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Action of luteinizing hormone-releasing hormone in rat ovarian cells : hormone production and signal… Wang, Jian 1989

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ACTION OF LUTEINIZING HORMONE-RELEASING HORMONE IN RAT OVARIAN CELLS: HORMONE PRODUCTION AND SIGNAL TRANSDUCTION  by  JIAN WANG M.B. ( M e d i c i n e ) , Harbin M e d i c a l U n i v e r s i t y , China, 1982  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE  FACULTY OF GRADUATE STUDIES (Department o f Physiology)  We accept t h i s t h e s i s as conforming t o t h e r e q u i r e d standard  THE  UNIVERSITY OF BRITISH COLUMBIA March 1989 (c) J i a n Wang, 1989  In  presenting  degree  at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be her  representatives.  permission.  Department The University of British Columbia Vancouver, Canada  for  an advanced  Library shall make  it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  DE-6 (2/88)  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  head of copying  my or  be allowed without my written  Abstract  The  present  study  was  hypothesis  that  membrane  participate  i n the actions  conducted  to  i n v e s t i g a t e the  phosphoinositide  breakdown  of l u t e i n i z i n g  may  hormone-releasing  hormone (LHRH) on hormone p r o d u c t i o n i n t h e r a t ovary. In granulosa  3 . . . [ H]-arachidonic acid  c e l l s prelabeled with  3 or  [ H]-inositol,  accumulation and  free  cholera  treatment  of r a d i o l a b e l e d i n o s i t o l  arachidonic  acid,  t o x i n d i d not e x e r t  protein  with  kinase  C  phorbol-13-acetate phosphoinositide  by  LHRH  increased  lipids,  but l u t e i n i z i n g  diacylglycerol  hormone  t h e same e f f e c t .  t h e phorbol  ester,  the  (LH) o r  A c t i v a t i o n of  12-0-tetradecanoyl  (TPA) had a s t i m u l a t o r y a c t i o n on membrane breakdown.  I n a d d i t i o n , TPA d i d not a l t e r  a r a c h i d o n i c a c i d r e l e a s e but p o t e n t i a t e d t h e A23187 s t i m u l a t e d l i b e r a t i o n of arachidonic acid. Changes i n t h e c y t o s o l i c f r e e c a l c i u m i o n c o n c e n t r a t i o n s , 2+ [Ca  ] i , induced by LHRH were s t u d i e d i n i n d i v i d u a l c e l l s u s i n g 2+ . fura-2 m i c r o s p e c t r o f l u o r i m e t r y . The r e s t i n g [Ca ] i was 96.7 ± 2+ . 2.9  nM  were  (n= 115).  transient  second  (n=64).  The a l t e r a t i o n s  and r e t u r n e d  there  others are  Sustained the  [Ca  A23187.  2 +  ] i induced  to resting  levels  A potent LHRH a n t a g o n i s t completely 2+  e f f e c t o f LHRH on [Ca whereas  i n [Ca  within  84±3  blocked the  ] i . Some c e l l s responded t o LHRH alone,  responded  different  by LHRH  t o angiotensin  subpopulations  I I , suggesting  of  granulosa  that cells.  p e r i f u s i o n o f LHRH r e s u l t e d i n a d e s e n s i t i z a t i o n o f ]i  response t o LHRH but not t o t h e c a l c i u m ionophore 2+ LHRH treatment a c c e l e r a t e d [Ca ] i d e p l e t i o n i n  iii granulosa the  cells  p e r i f u s e d with  involvement  changes induced  Ca  2+  f r e e medium,  of i n t r a c e l l u l a r  Ca  2+  indicating 2+ i n [Ca ] i  pool(s)  by LHRH.  The complex i n t e r a c t i o n s between t h e s i g n a l t r a n s d u c t i o n pathways  involved  in  the  were  also  prostaglandin  E  progesterone  level  2  (5  regulation  of  examined.  LHRH  and  24h  progesterone p r o d u c t i o n  induced  (FSH) o r c h o l e r a  (24h) .  and  toxin  and  increased  culture)  and  basal  attenuated  by f o l l i c l e - s t i m u l a t i n g hormone On t h e o t h e r  FSH o r c h o l e r a t o x i n s t i m u l a t e d  a c t i v a t o r o f phospholipase  hand, both  prostaglandin  were i n c r e a s e d by LHRH (5 and 24h) . (an  progesterone  E  basal  formation  2  A23187, TPA and m e l i t t i n  A )  were used  2  t o examine t h e  2+ roles  o f Ca  , protein  respectively,  in  kinase  LHRH  C  action.  progesterone and p r o s t a g l a n d i n  E  stimulation  E  of  and  prostaglandin  free  arachidonic  acid,  stimulated  basal  Melittin 2  production, by  2  LHRH,  and enhanced the A23187  and  TPA,  i n d i c a t i n g t h a t LHRH a l t e r s cyclooxygenase a c t i v i t y .  A23187 o r  TPA attenuated  by FSH o r  cholera  t h e formation  toxin  augmented  (5  cholera  formation.  The  prostaglandin  E  2  and  o f progesterone induced  24h).  toxin  or  stimulatory were  production induced  in a  progesterone  effects  acid  Arachidonic  dose  induced of  A23187  and  prostaglandin A23187  whether  or  TPA E  2  and  TPA  on  not  FSH  or  during the incubation.  The r o l e o f a r a c h i d o n i c investigated.  FSH  contrast,  synergistic,  c h o l e r a t o x i n was present  further  In  dependent  production.  a r a c h i d o n i c a c i d was blocked  by  i n t h e a c t i o n o f LHRH was a c i d enhanced progesterone  manner The  and  potentiated  stimulatory  TPA  effect  nordihydroguaiaretic  of  acid,  iv whereas  monohydroxyeicosatetraenoic  hydroperoxyeicosatetraenoic arachidonic  acid,  metabolites  i n LHRH  partially FSH acid,  suggesting  reversed  induced  and  arachidonic  LHRH  acid  only  These  metabolites  results  the  In a d d i t i o n ,  the i n h i b i t o r y  progesterone p r o d u c t i o n TPA.  mimicked  t h e involvement  action.  progesterone  TPA  acid  acids  action  production.  stimulated  of  Although  elevated  have a s t i m u l a t o r y  that  of  lipoxygenase acid  o f LHRH and TPA on arachidonic production,  20-alpha-hydroxy-  as compared t o t h a t induced suggest  effect  arachidonic  progesterone  slightly  and  arachidonic  by LHRH and acid  or i t s  r o l e i n t h e a c t i o n o f LHRH on  t h e de novo s y n t h e s i s o f o v a r i a n s t e r o i d hormones. Collectively, the  actions  production  these f i n d i n g s support  o f LHRH o r LHRH l i k e  peptides  the hypothesis on o v a r i a n  hormone  a r e mediated by m u l t i p l e second messengers i n v o l v i n g  2+ Ca  that  , p r o t e i n kinase C and a r a c h i d o n i c a c i d  metabolites.  V  T a b l e o f Contents  Abstract  Page  i i  List  of Tables  viii  List  of Figures  List  of Abbreviations  ix  Acknowledgements  Chapter 1. General  xiv xvi  Introduction  I. Ovary  1  A. I n t r o d u c t i o n  1  B. H i s t o l o g y  2  C. L i f e c y c l e o f t h e o v a r i a n f o l l i c l e  4  I I . S y n t h e s i s o f Sex S t e r o i d Hormones and Prostaglandins A. S y n t h e s i s o f sex s t e r o i d s B. S y n t h e s i s o f p r o s t a g l a n d i n s and l e u k o t r i e n e s I I I . R e g u l a t i o n o f o v a r i a n hormone S y n t h e s i s A. Role o f gonadotropins  6 6 10 13 13  B. I n t r a o v a r i a n r e g u l a t i o n by f o l l i c u l a r steroids  18  C. Role o f n e u r o t r a n s m i t t e r s on o v a r i a n steroidogenesis  21  D. R e g u l a t i o n o f o v a r i a n s t e r o i d o g e n e s i s and o v a r i a n f u n c t i o n by p r o s t a g l a n d i n s  22  E. Role o f l o c a l n o n s t e r o i d a l r e g u l a t o r s on ovarian function  23  vi  IV. S i g n a l T r a n s d u c t i o n Systems and Hormone A c t i o n  28  A. I n t r o d u c t i o n  28  B. C y c l i c AMP  29  C. Calcium and p r o t e i n k i n a s e C  32  V. The aim o f t h e p r e s e n t study  37  Chapter 2. I n d u c t i o n o f P o l y p h o s p h o i n o s i t i d e and A r a c h i d o n i c A c i d Release  Turnover  by LHRH  I. Introduction  38 38  I I . M a t e r i a l s and Methods  40  I I I . Results  45  IV. D i s c u s s i o n  56  Chapter 3. E f f e c t o f LHRH on Changes o f C y t o s o l i c f r e e Calcium  Ion C o n c e n t r a t i o n  Granulosa  i n Individual  cells  69  I. Introduction  69  I I . M a t e r i a l s and Methods  71  I I I . Results  75  IV. D i s c u s s i o n  95  Chapter 4. LHRH A c t i o n on Ovarian Hormone P r o d u c t i o n ; a l t e r a t i o n o f Progesterone accumulation  by Calcium  P r o t e i n Kinase C  and P r o s t a g l a n d i n  Ionophore and 106  vii  I. I n t r o d u c t i o n  106  I I . M a t e r i a l s and Methods  107  III. Results  110  IV. D i s c u s s i o n  Chapter 5. Role o f A r a c h i d o n i c  I. I n t r o d u c t i o n I I . M a t e r i a l s and Methods I I I . Results IV. D i s c u s s i o n  130  A c i d i n LHRH A c t i o n  142  142 143 146 179  General summary  195  References  199  viii L i s t of Tables  page  T a b l e I . Lowest hormone c o n c e n t r a t i o n s r e q u i r e d by g r a n u l o s a c e l l s f o r i n i t i a t i n g c y t o s o l i c c a l c i u m change.  80  2+ T a b l e I I . Average peak v a l u e o f [Ca by d i f f e r e n t doses o f LHRH.  ] i induced  80  I X  L i s t of Figures  Page  F i g . 1. The p r i n c i p a l b i o s y n t h e t i c pathway i n t h e ovary f o r p r o d u c t i o n o f t h e p r o g e s t i n s , androgens and estrogens. Fig.  2.  Key pathways i n a r a c h i d o n i c a c i d m e t a b o l i t e s .  7  11  F i g . 3. Diagram o f t h e "two c e l l , two gonadotropin theory" of f o l l i c l e steroidogenesis.  15  F i g . 4. General model o f cAMP mediated hormone response.  31  F i g . 5. I n o s i t o l p h o s p h o l i p i d t u r n o v e r and s i g n a l transduction.  33  F i g . 6. S t i m u l a t o r y e f f e c t s o f LHRH on t h e formation o f i n o s i t o l phosphates (IP ), DG, and t h e r e l e a s e o f u n e s t e r i f i e d AA i n r a t g r a n u l o s a c e l l s .  46  Fig. (DG)  3  7. E f f e c t o f LHRH on [ H ] - l a b e l e d d i a c y l g l y c e r o l formation.  n F i g . 8. Time response of stimulation of [ H]-labeled d i a c y l g l y c e r o l formation by LHRH. 3  . 3 F i g . 9. Comparison o f LH and LHRH on [ H ] - l a b e l e d i n o s i t o l phosphates.  47 49  50  F i g . 10. Comparison o f LH and LHRH on d i a c y l g l y c e r o l formation and a r a c h i d o n i c a c i d r e l e a s e .  51  F i g . 11. E f f e c t o f c h o l e r a t o x i n (CT) and LHRH on [ H ] - l a b e l e d i n o s i t o l phosphate formation.  53  3  F i g . 12. E f f e c t o f phospholipase C (PLC) on [ H ] labeled d i a c y l g l y c e r o l formation.  54  F i g . 13. A c t i o n o f t h e p h o r b o l e s t e r TPA on i n o s i t o l phosphate formation.  55  F i g . 14. S p e c i f i c i t y o f t h e p h o r b o l e s t e r TPA a c t i o n on d i a c y l g l y c e r o l f o r m a t i o n .  57  F i g . 15. I n t e r a c t i o n o f t h e c a l c i u m ionophore A23187 and the phorbol e s t e r TPA on a r a c h i d o n i c a c i d r e l e a s e .  58  F i g . 16. Scheme showing proposed mechanisms i n v o l v e d i n arachidonic acid release.  67  Fig.  77  17.  The c a l c i u m ionophore  A23187-induced r a p i d and  t r a n s i e n t increase i n c y t o s o l i c  calcium.  F i g . 18. LHRH-induced r a p i d and t r a n s i e n t i n c r e a s e i n c y t o s o l i c calcium. F i g . 19. The blockade o f LHRH-induced c y t o s o l i c c a l c i u m a l t e r a t i o n by LHRH a n t a g o n i s t . F i g . 20. E x i s t e n c e o f s u b p o p u l a t i o n o f g r a n u l o s a c e l l s : [Ca ] i changes induced by LHRH and A n g i o t e n s i n I I (Ang I I ) . 2+ . . F i g . 21. Increase i n [Ca ] i induced by separate i n j e c t i o n s o f LHRH t o two i n d i v i d u a l g r a n u l o s a c e l l s . 2+ F i g . 22. D e s e n s i t i z a t i o n o f [Ca ] i response induced by continuous exposure t o LHRH. 2+ F i g . 23. A l t e r a t i o n s i n [Ca ] i induced by d i f f e r e n t doses o f LHRH. 2+ F i g . 24. D e p l e t i o n o f i n t r a c e l l u l a r Ca i n calcium f r e e medium. F i g . 25. LHRH-accelerated f r e e medium.  [Ca  2 +  ] i depletion i n C a  2 +  2+ F i g . 26. Role o f _ e . x t r a c e l l u l a r Ca i n LHRH-induced a l t e r a t i o n o f [Ca ] i . F i g . 27. Comparision alteration. F i g . 28. Comparision alteration.  o f FSH w i t h LHRH on [ C a  2 +  ]i  2+ o f LH w i t h LHRH on [Ca ] i  F i g . 29. I n t e r a c t i o n o f m e l i t t i n (Mel, M; 3x10 M), w i t h LHRH (L; 10 M) o r t h e phorbol e s t e r TPA (T; 10~ M) on progesterone (PROG) p r o d u c t i o n (upper panel) and PGE. formation (lower panel) d u r i n g a 5h c u l t u r e . F i g . 30. E f f e c t s o f m e l i t t i n and/or t h e c a l c i u m ionophore A23187 on progesterone and PGE_ p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d . F i g . 31. E f f e c t s o f t h e p h o r b o l e s t e r TPA and/or i n c r e a s i n g c o n c e n t r a t i o n s o f t h e c a l c i u m ionophore A23187 on progesterone and PGE_ p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d . F i g . 32. E f f e c t s o f t h e c a l c i u m ionophore A23187 and/or i n c r e a s i n g c o n c e n t r a t i o n s o f t h e phorbol e s t e r  xi TPA on progesterone culture period.  and PGE, p r o d u c t i o n d u r i n g a 5h  F i g . 33. E f f e c t s o f the c a l c i u m ionophore A23187 and/or t h e phorbol e s t e r TPA on PGE_ p r o d u c t i o n , e i t h e r i n t h e absence (open bars) or presence (hatched bars) o f m e l i t t i n d u r i n g a 5h c u l t u r e p e r i o d .  117  F i g . 34. E f f e c t s of c h o l e r a t o x i n (CT) and/or LHRH on progesterone and PGE_ p r o d u c t i o n d u r i n g a 5h c u l t u r e period.  120  F i g . 35. E f f e c t s o f the c a l c i u m ionophore A23187 and/or c h o l e r a t o x i n (CT) on progesterone and PGE_ p r o d u c t i o n d u r i n g a 5h i n c u b a t i o n p e r i o d .  121  F i g . 36. E f f e c t s o f the c a l c i u m ionophore A23187 and/or t h e phorbol e s t e r TPA on b a s a l (open bars) o r FSH s t i m u l a t e d (hatched bars) p r o d u c t i o n o f progesterone and PGE p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d .  122  F i g . 37. E f f e c t s o f the c a l c i u m ionophore A23187 and/or t h e phorbol e s t e r TPA on b a s a l (open bars) o r C T - s t i m u l a t e d (hatched bars) PGE- p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d .  125  F i g . 38. I n t e r a c t i o n o f FSH and LHRH on t h e formation of progesterone (PROG) (panel, A ) , PGE- (panel, B), and P G F a l p h a (panel, C) d u r i n g a 24h c u l t u r e p e r i o d .  126  F i g . 39. I n t e r a c t i o n o f FSH and t h e phorbol e s t e r TPA on progesterone and PGE, f o r m a t i o n d u r i n g a 24h culture period.  127  F i g . 40. I n t e r a c t i o n o f FSH, t h e p h o r b o l e s t e r TPA and t h e c a l c i u m ionophore A23187 on progesterone and PGE formation d u r i n g a 24h c u l t u r e p e r i o d .  129  F i g . 41. S t i m u l a t o r y e f f e c t s o f m e l i t t i n , LHRH and a r a c h i d o n i c a c i d (AA) on progesterone (PROG) p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d .  148  F i g . 42. E f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n o f a r a c h i d o n i c a c i d (AA) on progesterone p r o d u c t i o n a 5h c u l t u r e p e r i o d .  149  2  2  2  F i g . 43. E f f e c t s o f unsaturated progesterone p r o d u c t i o n .  during  f a t t y a c i d s on  F i g . 44. E f f e c t s o f treatment o f g r a n u l o s a c e l l s w i t h a r a c h i d o n i c a c i d (AA) and LHRH o r a LHRH a g o n i s t (LHRHa) on progesterone p r o d u c t i o n .  150 152  xii F i g . 45. Time course o f s t i m u l a t i o n o f progesterone p r o d u c t i o n by a r a c h i d o n i c a c i d (AA), LHRH o r LHRH p l u s AA.  153  F i g . 46. E f f e c t s o f t h e phorbol e s t e r TPA and i n c r e a s i n g c o n c e n t r a t i o n s of a r a c h i d o n i c a c i d (AA) on progesterone p r o d u c t i o n .  155  F i g . 47. E f f e c t s o f a r a c h i d o n i c a c i d (AA) and i n c r e a s i n g c o n c e n t r a t i o n s o f t h e p h o r b o l e s t e r TPA on progesterone p r o d u c t i o n .  156  F i g . 48.  157  Role o f a r a c h i d o n i c a c i d  (AA) metabolism.  F i g . 49. E f f e c t s o f n o r d i h y d r o g u a i a r e t i c a c i d (NDGA) or indomethacin (INDO) on progesterone p r o d u c t i o n induced by LHRH and/or a r a c h i d o n i c a c i d (AA).  159  F i g . 50. E f f e c t s o f h y d r o x y e i c o s a t e t r a e n o i c a c i d s (HETEs) and h y d r o p e r o x y e i c o s a t e t r a e n o i c a c i d s (HPETEs) on progesterone p r o d u c t i o n .  160  F i g . 51. E f f e c t s o f h y d r o x y e i c o s a t e t r a e n o i c a c i d s (HETEs) on progesterone (upper panel) and PGE, (lower panel) p r o d u c t i o n .  163  F i g . 52. I n t e r a c t i o n s o f h y d r o x y e i c o s a t e t r a e n o i c a c i d s (HETEs) o r h y d r o p e r o x y e i c o s a t e t r a e n o i c a c i d s (HPETEs) w i t h LHRH on progesterone (upper panel) and PGE (lower panel) p r o d u c t i o n .  164  F i g . 53. I n t e r a c t i o n s o f h y d r o x y e i c o s a t e t r a e n o i c a c i d s (HETEs) o r h y d r o p e r o x y e i c o s a t e t r a e n o i c a c i d s (HPETEs) w i t h the p h o r b o l e s t e r TPA on progesterone (upper panel) and PGE, (lower panel) p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d .  165  F i g . 54. E f f e c t o f LHRH on FSH-induced progesterone p r o d u c t i o n : time response.  167  F i g . 55. E f f e c t s o f treatment o f r a t g r a n u l o s a c e l l s with a r a c h i d o n i c a c i d (AA) and/or FSH on progesterone production.  168  F i g . 56. Response t o a r a c h i d o n i c a c i d pretreatment with FSH and LHRH.  (AA) a f t e r  170  F i g . 57. Response t o a r a c h i d o n i c a c i d (AA) a f t e r pretreatment with FSH and t h e phorbol e s t e r TPA.  171  F i g . 58. Response t o a r a c h i d o n i c a c i d (AA) a f t e r pretreatment with c h o l e r a t o x i n (CT) and t h e p h o r b o l e s t e r TPA.  173  2  xiii F i g . 59. Response t o a r a c h i d o n i c a c i d (AA) a f t e r p r e t r e a t m e n t w i t h t h e phorbol e s t e r TPA and LHRH alone.  174  F i g . 60. E f f e c t s o f LHRH, t h e p h o r b o l e s t e r TPA and/or a r a c h i d o n i c a c i d (AA) on p r o g e s t i n p r o d u c t i o n d u r i n g a 5h i n c u b a t i o n .  177  F i g . 61. E f f e c t o f a r a c h i d o n i c a c i d (AA), t h e phorbol e s t e r TPA and LHRH on 25-hydroxycholesterol-enhanced s t e r o i d o g e n e s i s d u r i n g a 5h i n c u b a t i o n .  178  F i g . 62. I l l u s t r a t i o n o f t h e i n t e r a c t i o n s between LHRH and gonadotrophin second messenger pathways. R, receptor.  197  L i s t of Abbreviations  AA  arachidonic acid  GABA  gamma-aminobutyric a c i d  °C [Ca  degree 2 +  ]i  Celsius  i n t r a c e l l u l a r calcium i o n concentration  CAMP  3 1 5 ' - c y c l i c adenosine monophosphate  CT  cholera  DG  1,2-diacylglycerol  DPM  d i s i n t e g r a t i o n p e r minute  ER  endoplasmic  EBSS  E a r l ' s Balanced S a l t  FBS  f e t a l bovine serum  FSH  f o l l i c l e stimulating  G  guanine n u c l e o t i d e  GDP  guanosine diphosphate  GTP  guanosine t r i p h o s p h a t e  h  hour  hCG  human c h o r i o n i c  HDL  high density  HETE  hydroxyeicosatetraenoic acid  HPETE  hydroperoxyeicosatetraenoic acid  20-alpha-HSD  2O-alpha-hydroxysteroid  3-beta-rHSD  3-beta-hydroxysteroid  17-beta-HSD  17-beta-hydroxysteroid  I P  inositol  3  IU  toxin  reticulum Solution  hormone  regulatory  gonadotrophin  lipoprotein  dehydrogenase  dehydrogenase dehydrogenase  1,4,5-trisphosphate  international  protein  unit  LDL  low d e n s i t y  lipoprotein  LH  luteinizing  hormone  LHRH  luteinizing  hormone r e l e a s i n g hormone  LT  leukotriene  LX  lipoxin  min  minute  M  molar  NADPH  n i c o t i n a m i d e adenine d i n u c l e o t i d e  NDGA  nordihydroguaiaretic  20-alpha-OH-P  20-alpha-hydroxypregn-4-en-3-one  P  statistical probability  P  4  progesterone  PG  prostaglandin  PI  phosphatidylinositol  PIP  phosphatidylinositol  4-phosphate  phosphatidylinositol  4,5-phosphate  PIP  2  acid  protein kinase c  PKC PLA  phosphate  2  phospholipase A  2  PLC  phospholipase C  PMSG  pregnant mare's serum  PRL  prolactin  RIA  radio-immunoassay  sec  side-chain  sec  second  SE  standard  TPA  12-0-tetradecanolyphorbol-13-acetate  TX  thromboxane  gonadotrophin  cleavage  error  xvi  Acknowledgements  This  t h e s i s would  h e l p from many I Leung  am  grateful  thank  him  t o my  Drs.  H.  R.W.Brownsey  supervisor,  for his  f r i e n d s h i p throughout my study. to  completed  without  McLennan,  N.W.  advice,  Dr. P e t e r  t o express  Kasting,  R. A.  Pederson  appreciation  and  guidance.  thanks t o Dr. K.G. B a i n b r i d g e  my  and  My deep a p p r e c i a t i o n i s g i v e n  great a s s i s t a n c e with f u r a - 2 m i c r o s p e c t r o f l u o r i m e t r y . like  C.K.  encouragement  f o r s e r v i n g on my committee and t h e i r  I wish t o g i v e s p e c i a l  also  the  people.  very  and  not have been  t o Dr. K.H.  for his I would Chen,  M.  Rodway and D. S i d p r a f o r r e a d i n g and c o r r e c t i n g my t h e s i s . I parents and  take  this  and other  opportunity  t o express  f a m i l y members  my  for their  g r a t i t u d e t o my  emotional  support  encouragement. Finally,  program  I would  like  t o thank  a t the U n i v e r s i t y o f B r i t i s h  the p r e d o c t r a l f e l l o w s h i p .  the K i l l a m Columbia  Fellowship  f o r providing  1 Chapter 1. G e n e r a l I n t r o d u c t i o n  I . Ovary  A.  Introduction The  f u n c t i o n o f t h e ovary i s t o produce mature eggs  s e c r e t e o v a r i a n hormones.  The l a t t e r e x e r t a range o f e f f e c t s  i n c l u d i n g r e g u l a t i o n o f the r e p r o d u c t i v e system, characters,  the  gonadotropin  mating  release  and  behavior  and  of  some  secondary sex  species,  metabolic e f f e c t s .  pituitary  The  gametogenic  and endocrine f u n c t i o n s o f the ovary i n t h e female are  cyclic  p r o c e s s e s e x h i b i t i n g r e g u l a r peaks o f a c t i v i t y d u r i n g the o f the i n d i v i d u a l , and may  life  be regarded as p e r i o d i c p r e p a r a t i o n s  f o r f e r t i l i z a t i o n and pregnancy.  The p e r i o d i c i t y i s c a l l e d the  e s t r o u s c y c l e i n subprimate s p e c i e s and the menstrual c y c l e i n primates.  The  cyclical  changes  o c c u r as a r e s u l t  of  complex  i n t e g r a t e d a c t i v i t y o f t h e hypothalamus,  p i t u i t a r y and o v a r i e s .  The  of  most  important  luteinizing hypothalamus,  hormone  hormone-releasing follicle  signals hormone  stimulating  this  system  are  (LHRH)  from  the  (FSH)  and  hormone  l u t e i n i z i n g hormone  (LH) from the a n t e r i o r p i t u i t a r y g l a n d and  the o v a r i a n  hormones such as androgens,  progesterone  steroid (P ). 4  The  gonadotroph  cells  estrogens and of  pituitary  s y n t h e s i z e and s e c r e t e LH and FSH i n response t o LHRH. FSH a r r i v e a t t h e ovary v i a the c i r c u l a t o r y system.  LH and  FSH causes  o v a r i a n f o l l i c u l a r growth, w h i l e the LH surge induces o v u l a t i o n and r e g u l a t e s corpus luteum formation and  function.  Both  FSH  2  and  LH  a r e necessary  Ovarian  to stimulate  4  FSH, LH  negative  feedback  and LHRH  release  mechanism  e s t r o u s o r menstrual c y c l e . of peptides  selectively,  known  and t h a t  c o n t r o l l e d by FSH. closed  feedback  loop  regulatory  by e i t h e r  depending Recently,  as  on  a p o s i t i v e or  t h e stage  o f the  i t has been shown t h a t a  inhibin  regulates  FSH  the production  o f these  peptides  The i n h i b i n s thus r e p r e s e n t  regulate reproductive local  steroidogenesis.  s t e r o i d hormones, e s p e c i a l l y e s t r a d i o l and P , i n t u r n ,  regulate  family  ovarian  between  the p i t u i t a r y  functions  release  an a d d i t i o n a l and  ovary  ( R i v i e r e t a l . , 1986).  f a c t o r s such as p r o s t a g l a n d i n s  is  to  Other  and LHRH-like  p e p t i d e s may a l s o be i n v o l v e d i n t h e r e g u l a t i o n o f r e p r o d u c t i v e functions.  B.  Histology The  o v a r i e s a r e p a i r e d organs s i t u a t e d on e i t h e r s i d e o f  the u t e r u s .  Each ovary i s covered  by a continuous  composed o f a s i n g l e l a y e r o f c u b o i d a l  mesothelium  epithelium.  The ovary  i s r o u g h l y d i v i d e d i n t o a p e r i p h e r a l c o r t e x and a medulla. cortex primary  contains oocyte  granulosa  cortex,  enveloped  cells  connective  numerous o v a r i a n  i n various  stages  blood  lymph vessels  extend  spindle-shaped and a dense  The medulla i s s m a l l compared t o the  i t s connective  vessels  c o n s i s t of a  o f development  tissue  Embedded w i t h i n t h e l o o s e c o n n e c t i v e nerves,  that  by a s i n g l e l a y e r o f  t i s s u e stroma.  and  follicles  The  and many from  loosely  arranged.  t i s s u e o f t h e medulla a r e  large  the  is  blood  medulla  vessels.  into  the  Small cortex.  Cortical  stroma  connective  tissue  functions, and  consists  of  cells  contractile  at  least  performing  cells  three the  types  customary  s c a t t e r e d i n the  3 cells:  of  support  cortical  stroma  i n the w a l l s o f p r e o v u l a t o r y f o l l i c l e s , and c l o s e l y packed  spindle-shaped i n t e r s t i t i a l c e l l s . Four  major  identified: secondary  classes  1) primary  of  interstitial  cells  i n t e r s t i t i a l ; 2) t h e c a  interstitial;  and  4)  hilus  have  been  i n t e r s t i t i a l ; 3)  interstitial  cells.  Although these c e l l s a r e l o c a t e d i n the l o o s e c o n n e c t i v e t i s s u e of both the c o r t e x and medulla,  a l l a r i s e from a p o p u l a t i o n of  u n s p e c i a l i z e d mesenchymal c e l l s  i n the stroma compartment.  principal and  f u n c t i o n of t h e  secrete  steroids,  interstitial cells most  notably  The  i s to synthesize  androstenedione  and  testosterone. It  appears  certain  cells  resemble  that granulosa within  granulosa  cells  m i c r o f i l a m e n t s (Byskov, differentiation  the  organized  into  cells  terms  can  be  of  rete their  from  ovarii  which  organelles  Byskov and Rasmussen, 1973). cells  i s not  uniform  in a  The given  distinguishable  regions  with  At l e a s t t h r e e d i f f e r e n t p o p u l a t i o n s of distinguished.  The  antral  granulosa  c e l l s are c l o s e r t o the a n t r a l c a v i t y , w h i l e the cumulus surround oocyte growth.  the  oocyte.  w i t h i n the They a l s o  and  f o l l i c l e develops, t h e s e c e l l s become  morphologically  specialized functions. granulosa  in  of g r a n u l o s a  As the a n t r a l  are d e r i v e d mainly  intraovarian  1978;  follicle.  cells  Cumulus  f o l l i c l e and probably  cells  physically  provide  support  nutrients for  exchange s i g n a l s w i t h  cells  the  the  oocyte oocyte  f o r t h e c o o r d i n a t e d maturation  4 and t h e oocyte.  o f the f o l l i c l e  The m a j o r i t y o f g r a n u l o s a c e l l s a r e mural o r p a r i e t a l c e l l s l i n i n g the f o l l i c u l a r  granulosa  cavity.  C. L i f e c y c l e o f t h e o v a r i a n f o l l i c l e The oocyte  primordial f o l l i c l e s  are present before b i r t h .  and a s s o c i a t e d s p i n d l e - s h a p e d  cells  are separated  the surrounding stroma by t h e b a s a l membrane. events  w i t h i n t h e ovary  p r o c e s s , which r e s u l t s  i s follicular  growth, an  i n ovulation or a t r e s i a .  are subject t o intra-ovarian  1975). shaped  Follicular granulosa  maturation  cell  controls  i s initiated  irreversible A t t h e onset  Simpson,  granulosa  1965) .  when t h e s p i n d l e -  precursors d i f f e r e n t i a t e  cells  The oocyte  proliferate  increases  into  the  zona  pellucida  that  surrounds  follicle  cells, interna  becomes e n c a p s u l a t e d  t h e theca  interna  i s separated  membrane. externa  Blood but  granulosa ovulation.  do  After  cells  penetrate  a r e without  the  direct  the  follicles,  l a y e r s o f theca  externa.  The  theca  l a y e r by t h e basement  v e s s e l s and lymphatics not  cells  which g i v e r i s e t o  by d i s t i n c t  from t h e g r a n u l o s a  and the  Granulosa  the oocyte.  and t h e theca  single  (Van Wagenen  g r a n u l o s a c e l l s b e g i n t o p r o l i f e r a t e i n t h e primary the  a  i n size  mitotically.  s y n t h e s i z e and s e c r e t e mucopolysaccharides,  follicles,  (Peters e t a l . ,  l a y e r o f c u b o i d a l c e l l s t h a t then begin t o d i v i d e and  from  One o f t h e b a s i c  o f puberty, p r i m o r d i a l f o l l i c l e s mature i n t o primary which  The  penetrate  basement blood  t h e theca  membrane  supply  until  thus after  As t h e f o l l i c l e grows, t h e g r a n u l o s a c e l l s i n c r e a s e  in  number and s i z e .  follicle Antral  Follicular  and c o a l e s c e s formation  Graafian  accumulation  t o form  transforms  follicle.  fluid  Within  of granulosa  a single  the  this cells  5 w i t h i n the  accumulates cavity,  primary  t h e antrum.  follicle  i s the cumulus  into  a  oophorus, an  containing the oocyte.  This  oocyte i s l i b e r a t e d when t h e mature f o l l i c l e r u p t u r e s f o l l o w i n g the LH surge i n a p r o c e s s c a l l e d o v u l a t i o n . Following both  o v u l a t i o n , t h e corpus  the granulosa  membrane breaks  and t h e c a  luteum  interna  i s formed  cells.  from  The  basement  down, and c a p i l l a r i e s and f i b r o b l a s t s  from t h e  t h e c a i n t e r n a invade t h e c a v i t y o f t h e r u p t u r e d f o l l i c l e . granulosa  cells  do  increase  i n volume  increase  i n masses  not  divide  and undergo of  lipid  r e t i c u l u m , and m i t o c h o n d r i a . luteinization.  after  Since  ovulation,  morphologic droplets,  but  changes  smooth  of  t h e corpus  d e r i v e d from both g r a n u l o s a and theca c e l l s ,  cells,  luteum  t h e corpus  an  are  luteum  which a r e  t h e l a r g e l u t e a l c e l l s and t h e s m a l l  These c e l l s , t o g e t h e r w i t h t h e s u r r o u n d i n g theca  c a p i l l a r i e s and b l o o d v e s s e l s form t h e corpus  temporary  with  endoplasmic  c o n s i s t s o f two types o f s t e r o i d o g e n i c l u t e a l c e l l s ,  luteal cells.  they  These changes a r e r e f e r r e d t o as  the c e l l s  morphologically d i s t i n c t ,  The  endocrine  s t e r o i d hormones.  gland  that  secretes  large  luteum, a  amounts  of  6  II.  S y n t h e s i s o f sex s t e r o i d hormones and  prostaglandins  A. S y n t h e s i s o f sex s t e r o i d s O v a r i e s have t h e c a p a c i t y t o s y n t h e s i z e a l l t h r e e c l a s s e s of  sex  steroid  hormones  cholesterol  (Fig.  lipoproteins  (LDL)  1).  to  be  their  Cholesterol  as p r e c u r s o r  droplets  or  from  plasma  synthesized  al.,  1981).  the  availability  i n rodents,  is  of  synthetase  de  serum  by  and  the  has been  While HDL  appears  c h o l e s t e r o l from LDL  from  novo i n o v a r i a n  cytoplasmic cells  lipid  (Strauss  the  et  lipoprotein  C h o l e s t e r o l can be s t o r e d i n  long-chain  fatty acid  and  relative  activities  of  c h o l e s t e r o l esterase.  this  process  cholesterol  Additionally,  de  novo  s y n t h e s i s o f c h o l e s t e r o l i s dependent on the a c t i v i t i e s of rate-limiting  is  from plasma i s r e g u l a t e d by  l i p o p r o t e i n s and  uptake system.  as e s t e r s o f  regulated  low-density  C e l l u l a r c h o l e s t e r o l may  lipoprotein,  Uptake o f l i p o p r o t e i n  receptor-dependent the c e l l s  both  from  (Gwynne and S t r a u s s , 1982).  the major p r e c u r s o r  derived  precursor,  f o r s t e r o i d o g e n e s i s i n the  the major p r e c u r s o r i n other s p e c i e s . be  common  and h i g h - d e n s i t y l i p o p r o t e i n s (HDL)  demonstrated t o s e r v e ovarian f o l l i c l e  from  3-hydroxy-methylglutaryl  coenzyme  A  the  reductase  (Brown e t a l . , 1981). Granulosa important  cells  are the c e l l u l a r  ovarian steroids,  estradiol  source and  P^.  o f the two The  first  most step  i n the c o n v e r s i o n o f c h o l e s t e r o l t o s t e r o i d s i s b e l i e v e d t o  be  r a t e l i m i t i n g i n s t e r o i d o g e n e s i s , and  of  the c h o l e s t e r o l  s i d e - c h a i n by  the  i n v o l v e s the cleavage side-chain  cleavage P-450  17alpha-hydroxypregnenolone  Dehydroepiandrosterone  •  17alpha-hydroxyprogesterone  • A n d r o s t e n e d i o n e — • Estrone  Testosterone  • Estradiol-17beta  F i g . 1. The p r i n c i p a l b i o s y t h e t i c pathway i n the ovary p r o d u c t i o n o f the p r o g e s t i n s , androgens and estrogens. 1: c h o l e s t e r o l s i d e - c h a i n c l e a v a g e P 4 5 0  2: 3 :  17-alpha-hydroxylase C  17,  2<T Y 1  a S e  4: 1 7 - b e t a - h y d r o x y s t e r o i d dehydrogenase 5  MA  5: 3 - b e t a - h y d r o x y s t e r o i d dehydrogenase/d '^1 6: Aromatase 7: 2 0 - a l p h a - h y d r o x y s t e r o i d dehydrogenase 2Oalpha-OH-P: 2Oalpha-hydroxypregn-4-en-3-one  isomerase.  for  8  enzyme (SCC) r e s u l t i n g i n t h e C  compound, pregnenolone.  2 1  including cholesterol  22-hydroxylase,  hydroxylase  2 2  and  C  2 Q  -lyase,  are  cholesterol located  in  SCC,  20-alphathe  inner  m i t o c h o n d r i a l membrane. Pregnenolone i s t h e key s t e r o i d o g e n i c i n t e r m e d i a t e common to  a l l c l a s s e s of s t e r o i d  Both P, 4  granulosa but  and  granulosa  ( B j e r s i n g , 1967).  hormones produced by t h e  theca cells  cells are  convert  more  follicles.  pregnenolone  active  to  in  this  regard  Pregnenolone i s converted t o P  by a  complex  4  of two enzymes 3 - b e t a - h y d r o x y s t e r o i d dehydrogenase  (3-beta-HSD)  and an isomerase (Samuels e t a l . , 1951; Cheatum e t a l . , Both enzymes r e q u i r i n g n i c o t i n a m i d e adenine  1966).  dinucleotide  (NAD)  as a c o f a c t o r a r e l o c a t e d i n t h e microsomal f r a c t i o n , although 3-beta-HSD may (Sulimovici  a l s o be p r e s e n t i n t h e m i t o c h o n d r i a o f t h e ovary  and  Boyd  1969;  Haksar  and  Romanoff,  and Campbell, 1976).  S i n c e isomerase a c t i v i t y  excess  Peron,  (Philpott  and  1971),  1971;  Dimino  appears t o be i n  the p r o d u c t i o n  of P  4  from  pregnenolone i s mainly r e g u l a t e d by 3-beta-HSD. The  rate-limiting  i n the f o l l i c l e C._  o f the c e l l s  dinucleotide action.  i n the biosynthesis  phosphate  which  i g  and which (NADPH)  Hydroxylation at the C  steroids  pregnenolone  androgens  i s l o c a t e d i n t h e microsomal r e q u i r e s n i c o t i n a m i d e adenine  and 1 7  and  (androgens). P.  as  m o l e c u l a r oxygen  for i t s  p o s i t i o n i s e s s e n t i a l before  s i d e c h a i n i s c l e a v e d from t h e C  form C  of  i s t h a t c a t a l y z e d by the 17-alpha-hydroxylase/  . . - l y a s e enzyme complex  fraction  the  step  The  2 1  steroids  (progestins) to  r e a c t i o n can u t i l i z e both  substrates  resulting  in  dehydroepiandrosterone  o r androstenedione,  enzymatic  i s under  step  which  the control  feedback r e g u l a t i o n by t h e end products  9 This  respectively.  o f hormones and  of steroidogenesis, i s  one o f t h e key p o i n t s f o r t h e p h y s i o l o g i c c o n t r o l o f f o l l i c u l a r steroid secretion. In c o n t r a s t t o t h e n e i g h b o r i n g theca c e l l s , t h e granulosa cells  contain  hydroxylase  very  and C  1 7  low 2 Q  of  t h e enzymes,  17-alpha-  - l y a s e , which mediate t h e c o n v e r s i o n o f  p r o g e s t i n s t o androgens 1967).  levels  (Short, 1962; B j e r s i n g and Carstensen,  The d e f i c i e n c y o f these  enzymes  i n granulosa  i n d i c a t e s t h a t both g r a n u l o s a c e l l s and t h e c a c e l l s  cells  participate  i n androgen and e s t r o g e n b i o s y n t h e s i s . The estrone  conversion and  o f androstenedione  estradiol-17-beta  and t e s t o s t e r o n e t o  i s catalyzed  by  an  enzyme  complex, r e f e r r e d t o as aromatase, l o c a t e d i n t h e membranes o f the  agranular  types.  endoplasmic  The  oxygens.  reaction  Two  hydroxylation  of  reticulum  requires  these  are  of several  NADPH,  and t h r e e  involved  a t C-19, and t h e o v e r a l l  consequent  reaction  involves a  of this  i s not  clear  (Kantsky and Hagerman, 1980; Brodie e t a l . , 1976). 4  by o v a r i a n  changes i n t h e c o n v e r s i o n o f P route  of P  4  breakdown  4  cells  by  yet  i s modulated by  t o i t s metabolites.  i s mediated  of  two  hydroxylation,  secretion of P  site  in  cell  moles  third  The  but t h e exact  ovarian  The main  20-alpha-hydroxysteroid  dehydrogenase (20-alpha-HSD), l o c a t e d i n t h e c y t o s o l p o r t i o n o f ovarian  cells  reversibly  utilizing  converts  NADPH  as  a  hydrogen  P. t o i t s i n a c t i v e  donor,  metabolite,  which  20alpha-  hydroxy-pregn-4-en-3-one considerably precursor  less  P .  (20-alpha-OH-P).  