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Prostaglandin F[sub 2⍺]-mediated luteolytic and luteotrophic effects on the human granulosa-luteal cell 1997

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Prostaglandin F2a-Mediated Luteolytic and Luteotrophic Effects on the Human Granulosa-Luteal Cell by Jeffrey Eric Vaananen B.Sc, Simon Fraser University, 1991 M.Sc, The University of British Columbia, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Obstetrics and Gynaecology, Reproductive and Developmental Sciences Program) We accept this thesis as conforming ~^tp l^ie retired standard THE UNIVERSITY OF BRITISH COLUMBIA March 1997 © Jeffrey Eric Vaananen, 1997 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. v Department of OB>^l^TK(CS + (Zlf^AeCPtoC^ The University of British Columbia Vancouver, Canada Date ? - 1 fj-PAjC DE-6 (2788) ABSTRACT These studies examined the effects of prostaglandin^ a (PGF2a) on progesterone and 178-estradiol (estradiol) production, as well as D N A and PGF 2 a-receptor ( P G F 2 a - R ) mRNA levels, in the human granulosa-luteal cell (GLC). Additionally, the interactions of P G F 2 o ; with human chorionic gonadotrophin (hCG), gonadotrophin-releasing hormone (GnRH) and prostaglandin E 2 (PGE2) were examined, with respect to progesterone and estradiol production. In one study, cells were collected from small (<12 mm) and large (>12 mm) follicles separately, permitting the examination of follicle size-dependent alterations in steroidogenesis. Pharmacological techniques were utilized to elucidate the signal transduction pathways involved in the anti-gonadotrophic effects of PGF2«- Moreover, these experiments were performed on GLCs cultured for one (Di), eight (Dg) and/or twelve to fourteen days (D12-14), in order to reveal culture time-dependent alterations in cellular response. Briefly, GLCs collected from patients undergoing in vitro fertilization (IVF), were cultured for the time periods described above, followed by a 24 h treatment period. After the treatment period media were collected and assayed for progesterone and estradiol, while cells were extracted for D N A or total RNA. It was found that human G L C responses to P G F 2 a are culture time- and concentration- dependent, with PGF201 being either luteolytic or luteotrophic, depending on culture and treatment conditions. Moreover, G L C responses to hCG and P G F 2 a varied with follicle size, suggesting that these hormones' actions are targeted toward more mature follicles. Furthermore, GnRH potentiates the luteolytic effects of P G F 2 a , while it acts as a permissive factor for the luteotrophic effects. A complex interaction between P G F 2 a and PGE2 was also seen. The luteolytic effects of P G F 2 a are mediated through a pertussis toxin-sensitive G-protein (possibly Gj, G p or both). P G F 2 a inhibits cholera toxin-, isoproterenol- and forskolin-, but not db-cAMP-stimulated progesterone production suggesting that this G-protein is exerting its actions on the adenylate cyclase pathway at the level of adenylate cyclase, but not distal to it. Additionally, P G F 2 a is capable of autoregulating its receptor mRNA levels, and thus its ability to regulate steroidogenesis in the human G L C . Prostaglandin F 2 a - R mRNA levels were found to be inversely related to progesterone and estradiol production. In conclusion, P G F 2 a is a multi-functional hormone which acts through complex signal transduction pathways and interactions with confounding hormones, to exert both luteotrophic and luteolytic effects. T A B L E OF CONTENTS iii Page ABSTRACT ii T A B L E OF CONTENTS iii LIST OF TABLES viii LIST OF FIGURES ix LIST OF ABBREVIATIONS xiii ACKNOWLEDGMENTS xviii DEDICATION xix I - BACKGROUND 1 A. The Classical Neuro-Endocrine Pathway of Gonadal Regulation 1 B. Pregnancy 4 C. The Sex Steroids 6 The Progestins and Estrogens 6 The Synthesis of Progesterone and Estradiol 6 The Two Cell Model of Steroid Biosynthesis 6 Sex Steroid Receptors 9 Sex Steroid Sites of Action 10 1. The Fallopian Tubes 10 2. The Uterus 10 3. The Vagina 11 4. The Breasts 11 5. Other Progesterone-Dependent Actions 11 D. The Eicosanoids 12 Prostaglandins, Thromboxanes and Leukotrienes 12 Phospholipases and Arachidonic Acid 12 Eicosanoid Production from Arachidonic Acid 14 Prostanoid Receptors 18 Prostaglandins as Autocrine/Paracrine Factors 18 Inhibition and Degradation of Prostaglandins 19 IV E. Prostaglandin F2a in Reproduction 20 Localization ofPGF2a 20 Regulation ofPGF2a Production in the Ovary 20 Functions ofPGF2a 20 PGF2 a in Pregnancy 21 Prostanoid Receptors in Reproductive Tissues 22 PGF2a Signal Transduction 22 Clinical Applications of PGF2a 23 F. Gonadotrophin-Releasing Hormone 25 GnRH Functions 25 GnRH Localization 25 GnRH Receptor 25 GnRH Signal Transduction 26 GnRH Mechanism of Action 27 Clinical Applications of GnRH 27 II - HYPOTHESIS 28 III - SPECIFIC OBJECTIVES 28 IV - RATIONALE 30 A. The Effects of PGF2a on Steroidogenesis 30 B. PGF200 and GnRH Interaction Studies 31 C. PGF2a and PGE2 Interaction Studies 31 D. Signal Transduction Studies 32 E. PGF2orR mRNA Studies 33 V - MATERIALS AND METHODS 34 A. Granulosa-Luteal Cell Collection and Culture 34 B. Static Incubation Experiments 36 C. Microscopy 39 D. Radioimmunoassay of Progesterone and Estradiol 40 E. Hoechst Dye DNA Assay 42 F. RNA Extraction Procedure 44 G. RNA Gel 45 H. Reverse Transcription of RNA to cDNA 45 /. Polymerase Chain Reaction (PCR) 46 J. DNA Gel 46 K. Southern Blot Hybridization 50 L. Densitometry of Photographs 50 M. Analysis of Results 53 VI - RESULTS 54 Preliminary Results 54 Basal and hCG-Stimulated Steroidogenesis from human GLCs 1. Basal Steroid Secretion per Cell or Level of DNA/Well 54 2. hCG-Stimulated Progesterone Production, in Cells from Three Different Patients 54 Human GLC Morphology with Culture Time 60 A. The Effects of PGF201 on Steroidogenesis in the Absence and Presence of hCG 64 Effects ofPGF2 a on Steroidogenesis 64 1. Progesterone and Estradiol Production in Response to PGF 2 a 64 2. DNA Levels in Response to P G F 2 a 64 Effects ofPGF2 a on hCG-Stimulated Steroidogenesis 68 1. Progesterone Production in Response to hCG Treatment 68 2. Follicle Size-Dependent Regulation of Steroidogenesis by hCG and PGF 2 a 68 3. The Effects of PGF 2 a on hCG-Stimulated Steroidogenesis 68 Effects of GnRH on hCG-Stimulated Steroidogenesis 74 B. The Interaction of PGF2a with GnRH 77 Progesterone Response to GnRH and/or PGF2a, with or without hCG 11 Estradiol Response to GnRH and/or PGF2a, with or without hCG. 11 Progesterone Response to GnRH with or without PGF2a 11 Estradiol Response to GnRH with or without PGF2a 78 DNA Levels in Response to GnRH and PGF2a Treatment 78 Effects of Indomethacin on PGF2a and GnRH Stimulated Progesterone Production 19 vi C. Progesterone Response to PGF2a plus PGE2 92 D. Signal Transduction of PGF2a-Mediated Luteolysis 97 Effects ofPGF2a on hCG-Stimulated Steroidogenesis 97 Effects ofPGF2a on Isoproterenol Stimulated Progesterone Production 97 Effects of PTX on And-gonadotrophs Actions of PGF2a 97 Effects ofPGF2a on CTX Stimulated Steroidogenesis 98 Effects ofPGF2aon Forskolin Stimulated Progesterone Production 98 Effects of PGF2 a on cAMP Stimulated Progesterone Production 98 The Effects of a PKC Inhibitor on PGF2a-Mediated Inhibition of hCG'-Stimulated Progesterone Production 98 E. Effects of hCG and PGF2a on PGF2a-R mRNA 107 Spectrophotometry Estimation of Known DNA Levels in Solution 107 RNA Integrity and Relative Quantity 107 PCR Cycle Experiment 107 Amplification ofPGF2a-R dndfi-Actin cDNAs in Human GLCs 108 Confirmation ofPGF2a-R cDNA in Human Granulosa-Luteal and Placental Cells 108 Regulation ofPGF2a-R cDNA by hCG and PGF2a 108 VII - DISCUSSION 117 Caveats of the Human Granulosa-Luteal Cell Model 117 Variability in Basal Steroidogenisis in the Human GLC Model 117 Cell Numbers and Low Level RNA Expression 118 A Question of Physiological Concentration? 119 Summary 119 Morphology of Human GLCs in Culture 120 A. Effects of PGF2a on Human Granulosa-Luteal Cells in the Absence and Presence of hCG 121 Follicle Size 121 Concentration and Culture Time Dependent Responses 122 Summary 123 B. Interaction of PGF2a with GnRH 125 Progesterone Response : 125 vii Estradiol Response 125 Implications 126 Experimental Model 128 Summary 128 C. Interaction of PGF2a with PGE2 131 D. Signal Transduction of PGF2crMediated Luteolysis 132 Pertussis Toxin Sensitive G-Protein 132 Adenylate Cyclase and cAMP 135 Protein Kinase C 135 De Novo Protein Synthesis 136 Summary 136 Regulation of PGF2crR mRNA 142 VIII - SYNOPSIS 143 A. Basic Physiological Responses to PGF2a 143 2?. Confounding Interactions of PGF2a with GnRH 144 C. Confounding Interactions of PGF2a with PGE2 145 D. Signal Transduction of the Luteolytic Effects of PGF2a 145 E. Regulation of PGF2a-R mRNA 146 IX - CONCLUSIONS 147 REFERENCES 148 viii LIST OF TABLES Number Title Page 1 The Eicosanoid Superfamily of Hormones 13 2 Hormones and pharmacological agents utilized in these studies 37 3 Primer combinations and expected product size following PCR 47 4 PCR conditions utilized for genes examined 48 5 Oligonucleotide sequences utilized for PCR and Southern blot hybridization 49 6 Southern Blot SSC Washes 52 7 Spectrophotometer estimation of known DNA levels in solution 110 LIST OF FIGURES ix Number Title Page Introduction 1 The hypothalamopituitary axis 2 2 Model of signal transduction pathway for hCG-stimulated steroidogenesis 5 3 The synthetic pathway of the female sex steroids 7 4 The two cell model of steroidogenesis 8 5 Phospholipase cleavage (hydrolysis) sites on phospholipids 15 6 Arachidonic acid production in a model system 16 7 Synthesis of P G F 2 a from arachidonic acid 17 8 A diagramatic depiction of the specific objectives 29 Methods 9 Schematic of methods 35 10 Typical progesterone and estradiol RIA standard curves 41 11 Typical hoechst dye DNA assay standard curve 43 12 Setup for overnight transfer of gel products to a nylon membrane 51 Results 13 Basal progesterone production versus cells/well 55 14 Basal progesterone production versus DNA content 56 15 Basal estradiol production versus cells/well 57 16 Basal estradiol production versus DNA content 58 17 Progesterone responses to hCG in cells from 3 different patients 59 18 Human granulosa-luteal cells that were freshly plated 61 19 Eight day cultures of human granulosa-luteal cells 62 2 0 Sixteen day cultured human granulosa-luteal cells 63 21 Progesterone production in response to PGF2a. D i and D12-14 GLCs 65 22 Progesterone production in response to P G F 2 a , Dg GLCs 66 23 A. Estradiol production in response to PGF2«, D i and Dg GLCs 67 B. D N A content in response to PGF2 a> in Dg GLCs 67 24 hCG stimulated progesterone production 69 2 5 Follicle size-dependent responses to hCG P G F 2 a 70 26 The effects of PGF2a on hCG-stimulated progesterone production from D x and Dg GLCs 71 2 7 The effects of P G F 2 a on hCG-stimulated progesterone production from Di2-14 GLCs 72 2 8 The effects of P G F 2 a on hCG-stimulated estradiol production from D i and D 8 GLCs 73 2 9 The effects of GnRH on hCG-stimulated progesterone production from D i and D 8 GLCs 75 30 A. The effects of GnRH on hCG-stimulated estradiol production from D i and D 8 GLCs 76 B. The effects of GnRH and hCG on DNA levels in D 8 GLCs 76 31 Progesterone production in response to GnRH, P G F 2 a and/or hCG 80 3 2 Estradiol production in response to GnRH, PGF2« and/or hCG 81 3 3 Three dimensional plot of GnRH and P G F 2 a interactions on progesterone 82 3 4 Contour plot of GnRH and P G F 2 a interactions on progesterone 83 3 5 Effects of P G F 2 a in the absence and presence of GnRH on progesterone 84 3 6 Effects of GnRH in the absence and presence of P G F 2 a on progesterone 85 3 7 Three dimensional plot of GnRH and P G F 2 a interactions on estradiol 86 3 8 Contour plot of GnRH and P G F 2 a interactions on estradiol 87 3 9 Effects of P G F 2 a in the absence and presence of GnRH on estradiol 88 4 0 Effects of GnRH in the absence and presence of P G F 2 a on estradiol 89 41 Progesterone response to P G F 2 a with/without indomethacin 90 4 2 Three dimensional plot of GnRH and P G F 2 a interactions on progesterone in the presence of indomethacin 91 xi 43 Three dimensional plot of P G F 2 a and P G E 2 interactions on progesterone 93 44 Contour plot of P G F 2 a and P G E 2 interactions, on progesterone 94 45 P G F 2 a and P G E 2 concentration response curves 95 46 The effects of P G F 2 a , in the presence of P G E 2 96 47 PGF 2 a-mediated inhibition of hCG-stimulated steroidogenesis 99 48 PGF 2 a-mediated inhibition of isoproterenol-stimulated progesterone production 100 49 Effects of pertussis toxin on PGF2 a-mediated inhibition of hCG-stimulated steroidogenesis 101 50 Effects of pertussis toxin, P G F 2 a and hCG on D N A levels 102 51 Effects of P G F 2 a on cholera toxin-stimulated steroidogenesis 103 52 Effects of P G F 2 a on forskolin-stimulated steroidogenesis 104 53 Effects of P G F 2 a on db-cAMP-stimulated steroidogenesis 105 54 Effects of a bisindolylmaleimide PGF2 a-mediated luteolysis 106 55 RNA integrity gel 111 56 PCR cycle experiments 112 57 PCR amplification of P G F 2 a - R and B-actin cDNA 113 58 PCR amplification of P G F 2 a - R cDNA from human GLCs, placenta and leukocytes 114 59 Effects hCG and P G F 2 a on P G F 2 a - R mRNA 115 6 0 Southern blot hybridization of a PCR experiment for P G F 2 a - R 116 Discussion 61 Dual actions of P G F 2 a on steroidogenesis 62 GnRH as a permissive or potentiatory factor for PGF 2 a-mediated effects (Model I) 6 3 GnRH as a permissive or potentiatory factor for PGF2 a-mediated effects (Model II) 6 4 Proposed positive feedback loop for P G F 2 a synthesis 124 129 130 137 xii 6 5 Pertussis toxin to blocks PGF2a-mediated inhibition of hCG- stimulated steroidogenesis 138 6 6 PGF 2 a-mecliated inhibition of hCG- and isoproterenol-stimulated steroidogenesis 139 6 7 PGF2a-mediated inhibition of cholera toxin- and forskolin-stimulated steroidogenesis 140 68 The inability of PGF2a to inhibit cAMP-stimulated steroidogenesis 141 LIST OF ABBREVIATIONS Category Abbreviation Meaning Standard Abbreviations ATP Adenosine triphosphate AA Arachidonic acid bp Base pairs C Celcius CA California cAMP Cyclic adenosine monophosphate cDNA Complimentary DNA CL Corpus luteum Cox-I Cyclooxygenase I (constitutive) Cox-II Cyclooxygenase II (inducible) CTX Cholera Toxin 2D Two dimensional 3 D Three dimensional Di Precultured for 1 day Dg Precultured for 8 days D12-14 Precultured for 12-14 fourteen days DAG Diacylglyceriolordiglyceride db-cAMP Dibutryl-cyclic-adenosine monophosphate ddhkO Double distilled water dCTP Deoxycytosine-triphosphate dNTPs Deoxynucleotide-triphosphate(s) DP Prostaglandin D 2 receptor dpi Dots per inch FSH Follicle stimulating hormone DEPC Diethylpyrocarbonate xiv D M E M Dulbecco's Modified Eagle's Medium DNA Deoxyribonucleic acid dNTPs Deoxynucleotide-triphosphates DTT Dithiothreitol E2 Estradiol EDTA Ethylenediaminetetraacetic acid EPi Prostaglandin E 2 receptor (isoform 1) E P 3 Prostaglandin E 2 receptor (isoform 3) Estradiol 176-estradiol FBS Foetal bovine serum For Forskolin FP Prostaglandin F 2 a receptor g Grams G L B Gel loading buffer G L C Granulosa-luteal cell GnRH Gonadotrophin-releasing hormone GTP Guanosine triphosphate G-protein GTP dependent protein G a G-protein alpha subunit(s) G a s G a stimulatory G«il,2 G a inhibitory (isoform 1 or 2) G a i3 G a inhibitory (isoform 3) G a p G a placental G a q G a placental (q isoform) G a l l G a placental (11 isoform) G aO G a olfactory GRB Gel running buffer GTC Guanosine thiocyanate Lysis buffer h Hours hCG Human chorionic gonadotrophin Indo Indomethacin IP Prostaglandin I 2 receptor IP 3 Inositol trisphosphate IsoP, or Iso Isoproterenol IU International units IVF In vitro fertilization K d Equilibrium dissociation constant K D a Kilodaltons 1 Liter L H Luteinizing hormone LT Leukotrienes M199 Medium 199 ml Milliliters min Minutes MD Maryland mRNA Messenger ribonucleic acid MO Missouri m. w. Molecular wei ght M Moles/Liter NADP+ Nicotinamine adenine dinucleotide phosphate Hydrogenated NADP+ New Hampshire Nanomolar New York Optical density Ontario Progesterone Phosphatidic acid Pituitary adenylate cyclase activating polypeptide PCR Polymerase chain reaction N A D P H N H nM NY CD ON P 4 PA PACAP PBS Phosphate-buffered saline P G Prostaglandin PGE2 Prostaglandin E 2 P G F 2 a Prostaglandin F 2 a P G F 2 a - R Prostaglandin F 2 a receptor P G G 2 Prostaglandin G 2 P G H 2 Prostaglandin H P G I 2 Prostaglandin I 2 PI Phosphoinositide P K A Protein kinase A P K C Protein kinase C P K C i Protein kinase C inhibitor Bisindolylmaleimide PL Prolactin PLAx Phospholipase A1 P L A 2 Phospholipase A 2 PLB Phospholipase B P L C Phospholipase C PLD Phospholipase D pM Picomolar pmole Pico moles P T X Pertussis toxin PJA Radioimmunoassay RNA Ribonucleic acid RT Reverse transcription s Seconds SCC P450-side chain cleavage enzyme SDS Sodium dodecyl sulfate Sigma Sigma Chemical Company, St. Louis, MO. ssc T B E TRIS Tx U V v/v w/v X x g PQ Abbreviations Starting with Greek Characters a 6 Y Sodium chloride and sodium cytrate buffer Tris borate EDTA Tris(hydroxymethyl)aminomethane Thromboxanes Ultraviolet Volume per volume Weight per volume Times (or multiplied by) Times gravity Quebec Alpha Beta Gamma Micromolar (IO 6 molar) ACKNOWLEDGMENTS XVlll M y gratitude to Dr. Leung for letting me explore my ideas while acting as a sounding board cannot be overstated. I would further like to thank him for allowing me the opportunity to gain experience by helping review papers, acting as chairperson for the departmental seminars and supervising three undergraduate students. This has truly been an educational experience. Many thanks to Dr. Ho Yuen for encouragement and instructive suggestions. I would also like to thank Dr. Auersperg for demonstrating so much enthusiasm after a good number of years of science. I hope that I may maintain such a passion for science. Dr. Rajamahendran's comments during my committee meetings have helped me tighten up my language. The chairman of my committee Dr. Lee helped strengthen this thesis by reinforcing my awareness of the some of the caveats of the human granulosa-luteal cell model. Additionally, Dr. Buchan, in the Department of Physiology, is largely responsible for the style and approach to science contained within this thesis, as her prolific advice during my Masters thesis still resides within my mind. I hope that she would be pleased with this manuscript. Suzie, Brenda and Ivan deserve thanks for assistance about the lab, and for acting as guinea pigs during my attempts at being a teacher. I hope that they took away as much as they gave. Pearly has provided some good advice, and a great deal of entertaining conversation. I appreciate Pearly's willingness to process cells on alternate days with me. The IVF staff have been incredibly supportive, and have provided me with cells literally every day that it was possible to provide them. Special words of praise should be reserved for Cindy and Pathma for their attention to detail and enthusiasm for research. I would like to thank the folks at the Center for Evaluation Sciences for the two talks on statistics that they gave during my tenure in the department, and for the free consultations reguarding my project. It was reassuring to talk to an expert in that discipline. M y friend Janine Senz was always willing to answer the phone at two in the morning and provide molecular biology tips- for this she must be thanked. Ted Urbanek, who provided me with diversions when I couldn't continue to work also deserves comment. Having worked for countless years as a journalist, both independently and for the Canadian Broadcasting Corporation (CBC), my uncle Garth Cochran, provided an endless source of suggestions, corrections and typesetting. My parents have instilled in me, determination and an interest in science, the two most important driving forces in the underlying studies. Without Celine, my tag team partner, I could not have finished this thesis. Celine has been a wonderful friend, playmate, helper, wife and mother to my child(ren). If everyone was as silently confident and wise as Celine the world would be a much more generous and peaceful place. Her comfort with herself is something that I shall forever envy. At our wedding, Celine's family showed me kindness and acceptance that I have never known. Their support has been crucial in this last three years.  I-BACKGROUND A. The Classical Neuro-Endocrine Pathway of Gonadal Regulation Classically, regulation of ovarian steroidogenesis was seen as a purely hypothalamo- pituitary axis phenomena (Fig. 1). The classical neuro-endocrine pathway acts as follows. Various inputs such as corticotrophin releasing hormone, dopamine, endorphin, estradiol, norepinephrine, pheromones, serotonin and the light/dark cycle are integrated in the arcuate and preoptic nuclei [Advis et al., 1978; Balthazart et al., 1981; Donham et al., 1993; Dufour et al., 1988; Laatikainen, 1991; Rotsztejn et al., 1976; Sawyer, 1975; Yen et al., 1977]. These influences regulate the secretion of gonadotrophin-releasing hormone (GnRH) from neuron-like cells, originating in these nuclei, and terminating in the anterior pituitary. Gonadotrophin- releasing hormone is a decapeptide that is clipped from a larger propeptide [Hsueh et al., 1983; Nillius et al., 1974]. Through a receptor-dependent mechanism, GnRH acts on gonadotrophs to stimulate the release of the gonadotrophins, follicle stimulating hormone (FSH) and lutenizing hormone (LH) [Baldwin et al., 1984; Joshi et al., 1993; Rommler et al., 1979]. Follicle stimulating hormone and L H are collected by the portal system of the anterior pituitary and distributed, via the efferent veins, into the general circulation where they eventually reach their target the ovaries [Sawyer, 1975]. Lutenizing hormone is secreted in pulses, with the period between peaks being 1 to 7 hours depending on the phase of the menstrual cycle [Filicori et al., 1979]. The pulsatile nature of L H release is probably due to pulsatile GnRH secretion or GnRH-receptor number fluctuations, rather than gonadal feedback [Baldwin et al., 1984; Inaudi et al, 1992; Schuiling and Gnodde, 1976]. During the follicular phase estradiol levels increase in response to FSH. When the developing follicle is fully mature, the estradiol levels reach a threshold which initiates an L H peak and triggers ovulation. Follicle stimulating hormone is released from a single pool, in a pulsatile manner, with a lower amplitude than L H [Filicori et al., 1979]. The release of FSH is less sensitive to GnRH than L H [Hall et al., 1992], and can further be regulated by estradiol. 2 Dopamine Norepinephrine Endorphins Inhibin Hypothalamus Inhibin- GnRH Pituitary FSH Inhibin Ovaries Progesterone & Estradiol Estradiol A •Estradiol A Figure 1. The hypothalamopituitary axis. Various stimulatory ( >•) and inhibitory ( »-) neural inputs regulate the secretion of gonadotrophin-releasing hormone (GnRH) from the hypothalamus. GnRH in turn stimulates the production and secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary. Circulating FSH and L H stimulate progesterone, estradiol and inhibin production from the ovaries. Inhibin exerts negative feedback on the pituitary and possibly the hypothalamus. Additionally, follistatin (an ovarian product) inhibits the actions of inhibin (not diagrammed). Estradiol can positively or negatively feedback on the pituitary and hypothalamus depending on temporal and concentration conditions. Both FSH and L H are glycoproteins which share a common a-subunit (m.w. 14,000; 96 A A ) . Additionally, FSH and L H each have a unique 6-subunit, which is noncovalently linked to the a-subunit [Combarnous, 1988; Gray , 1988; Ryan et al., 1987; Wierman et al., 1988]. These peptides possess carbohydrate moieties which account for 15 percent of their weight, and are involved in receptor binding [Combarnous, 1988; Gray , 1988; Ryan et al., 1987; Wierman et al., 1988]. Once FSH and L H reach their primary target in the female- the ovaries their actions diverge. In the granulosa cell FSH is responsible for stimulating mitosis, aromatase activity and inducing LH-receptor expression and membrane presentation. These actions serve to ripen or prepare the developing follicle for ovulation. This FSH-induced increase in LH-receptors primes the granulosa cell for the L H surge just prior to ovulation. Lutenizing hormones primary action on the granulosa cell is an increase in progesterone synthesis. Furthermore, in the theca cell L H promotes mitosis and progesterone and androgen synthesis. With the granulosa and theca cells working in concert, estradiol is released into the intracellular space where it feeds back on both cell types. Estradiol promotes FSH-receptor and estradiol-receptor expression on the granulosa cell, and LH-receptor expression on the theca cell, further enhancing the actions of these hormones. Additionally, estradiol feeds back on the anterior pituitary to increase GnRH secretion as well as the pituitary response to it [Burger, 1981]. The increase in GnRH and the pituitary's sensitivity to it, increases L H secretion and decreases FSH secretion. This feedback further promotes the ripening of the follicle in preparation for ovulation. Ovulation is induced by L H in concert with numerous peptides, steroids, prostaglandins, leukotrienes and neurotransmitters, including but not limited to: collagenase, epidermal growth factor, relaxin, GnRH, vasoactive intestinal polypeptide, progesterone, prostaglandin F2a, prostaglandin E 2 and possibly prostaglandin I 2 . For further information on the mechanisms of ovulation, see the following reviews: Channing et al., 1980; Haour and Lang, 1978; Leung and Steele, 1992; Suzuki and Takahashi, 1974; Turgeon, 1980; Wu and Prazak, 1974; Yen, 1977. B. Pregnancy Following ovulation, the ovum is transported down the fallopian tubes where fertilization occurs, usually within 12 to 24 hours post-ovulation. If fertilization has been successful the zygote will pass through the fallopian tubes (2-3 days) and implant in the uterus (approx. 