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Steroidogenesis and the role of steroids in the endocrine control of oogenesis and vitellogenesis in… Khoo, Khay Huat 1974

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STEROIDOGENESIS AND THE ROLE OF STEROIDS IN THE  ENDOCRINE CONTROL OF OOGENESIS AND VITELLOGENESIS IN THE GOLDFISH, CARASSIUS AURATUS  by KHAY HUAT KHOO B.Sc. (Hons) U n i v e r s i t y o f Malaya, 1968 M.Sc. U n i v e r s i t y o f Malaya, 1971  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF  THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Zoology  We accept t h i s t h e s i s as conforming to the required standard.  THE  UNIVERSITY OF BRITISH COLUMBIA J u l y , 1974  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  further  for  scholarly  by h i s of  agree  this  thesis  in p a r t i a l  fulfilment  of  at  University  of  Columbia,  the  make  it  that permission  available  for  It  financial  is  gain  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  by  the  Columbia  shall  not  requirements  reference copying of  I  agree  and  copying or  be a l l o w e d  for  that  study.  this  thesis  Head o f my D e p a r t m e n t  understood that  written permission.  Department  for  for extensive  p u r p o s e s may be g r a n t e d  representatives. thesis  freely  British  the  or  publication  without  my  ABSTRACT  I n t h i s t h e s i s I s t u d i e d t h e r o l e o f o v a r i a n s t e r o i d s i n oogenesis and  o v a r i a n development o f g o l d f i s h  ( C a r a s s i u s auratus L.) and examined  the i n t e r r e l a t i o n s h i p o f p i t u i t a r y gonadotropin  and o v a r i a n s t e r o i d s i n  the e n d o c r i n e  There a r e f o u r main  control of teleost reproduction.  p a r t s t o t h e i n v e s t i g a t i o n : ( i ) the c y t o l o g i c a l a n a l y s i s o f v i t e l l o g e n e sis,  ( i i ) the demonstration  t h e i r endocrine pus  control,  of s t e r o i d o g e n i c t i s s u e s i n the ovary and  (iii)  the development and f u n c t i o n s o f the c o r -  luteum, and ( i v ) the r o l e o f o v a r i a n s t e r o i d s on oogenesis  and v i t e l -  logenesis. The h i s t o l o g i c a l and h i s t o c h e m i c a l examination two  demonstrated t h a t  types o f y o l k i n c l u s i o n s a r e formed d u r i n g v i t e l l o g e n e s i s : y o l k  v e s i c l e s , comprised o f mucopolysaccharides, subsequently  by y o l k g r a n u l e s  neutral l i p i d s .  and f i r s t  formed, f o l l o w e d  composed o f p r o t e i n s , p h o s p h o l i p i d s and  N e i t h e r type o f y o l k develops  i n the absence o f the  p i t u i t a r y ; e s t r o g e n r e g u l a t e s the f o r m a t i o n o f y o l k v e s i c l e s w h i l e nenolone was found  to c o n t r o l d e p o s i t i o n of yolk granules.  cance o f these two k i n d s o f y o l k i s c o n s i d e r e d .  preg-  The s i g n i f i -  T h i s i s the f i r s t  demon-  s t r a t i o n of two y o l k types i n g o l d f i s h w i t h a s e p a r a t e e n d o c r i n e c o n t r o l f o r t h e f o r m a t i o n o f each o f them. The major s t e r o i d s y n t h e s i z i n g t i s s u e s i n g o l d f i s h o v a r i e s are the g r a n u l o s a c e l l s o f oocytes  and the corpus luteum.  whether p r e - o r p o s t - o v u l a t o r y p r o b a b l y  The corpus luteum,  synthesizes estrogens.  In t h i s  study, no f u n c t i o n a l d i f f e r e n c e s were e s t a b l i s h e d between p r e - and p o s t -  iii ovulatory corpora tritiated  lutea.  Treatment of p o s t - o v u l a t o r y  thymidine s t r o n g l y suggests t h a t the c o r p o r a l u t e a l  p r o l i f e r a t e to form another g e n e r a t i o n of oogonia. bably  g o l d f i s h with  a c t i v e hormones i n t h i s r e a c t i o n .  f i n d i n g s are  The  Estrogens  cells are  pro-  i m p l i c a t i o n s of these  new  discussed.  A d m i n i s t r a t i o n of exogenous estrogen  i n c r e a s e s oogonia  formation  i n p o s t - o v u l a t o r y g o l d f i s h whereas c h r o n i c a d m i n i s t r a t i o n o f or t e s t o s t e r o n e i n t o g r a v i d f i s h induces  atresia.  Treatment  o f g r a v i d f i s h w i t h p r o g e s t e r o n e o r c o r t i c o s t e r o i d s induces  ovulation.  As a working h y p o t h e s i s s i z e d by  extensive  estrogens  i t i s proposed t h a t s t e r o i d s are  the o v a r i e s under gonadotropin  stimulation.  P i t u i t a r y gonado-  t r o p i n r e g u l a t e s the whole o v a r i a n s t e r o i d s y n t h e t i c p r o c e s s s p e c i f i c r e a c t i o n i n the s t e r o i d m e t a b o l i c  pathways.  synthe-  The  and  not  any  s y n t h e s i s of  s p e c i f i c s t e r o i d s d u r i n g the r e p r o d u c t i v e c y c l e i s brought about by l o c a l i z e d i n h i b i t i o n of s t e r o i d o g e n i c enzymes by  s t e r o i d s , most  likely  estrogens. The  p o t e n t i a l p r a c t i c a l a p p l i c a t i o n s of endocrine  f i s h r e p r o d u c t i o n are  discussed.  manipulations  in  iv TABLE OF CONTENTS Page i i  Abstract L i s t of Tables  '  .  L i s t of Figures  .....  viii i  x  Acknowl ed gemen t s  xii  General I n t r o d u c t i o n  1  Section I :  The H i s t o l o g y and H i s t o c h e m i s t r y  of Ovarian  Development.  3  Introduction  4  M a t e r i a l s and Methods  6  ( i ) Maintenance o f G o l d f i s h  6  (a) N a t u r a l G o l d f i s h  6  (b) G r a v i d G o l d f i s h  6  (c) R e f r a c t o r y g o l d f i s h  6  ( i i ) H i s t o l o g y and H i s t o c h e m i s t r y  of Ovaries  Results  7 11  ( i ) H i s t o l o g y o f Oocyte Growth and Development (a) Oogonia  11 H  (b) F i r s t Growth Phase Oocytes  11  (c) Second Growth Phase Oocytes  13  ( i i ) Histochemistry  of V i t e l l o g e n e s i s  15  (a) D i s t r i b u t i o n of P o l y s a c c h a r i d e s  16  (b) D i s t r i b u t i o n o f P r o t e i n s . . . .  20  (c) D i s t r i b u t i o n o f L i p i d s  21  V  T a b l e o f Contents  (cont'd) Page  S e c t i o n I I : The Corpus Luteum and i t s F u n c t i o n . . . .  28  Introduction  29  M a t e r i a l s and Methods  ..  .  ( i ) Hypophysectomy  33 33  ( i i ) H i s t o l o g y o f p r e - and P o s t - o v u l a t o r y Corpus Luteum  35  ( i i i ) Histochemical Detection of Hydroxysteroid Dehydrogenases  35 3  ( i v ) I n c o r p o r a t i o n o f Thymidine- H I n t o O v a r i e s  37  Results  39  ( i ) H i s t o g e n e s i s of a t r e t i c oocytes  39  (a) a - s t a g e  39  (b) g-stage  39  (c) y - s t a g e .  40  (d) 6-stage  40  (e) e-stage.  ...  ( i i ) . ) H i s t o g e n e s i s of p o s t - o v u l a t o r y Corpus Luteum  40 42  (a) Stage I . . . . . . .  42  (b) Stage I I  42  (c) Stage I I I  43  ( i i i ) H y d r o x y s t e r o i d dehydrogenases i n Normal O v a r i e s . .  43  (a) 3 g - h y d r o x y s t e r o i d dehydrogenase  46  (b) 3 a - h y d r o x y s t e r o i d dehydrogenase  46  (c) 1 7 a - h y d r o x y s t e r o i d (d) 1 7 3 - h y d r o x y s t e r o i d  47 47  dehydrogenase dehydrogenase....  vi T a b l e o f Contents  (cont'd) Page  !• ( i v ) H y d r o x y s t e r o i d Dehydrogenases i n Hypophysectomized  Fish........  50  (v) H y d r o x y s t e r o i d Dehydrogenases i n Hypophysectomized  f i s h T r e a t e d w i t h LH.  53  ( v i ) H y d r o x y s t e r o i d Dehydrogenases i n P o s t ovulatory Ovaries  55  ( v i i ) The F a t e of P o s t - o v u l a t o r y Corpus Luteum as Determined by Autoradiography  57  Discussion  60  ( i ) Steroid Synthesis i n F i s h Ovaries  60  ( i i ) A t r e s i a of Second Growth Phase Oocytes...  61  ( i i i ) P o s t - o v u l a t o r y Corpora L u t e a .  63  ( i v ) L o c a l i z e d Hormone P r o d u c t i o n  64  (v) P o s t - o v u l a t o r y P r o l i f e r a t i o n of Obgonia........  65  ( v i ) Fate of Corpora Luteum C e l l s (via.) The R e p r o d u c t i v e  Section I I I :  66  C y c l e W i t h i n the Ovary.........  67  The E f f e c t s of O v a r i a n S t e r o i d s on Oogenesis and V i t e l l o g e n e s i s  70  Introduction  71  Methods and M a t e r i a l s  73  ( i ) E f f e c t s of S t e r o i d s on O o g o n i a l M i t o s i s ( i i ) E f f e c t s of S t e r o i d s on V i t e l l o g e n e s i s  -  73 74  ( i i i ) E f f e c t s of Long Term Treatment o f S t e r o i d s on Gravid F i s h  :  75  vii T a b l e o f Contents  (cont'd) Page  ( i v ) E f f e c t s o f S t e r o i d s on O v u l a t i o n Results  76 78  ( i ) E f f e c t s o f S t e r o i d s on O o g o n i a l M i t o s i s ( i i ) E f f e c t s of S t e r o i d s on V i t e l l o g e n e s i s  78 80  ( i i i ) E f f e c t s o f Long Term A d m i n i s t r a t i o n o f S t e r o i d s on G r a v i d F i s h ( i v ) E f f e c t s o f S t e r o i d s on O v u l a t i o n Discussion  86 91 94  ( i ) E f f e c t s of S t e r o i d s on O o g o n i a l M i t o s i s  94  ( i i ) E f f e c t o f S t e r o i d s on V i t e l l o g e n e s i s . . . . .  95  ( i i i ) I n d u c t i o n o f O v u l a t i o n by S t e r o i d s  98  ( i v ) The Mechanism o f E n d o c r i n e C o n t r o l o f Oogenesis General Discussion (i) Possible Practical Applications References  98 103 105 107  viii LIST OF TABLES Page TABLE 1  2  3  4  5  6  7  8  9  L i s t o f h i s t o l o g i c a l and h i s t o c h e m i c a l techniques used w i t h the a p p r o p r i a t e p r e p a r a t i v e techniques  8  A summary of the h i s t o c h e m i c a l p r o p e r t i e s o f y o l k inclusions  17  The degree of a t r e s i a i n v a r i o u s o v a r i e s a f t e r hypophysectomy ....  39a,  H y d r o x y s t e r o i d dehydrogenases d e t e c t e d i n the o v a r i e s of normal v i t e l l o g e n i c o v a r i e s .  45  H y d r o x y s t e r o i d dehydrogenase d e t e c t e d i n the o v a r i e s of hypophysectomized g o l d f i s h  51  H y d r o x y s t e r o i d dehydrogenases i n the o v a r i e s of hypophysectomized g o l d f i s h i n j e c t e d w i t h LH  54  Hydroxysteroid post-ovulatory  56  dehydrogenases i n the o v a r i e s o f fish....'  Summary of the e f f e c t s of s t e r o i d a d m i n i s t r a t i o n on oogonial mitosis  79  The e f f e c t s o f s t e r o i d s on v i t e l l o g e n e s i s i n hypophysectomized g o l d f i s h  82  10  Comparison of the h i s t o c h e m i c a l p r o p e r t i e s o f p r e g nenolone induced y o l k granules w i t h those of normal vitellogenic ovaries....... . 84  11  The long-term e f f e c t s o f s t e r o i d a d m i n i s t r a t i o n the o v a r i e s of g r a v i d g o l d f i s h  12  The  on  e f f e c t s o f s t e r o i d s on o v u l a t i o n i n g r a v i d f i s h . .  89 92  ix LIST OF FIGURES FIGURE  Page  1  An oogonia o c c u r i n g s i n g l y i n the ovary....  12  2  Oogonial cyst c o n t a i n i n g s e v e r a l oogonia.......  12  3  Chromatin  12  4  P e r i n u c l e o l a r stage o o c y t e s . . . .  5  I n i t i a l y o l k v e s i c l e stage o o c y t e . . . . . . . . . .  6  L a t e y o l k v e s i c l e stage o o c y t e  7  E a r l y y o l k g r a n u l e stage oocyte  8  L a t e y o l k g r a n u l e stage o o c y t e  9  Ovarian s e c t i o n stained with p e r i o d i c reaction....  n u c l e o l a r stage o o c y t e s  .........  12 14 14 ...  14 14  acid-Schiff's 19  10  Oocytes  11  O v a r i a n s e c t i o n s t a i n e d w i t h the t e t r a z o t i z e d dianisidine reaction for proteins  12  ..  stained with d i n i t r o f l u r o b e n z e n e r e a c t i o n . . . .  O v a r i a n s e c t i o n s t a i n e d w i t h the f e r r i c method f o r s u l p h y d r y l groups  19  o19  ferricyanide 19  13  C r y o s t a t s e c t i o n of ovary s t a i n e d w i t h Sudan B l a c k B.  19  14  O v a r i a n s e c t i o n s s t a i n e d w i t h a c i d haematin f o r phospholipids  19  A diagrammatic f i s h ovary  27  15  16  r e p r e s e n t a t i o n o f oogenesis i n g o l d •.-  Diagrammatic p r e s e n t a t i o n o f the h y d r o x y s t e r o i d dehydrogenase r e a c t i o n . . .  17  a-stage  18  (3-stage o f f o l l i c u l a r  19  Y~  20  Higher m a g n i f i c a t i o n of the y-stage a t r e t i c f o l l i c l e .  s t a  g  e  of f o l l i c u l a r  atretic  2>&o~  atresia  41  atresia...........  41  follicle  41 41  X  L i s t of F i g u r e s (cont'd) Page FIGURE 21  6-stage a t r e t i c f o l l i c l e  22  e-stage a t r e t i c f o l l i c l e , showing the d i f f e r e n t i a t i o n of oogonia '  41  Stage I p o s t - o v u l a t o r y corpus l u t e u m , w i t h e v i d e n c e of the r u p t u r e o f f o l l i c u l a r w a l l  44  Stage I p o s t - o v u l a t o r y corpus luteum w i t h o u t any s i g n of f o l l i c u l a r w a l l rupture  44  A h i g h m a g n i f i c a t i o n o f Stage I p o s t - o v u l a t o r y corpus luteum d e m o n s t r a t i n g h y p e r t r o p h y o f granulosa c e l l s . . . . . . . . . . . .  44  26  Stage I I p o s t - o v u l a t o r y corpus luteum....  44  27  A higher m a g n i f i c a t i o n of stage I I p o s t - o v u l a t o r y corpus luteum........  44  28  Stage I I I p o s t - o v u l a t o r y corpus luteum  44  29  L o c a l i z a t i o n o f 3 3 - h y d r o x y s t e r o i d dehydrogenase i n the granulosa c e l l s of oocytes  48  Lack of h y d r o x y s t e r o i d dehydrogenase l a t e y o l k granule stage oocytes  48  23  24  25  30  31  32  33 34  35  36  . ...  41  activity in  D e m o n s t r a t i o n o f 1 7 ( 3 - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n y s t a g e a t r e t i c o o c y t e s o f hypophysectomized g o l d f i s h  48  3 f ? - h y d r o x y s t e r o i d dehydrogenase i n the p o s t ovulatory f o l l i c l e s after ovulation  48  A u t o r a d i o g r a p h y of g o l d f i s h o v a r i e s showing the l a b e l l i n g of f o l l i c u l a r c e l l s by thymidine-^H  59  A u t o r a d i o g r a p h y o f g o l d f i s h o v a r i e s showing the l a b e l l i n g o f 6-stage corpus l u t e a c e l l s  59  A u t o r a d i o g r a p h y showing the l a b e l l i n g o f o o g o n i a 30 days a f t e r t h y m i d i n e - ^ H i n j e c t i o n  59  A u t o r a d i o g r a p h o f l a b e l l e d o o c y t e 30 days a f t e r the i n i t i a l t h y m i d i n e - 3 i n j e c t i o n  59  H  XX  L i s t of Figures  (cont'd)  37  A summary o f corpus luteum f o r m a t i o n i n g o l d f i s h . . . . . .  38  Y o l k v e s i c l e s oocytes of hypophysectomized treated w i t h estrogen  Page 69  fish 81  39  Ovary o f hypophysectomized g o l d f i s h  81  40  Pregnenolone induced y o l k g r a n u l e f o r m a t i o n i n the o o c y t e o f hypophysectomized g o l d f i s h  81  41  H i s t o g r a m of the e f f e c t s o f e s t r a d i o l , e s t r o n e , and e s t r i o l on y o l k v e s i c l e f o r m a t i o n i n hypophysectomized f i s h . . . . . . .  .  85  42  H i s t o l o g y o f g r a v i d ovary t r e a t e d w i t h e s t r o g e n  88  43  H i s t o l o g y of g r a v i d ovary t r e a t e d w i t h d e o x y c o r t i c o s t e r o l containing post-ovulatory corpora l u t e a . . . . . .  88  44  O v a r i e s o f f i s h t h a t are ready t o o v u l a t e  88  45  O v a r i e s o f f i s h w i t h immature o o c y t e s . . . . . . . .  88  46  Comparison of the e f f e c t s of e s t r o n e , e s t r a d i o l , e s t r i o l and t e s t o s t e r o n e on a t r e s i a o f y o l k y o o c y t e s i n gravid f i s h  90  47  Diagram showing the i n t e r r e l a t i o n s h i p between p i t u i t a r y g o n a d o t r o p i n , s t e r o i d o g e n e s i s and o o g e n e s i s .  48  The  s t e r o i d m e t a b o l i c pathway i n t e l e o s t o v a r i e s  99  xii ACKNOWLEDGEMENTS I t i s my great pleasure to acknowledge my indebtedness and g r a t i t u d e to my research s u p e r v i s o r , Dr. W.S.Hoar, f o r h i s i n t e r e s t , advise and encouragements  throughout the course of t h i s study. I would l i k e to thank  the members of my d o c t o r a l committee: Dr.E.M.Donaldson, Dr.H.D.Fisher, Dr.P.Ford, Dr.N.R.Li ley and Dr .A.M.Perks f o r t h e i r generous assistance and advise during the study and f o r reading the t h e s i s . I extend my g r a t e f u l a p p r e c i a t i o n to Miss.D.Hards, f o r the preparat i o n of e x c e l l e n t h i s t o l o g i c a l s e c t i o n s , Mrs. Cathy D r i e d z i c f o r her caref u l preparation of the i l l u s t r a t i o n s , and Mrs, Pat Waldron, f o r typing the f i n a l copy of the t h e s i s . I iam obligated to my fellow graduate students, Kenneth Chan, Ross Neuman, Jean-Guy Godin, B i l l Marshall and e s p e c i a l l y Norman Stacey for many s t i m u l a t i n g discussions. My thanks to those that plunged i n t o the i c y winter waters of Vancouver i n search of the e l u s i v e Coryphopterus n i c h o l s i • To everyone who have made my stay here i n Vancouver meaningful and enjoyable, I say thank you. F i n a n c i a l support f o r t h i s research was received from the National Research Council of Canada through g r a n t s - i n - a i d to Dr.W.S.Hoar and a Canadian Commonwealth Scholarship to myself. I a l s o wish to thank the U n i v e r s i t i Sains Malaysia f o r granting me a Fellowship i n t h e i r Academic S t a f f T r a i n i n g Scheme.  GENERAL INTRODUCTION I n t e l e o s t , the r e g u l a t i o n o f gametogenetic a c t i v i t y by p i t u i t a r y i s now 1957;  Dodd, 1960;  w e l l e s t a b l i s h e d (see r e v i e w s by P i c k f o r d and Hoar, 1965;  Donaldson, 1973).  1969;  L o f t s , 1968;  Reinboth,  (Yamazaki and Donaldson, 1968;  1972;  A d m i n i s t r a t i o n o f mammalian L i l e y and Donaldson, 1968;  to o v a r i a n r e g r e s s i o n and a t r e s i a , hypophysectomy i n h i b i t s i n t e l e o s t o v a r i e s (Yamazaki and Donaldson, 1969; a d m i n i s t r a t i o n of gonadotropin  a c t i v i t y i n hypophysectomized f i s h . whether g o n a d o t r o p i n  gonadotropin  In addition steroidogenesis  Wiebe, 1969)  increase 3g-hydroxysteroid However, i t i s s t i l l  and  Sundararaj  a, b) m i t i g a t e s the e f f e c t s o f hypophysectomy.  whether i t o p e r a t e s  Atz,  Hypophysectomy r e s u l t s i n r e g r e s s i o n o f o v a r i e s  a t r e s i a of y o l k - l a d e n o o c y t e s .  e t a l . , 1972  the  while  dehydrogenase uncertain  a c t s d i r e c t l y on o o g e n e s i s and v i t e l l o g e n e s i s o r t h r o u g h the p r o d u c t i o n o f a second h o r m o n e — p o s s i b l y  a s t e r o i d o r whether b o t h g o n a d o t r o p i n s and s t e r o i d s a r e d i r e c t l y i n volved. There i s o n l y s c a n t y i n f o r m a t i o n c o n c e r n i n g f o r m a t i o n and development. o o g o n i a i n f i s h (Phoxinus  Bullough  endocrinology  of ovum  (1942) observed a g r e a t e r number o f  phoxinus) t r e a t e d w i t h estrogens  but d i d not  d e s c r i b e the d e t a i l e d changes i n the ovary nor the i n t e r r e l a t i o n s h i p of gonadotropin  and s t e r o i d s i n the c o n t r o l s .  A g a i n s t u d i e s o f sex r e v e r s a l  u s i n g s t e r o i d s suggest an e f f e c t o f the s t e r o i d s on the p r i m o d i a l germ c e l l s but t h e c y t o l o g i c a l changes have not been w e l l d e s c r i b e d ; Yamamoto (1969) r e v i e w s  the l i t e r a t u r e .  F i n a l l y i n v e s t i g a t i o n s of a c t i o n s of  2 s t e r o i d s on y o l k f o r m a t i o n  have p r o v i d e d  rather c o n t r a d i c t o r y evidence  c o n c e r n i n g t h e i r p o s s i b l e r o l e i n y o l k s y n t h e s i s and d e p o s i t i o n . though e s t r o g e n s s t i m u l a t e the l i v e r t o s y n t h e s i z e y o l k (Egami, 1955; 1961;  B a i l e y , 1957;  P l a c k et^ a l . , 1971;  Ho and Vanstone, 1961;  precursors  U r i s t and  A i d a e t a l . , 1973), B u l l o u g h  The  Schjeide,  (1942), Tavolga  (1949) , B e r k o w i t z (1951) , Egami (1955) observed t h a t e s t r o g e n t i o n i n h i b i t e d v i t e l l o g e n e s i s and  Al-  administra-  caused f o l l i c u l a r a t r e s i a .  r o l e of s t e r o i d s i n i n d u c i n g o v u l a t i o n i n t e l e o s t i s w e l l  cumented s i n c e K i r s h e n b l a t t e r o n e and  (1952, 1959)  deoxycorticosterone  whereas e s t r o g e n s and  do-  f i r s t demonstrated t h a t p r o g e s -  i n d u c e d o v u l a t i o n i n Misgurnus f o s s i l i s ,  androgens had no e f f e c t .  tained w i t h Heterdpneustes f o s s i l i s  S i m i l a r r e s u l t s were  (Ramaswami, 1962).  Further,  ob-  Sundararaj  and Goswami (1966) observed t h a t c o r t i c o s t e r o i d s i n d u c e d o v u l a t i o n and  in  some i n s t a n c e s o v i p o s i t i o n i n b o t h i n t a c t and hypophysectomized f i s h .  In  a d d i t i o n to the i n v i v o s t u d i e s , Goswami and Hirose  S u n d a r a r a j (1971  a, b,)  and  (1972) demonstrated the i n d u c t i o n o f i n v i t r o o v u l a t i o n by s t e r o i d s .  T h i s b r i e f summary i n d i c a t e s the u n s a t i s f a c t o r y s t a t e of knowledge c o n c e r n i n g e n d o c r i n e c o n t r o l o f ovum f o r m a t i o n  and  development.  This  i n v e s t i g a t i o n i s a study of the e n d o c r i n e r e g u l a t i o n o f o o g e n e s i s o v a r i a n development i n g o l d f i s h , w i t h p a r t i c u l a r r e f e r e n c e of s t e r o i d hormones.  I t was  and  t o the r o l e  c o n v e n i e n t to d i v i d e the p r e s e n t a t i o n  t h r e e s e c t i o n s , the h i s t o l o g i c a l and h i s t o c h e m i c a l  a n a l y s i s of oogenesis  and v i t e l l o g e n e s i s , the p i t u i t a r y c o n t r o l of s t e r o i d o g e n e s i s e s p e c i a l l y the corpus l u t e u m , and on o o g e n e s i s and v i t e l l o g e n e s i s .  into  i n the o v a r y ,  f i n a l l y the e f f e c t s o f o v a r i a n  steroids  3  SECTION I : THE HISTOLOGY AND HISTOCHEMISTRY OF OVARIAN DEVELOPMENT  4  INTRODUCTION  The p r e s e n t i n v e s t i g a t i o n i s an a n a l y s i s o f c y t o l o g i c a l  changes  d u r i n g the m a t u r a t i o n o f o o c y t e s o f g o l d f i s h , C a r a s s i u s a u r a t u s .  Such  an a n a l y s i s s h o u l d p r o v i d e a b a s i s f o r study of the e n d o c r i n e c o n t r o l of  o o g e n e s i s and v i t e l l o g e n e s i s i n t e l e o s t o v a r i e s .  Although early  stages o f o o c y t e development have been w e l l documented, phase has n o t p r e v i o u s l y been d e t a i l e d .  t-h& second growth  I n t h i s study a t t e n t i o n i s  d i r e c t e d to the second growth p h a s e — e s p e c i a l l y the f o r m a t i o n and c h e m i c a l c o m p o s i t i o n of y o l k  inclusions.  There a r e numerous s t u d i e s o f o v a r i a n h i s t o l o g y d u r i n g o o c y t e growth and development.  B a r r (1963) , L e h r i  (1968) and Lambert  v i d e d good d e s c r i p t i o n s o f t e l e o s t o o c y t e growth. an e x c e l l e n t d e s c r i p t i o n o f t h e h i s t o l o g i c a l  (1970) have p r o -  Yamazaki (1965) gave  changes i n the o v a r i e s o f  g o l d f i s h , while e a r l i e r observations of ovarian histology i n g o l d f i s h were p r o v i d e d by Stomsten (1931) and Beach (1959). It  i s g e n e r a l l y r e c o g n i z e d t h a t oocyte development o c c u r s i n two  phases. trol, (see  The f i r s t growth phase, which i s independent o f p i t u i t a r y con-  i n v o l v e s the i n c r e a s e i n s i z e o f oocytes w i t h some n u c l e a r changes r e v i e w by Hoar, 1969).  The second phase i s c h a r a c t e r i z e d by t h e  d e p o s i t i o n o f y o l k ; t h e r e i s much c o n f u s i o n c o n c e r n i n g t h i s phase especially  f o r m a t i o n o f the y o l k i n c l u s i o n s  Malone and H i s a o k a , 1963).  (Anderson, 1968; Guraya, 1965;  One o f t h e reasons f o r the c o n f l i c t i n g  opin-  i o n s may be a t t r i b u t e d t o a f a i l u r e to d i s t i n g u i s h t h e v a r i o u s y o l k components.  5 I n i t i a l h i s t o c h e m i c a l s t u d i e s o f v i t e l l o g e n e s i s were devoted t o the d i s t r i b u t i o n o f p o l y s a c c h a r i d e s 1954;  (Ihnumaxa and Tsukuda, 1952; A k e t a ,  and Yamamoto, 1955; 1956; 1958) b u t few attempts were made t o  c o r r e l a t e the histochemical d i s t r i b u t i o n o f polysaccharides w i t h chemical  components.  L a t e r , t h e emphasis s h i f t e d t o t h e l i p i d  other  composi-  t i o n o f o o c y t e s and t h e o r i g i n o f y o l k (Chopra, 1958; Yamamoto, 1958; N a t h , 1960).  These l a t e r w o r k e r s have been concerned about t h e o r i g i n  of y o l k and d i r e c t e d t h e i r a t t e n t i o n t o t h e " y o l k n u c l e u s " now r e c o g n i z e d as a d i f f u s e d c o n c e n t r a t i o n o f m i t o c h o n d r i a . y o l k n u c l e u s s t i l l remains (Guraya, 1963; 1968). been made t o sCtidy t h e h i s t o c h e m i c a l c o m p o s i t i o n teleost ovaries.  Some i n t e r e s t i n R e c e n t l y a t t e m p t s have  of yolk inclusions i n  Malone and H i s a o k a (1963), Guraya (1965) and Anderson  (1968) s t u d i e d t h e h i s t o c h e m i c a l d i s t r i b u t i o n o f p o l y s a c c h a r i d e s , and p r o t e i n s i n t e l e o s t o v a r i e s . chemical defined.  composition  lipids,  I n s p i t e o f t h e s e s t u d i e s , however, t h e  of the v a r i o u s y o l k i n c l u s i o n s i s s t i l l  poorly  6  METHODS  MAINTENANCE OF GOLDFISH . G o l d f i s h ( C a r a s s i u s a u r a t u s L.) o f the common comet v a r i e t y (1015 cm s t a n d a r d l e n g t h ) were o b t a i n e d from a commercial s u p p l i e r (Hartz Mountain Pet S u p p l i e r s L i m i t e d , Richmond, B r i t i s h  Columbia).  They were  kept i n f i b e r g l a s s tanks s u p p l i e d w i t h f l o w i n g d e c h l o r i n a t e d f r e s h water which was a r e a t e d w i t h compressed  air.  G o l d f i s h were f e d once a day  w i t h " C l a r k ' s New Age T r o u t Feed" (Moore-Clark Co., S a l t Lake C i t y , U t a h ) . T h i s d i e t was supplemented, once a week, w i t h f r o z e n b r i n e "Natural" goldfish.  shrimp.  The b a s i c s t o c k o f f i s h was kept under  natural  p h o t o p e r i o d and temperature, except i n w i n t e r when the temperature was r a i s e d to about 10°C w i t h immersion h e a t e r s . Gravid g o l d f i s h .  Another group o f g o l d f i s h was kept a t 10-13°C  with a  d a i l y p h o t o p e r i o d o f 16 h o u r s .  F l u o r e s c e n t 40 watt lamps,  trolled  by time c l o c k s , s u p p l i e d the l i g h t .  A f t e r about 3 months o f such  treatment most o f t h e females become g r a v i d . not  Refractory goldfish. to  They remain g r a v i d and w i l l  o v u l a t e i f kept below 13°C (Yamamoto et a l . , 1966).  used i n experiments r e q u i r i n g g r a v i d  These f i s h were  goldfish.  A group of g r a v i d g o l d f i s h was  a q u a r i a w i t h d e c h l o r i n a t e d water a t 13°C.  transferred  A f t e r t r a n s f e r , the water  temperature was s l o w l y r a i s e d to room temperature induced o v u l a t i o n .  con-  (22-25°C)  and t h i s  The f i s h were allowed t o o v u l a t e a t random.  They  were kept a t room temperature and 16 hours d a i l y p h o t o p e r i o d f o r 3-4 months when they became r e f r a c t o r y and had o v a r i e s s i m i l a r t o those o f  7  hypophysectomized g o l d f i s h . r e f r a c t o r y i f maintained  Under t h e s e c o n d i t i o n s , g o l d f i s h r e m a i n  at room temperature and cease t o be r e f r a c t o r y  o n l y when p l a c e d i n water below 20°C. HISTOLOGY AND  HISTOCHEMISTRY OF OVARIES  The h i s t o l o g y and h i s t o c h e m i s t r y o f g o l d f i s h o v a r i e s were s t u d i e d a t v a r i o u s s t a g e s of m a t u r i t y .  F i s h from the " n a t u r a l " s t o c k were sam-  p l e d throughout the y e a r t o ensure t h a t a l l stages o f o o g e n e s i s were i n cluded.  The h i s t o l o g y of f r e s h l y o v u l a t e d eggs were a l s o examined.  The  o v a r i e s of hypophysectomized f i s h were examined h i s t o l o g i c a l l y i n s t u d i e s on  the enzymic a c t i v i t y o f h y d r o x y s t e r o i d dehydrogenases.  o f p o s t - o v u l a t o r y f i s h were a l s o examined h i s t o l o g i c a l l y . techniques  The The  ovaries various  used a r e summarized i n T a b l e 1.  I n a l l c a s e s , the f i s h were d e c a p i t a t e d and t h e i r o v a r i e s p l a c e d i n v a r i o u s f i x a t i v e s i n c l u d i n g B o u i n ' s f l u i d , Baker's n e u t r a l f o r m a l i n , Smith's d i c h r o m a t e and Carnoy's f l u i d  ( G u r r , 1962).  o v a r i e s f o r h i s t o c h e m i c a l examination  was  A p o r t i o n o f the  f r o z e n immediately  w i t h an ace-  tone-dry  i c e m i x t u r e o r " C r y o w i c k " ( I n t e r n a t i o n a l Equipment Co.,  setts) .  These u n f i x e d but f r o z e n o v a r i e s were s e c t i o n e d a t 10 y w i t h  c r y o s t a t and mounted on a l b u m i n i z e d  Massachuthe  slides for histochemical staining.  O v a r i e s w i t h o n l y e a r l y stage oocytes were embedded r o u t i n e l y i n paraffin.  Y o l k l a d e n t i s s u e s are v e r y b r i t t l e and d i f f i c u l t t o s e c t i o n  when embedded r o u t i n e l y i n p a r a f f i n .  A t e c h n i q u e w h i c h uses Smith's  d i c h r o m a t e f i x a t i v e f o l l o w e d by double embedding of the o v a r i e s was veloped  t o overcome t h i s problem.  D e t a i l s a r e as f o l l o w s ;  de-  T a b l e 1:  L i s t o f t h e v a r i o u s h i s t o l o g i c a l and h i s t o c h e m i c a l t e c h n i q u e s p r e p a r a t i v e methods used.  TECHNIQUES  w i t h the a p p r o p r i a t e  FIXATIVES  SECTIONS  H a e m a t o x y l i n and e o s i n ( G u r r , 1962)  B, S, NF  Paraffin  M a l l o r y ' s trichrome  B, S, NF  Paraffin  HISTOLOGY  ( G u r r , 1962)  POLYSACCHARIDES Best's  carmine ( P e a r s e , 1968)  Paraffin  P e r i o d i c a c i d - S c h i f f ' s (McManus, 1948)  B, S, NF  Paraffin  D i a l y s e d i r o n f o r a c i d m u c o p o l y s a c c h a r i d e s ( H a l e , 1946)  B, S, NF  Paraffin  A l c i a n B l u e , pH 2.5 ( P e a r s e , 1968)  B, S, NF  Paraffin  D i n i t r o f l u r o b e n z e n e method ( B u r s t o n e , 1955)  S, NE, F  P a r a f f i n , Frozen  T e t r a - a z o t i z e d o - d i a n i s i d i n e ( D a n i e l l i , 1953)  S, NE, F  P a r a f f i n , Frozen  S a k a g u c h i ' s r e a c t i o n (Baker, 1953)  S, NF, F  P a r a f f i n , Frozen  M i l l o n ' s r e a c t i o n (Baker, 1956)  S, NF, F  P a r a f f i n , Frozen  B, S, NF  Paraffin  PROTEINS  F e r r i c f e r r i c y a n i d e method f o r SH (Chevremont & F r e d e r i c k , 1943) DDD r e a c t i o n f o r SH ( B a r n e t t & Seligman, 1952)  Continued  00  TABLE 1 (Cont'd) TECHNIQUES  FIXATIVES  SECTIONS  Sudan B l a c k B (Chayen et a l . , 1969)  NF,  F  Frozen  O i l Red  NF,  F  Frozen  LIPIDS  0 (Lillie,  1944)  A c i d haematin ( B a k e r , Copper p h t h a l o c y a n i n  Footnotes: -  Key  1946)  B, S, NF,  method ( K l u v e r & B a r r e r a ,  1953)  NF,  F  P a r a f f i n , Frozen  F  FFrozen  to a b b r e v i a t i o n o f f i x a t i v e s , B, B o u i n ' s f l u i d ; S, Smith's d i c h r o m a t e  f i x a t i v e ; NF,  Baker's n e u t r a l f o r m a l i n ; C, Carnoy's f l u i d ; F, u n f i x e d  and  frozen ovaries. 2 P a r a f f i n s e c t i o n s (7 y) were s e c t i o n e d on r o t a r y microtome. were cut on the c r y o s t a t at -15°C  Frozen sections  (10-12 u) .  VO  10  The o v a r i e s were f i x e d i n Smith's f i x a t i v e f o r 12-24 h r and t h e n washed i n r u n n i n g w a t e r f o r 6-12 h r .  The washed o v a r i e s were d e h y d r a t e d  t h r o u g h a s e r i e s o f a l c o h o l s up t o 95% a l c o h o l .  O v a r i e s were n o t com-  p l e t e l y d e h y d r a t e d s i n c e some r e s i d u a l water gave b e t t e r h i s t o l o g i c a l r e s u l t s w i t h y o l k l a d e n eggs (Rugh, 1968). m e t h y l benzonate f o r up t o 5 h r .  The t i s s u e s were c l e a r e d i n  A f t e r c l e a r i n g t h e y were i n i t i a l l y i n -  f i l t r a t e d w i t h 1% c e l l u l o i d i n i n m e t h y l benzoate and t h e n p l a c e d  over-  n i g h t i n c h l o r o f o r m b e f o r e i n f i l t r a t i n g w i t h p a r a f f i n ( P a r a p l a s t , m.p. 56-58°C) f o r s e c t i o n i n g . A s m a l l p i e c e of some o f neutral formalin.  t h e o v a r i e s was f i x e d o v e r n i g h t  i n Baker's  These t i s s u e s were washed i n r u n n i n g w a t e r f o r 12-24 h r ,  f r o z e n i n e i t h e r acetone-dry i c e mixture  o r w i t h Cryowick and mounted on  specimen h o l d e r s w i t h "O.C.T. compound" (Ames Co., I n d i a n a ) — s o l u b l e , embedding medium f o r f r o z e n t i s s u e s .  a water  S e c t i o n s o f 10-12 y t h i c k -  ness were c u t on t h e c r y o s t a t f o r h i s t o l o g i c a l and h i s t o c h e m i c a l s t a i n i n g . The s p e c i f i c i t y o f p o l y s a c c h a r i d e  s t a i n s was c o n f i r m e d  by s t a i n i n g  s e r i a l s e c t i o n s w h i c h were p r e t r e a t e d w i t h 0.5% d i a s t a s e (Chayen e t a l . , 1969).  The p e r i o d i c a c i d - S c h i f f ' s p o l y s a c c h a r i d e  confirmed  r e a c t i o n was f u r t h e r  by a c e t y l a t i n g s e r i a l s e c t i o n s b e f o r e s t a i n i n g ( L i l l i e ,  1954).  The s p e c i f i c i t y of the r e a c t i o n s f o r s u l p h y d r y l groups was confirmed t r e a t i n g s e r i a l s e c t i o n s w i t h 0.1 M, N - e t h y l - m a l e i m i d e ( P e a r s e , The o c c u r r e n c e of s i m p l e l i p i d s was confirmed  by  1968).  by p y r i d i n e e x t r a c t i o n o f  s e r i a l s e c t i o n s ( B a k e r , 1946) b e f o r e s t a i n i n g i n e i t h e r Sudan B l a c k B o r O i l Red 0.  11  RESULTS  HISTOLOGY OF OOCYTE GROWTH AND DEVELOPMENT Oogonia.  These c e l l s o c c u r r e d s i n g l y o r i n s m a l l n e s t s i n a l l  o v a r i e s examined ( F i g . 1 & 2 ) .  They were most numerous i n p o s t - o v u l a -  t o r y f i s h and i n o v a r i e s w i t h numerous c o r p o r a l u t e a .  Oogonial  were l e s s abundant i n maturing o v a r i e s w i t h y o l k y o o c y t e s . ium was about  Each oogon-  10-15 u i n diameter w i t h a s i n g l e n u c l e u s and one l a r g e  n u c l e o l u s which s t a i n e d deeply w i t h haematoxylin.  In sections of ovaries  s t a i n e d w i t h M a l l o r y ' s t r i c h r o m e the n u c l e o l i were deep F i r s t Growth Phase. oogonia.  cysts  orange.  F i r s t growth phase oocytes were l a r g e r  than  Primary oocytes were d i s t i n g u i s h e d from oogonia by the presence  of d i s t i n c t chromosomes i n v a r i o u s s t a g e s o f m e i o t i c prophase.  T h i s phase  was s u b - d i v i d e d i n t o two d e f i n i t e stages s e p a r a t e d a c c o r d i n g to t h e f e a tures o f the nucleus. and  These two s t a g e s were t h e c h r o m a t i n n u c l e o l a r stage  the p e r i - n u c l e o l a r s t a g e . The  first  stage a f t e r the development o f o o c y t e s i s the chromatin  n u c l e o l a r stage.  The oocytes i n t h i s stage had a n u c l e u s w i t h a s i n g l e  conspicuous n u c l e o l u s ( F i g . 3 ) . Chromatin nucleolus.  threads were a t t a c h e d t o the  06cyte a t t h i s stage were s l i g h t l y l a r g e r than oogonia and  had diameters o f 12-20 y. The stage.  chromatin n u c l e o l a r s t a t e i s f o l l o w e d by the p e r i - n u c l e o l a r The p e r i n u c l e o l a r stage oocytes were e a s i l y i d e n t i f i e d by the  p e r i p h e r a l arrangement o f a l a r g e number o f s m a l l n u c l e o l i on t h e i n n e r s i d e o f the n u c l e a r membrane ( F i g . 4 ) . The n u c l e u s was e n l a r g e d and t h e  12  Figure  1:  An oogonium, as i n d i c a t e d by the arrow, singly.  Figure  2:  Haematoxylin  Oogonial cyst  and  eosin.  (arrow) c o n t a i n i n g  several  w i t h large n u c l e i deeply stained with  Figure  3:  Chromatin  nucleolar  Haematoxylin  Figure  4;  and  stage oocytes  oogonia  haematoxylin.  (arrows).  eosin.  P e r i n u c l e o l a r stage o o c y t e s . stage o o c y t e s  occuring  The e a r l y p e r i n u c l e o l a r  (Arrow 1) are s m a l l e r  i n size  and  s t a i n e d d e e p l y w i t h haematoxylin  as compared w i t h  l a t e p e r i n u c l e o l a r stage oocytes  (Arrow  2).  13  chromosomes l o s t  t h e i r d i s t i n c t nature.  Together w i t h the  enlargement  of  the n u c l e u s , the cytoplasm i n c r e a s e d d r a s t i c a l l y i n volume.  at  the p e r i n u c l e o l a r s t a g e , had diameters o f 20-150 y.  of  o o c y t e s a t t h i s stage s t a i n e d deeply w i t h haematoxylin.  c o n t r a s t to o t h e r stages where the cytoplasm was toxylin.  The  Oocytes,  cytoplasm T h i s was  not s t a i n e d by haema-  M a l l o r y ' s t r i c h r o m e s t a i n e d the cytoplasm deep r e d .  The  n u c l e o l i were v e r y conspicuous w i t h M a l l o r y ' s t r i c h r o m e and appeared orange ( F i g . 5 ) .  A t the end o f the p e r i n u c l e o l a r stage the  lost i t s affinity  f o r haematoxylin.  Second growth phase.  The  second  cytoplasm  Two  types o f y o l k  i n c l u s i o n s were e a s i l y d i s t i n g u i s h e d by t h e i r s i z e and s t a i n i n g (10-15 y i n diameter)  with Mallory's trichrome.  These two  properties.  stained blue with Mallory's  t r i c h r o m e ; the s m a l l e r y o l k g r a n u l e s (about 1 y i n diameter) red  deep  growth phase oocytes were c h a r a c -  t e r i z e d by the f o r m a t i o n and accumulation o f y o l k .  The l a r g e r y o l k v e s i c l e  in  s t a i n e d deep  types o f y o l k were formed sequen-  t i a l l y with yolk v e s i c l e s deposited before yolk granules. Yolk v e s i c l e stage. presence (Fig.  5).  The  first  the  o f a r i n g o f v e s i c l e s i n the p e r i p h e r y o f the cytoplasm o f oocytes There was  no p a t t e r n i n the l a t e r d e p o s i t i o n of y o l k v e s i c l e s .  The v e s i c l e s were formed randomly. 150  i n d i c a t i o n o f y o l k f o r m a t i o n was  y i n diameter.  The  oocyte at t h i s stage was  about  I n i t i a l l y , each v e s i c l e formed as a minute body but  g r a d u a l l y i n c r e a s e d i n s i z e u n t i l i t reached a diameter o f 10-15  y.  These  y o l k v e s i c l e s i n c r e a s e d b o t h i n s i z e and numbers u n t i l they o c c u p i e d the whole cytoplasm o f o o c y t e s ( F i g . 6 ) .  In o v a r i a n s e c t i o n s s t a i n e d w i t h  14  F i g u r e 5:  Oocyte i n the i n i t i a l y o l k v e s i c l e stage w i t h characteristic ring  of y o l k v e s i c l e s  (v).  the  Mallory's  trichrome.  F i g u r e 6:  L a t e y o l k v e s i c l e stage oocyte trichrome. The  F i g u r e 7:  The y o l k v e s i c l e s  zona r a d i a t a  stained with  (v) are more abundant.  (z) becomes t h i c k e n e d and  red w i t h M a l l o r y ' s  trichrome.  E a r l y y o l k granule  stage oocyte  trichrome.  The  Mallory's  i s stained  stained with  red y o l k g r a n u l e s  Mallory's  (g) accumulate between  the b l u e y o l k v e s i c l e s ( v ) .  F i g u r e 8:  Late yolk granule trichrome.  The  stage oocytes  Mallory's  r e d y o l k g r a n u l e s have accumulated toward  the c e n t e r o f oocytes t o the  stained with  periphery.  and  d i s p l a c i n g the y o l k v e s i c l e s  15 h a e m a t o x y l i n and e o s i n . t h e y o l k v e s i c l e s appeared t h i s proved t o be an a r t i f a c t .  t o be v a c u o l e s b u t  With M a l l o r y ' s t r i c h r o m e , these v e s i c l e s  stained l i g h t blue. A t t h i s s t a g e o f o o c y t e development t h e f o l l i c u l a r f a i r l y w e l l developed. able.  l a y e r s were  The t h e c a l and g r a n u l o s a c e l l s were d i s t i n g u i s h -  The zona r a d i a t a s t a r t e d t o develop d u r i n g t h e y o l k v e s i c l e s t a g e .  I n i t i a l l y t h e zona r a d i a t a was t h i n and s t a i n e d d a r k r e d w i t h M a l l o r y ' s trichrome.  By t h e l a t e s t a g e s of y o l k v e s i c l e f o r m a t i o n , however, t h e  zona r a d i a t a was almost f u l l y d e v e l o p e d and i t s r a d i a t e n a t u r e was apparent. Yolk granule stages.  The f o r m a t i o n o f y o l k g r a n u l e s c h a r a c t e r i z e d  the f i n a l s t a g e of v i t e l l o g e n e s i s and o o c y t e development. formed o n l y i n o o c y t e s w i t h f u l l y d e v e l o p e d y o l k v e s i c l e s . g r a n u l e s f i r s t appeared  Yolk granules The y o l k  c l o s e t o t h e zona r a d i a t a ( F i g . 7) and were e a s i l y  d i s t i n g u i s h a b l e as r e d g r a n u l e s a f t e r s t a i n i n g w i t h M a l l o r y ' s t r i c h r o m e ; they were n o t v e r y e v i d e n t a f t e r s t a i n i n g w i t h h a e m a t o x y l i n and e o s i n . A t a l a t e r s t a g e y o l k g r a n u l e s were observed between t h e y o l k v e s i cles.  However, even at t h i s s t a g e t h e y o l k g r a n u l e s were a l s o  observed  on the i n n n e r s u r f a c e o f the zona r a d i a t a ( F i g . 8 ) . The o o c y t e a t t h i s s t a g e was about 350-500 u i n d i a m e t e r w h i l e t h e i n d i v i d u a l g r a n u l e s were about 1 y o r l e s s i n d i a m e t e r . t h e y m i g r a t e d and accumulated  As more and more y o l k g r a n u l e s were towards  the center of the oocytes.  formed,  At the  same time the y o l k v e s i c l e s were d i s p l a c e d towards t h e p e r i p h e r y o f o o c y t e s .  HISTOCHEMISTRY OF VITELLOGENESIS H i s t o c h e m i c a l a n a l y s e s f o c u s s e d on t h e c h e m i c a l c o m p o s i t i o n o f y o l k inclusions.  F i n d i n g s f o r t h e two types o f y o l k i n c l u s i o n s a r e summarized  16 i n Table  2.  D i s t r i b u t i o n of p o l y s a c c h a r i d e s . i n the g e n e r a l c y t o p l a s m  P o l y s a c c h a r i d e s were not  detected  o f o o g o n i a o r p r i m a r y o o c y t e s , but o n l y i n the  y o l k v e s i c l e s w h i c h r e a c t e d w i t h a l l the p o l y s a c c h a r i d e s t a i n s .  The  v e s i c l e s s t a i n e d r e d w i t h B e s t ' s C a r m i n e — a n e m p i r i c a l method f o r d e t e c t i n g glycogen w h i c h i s s t i l l w i d e l y used by h i s t o c h e m i s t s ( P e a r s e , 1968). C o n t r o l s e c t i o n s d i g e s t e d w i t h d i a s t a s e b e f o r e s t a i n i n g gave a n e g a t i v e r e a c t i o n ; a b o l i t i o n o f s t a i n i n g by d i a s t a s e suggested contained glycogen. not s t a i n e d by B e s t ' s  I n c o n t r a s t to the y o l k v e s i c l e s , y o l k g r a n u l e s were carmine.  The y o l k v e s i c l e s and S c h i f f ' s reagent  t h a t the v e s i c l e s  zona r a d i a t a r e a c t e d w i t h p e r i o d i c a c i d -  t o form a dark r e d c o l o u r ( F i g . 9 ) .  a b o l i s h e d i n a c e t y l a t e d s e c t i o n s and t h i s c o n f i r m e d n a t u r e of y o l k v e s i c l e s and zona r a d i a t a .  T h i s r e a c t i o n was the p o l y s a c c h a r i d e  Since sections digested with  diastase reacted p o s i t i v e l y with p e r i o d i c a c i d - S c h i f f ' s reagents, c o n t e n t s of y o l k v e s i c l e s are p r o b a b l y complex p o l y s a c c h a r i d e s and simple polysaccharides.  The y o l k g r a n u l e s and ooplasm o f p r i m a r y  the not oocytes  were not s t a i n e d by the p e r i o d i c a c i d - S c h i f f s r e a c t i o n . The y o l k v e s i c l e s a l s o s t a i n e d w i t h d i a l y s e d i r o n ( H a l e , 1 9 4 8 ) , and t h i s i n d i c a t e d t h a t the y o l k v e s i c l e s c o n t a i n e d a c i d The  general cytoplasm  o f p r i m a r y o o c y t e s s t a i n e d g r e e n i s h b l u e as com-  pared t o the b l u e s t a i n i n g of the y o l k v e s i c l e s . also stained greenish blue. i n b o t h the c y t o p l a s m of  mucopolysaccharides.  The y o l k g r a n u l e s were  S i n c e t h i s g r e e n i s h b l u e s t a i n was  and y o l k g r a n u l e s ; i t was  s t a i n i n g s o l u t i o n and not due  p r o b a b l y due  present  to r e t e n t i o n  to. a h i s t o c h e m i c a l r e a c t i o n .  There  no change i n d i a l y s e d i r o n r e a c t i o n i n y o l k v e s i c l e s at v a r i o u s s t a g e s  was of  T a b l e 2:  A summary of the h i s t o c h e m i c a l  properties  o f t h e two types o f y o l k  TECHNIQUES  YOLK VESICLES  inclusions.  YOLK GRANULES  POLYSACCHARIDES B e s t ' s Carmine ( P e a r s e , 1968) D i a s t a s e - B e s t ' s Carmine P e r i o d i c a c i d - S c h i f f ' s (McManus, 1948) Diastase- Periodic acid- Schiff's Acetylation- Periodic acid- Schiff's Dialysed  + +  i r o n ( H a l e , 1946)  A l c i a n B l u e , pH 2.5 ( P e a r s e , 1968) PROTEINS Dinitroflurobenzene Tetra-azotized  ++  ( B u r s t o n e , 1955)  o-dianisidine  Sakaguchi's r e a c t i o n  ( D a n i e l l i , 1953)  +  ( B a k e r , 1956)  Ferric ferricyanide reaction  (Chevremont & F r e d e r i c k ,  N - e t h y l maleimide- F e r r i c f e r r i c y a n i d e DDD r e a c t i o n  (Barnett  +++  (Baker, 1947) ——  Millon's reaction  I I II  & Seligman, 1952)  N - e t h y l maleimide - DDD r e a c t i o n  1943)  I I  -  ++  +++  +  ++ Continued  T a b l e 2 (Cont'd)  TECHNIQUE  YOLK VESICLES  YOLK GRANULES  LIPIDS Sudan B l a c k B (Chayen et a l . , 1969)  4-f  P y r i d i n e e x t r a c t i o n - Sudan B l a c k B O i l Red 0 ( L i l l i e , 1944)  +4-  P y r i d i n e e x t r a c t i o n - O i l Red 0 A c i d haematin (Baker, 1946)  ++++  P y r i d i n e e x t r a c t i o n - A c i d haematin  +44+  Copper t h i o c y a n i n  ( K l u v e r & B a r r e r a , 1953)  +4-  t-  1  oo  19  F i g u r e 9:  Y o l k v e s i c l e stage oocytes s t a i n e d f o r p o l y s a c c h a r i d e s periodic acid-Schiff's reaction. a strong  Figure  10:  reaction.  11:  method f o r  The y o l k g r a n u l e s (g) s t a i n e d r e d d i s h whereas the  y o l k v e s i c l e s (v) s t a i n e d Figure  Y o l k v e s i c l e s (v) showed  Oocytes s t a i n e d w i t h the d i n i t r o f l u r o b e n z e n e proteins.  with  yellowish.  Oocytes s t a i n e d w i t h t e t r a z o t i s e d o - d i a n i s i d i n e r e a c t i o n for  proteins.  The y o l k g r a n u l e s (g) s t a i n e d dark r e d w h i l e  the y o l k v e s i c l e s (v) s t a i n e d brownish r e d , i n d i c a t i n g t h a t the y o l k v e s i c l e s c o n t a i n e d  l e s s p r o t e i n s than the y o l k  granules.  Figure  12;  Oocytes s t a i n e d w i t h the f e r r i c s u l p h y d r y l groups.  f e r r i c y a n i d e method f o r  Y o l k v e s i c l e s (v) r e a c t e d p o s i t i v e l y  though y o l k g r a n u l e s (g) r e t a i n e d background s t a i n i n g o n l y .  Figure  13:  Cryostat  s e c t i o n s of ovary s t a i n e d w i t h Sudan B l a c k B.  the y o l k g r a n u l e s they c o n t a i n e d  Figure  14:  Only  (g) r e t a i n e d the s t a i n i n d i c a t i n g t h a t  lipids.  Oocytes s t a i n e d w i t h a c i d haematin. reacted p o s i t i v e l y i n d i c a t i n g  The y o l k g r a n u l e s (g)  phospholipids.  20 vitellogenesis. A l c i a n blue  (8GX)  b l u i s h - g r e e n a t pH 2.5.  s t a i n e d the y o l k v e s i c l e s and zona r a d i a t a The y o l k g r a n u l e s were not s t a i n e d .  