a c t i v e as a p r o g e s t a t i o n a l agent  I t has been  4  20alpha-OH-P may  play  a  amount o f C ^ s u b s t r a t e follicular  cells,  substrates for C  1 7  2 Q  suggested  significant  that  role  since  i n determining  -lyase  20alpha-reduced  10 is  than i t s  the a c t i v i t y  a v a i l a b l e f o r conversion  2  in  20-alpha-OH-P  of the  t o androgens  s t e r o i d s a r e poor  ( G o l d r i n g and O r l y , 1985).  B. S y n t h e s i s o f P r o s t a g l a n d i n s and L e u k o t r i e n e s Prostaglandins  (PGs), which were f i r s t d i s c o v e r e d by Von  E u l e r i n t h e 1930s as a b i o l o g i c a l l y a c t i v e component o f human seminal  fluid,  are also  important  secretory  products  of the  o v a r i a n c e l l s and t h e s e c r e t i o n o f p r o s t a g l a n d i n s may be under hormonal 1978) .  control  The p r e c u r s o r  (AA) , which mammalian the  (Triebwasser  2-acyl  e t a l . , 1978; C l a r k  f o r PGs s y n t h e s i s  i s a C 20:4 p o l y u n s a t u r a t e d  cells  i s normally  position  esterified  to glycerol  et a l . ,  i s arachidonic fatty  almost  acid.  acid AA i n  exclusively i n  i n the phospholipids  of the  c e l l membrane and i s r e l e a s e d through a p h o s p h o l i p a s e - c a t a l y z e d reaction.  The c o n c e n t r a t i o n o f f r e e AA i n c e l l s  i s less  than  6  10~ M, and t h e f r e e a c i d l e v e l i n a t i s s u e r e p r e s e n t s a balance between t h e l i b e r a t i o n esterification.  o f t h e a c i d by h y d r o l y s i s and i t s r e -  Free AA can undergo two o x i d a t i v e pathways o f  metabolism as o u t l i n e d i n F i g u r e 2.  The cyclooxygenase pathway  leads  endoperoxide  to  the  prostaglandin  formation H , 2  which  of  the  i s then  converted  intermediate  by t h e a c t i o n o f  isomerases t o a number o f b i o l o g i c a l l y a c t i v e molecules,  11 II  O C-O-C-R, it i CH (CH ) (CH = CHCH ) (CH ) -CO-C 0 i II C-O-P-O-Base 3  ?  4  2  4  2  2  PHOSPHOLIPIDS  'A  ARACHIDONIC ACID  i 5-lipoxygenase  15-lipoxygenase  12-lipoxygenase  cyclooxygenase  F i g . 2. Key pathways i n a r a c h i d o n i c a c i d m e t a b o l i t e s . PG: p r o s t a g l a n d i n LT: l e u k o t r i e n e LX: l i p o x i n HETE: h y d r o x y e i c o s a t e t r a e n o i c a c i d HPETE: h y d r o p e r o x y e i c o s a t e t r a e n o i c a c i d  prostaglandin E F  2alpha  ^  (TXA ).  (PGE ), p r o s t a g l a n d i n D 2  r o s t a  2alpha^' P  glandin I  2  12 (PGD ), p r o s t a g l a n d i n 2  (PGI ) and thromboxane A  2  2  2  The l e t t e r s f o l l o w i n g t h e a b b r e v i a t i o n PG i n d i c a t e t h e  2  nature  P G F  2  and  position  of  the  oxygen-containing  p r e s e n t i n t h e cyclopentane r i n g .  substituents  The 2 - s e r i e s PGs a r e formed  from AA, and t h e 1 - s e r i e s and 3 - s e r i e s PGs a r e s y n t h e s i z e d from 8,11,14-eicosatrienoic respectively. is  provided  lipoxygenase (HPETEs)  and 5,8,11,14,17-eicosapentaenoic  An a l t e r n a t i v e pathway f o r t h e oxygenation o f AA by  lipoxygenase  enzymes  are  which  enzymes.  The p r o d u c t s  can  then  be  ovarian  and  lipoxygenase  activity  administration  o f human  both  converted  into  l e u k o t r i e n e s (LTs), and  follicular that  homogenates  increases  chorionic  after  gonadotropin  possess in  in  granulosa  and  (Hedin e t a l . , 1987).  can be i n h i b i t e d aspirin  Inhibition  cell  cells  of  t o occur  preovulatory  The a c t i v i t y o f cyclooxygenase  by n o n s t e r o i d a l a n t i - i n f l a m m a t o r y drugs  and of  theca  vivo  (hCG) (Reich,  The i n d u c t i o n by hCG o f PGs i s demonstrated  follicles  as  acids  (Fig. 2).  Rat  1985) .  o f the  hydroperoxyeicosatetraenoic  h y d r o x y e i c o s a t e t r a e n o i c a c i d s (HETEs), lipoxins  acid,  PG  indomethacin  (Flower  cyclooxygenase  s y n t h e s i s o f a l l cyclooxygenase  and  effectively  series.  Vane,  1974).  blocks  Lipoxygenase  such  the  activity  can be i n h i b i t e d by compounds such as n o r d i h y d r o g u a i a r e t i c a c i d (NDGA) ( S a l a r i e t a l . ,  1984).  13  III.  R e g u l a t i o n o f o v a r i a n hormone s y n t h e s i s  A. R o l e o f gonadotropins Ovarian are  mainly  and FSH.  follicle  growth  under t h e c o n t r o l  and s t e r o i d  o f two gonadotropin  r e l e a s i n g hormone (LHRH).  i n response t o l u t e i n i z i n g hormoneLHRH i s a decapeptide ( p y r o - G l u - H i s -  Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2)  the  hormones LH  LH and FSH a r e s y n t h e s i z e d and s t o r e d i n t h e a n t e r i o r  p i t u i t a r y and a r e r e l e a s e d  of  hormone p r o d u c t i o n  found  a l l mammalian s p e c i e s so f a r s t u d i e d . hypophyseal  portal  system  i n t h e hypothalamus LHRH i s s e c r e t e d i n t o  i n a pulsatile  fashion  and i s  t r a n s p o r t e d along t h e p i t u i t a r y s t a l k t o t h e p i t u i t a r y . pituitary,  LHRH  controls  the  synthesis  and  gonadotropins by a r e c e p t o r dependent mechanism. are  likewise  released  in a pulsatile  r e l e a s e and c y c l i c v a r i a t i o n of  gonadotropins  control  pattern.  In the  secretion  of  Gonadotropins The p u l s a t i l e  i n the c i r c u l a t i n g concentrations  ovarian  f u n c t i o n s by  s e n s i t i v i t y o f o v a r i a n c e l l s through  altering  the  i n c r e a s i n g and d e c r e a s i n g  r e c e p t o r f o r m a t i o n and a c t i v i t i e s o f c e l l u l a r enzymes. Although  the i n i t i a t i o n  of primordial  follicle  growth  o c c u r s independently o f p i t u i t a r y gonadotropins, once r e a c h i n g the primary f o l l i c l e stage f u r t h e r growth and m a t u r a t i o n o f t h e f o l l i c l e becomes completely dependent on LH and FSH. FSH  induces  responsible  for  ovarian the  responsiveness t o several ovarian  cells  through  follicle  development  maturation of  o t h e r hormones.  specific  and  is  granulosa  cell  FSH i n t e r a c t s  with  plasma membrane r e c e p t o r s . I n  the  female,  ovarian  FSH  binds  follicles.  biosynthesis  by  only  FSH  modulating  a l . , 1983).  preovulatory  FSH  granulosa  the  induces  The  granulosa the  maintenance  on  and  theca  of  the  FSH  various  LH  (Toaff  stimulates regulates  o f a c t i o n o f LH  on  P  4  (Armstrong e t a l . , 1970;  induction  cells  apparently and  C  1 7  Previous  of  LH  2 Q  involved  in  secretory  cells:  receptors  -lyase  et  in  the  action  activities  i n ovaries  1981;  of  of 17-  (Fukuda e t a l . ,  1982).  the  the  cell-type,  theca  (1)  interna  interstitial  LH-responsive cells  cells  of  (2) FSH-responsive c e l l s which are granulosa  t o t h i s model, t h e c a  two  There are p r i n c i p a l c e l l types  steroidogenesis:  comprising and  al.,  steroidogenic  increases  ( F i g . 3) .  follicular  envelope  stroma, and  The  (Wang  s t u d i e s have demonstrated a "two  gonadotropin theory"  follicular  activity  1979).  Bogovich and R i c h a r d s ,  According  of  o f c h o l e s t e r o l t o pregnenolone,  aromatase  Armstrong,  alpha-hydroxylase 1979;  progestin  c e l l s , t h e s e c e l l s are c a p a b l e o f responding t o LH i n  Dorrington LH  After  14 the  of  20-alpha-HSD  aromatase.  major s i t e  i s the c o n v e r s i o n  1980).  cell  activities  although 3-beta-HSD i s a l s o s t i m u l a t e d Madej,  cells  growth, induces o v u l a t i o n , and  corpus luteum f u n c t i o n . biosynthesis  granulosa  3-beta-HSD and  also  follicle  the  regulates  s t e r o i d o g e n i c enzymes, SCC, et  to  of  the  ovarian cells.  i n t e r n a c e l l s are s t i m u l a t e d  by  LH t o produce androgen from c h o l e s t e r o l , which d i f f u s e s across the basement membrane t o be used f o r estrogen FSH-stimulated 1975;  reaction in  granulosa  Fortune and Armstrong, 1977;  cells  synthesis  in  an  ( Makris and Ryan,  Tsang and Armstrong  1980;  15  THECA CELLS  1  Cholesterol  Progesterone  4-  e  ATP CAMP  Androstenedione  Estrogen (circulation)  Androstenedione \ v X Basement membrane Progesterone 3ST 0  Aromatase Androstenedione — — • Estrogen  Cholesterol^"^ - ^  ^ '"'''© CAMP''  FSH Estrogen (Follicular fluid) GRANULOSA CELLS  F i g . 3 . Diagram o f t h e " t w o - c e l l , two gonadotropin t h e o r y " o f f o l l i c l e steroidogenesis.  Erickson, to  1978).  Since granulosa  gonadotropins,  may  diffuse  androgen  cells  secrete P  i t i s also possible that  into  theca  cells  biosynthesis.  as  interna  cells  androstenedione by 17-alpha-hydroxylase and C contrast, granulosa C  side-chain  2 1  androgens from e i t h e r P  4  enzymes  other  hand,  cell  P  4  substrate f o r convert  1 7  2 Q  P  4  -lyase.  to In  a c t i v i t i e s of  and s y n t h e s i z e  little  o r no  o r pregnenolone ( L a c r o i x e t a l . , 1974;  Hamberger e t a l . , 1978; Short, the  a  c e l l s do not have s i g n i f i c a n t  cleavage  4  granulosa  t o serve  Theca  16 i n response  granulosa  1962; Fowler e t a l . , 1978).  cells  do possess  considerable  On 17-  beta-HSD a c t i v i t y  (Makris  and Ryan, 1980; Nimrod e t a l . , 1980;  Moon  1982),  which  and  estrone  Duleba t o form  testosterone  acts  on  androstenedione  and e s t r a d i o l ,  and  respectively.  Although androstenedione i s t h e major o v a r i a n androgen i n most species,  t h e 17-beta-HSD  estradiol  as t h e major estrogen.  and  FSH  together  with  reaction  the  favors  the production  of  These i n t e r a c t i o n s between LH cyclical  changes  in  plasma  c o n c e n t r a t i o n o f LH and FSH p r o v i d e a mechanism t o account f o r the  regulation  of  ovarian  steroidogenesis  and  follicular  growth. While t h e f o r e g o i n g account has f o c u s s e d LH  and FSH i n r e g u l a t i n g a c t i v i t y  i s a l s o evidence  of ovarian  that a t h i r d pituitary  on t h e r o l e s o f functions,  gonadotropin,  there  prolactin  (PRL), may a l s o r e g u l a t e o v a r i a n a c t i v i t y a t t h e o v a r i a n  level.  PRL  r e c e p t o r s have been demonstrated i n t h e human ovary  (Saito  and  Saxena  luteal  cells  1975),  and i n r a t and p o r c i n e  granulosa  c e l l s and  (Richards and W i l l i a m s , 1976; R o l l a n d and Hammond,  1975;  Rolland  cells  to  e t a l . , 1976).  promote t h e i r  maximum p r o d u c t i o n in  vitro  of P  studies agent  1981;  1980)  Smith,  receptor the  by  of  like  those  with et  al.,  1973)  as by  P  PRL  available  and  FSH,  does not no  stimulate  second  level  FSH,  a  of  LH  may  influence  P  synthesis  for  appear t o be  as  (Rothchild,  the  Behrman e t a l . , 1970).  cells in  acts  production  4  maintaining  precursors  and  i s essential for  a l . , 1974).  i n c o n t r a s t t o LH and  i t s receptor  PRL  (Holt e t a l . , 1976), and  f o r LH  c e l l membrane but  since  stimulating  (Armstrong e t a l . , 1970; PRL,  et  as w e l l  steroid  17 granulosa  i n t e r a c t with  by human l u t e i n i z e d g r a n u l o s a  4  i n r a t ovary  pool  may  maturation  (McNatty  luteotrophic  PRL  4  Receptors f o r located  on  the  i n t e r a c t i o n of  adenylate  cyclase  messenger  for  granulosa  cells  PRL  PRL  (Mason  has  been  c o n v i n c i n g l y documented. In PGs.  addition  PG  to  synthesis  steroids,  i s stimulated  increased production Marsh e t a l . , 1974;  of PGE  in  (Richards  granulosa  cells  is  hCG  used t o  of  al.,  Doses  i n c r e a s e PG slightly The  increase  et  2 ipha a  (  FSH c l a r k  of  PG  PGs  resulting e  t  a l  »/  of  hCG  The  1978;  proportional  stimulate capable  of  the  synthesis induced  a l . , 1987).  The  by  and hCG  do  cells  of  to  doses o f hCG induce  of  the  PGs  et  ovulation only  ovulation.  i s transient in rat  concentrations  PGs  (Hedin.,  stimulating  not  An  content  r a t e of p r o d u c t i o n  directly  in  Zor e t a l . , 1983).  s y n t h e s i s , whereas s u b - t h r e s h o l d  increase  (Hedin  P G F  and  secrete  d i r e c t l y i n c r e a s e s the f o l l i c u l a r  e t a l . , 1982).  concentration 1987).  and  LH  Knazek e t a l . , 1981;  o v u l a t o r y dose o f hCG o f PGs  2  by  also  ovary  reach  a  18 maximum p r i o r t o o v u l a t i o n and r e t u r n t o low l e v e l s w i t h i n 2448 h f o l l o w i n g t h e LH/hCG surge.  B. I n t r a o v a r i a n r e g u l a t i o n by f o l l i c u l a r s t e r o i d s  Role o f progestins Granulosa o f P , which cell  administration small  synthesize  antral  function. of P  In  facilitates  4  follicles  and Bogovich,  secretion  by t h e p r e o v u l a t o r y  1982).  l e v e l o f LH s e c r e t i o n (Uchida unilateral  follicular  growth  contralateral  ovarian without  ovary,  concentration  of P  Hodgen, 1979). fall  4  P  follicle  estrogen  implants  of P  affecting  Administration  that  of P  4  receptors  have  granulosa  cells  Similar  P  rabbit,  cow  4  been  identified  (Schreiber  receptors  o f the  locally  identified  (Philibert  high  (Goodman and in a  a change i n serum reversed  i n d i c a t i n g that  (Greenwald, 1974) .  and E r i c k s o n ,  have been  and human  function  i s not  i n the  4  inhibit  a  concomitant a d m i n i s t r a t i o n o f t e s t o s t e r o n e ,  P  directly  t o hamsters r e s u l t s  decline  a c t s a t t h e l e v e l o f t h e aromatase  ovarian  In contrast, i n  4  the  growth o f  a f f e c t i n g the  folliculogenesis  This  exogenous  biosynthesis  without  e t a l . , 1972).  inhibit  o f gonadotropins.  rats,  a l s o enhances  4  suggesting  may  growth and  t h e hCG-stimulated  i n b l o o d e s t r a d i o l c o n c e n t r a t i o n without  levels  large quantities  prepubertal  and hCG-induced  (Richards  monkeys,  and s e c r e t e  may e x e r t some e f f e c t s on f o l l i c u l a r  4  granulosa  cells  cytoplasm 1979; Naess,  by P  4  The P  4  of rat 1981).  i n the ovaries of  e t a l . , 1977; Jacobs and  Smith, 1980; receptors for  Jacobs e t a l . , 1980).  suggests  P.. *  Other  autonomy  of  an  presence o f o v a r i a n  intracellular  regulatory  s t u d i e s have demonstrated a r o l e o f  luteal  cell  control  mechanism  exerts  ultra-short  production  important  The  in  P  biosynthesis  4  which loop  the  P  the  autocrine  of  regulation  4  role  P„ i n 4  i n an  production  4  feedback  (Goff e t a l . , 1979;  and  19 P  the  of  cells  its  own  F a n j u l e t a l . , 1983).  R o l e o f androgens In  addition  to  serving  enzymes t o form estrogens, in  granulosa  androgen  cells  substrates  interaction  Pretreatment  dihydrotestosterone  p r e v e n t s the FSH  in  and  granulosa  cells  estrogen  treatment  treatment  induces  this  in vitro  ovarian  effect  (Farookhi, atresia  in  augment g o n a d o t r o p i n - s t i m u l a t e d and  the  can  absence  activity  with  intracellular  intact  be  (Katz  and  FSH  to stimulate progestin production  rats  receptors  Although of  with  antagonized  FSH,  steroidogenesis.  Armstrong, 1980).  The  aromatase  i n d u c t i o n o f LH  1980).  and  cells.  of  experiments have shown t h a t  aromatase  for  androgens e x e r t a v a r i e t y o f a c t i o n s  through  receptors.  as  androgen androgens  Both i n v i v o  androgens  Armstrong,  stimulate  1976;  Daniel  Androgens a l s o a c t s y n e r g i s t i c a l l y  stimulatory  effect  of  i n cultured rat androgens  on  3-beta-HSD (Nimrod, 1977;  Welsh e t a l . , 1982).  with  granulosa progestin  b i o s y n t h e s i s appears t o be the r e s u l t o f the s t i m u l a t i o n of and  by  SCC  20 Role o f estrogens Estrogens exert  maintain  feedback  action  secondary on  the  sexual  hypothalamic-pituitary  Moreover, estrogens p l a y a modulating formation. atretic with  Estrogens  effect.  a  loss  (Richards,  c h a r a c t e r i s t i c s and  r o l e a t the s i t e  estradiol  1975;  of i t s  have been known t o e x e r t a d i r e c t  The i n d u c t i o n o f a t r e s i a of  unit.  Harman  receptors  et  may  in  a l . , 1975;  anti-  be a s s o c i a t e d  granulosa Ingraham,  cells 1959).  Estrogens a l s o r e g u l a t e e s t r o g e n p r o d u c t i o n o f g r a n u l o s a by  augmenting  minimal  the  effective  aromatase  FSH-induced  10  dose  activity  aromatase  (3.7xl0~ M)  i s within  activity,  cells  and t h e  o f e s t r a d i o l - 1 7 - b e t a on  t h e range  of estradiol-17-beta  measured i n t h e f o l l i c u l a r f l u i d o f r a t p r e o v u l a t o r y f o l l i c l e s . T h i s suggests t h a t o f e s t r o g e n p l a y s a p h y s i o l o g i c a l r o l e as an end  product  amplifier  synergistic Adashi  effect  and  stimulated  o f aromatase  of  Hsueh,  androgens  1982).  granulosa  cell  (Goff  Estrogen aromatase  maintenance o f dominant f o l l i c l e s hand,  estradiol  may  inhibit  estrogen  androgen  may  and  before  concentrations  activity  may  of  provide  ovulation. estrogen  determine  which  or  ratio  follicle(s)  (Leung e t  intraovarian  i n l i m i t i n g the  time  Local the  FSH-  i t s precursor  Such an  adequate  of  e x p l a i n the  on t h e theca c e l l s 1979).  1979;  On t h e o t h e r  of  mechanism may be s i g n i f i c a n t  production  maturation  Henderson,  enhancement  production  1978; Leung and Armstrong,  n e g a t i v e feedback  and  t o enhance t h e  i n the ovary.  androgen through n e g a t i v e feedback al.,  activity  for  oocyte  intrafollicular of  estrogen  i n one  cycle  and will  escape  atresia  21 (Harmon e t a l . , 1975;  and go on t o o v u l a t i o n  H i l l i e r e t a l . , 1980).  C. R o l e o f n e u r o t r a n s m i t t e r s The  innervation  documented. ovary  on o v a r i a n  o f t h e mammalian  The dense a d r e n e r g i c  suggests  a  role  f o r the  regulation of ovarian functions Lawrence  and  steroidogenesis  Burden,  ovary  has been  well  i n n e r v a t i o n o f t h e mammalian adrenergic  system  i n the  (Moshin and Pennefather, 1979;  1980).  The  possible  role  of  catecholamines i n t h e d i r e c t r e g u l a t i o n o f s t e r o i d b i o s y n t h e s i s by  f o l l i c l e c e l l s has been s t u d i e d both i n v i v o and i n v i t r o .  Catecholamines s t i m u l a t e granulosa  cells,  beta -adrenergic 2  P  4  production  i n c u l t u r e d l u t e a l and  and t h e s t i m u l a t i o n c o u l d antagonist  be blocked  by t h e  (IPS339), b u t not p r a c t o l o l  (beta ~  adrenergic  antagonist)  antagonist)  (Bahr e t a l . , 1974; Condon and Black,  vivo  s t u d i e s have  adrenergic ovary been  or  shown t h a t  agonists  result  (Bahr e t a l . , 1974). extensively  acid  (GABA)  phentolamine  examined  (Erdo  (alpha-adrenergic  beta-adrenergic,  i n increased  P  4  1976).  and L a p i s ,  1982).  In  but n o t a l p h a -  production  Another n e u r o t r a n s m i t t e r i n t h e ovary  1  by t h e  t h a t has  i s gamma-aminobutyric I n whole  ovary,  GABA  c o n c e n t r a t i o n i s comparable t o b r a i n l e v e l s and i s 5 t o 6 f o l d higher  than  any o t h e r  non-neuronal  tissues  studied.  b i n d i n g s i t e s a r e e l u c i d a t e d by measuring t h e s p e c i f i c  GABA binding  3  o f a GABA a g o n i s t ,  [ H]-muscimol  ( S c h a e f f e r and Hsueh,  1982).  Although t h e p h y s i o l o g i c a l r o l e o f GABA i n t h e o v a r i a n t i s s u e s remains t o be e l u c i d a t e d , p r o d u c t i o n  o f cAMP i n s l i c e s o f r a t  ovary  i s increased  GABA r e c e p t o r Lapis,  blockers  induces  action  of ovarian  resumption  steroidogenesis  cAMP,  of  Although  o f LH and PGE  presence  of  1980).  estrogen  meiotic  PGE  (Erdo and  and o v a r i a n  function  of  t h e oocyte  and  LH  In the  stimulates  cAMP  ( L i n d e r e t a l . , 1974; L i n d e r e t  Although LH i n d u c e s t h e p r o c e s s o f o v u l a t i o n , t h e  absence o f P G E required  2<  This  rupture,  I n h i b i t i o n o f PG  o f indomethacin b l o c k s  ovulation  Grinwich, 1972; Armstrong e t a l . , 1974).  approaches  Ratwardhan  and  synthesis  by  (Armstrong and  The c o n c e n t r a t i o n o f  PGs i n t h e o v a r i e s , f o l l i c l e s and f o l l i c u l a r of o v u l a t i o n  does n o t o c c u r i n  i n d i c a t e s t h a t t h e presence o f PGs  f o r ovulation.  administration  f l u i d r i s e as time  ( L i n d e r e t a l . , 1980; Murdoch e t a l . ,  Lanthier,  1981).  plasminogen a c t i v a t o r and p r o t e o g l y c a n cells  and  and p a r a l l e l .  inhibitors,  f i n a l phase o f o v u l a t i o n , f o l l i c u l a r  1981;  production,  4  some e f f e c t s o f LH, t h e  a r e independent  2  production  4  and P  division  c a n mimic  2  cyclooxygenase  accumulation and P  is  i s a n t a g o n i z e d by  ( p i c r o t o x i n and b i c u c u l l i n e )  stimulates  2  ovulation.  the  effect  prostaglandins PGE  al  and t h i s  1982).  D. R e g u l a t i o n by  by GABA  The  stimulation  production  of  i n granulosa  f u r t h e r supports t h e involvement o f PG i n t h e p r o c e s s o f  ovulation.  Plasminogen  is a  glycoprotein  contained  i n the  plasma and i s converted t o t h e a c t i v e s e r i n e p r o t e a s e by two different  plasminogen  PGF_ , .  and PGE_.  activators  which  can be  stimulated  by  Plasmin, which i s produced by t h e a c t i o n  of  plasminogen a c t i v a t o r on  steps  i n the p o s t u l a t e d  plasminogen,  cascade.  The  23 various  t r i g g e r s the  net e f f e c t i s t o decrease  the s t r e n g t h of the f o l l i c l e w a l l t o the p o i n t a t which occurs  (Ossowski  1980).  Because  stimulate  a d e n y l a t e c y c l a s e , cAMP i s p r o b a b l y  activities  et  of  production  a l . , 1979;  the  gonadotropins  protein  of  plasminogen  has  been  demonstrated  reversal  of  ovum m a t u r a t i o n  blockade of o v u l a t i o n by  in  and  the  that P G F  that  indomethacin  animals  i n the f o l l i c l e s  of  PG  on  and  ovulation,  p  smooth-muscle  i s responsible P G F  but  elements  2alpha  of  the  m a  to  is  2  2alpha  ^  GE  glands,  t h y r o i d glands, blood  ^  in  overcome  a l . , 1972),  i s the  and  predominant  f o r most of the e f f e c t s exert  follicle  a  n  wall  e f f e c t on  the  (Diaz-Infante  et  stream  of  such  as  and  thereby  pituitary,  Paracrine  hormones  to  a f f e c t other control  their  e n t e r i n g the c i r c u l a t o r y system regulatory  the  function ovary  and  r e l e a s e hormones which reach t h e i r t a r g e t  functions.  diffusion  the  Beers,  1974).  Endocrine  body  and  Longo, 1982;,  E. R o l e o f l o c a l n o n s t e r o i d a l r e g u l a t o r s on o v a r i a n  the  increased  affects ovulation  2  et 2  i n the  involved  m a  (Downs and  thus p l a y s a major r o l e i n o v u l a t i o n . PG  involved  PGE  (Tsafriri  Espey,  prostaglandins  (Strickland  However, i t should be noted t h a t PGE  indomethacin-blocked  al.,  a l . , 1975;  leading  activator  It  et  and  synthesis,  1976).  1983).  Beers  rupture  function  of  t i s s u e s , organs  mechanisms  neighboring  involve cells  (Roth e t a l . , 1983).  some hormones  is  via or  local  without Finally,  autocrine  since  24 e f f e c t s a r e e x e r t e d on t h e c e l l s which produce t h e hormones. There  i s increasing  nonsteroidal paracrine  regulators  or autocrine  evidence  play  to  important  suggest  roles  c o n t r o l mechanisms.  that  local  i n t h e ovary by  These  nonsteroidal  r e g u l a t o r s i n c l u d e LHRH (Hsueh and Jones, 1981), growth f a c t o r s (Gospodarowicz insulin-like al,  et  al.,  1977a;  growth f a c t o r s  (Veldhuis  1985), o v a r i a n angiogenic  angiotensin  (Culler  bradykinin  1979),  insulin  1984),  1984)  and  oocyte  f a c t o r s (Koos and LeMaire, 1983),  al.,  1986;  maturation  neurohypophyseal  These l o c a l  and  e t a l . , 1983; Adashi e t  Husain  et  al.,  (Smith and Perks, 1983), n e u r o t r a n s m i t t e r s  al.,  1984).  et  1977b;  inhibitor  hormones  nonsteroidal  (Hsueh e t  (Tsafriri  (Sheldrick  1987),  and Braw, and  Flint,  r e g u l a t o r s may i n t e r a c t with  gonadotropins,  steroid  hormone  steroidogenesis,  oocyte m a t u r a t i o n  and  PGs  to  regulate  and o v u l a t i o n by p a r a c r i n e  o r a u t o c r i n e mechanisms. The  effects  extensively was r e p o r t e d hCG  of  studied.  LHRH  on  A direct  by R i p p e l  ovarian  function  have  been  f u n c t i o n o f LHRH i n t h e ovary  and Johnson who observed a decrease i n  augmented o v a r i a n weight i n immature hypophysectomized r a t s  t r e a t e d with confirmed mare's 1979;  LHRH (Rippel and Johnson 1976) .  i n hypophysectomized  serum gonadotropin  rats  T h i s f i n d i n g was  stimulated  (PMSG) o r FSH (Ying  Hsueh and E r i c k s o n , 1979).  with  and G u i l l e m i n ,  I n v i t r o s t u d i e s have shown a  d i r e c t e f f e c t o f LHRH on primary c u l t u r e s o f g r a n u l o s a well.  Treatment  stimulated  with  progestin  LHRH and  or  pregnant  i t s agonists  estrogen  production  c e l l s as  inhibits (Hsueh  FSHand  Erickson,  1979; Arimura e t a l . , 1979).  granulosa  cell  including  steroidogenesis  inhibition  of  25 The a c t i o n o f LHRH on  i s exerted  at multiple  FSH-stimulated  cAMP  sites  production,  i n h i b i t i o n o f aromatase, SCC and 3-beta-HSD and s t i m u l a t i o n o f 20-alpha-HSD. and  Jones,  inhibitory ovarian  P  4  action  basal  o f LHRH  on t h e ovary  and LH-stimulated  interstitial cells  Erickson,  (Hsueh  1981; Hsueh e t a l . , 1981; Gore-Langton 1981). i s exerted  compartments i n a d d i t i o n t o t h e g r a n u l o s a  inhibits ovarian  LHRH a l s o suppresses LH and FSH r e c e p t o r s  1982).  androgen  (Magoffin  on  other  cells.  LHRH  synthesis  by r a t  e t a l . , 1981; M a g o f f i n and  A d d i t i o n a l l y , LHRH i n h i b i t s LH/hCG-stimulated  s e c r e t i o n by r a t l u t e a l c e l l s i n v i v o and i n v i t r o  et  The  a l . , 1979; Jones  and Hsueh,  1980).  (Clayton  In contrast  t o the  i n h i b i t o r y e f f e c t s o f LHRH, s t i m u l a t o r y e f f e c t s f o l l o w i n g acute administration effects and al.,  o f LHRH alone have a l s o been observed.  i n c l u d e t h e s t i m u l a t i o n o f estrogen,  PGs p r o d u c t i o n 1981; C l a r k  inhibitory  o f LHRH c o u l d  4  1982).  be b l o c k e d  S t i m u l a t o r y and by treatment  with  (Jones and Hsueh 1981; Hsueh and L i n g , 1979;  N a v i c k i s e t a l . , 1982). o f LHRH on o v a r i a n follicles.  P , 20-alpha-OH-P  e t a l . , 1982; Gore-Langton e t  e t a l . , 1980; C l a r k ,  effects  LHRH a n t a g o n i s t s  (Dorrington  These  The most c o n s i s t e n t s t i m u l a t o r y  function  i s exerted  LHRH induces o v u l a t i o n  on mature  and t h i s  blocked  by LHRH a n t a g o n i s t s  1983).  The a c t i o n o f LHRH on f o l l i c u l a r  appears  t o be r e l a t e d  plasminogen a c t i v i t o r ,  action  preovulatory  a c t i o n o f LHRH i s  (Ekholm e t a l . , 1982; Dekel e t a l . ,  to i t s ability  rupture  at ovulation  t o stimulate  PGs and  both o f them have been shown p r e v i o u s l y  to  be i n v o l v e d  in follicular  rupture  Wang 1983; R e i c h e t a l , 1985).  Additionally,  in  maturation  the  resumption  dispersion  of  the action  indomethacin  and  1981).  proposed  involves the  that  protein  lipoxygenase  meiosis  induced  does  not  involvement  kinase C  (PKC).  pathway  o f AA  of this  maturation  the action  by  LHRH,  but  pathway  (Tsafriri  seem  i s not b l o c k e d by to  LHRH i n t h e r a t oocyte  o f LHRH  on  inhibits not  by  The f i n d i n g  ovum  maturation  indicating  LHRH a c t i o n  suggests  studies  t h e resumption LH,  by  i n h i b i t o r s of of the  on ovum  and Dekel, 1985;  of specific  LHRH on oocyte maturation (Dekel e t a l . ,  mediated  Recent  Furthermore,  i n mediating  strongly  be  1982).  e t a l . , 1986; Aberdam  Ekholm e t a l . , 1982).  cell  and LeMaire, 1980;  on t h e ovum  f o l l i c u l a r PG p r o d u c t i o n (Ekholm e t a l . , have  cumulus  U n l i k e i t s e f f e c t on f o l l i c u l a r  o f LHRH  hence  LHRH i s i n v o l v e d  and  (Dekel e t a l . , 1981; H i l l e n s j t t  Magnusson and LeMaire, rupture,  ovum  26 e t a l . , 1982  (Hillensjtt  receptors f o r  a direct  e f f e c t of  1988).  The d i r e c t e f f c t s o f LHRH on o v a r i a n s t e r o i d o g e n e s i s a r e mediated  by i t s s p e c i f i c r e c e p t o r s .  in luteal,  These r e c e p t o r s a r e found  t h e c a and g r a n u l o s a c e l l s a t a l l stages o f c e l l u l a r  differentiation  (Pelletier  et  al.,  1982).  Photoaffinity  l a b e l i n g o f o v a r i a n LHRH r e c e p t o r s has i d e n t i f i e d two s p e c i f i c components w i t h apparent MW o f 60,000 and 54,000 d a l t o n s (Hazum and Nimrod, 1982; Hazum, 1984). its  own  depletion  r e c e p t o r , whereas  gonadotropins  ( C l a y t o n and C a t t ,  regulation,  LHRH i n c r e a s e s t h e amount o f  1981).  cause  Apart  t h e o v a r i a n LHRH r e c e p t o r might  LHRH r e c e p t o r  from also  t h e hormonal be under a  d i r e c t n e u r a l c o n t r o l ( M a r c h e t t i and C i o n i , one  type  o f LHRH r e c e p t o r  i s identified  1988).  27 only  Since  i n the p i t u i t a r y , the  e x t r a component o f t h e o v a r i a n r e c e p t o r s may be r e l a t e d t o t h e different ovary. study  and s p e c i f i c Although  the direct  functions  o f LHRH-like p e p t i d e  t h e r a t model has been effects  of  rabbit  1980),  (Koos and LeMaire,  cow  (Milvae  Hertelendy, (Tureck  1982),  e x t e n s i v e l y used t o  o f LHRH on gonadal  s t u d i e s have demonstrated d i r e c t  et  1985),  al.,  monkey  effects  (Knecht  e t a l . , 1982).  function,  chicken  et a l . ,  (Takats  e t a l . , 1983),  The h i g h  other  o f LHRH on t h e ovary  p i g (Massicotte  1984),  i n the  affinity  and  and human  ovarian  LHRH  r e c e p t o r s have been demonstrated i n r a t , b u t not i n sheep, p i g , and  cow (Brown and Reeves,  and  human (Clayton and Huhtaniemi,  low  affinity  luteum  LHRH r e c e p t o r s  (Popkin  affinity  1983), monkey  were  e t a l . , 1983).  LHRH b i n d i n g  the poor a b i l i t y  sites  1982).  e t a l . , 1981),  On t h e o t h e r  documented  i n human  hand, corpus  The f a i l u r e t o demonstrate h i g h i n other  s p e c i e s might be due t o  o f t h e l a b e l e d LHRH analogs used t o i n t e r a c t  w i t h t h e o v a r i a n LHRH r e c e p t o r s i n these However,  (Asch  the  low  level  of  species.  LHRH  in  systemic  i n d i c a t e s t h a t LHRH may not be t h e endogenous l i g a n d t h a t  blood binds  t o t h e LHRH r e c e p t o r s i n t h e r a t ovary (Aten e t a l . , 1986). demonstrate ovarian  the  actions  physiological o f LHRH,  significance  i t i s necessary  r a t , bovine  and ovine  ovaries  the  direct  t o e s t a b l i s h the  presence o f an o v a r i a n LHRH-like substance. shown t h a t  of  To  Recently,  contain  i t was  a LHRH-like  p e p t i d e t h a t competes w i t h LHRH f o r b i n d i n g t o o v a r i a n membrane  28 different  r e c e p t o r s b u t w i t h immunoreactive a c t i v i t y d i s t i n c t l y from  those  o f LHRH  Interestingly, been  isolated  a  (Aten  e t a l . , 1986; Aten  separate  from  e t a l . , 1987).  gonadotropin-releasing  human f o l l i c u l a r  fluid  p e p t i d e has  ( L i e t a l . , 1987).  The amino a c i d composition and sequence o f t h i s l a t t e r p e p t i d e differ  from  those  structure of Phe-Asn-OH)  hypothalamic  LHRH  (with  the  primary  H-Thr-Asp-Thr-Ser-His-His-Asp-Gln-Asp-His-Pro-Thrand t h i s  stimulating pituitary  of  the  peptide  release  i n vitro.  i s considerably less  of  gonadotropins  from  potent i n t h e mouse  The LHRH r e c e p t o r s i n t h e r a t ovary  may  r e p r e s e n t r e c e p t o r s f o r one o r more o f t h e s e LHRH-like p e p t i d e s found  endogenously  i n the  r a t ovary.  The  presence  of  e q u i v a l e n t l e v e l s o f LHRH-like p e p t i d e i n t h e o v i n e , bovine and human  ovary  suggests  that  LHRH-like  peptide  might  serve  a  p a r a c r i n e o r a u t o c r i n e r o l e i n these t i s s u e s v i a t h e r e c e p t o r s specific has acid  f o r LHRH-like p e p t i d e .  suggested  Additionally,  that porcine i n h i b i n  suppresses  FSH-induced  a recent report  alpha-subunit  production  of  o f 134 amino  cAMP,  e s t r a d i o l v i a a LHRH r e c e p t o r i n r a t g r a n u l o s a c e l l s , further  interest  (Hillier et al.,  i n t h e nature  P  4  and  raising  o f LHRH r e c e p t o r s i n the ovary  1987).  IV. S i g n a l t r a n s d u c t i o n systems i n ovary  A. I n t r o d u c t i o n Ovarian  cellular  functions  are regulated  by  peptide  hormones, n e u r o t r a n s m i t t e r s and n o n s t e r o i d a l f a c t o r s and these  hormones  regulate  Generally,  the  of  the  via  second  29 messengers.  t o respond t o a  presence o r absence o f t h e  given  receptor  in  I t i s w e l l r e c o g n i z e d t h a t t h e r e are s e v e r a l c l a s s e s  hormone r e c e p t o r s ,  hormones,  which when occupied  stimulate  diffusion  enables  different  the  throughout the c e l l . One  cells  capacity of a given c e l l  hormone depends on the c e l l .  ovarian  hormonal  Two  by  their  specific  second  messengers,  signal  to  spread  whose rapidly  major s i g n a l pathways a r e now  known.  employs the second-messenger c y c l i c adenosine monophosphate  (cAMP).  The  other  employs a combination of second messengers 2+  t h a t i n c l u d e s c a l c i u m i o n s (Ca (IP ) 3  ), i n o s i t o l  and s n - d i a c y l g l y c e r o l (DG).  B. C y c l i c A  AMP large  number  of  hormones  exert  i n c r e a s i n g the c o n c e n t r a t i o n of cAMP. by  effects  by  cAMP i s formed from  ATP  the membrane bound enzyme adenylate  molecule  results  cAMP.  Therefore,  amplified  i n increased the  formation  initial  cAMP  allosteric  receptor,  cyclase.  Each hormone  o f many molecules  hormone  interacts the  with  a  regulatory  protein  kinase,  and  upon  subunit  induces  the  phosphorylation  cAMP  their  signal  is  of  greatly  f o l l o w i n g i t s i n t e r a c t i o n w i t h plasma membrane-bound  receptors.  give  1,4,5-trisphosphate  and  degradation  subsequent of  cAMP  subunit  dissociation  further amplification.  The  cellular to  specific  of  of  of the  intracellular cAMP-dependent free  catalytic  substrate  proteins  to  agonist-induced  increase  in  response 5'-AMP  by  is the  terminated  by  action  of  phosphodiesterase, removal  of  hydrolysis  phosphate  of  groups  phosphatase enzymes.  GTP  to  from  GDP  substrate  I t i s believed that  GTPase  30 and  proteins  by  by  cAMP i s the  messenger f o r the a c t i o n of both gonadotropins, LH ovarian  cells,  elicited  by  Channing, 1966;  and  1972;  Goff  and  Tsang e t a l . , 1979;  cells,  functions  cAMP analogs and  a l s o induces the  PGs  cAMP-inducing  Armstrong,  agents  1979;  also  Marsh  affect  and Channing, 1972;  FSH,  in  can  be  cells  (Kolena and  and  Savard,  Since  i n cAMP l e v e l s i n c u l t u r e d may  (Kolena  of ovarian  and  Dennefors e t a l . , 1980).  increase  endogenous  differentiation 1977;  multiple  second  PGE  granulosa  granulosa  G o f f and  2  cell  Armstrong,  Behrman, 1979). Studies  performed  that  the  r e g u l a t i o n of  far  more  complicated  presents  in  aspects  a  of  hormones.  during  many  adenylate  decade have  originally of  the  cyclase  Adenylate  past  hormone-sensitive adenylate than  scheme  the  cyclase  activity is  cyclase  suspected.  structural by  only  and  revealed  Fig.  of  4  functional  nucleotides  part  is  a  and  complex  r e g u l a t o r y system t h a t mediates the a c t i o n of hormones on  their  target c e l l s .  three  The  enzyme system i s composed o f a t l e a s t  c l a s s e s o f components. is  the  outer  At the i n n e r f a c e of the membrane are  the  catalytic  and  of  (C)  the  (Rodbell, 1980).  homologous guanine  guanine  specific  nucleotide  regulatory  Receptors communicate w i t h a p a i r  proteins.  s t i m u l a t i o n o f adenylate  a  surface  b i n d i n g o f hormones.  (G)  component c o n t a i n i n g  membrane  for  unit  (R)  a t the  site  protein  receptor  Located  One  of  which  (Gs)  mediates  c y c l a s e a c t i v i t y , w h i l e the o t h e r  (Gi)  31 signal  signal  •  inactive cAMP-dependent p r o t e i n kinase  + ®  1  5 -AMP  active cAMP-dependent protein kinase  •  biologic  effect  F i g . 4. General model o f cAMP mediated hormone response. R, receptor; Gs, stimulatory guanine-binding protein; Gi, inhibitory guanine-binding protein; GTP, guanosine triphosphate; GDP, guanosine diphosphate; ATP, adenosine t r i p h o s p h a t e ; cAMP, c y c l i c adenosine monophosphate; 5'-AMP, adenosine 5'-phosphate. is  responsible  for inhibition  They a r e both  formed  alpha-subunits  bind  analogs.  (Rodbell,  1984).  o f alpha, b e t a and gamma s u b u n i t s , both guanosine  When hormone b i n d s  triphosphate t o receptor  r a p i d i n t e r a c t i o n o f H*R w i t h G t o form H»R»G complex a f f e c t s G  activity,  to  site  i t s specific  1980; Gilman,  binding  (GTP) and i t s  (H*R),  H^'G.  there  isa  Formation o f  a l l o w i n g t h e b i n d i n g o f GTP  t o form H»R»G»GTP.  G i s active  only  when  a  H R*G  GTP  i s bound;  complex  diphosphate  increases  removal  (GDP) and f a c i l i t a t e s  activation  i s represented  GDP  32 i s bound.  inhibitory  guanosine  i t i s inactive of  when  GTP b i n d i n g .  by concomitant  GTP-dependent  subunit  dissociation  t o g i v e a GTP»alpha complex, which i n t e r a c t s w i t h C t o enhance or  decrease  catalytic  activity  depending  on  t h e type  of G  8  p r o t e i n , and a beta gamma complex, which does n o t i t s e l f appear to  dissociate.  results  from  protein  activition.  to  be  GTP  Reversal  completed  4  beta gamma  of  adenylate  cyclase  h y d r o l y s i s by GTPase,  which  stimulation  terminates  G-  GTPase-dependent d e a c t i v a t i o n i s assumed  upon  reassociation of  complexes  (Rodbell,  1980;  alpha  Jakobs  subunits et  with  a l , 1984;  Gilman, 1984; 1987).  C. Calcium and p r o t e i n k i n a s e C pathway In a d d i t i o n t o t h e cAMP pathway, t h e r e  i s another  major  s i g n a l l i n g pathway t h a t u t i l i z e s the membrane p h o s p h o i n o s i t i d e s (Fig.  5).  So f a r , t h e c o l l e c t i v e  term p h o s p h o i n o s i t i d e s has  been used t o d e s c r i b e t h e t h r e e a n i o n i c p h o s p h o i n o s i t i d e s t h a t contain The  myo-inositol  most  contains hydroxyl  abundant  i n their form  myo-inositol  head  groups  (Berridge,  1981).  i s p h o s p h a t i d y l i n o s i t o l (PI) t h a t  attached  to  phosphate  on t h e 1 - p o s i t i o n o f i t s i n o s i t o l  head  through  the  group.  The  o t h e r two members a r e formed by s e q u e n t i a l p h o s p h o r y l a t i o n o f hydroxyl  groups  on  the  4-  and  5-posit ions  to  form  p h o s p h a t i d y l i n o s i t o l 4-phosphate (PIP) and p h o s p h a t i d y l i n o s i t o l 4,5-bisphosphates (PIP,) t h a t i s the immediate p r e c u r s o r  33 Agonist  receptor  PI  • PIP  [Ca  2 +  ]i  C e l l u l a r response F i g . 5. I n o s i t o l p h o s p h o l i p i d t u r n o v e r and s i g n a l t r a n s d u c t i o n . Abbreviations: G, guanine nucleotide-binding p r o t e i n ; PI, phosphatidylinositol; PIP, phosphatidylinositol-4-phosphate; P I P , p h o s p h a t i d y l i n o s i t o l - 4 , 5 - b i s p h o s p h a t e ; PLC, phospholipase C; DG, 1,2-diacylglycerol; PA, phosphatidic acid; AA, a r a c h i d o n i c a c i d ; PGs, p r o s t a g l a n d i n s ; LTs, l e u k o t r i e n e s . 2  located  within  the  plasma  mechanism t o r e l e a s e i n o s i t o l cytosol,  leaving  DG w i t h i n  membrane  used  by  1,4,5-trisphosphate t h e plane  the  ( I P ) t o the 3  o f t h e membrane.  i n o s i t o l phosphates a r e r a p i d l y degraded t o i n o s i t o l , utilized  receptor  f o r resynthesis of phosphoinositides,  The  which i s  by a complex o f  phosphatases,  whereas  phosphatidic (AA) .  acid  cytidine  of  reticulum  PI  occurs  where  triphosphate  then  carried  back  arachidonic  i n t h e membranes  give  acid  acid  of the  interacts  cytidine  d i a c y l g l y c e r o l and t h i s combines w i t h is  plus  phosphatadic  to  34 to either  i s converted  or monoacylglycerol  Resynthesis  endoplasmic  diacylglycerol  with  diphosphate-  i n o s i t o l to give PI.  t o t h e plasma  membrane  by  a transfer  p r o t e i n t o complete t h e c y c l e o f breakdown and r e s y n t h e s i s . can be d e r i v e d from membrane p h o s p h o i n o s i t i d e s the  sn-2 p o s i t i o n o f o t h e r  availability most  AA  as w e l l as from  phospholipids.  Since  AA  l i m i t s t h e r a t e o f s y n t h e s i s o f AA m e t a b o l i t e s i n  tissues,  lipoxygenase  membrane  PI  the r e a c t i o n s  and  that  cyclooxygenase  produce  pathways  AA  can  thereby  stimulate generating  other s i g n a l s , f o r example, PGs, TXs and LTs. The h y d r o l y s i s o f . . . . l i p i d i s mainly  inositol mobilizing receptors sources  agonists, and g a i n  of  hypothesis  which  access  2+ Ca .  has been  . confined bind  t o both  Evidence obtained  to  the action  to  3  2+  specific  cell-surface  intracellular  and e x t e r n a l  for  the  by s t u d y i n g  IP /Ca  2+  3  -mobilizing  the e f f e c t  p u t a t i v e second messenger on v a r i o u s p e r m e a b i l i z e d IP  o f Ca  of this  c e l l s where  2+ c o u l d g a i n access t o t h e i n t r a c e l l u l a r Ca s t o r e s , such as  endoplasmic r e t i c u l u m  (ER) (Streb e t a l , 1983; Burgess e t a l , 2+  1984). It  has  Another p o s s i b l e source been  participates  demonstrated  o f Ca that  i s from isolated  mitochondria. mitochondria  i n t h e r e l e a s e and uptake o f l a r g e amounts o f 2+ i n t r a c e l l u l a r Ca (Lehninger, 1970; C a r a f o l i and Crompton, 2+ 1978). I P , a c t s through a s p e c i f i c r e c e p t o r t o r e l e a s e Ca by  opening a channel Irvine cause  across  e t a l , 1984).  t h e ER membrane  The i n i t i a l  35 e t a l , 1985;  (Smith  response  t o agonists  2+ Ca - m o b i l i z a t i o n i s a r e l e a s e o f i n t e r n a l  2+ Ca  that (phase  2+ 1) , which i s soon f o l l o w e d by e n t r y o f Ca membrane  across  t h e plasma  (phase I I ) (Kojima e t a l , 1985; Reynolds and Dubyak,  1985). the  Most a t t e n t i o n has focused on i t s r o l e i n s t i m u l a t i n g 2+ r e l e a s e o f Ca d u r i n g c e l l a c t i v a t i o n , b u t I P may serve 2+ 3  t o r e g u l a t e the r e s t i n g o r b a s a l l e v e l Ca al,  1985).  DG  that  remains  within  as w e l l  the plane  (Prentki et  o f t h e plasma  membrane f u n c t i o n s as a second messenger by a c t i v a t i n g p r o t e i n 2+ kinase  C  (PKC) .  PKC  has  been  shown  phospholipid-dependent f o r i t s a c t i v i t y o f t h e important  aspects  to  be  (Nishizuka  Ca  -  1984).  o f the a c t i v a t i o n process  and One  appears t o  be  a t r a n s l o c a t i o n o f PKC from t h e c y t o s o l i n t o t h e membrane, 2+ and t h i s process might be t h e r o l e o f Ca (Wolf e t a l , 1985). Although t h e a c t i v a t i o n o f PKC i s thought t o be b i o c h e m i c a l l y 2+ dependent upon Ca , i t can be p h y s i o l o g i c a l l y a c t i v a t e d 2+ . . . independence of Ca under some c o n d i t i o n s . I t i s now c l e a r that there  i s more than one s p e c i e s o f PKC molecule, and seven  subspecies  o f PKC have been i d e n t i f i e d  v a r i o u s subspecies The  (Nishizuka,  1988).  The  o f PKC have d i f f e r e n t enzymatic p r o p e r t i e s .  gamma and a l p h a - s u b s p e c i e s  o f PKC a r e much l e s s a c t i v a t e d  by DG i n t h e presence o f p h o s p h a t i d y l s e r i n e than i s t h e mixture of  beta-1  and  beta-2  subspecies, 2+  a c t i v i t y i n t h e absence o f Ca been  proposed  activated  that  different  which  shows  substantial  (Nishizuka, 1988).  I t has a l s o  subspecies  by t h e s e r i e s o f p h o s p h o l i p i d  of  PKC  metabolites,  are  also  such as  DG, AA and l i p o x i n  A  (Hansson e t a l , 1986, N i s h i z u k a ,  36 1988) .  Once PKC has been a c t i v a t e d through t h e c o n c e r t e d a c t i o n o f DG 2+ and Ca  , i t begins t o phosphorylate s p e c i f i c p r o t e i n s t h a t a r e  thought  t o contribute t o the control  m e t a b o l i c and other p r o c e s s e s IP  o f many  ( N i s h i z u k a , 1986).  and DG a r e r e l e a s e d from  3  o r modulation  membrane p h o s p h o i n o s i t i d e s  by a p h o s p h o i n o s i t i d e - s p e c i f i c p h o s p h o l i p a s e C (PLC). convincing role  experimental  f o r GTP-binding  PLC.  An example  protein  evidence  protein  a t present  which  s e r v i n g t o couple  o f t h e evidence  indicating  There i s  suggests  a  receptors to  a role  i n the coupling various receptors to PIP  2  f o r G-  hydrolysis  2+ and  Ca  m o b i l i z a t i o n i s the f i n d i n g  analogues  that  o f GTP s t i m u l a t e breakdown o f P I P  (MaJerus e t a l , 1986).  nonhydrolyzable and PLC a c t i v i t y  2  The i d e n t i t y o f t h i s G - p r o t e i n and i t s  r e l a t i o n s h i p t o o t h e r G - p r o t e i n s i s unknown. LHRH i s a p e p t i d e hormone and i t s e f f e c t s by  specific  receptors.  The mechanism  a r e mediated  o f LHRH a c t i o n  on t h e  ovary has been i n v e s t i g a t e d i n t h e p a s t few y e a r s .  There i s no  convincing  the  messenger LHRH  and  evidence  suggesting  f o r LHRH a c t i o n i t s agonists  that  cAMP  i n the ovary. have  been  is  On t h e other  shown  to  second hand,  stimulate the  breakdown o f p o l y p h o s p h o i n o s i t i d e s i n t o i n o s i t o l phosphates and DG i n t h e ovary  (Leung  e t a l , 1983; Naor and Yavin, 2+  and Leung, 1985; M i n e g i s h i and Leung, 1985). in  the action  1983)  o f LHRH i n t h e g r a n u l o s a  Ca  cells  1982; Ma  i s required (Ranta  et  al,  and p r o t e i n k i n a s e C has been c h a r a c t e r i z e d i n t h e ovary  (Noland  and Dimino,  1986; Davis  and C l a r k , 1983).  Recently,  the  e f f e c t o f LHRH on [ H]AA r e l e a s e  a l s o been examined level  (Minegishi  of the ovarian  cell,  and Leung, 1985) .  the hydrolysis  may immediately f o l l o w LHRH r e c e p t o r rapid  generation  of I P  i n r a t ovarian  has  Thus, a t t h e  of inositol  lipids  occupancy and l e a d t o the  and DG, and t h e r e l e a s e  3  cell  o f AA.  The  2+ r e s u l t a n t changes i n Ca  m o b i l i z a t i o n and/or PKC a c t i v i t y and  AA  be  metabolism  may  well  e f f e c t s o f LHRH on o v a r i a n V. The aim o f t h e p r e s e n t  substance  mechanism Since  have i n d i c a t e d t h a t LHRH o r LHRH-  affect  o f LHRH  r a t ovarian  function,  i s n o t completely  LHRH has been shown t o induce  breakdown,  t h e modulatory  study  directly  of action  with  steroidogenesis.  Although many r e p o r t s like  correlated  membrane  the o v e r a l l aim o f t h e p r e s e n t  the  understood.  phosphoinositide  study i s t o f u r t h e r  t e s t t h e e f f e c t s o f LHRH on hormone p r o d u c t i o n  i n rat  granulosa  c e l l s and i n v e s t i g a t e t h e p o s s i b l e s i g n a l t r a n s d u c t i o n r o l e s o f PKC,  2+ Ca , AA  Specifically,  . . and i t s m e t a b o l i t e s the a c t i o n  gonadotropins  and  phosphoinositide  o f LHRH was  cAMP-stimulating  turnover.  . i n the a c t i o n  Other  compared  agents  on  experiments  o f LHRH.  with  that of  t h e membrane was  examined  2+ LHRH-induced as  well  as  transduction production. to  [Ca  ] i a l t e r a t i o n i n i n d i v i d u a l granulosa  the  interactions  pathways  on  the  among  the  regulation  The o b j e c t i v e o f t h e p r e s e n t  understand,  more  completely,  of  P  4  of  signal  and  study was  the role  paracrine or autocrine regulator o f ovarian  putative  cells,  LHRH  functions.  PGE  2  therefore as a  Chapter  2.  Induction  of Polyphosphoinositide  Turnover  38 and  Arachidonic A c i d Release by LHRH  I. I n t r o d u c t i o n Numerous studies have shown that LHRH and i t s s y n t h e t i c agonists the  could d i r e c t l y  ovary  affect  (Hsueh and Jones,  steroid  hormone production i n  1981; Leung,  1985).  The d i r e c t  e f f e c t s of LHRH on the ovary are mediated by s p e c i f i c receptors (Pelletier  e t a l . , 1982).  These e x t r a p i t u i t a r y i n t r a o v a r i a n  a c t i o n s are e i t h e r stimulatory or i n h i b i t o r y , depending on the duration of LHRH treatment  as w e l l as the simultaneous  presence  of other ovarian c e l l regulators (such as gonadotropins) the  c u l t u r e period.  during  While the i n f l u e n c e of LHRH on ovarian  hormone production i s well documented, i t s mechanism of a c t i o n at the postreceptor l e v e l i s s t i l l past  few years,  LHRH  l a r g e l y unresolved.  and i t s agonists  have  been  stimulate the breakdown of polyphosphoinositides i n t o phosphates and DG i n the ovary  In the shown t o inositol  (Leung e t a l . , 1983; Naor and  Yavin, 1982; Davis e t a l . , 1986; Ma and Leung, 1985; Minegishi and  Leung,  1985; Leung e t a l . , 1986).  Inositol  phosphates,  e s p e c i a l l y I P are known t o induce m o b i l i z a t i o n of calcium ions 3  from i n t r a c e l l u l a r stores (Burgess e t a l . , 1984).  On the other  hand, DG i s now widely accepted t o be a potent a c t i v a t o r of PKC 2+ (Nishizuka e t a l . , 1984). the  action  Dorflinger  of LHRH et  Calcium  i n ovarian  a l . , 1984),  c h a r a c t e r i z e d i n the ovary  cells  and  (Noland  i o n (Ca ) i s required i n  PKC  (Ranta has  e t a l . , 1983; recently  been  and Dimino, 1986; Davis and  Clark,  1983).  Recently,  a  LHRH-like  peptide  has  demonstrated i n r a t , bovine, ovine and human ovaries, strengthening a  role  ovarian  cells,  immediately rapid  the  follow  generation  calcium  with  breakdown  functions.  hydrolysis  of I P  3  Thus,  inositol  occupancy  and DG.  and/or  the a c t i v i t y effects  proposed  lipids  on  involving  f o r LHRH  may  t o the  changes i n  of PKC  of LHRH  plays  w i t h i n the  and lead  The r e s u l t a n t  A s i m i l a r mechanism  has been  gonadotropin release  of  receptor  the modulatory  hormone production. lipid  ovarian  LHRH  mobilization  correlated  further  the concept that LHRH or LHRH-like peptide  i n mediating  39 been  may  be  ovarian inositol  s t i m u l a t i o n of  i n the a n t e r i o r p i t u i t a r y gland  (Raymond  et a l . , 1984; Huckle and Conn, 1987; H a r r i s e t a l . , 1985; Conn et a l . , 1985). In  the present  study,  the actions  of LHRH on  phosphates, d i a c y l g l y c e r o l and arachidonic further  investigated.  Specifically,  inositol  a c i d formation  the a c t i o n  were  of LHRH was  compared with that of gonadotropins and cAMP-stimulating agents on  the membrane phosphoinositide  turnover.  a c t i v a t i o n i n r e g u l a t i n g production and AA was emphasized i n t h i s study.  The r o l e of PKC  of i n o s i t o l phosphates, DG  40 I I . M a t e r i a l s and Methods  Animals Immature  Sprague-Dawley  female  rats  purchased  from  C h a r l e s R i v e r Canada, I n c . (Montreal, Canada,) o r Animal Care (University on  of B r i t i s h  the 23th  day a f t e r b i r t h w i t h  gonadotropins stimulate provide cells  Columbia) were  injected  subcutaneously  12 IU pregnant  mare's serum  (PMSG) between 09:00 and 10:00 i n t h e morning t o  the formation large  numbers  o f m u l t i p l e p r e a n t r a l f o l l i c l e s and of  relatively  homogenenous  a t t h e same stage o f development.  granulosa  The r a t s were k i l l e d  by c e r v i c a l d i s l o c a t i o n a f t e r 48h and t h e o v a r i e s were removed by  surgery.  Preparation of granulosa Granulosa microscope, gauge  by  cells  granulosa Eagle's  needle  1978). cells  salts  were  puncturing  hypodermic  Armstrong,  cells harvested  the ovarian as  into  dissecting  f o l l i c l e s with  squeezed  Minimum  and supplemented w i t h  units/ml of p e n i c i l l i n ,  the  previously described  The o v a r i e s were  released  under  27Gi  (Leung  and  g e n t l y and the  Essential  2 mM  a  Medium  o f L-glutamine,  o f n o n e s s e n t i a l amino a c i d s  NY) .  A f t e r removal from t h e o v a r i e s , t h e c e l l s were a fine  centrifugation MEM.  100  100 ug/ml o f s t r e p t o m y c i n s u l f a t e , and  5 ml  through  with  sterilized  mesh.  (MEM;  Gibco,  The c e l l s  Grand I s l a n d , expressed  were recovered by  (5 min a t 200xg), washed once, and suspended i n  41 R a d i o l a b e l e d d i a c y l q l y c e r o l and a r a c h i d o n i c a c i d  liberation 5  In  some  experiments,  granulosa  cells  (5x10  cells/ml)  were added t o 24 w e l l c u l t u r e p l a t e s (Falcon) and were l a b e l e d by  i n c u b a t i o n f o r 24h  0.5  England  bovine  Nuclear,  Boston,  MA)  (FBS).  The  cells  serum  and  without FBS. various  juCi/ml or  [5,6,8,9,11,12,14,14,15,- H]Arachidonic  Ci/mmol; New  thoroughly  0.2  3  uci/ml  fetal  i n medium c o n t a i n i n g w i t h  incubated  for a  i n MEM  further  to  (60  c o n t a i n i n g 5%  were  30  acid  then 60  washed  min  in  MEM  A t t h i s time, d i f f e r e n t hormones were added.  preparations  intervals.  At  removed and  the  were end  incubated  of  the  for  c o l d methanol.  The  lipids  different  incubation,  the c e l l s were scraped d i r e c t l y  The  the  into  time  medium 1 ml  was  of i c e -  i n t h e c e l l s were e x t r a c t e d by  method o f F o l c h e t a l . (1957) .  Briefly,  1 ml  of methanol  the was  mixed w i t h 2 ml of c h l o r o f o r m and 0.6 ml o f water, and mixed on a vortex vigorously. and  1 ml  The  of  pooled  The  chloroform chloroform  was  to  dryness  redissolved  i n chloroform (TLC).  c h l o r o f o r m phase was  added  layer  evaporated  chromatography  lower  of  under and  The  f o r the the  two  nitrogen methanol  fatty  acids  were e x t r a c t e d by the method o f Borgeat  second  removed  extraction.  extractions and  (2:1) i n the  the  was  residue  for thin  layer  c u l t u r e medium  and Samuelsson  (1979).  A f t e r a d d i t i o n o f 1.5 ml o f methanol t o the medium, the b u l k of the p r e c i p i t a t e d m a t e r i a l was collected,  and  the  methanol.  The  pooled methanol supernatants  pH  3  and  mixed  pellets  centrifuged.  with  6 ml  were  of  washed  diethyl  The  once  supernatant ml  of  were a c i d i f i e d  to  ether.  with  0.5  was  Then  4 ml  of  42 A f t e r s e p a r a t i o n of the  d i s t i l l e d water were added and mixed. phases,  the  water-methanol  phase was  evaporated  dissolved  i n a 2:1  labeled  AA  was  (Merck,  Rahway,  mixture  mixture  isolated  acetate-water-acetic  acid  TLC  diacylglycerol  ether  pure was  on  (5:11:10:2) Leung  standards separated  containing  silica  gel  The  60F-254  [ H]plates  solvent containing iso-octane-ethyl  p r e v i o u s l y by M i n e g i s h i and with  The  o f chloroform-methanol.  by  with  0.85  removed.  t o dryness under n i t r o g e n and the r e s i d u e  NJ)  was  was  vol/vol,  (1985) .  as  by  as  The  R^  v a l u e of  reference.  TLC  with  a  described AA  Radiolabeled solvent  system  benzene-diethylether-ethanol-ammonia-water  (50:40:2:0.1) v o l / v o l ,  as d e s c r i b e d by K a i b u c h i e t a l . (1983).  The areas o f the p l a t e c o r r e s p o n d i n g t o DG out and t h e i r r a d i o a c t i v i t y determined  (R  f  =0.72) were cut  by l i q u i d  scintillation  spectrometry.  Analysis of i n o s i t o l Granulosa  phosphates 5 cells (5x10 cells/ml)  were  prelabeled  by  c o n t a i n i n g myo-[2- H ] i n o s i t o l  (5  3  incubation jiCi/ml)  for  (New  24h  in  MEM  England N u c l e a r ; 16.5  The  cells  were  then  min  i n r a d i o t r a c e r - f r e e MEM.  ( i n a 10 u l volume), times.  washed  treatment, Incubation was  was  incubated  f o r an  f o r 24h.  initial  10  At t h i s time, hormones were added  and the c e l l s were i n c u b a t e d f o r d i f f e r e n t  Lithium c h l o r i d e  1-phosphatase,  and  Ci/mmol) and 5% FBS  added  enhancing  +  ( L i ; 10 mM), to  the  inositol  which i n h i b i t s  medium  prior  phosphate  to  inositolhormonal  accumulation.  t e r m i n a t e d by s c r a p i n g c e l l s d i r e c t l y  i n t o 1 ml  of  ice-cold  methanol.  c h l o r o f o r m and ratio  of  was  added and  the  aqueous phase were disposable (BioRad,  washed  with  Aliquots  (2ml)  inositol  was  whereas 0.2, IP  2  0.4, and  (1981). each  analyzed  200-400 M  of  the  sequential and  1.0  M  with  Hormone and  drug  Granulosa  water 0.1  (2.5 ml) counted  0.5  extractions  ml  mM  of  Dowex AG1-X8 The  resin  inositol  (10 M  use.  4°C.  Free  at  volume  formic  acid  the  (Fisher S c i e n t i f i c ,  of  resin)  containing eluted  Downes and  were c o l l e c t e d and  was  before  bed  by  the  IP,  Michell  r a d i o a c t i v i t y of  addition  of  15  ml  of  USA).  preparation cells  LHRH and  CT  were d i s s o l v e d  were t r e a t e d  with  were d i s s o l v e d  in  and  i n ethanol.  i n 5 ;al a l i q u o t s t o incubations  various saline.  hormones AA,  and  4-alpha-  12-O-tetradecanoylphorbol-13-acetate A l l drugs were d i l u t e d  t h e i r r e s p e c t i v e working c o n c e n t r a t i o n s  Control  vortex  chromatography  form).  described  following  final  ml o f water  ammonium formate p r o g r e s s i v e l y  12,13-didecanoate  added  The  two  l y s a t e s were loaded  I P 3 / r e s p e c t i v e l y , as Fractions  The  43 of  ml  After  exchange  formate  acid/5  cell  out  scintillation fluid  (TPA)  containing mesh,  2  i n o s i t o l phosphates i n  anion  washes w i t h  f r a c t i o n was  drugs.  by  formic  washed  repeated.  radiolabeled  columns  0.1  2:1:0.6.  l a y e r (aqueous), another 0.6  the e x t r a c t i o n was  were combined, and  another  were added.  chloroform/methanol/water was  removal of the top  resin  extraction,  5 jal of c o n c e n t r a t e d HC1  and  using  For  a total  received  the  w i t h MEM  incubation  b e f o r e use volume of  same volume of e t h a n o l .  1  to and ml. The  final  concentration of  exceed  0.5%,  phospholipid  and  ethanol  ethanol  in  did  the  44 not  incubations d i d  not  influence  membrane  metabolism.  Reagents The  following  were  chloride, myo-inositol, 4-alpha-phorbol formate was  13-didecanoate,  PMSG were  Program  from  BDH  Inc.  LHRH Inc.  from  NIH.  Sigma:  and Ovine  and  Acetic  CT. LH  the N a t i o n a l  Iso-octane,  methanol  (Canada) .  purchased from Canlab  Statistical  gifts  NIDDKD,  benzene, d i e t h y l e t h e r ,  from  lithium  f o r m i c a c i d , p h o s p h o l i p a s e C, AA,  from F i s h e r S c i e n t i f i c  25) , LHRH and Pituitary  12,  purchased  TPA,  Ammonium (NIDDK  oLH-  Hormone  and  ethyl  acetate,  c h l o r o f o r m were  purchased  acid  and  ammonia water  were  (Travenol Canada I n c . ) .  analysis  Statistical  significance  o f the  d a t a was  determined  by  Student's T - t e s t o r a n a l y s i s o f v a r i a n c e f o l l o w e d by S c h e f f e ' s multiple results  range were  experiments. determinations  test.  observed  i n at  a l l cases, least  identical  t h r e e o r more  or  similar  independent  A l l r e s u l t s were presented as the mean ± SE from  treatment group. significant.  In  triplicate  cultures  of  A P v a l u e o f l e s s than  cells  0.05  was  within  of  each  considered  45 III.  Results  Effects acid  o f LHRH on I n o s i t o l  lipid  breakdown  and  arachidonic  release 3  In  granulosa  cells  prelabeled  with  w i t h LHRH caused a s i g n i f i c a n t i n c r e a s e of  radiolabeled  illustrated stimulated from  DG  and AA  i n Fig.  6,  prelabeled  respectively,  o f LHRH  3  treatment  (P<0.01), i n t h e l e v e l s  i n the c e l l u l a r  addition  the liberation  [ H]-AA,  extracts.  As  6  (10~" M) f o r 5 min 3  o f [ H]-DG and u n e s t e r i f i e d [ H] -AA  phospholipids,  compared with  by  about  4  and  control incubations.  2.7  fold,  Furthermore,  3  i n c e l l s p r e l a b e l e d with  [ H ] - i n o s i t o l , treatment w i t h LHRH f o r  5 min caused a s i g n i f i c a n t i n c r e a s e i n accumulation o f i n o s i t o l phosphates  (P<0.01). 3  E f f e c t o f LHRH on \ H ] - l a b e l e d d i a c y l q l y c e r o l f o r m a t i o n 3  The  effect  o f LHRH  on  [ H] -DG  formation  was  further  prelabeled  with  [ H]-AA  3  examined and the  i n cultured  granulosa  l a t e r exposed t o LHRH. intracellular  cells  As shown i n F i g . 7, LHRH enhanced  DG formation  by 1.79 f o l d .  The b a s a l  level  3  of  [ H]-DG i n t h e medium was much lower than t h a t i n t h e c e l l s 3  and  [ H] -DG  level  d i d not i n c r e a s e  treatment o f g r a n u l o s a  i n t h e medium a f t e r the  c e l l s w i t h LHRH f o r 3 min.  46  I  1 Control Effig  8  LHRH (K)" M)  Fig. 6. S t i m u l a t o r y e f f e c t s o f LHRH on the f o r m a t i o n o f i n o s i t o l phosphates (IP_), d i a c y l g l y c e r o l (DG), and t h e r e l e a s e of u n e s t e r i f i e d a r a c h i d o n i c a c i d (AA) i n r a t granulosa c e l l s . The cells were prelabeled with either [ H]-inositol or [ H ] - a r a c h i d o n i c a c i d , as d e s c r i b e d i n M a t e r i a l s and Methods, and t r e a t e d w i t h LHRH f o r 5 min.  47  2.5 6**  medium  cellular  b X 2  Q. Q c o  1.5  T  <->  (0 E  1  i Q  0.5  CO  LHRH  C  LHRH  6  LHRH (10' M)  3 F i g . 7. E f f e c t o f LHRH on [ H ] - d i a c y l g l y c e r o l (DG) formation. The c e l l s were t r e a t e d w i t h LHRH f o r 3 min and d i a c y l g l y c e r o l (DG) formation was d e t e c t e d from t h e c e l l s and t h e medium. In t h i s and subsequent f i g u r e s , t h e absence o f standard e r r o r bars i n some o f t h e data p o i n t s i n d i c a t e s v a l u e s t o o s m a l l t o be shown.  Time response o f [ H ] - l a b e l e d d i a c y l g l y c e r o l t o LHRH Fig. granulosa  8 shows cells  significant  t h e time  i n response  t o LHRH.  DG  levels  continued  about 197% above t h e c o n t r o l l e v e l a t 5 min. about 40% o f t h e 5 min l e v e l a t 10 min. t H]-DG was s t i l l level  (P<0.01)  levels  o f DG  considerably  higher  a t 10 min a f t e r d i d n o t change  which  caused a  In  cells,  formation,  6  (10~ M)  in  observed as e a r l y as 15 sec a f t e r LHRH a d d i t i o n (P<0.05). treated  [ H]-DG  LHRH  formation  be  LHRH  in  o f [ H]-DG  could  the  increase  course  t o increase to This declined to  However, t h e l e v e l o f  (190%) t h a n t h e c o n t r o l  t h e treatment.  The c o n t r o l  during  t h e 10 min experiment  E f f e c t s o f LH and LHRH on [ H ] - l a b e l e d  i n o s i t o l phosphates and  period.  3  d i a c y l g l y c e r o l f o r m a t i o n and a r a c h i d o n i c a c i d r e l e a s e 6  F i g . 9 i l l u s t r a t e s t h a t t h e presence o f 10~ M LHRH f o r 3 minutes IP,  markedly  I P , and I P 2  stimulated 3  t h e accumulation  of radiolabeled  t o 155%, 545% and 100%, r e s p e c t i v e l y , when  compared w i t h u n t r e a t e d  control levels.  In c o n t r a s t , LH (1 jig)  d i d n o t s t i m u l a t e t h e formation o f i n o s i t o l phosphate from 3 [ H]-inositol prelabeled granulosa cells in t h e same experiment. A s i m i l a r r e s u l t was a l s o observed f o r the 3 , formation o f [ H]-DG ( F i g . 10, panel A) . In a d d i t i o n , LHRH 6  (10~ M) s i g n i f i c a n t l y  s t i m u l a t e d AA r e l e a s e , whereas LH (1 jug)  d i d n o t a f f e c t AA r e l e a s e ( F i g . 10, panel B).  £2 0  I — ' — i — ' — i — i — i — i — i — i — i — i  0  11  2  i  3  i  i  i  i  i  4  i  •  »  6  '  '  '  /  j  i  i  i  i  10  Time (min)  F i g . 8. Time response o f s t i m u l a t i o n o f [ H ] - d i a c y l g l y c e r o l (DG) formation by LHRH. A significant increase i n DG f o r m a t i o n was observed as e a r l y as 15 second a f t e r LHRH addition.  50  0.8 IP  IP3 •  Control LH (10 jug)  0.6  LHRH ("KT* M)  i O  y—  X  I s  0.4  X  i 0.2  C  LH  LHRH  LH  LHRH  LH  LHRH  Fig. 9. Comparison o f LH and LHRH on [ H]-labeled i n o s i t o l phosphates. The c e l l s were t r e a t e d w i t h LH and LHRH f o r 3 min. LHRH markedly s t i m u l a t e d t h e formation o f i n o s i t o l phosphates, whereas LH was i n e f f e c t i v e .  51  Control  LH  (1 ug)  LHRH <10' M) 6  F i g . 10. Comparison o f LH and LHRH on d i a c y l g l y c e r o l (DG) formation and a r a c h i d o n i c a c i d (AA) r e l e a s e . The c e l l s were t r e a t e d w i t h LH and LHRH f o r 3 min. The a d d i t i o n o f LHRH caused significant diacylglycerol (DG) formation and AA r e l e a s e , whereas LH d i d not a l t e r t h e formation o f these compounds.  Effects  of cholera  toxin  and  on  TJTRH  \ HI - l a b e l e d  inositol  phosphates f o r m a t i o n As  shown i n F i g . 11, i n p r e l a b e l e d g r a n u l o s a  cells  with  3  [ H]inositol,  the  addition  cellular  IP  and  IP,  2  IP  of  LHRH  (about  3  produced 380%,  increases i n  660%  and 191%,  r e s p e c t i v e l y ) , d u r i n g a 5 min p e r i o d , w h i l e a d d i t i o n o f CT (100 ng)  f a i l e d t o a f f e c t t h e formation o f i n o s i t o l  phosphates.  3  E f f e c t o f p h o s p h o l i p a s e C on r H ] - d i a c y l g l y c e r o l  formation 3  The DG  effect  accumulation  o f exogenous phospholipase was  investigated  in a  C on [ H ] - l a b e l e d  separate  experiment.  L i k e LHRH, a d d i t i o n o f 100 mU PLC r e s u l t e d i n a marked i n c r e a s e in  intracellular  whereas  a  DG  maximal  (5.6 f o l d dose  as compared -6  o f LHRH  (10 M)  i n c r e a s e i n t h e f o r m a t i o n o f DG ( F i g .  Effects  o f phorbol  ester  TPA  to control  on  caused  a  level), 1.8  fold  12).  inositol  phosphate  and DG  formation To determine whether t h e a c t i v a t i o n o f PKC by LHRH e x e r t s a  possible  feedback  effect  phosphatidylinositides, with  a  specific  challenged with  PKC  the hydrolysis  the granulosa activator,  TPA,  6  cells  2  and I P  3  o f membrane  were  for 5  10~ M LHRH f o r a f u r t h e r  i n c r e a s e s o f IP, I P cells  on  pretreated  min  3 min.  and  Significant  f o r m a t i o n were observed  were s t i m u l a t e d with TPA.  As demonstrated  6  LHRH (10" M) s t i m u l a t e d formation o f IP, I P response was u n a f f e c t e d by pretreatment  2  then  when t h e  i n F i g . 13,  and I P  3  and t h i s  o f c e l l s w i t h TPA.  53  (0  O K  2 02 Q. Q  9  009 go.i  M OL  o 0.2  0  u _ J  1  C  1  1  . CT (I00no>  1  1  '  •  LHRH (10" M) 9  3  F i g . 11. E f f e c t o f c h o l e r a t o x i n (CT) and LHRH on [ H ] - l a b e l e d i n o s i t o l phosphate formation. The c e l l s were t r e a t e d w i t h CT and LHRH f o r 5 min. The formation o f i n o s i t o l phosphates was s t i m u l a t e d by LHRH but n o t CT.  54  Control  LHRH (10- M) 6  PLC (IQOmU)  Fig. 12. Effect o f phospholipase C (PLC) on [ H]d i a c y l g l y c e r o l (DG) f o r m a t i o n . The c e l l s were t r e a t e d w i t h PLC and LHRH f o r 3 min. Both PLC and LHRH s i g n i f i c a n t l y i n c r e a s e d the f o r m a t i o n of DG.  55  x 2E Q. O  • » "  s 8  C  TPA (10~ M) 7  LHRH (10-6M)  LHRH TPA  F i g . 13. A c t i o n o f t h e phorbol e s t e r TPA on i n o s i t o l phosphate formation. The c e l l s were f i r s t t r e a t e d w i t h TPA f o r 5 min, and then t r e a t e d w i t h LHRH f o r f u r t h e r 3 min. TPA alone s t i m u l a t e d i n o s i t o l phosphate formation, b u t t h e pretreatment of TPA d i d n o t a l t e r t h e response o f t h e c e l l s t o LHRH.  The another  effect  o f TPA  experiment  on  DG  i n which  formation  granulosa  was  cells  56 in  determined  were  prelabeled  3  with  [ H] -AA.  TPA  increased  a c t i o n o f TPA was s p e c i f i c , alpha-phorbol  12,  the f o r m a t i o n  since  of  DG  and  this  another phobol congener,  13-didecanoate,  did  not  stimulate  4the  formation o f DG ( F i g . 14). Interaction  o f the calcium  e s t e r TPA on a r a c h i d o n i c  ionophor  acid  A23187  and  t h e phorbol  release  To determine t h e p o s s i b l e i n t e r a c t i o n between t h e calcium ionophore A23187 and t h e p h o r b o l e s t e r TPA on AA r e l e a s e ,  [ H]-  AA p r e l a b e l e d g r a n u l o s a c e l l s were t r e a t e d w i t h TPA and A23187, following  a  5  min  incubation,  A23187  at  10  M  caused  a  3  significant  stimulation  P<0.05).  However,  TPA  of  [ H]-AA  used  release  alone  i n similar  showed no such e f f e c t on AA r e l e a s e . A23187  and  TPA  were  present,  (80% o f  control,  concentration  I n t e r e s t i n g l y , when both  the  effect  of  A23187  was  3  potentiated  (P<0.05), w i t h t h e l e v e l o f [ H]AA r e l e a s e  130%  o f c o n t r o l l e v e l s ( F i g . 15).  IV.  Discussion The  previous  present findings  results that  ( F i g . 6)  LHRH  further  causes  a  i n o s i t o l l i p i d s i n r a t ovarian  cells.  proposed  t o cause  when LHRH was  shown  rapid  reaching  strengthen breakdown  T h i s mechanism was a rapid  the of  first  and s e l e c t i v e  3 2  incorporation of  P i n t o p h o s p h a t i d y l i n o s i t o l and  a c i d i n r a t granulosa c e l l s  phosphatidic  (Naor and Y a v i n 1982; M i n e g i s h i and  57  F i g . 14. S p e c i f i c i t y of t h e phorbol e s t e r TPA a c t i o n on diacylglycerol (DG) formation. The a c t i o n o f TPA on DG f o r m a t i o n was s p e c i f i c , s i n c e another phorbol congener, 4alphap h o r b o l 12, 13-didecanoate (4alpha-PDD), d i d not change the f o r m a t i o n of DG.  58  Control  A23187 (10" M) 7  TPA (10 " M) 7  A23187 TPA  F i g . 15. I n t e r a c t i o n o f t h e c a l c i u m ionophore A23187 and the p h o r b o l e s t e r TPA on a r a c h i d o n i c a c i d (AA) r e l e a s e . TPA alone d i d n o t a l t e r the r e l e a s e o f AA, b u t p o t e n t i a t e d t h e a c t i o n o f A23187.  Leung, 1985; Leung e t a l . , 1983). demonstrated  that  the  breakdown  products,  following  the addition  Leung,  1985; Davis  produced  since  radiolabeled  of  and I P  2  o f LHRH  t o LHRH  LHRH  inositol  i s markedly  3  a  from  cells  (Ma and  phosphates  polyphosphoinositol  decrease  polyphosphoinositides,  lipid  increased  The i n o s i t o l  were  caused  the  t o granulosa  e t a l . , 1986).  i n response  hydrolysis,  accumulation  IP, I P  59 i t has been  Subsequently,  i n the l e v e l  while  of  increasing  32 P  labeling  to phosphatidylinositol  (Leung e t a l . , 1986). phosphate  formation  The a c t i o n o f LHRH on o v a r i a n i s similar  p i t u i t a r y gonadotropes. with  [ H] i n o s i t o l  increase  to  f o r 5h, a d d i t i o n 3  turnover  Thus, p r o d u c t s o f p o l y p h o s p h o i n o s i t i d e mediators  transduction formation  of  the  3  may be r e s p o n s i b l e  c e l l u l a r b i o l o g i c a l responses.  acid  inositol  of  cultures  o f LHRH  LHRH  on  prelabeled  resulted  i n an  (Huckle and Conn,  1987).  breakdown may serve as  early  f o r LHRH both i n o v a r i a n  of I P  the action  In p i t u i t a r y c e l l  i n the rate of I P  primary  and p h o s p h a t i d i c  intracellular  signal  and p i t u i t a r y c e l l s .  The  f o r some o f LHRH-induced  In f a c t , I P  3  has been shown t o  2+ induce Ca 1984; role  mobilization  Burgess for IP  membrane. evidence  3  intracellular  e t a l . , 1984). i n regulating  There calcium  However, p r e l i m i n a r y that  inositol  role i n the stimulation is  from  formed from I P  3  pools  apparently entry  (Nishizuka,  i s no d i r e c t  across  t h e plasma  s t u d i e s have p r o v i d e d  indirect  1,3,4,5-tetrakisphosphate 2+  ( I P ) has a  o f Ca  1987) .  i n f l u x (Berridge,  by a I P ~ k i n a s e t h a t  from ATP t o t h e 3 - p o s i t i o n  3  o f IP..  4  IP  4  t r a n s f e r s a phosphate  I n t h e ovary,  both t h e  inhibitory  and  the s t i m u l a t o r y  a c t i o n s of LHRH on P  60 production  4  2+ have been shown t o  be  Ca  dependent  (Erickson  et  al.,  1986;  Leung and Wang, i n p r e s s ) . Since known  phosphodiesterase  mechanism  induced  PIP  2  for  IP  breakdown  formation  3  must  proposed  that  of  in  occur  polyphosphoinositide-specific been  cleavage  PIP  mammalian  through  the  phospholipase  thyrotropin-releasing  C  a c t i v a t i o n (Conn et a l . , 1987).  addition P G F  of  2alpha  al., study  on  1986).  the  PLC  formation  of  cells  of  LHRH  on  inositol  is specific.  LHRH-induced  completely  The  formation  b l o c k e d by  mediated mechanism.  of  action  a has on  cells,  the  LHRH  and  (Leung  et  of  phosphates  present  of  phosphate  earlier  studies  inositol  LHRH a n t a g o n i s t s ,  formation  phosphate  suggesting  can  a  ( F i g . 8-10). any  effect  phosphatidylinositide. ganglioside  mediated  n o t o r i o u s l y slow. other  hand,  be  receptor  Furthermore, gonadotropin hormones, which  breakdown o f membrane p o l y p h o s p h a t i d y l i n o s i t i d e s studies  in  have shown  a c t through i n c r e a s i n g cAMP i n the ovary, d i d not r e s u l t  the  LHRH  It  S i m i l a r r e s u l t s were a l s o observed i n the  action  granulosa  have  only  c e l l s occurs v i a  action  inositol  e  action  hormone  the  n  cells,  In r a t l u t e a l  mimicked  t  ( F i g . 12). The  that  exogenous  s  (PLC).  