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Seasonal changes in the cytology of the pituitary gland and the ovary of the goldfish. Beach, Alan Winston 1956

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SEASONAL CHANGES I N THE CYTOLOGY OF THE PITUITARY GLAND AND THE OYARY OF THE GOLDFISH by A l a n Winston Beach B.A., U n i v e r s i t y o f B r i t i s h Columbia, 1953 A t h e s i s submitted 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 t h e degree o f MASTER OF ARTS i n t h e Department of Zoology  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the s t a n d a r d r e q u i r e d from c a n d i d a t e s f o r the degree o f MASTER OF ARTS.  Members o f the Department o f Z o o l o g y  The U n i v e r s i t y o f B r i t i s h A p r i l , 1956  Columbia  ABSTRACT G o l d f i s h ova b e g i n r a p i d m a t u r a t i o n  i n November.  M a t u r a t i o n i s completed i n June and J u l y .  Seasonal cycles i n  mature and r e s o r b i n g f o l l i c l e s were c o r r e l a t e d w i t h changes i n b a s o p h i l c o u n t s o f the p i t u i t a r y g l a n d . evidence confirms ovarian a c t i v i t y .  Cytological  the expected p i t u i t a r y c o n t r o l o f c y c l i c Resorbing  f o l l i c l e s a r e found a l l y e a r  but are more abundant d u r i n g s e x u a l m a t u r i t y , and  are  p a r t i c u l a r l y numerous i n f i s h w h i c h f a i l e d to spawn.  Gold-  f i s h i n j e c t e d w i t h e x t r a c t s o f salmon p i t u i t a r y f o r twelve d a y s showed no c y t o l o g i c a l changes i n the o v a r y . concluded  It is  t h a t the g r a n u l o s a i s r e s p o n s i b l e f o r removal o f  r e s o r b i n g ova and  t h a t these r e s o r b i n g ova a r e the means o f  removing s u r p l u s eggs.  TABLE OF CONTENTS Page INTRODUCTION ACKNOWIJEGEMENTS  i..1 ....  2  MATERIALS AND METHODS  .  3  E x p e r i m e n t a l c o n d i t i o n s and s a m p l i n g  ........  H i s t o l o g i c a l methods A n a l y t i c a l methods  3 5  ,  3  E x p e r i m e n t a l work  ,  11  RESULTS  12 G e n e r a l d e s c r i p t i o n o f o v a r y and o o g e n e s i s  Histogenesis of a t r e t i c f o l l i c l e s  18  S e a s o n a l changes i n the o v a r y  ,  26  C o r r e l a t i o n s between p i t u i t a r y and o v a r y  .... 30  R e s u l t s from I n j e c t i o n experiments. LITERATURE REVIEW  The o v a r i a n f o l l i c l e  41  ,.  DISCUSSION  42 ,  ,  .. 12  46 46  The a t r e t i c f o l l i c l e  48  P i t u i t a r y and o v a r y c o r r e l a t i o n s  50  I n j e c t i o n experiments  51  SUMMARY  52  LITERATURE CITED  53  APPENDIX  57  ILLUSTRATIONS  76  L I S T OF FIGURES Page  1.  Apparatus f o r m i c r o p r o j e c t i o n drawings o f pituitaries.  2.  Drawing o f t r a n s v e r s e s e c t i o n through v i s c e r a l c a v i t y showing r e l a t i o n s o f organs. 13  3.  An a-stage degenerate f o l l i c l e .  21  4.  A. b-stage degenerate f o l l i c l e .  21  5.  Hypertrophied granulosa c e l l .  23  6.  A c-stage degenerate f o l l i c l e .  23  7.  Graph showing development o f ova throughout the y e a r .  27  8.  Graph comparing a t r e t i c egg mumher and i n c r e a s e i n b a s o p h i l s t h r o u g h o u t the y e a r .  29  9.  Graph comparing ova growth and i n c r e a s e i n b a s o p h i l s throughout the y e a r .  31  C o r r e l a t i o n s between p i t u i t a r y b a s o p h i l s and egg d i a m e t e r ( i n f o u r s e a s o n s ) .  32-35.  Graph snowing d i s t r i b u t i o n of p o i n t s r e l a t i n g i t u i t a r y b a s o p h i l s and a t r e t i c egg number. I n f o u r seasons)  P  37  15.  Immature o v a r y .  76  16.  Three Immature f o l l i c l e s showing v a r i o u s s i z e s and shapes o f n u c l e o l i .  76  Immature ova showing oolemma and c e l l s stained w i t h Feulgen's.  77  10-13. 14.  17.  10  follicle  18.  P h o t o m i c r o g r a p h o f mature ovum showing zona pellucida with radial s t r i a t i o n s .  77  19.  S u r f a c e o f t h e c a e x t e r n a showing squamous e p i t h e l i a l - l i k e s t r u c t u r e of c e l l s .  78  20.  Ovary showing l o n g i t u d i n a l s e e t i o n o f p r i n c i p l e branch of ovarian a r t e r y .  78  21.  T r a n s v e r s e s e c t i o n through, t h e o v a r i a n a r t e r y .  79  22.  Immature o v a r y c o n t a i n i n g a v e r y young a t r e t i c . follicle. 79  23i  Young a t r e t i c f o l l i c l e from immature ovum showing I n t e n s e g r a n u l a t i o n i n s t r a t u m granulosum.  80  24.  Y e r y young a t r e t i c f o l l i c l e .  80  2:5.  A a-stage degenerate f o l l i c l e showing hut eroded zona p e l l u c i d a .  Intact 81  26.  Small a t r e t i c f o l l i c l e contrasted to large a t r e t i c f o l l i c l e , b o t h I n t h e same o v a r y .  81  27.  A t r e s i a o f a l l bound o r unspawned eggs; t h e d-stage a t r e t i c f o l l i c l e .  82  28.  A brstage a t r e t i c  follicle.  82  29.  A l a t e b-stage a t r e t i c f o l l i c l e showing broken and c o l l a p s i n g zona p e l l u c i d a .  83  A t r e t i c f o l l i c l e showing zona p e l l u c i d a r e m a i n i n g a l m o s t t o the-- l a s t .  83  A c-stage degenerate f o l l i c l e of p h a g o c y t i c e e l l s .  84  30 ; ;  31. 32.  showing I n v a s i o n  Transverse s e c t i o n through p o s t e r i o r r e g i o n o f a male g o l d f i s h p i t u i t a r y .  84  33.  Median s a g i t t a l s e c t i o n o f g o l d f i s h showing d i f f e r e n t r e g i o n s .  85.  34.  G o l d f i s h p i t u i t a r y w i t h ffiew b a s o p h i l s .  86  35.  G o l d f i s h p i t u i t a r y w i t h many b a s o p h i l s .  86  pituitary  L I S T OF TABLES Page I. II.  IIIpVI, YII.  Correlations results f o r p i t u i t a r y b a s o p h i l s and egg d i a m e t e r . Correlations  36  results f o r pituitary  b a s o p h i l s and a t r e t i c egg number.  38  Variated  70-73  f o r correlations  ( i n f o u r seasons)  Values f o r c o r r e l a t i o n equation.  74  -1-  INTRODTJ C TION S e a s o n a l changes i n the p i t u i t a r y gland were d e s c r i b e d by S c r u g g s (1939), breeder,  of t h e g o l d f i s h  The g o l d f i s h i s a  seasonal  i s i n other vertebrates the seasonal cycles i n  r e p r o d u c t i o n are p r o b a b l y c o n t r o l l e d by the p i t u i t a r y  gland.  Concomitant w i t h egg p r o d u c t i o n , the o v a r y may be e x p e c t e d t o produce g o n a d a l hormones.  However, the c y t o l o g i c a l  o f these o v a r i a n hormones has not been determined  source  although  s e v e r a l w r i t e r s have s p e c u l a t e d on the c e l l s r e s p o n s i b l e f o r hormone p r o d u c t i o n .  B r e t s c h n e i d e r & de W i t (.1941, 1947) and  de Groot (1949) attempted t o e s t a b l i s h hormone  sources(s)  p h y s i o l o g i c a l l y w h i l e s e v e r a l workers have e x t r a c t e d  estrogens  from the o v a r i e s o f t e l e o s t f i s h (Hoar, 1955). I n t h i s s t u d y , c y t o l o g i c a l and h i s t o l o g i c a l d e t a i l s o f d e v e l o p i n g and r e s o r b i n g o v a r i a n f o l l i c l e s w i l l be d e s c r i b e d and  illustrated.  P i t u i t a r y c y t o l o g y w i l l a l s o be examined.  I n t e n s e l y s t a i n i n g p i t u i t a r y b a s o p h i l c e l l s s e c r e t e gonadot r o p i n w h i c h govern s e x u a l c y c l e s ( A t z , 1953).  Correlations  were made between these p i t u i t a r y b a s o p h i l s and d i f f e r e n t stages o f b o t h d e v e l o p i n g and r e s o r b i n g f o l l i c l e s .  In this  way, i t i s hoped t o shed new l i g h t on the p i t u i t a r y - o v a r y relationship. G o n a d o t r o p i n s were a d m i n i s t e r e d or a e c e l a r a t e o v a r i a n  maturation.  i n an attempt t o i n d u c e  - 2 -  ACKNOWLEDGEMENTS S i n c e r e thanks  a r e due D r . W.S. H o a r , o f t h e D e p a r t m e n t  Of Z o o l o g y , 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 , o f t h i s problem.  f o r suggestion  and  guidance  his  g r a t i t u d e t o D a v i d P. S c o t t f o r s u g g e s t i o n s  on s t a t i s t i c a l  analysis.  t o express and a d v i c e  A s s i s t a n c e was g r a t e f u l l y  from D r . P e t e r F o r d and M r s . h i s t o l o g i c a l technique.  The a u t h o r w i s h e s  received  K a t h e r i n e Newman f o r h e l p w i t h  T h a n k s a r e a l s o due t o colleagues a n d  a l l t h o s e who k i n d l y o f f e r e d c o u n s e l a n d e n c o u r a g e m e n t d u r i n g this  study. F i n a n c i a l a s s i s t a n c e through a r e s e a r c h grant from t h e  Research  Committee o f the U n i v e r s i t y  without which gratefully  of B r i t i s h Golumbia,  t h i s work would have been i m p o s s i b l e , i s  acknowledged.  3 -  MATERIALS AND METHODS Experimental  Conditions  Three hundred g o l d f i s h ( S t o c k 1, Appendix C) were used for  t h i s study.  Three a q u a r i a , each of about 35 g a l l o n s  c a p a c i t y , c o n t a i n e d the f i s h . was  D e c h l o r i n a t e d , a e r a t e d water o  t h e r m o s t a t i c a l l y m a i n t a i n e d a t 19,4  o f not more than - 2°,  C. w i t h a f l u c t u a t i o n  No v e g e t a t i o n was  present.  Tanks  were c l e a n e d a f t e r the s h r i m p f e e d p o r t i o n o f the d i e t to prevent p r o t e i n p u t r e f a c t i o n of s u r p l u s food.  Tanks were  c l e a n e d e v e r y f i f t h day, care b e i n g t a k e n t o ensure t h a t f r e s h water be a p p r o x i m a t e l y a t the c o n d i t i o n e d  temperature:.  D i e t c o n s i s t e d of Pablum f o r f o u r c o n s e c u t i v e days and ground shrimp f e e d on the f i f t h day.  Manufacturers  o f Pablum,  Mead Johnson & Co. o f Canada L t d . , B e l l e v i l l e , O n t a r i o , g i v e t h i s composition f o r t h e i r product Carbohydrate (by d i f f e r e n c e ) Protein Moisture Fat Crude F i b r e M i n e r a l s (ash) i n c l u d i n g : Calcium Phosphorus Iron Copper Calories/gnu The  69.6$ 15.0 7.0 3,0 0,9 4.2 0.78 0.65 0.03 0.001 3.7  c o m p o s i t i o n o f the s h r i m p food i s unknown.  A close  a p p r o x i m a t i o n , however, ean p r o b a b l y be o b t a i n e d f r o m B a i l e y ' s (1942) a n a l y s i s of d r i e d canned shrimp.  H i s a n a l y s i s i s aa  - 4 -  follows:20.0$ 1.8$  Protein O i l or f a t Carbohydrate Vitamin A Vitamin B l Riboflavin N i c o t i n i c a c i d mg./lb. Vitamin D Calcium Phosphorua Iodine mg./lb. Iron Copper mg./lb.  3.5  0.10 6.0  D a i l y r a t i o n of f o o d , e i t h e r Pablum o r shrimp food was ©.15 gm. per f l a n . L i g h t was not c o n t r o l l e d .  There was i r r e g u l a r  ceiling  incandescent l i g h t i n a d d i t i o n t o n a t u r a l l i g h t from the windows. experiment extremely  F o r p a r t o f t h e time, i l l u m i n a t i o n from was continuous.  another  Thus, the houra o f l i g h t were  erratic.  Sampling Sampling  was done every other F r i d a y .  F i s h were k i l l e d  by chordotomy j u s t p o s t e r i o r t o the medulla oblongata. l e n g t h measurements were taken. c a r e f u l l y and immediately  fixed.  Fork  Gonads were d i s s e c t e d out In general r i g h t ovaries  were f i x e d and preserved i n Bouin's  fluid  (Appendix A2).  A l ) , and  l e f t o v a r i e s i n formol-sublimate  (Appendix  were f i x e d i n f o r m o l - s u b l i m a t e .  T h i s l a t t e r f i x i t i v e pene-  t r a t e s w e l l and exposure of p i t u i t a r y r e g i o n was  The heads  unnecessary.  A l l d a t a were f i l e d on 3" x 5" c a r d s . prepared f o r each p i e c e o f t i s s u e .  One c a r d was  As subsequent  treatments  were performed on t i s s u e s , the c o r r e s p o n d i n g c a r d was i n t o the a p p r o p r i a t e f i l e , v i z . ,  advanced  f i x e d m a t e r i a l s , wax b l o c k s ,  s e c t i o n s , and a l l i d e s . H i s t o l o g i c a l Methods B e f o r e w a s h i n g , heads were s t r i p p e d o f a l l e x t r a n e o u s m a t e r i a l e x c e p t f o r a s m a l l p a r t c o n t a i n i n g the p i t u i t a r y . The l o w e r jaw was removed f i r s t and t h e n the topmost p a r t o f the  cranium.  S i d e s o f the c r a n i u m , g i l l s , and s n o u t were  cut  away i n t h a t o r d e r .  The r e m a i n i n g p i e c e o f t i s s u e i s an  e l o n g a t e p i e c e o f p a r a s p h e n o i d bone, from w h i c h extend the eyes.  L y i n g i n t h i s bone, j u s t p o s t e r i o r t o the p o s t e r i o r  m a r g i n o f the eyes i s t h e p i t u i t a r y .  