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The role of adenosine triphosphate in human granulosa-luteal cells Tai, Chen-Jei 2001

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THE ROLE OF ADENOSINE TRIPHOSPHATE IN HUMAN GRANULOSA-LUTEAL CELLS BY CHEN-JEI TAI M.D., K A O H S I U N G M E D I C A L C O L L E G E , TAIWAN, 1990 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY IN THE F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF OBSTETRICS A N D G Y N A E C O L O G Y P R O G R A M OF REPRODUCTIVE A N D D E V E L O P M E N T A L SCIENCES WE A C C E P T THIS THESIS A S CONFORMING TO THE REQUIRED STANDARD.  THE UNIVERSITY OF BRITISH C O L U M B I A APRIL 2001 ©CHEN-JEI TAI, 2001  In  presenting this  degree at the  thesis in  University of  partial  fulfilment  of  the  requirements  for  an advanced  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 department  or  by  his  or  scholarly purposes may be granted her  representatives.  It  is  by the head of  understood  that  copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT Adenosine triphosphate (ATP) is released from cells such as platelets and co-released with neurotransmitter  granules from autonomic nerves by exocytosis.  Extracellular ATP binds to a G protein-coupled P2 purinoceptor that activates phospholipase C and phosphatidylinositol hydrolysis, generating diacylglycerol and inositol 1,4,5-trisphosphate, which stimulate protein kinase C (PKC) and cytosolic calcium mobilization, respectively. Autonomic nerves have been shown to innervate the ovary and may be involved in regulating steroidogenesis.  It is tempting to speculate that the co-released ATP from  autonomic nerve endings in the ovary may play a role in regulating ovarian function. A series of experiments has been performed in this study to examine (1) the expression and regulation of P2U purinergic receptor (P2UR) in human granulosa-luteal cells (hGLCs), (2) the role of P K C in ATP-induced calcium oscillations, (3) the action and mechanism of antigonadotropic effect of ATP on hGLCs, (4) the functional role of extracellular ATP in the ovary, and (5) the effect of ATP on the activation of the mitogen-activated protein kinase (MAPK) signaling pathway and its physiological role in hGLCs. This study demonstrated for the first time the expression of P2UR mRNA in hGLCs, and the regulation of P2UR mRNA by hCG, c A M P and forskolin.  The P2UR  was functional in hGLCs, since activation of the P2UR by ATP or UTP resulted in rapid and transient mobilization of cytosolic calcium at the single cell level. It appears that P K C may have dual actions by providing positive forward actions as well as negative feedback in controlling various signaling steps. As shown in this ii  study, ATP was capable of inducing calcium mobilization, which was negatively regulated by P K C , from both intracellular stores and extracellular influx in cultured hGLCs.  The antigonadotropic effect of extracellular ATP was revealed as it significantly  reduced hCG-stimulated c A M P production. The inhibitory effect of ATP was reversed by P K C inhibitors, staurosporin and bisindolylmaleimide I, indicating the involvement of P K C in mediating the antigonadotropic action of ATP in hGLCs. Further, our data demonstrated that ATP was able to activate the M A P K signaling pathway in hGLCs.  After binding to P2-purinoceptor, ATP activated M A P K subsequent  to P L C and P K C activation through a PTX-insensitive G-protein in hGLCs. mediated  the  anti-gonadotropic  action of ATP in steroidogenesis  by  MAPK reducing  hCG-stimulated progesterone production. These findings support a potential role of ATP in regulating ovarian function.  iii  TABLE OF CONTENTS ABSTRACT  n  T A B L E OF C O N T E N T S  iv  LIST OF T A B L E S  vii  L I S T OF F I G U R E S  viii  L I S T OF A B B R E V I A T I O N S  xi  P U B L I C A T I O N LIST  xiv  ACKNOWLEDGEMENTS  xvii  P A R T 1. B A C K G R O U N D  1  1. 1 I N T R O D U C T I O N  1  1.2  H O R M O N A L R E G U L A T I O N OF T H E O V A R Y  3  1.3  I N N E R V A T I O N OF T H E O V A R Y  13  1.4  A D E N O S I N E 5'-TRIPHOSPHATE (ATP) A N D PURINERGIC RECEPTOR  15  1.5  SIGNAL TRANSDUCTION VIA P2U-PURINERGIC (G-PROTEIN-COUPLED SYSTEM)  RECEPTOR 29  1.5.1  G T P - B I N D I N G PROTEINS (G-PROTEINS)  29  1.5.2  PHOSPHOLIPASE C  31  1.5.3  CALCIUM  35  1.5.4  PROTEIN KINASE C  39  1.5.5  MITOGEN-ACTIVATED PROTEIN K I N A S E (MAPK)  45  1.5.6  CYCLIC A M P / P K A SIGNAL TRANSDUCTION  49  HYPOTHESIS  52  SPECIFIC OBJECTIVES  52 iv  PART 2. G E N E R A L M A T E R I A L S A N D M E T H O D S  53  2. 1 H U M A N G R A N U L O S A - L U T E A L C E L L S C U L T U R E  53  2.2  54  I S O L A T I O N OF T O T A L R N A  2. 3 R E V E R S E TRANSCRIPTION OF R N A TO FIRST-STRAND c D N A  55  2.4  55  SOUTHERN BLOT ANALYSIS  2. 5 S U B C L O N I N G A N D P L A S M I D I S O L A T I O N  59  2.6  60  SEQUENCE ANALYSIS  2. 7 N O R T H E R N B L O T A N A L Y S I S  62  2.8  WESTERN BLOT ANALYSIS  63  2.9  MICROSPECTROFLUORIMETRY  67  2. 10 R A D I O I M M U N O A S S A Y F O R P R O G E S T E R O N E  69  2. 11 R A D I O I M M U N O A S S A Y F O R c A M P  71  PART 3. EXPRESSION A N D REGULATION OF P2U-PURINERGIC RECEPTOR IN HUMAN GRANULOSA-LUTEAL CELLS  73  3. 1 A B S T R A C T  73  3.2  INTRODUCTION  74  3.3  MATERIALS AND METHODS  76  3.4  RESULTS  81  3.5  DISCUSSION  96  P A R T 4. A D E N O S I N E T R I P H O S P H A T E - E V O K E D C Y T O S O L I C C A L C I U M OSCILLATIONS IN H U M A N G R A N U L O S A - L U T E A L C E L L S : R O L E OF PROTEIN KINASE C  100  4. 1 A B S T R A C T  100  4.2  INTRODUCTION  102  4.3  MATERIALS AND METHODS  103  4.4  RESULTS  106  4.5  DISCUSSION  116  PART 5. A C T I O N OF ATP IN H C G - I N D U C E D C Y C L I C A M P P R O D U C T I O N IN H U M A N G R A N U L O S A - L U T E A L C E L L S : I N V O L V E M E N T OF P K C a  122  5. 1 A B S T R A C T  122  5.2  INTRODUCTION  124  5.3  MATERIALS AND METHODS  126  5.4  RESULTS  131  5.5  DISCUSSION  143  PART 6. ATP ACTIVATES MITOGEN-ACTIVATED PROTEIN K I N A S E IN H U M A N GRANULOSA-LUTEAL CELLS  148  6. 1 A B S T R A C T  148  6.2  INTRODUCTION  150  6.3  MATERIALS AND METHODS  151  6.4  RESULTS  158  6.5  DISCUSSION  177  P A R T 7. S U M M A R Y  183  P A R T 8. F U T U R E S T U D I E S  188  BIBLIOGRAPHY  191  vi  LIST OF TABLES Table 1 Pharmacological classification of P2-purinergic receptors. Table 2 Characteristics of IP3 receptors.  vii  LIST OF FIGURES Fig.l.  Hypothalamus-pituitary-ovary axis and feedback loop.  4  Fig.2.  The structure o f p r e - o v u l a t o r y f o l l i c l e .  7  Fig.3.  Hormone-stimulated steroidogenesis.  11  Fig.4.  S t e r o i d o g e n e s i s i n the o v a r y .  12  Fig.5.  C h e m i c a l structure o f adenosine triphosphate ( A T P ) .  16  Fig.6.  Schematiic representation of synthesis, storage, release and inactivation of ATP in purinergic nerve.  21  Fig.7.  Scheme o f the P 2 U p u r i n o c e p t o r .  25  Fig.8.  The m o l e c u l a r d o m a i n o f P L C i s o z y m e s .  32  Fig.9.  Summary of two major receptor-mediated pathways for the formation of IP3 and DAG.  34  Fig.10. P K C s t r u c t u r e .  