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Action of luteinizing hormone-releasing hormone in rat ovarian cells : hormone production and signal.. 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. (Medicine), Harbin Medical University, 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 of Physiology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1989 (c) Jian Wang, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Department DE-6 (2/88) Abstract The present study was conducted to investigate the hypothesis that membrane phosphoinositide breakdown may pa r t i c i p a t e i n the actions of l u t e i n i z i n g hormone-releasing hormone (LHRH) on hormone production i n the r a t ovary. 3 . . . In granulosa c e l l s prelabeled with [ H]-arachidonic acid 3 or [ H ] - i n o s i t o l , treatment with LHRH increased the accumulation of radiolabeled i n o s i t o l l i p i d s , d i a c y l g l y c e r o l and free arachidonic acid, but l u t e i n i z i n g hormone (LH) or cholera toxin did not exert the same e f f e c t . A c t i v a t i o n of protein kinase C by the phorbol ester, 12-0-tetradecanoyl phorbol-13-acetate (TPA) had a stimulatory action on membrane phosphoinositide breakdown. In addition, TPA did not a l t e r arachidonic acid release but potentiated the A23187 stimulated l i b e r a t i o n of arachidonic acid. Changes in the c y t o s o l i c free calcium ion concentrations, 2+ [Ca ] i , induced by LHRH were studied i n in d i v i d u a l c e l l s using 2+ . fura-2 microspectrofluorimetry. The rest i n g [Ca ] i was 96.7 ± 2+ . 2.9 nM (n= 115). The alt e r a t i o n s i n [Ca ] i induced by LHRH were transient and returned to rest i n g l e v e l s within 84±3 second (n=64). A potent LHRH antagonist completely blocked the 2 + e f f e c t of LHRH on [Ca ] i . Some c e l l s responded to LHRH alone, whereas others responded to angiotensin I I , suggesting that there are d i f f e r e n t subpopulations of granulosa c e l l s . Sustained perifusion of LHRH resulted i n a desensitization of the [ C a 2 + ] i response to LHRH but not to the calcium ionophore 2+ A23187. LHRH treatment accelerated [Ca ] i depletion i n i i i 2+ granulosa c e l l s perifused with Ca free medium, i n d i c a t i n g 2+ 2+ the involvement of i n t r a c e l l u l a r Ca pool(s) i n [Ca ] i changes induced by LHRH. The complex interactions between the s i g n a l transduction pathways involved i n the regulation of progesterone and prostaglandin E 2 were also examined. LHRH increased basal progesterone l e v e l (5 and 24h culture) and attenuated progesterone production induced by f o l l i c l e - s t i m u l a t i n g hormone (FSH) or cholera toxin (24h) . On the other hand, both basal and FSH or cholera toxin stimulated prostaglandin E 2 formation were increased by LHRH (5 and 24h) . A23187, TPA and m e l i t t i n (an a c t i v a t o r of phospholipase A 2) were used to examine the 2+ roles of Ca , protein kinase C and free arachidonic acid, respectively, i n LHRH action. M e l i t t i n stimulated basal progesterone and prostaglandin E 2 production, and enhanced the stimulation of prostaglandin E 2 by LHRH, A23187 and TPA, in d i c a t i n g that LHRH a l t e r s cyclooxygenase a c t i v i t y . A23187 or TPA attenuated the formation of progesterone induced by FSH or cholera toxin (5 and 24h). In contrast, A23187 and TPA augmented cholera toxin or FSH induced prostaglandin E 2 formation. The stimulatory e f f e c t s of A23187 and TPA on prostaglandin E 2 were syn e r g i s t i c , whether or not FSH or cholera toxin was present during the incubation. The r o l e of arachidonic acid i n the action of LHRH was further investigated. Arachidonic acid enhanced progesterone production i n a dose dependent manner and potentiated TPA induced progesterone production. The stimulatory e f f e c t of arachidonic acid was blocked by nordihydroguaiaretic acid, i v whereas monohydroxyeicosatetraenoic acids and hydroperoxyeicosatetraenoic acid mimicked the e f f e c t of arachidonic acid, suggesting the involvement of lipoxygenase metabolites i n LHRH action. In addition, arachidonic acid p a r t i a l l y reversed the i n h i b i t o r y action of LHRH and TPA on FSH induced progesterone production. Although arachidonic acid, TPA and LHRH stimulated progesterone production, arachidonic acid only s l i g h t l y elevated 20-alpha-hydroxy- progesterone production as compared to that induced by LHRH and TPA. These results suggest that arachidonic acid or i t s metabolites have a stimulatory r o l e i n the action of LHRH on the de novo synthesis of ovarian s t e r o i d hormones. C o l l e c t i v e l y , these findings support the hypothesis that the actions of LHRH or LHRH l i k e peptides on ovarian hormone production are mediated by multiple second messengers involving 2+ Ca , protein kinase C and arachidonic acid metabolites. V Table of Contents Page Abstract i i L i s t of Tables v i i i L i s t of Figures i x L i s t of Abbreviations x iv Acknowledgements x v i Chapter 1. General Introduction I. Ovary 1 A. Introduction 1 B. Histology 2 C. L i f e c y c l e of the ovarian f o l l i c l e 4 I I . Synthesis of Sex Steroid Hormones and Prostaglandins 6 A. Synthesis of sex steroids 6 B. Synthesis of prostaglandins and leukotrienes 10 I I I . Regulation of ovarian hormone Synthesis 13 A. Role of gonadotropins 13 B. Intraovarian regulation by f o l l i c u l a r steroids 18 C. Role of neurotransmitters on ovarian steroidogenesis 21 D. Regulation of ovarian steroidogenesis and ovarian function by prostaglandins 22 E. Role of l o c a l nonsteroidal regulators on ovarian function 23 v i IV. Signal Transduction Systems and Hormone Action 28 A. Introduction 28 B. C y c l i c AMP 29 C. Calcium and protein kinase C 32 V. The aim of the present study 37 Chapter 2. Induction of Polyphosphoinositide Turnover and Arachidonic Acid Release by LHRH 38 I. Introduction 38 I I . Materials and Methods 40 I I I . Results 45 IV. Discussion 56 Chapter 3. E f f e c t of LHRH on Changes of Cytosolic free Calcium Ion Concentration i n Individual Granulosa c e l l s 69 I. Introduction 69 II . Materials and Methods 71 I I I . Results 75 IV. Discussion 95 Chapter 4. LHRH Action on Ovarian Hormone Production; a l t e r a t i o n of Progesterone and Prostaglandin accumulation by Calcium Ionophore and Protein Kinase C 106 v i i I. Introduction 106 I I . Materials and Methods 107 II I . Results 110 IV. Discussion 130 Chapter 5. Role of Arachidonic Acid i n LHRH Action 142 I. Introduction 142 II. Materials and Methods 143 I I I . Results 146 IV. Discussion 179 General summary 195 References 199 v i i i L i s t of Tables page Table I. Lowest hormone concentrations required by 80 granulosa c e l l s for i n i t i a t i n g c y t o s o l i c calcium change. 2 + Table I I . Average peak value of [Ca ] i induced 80 by d i f f e r e n t doses of LHRH. I X L i s t of Figures Page Fi g . 1. The p r i n c i p a l biosynthetic pathway i n the 7 ovary for production of the progestins, androgens and estrogens. F i g . 2. Key pathways i n arachidonic acid metabolites. 11 Fi g . 3. Diagram of the "two c e l l , two gonadotropin 15 theory" of f o l l i c l e steroidogenesis. F i g . 4. General model of cAMP mediated hormone 31 response. F i g . 5. I n o s i t o l phospholipid turnover and signal 33 transduction. F i g . 6. Stimulatory e f f e c t s of LHRH on the formation of 46 i n o s i t o l phosphates (IP ), DG, and the release of un e s t e r i f i e d AA i n rat granulosa c e l l s . F i g . 7. E f f e c t of LHRH on [ 3H]-labeled d i a c y l g l y c e r o l 47 (DG) formation. F i g . 8. Time n di a c y l g l y c e r o l formation by LHRH. response of stimulation of [ 3H]-labeled 49 . 3 F i g . 9. Comparison of LH and LHRH on [ H]-labeled 50 i n o s i t o l phosphates. F i g . 10. Comparison of LH and LHRH on d i a c y l g l y c e r o l 51 formation and arachidonic a c i d release. F i g . 11. E f f e c t of cholera toxin (CT) and LHRH on 53 [ H]-labeled i n o s i t o l phosphate formation. F i g . 12. E f f e c t of phospholipase C (PLC) on [ 3H]- 54 labeled d i a c y l g l y c e r o l formation. F i g . 13. Action of the phorbol ester TPA on i n o s i t o l 55 phosphate formation. F i g . 14. S p e c i f i c i t y of the phorbol ester TPA action 57 on d i a c y l g l y c e r o l formation. F i g . 15. Interaction of the calcium ionophore A23187 58 and the phorbol ester TPA on arachidonic acid release. F i g . 16. Scheme showing proposed mechanisms involved i n 67 arachidonic acid release. F i g . 17. The calcium ionophore A23187-induced rapid and 77 transient increase i n c y t o s o l i c calcium. Fi g . 18. LHRH-induced rapid and transient increase i n c y t o s o l i c calcium. F i g . 19. The blockade of LHRH-induced c y t o s o l i c calcium a l t e r a t i o n by LHRH antagonist. Fi g . 20. Existence of subpopulation of granulosa c e l l s : [Ca ] i changes induced by LHRH and Angiotensin II (Ang I I ) . 2+ . . Fig . 21. Increase i n [Ca ] i induced by separate in j e c t i o n s of LHRH to two i n d i v i d u a l granulosa c e l l s . 2+ Fig . 22. Desensitization of [Ca ] i response induced by continuous exposure to LHRH. 2+ Fig . 23. Alterations i n [Ca ] i induced by d i f f e r e n t doses of LHRH. 2 + Fig . 24. Depletion of i n t r a c e l l u l a r Ca i n calcium free medium. Fig . 25. LHRH-accelerated [ C a 2 + ] i depletion i n C a 2 + free medium. 2+ Fig. 26. Role of_e.xtracellular Ca i n LHRH-induced a l t e r a t i o n of [Ca ] i . Fig . 27. Comparision of FSH with LHRH on [ C a 2 + ] i a l t e r a t i o n . 2+ Fig . 28. Comparision of LH with LHRH on [Ca ] i al t e r a t i o n . F i g . 29. Interaction of m e l i t t i n (Mel, M; 3x10 M), with LHRH (L; 10 M) or the phorbol ester TPA (T; 10~ M) on progesterone (PROG) production (upper panel) and PGE. formation (lower panel) during a 5h culture. F i g . 30. Effects of m e l i t t i n and/or the calcium ionophore A23187 on progesterone and PGE_ production during a 5h culture period. F i g . 31. Effects of the phorbol ester TPA and/or increasing concentrations of the calcium ionophore A23187 on progesterone and PGE_ production during a 5h culture period. Fi g . 32. Effects of the calcium ionophore A23187 and/or increasing concentrations of the phorbol ester x i TPA on progesterone and PGE, production during a 5h culture period. F i g . 33. E f f e c t s of the calcium ionophore A23187 117 and/or the phorbol ester TPA on PGE_ production, e i t h e r i n the absence (open bars) or presence (hatched bars) of m e l i t t i n during a 5h culture period. F i g . 34. E f f e c t s of cholera toxin (CT) and/or LHRH on 120 progesterone and PGE_ production during a 5h culture period. F i g . 35. E f f e c t s of the calcium ionophore A23187 121 and/or cholera toxin (CT) on progesterone and PGE_ production during a 5h incubation period. F i g . 36. E f f e c t s of the calcium ionophore A23187 122 and/or the phorbol ester TPA on basal (open bars) or FSH stimulated (hatched bars) production of progesterone and PGE 2 production during a 5h culture period. F i g . 37. E f f e c t s of the calcium ionophore A23187 125 and/or the phorbol ester TPA on basal (open bars) or CT-stimulated (hatched bars) PGE- production during a 5h culture period. F i g . 38. Interaction of FSH and LHRH on the formation 126 of progesterone (PROG) (panel, A), PGE- (panel, B), and PGF 2alpha (panel, C) during a 24h culture period. F i g . 39. Interaction of FSH and the phorbol ester TPA 127 on progesterone and PGE, formation during a 24h culture period. F i g . 40. Interaction of FSH, the phorbol ester TPA 129 and the calcium ionophore A23187 on progesterone and PGE 2 formation during a 24h culture period. F i g . 41. Stimulatory effects of m e l i t t i n , LHRH and 148 arachidonic acid (AA) on progesterone (PROG) production during a 5h culture period. F i g . 42. E f f e c t of increasing concentration of 149 arachidonic acid (AA) on progesterone production during a 5h culture period. F i g . 43. E f f e c t s of unsaturated f a t t y acids on 150 progesterone production. F i g . 44. E f f e c t s of treatment of granulosa c e l l s with 152 arachidonic acid (AA) and LHRH or a LHRH agonist (LHRHa) on progesterone production. x i i F i g . 45. Time course of stimulation of progesterone 153 production by arachidonic acid (AA), LHRH or LHRH plus AA. Fi g . 46. E f f e c t s of the phorbol ester TPA and 155 increasing concentrations of arachidonic a c i d (AA) on progesterone production. F i g . 47. Ef f e c t s of arachidonic acid (AA) and 156 increasing concentrations of the phorbol ester TPA on progesterone production. F i g . 48. Role of arachidonic acid (AA) metabolism. 157 Fig. 49. E f f e c t s of nordihydroguaiaretic a c i d (NDGA) 159 or indomethacin (INDO) on progesterone production induced by LHRH and/or arachidonic acid (AA). Fig. 50. Ef f e c t s of hydroxyeicosatetraenoic acids 160 (HETEs) and hydroperoxyeicosatetraenoic acids (HPETEs) on progesterone production. F i g . 51. E f f e c t s of hydroxyeicosatetraenoic acids 163 (HETEs) on progesterone (upper panel) and PGE, (lower panel) production. Fig . 52. Interactions of hydroxyeicosatetraenoic 164 acids (HETEs) or hydroperoxyeicosatetraenoic acids (HPETEs) with LHRH on progesterone (upper panel) and PGE 2 (lower panel) production. Fig . 53. Interactions of hydroxyeicosatetraenoic 165 acids (HETEs) or hydroperoxyeicosatetraenoic acids (HPETEs) with the phorbol ester TPA on progesterone (upper panel) and PGE, (lower panel) production during a 5h culture period. Fig. 54. E f f e c t of LHRH on FSH-induced progesterone 167 production: time response. Fig . 55. E f f e c t s of treatment of r a t granulosa c e l l s 168 with arachidonic acid (AA) and/or FSH on progesterone production. Fig . 56. Response to arachidonic acid (AA) a f t e r 170 pretreatment with FSH and LHRH. Fig. 57. Response to arachidonic acid (AA) a f t e r 171 pretreatment with FSH and the phorbol ester TPA. Fig. 58. Response to arachidonic acid (AA) a f t e r 173 pretreatment with cholera toxin (CT) and the phorbol ester TPA. x i i i F i g . 59. Response to arachidonic a c i d (AA) a f t e r 174 pretreatment with the phorbol ester TPA and LHRH alone. F i g . 60. E f f e c t s of LHRH, the phorbol ester TPA and/or 177 arachidonic acid (AA) on progestin production during a 5h incubation. F i g . 61. E f f e c t of arachidonic a c i d (AA), the phorbol 178 ester TPA and LHRH on 25-hydroxycholesterol-enhanced steroidogenesis during a 5h incubation. F i g . 62. I l l u s t r a t i o n of the inte r a c t i o n s between 197 LHRH and gonadotrophin second messenger pathways. R, receptor. L i s t of Abbreviations AA GABA °C [ C a 2 + ] i CAMP CT DG DPM ER EBSS FBS FSH G GDP GTP h hCG HDL HETE HPETE 20-alpha-HSD 3-beta-rHSD 17-beta-HSD I P 3 IU arachidonic acid gamma-aminobutyric acid degree Celsius i n t r a c e l l u l a r calcium ion concentration 31 5 ' - c y c l i c adenosine monophosphate cholera toxin 1,2-diacylglycerol d i s i n t e g r a t i o n per minute endoplasmic reticulum E a r l ' s Balanced S a l t Solution f e t a l bovine serum f o l l i c l e stimulating hormone guanine nucleotide regulatory protein guanosine diphosphate guanosine triphosphate hour human chorionic gonadotrophin high density l i p o p r o t e i n hydroxyeicosatetraenoic acid hydroperoxyeicosatetraenoic acid 2O-alpha-hydroxysteroid dehydrogenase 3-beta-hydroxysteroid dehydrogenase 17-beta-hydroxysteroid dehydrogenase i n o s i t o l 1,4 ,5-trisphosphate int e r n a t i o n a l u n i t LDL low density l i p o p r o t e i n LH l u t e i n i z i n g hormone LHRH l u t e i n i z i n g hormone relea s i n g hormone LT leukotriene LX l i p o x i n min minute M molar NADPH nicotinamide adenine dinucleotide phosphate NDGA nordihydroguaiaretic a c i d 20-alpha-OH-P 20-alpha-hydroxypregn-4-en-3-one P s t a t i s t i c a l p r o b a b i l i t y P4 progesterone PG prostaglandin PI phosphatidylinositol PIP phosphatidylinositol 4-phosphate PIP 2 phosphatidylinositol 4,5-phosphate PKC protein kinase c PLA 2 phospholipase A 2 PLC phospholipase C PMSG pregnant mare's serum gonadotrophin PRL p r o l a c t i n RIA radio-immunoassay sec side-chain cleavage sec second SE standard error TPA 12-0-tetradecanolyphorbol-13-acetate TX thromboxane xvi Acknowledgements This thesis would not have been completed without the help from many people. I am very gra t e f u l to my supervisor, Dr. Peter C.K. Leung and thank him for h i s advice, encouragement and friendship throughout my study. My deep appreciation i s given to Drs. H. McLennan, N.W. Kasting, R. A. Pederson and R.W.Brownsey for serving on my committee and t h e i r guidance. I wish to give special thanks to Dr. K.G. Bainbridge for his great assistance with fura - 2 microspectrofluorimetry. I would also l i k e to express my appreciation to Dr. K.H. Chen, M. Rodway and D. Sidpra for reading and correcting my thes i s . I take t h i s opportunity to express my gratitude to my parents and other family members for t h e i r emotional support and encouragement. F i n a l l y , I would l i k e to thank the Killam Fellowship program at the University of B r i t i s h Columbia for providing the predoctral fellowship. Chapter 1. General Introduction 1 I. Ovary A. Introduction The function of the ovary i s to produce mature eggs and secrete ovarian hormones. The l a t t e r exert a range of effects including regulation of the reproductive system, secondary sex characters, the mating behavior of some species, p i t u i t a r y gonadotropin release and metabolic e f f e c t s . The gametogenic and endocrine functions of the ovary i n the female are c y c l i c processes e x h i b i t i n g regular peaks of a c t i v i t y during the l i f e of the i n d i v i d u a l , and may be regarded as peri o d i c preparations fo 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 estrous cycle i n subprimate species and the menstrual cycle i n primates. The c y c l i c a l changes occur as a r e s u l t of complex integrated a c t i v i t y of the hypothalamus, p i t u i t a r y and ovaries. The most important hormone signals of t h i s system are l u t e i n i z i n g hormone-releasing hormone (LHRH) from the hypothalamus, f o l l i c l e stimulating hormone (FSH) and l u t e i n i z i n g hormone (LH) from the anterior p i t u i t a r y gland and the ovarian s t e r o i d hormones such as androgens, estrogens and progesterone ( P 4 ) . The gonadotroph c e l l s of p i t u i t a r y synthesize and secrete LH and FSH i n response to LHRH. LH and FSH arrive at the ovary v i a the c i r c u l a t o r y system. FSH causes ovarian f o l l i c u l a r growth, while the LH surge induces ovulation and regulates corpus luteum formation and function. Both FSH 2 and LH are necessary to stimulate ovarian steroidogenesis. Ovarian 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 4, i n turn, regulate FSH, LH and LHRH release by either a p o s i t i v e or negative feedback mechanism depending on the stage of the estrous or menstrual cycle. Recently, i t has been shown that a family of peptides known as i n h i b i n regulates FSH release s e l e c t i v e l y , and that the production of these peptides i s con t r o l l e d by FSH. The inhibins thus represent an additional closed feedback loop between the p i t u i t a r y and ovary to regulate reproductive functions (Rivier et a l . , 1986). Other l o c a l regulatory factors such as prostaglandins and LHRH-like peptides may also be involved i n the regulation of reproductive functions. B . Histology The ovaries are paired organs situated on eithe r side of the uterus. Each ovary i s covered by a continuous mesothelium composed of a single layer of cuboidal epithelium. The ovary i s roughly divided into a peripheral cortex and a medulla. The cortex contains numerous ovarian f o l l i c l e s that consist of a primary oocyte enveloped by a si n g l e layer of spindle-shaped granulosa c e l l s i n various stages of development and a dense connective t i s s u e stroma. The medulla i s small compared to the cortex, and i t s connective t i s s u e i s loosely arranged. Embedded within the loose connective t i s s u e of the medulla are nerves, lymph vessels and many large blood vessels. Small blood vessels extend from the medulla into the cortex. 3 C o r t i c a l stroma consists of at l e a s t three types of c e l l s : connective tissue c e l l s performing the customary support functions, c o n t r a c t i l e c e l l s scattered i n the c o r t i c a l stroma and i n the walls of preovulatory 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 classes of i n t e r s t i t i a l c e l l s have been i d e n t i f i e d : 1) primary i n t e r s t i t i a l ; 2) theca i n t e r s t i t i a l ; 3) secondary i n t e r s t i t i a l ; and 4) h i l u s i n t e r s t i t i a l c e l l s . Although these c e l l s are located i n the loose connective tissue of both the cortex and medulla, a l l a r i s e from a population of unspecialized mesenchymal c e l l s i n the stroma compartment. The p r i n c i p a l function of the i n t e r s t i t i a l c e l l s i s to synthesize and secrete steroids, most notably androstenedione and testosterone. I t appears that granulosa c e l l s are derived mainly from c e r t a i n c e l l s within the intraovarian rete o v a r i i which resemble granulosa c e l l s i n terms of t h e i r organelles and microfilaments (Byskov, 1978; Byskov and Rasmussen, 1973). The d i f f e r e n t i a t i o n of granulosa c e l l s i s not uniform i n a given f o l l i c l e . As the a n t r a l f o l l i c l e develops, these c e l l s become organized into morphologically distinguishable regions with speci a l i z e d functions. At least three d i f f e r e n t populations of granulosa c e l l s can be distinguished. The antral granulosa c e l l s are closer to the antral cavity, while the cumulus c e l l s surround the oocyte. Cumulus c e l l s p h y s i c a l l y support the oocyte within the f o l l i c l e and provide nutrients for oocyte growth. They also probably exchange signals with the oocyte 4 for the coordinated maturation of the f o l l i c l e and the oocyte. The majority of granulosa c e l l s are mural or p a r i e t a l granulosa c e l l s l i n i n g the f o l l i c u l a r c a v i t y . C. L i f e cycle of the ovarian f o l l i c l e The primordial f o l l i c l e s are present before b i r t h . The oocyte and associated spindle-shaped c e l l s are separated from the surrounding stroma by the basal membrane. One of the basic events within the ovary i s f o l l i c u l a r growth, an i r r e v e r s i b l e process, which r e s u l t s i n ovulation or a t r e s i a . At the onset of puberty, primordial f o l l i c l e s mature into primary f o l l i c l e s , which are subject to intra-ovarian controls (Peters et a l . , 1975). F o l l i c u l a r maturation i s i n i t i a t e d when the spindle- shaped granulosa c e l l precursors d i f f e r e n t i a t e into a single layer of cuboidal c e l l s that then begin to divide (Van Wagenen and Simpson, 1965) . The oocyte increases i n s i z e and the granulosa c e l l s p r o l i f e r a t e m i t o t i c a l l y . Granulosa c e l l s synthesize and secrete mucopolysaccharides, which give r i s e to the zona p e l l u c i d a that surrounds the oocyte. Afte r the granulosa c e l l s begin to p r o l i f e r a t e i n the primary f o l l i c l e s , the f o l l i c l e becomes encapsulated by d i s t i n c t layers of theca c e l l s , the theca interna and the theca externa. The theca interna i s separated from the granulosa layer by the basement membrane. Blood vessels and lymphatics penetrate the theca externa but do not penetrate the basement membrane thus granulosa c e l l s are without d i r e c t blood supply u n t i l a f t e r ovulation. As the f o l l i c l e grows, the granulosa c e l l s increase 5 i n number and si z e . F o l l i c u l a r f l u i d accumulates within the f o l l i c l e and coalesces to form a single cavity, the antrum. Antral formation transforms the primary f o l l i c l e into a Graafian f o l l i c l e . Within t h i s i s the cumulus oophorus, an accumulation of granulosa c e l l s containing the oocyte. This oocyte i s li b e r a t e d when the mature f o l l i c l e ruptures following the LH surge i n a process c a l l e d ovulation. Following ovulation, the corpus luteum i s formed from both the granulosa and theca interna c e l l s . The basement membrane breaks 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 the theca interna invade the cavi t y of the ruptured f o l l i c l e . The granulosa c e l l s do not divide a f t e r ovulation, but they increase i n volume and undergo morphologic changes with an increase i n masses of l i p i d droplets, smooth endoplasmic reticulum, and mitochondria. These changes are referred to as l u t e i n i z a t i o n . Since the c e l l s of the corpus luteum are derived from both granulosa and theca c e l l s , the corpus luteum consists of two types of steroidogenic l u t e a l c e l l s , which are morphologically d i s t i n c t , the large l u t e a l c e l l s and the small l u t e a l c e l l s . These c e l l s , together with the surrounding theca c e l l s , c a p i l l a r i e s and blood vessels form the corpus luteum, a temporary endocrine gland that secretes large amounts of st e r o i d hormones. I I . Synthesis of sex s t e r o i d hormones and prostaglandins 6 A. Synthesis of sex steroids Ovaries have the capacity to synthesize a l l three classes of sex ster o i d hormones from t h e i r common precursor, cholesterol (Fig. 1). Cholesterol from both low-density lipoproteins (LDL) and high-density l i p o p r o t e i n s (HDL) has been demonstrated to serve as precursor f o r steroidogenesis i n the ovarian f o l l i c l e (Gwynne and Strauss, 1982). While HDL appears to be the major precursor i n rodents, c h o l e s t e r o l from LDL i s the major precursor i n other species. C e l l u l a r cholesterol may be derived from plasma lip o p r o t e i n , from cytoplasmic l i p i d droplets or synthesized de novo i n ovarian c e l l s (Strauss et a l . , 1981). Uptake of l i p o p r o t e i n from plasma i s regulated by the a v a i l a b i l i t y of serum lipoproteins and the lipoprotein receptor-dependent uptake system. Cholesterol can be stored i n the c e l l s as esters of long-chain f a t t y a c i d and t h i s process i s regulated by the r e l a t i v e a c t i v i t i e s of cholesterol synthetase and cholesterol esterase. A d d i t i o n a l l y , de novo synthesis of cholesterol i s dependent on the a c t i v i t i e s of the r a t e - l i m i t i n g 3-hydroxy-methylglutaryl coenzyme A reductase (Brown et a l . , 1981). Granulosa c e l l s are the c e l l u l a r source of the two most important ovarian steroids, e s t r a d i o l and P^. The f i r s t step i n the conversion of cholesterol to steroids i s believed to be rate l i m i t i n g i n steroidogenesis, and involves the cleavage of the cholesterol side-chain by the side-chain cleavage P-450 17alpha-hydroxypregnenolone • 17alpha-hydroxyprogesterone Dehydroepiandrosterone • Androstenedione—• Estrone Testosterone • Estradiol-17beta Fi 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 for production of the progestins, androgens and estrogens. 1: cholesterol side-chain cleavage P 4 5 0 2: 17-alpha-hydroxylase 3 : C17, 2 < T 1 Y a S e 4: 17-beta-hydroxysteroid dehydrogenase 5 MA 5: 3-beta-hydroxysteroid dehydrogenase/d '^1 isomerase. 6: Aromatase 7: 20-alpha-hydroxysteroid dehydrogenase 2Oalpha-OH-P: 2Oalpha-hydroxypregn-4-en-3-one 8 enzyme (SCC) r e s u l t i n g i n the C 2 1 compound, pregnenolone. SCC, including cholesterol 22-hydroxylase, cholesterol 20-alpha- hydroxylase and C 2 Q 2 2 - l y a s e , are located i n the inner mitochondrial membrane. Pregnenolone i s the key steroidogenic intermediate common to a l l classes of s t e r o i d hormones produced by the f o l l i c l e s . Both granulosa and theca c e l l s convert pregnenolone to P 4, but granulosa c e l l s are more active i n t h i s regard (Bjersing, 1967). Pregnenolone i s converted to P 4 by a complex of two enzymes 3-beta-hydroxysteroid dehydrogenase (3-beta-HSD) and an isomerase (Samuels et a l . , 1951; Cheatum et a l . , 1966). Both enzymes requiring nicotinamide adenine dinucleotide (NAD) as a cofactor are located i n the microsomal f r a c t i o n , although 3-beta-HSD may also be present i n the mitochondria of the ovary (Sulimovici and Boyd 1969; Haksar and Romanoff, 1971; Dimino and Campbell, 1976). Since isomerase a c t i v i t y appears to be i n excess (Philpott and Peron, 1971), the production of P 4 from pregnenolone i s mainly regulated by 3-beta-HSD. The r a t e - l i m i t i n g step i n the biosynthesis of androgens i n the f o l l i c l e i s that catalyzed by the 17-alpha-hydroxylase/ C._ ..-lyase enzyme complex which i s located i n the microsomal f r a c t i o n of the c e l l s and which requires nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen for i t s action. Hydroxylation at the C 1 7 position i s e s s e n t i a l before the side chain i s cleaved from the C 2 1 steroids (progestins) to form C i g steroids (androgens). The reaction can u t i l i z e both pregnenolone and P. as substrates r e s u l t i n g i n 9 dehydroepiandrosterone or androstenedione, respectively. This enzymatic step which i s under the control of hormones and feedback regulation by the end products of steroidogenesis, i s one of the key points for the physiologic control of f o l l i c u l a r s t e r o i d secretion. In contrast to the neighboring theca c e l l s , the granulosa c e l l s contain very low l e v e l s of the enzymes, 17-alpha- hydroxylase and C 1 7 2 Q - l y a s e , which mediate the conversion of progestins to androgens (Short, 1962; Bjersing and Carstensen, 1967). The deficiency of these enzymes i n granulosa c e l l s indicates that both granulosa c e l l s and theca c e l l s p a r t i c i p a t e i n androgen and estrogen biosynthesis. The conversion of androstenedione and testosterone to estrone and estradiol-17-beta i s catalyzed by an enzyme complex, re f e r r e d to as aromatase, located i n the membranes of the agranular endoplasmic reticulum of several ovarian c e l l types. The reaction requires NADPH, and three moles of oxygens. Two of these are involved i n two consequent hydroxylation at C-19, and the o v e r a l l reaction involves a t h i r d hydroxylation, but the exact s i t e of t h i s i s not yet clear (Kantsky and Hagerman, 1980; Brodie et a l . , 1976). The secretion of P 4 by ovarian c e l l s i s modulated by changes i n the conversion of P 4 to i t s metabolites. The main route of P 4 breakdown i s mediated by 20-alpha-hydroxysteroid dehydrogenase (20-alpha-HSD), located i n the cytosol portion of ovarian c e l l s u t i l i z i n g NADPH as a hydrogen donor, which re v e r s i b l y converts P. to i t s inactive metabolite, 20alpha- 10 hydroxy-pregn-4-en-3-one (20-alpha-OH-P). 20-alpha-OH-P i s considerably l e s s active as a progestational agent than i t s precursor P 4. I t has been suggested that the a c t i v i t y of 20alpha-OH-P may play a s i g n i f i c a n t r o l e i n determining the amount of C 2 ̂ s u b s t r a t e a v a i l a b l e f o r conversion to androgens i n f o l l i c u l a r c e l l s , since 20alpha-reduced steroids are poor substrates for C 1 7 2 Q - l y a s e (Goldring and Orly, 1985). B. Synthesis of Prostaglandins and Leukotrienes Prostaglandins (PGs), which were f i r s t discovered by Von Euler i n the 1930s as a b i o l o g i c a l l y active component of human seminal f l u i d , are also important secretory products of the ovarian c e l l s and the secretion of prostaglandins may be under hormonal control (Triebwasser et a l . , 1978; Clark et a l . , 1978) . The precursor for PGs synthesis i s arachidonic acid (AA) , which i s a C 20:4 polyunsaturated f a t t y a c i d . AA i n mammalian c e l l s i s normally e s t e r i f i e d almost excl u s i v e l y i n the 2-acyl p o s i t i o n to g l y c e r o l i n the phospholipids of the c e l l membrane and i s released through a phospholipase-catalyzed reaction. The concentration of free AA i n c e l l s i s les s than 10~6M, and the free acid l e v e l i n a tissue represents a balance between the l i b e r a t i o n of the aci d by hydrolysis and i t s re- e s t e r i f i c a t i o n . Free AA can undergo two oxidative pathways of metabolism as outlined i n Figure 2. The cyclooxygenase pathway leads to the formation of the endoperoxide intermediate prostaglandin H 2, which i s then converted by the action of isomerases to a number of b i o l o g i c a l l y active molecules, 11 II O C-O-C-R, it i CH 3(CH ?) 4(CH = CHCH 2) 4(CH 2) 2-CO-C 0 i II C-O-P-O-Base PHOSPHOLIPIDS 'A ARACHIDONIC ACID i 5-lipoxygenase 15-lipoxygenase 12-lipoxygenase cyclooxygenase F i g . 2. Key pathways i n arachidonic acid metabolites. PG: prostaglandin LT: leukotriene LX: l i p o x i n HETE: hydroxyeicosatetraenoic acid HPETE: hydroperoxyeicosatetraenoic acid 12 prostaglandin E 2 (PGE 2), prostaglandin D 2 (PGD 2), prostaglandin F2alpha ^ P G F 2 a l p h a ^ ' P r o s t a g l a n d i n I 2 (PGI 2) and thromboxane A 2 (TXA 2). The l e t t e r s following the abbreviation PG indicate the nature and p o s i t i o n of the oxygen-containing substituents present i n the cyclopentane r i n g . The 2-series PGs are formed from AA, and the 1-series and 3-series PGs are synthesized from 8,11,14-eicosatrienoic and 5,8,11,14,17-eicosapentaenoic acid, respectively. An al t e r n a t i v e pathway f o r the oxygenation of AA i s provided by lipoxygenase enzymes. The products of the lipoxygenase enzymes are hydroperoxyeicosatetraenoic acids (HPETEs) which can then be converted into hydroxyeicosatetraenoic acids (HETEs), leukotrienes (LTs), and lipo x i n s (Fig. 2). Rat ovarian and f o l l i c u l a r homogenates possess lipoxygenase a c t i v i t y that increases a f t e r i n vivo administration of human chorionic gonadotropin (hCG) (Reich, 1985) . The induction by hCG of PGs i s demonstrated to occur both i n granulosa c e l l and theca c e l l s of preovulatory f o l l i c l e s (Hedin et a l . , 1987). The a c t i v i t y of cyclooxygenase can be i n h i b i t e d by nonsteroidal anti-inflammatory drugs such as a s p i r i n and indomethacin (Flower and Vane, 1974). In h i b i t i o n of PG cyclooxygenase e f f e c t i v e l y blocks the synthesis of a l l cyclooxygenase s e r i e s . Lipoxygenase a c t i v i t y can be i n h i b i t e d by compounds such as nordihydroguaiaretic acid (NDGA) (S a l a r i et a l . , 1984). I I I . Regulation of ovarian hormone synthesis 13 A. Role of gonadotropins Ovarian f o l l i c l e growth and st e r o i d hormone production are mainly under the control of two gonadotropin hormones LH and FSH. LH and FSH are synthesized and stored i n the anterior p i t u i t a r y and are released i n response to l u t e i n i z i n g hormone- releasing hormone (LHRH). LHRH i s a decapeptide (pyro-Glu-His- Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) found i n the hypothalamus of a l l mammalian species so f a r studied. LHRH i s secreted into the hypophyseal p o r t a l system i n a p u l s a t i l e fashion and i s transported along the p i t u i t a r y s t a l k to the p i t u i t a r y . In the p i t u i t a r y , LHRH controls the synthesis and secretion of gonadotropins by a receptor dependent mechanism. Gonadotropins are likewise released i n a p u l s a t i l e pattern. The p u l s a t i l e release and c y c l i c v a r i a t i o n i n the c i r c u l a t i n g concentrations of gonadotropins control ovarian functions by a l t e r i n g the s e n s i t i v i t y of ovarian c e l l s through increasing and decreasing receptor formation and a c t i v i t i e s of c e l l u l a r enzymes. Although the i n i t i a t i o n of primordial f o l l i c l e growth occurs independently of p i t u i t a r y gonadotropins, once reaching the primary f o l l i c l e stage further growth and maturation of the f o l l i c l e becomes completely dependent on LH and FSH. FSH induces ovarian f o l l i c l e maturation and i s responsible for the development of granulosa c e l l responsiveness to several other hormones. FSH i n t e r a c t s with ovarian c e l l s through s p e c i f i c plasma membrane receptors. In 14 the female, FSH binds only to the granulosa c e l l s of the ovarian f o l l i c l e s . FSH regulates granulosa c e l l progestin biosynthesis by modulating the a c t i v i t i e s of various steroidogenic enzymes, SCC, 3-beta-HSD and 20-alpha-HSD (Toaff et a l . , 1983). FSH also induces aromatase. LH stimulates preovulatory f o l l i c l e growth, induces ovulation, and regulates corpus luteum function. The major s i t e of action of LH on P 4 biosynthesis i s the conversion of cholesterol to pregnenolone, although 3-beta-HSD i s also stimulated (Armstrong et a l . , 1970; Madej, 1980). A f t e r the FSH induction of LH receptors i n granulosa c e l l s , these c e l l s are capable of responding to LH i n the maintenance of aromatase a c t i v i t y (Wang et a l . , 1981; Dorrington and Armstrong, 1979). The steroidogenic action of LH on theca c e l l s apparently increases the a c t i v i t i e s of 17- alpha-hydroxylase and C 1 7 2 Q - l y a s e i n ovaries (Fukuda et a l . , 1979; Bogovich and Richards, 1982). Previous studies have demonstrated a "two c e l l - t y p e , two gonadotropin theory" (Fig. 3) . There are p r i n c i p a l c e l l types involved i n f o l l i c u l a r steroidogenesis: (1) LH-responsive secretory c e l l s : comprising the theca interna c e l l s of the f o l l i c u l a r envelope and the i n t e r s t i t i a l c e l l s of ovarian stroma, and (2) FSH-responsive c e l l s which are granulosa c e l l s . According to t h i s model, theca interna c e l l s are stimulated by LH to produce androgen from cholesterol, which d i f f u s e s across the basement membrane to be used f o r estrogen synthesis i n an FSH-stimulated reaction i n granulosa c e l l s ( Makris and Ryan, 1975; Fortune and Armstrong, 1977; Tsang and Armstrong 1980; 15 THECA CELLS Cholesterol 1 Progesterone 4- Androstenedione e ATP C A M P Androstenedione Estrogen (circulation) \ v X Basement membrane Progesterone Aromatase 0 Androstenedione — — • Estrogen Cholesterol^"^ - ̂  ^ '"'''© C A M P ' ' 3ST FSH Estrogen ( F o l l i c u l a r f l u i d ) GRANULOSA CELLS Fi g . 3 . Diagram of the "two-cell, two gonadotropin theory" of f o l l i c l e steroidogenesis. 