The  dis-  t r i b u t i o n s of the v e s i c l e s were s i m i l a r t o those observed d u r i n g p e r i o d i c acid-Schiff's staining (Fig. 9). I t i s c o n c l u d e d from p o l y s a c c h a r i d e  s t a i n i n g t h a t the y o l k v e s i c l e s  c o n t a i n g l y c o g e n and complex a c i d i c p o l y s a c c h a r i d e s . granules  do not c o n t a i n  By c o n t r a s t , y o l k  polysaccharides.  D i s t r i b u t i o n of p r o t e i n s .  O r i g i n a l l y i n t r o d u c e d by Sanger (1945)  as a r e a g e n t f o r N - t e r m i n a l - a m i n o a c i d a n a l y s i s , d i n i t r o f l u r o b e n z e n e a  has been much employed as a reagent f o r d e t e c t i n g p r o t e i n end  groups.  For  h i s t o c h e m i c a l d e t e c t i o n , the y e l l o w N l ^ - d i n i t r o p h e n y l compound i s r e d u c e d , d i a z o t i z e d and f i n a l l y coupled w i t h l - a m i n o - 8 - n a p h t h o l - 3 , a c i d ( H - a c i d ) i n an a l k a l i n e s o l u t i o n .  6-disulphonic  Both the y o l k g r a n u l e s  and  the zona  r a d i a t a r e a c t e d w i t h d i n i t r o f l u r o b e n z e n e r e a g e n t s t o g i v e a deep r e d ( F i g . 10).  colour  The y o l k v e s i c l e s on the o t h e r hand s t a i n e d b r o w n i s h - r e d w i t h  the d i n i t r o f l u r o b e n z e n e method. Yolk granules  a l s o s t a i n e d deep red w i t h the t e t r a z o t i z e d 0 — d i a n i s i d i n e  r e a c t i o n , a g e n e r a l s t a i n f o r p r o t e i n s ( F i g . 11). stained l i g h t red.  1947)  yolk vesicles  T h i s r e a c t i o n i s s t i o c h i o m e t r i c (Chayen et a l . 1969).  T h i s suggested t h a t the y o l k v e s i c l e s c o n t a i n e d granules.  The  l e s s p r o t e i n t h a n the y o l k  Sakaguchi r e a g e n t s , w h i c h are s p e c i f i c f o r a r g i n i n e  (Baker,  gave a p o s i t i v e r e a c t i o n w i t h y o l k g r a n u l e s w h i l e the y o l k v e s i c l e s  were h a r d l y s t a i n e d at a l l a p p e a r i n g  l i g h t yellow i n colour.  21  Yolk granules reagent  appeared dark r e d a f t e r t r e a t i n g w i t h M i l l o n ' s  which i s s p e c i f i c  f o r the hydroxy-phenyl group.  amino a c i d c o n t a i n i n g hydroxy-phenyl group i s t y r o s i n e . c l e s , by c o n t r a s t , s t a i n e d y e l l o w to orange w i t h M i l l o n ' s  The o n l y  common  The y o l k v e s i reagent.  Y o l k v e s i c l e s appeared b l u e i n c o l o u r a f t e r r e a c t i n g w i t h  Chevremont-  F r e d e r i c k ' s method ( F i g . 12). T h i s method f o r s u l p h y d r y l groups i s based on the r e d u c t i o n o f f e r r i c y a n i d e t o f e r r o c y a n i d e .  The r e s u l t i n g f e r r o c y a -  n i d e combined w i t h f e r r i c i o n s to form an i n s o l u b l e P r u s s i a n b l u e pitate.  T h i s r e a c t i o n i s n o t s p e c i f i c by i t s e l f ,  substances  reduce f e r r i c y a n i d e .  preci-  s i n c e many r e d u c i n g  C o n t r o l s e c t i o n s immersed i n 0.1 M,  N - e t h y l maleimide b e f o r e r e a c t i n g w i t h Chevremont-Frederick reagents appeared  green and showed no P r u s s i a n b l u e p r e c i p i t a t e i n the y o l k  N - e t h y l maleimide s p e c i f i c a l l y b l o c k e d  s u l p h y d r y l groups.  i s n o n - s p e c i f i c and due to r e t e n t i o n o f reagents  vesicles;  The green c o l o u r  by the y o l k  granules.  Yolk v e s i c l e s stained blue with dihydroxy-dinaphthyldisulphide method.  (DDD)  The DDD method i s based on the s p e c i f i c o x i d a t i o n o f s u l p h y d r y l s  (Pearse, 1968).  No b l u e c o l o u r was e v i d e n t i n y o l k v e s i c l e s o f c o n t r o l  s e c t i o n s where s u l p h y d r y l groups were b l o c k e d w i t h N - e t h y l maleimide. y o l k g r a n u l e s were n o t s t a i n e d by d i h y d r o x y - d i n a p h t h y l - d i s u l p h i d e  The  reagent.  In summary, the v a r i o u s p r o t e i n s t a i n s demonstrated t h a t both the y o l k v e s i c l e s and y o l k g r a n u l e s c o n t a i n e d p r o t e i n s .  Yolk v e s i c l e s contain  l e s s p r o t e i n s than y o l k g r a n u l e s ; s u l p h y d r y l groups a r e p r e s e n t  i n yolk  v e s i c l e s but n o t i n g r a n u l e s .  Histochemistry  of l i p i d s .  Y o l k granules but not y o l k v e s i c l e s s t a i n e d  w i t h Sudan B l a c k B ( F i g . 1 3 ) . Sudan B l a c k B was removed from y o l k  granules  22  by p y r i d i n e e x t r a c t i o n . Black  The zona r a d i a t a was not s t a i n e d by Sudan  B. The d i s t r i b u t i o n o f O i l Red 0 was s i m i l a r t o Sudan B l a c k B.  g r a n u l e s but n o t y o l k v e s i c l e s absorbed O i l Red 0. ing  Yolk  T h i s O i l Red 0 s t a i n -  o f y o l k g r a n u l e s was not e v i d e n t i n c o n t r o l s e c t i o n s e x t r a c t e d  with  pyridine. Yolk  granules  and zona r a d i a t a s t a i n e d b l u e - b l a c k  w i t h a c i d haematin ( F i g . 14).  t o almost  black  The d i s t r i b u t i o n o f a c i d haematin s t a i n i n g  was n o t changed by p y r i d i n e e x t r a c t i o n ; y o l k v e s i c l e s s t a i n e d l i g h t  yellow,  i n d i c a t i n g the l a c k o f p h o s p h o l i p i d s . L u x o l F a s t Blue MBS  stained yolk granules  and zona r a d i a t a dark b l u e .  T h i s copper t h i o c y a n i n method i s s p e c i f i c f o r p h o s p h o l i p i d s . c o l o u r o f y o l k g r a n u l e s was s t i l l p r e s e n t with p y r i d i n e .  The b l u e  i n control sections extracted  The y o l k v e s i c l e s were not s t a i n e d by the copper t h i o c y a n i n  method. In c o n c l u s i o n , the l i p i d h i s t o c h e m i s t r y o f v i t e l l o g e n e s i s i n d i c a t e d t h a t y o l k v e s i c l e s d i d not c o n t a i n any l i p i d w h i l e y o l k g r a n u l e s n e u t r a l f a t s and p h o s p h o l i p i d s . but n o t n e u t r a l f a t s .  The zona r a d i a t a c o n t a i n e d  had b o t h  phospholipid  23 DISCUSSION  The  h i s t o l o g i c a l and h i s t o c h e m i c a l f i n d i n g s demonstrated t h a t two  types o f y o l k i n c l u s i o n s ( y o l k v e s i c l e s and y o l k g r a n u l e s ) during v i t e l l o g e n e s i s i n g o l d f i s h .  a r e formed  These two y o l k i n c l u s i o n s d i f f e r  t i n c t i v e l y i n t h e i r morphology, t i n c t o r i a l p r o p e r t i e s , and c h e m i c a l they are deposited  disnature;  s e q u e n t i a l l y — f i r s t t h e y o l k v e s i c l e s and then the y o l k  granules. The  o c c u r r e n c e of two t y p e s o f y o l k i n c l u s i o n s i n t e l e o s t o o c y t e s  was p r e v i o u s l y d e s c r i b e d by Yamamoto (1956, 1958).  He found b o t h y o l k  v e s i c l e s and y o l k g r a n u l e s i n L i o p s e t t a o b s c u r a and C l u p e a p a l l a s i i , based on h i s t o l o g i c a l e v i d e n c e .  S i m i l a r l y , Malone and H i s o a k a (1963) d i s -  t i n g u i s h e d two types o f y o l k , d e s i g n a t e d  as e x t r a v e s i c u l a r and i n t r a v e s i -  c u l a r , i n the z e b r a f i s h Brachydanio r e r i o . However, t h e o c c u r r e n c e o f two types o f y o l k i n c l u s i o n s i s n o t u n i v e r s a l among t e l e o s t s .  I n some s p e c i e s , t h r e e types o f y o l k have been ob-  s e r v e d ; Yamamoto (1956) d e s c r i b e d y o l k v e s i c l e s , y o l k g l o b u l e s , and l i p i d g l o b u l e s , i n t h e s m e l t , Hypomesus j a p o n i c u s w h i l e Guraya (1965) noted t h r e e types o f y o l k i n c l u s i o n s i n Channa m a r u l i u s . The  f i r s t yolk i n c l u s i o n s deposited  i n g o l d f i s h oocytes are the y o l k  v e s i c l e s which form as minute o v a l b o d i e s t h a t grow u n t i l they r e a c h e d the s i z e o f 15 y.  These y o l k v e s i c l e s have been d e s c r i b e d by many i n v e s t i -  g a t o r s u s i n g d i f f e r e n t names i n c l u d i n g c o r t i c a l a l v e o l i , i n t r a v e s i c u l a r y o l k , v a c u o l e s , vacuome, y o l k g l o b u l e s , y o l k spheres and y o l k v e s i c l e s . I n s p i t e o f v a r y i n g n o m e n c l a t u r e , y o l k v e s i c l e s always c o n s i s t o f mucopolysaccharides  and r e a c t p o s i t i v e l y t o t h e p e r i o d i c a c i d - S c h i f f ' s r e a c t i o n  24 ( A k e t a , 1954; 1965;  Yamamoto, 1956  Y a m a z a k i , 1965).  a, b, c; Malone & H l s o a k a ,  1963;  Guraya,  Yolk v e s i c l e s i n oocytes of Clupea p a l l a s i i  and  L i o p s e t t a o b s c u r a c o n t a i n s i m p l e m u c o p o l y s a c c h a r i d e s (Yamamoto, 1956  a,  b) w h i l e the g o l d f i s h examined i n t h i s i n v e s t i g a t i o n have a c i d mucopolys a c c h a r i d e s ; Hypomesus j a p o n i c u s a l s o has y o l k v e s i c l e s w i t h m u c o p o l y s a c c h a r i d e s (Yamamoto, 1956  c).  acidic  A k e t a suggested t h a t y o l k v e s i -  c l e s o f O r y z i a s l a t i p e s c o n s i s t e d of s u l p h a t e d  mucopolysaccharides.  Towards the l a t t e r p a r t o f g o l d f i s h v i t e l l o g e n e s i s , the y o l k v e s i c l e s a r e d i s p l a c e d towards the p e r i p h e r y of o o c y t e s and alveioli.  The  c o r t i c a l a l v e o l i p l a y an i m p o r t a n t  r e a c t i o n a t the time of f e r t i l i z a t i o n The  develop i n t o c o r t i c a l r o l e i n the  (Yamamoto, 1961;  cortical  Manroy, 1965).  o r i g i n of y o l k v e s i c l e s had been a t t r i b u t e d to v a r i o u s  components.  cell  A k e t a (1954) suggested t h a t y o l k v e s i c l e s o r i g i n a t e from  the " p r e - c o r t e x " w h i l e Yamamoto (1961) suggested t h a t y o l k v e s i c l e s o r i g i n a t e from the ground c y t o p l a s m .  A c t u a l l y , t h e r e i s no r e a l d i f f e r e n c e  between t h e s e two p o s t u l a t i o n s .  By c o n t r a s t , Nath (1960) and Guraya (1965)  proposed the " y o l k n u c l e u s "  as the p r e c u r s o r of y o l k v e s i c l e s .  The  pre-  c u r s o r s of y o l k v e s i c l e s , because of t h e i r s m a l l s i z e , are beyond the r e s o l v i n g power of the l i g h t m i c r o s c o p e .  A l t h o u g h no one has  examined  t h e y o l k n u c l e u s i n t e l e o s t w i t h the e l e c t r o n m i c r o s c o p e , B a l i n s k y  and  D a v i s (1963) demonstrated t h a t the y o l k n u c l e i of amphibians were concent r a t i o n s of m i t o c h o n d r i a  w h i c h p l a y e d no p a r t i n y o l k f o r m a t i o n .  At  the  e l e c t r o n m i c r o s c o p e l e v e l , the b e s t e v i d e n c e of the o r i g i n of y o l k v e s i c l e s was  p r o v i d e d by Yamamoto and  Onozato (1965), and Yamamoto and  Oota  (1967).  Yamamoto and Onozato (1965) demonstrated t h a t G o l g i complexes  25 gave r i s e t o y o l k v e s i c l e s i n g o l d f i s h and t h e s e f i n d i n g s have been c o n f i r m e d by Yamamoto and Oota (1967) i n Brachydanio  r e r i o , and by  Gupta and Yamamoto (1972) i n g o l d f i s h , C a r a s s i u s a u r a t u s . The y o l k v e s i c l e s i n g o l d f i s h were d i s t i n g u i s h e d from y o l k g r a n u l e s by t h e i r l a r g e r s i z e and t i n c t o r i a l p r o p e r t i e s .  C h e m i c a l l y the y o l k  v e s i c l e s a r e p o l y s a c c h a r i d e s and p r o t e i n s r i c h i n s u l p h y d r y l groups w h i l e the y o l k g r a n u l e s c o n s i s t o f p r o t e i n s , p h o s p h o l i p i d s and n e u t r a l l i p i d s . The  c h e m i c a l c o m p o s i t i o n o f y o l k g r a n u l e s as determined by h i s t o c h e m i c a l  s t a i n i n g i s v e r y s i m i l a r t o the c o m p o s i t i o n o f y o l k as d e t e r m i n e d by b i o chemical s t u d i e s .  P l a c k and F r a s e r (1970), i n a b i o c h e m i c a l s t u d y , demon-  s t r a t e d t h a t t h e y o l k o f cod c o n t a i n s two major l i p o p r o t e i n s o f s i m i l a r m o l e c u l a r w e i g h t (400,000) and c h e m i c a l c o m p o s i t i o n ) . about 79% p r o t e i n and 21% l i p i d w h i l e a p p r o x i m a t e l y  Eachhlipoprotein i s  65% o f t h e l i p i d  frac-  tion i s phospholipid. The y o l k g r a n u l e s were d e p o s i t e d i n d e p e n d e n t l y and o n l y appeared i n oocytes  of the yolk vesicles  a f t e r t h e y o l k v e s i c l e s were f u l l y i i d e v e l o p e d .  There a r e s t i l l many c o n f l i c t i n g o p i n i o n s about t h e o r i g i n o f y o l k g r a n u l e s . D r o l l e r and Roth (1966) c o n c l u d e d  from e l e c t r o n m i c r o s c o p i c s t u d i e s o f  P o e c i l i a r e t i c u l a t a o v a r i e s t h a t y o l k g r a n u l e s were formed by t h e e n d o p l a s mic r e t i c u l u m and G o l g i b o d i e s .  These w o r k e r s d e s c r i b e d m i c r o p i n o c y t o s i s  i n the p e r i p h e r y o f o o c y t e s ; the g r a n u l e s formed by p i n o c y t o s i s accumulated w i t h i n t h e endoplasmic r e t i c u l u m and G o l g i complex t o form y o l k g r a n u l e s . Yamamoto and Oota (1967), and Gupta and Yamamoto (1972) d i s p u t e d t h e suggesti o n s o f D r o l l e r and R o t h ; Yamamoto and Oota p o s t u l a t e d t h a t were t h e p r e c u r s o r s o f y o l k g r a n u l e s .  mitochondria  They argued t h a t p i n o c y t o t i c b o d i e s  26 were t a k e n i n t o o o c y t e s and yolk granules.  resynthesized w i t h i n mitochondria  This suggestion  functions of mitochondria  recognized  i n c e l l r e s p i r a t i o n . F u r t h e r work, e s p e c i a l l y  at the e l e c t r o n m i c r o s c o p i c o p i n i o n s can be  seems u n l i k e l y i n v i e w o f the  t o form  l e v e l , i s required before these c o n f l i c t i n g  resolved.  I n s p i t e of t h e s e d i f f e r e n c e s i n o p i n i o n s , a l l i n v e s t i g a t o r s agreed t h a t p a r t of y o l k g r a n u l e s were d e r i v e d from an exogenous s o u r c e t h a t y o l k p r e c u r s o r were d e p o s i t e d i n o o c y t e s by p i n o c y t o s i s .  and  Anderson  (1968) and Guraya (1965) have a l s o p r o v i d e d e v i d e n c e o f p i n o c y t o s i s w i t h i n o o c y t e s o f Fundulus h e t e r o c l i t u s and Channa m a r u l i u s , r e s p e c t i v e l y . h i s t o g e n e s i s of y o l k g r a n u l e source of y o l k  The  f o r m a t i o n s u p p o r t e d the i d e a of an exogenous  granules.  I t i s w e l l known t h a t i n many t e l e o s t the l i v e r accumulates l i p i d s and p r o t e i n s d u r i n g the r e p r o d u c t i v e p e r i o d ( B a i l e y , 1957* Ho and 1961;  Cedard e_t al_. , 1961,  P l a c k and F r a s e r , 1970).  Vanstone,  Very r e c e n t i n v e s t i g a -  t i o n s by A i d a e_t a l . (1973) a l s o s u p p o r t the e a r l i e r p o s t u l a t i o n s t h a t t e l e o s t l i v e r i s the s i t e of y o l k p r o t e i n s y n t h e s i s .  I t seems l i k e l y  the y o l k p r o t e i n s s y n t h e s i z e d by the l i v e r are t r a n s p o r t e d by b l o o d t o the o v a r y and d e p o s i t e d w i t h i n o o c y t e s by p i n o c y t o s i s .  that  plasma  27  F i g u r e 15:  A d i a g r a m a t i c r e p r e s e n t a t i o n o f oogenesis i n g o l d f i s h ovaries.  The  stages i n oocyte development d u r i n g oogenesis are -  (I) oogonia w i t h i n c y s t s ;  ( I I ) chromatin n u c l e o l a r stage  o o c y t e s ; ( I I I ) p e r i n u c l e o l a r stage oocyte;  (IV) e a r l y  y o l k v e s i c l e s t a g e o o c y t e ; (V) i n t e r m e d i a t e y o l k v e s i c l e stage o o c y t e ; (VI) l a t e y o l k v e s i c l e stage o o c y t e ;  (VII)  i n i t i a l y o l k g r a n u l e stage o o c y t e ; ( V I I I ) l a t e y o l k g r a n u l e stage o o c y t e ; and  (IX) mature o o c y t e .  DIAGRAMATIC  REPRESENTATION OF O O C Y T E  GROWTH  SECTION I T :  The Corpus Luteum and i t s  Functions  29  INTRODUCTION Not a l l o f t h e d e v e l o p i n g o v a r i a n f o l l i c l e s complete t h e f o r m a t i o n of  an ovum; many o f them become a t r e t i c a t some s t a g e d u r i n g t h e i r de-  velopment.  These a t r e t i c f o l l i c l e s have many s i m i l a r i t i e s t o t h e p o s t -  o v u l a t o r y f o l l i c l e s and t h e r e has l o n g been a l i v e l y i n t e r e s t i n t h e i r function. the  A t present n e i t h e r the p h y s i o l o g y of the a t r e t i c f o l l i c l e s nor  p o s t - o v u l a t o r y corpus luteum i s f u l l y u n d e r s t o o d . A t r e t i c o o c y t e s o c c u r i n a wide v a r i e t y o f t e l e o s t as e v i d e n t by t h e  r e v i e w s by P i c k f o r d and A t z ( 1 9 5 7 ) , B a l l 1969) and C h i e f f i (1970).  ( 1 9 6 0 ) , Dodd ( 1 9 6 0 ) , Hoar (1965,  They have been d e s c r i b e d f r e q u e n t l y s i n c e  B r e t s c h n e i d e r and Duyvene de W i t (1947) f i r s t d i s c u s s e d t h e i r h i s t o g e n e s i s i n Rhbdeus amarus.  F o l l i c u l a r a t r e s i a has been d e s c r i b e d i n P o e c i l i a  r e t i c u l a t a ( S t o l k , 1951; Lambert, 1970), C a r a s s i u s a u r a t u s (Beach, 1959), G a s t e r o s t e u s a c u l e a t u s (Tromp-Blom, 1959) , Scomber scomber ( B a r a , 1960), Mystus s e e n h g a l a (Sa'thyanesan ( 1 9 6 1 ) , H e t e r o p n e u s t e s f o s s i l i s P l e u r o n e c t e s p l a t e s s a ( B a r r , 1963), Xenenotodon Monopterus  ( N a i r , 1963),  c a n c i l a ( R a s t o g i , 1966),  a l b a (Chan e t a l . , 1967), and Sebastodes p a u c i s p i n i s  (Moser,  1967) . The p o s t - o v u l a t o r y f o l l i c u l a r  s t r u c t u r e s w h i c h resemble mammalian  c o r p o r a l u t e a , have been r e p o r t e d i n Scomber scomber ( B a r a , 1960), Mystus s e e n g h a l a ( S a t h y a n e s a n , 1962), H e t e r o p n e u s t e s f o s s i l i s  ( N a i r , 1963),  E u c a l i a i n c o s t a n s ( B r a e k e v e l t and M c M i l l a n , 1967), and C l a r i u s b a t r a c h u s ( L e h r i , 1968).  A l t h o u g h B a r r ( 1 9 6 3 ) , R a j a l a k s h m i ( 1 9 6 6 ) , and Moser  (1967)  30  r e p o r t e d an absence of 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 i n t e l e o s t o v a r i e s , t h i s may have been due to random s a m p l i n g o r the o v a r i e s were not  sampled  soon enough a f t e r o v u l a t i o n . The s i m i l a r i t y i n morphology and o r i g i n o f t h e s e s t r u c t u r e s , whether pre-  o r p o s t - o v u l a t o r y , t o mammalian c o r p o r a l u t e a l e d B r e t s c h n e i d e r &  Duyvene de Wit (1947) t o c o n s i d e r them comparable  t o the mammalian corpus  luteum and t o suggest an e n d o c r i n e f u n c t i o n based on the q u e s t i o n a b l e " B i t t e r l i n g o v i p o s i t o r growth t e s t " .  T h i s work has s t i m u l a t e d many d i s -  c u s s i o n s of the f u n c t i o n o f the p r e - and p o s t - o v u l a t o r y o v a r i a n f o l l i c l e s but no d e f i n i t e c o n c l u s i o n s have been reached due t o the l a c k of  critical  e v i d e n c e of t h e i r e n d o c r i n e f u n c t i o n . Many  a u t h o r s , w h i l e r e c o g n i z i n g t h e l i m i t a t i o n s o f the  "Bitterling  o v i p o s i t o r growth t e s t " , s t i l l c o n s i d e r t h e s e s t r u c t u r e s comparable mammalian corpus luteum (see r e v i e w s by B a l l , 1960; Hoar, 1965).  t o the  In addi-  t i o n to the s i m i l a r i t y i n morphology and o r i g i n , p r e - and p o s t - o v u l a t o r y b o d i e s o f t e n have the appearance 1969)  of t r u e e p i t h e l i a n g l a n d s .  Hoar  (1965,  suggested t h a t t e l e o s t c o r p o r a l u t e a might produce e s t r o g e n s r a t h e r  than p r o g e s t e r o n e . The a p p r o p r i a t e n e s s of the term "corpuca l u t e u m " i n t e l e o s t s i s not u n i v e r s a l l y accepted.  The main argument a g a i n s t i t i s the l a c k of c r i t i c a l  e v i d e n c e o f the e n d o c r i n e f u n c t i o n of the a t r e t i c f o l l i c l e s .  The two b a s i c  e n d o c r i n e f u n c t i o n s of t h e corpus luteum are (a) a c t i v e s e c r e t i o n o f the hormone p r o g e s t e r o n e and (b) the p i t u i t a r y c o n t r o l .  I n c o n t r a s t t o the  mammalian corpus luteum, s u c c e s s f u l g e s t a t i o n s have been e s t a b l i s h e d i n the absence of t h e s e b o d i e s i n ' t e l e o s t s (Lambert and Van O o r d t , Lambert, 1970).  Lambert (1970) suggested t h a t i f t h e a t r e t i c  1965; follicles  31 were Important hormone p r o d u c i n g glands they s h o u l d be r e g u l a r l y p r e s e n t during gestation.  However, the absence o f a t r e t i c f o l l i c l e s d u r i n g ges-  t a t i o n does not r u l e out an e n d o c r i n e r o l e o f t h e s e s t r u c t u r e s but shows o n l y t h a t they p l a y no p h y s i o l o g i c a l r o l e i n g e s t a t i o n ; t h e y may,  however,  produce hormones and p l a y some o t h e r r e p r o d u c t i v e r o l e . Some workers  f e e l t h a t the l a c k of p i t u i t a r y c o n t r o l i n the f o r m a t i o n  and maintenance of a t r e t i c f o l l i c l e s enhances doubts o f an e n d o c r i n e f u n c t i o n (Hisaw, 1963).  Corpora l u t e a r e g u l a r l y form subsequent  t o hypo-  physectomy ( B r e t s c h n e i d e r and Duyvenne de W i t , 1947; B a r r , 1963)  and some  a u t h o r s who  doubt the e n d o c r i n e s t a t u s of t e l e o s t c o r p o r a l u t e a , m a i n t a i n  t h a t a t r e t i c o o c y t e s a r e p r o b a b l y a d a p t i v e d e v i c e s f o r d i s p o s i n g moribund ova (Dodd, 1960; P o l d e r , 1964).  They c o n s i d e r the absence o f p r o g e s t e r o n e  s e c r e t i o n and the l a c k of a r o l e i n g e s t a t i o n s t r o n g arguments f o r u s i n g a d i f f e r e n t d e s c r i p t i v e term from the mammalian corpus luteum.  However, the  a p p r o p r i a t e n e s s o f the term " c o r p o r a l u t e a " depends on i t s d e f i n i t i o n .  The  d e f i n i t i o n o f corpus luteum, as an e n d o c r i n e g l a n d t h a t s e c r e t e s p r o g e s t e r one and m a i n t a i n s g e s t a t i o n , i s too r e s t r i c t i v e .  In vertebrates, especially  t e l e o s t s , s t r u c t u r e s s i m i l a r t o c o r p o r a l u t e a are not r e s t r i c t e d t o v i v i p a r a (Hisaw, 1963; B r a m b e l l , 1960)  and a narrow d e f i n i t i o n p r e v e n t s compara-  t i v e s t u d i e s of these s t r u c t u r e s i n oviparous s p e c i e s .  From a comparative  v i e w p o i n t i t may be more a p p r o p r i a t e t o c o n s i d e r t h e "corpus luteum" as an e n d o c r i n e g l a n d t h a t s e c r e t e s s t e r o i d hormones and p l a y a r o l e i n r e p r o d u c t i o n but which need n o t n e c e s s a r i l y be concerned w i t h the maintenance o f gestation.  32  The broader t u r e s w i l l be  d e f i n i t i o n w i l l be adopted f o r t h i s study.  termed c o r p o r a l u t e a and  The s t r u c -  an attempt made to e s t a b l i s h  p h y s i o l o g i c a l f u n c t i o n o f both a t r e t i c oocytes and p o s t - o v u l a t o r y c l e s i n the g o l d f i s h — a n o v i p a r o u s  teleost.  folli-  T h i s study w i l l i n v e s t i g a t e  the corpus luteum as an organ capable o f s y n t h e s i z i n g s t e r o i d s by ing  the  the h i s t o c h e m i c a l l o c a t i o n o f v a r i o u s h y d r o x y s t e r o i d  examin-  dehydrogenases.  Dorfman and Ungar (1965) e s t a b l i s h e d the r o l e o f h y d r o x y s t e r o i d dehydrogenases i n s t e r o i d b i o s y n t h e s i s . p r e - or p o s t - o v u l a t o r y , w i l l be  The  f a t e of the corpus luteum, e i t h e r  f o l l o w e d p a s t the  6-stage.  