41  Fig.l 1. Signal transduction pathway for the activation o f P K C  44  Fig.12. Regulation of sequential kinase pathways that activate M A P K s .  47  Fig. 13. Signal-transduction pathways of receptors or stress-activated M A P K s .  48  Fig.14. The c A M P signal transduction pathway.  51  Fig. 15. The apparatus for D N A transferring.  58  Fig. 16. Standard curve for protein assay.  66  Fig. 17. Standard curves for radioimmunoassay.  70  Fig. 18. Standard curve for c A M P assay.  72  Fig. 19. Expression of P2UR mRNA in human granulosa-luteal cells (hGLCs).  83  Fig.20. Validation of semiquantitative P C R for P2UR in hGLCs.  87  Fig.21. The effect of different reagents on the regulation of P2UR mRNA in cultured hGLCs.  89  Fig.22. The dose effect of human chorionic gonadotropin (hCG) on the regulation of P 2 U R m R N A in cultured h G L C s .  90  Fig.23. The time effect of human chorionic gonadotropin (hCG) on the regulation of P 2 U R m R N A in cultured h G L C s .  91  Fig.24. The effects of 8-bromo-cAMP (cAMP) and forskolin on the regulation of P2UR,  m R N A in cultured h G L C s .  92  Fig.25. Effects of ATP and UTP on inducing cytosolic calcium mobilization in cultured h G L C s using microspectrofluorimetry.  94  Fig.26. Dose effects of ATP and UTP on inducing cytosolic calcium mobilization in cultured h G L C s using microspectrofluorimetry.  95  Fig.27. A . Effects of ATP on inducing cytosolic calcium oscillations in cultured hGLCs.  107  B . Effects of 10 p M A T P on h G L C s cultured for various days (Day 3D a y 7).  108  Fig.28. Dose-dependent effects of P M A on ATP-evoked cytosolic calcium oscillations in cultured h G L C s .  110  Fig.29. The role of P K C in ATP induced-calcium oscillations in cultured hGLCs.  111  Fig.30. The effect of P M A on ATP-evoked cytosolic calcium oscillations in cultured hGLCs.  112  Fig.31. A . The role of P K C in homologous desensitization of ATP induced-calcium oscillations in cultured h G L C s .  114  B . and C. The effect of P K C inhibitor.  115  Fig.32. A proposed model of the potential cross-talk between ATP-activated protein kinase C (PKC) and cytosolic calcium oscillations in hGLCs.  120  Fig.33. The effect of ATP on hCG-stimulated intracellular c A M P production in human granulosa-luteal cells (hGLCs).  132  Fig.34. The effect of P M A on hCG-stimulated intracellular c A M P production in human granulosa-luteal cells (hGLCs).  134  Fig.35. The role of staurosporin (ST) in inhibitory effect of ATP on hCG-stimulated c A M P production.  135  Fig.36. The role of Bisindolylmaleimide I (Bis) in inhibitory effect of A T P on hCG-stimulated c A M P production. Fig.37. The presence of P K C isoforms ih hGLCs.  136 138  Fig.38. P C R product showing the absence of P K C y in h G L C s in three different patients.  139  Fig.39. Translocation of P K C a from cytosolic to membrane fraction after A T P IX  treatment i n h G L C s .  141  Fig.40. Down-regulation of P K C a in hGLCs achieved by prolonged treatment with 1 u M P M A for 18 hours.  142  Fig.41. A proposed model of the potential cross-talk between ATP-activated protein kinase C a (PKCa) and hCG-induced c A M P production in hGLCs.  147  Fig.42. The dose-response of ATP on M A P K activation in human granulosa-luteal cells ( h G L C s ) .  160  Fig.43. The time course of ATP on M A P K activation in human granulosa-luteal cells (hGLCs). Fig.44. M A P kinase activity in hGLCs detected using a M A P kinase assay kit.  161 162  Fig.45. The effect of suramin, a P2 purinoceptor inhibitor, on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs).  164  Fig.46. The effect of pertussis toxin (PTX), a G i protein inhibitor, on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs).  165  Fig.47. The effect of neomycin, a PLC inhibitor, on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs).  168  Fig.48. The effect of staurosporin, a P K C inhibitor (PKCI), on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs).  169  Fig.49. The effect of PD98059, a M E K inhibitor (MEKI), on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs).  170  Fig.50. The effect of ATP on intracellular c A M P production in human granulosa-luteal cells ( h G L C s ) .  172  Fig.51. The effect of M A P K on progesterone production in human granulosa-luteal cells (hGLCs).  173  Fig.52. A . The effect of h C G on M A P K activation in human granulosa-luteal cells (hGLCs).  175  B. The effect of PD98059, a M E K inhibitor (MEKI), on hCG-induced progesterone production in human granulosa-luteal cells (hGLCs).  175  Fig.53. The effect of M E K I in inhibitory effect of ATP on hCG-stimulated c A M P production. Fig.54. Proposed intracellular signaling cascades of ATP in hGLCs.  176 182  LIST OF ABBREVIATIONS  ATP 33-HSD bp °C Ca cAMP cDNA Ci cpm DAG DDT DEPC DMEM dNTP DNA DNase EDTA E2 EGF ELISA ER ERK1/2 FBS FSH g (as in xg) GDP GnRH G-protein GTP h hCG hGLCs IP3 IU IVF-ET JNK/SAPK Kb kDa LH i +  P MAPK  Adenosine 5'-triphosphate 3(3-hydroxysteroid dehydrogenase Base pairs Degree Celsius Calcium Cyclic adenosine monophosphate Complementary deoxyribonucleic acid Curie Counts per minute Diacylglycerol Dithiothreitol Diethylpyrocarbonate Dulbecco's Modified Eagle Medium Deoxynucleoside triphosphate Deoxyribonucleic acid Deoxyribonuclease Ethylene diaminetetraacetic acid 17p-estradiol Epidermal growth factor Enzyme-linked immunosorbant assay Endoplasmic reticulum Extracellular signal-regulated kinase 1/2 Fetal bovine serum Follicle stimulating hormone Acceleration of gravity Guanosine diphosphate Gonadotropin-releasing hormone GTP-binding protein Guanosine triphosphate Hour Human chorionic gonadotropin Human granulosa-luteal cells Inositol 1,4,5-trisphosphate International unit In vitro fertilization-embryo transfer c-jun terminal kinase/stress-activated protein kinase Kilobases Kilodaltons Luteinizating hormone Micro Mitogen-activated protein kinase xi  MAPKKs(=MKK) MAPKKKs MAPKKKKs M E K 1/2 ml min mRNA MW n (as in nM) P2UR P4 P450arom P450cl7 P450scc PAGE PBS PBS-G PCR PDK-1 PGF2a PIP2 PIP3 PKA PKC aPKC cPKC nPKC PLA PLC PLD PMA PMSF PTX RIA rpm RT RT-PCR sec SD SE SDS StAR Taq TE TEMED  M A P K kinases M A P K K kinases M A P K K K kinases M A P K / E R K kinase 1/2 Milliliter Minute Messenger ribonucleic acid Molecular weight NM P2U purinergic receptor Progesterone Cytochrome P450 aromatase Cytochrome P450 17alpha hydroxylase/ C17-20 lyase Cytochrome 450 side chain cleavage enzyme Polyacrylamide gel electrophoresis Phosphate buffered saline Phosphate buffered saline-gelatin Polymerase chain reaction Phosphoinositide-dependent-kinase-1 Prostaglandin F2a Phosphatidylinositol 4,5-bisphosphate Phosphatidylinositol 3,4,5-triphosphate Protein kinase A Protein kinase C Atypical protein kinase C Conventional protein kinase C Novel protein kinase C Phospholipase A Phospholipase C Phospholipase D Phorbol 12-myristate 13-acetate Phenylmethylsulfonyl fluoride Pertussis toxin Radioimmunoassay Revolutions per minute Room temperature Reverse transcription polymerase chain reaction Second Standard deviation Standard error Sodium dodecyl sulphate Steroidogenic acute regulatory protein Thermus aquaticus, source of a D N A polymerase Tris-EDTA N , N , N ' , N ' -tetramethylethlenediamine xii  Tris v/v w/v  Tris(hydroxyl methyl) aminomethane Volume per volume Weight per volume  xiii  PUBLICATION LIST PAPERS IN REFERRED JOURNALS 1.  