I n l a t e r samples,  d i s s e c t i o n was completed b e f o r e f i x a t i o n .  this  Values of t h i s  d i s s e c t i o n a r e : (a) b e t t e r and f a s t e r p e n e t r a t i o n o f the f i x i t i v e , (b) e a s i e r d e c a l c i f i c a t i o n , (c); more complete replacement o f d e h y d r a t i n g agents by antemedium and p a r a f f i n , and (d) g r e a t l y reduced number o f s l i d e s s a v i n g time i n s e c t i o n i n g , s t a i n i n g , and a n a l y s i s . F o r m o l - s u b l i m a t e f i x e d t i s s u e s were washed I n r u n n i n g water o v e r n i g h t . Bouin's f l u i d  fixed tissues required, i n  a d d i t i o n t o washing i n w a t e r , immersion i n a d i l u t e b a t h o f l i t h i u m c a r b o n a t e and ammonium h y d r o x i d e j t o i n s u r e r e m o v a l  of p i c r i c  acid.  Cowdry (1943) c l a i m s t h a t Bouin's f l u i d  acts  as a s u i t a b l e mordant.  Nevertheless, this f i x i t i v e  much s t a t i c e l e c t r i c i t y  d u r i n g s e c t i o n i n g and i t s complete  removal i s d e s i r g a b l e .  F o r some r e a s o n , s t a i n i n g i s l e s s  brilliant  causes  i n Bouin's f l u i d f i x e d t i s s u e s ( B a k e r , 1 9 4 5 ) .  This  was found t o be t h e case f o r b o t h s t a i n s used i n t h i s work. S e v e r a l methods o f d e h y d r a t i o n were u s e d .  The f i r s t  c o n s i s t e d o f a s e r i e s o f e t h y l a l c o h o l s u s i n g benzene as antemedium (Appendix A 3 ) .  T h i s method, w h i l e s i m p l e t o u s e ,  was found t o harden t i s s u e s and produce poor i n f i l t r a t i o n paraffin.  with  More s a t i s f a c t o r y i m p r e g n a t i o n i s a c c o m p l i s h e d  w i t h t e r p i n e o l (Appendix A 4 ) . T h i s t e c h n i q u e a v o i d s dehgrd-^ r a t i o n w i t h a b s o l u t e a l c o h o l b u t , a s mentioned i n Appendix A 4 , t e r p i n e o l i s removed f r o m t i s s u e s w i t h d i f f i c u l t y o f i t r e n d e r p a r a f f i n s o f t and c r u m b l y . n o t appear t o be s o f t e n e d s i g n i f i c a n t l y .  and t r a c e s  Yolky tissues d i d T h i s p r o c e d u r e was  d i s c a r d e d f o r Smith's (1940) n - b u t y l a l c o h o l s e r i e s A5).  (Appendix  T h i s g i v e s e x c e e d i n g l y good p e n e t r a t i o n o f a l c o h o l s and  p a r a f f i n , e s p e c i a l l y when u s i n g vacuum embedding (Appendix A 6 ) . For  t i s s u e s c o n t a i n i n g bone, t h e s e r i e s o f n - b u t y l a l c o h o l s  i s proceeded by a d e c a l c i f y i n g s o l u t i o n o f 20$ n i t r i c  acid  i n 70f e t h a n o l f o r t h r e e d a y s . 0  A p p a r a t u s f o r vacuum embedding i s r e m a r k a b l y s i m p l e . The vacuum I s s u p p l i e d by a w a t e r tap- o r pimp type e v a c u a t o r  7  w i t h the vacuum l i n e e x t e n d i n g i n t o the embedding oven. end  o f the l i n e i s f i t t e d w i t h a l a r g e Neoprene No.  stopper.  The  14  S e v e r a l p i n t - s i z e d "Kerr"" p r e s e r v i n g j a r s c o n t a i n  the v a r i o u s p a r a f f i n s ;  ,  To a p p l y vacuum t o j a r s , the Neoprene  s t o p p e r i s p l a c e d on top of j a r and  suction holds i t f i r m l y  in place. S e c t i o n i n g was a standard  o f 200  done r o u t i n e l y a t 10 m i c r a .  s e r i a l s e c t i o n s was  o v a r i e s were s e c t i o n e d  completely.  ribboned.  For A  ovaries, few  Heads were s e c t i o n e d  e v e r y t e n t h s e c t i o n used I n o r d e r t o r e d u c e l a b o u r and  and cost.  G o l d f i s h p l t u i t a r i e s are l a r g e enough to o b t a i n about t e n s e c t i o n s mounting o n l y one was  sectioned The  One  pituitary  t r a n s v e r s e l y t o s t u d y i t s morphology.  a d h e s i v e used f o r p l a c i n g s e c t i o n s on s l i d e s  Mayer's albumen d e s c r i b e d The  section i n ten.  by Baker ( 1 9 4 5 ) .  f i r s t s t a i n i n g method was  e o s i n (Appendix A 8 ) , haemalum (Appendix A9)  was  I t was  H a r r i s ' haematoxylin  and  s u b s e q u e n t l y found t h a t Mayer's  gave b e t t e r s t a i n i n g .  ( P e r i o d i c a c i d S c h i f f , Appendix A10)  w h i c h was  McManu3 ( 1 9 4 8 ) , P u r v e s & G r i e s b a c h (1951  The FAS  reaction  employed  by  a,b>c, 1 9 5 5 ) , and  A t z (1953) i n t h e i r s t u d i e s o f p i t u i t a r y b a s o p h i l s ,  was  chosen i n the hope o f d i s t i n g u i s h i n g between g o n a d o t r o p h i c and  thyfotrophic basophils.  G r i e s b a c h (1951  a,b,c, 1955)  I t seems l i k e l y t h a t Purves.& have a more r e l i a b l e method i n  - 8 -  u t i l i z i n g a c o m b i n a t i o n o f s t a i n s , t h e PAS and G o m o r i , w i t h photomicrography.  T h i s , however, was n o t t r i e d w i t h the  goldfish.. Ovary A n a l y s e s Por q u a n t i t a t i v e a n a l y s i s o f o v a r i e s , d i f f e r e n t i a l  counts  were made o f a l l m a t u r i n g and mature o v a i n e v e r y t e n t h t o f i f t e e n t h s e c t i o n on the s l i d e u n t i l a t o t a l o f t e n s e c t i o n s had been counted.  F o r convenience ova were d i v i d e d  into  a r b i t r a r y stages. P i t u i t a r y Analyses S a t i s f a c t o r y d i f f e r e n t i a t i o n o f gonadotrophs t r o p h s was n o t a c h i e v e d .  and t h y r o -  However, d e f i n i t e c e l l n e s t s o f  deeply s t a i n i n g p i t u i t a r y b a s o p h i l s stand out d i s t i n c t l y a g a i n s t a c i d o p h i l s and chromophobes. p h i l s appear a p a l e b l u e .  A l l c e l l s except  baso-  S i n c e i t was i m p o s s i b l e t o  d i s t i n g u i s h between gonadotroph!c  and t h y r o t r o p h i c b a s o p h i l s ,  the c o r r e l a t i o n s made here a r e based on t o t a l b a s o p h i l a r e a . The p a r a s a g i t t a l s e c t i o n n e a r e s t t o the median l i n e , n o t i n c l u d i n g much p a r s n e r v o s a , was chosen f o r a n a l y s i s . A b s o l u t e b a s o p h i l a r e a was found t o be t h e most r e l i a b l e c r i t e r i o n o f b a s o p h i l c o n t e n t because of t h e morphology o f the; p i t u i t a r y . A t e c h n i q u e was d e v i s e d f o r making p r o j e c t i o n from t h e m i c r o s c o p e onto squared t r a c i n g paper.  drawings  U s i n g the  - 9 -  a n g l e i n c l i n a t i o n draw tube, 4x o b j e c t i v e , and lOx o c u l a r , a b r i l l i a n t image was  p r o j e c t e d a d i s t a n c e o f 25" o n t o a  s h e e t of p l a t e g l a s s m a i n t a i n e d p e r p e n d i c u l a r t o the c o n i c a l projection.  When graphed t r a c i n g p a p e r was  p l a t e g l a s s , the m i c r o s c o p i c image was onto i t .  h e l d o n t o the  p r o j e c t e d and  focused  F a c i n g the p r o j e c t i o n s c r e e n , b a s o p h i l c e l l n e s t s  and p i t u i t a r y o u t l i n e s were t r a c e d d i r e c t l y onto the paper ( F i g u r e 1 . ) .  squared  A r e a s of b a s o p h i l s were d e t e r m i n e d  a p o l a r p l a n i m e t e r f o r l a r g e r n e s t s and by c o u n t i n g for smaller nests.  with  squares  By l i n k i n g adjaeent n e s t s w i t h a s i n g l e  s t r a i g h t l i n e , a r e a s of  these a d j a c e n t nests, c o u l d then be  summed as l o n g as the t r a c i n g s t y l u s f o l l o w e d back e x a c t l y on t h e c o n n e c t i n g l i n e . e q u i v a l e n t t o counted  S i n c e p l a n i m e t e r areas a r e  s q u a r e s , the p l a n i m e t e r has t o be  c a l i b r a t e d according to the Statistical  not  squares.  Analyses  To c o r r e l a t e  changes w i t h i n the p i t u i t a r y w i t h changes  i n the o v a r y , d a t a f o r the y e a r was q u a r t e r s , b e g i n n i n g i n November.  divided i n t o four  Two  a n a l y s e s were made  c o r r e l a t i n g a r e a o f p i t u i t a r y b a s o p h i l s w i t h egg d i a m e t e r the o v a r y and a r e a o f p i t u i t a r y b a s o p h i l s w i t h a t r e t i c number. for  The e q u a t i o n by C r o x t o n & Cowden (1945) was  c o r r e l a t i o n . (See page 1 1 ) .  egg  used  in  -  F i g u r e 1.  10  -  Apparatus f o r m i c r o p r o j e c t i o n of  pituitaries.  drawings  - 11 -  [NSX^ - ( S X ) ] [ N S X ^ 2  2  X  where:-  (SX^) ] 2  X^ - i s a r e a o f p i t u i t a r y b a s o p h i l s X ^ j - (2) d i a m e t e r o f eggs o r (3) a t r e t i c e g g number.  Significance  o f t h e c o r r e l a t i o n c o e f f i c i e n t was d e t e r m i n e d  by a p p l y i n g t h e t - t e s t and r e a d i n g t h e P o r p r o b a b i l i t y v a l u e t h a t an e v e n t w i l l o c c u r a c c o r d i n g t o the c o r r e l a t i o n . P ' o o 5 was chosen as s i g n i f i c a n t . #  Experimental  Work  Preparations o f e x t r a c t s are d e s c r i b e d i n Appendices B l t o B5.  A l l i n j e c t i o n s were i n t r a p e r i t o n e a l u s i n g  t u b e r c u l a r s y r i n g e s a n d No; 26 hypodermic n e e d l e s .  Injec-  t i o n s were v i a the a n a l r e g i o n i n t h e s c a l e l e s a p a r t o f the body.  No m o r t a l i t i e s were s u f f e r e d by i n j e c t i n g I n t h i s  area.  Anaesthesia  o f f i s h was found u n n e c e s s a r y f o r t h e  o p e r a t i o n was r a p i d .  - 12  -  RESULTS G e n e r a l D e s c r i p t i o n o f Ovary Embryologically,  the o v a r y o r i g i n a t e s beneath the  peritoneum, of the body c a v i t y .  I t i s thread-like at  and. l i e s i n a l o n g i t u d i n a l p e r i t o n e a l f o l d .  The  dorsal  first  ovary  e x t e n d s through most of the body c a v i t y l e n g t h a t an e a r l y age  and g r o w t h i s seen as an i n c r e a s e i n t h i c k n e s s . Gradually., t h e o v a r y assumes a s p i n d l e shape a l t h o u g h  triangular i n cross-section.  I t s c r o s s - s e c t i o n resembles a  f l a t s y m m e t r i c a l t r i a n g l e w i t h i t s apex j u t t i n g out i n t o the coelome ( F i g u r e 2 ) .  A p p r o x i m a t e l y one h a l f way  along  i t s l e n g t h , an enlargement forms where the ovary f i l l s space between t h e a n t e r i o r and swim-bladder.  The  p o s t e r i o r d i v i s i o n s of  tbe the  o v a r y i s never a b s o l u t e l y f r e e i n t h e  coelome s i n c e i t remains i n t h e pericboneal f u r r o w . o v a r i a n growth c o n t i n u e s ,  As  the p e r i t o n e a l f u r r o w i s s t r e t c h e d  t o accomodate e x p a n s i o n . P a r a l l e l t o each o v a r y and m e d i o - d o r s a l l y body.  No p a r t i c u l a r a t t e n t i o n was  paid  lies a fat  to t h i s a l t h o u g h i t s  mass appeared to be g r e a t e r d u r i n g w i n t e r and  less  during  summer. Oogenesis Oogenesis I s the p r o c e s s o f egg  o r ova f o r m a t i o n .  Young  ova o r i g i n a t e i n the g e r m i n a l e p i t h e l i u m which s u r r o u n d s the o v a r y p e r i p h e r a l l y and  extends as f o l d s i n t o the body  - 13 -  Figure  2.  Drawing of t r a n s v e r s e s e c t i o n through v i s c e r a l c a v i t y showing r e l a t i o n s o f organs. G. g o n a d s , F.B. f a t body, S.B. swim b l a d d e r , I. i n t e s t i n e , P. c o e l o m e .  - 14 -  of  the organ ( F i g u r e 15)'.  D u r i n g the p e r i o d s o f i n t e n s e  o o g e n e s i s young ova a r e formed u n i f o r m l y throughout g e r m i n a l e p i t h e l i u m . New d u r i n g egg m a t u r a t i o n .  egg f o r m a t i o n i s a t a minimum  Presumably,  a f t e r spawning t h i s  a t i v e p r o c e s s would be enhanced a g a i n . was  the  form-  Unfortunately,i t  i m p o s s i b l e t o observe t h i s because the s t o c k o f g o l d f i s h  f a i l e d t o spawn. In oogenesis, p a r t i c u l a r e p i t h e l i a l c e l l s begin to e n l a r g e and a r e known as p r i m a r y oocj/tes.  As these p r i m a r y  oocytes e n l a r g e one o f the I m m e d i a t e l y s u r r o u n d i n g g e r m i n a l e p i t h e l i a l c e l l s b e g i n s t o d i v i d e t o e n c l o s e the o o c y t e . T h i s d i v i d i n g c e l l I s d e s t i n e d t o form g r a n u l o s a .  After  f i b r o b l a s t s from, the ovary w a l l m i g r a t e t o s u r r o u n d  this,  the  g r a n u l o s a , thus c o m p l e t i n g the young f o l l i c l e s ( B r e t s c h n e i d e r & de W i t , 1947)..  A t t h i s s t a g e , ova c y t o p l a s m i s q u i t e c l e a r  w i t h s t r a n d s o f m a t e r i a l between the c e l l membrane and nucleus. 23 m i c r a .  The mean d i a m e t e r o f eggs o f t h i s type i s about The nucleus, does n o t appear t o d i f f e r  from o l d e r eggs i n i t s s t a i n i n g .  