16 Erickson, 1978). Since granulosa c e l l s secrete P 4 i n response to gonadotropins, i t i s also possible that granulosa c e l l P 4 may d i f f u s e into theca c e l l s to serve as a substrate for androgen biosynthesis. Theca interna c e l l s convert P 4 to androstenedione by 17-alpha-hydroxylase and C 1 7 2 Q - l y a s e . In contrast, granulosa c e l l s do not have s i g n i f i c a n t a c t i v i t i e s of C 2 1 side-chain cleavage enzymes and synthesize l i t t l e or no androgens from eit h e r P 4 or pregnenolone (Lacroix et a l . , 1974; Hamberger et a l . , 1978; Short, 1962; Fowler et a l . , 1978). On the other hand, granulosa c e l l s do possess considerable 17- beta-HSD a c t i v i t y (Makris and Ryan, 1980; Nimrod et a l . , 1980; Moon and Duleba 1982), which acts on androstenedione and estrone to form testosterone and e s t r a d i o l , respectively. Although androstenedione i s the major ovarian androgen i n most species, the 17-beta-HSD reaction favors the production of es t r a d i o l as the major estrogen. These i n t e r a c t i o n s between LH and FSH together with the c y c l i c a l changes i n plasma concentration of LH and FSH provide a mechanism to account for the regulation of ovarian steroidogenesis and f o l l i c u l a r growth. While the foregoing account has focussed on the roles of LH and FSH i n regulating a c t i v i t y of ovarian functions, there i s also evidence that a t h i r d p i t u i t a r y gonadotropin, p r o l a c t i n (PRL), may also regulate ovarian a c t i v i t y at the ovarian l e v e l . PRL receptors have been demonstrated i n the human ovary (Saito and Saxena 1975), and i n r a t and porcine granulosa c e l l s and l u t e a l c e l l s (Richards and Williams, 1976; Rolland and Hammond, 17 1975; Rolland et a l . , 1976). PRL may i n t e r a c t with granulosa c e l l s to promote t h e i r maturation since PRL i s e s s e n t i a l f o r maximum production of P 4 by human l u t e i n i z e d granulosa c e l l s i n i n v i t r o studies (McNatty et a l . , 1974). PRL acts as a luteotrophic agent by stimulating P 4 production (Rothchild, 1981; Smith, 1980) as well as by maintaining the l e v e l of LH receptor i n r a t ovary (Holt et a l . , 1976), and may influence the pool of s t e r o i d precursors a v a i l a b l e for P 4 synthesis (Armstrong et a l . , 1970; Behrman et a l . , 1970). Receptors for PRL, l i k e those f o r LH and FSH, appear to be located on the c e l l membrane but i n contrast to LH and FSH, i n t e r a c t i o n of PRL with i t s receptor does not stimulate adenylate cyclase (Mason et a l . , 1973) and no second messenger for PRL has been convincingly documented. In addition to steroids, granulosa c e l l s also secrete PGs. PG synthesis i s stimulated by LH and FSH r e s u l t i n g i n increased production of PGE 2 and P G F 2 a i p h a ( c l a r k e t a l » / 1978; Marsh et a l . , 1974; Knazek et a l . , 1981; Zor et a l . , 1983). An ovulatory dose of hCG d i r e c t l y increases the f o l l i c u l a r content of PGs (Richards et a l . , 1982). The rate of production of PGs i n granulosa c e l l s i s d i r e c t l y proportional to the concentration of hCG used to stimulate the c e l l s (Hedin., et a l . , 1987). Doses of hCG capable of stimulating ovulation increase PG synthesis, whereas sub-threshold doses of hCG only s l i g h t l y increase PG synthesis and do not induce ovulation. The increase of PGs induced by hCG i s transient i n r a t ovary (Hedin et a l . , 1987). The concentrations of PGs reach a 18 maximum p r i o r to ovulation and return to low l e v e l s within 24- 48 h following the LH/hCG surge. B. Intraovarian regulation by f o l l i c u l a r steroids Role of progestins Granulosa c e l l s synthesize and secrete large quantities of P 4, which may exert some e f f e c t s on f o l l i c u l a r growth and granulosa c e l l function. In prepubertal r a t s , exogenous administration of P 4 f a c i l i t a t e s the hCG-stimulated growth of small a n t r a l f o l l i c l e s and hCG-induced estrogen biosynthesis (Richards and Bogovich, 1982). P 4 also enhances ovarian P 4 secretion by the preovulatory f o l l i c l e without a f f e c t i n g the l e v e l of LH secretion (Uchida et a l . , 1972). In contrast, i n monkeys, u n i l a t e r a l ovarian implants of P 4 d i r e c t l y i n h i b i t f o l l i c u l a r growth without a f f e c t i n g the function of the co n t r a l a t e r a l ovary, suggesting that a l o c a l l y high concentration of P 4 may i n h i b i t f o l l i c u l o g e n e s i s (Goodman and Hodgen, 1979). Administration of P 4 to hamsters r e s u l t s i n a f a l l i n blood e s t r a d i o l concentration without a change i n serum l e v e l s of gonadotropins. This decline i s not reversed by concomitant administration of testosterone, i n d i c a t i n g that P 4 acts at the l e v e l of the aromatase (Greenwald, 1974) . The P 4 receptors have been i d e n t i f i e d i n the cytoplasm of rat granulosa c e l l s (Schreiber and Erickson, 1979; Naess, 1981). Similar P 4 receptors have been i d e n t i f i e d i n the ovaries of rabbit, cow and human ( P h i l i b e r t et a l . , 1977; Jacobs and 19 Smith, 1980; Jacobs et a l . , 1980). The presence of ovarian P 4 receptors suggests an important i n t r a c e l l u l a r regulatory role fo r P.. Other studies have demonstrated a r o l e of P„ i n the * 4 autonomy of l u t e a l c e l l P 4 biosynthesis and i n an autocrine control mechanism i n which the P 4 production of the c e l l s exerts u l t r a - s h o r t loop feedback regulation of i t s own production (Goff et a l . , 1979; Fanjul et a l . , 1983). Role of androgens In addition to serving as substrates f o r aromatase enzymes to form estrogens, androgens exert a v a r i e t y of actions i n granulosa c e l l s through i n t e r a c t i o n with i n t r a c e l l u l a r androgen receptors. Pretreatment of i n t a c t rats with dihydrotestosterone prevents the FSH induction of LH receptors i n granulosa c e l l s and t h i s e f f e c t can be antagonized by estrogen treatment (Farookhi, 1980). Although androgen treatment induces a t r e s i a i n the absence of FSH, androgens augment gonadotropin-stimulated steroidogenesis. Both i n vivo and i n v i t r o experiments have shown that androgens stimulate ovarian aromatase a c t i v i t y (Katz and Armstrong, 1976; Daniel and Armstrong, 1980). Androgens also act s y n e r g i s t i c a l l y with FSH to stimulate progestin production i n cultured r a t granulosa c e l l s . The stimulatory e f f e c t of androgens on progestin biosynthesis appears to be the r e s u l t of the stimulation of SCC and 3-beta-HSD (Nimrod, 1977; Welsh et a l . , 1982). 20 Role of estrogens Estrogens maintain secondary sexual c h a r a c t e r i s t i c s and exert feedback action on the hypothalamic-pituitary unit. Moreover, estrogens play a modulating role at the s i t e of i t s formation. Estrogens have been known to exert a d i r e c t a n t i - a t r e t i c e f f e c t . The induction of a t r e s i a may be associated with a loss of e s t r a d i o l receptors i n granulosa c e l l s (Richards, 1975; Harman et a l . , 1975; Ingraham, 1959). Estrogens also regulate estrogen production of granulosa c e l l s by augmenting the FSH-induced aromatase a c t i v i t y , and the minimal e f f e c t i v e dose (3.7xl0~ 1 0M) of estradiol-17-beta on aromatase a c t i v i t y i s within the range of estradiol-17-beta measured i n the f o l l i c u l a r f l u i d of r a t preovulatory f o l l i c l e s . This suggests that of estrogen plays a physiological r o l e as an end product amplifier of aromatase a c t i v i t y to enhance the syn e r g i s t i c e f f e c t of androgens (Goff and Henderson, 1979; Adashi and Hsueh, 1982). Estrogen enhancement of FSH- stimulated granulosa c e l l aromatase a c t i v i t y may explain the maintenance of dominant f o l l i c l e s i n the ovary. On the other hand, es t r a d i o l may i n h i b i t production of i t s precursor androgen through negative feedback on the theca c e l l s (Leung et a l . , 1978; Leung and Armstrong, 1979). Such an intraovarian negative feedback mechanism may be s i g n i f i c a n t i n l i m i t i n g the estrogen production and provide adequate time f o r oocyte maturation before ovulation. Local i n t r a f o l l i c u l a r concentrations of estrogen or the r a t i o of estrogen and androgen may determine which f o l l i c l e ( s ) i n one cycle w i l l 21 escape a t r e s i a and go on to ovulation (Harmon et a l . , 1975; H i l l i e r et a l . , 1980). C. Role of neurotransmitters on ovarian steroidogenesis The innervation of the mammalian ovary has been well documented. The dense adrenergic innervation of the mammalian ovary suggests a r o l e f o r the adrenergic system i n the regulation of ovarian functions (Moshin and Pennefather, 1979; Lawrence and Burden, 1980). The possible r o l e of catecholamines i n the d i r e c t regulation of st e r o i d biosynthesis by f o l l i c l e c e l l s has been studied both i n vivo and i n v i t r o . Catecholamines stimulate P 4 production i n cultured l u t e a l and granulosa c e l l s , and the stimulation could be blocked by the beta 2-adrenergic antagonist (IPS339), but not p r a c t o l o l (beta 1~ adrenergic antagonist) or phentolamine (alpha-adrenergic antagonist) (Bahr et a l . , 1974; Condon and Black, 1976). In vivo studies have shown that beta-adrenergic, but not alpha- adrenergic agonists r e s u l t i n increased P 4 production by the ovary (Bahr et a l . , 1974). Another neurotransmitter that has been extensively examined i n the ovary i s gamma-aminobutyric acid (GABA) (Erdo and Lapis, 1982). In whole ovary, GABA concentration i s comparable to brain l e v e l s and i s 5 to 6 f o l d higher than any other non-neuronal tissues studied. GABA binding s i t e s are elucidated by measuring the s p e c i f i c binding 3 of a GABA agonist, [ H]-muscimol (Schaeffer and Hsueh, 1982). Although the phys i o l o g i c a l r o l e of GABA i n the ovarian tissues remains to be elucidated, production of cAMP i n s l i c e s of rat ovary i s increased by GABA and t h i s e f f e c t i s antagonized by GABA receptor blockers (picrotoxin and bi c u c u l l i n e ) (Erdo and Lapis, 1982). D. Regulation of ovarian steroidogenesis and ovarian function by prostaglandins PGE 2 stimulates cAMP, estrogen and P 4 production, and induces resumption of meiotic d i v i s i o n of the oocyte and ovulation. Although PGE 2 can mimic some e f f e c t s of LH, the action of LH and PGE 2 are independent and p a r a l l e l . In the presence of cyclooxygenase i n h i b i t o r s , LH stimulates cAMP accumulation and P 4 production (Linder et a l . , 1974; Linder et a l 1980). Although LH induces the process of ovulation, the f i n a l phase of ovulation, f o l l i c u l a r rupture, does not occur i n the absence of PGE 2 < This indicates that the presence of PGs i s required for ovulation. I n h i b i t i o n of PG synthesis by administration of indomethacin blocks ovulation (Armstrong and Grinwich, 1972; Armstrong et a l . , 1974). The concentration of PGs i n the ovaries, f o l l i c l e s and f o l l i c u l a r f l u i d r i s e as time of ovulation approaches (Linder et a l . , 1980; Murdoch et a l . , 1981; Ratwardhan and Lanthier, 1981). The stimulation of plasminogen acti v a t o r and proteoglycan production i n granulosa c e l l s further supports the involvement of PG i n the process of ovulation. Plasminogen i s a glycoprotein contained i n the plasma and i s converted to the active serine protease by two d i f f e r e n t plasminogen ac t i v a t o r s which can be stimulated by PGF_ , . and PGE_. Plasmin, which i s produced by the action 23 of plasminogen a c t i v a t o r on plasminogen, t r i g g e r s the various steps i n the postulated cascade. The net e f f e c t i s to decrease the strength of the f o l l i c l e wall to the point at which rupture occurs (Ossowski et a l . , 1979; Beers et a l . , 1975; Espey, 1980). Because the gonadotropins and the prostaglandins stimulate adenylate cyclase, cAMP i s probably involved i n the a c t i v i t i e s of p r o t e i n synthesis, leading to increased production of plasminogen a c t i v a t o r (Strickland and Beers, 1976). I t has been demonstrated that PGE 2 i s involved i n reversal of ovum maturation and that P G F 2 a l p h a m a ^ overcome blockade of ovulation by indomethacin (Downs and Longo, 1982;, 1983). However, i t should be noted that PGE 2 a f f e c t s ovulation i n indomethacin-blocked animals ( T s a f r i r i et a l . , 1972), and thus plays a major r o l e i n ovulation. pGE 2 i s the predominant PG i n the f o l l i c l e s and i s responsible for most of the e f f e c t s of PG on ovulation, but P G F 2 a l p h a m a ^ exert a n e f f e c t on the smooth-muscle elements of the f o l l i c l e wall (Diaz-Infante et a l . , 1974). E. Role of l o c a l nonsteroidal regulators on ovarian function Endocrine glands, such as the p i t u i t a r y , ovary and thyroid glands, release hormones which reach t h e i r target v i a the blood stream and thereby a f f e c t other tissues, organs or body functions. Paracrine control mechanisms involve l o c a l d i f f u s i o n of hormones to t h e i r neighboring c e l l s without entering the c i r c u l a t o r y system (Roth et a l . , 1983). F i n a l l y , the regulatory function of some hormones i s autocrine since 24 e f f e c t s are exerted on the c e l l s which produce the hormones. There i s increasing evidence to suggest that l o c a l nonsteroidal regulators play important roles i n the ovary by paracrine or autocrine control mechanisms. These nonsteroidal regulators include LHRH (Hsueh and Jones, 1981), growth factors (Gospodarowicz et a l . , 1977a; 1977b; 1979), i n s u l i n and i n s u l i n - l i k e growth factors (Veldhuis et a l . , 1983; Adashi et a l , 1985), ovarian angiogenic factors (Koos and LeMaire, 1983), angiotensin (Culler et a l . , 1986; Husain et a l . , 1987), bradykinin (Smith and Perks, 1983), neurotransmitters (Hsueh et a l . , 1984), oocyte maturation i n h i b i t o r ( T s a f r i r i and Braw, 1984) and neurohypophyseal hormones (Sheldrick and F l i n t , 1984). These l o c a l nonsteroidal regulators may i n t e r a c t with gonadotropins, ster o i d hormone and PGs to regulate steroidogenesis, oocyte maturation and ovulation by paracrine or autocrine mechanisms. The e f f e c t s of LHRH on ovarian function have been extensively studied. A d i r e c t function of LHRH i n the ovary was reported by Rippel and Johnson who observed a decrease i n hCG augmented ovarian weight i n immature hypophysectomized rats treated with LHRH (Rippel and Johnson 1976) . This finding was confirmed i n hypophysectomized rats stimulated with pregnant mare's serum gonadotropin (PMSG) or FSH (Ying and Guillemin, 1979; Hsueh and Erickson, 1979). In v i t r o studies have shown a d i r e c t e f f e c t of LHRH on primary cultures of granulosa c e l l s as we l l . Treatment with LHRH or i t s agonists i n h i b i t s FSH- stimulated progestin and estrogen production (Hsueh and 25 Erickson, 1979; Arimura et a l . , 1979). The action of LHRH on granulosa c e l l steroidogenesis i s exerted at multiple s i t e s including i n h i b i t i o n of FSH-stimulated cAMP production, i n h i b i t i o n of aromatase, SCC and 3-beta-HSD and stimulation of 20-alpha-HSD. LHRH also suppresses LH and FSH receptors (Hsueh and Jones, 1981; Hsueh et a l . , 1981; Gore-Langton 1981). The i n h i b i t o r y action of LHRH on the ovary i s exerted on other ovarian compartments i n addition to the granulosa c e l l s . LHRH i n h i b i t s basal and LH-stimulated androgen synthesis by rat ovarian i n t e r s t i t i a l c e l l s (Magoffin et a l . , 1981; Magoffin and Erickson, 1982). A d d i t i o n a l l y , LHRH i n h i b i t s LH/hCG-stimulated P 4 secretion by r a t l u t e a l c e l l s i n vivo and i n v i t r o (Clayton et a l . , 1979; Jones and Hsueh, 1980). In contrast to the in h i b i t o r y e f f e c t s of LHRH, stimulatory e f f e c t s following acute administration of LHRH alone have also been observed. These ef f e c t s include the stimulation of estrogen, P 4, 20-alpha-OH-P and PGs production (Dorrington et a l . , 1982; Gore-Langton et a l . , 1981; Clark et a l . , 1980; Clark, 1982). Stimulatory and in h i b i t o r y e f f e c t s of LHRH could be blocked by treatment with LHRH antagonists (Jones and Hsueh 1981; Hsueh and Ling, 1979; Navickis et a l . , 1982). The most consistent stimulatory action of LHRH on ovarian function i s exerted on mature preovulatory f o l l i c l e s . LHRH induces ovulation and t h i s action of LHRH i s blocked by LHRH antagonists (Ekholm et a l . , 1982; Dekel et a l . , 1983). The action of LHRH on f o l l i c u l a r rupture at ovulation appears to be r e l a t e d to i t s a b i l i t y to stimulate PGs and plasminogen a c t i v i t o r , both of them have been shown previously 26 to be involved i n f o l l i c u l a r rupture (Hillensjtt et a l . , 1982 Wang 1983; Reich et a l , 1985). A d d i t i o n a l l y , LHRH i s involved i n the resumption of ovum maturation and cumulus c e l l dispersion (Dekel et a l . , 1981; Hillensjtt and LeMaire, 1980; Magnusson and LeMaire, 1981). Unlike i t s e f f e c t on f o l l i c u l a r rupture, the action of LHRH on the ovum i s not blocked by indomethacin and hence does not seem to be mediated by f o l l i c u l a r PG production (Ekholm et a l . , 1982). Recent studies have proposed that the action of LHRH on ovum maturation involves protein kinase C (PKC). Furthermore, i n h i b i t o r s of the lipoxygenase pathway of AA i n h i b i t s the resumption of meiosis induced by LHRH, but not by LH, i n d i c a t i n g the involvement of t h i s pathway i n mediating LHRH action on ovum maturation ( T s a f r i r i et a l . , 1986; Aberdam and Dekel, 1985; Ekholm et a l . , 1982). The fi n d i n g of s p e c i f i c receptors for LHRH i n the rat oocyte strongly suggests a d i r e c t e f f e c t of LHRH on oocyte maturation (Dekel et a l . , 1988). The d i r e c t e f f c t s of LHRH on ovarian steroidogenesis are mediated by i t s s p e c i f i c receptors. These receptors are found i n l u t e a l , theca and granulosa c e l l s at a l l stages of c e l l u l a r d i f f e r e n t i a t i o n ( P e l l e t i e r et a l . , 1982). Photoaffinity l a b e l i n g of ovarian LHRH receptors has i d e n t i f i e d two s p e c i f i c components with apparent MW of 60,000 and 54,000 daltons (Hazum and Nimrod, 1982; Hazum, 1984). LHRH increases the amount of i t s own receptor, whereas gonadotropins cause LHRH receptor depletion (Clayton and Catt, 1981). Apart from the hormonal regulation, the ovarian LHRH receptor might also be under a 27 d i r e c t neural control (Marchetti and Cio n i , 1988). Since only one type of LHRH receptor i s i d e n t i f i e d i n the p i t u i t a r y , the extra component of the ovarian receptors may be re l a t e d to the d i f f e r e n t and s p e c i f i c functions of LHRH-like peptide i n the ovary. Although the r a t model has been extensively used to study the d i r e c t e f f e c t s of LHRH on gonadal function, other studies have demonstrated d i r e c t e f f e c t s of LHRH on the ovary of rabbit (Koos and LeMaire, 1985), p i g (Massicotte et a l . , 1980), cow (Milvae et a l . , 1984), chicken (Takats and Hertelendy, 1982), monkey (Knecht et a l . , 1983), and human (Tureck et a l . , 1982). The high a f f i n i t y ovarian LHRH receptors have been demonstrated i n r a t , but not i n sheep, pig, and cow (Brown and Reeves, 1983), monkey (Asch et a l . , 1981), and human (Clayton and Huhtaniemi, 1982). On the other hand, low a f f i n i t y LHRH receptors were documented i n human corpus luteum (Popkin et a l . , 1983). The f a i l u r e to demonstrate high a f f i n i t y LHRH binding s i t e s i n other species might be due to the poor a b i l i t y of the labeled LHRH analogs used to intera c t with the ovarian LHRH receptors i n these species. However, the low l e v e l of LHRH i n systemic blood indicates that LHRH may not be the endogenous ligand that binds to the LHRH receptors i n the rat ovary (Aten et a l . , 1986). To demonstrate the phy s i o l o g i c a l s i g n i f i c a n c e of the d i r e c t ovarian actions of LHRH, i t i s necessary to e s t a b l i s h the presence of an ovarian LHRH-like substance. Recently, i t was shown that rat, bovine and ovine ovaries contain a LHRH-like peptide that competes with LHRH for binding to ovarian membrane 28 receptors but with immunoreactive a c t i v i t y d i s t i n c t l y d i f f e r e n t from those of LHRH (Aten et a l . , 1986; Aten et a l . , 1987). Inter e s t i n g l y , a separate gonadotropin-releasing peptide has been i s o l a t e d from human f o l l i c u l a r f l u i d ( L i et a l . , 1987). The amino ac i d composition and sequence of t h i s l a t t e r peptide d i f f e r from those of hypothalamic LHRH (with the primary structure of H-Thr-Asp-Thr-Ser-His-His-Asp-Gln-Asp-His-Pro-Thr- Phe-Asn-OH) and t h i s peptide i s considerably l e s s potent i n stimulating the release of gonadotropins from the mouse p i t u i t a r y i n v i t r o . The LHRH receptors i n the r a t ovary may represent receptors for one or more of these LHRH-like peptides found endogenously i n the rat ovary. The presence of equivalent l e v e l s of LHRH-like peptide i n the ovine, bovine and human ovary suggests that LHRH-like peptide might serve a paracrine or autocrine r o l e i n these tissues v i a the receptors s p e c i f i c f o r LHRH-like peptide. A d d i t i o n a l l y , a recent report has suggested that porcine i n h i b i n alpha-subunit of 134 amino acid suppresses FSH-induced production of cAMP, P 4 and e s t r a d i o l v i a a LHRH receptor i n r a t granulosa c e l l s , r a i s i n g further i n t e r e s t i n the nature of LHRH receptors i n the ovary ( H i l l i e r et a l . , 1987). IV. Signal transduction systems i n ovary A. Introduction Ovarian c e l l u l a r functions are regulated by peptide hormones, neurotransmitters and nonsteroidal factors and these 29 hormones regulate ovarian c e l l s v i a second messengers. Generally, the capacity of a given c e l l to respond to a given hormone depends on the presence or absence of the receptor i n the c e l l . I t i s well recognized that there are several classes of hormone receptors, which when occupied by t h e i r s p e c i f i c hormones, stimulate d i f f e r e n t second messengers, whose d i f f u s i o n enables the hormonal signal to spread r a p i d l y throughout the c e l l . Two major signal pathways are now known. One employs the second-messenger c y c l i c adenosine monophosphate (cAMP). The other employs a combination of second messengers 2+ that includes calcium ions (Ca ), i n o s i t o l 1,4,5-trisphosphate (IP 3) 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 AMP A large number of hormones exert t h e i r e f f e c t s by increasing the concentration of cAMP. cAMP i s formed from ATP by the membrane bound enzyme adenylate cyclase. Each hormone molecule r e s u l t s i n increased formation of many molecules of cAMP. Therefore, the i n i t i a l hormone signal i s greatly amplified following i t s i n t e r a c t i o n with plasma membrane-bound receptors. cAMP in t e r a c t s with a s p e c i f i c i n t r a c e l l u l a r a l l o s t e r i c receptor, the regulatory subunit of cAMP-dependent protein kinase, and upon d i s s o c i a t i o n of the free c a t a l y t i c subunit induces the phosphorylation of substrate proteins to give further a m p l i f i c a t i o n . The agonist-induced increase i n cAMP and subsequent c e l l u l a r response i s terminated by degradation of cAMP to 5'-AMP by the action of 30 phosphodiesterase, hydrolysis of GTP to GDP by GTPase and removal of phosphate groups from substrate proteins by phosphatase enzymes. I t i s believed that cAMP i s the second messenger fo r the action of both gonadotropins, LH and FSH, i n ovarian c e l l s , and multiple functions of ovarian c e l l s can be e l i c i t e d by cAMP analogs and cAMP-inducing agents (Kolena and Channing, 1972; Goff and Armstrong, 1979; Marsh and Savard, 1966; Tsang et a l . , 1979; Dennefors et a l . , 1980). Since PGE 2 also induces the increase i n cAMP l e v e l s i n cultured granulosa c e l l s , endogenous PGs may also a f f e c t granulosa c e l l d i f f e r e n t i a t i o n (Kolena and Channing, 1972; Goff and Armstrong, 1977; Behrman, 1979). Studies performed during the past decade have revealed that the regulation of hormone-sensitive adenylate cyclase i s f a r more complicated than o r i g i n a l l y suspected. F i g . 4 presents i n a scheme many of the s t r u c t u r a l and functional aspects of adenylate cyclase a c t i v i t y by nucleotides and hormones. Adenylate cyclase i s only part of a complex regulatory system that mediates the action of hormones on t h e i r target c e l l s . The enzyme system i s composed of at l e a s t three classes of components. Located at the outer membrane surface i s the receptor (R) component containing a s p e c i f i c s i t e f o r binding of hormones. At the inner face of the membrane are the c a t a l y t i c u n i t (C) and the guanine nucleotide regulatory protein (G) (Rodbell, 1980). Receptors communicate with a p a i r of homologous guanine proteins. One of which (Gs) mediates stimulation of adenylate cyclase a c t i v i t y , while the other (Gi) signal signal 31 • 51-AMP inactive + ® active cAMP-dependent • cAMP-dependent protein kinase protein kinase b i o l o g i c e f f e c t F i g . 4. General model of cAMP mediated hormone response. R, receptor; Gs, stimulatory guanine-binding protein; Gi, i n h i b i t o r y guanine-binding protein; GTP, guanosine triphosphate; GDP, guanosine diphosphate; ATP, adenosine triphosphate; cAMP, c y c l i c adenosine monophosphate; 5'-AMP, adenosine 5'-phosphate. i s responsible for i n h i b i t i o n (Rodbell, 1980; Gilman, 1984). They are both formed of alpha, beta and gamma subunits, both alpha-subunits bind guanosine triphosphate (GTP) and i t s analogs. When hormone binds to receptor (H*R), there i s a rapid i n t e r a c t i o n of H*R with G to form H^'G. Formation of H»R»G complex affects G a c t i v i t y , allowing the binding of GTP to i t s s p e c i f i c binding s i t e to form H»R»G»GTP. G i s active 32 only when GTP i s bound; i t i s inactive when GDP i s bound. HaR*G complex increases removal of i n h i b i t o r y guanosine diphosphate (GDP) and f a c i l i t a t e s GTP binding. GTP-dependent ac t i v a t i o n i s represented by concomitant subunit d i s s o c i a t i o n to give a GTP»alpha complex, which interacts with C to enhance or decrease c a t a l y t i c a c t i v i t y depending on the type of G protein, and a beta8gamma complex, which does not i t s e l f appear to dis s o c i a t e . Reversal of adenylate cyclase stimulation r e s u l t s from GTP hydrolysis by GTPase, which terminates G- protein a c t i v i t i o n . GTPase-dependent deactivation i s assumed to be completed upon reassociation of alpha subunits with beta4gamma complexes (Rodbell, 1980; Jakobs et a l , 1984; Gilman, 1984; 1987). C. Calcium and protein kinase C pathway In addition to the cAMP pathway, there i s another major s i g n a l l i n g pathway that u t i l i z e s the membrane phosphoinositides (Fig. 5). So f a r , the c o l l e c t i v e term phosphoinositides has been used to describe the three anionic phosphoinositides that contain myo-inositol i n t h e i r head groups (Berridge, 1981). The most abundant form i s phosphatidylinositol (PI) that contains myo-inositol attached to phosphate through the hydroxyl on the 1-position of i t s i n o s i t o l head group. The other two members are formed by sequential phosphorylation of hydroxyl groups on the 4- and 5-posit ions to form phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphates (PIP,) that i s the immediate precursor 33 Agonist receptor PI • PIP [ C a 2 + ] i C e l l u l a r response Fig. 5. I n o s i t o l phospholipid turnover and sign a l transduction. Abbreviations: G, guanine nucleotide-binding protein; PI, phosphatidylinositol; PIP, phosphatidylinositol-4-phosphate; PIP 2, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C; DG, 1,2-diacylglycerol; PA, phosphatidic acid; AA, arachidonic acid; PGs, prostaglandins; LTs, leukotrienes. located within the plasma membrane used by the receptor mechanism to release i n o s i t o l 1,4,5-trisphosphate (IP 3) to the cytosol, leaving DG within the plane of the membrane. The i n o s i t o l phosphates are rapidl y degraded to i n o s i t o l , which i s u t i l i z e d f or resynthesis of phosphoinositides, by a complex of 34 phosphatases, whereas d i a c y l g l y c e r o l i s converted to eithe r phosphatidic acid or monoacylglycerol plus arachidonic acid (AA) . Resynthesis of PI occurs i n the membranes of the endoplasmic reticulum where phosphatadic acid interacts with cyt i d i n e triphosphate to give c y t i d i n e diphosphate- d i a c y l g l y c e r o l and t h i s combines with i n o s i t o l to give PI. PI i s then c a r r i e d back to the plasma membrane by a tra n s f e r protein to complete the cycle of breakdown and resynthesis. AA can be derived from membrane phosphoinositides as well as from the sn-2 p o s i t i o n of other membrane phospholipids. Since AA a v a i l a b i l i t y l i m i t s the rate of synthesis of AA metabolites i n most ti s s u e s , the reactions that produce AA can stimulate lipoxygenase and cyclooxygenase pathways thereby generating other signals, for example, PGs, TXs and LTs. The hydrolysis of . . . . . 2+ i n o s i t o l l i p i d i s mainly confined to the action of Ca mobilizing agonists, which bind to s p e c i f i c c e l l - s u r f a c e receptors and gain access to both i n t r a c e l l u l a r and external 2+ 2+ sources of Ca . Evidence f o r the IP 3/Ca -mobilizing hypothesis has been obtained by studying the e f f e c t of t h i s putative second messenger on various permeabilized c e l l s where 2+ IP 3 could gain access to the i n t r a c e l l u l a r Ca stores, such as endoplasmic reticulum (ER) (Streb et a l , 1983; Burgess et a l , 2+ 1984). Another possible source of Ca i s from mitochondria. I t has been demonstrated that i s o l a t e d mitochondria p a r t i c i p a t e s i n the release and uptake of large amounts of 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). IP, acts through a s p e c i f i c receptor to release Ca by 35 opening a channel across the ER membrane (Smith et a l , 1985; Irvine et a l , 1984). The i n i t i a l response to agonists that 2+ 2+ cause Ca -mobilization i s a release of i n t e r n a l Ca (phase 2+ 1) , which i s soon followed by entry of Ca across the plasma membrane (phase II) (Kojima et a l , 1985; Reynolds and Dubyak, 1985). Most attention has focused on i t s r o l e i n stimulating 2+ the release of Ca during c e l l activation, but IP 3 may serve 2+ to regulate the r e s t i n g or basal l e v e l Ca as well (Prentki et a l , 1985). DG that remains within the plane of the plasma membrane functions as a second messenger by a c t i v a t i n g protein 2 + kinase C (PKC) . PKC has been shown to be Ca - and phospholipid-dependent f o r i t s a c t i v i t y (Nishizuka 1984). One of the important aspects of the act i v a t i o n process appears to be a translocation of PKC from the cytosol into the membrane, 2+ and t h i s process might be the r o l e of Ca (Wolf et a l , 1985). Although the a c t i v a t i o n of PKC i s thought to be biochemically 2+ dependent upon Ca , i t can be p h y s i o l o g i c a l l y activated 2+ . . . independence of Ca under some conditions. I t i s now clear that there i s more than one species of PKC molecule, and seven subspecies of PKC have been i d e n t i f i e d (Nishizuka, 1988). The various subspecies of PKC have d i f f e r e n t enzymatic properties. The gamma and alpha-subspecies of PKC are much l e s s activated by DG i n the presence of phosphatidylserine than i s the mixture of beta-1 and beta-2 subspecies, which shows substantial 2+ a c t i v i t y i n the absence of Ca (Nishizuka, 1988). I t has also been proposed that d i f f e r e n t subspecies of PKC are also activated by the serie s of phospholipid metabolites, such as 36 DG, AA and l i p o x i n A (Hansson et a l , 1986, Nishizuka, 1988) . Once PKC has been activated through the concerted action of DG 2+ and Ca , i t begins to phosphorylate s p e c i f i c proteins that are thought to contribute to the control or modulation of many metabolic and other processes (Nishizuka, 1986). I P 3 and DG are released from membrane phosphoinositides by a phosphoinositide-specific phospholipase C (PLC). There i s convincing experimental evidence at present which suggests a role f o r GTP-binding protein serving to couple receptors to PLC. An example of the evidence i n d i c a t i n g a role for G- protein i n the coupling various receptors to PIP 2 hydrolysis 2+ and Ca mobilization i s the fi n d i n g that nonhydrolyzable analogues of GTP stimulate breakdown of PIP 2 and PLC a c t i v i t y (MaJerus et a l , 1986). The i d e n t i t y of t h i s G-protein and i t s r e l a t i o n s h i p to other G-proteins i s unknown. LHRH i s a peptide hormone and i t s e f f e c t s are mediated by s p e c i f i c receptors. The mechanism of LHRH action on the ovary has been investigated i n the past few years. There i s no convincing evidence suggesting that cAMP i s the second messenger f o r LHRH action i n the ovary. On the other hand, LHRH and i t s agonists have been shown to stimulate the breakdown of polyphosphoinositides into i n o s i t o l phosphates and DG i n the ovary (Leung et a l , 1983; Naor and Yavin, 1982; Ma 2+ and Leung, 1985; Minegishi and Leung, 1985). Ca i s required i n the action of LHRH i n the granulosa c e l l s (Ranta et a l , 1983) and protein kinase C has been characterized i n the ovary (Noland and Dimino, 1986; Davis and Clark, 1983). Recently, the e f f e c t of LHRH on [ H]AA release i n rat ovarian c e l l has also been examined (Minegishi and Leung, 1985) . Thus, at the l e v e l of the ovarian c e l l , the hydrolysis of i n o s i t o l l i p i d s may immediately follow LHRH receptor occupancy and lead to the rapid generation of IP 3 and DG, and the release of AA. The 2+ resultant changes i n Ca mobilization and/or PKC a c t i v i t y and AA metabolism may well be correlated with the modulatory e f f e c t s of LHRH on ovarian steroidogenesis. V. The aim of the present study Although many reports have indicated that LHRH or LHRH- l i k e substance d i r e c t l y a f f e c t r a t ovarian function, the mechanism of action of LHRH i s not completely understood. Since LHRH has been shown to induce membrane phosphoinositide breakdown, the o v e r a l l aim of the present study i s to further t e s t the ef f e c t s of LHRH on hormone production i n rat granulosa c e l l s and investigate the possible s i g n a l transduction r o l e s of 2+ . . . PKC, Ca , AA and i t s metabolites i n the action of LHRH. S p e c i f i c a l l y , the action of LHRH was compared with that of gonadotropins and cAMP-stimulating agents on the membrane phosphoinositide turnover. Other experiments was examined 2+ LHRH-induced [Ca ] i a l t e r a t i o n i n ind i v i d u a l granulosa c e l l s , as well as the interactions among the putative signal transduction pathways on the regulation of P 4 and PGE2 production. The objective of the present study was therefore to understand, more completely, the role of LHRH as a paracrine or autocrine regulator of ovarian functions. 38 Chapter 2. Induction of Polyphosphoinositide Turnover and Arachidonic Acid Release by LHRH I. Introduction Numerous studies have shown that LHRH and i t s synthetic agonists could directly affect steroid hormone production in the ovary (Hsueh and Jones, 1981; Leung, 1985). The direct effects of LHRH on the ovary are mediated by specific receptors (Pelletier et a l . , 1982). These extrapituitary intraovarian actions are either stimulatory or inhibitory, depending on the duration of LHRH treatment as well as the simultaneous presence of other ovarian c e l l regulators (such as gonadotropins) during the culture period. While the influence of LHRH on ovarian hormone production i s well documented, i t s mechanism of action at the postreceptor level i s s t i l l largely unresolved. In the past few years, LHRH and i t s agonists have been shown to stimulate the breakdown of polyphosphoinositides into i n o s i t o l phosphates and DG in the ovary (Leung et a l . , 1983; Naor and Yavin, 1982; Davis et a l . , 1986; Ma and Leung, 1985; Minegishi and Leung, 1985; Leung et a l . , 1986). Inositol phosphates, especially IP 3 are known to induce mobilization of calcium ions from i n t r a c e l l u l a r stores (Burgess et a l . , 1984). On the other hand, DG i s now widely accepted to be a potent activator of PKC 2 + (Nishizuka et a l . , 1984). Calcium ion (Ca ) i s required in the action of LHRH in ovarian c e l l s (Ranta et a l . , 1983; Dorflinger et a l . , 1984), and PKC has recently been characterized in the ovary (Noland and Dimino, 1986; Davis and 39 Clark, 1983). Recently, a LHRH-like peptide has been demonstrated in rat, bovine, ovine and human ovaries, further strengthening the concept that LHRH or LHRH-like peptide plays a role in mediating ovarian functions. Thus, within the ovarian c e l l s , the hydrolysis of i n o s i t o l l i p i d s may immediately follow LHRH receptor occupancy and lead to the rapid generation of IP 3 and DG. The resultant changes in calcium mobilization and/or the a c t i v i t y of PKC may be correlated with the modulatory effects of LHRH on ovarian hormone production. A similar mechanism involving i n o s i t o l l i p i d breakdown has been proposed for LHRH stimulation of gonadotropin release i n the anterior pituitary gland (Raymond et a l . , 1984; Huckle and Conn, 1987; Harris et a l . , 1985; Conn et a l . , 1985). In the present study, the actions of LHRH on i n o s i t o l phosphates, diacylglycerol and arachidonic acid formation were further investigated. S p e c i f i c a l l y , the action of LHRH was compared with that of gonadotropins and cAMP-stimulating agents on the membrane phosphoinositide turnover. The role of PKC activation in regulating production of in o s i t o l phosphates, DG and AA was emphasized in this study. I I . Materials and Methods 40 Animals Immature Sprague-Dawley female rats purchased from Charles River Canada, Inc. (Montreal, Canada,) or Animal Care (University of B r i t i s h Columbia) were injected subcutaneously on the 23th day a f t e r b i r t h with 12 IU pregnant mare's serum gonadotropins (PMSG) between 09:00 and 10:00 i n the morning to stimulate the formation of multiple preantral f o l l i c l e s and provide large numbers of r e l a t i v e l y homogenenous granulosa c e l l s at the same stage of development. The rats 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 the ovaries were removed by surgery. Preparation of granulosa c e l l s Granulosa c e l l s were harvested under the dissecting microscope, by puncturing the ovarian f o l l i c l e s with a 27Gi gauge hypodermic needle as previously described (Leung and Armstrong, 1978). The ovaries were squeezed gently and the granulosa c e l l s released into Minimum E s s e n t i a l Medium with Eagle's s a l t s and supplemented with 2 mM of L-glutamine, 100 units/ml of p e n i c i l l i n , 100 ug/ml of streptomycin sulfate, and 5 ml of nonessential amino acids (MEM; Gibco, Grand Island, NY) . A f t e r removal from the ovaries, the c e l l s were expressed through a fine s t e r i l i z e d mesh. The c e l l s were recovered by centrifugation (5 min at 200xg), washed once, and suspended i n MEM. 41 Radiolabeled d i a c y l q l y c e r o l and arachidonic a c i d l i b e r a t i o n 5 In some experiments, granulosa c e l l s (5x10 cells/ml) were added to 24 well culture plates (Falcon) and were labeled by incubation for 24h i n medium containing with 0.2 juCi/ml or 0.5 uci/ml [5,6,8,9,11,12,14,14,15,- 3H]Arachidonic acid (60 Ci/mmol; New England Nuclear, Boston, MA) i n MEM containing 5% f e t a l bovine serum (FBS). The c e l l s were then washed thoroughly and incubated for a further 30 to 60 min i n MEM without FBS. At t h i s time, d i f f e r e n t hormones were added. The various preparations were incubated for d i f f e r e n t time i n t e r v a l s . At the end of the incubation, the medium was removed and the c e l l s were scraped d i r e c t l y into 1 ml of i c e - cold methanol. The l i p i d s i n the c e l l s were extracted by the method of Folch et a l . (1957) . B r i e f l y , 1 ml of methanol was mixed with 2 ml of chloroform and 0.6 ml of water, and mixed on a vortex vigorously. The lower chloroform phase was removed and 1 ml of chloroform was added f o r the second extraction. The pooled chloroform layer of the two extractions was evaporated to dryness under nitrogen and the residue redissolved i n chloroform and methanol (2:1) f o r t h i n layer chromatography (TLC). The f a t t y acids i n the culture medium were extracted by the method of Borgeat and Samuelsson (1979). A f t e r addition of 1.5 ml of methanol to the medium, the bulk of the p r e c i p i t a t e d material was centrifuged. The supernatant was c o l l e c t e d , and the p e l l e t s were washed once with 0.5 ml of methanol. The pooled methanol supernatants were a c i d i f i e d to pH 3 and mixed with 6 ml of d i e t h y l ether. Then 4 ml of 42 d i s t i l l e d water were added and mixed. A f t e r separation of the phases, the water-methanol mixture was removed. The ether phase was evaporated to dryness under nitrogen and the residue dissolved i n a 2:1 mixture of chloroform-methanol. The [ H]- labeled AA was i s o l a t e d by TLC on s i l i c a gel 60F-254 plates (Merck, Rahway, NJ) with solvent containing iso-octane-ethyl acetate-water-acetic acid (5:11:10:2) v o l / v o l , as described previously by Minegishi and Leung (1985) . The R^ value of AA was 0.85 with pure standards as reference. Radiolabeled d i a c y l g l y c e r o l was separated by TLC with a solvent system containing benzene-diethylether-ethanol-ammonia-water (50:40:2:0.1) v o l / v o l , as described by Kaibuchi et a l . (1983). The areas of the p l a t e corresponding to DG (R f =0.72) were cut out and t h e i r r a d i o a c t i v i t y determined by l i q u i d s c i n t i l l a t i o n spectrometry. Analysis of i n o s i t o l phosphates 5 Granulosa c e l l s (5x10 cells/ml) were prelabeled by 3 incubation f o r 24h i n MEM containing myo-[2- H ] i n o s i t o l (5 jiCi/ml) (New England Nuclear; 16.5 Ci/mmol) and 5% FBS for 24h. The c e l l s were then washed and incubated f o r an i n i t i a l 10 min i n radiotracer-free MEM. At t h i s time, hormones were added (in a 10 u l volume), and the c e l l s were incubated for d i f f e r e n t times. Lithium c h l o r i d e ( L i + ; 10 mM), which i n h i b i t s i n o s i t o l - 1-phosphatase, was added to the medium p r i o r to hormonal treatment, enhancing i n o s i t o l phosphate accumulation. Incubation was terminated by scraping c e l l s d i r e c t l y into 1 ml 43 of i c e - c o l d methanol. For extraction, another 2 ml of chloroform and 5 jal of concentrated HC1 were added. The f i n a l r a t i o of chloroform/methanol/water was 2:1:0.6. Af t e r vortex and removal of the top layer (aqueous), another 0.6 ml of water was added and the extraction was repeated. The two extractions were combined, and the radiolabeled i n o s i t o l phosphates i n the aqueous phase were analyzed by anion exchange chromatography using disposable columns containing 0.5 ml of Dowex AG1-X8 r e s i n (BioRad, 200-400 mesh, formate form). The r e s i n was washed with 0.1 M formic acid/5 mM i n o s i t o l before use. Aliquots (2ml) of the c e l l lysates were loaded at 4°C. Free i n o s i t o l was washed out with water (10 bed volume of resin) whereas sequential washes with 0.1 M formic acid containing 0.2, 0.4, and 1.0 M ammonium formate progressively eluted IP, I P 2 and I P 3 / respectively, as described by Downes and Michell (1981). Fractions (2.5 ml) were c o l l e c t e d and r a d i o a c t i v i t y of each f r a c t i o n was counted following the addition of 15 ml of s c i n t i l l a t i o n f l u i d (Fisher S c i e n t i f i c , USA). Hormone and drug preparation Granulosa c e l l s were treated with various hormones and drugs. LHRH and CT were dissolved i n s a l i n e . AA, 4-alpha- 12,13-didecanoate and 12-O-tetradecanoylphorbol-13-acetate (TPA) were dissolved i n ethanol. A l l drugs were d i l u t e d to t h e i r respective working concentrations with MEM before use and added i n 5 ;al aliquots to a t o t a l incubation volume of 1 ml. Control incubations received the same volume of ethanol. The 44 f i n a l concentration of ethanol i n the incubations d i d not exceed 0.5%, and ethanol d i d not influence membrane phospholipid metabolism. Reagents The following were purchased from Sigma: lithium chloride, myo-inositol, formic acid, phospholipase C, AA, TPA, 4-alpha-phorbol 12, 13-didecanoate, LHRH and CT. Ammonium formate was from Fisher S c i e n t i f i c Inc. Ovine LH (NIDDK oLH- 25) , LHRH and PMSG were g i f t s from the National Hormone and P i t u i t a r y Program NIDDKD, NIH. Iso-octane, ethyl acetate, benzene, diethylether, methanol and chloroform were purchased from BDH Inc. (Canada) . Acetic acid and ammonia water were purchased from Canlab (Travenol Canada Inc.). S t a t i s t i c a l analysis S t a t i s t i c a l s i g n i f i c a n c e of the data was determined by Student's T-test or analysis of variance followed by Scheffe's multiple range t e s t . In a l l cases, i d e n t i c a l or s i m i l a r r e s u l t s were observed i n at le a s t three or more independent experiments. A l l r e s u l t s were presented as the mean ± SE of determinations from t r i p l i c a t e cultures of c e l l s within each treatment group. A P value of less than 0.05 was considered s i g n i f i c a n t . 