p h o s p h a t i d y l i n o s i t i d e breakdown i n c u l t u r e d GH PLC  ^  2  cAMP-inducing on  the  Since  i n the  CT,  this  A l o n g e r time may  toxin its  of  enters  onset  present  also  hydrolysis  mechanism,  gonadotropin  agents,  i n the  did  membrane  cells  of  via  action  need f o r f u r t h e r study.  hormones  which  are  not  the  a is On  major  hormones r e g u l a t i n g o v a r i a n f u n c t i o n s , and plays a l o c a l paracrine regulatory role, other  via  their  Interactions  different  between  adenylate  p h o s p h o i n o s i t i d e breakdown types. the  For  reticulum  of  in  preparations  of  cells  have been r e p o r t e d i n v a r i o u s  cell  rabbit  have been shown t o enhance  induced  (Bone  et  turnover  heart  and  in  sarcoplasmic  pig  granulocytes  e t a l . , 1984), but inositol  significantly  phosphate  a l . , 1986).  s t i m u l a t e s cAMP accumulation, s t i m u l a t e d PI  cyclase  accumulation  Prostacyclin,  (Watson e t a l . , 1984).  More r e c e n t l y ,  i n o s i t o l phosphate, but FSH  in  a l . , 1988). contrast  stimulated  The  with  present a  results  previous  report  be  the  which  (Monaco  showed  that  i n r a t granulosa  different  research  explained. approaches  example t h e l o n g exposure of the c e l l s may  cells  are LH  cells  The d i s c r e p a n c y between the p r e v i o u s  p r e s e n t s t u d i e s cannot be e a s i l y could  effect  ( F i g . 9-11), however,  i n o s i t o l phosphate formation  (Davis e t a l . , 1986).  accumulation  i t s e l f has no s i g n i f i c a n t  on the f o r m a t i o n of i n o s i t o l phosphate i n S e r t o l i et  which  has been shown t o b l o c k thrombin  FSH has been shown t o i n h i b i t the serum s t i m u l a t e d of  pathways. and  Farkas  norepinephrine  FRTL-5  signal  polyphosphoinositides  (Enyedi e t a l . , 1984; inhibit  i n t e r a c t with each  pathway  i n s t a n c e , cAMP analogs  formation  may  intracellular  the  61 probably  LHRH which  and  A p o s s i b l e reason undertaken.  i n Davis's  For  study  to  LH  f a c i l i t a t e the s y n t h e s i s of membrane p h o s p h o i n o s i t i d e s . I n response  t o LHRH, o t h e r p r o d u c t s  l i p i d h y d r o l y s i s such study  ( F i g . 6-8  and  as DG 10).  of membrane  were a l s o d e t e c t e d  i n the  inositol present  Geison e t a l . (1976) found t h a t the  sn-2  position  o f p h o s p h a t i d y l i n o s i t i d e s was r i c h  62 This  i n AA.  knowledge was used i n t h e p r e s e n t study t o determine t h e e f f e c t of  LHRH  on DG p r o d u c t i o n by l a b e l l i n g  3  .  [ H]AA. to  observed  study,  1986).  demonstrated  LHRH-induced  as 15 s e c .  f o r LHRH-induced  Davis  that  DG  T h i s time was v e r y  increase i n I P  Since  LHRH o n l y  the  suggests  likely  have  that  resulted  phosphoinositides  (Naor  M i n e g i s h i and Leung, It  from  studies  have  into  similarity i n  o f [ H]DG must  phospholipase  and Yavin,  similar to  P incorporation  the increased l e v e l  was  (Ma and Leung,  3  previous 32  increases  According  formation  p h o s p h a t i d y l i n o s i t o l and p h o s p h a t i d i c a c i d , t h i s time  with  3  response  as e a r l y  found  1985;  cells  The p r o d u c t i o n o f [ H]DG was then measured.  t h e time  that  granulosa  C  most  hydrolysis  1982; Davis  of  e t a l . , 1983;  1985).  i s interesting  t o note  that  most o f t h e [ H] DG was  r e c o v e r e d from t h e i n t r a c e l l u l a r space  ( F i g . 7 ) . Although LHRH 3  did  alter  t h e amount  significantly  change  of i n t r a c e l l u l a r the l e v e l  o f DG  [ H]DG, LHRH  d i d not  i n t h e medium.  This  f i n d i n g was c o n s i s t e n t w i t h t h e c h a r a c t e r o f DG as a membrane hydrophobic  metabolite. 3  Interestingly,  been observed w i t h [ H ] I P results  may  suggest  3  as w e l l  that  t h e DG  a similar  (Naor e t a l . , and  IP  3  r e s u l t has  1986).  formed  by  These LHRH  i n d u c t i o n may have b i o l o g i c a l r o l e s w i t h i n t h e c e l l s where they a r e produced r a t h e r than h a v i n g an i n f l u e n c e on o t h e r c e l l s . DG may  activating  play  a potent  PKC, which  is a  dependent p r o t e i n k i n a s e .  role 2+  i n the a c t i o n  Ca  activated  o f LHRH by  and p h o s p h o l i p i d  The a c t i v i t y o f t h i s enzyme has been  63  demonstrated Clark,  i n the  1983).  ovary  DG  and  (Noland and DG-like  Dimino,  phorbol  1986;  Davis  esters,  and  i.e.  TPA,  2+ stimulate  PKC  required The  by  reducing  for activation  dependence of PKC  activation  may  amounts o f Ca  (Nishizuka,  1984;  on p h o s p h o l i p i d  involve  phospholipid-rich  the  cell  LHRH, both i n h i b i t o r y and ovarian steroidogenesis  Takai  phospholipid et  al  1984).  i n d i c a t e s t h a t the enzyme  association  membranes.  and  of  the  Similar  stimulatory  to  enzyme the  with  effects  of  and  on  e f f e c t s o f DG  TPA  have been demonstrated, i n d i c a t i n g t h a t  a c t i v a t i o n of PKC  by endogenous d i a c y l g l y c e r o l s may  amplifier  of  the  LHRH-stimulated  Shinohara  et  a l . , 1985;  serve as  s i g n a l . (Welsh e t a l . ,  Kawai and  Clark,  1985).  DG  i n the p i t u i t a r y (Naor and C a t t , 1981; In a d d i t i o n t o IPs and DG, the  1984;  and  have a l s o been shown t o mimic the a c t i o n o f LHRH on LH  an  TPA  release  Conn e t a l . , 1985). t h i r d compound t h a t  was  3  measured i n the A previous  present  study has  study was  [ H]AA  ( F i g . 6,  10  demonstrated t h a t LHRH causes an  and  15) .  increase  3 in  the  level  as 15 min  of  [ H]AA  release  i n the  a f t e r LHRH a d d i t i o n (Minegishi and  stimulatory  e f f e c t of  LHRH can  be  blocked  presence of a potent LHRH a n t a g o n i s t . AA  i n the  actione  LHRH-stimulated production (Naor and 1986) . itself  c u l t u r e medium as  On  o f hormones, i t has LH  release  is  Catt, the  1981; other  r a t h e r than i t s m e t a b o l i t e s  by  The  concomitant  To e v a l u a t e d  the r o l e of  a l s o been observed coupled  that  with  Abou-Samra e t a l . ,  a l s o been suggested t h a t may  the  i n the a n t e r i o r p i t u i t a r y  Snyder e t a l . , 1983; hand, i t has  Leung, 1986). the  closely  of o x i d i z e d AA m e t a b o l i t e s  early  be  a cellular  AA  regulator  of  PRL  Since  secretion  from  GH  the i n t r a c e l l u l a r  study  with  f r e e AA  3  64 e t a l . , 1984).  (Kolesnick  concentration  s y n t h e s i s o f PGs and LTs, intracellular  cells  of free  AA  l i m i t s the  t h e demonstration o f t h e i n c r e a s e i n  i s clearly  r a t granulosa  important.  cells,  LHRH  In the present 3  stimulated  [ H]AA t o  i n c r e a s e by about 170% 5 min a f t e r t h e a d d i t i o n o f LHRH ( F i g . 6) .  The d a t a thus i n d i c a t e t h a t LHRH a c t i o n may be mediated by  its  induction  induced  o f AA  AA r e l e a s e  AA has indeed consecutive  release.  The mechanism  i n granulosa  cells  been made from i n o s i t o l p h o s p h o l i p i d s  apparently  radiolabeled inositol  through two  C f o l l o w e d by  l i p a s e , which has been shown i n p l a t e l e t s ( B e l l  e t a l . , 1979; Dixon and Hokin, 1984). an  LHRH-  i s , however, n o t c l e a r ,  r e a c t i o n s c a t a l y z e d by p h o s p h o l i p i a s e  diacylglycerol  cause  of t h i s  PI,  selective  suggesting  phospholipids  transient  formation  breakdown  o f membrane  LHRH has been found t o  depletion  that  (Minegishi  AA  i n the  may  be  level  derived  and Leung, 1985).  o f DG as a r e s u l t phosphoinositides  of from  Thus the  of agonist  stimulated  represents  a  major  pathway l e a d i n g t o l i b e r a t i o n o f AA f o r PGs and LTs s y n t h e s i s . On  the other  hand,  the release  of i n t r a c e l l u l a r  a l s o be due t o the a c t i v a t i o n o f PLA the  sn-2  position  of  one  or  2  f r e e AA may  which h y d r o l y z e s  several  AA from  phospholipids.  The  2+ activation  o f PLA  2  i s Ca  dependent.  Verapamil,  a  calcium-  channel b l o c k e r prevents both t h e enhancement o f AA r e l e a s e and the d e p l e t i o n i n t h e l e v e l o f r a d i o l a b e l e d PI i n r a t granulosa 2+ cells.  I n a d d i t i o n , t h e omission  o f Ca  from t h e i n c u b a t i o n  medium c o u l d a l s o d i m i n i s h LHRH-induced [ H]AA r e l e a s e i n these  cells  ( M i n e g i s h i and  Leung,  1985).  65 strengthen  These f i n d i n g s  2+ the concept  t h a t Ca  as suggested Forder  et  i s r e q u i r e d a t a s t e p b e f o r e AA  release,  p r e v i o u s l y by o t h e r s t u d i e s ( F o l k e r t e t a l . , 1984; a l . , 1985;  Naor  and  Catt,  1981).  Additionally,  2+ [Ca  ] i mobilization  phosphoinositide  induced  via  PLA  by  IP  could  3  activation.  2  liberate  However,  AA  from  neither  the  r e l a t i o n s h i p between the two  pathways, nor the r e l a t i v e amounts  that  l i b e r a t i o n was  each c o n t r i b u t e d t o AA  i n granulosa  cells.  Recently,  been proposed s u g g e s t i n g  another  t h a t PLA  clearly  p o s s i b l e mechanism  is itself  2  understood  r e g u l a t e d by  has both  2+ Ca  and  DG.  According  suppressed  by  leads  an  to  concomitant  a  increase  production  phosphorylation  and  activity  of  present ionophore  A  that  not  is  2  the  from the  lipocortin,  presence evoked release  was  of  enhanced (10  AA  TPA  mediated  increased  (Touqui  M)  AA by  et  by  by DG  and  [Ca  membrane  by  concomitant  ] i , optimal In  the  treatment  r e l e a s e i n human p l a t e l e t  has  of  related  release i n granulosa calcium  the  calcium  ( F i g . 15), presumably  p o t e n t i a t i o n of the  the  anti-PLA_  a l . , 1986).  induced  the  induces  its 2+  is  agonists  calcium  suppressing  of  activity  2  breakdown of  a c t i v a t i o n of PKC  a c t i v a t e d mechanism of AA  e t a l . , 1985). has  the  c e l l s w i t h TPA  similar  induced  PLA  A23187  granulosa t o a PKC  of  PLA  activation  intracellular  DG,  The  mechanism,  Receptor  in of  i n the  study,  this  lipocortin.  phosphatidylinositide.  activity,  to  cells.  ionophore  been r e p o r t e d  (Volpi  However, t h e nature of t h i s proposed mechanism  been e l u c i d a t e d i n granulosa c e l l s , 2+ Ca p l a y s a major r o l e . The t h r e e  although  i t seems  p o s s i b l e pathways  66 i n v o l v e d i n AA r e l e a s e a r e summarized i n F i g . 16. As action  mentioned on  involves role  earlier,  hormone  phorbol  production  in  ovary  a c t i v a t i o n of the c e l l u l a r  PKC.  i n control of c e l l u l a r  cells,  esters  controls  the  al.,  of the c e l l  1985).  sensitivity  membrane  Specifically,  capable o f s t i m u l a t i n g P I P i n c r e a s e t h e amounts o f I P to receptor  occupation  this  of  the  same  kinase  synthesis of PIP and  their  regulated  may 2  synthesis  function or the PIP  and c o u l d  and t h e formation  exert  a  by PKC a c t i v a t i o n .  DG i s  theoretically  block  synthesis,  2  control  In a d d i t i o n ,  on the  receptors  t o PLC can a l s o be  I t has been  a c t i v a t i o n o f PKC may completely  by  o f DG i n response  negative  t o f u n c t i o n a l l y couple  p r o d u c t i o n by d e c r e a s i n g  2  de C o u r c e l l s e t a l . , 1984).  (Aloy e t a l . , 1983).  ability  i n some  ( T a y l o r e t a l . , 1984; Cooper e t  (De C a f f o y  also  its  phosphoinositide-  In c o n t r a s t t o the s t i m u l a t o r y e f f e c t o f PKC on P I P the  probably  In a d d i t i o n t o  t h e a c t i v a t i o n o f PKC  2  3  and  t h e LHRH  s e c r e t i o n , PKC, a t l e a s t  s i g n a l i n g pathway by r e g u l a t i n g r e c e p t o r content  mimicked  observed  the agonist  t h a t the  induced  IP  3  t h e number o f r e c e p t o r s and r e g u l a t i n g  the c o u p l i n g o f r e c e p t o r s t o PLC through a G p r o t e i n (Cooper e t al.,  1985;  treatment basal of  Lynch  et  o f granulosa  al.,  1985).  cells  with  i n o s i t o l phosphate formation,  granulosa  attenuate  cells  inositol  with  LHRH  phosphate  In TPA  t h e present  (10  M)  study,  stimulated the  b u t t h e combined treatment  and TPA d i d not p o t e n t i a t e o r formation  induced  by LHRH  alone  ( F i g . 13). The s t i m u l a t o r y a c t i o n o f TPA on i n o s i t o l phosphate formation was s p e c i f i c , s i n c e an i n a c t i v e form o f t h e phorbol  67  ——lipase  > A r a c h i d o n i c a c i d <-  Fig. 16. Scheme showing arachidonic acid release.  ester,  4-alpha-phorbol  proposed  12, 13-didecanoate,  formation o f i n o s i t o l phosphate this  action  demonstrated cytosol  i n granulosa c e l l s that  TPA  t o the c e l l  mechanism  causes membrane  ( F i g . 14). was  in  d i d not a l t e r the So f a r , t h e s i t e o f  not c l e a r .  translocation (Kraft  involved  I t has been  o f PKC  from the  e t a l . , 1982).  Rapid  68 the onset o f LH r e l e a s e  r e d i s t r i b u t i o n o f PKC a c t i v i t y d u r i n g in  pituitary  i n response t o LHRH  ( N i r o t a e t a l . , 1985)  suggests t h a t t h e membrane l o c a l i z a t i o n  o f the a c t i v e PKC may  lead  cells  t o the a c t i v a t i o n o f p h o s p h o l i p a s e  Since  PLC  i s t h e enzyme  that  membrane p h o s p h a t i d y l i n o s i t i d e ,  C and A  catalyzes  directly.  2  the h y d r o l y s i s  of  i t might be concluded t h a t PKC  a c t i v a t i o n i s a f f e c t i n g , i n some manner, t h e PLC h y d r o l y s i s o f the PKC  phosphoinositides. activation  may  phosphate t o i n o s i t o l , the  source  quantity  of  However, decrease  i t cannot be r u l e d the  or increase  membrane  hydrolysis  the synthesis  phospholipid  of phosphatidylinositides  i n o s i t o l phosphate formation  of  i s the  out t h a t inositol  o f PIP «  As  2  same  and t h e  i s l i m i t e d , t h e TPA induced  was o v e r r i d d e n  by t h e concomitant  presence o f LHRH ( F i g . 13). Taken together,  these data suggest  that  the a c t i v a t i o n o f PKC p l a y s  IP ,  DG and AA.  3  In  summary,  membrane s p e c i f i c  the i n t e r a c t i o n receptors  membrane p h o s p h o i n o s i t i d e s , and  AA.  The  activation  a role  subsequent  o f LHRH  with  of  i t s plasma  r e s u l t s i n the r a p i d breakdown o f leading changes  and the metabolism  s e c r e t i o n o f granulosa  i n t h e formation  cells.  the p r o d u c t i o n 2+ o f Ca  o f I P , DG 3  mobilization,  PKC  o f AA may c o n t r o l t h e c e l l u l a r In a d d i t i o n , t h e a c t i v a t i o n o f  2+ PKC and Ca IP , 3  mobilization  may  also  control  DG and AA by r e g u l a t i n g r e c e p t o r s ,  synthesis.  t h e formation  PLC a c t i v i t y ,  of  and P I P  2  Chapter  3.  Effect  Concentrations  I.  of  LHRH  on  Cytosolic  Free  69 Ion  Calcium  i n I n d i v i d u a l Granulosa C e l l s  Introduction I t has been documented t h a t LHRH e x e r t s d i r e c t a c t i o n s on  rat 1981;  ovarian  cells  Clark,  mechanism  membrane  1979; Hsueh and Jones,  1982; H i l l e n s j o e t a l . , 1982; Leung, 1985).  of  stimulation  (Hsueh and E r i c k s o n ,  actions of  o f LHRH  on  the ovary  polyphosphoinositide  (Ma and Leung,  1,4,5-trisphosphate  (  phosphatidylinositol  i s due  breakdown  3  )/  a  product  4,5 biphosphate  of  (PIP ), 2  t o the  i n the  1985; Davis e t a l . , 1986).  I p  The  cell  Inositol  hydrolysis  of  has been proposed  2+ t o induce i n t r a c e l l u l a r Ca m o b i l i z a t i o n ( B e r r i d g e , 1984).  In  addition,  be  products  of phosphoinositide  turnover  may  also  2+ i n v o l v e d i n r e g u l a t i n g Ca (Berridge,  cytosolic has  also  from t h e e x t r a c e l l u l a r f l u i d  1987).  Many c e l l u l a r secretion  entry  and  f u n c t i o n s such as c e l l  activation  calcium been  depend  i o n concentrations demonstrated  to  on  movement, d i v i s i o n ,  changes  To  the  (Cheung, 1987). be  an  important  t r a n s d u c t i o n agent i n numerous t i s s u e s and c e l l s Barrett,  in  free  Calcium signal  (Rasmussen and  1984). evaluate  the r o l e  of calcium  as  an  intracellular  second messenger, q u a n t i t a t i v e measurement o f c y t o s o l i c f r e e 2+ . 2+ . Ca concentration ([Ca ] i ) i s r e q u i r e d . The most popular 2+ method f o r measuring [Ca ] i i s t o monitor t h e s h i f t s i n 2+ 2+ wavelength o f f l u o r e s c e n t Ca i n d i c a t o r s when they b i n d Ca .  These  i n d i c a t o r s are  tetracarboxylic acid  c a l c i u m c h e l a t o r EGTA [ e t h y l e n e g l y c o l N,N'-tetraacetic]. cells  using  the  A r e c e n t study  calcium  on  indicator  70 the  d e r i v a t i v e s of  bis(6-aminoethylether)suspensions  dye,  quin-2,  of  granulosa  measured  only  2+ average method  free was  Ca  not  changes designed  (Davis  to  et  a l . , 1986).  examine  responses  of  This  dye  individual 2+  cells due  and  t o an  i t i s u n c e r t a i n whether the 2+ i n c r e a s e d e n t r y o f Ca  i n c r e a s e of  [Ca  a c r o s s the c e l l  ] i was  membrane or  2+ t o Ca  r e l e a s e from an i n t r a c e l l u l a r  site. 2+  The  r e c e n t l y developed  2-acetoxy-methyl properties  ester  f l u o r e s c e n t Ca  (fura-2AM)  more a p p r o p r i a t e  indicator,  possesses  for intracellular  fura-  fluorescent  studies.  This 2+  has i n c r e a s e d the p r e c i s i o n o f the measurement of Ca by r e d u c i n g the e f f e c t s of instrument d r i f t , and more i m p o r t a n t l y 2+ has  permitted  the measurement o f Ca  without  i n t r a c e l l u l a r c o n c e n t r a t i o n of the dye. the  greater  fluorescence  buffering effect. been  The  s l i g h t l y improved  the  intracellular  thus a v o i d i n g a c a l c i u m 2+  s e l e c t i v i t y o f fura-2 (Grynkiewicz  the  Moreover, because of  intensity,  c o n c e n t r a t i o n of the dye can be reduced  determining  f o r Ca  has  also  e t a l . , 1985).  2+ . U s i n g fura-2, the p r e s e n t study examined: (1) the [Ca ] i l e v e l i n i n d i v i d u a l c e l l s i n primary c u l t u r e s of d i s p e r s e d 2+ granulosa  cells;  (2) the a c t i o n s of LHRH on  [Ca  c o n t r i b u t i o n s of i n t r a c e l l u l a r and e x t r a c e l l u l a r 2+ 2+ . Ca i n the LHRH-mduced [Ca ] i changes.  ] i and  (3) the  source(s)  of  71 I I , M a t e r i a l s and  Preparation  Methods  o f animals and g r a n u l o s a  Animals and i n the Chapter  cells  granulosa c e l l s  were p r e p a r e d  as  described  2. 5  Granulosa  cells  (10  diameter uncoated g l a s s incubated  in  incubation  MEM  at  coverslips  containing  37 °C  i n an  were loaded w i t h fura-2AM as d e s c r i b e d Preparation  previously and  were  was  placed  onto  18  i n 6-well c u l t u r e d i s h  FBS.  After  atmosphere o f (Molecular  5%  2  C0  to  3  Probes Inc.,  and  days  in air,  2  mm  of  cells  Eugene,  OR),  (Grynkiewicz e t a l . , 1985).  obtained  dissolved  were p i p e t t e d  5%  were p l a t e d  l o a d i n g o f the fura-2AM i n d i c a t o r  Fura-2AM amount was  cells/ml)  into in  a  in  i n 1 ml 20  small  1  mg  quantities.  The  total  of c h l o r o f o r m and  50 jal a l i q u o t s  plastic  ampules.  These  vacuum-dried  for  dessicator,  and  ampoules 3h.  The  d r i e d a l i q u o t s were s t o r e d at -70°C. For  each  Balanced pipetted  Salt  culture Solution  into a plastic  as  follows:  mM  HEPES, 2 6 mM  CaC1.2H 0.  117  used,  mM  1  (EBSS) tube.  NaCl,  1 mM  NaHC0 , 5mM 3  KC1,  s o l u t i o n (ImM).  at  5%  stabilize  in the  a  pH  C0 at  2  aliquots  fura-2AM  of  Earl's  loading  were  components of the EBSS were 2  0.8  4  2  mM  mM  glucose,  MgCl .6H 0 and 2  1.8  2  10 mM  d i s s o l v e d i n 50 jal of DMSO, t o EBSS medium was  environment 7.4.  ml  NaH P0 .H 0, 5.6  produce a s t o c k 37 °C  3  for  The  Fura-2AM (50 ;ag) was  2  to  Fura-2AM  for was  at  pre-incubated  least  added  to  15  min  EBSS  to  while  vigorously  agitating  concentration EBSS (1 ml)  of  the  medium  10 juM fura-2AM  culture  was  hydrophobic without with  therefore  difficulty.  The  fura-2AM  for  1  immediately  i n the  penetrates  to  allow  Once i n s i d e the c e l l ,  acetoxymethyl  fura-2  which  cells.  At  from  i s impermeable  the  were r i n s e d  groups  end  by  o f the  placing  producing  o f EBSS) .  Fura-2AM  membrane  then  incubated  uptake  to  indicator  to  reach  release  t h e r e f o r e trapped  fura-2  an  of  f u r t h e r h a l f - h o u r t o wash out excess  f r e s h EBSS  free  inside  "loading" incubation,  in a dish  is  c y t o s o l i c esterases cleave  the  and  granulosa  plasma  were  the  72 a  added i n t o a 6  well. the  cultured cells  hour  mixer,  1 ml o f EBSS, and the  face-up  and  equilibrium. the  placed  a  (10 jul fura-2AM/ml  c o n t a i n i n g fura-2AM was  w e l l c u l t u r e d i s h w i t h another cell  on  the  cells  (2 ml)  for a  fura-2AM.  F l u o r e s c e n c e Measurement Individual  coverslips  laminar flow-through was  used  to  were  mounted  chamber (volume 350  complete  a  water-tight  face-down .  Silicone  grease chamber  i n s e r t e d i n t o a s t a i n l e s s s t e e l h o l d e r and the e n t i r e  assembly  mounted onto the stage of a Z e i s s Jenalumar microscope  equipped  mercury a r c lamp powered by first  passed  filters  (350,  through 365  one  or 380  a DC  of nm,  The  and  a  the  with epifluorescence detector.  seal  onto  l i g h t souce was  power supply.  three  a 200  The  light  Watt was  differential  interference  bandwidths = 10 nm)  mounted i n a  t u r r e t which c o u l d be r o t a t e d by a c o m p u t e r - c o n t r o l l e d s t e p p i n g motor.  The  light  was  then  passed  through  a  410  nm  dichroic  mirror  and  a  lOOx  apochromatic  numerical a p e r t u r e o f 1.4 the l i g h t i n t e n s i t y . to  the  dichroic  illumination  to  lens  with  and an a d j u s t a b l e diaphragm t o  A field  mirror the  o i l immersion  size  reduce  diaphragm i n the l i g h t p a t h  was  used  of  a  to  reduce  single  the  granulosa  73 a  prior  area  cell.  of All  f l u o r e s c e n t l i g h t passed back through the d i c h r o i c m i r r o r and a 450  nm band pass f i l t e r t o reduce background f l u o r e s c e n c e .  emitted  fluorescence taken  at  350  nm  (indicator  The  fluorescence  2+ increased  maximally  with  Ca  binding)  and  380  nm  (decreased  2+ w i t h Ca a  b i n d i n g ) was  camera  port.  d e f l e c t e d e i t h e r t o the e y e p i e c e s or t o  Onto  the  p h o t o m u l t i p l i e r tube which was i n t o DC v o l t a g e . form  by  an  background  of  and  on  measurements were made a t EBSS mM 1.8  (0.8 mM  4  NaHC0 , 1 mM mM  CaCL .2H 0) 2  throughout  the  2  2  2  4  2  or  were 5  sec  mM 5.6  K S0 , 2  mM  4  117  a  digital computer.  corrected time  mM  glucose,  base. low  for The  chloride  I s e t h i o n a t e , 26 10 mM  rate of  HEPES 4  and  ml/min  experiment.  found  t o be  fura-2 uniformly d i s t r i b u t e d  the c y t o s o l and n u c l e u s o f granulosa c e l l s . 2+ of  mounted  the  with  constantly flowing at a  C e l l u l a r l o c a t i o n o f entrapped Fura-2 was  1.8  in  room temperature  NaH P0 .H 0,  3  Converter ratios  a  MgS0 .2H 0, 2.7  was  then converted t o  Digital  fluorescence  obtained  port  used t o c o n v e r t the f l u o r e s c e n c e  T h i s v o l t a g e was  Analogue-II  Measurements  camera  t h e fura-2 t o changes i n c y t o s o l i c  [Ca  The  ] i was  throughout  responsiveness  confirmed  by  d i r e c t i n j e c t i o n i n t o t h e laminar flow chamber of 50 ;ul o f 5 pM.  Br-A23187,  a  non-fluorescent  calcium  Development C o r p o r a t i o n , Toronto,  ionophore  Canada).  74 (HSC Reseach  The c e l l s c o u l d be  used f o r up t o 4 t o 5h a f t e r l o a d i n g w i t h o n l y minimal s i g n s o f leakage  o f fura-2.  Calculation of c y t o s o l i c calcium concentration The  c y t o s o l i c calcium  the f o l l o w i n g formula [Ca  2 +  c o n c e n t r a t i o n was c a l c u l a t e d  (Grynkiewicz  ] i = kd x fl x  using  e t a l . , 1985)  R - P W ^ - R  Where: kd = t h e e q u i l i b r i u m d i s s o c i a t i o n constant f o r t h e a s s o c i a t i o n of f u r a - 2 with c y t o s o l i c free 224  calcium:  JJM.  B = r a t i o of the values: the fluorescence i n t e n s i t y at 380nm w i t h zero  2+  2 +  [Ca ]/380nm w i t h i n f i n i t e  [Ca ].  R = e x p e r i m e n t a l l y determined r a t i o o f t h e f l u o r e s c e n c e i n t e n s i t y a t 350nm/380nm R^^  r a t i o of the values: the fluorescence of i n t e n s i t y 2+  a t 350nm/380nm w i t h zero R  m a x  [Ca ] .  = r a t i o of the values: the fluorescence of i n t e n s i t y a t 350nm/380nm w i t h i n f i n i t e  For t h e present study,  B =10.07;  2 +  [Ca ]. = 0.51; R  m a x  =4.83  were determined u s i n g t h e same g r a n u l o s a c e l l c u l t u r e s . 2+ l e a s t 50 nM [Ca ] i change was c o n s i d e r e d s i g n i f i c a n t .  At  C a l i b r a t i o n o f t h e system Each o f t h e two c u l t u r e s was f i r s t p l a c e d i n a measuring chamber  with  standard  EBSS  (1.8 mM  2+ Ca , 0.8  mM  Mg  2+  ) and  [Ca  ] i was determined i n 20 t o 40 c e l l s i n each c u l t u r e .  This  was done t o assess t h e h e a l t h o f t h e c u l t u r e and t o p r o v i d e a control  f o r further  values,  calibration.  After  measuring  control  one c u l t u r e was r i n s e d w i t h EBSS c o n t a i n i n g 5 mM EGTA 2+  and  no Ca  .  I t was then  p l a c e d onto  a chamber which was  2+ filled  w i t h Ca  - f r e e EBSS c o n t a i n i n g  5 mM EGTA and Br-A23187  2+ (10 uM).  [Ca ] i measurements were taken immediately and a f t e r  15 min.  After  t h e second  control  values, the c e l l s  chamber values  with were  culture  to a 2+ standard EBSS c o n t a i n i n g Br-A23187. [Ca ]i measured immediately (within seconds), as 2+  e x t r a c e l l u l a r Ca  were  c o v e r s l i p was measured f o r  immediately  quickly entered the c e l l .  transferred  B, R ^ Jn  n  and R  m a x  c o n s t a n t s were then c a l c u l a t e d from t h e averages o f t h e v a l u e s o b t a i n e d from these experiments. Reagents LHRH was purchased Ac-D-Nal  1  from Sigma. A potent LHRH a n t a g o n i s t , 2  3  10  ( 2 ) , 4 Cl-D-Phe , D-Trp , D-Ala -LHRH, was obtained  as a g i f t (oLH-25),  from Dr. M.V. Nekola o f Tulane U n i v e r s i t y . ovine  FSH  (oFSH-16)  and pregnant  Ovine LH  mare's  serum  gonadotropin were o b t a i n e d from t h e NIDDK and N a t i o n a l Hormone and  Pituitary  Medicine).  Program  (University  o f Maryland  School  Other chemicals were o b t a i n e d from Sigma.  of  76 Ill.  Results  Rapid and t r a n s i e n t e f f e c t s o f Br-A23187 on c y t o s o l i c c a l c i u m After challenged calcium  loading  with  by i n j e c t i o n  ionophore  was  a rapid  into  granulosa  t h e flow  Br-A23187.  example o f t h e e f f e c t There  fura-2,  cells  were  through-chamber o f the  F i g . 17 shows  a representative  o f a 50 ;ul o f i n j e c t i o n  and t r a n s i e n t i n c r e a s e  o f Br-A23187.  2+ i n [Ca ] i 18 sec  a f t e r t h e i n j e c t i o n o f Br-A23187 w i t h a peak v a l u e o f c y t o s o l i c 2+ [Ca  ] i approximately  time  delay  was  volume t o flow  8-10 f o l d above t h e r e s t i n g l e v e l .  due t o t h e time t o t h e observed  required  cell  This  f o r the injection  and t h e r e l a t i v e l y  slow  i n c o r p o r a t i o n o f t h e ionophore i n t o t h e membrane o f t h e c e l l s . LHRH-induced t r a n s i e n t i n c r e a s e i n c y t o s o l i c calcium Each  o f t h e 115 r a t granulosa  preparations  were  treated  with  cells  LHRH.  from  27  different  The average  resting  2+ l e v e l o f [Ca ] i o f these c e l l s was 96.7 ± 2.9 nM. c e l l s of the t o t a l  115 responded t o LHRH.  Table  Eighty-six  I illustrates  2+ t h a t t h e hormone c o n c e n t r a t i o n r e q u i r e d t o i n c r e a s e [Ca ] i was in  t h e range  _9  of 10 M  5  t o 10~ M.  LHRH  _5  at 10 M  increased  2+ [Ca  ] i i n a l l of the c e l l s  hormone.  The  cells  which  which  were  sensitive  d i d n o t respond  to  to  LHRH  this were  s e n s i t i v e t o t h e c a l c i u m ionophore A23187 o r A n g i o t e n s i n I I . Fig.  18  shows a r e p r e s e n t a t i v e example o f LHRH-induced 2+ rapid and t r a n s i e n t [Ca ] i a l t e r a t i o n i n a single rat 2+ granulosa c e l l . The determinations o f [Ca ] i were made a t 1.8  77  F i g . 17. Br-A23187 induced r a p i d and t r a n s i e n t i n c r e a s e i n c y t o s o l i c c a l c i u m . C o v e r s l i p s w i t h fura-2 loaded g r a n u l o s a c e l l s were mounted on a s p e c i a l l y designed laminar flow-through chamber a t room temperature. At the time (0 time) i n d i c a t e d by the symbol (A) , 50 jul of A23187 (5x10 M) was i n j e c t e d . The resulting images were measured by fluoroscence microscope microcomputer system a t a 5 sec base (12 r e c o r d i n g per min). S i m i l a r r e s u l t s were obtained from 9 i n d i v i d u a l c e l l s i n 6 separate experiments.  78  Fig. 18. LHRH induced rapid and t r a n s i e n t increase i n c y t o s o l i c calcium. A t t h e time (0 time) i n d i c a t e d by the symbol (A) , 25 j i l o f LHRH was i n j e c t e d . The r e s u l t i n g images were measured a t a 1.8 s e c base (33 r e c o r d i n g p e r min). The o t h e r experimental c o n d i t i o n s were t h e same as those d e s c r i b e d i n t h e legend o f F i g . 17. S i m i l a r r e s u l t s were o b t a i n e d from 21 i n d i v i d u a l c e l l s o f 8 experiments.  sec  intervals.  The  average  latency  of  79 intracellular  the  c a l c i u m response a f t e r t h e i n j e c t i o n o f LHRH was 21 ± 0.09 sec (n=70) and t h e average peak v a l u e induced by d i f f e r e n t doses o f LHRH  i s shown  increase  i n Table  induced  significantly  by  II.  The amplitudes  the d i f f e r e n t  different  from  each  doses  of the  o f LHRH  other.  Within  2 +  [Ca ]i  were not  84 ± 3 sec  2+ (n=64) a f t e r LHRH s t i m u l a t i o n , [Ca  ] i returned t o the r e s t i n g  level. The  blockade  o f LHRH-induced  cytosolic  calcium  a l t e r a t i o n by  LHRH a n t a g o n i s t To determine whether o r not a receptor-mediated  mechanism  2+ was i n v o l v e d i n t h e a c t i o n o f LHRH on [Ca potent  LHRH a n t a g o n i s t was examined  cells,  an i n i t i a l  ] i , the e f f e c t of a  ( F i g . 19) .  I n each o f 10  6  i n j e c t i o n o f 25 j i l o f 10~ M LHRH r e s u l t e d i n 2+ .  a r a p i d and t r a n s i e n t i n c r e a s e of [Ca  ] i . LHRH a n t a g o n i s t (25  —5 ;al o f a 10 M s o l u t i o n ) was then injected into the c e l l chamber. The LHRH a n t a g o n i s t by i t s e l f had no d i r e c t e f f e c t on 2+ the r e s t i n g [Ca ] i . On the o t h e r hand, two subsequent injections o f LHRH, at 2 and 4.5 min f o l l o w i n g the administration  of  t h e LHRH  antagonist,  failed  to  increase  2+ [Ca  ] i . These c e l l s nonetheless  f o l l o w i n g t h e treatments  still  responded t o Br-A23187  with LHRH and LHRH a n t a g o n i s t .  80  T a b l e I . lowest hormone c o n c e n t r a t i o n s r e q u i r e d t o i n i t i a t e a change i n c y t o s o l i c c a l c i u m i n g r a n u l o s a c e l l s .  LHRH  Cell  number  9  3  8  3  10" M. 10~ M _7  21  -6  37  10 M 10 M 5  10~ M  22  No response  29  Total  115  T a b l e I I . Average peak v a l u e o f [Ca doses o f LHRH.  LHRH _7  10 M  2+  fCa. M  ] i induced by d i f f e r e n t  Change  (fold)  4.97 ± 0.69 (n=21)  6  4.54 ± 0.32 (n=37)  5  4.51 ± 0.55 (n=22)  10~ M 10" M  Existence  of  a  subpopulation  of  granulosa  cells:  r c a |1  changes induced by d i f f e r e n t hormones 6  As i l l u s t r a t e d  i n F i g . 20 (panel A ) , i n j e c t i o n o f 10~ M 2+  LHRH caused  a rapid  and t r a n s i e n t  i n c r e a s e i n [Ca  5  ] i , whereas  4  two i n j e c t i o n s o f Ang I I a t 10~ M and 10" M, r e s p e c t i v e l y , d i d 2+ not a f f e c t t h e r e s t i n g l e v e l o f [Ca ] i i n t h e same c e l l . 2+ However, LHRH-induced [Ca ] i a l t e r a t i o n was n o t i n f l u e n c e d by 2+ Ang I I and an i n c r e a s e i n [Ca a f t e r Ang I I .  ] i induced by LHRH was observed  I n c o n t r a c t , t h e d i f f e r e n t r e s u l t was  from d i f f e r e n t i n d i v i d u a l g r a n u l o s a c e l l s . shows  a  r e p r e s e n t a t i v e example 2+  i n c r e a s e i n [Ca  ]i.  However,  o f Ang  observed  F i g . 20 (panel B) II  5  (10~ M)  induced  t h e same c e l l d i d n o t respond  5  t o LHRH (10~ M). 2+ Desensitization  of  fCa  ] i response  induced  bv  continuous  exposure t o LHRH The upper p a n e l o f F i g . 21 shows a r e p r e s e n t a t i v e example of  the c y t o s o l i c  injections  calcium  i n c r e a s e s t i m u l a t e d by  3  separate  6  The i n t e r v a l between the 2+ i n j e c t i o n s was 5 min. The i n c r e a s e i n [Ca ] i induced by these consecutive injections o f LHRH reached similar maximum amplitudes.  o f 25 jul o f 10~ M LHRH.  T h i s p a t t e r n was seen i n each o f 14 c e l l s ,  albeit  2+ the peak [Ca ] i responses o f t h e c e l l s t o t h e same dose of LHRH v a r i e d between 250 t o 600 nM. The lower p a n e l o f F i g . 21 2+ illustrates alterations  another  representative  induced by repeated doses  example  of  [Ca ] i 6  o f 25 )il o f 10~ M LHRH  g i v e n a t i n t e r v a l s o f l e s s than 2 min. A g r a d u a l decrease i n  82  Fig. 19. The blockade o f LHRH-induced c y t o s o l i c calcium a l t e r a t i o n by LHRH a n t a g o n i s t . The experimental c o n d i t i o n s were t h e same as those d e s c r i b e d under t h e legend o f F i g . 17, but with 25 jul i n j e c t i o n s o f LHRH, LHRH a n t a g o n i s t (LHRH anta), or Br-A23187 a t t h e times i n d i c a t e d by (•) . Similar results were obtained from 10 i n d i v i d u a l c e l l s o f 10 experiments.  -2  - 1 0  1 2  3  4  6  6  7  8  9  11  12  Time (min) 600  B 6  Ang II (K)" M)  LHRH (10~*M)  400  1  300  •  CM « 200  100  -1  -0.6  0  0.6  1  1.6  2  2.6  3.6  4.6  Time (min) Fig. •2+ Existence of subpopulations of granulosa c e l l s : [Ca ] i changes induced by LHRH and Ang I I . Upper panel and lower panel show t h e r e p r e s e n t a t i v e examples o f t h e c e l l s responded t o e i t h e r LHRH o r Ang I I , r e s p e c t i v e l y . (A) i n d i c a t e s t h e i n j e c t i o n o f LHRH o r Ang I I . Similar results were obtained from 11 i n d i v i d u a l cells i n 4 different experiments. 2 0  84  -2  4  6  8  10  Time (min) 2+ F i g . 21. Increase i n [Ca ] i induced by separate i n j e c t i o n s o f LHRH t o two i n d i v i d u a l granulosa c e l l s . The upper panel shows the cytosolic calcium i n c r e a s e s t i m u l a t e d by 3 separate i n j e c t i o n s o f LHRH. The. lower p a n e l shows a gradual decrease i n t h e amplitude o f [Ca ] i induced by LHRH a t i n t e r v a l s o f l e s s than 2 min. S i m i l a r r e s u l t s were obtained from 14 i n d i v i d u a l c e l l s i n 5 separate expriments.  the  amplitude o f [Ca  desensitization experiment _7  10 M  A s t r i k i n g example o f  induced by LHRH i s shown i n F i g .  a granulosa c e l l  LHRH  f o r 10  min.  was p e r i f u s e d  In t h i s  continuously  The p e r i f u s i o n  o f LHRH  induced  by a p u l s e  injection  o f LHRH.  2+ i n c r e a s e i n [Ca ] i r e t u r n e d t o t h e r e s t i n g c o n t i n u e d presence o f LHRH. 25  22.  with  caused  a  2+ 2+ i n c r e a s e i n [Ca ] i which was not u n l i k e t h e [Ca ] i  transient change  ] i c o u l d be seen.  5  ; i l o f 10" M  However, t h e  l e v e l despite the  Furthermore, a p u l s e i n j e c t i o n o f  LHRH 5 min a f t e r  the i n i t i a t i o n  o f t h e LHRH  2+ infusion  period  failed  to  increase  [Ca  following the cessation of the infusion of  8 min,  transient  t h e same i n j e c t i o n 2+ increase  i n [Ca  In  contrast,  and a f t e r a wash p e r i o d  o f 10  ] i , albeit  ]i.  M  LHRH r e s u l t e d  to a lesser  in a  amplitude  7  than t h e i n i t i a l e f f e c t o f 10" M LHRH. 2+ E f f e c t o f d i f f e r e n t doses o f LHRH on \Ca. J_i 2+ Fig.  23 shows t h e change o f [Ca  ] i induced by d i f f e r e n t  doses o f LHRH i n a s i n g l e g r a n u l o s a c e l l . with sequential interval any  injections  desensitization. 2+  peak l e v e l s o f [Ca different  8  4  o f LHRH from 10~ M t o 10"" M.  between t h e i n j e c t i o n s  possible  The c e l l was t r e a t e d  was a t l e a s t No  The  10 min t o a v o i d  significant  difference  in  ] i was observed f o l l o w i n g t h e i n j e c t i o n o f  doses o f LHRH i n s i n g l e  cells,  o r when t h e response  o f d i f f e r e n t c e l l s was analyzed t o g e t h e r (Table I I ) .  