significantly  This condition i s quite  t r a n s i t o r y f o r c y t o p l a s m soon becomes b a s o p h i l i c  (basophilic  i n t h i s t h e s i s r e f e r s t o t i s s u e s h a v i n g an a f f i n t i y f o r haematin).  N u c l e i o f these youngest ova a r e homogeneous,  a c o n d i t i o n w h i c h p e r s i s t s i n a l l but most mature o v a . However* j u s t i n s i d e and s e e m i n g l y a t t a c h e d t o the n u c l e a r  - 15 -  membrane i s one g i a n t n u c l e o l u s a v e r a g i n g seven m i e r a i n diameter.  Calderwood (1891-2) g i v e s an e x c e l l e n t d e s c r i p t i o n  o f n u c l e o l a r changes.  He r e p o r t e d t h a t these g i a n t n u c l e o l i  i n youngest ova seem t o bud o f f s m a l l e r ones towards t h e n u c l e a r membrane.  Daughter n u c l e o l i . ..probably do n o t m i g r a t e  c e n t r a l l y but take t h e i r place alongside others j u s t i n s i d e the n u c l e a r membrane.  F i g u r e 16 snows t h r e e s m a l l o v a . Ovum  No. 1 has numerous s p h e r i c a l n u c l e o l i .  I n ovum No. 2, t h r e e  n u c l e o l i appear t o be m i g r a t i n g from t h e n u c l e a r membrane. However, i t seems t h a t a s m a l l space e x i s t s between the n u c l e a r membrane and t h e " s h e l l " o f n u c l e o l i .  I f this i s  c o r r e c t , t h e apparent c e n t r a l m i g r a t i o n o f n u c l e o l i c a n be e x p l a i n e d by t h e p l a n e o f s e c t i o n i n g t h r o u g h t h e egg s i n c e a s e c t i o n t a k e n o f f t h e t o p o f t h e n u c l e u s eould  leave  behind any n u c l e o l i j u s t under t h e c a p . Thus, the c e n t r a l p o s i t i o n o f these n u c l e o l i c a u l d be e x p l a i n e d n o t by c e n t r a l m i g r a t i o n , b u t by p l a n e o f s e c t i o n .  Ovum No. 3 i n F i g u r e 16  i l l u s t r a t e s n u c l e o l i i n an o l d e r ovum.  Note t h a t they  still  form a s h e l l j u s t w i t h i n t h e - n u c l e a r membrane and a r e much reduced i n s i z e .  O b v i o u s l y , l i t t l e o r no c e n t r a l m i g r a t i o n  o f daughter n u c l e o l i has o c c u r r e d i n t h e ovum. A t t h i s stage o f development, f i r s t t r a c e s o f an e x c e e d i n g l y t h i n zona p e l l u c i d a a r e e n c o u n t e r e d .  I t apparently  undergoes a t r a n s f o r m a t i o n f r o m b a s o p h i l i a t o f e u l g e n - p o s i t i v e  - 16 -  s h o r t l y b e f o r e appearance o f y o l k g r a n u l e s  (Figure 17).  Cytoplasm g r a d u a l l y becomes l e s s i n t e n s e l y b a s o p h i l i c w h i l e s t i l l remaining  homogeneous.  The f i r s t appearance o f y o l k i s i n eggs o f about 150' miera.  P r i m a r y y o l k g l o b u l e s appear i n a r i n g n e a r t h e c e l l  membrane about o n e - q u a r t e r nucleus  o f the d i s t a n c e towards the  ( F i g u r e 3 1 ) , Such g l o b u l e s a r e q u i t e c l e a r and  c o l o r l e s s a t f i r s t t u r n i n g a c i d o p h i l i c and f e u l g e n - p o s i t i v e when a secondary l a y e r o f y o l k g l o b u l e s forms. and  Secondary  t e r t i a r y l a y e r s o f y o l k form c e n t r a l l y from the p r i m a r y  layer.  A f t e r t h a t , f o r m a t i o n i s q u i t e random.  As the amount  o f y o l k i n c r e a s e s , n u c l e a r membranes seem t o d i s a p p e a r l e a v i n g behind  i l l - d e f i n e d n u c l e a r masses w i t h s c a t t e r i n g s  o f minute n u c l e o l i .  By t h i s t i m e , an egg has reached an  average s i z e o f 300-400 m i c r a . undergo a m a t u r a t i o n  From now o n , ova r a p i d l y  c h a r a c t e r i z e d by (.1) e x t e n s i v e y o l k  f o r m a t i o n accompanied by c o n s i d e r a b l e i n c r e a s e i n s i z e , (2) r e l a t i v e d e c r e a s e I n n u c l e a r s i z e , (3) pronounced t h i c k e n i n g of zona p e l l u c i d a o r oolemma, and (4) i n c r e a s e d prominence of f o l l i c u l a r  c e l l s presumably because o f t h e i r r o l e i n  b r i n g i n g n u t r i e n t s to t h e ovuia. A s t r i k i n g change t a k e s p l a c e I n the zona p e l l u c i d a during d i f f e r e n t i a t i o n .  Along with i t s t h i c k e n i n g , r a d i a l  s t r i a t i o n s appear and t h e o u t e r s u r f a c e becomes q u i t e  - 17 -  irregular.  I t r e s e m b l e s v a s t numbers o f t i n y s t i c k s w i t h  rounded ends and o f s l i g h t l y d i f f e r e n t l e n g t h , a l l s t a n d i n g t o g e t h e r s i d e by s i d e ,  (Figure 18 photographed u n d e r o i l  immersion and o b l i q u e l i g h t i n g i l l u s t r a t e s t h i s v e r y w e l l . The oolemma a t t a i n s a t h i c k n e s s o f about 9 m i e r a . V a s c u l a r i z a t i o n i n the ovary is. through the o v a r i a n a r t e r y , a b r a n c h of the d o r s a l a o r t a .  The main branches  course l e n g t h w i s e a l o n g the o v a r y s e n d i n g s m a l l e r branches i n t o the o v a r i a n stroma.  F i g u r e 21 i l l u s t r a t e s a t r a n s v e r s e  s e c t i o n of t h e branch of the o v a r i a n a r t e r y I n a collapsed state. smooth m u s c l e .  moderately  Note t h i c k n e s s of w a l l s and abundance o f Branches o f the o v a r i a n a r t e r y a r e s i n u s o i d a l  b e i n g q u i t e l a r g e and h a v i n g v e r y t h i n w a l l s .  Bigure  20  shows a l o n g i t u d i n a l s e c t i o n o f a l a r g e branch o f the ovarian artery.  C a p i l l a r i e s course t h r o u g h  Interstitial  t i s s u e s w i t h many h a v i n g t e r m i n a l branches between t h e c a e e x t e r n a and i n t e r n a . o f f o l l i c l e s . F o l l i c l e s c o n s i s t of t h r e e l a y e r s of e e l l s an ovum.  surrounding  These l a y e r s a r e t h e c a e x t e r n a , t h e c a i n t e r n a , and  zona g r a n u l o s a .  The  e p i t h e l i u m o n l y one  theca e x t e r n a resembles s i m p l e squamous c e l l thick.  and a t t a c h e d t o i t s n e i g h b o u r  Each c e l l i s disc-shaped  by i t s edge.  F i g u r e 19 i s a  s e c t i o n from t h e v e r y t o p o f a f o l l i c l e , s e c t i o n e d so t h a t the ovum i s missed  entirely.  The squamous s t r u c t u r e o f  - 18 -  theca externa c e l l s w i t h t h e i r l a r g e disc-shaped n u c l e i i s evident.  Theca i n t e r n a seems m o r p h o l o g i c a l l y i d e n t i c a l to  theca externa.  F u n c t i o n s may d i f f e r . • The zona g r a n u l o s a i s  s l i g h t l y t h i c k e r and s y n c i t i a l . F o r q u a n t i t a t i v e a n a l y s i s , an a r b i t r a r y of  ova has been drawn up.  classification  Immature ova a r e those which have  b a s o p h i l i c c y t o p l a s m c o n t a i n i n g a b s o l u t e l y no y o l k . the n u c l e u s has a membrane w i t h a t t a c h e d n u c l e o l i .  I n these Maturing  ova a r e t h o s e w h i c h have v a r y i n g amounts o f y o l k , s t i l l p o s s e s s a n u c l e a r membrane, and do n o t exceed about 500 mi era  diameter.  M a t u r e ova ( g r e a t e r than 500 mi era)  have  much y o l k , no. n u c l e a r membrane, n u c l e o l i e i t h e r a l m o s t or c o m p l e t e l y a b s e n t , and possess a prominent Histogenesis of A t r e t i c  zona p e l l u c i d a .  Follicles  A t r e s i a may o c c u r i n f o l l i c l e s o f any age and s i z e . F i g u r e 26 shows two s i m i l a r s t a g e s of a t r e t i c f o l l i c l e s d i f f e r e n t developmental s t a g e s . are r e s o r t e d s i m i l a r l y .  from  Fundamentally, a l l f o l l i c l e s  D i f f e r e n c e s i n process o f a t r e s i a  are a t t r i b u t e d n o t t o method, but t o p r o b a b l e d i f f e r e n c e s i n p h y s i c o - c h e m i c a l s t r u c t u r e o f younger and o l d e r eggs. F o l l i c u l a r a t r e s i a w i l l be d e s c r i b e d i n t h r e e c l a s s e s c o r r e s p o n d i n g t o f o l l i c l e * ; age, v i z . ,  immature, m a t u r i n g ,  and mature eggs. A t r e s i a o f Immature F o l l i c l e s In immature f o l l i c l e s , e a r l y s t a g e s o f a t r e s i a , a r e  - 19  difficult  to i d e n t i f y .  found i s i n F i g u r e 24. this f o l l i c l e .  -  The youngest d e g e n e r a t i n g No e v i d e n c e  follicle  of c y t o p l a s m e x i s t s i n  Presumably, c y t o p l a s m i s removed f i r s t .  The  zona g r a n u l o s a , a p p e a r i n g as a r i n g o f l a r g e c e l l s about the nuclear m a t e r i a l , i s heavily granulated. the f i r s t  The g r a n u l o s a shows  changes a s s o c i a t e d w i t h r e s o r p t i o n .  The  syncitial  g r a n u l o s a , which I s a l m o s t as t h i n as the thecae i n d e v e l o p ing  follicles,  begins to hypertrophy.  Shortly, granulosal  c e l l membranes a p p e a r t e r m i n a t i n g the s y n c i t i u m . d i s i n t e g r a t i o n now disappears.  begins.  Nuclear  F i n a l l y cytoplasmic granulation  Granules a r e presumably passed i n t o the  blood.  A n o t h e r example of e a r l y f o l l i c u l a r a t r e s i a i s i n F i g u r e s 22 and 23.  This f o l l i c l e  l a r g e r t h a n the one i n F i g u r e 24. mature eggs s u r r o u n d i n g i t . hypertrophy  i s s l i g h t l y o l d e r and Compare i t s s i z e t o  In this larger f o l l i c l e  extreme  o f zona g r a n u l o s a i s accompanied by v e r y i n t e n s e  granulation of granulosa c e l l s .  On one s i d e o f t h i s  f o l l i c l e , i n v a s i o n i n t o n u c l e a r substance g r a n u l o s a c e l l s has begun. A t r e s i a of M a t u r i n g  by  ova.  vary greatly.  atretic  hypertrophied  A t r e s i a w i l l soon be  complete.  Follicles  The n e x t c a t e g o r y o f a t r e t i c f o l l i c l e s i n maturing  im-  i s that occurring  I n t h i s g r o u p , amounts o f y o l k i n  cytoplasm  Where t h e r e i s l i t t l e y o l k , t h e r e i s c o n s i d e r -  able cytoplasmic s t r u c t u r e .  Where t h e r e i s much y o l k , t h e r e  - 30  i a sparse  -  cytoplasmic s t r u c t u r e *  Consider  first, follicles  which c o n t a i n l i t t l e y o l k .  r e c a p i t u l a t e , an ovum c l a s s i f i e d as b e g i n n i n g  t o mature  To  has  (1) one o r two c o n c e n t r i c l a y e r s o f y o l k g l o b u l e s , (2) much homogeneously a p p e a r i n g n u c l e a r membrane, and quickly,presumably granulosa.  b a s o p h i l i c cytoplasm,  (4) many s m a l l n a o l e o l i .  through the a c t i v i t y o f the  The r e m a i n i n g  atretic  those o c c u r r i n g w i t h immature Older "maturing" of a t r e t i c follicles  changes.  (3) d e f i n i t e  hypertrophied  s t a g e s do n o t  differ  from  present a d i f f e r e n t s e r i e s  Morphological  c h a r a c t e r i s t i c s of older  zona g r a n u l o s a a r e  (.1) t h i c k e r  zona p e l l u c i d a , (2) i n c r e a s e d y o l k d e p o s i t i o n , and d i s a p p e a r i n g n u c l e a r membrane. much t h i c k e r , , a t r e s i a o f egg  disappears  follicles.  follicles  encountered by the  Yolk  (3)  a  S i n c e the zona p e l l u c i d a i s  contents  i s delayed.  The  of a t r e s i a i s a r b i t r a r i l y d i v i d e d i n t o f o u r s t a g e s but i t must be remembered t h a t each s t a g e  cycle  (a,b,c,d),  i s a continuum  l e a d i n g i n t o the n e x t by d e g r e e s . The  a-stage of a t r e t i c  (Figure 3).  f o l l i c l e i s quite" t r a n s i t o r y  I t i s c h a r a c t e r i z e d by the f i r s t s i g n s  of  g r a n u l o s a l h y p e r t r o p h y and t e r m i n a t i o n o f i t s s y n c i t i u m . I n t h i s , g r a n u l o s a l c e l l s become l o w c u b o i d a l s e p a r a t i n g t h e c a l c e l l s from the oolemma; c o l l e c t s between the g r a n u l o s a The  Sometimes a c o l l o i d a l and  oolemma i n the  fluid  a-stage.  r u p t u r e o f the zona p e l l u c i d a marks the commencement of  -  Figure 4 .  21  -  A b-stage degenerate f o l l i c l e . Th. t h e c a l c e l l s , G . g r a n u l o s a , ZP zona p e l l u c i d a , Y y o l k , PG c e l l u l a r g r a n u l a t i o n .  - as -  the next s t a g e .  In  the a - s t a g e , h o w e v e r ,  oolemma e r o s i o n do o c c u r . oolemma i r r e g u l a r i t y .  Figure  d e f i n i t e signs  25 i l l u s t r a t e s  and c l e a r .  