e r i m e n t a l • g r o u p s f o r F e b r u a r y 1962, M a r c h 19 6 3 , and May 1963 ( F i g . 8 ) . D i f f e r e n c e s b e t w e e n t h e GSI o f h y p o p h y s e c t o m i s e d and sham o p e r a t e d c o n t r o l s a r e l e a s t i n J u l y 1962, A u g u s t 1962, and J u l y 1963 e x p e r i m e n t s , as c o m p a r e d t o o t h e r g r o u p s . F i s h b e l o n g i n g t o t h e F e b r u a r y 1963, M a r c h 1963 and May 1963 e x p e r i m e n t s were k i l l e d b e t w e e n l a t e M a r c h and end o f J u n e . T h i s i s t h e p e r i o d o f maximum t e s t i c u l a r a c t i v i t y i n n a t u r e and t h e r e m o v a l o f t h e p i t u i t a r y g l a n d a t t h i s p h a s e has a more d e l e t e r i o u s e f f e c t on t h e t e s t e s t h a n d u r i n g t h e r e l a t i v e l y i n a c t i v e p e r i o d . V i v i e n ( 1 9 4 1) a l s o n o t e d most a c u t e r e g r e s s i o n o f t h e g onads i n f i s h o p e r a t e d a t t i m e s o f t h e i r g r e a t e s t r e p r o d u c t i v e a c t i v i t y . F i s h i n t h e l a t t e r s e t o f e x p e r i m e n t s q u o t e d above w ere s a c r i f i c e d i n l a t e A u g u s t and O c t o b e r . D u r i n g t h e s e months t h e c o n t r o l s a r e most r e g r e s s e d as i s t r u e a l s o u n d e r n a t u r a l c o n d i t i o n s and f o r t h i s r e a s o n t h e GSI o f t h e h y p o p h y s e c t o m i s e d and sham o p e r a t e d c o n t r o l i n J u l y and A u g u s t e x p e r i m e n t s a r e n o t w i d e l y a p a r t . H i s t o l o g i c a l C hanges At a l l t i m e s o f t h e y e a r t h e u n t r e a t e d hypophysectomised f i s h p r e s e n t a r a t h e r u n i f o r m p i c t u r e o f s p e r m a t o g e n e t i c a r r e s t w i t h t e s t i c u l a r r e g r e s s i o n a f f e c t i n g b o t h t h e r e l a t i v e g onad w e i g h t and t h e h i s t o l o g i c a l a p p e a r a n c e . The s e m i n i f e r o u s l o b u l e s s h r a n k t o a r a t h e r u n i f o r m s i z e and s h a p e c o m p a r a b l e t o s t a g e V o r I o f t h e a n n u a l c y c l e - 38 -as d e s c r i b e d on page 27. t h e m a j o r i t y o f s p e r m a t o g e n e t i c s t a g e s were composed o f p r i m a r y germ c e l l s and r e s t i n g p r i m a r y s p e r m a t o g o n i a ( F i g . 1 0 ) . These n e s t s a c t u a l l y c o n s i s t o f a g r e a t e r p r o p o r t i o n o f r e s t i n g n u c l e i w i t h o n l y a drew s h o w i n g some m i t o t i c a c t i v i t y . The r e s t i n g c e l l s c o n s t i t u t e 4 2 % o f t h e t o t a l mass o f t h e t e s t i s , s e c o n d a r y s p e r m a t o g o n i a , a r e s c a r c e c o n s t i t u t i n g an a v e r a g e o f 5.9% o f t h e s p e r m a t o g e n e t i c s t a g e s ( T a b l e I I ) . . M i t o t i c f i g u r e s a r e p r e s e n t b u t r a r e . The h i s t o l o g y o f t h e t e s t i s f r o m an u n t r e a t e d h y p o p h y s e c t o m i s e d f i s h i s shown i n F i g u r e 16A. I t a p p e a r s t h a t t h e p r i m a r y s p e r m a t o g o n i a l d i v i s i o n i s s u p p r e s s e d a l t h o u g h n o t t o t a l l y s t o p p e d . D i v i s i o n i n t h e s e c o n d a r y s p e r m a t o g o n i a l s t a g e s i s r a r e l y s e e n . I f l a t e r s t a g e s w e r e p r e s e n t a t t h e b e g i n n i n g o f t h e e x p e r i m e n t , t h e y a r e e i t h e r e x p e l l e d o r d e g e n e r a t e . Some p h a g o c y t i c d e g e n e r a t i o n and c l u m p i n g o f c e l l s o c c u r s d u r i n g f a l l and w i n t e r ; t h e s t a g e s j D h a g o c y t i s e d a r e l a t e r s p e r m a t o g o n i a and s p e r m a t o c y t e s . However p y c n o s i s i n h y p o p h y s e c t o m i s e d f i s h i s r a r e . Remnant sperms a r e n e v e r f o u n d * S i n c e c l u m p e d d e g e n e r a t i n g c e l l s a r e s e e n i n t h e t e r m i n a l p a r t s o f t h e d u c t s i n some e x p e r i m e n t s i t a p p e a r s t h a t l a t e r s t a g e s a r e m o s t l y e x p e l l e d t h r o u g h t h e d u c t s . S p e r m i a t i o n i s n o t i m p e d e d by t h e r e m o v a l o f t h e p i t u i t a r y g l a n d . F i s h o p e r a t e d d u r i n g t h e p r e s p a w n i n g and s p a w n i n g p h a s e o f t h e i r a n n u a l c y c l e a p p e a r t o s p e r m i a t e n o r m a l l y as r e m n a n t s sperms a r e r a r e l y s e e n . V i v i e n (1941) f o u n d t h a t an a p p r e c i a b l e number o f h y p o p h y s e c t o m i s e d G o b i u s p a o a n e l l u s a r e a b l e t o e m i t m i l t . B a r r ( 1 9 6 3 c ) has a l s o o b s e r v e d t h a t s p e r m i a t i o n o c c u r s n o r m a l l y i n t h e h y p o p h y s e c t o m i s e d f i s h . - 39 -However,he n o t e d t h e r e t e n t i o n o f r e m n a n t sperms f o r a c o n s i d e r a b l e t i m e . T h e r e i s an a p p a r e n t i n c r e a s e i n t h e number o f f i b r o b l a s t s i n o p e r a t e d f i s h t e s t e s . An i n c r e a s e i n t h e numbers o f t h e s e c e l l s s o m e t i m e s g i v e s t h e a p p e a r a n c e o f f i b r o s i s i n f i s h k e p t f o r l o n g e r p e r i o d s ( F i g . 1 6 A ) . S u c h f i b r o t i c d e v e l o p m e n t has a l s o b een n o t e d by B a r r ( 1 9 63) i n f i s h k e p t f o r a b o u t a y e a r . I n h i s f i s h f i b r o s i s seems t o have p r o g r e s s e d f a r t h e r , a p p a r e n t l y due t o t h e l e n g t h o f t h e t i m e f i s h were o b s e r v e d . No o b v i o u s e f f e c t s o f h y p o p h y s e c t o m y were s e e n i n t h e l o b u l e b o u n d a r y c e l l s h i s t o l o g i c a l l y , b u t t h e i r s e c r e t o r y a c t i v i t y was c o m p l e t e l y s u p p r e s s e d as e v i d e n c e d by n e g a t i v e c h o l e s t e r o l t e s t s ( T a b l e I I I ) . I n t h e p r e s e n t s t u d y m a r k e d r e g r e s s i o n was e v i d e n c e d w i t h i n 46 d a y s a f t e r t h e h y p o p h y s e c t o m y . B o t h V i v i e n ( 1 9 4 1) and B a r r ( 1 9 6 3 c ) seem t o have f o u n d a much s l o w e r c h a n g e . V i v i e n ( 1 9 4 1 ) d i d n o t n o t i c e any e v i d e n c e o f i n v o l u t i o n i n G o b i u s  p a q a n e l l u s f o r a l m o s t t h r e e months a f t e r t h e o p e r a t i o n . T a v o l g a ( 1 9 5 5) h o w e v e r on t h e same f i s h f o u n d u n m i s t a k a b l e s i g n s o f r e g r e s s i o n 30 d a y s a f t e r h y p o p h y s e c t o m y . S i n c e T a v o l g a ' s o p e r a t e d f i s h were m a i n t a i n e d a t t e m p e r a t u r e s h i g h e r t h a n t h o s e o f V i v i e n ' s , i t i s p o s s i b l e t h a t an a c c e l e r a t i o n i n r e g r e s s i o n f o l l o w i n g p i t u i t a r y r e m o v a l i s c a u s e d by h i g h e r w a t e r t e m p e r a t u r e . E v i d e n c e w i l l be p r e s e n t e d l a t e r w h i c h show t h a t r e g r e s s i o n i s s p e e d e d a t h i g h e r t e m p e r a t u r e s i n C o u e s i u s p l u m b e u s . C h o l e s t e r o l and L i p i d T h e r e i s l i t t l e c hange i n t h e l i p i d c o n t e n t o f t h e t e s t e s due t o h y p o p h y s e c t o m y . The l o b u l e b o u n d a r y c e l l s r e t a i n - 40 -t h e i r l i p o i d a l n a t u r e a l t h o u g h t h e s t a i n i n g seems t o be l e s s i n t e n s e t h a n i n t h e sham o p e r a t e d c o n t r o l s . C h o l e s t e r o l t e s t s a r e a l w a y s n e g a t i v e ( T a b l e I I I ) . I n t h e n o r m a l fish',.; a p o s i t i v e c h o l e s t e r o l t e s t ^'s o b t a i n e d o n l y d u r i n g t h e O c t o b e r t o M a r c h p a r t o f t h e a n n u a l c y c l e . A n e g a t i v e t e s t i n t h e hypophysectomised f i s h d u r i n g t h i s p e r i o d . i n d i c a t e s a r e d u c t i o n i n t h e a c t i v i t y o f t h e l o b u l e b o u n d a r y c e l l s due t o p i t u i t a r y r e m o v a l . S i n c e c h o l e s t e r o l t e s t s i n n o r m a l f i s h d u r i n g t h e r e s t o f t h e i r a n n u a l c y c l e i s n e g a t i v e i t i s n o t p o s s i b l e t o show any s e a s o n a l v a r i a t i o n i n t h e r e s p o n s e o f t h e s e c e l l s t o h y p o p h y s e c t o m y . The r e m a i n d e r o f t h e t e s t i s shows n e i t h e r a m a r k e d l i p o i d a l c o n t e n t n o r g i v e s any i n d i c a t i o n o f t h e p r e s e n c e o f c h o l e s t e r o l . I t w o u l d a p p e a r t h e r e f o r e t h a t s t e a t o g e n e s i s o r a c c u m u l a t i o n o f l i p i d i n t h e t e s t e s i n C. p l u m b e u s does n o t o c c u r due t o h y p o p h y s e c t o m y as i s t h e c a s e i n f r o g s ( L o f t s , . 1 9 6 1 ) . A l t h o u g h t h e h y p o p h y s e c t o m i s e d f i s h f e d w e l l d u r i n g t h e e x p e r i m e n t t h e y a l l showed a s l i g h t d e c r e a s e i n body w e i g h t when c o m p a r e d w i t h t h e sham o p e r a t e d c o n t r o l s o f s i m i l a r f o r k l e n g t h s . REPLACEMENT THERAPY E f f e c t o f Mammalian F o l l i c l e S t i m u l a t i n g Hormone (FSH) The GSI o f FSH t r e a t e d f i s h a r e s l i g h t l y b u t n o t s t a t i s t i c a l l y d i f f e r e n t f r o m t h o s e o f t h e h y p o p h y s e c t o m i s e d f i s h . T h e r e a r e s l i g h t v a r i a t i o n s i n t h e GSI o f t h e v a r i o u s e x p e r i m e n t s ( F i g . 9 ) , b u t t h e s e a r e p r o b a b l y due t o c o n t a m i n a n t LH. The s p e r m a t o g e n e t i c p i c t u r e : i s - ; more u n i f o r m t h a n t h e GSI ( F i g . 1 1 ) . The t e s t i c u l a r h i s t o l o g y o f t h e FSH t r e a t e d f i s h i s - 41 -F i g . 9 . C o m p a r i s o n o f t h e GSI o f FSH-, and LH- t r e a t e d f i s h w i t h u n t r e a t e d h y p o p h y s e c t o m i s e d and sham o p e r a t e d c o n t r o l s % • u n t r e a t e d h y p o p h y s e c t o m i s e d c o n t r o l s 0 0 FSH- t r e a t e d f i s h t » LH- t r e a t e d f i s h 0 0 sham o p e r a t e d c o n t r o l s - 4 2 -s i m i l a r t o t h a t o f t h e u n t r e a t e d c o n t r o l s . P r i m a r y s p e r m a t o g o n i a a r e ~tote most p r e d o m i n a n t ^ c o n s t i t u t i n g 3 9 9 9 % o f t h e s p e r m a t o g e n e t i c s t a g e . R e s t i n g germ c e l l s c o n s t i t u t e 1 4 % - 5 1 % b u t a r e l e s s numerous t h a n i n t h e u n t r e a t e d c o n t r o l s . D i v i d i n g p r i m a r y s p e r m a t o g o n i a a r e common. S e c o n d a r y s p e r m a t o g o n i a a r e p r e s e n t b u t c o n s t i t u t e o n l y a v e r y s m a l l p r o p o r t i o n ( 3 . 5 % - 17.7%) o f t h e s p e r m a t o g e n e t i c s t a g e s ( F i g . 1 1 ) . D i v i s i o n i n t h e s e c o n d a r y s p e r m a t o g o n i a a r e r a r e . FSH t r e a t e d f i s h show t e s t e s c o m p a r a b l e t o t h o s e a t S t a g e I o f t h e a n n u a l c y c l e . T h e r e i s ^ t h u s ? a n '. i n c r e a s e i n t h e s e c o n d a r y s p e r m a t o g o n i a i n t h e FSH t r e a t e d f i s h . T h i s i n c r e a s e i s d e f i n i t e o n l y i n e x p e r i m e n t s commenced d u r i n g t h e months o f F e b r u a r y , M a r c h and May. The maximum e f f e c t o f FSH i s . s l i g h t w hen/compared w i t h t h e l e v e l o b t a i n e d f r o m r e p l a c e m e n t t h e r a p y w i t h o t h e r hormones ( F i g . 1 9 ) . T h i s may be c a u s e d by a s m a l l amount o f L u t e i n i z i n g hormone (LH) p r e s e n t as an i m p u r i t y i n t h e FSH, S i n c e t h e p e r i o d f r o m M a r c h t o J u n e i s t h e most r e c e p t i v e p h a s e i n t h e r e p r o d u c t i v e c y c l e o f t h i s f i s h , t h e e f f e c t o f t h i s i m p u r i t y w o u l d be e x p e c t e d t o be maximum d u r i n g t h i s p e r i o d . . E x p e r i m e n t s d u r i n g 1963 show a more c o n s i s t e n t p i c t u r e w i t h t h e GSI o f t h e t r e a t e d f i s h b e i n g c l o s e t o t h o s e o f t h e u n t r e a t e d h y p o p h y s e c t o m i s e d c o n t r o l s ( F i g . 9 ) , i n some e x p e r i m e n t s t h e y a r e l o w e r t h a n i n t h e s e c o n t r o l s ' . None o f t h e s e d i f f e r e n c e s a r e l a r g e . Of a l l t h e f i s h r e c e i v i n g r e p l a c e m e n t t h e r a p y , gonad w e i g h t was l o w e s t among t h e FSH t r e a t e d o n e s . I n g e n e r a l , t h e r e i s no c l e a r e v i d e n c e t h a t FSH was h a v i n g any e f f e c t on e i t h e r t h e GSI o r t h e s p e r m a t o g e n e s i s i n t h i s f i s h . - 43 -C h o l e s t e r o l and L i p i d C h o l e s t e r o l t e s t s were a l w a y s n e g a t i v e ( T a b l e I V ) . T h e r e was v e r y l i t t l e l i p i d m a t e r i a l . ' i n t h e t e s t e s and o v e r a l l p i c t u r e o f V S u d a n s t a i n e d t e s t e s o f FSH t r e a t e d f i s h was s i m i l a r t o t h e u n t r e a t e d c o n t r o l s . FSH t h u s c l e a r l y has no s t i m u l a t i n g a c t i o n on t h e l o b u l e b o u n d a r y c e l l s . E f f e c t o f Mammalian L u t e i n i z i n g Hormone ( L H ) . The g o n o s o m a t i c i n d i c e s o f LH t r e a t e d f i s h a r e m a r k e d l y h i g h e r t h a n t h o s e o f t h e u n t r e a t e d c o n t r o l s ( F i g . 9 ) . I n t h e e a r l i e r two e x p e r i m e n t s o f 1962, f e w e r f i s h ( t h r e e i n J u l y and two i n A u g u s t e x p e r i m e n t s ) were i n j e c t e d w i t h LH due' t o c a s u a l t i e s i n t h e o p e r a t e d f i s h c a u s e d by f u n g a l and ' I c h ' i n f e c t i o n s . The GSI o f : LH t r e a t e d f i s h i n t h e s e two e x p e r i m e n t s a r e somewhat h i g h e r t h a n t h o s e o f t h e f o l l o w i n g months b u t t h e d i f f e r e n c e s may be due t o s m a l l e r s a m p l e s i z e . A t r e n d t o w a r d s i n c r e a s e i n GSI o f LH t r e a t e d f i s h i s a p p a r e n t f r o m J a n u a r y t o M a r c h f o l l o w e d by a m a r k e d r e d u c t i o n i n May. T h i s t r e n d has a l r e a d y b e e n p o i n t e d o u t i n t h e c a s e o f FSH t r e a t e d f i s h . The p e r i o d f r o m F e b r u a r y t o A p r i l t h u s a p p e a r s t o be t h e most r e s p o n s i v e p e r i o d i n . t h e : a n n u a l t e s t i c u l a r c y c l e o f t h i s f i s h i n t h e m a t t e r o f i t s s u s c e p t i b i l i t y t o LH t r e a t m e n t . I p 1963 e x p e r i m e n t s t h e f i s h were s i m i l a r l y s t i m u l a t e d i n e a c h e x p e r i m e n t e x c e p t f o r t h e J u l y e x p e r i m e n t s where t h e e f f e c t was l e s s m a r k e d . The GSI o f LH t r e a t e d f i s h i s a l w a y s h i g h e r t h a n t h o s e o f t h e FSH t r e a t e d o r o f t h e u n t r e a t e d o p e r a t e d c o n t r o l s b u t , e x c e p t f o r t h e J u l y and A u g u s t e x p e r i m e n t s i n 1 962, f a l l s somewhat s h o r t o f t h e l e v e l o f t h e sham o p e r a t e d c o n t r o l s ( F i g . 9 ) . The GSI o f LH t r e a t e d f i s h k e p t a t l o w - 44 -F i g . 1 0 . C y c l i c a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f u n t r e a t e d h y p o p h y s e c t o m i s e d f i s h - P r i m a r y germ c e l l s ; I .1 - P r i m a r y s p e r m a t o g o n i a — R e s t i n g S e c o n d a r y s p e r m a t o g o n i a U - D i v i d i n g s e c o n d a r y s p e r m a t o g o n i a ( r a r e ) SPERMATOGENETIC COMPOSITION OF UNTREATED HYPOPHYSECTOMI ZED FISH J A S O N J F M M J J A S O EXPERIMENTS - 45 -F i g . 1 1 . S e a s o n a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f FSH t r e a t e d f i s h . (See F i g . 10 f o r e x p l a n a t i o n s ) SPERMATOGENETIC COMPOSITION OF FSH TREATED FISH >»»»», V»W»m J2222222222. - 46 -t e m p e r a t u r e was m a r k e d l y h i g h e r i n J u n e , J u l y and A u g u s t , 1963 e x p e r i m e n t s . H o w e v e r , as p o i n t e d o u t l a t e r t h e s p e r m a t o g e n e t i c c o n d i t i o n o f J u l y and A u g u s t 1963 e x p e r i m e n t s i s n o t much d i f f e r e n t , w h e r e a s d i f f e r e n c e s a r e s e e n i n S e p t e m b e r 1963 e x p e r i m e n t w h i c h become m a r k e d i n O c t o b e r 1963 g r o u p . H i s t o l o g i c a l c h a n g e s . A g r a p h i c a l r e p r e s e n t a t i o n o f t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f LH t r e a t e d t e s t e s i s g i v e n i n F i g u r e 12. The p r o p o r t i o n o f r e s t i n g germ c e l l s i n t h e s e f i s h i s l o w ( r a n g i n g f r o m 0.6% t o 14.0%) i n d i c a t i n g a c o n v e r s i o n o f t h e s e i n t o s p e r m a t o g o n i a l f o r m s . P r i m a r y s p e r m a t o g o n i a a r e s t i l l : t h e most p r e d o m i n a n t f o r m a c c o u n t i n g f o r 2 2 . 8 % t o 6 3 % o f t h e v a r i o u s s t a g e s ( T a b l e V ) . P r i m a r y s p e r m a t o g o n i a l n e s t s i n LH t r e a t e d f i s h d i f f e r f r o m f i s h b e l o n g i n g t o FSH and h y p o p h y s e c t o m i s e d g r o u p s i n t h e i r g r e a t e r u n i f o r m i t y . The m a j o r i t y o f n u c l e i b e l o n g i n g t o t h i s s t a g e were u n d e r g o i n g d i v i s i o n , t h a t i s t h e d i v i d i n g p r i m a r y s p e r m a t o g o n i a were i n a c t i v e m i t o t i c p h a s e . i LH t r e a t e d f i s h a l s o show a m a r k e d d i f f e r e n c e f r o m t h e FSH t r e a t e d o n e s i n an i n c r e a s e i n t h e s e c o n d a r y s p e r m a t o g o n i a b o t h r e s t i n g and d i v i d i n g . The r e s t i n g s e c o n d a r y s p e r m a t o g o n i a r a n g e i n numbers f r o m a l o w o f 1 7 . 7 % t o a maximum o f 4 6 . 0 % . The d i v i d i n g f o r m s r a n g e f r o m 1.9% t o 3 2 . 5 % . P r i m a r y s p e r m a t o c y t e s a r e p r e s e n t i n a l l b u t two e x p e r i m e n t s r e a c h i n g a maximum o f 5.7% i n J a n u a r y e x p e r i m e n t . D i v i d i n g s p e r m a t o c y t e s a r e r a r e and s e e n o n l y i n f o u r e x p e r i m e n t s ; o c c a s i o n a l d i v i d i n g p r i m a r y s p e r m a t o c y t e s may be p r e s e n t . T e s t i c u l a r s t i m u l a t i o n o f Couesius t e s t i s w i t h mammalian LH a p p e a r s t o r e a c h a c l i m a x w i t h t h e d e v e l o p m e n t o f p r i m a r y s p e r m a t o c y t e s and i s c o m p a r a b l e t o - 47 -F i g . 1 2 . S e a s o n a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f mammalian LH- t r e a t e d f i s h . The r i g h t b a r i n J u n e 1963 and s u b s e q u e n t e x p e r i m e n t s i n d i c a t e c o n d i t i o n a t h i g h t e m p e r a t u r e ; t h e l e f t t h a t f o u n d a t l o w t e m p e r a t u r e . Y////M — S e c o n d a r y s p e r m a t o g o n i a ( d i v i d i n g ) HH — P r i m a r y s p e r m a t o c y t e s ( r e s t i n g and d i v i d i n g ) O t h e r e x p l a n a t i o n s as i n F i g . 10. SPERMATOGENETIC COMPOSITION OF FISH TREATED WITH MAMMALIAN LH J A S O N J F M M J J J J A A S S O O EXPTS - 48 -S t a g e I I I o f t h e n o r m a l c y c l e . I t i s o b v i o u s t h a t LH t r e a t m e n t i n c r e a s e s m i t o t i c a c t i v i t y i n t h e p r i m a r y s p e r m a t o g o n i a and p r o b a b l y t h e r e s t i n g germ c e l l s f o r m i n g l a t e r g e n e r a t i o n s o f s p e r m a t o g o n i a . I t a l s o s t i m u l a t e s d i v i s i o n i n t h e s e c o n d a r y s p e r m a t o g o n i a l e a d i n g t o t h e f o r m a t i o n o f p r i m a r y s p e r m a t o c y t e s . A l t h o u g h no s t r i k i n g s e a s o n a l v a r i a t i o n i n t h e r e s p o n s e o f LH t r e a t m e n t c a n be s e e n , t h i s t r e a t m e n t e l i c i t s a somewhat g r e a t e r r e s p o n s e d u r i n g t h e F e b r u a r y - A p r i l p e r i o d . T e m p e r a t u r e e f f e c t s on LH t r e a t e d f i s h . I n J u n e e x p e r i m e n t s , a s i g n i f i c a n t s p e r m a t o g e n e t i c d i f f e r e n c e i s o b s e r v e d i n t h e l o w and h i g h t e m p e r a t u r e LH t r e a t e d f i s h ( F i g . 