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

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Prostaglandin F2 -Mediated Luteolytic and Luteotrophic Effects on the Human Granulosa-Luteal Cell a  by Jeffrey Eric Vaananen B.Sc, Simon Fraser University, 1991 M.Sc, The University of British Columbia, 1993  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in  T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Obstetrics and Gynaecology, Reproductive and Developmental Sciences Program)  We accept this thesis as conforming ~^tp l^ie r e t i r e d standard  THE UNIVERSITY OF BRITISH C O L U M B I A 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 + The University of British Columbia Vancouver, Canada Date  DE-6 (2788)  ?-1  fj-PAjC  (Zlf^AeCPtoC^  ABSTRACT These studies examined the effects of prostaglandin^ (PGF2a) on progesterone and a  178-estradiol (estradiol) production, as well as D N A and PGF -receptor ( P G F - R ) mRNA 2a  2a  levels, in the human granulosa-luteal cell (GLC). Additionally, the interactions of P G F ; with 2o  human chorionic gonadotrophin (hCG), gonadotrophin-releasing hormone (GnRH) and prostaglandin E (PGE2) were examined, with respect to progesterone and estradiol production. In 2  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 , while it acts as a permissive factor for the luteotrophic effects. A 2 a  complex interaction between P G F  2 a  and PGE2 was also seen. The luteolytic effects of P G F  mediated through a pertussis toxin-sensitive G-protein (possibly Gj, G or both). P G F p  2 a  2 a  are  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 is capable of autoregulating its receptor a  mRNA levels, and thus its ability to regulate steroidogenesis in the human G L C . Prostaglandin F - R mRNA levels were found to be inversely related to progesterone and estradiol production. 2 a  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.  iii  T A B L E OF CONTENTS Page  ABSTRACT  ii  T A B L E O F CONTENTS  iii  LIST OF TABLES  viii  LIST O F FIGURES  ix  LIST O F ABBREVIATIONS  xiii  ACKNOWLEDGMENTS  xviii  DEDICATION  xix  I - BACKGROUND A. The Classical Neuro-Endocrine  1  Pathway of Gonadal  Regulation B.  Pregnancy  C. The Sex Steroids  1 4 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  D. The  10  1. The Fallopian Tubes  10  2. The Uterus  10  3. The Vagina  11  4. The Breasts  11  5. Other Progesterone-Dependent Actions  11  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 ofProstaglandins  19  IV  F2a in Reproduction  E. Prostaglandin  20 20  Localization ofPGF2a Regulation ofPGF  2a  20  Production in the Ovary  Functions ofPGF  20  PGF  21  2a  2  a  in Pregnancy  22  Prostanoid Receptors in Reproductive Tissues PGF  22  Signal Transduction  2a  23  Clinical Applications of PGF  2a  F. Gonadotrophin-Releasing  25  Hormone  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 - R A T I O N A L E  30  A. The Effects of B. PGF200  an  C. PGF2a  and PGE2  D. Signal  d  PGF2a  GnRH Interaction Studies Interaction Studies  Transduction  E. PGF rR 2o  on Steroidogenesis  34  Cell Collection and Culture  34 36  Experiments  39  C. Microscopy D. Radioimmunoassay  31  33  V - MATERIALS AND METHODS B. Static Incubation  31  32  Studies  mRNA Studies  A. Granulosa-Luteal  30  of Progesterone  and Estradiol  40  E. Hoechst Dye DNA Assay  42  F. RNA Extraction  44  G. RNA  Procedure  Gel  H. Reverse Transcription of RNA to cDNA  45  45  /. Polymerase J. DNA  Chain Reaction (PCR)  46  Gel  K. Southern  46 Blot Hybridization  L. Densitometry M. Analysis  50  of Photographs  50  of Results  53  VI - RESULTS Preliminary  54 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 Effects ofPGF  2  a  64  on Steroidogenesis  64  1. Progesterone and Estradiol Production in Response to P G F 2. D N A Levels in Response to P G F Effects ofPGF  2  a  2a  64 64  2 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 P G F  68  2 a  3. The Effects of P G F on hCG-Stimulated Steroidogenesis 2a  Effects of GnRH on hCG-Stimulated Steroidogenesis B. The Interaction of PGF  74  with GnRH  2a  Progesterone Response to GnRH and/or PGF , 2a  Estradiol Response to GnRH and/or PGF ,  77 with or without hCG  with or without hCG.  2a  68  11 11  Progesterone Response to GnRH with or without PGF  11  Estradiol Response to GnRH with or without PGF  78  2a  2a  DNA Levels in Response to GnRH and PGF  2a  Effects of Indomethacin on PGF  2a  Production  Treatment  78  and GnRH Stimulated Progesterone 19  vi C. Progesterone Response to PGF2a D. Signal  Transduction  Effects ofPGF  92  plus PGE2  of PGF2a-Mediated  Luteolysis  97  on hCG-Stimulated Steroidogenesis  2a  Effects ofPGF2a  97  on Isoproterenol Stimulated Progesterone Production  97  Effects of PTX on And-gonadotrophs Actions of PGF  97  Effects ofPGF2a  98  2a  on CTX Stimulated Steroidogenesis  Effects ofPGF on  Forskolin Stimulated Progesterone Production  2a  Effects of PGF2  a  on cAMP Stimulated Progesterone Production  The Effects of a PKC Inhibitor on PGF -M diated e  2a  2a  98  Inhibition of 98  hCG'-Stimulated Progesterone Production E. Effects of hCG and PGF  98  on PGF -R  107  mRNA  2a  Spectrophotometry Estimation of Known DNA Levels in Solution  107  RNA Integrity and Relative Quantity  107  PCR Cycle Experiment  107  Amplification ofPGF2 -R  dndfi-Actin cDNAs in Human GLCs  Confirmation ofPGF2a-R  cDNA in Human Granulosa-Luteal and  a  108  108  Placental Cells Regulation ofPGF -R  108  cDNA by hCG and PGF  2a  2a  117  VII - DISCUSSION 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 A. Effects  120  of Human GLCs in Culture of PGF2a  on Human Granulosa-Luteal  Cells 121  in the Absence and Presence of hCG  121  Follicle Size  122  Concentration and Culture Time Dependent Responses  123  Summary B. Interaction  of PGF  2a  Progesterone Response  125  with GnRH :  125  vii  Estradiol Response  125  Implications  126  Experimental Model  128  Summary  128  C. Interaction  of PGF  2a  131  with PGE  2  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 PGF rR 2c  142  mRNA  143  VIII - SYNOPSIS A. Basic Physiological Responses to PGF2a  143  2?. Confounding Interactions of PGF2a with GnRH  144  C. Confounding Interactions of PGF2a  145  with PGE2  D. Signal Transduction of the Luteolytic Effects of 145  PGF  2a  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  6  Southern Blot SSC Washes  7  Spectrophotometer estimation of known DNA levels in solution  49 52 110  ix  LIST OF FIGURES 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  17  8  A diagramatic depiction of the specific objectives  29  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  13  Basal progesterone production versus cells/well  55  14  Basal progesterone production versus D N A 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 a  from arachidonic acid  Methods  Results  20  Sixteen day cultured human granulosa-luteal cells  63  21  Progesterone production in response to P G F 2 a . D i and D12-14 GLCs  65  22  Progesterone production in response to P G F , 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 > in Dg GLCs  67  24  hCG stimulated progesterone production  69  25  Follicle size-dependent responses to hCG P G F  26  The effects of PGF2a on hCG-stimulated progesterone production from  2 a  a  70  2 a  D and Dg GLCs  71  x  27  The effects of P G F  2 a  on hCG-stimulated progesterone production from  Di -14 GLCs  72  2  28  The effects of P G F  2 a  on hCG-stimulated estradiol production from  D i and D GLCs  73  8  29  The effects of GnRH on hCG-stimulated progesterone production from D i and D GLCs  75  8  30  A. The effects of GnRH on hCG-stimulated estradiol production from D i and D GLCs  76  8  B. The effects of GnRH and hCG on DNA levels in D GLCs  76  31  Progesterone production in response to GnRH, P G F 2 and/or hCG  80  32  Estradiol production in response to GnRH, PGF2« and/or hCG  81  33  Three dimensional plot of GnRH and P G F  82  34  Contour plot of GnRH and P G F 2 interactions on progesterone  83  35  Effects of P G F  84  36  Effects of GnRH in the absence and presence of P G F  37  Three dimensional plot of GnRH and P G F 2 interactions on estradiol  86  38  Contour plot of GnRH and P G F 2 interactions on estradiol  87  39  Effects of P G F  in the absence and presence of GnRH on estradiol  88  40  Effects of GnRH in the absence and presence of P G F 2 on estradiol  89  41  Progesterone response to P G F 2 with/without indomethacin  90  42  Three dimensional plot of GnRH and P G F  8  a  2 a  interactions on progesterone  a  2 a  in the absence and presence of GnRH on progesterone 2 a  on progesterone  a  a  2 a  a  a  in the presence of indomethacin  2 a  85  interactions on progesterone 91  xi 43  Three dimensional plot of P G F  44  Contour plot of P G F  45  PGF  46  The effects of P G F , in the presence of P G E  47  PGF -mediated inhibition of hCG-stimulated steroidogenesis  48  PGF -mediated inhibition of isoproterenol-stimulated progesterone  2 a  and P G E interactions on progesterone  2 a  2  and P G E interactions, on progesterone  2 a  2  2  96  2  2a  99  2a  100  production 49  94 95  and P G E concentration response curves 2 a  93  Effects of pertussis toxin on PGF -mediated inhibition of 2a  101  hCG-stimulated steroidogenesis Effects of pertussis toxin, P G F  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 PGF -mediated luteolysis  106  55  RNA integrity gel  111  56  PCR cycle experiments  112  57  PCR amplification of P G F - R and B-actin cDNA  58  PCR amplification of P G F - R cDNA from human GLCs,  2 a  and hCG on D N A levels  102  50  2a  113  2 a  2 a  placenta and leukocytes  114  59  Effects hCG and P G F  60  Southern blot hybridization of a PCR experiment for P G F - R  2 a  on P G F - R mRNA  115  2 a  2 a  116  Discussion  124  61  Dual actions of P G F  62  GnRH as a permissive or potentiatory factor for PGF -mediated  2 a  on steroidogenesis 2a  129  effects (Model I) 63  GnRH as a permissive or potentiatory factor for PGF -mediated 2a  130  effects (Model II) 64  Proposed positive feedback loop for P G F  2 a  synthesis  137  xii 65  Pertussis toxin to blocks PGF2 -mediated inhibition of hCGa  stimulated steroidogenesis 66  PGF -mecliated inhibition of hCG- and isoproterenol-stimulated 2a  steroidogenesis 67  139  PGF2a-mediated inhibition of cholera toxin- and forskolin-stimulated steroidogenesis  68  138  The inability of PGF2a to inhibit cAMP-stimulated steroidogenesis  140 141  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 D N A  CL  Corpus luteum  Cox-I  Cyclooxygenase I (constitutive)  Cox-II  Cyclooxygenase II (inducible)  CTX  Cholera Toxin  2D  Two dimensional  3D  Three dimensional  Di  Precultured for 1 day  Dg  Precultured for 8 days  D12-14  Precultured for 12-14 fourteen days  DAG db-cAMP  Diacylglyceriolordiglyceride Dibutryl-cyclic-adenosine monophosphate  ddhkO  Double distilled water  dCTP  Deoxycytosine-triphosphate  dNTPs  Deoxynucleotide-triphosphate(s)  DP  Prostaglandin D receptor  dpi  Dots per inch  FSH  Follicle stimulating hormone  DEPC  2  Diethylpyrocarbonate  xiv DMEM  Dulbecco's Modified Eagle's Medium  DNA  Deoxyribonucleic acid  dNTPs  Deoxynucleotide-triphosphates  DTT  Dithiothreitol  E2  Estradiol  EDTA  Ethylenediaminetetraacetic acid  EPi  Prostaglandin E receptor (isoform 1)  EP  Prostaglandin E receptor (isoform 3)  3  2  2  Estradiol  176-estradiol  FBS  Foetal bovine serum  For  Forskolin  FP  Prostaglandin F  g  Grams  GLB  Gel loading buffer  GLC  Granulosa-luteal cell  GnRH  Gonadotrophin-releasing hormone  GTP  Guanosine triphosphate  G-protein  GTP dependent protein  G  G-protein alpha subunit(s)  a  2 a  receptor  G s  G  G«il,2  G inhibitory (isoform 1 or 2)  G i3  G inhibitory (isoform 3)  Gap  G placental  G  G placental (q isoform)  a  a  a q  a  stimulatory  a  a  a  a  Gall  G placental (11 isoform)  G O  G  GRB  Gel running buffer  GTC  Guanosine thiocyanate Lysis buffer  h  Hours  hCG  Human chorionic gonadotrophin  Indo  Indomethacin  a  a  a  olfactory  IP  Prostaglandin I receptor 2  IP  3  Inositol trisphosphate  IsoP, or Iso  Isoproterenol  IU  International units  IVF  In vitro fertilization  Kd  Equilibrium dissociation constant  KDa  Kilodaltons  1  Liter  LH  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  NADPH  Hydrogenated NADP+  NH  New Hampshire  nM  Nanomolar  NY  New York  CD  Optical density  ON  Ontario  P  Progesterone  4  PA  Phosphatidic acid  PACAP  Pituitary adenylate cyclase activating polypeptide  PCR  Polymerase chain reaction  PBS  Phosphate-buffered saline  PG  Prostaglandin  PGE2  Prostaglandin E  PGF  Prostaglandin F  2 a  2  PGF -R  Prostaglandin F  PGG  2  Prostaglandin G  PGH  2  Prostaglandin H  2a  PGI  Prostaglandin I  2  2 a  receptor  2 a  2  2  PI  Phosphoinositide  PKA  Protein kinase A  PKC  Protein kinase C  PKCi  Protein kinase C inhibitor Bisindolylmaleimide  PL  Prolactin  PLAx  Phospholipase A  PLA  Phospholipase A  2  1  2  PLB  Phospholipase B  PLC  Phospholipase C  PLD  Phospholipase D  pM  Picomolar  pmole  Pico moles  PTX  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, M O .  ssc  Sodium chloride and sodium cytrate buffer  TBE  Tris borate EDTA  TRIS  Tris(hydroxymethyl)aminomethane  Tx  Thromboxanes  UV  Ultraviolet  v/v  Volume per volume  w/v  Weight per volume  X  Times (or multiplied by)  x g  Times gravity  PQ  Quebec  Abbreviations Starting with Greek Characters a  Alpha  6  Beta  Y  Gamma Micromolar (IO molar) 6  XVlll  ACKNOWLEDGMENTS 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 I V F 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. M y 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. A t 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 hypothalamopituitary 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. Gonadotrophinreleasing 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 G n R H 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 Endorphins  Inhibin  Norepinephrine  Hypothalamus  Estradiol  A  GnRH  Pituitary  Inhibin-  •Estradiol  A  FSH  Inhibin  Ovaries  Progesterone & Estradiol  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 F S H 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 F S H and L H are glycoproteins which share a common a-subunit (m.w. 14,000; 96 A A ) . Additionally, F S H 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 F S H 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 F S H 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, G n R H , vasoactive intestinal polypeptide, progesterone, prostaglandin F2 , prostaglandin E a  2  and possibly  prostaglandin I . For further information on the mechanisms of ovulation, see the following 2  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 h C G , 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 17Bestradiol (commonly known as estradiol or E ) and estrone, of which estradiol is the most potent. 2  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 > -isomerase. Through a A4  5  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  Progesterone  1 7-OH-Pregnenolone  1 7-OH-Progesterone  Dihydro  if  I  Androstenedione  Estrone  Testosterone  Estradiol  Epiandrosterone Androstenediol -  jo  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 - -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. Figure 3.  A4  5  8  Theca Interna Cell  Granulosa Cell  Endocrine  Figure 4. The two cell model of steroidogenesis in the human ovary. Luteinizing hormone (LH) stimulates c A M P production in the theca interna and granulosa cells, while follicle stimulating hormone (FSH) stimulates c A M P 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 F S H 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 D N A 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 R N A polymerase to the chromosome, and results in the production of R N A 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 progesteronemediated 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 Fatty Acid 8,11,14Arachidonic acid Precursor Eicosatrienoate Lipox COX Lipox COX Enzyme* Eicosanoid P G E i TxA TxA LTA LTA PGD PGFx PGE LTC LTB PGF LTD LTC PGI LTD LTE l  2  4  3  2  3  2  4  3  2 a  4  2  Group I I I 5,8,11,14,17Eicosapentaenoate Lipox COX TxA LTA PGD PGE LTB PGF3 LTC5 3  3  3  5  5  4  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 involves a 2  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 , via removal of tonic inhibition 2  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 activity [Waite 1985] and 2  A A production. Finally, anti-inflammatory corticoids can block the P L A activity. 2  Eicosanoid ProductionfromArachidonic Acid  Two isoforms of cyclooxygenase (COX-I, constitutive and COX-II, inducible) are capable of converting arachidonic acid to prostaglandin G ( P G G ; Fig. 7). Cyclooxygenase I 2  2  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 to prostaglandin H (PGH ), the precursor to 2  2  2  group II or double bonded prostaglandins and thromboxanes. P G H - P G E isomerase converts PGFf to P G E , which can be further converted P G F 2  PGF  2ot  2  2 a  by E-2-9 ketoreductase. Theoretically,  could be produced directly from P G H by a reductase, although this pathway has not been  demonstrated [Smith 1985].  2  15  PLC  PLA,  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 ( P L A i ) or PLB , while the number two acyl bond is hydrolysed by phospholipase A or B ( P L A or PLB). The phosphodiesterases, phospholipase C (PLC) and phospholipase D (PLD) hydrolyse the glycerophosphate bond and base group, respectively. 