45 I I I . Results E f f e c t s of LHRH on I n o s i t o l l i p i d breakdown and arachidonic a c i d release 3 In granulosa c e l l s prelabeled with [ H]-AA, treatment with LHRH caused a s i g n i f i c a n t increase (P<0.01), i n the levels of radiolabeled DG and AA i n the c e l l u l a r extracts. As i l l u s t r a t e d i n Fig . 6, addition of LHRH (10~"6M) for 5 min 3 3 stimulated the l i b e r a t i o n of [ H]-DG and u n e s t e r i f i e d [ H] -AA from prelabeled phospholipids, by about 4 and 2.7 fol d , respectively, compared with control incubations. Furthermore, 3 i n c e l l s prelabeled with [ H ] - i n o s i t o l , treatment with LHRH for 5 min caused a s i g n i f i c a n t increase i n accumulation of i n o s i t o l phosphates (P<0.01). 3 E f f e c t of LHRH on \ H]-labeled d i a c y l q l y c e r o l formation 3 The e f f e c t of LHRH on [ H] -DG formation was further 3 examined i n cultured granulosa c e l l s prelabeled with [ H]-AA and l a t e r exposed to LHRH. As shown i n F i g . 7, LHRH enhanced the i n t r a c e l l u l a r DG formation by 1.79 f o l d . The basal l e v e l 3 of [ H]-DG i n the medium was much lower than that i n the c e l l s 3 and [ H] -DG l e v e l did not increase i n the medium a f t e r the treatment of granulosa c e l l s with LHRH for 3 min. 46 I 1 Control Effig LHRH (K)"8M) F i g . 6. Stimulatory effects of LHRH on the formation of 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 the release of u n e s t e r i f i e d arachidonic acid (AA) i n rat granulosa c e l l s . The c e l l s were prelabeled with e i t h e r [ H ] - i n o s i t o l or [H]-arachidonic acid, as described i n Materials and Methods, and treated with LHRH for 5 min. 47 2.5 6** b X 2 Q. Q c o <-> (0 E 1.5 1 i Q 0 . 5 CO cellular T medium LHRH C LHRH (10' 6M) LHRH 3 Fig. 7. E f f e c t of LHRH on [ H]-diacylglycerol (DG) formation. The c e l l s were treated with LHRH f o r 3 min and d i a c y l g l y c e r o l (DG) formation was detected from the c e l l s and the medium. In t h i s and subsequent figures, the absence of standard error bars i n some of the data points indicates values too small to be shown. Time response of [ H]-labeled d i a c y l g l y c e r o l to LHRH Fig. 8 shows the time course of [ H]-DG formation i n granulosa c e l l s i n response to LHRH. LHRH (10~6M) caused a s i g n i f i c a n t increase i n [ H]-DG formation, which could be observed as early as 15 sec af t e r LHRH addition (P<0.05). In the LHRH treated c e l l s , DG l e v e l s continued to increase to about 197% above the control l e v e l at 5 min. This declined to about 40% of the 5 min l e v e l at 10 min. However, the l e v e l of t H]-DG was s t i l l considerably higher (190%) than the control l e v e l (P<0.01) at 10 min after the treatment. The control l e v e l s of DG did not change during the 10 min experiment period. 3 E f f e c t s of LH and LHRH on [ H]-labeled i n o s i t o l phosphates and d i a c y l g l y c e r o l formation and arachidonic acid release Fig. 9 i l l u s t r a t e s that the presence of 10~6M LHRH for 3 minutes markedly stimulated the accumulation of radiolabeled IP, IP 2, and IP 3 to 155%, 545% and 100%, respectively, when compared with untreated control l e v e l s . In contrast, LH (1 jig) d i d not stimulate the formation of i n o s i t o l phosphate from 3 [ H ] - i n o s i t o l prelabeled granulosa c e l l s i n the same experiment. A s i m i l a r r e s u l t was also observed f o r the 3 , formation of [ H]-DG (Fig. 10, panel A) . In addition, LHRH (10~6M) s i g n i f i c a n t l y stimulated AA release, whereas LH (1 jug) did not a f f e c t AA release (Fig. 10, panel B). £2 0 I — ' — i — ' — i — i — i — i — i — i — i — i i i i i i i i • » ' ' ' / j i i i i 0 11 2 3 4 6 10 Time (min) F i g . 8. Time response of stimulation of [ H]-diacylglycerol (DG) formation by LHRH. A s i g n i f i c a n t increase i n DG formation was observed as early as 15 second a f t e r LHRH addition. 50 0.8 IP 0.6 i O y— X I 0.4 s i 0.2 X IP3 • Control LH (10 jug) LHRH ("KT* M) C LH LHRH LH LHRH LH LHRH F i g . 9. Comparison of LH and LHRH on [ H]-labeled i n o s i t o l phosphates. The c e l l s were treated with LH and LHRH for 3 min. LHRH markedly stimulated the formation of 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 <10'6M) F i g . 10. Comparison of LH and LHRH on d i a c y l g l y c e r o l (DG) formation and arachidonic acid (AA) release. The c e l l s were treated with LH and LHRH for 3 min. The addition of LHRH caused s i g n i f i c a n t d i a c y l g l y c e r o l (DG) formation and AA release, whereas LH did not a l t e r the formation of these compounds. E f f e c t s of cholera t o x i n and TJTRH on \ HI -labeled i n o s i t o l phosphates formation As shown i n F i g . 11, i n prelabeled granulosa c e l l s with 3 [ H ] i n o s i t o l , the addition of LHRH produced increases i n c e l l u l a r IP, IP 2 and IP 3 (about 380%, 660% and 191%, res p e c t i v e l y ) , during a 5 min period, while addition of CT (100 ng) f a i l e d to a f f e c t the formation of i n o s i t o l phosphates. 3 E f f e c t of phospholipase C on r H]-diacylglycerol formation 3 The e f f e c t of exogenous phospholipase C on [ H]-labeled DG accumulation was investigated i n a separate experiment. Like LHRH, addition of 100 mU PLC resulted i n a marked increase i n i n t r a c e l l u l a r DG (5.6 f o l d as compared to control l e v e l ) , whereas a maximal dose of LHRH (10 - 6M) caused a 1.8 f o l d increase i n the formation of DG (Fig. 12). E f f e c t s of phorbol ester TPA on i n o s i t o l phosphate and DG formation To determine whether the a c t i v a t i o n of PKC by LHRH exerts a possible feedback e f f e c t on the hydrolysis of membrane phosphatidylinositides, the granulosa c e l l s were pretreated with a s p e c i f i c PKC activator, TPA, f o r 5 min and then challenged with 10~6M LHRH for a further 3 min. S i g n i f i c a n t increases of IP, I P 2 and IP 3 formation were observed when the c e l l s were stimulated with TPA. As demonstrated i n Fig. 13, LHRH (10"6M) stimulated formation of IP, I P 2 and IP 3 and t h i s response was unaffected by pretreatment of c e l l s with TPA. 53 (0 O K 2 02 Q . Q 9 0 0 9 go.i M OL o 0.2 0 u _ J 1 1 1 . 1 1 ' • C CT LHRH (I00no> (10" 9 M) F i g . 11. E f f e c t of cholera toxin (CT) and LHRH on [ 3H]-labeled i n o s i t o l phosphate formation. The c e l l s were treated with CT and LHRH f o r 5 min. The formation of i n o s i t o l phosphates was stimulated by LHRH but not CT. 54 Control LHRH (10-6 M) PLC (IQOmU) F i g . 12. E f f e c t of phospholipase C (PLC) on [ H]- di a c y l g l y c e r o l (DG) formation. The c e l l s were treated with 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 increased the formation of DG. 55 x 2E Q. O • » " s 8 C TPA LHRH LHRH (10~7M) (10-6M) TPA F i g . 13. Action of the phorbol ester TPA on i n o s i t o l phosphate formation. The c e l l s were f i r s t treated with TPA f o r 5 min, and then treated with LHRH for further 3 min. TPA alone stimulated i n o s i t o l phosphate formation, but the pretreatment of TPA did not a l t e r the response of the c e l l s to LHRH. 56 The e f f e c t of TPA on DG formation was determined i n another experiment i n which granulosa c e l l s were prelabeled 3 with [ H] -AA. TPA increased the formation of DG and t h i s action of TPA was s p e c i f i c , since another phobol congener, 4- alpha-phorbol 12, 13-didecanoate, did not stimulate the formation of DG (Fig. 14). Interaction of the calcium ionophor A23187 and the phorbol ester TPA on arachidonic a c i d release To determine the possible i n t e r a c t i o n between the calcium ionophore A23187 and the phorbol ester TPA on AA release, [ H]- AA prelabeled granulosa c e l l s were treated with TPA and A23187, following a 5 min incubation, A23187 at 10 M caused a 3 s i g n i f i c a n t stimulation of [ H]-AA release (80% of control, P<0.05). However, TPA used alone i n s i m i l a r concentration showed no such e f f e c t on AA release. Interestingly, when both A23187 and TPA were present, the e f f e c t of A23187 was 3 potentiated (P<0.05), with the l e v e l of [ H]AA release reaching 130% of control l e v e l s (Fig. 15). IV. Discussion The present r e s u l t s (Fig. 6) further strengthen the previous findings that LHRH causes a rapid breakdown of i n o s i t o l l i p i d s i n rat ovarian c e l l s . This mechanism was f i r s t proposed when LHRH was shown to cause a rapid and selec t i v e 3 2 incorporation of P into phosphatidylinositol and phosphatidic acid i n r a t granulosa c e l l s (Naor and Yavin 1982; Minegishi and 57 F i g . 14. S p e c i f i c i t y of the phorbol ester TPA action on d i a c y l g l y c e r o l (DG) formation. The action of TPA on DG formation was s p e c i f i c , since another phorbol congener, 4alpha- phorbol 12, 13-didecanoate (4alpha-PDD), did not change the formation of DG. 58 Control A23187 (10"7M) TPA (10 "7M) A23187 TPA F i g . 15. Interaction of the calcium ionophore A23187 and the phorbol ester TPA on arachidonic a c i d (AA) release. TPA alone did not a l t e r the release of AA, but potentiated the action of A23187. 59 Leung, 1985; Leung et a l . , 1983). Subsequently, i t has been demonstrated that the accumulation of the i n o s i t o l l i p i d breakdown products, IP, I P 2 and IP 3 i s markedly increased following the addition of LHRH to granulosa c e l l s (Ma and Leung, 1985; Davis et a l . , 1986). The i n o s i t o l phosphates produced i n response to LHRH were from polyphosphoinositol hydrolysis, since LHRH caused a decrease i n the l e v e l of radiolabeled polyphosphoinositides, while increasing 32 P l a b e l i n g to phosphatidylinositol and phosphatidic acid (Leung et a l . , 1986). The action of LHRH on ovarian i n o s i t o l phosphate formation i s s i m i l a r to the action of LHRH on p i t u i t a r y gonadotropes. In p i t u i t a r y c e l l cultures prelabeled with [ H] i n o s i t o l f or 5h, addition of LHRH resu l t e d i n an increase i n the rate of I P 3 turnover (Huckle and Conn, 1987). Thus, products of polyphosphoinositide breakdown may serve as primary mediators of the early i n t r a c e l l u l a r signal transduction f o r LHRH both i n ovarian and p i t u i t a r y c e l l s . The formation of IP 3 may be responsible f o r some of LHRH-induced c e l l u l a r b i o l o g i c a l responses. In fa c t , IP 3 has been shown to 2+ induce Ca mobilization from i n t r a c e l l u l a r pools (Nishizuka, 1984; Burgess et a l . , 1984). There apparently i s no d i r e c t r o l e for IP 3 i n regulating calcium entry across the plasma membrane. However, preliminary studies have provided i n d i r e c t evidence that i n o s i t o l 1,3,4,5-tetrakisphosphate (IP 4) has a 2+ r o l e i n the stimulation of Ca i n f l u x (Berridge, 1987) . IP 4 i s formed from IP 3 by a IP 3~kinase that transfers a phosphate from ATP to the 3-position of IP.. In the ovary, both the 60 i n h i b i t o r y and the stimulatory actions of LHRH on P 4 production 2+ have been shown to be Ca dependent (Erickson et a l . , 1986; Leung and Wang, i n press). Since phosphodiesterase cleavage of PIP 2 ^ s t n e only known mechanism fo r IP 3 formation i n mammalian c e l l s , LHRH induced PIP 2 breakdown must occur through the action of a polyphosphoinositide-specific phospholipase C (PLC). I t has been proposed that thyrotropin-releasing hormone action on phosphatidylinositide breakdown i n cultured GH c e l l s occurs v i a PLC a c t i v a t i o n (Conn et a l . , 1987). In rat l u t e a l c e l l s , the addition of exogenous PLC mimicked the action of LHRH and P G F 2 a l p h a on the formation of i n o s i t o l phosphates (Leung et a l . , 1986). Similar r e s u l t s were also observed i n the present study (Fig. 12). The action of LHRH on i n o s i t o l phosphate formation i n granulosa c e l l s i s s p e c i f i c . The e a r l i e r studies have shown that LHRH-induced formation of i n o s i t o l phosphate can be completely blocked by LHRH antagonists, suggesting a receptor mediated mechanism. Furthermore, gonadotropin hormones, which act through increasing cAMP i n the ovary, did not r e s u l t i n the breakdown of membrane polyphosphatidylinositides i n the present studies (Fig. 8-10). cAMP-inducing agents, CT, also did not have any e f f e c t on the hydrolysis of membrane phosphatidylinositide. Since t h i s toxin enters c e l l s v i a a ganglioside mediated mechanism, i t s onset of action i s notoriously slow. A longer time may need for further study. On the other hand, gonadotropin hormones which are the major 61 hormones regulating ovarian functions, and LHRH which probably plays a l o c a l paracrine regulatory r o l e , may inte r a c t with each other v i a t h e i r d i f f e r e n t i n t r a c e l l u l a r signal pathways. Interactions between the adenylate cyclase pathway and phosphoinositide breakdown have been reported i n various c e l l types. For instance, cAMP analogs have been shown to enhance the formation of polyphosphoinositides i n sarcoplasmic reticulum preparations of rabbit heart and p i g granulocytes (Enyedi et a l . , 1984; Farkas et a l . , 1984), but s i g n i f i c a n t l y i n h i b i t norepinephrine induced i n o s i t o l phosphate accumulation i n FRTL-5 c e l l s (Bone et a l . , 1986). Prostacyclin, which stimulates cAMP accumulation, has been shown to block thrombin stimulated PI turnover (Watson et a l . , 1984). More recently, FSH has been shown to i n h i b i t the serum stimulated accumulation of i n o s i t o l phosphate, but FSH i t s e l f has no s i g n i f i c a n t effect on the formation of i n o s i t o l phosphate i n S e r t o l i c e l l s (Monaco et a l . , 1988). The present results (Fig. 9-11), however, are i n contrast with a previous report which showed that LH stimulated i n o s i t o l phosphate formation i n r a t granulosa c e l l s (Davis et a l . , 1986). The discrepancy between the previous and present studies cannot be e a s i l y explained. A possible reason could be the d i f f e r e n t research approaches undertaken. For example the long exposure of the c e l l s i n Davis's study to LH may f a c i l i t a t e the synthesis of membrane phosphoinositides. In response to LHRH, other products of membrane i n o s i t o l l i p i d hydrolysis such as DG were also detected i n the present study (Fig. 6-8 and 10). Geison et a l . (1976) found that the 62 sn-2 p o s i t i o n of phosphatidylinositides was r i c h i n AA. This knowledge was used i n the present study to determine the e f f e c t of LHRH on DG production by l a b e l l i n g granulosa c e l l s with 3 . 3 [ H]AA. The production of [ H]DG was then measured. According to the time response study, LHRH-induced DG formation was observed as early as 15 sec. This time was very s i m i l a r to that found for LHRH-induced increase i n IP 3 (Ma and Leung, 1985; Davis 1986). Since the previous studies have 32 demonstrated that LHRH only increases P incorporation into phosphatidylinositol and phosphatidic acid, t h i s s i m i l a r i t y i n time suggests that the increased l e v e l of [ H]DG must most l i k e l y have resulted from phospholipase C hydrolysis of phosphoinositides (Naor and Yavin, 1982; Davis et a l . , 1983; Minegishi and Leung, 1985). It i s interesting to note that most of the [ H] DG was recovered from the i n t r a c e l l u l a r space (Fig. 7). Although LHRH 3 d i d a l t e r the amount of i n t r a c e l l u l a r [ H]DG, LHRH d i d not s i g n i f i c a n t l y change the l e v e l of DG i n the medium. This finding was consistent with the character of DG as a membrane hydrophobic metabolite. Interestingly, a s i m i l a r r e s u l t has 3 been observed with [ H]IP 3 as well (Naor et a l . , 1986). These r e s u l t s may suggest that the DG and IP 3 formed by LHRH induction may have b i o l o g i c a l r o l e s within the c e l l s where they are produced rather than having an influence on other c e l l s . DG may play a potent r o l e i n the action of LHRH by 2+ ac t i v a t i n g PKC, which i s a Ca activated and phospholipid dependent protein kinase. The a c t i v i t y of t h i s enzyme has been 63 demonstrated i n the ovary (Noland and Dimino, 1986; Davis and Clark, 1983). DG and DG-like phorbol esters, i . e . TPA, 2+ stimulate PKC by reducing the amounts of Ca and phospholipid required for a c t i v a t i o n (Nishizuka, 1984; Takai et a l 1984). The dependence of PKC on phospholipid indicates that the enzyme ac t i v a t i o n may involve association of the enzyme with phospholipid-rich c e l l membranes. Similar to the e f f e c t s of LHRH, both i n h i b i t o r y and stimulatory e f f e c t s of DG and TPA on ovarian steroidogenesis have been demonstrated, i n d i c a t i n g that 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 serve as an amplifier of the LHRH-stimulated sig n a l . (Welsh et a l . , 1984; Shinohara et a l . , 1985; Kawai and Clark, 1985). DG and TPA have also been shown to mimic the action of LHRH on LH release i n the p i t u i t a r y (Naor and Catt, 1981; Conn et a l . , 1985). In addition to IPs and DG, the t h i r d compound that was 3 measured i n the present study was [ H]AA (Fig. 6, 10 and 15) . A previous study has demonstrated that LHRH causes an increase 3 i n the l e v e l of [ H]AA release i n the culture medium as early as 15 min a f t e r LHRH addition (Minegishi and Leung, 1986). The stimulatory e f f e c t of LHRH can be blocked by the concomitant presence of a potent LHRH antagonist. To evaluated the r o l e of AA i n the actione of hormones, i t has also been observed that LHRH-stimulated LH release i s c l o s e l y coupled with the production of oxidized AA metabolites i n the anterior p i t u i t a r y (Naor and Catt, 1981; Snyder et a l . , 1983; Abou-Samra et a l . , 1986) . On the other hand, i t has also been suggested that AA i t s e l f rather than i t s metabolites may be a c e l l u l a r regulator 64 of PRL secretion from GH3 c e l l s (Kolesnick et a l . , 1984). Since the i n t r a c e l l u l a r concentration of free AA l i m i t s the synthesis of PGs and LTs, the demonstration of the increase i n i n t r a c e l l u l a r free AA i s c l e a r l y important. In the present study with r a t granulosa c e l l s , LHRH stimulated [ 3H]AA to increase by about 170% 5 min a f t e r the addition of LHRH (Fig. 6) . The data thus indicate that LHRH action may be mediated by i t s induction of AA release. The mechanism of t h i s LHRH- induced AA release i n granulosa c e l l s i s , however, not clear, AA has indeed been made from i n o s i t o l phospholipids through two consecutive reactions catalyzed by phospholipiase C followed by d i a c y l g l y c e r o l lipase, which has been shown i n p l a t e l e t s (Bell et a l . , 1979; Dixon and Hokin, 1984). LHRH has been found to cause an apparently s e l e c t i v e depletion i n the l e v e l of radiolabeled PI, suggesting that AA may be derived from i n o s i t o l phospholipids (Minegishi and Leung, 1985). Thus the transient formation of DG as a r e s u l t of agonist stimulated breakdown of membrane phosphoinositides represents a major pathway leading to l i b e r a t i o n of AA for PGs and LTs synthesis. On the other hand, the release of i n t r a c e l l u l a r free AA may also be due to the ac t i v a t i o n of PLA 2 which hydrolyzes AA from the sn-2 p o s i t i o n of one or several phospholipids. The 2+ ac t i v a t i o n of PLA 2 i s Ca dependent. Verapamil, a calcium- channel blocker prevents both the enhancement of AA release and the depletion i n the l e v e l of radiolabeled PI i n r a t granulosa 2+ c e l l s . In addition, the omission of Ca from the incubation medium could also diminish LHRH-induced [ H]AA release i n these 65 c e l l s (Minegishi and Leung, 1985). These findings strengthen 2+ the concept that Ca i s required at a step before AA release, as suggested previously by other studies (Folkert et a l . , 1984; Forder et a l . , 1985; Naor and Catt, 1981). Additionally, 2 + [Ca ] i mobilization induced by IP 3 could l i b e r a t e AA from phosphoinositide v i a PLA 2 a c t i v a t i o n . However, 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 each contributed to AA l i b e r a t i o n was c l e a r l y understood i n granulosa c e l l s . Recently, another possible mechanism has been proposed suggesting that PLA 2 i s i t s e l f regulated by both 2+ Ca and DG. According to t h i s mechanism, PLA 2 a c t i v i t y i s suppressed by a l i p o c o r t i n . Receptor a c t i v a t i o n by agonists leads to an increase i n i n t r a c e l l u l a r calcium and the concomitant production of DG, from the breakdown of membrane phosphatidylinositide. The a c t i v a t i o n of PKC by DG induces the phosphorylation of the l i p o c o r t i n , suppressing i t s anti-PLA_ 2 + a c t i v i t y , and i n the presence of increased [Ca ] i , optimal a c t i v i t y of PLA 2 i s evoked (Touqui et a l . , 1986). In the present study, the release of AA induced by the calcium ionophore A23187 was enhanced by concomitant treatment of granulosa c e l l s with TPA (10 M) (Fig. 15), presumably related to a PKC activated mechanism of AA release i n granulosa c e l l s . A s i m i l a r TPA mediated potentiation of the calcium ionophore induced AA release i n human p l a t e l e t has been reported (Volpi et a l . , 1985). However, the nature of t h i s proposed mechanism has not been elucidated i n granulosa c e l l s , although i t seems 2+ that Ca plays a major r o l e . The three possible pathways 66 involved i n AA release are summarized i n F i g . 16. As mentioned e a r l i e r , phorbol esters mimicked the LHRH action on hormone production i n ovary and t h i s probably involves a c t i v a t i o n of the c e l l u l a r PKC. In addition to i t s ro l e i n control of c e l l u l a r secretion, PKC, at least i n some c e l l s , controls the s e n s i t i v i t y of the phosphoinositide- sig n a l i n g pathway by regulating receptor function or the PIP 2 content of the c e l l membrane (Taylor et a l . , 1984; Cooper et a l . , 1985). S p e c i f i c a l l y , the a c t i v a t i o n of PKC by DG i s capable of stimulating PIP 2 synthesis and could t h e o r e t i c a l l y increase the amounts of IP 3 and the formation of DG i n response to receptor occupation (De Caffoy de Courcells et a l . , 1984). In contrast to the stimulatory e f f e c t of PKC on PIP 2 synthesis, the same kinase may also exert a negative control on the synthesis of PIP 2 (Aloy et a l . , 1983). In addition, receptors and t h e i r a b i l i t y to f u n c t i o n a l l y couple to PLC can also be regulated by PKC ac t i v a t i o n . I t has been observed that the ac t i v a t i o n of PKC may completely block the agonist induced IP 3 production by decreasing the number of receptors and regulating the coupling of receptors to PLC through a G protein (Cooper et a l . , 1985; Lynch et a l . , 1985). In the present study, treatment of granulosa c e l l s with TPA (10 M) stimulated the basal i n o s i t o l phosphate formation, but the combined treatment of granulosa c e l l s with LHRH and TPA d i d not potentiate or attenuate i n o s i t o l phosphate formation induced by LHRH alone (Fig. 13). The stimulatory action of TPA on i n o s i t o l phosphate formation was s p e c i f i c , since an i n a c t i v e form of the phorbol 67 — — l i p a s e > Arachidonic acid <- Fi g . 16. Scheme showing proposed mechanism involved i n arachidonic acid release. ester, 4-alpha-phorbol 12, 13-didecanoate, did not a l t e r the formation of i n o s i t o l phosphate (Fig. 14). So fa r , the s i t e of t h i s action i n granulosa c e l l s was not c l e a r . I t has been demonstrated that TPA causes translocation of PKC from the cytosol to the c e l l membrane (Kraft et a l . , 1982). Rapid 68 r e d i s t r i b u t i o n of PKC a c t i v i t y during the onset of LH release i n p i t u i t a r y c e l l s i n response to LHRH (Nirota et a l . , 1985) suggests that the membrane l o c a l i z a t i o n of the active PKC may lead to the a c t i v a t i o n of phospholipase C and A 2 d i r e c t l y . Since PLC i s the enzyme that catalyzes the hydrolysis of membrane phosphatidylinositide, i t might be concluded that PKC act i v a t i o n i s a f f e c t i n g , i n some manner, the PLC hydrolysis of the phosphoinositides. However, i t cannot be ruled out that PKC a c t i v a t i o n may decrease the hydrolysis of i n o s i t o l phosphate to i n o s i t o l , or increase the synthesis of PIP 2« As the source of membrane phospholipid i s the same and the quantity of phosphatidylinositides i s limited, the TPA induced i n o s i t o l phosphate formation was overridden by the concomitant presence of LHRH (Fig. 13). Taken together, these data suggest that the a c t i v a t i o n of PKC plays a role i n the formation of IP 3, DG and AA. In summary, the i n t e r a c t i o n of LHRH with i t s plasma membrane s p e c i f i c receptors r e s u l t s i n the rapid breakdown of membrane phosphoinositides, leading the production of IP 3, DG 2+ and AA. The subsequent changes of Ca mobilization, PKC act i v a t i o n and the metabolism of AA may control the c e l l u l a r secretion of granulosa c e l l s . In addition, the a c t i v a t i o n of 2 + PKC and Ca mobilization may also control the formation of IP 3, DG and AA by regulating receptors, PLC a c t i v i t y , and PIP 2 synthesis. 69 Chapter 3. E f f e c t of LHRH on Cy t o s o l i c Free Calcium Ion Concentrations i n Individual Granulosa C e l l s I. Introduction I t has been documented that LHRH exerts d i r e c t actions on rat ovarian c e l l s (Hsueh and Erickson, 1979; Hsueh and Jones, 1981; Clark, 1982; H i l l e n s j o et a l . , 1982; Leung, 1985). The mechanism of actions of LHRH on the ovary i s due to the stimulation of polyphosphoinositide breakdown i n the c e l l membrane (Ma and Leung, 1985; Davis et a l . , 1986). I n o s i t o l 1,4,5-trisphosphate ( I p 3 ) / a product of hydrolysis of phosphatidylinositol 4,5 biphosphate (PIP 2), has been proposed 2+ to induce i n t r a c e l l u l a r Ca mobilization (Berridge, 1984). In addition, products of phosphoinositide turnover may also be 2+ involved i n regulating Ca entry from the e x t r a c e l l u l a r f l u i d (Berridge, 1987). Many c e l l u l a r functions such as c e l l movement, d i v i s i o n , secretion and a c t i v a t i o n depend on changes i n the free c y t o s o l i c calcium ion concentrations (Cheung, 1987). Calcium has also been demonstrated to be an important signal transduction agent i n numerous tissues and c e l l s (Rasmussen and Barrett, 1984). To evaluate the ro l e of calcium as an i n t r a c e l l u l a r second messenger, quantitative measurement of c y t o s o l i c free 2+ . 2+ . Ca concentration ([Ca ]i) i s required. The most popular 2+ method f o r measuring [Ca ] i i s to monitor the s h i f t s i n 2+ 2+ wavelength of fluorescent Ca indicators when they bind Ca . 70 These indicators are tetracarboxylic acid derivatives of the calcium chelator EGTA [ethylene g l y c o l bis(6-aminoethylether)- N,N'-tetraacetic]. A recent study on suspensions of granulosa c e l l s using the calcium i n d i c a t o r dye, quin-2, measured only 2+ average free Ca changes (Davis et a l . , 1986). This dye method was not designed to examine responses of individual 2+ c e l l s and i t i s uncertain whether the increase of [Ca ] i was 2+ due to an increased entry of Ca across the c e l l membrane or 2+ to Ca release from an i n t r a c e l l u l a r s i t e . 2+ The recently developed fluorescent Ca indicator, fura- 2-acetoxy-methyl ester (fura-2AM) possesses fluorescent properties more appropriate f o r i n t r a c e l l u l a r studies. This 2+ has increased the precision of the measurement of Ca by reducing the e f f e c t s of instrument d r i f t , and more importantly 2+ has permitted the measurement of Ca without determining the i n t r a c e l l u l a r concentration of the dye. Moreover, because of the greater fluorescence i n t e n s i t y , the i n t r a c e l l u l a r concentration of the dye can be reduced thus avoiding a calcium 2+ buffering e f f e c t . The s e l e c t i v i t y of fura-2 for Ca has also been s l i g h t l y improved (Grynkiewicz et a l . , 1985). 2+ . Using fura-2, the present study examined: (1) the [Ca ] i l e v e l i n individual c e l l s i n primary cultures of dispersed 2+ granulosa c e l l s ; (2) the actions of LHRH on [Ca ] i and (3) the contributions 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 source(s) of 2+ 2+ . Ca i n the LHRH-mduced [Ca ] i changes. I I , Materials and Methods 71 Preparation of animals and granulosa c e l l s Animals and granulosa c e l l s were prepared as described i n the Chapter 2. 5 Granulosa c e l l s (10 cells/ml) were plated onto 18 mm diameter uncoated glass coverslips i n 6-well culture dish and incubated i n MEM containing 5% FBS. A f t e r 2 to 3 days of incubation at 37 °C i n an atmosphere of 5% C0 2 i n a i r , c e l l s were loaded with fura-2AM (Molecular Probes Inc., Eugene, OR), as described previously (Grynkiewicz et a l . , 1985). Preparation and loading of the fura-2AM i n d i c a t o r Fura-2AM was obtained i n 1 mg quantities. The t o t a l amount was dissolved i n 1 ml of chloroform and 50 jal aliquots were pipetted into 20 small p l a s t i c ampules. These ampoules were placed i n a dessicator, and vacuum-dried for 3h. The dried aliquots were stored at -70°C. For each culture used, 1 to 3 ml aliquots of Earl's Balanced S a l t Solution (EBSS) for fura-2AM loading were pipetted i n t o a p l a s t i c tube. The components of the EBSS were as follows: 117 mM NaCl, 1 mM NaH 2P0 4.H 20, 5.6 mM glucose, 10 mM HEPES, 2 6 mM NaHC03, 5mM KC1, 0.8 mM MgCl 2.6H 20 and 1.8 mM CaC1.2H20. Fura-2AM (50 ;ag) was dissolved i n 50 jal of DMSO, to produce a stock solution (ImM). EBSS medium was pre-incubated at 37 °C i n a 5% C0 2 environment fo r at l e a s t 15 min to s t a b i l i z e the pH at 7.4. Fura-2AM was added to EBSS while 72 vigorously a g i t a t i n g the medium on a mixer, producing a concentration of 10 juM fura-2AM (10 jul fura-2AM/ml of EBSS) . EBSS (1 ml) containing fura-2AM was immediately added into a 6 well culture dish with another 1 ml of EBSS, and the granulosa c e l l culture was placed face-up i n the wel l . Fura-2AM i s hydrophobic and therefore penetrates the plasma membrane without d i f f i c u l t y . The cultured c e l l s were then incubated with fura-2AM for 1 hour to allow the uptake to reach an equilibrium. Once inside the c e l l , c y t o s o l i c esterases cleave the acetoxymethyl groups from the indicator to release free fura-2 which i s impermeable and therefore trapped inside the c e l l s . At the end of the fura-2 "loading" incubation, c e l l s were rinsed by placing i n a dis h of fresh EBSS (2 ml) f o r a further half-hour to wash out excess fura-2AM. Fluorescence Measurement Individual coverslips were mounted face-down onto a laminar flow-through chamber (volume 350 . S i l i c o n e grease was used to complete a water-tight seal and the chamber inserted into a s t a i n l e s s s t e e l holder and the ent i r e assembly mounted onto the stage of a Zeiss Jenalumar microscope equipped with epifluorescence detector. The l i g h t souce was a 200 Watt mercury arc lamp powered by a DC power supply. The l i g h t was f i r s t passed through one of three d i f f e r e n t i a l interference f i l t e r s (350, 365 or 380 nm, bandwidths = 10 nm) mounted i n a t u r r e t which could be rotated by a computer-controlled stepping motor. The l i g h t was then passed through a 410 nm di c h r o i c 73 mirror and a lOOx apochromatic o i l immersion lens with a numerical aperture of 1.4 and an adjustable diaphragm to reduce the l i g h t i n t e n s i t y . A f i e l d diaphragm i n the l i g h t path p r i o r to the d i c h r o i c mirror was used to reduce the area of il l u m i n a t i o n to the s i z e of a sing l e granulosa c e l l . A l l fluorescent l i g h t passed back through the d i c h r o i c mirror and a 450 nm band pass f i l t e r to reduce background fluorescence. The emitted fluorescence taken at 350 nm (indicator fluorescence 2+ increased maximally with Ca binding) and 380 nm (decreased 2+ with Ca binding) was deflected e i t h e r to the eyepieces or to a camera port. Onto the camera port was mounted a photomultiplier tube which was used to convert the fluorescence into DC voltage. This voltage was then converted to d i g i t a l form by an Analogue-II D i g i t a l Converter i n the computer. Measurements of fluorescence r a t i o s were corrected for background and obtained on a 1.8 or 5 sec time base. The measurements were made at room temperature with low chloride EBSS (0.8 mM MgS04.2H20, 2.7 mM K 2S0 4, 117 mM Isethionate, 26 mM NaHC03, 1 mM NaH 2P0 4.H 20, 5.6 mM glucose, 10 mM HEPES and 1.8 mM CaCL 2.2H 20) constantly flowing at a rate of 4 ml/min throughout the experiment. C e l l u l a r l o c a t i o n of entrapped fura-2 Fura-2 was found to be uniformly d i s t r i b u t e d throughout the cytosol and nucleus of granulosa c e l l s . The responsiveness 2+ of the fura-2 to changes i n c y t o s o l i c [Ca ] i was confirmed by d i r e c t i n j e c t i o n into the laminar flow chamber of 50 ;ul of 5 pM. 74 Br-A23187, a non-fluorescent calcium ionophore (HSC Reseach Development Corporation, Toronto, Canada). The c e l l s could be used for up to 4 to 5h a f t e r loading with only minimal signs of leakage of fura-2. Calcu l a t i o n of c y t o s o l i c calcium concentration The c y t o s o l i c calcium concentration was calculated using the following formula (Grynkiewicz et a l . , 1985) [ C a 2 + ] i = kd x fl x R - P W ^ - R Where: kd = the equilibrium d i s s o c i a t i o n constant f o r the association of f u r a - 2 with c y t o s o l i c free calcium: 2 2 4 J J M . B = r a t i o of the values: the fluorescence i n t e n s i t y at 380nm with zero [Ca 2 +]/380nm with i n f i n i t e [ C a 2 + ] . R = experimentally determined r a t i o of the fluorescence in t e n s i t y at 350nm/380nm R ^ ^ r a t i o of the values: the fluorescence of in t e n s i t y 2 + at 350nm/380nm with zero [Ca ]. R m a x= r a t i o of the values: the fluorescence of in t e n s i t y at 350nm/380nm with i n f i n i t e [ C a 2 + ] . For the present study, B =10.07; = 0.51; R m a x = 4 . 8 3 were determined using the same granulosa c e l l cultures. At 2+ lea s t 50 nM [Ca ] i change was considered s i g n i f i c a n t . C a l i b r a t i o n of the system Each of the two cultures was f i r s t placed i n a measuring 2+ 2+ chamber with standard EBSS (1.8 mM Ca , 0.8 mM Mg ) and [Ca ] i was determined i n 20 to 40 c e l l s i n each culture. This was done to assess the health of the culture and to provide a control f o r further c a l i b r a t i o n . A f t e r measuring control values, one culture was rinsed with EBSS containing 5 mM EGTA 2+ and no Ca . I t was then placed onto a chamber which was 2+ f i l l e d with Ca -free EBSS containing 5 mM EGTA and Br-A23187 2+ (10 uM). [Ca ] i measurements were taken immediately and a f t e r 15 min. Af t e r the second culture c o v e r s l i p was measured f o r control values, the c e l l s were immediately transferred to a 2 + chamber with standard EBSS containing Br-A23187. [Ca ] i values were measured immediately (within seconds), as 2+ e x t r a c e l l u l a r Ca quickly entered the c e l l . B, RJn^n and R m a x constants were then calculated from the averages of the values obtained from these experiments. Reagents LHRH was purchased from Sigma. A potent LHRH antagonist, Ac-D-Nal ( 2 ) 1 , 4 Cl-D-Phe 2, D-Trp 3, D-Ala 1 0-LHRH, was obtained as a g i f t from Dr. M.V. Nekola of Tulane University. Ovine LH (oLH-25), ovine FSH (oFSH-16) and pregnant mare's serum gonadotropin were obtained from the NIDDK and National Hormone and P i t u i t a r y Program (University of Maryland School of Medicine). Other chemicals were obtained from Sigma. I l l . Results 76 Rapid and transient e f f e c t s of Br-A23187 on c y t o s o l i c calcium A f t e r loading with fura-2, granulosa c e l l s were challenged by i n j e c t i o n into the flow through-chamber of the calcium ionophore Br-A23187. F i g . 17 shows a representative example of the e f f e c t of a 50 ;ul of i n j e c t i o n of Br-A23187. 2+ There was a rapid and transient increase i n [Ca ] i 18 sec a f t e r the i n j e c t i o n of Br-A23187 with a peak value of cy t o s o l i c 2+ [Ca ] i approximately 8-10 f o l d above the r e s t i n g l e v e l . This time delay was due to the time required f o r the i n j e c t i o n volume to flow to the observed c e l l and the r e l a t i v e l y slow incorporation of the ionophore into the membrane of the c e l l s . LHRH-induced transient increase i n c y t o s o l i c calcium Each of the 115 rat granulosa c e l l s from 27 di f f e r e n t preparations were treated with LHRH. The average resting 2+ l e v e l of [Ca ] i of these c e l l s was 96.7 ± 2.9 nM. Eighty-six c e l l s of the t o t a l 115 responded to LHRH. Table I i l l u s t r a t e s 2 + that the hormone concentration required to increase [Ca ] i was i n the range of 10 _ 9M to 10~5M. LHRH at 10 _ 5M increased 2+ [Ca ] i i n a l l of the c e l l s which were s e n s i t i v e to t h i s hormone. The c e l l s which did not respond to LHRH were se n s i t i v e to the calcium ionophore A23187 or Angiotensin I I . F i g . 18 shows a representative example of LHRH-induced 2+ rapid and transient [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 of [Ca ] i were made at 1.8 77 Fig. 17. Br-A23187 induced rapid and transient increase i n c y t o s o l i c calcium. Coverslips with fura-2 loaded granulosa c e l l s were mounted on a s p e c i a l l y designed laminar flow-through chamber at room temperature. At the time (0 time) indicated by the symbol (A) , 50 jul of A23187 (5x10 M) was injected. The r e s u l t i n g images were measured by fluoroscence microscope microcomputer system at a 5 sec base (12 recording per min). Similar 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 F i g . 18. LHRH induced rapid and transient increase i n c y t o s o l i c calcium. At the time (0 time) indicated by the symbol (A) , 25 j i l of LHRH was injected. The r e s u l t i n g images were measured at a 1.8 sec base (33 recording per min). The other experimental conditions were the same as those described i n the legend of Fig . 17. Similar r e s u l t s were obtained from 21 i n d i v i d u a l c e l l s of 8 experiments. 79 sec i n t e r v a l s . The average latency of the i n t r a c e l l u l a r calcium response a f t e r the i n j e c t i o n of LHRH was 21 ± 0.09 sec (n=70) and the average peak value induced by d i f f e r e n t doses of LHRH i s shown i n Table I I . The amplitudes of the [ C a 2 + ] i increase induced by the d i f f e r e n t doses of LHRH were not s i g n i f i c a n t l y d i f f e r e n t from each other. Within 84 ± 3 sec 2+ (n=64) af t e r LHRH stimulation, [Ca ] i returned to the resting l e v e l . The blockade of LHRH-induced c y t o s o l i c calcium a l t e r a t i o n by LHRH antagonist To determine whether or not a receptor-mediated mechanism 2 + was involved i n the action of LHRH on [Ca ] i , the e f f e c t of a potent LHRH antagonist was examined (Fig. 19) . In each of 10 c e l l s , an i n i t i a l i n j e c t i o n of 25 j i l of 10~6M LHRH resulted i n 2+ . a rapid and transient increase of [Ca ] i . LHRH antagonist (25 —5 ;al of a 10 M solution) was then injected into the c e l l chamber. The LHRH antagonist by i t s e l f had no d i r e c t e f f e c t on 2+ the resting [Ca ] i . On the other hand, two subsequent injec t i o n s of LHRH, at 2 and 4.5 min following the administration of the LHRH antagonist, f a i l e d to increase 2+ [Ca ] i . These c e l l s nonetheless s t i l l responded to Br-A23187 following the treatments with LHRH and LHRH antagonist. 