1 5 ) . I n J u l y and A u g u s t e x p e r i m e n t a l t h o u g h t h e GSI a r e d i s t i n c t l y d i f f e r e n t , s p e r m a t o g e n e t i c p i c t u r e o f t h e f i s h k e p t a t l o w and h i g h t e m p e r a t u r e . i s p r a c t i c a l l y s i m i l a r . I n S e p t e m b e r 1963 experiment t h e l o w t e m p e r a t u r e f i s h p o s s e s s a l o w e r GSI as c o m p a r e d t o h i g h t e m p e r a t u r e , a l t h o u g h t h e f o r m e r shows a c l e a r s i g n o f s p e r m a t o g e n e t i c a d v a n c e o v e r t h e l a t t e r ( T a b l e V . ) . T h i s i s more m a r k e d i n O c t o b e r 1963 e x p e r i m e n t . Low t e m p e r a t u r e LH t r e a t e d f i s h i n t h i s e x p e r i m e n t show a d e g r e e o f s t i m u l a t i o n s i m i l a r t o M a r c h 1963 e x p e r i m e n t w h e r e a s t h e GSI do n o t show t h i s . I t a p p e a r s t h a t t h e GSI i s n o t a l w a y s a t r u e i n d e x o f g o n a d a l a c t i v i t y . C h o l e s t e r o l and l i p i d . C h o l e s t e r o l t e s t s r e s u l t s were a l w a y s p o s i t i v e i n LH e x p e r i m e n t s a l t h o u g h t h e i n t e n s i t y o f t h e r e a c t i o n seeme-d' t o v a r y s l i g h t l y . These v a r i a t i o n s were i m p r a c t i c a l f o r t h e p u r p o s e s o f t h e p r e s e n t s t u d y . t o q u a n t i f y . The l o b u l e b o u n d a r y c e l l s were t h e o n l y a r e a s w h i c h gave - 49 -p o s i t i v e r e s u l t s f o r c h o l e s t e r o l . These a g a i n were t h e o n l y a r e a s w h i c h showed any a c c u m u l a t i o n o f s u d a n o p h i l i c l i p i d m a t e r i a l b e s i d e s t h e b l o o d v e s s e l s . The l i p i d c o n t e n t o f o t h e r a r e a s o f LH t r e a t e d t e s t s i s meagre and t h e r e f o r e s i m i l a r t o FSH t r e a t e d o n e s . These r e s u l t s s t r e n g t h e n t h e a r gument t h a t t h e l o b u l e b o u n d a r y c e L l s a r e t h e p h y s i o l o g i c a l homologue of. t h e L e y d i g c e l l s o f h i g h e r v e r t e b r a t e s w h i c h s e c r e t e a n d r o g e n s . Mammalian .I LH seem t o c o n s t a n t l y . j t i m u l a t e t h e s e c e l l s a l m o s t i r r e s p e c t i v e o f t h e s e a s o n . . I t e v i d e n t l y p o s s e s s e s b o t h t h e i n t e r s t i t i a l c e l l and t h e s p e r m a t o g e n e s i s s t i m u l a t i n g p r o p e r t y when a d m i n i s t e r e d t o C o u e s i u s . A s i d e f r o m t h e e f f e c t - o n t h e l o b u l e b o u n d a r y c e l l s -LH. t r e a t m e n t has no e f f e c t on th e i l i p i d c o n t e n t o f t h e t e s t i c u l a r l o b u l e s and so u n l i k e - E s o x l u o i u s and some a m p h i b i a n s d o es n o t c a u s e an a r t i f i c i a l s t . e a t o g e n e s i s i n t h i s f i s h ( L o f t s , 1 9 6 2 ) . E f f e c t o f F r a c t i o n a t e d S a l m o n P i t u i t a r y G o n a d o t r o p i n The GSI o f f i s h r e c e i v i n g f r a c t i o n a t e d s a l m o n p i t u i t a r y g o n a d o t r o p i n were h i g h e r - t h a n t h o s e o f f i s h r e c e i v i n g mammalian h o r m o n e s . The l e v e l o f t h e GSI was s i m i l a r i n 1962 and 1963 e x p e r i m e n t s , e x c e p t f o r m a r k e d i n c r e a s e s i n F e b r u a r y , M a r c h and J u n e e x p e r i m e n t s i n 1963. E x p e r i m e n t s - i n 1963 were commenced i n May and f i s h were k e p t a t room t e m p e r a t u r e s ( 1 9 . 5 - 1.5°C) and a t l o w e r t e m p e r a t u r e s (10 - 1 . 5 q C ) . H i g h T e m p e r a t u r e F i s h GSI o f t h e i n j e c t e d f i s h r e m a i n e d h i g h d u r i n g May and J u n e e x p e r i m e n t b u t f e l l s i g n i f i c a n t l y d u r i n g J u l y e x p e r i m e n t r e m a i n i n g t h e same as i n t h e c o r r e s p o n d i n g e x p e r i m e n t s o f 1962, - 50 -some i n c r e a s e b e i n g n o t i c e d i n S e p t e m b e r e x p e r i m e n t . I t i s o n l y d u r i n g t h e s e p e r i o d s ( M a r c h - J u l y ) t h a t t h e GSI o f t h e t r e a t e d f i s h a t h i g h t e m p e r a t u r e i s g r e a t e r t h a n t h e sham o p e r a t e d c o n t r o l s . The l e v e l o f s t i m u l a t i o n ( a s j u d g e d by GSI a l o n e ) i s o f t h e same d e g r e e as t h a t by mammalian LH e x c e p t f o r F e b r u a r y , M a r c h , May and J u n e e x p e r i m e n t , where s t i m u l a t i o n i s m a r k e d l y g r e a t e r i n s a l m o n g o n a d o t r o p i n t r e a t e d f i s h ( F i g . 1 3 ) . An i n c r e a s e o f a l e s s e r d e g r e e i s n o t e d a l s o i n S e p t e m b e r and O c t o b e r e x p e r i m e n t s o f 1963. H i s t o l o g i c a l - C h a n g e s The s p e r m a t o g e n e t i c c o n d i t i o n i s shown i n F i g u r e 14. The r e s t i n g germ c e l l s c o n s t i t u t e a v e r y s m a l l p r o p o r t i o n o f t h e t e s t e s (0.4 - 8.7%) w h i l e n e s t s o f d i v i d i n g p r i m a r y s p e r m a t o g o n i a a r e p r e d o m i n a n t ( 3 4 . 6 % - 6 4 . 9 % ) . These n e s t s show a g r e a t e r u n i f o r m i t y o f a p p e a r a n c e and some d e g r e e o f s y n c h r o n y i n d i v i s i o n . ' The i n c r e a s e i n t h e p r o p o r t i o n o f s e c o n d a r y s p e r m a t o g o n i a i s m a r k e d i n c o m p a r i s o n w i t h f i s h t r e a t e d w i t h mammalian LH w i t h 1 4 . 6 % - 4 0 . 4 % r e s t i n g and 1.4% - 3 5 . 6 % i n d i v i d i n g p h a s e . P r i m a r y s p e r m a t o c y t e s a r e p r e s e n t i n a l l e x p e r i m e n t s e x c e p t i n A u g u s t 1 9 6 2 , A u g u s t 1963 and May 1963 e x p e r i m e n t . I n s p i t e o f t h e f a c t t h a t s a l m o n g o n a d o t r o p i n t r e a t e d f i s h h ave h i g h e r GSI t h a n t h e LH t r e a t e d f i s h , p r i m a r y s p e r m a t o c y t e s a r e more numerous i n t h e L a t t e r g r o u p ( T a b l e s V. & V I ) . A l s o , d i v i d i n g p r i m a r y s p e r m a t o c y t e s a r e much l e s s common. I t a p p e a r s t h a t s a l m o n p i t u i t a r y e x t r a c t c a u s e s a g r e a t e r b u i l d up o f t h e s e c o n d a r y s p e r m a t o g o n i a , w h e r e a s i n t h e LH f i s h some n e s t s a r e s t i m u l a t e d b e y o n d s p e r m a t o g o n i a l s t a g e b e f o r e a s u f f i c i e n t s t o r e o f g o n i a l c e l l s i s a c c u m u l a t e d . T h i s i s obvious - 51 -f r o m t h e g r e a t e r e x t e n t o f s e c o n d a r y s p e r m a t o g o n i a l n e s t s i n t h e s a l m o n g o n a d o t r o p i n t r e a t e d f i s h ( F i g . 1 4 ) . I n t h e p r e s e n t e x p e r i m e n t t h e r e i s some d i s c r e p a n c y b e t w e e n the, s t i m u l a t i o n as i n d i c a t e d by t h e GSI and t h e h i s t o l o g y . I n May 1963 e x p e r i m e n t t h e GSI i s h i g h ( 0 . 9 0 3 2 ) a t h i g h e r t e m p e r a t u r e w h e r e a s t h e t e s t e s shows a g r e a t e r p r e p o n d e r a n c e o f p r i m a r y and s e c o n d a r y s p e r m a t o g o n i a w i t h f e w d i v i d i n g s e c o n d a r y s p e r m a t o g o n i a l n e s t s ( T a b l e V ) . The GSI a t l o w t e m p e r a t u r e i s l o w e r ( 0 . 6 4 9 7 ) w h i c h f i t s i n w i t h t h e h i s t o l o g i c a l c o n d i t i o n o f t h e t e s t e s i n t h i s s i t u a t i o n . The s p e r m a t o g e n e t i c c o m p o s i t i o n o f b o t h l o w . a n d h i g h t e m p e r a t u r e f i s h i s m a r k e d l y s i m i l a r . A d e c r e a s e i n t h e d e g r e e o f s t i m u l a t i o n i s n o t e d i n May e x p e r i m e n t as i n a l l p r e v i o u s e x p e r i m e n t s b o t h on t h e b a s i s o f t h e GSI and t h e s p e r m a t o g e n e t i c c o m p o s i t i o n . Low T e m p e r a t u r e F i s h The GSI o f l o w t e m p e r a t u r e f i s h i s m a r k e d l y l o w e r t h a n t h a t k e p t i n warmer w a t e r i n May e x p e r i m e n t . I n J u n e , J u l y , and A u g u s t , t h e l o w t e m p e r a t u r e f i s h show a h i g h e r G S I . I n September and O c t o b e r e x p e r i m e n t s GSI o f f i s h a t l o w e r t e m p e r a t u r e i s a g a i n l e s s t h a n t h a t a t t h e h i g h e r , t h e d i f f e r e n c e b e i n g more m a r k e d i n O c t o b e r e x p e r i m e n t ( F i g . 1 5 ) . I t i s o b v i o u s , t h a t maximum s t i m u l a t i o n o f t h e t e s t e s i s o b t a i n e d d u r i n g t h e p r e s p a w n i n g p e r i o d i n F e b r u a r y and M a r c h e x p e r i m e n t s . F i s h seem t o r e m a i n r e c e p t i v e t i l l t h e end o f t h e s p a w n i n g i n J u l y . A d r o p i n J u l y e x p e r i m e n t may r e f l e c t t h e r e d u c e d r e c e p t i v i t y o f t h e t e s t i c u l a r t i s s u e t o g o n a d o t r o p h i c s t i m u l a t i o n i m m e d i a t e l y a f t e r s p a w n i n g . I t i s p o s s i b l e t h a t - 52 -F i g . 1 3 . S e a s o n a l c h a n g e s i n t h e GSI o f F r a c t i o n a t e d S a l m o n G o n a d o t r o p i n (-JFSG)- t r e a t e d f i s h , as c o m p a r e d t o t h o s e w i t h mammalian LH. GSI o f u n t r e a t e d h y p o p h y s e c t o m i s e d and sham o p e r a t e d c o n t r o l s i n c l u d e d f o r c o m p a r i s o n . - 53 -i n c r e a s e d w e i g h t - o f t e s t e s i n May e x p e r i m e n t may be due t o i n c r e a s e d h y d r a t i o n o f t h e t e s t e s p r o b a b l y c a u s e d by i n f e c t i o n . The l o w t e m p e r a t u r e G S I , t h e r e f o r e , r e f l e c t s t h e c o r r e c t p i c t u r e o f t e s t e s i n May e x p e r i m e n t . Low t e m p e r a t u r e s i n t h e i n i t i a l e x p e r i m e n t s show a s l i g h t l y more s t i m u l a t o r y e f f e c t , b u t i n S e p t e m b e r and O c t o b e r e x p e r i m e n t s h i g h t e m p e r a t u r e s c a u s e an i n c r e a s e i n t h e G S I , w h i c h i s a l s o r e f l e c t e d i n an i n c r e a s e i n s e c o n d a r y s p e r m a t o g o n i a l n e s t s and t h e p r i m a r y s p e r m a t o c y t e s ( p a r t i c u l a r l y i n O c t o b e r e x p e r i m e n t , F i g u r e 1 4 ) . A c o m p a r i s o n o f F i g u r e 12 and F i g u r e 14 shows t h a t t h e s p e r m a t o g e n e t i c p i c t u r e i n t h e LH t r e a t e d f i s h and t h e salmon g o n o d a t r o p i n t r e a t e d i s s i m i l a r e x c e p t f o r a s l i g h t i n c r e a s e i n t h e o c c u r r e n c e o f p r i m a r y s p e r m a t o c y t e s i n t h e f o r m e r . C h o l e s t e r o l and L i p i d C h o l e s t e r o l t e s t s showed l e s s u n i f o r m i t y i n t h i s t r e a t m e n t t h a n i n f i s h t r e a t e d w i t h LH ( T a b l e V ) . P o s i t i v e t e s t s f o r c h o l e s t e r o l were o b t a i n e d i n a l l e x p e r i m e n t s e x c e p t A u g u s t , 1962, and May, 1 9 6 3 , e x p e r i m e n t s . The l i p i d c o n t e n t o f t h e t e s t e s d i d n o t c h a n g e . S u d a n o p h i l y was u n i f o r m l y n o t e d o n l y i n t h e r e g i o n o f t h e t e s t e s o c c u p i e d by t h e l o b u l e b o u n d a r y c e l l s . I n s h o r t , f r a c t i o n a t e d s a l m o n p i t u i t a r y e x t r a c t s t i m u l a t e d t e s t e s o f h y p o p h y s e c t o m i s e d t e s t e s t o t h e l e v e l o f s e c o n d a r y s p e r m a t o g o n i a l n e s t s w i t h t h e a p p e a r a n c e o f some p r i m a r y s p e r m a t o c y t e s . T h i s s t i m u l a t o r y e f f e c t was more m a r k e d d u r i n g t h e r e c e p t i v e p e r i o d o f t h e a n n u a l t e s t i c u l a r c y c l e o f t h e f i s h f a l l i n g b e t w e e n F e b r u a r y t o April„ w h i c h i s d e m o n s t r a t e d by t h e i n c r e a s e d s t i m u l a t i o n o f t h e t e s t e s i n J a n u a r y , F e b r u a r y - 54 -and M a r c h e x p e r i m e n t s . The r e f r a c t o r y p e r i o d seems t o f a l l i n A u g u s t and S e p t e m b e r ; c o n s e q u e n t l y s t i m u l a t i o n i s l e s s i n J u l y and A u g u s t e x p e r i m e n t s . The f i s h a r e most s u s c e p t i b l e t o p i t u i t a r y r e m o v a l d u r i n g May, t h e r e g r e s s i o n f o l l o w i n g t h e h y p o p h y s e c t o m y i s v e r y c o m p l e t e , so much s o , t h a t r e p l a c e m e n t t h e r a p y ( e i t h e r w i t h mammalian LH o r f r a c t i o n a t e d s a l m o n p i t u i t a r y e x t r a c t ) i s o n l y p a r t l y s u c c e s s f u l i n e f f e c t i n g a r e c o v e r y . - The c l o s e p a r a l l e l shown by t h e f r a c t i o n a t e d s a l m o n p i t u i t a r y e x t r a c t and t h e mammalian LH show t h a t t h e g o n a d o t r o p i c f a c t o r i n t h e f o r m e r w h i c h i s r e s p o n s i b l e f o r s t i m u l a t i o n i s p h y s i o l o g i c a l l y s i m i l a r t o mammalian LH. T h i s i s b o r n e o u t b o t h by h i s t o l o g i c a l and h i s t o c h e m i c a l c h a r a c t e r i s a t i o n o f t h e s e , two h o r m o n a l p r e p a r a t i o n s ( T a b l e s V and V I ) . E f f e c t o f C r u d e S a l m o n P i t u i t a r y E x t r a c t D i f f i c u l t i e s were e n c o u n t e r e d i n t h e a d m i n i s t r a t i o n o f c r u d e p i t u i t a r y e x t r a c t s t o o p e r a t e d f i s h . H owever, experiments w i t h i t were c o n d u c t e d f r o m S e p t e m b e r 1962 u n t i l t h e end o f t h e s t u d y . I n two e x p e r i m e n t s a l l f i s h i n j e c t e d w i t h c r u d e e x t r a c t d i e d a p p a r e n t l y due t o i n f e c t i o n . Red p a t c h e s on b e l l y and f l u i d i n t h e p e r i t o n e a l c a v i t y were t h e o n l y o b v i o u s p a t h o l o g i c a l s i g n s . A b s o r p t i o n seemed s l o w e r i n t h i s t r e a t m e n t w h i c h . o f t e n c a u s e d p r o l o n g e d d i s t e n s i o n o f t h e b e l l y . S u r v i v a l was i n c r e a s e d by m a i n t a i n i n g f i s h d u r i n g t h e - i n j e c t i o n a t l o w e r t e m p e r a t u r e s (10°C - 1°C) i n May 1963 and s u c c e e d i n g e x p e r i m e n t s . I n s p i t e o f t h e m o r t a l i t i e s , t h e GSI o f f i s h t r e a t e d w i t h t h e c r u d e e x t r a c t was a l w a y s h i g h e r t h a n t h a t o f any o t h e r t r e a t m e n t s , s h o w i n g i t s most m a r k e d i n c r e a s e i n t h e F e b r u a r y and - 55 -M a r c h e x p e r i m e n t s and a l o w a c t i v i t y i n J u l y and A u g u s t ( F i g . 18) - t h e same p a t t e r n o f r e s p o n s e e x h i b i t e d by t h e LH and f r a c t i o n a t e d s a l m o n e x t r a c t t r e a t e d - f i s h . The GSI o f June e x p e r i m e n t f i s h i s much l o w e r t h a n t h e r e s t p r o b a b l y b e c a u s e t h e f i s h were a u t o p s i e d a f t e r o n l y t h r e e i n j e c t i o n s . H i s t o l o g i c a l c h a n g e s . F i s h t r e a t e d w i t h c r u d e e x t r a c t show t h e most a c t i v e t e s t e s o f a l l f i s h e s r e c e i v i n g r e p l a c e m e n t t h e r a p y . A g r e a t i n c r e a s e i n m i t o t i c a c t i v i t y i s o b s e r v e d i n t h i s t r e a t m e n t c o u p l e d w i t h a r e d u c t i o n i n t h e p r o p o r t i o n s o f p r i m a r y s p e r m a t o g o n i a ( 1 6 . 5 - 4 4 . 8 % ) and p r i m a r y germ c e l l s ( 1 . 2 - 9.2%) F i g u r e 20. S e c o n d a r y s p e r m a t o g o n i a ( b o t h d i v i d i n g and r e s t i n g ) - a r e p r e d o m i n a n t , e x c e p t f o r May, J u l y and A u g u s t 1963 e x p e r i m e n t . Most m a r k e d p r o g r e s s i n t h e t e s t e s i s f o u n d i n t h e i n c r e a s e i n t h e e x t e n t o f p r i m a r y s p e r m a t o c y t e s b o t h d i v i d i n g ( 0 . 0 % - 8.9%) and r e s t i n g ( 0 . 2 % - 1 3 . 3 % ) . Some s e c o n d a r y s p e r m a t o c y t e s a r e p r e s e n t i n t h e S e p t e m b e r 1962 and F e b r u a r y 1963 e x p e r i m e n t ( F i g . 2 0 ) . The h i s t o l o g i c a l a p p e a r a n c e o f t h e t e s t i s o f a t y p i c a l c r u d e s a l m o n e x t r a c t t r e a t e d i s g i v e n i n F i g u r e 33A. C h o l e s t e r o l and L i p i d E x c e p t f o r May e x p e r i m e n t , t e s t s f o r c h o l e s t e r o l were p o s i t i v e ( T a b l e V I I ) . C h o l e s t e r o l t e s t was n o t a t t e m p t e d i n J u n e e x p e r i m e n t . T h e r e was no change i n t h e l i p i d c o n t e n t o f t h e t e s t e s w h i c h a l w a y s r e m a i n e d l o w . The d e g r e e o f s t i m u l a t i o n a t t a i n e d by c r u d e e x t r a c t t r e a t e d f i s h i s h i g h e r t h a n any o t h e r g r o u p o f t r e a t e d f i s h b o t h i n t h e m a t t e r o f t h e i r GSI and s p e r m a t o g e n e t i c c o n d i t i o n . Compared t o mammalian LH and f r a c t i o n a t e d s a l m o n e x t r a c t t r e a t e d f i s h , s t i m u l a t i o n w i t h c r u d e e x t r a c t a p p e a r s t o be f u l l e r and - 56 -F i g . 1 4 . S e a s o n a l c h a n g e s i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f F r a c t i o n a t e d S a l m o n G o n a d o t r o p i n t r e a t e d f i s h . L e f t b a r i n May 1963* and s u b s e q u e n t e x p e r i m e n t s i n d i c a t e c o n d i t i o n a t l o w t e m p e r a t u r e , r i g h t d e n o t e s t h a t a t h i g h . E x p l a n a t i o n s same as F i g u r e 12. F FRACTIONATED SALMON PITUITARY TREATED FISH EXPERIMENTS - 57 -F i g . 1 5 . E f f e c t o f l o w and h i g h t e m p e r a t u r e s on t h e F r a c t i o n a t e d S a l m o n G o n a d o t r o p i n and mammalian L H - t r e a t e d f i s h i n May 1963 and s u b s e q u e n t e x p e r i m e n t s . • • Mammalian LH- t r e a t e d ( h i g h t e m p e r a t u r e ) 0 0 Mammalian L H - t r e a t e d ( l o w t e m p e r a t u r e ) • 1 F r a c t i o n a t e d S a lmon G o n a d a t r o p i n ( h i g h temperature) X X F r a c t i o n a t e d S a l m o n G o n a d o t r o p i n ( l o w t e m p e r a t u r e ) M J J A S O EXPERIMENTS - 58 -F i g . l 6 A . P h o t o m i c r o g r a p h o f a t e s t i s f r o m h y p o p h y s e c t o m i s e d f i s h s h o w i n g a b u n d a n t f i b r o b l a s t s s i m u l a t i n g f i b r o t i c d e g e n e r a t i o n . P r i m a r y germ c e l l s a r e most p r e d o m i n a n t . Some p r i m a r y s p e r m a t o g o n i a a r e a l s o s e e n (X 700 ). B. P h o t o m i c r o g r a p h o f an FSH t r e a t e d f i s h t e s t i s s h o w i n g m o s t l y p r i m a r y germ c e l l s , and p r i m a r y s p e r m a t o g o n i a . A s m a l l n e s t o f s e c o n d a r y s p e r m a t o g o n i a on t h e l e f t c e n t r e c a n be s e e n (X 7 0 0 ) - 59 -more r e g u l a r , as e v i d e n c e d by t h e g r e a t b u i l d - u p o f s e c o n d a r y s p e r m a t o g o n i a d u r i n g t h e n o n - r e f r a c t o r y p e r i o d o f t h e a n n u a l c y c l e . C r u d e s a l m o n p i t u i t a r y e x t r a c t s e f f e c t a g r e a t e r s t i m u l a t i o n o f t h e h y p o p h y s e c t o m i s e d C o u e s i u s t e s t e s as compared t o r e p l a c e m e n t t h e r a p i e s w i t h mammalian LH and f r a c t i o n a t e d s a l m o n p i t u i t a r y e x t r a c t s . T h i s i n c r e a s e d s t i m u l a t o r y e f f e c t i s e x h i b i t e d b o t h i n t h e t e s t e s w e i g h t and i n t h e i r s p e r m a t o g e n e t i c c o m p o s i t i o n . The s e a s o n a l v a r i a t i o n i n r e s p o n s e t o t h e c r u d e e x t r a c t i s s i m i l a r t o t h o s e o b t a i n e d w i t h t h e LH and t h e f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n . E f f e c t o f E x p e r i m e n t a l C o n d i t i o n s on Sham O p e r a t e d U n t r e a t e d  C o n t r o l s Sham o p e r a t e d c o n t r o l s were k e p t f o r t h e d u r a t i o n o f e a c h e x p e r i m e n t . A s l i g h t r e g r e s s i v e e f f e c t i s n o t e d i n t h e s e c o n t r o l s d u r i n g t h e summer when comp a r e d w i t h n o r m a l s f r o m t h e l a k e . R e g r e s s i o n i s a l s o s l i g h t l y more m a r k e d d u r i n g t h e p r e s p a w n i n g w i n t e r m o n t h s . D u r i n g t h e p o s t s p a w n i n g p e r i o d o f l a t e J u l y ( J u n e 1963 e x p e r i m e n t ) t h e t e s t e s i n t h e s h a m - o p e r a t e d c o n t r o l do n o t r e g r e s s t o t h e same d e g r e e as t h e f i s h i n n a t u r e ; t h i s i s p r o b a b l y due t o t h e p r e v e n t i o n o f t h e c o n t r o l s f r o m n a t u r a l s p a w n i n g and c o n s e q u e n t l y some a d v a n c e d n e s t s and sperms a r e r e t a i n e d s h o w i n g a h i g h e r GSI f o r s-uch, \f i s h ( F i g . 8 ) . S p a w n i n g i n 1962 o c c u r r e d e a r l i e r t h a n i n 1 9 6 3 , and was l e s s e x t e n d e d as e v i d e n c e d f r o m t h e h i s t o l o g i e s o f n o r m a l f i s h f r o m J u l y and A u g u s t f o r t h e s e y e a r s . S p e r m i a t i o n was w e l l on t h e way i n J u l y 1962 n o r m a l s and i n A u g u s t a l l sperms had gone, w h e r e a s i n 1 9 6 3 , J u l y n o r m a l s show a g r e a t p r o p o r t i o n o f a c t i v e - 60 -t e s t e s f u l l o f s p e r m s , and some r e m n a n t s were p r e s e n t e v e n i n • A u g u s t . . • J u l y and A u g u s t n o r m a l s d u r i n g 1962 were more r e g r e s s e d ( t o b e g i n w i t h ) t h a n t h o s e o f 1963. T h i s a l s o a p p e a r s i n t h e G S I , ( F i g . 