33 METHODS AND  MATERIALS  HYPOPHYSECTOMY OF GOLDFISH G o l d f i s h were hypophysectomized u s i n g a m o d i f i c a t i o n of Yamazaki's (1965) t e c h n i q u e . s o l u t i o n of 0.01  The  f i s h were f i r s t  anaesthesized i n a cold  The  "operating table" f o r  g o l d f i s h , c o n s i s t e d of a sponge w i t h a groove i n i t . t h i s groove, w i t h the l e f t  bands of 2.5  C)  % t r i c a n e methane s u l f o n a t e ('MS222', Sandoz) u n t i l  lost their reflex activity.  in  (4-8  cm w i d t h .  The  hypophysectomizing  G o l d f i s h were p l a c e d  s i d e uppermost and h e l d i n p l a c e by  o p e r a t i n g t a b l e was  they  elastic  kept c o o l w i t h crushed i c e  d u r i n g the e n t i r e o p e r a t i o n . The  left  operculum t o g e t h e r w i t h the f i r s t two  t r a c t e d w i t h hooked p i n s a t t a c h e d to e l a s t i c c o r d s . p a r a l l e l to the second  gill  a r c h , was  i n c i s i o n v a r i e d from 5-10  oid  bone, beneath which the p i t u i t a r y i s s i t u a t e d . the parasphenoid  the p i t u i t a r y . p t e r y g o i d bone. of  An o b l i q u e c u t ,  immediately  A h o l e was  d e n t a l b u r r (No.  nerves  then  drilled  3) t o expose  i n f r o n t of the i n n e r t i p o f the  Another landmark f o r the p o i n t of d r i l l i n g  a p a i r of conspicuous  re-  i n l e n g t h ; t h i s cut exposed the parasphen-  bone w i t h a round  T h i s h o l e was  arches was  made i n the d o r s a l b u c c a l mucosa.  The  through  mm  gill  from the cranium.  i s the  origin  These nerves were i n -  e v i t a b l y cut d u r i n g hypophysectomy w h i l e i n the sham hypophysectomized they were i n t e n t i o n a l l y cut d u r i n g the d r i l l i n g of the parasphenoid in  sham hypophysectomized f i s h the p i t u i t a r y was  B l e e d i n g was  bone;  exposed but not removed.  To e f f e c t hypophysectomy, the exposed p i t u i t a r y was w i t h a curved p i p e t t e .  fish  removed by s u c t i o n  much reduced w i t h c o l d a n e s t h e t i c s  34  and ceased q u i c k l y i n most i n s t a n c e s . mouth h e a l e d w i t h i n a week. with connective  The i n c i s i o n i n the r o o f of the  The exposed area of the s k u l l was  replaced  tissues.  D u r i n g r e c o v e r y hypophysectomized g o l d f i s h were p l a c e d i n c o l d d i l u t e d sea water (4-8°C) prepared by d i l u t i n g w i t h t h r e e times i t s volume o f d e c h l o r i n a t e d water.  A temperature  shock a f t e r hypophysectomy r e s u l t s  i n h i g h m o r t a l i t y and consequently water temperatures to room temperature.  were r a i s e d s l o w l y  High s u r v i v a l r a t e s , a v e r a g i n g w e l l over 80%, were  o b t a i n e d w i t h the above t e c h n i q u e s ; most o f the m o r t a l i t y was damage.  due  to b r a i n  35 HISTOLOGY OF PRE- AND POST-OVULATORY CORPUS LUTEUM  P r e - o v u l a t o r y c o r p o r a l u t e a were induced hypophysectomy. e i g h t weeks. amination  i n g r a v i d g o l d f i s h by  Four hypophysectomized f i s h were k i l l e d each week f o r  A p o r t i o n o f each ovary was f r o z e n f o r h i s t o c h e m i c a l e x -  o f h y d r o x y s t e r o i d dehydrogenases w h i l e a n o t h e r p o r t i o n was f i x e d  i n B o u i n ' s f l u i d t o observe t h e h i s t o g e n e s i s o f a t r e t i c o o c y t e .  The f a t e  o f p o s t - o v u l a t o r y f o l l i c l e s was observed i n f i s h k i l l e d 1, 7 and 30 days after ovulation.  L i k e w i s e i n t h e s e experiments,  one p o r t i o n of each  ovary was f r o z e n f o r t h e d e t e c t i o n o f h y d r o x y s t e r o i d dehydrogenases and the o t h e r f i x e d i n Bouin's f l u i d .  Ovaries  embedded i n p a r a f f i n , s e c t i o n e d s e r i a l l y t o x y l i n and e o s i n , o r M a l l o r y ' s t r i c h r o m e  f i x e d i n B o u i n ' s f l u i d were  a t 7 y and s t a i n e d w i t h haema( G u r r , 1962).  HISTOCHEMICAL DEMONSTRATION OF HYDROXYSTEROID DEHYDROGENASE Hydroxysteroid  dehydrogenases i n o v a r i e s o f v i t e l l o g e n i c g o l d f i s h  were examined h i s t o c h e m i c a l l y w i t h a m o d i f i c a t i o n o f W a r t t e n b e r g (1958) technique.  H y d r o x y s t e r o i d dehydrogenases i n o v a r i e s o f hypophysectomized  g o l d f i s h were examined a t w e e k l y i n t e r v a l s f o r e i g h t c o n s e c u t i v e weeks a f t e r hypophysectomy and the f i n d i n g s were compared w i t h those i n t h e ovaries of ovulated g o l d f i s h D e t a i l s of the techniques  t e s t e d 1, 7 and 30 days a f t e r o v u l a t i o n .  a r e as f o l l o w s :  G o l d f i s h were d e c a p i t a t e d and  a p o r t i o n o f each ovary was f r o z e n w i t h " C r y o w i c k " (IEC Company), w h i l e a second p o r t i o n was f i x e d i n B o u i n ' s f i x a t i v e f o r r o u t i n e h i s t o l o g y . F r o z e n o v a r i e s were s e c t i o n e d w i t h a c r y o s t a t ( H a r r i s I n t e r n a t i o n a l ) a t  36 10-16  y; the temperature o f the c r y o s t a t , w h i c h was  s e c t i o n i n g , was m a i n t a i n e d  a t -10°C  t o -15°C.  c r i t i c a l f o r proper  C r y o s t a t s e c t i o n s were  2 mounted on 22 m  c o v e r s l i p s , d r i e d f o r about 10 min a t room t e m p e r a t u r e  and r i n s e d i n 0.1 M T r i s - H C l b u f f e r , pH 7.5 the r e a c t i o n medium a t 30°C.  The  r e a c t i o n mixture  tetrazolium salt  ( N i t r o BT o r MTT),  i n 1.6  M, T r i s - H C l b u f f e r , pH 7.5  ml o f 0.1  c o n t r a i n i n g 50-100 yg s t e r o i d .  before being incubated i n  MTT  2 mg NAD  was  c o n s i s t e d of 1  and 10 mg M g C l and 0.4  2  mg  dissolved  ml d i m e t h y l formamide  r o u t i n e l y used as N i t r o BT gave  a more i n t e n s e d i a p h o r a s e r e a c t i o n . S t e r o i d s used as s u b s t r a t e s i n c l u d e d d e h y d r o e p i a n d r o s t e r o n e c i f i c substrate f o r 3 3-hydroxysteroid  dehydrogenases), androsterone  s p e c i f i c substrate f o r 3 a-hydroxysteroid progesterone  (a spe-  dehydrogenases) , 17  (a' s p e c i f i c s u b s t r a t e f o r 17 a - h y d r o x y s t e r o i d  (a  cx-hydro-  dehydrogenases),  and t e s t o s t e r o n e ( a s p e c i f i c s u b s t r a t e f o r 17 ^ - h y d r o x y s t e r o i d dehydrogenases) .  Each o f t h e s e s u b s t r a t e s has one h y d r o x y l group f o r t h e h y d r o x y -  s t e r o i d dehydrogenase r e a c t i o n ( F i g . 1 6 ) .  Other o v a r i a n s t e r o i d s , 17  a-  h y d r o x y p r e g e n o l o n e , p r e g n e n o l o n e , and e s t r a d i o l were used as s u b s t r a t e s for  u n s p e c i f i c h y d r o x y s t e r o i d dehydrogenase r e a c t i o n s . To d i s t i n g u i s h h y d r o x y s t e r o i d dehydrogenases from d i a p h o r a s e  o c c u r s i n most t i s s u e , s e c t i o n s were i n c u b a t e d out any s t e r o i d s u b s t r a t e .  i n a r e a c t i o n mixture w i t h -  A t the end o f the i n c u b a t i o n p e r i o d , s e c t i o n s  were f i x e d i n 10% n e u t r a l f o r m a l i n ( B a k e r , 1944) zymes.  which  S e c t i o n s were then washed i n two  mide t o remove excess s t e r o i d s , and  which i n h i b i t e d the  changes o f 25% d i m e t h y l  finally  i n d i s t i l l e d water.  en-  formaThe  36a  3-16:  HYDROXYSTEROID DEHYDROGENASE REACTION  BluePpt.  37  washed s e c t i o n s were mounted i n F e r r a n t ' s media and examined w i t h i n a day o r two a f t e r  staining.  INCORPORATION OF THYMIDINE- H INTO GOLDFISH OVARIES 3  A group o f g r a v i d g o l d f i s h was t r a n s f e r r e d t o e x p e r i m e n t a l a q u a r i a c o n t a i n i n g d e o J a l o r i n a t e d water a t 10°C. r a i s e d t o room temperature  (22-25°C).  o f t h e s e f i s h o v u l a t e d a f t e r two days.  The water temperature  slowly  W i t h t h i s t r e a t m e n t the m a j o r i t y F i s h which d i d n o t o v u l a t e were  removed w h i l e the o v u l a t e d f i s h were spawned w i t h " a c t i v e " males. Two days a f t e r spawning  t h e females were i n j e c t e d w i t h 1 yc t h y m i d i n e -  3  H (360 mc/mM, Amersham) p e r gram body w e i g h t .  A f t e r the i n t r a p e r i t o n e a l  3  i n j e c t i o n of t h y m i d i n e - H i n 0.1 ml o f s a l i n e t h e f i s h were k i l l e d , i n 3  groups o f f o u r , a t 1, 2, 4, 8, 16, and 32 days a f t e r t h e t h y m i d i n e - H injection.  A p o r t i o n o f each o v a r y was f i x e d i n Bouin's f l u i d and embedded  i n p a r a f f i n using routine h i s t o l o g i c a l techniques.  P a r a f f i n s e c t i o n s (7 y  t h i c k ) were c u t and mounted on albumen coated s l i d e s . dewaxed and h y d r a t e d through a s e r i e s o f a l c o h o l .  These s e c t i o n s were  Hydrated s e c t i o n s were  d i p - c o a t e d w i t h n u c l e a r t r a c k e m u l s i o n s (Kodak NTB 2) i n the dark (Kopriwa and L e B l a n d , 1962).  The s l i d e s were a i r - d r i e d and t h e n kept i n  l i g h t p r o o f s l i d e boxes i n the r e f r i g e r a t o r (0-5°C) f o r a month, a f t e r w h i c h t h e s l i d e s were p r o c e s s e d w i t h p h o t o g r a p h i c d e v e l o p e r (Kodak D 19) and f i x e r (Kodak F 5 f i x i n g b a t h ) .  The a u t o r a d i o g r a p h s were s t a i n e d w i t h  Mayer's h a e m a t o x y l i n and e o s i n (Beserga and Malamud, 1969), d e h y d r a t e d i n a l c o h o l , c l e a r e d i n x y l e n e , and mounted i n DPX ( K i l k p a t r i c k and Lendrum, 1941).  38  A u t o r a d i o g r a p h s of f i v e s e c t i o n s from each ovary were examined 3 f o r the i n c o r p o r a t i o n of t h y m i d i n e - H i n t o the v a r i o u s the o v a r i e s .  c e l l types i n  39  RESULTS  HISTOGENESIS OF ATRETIC OOCYTES Y o l k y o o c y t e s became a t r e t i c a f t e r hypophysectomy. a t r e s i a was  The p a t t e r n o f  the same i n a l l o o c y t e s a l t h o u g h the r a t e s a t which v a r i o u s  o o c y t e s , w i t h i n an o v a r y , became a t r e t i c was v a r i a b l e ( T a b l e 3 ) . of  t h i s v a r i a b i l i t y i t was  f o l l o w i n g hypophysectomy.  Because  i m p o s s i b l e t o q u a n t i f y the r a t e o f a t r e s i a However, o o c y t e s a t t h e y o l k g r a n u l e s t a g e  were c l e a r l y t h e f i r s t t o become a t r e t i c and, i n g e n e r a l , o o c y t e s w i t h more y o l k became a t r e t i c much f a s t e r t h a n t h o s e w i t h l e s s y o l k .  For  d e s c r i p t i v e p u r p o s e s , a t r e s i a i n g o l d f i s h can be s u b - d i v i d e d i n t o f o u r c o n s e c u t i v e s t a g e s a c c o r d i n g t o the s u b d i v i s i o n s o f B r e t s c h n e i d e r and Duyvene de Wit (1947) and Beach (1959) w i t h an a d d i t i o n a l f i f t h s t a g e desc r i b e d here. -stage  tt  atresia.  (Fig.  17).  The h y p e r t r o p h y o f the g r a n u l o s a i n i t i a t e s  The g r a n u l o s a c e l l s change from a squamous t o a columnar  e p i t h e l i u m and, i n most i n s t a n c e s , y o l k v e s i c l e s r u p t u r e t o form a c o n t i n u o u s mass of c o l l o i d ; the e n t i r e o o c y t e shows gigns of degeneration.  The p u n c t u r e o f t h e zona r a d i a t a by  numerous p o r e s marks the end o f t h e <* - s t a g e a t r e t i c  g-stage  ( F i g . 18).  oocyte.  Hypertrophied granulosa c e l l s invade the  c y t o p l a s m o f the a t r e t i c o o c y t e t h r o u g h the p o r e s i n the zona radiata.  The i n v a d i n g g r a n u l o s a c e l l s d i g e s t and r e s o r b the  y o l k i n c l u s i o n s ; the zona r a d i a t a d i s i n t e g r a t e s towards the end of  the (3-stage.  3W  Table 3: The degree of a t r e s i a i n various ovaries a t various time i n t e r v a l s a f t e r hypophysectomy. Only second growth phase (yolky) oocytes were counted.  Days a f t e r  No. of y o l k y  hypophysectomy  oocytes counted  NO. OF OOCYTES IN EACH STAGE Yolk v e s i c l e  Yolk granule  Atretic  7 7  75 52  63  14  1  31  4  7  17  42  22  7  64  17  46  3 2  14  54  14  57 96  35 16  5 11  96  0 24 2  44  0  45  36 14 30  14 14 21  64  38  61  21  63  15 18  21 21  45 52  4 18  28  0 7  46  41 27  0  28  0  36  46  28  0  65  0 2  36  28  63  73 61  0 0 17 26 22  28(sham) 28 (sham) 28(sham) 28(sham)  69 34 81  0 44 43 6 66  0 2  73  15  : sham hypophysectomized g o l d f i s h , which serves as the c o n t r o l  0 0 6 0  40  y-stage  ( F i g . 19).  Hypertrophy  continues during t h i s stage. all  o f the g r a n u l o s a c e l l s  By the end o f this s t a g e  remnants of oocytes are c o m p l e t e l y r e s o r b e d and  h y p e r t r o p h i e d g r a n u l o s a c e l l s surround the a t r i u m ; t h i s marks the space the o o c y t e . membrane, one  The  entire  a small  cavity,  formerly occupied  structure  i s surrounded  the  by  by  a  c e l l t h i c k , which i s s i m i l a r to the t h e c a  of the normal oocyte  ( F i g . 20).  T h i s stage appears  have been o v e r l o o k e d by many i n v e s t i g a t o r s , described a y s t a g e  to  although  they  d i f f e r e n t from the ^ - s t a g e d e s c r i b e d  here.  6-stage ( F i g . 21). collapse  During t h i s s t a g e , the g r a n u l o s a c e l l s  i n t o the a t r i u m to form an i r r e g u l a r c e l l u l a r mass  and y e l l o w l u t e i n pigments are observed T h i s stage p e r s i s t s  among the  f o r long p e r i o d s ; Barr  6-stage p r e o v u l a t o r y c o r p o r a l u t e a  cells.  (1963) observed  s i x months a f t e r hypo-  physectomy.  e-stage  ( F i g . 22).  atresia a fifth  and  In a d d i t i o n  t o these f o u r s t a g e s o f  f i n a l stage was  recognized.  In the l a t e  s t a g e s o f a t r e s i a some c e l l s of the 6-stage corpus appear t o d i f f e r e n t i a t e i n t o oogonia. d i f f e r e n t i a t i o n were observed  luteum  V a r i o u s phases o f  and the b e s t evidence  for a  41  F i g u r e 17:  a-stage o f a t r e t i c oocyte s t a i n e d w i t h haematoxylin and e o s i n .  The g r a n u l o s a c e l l s o f the a t r e t i c  (arrow 1) h y p e r t r o p h i e d as compared w i t h the granulosa c e l l s  (arrow 2 ) .  y o l k v e s i c l e s was  oocytes  normal  Extensive degeneration of  e v i d e n t (homogeneous a r e a o p p o s i t e  arrow 1 ) . F i g u r e 18:  3-stage o f a t r e t i c o o c y t e s t a i n e d w i t h haematoxylin. Masses o f g r a n u l o s a and t h e c a l c e l l s invade and  digest  the a t r e t i c o o c y t e s . F i g u r e 19:  A s e c t i o n o f the ovary o f a hypophysectomized c o n t a i n i n g many y - s t a g e a t r e t i c f o l l i c l e s  goldfish  ( f r e e arrow)  w i t h the c h a r a c t e r i s t i c empty c a v i t y w i t h i n i t . F i g u r e 20:  A d e t a i l e d photomicrograph cle.  of the y s t a g e a t r e t i c  folli-  The oocyte i s c o m p l e t e l y reabsorbed and the colum-  nar g r a n u l o s a c e l l s have the c h a r a c t e r i s t i c s of p h y s i o l o g i c a l l y active F i g u r e 21:  cells.  6-stage a t r e t i c f o l l i c l e  (mass o f c e l l s on r i g h t )  w i t h haematoxylin and e o s i n .  stained  The g r a n u l o s a c o l l a p s e d  i n t o the space f o r m e r l y o c c u p i e d by the egg cytoplasm t o form an i r r e g u l a r mass of F i g u r e 22:  e-stage a t r e t i c f o l l i c l e s .  cells. T h i s photomicrograph  the d i f f e r e n t i a t i o n o f one h a l f o f the a t r e t i c  shows follicles  i n t o oogonia (arrow) w h i l e the o t h e r h a l f remain entiated.  undiffer-  42  s e p a r a t e e-stage corpus luteum was the presence o f t h e partially differentiated posed  c y s t s which show one h a l f com-  of oogonia w h i l e the o t h e r h a l f looked l i k e a 6-  stage corpus luteum w i t h y e l l o w l u t e i n  pigments.  HISTOGENESIS OF POST OVULATORY CORPUS LUTEUM  At o v u l a t i o n the f o l l i c u l a r membranes o f f u l l y developed r u p t u r e and r e l e a s e the eggs.  The f o l l i c u l a r membrane i s not r e l e a s e d  but r e t a i n e d w i t h i n the ovary where i t h y p e r t r o p h i e s .  Three  phases i n h i s t o g e n e s i s can be d i s t i n g u i s h e d . Stage I ( F i g . 23, 24, 25).  A f t e r o v u l a t i o n both t h e c a  and g r a n u l o s a c e l l s become h y p e r t r o p h i e d w i t h the more marked response i n the g r a n u l o s a ( F i g . 25).  The morpho-  l o g i c a l appearance of t h i s s t a g e i s the same as the ystage a t r e t i c f o l l i c l e . broken  Sometimes the remains  o f the  f o l l i c u l a r w a l l i s e v i d e n t ( F i g . 23) and appears  as a d i s t i n c t i v e  f e a t u r e but the m a j o r i t y o f p o s t - o v u l a -  t o r y b o d i e s l o o k l i k e y-stage a t r e t i c oocytes w i t h no indication due  of the r e l e a s e o f eggs ( F i g . 24); t h i s may be  to the way the o v a r i e s were s e c t i o n e d .  Stage I I ( F i g . 26, 27).  oocytes  There were no d i f f e r e n c e s be-  tween the <5-stage corpus luteum and the stage I I p o s t -  distinct  43  o v u l a t o r y corpus luteum.  The g r a n u l o s a c e l l s converged  i n t o an i r r e g u l a r mass as i n t h e 6-stage a t r e t i c  oocyte  and l o s t t h e i r g l a n d u l a r n a t u r e ; as the t h e c a s u r r o u n d s the compact mass o f g r a n u l o s a c e l l s .  As i n t h e 6-stage  a t r e t i c o o c y t e , y e l l o w l u t e i n pigments i n t e r m i n g l e w i t h granulosa  cells.  Stage I I I ( F i g . 2 8 ) .  The i r r e g u l a r mass o f c e l l s i n  s t a g e I I p o s t - o v u l a t o r y f o l l i c l e s seem t o d i f f e r e n t i a t e i n t o o o g o n i a l c y s t s as i n t h e 6-stage a t r e t i c  oocyte.  These o o g o n i a l c y s t s c o n s t i t u t e t h e s t a g e I I I o r e - s t a g e corpus luteum.  Again, p a r t i a l l y d i f f e r e n t i a t e d  oogonial  c y s t s were o b s e r v e d w i t h one h a l f composed o f u n d i f f e r e n t i a t e d s t a t e I I c e l l s c o n t a i n i n g y e l l o w l u t e a l pigments w h i l e the other h a l f c o n s i s t s of w e l l defined This evidence  oogonia.  prompted t h e p o s t u l a t i o n o f t h e d i f f e r e n t i a -  t i o n of stage I I c e l l s i n t o oogonial c y s t s .  H i s t o c h e m i c a l l o c a l i z a t i o n o f h y d r o x y s t e r o i d dehydrogenases.  The  r e s u l t s o f the h i s t o c h e m i c a l l o c a l i z a t i o n o f h y d r o x y s t e r o i d dehydrogenase are presented  i n T a b l e 4.  t r i b u t i o n o f diaphorase  Formazan d e p o s i t s demonstrated a g e n e r a l  a c t i v i t y i n sections of ovaries incubated i n a  medium c o n t a i n i n g N i t r o - B T and NAD b u t l a c k i n g s t e r o i d s u b s t r a t e . diaphorase  a c t i v i t y was o b s e r v e d i n oocytes  w i t h i n oocytes,  dis-  as w e l l as f o l l i c u l a r  t h i s a c t i v i t y increased w i t h oocyte diameter.  The cells;  I t was  Stage I p o s t - o v u l a t o r y corpus luteum s t a i n e d w i t h Mallory's trichrome. the  first  T h i s photograph documents f o r  time evidence of the r u p t u r e f o l l i c u l a r w a l l  (arrow) f o l l o w i n g  ovulation.  Stage I p o s t - o v u l a t o r y corpus luteum a l s o s t a i n e d w i t h Mallory's trichrome.  Here t h e r e i s no evidence o f the  r u p t u r e o f the f o l l i c l e a f t e r o v u l a t i o n .  This i s mainly  due to the s e c t i o n i n g o f the o v a r i a n t i s s u e . A d e t a i l e d photograph o f the stage I p o s t - o v u l a t o r y corpus luteum showing the h y p e r t r o p h y o f g r a n u l o s a c e l l s  (c) as  compared t o the f o l l i c u l a r c e l l s , b o t h t h e c a l and granul o s a o f the normal ovary ( f ) .  The r a d i a t e n a t u r e o f t h e  zona r a d i a t a (z) i s e v i d e n t . Stage I I p o s t - o v u l a t o r y corpus luteum ( a r r o w s ) . l o s a and t h e c a l c e l l s  The granu-  c o l l a p s e d i n t o an i r r e g u l a r mass o f  cells. A d e t a i l e d photograph o f t h e s t a g e I I corpus luteum.  The  c e l l s e s p e c i a l l y t h e n u c l e i a r e l e s s h y p e r t h r o p h i e d than those o f the s t a g e I corpus luteum ( F i g . 2 5 ) . The  stage I I I p o s t - o v u l a t o r y corpus luteum s t a i n e d w i t h  haematoxylin and e o s i n . the  T h i s stage i s c h a r a c t e r i z e d by  d i f f e r e n t i a t i o n o f some c e l l s i n t o o o g o n i a (arrow).  T a b l e 4:  A summary o f the h i s t o c h e m i c a l r e a c t i o n o f h y d r o x y s t e r o i d dehydrogenases i n o v a r i a n follicles.  STEROID SUBSTRATE  STAGES IN OOGENESIS  ENZYME  1 s t growth Vesicle  Early yolk Vesicle  Late yolk Vesicle  Yolk  granule  DtUycU ue . i •T  Control  Diaphorase  Dehydroepiandrosterone  33-HSD  Androsterone  3a-HSD  17a-hydroxy pregnenolone  33-HSD o r 17a-HSD  17a-hydroxyprogesterone  17a-HSD  Estradiol  173-HSD  Testosterone  176-HSD  +  +  4-  4-4-44  4-  ++7  4-4-  I n t e n s i t y o f r e a c t i o n s were graded (-) t o ( M i l ) ; (-) denotes no demonstrable a c t i v i t y and a maximal a c t i v i t y . 1  HSD i s h y d r o x y s t e r o i d T  dehydrogenase.  (MM)  46 much reduced when t h e t e t r a z o l i u m s a l t , MTT, was used a s t h e f i n a l electron acceptor. No 3 3 - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y was e v i d e n t i n ooc y t e s a t the l a t e y o l k g r a n u l e stages activity  ( F i g . 30) although some enzyme  was d i s c e r n a b l e i n e a r l y y o l k g r a n u l e stage o o c y t e s .  3 3 - h y d r o x y s t e r o i d dehydrogenase. i n a medium  used f o r the demonstration  O v a r i e s r e a c t e d when i n c u b a t e d of 33-hydroxysteroid  dehydro-  genase w i t h e i t h e r NAD o r NADP as a c o - f a c t o r b u t the r e a c t i o n was weaker w i t h NADP as t h e c o - f a c t o r . d r o s t e r o n e o r pregnenolone. follicular  c e l l s of f i r s t  i m p o s s i b l e t o determine  The s u b s t r a t e was  dehydroepian-  T h i s r e a c t i o n was c l e a r l y l o c a l i z e d i n  growth phase oocytes  ( F i g . 29) but i t was  whether the r e a c t i o n was o f t h e c a l o r granu-  l o s a l o r i g i n s i n c e these t i s s u e s a r e not w e l l d i f f e r e n t i a t e d . s m a l l e r second  The  growth phase o o c y t e s , i n the y o l k v e s i c l e s t a g e , gave  p o s i t i v e r e s u l t s w i t h the r e a c t i o n l o c a t e d i n t h e g r a n u l o s a  cells;  o c c a s i o n a l l y 3 3 - h y d r o x y s t e r o i d dehydrogenase was a l s o d e t e c t e d i n t h e theca c e l l s during t h i s  stage.  No 3 3 - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y was e v i d e n t i n o o c y t e s a t the l a t e y o l k g r a n u l e s t a g e s  ( F i g . 30) a l t h o u g h some enzyme  a c t i v i t y was d i s c e r n i b l e i n e a r l y y o l k g r a n u l e stage o o c y t e s .  3ot-Hydroxysteroid dehydrogenase a c t i v i t y .  The d i s t r i b u t i o n o f  3 a - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n g o l d f i s h o v a r i e s , was s i m i l a r t o t h a t o f 3 3 - h y d r o x y s t e r o i d dehydrogenase.  The b e s t r e s u l t s were  o b t a i n e d w i t h NAD as a c o - f a c t o r ; NADP gave a much weaker r e a c t i o n .  47  T h i s 3 a - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y was periphery of f i r s t  growth phase oocyte as was  s t e r o i d dehydrogenase ( F i g . 29).  