80 Table I. lowest hormone concentrations required to i n i t i a t e a change i n c y t o s o l i c calcium i n granulosa c e l l s . LHRH C e l l number 10"9M. 3 10~8M 3 10 _ 7M 21 10 - 6M 37 10~5M 22 No response 29 Total 115 Table I I . Average peak value of [Ca ] i induced by d i f f e r e n t doses of LHRH. LHRH fCa.2+M Change (fold) 10 _ 7M 4.97 ± 0.69 (n=21) 10~6M 4.54 ± 0.32 (n=37) 10"5M 4.51 ± 0.55 (n=22) Existence of a subpopulation of granulosa c e l l s : rca |1 changes induced by d i f f e r e n t hormones 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 of 10~6M 2 + LHRH caused a rapid and transient increase i n [Ca ] i , whereas two i n j e c t i o n s of Ang II at 10~5M and 10"4M, respectively, did 2 + not a f f e c t the r e s t i n g l e v e l of [Ca ] i i n the same c e l l . 2+ However, LHRH-induced [Ca ] i a l t e r a t i o n was not influenced by 2+ Ang II and an increase i n [Ca ] i induced by LHRH was observed a f t e r Ang I I . In contract, the d i f f e r e n t r e s u l t was observed from d i f f e r e n t i n d i v i d u a l granulosa c e l l s . F i g . 20 (panel B) shows a representative example of Ang II (10~5M) induced 2+ increase i n [Ca ] i . However, the same c e l l d i d not respond to LHRH (10~ 5M). 2+ Desensitization of fCa ] i response induced bv continuous exposure to LHRH The upper panel of F i g . 21 shows a representative example of the c y t o s o l i c calcium increase stimulated by 3 separate injections of 25 jul of 10~6M LHRH. The i n t e r v a l between the 2+ inject i o n s was 5 min. The increase i n [Ca ] i induced by these consecutive i n j e c t i o n s of LHRH reached s i m i l a r maximum amplitudes. This pattern was seen i n each of 14 c e l l s , a l b e i t 2+ the peak [Ca ] i responses of the c e l l s to the same dose of LHRH varied between 250 to 600 nM. The lower panel of Fig. 21 2+ i l l u s t r a t e s another representative example of [Ca ] i alt e r a t i o n s induced by repeated doses of 25 )il of 10~6M LHRH given at i n t e r v a l s of les s than 2 min. A gradual decrease i n 82 F i g . 19. The blockade of LHRH-induced c y t o s o l i c calcium a l t e r a t i o n by LHRH antagonist. The experimental conditions were the same as those described under the legend of Fig . 17, but with 25 jul i n j e c t i o n s of LHRH, LHRH antagonist (LHRH anta), or Br-A23187 at the times indicated by (•) . Similar results were obtained from 10 i n d i v i d u a l c e l l s of 10 experiments. -2 - 1 0 1 2 3 4 6 6 7 8 9 Time (min) 11 12 600 400 1 300 • CM « 200 100 Ang II (K)"6M) B LHRH (10~*M) -1 -0.6 0 0.6 1 1.6 2 2.6 Time (min) 3.6 4.6 F i g . 2 0 • 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 the representative examples of the c e l l s responded to either LHRH or Ang I I , respectively. ( A ) indicates the i n j e c t i o n of LHRH or Ang I I . Similar results were obtained from 11 in d i v i d u a l c e l l s i n 4 d i f f e r e n t experiments. 84 -2 4 6 8 10 Time (min) 2+ Fig. 21. Increase i n [Ca ] i induced by separate i n j e c t i o n s of LHRH to two in d i v i d u a l granulosa c e l l s . The upper panel shows the c y t o s o l i c calcium increase stimulated by 3 separate i n j e c t i o n s of LHRH. The. lower panel shows a gradual decrease i n the amplitude of [Ca ] i induced by LHRH at in t e r v a l s of less than 2 min. Similar 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 of [Ca ] i could be seen. A s t r i k i n g example of desen s i t i z a t i o n induced by LHRH i s shown i n Fi g . 22. In t h i s experiment a granulosa c e l l was perifused continuously with 10 _ 7M LHRH f o r 10 min. The pe r i f u s i o n of LHRH caused a 2+ 2+ transient increase i n [Ca ] i which was not unlike the [Ca ] i change induced by a pulse i n j e c t i o n of LHRH. However, the 2+ increase i n [Ca ] i returned to the resting l e v e l despite the continued presence of LHRH. Furthermore, a pulse i n j e c t i o n of 25 ; i l of 10"5M LHRH 5 min a f t e r the i n i t i a t i o n of the LHRH 2+ infusion period f a i l e d to increase [Ca ] i . In contrast, following the cessation of the infusion and a f t e r a wash period of 8 min, the same i n j e c t i o n of 10 M LHRH resulted i n a 2+ transient increase i n [Ca ] i , a l b e i t to a lesser amplitude than the i n i t i a l e f f e c t of 10"7M LHRH. 2+ E f f e c t of d i f f e r e n t doses of LHRH on \Ca. J_i 2+ Fig. 23 shows the change of [Ca ] i induced by d i f f e r e n t doses of LHRH i n a single granulosa c e l l . The c e l l was treated with sequential inj e c t i o n s of LHRH from 10~8M to 10""4M. The in t e r v a l between the in j e c t i o n s was at least 10 min to avoid any possible d e s e n s i t i z a t i o n . No s i g n i f i c a n t difference i n 2 + peak le v e l s of [Ca ] i was observed following the i n j e c t i o n of d i f f e r e n t doses of LHRH i n single c e l l s , or when the response of d i f f e r e n t c e l l s was analyzed together (Table I I ) . 86 Fi g . 22. Desensitization of [Ca ] i response induced _by continuous exposure to LHRH. F i r s t ( A ) : LHRH (10~7M) pe r i f u s i o n for 10 min; second and t h i r d (A): 25 jal LHRH (10 M) in j e c t i o n s . Similar r e s u l t s were obtained from 6 in d i v i d u a l c e l l s of 4 experiments. 87 600 600 2 4 0 0 ss. 300 • CM a) O 200 LHRH LHRH (10-8M) (10-7M) LHRH (10~«M> LHRH 0 0 - « M ) 10 16 20 Time (min) LHRH <10-«M) 26 30 36 40 F i g . 23. Al 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 treated with sequential i n j e c t i o n s of LHRH from 10~ to 10~ M and no s i g n i f i c a n t difference i n peak l e v e l was observed. Similar r e s u l t s were obtained from 11 individual c e l l s i n 4 separate experiments. (A) indicates the i n j e c t i o n Of LHRH. 2+ 2+ 8 8 Influence of Ca free medium on \Ca ] i a l t e r a t i o n 2+ 2+ To determine the influence of Ca free medium on [Ca ] i 2 + a l t e r a t i o n , granulosa c e l l s were perifused with Ca free EBSS 2+ following the rapid and transient increase i n [Ca ] i induced —6 2 + by LHRH (10 M) i n normal Ca EBSS. F i f t e e n minutes a f t e r the 2+ Ca free EBSS pe r i f u s i o n , two sequential i n j e c t i o n s of LHRH —6 2+ 2+ (10 M dissolved i n Ca free EBSS) were made and [Ca ] i did not increase i n response to LHRH. Interestingly, granulosa c e l l s responded to LHRH (10~6M) normally again a f t e r 7 min of washing with normal EBSS. F i g 24. shows a representative example of 14 c e l l s tested i n the s i m i l a r condition. The washing time required to e s t a b l i s h a completely non-responsive 2+ condition i n Ca free EBSS varied from 8 min to 20 min i n the d i f f e r e n t granulosa c e l l s studied. 2+ 2+ Role of i n t r a c e l l u l a r Ca i n LHRH-induced fCa ] i alt e r n a t i o n The source(s) of calcium which contributed to the 2+ increase of [Ca ] i induced by LHRH was further examined. As i l l u s t r a t e d i n upper panel of F i g . 25, a f t e r the i n i t i a l 2+ —6 increase of [Ca ] i induced by 10 M LHRH i n normal EBSS, 2+ granulosa c e l l s were perifused with Ca free EBSS medium. 2+ Aft e r washing with Ca free EBSS for 8 min, the i n j e c t i o n of LHRH (10~6M) resulted i n a rapid and transient increase of 2 + [Ca ] i but with a s i g n i f i c a n t l y decreased amplitude (about 35% 2+ of the amplitude of [Ca ] i increase i n normal EBSS medium). 2+ In addition, a notable decrease of basal [Ca ] i was also 2+ observed a f t e r the f i r s t i n j e c t i o n of LHRH i n Ca free EBSS, 89 600 600 - <| 400 sf 300 CM 200 100 0 Ca2* tree medium LHRH (lO" 6**) LHRH <*r 6 M) LHRH dO" e M) LHRH (10 _ 6M) -6 10 16 20 Time (min) 26 30 2+ Fi g . 24. Depletion of i n t r a c e l l u l a r Ca_ + i n calcium free medium. Af t e r the i n i t i a l increase of [Ca ] i induced Jay LHRH i n normal medium, the c e l l s were per i f used with Ca free medium. F i f t e e n minutes a f t e r the Ca free medium perifusion, [Ca ] i d i d not increase i n response to LHRH. The c e l l responded to LHRH again a f t e r washing with normal medium. Similar 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 indicates the entry of calcium free medium. 90 600 600 LHRH 10" 8M) Ga2* free medium -4 4 6 8 10 12 14 16 18 20 22 24 Time (min) F i g . 25. LHRH accelerated [Ca ] i depletion i n Ca free medium. The f i r s t (•) indicates the i n j e c t i o n of LHRH i n normal medium;_^the second and t h i r d (A) indicate the i n j e c t i o n s of LHRH i n Ca free medium (upper and lower panel). Similar r e s u l t s were obtained from 6 i n d i v i d u a l c e l l s i n 4 experiments. 0 time indicates the entry of calcium free medium. 2+ 9 1 and [Ca ] i d i d not subsequently respond to the i n j e c t i o n of LHRH at 13 min. When LHRH (10~6M) was injected at 13 min instead of 8 min 2+ a f t e r washing with Ca -free EBSS, a transient increase of 2 + [Ca ] i was observed, a l b e i t with a smaller amplitude than that 2+ induced by LHRH at 8 min i n Ca free EBSS medium (Fig. 25). 2+ Role of 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 X C a 2 + U 2+ Fi g . 26 shows the increases i n [Ca ] i i n a single granulosa c e l l following the i n j e c t i o n of LHRH at 0 time, 2+ followed by continuous washing the c e l l with Ca free EBSS. Af t e r entry of C a 2 + free EBSS medium, LHRH-induced [ C a 2 + ] i change was f i r s t decreased and eventually completely abolished. Subsequently, four separate i n j e c t i o n s of LHRH, which were dissolved i n EBSS medium with 2 mM, 5 mM, 10 mM and 20 mM 2+ calcium, d i d not r e s u l t i n the change of [Ca ] i . LHRH caused 2+ the increase of [Ca ] i again i n the same c e l l following the p e r i f u s i o n of normal EBSS. Comparison of gonadotropins with LHRH on TCa ] i a l t e r a t i o n F i g . 27 i l l u s t r a t e s that when a single granulosa c e l l was stimulated by two separate 25 pi i n j e c t i o n s of 50 pg FSH, the [ C a 2 + ] i was not altered. The i n j e c t i o n 25 pi of LHRH (10~6M) 2+ following FSH resulted i n the expected increase i n [Ca ] i . Similar r e s u l t s were obtained with 8 i n d i v i d u a l c e l l s . In addition, as shown i n F i g . 28, a granulosa c e l l which responded 92 800 600 2 ss. 400 CM 200 h Ca2* free medium LHRH dO _ 8 M) Ca2* (mM) 2 6 10 20 • • A A A A •10 10 20 30 40 Time (min) 60 60 2+ Fig. 26, Role of 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 for a l l the 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 inject i o n s i n calcium free medium; fourth to seventh (A) : LHRH plus d i f f e r e n t concentrations of Ca injec t i o n s i n calcium free medium; eighth ( A ) : LHRH i n j e c t i o n i n normal medium. Similar r e s u l t s were obtained from 4 c e l l s of 4 experiments. 93 F i g . 27. Comparison of FSH with LHRH on [ C a ^ j i a l t e r a t i o n . LHRH resulted i n rapid and transient [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 of i n j e c t i o n of FSH; second and fourth ( ) : LHRH. Similar r e s u l t s were obtained 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 a l t e r a t i o n . The c e l l which responded to LHRH d i d not respond to LH. Sim i l a r r e s u l t s were obtained from 8 i n d i v i d u a l c e l l s i n 4 experiments. to LHRH (10 M) did not respond to 10 ug of LH, nor d i d the two separate i n j e c t i o n s of LH have any influence on the subsequent 2+ LHRH-induced increase i n [Ca ] i . Identical r e s u l t s were seen i n 7 other c e l l s . IV. Discussion The calcium-sensitive fluorescent indicator, fura-2 has 2 + been used to study the e f f e c t of LHRH on the [Ca ] i of in d i v i d u a l r a t granulosa c e l l s . LHRH caused a rapid and 2+ transient increase i n [Ca ] i i n the majority of c e l l s tested (Fig. 17). Since an LHRH antagonist completely blocked the [ C a 2 + ] i response of the c e l l s to LHRH (Fig. 19), i t could be 2+ concluded that the e f f e c t s of LHRH on [Ca ] i are mediated by i t s s p e c i f i c receptors. However, the concentrations of LHRH required to produce a 2+ . -9 -5 [Ca ] i response varied considerably (10 M to 10 M) from c e l l to c e l l (Table I) . This i s not l i k e l y to have resulted from the design of the laminar flow-through chamber. I t has been estimated that each dose of LHRH would only be d i l u t e d by no more than a factor of 2-5, depending upon the flow rate used and the p o s i t i o n of the c e l l r e l a t i v e to the input of the chamber. Therefore, the varied concentrations of LHRH required by the in d i v i d u a l granulosa c e l l s may be due to the d i f f e r e n t threshold of the c e l l s (see following discussion). Most c e l l s responded to LHRH i n the range of 10 M to 10~5M, but for any single c e l l there was no c l e a r dose-related response to LHRH (Fig. 22; Table I I ) . The lowest dose of LHRH which resulted i n an increase i n [Ca ] i appeared to y i e l d a maximum response since higher LHRH concentrations given to the 2+ same c e l l d i d not r e s u l t i n additional increases i n [Ca ] i . Thus, singl e granulosa c e l l s seem to respond i n an " a l l or none" fashion. Previous studies have, however, shown that LHRH and i t s agonists do produce dose-dependent stimulatory and i n h i b i t o r y e f f e c t on progesterone production ( H i l l e n s j o et a l . , 1982; Knecht et a l . , 1985;). LHRH-induced arachidonic acid l i b e r a t i o n from the c e l l membranes (Minegishi and Leung, 1985) and LHRH-stimulated i n o s i t o l phosphate formation i n rat granulosa c e l l s are also dose-dependent (Ma and Leung, 1985; Davis et a l . , 1987). In addition, LH-induced [ C a 2 + ] i a l t e r a t i o n i n bovine l u t e a l c e l l s has been shown to be dose- dependent as well (Davis et a l . , 1987). I t i s important to note, however, that i n a l l these e a r l i e r studies populations of c e l l s were used rather than the i n d i v i d u a l c e l l s which were used i n the present study. In t h i s regard, i t can be speculated that 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 fashion to LHRH, but that the threshold concentrations of LHRH required to stimulate d i f f e r e n t c e l l s may d i f f e r . Hence when a mixed population of such c e l l s i s stimulated with LHRH, a dose response r e l a t i o n w i l l be observed as progressively more c e l l s "turn on". These data support the hypothesis of a quantal ( i . e . all-or-none) response of hormonal control mechanism, since i t has been suggested that a l l c e l l s of a given type may not be equal i n terms of hormonal responsiveness (Moyle et a l . , 1985). 97 The differences i n c e l l - t o - c e l l responsiveness were found to be randomly d i s t r i b u t e d i n t h i s study. The d i f f e r e n t minimum concentrations of LHRH required f o r i n i t i a t i n g the 2+ c y t o s o l i c [Ca ] i change may i n part be re l a t e d to the d i f f e r e n t functional states of the LHRH receptor i n these c e l l s . While some i n d i v i d u a l granulosa c e l l s responded to LHRH, others responded to d i f f e r e n t hormones such as Ang II (Fig. 20) . Previous studies have shown that subpopulations of granulosa c e l l s may e x i s t with respect to d i f f e r e n t i a l s e n s i t i v i t y to FSH and vasoactive i n t e s t i n a l peptide (Kasson et a l . , 1985). In addition, PRL receptors have been shown to be more abundant i n antral granulosa c e l l s than i n mural granulosa c e l l s (Dunaif et at., 1982). The use of fura-2 microspectrofluorimetry techniques f a c i l i t a t e d the inve s t i g a t i o n on the subpopulations of granulosa c e l l s by allowing the study of i n d i v i d u a l granulosa c e l l s . The present finding that d i f f e r e n t granulosa c e l l s responded to d i f f e r e n t hormones may indicate that there are d i f f e r e n t subpopulations of granulosa c e l l s which play d i f f e r e n t r o l e s i n response to d i f f e r e n t regulator-mediated ovarian functions. One i n t e r e s t i n g observation made i n t h i s study was that with the decreasing time i n t e r v a l s between i n d i v i d u a l LHRH in j e c t i o n s , the magnitude of the LHRH-stimulated increase in 2+ [Ca ] i declined (Fig. 21). Furthermore, continuous exposure to a r e l a t i v e l y low concentration of LHRH (10 M) resulted in —5 desens i t i z a t i o n of granulosa c e l l s to higher (10 M) doses of 98 LHRH (Fig. 22). This may r e f l e c t the well-known down- regulation phenomenon of LHRH surface receptors, which may be due to massive i n t e r n a l i z a t i o n of the LHRH-receptor complex into endocytic v e s i c l e s (Hazum and Nimrod, 1982) and subsequent degradation of t h i s complex. Peptide hormones, i . e i n s u l i n , LHRH (on gonadotrophes) , and hCG, have been shown to induce m o t i l i t y , aggregation, and i n t e r n a l i z a t i o n of t h e i r receptors (Terris et a l . , 1979; Amsterdam et a l . , 1979; Hopkins and Gregory, 1977). In the present study, the decrease i n receptor numbers may protect against intense stimulation by inappropriately high LHRH l e v e l s . On the other hand, t h i s desensitization may be due to a receptor-mediated mechanism that i s not r e l a t e d to the i n t e r n a l i z a t i o n of receptors. As i t has been shown that only a f t e r 2 h of exposure, a s i g n i f i c a n t proportion (10%) of the labeled hCG can be found i n lysosome- l i k e structures of r a t granulosa c e l l s , i t i s i n f e r r e d that i n t e r n a l i z a t i o n i s i n i t i a t e d within t h i s time (Amsterdam et a l . , 1979). Moreover, i n the present study, i t was observed that after washing with fresh medium, the c e l l s regained t h e i r 2+ responsiveness to LHRH i n terms of [Ca ] i (Fig. 22). The transient and r e v e r s i b l e nature of t h i s d e s e n s i t i z a t i o n process may be more compatible with alt e r n a t i v e mechanisms of desensitization, possibly at the l e v e l of s i g n a l transduction. I t i s i n t e r e s t i n g to note that, whatever the mechanism of t h i s desensitization, fluctuations i n LHRH l e v e l s may be more 2+ e f f e c t i v e i n stimulating a [Ca ] i response i n granulosa c e l l s than a sustained elevation i n LHRH concentration. 99 Recent studies have c l e a r l y shown that LHRH stimulates the formation of IP 3 i n ovarian granulosa c e l l s (Ma and Leung, 1985; Davis et a l . , 1986). I P 3 has been proposed as a mediator 2+ fo r i n t r a c e l l u l a r Ca mob i l i z a t i o n (Nishizuka et a l . , 1984). Phosphoinositide turnover i s also believed to be involved i n 2+ the regulation of Ca entry from the external environment (Berridge, 1987) . M i c r o i n j e c t i o n of IP 3 into some c e l l s 2+ r e s u l t s i n the [Ca ] i mob i l i z a t i o n and mimics calcium- dependent processes (Oron et a l . , 1985). Based on above 2+ observations, i t appears that the [Ca ] i changes stimulated by LHRH might be d i r e c t l y c o r r e l a t e d to IP 3 formation. This hypothesis i s supported by evidence obtained from the present and previous studies. LHRH antagonist can block LHRH-induced 2+ c e l l u l a r responses including both I P 3 formation and [Ca ] i mobilization (Ma and Leung, 1985; F i g . 19). Similar temporal 2+ relat i o n s h i p s between LHRH-induced I P 3 formation and [Ca ] i mobilization has been found by Davis et a l . (1986). Although LHRH resulted i n the rapid and transient 2+ 2+ increase of [Ca ] i , the precise source (s) of Ca which contributed to the c y t o s o l i c calcium a l t e r a t i o n has to be resolved. In the present studies, the LHRH-induced changes i n 2+ [Ca ] i were completely abolished by washing with calcium free medium between 8 min to 20 min i n 14 c e l l s tested. Following 2+ re-perifusion with medium containing normal Ca , the LHRH- induced increase i n [ C a 2 + ] i was again observed (Fig. 24). When the i n j e c t i o n of LHRH was performed several minutes a f t e r the entry of C a 2 + free medium int o the chamber, a s i g n i f i c a n t 2+ 1 0 0 decrease i n [Ca ] i amplitude was observed as compared to the 2+ peak l e v e l of [Ca ] i i n the normal medium (Fig. 25). This decrease can be due to eithe r the depletion of i n t r a c e l l u l a r 2+ 2+ Ca or the lack of Ca i n the e x t r a c e l l u l a r f l u i d . Based on the above r e s u l t s , i t can be estimated that the i n t r a c e l l u l a r 2+ Ca pool i s depleted by passive d i f f u s i o n when the external 2+ medium reaches a "Ca free" condition (approx. 8 min) . 2+ However, the complete depletion of [Ca ] i was a gradual 2+ process, and a smaller increase of [Ca ] i could s t i l l be 2+ observed i n e x t r a c e l l u l a r Ca free condition, suggesting that 2+ . . . LHRH does induce [Ca ] l mobilization from an i n t r a c e l l u l a r 2+ pool (Fig. 25). Ca which i s mobilized 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 solution. Therefore, with time the 2+ i n t r a c e l l u l a r pool of Ca would be exhausted, the c e l l would eventually lose responsiveness to LHRH. The LHRH-induced 2+ 2+ depletion of i n t r a c e l l u l a r Ca i n Ca free medium (Fig. 25) 2 + supports the concept that LHRH-induced increase of [Ca ] i i s , at l e a s t p a r t i a l l y , from i n t r a c e l l u l a r stores. The marked 2+ decline of the basal [Ca ] i a f t e r the i n j e c t i o n of LHRH i n 2+ Ca free medium further strengthens t h i s notion (Fig. 25). Since agonist-induced I P 3 i s believed to be responsible 2+ for i n t r a c e l l u l a r Ca release, many studies have been made to elucidate which i n t r a c e l l u l a r pool i s responsible for the 2+ [Ca ] i mobilization. The s i t e at which I P 3 acts has been 2+ shown to be a ATP-dependent non-mitochondrial Ca pool, probably ER. The experimental data obtained from previous studies have indicated that normal responses to IP 3 are 2+ 1 0 1 observed at free Ca concentrations below the threshold for mitochondrial uptake or i n the presence of mitochondrial i n h i b i t o r s (Burgess et a l . , 1984). Whereas IP 3 releases C a 2 + when added d i r e c t l y to microsomes obtained from a va r i e t y of 2+ tiss u e s , i t f a i l s to a l t e r Ca release from mitochondrial f r a c t i o n s (Streb et a l . , 1983; Prentki et a l . , 1984). Although s u b c e l l u l a r f r a c t i o n a t i o n studies have attributed a large 2+ portion of i n t r a c e l l u l a r Ca pool to mitochondria (Claret- Berthon et a l . , 1977), recent studies have demonstrated far 2+ less Ca i n mitochondria than i n ER (Reinhart et a l . , 1984; Shears and Kirk, 1984). Electron probe X-ray microanalysis study of r a p i d l y frozen l i v e r also indicates that only 5% of 2+ c e l l Ca i s present i n mitochondria, whereas 14-23% i s within rough ER (Somlyo et a l . , 1985). The present r e s u l t s indicate 2+ that the i n t r a c e l l u l a r Ca pools are probably responsible for 2+ LHRH-induced increase of. [Ca ] i . The r e l a t i v e importance of mitochondria and ER i n granulosa c e l l c y t o s o l i c calcium regulation remains uncertain. 2 + The possible contribution of e x t r a c e l l u l a r Ca to LHRH- 2+ induced increase of [Ca ] i was next examined. LHRH dissolved 2+ i n high concentration of Ca (2 mM to 20 mM) was given to 2+ granulosa c e l l s being perifused with Ca free medium and i n 2+ 2+ which the i n t r a c e l l u l a r Ca had been depleted. LHRH plus Ca 2+ f a i l e d to evoke the increase of [Ca ] i i n the granulosa c e l l , 2+ suggesting that LHRH-induced [Ca ] i a l t e r a t i o n may not be due 2+ to the immediate Ca i n f l u x across the c e l l membrane (Fig. 26). A l t e r n a t i v e l y , these r e s u l t s could suggest that even when 2+ l f& the i n t r a c e l l u l a r Ca pools are empty, the e x t r a c e l l u l a r Ca cannot quickly enter the cytosol. This i s despite the existence of a gradient p o t e n t i a l and the presence of LHRH to 2+ ensure the opening of the Ca channels on the c e l l membrane. 2+ An early suggestion was that the Ca content of i n t r a c e l l u l a r 2+ pools regulated the entry of Ca from the e x t r a c e l l u l a r f l u i d ; 2+ when the pools were empty, i t was open to e x t r a c e l l u l a r Ca entry, and when the pools were f i l l e d , i t was closed to the 2+ e x t r a c e l l u l a r Ca (Aub et a l . , 1982; Putney, 1986). Two 2+ phases of Ca mobilization 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 to agonists, and i n the second 2+ phase, entry of Ca across the plasma membrane following the f i r s t phase (Kojima et a l . , 1985 Reynolds and Dubyak., 1985). 2 + In p i t u i t a r y c e l l s , LHRH elevates [Ca ] i p a r t l y by releasing 2+ Ca from i n t r a c e l l u l a r pools and par t l y by t r i g g e r i n g i n f l u x across the c e l l membrane. I t has been shown that the elevation 2+ i n [Ca ] i induced by LHRH i s composed of a rapid f i r s t phase 2+ followed by a prolonged increase i n [Ca ] i i n the second phase (Clapper and Conn, 1985; Limor et a l . , 1987). Furthermore, Naor et a l . (1988) have recently demonstrated that 2 + LHRH induces a rapid mobilization of i n t r a c e l l u l a r Ca pool, 2+ and a second component of Ca i n f l u x v i a voltage s e n s i t i v e and 2+ in s e n s i t i v e changes contributes to further elevation of [Ca ] i 2+ i n p i t u i t a r y c e l l s . Although the increase i n [Ca ] i induced by LHRH i n individual granulosa c e l l s did not obviously show 2+ two phases, that e x t r a c e l l u l a r Ca might also be involved i n 2+ 1 0 3 LHRH-induced [Ca ] i changes, with an i n i t i a l i n t r a c e l l u l a r 2+ 2+ Ca mobilization t r i g g e r i n g the i n f l u x of Ca , cannot be excluded. In addition, a f t e r washing the c e l l s with medium 2+ containing normal Ca , granulosa c e l l s regained t h e i r response to LHRH (Fig. 24) , which implied that a continued p o s i t i v e 2+ d i f f u s i o n of Ca from e x t r a c e l l u l a r f l u i d was necessary to r e f i l l i n t r a c e l l u l a r pools without a rapid change i n c y t o s o l i c 2+ Ca . Although i t has been suggested that phosphoinositide 2+ turnover may be involved i n the regulation of Ca entry from e x t r a c e l l u l a r f l u i d (Berridge 1984), experimental evidence 2+ indicates that eit h e r I P 3 or PKC regulates Ca i n f l u x by a d i r e c t action at the plasma membrane (Streb et a l . , 1984; Cooper et a l . , 1985; Garrison et a l . , 1984). I t appears that a decrease i n the PIP 2 content of the c e l l membrane may i n h i b i t 2+ 2 + the Ca -ATPase and therefore cause an increase i n [Ca ] i (Berridge, 1982). This, however, i s not a s u f f i c i e n t 2+ . explanation f o r the rapid changes of [Ca ] i induced by LHRH. . 2 + The e f f e c t s of gonadotropins on granulosa c e l l [Ca ] i were also investigated i n the present study. Unlike LHRH, neither FSH nor LH, even at very high doses (10 ug of LH or 50 ug of FSH), had any e f f e c t on [ C a 2 + ] i (Fig. 27; 28). In is o l a t e d bovine l u t e a l c e l l s , i t has been reported that LH provokes a rapid increase i n the accumulation of IP 3 and 2+ increase i n [Ca ] i (Davis et a l . , 1987). Treatment with LH, f o r s k o l i n and cAMP may also cause Ca e f f l u x i n avian granulosa c e l l s (Asem et a l . , 1987). In addition, exogenous 2+ 1 0 4 cAMP and f o r s k o l i n have been shown to increase [Ca ] i i n Leydig c e l l s (Sallivan and Cooke, 1986). I t i s possible that 2+ the action of gonadotropins on [Ca ] i may be both species and c e l l s p e c i f i c . The present and previous studies demonstrate that, i n r a t granulosa c e l l s , administration of LHRH leads to a rapid breakdown of i n o s i t o l l i p i d s and an increase i n [ C a 2 + ] i , whereas gonadotropins consistently have no e f f e c t on either parameter (Ma and Leung, 1985). 2+ A study of 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 i n [Ca ] i a f t e r LHRH was added to suspensions of gonadotroph-enriched p i t u i t a r y c e l l s could be correlated to the LH release (Naor et a l . , 1988). This 2+ suggests that [Ca ] i plays an intermediary r o l e when LHRH stimulates LH release from the p i t u i t a r y . In the ovary, . . . . 2+ . si m i l a r increases i n [Ca ] i may serve to modulate the stimulatory or i n h i b i t o r y e f f e c t s of LHRH on P 4 and PGE 2 accumulation (see Chapter 4). In summary, the present study strongly indicates that 2 + LHRH causes a rapid and transient increase i n c y t o s o l i c [Ca ] i i n i n d i v i d u a l r a t granulosa c e l l s . The action of LHRH on the 2+ cy t o s o l i c [Ca ] i change i s mediated by i t s s p e c i f i c receptors. By investigating i n d i v i d u a l granulosa c e l l s , i t i s possible to demonstrate that d i f f e r e n t granulosa c e l l s require d i f f e r e n t concentrations of LHRH to i n i t i a t e what appears to be an " a l l or none" response and that d i f f e r e n t subpopulations of granulosa c e l l s may also e x i s t . Another i n t e r e s t i n g 2+ observation i s the down-regulation of the [Ca ] i response 105 induced by LHRH which has not been studied previously i n 2+ in d i v i d u a l ovarian c e l l s . F i n a l l y , the i n t r a c e l l u l a r Ca . 2+ sources are c l e a r l y involved i n LHRH-induced [Ca ] i changes, 2+ whereas the r o l e of e x t r a c e l l u l a r Ca needs to be further investigated. These r e s u l t s indicate that LHRH may function as a paracrine or autocrine mediator i n the r a t ovary with calcium functioning as a second messenger f o r LHRH. 106 Chapter 4 . LHRH Action on Ovarian Hormone Production; A l t e r a t i o n s of Progesterone and Prostaglandins Accumulation by Calcium Ionophore and Protein Kinase C Activa t o r I. Introduction Several laboratories have already reported that a c t i v a t i o n of protein kinase C stimulates basal P 4 production i n r a t granulosa c e l l s , but i n h i b i t s the P 4 response to stimulation by gonadotropins or cAMP derivatives (Shinohara et a l . , 1986; Kawai and Clark, 1985; Welsh et a l . , 1984; Leung et a l . , 1988). The steroidogenic e f f e c t of LHRH i s p a r t i a l l y blocked by a potent i n h i b i t o r of PKC (Wang and Leung, 1987). Recently, PKC a c t i v i t y has been characterized i n ovarian t i s s u e s (Noland and Dimino, 1986; Davis and Clark, 1983; Veldhuis and Demers, 1986). The highest s p e c i f i c enzyme a c t i v i t i e s are found i n the cytosol, followed by microsomes and mitochondria, respectively. In addition, i t has been observed 2+ that LHRH and i t s agonists rapi d l y increase c y t o s o l i c free Ca l e v e l i n populations of granulosa c e l l s as measured by quin 2 (Davis et a l . , 1986), and i n in d i v i d u a l granulosa c e l l s by fura-2 fluorescence (chapter 2). Although the addition of the calcium ionophore A23187 by i t s e l f s l i g h t l y enhances basal P 4 production i n granulosa c e l l s , the calcium ionophore markedly antagonizes the stimulation of P 4 by gonadotropins or CT or cAMP derivatives (Leung et a l . , 1988). Further, calcium i s required i n the i n h i b i t o r y and stimulatory actions of LHRH on 107 cAMP and s t e r o i d production during long-term incubation of ovarian c e l l s (Ranta et a l . , 1983; Dorflinger et a l . , 1984; Eckstein et a l . , 1986). Thus, at the l e v e l of the ovarian c e l l , the hydrolysis of i n o s i t o l l i p i d s may immediately follow LHRH receptor occupancy and lead to the rapid generation of IP 3 and DG. The resultant changes i n calcium mobilization and/or PKC a c t i v i t y may well be involved i n the modulatory effects of LHRH on ovarian hormone synthesis. The present study was performed to elucidate the mechanism of LHRH action on P 4 and PGs synthesis during the d i f f e r e n t c u l t u r e periods, the r o l e of calcium and PKC i n the 2+ LHRH action, and the inte r a c t i o n between IP 3/Ca , DG/PKC and cAMP pathways on ovarian hormone production i n r a t granulosa c e l l s . I I . Materials and Methods Preparation of animals and granulosa c e l l s Animals and granulosa c e l l s were prepared as those described i n the Chapter 2. Hormone and drug preparation Granulosa c e l l s were treated with various hormones and drugs. M e l i t t i n , CT, LHRH and FSH were dissolved i n saline. AA was dissolved i n ethanol. 12-0-tetradecanoylphorbol-13- acetate (TPA) was dissolved i n dimethylsulfoxide (DMSO). A l l drugs were d i l u t e d to t h e i r respective working concentrations 108 with MEM before use and added i n 5 jal aliquots to a t o t a l incubation volume of 1 ml. Control incubations received the same volume of ethanol and DMSO. The f i n a l concentration of ethanol or DMSO i n the incubations did not exceed 0.5%. At the end of a 5h incubation period, the culture medium was co l l e c t e d and stored at -20°C u n t i l assay. C e l l v i a b i l i t y , as determined by trypan blue exclusion, was not affected by the various treatments. Progesterone assay The P. concentration i n the culture medium was determined 4 by a s p e c i f i c RIA with an antiserum kindly provided by Dr. D.T. Armstrong of the Un i v e r s i t y of Western Ontario (Leung and Armstrong, 1978). The intra-assay c o e f f i c i e n t of v a r i a t i o n was 5.0%, and c o e f f i c i e n t of inter-assay v a r i a t i o n was 5.9% (n=25). Prostaglandin assay The PGE 2 and P G F 2 a l p h a c o n c e n t r a - t i ° n s i n t n e culture medium were determined by RIA with an antiserum kindly provided by Dr. T.G. Kennedy of the University of Western Ontario. The RIA procedure was s i m i l a r to that described previously (Kennedy, 1979), except that aliquots of the culture medium were assayed without extraction (Hirst et a l . , 1988). The intra-assay c o e f f i c i e n t of v a r i a t i o n of PGE 2 was 6.7% and c o e f f i c i e n t of inter-assay v a r i a t i o n was 9.6% (n=20). The c o e f f i c i e n t of intra-assay and inter-assay v a r i a t i o n for the PGF, , . assay were 6.8% and 5.7% (n=5), respectively. 109 Reagents The following drugs and hormones were from Sigma: AA, A23187, m e l i t t i n , TPA, and LHRH. Ovine FSH (NIH-oFSH-16) and pregnant mare's serum gonadotropin were g i f t s from the NIDDK and the National Hormone and P i t u i t a r y Program (University of Maryland School of Medicine). Penicillin-streptomycin, L- glutamine, nonessential amino acids, trypan blue were obtained 3 from Gibco. [1/2- H(N)]Progesterone ( s p e c i f i c a c t i v i t y 115.0 Ci/mmol), [5,6,8,11,12,14,15,- 3H(N)]Prostaglandin-F 2 a l p h a ( s p e c i f i c a c t i v i t y 100-200 Ci/mmol) and [5,6,8,11,14,15-3H(N)] Prostaglandin-E 2 ( s p e c i f i c a c t i v i t y 100-200 Ci/mmol) were purchased from New England Nuclear Inc. S t a t i s t i c a l analysis S t a t i s t i c a l s i g n i f i c a n c e among groups was calculated by analysis of variance followed by Scheffe's multiple range t e s t . A l l r e s u l t s were represented as the mean ± SE of determinations of quadruplicate c e l l cultures of i n d i v i d u a l treatments i n each experiment. In a l l cases, i d e n t i c a l or s i m i l a r r e s u l t s were observed i n at l e a s t two or more independent experiments. P<0.05 was considered s i g n i f i c a n t . I l l . Results 110 E f f e c t s of m e l i t t i n . LHRH and TPA on progesterone and PGE- production To determine how LHRH and TPA stimulate ovarian hormone production, especially PGE 2 formation, a phospholipase A 2 stimulator, m e l i t t i n , was added to the medium of granulosa c e l l c ulture to increase i n t r a c e l l u l a r free AA. As shown i n the upper panel of F i g . 29, m e l i t t i n (3xl0~ 7M), LHRH (10~6M) and TPA (10~7M) alone stimulated P 4 accumulation 2 fo l d , 4 f o l d and 4.1 f o l d , respectively, during a 5h granulosa c e l l culture (P<0.01). Concomitant treatment of granulosa c e l l s with m e l i t t i n with LHRH did not further increase P 4 production. To examine i f endogenous AA could synergize with protein kinase C, m e l i t t i n was added with TPA to granulosa c e l l . Again, m e l i t t i n and TPA f a i l e d to further enhance the accumulation of P 4 when compared with TPA alone. As shown i n the lower panel of F i g . 29, the PGE 2 concentrations i n the culture medium was also determined i n the same experiments. M e l i t t i n induced a 2.6 f o l d increase i n PGE 2 compared with control (51.9 pg/ml). LHRH caused a 3.2 f o l d increase i n PGE2 production and TPA also increased PGE 2 production 1.9 f o l d when compared with control. Interestingly, concomitant presence of m e l i t t i n with LHRH or with TPA further enhanced the production of PGE 2 (P<0.01), which was d i f f e r e n t from the e f f e c t s of m e l i t t i n with LHRH or TPA on P. production. I l l Sh Ul X 0.4 o.a E 0.2 SI 2 0.1 X C M«l LHRH L*M TR*. T**4 F i g . 29 g Interaction of m e l i t t i n (Mel, M;_3xlo" M), with LHRH (L; 10~ M) or the phorbol ester TPA (T; 10~ M) on progesterone (PROG) production (upper panel) and PGE, formation (lower panel) during a 5h culture. Concomitant presence of m e l i t t i n with LHRH or with TPA further enhanced the production of PGE_, while m e l i t t i n plus LHRH or TPA had no s y n e r g i s t i c e f f e c t on progesterone production. 112 E f f e c t s of m e l i t t i n and the calcium ionophore A23187 on progesterone and PGE^ production To further investigate the i n t r a c e l l u l a r mechanisms regulating P 4 and PGE 2 formation, granulosa c e l l s were treated with m e l i t t i n , A23187 and m e l i t t i n plus A23187 f o r 5h. As i l l u s t r a t e d i n the lower panel of F i g . 30, treatment of the c e l l s with m e l i t t i n (3xl0~ 7M) or A23187 (10"7M) alone stimulated PGE 2 formation by 1.9 f o l d and 3 f o l d , respectively. When both m e l i t t i n and A23187 were present together, PGE2 formation was stimulated by 5.2 f o l d . Interestingly, m e l i t t i n or A23187 alone also increased P 4 production (upper panel of Fi g . 30). However, when both m e l i t t i n and A23187 were present i n the same incubations, P 4 production was not s i g n i f i c a n t l y affected when compared with the response to either treatment alone. Interaction of the nalrHim ionophore A23187 and TPA; dose response The possible i n t e r a c t i o n between calcium and protein kinase C pathways was further examined. The lower panel of Fi g . 31 i l l u s t r a t e s the synergistic e f f e c t s of a single dose of —7 -9 TPA (10 M) and increasing concentrations of A23187 (10 M -7 —7 to 10 M). At 10 M, the phorbol ester TPA alone stimulated P 4 and PGE 2 production. There was no further enhancement of P 4 accumulation when both TPA and A23187 were present together as compared with the e f f e c t of TPA by i t s e l f . In contrast, the presence of TPA s i g n i f i c a n t l y augmented the stimulation of PGE- 2.75 E 2.2 c 6h 113 1.66 1.1 - o LU fc LU O ffiO.56 a. 0 0.12 0.09 c CM LU (D CL 0.06 0.03 - >!<! jM: • ISli iir i l l I r i Mf M i l m (TRT MM ii f Iff ill II m I1! iill «M Melittin (3x10- ̂ M) A23187 (10-7M) H B P A*M F i g . 30. E f f e c t s of m e l i t t i n and/or A23187 on progesterone and PGE_ production during a 5h culture period. "A+M" denotes c e l l s treated with both A23187 and m e l i t t i n . Treatment of the c e l l s with m e l i t t i n or A23187 alone stimulated both progesterone and PGE,. When both m e l i t t i n and A23187 were present, PGE 2 formation was further increased. 114 0.8 0.6 - 30.41-<N UJ O O. 0.2 • with TPA 0 without TPA TPA (10-1M) -8 A23187 (log M) Fig. 31. E f f e c t s of the phorbol ester TPA and/or increasing concentrations of the calcium ionophore A23187 on progesterone and PGE, production during a 5h culture period. A dose dependent increase i n both progesterone and PGE 0 was observed with A23187 treatment. The presence of TPA markedly potentiated the stimulatory action of A23187 on PGE_ production (lower panel) but not on progesterone (upper p a n e l ) 2 115 production by A23187. Fi g . 32 shows the e f f e c t s of A23187 (10~8M) and —10 —8 increasing concentrations of TPA (10 M to 10 M) on P 4 and —8 PGE 2 production. A23187 at 10 M d i d not a f f e c t P 4 production (upper panel of F i g . 32) but s i g n i f i c a n t l y increased PGE2 accumulation. A dose dependent increase i n both P 4 and PGE 2 was observed with TPA treatment. When A23187 and TPA were present together i n the culture medium, there was no s i g n i f i c a n t a l t e r a t i o n i n P 4 l e v e l s when compared with TPA or A23187 treatment alone. In contrast, the presence of A23187 markedly potentiated the stimulatory action of the d i f f e r e n t doses of TPA on PGE 2 production (lower panel of F i g . 