5) and e x p l a i n s why J u l y and A u g u s t c o n t r o l s . i n 1962 a r e more r e g r e s s e d t h a n t h o s e o f 1963 when compared t o t h e i r r e s p e c t i v e n o r m a l c o u n t e r p a r t . The. s p e r m a t o g e n e t i c c o n d i t i o n o f t h e sham o p e r a t e d c o n t r o l t e s t e s a r e g i v e n i n T a b l e I I , and show a g e n e r a l c o r r e s p o n d e n c e w i t h t h e GSI ( F i g . 8 ) . G e n e r a l Summary o f R e s u l t s o f R e p l a c e m e n t T h e r a p y P o s i t i v e r e s p o n s e t o r e p l a c e m e n t t h e r a p y was a c h i e v e d w i t h C r u d e S a l m o n P i t u i t a r y e x t r a c t , f r a c t i o n a t e d s a l m o n g o n a d a t r o p i n and p u r i f i e d mammalian LH. FSH and s a l i n e i n j e c t i o n s f a i l e d t o p r o d u c e any c h a n g e . The s l i g h t s t i m u l a t o r y e f f e c t shown w i t h FSH t r e a t m e n t m i g h t p o s s i b l y be due t o c o n t a m i n a n t LH. I n : C . p l u m b e u s t h e r e r e m a i n s l i t t l e d o u b t t h a t r e p l a c e m e n t t h e r a p y on h y p o p h y s e c t o m i s e d f i s h r e s t o r e s t e s t i c u l a r f u n c t i o n s . R e s u l t s on t h e e f f e c t s o f mammalian g o n a d o t r o p i n s l e a d t o t h e c o n c l u s i o n t h a t w h i l e LH r e s t o r e s and r e i n i t i a t e s s p e r m a t o g e n e s i s , FSH i s i n e f f e c t i v e (LH c o n t a m i n a n t s a r e r e s p o n s i b l e f o r t h e s l i g h t p o s i t i v e e f f e c t i n some e x p e r i m e n t s ) . C o u e s i u s p i t u i t a r y t h u s a p p e a r s t o p r o d u c e a g o n a d o t r o p i c f a c t o r s i m i l a r t o mammalian LH, b u t s u b s e r v i n g t h e f u n c t i o n s o f b o t h LH and FSH. Among t h e t r e a t m e n t s w h i c h p r o d u c e d p o s i t i v e r e s u l t s , maximum s t i m u l a t i o n o f t h e t e s t e s was o b t a i n e d w i t h c r u d e w h o l e s a l m o n p i t u i t a r y e x t r a c t . The e f f e c t i v e n e s s o f hormones i n r e p l a c e m e n t t h e r a p y seemed t o v a r y somewhat w i t h t h e s e a s o n s . S u c h v a r i a t i o n s i n r e s p o n s e has been w e l l known t o c o m t e m p o r a r y - 61 -F i g . l 7 A . P h o t o m i c r o g r a p h o f an LH- t r e a t e d t e s t i s s h o w i n g t h e s p e r m a t o g e n e t i c c o m p o s i t i o n o f a t y p i c a l LH- t r e a t e d f i s h . (X 7 0 0 ) B. P h o t o m i c r o g r a p h o f a f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n t r e a t e d t e s t i s s h o w i n g s p e r m a t o g e n e t i c c o n d i t i o n o f a t y p i c a l t r e a t e d f i s h (X 700) - 62 -F i g . 1 8 . S e a s o n a l c h a n g e s i n t h e GSI o f c r u d e w h o l e s a l m o n p i t u i t a r y e x t r a c t (CSE) t r e a t e d f i s h as c o m p a r e d t o t h o s e t r e a t e d w i t h f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n ( F S G ) . GSI o f u n t r e a t e d h y p o p h y s e c t o m i s e d (HPX.) and sham o p e r a t e d c o n t r o l s (SHAM) a r e i n c l u d e d f o r c o m p a r i s o n . J A S O N 3 F M M J "j A S O EXPERIMENTS - 63 -w o r k e r s ( B a l l and B a c o n , 1954, P a l m e r e t a l , 1954; P i c k f o r d and A t z , 1 9 5 7 ) . F i s h i n t h e p r e s e n t s t u d y were more r e s p o n s i v e during ( i ) The p r e s p a w n i n q p e r i o d when s t i m u l a t i o n was m a x i m a l . T h i s p e r i o d r a n g e s f r o m F e b r u a r y t o May o f t h e c a l e n d a r y e a r c o r r e s p o n d i n g t o t h e J a n u a r y , F e b r u a r y and M a r c h e x p e r i m e n t s o f t h e p r e s e n t i n v e s t i g a t i o n . ( i i ) S u s c e p t i b l e p e r i o d ( s p a w n i n g p e r i o d ) . R e g r e s s i o n was m a x i m a l d u r i n g t h i s p e r i o d f a l l i n g i n J u n e ( c o r r e s p o n d i n g t o May e x p e r i m e n t ) and r e p l a c e m e n t t h e r a p i e s p r o d u c e d r e c o v e r i e s o f l o w e r d e g r e e . ( i i i ) R e f r a c t o r y p e r i o d ( e a r l y p o s t - s p a w n i n g p e r i o d ) . Though r e g r e s s i o n i s n o t maximum, r e p l a c e m e n t t h e r a p i e s p r o d u c e o n l y a l i m i t e d s t i m u l a t i o n . T h i s p e r i o d o c c u r s i n A u g u s t ( J u l y e x p e r i m e n t ) . ( i v ) P e r i o d o f r e c o n s t i t u t i o n ( l a t e p o s t - s p a w n i n g p e r i o d ) . E x t e n d s f r o m S e p t e m b e r t o J a n u a r y ( c o r r e s p o n d i n g t o A u g u s t , S e p t e m b e r , O c t o b e r and November e x p e r i m e n t s ) . R e p l a c e m e n t t h e r a p i e s e l i c i t a g r e a t e r d e g r e e o f s t i m u l a t i o n d u r i n g t h i s p e r i o d t h a n d u r i n g t h e r e f r a c t o r y p e r i o d , b u t l e s s e r t h a n t h e p r e s p a w n i n g p e r i o d . A summary o f t h e r e p l a c e m e n t t h e r a p y e x p e r i m e n t s i s g i v e n i n F i g u r e 30. ENVIRONMENTAL CONTROL OF TESTICULAR CYCLE P h o t o p e r i o d - t e m p e r a t u r e E x p e r i m e n t 1. E x p l o r a t o r y E x p e r i m e n t F i s h p u t u n d e r l o n g and s h o r t p h o t o p e r i o d s were k e p t a t l o w t e m p e r a t u r e s (6°C - 8°C) f r o m J a n u a r y 1, 1963 t o A p r i l 1, 1963. As shown i n T a b l e V I I I t h e r e i s a l m o s t no d i f f e r e n c e i n t h e g o n o s o m a t i c i n d i c e s o f t h e f i s h a u t o p s i e d i n F e b r u a r y 15, 45 d a y s l a t e r . The GSI o f t h e l o n g p h o t o p e r i o d f i s h i s 1.12 and - 64 -F i g . 1 9 . S e a s o n a l c h a n g e s i n t h e GSI o f f r a c t i o n a t e d s a l m o n g o n a d o t r o p i n (FSG) t r e a t e d and FSH- t r e a t e d f i s h . , GSI o f u n t r e a t e d h y p o p h y s e c t o m i s e d (HPX) and sham o p e r a t e d c o n t r o l s (SHAM) a r e i n c l u d e d f o r c o m p a r i s o n . J A S O N J F M M J J A S O EXPERIMENTS - 65 -F i g . 2 0 . The s p e r m a t o g e n e t i c c o m p o s i t i o n o f c r u d e w h o l e s a l m o n p i t u i t a r y e x t r a c t t r e a t e d f i s h — P y c n o t i c n e s t s — S e c o n d a r y s p e r m a t o c y t e s and s p e r m a t i d s FSSS^ I — D i v i d i n g p r i m a r y s p e r m a t o c y t e s . O t h e r e x p l a n a t i o n s as i n F i g u r e 10. SPERMATOGENETIC CONDITION OF CRUDE SALMON PITUITARY EXTRACT TREATED FISH S N F M M J J A S O - 66 -t h a t o f t h e s h o r t p h o t o p e r i o d 1.19. The t e s t e s i n b o t h g r o u p s a r e composed p r e d o m i n a n t l y o f p r i m a r y s p e r m a t o c y t e s ( 4 5 % - 6 0 % ) ; p y c n o t i c c y s t s c o n s t i t u t e 5%. The r e m a i n d e r o f t h e t e s t e s a r e s e c o n d a r y s p e r m a t o g o n i a u n d e r g o i n d s l o w m i t o t i c a c t i v i t y ( 2 0 % ) , r e s t i n g s e c o n d a r y s p e r m a t o g o n i a ( 1 0 % ) and some p r i m a r y germ c e l l . The s e c o n d a u t o p s y 75 d a y s a f t e r t h e s t a r t o f t h e e x p e r i m e n t d i d n o t r e v e a l any s i g n i f i c a n t d i f f e r e n c e i n t h e t e s t i c u l a r a c t i v i t y o f t h e f i s h i n t h e two p h o t o p e r i o d r e g i m e s . T h i s e x p e r i m e n t s u g g e s t e d t h a t p h o t o p e r i o d control i n i t i a t e d i n J a n u a r y d o es n o t c a u s e s i g n i f i c a n t c h a n g e s a t l o w t e m p e r a t u r e s . I t seemed p o s s i b l e t h a t a c o m b i n a t i o n o f p h o t o p e r i o d and t e m p e r a t u r e m i g h t p r o d u c e s i g n i f i c a n t e f f e c t s and l e a d t o a c l e a r e r u n d e r - s t a n d i n g o f t h e c o n t r o l l i n g m e c h a n i s m . Baggerman ( 1 9 5 7 ) and H a r r i n g t o n ( 1 9 57) have d e m o n s t r a t e d some c y c l i c v a r i a b i l i t y i n t h e r e s p o n s e o f f i s h e s t o e x p e r i m e n t a l m a n i p u l a t i o n o f d a y l e n g t h s and t e m p e r a t u r e s . C o n s e q u e n t l y , f i s h were m a i n t a i n e d u n d e r f o u r c o m b i n a t i o n s ' o f p h o t o p e r i o d s and t e m p e r a t u r e s w i t h t h e e x t r e m e s r o u g h l y t h o s e t h a t a r e e x p e r i e n c e d by t h i s s p e c i e s i n n a t u r e . 2 . P h o t o p e r i o d - t e m p e r a t u r e E x p e r i m e n t w i t h P r e s p a w n i n q S t o c k F i s h c o l l e c t e d f r o m t h e l a k e were p l a c e d u n d e r t h e f o l l o w i n g f o u r c o m b i n a t i o n s o f p h o t o p e r i o d and t e m p e r a t u r e c o n d i t i o n s on A p r i l 3 0, 1 9 6 3 : -1) Long p h o t o p e r i o d and l o w t e m p e r a t u r e 2) S h o f t p h o t o p e r i o d and l o w t e m p e r a t u r e 3) Long p h o t o p e r i o d and h i g h t e m p e r a t u r e 4 ) S h o r t p h o t o p e r i o d and h i g h t e m p e r a t u r e F o u r s a m p l i n g s were made d u r i n g t h e c o u r s e o f t h i s experiment (Table IX). F i r s t S a m p l i n g ( J u n e 14, 1963) A l l e x p e r i m e n t a l g r o u p s show a d e c l i n e i n t h e GSI - 67 -F i g . 2 1 . G SI o f ' p r e s p a w n i n g f i s h ' u n d e r f o u r d i f f e r e n t photoperiod - t e m p e r a t u r e c o m b i n a t i o n s , a t t h e t i m e o f t h e i n d i c a t e d s a m p l i n g s c o m p a r e d w i t h t h o s e o f t h e n o r m a l . The p o i n t o f c o n v e r g e n c e a t ^ l e f t i n d i c a t e s t h e GSI o f t h e n o r m a l f i s h a t t h e s t a r t o f t h e e x p e r i m e n t t-0- -0 S h o r t p h o t o p e r i o d (8 h r ) S h o r t p h o t o p e r i o d (8 h r ) Long p h o t o p e r i o d (16 h r ) Lbr>g • p h o t o p e r i o d (16 h r ) and h i g h t e m p e r a t u r e and l o w t e m p e r a t u r e and h i g h t e m p e r a t u r e and l o w t e m p e r a t u r e (19°±1.5°C) ( 9°il.5°C) (19°±1.5°C) ( 9°+1.5. C) X X N o r m a l . JUN JULY JUL DEC - 68 -w h i c h i s o b v i o u s w i t h i n 45 d a y s o f e x p e r i m e n t a l c o n t r o l . T h i s d e c r e a s e i s most m a r k e d a t t h e h i g h e r t e m p e r a t u r e w i t h t h e f a s t e s t i n i t i a l d e c l i n e u n d e r h i g h t e m p e r a t u r e and s h o r t p h o t o p e r i o d . B o t h g r o u p s o f f i s h a t l o w t e m p e r a t u r e s show a l e s s e r d e c r e a s e i n t h e G S I . Under t h e l o w e r t e m p e r a t u r e t h e GSI d e c l i n e s f a s t e r i n l o n g p h o t o p e r i o d a l t h o u g h t h e s e p h o t o p e r i o d d i f f e r e n c e s a r e n o t s i g n i f i c a n t . I t appears t h a t a s h o r t p h o t o p e r i o d and a l o w t e m p e r a t u r e may be e x p e c t e d t o r e t a r d t e s t i c u l a r c h a n g e s . S e c o n d S a m p l i n g ( J u l y 6, 1963) A f u r t h e r d e c l i n e i n GSI was f o u n d i n a l l g r o u p s i n c l u d i n g c o n t r o l s a t t h e t i m e o f t h e s e c o n d s a m p l i n g . T h e r e i s a l m o s t no d i f f e r e n c e b e t w e e n t h e two p h o t o p e r i o d g r o u p s m a i n t a i n e d a t h i g h t e m p e r a t u r e s where t h e GiSI r e a c h e s t h e l o w e s t p o i n t . A l t h o u g h t h e l o n g p h o t o p e r i o d f i s h show a h i g h e r GSI a t l o w t e m p e r a t u r e s t h e d i f f e r e n c e i s n o t s i g n i f i c a n t . T h i r d S a m p l i n g ( J u l y 30, 1963) The GSI o f t h e h i g h t e m p e r a t u r e g r o u p s a r e s i m i l a r and r e m a i n v i r t u a l l y u n a l t e r e d f r o m t h e p r e v i o u s s a m p l i n g and a r e c l o s e l y c o m p a r a b l e t o t h o s e f o u n d a t t h i s t i m e i n n a t u r e . The GSI o f b o t h p h o t o p e r i o d g r o u p s k e p t a t l o w t e m p e r a t u r e was much h i g h e r t h a n t h e n o r m a l f i s h ( F i g . 2 1 ) , and a g a i n t h e l o n g p h o t o p e r i o d g r o u p was o n l y s l i g h t l y a d v a n c e d o v e r t h e s h o r t p h o t o p e r i o d f i s h . F o u r t h S a m p l i n g (December 16, 1963) The f i n a l s a m p l i n g on December 16, 1963 a g a i n showed a d i s t i n c t s e p e r a t i o n b e t w e e n t h e two t e m p e r a t u r e s i t u a t i o n s . The GSI o f t h e two h i g h t e m p e r a t u r e g r o u p s r e m a i n v e r y s i m i l a r - 69 -F i g . 22. Sperm-a t o g e n e t i c c o m p o s i t i o n o f ' p r e s p a w n i n g f i s h ' u n d e r f o u r c o m b i n a t i o n s o f p h b t o p e r i o d - t e m p e r a t u r e a t t h e t i m e o f t h e v a r i o u s s a m p l i n g s ( i n d i c a t e d by Roman n u m e r a l s ) T.N = L-L°= S-L° = L-H°=. S-H°= N = I n i t i a l N o r m a l L b n g p h o t o p e r i o d , l o w t e m p e r a t u r e S h o r t p h o t o p e r i o d , l o w t e m p e r a t u r e Long p h o t o p e r i o d , h i g h t e m p e r a t u r e -S h o r t p h o t o p e r i o d , h i g h t e m p e r a t u r e N o r m a l = P r i m a r y germ c e l l s ; [ ) = P r i m a r y s p e r m a t o g o n i a ; = S e c o n d a r y s p e r m a t o g o n i a ; = P r i m a r y s p e r m a t o c y t e s ; t ^ j ^ l = S e c o n d a r y s p e r m a t o c y t e s ; = S p e r m a t i d s and sperms; |||||i||H = P y c n o t i c n e s t s . The l o w e r b l o c k i n e a c h r e p r e s e n t s r e s t i n g n e s t s , t h e u p p e r d e n o t e s t h o s e d i v i d i n g . - 70 -b u t a r e s l i g h t l y more a d v a n c e d t h a n a t t h e p r e v i o u s a u t o p s y . At l o w t e m p e r a t u r e s t h e GSI i s much g r e a t e r t h a n a t h i g h t e m p e r a t u r e , t h e s h o r t p h o t o p e r i o d f i s h show s l i g h t l y g r e a t e r t e s t i c u l a r g r o w t h t h a n t h e l o n g p h o t o p e r i o d g r o u p . T h u s , t h e maximum GSI was f o u n d i n t h e l o w t e m p e r a t u r e l o n g p h o t o p e r i o d g r o u p d u r i n g t h e p e r i o d o f t h e a n n u a l c y c l e when d a y s a r e l o n g e s t i n n a t u r e ; and when t h e y s t a r t s h o r t e n i n g t h e s h o r t p h o t o p e r i o d f i s h show an i n c r e a s e i n t h e gonad w e i g h t ( F i g . 2 1 ) . The GSI o f f i s h b e l o n g i n g t o b o t h t h e p h o t o p e r i o d s a t l o w t e m p e r a t u r e i s h i g h e r t h a n t h o s e o f t h e n o r m a l s . The gonads o f f i s h a t h i g h e r t e m p e r a t u r e show a s l i g h t g a i n i n w e i g h t , t h e i n c r e a s e b e i n g i d e n t i c a l i n b o t h p h o t o p e r i o d c o n d i t i o n s . The GSI o f t h e s e f i s h a r e much l o w e r t h a n t h o s e o f t h e n o r m a l s . I t a p p e a r s t h a t h i g h e r t e m p e r a t u r e s have a r e g r e s s i v e e f f e c t on t h e t e s t e s d u r i n g t h i s p e r i o d o f t h e i r c y c l e , w h e r e a s l o w t e m p e r a t u r e seems t o s t a l l n o r m a l r e g r e s s i o n . . I t i s i n t e r e s t i n g t o n o t e t h a t a d e c r e a s e i n t h e GSI e v e n i n t h e l o w t e m p e r a t u r e g r o u p s i s n o t e d i n t h e s e c o n d s a m p l e i n e a r l y J u l y d u r i n g a p e r i o d when t h e f i s h s pawns; b u t t h e t e s t e s i n s t e a d o f r e g r e s s i n g c o m p l e t e l y as i n t h e n o r m a l and h i g h t e m p e r a t u r e g r o u p s , q u i c k l y r e c o v e r s so t h a t a t t h e n e x t s a m p l i n g t h e GSI : o f t h e s e g r o u p s a r e t h e same as a t t h e f i r s t a u t o p s y . I n s h o r t , , d e s p i t e an e x p o s u r e t o an u n s e a s o n a l l o w t e m p e r a t u r e b e f o r e s p a w n i n g t h e f i s h show a f a l l i n t h e GSI a t a t i m e c o n f o r m i n g t o t h e n o r m a l s p a w n i n g p e r i o d . An i n h e r e n t r h y t h m may be r e s p o n s i b l e f o r t h i s phenomenon w h i c h t h o u g h s u p p r e s s e d by an a b n o r m a l e n v i r o n m e n t a l c o n d i t i o n e x e r t s i t s e l f i n a s m a l l m e a s u r e . A m i l d d e p r e s s i o n due t o p h o t o p e r i o d may be n o t i c e d i-n tk& - 71 -l o w t e m p e r a t u r e g r o u p s w i t h s h o r t p h o t o p e r i o d d u r i n g t h e . p a r t o f t h e a n n u a l c y c l e where l i g h t p e r i o d i s l o n g ( i . e . J u l y ) . L a t e r , f r o m A u g u s t o n w a r d s , s h o r t p h o t o p e r i o d s seem t o be s l i g h t l y s t i m u l a t o r y w i t h t h e f i s h a t t a i n i n g a much h i g h e r l e v e l t h a n t h o s e i n t h e n a t u r e . I n summary, i t seems a p p a r e n t t h a t t e m p e r a t u r e has a f a r more i m p o r t a n t c o n t r o l o f t e s t i c u l a r m a t u r a t i o n t h a n p h o t o p e r i o d . Low t e m p e r a t u r e s a c c e l e r a t e t e s t i c u l a r d e v e l o p m e n t and r e c o n s t i t u t i o n , w h e r e a s h i g h e r t e m p e r a t u r e s s p e e d up r e g r e s s i o n and p r e v e n t r e c o v e r y o f t h e t e s t e s d u r i n g t h e J u l y -December p e r i o d . S p r e r m a t o q e n e t i c C o n d i t i o n Low t e m p e r a t u r e c o n t r o l l e d p h o t o p e r i o d s . T h e s p e r m a t o g e n e t i c p i c t u r e o f b o t h t h e l o n g and s h o r t p h o t o p e r i o d s i s s i m i l a r a t t h e f i r s t s a m p l i n g . U n l i k e t e s t e s i n n a t u r a l c o n d i t i o n a t s i m i l a r p e r i o d o f t h e a n n u a l c y c l e , b o t h m i t o t i c and m e i o t i c a c t i v i t y seem t o be s u p p r e s s e d ( F i g . 2 5 B ) ; a b o u t 3 5 % - 50% o f t h e e a r l i e r s t a g e s a r e i n r e s t i n g c o n d i t i o n ( T a b l e I X ) . C e l l d i v i s i o n i s s o m e t i m e s c o m p l e t e l y s u p p r e s s e d b u t c o p i o u s sperms may f i l l t h e l o b u l e s and t h e d u c t s . I n some f i s h t h e r e i s e v i d e n c e o f e v a c u a t i o n o f t h e l o b u l e s and d u c t s . The c e l l s l i n i n g t h e t e r m i n a l b r a n c h e s o f t h e d u c t a r e p a r t l y - s p e n t a l t h o u g h u n s p e n t c o l u m n a r c e l l s a r e a l s o n o t i c e d . Sperms o c c u p y 5 0 . 3 % - 5 4 . 8 % o f t h e l o b u l a r s p a c e s . O t h e r s t a g e s i n t h e o r d e r o f t h e i r p r e d o m i n a n c e a r e : I . SPG" ( 1 7 . 7 - 2 0 . 2 2 0 , I I . SPG"x ( 8 . 1 - 1 2 . 5 % ) , I I I . SPC'x ( 4 . 8 - 6 . 6 % ) , I V . SPGR (3.5-4.5%) , V. S P C ( 7 . 0 - 1 . 3 ) I t w o u l d ajppqar t h a t t h e m a j o r i t y o f t h e r e s t i n g c y s t s - 72 -b e l o n g t o e a r l y s p e r m a t o g e n e t i c s t a g e s w i t h no m a r k e d d i f f e r e n c e s a v e t h a t u n d e r l o n g p h o t o p e r i o d s , s p e r m a t o g e n e t i c a c t i v i t y i s a l m o s t c o m p l e t e l y s t o p p e d i n two f i s h ; no s u c h c o m p l e t e c e s s a t i o n was s e e n u n d e r t h e s h o r t p h o t o p e r i o d s . I t w o u l d a l s o a p p e a r t h a t l o w t e m p e r a t u r e s have a l i m i t e d r e s t r i c t i v e i n f l u e n c e on t h e d e v e l o p m e n t o f l a t e r m a t u r a t i o n s t a g e s when t h e t e s t e s a r e i n a c o n d i t i o n o f n o r m a l a c t i v i t y . Once t h e s e c o n d a r y s p e r m a t o c y t e s a r e f o r m e d t h e y c o n t i n u e d e v e l o p m e n t u n d i s t u r b e d by ; a b n o r m a l c o n d i t i o n s . H i g h T e m p e r a t u r e w i t h C o n t r o l l e d P h o t o p e r i o d s S p e r m a t o g e n e t i c c o n d i t i o n i n t h e two p h o t o p e r i o d g r o u p s i s a l m o s t s i m i l a r . I n t h e l o n g p h o t o p e r i o d f i s h b e s i d e s t h e sperms and s p e r m a t i d s o t h e r s t a g e s r e p r e s e n t e d a r e : S P G , J x ( 1 1 . 6 - 1 9 . 3 % ) , S P G " R ( 1 3 . 3 ~ 1 7 . 4 % ) , SPG 1 x ( 2 2 . 4 - 4 4 . 5%) and SPGR ( 1 1 . 6 - 1 8 . 