3 more days). The key hormones in promoting and maintaining pregnancy are estradiol and progesterone (reviewed below, p. 4). The post-ovulatory follicle differentiates into the corpus luteum following the ovulatory phase. Granulosa cells differentiate into luteal cells account for about 80 percent of the corpus luteum (large luteal cells), with the remainder of luteal cells being derived from the theca interna (small luteal cells). The corpus luteum is the primary source of sex steroids during the luteal phase. Moreover, if fertilization occurs, the luteal phase is maintained beyond its 14 day lifespan by conceptus and/or placental derived hCG, which stimulates steroidogenesis through a c A M P dependent mechanism (Fig. 2). The corpus luteum is maintained until placental derived progesterone levels are adequate to maintain pregnancy, after which time it regresses. The regressed corpus luteum either in pregnancy or in the menstrual cycle is called the corpus albicans. hCG Figure 2. Model of signal transduction pathway for human chorionic gonadotrophin (hCG) stimulated steroidogenesis, in human luteal cells. G - stimulatory G-protein; AC - adenylate cyclase (AC); cAMP - cyclic adenosine monophosphate; and PKA - protein kinase A. C. The Sex Steroids The Progestins and Estrogens The key sex steroid hormones are the progestins and the estrogens. Progestins are known as the pro-gestational hormones for their ability to maintain, prepare for and promote pregnancy. The progestins include progesterone, l7a-OH-progesterone and 20a-OH-progesterone, of which progesterone is the most potent. The estrogens are responsible for the secondary sex characteristics of the female, follicle maturation, and are behavioural modifiers in animals and possibly humans. In animals, the estrogens are reported to promote estrous behaviour (or mating behaviour), hence the name estrogen (a derivative of 'estrous-genic'). The estrogens include 17B- estradiol (commonly known as estradiol or E 2 ) and estrone, of which estradiol is the most potent. The Synthesis of Progesterone and Estradiol The sex steroids are synthesized in the ovarian granulosa, luteal and thecal cells where they are known to have paracrine and/or autocrine actions in addition to their peripheral endocrine effects. Progesterone and estradiol are synthesized from cholesterol, which may be obtained from dietary sources or synthesized from two acetyl-CoA molecules, by a series of enzymatic reactions [Stryder 1988; Schroepfer 1982; Fielding 1985; Nebert and Gonzales 1987; Granner 1988]. The side chain of cholesterol is cleaved by P450-side chain cleavage enzyme (P450-SCC) or 20,22-desmolase to produce pregnenolone (Fig. 3). Pregnenolone may then be converted to progesterone by a complex of 3B-ol-dehydrogenase and A4> 5-isomerase. Through a series of enzymatic reactions, progesterone or pregnenolone may be converted to estradiol. One of the key enzymes in this conversion is aromatase. Aromatase and P450-SCC are highly regulated enzymes, as discussed below. For a more complete description of these synthetic pathways, please refer to Figure 3. The Two-Cell Model of Steroid Biosynthesis In the human ovary it requires the co-operation of two different cell types, the theca interna cell and the granulosa cell, to produce estrogen [Moon et al., 1978; Moon et al., 1981; Tsang et al., 1982; Moon and Duleba 1982; Takahashi et al., 1984]. This two-cell model of steroidogenesis is depicted in Figure 4. Briefly, L H stimulates c A M P production in the theca 7 Cholesterol Pregnenolone 1 7-OH-Pregnenolone Dihydro Epiandrosterone Androstenediol - Progesterone if 1 7-OH-Progesterone I Androstenedione Testosterone Estrone jo Estradiol Figure 3. The synthetic pathway of the female sex steroids progesterone and estradiol, from cholesterol. The enzymes involved in sex steroidogenesis include: 1) P450 side chain cleavage enzyme or 20,22-desmolase, 2) 17-hydroxylase, 3) 17,20-desmolase, 4) 178-OH-steroid dehydrogenase, 5) 38-d-dehydrogenase and A 4 - 5-isomerase, and 6) aromatase. In the studies presented herewithin, androstenedione is added to the culture medium to provide an aromatizable substrate for the production of estradiol. 8 Theca Interna Cell Granulosa Cell Endocr ine Figure 4. The two cell model of steroidogenesis in the human ovary. Luteinizing hormone (LH) stimulates cAMP production in the theca interna and granulosa cells, while follicle stimulating hormone (FSH) stimulates cAMP production in the granulosa cell. Progesterone (P4) is produced via a cAMP-mediated increase in desmolase (1) activity, in both cell types. However only the theca interna cells are able to convert progesterone to aromatizable androgens. In order for the granulosa cell to produce estrogens an exogenous source of aromatizable androgens is necessary. These exogenous androgens are provided through diffusion from the theca interna cells to the granulosa cells. The conversion of androgens to estrogens is achieved by cAMP-mediated increase in aromatase (2) activity. interna and granulosa cells, while FSH stimulates c A M P production in the granulosa cell. Progesterone is produced in both cell types via a cAMP-mediated increase in desmolase activity. However, only the theca interna cells are able to convert progesterone to aromatizable androgens. In order for the granulosa cell to produce estrogens, an exogenous source of aromatizable androgens is necessary. These exogenous androgens are provided through diffusion from the theca interna cells to the granulosa cells. In an in vitro culture system, it is necessary to provide granulosa cells with exogenous androgens (usually androstenedione or testosterone), if one wishes to measure estradiol production in response to stimuli. The conversion of androgens to estrogens is achieved by cAMP-mediated increase in aromatase activity. Sex Steroid Receptors Following synthesis, the sex steroids have local effects within the ovary, as well as endocrine effects throughout the body and hypothalmo-pituitary axis [Goebelsmann 1979; McCarty et al., 1983; McNatty et al., 1979a and b; Schroepfer 1982; Rasmussen and Yen 1983; Nebert and Gonzalez 1987]. The majority of progestins and estrogens circulate bound to binding proteins including albumin, Cortisol binding protein and sex steroid binding protein. Only one to two percent of these steroids circulate in their free form. Due to the hydrophobic nature of steroid hormones, they readily pass though cellular membranes, both from their sites of production and into their sites of action. Thus, these hormones do not have membrane receptors. This has the advantage that it eliminates the need for a secondary messenger system. Steroid receptors belong to a super-family of receptors which also include the thyroid hormone, retinoic acid and vitamin D receptors [McCarty et al., 1983]. The progestin and estrogen receptors each possess a DNA binding domain and a ligand binding domain. Following binding of the steroid to its receptor, the receptor-steroid complex attaches to its D N A acceptor site. This complex forms a site for the binding of RNA polymerase to the chromosome, and results in the production of RNA transcripts and their associated proteins. These de novo proteins are responsible for steroid-mediated cellular actions. Sex Steroid Sites of Action The regulation of the human menstrual cycle, conception and pregnancy by progesterone and estradiol is a body-wide process involving the brain, pituitary, ovary, uterus, fallopian tubes, vagina, breasts and other tissues. The following is a brief review of the effects of progesterone and estradiol on these tissues. As the hypothalamopituitary-gonadal axis has already been reviewed, this section will not discuss them further [see Mahesh 1985; Franz 1988; Tonetta 1989; and Genuth 1988 for further review]. Likewise, there are too many sex steroid-dependent functions throughout the body to discuss them all in the context of this thesis. 1. The Fallopian Tubes Following ovulation estradiol assists in the capture and transportation of the ovum down the fallopian tube [Spilman and Harper 1975; Genuth 1988; Janzen 1995]. Estradiol is responsible for the widening and undulatory movement of the fimbria which assists in catching the ovum and directing it into the fallopian tube. The number of cilia on the surface of fallopian tube epithelial cells is increased by estradiol. Once in one of the fallopian tube, the ovum is transported toward the uterus by an estradiol-dependent beating of epithelial cilia and fallopian tube contractions. During the luteal phase, progesterone maximizes the cilliary beating and increases nutrient secretion into the lumen of the fallopian tubes. These nutrients may help to maintain the viability of both the ovum, sperm and eventually the zygote if fertilization occurs. 2. The Uterus Elevated estradiol levels during the follicular phase are responsible for an increase in endometrial thickness (3- to 5-fold), and elevated levels of watery, strand-like mucus [Bazer et al., 1979; Janne 1981]. The increase in endometrial thickness may be in preparation for implantation, and establishes a nutritive base for the new conceptus. Elevated levels of fluid, strand-like mucous create channels to allow sperm to pass freely through the cervix into the uterus. Thus, estradiol is responsible for creating a uterine environment conducive to fertilization and implantion. On the contrary, elevated progesterone levels reduce mitotic activity and the proliferation of the endometrium, although it is responsible for maintenance of the decidual lining [Genuth 1988]. Progesterone increases glycogen accumulation in vacuoles at the base of endometrial cells, and stimulates the movement of these vacuoles towards the lumen during the luteal phase. These glycogen stores provide an energy rich environment for the zygote within the lumen during implantation. Progesterone reduces the levels of mucus, and changes the mucus from fluid to viscous. These changes assist in implantation of the conceptus within the uterus. 3. The Vagina Estradiol assists in successful copulation by improving vaginal conditions such as increasing mucous secretions, mucus fluidity, epithelial thickness (protective), vaginal plasticity and external genitalia size [Genuth 1988]. Following the ovulatory phase when it would be less appropriate for copulation to occur, progesterone reduces secretions, secretion fluidity and the numbers of cornified cells [Genuth 1988]. 4. The Breasts In preparation for pregnancy, estradiol promotes the development of the breasts by increasing fat deposits (i.e. energy stores) and the number of lobules [Mauvais et al., 1986; Mauvais et al., 1986; Mauvais et al., 1987]. These changes are in concert with progesterone- mediated alveoli formation. Thus, should pregnancy occur, the breasts will be partially prepared to fulfill their role as a primary nutrient dispensary for the neonate. 5. Other Progesterone-Dependent Actions A number of other tissues are dependent on the sex steroids for their reproductive functions [Siiteri 1987]. Progesterone acts as a primary substrate for the production of Cortisol and aldosterone by the foetal adrenal gland. Additionally, the crucial inhibition of the maternal immune response to foetal antigens is regulated by progesterone [Genuth 1988]. Progesterone also suppresses uterine contractions and expulsion of the foetus from the uterus. Progesterone also acts as a pyrogen, through a thyroid gland mediated increase in metabolism, which elevates body temperature. Behavioural effects have also been reported [Barfield et al., 1984]. 12 D. The Eicosanoids Prostaglandins, Thromboxanes and Leukotrienes Membrane phospholipids can be metabolized into a class of hormones called the eicosanoids [Smith 1985; Mayes 1988]. The eicosanoids are further broken up into one of three sub-families, including the prostaglandins (PG), thromboxanes (Tx) and leukotrienes (LT). These hormone sub-families contain a number of hormones each designated by a letter such as A , B , C, et cetera. This character is further followed by a subscript number indicating the number of double bonds contained in the hormone. Furthermore, there are three groups within each of these three eicosanoid sub-families: those with one, two or three double bonds (Table 1). For example, the double bonded form of prostaglandin E is abbreviated PGE2. Phospholipases and Arachidonic Acid The main precursor to eicosanoid synthesis is a twenty carbon, four double-bond fatty acid called 5, 8, 11, 14-eicosatetraenoic acid, commonly known as arachidonic acid (AA). The primary enzyme responsible for the production of A A is phospholipase A2 ( P L A 2 ) , although a number of other lipases are capable of producing A A from glycerophospholipid precursors [Waite 1985; Dennis 1983]. Phospholipase A2 is a hydrophobic, membrane-bound esterase which is active at the water-lipid interphase [Waite 1985]. The family of phospholipases consists of at least five members including phospholipase A1 , A 2 (B), C and D, each of which cleaves phospholipids at a unique site (Fig. 5) [Mayes 1988]. Normal saturation kinetics do not apply to membrane-bound phospholipases, as they do to soluble esterases. Compared to soluble esterases, phospholipases are exposed to extremely high concentrations of substrate molecules (phosphlipids), which are pre-oriented toward the catalytic site due to their polarity [Waite 1985]. Moreover, phospholipase enzyme products are hydrophilic, a property which enhances their diffusion away from the enzyme and the hydrophobic membrane, thus reducing product inhibition of substrate catalysis. Phospholipases can be greater than 1000 times more active than soluble esterases, due to their aforementioned properties. 13 Table 1. The Eicosanoid Superfamily of Hormones. Group I Group I I Group I I I Fatty Acid Precursor 8,11,14- Eicosatrienoate Arachidonic acid 5,8,11,14,17- Eicosapentaenoate Enzyme* COX Lipox COX Lipox COX Lipox Eicosanoid PGEi TxAl LTA 3 PGD 2 TxA 2 LTA 4 PGD 3 TxA 3 LTA 5 PGFx LTC 3 PGE 2 L T B 4 PGE 3 L T B 5 LTD 3 PGF 2 a L T C 4 PGF3 LTC5 PGI 2 LTD 4 LTE 2 P G - Prostaglandin; Tx - thromboxanes; L T - leukotrienes. Group I, II, and III possess 1, 2 and 3 double bonds, respectively. * the key enzyme responsible for metabolism from the above fatty acid precursor, including: cyclooxygenase (COX) and lipoxygenase (Lipox). In a number of systems, the reported pathway for the activation of P L A 2 involves a receptor mediated rise in intracellular calcium, which activates phospholipase C (PLC). Phosphatidyl inositol (PI) cleavage by P L C produces diacylglyceride which can either be converted directly to A A by glyceride lipase(s), or may stimulate diacylglyceride (diglyceride) dependent-protein kinase C (PKC) which in turn activates P L A 2 , via removal of tonic inhibition by a protein inhibitor (Fig. 6) [Waite 1985]. Other factors influencing the activation of P L A 2 include membrane charge (and associated enzyme pH), density of phospholipids and membrane fluidity. Factors which affect these three parameters will alter P L A 2 activity [Waite 1985] and A A production. Finally, anti-inflammatory corticoids can block the P L A 2 activity. Eicosanoid Production from Arachidonic Acid Two isoforms of cyclooxygenase (COX-I, constitutive and COX-II , inducible) are capable of converting arachidonic acid to prostaglandin G 2 (PGG 2 ; Fig. 7). Cyclooxygenase I and II are selectively inhibitable by numerous anti-inflammatory agents. Inhibitors of COX-I include acetylsalicyclate and indomethacin [Vane 1971; Roth and Siok 1978], while dexamethasone and other modern nonsteroidal anti-inflammatory agents inhibit COX-II [McCarthy 1995]. Hydroperoxidase converts P G G 2 to prostaglandin H 2 (PGH 2), the precursor to group II or double bonded prostaglandins and thromboxanes. PGH-PGE isomerase converts PGFf 2 to P G E 2 , which can be further converted P G F 2 a by E-2-9 ketoreductase. Theoretically, P G F 2 o t could be produced directly from P G H 2 by a reductase, although this pathway has not been demonstrated [Smith 1985]. P L C P L A , 15 Figure 5 . Phospholipase cleavage (hydrolysis) sites on phospholipids. Phospholipases are capable of hydrolysing the number one acyl bond, number two acyl bond, glycerophosphate bond or the base group. The number one acyl bond is hydrolysed by phospholipase A i (PLAi) or PLB , while the number two acyl bond is hydrolysed by phospholipase A 2 or B (PLA 2 or PLB). The phosphodiesterases, phospholipase C (PLC) and phospholipase D (PLD) hydrolyse the glycerophosphate bond and base group, respectively. 16 Calcium Mobilization Phosphatidyl Inositol Diglyceride 4 PLA Glyceride Lipase Arachidonic Acid KA c\ r i f\ c\ I \ § f P r I f i Phosphatidyl Choline Arachidonic Acid Figure 6. Arachidonic acid production in a model system. This model is based on research performed on platelets. In platelets calcium is mobilized via some external stimuli. Elevated calcium levels activate phospholipase C (PLC) which liberates diglyceride from phosphatidyl inositol. Glyceride lipase can convert diglyceride to arachidonic acid directly. Alternately, diglyceride may activate phospholipase A 2 (PLA 2), which converts phosphatidyl choline to arachidonic acid. 17 Arachidonic Acid C O O H C y c l o o x y g e n a s e I o r II PGG COOH H y d r o p e r o x i d a s e PGH. C O O H C O O H Figure 7. Synthesis of prostaglandin F 2 a ( P G F 2 a ) from arachidonic acid. Enzymes are in red. Arachidonic acid may be converted to prostaglandin G 2 (PGG 2) by cyclooxygenase I (constitutive) or cyclooxygenase II (inducible). Hydroperoxidase converts PGG 2 into PGH 2. Prostaglandin E 2 is produced from PGH 2 by PGH-PGE isomerase. The enzyme E - 2 - 9 ketoreductase converts PGE 2 to prostaglandin F 2 a (PGF2a) by hydroxylation of the ketone group of PGE 2. Theoretically, a reductase could produce P G F 2 a by reducing PGH 2, although this pathway has never been demonstrated. Prostanoid Receptors Numerous prostanoid receptors have been cloned from mammalian tissues. These receptors include the P G D 2 receptor (DP), the PGE2 receptors ( E P l 5 E P 2 and EP3-family), the P G F 2 o t receptor (FP) and the prostacyclin or P G I 2 receptor (IP) [Lake et al., 1994; Abramovitz et al., 1994; Adam et al., 1994; Boie et al., 1994 and 1995; Funk et al., 1993]. Based on sequence analysis, these receptors all appear to belong to the seven-transmembrane G-protein coupled receptor family. The DP, IP and EP3-family of receptors are all coupled to c A M P regulation [Adam et al., 1994; Boie et al., 1994 and 1995; An et al., 1994], while the DP, FP, EPx and EP 3-family of receptors are coupled to rises in intracellular calcium [Abramovitz et al., 1994b; Adam et al., 1994; Boie et al., 1995; Funk et al., 1993; An et al., 1994]. Additionally, the human EP3-family of receptors is capable of inhibiting c A M P production through a pertussis toxin-sensitive G - protein [An et al., 1994]. Prostaglandins as Autocrine/Paracrine Factors Prostaglandins are believed to be autocrine or paracrine hormones. There are numerous lines of evidence pointing to the local nature of prostaglandin actions, these include the following [Smith et al., 1985]: 1) Prostaglandins have a short half life (minutes) in vivo, which probably prevents them from having effects systemically. This short half life is mainly due to local degradation by prostaglandin dehydrogenase(s), and systemic degradation by the lung. 2) Prostaglandins are secreted in short (1-5 min) bursts, likely preventing systemic hormone levels from becoming elevated. 3) Most cells that secrete prostaglandins also possess receptors for these hormones, suggesting that they are acting locally. 4) Almost every tissue produces prostaglandins and prostanoid receptors, although these prostanoids produce radically different actions from one tissue to another. Inhibition and Degradation of Prostaglandins Cyclooxygenase I and II are short lived enzymes as they are capable of undergoing self- catalyzed destruction. Thus they have been dubbed a suicide enzymes [Smith and Borgeat 1985]. This self catalyzed destruction acts as a negative feedback mechanism on prostaglandin synthesis. As mentioned above, prostaglandins have very short half lives. The rapid degradation of prostaglandins is due to molecular instability, local degradation by tissue specific hydoxyprostaglandin dehydrogenases and systemic degradation in the lung and kidney [Smith and Borgeat 1985]. It has been reported that circulating P G E i , P G E 2 and P G F 2 a are degraded on their first pass through the lung. The lung acts as a filter, by removing virtually all active prostaglandins from the circulatory system. Degradation is achieved by removal of the hydroxyl group at carbon 15 by a NADPH-dependent 15-OH prostaglandin dehydrogenase. Removal of this hydroxyl group reduces the biological activity to ten percent of its original level. Prostaglandin D 2 and P G I 2 are dehydroxylated by another 15-OH dehydrogenase which is specific to these prostaglandins. This second enzyme is found in the kidney. Further degradation of these prostaglandins occurs via the reduction of the A 1 3 double bond by an NADPH-dependent A 1 3 reductase, resulting in 15-keto-13, dihydroprostaglandins which are biologically inactive. Oxidation in the liver and excretion in the urine complete the process. E. Prostaglandin F 2a in Reproduction Localization of Prostaglandin F2a Prostaglandin F 2 a has been detected in the human decidua, amnion, pregnant myometrium and ovary [Satoh et al., 1981; Aksel et al., 1977]. In the human ovary, P G F 2 a has been localized to the follicle and theca-, granulosa- and luteal-cells [Aksel et al., 1977; Patwardhan and Lanthier, 1981; Plunkett et al., 1975]. Further, the presence of P G F 2 a has been detected in the human follicle at all stages of the reproductive cycle [Patwardhan and Lanthier, 1981]. Additionally, P G F 2 a synthesis has been detected in human luteal and stromal tissues where arachidonic acid derived P G E 2 is converted to P G F 2 a , via E-2-9-ketoreductase [Watson et al.,1979; Endo et al., 1988]. Regulation ofPGF2a Production in the Ovary In the ovary, P G F 2 a production is regulated by a number of ovarian hormones including luteinizing hormone, human chorionic gonadotrophin (hCG), interleukin-1, and tumor necrosis factor [Patwardhan and Lanthier, 1981; Plunkett et al., 1975; Mitsuhashi, 1981; Watanabe et al., 1993; Zolti et al., 1990]. In the rabbit, oxytocin has been suggested as another secretagogue [Fuchs, 1988]. Thus far, propranolol and norepinephrine are known to be receptor-mediated inhibitors of P G F 2 a production in the human ovary [Bennegard et al., 1984]. However, hCG or c A M P pretreatment has been shown to inhibit the antigonadotrophic actions of cloprostenol ( P G F 2 a analogue) in the luteal cell [Michael and Webley, 1991b]. Functions ofPGF2a Prostaglandin F2« has been shown to mediate functional luteolysis and luteal regression, in the mammalian ovary [Michael and Webley, 1991b; Jalkanen et al., 1987; Korda et al., 1975; Grinwich et al., 1976; Moon et al., 1986; Hanzen, 1984; Richardson and Masson, 1980]. However, the presence of PGF2« in the ovary only roughly correlates with this action, as P G F 2 a levels are highest in mid- rather than late luteal-phase in the human. This discrepancy has been accounted for with the examination of P G E 2 , which is known to counteract P G F 2 a induced luteolysis. Prostaglandin E2 levels in mid-luteal phase are high while they are not in late-luteal phase. Thus it is postulated that during the mid-luteal phase, the ratio of P G F 2 a : P G E 2 is low and not suitable for luteolysis, although in the late-luteal phase this ratio is high allowing for luteolysis in the human [Pathwardhan and Lanthier, 1985]. Prostaglandin F 2 a is known to inhibit L H - , hCG- and PGE2-stimulated progesterone production (functional luteolysis). Potential mechanisms for functional luteolysis include the inhibition of LH/hCG receptor levels and/or binding [Luborsky et al., 1984], a reduction in adenylate cyclase activation [Dorflinger et al., 1984], increased progesterone catabolism through 20-alpha-hydroxysteroid dehydrogenase [Moon et al., 1986] and possibly an increase in c A M P phosphodiesterase activity via P K C [Lahav et al., 1989; Michael and Webley, 1991a]. Luteal regression is believed to be effected through a PGF2 a-mediated reduction in blood flow to the corpus luteum and apoptotic cell resorption [Hanzen, 1984; Khan et al., 1989; Richardson and Masson 1980; Quirk et al., 1995]. The luteotrophic action of P G F 2 a appears to be time-, concentration- and species- dependent. These actions are reported to be strongest in the mid-luteal phase and during pregnancy of investigated species [Khan et al., 1989; Michael and Webley, 1993; Webley et al., 1989; Suginami et al., 1976]. Moreover, in vitro and in vivo studies have demonstrated the luteotrophic effects of P G F 2 a in the presence of gonadotropin [Suginami et al., 1976], suggesting that the mere presence of gonadotrophins is not sufficient to initiate a luteolytic response from P G F 2 a - PGF2a in Pregnancy Studies have demonstrated that temporal and confounding relationships of ovarian hormones may be important in preventing C L regression, should pregnancy occur [Michael and Webley, 1991b]. For example, P G F 2 a is well accepted as being able to inhibit hCG-stimulated progesterone production in studies where these two hormones are administered together. However, when hCG treatment preceeds PGF201, this luteolytic effect is not seen [Michael and Webley, 1991b]. Similarily, prolactin, L H and FSH, alone and in combination, were not capable of blocking PGF2 a-induced luteolysis. However, pretreatment with prolactin, FSH plus L H prevented PGF2 a-induced luteolysis in 11/14 hamsters [Harris and Murphy, 