2  2  16  Calcium Mobilization  Phosphatidyl Inositol  Diglyceride Glyceride Lipase  Arachidonic Acid  4  KA c\ ri f\ c\ I \ § f  P  r Ifi  PLA  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 ( P L A ) , which converts phosphatidyl choline to arachidonic acid. 2  2  17  Arachidonic Acid COOH  C y c l o o x y g e n a s e I o r II  PGG COOH  Hydroperoxidase  PGH. COOH  COOH  Figure 7. Synthesis of prostaglandin F ( P G F 2 ) from arachidonic acid. Enzymes are in red. Arachidonic acid may be converted to prostaglandin G ( P G G ) by cyclooxygenase I (constitutive) or cyclooxygenase II (inducible). Hydroperoxidase converts P G G into PGH . Prostaglandin E is produced from P G H by PGH-PGE isomerase. The enzyme E - 2 - 9 ketoreductase converts P G E to prostaglandin F (PGF2a) by hydroxylation of the ketone group of PGE . Theoretically, a reductase could produce P G F by reducing P G H , although this pathway has never been demonstrated. a  2 a  2  2  2  2  2  2  2  2 a  2 a  2  2  Prostanoid Receptors Numerous prostanoid receptors have been cloned from mammalian tissues. These receptors include the P G D receptor (DP), the PGE2 receptors ( E P 2  PGF  2ot  l5  E P and EP3-family), the 2  receptor (FP) and the prostacyclin or P G I receptor (IP) [Lake et al., 1994; Abramovitz et 2  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; A n et al., 1994], while the DP, FP, EPx and EP -family of 3  receptors are coupled to rises in intracellular calcium [Abramovitz et al., 1994b; Adam et al., 1994; Boie et al., 1995; Funk et al., 1993; A n 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 selfcatalyzed 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 and P G F 2  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 and P G I are dehydroxylated by another 15-OH dehydrogenase which is 2  2  specific to these prostaglandins. This second enzyme is found in the kidney. Further degradation of these prostaglandins occurs via the reduction of the A bond by an NADPH-dependent A  1 3  1 3  double  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 F  2a  Prostaglandin F 2 has been detected in the human decidua, amnion, pregnant a  myometrium and ovary [Satoh et al., 1981; Aksel et al., 1977]. In the human ovary, P G F 2 has a  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 has been a  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 , via E-2-9-ketoreductase [Watson et a  al.,1979; Endo et al., 1988]. Regulation ofPGF2  a  In the ovary, P G F  2 a  Production in the Ovary  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 production in the human ovary [Bennegard et al., 1984]. However, h C G or a  c A M P pretreatment has been shown to inhibit the antigonadotrophic actions of cloprostenol (PGF  2 a  analogue) in the luteal cell [Michael and Webley, 1991b]. Functions  ofPGF  2a  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 induced a  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 : P G E 2 is low and a  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 - , h C G - and PGE2-stimulated progesterone production (functional luteolysis). Potential mechanisms for functional luteolysis include the inhibition of L H / h C G 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 -mediated reduction in blood flow to the corpus luteum and apoptotic cell a  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 in the presence of gonadotropin [Suginami et al., 1976], a  suggesting that the mere presence of gonadotrophins is not sufficient to initiate a luteolytic response from P G F 2a  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 h C G 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 -induced luteolysis. However, pretreatment with prolactin, F S H plus L H a  prevented PGF2 -induced luteolysis in 11/14 hamsters [Harris and Murphy, 1981]. The blockade a  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 are both present and active in the human granulosa and luteal cells [Grinwich et al., 1976; 2  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 crossreactivity with P G E and P G F 2  2 a  [Lake et al., 1994; Abramovitz et al., 1994; Adam et al., 1994;  Boie et al., 1994 and 1995; Funk et al., 1993; A n et al., 1994]. Ligand binding studies have demonstrated that the human PGF -receptor binds P G F 2a  2 a  with an equilibrium dissociation constant (Kd) of approximately 1 to 1.63 n M [Abramovitz et al., 1994; Lake et al., 1994]. The binding characteristics of the rat P G F - R suggest a two site model, 2 a  with a high affinity site (Kd = 3.9 nM) and a lower affinity site (Kd = 34 nM) [Lake et al., 1994].  PGF  2a  Signal Transduction  Prostaglandin F -receptor cDNA sequences appear to suggest a G-protein coupled 2a  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 i , while rises in inositol phosphates and intracellular calcium appear to be  regulated by G  a  a p  (namely G  a q  and G n ) and G j [Lopez et al., 1995]. a  a  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 c A M P - 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 -mediated luteolysis in the 2a  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 PGF2 -stimulated IP3 a  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 , as cholera toxin stimulated progesterone production is blocked by P G F . s  2 a  Clinical Applications In the female, P G F  2 a  ofPGF2a  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 PGF2 -receptors on the a  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 -mediated inhibition of delta 5,3 beta-hydroxysteroid dehydrogenase 2a  activity [Bilinskaand Wojtusiak, 1988].  25  F. Gonadotrophin-Releasing Hormone 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]. G n R H is a decapeptide that was first discovered in the hypothalmo-pituitary axis. As mentioned above G n R H is the primary mediator of gonadotrophin release. Gonadotropinnreleasing 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 P G F - i n h i b i t i o n of c A M P production [Massicotte, 1984]. On the contrary, 2a  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 G n R H 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 G n R H 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 G n R H 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 G n R H - and G n R H 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 G n R H receptor mRNA levels. Messenger R N A 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 G n R H 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, G n R H 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 timedependently (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 G n R H in the alpha T3-1 gonadotroph cell line, although G n R H 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 n M for G n R H 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 G n R H 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 G n R H 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 PGF2 -mediated a  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 - R mRNA levels by P G F . 2 a  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 PGF2 -mediated luteolysis; and E) the effects of PGF2 on PGF2 -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. a  a  a  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 should reveal, in a very real sense, its effects on a  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 F -receptors have been demonstrated in and have been recently cloned 2a  from human ovarian cells. These findings suggest that P G F 2 may play an important role in the a  regulation of ovarian function. However, very few functional studies have been performed in the human granulosa cell. Thus the role of P G F  2ot  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 n M 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 is not only important in corpus luteum regression, but also that its a  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.  31 B. PGF200 and GnRH Interaction Studies 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 sideeffects 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 - As the 2a  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  is reported to be at its highest concentration during the mid-luteal  2 a  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 during these two 2  phases. It has been suggested that high levels of P G E during the mid-luteal phase may prevent 2  premature corpus luteum regression. However, this explanation fails to account for the fact that PGF -levels are (perhaps 'unnecessarily') at their highest during the mid-luteal phase when 2a  conception and implantation occur. A more comprehensive explanation for the elevated levels of PGF  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 with respect to steroidogenesis in human G L C in vitro. 2  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 -receptor [Lake et al., 1994; Abramovitz et al., 1994] is known to belong to 2a  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 PGE -stimulated progesterone production (through a lowering of 2  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 . Pertussis-toxin (PTX) and 2 a  cholera-toxin (CTX) were utilized to elucidate the potential role of G-proteins in the antigonadotrophic actions of P G F . In order to determine the action(s) of P G F 2 a  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 , although an 2 a  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 - R levels is as important as the regulation of P G F 2 a  2 a  itself. Receptor binding  studies have previously demonstrated the presence of P G F - R in the rat and bovine luteal cell 2 a  [Brambaifa et al., 1984; Bussmann et al., 1989]. Moreover, the existence of P G F - R mRNA has 2 a  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 - R mRNA levels in response to P G F . 2 a  Thus these studies examined the ability of P G F  2 a  2 a  to regulate P G F - R mRNA levels. 2 a  34  V - MATERIALS AND METHODS 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. Granulosaluteal 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 downregulation with a GnRH analogue (Synarel, Syntex; Montreal, PQ) and when estradiol levels were less than 150 pmol/1, follicular development was stimulated with h M G (Humegon 75 IU F S H and 75 IU L H , Organon, Scarborough, O N ; or Fertinorm 75 I U F S H , 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 h C G (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 x g ) 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, M O ) diluted in M199. This gradient was centrifuged (1,700 x g ) , for 10 min at 22 C. Following collection from the M199/Percoll interphase, granulosa cells were washed and resuspended ( l O ^ l O cells/0.5 ml) in 6  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 ) and twelve to fourteen-day (D12-14) pre8  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 ( 4 8 Well; M l 9 9 ;  I  1 0 % FBS)  Static Incubation ( 2 4 h; M l 9 9 ; Androstenedione 5 x l O ~ M ) 7  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 4 0 % Percoll density gradient (in Medium 199), after which cells were washed twice and plated at 10 to 10 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 M). Supernatant was then collected and stored (-20 C) until assayed for progesterone and estradiol. Cells were either extracted for D N A or total R N A which were assayed with a Hoechst dye D N A assay or revefse-tfanscfiption/semi-quantitative polymerase chain reaction, respectively. 3  4  7  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* M; precursor for 7  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 downregulation 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  PGFa,  (10-" to IO M). A similar experiment was 6  performed with GnRH in place of PGF2 a  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 P G F ^  (1CH  1  to), at D i . Ideally, follicles should have been separated into more categories. However due to clinical limitations this was not possible.  Table 2. Hormones and pharmacaogical agents utilized in these studies. Concentration (s) Abbrev. Class Target(s) # Name Estradiol 1 Androstenedione None used Steroid 5x 10"M Biosynthetic Hormone Pathway Precursor Protein kinase-C 2 Bisindolylmaleimide PKCi Enzyme 50 n M * Antagonist CTX l^g/ml Bacterial Toxin G Protein 2 Cholera Toxin a-subunit Gs Protein 3 Dibutryldb-cAMP Second 10- M Cyclic-Adenosine Kinase A Messenger Monophosphate Analogue Enzyme Adenylate Cyclase 10"M 4 Forskolin For Activator 5 Gonadotrophin GnRH Peptide GnRH Receptor l(r to 10Releasing hormone Hormone 6 Human Chorionic hCG Peptide LH/hCG Receptor 0.001 to 1 IU/ml Gonadotrophin Hormone Enzyme Cyclooxygenase I; IO" M 7 Indomethacin Indo Activator Prostaglandin Dehydrogenase 8 Isoproterenol Iso or IsoP Catecholamine 6-adrenergic 10"M Receptor Hormone Antagonist 50 ng/ml 9 Pertussis Toxin PTX Bacterial Toxin G Protein a-subunit(s): Gi.Gp PGE Receptor and 10" tolO- M 10 Prostaglandin E PGE Eicosanoid Hormone other Prostanoid Receptors 11 Prostaglandin F PGF Eicosanoid PGF Receptor 10- tolO" M Hormone and other Prostanoid Receptors * Toullec et al., 1991; McCarthy 1995. 7  5  5  10  5  6  5  12  2  12  2 a  6  2  2  2a  2a  6  4. Interaction of P G F vehicle, P G F  and GnRH: Dayi and D G L C s were treated with 8  2 a  ( I O M), GnRH (IO" M) or P G F 9  2 a  6  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-  to I O  11  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 G n R H , 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  each  2 a  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 P G F PGF  2 a  (10-  11  2 a  and PGE : Day GLCs were treated with vehicle, 8  2  to I O M) and PGF^ ( I O 6  1 0  to IO" M) concentration-response 5  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 and progesterone-response each on a separate axis. Similarily, results were 2  also plotted as a contour map, with P G F  2ot  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 P G F ^ Mediated Luteolysis: Dayi and D cells 8  used for G-protein studies were pre-treated (18 h) with M199 supplemented with vehicle, P T X (50 ng/ml), C T X (1 //g/ml), or P T X plus C T X . Following the pretreatment period, cells were exposed to M199 containing vehicle, P T X , C T X or P T X 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 M ) , P G F s  (10" M ) , or IsoP plus P G F . Finally, cells were 6  2 a  exposed to M199 containing vehicle or P G F (10 M ) or Db-cAMP (10 M). s  s  2 a  (10- M ) , plus or minus forskolin 6  2 a  39 7. Forskolin and Db-cAMP: Dayg cultured human GLCs were treated for 24 hours with vehicle and PGF2 ( I O M) with and without forskolin ( I O M) or 6  6  a  dibutryl c A M P ( d b - c A M P ; 10" M). 5  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 G L C s 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 PGF . 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 T M S 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.  40  D. Radioimmunoassay of Progesterone and Estradiol  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 H P 0 (0.04 M) and N a H P 0 (0.04 M) at pH 7.4. Typical standard curves 2  4  2  4  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 H-progesterone at 10,000 cpm/tube 3  (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 R I A 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 estratriene3,176-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 H-estradiol (Amersham, 3  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).  42  E. Hoechst Dye DNA Assay  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 T R T P K (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 D N A . A t 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 0 ; Sigma) was thawed (from -20 C) and diluted (lOx in PBS). Following the 2  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, M D ) for fluorescence. Excitation and emission wavelengths were 354 and 458 n M , 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 H P 0 (7.1 g), NaCl (116.88 g), 2  4  and E D T A (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 R N A with an RNaid kit (Bio 101, L a 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. R N A 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 R N A 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 R N A . A final spin (1 min; 10,000 g) was performed to pellet the R N A Matrix while leaving the R N A 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 R N A extraction procedure and the integrity of the R N A , the extraction products were run on an RNA gel. The R N A gel was composed of agarose (1.0%) dissolved in d H 0 (21.6 ml). Additionally, R N A gel-running buffer (GRB-R; 3 2  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. R N A 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 R N A 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%), E D T A (1 mM), bromophenol blue (0.4%), xylene cyanol (0.4%?) and ethidium bromide. The G R B (lOx) consisted of M O P S (0.2 M ) , NaOAc (80 mM) and EDTA (10 mM) in d H 0 (total volume 1.01). 2  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 R N A 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 c D N A K i t , 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 -receptor expression could be examined. The procedure 2a  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 M i x (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 P C R buffer (1/10 vol) plus deoxynucleotidetriphosphates (dNTPs; 0.179 ^mol/ml). Ten times PCR buffer consisted of Tris-HCl (100 m M ; pH 8.3), KC1 (500 nM), M g C l (15 mM) and gelatin (0.1%) in d d H 0 . Radiolabeled PCR 2  2  contained 4.0 nCi of P-dCTP. 32  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 c D N A samples (10 jA, with 5-20 \i% DNA) mixed with D N A gel-loading-buffer ( G L B 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. P C R products appear as fluorescent bands. T B E (5x) was composed of TRIS-base (10.8 g), boric acid (5.5 g) and E D T A (0.5 M ; pH 8.0) dissolved in d H 0 (final volume 1 1). Furthermore, G L B - D consists of glycerol (50 ml), 2  E D T A (0.5 M ; 20 ml), bromophenol blue (0.1 g), xylene cyanol (0.1 g) and H 0 (20 ml). 