Granulosa n u c l e i possess d e f i n i t e  nuclei  t o l i e n e a r the  tend  quently greater  pronounced  During i n i t i a l hypertrophy there  no g r a n u l a t i o n i n t h e z o n a g r a n u l o s a , and i t  of  appears  is  vacuolar  nucleoli  and  o o l e m m a . Oolemma e r o s i o n i s  i n s i t e s of s i n g l e highly active  fre-  granulosa  cells. The b - s t a g e o f a t r e t i c  f o l l i c l e seems t o p e r s i s t  some t i m e ( F i g u r e s 4, 2 8 ) , it  p e r s i s t s u n t i l most of  A l t h o u g h t h e oolemma i s the e g g d i s a p p e a r s .  o f t e n seems t o f o r m a s t r u c t u r a l of  the r e s o r b i n g f o l l i c l e .  collapse  virtually  removed a n d t h e oolemma t o g e t h e r w i t h c y t o p l a s m i c have m a i n t a i n e d a s p h e r i c a l s h a p e . degree o f c o l l a p s e does o c c u r , follicle  i n Figure  30.  More o f t e n ,  as i n the b - s t a g e  The oolemma I s  ruptured  The oolemma  frame p r e v e n t i n g  Y o l k i s sometimes  for  support  though,  some  atretic  t h i c k and b r o k e n  in  several places. The c - s t a g e i s v a r i e d m o r p h o l o g i c a l l y D i f f e r e n c e s between the b -  changes, i . e . , the c - s t a g e  Some f o l l i c l e s I n t h e  shape w h i l e o t h e r s  6).  and c - s t a g e s a r e d i f f e r e n c e s  degree r a t h e r than d e f i n i t e smaller.  (Figure  collapse.  in  is  c-stage r e t a i n a spherical  Oolemma t r a c e s a n d a c y t o p l a s m i c  r e t i c u l u m are sometimes o b v i o u s .  In  the d - s t a g e  complete  - 23 -  F i g u r e 5.  Hypertrophied granulosa c e l l . i . y . ingested y o l k (?) i n v a c u o l e , c. cytoplasm, e.g. cytoplasmic g r a n u l e s , n. nucleus, n.c. n u c l e o l u s , z.p. zona pellucida.  Figure 6.  A c-stage degenerate f o l l i c l e . Cp c a p i l l a r y , Th t h e c a l c e l l s , Y y o l k , PG cytoplasmic g r a n u l e s .  -  -  34  c o l l a p s e of f o l l i c l e s l e a v e s o n l y a s c a r . f o l l i c l e approaching t h i s c o n d i t i o n . egration i s generally gradual. i a invaded by g r a n u l o s a  The manner o f d i s i n t -  The a t r e t i c ovum i n F i g u r e  c e l l s (and  the " c e l l p e a r l " of B r e t s c h n e i d e r granulosa  F i g u r e 30 shows a  31  thecal c e l l s ? ) resembling and  de Wit  (1947),  Invading  c e l l s are a s s o c i a t e d w i t h d i s a p p e a r a n c e of ovum and  c o l l a p s e o f the whole s t r u c t u r e l e a v i n g o n l y the  follicular  sheath. The  ovum a t t h i s s t a g e i s s i n g u l a r .  No  a r e apparent and  t h e c y t o p l a s m appears as two  amorphous mass.  On one  yolk granules types of  side i t i s acidophilic containing  giant intensely basophilic granules scattered In defined  clumps.  an  poorly-  Cytoplasm on the o t h e r s i d e o f the egg  is  definitely basophilic. The  s t r a t u m granulosum i s t y p i c a l o f i t s advanced  h y p e r t r o p h y composed o f t a l l s t r a t i f i e d  columnar c e l l s .  j a c e n t to the a c i d o p h i l i c s i d e o f the ovum, the  Ad-  granulosal  c e l l s c o n t a i n a c i d o p h i l i c heavy g r a n u l a r i n c l u s i o n s which may  be i n g e s t e d y o l k .  Granulosal  p a r t i c l e s , on the  s i d e of the ovum are b a s o p h i l i c and may c y t o s i s of adjacent b a s o p h i l i c y o l k .  represent  Nuclei of  opposite phggo-  granulosa  c e l l s i n this active condition l i e i n close apposition yolk contents.  The  to  g r a n u l o s a l c e l l s have g r e a t v a c u o l e s about 7  t e n times n u c l e a r s i z e w h i c h seem t o c o n t a i n y o l k ;  Yolk  - 25  -  seems t o by-pass the n u c l e u s to be accumulated and the p e r i p h e r a l end o f such a  of g r a n u l o s a l c e l l s .  stored  on  Figure 5 i s a sketch  cell.  Considerable  v a s c u l a r i z a t i o n i s seen t h r o u g h o u t the  f o l l i c u l a r c e l l s at t h i s  time.  The d-stage o f a t r e t i c f o l l i c l e  ( F i g u r e 27)  s c a r t i s s u e composed o f few f o l l i c l e remains and oolemma remnants.  is virtually often a  E v e n t u a l l y n o t h i n g but c o n n e c t i v e  few  tissue  remains. A t r e s i a of Mature The  Ova  l a s t category  removal of mature ova.  of d e g e n e r a t i n g  f o l l i c l e s i s that of  B r i e f l y , mature eggs have the maximum  i n oolemma t h i c k n e s s , t h e g r e a t e s t d e p o s i t i o n o f y o l k and a n u c l e u s . w i t h  granules,  no e v i d e n c e o f a n u c l e a r membrane.  Figure  27 shows a low power v i e w o f a t r e t i c mature f o l l i c l e s . o f r e s o r p t i o n are e s s e n t i a l l y  the same as those o f  Stages,  late  m a t u r i n g f o l l i c l e s except t h a t t h e l a s t s t r u c t u r e s t o be resorbed  are n o t c y t o p l a s m and  g r a n u l e s and  oolemma.  The  oolemma but r a t h e r y o l k  a t r e t i c b- and  c - s t a g e s are l i k e  l a t e m a t u r i n g f o l l i c l e s by b e i n g d i s t i n g u i s h a b l e o n l y i n s i z e . I n e x a m i n a t i o n o f a t r e t i c f o l l i c l e s , t h e r e was of theca i n t e r n a hypertrophy as Bretschneider (1947) d e s c r i b e d  I n the b i t t e r l i n g .  The  & de  no  sign  Wit  only observeablp  d i f f e r e n c e between thecae i n t e r n a and e x t e r n a l i e s i n the  - 26 -  g r e a t e r degree o f v a s c u l a r i z a t i o n i n the l a t t e r . S e a s o n a l Changes i n t h e Ovary G o l d f i s h o v a r i e s show a p e r i o d o f maximal development d u r i n g the s p r i n g and e a r l y summer ( F i g u r e 7"), greatest immaturity occurs during the f a l l . mature o v a are p r e s e n t .  The p e r i o d o f  A t a l l times i m -  D u r i n g May and J u n e , c e r t a i n immature  f i s h begin a slow process of maturation.  The l a r g e r immature  ova ( d o t s i n F i g u r e 7) b e g i n t o c o l l e c t a r i n g o f o i l d r o p l e t s j u s t w i t h i n the egg membrane ( t r i a n g l e s i n F i g u r e 55), Through-* out the summer, t h e r e i s p r a c t i c a l l y no change i n these eggs. They remain i n t h i s a l m o s t dormant s t a t e u n t i l September when the m a t u r a t i o n p r o c e s s b e g i n s t o a c c e l e r a t e . The e a r l y m a t u r i n g ova ( t r i a n g l e s i n F i g u r e 7) g r a d u a l l y i n c r e a s e i n d i a m e t e r f r o m a mean o f a p p r o x i m a t e l y 200 t o 300 micra.  I n c r e a s e i n y o l k , I f any, i s v e r y s l i g h t  the p e r i o d from May t o O c t o b e r .  However, f r o m  throughout  October,through  January, i n d i v i d u a l f i s h begin a period of r a p i d formation of y o l k i n ova.  T h i s i s r e p r e s e n t e d by a change from  t o squares I n F i g u r e 7. p r o c e s s a t t h e same t i m e .  triangles  A l l f i s h do n o t b e g i n t h i s m a t u r a t i o n The d e v e l o p m e n t a l r a t e a t t h i s , time  i s more o r l e s s u n i f o r m and r e t a r d s a s m a t u r a t i o n n e a r s comp l e t i o n (change from squares t o c i r c l e s ) .  The end o f t h i s  r a p i d y o l k f o r m a t i o n o c c u r s sometime between J a n u a r y and June. The maximum range o f t h i s r a p i d development i s from  October  ' 12 26 10 24 7 21 4 18 4 18 15 28 14 27 10 23 25 5 19 2—17 30 6  I  NOV  F i g u r e 7.  DEC  JAN  FEB  MAR  APR  MAY  JUN  JUL  AUG  SEPT  Graph showing development o f ova throughout the y e a r .  OCT  -  28  t o . J u n e , a p e r i o d o f seven months.  The time r e q u i r e d f o r any  p a r t i c u l a r f i s h t o pass t h r o u g h t h i s s t a g e i s f o u r t o f i v e months, as observed by the s l o p e o f the band between t h e s i g m o i d c u r v e s on the g r a p h ,  i f t h e r a t e o f m a t u r a t i o n were  u n i f o r m and s i m u l t a n e o u s , d i s t r i b u t i o n o f p o i n t s r e p r e s e n t i n g egg d i a m e t e r would d e s c r i b e a narrow band.  T h e r e f o r e , because  t h i s i s a wide band, and the range o f the p e r i o d o f m a t u r a t i o n q u i t e extended, time when any g o l d f i s h reaches m a t u r i t y must vary g r e a t l y .  As a consequence, range f o r spawning must be  e x t e n s i v e , d e p e n d e n i t upon the time a t which such ova mature. The f i n a l s t a g e of ova m a t u r a t i o n i s not so much a p r o c e s s o f i n c r e a s e i n egg s i z e , as i t i s a p r o c e s s o f r i p e n i n g ( c i r c l e s i n F i g u r e 7).  R i p e n i n g i n v o l v e s changes i n  the n u c l e u s and y o l k . N o r m a l l y , a f t e r g o l d f i s h spawn, mean egg d i a m e t e r  of  the most mature ova l e f t i n the o v a r y i s much l e s s t h a n t h a t o f spawned eggs.  I t i s o b v i o u s , t h e n , t h a t an a b r u p t break i n  the graph would o c c u r a t t h e end o f the spawning p e r i o d . p o p u l a t i o n o f g o l d f i s h d i d not spawn and break i n t h e g r a p h d i d not  This  c o n s e q u e n t l y the  appear.  A t r e t i c f o l l i c l e s a r e found the y e a r f o u n d , and d e v e l o p from ova of any age or s i z e .  may  The number o f a t r e t i c  f o l l i c l e s shows a p e r i o d o f i n c r e a s e s t a r t i n g i n A p r i l ( F i g u r e 8).  U n t i l the m i d d l e o f O c t o b e r , t h e number of t h e s e  follicles  NOV-JAN  FEB-APR  MAY-JUL  AUG-OCT  Figure 8 . Graph comparing atretic egg number and increase in basophils throughout the year. Key:basophils; atretic egg number.  - 30 -  c o n t i n u e d t o surge g i v i n g no i n d i c a t i o n o f r e d u c t i o n .  As Jlshe  number o f f o l l i c l e s i n c r e a s e d , t h e y were from l a r g e r and more mature ova and. the a t r e t i c p r o c e s s of t h e s e l a r g e r would presumably be l o n g e r .  follicles  I t must be remembered t h a t these  g o l d f i s h d i d not spawn and i n c r e a s e d numbers of r e s o r b i n g f o l l i c l e s aire I n e v i t a b l e .  D e g e n e r a t i n g f o l l i c l e s were found t o  i n c r e a s e m a r k e d l y i n number and s i z e d u r i n g the time when g o l d f i s h n o r m a l l y spawn and a f t e r the normal spawning the a t r e t i c f o l l i c l e s number Soared ( F i g u r e 8 ) .  period,  Thus, the  more the d e g e n e r a t i n g f o l l i c l e s , the l a r g e r they were and l a r g e r ova.  from  D e g e n e r a t i n g f o l l i c l e s , t h e n , a r e m a i n l y from  unspawned eggs.  These f i n d i n g s n e i t h e r deny tfor s u p p o r t  endocrine f u n c t i o n of these s t r u c t u r e s . C o r r e l a t i o n s between P i t u i t a r y and  Ovary  The a s s o c i a t i o n between the p i t u i t a r y and o v a r y i s i n d i c a t e d by c o r r e l a t i n g changes i n the g o l d f i s h . was  Egg d i a m e t e r  chosen as a c r i t e r i o n f o r m a t u r a t i o n and a r e a o f PAS  p o s i t i v e c e l l s as a b a s i s for- g o n a d o t r o p h i c o u t p u t from the t r a n s i t i o n a l lobe o f the p i t u i t a r y ( F i g u r e s 32-35). two f a c t o r s were c o r r e l a t e d seasohs  ( F i g u r e s 9-13)  These  (Appendix D, and Table I ) i n f o u r  throughout the y e a r .  I n each .  season, t h e r e i s a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between i n c r e a s e i n amount o f p i t u i t a r y b a s o p h i l s and i n c r e a s e i n egg diameter.  There i s s t r o n g s u g g e s t i o n t h a t the p i t u i t a r y  Figure 9 . Graph comparing ova growth a n d i n c r e a s e i n b a s o p h i l s throughout the y e a r . Key: basophilsj egg diameter.  >  • •  . • •  • •  o  • •  A.  * 100  300  • 300 Egg  F i g u r e 10;  *  40 0  500  500  700  800  900  Diameter i n m i c r o n s  C o r r e l a t i o n s between p i t u i t a i - i e c b a s o p h i l s and egg diaiaet November - January  I  I  I  I  I  I  f  I  o  o  A)  o  3  o  J4  O  o o  a  •  100  A  800  300  Eg£  Figure  11.  Correlations  400  500  Diameter  600  700  SOO  90G  i n microns  between p i t u i t a r y b a s o p h i l s February - A p r i l  and  egg  diameter.  3  to o  •  rH  M  CD  3  8  o  n 5!  1  •  100 •  -.1  l i g u r a 18.  BOO  300  400  500  600  700  800  900  E g g D i a m e t e r - i n microns  C o r r e l a t i o n s between basophils, and egg d i a m e t e r . • • May - J u l y  100  £00  300 Egg  F i e t i r e 13.  Correlation*  400  500  600  700  800  900  Diameter i n m i c r o n s  between p i t u i t a r y August  -  basophils  October  ana egg  diameter,  Table I . Season  C o r r e l a t i o n s R e s u l t s f o r P i t u i t a r y B a s o p h i l s and Egg D i a m e t e r . Mean B a s o p h i l A r e a i n mm *L 2  Mean Egg Diameter *2  r  t  P  N.  Nov-^Jan  1064.4  307.4  +.4755  2.535  0.02  24  Feb-Apr  1567.3  358.8 .  +.5622  3.248  0.001  30  May-Jul  1899.0  360.0  +.8320  5.996  0.001  18  Aug-Oet  1648.0  400.7  +.5744  3.656  0.001  29  E E  < UJ < O.  o  <  a  to  to  o  to  to  NUMBERS OF ATRETIC EOOS NOV•JAN  FEB  APR  MAY J U L  AUG  OCT  F i g u r e 14. Graph showing d i s t r i b u t i o n o f j o i n t s r e l a t i n g p i t u i t a r y basophils* and a t r e t i c egg number. R e g r e s s i o n l i n e s a r e drawn through p o i n t s .  Table I I . C o r r e l a t i o n s R e s u l t s f o r P i t u i t a r y B a s o p h i l s and A t r e t i c Egg Number.  Season  Mean B a s o p h i l A r e a i n mm 2  Mean A t r e t i c Egg Number *3  r  t  P  N  Nov-Jan  1064.4  0.59  +.4442  2.324  0.02  24  Feb-Apr  1567.3  0.63  -.7708  6.408  0.001  30  May-Jul  1899.0  6.97  -.2184  0.895  0.4  18  Aug-Oct  1648.0  14.54  +.0634  0.331  0.75  29  -  39  -  b a s o p h i l s do govern m a t u r i t y o f the o v a r y i n g o l d f i s h ; S i n c e the p i t u i t a r y i s r e s p o n s i b l e f o r many r e g u l a t o r y functions i n governing  s e x u a l f a c t o r s , i t was d e c i d e d t o  c o r r e l a t e a t r e t i c egg numbers w i t h p i t u i t a r y b a s o p h i l s . A graph was drawn f o r the p a i r e d v a r i a t e s f o r the f u l l year i n f o u r seasons ( F i g u r e 1 4 ) . T h i s shows a g r o u p i n g  around t h e  v e r t i c a l axes from November t o A p r i l and a random s c a t t e r from May to O c t o b e r .  A p p a r e n t l y p i t u i t a r y b a s o p h i l s do n o t c o n t r o l  a t r e t i c egg number.  To support  t h i s hypothesis  statistically,  c o r r e l a t i o n s were determined i n f o u r s e a s o n s , the same as used for  c o r r e l a t i n g egg d i a m e t e r and b a s o p h i l s (Table I I ) . C o r -  r e l a t i o n s f o r the t h i r d and f o u r t h q u a r t e r s when a t r e t i c egg number i a i n c r e a s i n g , i . e . M a y - J u l y and A u g u s t - O c t o b e r , a r e b o t h low s u g g e s t i n g  e i t h e r no o r n e u t r a l p i t u i t a r y i n f l u e n c e .  But on the o t h e r hand, the c o r r e l a t i o n f o r t h e f i r s t  quarter  i s + .44 and f o r t h e second q u a r t e r i s -.77, b o t h which a r e s i g n i f i c a n t and o p p o s i t e i n s i g n .  During  the f i r s t  quarter,  there i s a v e r y low p r o b a b i l i t y that increase i n p i t u i t a r y b a s o p h i l s i s n o t a s s o c i a t e d w i t h an i n c r e a s e i n a t r e t i c egg number ( N u l l h y p o t h e s i s and p o s i t i v e c o r r e l a t i o n ) .  During the  second q u a r t e r , when the c o r r e l a t i o n c o e f f i c i e n t i s n e g a t i v e r a t h e r than p o s i t i v e , t h e r e i s a l o w p r o b a b i l i t y t h a t i n c r e a s e i n p i t u i t a r y basophils i s not associated with suppression i n a t r e t i c egg number.  Hence, d u r i n g t h e f i r s t and second  quarter,  - 40 -  the p r o b a b i l i t y t h a t the p i t u i t a r y b a s o p h i l s b o t h do and do not s t i m u l a i e a t r e t i c egg number i s l o w , and i f t h e r e i s an effect,  i t i s o p p o s i t e f o r the two s e a s o n s .  egg number shows l i t t l e  However, a t r e t i c  change throughout t h i s p e r i o d when  basophils are s t e a d i l y i n c r e a s i n g ; I n F i g u r e 3 two r e l a t i o n s h i p s a r e p l o t t e d .  Number o f  a t r e t i c eggs are low f o r most o f t h e y e a r and show a p r o nounced r i s e d u r i n g the l a s t quarter..  The number o f  b a s o p h i l s r i s e s t e a d i l y t o the end o f t h e t h i r d q u a r t e r and b e g i n to f a l l o f f .  F i g u r e 14 shows r e g r e s s i o n l i n e s  r e p r e s e n t i n g a g r e a t s c a t t e r d u r i n g these l a s t two s e a s o n s . O b v i o u s l y p i t u i t a r y b a s o p h i l s a r e independent o f a t r e t i c egg number d u r i n g summer and autumn. Throughout t h e t h i r d and f o u r t h seasons, t h e r e seems t o be no p i t u i t a r y b a s o p h i l i n f l u e n c e on a t r e t i c egg number. However, d u r i n g t h e season f r o m F e b r u a r y  to A p r i l ,  there i s  a p p a r e n t l y some f a c t o r c a u s i n g s u p p r e s s i o n o f a t r e t i c  eggs.  T h i s s u p p r e s s i o n i s absent f o r the r e s t o f the y e a r ;  On the  o t h e r hand, sudden r e v e r s a l o c c u r s from t h e f i r s t s e a s o n when a t r e t i c egg p r o d u c t i o n i s enhanced.  Possible explanations  of these c o r r e l a t i o n s are that l e s s p i t u i t a r y  gonadotrophin  i s produced a t t h a t t i m e , o r t h e c o r r e l a t i o n i s s p u r i o u s . Hormones i n f l u e n c i n g a t r e t i c egg number, o f c o u r s e , do n o t c o n f i r m n o r deny the p o s s i b i l i t y t h a t a t r e t i c eggs may  - 41 -  themselves produce hormones. R e s u l t s from I n j e c t i o n E x p e r i m e n t s G o l d f i s h were i n j e c t e d w i t h s a l i n e e x t r a c t o f salmon p i t u i t a r y e v e r y two days f o r t w e l v e days. a r e known t o c o n t a i n  oestrogens.  f i s h showed l i t t l e movement,  The p i t u l t a r l e s  Just a f t e r i n j e c t i o n s  f e e d i n g was not p r e v e n t e d .  The f o l l o w i n g t a b l e shows r e s u l t s of o v a r y a n a l y s i s . Table I I I R e s u l t s tram. I n j e c t i o n s o f Salmon Pituitaries.  Average V a l u e s f o r Three  Fish  Immature Eggs  Mature Eggs  Atretic Eggs;  R a t i o Mature t o Immature Eggs.  3-day f i s h  1165  48  0  0.024  l2-*lay f i s h  957  46  0  0.021  There appears t o be no s i g n i f i c a n t d i f f e r e n c e r a t i o s o f the 3-day f i s h and  between t h e s e  the 12-day f i s h .  S e v e r a l g o l d f i s h were i n j e c t e d w i t h P r e g n a n t ^ a r e s * Serum (PMS).  H i s t o l o g i c a l e x a m i n a t i o n was not done.  i n j e c t i o n b e h a v i o u r was as f o l l o w s : -  Post-  F i s h showed v i o l e n t  b l i n d d a r t i n g movements, even jumping out o f the w a t e r , then sudden q u i e t p e r i o d s .  This a c t i v i t y  c y c l e was  repeated  s e v e r a l t i m e s , and was not a p p a r e n t the f o l l o w i n g  day.  - 42 -  LITERATURE REVIEW Numerous i n v e s t i g a t o r s have g i v e n e x c e l l e n t c l a s s i c a l descriptions of ovarian s t r u c t u r e .  Walace (1903) d e s c r i b e d  c y t o l o g y o f ova i n e l a s m o b r a n c h s , S t r o m a t e n (1931) ova i n g o l d f i s h , and Calderwood (1891) ova I n P l e u r o n e c t i d a e . Seasonal  changes i n t h e o v a r y were d e s c r i b e d I n  Gasterosteus  a c u l e a t u s ( C r a i g - B e n n e t t , 1 9 3 0 ) , and i n C o t t u s b a i r d i  (Hann,  1927). In  immature ova, n u c l e o l i a r e v e r y prominent b e i n g more  numerous I n the l a r g e r immature ova.  The  pjmpose i n m u l t i -  p l i c a t i o n o f n u c l e o l i i s unknown j u s t as i s t h e i r f u n c t i o n . S c h a r f f (1887)- does not c o n s i d e r them i m p o r t a n t m o r p h o l o g i c a l l y but Calderwood (1891) b e l i e v e s them h i g h l y i m p o r t a n t b e i n g "the f o u n t a i n - h e a d  o f the e n t i r e system."'  B o t h of  these c y t o l o g i s t s a r e e x t r e m i s t s . Mature ova have much t h i c k e n e d egg membranes.  Wallace  (1903) d e s c r i b e s t h r e e t y p e s of ova based on egg membranes. G e n e r a l l y , the egg membranes a r e b e l i e v e d to be s e c r e t e d f r o m t h e egg a l t h o u g h some m a i n t a i n t h a t i t i s a s e c r e t i o n ( W a l l a c e , 1903).  follicular  D i f f e r e n t i n v e s t i g a t o r s have  c l a i m e d up t o t h r e e s e p a r a t e egg membranes (Cunningham, 1886). Some have d e s c r i b e d o n l y one membrane wl t h a h o s t o f names i n c l u d i n g Zona r a d i a t a , v i t e l l i n e oolemma, zona p e l l u c i d a , and  membrane,  egg-capsule,  e g g - s h e l l ( S c h a r f f , 1887).  The  - 43 -  t h i c k rough s u r f a c e o f t h e zona p e l l u c i d a has been c a l l e d o c c a s i o n a l l y a t h i r d l a y e r ( K u p f f e r i n S c h a r f f , 1887). The l i t e r a t u r e on c o r p o r a l u t e a and c o r p o r a a t r e t l c a i s becoming r a p i d l y v o l u m i n o u s .  I n f i s h and the r e s t o f the  v e r t e b r a t e s e r i e s e x c l u d i n g mammals, t h e r e i s l i t t l e o r no agreement on naming o f c e r t a i n c e l l u l a r masses I n o v a r i e s for  some c a l l them c o r p o r a l u t e a , some c o r p o r a a t r e t i c a , and  some by b o t h names. The corpus luteum was d i s c o v e r e d i n the mammal by V a l c h e r i u s C o i t e r (1573) and named by M a l p i g h i i n 1686 ( r e f . Leonardo).  B o r n and h i s p u p i l Loeb (1906) d i s c o v e r e d the  e n d o c r i n e f u n c t i o n o f t h e organ i n the g u i n e a p i g .  Harrison  (1948) d e f i n e s corpus luteum as:-* "a term a p p l i e d t o the e n d o c r i n e g l a n d w h i c h develops i n the ruptured ovarian f o l l i c l e a f t e r m a t u r a t i o n and d i s c h a r g e o f t h e ovum. The corpus luteum i s so c a l l e d because o f the y e l l o w c o l o r which t h e g l a n d e x h i b i t s i n i t s f r e s h s t a t e I n some mammals...." However, he r e c o g n i z e s a p o s s i b l e d i s t i n c t i o n I n these c e l l u l a r masses between v i v i p a r o u s and o v i p a r o u s f i s h e s . H a i l e y (1933) i s s k e p t i c a l i n the use o f t h e term corpus l u t e u m a l s o f o r be r e p o r t s t h a t : "the s p e n t f o l l i c l e o f t h e t e l e o s t s i s a t t i m e s compared w i t h the corpus l u t e u m o f t h e mammalia and i s even s o c a l l e d by some a u t h o r s . I t i s customary t o l i m i t t h e t e r m t o s u c h o v u l a t e d f o l l i c l e s as contain that p a r t i c u l a r s o r t o f  -v44  -  i r r e g u l a r s e c r e t o r y c e l l s known as l u t e i n  cells".  R a i l e y g i v e s the example t h a t : "...XIphophorus agrees w i t h o t h e r t e l e o s t s i n t h a t i t s s p e n t f o l l i c l e s cannot p r o p e r l y he termed corpora l u t e a " . I t would appear t h a t t h e term " c o r p o r a l u t e a " i s j u s t ified if  (1) f o r p o s t - o v u l a t o r y f o l l i c l e s w i t h i n v i v i p a r o u s f i s h  these f o l l i c l e s produce hormones, (2) f o r p o s t - o v u l a t o r y  f o l l i c l e s where a subsequent p a r e n t a l c a r e i s demanded on p a r t o f t h e female assuming- p a r e n t a l c a r e I s governed  the  by  hormonal p r o d u c t i o n from these b o d i e s , and p o s s i b l y (3). f o r s u c h p o s t - o v u l a t o r y s t r u c t u r e s i n o v i p a r o u s f i s h e s , i f they p e r s i s t f o r some time a f t e r o v u l a t i o n w i t h hormonal f u n c t i o n . A l l o t h e r s t r u c t u r e s (and t h a t l e a v e s o n l y p r e - o v u l a t o r y c e l l u l a r masses) d e r i v e d f r o m eggs s h a l l be c a l l e d  corpora  atretica i n this thesis; I t I s p o s s i b l e that corpora a t r e t i c a (pre-ovulatory degenerate masses) have e n d o c r i n e f u n c t i o n s .  Craig-Bennett  (1930) d e s c r i b e d f o r G a s t e r o s t e u s a c u l e a t u a . p o s t - o v u l a t o r y c o r p o r a l u t e a which were " s h o r t - l i v e d " .  He i m p l i e d from t h i s  that endocrine f u n c t i o n i s b r i e f or l a c k i n g w i t h p o s t ovulatory corpora l u t e a .  B r e t s c h n e i d e r & de Wit  (1947)  describe a corpora a t r e t i c a I n which there i s t h e c a l hypert r o p h y and n o t g r a n u l o s a l h y p e r t r o p h y . not found I n g o l d f i s h .  Such s t r u c t u r e s a r e  However, B r e t s c h n e i d e r &. de Wit  (1947)  - 45 -  d e s c r i b e d a p r e - o v u l a t o r y corpus luteum t h a t has s i m i l a r c y t o l o g y t o the c o r p o r a a t r e t i c a o f t h i s t h e s i s , v i z . , p r e ovulatory s t r u c t u r e s w i t h g r a n u l o s a l hypertrophy.  These  i n v e s t i g a t o r s m a i n t a i n t h a t c o r p o r a l u t e a and c o r p o r a  atretica  are endocrine s t r u c t u r e s having hypertrophied s e c r e t o r y g l a n d u l a r t i s s u e , t h e g r a n u l o s a and t h e c a I n t e r n a r e s p e c t i v e l y . T h e i r c r i t e r i a f o r e n d o c r i n e l i b e r a t i o n I s o v i p o s i t o r growth s t i m u l a t e d by o v l d u c t i n , t h e " c o r p o r a l u t e a hormone". B r e t s c h n e i d e r and de W i t (1947) f a i l e d  What  t o do was i s o l a t e  these s u p p o s e d l y e n d o c r i n e g l a n d s f r o m f i s h and cause o v i p o s i t o r g r o w t h , and o t h e r more s p e c i f i c r e a c t i o n s by i n j e c t i o n s o f e x t r a c t s o f these ^ g l a n d s " . a v o i d the e n d o c r i n e i s s u e .  