1981]. The blockade of luteolysis by pretreatment with hCG is suggested as being a means by which the placenta rescues the corpus luteum (CL) from PGF2a-mediated regression [Webley et al., 1991], thus allowing pregnancy to proceed. Prostanoid Receptors in Reproductive Tissues Prostaglandin F 2 a lowers both gonadotropin- and prostaglandin E2-stimulated rises in c A M P , as well as increases intracellular calcium and inositol phosphates in reproductive tissues [Davis et al., 1989; Currie et al., 1992; Pepperell et al., 1989; Lahav et al., 1987]. It is unknown if the actions of P G F 2 a are exerted through a single or multiple-receptors. Prostaglandin F 2 a and P G E 2 are both present and active in the human granulosa and luteal cells [Grinwich et al., 1976; Richardson and Masson, 1980; Pathwardhan and Lanthier, 1985; Satoh et al., 1981; Watson et al., 1979]. Thus, it is probable that multiple prostanoid receptors exist in these cells. Furthermore, the currently cloned prostanoid receptors all possess varying degrees of cross- reactivity with P G E 2 and P G F 2 a [Lake et al., 1994; Abramovitz et al., 1994; Adam et al., 1994; Boie et al., 1994 and 1995; Funk et al., 1993; An et al., 1994]. Ligand binding studies have demonstrated that the human PGF 2 a-receptor binds P G F 2 a with an equilibrium dissociation constant (Kd) of approximately 1 to 1.63 nM [Abramovitz et al., 1994; Lake et al., 1994]. The binding characteristics of the rat P G F 2 a - R suggest a two site model, with a high affinity site (Kd = 3.9 nM) and a lower affinity site (Kd = 34 nM) [Lake et al., 1994]. PGF2a Signal Transduction Prostaglandin F 2 a-receptor cDNA sequences appear to suggest a G-protein coupled receptor [Lake et al., 1994; Abramovitz et al., 1994], as with other cloned prostanoid receptors [Adam et al., 1994; Boie et al., 1994 and 1995; Funk et al., 1993], although pharmacological studies toward this end have not been done in the human ovary. Immunocytochemical studies have localized four different G-protein alpha subunits to the human granulosa-luteal cell including G a S , G a i 3 , G a i l 2 and G a p (namely G a q and G a l l ) , but not G a G [Lopez et al., 1995]. Furthermore, it has been demonstrated in these cells that c A M P production is regulated by the ratio of G a S and G a i , while rises in inositol phosphates and intracellular calcium appear to be regulated by G a p (namely G a q and G a n ) and G a j [Lopez et al., 1995]. Exposure of mammalian granulosa or luteal cells to PGF2a has been shown to stimulate phospolipase-C and its downstream pathways [Dorflinger et al., 1984; Abayasekara et al., 1993; Davis et al., 1989; Currie et al., 1992; Michael et al., 1993]. It has been suggested that P G F 2 a is inhibiting cAMP- and progesterone-production via this rise in inositol phosphates and/or calcium [Leung, 1985; Steele and Leung, 1993]. A direct link between these two pathways has not been clearly established, as numerous reports have demonstrated PGF 2 a-mediated luteolysis in the presence of inositol phosphate, calcium and calmodulin inhibitors [Jalkanen, 1987; Michael and Webley, 1993; Pepperell et al., 1989; Lahav et al., 1987]. Moreover, P G F 2 a and GnRH stimulate phospholipase-C (PLC) in young, and mid but not in old corpora lutea, suggesting that inositol phospholipid metabolism by itself is not sufficient to explain the luteolytic effects of these hormones [Lahav et al., 1988; Endo et al., 1992]. Further confusing the issue, there are reports of P L C products stimulating progesterone production. Luteinizing hormone can stimulate [Davis et al., 1989; Richards et al., 1995], and has been shown to even potentiate PGF2a-stimulated IP3 production [Davis et al., 1989]. Thus, the possibility of these messengers being responsible for the luteotrophic effects of P G F 2 a also exists. Prostaglandin-F2a is known to increase P K C [Abayasekara et al., 1993a,b] and intracellular calcium levels [Currie et al., 1992]. Additionally, P K C activators have been shown to reduce hCG-stimulated c A M P levels. These results suggest that P G F ^ exerts its inhibition of hCG-stimulated c A M P and progesterone production via P K C [Abayasekara et al., 1993a,b]. Furthermore, it is believed that inhibition of hCG-stimulated c A M P levels may occur at the level of G s , as cholera toxin stimulated progesterone production is blocked by P G F 2 a . Clinical Applications ofPGF2a In the female, P G F 2 a has been utilized for contraception and the induction of abortion or parturition [Concannon and Hansel 1977; Lau et al., 1980; Cameron and Baird 1988; Baird et al., 1988]. Conversely, cyclooxygenase inhibitors such as indomethacin have been used effectively to arrest premature labour and delivery [Manaugh and Novoy 1976; Fuchs et al., 1976]. Prostaglandin F 2 a is capable of contraceptive effects in the human as well as in some other mammals [Singh and Dominic, 1986; Bilinska and Wojtusiak, 1988; Orlicky and Williams, 1992; Chinoy et al., 1980]. Investigation has revealed the presence of PGF2a-receptors on the Leydig cell, although not on cells of the tunica albuginea, subcapsular- or peritubular-stroma, peritubular boundary tissue, vasculature, spermatogonia, spermatocytes, spermatids, spermatozoa or Sertoli cells [Orlicky and Williams, 1992]. In the mouse, suppressed spermatogenesis and a significant reduction in the weights of the testis, epididymis and accessory sex glands have been reported following P G F 2 a administration [Singh and Dominic, 1986]. Moreover, seminiferous tubules were found to be devoid of spermatazoa, while Leydig cells showed atrophy. Interestingly, these regressive changes were reversible, as 56 days after drug withdrawl a normal state was achieved [Singh and Dominic, 1986]. Prostaglandin F 2 a treated rats exhibited reduced testicular- and epididymal- weight, while the weight of their seminal vesicle and ventral prostate increased. Additionally, altered morphology and reduced density- and motility-spermatazoa were seen [Chinoy et al., 1980]. Aside from morphological changes, Leydig cell-androgen production has been reported to be reduced by a PGF 2 a-mediated inhibition of delta 5,3 beta-hydroxysteroid dehydrogenase activity [Bilinskaand Wojtusiak, 1988]. F. Gonadotrophin-Releasing Hormone 25 GnRH Functions GnRH- and GnRH-receptor mRNA have recently been isolated in the human granulosa cell, indicating that GnRH probably has important local actions within the ovary [Peng et al., 1994]. GnRH is a decapeptide that was first discovered in the hypothalmo-pituitary axis. As mentioned above GnRH is the primary mediator of gonadotrophin release. Gonadotropinn- releasing hormone has also been shown to have luteolytic as well as luteotrophic effects [Leung 1985] in some mammals. Buserelin (a GnRH agonist) has been reported to block hCG, P G E 2 epinephrine and cholera-toxin stimulated progesterone production, as well as potentiating PGF 2 a - inhibi t ion of c A M P production [Massicotte, 1984]. On the contrary, GnRH administration has been utilized to maintain pregnancy or enhance fertility in the cow [Farin and Estill 1993; Funston and Seidel 1995]. GnRH Localization In humans and other mammals, at least two molecular forms of GnRH have been demonstrated in the brain, ovary and other tissues [King et al., 1990; Ireland et al., 1988; Aten et al., 1987; Behrman et al., 1989; King et al., 1989]. The amount of GnRH in luteal tissues is reported as being proportional to the weight of these tissues, although the concentration of GnRH peptides drops as the corpus luteum develops. While GnRH and/or GnRH peptides are found in numerous nonovarian tissues, in cattle they appear to be relatively concentrated in granulosa cells [Ireland et al., 1988] and pituitary. GnRH Receptor Gonadotrophin-releasing hormone is capable of reducing progesterone production and interrupting reproductive cycles and pregnancy in the rat [Clayton et al., 1979]. These actions have been attributed to specific high-affinity receptors present in luteal cell membranes [Clayton et al., 1979; Latouche et al., 1989]. Additionally, this action appears to be autocrine in nature as both GnRH- and GnRH receptor (GnRH-R)-mRNA have been detected within the human granulosa-luteal cell [Peng et al., 1994]. Moreover, GnRH is reported to autoregulate its own mRNA level as well as those of GnRH-R. Conversely, hCG has been shown to down-regulate GnRH receptor mRNA levels. Messenger RNA for GnRH has also been cloned from the rat corpus luteum, where it was found to have an identical sequence to the rat anterior pituitary GnRH receptor [Whitelaw et al., 1995]. Furthermore, the expression of GnRH-R gene in granulosa cells is purported to be individually regulated for each follicle, to persist in the corpus luteum and is expressed in atretic follicles [Whitelaw et al., 1995; Minaretzis et al., 1995]. In fact atretic follicles appear to exhibit the greatest degree of GnRH-R gene expression, suggesting that GnRH is important in the induction of follicular atresia [Bauer and Jameson, 1995]. GnRH Signal Transduction In the pituitary gonadotroph, GnRH is known to stimulate polyphosphoinositide breakdown [Kiesel et al., 1986]. On the other hand, both GnRH and NaF-stimulated L H release can occur in the absence of inositol phosphate production [Hawes et al., 1992], suggesting that inositol triphosphate is not an essential second messenger for the release of L H . The question remains which second messengers are necessary for the release of L H from the gonadotroph. Phosphatidic acid, a phospholipase D product, has been reported to increase dose- and time- dependently (2-3 fold; 1-2 min) following GnRH analogue administration in alpha T3-1 cells [Netiv etal., 1991]. Pituitary adenylate cyclase activating polypeptide (PACAP)-stimulated c A M P production is inhibited by GnRH in the alpha T3-1 gonadotroph cell line, although GnRH did not inhibit PA C A P binding to gonadotrophs nor forskolin- or cholera toxin-stimulated c A M P production. Thus it has been suggested that the inhibitory effects are exerted at early stages in the signal transduction pathway distal to receptor occupancy but preceeding c A M P production [McArdle et al., 1994], possibly at the level of a G-protein. Gonadotrophin-releasing hormone and P G F 2 a both inhibit c A M P production in the corpus luteum. Phosphatidyl inositol (PI) and phosphatidic acid (PA) turnover occurs rapidly (2 and 5 min respectively) with a mean effective dose of 15 and 100 nM for GnRH and P G F 2 a , respectively [Leung, 1985; Davis et al., 1984; Davis et al., 1986]. When co-treatment with the hormones is performed, their effects appear to be additive. Incidentally, A23187 (a pore-forming calcium ionophore) also causes a dramatic increase in PA and PI turnover. Dibutryl-cAMP and 8-Br-cAMP attenuate GnRH and P G F 2 a stimulated PA and PI turnover. The biproducts of PLC activity (IP3 and DAG) mobilize intracellular calcium, activate P K C and release arachidonic acid [Davis et al., 1986; Shinohara et al., 1985]. The similarity of GnRH and P G F 2 a responses has led to the suggestion that they may share post-receptor signalling mechansisms [Leung, 1985]. GnRH Mechanism of Action Studies in the rat have demonstrated GnRH-mediated inhibition of progesterone production through increased activity of 20-alpha-hydroxysteroid dehydrogenase, inhibition of pregnenolone production and reduced activity of P450SCC and 3-beta-hydroxysteroid dehydrogenase activity [Jones et al, 1983; Srivastava et al., 1994]. The mechanisms by which GnRH exerts its luteotrophic effects are not reported in the literature. Clinical Applications of GnRH Potent and long-lasting GnRH analogues (super-active agonists) originally developed with fertility promotion in mind have, in fact, proven to have anti-fertility properties in the male and female [Molcho et al., 1984; Bhasin et al, 1984; Nillius, 1985]. These compounds have been applied to numerous therapeutic applications in the female including contraception, treatment of central precocious puberty, and sex steroid-dependent benign and malignant diseases of the reproductive organs [Nillius, 1985]. One of the most common uses of GnRH agonists is the down-regulation of pituitary function in preparation for IVF treatment. Inhibition of ovulation by continuous GnRH agonist administration appears to be safe, reliable and reversible in women [Nillius, 1985]. However, attempts to inhibit luteal function, induce luteolysis or early abortion have not been very successful [Nillius, 1985]. In the human male, high dose GnRH administration interrupts testicular function leading to azoospermia. However, the incompleteness of this azoospermia and unacceptable side effects (loss of libido and potency) rule out the use of GnRH as a male contraceptive [Nillius, 1985]. There have, however, been reports of reduced side effects with co-administration of testosterone [Bhasin et al, 1984; Nillius, 1985]. Interestingly, GnRH is also capable of improving rather than impairing fertility in some species such as the bovine [Farin and Estill 1993; Funston and Seidel 1995]. 28 II-HYPOTHESIS Prostaglandin F 2 o t is a multi-functional hormone capable of luteolytic and luteotrophic effects in the human granulosa-luteal cell. Moreover, these effects are time-, concentration- and confounding factor-dependent. Ill - SPECIFIC OBJECTIVES A . To define the steroidogenic response of human GLCs to P G F 2 a with respect to the effects of time in culture, hormone concentration and follicle-size. B. To examine the potential interactions of P G F 2 a and GnRH with respect to steroidogenesis. C. To examine the potential interactions of P G F 2 a and PGE^ with respect to steroidogenesis. D. To define the signal transduction pathways involved in PGF2a-mediated luteolysis. Additionally, to define the signal transduction pathway(s) or mechanism(s) by which P G F 2 a exerts its luteotrophic actions. E. To examine the regulation of P G F 2 a - R mRNA levels by P G F 2 a . For a diagramatic depiction of the specific objectives which these studies sought to satisfy please refer to Figure 8. For the rationale (p. 3), results (p. 54), discussion (p. 117) and a synopsis of the findings (p. 143) for each of these objectives refer to the corresponding character (i.e. A , B , C, D & E) in the respective section. r Figure 8. A diagramatic depiction of the specific objectives to be satisfied in these studies. Note that the characters A, B, C, D and E refer to the specific objectives presented above (p. 1). These studies sought to examine the following: A) the effects of PGF2« on progesterone and estradiol production; B) the potential interactions of GnRH and PGF2a on steroidogenesis; C) the potential interactions of PGE2 and PGF2« on steroidogenesis; D) the signal transduction pathways involved in PGF2a-mediated luteolysis; and E) the effects of PGF2a on PGF2a-receptor mRNA levels. For the rationale (p. 3), results (p. 54), discussion (p. 117) and a synopsis of the findings (p. 143) for each of these objectives please refer to the corresponding character (i.e. A, B, C, D & E) in the respective section. 30 IV-RATIONALE Progesterone and estradiol are key hormones in the regulation of all aspects of the reproductive cycle and pregnancy (as reviewed above, p. 12). Thus the examination of the regulation of these two hormones by P G F 2 a should reveal, in a very real sense, its effects on reproduction as a whole. If PGF201 were to regulate either of these two hormones in any significant fashion, this would suggest that this hormone is a very important regulator of the human female reproductive system. Reports on the effects of PGF2« on estradiol production are scant to non-existent. Thus the underlying studies report estradiol in addition to progesterone responses wherever possible (i.e. sample volume permitting). The rationale for each group of studies corresponding to the specific objectives follows. A. The Effects of PGF2a on Steroidogenesis Prostaglandin F2a-receptors have been demonstrated in and have been recently cloned from human ovarian cells. These findings suggest that P G F 2 a may play an important role in the regulation of ovarian function. However, very few functional studies have been performed in the human granulosa cell. Thus the role of P G F 2 o t remains unclear. The conditions under which the luteotrophic and luteolytic functions of P G F 2 a exist have not been adequately defined. Furthermore, the majority of previous reports examined the effects of P G F 2 a in the piM range of concentrations, while the reported equilibrium dissociation constants (Kd) of cloned prostanoid receptors fall within the nM range [Abramovitz et al., 1994; Lake et al., 1994]. Therefore, these studies utilized P G F 2 a at concentrations ranging from 1 p M to 1 JAM in order to provide a more complete understanding of the nature of estradiol and progesterone responses to PGF2«. There exists the potential that P G F 2 a is not only important in corpus luteum regression, but also that its temporal relationship to hCG may play a role in the maintenance of early pregnancy. Not only is an understanding of P G F 2 a important for basic science, but it could also be important clinically. B. PGF200 and GnRH Interaction Studies 31 Historically, GnRH has been considered a modulator of gonadotrophin secretion from the gonadotroph. As such, GnRH analogs have been used extensively in both experimental and clinical settings for the modulation of the hypothalamopituitary axis in various situations including: I V F [Pellicer et al., 1992; Gonen et al., 1991; Segars et al., 1990], contraception [Fraser, 1993] and control of amenorrhoea [Martin et al., 1990]. It is only recently that GnRH has been identified in the human ovary, and suggested as a potential local regulator of human ovarian function [Oikawa et al., 1990; Peng et al., 1994]. In order to understand any unwanted side- effects of GnRH use in these applications, it is important to further elucidate the local actions of GnRH in the ovary and human granulosa cell. Gonadotrophin-releasing hormone is believed to share common functions (both luteolytic and luteotrophic actions) and signal transduction pathways (IP3 and PKC) with P G F 2 a - As the focus of these studies has been to examine the effects of P G F 2 a in the human ovary, GnRH has been examined primarily in its relationship to potential interactions with P G F 2 a . C.PGF2a and PGE2 Interaction Studies As described above, in the human granulosa-luteal cell P G F 2 a and PGE^ exert opposing actions on cAMP-levels and progesterone-production [Grinwich et al., 1976; Richardson and Masson, 1980; Pathwardhan and Lanthier, 1985; Satoh et al., 1981; Watson et al., 1979]. Prostaglandin F 2 a and PGE^ can decrease or increase cAMP-levels and progesterone-production, respectively. Prostaglandin F 2 a is reported to be at its highest concentration during the mid-luteal phase, although it is reported to be luteolytic during the late-luteal phase. The temporal discrepancy between these two events is accounted for by the levels of P G E 2 during these two phases. It has been suggested that high levels of P G E 2 during the mid-luteal phase may prevent premature corpus luteum regression. However, this explanation fails to account for the fact that PGF 2 a -levels are (perhaps 'unnecessarily') at their highest during the mid-luteal phase when conception and implantation occur. A more comprehensive explanation for the elevated levels of P G F 2 a during the mid-luteal phase may be necessary. Thus, these studies examined the interactions of P G F 2 a and P G E 2 with respect to steroidogenesis in human G L C in vitro. D. Signal Transduction Studies In order to fully understand the actions of a hormone, it is essential to know the mode of these actions. Therefore, these studies undertook to examine the signal transduction pathways involved in PGF2a-mediated luteolysis and luteotrophism. The post-receptor events involved in the luteotrophic and luteolytic actions of P G F 2 a are at present speculative. As the PGF 2 a-receptor [Lake et al., 1994; Abramovitz et al., 1994] is known to belong to the seven transmembrane G-protein coupled receptor family, studies focused on the potential role of G-proteins in the mediation of luteolysis and luteotrophism. Prostaglandin F 2 a has been shown to lower gonadotrophin- and PGE2-stimulated progesterone production (through a lowering of c A M P levels), and G-proteins are known to regulate c A M P levels within these cells. This study examined the role of G-proteins in mediating the effects of P G F 2 a . Pertussis-toxin (PTX) and cholera-toxin (CTX) were utilized to elucidate the potential role of G-proteins in the anti- gonadotrophic actions of P G F 2 a . In order to determine the action(s) of P G F 2 a distal to G- proteins in the signal transduction cascade, these studies examined the ability of P G F 2 a to inhibit progesterone production induced by activators of the adenylate-cyclase, and by cyclic adenosine monophosphate (cAMP) analogues. Previous studies have demonstrated a correlation between the effects of P G F 2 a and a rise in inositol phosphate metabolism [Leung, 1985; Steele and Leung, 1993]. Moreover, a number of studies have demonstrated altered responses to P G F 2 a in the presence of P K C modulators. However, there is much controversy in the literature over the importance of inositol phosphates and P K C in the luteolytic effects of P G F 2 a [Jalkanen, 1987; Michael and Webley, 1993; Pepperell et al., 1989; Lahav et al., 1987]. The underlying studies sought to confirm or disaffirm the existence of of PKC-mediated alteration in the luteolytic effects of P G F 2 a , although an exhaustive examination of this pathway was not performed. A n explanation for the apparent discrepancies in the literature is proposed based on these studies and the known pathways by which prostaglandins are known to act in other systems. E. PGF2orR mRNA Studies Prostaglandin F 2 a is known to act though receptor mediated mechanisms. Thus the regulation of P G F 2 a - R levels is as important as the regulation of P G F 2 a itself. Receptor binding studies have previously demonstrated the presence of P G F 2 a - R in the rat and bovine luteal cell [Brambaifa et al., 1984; Bussmann et al., 1989]. Moreover, the existence of P G F 2 a - R mRNA has recently been demonstrated in the human granulosa-luteal cell [Ristimaki et al., 1997]. However, there have been no reports on the regulation of P G F 2 a - R mRNA levels in response to P G F 2 a . Thus these studies examined the ability of P G F 2 a to regulate P G F 2 a - R mRNA levels. V - MATERIALS AND METHODS 34 A. Granulosa-Luteal Cell Collection and Culture The use of human G L C was approved by the Clinical Screening Committee for Research and Other Studies Involving Human Subjects of the University of British Columbia. Granulosa- luteal cells were harvested in conjunction with oocyte collection in the University of British Columbia's in vitro fertilization program. Throughout the pre-collection period, follicular development was monitored using estradiol assays and ultrasonography. After pituitary down- regulation with a GnRH analogue (Synarel, Syntex; Montreal, PQ) and when estradiol levels were less than 150 pmol/1, follicular development was stimulated with hMG (Humegon 75 IU FSH and 75 IU L H , Organon, Scarborough, O N ; or Fertinorm 75 IU FSH, Serono, Oakville, ON). When three or more follicles reached a diameter greater than 16-18 mm, and estradiol levels were greater than 5000 pmole/1, final maturation was induced with hCG (10,000 IU; Serono). Thirty-two to thirty-six hours later oocytes were harvested using a transvaginal approach. Granulosa-luteal cells were harvested from the follicular fluid following oocyte identification and removal. Following centrifugation (1,000 xg) of the follicular contents, the supernatant was decanted and cells were resuspended in medium 199 (M199; Gibco-BRL Life Technologies, Burlington, ON) supplemented with 10% fetal bovine serum (FBS, Gibco). This step was repeated to provide a second wash. Following the second wash, the resuspended cells were layered on top of a mixture of Percoll (40%; Sigma, St. Louis, MO) diluted in M199. This gradient was centrifuged (1,700 xg) , for 10 min at 22 C. Following collection from the M199/Percoll interphase, granulosa cells were washed and resuspended ( l O ^ l O 6 cells/0.5 ml) in M199, supplemented with 10% FBS, sodium penicillin (100 IU/ml; Gibco) and streptomycin (100 fAg/ml; Gibco), and plated on 48-well plates (Corning, N Y ; 0.5 ml cell suspension/well). Cells to be used in one-day (Di) pre-cultured experiments were cultured for 24 h and then used. However, cells to be used in eight-day (D 8) and twelve to fourteen-day (D12-14) pre- culture experiments had media changed every 2-3 days until the cells had been cultured for a total of 8 or 12-14 days, respectively. A pre-incubation (wash; 1 h) with fresh M199 was performed prior to experimental incubations in order to rinse the cells. A l l incubations were at 37 C, in a humidified, water-jacketed incubator (5% C02 ; ; Forma Scientific Inc, Mississauga, ON). The methods utilized in these studies are depicted in Figure 9. Methods GLC Collection Wash l 40% Percoll I Wash Plating (48 Well ; M l 9 9 ; 1 0 % FBS) I Static Incubation (24 h; M l 9 9 ; Androstenedione 5 x l O ~ 7 M ) Media """"""(Jiis*'^ Cells R I A ( E 2 ' P < } DNA R N A Figure 9. Schematic of methods utilized in studies presented herewithin. In short, human granulosa luteal cells (GLC) are collected during oocyte collection from patients undergoing in vitro fertilization. Cells were washed twice and then separated from red blood cells on a 40% Percoll density gradient (in Medium 199), after which cells were washed twice and plated at 10 3 to 10 4 cells/well (on a 48 well tissue culture plate), in medium supplemented with 10 % foetal bovine serum (FBS). After culturing for one, eight or twelve to fourteen days of culture cells were preincubated (washed) for 1 h in fresh medium and then subjected to hormonal or pharmacological treatments in medium (24 h), supplemented with substrate for estradiol production (androstenedione 5 x I O 7 M). Supernatant was then collected and stored (-20 C) until assayed for progesterone and estradiol. Cells were either extracted for DNA or total RNA which were assayed with a Hoechst dye DNA assay or revefse-tfanscfiption/semi-quantitative polymerase chain reaction, respectively. B. Incubation Experiments A l l treatment regimens were performed in serum free Medium-199 or Dulbecco's Minimum Essential Medium) supplemented with androstenedione (5 x IO*7 M; precursor for estradiol formation). Following a 24 hour treatment, media were removed and stored at -20 C until assayed for progesterone or estradiol concentrations. The hormones and pharmacological agents utilized in these studies are presented in Table 2. The concentrations of these agents utilized were selected based on their known pharmacology. The duration of these release experiments posses potential problems with receptor down- regulation or desensitization, however, this treatment duration was chosen to increase the probability of attaining measurable steroid levels in the release media. Viability was checked post-experiment by the ability of cells to exclude trypan blue. Viability as approximated by this method was greater than 95% at all culture-time periods and under all treatment regimens. The following experiments were performed: 1. Human Chorionic Gonadotrophs Concentration Response Curve: D 8 cultured GLCs were treated with vehicle or hCG (0.001 to 10 IU/ml). 