2  Table 3. Primer combinations and expected product size following PCR. Sense Antisense Predicted Product Size (bp) HPGF+ hPGF802 rPGF+ rPGF720 Act+ Act524 bp - base pairs  48  Table 4. P C R conditions utilized for genes examined. Gene Denaturing Annealing Polymerization Temp  Time  Temp  Time  Temp  Cycles  Time  96 30 57 30 72 1:30 40 hPGF2«-R rPGF^-R 96 30 50 30 72 1:30 40 B-Actin 96 30 55 30 72 1:30 30 All temperatures are given in degrees C, while times are in minutes: seconds  Extension Time  Cycle Expt Figure  7:00 7:00 7:00  56A None 56B  Table 5. Oligonucleotide sequences utilized for PCR and Southern blot hybridization. Gene Primer sequence (5* to 3') Name MW Ref human + CTC A T G A A G G C A TAT C A G A G hPGF+ 6127 1 hPGF5831 PGF^ - GTT G C C A T T C G G A G A G C A A Receptor hPGFP+ 7955 GCT TCT G A T A A A G A A T G G A T C C G C T T Rat + CCA TTG C C A TCC TCA TGA A G G rPGF+ 6407 2 PGF^ rPGF6120 - A G C GTC GTC T C A C A G GTC A C Receptor C A G T A C G A T G G C C A T T G A G A G G T G C A T rPGFP+ 8399 B-Actin + T G A T C C A C A TCT GCT G G A A G Act+ 6117 3 Control Act6037 - G A C CTG ACT G A C TCA CTC A T + = 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 P C R 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 0 (pH7.0). 2  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 kinase buffer (1 JAI; lOx), d H 0 (2 pil), y ^ P - A T P (5 }AI) and T kinase 4  2  4  (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 R N A 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.  Paper \vw-w-- T o w e l s \vsv^^~  Membrane  Transfer Membrane Nylon Membrane Gel  ssc Buffer  EES Support  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 cultured human G L C s (n=17). Furthermore, up to 5000-fold differences in basal 8  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 D N A 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 cells/well. For example, cells from patient 1 4  (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  IOOOO-I  05  _  ooo  1  O a) -t->  ^  1 00-J  <D E a.  o o  o o  Cells/well  B 10000  „  1000  a? ?  c ** t-t->  «0  g  100  " -v.  ^) ^ O "v. (_ o>  10-J i -4  0  n:-j-i  I:-:I  t:vi  [j]  t--I  IXI  KjH e  K : < K : I K - I I : J I I:---I K M  s  e  s  s  e  s  V.-.  e  Patient Figure 13. A. Basal progesterone production (ng/ml; over 24 h) versus cells/well, in Dg precultured 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 G L C s . Note that no correlation was seen between plated cell numbers and progesterone production between patients.  56  10000-7 o c o  • •  •  1 OOOd  +i E CO ^ <L> =  •  •  •  • •  •  1 00  O L> Q.  •  •  D  10  Csl  DNA (ng/ml)  B  10000 *3  22 1000J to  ^  O E L. ^  1 00^ 10  v  i  I  {  \  i'  i  I  i  I  i V  I  y  I  r  I  i  I  •-CMIOW>tDr>»0>0<-tMrO^U)yOr-009t  Patient Figure 14. A. Basal progesterone production (ng/ml; over 24 h) versus D N A content per well (ng/ml), in D 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 pre-cultured human GLCs. Note that no correlation was seen between extracted D N A levels and progesterone production. 8  8  57  1 000  E  i oo 10-J  (0  1 -J  0.1  I I I I III  o o  I  I  I I I I III  o o  Cells/Well  B 100  _ u  Oo ra (_  LU =  Patient Figure 15. A. Basal estradiol production (ng/ml; over 24 h) versus cells/well, in D pre-cultured human granulosa-luteal cells (GLCs). B. Basal estradiol production of individual patients plotted in ng/ml per 1000 cells plated, in D pre-cultured human GLCs. Note that no correlation was seen between plated cell numbers and estradiol production. 8  8  58  1000  .2-  100  2^  DNA (ng/ml)  B 1 000  100-J PS t_ CO LU  Q  10-J  E  1 -J  O.i  *i'* " i " r " I " ' V r ' " r ' 'r' '"i"' r''T" "i "i" ' r "i 1  r  1r  C M h o ^ L n v o r ^ - e o o ^ o c s i ^  11  m  1  vo  oo  ON  Patient Figure 16. A. Basal estradiol production (ng/ml; over 24 h) versus D N A content per well (in ng/ml), in D 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 pre-cultured human GLCs. Note that no correlation was seen between extracted D N A levels and estradiol production. 8  8  59  o 3*  |§ o CM  Figure 17. Comparison of progesterone responses to hCG treatment (0.001 IU/ml for 24 h), in D 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 cells/well. 8  4  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 cells per well and cultured as described above. The morphology of 4  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 ( D i ) resulted in cells that were even more associated and 2  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 G L C s 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 G L C s , 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 in D i cultured GLCs (Fig. 21A; n=4). Conversely, in D12-14 cultured a  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 to 1 0 -8  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 had no effect and stimulated a  estradiol production, respectively (Fig. 23A). The stimulatory response was significant at low (10- to IO" M ; a*b; p<0.05) and high concentrations of P G F « ( I O to I O M ; a*c; p<0.0001). 12  8  7  6  2  2. D N A 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  100H  ab  C  o~ 5r « •*->  CD CU  T  a  b  be be  T  75 A  =  e u  O ~ £_ CL  50 ^ 25 J  0 Q  on  CM I  O  PGF^ (10 M) X  o  B  i  200 H b C  T  b C  J,  b  c T  b  150 a  o~ ••-» "£  (0 e O ~ t_  100H,  50H  i  ns e  PGF^ (10 M)  Figure 21. Progesterone production in response to  X  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, inhibited and stimulated progesterone production, respectively.  PGF2«  66  250  P G F ^ (10  X  H)  Figure 22. Progesterone production in response to PGF2« treatment (for 24 h), in eight day precultured 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  a.  LU  100-1  i  »ntr  ©  ^  a  i  i  1  u  oo  CT.  PGF^(10  X  tl)  B 200  150H  a  g  a  a  a  a  100H  50-^  4 •  a i  ns  £a  r*>  t  PGF^  ( 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 P G F , in eight day pre-cultured human granulosa-luteal cells. 2a  68  Effects ofPGF2a  on hCG-Stimulated Steroidogenesis  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 G L C s . 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 PGF2  a  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 PGF2 ( I O M) treatment at D i 6  a  (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, h C G stimulated progesterone (p<0.001) and estradiol (p<0.02) production in GLCs collected from large follicles. In addition, PGF2 inhibited hCG-stimulated progesterone and estradiol production in GLCs tt  from large follicles (p<0.03), while it did not in cells from small follicles.  3. The Effects of P G F  2a  on hCG-Stimulated Steroidogenesis  In the presence of hCG, culture-time dependent changes in progesterone responses to PGF  2 a  were observed. Prostaglandin F  (IO" M) inhibited hCG-stimulated progesterone 6  2 a  production in Di (Fig. 26A; p<0.05; n=5) and Dg (Fig. 26B; p<0.01; n=6), although not in D (Fig. 27; n=4) cultured G L C s . Alternately, P G F  1 4  (lO^ M) potentiated hCG-stimulated 9  2ot  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 F2 (10" M) inhibited 6  W  hCG-stimulated estradiol production in D i (Fig. 28A; p<0.05; n=8) and Dg (Fig. 28B; p<0.05; n=5) cultured G L C s . Alternately, PGF2 (10^M) potentiated hCG-stimulated estradiol a  production in Dg (p<0.01; n=5; 1.5 fold), although not in D i (n=4) cultured GLCs.  69  1000  to  be  750 4  o— 500 4  gS  b  250 4  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  Control  hCG  hCG & PGF  B 300  Figure 25. Follicle size-dependent responses to human chorionic gonadotrophin (hCG; 1 IU/ml) and prostaglandin F 2 (PGF20J treatment (for 24 h). A. In 1 day pre-cultured human granulosaluteal cells, P G F inhibited (n=4; a*b, p<0.001 and b*c, p<0.03 by A N O V A ) 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). a  2 a  71  300 J  b  c  d  o  T  200 H  &- *  . cd  ac cd  ^ .b  ¥  i  •<-> e (0 e  ac  100 4  <b u  o~ L.  CL  OS  CQ  PGF-^ ( 1 0  I  X  M)  hCG (1 iu/ml)  B IOOO H  be  05  £  be  c  o  o£ (0 e <U u O L.  i]  a 100 4  ^  a.  10 i  CQ  I  PGF^ ( 1 0 M) hCG (1 IU/ml) X  Figure 26. The effects of prostaglandin F ( P G F ) on hCG-stimulated progesterone production (over 24 h) from one-day (A; n=5; a*b*c*d, p<0.05 by A N O V A ) and eight-day (B; n=6; a*b*c, p<0.01 by A N O V A ) pre-cultured human granulosa-luteal cells. 2 a  2a  72  300  •=  200-1  100H  hCG ( 1 iu/ml)  Figure 27. The effects of prostaglandin F ( P G F ) on hCG-stimulated progesterone production (over 24 h) from twelve to fourteen-day (n=4; a*b*c, p<0.05 by A N O V A ) pre-cultured human granulosa-luteal cells. 2 a  2a  73  250  200  c  .25  150H  to 6fi  100-1  c  ac  ac  50 H  ^  O  0>  PGF^ ( 1 0  X  M)  hCG ( 1 I U / m l )  B 800  600  ii  CO =  t-  5  400 H  cn be LU  k  v  200  J  0  OS  CQ  I  I  PGF^ ( 1 0  X  I i  a  i  M)  hCG (1 iu/ml) Figure 28. The effects of prostaglandin F 2 ( P G F 2 ) on hCG-stimulated estradiol production (over 24 h) from one-day (A; n=8; a*b;*c, p<0.05 by A N O V A ) and eight-day (B; n=5; a*b*c, p<0.05 by A N O V A ) pre-cultured human granulosa-luteal cells. a  a  Effects of GnRH on hCG-Stimulated Steroidogenesis  Gonadotrophin-releasing hormone ( l O ^ M ) inhibited hCG-stimulated progesterone production in D (Fig. 29A; n=6; p<0.05) and D (Fig. 29B; n=5; p<0.05) cultured GLCs. :  8  Alternately, G n R H ( I O M) potentiated hCG-stimulated progesterone production in D (Fig. 8  8  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- M) inhibited hCG-stimulated estradiol production in D (Fig. 30A; n=4; p<0.05) cultured 6  8  G L C s . Alternately, G n R H (10- M ) potentiated hCG-stimulated estradiol production in D 9  8  cultured G L C s (n=5; p<0.01). D N A levels were unaltered by any of the treatments (Fig. 30B; n=3;p>0.05).  75  S C O  C o  b  400 H  b  T  ~  ••-» c (0 e <D U O L. CI-  H  ~  300 4 200 4 100 4 0 CO  I" s  VO  •  GnRH ( 1 0 M) X  hCG (1 IU/ml)  B 800  £ o  be  600 H  b  c T  e  ~  t- ' o  <n o  400 4 rj  o L. C L  ~ 200 4 a  OS  GnRH ( 1 0 M) x  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 A N O V A ) and eight-day (B; n=5; a*b*c;<id, p<0.05 by A N O V A ) pre-cultured human granulosa-luteal cells.  76  be  c  3r  rfi  1000H  bed .  .  T  .25  •o £ 100 J  10  -r—-i \  ,_ <—  1  a »—  1  as i  1  1  i  i  CD  GnRH ( 1 0  r  ta i  f  M)  X  hCG (1 IU/ml)  B 200  150H  "y e  100  a  a  a  a  a  a 50 J  ea i  GnRH ( 1 0  i  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 A N O V A ) 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 PGF  2a>  Neither P G F  (IO" M) nor G n R H ( I O M) significantly altered progesterone 9  2 a  with or without hCG.  6  production, in D i human GLCs (Fig. 31A; n=5). However, the combination of PGF2« plus G n R H 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 hCGstimulated progesterone production, although it did potentiate PGF -mediated inhibition 2a  (a*b*c, p<0.05). In Dg pre-cultured GLCs a significant luteotrophic response to P G F  ( I O M) was 9  2 a  present (Fig. 3IB; n=4; p<0.05). However, no luteotrophic response to G n R H (IO M) was -6  observed, although GnRH potentiated the PGF2 -mediated luteotrophic response (p<0.05). Both a  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 (IO  6  (10- M) nor GnRH 9  2 a  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 -niediated inhibition (p<0.05). 2a  Progesterone Response to GnRH with or without PGF2a  Vehicle, P G F  (IO" to IO" M) and GnRH (IO 11  2 a  6  -10  to IO" M) concentration-response 5  curves were crossed into a matrix of 49 separate treatments. Results were plotted in three dimensions, with GnRH, P G F  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 P G F 2  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  (IO M; p<0.05) interacted with high concentrations -9  2 a  of G n R H ( I O to I O M). In the presence of high concentrations of G n R H (IO" M), P G F 6  5  6  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  ( I O M), G n R H significantly stimulated 9  2 a  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 (IO  -6  (10" M; p<0.05) interacted with high concentrations of GnRH 6  2 a  to lCr M). These data are presented in three dimensional graph (Fig. 37; n=6) and contour 5  format (Fig. 38), as for progesterone data above. In the presence of high concentrations of GnRH (Fig. 39; 1 0 M ) , P G F 5  although P G F  2 a  2 a  significantly and linearly stimulated estradiol production (p<0.05),  was ineffective in the absence of GnRH. On the other hand, in the absence and  presence of P G F  (10- M), GnRH significantly stimulated estradiol production (Fig. 40A and 6  2 a  B ; p<0.05). The nature of GnRH stimulated estradiol production was, however, different in the presence and absence of P G F , as the response shifted from a bell curve-like stimulation to a 2ot  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 G n R H ( I O (IO  11  1 0  to IO" M), P G F 5  2ct  to 10~ M ) or hCG, suggesting that responses seen were due to alterations in steroid 6  production rather than changes in cell numbers (data not shown).  79 Effects of Indomethacin on PGF2  a  and GnRH Stimulated Steroidogenesis  In Dg cultured human GLCs, progesterone was significantly stimulated in a bell curvelike fashion by P G F 2 (Fig. 41). Maximal stimulation of progesterone production was at 1 n M of a  PGF2a  (p<0.05). However, co-incubation with indomethacin (IO" M) reversed this effect, and  PGF2a  instead inhibited or had no effect on progesterone production (depending on  6  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 M ) and/or P G F 6  2 a  (IO  11  to I O ) when cells were co-incubated with 6  indomethacin (IO" M ; n=4; Fig. 42). Cells remained viable in the presence of indomethacin, as 6  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  400  300 ^  C  o~ CO <D  e  u  200 H  O  (_  100-^  CL  0 e  o  Of  e  is  C9 Q.  T L_  0.  e  T  CD C3 Q. Q. e  is  CD  hCG (i iu/ml)  B 2000 H  cn  e  <D «  1000H 500 H  CL  il  ^ a  e  19 Q.  CL  C9  o  19 Q.  C9  a.  C9  hCG (1 IU/ml)  Figure 31. Progesterone production in response to vehicle (Cont), gonadotrophin-releasing hormone (GnRH; I O M), prostaglandin F ( P G F ; I O M) and GnRH plus P G F 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 ± S E M of experiments performed on separate patients (a*b*c, p<0.05 by A N O V A ) . 6  9  2 a  2a  2 a  81  1000 4 be  .2  o  •5  t  I  UP  1004,  10  1  T  X  e  CJ  UCS  «  o.  e  CS Ot >H  e cs  in  I Z  a e  CD  I '  lb  cs a.  I CS  oe  CS  hCG (1 IU/ml)  Figure 32. Estradiol production in response to vehicle (Cont), gonadotrophin-releasing hormone (GnRH; 10- M), prostaglandin F (PGF; IO" M) and GnRH plus P G F treatment (over 24 h), in the presence and absence of human chorionic gonadotrophin (hCG), in one day (n=3) precultured human granulosa-luteal cells. Graph bars represent mean ± S E M of experiments performed on separate patients (a*b5*c, p<0.05 by ANOVA). 6  9  2 a  82  (10~ M) X  Figure 33. Three dimensional plot of progesterone production in response to vehicle (C), gonadotrophin-releasing hormone (GnRH; I O to I O M) and/or prostaglandin F (PGF ; 1 0 " to IO" 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. 1 0  5  2 a  6  2a  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 to I O M) and/or prostaglandin F ( P G F ^ ; I O to IO" 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 1 0  1 1  5  6  2 a  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« < 1 G  X  h)  Figure 35. Effects of prostaglandin F (PGF2 ; I O to I O M), in the absence ( D ) and presence ( -o- ) of gonadotrophin-releasing hormone (GnRH; 10" M) treatment (over 24 h), in one day pre-cultured human granulosa-luteal cells. In the presence of G n R H , PGF2 stimulated progesterone production in a bell curve-like fashion, with significant stimulation at middle concentrations (IO and IO" M P G F ; 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 ( I O 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. 1 1  6  a  2 a  6  a  -9  8  2 a  6  85  Figure 36. Effects of gonadotrophin-releasing hormone (GnRH; I O to IO M), in the absence ( • ) and presence ( «- ) of prostaglandin F2« (PGF2 ; I O M) treatment (over 24 h), in one day pre-cultured human granulosa-luteal cells. In the presence of PGF > G n R H stimulated progesterone production in a linear concentration-dependent fashion, with significant stimulation at upper concentrations (IO to IO M: a^b, p<0.05 by A N O V A ) . While in the absence of PGF2 , GnRH had no effect on progesterone production. Progesterone production in response to PGF2 alone ( I O M) was not significantly different from the control response. This Figure represents the mean ± S E M of seven separate experiments performed on seven separate patients, and is a two dimensional slice of three dimensional matrix presented in Figure 33A 1 0  -5  9  a  2a  -8  a  9  a  -5  PGF  2a  (10" M) X  Figure 37. Three dimensional plot of estradiol production (over 24 h) in response to vehicle (C), gonadotrophin-releasing hormone (GnRH; 10" to lf> M) and/or prostaglandin F (PGF ; 1 0 to IO- M), in one day pre-cultured human granulosa-luteal cells. This Figure represents the mean of six separate experiments performed on six separate patients. 