32). Interaction of the calcium ionophore A23187. TPA and m e l i t t i n on_PGE2 production Based on the above observations, the i n t e r a c t i o n between m e l i t t i n , TPA and/or A23187 on PGE 2 production was further examined. As expected, treatment of granulosa c e l l s with TPA —8 —7 (10 M) or A23187 (10 M) alone resulted i n a s i g n i f i c a n t increase of PGE2 formation (Fig. 33) . M e l i t t i n at 3x10 M increased PGE 2 formation on i t s own, and further enhanced PGE 2 formation induced by TPA or A23187. Interestingly, while the combined treatment of the c e l l s with TPA plus A23187 exerted s y n e r g i s t i c stimulation of PGE 2 formation, the addition of m e l i t t i n to these c e l l s did not further increase the high l e v e l s of PGE- accumulation. 116 5h 4.51 Q I I I 1 I I L_ C A23187 -10 -9 -8 (10-8M) TPA (log M) Fig. 32. E f f e c t s of the calcium ionophore A23187 and/or increasing concentrations of the phorbol ester TPA on progesterone and PGE_ production. A23187 alone did not affect progesteron production (upper panel) but increased PGE_ production (lower panel). The presence of A23187 markedly potentiated the dose-dependent stimulatory action of TPA on PGE, (lower panel). 117 6h Melittin (3x10' 7M) (10-8M) (lO-^M) A23187 F i g . 33. E f f e c t s of the calcium ionophore A23187 and/or the phorbol ester TPA on PGE. production, e i t h e r i n the absence (open bars) or presence (hatched bars) of m e l i t t i n during a 5h culture. The addition of m e l i t t i n to the c e l l s did not further increase the PGE, production induced by A23187 plus TPA. 118 E f f e c t s of cholera t o x i n and LHRH on progesterone and PGE^ production during a 5h cultur e period Granulosa c e l l s were incubated i n the presence of CT (100 ng/ml), LHRH (10 _ 6M) or with CT plus LHRH f o r 5h. As expected, CT induced a marked increase (14 fold) i n P 4 formation, while LHRH caused a r e l a t i v e l y smaller (3.5 fold) elevation i n P 4 production when compared with control lev e l s (Fig. 34, upper panel). There was no s i g n i f i c a n t difference between the P 4 l e v e l s induced by CT alone or by CT plus LHRH. In addition to P 4, the accumulation of PGE 2 was also determined i n the same experiments (Fig. 34, lower panel). Addition of ei t h e r CT or LHRH stimulated PGE 2 formation, by 3.8 f o l d and 5.5 f o l d , respectively. An additive e f f e c t on PGE 2 production was observed when both CT and LHRH were present during the incubation. E f f e c t s of the calcium ionophore A23187 and/or cholera toxin on P_4 and PGE 2 production during a 5h incubation period A calcium ionophore, A23187 (10 M) , was used to mimic the action of LHRH on hormone production. As i l l u s t r a t e d i n Fi g . 35 (upper panel), CT caused a 11 f o l d increase of P 4 accumulation, whereas only a 94% increase (P<0.05) of P 4 l e v e l was observed with the A23187 treatment. Combined treatment of granulosa c e l l s with CT plus A23187 s i g n i f i c a n t l y attenuated P 4 accumulation compared with the e f f e c t of CT alone. The lower panel of F i g . 35 shows the e f f e c t of CT and/or A23187 on PGE_ formation. In the same incubations, treatment 119 with e i t h e r CT or A23187 alone s i g n i f i c a n t l y stimulated PGE 2 formation, by 6.5 f o l d and 3.1 f o l d , respectively. Combined treatment of the c e l l s with CT plus A23187 induced a further increase i n PGE2 l e v e l s , to 10.9 f o l d when compared with control l e v e l s at the end of 5h culture period. Interaction of the calcium ionophore A23187. TPA and FSH on progesterone and PGE 2 production during a 5h culture period To further investigate the in t e r a c t i o n of calcium, protein kinase C and cAMP pathways on P 4 and PGE 2 synthesis, granulosa c e l l s were treated with A23187 (10 _ 7M), TPA (10 - 7M) and FSH (lOOng) alone or with d i f f e r e n t combinations. FSH treatment of the c e l l s caused a 17 f o l d increase of P. 4 production (Fig. 36, upper panel). Concomitant presence of FSH with A23187 or TPA resulted i n the decrease of P 4 production, 36% and 20% (P<0.05), resp e c t i v e l y . There was no sy n e r g i s t i c i n h i b i t o r y actions on P 4 production with the combined FSH plus A23187 and TPA treatment of the c e l l s . Moreover, the synthesis of PGE 2 i n the same experiment was measured (lower panel of Fi g . 36) , A23187 or TPA could s i g n i f i c a n t l y enhance FSH- induced PGE 2 formation. Most i n t e r e s t i n g l y , the presence of A23187, TPA and FSH together could even further stimulate PGE 2 accumulation (P<0.01). 120 20 1 g l 6 UJ Z SS UJ 8 0. 0 0.7 0.6 O a 6 r E g0 .4 | - UJ 0.3 0 0. 0.2 0.1 0 CT (100ng) I LHRH (10-6M) F i g . 34. E f f e c t s of cholera toxin (CT) and/or LHRH on progesterone and PGE, production during a 5h culture period. Progesterone and PGE_ production were stimulated by CT or LHRH and an additive e f f e c t on PGE_ production was observed when both CT and LHRH were present. 121 5h 25 i 1 OOOng) (10-7M) A23187 F i g . 35. E f f e c t of the calcium ionophore A23187 and/or cholera toxin (CT) on progesterone and PGE_ production during a 5h incubation period. Combined treatment of granulosa c e l l s with CT plus A23187 induced a further increase i n PGE, production compared with each treatment alone. 2 20 FSH 100ng 6h 122 o» c UJ z o DC UJ UJ o O QC 0. 15 10 0 0.7 0.6 0.5 B» 0.4 e a 0.3 (9 0.2 0.1 T C A23187 TPA A*T (10-7M) (10-7M) F i g . 36. E f f e c t s of the calcium ionophore A23187 and/or the phorbol ester TPA on basal (open bars) or FSH stimulated (hatched bars) progesterone and PGE_ production during a 5h culture period. "A+T" denotes c e l l s treated with A23187 and TPA concomitantly. A23187 and/or TPA enhanced FSH stimulated PGE, production (lower panel), while s l i g h t l y i n h i b i t i n g FSH induced P 4 production (upper panel). 123 Interaction of the calcium ionophore A23187. TPA and cholera to x i n on PGE 2 production The c e l l s were treated with A23187 and/or TPA for 5h, and with or without the concomitant presence of CT (Fig. 37) . In the absence of CT, A23187 or TPA by i t s e l f stimulated PGE2 production. Combined treatment of A23187 plus TPA showed syn e r g i s t i c stimulation of PGE2- CT alone induced a 6.4 f o l d increase i n PGE 2 production. In the presence of CT, TPA or A23187 further augmented PGE 2 formation, by 70% and 25%, respectively, when compared with CT treatment alone. Addition of A23187 plus TPA to the CT-treated c e l l s resulted i n the highest increase i n PGE 2 formation. Interaction of FSH and LHRH on the formation of progesterone. PGE_2 and PG F 2alpha ^ u r^- n cF a 24h culture period As shown i n the panel A of F i g . 38, FSH s i g n i f i c a n t l y stimulated P 4 production during a 24h culture. The production of P 4 induced by FSH was decreased 50% (P<0.01) by the concomitant treatment of LHRH (10~ 6M). LHRH treatment alone s l i g h t l y stimulated P 4 production but was much less potent than FSH-induced P 4 production. Panel B shows the accumulation of PGE 2 during a 24h culture i n the same experiments. Both FSH and LHRH could stimulate PGE 2 production, at l e a s t 10 fo l d , when compared with control; the e f f e c t of LHRH was as potent as FSH. Interestingly, addition of LHRH to FSH-treated c e l l s further stimulated PGE 2 formation (P<0.01), which was contrary to the e f f e c t of LHRH on FSH-induced P. production. Panel C of 124 Fi g . 38. shows the e f f e c t s of LHRH and FSH on the production of P G F 2 a l p h a * The basal l e v e l of PG F 2alpha i n c o n t r o 1 cultures was below the s e n s i t i v i t y of the assay. Treatment with FSH (lOOng) or LHRH (10~6M) caused a s i g n i f i c a n t increase of PGF 2 a^p n a i n the culture medium. Moreover, l i k e t h e i r e f f e c t s on PGE 2, FSH and LHRH s y n e r g i s t i c a l l y stimulated the production 2alpha* J2 of PGF Interaction of FSH and TPA on progesterone and PGE 2 formation during a 24h culture period. As the e f f e c t s of LHRH can be mimicked by the phorbol ester TPA, the action of TPA on FSH-induced P. and PGE_ 4 2 production was examined. Treatment of r a t granulosa c e l l s with TPA, l i k e LHRH, was shown to i n h i b i t FSH-induced P 4 production. The i n h i b i t o r y e f f e c t of TPA was dose dependent, and 10~ 1 0M TPA caused a 50% i n h i b i t i o n of P 4 production during a 24h c e l l culture as shown i n the upper panel of F i g . 39. Unlike the i n h i b i t o r y e f f e c t of TPA on FSH-induced progesterone accumulation, TPA resulted i n a dose dependent enhancement of PGE 2 production by FSH (Fig. 39, lower panel). The lowest e f f e c t i v e dose of TPA was at 10 - 1 0M (P<0.01). 125 Fi g . 37. E f f e c t s of the calcium ionophore A23187 and/or the phorbol ester TPA on basal (open bars) or CT stimulated (hatched bars) PGE_ production during a 5h culture period. CT, A23187 and TPA by T.tself stimulated PGE, production. Addition of A23187 plus TPA to the CT treated c e l l s resulted i n the highest increase i n PGE 5 formation. 126 86 24h 0 i.e 1.2 I 0.8 CM 0.4 0 0.4 0.3 1 0.2 0.1 6 i i i i i i IJJjIII 1 1 MHI Control FSH (100 n«) LHRH do-HO -J-^'-VJ'""'- -,«5»-yjU"- •.Vess&jigr,,-, vgAS;./-.' • v -J F i g . 38. Interaction of FSH and LHRH on the formation of progesterone (PROG) (panel, A), PGE_ (panel, B) , and PGF alpha (panel, C) during a 24h culture period. While LHRH decreased the production of progesterone induced by FSH, LHRH had additive e f f e c t s on FSH induced PGE 2 and PGF2 1 n production. 127 n 8 ) TPA (log M) F i g . 39. Interaction of FSH and the phorbol ester TPA on progesterone and PGE_ formation during a 24h culture period. Treatment of r a t granulosa c e l l s with TPA, l i k e LHRH, was shown to i n h i b i t FSH induced progesterone production (upper panel) and enhance PGE 5 production induced by FSH (lower panel). 128 Interaction of FSH. TPA and the calcium Ionophore A23187 on progesterone and PGE 2 formation during a 24h culture period FSH treatment of granulosa c e l l s caused 18.3 f o l d increase i n P 4 production during a 24h c e l l culture period (Fig. 40, upper panel). Concomitant presence of A23187 with FSH resulted i n a s i g n i f i c a n t decrease i n P 4 production, by 40%, when compared with FSH treatment alone. Granulosa c e l l s were also treated with TPA (10 - 1 1M to 10 - 9M) . TPA alone at -9 10 M s l i g h t l y decreased the basal l e v e l of P 4« With the presence of FSH, TPA induced a concentration dependent decreases i n P 4 production but the addition of A23187 (10 M) did not further potentiate the i n h i b i t o r y e f f e c t of TPA on FSH- induced P 4 production. In contrast, FSH-stimulated PGE 2 production was further enhanced by the concomitant presence of A23187, from 3.6 f o l d to 6.4 f o l d , when compared with the control l e v e l of PGE 2. Additionaly, TPA alone caused the concentration dependent increases of basal l e v e l of PGE 2. The s i g n i f i c a n t e f f e c t i v e dose was at 10"10M. Furthermore, FSH-induced PGE 2 production was enhanced by the concomitant presence TPA and was further —7 augmented by the addition of 10 M A23187 i n the same experiment. F i g . 40. Interaction of FSH, the phorbol ester TPA and A23187 on progesterone and PGE, formation during a 24h culture period. FSH induced progesterone production was i n h i b i t e d by TPA and/or A23187 (upper panel), while FSH induced PGE 2 production was enhanced by TPA and/or A23187 (lower panel). 130 IV. Discussion In addition to i t s well known stimulatory action on p i t u i t a r y gonadotropics, LHRH can exert d i f f e r e n t i a l e f f e c t s on hormonogenesis i n the ovary (Hsueh and Jones 1981; Clark, 1984) . While the mechanism of action of LHRH at the l e v e l of the ovarian c e l l i s not completely understood, there i s increasing evidence that i n o s i t o l l i p i d may play a role i n i t s 2+ . e f f e c t on PKC and [Ca ] i . Therefore, the hydrolysis of phosphoinositides may immediately follow LHRH receptor occupancy and lead to the rapid generation of d i a c y l g l y c e r o l and i n o s i t o l phosphates (Ma and Leung, 1985; Davis et a l . , 1986; Leung et a l . , 1986). The resultant changes i n PKC . . 2+ a c t i v i t y , and [Ca ] i l e v e l may well be correlated with the modulatory e f f e c t s of LHRH on P 4 production. The ac t i v a t i o n of PKC by TPA has been shown to i n h i b i t FSH-stimulated estrogen, P 4 and 20-alpha-OH-P production. This i n h i b i t o r y action induced by TPA appears to be s p e c i f i c , as the phorbol congener 4-alpha-phorbol-12,13-didecanoate i s i n e f f e c t i v e on st e r o i d hormone production (Welsh et a l . , 1984). TPA and two synthetic d i a c y l g l y c e r o l s , sn-1,2-dioctanoyl g l y c e r o l and l-ole o y l - 2 - acetoyl-sn-3-glycerol have been shown to i n h i b i t cAMP dependent granulosa c e l l d i f f e r e n t i a t i o n (Shinohara et a l . , 1985). Structural s i m i l a r i t i e s between DG and TPA suggest that these compounds may stimulate the a c t i v a t i o n of PKC i n the same manner (Hsueh, 1979), and endogenous DG formation induced by LHRH may have s i m i l a r action as exogenous DG or TPA. In contrast to t h e i r i n h i b i t o r y e f f e c t , TPA and DG stimulate the 131 production of P 4 and PGs without the presence of gonadotropins (Shinohara et a l . , 1985; Kawai and Clark, 1985; Wang and Leung, 1987) . The stimulatory e f f e c t s of LHRH on P 4 and PGE2 production i n the present study confirmed those of e a r l i e r studies (Fig. 29, 31 and 32). In addition, the proposed involvement of PKC a c t i v a t i o n i n mediating the production of P 4 as well as the proposed s p e c i f i c i t y of the phorbol ester for ac t i v a t i o n of PKC have been further supported by the i n h i b i t o r y e f f e c t of the PKC i n h i b i t o r H-7 on LHRH- or TPA- induced P 4 production (Wang and Leung, 1987). S i m i l a r l y , the stimulatory e f f e c t of TPA on the plasminogen a c t i v a t o r a c t i v i t y of hen granulosa c e l l i s also blocked by H-7 ( T i l l y and Johnson, 1988). Although H-7 may i n h i b i t adenylate cyclase as well as PKC, the stimulatory e f f e c t of exogenous cAMP on H-7 treated granulosa c e l l s indicate the inta c t responsiveness of the c e l l s at a post-cAMP step (Wang and Leung, 1987). Concerning the ro l e of AA i n LHRH action, i t has been shown that LHRH could induce AA l i b e r a t i o n from membrane phospholipids i n [ H] AA prelabeled granulosa c e l l s (Minegishi and Leung, 1985; Kawai and clark, 1986). Thus, LHRH stimulated PGE 2 increase could be related to the accumulation of i n t r a c e l l u l a r free AA which serves as the substrate for PGs and leukotrienes (LTs) formation. The e f f e c t of AA may be due to one or more of i t s metabolites (Hirst et a l . , 1988). Although AA i s extensively present i n the body, p r a c t i c a l l y a l l of i t i s e s t e r i f i e d on phospho or neutral l i p i d (Irvine, 1982). Therefore the hydrolysis of e s t e r i f i e d AA provides the f i r s t 132 r a t e - c o n t r o l l i n g step i n PGs and LTs formation by l i m i t i n g the amount of substrate a v a i l a b l e to cyclooxygenase and lipoxygenase. M e l i t t i n , a polypeptide from the venom of honey bee which can activate PLA 2, has been used to enhance i n t r a c e l l u l a r free arachidonic acid concentrations (Haberman, 1972). In the present study, the e f f e c t of m e l i t t i n on P 4 production was measured a f t e r 5h incubation, and m e l i t t i n induced a 2 f o l d increase i n P 4 (Fig. 29). This r e s u l t suggests that endogenous AA release may also be involved i n the regulation of P 4 production. To further examine i f the endogenous AA release i n t e r a c t s with PKC pathway on ovarian hormone production, m e l i t t i n and LHRH were added concomitantly to the c e l l culture, and they d i d not further increase P 4 production (Fig. 29, upper panel) . I t appears that the e f f e c t of LHRH on P 4 production already includes the action of endogenous AA. AA induced by LHRH could be lib e r a t e d from membrane phospholipids by two d i f f e r e n t mechanisms to form i t s metabolites. AA can be +2 hydrolyzed from phospholipids by a Ca dependent process involving phospholipase A 2, or a l t e r n a t i v e l y from i n o s i t o l l i p i d s through two consecutive reactions catalyzed by phospholipase C and DG li p a s e (Lapetina et a l . , 1981; B e l l and Majerus, 1980). I f LHRH stimulated PLA 2 and PKC separately, the combined treatment of m e l i t t i n and TPA should enhance the accumulation of progesterone to a greater extent than each agent alone (Fig. 29, upper panel). However the formation of P. remained at the same l e v e l i n the presence of LHRH plus TPA 133 as that caused by TPA alone. This suggests that the l i b e r a t i o n of AA by LHRH could be d i s t a l to the a c t i v a t i o n of PKC. In addition, the previous observations of the stimulatory e f f e c t s of m e l i t t i n on LH and ACTH release further support the concept that the a c t i v a t i o n of PLA 2 could p a r t i c i p a t e i n the action of hormones (Chang et a l . , 1986; Aboutsamra et a l . , 1986). LHRH not only stimulated basal P 4 production, but also enhanced the formation of PGE 2 (Fig. 29, lower panel). In the present study, LHRH and TPA were found to act s y n e r g i s t i c a l l y with m e l i t t i n on the accumulation of PGE 2. Since the e f f e c t of m e l i t t i n on the formation of PGE 2 i s probably due to i t s a b i l i t y to increase 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, the present r e s u l t s indicate that LHRH could further regulate the a c t i v i t y of cyclooxygenase to e l i c i t the formation of PGE 2 when the u n e s t e r i f i e d AA substrate i s increased by m e l i t t i n . TPA could mimic the e f f e c t of LHRH, in d i c a t i n g involvement of a PKC dependent mechanism. On the other hand, i t has been observed that TPA potentiates A23187 induced AA release (chapter 2) . These r e s u l t s indicate that LHRH-induced a c t i v a t i o n of PKC i s involved i n a two-step process of PGE 2 production: f i r s t AA i s released from membrane phospholipids and second cyclooxygenase i s activated. In the present study, the granulosa c e l l s were cultured with d i f f e r e n t treatments for hours to detect the hormone production, which was much longer than that taken up (seconds or minutes) for the measurements of the i n o s i t o l phosphate 2 + formation and the [Ca ] i change. E a r l i e r time points have not 134 been explored because the production of P 4 and PGE 2 were below the s e n s i t i v i t y of RIA. On the other hand, i t has become apparent that i n many c e l l s i n which the calcium messenger system controls sustained c e l l u l a r response there i s no simple 2+ c o r r e l a t i o n between the increase i n the [Ca ] i and the c e l l u l a r response (Rink et a l . , 1982; O'Doberty et a l . , 1980; Charest et a l . , 1983). In some systems, t h i s phenomenon i s c a l l e d "hysteresis" (Rasmussen, 1983). One explanation i s that 2+ 2 + a larger increase i n [Ca ] i i s required to activate Ca dependent processes, such as PKC a c t i v i t y , than to control sustained c e l l u l a r response. I t has been suggested that PKC 2+ and Ca pathway may have d i s t i n c t functional r o l e s . For 2+ example, i n pancreatic i s l e t s , Ca i s l a r g e l y responsible for i n i t i a t i n g the c e l l u l a r response, whereas PKC i s largely responsible f o r maintaining c e l l u l a r response (Rasmussen et 2 + a l . , 1984). I t has also been observed that addition of Ca to 2+ the cultured p i t u i t a r y c e l l s i n Ca free medium causes an increase i n PRL production. Increase i n PRL synthesis i s not observed for several hours, but once synthesized, t h i s increase i n PRL may be sustained for days (Gick and Bancroft, 1985). In a s i m i l a r manner, LHRH induces rapid and transient increase i n 2 + [Ca ] i which may then go on to control the sustained ovarian hormone production. In an attempt to modify i n t r a c e l l u l a r l e v e l s of calcium more d i r e c t l y , A23187, a divalent cation ionophore which f a c i l i t a t e s the transmembrane transport of calcium, was used i n the present study. A23187 alone stimulated both P 4 and PGE, 135 production during a 5h culture period (Fig. 30). The stimulatory e f f e c t of A23187 on the production of P 4 was probably due to the conversion of pregnenolone to progesterone but not the synthesis of t h i s intermediate from cholesterol as previously proposed (Carnegie and Tsang, 1987). Interestingly, treatment of granulosa c e l l s with m e l i t t i n plus A23187 further enhanced PGE 2 production (Fig. 30, lower panel). A23187 could also stimulate AA release from r a t granulosa c e l l s (Minegishi et a l . , 1987). These data imply that calcium plays modulatory r o l e s i n both the release of AA by the a c t i v i t y of phospholipase A 2 and the conversion of AA to PGE 2 by cyclooxygenase. In many systems, a dual signal transduction mechanism +2 ex i s t s that involves the Ca and PKC pathways acting either cooperatively or s y n e r g i s t i c a l l y to give the maximal response to a given hormonal sign a l (Nishizuka et a l . , 1984; Berridge, 1987). Huckle and Conn have observed that both calcium and PKC are involved i n the action of LHRH on gonadotropins release from the anterior p i t u i t a r y (Huckle and Conn, 1987). The sy n e r g i s t i c e f f e c t s on LH release are noted with the calcium ionophore A23187 and PKC act i v a t o r s such as the phorbol ester TPA, possibly through enhanced a c t i v a t i o n of PKC (Harris et a l . , 1985). Combination of TPA and A23187 causes larger amounts of sustained release of PRL compared to each compound alone (Delbeke et a l . , 1984). The present study c l e a r l y demonstrated that TPA f a i l e d to act s y n e r g i s t i c a l l y with A23187 on P. production, whereas TPA and A23187 were syn e r g i s t i c on 136 PGE 2 production (Fig. 31-33). While A23187 has been shown to enhance acute TPA-induced production of P G F 2 a l p h a * n s w * n e granulosa c e l l s (Veldhuis and Demers, 1987), a report on the i n h i b i t o r y e f f e c t s of TPA and A23187 on LH induced P 4 production i n rat l u t e a l c e l l s has not described t h i s potential synergism (Baum and Rosberg, 1987). I t has been shown that i n parotid gland, where PKC and 2+ 2 + Ca s y n e r g i s t i c a l l y induce enzyme secretion, Ca alone i s responsible for control of plasma membrane K + channels (Putney et a l . , 1984). In the present study, TPA did not act s y n e r g i s t i c a l l y with A23187 on P 4 production but d i d act s y n e r g i s t i c a l l y with A23187 on PGE2 formation (Fig. 31 and 32). Taken together, these r e s u l t s suggest the po t e n t i a l for a degree of independent control of the c e l l responses that are 2 + unaffected by the synergism of PKC and Ca . As proposed previously, addition of EDTA to the granulosa c e l l s p a r t i a l l y reversed the i n h i b i t o r y e f f e c t of LHRH and TPA upon FSH-induced P 4 production, while the reversal e f f e c t of EDTA could be completely abolished by the simultaneous addition 2+ of Ca (Leung and Wang, 1988). These r e s u l t s indicate that the action of LHRH on ovarian P 4 steroidogegesis i s mediated at 2+ leas t i n part by a Ca dependent PKC. The present studies also showed that the formation of P 4 was not coupled t i g h t l y to the production of PGE 2« The treatments of granulosa c e l l s with m e l i t t i n plus A23187, TPA plus m e l i t t i n and TPA plus A23187 only increased PGE 2 formation but not P 4 production to higher amounts (Fig. 31 and 32) . 137 Furthermore, i n the presence of both TPA and A23187, the formation of PGE 2 was not further augmented by m e l i t t i n . This perhaps r e f l e c t s the already maximal conversion of AA to PGE2 under the influence of TPA plus A23187 (Fig. 33). 2+ The interactions between Ca -PKC and cAMP pathways were also investigated i n the present study. Treatment of rat granulosa c e l l s with LHRH further enhanced CT-stimulated PGE2 production but f a i l e d to a f f e c t the production of P 4 induced by CT during a 5h incubation period (Fig. 34) . This r e s u l t supports the previous observation that the mechanism of LHRH on PGs production i s distinguishable from that of gonadotropins (Clark, 1982). While LH increases PG production, i t does not 3 increase [ H]AA release (see Chapter 2). Therefore, the e f f e c t of LH on PG production i n granulosa c e l l s i s presumably at a step i n the PG synthesis a f t e r hydrolysis of AA, and may involve an increase i n PG synthetase a c t i v i t y (Clark et a l . , 1980; Koos and Clark, 1982). A23187 and TPA were used to mimic the action of LHRH on PGE 2 formation with the presence of FSH and CT i n the present study. Interestingly, TPA suppressed FSH-induced P 4 production but enhanced PGE 2 formation induced by FSH or CT during a 5h culture (Fig. 36 and 37) . Likewise, the r o l e of calcium i n ovarian hormone production appears to be complex and may be affected by the presence of gonadotropins or cAMP induced agents. While basal P 4 formation was increased only marginally by 10 M A23187, PGE 2 production was stimulated by 4.5 f o l d i n the same incubations (Fig. 35 and 36). Moreover, i n the presence of FSH or CT, the production of P. 138 was attenuated by the concomitant presence of A23187, whereas PGE 2 formation was further augmented (Fig. 35 and 36). In long term culture (24 h to 48 h), LHRH has been reported to reduce FSH-induced P 4 production (Hsueh and Jones, 1981). In contrast, LHRH acted s y n e r g i s t i c a l l y with FSH on the formation of PGE 2 and p G F 2 a l p n a i n the present study (Fig. 38). Since the increase i n PGE 2 concentration (panel B of Fig . 38) was about four times greater than that of P G F 2 a l p h a ( P a n e ^ c °^ Fi g . 38), only the formation of PGE2 was determined i n the subsequent experiments. Very s i m i l a r e f f e c t s were observed with the PKC activ a t o r TPA on FSH-induced P. and PGE„ formation 4 2 (Fig. 39). A23187 potentiated FSH-induced PGE 2 production, and further enhanced PGE 2 formation induced by TPA plus FSH (Fig. 40, lower panel), whereas A23187 i n h i b i t e d FSH-induced P 4 production (Fig. 40, upper panel). The present short and long term studies c l e a r l y strengthen the multiple r o l e s of protein 2+ kinase C and Ca on hormone production, e i t h e r stimulatory or in h i b i t o r y , depending on the time of culture and the nature of the hormones. 2+ The i n t e r a c t i o n between cAMP and Ca -PKC pathways can occur at d i f f e r e n t l e v e l s . The f i r s t i s at the l e v e l of receptors; signals produced by acti v a t i o n of one pathway may a f f e c t the a b i l i t y of a receptor functioning v i a the other pathway to generate an i n t r a c e l l u l a r s i g n a l i n response to an agonist. Secondly, these i n t r a c e l l u l a r messengers may interact i n producing the c e l l response. F i n a l l y , these interactions may extend to neighboring c e l l s ; thus AA produced a f t e r 139 a c t i v a t i o n of PLA 2 or PLC followed by DG lipase i s the substrate for synthesis of PGs and/or LTs. These metabolites might be released from the c e l l where they are produced and function as l o c a l hormones to mediate the functions of t h e i r target c e l l s . Although the i n h i b i t o r y e f f e c t of LHRH and i t s agonists on P 4 production have been studied extensively, t h i s i s the f i r s t demonstration of a s y n e r g i s t i c e f f e c t between LHRH with FSH on ovarian PGE2 formation, while at the same time i n h i b i t i n g P 4 accumulation during a 24h culture period (Fig. 38). The i n h i b i t o r y e f f e c t of LHRH, A23187 and TPA on FSH- induced P 4 accumulation may be p a r t i a l y due to a reduction i n cAMP formation (Shinohara et a l . , 1985; Knecht et a l . , 1981; Jones and Hsueh, 1981). Previous studies have shown that i n cultured theca and granulosa c e l l s , FSH-stimulated PGE production could be mimicked by cAMP i n both c e l l types (Zor et a l . , 1983; Clark et a l . , 1978). However, the present observation that LHRH, TPA and A23187 d i d not antagonize the FSH-induced PGE 2 production, but rather augmented the production of PGs raises the p o s s i b i l i t y that the stimulatory action of LHRH on PGE 2 production v i a the ac t i v a t i o n of PKC and 2+ the change of [Ca ] i i s at a post-cAMP step (Fig. 38-40). These data also support the concept that TPA and A23187 i n h i b i t FSH-induced P 4 production, i n part at a step or steps beyond cAMP generation and degradation (Leung et a l . , 1988). Taken together, i t can be concluded that cAMP, calcium and protein kinase C pathways are a l l involved i n PGs synthesis, and each pathway i s distinguishable from the others i n the way that PG 140 i s produced. Nishizuka (1984) has proposed that two classes of 2+ receptors are coupled with cAMP and Ca -PKC induced i n t r a c e l l u l a r cascades. In some tissue s , the two classes of receptors function i n s i m i l a r d i r e c t i o n to cause a f u l l p h y s iological c e l l u l a r response. In other t i s s u e s , the two types of receptor-linked cascades counteract each other, e.g., an e x t r a c e l l u l a r signal that increases cAMP blocks PKC 2+ acti v a t i o n , Ca mobilization and AA release (Nishizuka, 1984). The present study supports t h i s notion and further indicates that the i n t r a c e l l u l a r process i n ovarian c e l l s induced by two major second messenger pathways can eithe r cooperate with or antagonize each other. The ph y s i o l o g i c a l r o l e of LHRH and i t s i n t e r a c t i o n with FSH on P 4 and PGE 2 formation i s not yet c l e a r at present. A rol e of PGE 2 to increase plasminogen a c t i v a t i o n has been proposed (Strickland and Beers, 1976). Furthermore, prostaglandins may be important i n f o l l i c l e growth, oocyte maturation and ovulation (Clark et a l . , 1978; Naor et a l . , 1984) . On the other hand, i t i s known that the maturation of rat oocytes could be blocked by cAMP (Dekel and Beers, 1978). LHRH could decrease the formation of cAMP induced by gonadotropins and r e s u l t i n the maturation of oocytes (Hi l l e n s j o and Lemaire, 1980; Dekel et a l . , 1983; Dekel and Aberdam, 1985). The present studies further strengthen the concept that LHRH does not exert an o v e r a l l i n h i b i t o r y action on ovary but rather causes s p e c i f i c stimulation of P. and PG 141 synthesis, suggesting that LHRH may exert multiple e f f e c t s on the ovarian hormone production and the multiple pathways are intimately involved i n the actions of LHRH. 142 Chapter 5. Role of Arachidonic Acid i n L u t e i n i z i n g Hormone- Releasing Hormone Action. I. Introduction While the influence of LHRH on ovarian hormone production has been well documented, i t s mechanism of action at the post- receptor l e v e l i s s t i l l l a r g e l y unresolved. In the past few years, LHRH and i t s agonists have been shown to stimulate the breakdown of polyphosphoinositides into i n o s i t o l phosphates and 1,2 d i a c y l g l y c e r o l i n the ovary. Phospholipid turnover was 2+ usually accompanied by the mobilization of [Ca ] i , and may 2+ cause the a c t i v a t i o n of Ca dependent PLA 2, leading to the l i b e r a t i o n of AA. The above hypothesis notwithstanding, i t has been proposed that the action of LHRH i n the stimulation of LH release from gonadotrophs also involves the release of AA from membrane phospholipids. One or more of the lipoxygenated metabolites or epoxygenated products of AA might be a component of the cascade of reactions i n i t i a t e d by LHRH that ultimately r e s u l t i n the secretion of LH (Naor and Catt, 1981; Snyder et a l . , 1983). To ascertain whether s i m i l a r signal transduction mechanisms are evoked a f t e r the binding of LHRH to i t s ovarian receptors, the e f f e c t of LHRH on AA release i n rat ovarian c e l l s has been recently examined (Minegishi and Leung, 1985). In cultured granulosa c e l l s prelabeled with [ H]AA, LHRH caused a rapid increase i n the l e v e l of AA release from phospholipids. This increase i n AA release was also induced by an agonistic LHRH 143 analog, but completely blocked by the concomitant presence of a potent LHRH antagonist, suggesting a s p e c i f i c receptor mediated mechanism (Minegishi and Leung, 1985). While these data support the notion that AA l i b e r a t i o n from phospholipids i s enhanced by LHRH, i t s re l a t i o n s h i p to s t e r o i d hormone production i n the ovary i s not understood. Treatment of granulosa c e l l s with m e l i t t i n resulted i n the elevation of basal l e v e l of P 4 and PGs (chapter 4), suggesting endogenous AA not only serves as the precursor for PGs synthesis but also exerts i t s e f f e c t on steroidogenesis. The present study was designed to determine i f AA could be a mediator of LHRH action during the d i f f e r e n t culture periods. The involvement of various pathways of AA metabolism i n the control of P 4 production was also investigated. I I . Materials and Methods Animals and granulosa c e l l s preparation The preparation of animals and granulosa c e l l s were the same as those described i n Chapter 2. Hormone and drug preparation Granulosa c e l l s were treated with various hormones and drugs. LHRH, FSH, CT and m e l i t t i n were dissolved i n saline. 25-Hydroxycholesterol (25-OH-cholesterol), AA and a l l hydroperoxy acids were prepared freshly i n ethanol. After opening the ampules of AA and hydroperoxy acid, they were 144 stored under nitrogen at -70°C. TPA, indomethacin, and nordihydroguaiaretic acid (NDGA) were dissolved i n DMSO. A l l drugs were d i l u t e d to t h e i r respective working concentrations with MEM before use and added i n 5 jul aliquots to a t o t a l incubation volume of 1 ml. Control incubations received the same volume of ethanol and DMSO. The f i n a l concentration of ethanol or DMSO i n the incubations d i d not exceed 0.5%. At the end of d i f f e r e n t incubation periods, the culture medium was co l l e c t e d and stored at -20°C u n t i l assay. For i n t r a c e l l u l a r P 4 production, 0.5 ml of 100% ethanol was added into the culture dishes, and the c e l l s were scraped out with a rubber policeman. The c e l l suspension was transfered into 10 x 75 mm te s t tube and mixed vigorously on a vortex. Then the suspension was centrifuged at 3000xg for 10 min at 4°C. Ethanol was poured out into another t e s t tube, dried under nitrogen and redissolved with 200 u l of MEM for RIA. Progestin assay The P 4 and 20alpha-hydroxy-4-pregnen-3-one (20alpha-OH-P) concentrations i n the culture medium were determined by a s p e c i f i c RIAs with antisera kindly provided by Dr. D.T. Armstrong of the University of Western Ontario. The lowest detectable concentration was 0.08 ng/ml f o r both P 4 and 20alpha-OH-P. The intra-assay c o e f f i c i e n t of v a r i a t i o n was 5.0%, and c o e f f i c i e n t of inter-assay v a r i a t i o n was 5.9% for P 4 assay (n=25). The intra-assay c o e f f i c i e n t 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 of inter-assay v a r i a t i o n was 10% (n=6). 145 Prostaglandin E 2 assay The PGE 2 concentrations i n the culture medium were determined by RIA with an antiserum kindly provided by Dr. T.G. Kennedy of the u n i v e r s i t y of Western Ontario. The RIA procedure was s i m i l a r to that described previously (Kennedy, 1979), except that aliquots of the culture medium were assayed without extraction (Hirst et a l . , 1988). The intra-assay c o e f f i c i e n t of v a r i a t i o n was 6.7% and c o e f f i c i e n t of i n t e r - assay v a r i a t i o n was 9.6% (n=20). Materials The following were purchased from Sigma (St. Louis, USA): o l e i c acid (C18:l), 11,14 eicosadenoic acid (C18:2), l i n o l e i c a cid (C18:2), homo-gamma-linolenic acid (C18:3) (8,11,14 eic o s a t r i e n o i c a c i d ) , gamma-linolenic acid (C18:3), arachidonic acid (C20:4), TPA, LHRH, CT, 25-OH-cholesterol indomethacin, and NDGA. Ovine FSH (NIDDK oFSH-16) and PMSG were g i f t s from the National Hormone and P i t u i t a r y Program NIDDKD, NIH. The LHRH agonist [d-Trp 6,Des-Gly 1 0,Pro 9-NHEt]LHRH was kindly supplied by Dr. Nicholas Ling of the Salk I n s t i t u t e (La J o l l a , CA) . A l l hydroperoxy f a t t y acids were purchased from BIOMOL 3 Laboratories (Philadelphia, PA). [1,2- H(N)]Progesterone ( s p e c i f i c a c t i v i t y 115.0 Ci/mmol), 20alpha-[l,2- 3H(N)]- hydroxypregn-4-ene-3-one ( s p e c i f i c a c t i v i t y 40-60 Ci/mmol) and [5,6,8,11,14,15- 3H(N)]Prostaglandin E 2 ( s p e c i f i c a c t i v i t y 100- 146 200 Ci/mmol) were purchased from the New England Nuclear Corporation (Ontario, Canada). S c i n t i l l a t i o n f l u i d was obtained from the Fisher S c i e n t i f i c Company. S t a t i s t i c a l analysis S t a t i s t i c a l s i g n i f i c a n c e of the data was determined by analysis of variance and followed by Scheffe's multiple range t e s t . In a l l cases, i d e n t i c a l or s i m i l a r r e s u l t s were observed i n at le a s t three or more independent experiments. A l l res u l t s were presented as the mean ± SE of determinations of c e l l s from quadruplicate cultures within each treatment group. A P value of l e s s than 0.05 was considered s i g n i f i c a n t . I I I . Results E f f e c t s of m e l i t t i n . LHRH and arachidonic a c i d on progesterone production To compare the e f f e c t s of endogenous AA, exogenous AA-and LHRH on P 4 production, granulosa c e l l s were treated with m e l i t t i n , AA and LHRH for 5h (Fig. 41). Treatment of the c e l l s -7 with a maximal e f f e c t i v e dose of m e l i t t i n (3x10 M) caused a more than 2 f o l d increase i n P. i n the culture medium. In the 4 same experiment, the degree of stimulation of P 4 production achieved with the maximal dose of exogenous AA and LHRH was s i g n i f i c a n t l y larger than that induced by m e l i t t i n , 5.1 f o l d and 4 f o l d , respctively, as compared with the untreated control c e l l s . 147 S e n s i t i v i t y of progesterone response to arachidonic a c i d The e f f e c t s of increasing concentration of AA on P 4 production were determined 5h a f t e r the addition of AA to the granulosa c e l l s (Fig. 42). A maximal enhancement of P 4 production was observed at AA concentrations above 10~5M. The -7 minimal e f f e c t i v e dose of AA was 3x10 M (P<0.05). In addition -5 to enhancing P 4 accumulation i n the culture medium, 10 M AA caused a s i g n i f i c a n t increase i n the i n t r a c e l l u l a r P 4 5 concentration (0.53 ± 0.02 vs. 0.12 ± 0.01 ng/2xl0 c e l l s i n control incubations; P<0.01). E f f e c t of unsaturated f a t t y acids on progesterone production In t h i s experiment, the s p e c i f i c i t y of other unsaturated f a t t y acids as well as AA action on P 4 production was investigated. A l l f a t t y acids were added to the culture medium -5 . . . . at the concentration of 10 M. 11,14 eicosadienoic acid, homo- gamma-linolenic acid (C18:3), gamma-linolenic acid (C18:3), l i n o l e i c acid (C18:2) and AA increased P 4 production during a 5h incubation period. Interestingly, these f a t t y acids showed d i f f e r e n t potencies i n stimulating P 4 production i n the following order: AA> homo-gamma-linolenio gamma-linolenio l i n o l e i o 11,14 eicosadienoic acid. Another unsaturated f a t t y acid, o l e i c acid, f a i l e d to stimulate P. production (Fig. 43). 148 F i g . 41. Stimulatory e f f e c t s of m e l i t t i n , LHRH and arachidonic acid (AA) on progesterone (PROG) accumulation during a 5h culture period. C, control; Mel, m e l i t t i n . 149 Fi g . 42. E f f e c t of increasing concentration of arachidonic acid (AA) on progesterone production during a 5h culture period. AA caused a dose dependent increase i n progesterone production. 150 UJ M - a> - en o cc UJ H CO UJ o Q. o - 6h X c o o o '5 O o o c © *o CO CO o o o '5 o o c © o c T o c T •r o £ o X o c o •g o co Fatty acid (10" 5M) F i g . 43. Ef f e c t s of unsaturated f a t t y acids on progesterone production. Arachidonic, homo-gamma-linolenic, gamma- l i n o l e n i c , l i n o l e i c , and 11, 14 eicosadienoic acids stimulated progesterone production during a 5h culture period, whereas o l e i c a c i d f a i l e d to stimulate progesterone production. 151 E f f e c t s of LHRH and arachidonic a c i d on progesterone production As expected, addition of LHRH (lo" 6M) to rat granulosa c e l l s enhanced P 4 production during a 5h incubation (P<0.01) (Fig. 44A) . Addition of AA (10 - 5M) also s i g n i f i c a n t l y enhanced the P 4 production to about 2.9 f o l d of the control l e v e l (P<0.01). The concomitant presence of LHRH and AA further stimulated P. l e v e l s to about 4.5 f o l d of the control P, values (P<0.01). As i l l u s t r a t e d i n F i g . 