2. Culture Time- and Concentration-Dependent Responses to P G F 2 a and GnRH: Dayi, Dg and D12-14 cultured human GLCs were treated with vehicle, hCG (1 IU/ml) or hCG plus P G F a , (10-" to IO 6 M). A similar experiment was performed with GnRH in place of PGF2a- 3. Follicle Size Dependent Changes in hCG and P G F 2 a Responses: Cells were also separated based on follicle size (> and < 12 mm in diameter) and subjected treatment with vehicle, hCG (1 IU/ml), PGF^ ( 10* M), or hCG plus PGF^ (1CH1 to), at Di. Ideally, follicles should have been separated into more categories. However due to clinical limitations this was not possible. Table 2. Hormones and pharmaca ogical agents utilized in these studies. # Name Abbrev. Class Target(s) Concentration (s) 1 Androstenedione None used Steroid Hormone Precursor Estradiol Biosynthetic Pathway 5x 10"7M 2 Bisindolylmaleimide PKCi Enzyme Antagonist Protein kinase-C 50 n M * 2 Cholera Toxin CTX Bacterial Toxin G Protein a-subunit Gs l^g/ml 3 Dibutryl- Cyclic-Adenosine Monophosphate db-cAMP Second Messenger Analogue Protein Kinase A 10-5M 4 Forskolin For Enzyme Activator Adenylate Cyclase 10"5M 5 Gonadotrophin Releasing hormone GnRH Peptide Hormone GnRH Receptor l(r1 0 to 10-5 6 Human Chorionic Gonadotrophin hCG Peptide Hormone LH/hCG Receptor 0.001 to 1 IU/ml 7 Indomethacin Indo Enzyme Activator Cyclooxygenase I; Prostaglandin Dehydrogenase IO"6 M 8 Isoproterenol Iso or IsoP Catecholamine Hormone Antagonist 6-adrenergic Receptor 10"5M 9 Pertussis Toxin PTX Bacterial Toxin G Protein a-subunit(s): Gi.Gp 50 ng/ml 10 Prostaglandin E 2 PGE2 Eicosanoid Hormone PGE2 Receptor and other Prostanoid Receptors 10"12tolO-6M 11 Prostaglandin F 2 a PGF2 a Eicosanoid Hormone PGF2a Receptor and other Prostanoid Receptors 10-12tolO"6M * Toullec et al., 1991; McCarthy 1995. 4. Interaction of PGF 2 a and GnRH: Dayi and D 8 GLCs were treated with vehicle, P G F 2 a ( IO 9 M), GnRH (IO"6 M) or P G F 2 a plus GnRH, in the absence or presence of human chorionic gonadotrophin (hCG). In a second experiment (D \ cells), vehicle, P G F 2 a (10- 1 1 to I O 6 M) and G n R H ( I O 1 0 to I O 5 M ) concentration-response curves were crossed into a matrix of 49 separate treatments which were assayed for progesterone. Results were plotted in three dimensions with GnRH, P G F 2 a and progesterone-response each on a axis. Similarily, results were also plotted as a contour map with GnRH and P G F 2 a each on a separate axis and progesterone response represented by shading and colour. Moreover, 'slices' of the three dimensional matrix were plotted in two dimenstions and analyzed statistically. 5. Interaction of PGF 2 a and PGE 2: Day 8 GLCs were treated with vehicle, P G F 2 a (10- 1 1 to I O 6 M) and PGF^ ( I O 1 0 to IO"5 M) concentration-response curves which were crossed into a matrix of 49 separate treatments. Media were assayed for progesterone. Results were plotted in three dimensions, with P G F 2 a , P G E 2 and progesterone-response each on a separate axis. Similarily, results were also plotted as a contour map, with P G F 2 o t and PGE^ each on a separate axis and progesterone response represented by shading and colour. Moreover, 'slices' of the three dimensional matrix were plotted in two dimenstions and analyzed statistically, as above. 6. PTX and CTX Effects on PGF^ Mediated Luteolysis: Dayi and D 8 cells used for G-protein studies were pre-treated (18 h) with M199 supplemented with vehicle, PTX (50 ng/ml), C T X (1 //g/ml), or PTX plus C T X . Following the pre- treatment period, cells were exposed to M199 containing vehicle, PTX, C T X or PTX plus C T X ; plus vehicle, hCG (1 IU/ml), P G F ^ (10* M ) , or hCG plus P G F 2 a , for 24 h. In another set of experiments cells were treated with M l 9 9 containing vehicle, IsoP (10 s M ) , P G F 2 a (10"6 M ) , or IsoP plus P G F 2 a . Finally, cells were exposed to M199 containing vehicle or P G F 2 a (10-6 M ) , plus or minus forskolin (10 s M ) or Db-cAMP (10 s M). 39 7. Forskolin and Db-cAMP: Dayg cultured human GLCs were treated for 24 hours with vehicle and PGF2a ( I O 6 M) with and without forskolin ( I O 6 M) or dibutryl cAMP(db-cAMP; 10" 5M). 8. Progesterone and Estradiol Production per Cell or DNA Level: Plots were made of the basal progesterone- and estradiol-production from human G L C versus total cell numbers plated or DNA levels per well. This experiment was performed to determine if there was any correlation between steroid production and cell numbers or DNA levels. 9. Morphology of Human GLCs with Culture Time: Photographs of human GLCs at day zero, one, eight, twelve and sixteen were taken, in order to present the general morphology of cells at these culture times. Following studies 2 and 3, it was apparent that culture-time radically altered the responses to hormone treatment. Thus, particular attention was paid to culture-time when deciding which response was to be examined with a particular experiment. For example, a luteotrophic response to P G F 2 a was absent in D i cultured GLCs. Therefore, this time period was particularly appropriate for examining the ability of GnRH to elicit a luteotrophic response to P G F 2 a . C. Microscopy Cells were routinely checked following plating, prior to experiments and following experiments for viability (as described above) and general appearance with a Nikon TMS inverted tissue culture microscope. Moreover, photographs of cells at different culture periods were taken with either a Nikon N2000 or Contax 167 M T camera body mounted on this microscope, using Fuji Provia (100 ASA) or Fujichrome Tungsten (400 ASA) film. Slides were scanned with a Power Macintosh 6100AV (72 M B R A M ) using a Nikon Coolscan II and printed on a photoenhanced Macintosh Colour Stylewriter 2500 using photograde paper (at > 720 dpi). Colour synchronization was set to automatic photograde. D. Radioimmunoassay of Progesterone and Estradiol 40 The progesterone and estradiol concentrations in culture media were determined by specific RIAs, as previously described [Li et al., 1993; Rodway et al., 1990; Leung & Armstrong, 1979], with the following modification: phosphate buffered saline was replaced by a phosphate buffer containing N a 2 H P 0 4 (0.04 M) and N a H 2 P 0 4 (0.04 M) at pH 7.4. Typical standard curves for these progesterone and estradiol assays are presented in Figure 10. Progesterone-RIA was performed as follows. Briefly, the assay used rabbit progesterone antiserum (R4 -2 ; Kindly provided by D.T. Armstrong, University of Western Ontario) raised against 4-pregnen-6B-ol-3,20-dione hemisuccinate:bovine serum albumin conjugate (Steraloids, Wilton, NH). The final antiserum concentration was 50 pig/ml. A standard competition method was employed utilizing progesterone (Sigma) standards, and 3H-progesterone at 10,000 cpm/tube (Amersham, Oakville, ON). The range of the assay standards was from 1 to 128 ng/ml. A 0.04 M phosphate buffer (pH 7.4) was used for diluting samples and controls with a final assay volume of 600 /4/tube. Phosphate buffer with dextran (0.025% w/v) and charcoal (0.25% w/v) was used to separate free progesterone from bound. Free progesterone in the supernatant was diluted in 3.0 ml of scintiverse (Fisher) scintillation cocktail and counted for 60 sec on a Wallac 1217 Rackbeta-counter. The RIA was sensitive to 1.5 ng/ml, as determined by taking the progesterone concentration two times the standard deviation below the zero-binding value. Samples were assayed in duplicate. Intra- and inter-assay coefficients of variation were less than 11%. Estradiol-RIA used specific rabbit antiserum (D.T. Armstrong) raised against estratriene- 3,176-diol-6-carboxymethyl-oxime:BSA conjugate (Steraloids). The final antiserum dilution was 1:200,000 w/v in phosphate buffer. As with the progesterone-RIA, a standard competition method was employed, utilizing estradiol (Sigma) standards and 3H-estradiol (Amersham, Oakville, ON) at 10,000 cpm/tube. The estradiol-RIA was performed as described above for the progesterone-RIA. Furthermore, the range and sensitivity was similar to the progesterone-RIA. Intra- and inter-assay coefficients of variation were less than 10%. 41 CPM Figure 10. Typical progesterone (A) and estradiol (B) radioimmunoassay standard curves. Counts per minute (CPM). E. Hoechst Dye DNA Assay 42 D N A quantification was performed using a modified version of Mates method [1986]. Briefly, following the treatment period, media were removed and replaced with trypsin TRTPK (50 ^g/ml; Sigma) in a final volume of 500 ]x\ in phosphate buffered saline (PBS as defined below). The plate was stored frozen at (-70 C) until assayed for DNA. At the time of assay, the plate was thawed at room temperature and incubated for 30 min to allow the trypsin to lyse the cells. During this incubation period, pre-prepared (see below) Hoechst dye stock (Bisbenzimide; 20 ><g/ml in H 2 0 ; Sigma) was thawed (from -20 C) and diluted (lOx in PBS). Following the incubation period, Hoechst dye solution was added to each well (at 500 ptl/well), mixed and incubated for 5 min before well contents were measured with a spectrofluorometer (Aminco Rowman Spectrophoto Fluorometer, American Instrument Co., Silver Springs, MD) for fluorescence. Excitation and emission wavelengths were 354 and 458 nM, respectively. D N A was quantified by extrapolation from known standards (calf thymus D N A ; Sigma) which were prepared by serial dilution (in phosphate buffered saline) over a range of 2.5 to 1000 ng/ml. Standards (1 ml) contained Hoechst dye diluted in similar fashion to samples above. Fluorescence was measured as above, with standards being measured in triplicate. Hoechst dye stock (20 /<g/ml) was slowly dissolved in distilled water, aliquotted (5 ml), wrapped in foil, and then stored at -20 C until use. Foil wrapping was necessary as bisbenzimide is light sensitive and will quench with time. Phosphate buffered saline (PBS) was composed of N a 2 H P 0 4 (7.1 g), NaCl (116.88 g), and EDTA (0.84 g), dissolved in 750 ml of water, and then made up to final volume (1.0 1) and pH (7.4). PBS was stored at room temperature until use. A typical standard curve for this assay is presented in Figure 11. Figure 11. Typical hoechst dye deoxyribonucleic acid (DNA) assay standard curve. Optical density (OD). F. RNA Extraction Procedure Following experiments, some plates were stored (at -70C) until extracted for total RNA with an RNaid kit (Bio 101, La Jolla, CA). The extraction procedure was performed as outlined in the kits instructions. Lysis buffer (100 jA, as defined below) was added to each well, mixed with a pipette tip and left on ice for 5 min. The buffer with lysed cells was then transferred to a microcentrifuge tube (1.5 ml; Canlab). Sodium acetate (0.2M; 10]A\ pH 4.0) and phenol (100 pi\) were added and vortexed. Chloroform:isoamyl alcohol (24:1; 100 JAI) was added, vortexed and the preparation was then left on ice (15 min). Following this incubation, the tubes were spun (10,000 g; 20 min; 4 C) with the top phase being collected afterwards. RNA was present in the top phase, while protein and D N A remained in the lower phases. Thus, care was taken not to remove any of the interphase as this would introduce contamination into the R N A extract. A second extraction with chloroform:isoamyl alcohol (24:1; 100 pt\) was performed and spun (2 min) with the top phase again being carefully removed and placed in a new microtube. Vortexed RNAMatrix (10 yt\) was added to each tube, vortexed (30 s) and incubated (5 min; RT) with occasional mixing to allow RNA adsorption. Tubes were then centrifuged (1 min; 10,000 g) to pellet the RNA/RNAMatrix complex. Supernatant was removed and saved for possible readsorption. Tubes with the pellet were briefly re-centrifuged and the remaining supernatant was carefully removed with a small bore pipette tip. Following this, the pellet was resuspended in the provided RNA wash solution (500 ftl), spun (1 min; 10,000 g), supernatant was removed, and this step was repeated 1 more time. The microfuge-tubes with the pellets were then placed in the speed-vac micro centrifuge(l min). Finally, the pellet was resuspended in DEPC treated water (15-100 pi\) and incubated (55 C; 5 min) to elute RNA. A final spin (1 min; 10,000 g) was performed to pellet the RNA Matrix while leaving the RNA in solution which was transferred to a final microfuge tube (0.5 ml). The solution was then subjected to spectrophotometric analysis to quantify total RNA. Lysis buffer was composed of guanidine thyocyanate (4.0 M), sodium citrate (pH 7.0; 5 mM), sarcosyl (0.5 % w/v) and 6-mercapto-ethanol (0.7% v/v) in diethylpyrocarbonate (DEPC) treated water. G. RNA Gel In order to check the relative efficacy of the RNA extraction procedure and the integrity of the R N A , the extraction products were run on an RNA gel. The RNA gel was composed of agarose (1.0%) dissolved in d H 2 0 (21.6 ml). Additionally, RNA gel-running buffer (GRB-R; 3 ml; as defined below) and formaldehyde (5.34 ml) were added, and the solution was allowed to cool (5 min) before pouring into a gel tray. RNA samples were loaded (1-2 yg in 10 yX) along with G L B - R (3 ptl), and the gel was run (100 V ; 50 min). Staining of the gel with ethidium bromide revealed two RNA bands (18 and 28 S). The gel was then photographed with polaroid 665 positive/negative film. G L B - R was composed of glycerol (50%), EDTA (1 mM), bromophenol blue (0.4%), xylene cyanol (0.4%?) and ethidium bromide. The GRB (lOx) consisted of MOPS (0.2 M), NaOAc (80 mM) and EDTA (10 mM) in d H 2 0 (total volume 1.01). H. Reverse Transcription of RNA to cDNA A fixed quantity of total R N A , between 1-3 yg depending on the amount available (following RNA extraction) was made up in DEPC treated water (8 yl), heated (70 C; 10 min) and then spun down (5 min; 10,000 g). DTT (1/4), oligo-dT (lyl) and bulk mixture (5 yl) were added, followed by an incubation (37 C; 1 h; Pharmacia First Strand cDNA Kit , Upsala, Sweden). The preparation was boiled for (10 min), spun down and frozen (-20 C) until use. Total R N A levels were determined by spectrophotometric estimation. The spectrophotometer was validated by repeatedly measuring a known quantity of D N A and calculating the error between measurements (see results, p. 110). /. Polymerase Chain Reaction (PCR) Complementary D N A obtained from reverse transcription reactions were amplified by PCR such that relative changes in PGF 2 a-receptor expression could be examined. The procedure was performed as follows. A fixed quantity of complementary D N A (cDNA) between 1 to 5 }A depending on availability for each experiment was mixed with a sense and antisense primer (1 ptl of each; Table 3), Master Mix (22 }A\ as defined below) and Taq polymerase (0.2 pil) in a microcentrifuge tube (0.5 ml; Canlab). Vegetable oil was then dropped on top of the mixture and the tube was capped. PCR was performed for each gene as specified in Table 4, with the primers described in Table 5. Master M i x was composed of lOx PCR buffer (1/10 vol) plus deoxynucleotide- triphosphates (dNTPs; 0.179 ^mol/ml). Ten times PCR buffer consisted of Tris-HCl (100 mM; pH 8.3), KC1 (500 nM), M g C l 2 (15 mM) and gelatin (0.1%) in ddH 2 0 . Radiolabeled PCR contained 4.0 nCi of 3 2 P-dCTP. J. DNA Gel Polymerase chain reaction products were run on an agarose gel composed of the following. Agarose (1.0%) was dissolved in a Tris-Borate-EDTA buffer (TBE) by boiling for 2 minutes. When the agarose solution had cooled (5-10 min) it was poured into a gel tray and a comb was inserted until the gel had solidified (approx. 20 min). The gel was then submersed in T B E , and cDNA samples (10 jA, with 5-20 \i% DNA) mixed with D N A gel-loading-buffer (GLB- D; 3 pA) were loaded. After loading, a DNA ladder (Gibco BRL) was loaded on the outside lanes of the gel, and the gel was run (120-140 V). The gel was removed, stained with ethidium bromide and photographed with a Polaroid camera under ultraviolet light. PCR products appear as fluorescent bands. T B E (5x) was composed of TRIS-base (10.8 g), boric acid (5.5 g) and EDTA (0.5 M ; pH 8.0) dissolved in d H 2 0 (final volume 1 1). Furthermore, G L B - D consists of glycerol (50 ml), EDTA (0.5 M ; 20 ml), bromophenol blue (0.1 g), xylene cyanol (0.1 g) and H 2 0 (20 ml). Table 3. Primer combinations and expected product size following PCR. Sense Antisense Predicted Product Size (bp) HPGF+ hPGF- 802 rPGF+ rPGF- 720 Act+ Act- 524 bp - base pairs 48 Table 4. PCR conditions utilized for genes examined. Gene Denaturing Annealing Polymerization Cycles Extension Time Cycle Expt Figure Temp Time Temp Time Temp Time hPGF2«-R 96 30 57 30 72 1:30 40 7:00 56A rPGF^-R 96 30 50 30 72 1:30 40 7:00 None B-Actin 96 30 55 30 72 1:30 30 7:00 56B All temperatures are given in degrees C, while times are in minutes: seconds Table 5. Oligonucleotide sequences utilized for PCR and Southern blot hybridization. Gene Primer sequence (5* to 3') Name MW Ref human PGF^ Receptor + C T C A T G A A G G C A TAT C A G A G hPGF+ 6127 1 - GTT GCC ATT C G G A G A G C A A hPGF- 5831 GCT TCT GAT A A A G A A TGG A T C C G C TT hPGFP+ 7955 Rat PGF^ Receptor + C C A TTG C C A TCC TCA T G A A G G rPGF+ 6407 2 - A G C GTC GTC TCA C A G GTC A C rPGF- 6120 C A G T A C GAT G G C CAT TGA G A G GTG CAT rPGFP+ 8399 B-Actin Control + T G A T C C A C A TCT GCT G G A A G Act+ 6117 3 - G A C CTG ACT G A C TCA CTC A T Act- 6037 + = sense; - = antisense. * - utilized as an internal probe for blots. M W - Molecular weight. 1 - Abramovitz et al., 1994. 2 - Lake et al., 1994. 3 - Ng et al., 1985, K. Southern Blot Hybridization Southern blot hybridization allows for the verification of PCR products by hybridizing a probe designed to bind to the internal portion of the predicted PCR product. The procedure used was as follows. A n agarose gel containing the expected PCR product was denatured by immersion and agitation (15 min; RT) in a solution containing NaCl (1.5 M) and NaOH (0.5 M). Sodium hydroxide was then neutralized with a solution of NaCl (3 M) and Tris (0.5 M) at pH 8.0. Three washes with a sodium chloride/sodium citrate buffer (SSC; as defined below) followed (5 min each), after which an overnight transfer to a nylon membrane was performed (Fig. 12). The SSC buffer was composed of sodium chloride (26.3 g/1), and sodium citrate (13.2 g/l),in d H 2 0 (pH7.0). Following transfer of the gel to a nylon membrane, the membrane was washed (SSC), dried wrapped in Saran Wrap™, and exposed to U V light (2 min). The membrane then was stored (4 C) until hybridization, which was performed with a radiolabelled oligonucleotide, specific to the inner sequence of the predicted PCR product (Table 5). Radiolabelling of the oligonucleotide was performed by a kination reaction, as follows. Primer (10 pmol; 1 JAI), T 4 kinase buffer (1 JAI; lOx), d H 2 0 (2 pil), y^P-ATP (5 }AI) and T 4 kinase (10 U ; lyd) were mixed and incubated (1 h; 37 C). The probe was then boiled (2 min), and spun (1 min; 10,000 g). Just prior to hybridization the nylon membrane was removed from the refrigerator and preincubated in a prehybridization solution. The probe was then diluted in a hybridization solution and hybridized (40 C; over night). The following day the membrane was washed repeatedly in SSC (Table 6). The washed membrane was blotted, re-wrapped in Saran Wrap™, and then autoradiographed for 20 min to several days (at -70 C) depending on signal strength. L. Densitometry of Photographed Gels and Autoradiographs RNA and D N A gels stained with ethidium bromide (200 /<g/100 ml gel; Sigma) could be visualized with U V illumination (Photoprep, Bio/Can Scientific, Mississauga, ON). However, quantification of products required gels to be photographed with a negative film (polaroid, 665). Negatives and autoradiographs from Southern blotting were scanned with a transluminescence video densitometer (Model 620, Bio-Rad Laboratories Inc.). Scanning software was utilized to calculate the relative optical density of each product band. In order to reduce variability, three scans of each film were performed and the means of the three scans were plotted. Pape r \vw-w-- Towels \vsv^^~ Transfer Membrane Nylon Membrane Gel ssc Buffer EES Support Membrane Figure 12. Setup for overnight transfer of gel products to a nylon membrane for further Southern blot hybridization. Table 6. Southern Blot SSC Washes. Wash Duration Temperature SSC (Minutes) (Celcius) Dilution 1 20 min 40 l.Ox 2 10 min 50 O.lx 3 10 min 50 O.lx SSC-sodium chloride/sodium citrate buffer. M. Analysis of Results The results were presented as percentage of control values or by representative experiment. Graph bars represent the mean ± S E M of experiments performed on cells from different patients ('n' refers to patient numbers). Statistical analysis utilized one-way A N O V A followed by a Fischer or Scheffe post-hoc test. Statistical analysis was performed on mean standard score data and plotted in percentage of control data [Lewis 1984; Fisher and van Belle 1993; Grimm and Yarnold 1996; Porkess 1991]. Different characters above graph bars signify statistical difference. 54 VI - RESULTS Preliminary Results Basal and hCG-Stimulated Steroidogenesis from human GLCs 1. Basal Steroid Secretion per Cell or Level of DNA/Well No correlation between cell plating number and basal progesterone production was seen in D 8 cultured human GLCs (n=17). Furthermore, up to 5000-fold differences in basal progesterone production from individual patients were observed (Fig. 13A and B). Moreover, when progesterone production was plotted against extracted D N A levels, no correlation was seen between culture-well DNA content and progesterone production (n=19; Fig. 14A and B). Similar results were seen when basal estradiol production was plotted against cell plating numbers (Fig. 15A and B; n=ll) or extracted DNA levels (Fig. 16A and B; n=17). Please see the discussion section for possible explanations for these results (p. 117). Cell viability as determined by trypan blue dye exclusion was greater than 95% in these experiments, a result further supported by the ability of these cells to respond (with steroid production) to experimental stimuli such as hCG and PGF2« (not shown). 2. hCG-Stimulated Progesterone Production in Cells from 3 Different Patients Progesterone responses to hCG (0.001 IU/ml) in Dg precultured human GLCs from three different patients produced significant stimulatory responses (p<0.05) of similar magnitude (approximately 4-fold; Fig. 17). However, the basal concentrations of progesterone varied up to 100-fold between experiments performed on cells from different patients, although all three experiments were performed on cells plated at 10 4 cells/well. For example, cells from patient 1 (Fig. 17A) produced basal progesterone levels of approximately 10 ng/ml, while hCG-stimulated progesterone levels were approximately 45 ng/ml. Basal and hCG-stimulated progesterone production were approximately 1 and 4 ng/ml, respectively in cells from patient 2 (Fig. 17B). Finally, basal and hCG-stimulated progesterone production were approximately 110 and 420 ng/ml, respectively in cells from patient 3 (Fig. 17C). Thus, although very different basal levels were seen in all three cases, the relative responses to hCG were similar. (see p. 117 for relevant discussion) 55 05 O a) -t-> ^ <D E a . IOOOO-I _ 1 ooo 1 0 0 - J o o o o B C e l l s / w e l l 1 0 0 0 0 „ 1 0 0 0 a? ? c ** t- g 1 0 0 -t-> " «0 - v . )̂ ^ O " v . (_ o> 10-J i -4 0 n:-j-i I : - : I t:vi [j] t--I I X I KjH K : < K : I K - I I : J I I:---I K M V.-. e s e s s e s e Pat ien t Figure 13. A. Basal progesterone production (ng/ml; over 24 h) versus cells/well, in Dg pre- cultured human granulosa-luteal cells (GLCs). B. Basal progesterone production of individual patients plotted in ng/ml per 1000 cells plated, in Dg pre-cultured human GLCs. Note that no correlation was seen between plated cell numbers and progesterone production between patients. 56 1 0 0 0 0 - 7 o c o +i E CO ^ <L> = O L> Q. 1 OOOd 1 0 0 1 0 • • • • • • • • • • D • Csl B 1 0 0 0 0 *3 2 2 1 0 0 0 J to ^ O E L. ^ DNA ( n g / m l ) 1 0 0 ^ 1 0 v i I { \ i' i I i I i V I y I I r i I • - C M I O W > t D r > » 0 > 0 < - t M r O ^ U ) y O r - 0 0 9 t Patient Figure 14. A. Basal progesterone production (ng/ml; over 24 h) versus DNA content per well (ng/ml), in D 8 pre-cultured human granulosa-luteal cells (GLCs). B. Basal progesterone production of individual patients plotted in ng/ml per 100 ng of DNA, in D 8 pre-cultured human GLCs. Note that no correlation was seen between extracted DNA levels and progesterone production. 