10  5  2 o t  11  6  2a  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; I O to I O M) and/or prostaglandin F2« (PGF2„; I O to I O 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 m^land IB L i , respectively. These Figures represent the mean of six separate experiments performed on six separate patients. -10  1 1  ^B.  -6  5  88  Figure 39. The interaction of gonadotrophin-releasing hormone ( G n R H ; I O M ) and prostaglandin F (PGF2a) on estradiol response (over 24 h), in one day pre-cultured human granulosa-luteal cells. In the presence ( — o - - - ) of G n R H (IO" M ) , P G F significantly stimulated estradiol production (n=6; a*b, p<0.05 by A N O V A ) , however P G F was ineffective in the absence ( • ) of GnRH. These data represent a two dimensional slice of the three dimensional graph presented in Figure 37. 5  2 a  5  2 a  2 a  89  500  400 H  .25 300 H to K 200 H  100H  B 500  400^  o -3 300H  200H i OOH  GnRH (10 M) X  Figure 40. Estradiol response (over 24 h) to gonadotrophin-releasing hormone (GnRH; 10 M) in the absence (A) and presence (B) of prostaglandin F2 ( P G F ; IO M), in one day precultured human granulosa-luteal cells. In the presence of PGF2 , 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. -5  -6  a  2a  a  90  1250  1000-1  C  o •+->  750 =  OS — O £_ Q.  500-1 li  250  12  11  10  9  P G F ^ ( 1 0"  8 x  7  6  M)  Figure 41. Progesterone response (over 24 h) to PGF2« in the absence ([3) and presence (H) of indomethacin (IO 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 PGF2 , I O M). However, in the presence of indomethacin, P G F either inhibited (p<0.05; control vs P G F , 1 0 to IO" 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. -6  9  a  1 0  2 a  2a  6  91  Figure 42. Three dimensional plot of progesterone production (over 24 h) in response to vehicle (C), gonadotrophin-releasing hormone (GnRH; I O to I O M) and/or prostaglandin F2« (PGF2 ; IO to I O M) in the presence of indomethacin (IO 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. 1 0  -11  a  6  6  -6  C. Progesterone Response to PGF2a plus PGE2  The following study reveals a complex regulation of progesterone production in response to vehicle P G F  (IO" to I O M) and/or PGE2 ( I O 11  2 a  6  in eight day cultured human GLCs. Prostaglandin F  11  2 a  to I O M) concentration-response curves 6  and P G E concentration-response curves 2  were crossed into a matrix of 49 separate treatments. Results were plotted in three dimensions with P G F , PGE2 and progesterone-response each on a separate axis (Fig. 43). Additionally, 2 a  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 (p<0.05). A similar response to P G E  2 a  with maximal stimulation at 1 n M  was seen although the bell curve was shifted right.  2  Maximal PGE^-mediated stimulation of progesterone production was seen at 10 to 100 n M (Fig. 45). However, in the presence of P G E (1CK M ) , P G F 7  2  2 a  significantly inhibited progesterone  production (p<0.05) in an inverse bell curve-like manner, with maximal inhibition at ( I O 10-8M,PGF ;Fig.46). 2a  (see p. 131 for relevant discussion)  -10  to  93  Figure 43. Progesterone production (over 24 h) in response to vehicle, prostaglandin F ( P G F ; 10-" to 1(K> M) and/or prostaglandin fc^ ( P G E ; I O " to IO 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.  2 c t  -6  2a  2  94  Figure 44. Black and white (A) and colour (B) contour plots of progesterone production (over 24 h) in response to vehicle, prostaglandin F (PGF2«; I O to IO" M) and/or prostaglandin E ( P G E ; I O to 10" 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 1 1  2 a  -11  6  2  6  2  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  400  C  o  A  300  cu e CO  CU ° O — (_ CL  200 H  100-1  B 350  ccu  o ~ 5r « •*•> E (0 e CD (-> O ~ L. CL  c  300  c  250 200 150 100 50  T -  c  ~i  1—-1  11-10-9  1  1  -8  -7  PGEj ( 1 0  x  r  -6  M)  Figure 45. Prostaglandin F2a( A ; P G F ) and prostaglandin E2 ( B ; PGE2) concentration response curves ( 1 0 to I O 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 n M (a*b*c, p<0.05 by A N O V A ) . A similar response to PGE2 was seen although the bell curve was shifted right. Maximal PGE -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. 2a  1 1  6  2  96  Figure 46. The effects of prostaglandin F ( P G F ; IO" to I O M), in the presence of prostaglandin E (PGE ; 10 M). Progesterone production (over 24 h) was significantly inhibited (a*b, p<0.05 by A N O V A ) in an inverse bell curve4ike manner by P G F ( I O to IO" M), in eight day pre-cultured human granulosa-luteal cells. These data represent a two dimensional slice of the data presented in Figure 43. 11  2 a  6  2a  7  2  2  4 0  2 a  8  97  D. Signal Transduction of PGF'^Mediated Lute olysis 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 P G F ( 1 0 - M ; p>0.05 control vs hCG plus PGF ). 6  2ot  2ot  Effects of PGF  2a  on Isoproterenol Stimulated Progesterone Production  The 6-adrenergic agonist isoproterenol ( I O M) was capable of stimulating progesterone -5  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  ( I O M) to culture media (p>0.05; control vs. isoproterenol/PGF ; -6  2 a  2a  p<0.05 isoproterenol vs isoproterenol/PGF ). Isoproterenol also stimulated estradiol production 2a  from human granulosa-luteal cells (Fig. 48B; p<0.05, control vs. isoproterenol). The ability of PGF  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 ofPGF  2a  Treatment of eight-day cultured human granulosa luteal cells with h C G (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  (IO M ; p<0.05 versus -6  2 a  hCG and p>0.05 versus control). However, in the presence of P T X (50 ng/ml), PGF -mediated 2a  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). D N A levels remained unchanged by this treatment regime (Fig. 50) suggesting that steroid responses were not due to altered cell numbers in these experiments.  98 Effects of PGF2a on CTX Stimulated Steroidogenesis  Cholera toxin (1 pg/ml) significantly stimulated progesterone production from eight-day cultured human granulosa-luteal cells (Fig. 51A; p<0.05, C T X versus control). Subsequently, PGF « (10- M) was able to block the stimulatory effect of C T X (p<0.05, C T X versus C T X plus 6  2  P G F ; and p>0.05 for control versus CTX/PGF ). However, co-treatment with P T X (50 ng/ml) 2a  2ot  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 M ) significantly stimulated progesterone production from eight-day s  cultured human granulosa-luteal cells (Fig. 52A and 52B; p<0.05, forskolin vs. control). P G F  2 a  (10 M) was able to block the stimulatory effect of forskolin (p<0.05, forskolin vs. forskolin -6  plus P G F ; p>0.05 control vs. forskolin plus P G F ) . Please note that P G F 2a  2a  2 a  alternately  stimulated (p<0.05, control vs. P G F ) or did not stimulate progesterone production on its own 2a  (compare Fig. 47A and 48B), although this transience did not alter the inhibitory properties of PGF . 2a  Effects of PGF  2a  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 PGF -Mediated Inhibition of hCG-Stimulated Progesterone Production. 2a  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) PGF -mediated inhibition of hCG-stimulated progesterone production in these cells. 2a  (see p. 132 for relevant discussion)  A 800  600 4  c  o L.  ^  °1  400 4  o  200 4  L.  CL T  hCG  P G R2s  hCG P G F 2s  B  hCG  P G F2d  hCG P G F 2s  Figure 47. Prostaglandin F ( P G F ; IO 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 ± S E M of triplicate measures (a*b; p<0.05; by A N O V A ) . Similar results were seen in fourteen separate experiments performed on cells from fourteen other patients. -6  2 a  2a  100  1000  fc c o  750H  L. fc ••-»  ^ •= =  fc  e  O (_ CL  500H  250 H  Figure 48. Prostaglandin F (PGF2a; IO' M)-mediated inhibition of isoproterenol (Iso; IO" M)-stimulated progesterone 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 three separate experiments performed on cells from three other patients. 6  2 a  5  101  Figure 49. The effects of pertussis toxin (PTX 50 ng/ml) on prostaglandin F (PG; lO^ 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. 6  2 o t  102  Figure 50. The effects of pertussis toxin (FTX 50 ng/ml), prostaglandin F ( P G F a ; lO^M) and human chorionic gonadotrophin (hCG; 1 IU/ml) on D N A levels (over 24 h), in eight day precultured human granulosa-luteal cells. Data represent the mean ± S E M of triplicate measures (p>0.05). Similar results were seen in two separate experiments performed on cells from two other patients. 2 a  2  103  A 150-r—  ,  b  0)  c o L. ~  100  -j  CD •=  *J  =  CD  0>  O L CL  s  v  50  a a  a i  :•  ' C  1  PGF  J•  1  C  Vehicle  ' J PGF  CTX  C  PGF  CTX/PTX  B 30 25 20-  "2  1  50  •f ••••••T C PGF  ,V|  C  PGF  C  Vehicle  PGF  CTX  CTX/PTX  Figure 51. The effects of prostaglandin F (PGF ; IO M) on cholera toxin (CTX; 1 //g/ml) and C T X 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 ± S E M 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. -6  2 a  2a  104  1000  750  0>  c  -i  o  fee  +* § CO  500 4  ^  O L  250 4  a.  ,1  a  a  1  1  C  PGF^  Vehicle  C  PGF^  Forskolin  B 500 4 0>  400 4  c  o  to ^  05  S  rjh *»"  O L CL  300 4  c  200 4  ac  i  ioo4 I  1  1  C  J i  PGF^  Vehicle  '  C  1  ;  PGFj,  Forskolin  Figure 52. Prostaglandin F ( P G F ; IO" M)-mediated inhibition of forskolin (IO" M)stimulated progesterone production (over 24 h), in eight day pre-cultured human granulosa-luteal cells (A and B). Data represent the mean ± S E M 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 (IO" M, A vs B) 6  2 a  6  2a  6  2 a  105  800 0)  c  o  600  (0  400 4  L. ,  o L.  CL  200 4  Db-cAMP P G F ^ Db-cAMP  Figure 53. The effects of prostaglandin F « ( P G F ; I O M) on dibutryl c A M P (Db-cAMP; IO" M)-stimulated progesterone 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 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. 6  2  5  2a  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 (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. 2 a  107  E. Effects ofhCG and PGF2a on PGF 2a-R-mRNA 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 R N A 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. A t high concentrations (1250 - 5000 ng/ml), this estimation improved to 100.8 ± 0.2% of the actual D N A concentration. See Table 7 for a complete listing of the results.  RNA Integrity and Relative Quantity The integrity and relative quantity of total R N A 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 R N A 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 - R and 6-Actin c D N A were 2 a  performed in order determine the optimal number of cycles for a given concentration range and species of c D N A (Fig. 56). A t 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 - R and 6-Actin was performed using 40 and 30 cycles, respectively, based 2a  on the results of these experiments.  108  Amplification ofPGF2 R a  andfi-ActincDNAs in Human GLCs  Prostaglandin F - R cDNA was amplified from human GLCs (obtained from 2 different 2 a  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 c D N A (Act+/-) successfully amplified a product of the expected size (524 bp) from human GLCs obtained from 3 different patients (Fig. 57C).  Confirmation ofPGF2 -R cDNA in Human Granulosa-Luteal and Placental Cells a  Amplification of P G F - R c D N A using P C R incorporating P - d C T P revealed the 32  2 a  presence of products in samples obtained from human GLCs from three separate patients and in placental cells from two separate patients (Fig. 58A). However, P C R failed to detect P G F - R 2a  c D N A 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 ofPGF R  cDNA by hCG and PGF 2a  2a  One-day cultured human GLCs were incubated with vehicle, hCG (1 IU/ml) or hCG plus PGF  2 a  (10  1 1  to I O M). The effects of these treatments on P G F - R and 6-Actin cDNA levels 6  2 a  were examined by RT-PCR (Fig. 59A), densitometry (Fig. 59B) and Southern blot hybridization (Fig. 60A and B). Briefly, P G F - R message was down-regulated by hCG. However, PGF2a at low (10"  n  2 a  M) and high ( I O M ) concentrations prevented this down-regulation. On the contrary, the middle 6  concentration of P G F  ( I O M) potentiated hCG-mediated down-regulation of P G F - R 9  2 a  2 a  message (Fig. 59A). Densitometric analysis revealed significant inhibition of P G F - R mRNA 2 a  levels in cells treated with hCG and hCG plus P G F  (n=3; p<0.05 by A N O V A ; IO' M ; Fig. 9  2 a  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 P C R 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 - R mRNA message in this 2 a  experiment. Additionally, in presence of hCG, P G F  2 a  (IO  1 1  to I O M ) inhibited P G F - R 7  m R N A message, potentiating hCG-mediated inhibition at concentrations of ( I O PGF ). 2a  (see p. 142 for relevant discussion)  2 a  -10  to IO M , -8  Table 7. Spectrophotometer estimation of known D N A levels in solution. DNA Spectrophotometer by Weight * Estimation (ng/ml) (% 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 R N A 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 0.32 y = O.OOIx 0.30-1 r = 0.986 1  I  E  o  o  •  CL  4 8 9  •  0.27 0.25  ~  0.22 /  3  0.20 0.17 in  in  Cycle  B 0.40 y = 0.880L0G(x) - 1.129 r = 0.990 0.30  ' i z  0 E 1 o  0.20H  0.10-^  0.00 in  in  Cycle * Figure 56. Polymerase chain reaction (PCR) cycle experiments for prostaglandin F -receptor (A; P G F - R ) and 6-Actin (B) complementary D N A (cDNA). A t 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 P C R amplification of c D N A for and P G F - R and 6-Actin was performed using 40 and 30 cycles, respectively. 2a  2a  2 a  113  bp  LD GLC1 GLC2  4072! 305420361636-  1018-1 506/17-  B  bp  LD  GLC1 GLC2  I636i 1018'  506/17'  bp  LD  720 "bp  GLC1 GLC2 GLC3  2036H 1636' 1018' 506/171 39641  524 "bp  Figure 57. Polymerase chain reaction amplification of P G F 2 R and 6-actin c D N A . Two different sets of oligonucleotide primers were utilized to amplify prostaglandin F^-receptor c D N A , 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. a  114  802 bp"  LD GLC GLC GLC PL PL Leu Leu LD 1 2 3 1 2 1 2  B 4000  3000H  S 2000H  1 000H  C J  —I  CP  CM  co  C J  C J  ca  ca  —I  I  — —I  o_  —i  o.  T  r  —  CM  S =»  Figure 58. Polymerase chain reaction amplification of PGF^-receptor c D N A from human granulosa-luteal cells, placenta and leukocytes. A. Amplification of the prostaglandin F 2 receptor c D N A with the oligonucleotides hPGF+ and hPGF-, in the presence of 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) c D N A samples. Conversely, gel lanes loaded with molecular weight ladder (LD) or two different Leu c D N A samples (uncultured samples), did not show visible amplification of product. B. Gel bands (from A) separated and counted with a 8counter. a  32  115 1018bp  hCG (1 IU/ml) 2.0  1.5  <  ab  I cCj •s  ab  10 4  I  O  0.5  CL  4  0.0 0  HS  e  e  u  PGF- ( 1 0 hCG (1  X  M)  IU/ml)  Figure 59. The effects of vehicle, human chorionic gonadotrophin (hCG) and prostaglandin  F2  a  (PGF2 ) 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). PGF2 -R message was down regulated by hCG, however, PGF2ct at low ( l o M) and high ( l o M) concentrations prevented this down-regulation. On the contrary, the middle concentration of PGF2a ( I O M) potentiated hCG-mediated down-regulation of PGF2 -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 PGF2 -R mRNA levels was seen in cells treated with hCG and hCG plus P G F (a*b*c. p<0.05 by ANOVA; 10 M). a  a  1 1  -6  9  a  a  9  2a  116  hCG (1 IU/ml)  B  PGFa, ( 1 0  x  M)  hCG ( 1 I U / m l )  Figure 60. A. Southern blot hybridization of a semi-quantitative P C R experiment, with an oligonucleotide probe for P G F ^ R , in cells from one day pre-cultured human granulosa-luteal cells. These data confirm the identity of the P C R 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 ( 1 0 to 10- M ) inhibited PGF2«-R m R N A message, potentiating hCG-mediated inhibition at concentrations of ( 1 0 to IO" M, P G F 2 ) . 1 1  7  1 0  8  a  117  VII-DISCUSSION 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 -mediated responses of luteal cells may further complicate matters, 2a  should this exist. Epithelial cells are reported to permit or even enhance PGF2 -mediated a  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 to 10 cells/well. 3  4  Within this range of cell densities, no significant density-dependent difference was seen in the steroidogenic responses to h C G 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 cells/well could increase progesterone 4  production 1.3-fold. A further increase in cell density from l x l O to 8X10 cells/well could 4  4  decrease progesterone production 3.7-fold. They also reported density and culture timedependent 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 R N A 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 - R mRNA did not permit this procedure. 2 a  Rather than not study the regulation of P G F - R mRNA, RT-PCR was utilized to provide 2 a  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 , PGF^ and GnRH ranged from pM to piM. levels. This range of 2 a  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.  