86  Fig. 22. Desensitization o f [Ca ] i response induced _by c o n t i n u o u s exposure t o LHRH. First ( A ) : LHRH (10~ M) p e r i f u s i o n f o r 10 min; second and t h i r d (A): 25 jal LHRH (10 M) injections. S i m i l a r r e s u l t s were o b t a i n e d from 6 i n d i v i d u a l c e l l s o f 4 experiments. 7  87  600 LHRH <10-«M)  LHRH 00-«M)  LHRH (10~«M>  LHRH LHRH (10-8M) (10-7M) 600  2  4 0 0  ss. 300  •  CM a)  O 200  10  16  20  26  30  36  40  Time (min)  F i g . 23. A l t e r a t i o n s i n [Ca ] i induced by d i f f e r e n t doses of LHRH. The c e l l w a s t r e a t e d w i t h s e q u e n t i a l i n j e c t i o n s o f LHRH from 10~ t o 10~ M and no s i g n i f i c a n t d i f f e r e n c e i n peak l e v e l was observed. S i m i l a r r e s u l t s were o b t a i n e d from 11 i n d i v i d u a l c e l l s i n 4 s e p a r a t e experiments. (A) i n d i c a t e s t h e i n j e c t i o n Of LHRH.  I n f l u e n c e o f Ca  2+  8  2+ f r e e medium on \Ca ] i a l t e r a t i o n  To determine t h e i n f l u e n c e  o f Ca  2+  8  2+ f r e e medium on [Ca ] i 2+  a l t e r a t i o n , g r a n u l o s a c e l l s were p e r i f u s e d following by  the r a p i d —6  LHRH (10  and t r a n s i e n t 2+  M) i n normal Ca  w i t h Ca  f r e e EBSS 2+ i n [Ca ] i induced  increase  EBSS.  F i f t e e n minutes a f t e r t h e  2+ Ca f r e e EBSS p e r i f u s i o n , two s e q u e n t i a l i n j e c t i o n s o f LHRH —6 2+ 2+ (10 M d i s s o l v e d i n Ca f r e e EBSS) were made and [Ca ] i d i d not  increase  cells  i n response  Interestingly,  granulosa  6  responded t o LHRH (10~ M) normally a g a i n a f t e r 7 min o f  washing  with  example  o f 14  normal cells  washing time r e q u i r e d 2+ condition  i n Ca  EBSS.  F i g 24.  tested  i n the s i m i l a r  f r e e EBSS v a r i e d  R o l e o f i n t r a c e l l u l a r Ca The increase  source(s)  a  condition.  i n upper  of  granulosa  non-responsive  from 8 min t o 20 min i n the  2+ i n LHRH-induced fCa ] i a l t e r n a t i o n calcium  panel  2+ [Ca ] i induced  cells  were  6  (10~ M)  of  which  contributed  resulted  F i g . 25,  after  to  the  examined.  As  the  initial  —6 by  perifused 2+  A f t e r washing w i t h Ca  The  studied.  2+  of  representative  2+ o f [Ca ] i induced by LHRH was f u r t h e r  illustrated increase  shows  t o e s t a b l i s h a completely  d i f f e r e n t granulosa c e l l s  LHRH  t o LHRH.  10  M  with  LHRH i n normal EBSS, 2+ Ca f r e e EBSS medium.  f r e e EBSS f o r 8 min, the i n j e c t i o n o f in a  rapid  and t r a n s i e n t  increase  of  2+  [Ca  ] i but w i t h a s i g n i f i c a n t l y decreased amplitude (about 35% 2+ of t h e amplitude o f [Ca ] i i n c r e a s e i n normal EBSS medium). 2+ In a d d i t i o n , a n o t a b l e decrease o f b a s a l [Ca ] i was a l s o 2+ observed a f t e r t h e f i r s t i n j e c t i o n o f LHRH i n Ca f r e e EBSS,  89  600  C a 2 * tree medium  600 -  <|  e  LHRH d O " M )  6  LHRH < * r M )  6  LHRH (lO" **) 400  _6  LHRH (10 M)  sf  300  CM 200  100  0 -6  10  16  20  26  30  Time (min)  2+ F i g . 24. D e p l e t i o n o f i n t r a c e l l u l a r Ca_ i n calcium free medium. A f t e r t h e i n i t i a l i n c r e a s e o f [Ca ] i induced Jay LHRH i n normal medium, t h e c e l l s were per i f used w i t h Ca free medium. F i f t e e n minutes a f t e r t h e Ca f r e e medium p e r i f u s i o n , [Ca ] i d i d not i n c r e a s e i n response t o LHRH. The c e l l responded t o LHRH again a f t e r washing w i t h normal medium. S i m i l a r r e s u l t s were obtained from 9 i n d i v i d u a l c e l l s i n 5 experiments. 0 time i n d i c a t e s t h e e n t r y o f c a l c i u m free medium. +  90 600 8  LHRH 10" M) 600  -4  Ga2* free medium  4  6  8  10  12  14  16  18  20  22  24  Time (min) Fig. 25. LHRH accelerated [Ca ] i d e p l e t i o n i n Ca f r e e medium. The f i r s t (•) i n d i c a t e s t h e i n j e c t i o n o f LHRH i n normal medium;_^the second and t h i r d (A) i n d i c a t e t h e i n j e c t i o n s of LHRH i n Ca f r e e medium (upper and lower p a n e l ) . Similar r e s u l t s were o b t a i n e d from 6 i n d i v i d u a l c e l l s i n 4 experiments. 0 time i n d i c a t e s t h e entry o f c a l c i u m f r e e medium.  and  9  2+ [Ca ] i d i d not subsequently  respond  1  t o the i n j e c t i o n of  LHRH a t 13 min. 6  When LHRH (10~ M) was i n j e c t e d a t 13 min i n s t e a d o f 8 min 2+ after  washing  with  Ca  -free  EBSS,  a  transient  increase of  2+ ] i was observed, a l b e i t w i t h a s m a l l e r amplitude than t h a t 2+ induced by LHRH a t 8 min i n Ca f r e e EBSS medium ( F i g . 25). [Ca  2+ Role XCa  of 2 +  extracellular  Ca  in  LHRH-induced  alteration  of  U 2+ Fig.  26  granulosa  cell  followed After  shows  the increases  following  by continuous  entry  of C a  2 +  in  the i n j e c t i o n  washing t h e c e l l free  EBSS  medium,  [Ca  ] i in a  single  o f LHRH a t 0 2+ with  Ca  time,  f r e e EBSS.  LHRH-induced  2 +  [Ca ]i  change was f i r s t decreased and e v e n t u a l l y completely a b o l i s h e d . Subsequently, dissolved  four  separate  injections  i n EBSS medium w i t h  2 mM,  of  LHRH,  which  were  5 mM,  10 mM and 20 mM 2+ c a l c i u m , d i d not r e s u l t i n t h e change o f [Ca ] i . LHRH caused 2+  the i n c r e a s e o f [Ca ] i again i n t h e same c e l l f o l l o w i n g the Comparison o f gonadotropins w i t h LHRH on TCa ] i a l t e r a t i o n p e r i f u s i o n o f normal EBSS. F i g . 27 i l l u s t r a t e s t h a t when a s i n g l e granulosa c e l l was s t i m u l a t e d by two separate 25 pi i n j e c t i o n s o f 50 pg FSH, the [Ca  2 +  ] i was not a l t e r e d .  The i n j e c t i o n  6  25 pi o f LHRH (10~ M) 2+  following Similar  FSH r e s u l t e d results  were  i n t h e expected obtained  with  8  i n c r e a s e i n [Ca ] i . individual  cells.  In  a d d i t i o n , as shown i n F i g . 28, a g r a n u l o s a c e l l which responded  92  800 Ca2* free medium  600 _8  LHRH d O M )  2  ss. 400 CM  2  Ca2* (mM) 6 10 20  A  A  200 h  •  •10  •  A  10  20  A  30  40  60  60  Time (min)  2+ F i g . 26, R o l e o f e x t r a c e l l u l a r Ca i n LHRH-induced a l t e r a t i o n of [Ca ] i . LHRH was 10 M f o r a l l t h e treatments. F i r s t ( A ) : LHRH i n j e c t i o n i n normal medium; second and t h i r d ( A ) : LHRH i n j e c t i o n s i n c a l c i u m f r e e medium; f o u r t h t o seventh (A) : LHRH p l u s d i f f e r e n t c o n c e n t r a t i o n s o f Ca i n j e c t i o n s i n calcium f r e e medium; e i g h t h ( A ) : LHRH i n j e c t i o n i n normal medium. S i m i l a r r e s u l t s were o b t a i n e d from 4 c e l l s o f 4 experiments.  93  Fig. 27. Comparison of FSH with LHRH on [Ca^ji alteration. LHRH r e s u l t e d i n rapid and t r a n s i e n t [Ca ] i a l t e r a t i o n , whereas FSH had no e f f e c t . F i r s t and t h i r d ( ): 25 u l o f i n j e c t i o n o f FSH; second and f o u r t h ( ) : LHRH. Similar r e s u l t s were o b t a i n e d from 8 i n d i v i d u a l c e l l s i n 5 experiments.  94  Fig. 28. Comparison of LH with LHRH on [Ca ] i alteration. The c e l l which responded t o LHRH d i d not respond to LH. S i m i l a r r e s u l t s were o b t a i n e d from 8 i n d i v i d u a l c e l l s i n 4 experiments.  to  LHRH (10  separate  M) d i d n o t respond t o 10 ug o f LH, nor d i d t h e two  i n j e c t i o n s o f LH have any i n f l u e n c e on t h e subsequent 2+  LHRH-induced i n c r e a s e i n [Ca i n 7 other IV.  ] i . I d e n t i c a l r e s u l t s were seen  cells.  Discussion The  been  calcium-sensitive  used  t o study  individual  f l u o r e s c e n t i n d i c a t o r , fura-2 has  the e f f e c t  r a t granulosa  o f LHRH  cells.  LHRH  on  2+ t h e [Ca ] i o f  caused  a  r a p i d and  2+ transient (Fig. [Ca  2 +  increase  17). ]i  Since  to  of c e l l s  completely  t o LHRH  blocked the  ( F i g . 19), i t c o u l d be 2+ ] i a r e mediated by  receptors.  (Table  design  estimated  I) .  This  i s n o t l i k e l y t o have r e s u l t e d from  o f t h e laminar  flow-through  chamber.  t h a t each dose o f LHRH would only be  I t has been d i l u t e d by no  more than a f a c t o r o f 2-5, depending upon t h e flow and  the p o s i t i o n o f the c e l l  chamber. by  tested  However, t h e c o n c e n t r a t i o n s o f LHRH r e q u i r e d t o produce a . -9 -5 ] i response v a r i e d c o n s i d e r a b l y (10 M t o 10 M) from c e l l  cell  the  an LHRH a n t a g o n i s t  t h a t t h e e f f e c t s o f LHRH on [Ca  its specific  [Ca  ] i i n the majority  response o f t h e c e l l s  concluded  2+  i n [Ca  Therefore,  t o the input  o f the  t h e v a r i e d c o n c e n t r a t i o n s o f LHRH r e q u i r e d  t h e i n d i v i d u a l granulosa  threshold of the c e l l s Most c e l l s  relative  r a t e used  cells  may be due t o t h e d i f f e r e n t  (see f o l l o w i n g d i s c u s s i o n ) .  responded t o  LHRH i n t h e range o f 10  M to  5  10~ M, but f o r any s i n g l e c e l l t h e r e was no c l e a r d o s e - r e l a t e d response t o LHRH ( F i g . 22; T a b l e  II).  The lowest  dose o f LHRH  which r e s u l t e d i n an  increase  i n [Ca  ] i appeared  to y i e l d  maximum response s i n c e h i g h e r LHRH c o n c e n t r a t i o n s g i v e n t o  a the  2+ same c e l l Thus,  d i d not  single  granulosa  none" f a s h i o n . and  result  i n additional increases  cells  seem  do  produce  dose-dependent  i n h i b i t o r y e f f e c t on progesterone Knecht  respond  i n an  et  production  a l . , 1985;).  LHRH-stimulated  granulosa Davis  cells  et  are  al.,  alteration  as  a l s o dose-dependent  1987).  well  phosphate  In  luteal  (Davis  et  cells  has  used  were used in  the  rather  present  (Ma  than  the  study.  In  Leung,  and  acid 1985)  in  Leung,  LH-induced  rat 1985;  [Ca  been shown t o be  a l . , 1987).  and  arachidonic  formation  addition,  note, however, t h a t i n a l l these e a r l i e r cells  " a l l or  ( H i l l e n s j o et a l . ,  LHRH-induced  inositol  i n bovine  dependent  ]i.  stimulatory  l i b e r a t i o n from the c e l l membranes ( M i n e g i s h i and and  [Ca  P r e v i o u s s t u d i e s have, however, shown t h a t LHRH  i t s agonists  1982;  to  in  2 +  ]i  dose-  I t i s important  to  studies populations of  individual this  cells  regard,  which  were  i t can  be  s p e c u l a t e d t h a t i n d i v i d u a l granulosa c e l l s respond i n an a l l or none f a s h i o n t o LHRH, but t h a t the t h r e s h o l d c o n c e n t r a t i o n s LHRH r e q u i r e d t o s t i m u l a t e d i f f e r e n t when a mixed p o p u l a t i o n of such c e l l s a  dose  response  more c e l l s quantal  " t u r n on".  (i.e.  mechanism,  relation  be  These data  all-or-none)  since  g i v e n type may  will  i t has  been  cells  may  differ.  Hence  i s s t i m u l a t e d with LHRH,  observed support  response suggested  as p r o g r e s s i v e l y the hypothesis  of  hormonal  that  of a  control  a l l cells  not be equal i n terms o f hormonal  (Moyle e t a l . , 1985).  of  of  a  responsiveness  97 The d i f f e r e n c e s i n c e l l - t o - c e l l r e s p o n s i v e n e s s were found to  be  randomly  minimum  distributed  concentrations  i n this  o f LHRH  study.  required  The  different  for initiating  the  2+ cytosolic different  [Ca  ] i change  functional  may  states  i n part  be  o f t h e LHRH  related  t o the  receptor  i n these  responded  t o LHRH,  cells. While  some i n d i v i d u a l g r a n u l o s a c e l l s  others  responded  to different  20) .  Previous  studies  granulosa  cells  sensitivity al.,  may  have  exist  hormones shown with  such  that  In a d d i t i o n ,  (Fig.  subpopulations  of  respect  t o FSH and v a s o a c t i v e i n t e s t i n a l  1985).  as Ang I I  to  differential  peptide  (Kasson e t  PRL r e c e p t o r s have been shown t o be  more abundant i n a n t r a l g r a n u l o s a c e l l s than i n mural g r a n u l o s a cells  (Dunaif  et  at.,  microspectrofluorimetry investigation  on  1982).  The  techniques  use  of  facilitated  the subpopulations  of granulosa  a l l o w i n g t h e study o f i n d i v i d u a l g r a n u l o s a c e l l s . finding  that different  granulosa  cells  responded  hormones may i n d i c a t e t h a t t h e r e a r e d i f f e r e n t of  granulosa  cells  fura-2  which p l a y d i f f e r e n t  roles  cells  the by  The present to different subpopulations  i n response t o  d i f f e r e n t regulator-mediated ovarian f u n c t i o n s . One with  interesting  the decreasing  o b s e r v a t i o n made i n t h i s time  intervals  between  study was t h a t individual  LHRH  i n j e c t i o n s , t h e magnitude o f t h e LHRH-stimulated i n c r e a s e i n 2+ [Ca ] i d e c l i n e d ( F i g . 2 1 ) . Furthermore, continuous exposure t o a r e l a t i v e l y low c o n c e n t r a t i o n o f LHRH (10 M) r e s u l t e d i n —5 d e s e n s i t i z a t i o n o f g r a n u l o s a c e l l s t o h i g h e r (10 M) doses o f  LHRH  (Fig.  regulation due  to  22).  This  phenomenon of  massive  degradation (on  this  et  al.,  Gregory, 1977). numbers  may  desensitization  may  has  related  like  (10%)  internalization al.,  be  due  1979).  to  of  al.,  subsequent  i.e  insulin,  their  1979;  induce  receptors  Hopkins  stimulation  the  other  rat  granulosa  is  initiated the  of  hCG  exposure, a  can  be  cells,  within  this  present  t h a t a f t e r washing w i t h f r e s h medium, the  As i t  significant lysosome-  inferred  time  study,  this  mechanism  found i n  i t is  by  hand,  receptor-mediated  2 h  and  decrease i n r e c e p t o r  i n t e r n a l i z a t i o n of r e c e p t o r s .  of  in  On  a  labeled  Moreover,  and  be  complex  shown t o  intense  levels.  the  structures  et  against  t o the  of  have been  internalization  LHRH  which may  P e p t i d e hormones,  been shown t h a t o n l y a f t e r  proportion  Nimrod, 1982)  hCG,  98 down-  well-known  LHRH-receptor  p r e s e n t study, the  protect high  the  Amsterdam  In the  inappropriately  t h a t i s not  and  1979;  of  (Hazum and  complex.  aggregation,  the  LHRH s u r f a c e r e c e p t o r s ,  gonadotrophes) , and  motility, (Terris  of  reflect  internalization  into endocytic v e s i c l e s  LHRH  may  that  (Amsterdam  i t was  et  observed  c e l l s regained  their  2+ responsiveness transient  and  may  more  be  to  LHRH  reversible  in  of  [Ca  ]i  (Fig.  22).  nature of t h i s d e s e n s i t i z a t i o n  compatible  desensitization,  terms  possibly  with a t the  alternative l e v e l of  t o note t h a t , whatever the  desensitization,  fluctuations  in  LHRH  process  mechanisms  signal  It i s interesting  The  transduction.  mechanism of  levels  of  may  this  be  more  ] i response i n g r a n u l o s a  cells  2+ effective  in stimulating a  than a s u s t a i n e d e l e v a t i o n  [Ca  i n LHRH c o n c e n t r a t i o n .  Recent the  studies  formation o f I P  1985; for  clearly  i n ovarian  3  Davis e t a l . , 1986).  Phosphoinositide  (Berridge,  turnover 2+ o f Ca  regulation  IP  3  shown t h a t  1987) .  (Ma and Leung,  has been proposed as a mediator  i s also entry  99 stimulates  LHRH  granulosa c e l l s  2+ Ca mobilization  intracellular  the  have  (Nishizuka believed  from  Microinjection  e t a l . , 1984).  t o be i n v o l v e d i n  the external  environment  of  some  IP  3  into  cells  2+ results  in  dependent  the  processes  observations, LHRH  might  hypothesis and  [Ca  ] i mobilization  (Oron  et a l . ,  1985). 2+  i t appears t h a t t h e [Ca be d i r e c t l y  correlated  and  Based  studies.  calciumon  above  ] i changes s t i m u l a t e d by  to IP  3  i s supported by e v i d e n c e obtained  previous  mimics  LHRH a n t a g o n i s t  formation.  This  from t h e p r e s e n t  can b l o c k  LHRH-induced 2+ c e l l u l a r responses i n c l u d i n g b o t h I P formation and [Ca ] i m o b i l i z a t i o n (Ma and Leung, 1985; F i g . 19). S i m i l a r temporal 2+ 3  relationships  between  LHRH-induced  IP  3  formation  and [Ca ] i  m o b i l i z a t i o n has been found by D a v i s e t a l . (1986). Although LHRH r e s u l t e d i n the rapid and t r a n s i e n t 2+ 2+ increase o f [Ca ] i , t h e p r e c i s e source (s) o f Ca which contributed t o the c y t o s o l i c calcium a l t e r a t i o n has t o be resolved. I n t h e p r e s e n t s t u d i e s , t h e LHRH-induced changes i n 2+ [Ca  ] i were completely a b o l i s h e d  by washing w i t h c a l c i u m  medium between 8 min t o 20 min i n 14 c e l l s t e s t e d .  free  Following  2+ re-perifusion  with  induced i n c r e a s e the  medium c o n t a i n i n g  in [Ca  2 +  normal  Ca  , t h e LHRH-  ] i was a g a i n observed ( F i g . 2 4 ) . When  i n j e c t i o n o f LHRH was performed s e v e r a l minutes a f t e r t h e  entry  of C a  2 +  free  medium  into  t h e chamber,  a significant  2+ decrease i n [Ca ] i amplitude was observed  2+ o f [Ca ] i i n t h e normal medium  peak l e v e l decrease Ca  2+  the  1  ( F i g . 25).  can be due t o e i t h e r t h e d e p l e t i o n  0  This  of intracellular  2+ o r t h e l a c k o f Ca i n the e x t r a c e l l u l a r f l u i d . above r e s u l t s , i t can be e s t i m a t e d t h a t  0  as compared t o t h e  Based on  the i n t r a c e l l u l a r  2+ Ca  pool  i s depleted  by p a s s i v e d i f f u s i o n when t h e e x t e r n a l 2+ medium reaches a "Ca free" condition (approx. 8 min) . 2+ However, t h e complete d e p l e t i o n o f [Ca ] i was a gradual 2+ p r o c e s s , and a s m a l l e r i n c r e a s e o f [Ca ] i c o u l d s t i l l be 2+ observed i n e x t r a c e l l u l a r Ca f r e e c o n d i t i o n , suggesting t h a t 2+ . .. LHRH does induce [Ca ] l m o b i l i z a t i o n from an i n t r a c e l l u l a r 2+ p o o l ( F i g . 25). Ca which i s m o b i l i z e d may e a s i l y d i f f u s e into the e x t r a c e l l u l a r s o l u t i o n . Therefore, w i t h time t h e i n t r a c e l l u l a r pool eventually  lose  2+ o f Ca would be exhausted, t h e c e l l  responsiveness  to  LHRH.  The  would  LHRH-induced  2+ d e p l e t i o n o f i n t r a c e l l u l a r Ca  2+ i n Ca f r e e medium ( F i g . 25) 2+ supports t h e concept t h a t LHRH-induced i n c r e a s e o f [Ca ] i i s , at  least  partially,  from  intracellular  stores.  The marked  2+ decline  of the basal  [Ca  ] i a f t e r t h e i n j e c t i o n o f LHRH i n  2+ Ca  f r e e medium f u r t h e r strengthens t h i s n o t i o n Since  agonist-induced  IP  i s believed  3  ( F i g . 25).  t o be  responsible  2+ f o r i n t r a c e l l u l a r Ca r e l e a s e , many s t u d i e s have been made t o elucidate  which  intracellular  pool  i s responsible  f o r the  2+ [Ca  ] i mobilization.  shown  to  probably studies  be ER.  have  a  The s i t e  ATP-dependent  that  IP  3  acts  non-mitochondrial  The experimental indicated  a t which  data  normal  obtained responses  has been 2+  Ca  from to  pool, previous IP  3  are  2+ a t f r e e Ca concentrations  observed  mitochondrial inhibitors  uptake  (Burgess  or  i n the  presence  e t a l . , 1984).  when added d i r e c t l y  1  0  1  below t h e t h r e s h o l d f o r of  Whereas I P  t o microsomes o b t a i n e d  mitochondrial releases  3  from  Ca  2 +  a v a r i e t y of  2+ tissues,  i t fails  fractions  to alter  fractionation  portion  of i n t r a c e l l u l a r  Berthon  e t a l . , 1977),  from  mitochondrial  of rapidly  s t u d i e s have a t t r i b u t e d 2+ Ca p o o l t o mitochondria  recent  2+ l e s s Ca i n mitochondria Shears and K i r k , 1984).  studies  have  i n mitochondria,  LHRH-induced i n c r e a s e of. [Ca ER  in  large  (Claret-  demonstrated f a r  The p r e s e n t  results indicate responsible f o r  ] i . The r e l a t i v e  granulosa  5% o f  whereas 14-23% i s w i t h i n  2+ t h a t t h e i n t r a c e l l u l a r Ca p o o l s a r e probably 2+  and  a  a l s o indicates that only  rough ER (Somlyo e t a l . , 1985).  mitochondria  Although  than i n ER (Reinhart e t a l . , 1984; E l e c t r o n probe X-ray m i c r o a n a l y s i s  frozen l i v e r  2+ Ca i s present  cell  release  (Streb e t a l . , 1983; P r e n t k i e t a l . , 1984).  subcellular  study  Ca  cell  importance o f  cytosolic  calcium  r e g u l a t i o n remains u n c e r t a i n . 2+ p o s s i b l e c o n t r i b u t i o n o f e x t r a c e l l u l a r Ca t o LHRH2+ induced i n c r e a s e o f [Ca ] i was next examined. LHRH d i s s o l v e d 2+ i n h i g h c o n c e n t r a t i o n o f Ca (2 mM t o 20 mM) was g i v e n t o 2+ g r a n u l o s a c e l l s b e i n g p e r i f u s e d w i t h Ca f r e e medium and i n 2+ 2+ which t h e i n t r a c e l l u l a r Ca had been d e p l e t e d . LHRH p l u s Ca 2+ f a i l e d t o evoke t h e i n c r e a s e o f [Ca ] i i n t h e g r a n u l o s a c e l l , 2+ s u g g e s t i n g t h a t LHRH-induced [Ca ] i a l t e r a t i o n may not be due 2+ The  to  t h e immediate  26).  Ca  influx  across  the c e l l  A l t e r n a t i v e l y , these r e s u l t s c o u l d suggest  membrane ( F i g . t h a t even when  the  lf  2+ i n t r a c e l l u l a r Ca pools  cannot  quickly  enter  the  a r e empty, t h e e x t r a c e l l u l a r Ca cytosol.  This  i s despite  &  the  e x i s t e n c e o f a g r a d i e n t p o t e n t i a l and t h e presence o f LHRH t o 2+ ensure t h e opening o f t h e Ca channels on t h e c e l l membrane. 2+ An e a r l y suggestion was t h a t t h e Ca content o f i n t r a c e l l u l a r 2+ p o o l s r e g u l a t e d t h e e n t r y o f Ca from t h e e x t r a c e l l u l a r f l u i d ; 2+  when t h e p o o l s entry,  were empty,  and when the p o o l s  i t was open t o e x t r a c e l l u l a r Ca were  filled,  i t was c l o s e d  t o the  2+ extracellular  Ca  (Aub e t a l . , 1982; Putney,  1986).  Two  2+ phases o f Ca m o b i l i z a t i o n have been demonstrated i n the previous studies. In the f i r s t phase, a release of 2+ i n t r a c e l l u l a r Ca i n response t o a g o n i s t s , and i n t h e second 2+ phase, e n t r y o f Ca a c r o s s t h e plasma membrane f o l l o w i n g the f i r s t phase (Kojima e t a l . , 1985 Reynolds and Dubyak., 1985). 2+ In p i t u i t a r y  cells,  LHRH e l e v a t e s  [Ca  ] i p a r t l y by r e l e a s i n g  2+ Ca  from  intracellular  pools  a c r o s s the c e l l membrane. in  2+ [Ca ] i induced  followed phase  and  influx  I t has been shown t h a t t h e e l e v a t i o n  by LHRH i s composed o f a r a p i d f i r s t 2+  by a prolonged (Clapper  and p a r t l y by t r i g g e r i n g  increase  Conn,  1985;  i n [Ca Limor  phase  ] i i n t h e second et  a l . , 1987).  Furthermore, Naor e t a l . (1988) have r e c e n t l y demonstrated t h a t 2+ LHRH induces a r a p i d m o b i l i z a t i o n o f i n t r a c e l l u l a r Ca pool, 2+ and a second component o f Ca i n f l u x v i a v o l t a g e s e n s i t i v e and 2+ i n s e n s i t i v e changes c o n t r i b u t e s t o f u r t h e r e l e v a t i o n o f [Ca ] i 2+ in pituitary cells. A l t h o u g h t h e i n c r e a s e i n [Ca ] i induced by LHRH i n i n d i v i d u a l g r a n u l o s a c e l l s d i d n o t o b v i o u s l y show 2+ two phases, t h a t e x t r a c e l l u l a r Ca might a l s o be i n v o l v e d i n  Ca  2+  1  2+ [Ca ] i changes,  LHRH-induced  mobilization triggering  with  an  initial  2+ o f Ca , cannot  the i n f l u x  0  3  intracellular be  excluded. In  addition,  after  washing  the  cells  with  medium  2+ c o n t a i n i n g normal Ca , g r a n u l o s a c e l l s r e g a i n e d t h e i r  response  to  positive  LHRH  ( F i g . 24) , which  diffusion refill  2+ o f Ca  from  implied  that  extracellular  i n t r a c e l l u l a r p o o l s without  a  continued  fluid  was necessary t o  a r a p i d change i n c y t o s o l i c  2+ Ca  .  Although  that phosphoinositide 2+ t u r n o v e r may be i n v o l v e d i n t h e r e g u l a t i o n o f Ca e n t r y from extracellular  i t has been  fluid  suggested  (Berridge  1984),  experimental  evidence  2+ indicates direct  that  either  action  Cooper e t a l . ,  IP  3  o r PKC r e g u l a t e s Ca  a t t h e plasma  membrane  1985; G a r r i s o n e t a l . ,  (Streb  1984).  influx  by a  e t a l . , 1984;  I t appears  that a  decrease i n t h e P I P content o f t h e c e l l membrane may i n h i b i t 2+ 2+ the Ca -ATPase and t h e r e f o r e cause an i n c r e a s e i n [Ca ] i (Berridge, 1982). This, however, i s not a sufficient 2  2+ . e x p l a n a t i o n f o r t h e r a p i d changes o f [Ca ] i induced by LHRH. . 2+ The e f f e c t s o f gonadotropins on g r a n u l o s a c e l l [Ca ] i were  also  neither 50  investigated FSH n o r LH,  i n the present  even a t v e r y h i g h doses  ug o f FSH), had any e f f e c t  isolated  bovine  provokes  a  luteal  rapid  study.  cells,  increase  on [ C a  2 +  ]i  Unlike  LHRH,  (10 ug o f LH o r  ( F i g . 27; 28).  i t has been  reported  i n t h e accumulation  of  that IP  3  In LH and  2+ i n c r e a s e i n [Ca forskolin  and  granulosa  cells  ] i (Davis e t a l . , 1987).  cAMP  may  also  cause  (Asem e t a l . , 1987).  Ca  Treatment w i t h LH, efflux  i n avian  In addition,  exogenous  1  cAMP  and f o r s k o l i n have  Leydig  cells  been  shown  t o increase  ( S a l l i v a n and Cooke, 1986).  0  4  2+ [Ca ] i i n  I t i s possible  that  2+ the a c t i o n o f gonadotropins on [Ca cell  specific.  The p r e s e n t  t h a t , i n r a t granulosa  and p r e v i o u s  studies  c e l l s , administration  r a p i d breakdown o f i n o s i t o l whereas  ] i may be both s p e c i e s and  gonadotropins  demonstrate  o f LHRH l e a d s t o a  l i p i d s and an i n c r e a s e  consistently  have  in [Ca  no e f f e c t  2 +  ]i,  on e i t h e r  parameter (Ma and Leung, 1985). 2+ A study o f LHRH e f f e c t on [Ca ] i i n p i t u i t a r y c e l l s has 2+ shown  that  the increase  suspensions  i n [Ca  ] i after  o f gonadotroph-enriched  LHRH was added t o  pituitary  cells  could  be  correlated  t o t h e LH r e l e a s e (Naor e t a l . , 1988). This 2+ suggests t h a t [Ca ] i p l a y s an i n t e r m e d i a r y r o l e when LHRH stimulates LH r e l e a s e from t h e p i t u i t a r y . I n t h e ovary, . . . . 2+ . similar increases i n [Ca ] i may serve t o modulate t h e stimulatory accumulation In  or  inhibitory  effects  o f LHRH  on  P  4  and  PGE  2  (see Chapter 4 ) .  that 2+ LHRH causes a r a p i d and t r a n s i e n t i n c r e a s e i n c y t o s o l i c [Ca ] i in  summary,  the present  i n d i v i d u a l r a t granulosa  study  cells.  strongly  indicates  The a c t i o n o f LHRH on t h e  2+ cytosolic By  [Ca  i n v e s t i g a t i n g i n d i v i d u a l granulosa  demonstrate  that  concentrations or  ] i change i s mediated by i t s s p e c i f i c  none"  granulosa observation  d i f f e r e n t granulosa  receptors.  cells,  i t i s possible to  cells  require d i f f e r e n t  o f LHRH t o i n i t i a t e what appears t o be an " a l l  response cells  and  may  that  also  different exist.  i s t h e down-regulation  subpopulations  Another  of  interesting 2+ o f t h e [Ca ] i response  induced  by  individual  LHRH  which  ovarian  has n o t been  studied  previously  105 in  2+ F i n a l l y , t h e i n t r a c e l l u l a r Ca . 2+ i n v o l v e d i n LHRH-induced [Ca ] i changes,  cells.  sources a r e c l e a r l y  2+ whereas  the r o l e  investigated. a paracrine  o f e x t r a c e l l u l a r Ca  needs  t o be  further  These r e s u l t s i n d i c a t e t h a t LHRH may f u n c t i o n as  o r a u t o c r i n e mediator i n t h e r a t ovary w i t h  f u n c t i o n i n g as a second messenger f o r LHRH.  calcium  106 Chapter 4 . LHRH A c t i o n on O v a r i a n Hormone P r o d u c t i o n ; A l t e r a t i o n s o f Progesterone Accumulation by Calcium  and P r o s t a g l a n d i n s  Ionophore and P r o t e i n  Kinase C A c t i v a t o r  I. Introduction Several activation in  laboratories  o f p r o t e i n kinase  r a t granulosa  cells,  al.,  already  reported  C stimulates basal P  but  s t i m u l a t i o n by gonadotropins al.,  have  inhibits  the P  that  production  4  response  4  to  o r cAMP d e r i v a t i v e s (Shinohara e t  1986; Kawai and C l a r k , 1985; Welsh e t a l . , 1984; Leung e t 1988).  blocked  The s t e r o i d o g e n i c  by a potent  Recently, tissues  inhibitor  PKC  activity  (Noland  and  Veldhuis  and  mitochondria,  o f PKC been  Dimino,  Demers,  a c t i v i t i e s a r e found  has  effect  o f LHRH  (Wang and Leung,  characterized  1986; Davis  1986).  i s partially  The  and  highest  1987).  in  ovarian  Clark,  specific  1983; enzyme  i n t h e c y t o s o l , f o l l o w e d by microsomes and  respectively.  In a d d i t i o n , i t has been  observed 2+  t h a t LHRH and i t s a g o n i s t s r a p i d l y i n c r e a s e c y t o s o l i c f r e e Ca level  i n populations  o f granulosa  (Davis  e t a l . , 1986),  fura-2  fluorescence  calcium  (chapter 2 ) .  antagonizes  i n granulosa  Although  cells,  4  cells  by  the a d d i t i o n of the enhances b a s a l  P  4  ionophore markedly  by gonadotropins  e t a l . , 1988).  i n the i n h i b i t o r y  granulosa  slightly  the calcium  the stimulation of P  cAMP d e r i v a t i v e s (Leung  as measured by quin 2  and i n i n d i v i d u a l  ionophore A23187 by i t s e l f  production  required  cells  Further,  o r CT o r calcium i s  and s t i m u l a t o r y a c t i o n s o f LHRH on  cAMP  and s t e r o i d  ovarian  cells  Eckstein cell,  production  (Ranta  during  long-term  incubation  107 of  e t a l . , 1983; D o r f l i n g e r e t a l . , 1984;  e t a l . , 1986).  Thus,  at the l e v e l  the h y d r o l y s i s o f i n o s i t o l  of the ovarian  l i p i d s may immediately  follow  LHRH r e c e p t o r occupancy and l e a d t o t h e r a p i d g e n e r a t i o n o f I P and  DG.  The r e s u l t a n t changes i n c a l c i u m  3  m o b i l i z a t i o n and/or  PKC a c t i v i t y may w e l l be i n v o l v e d i n t h e modulatory e f f e c t s o f LHRH on o v a r i a n hormone s y n t h e s i s . The  present  study  was  performed  mechanism o f LHRH a c t i o n on P different  4  to  elucidate  and PGs s y n t h e s i s  c u l t u r e periods, the r o l e  of calcium  the  during the  and PKC i n t h e 2+  LHRH a c t i o n , and t h e i n t e r a c t i o n between I P / C a 3  cAMP pathways on o v a r i a n  hormone  production  , DG/PKC and  i n r a t granulosa  cells. I I . M a t e r i a l s and Methods  P r e p a r a t i o n o f animals and g r a n u l o s a Animals  and  granulosa  cells  cells were  prepared  as  those  d e s c r i b e d i n t h e Chapter 2.  Hormone and drug p r e p a r a t i o n Granulosa  cells  were t r e a t e d w i t h  various  hormones and  drugs.  Melittin,  CT, LHRH and FSH were d i s s o l v e d i n s a l i n e .  AA  dissolved  i n ethanol.  was  acetate  12-0-tetradecanoylphorbol-13-  (TPA) was d i s s o l v e d i n d i m e t h y l s u l f o x i d e  drugs were d i l u t e d  to their  r e s p e c t i v e working  (DMSO). A l l concentrations  with  MEM before  incubation  use and added  volume o f 1 ml.  same volume o f e t h a n o l ethanol  i n 5 jal a l i q u o t s  Control  and DMSO.  incubations  The f i n a l  o r DMSO i n the i n c u b a t i o n s  108 total  to a  received the  concentration of  d i d not exceed 0.5%.  At the  end  o f a 5h i n c u b a t i o n p e r i o d , the c u l t u r e medium was c o l l e c t e d  and  s t o r e d a t -20°C u n t i l assay.  by  trypan  blue  exclusion,  Cell viability,  was not a f f e c t e d  as determined  by t h e v a r i o u s  treatments.  Progesterone assay The by  P. c o n c e n t r a t i o n 4  a specific  D.T.  RIA w i t h  i n the c u l t u r e medium was determined an antiserum  kindly  provided  Armstrong o f the U n i v e r s i t y o f Western O n t a r i o  Armstrong, 1978). 5.0%,  by Dr.  (Leung and  The i n t r a - a s s a y c o e f f i c i e n t o f v a r i a t i o n was  and c o e f f i c i e n t o f i n t e r - a s s a y v a r i a t i o n was 5.9% (n=25).  Prostaglandin The  assay  PGE  2  and  P G F  2alpha  c  o  n  c  e  n  t  r  a  -  t  i°  n  s  i  n  t  n  e  culture  medium were determined by RIA w i t h an antiserum k i n d l y by  Dr.  RIA  T.G. Kennedy o f the U n i v e r s i t y o f Western O n t a r i o .  procedure  (Kennedy, were  1979),  assayed  was  similar  except  without  that  coefficient  coefficient  of inter-assay  coefficient  of intra-assay  , .  to  that  aliquots  extraction  intra-assay  PGF,  provided  (Hirst  of variation  described  previously  of the culture e t a l . , 1988). o f PGE  2  medium The  was 6.7% and  v a r i a t i o n was 9.6% (n=20). and i n t e r - a s s a y  The  The  v a r i a t i o n for the  assay were 6.8% and 5.7% (n=5), r e s p e c t i v e l y .  109 Reagents The  following  A23187, m e l i t t i n , pregnant and  drugs  and hormones  TPA, and LHRH.  Ovine FSH  from  School  of  Medicine).  Sigma:  AA,  (NIH-oFSH-16) and  mare's serum gonadotropin were g i f t s  t h e N a t i o n a l Hormone and P i t u i t a r y  Maryland  were  from  the NIDDK  Program ( U n i v e r s i t y o f  Penicillin-streptomycin,  L-  glutamine, n o n e s s e n t i a l amino a c i d s , t r y p a n b l u e were o b t a i n e d 3 from  Gibco.  [1/2- H(N)]Progesterone  (specific  activity  115.0  3  Ci/mmol),  [5,6,8,11,12,14,15,- H(N)]Prostaglandin-F  (specific activity Prostaglandin-E  2  2alpha  3  100-200 Ci/mmol) and [5,6,8,11,14,15- H(N)] (specific  activity  100-200  Ci/mmol)  were  purchased from New England N u c l e a r I n c . Statistical  analysis  Statistical  significance  among groups  was  calculated  by  a n a l y s i s o f v a r i a n c e f o l l o w e d by S c h e f f e ' s m u l t i p l e range t e s t . A l l r e s u l t s were r e p r e s e n t e d as t h e mean ± SE o f d e t e r m i n a t i o n s o f q u a d r u p l i c a t e c e l l c u l t u r e s o f i n d i v i d u a l treatments i n each experiment. observed  In a l l cases, i d e n t i c a l  i n at  least  two  or  more  P<0.05 was c o n s i d e r e d s i g n i f i c a n t .  or similar independent  results  were  experiments.  110 Ill.  Results  Effects  of melittin.  LHRH and TPA  on p r o g e s t e r o n e  and PGE-  production To determine production,  how LHRH and TPA s t i m u l a t e o v a r i a n hormone  especially  PGE  formation,  2  a  phospholipase  s t i m u l a t o r , m e l i t t i n , was added t o t h e medium o f g r a n u l o s a  A  2  cell  c u l t u r e t o i n c r e a s e i n t r a c e l l u l a r f r e e AA. As  shown  7  (3xl0~ M),  i n t h e upper 6  LHRH  accumulation  (10~ M)  2 fold,  panel  and TPA  of  F i g . 29,  7  (10~ M)  4 f o l d and 4.1 f o l d ,  a 5h g r a n u l o s a c e l l c u l t u r e  (P<0.01).  alone  melittin  stimulated  respectively,  Concomitant  P  4  during  treatment o f  g r a n u l o s a c e l l s w i t h m e l i t t i n w i t h LHRH d i d not f u r t h e r i n c r e a s e P  4  production.  To examine  i f endogenous AA  could synergize  w i t h p r o t e i n k i n a s e C, m e l i t t i n was added w i t h TPA t o g r a n u l o s a cell.  Again,  melittin  accumulation o f P As  shown  4  and TPA f a i l e d  to further  enhance t h e  when compared w i t h TPA a l o n e .  i n the  lower  panel  of  F i g . 29,  the  c o n c e n t r a t i o n s i n the c u l t u r e medium was a l s o determined same experiments. compared w i t h increase  in  2  (51.9 pg/ml).  production  and  LHRH caused TPA  also  p r o d u c t i o n 1.9 f o l d when compared w i t h c o n t r o l . concomitant  2  i n the  M e l i t t i n induced a 2.6 f o l d i n c r e a s e i n PGE  control PGE  PGE  a 3.2  increased  2  fold PGE  2  Interestingly,  presence o f m e l i t t i n w i t h LHRH o r w i t h TPA f u r t h e r  enhanced t h e p r o d u c t i o n o f PGE  2  (P<0.01), which was  different  from the e f f e c t s o f m e l i t t i n w i t h LHRH o r TPA on P. p r o d u c t i o n .  Ill  Sh  X Ul  0.4  X  o.a  E 0.2  SI  2 0.1  C  M«l  LHRH  L*M  TR*.  T**4  Fig. 29 I n t e r a c t i o n o f m e l i t t i n (Mel, M;_3xlo" M), w i t h LHRH (L; 10~ M) o r t h e p h o r b o l e s t e r TPA (T; 10~ M) on progesterone (PROG) p r o d u c t i o n (upper panel) and PGE, f o r m a t i o n (lower panel) d u r i n g a 5h c u l t u r e . Concomitant presence o f m e l i t t i n w i t h LHRH o r w i t h TPA f u r t h e r enhanced t h e p r o d u c t i o n o f PGE_, w h i l e m e l i t t i n p l u s LHRH o r TPA had no s y n e r g i s t i c e f f e c t on progesterone p r o d u c t i o n . g  Effects  of  melittin  progesterone To  further  illustrated  2  A23187  the  panel  plus  or A23187 a l o n e  A23187  f o r 5h.  7  (3xl0~ M)  or  and  A23187  were  also increased P  4  o f the  7  A23187  (10" M)  present  alone  respectively.  together,  Interestingly,  production  As  (upper  PGE  2  melittin panel o f  However, when both m e l i t t i n and A23187 were present  t h e same  affected  112 on  mechanisms  o f F i g . 30, treatment  f o r m a t i o n was s t i m u l a t e d by 5.2 f o l d .  