57 1 0 0 0 E i oo (0 1 0 - J 1 -J 0.1 I I I I III I I I I I I III o o o o C e l l s / W e l l B 1 0 0 _ u O o ra - (_ LU = Pat ien t Figure 15. A. Basal estradiol production (ng/ml; over 24 h) versus cells/well, in D 8 pre-cultured human granulosa-luteal cells (GLCs). B. Basal estradiol production of individual patients plotted in ng/ml per 1000 cells plated, in D 8 pre-cultured human GLCs. Note that no correlation was seen between plated cell numbers and estradiol production. 58 1 0 0 0 . 2 - 2^ 1 0 0 B 1 0 0 0 PS - t_ CO Q LU E 1 0 0 - J 1 0 - J 1 -J D N A ( n g / m l ) O.i *i'* " i " 1 r" I"'V r r ' " r ' 'r' '"i"'1 rr''T" "i 1 1"i" ' r 1 "i C M h o ^ L n v o r ^ - e o o ^ o c s i ^ m vo oo O N Pat ien t Figure 16. A. Basal estradiol production (ng/ml; over 24 h) versus DNA content per well (in ng/ml), in D 8 pre-cultured human granulosa-luteal cells (GLCs). B. Basal estradiol production of individual patients plotted in ng/ml per 100 ng of DNA, in D 8 pre-cultured human GLCs. Note that no correlation was seen between extracted DNA levels and estradiol production. 59 o 3* | § o CM Figure 17. Comparison of progesterone responses to hCG treatment (0.001 IU/ml for 24 h), in D 8 pre-cultured human granulosa-luteal cells from three different patients (A, B and Q. Progesterone production was significantly stimulated in all three experiments (a*b, p<0.05). Moreover, all three experiments responded with similar amplitudes (approximately 4 fold). However, the magnitude of progesterone concentrations varied up to 100 fold between experiments performed on cells from different patients. A l l three experiments were performed on cells plated at 10 4 cells/well. Human GLC Morphology with Culture Time Morphology slides presented within this section were taken from cells of a single patient which were plated at 10 4 cells per well and cultured as described above. The morphology of cultured human GLCs plated on 48-well plates changed with culture time. Cells that were freshly plated (2-3 h of culture) appeared to be evenly distributed about the surface of the well (Fig. 18A; 66 x mag). Additionally, cells did not appear to be highly associated with one another, although some cell aggregates were present. Individual cells appeared round and smooth. Following one day of culture (Di), GLCs were unevenly distributed throughout the well with clusters of cells being present and empty unpopulated regions throughout the well (Fig. 18B; 66 x mag). Cells were either round and smooth or elongated. Many cells possessed cytoplasmic projections which appeared to form associations with neighbouring cells. Long term cultures (Dg) of G L C resulted in highly associated cells which were primarily present in clusters with very few cells existing outside of these aggregates (Fig. 19A; 200 x mag). Cells that were not part of a tightly associated aggregate formed contacts with aggregated cells with cytoplasmic projections. Individual cells again appeared round, although not smooth. Cells appeared luteinized, as they were highly irregular and granulated, when compared to early cultures. Even longer term cultures (Di 2 ) resulted in cells that were even more associated and irregular in shape than those of Dg cultures (Fig. 19B; 200 x mag). Cells cultured for 1 to 12 days remained viable as evidenced by trypan blue dye exclusion (> 95%) and their ability to respond to experimental stimuli with steroid production. However, cells maintained in culture for sixteen or more days were no longer viable or responsive to stimuli and appeared to be luteolysed (Fig. 20; 66 x mag). Similar culture-time dependent morphological changes were witnessed in all other cultures of human GLCs presented herewithin. (see p. 120 for relevant discussion) 61 A Figure 18. A. Human granulosa-luteal cells (GLCs) that were freshly plated (2-3 h of culture) appeared to be evenly distributed throughout the surface of the culture well (40 x mag). Additionally, cells did not appear to be highly associated with one another, although some cell aggregates were present. Individual cells appeared round and smooth. B. Following one day of culture GLCs were unevenly distributed, with cell clusters and empty unpopulated regions being distributed throughout the well (66 x mag). Cells appeared to be smooth. Many cells possessed cytoplasmic projections which appeared to form associations with neighbouring cells. 62 Figure 19. A. Eight day cultures of human granuosa-luteal cells (GLCs) resulted in highly associated cells, which were primarily present in clusters, with very few cells existing outside of these aggregates (200 x mag). Cells that were not part of a tighti / associated aggregate formed contacts with aggregated cells with cytoplasmic projections. Inciviaual cells again apppeared round, although not smooth. Cells appeared luteinized, as they appeared blebbed and granulated (or vacuolated), when compared to early cultures. B. Twelve day cultured GLCs were highly associated and irregular in shape (200 x mag). Figure 20. Sixteen day cultured human granulosa-luteal cells were no longer viable or responsive to stimuli and appeared to be luteolysed (66 x mag). 64 A. The Effects of PGF2a on Steroidogenesis in the Absence and Presence of hCG Effects ofPGF2a on Steroidogenesis 1. Progesterone and Estradiol Production in Response to P G F 2 a Briefly, progesterone production in response to PGF2a changed with culture time from inhibition (Fig. 21 A) to stimulation (Fig. 21B; biphasic) in D i and D12-14 cultured GLCs, respectively. While cells at Dg of culture were in a state of transition with inhibition, stimulation or intermediate responses being possible (Fig. 22). Progesterone production was significantly inhibited (50% of control; a;*c, p<0.001) in a linear fashion by P G F 2 a in D i cultured GLCs (Fig. 21A; n=4). Conversely, in D12-14 cultured GLCs PGF2a significantly stimulated progesterone production (Fig. 21B; 200% of control; a*c; p<0.001; n=5), with maximal stimulation at mid-range concentrations (IO - 8 to 1 0 1 0 M) . Dayg cultured GLCs were in a state of transition between D i and D14 cells, with four responses being present (Fig. 22; total n=9): inhibition (n=2), no response (not shown; n=l), linear stimulation (n=3) and bimodal stimulation (n=3). In D i (n=6) and Dg (n=5) pre-cultured human GLCs, P G F 2 a had no effect and stimulated estradiol production, respectively (Fig. 23A). The stimulatory response was significant at low (10-1 2 to IO"8 M ; a*b; p<0.05) and high concentrations of PGF 2 « ( I O 7 to I O 6 M ; a*c; p<0.0001). 2. DNA Levels in Response to P G F 2 a D N A levels of G L C s remained unchanged by P G F 2 a treatment in Dg GLCs (Fig. 23B; n=3), suggesting that responses were due to alterations in steroid production rather than changes in the number of cells per well. (see p. 121 for relevant discussion) 65 A 125-, 1 C o ~ 5r « •*-> = CD e CU u O ~ £_ CL B 100H 75 A 50 ^ 25 J 0 ab a b T T be be Q O o CM I on i PGF^ (10X M) C o ~ ••-» "£ (0 e O ~ t_ 200 H 150 100H, 50H b i T b C J , b T c b ns a e PGF^ (10X M) Figure 21. Progesterone production in response to PGF2« treatment (for 24 h), in one-day (A; n=4; a*c, p<0.001 by A N O V A ) and twelve to fourteen-day (B; n=4; a*c, p<0.001 by A N O V A ) pre-cultured human granulosa-luteal cells (GLCs). In one and twelve to fourteen day pre-cultured human GLCs, PGF2« inhibited and stimulated progesterone production, respectively. 66 250 P G F ^ (10 X H) Figure 22. Progesterone production in response to PGF2« treatment (for 24 h), in eight day pre- cultured human granulosa-luteal cells, four different progesterone-responses to PGF2a were seen in nine separate experiments, including: linear stimulation (fx]; n=3), bell curve-like stimulation (HI; n=3), inhibition ( ^ ; n=2) and no response (not shown; n=l). 67 300 LU 100-1 B a. i © ^ a CT. oo »n tr  1 i i u P G F ^ ( 1 0 X tl) 200 1 5 0 H 1 0 0 H 5 0 - ^ a g a a a a 4 • a ns £a r*> i t P G F ^ ( 10 X Ii) Figure 23. A. Estradiol production in response to PGF2« treatment (for 24 h) in one (£3; n=6)- and eight-day (U; n=5; a*b, p<0.04 and a*c, p<0.0001 by ANOVA) pre-cultured human granulosa-luteal cells. DNA content (B; n=3) in response to PGF 2 a , in eight day pre-cultured human granulosa-luteal cells. Effects ofPGF2a on hCG-Stimulated Steroidogenesis 68 1. Progesterone Production in Response to hCG Treatment As shown in Figure 24, human chorionic gonadotrophin (0.001 to 10 IU/ml) significantly stimulated progesterone production up to six fold from human GLCs. The highest level of statistical significance was seen with 0.01 to 1 IU/ml (hCG) treated cells (p<0.001; n=4), However, cells treated with lower (0.001 IU/ml) and higher (10 IU/ml) concentrations of hCG still responded significantly (p<0.05). 2. Follicle Size-Dependent Regulation of Steroidogenesis by hCG and PGF2a Cells from four patients were separated into small (< 12 mm) and large (> 12 mm)- follicle size groups and subjected to hCG (1 IU/ml) and hCG plus PGF2a ( IO 6 M) treatment at D i (Fig. 25; n=4). Human chorionic gonadotrophin failed to significantly stimulate progesterone or estradiol production in GLCs collected from small follicles (p>0.05). In contrast, hCG stimulated progesterone (p<0.001) and estradiol (p<0.02) production in GLCs collected from large follicles. In addition, PGF2tt inhibited hCG-stimulated progesterone and estradiol production in GLCs from large follicles (p<0.03), while it did not in cells from small follicles. 3. The Effects of P G F 2 a on hCG-Stimulated Steroidogenesis In the presence of hCG, culture-time dependent changes in progesterone responses to P G F 2 a were observed. Prostaglandin F 2 a (IO"6 M) inhibited hCG-stimulated progesterone production in Di (Fig. 26A; p<0.05; n=5) and Dg (Fig. 26B; p<0.01; n=6), although not in D 1 4 (Fig. 27; n=4) cultured GLCs . Alternately, P G F 2 o t (lO^9 M) potentiated hCG-stimulated progesterone production in Dg (p<0.01; n=6; 3 fold) and D14 (p<0.05; n=4; 1.5 fold), although not in Di (n=4) cultured GLCs. A similar trend was seen with estradiol production. Prostaglandin F2W (10"6 M) inhibited hCG-stimulated estradiol production in D i (Fig. 28A; p<0.05; n=8) and Dg (Fig. 28B; p<0.05; n=5) cultured GLCs . Alternately, PGF2a (10^M) potentiated hCG-stimulated estradiol production in Dg (p<0.01; n=5; 1.5 fold), although not in D i (n=4) cultured GLCs. 69 1000 to o — gS 750 4 500 4 250 4 be b a •f v 9 o — o o hCG (IU/ml) Figure 24. Human chorionic gonadotrophin (hCG) stimulated progesterone production from human granulosa-luteal cells treated for 24 h following eight days of culture (n=4; a*b or b*c, p<0.05; a*c, p<0.001 by A N O V A ) . 70 A Control hCG hCG & PG B 3 0 0 Control hCG hCG & PGF Figure 25. Follicle size-dependent responses to human chorionic gonadotrophin (hCG; 1 IU/ml) and prostaglandin F 2 a (PGF20J treatment (for 24 h). A. In 1 day pre-cultured human granulosa- luteal cells, PGF 2 a inhibited (n=4; a*b, p<0.001 and b*c, p<0.03 by ANOVA) hCG-stimulated progesterone production from cells collected from large follicles ( H : > 12 mm). However, cells collected from small follicles ( E 3 ; < 12 mm), were unable to respond to hCG. B. Similar results were seen for hCG-stimulated estradiol production (n=4; a^b, p<0.03 by ANOVA). 71 300 J b c o &- * ^ .b •<-> e (0 e <b u o ~ L. CL 200 H 100 4 d . T cd ac cd i ¥ ac OS CQ PGF-^ ( 1 0 X M) hCG (1 iu/ml) B IOOO H 05 c o o £ (0 e <U u O ^ L. a. 100 4 10 be £ a i ] be i CQ PGF^ ( 1 0 X M) hCG (1 IU/ml) I I Figure 26. The effects of prostaglandin F 2 a (PGF 2 a) on hCG-stimulated progesterone production (over 24 h) from one-day (A; n=5; a*b*c*d, p<0.05 by ANOVA) and eight-day (B; n=6; a*b*c, p<0.01 by ANOVA) pre-cultured human granulosa-luteal cells. 72 300 •= 200-1 100H hCG ( 1 iu/ml) Figure 27. The effects of prostaglandin F 2 a (PGF 2 a) on hCG-stimulated progesterone production (over 24 h) from twelve to fourteen-day (n=4; a*b*c, p<0.05 by ANOVA) pre-cultured human granulosa-luteal cells. 73 .25 to 6fi B 250 200 150H 100-1 50 H c c ac ^ O 0> PGF^ ( 1 0 X M) hCG ( 1 IU/ml) 800 600 CO = t- 5 400 H cn be LU v 200 J 0 i i OS I CQ I PGF^ ( 1 0 X M) hCG (1 iu/ml) ac k I a i i Figure 28. The effects of prostaglandin F 2 a (PGF2 a ) on hCG-stimulated estradiol production (over 24 h) from one-day (A; n=8; a*b;*c, p<0.05 by ANOVA) and eight-day (B; n=5; a*b*c, p<0.05 by ANOVA) pre-cultured human granulosa-luteal cells. Effects of GnRH on hCG-Stimulated Steroidogenesis Gonadotrophin-releasing hormone ( lO^M) inhibited hCG-stimulated progesterone production in D : (Fig. 29A; n=6; p<0.05) and D 8 (Fig. 29B; n=5; p<0.05) cultured GLCs. Alternately, GnRH ( I O 8 M) potentiated hCG-stimulated progesterone production in D 8 (Fig. 29B; n=5; p<0.05; a*b*c), although not in D i (n=6) cultured GLCs. A similar trend was seen with estradiol production. Gonadotrophin-releasing hormone (10-6 M) inhibited hCG-stimulated estradiol production in D 8 (Fig. 30A; n=4; p<0.05) cultured GLCs . Alternately, GnRH (10-9 M) potentiated hCG-stimulated estradiol production in D 8 cultured GLCs (n=5; p<0.01). D N A levels were unaltered by any of the treatments (Fig. 30B; n=3;p>0.05). 75 B C o ~ ••-» c (0 e <D U O ~ L. CI- S C O H 400 H 300 4 200 4 100 4 0 b b T CO Is" VO • GnRH (10 X M) hCG (1 IU/ml) 800 £ 600 H o ~ t - ' o <n o 400 4 o ~ L. C L 200 4 a be b T c e O S rj GnRH ( 1 0 x M) hCG (1 IU/ml) Figure 29. The effects of gonadotrophin-releasing hormone (GnRH) on hCG-stimulated progesterone production (over 24 h) from one-day (A; n=4; a*b*c, p<0.05 by ANOVA) and eight-day (B; n=5; a*b*c;<id, p<0.05 by ANOVA) pre-cultured human granulosa-luteal cells. 76 1000H .25 •o £ B "y e a 100 J 10 be rfi c bed . . 3r T - r — - i 1 1 1 1 r \ , _ a as CD ta f <— »— i i i i GnRH ( 1 0 X M) hCG (1 IU/ml) 200 150H 100 50 J a a a a a ea i i GnRH ( 1 0 x M) hCG (1 IU/ml) Figure 30. A. The effects of gonadotrophin-releasing hormone (GnRH) on hCG-stimulated estradiol production (over 24 h) from eight-day (n=4; a*b*c, p<0.05 by ANOVA) pre-cultured human granulosa-luteal cells. B. DNA levels were unaltered by any of the above treatments (n=2; p>0.05 by ANOVA). 77 B. The Interaction of PGF 2a with GnRH Progesterone Response to GnRH and/or PGF2a> with or without hCG. Neither P G F 2 a (IO"9 M) nor GnRH (IO 6 M) significantly altered progesterone production, in Di human GLCs (Fig. 31A; n=5). However, the combination of PGF2« plus GnRH significantly stimulated progesterone production (a*b, p<0.05). Human chorionic gonadotrophin (1 IU/ml) also significantly stimulated progesterone production (2.5-3 fold; a*b, p<0.05). Conversely, gonadotrophin-releasing hormone alone was unable to inhibit hCG- stimulated progesterone production, although it did potentiate PGF 2 a-mediated inhibition (a*b*c, p<0.05). In Dg pre-cultured GLCs a significant luteotrophic response to P G F 2 a ( I O 9 M) was present (Fig. 3IB; n=4; p<0.05). However, no luteotrophic response to GnRH (IO - 6 M) was observed, although GnRH potentiated the PGF2a-mediated luteotrophic response (p<0.05). Both GnRH and PGF2a significantly inhibited hCG-stimulated progesterone production (b*c, p<0.05), while their combination potentiated inhibition beyond levels of either hormone alone (p<0.05). Estradiol Response to GnRH and/or PGF2a, with or without hCG. In Di human granulosa luteal cells (Fig. 32; n=3), neither P G F 2 a (10-9 M) nor GnRH (IO 6 M) significantly altered estradiol production. However, the combination of PGF2a plus GnRH significantly stimulated estradiol production (p<0.01). Human chorionic gonadotrophin (1 IU/ml) also significantly stimulated estradiol production (p<0.05). Gonadotrophin-releasing hormone alone was unable to inhibit hCG-stimulated progesterone production, although it did potentiate PGF 2 a-niediated inhibition (p<0.05). Progesterone Response to GnRH with or without PGF2a Vehicle, P G F 2 a (IO"11 to IO"6 M) and GnRH (IO - 1 0 to IO"5 M) concentration-response curves were crossed into a matrix of 49 separate treatments. Results were plotted in three dimensions, with GnRH, PGF 2 a and progesterone-response each on one axis (Fig. 33A and B [mirror image of A]). Results were also plotted and as a contour map with GnRH and PGF2 a each on a separate axis and progesterone response represented by shading (Fig. 34A) and colour (Fig. 34B). In D i human GLCs, maximal stimulation of progesterone-production (2-3 fold) was seen when middle concentrations of P G F 2 a (IO - 9 M; p<0.05) interacted with high concentrations of GnRH ( I O 6 to I O 5 M). In the presence of high concentrations of GnRH (IO"6 M) , P G F 2 a stimulated progesterone production in a bell curve-like fashion as middle concentrations significantly stimulated while low and high concentrations did not (Fig. 35; p<0.05). In the presence of middle concentrations of P G F 2 a ( I O 9 M), GnRH significantly stimulated progesterone-production in a linear concentration-dependent manner (Fig. 36; p<0.05). Estradiol Response to GnRH with or without PGF2a In Di human GLCs, maximal stimulation of estradiol-production (4-fold) was seen when high concentrations of P G F 2 a (10"6 M; p<0.05) interacted with high concentrations of GnRH (IO - 6 to lCr 5 M). These data are presented in three dimensional graph (Fig. 37; n=6) and contour format (Fig. 38), as for progesterone data above. In the presence of high concentrations of GnRH (Fig. 39; 1 0 5 M ) , P G F 2 a significantly and linearly stimulated estradiol production (p<0.05), although P G F 2 a was ineffective in the absence of GnRH. On the other hand, in the absence and presence of P G F 2 a (10-6 M), GnRH significantly stimulated estradiol production (Fig. 40A and B ; p<0.05). The nature of GnRH stimulated estradiol production was, however, different in the presence and absence of P G F 2 o t , as the response shifted from a bell curve-like stimulation to a linear one with the addition of P G F 2 a . DNA Levels in Response to GnRH and PGF2a Treatment D N A Levels were unaltered by treatment with either GnRH ( I O 1 0 to IO"5 M) , P G F 2 c t ( I O 1 1 to 10~6 M) or hCG, suggesting that responses seen were due to alterations in steroid production rather than changes in cell numbers (data not shown). Effects of Indomethacin on PGF2a and GnRH Stimulated Steroidogenesis 79 In Dg cultured human GLCs, progesterone was significantly stimulated in a bell curve- like fashion by P G F 2 a (Fig. 41). Maximal stimulation of progesterone production was at 1 nM of PGF2a (p<0.05). However, co-incubation with indomethacin (IO"6 M) reversed this effect, and PGF2a instead inhibited or had no effect on progesterone production (depending on concentration). Similar results were seen in cells from two other patients. Cells remained viable in the presence of indomethacin, as suggested by their ability to exclude tryphan blue. Progesterone production in D j cultured human GLCs was uneffected (p>0.05) by vehicle, GnRH ( I O 1 0 to I O 6 M) and/or P G F 2 a ( I O 1 1 to IO 6 ) when cells were co-incubated with indomethacin (IO"6 M ; n=4; Fig. 42). Cells remained viable in the presence of indomethacin, as suggested by their ability to exclude tryphan blue. Compare these results and those presented in Figure 33. Note that hCG-stimulated progesterone production was seen in these cells (not shown). (see p. 125 for relevant discussion) 80 B C o ~ CO e <D u O (_ CL 400 300 ^ 200 H 100-^ 0 e o Of e is C9 Q. 0. T T e is L_ CD C3 Q. Q. e CD hCG (i iu/ml) 2000 H cn e <D « 1000H C L 500 H ^ a i l e o 19 Q. CL 19 Q. C9 C9 a. C9 hCG (1 IU/ml) Figure 31. Progesterone production in response to vehicle (Cont), gonadotrophin-releasing hormone (GnRH; I O 6 M), prostaglandin F 2 a (PGF 2 a; IO 9 M) and GnRH plus PGF 2 a treatment (over 24 h), in the presence and absence of human chorionic gonadotrophin (hCG), in one-day (A; n=5) and eight-day (B; n=4) pre-cultured human granulosa-luteal cells. Graph bars represent mean ± SEM of experiments performed on separate patients (a*b*c, p<0.05 by ANOVA) . 81 1000 4 .2 o •5 t 1004, 10 e CJ UP I 1 T X U- CS « CS Ot e o. >H in e cs be I I ' I Z lb CS a cs o- e a. CD e CS hCG (1 IU/ml) Figure 32. Estradiol production in response to vehicle (Cont), gonadotrophin-releasing hormone (GnRH; 10-6 M), prostaglandin F 2 a (PGF; IO"9 M) and GnRH plus PGF treatment (over 24 h), in the presence and absence of human chorionic gonadotrophin (hCG), in one day (n=3) pre- cultured human granulosa-luteal cells. Graph bars represent mean ± SEM of experiments performed on separate patients (a*b5*c, p<0.05 by ANOVA). 82 (10~XM) Figure 33. Three dimensional plot of progesterone production in response to vehicle (C), gonadotrophin-releasing hormone (GnRH; I O 1 0 to I O 5 M) and/or prostaglandin F 2 a ( P G F 2 a ; 1 0 " to IO"6 M) treatment (over 24 h), in one day pre-cultured human granulosa-luteal cells. A mirror image (B) provides a view of the back side of the image (A). These Figures represent the mean of seven separate experiments performed on seven separate patients. 83 Figure 34. Black and white (A) and colour (B) contour plot of progesterone production in response to vehicle (C), gonadotrophin-releasing hormone (GnRH; I O 1 0 to IO 5 M) and/or prostaglandin F 2 a (PGF^ ; I O 1 1 to IO"6 M) treatment (over 24 h), in one day pre-cultured human granulosa-luteal cells. Progesterone production of 50, 100, 200 and 300% of the control level are symbolized by: • H , [I] fl, H O and S B L3 respectively. These Figures represent the mean of seven separate experiments performed on seven separate patients. 84 400 PGF2« <1G X h ) Figure 35. Effects of prostaglandin F 2 a (PGF2a; I O 1 1 to I O 6 M) , in the absence ( D ) and presence ( -o- ) of gonadotrophin-releasing hormone (GnRH; 10"6 M) treatment (over 24 h), in one day pre-cultured human granulosa-luteal cells. In the presence of GnRH, PGF2a stimulated progesterone production in a bell curve-like fashion, with significant stimulation at middle concentrations (IO - 9 and IO"8 M P G F 2 a ; a*b, p<0.05, by A N O V A ) . While in the absence of GnRH, PGF2« did not significantly alter progesterone production. Progesterone production in response to GnRH alone ( IO 6 M) was not significantly different from the control response. This Figure represents the mean ± sem of seven separate experiments performed on seven separate patients, and is a two dimensional slice of three dimensional matrix presented in Figure 33A. 85 Figure 36. Effects of gonadotrophin-releasing hormone (GnRH; I O 1 0 to IO-5 M), in the absence ( • ) and presence ( «- ) of prostaglandin F2« (PGF2a; IO 9 M) treatment (over 24 h), in one day pre-cultured human granulosa-luteal cells. In the presence of PGF 2 a> GnRH stimulated progesterone production in a linear concentration-dependent fashion, with significant stimulation at upper concentrations (IO - 8 to IO - 5 M: a^b, p<0.05 by ANOVA). While in the absence of PGF2a, GnRH had no effect on progesterone production. Progesterone production in response to PGF2a alone ( I O 9 M) was not significantly different from the control response. This Figure represents the mean ± SEM of seven separate experiments performed on seven separate patients, and is a two dimensional slice of three dimensional matrix presented in Figure 33A PGF 2 a (10" XM) Figure 37. Three dimensional plot of estradiol production (over 24 h) in response to vehicle (C), gonadotrophin-releasing hormone (GnRH; 10"10 to lf>5 M) and/or prostaglandin F 2 o t ( P G F 2 a ; 10 1 1 to IO-6 M), in one day pre-cultured human granulosa-luteal cells. This Figure represents the mean of six separate experiments performed on six separate patients. 87 Figure 38. Black and white (A) and colour (B) contour plot of estradiol production (over 24 h) in response to vehicle (C), gonadotrophin-releasing hormone (GnRH; IO - 1 0 to I O 5 M) and/or prostaglandin F2« (PGF2„; I O 1 1 to IO - 6 M) , in one day pre-cultured human granulosa luteal cells. Estradiol production of 100, 200, 300 and 400% of the control level are symbolized by: 1 I I v>i ̂ B. m^land IB L i , respectively. These Figures represent the mean of six separate experiments performed on six separate patients. 88 Figure 39. The interaction of gonadotrophin-releasing hormone (GnRH; I O 5 M) and prostaglandin F 2 a (PGF2a) on estradiol response (over 24 h), in one day pre-cultured human granulosa-luteal cells. In the presence ( — o - - - ) of GnRH (IO"5 M) , P G F 2 a significantly stimulated estradiol production (n=6; a*b, p<0.05 by ANOVA) , however P G F 2 a was ineffective in the absence ( • ) of GnRH. These data represent a two dimensional slice of the three dimensional graph presented in Figure 37. 89 .25 to K 500 400 H 300 H 200 H 100H B o -3 5 0 0 4 0 0 ^ 300H 200H i OOH GnRH (10XM) Figure 40. Estradiol response (over 24 h) to gonadotrophin-releasing hormone (GnRH; 10 - 5 M) in the absence (A) and presence (B) of prostaglandin F2 a (PGF 2 a; IO - 6 M), in one day pre- cultured human granulosa-luteal cells. In the presence of PGF2a, GnRH significantly stimulated estradiol production (n=6; a*b*c, p<0.05 by ANOVA). These data represent a 2D slice those in Figure 37. 