Tai C J , Kang SK, Cheng KW, Choi K - C , S. Nathwani PS and Leung P C K 2000 Expression and regulation of P2U-purinergic receptor in human granulosa-luteal cells. J Clin Endocrinol Metab 85: 1591-1597  2.  Tai CJ, Kang SK, and Leung P C K 2001 Adenosine triphosphate-evoked cytosolic calcium oscillations in human granulosa-luteal cells: role of protein kinase C. J Clin Endocrinol Metab 86: 773-777  3.  Tai CJ, Kang SK, Choi K - C , Tzeng CR, and Leung P C K 2001 Antigonadotropic action of A T P in human granulosa-luteal cells: involvement of P K C a . J Clin Endocrinol Metab (In press)  4.  Tai CJ, Kang SK, Tzeng C R and Leung P C K 2001 ATP activates mitogen-activated protein kinase in human granulosa-luteal cells. Endocrinology (In press)  5.  Tai C J , Kang SK, Choi K - C , Tzeng CR, and Leung P C K 2001 Role of mitogen-activated  protein  kinase  in  prostaglandin  F2a action  in  human  granulosa-luteal cells. J Clin Endocrinol Metab 86: 375-380 6.  Kang SK, Tai CJ, Cheng K W Leung P C K 2000 Gonadotropin-releasing hormone activates mitogen-activated protein kinase in human ovarian and placental cells. M o l Cell Endocrinol 170: 143-151  7.  Kang SK, Tai CJ, Nathwani PS, Leung P C K 2001 Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid in human granulosa-luteal cells. Endocrinology 142: 182-192  8.  Kang SK, Tai CJ, Nathwani PS, Choi K - C , Leung P C K 2001 Stimulation of xiv  mitogen-activated protein kinase by gonadotropin-releasing hormone in human granulosa-uteal cells. Endocrinology (In press) 9.  Kang SK, Choi K - C , Tai CJ, Auersperg N , Leung P C K 2001 Estradiol regulates gonadotropin-releasing hormone (GnRH) and its receptor gene expression and modulates the growth inhibitory effects of GnRH in human ovarian surface epithelial and ovarian cancer cells. Endocrinology (In press)  10. Woo M M M , Tai CJ, Kang SK, Nathwani PS, Pang SF, Leung P C K 2001 Direct action of melatonin in human granulosa-luteal cells. J Clin Endocrinol Metab, submitted 11. Choi K - C , Kang SK, Tai CJ, Auersperg N , Leung P C K 2001 The regulation of apoptosis by activin and TGF-P in normal, early neoplastic and tumorigenic ovarian surface epithelium (OSE). J Clin Endocrinol Metab (In press) 12. Choi K - C , Kang SK, Tai CJ, Auersperg N , Leung P C K 2001 Estradiol up-regulates anti-gonadotropic  bcl-2 mRNA  and protein in tumorigenic  ovarian  surface  epithelium (OSE). Endocrinology (In press) 13. Choi K - C , Kang SK, Tai CJ, Auersperg N , Leung P C K 2001 Follicle-stimulating hormone activates Mitogen-activated protein kinase in normal and neoplastic ovarian surface epithelial cells. Endocrinology, submitted  ABSTRACTS AND PRESENTATIONS 1.  Tai CJ, Cheng KW, Nathwani PS, Leung P C K 1999 The role of protein kinase C in regulating  ATP-evoked intracellular calcium oscillations in cultured  XV  human  granulosa-luteal cells. 32  nd  Annual meeting of the Society for the Study of  Reproduction. Pullman, Washington, U S A 60 (1): 131 2.  Tai CJ, Kang SK, Cheng K W , Choi K - C , Nathwani PS, Leung P C K 1999 Expression  of purinergic  receptor  (P2U) in human  granulosa-luteal  cells.  A S R M / C F A S Conjoint Annual Meeting, Toronto, Ontario, Canada, Sep. 25-30, 1999, S-47 3.  Tai CJ, Kang SK, Choi K - C , Leung P C K . 2000 Prostaglandin F2a activates mitogen-activated protein kinase in human granulosa-luteal cells. A S R M Annual Meeting, San Diego, California, USA, Oct. 21-26, 2000, Fertil Steril 74: 3S: S2  4.  Choi K - C , Kang SK, Tai CJ, Auersperg N , Leung P C K 2000 Endocrine influences on normal and neoplastic ovarian surface epithelium (OSE) cell growth. A S R M Annual Meeting, San Diego, California, USA, Oct. 21-26, 2000, Fertil Steril 74: 3S: S5  5.  Cheng K W , Kang SK, Tai CJ, Nathwani PS, Leung P C K 1999 Transcriptional regulation of human gonadotropin-releasing hormone receptor (GnRHR) in human placental cells. 32  nd  Annual meeting of the Society for the Study of Reproduction.  Pullman, Washington, U S A 60(1): P-2 6.  Choi K - C , Kang SK, Nathwani PS, Cheng KW, Tai CJ, Auersperg N , Leung P C K 1999 The expression levels of activin/inhibin subunits and activin receptors: autocrine functions of activin in ovarian surface epithelium and ovarian cancer cells. Health Scicences Research Forum, University of British Columbia, Vancouver, Canada  xvi  ACKNOWLEDGEMENTS I would like to express my most sincere appreciation to my supervisor Dr. Peter C.K. Leung. Only under his guidance and assistance was this thesis made possible. I am also extremely indebted to my supervisory committee members, Dr. Rurak, Dr. Auersperg, Dr. Buchan and Dr. Fluker. Sincerely, I would like to express my gratitude to all my colleagues, K.W. Cheng, S.K. Kang, K - C Choi, P.S. Nathwani, M . Woo, CS Chou and X M Zhu, and members of Dr. Auersperg's lab for their invaluable advice and whole-hearted support. Grateful acknowledgement must be extended to Dr. C R . Tzeng, Taipei Medical University Hospital, for introducing me into this field of reproductive sciences. I sincerely thank the staff of the Genesis Fertility Centre, Vancouver, Canada for the generous provision of human granulosa-luteal cells, which are indispensable for this study. I also thank the Medical Research Council of Canada and the Canadian Institutes of Health Research for supporting my research. A special note of appreciation is extended to my parents and parent-in-laws for their countless support and backing throughout my study. Over the past several years in Canada, I have been blessed to have my dear wife and beloved daughters accompanying me, with their smiling faces, and providing me the source of strength and motivation required for fulfilling this study. Thank you, Lynn, Felicia and Eunice.  xvii  PARTI  BACKGROUND  1.1 INTRODUCTION  Granulosa  cells  exhibit  increasing  secretory  levels  of  estrogen  during  folliculogenesis. After the endogenous L H surge or subsequent to the administration of L H / H C G , granulosa cells of the Graafian follicle alter their main steroidogenic secretory products from estrogens to progestin [Charming et al., 1980]. Evidence suggests that growth factors [Pully and Marone, 1986; Veldhuis and Gwynne, 1989; Kamada et al., 1992], cytokines [Fukuoka et al, 1988], and vasoconstrictive peptides [Pucell et al., 1991] play a role in regulating the differentiation and proliferation of ovarian granulosa cells in an autocrine and / or paracrine manner. In addition, adrenergic and cholinergic nerves innervating the ovary may also be involved in regulating steroidogenesis [Mohsin and Pennefather,  1979;  Burden  and  Lawrence,  1978;  Stefenson  et  al,  1981].  Neurotransmitters such as epinephrine and norepinephrine have been shown to stimulate progesterone (P4) secretion in human granulosa cells via interaction with adrenergic receptors [Webley et al., 1988].  