1 % ) S i m i l a r s t a g e s a r e a l s o s e e n i n t h e s h o r t p h o t o p e r i o d f i s h a l t h o u g h t h e i r p r o p o r t i o n s a r e s l i g h t l y h i g h e r b e c a u s e o f a g r e a t e r d e g r e e o f e v a c u a t i o n and r e g r e s s i o n o f t h e t e s t e s . I t i s i n t e r e s t i n g t o n o t e t h a t i n h i g h e r t e m p e r a t u r e g r o u p s p r i m a r y and s e c o n d a r y s p e r m a t o c y t e s a r e r a r e l y s e e n . They a r e e i t h e r c o n v e r t e d i n t o s perms o r d e g e n e r a t e and s i n c e p y c n o s i s i s n o t common i t w o u l d a p p e a r t h a t t h e y -do n o t d e g e n e r a t e b u t a r e r a p i d l y t r a n s f o r m e d t o sperms and e x p e l l e d . S p e r m a t o c y t e s a r e q u i t e f r e q u e n t i n t h e l o w e r t e m p e r a t u r e g r o u p s and some c o n t i n u e d i v i d i n g . I t m i g h t be i n f e r r e d f r o m t h i s o b s e r v a t i o n t h a t t h e l a t t e r s t a g e s a r e r a t h e r i n d e p e n d e n t o f e n v i r o n m e n t a l c h a n g e s . - 73 -S e c o n d S a m p l i n g Low t e m p e r a t u r e w i t h c o n t r o l l e d p h o t o p e r i o d s . Sperms a r e s t i l l a b u n d a n t i n b o t h p h o t o p e r i o d g r o u p s a l t h o u g h t h e r e i s some c o n t i n u e d e v i d e n c e o f e v a c u a t i o n . S l o w m i t o t i c a c t i v i t y i s e v i d e n t i n a l l f i s h , and a m a r k e d f e a t u r e o f t h e t e s t e s i n t h i s g r o u p i s t h e f o r m a t i o n o f a p p a r e n t l y f r e s h n e s t s o f s e c o n d a r y s p e r m a t o g o n i a . Few l a t e r s t a g e s ( b e s i d e s s p e r m s ) a r e p r e s e n t ; s p e r m a t o c y t e s a r e s e e n i n o n l y a few o f t h e s e s h o r t p h o t o p e r i o d f i s h . P y c n o s i s i s r a r e ; e v i d e n t l y t h e s p e r m a t o c y t e s t r a n s f o r m f i n a l l y i n t o sperms and a r e c a s t o f f . D i v i d i n g s e c o n d a r y s p e r m a t o g o n i a a r e t h e most p r e d o m i n a n t s t a g e c o n s t i t u t i n g 14.9 - 1 9 . 3 % and r e s t i n g s e c o n d a r y s p e r m a t o g o n i a a r e n e x t i n o r d e r ( 14.0 - 1 4 . 6 % ) . The c e l l s l i n i n g t h e s p e r m a t i c d u c t s a r e a l l e x h a u s t e d and r a t h e r squamose. Some m i g r a t i n g c e l l s a r e p r e s e n t i n a few f i s h b e l o n g i n g t o b o t h p h o t o p e r i o d s . H i g h T e m p e r a t u r e w i t h C o n t r o l l e d P h o t o p e r i o d s B o t h t h e p h o t o p e r i o d g r o u p s a r e s p e n t w i t h o n l y a few f i s h s h o w i n g c l u m p e d sperms i n some l o b u l e s and d u c t . P y c n o s i s i s ' r a r e . D i v i d i n g p r i m a r y s p e r m a t o g o n i a p r e d o m i n a t e ( 5 9 . 6 - 6 7 . 9 % ) . S e c o n d a r y s p e r m a t o g o n i a l n e s t s a r e f e w e r ( 1 9 . 5 - 2 2 . 0 % ) ; r e s t i n g germ c e l l s a c c o u n t f o r t h e r e s t . D i v i s i o n i n t h e s e c o n d a r y s p e r m a t o g o n i a i s r a r e and p r i m a r y s p e r m a t o c y t e s a r e n o t s e e n . M i g r a t i n g c e l l s a r e much i n e v i d e n c e i n b o t h p h o t o p e r i o d s . S i n c e t h e s e c e l l s i n d i c a t e t h e b e g i n n i n g o f t h e r e c o n s t i t u t i o n o f t h e t e s t e s t t f g e t h e r w i t h some m i t o t i c a c t i v i t y i n t h e r e s t i n g germ c e l l s and p r i m a r y s p e r m a t o g o n i a ; i t a p p e a r s t h a t t h e s e f i s h have a l r e a d y commenced t h e i r r e c o v e r y f o l l o w i n g a c o m p l e t e r e g r e s s i o n . I n s h o r t , no s p e r m a t o g e n i c d i f f e r e n c e c a n be s e e n - 74 -i n t h e two p h o t o p e r i o d g r o u p s a t h i g h t e m p e r a t u r e . However, r e g r e s s i o n w h i c h was s l o w e r u n d e r l o n g e r p h o t o p e r i o d s up t o t h e f i r s t s a m p l i n g on J u n e 14, 1963, p r o c e e d e d a t a ; f a s t e r r a t e t h e r e a f t e r as compared t o t h e s h o r t e r p h o t o p e r i o d s r e s u l t i n g i n a v e r y s i m i l a r l e v e l o f t h e GSI and s p e r m a t o g e n e t i c c o n d i t i o n a t t h i s s a m p l i n g i n e a r l y J u l y . T h i s , h o w e v e r , i s i n m a r k e d c o n t r a s t t o t h o s e o f t h e n o r m a l s d u r i n g t h i s p a r t o f t h e y e a r . I t i s o b v i o u s t h a t h i g h e r t e m p e r a t u r e i r r e s p e c t i v e o f t h e p h o t o p e r i o d c a u s e s a m a r k e d and q u i c k r e g r e s s i o n i n t h e t e s t e s o f C o u e s i u s p l u m b e u s . T h i r d S a m p l i n g F i s h were k i l l e d and a u t o p s i e d on J u l y 30, 1 9 6 3 , a t a p e r i o d when t e s t e s u n d e r n a t u r a l c o n d i t i o n s a r e a t t h e i r minimum a c t i v i t y . These f i s h show t h e g r e a t e s t c o n t r a s t b e t w e e n t h e l o w and h i g h t e m p e r a t u r e c o n d i t i o n s a p p a r e n t l y i r r e s p e c t i v e o f t h e p h o t o p e r i o d s . Low T e m p e r a t u r e and C o n t r o l l e d P h o t o p e r i o d s The t e s t e s o f b o t h p h o t o p e r i o d g r o u p s p r e s e n t a r a t h e r s i m i l a r h i s t o l o g i c a l p i c t u r e . Sperms a r e p r e s e n t i n a l l , a l t h o u g h t h e r e seems t o be some f u r t h e r e v i d e n c e o f e v a c u a t i o n , sperms o c c u p y i n g 4 2 . 1 - 4 6 % o f t h e l o b u l a r s p a c e s . M i t o t i c a c t i v i t y t h o u g h p r e s e n t i s s t i l l s l o w . S e c o n d a r y s p e r m a t o g o n i a a r e t h e n e x t d o m i n a n t s t a g e a f t e r t h e s p e r m s , a c c o u n t i n g f o r 1 3 . 0 - 1 6 4 % o f t h e s e s t a g e s . D i v i d i n g s p e r m a t o g o n i a c o n s t i t u t e 1 4 . 7 - 1 5 . 3 % . Newly f o r m e d p r i m a r y s p e r m a t o c y t a l n e s t s a r e p r e s e n t i n f i s h o f b o t h g r o u p s . S i n c e t h e s e s t a g e s were c o m p l e t e l y l a c k i n g i n p r e v i o u s s a m p l i n g i t i s o b v i o u s t h a t t h e y a r e f o r m e d anew f r o m t h e r e c e n t l y f o r m e d s e c o n d a r y s p e r m a t o g o n i a . P y c n o s i s - 75 -i s s o m e t i m e s n o t i c e d . S i n c e sperms a r e nnuraerous i n b o t h g r o u p s i t may be c o n c l u d e d t h a t l o w t e m p e r a t u r e c a u s e s r e t e n t i o n o f s p e r m s . H i g h T e m p e r a t u r e and C o n t r o l l e d P h o t o p e r i o d s The' s p e r m a t o g e n e t i c c o n d i t i o n i n t h e two p h o t o p e r i o d g r o u p s i s s i m i l a r t o t h a t o f t h e p r e v i o u s s a m p l i n g and r e s e m b l e s t h e c o n d i t i o n o f n o r m a l t e s t e s w h i c h i s e x t r e m e l y r e g r e s s e d a t t h i s t i m e o f t h e y e a r . S l i g h t d i f f e r e n c e s f r o m t h e p r e v i o u s s a m p l i n g s a r e n o t e d i n a g e n e r a l i n c r e a s e o f s e c o n d a r y s p e r m a t o g o n i a ; t h e s e a c c o u n t f o r 2 8 . 1 - 2 9 . 5 % o f a l l t h e s t a g e s and a l s o i n c l u d e some d i v i d i n g n e s t s ( 1 . 1 - 7 . 9 % ) . P r i m a r y s p e r m a t o g o n i a s t i l l p r e d o m i n a t e ( 5 8 . 2 - 5 8 . 5 % ) . Some m i g r a t i n g c e l l s 1 a r e a l s o p r e s e n t . I t has been s u g g e s t e d e a r l i e r t h a t l o w t e m p e r a t u r e s f a v o u r r e t e n t i o n o f s p e r m , i t i s now o b v i o u s t h a t h i g h t e m p e r a t u r e s a c c e l e r a t e s p e r m i a t i o n . A l l t h e h i g h t e m p e r a t u r e f i s h a r e a l m o s t c o m p l e t e l y s p e n t , i n n a t u r e a l s o , s p e r m i a t i o n p e a k c o i n c i d e s w i t h t h e p e a k i n w a t e r t e m p e r a t u r e . H o wever, s p e r m i a t i o n may a l s o d e p e n d on o t h e r f a c t o r s , n o t a b l y t h e b e h a v i o u r a l , t h e p r e s e n c e o f m a t e s , and s u i t a b l e e n v i r o n m e n t a l c o n d i t i o n . Sperm r e t e n t i o n a t l o w t e m p e r a t u r e s s h o u l d be v i e w e d w i t h t h e s e f a c t o r s i n m i n d . N e v e r t h e l e s s , t h e a c c e l e r a t i o n o f s p e r m i a t i o n a t h i g h e r t e m p e r a t u r e shows some t e m p e r a t u r e d e p e n d e n c e o f t h i s phenomenon. F i n a l S a m p l i n g The f o u r t h and f i n a l s a m p l i n g o f t h e p r e s p a w n i n g s t o c k o f f i s h was on December 16, 1963. These f i s h a l s o showed t h e same t e m p e r a t u r e b a s e d v a r i a t i o n s n o t e d i n p r e v i o u s s a m p l i n g s . - 76 -Low T e m p e r a t u r e and C o n t r o l l e d P h o t o p e r i o d s S p e r m i a t i o n i s now c o m p l e t e i n b o t h p h o t o p e r i o d g r o u p s w i t h o n l y a few sperm r e m n a n t s s e e n p a r t i c u l a r l y i n t h e l o n g p h o t o p e r i o d g r o u p s . M i t o t i c a c t i v i t y has i n c r e a s e d s l i g h t l y . S e c o n d a r y s p e r m a t o g o n i a a r e t h e most i m p o r t a n t c e l l t y p e i n t h e l o n g p h o t o p e r i o d c o n t r i b u t i n g up t o 4 9 . 7 % o f t h e t o t a l o f t h e v a r i o u s - s t a g e s w h i l e p r i m a r y s p e r m a t o c y t e s ( 5 9 . 4 % ) d i s t i n c t l y o u t n u m b e r t h e s e c o n d a r y s p e r m a t o g o n i a ( 3 4 . 0 % ) u n d e r s h o r t p h o t o p e r i o d r e g i m e s ( T a b l e I X ) . The i n c r e a s e i n t h e e x t e n t o f p r i m a r y s p e r m a t o c y t e s and i n p y c n o s i s i s a l s o a d i s t i n c t i v e f e a t u r e o f t h e s h o r t p h o t o p e r i o d f i s h a t l o w t e m p e r a t u r e . S h o r t p h o t o p e r i o d f i s h , i n s p i t e o f a s l o w e r m i t o t i c a c t i v i t y , a r e s p e r m a t o g e n e t i c a l l y s l i g h t l y more a d v a n c e d t h a n t h e l o n g g r o u p as e v i d e n c e d by l a r g e r p r o p o r t i o n o f p r i m a r y s p e r m a t o c y t e s . A l t h o u g h t h e s e d i f f e r e n c e s a r e s l i g h t i t i s o b v i o u s t h a t t e s t i c u l a r m a t u r a t i o n d u r i n g t h i s p a r t o f t h e a n n u a l c y c l e i s r e t a r d e d u n d e r l o n g p h o t o p e r i o d ; u n d e r s h o r t p h o t o p e r i o d s , t h e t e s t e s r e a c h t h e i r maximum s e a s o n a l l i m i t f a s t e r and t h e r e f o r e t h e p a c e o f d e v e l o p m e n t i s s l a c k e n e d e a r l i e r . The h i s t o l o g i c a l p i c t u r e o f b o t h t h e s e g r o u p s i s somewhat more a d v a n c e d t h a n t h e n o r m a l f i s h d u r i n g t h e same p e r i o d o f t h e y e a r . H i g h T e m p e r a t u r e and C o n t r o l l e d P h o t o p e r i o d s The two p h o t o p e r i o d g r o u p s a t h i g h t e m p e r a t u r e s show no d e f i n i t e d i f f e r e n c e and a r e b o t h much r e t a r d e d as compared t o t h e n o r m a l s ( T a b l e I X ) . However, t h e y do show a s l i g h t i n c r e a s e i n s p e r m a t o g e n e t i c a c t i v i t y o v e r t h e p r e v i o u s s a m p l e w i t h i n c r e a s e d a p p e a r a n c e o f t h e p r i m a r y s p e r m a t o c y t e s ( 3 . 8 - 4 , 6 % ) and a l s o i n t h e s e c o n d a r y s p e r m a t o g o n i a ( F i g . 2 2 ) . M i g r a t i n g - 7 7 -c e l l s a r e n o t s e e n . F i s h a t h i g h e r t e m p e r a t u r e u n d e r e i t h e r l i g h t . . c o n d i t i o n s t h u s show a v e r y s l o w i n c r e a s e i n t h e s p e r m a t o g e n e t i c a c t i v i t y o v e r t h e p r e v i o u s s a m p l e . F i g u r e 21 shows t h a t t h e r e i s a g r a d u a l b u i l d up o f t h e n o r m a l t e s t e s a f t e r c o m p l e t e : r e g r e s s i o n i n e a r l y A u g u s t . The same t r e n d i s shown t o a l i m i t e d e x t e n t i n t h e s e two e x p e r i m e n t a l g r o u p s . I n summary, by t h e end o f t h e e x p e r i m e n t b o t h t h e GSI and t h e t e s t i c u l a r d e v e l o p m e n t o f t h e h i g h t e m p e r a t u r e f i s h i s much b e l o w t h a t o f t h e n o r m a l f i s h . On t h e c o n t r a r y , a t l o w t e m p e r a t u r e s , a t e s t i c u l a r r e g r e s s i o n n e v e r o c c u r s ; t h e r e i s a s u s t a i n e d m i t o t i c a c t i v i t y o v e r t h e e n t i r e p e r i o d o f t h e e x p e r i m e n t e n d i n g i n a h i s t o l o g i c a l c o n d i t i o n more a d v a n c e d t h a n t h o s e o b t a i n i n g i n f i s h i n n a t u r e . I t i s i n t e r e s t i n g t o n o t e t h a t s p e r m a t o g e n e t i c p r o g r e s s e v e n i n t h e l o w t e m p e r a t u r e g r o u p s i s b l o c k e d a t t h e l e v e l o f p r i m a r y s p e r m a t o c y t e p r o d u c t i o n d u r i n g t h e p r e - f r e e z e up p e r i o d o f t h e y e a r , and t h e t e s t e s a r e p r e v e n t e d f r o m p a s s i n g i n t o t h e m e i o t i c p h a s e o f d e v e l o p m e n t . I 3 . T . P h o t o p e r i o d - T e m p e r a t u r e E x p e r i m e n t s w i t h t h e P o s t - S p a w n i n g  S t o c k T h i s e x p e r i m e n t commenced on A u g u s t 9, 1 9 6 3 , and f i s h were s a m p l e d on t h r e e o c c a s i o n s w i t h t h e f i n a l s a m p l i n g on F e b r u a r y 2, 1963 ( T a b l e X ) . At t h e f i r s t s a m p l i n g ( S e p t e m b e r 10, 1 9 6 3 ) , c h a n g e s i n t h e l o w t e m p e r a t u r e p h o t o p e r i o d g r o u p s a p p e a r t o be s i m i l a r t o t h o s e f o u n d i n n a t u r e ; t h e GSI o f t h e l o n g p h o t o p e r i o d f i s h i s s i g n i f i c a n t l y h i g h e r t h a n t h a t o f t h e n o r m a l s w h i c h c l o s e l y a p p r o x i m a t e d t o t h e s h o r t p h o t o p e r i o d G S I . T e s t i c u l a r development d o e s n o t seem t o a d v a n c e s i g n i f i c a n t l y i n t h e h i g h t e m p e r a t u r e g r o u p s . I n f a c t , GSI o f b o t h g r o u p s i s l o w e r t h a n t h e n o r m a l s ; - 78 -F i g . 2 3 , GSI o f ' p o s t s p a w n i n g ' f i s h u n d e r f o u r d i f f e r e n t p h o t o -o e r i o d - t e m p e r a t u r e c o m b i n a t i o n a t t h e t i m e s o f t h e i n d i c a t e d s a m p l i n g s c o m p a r e d w i t h t h o s e o f t h e n o r m a l s . F o r e x p l a n a t i o n s see F i g u r e 21. - 79 -F i g . 2 4 A . P h o t o m i c r o g r a p h o f a S t a g e V o r e a r l y S t a g e I t e s t i s s h o w i n g l o b u l e s c o n s i s t i n g m o s t l y o f p r i m a r y germ c e l l s . M i g r a t i n g c e l l s a r e shown i n t h e i n t e r l o b u l a r s p a c e , some o f w h i c h have e n t e r e d t h e l o b u l e s w h i c h l a c k a l u m e n . (X 700) B. P h o t o m i c r o g r a p h o f a s e c t i o n o f t e s t i s f r o m p h o t o p e r i o d - t e m p e r a t u r e f i s h s h o w i n g p r e m a t u r e f o r m a t i o n o f primary s p e r m a t o c y t e s and t h e s t o p p i n g o f any f u r t h e r s p e r m a t o g e n e t i c p r o g r e s s a t l o w e r t e m p e r a t u r e s ( X 700) - 80 -F i g . 25A. P h o t o m i c r o g r a p h o f a p o r t i o n o f an LS o f a n o r m a l s p a w n i n g t e s t i s s h o w i n g d i v i d i n g n e s t s o f s p e r m a t o g o n i a and s p e r m a t o c y t e s l i n i n g t h e l o b u l e f i l l e d w i t h s perms (X 7 0 0 ) B. P h o t o m i c r o g r a p h o f a p o r t i o n o f an LS o f a t e s t i s o f s i m i l a r f i s h u n d e r l o w t e m p e r a t u r e s ( e i t h e r p h o t o p e r i o d ) s h o w i n g s u p p r e s s i o n o f s p e r m a t o g e n e t i c a c t i v i t y . (X 700 ) - i 81 -F i g . 2 6 A . P h o t o m i c r o g r a p h o f a p o r t i o n o f t e s t i s s h o w i n g l o b u l e b o u n d a r y c e l l s (X 1260) B. P h o t o m i c r o g r a p h o f a t e s t i s o f C. p l u m b e u s s t a i n e d w i t h Sudan B l a c k B s h o w i n g l i p o i d a l l o b u l e b o u n d a r y c e l l a r e a s , and a b s e n c e o f any i n t r a l o b u l a r l i p i d a c c u m u l a t i o n (X 1 3 5 ) . - 82 -t h e l o n g p h o t o p e r i o d f i s h show s l i g h t l y l e s s r e g r e s s i o n . I n t h e s e c o n d s a m p l i n g t h e l o w t e m p e r a t u r e g r o u p s show a c o n s i d e r a b l y h i g h e r GSI t h a n t h e n o r m a l s w i t h t h e l o n g p h o t o p e r i o d f i s h s t i l l s l i g h t l y h i g h e r i n GSI t h a n t h e s h o r t p h o t o p e r i o d a n i m a l s . F i s h i n t h e h i g h t e m p e r a t u r e g r o u p s a l s o show an i n c r e a s e i n t h e i r GSI a l t h o u g h t h e change i s more g r a d u a l ; t h e s h o r t e r p h o t o p e r i o d GSI i s h i g h e r t h a n t h e l o n g a t t h e s e t e m p e r a t u r e s ( F i g . 2 3 ) . A t t h e f i n a l s a m p l i n g t h e GSI o f t h e l o w t e m p e r a t u r e g r o u p s r e m a i n h i g h e r t h a n t h e n o r m a l s a l t h o u g h t h e GSI o f t h e s h o r t p h o t o p e r i o d g r o u p now e x c e e d s t h a t o f t h e l o n g p h o t o p e r i o d f i s h . T h i s change i s s i m i l a r t o t h e s i t u a t i o n s e e n i n t h e p r e v i o u s e x p e r i m e n t ( F i g . 2 1 ) . T h u s , i n b o t h e x p e r i m e n t s t e s t i c u l a r d e v e l o p m e n t was a t f i r s t f a s t e r u n d e r l o n g p h o t o p e r i o d and l o w t e m p e r a t u r e b u t l a t e r i t p r o c e e d e d a t more r a p i d r a t e u n d e r t h e s h o r t p h o t o p e r i o d s ( l o w t e m p e r a t u r e ) . F i s h i n t h e two p h o t o p e r i o d g r o u p s a t h i g h t e m p e r a t u r e have s i m i l a r GSI w h i c h i n d i c a t e s t h a t t h e l o n g p h o t o p e r i o d g r o u p has c o n t i n u e d t o d e v e l o p a t a s l i g h t l y f a s t e r r a t e t h a n t h e short p h o t o p e r i o d o nes e n d i n g up a t l e v e l s w i t h them. A c t u a l l y , t h e h i g h t e m p e r a t u r e g r o u p s have c h a n g e d o n l y s l i g h t l y f r o m t h e i r i n i t i a l c o n d i t i o n . I t i s c o n c l u d e d t h a t i n t h e p o s t - s p a w n i n g s t o c k t e s t i c u l a r r e g r e s s i o n a t h i g h t e m p e r a t u r e s o c c u r s i r r e s p e c t i v e o f t h e p h o t o p e r i o d . On t h e o t h e r h a n d , m a t u r a t i o n a t l o w t e m p e r a t u r e i s f a s t e r and c o n s i d e r a b l y g r e a t e r t h a n t h e n o r m a l s w i t h l i t t l e d i f f e r e n c e i n t h e GSI o f t h e two p h o t o p e r i o d g r o u p s . The s m a l l p h o t o p e r i o d e f f e c t seems t o be c o n f i n e d t o t h e s t i m u l a t o r y n a t u r e o f t h e l o n g p h o t o p e r i o d i n t h e i n i t i a l - 83 -F i g . 2 7 . S p e r m a t o g e n e t i c c o m p o s i t i o n o f ' p o s t s p a w n i n g f i s h * u n d e r f o u r c o m b i n a t i o n s o f p h o t o p e r i o d - t e m p e r a t u r e a t t h e t i m e o f t h e v a r i o u s s a m p l i n g s i n d i c a t e d by Roman n u m e r a l s . O t h e r e x p l a n a t i o n s as i n F i g u r e 22. - 84 -s t a g e s o f t h e e x p e r i m e n t . S p e r m a t o g e n e t i c C o n d i t i o n o f P o s t - s p a w n i n g F i s h u n d e r Experimental  C o n d i t i o n s F i r s t S a m p l i n g Low t e m p e r a t u r e w i t h c o n t r o l l e d p h o t o p e r i o d s . At t h e f i r s t s a m p l i n g , some m i t o t i c a c t i v i t y i s n o t i c e d i n f i s h u n d e r b o t h p h o t o p e r i o d r e g i m e s a t l o w t e m p e r a t u r e . D i v i d i n g n e s t s c o n s t i t u t e 4 3 . 9 - 4 4 . 0 % o f a l l s t a g e s , and d i v i d i n g s e c o n d a r y s p e r m a t o g o n i a a r e t h e most p r e d o m i n a n t s t a g e i n t h e l o n g p h o t o p e r i o d f i s h ( 3 0 . 8 % ) , f o l l o w e d c l o s e l y by r e s t i n g s e c o n d a r y s p e r m a t o g o n i a ( 2 9 . 4 % ) . D i v i d i n g p r i m a r y s p e r m a t o g o n i a c o n s t i t u t e 1 2 . 6 % o f t h e t o t a l and t h e r e s t i s made up by p r i m a r y s p e r m a t o c y t e s 6.7% ( T a b l e X ) l . A f e w s e c o n d a r y s p e r m a t o c y t e s a r e a l s o s e e n . F i s h u n d e r t h e s h o r t p h o t o p e r i o d show a s i m i l a r p i c t u r e e x c e p t t h a t t h e y show a g r e a t e r p r o p o r t i o n o f s p e r m a t o c y t a l s t a g e s ( F i g . 2 7 ) . P y c n o s i s i s n o t common. H i g h T e m p e r a t u r e w i t h C o n t r o l l e d P h o t o p e r i o d s The two p h o t o p e r i o d s seem t o p r o d u c e some d i f f e r e n c e i n f i s h a t h i g h t e m p e r a t u r e ( F i g . 