90 C o •+-> = OS — O £_ Q. 1250 1000-1 750 500-1 250 l i 12 11 10 9 8 7 6 P G F ^ ( 1 0" x M) Figure 41. Progesterone response (over 24 h) to PGF2« in the absence ([3) and presence (H) of indomethacin (IO - 6 M), in eight day pre-cultured human granulosa-luteal cells. In the absence of indomethacin, PGF2a significantly and in a bell curve-like fashion stimulated progesterone production (p<0.05; control vs PGF2a, I O 9 M). However, in the presence of indomethacin, P GF 2 a either inhibited (p<0.05; control vs PGF 2 a, 1 0 1 0 to IO"6 M) or had no effect on progesterone production. This Figure represents the response of cells from one patient. Similar results were seen in cells from two other patients. Cells remained viable in the presence of indomethacin, as suggested by their ability to exclude tryphan blue. 91 Figure 42. Three dimensional plot of progesterone production (over 24 h) in response to vehicle (C), gonadotrophin-releasing hormone (GnRH; I O 1 0 to IO 6 M) and/or prostaglandin F2« (PGF2 a; IO - 1 1 to IO 6 M) in the presence of indomethacin (IO - 6 M), in one day pre-cultured human granulosa-luteal cells. This Figure represents the mean of four separate experiments performed on cells from four separate patients. No significant difference between treatments was seen (p>0.05). Cells remained viable in the presence of indomethacin, as suggested by their ability to exclude tryphan blue. C. Progesterone Response to PGF2a plus PGE2 The following study reveals a complex regulation of progesterone production in response to vehicle P G F 2 a (IO"11 to I O 6 M) and/or PGE2 ( IO 1 1 to I O 6 M) concentration-response curves in eight day cultured human GLCs. Prostaglandin F 2 a and P G E 2 concentration-response curves were crossed into a matrix of 49 separate treatments. Results were plotted in three dimensions with P G F 2 a , PGE2 and progesterone-response each on a separate axis (Fig. 43). Additionally, data were plotted in contour map form (Fig. 44). Moreover, two dimensional slices of the three dimensional matrix were plotted and analyzed statistically (Fig. 45). Briefly, progesterone was significantly stimulated in a bell curve-like manner by P G F 2 a with maximal stimulation at 1 nM (p<0.05). A similar response to P G E 2 was seen although the bell curve was shifted right. Maximal PGE^-mediated stimulation of progesterone production was seen at 10 to 100 nM (Fig. 45). However, in the presence of P G E 2 (1CK7 M), P G F 2 a significantly inhibited progesterone production (p<0.05) in an inverse bell curve-like manner, with maximal inhibition at ( IO - 1 0 to 1 0 - 8 M , P G F 2 a ; F i g . 4 6 ) . (see p. 131 for relevant discussion) 93 Figure 43. Progesterone production (over 24 h) in response to vehicle, prostaglandin F 2 c t ( P G F 2 a ; 10-" to 1(K> M) and/or prostaglandin fc^ (PGE 2 ; I O " to IO - 6 M) concentration response curves, in eight day pre-cultured human granulosa-luteal cells. A mirror image (B) provides a view of the back side of the image (A). This Figure represents the mean of four separate experiments performed on cells from four different patients. 94 Figure 44. Black and white (A) and colour (B) contour plots of progesterone production (over 24 h) in response to vehicle, prostaglandin F 2 a (PGF2«; I O 1 1 to IO"6 M) and/or prostaglandin E 2 (PGE 2 ; IO - 1 1 to 10"6 M) concentration response curves (n=4), in eight day pre-cultured human granulosa-luteal cells. Progesterone production of 100, 200, and 300% of control level are symbolized by: CU IH' HII Hl^ and | 9 respectively.This Figure represents the mean of four separate experiments performed on cells from four different patients, and is derived from the same data as those presented in Figure 43. 95 C o cu 400 A 300 CO e CU ° 200 H O — (_ CL 100-1 B cu c o ~ 5r « •*•> E (0 e CD (-> O ~ L. CL 350 300 250 200 150 100 50 c c T - c ~ i 1—-1 1 1 r 1 1 - 1 0 - 9 -8 -7 -6 PGEj ( 1 0 x M) Figure 45. Prostaglandin F2a( A ; PGF 2 a) and prostaglandin E2 (B ; PGE2) concentration response curves ( 1 0 1 1 to IO 6 M), in eight day pre-cultured human granulosa-luteal cells. Progesterone was significantly stimulated (over 24 h) in a bell curve-like manner by PGF2a with maximal stimulation at 1 nM (a*b*c, p<0.05 by ANOVA) . A similar response to PGE2 was seen although the bell curve was shifted right. Maximal PGE2-mediated stimulation of progesterone production was seen at 10 to 100 nM. These data represent a two dimensional slice of the data presented in Figure 43. 96 Figure 46. The effects of prostaglandin F 2 a (PGF 2 a; IO"11 to IO 6 M), in the presence of prostaglandin E 2 (PGE 2; 10 7 M). Progesterone production (over 24 h) was significantly inhibited (a*b, p<0.05 by ANOVA) in an inverse bell curve4ike manner by PGF 2 a ( I O 4 0 to IO"8 M), in eight day pre-cultured human granulosa-luteal cells. These data represent a two dimensional slice of the data presented in Figure 43. D. Signal Transduction of PGF'^Mediated Lute oly sis 97 Effects of PGF2a on hCG-Stimulated Steroidogenesis Progesterone (Fig. 47A) and estradiol (Fig. 47B) production were stimulated by hCG (1 IU/ml; p<0.05 control vs. hCG) in eight-day cultured human granulosa-luteal cells, although hCG-stimulated progesterone and estradiol production were both attenuated in the presence of PGF 2 o t (10 - 6 M ; p>0.05 control vs hCG plus PGF 2 o t ) . Effects of PGF2a on Isoproterenol Stimulated Progesterone Production The 6-adrenergic agonist isoproterenol ( IO - 5 M) was capable of stimulating progesterone production from eight-day cultured human granulosa-luteal cells (Fig. 48A; p<0.05; control vs isoproterenol). As with hCG, isoproterenol-stimulated progesterone production was blocked by the addition of P G F 2 a ( IO - 6 M) to culture media (p>0.05; control vs. isoproterenol/PGF 2 a; p<0.05 isoproterenol vs isoproterenol/PGF2a). Isoproterenol also stimulated estradiol production from human granulosa-luteal cells (Fig. 48B; p<0.05, control vs. isoproterenol). The ability of P G F 2 a to inhibit isoproterenol-stimulated estradiol production was not examined in these studies. Please note that P G F 2 a transiently stimulated progesterone production (compare Fig. 47A with Fig. 48A). The cause of this transience is under study in another project. Effects of PTX on Anti-gonadotrophs Actions ofPGF2a Treatment of eight-day cultured human granulosa luteal cells with hCG (1 IU/ml) significantly stimulated progesterone production (Fig. 47A and Fig. 49A; p<0.05 versus control). Furthermore, this stimulation was inhibited by co-treatment with P G F 2 a (IO - 6 M ; p<0.05 versus hCG and p>0.05 versus control). However, in the presence of PTX (50 ng/ml), PGF 2 a-mediated inhibition of hCG-stimulated progesterone production was blocked (p<0.05 versus control and p>0.05 versus PTX/hCG treated cells). Similar progesterone responses were seen from one-day cultured cells under the same conditions (not shown). Estradiol production from eight-day cultured human granulosa-luteal cells paralleled progesterone responses under the above treatment conditions (Fig. 49B). DNA levels remained unchanged by this treatment regime (Fig. 50) suggesting that steroid responses were not due to altered cell numbers in these experiments. Effects of PGF2a on CTX Stimulated Steroidogenesis 98 Cholera toxin (1 pg/ml) significantly stimulated progesterone production from eight-day cultured human granulosa-luteal cells (Fig. 51A; p<0.05, CTX versus control). Subsequently, PGF 2« (10-6 M) was able to block the stimulatory effect of CTX (p<0.05, CTX versus CTX plus PGF 2 a; and p>0.05 for control versus CTX/PGF2ot). However, co-treatment with PTX (50 ng/ml) partially reversed this effect. Estradiol production in response to these treatments followed a similar profile (Fig. 51B). Effects of PGF2a on Forskolin Stimulated Progesterone Production Forskolin (10 s M) significantly stimulated progesterone production from eight-day cultured human granulosa-luteal cells (Fig. 52A and 52B; p<0.05, forskolin vs. control). PGF 2 a (10 - 6 M) was able to block the stimulatory effect of forskolin (p<0.05, forskolin vs. forskolin plus PGF 2 a; p>0.05 control vs. forskolin plus PGF 2 a). Please note that P G F 2 a alternately stimulated (p<0.05, control vs. PGF 2 a) or did not stimulate progesterone production on its own (compare Fig. 47A and 48B), although this transience did not alter the inhibitory properties of PGF 2 a. Effects of PGF2a on cAMP Stimulated Progesterone Production Db-cAMP was capable of stimulating progesterone production from eight-day cultured human granulosa-luteal cells (p<0.05, control vs. Db-cAMP), although in these experiments, Db-cAMP-stimulated progesterone production was not inhibited by P G F 2 a (Fig. 53). The Effects of a PKC Inhibitor on PGF2a-Mediated Inhibition of hCG-Stimulated Progesterone Production. Human chorionic gonadotrophin (1 IU/ml) significantly stimulated progesterone production in Dg cultured human granulosa-luteal cells (Fig. 54; n=4). Moreover, the highly specific protein kinase-C inhibitor bisindolylmaleimide (50 nM) significantly inhibited (p<0.05) PGF2a-mediated inhibition of hCG-stimulated progesterone production in these cells. (see p. 132 for relevant discussion) A 800 c o L . ^ ° 1 o L . CL 600 4 400 4 200 4 T h C G P G R 2s h C G P G F 2s B h C G P G F 2d h C G P G F 2s Figure 47. Prostaglandin F 2 a (PGF 2 a; IO - 6 M)-mediated inhibition of human chorionic gonadotrophin (hCG; 1 IU/ml)-stimulated progesterone (A) and estradiol (B) production (over 24 h), in eight day pre-cultured human granulosa-luteal cells. Data represent the mean ± SEM of triplicate measures (a*b; p<0.05; by ANOVA) . Similar results were seen in fourteen separate experiments performed on cells from fourteen other patients. 100 fc c o L . ^ fc •= ••-» = fc e O (_ CL 1000 750H 500H 250 H Figure 48. Prostaglandin F 2 a (PGF2a; IO'6 M)-mediated inhibition of isoproterenol (Iso; IO"5 M)-stimulated progesterone production (over 24 h), in eight day pre-cultured human granulosa-luteal cells. Data represent the mean ± SEM of triplicate measures (a*b; p<0.05; by ANOVA). Similar results were seen in three separate experiments performed on cells from three other patients. 101 Figure 49. The effects of pertussis toxin (PTX 50 ng/ml) on prostaglandin F 2 o t (PG; lO^6 M)- mediated inhibition of human chorionic gonadotrophin (hCG; 1 IU/mi)-stimulated progesterone (A) and estradiol (B) production (over 24 h), in eight day pre-cultured human granulosa-luteal cells. Data represent the mean ± S E M of triplicate measures (a*b; p<0.05; by A N O V A ) . Similar results were seen in five separate experiments performed on cells from five other patients. 102 Figure 50. The effects of pertussis toxin (FTX 50 ng/ml), prostaglandin F 2 a(PGF 2 a ; lO^M) and human chorionic gonadotrophin (hCG; 1 IU/ml) on DNA levels (over 24 h), in eight day pre- cultured human granulosa-luteal cells. Data represent the mean ± SEM of triplicate measures (p>0.05). Similar results were seen in two separate experiments performed on cells from two other patients. 103 A 1 5 0 - r — , B 0) c o L. ~ CD •= * J = CD s 0 > v O L CL "2 1 100 - j 50 a a b a i :• ' 1 J C PGF • 1 ' J C PGF C PGF Vehicle CTX CTX/PTX 30 25 - 2 0 - 5 - 0 C PGF Vehicle C PGF CTX •f ••••••T ,V| C PGF CTX/PTX Figure 51. The effects of prostaglandin F 2 a (PGF 2 a; IO - 6 M) on cholera toxin (CTX; 1 //g/ml) and CTX plus pertussis toxin (PTX; 50 ng/ml) stimulated progesterone (A) and estradiol (B) production (over 24 h), in eight day pre-cultured human granulosa-luteal cells. Data represent the mean ± SEM of triplicate measures (a*b; p<0.05; by ANOVA). Similar results were seen in five separate experiments performed on cells from five other patients. 104 0> c o fee +* § CO ^ O L a. 1000 750 - i 500 4 2 5 0 4 a a ,1 1 1 C PGF^ C PGF^ Vehicle Forskolin B 0> c o to ^ 05 S rjh *»" O L CL 500 4 4 0 0 4 300 4 200 4 ioo4 i c ac I 1 1 J C PGF^ i ' 1 ; C PGFj, Vehicle Forskolin Figure 52. Prostaglandin F 2 a (PGF 2 a; IO"6 M)-mediated inhibition of forskolin (IO"6 M)- stimulated progesterone production (over 24 h), in eight day pre-cultured human granulosa-luteal cells (A and B). Data represent the mean ± SEM of triplicate measures (a*b*c; p<0.05; by ANOVA). Similar results were seen in three separate experiments performed on cells from three other patients. Note: transient stimulatory effect of P G F 2 a (IO"6 M, A vs B) 105 0) c o L. , (0 o L. CL 800 600 400 4 200 4 Db-cAMP PGF^ Db-cAMP Figure 53. The effects of prostaglandin F 2« (PGF 2 a; IO 6 M) on dibutryl cAMP (Db-cAMP; IO"5 M)-stimulated progesterone production (over 24 h), in eight day pre-cultured human granulosa-luteal cells. Data represent the mean ± SEM of triplicate measures (a*b; p<0.05; by ANOVA). Similar results were seen in three separate experiments performed on cells from three other patients. Additionally, similar results seen with experiments utilizing 8-bromo-cAMP, on cells from two other patients. 106 Figure 54. The effects of a protein kinase-C inhibitor (PKCi; bisindolylmaleimide 50 nM) on vehicle (E3), human chorionic gonadotrophin (hCG; fD or hCG plus prostaglandin F 2 a (h/P; • ) - mediated inhibition of hCG-stimulated progesterone production (over 24 h; n=4; a^b^c, p<0.05 by A N O V A ) , in eight day pre-cultured human granulosa-luteal cells. E. Effects ofhCG and PGF2a on PGF 2a-R-mRNA 107 Spectrophotometric Estimation of Known DNA Levels in Solution Known quantities of D N A were estimated with spectrophotometric analysis in order to validate the spectrophotometer as a tool for approximating D N A and/or RNA levels in samples to be reverse transcribed. Concentrations of D N A between 5 and 5000 ng/ml were sampled. Overall, the spectrophotometric estimation of DNA levels were within 133.8 ± 5.0% of the actual D N A concentration. At high concentrations (1250 - 5000 ng/ml), this estimation improved to 100.8 ± 0.2% of the actual DNA concentration. See Table 7 for a complete listing of the results. RNA Integrity and Relative Quantity The integrity and relative quantity of total RNA samples extracted from human GLCs were checked by denaturing (formaldehyde) agarose (1.5%) gel electrophoresis. The presence of 28 and 18 S bands suggested that RNA was intact. Moreover, the apparent consistency of signal strength from one sample to the next suggested that similar efficiency of extraction was obtained for all samples. Data from two different experiments are presented here (Fig. 55 A and B). Similar results were found in other experiments. PCR Cycle Experiment Polymerase chain reaction cycle experiments for P G F 2 a - R and 6-Actin cDNA were performed in order determine the optimal number of cycles for a given concentration range and species of cDNA (Fig. 56). At the concentrations of cDNA utilized in these experiments, PCR amplification of product was relatively linear over the range of cycles tested. Amplification of cDNA for and P G F 2 a - R and 6-Actin was performed using 40 and 30 cycles, respectively, based on the results of these experiments. Amplification ofPGF2aR and fi-Actin cDNAs in Human GLCs 108 Prostaglandin F 2 a - R cDNA was amplified from human GLCs (obtained from 2 different patients) with two different sets of oligonucleotide primers (hPGFf/- and rPGF+/-). Products of the expected size (802 and 720 bp) were amplified by both primers (hPGF+/- and rPGFW-; Fig. 57A and B). Additionally, oligonucleotide primers for B-actin cDNA (Act+/-) successfully amplified a product of the expected size (524 bp) from human GLCs obtained from 3 different patients (Fig. 57C). Confirmation ofPGF2a-R cDNA in Human Granulosa-Luteal and Placental Cells Amplification of P G F 2 a - R cDNA using PCR incorporating 3 2 P - d C T P revealed the presence of products in samples obtained from human GLCs from three separate patients and in placental cells from two separate patients (Fig. 58A). However, PCR failed to detect P G F 2 a - R cDNA in human leukocyte cDNA samples from two patients. The photograph of this gel was further validated when lanes from this experiment were cut and counted with a 6-counter. Similar results were demonstrated using this technique. Regulation ofPGF2aR cDNA by hCG and PGF 2a One-day cultured human GLCs were incubated with vehicle, hCG (1 IU/ml) or hCG plus P G F 2 a ( 1 0 1 1 to I O 6 M). The effects of these treatments on P G F 2 a - R and 6-Actin cDNA levels were examined by RT-PCR (Fig. 59A), densitometry (Fig. 59B) and Southern blot hybridization (Fig. 60A and B). Briefly, P G F 2 a - R message was down-regulated by hCG. However, PGF2a at low (10" n M) and high ( I O 6 M ) concentrations prevented this down-regulation. On the contrary, the middle concentration of P G F 2 a ( I O 9 M) potentiated hCG-mediated down-regulation of P G F 2 a - R message (Fig. 59A). Densitometric analysis revealed significant inhibition of P G F 2 a - R mRNA levels in cells treated with hCG and hCG plus P G F 2 a (n=3; p<0.05 by A N O V A ; IO' 9 M ; Fig. 59B). The housekeeping gene B-actin was unaffected by any of the above treatments (not shown). Southern blot hybridization of a semi-quantitative PCR experiment (presented in Figure 59A), with an oligonucleotide probe confirmed the identity of the PCR products (Fig. 60A). Moreover, densitometric analysis of the autoradiogram revealed a pattern of mRNA regulation similar to that found in Figure 59B. In short, hCG inhibited P G F 2 a - R mRNA message in this experiment. Additionally, in presence of hCG, P G F 2 a ( I O 1 1 to I O 7 M) inhibited P G F 2 a - R mRNA message, potentiating hCG-mediated inhibition at concentrations of ( IO - 1 0 to IO - 8 M , P G F 2 a ) . (see p. 142 for relevant discussion) Table 7. Spectrophotometer estimation of known DNA levels in solution. DNA by Weight * (ng/ml) Spectrophotometer Estimation (% Actual Conc.± SEM) 5 to 5000 133.8 ± 5.0 5 to 40 183.6 ± 12.0 80 to 625 111.1 ± 1.6 1250 to 5000 100.8 ± 0.2 * Salmon sperm D N A serially diluted in 2x dilutions) from 5000 to 5 ng/ml. i — i — i — i — i — i — i — r 1 2 3 4 5 6 7 8 B i — i — i — i — i — i — i — r 1 2 3 4 5 6 7 8 Figure 55. R N A integrity gel. Agarose (1.5 %) and formaldehyde denaturing gel of RNA samples (1 /*g/sample) extracted from one day pre-cultured human granulosa-luteal cells. The presence of 28 and 18 S bands suggests that R N A integrity was good. R N A from two experiments is presented here (A and B). 112 A I E • o o CL ~ 0.32 0.30-1 0.27 0.25 0.22 0.20 0.17 y = O . O O I x 1 - 4 8 9 r = 0.986 • / 3 in in B ' z i 0 E 1 o 0.40 0.30 0.20H 0.10-^ 0.00 Cycle y = 0.880L0G(x) - 1.129 r = 0.990 in in Cycle * Figure 56. Polymerase chain reaction (PCR) cycle experiments for prostaglandin F2 a-receptor (A; P G F 2 a - R ) and 6-Actin (B) complementary D N A (cDNA). At the concentrations of cDNA utilized in these experiments PCR amplification of product was relatively linear over the range of cycles tested. Based on these experiments PCR amplification of cDNA for and P G F 2 a - R and 6-Actin was performed using 40 and 30 cycles, respectively. 113 bp 4072! 3054- 2036- 1636- 1018-1 506/17- LD GLC1 GLC2 B bp L D G L C 1 G L C 2 I636i 1018' 506/17' 720 "bp bp L D G L C 1 G L C 2 G L C 3 2036H 1636' 1 0 1 8 ' 506/171 3 9 6 4 1 524 "bp Figure 57. Polymerase chain reaction amplification of P G F 2 a R and 6-actin cDNA. Two different sets of oligonucleotide primers were utilized to amplify prostaglandin F^-receptor cDNA, and one set was utilized to amplify 6-actin cDNA, from one day pre-cultured human granulosa-luteal cells. These primers were hPGF+ and hPGF- (A), rPGF-i- and rPGF+ (B) and Act+ and Act- (C). A l l three sets of oligonucleotides were able to amplify products of the predicted size, from human granulosa-luteal cells (GLC) from up to three different patients. 114 802 bp" LD GLC GLC GLC PL PL 1 2 3 1 2 Leu Leu LD 1 2 B S 4 0 0 0 3 0 0 0 H 2 0 0 0 H 1 0 0 0 H CM co — C J C J C J —I —I —I I o_ CP ca ca —i o. T r — CM S =» Figure 58. Polymerase chain reaction amplification of PGF^-receptor cDNA from human granulosa-luteal cells, placenta and leukocytes. A. Amplification of the prostaglandin F2 a - receptor cDNA with the oligonucleotides hPGF+ and hPGF-, in the presence of 3 2 P-dCTP. Polymerase chain reaction products of the predicted size were amplified from three different human granulosa-luteal cell (GLC1, 2 and 3; uncultured samples) and two different human placenta (PL1 and 2; uncultured samples) cDNA samples. Conversely, gel lanes loaded with molecular weight ladder (LD) or two different Leu cDNA samples (uncultured samples), did not show visible amplification of product. B. Gel bands (from A) separated and counted with a 8- counter. 115 1018- bp hCG (1 IU/ml) 2.0 < I cCj •s I O CL 1.5 10 4 0 .5 4 0.0 e e u ab ab 0 HS PGF- ( 1 0 X M) hCG (1 I U / m l ) Figure 59. The effects of vehicle, human chorionic gonadotrophin (hCG) and prostaglandin F2 a (PGF2a) on PGF2a-receptor (PGF2a-R) mRNA levels (over 24 h), in one day pre-cultured human granulosa-luteal cells from three separate patients (A, B and C). Following the treatment period cells were extracted for RNA, which was reverse transcribed (RT) to cDNA and subjected to semi-quantitative polymerase chain reaction (PCR). PGF2a-R message was down regulated by hCG, however, PGF2ct at low ( l o 1 1 M) and high ( lo - 6 M) concentrations prevented this down-regulation. On the contrary, the middle concentration of PGF2a ( IO 9 M) potentiated hCG-mediated down-regulation of PGF2a-R message. The house keeping gene 6-actin was uneffected by any of the above treatments (not shown). Photographs were subjected to densitometric analysis, and normalized to 6-actin responses and averaged (D). Significant inhibition of PGF2a-R mRNA levels was seen in cells treated with hCG and hCG plus P G F 2 a (a*b*c. p<0.05 by ANOVA; 10 9 M). 116 hCG (1 IU/ml) B PGFa, ( 1 0 x M) hCG ( 1 IU /m l ) Figure 60. A. Southern blot hybridization of a semi-quantitative PCR experiment, with an oligonucleotide probe for PGF^R, in cells from one day pre-cultured human granulosa-luteal cells. These data confirm the identity of the PCR products presented in (Fig. 59 A). B. Densitometric analysis of the autoradiogram presented in A. Human chorionic gonadotrophin (hCG) inhibited PGF^-R mRNA message in this experiment. Moreover, in the presence of hCG, PGF2a (10 1 1 to 10-7 M) inhibited PGF2«-R mRNA message, potentiating hCG-mediated inhibition at concentrations of (10 1 0 to IO"8 M, P G F 2 a ) . VII-DISCUSSION 117 Caveats of the Human Granulosa-Luteal Cell Model Variability in Basal Steroidogenesis in the Human GLC Model Granulosa-luteal cells obtained during oocyte collection from superovulated in vitro fertilization patients are a very difficult model to work with as they exhibit a high degree of variability in their basal levels of steroidogenesis (Fig. 13-16). In part, these differences can be accounted for by varying proportions of cells obtained from different-sized follicles (Fig. 25) since the number and maturity of follicles punctured varies greatly with patients. Additionally, the possiblity of contaminant epithelial or immune cell involvement in the PGF 2 a-mediated responses of luteal cells may further complicate matters, should this exist. Epithelial cells are reported to permit or even enhance PGF2a-mediated responses in luteal cells in the placenta [Alecozay et al., 1991]. Thus, differences in follicle puncture, cell collection and purification can all contribute to variability in steroidogenic responses. Other potential sources of variability include the following: 1) the population of patients from which cells are collected have a much higher probability of infertility than the general population; 2) patient responses to super-ovulation are highly variable, suggesting important biological differences; and 3) the differences in time between follicle puncture and cell plating. Throughout these experiments cells were plated at a density of 10 3 to 10 4 cells/well. Within this range of cell densities, no significant density-dependent difference was seen in the steroidogenic responses to hCG or hCG plus P G F 2 a between patients (differences within a patient were not examined). Moreover, basal steroidogenesis did not change in response to cell density. However, in a report by Bari-Ami and Gitay-Goren [1993], basal steroidogenesis showed cell density and culture-time dependent changes in progesterone and estradiol production such that increases in cell density from 2.5xlCP to l x l O 4 cells/well could increase progesterone production 1.3-fold. A further increase in cell density from l x l O 4 to 8X10 4 cells/well could decrease progesterone production 3.7-fold. They also reported density and culture time- dependent changes in estradiol production. Culture condition, cell manipulation or other factors may account for the discrepancies in these two studies. It should be noted, however, that in most cases, although the basal levels of steroidogenesis varied greatly, the relative responses to stimuli were quite similar when comparing responses from different patients (see Fig. 17). Thus, although the problem of variability exists, standardization of data can still render this model useful for determining relative trends and mechanisms of action. Data standardization can be achieved by either experimental or statistical means. Experimental methods normally include taking the ratio of the data from one physiological parameter over some other form of data. For example: peptide secretion from endocrine cells is often represented over the total amount of stored peptide. Taking the ratio of a response over D N A levels or cellular protein content is also common. In this model, experimental methods of standardization are not satisfactory, as response per cell or D N A level is too variable. Therefore, a statistical method of standardization is required. Statistical standardization methods include converting data to a percentage of the control level, log transformations or conversion to standard mean scores [Lewis 1984]. A l l of these methods have advantages and disadvantages. Log transformations are not completely satisfactory with respect to this model as basal levels can vary many-fold. Thus, these studies utilized percentage of control and standard mean score transformations. Although the human G L C model is quite variable in the basal levels of steroidogenesis, the relative responses to stimuli are similar in cells from different patients. Thus, this model can be effectively utilized to determine relative responses to stimuli and the mechanisms of these responses. Moreover, this model provides the only source of human granulosa-luteal cells ethically available in high enough quantity to study effectively. Cell Numbers and Low Level RNA Expression Although human GLCs are readily available in high enough quantity for morphological or pharmacological study, the numbers of cells obtained are barely adequate for molecular