Intracellularly, ATP is a major energy source for diverse reactions and does not cross the plasma membrane of viable cells. A T P and its metabolites are released from cells such as platelets and purinergic nerves, and are coreleased with neurotransmittter granules from autocrine nerves by exocytosis [Gordon, 1986, Burnstock, 1977]. Extracellular A T P binds to a G protein-coupled P2 purinoceptor that activates 1  phospholipase C (PLC) and phosphatidylinositide hydrolysis, generating diacylglycerols and inositol 1,4,5-triphosphate, which stimulate protein kinase C (PKC) and intracellular calcium mobilization, respectively [Berridge, 1984]. Thereafter, ATP may participate in various types of physiological responses including secretion, change in membrane potential, cell proliferation, platelet aggregation, neurotransmission, cardiac function, and muscle contraction [el-Moatassim et al, 1992; Burnstock, 1990].  Pharmacologically,  the  P2U purinoceptor  has  been  identified  in  human  granulosa-luteal cells (hGLCs) using microspectrofluorimetry [Lee et al, 1996; Kamada et al, 1994]. Furthermore, A T P has also been shown to regulate basal progesterone and estradiol secretion from luteinized human granulosa cells [Kamada et al, 1994]. It is tempting to speculate that the coreleased A T P from nerve endings innervating the ovary may play a role in regulating ovarian function. The following studies are designed to examine the expression and regulation of A T P receptor, the signaling pathway, and functional role of ATP in hGLCs.  2  1.2 Hormonal regulation ofthe ovary  1.2.1  Hypothalamo-pituitary-gonadal axis (Fig. 1) The hypothalamo-pituitary-gonadal axis plays an important role in the regulation of  sexual maturation and reproductive functions.  Gonadotropin-releasing hormone (GnRH),  synthesized in the hypothalamic neurons and secreted in a pulsatile manner, acts as a key regulator of the hormonal cascade [Braden and Conn, 1993; Conn, 1994]. Through the hypothalamo-hypophyseal portal system, GnRH is transported to the anterior lobe of the pituitary gland, where it stimulates the release of the gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH) [Braden and Conn, 1993; Leung and Steele, 1992]. F S H and L H then regulate the production of steroid hormones from the ovary.  Steroid hormones such as progesterone and estrogen negatively or positively  regulate the secretion of GnRH and/or gonadotropins.  3  4  1.2.2 Hormonal regulation of menstrual cycle The understanding of the regulation of the ovarian cycle can be divided into three phases: the follicular phase, ovulation and the luteal phase. During the follicular phase a series of events takes place to ensure the recruitment and selection of a dominant follicle destined for ovulation. The reduced steroidogenesis and inhibin A secretion during the luteal phase give rise to an increase of FSH level, beginning several days before menses [Vermesh and Kletzky, 1987; Welt et al., 1997].  Aromatization, which converts  androgens to estrogens, is induced or activated through the action of FSH.  Thus, F S H  initiates steroidogenesis in granulosa cells and stimulates granulosa cell growth [Yong et al., 1992].  Under the synergistic effect of estrogen and F S H , there is an increased  production of follicular fluid, which accumulates in the intercellular spaces between granulosa cells. In the presence of FSH, estrogen becomes the dominant substance in the follicular fluid, whereas androgens predominate in the absence of F S H [McNatty et al., 1979; McNatty et al., 1980].  The two-cell/ two-gonadotropin system is an integrative process, between granulosa and theca cells, required for the production of steroid hormones in the follicular phase. The aromatase activity of granulosa cells is much higher than that of theca cells. In human preantral and antral follicles (Fig. 2), L H receptors are present only on theca cells and F S H receptors are present only on granulosa cells. Stimulated by L H , theca cells produce androgens which will pass through basement membrane and enter granulosa cells.  Androgens are converted to estrogens under the effect of FSH-induced  aromatization [Kobayashi et al., 1990; Yamoto et al., 1992]. The successful conversion to an estrogen dominant follicle marks the selection of a follicle destined to ovulate, and 5  only a single follicle succeeds.  The negative feedback of estrogen on F S H secretion  from the pituitary gland inhibits the development of all but the dominant follicle [Goodman and Hodgen, 1983]. The combination of FSH and a high local estrogen level in the dominant follicle provides the optimal environment for L H receptor development [Jia X C and Hsueh AJW, 1984; Kessel et al., 1985]. During the late follicular phase, estrogens rise rapidly to reach a peak approximately 24-36 hours prior to ovulation. The high level of estrogens positively feedback to induce the L H surge from anterior pituitary gland [Pauerstein et al., 1978; Fritz et al., 1992].  The L H surge initiates the continuation of meiosis in the oocyte, luteinization of granulosa cells, the rise of progesterone production, expansion of the cumulus and the synthesis of prostaglandins, which are required for ovulation [Tedeschi et al., 1992; O'Grady et al., 1972; Killick et al., 1987; Miyazaki et al., 1991].  F S H , L H and  progesterone stimulate the activity of proteolytic enzymes responsible, together with prostaglandins, for digestion and rupture of the follicular wall [Yoshimura et al., 1987; Peng etal., 1992].  Normal luteal function requires optimal preovulatory follicular development and continuous L H support.  Insufficient F S H during the follicular phase is related with  lower preovulatory estrogens level, reduced midluteal progesterone production and a decreased luteal cell mass [Smith et al., 1986].  Progesterone exerts actions both  centrally and locally to suppress the growth of any new follicle [diZerega and Hodgen, 1982; Gougeon and Lefevre, 1984]. Regression of the corpus luteum may be the action of luteolysis, mediated by estrogen, prostaglandin and endothelin-1 [Girsh et al., 1996a; 6  Girsh et al., 1996b; Auletta and Flint, 1988].  Fig. 2 The structure of preovulatory follicle.  7  1.2.3 Human granulosa cells After ovulation, the ruptured follicle reorganizes to become the corpus luteum [Drews, 1995]. Collectively, granulosa-lutein cells, theca interna cells, and the cells of the microvasculature interact with one another to give rise to this transient endocrine organ [Drews, 1995]. Concurrently, the granulosa cells begin to undergo the process of luteinization, which is characterized by both morphologic and functional differentiation [Amsterdam and Rotmensch, 1987; Zeleznik, 1991]. expressed  histologically by an increase  in cell  Morphological luteinization is size, and ultrastructurally by  well-developed Golgi apparatus, endoplasmic reticulum and mitochondria [Rotmensch et al., 1986]. Functionally, luteinization is associated with an increase in the production and secretion of progesterone [Schipper et al, 1993]. The differential expression of the enzymes involved in the production of gonadal steroids is regulated by the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). If implantation does not occur, the corpus luteum regresses rapidly, allowing resumption of the next cycle of folliculogenesis [Drews, 1995].  1.2.4 Steroidogenesis in the ovary The overall steroid biosynthesis pathway shown here is based on the findings of Kenneth J Ryan et al. [Ryan, 1959; Ryan and Smith, 1965]. The human ovary produces all 3 classes of sex steroids: estrogens, progestins and androgens.  