120  Morphology of Human Granulosa-Luteal Cells in Culture 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. A n 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  under these conditions. Profoundly  a  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 cells. These findings emphasize the importance of maintaining awareness of culture 8  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 earlyand mid-luteal phase cells [Khan et al., 1989], The effects of P G F 2 on progesterone production a  in G L C s differed with culture time. Prostaglandin F 2 inhibited in day D i , but stimulated a  progesterone production in D12-14 cultured GLCs. The cells were found to be less defined in their responses to P G F 2 in Dg, as they appeared to be in a state of transition between their inhibitory a  and stimulatory responses. Early-luteal and D i GLCs both demonstrated inhibition, while midluteal and Dg G L C s demonstrated stimulation of basal-progesterone production in response to PGF  2 a  [Khan et al., 1989]. This further supports the suitability of I V F 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 nonhomogenous population in which GLCs from follicles yielding type A - B C O C (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 and PGEj have been reported, respectively [Cohen 2  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 can act in a glycogenolytic and in a antiglycogenolytic fashion at concentrations of 10 JAM 2  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 ii,2 and G a  a  q  G j3, a  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 PGF2 and GnRH have also been observed. The general trend of a  the concentration-dependent response to PGF2 or G n R H was retained with culture time a  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 hCGstimulated progesterone production was seen D GLCs cultured in the presence of P G F 8  G n R H (at 1 nM; Fig. 3B and 6B). The ability of P G F  2 a  2 a  or  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 PGF2 [Michael and Webley, 1991b]. It a  has been suggested that h C G 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 PGF2 -mediated luteolysis. Perhaps a  PGE -mediated antagonism of PGF2 -mediated luteolysis enhances the luteotrophic 2  a  responsiveness of these cells.  Summary  Prostaglandin F2 and G n R H appear to be bimodal regulators of steroidogenesis in the a  human ovarian cell. In addition to the antisteroidogenic abilities of PGF2 and GnRH (IO M; D i -6  a  and D ) , these results suggest that P G F ( I O M ; Dg and D12-14) and GnRH (10" M ; Dg) may 9  8  9  2 a  play a role in the maintenance of the corpus luteum through their potentiation of hCG-stimulated progesterone production (Fig. 61).  124  PGR  hCG  Progesterone & Estradiol Production  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 in the presence and absence of a  hCG. In general, at the concentrations tested, neither GnRH nor P G F 2 altered progesterone or a  estradiol production in D i cultures. However, when G n R H and P G F 2 were co-applied, a 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, G n R H potentiated  PGF2a-stimulated progesterone production, but was again ineffective on its own. These results suggest that P G F 2 requires GnRH as a permissive factor in order for its luteotrophic action to be a  present, and that GnRH on its own is not a luteotrophin. The ability of P G F progesterone production in D  8  2 a  to stimulate  cultures suggests that endogenous G n R H 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 G n R H and . PGF  2 a  are intriguing. A t optimal PGF201 concentrations, G n R H stimulated progesterone  production appears linear and concentration dependent. A t optimal G n R H 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 -mediated luteolysis in D i a  and Dg pre-cultured human GLCs.  Estradiol Response Estradiol production was also regulated in a remarkable fashion by the co-application of G n R H and P G F 2 - In the presence of high concentrations of G n R H , P G F 2 a  a  linearly and  concentration dependency stimulated estradiol production. These effects of GnRH appeared to  be permissive, as PGF -mediated stimulation of estradiol production was not present in the 2a  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 eightday cultures (in the absence of exogenous GnRH; not shown). Perhaps the levels of endogenous G n R H 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 G n R H , 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 G n R H 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 G n R H 10~ M) estradiol production is relatively low, while under conditions optimal for 5  luteolysis (ie. low progesterone production; PGF 10~ and GnRH 10~ M) estradiol production is 6  5  high. The present results further support the hypothesis that PGF2a and G n R H 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 from the luteal cell, and PGF production in the placenta [Kawai and Clark, 1985 and 2  1986; Hillensjo et al., 1982; Siler et al., 1986]. It is unknown if P G F production in these cells. The bell curve-like response to P G F  2 a  2 a  stimulates GnRH  could act as a fine tuning or  switching mechanism, allowing a luteotrophic response to turn into a luteolytic one should PGF  2 a  levels increase much beyond n M 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 PGE -mediated inhibition and stimulation 2  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 -mediated luteolysis when cellular exposure to hCG or prolactin preceeds P G F 2a  [Harris and Murphy, 1981; Suginami et al., 1976; Michael and Webley, 1991b].  2 a  exposure  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, G n R H may provide a missing component in a PGF -affected signal transduction system, or it may promote the 2a  production of de novo P G F . Further treatment of this subject may be found below in the signal 2 a  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 G n R H analogues is the down-regulation of pituitary function in preparation for superovulation as part of an in vitro fertilization protocol. Conversely, attempts to inhibit luteal function and induce luteolysis or early abortion with G n R H 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 multiconcentration 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  PGF  GnRH  2a  Progesterone & Estradiol Production  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 functions. a  130  GnRH  PGF  Progesterone & Estradiol Production  Figure 63. Gonadotrophin-releasing hormone (GnRH) acts as a permissive or potentiatory factor for prostaglandin F 2 (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]. a  C. Interaction of PGF2a with PGE2.  In eight-day precultured G L C s , both PGF2 and PGE2 stimulated progesterone a  production in a bell curve-like manner. As with previous experiments PGF2 exerted its maximal a  luteotrophic effect at a concentration of 1 n M . Interestingly, the bell curve-like stimulation mediated by PGE2 was shifted to the right (when compared to that of P G F ) such that maximal 2a  stimulation of progesterone production was seen at a concentrations of 10 to 100 n M . 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 PGF2 ; 3) receptor cross reactivity; 4) the opposing K  actions of these hormones on common signal transduction pathways; and 5) the combination PGF -mediated luteolytic and luteotrophic effects in the presence of P G E . It is likely that all of 2a  2  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 PGF2 to PGE2 can profoundly alter K  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 PGF2 to PGE2 is known to change in vitro. Taken a  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.  132  D. Signal Transduction of PGF ^Mediated Luteolysis This study has examined the signal transduction pathways utilized in the antigonadotrophic (or luteolytic) actions of PGF2 in the human granulosa-luteal cell. Specifically, a  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 PGF2 were quite variable (Fig. 47,49, and 51-53). a  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 P T X . These data also suggest that PGF2« rnay exert its antigonadotrophic 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 . Firstly, human granulosa-luteal cells have been examined immunocytochemically to 2 a  reveal a number of G-protein alpha subunits, including, G , G j3, G ii2 and G a s  a  a  a p  (namely, G  a q  and G i i ) , while G was undetectable by three different antibodies [Lopez et al., 1995]. a  Q  Intracellular c A M P levels in human granulosa cells appear to be regulated by the ratio of G G j-subunits, while G a  a q  a s  and  n and G j levels regulate the accumulation of inositol phosphates a  [Lopez et al., 1995]. Coupling of one or both of these identified G i-subunits to the P G F a  2 a  receptor could explain the P T X sensitivity of the anti-gonadotrophic action of PGF2 , as well as a  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 are the a  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 gonadotrophinreleasing 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; A n 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 and PGE2 both being present and active in the human granulosa a  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 [Lake et al., 1994; Abramovitz et al., 1994; a  Adam et al., 1994; Boie et al., 1994 and 1995; Funk et al., 1993; A n 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 both. The G-protein alpha-subunit designated G  a p  a  p  or  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 pathway often exists within a single cell type, with distinct p  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 toxinsensitive and a pertussis toxin-insensitive G , both of which are involved in P L C regulation p  [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 G proteins [Langois et al., 1990]. This example demonstrates the ability of a P  single receptor to be coupled to multiple-forms of G , providing for a complex regulation of the P  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 antigonadotrophic 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 -receptor is coupled to more than one G-protein,. Finally it has been suggested that a  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 P K C 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 on a  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, G n R H 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 c A M P . 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 PGF  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 PGF2 . The ability of PGF2 to inhibit isoproterenol-stimulated estradiol production was not a  a  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 PGF2 -mediated luteolysis. These studies have confirmed this result. The a  inhibitory actions of P K C inhibitors are not as complete as those of P T X , 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 -mediated luteolysis, as de novo prostaglandin synthesis 2a  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 , as GnRH is known to stimulate the calcium-diglyceride-PKC pathway as well 2 a  (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 . As indomethacin is a blocker of de 2 a  novo prostaglandin production, this effect suggests that in order for PGF2 to exert its actions in a  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 G i , G  p  or both; Fig. 65). Prostaglandin F2 is capable of inhibiting a  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 P T X 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  PGF.  GnRH  Progesterone & Estradiol Production  Figure 64. Proposed positive feedback loop for prostaglandin F 2 synthesis ( P G F ) Gonadotrophin-releasing hormone (GnRH) and P G F are both known to stimulate the calciumdiglyceride-protein kinase C (PKC) pathway. Moreover, P K C pathway is reported to stimulate de novo prostaglandin synthesis in some systems. Thus, there exists the possiblity that P G F and GnRH may provide postive feedback on de novo P G F 2 synthesis in the human granulosaluteal cell. a  2a  2 a  2 a  a  138  Figure 65. Pertussis toxin blocks prostaglandin F (PGF )-mediated inhibition of human chorionic gonadotrophin (hCG)-stimulated steroidogenesis in the human luteal ceil. G - stimulatory G-protein; - pertussis toxin sensitive G-protein; A C - adenylate cyclase (AC); c A M P - cyclic adenosine monophosphate; and P K A - protein kinase A 2 a  2a  139  Isoproterenol hCG  PGR  Progesterone & Estradiol Production  Figure 66. Prostaglandin F 2 (PGF2 )-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 a  a  140  PGF.  Figure 67. Prostaglandin F (PGF )-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 2 a  2a  Figure 6 8 . The inability of prostaglandin F2« (PGF2o) to inhibit dibutryl cyclic adenosine monophosphate (cAMP) stimulated progesterone and estradiol production in human granulosaluteal cells. P K A - protein kinase A  E. Regulation ofPGF2a-R mRNA A n inverse relationship between progesterone production and P G F 2 - R m R N A levels a  was revealed in the present studies. Human chorionic gonadotrophin and P G F 2 both inhibited a  P G F - R mRNA levels. Maximal inhibition of P G F 2 - R mRNA levels was seen at 1 n M P G F 2 a  a  2 a  in the presence of h C G . A s 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 - R 2a  mRNA regulation in the literature. Moreover, the effects of PGF2« on P G F 2 - R mRNA levels a  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 - R mRNA levels- a 2 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 - R mRNA and presumably P G F - R would reduce the effectiveness 2 a  2 a  of PGF2 -mediated luteolytic effects. Thus, the inverse bell curve-like autoregulation of a  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 h C G and P G F  2ct  (1 nM) occurred when P G F 2 - R mRNA levels were at their lowest. Thus, rather than a  potentiating h C G in a true sense, P G F 2 is inhibiting its own luteolytic effects allowing more a  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  back on its steroidogenic effects through this autoregulation.  2 a  feeds  143  VIII-SYNOPSIS The aforementioned studies examined the effects of prostaglandin^,* ( P G F ) on 2ot  progesterone and estradiol production, as well as DNA and P G F - R mRNA levels in the human 2a  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 . Moreover, these experiments were performed on one^Di), eight-(Dg) and/or 2 a  twelve- to fourteen-day  (D .i4) 12  cultured GLCs in order to reveal culture time-dependent  alterations in cellular response. Briefly, G L C s 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 R N A .  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 PGF2 a  and GnRH-mediated regulation of human G L C 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 to stimulation in Di and  D _i4 12  2 a  changed with culture-time from inhibition  cultured GLCs, respectively. Cells at D of culture were in a state 8  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 cultured 8  GLCs, respectively. D N A levels were unaltered by PGF2 treatment. tt  In the presence of hCG similar culture-time dependent changes were observed. PGF2« (IO M) inhibited hCG-stimulated progesterone production in D i and Dg, but not in -6  cultured GLCs. In contrast, P G F in Dg and  D1244,  2 a  D12-14  (10* M) potentiated hCG-stimulated progesterone production  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 G L C s 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 M) in D i and Dg cultured GLCs. -6  In a fashion similar to P G F , GnRH (10" ) was capable of potentiating hCG-stimulated 9  2 a  stimulated progesterone production in D human GLCs. Similar results were seen for estradiol 8  production in Dg GLCs. D N A levels were unaltered by these treatments.  B. Confounding Interactions of PGF2a with GnRH A second study examined the effects of PGF2« and G n R H and their interactions on progesterone- and estradiol-production from D i and Dg cultured human GLCs. In a preliminary experiment, G L C s were treated with vehicle, P G F  ( I O M), GnRH ( I O M) or P G F 9  2 a  6  GnRH in the absence or presence of hCG. It was demonstrated that P G F  2 a  plus  and G n R H alone had  2 a  no significant effect on progesterone or estradiol production in D i G L C s . 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, G n R H did not inhibit hCG-stimulated progesterone- or estradiol-production, although it did potentiate PGF2 -mediated inhibition of hCG-stimulated steroidogenesis. a  In a second experiment (n=7 patients), vehicle, P G F  (IO" to IO" M) and GnRH (IO" 11  2 a  6  10  to IO" M) concentration-response curves were crossed into a matrix of 49 separate treatments. 5  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* to I O M). In the 6  5  presence of high concentrations of GnRH ( I O M), P G F 6  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  ( I O M), GnRH 9  2 a  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 G n R H ( I O M), a significant concentration-dependent stimulation was seen. 5  Maximal stimulation of estradiol production was seen when high concentrations of P G F  2 a  (IO  -6  M) and G n R H ( I O M) were co-applied. Gonadotrophin-releasing hormone alone stimulated 5  estradiol production in a bell curve-like manner, although in the presence of high concentrations of P G F  (IO" M ) , estradiol was stimulated in a linear concentration-dependent manner. 6  2 a  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  In D cultured GLCs, P G F 8  2 a  PGE2  and P G E concentration response curves were crossed and 2  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 was seen, 2  although the bell curve was shifted right. Maximal PGE -mediated stimulation of progesterone 2  production was seen at 10 to 100 nM. However, in the presence of P G E (IO" M ) , P G F 7  2  2 a  significantly inhibited progesterone production in an inverse bell curve-like manner with maximal inhibition at ( I O to I O M , P G F ) . 