l o c a l i z e d i n the  the case f o r 33-hydroxy-  Formazan d e p o s i t s w i t h  androsterone  as s u b s t r a t e were d e t e c t e d i n the g r a n u l o s a o f y o l k v e s i c l e stage ooc y t e s b u t t h e a c t i v i t y o f 3 a - h y d r o x y s t e r o i d dehydrogenase was much weaker than 3 3 - h y d r o x y s t e r o i d dehydrogenase.  As p r e v i o u s l y d e s c r i b e d f o r 33-  h y d r o x y s t e r o i d dehydrogenase, no 3 a - h y d r o x y s t e r o i d dehydrogenase ty was  d e t e c t e d i n the f o l l i c u l a r c e l l s o f y o l k g r a n u l e stage  17a^-Hydroxysteroid  dehydrogenase.  activi-  oocytes.  17a-hydroxy p r o g e s t e r o n e  was  s u c c e s s f u l l y used as a s u b s t r a t e f o r the h y d r o x y s t e r o i d dehydrogenase reaction.  T h i s r e a c t i o n i n d i c a t e s the presence  of 1 7 a - h y d r o x y s t e r o i d  dehydrogenase enzymes i n g o l d f i s h o v a r i e s . The d i s t r i b u t i o n of t h i s enzyme was 3 3 - h y d r o x y s t e r o i d dehydrogenases.  s i m i l a r t o t h a t o f 13a- and  In s h o r t , 17a-hydroxysteroid  dehydro-  genase o c c u r r e d i n the p e r i p h e r y o f f i r s t growth phase o o c y t e s and i n the g r a n u l o s a o f y o l k v e s i c l e stage oocytes but y o l k g r a n u l e stage showed no s i g n o f 1 7 a - h y d r o x y s t e r o i d  dehydrogenase a c t i v i t y .  oocytes  The 17a-  h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n the o v a r i e s i s l e s s i n t e n s e than 3 3 - h y d r o x y s t e r o i d dehydrogenase and s i m i l a r i n i n t e n s i t y w i t h 3a-hydroxyteroid  dehydrogenase.  173-Hydroxysteroid  dehydrogenase.  E s t r a d i o l and t e s t o s t e r o n e were  s u c c e s s f u l l y used as s u b s t r a t e s f o r the demonstration dehydrogenase a c t i v i t y i n the ovary. ase r e a c t i o n s NAD  of  173-hydroxysteroid  As i n the h y d r o x y s t e r o i d dehydrogen-  gave a b e t t e r r e a c t i o n than NADP.  48  F i g u r e 29:  Histochemical demonstration  of 33-hydroxysteroid  genase i n o v a r i e s o f n o r m a l f i s h .  dehydro-  The formazan d e p o s i t s  (arrow) a r e l o c a l i z e d i n t h e g r a n u l o s a c e l l s o f o o c y t e s . The n u c l e i (n) a r e l a b e l l e d t o p r o v i d e e a s i e r i n t e r p r e t a t i o n of t h e i l l u s t r a t i o n .  F i g u r e 30:  O v a r i e s o f g r a v i d f i s h s t a i n e d w i t h 3 ( 3 - h y d r o x y s t e r o i d dehydrogenase.  There were no a c t i v i t i e s i n t h e f o l l i c u l a r  c e l l s ( f r e e arrow) o f t h e y o l k granulestage  F i g u r e 31:  oocytes.  O v a r i e s o f a hypophysectomized g o l d f i s h (two weeks a f t e r hypophysectomy) s t a i n e d f o r 1 7 3 - h y d r o x y s t e r o i d ase.  dehydrogen-  A c t i v i t y , as i n d i c a t e d by t h e d e p o s i t i o n o f formazan  (arrow) was l o c a l i z e d i n y - s t a g e a t r e t i c f o l l i c l e s .  No  enzyme a c t i v i t y was p r e s e n t i n f o l l i c u l a r c e l l s of t h e f i r s t growth phase o o c y t e s .  F i g u r e 32:  Histochemical demonstration  o f 3 [ 3 - h y d r o x y s t e r o i d dehydro-  genase i n the p o s t - o v u l a t o r y f o l l i c l e s o f r e c e n t l y o v u l a t e d goldfish. luteum  H i g h enzyme a c t i v i t y was d e t e c t e d i n t h e corpus  (arrow).  49  The distribution of 173-hydroxysteroid dehydrogenases in the ovaries was similar to other hydroxysteroid dehydrogenases.  The reac-  tion with either estradiol or testosterone was restricted to the periphery of first growth phase oocytes and the granulosa of yolk vesicle stage oocytes.  It was impossible to determine the precise  location of 173-hydroxysteroid dehydrogenase i n the f i r s t growth phase oocytes as the follicular tissues were poorly differentiated.  As in  the other hydroxysteroid dehydrogenases examined, there was a gradual reduction of enzyme activity as the oocyte developed into the yolk granule stage which showed no evidence of 173-hydroxysteroid dehydrogenase activity.  50 HYDROXYSTEROID DEHYDROGENASES IN HYPOPHYSECTOMIZED FISH T a b l e 5 summarizes  the d i s t r i b u t i o n  o f the v a r i o u s h y d r o x y s t e r o i d  dehydrogenases i n the o v a r i e s of hypophysectomized One week a f t e r hypophysectomy, observed  but no q u a n t i f i c a t i o n  goldfish.  o n l y a-stage a t r e t i c oocytes were  o f the r a t e o f a t r e s i a was  because of the g r e a t v a r i a b i l i t y as i n d i c a t e d e a r l i e r .  attempted  Hydroxysteroid  dehydrogenases were not d e t e c t e d i n c e l l s o f e i t h e r a-stage oocytes in  or i n f i r s t  growth phase o o c y t e s .  the f o l l i c l e s o f f i r s t  The l a c k o f enzyme a c t i v i t y  growth phase oocytes  of hypophysectomized g o l d f i s h  atretic  c h a r a c t e r i z e d the o v a r i e s  at a l l times.  Most y o l k y oocytes have become a t r e t i c by the end o f the second o r t h i r d weeks of hypophysectomy. Y-stage d u r i n g t h i s p e r i o d . oocytes,  they were mainly  A t r e s i a progressed  Although  as f a r as the  t h e r e were some a-stage  atretic  i n t h e 3 - or y-stages and no s i g n i f i c a n t  dif-  f e r e n c e s were noted i n t h e o v a r i e s o f f i s h hypophysectomized f o r two o r t h r e e weeks. Hydroxysteroid  dehydrogenase a c t i v i t y was  f i r s t growth phase oocytes In s t r i k i n g  as w e l l as « - and  was  granulosa  ( F i g . 31).  oocytes.  This  was used as  The o t h e r h y d r o x y s t e r o i d dehydrogenases, 3a-,  17a-hydroxysteroid  oocytes.  173-hydroxysteroid  o b t a i n e d whether t e s t o s t e r o n e or e s t r a d i o l  the s u b s t r a t e .  atretic  i n f o l l i c l e s of  3-stage a t r e t i c  c o n t r a s t , y-stage a t r e t i c oocytes had  dehydrogenase, a c t i v i t y i n the h y p e r t r o p h i e d activity  absent  33-,  dehydrogenases, were n o t d e t e c t e d i n t h e y-stage  and  T a b l e 5.  H i s t o c h e m i c a l R e a c t i o n o f H y d r o x y s t e r o i d Dehydrogenase i n O v a r i e s o f Hypophysectomized Goldfish.  STEROID SUBSTRATE  ENZYME  Control  Diaphorase  Dehydroepiandrosterone  33-HSD  Androsterone  3a-HSD  17a-hydroxyprogesterone  17a-HSD  Estradiol  173-HSD  Testosterone  173-HSD  1 s t growth phase o o c y t e  a  corpus l u t e u m s t a g e s 3 y  6  1  I n t e n s i t y o f h y d r o x y s t e r o i d dehydrogenase r e a c t i o n and ( M M ) maximal.  a c t i v i t y graded (-) t o  ^HSD denotes h y d r o x y s t e r o i d dehydrogenase  enzymes.  ( M i l ) ;  (-) denotes no demonstrable-  2 No enzyme a c t i v i t y was d e t e c t e d i n second growth phase o o c y t e s when t h e y were p r e s e n t . Ln  52  No intact second growth phase oocytes were found i n ovaries of fish  k i l l e d four weeks after hypophysectomy.  A l l yolk oocytes had  become a t r e t i c and some of them reached the 6-stage while others were at the a-stage.  These a-stage a t r e t i c oocytes were much smaller than  those observed e a r l i e r ; the majority of oocytes were at the 3- or ystages.  As i n the ovaries of f i s h hypophysectomized f o r two and three  weeks hydroxysteroid dehydrogenases were absent from f i r s t growth phase oocytes and from the a-, 3- or 6-stages a t r e t i c f o l l i c l e s .  Although  173-hydroxysteroid dehydrogenase a c t i v i t y was detected i n y-stage atret i c oocytes, 3a-,  33-> and 17a-hydroxysteroid dehydrogenases were not  evident. The ovaries of f i s h k i l l e d i n the f i f t h to eight week a f t e r hypophysectomy had only f i r s t growth phase oocytes and 6-stage a t r e t i c cles.  folli-  No hydroxysteroid dehydrogenases were found i n these ovaries. In summary, hypophysectomy inhibited  a l l hydroxysteroid dehydrogen-  ases except 17 3-hydroxy steroid dehydrogenase i n the y-stage a t r e t i c follicles.  53  HYDROXYSTEROID DEHYDROGENASES IN HYPOPHYSECTOMIZED GOLDFISH INJECTED WITH PORCINE LUTEINIZING HORMONE  F i s h hypophysectomized f o r one week were i n j e c t e d p o r c i n e l u t e i n i z i n g hormone (NIH-LH-S8). toneal injections, dehydrogenases. (Table 6 ) .  w i t h 100  ug/g  Two days a f t e r t h e i n t r a p e r i -  t h e f i s h were k i l l e d and examined f o r h y d r o x y s t e r o i d  T h e i r o v a r i e s gave s t r o n g r e a c t i o n s f o r a l l f o u r enzymes  Apparently,  the l u t e i n i z i n g hormone s t i m u l a t e s f o r m a t i o n o f  t h e s e enzymes s i n c e h y d r o x y s t e r o i d dehydrogenases were not d e t e c t e d i n u n t r e a t e d hypophysectomized g o l d f i s h . f i s h the d i s t r i b u t i o n  I n l u t e i n i z i n g hormone-treated  o f the formazan d e p o s i t s was normal e x c e p t f o r the  presence of a l l f o u r h y d r o x y s t e r o i d dehydrogenases i n y-stage  atretic  oocytes. As i n the o v a r i e s o f hypophysectomized g o l d f i s h , dehydrogenase enzymes were d e t e c t e d i n the a-, oocytes  o f t h e hormoner:treated f i s h .  no  hydroxysteroid  and 6-stage  atretic  By c o n t r a s t , t h e y-stage  atretic  o o c y t e s had a c t i v i t i e s o f 3 a - , 3(3-, 17a-, and 1 7 3 - h y d r o x y s t e r o i d  dehydro-  genases; o f t h e s e , 1 7 3 - h y d r o x y s t e r o i d  active.  This i s d i f f e r e n t steroid  dehydrogenase was  from hypophysectomized f i s h where o n l y  the most  173-hydroxy-  dehydrogenase was d e t e c t e d i n t h e y-stage a t r e t i c o o c y t e and  a g a i n argues f o r s t i m u l a t i o n o f enzyme f o r m a t i o n by LH.  T a b l e 6.  E f f e c t o f LH Treatment on H i s t o c h e m i c a l l y Demonstrable H y d r o x y s t e r o i d Dehydrogenases i n O v a r i e s o f Hypophysectomized G o l d f i s h  Steroid Substrate  Enzyme  Control  diaphorase  Dehydroepiandrosterone  33-HSD  Androsterone  3a-HSD  17a-hydroxyprogesterone  17a-HSD  Estradiol  176-HSD  Testosterone  176-HSD  1 s t growth phase oocytes  Stages o f a t r e s i a  a  g  y  * •6  +  ++++  Ul  55  HYDROXYSTEROID DEHYDROGENASES IN POST-OVULATORY CORPUS LUTEUM  The  h i s t o c h e m i c a l d e t e c t i o n o f h y d r o x y s t e r o i d dehydrogenases was  c a r r i e d out a t t h r e e times a f t e r o v u l a t i o n : on the day o f o v u l a t i o n and on the seventh and t h i r t i e t h  days a f t e r o v u l a t i o n .  a f t e r o v u l a t i o n showed v e r y a c t i v e h y d r o x y s t e r o i d i n their ovaries.  c e l l s although  The formazan d e p o s i t s were m a i n l y i n  o c c a s i o n a l l y the t h e c a l c e l l s  Except f o r t h i s v e r y a c t i v e h y d r o x y s t e r o i d post-ovulatory  dehydrogenase r e a c t i o n s  B l u e formazan d e p o s i t s developed i n t h e 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 a l l the s u b s t r a t e s . the g r a n u l o s a  Fish k i l l e d shortly  stained also.  dehydrogenase r e a c t i o n i n t h e  c o r p o r a l u t e a ( F i g . 32) t h e ovary l o o k s l i k e a normal  v i t e l l o g e n i c ovary.  First  growth phase oocytes  developed formazan i n  the f o l l i c u l a r c e l l s w i t h a l l the s t e r o i d s u b s t r a t e s .  Likewise,  i n the y o l k v e s i c l e stages had  dehydrogenases  but  a l l the hydroxysteroid  these were c l e a r l y c o n f i n e d t o the g r a n u l o s a  o v a r i e s , no h y d r o x y s t e r o i d  dehydrogenase a c t i v i t y  l a r c e l l s during the y o l k granules The  cells.  As i n normal  occured  i n the f o l l i c u -  stages.  d i s t r i b u t i o n o f the v a r i o u s h y d r o x y s t e r o i d  seem t o change d u r i n g the f i r s t  oocytes  dehydrogenases d i d not  seven days a f t e r o v u l a t i o n .  p o s t - o v u l a t i o n , t h e r e were v e r y few stage I p o s t - o v u l a t o r y  A t seven days corpora l u t e a  but most o f t h e post o v u l a t o r y f o l l i c l e s had reached stage I I . The  stage I p o s t o v u l a t o r y corpus luteum showed a l l f o u r hydroxy-  s t e r o i d dehydrogenases (Table 7) w i t h formazan d e p o s i t s i n the g r a n u l o s a cells. strates.  These d e p o s i t s were observed w i t h Only s l i g h t  a l l s t e r o i d s t e s t e d as sub-  a c t i v i t y was observed i n t h e stage  I I post  ovulatory  T a b l e 7:  H i s t o c h e m i c a l L o c a l i z a t i o n o f H y d r o x y s t e r o i d Dehydrogenases i n the O v a r i e s o f Ovulated G o l d f i s h  Steroid Substrate  Enzyme  Stages l  g  t  Y  growth  Control  Diaphorase  o  l  i n Oogenesis k  vesicle  Y  o  l  k  Post O v u l a t o r y g  t  a  g  e  I  g  t  Follicles ^  a  g  e  Granule  +  +  +  +  4-  +++  4+++  +  4-H-  +  4+  +  Dehydroepiand androsterone  33-HSD  Androsterone  3a-HSD  +4-  444-  +  17a-hydroxyprogesterone  17a-HSD  4-4-  4+  +•  4-  +•  Estradiol  17B-HSD  4+4-  +4-f  +  +4+  +  ON  57  c o r p u s luteum.  The r e a c t i o n was  n o t much g r e a t e r t h a n i n t h e c o n t r o l  s e c t i o n s and suggested t h a t the r e a c t i o n was  due  to diaphorase  The h i s t o c h e m i c a l d i s t r i b u t i o n o f these enzymes i n the  activity.  other  o v a r i a n t i s s u e s i s s i m i l a r t o the d i s t r i b u t i o n i n the normal v i t e l l o genic ovary.  The  s m a l l oocytes  i n the f i r s t growth phase had  h y d r o x y s t e r o i d dehydrogenase a c t i v i t y . with a l l steroid substrates.  T h i s h i g h a c t i v i t y was  Y o l k v e s i c l e stage oocytes  36-, 17a- and 1 7 3 - h y d r o x y s t e r o i d  high  showed  3a-,  dehydrogenase a c t i v i t i e s i n the granu-  l o s a ; r e a c t i o n s were comparable to those o f the normal o v a r y . o t h e r o v a r i e s examined, t h e r e was  obtained  As i n the  no h y d r o x y s t e r o i d dehydrogenase a c t i v i -  t y i n the f o l l i c l e s o f y o l k g r a n u l e s s t a g e o o c y t e s .  Only stage I I 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 are found t h i r t y days a f t e r o v u l a t i o n .  These  are m o r p h o l o g i c a l l y s i m i l a r t o the 6-stage p r e - o v u l a t o r y c o r p o r a l u t e a and h y d r o x y s t e r o i d dehydrogenase a c t i v i t y was  d e t e c t e d i n them.  The  distribu-  t i o n o f h y d r o x y s t e r o i d dehydrogenase i n the o t h e r o v a r i a n t i s s u e s was s i m i l a r t o t h a t of the e a r l i e r stages w i t h r e a c t i o n s i n the c e l l s o f f i r s t growth phase oocytes c l e stage o o c y t e s .  as w e l l as the g r a n u l o s a o f y o l k v e s i -  A g a i n , no h y d r o x y s t e r o i d dehydrogenase a c t i v i t y  c u r r e d i n y o l k g r a n u l e stage I t i s concluded  follicular  oocytes.  t h a t the p o s t - o v u l a t o r y corpus l u t e u m i s c a p a b l e  s t e r o i d hormone s y n t h e s i s .  oc-  of  V e r y a c t i v e s t e r o i d s y n t h e s i s o c c u r s i n the  c o r p u s l u t e u m as w e l l as i n the g r a n u l o s a c e l l s o f p o s t - o v u l a t o r y o v a r i e s . The Fate o f P o s t o v u l a t o r y Corpus Luteum as Determined by T r i t i a t e d thymidine  Autoradiography  l a b l e s were d e t e c t e d i n f o l l i c u l a r c e l l s o f  3 k i l l e d one day a f t e r t h y m i d i n e - H i n j e c t i o n ( F i g . 3 3 ) .  More o f t h e s e  fish cells  58 were l a b e l l e d n e a r t h e o v a r i a n p e r i p h e r y t h a n i n the c e n t e r o f o v a r i e s . Follicular ment.  c e l l s were l a b e l l e d i r r e s p e c t i v e o f t h e s t a g e o f ovum d e v e l o p -  None o f t h e abundant s t a g e I 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 were  labelled. 3  The p a t t e r n o f t h y m i d i n e - H i n c o r p o r a t i o n was s i m i l a r i n o v a r i e s o f 3  f i s h k i l l e d two days a f t e r t h y m i d i n e - H i n j e c t i o n w i t h f o l l i c u l a r  cells 3  l a b e l l e d randomly i n the o o c y t e s .  On t h e f o u r t h day a f t e r t h y m i d i n e - H  i n j e c t i o n , however, l a b e l s were observed i n t h e c e l l s ovulatory corpora l u t e a  o f stage I I post-  ( F i g . 34) as w e l l as the f o l l i c u l a r c e l l s .  none of s t a g e I c o r p o r a l u t a c e l l s were l a b e l l e d .  Again,  The m a j o r i t y o f t h e  s t a g e IT c o r p o r a l u t e a were l a b e l l e d i n o v a r i e s on f i s h k i l l e d e i g h t days 3  a f t e r t h y m i d i n e - H i n j e c t i o n . The i n c r e a s e i n number o f s t a g e I I c o r p o r a l u t e a . was accompanied by a r e d u c t i o n i n s t a g e I c o r p o r a l u t e a . 3  The d i s t r i b u t i o n o f t h y m i d i n e - H l a b e l s i n o v a r i e s o f f i s h 16 days a f t e r t r i t i a t e d  t h y m i d i n e i n j e c t i o n was e s s e n t i a l l y s i m i l a r t o  t h a t o f f i s h k e p t f o r 8 days w i t h some l a b e l l e d c e l l s s t a g e IT c o r p o r a l u t e a .  killed  The f o l l i c u l a r  of t h e s t a g e o f t h e o o c y t e .  i n the majority of  c e l l s were l a b e l l e d ,  irrespective  The o n l y d i f f e r e n c e was t h e absence o f stage  I corpora l u t e a i n the o v a r i e s of these f i s h .  The f o l l i c u l a r c e l l s o f  o o c y t e s as w e l l as c e l l s o f s t a g e IT c o r p o r a l u t e a were l a b e l l e d i n o v a r 3 i e s o f f i s h k i l l e d 32 days a f t e r t h y m i d i n e - H i n j e c t i o n . many o o g o n i a ( F i g . 35) and some o o c y t e s were l a b e l l e d  In addition  ( F i g . 36).  The c o n c l u s i o n o f t h e experiments d e s c r i b e d i n t h i s s e c t i o n i s t h a t both f o l l i c u l a r  and s t a g e I I c o r p o r a l u t e a c e l l s  are a c t i v e l y  multiplying.  One o f t h e s e two c e l l types d i f f e r e n t i a t e s i n t o o o g o n i a and u l t i m a t e l y oocytes.  59  F i g u r e 33:  A u t o r a d i o g r a p h y o f the o v a r y seven.days  a f t e r the  3  i n j e c t i o n o f t h y m i d i n e - H.  The f o l l i c u l a r  cells  (arrow) were l a b e l l e d . F i g u r e 34:  A u t o r a d i o g r a p h y o f the o v a r y , seven days a f t e r the 3  i n j e c t i o n o f t h y m i d i n e - H.  In addition  to the l a b e l -  l i n g o f f o l l i c u l a r c e l l s ( F i g . 33) c e l l s o f 5-stage corpus luteum were a l s o l a b e l l e d ( a r r o w s ) . F i g u r e 35:  A u t o r a d i o g r a p h y o f g o l d f i s h o v a r i e s 30 days a f t e r t h y 3  midine- H i n j e c t i o n .  Some o o g o n i a w i t h i n o o g o n i a l c y s t s 3  were l a b e l l e d by t h y m i d i n e - H ( a r r o w ) .  3  F i g u r e 36:  A u t o r a d i o g r a p h y o f o o c y t e l a b e l l e d w i t h t h y m i d i n e - H. t h i s o o c y t e was from t h e o v a r y o f g o l d f i s h i n j e c t e d w i t h 3  t h y m i d i n e - H f o r 30 days.  60  DISCUSSION  This investigation indicated that the f o l l i c u l a r c e l l s , especiall y the granulosa, serve many functions. They are the major endocrine tissue i n the ovary, either as f o l l i c u l a r c e l l s of normal oocytes, or as corpora lutea.  In addition to steroid hormone synthesis, the f o l l i -  cular c e l l s are responsible f o r the removal of a t r e t i c oocytes by phagocytosis . After a t r e s i a or ovulation the f o l l i c u l a r c e l l s developed into corpora lutea which l a t e r d i f f e r e n t i a t e into oogonia.  Steroid Synthesis i n Fish Ovaries In the present study, hydroxysteroid dehydrogenases have only been found i n the ovarian granulosa.  The a c t i v i t i e s of 3a-, 33-, 17a-, and  173-hydroxysteroid dehydrogenases increased with oocyte development or d i f f e r e n t i a t i o n , but at the yolk granule stage, a l l four hydroxysteroid dehydrogenases decreased so dramatically that during the l a t t e r part of t h i s stage, no hydroxysteroid dehydrogenases could be detected i n the granulosa. In post-ovulatory ovaries hydroxysteroid dehydrogenases were evident in  f i r s t growth phase oocytes and i n corpora lutea, but were not detec-  table i n the granulosa of large unovulated oocytes.  In summary, these  histochemical findings suggested that the granulosa c e l l s of developing oocytes are capable of steroid synthesis although the actual steroid hormones synthesized are not known. The absence of hydroxysteroid dehydrogenase a c t i v i t y during the yolk granule stage was surprising.  I t indicated an i n h i b i t i o n of steroid  61 synthesizing enzyme a c t i v i t y when oocytes reached the yolk granule stage.  The steroid enzymes were evidently re-activated after ovulation.  Hydroxysteroid dehydrogenase a c t i v i t y seems to depend on the p i t u i tary gland.  Following hypophysectomy, hydroxysteroid dehydrogenases were  not detected i n granulosa of either f i r s t growth phase or yolk v e s i c l e stage oocytes.  Restoration of a c t i v i t y with bovine l u t e i n i z i n g hormone  supports this argument.  Although there are no comparable studies of  hypophysectomy on hydroxysteroid dehydrogenases i n the ovaries of t e l e o s t s , Yamazaki and Donaldson (1968) demonstrated a decrease i n the 3g-hydroxysteroid dehydrogenase of i n t e r s t i t i a l c e l l s of goldfish t e s t i s a f t e r hypophysectomy.  In their experiments, 33-hydroxysteroid dehydrogenase  a c t i v i t y was restored with salmon gonadotropin, again confirming the p i t u i t a r y involvement.  S i m i l a r l y , Yaron (1966) observed that 3B-hydroxy-  steroid dehydrogenase decreased i n t e s t i s of T i l a p i a mossembica  after  hypophysectomy, while Wiebe (1969) found that methallibure administration decreased 33-hydroxysteroid dehydrogenase a c t i v i t y i n i n t e r s t i t i a l and Suertoli c e l l s of Cymatogaster aggregata;  methallibure inhibited  gonado-  tropin function i n this teleost (Hoar et a l . , 1967). In conclusion, i t i s evident that steroidogenesis i n the ovaries and testes of teleosts i s under the control of p i t u i t a r y gonadotropin.  In  the absence of gonadotropin a l l hydroxysteroid dehydrogenase a c t i v i t y ceases. A t r e s i a of Second Growth Phase Oocytes In this study, a t r e s i a was induced by hypophysectomy; i n nature, adverse environmental or physiological conditions probably induce a t r e s i a  62 i n y o l k y o o c y t e s ( Y a r o n , 1971).  The h i s t o l o g y o f a t r e t i c  follicles  i n g o l d f i s h i s s i m i l a r to that described f o r other t e l e o s t (Bretschneider and Duyvene de W i t , 1947; S t o l k , 1951; Beach, 1959; B a r r , 1963; L e h r i , 1968; Lambert, 1970).  I t i s c h a r a c t e r i z e d by t h e h y p e r t r o p h y o f granu-  l o s a c e l l s , o r t h e c a l c e l l s , o r b o t h as demonstrated (1967).  by Chan e t a l .  The e a r l y s t a g e s ( a - and 3-stages) i n v o l v e d t h e breakdown and  r e s o r p t i o n o f moribund o o c y t e s .  The p h a g o c y t i c a c t i o n o f f o l l i c u l a r  c e l l s i n a t r e t i c o o c y t e s was g e n e r a l l y r e c o g n i z e d (see r e v i e w by B a l l , 1960; Dodd, 1960; Hoar, 1965; C h i e f f i , 1970), t h i s f u n c t i o n o f t h e a t r e t i c f o l l i c l e has been observed i n a l l v e r t e b r a t e groups i n c l u d i n g mammals (Brambell,  1955).  Hypertrophy  o f g r a n u l o s a c e l l s i n g o l d f i s h c o n t i n u e s even a f t e r the  y o l k i s completely resorbed.  The y-stage a t r e t i c f o l l i c l e s have one o r  two l a y e r s o f h y p e r t r o p h i e d c e l l s s u r r o u n d i n g an i n n e r c a v i t y , t h e a t r i u m ( F i g . 19).  Though many i n v e s t i g a t o r s r e c o g n i z e d a y-rstage d u r i n g a t r e s i a  (Beach, 1959; B a r r , 1963; Lambert, 1970), t h e i r ^ d e s c r i p t i o n o f y-stage was  s i g n i f i c a n t l y d i f f e r e n t from o u r s and I n v o l v e o n l y t h e i r r e g u l a r mass  o f c e l l s n o t e d i n t h e 6-stage.  These a u t h o r s may have f a i l e d t o observe  an i n t e r m e d i a t e s t a g e b e f o r e the c o l l a p s e o f t h e a t r e t i c f o l l i c l e s .  