in  A23187  c e l l s were t r e a t e d  f o r m a t i o n by 1.9 f o l d and 3 f o l d ,  2  melittin  F i g . 30).  ionophore  intracellular  and m e l i t t i n  melittin  s t i m u l a t e d PGE both  calcium  formation, g r a n u l o s a  i n t h e lower  with  When  investigate  and PGE  4  melittin,  cells  the  and PGE^ p r o d u c t i o n  regulating P with  and  incubations, P  when compared  4  with  production  was  t h e response  not s i g n i f i c a n t l y  to either  treatment  alone.  Interaction  of  t h e nalrHim  ionophore  A23187  and  TPA;  dose  response The kinase  possible  interaction  C pathways was  between  calcium  f u r t h e r examined.  and  The lower  protein panel o f  F i g . 31 i l l u s t r a t e s t h e s y n e r g i s t i c e f f e c t s o f a s i n g l e dose o f TPA  —7 (10 M)  and  -7 t o 10 and  increasing concentrations  -9 o f A23187 (10 M  —7 M).  PGE  2  A t 10  production.  accumulation compared w i t h presence  M, t h e phorbol e s t e r TPA alone s t i m u l a t e d P There was no f u r t h e r enhancement o f P  4  4  when both TPA and A23187 were p r e s e n t t o g e t h e r as the e f f e c t  o f TPA by i t s e l f .  In c o n t r a s t , the  o f TPA s i g n i f i c a n t l y augmented t h e s t i m u l a t i o n o f PGE-  2.75  E  113  6h  2.2  c  m (TRT  1.66  o  >!<! jM:  LU  • ISli iir f  1.1 -  fc  MM  LU  O  i l l I Iff  ffiO.56  ill  r i Mf  a.  II  M i l ii  0 0.12  0.09  H B P  c  m  0.06  CM LU  1  I!  (D  iill  CL 0.03 -  «M Melittin (3x10- ^M)  A23187  A*M  7  (10- M)  F i g . 30. E f f e c t s o f m e l i t t i n and/or A23187 on progesterone and PGE_ p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d . "A+M" denotes c e l l s t r e a t e d w i t h both A23187 and m e l i t t i n . Treatment o f t h e cells with melittin o r A23187 alone stimulated both p r o g e s t e r o n e and PGE,. When both m e l i t t i n and A23187 were p r e s e n t , PGE f o r m a t i o n was f u r t h e r i n c r e a s e d . 2  114  0.8 •  with TPA  0 without TPA  0.6 -  30.41<N UJ O O.  0.2  TPA  (10-1M)  -8 A23187 (log M)  F i g . 31. E f f e c t s o f t h e phorbol e s t e r TPA and/or i n c r e a s i n g c o n c e n t r a t i o n s o f t h e c a l c i u m ionophore A23187 on progesterone and PGE, p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d . A dose dependent i n c r e a s e i n both progesterone and PGE was observed with A23187 treatment. The presence o f TPA markedly p o t e n t i a t e d t h e s t i m u l a t o r y a c t i o n o f A23187 on PGE_ p r o d u c t i o n (lower panel) but not on progesterone (upper p a n e l ) 0  2  115 p r o d u c t i o n by A23187. Fig.  32  increasing PGE  2  panel  F i g . 32)  with  significant  but  in  the  alteration  in P  A23187 treatment  alone.  —10 —8 (10 M t o 10 M)  medium,  P  and  production  4  4  PGE  2  and PGE  2  and TPA were  there  was  l e v e l s when compared w i t h  I n c o n t r a s t , t h e presence  2  production  no  TPA o r  o f A23187  (lower panel o f F i g . 32).  I n t e r a c t i o n o f t h e c a l c i u m ionophore 2  4  increased  When A23187  culture  on P  and  potentiated the stimulatory a c t i o n of the d i f f e r e n t  doses o f TPA on PGE  on_PGE  (10~ M)  i n c r e a s e i n both  treatment.  4  8  A23187  significantly  TPA  together  of  M d i d not a f f e c t P  A dose dependent  observed  markedly  effects  A23187 a t 10  of  accumulation.  present  the  c o n c e n t r a t i o n s o f TPA —8  production.  (upper  was  shows  A23187. TPA and m e l i t t i n  production  Based on the above o b s e r v a t i o n s , t h e i n t e r a c t i o n between melittin,  TPA  examined.  and/or  As expected,  —8 (10  A23187  on  PGE  treatment  2  production  of granulosa  was  further  c e l l s w i t h TPA  —7 M)  or  increase  A23187  o f PGE  i n c r e a s e d PGE formation combined  M)  formation  treatment  resulted  ( F i g . 33) .  by TPA o r A23187. of the c e l l s  stimulation of  t o these  l e v e l s o f PGE-  alone  in a  Melittin  significant a t 3x10  M  formation on i t s own, and f u r t h e r enhanced PGE  induced  synergistic melittin  2  2  (10  cells  PGE  with 2  d i d not  accumulation.  Interestingly,  while the  TPA p l u s A23187  formation, further  2  exerted  the a d d i t i o n of increase  the high  116 5h 4.51  QI  I  I  C  F i g . 32. increasing progesterone progesteron production potentiated PGE, (lower  1 A23187 (10-8M)  I  I  -10  -9 TPA (log M)  L_  -8  E f f e c t s o f t h e c a l c i u m ionophore A23187 and/or concentrations of the phorbol ester TPA on and PGE_ p r o d u c t i o n . A23187 alone d i d not a f f e c t production (upper panel) but increased PGE_ (lower p a n e l ) . The presence o f A23187 markedly t h e dose-dependent s t i m u l a t o r y a c t i o n o f TPA on panel).  117  7  6h  (10-8M)  Melittin (3x10' M)  (lO-^M)  A23187  F i g . 33. E f f e c t s o f the c a l c i u m ionophore A23187 and/or t h e p h o r b o l e s t e r TPA on PGE. p r o d u c t i o n , e i t h e r i n t h e absence (open bars) o r presence (hatched bars) o f m e l i t t i n d u r i n g a 5h culture. The a d d i t i o n o f m e l i t t i n t o t h e c e l l s d i d not f u r t h e r i n c r e a s e t h e PGE, p r o d u c t i o n induced by A23187 p l u s TPA.  Effects  of cholera  toxin  118 and PGE^  and LHRH on p r o g e s t e r o n e  p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d Granulosa ng/ml),  c e l l s were incubated i n t h e presence o f CT (100 _6  LHRH  (10 M)  expected,  CT  induced  formation,  while  elevation (Fig.  in P  4  or a  In  LHRH caused production  plus  LHRH  increase  a relatively  P  smaller  when compared  with  t h e accumulation  i n t h e same  production  fold)  in  4  (3.5 f o l d )  control  levels  l e v e l s induced by CT alone o r by CT p l u s LHRH.  4  4  and 5.5  As  (14  experiments  o f PGE  ( F i g . 34,  A d d i t i o n o f e i t h e r CT o r LHRH s t i m u l a t e d PGE fold  f o r 5h.  There was no s i g n i f i c a n t d i f f e r e n c e  addition to P ,  determined  CT  marked  34, upper p a n e l ) .  between t h e P  with  fold,  was  respectively.  observed  when  both  2  2  was  lower  panel).  formation, by 3.8  An a d d i t i v e e f f e c t CT  also  on PGE  and LHRH were  2  present  during the incubation.  E f f e c t s o f t h e c a l c i u m ionophore A23187 and/or c h o l e r a t o x i n on P_ and PGE 4  A the Fig.  2  p r o d u c t i o n d u r i n g a 5h i n c u b a t i o n p e r i o d  calcium  ionophore,  A23187  (10  M) , was used  a c t i o n o f LHRH on hormone p r o d u c t i o n . 35  (upper  accumulation, was observed  panel),  CT  caused  a  11  whereas o n l y a 94% i n c r e a s e w i t h t h e A23187 treatment.  t o mimic  As i l l u s t r a t e d i n fold  increase  (P<0.05) o f P  of 4  The  4  level  Combined treatment  granulosa c e l l s w i t h CT p l u s A23187 s i g n i f i c a n t l y attenuated accumulation  P  of P  4  compared w i t h t h e e f f e c t o f CT a l o n e .  lower p a n e l o f F i g . 35 shows t h e e f f e c t o f CT and/or  A23187 on PGE_  formation.  I n the same i n c u b a t i o n s ,  treatment  with  either  formation,  CT o r A23187 a l o n e by 6.5  fold  treatment  o f the c e l l s  increase  i n PGE  and 3.1 with  levels,  2  significantly fold,  respectively.  CT p l u s A23187 to  stimulated  10.9  fold  2  Combined  induced  when  119 PGE  a  further  compared  with  c o n t r o l l e v e l s a t t h e end o f 5h c u l t u r e p e r i o d .  Interaction  o f the calcium  p r o g e s t e r o n e and PGE To protein  further  A23187. TPA and FSH on  p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d  investigate  the  interaction  k i n a s e C and cAMP pathways on P  granulosa and  2  ionophore  FSH  4  treatment  of  alone  the  or with  cells  a  17  2  synthesis,  ( 1 0 M ) , TPA  different  caused  and PGE  calcium,  _7  c e l l s were t r e a t e d w i t h A23187 (lOOng)  of  -7  (10 M)  combinations.  fold  increase  FSH of  P. 4  production  ( F i g . 36, upper p a n e l ) .  w i t h A23187 o r TPA r e s u l t e d 36%  Concomitant presence o f FSH  i n t h e decrease  and 20% (P<0.05), r e s p e c t i v e l y .  i n h i b i t o r y a c t i o n s on P  4  PGE  Fig.  2  i n t h e same  production  36) ,  induced  PGE  A23187 2  experiment o r TPA  formation.  Most  synergistic  w i t h t h e combined FSH p l u s  was  could  production,  4  There was no  A23187 and TPA treatment o f t h e c e l l s . of  of P  Moreover, t h e s y n t h e s i s measured  (lower  significantly  interestingly,  panel o f  enhance  FSH-  t h e presence  of  A23187, TPA and FSH t o g e t h e r c o u l d even f u r t h e r s t i m u l a t e PGE accumulation  (P<0.01).  2  120 20  1 gl6  UJ Z  SS UJ  8  0.  0 0.7 0.6  O E  a  6  r  g0.4|UJ 0.3 0 0. 0.2 0.1 0  I  CT (100ng)  LHRH (10-6M)  Fig. 34. E f f e c t s o f c h o l e r a t o x i n (CT) and/or LHRH on progesterone and PGE, p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d . Progesterone and PGE_ p r o d u c t i o n were s t i m u l a t e d by CT o r LHRH and an a d d i t i v e e f f e c t on PGE_ p r o d u c t i o n was observed when both CT and LHRH were p r e s e n t .  121 5h 25 i  1  OOOng)  (10-7M)  A23187  F i g . 35. E f f e c t o f t h e c a l c i u m ionophore A23187 and/or c h o l e r a t o x i n (CT) on progesterone and PGE_ p r o d u c t i o n d u r i n g a 5h incubation period. Combined treatment o f g r a n u l o s a c e l l s with CT p l u s A23187 induced a f u r t h e r i n c r e a s e i n PGE, p r o d u c t i o n compared w i t h each treatment alone. 2  FSH 100ng  6h  122  20  o» 15 c  UJ  o z  DC 10 UJ UJ  o O  QC 0.  0 0.7 T 0.6 0.5 B» 0.4 e  a  0.3  (9  0.2 0.1  C  A23187 TPA A*T (10-7M) (10-7M) F i g . 36. E f f e c t s o f t h e c a l c i u m ionophore A23187 and/or t h e p h o r b o l e s t e r TPA on b a s a l (open bars) o r FSH s t i m u l a t e d (hatched bars) progesterone and PGE_ p r o d u c t i o n d u r i n g a 5h culture period. "A+T" denotes c e l l s t r e a t e d w i t h A23187 and TPA c o n c o m i t a n t l y . A23187 and/or TPA enhanced FSH s t i m u l a t e d PGE, p r o d u c t i o n (lower p a n e l ) , w h i l e s l i g h t l y i n h i b i t i n g FSH induced P p r o d u c t i o n (upper p a n e l ) . 4  Interaction  o f t h e c a l c i u m ionophore  t o x i n on PGE  2  123 A23187. TPA and c h o l e r a  production  The c e l l s were t r e a t e d w i t h A23187 and/or TPA f o r 5h, and w i t h o r without the  absence  Combined  synergistic  A23187  presence  o f CT  o f CT, A23187 o r TPA by i t s e l f  production.  increase  t h e concomitant  treatment  o f A23187  s t i m u l a t i o n o f PGE -  further  2  production. augmented  plus  In t h e presence  PGE  formation,  2  TPA  by  70%  of  resulted  TPA  t o the CT-treated  h i g h e s t i n c r e a s e i n PGE  2  showed  cells  fold  and  alone.  plus  2  o f CT, TPA o r  r e s p e c t i v e l y , when compared w i t h CT treatment A23187  In  s t i m u l a t e d PGE  CT a l o n e induced a 6.4  2  i n PGE  ( F i g . 37) .  25%,  Addition i n the  formation.  I n t e r a c t i o n o f FSH and LHRH on t h e f o r m a t i o n o f progesterone. F  ur  nc  PGE_ and P G a l p h a ^ ^ - F a 24h c u l t u r e p e r i o d 2  2  As  shown  stimulated P of  P  4  i n t h e panel  by  FSH  treatment  s l i g h t l y stimulated P FSH-induced P PGE  2  and  o f F i g . 38,  FSH  p r o d u c t i o n d u r i n g a 24h c u l t u r e .  4  induced  concomitant  A  4  was  6  o f LHRH 4  (10~ M).  50%  The p r o d u c t i o n (P<0.01)  by  LHRH treatment  the alone  p r o d u c t i o n but was much l e s s potent than  production.  d u r i n g a 24h c u l t u r e LHRH c o u l d  decreased  significantly  Panel B shows t h e accumulation o f i n t h e same experiments.  s t i m u l a t e PGE  2  production,  at least  Both 10  FSH fold,  when compared w i t h c o n t r o l ; t h e e f f e c t o f LHRH was as potent as FSH.  Interestingly,  f u r t h e r s t i m u l a t e d PGE  addition 2  o f LHRH t o FSH-treated  formation  cells  (P<0.01), which was c o n t r a r y  t o the e f f e c t o f LHRH on FSH-induced P. p r o d u c t i o n .  Panel C o f  124 o f LHRH and FSH on t h e p r o d u c t i o n o f  F i g . 38. shows t h e e f f e c t s P G F  2alpha*  was  below  (lOOng) PGF  2 a  n a  of PG alpha  i  n  6  LHRH  (10~ M)  c  o  n  t  r  o  1  cultures  2  t h e s e n s i t i v i t y o f t h e assay.  or  ^p  F  The b a s a l l e v e l  caused  i n t h e c u l t u r e medium.  a  Treatment  significant  with  FSH  increase of  Moreover, l i k e t h e i r  effects  on PGE , FSH and LHRH s y n e r g i s t i c a l l y s t i m u l a t e d t h e p r o d u c t i o n 2 J 2  of PGF  2alpha*  I n t e r a c t i o n o f FSH and TPA on progesterone  and PGE  2  formation  d u r i n g a 24h c u l t u r e p e r i o d . As ester  the effects  TPA,  the  o f LHRH can be mimicked  action  p r o d u c t i o n was examined. TPA,  like  LHRH,  production.  of  TPA  on  FSH-induced  was  shown  The i n h i b i t o r y  to  effect  inhibit  culture  and  as shown  FSH-induced  o f TPA was dose  10  24h c e l l  P. 4  PGE_ 2  Treatment o f r a t g r a n u l o s a c e l l s w i t h  and 1 0 ~ M TPA caused a 50% i n h i b i t i o n o f P a  by t h e phorbol  4  P  4  dependent,  production during  i n t h e upper p a n e l  o f F i g . 39.  U n l i k e t h e i n h i b i t o r y e f f e c t o f TPA on FSH-induced progesterone accumulation, PGE  2  TPA r e s u l t e d  p r o d u c t i o n by  FSH  i n a dose dependent  enhancement o f  ( F i g . 39, lower p a n e l ) .  e f f e c t i v e dose o f TPA was a t 1 0  - 1 0  M  (P<0.01).  The  lowest  125  F i g . 37. E f f e c t s o f t h e c a l c i u m ionophore A23187 and/or the phorbol e s t e r TPA on b a s a l (open bars) o r CT s t i m u l a t e d (hatched bars) PGE_ p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d . CT, A23187 and TPA by T . t s e l f s t i m u l a t e d PGE, p r o d u c t i o n . Addition o f A23187 p l u s TPA t o t h e CT t r e a t e d c e l l s r e s u l t e d i n the h i g h e s t i n c r e a s e i n PGE formation. 5  126 24h  86  iiiiii  0 i.e  6  -J-^'-VJ'""'-  -,«5»-yjU"-  •.Vess&jigr,,-,  1.2  vgAS;./-.'  I 0.8 CM • v  -J  0.4  0 0.4  0.3  1 0.2  IJJjIII  0.1  Control  11 FSH (100 n«)  MHI LHRH  do-HO  F i g . 38. I n t e r a c t i o n o f FSH and LHRH on t h e formation of p r o g e s t e r o n e (PROG) (panel, A ) , PGE_ (panel, B) , and PGF alpha (panel, C) d u r i n g a 24h c u l t u r e p e r i o d . While LHRH decreased the p r o d u c t i o n o f progesterone induced by FSH, LHRH had a d d i t i v e e f f e c t s on FSH induced PGE and PGF2 production. 2  1  n  127  n 8 )  TPA (log M)  F i g . 39. I n t e r a c t i o n o f FSH and t h e p h o r b o l e s t e r TPA on progesterone and PGE_ formation d u r i n g a 24h c u l t u r e p e r i o d . Treatment o f r a t g r a n u l o s a c e l l s w i t h TPA, l i k e LHRH, was shown t o i n h i b i t FSH induced progesterone p r o d u c t i o n (upper panel) and enhance PGE p r o d u c t i o n induced by FSH (lower p a n e l ) . 5  Interaction  o f FSH. TPA  p r o g e s t e r o n e and PGE FSH increase (Fig. FSH  treatment in P  of  d u r i n g a 24h c u l t u r e p e r i o d  granulosa  production  4  40, upper p a n e l ) .  resulted  40%,  formation  2  128 Ionophore A23187 on  and t h e c a l c i u m  during  cells  caused  a  24h c e l l  Concomitant  presence  in a significant  decrease  in P  when compared w i t h FSH treatment alone.  were a l s o t r e a t e d w i t h TPA  (10  - 1 1  18.3  culture  fold period  o f A23187  with  p r o d u c t i o n , by  4  Granulosa  -9  M t o 10 M) .  cells  TPA alone a t  -9 10  M  slightly  decreased  presence  of  FSH,  decreases  in P  4  TPA  the basal induced  level  a  of P «  With the  4  concentration  dependent  p r o d u c t i o n b u t t h e a d d i t i o n o f A23187  (10  M)  d i d not f u r t h e r p o t e n t i a t e t h e i n h i b i t o r y e f f e c t o f TPA on FSHinduced P In  production.  4  c o n t r a s t , FSH-stimulated  enhanced by the concomitant to  6.4  fold,  Additionaly, increases  when compared TPA  alone  of basal l e v e l 10  dose was a t 10" M. was  presence with  caused  experiment.  by  the  the 2  Furthermore,  addition  of  p r o d u c t i o n was  2  level  3.6 of  fold PGE . 2  concentration  dependent  The s i g n i f i c a n t  effective  FSH-induced PGE presence —7 10  further  o f A23187, from  the c o n t r o l  o f PGE .  enhanced by t h e concomitant  augmented  PGE  M  2  production  TPA and was  A23187  in  further  the  same  F i g . 40. I n t e r a c t i o n o f FSH, t h e p h o r b o l e s t e r TPA and A23187 on progesterone and PGE, f o r m a t i o n d u r i n g a 24h c u l t u r e period. FSH induced progesterone p r o d u c t i o n was i n h i b i t e d by TPA and/or A23187 (upper p a n e l ) , w h i l e FSH induced PGE p r o d u c t i o n was enhanced by TPA and/or A23187 (lower p a n e l ) . 2  130 IV. D i s c u s s i o n In  addition  to i t s well  p i t u i t a r y gonadotropics, hormonogenesis 1984) . the  known  stimulatory  a c t i o n on  LHRH can e x e r t d i f f e r e n t i a l e f f e c t s on  i n t h e ovary  (Hsueh  and Jones  1981; C l a r k ,  While t h e mechanism o f a c t i o n o f LHRH a t t h e l e v e l o f  ovarian  cell  i s not  i n c r e a s i n g evidence  completely  that i n o s i t o l  understood,  there  is  l i p i d may p l a y a r o l e i n i t s  2+ . effect  on  PKC  and  phosphoinositides occupancy and  [Ca may  ]i.  Therefore,  immediately  and l e a d t o the r a p i d  inositol  phosphates  the h y d r o l y s i s of  follow  generation  (Ma and Leung,  LHRH  receptor  of diacylglycerol  1985; Davis  et a l . ,  1986; Leung e t a l . , 1986). The r e s u l t a n t changes i n PKC . . 2+ a c t i v i t y , and [Ca ] i l e v e l may w e l l be c o r r e l a t e d w i t h t h e modulatory e f f e c t s o f LHRH on P  production.  4  The a c t i v a t i o n o f  PKC  by TPA has been shown t o i n h i b i t  FSH-stimulated  P  and  This  4  20-alpha-OH-P  production.  induced by TPA appears t o be s p e c i f i c , 4-alpha-phorbol-12,13-didecanoate hormone p r o d u c t i o n diacylglycerols,  (Welsh e t a l . ,  inhibitory  as t h e phorbol  i s ineffective 1984).  sn-1,2-dioctanoyl  estrogen, action congener  on  steroid  TPA and two s y n t h e t i c  glycerol  and  l-oleoyl-2-  a c e t o y l - s n - 3 - g l y c e r o l have been shown t o i n h i b i t cAMP dependent granulosa  cell  Structural  similarities  compounds manner LHRH  may  (Hsueh,  may  have  differentiation  similar  contrast t o their  et  al.,  between DG and TPA suggest  stimulate 1979),  (Shinohara  the a c t i v a t i o n  o f PKC  as exogenous  inhibitory effect,  DG  that  these  i n t h e same  and endogenous DG formation action  1985).  induced  o r TPA.  by In  TPA and DG s t i m u l a t e t h e  production of P  and PGs without  4  (Shinohara e t a l . , 1987) .  The  production studies  1985; Kawai and C l a r k , 1985; Wang and Leung,  stimulatory  i n the present  ( F i g . 29,  involvement  31  and  effects study  of  LHRH  on  confirmed  32).  P  those  In a d d i t i o n ,  and  4  of  the  activation  of  PKC  effect  induced  production  4  specificity  have  inhibitory  been  of the phorbol  further  o f t h e PKC i n h i b i t o r  supported  H-7  hen  granulosa  Johnson, 1988). well  i s also  blocked  proposed 4  by t h e  S i m i l a r l y , the  by  H-7  activity  (Tilly  and  Although H-7 may i n h i b i t a d e n y l a t e c y c l a s e as  as PKC, t h e s t i m u l a t o r y e f f e c t  treated  earlier  on LHRH- o r TPA-  (Wang and Leung, 1987).  cell  2  ester f o r  s t i m u l a t o r y e f f e c t o f TPA on t h e plasminogen a c t i v a t o r of  PGE  o f PKC a c t i v a t i o n i n mediating t h e p r o d u c t i o n o f P  as w e l l as t h e proposed  P  131 o f gonadotropins  t h e presence  granulosa  cells  indicate  o f exogenous cAMP on H-7  the i n t a c t  responsiveness of  the c e l l s a t a post-cAMP s t e p (Wang and Leung, 1987). Concerning shown  that  the role  LHRH  could  o f AA induce  i n LHRH a c t i o n , AA  liberation  i t has been  from  p h o s p h o l i p i d s i n [ H] AA p r e l a b e l e d g r a n u l o s a c e l l s and Leung, 1985; Kawai and c l a r k , 1986). PGE  2  increase  could  be  related  to  membrane (Minegishi  Thus, LHRH s t i m u l a t e d the  accumulation  of  i n t r a c e l l u l a r f r e e AA which s e r v e s as t h e s u b s t r a t e f o r PGs and leukotrienes one AA  (LTs) f o r m a t i o n .  o r more o f i t s m e t a b o l i t e s  The e f f e c t  o f AA may be due t o  ( H i r s t e t a l . , 1988).  Although  i s e x t e n s i v e l y p r e s e n t i n t h e body, p r a c t i c a l l y a l l o f i t i s  esterified Therefore  on  phospho  or  neutral  the h y d r o l y s i s o f e s t e r i f i e d  lipid  (Irvine,  AA p r o v i d e s  1982).  the f i r s t  132 r a t e - c o n t r o l l i n g s t e p i n PGs and LTs formation by l i m i t i n g t h e amount  of  substrate  available  to  cyclooxygenase  and  lipoxygenase. Melittin,  a p o l y p e p t i d e from t h e venom o f honey bee which  can a c t i v a t e PLA , has been used t o enhance i n t r a c e l l u l a r 2  arachidonic present  acid  study,  measured  AA  the e f f e c t  after  increase i n P release  interacts  (Haberman,  of m e l i t t i n  5h i n c u b a t i o n ,  1972).  on P  and m e l i t t i n  In the  production  4  induced  a 2  also  be  involved  To f u r t h e r examine with  PKC  pathway  i n the regulation  on  ovarian  and they d i d not f u r t h e r i n c r e a s e P  already LHRH  hormone  production  4  I t appears t h a t t h e e f f e c t  different  be  liberated  mechanisms  to  of  P  4  production, culture,  ( F i g . 29, upper  o f LHRH on P  i n c l u d e s t h e a c t i o n o f endogenous AA.  could  fold  i f t h e endogenous AA r e l e a s e  m e l i t t i n and LHRH were added c o n c o m i t a n t l y t o t h e c e l l  panel) .  was  ( F i g . 29). T h i s r e s u l t suggests t h a t endogenous  4  may  production.  concentrations  free  production  4  AA induced by  from  membrane  phospholipids  form  i t s metabolites.  by two  AA  can be  dependent  process  +2 hydrolyzed involving lipids  from  phospholipase  through  phospholipase Majerus,  phospholipids  two  or  a  Ca  alternatively  consecutive  reactions  of m e l i t t i n  o f progesterone  to a  ( F i g . 29, upper p a n e l ) .  P. remained a t t h e same l e v e l  from  inositol  catalyzed  by  ( L a p e t i n a e t a l . , 1981; B e l l and  I f LHRH s t i m u l a t e d PLA  t h e combined treatment  agent alone  2  C and DG l i p a s e  1980).  accumulation  A ,  by  2  and PKC s e p a r a t e l y ,  and TPA should greater  extent  enhance t h e than  each  However t h e formation o f  i n t h e presence  o f LHRH p l u s TPA  as t h a t caused by TPA o f AA  by  LHRH c o u l d  addition,  alone. be  the p r e v i o u s  o f m e l i t t i n on LH and  distal  enhanced the present  only  2  of PGE  LHRH and  ability  on  the  PKC.  o f the s t i m u l a t o r y  TPA  basal  P  production,  4  ( F i g . 29,  2  of  o f PGE . is  2  u n e s t e r i f i e d AA mimic  the  elicit  the  probably  of  LHRH,  dependent mechanism.  On  that  A23187  These is  TPA  potentiates  results  formation  indicating  indicate  the  that  released  from  AA  o f PGE  membrane  the  to  the  present  PGE  release  2  its  activity when  2  involvement  LHRH-induced  i n v o l v e d i n a two-step process  is  of  other hand, i t has induced  In  due  s u b s t r a t e i s i n c r e a s e d by m e l i t t i n .  effect  also  S i n c e the e f f e c t of  2  PGE  but  synergistically  t o i n c r e a s e i n t r a c e l l u l a r u n e s t e r i f i e d AA,  to  concept  lower p a n e l ) .  were found t o a c t  formation  cyclooxygenase  effects  the  r e s u l t s i n d i c a t e t h a t LHRH c o u l d f u r t h e r r e g u l a t e the of  In  Aboutsamra e t a l . , 1986).  w i t h m e l i t t i n on the accumulation melittin  a c t i v a t i o n of  c o u l d p a r t i c i p a t e i n the a c t i o n of  stimulated  formation  study,  the  ACTH r e l e a s e f u r t h e r support  hormones (Chang e t a l . , 1986; LHRH not  to  observations  t h a t the a c t i v a t i o n o f PLA  133 liberation  T h i s suggests t h a t the  TPA of  the  could a  PKC  been observed (chapter  2) .  a c t i v a t i o n of  PKC  production:  phospholipids  and  first  AA  second  cyclooxygenase i s a c t i v a t e d . In the with  different  production, or  present  minutes)  study,  treatments  which was for  the  the for  granulosa  c e l l s were c u l t u r e d  hours  detect  much longer measurements  to  the  than t h a t taken up of  the  inositol  hormone (seconds phosphate  2+ formation  and the  [Ca  ] i change.  E a r l i e r time p o i n t s have not  been e x p l o r e d the  because the p r o d u c t i o n  sensitivity  apparent  that  of in  RIA.  many  On  the  cells  system c o n t r o l s s u s t a i n e d  in  of P other  which  and  4  PGE  hand, the  134 were below  2  i t has  calcium  c e l l u l a r response t h e r e  become  messenger  i s no  simple  2+ correlation cellular Charest  between  response et  the  (Rink  increase  in  e t a l . , 1982;  a l . , 1983).  In  the  and  the  O'Doberty e t a l . ,  1980;  some systems,  [Ca  this  ]i  phenomenon i s  c a l l e d " h y s t e r e s i s " (Rasmussen, 1983). One e x p l a n a t i o n i s t h a t 2+ 2+ a l a r g e r i n c r e a s e i n [Ca ] i i s r e q u i r e d t o a c t i v a t e Ca dependent p r o c e s s e s , such as PKC a c t i v i t y , than to c o n t r o l sustained  cellular  response.  I t has  been suggested  that  PKC  2+ and  Ca  pathway  may  have  example, i n p a n c r e a t i c initiating  the for  al.,  I t has  the  1984). cultured  increase  islets,  cellular  responsible  Ca  functional  cellular  whereas  PKC  For  is  for  largely  (Rasmussen et 2+ a l s o been observed t h a t a d d i t i o n of Ca to 2+ cells  production.  in  response  Ca  Increase  free i n PRL  medium  be s u s t a i n e d  causes  synthesis  observed f o r s e v e r a l hours, but once s y n t h e s i z e d , i n PRL may  roles.  i s l a r g e l y responsible  response,  maintaining  pituitary  i n PRL  distinct 2+  this  f o r days (Gick and B a n c r o f t ,  a s i m i l a r manner, LHRH induces r a p i d and  an  i s not increase  1985).  In  t r a n s i e n t increase i n  2+ [Ca  ] i which may  then go  on t o c o n t r o l the  sustained  ovarian  attempt t o modify i n t r a c e l l u l a r  l e v e l s of  calcium  hormone p r o d u c t i o n . In an more  directly,  A23187,  a  divalent  cation  ionophore  f a c i l i t a t e s the transmembrane t r a n s p o r t o f c a l c i u m , was the  present  study.  A23187 alone  stimulated  both P  4  which used i n  and  PGE,  production  during  stimulatory probably  a  effect  5h  culture  o f A23187  on  due t o the c o n v e r s i o n  but n o t t h e s y n t h e s i s o f t h i s  period  the production  o f granulosa  enhanced PGE also  intermediate  from c h o l e s t e r o l as  roles  in  These d a t a  both  phospholipase  the  A  and  2  Interestingly,  ( F i g . 30, lower p a n e l ) .  s t i m u l a t e AA r e l e a s e from r a t granulosa  e t a l . , 1987).  was  4  c e l l s w i t h m e l i t t i n p l u s A23187 f u r t h e r  production  2  of P  o f pregnenolone t o progesterone  p r e v i o u s l y proposed (Carnegie and Tsang, 1987). treatment  135 The  ( F i g . 30).  A23187 c o u l d  cells  (Minegishi  imply t h a t c a l c i u m p l a y s modulatory  release the  of  AA  by  conversion  the  of  AA  activity  of  to  by  PGE  2  cyclooxygenase. In  many  systems,  a  dual  signal  transduction  mechanism  +2 exists  that  i n v o l v e s t h e Ca  cooperatively  or s y n e r g i s t i c a l l y  t o a g i v e n hormonal s i g n a l 1987). are  t o g i v e t h e maximal  effects  response  t h a t both c a l c i u m and PKC  i n t h e a c t i o n o f LHRH on gonadotropins  the a n t e r i o r p i t u i t a r y  synergistic  either  ( N i s h i z u k a e t a l . , 1984; B e r r i d g e ,  Huckle and Conn have observed  involved  from  and PKC pathways a c t i n g  (Huckle  and Conn,  on LH r e l e a s e a r e noted  release  1987).  with  the calcium  ionophore A23187 and PKC a c t i v a t o r s such as t h e phorbol TPA,  p o s s i b l y through  al.,  1985).  enhanced  Combination  activation  o f TPA  o f PKC  and A23187  The  ester  (Harris et  causes  larger  amounts o f s u s t a i n e d r e l e a s e o f PRL compared t o each compound alone  (Delbeke  e t a l . , 1984).  The p r e s e n t  study  clearly  demonstrated t h a t TPA f a i l e d t o a c t s y n e r g i s t i c a l l y w i t h A23187 on  P. p r o d u c t i o n ,  whereas TPA and A23187 were s y n e r g i s t i c on  PGE  production  2  enhance  acute  granulosa  ( F i g . 31-33). TPA-induced  cells  inhibitory  136 A23187 has been shown t o  While  production  P G F  of  2alpha  ( V e l d h u i s and Demers, 1987),  effects  of  TPA  and  A23187  n  *  s w  *  n e  a r e p o r t on the  on  LH  induced  P  4  p r o d u c t i o n i n r a t l u t e a l c e l l s has n o t d e s c r i b e d t h i s p o t e n t i a l synergism It  (Baum and Rosberg, 1987). has been shown t h a t  i n parotid  gland,  where PKC and  2+  2+  Ca  synergistically  induce  enzyme  secretion,  r e s p o n s i b l e f o r c o n t r o l o f plasma membrane K et  a l . , 1984).  In  synergistically  with  t h e present  A23187  on  P  s y n e r g i s t i c a l l y w i t h A23187 on PGE Taken  together,  these  degree o f independent  results control  2  study,  cells partially  TPA  formation  suggest  alone i s  channels  production  4  (Putney  d i d not a c t but  d i d act  ( F i g . 31 and 32).  the p o t e n t i a l  o f t h e c e l l responses 2+  u n a f f e c t e d by t h e synergism o f PKC and Ca As proposed  +  Ca  for a  that are  .  p r e v i o u s l y , a d d i t i o n o f EDTA t o t h e granulosa  r e v e r s e d t h e i n h i b i t o r y e f f e c t o f LHRH and TPA  upon FSH-induced  P  4  production, while  the r e v e r s a l  e f f e c t of  EDTA c o u l d be completely a b o l i s h e d by t h e simultaneous  addition  2+ of Ca (Leung and Wang, 1988). These r e s u l t s i n d i c a t e t h a t the a c t i o n o f LHRH on o v a r i a n P s t e r o i d o g e g e s i s i s mediated a t 2+ 4  l e a s t i n p a r t by a Ca The was  not  dependent PKC.  p r e s e n t s t u d i e s a l s o showed t h a t t h e formation o f P coupled  treatments  tightly  of granulosa  to cells  the p r o d u c t i o n with m e l i t t i n  of  PGE «  not P  4  production  t o higher  amounts  The  p l u s A23187, TPA  p l u s m e l i t t i n and TPA p l u s A23187 o n l y i n c r e a s e d PGE but  2  4  2  ( F i g . 31  formation and  32) .  Furthermore,  in  the  f o r m a t i o n of PGE  was  2  perhaps r e f l e c t s  presence  of  both  TPA  and  not f u r t h e r augmented by m e l i t t i n .  the a l r e a d y maximal c o n v e r s i o n  under the i n f l u e n c e of TPA  137 the  A23187,  o f AA  This  to  PGE  2  p l u s A23187 ( F i g . 33). 2+  The also  i n t e r a c t i o n s between Ca  investigated  granulosa  cells  in  with  the  -PKC  present  and  cAMP pathways were  study.  Treatment  LHRH f u r t h e r enhanced C T - s t i m u l a t e d  p r o d u c t i o n but f a i l e d t o a f f e c t the p r o d u c t i o n of P CT  during  a  5h  of  incubation  period  (Fig.  34) .  rat PGE  induced  4  This  2  by  result  supports the p r e v i o u s o b s e r v a t i o n t h a t the mechanism o f LHRH on PGs  production  (Clark,  i s d i s t i n g u i s h a b l e from  1982).  While LH  i n c r e a s e s PG  that  of  gonadotropins  production,  i t does  not  3 increase of LH step  [ H]AA  on  PG  in  involve 1980;  production  the an  r e l e a s e (see Chapter 2 ) .  PG  i n granulosa  synthesis  i n c r e a s e i n PG  CT  i n the  FSH-induced P by FSH  o r CT  present  2  effect  i s presumably a t  hydrolysis  of  activity  AA,  and  a may  (Clark et a l . ,  A23187 and TPA were used t o mimic f o r m a t i o n w i t h the presence of  study.  p r o d u c t i o n but  4  cells  synthetase  Koos and C l a r k , 1982).  the a c t i o n o f LHRH on PGE and  after  T h e r e f o r e , the  I n t e r e s t i n g l y , TPA enhanced PGE  2  suppressed  formation  d u r i n g a 5h c u l t u r e ( F i g . 36 and  37) .  FSH  induced Likewise,  the r o l e o f c a l c i u m i n o v a r i a n hormone p r o d u c t i o n appears t o be complex and may cAMP induced  be a f f e c t e d by the presence of gonadotropins  agents.  o n l y m a r g i n a l l y by 10 by  4.5  Moreover,  fold  in  i n the  the  While b a s a l  P  M A23187, PGE same  presence o f  2  4  formation  p r o d u c t i o n was  incubations FSH  or  was  CT,  (Fig.  35  or  increased stimulated and  the p r o d u c t i o n  36). of  P.  was a t t e n u a t e d PGE  by t h e concomitant  138 o f A23187, whereas  presence  f o r m a t i o n was f u r t h e r augmented ( F i g . 35 and 36).  2  In long term c u l t u r e (24 h t o 48 h ) , LHRH has been r e p o r t e d to  reduce  In  FSH-induced  contrast,  LHRH  formation o f PGE  P  production  4  acted  synergistically  p  2  and G F  concentration  2  38), o n l y  subsequent  the formation  experiments.  o f PGE  Very  2  similar  1981).  FSH  on t h e  ( F i g . 38).  (panel B o f F i g . 38)  was about f o u r times g r e a t e r than t h a t o f Fig.  with  i n t h e p r e s e n t study  2 a l p n a  S i n c e t h e i n c r e a s e i n PGE  (Hsueh and Jones,  P G F  2alpha  (P  a n e  was determined effects  were  ^  c  °^  i n the observed  w i t h t h e PKC a c t i v a t o r TPA on FSH-induced P. and PGE„ formation 4 2 (Fig.  39). A23187 p o t e n t i a t e d FSH-induced PGE  f u r t h e r enhanced PGE 40,  lower  panel),  production  formation  2  whereas  induced  A23187  k i n a s e C and Ca  strengthen  p r o d u c t i o n , and  by TPA p l u s FSH ( F i g .  inhibited  ( F i g . 40, upper p a n e l ) .  term s t u d i e s c l e a r l y 2+  2  The p r e s e n t  FSH-induced  P  4  s h o r t and long  the m u l t i p l e r o l e s  of protein  on hormone p r o d u c t i o n , e i t h e r s t i m u l a t o r y or  i n h i b i t o r y , depending on t h e time o f c u l t u r e and t h e nature o f the hormones. 2+ The occur  interaction  at different  receptors; affect  signals  the a b i l i t y  pathway t o generate agonist. in may  Secondly,  producing extend  levels. produced  cAMP and Ca The f i r s t  -PKC pathways can  i s a t the level  by a c t i v a t i o n  of a receptor  of  o f one pathway may  f u n c t i o n i n g v i a t h e other  an i n t r a c e l l u l a r  signal  i n response  t o an  these i n t r a c e l l u l a r messengers may i n t e r a c t  the c e l l to  between  response.  neighboring  Finally,  cells;  thus  these AA  interactions  produced  after  activation substrate might  of  PLA  PLC  from t h e c e l l  as l o c a l  target c e l l s .  inhibiting  P  PGE  The i n h i b i t o r y P  2  cAMP f o r m a t i o n  metabolites  a r e produced and  effect  o f LHRH and i t s  during  while  a t t h e same  time  a 24h c u l t u r e p e r i o d ( F i g .  o f LHRH, A23187 and TPA on FSH-  may be p a r t i a l y due t o a r e d u c t i o n i n  (Shinohara  Jones and Hsueh,  1981).  cultured  and  theca  formation,  effect  accumulation  4  These  139 i s the  o f a s y n e r g i s t i c e f f e c t between LHRH  accumulation  4  lipase  p r o d u c t i o n have been s t u d i e d e x t e n s i v e l y , t h i s  4  FSH on o v a r i a n  induced  DG  where they  the i n h i b i t o r y  i s t h e f i r s t demonstration  38).  by  hormones t o mediate t h e f u n c t i o n s o f t h e i r  Although  a g o n i s t s on P  with  followed  f o r s y n t h e s i s o f PGs and/or L T s .  be r e l e a s e d  function  or  2  e t a l . , 1985; Knecht e t a l . , 1981; Previous  granulosa  s t u d i e s have shown t h a t i n cells,  FSH-stimulated  PGE  p r o d u c t i o n c o u l d be mimicked by cAMP i n both c e l l types  (Zor e t  al.,  present  1983;  observation  Clark  et  al.,  1978).  However,  the  t h a t LHRH, TPA and A23187 d i d not antagonize t h e  FSH-induced  PGE  production,  2  but  rather  p r o d u c t i o n o f PGs r a i s e s t h e p o s s i b i l i t y a c t i o n o f LHRH on PGE  2  augmented  the  that the stimulatory  p r o d u c t i o n v i a t h e a c t i v a t i o n o f PKC and  2+ the  change o f [Ca  ] iis  a t a post-cAMP  These d a t a a l s o support t h e concept FSH-induced P  4  cAMP g e n e r a t i o n together,  production,  i n part  and degradation  i t can be concluded  step  ( F i g . 38-40).  t h a t TPA and A23187 i n h i b i t a t a step  o r steps  beyond  (Leung e t a l . , 1988).  Taken  t h a t cAMP, c a l c i u m  and p r o t e i n  k i n a s e C pathways a r e a l l i n v o l v e d i n PGs s y n t h e s i s , and each pathway i s d i s t i n g u i s h a b l e from t h e o t h e r s i n t h e way t h a t PG  140 is  produced. Nishizuka  (1984)  has  proposed  that  two  classes  of  2+ receptors  are  intracellular receptors  coupled  with  cascades.  function  physiological  and  I n some t i s s u e s ,  in  cellular  cAMP  similar  Ca  induced  t h e two c l a s s e s o f  direction  response.  -PKC  to  In other  cause  a  tissues,  full  t h e two  types o f r e c e p t o r - l i n k e d cascades  c o u n t e r a c t each other, e.g.,  an  increases  extracellular  signal  that  cAMP  blocks  PKC  2+ a c t i v a t i o n , Ca The  present  m o b i l i z a t i o n and AA r e l e a s e ( N i s h i z u k a , 1984).  study  supports  this  n o t i o n and f u r t h e r  that the i n t r a c e l l u l a r process i n ovarian c e l l s major  second  messenger pathways can e i t h e r  indicates  induced by two  cooperate  with or  antagonize each o t h e r . The FSH  on P  role  physiological and PGE  4  of  PGE  proposed  to  2  increase  maturation  may  o f LHRH and i t s i n t e r a c t i o n  formation i s not y e t c l e a r  (Strickland  prostaglandins  1984) .  2  role  be  plasminogen  and  Beers,  important  and o v u l a t i o n  (Clark  activation  has  A  been  Furthermore,  in follicle  growth,  e t a l . , 1978; Naor  oocyte et a l . ,  On t h e o t h e r hand, i t i s known t h a t t h e maturation o f  could  decrease  gonadotropins (Hillensjo Aberdam, concept on  a t present.  1976).  r a t oocytes c o u l d be b l o c k e d by cAMP LHRH  with  ovary  and  result  and Lemaire,  1985). that  the  (Dekel and Beers,  formation in  the  1980; Dekel  The p r e s e n t  of  maturation  studies further  specific  induced of  by  oocytes  e t a l . , 1983; Dekel and  LHRH does n o t e x e r t an o v e r a l l  but r a t h e r causes  cAMP  1978).  s t r e n g t h e n the  inhibitory  stimulation  action  o f P. and PG  synthesis, the  ovarian  suggesting that  LHRH may e x e r t  hormone p r o d u c t i o n  multiple  and t h e m u l t i p l e  i n t i m a t e l y i n v o l v e d i n t h e a c t i o n s o f LHRH.  141 e f f e c t s on  pathways a r e  5.  Chapter  Role  o f Arachidonic  Acid  142 i n L u t e i n i z i n g Hormone-  R e l e a s i n g Hormone A c t i o n .  I.  Introduction While t h e i n f l u e n c e o f LHRH on o v a r i a n hormone p r o d u c t i o n  has  been w e l l documented, i t s mechanism o f a c t i o n a t t h e p o s t -  receptor years,  level  is still  l a r g e l y unresolved.  LHRH and i t s a g o n i s t s  have been shown t o s t i m u l a t e the  breakdown o f p o l y p h o s p h o i n o s i t i d e s 1,2  i n t o i n o s i t o l phosphates and  d i a c y l g l y c e r o l i n t h e ovary.  usually cause  accompanied  I n t h e p a s t few  Phospholipid  by t h e m o b i l i z a t i o n 2+  t h e a c t i v a t i o n o f Ca  dependent  turnover  was  2+ o f [Ca ] i , and may PLA , 2  leading  t o the  l i b e r a t i o n o f AA. The  above h y p o t h e s i s  notwithstanding,  i t has been proposed  t h a t t h e a c t i o n o f LHRH i n t h e s t i m u l a t i o n o f LH r e l e a s e gonadotrophs  also  phospholipids.  involves  the release  o f AA  from  One o r more o f t h e lipoxygenated  from  membrane  metabolites or  epoxygenated p r o d u c t s o f AA might be a component o f t h e cascade of  reactions  initiated  by LHRH t h a t  ultimately result  i n the  s e c r e t i o n o f LH (Naor and C a t t , 1981; Snyder e t a l . , 1983). ascertain  whether  similar signal  transduction  To  mechanisms a r e  evoked a f t e r t h e b i n d i n g o f LHRH t o i t s o v a r i a n r e c e p t o r s , the effect  o f LHRH on AA r e l e a s e  recently granulosa  examined cells  (Minegishi  prelabeled  i n r a t ovarian and Leung,  with  increase  i n the level  increase  i n AA r e l e a s e was a l s o  cells  1985).  In cultured  [ H]AA, LHRH caused  o f AA r e l e a s e  has been  a rapid  from p h o s p h o l i p i d s .  induced  This  by an a g o n i s t i c LHRH  analog, b u t completely  143 b l o c k e d by t h e concomitant presence o f a  potent LHRH a n t a g o n i s t , suggesting a s p e c i f i c r e c e p t o r mediated mechanism support  (Minegishi  the notion  enhanced  by  production granulosa  only  exerts  cells  with  during  1985).  While  liberation  from  i t s relationship  to  resulted  data  phospholipids i s steroid  i s not understood.  melittin  these  hormone  Treatment  of  i n the e l e v a t i o n of  and PGs (chapter 4 ) , s u g g e s t i n g endogenous AA  4  serves  Leung,  AA  i n t h e ovary  as t h e p r e c u r s o r  i t s effect  designed  that  LHRH,  basal l e v e l of P not  and  f o r PGs s y n t h e s i s but a l s o  on s t e r o i d o g e n e s i s .  The p r e s e n t  study  was  t o determine i f AA c o u l d be a mediator o f LHRH a c t i o n the d i f f e r e n t  various  pathways  of  culture periods. AA  metabolism  The involvement i n the  control  of  of  P  4  p r o d u c t i o n was a l s o i n v e s t i g a t e d .  I I . M a t e r i a l s and Methods  Animals and g r a n u l o s a c e l l s p r e p a r a t i o n The  p r e p a r a t i o n o f animals  and g r a n u l o s a  c e l l s were t h e  same as those d e s c r i b e d i n Chapter 2.  Hormone and drug p r e p a r a t i o n Granulosa drugs.  cells  were  t r e a t e d with  LHRH, FSH, CT and m e l i t t i n  various  were d i s s o l v e d i n s a l i n e .  25-Hydroxycholesterol  (25-OH-cholesterol),  hydroperoxy  prepared  opening  acids  were  t h e ampules  o f AA  hormones and  freshly  AA  and  i n ethanol.  and hydroperoxy  acid,  they  a l l After were  stored  under  nitrogen  nordihydroguaiaretic  a t -70°C.  acid  MEM b e f o r e  incubation  ethanol end  volume o f 1 ml.  of d i f f e r e n t  4  i n 5 jul a l i q u o t s  Control  and DMSO.  incubations  The f i n a l  incubation  culture dishes,  to a  and t h e c e l l s  The c e l l  test  and mixed  tube  suspension  was  assay.  concentration of At the  medium was  For i n t r a c e l l u l a r  was added  were scraped  i n t o the  out with  a rubber  suspension was t r a n s f e r e d i n t o 10 x 75 mm vigorously  centrifuged  was poured  total  received the  the c u l t u r e  0.5 ml o f 100% ethanol  policeman.  Ethanol  periods,  and s t o r e d a t -20°C u n t i l  production,  144 and  concentrations  o r DMSO i n the i n c u b a t i o n s d i d not exceed 0.5%.  collected P  r e s p e c t i v e working  use and added  same volume o f ethanol  indomethacin,  (NDGA) were d i s s o l v e d i n DMSO. A l l  drugs were d i l u t e d t o t h e i r with  TPA,  on  a  a t 3000xg  out i n t o  another  vortex.  Then t h e  f o r 10 min a t 4°C.  test  tube,  dried  under  n i t r o g e n and r e d i s s o l v e d w i t h 200 u l o f MEM f o r RIA.  P r o g e s t i n assay The  P  and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-P)  4  concentrations  i n the culture  specific  with  Armstrong detectable  RIAs  of the University concentration  20alpha-OH-P. 5.0%, assay  antisera  medium kindly  were  provided  o f Western  was  0.08  The i n t r a - a s s a y  determined  Ontario.  ng/ml  coefficient  by  by a  Dr. D.T. The lowest  f o r both  P  4  and  o f v a r i a t i o n was  and c o e f f i c i e n t o f i n t e r - a s s a y v a r i a t i o n was 5.9% f o r P (n=25).  The i n t r a - a s s a y  coefficient  4  of v a r i a t i o n of  20alpha-OH-P was 7.2% and c o e f f i c i e n t o f i n t e r - a s s a y v a r i a t i o n  145 was 10%  (n=6).  Prostaglandin E The  PGE  assay  2  concentrations  2  in  the  culture  medium  were  determined by RIA w i t h an antiserum k i n d l y p r o v i d e d by Dr. T.G. Kennedy  of  procedure 1979),  the  was  university  similar  of  to that  Western  Ontario.  described  The  previously  RIA  (Kennedy,  except t h a t a l i q u o t s o f t h e c u l t u r e medium were assayed  without  extraction  coefficient  (Hirst  of v a r i a t i o n  assay v a r i a t i o n was 9.6%  et was  a l . , 1988). 6.7%  and  The  intra-assay  coefficient  of  inter-  (n=20).  Materials The f o l l o w i n g were purchased from Sigma ( S t . L o u i s , USA): oleic  acid  (C18:l),  acid  (C18:2),  11,14  eicosadenoic acid  homo-gamma-linolenic  acid  e i c o s a t r i e n o i c a c i d ) , gamma-linolenic a c i d acid  (C20:4),  and NDGA.  LHRH,  Ovine FSH  the N a t i o n a l LHRH  TPA,  CT,  linoleic  (C18:3)  (8,11,14  (C18:3), a r a c h i d o n i c  25-OH-cholesterol  indomethacin,  (NIDDK oFSH-16) and PMSG were g i f t s  Hormone and P i t u i t a r y 6  agonist  (C18:2),  Program NIDDKD, NIH.  10  9  [d-Trp ,Des-Gly ,Pro -NHEt]LHRH  was  s u p p l i e d by Dr. N i c h o l a s L i n g o f t h e S a l k I n s t i t u t e CA) .  A l l hydroperoxy  Laboratories (specific  fatty  (Philadelphia, activity  115.0  hydroxypregn-4-ene-3-one 3  a c i d s were purchased 3 PA).  [5,6,8,11,14,15- H(N)]Prostaglandin  kindly  (La J o l l a , from  BIOMOL  3  20alpha-[l,2- H(N)]-  activity E  The  [1,2- H(N)]Progesterone  Ci/mmol),  (specific  from  2  40-60 Ci/mmol) and  (specific  activity  100-  200  Ci/mmol)  Corporation  were  purchased  (Ontario,  from  Canada).  the  New  146 Nuclear  England  Scintillation  fluid  was  o b t a i n e d from the F i s h e r S c i e n t i f i c Company.  S t a t i s t i c a l analysis Statistical analysis test.  significance  o f t h e data  was  determined  of v a r i a n c e and f o l l o w e d by S c h e f f e ' s m u l t i p l e range  In a l l cases, i d e n t i c a l o r s i m i l a r r e s u l t s were  i n a t l e a s t t h r e e o r more independent experiments. were presented  as the mean ± SE o f d e t e r m i n a t i o n s  q u a d r u p l i c a t e c u l t u r e s w i t h i n each treatment o f l e s s than 0.05 was c o n s i d e r e d  III.  by  observed  A l l results o f c e l l s from  group.  A P value  significant.  Results  E f f e c t s o f m e l i t t i n . LHRH and a r a c h i d o n i c a c i d on  progesterone  production To compare the e f f e c t s o f endogenous AA, exogenous AA-and LHRH  on  P  4  production,  granulosa  cells  m e l i t t i n , AA and LHRH f o r 5h ( F i g . 41).  were  treated  with  Treatment o f the c e l l s -7  with  a maximal e f f e c t i v e  dose o f m e l i t t i n  (3x10  M)  caused a  more than 2 f o l d i n c r e a s e i n P. i n the c u l t u r e medium. 4 same experiment, achieved  with  significantly and  4  control  fold, cells.  the degree  the maximal l a r g e r than  of stimulation of P  dose that  respctively,  as  o f exogenous AA induced  4  with  production  and LHRH  by m e l i t t i n ,  compared  I n the  the  5.1  was fold  untreated  147 S e n s i t i v i t y o f p r o g e s t e r o n e response t o a r a c h i d o n i c a c i d The  effects  of  increasing  production  were determined 5h  granulosa  cells  production  was  (Fig.  concentration  a f t e r the  42).  A  observed a t AA  AA  on  P  a d d i t i o n of AA  to  the  of  P  maximal  of  enhancement 5  concentrations  above 10~ M.  4  4  The  -7 minimal e f f e c t i v e dose o f AA was to  enhancing  caused  a  P  accumulation  4  significant  3x10  i n the  increase  M  In a d d i t i o n -5 c u l t u r e medium, 10 M AA  in  (P<0.05).  the  intracellular  P  4  5 concentration  (0.53  ±  control incubations;  0.02  vs.  0.12  ±  0.01  ng/2xl0  f a t t y a c i d s on p r o g e s t e r o n e  production  In t h i s experiment, the s p e c i f i c i t y o f o t h e r acids  as  investigated.  well  as  gamma-linolenic  5h  acid  acid  different  potencies  following  order: 11,14  action  AA>  -5  M.  11,14  (C18:3),  (C18:2) and  incubation period.  linoleio  AA  on  P  4  unsaturated  production  was  A l l f a t t y a c i d s were added t o the c u l t u r e medium  a t the c o n c e n t r a t i o n o f 10  linoleic  in  P<0.01).  E f f e c t of unsaturated  fatty  cells  AA  . . . . e i c o s a d i e n o i c a c i d , homo-  gamma-linolenic  increased  P  4  acid  production  (C18:3), during  a  I n t e r e s t i n g l y , t h e s e f a t t y a c i d s showed in  stimulating  P  4  production  homo-gamma-linolenio  eicosadienoic acid.  in  the  gamma-linolenio  Another unsaturated  a c i d , o l e i c a c i d , f a i l e d t o s t i m u l a t e P. p r o d u c t i o n  fatty  ( F i g . 43).  148  Fig. 41. Stimulatory effects of m e l i t t i n , LHRH and arachidonic acid (AA) on progesterone (PROG) accumulation d u r i n g a 5h c u l t u r e p e r i o d . C, c o n t r o l ; Mel, m e l i t t i n .  149  F i g . 42. E f f e c t of increasing concentration of arachidonic acid (AA) on progesterone p r o d u c t i o n d u r i n g a 5h c u l t u r e period. AA caused a dose dependent i n c r e a s e i n progesterone production.  150  6h M -  X  a> -  UJ  o  en  X  cc  UJ H CO UJ  o -  o  Q.  o c o o  '5 O  o o c ©  *o CO CO  o o  '5  o c ©  o c  o c  T  T •r  o o  o c o •g o co  o £ o  5  Fatty acid (10" M)  F i g . 43. E f f e c t s o f unsaturated f a t t y a c i d s on progesterone production. Arachidonic, homo-gamma-linolenic, gammal i n o l e n i c , l i n o l e i c , and 11, 14 e i c o s a d i e n o i c a c i d s s t i m u l a t e d p r o g e s t e r o n e p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d , whereas o l e i c a c i d f a i l e d t o s t i m u l a t e progesterone p r o d u c t i o n .  151 E f f e c t s o f LHRH and a r a c h i d o n i c a c i d on progesterone p r o d u c t i o n As cells  expected,  enhanced  P  (P<0.01).  (lo" M)  production during  4  t o r a t granulosa  a 5h i n c u b a t i o n (P<0.01)  A d d i t i o n o f AA (10 M) a l s o s i g n i f i c a n t l y enhanced  production  4  6  o f LHRH  -5  ( F i g . 44A) . the  P  addition  t o about  The concomitant  2.9  fold  presence  o f the c o n t r o l o f LHRH and AA  level further  s t i m u l a t e d P. l e v e l s t o about 4.5 f o l d o f t h e c o n t r o l P, v a l u e s (P<0.01).  As i l l u s t r a t e d i n F i g . 44 p a n e l B, a d d i t i o n o f AA t o  an a g o n i s t i c analog o f LHRH f u r t h e r enhanced P t h a t induced by t h e LHRH a g o n i s t a l o n e  Time  course  of  effects  of  LHRH  p r o d u c t i o n over  4  (P<0.01).  and  arachidonic  acid  on  progesterone production As  shown i n F i g . 45, P  above t h e c o n t r o l 6  o f 10~ M LHRH. onset;  incubations  The e f f e c t  markedly  P  4  5  addition,  P  4  compared  p r o d u c t i o n was i n c r e a s e d with  A t 5h, t h e l e v e l o f P  from  that  presence  production  potentiated  as l h a f t e r t h e a d d i t i o n  o f 10~ M AA was somewhat slower i n  fold)  (P<0.05).  the concomitant enhance  AA  (1.9  was n o t d i f f e r e n t  p r o d u c t i o n was s t i m u l a t e d , 82%  (P<0.05), as e a r l y  by 3h a f t e r  significantly  4  induced  in  control  s t i m u l a t e d by AA  4  by LHRH.  Interestingly,  o f AA and LHRH a t l h d i d not f u r t h e r induced  P  that  4  by  production  LHRH at  alone 3h  and  (P<0.05), but 5h  (P<0.01)  compared w i t h t h a t a f t e r treatment w i t h AA o r LHRH a l o n e .  152  C  LHRH AA LHRH (*r tyD(io-8M) AA 8  C  LHRHa AA LHRHa (10-7*1) (10- M) AA 6  Fig. 44. Effects o f treatment o f granulosa cells with a r a c h i d o n i c a c i d (AA) and LHRH o r a LHRH a g o n i s t (LHRHa) on progesterone p r o d u c t i o n . LHRH (panel A) o r LHRHa (panel B) s t i m u l a t e d progesterone p r o d u c t i o n was f u r t h e r enhanced by AA d u r i n g a 5h c u l t u r e p e r i o d .  153  0  1  3  6  T i m e (hour)  Fig. 45. Time course of stimulation o f progesterone p r o d u c t i o n by a r a c h i d o n i c a c i d (AA) , LHRH o r LHRH p l u s AA. Progesterone p r o d u c t i o n was s t i m u l a t e d as e a r l y as l h a f t e r t h e a d d i t i o n o f LHRH, whereas t h e e f f e c t o f AA was s i g n i f i c a n t a t 3h. Progesterone p r o d u c t i o n was p o t e n t i a t e d by t h e presence o f both AA and LHRH a t 3h and 5h compared with t h a t after treatment w i t h AA o r LHRH alone.  Interaction  between a r a c h i d o n i c  acid  and TPA on  154 progesterone  production A d d i t i o n o f t h e phorbol  e s t e r , TPA (10 M) , t o granulosa  c e l l s r e s u l t e d i n a 93% i n c r e a s e i n P compared  with  concomitant enhanced 10  —5  untreated  presence  control  o f TPA  (10 M)  M) on P  . production.  4  of  .  -5 (10 M)  AA  . Likewise, to  ( F i g . 46) as  A d d i t i o n a l l y , the and  AA  significantly  o f AA  ( a t 10~ M o r  6  . i n F i g . 47,  as shown  TPA-treated  potentiated the stimulation of P - 8  cells.  (P<0.01) t h e s t i m u l a t o r y e f f e c t  addition  lO  production  4  cells  the  markedly -9  production  by TPA ( a t 10 ,  To i n v e s t i g a t e t h e p o s s i b l e involvement  o f AA m e t a b o l i t e s  4  _7  ,  o r 10 M) alone.  Role o f a r a c h i d o n i c a c i d metabolism  in  P  production,  4  granulosa  indomethacin and NDGA with As  cells  were  treated  t h e presence o f e i t h e r LHRH o r AA.  shown i n F i g . 48, a d d i t i o n o f t h e AA metabolism -5  alone  .  same  on P dose  production  4  production.  o f NDGA induced  inhibitors  . . .  (10 M) had a s l i g h t b u t s i g n i f i c a n t  effect  with  More  partially  (P<0.05) s t i m u l a t o r y  importantly,  suppressed  6  by 10~ M LHRH  a d d i t i o n o f the  (by about  50%) P  4  ( F i g . 48, upper p a n e l ) ; the  same molar c o n c e n t r a t i o n o f indomethacin was i n e f f e c t i v e .  On  the  not  other  hand,  indomethacin, level  as t h a t  panel).  t h e concomitant  inhibited caused  presence  AA-induced  by NDGA alone  P  4  o f NDGA,  production (P<0.01)  but  t o t h e same  ( F i g . 48, lower  155  F i g . 46. E f f e c t s o f the phorbol e s t e r TPA and i n c r e a s i n g concentrations of arachidonic a c i d (AA) on progesterone production. The presence of TPA enhanced the s t i m u l a t o r y e f f e c t o f AA on progesterone p r o d u c t i o n d u r i n g a 5h c u l t u r e period.  156  F i g . 47. E f f e c t s of a r a c h i d o n i c a c i d (AA) and increasing concentrations of the phorbol ester TPA on progesterone production. The addition of AA to TPA treated cells p o t e n t i a t e d the s t i m u l a t i o n of progesterone p r o d u c t i o n by TPA alone d u r i n g a 5h c u l t u r e p e r i o d .  157  Control  NDGA  INOO  5  («T M)  («r M)  5  Fig. 48. Role of arachidonic acid (AA) metabolism. Addition of nordihydroguaiaretic acid (NDGA) partially suppressed progesterone p r o d u c t i o n induced by LHRH (upper panel) and i n h i b i t e d AA induced progesterone p r o d u c t i o n t o the same l e v e l as t h a t caused by NDGA alone (lower p a n e l ) , whereas indomethacin (INDO) was i n e f f e c t i v e .  The P  4  e f f e c t s o f NDGA o r indomethacin  induced  by LHRH p l u s AA were f u r t h e r examined (Fig. 4 9 ) .  experiment marked  increase  presence  158 on t h e p r o d u c t i o n o f  in P  o f LHRH  suppressed  the P  Dose response  While indomethacin  4  production  4  _6  (10 M) , response  due 5  AA  (10" M)  i n another  f a i l e d t o a f f e c t the to  the  and  concomitant  NDGA  5  (10" M)  d r a m a t i c a l l y (P<0.01).  o f HETEs and HPETEs on progesterone  production  S i n c e t h e lipoxygenase m e t a b o l i t e s o f AA may be i n v o l v e d i n t h e a c t i o n o f LHRH on s t e r o i d o g e n e s i s , t h e e f f e c t s o f these lipoxygenase  metabolites  acids  and h y d r o p e r o x y e i c o s a t e t r a e n o i c  (HETEs)  including  hydroxyeicosatetraenoic acids  (HPETEs),  on o v a r i a n s t e r o i d hormone were f u r t h e r examined. Rat  g r a n u l o s a c e l l s were incubated f o r 5h i n t h e absence  o r presence  of increasing concentration of  12-HETE, 15-HETE o r 15-HPETE was i n c r e a s e d by these 6  10~ M,  a l l treatments  7  _5  5-HETE,  (10~ M t o 1 0 M ) .  P  4  5-HPETE,  production  a c i d s i n a dose dependent manner. resulted  in a  slight  At  but s i g n i f i c a n t  —5 increase i n P  4  formation.  A t 10  HPETE) f u r t h e r s t i m u l a t e d P o f potency  was observed:  15-HPETE ( F i g . 50).  4  M, a l l compounds  production.  (except 15-  The f o l l o w i n g order  12-HETE > 5-HETE > 5-HPETE = 15-HETE >  159  12  Fig. 49. Effects of nordihydroguaiaretic acid (NDGA) o r indomethacin (INDO) on progesterone p r o d u c t i o n induced by LHRH and/or a r a c h i d o n i c a c i d (AA). Whereas indomethacin d i d a f f e c t the i n c r e a s e i n progesterone p r o d u c t i o n due t o t h e presence o f both LHRH and AA, NDGA d r a m a t i c a l l y suppressed progesterone production.  160  Fig. 50. production. fatty acids period.  Effects o f HETEs and HPETEs on progesterone Progesterone p r o d u c t i o n was i n c r e a s e d by these i n a dose dependent manner d u r i n g a 5h c u l t u r e  161 E f f e c t s o f H E T E S on p r o g e s t e r o n e  and PGE^ p r o d u c t i o n  Granulosa c e l l s were t r e a t e d w i t h 5-, 12- o r 15-HETE and the e f f e c t s on P seen  as w e l l as PGE  4  i n F i g . 51  (upper  and  15-HETE  resulted  accumulation,  p r o d u c t i o n were examined.  panel) , a t 5 x l O * " M , increase i n P  i n 3.5  and  respectively,  2.4  when  4  As  12-HETE was most  6  a 4.1 f o l d  potent and caused  2  formation.  fold  5-HETE  increase  o f P„  with  control  compared  incubations. I n t e r e s t i n g l y , these AA m e t a b o l i t e s a l s o s t i m u l a t e d PGE production Unlike  i n t h e same  their  experiment  a c t i o n s on P  4  production,  was as potent as 12-HETE on PGE caused  an approximate 15 f o l d  the c u l t u r e medium.  ( F i g . 51.  2  lower  the e f f e c t  formation.  increase  panel).  o f 15-HETE  15-HETE o r 12-HETE  i n c r e a s e i n PGE  2  accumulation i n  In c o n t r a s t , 5-HETE was c o n s i d e r a b l y l e s s  potent when compared w i t h 12- o r 15-HETE, but s t i l l significant  2  i n PGE  2  formation,  about  resulted i n  6  fold,  when  compared w i t h the c o n t r o l i n c u b a t i o n s .  Interaction PGE  o f HETEs o r HPETEs w i t h  LHRH on progesterone  and  production  2  S i n c e lipoxygenase m e t a b o l i t e s o f AA were b e l i e v e d t o be involved on  the  i n t h e a c t i o n o f AA, t h e e f f e c t s o f HETEs and HPETEs stimulation of  investigated dose  P  4  production  ( F i g . 52, upper p a n e l ) .  (i.e 10~ M), 6  production s l i g h t l y . observed w i t h 1 0  - 6  the  AA  LHRH  were  A t t h e minimum  metabolites  A more e f f e c t i v e  M LHRH.  by  stimulated  further effective  basal  stimulation of P  4  P  4  was  Concomitant treatment w i t h LHRH and  the  various  AA  accumulation,  metabolites  caused  further  increase  in  162 P. 4  by 20% t o 93%, when compared w i t h t h e e f f e c t o f  LHRH a l o n e . PGE  production  2  determined  in  the  same  ( F i g . 52, lower p a n e l ) .  experiments  was  5-HETE and 5-HPETE, a t the  minimum e f f e c t i v e dose which s t i m u l a t e d P, d i d not a l t e r basal  o r LHRH induced  15-HETE  o r 15-HPETE  compared  with  PGE  formation.  2  significantly  the control  i n c r e a s e d PGE  incubation.  PGE  production  2  by  2  fold,  2.9  either  In c o n t r a s t , 12-HETE, levels  2  Furthermore,  fold  and  when  12-HETE,  15-HETE and 15-HPETE augmented t h e s t i m u l a t o r y e f f e c t on  also  o f LHRH  2.5  fold,  r e s p e c t i v e l y , when compared w i t h t h e LHRH treatment alone.  Interactions PGE  and  production  2  The 10  o f HETEs o r HPETEs w i t h TPA on progesterone  M,  a d d i t i o n o f the p r o t e i n k i n a s e C a c t i v a t o r  to  production  granulosa  cells  caused  ( F i g . 53, upper p a n e l ) .  a  marked  (TPA), a t  increase  in  P  4  A l l HETEs and HPETEs t e s t e d  s i g n i f i c a n t l y augmented t h e s t i m u l a t o r y e f f e c t o f TPA, by 55 t o 83%, when compared w i t h P In increase HETE  t h e same i n PGE  o r 5-HPETE  2  4  l e v e l s induced by TPA alone.  experiment,  TPA  alone  p r o d u c t i o n ( F i g . 53, lower d i d not s i g n i f i c a n t l y  induced by TPA, concomitant  treatment  caused  panel).  affect with  a  PGE  2  4.9 While  fold 5-  production  12-HETE, 15HETE o r  15-HPETE f u r t h e r enhanced T P A - s t i m u l a t e d PGE. accumulation.  163  6h  6  VUJ  z  8» UJ UJ ce  1.6 -  a.  o 0.6  0.4  ^ 0.3 h  1  UJ 0.2 © a.  0.1  _1_  c  6HE  12HE  16HE  e  (6x10"M)  F i g . 51. E f f e c t s o f HETEs on progesterone (upper panel) and PGE_ (lower panel) p r o d u c t i o n . Both progesterone and PGE_ were s t i m u l a t e d by HETEs d u r i n g a 5h c u l t u r e p e r i o d . 5HE, 5-HETE; 12HE, 12-HETE; 15HE, 15-HETE.  164  (10-»M)  Fig. 52. I n t e r a c t i o n s o f HETEs o r HPETEs w i t h LHRH on progesterone (upper panel) and PGE^ (lower panel) p r o d u c t i o n . A t t h e minimum e f f e c t i v e dose (10~T1), t h e v a r i o u s a r a c h i d o n i c a c i d m e t a b o l i t e s enhanced LHRH induced progesterone p r o d u c t i o n and 12-HETE, 15-HETE and 15-HPETE augmented t h e s t i m u l a t o r y e f f e c t o f LHRH on PGE, p r o d u c t i o n d u r i n g a 5h c u l t u r e p e r i o d .  165  F i g . 53. I n t e r a c t i o n s o f HETEs o r HPETEs w i t h the phorbol e s t e r TPA on progesterone (upper panel) and PGE, (lower panel) production. TPA induced progesterone p r o d u c t i o n was enhanced by a l l HETEs and HPETEs t e s t e d , w h i l e TPA induced PGE, p r o d u c t i o n was augmented by 12-HETE, 15-HETE and 15-HPETE d u r i n g a 5h c u l t u r e p e r i o d .  Effect  o f LHRH  on FSH-induced  progesterone  production;  166 time  response To  examine  granulosa  cells,  granulosa FSH  the action  cells  FSH-  8h,  16h  and  stimulated cells,  4  P  LHRH-  were c u l t u r e d  (100 ng) alone  increase i n P  or  o f gonadotrophin  resulted  accumulation,  or  production  i n a significant  induced by FSH.  LHRH  The concomitant  significant  and  P  4  decrease  production  in P  was  time  alone  with  presence  the c u l t u r e medium d i d not a l t e r P a  LHRH-treated  dependent  also  untreated  markedly culture  p r o d u c t i o n was much l e s s than  4  on  20 f o l d , 21.5 f o l d and 30 f o l d , a t  as compared  b u t LHRH-induced P  plus  LHRH  f o r 8h, 16h and 24h ( F i g . 54) .  24h, r e s p e c t i v e l y . 4  FSH  and  o f FSH w i t h LHRH i n  p r o d u c t i o n a t 8h.  4  p r o d u c t i o n was observed  4  further  reduced  that  a t 24h  However, a t 16h,  i n combined  treatment o f g r a n u l o s a c e l l s w i t h FSH p l u s LHRH.  Effects  of  arachidonic  acid  and/or  FSH  on  progesterone  p r o d u c t i o n d u r i n g a 24h c u l t u r e To examine t h e r o l e o f AA on P  4  p r o d u c t i o n , r a t granulosa  —5 c e l l s were t r e a t e d w i t h 10 FSH  (lOOng),  stimulated P control  f o r 24h 4  ( F i g . 55).  of P  4  AA alone  caused  with  FSH  accumulation.  a slight  p r o d u c t i o n , 4.1 f o l d  with the untreated c o n t r o l c e l l s . AA  As expected,  FSH markedly  p r o d u c t i o n (23 f o l d ) compared w i t h t h e u n t r e a t e d  (P<0.01).  stimulation  M AA, i n t h e absence o r presence o f  d i d not  affect  but s i g n i f i c a n t  (P<0.05) when compared  The concomitant FSH-induced  presence o f  increase  in  P  4  167  50  0  8  16  Hours  Fig. 54. Effect o f LHRH on FSH induced progesterone p r o d u c t i o n : time response. LHRH alone s t i m u l a t e d progesterone p r o d u c t i o n as e a r l y as 4h, b u t p r o d u c t i o n was much l e s s than t h a t induced by FSH. FSH s t i m u l a t e d progesterone p r o d u c t i o n was reduced by LHRH a f t e r 16h.  168  F i g . 55. E f f e c t s of a r a c h i d o n i c a c i d (AA) and/or FSH on progesterone p r o d u c t i o n . AA alone caused a s l i g h t i n c r e a s e i n progesterone p r o d u c t i o n . Concomitant presence of AA w i t h FSH did not affect the FSH induced increase i n progesterone p r o d u c t i o n a f t e r 24h c u l t u r e . C o n t r o l (C) and FSH treated c e l l s r e c e i v e d the a p p r o p r i a t e amount o f s o l v e n t f o r AA.  Effect  of arachidonic  progesterone  acid  cells  were  ng/ml), w i t h o r without of  18h,  the  significantly cells  LHRH  induced  given  treated  FSH a l o n e .  with 6  P  4  treatment  production  At t h i s  time,  shown i n F i g . 56, LHRH decreased  of  LHRH  during  FSH-induced  P  the l a s t was  4  FSH  with the  AA was added t o two o f f o r a f u r t h e r 6h.  FSH-induced P  6h, t h e i n h i b i t o r y  partially  At the  plus  when compared  reversed,  When AA was  effect  by  As  p r o d u c t i o n , by  4  a t t h e end o f t h e 24h i n c u b a t i o n p e r i o d .  present  169 of  FSH (100  t h e presence o f LHRH (10~ M).  combined  decreased  f o r 18h  the groups and t h e c u l t u r e was c o n t i n u e d  47%,  inhibition  production  Granulosa  end  on  about  o f LHRH on 42%, when  compared with  the cultured c e l l s  t r e a t e d with  LHRH p l u s FSH.  AA  d i d not a f f e c t  accumulation  induced  by i t s e l f  P  4  by FSH  d u r i n g t h e l a s t 6h.  Effect  of  arachidonic  progesterone  acid  on  TPA-induced  inhibition  of  production  F i g . 57 i l l u s t r a t e s AA r e v e r s a l o f the i n h i b i t o r y a c t i o n o f TPA on P (100 was  4  production.  10" M TPA f o r 18h.  g i v e n t o one o f t h e groups. 6h.  FSH-induced  P  untreated  4  As expected, production,  group.  c e l l s were t r e a t e d w i t h FSH  9  ng/ml) with o r without  f o r another  FSH  Granulosa  A f t e r t h a t , AA  A l l i n c u b a t i o n s were c o n t i n u e d  TPA caused a marked i n h i b i t i o n on  69% (P<0.01) as compared w i t h t h e  A d d i t i o n o f AA t o t h e group g i v e n  resulted i n a partial  reversal of P  4  production  when compared w i t h t h e TPA p l u s FSH treatment  alone  TPA p l u s by 71%,  (P<0.01).  170  F i g . 56. Response t o a r a c h i d o n i c a c i d (AA) a f t e r pretreatment w i t h FSH and LHRH. Granulosa c e l l s p r e t r e a t e d w i t h s a l i n e (C) or w i t h FSH ± LHRH f o r 18h, a t which time AA (or s o l v e n t ) was added. A l l groups were incubated f o r a f u r t h e r 6h. The inhibitory effect o f LHRH on FSH induced progesterone p r o d u c t i o n was p a r t i a l l y r e v e r s e d by t h e presence o f AA.  171  Fig. 57. Response to arachidonic acid (AA) after pretreatment w i t h FSH and the p h o r b o l e s t e r TPA. Granulosa c e l l s were p r e t r e a t e d w i t h DMSO (C) o r w i t h FSH ± TPA f o r 18h p r i o r t o t h e a d d i t i o n o f AA (or s o l v e n t ) . Then, t h e c e l l s were incubated f o r 6h. TPA caused i n h i b i t o r y e f f e c t on progesterone p r o d u c t i o n by FSH was p a r t i a l l y r e v e r s e d by t h e a d d i t i o n o f AA.  Response t o a r a c h i d o n i c  acid  after  pretreatment  172 cholera  with  t o x i n and TPA Addition  o f CT  resulted  in a  production  and TPA s i g n i f i c a n t l y  P  by CT.  4  induced  p r e c u l t u r e d with  o f AA  caused  increase  in P  production,  significantly  CT  i n P, 4  the production of  CT o r CT p l u s TPA f o r 18h.  presence  TPA p l u s  increase  AA was added t o two groups t h a t had been  f o r a f u r t h e r 6h.  with  fold  attenuated  were c o n t i n u e d  4  29  a partial  alone.  when  A l l incubations  As shown i n F i g . 58, the reversal, compared  CT-induced  P  4  i . e . 39% (P<0.01) with  the  production  a f f e c t e d by t h e a d d i t i o n o f AA alone  treatment was not  d u r i n g the  l a s t 6h c u l t u r e p e r i o d .  E f f e c t o f a r a c h i d o n i c a c i d on P  4  production a f t e r  pretreatment  w i t h TPA and LHRH 10 the  -9  -6 M TPA and 10 M LHRH were added t o g r a n u l o s a c e l l s a t  beginning  gonadotropins groups  of  the c u l t u r e  o r CT.  of  exogenous  A f t e r 18h, AA was added t o some o f the  f o r a f u r t h e r 6h t o determine t h e P  cells.  4  response o f the  A t t h e end o f t h e 24h c u l t u r e , TPA d i d n o t s t i m u l a t e P  production increase  ( F i g . 59). in  controls.  P  4  accumulation  when  compared  A d d i t i o n o f AA alone d u r i n g t h e l a s t  pretreated c e l l s ,  P  4  4  In c o n t r a s t , LHRH caused a 55% (P<0.01)  a 160% i n c r e a s e i n P. formation. 4  alone.  i n the absence  with  untreated  6h r e s u l t e d i n  When AA was added t o the TPA-  p r o d u c t i o n was same as t h a t caused by AA  In c o n t r a s t , when AA was g i v e n t o t h e LHRH-pretreated  c e l l s , t h e r e was an a d d i t i v e e f f e c t on P. p r o d u c t i o n .  173  Fig. 58. Response to arachidonic acid (AA) after pretreatment w i t h c h o l e r a t o x i n (CT) and the p h o r b o l e s t e r TPA. Granulosa c e l l s were p r e t r e a t e d w i t h DMSO (C) o r w i t h CT ± TPA. A t 18h, AA (or s o l v e n t ) was added. A l l i n c u b a t i o n s were stopped 6h l a t e r . The presence o f AA p a r t i a l l y r e v e r s e d t h e i n h i b i t o r y e f f e c t of TPA on CT induced progesterone p r o d u c t i o n .  174  F i g . 59. Response t o a r a c h i d o n i c a c i d (AA) a f t e r pretreatment w i t h t h e phorbol e s t e r TPA and LHRH a l o n e . Granulosa c e l l s were p r e t r e a t e d w i t h dimethyl s u l f o x i d e (C), TPA o r LHRH f o r 18h. A t t h i s time, AA (or s o l v e n t ) was added. A l l groups were incubated f o r a f u r t h e r 6h. Treatment o f t h e c e l l s w i t h TPA d i d not a f f e c t t h e response t o AA.  Effects  o f LHRH.  TPA  and/or  arachidonic  175 on p r o g e s t i n  acid  p r o d u c t i o n d u r i n g a 5h I n c u b a t i o n Granulosa  cells  6  LHRH  (10~ M) , w i t h  were t r e a t e d  o r without  f o r 5h w i t h TPA (10 M) o r  t h e concomitant  presence  o f AA  —5 (10  M) .  TPA  or  As shown i n A panel o f F i g . 60, t h e presence LHRH  alone  caused  p r o d u c t i o n , by 4.8 f o l d , when  compared  with  significant  7.8 f o l d  control  increases  and 7.1 f o l d ,  levels.  AA  o f AA, in  P  4  respectively,  exerted  an  additive  e f f e c t w i t h TPA and LHRH on P. f o r m a t i o n . 4  The  p r o d u c t i o n o f 20alpha-OH-P i s shown a l s o  (B p a n e l ) . by  1.5  AA, TPA and LHRH s t i m u l a t e d 20alpha-OH-P p r o d u c t i o n  fold,  magnitude  4.9  fold  and  o f AA-induced  4.7  fold,  20alpha-OH-P  respectively.  accumulation  lower than t h a t induced by e i t h e r TPA o r LHRH. the  i n F i g . 60  additive  effects  observed  for P  4  The  was  much  In c o n t r a s t t o  formation,  AA  d i d not  a l t e r t h e e f f e c t o f TPA o r LHRH on t h e accumulation o f 20alphaOH-P.  On t h e o t h e r hand, t o t a l p r o g e s t i n accumulation  p l u s 20alpha-OH-P) was i n c r e a s e d by treatment  (i.e. P  4  w i t h AA, TPA o r  LHRH alone and f u r t h e r i n c r e a s e d by combined treatment w i t h TPA p l u s AA, o r w i t h LHRH p l u s AA ( F i g . 60, Effects  of  arachidonic  hydroxycholesterol  enhanced  acid.  TPA  C panel). and  steroidogenesis  LHRH  on  during  25a  5h  incubation To determine  i f AA a f f e c t s t h e a c t i v i t y o f t h e s i d e - c h a i n  cleavage enzyme, a water s o l u b l e c h o l e s t e r o l d e r i v a t i v e , 25-OHcholesterol,  was used.  I n c l u s i o n o f 25-OH-cholesterol  i n the  176 c u l t u r e medium s i g n i f i c a n t l y enhanced t h e accumulation o f P i n 4  t h e c o n t r o l c e l l s by 4.1 f o l d d u r i n g a 5h i n c u b a t i o n ( F i g . 61). The  concomitant  presence  markedly i n c r e a s e d P respectively, cholesterol also  when  alone.  4  o f TPA o r LHRH with  p r o d u c t i o n by about compared  with  The presence  s i g n i f i c a n t l y increased P  compared  to  the  effect  formation  o f AA w i t h production,  of  by  25-OH-cholesterol about  34%, when  25-OH-cholesterol  p r o d u c t i o n i n t h e presence o f 2 5 - O H - c h o l e s t e r o l , from  the e f f e c t  without t h e added c h o l e s t e r o l .  enhance  o f AA  TPA-  on P  4  and  alone.  stimulated P  was d i f f e r e n t  further  25-OH-  AA  which  to  4  56% and 84% (P<0.01)  Nevertheless, 4  failed  4  P  25-OH-cholesterol  LHRH-  production  F i g . 60. E f f e c t s o f LHRH, t h e phorbol e s t e r TPA and/or arachidonic acid (AA) on p r o g e s t i n p r o d u c t i o n d u r i n g a 5h incubation. AA, TPA o r LHRH alone caused s i g n i f i c a n t i n c r e a s e i n progesterone p r o d u c t i o n , and AA e x e r t e d an a d d i t i v e e f f e c t w i t h TPA and LHRH, whereas t h e magnitude o f AA induced 20alpha-OH-P accumulation was much lower than t h a t induced by e i t h e r TPA o r LHRH, and AA d i d n o t a l t e r t h e e f f e c t o f TPA o r LHRH on t h e accumulation o f 20-alpha-OH-P.  178  F i g . 61. E f f e c t o f a r a c h i d o n i c a c i d (AA) , t h e phorbol e s t e r TPA and LHRH on 25-hydroxycholesterol enhanced s t e r o i d o g e n e s i s d u r i n g a 5h i n c u b a t i o n . Although t h e presence of AA increased 25-OH-cholesterol induced progesterone production, AA f a i l e d t o f u r t h e r enhance TPA and/or LHRH s t i m u l a t e d progesterone p r o d u c t i o n i n t h e presence o f 25-OHcholesterol. Incubations c o n t a i n i n g 2 5 - h y d r o x y c h o l e s t e r o l were denoted by t h e hatched b a r s .  179 IV.  Discussion The  release  intracellular i n granulosa  pathway  cells  (chapter  LHRH  stimulated  was n o t c l e a r .  mechanisms f o r the l i b e r a t i o n been proposed  by which  2) .  