2 7 ) . I n p a r t i c u l a r , t h e l o n g p h o t o p e r i o d f i s h show v e r y l i t t l e m i t o t i c a c t i v i t y ( d i v i d i n g s t a g e s c o n s t i t u t i n g o n l y 1.0%) as c o m p a r e d t o t h o s e u n d e r s h o r t p h o t o p e r i o d ( d i v i d i n g s t a g e s 10.8%) l e a v i n g a s i d e t h e p r i m a r y s p e r m a t o g o n i a whose d i v i s i o n i n t h e s e f i s h i s r e s t r i c t e d t o a few n u c l e i h e r e and t h e r e . T h i s a c t i v i t y d i f f e r e n c e i s a l s o r e f l e c t e d i n t h e s p e r m a t o g e n e t i c c o m p o s i t i o n . I n l o n g photoperiod f i s h , s l o w l y and s p a r s e l y d i v i d i n g p r i m a r y s p e r m a t o g o n i a p r e d o m i n a t e ( 6 1 . 7 % ) ; t h e r e a r e f e w e r s e c o n d a r y s p e r m a t o g o n i a ( 2 5 . 3 % ) ; t h e r e m a i n d e r a r e p r i m a r y germ c e l l s ( 1 2 . 0 % ) . C o n t r a s t e d - 85 -t o t h i s , s e c o n d a r y s p e r m a t o g o n i a a r e t h e most common s t a g e ( 3 7 . 0 % ) i n t h e s h o r t p h o t o p e r i o d g r o u p . T h e r e a r e a l s o some d i v i d i n g n e s t s o f t h i s s t a g e ( 2 . 0 % ) . D i v i d i n g p r i m a r y s p e r m a t o g o n i a c o n s t i t u t e o n l y 3 1 . 7 % o f t h e s p e r m a t o g e n e t i c s t a g e s . k . P y c n o t i c n e s t s a r e n o t i c e d o n l y i n t h e s h o r t p h o t o p e r i o d g r o u p . I t a p p e a r s t h a t t h e s e r e p r e s e n t t h e d e g e n e r a t i n g l a t e r g e n e r a t i o n s o f s e c o n d a r y s p e r m a t o g o n i a and p r i m a r y s p e r m a t o c y t e s . S e c o n d S a m p l i n g Low t e m p e r a t u r e w i t h c o n t r o l l e d p h o t o p e r i o d s . The s p e r m a t o g e n e t i c c o n d i t i o n o f f i s h u n d e r t h e two p h o t o p e r i o d s i t u a t i o n s a r e much more s i m i l a r . M i t o t i c a c t i v i t y i s o f t h e same o r d e r as i n t h e f i r s t s a m p l e w i t h p r i m a r y s p e r m a t o c y t e s t h e p r e d o m i n a n t f o r m i n b o t h g r o u p s ( 2 9 . 6 - 3 2 . 5 % ) . P y c n o s i s i s p r e s e n t ( 1 . 9 % i n t h e l o n g p h o t o p e r i o d and 1.6% i n t h e s h o r t ) . S e c o n d a r y s p e r m a t o g o n i a a r e o n l y s l i g h t l y l e s s common t h a n t h e p r i m a r y s p e r m a t o c y t e s . H i g h T e m p e r a t u r e w i t h C o n t r o l l e d P h o t o p e r i o d s The two p h o t o p e r i o d g r o u p s show some s p e r m a t o g e n e t i c d i f f e r e n c e . M i t o t i c a c t i v i t y i s l o w ( 2 . 8 - 1 1 . 5 % ) i n b o t h , b u t t h e s h o r t p h o t o p e r i o d g r o u p s i s s p e r m a t o g e n e t i c a l l y r a t h e r more a d v a n c e d t h a n t h e l o n g p h o t o p e r i o d g r o u p . A l t h o u g h d i v i d i n g p r i m a r y s p e r m a t o g o n i a a r e t h e m a j o r c e l l t y p e i n b o t h g r o u p s ( T a b l e X ) , t h e r e i s a h i g h e r p r o p o r t i o n o f s e c o n d a r y spermatogonia i n t h e s h o r t p h o t o p e r i o d f i s h . A l s o , i n t h e s e f i s h , some p r i m a r y s p e r m a t o c y t e s a r e s e e n ( 3 . 6 % ) ; t h e s e a r e l e s s ( 1 . 5 % ) . i n t h e l o n g e r g r o u p . P y c n o t i c n e s t s a r e n o t s e e n i n f i s h i n t h e s e g r o u p s . The n o r m a l f i s h d u r i n g a s i m i l a r p e r i o d o f t h e y e a r show a h i g h e r p r o p o r t i o n o f p r i m a r y s p e r m a t o c y t e s ( 3 5 . 4 % ) and - 86 -secondary spermatogonia (34.3%). These are thus much more advanced than the high temperature fish, but compared to the low temperature fish they are less advanced and less active. Fina^ ..Sampling Low temperature with controlled photoperiods. At the final sampling the spermatogenetic condition in the two photoperiod groups at low temperature is rather similar, although the short photoperiod fish are slightly advanced over the long photoperiod group. In the latter, most of the testes is composed of primary spermatocytes (52.2%). Next are the secondary spermatogonia (21.3%) and their dividing stage (17.4%). Some secondary spermatocytes appear for the f i r s t time in this sample in small numbers (0,5%). Pycnosis is common, with the long photoperiod group showing more pycnotic nests (3.8%). In only one fish under long photoperiod a few nests of spermatids and some sperms were noticed. The sihort photoperiod fish have slightly more active testes. In 50% of them sperms are present in small quantity. The bulk of the testes is composed of primary spermatocytes (63.2%) with about half of these nests undergoing division. Dividing secondary spermatogonia constitute 16,2% of the stages. Some resting secondary spermatogonial nests are present. Sperms and a few resting germ cells complete the spermatogenetic picture. In those fish which do not show sperms, primary spermatocytes are the most predominant stage. The short photoperiod fish thus show a more advanced testicular development than the longer photoperiod. The spermatogenetic situation even in the long photoperiod group is in advance over that of the normals from - 87 -same p e r i o d o f t h e y e a r . H i g h T e m p e r a t u r e w i t h C o n t r o l l e d P h o t o p e r i o d s F i s h b e l o n g i n g t o t h e two p h o t o p e r i o d s show s i m i l a r s p e r m a t o g e n e t i c a p p e a r a n c e . M i t o t i c a c t i v i t y i n b o t h i s l o w . S e c o n d a r y s p e r m a t o g o n i a p r e d o m i n a t e ( 4 2 . 0 - 4 2 . 6 % ) , f o l l o w e d by d i v i d i n g p r i m a r y s p e r m a t o g o n i a l n e s t s ( 2 4 . 5 - 2 7 . 2 % ) . D i v i d i n g s e c o n d a r y s p e r m a t o g o n i a a r e f e w e r ( 1 1 . 6 - 2 0 . 9 % ) . I n some f i s h , p r i m a r y s p e r m a t o c y t e s a r e a l s o s e e n . P y c n o t i c n e s t s a r e much f e w e r i n t h ^ s e f i s h t h a n t h e l o w t e m p e r a t u r e o n e s . F i s h k e p t u n d e r n o r m a l l y c h a n g i n g d a y l e n g t h s a t l o w t e m p e r a t u r e have GSI l o w e r t h a n t h o s e o f t h e e x p e r i m e n t a l f i s h a t s i m i l a r t e m p e r a t u r e s , a n d . t h e s p e r m a t o g e n e t i c c o n d i t i o n i s a l s o l e s s a d v a n c e d . These t e s t e s a r e composed o f p r i m a r y s p e r m a t o c y t e s and s e c o n d a r y s p e r m a t o g o n i a , and l a t e r s t a g e s a r e a b s e n t . M i t o t i c a c t i v i t y i s l e s s t h a n t h e s h o r t p h o t o p e r i o d l o w t e m p e r a t u r e f i s h . P y c n o t i c n e s t s i n t h e n o r m a l s a r e a b s e n t . Summary o f E f f e c t o f P h o t o p e r i o d - t e m p t e r a t u r e C o m b i n a t i o n on P o s t - s p a w n i n g F i s h Low t e m p e r a t u r e s seem t o be s t i m u l a t o r y f o r t h e t e s t i c u l a r m a t u r a t i o n i n C o u e s i u s d u r i n g t h e p o s t - s p a w n i n g p e r i o d o f t h e a n n u a l c y c l e . L ong p h o t o p e r i o d i s s l i g h t l y more a c t i v a t i n g t h a n s h o r t p h o t o p e r i o d i n t h e e a r l i e r p a r t o f t h i s p e r i o d b u t i n t h e w i n t e r months t h e s h o r t p h o t o p e r i o d a p p e a r s t o be more e f f i c a c i o u s . C o n t i n u e d h i g h t e m p e r a t u r e s have a r e g r e s s i v e e f f e c t on t h e t e s t e s . -4 • P h o t o p e r i o d - t e m p e r a t u r e E x p e r i m e n t s w i t h t h e ' P r e - f r e e z e Up'  S t o c k I n t h i s e x p e r i m e n t , s t a r t e d on November 9, 1963, t h e f i s h were s a m p l e d t w i c e , w i t h t h e f i n a l s a m p l i n g on A p r i l 1, 1964. - 88 -At t h e f i r s t s a m p l i n g (December 6, 1963) t h e GSI o f t h e l o n g p h o t o p e r i o d g r o u p was h i g h e r t h a n t h e i n i t i a l v a l u e s o r t h e n o r m a l s a t t h i s s a m p l i n g . S h o r t p h o t o p e r i o d f i s h showed o n l y a s l i g h t i n c r e a s e i n t h e GSI ( F i g . 2 8 ) . At t h e h i g h t e m p e r a t u r e s , b o t h p h o t o p e r i o d g r o u p s e x h i b i t e d a m a r k e d b u t s i m i l a r d e c r e a s e i n t h e 'GSI. At t h e f i n a l s a m p l i n g , t h e s h o r t p h o t o p e r i o d g r o u p a t l o w t e m p e r a t u r e showed a much h i g h e r GSI t h a n t h o s e u n d e r t h e l o n g p h o t o p e r i o d ( a g a i n s i m i l a r t o e a r l i e r e x p e r i m e n t s ) , w h i l e t h e GSI o f b o t h g r o u p s were s i g n i f i c a n t l y h i g h e r t h a n t h e n o r m a l s . The h i g h t e m p e r a t u r e p h o t o p e r i o d g r o u p s showed no change i n t h e i r G S I , t h e GSI o f t h e l o n g o h o t o p e r i o d f i s h i s s l i g h t l y h i g h e r t h a n t h e s h o r t p h o t o p e r i o d a n i m a l s b u t t h e r e i s r e l a t i v e l y l i t t l e c h ange f r o m t h e f i r s t s a m p l i n g . T h u s , h i g h t e m p e r a t u r e s seem t o c o m p l e t e l y b l o c k t e s t i c u l a r g r o w t h d u r i n g t h e ' f r e e z e up' months o f t h e a n n u a l c y c l e i n C o u e s i u s . S p e r m a t o g e n e t i c C o n d i t i o n  F i r s t S a m p l i n g Low t e m p e r a t u r e s w i t h c o n t r o l l e d p h o t o p e r i o d s . H i s t o l o g i c a l , a p p e a r a n c e s o f t h e two c o n t r a s t i n g p h o t o p e r i o d g r o u p s a r e s i m i l a r w i t h a s l i g h t l y i n c r e a s e d c e l l d i v i s i o n i n t h e l o n g e r p h o t o p e r i o d f i s h ( F i g . 2 9 ) . The t e s t e s i n t h e s h o r t p h o t o p e r i o d g r o u p have s i m i l a r amounts o f d i v i d i n g s e c o n d a r y s p e r m a t o g o n i a {22.6%) and p r i m a r y s p e r m a t o c y t e s ( 2 5 . 2 % ) . D i v i d i n g n e s t s a r e r a t h e r f e w e r i n i;he s h o r t p h o t o p e r i o d t h a n i n t h e l o n g . P y c n o s i s i s s l i g h t l y g r e a t e r i n t h e l o n g p h o t o p e r i o d w h i c h a l s o has a g r e a t e r p r o p o r t i o n o f p r i m a r y s p e r m a t o c y t e s ( 3 1 . 0 % ) . Some d i v i d i n g p r i m a r y s p e r m a t o c y t e s a r e s e e n o n l y i n t h e l o n g p h o t o p e r i o d f i s h ( T a b l e XI). As p y c n o s i s o c c u r s i n l a t e r g e n e r a t i o n s o f s e c o n d a r y - 89 -F i g . 2 8 . GSI o f ' p r e - f r e e z e up' f i s h u n d e r f o u r d i f f e r e n t p h o t a p e r i o d - L t e m p e r a t u r e c o m b i n a t i o n s a t t h e t i m e o f t h e i n d i c a t e d s a m p l i n g s . The c o n v e r g i n g p o i n t a t l e f t i n d i c a t e s t h e GSI o f t h e n o r m a l s a t t h e s t a r t o f t h e e x p e r i m e n t . • t N o r m a l s . . O t h e r e x p l a n a t i o n s same as i n F i g u r e 21. D E C APR - 9 0 : -F i g . 2 9 . S p e r m a t o g e n e t i c c o m p o s i t i o n o f ' p r e - f r e e z e up' f i s h u n d e r f o u r c o m b i n a t i o n s o f p h o t o p e r i o d and t e m p e r a t u r e a t t h e t i m e o f t h e v a r i o u s s a m p l i n g s i n d i c a t e d by Roman n u m e r a l s . E x p l a n a t i o n s same as i n F i g u r e 22. - 91 -s p e r m a t o g o n i a and p r i m a r y s p e r m a t o c y t e s , an i n c r e a s e i n t h e e x t e n t o f p y c n o s i s i n t h e l o n g p h o t o p e r i o d g r o u p w h i c h a l s o s h o w s g r e a t e r a c t i v i t y may i n d i c a t e t h a t s u p e r f l u o u s o r e x c e s s n e s t s o f t h e s e s t a g e s a r e d e g e n e r a t e d by an i n h e r e n t r e g u l a t o r y m e c h a n i s m . H i g h T e m p e r a t u r e s w i t h C o n t r o l l e d P h o t o p e r i o d s • S p e r m a t o g e n e t i c c o n d i t i o n i n t h e two p h o t o p e r i o d g r o u p s i s v e r y s i m i l a r . T estes a r e m o s t l y composed o f s e c o n d a r y s p e r m a t o g o n i a ( 3 5 . 6 - 4 2 . 2 % ) and d i v i d i n g p r i m a r y s p e r m a t o g o n i a ( 3 5 . 1 - 3 6 . 8 % ) . T h e r e a r e some d i v i d i n g s e c o n d a r y s p e r m a t o g o n i a and a f e w s p e r m a t o c y t a l n e s t s ( F i g . 2 9 ) . No l a t e r s t a g e s a r e s e e n . F a i r l y e x t e n s i v e p y c n o t i c n e s t s a r e o b s e r v e d i n b o t h groups, and m i t o t i c a c t i v i t y i s l o w . These g r o u p s t h u s show u n m i s t a k a b l e s i g n s o f r e g r e s s i o n . F i n a l S a m p l i n g ( A p r i l 1, 1964) Low t e m p e r a t u r e w i t h c o n t r o l l e d p h o t o p e r i o d s . B o t h p h o t o p e r i o d g r o u p s a r e more a d v a n c e d t h a n the. n o r m a l a n i m a l s k i l l e d a t t h e same p e r i o d . The s h o r t p h o t o p e r i o d f i s h a r e t h e more a d v a n c e d o f t h e two e x p e r i m e n t a l g r o u p s . A s m a l l number o f m a t u r e sperms a r e s e e n i n f i s h a t t h i s a u t o p s y , w i t h 4 0 % o f t h e l o n g p h o t o p e r i o d f i s h and 8 0 % o f t h e s h o r t p h o t o p e r i o d f i s h s h o w i n g f e w l o b u l e s c o n t a i n i n g s p e r m s . The d u c t s a r e s t i l l c o l l a p s e d and empty. S p e r m a t o c y t e s a r e t h e d o m i n a n t f o r m i n b o t h g r o u p s , w i t h t h e s h o r t p h o t o p e r i o d f i s h s h o w i n g a m a r k e d i n c r e a s e i n t h e s e c o n d a r y s p e r m a t o c y t e s ( 2 7 . 7 % ) , F i g u r e 28. An i n t e r e s t i n g f e a t u r e o f t h e s e two g r o u p s o f f i s h a t t h i s a u t o p s y i s t h e c o m p l e t e a b s e n c e o f p y c n o t i c n e s t s . S h o r t p h o t o p e r i o d f i s h a r e - 92 -s i m i l a r i n a c t i v i t y ( 7 2 . 1 % d i v i d i n g s t a g e s ) t o t h e l o n g g r o u p ( 7 1 . 6 % d i v i d i n g s t a g e s ) b u t i n c l u d e a s l i g h t l y g r e a t e r p r o p o r t i o n o f a d v a n c e d s t a g e s . T h e r e i s no change i n t h e c e l l s l i n i n g t h e s p e r m a t i c d u c t s ; t h e y r e m a i n squamose. N o r m a l f i s h a t t h i s a u t o p s y show a u n i f o r m p i c t u r e . Sperms a r e s e e n i n o n l y one f i s h o u t o f e i g h t . L o b u l e s a r e composed o f a c t i v e l y d i v i d i n g n e s t s , w i t h a p r e p o n d e r a n c e o f t h e p r i m a r y s p e r m a t o c y t e s ( 5 3 . 3 % ) o v e r o t h e r s t a g e s . D i v i d i n g s e c o n d a r y s p e r m a t o g o n i a make up f o r 2 0 . 3 % o f t h e v a r i o u s s t a g e s . S e c o n d a r y s p e r m a t o c y t e s , c o n s t i t u t e 1 0 . 6 % o f t h e t o t a l s t a g e s . T hese f i s h a r e t h u s l e s s a c t i v e t h a n any o f t h e two l o w t e m p e r a t u r e g r o u p s , b u t much more a d v a n c e d t h a n any o f t h o s e a t h i g h t e m p e r a t u r e . H i g h T e m p e r a t u r e w i t h C o n t r o l l e d P h o t o p e r i o d s F i s h b e l o n g i n g t o b o t h p h o t o p e r i o d g r o u p s show r e g r e s s e d t e s t e s . M i t o t i c a c t i v i t y i s r a r e . L o b u l e s a r e n a r r o w and t h e n e s t s a r e s m a l l . S e c o n d a r y s p e r m a t o g o n i a (52.1-52 . 9 % ) and d i v i d i n g p r i m a r y s p e r m a t o g o n i a l n e s t s ( 3 5 . 1 - 3 6 . 8 % ) a r e t h e commoner c e l l t y p e s . P r i m a r y s p e r m a t o c y t e s a r e s e e n i n some f i s h . No l a t e r s t a g e s a r e f o u n d , and t h e c e l l s l i n i n g t h e d u c t s r e m a i n squamose. Summary o f E f f e c t s o f P h o t o p e r i o d - t e m p e r a t u r e C o m b i n a t i o n s on " P r e - f r e e z e Up" F i s h When C o u e s i u s p l u m b eus i s s u b j e c t e d t o t e m p e r a t u r e -p h o t o p e r i o d m a n i p u l a t i o n , n o r m a l m a t u r a t i o n o c c u r s o n l y a t l o w t e m p e r a t u r e . The s p e e d o f m a t u r a t i o n i s f a s t e r ( i n b o t h l o n g and s h o r t p h o t o p e r i o d g r o u p ) t h a n i n t h e n o r m a l s k e p t u n d e r n o r m a l l y c h a n g i n g d a y l e n g t h a t l o w t e m p e r a t u r e s . S h o r t p h o t o p e r i o d i s s l i g h t l y more s t i m u l a t o r y i n t h e l a t e r p a r t o f t h e w i n t e r ; whereas - 93 -i n t h e e a r l y w i n t e r l o n g p h o t o p e r i o d seems t o be more e f f i c a c i o u s . P h o t o p e r i o b l d i f f e r e n c e s a r e n o t a p p a r e n t a t h i g h t e m p e r a t u r e s whefe b o t h g r o u p s r e g r e s s e d t o t h e same l e v e l . G e n e r a l Summary o f P h o t o p e r i o d - t e m p e r a t u r e E x p e r i m e n t s The r e s u l t s o f t h e p h o t o p e r i o d - t e m p e r a t u r e e x p e r i m e n t s i n d i c a t e t h a t t e m p e r a t u r e i s a f a r more i m p o r t a n t e n v i r o n m e n t a l f a c t o r f o r t h e s p e r m a t o g e n e t i c p r o c e s s o f C. p l u m b e u s t h a n l i g h t . Low t e m p e r a t u r e h a s a r e t a r d i n g e f f e c t on t h e s p e r m i o g e n e s i s and s p e r m i a t i o n 1 i n t h e p r e s p a w n i n g f i s h ; i n t h e p o s t s p a w n i n g and p r e -f r e e z e up f i s h l o w t e m p e r a t u r e s s t i m u l a t e e a r l i e r s p e r m a t o g e n e t i c d e v e l o p m e n t , and f i s h u n d e r t h e s e e x p e r i m e n t a l c o n d i t i o n s o u t s t r i p t h e s p e r m a t o g e n e t i c p r o g r e s s o f t h e f i s h u n d e r n o r m a l e n v i r o n m e n t a l c o n d i t i o n s . H i g h e r t e m p e r a t u r e s s p e e d s p e r m i a t i o n and i n d u c e q u i c k e r r e g r e s s i o n i n p r e s p a w n i n g f i s h . They r e s t r i c t t h e e a r l i e r d e v e l o p m e n t o f t e s t e s i n f i s h d u r i n g a l l t h e t h r e e p h a s e s o f t h e c y c l e s t u d i e d . A d i a g r a m m a t i c summary o f t h e p h o t o p e r i o d -t e m p e r a t u r e e x p e r i m e n t i s g i v e n i n F i g u r e 3 1 . A fee±>le p h o t o p e r i o d i c e f f e c t i s a l s o n o t i c e d . I n t h e i n i t i a l p h a s e o f e a c h e x p e r i m e n t a t l o w t e m p e r a t u r e s t h e l o n g p h o t o p e r i o d i s s l i g h t l y s t i m u l a t o r y , b u t l a t e r s h o r t p h o t o p e r i o d seems more a c t i v a t i n g . A t t h e f i n a l s a m p l i n g of. t h e p r e - f r e e z e up f i s h , p o s t s p e r m a t o c y t a l c h a n g e s ( a t l o w t e m p e r a t u r e s o n l y ) a r e n o t i c e d u n d e r b o t h p h o t o p e r i o d s ; t h e t e s t e s o f t h e s h o r t p h o t o p e r i o d f i s h i s s l i g h t l y more a d v a n c e d t h a n t h o s e o f t h e l o n g p h o t o p e r i o d f i s h . T h i s t r e n d t h o u g h c o n s i s t e n t i n a l l t h e t h r e e s t o c k s o f f i s h i s n o t s t a t i s t i c a l l y s i g n i f i c a n t ( p r e s p a w n i n g f i s h v a l u e o f t ~ 1.468, p o s t - s p a w n i n g f i s h t = 0.818 and p r e - f r e e z e up f i s h t = 1 . 0 3 6 ) . - 94 -F i g . 3 0 . Summary o f t h e r e p l a c e m e n t t h e r a p y e x p e r i m e n t s s h o w i n g s e a s o n a l e f f e c t . The smooth c u r v e i n d i c a t e s t h e seasonal c h a n g e i n t h e a c t i v i t y o f t h e n o r m a l t e s t e s . Roman n u m e r a l s i n d i c a t e t h e f i v e s t a g e s o f t h e a n n u a l t e s t i c u l a r c y c l e . S i g n s a t t h e t o p o f t h e f i g u r e i n d i c a t e t h e t i m i n g o f t h e v a r i o u s e x p e r i m e n t s w i t h r e f e r e n c e t o t h e a n n u a l c y c l e , and t h e i r e f f e c t s . A (-) s i g n d e n o t e s r e g r e s s i o n ; and a (+) s i g n s t i m u l a t i o n . I n c r e a s e s i n s t i m u l a t o r y and r e g r e s s i v e e f f e c t i s i n d i c a t e d by i n c r e a s e i n number o f t h e s e s i g n s . 1 1 l 1 • i i 1 I i 4-i 4-i 4-1 + I +,++(+ +,++ + 4 I + 1 4-i 4-1 4-1 4-1 1 r i +,+ +,+ +1+ 4-| 4-i 4-1 4-1 4-1 +,+ +,++#• 4-i + I 4-i i i i i T 7 - Y + i J L . 1 , HX CSE FSC LH FSH 1Z - 95 -F i g . 3 1 . Summary o f t h e p h o t o p e r i o d - t e m p e r a t u r e e x p e r i m e n t s . S y m b o l s above t h e c u r v e i n d i c a t e t h e c o n d i t i o n o f f i s h a t l a t e r s a m p l i n g s a t h i g h e r t e m p e r a t u r e , t h e on e s b e l o w t h e c u r v e d e n o t e t h o s e a t l o w t e m p e r a t u r e , t h e s p o t s on t h e c u r v e where t h e s e s y m b o l s a r e plac e d i n d i c a t e t h e l e v e l o f s t i m u l a t i o n o r r e g r e s s i o n e x h i b i t e d by t h e f i s h a t t h e f i n a l s a m p l i n g . O t h e r e x p l a n a t i o n s same as i n F i g u r e 30. I—POST SPAWNING FISH - 96 -F i g . 3 2 . P h o t o m i c r o g r a p h s h o w i n g c e l l s l i n i n g t h e d i s t a l s p e r m a t i c d u c t s o f s p a w n i n g f i s h . N o t e t h r e e c e l l s i n t h e c e n t r e w i t h e l o n g a t e d n u c l e i i n t h e i r b a s a l h a l f and g r a n u l e f i l l e d l u m e n - h a l f ; a d j a c e n t c e l l s on l e f t a r e s p e n t (X 1260) - 97 -F i g . 