Most i n v e s t i g a t o r s seem t o  Craig-Bennett  & Smart ( 1 9 3 4 ) , S t o l k (1951)  ( 1 9 3 0 ) , Cunningham  d e s c r i b e corpora l u t e a but  avoid d i s c u s s i n g endocrine production.  Dodd (1955) warns  a g a i n s t u s i n g the name c o r p o r a l u t e a u n l e s s p r o v e n homologous t o the mammalian s t r u c t u r e .  Wallace  (1903) p r e f e r s t o use  the term a b o r t i v e f o l l i c l e , an e x c e l l e n t name f o r f o l l i c l e s which f a i l e d t o r e a c h m a t u r i t y .  S i n c e w o r k i n g on g o l d f i s h ,  the b e s t c o n c l u s i o n t h a t can be made a t the p r e s e n t time i s t h a t g r e a t c a u t i o n s h o u l d be e x e r c i s e d i n assuming t h e f i s h corpora a t r e t i c a are endocrine  bodies.  46  -  DISCUSSION The O v a r i a n The  Follicle;  f o l l i c l e i s the f u n c t i o n a l u n i t i n the o v a r y .  i n t e g r i t y i s e s s e n t i a l I n perpetuating the s p e c i e s .  Its  Maximum;  egg s i z e seems to he governed by i n h e r e n t mechanisms because a l l mature eggs a r e a p p r o x i m a t e l y the same s i z e . egg number i s h i g h l y v a r i a b l e .  However,,  A few o v a r i e s c o n t a i n e d  a few mature ova w h i l e o t h e r s c o n t a i n e d more t h a n one I t i s b e l i e v e d that, t h e n u t r i t i o n a l s t a t e o f the  hundred.  individual  governs the number o f eggs t h a t mature ( S c o t t , 1 9 5 6 ) . there i s i n s u f f i c i e n t or inadequate  only  food a v a i l a b l e ,  If  egg  number p r o b a b l y would be l i m i t e d e i t h e r by r e s t r i c t i n g  the  numbers o f eggs b e g i n n i n g t o mature and/or by r e s o r b i n g e x t r a f o l l i c l e s already forming.  In a d d i t i o n , f i s h size obviously  r e s t r i c t s egg- number. The problem o f n u c l e a r d i s i n t e g r a t i o n on  approaching  m a t u r a t i o n has s t i m u l a t e d the; q u e s t i o n c o n c e r n i n g i t s n e c e s s i t y i n governing i t s c e l l u l a r f u n c t i o n s .  Although  the  n u c l e a r membrane d i s a p p e a r s and n u c l e o p l a s m  disperses, i t i s  not b e l i e v e d that n u c l e a r c o n t r o l i s l o s t .  C o n t r o l by  n u c l e u s m i g h t be b e t t e r when n u c l e o p l a s m  the  Is dispersed.  Perhaps  break-up and subsequent a g g r e g a t i o n of t h e n u c l e u s i s a s t e p i n i t s m i g r a t i o n from a c e n t r a l p o s i t i o n t o a p e r i p h e r a l p o s i t i o n , thus a v o i d i n g g r e a t m e c h a n i c a l  difficulties.  At  the  - 47 -  p r e s e n t t i m e e x p l a n a t i o n o f n u c l e a r break-up i s h y p o t h e t i c a l . The o r i g i n o f y o l k i s unknown. appear de novo i n c y t o p l a s m .  O i l g l o b u l e s seem t o  The g r a n u l o s a p r o b a b l y s e c r e t e s  y o l k y n u t r i e n t s i n t o the egg s i n c e i t i s a c o n t i n u o u s l a y e r completely surrounding i t .  Y o l k a p p a r e n t l y condenses out o f  the c y t o p l a s m much l i k e a c o a l e s c i n g e m u l s i o n .  Early yolk i s  c l e a r and c o l o r l e s s and, t h e r e f o r e , i s l i k e l y composed o f o i l s h a v i n g l o w m o l e c u l a r w e i g h t f a t t y acid's. y o l k seems t o change from a t h i n l i q u i d state.  T h i s may  As ova mature  to a much h a r d e r  be due t o imbibed p r o t e i n o r i n c r e a s e i n  s i z e o f the f a t t y a c i d p a r t i o n o f l i p i d m o l e c u l e s .  Either  way would i n c r e a s e v i s c o s i t y of y o l k . I t i s unknown how l o n g i t takes for. a p r i m a r y o o c y t e to d e v e l o p i n t o l a r g e "Immature ova"" t h a t have homogeneous b a s o p h i l i c cytoplasm.  From the time when y o l k f i r s t b e g i n s  to form u n t i l t h e time when eggs a r e r e a d y t o spawn, about 12-14 months have e l a p s e d r a n g i n g from May May  o f one y e a r t o  t o J u l y of the f o l l o w i n g y e a r ( F i g u r e 7 ) .  Thus, i n  m a t u r i n g g o l d f i s h , t h e r e l a an o v e r l a p o f s t g g e s o f m a t u r i n g eggs, v i z . , (1) o o g e n e s i s from g e r m i n a l e p i t h e l i u m , (2) eggs w i t h p r i m a r y y o l k f o r m a t i o n w h i c h w i l l not be r i p e u n t i l the f o l l o w i n g y e a r , and  (3) mature eggs ready to be spawned.  It  seems, t h e r e f o r e , t h a t the e n t i r e o o g e n i c p r o c e s s f r o m p r i m a r y oocytes t o mature o o c y t e s t a k e s something l i k e two y e a r s ,  -  48  about twelve months o f t h i s f o r y o l k d e p o s i t i o n . The A t r e t i c  Follicle  One o f the f u n c t i o n s o f the s t r a t u m granulosum I n g o l d f i s h i s the removal o f s u r p l u s eggs.  I f f o r any o f many  r e a s o n s , eggs become s u r p l u s , they become a t r e t i c , o r as W a l l a c e  (1903) s a i d , a b o r t i v e .  degenerate,  Such causes o f degener-  a t i o n may be (1) i n s u f f i c i e n t nourishment t o b r i n g a l l f o l l i c l e s t o m a t u r i t y , (2) malformed f o l l i c l e s , o r (3) i n a b i l i t y t o spawn because o f l a c k o f the a p p r o p r i a t e s t i m u l u s light, etc.  temperature,  I n a b i l i t y t o spawn causes a l l mature and m a t u r i n g  ova t o become a t r e t i c .  These a t r e t i c f o l l i c l e s  probably  p e r s i s t f o r some time a f t e r t h e normal spawning p e r i o d ( F i g u r e 27).  B a r f u r t h ( i n W a l l a c e , 1903) r e p o r t s t h a t t h e " e n t i r e  c r o p " o f eggs may become a b o r t i v e i n t h e t r o u t . F o l l i c u l a r d e g e n e r a t i o n has p r e s e n t e d a n I n t e r e s t i n g question.  Degeneratioh  granulosal c e l l s .  i s brought about by a c t i o n o f t h e  H i s t o l o g i c a l l y , these c e l l s f i r s t  becoming vacuolated, and: t h e n show many d a r k inclusions.  enlarge  cytoplasmic  Oolemma, y o l k and o t h e r c y t o p l a s m i c elements a r e  broken down and make t h e i r way i n t o the h y p e r t r o p h y l n g granulosal c e l l s .  L i t e r a t u r e on t h i s r e g u l a r l y uses the  word " p h a g o c y t o s i s " i m p l y i n g amoeboid movements w h i c h s u r r o u n d , i n g e s t , o r i n c l u d e p a r t i c l e s l i k e macrophagic action.  T h i s may be w h o l l y o r p a r t i a l l y t r u e .  I t was  -  suggested to  49  -  by H o a r t h a t t h i s d i g e s t i v e p r o c e s s m i g h t  enzyme a c t i o n .  Enzymes may  the g r a n u l o s a d i s s o l v i n g egg t h e n be a b s o r b e d  be p r o d u c e d  elements.  and  R e c o n s t i t u t i o n may  by  and  28),  i n t e r e s t r a t h e r than o f p r a c t i c a l Importance  that  question  to determine.  s i z e and  also larger f o l l i c l e s  persists  t h a t the p r o c e s s i s b r i e f , a b o u t 2-|—4 m o n t h s , and  The  a t r e t i c process l i k e l y  l a r g e f o l l i c l e s and  This, i s because  a-stage  c h a n g e s and b - s t a g e and  the d - s t a g e  No p r e c i s e m e a s u r e c a n be made.  for  The  over a v e r y b r i e f p e r i o d .  the s t r u c t u r e suggests s l i g h t  complete  this  have more m a t u r e  t h a t a r e p r o b a b l y h a r d e r t o remove.  to  I t i s of  Very s m a l l f o l l i c l e s o b v i o u s l y  d i s a p p e a r more r a p i d l y t h a n l a r g e r f o l l i c l e s .  of  appearance  d u r a t i o n o f t h e a t r e t i c p r o c e s s i s u n c e r t a i n and  difficult  follicle  interior.  arises.  The  of  would  to t h e g r a n u l o s a c e l l  ( F i g g u r e s 23, 24,  by  further  then occur t o g i v e t h e g r a n u l a r  t y p i c a l of these c e l l s  due  liberated  This digest  e i t h e r by d i f f u s i o n a n d / o r  chemical transformation across  be  The  elements  of degenerating definition  i t is. expected c-stage l a s t  up  considerably longer.  H o w e v e r , a s an e s t i m a t e , t h e t a k e s more t h a n s i x m o n t h s  p o s s i b l y as s h o r t as  two m o n t h s f o r  the s m a l l e s t f o l l i c l e s , R e s o r p t i v e p r o c e s s e s g e n e r a l l y remove s o f t e s t first.  Once t h e oolemma i s b r o k e n , i t seems t o be  materials spared'and  - 50 -  f r e q u e n t l y remains as one o f the l a s t elements to be removed. from the a t r e t i c f o l l i c l e .  Y o l k from mature f o l l i c l e s  seems  v e r y g r a n u l a r and tends t o escape r e s o r p t i o n u n t i l the l a s t . T h e r e f o r e , I n mature f o l l i c l e s ,  a d e f i n i t e c y c l e occurs, i n  the breakdown events. J u n c t i o n o f a t r e t i c eggs has not been d i s c o v e r e d except f o r removing unspawned eggs.  To determine p o s s i b l e endocrine  s e c r e t i o n , c a r e f u l l y c o n t r o l l e d experiments sBaould be r u a , e.g. hypophysectomy to determine i f c o r p o r a a t r e t i c a  will  i n c r e a s e o r decrease i n number ot s i z e , followed by hormone i n j e c t i o n s to c o u n t e r a c t p i t u i t a r y a b l a t i o n .  Extirpation of  c o r p o r a a t r e t i c a and e x t r a c t s made from them could be used f o r i n j e c t i o n i n t o hypophysectomized effect.  f i s h t o observe the o  I t Is p o s s i b l e t h a t environmental and i n t e r n a l  c o n t r o l numbers of c o r p o r a a t r e t i c a .  factors  Function of a t r e t i c  eggs can be determined o n l y by c a r e f u l l y c o n t r o l l e d  physiol-  o g i c a l experiments. P i t u i t a r y and Ovary G o r r e l a t i ons From c o r r e l a t i o n s between p i t u i t a r y b a s o p h i l s and o v a r y , i t i s suggested that p i t u i t a r y b a s o p h i l s produce which s t i m u l a t e f o l l i c u l a r development. I n t e r e s t e n d o c r i n o l o g i s t s who  gonadotrophins  T h i s f a c t should  are using p i t u i t a r y  extracts  f o r they should choose animals which are n e a r i n g m a t u r a t i o n if  they are p r e p a r i n g gonadotrophins.  - 51  C o r r e l a t i o n s show a t r e t i c egg number does n o t change w h i l e p i t u i t a r y b a s o p h i l s a r e i n c r e a s i n g from November through A p r i l .  A l t h o u g h from May  through O c t o b e r , a t r e t i c  egg numbers do i n c r e a s e when b a s o p h i l s i n c r e a s e , t h e r e i a no c o r r e l a t i o n . I t i s t h e r e f o r e c o n c l u d e d t h a t the p i t u i t a r y b a s o p h i l s do n o t a f f e c t a t r e t i c egg number.  The  two c o r r e l a t i o n s which a r e s i g n i f i c a n t must, t h e r e f o r e , be spurious• Injection  Experiments  The r e s u l t s from i n j e c t i o n s a r e i n c o n c l u s i v e because v e r y few animals, were u s e d .  In addition,  f i s h used f o r  i n j e c t i o n o f salmon p i t u i t a r y e x t r a c t s showed i n d i v i d u a l v a r i a b i l i t y i n s e x u a l m a t u r i t y and were a mixed s t o c k . were h e l d t o be n e g a t i v e i n v i e w o f t h e s e f a c t s ,  Results  However,  the r e s u l t s can by used f o r d e s i g n i n g f u t u r e e x p e r i m e n t s . H o m o p l a s t i c i n j e c t i o n s have been a d v i s e d by Houssay (1929) and  t h e r e f o r e , the mammalian pregnant m a r e s  ?  serum can  h a r d l y be expected t o y i e l d p r o f i t a b l e r e s u l t s i n i n d u c i n g sexual maturity.  - 52  -  SUMMARY 1. O v a r i a n s t r u c t u r e i s d e s c r i b e d I n d e t a i l f o r a l l s t a g e s o f d e v e l o p i n g ova.  Cytology of resorbing f o l l i c l e s  (corpora a t r e t i c a ) i s described f o r s e v e r a l d i f f e r e n t s t a g e s w h i c h were d e r i v e d from c o r r e s p o n d i n g d e v e l o p i n g follicles. 2. Oogenesis begins i n autumn.  About O c t o b e r , t h e f i r s t y o l k  i s d e p o s i t e d and t h i s c o n t i n u e s r a p i d l y u n t i l  follicles  are mature sometime between J a n u a r y and June.  Up t o t h r e e  d i f f e r e n t crops o f d e v e l o p i n g ova may one time i n a s i n g l e 3. D e g e n e r a t i o n Atretic maturity.  be p r e s e n t a t any  ovary.  of f o l l i c l e s may  oceur any time i n the y e a r .  f o l l i c l e s are more numerous as the ovary One  reaches  f u n c t i o n o f r e s o r b i n g f o l l i c l e s i s the  removal o f s u r p l u s eggs. 4. C o r r e l a t i o n between i n c r e a s i n g p i t u i t a r y b a s o p h i l s and increas© i n d i a m e t e r o f eggs i s p o s i t i v e and  significant.  