biological techniques. This is especially true when examining genes which are expressed at low levels. Reverse transcription-PCR allowed for semi-quantitative examination of genes of low level expression, due to the amplification obtained through PCR. Even with this amplification PGF2«-R mRNA was difficult to detect. Total RNA levels extracted from cells were normally between 1 to 2 ptg per sample, a level too low to be useful for Northern blot hybridization. Thus, although Northern blot analysis would have been an easier and more direct quantification method, the levels of P G F 2 a - R mRNA did not permit this procedure. Rather than not study the regulation of P G F 2 a - R mRNA, RT-PCR was utilized to provide some useful insights that would not otherwise be possible. A Question of Physiological Concentration? What is a physiological concentration? This is a difficult question to answer. In these studies, concentrations of P G F 2 a , PGF^ and GnRH ranged from pM to piM. levels. This range of concentrations was utilized because in vivo data in the human is unobtainable. Moreover, the concentrations which a receptor 'sees' are virtually impossible to determine. Tight junctions, secretion patterns, local degradation, binding proteins, receptor affinity and other factors can greatly alter the effective concentration of a hormone at its site of action. The female reproductive system is not a homeostatic system, it is a cycling system. Herein lies its appeal to many scientists. In a cycling system, one physiological concentration cannot be assigned to most hormones. For example, progesterone and estradiol concentrations vary greatly throughout the menstrual cycle. Which concentration is physiological? At any given point in time, the physiological concentration of a hormone may change. Additionally, functions can be attributed to a hormone's absence as well as its presence. Finally, as the affinty of the PGF2ct-R is quite high (Kd of 1 nM), it is reasonable to expect that this hormone may have physiological effects at concentrations which are several-fold lower or higher than this Kd. This turned out to be the case. Summary Notwithstanding these limitations, it must be noted that this model is a valuable one. There are very few human tissues which are so obtainable for scientific examination. The author believes that it is a scientist's responsibility to work around the limitations of such a valuable model and learn as much as one can from it. Morphology of Human Granulosa-Luteal Cells in Culture 120 The morphological characteristics of human GLCs change dramatically with culture time. Moreover, these changes correspond well with those reported for cells undergoing luteinization. Granulosa cells exposed to luteotrophins change from their characteristic polygonal shape to a round one, occasionally projecting finger-like processes which may attach to adjacent cells [Soto et al., 1986]. These characteristics are very similar to those of freshly cultured and one-day cultured human G L C , as seen in these studies. As cells luteinize, they are reported to form tighter associations [Ratamales et al., 1994], increased vacuolation and blebbing [Quirk et al., 1995]. A l l of these reported characteristics were seen in eight-day and twelve-day cultures of human GLCs, suggesting that these cells are luteinizing in culture. This notion is certainly supported by the functional differentiation seen with culture time. An increase in luteal cell blebbing, characteristic of cells undergoing apoptosis, has also been reported in human granulosa-luteal cells [Quirk et al., 1995]. Interestingly, in these studies it was found that an increase in cell irregularity and blebbing was seen as culture time progressed. Moreover, cells disrupted by 16 to 18 days of culture appeared to have undergone apoptosis. This disruption was associated with a loss in functional response. Similar morphological characteristics have been reported in other mammals including porcine, bovine, feline and rat models [Gregoraszczuk and Krzysztofowicz 1989; Roth et al., 1995; Chegini et al., 1984; Fields et al., 1992; Meidan et al., 1990; Fields et al., 1985; Yuh et al., 1986; Nelson et al., 1992]. Luteal cell vacuolation, blebbing of various sizes, ruffles and lipid vacuoles are also reported in these species. In the rat, differences between small and large luteal cells are also reported, such that small luteal cells appear stellate while large luteal cells do not flatten out completely (probably due to large lipid droplets) [Nelson et al., 1992]. In one-day human GLCs, two cell populations appeared to be present which corresponded remarkably well with those reported in the rat. In summary, human GLCs appeared to undergo morphological luteinization and possibly apoptosis with culture time. These results support the human granulosa-luteal cell as a good model for the study of luteinization. 121 A Effects of PGF2a on Human Granulosa-Luteal Cells in the Absence and Presence of hCG Wide ranging concentration-response studies (1 pM to lyM PGF2« or GnRH) were performed in both the presence and absence of hCG, in short, medium and long term cultures of human GLCs. The importance of these parameters was highlighted by the concentration- and culture time-dependent differential responses to P G F 2 a under these conditions. Profoundly different progesterone and estradiol production in response to P G F 2 a was seen when comparing D i , Dg and D12-14 cultured human GLCs. In the case of GnRH, differences were observed from D i to D 8 cells. These findings emphasize the importance of maintaining awareness of culture time in experiments using highly differentiated GLCs. Further, the basal effects of P G F 2 a suggest that superovulation-derived human GLCs continue to undergo luteinization in vitro, as they paralleled previous results examining early- and mid-luteal phase cells [Khan et al., 1989], The effects of P G F 2 a on progesterone production in GLCs differed with culture time. Prostaglandin F 2 a inhibited in day D i , but stimulated progesterone production in D12-14 cultured GLCs. The cells were found to be less defined in their responses to P G F 2 a in Dg, as they appeared to be in a state of transition between their inhibitory and stimulatory responses. Early-luteal and D i GLCs both demonstrated inhibition, while mid- luteal and Dg GLCs demonstrated stimulation of basal-progesterone production in response to P G F 2 a [Khan et al., 1989]. This further supports the suitability of IVF derived cells as a model to study human ovarian cell function. Thus, P G F 2 a was capable of either inhibiting or stimulating progesterone production depending on concentration and culture conditions. Follicle Size Differential responses to hCG and PGF2« based on follicle size were seen in D i GLCs. When the results from four separate experiments were pooled, it became clear that the magnitude of the hCG-induced steroidogenic response was reduced in small versus large follicles. The response was significant in large follicles, but not in small follicles. Previous studies have demonstrated that the number of hCG receptors increases with follicle size [Kammerman and Ross, 1975; May and Schomberg, 1984; Hillier et al., 1980]. These results suggest that there could be differential steroidogenic responses in cells from different patients, due to differing proportions of small and large follicles. This hypothesis is supported by a previous report which found that follicles yielding mature cumulus-oocyte complexes (COC) represent a non- homogenous population in which GLCs from follicles yielding type A - B COC (cumulus cells aggregated into clumps) are less luteinized than GLCs from follicles yielding type C-D COCs (cumulus cells homogeneously spread out) [Gitay-Goren et al., 1990]. Concentration and Culture Time Dependent Responses The concentration range of PGF201 and GnRH used in these studies resulted in bell curve like inhibition of hCG-stimulated progesterone and estradiol production (see Fig. 26, 28, 30-31). This bimodal nature is not unusual for prostaglandin actions [Cohen and Rimon, 1992; Sano and Shichi, 1993; Puschel et al., 1993; Hargrove et al., 1975]. For example, in the rat and rabbit testicular tissues, bimodal responses to P G E 2 and PGEj have been reported, respectively [Cohen and Rimon, 1992; Hargrove et al., 1975]. Rat epididymal adipocytes displayed a PGE2-mediated inhibition and stimulation of c A M P at concentrations of 10 mM and >10 mM, respectively. In the rabbit, testicular contractions were stimulated and inhibited by PGEj at concentrations of 1-10 nM and 100 nM, respectively. Non-reproductive tissues also have been shown to exhibit bimodal responses to prostaglandins [Sano and Shichi, 1993; Puschel et al., 1993]. In rat hepatocytes, P G E 2 can act in a glycogenolytic and in a antiglycogenolytic fashion at concentrations of 10 JAM and 1 nM, respectively. It is reported that these glycogenolytic and antiglycogenolygic actions are likely mediated through the inositol triphosphate and c A M P pathways, respectively. The potential for multiple G-protein coupling to P G F 2 a receptors is also present as seen in the gonadotroph [Hawes et al., 1993; Barnes and Conn, 1993]. This possiblity is supported by the identification of four different G-protein alpha subunits in the human G L C , including G a S , G a j3 , G a i i ,2 and G a q n [Lopez et al., 1995]. Similarity, multiple-receptors have been suggested as an explanation for bimodal prostaglandin responses in the porcine ciliary epithelium [Sano and Shichi, 1993]. In the presence of hCG, culture time (presumably luteinization)-dependent alterations in the steroidogenic responses to PGF2a and GnRH have also been observed. The general trend of the concentration-dependent response to PGF2a or GnRH was retained with culture time although it shifted in an upward (stimulatory) fashion, retaining its anti-gonadotrophic effects only at the highest concentration tested (1 ptM), in Dg GLCs. Potentiation (1.5- to 3-fold) of hCG- stimulated progesterone production was seen D 8 GLCs cultured in the presence of P G F 2 a or G n R H (at 1 nM; Fig. 3B and 6B). The ability of P G F 2 a to potentiate hCG-stimulated progesterone production in Dg (presumably mid-luteal like) cells, may have implications for early pregnancy. Further support for this idea resides in the literature, as hCG pretreatment has been shown to prevent the anti-gonadotrophic actions of PGF2a [Michael and Webley, 1991b]. It has been suggested that hCG produced by the new conceptus may prevent corpus luteum regression by this mechanism. Furthermore, P G F 2 a concentrations in the human luteal cell are at their highest levels in mid-luteal phase, the time when it would be least appropriate to undergo luteolysis. This potentiation may have an important biological function at this stage, and may involve the interaction of P G F 2 a and other ovarian hormones such as PGF^. Prostaglandin E2 is at its highest concentrations during the mid-luteal phase [Patwardhan and Lanthier, 1980 and 1985] and is reported to have antagonistic actions against PGF2a-mediated luteolysis. Perhaps PGE 2-mediated antagonism of PGF2a-mediated luteolysis enhances the luteotrophic responsiveness of these cells. Summary Prostaglandin F2 a and GnRH appear to be bimodal regulators of steroidogenesis in the human ovarian cell. In addition to the antisteroidogenic abilities of PGF2a and GnRH (IO - 6 M; D i and D 8 ) , these results suggest that P G F 2 a ( I O 9 M ; Dg and D12-14) and GnRH (10"9 M; Dg) may play a role in the maintenance of the corpus luteum through their potentiation of hCG-stimulated progesterone production (Fig. 61). 124 hCG PGR P r o g e s t e r o n e & E s t r a d i o l P r o d u c t i o n Figure 61. Dual (bimodal) actions of Prostaglandin F2« on progesterone and estradiol production, in human luteal cells. 125 B. Interaction of PGF 2a with GnRH Progesterone Response This study examined the interactions of GnRH and P G F 2 a in the presence and absence of hCG. In general, at the concentrations tested, neither GnRH nor P G F 2 a altered progesterone or estradiol production in D i cultures. However, when GnRH and P G F 2 a were co-applied, a significant stimulation of progesterone and estradiol production was seen. Furthermore, in Dg cultures where a weak luteotrophic action was seen with P G F 2 a treatment, GnRH potentiated PGF2a-stimulated progesterone production, but was again ineffective on its own. These results suggest that P G F 2 a requires GnRH as a permissive factor in order for its luteotrophic action to be present, and that GnRH on its own is not a luteotrophin. The ability of P G F 2 a to stimulate progesterone production in D 8 cultures suggests that endogenous GnRH may be present. Alternative explanations would be that a second permissive factor exists, or that P G F 2 a does not require one in later cultures. Interestingly, the magnitude of the response is much greater in Dg cultures. When viewed in three dimensions, the nature of the interactions between GnRH and . P G F 2 a are intriguing. At optimal PGF201 concentrations, GnRH stimulated progesterone production appears linear and concentration dependent. At optimal GnRH conditions, PGF2«- stimulated progesterone production is bell curve-like. One remarkable characteristic of GnRH plus PGF2a-mediated progesterone production is the consistency of the response. In this laboratory, the luteotrophic response to P G F 2 a alone is only present in cells from about 50% of patients, while in these experiments, 100% of patients demonstrated a luteotrophic response with co-application of these two hormones. In Di cultures and at the concentrations tested, GnRH was not luteolytic, although it was in Dg cultures. Gonadotrophin-releasing hormone potentiated PGF2 a-mediated luteolysis in D i and Dg pre-cultured human GLCs. Estradiol Response Estradiol production was also regulated in a remarkable fashion by the co-application of GnRH and P G F 2 a - In the presence of high concentrations of GnRH, P G F 2 a linearly and concentration dependency stimulated estradiol production. These effects of GnRH appeared to be permissive, as PGF 2 a-mediated stimulation of estradiol production was not present in the absence of GnRH. Moreover, the linear stimulation of estradiol production by PGF2a (in the presence of high concentrations of GnRH) appeared to be similar to the response seen in eight- day cultures (in the absence of exogenous GnRH; not shown). Perhaps the levels of endogenous GnRH in the culture system increase with culture time, with the effect of modulating the response over culture-time. Gonadotrophin-releasing hormone alone, significantly stimulated estradiol production in a bell curve-like manner. However, in the presence of high concentrations of PGF2a, GnRH-mediated stimulation of estradiol production shifted from being bell curve-like to linear. The effects of PGF2« on the response to GnRH were modulatory rather than permissive. Implications The striking difference in the progesterone and estradiol responses to PGF2« and/or GnRH, may play an important role in the regulation of the luteal phase or even the menstrual cycle. If one superimposes the three dimensional plots of these two hormones, one can see that as the concentrations of PGF2a and/or GnRH change, so does the ratio of progesterone to estradiol production. For example, at high concentrations of GnRH and middle concentrations of P G F 2 a estradiol production is low relative to progesterone production, which is at its highest. However, at high concentrations of GnRH and P G F 2 a estradiol production is at its highest, while progesterone production is at its lowest level. Thus, due to the co-operative nature of P G F 2 a and GnRH, subtle changes in the concentrations of these hormoens can have profound effects on the ratio of progesterone to estradiol production. With this in mind, it is interesting to note that under conditions conducive to a luteotrophic response (ie. high progesterone production; P G F 2 a 10"9 and GnRH 10~5 M) estradiol production is relatively low, while under conditions optimal for luteolysis (ie. low progesterone production; PGF 10~6 and GnRH 10~5 M) estradiol production is high. The present results further support the hypothesis that PGF2a and GnRH have very similar and/or complementary roles in the ovary. Teleologically speaking, the advantages of a dual hormone system over a single hormone system seem obvious. Should one system be deficient or fail, the second system would provide a backup. Conversely, when both systems are working amplification and fine tuning of the signal are improved. Interestingly, a positive feedback loop may exist in this system, as GnRH has been shown to stimulate arachidonic acid and P G E 2 from the luteal cell, and PGF production in the placenta [Kawai and Clark, 1985 and 1986; Hillensjo et al., 1982; Siler et al., 1986]. It is unknown if P G F 2 a stimulates GnRH production in these cells. The bell curve-like response to P G F 2 a could act as a fine tuning or switching mechanism, allowing a luteotrophic response to turn into a luteolytic one should P G F 2 a levels increase much beyond nM concentrations. This bimodal response to prostaglandins is not unique to the corpus luteum [Cohen and Rimon, 1992; Sano and Shichi, 1993; Puschel et al., 1993; Hargrove et al., 1975]. For example, in the rat and rabbit testicular tissues, bimodal responses to PGE2 and P G E i have been reported, respectively [Hillensjo et al., 1982; Sano and Shichi, 1993]. Rat epididymal adipocytes displayed a PGE2-mediated inhibition and stimulation of c A M P at concentrations of 10 mM and >10 mM, respectively. In the rabbit, testicular contractions were stimulated and inhibited by P G E i , at concentrations of 1-10 nM and 100 nM, respectively. Non-reproductive tissues also have been shown to exhibit bimodal responses to prostaglandins [Siler et al., 1986; Cohen and Rimon, 1992]. In rat hepatocytes, PGE2 can act in a glycogenolytic and in a antiglycogenolytic fashion at concentrations of 10 piM and 1 nM, respectively. As mentioned previously, these glycogenolytic and antiglycogenolygic actions are likely mediated through the inositol triphosphate and c A M P pathways, respectively. Controversy over which effect (luteotrophic or luteolytic) is physiological are bound to arise from these results and those of others. The reported K d for the P G F 2 a receptor is in the nano-molar range [Lake et al., 1994], close to the concentrations at which the luteotrophic response is present. It is likely that both responses are physiological and that their temporal relationship to luteolysis and early pregnancy is important. As mentioned above, studies have demonstrated the predominance of the luteotrophic response in the mid-luteal phase [Khan et al., 1989; Richardson and Masson, 1980; Michael and Webley, 1993], corresponding well with a potential role in the promotion of early pregnancy. In support of this notion, there have been reports of enhanced progesterone production in the presence of hCG plus P G F 2 a compared with hCG alone [Suginami et al., 1976], as well as studies that have demonstrated the abrogation of PGF 2 a-mediated luteolysis when cellular exposure to hCG or prolactin preceeds P G F 2 a exposure [Harris and Murphy, 1981; Suginami et al., 1976; Michael and Webley, 1991b]. Two models by which GnRH may be permissive or potentiatory with respect to P G F 2 a - mediated steroidogenesis are depicted in Figures 62 and 63. In short, GnRH may provide a missing component in a PGF 2 a-affected signal transduction system, or it may promote the production of de novo P G F 2 a . Further treatment of this subject may be found below in the signal transduction section (p. 132). With enhanced magnitude and consistency of both the luteotrophic and luteolytic actions of P G F 2 a and GnRH when co-applied, the potential for improving the clinical applications of these two hormones exists. Currently, potent, long-lasting GnRH analogues have been applied to numerous therapeutic applications in the female including: contraception; treatment of central precocious puberty; and sex steroid-dependent benign and malignant diseases of the reproductive organs [Molcho et al., 1984; Bhasin et al., 1984; Nillius, 1985]. One of the most common uses of GnRH analogues is the down-regulation of pituitary function in preparation for super- ovulation as part of an in vitro fertilization protocol. Conversely, attempts to inhibit luteal function and induce luteolysis or early abortion with GnRH have not been very successful [Nillius, 1985]. The contraceptive effects of GnRH appear to be safe, reliable and reversible in women [Nillius, 1985]. Experimental Model When examining the interactions of two or more hormones, the author has found multi- concentration experiments to be much more revealing than single concentration studies. The difficulties with these experimental models lie in visualization of the results. Plotting data in three dimensions with the two interacting agents on the horizonal axes and the response on the vertical axis simplifies interpretation of the results. Further enhancement of visualization of the results with a contour plot of the data was achieved. This design and the improved speed of modern computers in plotting such data has revealed a more complex interaction between these two hormones than would be visible with a standard two dimensional experimental design. Certainly numerous other hormones are interacting in similar or even more complex manners. Summary These studies reveal the complex luteolytic and luteotrophic actions of GnRH and PGF2a. 129 GnRH PGF 2a Progesterone & Es t rad io l Product ion Figure 62. Gonadotrophin-releasing hormone (GnRH) acts as a permissive or potentiatory factor for prostaglandin F2« (PGF2o)-mediated luteotrophic and luteolytic effects, respectively. This model suggests that GnRH may provide a missing signal transduction factor, thus completing or enhancing the signal transduction pathways by which P G F 2 a functions. 130 GnRH PGF Progesterone & Es t rad io l Product ion Figure 63. Gonadotrophin-releasing hormone (GnRH) acts as a permissive or potentiatory factor for prostaglandin F 2 a (PGF2a)-mediated luteotrophic and effects luteolytic, respectively. This model suggests that GnRH may stimulate de novo synthesis of PGF2«, through a PKC-dependent stimulation of arachidonic acid (AA) production, as is seen in some other systems [Smith and Borgeat 1988]. C. Interaction of PGF2a with PGE2. In eight-day precultured GLCs , both PGF2a and PGE2 stimulated progesterone production in a bell curve-like manner. As with previous experiments PGF2a exerted its maximal luteotrophic effect at a concentration of 1 nM. Interestingly, the bell curve-like stimulation mediated by PGE2 was shifted to the right (when compared to that of P G F 2 a ) such that maximal stimulation of progesterone production was seen at a concentrations of 10 to 100 nM. In both cases, maximal luteotrophic effects were present at or near the Kds of their receptors [Abramovitz et al., 1994; Abramovitz et al., 1994b; Lake et al., 1994]. The combination of these hormones resulted in an elaborate regulation of progesterone production whereby the bell curve was inverted. There are a number of possible explanations for the complexity of this regulation, including: 1) the non-standard saturation kinetics of PLA2; 2) product inhibition of PGE2 production by PGF2K; 3) receptor cross reactivity; 4) the opposing actions of these hormones on common signal transduction pathways; and 5) the combination PGF 2 a-mediated luteolytic and luteotrophic effects in the presence of P G E 2 . It is likely that all of these factors play a role in these findings. The physiological significance of these findings is not obvious at first glance. However, these results provide a potential mechanism for the changing functional role of the corpus luteum as luteinization progresses. Changing the ratio of PGF2K to PGE2 can profoundly alter progesterone production, a result seen in other reports which compared luteal cells in early, mid and late luteal stages [Pathwardhan and Lanthier, 1985]. Although complex in nature, these results should not be ignored as the ratio of PGF2a to PGE2 is known to change in vitro. Taken with the confounding effects of PGF2« and GnRH, there exists more than adequate room for explaining the changing responses of granulosa and luteal cells with differentiation. Further, these results suggest that PGF2« interactions with PGE2 form a sophisticated means of regulating progesterone production in human granulosa-luteal cells. D. Signal Transduction of PGF ̂ Mediated Luteolysis 132 This study has examined the signal transduction pathways utilized in the anti- gonadotrophic (or luteolytic) actions of PGF2a in the human granulosa-luteal cell. Specifically, the ability of P G F 2 a to inhibit both hCG- and isoproterenol-stimulated progesterone production was examined, as well as hCG-stimulated estradiol production. Furthermore, the signal transduction pathways involved in this effect were examined with pertussis- and cholera-toxin, as well as with forskolin and db-cAMP. As seen above, the basal responses to PGF2a were quite variable (Fig. 47,49, and 51-53). This may be due to differences in the endogenous levels of PGF2« within the culture media, differences in cellular differentiation state or differences in endogenous levels of interacting hormone levels. Further studies are underway to determine the exact nature of this phenomenon. Pertussis Toxin Sensitive G-Protein It was found that P G F 2 a is exerting its anti-gonadotrophic (specifically anti-hCG) actions through a pertussis toxin sensitive G-protein. These data were supported by the ability of PGF2« to inhibit CTX-stimulated progesterone and estradiol production, and by the reversal of this inhibition by the addition of PTX. These data also suggest that PGF2« rnay exert its anti- gonadotrophic actions at an early step in the