3 3 A . P h o t o m i c r o g r a p h o f a p o r t i o n o f an LS o f t e s t i s o f f i s h t r e a t e d w i t h c r u d e w h o l e s a l m o n p i t u i t a r y e x t r a c t . N o t e t h e l o b u l e on l e f t u p p e r c o r n e r a l m o s t f u l l o f p r i m a r y s p e r m a t o c y t e s . Some s e c o n d a r y s p e r m a t o c y t e s a r e s e e n d i v i d i n g a t r i g h t u p p e r c o r n e r (X 7 0 0 ) . B. P h o t o m i c r o g r a p h o f a p o r t i o n o f an LS o f a n o r m a l S t a g e I I t e s t i s s h o w i n g p y c n o t i c n e s t s among h e a l t h y s p e r m a t o g e n e t i c s t a g e s (X 7 0 0 ) - 9.8 -; i . i DISCUSSION S e a s o n a l T e s t i c u l a r C y c l e The t e s t e s o f C o u e s i u s p l u m b e u s u n d e r g o r e g u l a r c y c l i c a l c h a n g e s . The c h a n g e s a r e s i m i l a r t o t h o s e d e s c r i b e d i n d e t a i l by B u l l o u g h ( 1 9 39) i n a c l o s e l y r e l a t e d f i s h , P h o x i n u s l a e v i s and H a r r i n g t o n ( 1 9 5 7 ) i n N o t r o p i s b i f r e n a t u s a n o t h e r a l l i e d s p e c i e s . The s p e r m a t o g e n e t i c c y c l e i n C. p l u m b e u s c a n be d i v i d e d i n t o two d i s t i n c t p h a s e s on t h e b a s i s o f t h e k i n d o f t h e m u l t i p l i c a t o r y p r o c e s s e s : ( i ) The m i t o t i c p h a s e - t h i s i s v e r y s l o w and commences i n S e p t e m b e r , p r o g r e s s i n g s l o w l y t i l l A p r i l . ( i i ) The m e i o t i c p h a s e - t h i s i s r a p i d and s h o r t e r i n d u r a t i o n . D u r i n g t h i s p e r i o d , w h i c h b e g i n s i n A p r i l , s e c o n d a r y s p e r m a t o c y t e s a r e f o r m e d w h i c h p r o c e e d t p w a r d s f i n a l m a t u r a t i o n and f o r m a t i o n o f sperm. T h e r e i s no t i m e l a p s e b e t w e e n t h e s e two p h a s e s , and a s i m i l a r scheme c a n be f o u n d i n t h e t e s t i c u l a r c y c l e o f a l l o t h e r c y p r i n i d f i s h e s s t u d i e d ( B u l l o u g h , 1939; S c o t t , 1952; H a r r i n g t o n , 1 9 5 7 ) . I n C o u e s i u s s p a w n i n g commences b e f o r e t h e l o b u l e s become f u l l y p a c k e d w i t h s p e r m s ; s p e r m i o g e n e s i s c o n t i n u e s d u r i n g t h e s p a w n i n g p e r i o d . T h e r e i s no i n d i c a t i o n o f any s p e r m a t o g e n e t i c a r r e s t d u r i n g s p a w n i n g , and s i n c e s p e r m i a t i o n and s p e r m i o g e n e s i s o c c u r s i m u l t a n e o u s l y r e p e a t e d s perm d i s c h a r g e s a r e p o s s i b l e . I t i s i n t e r e s t i n g t h a t a l o w r a t i o o f m a l e s t o f e m a l e s was f o u n d i n a l l c a t c h e s f r o m t h e l a k e s ; r e p e a t e d s p e r m i a t i o n m i g h t c o n s e q u e n t l y be o f a d e c i d e d a d v a n t a g e i n t h i s s p e c i e s . A f t e r s p e r m i a t i o n r e c o n s t i t u t i o n o f t h e t e s t e s commences s u b s e q u e n t t o . a . b r i e f - 99 -s p e r m a t o g e n e t i c i n a c t i v i t y i n A u g u s t . The a n n u a l t e s t i c u l a r c y c l e i n t h i s f i s h i s c l o s e l y c o r r e l a t e d w i t h t h e c h a n g e s i n i t s e n v i r o n m e n t . When t h e i c e goes f r o m t h e l a k e and t h e w a t e r t e m p e r a t u r e and d a y l e n g t h s t a r t i n c r e a s i n g t h e r e i s a b u r s t o f s p e r m a t o g e n e t i c a c t i v i t y l e a d i n g t o t h e f o r m a t i o n o f sperms and e n d i n g w i t h s p a w n i n g . S p a w n i n g o c c u r s i n J u n e and e a r l y J u l y ( a s e v i d e n c e d f r o m t h e t e s t i c u l a r h i s t o l o g y ) when w a t e r t e m p e r a t u r e i s s t i l l r i s i n g b u t t h e d a y l e n g t h i s maximum. I n A u g u s t when t e m p e r a t u r e i s maximum s p e r m i a t i o n i s c o m p l e t e d , and d u r i n g t h i s p e r i o d v e r y l i t t l e m i t o t i c a c t i v i t y i s p r e s e n t . R e c o n s t i t u t i o n commences i n l a t e A u g u s t and d u r i n g t h e p e r i o d o f d e c r e a s i n g l i g h t and t e m p e r a t u r e s l o w m i t o t i c a c t i v i t y c o n t i n u e s . F i s h i m m e d i a t e l y b e f o r e t h e " f r e e z e - u p " have d e v e l o p e d a s t o c k o f p r i m a r y s p e r m a t o c y t e s and c o n s i d e r a b l e s p e r m a t o g o n i a l r e s e r v e . D u r i n g t h e p e r i o d o f i c e c o v e r l i t t l e c h ange o c c u r s , a l t h o u g h s l o w m i t o t i c a c t i v i t y c o n t i n u e s . L a t e r d e v e l o p m e n t o c c u r s o n l y when t h e t e m p e r a t u r e o f w a t e r r i s e s i n m i d - A p r i l ; d a y l e n g t h h a s b e e n c o n t i n u a l l y i n c r e a s i n g s i n c e l a t e December. I n s h o r t , t h e f i r s t p h a s e o f t e s t i c u l a r chang.es o c c u r d u r i n g t h e f a l l and " f r e e z e o v e r " months and t h e f i n a l p h a s e commences i m m e d i a t e l y on t h e w a r m i n g o f t h e l a k e i n t h e s p r i n g t i m e . S e a s o n a l 1 F a c t o r s a f f e c t i n g T e s t i c u l a r C y c l e I t i s n a t u r a l t o e x p e c t t h a t l i k e many o t h e r a n i m a l s g o n a d a l m a t u r a t i o n i n C o u e s i u s p l u m b e u s i s r e g u l a t e d and c o n t r o l l e d by t h e e n v i r o n m e n t a l ' v a r i a b l e s n o t a b l y l i g h t and t e m p e r a t u r e . T e s t i c u l a r c y c l e s , i n C. p l u m b e u s have been c l o s e l y c o r r e l a t e d w i t h c h a n g e s i n t h e e n v i r o n m e n t a l f a c t o r s i n t h e p r e s e n t s t u d y . I t i s - 100 -s u p p o s e d t h a t t h e s e c o r r e l a t i o n s have a c a u s e - e f f e c t r e l a t i o n s h i p . T h i s b e l i e f i s s u p p o r t e d by s i m i l a r e a r l i e r s t u d i e s on s e a s o n a l c h a n g e s i n t e l e o s t t e s t i c u l a r c y c l e . Out o f v a r i o u s p o s s i b l e f a c t o r s , p h o t o p e r i o d and t e m p e r a t u r e a r e t h e most l i k e l y c a n d i d a t e s f o r t h e r e g u l a t i o n o f t h e t e s t i c u l a r c y c l e . S t u d i e s i n t h i s f i e l d a r e numerous. The l i t e r a t u r e has been s u m m a r i z e d by P i c k f o r d and A t z (1957) and i n r e v i e w s e d i t e d by W i n t h r o w ( 1 9 5 9 ) . Some o f t h e s e w o r k s f a v o u r c h a n g i n g p h o t o p e r i o d s as t h e m a j o r r e g u l a t o r , w h e r e a s o t h e r s deny i t . A f e w have shown t e m p e r a t u r e as t h e s o l e r e g u l a t i n g f a c t o r , w h i l e o t h e r s have c o m p r o m i s e d by i n c l u d i n g b o t h l i g h t and t e m p e r a t u r e as i m p o r t a n t i n t h e c o n t r o l o f t e s t i c u l a r c h a n g e s . Some w o r k e r s have r e p o r t e d d i s c o r d a n t r e s u l t s i n t h e same s p e c i e s . I t i s p o s s i b l e t h a t some o f t h e d i s c r e p a n c y i n t h e e x p e r i m e n t a l w o r k i s due t o d i f f e r e n c e s i n t h e r e s p o n s e t o m a n i p u l a t i o n s o f p h o t o p e r i o d and t e m p e r a t u r e a t t h e v a r i o u s phase's o f t h e l i f e c y c l e ; a l s o p o s s i b l y t o s h o r t - t i m e o f t h e e x p e r i m e n t a l c o n t r o l s . To t e s t t h e r e l a t i v e s i g n i f i c a n c e o f t h e s e two f a c t o r s i n C o u e s i u s . f i s h a t t h r e e d i s t i n c t p e r i o d s o f t h e a n n u a l c y c l e were s u b j e c t e d t o f o u r c o m b i n a t i o n s o f p h o t o p e r i o d s and t e m p e r a t u r e s and t h e e x p e r i m e n t s c o n d u c t e d f o r an e x t e n d e d p e r i o d . R o l e o f T e m p e r a t u r e and L i g h t When p r e s p a w n i n g f i s h s h o w i n g a d v a n c e d s p e r m i o g e n e s i s were s u b j e c t e d t o d i f f e r e n t p h o t o p e r i o d - t e m p e r a t u r e c o n d i t i o n s , n o r m a l t e s t i c u l a r a c t i v i t y was i n t e r r u p t e d a t l o w t e m p e r a t u r e s i r r e s p e c t i v e o f t h e p h o t o p e r i o d s . At h i g h e r t e m p e r a t u r e s , on t h e c o n t r a r y , s p e r m i o g e n e t i c c h a n g e s were a c c e l e r a t e d and t h e f i s h showed s p e n t t e s t e s a t l e a s t 20 d a y s e a r l i e r t h a n t h e f i s h i n - 101 -n a t u r e . D i f f e r e n c e b e t w e e n t h e two h i g h e r t e m p e r a t u r e g r o u p s on t h e b a s i s o f p h o t o p e r i o d was a l m o s t a b s e n t . At l o w t e m p e r a t u r e s p e r m i a t i o n was r e t a r d e d f o r c o n s i d e r a b l e t i m e and o n l y m i l d a c t i v i t y was r e t a i n e d i n t h e f i s h , a g r e a t e r p r o p o r t i o n o f s p e r m a t o g o n i a l and s p e r m a t o c y t a l n e s t s s t o p p e d d i v i d i n g . T h i s t e m p e r a t u r e e f f e c t was a g a i n e m p h a s i z e d i n t h e l a t e r p a r t o f t h e e x p e r i m e n t s w i t h t h e p r e s p a w n i n g f i s h . S i m i l a r c h a n g e s were n o t e d w i t h t h e p o s t - s p a w n i n g s t o c k , and t h e " p r e - f r e e z e up" s t o c k o f f i s h . I n b o t h c a s e s t e m p e r a t u r e and n o t p h o t o p e r i o d i s t h e p r o m i n e n t r e g u l a t o r . H owever, two p e r t i n e n t f a c t s were a p p a r e n t : ( i ) A l t h o u g h t h e r e was l i t t l e d i f f e r e n c e i n t h e l o n g and s h o r t p h o t o p e r i o d f i s h a t h i g h e r t e m p e r a t u r e , ] f i s h a t l o W e r t e m p e r a t u r e h e l d u n d e r s h o r t p h o t o p e r i o d a l w a y s showed a s l i g h t l y a d v a n c e d t e s t i c u l a r c o m p o s i t i o n a t t h e f i n a l s a m p l i n g . ( i i ) Some p o s t - s p e r m a t o c y t a l c h a n g e s o c c u r r e d i n t h e l o w t e m p e r a t u r e f i s h u n d e r b o t h c o n d i t i o n s o f p h o t o p e r i o d s when e x p e r i m e n t s were e x t e n d e d u n t i l e a r l y A p r i l , t h e n o r m a l p r e s p a w n i n g p e r i o d . These two f a c t o r s s u g g e s t a m i l d e f f e c t o f p h o t o p e r i o d and an e n d o g e n o u s r h y t h m w h i c h c a n n o t be c o m p l e t e l y s u p p r e s s e d by e i t h e r t h e l o w t e m p e r a t u r e o r t h e p h o t o p e r i o d . The p r o b a b i l i t y o f l i g h t as a t r i g g e r i n t h e c o n t r o l o f t e s t i c u l a r c y c l e i s f u r t h e r r e d u c e d by t h e i r r e g u l a r i t y o f i l l u m i n a t i o n r e a c h i n g t h e f i s h d u r i n g t h e c r i t i c a l p r e s p a w n i n g p h a s e . I n m i d w i n t e r t h e t h i c k n e s s o f i c e c o v e r i n t h e l a k e s i s a b o u t 18 i n c h e s w h i c h i s o f t e n c o v e r e d by a l a y e r o f snow. The - 102 -p e r i o d o f snow c o v e r i s i r r e g u l a r . D u r i n g t h e t i m e when d r y snow c o v e r s t h e l a k e v e r y l i t t l e o r no l i g h t p e n e t r a t e s b e l o w t h e f r o z e n l a y e r . T h e r e a r e p e r i o d s when t h e r e i s no snow c o v e r , o r ^ i f p r e s e n t ^ i t i s w e t . U n d e r s u c h c o n d i t i o n s t h e r e i s some p e n e t r a t i o n o f l i g h t ; t h e amount v a r y i n g w i t h t h e s e v e r a l p a r a m e t e r s c o n c e r n i n g t h e i c e and snow. F i s h d u r i n g t h e p e r i o d o f i c e and snow c o v e r a r e s u b j e c t e d t o a f l u c t u a t i n g a c t i o n o f l i g h t w h i c h i s e x t r e m e l y i r r e g u l a r and n o t p r e d i c t a b l e . Had t h e snow c o v e r b e e n u n i f o r m l y p r e s e n t and a l s o d r y , l i g h t p e n e t r a t i o n would* have b e e n p r e v e n t e d . At t h e t i m e o f t h a w i n g , t h e n , t h e r e w o u l d have b e e n a s u d d e n o n s e t o f i l l u m i n a t i o n . I f i t were s o , an a b r u p t i n c r e a s e i n l i g h t w o u l d t h e n be a l o g i c a l c a n d i d a t e f o r a t r i g g e r i n g m e c h a n i s m . As t h e f i s h e x p e r i e n c e i r r e g u l a r p e r i o d s o f i n t e r m i t t e n t l i g h t and n e a r d a r k n e s s , a t r i g g e r r o l e c a n n o t be a t t r i b u t e d t o l i g h t . E v e n i f i t were p o s s i b l e t h a t l i g h t c o u l d t r i g g e r o f f c h a n g e s i n t e s t i c u l a r a c t i v i t y , t h e amount o f l i g h t w o u l d f a l l b e l o w t h e t h r e s h o l d l e v e l demanded f o r s t i m u l a t i o n . I n summary, i t i s c l e a r t h a t t e m p e r a t u r e i s t h e more i m p o r t a n t r e g u l a t o r q f t h e t e s t i c u l a r c y c l e , o f C o u e s i u s p l u m b e u s . When t e m p e r a t u r e s a r e l o w s p e r m a t o c y t a l s t a g e s l o s e "most o f t h e i r a c t i v i t y , and s p e r m i a t i o n i s h i n d e r e d ; w h i l e h i g h temperature a c c e l e r a t e s t h e s e c h a n g e s and f a c i l i t a t e s s p e r m i a t i o n . H i g h t e m p e r a t u r e s , m o r e o v e r , impede n o r m a l g o n i a l p r o l i f e r a t i o n as a r e s u l t o f w h i c h h i g h t e m p e r a t u r e f i s h n e v e r r e a c h t h e s p e r m a t o c y t a l s t a g e s . The i n h i b i t i o n i m p o s e d by t e m p e r a t u r e ( l o w i n t h e c a s e o f s p e r m i a t i o n and s p e r m i o g e n e s i s ; and h i g h f o r g o n i a l p r o l i f e r a t i o n ) i s n o t o v e r r i d d e n by e i t h e r p h o t o p e r i o d . - 103 -A n o t h e r s i g n i f i c a n t f a c t i s t h a t a t l o w t e m p e r a t u r e s b o t h p r e - and p o s t - s p a w n i n g s t o c k s r e a c h t h e p r e f r e e z e up s p e r m a t o g e n e t i c c o n d i t i o n much e a r l i e r , b u t d e v e l o p m e n t s t o p s a t t h e p r i m a r y s p e r m a t o c y t e l e v e l f o r a c o n s i d e r a b l y l o n g t i m e . I t i s o n l y i n t h e i n s t a n c e o f a f o r e s a i d s a m p l i n g w i t h p r e -f r e e z e up s t o c k t h a t t h i s b a r r i e r i s somewhat b r o k e n . The e n v i r o n m e n t a l r e g u l a t i o n o f t h e a n n u a l t e s t i c u l a r c y c l e may now be e x p l a i n e d . The f i r s t p h a s e o f s p e r m a t o g e n e t i c p r o c e s s commences and c o n t i n u e s when w a t e r t e m p e r a t u r e i s l o w , and as l a t e as M a r c h s p e r m a t o g e n e s i s p r o c e e d s o n l y up t o p r i m a r y s p e r m a t o c y t e s t a g e ( F i g . 6 ) . The s e c o n d p h a s e o f s p e r m a t o g e n e s i s o c c u r s when t h e l a k e t h a w s and w a t e r t e m p e r a t u r e s s t a r t r i s i n g r a p i d l y and f i n a l s t a g e s o f m a t u r a t i o n and s p e r m i a t i o n o c c u r a t h i g h e r t e m p e r a t u r e . G o n i a l p r o l i f e r a t i o n (= t h e f i r s t p h a s e ) commences o n l y when t h e t e m p e r a t u r e s a r e l o w e r and p r o g r e s s e s s t e a d i l y t i l l t h e f r e e z e up. End o g e n o u s r h y t h m T h e o r e t i c a l l y i f t h e t e m p e r a t u r e was t h e o n l y r e g u l a t o r no p o s t - s p e r m a t o c y t a l d e v e l o p m e n t s h o u l d have o c c u r r e d a t l o w t e m p e r a t u r e and no s p e r m a t o c y t e s f o r m e d a t t h e h i g h e r . S i n c e t h e s e c h a n g e s do o c c u r i n a m i l d way t h e r e i s some end o g e n o u s r h y t h m t o t h e a n n u a l t e s t i c u l a r c y c l e i n t h i s s p e c i e s . T h i s phenomenon h a s been e n c o u n t e r e d by some o t h e r w o r k e r s . H e n d e r s e n (1963) i n male S a l v e l i n u s f o n t i n a l i s h a s s u g g e s t e d t h a t t h e male germ c e l l s a r e more s t a b l e t h a n t h e f e m a l e i n s o f a r as m o d i f i c a t i o n by e x p e r i m e n t a l p h o t o p e r i o d and t e m p e r a t u r e c o n d i t i o n s a r e c o n c e r n e d . B u l l o u g h ( 1 9 4 1 ) f o u n d some m a t u r a t i o n i n P h o x i n u s  l a e v i s m a i n t a i n e d i n d a r k . - 104 -I t i s p r o p o s e d t h a t i n C o u e s i u s p l u m b e u s . t e m p e r a t u r e i s t h e m a j o r e n v i r o n m e n t a l f a c t o r r e s p o n s i b l e f o r t h e t r i g g e r i n g o f t h e s e c o n d p h a s e o f t h e a n n u a l t e s t i c u l a r c y c l e ; i t i s a l s o i m p o r t a n t i n t h e n o r m a l p r o g r e s s i o n o f t h e f i r s t p h a s e . The t e s t e s p o s s e s s an i n h e r e n t r h y t h m w h i c h i s more e f f e c t i v e l y s u p p r e s s e d by h i g h e r t e m p e r a t u r e ( h e n c e no n o r m a l g o n i a l p r o l i f e r a t i o n i n f i s h u n d e r s u c h s i t u a t i o n ) t h a n by t h e l o w e r ( h e n c e a l i m i t e d p o s t s p e r m a t o c y t a l d e v e l o p m e n t i s p o s s i b l e i n f i s h a t l o w e r t e m p e r a t u r e b u t a p p r o a c h i n g n o r m a l s p a w n i n g season). The n a t u r e o f t h i s e n d o g e n o u s r e g u l a t o r y r h y t h m i n t e l e o s t s and e v e n i n o t h e r v e r t e b r a t e s i s unknown, b u t i t i s r e a s o n a b l e t o s u p p o s e t h a t i t i s g e n e t i c and p e c u l i a r t o e a c h s p e c i e s . P h o t o p e r i o d d o e s n o t p l a y any s i g n i f i c a n t r o l e i n t h e r e g u l a t i o n o f t h e t e s t i c u l a r c h a n g e s i n t h i s f i s h . M e c h a n i s m o f C o n t r o l o f T e s t i c u l a r C y c l e The. r e l a t i o n o f p i t u i t a r y t o t h e g onads i s w e l l e s t a b l i s h e d i n t h e e n t i r e v e r t e b r a t e s e r i e s , and i t i s l o g i c a l t o assume t h a t i n C o u e s i u s a l s o t h e r e g u l a t o r y m e c h a n i s m o f t e s t i c u l a r a c t i v i t y o p e r a t e s t h r o u g h t h i s g l a n d . E v i d e n c e f r o m H y p o p h y s e c t o m y Out o f a number o f t e l e o s t s s u c c e s s f u l l y hypophysectomised t h e e f f e c t s o f p i t u i t a r y r e m o v a l on t h e gonads has been s t u d i e d i n o n l y n i n e s p e c i e s , and among t h e s e o n l y i n a few f o r m s have t h e h i s t o l o g i c a l c h a n g e s been f o l l o w e d p a r t i c u l a r l y i n t h e f e m a l e s ( V i v i e n , 1 9 4 1 , and B a r r , 1 9 6 3 ) . S e a s o n a l v a r i a t i o n s i n r e s p o n s e t o t h i s o p e r a t i o n have b e e n t a k e n i n t o a c c o u n t o n l y by M a t t h e w s ( 1 9 3 9 , 1 9 4 1 ) , V i v i e n (1941),Bur:geri(l94;ii£ariid B a r r (1963,a,b,c). From t h e s e e a r l i e r w o r k s and t h e p r e s e n t s t u d y t h e - 105 -f o l l o w i n g c o n c l u s i o n s may now be made: ( i ) R e m o v a l o f p i t u i t a r y g l a n d a f f e c t s t h e m i t o t i c a c t i v i t y o f t h e s p e r m a t o g o n i a l c e l l s w i t h c o m p l e t e s u p p r e s s i o n o f s p e r m a t o g o n i a l a c t i v i t y . T h u s , c o n v e r s i o n o f s p e r m a t o g o n i a i n t o . . s p e r m a t o c y t e s i s c o m p l e t e l y s t o p p e d . M i t o s e s may o c c u r . b u t a r e r a r e . ( i i ) I f , h o w e v e r , s p e r m a t o g e n e s i s i s w e l l u n d e r way h y p o p h y s e c t o m y does n o t seem t o h a l t a b r u p t l y t h e p r o c e s s . ( i i i ) S p e r m i a t i o n o c c u r s n o r m a l l y . S e a s o n a l v a r i a t i o n i n t h e r e s p o n s e t o p i t u i t a r y r e m o v a l i s e v i d e n t . I n t h i s s t u d y , i t was maximum d u r i n g t h e p e a k o f t h e spermatogenetic a c t i v i t y c l o s e l y f o l l o w e d by t h e e f f e c t on t h e f i s h i m m e d i a t e l y b e f o r e s p a w n i n g . H y p o p h y s e c t o m y p r o d u c e s i t s l e a s t e f f e c t i m m e d i a t e l y a f t e r s p a w n i n g . I t s h o u l d be n o t e d t h a t t h e s e s e a s o n a l e f f e c t s o f h y p o p h y s e c t o m y a f f e c t o n l y t h e s p e e d o f r e g r e s s i o n ; t h e end r e s u l t i s a l w a y s t h e same and i n a l l c a s e s t h e t e s t e s assume a r e s t i n g c o n d i t i o n . The e f f e c t s o f h y p o p h y s e c t o m y on s p e r m i a t i o n i s so f a r u n s e t t l e d . I n t h e p r e s e n t s t u d y a l l f i s h s p e r m i a t e d c o m p l e t e l y and t h e s p e r m a t i c d u c t and t h e i r b r a n c h e s were c o l l a p s e d and a l m o s t d e v o i d o f spermS. S p e r m i a t i o n t h e r e f o r e , a p p e a r s t o be i n d e p