The gonadotropic hormones from the anterior pituitary bind to G-protein coupled receptors, activate adenylate cyclase and increase intracellular c A M P level. Cyclic A M P activates protein kinase A and leads to gene transcription that encodes the steroidogenic 8  enzymes and accessory proteins.  Additionally, c A M P stimulates the hydrolysis of  cholesterol esters, resulting in the release of free cholesterol [Stocco, 1999]. Most ofthe cholesterol used for steroid synthesis is derived from the mobilization and transport of intracellular stores [Liscum and Dahl, 1992; Reaven et al., 1995].  Indeed, the  rate-limiting step in steroidogenesis is the transfer of cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane where fully active cytochrome P450 side chain cleavage enzyme (P450scc) waits for the substrate.  In  response to hormone stimulation, steroidogenic acute regulatory (StAR) protein was shown to stimulate the translocation of cholesterol from the sterol-rich outer mitochondrial membranes to the cholesterol-poor inner mitochondrial membrane [Stocco, 1998] (Fig. 3).  During steroidogenesis, the number of carbon atoms in cholesterol is always reduced, never increased (Fig. 4). Thus, the biosynthesis begins with the cleavage of a 6-carbon side-chain of the 27-carbon cholesterol molecule to form the first steroid, the 21-carbon-containing molecule pregnenolone.  This reaction is catalyzed by P450scc,  which is located on the matrix side of the inner mitochondrial membrane [Simpson and Boyd, 1966, 1967; Yago and Ichii, 1969; Churchill and Kimura, 1979].  Once  pregnenolone is formed, it may be metabolized within the mitochondria to progesterone by the enzyme 3P-hydroxysteroid dehydrogenase (3[3-HSD) [Sulimovici et al., 1973; Chapman et al., 1992; Cherradi et al., 1994, 1995, 1997; Sauer et al., 1994] (Fig. 3).  Progesterone is then hydroxylated, and the side chain is cleaved subsequently by  9  cytochrome P450 17alpha hydroxylase/C 17-20-lyase (P450cl7) to form androstenedione (19 carbons, C-19). Androstenedione (ketone) may be reduced to testosterone (hydroxyl) by  the  17(3-hydroxysteroid  dehydrogenase  reaction.  Both  C-19  steroids  (androstenedione and testosterone) are rapidly converted to the corresponding C-18 phenolic steroid estrogens (estrone and estradiol) by aromatization reaction.  This  process involves hydroxylation of the 19-methyl group, followed by oxidation, loss of the 19-carbon, and ring A aromatization (dehydrogenation).  Aromatization is mediated by  cytochrome P450 aromatase (P450arom) found in the endoplasmic reticulum [Simpson et. al., 1994].  10  Fig. 3 Hormone-stimulated steroidogenesis. StAR: steroidogenic acute regulatory protein P450scc: cytochrome P450 side chain cleavage enzyme 3(3-HSD: 3 P-hydroxy steroid dehydrogenase  11  Acetate Carbon Number  C27  Cholesterol  P450scc  C21  Pregnenolone  3-hydroxysteroid dehydrogenase  C21  Progesterone  P450cl7  C19  Androstenedione  Testosterone  P450arom  P450arom  C18  Estrone  Estradiol  Fig. 4 Steroidogenesis in the ovary. P450scc: cytochrome P450 side chain cleavage enzyme P450cl7: cytochrome P450 17alpha hydroxylase/C 17-20-lyase P450arom: cytochrome P450 aromatase  12  1.3 Innervation ofthe ovary According to Mitchell, the ovarian nerves originate from three sources [Mitchell, 1938]. The superior ovarian nerves come from the intermesenteric nerves and from the renal plexus and descend along the outer side of the ovarian blood vessels to the ovary. The middle ovarian nerves from the superior hypogastric plexus (pre-sacral nerve) or from the hypogastric nerve are usually paired and also supply the fallopian tube. The inferior ovarian nerves may be three or four in number and arise from the inferior hypogastric plexus or the lower end of the hypogastric nerve. About half of the ovarian nerves are post-ganglionic with their cell bodies in the inferior mesenteric or spinal ganglia. The remainders are preganglionic and synapse in ganglia located in or near the ovaries and oviduct [Marshall, 1970; Langley and Anderson, 1895a, 1895b; Brundin, 1965].  The presence of intraovarian nerves is reported on the basis of histological studies [ Frankenhauser, 1867; Waldeyer, 1870]. Thereafter, many studies have been performed to examine the pattern of the intrinsic innervation of the ovary. Not only the vascular bed but also other components, such as the follicle or its wall, are innervated by autonomic nerves [Guttmacher and Guttmacher, 1921]. Ovaries from cow, sheep, cat and guinea pig are richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in different stages of development.  In the follicular wall  the nerve terminals are located in the theca externa, running parallelly to the follicular surface.  Numerous adrenergic terminals also surround ovarian blood vessels.  The  adrenergic innervation is of intermediary density in the human ovary as well as the pig, 13  dog and cat ovaries. The cholinergic innervation is generally less developed, but has the same distribution as the adrenergic system around blood vessles and in the ovarian stroma, including follicular walls [Stefenson et al., 1981]. Some of the locations of adrenergic nerves in the ovary in all species investigated correspond to the distribution of the vascular system, but varicose nerve terminals are also found to run along various structures of the ovarian parenchyma [Owman et al., 1967].  The findings of a well-developed autonomic innervation of the ovary and its follicles, together with the notion that sympathetic activation contracts the follicular wall and increases intrafollicular pressure, have led to an hypothesis that peripheral neurogenic factors may also be involved in regulating follicular development, ovulation and ovarian function [Owman et al., 1975, 1979].  Evidence shows that neurotransmitters steroidogenesis.  play a role in regulating ovarian  Catacholamines have been demonstrated to modulate basal and  gonadotropin-stimulated steroid secretion in human granulosa cells [Webley et al., 1988; Papenfu(3 et al., 1993; Bodis et al., 1993]. Further, cholingeric effects are demonstrated using acetylcholine and carbachol in human granulosa cells, suggesting that cholingeric neurotransmission may have a physiological significance in the ovary [Bodis et al., 1993].  Considering that the ovary is a well-innervated organ, co-release of ATP and noradrenaline from neurons suggests a potential role of A T P in regulating ovarian  14  functions [von Kugelgen et al., 1994].  1.4 Adenosine 5'-triphosphate (ATP) and purinergic receptor  1.4.1 Chemical structure of ATP The molecule of ATP contains adenine, ribose and triphosphate [Albert et al, 1994] (Fig. 5).  15  NH2  3'  Phosphate  2'  Ribose sugar  Adenine  Fig. 5 Chemical structure of Adenosine Triphosphate (ATP).  16  1.4.2 The sources of ATP Intracellularly, A T P is produced during glycolysis, the citric acid cycle and via the electron transport chain and oxidative phosphorylation. Two molecules of A T P are formed from per molecule of glucose under anaerobic conditions.  In contrast, 38  molecules of A T P are formed per molecule of glucose under aerobic conditions [Mayes, 1993].  