44 panel B, addition of AA to an agonistic analog of LHRH further enhanced P 4 production over that induced by the LHRH agonist alone (P<0.01). Time course of e f f e c t s of LHRH and arachidonic acid on progesterone production As shown i n F i g . 45, P 4 production was stimulated, 82% above the control (P<0.05), as ear l y as l h af t e r the addition of 10~6M LHRH. The e f f e c t of 10~5M AA was somewhat slower i n onset; by 3h a f t e r AA addition, P 4 production was increased s i g n i f i c a n t l y (1.9 fold) compared with that i n control incubations (P<0.05). At 5h, the l e v e l of P 4 stimulated by AA was not d i f f e r e n t from that induced by LHRH. Interestingly, the concomitant presence of AA and LHRH at l h d i d not further enhance P 4 production induced by LHRH alone (P<0.05), but markedly potentiated P 4 production at 3h and 5h (P<0.01) compared with that a f t e r treatment with AA or LHRH alone. 152 C LHRH AA LHRH (*r8tyD(io-8M) AA C LHRHa AA LHRHa (10-7*1) (10-6M) AA F i g . 44. E f f e c t s of treatment of granulosa c e l l s with arachidonic acid (AA) and LHRH or a LHRH agonist (LHRHa) on progesterone production. LHRH (panel A) or LHRHa (panel B) stimulated progesterone production was further enhanced by AA during a 5h culture period. 153 0 1 3 6 T i m e (hour) F i g . 45. Time course of stimulation of progesterone production by arachidonic acid (AA) , LHRH or LHRH plus AA. Progesterone production was stimulated as early as l h a f t e r the addition of LHRH, whereas the e f f e c t of AA was s i g n i f i c a n t at 3h. Progesterone production was potentiated by the presence of both AA and LHRH at 3h and 5h compared with that a f t e r treatment with AA or LHRH alone. 154 Interaction between arachidonic a c i d and TPA on progesterone production Addition of the phorbol ester, TPA (10 M) , to granulosa c e l l s resulted i n a 93% increase i n P 4 production (Fig. 46) as compared with untreated control c e l l s . A d d i t i o n a l l y , the concomitant presence of TPA (10 M) and AA s i g n i f i c a n t l y enhanced (P<0.01) the stimulatory e f f e c t of AA (at 10~6M or —5 . . . . 10 M) on P 4 production. Likewise, as shown i n F i g . 47, the -5 addition of AA (10 M) to TPA-treated c e l l s markedly -9 potentiated the stimulation of P 4 production by TPA (at 10 , l O - 8 , or 10 _ 7M) alone. Role of arachidonic acid metabolism To investigate the possible involvement of AA metabolites i n P 4 production, granulosa c e l l s were treated with indomethacin and NDGA with the presence of ei t h e r LHRH or AA. As shown i n F i g . 48, addition of the AA metabolism in h i b i t o r s -5 . . . . alone (10 M) had a s l i g h t but s i g n i f i c a n t (P<0.05) stimulatory e f f e c t on P 4 production. More importantly, addition of the same dose of NDGA p a r t i a l l y suppressed (by about 50%) P 4 production induced by 10~6M LHRH (Fig. 48, upper panel); the same molar concentration of indomethacin was i n e f f e c t i v e . On the other hand, the concomitant presence of NDGA, but not indomethacin, i n h i b i t e d AA-induced P 4 production to the same l e v e l as that caused by NDGA alone (P<0.01) (Fig. 48, lower panel). 155 F i g . 46. E f f e c t s of the phorbol ester TPA and increasing concentrations of arachidonic acid (AA) on progesterone production. The presence of TPA enhanced the stimulatory e f f e c t of AA on progesterone production during a 5h culture period. 156 Fi g . 47. E f f e c t s of arachidonic a c i d (AA) and increasing concentrations of the phorbol ester TPA on progesterone production. The addition of AA to TPA treated c e l l s potentiated the stimulation of progesterone production by TPA alone during a 5h culture period. 1 5 7 Control NDGA («r5M) INOO («T 5 M) F i g . 48. Role of arachidonic acid (AA) metabolism. Addition of nordihydroguaiaretic acid (NDGA) p a r t i a l l y suppressed progesterone production induced by LHRH (upper panel) and i n h i b i t e d AA induced progesterone production to the same l e v e l as that caused by NDGA alone (lower panel), whereas indomethacin (INDO) was i n e f f e c t i v e . 158 The e f f e c t s of NDGA or indomethacin on the production of P 4 induced by LHRH plus AA were further examined i n another experiment (Fig. 4 9 ) . While indomethacin f a i l e d to a f f e c t the marked increase i n P 4 production due to the concomitant presence of LHRH (10 _ 6M) , AA (10"5M) and NDGA (10"5M) suppressed the P 4 response dramatically (P<0.01). Dose response of HETEs and HPETEs on progesterone production Since the lipoxygenase metabolites of AA may be involved i n the act i o n of LHRH on steroidogenesis, the e f f e c t s of these lipoxygenase metabolites including hydroxyeicosatetraenoic acids (HETEs) and hydroperoxyeicosatetraenoic acids (HPETEs), on ovarian s t e r o i d hormone were further examined. Rat granulosa c e l l s were incubated f o r 5h i n the absence or presence of increasing concentration of 5-HETE, 5-HPETE, 12-HETE, 15-HETE or 15-HPETE (10~7M to 10 _ 5M). P 4 production was increased by these acids i n a dose dependent manner. At 10~6M, a l l treatments resulted i n a s l i g h t but s i g n i f i c a n t —5 increase i n P 4 formation. At 10 M, a l l compounds (except 15- HPETE) further stimulated P 4 production. The following order of potency was observed: 12-HETE > 5-HETE > 5-HPETE = 15-HETE > 15-HPETE (Fig. 50). 159 1 2 F i g . 49. E f f e c t s of nordihydroguaiaretic a c i d (NDGA) or indomethacin (INDO) on progesterone production induced by LHRH and/or arachidonic a c i d (AA). Whereas indomethacin d i d a f f e c t the increase i n progesterone production due to the presence of both LHRH and AA, NDGA dramatically suppressed progesterone production. 160 Fig. 50. E f f e c t s of HETEs and HPETEs on progesterone production. Progesterone production was increased by these f a t t y acids i n a dose dependent manner during a 5h culture period. 161 E f f e c t s of H E T E S on progesterone and PGE^ production Granulosa c e l l s were treated with 5-, 12- or 15-HETE and the e f f e c t s on P 4 as well as PGE 2 production were examined. As seen i n Fi g . 51 (upper panel) , at 5xlO * " 6 M , 12-HETE was most potent and caused a 4.1 f o l d increase i n P 4 formation. 5-HETE and 15-HETE resulted i n 3.5 and 2.4 f o l d increase of P„ accumulation, respectively, when compared with control incubations. Interestingly, these AA metabolites also stimulated PGE2 production i n the same experiment (Fig. 51. lower panel). Unlike t h e i r actions on P 4 production, the e f f e c t of 15-HETE was as potent as 12-HETE on PGE 2 formation. 15-HETE or 12-HETE caused an approximate 15 f o l d increase i n PGE 2 accumulation i n the culture medium. In contrast, 5-HETE was considerably less potent when compared with 12- or 15-HETE, but s t i l l resulted i n s i g n i f i c a n t increase i n PGE 2 formation, about 6 f o l d , when compared with the control incubations. Interaction of HETEs or HPETEs with LHRH on progesterone and PGE 2 production Since lipoxygenase metabolites of AA were believed to be involved i n the action of AA, the e f f e c t s of HETEs and HPETEs on the stimulation of P 4 production by LHRH were further investigated (Fig. 52, upper panel). At the minimum e f f e c t i v e dose ( i . e 1 0 ~ 6 M ) , the AA metabolites stimulated basal P 4 production s l i g h t l y . A more e f f e c t i v e stimulation of P 4 was observed with 1 0 - 6 M LHRH. Concomitant treatment with LHRH and 162 the various AA metabolites caused further increase i n P. 4 accumulation, by 20% to 93%, when compared with the e f f e c t of LHRH alone. PGE 2 production i n the same experiments was also determined (Fig. 52, lower panel). 5-HETE and 5-HPETE, at the minimum e f f e c t i v e dose which stimulated P, d i d not a l t e r either basal or LHRH induced PGE2 formation. In contrast, 12-HETE, 15-HETE or 15-HPETE s i g n i f i c a n t l y increased PGE 2 l e v e l s when compared with the control incubation. Furthermore, 12-HETE, 15-HETE and 15-HPETE augmented the stimulatory e f f e c t of LHRH on PGE 2 production by 2 f o l d , 2.9 f o l d and 2.5 f o l d , r espectively, when compared with the LHRH treatment alone. Interactions of HETEs or HPETEs with TPA on progesterone and PGE 2 production The addition of the protein kinase C activ a t o r (TPA), at 10 M, to granulosa c e l l s caused a marked increase i n P 4 production (Fig. 53, upper panel). A l l HETEs and HPETEs tested s i g n i f i c a n t l y augmented the stimulatory e f f e c t of TPA, by 55 to 83%, when compared with P 4 l e v e l s induced by TPA alone. In the same experiment, TPA alone caused a 4.9 f o l d increase i n PGE 2 production(Fig. 53, lower panel). While 5- HETE or 5-HPETE did not s i g n i f i c a n t l y a f f e c t PGE 2 production induced by TPA, concomitant treatment with 12-HETE, 15HETE or 15-HPETE further enhanced TPA-stimulated PGE. accumulation. 163 V-6 UJ z 8 » UJ UJ 1.6 -ce a. o 0.6 6h 0.4 ^ 0.3 h 1 UJ 0.2 © a. 0.1 _1_ c 6HE 12HE (6x10"eM) 16HE F i g . 51. E f f e c t s of HETEs on progesterone (upper panel) and PGE_ (lower panel) production. Both progesterone and PGE_ were stimulated by HETEs during a 5h culture period. 5HE, 5-HETE; 12HE, 12-HETE; 15HE, 15-HETE. 164 (10-»M) F i g . 52. Interactions of HETEs or HPETEs with LHRH on progesterone (upper panel) and PGE^ (lower panel) production. At the minimum e f f e c t i v e dose (10~T1), the various arachidonic ac i d metabolites enhanced LHRH induced progesterone production and 12-HETE, 15-HETE and 15-HPETE augmented the stimulatory e f f e c t of LHRH on PGE, production during a 5h culture period. 165 Fig. 53. Interactions of HETEs or HPETEs with the phorbol ester TPA on progesterone (upper panel) and PGE, (lower panel) production. TPA induced progesterone production was enhanced by a l l HETEs and HPETEs tested, while TPA induced PGE, production was augmented by 12-HETE, 15-HETE and 15-HPETE during a 5h culture period. 166 E f f e c t of LHRH on FSH-induced progesterone production; time response To examine the action of gonadotrophin and LHRH on granulosa c e l l s , FSH- or LHRH- or FSH plus LHRH-treated granulosa c e l l s were cultured for 8h, 16h and 24h (Fig. 54) . FSH (100 ng) alone resulted i n a s i g n i f i c a n t time dependent increase i n P 4 accumulation, 20 f o l d , 21.5 f o l d and 30 f o l d , at 8h, 16h and 24h, respectively. LHRH alone also markedly stimulated P 4 production as compared with untreated culture c e l l s , but LHRH-induced P 4 production was much les s than that induced by FSH. The concomitant presence of FSH with LHRH i n the culture medium did not a l t e r P 4 production at 8h. However, a s i g n i f i c a n t decrease i n P 4 production was observed at 16h, and P 4 production was further reduced at 24h i n combined treatment of granulosa c e l l s with FSH plus LHRH. Eff e c t s of arachidonic acid and/or FSH on progesterone production during a 24h culture To examine the ro l e of AA on P 4 production, rat granulosa —5 c e l l s were treated with 10 M AA, i n the absence or presence of FSH (lOOng), f o r 24h (Fig. 55). As expected, FSH markedly stimulated P 4 production (23 fold) compared with the untreated control (P<0.01). AA alone caused a s l i g h t but s i g n i f i c a n t stimulation of P 4 production, 4.1 f o l d (P<0.05) when compared with the untreated control c e l l s . The concomitant presence of AA with FSH d i d not a f f e c t FSH-induced increase i n P 4 accumulation. 167 50 0 8 16 Hours F i g . 54. Ef f e c t of LHRH on FSH induced progesterone production: time response. LHRH alone stimulated progesterone production as early as 4h, but production was much les s than that induced by FSH. FSH stimulated progesterone production was reduced by LHRH af t e r 16h. 168 F i g . 55. E f f e c t s of arachidonic acid (AA) and/or FSH on progesterone production. AA alone caused a s l i g h t increase i n progesterone production. Concomitant presence of AA with FSH did not a f f e c t the FSH induced increase i n progesterone production a f t e r 24h culture. Control (C) and FSH treated c e l l s received the appropriate amount of solvent for AA. 169 E f f e c t of arachidonic a c i d on LHRH induced i n h i b i t i o n of progesterone production Granulosa c e l l s were treated for 18h with FSH (100 ng/ml), with or without the presence of LHRH (10~ 6M). At the end of 18h, the combined treatment of LHRH plus FSH s i g n i f i c a n t l y decreased P 4 production when compared with the c e l l s given FSH alone. At t h i s time, AA was added to two of the groups and the culture was continued for a further 6h. As shown i n Fig . 56, LHRH decreased FSH-induced P 4 production, by 47%, at the end of the 24h incubation period. When AA was present during the l a s t 6h, the i n h i b i t o r y e f f e c t of LHRH on FSH-induced P 4 was p a r t i a l l y reversed, by about 42%, when compared with the cultured c e l l s treated with LHRH plus FSH. AA by i t s e l f d id not a f f e c t P 4 accumulation induced by FSH during the l a s t 6h. E f f e c t of arachidonic a c i d on TPA-induced i n h i b i t i o n of progesterone production F i g . 57 i l l u s t r a t e s AA reversal of the i n h i b i t o r y action of TPA on P 4 production. Granulosa c e l l s were treated with FSH (100 ng/ml) with or without 10"9M TPA for 18h. After that, AA was given to one of the groups. A l l incubations were continued f o r another 6h. As expected, TPA caused a marked i n h i b i t i o n on FSH-induced P 4 production, 69% (P<0.01) as compared with the untreated group. Addition of AA to the group given TPA plus FSH resulted i n a p a r t i a l r e v ersal of P 4 production by 71%, when compared with the TPA plus FSH treatment alone (P<0.01). 170 F i g . 56. Response to arachidonic acid (AA) a f t e r pretreatment with FSH and LHRH. Granulosa c e l l s pretreated with s a l i n e (C) or with FSH ± LHRH for 18h, at which time AA (or solvent) was added. A l l groups were incubated for a further 6h. The in h i b i t o r y e f f e c t of LHRH on FSH induced progesterone production was p a r t i a l l y reversed by the presence of AA. 171 Fig. 57. Response to arachidonic acid (AA) a f t e r pretreatment with FSH and the phorbol ester TPA. Granulosa c e l l s were pretreated with DMSO (C) or with FSH ± TPA for 18h p r i o r to the addition of AA (or solvent). Then, the 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 production by FSH was p a r t i a l l y reversed by the addition of AA. 172 Response to arachidonic a c i d a f t e r pretreatment with cholera t o x i n and TPA Addition of CT resulted i n a 29 f o l d increase i n P, 4 production and TPA s i g n i f i c a n t l y attenuated the production of P 4 induced by CT. AA was added to two groups that had been precultured with CT or CT plus TPA for 18h. A l l incubations were continued for a further 6h. As shown i n Fig. 58, the presence of AA caused a p a r t i a l reversal, i . e . 39% (P<0.01) increase i n P 4 production, when compared with the treatment with TPA plus CT alone. CT-induced P 4 production was not s i g n i f i c a n t l y affected by the addition of AA alone during the l a s t 6h culture period. E f f e c t of arachidonic acid on P 4 production a f t e r pretreatment with TPA and LHRH -9 -6 10 M TPA and 10 M LHRH were added to granulosa c e l l s at the beginning of the culture i n the absence of exogenous gonadotropins or CT. Af t e r 18h, AA was added to some of the groups for a further 6h to determine the P 4 response of the c e l l s . At the end of the 24h culture, TPA d i d not stimulate P 4 production (Fig. 59). In contrast, LHRH caused a 55% (P<0.01) increase i n P 4 accumulation when compared with untreated controls. Addition of AA alone during the l a s t 6h resulted in a 160% increase i n P. formation. When AA was added to the TPA- 4 pretreated c e l l s , P 4 production was same as that caused by AA alone. In contrast, when AA was given to the LHRH-pretreated c e l l s , there was an additive e f f e c t on P. production. 173 Fi g . 58. Response to arachidonic acid (AA) a f t e r pretreatment with cholera toxin (CT) and the phorbol ester TPA. Granulosa c e l l s were pretreated with DMSO (C) or with CT ± TPA. At 18h, AA (or solvent) was added. A l l incubations were stopped 6h l a t e r . The presence of AA p a r t i a l l y reversed the i n h i b i t o r y e f f e c t of TPA on CT induced progesterone production. 174 Fig. 59. Response to arachidonic acid (AA) a f t e r pretreatment with the phorbol ester TPA and LHRH alone. Granulosa c e l l s were pretreated with dimethyl sulfoxide (C), TPA or LHRH for 18h. At t h i s time, AA (or solvent) was added. A l l groups were incubated for a further 6h. Treatment of the c e l l s with TPA did not a f f e c t the response to AA. 175 E f f e c t s of LHRH. TPA and/or arachidonic a c i d on progestin production during a 5h Incubation Granulosa c e l l s were treated f o r 5h with TPA (10 M) or LHRH (10~6M) , with or without the concomitant presence of AA —5 (10 M) . As shown i n A panel of F i g . 60, the presence of AA, TPA or LHRH alone caused s i g n i f i c a n t increases i n P 4 production, by 4.8 f o l d , 7.8 f o l d and 7.1 f o l d , respectively, when compared with control l e v e l s . AA exerted an additive e f f e c t with TPA and LHRH on P. formation. 4 The production of 20alpha-OH-P i s shown also i n Fig. 60 (B panel). AA, TPA and LHRH stimulated 20alpha-OH-P production by 1.5 f o l d , 4.9 f o l d and 4.7 f o l d , respectively. The magnitude of AA-induced 20alpha-OH-P accumulation was much lower than that induced by either TPA or LHRH. In contrast to the additive e f f e c t s observed for P 4 formation, AA did not a l t e r the e f f e c t of TPA or LHRH on the accumulation of 20alpha- OH-P. On the other hand, t o t a l progestin accumulation ( i . e . P 4 plus 20alpha-OH-P) was increased by treatment with AA, TPA or LHRH alone and further increased by combined treatment with TPA plus AA, or with LHRH plus AA (Fig. 60, C panel). E f f e c t s of arachidonic acid. TPA and LHRH on 25- hydroxycholesterol enhanced steroidogenesis during a 5h incubation To determine i f AA aff e c t s the a c t i v i t y of the side-chain cleavage enzyme, a water soluble cholesterol derivative, 25-OH- cholest e r o l , was used. Inclusion of 25-OH-cholesterol i n the 176 culture medium s i g n i f i c a n t l y enhanced the accumulation of P 4 i n the control c e l l s by 4.1 f o l d during a 5h incubation (Fig. 61). The concomitant presence of TPA or LHRH with 25-OH-cholesterol markedly increased P 4 production by about 56% and 84% (P<0.01) respectively, when compared with P 4 formation by 25-OH- cholesterol alone. The presence of AA with 25-OH-cholesterol also s i g n i f i c a n t l y increased P 4 production, about 34%, when compared to the e f f e c t of 25-OH-cholesterol alone. Nevertheless, AA f a i l e d to further enhance TPA- and LHRH- stimulated P 4 production i n the presence of 25-OH-cholesterol, which was d i f f e r e n t from the e f f e c t of AA on P 4 production without the added c h o l e s t e r o l . F i g . 60. Ef f e c t s of LHRH, the phorbol ester TPA and/or arachidonic acid (AA) on progestin production during a 5h incubation. AA, TPA or LHRH alone caused s i g n i f i c a n t increase i n progesterone production, and AA exerted an additive e f f e c t with TPA and LHRH, whereas the magnitude of AA induced 20- alpha-OH-P accumulation was much lower than that induced by either TPA or LHRH, and AA did not a l t e r the e f f e c t of TPA or LHRH on the accumulation of 20-alpha-OH-P. 178 F i g . 61. E f f e c t of arachidonic a c i d (AA) , the phorbol ester TPA and LHRH on 25-hydroxycholesterol enhanced steroidogenesis during a 5h incubation. Although the presence of AA increased 25-OH-cholesterol induced progesterone production, AA f a i l e d to further enhance TPA and/or LHRH stimulated progesterone production i n the presence of 25-OH- cho l e s t e r o l . Incubations containing 25-hydroxycholesterol were denoted by the hatched bars. 179 IV. Discussion The i n t r a c e l l u l a r pathway by which LHRH stimulated AA release i n granulosa c e l l s was not c l e a r . Three possible mechanisms f o r the l i b e r a t i o n of AA from plasma membrane have been proposed (chapter 2) . The increase of c e l l u l a r l e v e l of free or un e s t e r i f i e d radiolabeled AA was observed by treatment of granulosa c e l l s with LHRH within 5 min (chapter 2). This observation strengthens the previous proposal that LHRH stimulated AA l i b e r a t i o n from phospholipids might also be involved as an early step i n LHRH si g n a l transduction i n the ovarian c e l l s (Minegishi and Leung, 1985; chapter 2 ). I t has also been shown that AA release i n granulosa c e l l s i s enhanced by the calcium ionophore A23187 (Kawai and Clark, 1986; Minegishi et a l . , 1987), and potentiated by TPA (chapter 2), suggesting that LHRH-induced AA release i s calcium dependent, and i s regulated by the a c t i v a t i o n of PKC. Nevertheless, i t appears that i n many tissues a si n g l e e x t r a c e l l u l a r signal could induce a c t i v a t i o n of both phospholipase C and phospholipase A 2 reactions and as a r e s u l t , cause AA release from various phospholipids (Lapetina, 1982). In the present study the e f f e c t s of m e l i t t i n , AA and LHRH on P 4 production were further examined. M e l i t t i n could induce P 4 production, but m e l i t t i n stimulated P 4 production was lower than exogenous AA and LHRH stimulated P 4 formation i n the same experiment (Fig. 41) . This may r e f l e c t the fact that the quantity of endogenous AA was l i m i t e d . Additionally, the r e s u l t s suggested that the e f f e c t of LHRH cannot be only due to endogenous AA and i t s metabolites, and that Ca and PKC pathways were also involved. The previous experiment has shown that concomitant treatment of granulosa c e l l s with m e l i t t i n plus LHRH does not further increase P 4 production induced by LHRH alone, presumably implying that the e f f e c t of LHRH on P 4 production already included the action of endogenous AA (chapter 4). Taken together, P 4 production induced by m e l i t t i n suggested that the a c t i v a t i o n of PLA 2, an enzyme that cleaves AA from the 2-acyl p o s i t i o n of phospholipids, p a r t i c i p a t e d i n c o n t r o l l i n g P 4 production as well as PGE 2 i n granulosa c e l l s . More importantly, the present data c l e a r l y demonstrated that treatment of granulosa c e l l s with AA f o r 5h enhanced P 4 production (Fig. 41) . This stimulation was dose dependent, —7 —5 within a rather narrow range ( i . e . between 3x10 M to 10 M) (Fig. 42). The use of the exogenous AA was c l o s e l y r e l a t e d to the physiologic s i t u a t i o n since i t was converted to ovarian cyclooxygenase or lipoxygenase metabolites at the s i t e where the appropriate biosynthetic process was present. Both i n t r a c e l l u l a r P. concentration and the accumulation of P. i n 4 4 the culture medium were increased i n the presence of AA. C e l l v i a b i l i t y was not affected by these dosages of AA, as judged by trypan blue exclusion. In t h i s regard, s i m i l a r doses of AA have recently been reported to stimulate hormone production i n other endocrine tissues. For example, i n anterior p i t u i t a r y —5 —4 c e l l s , AA at 5x10 M or 10 M was a potent secretagogue for LH release (Chang et a l . , 1986); at 10"5M and 10~4M, AA stimulated ACTH release (Abou-Samra et a l . , 1986). PRL secretion from GH3 c e l l s was stimulated by AA at 3x10 M (Kolesnick et a l . , 1984). Likewise, AA at 10 to 10 M resulted i n the increase of oxytocin i n corpus luteam (Hirst et a l . , 1988), and AA at 10 M —5 to 10 M enhanced testosterone production by Leydig c e l l s (Lin, 1985). In the present study, the e f f e c t of AA on P 4 production was found to be s i m i l a r to that of LHRH, although the stimulation of P. due to AA was somewhat slower i n onset 4 compared with that due to LHRH. P 4 l e v e l s i n the culture medium were i d e n t i c a l 5h a f t e r the addition of LHRH or AA (Fig. 45). When present together, the eff e c t s of AA and LHRH or LHRH agonist became additive; t h i s could be seen as early as 3h af t e r treatment. Since AA also greatly potentiated the stimulation of P 4 production by TPA, the s y n e r g i s t i c e f f e c t of AA on LHRH-induced P 4 production perhaps r e f l e c t e d a potentiation by AA on LHRH-induced PKC a c t i v i t y (Fig. 44-47). The r e l a t i v e l y higher dose and longer time required for the action of AA on P 4 production may be due to the rate of penetration of AA into the c e l l membrane and the conversion of AA to i t s metabolites. Recently, PKC a c t i v i t y has been characterized i n ovarian tissues (Noland and Dimlno, 1986; Davis and Clark, 1983). The highest s p e c i f i c a c t i v i t i e s were found i n cytosol, followed by microsomes and mitochondria (Noland and Dimino, 1986). Several laboratories have reported that a c t i v a t i o n of PKC by phorbol esters, such as TPA, stimulates basal P 4 production i n rat granulosa c e l l s (Wang and Leung, 1987; Kawai and Clark, 1985; Shinohara et a l . , 1986). 182 Thus, the present r e s u l t s could be taken to suggest that the stimulatory action of LHRH on ovarian steroidogenesis i s mediated, i n part at le a s t , by PKC and potentiated by LHRH- induced AA release. Phosphorylation of cytochrome P-450, which i s responsible for choleste r o l side-chain cleavage, may also r e s u l t from a c t i v a t i o n of PKC i n steroidogenic tissues ( V i l g r a i n et a l . , 1984). I t seems pl a u s i b l e that LHRH-induced AA could play a second messenger r o l e by amplification of PKC a c t i v i t y , as has been proposed recently for other s i g n a l l i n g systems (McPhail et a l . , 1984; Murakami and Routtenberg, 1985). In support of t h i s theory, i t has recently been documented that AA and PKC s y n e r g i s t i c a l l y mediate the stimulation of gonadotrophin secretion by LHRH i n anterior p i t u i t a r y c e l l s (Chang et a l . , 1986). In the same experiment, the e f f e c t s of other unsaturated f a t t y acids on P 4 production were also examined (Fig. 43). A l l -5 the treatments were at the dose of 10 M which was the maximal e f f e c t i v e concentration f o r AA on stimulating the production of P 4« 11,14 eicosadenoic a c i d (C18:2), l i n o l e i c acid (C18:2), gamma-linolenic acid (C18:3), homo-gamma-linolenic acid (C18:3) l i k e AA, could increase P 4 production i n 5h c e l l incubation. Fatty acids stimulated P 4 production i n a order of AA> homo-gamma-linolenio gamma-linolenio 11,14 eicosadienoic aci d . Another unsaturated f a t t y acid, o l e i c acid (C18:l), f a i l e d to stimulate P 4 production. These data were s i m i l a r to the e f f e c t s of AA and other unsaturated f a t t y acids on PRL secretion i n human decidual t i s s u e (Handwerger et a l 1981). 183 The mechanism of these unsaturated f a t t y acids on P 4 production was not c l e a r . However, these unsaturated f a t t y acids are used for c e l l structures and can convert to AA cascade which could n a t u r a l l y e x i s t i n the c e l l membrane, or produce other series of PGs and LTs (Crawford, 1983). Therefore, a large membrane source of PGs and LTs precursor was provided. The mechanism by which AA stimulates P 4 production was not known. Previously, LHRH and i t s agonists have been shown to stimulate PG production i n rat granulosa c e l l s (Clark, 1982). The stimulatory e f f e c t of LHRH on PG synthesis was additive but apparently d i s t i n c t from that induced by LH. LH or hCG, as well, has been reported to stimulate ovarian lipoxygenase a c t i v i t y i n vivo and i n v i t r o . The a c t i v a t i o n of lipoxygenase might be correlated with f o l l i c u l a r rupture at ovulation (Reich et a l . , 1983; Reich et a l . , 1985). In' the present study, the possible involvement of PGs was examined using indomethacin that i n h i b i t s the cyclooxgenase pathway of AA metabolism. The addition of indomethacin to granulosa c e l l s d i d not a l t e r LHRH- or AA-induced P 4 production during a 5h culture period (Fig. 48 and 49) . Although the blockade of prostaglandin did not a f f e c t P 4 production, the i n h i b i t i o n of cyclooxygenase with indomethacin did block ovulation i n rat (Amrstrong and Grinwich, 1972), rabbit (Armstrong et a l , 1974) and marmoset monkeys (Mai et a l . , 1975). In addition, i n indomethacin-blocked rats, administration of PGE 2 can induce ovulation ( T s a f r i r i et a l . , 1972). Further, i n the absence of prostaglandins, the rupture of ovarian f o l l i c l e d i d not occur 184 (LeMaire and Marsh, 1975) . AA can be also converted by the lipoxygenase enzymes to a va r i e t y of HPETEs that would be rapidly reduced to t h e i r respective HETEs, and 5-HPETE gives r i s e to another series of products known as the leukotrienes. The contribution of the lipoxygenase pathways to the stimulation of P 4 synthesis was further investigated u t i l i z i n g NDGA, an e f f e c t i v e i n h i b i t o r of lipoxygenase pathway of AA in f o l l i c u l a r t i s s u e (Reich et a l . , 1983). AA-induced P 4 production was reduced to the l e v e l as that caused by NDGA, and LHRH-induced P 4 formation was only p a r t i a l l y suppressed (Fig. 48 and 49). Although NDGA reduced AA- and LHRH-stimulated P 4 production, the basal l e v e l of P 4 was increased by NDGA, which might r e s u l t from the increase i n the precursor f o r PGs synthesis. These r e s u l t s indicated that there may be a stimulatory r o l e for PGs i n steroidogenesis. Indeed, exogenous PGE 2 has been shown to stimulate cAMP, estrogen and P 4 production (Richards et a l . , 1976). Furthermore, the p a r t i a l l y i n h i b i t o r y e f f e c t of NDGA on LHRH-induced P 4 production supports the notion that multiple second messengers were involved i n the action of LHRH. Besides the release of AA from plasma membrane, LHRH also induced the formation of DG which 2+ leads the act i v a t i o n of PKC, and IP 3 which causes Ca mobilization. These d i f f e r e n t pathways cooperated each other and thus contributed to the action of LHRH. The finding that LHRH- or AA-stimulated P 4 production was not influenced by the presence of indomethacin supported the previous observations that LH induced P. production was not affected by the 185 indomethacin-blocked PG formation (Clark, 1982), and that the enhancement of FSH or CT-induced PGE2 production by LHRH or TPA di d not t i g h t l y couple to the production of progesterone (Chapter 4) . Taken together, LHRH-stimulated P 4 production, p a r t i a l l y by increasing free AA and by converting AA to i t s metabolites, d i d not r e s u l t from PG formation. Recently, AA has been implicated i n the secretion of oxytocin i n ovine corpus luteum. The r e s u l t s showed that PGE 2 and P G F 2 a ] _ p n a do not stimulate oxytocin secretion, and AA may have i t s e f f e c t v i a the lipoxygenase pathway (Hirst et a l . , 1988), further implicating the involvement of lipoxygenase metabolites i n regulating ovarian functions. The i n h i b i t o r y e f f e c t of NDGA on LHRH- or AA-induced P 4 formation indicated that lipoxygenase metabolites of AA had a ro l e i n the P 4 production induced by LHRH. This hypothesis was further investigated i n the present study using several HETEs and HPETEs from the 5-, 12-, and 15-lipoxygenase metabolism of AA. The r e s u l t s indicated that at least some lipoxygenase metabolites of AA were capable of enhancing the formation of P 4 by r a t granulosa c e l l s i n a dose dependent manner (Fig. 50) . The stimulatory e f f e c t s of 12-HETE on P 4 production appeared to be more potent than that of 5-HETE, 5-HPETE or 15-HPETE. In addition to P 4, the formation of PGE2 was also stimulated by several of the AA metabolites (Fig. 51) . This e f f e c t of AA metabolitws i s not due to cross-reaction of the metabolites i n the PGE2 assay, since at the concentrations used the metabolites do not cross-react. At 5xl0~ 6M, 12-HETE and 15- 186 HETE were more potent than 5-HETE i n t h i s regard. Like AA, 5- HETE, 5-HPETE, 12-HETE, 15-HETE and 15-HPETE increased basal P 4 production and further augmented the LHRH-induced P 4 production. Also, at 10~6M, 12-HETE and 15-HETE stimulated basal PGE 2 formation and potentiated the stimulation of PGE 2 formation induced by LHRH (Fig. 52). Since very s i m i l a r r e s u l t s were observed with TPA (Fig. 53), the f a c i l i t a t o r y e f f e c t s of HETEs and HPETEs on LHRH-induced P 4 and PGE 2 production may be due to the in t e r a c t i o n of these AA metabolites with LHRH-activated PKC. Since the present data showed the metabolites of lipoxygenase pathway of AA-stimulated PGE 2 production, one might speculate that HETEs or HPETEs act as i n t e r n a l regulators between the metabolites of lipoxygenase and cyclooxygenase pathway. Previous studies have shown that both LH and hCG regulate lipoxygenase a c t i v i t y , but the action of LHRH on these enzymes needs further in v e s t i g a t i o n . I t has been reported that both 5- and 15-lipoxygenase require calcium for a c t i v i t y i n p l a t e l e t (Pace-Asciak and Smith, 1986), thus, 2+ LHRH-induced rapid increase i n [Ca ] i might be related to the a c t i v i t y of these enzymes i n the ovarian c e l l s as well. There was increasing evidence to support the notion that lipoxygenase metabolites of AA were potent mediators of hormone production i n d i f f e r e n t endocrine ti s s u e s . One or more of the cyclooxygenated and/or lipoxygenated metabolites of AA might be a component of the cascade of reactions i n i t i a t e d by LHRH and ultimately r e s u l t i n LH secretion i n p i t u i t a r y (Kiesel et a l . , 1986; K i e s e l et a l . , 1987). I t has also been reported that 187 leukotrienes are e f f e c t i v e stimulators of LH release from dispersed r a t anterior p i t u i t a r y c e l l s (Kiesel et a l . , 1987; Ki e s e l et a l . , 1986; Hurling et a l . , 1985). Lipoxygenase products of AA metabolism have already been shown to stimulate PRL release (Kiesel et a l . , 1987). Yamamoto et a l . have reported that 5-HETE stimulates i n s u l i n release i n pancreatic i s l e t s (Yamamoto et a l . , 1983). In bovine corpus luteum, 5- HETE reduced the biosynthesis of P^ and 6 - k e t o - p G F i a j _ p n a f while the synthesis of P G F 2 a i p h a w a s u n a f f e c t e d (Milvae et a l . , 1986). I n h i b i t i o n of lipoxygenase a c t i v i t y with NDGA, BW 755C and FPL-55712 resulted i n p a r t i a l blockade of ovulation (Reich et a l , 1983; Reich et a l . , 1985). Although high concentration of 5-HETE had 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 p h y s i o l o g i c a l importance of those compounds as regulator of ovarian functions (Milvae et a l . , 1986), thus f a r , there was no evidence to show that the receptors of these hydroperoxy acids e x i s t on r a t granulosa c e l l . Whether HETEs and HPETEs prove to be i n t r a c e l l u l a r rather than e x t r a c e l l u l a r signals remains to be determined. The 5-lipoxygenase pathway i s of special i n t e r e s t because 5-HPETE can be r a p i d l y converted to leukotrienes that are presumably the most active of the lipoxygenase metabolites of AA (Samuelsson, 1983; Morris et a l . , 1982). In view of the present demonstration of stimulatory e f f e c t s of 5-HPETE on P 4 and PGE 2 production, the r o l e of leukotrienes on ovarian c e l l function warrants further inve s t i g a t i o n . In addition, the stimulatory r o l e of AA was further 188 examined i n the present study. The i n h i b i t o r y or stimulatory e f f e c t s of LHRH on P 4 accumulation with or without the presence of FSH during the d i f f e r e n t culture period (Fig. 54) further confirmed the previous studies that the i n h i b i t o r y action of LHRH on granulosa c e l l steroidogenesis was only observed a f t e r 24h i n the presence of exogenous gonadotropins, or other cAMP stimulating agents, whereas LHRH did not influence steroidogenesis induced by gonadotrophins i n short term incubations (Knecht et a l . , 1982; Hsueh and Schaeffer, 1985; H i l l e n s j o et a l . , 1982). The reason for the apparent delay i n the onset of t h i s i n h i b i t o r y e f f e c t was not known, although i t was believed that LHRH-induced membrane polyphosphoinositide breakdown l e d to the formation of IPs and DG, and the release of AA, might p a r t i c i p a t e i n the action of LHRH (Ma and Leung, 1985; Davis et a l . , 1986; Davis et a l . , 1987; Minegishi and Leung, 1985; Wang and Leung, 1987). Unlike the markedly i n h i b i t o r y e f f e c t of LHRH and TPA, AA d i d not a f f e c t the magnitude of P 4 production induced by FSH during a 24h incubation period (Fig. 55) . Since the e f f e c t of TPA was e s s e n t i a l l y s i m i l a r to that of LHRH during long term granulosa c e l l culture, a c t i v a t i o n of protein kinase C may p a r t i c i p a t e i n the i n h i b i t o r y action of LHRH at two d i s t i n c t s i t e s , the gonadotrophin receptor/adenylate cyclase complex and a s i t e d i s t a l to the generation of cAMP (Welsh et a l . , 1984; Barry et a l . , 1985). Likewise, calcium mobilization may also be involved i n the action of LHRH to i n h i b i t gonadotrophin induced cAMP and s t e r o i d formation i n granulosa c e l l s (Ranta et a l . , 189 1983; Leung et a l . , 1988). On the other hand, the findings with exogenous AA suggested that AA mediated a stimulatory, rather than i n h i b i t o r y r o l e i n the action of LHRH (Wang and Leung, 1988) . To further examine t h i s hypothesis, granulosa c e l l s were treated with FSH (with or without LHRH or TPA) for 18h, at which time the i n h i b i t o r y e f f e c t of LHRH or TPA was already evident. Addition of AA during a further 6h incubation p a r t i a l l y reversed the i n h i b i t o r y e f f e c t of LHRH or TPA on FSH- or CT-induced P 4 production (Fig. 56-58). AA was also added to some c e l l s which had been pretreated with LHRH or TPA alone ( i . e . i n the absence of FSH) f o r 18h, and the response of the c e l l s to AA was quite s i m i l a r to that of untreated granulosa c e l l s given AA during a 5h incubation (Fig. 59). The previous studies have reported that LHRH and TPA share a similar i n h i b i t o r y mechanism of action on FSH induced P 4 production, and the i n h i b i t o r y e f f e c t s of LHRH or TPA could not be reversed by the addition of FSH (Knecht et a l . , 1982; Shinohara et a l . , 1985). The present data suggested that P 4 production elevated by AA was mediated by a mechanism which was not suppressed by LHRH and TPA, and granulosa c e l l s s t i l l retained the a b i l i t y to respond to AA following LHRH or TPA pretreatment. The e f f e c t of AA on the enzymes involved i n progesterone synthesis and metabolism was further examined. Activation of . 5 .4 the side chain cleavage enzymes (SCC), and 3-beta-HSD/4 -A - isomerase convert cholesterol to P 4 v i a pregnenolone. On the other hand, P 4 i s converted by 20-alpha-HSD to i t s inactive form, 20-alpha-hydroxy-pregn-4-en-3-one (20-alpha-OH-P). 190 Previous studies have indicated that LHRH alone increases 3- beta-HSD a c t i v i t y by an increase i n the apparent V and i s max accompanied by increased accumulation of pregnenolone, P 4 and 20-alpha-OH-P production (Jones and Hsueh, 1981a; 1982b). Moreover, the i n h i b i t o r y e f f e c t of LHRH on FSH induced 3-beta- HSD a c t i v i t y , FSH induced pregnenolone and P 4 production have also been observed during 24h granulosa c e l l culture, and t h i s i n h i b i t o r y action of LHRH i s characterized by a decrease i n the apparent V m a x without an a l t e r a t i o n of the 1^ of the enzyme (Jones and Hsueh, 1981b; 1982a; 1982b). TPA exerted a s i m i l a r e f f e c t on enzyme a c t i v i t i e s and FSH induced hormone production to LHRH (Jones and Hsueh, 1982a; 1982b; Welsh et a l . , 1984). TPA alone stimulated P 4 production and the a c t i v i t i e s of 3- beta-HSD and 20-alpha-HSD, leading to increased production of progestin. In contrast, TPA i n h i b i t i o n of P 4 biosynthesis induced by gonadotrophin was accompanied by reduction of 3- beta-HSD a c t i v i t y . The increase i n 20-alpha-HSD a c t i v i t y resulted i n the conversion of b i o l o g i c a l active P 4 to 20-alpha- OH-P, a b i o l o g i c a l i nactive metabolites. The e f f e c t s of AA, LHRH and/or TPA on P 4 and 20-alpha-OH-P accumulation were compared i n the present study (Fig. 60). AA, LHRH, or TPA each stimulated the production of P 4 and AA enhanced the P 4 production induced by LHRH and TPA (Fig. 60; A panel). LHRH or TPA markedly increased 20-alpha-OH-P production, however AA was only marginally e f f e c t i v e i n increasing 20-alpha-OH-P (Fig. 60; panel B). In addition, AA d i d not show any sy n e r g i s t i c e f f e c t with LHRH or TPA on 20-alpha-OH-P production. These r e s u l t s 191 indicate that AA increases P 4 production by stimulating biosynthesis rather than s i g n i f i c a n t l y a l t e r i n g 20-alpha-HSD a c t i v i t y . To further examine the action of AA on SCC a c t i v i t y , a substrate for the SCC enzymes, 25-OH-cholesterol, has been used to increase P. formation. 