The  p r e s e n t s t u d y i n d i c a t e s t h a t t h i s s t a g e , c h a r a c t e r i z e d b y g l a n d u l a r morphology, i s very important t o the p h y s i o l o g i c a l f u n c t i o n s o f a t r e t i c follicles. These y-stage a t r e t i c f o l l i c l e s i n g o l d f i s h appear t o be t r u e e p i t h e l i a l g l a n d s ( F i g . 2 0 ) . H i s t o c h e m i c a l d a t a ( T a b l e 5, F i g . 31) show c o n s i d e r a b l e 1 7 3 - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y .  T h i s enzyme  63.  c a t a l y s e s e i t h e r the c o n v e r s i o n o f t e s t o s t e r o n e t o a n d r o s t e r o n e , e s t r a d i o l to e s t r o n e ( T a l a l a y , 1965)  and i t s presence  or  i n y-stage  c o r p o r a l u t e a a t r e t i c a s u p p o r t s Hoar's (1965) s u g g e s t i o n t h a t the  tele-  ost  In  corpus luteum i s r e s p o n s i b l e f o r the s y n t h e s i s of e s t r o g e n s .  c o n t r a s t t o 1 7 3 - h y d r o x y s t e r o i d dehydrogenase, t h e r e was  no evidence f o r  3 3 - h y d r o x y s t e r o i d dehydrogenase o r 3a-  and 1 7 a - h y d r o x y s t e r o i d dehydro-  genases i n y-stage a t r e t i c f o l l i c l e s .  These o b s e r v a t i o n s a r e i n p a r t i a l  agreement w i t h t h o s e o f Lambert (1970) and Y a r o n (1971) who absence of 3 g - h y d r o x y s t e r o i d  r e p o r t e d an  dehydrogenase i n a t r e t i c f o l l i c l e s  of  P o e c i l i a r e t i c u l a t a and T i l a p i a mossembica r e s p e c t i v e l y but d i d not for  3  17 ^-i,-. o r 1 7 3 - h y d r o x y s t e r o i d  test  dehydrogenases.  Hypophysectomized g o l d f i s h i n j e c t e d w i t h LH developed d r o x y s t e r o i d dehydrogenases i n y-stage c o r p o r a l u t e a .  a l l f o u r hy-  This s t i m u l a t i o n  o f h y d r o x y s t e r o i d dehydrogenases suggests t h a t p i t u i t a r y  gonadotropins  are r e s p o n s i b l e f o r t h e a c t i v i t y o f t h e s e h y d r o x y s t e r o i d dehydrogenase enzymes i n t h e a t r e t i c f o l l i c l e s .  Hypophysectomy i m p a i r e d p a r t o f t h e  s t e r o i d synthesizing c a p a b i l i t y of a t r e t i c P o s t - o v u l a t o r y Corpora  follicles.  Lutea  Stage I 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 are s i m i l a r i n appearance t o y-stage a t r e t i c f o l l i c l e s  and c o n s i s t o f h y p e r t r o p h i e d g r a n u l o s a  t h e c a l c e l l s s u r r o u n d i n g an empty c a v i t y . the presence  of an opening  f o l l i c l e ( F i g . 23  and 24).  and  The main d i f f e r e n c e noted  was  i n some, but not a l l o f the p o s t - o v u l a t o r y 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 w i t h t h i s open-  i n g w h i c h marks the p o i n t o f o v u l a t i o n have not been p r e v i o u s l y d e s c r i b e d .  64 In f a c t , most i n v e s t i g a t o r s have p a i d o n l y c u r s o r y a t t e n t i o n t o p o s t ovulatory f o l l i c l e s ,  r e p o r t i n g o n l y t h e i r r a p i d d i s i n t e g r a t i o n and  d i s a p p e a r a n c e ( B a i l e y , 1943; McMillan,  1967).  Barr  (1967) m a i n t a i n e d  Moser  in teleost.  B a r a , 1960;  R a s t o g i , 1966;  (1963), Lambert (1966), R a j a l a k s h m i (1966), and t h a t no p o s t - o v u l a t o r y corpus luteum e x i s t e d  T h e i r c o n c l u s i o n s were based on random sampling which d i d  not i n c l u d e o v a r i e s soon a f t e r o v u l a t i o n . fish  Braekevelt &  o v a r i e s from o v u l a t e d  fish  A detailed analysis of gold-  showed t h a t the p o s t - o v u l a t o r y  follicles  always e x i s t e d and had a d e f i n i t e p a t t e r n o f h i s t o g e n e s i s . L o c a l i z e d hormone p r o d u c t i o n .  The d i s t r i b u t i o n o f h y d r o x y s t e r o i d  dehydrogenases i n stage I p o s t - o v u l a t o r y stage  atretic follicles  i nfish  f o l l i c l e s was s i m i l a r t o y  i n j e c t e d w i t h LH.  I n s h o r t , the t e s t s  were p o s i t i v e f o r 173-hydroxysteroid dehydrogenase as w e l l as 3a-, and  17a-hydroxysteroid dehydrogenases.  -  33-  However, these stage I 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 w i t h complete s t e r o i d s y n t h e s i z i n g c a p a c i t i e s were r a t h e r t r a n s i e n t and l a s t e d no l o n g e r than a week. ovulation hydroxysteroid  Seven days a f t e r  dehydrogenase a c t i v i t y had d e f i n i t e l y d e c l i n e d .  B a r a (1965) demonstrated weak 33-hydroxysteroid dehydrogenases i n t h e theca of post-ovulatory gested  follicles  t h a t stage I p o s t - o v u l a t o r y  o f Scomber scomber and t h i s a l s o sugf o l l i c l e s were capable  of steroid  hormone s y n t h e s i s . The main d i f f e r e n c e between the p o s t - o v u l a t o r y b o d i e s  described  here and the mammalian corpus luteum i s the sparse v a s c u l a r i t y o f t h e t e l e o s t corpus luteum.  As i n stage 1 p o s t - o v u l a t o r y f o l l i c l e s ,  stage a t r e t i c f o l l i c l e s were p o o r l y v a s c u l a r i z e d .  Again,  the  t h i s poor  y  65 v a s c u l a r i z a t i o n suggested t h a t t h e hormone s y n t h e s i z e d , most l i k e l y e s t r o g e n , a c t e d l o c a l l y and were p r o b a b l y n o t r e l e a s e d i n l a r g e amounts i n t o t h e c i r c u l a t o r y system.  T h i s l o c a l i z a t i o n o f s t e r o i d s would r e s u l t  i n h i g h c o n c e n t r a t i o n o f s t e r o i d hormones i n the corpus luteum.  High  l e v e l s o f e s t r o g e n s w i t h i n t h e o v a r y might be r e q u i r e d t o induce  oogonial  d i f f e r e n t i a t i o n ; t h i s l o c a l i z a t i o n of estrogens  a t the p o i n t o f a c t i o n  would p r o v i d e a mechanism f o r d i r e c t a c t i o n o f e s t r o g e n on the t a r g e t tissues without  a s i m i l a r r i s e i n b l o o d e s t r o g e n w h i c h might i n h i b i t t h e  production of gonadotropin.  L o c a l i z e d a c t i o n s o f s t e r o i d hormone p r o v i d e  a p l a u s i b l e e x p l a n a t i o n o f t h e asynchronus development o f oocytes  i n the  ovary. I n h i s s t u d i e s on t h e t e s t i c u l a r c y c l e of a n o t h e r amniote, t h e r e p t i l e Naja naja,  L o f t s (1972) found t h a t t h e S e r t o l i c e l l s , a homologue  o f t h e g r a n u l o s a c e l l s , s y n t h e s i z e d androgens w h i c h were n o t r e l e a s e d i n t o t h e p e r i p h e r a l v a s c u l a r system b u t induced cells locally.  the p r o p a g a t i o n  o f germ  I n a d d i t i o n the S e r t o l i c e l l s o f r e p t i l e s p l a y e d an im-  p o r t a n t r o l e i n the s y n c h r o n i z a t i o n and maintenance o f t h e germ c e l l s . P o s t - o v u l a t o r y p r o l i f e r a t i o n of o o g o h i a .  Yamazaki (1965) observed  numerous o o g o n i a i n o v a r i e s o f g o l d f i s h w i t h c o r p o r a l u t e a .  Though  o o g o n i a a r e d e t e c t e d i n the o v a r i e s o f many d i f f e r e n t t e l e o s t s throughout the y e a r , a sharp r i s e i n number o f f r e q u e n t l y observed s h o r t l y a f t e r spawning ( C r a i g - B e n n e t t , 1930; H i c k l i n g , 1935; Matthews, 1938; Mendoza, 1943;  B u l l o u g h , 1942; B a r r , 1963; Yamazaki, 1965).  o f o o g o n i a a f t e r o v u l a t i o n and t h e o c c u r r e n c e  The sudden i n c r e a s e  o f h y d r o x y s t e r o i d dehydro-  genases i n p o s t - o v u l a t o r y f o l l i c l e s s u g g e s t s some r e l a t i o n s h i p between  66 the two processes and the p o s s i b i l i t y that the post-ovulatory f o l l i c l e s may secrete steroid.hormones  to  induce oogonial formation.  Bullough (1942) and Egami (1954) demonstrated  that estrogens i n -  duced an increase i n the number of oogonia i n ovaries of Phoxirius l a e v i s and Oryzias l a t i p e s respectively.  Yamamoto (1969) has reviewed  the many d i f f e r e n t studies on sex d i f f e r e n t i a t i o n i n t e l e o s t that demonstrated the potency of estrogens as inducers of oogonial formation. These findings seem to support Hoar's (1965) hypothesis that the teleost corpus luteum synthesizes estrogens rather than progesterone and that the estrogens may induce germ c e l l s to form oogonia. The Fate of Corpus Luteum C e l l s 3  The incorporation of thymidine- H into r e l a t i v e l y large numbers of stage I I post-ovulatory corpora lutea c e l l s indicated that these c e l l s were a c t i v e l y multiplying rather than being inactive and dying as some authors have suggested (Polder, 1964; Lambert, 1970). The autoradiographic evidence suggests that oogonia originated from either the f o l l i c u l a r c e l l s or corpora lutea c e l l s .  The h i s t o l o g i c a l  picture of intermediate stages between stage IT corpora lutea and oogonial cysts supports the suggestion that some c e l l s i n the stage IT corpora lutea d i f f e r e n t i a t e into oogonia. This postulation that oogonia were derived from corpora lutea c e l l s of recently ovulated or resorbed ova i s supported by the sudden increase i n oogonia after ovulation or resorption of ova.  Yamazaki (1965) ob-  served large numbers of oogonia i n ovaries with a t r e t i c oocytes.  Other  67 workers have also noted a sharp r i s e i n the number of oogonia shortly after spawning (Craig-Bennett, 1930; Hickling, 1930; Matthews, 1938; Mendoza, 1941; Bullough, 1942; Barr, 1963; Yamazaki, 1965). Goldfish are multiple breeders that spawn i n the spring (Yamazaki, 1968).  Like other teleosts that spawn more than once, ovulated eggs  are replaced i n a c y c l i c a l manner ( F i g . 37) a f t e r each spawning.  The reproductive cycle within the ovary.  During the i n i t i a l  ma-  turation, primodial germ c e l l s d i f f e r e n t i a t e into oogonia which form yolk-ladened oocytes. developmental paths.  A f t e r v i t e l l o g e n e s i s , oocytes follow one of two Under adverse conditions the yolk laden oocytes  become a t r e t i c and develop into ing  a  - stage corpora lutea a t r e t i c a .  Dur-  this stage, f o l l i c u l a r c e l l s hypertrophy and phagocytose the a t r e t i c  oocyte and form the g-stage corpora lutea which terminates the resorbtion of  the aberrant oocyte.  of the y s t a g e .  The absorption of the oocyte marks the beginning  Steroid (probably estrogen) synthesis occurs i n y-stage  corpora lutea. The steroid synthesizing c a p a b i l i t y i n y-stage f o l l i c l e s were part i a l l y impaired by hypophysectomy.  Under normal circumstances (with the  adverse conditions removed and the p i t u i t a r y intact) the f u l l steroid synthesizing apparatus would be functioning as suggested by the occurrence of  a l l four hydroxysteroid dehydrogenases i n corpora lutea of hypophysec-  tomized f i s h injected with LH. Estrogen synthesized bv y-stage corpora lutea i s probably l o c a l i z e d within the 6-stage corpora lutea concerned with the d i f f e r e n t i a t i o n of  68  oogonia i n t o e-stage c o r p o r a l u t e a . r i s e to new  I t i s suggested t h a t t h i s  gave  o o g o n i a l c y s t s t o r e p l a c e the germ c e l l s l o s t d u r i n g a t r e s i a .  The m a j o r i t y of f i s h a t t a i n e d t h e i r f u l l m a t u r i t y and o v u l a t e d regularly.  The o v u l a t e d f o l l i c l e s  formed the stage I 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 , comparable to the y-stage c o r p o r a l u t e a a t r e t i c a . f o l l i c l e s had  full  steroid synthesizing a b i l i t y .  s i z e d e s t r o g e n s which were s t o r e d l o c a l l y . t h e s i s was lutea.  completed  They p r o b a b l y  These synthe-  By the time the s t e r o i d  the c o r p o r a l u t e a developed  syn-  i n t o stage IT c o r p o r a  E s t r o g e n s t h a t were s y n t h e s i z e d e a r l i e r , induced some stage I I  c e l l s t o d i f f e r e n t i a t e i n t o oogonia.  The newly d i f f e r e n t i a t e d  oogonia  w i t h i n the stage I I I c o r p o r a l u t e a r e p l a c e d oocytes l o s t d u r i n g o v u l a t i o n .  69  F i g u r e 37:  Summary of the f a t e of p r e - and p o s t - o v u l a t o r y follicles  (1)  Adverse  i n goldfish ovaries.  c o n d i t i o n s cause y o l k y oocytes t o undergo  atresia.  F o l l i c u l a r c e l l s hypertrophy  and became  p h a g o c y t i c ; the h y p e r t r o p h i e d g r a n u l o s a a t the and  3-stages r e s o r b the a t r e t i c o o c y t e .  oocyte i s removed the a t r e t i c f o l l i c l e s i n t o the Y ~  s t a  S  e  corpus luteum.  a-  Once the develops  T h i s has  the appear-  ance of an e n d o c r i n e gland and most l i k e l y s y n t h e s i z e s estrogens.  The e s t r o g e n s s y n t h e s i z e d p r o b a b l y  some of the d-stage i a as observed  c e l l s to d i f f e r e n t i a t e i n t o  i n the  € - s t a g e corpus  induce oogon-  luteum.  (2) The y o l k y oocytes n o r m a l l y mature and a r e o v u l a t e d ; the o v u l a t e d f o l l i c l e s  (Stage I p o s t - o v u l a t o r y c o r p o r a  lutea) synthesize steroids. duct of s t e r o i d metabolism  I suspect t h a t the end  i s estrogens, l o c a l i z e d with-  i n the 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 .  These  steroids  p r o b a b l y induce some of the stage I I corpus luteum to d i f f e r e n t i a t e i n t o oogonia. some c e l l s i n t o oogonia e-stage corpus  luteum.  pro-  cells  This d i f f e r e n t i a t i o n of  g i v e r i s e to the s t a g e I I I or  THE FATE OF PRE-AND POST-OVULATORY FOLLICLES IN GOLDFISH OVARY  r  P Stage Atresia  SECTION I I I : THE EFFECTS OF OVARIAN STEROIDS ON OOGENESIS AND OVARIAN DEVELOPMENT  71 INTRODUCTION  There a r e v e r y few s t u d i e s on t h e e f f e c t s o f s t e r o i d s on o o g e n e s i s and o v a r i a n development i n s p i t e o f t h e e x t e n s i v e s t u d i e s o f t h e e f f e c t s o f s t e r o i d s on sex d i f f e r e n t i a t i o n ( s e e r e v i e w by Yamamoto, 1969), t h e secondary s e x u a l c h a r a c t e r s and s e x u a l b e h a v i o u r  ( s e e r e v i e w by L i l e y ,  1969). There i s up t o now no adequate study on t h e e f f e c t s o f s t e r o i d s on o o g o n i a l m i t o s i s , though B u l l o u g h  (1942) and Egami (1954) observed an  abundance o f oogonia i n f i s h t r e a t e d w i t h e s t r o g e n and s p e c u l a t e d  that  e s t r o g e n induces i n c r e a s e oogonia f o r m a t i o n . Estrogen  i n h i b i t e d y o l k d e p o s i t i o n i n oocytes  o f Phoxinus phoxinus  ( B u l l o u g h , 1942), Xiphophorus maculatus ( T a v o l g a , 1 9 4 9 ) , P o e c i l i a r e t i c u l a t a ( B e r k o w i t z , 1951), and O r y z i a s l a t i p e s (Egami, 1955).  The i n h i b i -  t i o n o f v i t e l l o g e n e s i s r e s u l t e d i n f o l l i c u l a r a t r e s i a and o v a r i a n r e g r e s sion.  R e c e n t l y , Yamazaki (1972) demonstrated t h a t t e s t o s t e r o n e a l s o  s u p p r e s s e d o v a r i a n development l e a d i n g t o f o l l i c u l a r a t r e s i a .  I n sharp  c o n t r a s t , Egami (1955), I s h i l and Yamamoto (1970), P l a c k et^ a l (1971) and A i d a ejt a l . (1973) demonstrated t h a t e s t r o g e n s  stimulated the l i v e r  to synthesize y o l k precursors. K i r s h e n b l a t (1952) f i r s t demonstrated t h a t p r o g e s t e r o n e  and deoxy-  c o r t i c o s t e r o n e induced o v u l a t i o n i n the t e l e o s t , Misgurnus f o s s i l i s , whereas e s t r o g e n s  and androgens gave a n e g a t i v e r e s p o n s e .  o f o v u l a t i o n , by c o r t i c o s t e r o i d s have been c o n f i r m e d  The i n d u c t i o n  by Ramaswami (1962) ,  S u n d a r a r a j and Goswami (1966), Goswami and S u n d a r a r a j (1971) and H i r o s e (1972). I n t h i s s e c t i o n , I have examined t h e e f f e c t s o f s t e r o i d hormones on o o g o n i a l m i t o s i s , v i t e l l o g e n e s i s , m a i n t a i n e n c e o f second phase o o c y t e s and o v u l a t i o n  o f mature eggs.  growth  73 METHODS AND MATERIALS  THE EFFECTS OF STEROIDS ON OOGONIAL MULTIPLICATION The e f f e c t s o f e s t r a d i o l , p r o g e s t e r o n e and d e o x y c o r t i c o s t e r o n e on m i t o s i s were s t u d i e d i n g o l d f i s h o v a r i e s .  Female f i s h were i n j e c t e d 3  two days a f t e r t h e y had spawned, w i t h 1 yc t h y m i d i n e - H p e r gram body weight.  The i n j e c t e d f i s h were then d i v i d e d i n t o f o u r groups o f f i v e  f i s h each.  F i s h i n each of t h e f i r s t . t h r e e groups were i n j e c t e d t h r e e  times per week f o r f o u r weeks w i t h 10 yg/g o f a m i c r o c r y s t a l l i n e s u s p e n s i o n of e i t h e r e s t r a d i o l , p r o g e s t e r o n e o r d e o x y c o r t i c o s t e r o n e .  The  f o u r t h group, t h e c o n t r o l s , were i n j e c t e d a t the same time i n t e r v a l s w i t h s a l i n e c o n t a i n i n g Tween 80. The f i s h were k i l l e d two days a f t e r t h e l a s t i n j e c t i o n and t h e i r o v a r i e s removed and f i x e d i n 10% n e u t r a l f o r m a l i n f o r a u t o r a d i o g r a p h y . O v a r i e s were r o u t i n e l y embedded i n p a r a f f i n , s e c t i o n e d ( a t 7 y) , mounted on albumen c o a t e d s l i d e s , dewaxed and h y d r a t e d t h r o u g h a s e r i e s o f alcohols.  Hydrated  s e c t i o n s were d i p - c o a t e d w i t h n u c l e a r t r a c k e m u l s i o n  (Kodak NTB 2) i n t h e dark (Kopriwa & L e B l a n d , 1962); s l i d e s were a i r d r i e d and k e p t i n a l i g h t p r o o f box i n t h e r e f r i g e r a t o r (0-5°C) f o r a month.  A f t e r one month the s l i d e s were p r o c e s s e d w i t h p h o t o g r a p h i c de-  v e l o p e r (Kodak D19) and f i x e r (Kodak F 5 f i x i n g bath) s t a i n e d w i t h Mayer's h a e m a t o x y l i n and e o s i n (Beserga & Malamud, 1969), d e h y d r a t e d i n a l c o h o l , c l e a r e d i n x y l e n e , and mounted i n "DPX" ( K i r k p a t r i c k & Lendrum, 1941).  74 The e s t i m a t i o n of p e r c e n t a g e o f o o g o n i a and c o r p o r a l u t e a  cells  3  l a b e l l e d by t h y m i d i n e - H was based on 50 random h i g h power (x 450) f i e l d s o f v i e w from each o v a r y .  The m i t o t i c f r e q u e n c y o f o o c y t e s was  based on the t o t a l number o f o o c y t e s w h i c h showed m i t o t i c f i g u r e s i n f i v e c r o s s s e c t i o n s of each o v a r y .  The m i t o t i c f r e q u e n c y i n f o l l i c u l a r  c e l l s was based on t o t a l number o f f o l l i c u l a r c e l l s showing f i g u r e s i n the t e n l a r g e s t o o c y t e s o f each o v a r y .  mitotic  The average  o f f o l l i c u l a r c e l l s counted p e r o v a r y was 505 + 207.  number  I n a d d i t i o n 790 +  248 c o r p o r a l u t e a c e l l s , 53 + 35 o o g o n i a , and 469 + 245 o o c y t e s were counted p e r o v a r y . variance.  The d a t a were a n a l y s e d s t a t i s t i c a l l y by a n a l y s i s o f  The mean m i t o t i c f r e q u e n c i e s o f t h e v a r i o u s e x p e r i m e n t a l  groups of f i s h were compared by the " l e a s t s i g n i f i c a n t d i f f e r e n c e " &_ p r i o r i t e s t ( S o k a l & R o h l f , 1969). EFFECTS OF STEROIDS ON VITELLOGENESIS Female g o l d f i s h were hypophysectomized a l l y o l k y o o c y t e s were r e a b s o r b e d .  and k e p t f o r 5-6 weeks u n t i l  A t t h e end o f t h i s p e r i o d , t h e y  were d i v i d e d i n t o groups o f s i x f i s h and i n j e c t e d i n t r a p e r i t o n e a l l y w i t h one of the gonadal s t e r o i d s ; an a d d i t i o n a l group was i n j e c t e d w i t h a m i x t u r e o f gonadal s t e r o i d s .  S t e r o i d s were i n j e c t e d as m i c r o c r y s t a l l i n e  s u s p e n s i o n s i n s a l i n e (0.7% NaCl) c o n t a i n i n g 2% Tween 80. was t r i t u r a t e d t o a f i n e powder i n a g l a s s homogenizer;  Each s t e r o i d  Tween 80 was  then added and f o l l o w e d by 0.7% N a C l t o form t h e m i c r o c r y s t a l l i n e sion.  suspen-  75  As i n the p r e v i o u s e x p e r i m e n t s , each g o l d f i s h was i n j e c t e d w i t h 10 yg/g o f s t e r o i d i n 0.1 ml o f s o l v e n t .  The s t e r o i d s s t u d i e d were  c h o l e s t e r o l , p r e g n e n o l o n e , 17a-hydroxypregnenolone,  p r o g e s t e r o n e , 17a-  hydroxyprogesterone, dehydroepiandrosterone, androstenedione, e s t r a d i o l , e s t r o n e , e s t r i o l and t e s t o s t e r o n e . A c o n t r o l group was the s o l v e n t o n l y .  injected with  The i n j e c t i o n s c h e d u l e was once e v e r y 3 days f o r a  p e r i o d of one month. A t the end of the momth, f i s h were s a c r i f i c e d and t h e i r o v a r i e s examined h i s t o l o g i c a l l y and h i s t o c h e m i c a l l y . sectomy was  Completeness  of hypophy-  ensured by h i s t o l o g i c a l e x a m i n a t i o n of t h e heads.  were f i x e d i n B o u i n ' s f i x a t i v e , d e c a l c i f i e d in paraffin.  (Wiebe, 1968)  The heads  and embedded  S e r i a l s e c t i o n s o f the heads through the p i t u i t a r y r e g i o n  were examined f o r p i t u i t a r y  tissue.  EFFECTS OF LONG TERM STEROID INJECTIONS ON GRAVID GOLDFISH I n t h i s group o f e x p e r i m e n t s , the e f f e c t s o f c h r o n i c i n j e c t i o n s o f the f o l l o w i n g s t e r o i d s were compared: pregnenolone, p r o g e s t e r o n e , dehydroepiandrosterone, androstenedione, e s t r a d i o l , estrone, e s t r i o l ,  testos-  t e r o n e , d e o x y c o r t i c o s t e r o n e , c o r t i s o n e , c o r t i c o s t e r o n e , and s a l i n e controls. C a l c u l a t i o n of t h e p e r c e n t a g e o f y o l k y o o c y t e s t h a t were a t r e t i c was based on the assumption t h a t a l l a t r e t i c o o c y t e s were d e r i v e d from yolky oocytes. variance.  The d a t a were t r e a t e d s t a t i s t i c a l l y by an a n a l y s i s o f  The mean p e r c e n t a g e of a t r e t i c o o c y t e s i n d u c e d by e s t r o n e ,  e s t r a d i o l , e s t r i o l , and t e s t o s t e r o n e were compared u s i n g Tukey's  W-  76  procedure  ( S o k a l & R o h l f , 1969).  T h i s i s an a p o s t e r i o r i t e s t as  the d e c i s i o n t o compare t h e p e r c e n t a g e o f a t r e s i a i n t h e s e f o u r groups of  e x p e r i m e n t a l f i s h were made a f t e r t h e r e s u l t s were known.  EFFECTS OF STEROIDS ON OVULATION IN GRAVID GOLDFISH I n t h i s s e r i e s o f e x p e r i m e n t s , each group of f i s h was i n j e c t e d w i t h one o f t h e f o l l o w i n g s t e r o i d s : c h o l e s t e r o l , p r e g n e n o l o n e , 17ahydroxypregnenolone,  progesterone, 17a-hydroxyprogesterone,  dehydroepiandro.  sterone, e s t r a d i o l , estrone, e s t r i o l , testosterone, deoxycorticosterone, c o r t i s o n e , and C o r t i s o l .  As i n t h e p r e v i o u s s t u d y , groups o f 6-8  gravid  g o l d f i s h were t r a n s f e r r e d t o 50 l i t e r e x p e r i m e n t a l t a n k s s u p p l i e d w i t h f l o w i n g d e c h l o r i n a t e d w a t e r (12+1°C) and a d a i l y p h o t o p e r i o d o f 16 h o u r s . Each f i s h was i n j e c t e d , i n t r a p e r i t o n e a l l y , w i t h 100 yg/g o f t h e approp r i a t e s t e r o i d suspended  i n 0.1 ml o f 0.7% NaCl c o n t a i n i n g 2% Tween 80.  A c o n t r o l group was i n j e c t e d w i t h 0.1 ml o f 0.7% N a C l c o n t a i n i n g 2% Tween 80.  The i n j e c t i o n s were c a r r i e d out a t noon. The t r e a t e d g o l d f i s h were examined f o r o v u l a t i o n a t 9:00 a.m. t h e  n e x t day.  