The sources of extracellular ATP are mainly neuronal in origin; either released from purinergic nerve endings, or coreleased with traditional neurotransmitter granules such as acetylcholine and noradenaline during neurotransmission [Morel and Meunier, 1981; Poisner and Trifaro, 1982]. sympathetic  nervous  The concentration of A T P in adrenergic granules of the  system  and  in  acetylcholine-containing  granules  of  the  parasympathetic nervous system can be as high as 150mM [Poisner and Trifaro, 1982]. Exocytotic release of ATP has also been found in nonneuronal cells, including platelets [Born and Kratzer, 1984], adrenal chromaffin cells [Cena and Rojas, 1990], mast cells [Osipchuk and Cahalan, 1992], and basophilic leukocytes [Osipchuk and Cahalan, 1992]. Another way to release cytosolic A T P is via intrinsic plasma membrane channels in the absence of irreversible cytolysis. The multidrug resistance (mdrl) gene product (or P glycoprotein) can function as a channel for ATP [Abraham et al, 1993].  Overexpression  of P glycoprotein in transfected Chinese hamster ovary cells was accompanied by a threefold increase in the steady-state release of ATP to the extracellular medium [Abraham et al, 1993].  Although A T P is present in millimolar level in the cytosol of all cell types, 17  extracellular levels of the nucleotide are normally maintained at extremely low levels by ubiquitous ecto-ATPase and ectophosphatase [Dubyak and el-Moatassim, 1993]. Extracellular ATP is quickly degraded by the ubiquitous ecto-ATPase and ecto-ATP diphosphohydrolase (Dombrowski et al., 1998; Dubyak and el-Moatassim, 1993; Zimmermann et al., 1998), there is no in vivo report about physiologically relevant time or condition with the release of ATP.  Considering that ATP is co-released with  neurotransmitter such as norepinephrine and acetylcholine from nerve endings (Gordon, 1986), and stress or acute exercise may induce the release of norepinephrine (De Cree et al., 1997; Paredes et al., 1998), it leads us to speculate that stress or exercise may induce the release of ATP and affect the ovarian function.  1.4.3 Biological roles of ATP Intracellular A T P plays fundamental and ubiquitous roles in energy metabolism, nucleic acid synthesis, and enzyme regulation [Albert et al, 1994]. A T P is therefore called the energy currency.  Biological responses to extracellular ATP have been shown in virtually every organ and /or tissue system.  Due to its ability to modulate contractility of most vascular  smooth muscles and cardiac myocytes, extracellular ATP can exert significant effects on cardiovascular function and regional blood flow [Dubyak and el-Moatssim, 1993; Olsson and Pearson, 1990].  18  In the nervous system, extracellular ATP can potentiate acetylcholine release at the neuromuscular synapses that develop during in vitro culture of Xenopus-derived neurons and muscle cells [Fu and Poo, 1991].  In the lung, A T P is a particularly efficacious agonist for stimulating surfactant release from type II alveolar pneumocytes [Rice et al, 1990].  In vitro studies with  tissue-cultured lymphocytes suggest that extracellular A T P might modulate D N A synthesis, cell-mediated killing, and apoptosis [Ikehara et al, 1981; Fillipini et al, 1990; Zheng etal., 1991].  In addition, extracellular A T P had been shown to act as a secretagogue in endocrine tissues or organs.  A T P stimulates the secretion of pancreatic hormones (insulin and  glucagon) from intact pancreas [Bertrand et al., 1990]. In porcine thyroid cells, A T P induces production of hydrogen peroxide, which is an essential process of iodide organization and a key reaction of Thyroid Stimulating Hormone (TSH)-induced thyroid hormone synthesis [Nakamura and Ohtaki, 1990].  Steroidogenesis in adrenocortical  fasciculata cells is stimulated when P2 receptors are occupied [Kawamura et al., 1991].  In the reproductive system, extracellular A T P has been shown to activate contraction in intact myometrium [Osa and Maruta, 1987]. In addition, amnion cells isolated from human placenta express A T P receptors that are coupled to inositol phospholipid breakdown and Ca  mobilization [Vander Kooy et al, 1989].  A T P can trigger the  acrosome reaction in vitro in human sperm [Foresta et al, 1992].  After binding to  P2-purinergic receptors, A T P increases the secretion of testosterone in rat Leydig cells 19  [Foresta et al., 1996].  Furthermore, extracellular A T P has been shown to regulate  steroidogenesis in human granulosa cells and porcine granulosa cells [Kamada et a l , 1994].  1.4.4 Purinergic receptor  1.4.4.1 Introduction The term "purinergic" was introduced by Burnstock [Burnstock, 1971] to represent nerves that use ATP or other purine nucleotides as transmitters (Fig. 6). The notion that one type of nerve may use ATP or a related purine as the transmitter is supported by an electrophysiological observation that a response to neuronal stimulation is demonstrated following blockade of adrenergic and cholinergic nerve transmission [Burnstock, 1981]. Evidence has been accumulated that ATP is the principal active substance released from purinergic nerves.  Such evidence includes (1) synthesis and storage of A T P in nerves;  (2) release of A T P from nerve endings while they are stimulated; (3) action of exogenous ATP close to the responses to non-adrenergic, non-cholinergic nerve stimulation; (4) the presence of Mg -activated ATPase, 5'-nucleotidase and adenosine deaminase, enzymes 2+  which inactivate ATP; (5) antagonists block the response to exogenous ATP; and (6) the identification of purinergic receptors [Burnstock, 1977; Su, 1983].  20  Fig. 6 Schematic representation of synthesis, storage, release and inactivation of ATP in purinergic nerve. (Modified from Burnstock, 1972)  21  1.4.4.2 Classification The purinergic receptors have been classified as the PI receptors and P2 receptors. Pharmacologically, the PI receptors have a high affinity for extracellular adenosine and A M P (Adenosine > A M P > A D P > ATP), whereas P2 receptors have high affinity for ATP and A D P (ATP > A D P > A M P > Adenosine) [Gordon, 1986; el-Moatassim et al, 1992; Burnstock, 1990].  The PI purinergic receptors have been subclassified into A l , A2a, A2b and A3 receptors on the basis of different effects of adenosine on adenylate cyclase [Gordon, 1986; el-Moatassim et al, 1992; Burnstock, 1990; Fredholm et al., 1994].  Six subtypes  of P2 purinergic receptors, P2X, P2Y, P2Z, P2U, P2T and P2D have been identified in pharmacological and functional studies and supported by cloning data [Fredholm et al., 1994]. These include four A T P receptor types, termed P2X, P2Y, P2Z, and P2U. The ADP-selective receptor expressed in platelets has been denoted as the P2T (for thrombocyte) purinergic receptor [Dubyak and el-Moatassim, 1993]. P2D purinoceptors can be activated by the diadenosine polyphosphates, Ap4A and Ap5A (diadenosine tetraphosphate and diadenosine pentaphosphate) [Miras-Portugal, 1996].  Functionally, P2X-type receptors act as ligand-gated ion channels.  P2Y-receptors  and P2T function as G-protein-coupled calcium mobilizing A T P receptors, and P2Z receptors are associated with ATP-induced nonselective pore formation [Dubyak and el-Moatassim, 1993; Fredholm et al., 1994]. UTP is a particularly potent and efficacious agonist for P2U receptors.  