C o r r e l a t i o n between p i t u i t a r y b a s o p h i l s and d e g e n e r a t i n g  ,  f o l l i c l e s i s spurious. 5. I n j e c t i o n s o f s a l i n e e x t r a c t s o f salmon p i t u i t a r i e s to a f f e c t g o l d f i s h o v a r i e s c y t o l o g i c a l l y . serum a f f e c t e d b e h a v i o u r of g o l d f i s h .  Pregnant  failed mares  1  LITERATURE CITED  Atz, E t h y l H., 1953. Experimental d i f f e r e n t i a t i o n of basophil c e l l types i n the t r a n s i t i o n a l lobe of the p i t u i t a r y of a teleost f i s h , Astyanax mexicanus. In F i s h Endocrinolgy* B u l l . Bingham Oceanograph. C o l l . Bailey, B.E.„ 1942. Chart of the n u t r i t i v e values of B r i t i s h Columbia f i s h e r y products. Prog. Rep. Pac. Coast Stns. of the F i s h , Res. Bd. Can. 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Eggs and l a r v a e of' t e l e o s t s . E d i n . 33 p a r t 1:97-135,  T r a n s . Roy. S o c .  Cunningham, J.T. & W.A.M. S m a r t , 1934. The s t r u c t u r e an4 o r i g i n o f c o r p o r a l u t e a i n some o f t h e l o w e r V e r t e b r a t e . P r o c . Roy. S o c , Lond. 116B:258-281. Dodd, J.M., 1955, V a r y i n g e f f e c t s o f s e x hormones i n - f i s h and l o w e r c h o r d a t e s . Mem. E n d o c r i n o l . No. 4. C l i c k , D., 1949. Techniques o f h i s t o - c y t o c h e m i s t r y . P u b l . N.Y.  Interscience  De G r o o t , B., & J . J . Duyvene de W i t , 1949. I I I . O v i p o s i t o r growth and the a d a p t a t i o n syndrome o f S e l y e . A c t a E n o f o c r i n o l o g i c a . 3:288-2,97; Guyer, M.E., 1906. A n i m a l M i c r o l o g y . U n i v . C h i c a g o . Hann, H.W., 1927. The h i s t o r y of the germ c e l l s o f C o t t u s G i r a r d . J . Mbrph. 43:427-497.  bairdil  Harrison, R.J., 1948. 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D.F., Personal  fishes.  communication.  Scruggs ,' F.M., 1939. The e p i t h e l i a l components of the t e l e o s t p i t u i t a r y gland as i d e n t i f i e d by a s t a n d a r d i z e d method o f s e l e c t i v e s t a i n i n g . J . Morph. 65:187-214.• Smith, 3.G., 1940. A new embedding schedule f o r i n s e c t S t a i n Tech. 15(4):175-176.  cytology.  -  56  S t o l k , A., 1951. Histoendocrinological. analysis.of. gestation phenomena l h t h e cyprinodont. L e b i s t e s r e t l c u l a t u s P e t e r s . I I , The c o r p u s l u t e u m c y c l e d u r i n g pregnancy. K: Ned'erland Wetenschap Amsterdam^ Proc. 540:550-578. S t r o n s t e h , F.A., 1931. The development o f the gonads i n t h e g o l d f i s h , G a r a s s i u s a u r a t u s ( L . ) . U n i v . Iowa S t u d i e s : S t u d i e s i n N a t u r a l H i s t o r y . 13(7):1^45. Wallace,"W.M., 1903. O b s e r v a t i o n s on o v a r i a n ova and f o l l i c l e s i n c e r t a i n T e l e o s t e a n and-Elasmobranch f i s h e s . Qjtiart. J . M I c r . S c i . 47:161-214.  -  57  -  APPENDIX A H i s t o l o g i c a l Procedures Al  Bouin's F l u i d (Cowdry, 1943) Saturated aqueous p i c r i c acid  75 ml.  Formalin, 37% commercial  25 ml.  Acetic acid  5 ml.  Cowdry (1943) suggested that washing a f t e r f i x a t i o n needn.not be too complete since p i c r i c acid acts as a mordant* However, i n this case, p i c r i c acid l e f t i n the tissues was found to cause much s t a t i c e l e c t r i c i t y during sectioning. A2 Formoib-sublimate (Purves & Greisbach, 1951) Eormalin, 37% commercial  10%  Mercuric chloride (HgGl )  90%  g  Author's Modification: Formalin, 37% commercial  10 ml.  Mercuric Chloride (HgClg)  2.5 gm.  Water, d i s t i l l e d  90 ml.  In 50 ml. of water heated almost to b o i l i n g i s d i s solved the mercuric chloride.  The remaining water i s added  cold, followed by formalin. The f i x i t i v e i s ready for Immediate use, A3  Dehydration with E t h y l Alcohol (Baker, 1945) Wash i n running water overnight, 50% ethanol  1 hr.  - 58 -  70$ e t h a n o l  1 nr.  80$ e t h a n o l  1 hr.  96$ e t h a n o l  1 hr.  Absolute ethanol  1 hr.  A b s o l u t e e t h a n o l + benzene  50/50  Benzene  % hr. ,  nr.  Benzene s a t u r a t e d w i t h p a r a f f i n ( i n oven) P a r a f f i n i n oven  l/2 hr. 2 hr.  Embed and c o o l r a p i d l y . D e h y d r a t i o n w i t h T e r p i n e o l ( L e e , 1948) 30$ e t h a n o l  1 hr.  60$ e t h a n o l  1 hr.  80$ e t h a n o l  1 hr.  90$ e t h a n o l  1 hr.  96$ e t h a n o l  1 hr.  96$ e t h a n o l + t e r p i n e o l (1:1) u n t i l tissues  sink.  96$ e t h a n o l + t e r p i n e o l (1:2)  1 hr.  96$ e t h a n o l + t e r p i n e o l (1:3)  1 hr.  Pure t e r p i n e o l - 3 changes  3 hr.  Pure p a r a f f i n . Change e v e r y h o u r u n t i l no l i l a c odour r e m a i n s . Because o f d i f f i c u l t y i n r e m o v i n g t e r p i n e o l t h i s method  - 59 -  was  abandoned, Lee  p e n e t r a t i n g and  (1948) r e p o r t e d  t e r p i n e o l as u s e f u l i n  s o f t e n i n g y o l k y t i s s u e s . T h i s was  not  found  so. A5  Dehydration with n-Butyl A l c o h o l (Smith,  1940)  Percentage Composition o f S o l u t i o n s Time (hours)  Water (dist)  Ethanol  n-Butyl Ale.  Phenol  1-3  30  50  20  -  24  11  50  35  4  1-3  5  40  55  mm  1-3  -  25  75  1  -  1 2  -  -  (oven)  l£(oven)  -  -  l|-( oven)  -  .  -  100  4  -  -  -  100  50  Paraffin  -  -  *•  50 100 100  I t has been suggested t h a t t h i s method i s good f o r yolky tissue.  I t i s very s a t i s f a c t o r y f o r both ovaries  and  d e c a l c i f i e d sections c o n t a i n i n g s o f t b r a i n t i s s u e . With embedded f i s h h e a d s , no c o l l a p s e of t i s s u e i s d i s c e r n a b l e . I n t h i s i n v e s t i g a t i o n , the. method was m o d i f i e d u s i n g t h r e e changes o f p a r a f f i n , e a c h under vacuum. to embedding w  tt, >  seetlon  A6.  by Refer  - 60 -  A6  Embedding F i l t e r e d F i s h e r T i s s u e Mat (m.p. 54-56°C.) was used i n  e a r l y work.  T h i s p a r a f f i n was found  t o h a r d e n t i s s u e , cause  severe s h r i n k a g e , and s e c t i o n s r i b b o n e d w i t h d i f f i c u l t y . Impregnation  o f p a r a f f i n was o f t e n p o o r .  On the s u g g e s t i o n o f D.P. S c o t t , a p a r a f f i n s a t u r a t e d w i t h beeswax was used.  T h i s m i x t u r e was much s t i c k i e r and  r i b b o n i n g was s u c c e s s f u l and f r e e o f s t a t i e e l e c t r i c i t y i n a l l cases.  T h i s work embodied use o f t h r e e changes o f  bees-wax-paraffin  o f two hours each and under g r a d u a l l y  i n c r e a s i n g vacuum u n t i l n o t f u r t h e r b u b b l i n g o f gasses came from t i s s u e s .  A t t h i s t i m e , i t was c o n s i d e r e d t h a t a l l  antemedium had v a p o r i z e d from wax and t i s s u e , no a i r l o c k s e x i s t e d , and i m p r e g n a t i o n A7  H a r r i s * Haematoxylin  complete. (Guyer, 1906)  Haematoxylin  1 gm.  Absolute ethanol  10 m l .  P o t a s s i u m aluminum s u l f a t e  20 gm.  Water ( d i s t i l l e d )  200 m l .  M e r c u r i c o x i d e (HgO)  0.5 gm.  acetic acid D i s s o l v e haematoxylin s o l u t i o n o f alum.  i n a l c p h o l and . add t o warm,  B r i n g t o b o i l ; add.mercuric  oxide.  f o r one minute and.plunge f l a s k . i n t o c o l d w a t e r .  Boil  Add a c e t i c  -  61-  "acid j u s t b e f o r e u s e . T h i s s t a i n d i d n o t keep as w e l l as i n d i c a t e d by Guyer (1906).  S t a i n i n g time was l e n g t h y and b a s o p h i l i c  were n o t a c l e a r AS  structures  blue.  Mayer's Haemalum ( B a k e r , 1946) Haematoxylin  0,1 gm.  Water, d i s t i l l e d  100 m l .  Sodium i o d a t e  0,03 gm.  (NalOg)  P o t a s s i u m aluminum s u l f a t e  5,0 gm.  B o i l h a e m a t o x y l i n w i t h w a t e r ; add i o d a t e and alum gradually,  S t a i n i s ready f o r use.  T h i s s t a i n was found t o be much s u p e r i o r preparation A9  i ni t s clarity  to Harris'  and ease o f s t a i n i n g .  F e u l g e n Reagent for- PAS ( G l i c k , 1949) Basic fuchsin  1.0 gm.  Water, d i s t i l l e d  200 m l .  Potassium m e t a b i s u l f i t e  2  2  5  2 gm,  1 N HC1  10 m l .  N o r i t ( a c t i v a t e d carbon)  0.5 gm.  Dissolve cool.  (K S 0 )  b a s i c f u c h s i n I n b o i l i n g w a t e r , f i l t e r , and  Add m e t a b i s u l f i t e and h y d r o c h l o r i c  acid.Bleach f o r  24 h o u r s , add N o r i t , shake f o r one minute and f i l t e r . Filtrate  s h o u l d be c o l o r l e s s .  P u r v e s & G r i e s b a c h (1951)  suggested t h e l a k e method o f s t a i n i n g .  I f t h e r e a r e many  - 62 -  s l i d e s , the l a k e method i s i m p r a c t i c a l and s t a i n i n g d i s h e s may he u s e d . method.  F e u l g e n ' s s t a i n hecomes reduced by t h i s  A d d i t i o n o f a s m a l l amount o f the b l e a c h i n g a g e n t ,  m e t a b i s u l f i t e , o x i d i z e s the s t a i n to i t s c o l o r l e s s s t a t e again. A10  H a e m a t o x y l i n & B o s i n : s t a i n i n g procedure X y l e n e dewax  5 min.  Absolute ethanol  2 min.  95% e t h a n o l  2 min.  90% e t h a n o l  2 min.  i o d i n e e t h a n o l used a f t e r m e r c u r i c f i x a t i o n u n t i l c r y s t a l s removed. 70% e t h a n o l  2 min.  50% e t h a n o l  2 min.  D i s t i l l e d water  2 min.  Haemalum  -j§->5 m i n .  Wash'  i min.  Acid ethanol ( d i f f e r e n t i a t i o n )  -J- m i n .  A l k a l i n e e t h a n o l (HG0 ")  5 min.  70% e t h a n o l  2 min.  90% e t h a n o l  2 min.  Alcoholic eosin  35 s e c . g  95% e t h a n o l  rinse  Absolute ethanol  rinse  3  X y l e n e and mount w i t h Permount.  - 63  All  Periodic  -  Acid S c h i f f Reaction  (PAS): S t a i n i n g  m o d i f i e d from P u r v e s & G r i e s h a c h  procedure  (1951).  X y l e n e dewax  5 min*  Absolute  2 min;  ethanol  95$  ethanol  2 min.  90$  ethanol  2 min.  70$ i o d i n e  ethanol a f t e r HgCl  g  25 min.  70$  ethanol  2 min.  50$  ethanol  2 min.  2$ aqueous t h i o s u l f a t e  solution  ±  1 min. 5 min.  Wash 0.5$. p e r i o d i c  acid  (HglOg)  Wash Schiff*s solution Wash i n s e v e r a l  (Feulgen)  changes o f water  Haemalum  <5  min.(Pearse, 1954)  rinse 25 min.  ±  1 ho.UE  4-  •§•-•10 m i n ,  Wash Alkaline ethanol  (HC0 ~) 3  5 min.  e thanoi  2 min,  ethanol  2 min,  90$  ethanol  2 min.  95$  ethanol  2 min.  Absolute  ethanol  X y l e n e , mount w i t h Permount.  2 min.  -  64  -  Time i n p e r i o d i c a c i d s h o u l d never exceed f i v e m i n u t e s ; one minute i s p r o b a b l y enough. p e r i o d i c a c i d destroys  Too much o x i d a t i o n by  a b i l i t y of S c h i f f a s o l u t i o n to  d i f f e r e n t i a t e between m u c o p r o t e i n s (Weak a f f i n i t y S c h i f f s ) and. g l y c o p r o t e i n s  for  (strong a f f i n i t y f o r S c h i f f s ) .  McManus (1948) observed t h a t the F e u l g e n s t a i n became enhanced d u r i n g the 5-10 the s t a i n .  minute w a s h i n g a f t e r i m m e r s i o n i n  However, i n t h i s s t u d y , t h i s phenomenon  was  observed t o o c c u r p r o g r e s s i v e l y over a 30 m i n u t e p e r i o d . More i m p o r t a n t ,  however, the s t a i n became even more b r i l l i a n t  some weeks a f t e r mounting i n Canada balsam.  To  counteract  t h i s change i n s t a i n i n g i n t e n s i t y , t i s s u e s were washed f o r p e r i o d s up t o two h o u r s ; the s t a i n seems to be permanent once mounted a f t e r complete w a s h i n g . Control sections should  always be r u n w i t h each b l o c k .  In several instances, p a r t i c u l a r blocks stained  exceedingly  more d e n s e l y when s t a i n e d under the r o u t i n e p r o c e d u r e . o n l y e x p l a n a t i o n o f t h i s occurrssnee p r o b a b l y l i e s i n n a t u r e or c o n c e n t r a t i o n s  of the f i x i t i v e u s e d .  the  The  -  65  -  APPENDIX B Preparation o f I n j e c t i o n Materials. Bl  S e r i e s H: P r e p a r a t i o n o f s a l i n e e x t r a c t s o f Coho salmon  (Onoorhynchua k i s u t o h ) p i t u i t a r i e s . were  P i t u i t a r i e s , which  p r e s e r v e d i n a b s o l u t e e t h a n o l and s t o r e d under  r e f r i g e r a t i o n (4°0.), were d r i e d on f i l t e r paper p r e p a r a t o r y to w e i g h i n g .  Two dosages were u s e d .  "DU^ * 1  (dosage u n i t 1)  c o n s i s t e d o f 25 mg. o f whole salmon p i t u i t a r i e s minced i n 1 ml. frog s a l i n e . saline.  