25-OH-cholesterol i s a water soluble 4 s t e r o i d which r e a d i l y enters c e l l s and i s metabolized to s t e r o i d hormones i n mitochondria (Toaff et a l . , 1982; Lino et a l . , 1985). Several steps i n the cholesterol SCC reaction such as uptake of cholesterol by mitochondria, the intramitochondrial access of cholesterol to the SCC enzyme complexes, and the modulation of the mitochondrial cytochrome P-450 l e v e l s , have been suggested to be under hormone control (Leaven and Boyd, 1981; Sulimovici and Boyd, 1968). In the present study, i t was observed that both LHRH and TPA enhanced P 4 production i n the presence of 25-OH-cholesterol (Fig. 61) . 5 * 4 Since 3-beta-HSD/ - -isomerase a c t i v i t y was not rate- l i m i t i n g i n granulosa c e l l s , the increase i n progesterone production i n the presence of 25-OH-cholesterol most l i k e l y r e f l e c t e d the increased a v a i l a b i l i t y of substrate to SCC, and the stimulation of P 4 production by 25-OH-cholesterol indicated that the SCC enzymes were substrate l i m i t e d as previously reported (Bagavandoss and Midgley, 1987; Toaff et a l . , 1982). Furthermore, AA increased P 4 production, i n the presence of 25- OH-cholesterol, but to a lesser extent than that induced by LHRH or TPA (Fig. 61), suggesting that the AA stimulation of P 4 also takes place at the l e v e l of SCC which enhanced substrate 192 uptake by mitochondria. Interestingly, AA f a i l e d to further enhance P 4 production i n the presence of cholesterol substrate. The combined treatment of granulosa c e l l s with AA plus LHRH or with AA plus TPA apparently caused maximal a c t i v i t y of the SCC enzymes; therefore addition of 25-OH-cholesterol f a i l e d to further enhance P 4 production. In addition, the i n vivo synthesis of ovarian pregnenolone i s from cholesterol that i s taken up from the plasma, l i b e r a t e d from cholesterol ester stored within cytoplasmic l i p i d droplets and synthesized i n the ovarian c e l l from 2 carbon components. As demonstrated i n granulosa c e l l s cultured i n serum free medium, choleste r o l could come from de novo biosynthesis, which i s dependent on the a c t i v i t i e s of the rate l i m i t i n g 3-hydroxy-3-methylglutaryl coenzyme A reductase (Dorrington and Armstrong, 1979; Wang and Hsueh, 1979). AA may also increase enzyme a c t i v i t y i n some steps p r i o r to pregnenolone synthesis. I t i s possible that AA-induced P 4 production i s due to either the increased endogenous synthesis of c h o l e s t e r o l , or the l i b e r a t i o n of cholesterol from cholesterol esters. There would also be a combination of the above reactions i n response to AA. However, any of these mechanisms could account for the observed increase i n AA- induced P 4 production. I t i s of interest that the i n h i b i t o r y e f f e c t of LHRH on FSH induced ovarian ster o i d hormone production was only observed a f t e r a r e l a t i v e l y long time i n culture (Fig. 54). One of the proposed mechanisms was that LHRH further enhanced 193 gonadotrophin induced 20-alpha-HSD a c t i v i t y by which LHRH diminished the gonadotrophin stimulation of production. The LHRH stimulation of 20-alpha-HSD i n gonadotrophin treated c e l l s was the r e s u l t of changes i n enzyme a c t i v i t y , rather than enzyme a f f i n i t y f o r the substrate ( P h i l l i p et a l . , 1980). Assuming that the calcium and PKC pathways can p a r t i a l l y mediate the i n h i b i t o r y action of LHRH on granulosa c e l l s , i t can be postulated that LHRH-induced l i b e r a t i o n of AA may somehow antagonize the i n h i b i t o r y component of LHRH action. This was suggested by the present findings that AA d i d not decrease FSH induced P 4 accumulation even a f t e r 24h (Fig. 53) and that acute addition of AA to the FSH and CT-pretreated c e l l s caused a p a r t i a l reversal of the i n h i b i t o r y e f f e c t of TPA or LHRH on P 4 production (Fig. 56-58). Moreover, AA mainly stimulated P 4 production, but TPA stimulated both P 4 and 20a- OH-P e f f e c t i v e l y (Fig. 60). These data suggest that a c t i v a t i o n of PKC may well mediate the long term i n h i b i t o r y action of LHRH on P 4 accumulation, by converting P 4 to 20-alpha-OH-P. In contrast, AA (or i t s active metabolites) most l i k e l y played a r o l e i n the short term stimulatory e f f e c t of LHRH on P 4 production by enhancing the a c t i v i t y of SCC. Taken together, the p o t e n t i a l i n h i b i t o r y e f f e c t s of LHRH (via a c t i v a t i o n of PKC) might have been prevented by AA during the 5h incubations. A f t e r that, the LHRH-induced- free AA may convert to some inac t i v e metabolites and the inh i b i t o r y component of LHRH action became dominant. In addition, study of the functions of TPA, A23187 and AA has provided evidence for a p a r t i c i p a t i o n of PKC, C a 2 + and metabolites of AA i n the mediation of LHRH action (chapter 4). However, i t was u n l i k e l y that c e l l u l a r responses involving PKC, calcium and metabolites of AA were j u s t l i m i t e d to the c e l l membrane and cytoplasm. There was accumulating evidence that extranuclear events were coordinated by nuclear components, including changes i n s p e c i f i c gene expression, i . e . the regulation of P450 g c c mRNA by FSH (Richards and Hedin, 1988). Therefore, the action of LHRH on FSH induced ovarian steroidogenesis i n long term culture may be due to the int e r a c t i o n of these hormones on gene expression. I t has already been shown that FSH administration to hypophysectomized rats causes the increase i n cytochrome P450o__ mRNA i n granulosa c e l l s and FSH induced gene expression which i s only c l e a r l y demonstrable a f t e r 7 to lOh. Thus far, no discussion of LHRH regulation of gene expression has been made i n granulosa c e l l s . In conclusion, the present r e s u l t s strongly support the hypothesis that AA and i t s lipoxygenase metabolites p a r t i a l l y mediate the action of LHRH by playing a stimulatory r o l e i n the d i r e c t e f f e c t s of LHRH on ovarian hormone production. Furthermore, i t indicates that the actions of LHRH or LHRH-like peptide on granulosa c e l l s are mediated by the d i f f e r e n t i n t r a c e l l u l a r signal pathways, and that the complex interplay between these pathways ultimately d i c t a t e s the time-dependent steroidogenic response of the ovary to LHRH or LHRH-like peptide. General Summary 195 Although gonadotropins are the major trop h i c hormones that regulate ovarian functions, increasing evidence suggests that l o c a l regulators p a r t i c i p a t e i n paracrine or autocrine control of ovarian functions. The d i r e c t actions of LHRH on rat ovarian c e l l s have been documented. Unlike gonadotropins, LHRH does not use cAMP as i t s second messenger. Increasing evidence shows that the i n i t i a l action of LHRH involves a rapid a l t e r a t i o n i n the metabolism of membrane i n o s i t o l l i p i d s i n the ovary. In the present study, the actions of LHRH on the breakdown of membrane 2 + phosphoinositides, changes of i n t r a c e l l u l a r Ca , production of s t e r o i d hormones and prostaglandins i n r a t granulosa c e l l s were extensively studied. In radiolabeled rat granulosa c e l l s , the rapid and s p e c i f i c formation of i n o s i t o l 1,4,5- trisphosphate and d i a c y l g l y c e r o l , and the release of arachidonic acid were observed shortly a f t e r addition of LHRH. 2 + LHRH also caused a rapid and transient increase i n [Ca ] i i n the majority of granulosa c e l l s as assessed by fura-2 microspectrofluorimetry. I n o s i t o l 1,4,5-trisphosphate, which i s produced simultaneously with d i a c y l g l y c e r o l by PLC hydrolysis of PIP 2/ m a v D e responsible f o r the LHRH induced 2+ rapid and transient a l t e r a t i o n s of [Ca ] i i n granulosa c e l l s . I t i s known that LHRH exerts e i t h e r stimulatory or i n h i b i t o r y actions on ovarian c e l l s depending on the culture period, the presence of other hormones such as gonadotropins, and the 196 nature of the hormone examined. To t e s t the hypothesis that the e f f e c t s of LHRH on granulosa c e l l s were mediated, at least i n part, by calcium and PKC, the e f f e c t s of the calcium ionophore A23187 and the phorbol ester TPA on the production of progesterone and PGE 2 have been examined. The present study demonstrated that LHRH i n h i b i t e d the production of progesterone stimulated by FSH, while simultaneously enhancing PGE2 production stimulated by FSH. These data suggest that the action of LHRH i s mainly at a step(s) following gonadotropin induced cAMP formation. TPA and A23187 can mimic the actions of LHRH. Interestingly, TPA acted s y n e r g i s t i c l y with A23187 on PGE 2 production but not on the production of progesterone, 2+ suggesting multiple aspects of PKC and Ca action on granulosa c e l l s . I t appears that a c t i v a t i o n of PKC and a l t e r a t i o n of 2+ [Ca ] i not only mediates c e l l u l a r processes, but also a l t e r s membrane phosphoinositide metabolism, thus providing a potent i a l feedback control mechanism. Increased free arachidonic a c i d l e v e l induced by LHRH serves as the precursor for the synthesis of cyclooxygenase and lipoxygenase metabolites of arachidonic acid. Prostaglandins are the cyclooxygenase metabolites of arachidonic a c i d which play a very important r o l e i n reproductive functions of the ovary. Although the production of prostaglandin may not be t i g h t l y coupled to progesterone production, i t i s c e r t a i n l y involved i n the ovulation process. In the present study, lipoxygenase pathway metabolites apparently p a r t i c i p a t e d i n the stimulatory action of LHRH probably by enhancing the action of protein 197 LHRH LH FSH cholesterol HPETEs -1- — f HETES I LTs I I J [Ca"']i mobilization L F i g . 62. I l l u s t r a t i o n of the interactions between l u t e i n i z i n g hormone-releasing hormone (LHRH) and gonadotrophin second messenger pathways. Abbreviations: LH, l u t e i n i z i n g hormone; FSH, f o l l i c l e stimulating hormone; R, receptor; DG, 1,2- d i a c y l g l y c e r o l ; cAMP, 3'5'-cyclic adenosine monophosphate; PKC, protein kinase C; ER, endoplasmic reticulum; AA, arachidonic acid; HPETE, hydroperoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic 2£cid; LT, leukotriene; PG, prostaglandin; [Ca ] i , i n t r a c e l l u l a r calcium ion concentration; IP 3, i n o s i t o l 1,4,5,-trisphosphate; P 4, progesterone. 198 kinase C on the enzymes involved i n steroidogenesis. The in t e r a c t i o n between the gonadotropins and LHRH are summarized schematically i n F i g . 62. The evidence f o r the possible paracrine or autocrine ro l e s of LHRH i n ovarian c e l l s i s strengthened by the demonstration of the presence of LHRH-like peptides i n human, rat , bovine and ovine ovaries i n other sdudies. The involvement of these d i f f e r e n t hormonal systems and multiple second messenger mechanisms i n the regulation of granulosa c e l l function ensures the optimal ovarian hormone synthesis and the growth of the ovarian f o l l i c l e s . In addition, r a t granulosa c e l l s serve as an id e a l model for studies on the mechanism of hormone action because of the presence of both cAMP and 2 + Ca -protein kinase C pathways. The present i n v i t r o findings should help future elucidation of the processes of ovarian hormone production and ovulation. References 199 Aberdam, E. and Dekel, N. 1985 Activators of protein kinase C stimulate meiotic maturation of rat oocytes. Biochem Biophys Res Commun 132:570-574 Abou-samra, A.B., Catt, K.J. and Aguilera, G. 1986 Role of arachidonic acid i n the regulation of adenocorticotropin release from r a t anterior p i t u i t a r y c e l l cultures. Endocrinology 119:1427-1431 Adashi, E.Y. and Hsueh, A.J.W. 1982 Estrogens augment the stimulation of ovarian aromatase a c t i v i t y by f o l l i c l e - stimulating hormone i n cultured r a t granulosa c e l l s . J B i o l Chem 257:6077-6083 Adashi, E.Y., Resnick, C.E., D'Ercole, A.J., Svoboda, M.E. and Van Wyk, J . J . 1985 I n s u l i n - l i k e growth factors as intraovarian regulators of granulosa c e l l growth and function. Endocr Rve 6:400-420 Aloyo, V.J., Zwiers, H. and Gispen, W.H. 1983 Phosphorylation of B-50 protein by calcium-activated, phospholipid-dependent protein kinase and B-50 protein kinase. J Neurochem 41:649-653 Amsterdam, A., Nimord, A., Lamprecht, S.A. and Burstein Linder, H.R. 1979 I n t e r n a l i z a t i o n and degradation of receptor-bound hCG i n granulosa c e l l culture. Am J Physiol 236:E129-138 Amsterdam, A. and Rotmensch, S. 1987 Structure-function rel a t i o n s h i p s during granulosa c e l l d i f f e r e n t i a t i o n . Endocr Rev 8:309-336 Arimura, A., S e r a f i n i , P., Talbot, S. and Schally, A.V. 1979 Reduction of t e s t i c u l a r l u t e i n i z i n g hormone/human chorionic gonadotropin receptors by [D-Trp 6 ] - l u t e i n i z i n g hormone releasing hormone i n hypophysectomized rat s . Biochem Biophys Res Commun 90:687-693 Armstrong, D.T., Lee, T.P. and M i l l e r , L.S. 1970 Stimulation of progesterone biosynthesis i n bovine corpora lutea by l u t e i n i z i n g hormone i n the presence of an i n h i b i t o r of cholesterol synthesis. B i o l Reprod 2:29-36 Armstrong, D.T., Knudsen, K.A. and M i l l e r , L.S. 1970 Ef f e c t of p r o l a c t i n upon cholesterol metabolism and progesterone biosynthesis i n corpora lutea of hypophysectomized rats during pseudopregnancy. Endocrinology 86:634-641 Armstrong, D.T. and Grinwich, D.L. 1972 Blockade of spontaneous and LH-induced ovulation i n rats by indomethacin, an i n h i b i t o r of prostaglandin biosynthesis. Prostaglandins 1:21-28 200 Armstrong, D.T., Grinwich, D.L., Moon, Y.S. and Zanecnik, J . 1974 I n h i b i t i o n of ovulation i n rabbit by i n t r a f o l l i c u l a r i n j e c t i o n of indomethacin and PGF anti-serum. L i f e S c i 14:129-140 zaipna Asch, R.H., S i c k l e , M.V., Ret t o r i , V., Balmaceda, J.P., Eddy, CP., Coy, D.H. and Schally, A.V. 1981 Absence of LHRH binding s i t e s i n corpora lutea from rhesus monkeys (Macaca mulatta). J C l i n Endocrinol Metab 53:215-217 Asem, E.K., Molnar, M. and Hertelendy, F. 1987 Lu t e i n i z i n g hormone-induced i n t r a c e l l u l a r calcium mobilization i n granulosa c e l l s : Comparison with f o r s k o l i n and 8-bromo-adenosine 3',5'- monophosphate. Endocrinology 120:853-859 Aten, R.F., Williams, A.T. and Behrman, H.R. 1986 Ovarian gonadotropin-releasing hormone-like protein (s): demonstrations and characterization. Endocrinology 118:961-967 Aten, R.F., Ireland, J . J . , Weems, C W. and Behrman, H.R. 1987 Presence of gonadotropin-releasing hormone-like protein i n bovine and ovine ovaries. Endocrinology 120:1727-1733 Aub, D.L., McKinney, J.S. and Putney, J.W., J r . 1982 Nature of the receptor-regulated calcium pool i n the rat parotid gland. J Physiol 331:557-565 Bagavandoss, P. and Midgley, A.R., J r . 1987 Lack of difference between r e t i n o i c a c i d and r e t i n a l i n stimulating progesterone production by l u t e i n i z i n g granulosa c e l l s i n v i t r o . Endocrinology 121:420-428 Bahr, J . , Kao, L. and Nalbandov, A.V. 1974 The ro l e of catecholamines and nerves i n ovulation. B i o l Reprod 10:273-290 Baum, M.S. and Rosberg, S. 1987 A phorbol ester, phorbol 12- myristate 13-acetate, and a calcium ionophore, A23187, can mimic the l u t e o l y t i c e f f e c t of prostaglandin F2alt5ha * n i s o l a t e d r a t l u t e a l c e l l s . Endocrinology 120:1019-1025 p Beers, M.H., Strickland, S. and Reich, E. 1975 Ovarian plasminogen a c t i v a t o r : Relationship to ovulation and hormonal regulation. C e l l 6:387-394 Behrman, H.R. 1979 Prostaglandins i n hypothalamo-pituitary and ovarian function. Ann Rev Physiol 41:685-700 B e l l , R.L. and Majerus, P.W. 1979 Thrombin-induced hydrolysis of phosphatidylinositol i n human p l a t e l e t s . J B i o l Chem 255:1790-1792 B e l l , R.L., Kennerly, D.A., Stanford, N. and Majerus, P.W. 1979 Diglyceride l i p a s e : a pathway for arachidonate release from 201 human p l a t e l e t s . Proc Natl Acad S c i USA 76:3238-3241 Ben-Ze'ev, A. and Amsterdam, A. 1987 In v i t r o regulation of granulosa c e l l d i f f e r e n t i a t i o n . J B i o l Chem 262:5366-5376 Berridge, M.J. 1981 Phosphatidylinositol h y d r o l y s i s : a multifunctional transducing mechanism. Mol C e l l Endocrinol 24:115-140 Berridge, M.J., Downes, C P . and Hanley, M.R. 1982 Lithium amplifies agonist-dependent phosphatidylinositol responses i n brain and s a l i v a r y glands. Biochem J 206:587-589 Berridge, M.J. 1983 Rapid accumulation of i n o s i t o l trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol. Biochem J 211:849-858 Berridge, M.J. 1984 I n o s i t o l triphosphate and d i a c y l g l y c e r o l as second messengers. Biochem J 820:345-360 Berridge, M.J. 1987 I n o s i t o l trisphosphate and d i a c y l g l y c e r o l : two i n t e r a c t i n g second messengers. Ann Rev Biochem 56:159-193 Bjersing, L. 1967 Histochemical demonstration of ^-3-beta- and 17-beta-hydroxysteroid dehydrogenase a c t i v i t i e s i n porcine ovary. Histochemie 10:295-304 Bjersing, L. and Carstensen, H. 1967 Biosynthesis of steroids by granulosa c e l l s of the porcine ovary i n v i t r o . J Reprod F e r t i l 14:101-111 Bone, E.A., A i l i n g , D.W. and Grollman, E.F. 1986 Norepinephrine and thyoroid-stimulating hormone induce i n o s i t o l phosphate accumulation i n FRTL-5 c e l l s . Endocrinology 119:2193-2200 Brodie, A.M., Schwarzel, W.C. and Brodie, H.J. 1976 Studies on the mechanism of estrogen biosynthesis i n the r a t ovary. J Steroid Biochem 7:787-793 Brown, J.L. and Reeves, J . J . 1983 Absence of s p e c i f i c LHRH receptors i n ovine, bovine, and porcine ovaries. B i o l Reprod 29:1179-1182 Brown, M.S., Kovanen, P.T. and Goldstein, J.L. 1981 Regulation of plasma cholesterol by l i p o p r o t e i n receptors. Science 212:628-635 Burgess, G.M., Irvine, R.F., Berridge, M.J., McKinney, J^S. and Putney, J.W. 1984 Actions of i n o s i t o l phosphates on Ca pools i n guinea p i g hepatocytes. Biochem J 224:741-746 Burgess, G.M., Godfrey, P.P., McKinney, J.S., Berridge, M.J., 202 Irvine, R.F. and Putney, J.W., J r . 1984 TJhe second messenger l i n k i n g receptor a c t i v a t i o n to i n t e r n a l Ca release i n l i v e r . Nature 309:63-66 Byskov, A.G. and Rasmussen, G. 1973 Ultrastructure studies of the developing f o l l i c l e . In: The Development and Maturaion of the Ovary and Its Function, International Congress Steries No. 2 67 edited by H. Peters, pp. 55-62 Byskov, A.G. 1978 The anatomy and u l t r a s t r u c t u r e of the rete system i n the f e t a l mouse ovary. B i o l Reprod 19:720-735 C a r a f o l i , E. and Crompton, M. 1978 The regulation of i n t r a c e l l u l a r calcium. Current Topics i n Membranes and Transport 10:151-216 Carlson, J.C., Nelson, S.E. and Bols, N.C. 1986 Progesterone secretion by primary cultures of rat l u t e a l c e l l s Endocrine Research 12:1-2 0 Change, J.P., Graeter, J . and Catt, K.J. 1986 Coordinate actions of arachidonic acid and protein kinase C i n gonadotropin-releasing hormone-stimulated secretion of l u t e i n i z i n g hormone. Biochem Biophys Res Commun 134:134-139 Charest, R., Blackmore, P.Fw, Berthon, B. and Exton, H. 1983 Changes i n free c y t o s o l i c Ca i n hepatocytes following alpha- adrenergic stimulation. J B i o l Chem 258:8769-8773 Cheatum, S.G. and Warren, J.C. 1966 P u r i f i c a t i o n and properties for 3-beta-hydroxysteroid dehydrogenase and 3-ketosteroids isomerase from bovine corpora lutea. Biochim Biophys Acta 122:1-6 Clapper, D.A. and Conn, P.M. 1985 GnRH stimulation of p i t u i t a r y gonadotrope c e l l s produces an increase i n i n t r a c e l l u l a r calcium. B i o l Reprod 32:269-278 Claret-Berthon, B., Claret, M. and Mazet, J.L. 1977 Fluxes and d i s t r i b u t i o n of calcium i n rat l i v e r c e l l s : K i n e t i c analysis and i d e n t i f i c a t i o n of pools. J Physiol 272:529-552 Clark, M.R. 1982 Stimulation of progesterone and prostaglandin E2 accumulation by l u t e i n i z i n g hormone-releasing hormone (LHRH) and LHRH analogs i n rat granulosa c e l l s . Endocrinology 110:146- 152 Clark, M.R., Marsh, J.M. and LeMaire, W.J. 1978 Mechanism of l u t e i n i z i n g hormone regulation of prostaglandin synthesis i n ra t granulosa c e l l s . J B i o l Chem 253:7757-7761 Clark, M.R., Thibier, C , Marsh, J.M. and LeMaire, M. 1980 Stimulation of prostaglandin accumulation by l u t e i n i z i n g 203 hormone-releasing hormone (LHRH) and LHRH analogs i n rat granulosa c e l l s i n v i t r o . Endocrinology 107:17-23 Clayton, R.N., Harwood, J.P. and Catt, K.J. 1979 Gonadotropin- releasing hormone analogue binds to l u t e a l c e l l s and i n h i b i t s progesterone production. Nature 282:90-92 Clayton, R.N. and Catt, K.J. 1981 Gonadotropin-releasing hormone receptors: characterization, p h y s i o l o g i c a l regulation, and r e l a t i o n s h i p to reproductive function. Endocrine Rev 2:186- 209 Clayton, R.N. and Huhtaniemi, I.T. 1982 Absence of gonadotropin-releasing hormone receptors i n human gonadal t i s s u e . Nature 299:56-59 Condon, W.A. and Black, D.L 1976 Catecholamine-induced stimulation of progesterone by the bovine corpus luteum i n v i t r o . B i o l Reprod 15:573-578 Conn, P.M., Ganong, B.R., Ebeling, J . , Staley, D., Neidel, J.E. and B e l l , R.M. 1985 Diacylglycerols release LH: Structure- a c t i v i t y r e l a t i o n s and protein kinase C. Biochem Biophys Res Comm 126:532-539 Conn, P.M., McArdle, C , Andrews, W.V. and Huckle, W. 1987 The molecular basis of gonadotropin-releasing hormone (GnRH) action i n the p i t u i t a r y gonadotrope. B i o l Reprod 36:17-35 Cooper, R.H., C o l l , K.E. and Williamson, J.R. 1985 D i f f e r e n t i a l e f f e c t s of phorbol ester on phenylephrine and vasopressin- induced Ca++ mobilization i n i s o l a t e d hepatocytes. J B i o l Chem 260:3281-3288 Corvera, S., Schwarz, K.R., Graham, R.M. and Garcia-Sainz, J.A. 1986 Phorbol ester i n h i b i t s alpha 1-adrenergic e f f e c t s and decrease the a f f i n i t y of l i v e r c e l l alpha 1-adrenergic receptors for (-)-epinephrine. J B i o l Chem 261:520-526 Crawford, M.A. 1983 Essen t i a l f a t t y acids and t h e i r prostanoid de r i v a t i v e s . Br Med B u l l 39:210-213 Cu l l e r , M.D., T a r l a t z i s , B.C., Lightman, A., Fernandez, L.A., De-Cherney, A.H., Negro-Vilar, A. and Na f t o l i n , F. 1986 Angiotensin I I - l i k e immunoreactivity i n human f o l l i c u l a r f l u i d . J C l i n Endocrinol Metab 62:613-615 Daniel, S.A.J. and Armstrong, D.T. 1980 Enhancement of f o l l i c l e - s t i m u l a t i n g hormone-induced aromatase a c t i v i t y by androgens i n cultured r a t granulosa c e l l s . Endocrinology 107:1027-1033 Darbon, J.M., Knecht, M., Ranta, R., Dufau, M. and Catt, K.J. 204 1984 Hormonal regulation of c y c l i c AMP-dependent protein kinase i n cultured ovarian granulosa c e l l s . J B i o l Chem 259:14778- 14782 Davidson, J . , King, J.A. and M i l l a r , R.P. 1987 Luteinizing hormone release from chicken p i t u i t a r y c e l l s : synergism between calcium and protein kinase C and i t s i n h i b i t i o n by calmodulin antagonists. Endocrinology 120:692-699 Davis, J.S. and Clark, M.R. 1983 Acti v a t i o n of protein kinase C i n the bovine corpus luteum by phospholipids and Ca2+. Biochem J 214:569-574 Davis, J.S., Farese, R.V. and Clark, M.R. 1983 Gonadotropin- releasing hormone (GnRH) stimulates phosphatidylinositol metabolism i n r a t granulosa c e l l s : mechanism of action of GnRH. Proc Natl Acad S c i USA 80:2049-2053 Davis, J.S., Weakland, L.L., West, L.A. and Farese, R.V. 1986 Lut e i n i z i n g hormone stimulates the formation of i n o s i t o l triphosphate and c y c l i c AMP i n rat granulosa c e l l s : evidence for phospholipase C generated second messengers i n the action of l u t e i n i z i n g hormone. Biochem J 238:597-604 Davis, J.S., West, L.A. and Farese, R.V. 1986 Gonadotropin- releasing hormone (GnRH) r a p i d l y stimulates the formation of 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 r a t granulosa c e l l s : further evidence f o r the involvement of Ca++ and PKC i n the action of GnRH. Endocrinology 118:2561-2571 Davis, J.S., Weakland, L.L., Farese, R.V. and West, L.A. 1987 Lutei n i z i n g hormone increases i n o s i t o l triphosphate and c y t o s o l i c free Ca2+ i n i s o l a t e d bovine l u t e a l c e l l s . J B i o l Chem 262:8515-8521 De Chaffoy de Courcelles , D., Roevens, P. and Van Belle, H. 1984 l - o l e o y l - 2 - a c e t y l - g l y c e r o l (OAG) stimulates the formation of phosphatidylinositol 4-phosphate i n i n t a c t human p l a t e l e t s . Biochem Biophys Res Commun 123:589-595 Dekel, N. and Beers, W.H. 1978 Rat oocyte maturation i n v i t r o : r e l i e f of c y c l i c AMP i n h i b i t i o n s by gonadotropins. C e l l B i o l 75:4369-4373 Dekel, N. and Aberdam, E. 1985 Activators of protein kinase C stimulate meiotic maturation of rat oocytes. Biochem Biophys Res Commun 132:570-574 Dekel, N., Lawrence, T.S., G i l u l a , N.B. and Beers, W.H. 1981 Modulation of c e l l - t o - c e l l communication i n the cumulus-oocyte complex and the regulation of oocyte maturation by LH. Dev B i o l 86:356-362 205 Dekel, N., Lewysohn, 0., Ayalon, D. and Hazum, E. 1988 Receptors for gonadotropin rel e a s i n g hormone are present i n rat oocytes. Endocrinology 123:1205-1207 Dekel, N., Sherizly, I., T s a f r i r i , A. and Naor, Z. 1982 A comparative study of the mechanism of action of LH and a GnRH analog on the ovary. B i o l Reprod 28:161-166 Dekel, N., Sherizly, I., T s a f r i r i , A. and Naor, Z. 1983 A comparative study of the mechanism of action of LH and a GnRH analog on the ovary. B i o l Reprod 28:161-166 Delbeke, D., Scammell, J.G. and Dannies, P.S. 1984 Differences i n calcium requirements f o r forskolin-induced release of p r o l a c t i n from normal p i t u i t a r y c e l l s and GH4C1 c e l l s i n culture. Endocrinology 114:1433-1400 Dennefors, B.L., Janson, P., Knutson, F. and Hamberger, L. 1980 Steroid production and responsiveness to gonadotropin i n is o l a t e d stromal t i s s u e of human nenopausal ovaries. Am J Obstet Gynecol 136:997-1002 Dexter, R.N., Fishman, L.M., Ney, R.L. and Liddle, G.W. 1967 In h i b i t i o n of adrenal c o r t i c o s t e r o i d synthesis by aminoglutethimide: studies on the mechanism of action. J C l i n Endocrinol Metab 27:473-480 Diaz-Infante, A., J r . , Wright, K.H. and Wallach, E.E. 1974 Ef f e c t s of indomethacin and prostaglandin F 2 a i r>ha o n o v u l a t i ° n and ovarian c o n t r a c t i l i t y i n the rabbit. Prostaglandins 5:567- 579 Dimino, M.J. and Campbell, M.D. 1976 Progesterone synthesis by l u t e a l mitochondria i n v i t r o . Pro Soc Exp B i o l Med 152:54-56 Dixon, J.F. and Hokin, L.E. 1984 Secretagogue-stimulated phosphatidylinositol breakdown i n the exocrine pancreas l i b e r a t e s arachidonic acid, s t e a r i c acid, and gly c e r o l by sequential actions of phospholipase C and d i g l y c e r o l l i p a s e . J B i o l Chem 259:14418-14425 Dorflinger, L.J., Albert, P.J., William, A.T. and Behrman, H.R. 1984 Calcium i s an i n h i b i t o r of l u t e i n i z i n g hormone-sensitive adenylate cyclase i n the l u t e a l c e l l . Endocrinology 114:1208- 1215 Dorrington, J.H. and Armstrong, D.T. 1979 E f f e c t of FSH on gonadal functions. Recent Prog Horm Res 35:301-342 Dorrington, J . , Mckeracher, H., Munshi, S. and Gore-langton, R. 1982 LHRH independently stimulates steroidogenic enzymes i n granulosa c e l l culture. Endocrinology 110 (Suppl):178 206 Downs, S.M. and Longo, F.J. 1982 E f f e c t s of Indomethacin on preovulatory f o l l i c l e s i n immature superovulated mice. Am J Anat 164: 265-274 Downs, S.M. and Longo, F.J. 1983 An u l t r a s t r u c t u r a l study of preovulatory a p i c a l development i n mouse ovarian f o l l i c l e s : E f f e c t s of indomethacin. Anat Rec 205: 159-168 Dunaif, A.E., Zimmerman, E.A., Friesen, H.G. and Frantz, A.G. 1982 I n t r a c e l l u l a r l o c a l i z a t i o n of p r o l a c t i n receptor and p r o l a c t i n i n the rat ovary by immunocytochemistry. Endocrinology 110:1465-1471 Eckstein, N., Eshel, A., E l i , Y., Ayalon, D. and Naor, Z. 1986 Calcium-dependent actions of gonadotropin-releasing hormone agonist and l u t e i n i z i n g hormone upon c y c l i c AMP and progesterone production i n r a t ovarian granulosa c e l l s Molecular and C e l l u l a r Endocrinology 47:91-98 Ekholm, C , Clark, M.R., Magnusson, C., Isaksson, 0. and LeMaire, W.J. 1982 Ovulation induction by a GnRH analog i n hypophysectomized rats involves prostaglandins. Endocrinology 110:288-290 Enyedi, A., Farago, Sarkadi, B. and +Gardos, G. 1984 C y c l i c AMP-dependent protein kinase and Ca -calmodulin stimulate the formation of polyphosphoinositides i n a sarcoplasmic reticulum preparation of rabbit heart. FEBS Lett 176:235-238 Erdo, S.L. and Lapis, E. 1982 B i c u c u l l i n e - s e n s i t i v e GABA receptors i n rat ovary. Eur J Pharmacol 85:243-246 Espey, L.L. 1980 Ovulation as an inflammatory reaction: a hypothesis. B i o l Reprod 22:73-106 Erickson G.F. 1978 Normal ovarian function. C l i n Obstet Gynecol 21:31-42 Fanjul, L.F., Ruiz de Galarreta, CM. and Hsueh, A.J.W. 1983 Progestin augmentation of gonadotropin-stimulated progesterone production by cultured rat granulosa c e l l s . Endocrinology 112:405-407 Farkas, G., Enyedi, A., Sarkadi, B. Gardos, G., Nagy, Z. and Farago, A. 1984 C y c l i c AMP-dependent protein kinase stimulates the phosphorylation of phosphatidylinositol to phosphatidylinositol 4-phosphate i n a plasma membrane preparation from p i g granulocytes. Biochem Biophys Res Commun 124:871-876 Farkash, Y., Timberg, R. and Orly, J . 1986 Preparation of antiserum to rat cytochrome P-450 cholesterol side chain cleavage, and i t s use for u l t r a s t r u c t u r a l l o c a l i z a t i o n of 207 immunoreactive enzyme by protein A-gold technique. Endocrinology 118:1353-1365 Farookhi, R. 1980 Ef f e c t s of androgen on induction of gonadotropin receptors and gonadotropin-stimulated adenosine 35'-monophosphate production i n rat ovarian granulosa c e l l s . Endocrinology 106:1216-1223 Flower, R.J. and Vane, J.R. 1974 Some pharmacologic and biochemical aspects of prostaglandin biosynthesis and i t s i n h i b i t i o n , In: Robinson, H.J. and Vane, J.R. (eds) , Prostaglandin Synthesis I n h i b i t o r s . Raven Press, New York, pp. 9-18 Folch, J . , Lees, M. and Sloane-Stanley, G.H. 1957 A simple method f o r the i s o l a t i o n and p u r i f i c a t i o n of t o t a l l i p i d s from animal t i s s u e . J B i o l Chem 226:497-509 Forder, J . , Scriabine, A. and Rasmussen, H. 1985 Plasma membrane calcium flux, protein kinase C ac t i v a t i o n , and smooth muscle contraction. J Pharmacol Exp Ther 235:267-273 Forkert, V.W., Yunis, M. and Schlondorff, D. 1984 Prostaglandin synthesis linked to phosphatidylinositol turnover i n is o l a t e d r a t glomeruli. Biochem Biophys Acta 794:206-217 Fortune, J.E. and Armstrong, D.T. 1977 Androgen production by theca and granulosa i s o l a t e d from proestrous r a t f o l l i c l e s . Endocrinology 100:1341-1347 Fowler, R.E., Fox, N.L., Edwards, R.G., Walters, D.E. and Steptoe, P.C. 1978 Steroidogenesis by cultured granulosa c e l l s aspirated from human f o l l i c l e s using pregnenolone and andrgen as precursors. J Endocrinol 77:171-183 Fukuda, S., Terakawa, N., Sato, B., Imori, T. and Matsumoto, K. 1979 Hormonal regulation of a c t i v i t i e s of 17beta- dehydrogenases, aromatase and 4-ene-5alpha-reductase i n immature ovaries. J Steroid Biochem 11:1421-1427 Garrison, J.C., Johnsen, D.E. and Campanile, C P . 1984 Evidence for the ro l e of phosphorylase kinase, protein kinase C, and other Ca -s e n s i t i v e protein kinases i n the response of hepatocytes to angiotensin II and vasopressin. J B i o l Chem 259:3283-3297 Geison, R.L., Banschbach, M.W., Sabeghian, K. and Hokin- Neaverson, M. 1976 Acetylcholine stimulation of se l e c t i v e increase i n s t e a r i c acid and arachidonic acids i n phosphatidic acid i n mouse pancreas. Biochem Biophys Res Comm 68:343-349 Gick, G.G. and Bancroft, C. 1985 Regulation by calcium of p r o l a c t i n and growth hormone mRNA sequences i n primary cultures of rat p i t u i t a r y c e l l s . J B i o l Chem 259:10675-10681 208 Gilman, A.G. 1984 Guanine nucleotide-binding regulatory proteins and dual control of adenylate cyclase. J C l i n Invest 73:1-3 Gilman, A.G. 1987 G proteins:transducters of receptor generated signals. Annu Rev Biochem 56:615-649 Goff, A.K. and Armstrong, D.T. 1977 Stimulatory action of gonadotropins and prostaglandins on adenosine 3',5"- monophosphate production by i s o l a t e d r a t granulosa c e l l s . Endocrinology 101:1461-1467 Goff, A.K. and Henderson, K.M. 1979 Changes i n f o l l i c u l a r f l u i d and serum concentrations of steroids i n PMSG treated immature rats following LH administration. B i o l Reprod 20:1153-1157 Goff, A.K., Leung, P.C.K. and Armstrong, D.T. 1979 Stimulatory e f f e c t of f o l l i c l e - s t i m u l a t i n g hormone and androgens on the responsiveness of r a t granulosa c e l l s to gonadotropins i n v i t r o . Endocrinology 104:1124-1129 Goldring, N.B. and Orly, G. 1985 Concerted metabolism of steroid hormone produced by co-cultured ovarian c e l l types. J B i o l Chem 260:913-920 Goodman, A. and Hodgen, G. 1979 Between-ovary i n t e r a c t i o n i n the regulation of f o l l i c l e growth: Corpus luteum function and gonadotropin secretion i n the primate ovarian cycle. Endocrinology 104:1310-1316 Gore-Langton, R.E., Lacroix, M. and Dorrington, J.H. 1981 D i f f e r e n t i a l e f f e c t s of l u t e i n i z i n g hormone-releasing hormone on f o l l i c l e - s t i m u l a t i n g hormone-dependent responses i n rat granulosa c e l l s and S e r t o l i c e l l s i n v i t r o . Endocrinology 108:812-818 Gospodarowicz, D., I I I , C.R. and Birdwell, C.R. 1977 E f f e c t of fi b r o b l a s t and epidermal growth factors on ovarian c e l l p r o l i f e r a t i o n i n v i t r o . I. Characterization of the response of granulosa c e l l s f o r FGF and EGF. Endocrinology 100:1108-1120 Gospodarowicz, D., I l l , C.R. and Birdwell, C.R. 1977 E f f e c t of f i b r o b l a s t and epidermal growth factors on ovarian c e l l p r o l i f e r a t i o n i n v i t r o . I I . P r o l i f e r a t i v e response of l u t e a l c e l l s to FGF but not EGF. Endocrinology 100:1121-1128 Gospodarowicz, D. and B i a l e c k i , I. 1979 Fibroblast and epidermal growth factors are mitogenic agents f o r cultured granulosa c e l l s of rodent, porcine and human o r i g i n . Endocrinology 104:757-764 Greenwald, G.S. 1974 Gonadotropin regulation of f o l l i c u l a r development. In: Gonadotropins and Gonadal Function, edited by N.R. Moudgal. pp. 205-212 209 Grynkiewicz, G., Poenie, M. and Tsien, Y. 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Bio l Chem 260:3440-3450 Gwynne, J.T. and Strauss I I I , J.F. 1982 The r o l e of lipoproteins i n steroidogenesis and cholesterol metabolism i n steroidogenic glands. Endocr Rev 3:299-329 Habermann, E. 1972 Bee and wasp venoms. Science 177:314-322 Haksar, A. and Romanoff, E.B. 1971 Electron transport and progesterone biosynthesis i n the bovine corpus luteum homogenate. Biochem Biophys Res Commun 43:246-251 Hamberger, L., H i l l e n s j o , T. and Ahren, K. 1978 Steroidogenesis i n i s o l a t e d c e l l s of preovulatory r a t f o l l i c l e s . Endocrinology 103:771-777 Handwerger, S., Barry, S., Barrett, J . , Markoff, E., Z e i t l e r , P., Cwikei, B. and Siegel, M. 1981 In h i b i t i o n of the synthesis and secretion of decidual p r o l a c t i n by arachidonic acid. Endocrinology 109:2016-2021 Hansson, A., Serhan, C.N., Haeggstrom, J . , Ingelman-Sundberg, M. and Samuelsson, B. 1986 Ac t i v a t i o n of protein kinase C by l i p o x i n A and other eicosanoids. I n t r a c e l l u l a r action of oxygenation products of arachidonic acid. Biochem Biophys Res Commun 134:1215-1222 Harman, S.M., Louvet, J-P. and Ross, G.T. 1975 Interaction of estrogen and gonadotrophins on f o l l i c u l a r a t r e s i a . Endocrinology 96:1145-1152 Harris, C.E., Staley, D. and Conn, P.M. 1985 Diacylglycerols and protein kinase C: po t e n t i a l amplifying mechanism for Ca- mediated gonadotropin-releasing hormone-stimulated l u t e i n i z i n g hormone release. Molecular Pharmacology 27:532-536 Hazum, E. and Nimrod, A. 1982 Pho t o a f f i n i t y - l a b e l i n g and fluorescence d i s t r i b u t i o n studies on GnRH receptors i n ovarian granulosa c e l l s . Proc Natl Acad S c i USA 79:1747-1750 Hazum, E. and Schvartz, I. 1984 Photoaffinity l a b e l i n g of gonadotropin releasing hormone receptors i n control and desensitized p i t u i t a r y c e l l s . Biochem Biophys Res Commun 125:532-538 Hedin, L., Gaddy-Kurten, G., Kurten, R., DeWitt, D.L., Smith, W.L. and Richards, J.S. 1987 Prostaglandin endoperoxide (PGH) synthase i n rat ovarian f o l l i c l e s : Content, c e l l u l a r d i s t r i b u t i o n and evidence f o r hormonal induction preceding 210 ovulation. Endocrinology 121:722-731 Hidaka, H., Inagaki, M., Kawamoto, S. and Sasaki, Y. 1984 Isoquinoline-sulfonamides, novel and potent i n h i b i t o r s of c y c l i c nucleotide dependent protein kinase and protein kinase C. Biochem 23:5036-5041 Higuchi, T., Kaneko, A. Abel, J.H. and Niswender, G.D. 1976 Relationship between membrane p o t e n t i a l and progesterone release i n ovine corpora l u t e a l . Endocrinology 99:1023-1039 H i l l e n s j o , T. and LeMaire, W.J. 1980 Gonadotropin-releasing hormone agonist stimulate meiotic maturation of f o l l i c l e - enclosed r a t oocytes i n v i t r o . Nature 287:145-146 H i l l e n s j o , T., LeMaire, W.J., Clark, M.R. and Ahren, K. 1982 E f f e c t of GnRH and GnRH agonists upon accumulation of progesterone, cAMP and prostaglandin i n iso l a t e d preovulatory r a t f o l l i c l e s . Acta Endocrinol Copenh 101:603-610 H i l l i e r , S.G., Zeleznik, A.J., Knazek, R.A. and Ross, G.T. 1980 Hormonal regulation of preovulatory f o l l i c l e maturation i n the r a t . J Reprod F e r t i l 60:219-229 H i r s t , J . J . , Ri i e , G.E., Jenkins, G. and Thorburn, G.D. 1988 Control of oxytoxin secretion by ovine corpora lutea: e f f e c t s of arachidonic acid, phospholipases, and prostaglandins. Endocrinology 122:774-781 H o l l , R.W., Thorner, M.O. and Leong, D.A. 1988 I n t r a c e l l u l a r calcium concentration and growth hormone secretion i n in d i v i d u a l somatotropes: e f f e c t s of growth hormone releasing fa c t o r (GRF) and somatostatin (SRIF). Endocrinology 122:2927- 2932 Holt, J.A., Richards, J.S., Midgley, A.R., J r . and Reichert L.E., J r . 