P2U also acts via a G-protein-coupled system; i.e., P2U  receptors are functionally similar to but pharmacologically distinct from the P2Y-receptor 22  class [Seifert and Schulz, 1989] (Table 1).  Table 1 Pharmacological classification of P2-purinergic receptors Subtype  Agonist Selective  Antagonist  Suramin  Signal Transduction  Intrinsic ion channel  P2X  AMP-C-PP=ATP=ADP>AMP  P2Y  2-MeSATP>ATP>ADP»=UTP  G-protein activation  P2Z  BzATP>ATP»ADP,AMP  Intrinsic channel/pore  P2U  UTP>ATP=ATPyS>ADP  P2T  2-MeSADP>ADP  Intrinsic ion channel ?  P2D  Ap4A>ADPpS>Ap5A>2-MeSATP  increase calcium  Suramin  2-MeSATP: 2-Methylthio A T P BzATP: Benzoylbenzoyl-ATP ATPyS: Adenosine 5'-0-(3-thiotriphosphate) 2-MeSADP: 2-Methylthio A D P ADPpS: Adenosine 5'-0-(3-thiodiphosphate)  23  G-protein activation  1.4.4.3 The structure of P2-purinergic receptor (G protein-coupled receptors) Reported cDNA and amino acid sequences of P2U receptors [Lustig et al., 1993; Parr et al., 1994] and P2Y receptors [Webb et al, 1993; Filtz et al., 1994] have shown that these receptors are members of the superfamily of G protein-coupled receptors.  This  superfamily contains seven putative transmembrane segments, with their amino termini located on the extracellular side and their carboxy termini located on the intracelllar side of the cell membrane [Harden et al., 1995] (Fig. 7).  The P2-purinoreceptors are the only known G protein-coupled receptors that have a histidine residue at the cytoplasmic region of the third transmembrane segment, instead of the usual aspartic acid.  This residue may be involved in transmission of ligand  binding to the activation of the G protein in this G protein-coupled receptors [Fraser et al., 1988].  24  NH2 '  '  AfP-binding site  Extracellular  m  A  Membrane  >./ W W W \J \J \  Intracellular COOH  Fig. 7 Scheme of the P2U purinoceptor. P2U purinoceptor contains seven transmembrane domains with an extracellular N-terminus and intracellular C-terminus. Stars (*) denote the locations of potential phosphorylation sites by protein kinases such P K A or P K C . A near consensus ATP-binding site is located close to the N-terminus.  25  1.4.4.4 Cloning of P2 purinoceptors The c D N A of P2Y and P2U purinoceptors have been cloned. Hydropathicity analyses of the predicted amino acid structures reveal that these receptors belong to the superfamily of seven transmembrane domain (TM), G-protein-coupled receptors. P2Y purinoceptors cloned from chick and turkey have about 30% homology with cloned murine and human P2U purinoceptors, and they share less identity with cloned PI purinoceptors [Webb et al., 1993; Filtz et al., 1994; Lustig et al., 1993; Par et al., 1994]. In addition, the cloning, structural prediction and functional studies of rat P2X purinoceptors confirm that these receptors  constitute  another  subfamily of P2  purinoceptors, the ligand-gated ion channel receptors [Valera et al., 1994; Brake et al., 1994].  Functional expression of cloned murine P2U purinoceptors in K562 human erythroleukemic cells has identified the P2U purinoceptors as a 53 kDa plasma membrane protein [Erb et al, 1993]. Considering that the predicted amino acid sequence of P2U purinoceptor indicates a molecular weight of approximately 42 kDa, this receptor appears to be glycosylated on one or both Asn (Asparagine) residues in its N-terminal extracellular domain [Boarder et al., 1995].  Sequence comparisons among P2Y and P2U purinoceptors with the adenosine A l receptor reveal that several positively charged amino acid residues in TM3, TM6 and TM7 are present in the two P2 purinoceptors but not in the adenosine A l receptor, suggesting that these positively charged residues are associated with the binding of the 26  negatively charged phosphate moieties of P2 purinoceptor agonists [Erb et al., 1995]. This suggestion is supported by site-directed mutagenesis of murine P2U purinoceptor cDNA followed by functional expression of the mutated receptors [Erb et al., 1995]. Neutralization of the positively charged amino acids Arg 262 and Arg 292 by substitution with Leu markedly reduced nucleotide potencies, indicating an inhibition of the binding of the negatively charged phosphates of P2U purinoceptor agonists.  Northern blot analysis demonstrates the existence of P2Y purinoceptor mRNA in chicken brain, spinal cord, gastrointestinal tract, spleen and leg muscle [Webb, 1993]. P2U purinoceptor mRNA is presented in mouse spleen, testis, kidney, liver, lung, heart and brain [Lustig et al., 1993] and in human heart, liver, lung, kidney, placenta and skeletal muscle [Parr et al., 1994].  1.4.5 Clinical application and development As an extracellular signaling molecule, ATP binds to P2 receptor, a G-protein coupled receptor, to regulate physiological functions. While eight possible members of the P2X receptor and more than 11 P2Y receptors have been reported, an array of studies are undergoing to explore the possible clinical applications [Burnstock et al., 1998; Williams, 1999; Willaims and Jarvis, 2000].  ATP is able to stimulate pancreatic insulin release via a glucose-dependent mechanism through activation of a P2Y receptor [Loubatrieres-Mariani et al., 1997]. Based on these studies, it has been suggested that a P2Y receptor agonist may improve 27  glucose tolerance and act as a potential antidiabetic drug.  Exogenous ATP and UTP are potent stimulants of chloride secretion in airway epithelium [Manson et al., 1993] and mucin glycoprotein production from epithelial goblet cells [Lethem et al., 1993] acting through the P2Y2 receptor. While both ATP and UTP are equipotent, UTP is being developed as an inhalation formulation to enhance mucociliary clearance, chloride secretion and sputum expectoration for the treatment of cystic fibrosis and chronic bronchitis [Connolly and Duley, 1999; Donaldson and Boucher, 1998].  Extracellular ATP may act as a trigger of apoptosis or programmed cell death [ Zheng et al., 1991].  The cytolytic action of ATP provides a potential approach to  treatment of cancers [Papaport, 1997].  28  1.5 Signal transduction via P2U-purinergic receptor (G protein- coupled system) After binding to a G-protein coupled P2 purinergic receptor, ATP stimulates phospholipase C. The resultant production of diacylglycerol and inositol triphosphate activate protein kinase C (PKC) and intracellular calcium [Ca2+]i mobilization, respectively.  1.5.1 GTP-binding proteins (G-proteins)  1.5.1.1 Composition and classification of G-proteins Hormones and neurotransmitters  interact with seven-transmembrane spanning  receptors to regulate cellular functions through G-proteins .[Post and Brown, 1996]. G-proteins are heterotrimeric in nature and are composed of a,p and y subunits encoded by distinct genes. Molecular cloning revealed the presence of at least 17 Goc isoforms, which can be divided into two subgroups- pertussis toxin (PTX) sensitive and P T X insensitive.  P T X sensitive G a proteins include G t l (t=transducin),  G t 2 , G i l (i=  inhibitory), Gi2, Gi3, Go, Ggust (gust= gustducin), G z , whereas G s (s= stimulatory), Golf  (olf= olfactory), Gq, G i l , G l 2 , G l 3 , G l 4 and G15/16 belong to PTX-insensitive group. In addition, five subtypes of p - subunit (Pi, P2, p3, P4 and P5) and 11 subtypes of y-subunit are identified [Downes and Gautam, 1999].  