Each f i s h was h e l d i n t h e l e f t hand so t h a t t h e abdomen was  not grasped.  O v u l a t e d eggs were s t r i p p e d out o f t h e o v i d u c t by g e n t l y  moving t h e f i r s t two f i n g e r s o f t h e r i g h t hand o v e r t h e abdomen i n t h e d i r e c t i o n of the cloaca.  E x c e s s i v e f o r c e was a v o i d e d as i t w i l l r u p t u r e  t h e o v a r i e s and e x p r e s s u n o v u l a t e d o o c y t e s .  I f nothing or only f l u i d  i s s u e d from t h e c l o a c a t h e f i s h were r e p l a c e d and t e s t e d a g a i n a f t e r 24 hours.  T h i s procedure was c a r r i e d o u t d a i l y f o r 5 days a f t e r the i n i t i a l  steroid injection.  O v u l a t e d eggs t h a t had been s t r i p p e d i n t h i s manner  were t h e n p l a c e d i n p l a s t i c t r a y s and a r t i f i c i a l l y f e r t i l i z e d .  At the  77  end o f the experiments the f i s h were k i l l e d by d e c a p i t a t i o n and the o v a r i e s removed f o r h i s t o l o g i c a l examination. of each f i s h was  determined.  The gonadosomatic  index  78  RESULTS  THE  EFFECTS OF  STEROIDS ON  MITOSIS  T a b l e 8 summarizes the  r e s u l t s of s t e r o i d a d m i n i s t r a t i o n  mitosis i n goldfish ovaries. c o r t i c o s t e r o n e was  injected  on  When e s t r a d i o l , p r o g e s t e r o n e , o r deoxyinto recently  ovulated f i s h together with  3  thymidine- H ( l u c / g ) , a c t i v e l y d i v i d i n g c e l l s incorporated i n the DNA.  I t s h o u l d be  emphasized t h a t the  this  c e l l s l a b e l l e d by  label thymi-  3  d i n e - H,  i n f i s h o v a r i e s are d i v i d i n g by m i t o s i s ;  the m e i o t i c d i v i s i o n  does not  o c c u r u n t i l o o c y t e s are  ready t o o v u l a t e .  f u l l y mature and  c e l l s l a b e l l e d were f o l l i c u l a r c e l l s , corpus l u t e u m c e l l s , o o g o n i a o o c y t e s ( F i g . 36, between t h e c a l  and  37,  38,  39).  I t was  not  g r a n u l o s a c e l l s o f the  p o s s i b l e to  The and  differentiate  f o l l i c u l a r layers  i n autora-  diography . About 10%  o f the  f o l l i c u l a r c e l l s were l a b e l l e d i n a l l f i s h — w h e t h e r  e x p e r i m e n t a l or c o n t r o l . p r o g e s t e r o n e , and  An  analysis  of v a r i a n c e i n d i c a t e d  d e o x y c o r t i c o s t e r o n e had  no  that estrogen,  s i g n i f i c a n t e f f e c t on  the  3  p e r c e n t a g e of f o l l i c u l a r c e l l s l a b e l l e d by The  s i t u a t i o n , however, was  where s t e r o i d s  quite different  twice weekly i n j e c t i o n s  f r e q u e n c i e s by the m i t o t i c  i n the  H. corpora  lutea,  i n d u c e d s i g n i f i c a n t i n c r e a s e i n m i t o s e s (p < 0.01).  h i g h e s t l e v e l of m i t o s e s i n c o r p o r a l u t e a the  thymidine-  the  (3.9  f o r f o u r weeks.  + 1.0%)  was  found  The  after  Comparison o f mean m i t o t i c  least significant difference  method i n d i c a t e d  that  f r e q u e n c y o f e s t r o g e n t r e a t e d f i s h were s i g n i f i c a n t l y h i g h e r  than t h o s e of p r o g e s t e r o n e o r d e o x y c o r t i c o s t e r o n e t r e a t e d f i s h (p <  0.01)  T a b l e 8:  The e f f e c t s of e s t r a d i o l , p r o g e s t e r o n e , and ovaries.  Treatment •(10 yg/g i n 0.1 ml. s a l i n e )  i n goldfish  % follicular cells labelled  % corpora l u t e a cells labelled  5  9.0  +.1.3  1.3  +  0.3  15.5  +  6.8  0.6  +  0.2  Estrogen  5  10.5  +  1.7  3.9  +  1.0  26.1  +  6.1  0.4  +  0.3  Progesterone  5  9.6  +  2.0  2.2  +  0.4  11.0  +  4.4  0.7  +  0.1  Deoxycorticosterone  5  9.7  +  1.0  2.5  +  0.7  7.9  +  3.0  0.7  +  0.3  Control  (Saline)  No. g o l d fish  d e o x y c o r t i c o s t e r o n e on m i t o s i s  % oogonia labelled  % oocyte labelled  Each f i s h was i n j e c t e d w i t h 1 uc/g t h y m i d i n e - H two days a f t e r o v u l a t i o n . F i s h was t h e n i n j e c t e d , i n t r a p e r i t o n e a l l y , w i t h 10 ug/g of the a p p r o p r i a t e s t e r o i d t w i c e a week, f o r f o u r weeks. A u t o r a d i o graphs of o v a r i a n s e c t i o n s were examined to c a l c u l a t e the p e r c e n t a g e o f v a r i o u s o v a r i a n c e l l t y p e s l a b e l l e d by t h y m i d i n e ^ H . * S t a t i s t i c a l s i g n i f i c a n c e a g a i n s t c o n t r o l f i s h a t 0.01 < p < 0.05. ** S t a t i s t i c a l s i g n i f i c a n c e at p < 0.01.  80 w i t h no d e t e c t a b l e d i f f e r e n c e between the l a t t e r two Comparisons o f m i t o t i c  steroids.  f r e q u e n c i e s i n oogonia o f t r e a t e d f i s h  a l s o showed a s i g n i f i c a n t e f f e c t o f e s t r o g e n .  Estrogen treated f i s h  had 26.1 + 6.1%  o f l a b e l l e d as compared t o 15.5 + 6.8%  group o f f i s h .  Progesterone  i n the  and d e o x y c o r t i c o s t e r o n e had no  e f f e c t on o o g o n i a l m i t o s i s w h i l e d e o x y c o r t i c o s t e r o n e may inhibitory.  significant  be  slightly  I n c o n t r a s t t o the oogonia, o o c y t e s showed no r e s p o n s e t o  any of the s t e r o i d s t e s t e d .  Only s m a l l p e r c e n t a g e o f o o c y t e s were l a -  3 b e l l e d by t h y m i d i n e - H (0.6%) a t any  THE  control  time.  EFFECTS OF STEROID ON VITELLOGENESIS IN HYPOPHYSECTOMIZED FISH A l l the e s t r o g e n s  ( e s t r a d i o l , e s t r o n e , and e s t r i o l ) induced  f o r m a t i o n of y o l k v e s i c l e s i n the o v a r i e s o f hypophysectomized (Table 9 ) .  I n some o o c y t e s  There was  randomly throughout the  no i n d i c a t i o n of y o l k g r a n u l e  i n the o v a r i e s o f e s t r o g e n t r e a t e d hypophysectomized E s t r a d i o l was E s t r a d i o l was t r o g e n s was  goldfish  the y o l k v e s i c l e s appeared i d e n t i c a l t o n o r -  mal v i t e l l o g e n e s i s ; the v e s i c l e s o f t e n developed ooplasm ( F i g . 3 8 ) .  the  goldfish.  the most p o t e n t i n d u c e r o f y o l k v e s i c l e  f o l l o w e d by e s t r o n e .  formation  formation.  The d i f f e r e n c e between the two  es-  e v i d e n t i n a r e s p o n s e o f f i v e out o f s i x f i s h w i t h e s t r a d i o l  compared to t h r e e out o f s i x f o r e s t r o n e and e s t r i o l . percentage of oocytes  Moreover, a g r e a t e r  (6.9 + 1 . 9 % ) i n any one o v a r y developed  yolk v e s i -  c l e s w i t h e s t r a d i o l ( F i g . 4 1 ) ; f i s h t r e a t e d w i t h s i m i l a r dose of e s t r i o l had  fewest o o c y t e s w i t h y o l k v e s i c l e s  (5.6 + 1.6%).  E s t r o g e n induced y o l k v e s i c l e  (v) f o r m a t i o n i n  oocytes o f hypophysectomized g o l d f i s h .  This section  was s t a i n e d w i t h M a l l o r y ' s t r i c h r o m e .  The  ovary o f hypophysectomized g o l d f i s h s t a i n e d  with Mallory's trichrome. first  The ovary c o n t a i n s o n l y  growth phase o o c y t e s .  Pregnenolone induced y o l k granule f o r m a t i o n i n the o o c y t e s o f hypophysectomized g o l d f i s h . granules ment.  Only y o l k  (g) were formed a f t e r pregnenolone t r e a t -  The o v a r i a n s e c t i o n was s t a i n e d w i t h M a l l o r y ' s  trichrome.  T a b l e 9:  The e f f e c t s o f v a r i o u s s t e r o i d s ( i . p . , 10 ug/g once e v e r y 3 days f o r a month) on v i t e l l o g e n e s i s i n hypophysectomized g o l d f i s h .  Steroids  Body wt. (g)  Saline  20.6  + 7.8  1.9  + 0.8  6  0  0  Cholesterol  24.2  + 6.9  2.0  + 0.4  6  0  0  Pregnenolone  21.0  + 7.3  1.6  + 0.6  5  0  4  Progesterone  28.1  + 4.4  1.9  + 0.5  6  0  0  17 a-hydroxypro ges t erone  27.4  + 6.3  1.6  + 0.8  6  0  0  Dehydroepiandrosterone  23.8  + 3.9  1.6  + 0.8  6  0  0  Androstenedione  21.7  + 4.2  2.2  + 0.4  5  0  0  Estradiol  23.6  + 4.3  1.5  + 0.6  6  5  0  Estrone  25.0  + 8.7  2.2  + 0.7  6  3  0  Estriol  23.9  + 2.9  1.8  + 0.8  6  3  0  M i x t u r e o f above s t e r o i d s 23.5  + 3.2  2.1  + 0.6  6  0  0  (Control  G.S. ,1.  No. fish  No. o v a r i e s w i t h yolk vesicles  No. o v a r i e s w i t h y o l k granules  ^  oo  83  Pregnenolone  induced the f o r m a t i o n o f y o l k g r a n u l e s without the  p r i o r f o r m a t i o n o f y o l k v e s i c l e s and t h i s c u r i o u s f i n d i n g i s i n sharp c o n t r a s t to normal v i t e l l o g e n e s i s where the v a r i o u s y o l k i n c l u s i o n s are formed i n sequence w i t h t h e y o l k v e s i c l e s always a p p e a r i n g b e f o r e yolk granules. physectomized  Y o l k v e s i c l e s were never observed i n o o c y t e s o f hypog o l d f i s h treated with  pregnenolone.  The y o l k g r a n u l e s induced by pregnenolone o u t e r membrane o f o o c y t e s  ( F i g . 40).  Although  were found c l o s e to the they were p o o r l y d i f f e r e n -  t i a t e d i n h e a m a t o x y l i n and e o s i n s t a i n e d s l i d e s , they were w e l l d e f i n e d as dark r e d g r a n u l e s w i t h M a l l o r y ' s t r i c h r o m e . showed these pregnenolone  Histochemical studies  induced y o l k g r a n u l e s to have the same c h e m i c a l  p r o p e r t i e s as normal y o l k g r a n u l e s ( T a b l e 10); i n s h o r t they c o n t a i n e d p r o t e i n s , p h o s p h o l i p i d s and t y p i c a l n e u t r a l l i p i d s . i n t e r e s t concerns  the v i t e l l i n e membrane.  A further point of  Under normal c o n d i t i o n s t h i s  i s w e l l developed by the time t h e y o l k g r a n u l e s appear.  However, i n con-  t r a s t w i t h normal v i t e l l o g e n e s i s , the v i t e l l i n e membrane was not e v i d e n t i n o o c y t e s w i t h y o l k g r a n u l e s induced by  pregnenolone.  None of the o t h e r s t e r o i d s t e s t e d appear to a f f e c t y o l k f o r m a t i o n . The o v a r i e s remain t y p i c a l of those i n hypophysectomized s i m i l a r l y the f i s h i n j e c t e d w i t h a complete showed no changes i n v i t e l l o g e n e s i s .  fish  mixture o f gonadal  ( F i g . 39); steroids  T a b l e 10.  Comparison of the h i s t o c h e m i c a l s t a i n i n g p r o p e r t i e s o f pregnenolone y o l k granules w i t h those of normal v i t e l l o g e n i c o v a r i e s  Techniques  Periodic  Normal Y o l k Granules  Pregnenolone-induced Granules  induced  Yolk  acid-Schiff's  D i n i t r o f l u r o b e n z e n e method  +++  +++  T e t r a z o t i z e d o - d i a n i s i d i n e method  +++  +++  ++  ++  +++  +++  Sudan Black B A c i d haematin  for phospholipids  CO  85  Figure 41:  Comparison o f t h e e f f e c t s o f e s t r a d i o l , e s t r o n e , and e s t r i o l on y o l k v e s i c l e f o r m a t i o n i n hypophysectomized g o l d f i s h .  The mean gonadosomatic i n d e x  of the groups o f f i s h from l e f t t o r i g h t a r e : 1.5 +0.6,  2 . 2 + 0 . 7 , and 1 . 8 + 0 . 8  A n a l y s i s of v a r i a n c e i n d i c a t e d significant differences ments .  t h a t t h e r e were no  between t h e d i f f e r e n t  treat-  10  8 CO 0> co  £  6  to cu  o O  4  cu  o 4>  u  cu  0_  Estradiol  Estrone  Estriol  86 E f f e c t s o f l o n g term a d m i n i s t r a t i o n o f s t e r o i d s i n t o g r a v i d  fish.  T a b l e 11 summarized t h e r e s u l t s o f t h e experiment t o d e t e r m i n e t h e l o n g term e f f e c t s o f s t e r o i d a d m i n i s t r a t i o n i n g r a v i d g o l d f i s h , e s p e c i a l l y on t h e maintenance o f second growth phase o o c y t e s .  Though e s t r o g e n s  i n d u c e t h e s y n t h e s i s and m o b i l i z a t i o n o f y o l k p r e c u r s o r s Ho & Vanstone, 1961; been r e p o r t e d  U r i s t & S c h j e i d e , 1961;  ( B a i l e y , 1957;  P l a c k e t a l . , 1971), i t has  t h a t e s t r o g e n s i n h i b i t v i t e l l o g e n e s i s and cause o v a r i a n  regression. Estrone, ug/g  e s t r a d i o l , e s t r i o l , and t e s t o s t e r o n e  administered  a t 10  t w i c e a week f o r f o u r weeks, i n d u c e d a t r e s i a i n o v a r i e s o f about  80% o f t h e s e f i s h .  A n a l y s i s of variance i n d i c a t e d that a l l three e s t r o -  g e n i c s t e r o i d s ( e s t r o n e , e s t r a d i o l , and e s t r i o l ) were e q u a l l y p o t e n t b u t t h a t t e s t o s t e r o n e was s i g n i f i c a n t l y l e s s a c t i v e than t h e e s t r o g e n s (p < 0.01).  About 54% o f y o l k y o o c y t e s were a t r e t i c i n t e s t o s t e r o n e  f i s h as compared t o 80% w i t h t h e e s t r o g e n s ( F i g . 4 6 ) .  treated  The a t r e s i a ap-  peared t o be s i m i l a r t o t h a t observed i n hypophysectomized f i s h b u t det a i l e d h i s t o g e n e s i s was n o t s t u d i e d . and  appeared c o m p l e t e l y  Some y o l k y o o c y t e s remained i n t a c t  normal ( F i g . 4 2 ) .  By c o n t r a s t , no e v i d e n c e o f a t r e s i a was found a f t e r treatment w i t h p r e g n e n o l o n e , p r o g e s t e r o n e , d e h y d r o e p i a n d r o s t e r o n e , a n d r o s t e n e d i o n e , deoxycorticosterone,  c o r t i s o n e , and c o r t i c o s t e r o n e .  However, some deoxy-  c o r t i c o s t e r o n e , c o r t i s o n e , and c o r t i c o s t e r o n e t r e a t e d f i s h had l e s s t h a n 5% o f p o s t - o v u l a t o r y corpora  corpora l u t e a i n t h e i r ovaries  (Table 1 1 ) , b u t t h e s e  l u t e a were d i s t i n c t from t h e a t r e s i a i n d u c e d b y e s t r a d i o l ,  e s t r i o l or t e s t o s t e r o n e .  The p o s t - o v u l a t o r y  corpora  estrone,  l u t e a were made up  87 o f i r r e g u l a r masses of c e l l s  ( F i g . 43) whereas the a t r e t i c  follicles,  e s p e c i a l l y those at the e a r l y s t a g e s , c o n t a i n e d p a r t i a l l y d i g e s t e d cytoplasmic i n c l u s i o n s  ( F i g . 42).  These f i n d i n g s i n d i c a t e t h a t estrogens  and t e s t o s t e r o n e induce  a t r e s i a and o v a r i a n r e g r e s s i o n p r o b a b l y by n e g a t i v e feedback on the p i t u i t a r y gonadotropin. discussion.  T h i s h y p o t h e s i s w i l l be c o n s i d e r e d i n the  88  F i g u r e 42:  S e c t i o n of an o v a r y o f a g o l d f i s h i n j e c t e d w i t h 10 ug/g o f e s t r a d i o l , t w i c e a week f o r f o u r weeks. Note t h e e x t e n s i v e a t r e s i a (arrows) i n t h e ovary.  F i g u r e 43:  S e c t i o n o f an ovary i n j e c t e d w i t h 10 ug/g o f deo x y c o r t i c o s t e r o n e , t w i c e a week f o r f o u r weeks. A l l y o l k y oocytes were i n t a c t ; o c c a s i o n a l l y t h e r e were some 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 ( a r r o w ) .  F i g u r e 44:  Ovary o f a g r a v i d g o l d f i s h t h a t i s ready t o o v u l a t e . The ovary c o n s i s t s o f w e l l developed o o c y t e s l a t e y o l k granule  F i g u r e 45:  i n the  stage.  Ovary o f g o l d f i s h where t h e oocytes have r e a c h e d o n l y t h e l a t e y o l k v e s i c l e s t a g e and hence were n o t ready f o r o v u l a t i o n .  T a b l e 11:  E f f e c t s of s t e r o i d s a d m i n i s t e r e d t o g o l d f i s h f o r one month. R e s u l t s were r e c o r d e d as t h e p r e s e n c e o r absence o f a t r e s i a , more d e t a i l e d d a t a a r e p r e s e n t e d i n F i g . 46.  Treatment  Body Wt. (g)  .G.S.I.  1  No. f i s h tested  No. f i s h w i t h - a t r e t i c oocytes  Saline (Control)  42.9 + 7.1  13.9 + 6.4  5  0  Pregnenolone  41.3 + 6.9  17.3 + -5.4  5  0  Progesterone  42.6+6.5  14.0 + 4.3  5  0  Dehydroepiandrosterone  41.7 + 6.5  12.0 + 4.3  5  0  Androstenedione  39.0 + 6.0  14.3 + 4 . 7  5  0  Estradiol  48.1 + 6.0  15.9 + 5.0  5  5  Estrone  41.1 + 7.4  13.5 + 3.3  5  5  Estriol  45.2 + 4.1  12.5 + 6.3  5  5  Testosterone  41.5 + 5.2  10.4 + 2.4  5  5  Deoxycorticosterone  42.7  +4.5  17.9 + 4.6  5  3  2  Cortisone  44.1 + 5.8  12.8 + 4 . 6  5  I  2  Corticosterone  40.1+7.5  13.3 + 7.1  5  2  2  "Gonadosomatic Index (G.S.I.) determined  a t the end o f experiment  'These f i s h had s t a g e I I o r 6-stage c o r p o r a l u t e a . These were p r o b a b l y t h e r e s u l t o f o v u l a t i o n . They were d i s t i n c t from e s t r o g e n induced a t r e s i a which c o n s i s t e d m a i n l y o f « - and g-stage c o r p o r a l u t e a .  ,90  F i g u r e 46:  Comparison o f t h e e f f e c t s o f e s t r o n e , e s t r a d i o l , and  estriol  t e s t o s t e r o n e on a t r e s i a o f y o l k y o o c y t e s i n g r a v i d  goldfish.  The mean gonadosomatic i n d e x o f t h e groups o f  f i s h from l e f t t o r i g h t a r e : 13.5 + 3.3, 15.9 + 5.0, 12.5  + 6.3, and 10.4 + 2.4.  Analysis of variance  i n d i c a t e d t h a t t h e r e were s i g n i f i c a n t  d i f f e r e n c e s i n some o f the t r e a t m e n t s ( p < 0 . 0 1 ) .  A com-  p a r i s o n o f the mean p e r c e n t a g e o f a t r e t i c o o c y t e s i n d u c e d by v a r i o u s s t e r o i d s b y Tukey's W-procedure i n d i c a t e d t h a t testosterone  induced s i g n i f i c a n t l y l e s s a t r e s i a than the  e s t r o g e n s (p < 0.01).  There were no s i g n i f i c a n t d i f f e r e n c e s  among t h e v a r i o u s e s t r o g e n  treatments.  Estrone  Estradiol  Estriol  Testosterone  91  THE  EFFECTS OF STEROIDS ON OVULATION IN GOLDFISH  The  e f f e c t i v e n e s s o f v a r i o u s s t e r o i d s u s p e n s i o n i n i n d u c i n g ovu-  l a t i o n i n g r a v i d g o l d f i s h , maintained are p r e s e n t e d i n T a b l e 12.  at 12 + 1°C and l o n g p h o t o p e r i o d ,  I t should be r e c a l l e d t h a t g o l d f i s h do not  o v u l a t e under these e x p e r i m e n t a l  conditions.  P r o g e s t e r o n e , d e o x y c o r t i c o s t e r o n e , c o r t i s o n e , and c o r t i c o s t e r o n e were found t o induce o v u l a t i o n i n g r a v i d f i s h .  C o r t i s o n e was  the most  p o t e n t of t h e s e s t e r o i d s w i t h f o u r out of the f i v e f i s h r e s p o n d i n g the f i r s t  day a f t e r i n j e c t i o n of 100 ug/g  c o r t i s o n e ; w i t h i n one  on  day,  o v u l a t e d f i s h developed o v a r i e s w i t h 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 ( F i g . 43).  The most advanced oocytes i n these f i s h were i n the e a r l y y o l k  granule stage.  The o v u l a t e d eggs were f u l l y developed as evidence by  the f a c t that when a r t i f i c i a l l y fry within five  f e r t i l i z e d they developed  the next day and another  injection.  p r o g e s t e r o n e , one o v u l a t e d  on the t h i r d day a f t e r the i n i t i a l  The o t h e r t h r e e d i d not o v u l a t e .  l a t e d f i s h had  Two  o v a r i e s w i t h incomplete v i t e l l o g e n e s i s .  The most advanced 45).  Three of the d e o x y c o r t i c o s t e r o n e t r e a t e d f i s h o v u l a t e d . o v u l a t e d each o f the f i r s t  steroid  out of the t h r e e unovu-  o o c y t e s had j u s t reached the l a t e y o l k v e s i c l e stage ( F i g l  o v u l a t i o n was  into  days.  Of the f i v e f i s h i n j e c t e d w i t h 100 ug/g on  and hatched  t h r e e days o f the experiment.  The  comparable to that i n c o r t i s o n e t r e a t e d f i s h .  eggs were a l s o s u c c e s s f u l l y f e r t i l i z e d and hatched.  One  One  fish  extent o f These o v u l a t e d  of the two  unovu-  l a t e d f i s h i n t h i s group had oocytes o n l y up to the l a t e y o l k v e s i c l e  T a b l e 12.  The response of g r a v i d g o l d f i s h to s t e r o i d s . Each f i s h was i n j e c t e d w i t h 100 ug/g o f s t e r o i d s u s p e n s i o n d a i l y . F i s h were examined f o r o v u l a t i o n b e f o r e each i n j e c t i o n . The experiment was t e r m i n a t e d a f t e r f o u r days.  Treatment  No. fish  Body Wt. (g)  G.S.I.  1  No. f i s h ovulated  No. f i s h w i t h immature o v a r ies  Saline (Control)  5  39.6  +5.3  24.3 + 6.6  0  1  Cholesterol  5  45.0 + 8.9  14.8 + 5.8  0  1  Pregnenolone  5  54.0  +7.2  20.0 + 4.3  0  0  Progesterone.  5  35.9 + 6.7  10.8 + 6.1  2  2  Dehydroepiandrosterone  5  48.1  +7.9  10.9 + 4.7  0  0  Androstenedione  5  43.5  +9.9  12.7 + 3.2  0  1  Estradiol  5  36.7 + 9.4  16.5 + 4.2  0  1  Estrone  5  57.1 + 9 . 1 .  19.9 + 6.9  0  1  Estriol  5  53.8 + 8.7  13.6 + 5.8  0  1  Testosterone  5  40.0 + 12.9  12.3 + 5.1  0  1  Deoxycorticosterone  5  48.8 + 7.4  12.4 + 4.3  3  1  Cortisone  5  40.4 + 7.6  7.6 +  47.  0  Corticosterone  5  32.0 + 6.9  12.9 + 5.9  2  1  "''G.S.I.: Gonadosomatic i n d e x , as  4.2  determined at t h e end o f the experiment. VO  hp  93 s t a g e s ; the o t h e r had oocytes i n the e a r l y y o l k granule s t a g e .  The  t h r e e f i s h t h a t o v u l a t e d had 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 i n t h e i r ovaries  ( F i g . 47).  Only two o f the f i s h i n j e c t e d w i t h c o r t i c o s t e r o n e o v u l a t e d .  One  o v u l a t e d on the f i r s t day a f t e r s t e r o i d i n j e c t i o n and the second on the f o l l o w i n g day.  None o f the o t h e r t h r e e showed any s i g n o f o v u l a t i o n  d u r i n g the experiment.  One of the t h r e e u n o v u l a t e d  f i s h had oocytes a t  the l a t e y o l k v e s i c l e stage o n l y ; the o t h e r two had oocytes i n the e a r l y y o l k granule stage w i t h a few i n the l a t e y o l k granule s t a g e . of  Ovaries  t h e o v u l a t e d f i s h were t y p i c a l of o v u l a t e d f i s h and c o n t a i n e d many  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 and o o c y t e s i n a l l stages o f o o c y t e ment except  the l a t e y o l k g r a n u l e  develop-  stage.  The m a t u r i t y o f oocytes i n u n o v u l a t e d f i s h o v a r i e s as p r e s e n t e d i n T a b l e 12, i n d i c a t e s t h a t although one of the c h o l e s t e r o l t r e a t e d f i s h had o o c y t e s i n the l a t e y o l k v e s i c l e s t a g e , the o t h e r f i s h t r e a t e d w i t h c h o l e s t e r o l had many f u l l y developed of b e i n g o v u l a t e d . one  oocytes  ( F i g . 44) which were  S i m i l a r l y one o f the androstenedion  e s t r a d i o l t r e a t e d f i s h , one e s t r i o l  capable  treated f i s h ,  t r e a t e d f i s h and one f i s h t r e a t e d  w i t h t e s t o s t e r o n e had i n c o m p l e t e l y mature o v a r i e s but the remainder o f t h e s e e x p e r i m e n t a l f i s h had o v a r i e s w i t h f u l l y developed l a t e y o l k granule In  oocytes a t the  stage.  summary, p r o g e s t e r o n e , d e o x y c o r t i c o s t e r o n e , c o r t i s o n e , and c o r t i -  c o s t e r o n e induced i n v i v o o v u l a t i o n i n g o l d f i s h .  This e f f e c t i f quite  s p e c i f i c as c h o l e s t e r o l , pregnenolone, d e h y d r o e p i a n d r o s t e r o n e , dione, e s t r a d i o l , estrone, e s t r i o l , ovulation.  androstene-  and t e s t o s t e r o n e had no e f f e c t on  94 III DISCUSSION T h i s study demonstrates t h a t , i n a d d i t i o n t o t h e i r w e l l known e f f e c t s on sex d i f f e r e n t i a t i o n (see r e v i e w by Yamamoto, 1969), s e x u a l b e h a v i o u r , and secondary s e x u a l c h a r a c t e r s (see r e v i e w by L i l e y , 1969), ovarian steroids also stimulate oogonial m i t o s i s , v i t e l l o g e n e s i s , ovulation.  U n l i k e male f i s h where t e s t o s t e r o n e s t i m u l a t e s complete  s p e r m a t o g e n e s i s i n hypophysectomized f i s h ( L o f t s e t a l . , 1966; and N a y y a r , 1967; their effects.  Sundararaj  Pandey, 1969), the o v a r i a n s t e r o i d s are s p e c i f i c i n  E s t r o g e n s w h i c h induced o o g o n i a l m i t o s i s had no  on o v u l a t i o n and i n s t e a d induced a t r e s i a o f y o l k y o o c y t e s . d i n g l y , progesterone  effect  Correspon-  and c o r t i c o s t e r o i d s w h i c h induced o v u l a t i o n had  e f f e c t on o o g o n i a l m i t o s i s .  