1976 E f f e c t of p r o l a c t i n on LH receptor i n rat l u t e a l c e l l s . Endocrinology 98:1005-1013 Hopkins, C.R. and Gregory, H. 1977 Topographical l o c a l i z a t i o n of the receptors for l u t e i n i z i n g hormone-releasing hormone on the surface of dissociated p i t u i t a r y c e l l s . J C e l l B i o l 75:528- 540 Hsueh, A.J.W. and Erickson, G.F. 1979 Ext r a p i t u i t a r y action of gonadotropin-releasing hormone: d i r e c t i n h i b i t i o n of ovarian steroidogenesis. Science 204:854-855 Hsueh, A.J.W. and Ling, N.C. 1979 E f f e c t of an antagonistic analog of gonadotropin-releasing hormone upon ovarian granulosa c e l l function. L i f e S c i 25:1223-1230 Hsueh, A.J.W., Wang, C. and Erickson, G.F. 1980 Direct 211 i n h i b i t o r y e f f e c t of gonadotropin-releasing hormone upon f o l l i c l e - s t i m u l a t i n g hormone induction of l u t e i n i z i n g hormone receptor and aromatase a c t i v i t y i n r a t granulosa c e l l s . Endocrinology 106:1697-1705 Hsueh, A.J.W. and Jones, P.B.C. 1981 E x t r a p i t u i t a r y actions of gonadotropin-releasing hormone. Endocr Rev 2:186-209 Hsueh, A.J.W. and Schaeffer, J.M. 1985 Gonadotropin-releasing hormone as a paracrine hormone and neurotransmitter i n ex t r a p i t u i t a r y s i t e s . J Steroid Biochem 23:757-764 Huckle, W. and Conn, P.M. 1987 The r e l a t i o n s h i p between gonadotropin-releasing hormone-stimulated l u t e i n i z i n g hormone release and i n o s i t o l phosphate production: studies with calcium antagonists and protein kinase C a c t i v a t o r s . Endocrinology 120:160-69 Hulting, A-L., Lindgren, J.A., Hokfelt, T., Eneroth, P., Werner, S., Patrono, C. and Samuelsson, B. 1985 Leukotriene C4 as a mediator of l u t e i n i z i n g hormone release from r a t anterior p i t u i t a r y c e l l s . Proc Natl Acad S c i USA 82:3834-3838 Husain, A., Bumpus, F.M., De S i l v a , P. and Speth, R.C. 1987 Loc a l i z a t i o n of angiotensin II receptors i n ovarian f o l l i c l e s and the i d e n t i f i c a t i o n of angiotensin II i n r a t ovaries. Proc Natl Acad S c i USA 84:2489-2493 Ingraham, D.L. 1959 The e f f e c t of oestrogen on the atr e s i a of ovarian f o l l i c l e s . J Endocrinol 19:123-125 Irvine, R.F. 1982 How i s the l e v e l of free arachidonic acid controlled i n mammalian c e l l s . Biochem J 204:3-16 Irvine, R.F., Brown, K.D. and Berridge, M.J. 1984 S p e c i f i c i t y of i n o s i t o l trisphosphate-induced calcium release from permeablized Swiss-mouse 3T3 c e l l s . Biochem J 222:269-272 Irvine, R.F., Letcher, A.J., Lands, D.J. and Downes, P. 1984 In o s i t o l trisphosphates i n carbachol-stimulated r a t parotid glands. Biochem J 223:237-243 Jacobs, B.R. and Smith, R.G. 1980 Evidence f o r a receptor-like protein f o r progesterone i n bovine ovarian cytosol. Endocrinology 106:1276-1282 components involved i n adenylate cyclase i n h i b i t i o n by hormones. Adv C y c l i c Nucleotide Protein Phosphorylat Res 17:135-143 Jones, P.B.C. and Hsueh, A.J.W. 1980 Direct i n h i b i t o r y effect of gonadotropin-releasing hormone upon l u t e a l l u t e i n i z i n g hormone receptor and steroidogenesis i n hypophysectomized rats. 212 components involved. i n adenylate cyclase i n h i b i t i o n by hormones. Adv C y c l i c Nucleotide Protein Phosphorylat Res 17:135-143 Jones, P.B.C. and Hsueh, A.J.W. 1980 Direct i n h i b i t o r y e f f e c t of gonadotropin-releasing hormone upon l u t e a l l u t e i n i z i n g hormone receptor and steroidogenesis i n hypophysectomized r a t s . Endocrinology 107:1930-1936 Jones, P.B.C. and Hsueh, A.J.W. 1981a Direct stimulation of ovarian progesterone-metabolizing enzyme by gonadotropin- releasing hormone i n cultured granulosa c e l l s . J B i o l Chem 256:1248-1254 Jones, P.B.C. and Hsueh, A.J.W. 1981b Direct e f f e c t s of gonadotropin-releasing hormone and i t s antagonist upon ovarian functions stimulated by FSH, p r o l a c t i n , and LH. B i o l Reprod 24:747-759 Jones, P.B.C. and Hsueh, A.J.W. 1982a Pregnenolone biosynthesis by cultured granulosa c e l l s : modulation by f o l l i c l e - s t i m u l a t i n g hormone and gonadotropin-releasing hormone. Endocrinology 111:713-721 Jones, P.B.C. and Hsueh, A. J . W. 1982b Regulation of ovarian 3 -hydroxysteroid dehydrogenase a c t i v i t y by gonadotropin- releasing hormone and F o l l i c l e - s t i m u l a t i n g Hormone i n cultured r a t granulosa c e l l s Endocrinology 1982:1663-1671 Judd, A.M., J a r v i s , W.D. and MacLeod, R.M. 1987 Attenuation of p i t u i t a r y polyphos[hoinositide metabolitsm by protein kinase C ac t i v a t i o n . Molecul C e l l u l Endocrinol 54:107-114 Kaibuchi,KY., Takai, Y, Ogawa, Y, Kimura, S, Nishizuka, Y, Nakamura, T, Tomomura, A and Ichihara, A. 1981 Inhibitory action of adenosine-3 1,5 1-monophosphate on phosphatidylinositol tunrover: difference i n t i s s u e response. Biochem Biophys Res Commun 104:105-112 Kasson, B.G., Conn, P.M. and Hsueh, A.J.W. 1985 I n h i b i t i o n of granulosa c e l l d i f f e r e n t i a t i o n by dioctanoylglycerol-a novel a c t i v a t o r of protein kinase C Molecul c e l l u l Endocrinol 42:29- 37 Kasson, B.G., Median, R., Davoren, J.B. and Huseh, A.J.W. 1985 I d e n t i f i c a t i o n of subpopulations of rat granulosa c e l l s : sedimentation properties and hormone responsiveness. Endocrinology 117:1027-1034 Katz, Y. and Armstrong, D.T. 1976 In h i b i t i o n of ovarian e s t r a d i o l secretion by l u t e i n i z i n g hormone i n prepubertal, pregnant mare serum-treated r a t s . Endocrinology 99:1442-1447 213 Kautsky, M.P. and Hagerman, D.D. 1980 K i n e t i c properties of ste r o i d 19-hydroxylase and estrogen synthetase from porcine ovary microsomes. J Steroid Biochem 13:1283-1290 Kawai, Y. and Clark, M.R. 1985 Phorbol ester regulation of rat granulosa c e l l prostaglandin and progesterone accumulation Endocrinology 116:2320-2326 Kawai, Y. and Clark, M.R. 1986 Mechanism of action of gonadotropin r e l e a s i i n g hormone on r a t granulosa c e l l s . Endocr Res 12:195-209 Kennedy, T.G. 1979 Prostaglandin and increased endometrial vascular permeability r e s u l t i n g from the ap p l i c a t i o n of an a r t i f i c i a l stimulus to the uterus of the r a t s e n s i t i z e d for the decidual c e l l reaction. B i o l Reprod 20:560-566 Kies e l , L., Prz y l i p i a k , A., Rabe, T. and Runnebaum, B. 1986 The ro l e of novel arachidonic acid metabolites i n GnRH action on gonadotrophin release i n v i t r o . Human Reprod 1:349-352 Ki e s e l , L., Pr z y l i p i a k , A., Rabe, T., Pr z y l i p i a k , M. and Runnebaum, B. 1987 Arachidonic a c i d and i t s lipoxygenase metabolites stimulate p r o l a c t i n release i n superfused p i t u i t a r y c e l l s . Human Reprod 2:281-285 Kies e l , L., Prz y l i p i a k , A., Rabe, T. and Runnebaum, B. 1987 Leukotrienes stimulate gonadotropin release i n v i t r o . Gynecol Endocrinol 1:23-35 Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. 1980 Actions of calcium and phospholipid-dependent protein kinase by d i a c y l g l y c e r o l , i t s possible r e l a t i o n to phosphatidylinositol turnover. J B i o l Chem 255:2273-2276 Knecht, M. and Catt, K. 1981 Gonadotropin-releasing hormone: regulation of adenosine 3 1,5 1-monophosphate i n ovarian granulosa c e l l s Science 214:1346-1348 Knecht, M., Katz, M. and Catt, K.J. 1981 GnRH i n h i b i t s c y c l i c nucleotide accumulation i n cultured r a t granulosa c e l l s . J B i o l Chem 256:34-36 Knecht, M., Amsterdam, A. and Catt, K. 1982 Inh i b i t i o n of granulosa c e l l d i f f e r e n t i a t i o n by gonadotropin-releasing hormone. Endocrinology 110:865-872 Knecht, M., Ranta, T., Naor, Z. and Catt, K.J. 1983 Direct e f f e c t s of GnRH on the ovary. In: Factors regulating Ovarian Function, edited by G.S. Greenwald and P.F. Terranova. pp. 225- 243 Knecht, M., Ranta, T., Feng, P., Shinohara, O. and Catt, K.J. 214 1985 Gonadotropin-releasing hormone as a modulator of ovarian functions. J Steroid Biochem 23:771-778 Kojima, I., Kojima, K. +and Rasmussen, H. 1985 Charact e r i s t i c s of angiotensin I I - , K -, and ACTH-induced calcium i n f l u x i n adrenal glomerulosa c e l l s . Evidence that angiotensin I I , K+-, and ACTH may open a common calcium channel. J B i o l Chem 260:9177-9184 Kolena, J . and Channing C P . 1972 Stimulatory e f f e c t s of LH, FSH and prostaglandin upon 3•,51-AMP l e v e l s i n porcine granulosa c e l l s . Endocrinology 90:1543-1550 Kolesnick, R.N., Musacchio, I. and Thaw, C. 1984 Arachidonic a c i d mobilizes calcium and stimulates p r o l a c t i n secretion from GH3 c e l l s . Am J Physiol 246:E458-462 Koos, R. and Clark, M.R. 1982 Production of 6-keto- prostaglandin F-l-alpha by r a t granulosa c e l l s i n v i t r o . Endocrinology 11:1513-1518 Koos, R.D. and LeMaire, W.J. 1983 Factors that may regulate the growth anf regression of blood vessels i n the ovary. Sem Reprod Endocrinol 1:295-307 Koos, R.D. and LeMaire, W.J. 1985 The e f f e c t s of gonadotropin- releasing hormone agonist on ovulation and steroidogenesis during perfusion of rabbit and r a t ovaries i n v i t r o . Endocrinology 116:628-632 Kraft, A.S., Anderson, W.B., Cooper, H.L. and Sando, J . J . 1982 Decreases i n c y t o s o l i c calcium/phospholipid-dependent protein kinase a c t i v i t y following phorbol ester treatment of EL4 thymoma c e l l s . J B i o l Chem 257:3193-3196 Lacroix, E., Eechaute, W and Leusen, I. 1974 The biosynthesis of oestrogen by cow f o l l i c l e s . Steroids 23:337-356 Lahav, M., West, L.A. and Davis, J.S. 1988 E f f e c t of prostaglandin F2alpha and a gonadotropin-releasing hormone agonist on inosotol phospholipid metabolism i n i s o l a t e d r a t corpora lutea of various ages Endocrinology 123:1044-1052 Lapetina, E.G., B i l l a h , M.M. and Cuatrecasa, P. 1981 The phosphadidylinositol cycle and the regulation of arachidonic acid production. Nature 292:367-369 Lapetina, E.G., B i l l a h , M.M. and Cuatrecasas, P. 1981 The i n i t i a l actions of thrombin on p l a t e l e t s . Conversion of phosphatidylinositol to phosphatidic acid preceding the production of arachidonic acid. J B i o l Chem 265:5037-5040 Lawrence, J r . I.E. and Burden, H.W. 1980 The o r i g i n of the 215 e x t r i n s i c adrenergic innervation to the r a t ovary. Anat Rec 196:51-59 Leaver, H.A. and Boyd, G.S. 1981 Control of steroidogenesis i n ra t corpus luteum: the rate of access of cho l e s t e r o l to the active s i t e of the c h o l e s t e r o l side-chian cleavage enzyme. J Endocrinol 91:123-133 Lehninger, A.L. 1970 Mitochondrial and calcium ion transport. Biochem J 119:129-138 LeMaire, W.J. and Marsh, J.M. 1975 Interrelationships between prostaglandins, c y c l i c AMP and steroids i n ovulation. J Reprod F e r t i l (Suppl) 22:53-74 Leung, P.C.K., Goff, A.K., Kennedy, T.G. and Armstrong, D.T. 1978 An intraovarian i n h i b i t o r y action of estrogen and androgen production i n vivo. B i o l Reprod 19:641-647 Leung, P.C.K. and Armstrong, D.T. 1979 Estrogen treatment of immature rats i n h i b i t s ovarian androgen production i n v i t r o . Endocrinology 104:1411-1417 Leung, P.C.K. 1985 Mechanism of gonadotropin-releasing hormone and prostaglandin action on l u t e a l c e l l s . Can J Physiol Pharmacol 63:249-256 Leung, P.C.K., Minegishi, T., Ma, F., Zhou, F.Z. and Ho Yuen, B. 1986 Induction of polyphosphoinositide breakdown i n rat corpus luteum by prostaglandin F2a. Endocrinology 119:12-18 Leung, P.C.K., Wang, J . and Baimbridge, G.K. 1989 Role of calcium i n the action of LHRH on ovarian progesterone production. VII Ovarian Workshop (in press) Leung, P.K.C., Raymond, V. and Labrie, F. 1983 Stimulation of phosphatidic acid and phosphatidylinositol l a b e l i n g i n l u t e a l c e l l s by l u t e i n i z i n g hormone-releasing hormone. Endocrinology 112:1138-1140 Leung, P.K.C., Minegishi, T. and Wang, J . 1988 I n h i b i t i o n of FSH and c y c l i c adenosine-3',5•-monophosphate induced progesterone production by calcium and protein kinase C i n the ra t ovary. Am J Obstet Gynecol 158:350-356 L i , C.H., Yamashiro, R.D. and Chung, D. 1987 Gonadotropin- releasing peptide from human f o l l i c u l a r f l u i d : i s o l a t i o n , characterization, and chemical synthesis. Proc Natl Acad Sci USA 84:959-962 Liebeman, S., Greenfield, N.J. and Wolfson, A. 1984 A h e u r i s t i c proposal f o r understanding steroidogenesis process. Endocr Rev 5:128-148 216 Limor, R., Ayalon, D., Capponi, A.M, Childs, G.V. and Naor, Z. 1987 C y t o s o l i c free calcium l e v e l s i n cultured p i t u i t a r y c e l l s separated by c e n t r i f i g a l e l u t r i a t i o n : e f f e c t of gonadotropin- rele a s i n g hormone. Endocrinology 120:497-503 Lin, T. 1985 Mechanism of action of gonadotropin-releasing hormone-stimulated Leydig c e l l steroidogenesis. I I I . The r o l e of arachidonic acid and calcium/phospholipid dependent protein kinase. L i f e S c i 36:1255-1264 Lindner, H.R., T s a f r i r i , A., Lieberman, M.E., Zor, U., Koch, Y., Bauminger, S. and Barne, A. 1974 Gonadotropin action on cultured granulosa c e l l s : Induction of maturationdivision of the mammalian oocyte and d i f f e r e n t i a t i o n of the l u t e a l c e l l . Rec Prog Hormone Res 30:79-138 Lindner, H.R., Zor, U., Kohen, F., Bauminger, S., Amsterdam, A., Lahar, M. and Salomon, Y. 1980 Significance of prostaglandins i n the regulation of c y c l i c events i n the ovary and uterus. Adv Prostaglandin Thromboxane Leukotriene Res 8:1371-1390 Lino, J . , Baranao, S. and Hammond, J.M. 1985 Multihormone regulation of steroidogenesis i n cultured porcine granulosa c e l l s : studies i n serum-free medium. Endocrinology 116:2143- 2151 Lynch, C.J., Charest, R., Bocckino, S.B., Exton, J.H. and Blackmore, P.F. 1985 I n h i b i t i o n of hepatic al-adrenergic e f f e c t s and binding by phorbol myristate acetate. J B i o l Chem 260:2844-2851 Ma, F. and Leung, P.C.K. 1985 L u t e i n i z i n g hormone-releasing hormone enhances polyphosphoinositide breakdown i n r a t granulosa c e l l s . Biochem Biophys Res Commun 130:1201-1208 Madej, E. 1980 E f f e c t of exogenous hormones on the a c t i c i t y of deltas,3beta-hydroxysteroid dehydrogenase i n cultured granulosa c e l l s from proestrous and preovulatory r a t ovarian f o l l i c l e s . Acta Histochem 67:253-260 Magnusson, C. and LeMaire, W.J. 1981 A gonadotropin-releasing hormone agonist stimulates oxygen consumption and maturation of f o l l i c l e - e n c l o s e d r at oocytes i n v i t r o . Acta Physiol Scand 111:377-379 Magoffin, D.A., Reynolds, D.S. and Erickson, G.F. 1981 Direct i n h i b i t o r y e f f e c t of GnRH on androgen secretion by ovarian i n t e r s t i t i a l c e l l s . Endocrinology 109:661-663 Magoffin, D.A. and Erickson, G.F. 1982 Mechanism by which GnRH i n h i b i t s androgen synthesis d i r e c t l y i n ovarian i n t e r s t i t i a l c e l l s . Mol C e l l Endocrinol 27:191-198 217 Makris, A. and Ryan, K.J. 1975 Progesterone, androstenedione, testosterone, estrone, and e s t r a d i o l synthesis i n hamster ovarian f o l l i c l e c e l l s . Endocrinology 96:694-701 Makris, A. and Ryan, K.J. 1980 The source of f o l l i c u l a r androgen i n the hamster f o l l i c l e . Steroids 35:53-64 Marchetti, B. and Cioni, M. 1988 Opposite changes of p i t u i t a r y and ovarian receptors for LHRH i n ageing r a t s : further evidence fo r a d i r e c t neural control of ovarian LHRH receptor a c t i v i t y . Neuroendocrinology 48:242-251 Marian, J . and Conn, P.M. 1983 Subcellular l o c a l i z a t i o n of the receptor for gonadotropin-releasing hormone i n p i t u i t a r y and ovarian t i s s u e . Endocrinology 112:104-112 Marsh, J.M. and Savard, K. 1966 The stimulation of progesterone synthesis i n bovine corpora lutea by adenosine-3 1,5'- monophosphate. Steroids 8:133-148 Marsh, J.M., Yang, N.S.T. and LeMaire, W.J. 1974 Stimulation of prostaglandin synthesis by LH i n graafian f o l l i c l e s i n v i t r o . Endocrinology (suppl) 56:A109 Mason, N.R., Schaffer, R.J. and Toomey, R.E. 1973 Stimulation of c y c l i c AMP accumulation i n rat ovaries i n v i t r o . Endocrinology 93:34-41 Massicotte, J . , Veilleux, R., Lavoie, M. and Labrie, F. 1980 An LHRH agonist i n h i b i t s FSH-induced c y c l i c AMP accumulation and steroidogenesis i n porcine granulosa c e l l s i n culture. Biochem Biophys Res Commun 94:1362-1366 McNatty, K.P., Sawers, R.S. and McNeilly, A.S. 1974 A possible r o l e for p r o l a c t i n i n control of steroi d secretion by the human Graafian f o l l i c l e . Nature 250:635-655 McPhail, L.C., Clayton, C.C. and Snyderman, R. 1984 A potential second messenger ro l e for unsaturated f a t t y acids: actions of Ca2+-dependent protein kinase. Science 224:622-625 Milvae, R.A., Murphy, B.D. and Hansel, W. 1984 Prolongation of the bovine estrous cycle with a gonadotropin-releasing hormone analog. B i o l Reprod 31:664-670 Milvae, R.A., A l i l a , H.W. and Hansel, W. 1986 Involvement of lipoxygenase products of arachidonic acid metabolism i n bovine l u t e a l function. B i o l Reprod 35:1210-1215 Minegishi, T. and Leung, P.C.K. 1985 Lu t e i n i z i n g hormone- releasing hormone stimulates arachidonic a c i d release i n rat granulosa c e l l s . Endocrinology 117:2001-2007 218 Minegishi, T., Wang, J . and Leung, P.C.K. 1987 L u t e i n i z i n g hormone-releasing hormone (LHRH)-induced arachidonic acid release i n rat granulosa c e l l s : r ole of calcium and protein kinase C. FEBS Lett 214:139-142 Mohsin, S. and Pennefather, J.N. 1979 The sympathetic innervation of the mammalian ovary. A review of pharmacological and h i s t o l o g i c a l studies. C l i n Exp Pharm Physiol 6:335-354 Monaco, L., Adamo, S. and Conti, M. 1988 F o l l i c l e - s t i m u l a t i n g hormone modulation of phosphoinositide turnover i n the immature r a t S e r t o l i c e l l i n culture. Endocrinology 123:2032-2039 Moon, Y.S. and Duleba, A.J. 1982 Comparative studies of androgen metabolism i n thecr and granulosa c e l l s of human f o l l i c l e s i n v i t r o . Steroids 39:419-430 Morris, H.R., Taylor, G.W., Jones, CM., Piper, P.H., Samhoun, M.N. and Tippins, J.R. 1982 Slow reacting substances (leukotrienes): enzymes involved i n t h e i r biosynthesis. Proc Natl Acad S c i USA 79:4838-4842 Moyle, W.R., Kuczek, T. and Bailey, CA. 1985 Potential of a quantal Response as a mechanism for o s c i l l a t o r y behavior: Implications for our concepts of hormonal control mechanisms. B i o l Reprod 32:43-69 Murakami, k. and Routtenberg, A. 1985 Direct action of p u r i f i e d protein kinase C by unsaturated f a t t y acids (oleate and arachidonate) i n the absence of phospholipids and Ca2+. FEBS Lett 192:189-193 Murdoch, W.J., Dailey, R.A. and Inskeep, E.K. 1981 Preovulatory changes i n prostaglandins E2 and F2 i n ovine f o l l i c l e s . J Animal S c i 53:192-205 Naess, O. 1981 Characterization of cytoplasmic progesterone receptors i n r a t granulosa c e l l s . Evidence f o r nuclear translocation. Acta Endocrinol (Capenh) 98:288-294 Naor, Z. and Catt, K.J. 1981 Mechanism of action of gonadotropin-releasing hormone: involvement of phospholipid turnover i n l u t e i n i z i n g hormonr release. J B i o l Chem 256:2226- 2229 Naor, Z. and Yavin, E. 1982 Gonadotropin-releasing hormone stimulates phospholipid l a b e l i n g i n cultured granulosa c e l l s . Endocrinology 111:1615-1619 Naor, Z., Azrad, A., Limor, R., Zakut, H. and Lotan, M. 1986 Gonadotropin-releasing hormone activates a rapid Ca2+- independent phosphodiester hydrolysis of polyphosphoinositides i n p i t u i t a r y gonadotrophs. J B i o l Chem 261:12506-12512 219 Naor, Z., Vanderhoek, J.Y., Lindner, H.R. and Catt, K.H. 1983 Arachidonic acid products as possible mediators of the action of gonadotropin-releasing hormone. In: Advances i n Prostaglandin, Thromboxane, and Leukotriene Research 12:259-263 Navickis, R.J., Jones, P.B.C. and Hsueh, A.J.W. 1982 Modulation of p r o l a c t i n receptors i n cultured r a t granulosa c e l l s by FSH, LH and GnRH. Mol C e l l Endocrinol 27:77-88 Nimrod, A. 1977 Studies on the s y n e r g i s t i c e f f e c t of androgen on the stimulation of progestin secretion by FSH i n cultured r a t granulosa c e l l s : A search f o r the mechanism of action. Mol C e l l Endocrinol 8:201-211 Nimrod, A., Rosenfield, R.L. and Otto, P. 1980 Relationship of androgen action to androgen metabolism i n i s o l a t e d r a t granulosa c e l l s . J Steroid Biochem 13:1015-1019 Nishizuka, T. 1984 The r o l e of protein kinase C i n c e l l surface transduction and tumor promotion Nature 308:693-698 Nishizuka, Y., Takai, Y., Kishimoto, A., Kikkawa, U. and Kaibuichi, K. 1984 Phospholipid turnover i n hormone action. Recent Prog Horm Res 40:301-341 Nishizuka, Y. 1986 Studies and perspectives of protein kinase C. Science 233:305-312 Noland, T.A. and Dimino, M.J. 1986 Characterization and d i s t r i b u t i o n of protein kinase C i n ovarian t i s s u e . B i o l Reprod 35:863-872 O'Doherty, J . , Youmans, S.J., Armstrong, W. McD. and Stark, R.J. 1980 Calcium regulation during stimulus secretion coupling: continuous measurement of i n t r a c e l l u l a r calcium a c t i v i t e s . Science 209:510-513 Orellana, S.A., So l s k i , P.A. and Brown, L.H. 1985 Phorbol ester i n h i b i t s phosphoinositide hydrolysis and calcium mobilization i n cultured astrocytoma c e l l s . J B i o l Chem 260:5236-5239 Oron, Y., Dascal, N., Nadler, E. and Lupu, M. 1985 I n o s i t o l 1,4,5-triphosphate mimics muscarinic response i n Xenopus oocytes. Nature 313:141-143 Ossowski, L., Biegel, O. and Reich, E. 1979 Mammary plasminogen activ a t o r : Correlation with involution, hormonal modulation and comparison between normal and neoplastic ti s s u e s . C e l l 16:929- 940 Pace-Asciak, CR. and Smith, W.L. 1986 Enzymes i n the biosynthesis and catabolism of the eicosanoids: prostaglandins, thromboxanes, leukotrienes and hydroxy f a t t y acids, Enzymes 16:543-603 220 Patwardhan, V.V. and Lanthier, A. 1981 Prostaglandin E and prostaglandin F i n human ovarian f o l l i c l e s : Endogenous contents and i n v i t r o formation by theca and granulosa c e l l s . Acta Endocrinol 97:543-550 P e l l e t i e r , G., Seguin, C , Dube, D. anf St.-Arnaud, R. 1982 Di s t r i b u t i o n of LHRH receptors i n the r a t ovary. B i o l Reprod (Suppl) 26:151A P e l l i c e r , A., Palumbo, A., DeCherney, A. H. and Nafto l i n , F. 1988 Blockage of ovulation by an angiotensin antagonist Science 240:1660-1661 Peters, H., Byskov, A.G., Himelstein-Braw, R. and Faber, M. 1975 F o l l i c u l a r , growth: the basic event i n the mouse and human ovary. J Reprod F e r t i l 45:559-566 P h i l i b e r t , D., Ojasoo, T. and Raynaud, J-P. 1977 Properties of the cytoplasmic progestin-binding protein i n the rabbit uterus. Endocrinology 101:1850-1861 P h i l l i p s , H.S., Hostetter, G., Kerdelhue, B. and Kozlowski, G.P. 1980 Immunocytochemical l o c a l i z a t i o n of LHRH i n central olfactory pathways of hamster. Brain Res 193:574-579 Philpott, J.E. and Peron, F.G. 1971 A microassay procedure for 3beta-hydroxysteroid dehydrogenase based on substrate depletion. Endocrinology 88:1082-1085 Popkin, R.M., Bramly, T.A., Currie, A.J., Shaw, R.W., Baird, D.T. and Fraser, H.M. 1983 S p e c i f i c binding of l u t e i n i z i n g hormone-releasing hormone to human l u t e a l t i s s u e . Biochem Biophys Res Commun 114:750-756 Prentki, M., Biden, T.J., Ja n j i c , D., Irvine, R.F., Beridge, M.J. and Wollheim, C.B. 1984 Rapid mobilization of Ca from rat insulinoma microsomes by inositol-1,4,5-trisphosphate. Nature 309:562-564 Prentki, M., Corkey, B.E. and Matschinsky, F.M. 1985 J n o s i t o l 1,4,5,-trisphosphate and the endoplasmic reticulum Ca cycle of a r a t insulinoma c e l l l i n e . J B i o l Chem 260:9185-9190 Putney, J.W., J r . , McKinney, J.S., Aub, D.L. and L e s l i e , B.A. 1984 Phorbol ester-induced protein secretion i n r a t parotid gland. Relationship to the role of i n o s i t o l l i p i d breakdown and protein kinase C a c t i v a t i o n i n stimulus-secretion coupling. Mol Pharmacol 26:261-266 Putney, J.W., J r . 1986 A model f o r receptor-regulated calcium entry. C e l l Calcium 7:1-12 221 Ranta, T., Baukal, A., Knecht, M., Korhonen, M. and Catt, K. J . 1983 I n h i b i t i o n actions of a gonadotropin-releasing hormone agonist on ovarian f o l l i c l e - s t i m u l a t i n g hormone receptors and adenylatte cyclase i n vivo. Endocrinology 112:956-963 Ranta, T., Knecht, M., Darbon, J-M., Baukal, A.J. and Catt, K.J. 1983 Calcium dependence of the i n h i b i t o r y e f f e c t of gonadotropin-releasing hormone on l u t e i n i z i n g hormone-induced c y c l i c AMP production i n r a t granulosa c e l l s . Endocrinology 113:427-429 Rasmussen, H. 1983 Pathways of Amplitude and S e n s i t i v i t y Modulation i n the Calcium Messenger System. In: Calcium and C e l l Function, edited by W.Y. Cheung, pp 1-63 Rasmussen, H. and Barrett, P.Q. 1984 Calcium messenger system: an integrated view. Physiol Rev 64:938-984 Raymond, V., Leung, P.C.K., Vei l l e u x , R., Lefevre, G. and Labrie, F. 1984 LHRH r a p i d l y stimulates phosphatidylinositol metabolism i n enriched gonadotrophs. Mol C e l l Endocrinol 36:157-164 Reich, R., Abisogun, A.O., Miskin, R. and T s a f r i r i , A. 1985 The mechanism(s) of LH and GnRH induced steroidogenesis and plasminogen activator i n preovulatory f o l l i c l e s . J Steroid Biochem (Suppl) 23:5 Reich, R., Kohen, F., Naor, Z. and T s a f r i r i , A. 1983 Possible involvement of lipoxygenase products of arachidonic acid pathway i n ovulation. Prostaglandin 26:1011-1020 Reich, R., Kohen, F., Slager, R. and T s a f r i r i , A. 1985 Ovarian lipoxygenase a c t i v i t y and i t s regulation by gonadotropin i n the ra t . Prostaglandins 30:581-590 Reynolds, E.E. and Dubyak, G.R. 1985 Ac t i v a t i o n of calcium mobilization and calcium i n f l u x by alpha 1-adrenergic receptors i n a smooth mulscle c e l l l i n e . Biochem Biophys Res Commun 130:627-632 Richards, J.S. 1975 E s t r a d i o l receptor content i n r a t granulosa c e l l s during f o l l i c u l a r development: modification by e s t r a d i o l and gonadotropins. Endocrinology 97:1174-1184 Richards, J.S., Ireland, J . J . , Rao, M.C. and Bernath, G.A. 1976 Ovarian f o l l i c u l a r development i n the rat : hormone receptor regulation by e s t r a d i a l , f o l l i c l e stimulating hormone and l u t e i n i z i n g hormone. Endocrinology 99:1562-1570 Richards, J.S. and Williams, J . J . 1976 Luteal c e l l receptor content for p r o l a c t i n (PRL) and l u t e i n i z i n g hormone (LH): Regulation by LH and PRL. Endocrinology 99:1571-1581 222 Richards, J.S. and Bogovich, K. 1982 E f f e c t of human chorionic gonadotropin and progesterone on f o l l i c u l a r development i n the immature r a t . Endocrinology 111:1429-1438 Richards, J.S. and Hedin, L. 1988 Molecular aspects of hormone action i n ovarian f o l l i c u l a r development, ovulation, and l u t e i n i z a t i o n . Ann Rev Physiol 50:441-463 Rink, T.J., Smith, S.W. and Tsien, R.Y. 1982 I n t r a c e l l u l a r free calcium i n p l a t e l e t e shape change and aggregation. J Physiol 324:53-54 Rippel, R.H. and Johnson, E.S. 1976 I n h i b i t i o n of hCG-induced ovarian and uterine weight augmentation i n the immature rat by analogs of GnRH. Proc Soc Exp B i o l Med 152:432-436 R i v i e r , C , R i v i e r , J . arid Vale, W. 1986 Inhibin-mediated feedback control of f o l l i c l e - s t i m u l a t i n g hormone secretion i n the female r a t . Science 234:205-208 Rodbell, M. 1980 The r o l e of hormone receptors and GTP- regulatory proteins i n mambrane transduction. Nature 284:17-22 Rolland, R. and Hammond, J.M. 1975 Demonstration of a s p e c i f i c receptor f o r p r o l a c t i n i n porcine granulosa c e l l s . Endocr Res Commun 2:281-298 Rolland, R., Hammond, J.M. and Schellekens, L.A. 1976 Pro l a c t i n and ovary function. In: The Endocrine function of the human ovary, edited by James, V.H. et a l . , 1976. pp.305-321 Roth, J . , LeRoith, D., Schiloach, J . and Rabinowitz, C. 1983 I n t e r c e l l u l a r communication: an attempt at a unifying hypothesis. C l i n Res 31:354-363 Rothchild, I. 1981 The regulation of the mammalian corpus luteum. Recent Prog Horm Res 37:183-298 Rush, J.S. and Waechter, C.J. 1987 Inhibitors of protein kinase C block a c t i v a t i o n of B lymphocytes by b a c t e r i a l lipopolysaccharide. Biochem Biophys Res Commun 145:1315-1320 S a l a r i , H., Braquet, P. and Borgeat, P. 1984 Comparative e f f e c t s of indomethacin, acetylenic acid, 15-HETE, nordihydroguaiaretic acid and BW 755C on the metabolism of arachidonic acid i n human leukocytes and p l a t e l e t s . Prostaglandins leukotriences Med 13:53-59 Samuelsson, B. 1983 Leukotrienes: mediators of immediate hy p e r s e n s i t i v i t y reactions and inflammation. Science 220:568- 575 Samuels, L.T., Helmreich, M.L., Lasater, M.B. and Reich, H. 223 1951 An enzyme i n endocrine t i s s u e s which Oxidizes deltas, 3beta hydroxysteroids to a,b nusaturated ketones. Science 113:490-491 Schaeffer, J.M. and Hsueh, A.J.W. 1982 I d e n t i f i c a t i o n of gamma- aminobutyric acid and i t s binding s i t e s i n the ovary. L i f e S c i 30:1599-1604 Schreiber, J.R. and Erickson, G.F. 1979 Progesterone receptor i n the rat ovary: further characterization and l o c a l i z a t i o n i n the granulosa c e l l . Steroids 34:459-469 Shears, S.B. and Kirk, C.J. 1984 Determination of mitochondrial calcium content i n hepatocytes by a rapid c e l l u l a r f r a c t i o n a t i o n technique. Vasopressin stimulates mitochondrial Ca++ uptake. Biochem J 220:417-421 Sheldrick, E.L. and F l i n t , A.P.F. 1984 Ovarian oxytocin. In: Gonadal Proteins and Peptides and Their B i o l o g i c a l Significance, edited by M.R. Sairam and L.E. Atkinson, pp. 257- 272 Shinohara, 0., Knecht, M., and Catt, K.J. 1985 I n h i b i t i o n of gonadotropin-induced granulosa c e l l d i f f e r e n t i a t i o n by act i v a t i o n of protein kinase C Proc Natl Acad S c i USA 82:8518- 8522 Shinohara, 0., Kencht, M., Feng, P. and Catt, K.J. 1986 Activ a t i o n of protein kinase C potentiates c y c l i c AMP production and stimulates steroidogenesis i n d i f f e r e n t i a t e d ovarian granulosa c e l l s . J Steroid Biochem 24:161-168 Short, R.V. 1962 Steroids i n the f o l l i c u l a r f l u i d and the corpus luteum of the mare: A 'two-cell type" theory of ovarian s t e r o i d synthesis. J Endocrinol 24:59-63 Smith C. and Perks A.M. 1983 The k i n i n system and ovulation: changes i n plasma kininogens, and i n kinin-forming enzymes i n the ovaries and blood of rats with 4-day estrous cycles. Can J Physiol Pharmacol 61:736-742 Smith, J.B., Smith, L. and Higgins, B.L. 1985 Temperature and nucleotide dependence of calcium release by myo-inositol 1,4,5- trisphosphate i n cultured vascular smooth muscle c e l l s . J B i o l Chem 260:14413-14416 Smith, M.S. 1980 Role of p r o l a c t i n i n mammalian reproduction. Int Rev Physiol 22:249-276 Snyder, G.D. and Bleasdale, J.E. 1982 Ef f e c t of LHRH on incorporation of [32P]orthophosphate into phosphatidylinositol by dispersed anterior p i t u i t a r y c e l l s . Mol C e l l Endocrinol 28:55-63 224 Somlyo, A.P., Bend, M. and Somlyo, A.V. 1985 Calcium content of mitochondria and endoplasmic reticulum i n l i v e r frozen rapidly i n vivo. Nature 314:622-625 Strauss I I I , J.F., Schuler, L.A., Rosenblum, M.F. and Tanaka, T. 1981 Cholesterol metabolism by ovarian t i s s u e . Adv L i p i d Res 18:99-157 Streb, H., Bayerdorfer, E., Haase, W., Irvine, R.F. and Schulz, I. 1984 E f f e c t of i n o s i t o l 1,4,5-trisphosphate on isolated s ubcellular fractions of r a t pancreas. J Membr B i o l 81:241-253 Streb, H., Irvine, R.F., Berridge, M.J. and Schulz, I. 1983 Release of Ca from a nonmitochondrial i n t r a c e l l u l a r store i n pancreatic acinar c e l l s by inositol-l,4,5-trisphosphate. Nature 306:67-69 Strickland, S. and Beers, W.H. 1976 Studies on the role of plasminogen activator i n ovulation. In v i t r o response of granulosa c e l l s to gonadotropin, c y c l i c nucleotides and prostaglandins. J B i o l Chem 251:5694-5702 Sulimovici, S. and Boyd, G.S. 1968 The cholesterol side-chain cleavage enzyme i n immature r a t ovary. Eur J Biochem 3:332-345 Sulimovici, S. and Boyd, G.S. 1969 The delta5, 3beta- hydroxysteroid dehydrogenase of r a t ovarian t i s s u e . Eur J Biochem 7:549-558 Sulliv a n , M.H. and Cooke, B.A. 1986 The r o l e of C a 2 + i n steroidogenesis i n Leydig c e l l s , stimulation of i n t r a c e l l u l a r free Ca by l u t r o p i n (LH), l u l i b e r i n (LHRH) agonist and cAMP. Biochem J 236:45-51 Takai, Y., Kikkawa, U., Kaibuchi, K. and Nishizuka, Y. 1984 Membrane phospholipid metabolism and signal transduction for protein phosphorylation. In: Advances i n C y c l i c Nuceotide and Protein Phosphorylation Research, edited by Greengard, P. and Robinson, G.A. pp. 119-157 Takats, A. and Hertelendy, F. 1982 Adenylate cyclase a c t i v i t y of avian granulosa: e f f e c t of gonadotropin-releasing hormone. Gen Comp Endocrinol 48:515-524 Taylor, M.V., Metcalfe, J.C., Hesketh, T.R., Smith, G.A. and Moore, J.P. 1984 Mitogens increase phosphorylation of phosphoinositides i n thymocytes. Nature 312:462-465 T e r r i s , S., Hofmanrij,-£. and Steiner, D.F. 1979 Mode of uptake and degradation of i - l a b e l e d i n s u l i n by i s o l a t e d hepatocytes and H4 hepatoma c e l l s . J C e l l B i o l 71:159-171 T i l l y , J.L. and Johnson, A.L. 1988 E f f e c t s of a phorbol ester, 225 a calcium ionophore, and 3 1,5'-adenosine monophosphate production on hen granulosa c e l l plasminogen a c t i v a t o r a c t i v i t y Endocrinology 123:1433-1440 Toaff, M.E., Schleyer, H. and Strauss, I I I . , J.F. 1982 Metabolism of 25-hydroxycholesterol by rat l u t e a l mitochondria and dispersed c e l l s . Endocrinology 111:1785-1790 Toaff, M.E., Strauss, J.F. I I I . and Hammond, J.M. 1983 Regulation of cytochrome P450 SCC i n immature porcine granulosa c e l l s by FSH and e s t r a d i o l . Endocrinology 112:1156-1158 Touqui, L., Rothhut, B., Shaw, A.M., Fradin, A., Vargaftig, B.B. and Russo-Marie, F. 1986 P l a t e l e t activation-a r o l e for a 40K anti-phospholipase A, protein indistinguishable from l i p o c o r t i n . Nature 321:177-180 Triebwasser, W.F., Clark, M.R., LeMaire, W.J. and Marsh, J.M. 1978 L o c a l i z a t i o n and i n v i t r o synthesis of prostaglandins i n components of rabbit preovulatory graafian f o l l i c l e s . Prostaglandins 16:621-632 T s a f r i r i , A., Lindner, H.R., Zor, U. and Lamprecht, S.A. 1972 Physiological r o l e of prostaglandins i n the induction of ovulation. Prostaglandins 2:1-10 T s a f r i r i , A., Lindner, H.R., Zor, U. and Lamprecht, S.A. 1972 In v i t r o induction of meiotic d i v i s i o n i n f o l l i c l e - e n c l o s e d rat oocytes by LH, c y c l i c AMP and prostaglandin E2. J Reprod F e r t i l 31:39-50 T s a f r i r i , A. and Braw, R. 1984 Experimental approaches to a t r e s i a i n mammals. Oxford Rev Reprod B i o l 6:226-265 T s a f r i r i , A., Reich, R. and Abisogun, A.O. 1986 Ovarian regulation of oocyte maturation. In: Gynecological Endocrinology, edited by Genazzani, A.R., Volpe, A. and F a c c h i n e t t i , F. pp 109-115 Tsang, B.K., Moon, Y.S., Simpson, C.W. and Armstrong, D.T. 1979 Androgen biosynthesis i n human ovarian f o l l i c l e s : c e l l u l a r source, gonadotropic control, and andenosine 3',5'- monophosphate mediation. J C l i n Endocrinol Metab 48:153-158 Tsang, B.K., Armstrong, D.T. and Whitfield, J.F. 1980 Steroid biosynthesis by isolated human ovarian f o l l i c u l a r c e l l s i n v i t r o . J C l i n Endocrinol Metab 51:1407-1411 Tureck, R.W., Mastroianni, L, J r . , Blasco, L. and Strauss, J.F. 1982 I n h i b i t i o n of human granulosa c e l l progesterone secretion by a GnRH agonist. J C l i n Endocrinol Metab 54:1078-1083 Uchida, K., Kadowaki, M., Miyake, T. and Wakabayashi, K. 1972 226 E f f e c t of exogenous progesterone on the ovarian progestin secretion and plasma LH and p r o l a c t i n l e v e l s i n c y c l i c r a t s . Endocrinol Jpn 19:323-333 Veldhuis, J . , Kolp, L.A., Toaff, M.E., Strauss, I I I , J.F. and Demmers, L.M. 1983 Mechanism subserving the t r o p i c actions of i n s u l i n on ovarian c e l l s . In v i t r o studies using swine granulosa c e l l s . J C l i n Invest 72:1046-1057 Veldhuis, J . D. and Demers, L. M. 1986 An i n h i b i t o r y r o l e of the protein kinase C pathway i n ovarian steroidogenesis Biochem. J . 239:505-511 Veldhuis, J.D. and Demers, L.M. 1987 Ac t i v a t i o n of protein kinase C i s coupled to prostaglandin F, synthesis i n the ovary: studeis with cultured swine granulosa c e l l s . Mol C e l l Endocrinol 49:249-254 V i l g r a i n , I., Defaye, G. and Chambaz, E.M. 1984 Adrenocortical cytochrome P-450 responsible for choleste r o l side chian cleavage (P-450 sec) i s phosphorylated by the calcium- activated, phospholipid-sensitive protein kinase (protein kinase C). Biochem Biophys Res Commun 125:554-561 Vol p i , M., Molski, T.E.P., Naccache, P.H., Feinstein, M.B. and Sha'afi, R.I. 1985 Phobol 12-myristate 13-acetate potentiates the action of the calcium ionophore i n stimulating arachidonic acid release and production of phosphatidic acid i n rabbit neutrophils. Biochem Biophys Res Commun 128:594-600 Wang, C. 1983 Lut e i n i z i n g hormone releasing hormone stimulates plasminogen act i v a t o r production by r a t granulosa c e l l s . Endocrinology 112:1130-1132 Wang, C. and Hsueh, A.J.W. 1979 Induction of functional p r o l a c t i n receptors by f o l l i c l e - s t i m u l a t i n g hormone i n rat granulosa c e l l s i n v i t r o . J B i o l Chem 254:11330-11336 Wang, C , Hsueh, A.J.W. and Erickson, G.F. 1981 LH stimulation of estrogen secretion i n cultured granulosa c e l l s . Mol C e l l Endocrinol 24:17-28 Wang, J. , Baimbridge, K.G. and Leung, P.C.K. 1989 Perturbation of i n t r a c e l l u l a r calcium ion concentration i n single rat granulosa c e l l s by angiotensin I I . Endocrinology ( i n press). Wang, J . and Leung, P.C.K. 1987 Role of arachidonic acid i n l u t e i n i z i n g hormone-releasing hormone actions: stimulations of progesterone production i n rat granulosa c e l l s . Endocrinology 122:906-911 Wang, J . and Leung, P.C.K. 1987 Role of protein kinase C i n l u t e i n i z i n g hormone-releasing hormone (LHRH)-stimulated 227 progesterone production i n rat granulosa c e l l s . Biochem Biophys Res Commun 146:939-944 Watson, S.P., McConnell, R.T. and Lapetina, E.G. 1984 The rapid formation of i n o s i t o l phosphate i n humane p l a t e l e t s by thrombin i s i n h i b i t i e d by pr o s t a c y c l i n . J B i o l Chem 259:13199-13203 Welsh, J r . T.H., Jones, P.B.C., Ruiz, de Galarreta, CM., Fanjul, L.F. and Hsueh, A.J.W. 1982 Androgen regulation of progestin biosynthetic enzymes i n FSH-treated r a t granulosa c e l l s i n v i t r o . Steroids 40:691-700 Welsh, J r . , T.H., Jones, P.B.C. and Hsueh, A.J.W. 1984 Phorbol ester i n h i b i t i o n of ovarian and t e s t i c u l a r steroidogenesis i n v i t r o . Cancer Research 44:885-892 Wolfe, M., LeVine, III H., May, S. J r . , Cuatrecacasa, P. and Sahyoun, N. 1985 A model for i n t r a c e l l u l a r t r a n s l o c a t i o n of protein kinase C involving synergism between calcium and phorbol esters. Nature 317:546-49 Yamamoto, S., I s h i i , M., Nakadate, T., Nakaki, T. and Kato, R. 1983 Modulation of i n s u l i n secretion by lipoxygenase productions of arachidonic acid. J B i o l Chem 258:12149-12152 Ying, S.Y. and Guillemin, R. 1979 (D-Trp6-Pro9-NEt)-luteinizing hormone-releasing factor i n h i b i t s f o l l i c u l a r development i n hypophysectomized rats. Nature 280:593-595 Z o l l e r , L.C and Weisz, J . 1978 I d e n t i f i c a t i o n of cytochrome P450 and i t s d i s t r i b u t i o n i n the menbrana granulosa of the preovulatory f o l l i c l e using quantitative cytochemistry. Endocrinology 103:310-313 Z o l l e r , L.C. and Weisz, J . 1979 A quantitative cytochemical study of glucose-6-phosphate dehydrogenase and 3beta- hydroxysteroid dehydrogenase a c t i v i t y i n membrana granulosa of the ovulable type of f o l l i c l e of the r a t . Histochemistry 62:125-135 Zor, U., S t r u l o v i c i , B., Braw, R., Lindner, H.R. and T s a f r i r i , A. 1983 F o l l i c l e - s t i m u l a t i n g hormone-induced prostaglandin E formation i n is o l a t e d r a t ovarian theca. J Endocrinology 97:43- 49 PUBLICATIONS Leung PCK, Minegishi T and Wang J 1988 I n h i b i t i o n of f o l l i c l e - stimulating hormone- and adenosine-3•,5 1-cyclic monophosphate-induced progesterone production by calcium and protein kinase C i n the rat ovary. Am J Obstet Gynecol 158:350-356 Leung PCK and Wang J 1989 I n o s i t o l l i p i d s and LHRH action i n the ovary. J Reprod F e r t i l (suppl) 37:287-293 Leung PCK and Wang J 1989 Role of calcium i n the action of LHRH on ovarian progesterone production. In: VII ovarian workshop: Paracrine Communication i n the ovary ( i n press). Leung PCK and Wang J 1989 The r o l e of i n o s i t o l l i p i d metabolism i n the ovary. B i o l Reprod (in press). Leung PCK, Wang J, and Baimbridge KG 1989 Mechanism of action of l u t e i n i z i n g hormone-releasing hormone i n r a t ovarian c e l l s Can J Physiol Pharmacol (in press). Minegishi T, Wang J and Leung PCK 1987 Lu t e i n i z i n g hormone- releasing hormone (LHRH)-induced arachidonic a c i d release i n rat granulosa c e l l s : Role of calcium and protein kinase C. FEBS Lett 214:139-142 Wang J , Baimbridge KG, and Leung PCK 1989 Perturbation of i n t e r a c e l l u l a r calcium ion concentrations i n single r a t granulosa c e l l s by angiotensin I I . Endocrinology 124:1094- 1096 Wang J , Baimbridge KG, and Leung PCK 1989 Changes i n c y t o s o l i c free calcium ion concentrations i n in d i v i d u a l r a t granulosa c e l l s : e f f e c t of l u t e i n i z i n g hormone-releasing hormone. Endocrinology 124:1912-1917. Wang J , Ho Yuen B, and Leung PCK 1989 Stimulation of progesterone and prostaglandin E_ production by lipoxygenase metabolites of arachidonic acid. FEBS Lett 244:154-158. Wang J, Lee V and Leung PCK 1989 D i f f e r e n t i a l r o l e of protein kinase C i n the action of LHRH on hormone production i n rat ovarian c e l l s . Am J Obstet Gynecol ( i n press). Wang J and Leung PCK 1987 Role of protein kinase C i n l u t e i n i z i n g hormone-releasing hormone (LHRH)-stimulated progesterone production i n rat granulosa c e l l s . Biochem. Biophys. Res. Comm. 146:939-944 Wang J and Leung PCK 1988 Role of arachidonic acid i n l u t e i n i z i n g hormone-releasing hormone action: stimulation of progesterone production i n rat granulosa c e l l s . Endocrinology 122:906-911 Wang J and Leung PCK 1989 Arachidonic acid as a stimulatory mediator of LHRH action i n the rat ovary. Endocrinology 124: 1973-1979 Wang J and Leung PCK 1989 Synergistic stimulation of prostaglandin E. production by calcium ionophore and protein kinase C a c t i v a t o r i n rat granulosa c e l l s . B i o l Reprod (in press).

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