n  D U " c o n s i s t e d o f 50 mg; p i t u i t a r i e s / m l b . g  T h i s s u s p e n s i o n was s t o r e d under r e f r i g e r a t i o n  u n t i l u s e d , and any unused s u s p e n s i o n was d i s c a r d e d afgjer f o u r days. Prog  saline:Watery d i s t i l l e d , . . , . , .  1 li.  NaGl  6.5 gu.  KOI  0.14 gm.  CaClg  0.12 gm.  NaHC0  NaH P0 g  0.2 gm.  3  4  0.01 gm.  I n j e c t i o n S c h e d u l e ( n e x t page)  -  66  I n j e c t i o n Schedule:- ( i n c l u d i n g  -  sampling)  I n j e c t i o n Dose/Fish Date  DTL  Control  12 Oct 1955 13 Oct 1955 14 Oct 1955 15 Oct 1955  0.2 ml.  0.2 m l .  0.2 m l .  Sampled 2 females - each group 0.2 m l .  0.2 ml,  0.2 m l .  Sampled 2 females -.each group  16 Oct 1955  0.2 m l .  0.2 m l .  18 Oct 1955  0.2 m l .  0.2 m l .  0.2 m l .  80G04t 1955  0.2: m l .  0.2 m l .  0.2 m l .  22 Oct 1955  0.2 m l .  0.2 m l .  0.2 m l .  24 Oct 1955 B2  v  0.2 m l .  Sampled 2 females - each group  !  1  S e r i e s I : "p.m.s,* o r P r e g n a n t Mares* Serum Gonado-  t r o p i n s u p p l i e d d r i e d by V e t e r i n a r y D i v i s i o n , A y e r s t , McKenna & H a r r i s o n L t d . , M o n t r e a l , Quebec. I n j e c t i o n Schedule:-  50 I.U. ( o . l ml.) o f P r e g n a n t  Mares* Serum G o n a d o t r o p h i n were i n j e c t e d i n t o each o f 20 g o l d f i s h e v e r y two days f o r 26 d a y s .  These f i s h were t a k e n  from S t o c k 4 (Appendix C ) . S a m p l i n g Schedule T h u r s . Nov. 3, 1955  S t a r t of E x p e r i m e n t  -  67  Sun.  Nov,  6,  1955  2- f e m a l e s  -  "3-day  fish"'  Sat,  Nov, 12,  1955  3 females  -  "9-day  fish"  Wed., Nov. 29,11955 B3  -  Series J : Pituitary  from f i x a t i o n pituitaries  6 females -  Alcohol Extract.  and p r e s e r v a t i o n o f  were  gonadotropin.  evacuation.  T h i s a l c o h o l was p a r t l y  its  Kisutch)  o r i g i n a l volume.  hydrated  An e f f i c i e n t  vacuum to  absolute to  water-tap of  considerably during  was hoped t o remove a l l  This hydrated  of  cause b o i l i n g  ethanol  o n l y the. a q u e o u s p o r t i o n c o n t a i n i n g p o s s i b l e extracts.  drainings  room t e m p e r a t u r e  T e m p e r a t u r e was l o w e r e d It  fish"  saved because o f p o s s i b l e washings  evaporator developed s u f f i c i e n t the a l c o h o l .  First  Coho s a l m o n ( 0 .  e t h a n o l and was vaeuum. e v a p o r a t e d a t about l / l O t h  "27-day  leaving  pituitary  e x t r a c t was i n j e c t e d  intraperiton-  eally, B4  S e r i e s K:  (0. kisutch)  Salmon P i t u i t a r y pituitaries  were d r i e d  paper,  ground i n a m o r t a r ,  saline  (Appendix B l ) .  4°C,required  This suspension,  Injection Schedule:-  whole p i t u i t a r i e s or 0.4  ..t.. whole  every  Coho s a l m o n  by b l o t t i n g  and l i q u i f i e d  shaking before  f i s h f r o m s t o c k 4 were  Suspension.  i n 25 m l .  filter frog  w h i c h was s t o r e d  at  injection.  B e g i n n i n g on N o v .  injected  with:-  two d a y s f o r  pituitaries  on  every  (a)  3,  0.1  1955, ml.  27 d a y s , and  two d a y s  gold-  or 0.2 (b)  0.2  f o r 27 d a y s .  -  .  ml.  - 68 -  Sampling Schedule:-  Group a ( o . l ml.)  Date  Group h (0;2 ml.)  Class  E x p e r i : oent s t a r t e d  Thurs.Nov.  3,1955  Sun.  Nov.  6,1955  2 females  2 females  "3-day f i s h "  Sat.  Nov.12,1955  3 females  3 females  "9-day f i s h "  Wed.  Nov.30,1955  1 female  7 females  "27-day f i s h "  B5  S e r i e s L:  F r e s h f r o z e n Coho salmon ( O . k i s u t c h )  p i t u i t a r i e s , c o l l e c t e d d u r i n g l a t e September, 1955, were s t o r e d n i n e p i t u i t a r i e s t o the v i a l a t - 15°C. o f t h e s u s p e n s i o n was as f o l l o w s : was  Preparation  Each v i a l o f p i t u i t a r i e s  thawed out and minced i n 10 m l . f r o g s a l i n e (Appendix B l ) .  20 m l . o f t h e s u s p e n s i o n was p r e p a r e d and s t o r e d a t ready f o r i n j e c t i o n .  4°G.  - 69 -  APPENDIX C Source o f G o l d f i s h G o l d f i s h f o r a l l experiments were o b t a i n e d f r o m a mixed population.  D i s t r i b u t o r s a r e the G o l d f i s h S u p p l y Co.,  S t o u f f v i l l e , Ontario. The f o l l o w i n g s t o c k s (average f o r k l e n g t h 7-9cm.) were u s e d : S t o c k 1. R e c e i v e d 29 O c t . 1954 S t o c k 2. R e c e i v e d 30 A p r . 1955 S t o c k 3. R e c e i v e d  J u l , 1955  S t o c k 4. ..Received 18 O c t , 1955  300 g o l d f i s h .  -  70  -  APPENDIX D Statistical  Records  rCorrelatlonsT a b l e I I I Y a r i a t e s f o r C o r r e l a t i o n s , November t h r o u g h J anuary.  No.  *2  *3  No. B16  B 1  2128  261  -  2  1542  394  1.1  17  *1  *2  X  1935  361  0.1  307  «*  M  690  3  18  1498  359  5.1  20  1322  419  0.1  263  0.3  21  1000  389  0.9  839  259  •a*  22  148  350  0.1  10  510  217  -  23  628  173  0.3  11  826  320  0.3  24  1477  315  12  1967  246  509  173  13  781  339  -  25 26  155  227  0.3  14  1974  46.5  2.4  27  1393  618  1.3  15  929  222  -  28 .  1883  337  1.9  3  328  133  5  408  231  6  581  7  =  -  -  A r e a o f p i t u i t a r y b a s o p h i l s i n mm .  Xg - Mean egg d i a m e t e r o f most mature eggs p e r s e c t i o n . X„ - Sum o f a l l a t r e t i c f o l l i c l e s i n each s e c t i o n .  - 71 -  T a b l e IY  No.  Variates  X  l  X  f o r C o r r e l a t i o n s , February through A p r i l .  2  *3  No.  -  B44  *2  X  1432  253  0.3  Z  l  3  B29  555  218  30  1032  173  46  3335  663  4.2  31  1174  473  1174  212  1.3  32  948  570  -  48 49  1232  177  33  303  167  0.1  50  780  151  -  34  722  546  51  1097  160  0.5  35  942  85  52  271  400  1.1  36  2922  737  -  53  1994  708  0.3  37  381  127  0.1  54  2206  702  38  1206  501  0.5  2251  385  39  1961  206  57  2754  540  1.4  40  909  146  -  56  -  5&  3354  562  0.8  41  2702  432  0.2  61  2090  730  5.0  42  2070  680  2.8  62  1006  104  -  43  2864  167  -  63  1361  290  0.2  -  -  Table V  Variates  No.  X  B64  72  -  f o r C o r r e l a t i o n s , May through J u l y ,  *2  X  3  No.  897  116  0.2  B75  3425  477  3.0  65  909  113  -  76  2864  728  0.3  66  2167  855  16.0  79  839  159  9.7  68  1735  270  10.2  80  2896  800  7.1  69  1548  317  7.4  82  980  181  8.3  70  3657  816  1.3  83  3973  696  2,8  71  851  175  2.4  84  2129  235  7.9  72  1367  122.  86  1683  153  18.6  73  1051  162  87  1213  205  20.2  l  mm  10.1  h  - 73 -  Table V I  Variates  No.  f o r C o r r e l a t i o n s , August through. O c t o b e r .  *2  X  3  No.  Z  l  X  2  X  3  B88  1877  724  3.1  B103  1490  775  5.6  89  2928  741  3.3  104  697  279  38.8  90  3173  194  15.7  105  1425  800  25.6  91  3431  883  20.2  106  426  149  1.9  92  993  238  15.1  107  1432  130  93  1522  179  5.2  108  1473  160  94  2070  220  2.5  109  901  238  23.0  95  355  227  7.0  110  3100  754  55,0  96  1954  238  17.0  111  2365  205  25.8  97  581  172  3.7  112  577  183  26.8  98  1819  793  30.4  113  644  184  0.9  99  1954  328  10.5  114  1504  380  24.2  100  3032  916  19.4  115  2500  918  21.8  101  1664  207  7.8  116  628  175  3.4  102  1277  231  8.0  -  Table V I I V a l u e s f o r C o r r e l a t i o n E q u a t i o n Taken from T a b l e s I I I . t o V I .  February - A p r i l  November - J a n u a r y  N  SX, SX, , SX, X »,3 %  sxf (sx,) S  X  8  8  0 r 4  E g g Diameter Analysis  Atretic Egg Analysis  E g g Diameter Analysis  Atretic Egg Analysis  24  24.  30  30.  25,451  25,451.  47,028  47,028.  7;378  14.2  8,554,162  15,800.7  11,265 20,982,184'  18.8 4,167.6  35,981,895  35,981,895.  97,678,130  97,678,130.  647,753,401  647,753,401.  2,211,632,784  2,211,632,784.  2,530,030  39.48  5,676,081  56.76  54,434,884  201.64  126,900,225  353.44  NSX.X,.,,  205,299,888  379,216.8  629,465,520  125,028.  (sx.Hsxj  187,777,478  361,404.2  529,770,420  884,126  NSX? - (SEX,)  215,818,079  8  NSX , 8  r  (SXJ  2  6,285,836  215,812,079. 745.88  718,711,116 43,382,205  #  718,711,116. 1,349.36  Table V I I (continued)  August - October  May - J u l y Egg D i a m e t e r Analysis N  SX,  X  L  I  R  :.16;  L  sxf (sx,)  2  SXJJ,,,  89  8 9 .  3 4 , 1 8 4  3 4 , 1 8 4 .  4 7 , 7 9 8  4 7 , 7 9 8 .  4 5 1 , 9 7 8  NSX?  -  NSX ,,2  3  (SJJ  )  (SX,  R  ( S X , )  2  ( S X , . /  1 2 5 . 5  1 1 , 6 8 1  4 2 1 . 7  2 1 3 , 1 3 3 . 2  8 3 * 4 9 5 , 8 9 5  7 9 8 , 3 6 2 . 6  8 3 , 4 0 9 , 9 3 4  8 3 , 4 0 9 , 9 3 4 .  1 0 1 , 3 3 8 , 1 1 8  1 0 1 , 3 3 2 , 1 1 8 .  1 * 1 6 8 , 5 4 5 , 8 5 6  1 , 1 6 8 , 5 4 5 , 8 5 6 .  8 , 8 8 4 , 0 7 5 , 8 6 4  2 , 2 8 4 , 0 7 5 , 2 6 4 .  4 1 , 9 9 0 , 4 0 0  Z  A t r e t i c Egg Analysis  1 8 .  3 , 6 7 5 , 4 7 8  NSX,X .  Egg Diameter Analysis  1 8  6 , 4 8 0  sx,  A t r e t i c Egg Analys i s  1 , 5 7 1 . 0 3 1 5 , 7 5 0 . 2 5  6 , 9 7 8 , 5 6 9 1 3 5 , 0 4 7 , 6 4 1  1 1 0 , 3 0 5 . 3 1 7 7 , 8 3 0 . 8 9  8 9 6 , 1 3 5 , 6 0 4  3 , 8 3 6 , 3 9 7 . 6  6 8 1 , 3 6 3 , 5 5 5  2 2 , 9 7 8 , 5 1 5 . 4  2 8 1 , 5 1 2 , 3 2 0  4 , 2 9 0 , 0 9 8 .  5 5 5 , 3 9 0 , 8 3 8  2 0 , 1 5 3 * 8 8 6 . 4  3 3 8 , 8 3 8 , 9 5 6 .  6 5 4 , 5 5 6 , 1 5 8  3 3 8 , 8 3 2 , 9 5 6 2 4 , 1 6 8 , 2 0 4  1 8 , 5 8 8 . 8 9  6 7 , 1 5 6 , 8 6 0  6 5 4 , 5 5 6 , 1 5 8 . 3 , 0 2 1 , 0 2 2 . 8  - 76 -  F i g u r e 15. Immature o v a r y . A l l f o l l i c l e s a r e "immature". Cytoplasm i s b a s o p h i l i c and has no y o l k . Note g e r m i n a l e p i t h e l i u m l i n i n g t r a b e c u l a e . x4Q.  F i g u r e 16. Three immature f o l l i c l e s showing v a r i o u s s i z e s and shapes o f n u c l e o l i . x40Q  - 77 -  F i g u r e 18, P h o t o m i c r o g r a p h o f mature ovum showing zona p e l l u c i d a w i t h r a d i a l s t r i a t i o n s ; T.E., theca e x t e r n a ; T . I . , t h e c a i n t e r n a ; z.p. zona p e l l u c i d a ; Cp, c a p i l l a r y ; : 0 g , o i l g l o b u l e . 3x900.  - 78 -  F i g u r e 19, Photomicrograph o f s u r f a c e o f theca externa showing squamous e p i t h e l i a l - l i k e s t r u c t u r e o f cells. x40Q,  F i g u r e 20. Ovary showing l o n g i t u d i n a l s e c t i o n o f p r i n c i p l e branch o f o v a r i a n a r t e r y . Note i t s t h i n w a l l s c h a r a c t e r i s t i c o f s i n u s o i d s . Much connective t i s s u e i s present i n t h i s ovary. xlOO.  Figure  Figure  21, T r a n s v e r s e s e c t i o n t h r o u g h the o v a r i a n artery. At t h i s place i t s w a l l s are t h i c k muscular, z400.  and  22, Immature o v a r y c o n t a i n i n g a v e r y y o u n g a t r e t i c follicle (a.f.). Note s i n g l e l a r g e n u c l e o l u s ( n . c . ) i n the s m a l l e s t f o l l i c l e s , zlOO.  - 80 -  F i g u r e 23. Young a t r e t i c f o l l i c l e from immature ovum (same as f i g u r e 22) showing i n t e n s e g r a n u l a t i o n i n s t r a t u m granulosum, c h a r a c t e r i s t i c o f p h a g o c y t o s i s . x40Q.  Cytoplasm a p p a r e n t l y i s removed f i r s t .  z400  - 81  -  F i g u r e 25. An a-stage degenerate f o l l i c l e , showing i n t a c t but eroded zona p e l l u c i d a (z.p.) and h y p e r t r o p h y l n g and s y n c i t i a l g r a n u l o s a l l a y e r , (g.l.) x400.  F i g u r e 26. S m a l l a t r e t i c f o l l i c l e (s) c o n t r a s t e d t o l a r g e a t r e t i c f o l l i c l e ( L ) , both i n t h e same o v a r y . Large a t r e t i c f o l l i c l e i s i n the as t a g e . x40.  F i g u r e 88. A b-stage a t r e t i c f o l l i c l e showing g r a n u l o s a l g r a n u l a t i o n and remnants o f oolemma. z400.  - 83 -  F i g u r e 29. A l a t e b-stage a t r e t i c f o l l i c l e (from l a t e maturing egg) showing broken and c o l l a p s i n g zona p e l l u c i d a . zlOO.  F i g u r e 30. A t r e t i c f o l l i c l e showing zona p e l l u c i d a remaining almost to the l a s t . This f o l l i c l e i s approaching the d-stage. XlOO.  -  84  -  F i g u r e 32. T r a n s v e r s e s e c t i o n through p o s t e r i o r r e g i o n of a male g o l d f i s h p i t u i t a r y . z40.  n s a g i t t a l s e c t i o n of  F i g u r e 33. Median s a g i t t a l s e c t i o n of g o l d f i s h p i t u i t a r y showing d i f f e r e n t r e g i o n s . The dark c e l l s i n the t r a n s i t i o n a l lobe are b a s o p h i l s . xl50.  -  86  -  F i g u r e 34. Median s e c t i o n through g o l d f i s h p i t u i t a r y . Note few b a s o p h i l s i n t r a n s i t i o n a l l o b e . The ovary f r o m t h i s f i s h was v e r y immature. x40.  F i g u r e 35. . T y p i c a l p a r a s a g i t t a l s e c t i o n o f g o l d f i s h p i t u i t a r y w i t h many b a s o p h i l s i n t r a n s i t i o n a l l o b e . The ovary (ffrom t h i s f i s h was q u i t e mature. x40.  

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