1 0  8  2a  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 GLCs. During 8  the final 18 h of the pre-culture period, the cells were cultured in media or media containing P T X (50 ng/ml) and/or C T X (1 ^g/ml). The cells were then treated with vehicle, P G F (1 IU/ml) or P G F  2 a  ( I O M ) , hCG 6  2 a  plus hCG in the presence of vehicle, P T X , C T X or P T X plus C T X . In another  experiment, cells were treated with vehicle, P G F ( I O 2 a  It was demonstrated that P G F  - 6  M), I s o P ( I O M ) , or P G F p l u s IsoP. 5  2a  caused a significant inhibition of hCG stimulated progesterone  2 a  and estradiol production, and that this inhibition was abolished by P T X . Similarly, cholera toxinstimulated progesterone and estradiol production was blocked by P G F , with P T X reversing this 2 a  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 G L C s (Di) were exposed to culture media containing either vehicle, h C G (1 IU/ml) or hCG plus P G F  (10" to IO" M), for 24 h. Following the treatment period, cells were n  2 a  6  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 semiquantitative PCR. Transcripts for P G F - R were detected by P C R with two different sets of 2 a  oligonucleotide primers based on the published human P G F - R sequence. PCR products were 2 a  run on a 1.5% agarose gel, stained with ethidium bromide and/or autoradiographed when [ P]dCTP was incorporated. PCR products were confirmed to be those of P G F - R by size and 32  2 a  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 - R mRNA was significantly down-regulated by hCG when compared to 2 a  the control. In contrast, progesterone and estradiol production were significantly stimulated by hCG. However, as described above, the addition of both low (10" M ) and high concentrations u  ( I O M ) of PGF2a restored P G F - R mRNA levels to those of the controls. A corresponding 6  2 a  change in progesterone and estradiol levels was seen, such that hCG-stimulated steroidogenesis was significantly inhibited by these concentrations of P G F . Finally, the strongest effect of 2 a  PGF  was seen at a concentration of I O M where P G F - R mRNA was barely detectable. As 9  2 a  2 a  before, progesterone and estradiol production were inversely related to P G F - R levels, as hCG2 a  stimulated progesterone and estradiol production were completely restored in the presence of 1 n M P G F - Messenger R N A levels for the 8-actin gene were unaltered by these treatments. 2 a  147  IX - CONCLUSIONS Human G L C s 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 , suggesting that 2 a  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 or both). Prostaglandin F p  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 Finally, P G F  2 a  2 a  were potentiated by GnRH.  was capable of autoregulating its receptor mRNA levels and, thus, its  ability to regulate steroidogenesis in the human G L C . A n inverse relationship between P G F - R 2a  mRNA levels and steroidogenesis exists.  148  REFERENCES Abayasekara DR, Michael AE, Webley GE, Flint AP. Mode of action of prostaglandin F  2 a  in  human luteinized granulosa cells: role of protein kinase C. Mol Cell Endocrinol 1993; 97:81-91.  Abayasekara DR, Jones PM, Persaud SJ, Michael A E and Hint AP, Prostaglandin F  2 a  activates  protein kinase C in human ovarian cells, Mol Cell Endocrinol 1993; Feb; 91(l-2):51-7.  Abramovitz M, Boie Y, Nguyen T, Rushmore TH, Bayne MA, Metters K M , Slipetz DM, Grygorczyk R. Cloning and expression of a cDNA for the human prostanoid FP receptor. J Biol Chem 1994; 269:2632-6.  Abramovitz M. Cloning and expression of three isoforms of the human EP3 prostanoid receptor. Febs Lett 1994b; 338:170-4.  Adam M, Boie Y, Rushmore TH, Muller G, Bastien L, McKee KT, Metters K M , Barak LS, Menard L, Ferguson SS, Colapietro A M , Caron MG. The conserved seven-transmembrane sequence NP(X)23Y of the G-protein-coupled receptor superfamily regulates multiple properties of the beta 2-adrenergic receptor, Biochemistry 1995; 34:15407-14.  Advis JP, Simpkins JW, Chen HT, Meites J. Relation of biogenic amines to onset of puberty in the female rat. Endocrinology 1978; 103(1): 11-6.  Ackland JF, Schwartz NB, Mayo K E , Dodson RE. Nonsteroidal signals originating in the gonads. Physiological Reviews 1992; 72(3):731-87.  Aksel S, Schomberg DW, Hammond CB. Prostaglandin F Obstet Gynecol 1977; 50:347-50.  2 a  production by the human ovary.  Alecozay A A , Harper M J , Schenken RS, Hanahan DJ. Paracrine interactions between plateletactivating factor and prostaglandins in hormonally-treated human luteal phase endometrium in vitro. J Reprod Fertil 1991; 91:301-12.  A n S, Yang J, So SW, Zeng L , Goetzl EJ. Isoforms of the E P 3 subtype of human prostaglandin E2 receptor transduce both intracellular calcium and c A M P signals. Biochemistry 1994; 33:14496-502.  Ashkenazi A , Peralta E G , Winslow JW, Ramachandran J, Capon D G . Functionally distinct G proteins selectively couple different receptors to PI hydrolysis in the same cell. Cell 1989; 56(3):487-93.  Aten RF, Ireland JJ, Weems CW, Behrman HR. Presence of gonadotrophin-releasing hormonelike proteins in bovine and ovine ovaries. Endocrinology 1987; 120(5): 1727-33.  Baird D T , Rodger M , Cameron IT, Roberts I. Prostaglandins and antigestagens for the interruption of early pregnancy. J Reprod Fertil Suppl 1988; 36:173-9.  Baldwin D M , Bourne G A , Marshall JC. Pituitary L H responsiveness to G n R H in vitro as related to G n R H receptor number. American Journal of Physiology 1984; 247:E651-6.  Balthazart J, Balthazart R C , Cheng M F. Hormonal control of the gonadal regression and recovery observed in short days in male and female doves. Journal of Endocrinology 1981; 89(l):79-89.  Barak L S , Menard L , Ferguson SS, Colapietro A M , Caron M G . The conserved seventransmembrane sequence NP(X)2,3Y of the G-protein-coupled receptor superfamily regulates multiple properties of the beta 2-adrenergic receptor. Biochemistry 1995; 34:15407-14.  Bar-Ami S, Gitay-Goren H . Altered steroidogenic activity of human granulosa-lutein cells at different cell densities in culture. Mol Cell Endocrinol 1993; 90(2): 157-64.  150 Barfield RJ, Glaser J H , Rubin BS,Etgen A M . Behavioral effects of progestin in the brain. Psychoneuroendocrinology 1984; 9:217-31 [Review].  Barnes SJ, Conn P M . Cholera toxin and dibutyryl cyclic adenosine 3',5'-monophosphate sensitize gonadotrophin-releasing hormone-stimulated inositol phosphate production to inhibition in protein kinase-C (PKC)-depleted cells: evidence for cross-talk between a cholera toxin-sensitive G-protein and P K C . Endocrinology 1993; 133:2756-60.  Barnett JV, Shamah S M , Lassegue B , Griendling K K , Galper JB. Development of muscariniccholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling. Biochem J 1990; 271(2):443-8.  Bauer D A , Jameson JL. Gonadotrophin-releasing hormone receptor messenger ribonucleic acid expression in the ovary during the rat estrous cycle. Endocrinology 1995; 136(10):4432-8.  Bazer F W , Roberts R M , Thatcher W W . Actions of hormones on the uterus and effect on conceptus development. J Anim Sci 1979; 2:35-45 [Review].  Behrman HR, Luborsky JL, Aten RF, Polan M L , Tarlatzis B C , Haseltine FP, Preston SL, Soodak L K , Mattson G F , Chi A S . Luteolytic hormones are calcium-mediated, guanine nucleotide antagonists of gonadotrophin-sensitive adenylate cyclase. Adv Prostaglandin Thrombaxane LeukotRes 1985; 15:601-4.  Behrman H R , Aten RF, Ireland JJ, Milvae RA. Characteristics of an antigonadotrophic GnRHlike protein in the ovaries of diverse mammals. J Reprod Fertil Suppl 1989; 37(189): 189-94.  Benhaim A , Bonnamy PJ, Papadopoulos V , Mittre H , Leymarie P. In vitro action of P G F  2 a  on  progesterone and c A M P synthesis in small bovine luteal cells. Prostaglandins 1987; 33(2):22739.  151 Bennegard B, Dennefors B , Hamberger L. Interaction between catecholamines and prostaglandin F2a in human luteolysis. Acta Endocrinol (Copenh) 1984; 106:532-7.  Bhasin S, Heber D , Steiner B , Peterson M , Blaisch B, Campfield L A , Swerdloff RS. Hormonal effects of G n R H agonist in the human male: II. Testosterone enhances gonadotrophin suppression induced by GnRH agonist. Clin Endocrinol (Oxf) 1984; 20(2): 119-28.  Bilinska B , Wojtusiak A . Effect of prostaglandins on hormonal function of cultured rat Leydig cells. Folia Histochem Cytobiol 1988; 26(2):53-9.  Boie Y , Sawyer N , Slipetz D M , Metters K M , Abramovitz M . Molecular cloning and characterization of the human prostanoid DP receptor. J Biol Chem 1995; 270:18910-6.  Boie Y , Rushmore T H , Darmon G A , Grygorczyk R, Slipetz D M , Metters K M , Abramovitz M . Cloning and expression of a cDNA for the human prostanoid IP receptor. J Biol Chem 1994; 269:12173-8.  Brambaifa N , Schillinger E. Binding of prostaglandin F  2 a  and 20 alpha hydroxysteroid-  dehydrogenase activity of immature rat ovaries throughout pseudopregnancy. Prostaglandins 1984; 14:225-234.  Bramley T A , Menzes GS, Baird DT. Specificity of gonadotrophin-releasing hormone binding sites of the human corpus luteum: comparison with receptors of rat pituitary gland. J Endocrinol 1986; 108:323-328.  Briggs M M , Lefkowitz RJ. The 8-adrenergic receptor system: a model for the transmembrane regulation of adenylate cyclase. In: Conn P M (eds.), Cellular regulation of secretion and release, Toronto, Ontario: Academic Press; 1982:23-50.  Buckler H M , Phillips SE, Kovacs GT, Burger H G , Healy DL. G n R H agonist administration in polycystic ovary syndrome. Clinical Endocrinology 1989; 31(2): 151-65.  152 Burger HG. Neuroendocrine Control of Human Ovulation. Int J of Fertility 1981; 26(3): 153-60.  Bussenot I, Azoulay-Barjonet C, Parinaud J. Modulation of steroidogenesis of cultured human granulosa-lutein cells by gonadotrophin-releasing hormone analogues. J Clin Endocrinol Metab 1993;76:1376-1379.  Bussmann L E . Prostaglandin F 2 receptors in corpora lutea of pregnant rats and relationship with a  induction of 20 alpha-hydroxysteroid dehydrogenase. J Reprod Fertil 1989; 85:331-41.  Cameron IT, Baird DT. The return to ovulation following early abortion: a comparison between vacuum aspiration and prostaglandin. Acta Endocrinol (Copenh) 1988; 118:161-7.  Casper RF, Cotterell M A . The effects of adrenergic and cholinergic agents on progesterone production by human corpus luteum in vitro. A m J Obstet Gynecol 1984; 148:663-9.  Carroll RS, Corrigan A Z , Gharib SD, Vale W, Chin W W . Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels. Molecular Endocrinology 1989; 3(12): 1969-76.  Channing CP, Schaerf FW, Anderson L D , Tsafriri A . Ovarian follicular and luteal physiology. International Review of Physiology 1980; 22( 117): 117-201.  Chegini N , Ramani N , Rao C V . Morphological and biochemical characterization of small and largebovine luteal cells during pregnancy. Mol Cell Endocrinol 1984; 37(1):89-102.  Chinoy N J , Sharma JD, Seethalakshmi L , Sanjeevan A G . Effects of prostaglandins on histophysiology of male reproductive organs and fertility in rats. Int J Fertil 1980; 25(4):267-74.  Clayton R N , Harwood JP, Catt K J . Gonadotrophin-releasing hormone analogue binds to luteal cells and inhibits progesterone production. Nature 1979; 282:90-2.  153 Clayton R N , Huhtaniemi IT. Absence of gonadotrophin-releasing hormone receptors in human gonadal tissue. Nature 1982; 299:56-59.  Cockcroft S, Stutchfield J. G-proteins, the inositol lipid signalling pathway, and secretion. Philos Trans R Soc Lond B Biol Sci 1988; 320(1199):247-65 [Review].  Cohen L R , Rimon G. Prostaglandin  can bimodally inhibit and stimulate the epididymal  adipocyte adenylyl cyclase activity. Cell Signal 1992; 4:331-5.  Combarnous Y . Structure and structure-function relationships in gonadotrophins. Reproduction, Nutrition, Development 1988; 28(2A):211-28 [Review]. Concannon PW, Hansel W. Prostaglandin F  2 a  induced luteolysis, hypothermia, and abortions in  beagle bitches. Prostaglandins 1977; 13:533-42.  Crumeyrolle A M , Latouche J, Laniece P, Charon Y , Tricoire H, Valentin L , Roux P, Mirambeau G, Leblanc P, Fillion G et al. "In situ" characterization of G n R H receptors: use of two radioimagers and comparison with quantitative autoradiography. J Recept Res 1994; 14:251-65.  Currie W D , L i W, Baimbridge K G , Ho Yuen B , Leung PCK. Cytosolic free calcium increased by prostaglandin F  2 a  ( P G F ) , gonadotrophin-releasing hormone, and angiotensin II in rat 2a  granulosa cells and P G F  2 a  in human granulosa cells. Endocrinology 1992; 130:1837-43.  Davis JS, West L A , Farese R V . Gonadotrophin-releasing hormone rapidly alters polyphosphoinositide metabolism in rat granulosa cells. Biochem Biophys Res Commun 1984; 122(3): 1289-95.  Davis JS, West L A , Farese R V . Gonadotrophin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacyglycerol in rat granulosa cells: further evidence for the involvement of C a 118:2561-71.  2 +  and protein kinase C in the action of GnRH. Endocrinology 1986;.  Davis JS, West L A , Farese R V . Gonadotrophin-releasing hormone (GnRH) rapidly alters polyphosphoinositide metabolism in rat granulosa cells. Biochem Biophys Res Commun 1984; 122(3): 1289-95.  Davis JS, Tedesco T A , West L A , Maroulis G B , Weakland L L . Effects of human chorionic gonadotrophin, prostaglandin F  2 a  and protein kinase C activators on the cyclic A M P and inositol  phosphate second messenger systems in cultured human granulosa-luteal cells. M o l Cell Endocrinol 1989;65:187-93.  Davis P D , H i l l C H , Lawton G , Nixon JS, Wilkinson S E , Hurst S A , Keech E , Turner SE. Inhibitors of protein kinase C. 1. 2,3-Bisarylmaleimides. J Med Chem 1992; 35:177-84.  Dennis E A . Phospholipases. In: Boyer PD (ed.), The enzymes, vol. 16, 3rd ed., Academic Press; 1983:307-353.  DePaolo L V , Mercado M , Guo Y , Ling N . Increased follistatin (activin-binding protein) gene expression in rat anterior pituitary tissue after ovariectomy may be mediated by pituitary activin. Endocrinology 1993; 132(5):2221-8.  Donham R S , Champney T H , Kerner T, and Stetson M H . Temporary anestrus induced by injection of luteinizing hormone-releasing hormone in hamsters. Biology of Reproduction 1993; 48(5): 1135-40.  Dong K - W , Y u K - L , Roberts JL. Identification of a major up-stream transcription start site for the human progonadotrophin-releasing hormone gene used in reproductive tissues and cell lines. Mol Endocrinol 7:1654-1666.  Dorflinger L J , Luborsky JL, Gore SD, Behrman HR. Inhibitory characteristics of prostaglandin F  2 a  in the rat luteal cell, Mol Cell Endocrinol 1983; 33(2-3):225-41.  155 Dorflinger L J , Albert PJ, Williams A T , Behrman HR. Calcium is an inhibitor of luteinizing hormone-sensitive adenylate cyclase in the luteal cell. Endocrinology 1984; 114(4): 1208-15.  Dufour S., Lopez E , Le M F , Le B N , Baloche S, Fontaine Y A . Stimulation of gonadotrophin release and of ovarian development, by the administration of a gonadoliberin agonist and of dopamine antagonists, in female silver eel pretreated with estradiol. General & Comparative Endocrinology 1988;70(l):20-30.  Endo T, Yamamoto H , Tanaka S. Effect of prostaglandins(PGs) on progesterone production by human cultured luteal cells and their ability of PGs production. Nippon Naibunpi Gakkai Zasshi 1988; 64:687-97.  Endo T, Watanabe H , Yamamoto H , Tanaka S. and Hashimoto M . Prostaglandin F2 - and W  phorbol 12-myristate-13-acetate-stimulated progesterone production by cultured human luteal cells in the mid-luteal phase: prostaglandin F  2 a  increases cytosolic C a  2 +  and inositol phosphates.  J Endocrinol 1992; 133(3):451-8.  Fan N C , Jeung E B , Peng C, Olofsson JI, Krisinger J, Leung PCK. The human gonadotrophinreleasing hormone (GnRH) receptor gene: cloning, genomic organization and chromosomal assignment. Mol Cell Endocrinol 1994; 103(l-3):Rl-6.  Farm PW, Estill CT. Infertility due to abnormalities of the ovaries in cattle. Vet Clin North A m Food Anim Pract 1993; 9:291-308 [Review].  Fields M J , Barros C M , Watkins WB, Fields PA. Characterization of large luteal cells and their secretory granulesduring the estrous cycle of the cow. Biol Reprod 1992; 46(4):535-45.  Fields M J , Dubois W , Fields P A . Dynamic features of luteal secretory  granules:  ultrastructuralchanges during the course of pregnancy in the cow. Endocrinology 1985; 117(4): 1675-82.  Fielding CJ, Fielding PE. Metabolism of Cholesterol and Lipoproteins. In: Vance DE, Vance J E (eds.),  Biochemistry of Lipids  and Membranes, Menlo  Park, California:  The  Benjamin/Cummings Publishing Co. Inc.; 1985:404-424.  Filicori M . , Bolelli G . , Franceschetti F, Lafisca S. The ultradian pulsatile release of gonadotrophins in normal female subjects. Acta Europaea Fertilitatis 1979; 10(l):29-33.  Findlay J K . A n update on the roles of inhibin, activin, and follistatin as local regulators of folliculogenesis. Biology of Reproduction 1993; 48(1): 15-23.  Findlay J K , Sai X , Shukovski L . Role of inhibin-related peptides as intragonadal regulators. Reproduction, Fertility, & Development 1990; 2(3):205-18.  Fisher L D , van Belle G. Biostatistics: a methodology for the health sciences. John Wiley & Sons, Inc. Toronto, 1993.  Fleming N , Sliwinski L E , Burke D N . G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland. Life Sci 1989; 44(15): 1027-35.  Fohr K J , Mayerhofer A , Sterzik K , Rudolf M , Rosenbusch B , Gratzl M . Concerted action of human chorionic gonadotrophin and norepinephrine on intracellular-free calcium in human granulosa-lutein cells: evidence for the presence of a functional alpha-adrenergic receptor. J Clin Endocrinol Metab 1993; 76:367-73  Franz W3. Basic review: endocrinology of the normal menstrual cycle. Prim Care 1988; 15:60716 [Review].  