e n d e n t o f p i t u i t a r y c o n t r o l . G e n e r a l i s a t i o n s s h o u l d , however, be made w i t h c a u t i o n as r e t e n t i o n o f sperms and c o m p l e t i o n o f sp e r m f o r m a t i o n h a s b e e n shown t o be a s p e c i e s p e c u l i a r i t y . I n a w i d e v a r i e t y o f n o n - c y p r i n i d f i s h e s s p e r m f o r m a t i o n i s completed b e f o r e a c t u a l s p a w n i n g ( B a r r , 1963% H a r r i n g t o n , 1956) and some - 106 -f i s h may show sperms t h r o u g h o u t t h e y e a r ( W e i s e l , 1 9 4 9 ) . I n C o u e s i u s . p h o t o p e r i o d and t e m p e r a t u r e s t u d i e s have shown t h a t h i g h t e m p e r a t u r e s a c c e l e r a t e s p e r m d i s c h a r g e w h e r e a s l o w t e m p e r a t u r e s c a u s e sperm r e t e n t i o n f o r a l o n g e r t i m e . I t may be p o s s i b l e t h a t h i g h e r t e m p e r a t u r e s p e r se a f f e c t t h e m o t i l i t y o f t h e sperms c a u s i n g t h e d i s c h a r g e and l o w e r t e m p e r a t u r e s r e d u c e i t i n t h i s f i s h . I f t h i s i s t r u e , i t w o u l d n o t be. s u r p r i s i n g t h a t s p e r m i a t i o n o c c u r r e d i n some f i s h n o r m a l l y i n t h e a b s e n c e o f t h e p i t u i t a r y . The p r e s e n t s t u d y i s i n f u l l a g r e e m e n t w i t h t h o s e o f B a r r (1963c) on a m a r i n e t e l e o s t P l e u r o n e c t e s p l a t e s s a and Dodd e_t a l ( i 9 6 0 ) on t h e e l a s m o b r a n c h S c v l i o r h i n u s c a n i c u l a . on t h e q u e s t i o n o f t h e p r e c i s e s t a g e o f s p e r m a t o g e n e s i s most a f f e c t e d by h y p o p h y s e c t o m y . W i t h d r a w a l o f p i t u i t a r y c o m p l e t e l y s u p p r e s s e s t h e v i t a l t r a n s f o r m a t i o n o f s e c o n d a r y s p e r m a t o g o n i a i n t o p r i m a r y s p e r m a t o c y t e s . I t i s w e l l known t h a t m e i o t i c d i v i s i o n s a r e much more e l a b o r a t e phenomenon t h a n t h e m i t o t i c . They i n v o l v e f a r r e a c h i n g b i o c h e m i c a l and g e n e t i c p r o c e s s e s . I n C o u e s i u s . f i r s t m e i o t i c d i v i s i o n o c c u r s a t t h e p r i m a r y s p e r m a t o c y t e s t a g e w h i c h i s t h e c l i m a c t i c s t a g e o f t h e f i r s t p h a s e o f t h e t e s t i c u l a r c y c l e . L i t t l e p o s t - s p e r m a t o c y t a l a c t i v i t y o c c u r s i n f i s h a t l o w \, j t e m p e r a t u r e , w h e r e a s p i t u i t a r y r e m o v a l b l o c k s d e v e l o p m e n t a t a ... s t a g e e a r l i e r . I t i s p r e s u m e d t h a t t h e change f r o m s e c o n d a r y s p e r m a t o g o n i a i n t o p r i m a r y s p e r m a t o c y t e s r e q u i r e s some, exceptionally s p e c i f i c t r i g g e r i n g so t h a t t h e s p e r m a t o c y t e s t h a t a r e f o r m e d c a n embark on m e i o t i c d i v i s i o n s . L o b u l e b o u n d a r y c e l l s a r e r e c o g n i s e d i n C. p l u m b e u s b o t h h i s t o l o g i c a l l y and h i s t o c h e m i c a l l y as homologue o f v e r t e b r a t e - 107 -L e y d i g c e l l s . They u n d e r g o a c y c l i c a l change i n a c t i v i t y , and t h e i r n o r m a l f u n c t i o n i n g a l s o i s s u p p r e s s e d by p i t u i t a r y w i t h d r a w a l . I n s h o r t , t h e p i t u i t a r y g l a n d i n C. p l u m b e u s i s n o t o n l y e s s e n t i a l f o r t h e m a i n t e n a n c e o f t h e f i r s t p h a s e o f s p e r m a t o g e n e s i s and s u c c e s s f u l c o m p l e t i o n o f t h e s e c o n d , i t i s a l s o n e c e s s a r y f o r t h e m a i n t e n a n c e o f t h e n o r m a l s e c r e t o r y a c t i v i t y o f t h e l o b u l e b o u n d a r y c e l l s . The q u e s t i o n now a r i s e s as t o w h i c h hormone o r hormones o f t h e p i t u i t a r y a r e r e s p o n s i b l e f o r t h i s c o n t r o l ? To an s w e r t h i s q u e s t i o n known e x t r a c t s f r o m t h i s g l a n d were u t i l i s e d i n r e p l a c m e n t t h e r a p y on hypophysectomised C o u e s i u s . R o l e o f G o n a d o t r o p i n ( s ) i n t h e C o n t r o l o f t h e T e s t i c u l a r C y c l e E x t r a c t s f r o m p i t u i t a r y g l a n d s o f v a r i o u s a n i m a l s have been u s e d i n f i s h e s t o a c c e l e r a t e g a m e t o g e n e s i s and i n d u c i n g s p a w n i n g . A l m o s t a l l o f t h e s e w o r k s i n v o l v e d homo- o r h e t e r o p l a s t i c i n j e c t i o n s o f p i t u i t a r y s u s p e n s i o n i n t o i n t a c t f i s h u s u a l l y a p p r o a c h i n g g o n a d a l m a t u r i t y . V a r y i n g d e g r e e s o f s u c c e s s were o b t a i n e d i n t h e s e s t u d i e s ( P i c k f o r d and A t z , 1 9 5 7 ) . These t e s t s a r e i n s t a n c e s o f a u g m e n t a t i o n t h e r a p y s i n c e no e f f o r t was made t o e l i m i n a t e e n d o g e n o u s g o n a d o t r o p i n s and a r e n o t c r i t i c a l s i n c e t h e i d e n t i t y o f hormones u s e d were n o t known. R e i n i t i a t i o n and r e s t o r a t i o n o f s p e r m a t o g e n e s i s was o b t a i n e d i n t h i s s t u d y w i t h p u r i f i e d s a l m o n g o n a d o t r o p i n , c r u d e w h o l e s a l m o n p i t u i t a r y e x t r a c t and p u r e m a m malian LH. Mammalian FSH f a i l e d t o p r o d u c e any s t i m u l a t i o n . S t i m u l a t i o n w i t h mammalian LH was a l m o s t as g r e a t as t h a t w i t h p u r i f i e d s a l m o n g o n a d o t r o p i n ; d u r i n g t h e r e c e p t i v e p r e s p a w n i n g p e r i o d s a l m o n g o n a d o t r o p i n was s l i g h t l y more e f f i c a c i o u s . The d i f f e r e n c e s - 108 -m i g h t have been due t o c o n t a m i n a n t s o r d o s a g e and do n o t p r o v i d e e v i d e n c e f o r any l a r g e d i f f e r e n c e s b e t w e e n them. I t i s s u g g e s t e d t h a t t h e s a l m o n p i t u i t a r y p o s s e s s e s a f a c t o r s i m i l a r t o mammalian LH w h i c h i s e f f e c t i v e i n t h e r e s t o r a t i o n o f s p e r m a t o g e n e s i s i n C o u e s i u s p l u m b e u s . T h e r e i s no s a t i s f a c t o r y e v i d e n c e , s o f a r , i n any f i s h o f an a c t i o n o f mammalian FSH i n t e s t i c u l a r m a t u r a t i o n o f f i s h e s . P i c k f o r d ( 1 9 5 3 ) c o n c l u d e d t h a t mammalian FSH was l / 5 t h t o 1 / l O t h as e f f i c a c i o u s as LH. She s u s p e c t e d t h a t t h e c o n t a i n e d 3% LH i m p u r i t y m i g h t have c a u s e d t h e s t i m u l a t i o n . W i t s c h i ( 1 9 5 3 ) and " K i r s h e f f l M a t „ ( 1 9 4 9 ) have f o u n d e v i d e n c e f o r an F S H - l i k e f a c t o r i n f i s h e s u s i n g s t a n d a r d a s s a y methods on mammals and b i r d s . O t s u k a ( 1 9 56) c l a i m s t o have s e p a r a t e d two g o n a d o t r o p i n s f r o m s a l m o n p i t u i t a r y e x h i b i t i n g FSH and LH p r o p e r t i e s when t e s t e d by m i c e b i o a s s a y . B a r r ( 1 9 6 3 b ) d e m o n s t r a t e d v i t e l l o g e n e s i s i n p r i m a r y o o c y t e s o f h y p o p h y s e c t o m i s e d p l a i c e w i t h F S H - l i k e p r e p a r a t i o n s . He, h o w e v e r , d i d n o t s t u d y t h e e f f e c t o f p u r i f i e d mammalian hormones on h i s f i s h ; nor d i d rhec s t u d y t h e e f f e c t o f r e p l a c e m e n t t h e r a p y i n ffcfoee nfrBi.e. B a l l ( 1 9 6 0 ) c a u t i o n s t h e r e l i a n c e on mammalian b i o a s s a y f o r d e t e r m i n i n g t h e n a t u r e o f f i s h g o n a d o t r o p i n s . The p o s i t i v e a s s a y s m i g h t have been o b t a i n e d by t h e e f f e c t o f i n c r e a s e d e n d o g e n o u s s e c r e t i o n o f g o n a d o t r o p i n s i n t h e mammals c a u s e d by t h e a c t i o n o f t h e r e l e a s i n g s u b s t a n c e s p o s s i b l y p r e s e n t i n t h e f i s h p i t u i t a r y . These r e l e a s i n g s u b s t a n c e s a c c o r d i n g t o M a r t i n i e_t a_l ( 1 9 5 9 ) a r e p r o b a b l y t h e p o s t e r i o r l o b e p o l y p e p t i d e s . A l t h o u g h O l i v e r e a u ( 1 9 62) d i f f e r e n t i a t e d two k i f l d s »bf g o n a d o t r o p h s i n s e v e r a l s p e c i e s o f f i s h , t h e n a t u r e o f t h e i r s e c r e t i o n s r e m a i n s y e t t o be - 109 -e s t a b l i s h e d . Ramaswami (1962) d i d n o t use any t e s t s p e c i f i c a l l y f o r FSH b u t f o u n d s p e c i f i c e v i d e n c e f o r LH i n t h e p i t u i t a r y o f H e t e r o p n u e s t e s f o s s i l i s . A h s a n and Hoar (1963) t e s t e d f o r FSH on p i t u i t a r y s u p p r e s s e d G a s t e r o s t e u s a c u l e a t u s and f o u n d n e g a t i v e i n d i c a t i o n s . I t i s p o s s i b l e t h a t f i s h p i t u i t a r i e s p r o d u c e a s u b s t a n c e w i t h an F S H - l i k e a c t i o n i n h i g h e r v e r t e b r a t e s , b u t t h e r e i s p r e s e n t l y no e v i d e n c e f o r any g o n a d o t r o p i c e f f e c t i n f i s h e s w h i c h c a n n o t be i n i t i a t e d by t h e L H - l i k e f a c t o r . T e s t i c u l a r a c t i v i t y i n C o u e s i u s i s p h y s i o l o g i c a l l y c o n t r o l l e d by g o n a d o t r o p i n ( s ) s i m i l a r t o mammalian LH. C o m p l e t e r e s t o r a t i o n o f s p e r m a t o g e n e s i s was n o t o b t a i n e d by r e p l a c e m e n t t h e r a p y d u r i n g t h e p r e s e n t i n v e s t i g a t i o n . A l t h o u g h t h e h u r d l e o f t h e s p e r m a t o c y t a l t r a n s f o r m a t i o n was c r o s s e d , t h e l a t e r s t a g e s were n o t numerous. T e s t i c u l a r s t i m u l a t i o n r e a c h e d o n l y up t o s e c o n d a r y s p e r m a t o c y t a l s t a g e a t b e s t . T h e r e a r e s e v e r a l p o s s i b l e e x p l a n a t i o n s . I n c o m p l e t e a c t i v a t i o n m i g h t e i t h e r be due t o s p e c i f i c d i f f e r e n c e s i n t h e q u a l i t y o f C o u e s i u s g o n a d o t r o p i n and t h e e x o g e n o u s g o n a d o t r o p i n s u s e d , o r t o an u n p h y s i o l o g i c a l d o s a g e , o r t o t h e d u r a t i o n o f t h e t r e a t m e n t . A l t h o u g h t h e f i s h g o n a d o t r o p i n d o s e m i g h t be l e s s ( t h e r e i s l i t t l e c h a n c e o f t h i s i f t h e d o s e i s c o n s i d e r e d i n t e r m s o f wt/wt and n o t i n numbers o f e q u i v a l e n t w h o l e p i t u i t a r i e s i n v o l v e d ) , t h e r e i s no d o u b t t h a t a d o s e o f 0.35 mgm mammalian LH and FSH f o r an a v e r a g e o f 2.5 gm f i s h i s s u f f i c i e n t when c o m p a r e d t o t h e d o s a g e s s u c c e s s f u l l y u s e d by A h s a n and Hoar ( 1 9 6 3 ) and P i c k f o r d ( 1 9 5 3 ) . G o n a d o t r o p i n s a r e e i t h e r g l y c o p r o t e i n . . (FSH) o r m u c o p r o t e i n (LH) i n mammals ( B a r r i n g t o n , 1 9 6 3 ) . The - 110 -;-_c c h e m i s t r y o f f i s h g o n a d o t r o p i n s h a s n o t been s t u d i e d b u t there iTn i n d i c a t i o n s t h a t t h e y a r e s i m i l a r ( P i c k f o r d and A t z . , 1 9 5 7 ) . a n i m a l s u s u a l l y r e a c t more s t r o n g l y t o p i t u i t a r y hormones •rem t h e i r own o r c l o s e l y r e l a t e d s p e c i e s . However, v a r y i n g •agrees o f r e s p o n s e have been n o t e d i n t h e gonads i n t h e case, o f : . : i v ' I o g e n e t i c a l l y r e m o t e d o n o r s and r e c i p i e n t ( P i c k f o r d and A t z , ,'357; G r e a s e r and Gorbman, 1939; Gorbman and B e r n , 1 9 6 3 ) . L i m i t e d r e s p o n s e t o r e p l a c e m e n t t h e r a p y i s p o s s i b l y due t o s p e c i e s s p e c i f i c i t y o f t h e hormones. I t i s assumed t h a t t h e g o n a d o t r o p i n s i n t h e v a r i o u s v e r t e b r a t e g r o u p s p o s s e s s c e r t a i n common p h y s i o l o g i c a l l y a c t i v e m o l e c u l a r s e c t i o n s w h i c h a r e t h e a c t i v e c o r e , and s p e c i e s d i f f e r e n c e s may be r e l a t e d t o t h e r e m a i n d e r o f t h e m o l e c u l e . Some m o l e c u l a r d i f f e r e n c e s have been f o u n d i n t h e LH (ICSH) o b t a i n e d f r o m v a r i o u s mammalian s o u r c e s ( L i , 1963'). I t may be b;.;..,f.,osed t h a t t h e d e g r e e o f s p e c i f i c i t y may be r e l a t e d t o t h e .iegree o f d i s s i m i l a r i t y b e t w e e n t h e i r g o n a d o t r o p i c m o l e c u l e s . Or' t h e b a s i s o f t h i s h y p o t h e s i s i t i s n o t s u r p r i s i n g t h a t most i n s t a n c e s of. s p e c i f i c i t y a r e o f r e l a t i v e n a t u r e and a b s o l u t e s p e c i f i c i t y i s r a r e . I t i s p o s s i b l e t h a t t h e g r e a t e r s t i m u l a t i o n o b t a i n e d w i t h c r u d e , w h o l e s a l m o n p i t u i t a r y e x t r a c t i s due t o t h e " / n e r g e s t i c a c t i o n o f o t h e r p i t u i t a r y hormones. F o n t a i n e (1961) i n t h e d i s c u s s i o n a f t e r Ramaswami's p a p e r r e p o r t e d t h a t t o t u r a t i o n o f gonads i n A n q u i l l a was o b t a i n e d w i t h TSH w h i c h had a l i t t l e LH as i m p u r i t y . Ramaswami (1961) h i m s e l f f o u n d TSH c o n t a i n i n g s m a l l LH i m p u r i t i e s e f f e c t i v e i n c o m p l e t i o n o f the m a t u r a t i o n o f gonads i n i n t a c t H e t e r o p n u e s t e s f o s s i l i s . A hsan and Hoar (1963) showed t h a t t e s t i c u l a r and o v a r i a n - I l l -m a t u r a t i o n was e n h a n c e d w i t h TSH c o n t a i n i n g LH i m p u r i t i e s . P i c k f o r d (195^-) has a l s o r e p o r t e d s i m i l a r s y n e r g i s t i c a c t i o n o f g o n a d o t r o p i n s w i t h TSH and STH i n F u n d u l u s . I n mammalian work a l s o , a d d i t i o n o f STH t o g o n a d o t r o p i n s and t e s t o s t e r o n e i n c r e a s e d t h e r e s p o n s e by t h e g o n a d s t o r e p l a c e m e n t t h e r a p y . (Woods and S i m p s o n , 196.1; B o c c a b e l l a , 1 9 6 3 ) . I t i s . s u g g e s t e d t h a t s p e r m a t o g e n e t i c p r o c e s s e s r e s p o n d s p e c i f i c a l l y t o g o n a d o t r o p i n s w h i l e o t h e r f a c t o r s l i k e TSH and STH, w i t h a g e n e r a l s t i m u l a t o r y a c t i o n on m e t a b o l i c , p r o c e s s e s ^ . , a c c e n t u a t e the e f f e c t o f t h e g o n a d o t r o p i n on t h e t e s t e s o f C o u e s i u s p l u m b e u s . A t h e s i s r e g a r d i n g t h e r e g u l a t o r y m e c h a n i s m s o f t h e a n n u a l t e s t i c u l a r c y c l e o f C o u e s i u s p l u m b e u s can now be presented on t h e b a s i s o f t h e s e e x p e r i m e n t a l f i n d i n g s . The c o n t r o l l i n g m e c h a n i s m o f t h e r e g u l a t i o n o f t h e t e s t i c u l a r c y c l e i n t h i s f i s h i s c o m p l e x . T e m p e r a t u r e v a r i a t i o n s a c t as t h e m a j o r f a c t o r w h i c h a f f e c t t h e s e c r e t o r y a c t i v i t y o f t h e p i t u i t a r y g l a n d ; l i g h t p l a y s no s i g n i f i c a n t r o l e . T h i s t e m p e r a t u r e e f f e c t i s i n t h e n a t u r e o f a t r i g g e r r a t h e r t h a n t h e g e n e r a l i n c r e a s e i n m e t a b o l i c a c t i v i t y commonly a s s o c i a t e d w i t h t e m p e r a t u r e c h a n g e s . The p i t u i t a r y g l a n d i s s o l e l y r e s p o n s i b l e f o r t h e m a i n t e n a n c e and c o n t i n u a t i o n o f t e s t i c u l a r c y c l e . I m p o r t a n c e o f t h i s g l a n d i n t h e t e s t i c u l a r r e g u l a t i o n i s u n d e r l i n e d by t h e f a c t t h a t i t i s n o t o n l y r e l a t e d t o t h e g o nads t h r o u g h g o n a d a t r o p i n s and s t e r o i d s , b u t a l s o a c t s as an i n t e r m e d i a r y b e t w e e n i t s t a r g e t o r g a n s and t h e a n i m a l ' s e x t e r n a l e n v i r o n m e n t t h r o u g h i t s c l o s e a s s o c i a t i o n s w i t h t h e n e r v o u s s y s t e m . T e m p e r a t u r e c h a n g e s a c t on t h e c e n t r a l n e r v o u s s y s t e m and a f f e c t t h e n e u r o s e c r e t o r y a c t i v i t y i n t h e hypothalamus - 112 -w h i c h i n t u r n r e g u l a t e t h e p r o d u c t i o n o f t h e g o n a d a t r o p i n ; t h i s i n t h e f i n a l a n a l y s i s c o n t r o l s t h e t e s t i c u l a r c h a n g e s . I n c r e a s i n g w a t e r t e m p e r a t u r e s i n s p r i n g c a u s e e n h a n c e d g o n a d a t r o p i n s e c r e t i o n w h i c h r e s u l t i n a s u d d e n b u r s t o f m e i o t i c a c t i v i t y i n t h e t e s t e s l e a d i n g t o t h e c o m p l e t i o n o f t e s t i c u l a r m a t u r a t i o n . Sperms a r e s h e d as t h e w a t e r t e m p e r a t u r e r i s e s and i t i s p o s s i b l e t h a t h i g h e r t e m p e r a t u r e may a c t d i r e c t l y upon sperms and a c c e l e r a t e s p e r m i a t i o n by i n c r e a s i n g t h e i r m o t i l i t y . R e c o n s t i t u t i o n o f t e s t e s a f t e r s p a w n i n g i s s l o w i n A u g u s t as g o n a d o t r o p i n s e c r e t i o n i s s t o p p e d a f t e r a c e r t a i n l i m i t . When w a t e r t e m p e r a t u r e f a l l s a g a i n , s m a l l amounts o f g o n a d o t r o p i n s a r e s e c r e t e d m a i n t a i n i n g t h e s l o w a c t i v i t y d u r i n g t h e f i r s t p h a s e o f t h e t e s t i c u l a r c y c l e i n t h i s f i s h . The g o n a d o t r o p i n r e s p o n s i b l e f o r t e s t i c u l a r m a t u r a t i o n i s s i m i l a r t o mammalian LH, and a f a c t o r l i k e mammalian FSH i s p r o b a b l y n o t p h y s i o l o g i c a l l y a c t i v e i n t h i s f i s h . A l t h o u g h g o n a d o t r o p i n i s p r i m a r i l y r e s p o n s i b l e f o r t e s t i c u l a r m a t u r a t i o n , e v i d e n c e i s p r e s e n t t h a t o t h e r f a c t o r s ( p o s s i b l y t h y r o t r o p i n and s o m a t o t r o p i n ) may have some s t i m u l a t o r y e f f e c t by a c t i n g s y n e r g e s t i c a l l y w i t h t h e g o n a d o t r o p i n . T e s t e s o f C o u e s i u s p o s s e s s an unknown b u t f e e b l e e n d o g e n o u s r h y t h m w h i c h i s a l m o s t c o m p l e t e l y s u p p r e s s e d by h i g h e r t e m p e r a t u r e s (1.9 - 1.5°C). I t i s p o s s i b l e t h a t t h i s r h y t h m i s g e n e t i c and p e c u l i a r t o t h e s p e c i e s p o s s e s s i n g i t . - 113 -SUMMARY 1. . The a n n u a l t e s t i c u l a r c y c l e i n C o u e s i u s p l u m b e u s i s i n harmony w i t h t h e c y c l i c a l c h a n g e s i n i t s e n v i r o n m e n t , and t h e s p e r m a t o g e n e t i c p r o c e s s e s a r e c o n t r o l l e d m o s t l y by t e m p e r a t u r e c h a n g e s . 2. H y p o p h y s e c t o m y c a u s e s m a r k e d r e g r e s s i o n i n t h e t e s t i s s u p p r e s s i n g s p e r m a t o g o n i a l m u l t i p l i c a t i o n and p r e v e n t i n g t r a n s f o r m a t i o n o f s p e r m a t o g o n i a i n t o s p e r m a t o c y t e s . T h e r e i s some s e a s o n a l v a r i a t i o n i n t h e e f f e c t o f h y p o p h y s e c t o m y , i t i s maximum i n p r e s p a w n i n g and s p a w n i n g p e r i o d . 3. L o b u l e b o u n d a r y c e l l s (- t h e homologue o f L e y d i g c e l l s of i e t r a p o d ) a l s o show c y c l i c a l c h a n g e s o f a c t i v i t y as e v i d e n c e d by S c h u l t z ' s c h o l e s t e r o l t e s t , and a r e m a r k e d l y d e p r e s s e d by p i t u i t a r y r e m o v a l . 4. S p e r m a t o g e n e s i s i s l a r g e l y r e s t o r e d by i n j e c t i o n s of' f r a c t i o n a t e d s a l m o n p i t u i t a r y g o n a d o t r o p i n , p u r e mammalian LH, and c r u d e w h o l e s a l m o n p i t u i t a r y e x t r a c t . I t i s p o s s i b l e t h a t o t h e r f a c t o r s s u c h as STH and TSH c o n t a i n e d t h e r e i n , a c t s y n e r g e s t i c a l l y and e n h a n c e t h e e f f e c t o f t h e g o n a d o t r o p i n . 5. F r a c t i o n a t e d s a l m o n p i t u i t a r y g o n a d o t r o p i n , mammalian LH and w h o l e s a l m o n e x t r a c t r e s t o r e t h e a c t i v i t y o f t h e l o b u l e b o u n d a r y c e l l s o f h y p o p h y s e c t o m i s e d f i s h . 