Three  AA  possible  o f AA from plasma membrane have  The i n c r e a s e  of c e l l u l a r  l e v e l of  f r e e o r u n e s t e r i f i e d r a d i o l a b e l e d AA was observed by treatment of granulosa  cells  observation  strengthens  stimulated involved  AA  with  LHRH w i t h i n the  liberation  ovarian c e l l s  previous  from  as an e a r l y step  5 min (chapter proposal  phospholipids  the  Minegishi suggesting and  ionophore  could  by t h e a c t i v a t i o n o f PKC.  i n many  induce  tissues  activation 2  4  production  production,  a  of  reactions  In t h e present  P  single both  (Lapetina,  quantity  i n the I t has  Clark,  1986;  (chapter 2 ) , dependent,  Nevertheless, i t  extracellular signal  phospholipase  and as a r e s u l t ,  C  and  cause AA r e l e a s e  1982).  study t h e e f f e c t s o f m e l i t t i n , AA and LHRH  were f u r t h e r examined.  but m e l i t t i n s t i m u l a t e d  than exogenous AA and LHRH s t i m u l a t e d experiment  and  i s calcium  from v a r i o u s p h o s p h o l i p i d s  4  (Kawai  t h a t LHRH-induced AA r e l e a s e  that  be  c e l l s i s enhanced  by TPA  phospholipase A  on P  A23187  LHRH  also  2 ).  e t a l . , 1987), and p o t e n t i a t e d  i s regulated  appears  might  (Minegishi and Leung, 1985; c h a p t e r  calcium  This  that  i n LHRH s i g n a l t r a n s d u c t i o n  a l s o been shown t h a t AA r e l e a s e i n g r a n u l o s a by  2).  ( F i g . 41) .  o f endogenous  This AA  may  was  M e l i t t i n could P  P  4  production  4  formation  reflect  limited.  induce  was lower  i n the same  the fact  that the  A d d i t i o n a l l y , the  r e s u l t s suggested t h a t t h e e f f e c t o f LHRH cannot be only due t o  endogenous  AA  and  i t s metabolites,  pathways were a l s o i n v o l v e d . that  concomitant  treatment  plus  LHRH does not  and  that  Ca  of  granulosa  further increase  P  cells  with  production  4  presumably i m p l y i n g t h a t the e f f e c t  production  already  suggested AA  from the  controlling More  P  action  induced  of  the  of phospholipids,  as w e l l as  present  granulosa  ( F i g . 41) .  data  cells This  PGE  clearly  with  AA  cleaves  demonstrated 5h  s t i m u l a t i o n was  a  r a t h e r narrow range  ( F i g . 42). the  The  use of the exogenous AA was  physiologic situation  cyclooxygenase the  or  appropriate  intracellular  ( i . e . between  since  lipoxygenase biosynthetic  that  enhanced  dose  dependent,  process  was  to  10  M)  to  the  ovarian  site  where  present.  Both  the c u l t u r e medium were i n c r e a s e d i n the presence o f AA.  Cell  blue  accumulation  to  P. i n 4  trypan  the  M  closely related  at  4  —5  converted  metabolites  and  3x10  P  of  v i a b i l i t y was  P. c o n c e n t r a t i o n 4  i t was  in  cells.  —7 within  AA  participated  for  4  melittin  i n granulosa  2  P  endogenous  p r o d u c t i o n induced by  4  by  o f LHRH on  2  production  4  of  production  the  melittin  a c t i v a t i o n o f PLA , an enzyme t h a t  2-acyl p o s i t i o n  importantly,  treatment  included  Taken t o g e t h e r , P  t h a t the  PKC  The p r e v i o u s experiment has shown  LHRH alone,  (chapter 4 ) .  and  not a f f e c t e d by these dosages o f AA, exclusion.  In  this  regard,  similar  as judged by doses  of  AA  have r e c e n t l y been r e p o r t e d t o s t i m u l a t e hormone p r o d u c t i o n i n other  endocrine  c e l l s , AA release  a t 5x10  tissues. —5 M or 10  For —4  example,  M was  in anterior pituitary  a potent 5  (Chang e t a l . , 1986); a t 10" M  ACTH r e l e a s e (Abou-Samra e t a l . , 1986).  secretagogue f o r LH 4  and 10~ M, AA PRL  stimulated  s e c r e t i o n from  GH  3  c e l l s was s t i m u l a t e d by AA a t 3x10 Likewise,  AA  a t 10  t o 10  o x y t o c i n i n corpus luteam  M  M ( K o l e s n i c k e t a l . , 1984).  resulted  i n the increase of  ( H i r s t e t a l . , 1988), and AA a t 10  M  —5 t o 10  M enhanced t e s t o s t e r o n e p r o d u c t i o n by L e y d i g c e l l s ( L i n ,  1985). In t h e p r e s e n t was  found  stimulation  compared  to  be  the e f f e c t  similar  o f P. 4  with  study,  to  that  due t o AA  that  o f AA on P  was  of  LHRH,  somewhat  due t o LHRH.  P  although  slower  levels  4  production  4  the  i n onset  i n the culture  medium were i d e n t i c a l 5h a f t e r t h e a d d i t i o n o f LHRH o r AA ( F i g . 45).  When p r e s e n t t o g e t h e r , t h e e f f e c t s o f AA and LHRH o r LHRH  agonist after  became  additive; this  treatment.  stimulation of P AA  on  Since 4  could  AA  also  be seen  as e a r l y as 3h  greatly  potentiated  p r o d u c t i o n by TPA, t h e s y n e r g i s t i c e f f e c t o f  LHRH-induced  P  production  4  perhaps  reflected  p o t e n t i a t i o n by AA on LHRH-induced PKC a c t i v i t y The  relatively  action  o f AA  the  higher on P  4  dose  and l o n g e r  production  may  time  be  a  ( F i g . 44-47).  required  for  the  due t o t h e r a t e o f  p e n e t r a t i o n o f AA i n t o t h e c e l l membrane and t h e c o n v e r s i o n o f AA  to  i t s metabolites.  characterized Davis  1983).  i n cytosol,  (Noland that  i n ovarian  and C l a r k ,  found  tissues  followed  activation  of 4  PKC  by  PKC  activity  (Noland  The h i g h e s t  and Dimino, 1986).  stimulates basal P Leung,  Recently,  and Dimlno,  specific  microsomes  has  been 1986;  a c t i v i t i e s were  and  mitochondria  S e v e r a l l a b o r a t o r i e s have r e p o r t e d by  such  as TPA,  production i n r a t granulosa c e l l s  (Wang and  1987; Kawai and C l a r k ,  phorbol  esters,  1985; Shinohara  e t a l . , 1986).  Thus,  t h e present  stimulatory mediated, induced is  action  i n part  from  of  LHRH  at least,  AA r e l e a s e .  responsible  result  results could  activity,  and  steroidogenesis  of  PKC  is  by LHRH-  o f cytochrome P-450, which  in  cleavage,  may  steroidogenic  also  tissues  I t seems p l a u s i b l e t h a t LHRH-induced  a second messenger r o l e by a m p l i f i c a t i o n o f PKC  systems (McPhail  AA  ovarian  by PKC and p o t e n t i a t e d  as has been proposed  In support  182 that the  t o suggest  f o r cholesterol side-chain activation  could play  on  Phosphorylation  ( V i l g r a i n e t a l . , 1984). AA  be taken  signalling  i t has r e c e n t l y been documented t h a t  synergistically  gonadotrophin  f o r other  e t a l . , 1984; Murakami and Routtenberg, 1985).  o f t h i s theory, PKC  recently  secretion  mediate  by LHRH  the  i n anterior  stimulation pituitary  of  cells  (Chang e t a l . , 1986). In t h e same experiment, t h e e f f e c t s o f o t h e r f a t t y a c i d s on P  4  unsaturated  p r o d u c t i o n were a l s o examined ( F i g . 43).  All  -5 the treatments were a t t h e dose o f 10 e f f e c t i v e concentration P«  f o r AA on s t i m u l a t i n g t h e p r o d u c t i o n o f  11,14 e i c o s a d e n o i c  4  gamma-linolenic (C18:3)  like  incubation.  acid AA,  acid  could  Another  4  fatty  production.  and o t h e r  i n human d e c i d u a l  (C18:2),  P  cell  4  4  production production  acid,  i n 5h  i n a order o f  11,14 e i c o s a d i e n o i c oleic  acid  (C18:l),  These data were s i m i l a r t o  unsaturated tissue  acid  acid  gamma-linolenio  unsaturated  e f f e c t s o f AA  secretion  increase  linoleic  homo-gamma-linolenic  Fatty acids stimulated P  f a i l e d t o stimulate P the  (C18:2),  (C18:3),  AA> homo-gamma-linolenio acid.  M which was t h e maximal  fatty  (Handwerger  acids  on PRL  e t a l 1981).  The mechanism of these u n s a t u r a t e d f a t t y a c i d s on P was  not c l e a r . However, t h e s e u n s a t u r a t e d  for c e l l  s t r u c t u r e s and  naturally o f PGs  exist  and  LTs  source o f PGs The  to  (Crawford,  1982).  1983).  which AA  The  production  stimulates P  in  rat  apparently d i s t i n c t  from  or  well,  lipoxygenase a c t i v i t y lipoxygenase ovulation present using AA  might  been  the  metabolism.  to  that i n h i b i t s The  addition  et  p r o s t a g l a n d i n d i d not cyclooxygenase (Amrstrong and  with  of  P  rats,  et  rupture  was  did  at  In' the examined  pathway of  to  granulosa  production during a  block  the blockade inhibition  administration of  of of  ovulation in rat  (Armstrong  a l . , 1975).  ( T s a f r i r i e t a l . , 1972).  p r o s t a g l a n d i n s , the  4  Although  1972), r a b b i t  (Mai  PGs  p r o d u c t i o n , the  4  ovarian  a l . , 1985). of  LH  a c t i v a t i o n of  indomethacin  49) .  indomethacin  monkeys  indomethacin-blocked ovulation  affect  and Grinwich,  marmoset  and  The  was  LH.  stimulate  the cyclooxgenase  c e l l s d i d not a l t e r LHRH- o r AA-induced P 5h c u l t u r e p e r i o d ( F i g . 48  synthesis  follicular  p o s s i b l e involvement  indomethacin  was  (Clark,  t h a t induced by  with  Reich  cells  PG  in vitro.  correlated  (Reich e t a l . , 1983;  study,  LHRH on  reported  i n v i v o and  be  production  4  granulosa  a d d i t i v e but  has  series  i t s a g o n i s t s have been shown  of  as  which c o u l d  provided.  stimulatory effect  hCG,  used  T h e r e f o r e , a l a r g e membrane  P r e v i o u s l y , LHRH and PG  cascade  membrane, o r produce o t h e r  and LTs p r e c u r s o r was  stimulate  4  f a t t y a c i d s are  convert t o AA  i n the c e l l  mechanism by  not known.  can  183 production  et a l ,  In  1974)  addition,  PGE  2  can  in  induce  F u r t h e r , i n the absence of  r u p t u r e of o v a r i a n f o l l i c l e  d i d not  occur  (LeMaire  and Marsh,  lipoxygenase rapidly  enzymes  reduced  series  contribution  stimulation of P  of  tissue  48  and 49).  4  r e s p e c t i v e HETEs,  o f products the  (Reich  synthesis.  gives  pathways  t o the  et  al.,  from These  pathway o f AA i n  1983).  AA-induced  P  4  t o t h e l e v e l as t h a t caused by NDGA, and  formation  Although  result  would be  known as t h e l e u k o t r i e n e s .  lipoxygenase  was o n l y p a r t i a l l y  NDGA reduced  production, the basal l e v e l of P might  that  and 5-HPETE  i n h i b i t o r o f lipoxygenase  p r o d u c t i o n was reduced LHRH-induced P  o f HPETEs  s y n t h e s i s was f u r t h e r i n v e s t i g a t e d u t i l i z i n g  4  NDGA, an e f f e c t i v e follicular  184 by the  AA can be a l s o converted  to a variety  to their  r i s e t o another The  1975) .  4  (Fig.  AA- and LHRH-stimulated P  4  was i n c r e a s e d by NDGA, which  the increase results  suppressed  i n the precursor  indicated  that  there  f o r PGs may  be a  s t i m u l a t o r y r o l e f o r PGs i n s t e r o i d o g e n e s i s .  Indeed, exogenous  PGE  estrogen  has been  2  production inhibitory supports  shown  to  stimulate  (Richards e t a l . , 1976). effect  of  the notion  NDGA that  on  LHRH-induced  induced  leads  PKC,  mobilization. and  thus  These d i f f e r e n t  presence that  LH  second  P  4  o f indomethacin induced  P.  P  4  4  production  messengers  t h e formation  and  IP  3  which  pathways cooperated  c o n t r i b u t e d t o t h e a c t i o n o f LHRH.  LHRH- o r AA-stimulated  P  were  Besides t h e r e l e a s e o f AA from  plasma membrane, LHRH a l s o  of  and  Furthermore, t h e p a r t i a l l y  multiple  i n v o l v e d i n t h e a c t i o n o f LHRH.  the activation  cAMP,  o f DG which 2+ causes each  The f i n d i n g  Ca other that  p r o d u c t i o n was n o t i n f l u e n c e d by the supported  production  the previous was  observations  not affected  by the  indomethacin-blocked  PG f o r m a t i o n  185 ( C l a r k , 1982), and t h a t t h e  enhancement o f FSH o r CT-induced PGE did  not  tightly  (Chapter  4) .  partially  by  Taken  been  corpus  to  the  together,  increasing free  metabolites, has  couple  d i d not r e s u l t  implicated  luteum.  2  p r o d u c t i o n by LHRH o r TPA  production  of  LHRH-stimulated AA  from PG f o r m a t i o n .  i n the secretion of  The r e s u l t s  and  2  s t i m u l a t e o x y t o c i n s e c r e t i o n , and AA may  via  t h e lipoxygenase the  involvement  (Hirst of  to i t s  Recently,  oxytocin  showed t h a t PGE  pathway  production,  4  and by c o n v e r t i n g AA  not  implicating  P  progesterone  P G  F  have  in 2 a  ]_p  ovine n a  do  i t s effect  e t a l . , 1988),  lipoxygenase  AA  further  metabolites  in  regulating ovarian functions. The  inhibitory  formation  indicated  r o l e i n the P  effect  o f NDGA on LHRH- o r AA-induced  that lipoxygenase  metabolites  p r o d u c t i o n induced by LHRH.  4  f u r t h e r i n v e s t i g a t e d i n the present  study  The  results  indicated  that  The be  r a t granulosa  cells  at least  addition to P , 4  several  than  metabolism o f  some  4  lipoxygenase  t h e formation o f P  i n a dose dependent manner  s t i m u l a t o r y e f f e c t s o f 12-HETE on P more potent  o f AA had a  u s i n g s e v e r a l HETEs  m e t a b o l i t e s o f AA were capable o f enhancing by  o f t h e AA m e t a b o l i t e s  o f PGE  2  4  ( F i g . 50) .  p r o d u c t i o n appeared t o  t h a t o f 5-HETE, 5-HPETE o r 15-HPETE.  the formation  4  T h i s h y p o t h e s i s was  and HPETEs from the 5-, 12-, and 15-lipoxygenase AA.  P  In  was a l s o s t i m u l a t e d by  ( F i g . 51) .  This effect  o f AA  metabolitws  i s not due t o c r o s s - r e a c t i o n o f t h e m e t a b o l i t e s i n  the  assay,  PGE  2  metabolites  since  at  the  do not c r o s s - r e a c t .  concentrations 6  At 5xl0~ M,  used  the  12-HETE and 15-  186 L i k e AA, 5-  HETE were more potent than 5-HETE i n t h i s r e g a r d .  HETE, 5-HPETE, 12-HETE, 15-HETE and 15-HPETE i n c r e a s e d b a s a l P  4  production  4  and  production. basal  PGE  a t 10~ M,  formation induced  results  were  effects  of  production  augmented 6  Also,  2  formation  further  12-HETE  the  and p o t e n t i a t e d t h e s t i m u l a t i o n o f PGE LHRH  ( F i g . 52).  observed  with  TPA  may  and HPETEs  be  metabolites with  P  and 15-HETE s t i m u l a t e d  by  HETEs  LHRH-induced  due  to  very  ( F i g . 53), t h e  on  LHRH-induced  the  LHRH-activated  Since  PKC.  Since  similar  facilitatory P  interaction  2  and  4  of  PGE  these  the present  2  AA data  showed t h e m e t a b o l i t e s o f l i p o x y g e n a s e pathway o f AA-stimulated PGE  p r o d u c t i o n , one might s p e c u l a t e t h a t HETEs o r HPETEs a c t  2  as i n t e r n a l r e g u l a t o r s between t h e m e t a b o l i t e s o f lipoxygenase and  cyclooxygenase  pathway.  P r e v i o u s s t u d i e s have shown t h a t  both LH and hCG r e g u l a t e l i p o x y g e n a s e a c t i v i t y , o f LHRH on these enzymes needs f u r t h e r  investigation.  been r e p o r t e d t h a t both 5- and 15-lipoxygenase for activity  i n platelet  (Pace-Asciak  but the a c t i o n I t has  require calcium  and Smith,  1986),  thus,  2+ LHRH-induced r a p i d i n c r e a s e i n [Ca  ] i might be r e l a t e d t o t h e  a c t i v i t y o f these enzymes i n t h e o v a r i a n c e l l s as w e l l . There was i n c r e a s i n g evidence t o support t h e n o t i o n t h a t l i p o x y g e n a s e m e t a b o l i t e s o f AA were potent mediators o f hormone p r o d u c t i o n i n d i f f e r e n t endocrine t i s s u e s . cyclooxygenated  One o r more o f t h e  and/or l i p o x y g e n a t e d m e t a b o l i t e s o f AA might be  a component o f t h e cascade  of reactions i n i t i a t e d  u l t i m a t e l y r e s u l t i n LH s e c r e t i o n i n p i t u i t a r y 1986;  Kiesel  e t a l . , 1987).  by LHRH and  (Kiesel et a l . ,  I t has a l s o been  reported that  leukotrienes dispersed Kiesel  are  rat  et  effective  anterior pituitary  a l . , 1986;  Hurling  p r o d u c t s o f AA metabolism PRL  release  reported islets  stimulators  (Kiesel  that  et  cells  et  a l . , 1985).  a l . , 1987).  synthesis  a l . , 1983).  1986). and et  P G F  of  2 a  al.,  w  ipha  a  s  u  n  a  I n h i b i t i o n of lipoxygenase  f  f  e  c  k e t o  t  e  p G F  -  d  al.  in  In bovine corpus -  stimulate  et  release  6  luteum,  i j_p a  n a  (Milvae  a c t i v i t y with  R e i c h et a l . , 1985).  o f 5-HETE had  f  importance  ovarian functions  of  those  compounds  et a l . ,  be  intracellular  be  determined.  concentration  (Milvae e t a l . , 1986), thus f a r , t h e r e was  no  leukotrienes  cell.  rather The  because that  In  are  Whether HETEs and HPETEs prove t o  can  of of  AA  pathway  be  presumably  view  r o l e o f l e u k o t r i e n e s on  o f these hydroperoxy a c i d s  5-lipoxygenase  5-HPETE  e f f e c t s of  as  than e x t r a c e l l u l a r s i g n a l s remains  metabolites  1982).  stimulatory  (Reich  of  e x i s t on r a t g r a n u l o s a  al.,  755C  regulator  evidence t o show t h a t the r e c e p t o r s  lipoxygenase  while  NDGA, BW  Although h i g h  5-  been found i n bovine l u t e a l t i s s u e , i n d i c a t i n g a  physiological  interest  have  pancreatic  FPL-55712 r e s u l t e d i n p a r t i a l blockade o f o v u l a t i o n a l , 1983;  1987;  Lipoxygenase  Yamamoto  insulin  187 from  release  (Kiesel et  HETE reduced the b i o s y n t h e s i s of P^ and the  LH  have a l r e a d y been shown t o  5-HETE s t i m u l a t e s  (Yamamoto e t  of  rapidly the  most  5-HPETE on  present P  4  ovarian c e l l  and  of  special  converted active  (Samuelsson, the  is  1983;  to  of  to the  Morris  et  demonstration  of  PGE  2  production,  the  f u n c t i o n warrants f u r t h e r  investigation. In  addition,  the  stimulatory  role  of  AA  was  further  examined i n t h e p r e s e n t study. e f f e c t s o f LHRH on P of  accumulation w i t h o r without t h e presence  4  FSH d u r i n g t h e d i f f e r e n t  confirmed  the previous  LHRH on g r a n u l o s a c e l l 24h  i n t h e presence  stimulating  incubations  Hillensjo et a l . ,  period  ( F i g . 54) f u r t h e r  the i n h i b i t o r y  action of  s t e r o i d o g e n e s i s was o n l y observed  o f exogenous gonadotropins, whereas  induced  (Knecht  culture  studies that  agents,  steroidogenesis  188 or stimulatory  The i n h i b i t o r y  by  LHRH  did  gonadotrophins  after  o r o t h e r cAMP  not in  influence short  term  e t a l . , 1982; Hsueh and S c h a e f f e r , 1985;  1982).  The reason f o r t h e apparent  delay i n  t h e onset o f t h i s i n h i b i t o r y e f f e c t was not known, a l t h o u g h i t was  believed  that  LHRH-induced  membrane p o l y p h o s p h o i n o s i t i d e  breakdown l e d t o t h e f o r m a t i o n o f IPs and DG, and t h e r e l e a s e of  AA, might p a r t i c i p a t e  1985;  Davis  Leung,  e t a l . , 1986; Davis  1985; Wang  inhibitory magnitude incubation  i n t h e a c t i o n o f LHRH (Ma and Leung,  effect of  P  4  period  and Leung, o f LHRH  e t a l . , 1987; M i n e g i s h i and 1987).  Unlike  and TPA, AA  production  induced  ( F i g . 55) .  Since  by  t h e markedly  d i d not a f f e c t FSH  during  the e f f e c t  a  the 24h  o f TPA 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 LHRH d u r i n g l o n g term  granulosa  c e l l c u l t u r e , a c t i v a t i o n o f p r o t e i n k i n a s e C may p a r t i c i p a t e i n the  inhibitory  gonadotrophin  action  o f LHRH  a t two  receptor/adenylate  cyclase  distinct  sites,  the  complex  and a  site  d i s t a l t o t h e g e n e r a t i o n o f cAMP (Welsh e t a l . , al.,  1985).  Likewise,  calcium  mobilization  1984; B a r r y e t may  i n v o l v e d i n t h e a c t i o n o f LHRH t o i n h i b i t gonadotrophin cAMP and s t e r o i d  formation  i n granulosa  cells  (Ranta  also  be  induced et a l . ,  1983;  Leung  with  e t a l . , 1988).  exogenous AA  rather  than  Leung,  1988) .  cells  suggested  inhibitory  t h a t AA  role  a t which  time  already evident.  189 the findings  hand,  mediated a s t i m u l a t o r y ,  i n t h e a c t i o n o f LHRH  To f u r t h e r examine t h i s  were t r e a t e d w i t h  18h,  On t h e o t h e r  hypothesis,  FSH (with o r without  the i n h i b i t o r y  (Wang and  effect  granulosa  LHRH o r TPA)  for  o f LHRH o r TPA was  A d d i t i o n o f AA d u r i n g a f u r t h e r 6h i n c u b a t i o n  p a r t i a l l y r e v e r s e d t h e i n h i b i t o r y e f f e c t o f LHRH o r TPA on FSHor CT-induced P some c e l l s  production  4  which  had been  ( F i g . 56-58).  AA was a l s o added t o  pretreated with  LHRH o r TPA  alone  (i.e.  i n t h e absence o f FSH) f o r 18h, and t h e response o f the  cells  t o AA was q u i t e s i m i l a r  t o that  of untreated  granulosa  c e l l s g i v e n AA d u r i n g a 5h i n c u b a t i o n ( F i g . 59). The previous studies  have  inhibitory  reported  mechanism  that  LHRH  and  TPA  share  o f a c t i o n on FSH induced  P  4  a  similar  production,  and t h e i n h i b i t o r y e f f e c t s o f LHRH o r TPA c o u l d not be reversed by t h e a d d i t i o n o f FSH (Knecht 1985).  e t a l . , 1982; Shinohara  The p r e s e n t data suggested  that P  4  production  et a l . , elevated  by AA was mediated by a mechanism which was not suppressed LHRH and TPA, and g r a n u l o s a c e l l s s t i l l respond  t o AA f o l l o w i n g LHRH o r TPA  The synthesis  the  effect  form,  pretreatment.  o f AA on t h e enzymes i n v o l v e d i n progesterone  cleavage  isomerase c o n v e r t other  retained the a b i l i t y to  and metabolism was f u r t h e r examined.  side chain  hand,  P  4  by  enzymes  cholesterol  i s converted  (SCC),  to P  4  A c t i v a t i o n of . 5 .4  and 3-beta-HSD/4 -A -  v i a pregnenolone.  On the  by 20-alpha-HSD t o i t s i n a c t i v e  20-alpha-hydroxy-pregn-4-en-3-one  (20-alpha-OH-P).  Previous  s t u d i e s have  beta-HSD a c t i v i t y  indicated  that  by an i n c r e a s e  i n t h e apparent  accompanied by i n c r e a s e d accumulation 20-alpha-OH-P  production  190 i n c r e a s e s 3-  LHRH alone  (Jones  V and i s max  o f pregnenolone,  and  Hsueh,  P  1981a;  1982b).  Moreover, t h e i n h i b i t o r y e f f e c t o f LHRH on FSH induced HSD a c t i v i t y ,  FSH induced  a l s o been observed  pregnenolone and P  d u r i n g 24h g r a n u l o s a  cell  4  3-beta-  p r o d u c t i o n have  c u l t u r e , and t h i s  i n h i b i t o r y a c t i o n o f LHRH i s c h a r a c t e r i z e d by a decrease apparent  V  without  m a x  an a l t e r a t i o n  to  LHRH  TPA  (Jones  alone  and Hsueh,  stimulated  P  4  i n the  o f t h e 1^ o f t h e enzyme  (Jones and Hsueh, 1981b; 1982a; 1982b). e f f e c t on enzyme a c t i v i t i e s  and  4  TPA exerted a s i m i l a r  and FSH induced hormone p r o d u c t i o n 1982a; 1982b; Welsh e t a l . , 1984).  production  and t h e a c t i v i t i e s  o f 3-  beta-HSD and 20-alpha-HSD, l e a d i n g t o i n c r e a s e d p r o d u c t i o n o f progestin. induced  In contrast,  by gonadotrophin  beta-HSD  activity.  TPA  inhibition  of P  was accompanied by r e d u c t i o n  The  increase  i n 20-alpha-HSD  resulted i n the conversion of b i o l o g i c a l active P OH-P, LHRH  a biological and/or  TPA  inactive  on  P  4  and  compared i n t h e p r e s e n t study stimulated  biosynthesis  4  the production  metabolites.  4  o f 3-  activity  t o 20-alpha-  The e f f e c t s  20-alpha-OH-P  o f AA,  accumulation  were  ( F i g . 60). AA, LHRH, o r TPA each of  P  4  and  AA  enhanced  p r o d u c t i o n induced by LHRH and TPA ( F i g . 60; A p a n e l ) .  the  P  4  LHRH o r  TPA markedly i n c r e a s e d 20-alpha-OH-P p r o d u c t i o n , however AA was o n l y m a r g i n a l l y e f f e c t i v e i n i n c r e a s i n g 20-alpha-OH-P ( F i g . 60; panel B). with  I n a d d i t i o n , AA d i d n o t show any s y n e r g i s t i c  LHRH o r TPA on 20-alpha-OH-P p r o d u c t i o n .  These  effect results  indicate  that  biosynthesis  AA  increases  rather  than  P  production  4  significantly  191 stimulating  by  altering  20-alpha-HSD  activity. To  further  examine the  s u b s t r a t e f o r the SCC  which  as  1985).  readily  of  enters  cells  and  P-450 l e v e l s , and  access  4  of  Boyd,  in  production reflected  1981;  the  reported  to  Lino et  the  cells,  OH-cholesterol,  enzymes  were  the  of  Boyd,  SCC  to  a  4  the enzyme  1968).  activity increase  in  In  enhanced  not  rate-  progesterone most  likely  of s u b s t r a t e t o SCC,  substrate 1987;  limited  and  indicated  as p r e v i o u s l y  T o a f f e t a l . , 1982).  p r o d u c t i o n , i n the presence of 25-  lesser  extent  than  that  induced  ( F i g . 61), s u g g e s t i n g t h a t the AA s t i m u l a t i o n of  a l s o takes p l a c e a t the  the  ( F i g . 61) .  was  25-OH-cholesterol  Midgley,  increased P but  the  p r o d u c t i o n by 25-OH-cholesterol  (Bagavandoss and AA  to  o f 25-OH-cholesterol  increased a v a i l a b i l i t y  SCC  Furthermore,  and  -isomerase  presence  4  mitochondria,  observed t h a t both LHRH and TPA  granulosa  i n the  LHRH o r TPA  i s metabolized  t o be under hormone c o n t r o l  Sulimovici  -  the stimulation of P that  by  have been suggested  3-beta-HSD/  limiting  a  o f the m i t o c h o n d r i a l cytochrome  p r o d u c t i o n i n the presence 5 *4  Since  and  cholesterol  the modulation  p r e s e n t study, i t was P  activity,  (Toaff e t a l . , 1982;  cholesterol  intramitochondrial  (Leaven  SCC  S e v e r a l s t e p s i n the c h o l e s t e r o l SCC r e a c t i o n such  uptake  complexes,  on  25-OH-cholesterol i s a water s o l u b l e  s t e r o i d hormones i n m i t o c h o n d r i a al.,  of AA  enzymes, 25-OH-cholesterol, has been used  t o i n c r e a s e P. f o r m a t i o n . 4 steroid  action  l e v e l o f SCC  by P  4  which enhanced s u b s t r a t e  uptake by enhance P The  mitochondria. production  4  I n t e r e s t i n g l y , AA  p l u s TPA  enzymes;  apparently  therefore  f u r t h e r enhance P In  plasma,  the  from  liberated  cytoplasmic  lipid  in  25-OH-cholesterol  vivo  failed  synthesis  cholesterol that from  is  cholesterol  d r o p l e t s and  from 2 carbon components. cultured  of  As  of  taken  ester  up  from  stored  the  within  synthesized  i n the o v a r i a n  demonstrated  i n granulosa  cell cells  i n serum f r e e medium, c h o l e s t e r o l c o u l d come from  limiting  3-hydroxy-3-methylglutaryl  (Dorrington and Armstrong, 1979; also  increase  pregnenolone production of  enzyme  i s due  cholesterol  or  the  in  induced  production.  It FSH  induced  observed One  i s of  to  for  AA.  the  i n t e r e s t t h a t the ovarian  after  of the  account  a  steroid  relatively  steps  that  de the  reductase AA  may  prior  to  AA-induced  P  4  i n c r e a s e d endogenous s y n t h e s i s  liberation  response  could  4  some  of  There would a l s o be  mechanisms P  in  i s possible  t o e i t h e r the  esters.  reactions  It  coenzyme A  Wang and Hsueh, 1979).  activity  synthesis.  cholesterol,  above  to  ovarian  novo b i o s y n t h e s i s , which i s dependent on the a c t i v i t i e s of rate  SCC  production.  4  is  c e l l s w i t h AA p l u s LHRH or  caused maximal a c t i v i t y o f the  addition  addition,  pregnenolone  192 further  to  i n the presence of c h o l e s t e r o l s u b s t r a t e .  combined treatment o f g r a n u l o s a  w i t h AA  failed  cholesterol  a combination of  However, observed  any  long  proposed mechanisms was  time  of  increase  inhibitory effect  hormone  from  production i n culture  the  these in  AA-  of LHRH on was  only  (Fig.  54).  t h a t LHRH f u r t h e r enhanced  gonadotrophin diminished  induced  20-alpha-HSD  which  193 LHRH  production.  The  activity  by  t h e gonadotrophin s t i m u l a t i o n o f  LHRH s t i m u l a t i o n o f 20-alpha-HSD i n gonadotrophin t r e a t e d was  the r e s u l t  enzyme  o f changes  affinity  Assuming  that  i n enzyme  f o r the substrate the calcium  and  mediate t h e i n h i b i t o r y a c t i o n can  activity,  (Phillip  PKC  rather  et a l . ,  pathways  can  postulated  that  somehow  antagonize  t h e i n h i b i t o r y component  o f LHRH  by t h e p r e s e n t  that  was  suggested  decrease FSH induced P and  that  acute  4  liberation  findings  1980).  cells, i t  be  This  than  partially  o f LHRH on granulosa  LHRH-induced  cells  o f AA  AA  may  action. did  not  accumulation even a f t e r 24h ( F i g . 53)  addition  o f AA t o t h e FSH  and  CT-pretreated  c e l l s caused a p a r t i a l r e v e r s a l o f t h e i n h i b i t o r y e f f e c t o f TPA or  LHRH on P  stimulated  P  4  production production,  4  OH-P e f f e c t i v e l y  ( F i g . 56-58).  Moreover,  b u t TPA s t i m u l a t e d  both P  AA 4  mainly  and 20a-  ( F i g . 60). These data suggest t h a t a c t i v a t i o n  of PKC may w e l l mediate t h e l o n g term i n h i b i t o r y a c t i o n o f LHRH on  P  4  accumulation,  contrast, role  After  term  stimulatory  by enhancing t h e a c t i v i t y  potential  PKC)  P  t o 20-alpha-OH-P.  4  (or i t s a c t i v e metabolites)  i n the short  production the  AA  by c o n v e r t i n g  most l i k e l y  effect o f SCC.  i n h i b i t o r y e f f e c t s o f LHRH  of  played  LHRH  Taken  on  In a P  4  together,  (via a c t i v a t i o n of  might have been prevented by AA d u r i n g t h e 5h i n c u b a t i o n s . that,  inactive  t h e LHRH-induced- f r e e  metabolites  and  AA may  the i n h i b i t o r y  convert  t o some  component  o f LHRH  a c t i o n became dominant. In a d d i t i o n , study o f t h e f u n c t i o n s o f TPA, A23187 and AA  has  provided  metabolites  evidence  fora participation  o f AA i n t h e mediation  o f PKC, C a  2 +  and  o f LHRH a c t i o n (chapter 4 ) .  However, i t was u n l i k e l y t h a t c e l l u l a r responses i n v o l v i n g PKC, calcium  and m e t a b o l i t e s  o f AA were j u s t  membrane and cytoplasm. extranuclear including  events  regulation of P 4 5 0 Therefore,  There was accumulating  were  changes  interaction  of  gene  i n long  o f these  LHRH  t o the c e l l evidence  by n u c l e a r  on  FSH  that  components,  expression,  mRNA by FSH (Richards  the action  steroidogenesis  coordinated  i n specific g c c  limited  i.e.  and Hedin, induced  term  culture  may  be  hormones  on gene  expression.  the  1988). ovarian  due t o t h e It  has  a l r e a d y been shown t h a t FSH a d m i n i s t r a t i o n t o hypophysectomized rats  causes  granulosa clearly of  the increase  cells  i n cytochrome  and FSH induced  regulation  granulosa  o f gene  o  mRNA i n  gene e x p r e s s i o n which i s only  demonstrable a f t e r 7 t o l O h .  LHRH  P450 __  Thus f a r , no d i s c u s s i o n  expression  has been  made i n  cells.  In c o n c l u s i o n , t h e p r e s e n t hypothesis  results  t h a t AA and i t s lipoxygenase  s t r o n g l y support t h e metabolites  partially  mediate t h e a c t i o n o f LHRH by p l a y i n g a s t i m u l a t o r y r o l e i n t h e direct  effects  of  LHRH  on  ovarian  hormone  production.  Furthermore, i t i n d i c a t e s t h a t t h e a c t i o n s o f LHRH o r LHRH-like peptide  on granulosa  cells  a r e mediated  by t h e d i f f e r e n t  intracellular  signal  between these  pathways u l t i m a t e l y d i c t a t e s t h e time-dependent  steroidogenic  response  peptide.  pathways, and t h a t t h e complex  o f t h e ovary  t o LHRH  interplay  o r LHRH-like  195 General  Summary  Although  gonadotropins  that  regulate ovarian  that  local  a r e t h e major  functions,  regulators  trophic  hormones  i n c r e a s i n g evidence  suggests  participate i n paracrine  or  autocrine  c o n t r o l of ovarian functions. The  d i r e c t a c t i o n s o f LHRH on r a t o v a r i a n c e l l s have been  documented. its  second  initial  Unlike  gonadotropins,  messenger.  action  o f LHRH  Increasing  evidence  involves  rapid  metabolism o f membrane i n o s i t o l present  LHRH does n o t use cAMP as  a  lipids  shows  t h a t the  alteration  i n the  i n t h e ovary.  In the  study, t h e a c t i o n s o f LHRH on t h e breakdown o f membrane 2+  phosphoinositides, of  steroid  changes o f i n t r a c e l l u l a r  hormones and p r o s t a g l a n d i n s  were e x t e n s i v e l y s t u d i e d . the  rapid  and  trisphosphate  ,  production  i n r a t granulosa  In r a d i o l a b e l e d r a t granulosa  specific  and  Ca  formation  of  diacylglycerol,  and  inositol the  cells cells, 1,4,5-  release  of  a r a c h i d o n i c a c i d were observed s h o r t l y a f t e r a d d i t i o n o f LHRH. 2+ LHRH a l s o caused a r a p i d and t r a n s i e n t i n c r e a s e the  majority  of  granulosa  microspectrofluorimetry. is  produced  hydrolysis  Inositol  simultaneously  of PIP /  m  a  v  2  cells  D  e  as  fura-2  1,4,5-trisphosphate,  which  with  responsible  i s known t h a t LHRH e x e r t s  a c t i o n s on o v a r i a n c e l l s presence  o f other  assessed  ] i in  by  diacylglycerol  by  f o r t h e LHRH 2+  induced  r a p i d and t r a n s i e n t a l t e r a t i o n s o f [Ca It  i n [Ca  ] i i n granulosa  PLC  cells.  e i t h e r stimulatory or inhibitory  depending on t h e c u l t u r e p e r i o d , the  hormones  such  as  gonadotropins,  and the  nature  o f t h e hormone examined.  To t e s t  the hypothesis  196 that  the e f f e c t s o f LHRH on g r a n u l o s a c e l l s were mediated, a t l e a s t in  part,  by  ionophore  calcium  and  PKC,  the e f f e c t s  o f t h e calcium  A23187 and t h e phorbol e s t e r TPA on t h e p r o d u c t i o n o f  progesterone  and PGE  have been examined.  2  The p r e s e n t  study  demonstrated t h a t LHRH i n h i b i t e d t h e p r o d u c t i o n o f progesterone stimulated  by  production action  stimulated  simultaneously  by FSH.  cAMP f o r m a t i o n .  of LHRH.  These  enhancing  data  a t a step(s)  suggest  following  PGE  2  that the  gonadotropin  TPA and A23187 can mimic t h e a c t i o n s  I n t e r e s t i n g l y , TPA acted s y n e r g i s t i c l y w i t h A23187 on  production  2  while  o f LHRH i s mainly  induced  PGE  FSH,  b u t not on t h e p r o d u c t i o n  of  progesterone,  2+ s u g g e s t i n g m u l t i p l e aspects o f PKC and Ca a c t i o n on granulosa cells.  I t appears  that  activation  o f PKC and a l t e r a t i o n o f  2+ [Ca  ] i n o t o n l y mediates c e l l u l a r p r o c e s s e s ,  membrane  phosphoinositide  potential  feedback  metabolism,  control  but a l s o  thus  mechanism.  alters  providing Increased  a free  arachidonic acid l e v e l  induced by LHRH s e r v e s as t h e p r e c u r s o r  for  of  the  synthesis  metabolites  of  cyclooxygenase very  important  Although  arachidonic metabolites role  o v u l a t i o n process.  acid.  and  lipoxygenase  Prostaglandins  of arachidonic  acid  which  are t h e play  a  i n r e p r o d u c t i v e f u n c t i o n s o f t h e ovary.  the production  coupled t o progesterone the  cyclooxygenase  o f p r o s t a g l a n d i n may  n o t be  tightly  production, i t i s c e r t a i n l y involved i n In t h e p r e s e n t  study,  pathway m e t a b o l i t e s a p p a r e n t l y p a r t i c i p a t e d a c t i o n o f LHRH p r o b a b l y by enhancing  the  lipoxygenase  i n the stimulatory  action  of protein  197  LHRH  LH FSH  cholesterol  HPETEs -1- — HETES LTs  f I I I  J  L  [Ca"']i mobilization  F i g . 62. I l l u s t r a t i o n o f t h e i n t e r a c t i o n s between l u t e i n i z i n g hormone-releasing hormone (LHRH) and gonadotrophin second messenger pathways. A b b r e v i a t i o n s : LH, l u t e i n i z i n g hormone; FSH, f o l l i c l e s t i m u l a t i n g hormone; R, r e c e p t o r ; DG, 1,2d i a c y l g l y c e r o l ; cAMP, 3 ' 5 ' - c y c l i c adenosine monophosphate; PKC, p r o t e i n k i n a s e C; ER, endoplasmic r e t i c u l u m ; AA, a r a c h i d o n i c acid; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic £cid; LT, leukotriene; PG, prostaglandin; [Ca ] i , i n t r a c e l l u l a r calcium ion concentration; IP , inositol 1,4,5,-trisphosphate; P , progesterone. 2  3  4  198 kinase  C  on  interaction  t h e enzymes  involved  i n steroidogenesis.  between t h e gonadotropins  The  and LHRH a r e summarized  s c h e m a t i c a l l y i n F i g . 62. The roles  evidence  of  LHRH  f o r the possible  in  ovarian  demonstration  o f t h e presence  rat,  and  bovine  involvement  ovine  o f these  cells  paracrine  or autocrine  i s strengthened  o f LHRH-like p e p t i d e s  ovaries  different  in  other  hormonal  by the i n human,  sdudies.  systems  and m u l t i p l e  second messenger mechanisms i n t h e r e g u l a t i o n o f g r a n u l o s a f u n c t i o n ensures  cell  t h e o p t i m a l o v a r i a n hormone s y n t h e s i s and t h e  growth o f t h e o v a r i a n cells  The  follicles.  I n a d d i t i o n , r a t granulosa  s e r v e as an i d e a l model f o r s t u d i e s on t h e mechanism of  hormone  action  because  of  t h e presence  of  both  cAMP and  2+ Ca  - p r o t e i n k i n a s e C pathways.  should  help  future  elucidation  hormone p r o d u c t i o n and o v u l a t i o n .  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