signal transduction cascade. Reports in the literature support the potential role of G-proteins in the signal transduction of P G F 2 a . Firstly, human granulosa-luteal cells have been examined immunocytochemically to reveal a number of G-protein alpha subunits, including, G a s , Gaj3, Gaii2 and G a p (namely, G a q and G a i i ) , while G Q was undetectable by three different antibodies [Lopez et al., 1995]. Intracellular c A M P levels in human granulosa cells appear to be regulated by the ratio of G a s and Gaj-subunits, while G a q n and G a j levels regulate the accumulation of inositol phosphates [Lopez et al., 1995]. Coupling of one or both of these identified Gai-subunits to the P G F 2 a - receptor could explain the PTX sensitivity of the anti-gonadotrophic action of PGF2a, as well as the regulation of both c A M P and inositol phosphates by P G F 2 a . Further supporting a role for G-proteins in the signal transduction of P G F 2 a are the sequences and predicted structure of the cloned prostanoid receptors. A l l of these receptors possess the seven-transmembrane domain structure characteristic of G-protein coupled receptors [Lake et al., 1994; Abramovitz et al., 1994; Adam et al., 1994; Boie et al., 1994 and 1995; Funk et al., 1993]. Additionally, the human EP3-family of receptors is capable of inhibiting c A M P production through a pertussis toxin-sensitive G-protein [An et al., 1994]. It is not known if PGF2a is acting through single or multiple G-proteins, as is seen in the actions of gonadotrophin- releasing hormone (GnRH) in the gonadotrope [Hawes et al., 1993]. Thus far, P G F 2 a has been demonstrated to lower gonadotrophin- and prostaglandin L V stimulated rises in c A M P , as well as increase intracellular calcium and inositol phosphates [Davis et al., 1989; Currie et al., 1992; Pepperell et al., 1989; Lahav et al., 1987]. The DP, IP and EP3-family of receptors are all coupled to c A M P regulation [Adam et al., 1994; Boie et al., 1994 and 1995; An et al., 1994], while the DP, FP, E P i and EP3-family of receptors are coupled to rises in intracellular calcium [Abramovitz et al., 1994; Adam et al., 1994; Boie et al., 1995; Funk et al., 1993; A n et al., 1994]. It is unknown if the actions of P G F 2 a are exerted through single or multiple-receptors. With P G F 2 a and PGE2 both being present and active in the human granulosa and luteal cells [Grinwich et al., 1976; Richardson and Masson, 1980; Pathwardhan and Lanthier, 1985; Satoh et al., 1981; Watson et al., 1979], it is probable that multiple prostanoid receptors exist in these cells. Furthermore, the currently cloned prostanoid receptors all possess varying degrees of cross-reactivity with P G E 2 and P G F 2 a [Lake et al., 1994; Abramovitz et al., 1994; Adam et al., 1994; Boie et al., 1994 and 1995; Funk et al., 1993; An et al., 1994]. Although the present results indicate that the anti-gonadotrophic effects of PGF2« are due to a pertussis toxin sensitive G-protein, it is unclear if they are mediated through Goi, G a p or both. The G-protein alpha-subunit designated G a p is not a single G-protein but is, in fact, a family of G-proteins capable of activating phosphoinositide phosphodiesterase [Cockcroft and Stutchfield, 1988]. More than one G p pathway often exists within a single cell type, with distinct proteins coupling different receptors to phosphatidyl inositide hydrolysis selectively, thus allowing for regulation of the magnitude of phosphatidyl inositide hydrolysis [Ashkenazi et al., 1989]. Within the Gp family of G-proteins there exists two sub-families, a pertussis toxin- sensitive and a pertussis toxin-insensitive G p , both of which are involved in P L C regulation [Martin et al., 1991]. For example, bovine adrenal fasiculata cells possess angiotensin-II receptors which are coupled to the phosphoinositide pathway through pertussis toxin-sensitive and insensitive GP proteins [Langois et al., 1990]. This example demonstrates the ability of a single receptor to be coupled to multiple-forms of GP, providing for a complex regulation of the phosphoinositide pathway. If the anti-gonadotrophic effects of P G F 2 a are mediated by GOJ, this would probably be through a direct effect of Goi on adenylate cyclase and/or through the 'mopping-up' of G a s - subunits by free beta/gamma-subunits freed when G ^ was released. Alternatively, if the anti- gonadotrophic effects of PGF2a are mediated through a pertussis toxin sensitive G a p , it is likely that inhibition of the c A M P pathway would be through elevated levels of inositol phosphates, calcium, diacylglyceride and P K C activity and through the actions of these messengers on the c A M P pathway. In the sheep, it has been demonstrated that elevated levels of phospholipase-C activity involves a pertussis toxin-sensitive protein [McCann and Flint, 1993]. It is also possible that the PGF2 a-receptor is coupled to more than one G-protein,. Finally it has been suggested that a single God-like G a p protein may be capable of multiple actions [Magnaldo et al., 1988]. In hamster fibroblasts, thrombin is capable of inhibiting adenylate cyclase via a G-protein, while the G-protein mediated-activation of PKC causes a stimulatory effect on adenylate cyclase. This indirect stimulatory effect is exerted by P K C action directly on an element of the adenylate cyclase-Gos complex [Magnaldo et al., 1988]. If a similar mechanism existed in the human granulosa-luteal cell, this might help to explain the transient stimulatory effects of P G F 2 a on basal progesterone production. In the gonadotrope, it has been demonstrated that GnRH may exert its actions through as many as three G-proteins [Hawes et al., 1993]. Briefly, GnRH has been demonstrated to stimulate IP production through a PTX-sensitive G-protein (Gp), while a distinct CTX-sensitive G protein can sensitize the gonadotrope to luteinizing hormone (LH) release through cAMP. Finally, a third CTX/PTX-insensitive G-protein can mediate L H release. Furthermore, there has been the suggestion of cross-talk between the CTX-sensitive G-protein and the P K C pathway [Barnes and Conn, 1993]. In view of the remarkable similarities (signal transduction and steroidogenic effects) between GnRH and P G F 2 a [Quirk et al., 1995; Leung and Steele, 1992; Stoljelkovics et al., 1994] and their receptors [Lake et al., 1994; Abramovitz et al., 1994; Stoljelkovics et al., 1994] in the ovary, similar complexities may play a role in the signal transduction of the actions of P G F 2 a in the human granulosa-luteal cells. 135 Adenylate Cyclase and cAMP In these studies, PGF201 inhibited forskolin- but not db-cAMP-stimulated progesterone production from human granulosa-luteal cells, suggesting that it is exerting its actions at or above the level of gonadotrophin-dependent adenylate cyclase. In previous studies, forskolin-stimulated progesterone production from large luteal cells (bovine CL) was inhibited by PGF2« only in the presence of endothelial cells [Girsh et al., 1995]. This action was attributed to the secretion of PGI2 by endothelial cells. In contrast to the present results, several other studies in rat and bovine luteal cells, have demonstrated the inhibition of progesterone production by PGF2« at sites distal to adenylate cyclase [Rajkumar et al., 1988; Benhaim et al., 1987; Dorflinger et al., 1983]. This inhibition may be mediated by a reduction in the sensitivity of the cells to c A M P . In light of the irreversibility of forskolin-activation of adenylate cyclase it is unlikely that P G F 2 a is exerting it's inhibitory actions solely on adenylate cyclase. These actions may be exerted in part through a phosphodiesterase family member (Michael and Webley 1991a). The 6-adrenergic receptor, one of the most studied and understood of receptors, has been well established as a seven transmembrane, G-protein coupled receptor which activates the production of c A M P from adenylate cyclase [Briggs 1982; Barak et al., 1995; O'Dowd et al., 1988]. Several studies have demonstrated that isoproterenol (a 8-adrenergic agonist) is capable of stimulating c A M P and progesterone production from granulosa and luteal cells in non-primate species [Leung, 1985]. However, thus far there have been conflicting reports regarding the effects of isoproterenol on progesterone production and adenylate cyclase activity in the granulosa-luteal cells of the human [Fohr et al., 1993; Casper and Cotterell, 1984; Hellensjo et al., 1985]. This study demonstrates that isoproterenol is capable of stimulating progesterone production in the human granulosa-luteal cell, and that this stimulation may be inhibited by PGF2a. The ability of PGF2a to inhibit isoproterenol-stimulated estradiol production was not examined in these studies, although isoproterenol was found to stimulate estradiol production (not shown). Protein Kinase C As mentioned above, previous studies have demonstrated the ability of P K C inhibitors to partially inhibit PGF2a-mediated luteolysis. These studies have confirmed this result. The inhibitory actions of P K C inhibitors are not as complete as those of PTX, suggesting that P K C inhibitors are blocking the effects of PGF2a through an indirect means. Based on the known pathways by which prostaglandin production is stimulated in other models [Smith and Borgeat 1988], the author would like to suggest that endogenous PGF2« production may be stimulated by the calcium-diglyceride-PKC pathway in these cells. This would help to explain the partiality of the P K C inhibitory effect on PGF 2 a-mediated luteolysis, as de novo prostaglandin synthesis would be blocked, although existing P G F 2 a could still be effective until degraded. Moreover, it would provide a mechanistic explanation for the ability of GnRH to potentiate or permit the effects of P G F 2 a , as GnRH is known to stimulate the calcium-diglyceride-PKC pathway as well (see Fig. 64). Additionally, the known stimulation of this signal transduction pathway by PGF2a could provide a positive feedback mechanism. In support of this idea is the ability of indomethacin to block the luteotrophic effects of P G F 2 a . As indomethacin is a blocker of de novo prostaglandin production, this effect suggests that in order for PGF2a to exert its actions in these cells a feedback loop is necessary. Given the short half-life of PGF2«, this would make sense. De Novo Protein Synthesis Interestingly, the effects of PGF2a are reported to be dependent on de novo protein synthesis, as actinomycin-D blocks them [Fitz et al., 1993]. It would not be surprising if this de novo protein is either an eicosogenic enzyme or GnRH. Summary The anti-gonadotrophic actions of PGF2a are mediated through a pertussis-toxin sensitive G-protein (possibly Gi, G p or both; Fig. 65). Prostaglandin F2a is capable of inhibiting progesterone production in response to hCG (Fig. 66), isoproternol (Fig. 66), C T X (Fig. 67A) and forskolin (Fig. 67B), but not db-cAMP (Fig. 67), strongly suggesting that PGF2« is exerting its anti-gonadotrophic actions at or above the level of adenylate-cyclase. Further supporting this conclusion is the ability of PTX to reinstate the stimulatory actions of C T X following P G F 2 a administration. There exists the potential that these actions are exerted through a member of the phosphodiesterase family of enzymes. 1 3 7 GnRH PGF. Progesterone & Es t rad io l Product ion Figure 64. Proposed positive feedback loop for prostaglandin F 2 a synthesis (PGF 2 a ) - Gonadotrophin-releasing hormone (GnRH) and P G F 2 a are both known to stimulate the calcium- diglyceride-protein kinase C (PKC) pathway. Moreover, PKC pathway is reported to stimulate de novo prostaglandin synthesis in some systems. Thus, there exists the possiblity that P G F 2 a and GnRH may provide postive feedback on de novo PGF2 a synthesis in the human granulosa- luteal cell. 138 Figure 65. Pertussis toxin blocks prostaglandin F 2 a (PGF2a)-mediated inhibition of human chorionic gonadotrophin (hCG)-stimulated steroidogenesis in the human luteal ceil. G - stimulatory G-protein; - pertussis toxin sensitive G-protein; AC - adenylate cyclase (AC); cAMP - cyclic adenosine monophosphate; and PKA - protein kinase A 139 Isoproterenol hCG PGR Progesterone & Estradiol Production Figure 66. Prostaglandin F2 a (PGF2a)-mediated inhibition of human chorionic gonadotrophin (hCG)- and isoproterenol-stimulated steroidogenesis, in the human granulosa-luteal cell. G - stimulatory G-protein; A C - adenylate cyclase (AC); c A M P - cyclic adenosine monophosphate; and P K A - protein kinase A 140 PGF. Figure 67. Prostaglandin F 2 a (PGF2a)-mediated inhibition of cholera toxin (A) and forskolin (B) stimulated progesterone and estradiol production in human granulosa4uteal cells. G - stimulatory G-protein; A C - adenylate cyclase (AC); c A M P - cyclic adenosine monophosphate; and P K A - protein kinase A Figure 68. The inability of prostaglandin F2« (PGF2o) to inhibit dibutryl cyclic adenosine monophosphate (cAMP) stimulated progesterone and estradiol production in human granulosa- luteal cells. PKA - protein kinase A E. Regulation ofPGF2a-R mRNA A n inverse relationship between progesterone production and P G F 2 a - R mRNA levels was revealed in the present studies. Human chorionic gonadotrophin and P G F 2 a both inhibited P G F 2 a - R mRNA levels. Maximal inhibition of P G F 2 a - R mRNA levels was seen at 1 nM P G F 2 a in the presence of hCG. As this receptor is only recently cloned in the human and rat [Abramovitz et al., 1994; Lake et al., 1994], there exists only one other report of P G F 2 a - R mRNA regulation in the literature. Moreover, the effects of PGF2« on P G F 2 a - R mRNA levels have not been examined. However, the effects of hCG have been examined [Ristimaki et al., 1997]. This report demonstrated an hCG-mediated upregulation of P G F 2 a - R mRNA levels- a result differing from the present results. The difference between these two reports may be explained by the fact that these experiments were performed on cells of different culture periods. Inhibition of P G F 2 a - R mRNA and presumably P G F 2 a - R would reduce the effectiveness of PGF2 a-mediated luteolytic effects. Thus, the inverse bell curve-like autoregulation of PGF2a-R mRNA by P G F 2 a may explain its bell curve-like effects on progesterone production. Notably, maximal stimulation of progesterone production in the presence of hCG and P G F 2 c t (1 nM) occurred when P G F 2 a - R mRNA levels were at their lowest. Thus, rather than potentiating hCG in a true sense, P G F 2 a is inhibiting its own luteolytic effects allowing more effective stimulation by gonadotrophins. The mechanism by which P G F 2 a autoregulates its receptor mRNA needs to be studied further. In summary, P G F 2 a negatively autoregulates its receptor mRNA. Moreover, P G F 2 a feeds back on its steroidogenic effects through this autoregulation. 143 VIII-SYNOPSIS The aforementioned studies examined the effects of prostaglandin^,* (PGF 2 o t ) on progesterone and estradiol production, as well as DNA and P G F 2 a - R mRNA levels in the human granulosa-luteal cell (GLC). Additionally, the interactions of P G F 2 a with human chorionic gonadotrophin (hCG), gonadotrophin-releasing hormone (GnRH) and prostaglandin E2 (PGE2) were examined with respect to progesterone and estradiol production. In one study, cells were collected from small (<12 mm) and large (>12 mm) follicles separately, permitting the examination of follicle size-dependent alterations in steroidogenisis. Pharmacological techniques were utilized to elucidate the signal transduction pathways involved in the anti-gonadotrophic effects of P G F 2 a . Moreover, these experiments were performed on one^Di), eight-(Dg) and/or twelve- to fourteen-day (D 1 2 . i4) cultured GLCs in order to reveal culture time-dependent alterations in cellular response. Briefly, GLCs collected from patients undergoing in vitro fertilization (IVF) were cultured for the time periods described above, followed by a 24 h treatment period, after which media were collected and assayed for progesterone and estradiol while cells were extracted for D N A or total RNA. A. Basic Physiological Responses to PGF2a Human GLCs undergo morphological luteinization and then luteolysis with increasing time in culture. Culture-time, concentration and/or follicle-size dependent alterations in PGF2a- and GnRH-mediated regulation of human GLC steroidogenesis in the presence and/or absence of human chorionic gonadotrophin (hCG) were investigated. This study clearly demonstrated functional differentiation of human granulosa-luteal cells in culture. Progesterone production in response to P G F 2 a changed with culture-time from inhibition to stimulation in Di and D 1 2_i4 cultured GLCs, respectively. Cells at D 8 of culture were in a state of transition, with inhibition, stimulation or intermediate responses being possible. Similarly, estradiol responses changed from no response to a stimulatory response in D i and D 8 cultured GLCs, respectively. DNA levels were unaltered by PGF2tt treatment. In the presence of hCG similar culture-time dependent changes were observed. PGF2« (IO -6 M) inhibited hCG-stimulated progesterone production in D i and Dg, but not in D12-14 cultured GLCs. In contrast, P G F 2 a (10* M) potentiated hCG-stimulated progesterone production in Dg and D1244, but not in Di cultured GLCs. A similar trend was seen with estradiol production. Human C G significantly stimulated progesterone and estradiol production from D i cultured GLCs collected from large follicles (> 12 mm), while it did not in cells collected from small follicles (< 12 mm). Consequently, PGF2a significantly inhibited hCG-stimulated progesterone and estradiol production in GLCs collected from large, but not small follicles. Human C G stimulated progesterone production was inhibited by high concentrations of GnRH (IO - 6 M) in D i and Dg cultured GLCs. Human CG-stimulated progesterone production was inhibited by high concentrations of GnRH (IO - 6 M) in D i and Dg cultured GLCs. In a fashion similar to P G F 2 a , GnRH (10"9) was capable of potentiating hCG-stimulated stimulated progesterone production in D 8 human GLCs. Similar results were seen for estradiol production in Dg GLCs. DNA levels were unaltered by these treatments. B. Confounding Interactions of PGF2a with GnRH A second study examined the effects of PGF2« and GnRH and their interactions on progesterone- and estradiol-production from D i and Dg cultured human GLCs. In a preliminary experiment, GLCs were treated with vehicle, P G F 2 a ( IO 9 M) , GnRH ( I O 6 M) or P G F 2 a plus GnRH in the absence or presence of hCG. It was demonstrated that P G F 2 a and GnRH alone had no significant effect on progesterone or estradiol production in D i GLCs . However, the combination of PGF2a plus GnRH caused a significant stimulation of progesterone and estradiol production. PGF2a partially inhibited hCG-stimulated progesterone- and estradiol-production. Conversely, GnRH did not inhibit hCG-stimulated progesterone- or estradiol-production, although it did potentiate PGF2a-mediated inhibition of hCG-stimulated steroidogenesis. In a second experiment (n=7 patients), vehicle, P G F 2 a (IO"11 to IO"6 M) and GnRH (IO"1 0 to IO"5 M) concentration-response curves were crossed into a matrix of 49 separate treatments. Steroidogenic responses were plotted in three dimensions and as a contour map. Moreover, 'slices' of the three dimensional matrix were plotted in two dimenstions and analyzed statistically. Maximal stimulation of progesterone-production (2-3-fold) was seen when medium concentrations of PGF2« interacted with high concentrations of GnRH (IO*6 to I O 5 M). In the presence of high concentrations of GnRH ( I O 6 M), P G F 2 a stimulated progesterone production in a bell curve-like fashion, as middle concentrations significantly stimulated while low and high concentrations did not. In the presence of middle concentrations of P G F 2 a ( I O 9 M) , GnRH significantly stimulated progesterone production in a linear concentration-dependent manner. Prostaglandin F 2 a alone elicited no estradiol response. However in the presence of high concentrations of GnRH ( IO 5 M), a significant concentration-dependent stimulation was seen. Maximal stimulation of estradiol production was seen when high concentrations of P G F 2 a ( IO - 6 M) and GnRH ( I O 5 M) were co-applied. Gonadotrophin-releasing hormone alone stimulated estradiol production in a bell curve-like manner, although in the presence of high concentrations of P G F 2 a (IO" 6 M) , estradiol was stimulated in a linear concentration-dependent manner. Inhibition of cyclooxygenase-I (by indomethacin) prevented the luteotrophic effects of P G F 2 a in the presence and absence of GnRH in D i and Dg cultured human GLCs. C. Confounding Interactions of PGF2a with PGE2 In D 8 cultured GLCs, P G F 2 a and P G E 2 concentration response curves were crossed and treated as with GnRH. Briefly, progesterone was significantly stimulated in a bell curve-like manner by P G F 2 a with maximal stimulation at 1 nM. A similar response to P G E 2 was seen, although the bell curve was shifted right. Maximal PGE2-mediated stimulation of progesterone production was seen at 10 to 100 nM. However, in the presence of P G E 2 (IO"7 M) , P G F 2 a significantly inhibited progesterone production in an inverse bell curve-like manner with maximal inhibition at ( I O 1 0 to I O 8 M , P G F 2 a ) . D. Signal Transduction of the Luteolytic Effects of PGF 2a The third study utilized the G-protein effectors pertussis toxin (PTX) and cholera toxin (CTX), the 6-adrenergic agonist (and known activator of the c A M P pathway) isoproterenol, forskolin, and the c A M P analogue dibutryl-cAMP (Db-cAMP) to examine the signal transduction pathways involved in the anti-gonadotrophic actions of P G F 2 a in D 8 GLCs. During the final 18 h of the pre-culture period, the cells were cultured in media or media containing PTX (50 ng/ml) and/or C T X (1 ̂ g/ml). The cells were then treated with vehicle, P G F 2 a ( IO 6 M ) , hCG (1 IU/ml) or P G F 2 a plus hCG in the presence of vehicle, PTX, C T X or PTX plus CTX. In another experiment, cells were treated with vehicle, P G F 2 a ( I O - 6 M), I soP(IO 5 M) , or P G F 2 a p l u s IsoP. It was demonstrated that P G F 2 a caused a significant inhibition of hCG stimulated progesterone and estradiol production, and that this inhibition was abolished by PTX. Similarly, cholera toxin- stimulated progesterone and estradiol production was blocked by P G F 2 a , with PTX reversing this effect. Finally, P G F 2 a also inhibited isoproterenol- and forskolin-stimulated, but not Db-cAMP stimulated progesterone production from eight day cultured human granulosa-luteal cells. E. Regulation ofPGF2a-R mRNA Human GLCs (Di) were exposed to culture media containing either vehicle, hCG (1 IU/ml) or hCG plus P G F 2 a (10"n to IO"6 M), for 24 h. Following the treatment period, cells were extracted for total R N A , which was confirmed to be intact by the presence of 18 and 28S bands revealed by R N A gel electrophoresis. A fixed quantity of mRNA (between 0.5 and 2 pig depending on yield) was reverse transcribed to cDNA and frozen (at -20 C) until used in semi- quantitative PCR. Transcripts for P G F 2 a - R were detected by PCR with two different sets of oligonucleotide primers based on the published human P G F 2 a - R sequence. PCR products were run on a 1.5% agarose gel, stained with ethidium bromide and/or autoradiographed when [ 3 2P]dCTP was incorporated. PCR products were confirmed to be those of P G F 2 a - R by size and by Southern blot hybridization with an internal oligonucleotide probe. Photographs and/or autoradiograms of the gels or Southern blots were quantified by densitometry. These experiments were performed a minimum of three times on cells from a minimum of three separate patients. Similar results were seen in all experiments performed. Prostaglandin F 2 a - R mRNA was significantly down-regulated by hCG when compared to the control. In contrast, progesterone and estradiol production were significantly stimulated by hCG. However, as described above, the addition of both low (10" u M) and high concentrations ( I O 6 M) of PGF2a restored P G F 2 a - R mRNA levels to those of the controls. A corresponding change in progesterone and estradiol levels was seen, such that hCG-stimulated steroidogenesis was significantly inhibited by these concentrations of P G F 2 a . Finally, the strongest effect of P G F 2 a was seen at a concentration of I O 9 M where P G F 2 a - R mRNA was barely detectable. As before, progesterone and estradiol production were inversely related to P G F 2 a - R levels, as hCG- stimulated progesterone and estradiol production were completely restored in the presence of 1 n M P G F 2 a - Messenger RNA levels for the 8-actin gene were unaltered by these treatments. IX - CONCLUSIONS 147 Human GLCs undergo morphological luteinization and luteolysis with culture-time. Steroidogenic responses to P G F 2 a were culture-time and concentration dependent, with P G F 2 a being either luteolytic or luteotrophic depending on these conditions. Cyclooxygenase-I inhibition prevented the luteotrophic effects of P G F 2 a , suggesting that de novo prostaglandin synthesis is required for this effect. Furthermore, the luteotrophic effects of P G F 2 a required GnRH as a permissive factor. Additionally, the luteotrophic effects of P G F 2 a could also be regulated in a complex manner by P G E 2 . The luteolytic effects of P G F 2 a were mediated through a pertussis-toxin sensitive G-protein (possibly Gj, G p or both). Prostaglandin F 2 a inhibited cholera-toxin, isoproterenol and forskolin (but not db-cAMP) stimulated progesterone production, suggesting that this G-protein exerts its actions proximal to P K A (possibly at the level of adenylate cyclase or phosphodiesterase). The luteolytic actions of P G F 2 a were potentiated by GnRH. 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