6. T e m p e r a t u r e i s a f a r more i m p o r t a n t f a c t o r t h a n l i g h t i n t h e r e g u l a t i o n o f t h e t e s t i c u l a r c y c l e and t h e v a r i a t i o n s i n t h i s , e n v i r o n m e n t a l p a r a m e t e r has d i f f e r e n t a c t i o n s a t d i f f e r e n t p h a s e s o f t h e s p e r m a t o g e n e t i c p r o c e s s . 7. S p e r m i a t i o n i s s p e e d e d by r i s e i n t e m p e r a t u r e and i s p r o b a b l y autonomous o f p i t u i t a r y c o n t r o l . 8. P y c n o t i c d e g e n e r a t i o n o f e x c e s s and p r e c o c i o u s l a t e r g e n e r a t i o n o f s e c o n d a r y s p e r m a t o g o n i a and o f ' p r i m a r y r.;).; j - ; : ^ - 1 1 4 -s p e r m a t o c y t e s i s a r e g u l a r f e a t u r e o f t h e a n n u a l c y c l e o f t h i s f i s h . T h i s c h a r a c t e r i s t i c c a n be u s e d i n f i n d i n g w h e t h e r a f i s h has spawned o r n o t . CONCLUSIONS I n t e g r a t i o n o f t h e p r e s e n t f i n d i n g s s u g g e s t t h a t t h e t e s t i c u l a r c y c l e i n C o u e s i u s p l u m b e u s has a d u a l b a s i s r e f l e c t e d i n i t s c o n t r o l l i n g m e c h a n i s m w h i c h i s p a r t l y endogenous ( g o n a d o t r o p i n , s i m i l a r i n c h a r a c t e r t o mjammalian LH) and i n p a r t e x o g e n o u s ( v a r i a t i o n ' s i n t h e w a t e r t e m p e r a t u r e ) . An i n t e r p l a y o f ' . b o t h t h e s e f a c t o r s i s e s s e n t i a l f o r s p e r m a t o g o n i a l m u l t i p l i c a t i o n , s p e r m a t o c y t e • f o r m a t i o n and c o m p l e t i o n o f t h e . m a t u r a t i o n s t a g e s . The a n n u a l t e s t i c u l a r c y c l e o f C o u e s i u s . i s c l o s e l y r e l a t e d t o c h a n g e s i n t h e e n v i r o n m e n t a l c o n d i t i o n s , and m e d i a t e d by t h e p i t u i t a r y g l a n d whose own s e c r e t o r y a q t i v i t y i s r e g u l a t e d by t h e s e c h a n g e s ( o f w h i c h t e m p e r a t u r e i s t h e most i m p o r t a n t ) . S t i m u l a t i o n o f t e s t e s o f h y p o p h y s e c t o m i s e d f i s h i s b r o u g h t a b o u t by f i s h g o n a d o t r o p i n and mammalian LH, b u t mammalian FSH i s i n e f f e c t i v e . R e s p o n s e t o r e p l a c e m e n t t h e r a p y i s maximum d u r i n g t h e p r e s p a w n i n g p e r i o d and l e a s t i m m e d i a t e l y a f t e r t h e s p a w n i n g ? ;,nc: maximum t e s t i c u l a r r e g r e s s i o n i s n o t e d when p i t u i t a r y i s r e m o v e d a t t h e p e a k o f g o n a d a l a c t i v i t y . T e m p e r a t u r e i s t h e most i m p o r t a n t e x o g e n o u s r e g u l a t o r o f t e s t i c u l a r c y c l e . Low t e m p e r a t u r e s f a c i l i t a t e s p e r m a t o g o n i a l m u l t i p l i c a t i o n and h i g h t e m p e r a t u r e s a r e e s s e n t i a l f o r s p e r m i o g e n e s i s and s p e r m i a t i o n . T e m p e r a t u r e e f f e c t s a r e r a r e l y o v e r r i d d e n by p h o t o p e r i o d . 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Newman. 1954, F u r t h e r s t u d i e s on t h e r e a c t i o n s o f a d u l t b l u e b l a c k s a l m o n t o i n j e c t e d s a l m o n and mammalian g o n a d o t r o p i n s . P r o g . F i s h . C u l t : 9 9 -107. . P i c k f o r d , G.E. 1953. A s t u d y o f h y p o p h y s e c t o m i s e d male F u n d u l u s h e t e r o c l i t u s . B u l l . B ingham. O c e a n o g . C o l l e c . 14: 5-41. , 1954. The r e s p o n s e o f h y p o p h y s e c t o m i s e d male k i l l i f i s h t o p u r i f i e d g r o w t h hormone as ; . compared, w i t h t h e r e s p o n s e t o p u r i f i e d b e e f g r o w t h hormone. E n d o c . 5 5 : 274-287. , and J.W. A t z . 1957. The p h y s i o l o g y o f t h e p i t u i t a r y g l a n d o f f i s h e s . New Y o r k Z o o l o g i c a l S o c i e t y , New Y o r k . Ramaswami, L.S. 1962. E n d o c r i n o l o g y o f r e p r o d u c t i o n i n f i s h and f r o g . Gen. Comp. E n d o c r i n o l . S u p p l . II-286-299. S c o t t , R.N. 1952. The t i m e o f s p a w n i n g , age, and s e c o n d a r y s e x c h a r a c t e r o f a d u l t c h u b s , M v l o c h e i l u s c a u r i n u m . M.S. T h e s i s M o n t a n a S t a t e U n i v . U n p u b l i s h e d . S o k o l , H.W. 1955. E x p e r i m e n t a l d e m o n s t r a t i o n o f t h y r o t r o p i c and g o n a d o t r o p i c a c t i v i t y i n t h e a d e n o h y p o p h y s i s o f t h e Guppy, L e b i s t e s r e t i c u l a t u s ( P e t e r s ) A b s t r a c t . A n a t . R e c . , 1 2 2 : 4 5 1 . - 121 -T u r n e r , C L . 1919. The s e a s o n a l c y c l e i n t h e s p e r m a r y o f t h e p e r c h . J . Morph. 32: 6 8 1 - 7 1 1 . - V a u p e l , J . 1929. The s p e r m a t o g e n e s i s o f L e b i s t e s r e t i c u l a t u s . J . . M o r p h . and P h y s i o l . 4 7 : 5 5 5 - 5 8 7 , V e r h o e v e n , B. and G.J. Van O o r d t . 1955. The i n f l u e n c e o f l i g h t and t e m p e r a t u r e on t h e s e x u a l c y c l e o„; o f t h e b i t t e r l i n g , Rhodeus amarus ( P r e l i m i n a r y c o m m u n i c a t i o n ) . K o n i n k l . N e d e r l , A k a d emie v a n W e t e n s c h a p p e n . Amsterdam C, 58 : 6 2 8 - 6 3 4 . V i v i e n , J-H. 1938, 1939. 1941 W e i s e l , G.F. 1943. 1949. W i t s c h i , E. 1955. S u r l e s e f f e t s de 1 ' h y p o p h y s e c t o m i e c h e z un t e l e o s t e e n m a r i n , G o b i u s p a q a n e l l u s L. Comp. Rend, A c a d , S c i . P a r i s , 2 0 7 : 1 4 5 2 - 1 4 5 5 . R o l e de l ' h y p o p h y s e dans l e d e t e r m i n i s m e du c y c l e g e n i t a l f e m e l l e d'un t e l e o s t e e n , G o b i u s p a a a n e l l u s L. Comp, Rend. A c a d . S c i . P a r i s , 208: 94 8 - 9 4 9 . C o n t r i b u t i o n a 1 ' e t u d e de l a p h y s i o l o g i e h y p o p h y s a i r e d a n s s e s r e l a t i o n s a v e c l . ! u l ' a p p a r e i l g e n i t a l , l a t h y r o i d e e t l e s c o r p s s u p r a r e n a u x c h e z l e s p o i s s o n s s e l a c i a n s e t t e l e o s t e e n s S c y l l i o r h i n u s c a n i c u l a e t G o b i u s p a q a n e l l u s . B u l l . B i o l , 7 5 : 2 5 7 -309. A h i s t o l o g i c a l s t u d y o f t h e t e s t e s o f S o c k e y e S a l m o n . J . Morph. 7 3 : 207-229. The s e m i n a l v e s i c l e s and t e s t e s o f G i l l i c h t h y s . a m a r i n e t e l e o s t . C o p e i a ( 1 9 4 9 ) : 101-110. V e r t e b r a t e g o n a d a t r o p i n s , 1 4 9-165. Mem.Soc.Endoc,4: W i t h r o w , R.B. 1959. E d : " P h o t o p e r i o d i s m and r e l a t e d phenomena i n p l a n t s and a n i m a l s " , AAAS, Washington-, D.C. Woods, M.C. and M.E. S i m p s o n . 1961. P i t u i t a r y c o n t r o l o f t h e t e s t i s o f t h e h y p o p h y s e c t o m i s e d r a t . E n d o c , 69: 9 1 - 1 2 5 . Y o u n g , J . Z. 1933. The p r e p a r a t i o n o f i s o t o n i c s o l u t i o n s f o r use i n e x p e r i m e n t s i n f i s h . P u b l . s t a z . z o o l , N a p o l i , 12: 4 2 5 - 4 3 1 . T a b l e I . Summary o f l i p i d s t a i n and c h o l e s t e r o l t e s t r e s u l t s on n o r m a l C o u e s i u s t e s t i s M o n t h 1963 J a n u a r y F e b r u a r y M a r c h May J u n e J u l y A u g u s t S e p t e m b e r O c t o b e r November L i p i d s Sudan B l a c k B S t a i n D i f f u s e l i p o i d a l g r a n u l e s L o b u l e b o u n d a r y c e l l l i p o i d a l C h o l e s t e r o l t e s t + + + L i t t l e e v i d e n c e o f l i p o i d a l s u b s t a n c e D i f f u s e l i p o i d a l g r a n u l e s L o b u l e b o u n d a r y c e l l s l i p o i d a l + + T a b l e I I . E f f e c t o f h y p o p h y s e c t o m y o n ' t h e t e s t e s i n C. p l u m b e u s . F i g u r e s u n d e r s p e r m a t o g e n e t i c c o n d i t i o n a r e t h e p e r c e n t a g e c o m p o s i t i o n o f v a r i o u s s p e r m a t o g e n e t i c s t a g e s f r o m p l a n i m e t r y . SPGR = p r i m a r y germ c e l l s ; SPG 1 = p r i m a r y . s p e r m a t o g o n i a ; SPG" = r e s t i n g S e c o n d a r y s p e r m a t o g o n i a ; SPG"x = d i v i d i n g s e c o n d a r y s p e r m a t o g o n i a ; S P C - r e s t i n g p r i m a r y s p e r m a t o c y t e s ; S P C x = d i v i d i n g p r i m a r y s p e r m a t o c y t e s ; SPC" = s e c o n d a r y s p e r m a t o c y t e s . SPD = s p e r m a t i d s . S = sperm s . . d = p y c n o t i c n e s t s . • S p e r m a t o g e n e t i c c o n d i t i o n S p e r m a t o g e n e t i c c o n d i t i o n o f D a t e h y p o - D a t e o f E x p t . ^ n h y p o p h y s e c t o m i s e d f i s h * , c o n t r o l f i s h  p h v s e c t o m i s e d A u t o p s y Davs!. SPGR SPG' x SPG" d SPGR S P C X SPG" SPG"x S P C C"x S d 1962 J u l y 14-19 Aug. •31 46 56.3 .33.1 9.8 0.3 "9.8 34.6 3 4 . 2 18.3 - - 3. 1 Aug. 15-19 O c t . 2 46 ; 49.6 4 9 . 2 0.7 0.5 8.0 46'. 8 2§". 4 16.7 - -S e p t . 3-7 Nov. 1 56 4 7 . 5 38.6 12.5 1.4 6.7 3 8 \ l 3-3.0 16.8 1.1 - - 4. 3 O c t . 23-26 Dec. 19 56 66.5 33.0 0.5 - 5.7 23.1 20.8 35.5 8.0 - - 6. 9 Nov. 23-25 J a n . 26 63 52.0 45.5 2.5 - 7.7 19.2 34.4 27/. 1 l f . ~ 5 - -1963 J a n . 3-5 Feb. 25 52 31.3 61.1 7.6 6.7 18.2 1-9.0 31.6 17.5 0.4 0.8 5.8 Feb. 3-6 Mar. 23 47 3,1.7 61.0 7.6 7.0 12.2 9.9 24.5 46.4 - 0.9 Mar.. . 5-7 May 2 58 39.0 56.8 4.2 8.2 11.0 15.7 39.6 21.0 2.8 1.7 May 2-4 „ : J u n e 30 57 40.7 53.1 6.2 13.5 39.6 11.2 0.2 - • -' 37.4 -J u n e 6-8 J u l y 31 54 24.8 68.2 7.0 7.1 33.6 23.7 27.1 5.1 0.5 - 2.9 J u l y 3-5 Aug. 27 53 32.0 60.0 8.0 21.0 27.8 27.6 8.8 7.4. - - -Aug. 12-14 O c t . 8 55 42.0 52,.8 5.2 - 11.8 36.0 35.1 11.5 4.5 - - 1.0 S e p t . 5-7 O c t . 28 5.2 30.3 61.7 8.0 8.1 25.5 36.8 23.0 6.6 - - -O c t . 11-13 Dec. 5 53 25.0 67.0 8.0 8.5 31.0 30.8 22.9 6.9 — • _ - 124 -T a b l e I I I . E f f e c t o f h y p o p h y s e c t o m y on t h e t e s t e s i n C. p l u m b e u s as m e a s u r e d by g o n o s o m a t i c i n d i c e s ( G S I) and c h o l e s t e r o l t e s t . E x p t s . 1962 J u l y A u g u s t S e p t . O c t . Nov. 1963 J a n . Feb. M a r c h May J u n e J u l y A u g u s t S e p t . O c t . G SI Hx 0.4701 0.5043 0.4934 0.4689 0.4916 0.4694 0.3856 0.3641 0.2104 0.411 0.525 0.404 0.460 0.4226 C o n t r o l 0,5423 0.5385 0.8121 0.7898 0.7525 0.9154 0.8317 0.8720 0.6903 0.7460 0.8568 0.8521 0.899 0.8573 C h o l e s t e r o l ' T e s t Hx C o n t r o l + + + + + + T a b l e I V . Summary o f e f f e c t o f r e p l a c e m e n t t h e r a p y w i t h mammalian p u r i f i e d s t i m u l a t i n g hormone ( F S H ) . S y m b o l s as i n p r e v i o u s t a b l e . f o l l i c l e M o n t h M o n t h o f E x p t . Days. G S I S p e r m a t o g e n e t i c c o n d i t i o n i n t r e a t e d f i s h ( o f E x p t . A u t o p s y Hx C o n t r o l T r e a t e d SPGR SPG'x SPG , J SPG"x S P C d • 1962 j J u l y Aug. 31 46 0.4701 0.5423 Aug. O c t . 46 0.5043 0.5385 0.5114 32.3 62.5 5.2 - -S e p t . Nov. 56 0.4934 0.8121 0.5776 49.6 39.9 9.9 0,6 -O c t . Dec. 56 0.4689 0.7898 0.6021 51.0 45 . 5 3.5 - . .- -Nov. 1963 J a n . 63 0.4916 0.7525 0.5110 48.6 45.1 5.9 — — 0.4 1963 J a n . F e b . 52 0.4694 0.9154 0.5449 27.3 60.0 11.5 1.2 • - -Feb. M a r c h 47 0.3856 0.8317 0.5023 16.4 70.6 11.4 1.4 -M a r c h May 58 0.3641 0.8720 0.5793 21.1 70.2 8.5. 0.2 - -May J u n e 30 57 0.2104 0.6903 0.3691 15.1 65.1 17.7 2.1 -J u n e J u l y 31 54 0.4110 0.7460 0.4280 26.2 64.7 8.3:. 0.8 -J u l y Aug. 27 53 0.5250 0.7568 0.4270 18.4 77.0 ' 4.6. - - -Aug. O c t . 55 0.4040 0.8521 0.4010 26.4 66.2 7.4 •- - -S e p t . O c t . 28 52 0.4600 0.89.90 0.4182 25.0 70.0 4.1 - 0.9 Oct.,:. Dec. 53 0.4226 0.8573 0.5210 13.5 75.0 10.5 1.0 _ C h o l e s -t e r o l t r e a t e d f i s h T a b l e V. Summary o f t h e e f f e c t o f r e p l a c e m e n t t h e r a p y w i t h mammalian p u r i f i e d l u t e i n i z i n g hormone ( L H ) . S y m b o l s as i n T a b l e I I . The u p p e r r o w d e n o t e d by H i n d i c a t e s h i g h t e m p e r a t u r e , t h e l o w e r r o w L t h a t a t l o w temperature. G S M p n t h M o n t h o f E x p t . o f E x p t . A u t o p s y Days Hx C b n t r o : 1962 J u l y Aug. 31 .46 0.4.701 .0.5423 Aug. O c t . 46 0.5043 0.5385 S e p t . Nov. 56 0.4934 0.8121 O c t . Dec. 56 0.4689 0.7898 Nov. J a n . 63 0.4916 .0.7525 1963 J a n . Feb. 52 0.4694 0.9154 F e b . M a r c h 47 0.3856 0.8317 M a r c h May 58 0.3641 0.8720 May J u n e 57 0.2104 0.6903 J u n e J u l y 31 54 0.4110 0.7460 J u l y Aug. 27 53 0.5250 0.7568 A u g / O c t . 55 0.4040 0.8521 S e p t . O c t . 28 5 2 0.4600 0.8990 O c t . Dec. 5 3 0.4226 0.8573 S p e r m a t o g e n e t i c c o n d i t i o n i n ' t r e a t e d f i s h . . SPGR SPG'X SPG""" "SPG"x S P C S P C 0.6930 0.6969 0.6282 0.6460 0.5779 0.6606 0.6708 0.7372 0.5108 H 0 . 6 9 4 0 L 0 . 7 8 8 0 ^0.5490 ^ . 6 6 8 0 TH0.6480 L 0 . 6950 H 0.5930 TH0.6464 C h o l e s t e r -t e r o l i n t r e a t e d x f i s h 4.8 3.8 4.4 3.8 5.0 52.5 53.0 47.7 4 0 . 2 57.0 30.7 32.7 27.3 4 6 . 0 34.S 12.0 10.5 16.7 10.1 4.2 3.0 0.9 + + + 2.5 50.0 18.6 22.7 5.7 1. 0 + 3.2 4 8 . 2 19.-7"'" 22.2 5.6 1. 1 + 4.2 46.0 22.3 22.6 5.0 + 14.1 53.7 23.7 8.0 0.5 + 14,8 . 47.0 17.8 16.2 2.4 1. 6 + 4.5. -22.8 30.6 32.5 6.1 3. 5 + 4.4 ; 57.7 ' 27.7 '2.4 5.6 46.4 30.3 9.1 6.3 + 5..7 57.4 31.2 5.6 _ + 3.9 53.5 33.0 9.3 0.6 + 1.9 63.6 32.5 1.9 — + 0.6 47.8 4 1 . 7 , 8.6 3.4 + ;4.6 62.3 22.3 8.0 0.8 + 3.5 4 4 . 2 36.5 13.8 2.2 + T a b l e ; V I . Summary o f t h e e f f e c t o f r e p l a c e m e n t t h e r a p y w i t h f r a c t i o n a t e d s a l m o n p i t u i t a r y g o n a d o t r o p i n . S y m b o l s as i n T a b l e I I . I n May 1963 and s u b s e q u e n t e x p e r i m e n t s t h e u p p e r r o w d e n o t e d by H i n d i c a t e s c o n d i t i o n o f t r e a t e d f i s h a t h i g h t e m p e r a t u r e , t h e l o w e r r o w L t h a t a t l o w t e m p e r a t u r e . • . : . • ' - M o n t h M o n t h o f E x p t , o f ••Expt. A u t o p s y Days S p e r m a t o g e n e t i c c o n d i t i o n i n t r e a t e d . • f i s h • -. ••: C h o l e s -t e r o l i n Hx C o n t r o l T r e a t e d SPGR' SPG'x SPG" SPG"X S P C SPC'x SPG" d t r e a t e d • •• r f i s h 1262. J u l y Aug. S e p t . Oct,:. Nov. 1963 J a n . Feb. May J u n e Aug. 31 46 0.4701 0.5423 0.6250 4.7 4 1 . 2 31.8 18.7 2.8 - - 0.8 -/+ O c t . 46 0.5043 0.5385 0.5616 8.3 55.4 31.6 5.0 - - - _/+ Nov. 56 0.4934 0.8121 0.6278 3.3 . 43,4 37,1 15.1 .1.1 - - - + Dec. 56 0.4689 0.7898 0.6892 2.0 ' 4 5 , 9 33.4 18.7 - - + Jan.. 1963 63 0.4916 0.7525 .0.7001 4.3 4 7 , 1 37.6 10.3 — — - - + Feb. 52 0.4694 0.9154 0.6684 3.4 4 9 . 2 23.1 21.6 2.7 •'- - + M a r c h : 47 0.3856 0.8317 0.0874 1.4 49.0 18.3 26.1 4.4 0.8 - + May 58 :0.3641 0.8720 1.0137 1.5 47.4 30.8 17.1 3.4 - _ _ + J u n e 30 57 J u l y 31 54 J u l y Aug. 27 53 Aug. O c t . 55 S e p t . O c t . 28 52 O c t . Dec. 53. 0.2104 0.6903 0.4110 0.7460 0.5250 0.7568 0.4040 0.8521 ,0.4600 0.8990 0.4252 0.8573 ."0.6497 L 0 . 9 0 3 2 H 1.0290 L 0 . 9 1 9 0 T*"b.6650 L 0 . 5 7 9 0 TH 0 . 7 4 9 0 L 0 . 7 0 3 0 ^0.8170 L0'.8700 TF0.5896 L 0 . 7 7 9 8 7.0 2.3 8.0 8.7 0.4 1.2 61.9 27.7 64.9 30.9 36.0 14.6 34.6 19,9 55.1 27.7 62.9 26.2 3.4 3.9 3.5.6 2.9 1.6 34.0 2.6 0.2 4.9 53.3 40.4 3.0 56,9 27.0 2.1 1.8 3.4 2.7 47.4 34.6 48.8 39.4 54.3 34.0 48.0 34.7 10.8 7.0 1.4 3.1 12,9 9.0 17.5 12.2 6.0 2.1 3.0 1.0 0.8 2.4 0.6 + - A -/+ •/•+ + + + + + + + T a b l e V I I . E f f e c t o f r e p l a c e m e n t t h e r a p y : Crude w h o l e s a l m o n p i t u i t a r y e x t r a c t (CSE) S y m b o l s as i n T a b l e I I . C h o l e s— M o n t h M o n t h o f E x p t . fi-JLJL S p e r m a t o q e n e t i c s t a q e s i n t r e a t e d f i s h t e r Q l o f E x p t . A u t o p s y Days. Hx C o n t r o l T r e a t e d SPGR SPG'x SPG" SPG"x S P C SPC'x SPC" SPD d t r e a t e d . - f i s h 1 962 S e p t . Nov. 56 0.4934 0.8121 0.01047 3.0 23. 2 2 1 . 0 34. 1 6.4 8.9 0.3 - 3.0 + Nov. 1963 J a n . 6 3 0.4916 0.7525 0.7932 2.3 29. 0 33. 0 24. 4 8.1 2.9 - - + 1963 J a n . F e b . 5 2 0.4694 0.9154 - - - - - -Feb. M a r c h 47 0.3856 0.8317 1.1395 1.7 20. 7 20. 8 35. 9 13.3 2.6 0.6 - 0.4 + M a r c h May .58 0.3641 0.8720 1.2210 2.0 25. 6 24. 1 32. 3 12.5 3.5 - - + May J u n e 30 57 0.2104 0.6903 1.0378 2.0 44. 8 25. 3 22. 7 4.4 0.8 - -J u n e J u l y 3 1 54 0.4110 0.7460 0.7170* L - 2 57. 0 26. 5 13. 0 1.2 1.1 • -J u l y Aug. 27 53 0.5250 0.7568: 0.8650 4.9 35. 9 22. 4 27. 5 8.7 0.4 • - - + Aug. O c t . 55 0.4040 0.8521' 0.7920 9.2 44. 6 36. 2 9. 8 0.2 - - - + S e p t . O c t . 28 5 2 0.4600 0.8990 1.0896 1.2 16. 5 4 0 . 6 33. 7 7.6 0,3 - - + Oct.„.. Dec. 53 0.4252 0.8573 0.7672* 2.2 19. 1 33. 0 35. 9 8.3 0.9 — - — - 129 -T a b l e V I I I . E x p l o r a t o r y P h o t o p e r i o d E x p e r i m e n t F i s h i n E x p e r i m e n t a l C o n d i t i o n J a n u a r y 1 9 1963. No. and d a t e o f A u t o p s y D u r a t i o n ' o f E x p e r i m e n t G o n o s o m a t i c I n d i c e s L ong S h o r t P h o t o p e r i o d P h o t o p . e r i o d N o r m a l I . 1963.-. Feb. 15 45 d a y s 1.12 •1.19 1.464 I I . M a r c h 15 75 d a y s 1.704 1.609 1.384 - 130 T a b l e I X . P h o t o p e r i o d - t e m p e r a t u r e E x p e r i m e n t : E f f e c t o f Bhotoper i o d - t e m p e r a t u r e C o m b i n a t i o n on P r e s p a w n i n g S t o c k - F i s h i n E x p e r i m e n t a l C o n d i t i o n - A p r i l 3 0 , 1963. L-L°, Long p h o t o p e r i o d , Low t e m p e r a t u r e ; L-H° S-L c S h o r t p h o t o p e r i o d , Low t e m p e r a t u r e ; L ong p h o t o p e r i o d , H i g h t e m p e r a t u r e ; S h o r t p h o t o p e r i o d , H i g h t e m p e r a t u r e ; O t h e r S y m b o l s as i n T a b l e I I , L-L° S-L° L-H° S-H° N o r m a l GSI 1.5095 1 .7876 Q.7311 0.5882 2.1374 SPGR 4.5 3.5 11.6 18.1 2.5 SPG* 3.2 1.6 22.4 44.5 4.7 SPG" 20.2 17.7 17.4 13.3 0.7 T SPG"x 8.1 12.5 . 19.3 11.6 12.6 1 . S P C 7.0 1.3 0.4 0.3 0.6 S P C x 4.8 6.6 3.2 - 15.1 SPC" 2.0 2.0 0.4 - 11.6 SPD & S 50,3 54.8 25.3 11.5 52.2 d - - 0.7 GSI 1.3205 1 .1922 0.4094 0.4369 1 1.721 SPGR 8.2 • 7.4 8.5 17.4 1.9 SPG:' X - - 6779 59". 6 1.2 T T SPG" 14.0 14.6 19.5 22.0 • 3.2 11 . SPG" 14.9 19.3 4.1 0.8 10.6 S P C 2.1 4.5 - - 2.8 SPC'x \ 0.8 4.0 - - 18.9 SPC" 0.1 0.3 - - 20.9 SPD & S 59.8 49.9 _ 0.2 40.8 d 0.1 - - -GSI 1.8220 1 .5970 0.4510 0.04520 0.5309 SPGR 2.5 3.1 10.8 5.4 62.3 SPG'x 0.8 jO.8 58.5 58,2 1.0 I I I . SPG" 16.4 13.0 29.5 28.1 21.0 SPG"x 14.7 15.3 1.1 7.9 2.0 S P C 10.0 1.2*2 - 0.4 8.5 SPC'x 8.0 • 12.3 - - 0.6 SPC" 1.6 1.2 - _ 1.2 SPD & s 46.0 42.1 - - 0.4 d - - - - 2.1 GSI 1.4422 1 .7185 0.8964 0.8725 1.223 SBfGR 2.5 1.4 4.8 6.6 4.4 SPG'x. 3.9 0.6 35.8 39.9 0.3 SPG"' 28.3 15.6 36.5 33.8 14.4 IV . SPG"x 21.4 18.4 15.0 13.8 31.1 S P C 41 . 3 59.4 3.8 4.6 a.7.5 SPC'x 2.1 2.4 - - 0.4 SPC" - - - - -SPD & s - - - _ d 0.5 2.2 4.1 1.3 1.9 -• 131 -T a b l e X. Summary o f t h e P h o t o p e r i o d - t e m p e r a t u r e E x p e r i m e n t : E f f e c t on P o s t p a w n e d S t o t k . F i s h i n E x p e r i m e n t a l C o n d i t i o n f r o m A u g u s t 9, 1963. S y m b o l s as i n T a b l e I I & I X . . L-L° S-L° L-H° S-H° N o r m a l GSI •'• '" 0.913 0.708 0 .4550 0.6190 0.7123 SPGR • 10.2 • 14.1 12.0 9.5 20.0 SPG'x 12.6 12.6 61.7 39.4 5.0 SPG" 29.4 24.9 25.3 37.0 18.5 T SPG"x 30.8 28.9 1.0 10.8 18.0 X . S P C 6.2 8.2 •• _ -- 2.0 28.0 SPC'x 0.5 1.3 - 0.5 0.8 SPC" - . 1.2 - - 2.0 SPD & S 10.3 8.8 - 0.4 d — — " — • 0.8 7.3 GSI 1.2680 1.1434 0.5812 0,8027 0.9295 SPGR 6.0 3.8 * - 9.8 3.2 13.7 SPG'x 20.3 10.4 53.0 40.1 6.5 SPG" 27.3 37.6 32.9 41.6 27.0 ' T SPG"x 14.8 14.1 2.8 11.5 7.3 . x, SPC' 29.6 32.5 • 1.5 3.6 25.4 SPC'x _ - - - 0.6 SPC" - - - - -SPD & S 0.1 - - - -d 1.9 1.6 - — — 16.5 GSI , 1.2834 1.4328 .-0.7360 0.7581 1.1921 SPGR 2.4 0.5 / ""7.5 4.8 3.8 -SPG'x 1.8 0.6 37.2 24.5 . 8.5 SPG" , 21.3 10.5 •• • 42.0 42.6 10.7 TT SPG"x L7.4 16.2 11.6 20.9 31.5 JLJ. t S P C 47.3 41.0 1.7 6.4 38.4 SPC'x 4 . 9 / 20.2 — _ 6.6 SPC" 0.5 4.9 - - 0.5 SPD & S 0.6 3.9 ! - - -a 3.8 2.2 - !' 0.8 -- 1 3 2 -T a b l e X I . Summary o f t h e P h o t o p e r i o d - t e m p e r a t u r e E x p e r i m e n t : E f f e c t on P r e - f r e e z e up S t o c k . F i s h i n E x p e r i m e n t a l C o n d i t i o n f r o m November 1, 1963 S y m b o l s as i n T a b l e I I and I X . L-L° S-L° L-H° S-H° N o r m a l G . S . I , 1.6722' 1 .4448 0.7291 0.6632 1.2223 SPGR 2.0 4.8^ :4:4 . 9.2 5.6 SPG'x . 5 . 8 9.9 39.9 38 ,2 2.0 SPG" : :..25.8 36.2 35.5 31.2 13.9 SPC'x 33.7 22.6 13.0 13.8 15.4 S P C ;31.0 25.2 1.7 3.0 55.0 SPC'x 0.4 - 0.4 SPC" _ - - - -SPD & S , 0.2 - - - -d 1.5 1.3 5.1 4.6 7.7 I I . G S I . 1.8235 2.1032 0.7488 0.6755 1.5904 SPGR 1.5 0.9 8.1 5.5 1.6 SPG'x - - ' 35.1 36.8 5.5 SPG" 6.4 4.5 35.6 4 2 . 2 7.2 SPG"x 14.5 9.2 17.3 9.9 20.3 S P C 15.7 18.2 3:0 ... 3.7 33.9 SPC'x 42.7 39.2 - — 19.4 SPC" 12.4 21.7 - - 10.6 SPD & S 5.9 6.3 - - 1.5 d 0.9 — 0.9 1.9 — 

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