and t h e f o r m a t i o n o f  y o l k v e s i c l e s , had no e f f e c t on y o l k g r a n u l e f o r m a t i o n .  Instead  s t e r o i d hormone, p r e g n e n o l o n e , induced the f o r m a t i o n o f y o l k These new  no  Estrogens, which s t i m u l a t e d the synthesis  o f y o l k p r e c u r s o r s by the l i v e r ( P l a c k et al_ 1971)  i n oocytes.  and  f i n d i n g s on the e n d o c r i n o l o g y  another  granules  of oogenesis  will  be d i s c u s s e d i n r e l a t i o n to the r e p r o d u c t i v e b i o l o g y of the female f i s h . E f f e c t o f s t e r o i d s on o o g o n i a l m i t o s i s I t has been shown f o r the f i r s t time t h a t e s t r o g e n s oogonial mitosis i n post-ovulatory goldfish. q u i t e s p e c i f i c s i n c e progesterone The  stimulated  T h i s e f f e c t appears t o be  and d e o x y c o r t i c o s t e r o n e had no  e x t e n t o f t h i s s p e c i f i c i t y i s not known as the p r e s e n t study  l i m i t e d t o these t h r e e s t e r o i d s .  effect. was  I n a d d i t i o n t o the s t i m u l a t i o n o f oogon-  i a l m i t o s i s , e s t r o g e n s induced m i t o s i s i n 6-stage c o r p o r a l u t e a c e l l s  95 which have been found to d i f f e r e n t i a t e i n t o o o g o n i a .  Our  findings  i n d i c a t e t h a t m i t o s i s w i t h i n the corpus luteum r e s u l t s i n a c o r r e s p o n d i n g i n c r e a s e i n oogonia.  I n s u p p o r t o f t h i s c o n c l u s i o n i t may be noted  t h a t B u l l o u g h (1942) and Egami (1954) observed an abundance o f o o g o n i a i n f i s h t r e a t e d w i t h e s t r o g e n s w h i l e Cedard e_t a l . (1961) found a s i x f o l d i n c r e a s e i n plasma e s t r o g e n a t the time o f spawning; i n plasma e s t r o g e n may  this increase  be r e l a t e d to the marked i n c r e a s e i n the number  o f oogonia w h i c h many workers have noted i n post-spawning Bennet, 1930; Matthews, 1938; B u l l o u g h , 1942; B a r r , 1963  fish and  (CraigYamazaki,  1965). As a w o r k i n g h y p o t h e s i s , i t i s proposed  that corpora l u t e a synthe-  s i z e e s t r o g e n s which i n d u c e the g e n e s i s o f oogonia from c o r p o r a l u t e a t o r e p l a c e o o c y t e s l o s t d u r i n g spawning.  T h i s may  be the p h y s i o l o g i c a l  f u n c t i o n of c o r p o r a l u t e a i n t e l e o s t where o o g o n i a l m i t o s i s goes on throughout the l i f e  cycle.  E f f e c t o f s t e r o i d s on v i t e l l o g e n e s i s .  The s t u d y on t h e e f f e c t s o f  s t e r o i d s on v i t e l l o g e n e s i s i n hypophysectomized  goldfish indicated that  o n l y c e r t a i n a s p e c t s o f v i t e l l o g e n e s i s were under t h e i n f l u e n c e o f steroids.  The two types o f y o l k i n c l u s i o n s were i n d u c e d by q u i t e d i f -  f e r e n t s t e r o i d s ; e s t r o g e n s i n d u c e d the f o r m a t i o n of y o l k v e s i c l e s w h i l e pregnenolone  i n d u c e d the f o r m a t i o n of t h e second type o f y o l k i n c l u s i o n s —  the y o l k g r a n u l e s .  The s e q u e n t i a l f o r m a t i o n o f t h e two t y p e s of y o l k  i n c l u s i o n , as i n normal v i t e l l o g e n e s i s , have not been e x p e r i m e n t a l l y achieved.  I t seems s t r a n g e t h a t the presence of a l l t h e o v a r i a n s t e r o i d s ,  as i n the experiment u s i n g a m i x t u r e o f o v a r i a n s t e r o i d s , had no  effect  96 on y o l k f o r m a t i o n .  However, t h e s e q u e n t i a l f o r m a t i o n o f y o l k i n c l u s i o n s  may depend on t h e s e q u e n t i a l s y n t h e s i s o f s t e r o i d s o r some p r e c i s e comb i n a t i o n of s p e c i f i c s t e r o i d s . Previous  s t u d i e s demonstrated t h a t e s t r o g e n  administration stimulated  the s y n t h e s i s o f a complex p r o t e i n i n t h e l i v e r (Egami, 1955; I s h i i & Yamamoto, 1970; P l a c k e t a l . , 1971; and A i d a e t a l . , 1973). i s normally blood  This p r o t e i n  absent i n males and immature females b u t appears i n t h e i r  f o l l o w i n g estrogen  administration.  T h i s complex p r o t e i n i n the  b l o o d i s t h e p r e s u m p t i v e y o l k p r o t e i n ( P l a c k e_t a l . , 1971) . The  uptake o f y o l k p r e c u r s o r s  been s t u d i e d i n t e l e o s t s .  from t h e plasma by o o c y t e s have n o t  I n comparable s t u d i e s o f v i t e l l o g e n e s i s i n  a m p h i b i a n s , F o l l e t t e t a l . (1968), W a l l a c e and J a r e d  (1968), and Redshaw  (1972) r e p o r t e d t h a t no a c t i v e u p t a k e o f y o l k p r e c u r s o r s by o o c y t e s o c curred i n the presence of high l e v e l s of estrogen.  T h i s r e s p o n s e was v e r y  d i f f e r e n t from normal v i t e l l o g e n e s i s where r a p i d uptake o f y o l k s o r s was o b s e r v e d .  Gonadotropin treatment,  u n l i k e estrogen  precur-  administration,  s t i m u l a t e d b o t h the s y n t h e s i s o f yolk p r e c u r s o r s by t h e l i v e r and the subsequent i n c o r p o r a t i o n o f t h e s e y o l k p r e c u r s o r s  i n t o oocytes.  In contrast to the synthesis o f y o l k precursors immature f i s h , t h e a d m i n i s t r a t i o n o f e s t r o g e n a t r e s i a i n second growth phase o o c y t e s .  i n the l i v e r of  to g r a v i d f i s h induces  Many w o r k e r s have l i k e w i s e ob-  served the i n h i b i t i o n of v i t e l l o g e n e s i s leading t o f o l l i c u l a r a t r e s i a i n v a r i o u s f i s h : P h o x i n u s phoxinus ( B u l l o u g h , 1942), Xiphophorus m a c u l a t u s ( T r a v o l g a , 1949), P e o c i l i a r e t i c u l a t a ( B e r k o w i t z , 1951), and O r y z i a s l a t i p e s (Egami, 1954, 1955).  I n a d d i t i o n , Yamazaki (1972) found t h a t  97 t e s t o s t e r o n e i n d u c e d a t r e s i a i n second growth phase o o c y t e s o f Oncorhynchus gorbuscha.  Our f i n d i n g s i n d i c a t e d t h a t t h e responses t o  e s t r o g e n s and t e s t o s t e r o n e were s p e c i f i c and n o t d u p l i c a t e d by pregnenol o n e , progesterone, dehydroepiandrosterone, androstenedione, d e o x y c o r t i c o s t e r o n e , c o r t i s o n e , and c o r t i c o s t e r o n e . D u r i n g v i t e l l o g e n e s i s , e s t r o g e n s appeared  t o have d i v e r s e e f f e c t s .  E s t r o g e n s s t i m u l a t e d the s y n t h e s i s o f y o l k p r e c u r s o r s by t h e l i v e r and i t s a c c u m u l a t i o n i n t h e plasma ( B a i l e y , 1957; Ho & Vanstone, U r i s t & S c h j e i d e , 1961).  We observed t h a t e s t r o g e n s t i m u l a t e d t h e f o r m a t i o n o f  yolk v e s i c l e s i n oocytes.  The f o r m a t i o n o f y o l k v e s i c l e s i s n o t r e l a t e d  to t h e s y n t h e s i s . o f y o l k p r e c u r s o r i n t h e l i v e r . of m u c o p o l y s a c c h a r i d e s of  Yolk v e s i c l e s  comprised  and n o t l i p o p r o t e i n which i s t h e major c o n s t i t u e n t  t h e y o l k p r e c u r s o r s y n t h e s i z e d by t h e l i v e r .  I t i s t h e second  type  of y o l k i n c l u s i o n , t h e y o l k g r a n u l e s , t h a t s h a r e s t h e same c h e m i c a l comp o s i t i o n as y o l k p r e c u r s o r s d e s c r i b e d by P l a c k et_ a l . (1971).  We found  t h a t the d e p o s i t i o n o f y o l k g r a n u l e s i n o o c y t e s i s n o t mediated by e s t r o gens b u t by another o v a r i a n hormone, pregnenolone.  Yamazaki and Donaldson  (1968) have i n d u c e d complete v i t e l l o g e n e s i s i n hypophysectomized with  salmon g o n a d o t r o p i n .  T h i s f i n d i n g was c o n f i r m e d i n P o e c i l i a  r e t i c u l a t a ( L i l e y and Donaldson, ( S u n d a r a r a j e t a l . , 1972b).  goldfish  1969) and H e t e r o p n e u s t e s  fossilis  The d e m o n s t r a t i o n t h a t b o t h g o n a d o t r o p i n and  o v a r i a n s t e r o i d s induced v i t e l l o g e n e s i s i n t e l e o s t s suggests that the g o n a d o t r o p i n i n d u c t i o n o f v i t e l l o g e n e s i s i s mediated by i t s r e g u l a t i o n of s t e r o i d o g e n e s i s .  98 The  I n d u c t i o n o f O v u l a t i o n by S t e r o i d s .  The p r e s e n t  indicated that progesterone, deoxycorticosterone,  findings  c o r t i s o n e , and  corti-  c o s t e r o n e were e f f e c t i v e i n i n d u c i n g i n v i v o o v u l a t i o n of g r a v i d g o l d fish.  These o b s e r v a t i o n s were s i m i l a r t o those o b t a i n e d by K i r s h e n b l a t  (1952, 1959)  Ramaswami (1962), and  Sundararaj and Goswami (1966).  In  a d d i t i o n t o i n v i v o o v u l a t i o n , Goswami and S u n d a r a r a j (1971) demonstrated i n v i t r o o v u l a t i o n o f o v a r i a n fragments by c o r t i c o s t e r o i d s . served that deoxycorticosterone  was  They ob-  the most p o t e n t i n d u c e r . o n  in vitro  ovulation. R e c e n t l y Colombo e_t a l . (1973) demonstrated c o r t i c o s t e r o i d s y n t h e s i s i n t e l e o s t ( G i l l i c h t h y s m i r a b i l i s ) o v a r i e s and suggested t h a t the e n d o c r i n e c o n t r o l of o v u l a t i o n a c t s by p i t u i t a r y g o n a d o t r o p i n s t i m u l a t i o n o f  the  s y n t h e s i s of c o r t i c o s t e r o i d s i n the ovary and not the i n t e r e n a l s as post u l a t e d by S u n d a r a r a j and Goswami (1966).  There i s no d e f i n i t i v e  proof  i n f a v o u r o f any o f t h e s e two p o s t u l a t i o n s . The Mechanism o f E n d o c r i n e C o n t r o l o f Oogenesis i n T e l e o s t .  The  i n t e r a c t i o n between p i t u i t a r y g o n a d o t r o p i n and s t e r o i d o g e n e s i s , as suggested by our f i n d i n g s , p r e s e n t  a p l a u s i b l e mechanism whereby one  t r o p i n , as i s w i d e l y a c c e p t e d i n t e l e o s t (Burzawa-Gerard and 1972;  Donaldson e t a l . , 1972)  or a t most two  The  Fontaine,  g o n a d o t r o p i n s , c o n t r o l the  d i v e r s e t e m p o r a l changes i n o v a r i a n f u n c t i o n s d u r i n g the cycle.  gonado-  reproductive  r e g u l a t i o n of s p e c i f i c s t e r o i d s y n t h e s i s through the complex  s t e r o i d metabolic  pathway ( F i g . 48) may  be brought about by the  t i o n o r s t i m u l a t i o n of s p e c i f i c s t e r o i d o g e n i c enzymes.  inhibi-  99  F i g u r e 48:  The major s t e r o i d m e t a b o l i c pathways i n t e l e o s t ovaries.  Figure  48i  The m a j o r  steroid  metabolic  pathways  i n teleost  ovaries. ft  Cholasftrol  Pwgwnolone  Progasterona  fro-Hy4-oxypre nenolon<> fforHydroxyprogasti 8  Oahydrcepiondrosterone  Androttenadiona  Oaoxycorticosferona  ll-Deoxycortijol  Tastostercna  Cortiewterone  Cortisol  ll^-Hydroxytestosterona  H 0  Estrona  Esfrodiol-17^  Ealriot  ll-KetotesteMerone  Dehydrocorticostarone  Cortisona  100 At the i n i t i a l  stages o f t e l e o s t r e p r o d u c t i v e c y c l e  induces the s y n t h e s i s o f e s t r o g e n s which f i r s t  gonadotropin  induce the  differentia-  t i o n o f germ c e l l s i n t o oocytes and l a t e r the f o r m a t i o n o f y o l k v e s i c l e s w i t h i n oocytes.  Some of the o v a r i a n e s t r o g e n s a r e r e l e a s e d i n t o  the c i r c u l a t o r y system where they induce the l i v e r to s y n t h e s i z e y o l k precursors.  E s t r o g e n s w i l l be s y n t h e s i z e d u n t i l they r e a c h a t h r e s h o l d  l e v e l which i s m a i n t a i n e d by n e g a t i v e feedback gonadotropin. and Marcus,  i n t e r a c t i o n with p i t u i t a r y  I f as i n mammalian and b a c t e r i a l enzyme systems  1955;  Goldman,  1965),  (Talalay  e s t r a d i o l i n h i b i t s 3g-hydroxysteroid  dehydrogenase a c t i v i t y i n f i s h o v a r i e s , then an a c c u m u l a t i o n o f pregnenol o n e , 17 <* -hydroxypregnenolone, The  and d e h y d r o e p i a n d r o s t e r o n e w i l l  i n c r e a s e d s y n t h e s i s of pregnenolone  result.  at t h i s stage w i l l induce  d e p o s i t i o n of the y o l k p r e c u r s o r s i n o o c y t e s as y o l k g r a n u l e s .  the  The l o -  c a l i z e d i n h i b i t i o n o f h y d r o x y s t e r o i d dehydrogenase by e s t r o g e n s i s supp o r t e d by our o b s e r v a t i o n s o f the l a c k o f h y d r o x y s t e r o i d dehydrogenase a c t i v i t i e s i n the g r a n u l o s a of y o l k g r a n u l e stage o o c y t e s . As the i n h i b i t i o n o f 3 g - h y d r o x y s t e r o i d dehydrogenase by a l s o p r e v e n t s the s y n t h e s i s of a d d i t i o n a l e s t r o g e n s .  The  estrogens  i n h i b i t i o n of  3 6 - h y d r o x y s t e r o i d dehydrogenase, as w e l l as the n e g a t i v e feedback of  e s t r o g e n s on p i t u i t a r y g o n a d o t r o p i n w i l l be removed when the  e s t r o g e n s are c a t a b o l i z e d .  The removal  of n e g a t i v e feedback  w i l l cause a surge o f g o n a d o t r o p i n s y n t h e s i s and s e c r e t i o n , i n c r e a s e d s t e r o i d o g e n e s i s throughout h i g h l e v e l s of pregnenolone, of  3 3 - h y d r o x y s t e r o i d dehydrogenase, w i l l J  .  effects stimulating The  during estrogen i n h i b i t i o n  result i n increased synthesis  "no snfi tcrtico&iicioids _ s 3 u l t i ^ ,  '»  existing  the whole m e t a b o l i c pathway.  which accumulated  effects  ^  101  pcLfle  doe*  Kerf  &fitsr<  102  o f p r o g e s t e r o n e and  c o r t i c o s t e r o i d s r e s u l t i n g i n o v u l a t i o n of mature  oocytes. A l l the s t e r o i d o g e n i c enzymes a r e a p p a r e n t l y tion.  The  post-ovulatory  f o l l i c l e s s y n t h e s i z e estrogens  to c o r t i c o s t e r o i d s . The  estrogens  l u t e a induce  of a new  the genesis  b a t c h of oogonia and  dehydrogenase r e p e a t s  This estrogen  ovula-  i n addition  s y n t h e s i z e d by p o s t - o v u l a t o r y  i n a c t i v a t e the s t e r o i d o g e n i c enzymes. hydroxysteroid  a c t i v e during  corpora  at the same time  i n h i b i t i o n of  the r e p r o d u c t i v e c y c l e .  33-  103  GENERAL DISCUSSION  The ' p i t u i t a r y i s t h e p r i m a r y e n d o c r i n e g l a n d r e g u l a t i n g r e p r o d u c t i o n s i n c e hypophysectomy e l i m i n a t e s b o t h v i t e l l o g e n e s i s and s t e r o i d o genesis.  However, t h i s s t u d y p r o v i d e s f u r t h e r e v i d e n c e t h a t , i n a d d i -  t i o n to p i t u i t a r y gonadotropin, t e l e o s t reproduction i s strongly regulated by s t e r o i d hormones. V i t e l l o g e n e s i s i n g o l d f i s h i n v o l v e s t h e s e q u e n t i a l f o r m a t i o n and d e p o s i t i o n o f two t y p e s o f y o l k i n c l u s i o n s .  Yolk v e s i c l e s are f i r s t  d e p o s i t e d , f o l l o w e d s u b s e q u e n t l y by y o l k g r a n u l e s .  The h i s t o c h e m i c a l  s t u d y demonstrates t h a t y o l k v e s i c l e s a r e comprised o f m u c o p o l y s a c c h a r i d e s whereas y o l k g r a n u l e s a r e more complex and c o n t a i n p r o t e i n s , p h o s p h o l i p i d s and n e u t r a l l i p i d s .  This  y o l k types i n g o l d f i s h .  i s t h e f i r s t c h e m i c a l c h a r a c t e r i z a t i o n o f two I t i s suggested t h a t t h e y o l k v e s i c l e s may be  i n v o l v e d i n t h e c o r t i c a l r e a c t i o n a t f e r t i l i z a t i o n as d e s c r i b e d by Yamamoto (1961) w h i l e the y o l k g r a n u l e s s e r v e as n u t r i e n t s f o r t h e d e v e l o p ing  embryo. A f t e r hypophysectomy a l l o o c y t e s c o n t a i n i n g y o l k become a t r e t i c  and a r e r e s o r b e d . of  The f o l l i c u l a r c e l l s may be i n v o l v e d i n t h e r e s o r p t i o n  dead o o c y t e s as has been suggested so o f t e n but t h i s s t u d y p r o v i d e d  no c o n v i n c i n g e v i d e n c e .  A f t e r the cytoplasmic i n c l u s i o n s o f a t r e t i c  o o c y t e s a r e c o m p l e t e l y removed, t h e f o l l i c u l a r c e l l s develop i n t o t h e p r e - o v u l a t o r y corpus luteum.  The p r e - o v u l a t o r y corpus luteum s y n t h e s i z e s  s t e r o i d hormones, p r o b a b l y e s t r o g e n s s i n c e o n l y 1 7 3 - h y d r o x y s t e r o i d dehydrogenase was d e t e c t e d i n t h e y - s t a g e .  104  T h i s study p r o v i d e s the f i r s t  d e f i n i t e evidence of the r u p t u r e  of t h e t e l e o s t o v a r i a n f o l l i c u l a r membrane d u r i n g o v u l a t i o n p r i o r to t h e i r f o r m a t i o n i n t o corpus luteum.  Post-ovulatory corpora lutea  have v e r y a c t i v e 3 a - , 3 3 - , 1 7 a - , and 1 7 3 - h y d r o x y s t e r o i d s u g g e s t i n g a c t i v e s t e r o i d hormone s y n t h e s i s .  dehydrogenases  A f t e r a stage o f a c t i v e  hormone s y n t h e s i s , t h e p o s t - o v u l a t o r y corpus luteum forms an i r r e g u l a r mass of c e l l s i n d i s t i n g u i s h a b l e from t h e 6-stage p r e - o v u l a t o r y corpus  luteum.  The s t e r o i d hormones s y n t h e s i z e d by e i t h e r the p r e - o r t h e p o s t o v u l a t o r y corpus luteum p r o b a b l y induce some of t h e c o r p o r a l u t e a l to form the next b a t c h o f oogonia.  cells  T h i s f o r m a t i o n o f oogonia from corpus  luteum c e l l s i s s t r o n g l y supported by a u t o r a d i o g r a p h i c evidence and has not p r e v i o u s l y been d e s c r i b e d . T h i s study a l s o demonstrated h y d r o x y s t e r o i d dehydrogenases  t h e presence o f 3 a , 3 3 - , 1 7 a - , and 173-  i n the g r a n u l o s a c e l l s o f a l l o o c y t e s except  those a t the y o l k g r a n u l e s t a g e s .  The presence o f these s t e r o i d o g e n i c  enzymes suggests t h a t the g r a n u l o s a i s i n v o l v e d i n the s y n t h e s i s o f s t e r o i d hormones.  Hypophysectomy i n a c t i v a t e s a l l these enzymes, they a r e  r e a c t i v a t e d by l u t e i n i z i n g hormone therapy demonstrating t h a t g e n e s i s i n t h e s e t i s s u e s i s r e g u l a t e d by p i t u i t a r y The s t e r o i d s y n t h e s i z e d by t h e f o l l i c u l a r  steroido-  gonadotropin.  c e l l s o f normal  oocytes  or the c o r p o r a l u t e a , b o t h p r e - and p o s t - o v u l a t o r y , a f f e c t many a s p e c t s o f oogenesis i n f i s h .  A d m i n i s t r a t i o n o f exogenous s t e r o i d s  indicates  t h a t e s t r o g e n s i n c r e a s e oogonia f o r m a t i o n i n p o s t - o v u l a t o r y g o l d f i s h . E s t r o g e n s a l s o induce the f o r m a t i o n o f y o l k v e s i c l e s i n hypophysectomized fish.  Further, y o l k granule d e p o s i t i o n i n o v a r i e s of  f i s h i s s t i m u l a t e d by pregnenolone.  hypophysectomized  A d i f f e r e n t series of steroids (pro-  105 g e s t e r o n e and t h e c o r t i c o s t e r o i d s ) i n d u c e d o v u l a t i o n i n g r a v i d  fish.  The v a r i o u s p r o c e s s e s i n o o g e n e s i s a r e s t i m u l a t e d by d i f f e r e n t s t e r o i d s s u g g e s t i n g t h a t a c e r t a i n degree o f c h e m i c a l s p e c i f i c i t y i s required f o r b i o l o g i c a l action.  This c h e m i c a l l s p e c i f i c i t y of s t e r o i d s  provides a p l a u s i b l e e x p l a n a t i o n f o r the endocrine c o n t r o l o f reproductive cycles.  I t i s proposed t h a t t h e s t e r o i d hormones s y n t h e s i z e d by  ovarian tissues act l o c a l l y . the asynchronous  T h i s p r o p o s a l p r o v i d e s an e x p l a n a t i o n o f  development o f o o c y t e s i n f i s h o v a r i e s .  T h i s demon-  s t r a t i o n o f gonadal s t e r o i d s a c t i n g d i r e c t l y on t h e o v a r i e s i s n o v e l but n o t u n i q u e ; i n comparable  s t u d i e s , L o f t s e t a l . ( 1 9 6 6 ) , Sundararaj  and Nayyar (1967) , Donaldson and Yamazaki (1969) and Pandey (1969) demons t r a t e d t h a t a d m i n i s t r a t i o n o f androgens i n male hypophysectomized restored  fish  spermatogenesis.  Possible Practical Applications.  T h i s s t u d y o f t h e e n d o c r i n e con-  t r o l o f o o g e n e s i s and v i t e l l o g e n e s i s s u g g e s t s two p o s s i b l e p r a c t i c a l a p p l i c a t i o n s . f o r the endocrine manipulations of f i s h r e p r o d u c t i o n i n a q u a c u l t u r e u s i n g s t e r o i d hormones.  The i n d u c t i o n o f o v u l a t i o n by p r o -  g e s t e r o n e and c o r t i c o s t e r o i d s o f f e r s a p o t e n t i a l l y easy and perhaps n o m i c a l method t o supplement  the current p r a c t i c e o f i n j e c t i n g  eco-  individual  f i s h w i t h g o n a d o t r o p i n o r p i t u i t a r y e x t r a c t s ( s e e r e v i e w s by P i c k f o r d and A t z , 1957; Clements, 1968; Shehadeh, 1970; Donaldson, 1973).  Pro-  g e s t e r o n e o r c o r t i c o s t e r o i d s can be a p p l i e d i n t h e w a t e r o r t h e food e n a b l i n g a g r e a t e r number o f f i s h t o be t r e a t e d s i m u l t a n e o u s l y . The economics o f t h i s type o f a p p l i c a t i o n s h o u l d be examined.  Further, ster-  o i d s a r e c h e m i c a l l y more s t a b l e than g o n a d o t r o p i n s and t h i s may be an i m p o r t a n t f a c t o r when c o n s i d e r i n g t h e i n t r o d u c t i o n o f t h e s e t e c h n i q u e s  106  into r u r a l aquaculture. F i s h kept i n c a p t i v i t y , e i t h e r i n ponds o r a q u a r i a , f r e q u e n t l y undergo o v a r i a n d e g e n e r a t i o n . t h e i r brood  Prowse (1966) r e p o r t e d t h a t 30-60% o f  s t o c k of grass c a r p , Ctenopharyngodon i d e l l a , had  o o c y t e s and were of no use as spawners. suggests used on  t h a t pregnenolone,  and perhaps  The p r e s e n t  atretic  investigations  the c o r t i c o s t e r o i d s , can be  to induce y o l k d e p o s i t i o n and o v a r i a n m a t u r a t i o n .  Our s t u d i e s  the long-term e f f e c t s o f s t e r o i d s on g r a v i d f i s h demonstrated  pregnenolone,  p r o g e s t e r o n e , and the c o r t i c o s t e r o i d s d i d not  that  induce  a t r e s i a whereas e s t r o g e n s and t e s t o s t e r o n e cause e x t e n s i v e a t r e s i a of yolky oocytes. trogens may  T h i s adverse e f f e c t o f l o n g term a d m i n i s t r a t i o n o f e s -  e x p l a i n the o v a r i a n d e g e n e r a t i o n observed by Funk e_t a l .  (1973) a f t e r the treatment  of j u v e n i l e salmon w i t h salmon  gonadotropin  and e s t r o g e n f o r an extended p e r i o d . T h i s study opens up a l o g y which may  new  area i n t e l e o s t reproductive endocrino-  have s e v e r a l p r a c t i c a l a p p l i c a t i o n s .  on the economics i s r e q u i r e d and  However, r e s e a r c h  the p o s s i b l e s p e c i e s d i f f e r e n c e should  be t h o r o u g h l y i n v e s t i g a t e d b e f o r e t h i s e n d o c r i n e m a n i p u l a t i o n o f r e p r o d u c t i o n can be a p p l i e d commercially. hormones on. metabolism  The p o s s i b l e e f f e c t s o f these  and growth s h o u l d a l s o be  investigated.  107  REFERENCES  Ahsan, S.N., and Hoar, W. S.  1963.  hormones on t h e t h r e e - s p i n e  Some e f f e c t s o f g o n a d o t r o p i c  s t i c k l e b a c k , Gasterosteus  aculeatus.  Can. J . Z o o l . 41: 1045-1053. A i d a , K., H i r o s e , K., Y o k o t e , M., and H i b i y a , T.  1973.  Physiological  s t u d i e s on gonadal m a t u r a t i o n o f f i s h e s - I I . 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