Fraser H M . G n R H analogues for contraception. British Medical Bulletin 1993, 49(1):62-72 [Review]. Fuchs A R , Smitasiri Y , Chantharaksri U . The effect of indomethacin on uterine contractility and luteal regression in pregnant rats at term. J Reprod Fertil 1976; 48:331-40.  157 Fuchs A R . Oxytocin and ovarian function. J Reprod Fertil Suppl 1988; 36:39-47.  Funk C D , Furci L , FitzGerald G A , Grygorczyk R, Rochette C, Bayne M A , Abramovitz M , Adam M , Metters K M . Cloning and expression of a c D N A for the human prostaglandin E receptor EP1 subtype. J Biol Chem 1993; 268:26767-72.  Funston R N , Seidel GJ. Gonadotropin-releasing hormone increases cleavage rates of bovine oocytes fertilized in vitro. Biol Reprod 1995; 53:541-5.  Genuth S M . The endocrine system. In: Berne R M , Levy M N (eds.), Physiology, 2nd ed., Toronto, Ontario: The C V Mosby Co.; 1988; 983-1024.  Girsh E, Greber Y , Meidan R. Luteotrophic and luteolytic interactions between bovine small and large luteal-like cells and endothelial cells. Biol Reprod 1995; 52(4):954-62.  Gitay-Goren H , Brandes J M , Bar-Ami S. Altered steroidogenic pattern of human granulosalutein cells in relation to cumulus cell culture morphology. J Steroid Biochem 1990; 36(5):45764.  Goebelsmann U . Protein and steroid hormones in pregnancy. J Reprod Med 1979; 23:166-77 [Review].  Gonen Y , Dirnfeld M , Goldman S, Koifman M , Abramovici H . The use of long-acting gonadotrophin-releasing hormone agonist (GnRH-a; decapeptyl) and gonadotrophins versus short-acting GnRH-a (buserelin) and gonadotrophins before and during ovarian stimulation for in vitro fertilization (IVF). Journal of in Vitro Fertilization & Embryo Transfer 1991; 8(5):254-9.  Goubau S, Bond C T , Adelman JP, Misra V , Hynes M F , Schultz G A , Murphy B D . Partial characterization of the gonadotrophin-releasing hormone (GnRH) gene transcript in the rat ovary. Endocrinology 1992; 130:3098-3100.  Granner D K . Hormones of the gonads. In: Murray R K , Granner D K , Mayes PA, Rodwell V W (eds.), Harper's Biochemistry, 21st ed., San Mateo, California, Appleton & Lange; 1988:536546.  Gray C J . Glycoprotein gonadotrophins.  Structure and synthesis.  [Review]. Acta  Endocrinologica, Supplement 1988; 288(20):20-7.  Gregoraszczuk E , Krzysztofowicz E. The corpus luteum of the pig. Scanning electron microscopic study of surface features at different times of incubation. Acta Biol Hung 1989; 40(1-2): 145-56.  Grinwich D L , Ham E A , Hichens M , Behrman HR. Binding of human chorionic gonadotrophin and response of cyclic nucleotides to luteinizing hormone in luteal tissue from rats treated with prostaglandin F2a- Endocrinology 1976; 98:146-50.  Grimm L G , Yarnold PR. Reading and understanding multivariate statistics. American Psychological Association, Washington DC, 1996.  Hall JE, Whitcomb RW, Rivier JE, Vale WW, Crowley WJ. Differential regulation of luteinizing hormone, follicle-stimulating hormone, and free alpha-subunit secretion from the gonadotrope by gonadotrophin-releasing hormone (GnRH): evidence from the use of two G n R H antagonists. Journal of Clinical Endocrinology & Metabolism 1990; 70(2):328-35.  Hanzen C. Prostaglandins and the physiology of human and animal reproduction. J Gynecol ObstetBiol Reprod (Paris) 1984; 13:351-61.  Haour F, Lang B . Role of hormonal receptors in the regulation of the corpus luteum (author's transl). [French]. Semaine Des Hopitaux De Paris 1978; 54(33-36): 1063-70.  Hargrove J L , Seeley RR, Ellis L C . Rabbit testicular contractions: bimodal interaction of prostaglandin E l with other agonists. A m J Physiol 1975; 228:810-4.  159 Harris K H , Murphy BD. Luteolysis in the hamster: abrogation by gonadotrophin and prolactin pretreatment. Prostaglandins 1981; 21(2): 177-87.  Harris K H , Murphy B D . Luteolysis in the hamster: abrogation by gonadotrophin and prolactin pretreatment. Prostaglandins 1981; 21(2): 177-87.  Harvey H A , Lipton A , Max DT, Pearlman H G , Diaz PR, de, la, Garza J. Medical castration produced by the GnRH analogue leuprolide to treat metastatic breast cancer. Journal of Clinical Oncology 1985; 3(8): 1068-72.  Hawes B E , Waters S B , Janovick J A , Bleasdale JE, Conn P M . Gonadotrophin-releasing hormone-stimulated intracellular Ca2+ fluctuations and luteinizing hormone release can be uncoupled from inositol phosphate production. Endocrinology 1992; 130(6):3475-83.  Hawes B E , Barnes S, Conn P M . Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotrophin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action. Endocrinology 1993; 132:2124-30.  Hillensjo T, LeMaire W J , Clark M R , Ahren K . Effect of gonadotrophin-releasing hormone (GnRH) and G n R H agonists upon accumulation of progesterone, c A M P and prostaglandin in isolated preovulatory rat follicles. Acta Endocrinol (Copenh) 1982; 101(4):603-10.  Hillensjo T, Sjogren A , Strander B , Nilsson L , Wikland M , Hamberger L , Roos P. Effect of gonadotrophins on progesterone secretion by cultured granulosa cells obtained from human preovulatory follicles, Acta Endocrinol (Copenh) 1985; 110(3):401-7.  Hillier S G , Zeleznik A J , Knazek R A , Ross GT. Hormonal regulation of preovulatory follicle maturation in the rat. J Reprod Fertil 1980; 60:219-29.  160 Hillier SG, Miro F. Inhibin, activin, and follistatin. Potential roles in ovarian physiology. Annals of the New York Academy of Sciences 1993; 687(29):29-38.  Hsueh A J W , Schaeffer J M . Gonadotrophin-releasing hormone as a paracrine hormone and neurotransmitter in extra-pituitary sites. J Steroid Biochem 1985; 23:757-764.  Hsueh M J , Adashi E Y , Tucker E, Valk C, Ling N C. Relative potencies of gonadotrophinreleasing hormone agonists and antagonists on ovarian and pituitary functions. Endocrinology 1983; 112(2):689-95.  Igarashi M . Mini review on inhibins and activins. [Japanese]. Nippon Naibunpi Gakkai Zasshi Folia Endocrinologica Japonica 1992; 68(2):71-80.  Inaudi P, Reymond M J , Rey F, Genazzani A D , Lemarchand B T . Pulsatile secretion of gonadotrophins and prolactin during the follicular and luteal phases of the menstrual cycle: analysis of instantaneous secretion rate and secretory concomitance. Fertility & Sterility 1992; 58(l):51-9.  Ireland JJ, Aten RF, Behrman HR. GnRH-like proteins in cows: concentrations during corpora lutea development and selective localization in granulosal cells. Biol Reprod 1988; 38(3):544-50.  Jalkanen J, Ritvos O, Huhtaniemi I, Stenman U H , Laatikainen T, Ranta T. Phorbol ester stimulates human granulosa-luteal cell cyclic adenosine 3', 5'-monophosphate and progesterone production. Mol Cell Endocrinol 1987; 51:273-6.  Janne OA. Progesterone action in mammalian uterus. Acta Obstet Gynecol Scand Suppl 1981; 101:11-6 [Review].  Jansen RP. Ultrastructure and histochemistry of acid mucus glycoproteins in the estrous mammal oviduct. Microsc Res Tech 1995; 32:24-49 [Review].  Jones P B , Valk C A , Hsueh A J . Regulation of progestin biosynthetic enzymes in cultured rat granulosa cells: effects of prolactin, beta 2-adrenergic agonist, human chorionic gonadotrophin and gonadotrophin-releasing hormone. Biol Reprod 1983; 29(3):572-85.  Joshi D , Lekhtman I, Billiar R B , Miller M . Gonadotrophin-releasing hormone induced luteinizing hormone responses in young and old female C57BL/6J mice. Proceedings of the Society for Experimental Biology & Medicine 1993; 204(2): 191-4,.  Kaiser U B , Lee B L , Carroll RS, Unabia G, Chin WW, Childs G V . Follistatin gene expression in the pituitary: localization in gonadotropes and folliculostellate cells in diestrous rats. Endocrinology 1992; 130(5):3048-56.  Kawai Y , Clark M R . Phorbol ester regulation of rat granulosa cell prostaglandin and progesterone accumulation. Endocrinology 1985; 116(6):2320-6.  Kakar SS, Musgrove L C , Devor D C , Sellers JC, Neill JD. Cloning, sequencing, and expression of human gonadotrophin-releasing hormone (GnRH) receptor. Biochem Biophys Res Commun 1992; 189:289-295.  Kakar SS, Grantham K , Musgrove L C , Devor D, Sellers JC, Neill JD. Rat gonadotrophinreleasing hormone (GnRH) receptor: tissue expression and hormonal regulation of its mRNA. Mol Cell Endocrinol 1994; 101:151-157.  Kawai Y , Clark M R . Mechanisms of action of gonadotrophin-releasing hormone on rat granulosa cells. EndocrRes 1986; 12(3): 195-209.  Kammerman S, Ross J. Increase in numbers of gonadotrophin receptors on granulosa cells during follicle maturation. J Clin Endocrinol Metab 1975; 41:546-50.  Khan DF, Huang JC, Dawood M Y . Effect of human chorionic gonadotrophin and prostaglandin F2a on progesterone production by human luteal cells. J Steroid Biochem 1989; 33:941-7.  Kiesel L , Bertges K , Rabe T, Runnebaum B. Gonadotrophin-releasing hormone enhances polyphosphoinositide hydrolysis in rat pituitary cells. Biochem Biophys Res Commun 1986; 134(2):861-7.  King JA, Mehl A E , Tyndale B C , Hinds L , Millar RP. A second form of gonadotrophin-releasing hormone (GnRH), with chicken GnRH II-like properties, occurs together with mammalian GnRH in marsupial brains. Endocrinology 1989; 125(5):2244-52.  King JA, Hinds L A , Mehl A E , Saunders NR, Millar RP. Chicken GnRH II occurs together with mammalian GnRH in a South American species of marsupial (Monodelphis domestica). Peptides 1990; ll(3):521-5.  Korda A R , Shutt D A , Smith ID, Shearman RP, Lyneham RC. Assessment of possible luteolytic effect on intra-ovarian injection of prostaglandin F 2 in the human. Prostaglandins 1975; 9:443a  9.  Laatikainen TJ. Corticotropin-releasing hormone and opioid peptides in reproduction and stress. Annals of Medicine 1991; 23(5):489-96 [Review].  Lahav M , Rennert H , Sabag K , Barzilai D. Calmodulin inhibitors and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate do not prevent the inhibitory effect of prostaglandin F  2 a  on  cyclic A M P production in isolated rat corpora lutea. J Endocrinol 1987; 113:205-12.  Lahav M , West L A , Davis JS. Effects of prostaglandin F  2 a  and a gonadotrophin-releasing  hormone agonist on inositol phospholipid metabolism in isolated rat corpora lutea of various ages. Endocrinology 1988; 123(2): 1044-52.  Lahav M , Davis JS, Rennert H. Mechanism of the luteolytic action of prostaglandin F rat. J Reprod Fertil Suppl 1989; 37(233):233-40.  2 a  in the  Lake S, Gullberg H , Wahlqvist J, Sjogren A M , Kinhult A , Lind P, Hellstrom L E , Stjernschantz J. Cloning of the rat and human prostaglandin F2 receptors and the expression of the rat a  prostaglandin F  2 a  receptor. Febs Lett 1994; 355:317-25.  Langlois D , Hinsch K D , Saez J M , Begeot M . Stimulatory effect of insulin and insulin-like growth factor I on G i proteins and angiotensin-II-induced phosphoinositide breakdown in cultured bovine adrenal cells. Endocrinology 1990; 126(4): 1867-72.  Latouche J, Crumeyrolle A M , Jordan D, Kopp N , Augendre F B , Ceddard L , Haour F. GnRH receptors in human granulosa cells: anatomical localization and characterization by autoradiographic study. Endocrinology 1989; 125(3): 1739-41. Lau IF, Hoogasian J, Wong SK.Saksena S K . Effects of prostaglandin F  2 a  and an  antiprogestational steroid, RMI 12,936, on rat pregnancy. Prostaglandins Med 1980; 4:121-32.  Lee B L , Unabia G , Childs G . Expression of follistatin m R N A by somatotropes and mammotropes early in the rat estrous cycle. Journal of Histochemistry & Cytochemistry 1993; 41(7):955-60.  Leung P C K , Armstrong DT. Stimulatory action of follicle-stimulating hormone and androgens on the responsiveness of rat granulosa cells to gonadotrophins in vitro. Endocrinology 1979; 104:1124-1129.  Leung PCK. Mechanisms of gonadotrophin-releasing hormone and prostaglandin action on luteal cells. Can J Physiol Pharmacol 1985; 63(3):249-56.  Leung P C K , Steele G L . Intracellular signaling in the gonads. Endocr Rev 1992; 13(3):476-98 [Review].  Lewis A E , Edwards A. Biostatistics. 1984; 2nd ed. Van Nostrand Reinhold. New York.  164 L i W, Khorasheh S, Ho Yuen B , Ling N , Leung PCK. Stimulation of progesterone secretion by recombinant follistatin-288 in human granulosa cells. Endocrinology 1993; 132:1750-1756.  Ling N , DePaolo L V , Bicsak T A , Shimasaki S. Novel ovarian regulatory peptides: inhibin, activin, and follistatin. Clinical Obstetrics & Gynecology 1990; 33(3):690-702.  Lopez B A , Bellinger J, Marshall J M , Phaneuf S, Europe F G , Asboth G, Barlow D H . G protein expression and second messenger formation in human granulosa cells. J Reprod Fertil 1995; 104:77-83.  Luborsky J L , Slater W T , Behrman HR. Luteinizing hormone (LH) receptor aggregation: modification of ferritin-LH binding and aggregation by prostaglandin F2 and ferritin-LH. a  Endocrinology 1984; 115(6):2217-26.  M a F, Leung P C K . Luteinizing hormone-releasing hormone enhances polyphosphoinositide breakdown in rat granulosa cells. Biochemical & Biophysical Research Communications 1985; 130(3): 1201-8.  Magnaldo I, Pouyssegur J, Paris S. Thrombin exerts a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an inhibition via a GTP-binding protein and a potentiation via activation of protein kinase C. Biochem J 1988; 253(3):711-9. Manaugh L C , Novy MJ. Effects of indomethacin on corpus luteum function and pregnancy in rhesus monkeys. Fertil Steril 1976; 27:588-98. Mahesh V B . The dynamic interaction between steroids and gonadotropins in the mammalian ovulatory cycle. Neurosci Biobehav Rev 1985; 9:245-60 [Review].  Martin K A , Liptrap R M . Half-life of 13,14dihydro-15-keto prostaglandin F plasma of the pig. Res Vet Sci 1981; 31(3):387-9.  2 a  in peripheral  165 Martin K , Santoro N , Hall J, Filicori M , Wierman M , Crowley W J . Clinical review 15: Management of ovulatory disorders with pulsatile gonadotrophin-releasing hormone. Journal of Clinical Endocrinology & Metabolism 1990; 71(5):1081A-1081G [Review].  Martin T F , Lewis JE, Kowalchyk JA. Phospholipase C-beta 1 is regulated by a pertussis toxininsensitive G-protein. Biochem J 1991; 280: 753-60.  Massicotte J, Lachance R, Labrie F. Modulation of cyclic A M P formation and progesterone secretion by human chorionic gonadotrophin, epinephrine, buserelin and prostaglandins in normal or human chorionic gonadotrophin desensitized rat immature luteal cells in monolayer culture. J Steroid Biochem 1984;. 21:745-54.  Mates G , Daniel M , Walker C. Factors affecting the reproducibility of a spectrofluoroimetric assay for the enumeration of human venous endothelium in culture. Cell Biol Int Rep 1986; 10:641-8.  Mauvais JP, Kuttenn F, Gompel A . Estradiol/progesterone interaction in normal and pathologic breast cells. Ann N Y Acad Sci 1986; 464:152-67 [Review].  Mauvais JP, Kuttenn F, Gompel A . Antiestrogen action of progesterone in breast tissue. Horm Res 1987; 28:212-8 [Review].  Mauvais JP, Kuttenn F,Gompel A . Antiestrogen action of progesterone in breast tissue. Breast Cancer Res Treat 1986; 8:179-88 [Review].  May JV, Schomberg DW. Developmental coordination of luteinizing hormone/human chorionic gonadotrophin (hCG) receptors and acute hCG responsiveness in cultured and freshly harvested porcine granulosa cells. Endocrinology 1984; 114:153-63.  Mayes P A . Lipids of Physiologic Significance. In: Murray R K , Granner D K , Mayes P A , Rodwell V W (eds.), Harper's Biochemistry, 21st ed., San Mateo, California, Appleton & Lange; 1988:130-137.  McArdle C A , Poch A , Schomerus E , Kratzmeier M . Pituitary adenylate cyclase-activating polypeptide effects in pituitary cells: modulation by gonadotrophin-releasing hormone in alpha T3-1 cells. Endocrinology 1994; 134(6):2599-605.  McCann T J , Flint A P . Use of pertussis toxin to investigate the mechanism of action of prostaglandin F a on the corpus luteum in sheep. J Mol Endocrinol 1993; 10(l):79-85. 2  McCarty K J , Lubahn D B , McCarty K S . Oestrogen and progesterone receptors: physiological and pathological considerations. Clin Endocrinol Metab 1983; 12:133-54.  McCarthy D M . Mechanisms of mucosal injury and healing: the role of non-steroidal antiinflammatory drugs. Scand J Gastroenterol Suppl 1995; 208:24-9.  McNatty K P , Smith D M , Makris A , Osathanondh R, Ryan K J . The microenvironment of the human antral follicle: interrelationships among the steroid levels in antral fluid, the population of granulosa cells, and the status of the oocyte in vivo and in vitro. J Clin Endocrinol Metab 1979; 49:851-60.  McNatty K P , Makris A , De G C , Osathanondh R, Ryan K J . The production of progesterone, androgens and oestrogens by human granulosa cells in vitro and in vivo. J Steroid Biochem 1979; 11:775-9.  Meidan R, Girsh E , Blum O, Aberdam E. In vitro differentiation of bovine theca and granulosa cells intosmall and large luteal-like cells: morphological and functional characteristics. Biol Reprod 1990;43(6):913-21.  167 Meldrum DR, Chang RJ, L u J, Vale W, Rivier J, Judd HL. Medical oophorectomy using a longacting G N R H agonist-a possible new approach to the treatment of endometriosis. Journal of Clinical Endocrinology & Metabolism 1982; 54(5): 1081-3.  Mercado M , Shimasaki S, Ling N , DePaolo L. Effects of estrous cycle stage and pregnancy on follistatin gene expression and immunoreactivity in rat reproductive tissues: progesterone is implicated in regulating uterine gene expression. Endocrinology 132(4): 1774-81, 1993.  Michael A E , Abayasekara DR, Webley GE. The luteotrophic actions of prostaglandins E and 2  F  2 a  on dispersed marmoset luteal cells are differentially mediated via cyclic A M P and protein  kinase C. J Endocrinol 1993; 138:291-8.  Michael A E , Webley GE. Roles of cyclic A M P and inositol phosphates in the luteolytic action of cloprostenol, a prostaglandin F  2 a  analogue, in marmoset monkeys (Callithrix jacchus). J Reprod  Fertil 1993;97:425-31.  Michael A E , Webley GE. Prostaglandin F  2 a  stimulates c A M P phosphodiesterase via protein  kinase C in cultured human granulosa cells. Mol Cell Endocrinol 1991a; 82:207-14.  Michael A E , Webley G E . Prior exposure to gonadotrophins prevents the subsequent antigonadotrophic actions of cloprostenol by a cyclic AMP-dependent mechanism in cultured human granulosa cells. J Endocrinol 1991b; 131:319-25.  Michel U , Albiston A , Findlay JK. Rat follistatin: gonadal and extragonadal expression and evidence for alternative splicing. Biochemical & Biophysical Research Communications 1990; 173(l):401-7.  Michel U , McMaster JW, Findlay JK. Regulation of steady-state follistatin mRNA levels in rat granulosa cells in vitro. Journal of Molecular Endocrinology 1992; 9(2): 147-56.  168 Minaretzis D , Jakubowski M , Mortola JF, Pavlou S N . Gonadotrophin-releasing hormone receptor gene expression in human ovary and granulosa-lutein cells. J Clin Endocrinol Metab 1995; 80(2):430-4.  Mitsuhashi N . Studies on the mechanism and the significance of prostaglandin biosynthesis by the ovary-ovulation block by the indomethacin and incubation of the follicle. Acta Obstet Gynaecol Jpn 1981; 33:479-88.  Molcho J, Zakut H , Naor Z. Gonadotrophin-releasing hormone stimulates phosphatidylinositol labeling and prostaglandin E production in Leydig cells. Endocrinology 1984; 114(3): 1048-50  Moon Y S , Duleba AJ. Comparative studies of androgen metabolism in theca and granulosa cells of human follicles in vitro. Steroids 1982; 39:419-30.  Moon Y S . The role of gonadotropins and testosterone in progesterone production by human ovarian granulosa cells. Mol Cell Endocrinol 1981; 23:115-22.  Moon Y S , Tsang B K , Simpson C, Armstrong DT. 17 beta-Estradiol biosynthesis in cultured granulosa and thecal cells of human ovarian follicles: stimulation by follicle-stimulating hormone. J Clin Endocrinol Metab 1978; 47:263-7.  Moon Y S , Duleba A J , K i m K S , Ho Yuen B . Effects of prostaglandins E  2  and F  2 a  on  progesterone metabolism by rat granulosa cells. Biochem Biophys Res Commun 1986; 135:7649. Nakatani A , Shimasaki S, Depaolo L V , Erickson G F , Ling N . Cyclic changes in follistatin messenger ribonucleic acid and its protein in the rat ovary during the estrous cycle. Endocrinology 1991; 129(2):603-11.  Nillius SJ, Gemzell C, Johansson ED, Wide L. Monitoring treatment with human gonadotrophins or the synthetic decapeptide LH-releasing hormone, pp. 753-75, In: Crosignani Pg, James Vh, ed. Recent progress in reproductive endocrinology. 1974; London, Academic Press, 4.  169 Nebert D W , Gonzales FJ. P450 genes: structure, evolution, and regulation. Ann Rev Biochem 1987; 56:945-993.  Netiv E , Liscovitch M , Naor Z. Delayed activation of phospholipase D by gonadotrophinreleasing hormone in a clonal pituitary gonadotrope cell line (alpha T3-1). Febs Lett 1991; 295(1-3): 107-9.  Nelson SE, McLean M P , Jayatilak PG, Gibori G. Isolation, characterization, and culture of cell subpopulationsforming the pregnant rat corpus luteum. Endocrinology 1992; 130(2):954-66.  Nillius SJ. Gonadotrophin-releasing hormone agonists for new approaches to contraception in man. Wien K l i n Wochenschr 1985; 97(23):865-73 [Review].  Ng sy, Gunning P, Eddy R, Ponte P, Leavitt J, Shows T, Kedes L . Evoloution of the functional human B-actin gene and its multipseudogene family: Conservation of noncoding regions and chromosomal dispersion of pseudogenes. Mol Cell Biol 1985; 5:2720-2732.  O'Dowd B F , Hnatowich M , Regan JW, Leader W M , Caron M G , Lefkowitz R J , Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling, J Biol Chem 1988; 263:15985-92.  Ohno T, Imai A , Furui T, Takahashi K , Tamaya T. Presence of gonadotrophin-releasing hormone and its messenger ribonucleic acid in human ovarian epithelial carcinoma. A m J Obstet Gynecol 1993; 169:605-610.  Oikawa M , Dargan C, Ny T, Hsueh A J . Expression of gonadotrophin-releasing hormone and prothymosin-alpha messenger ribonucleic acid in the ovary. Endocrinology 1990; 127(5):2350-6.  Olofsson JI, Conti C C , Leung C, Krisinger J, Leung P C K . Tissue-specific regulation of gonadotrophin-releasing hormone (GnRH) receptor gene expression during the periovulatory period. EndocrJ 1994;2:471-476.  Olsson J-H, Akesson I, Hillensjo T. Effects of a gonadotrophin-releasing hormone agonist on progesterone formation in cultured human granulosa cells. Acta Endocrinol (Copenh) 1990; 122:427-431.  Orlicky DJ, Williams SC. Immunohistochemical localization of PGF2 receptor in the mouse a  testis. Prostaglandins Leukot Essent Fatty Acids 1992; 47(4):247-52.  Parinaud J, Vieitez G , Beaur A , Pontonnier G , Boureau E. Effect of a luteinizing hormonereleasing hormone agonist (buserelin) on steroidogenesis of cultured human preovulatory granulosa cells. Fertil Steril 1988; 50:597-602.  Patwardhan V V , Lanthier A . Concentration of prostaglandins P G E and PGF, estrone, estradiol, and progesterone in human corpora lutea. Prostaglandins 1980; 20:963-9.  Patwardhan V V , Lanthier A . Prostaglandins P G E and P G F in human ovarian follicles: endogenous contents and in vitro formation by theca and granulosa cells. Acta Endocrinol (Copenh) 1981;97:543-50.  Patwardhan V V , Lanthier A . Luteal phase variations in endogenous concentrations of prostaglandins P G E and PGF and in the capacity for their in vitro formation in the human corpus luteum. Prostaglandins 1985; 30(l):91-8.  Pellicer A , Tarin JJ, Miro F, Sampaio M , De-los-Santos, M j , Remohi J. The use of gonadotrophin-releasing-hormone analogues (GnRHa), in in-vitro fertilization: some clinical and experimental investigations of a direct effect on the human ovary. Human Reproduction 1992. l(39):39-47.  Peng C , Fan N C , Ligier M , Vaananen JE, Leung P C K . Expression and regulation of gonadotrophin-releasing hormone (GnRH) and GnRH receptor messenger ribonucleic acids in human granulosa-luteal cells. Endocrinology 1994; 135(5): 1740-6.  171 Pepperell JR, Preston SL, Behrman HR. The antigonadotropic action of prostaglandin F  2 a  is not  mediated by elevated cytosolic calcium levels in rat luteal cells. Endocrinology 1989; 125:14451.  Plunkett E R , Moon Y S , Zamecnik J, Armstrong DT. Preliminary evidence of a role for prostaglandin F in human follicular function. A m J Obstet Gynecol 1975; 123:391-7.  Popkin R, Bramley T A , Currie A , Shaw RW, Baird DT, Fraser H M . Specific binding of luteinizing hormone releasing hormone to human luteal tissue. Biochem Biophys Res Commun 1983; 114:750-756.  Porkess. The Harper Collins dictionary of statistics. Harper Perennial. New York, 1991.  Puschel GP, Kirchner C, Schroder A , Jungermann K . Glycogenolytic and antiglycogenolytic prostaglandin E actions in rat hepatocytes are mediated via different signalling pathways. Eur J 2  Biochem 1993;218:1083-9.  Quirk S M , Cowan R G , Joshi SG, Henrickson K P . Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biol Reprod 1995; 52(2):279-87.  Rajkumar K , Ganguli S, Menon K M , Mead R A , Murphy B D . Studies on the mechanism of action of prostaglandin F  2 a  induced luteolysis in rats. Prostaglandins 1988; 36(4):547-64.  Rasmussen DD.Yen SS. Progesterone and 20 alpha-hydroxyprogesterone stimulate the in vitro release of G n R H by the isolated mediobasal hypothalamus. Life Sci 1983; 32:1523-30.  Retamales I, Carrasco I, Troncoso JL, Las Heras J, Devoto L, Vega M . Morpho-functional study of human luteal cell subpopulations. Hum Reprod 1994; 9(4):591-6.  Richards JS, Fitzpatrick S L , Clemens JW, Morris J K , Alliston T, Sirois J. Ovarian cell differentiation: a cascade of multiple hormones, cellular signals, and regulated genes. Recent Prog Horm Res 1995; 50:223-54 [Review].  Richardson M C , Masson G M . Progesterone production by dispersed cells from human corpus luteum: stimulation by gonadotrophins and prostaglandin F2«; lack of response to adrenaline and isoprenaline. J Endocrinol 1980; 87:247-54.  Ristimaki A , Jaatinen R and Ritvos O. Regulation of prostaglandin F  2 a  receptor expression in  cultured human granulosa-luteal cells. Endocrinology 1997; 138:191-195.  Rodway M R , Ho yuen B , Leung P C K . Inhibition of aromatase activity by 8-Br-cAMP in cultured first trimester human trophoblast. A m J Obst Gynecol. 1990; 163:1546-1551.  Rommler A , Viebahn C, Schwartz U , Hammerstein J. Short-term regulation of L H and F S H secretion in cyclic women. III. Effects of varying doses of two consecutive L H - R H injections on pituitary and ovarian response. Acta Endocrinologica 1979; 90(3):394-402.  Roth T L , Munson L , Swanson W F , Wildt D E . Histological characteristics of the uterine endometrium and corpusluteum during early embryogenesis and the relationship to embryonicmortality in the domestic cat. Biol Reprod 1995; 53(5): 1012-21.  Roth GJ, Siok CJ. Acetylation of the NH2-terminal serine of prostaglandin synthetase by aspirin. J Biol Chem 1978; 253:3782-4. Rotsztejn W H , Charli J L , Pattou E , Epelbaum J, Kordon C. In vitro release of luteinizing hormone-releasing hormone (LHRH) from rat mediobasal hypothalamus: effects of potassium, calcium and dopamine. Endocrinology 1976; 99(6): 1663-6.  Ryan RJ, Keutmann HT, Charlesworth M C , McCormick DJ, Milius RP, Calvo FO, Vutyavanich T. Structure-function relationships of gonadotrophins. Recent Progress in Hormone Research 1987; 43(383):383-429 [Review].  Sano N , Shichi H . Bimodal regulation of adenylate cyclase by prostaglandin E2 receptors in porcine ciliary epithelium. Prostaglandins Leukot Essent Fatty Acids 1993; 49:765-9.  173 Satoh K , Yasumizu T, Kawai Y , Ozaki A , Wu T, Kinoshita K , Sakamoto S. In vitro production of prostaglandins E , F, and 6-keto prostaglandin Fi« by human pregnant uterus, decidua and amnion. Prostaglandins Med 1981; 6:359-68.  Sawyer C H . First Geoffrey Harris Memorial lecture. Some recent developments in brainpituitary-ovarian physiology. Neuroendocrinology 1975; 17(2):97-124 [Review].  Schroepfer GJ Jr. Sterol biosynthesis. Ann Rev Biochem 1982; 51:555-585.  Schuiling G A , Gnodde HP. Site of origin of the pulsatile secretion of luteinizing hormone in long-term ovariectomized rats. Journal of Endocrinology 1976; 70(1):97-104.  Schwartzman M L , Falck JR, Yadagiri P, Escalante B. Metabolism of 20-hydroxeicosatetraenoic acid by cyclooxygenase. Formation and identification of novel endothelium-dependent vasoconstrictor metabolites. J Biol Chem 1989; 264(20): 11658-62.  Segars J H , H i l l G A , Bryan S H , Herbert C3, Osteen K G , Rogers B J , Wentz A C . The use of gonadotrophin-releasing hormone agonist (GnRHa) in good responders undergoing repeat in vitro fertilization/embryo transfer (IVF/ET). Journal of in Vitro Fertilization & Embryo Transfer 1990;7(6):327-31.  Shimasaki S, Koga M , Buscaglia M L , Simmons D M , Bicsak T A , Ling N . Follistatin gene expression in the ovary and extragonadal tissues. Molecular Endocrinology 1989; 3(4):651-9.  Shinohara O, Knecht M , Catt K J . Inhibition of gonadotrophin-induced granulosa cell differentiation by activation of protein kinase C. Proc Natl Acad Sci U S A 1985; 82(24):851822.  Shimonaka M , Inouye S, Shimasaki S, Ling N . Follistatin binds to both activin and inhibin through the common subunit. Endocrinology 1991; 128(6):3313-5.  Siler K T , Khodr GS, Valenzuela G , Harper M J , Rhode J. GnRH effects on placental hormones during gestation. III. Prostaglandin E, prostaglandin F, and 13,14-dihydro-15-keto-prostaglandin F. Biol Reprod 1986; 35:312-9.  Singh S K , Dominic CJ. Prostaglandin F -induced changes in the sex organs of the male 2a  laboratory mouse. Exp Clin Endocrinol 1986; 88(3):309-15.  Siiteri PK. Adipose tissue as a source of hormones. A m J Clin Nutr 1987 [Review].  Smith W L , Borgeat P. The Eicosanoids: Prostablandins, Thromboxanes, Leukotrienes, and Hydroxy-Eicosaenoic Acids. In: Vance D E , Vance J E (eds.), Biochemistry of Lipids and Membranes, Menlo Park, California: The Benjamin/Cummings Publishing Co. Inc.; 1985:325360.  Soto E A , Kliman HJ, Strauss JF 3d, Paavola L G . Gonadotrophins and cyclic adenosine 3',5'monophosphate (cAMP) alterthe morphology of cultured human granulosa cells. Biol Reprod 1986; 34(3):559-69.  Spilman C H , Harper M J . Effects of prostaglandins on oviductal motility and egg transport. Gynecol Invest 1975; 6:186-205 [Review].  Srivastava R K , Luu T V , Marrone B L , Harris HS, Sridaran R. Inhibition of steroidogenesis by luteal cells of early pregnancy in the rat in response to in vitro administration of a gonadotropinreleasing hormone agonist. J Steroid Biochem Mol Biol 1994; 49(l):73-9.  Steele G L , Leung PCK. Mechanism of prostaglandin F  2 a  action in the ovary. J Lipid Mediat  1993;6:509-13.  Stoljelkovics SS, Reinhart J, Catt K J . Gonadotropin-releasing hormone receptors: Structure and signal transduction pathways. Endocrine Reviews 1994; 15:462-499.  175 Stryder L. Biochemistry, New York: W H Freeman and Company; 1988:555-574, 991-1000.  Suginami H , Okamura H , Yogo I. In vitro steroidogenesis by human corpora lutea of pregnancy. Effects of human chorionic gonadotropin and prostaglandin F2«- Obstet Gynecol 1976; 47(2): 177-82.  Sugino H , Nakamura T, Hasegawa Y , Miyamoto K , Igarashi M , Eto Y , Shibai H , Titani K . Identification of a specific receptor for erythroid differentiation factor on follicular granulosa cell. Journal of Biological Chemistry 1988; 263(30): 15249-52.  Suzuki M , Takahashi K . Hypothalamo-hypophyseal control of ovulation, pp. 114-21. In: Hatotani N , ed. Psychoneuroendocrinology 1974; Basel, Karger [Review].  Takahashi H , Duleba A J , Yuen B H , Moon Y S . Steroidogenic capabilities of various compartments of rat ovarian follicles in culture. Steroids 1984; 44:337-48.  Tonetta S A , diZerega GS. Intragonadal regulation of follicular maturation. Endocr Rev 1989; 10:205-29 [Review].  Toullec D , Pianetti P, Coste H , Bellevergue P, Grand PT, Ajakane M , Baudet V , Boissin P, Boursier E, Loriolle F, et al. The bisindolylmaleimide G F 109203X is a potent and selective inhibitor of protein kinase. C J Biol Chem 1991; 266:15771-81.  Tsang B K , Moon Y S , Armstrong DT. Estradiol-17 beta and androgen secretion by isolated porcine ovarian follicular cells in vitro. Can J Physiol Pharmacol 1982; 60:1112-8.  Turgeon JL. Neural control of ovulation. Physiologist 1980; 23(3):56-62 [Review].  Tureck R W , Mastroianni Jr L , Blasco L , Strauss JR. Inhibition of human granulosa cells progesterone secretion by gonadotropin releasing hormone agonist. J Clin Endocrinol Metab 1982;54:1078-1080.  176 Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 1971; 231:232-5.  Waite M . Phospholipases. In: Vance D E , Vance J E (eds.), Biochemistry of Lipids and Membranes, Menlo Park, California: The Benjamin/Cummings Publishing Co. Inc.; 1985:299324.  Wallace CR, Kiser TE, Rampacek GB, Draeling RR. The relationship between 13, 14-dihydro15-keto P G F  2 a  and L H secretion in bulls. Prostaglandins 1985; 30(6):925-33.  Wang J, Leung PC. Role of arachidonic acid in luteinizing hormone-releasing hormone action: stimulation of progesterone production in rat granulosa cells. Endocrinology 1988; 122(3):90611.  Watanabe H , Nagai K , Yamaguchi M , Ikenoue T, Mori N . Interleukin-1 beta stimulates prostaglandin E2 and F  2 a  synthesis in human ovarian granulosa cells in culture. Prostaglandins  Leukot Essent Fatty Acids 1993; 49:963-7.  Watson J, Shepherd TS, Dodson K S . Prostaglandin E-2-9-ketoreductase in ovarian tissues. J Reprod Fertil 1979; 57:489-96.  Webley G E , Richardson M C , Summers P M , Given A , Hearn JP. Changing responsiveness of luteal cells of the marmoset monkey (Callithrix jacchus) to luteotrophic and luteolytic agents during normal and conception cycles. J Reprod Fertil 1989; 87:301-10.  Webley G E , Richardson M C , Given A , Harper J, Preincubation of human granulosa cells with gonadotrophin prevents the cloprostenol-induced inhibition of progesterone production. Hum Reprod 1991 ;6(6):779-82.  Whitelaw PF, Eidne K A , Sellar R, Smyth CD, Hillier SG. Gonadotropin-releasing hormone receptor messenger ribonucleic acid expression in rat ovary. Endocrinology 1995; 136(1): 172-9.  177 Wierman M E , Gharib SD, Chin WW. The structure and regulation of the pituitary gonadotrophin subunit genes. Baillieres Clinical Endocrinology & Metabolism 1988; 2(4):869-89 [Review].  Williams WF, Lewis GS, Thatcher WW, Underwood CS. Plasma 13,14-dihydro-15 keto P G F  i a  (PGFM) in pregnant and nonpregnant heifers prior to and during surgery and following intrauterine injection of P G F . Prostaglandins 1983; 25(6):891-9. 2 a  Wu C H , Prazak L M . Endocrine basis for ovulation induction. Clinical Obstetrics & Gynecology 1974; 17(4):65-78 [Review].  Yen SS. Regulation of the hypothalamic—pituitary—ovarian axis in women. Journal of Reproduction & Fertility 1977; 51(1): 181-91.  Ying S Y , Becker A , Swanson G , Tan P, Ling N , Esch F, Ueno N , Shimasaki S, Guillemin R. Follistatin specifically inhibits pituitary follicle stimulating hormone release in vitro. Biochemical & Biophysical Research Communications 1987; 149(1): 133-9.  Yuh K C , Possley R M , Brabec R K , Keyes PL. Steroidogenic and morphological characteristics of granulosa and thecal compartments of the differentiating rabbit corpus luteum in culture. J Reprod Fertil 1986; 76(l):267-77.  Xiao S, Robertson D M , Findlay JK. Effects of activin and follicle-stimulating hormone (FSH)suppressing protein/follistatin on F S H receptors and differentiation of cultured rat granulosa cells. Endocrinology 1992; 131(3): 1009-16.  Zolti M , Meirom R, Shemesh M , Wollach D, Mashiach S, Shore L , Rafael Z B . Granulosa cells as a source and target organ for tumor necrosis factor-alpha. Febs Lett 1990; 261:253-5.  

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