29  1.5.1.2 Activation of G-proteins Under basal conditions, G-proteins exist as heterotrimers with GDP bound to the a-subunit.  Union of agonist to G-protein coupled receptor induces the release of GDP,  the binding of GTP and the dissociation of GTP-Ga complex from G(3y dimer. GTP-Ga and G P Y then interact with their effectors such as ion channels, adenylate cyclase and phospholipase C (PLC). Endogenous GTPase hydrolyses GTP to G D P and leads to the re-association of GDP- G a and Gpy, thus terminating signal transduction [Lopez-Ilasac, 1998].  1.5.1.3 Expression of G-proteins in the human ovary It is demonstrated that human granulosa-luteal cells express PTX-insensitive G proteins, Gaq and G a l l , which are believed to exert biological function through PLC-p activation [Carrasco et al., 1997].  30  1.5.2 Phospholipase C  1.5.2.1 Introduction A n array of extracellular signals stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide specific phospholipase C (PLC) to yield two important second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. A large number of studies provided an insight into structures and regulation of P L C isozymes [Williams, 1999; Katan, 1998].  1.5.2.2 Classification and structure  Ten mammalian P L C isozymes have been reported and classified into three subtypes: P L C p l - 4 , P L C y l - 2 , PLC81-4.  The basic structure of P L C includes P H domain,  EF-hand, catalytic domain and C2 domain. It is thought that P H (pleckstrin homology) domain is associated with membrane binding [Lemmon et al., 1995].  The EF-hand  domain is a region of the enzyme that forms a flexible link to the P H domain, and there is no evidence that this domain of PLC binds calcium [Grobler and Hurley, 1998]. The catalytic  domain  hydrolyzes  the  substrate  through  a  two-step  mechanism:  phosphotransferase reaction and phosphohydrolase reaction [Williams, 1999]. The C2 domain, which is similar to the second conserved domain of protein kinase C, is present in all of the isozymes. The function of C2 domain, with three calcium-binding sites per domain, has been studied intensively. They suggest that the C2 domain may act as a calcium-dependent lipid membrane binding module [Nalefski and Falke, 1996] (Fig. 8).  J 1  PH domain  EF-hand  Catalytic domain  C2  c-terminal tail  PLCp  1  1  Ca 2+  substrate  SH2  SH2  SH  PH  PLCy Tyrosine kinase  PLCS  Fig. 8 The molecular domain of P L C isozymes. The p-isozymes have C-terminal extensions of about 400 residues. The y-class of isozyme has an insertion of about 500 residues in catalytic domain.  32  1.5.2.3 Activation of P L C (Fig. 9) The regulation of four mammalian PLCP isozymes (pi-(54) has been extensively studied. Activation of PLCp by many agonists such as histamine, vasopressin, GnRH and ATP is induced through G-protein coupled receptors, and the reaction is mediated by the a-subunit and/or by Py-subunits [Rhee and Bae, 1997; Morris and Scarlata, 1997; Jiang et al., 1997; K i m et al., 1997].  It is generally accepted that stimulation of PLCy involves receptor or non-receptor tyrosine kinases. Almost all polypeptide growth factor receptors, containing an intrinsic tyrosine kinase activity, have been linked to activation of PLCy. Some receptors such as T-cell antigen receptor and IgE receptor with no tyrosine kinase activity themselves are able to activate PLCy through non-receptor tyrosine kinase such as Src or Syk [Rhee and Bae, 1997, Kamat and Carpenter, 1997].  Regulation of the 5-class of isozymes has not been fully characterized.  Unlike  PLCP and PLCy, the activation of PLC5 is induced by an increase in calcium concentration in the absence of any other stimuli [Allen et al., 1997].  33  PDGF, EGF  Histamine, vasopressin, ATP, GnRH, PGF2a  Tyrosine kinase-linked receptor  G-protein-coupled receptor  LL  PIP  V  2  PLCp  PLCy  DAG  IP  I 3  IP3R  ER  Ca2+ Protein Kinase C  Fig. 9 Summary of two major receptor-mediated pathways for the formation of IP3 and D A G .  34  1.5.3 Calcium Calcium ions play a major role in transmitting extracellular stimuli into regulating a wide range of biological events such as muscle contraction, fertilization, neurotransmitter release, secretory processes, cell proliferation, gene expression and apoptosis [Evenaset al., 1998; Berridge, 1993].  1.5.3.1 C a  2 +  store and mobilization  In eukaryotic cells the cytoplasmic calcium concentration ranges from 100 n M in a resting (baseline) level to 1-10 uM, whereas the extracellular free C a around 1.2 m M .  The flow of C a  2 +  2 +  concentration is  can be regulated by voltage, receptor or  store-operated channels [Barritt, 1999].  Intracellular^, calcium is stored in the endoplasmic reticulum (ER) and/or sarcoplasmic reticulum (SR). Two major intracellular Ca -release channels in the E R 2+  and SR are identified- the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR).  Calcium release through IP3R is induced by IP3, while cyclic  ADP-ribose regulates the RyR [Mikoshiba, 1997]. Muscle tissue, especially skeletal and cardiac, provides the richest source of RyR, whereas IP3Rs are more evenly distributed.  At lease three isoforms of both IP3R and RyR have been characterized [Shoshan-Barmatz and Ashley, 1998; Joseph, 1996]. RyR is almost twice as large as the IP3R.  The skeletal R y R l and cardiac RyR2 share 66% homology. RyR3 has been  35  demonstrated in mink lung epithelium [Takeshima et al., 1989; Zorzato et al, 1990; Otsu et al, 1990].  Characteristics of IP3 receptors are shown in Table 2.  Various  pharmacological agents have been shown to modulate IP3R activity but not specific. For example, heparin acts as a competitive inhibitor, but it may also inhibit the generation of IP3. Caffeine is a potent inhibitor of the IP3R by abolishing IP3-induced calcium release, but it may activate RyRs [Parker and Ivorra, 1991; Missiaen et al., 1992].  Table 2 Characteristics of IP3 receptors Family % homology  Amino-acids Chromosomal  with type -I  Tissue distrubution  location  Type-I  -  2749  3p25-p26  Type-II  69%  2701  12pll  liver, lung, testis, spleen  Type-Ill  62%  2670  6p21  intestine, kidney, pancreatic islet  (Modified from Joseph, 1996)  36  Brain»peripheral  1.5.3.2 Intracellular Ca2 -binding proteins +  The main targets of C a  2 +  released from intracellular stores are Ca -binding 2+  proteins, many of which play important roles in regulating cellular functions.  These  proteins include calmodulin, troponin C, calpain and protein kinase C [James and Putney, 1998; Spyracopoulous et al., 1997; Blanchard et al., 1997; Essen et al., 1996].  1.5.3.3 Calcium channels The entry of calcium from extracellular source is regulated by calcium channels. There are three main channels classified on the basis of their regulatory mechanism including voltage-operated channels, receptor-operated channels and store-operated channels [Berridge, 1997]. Physiological and molecular studies have identified several different voltage-dependent calcium channels such as L - , N - , P-, Q-, R- and T-type [Jones, 1998].  Pharmacologically, L-type channels are highly sensitive to dihydropyridines  (DHPs), and N-type channels are blocked potently by co-conotoxin [Plummer et al., 1989]. P channels, originally characterized in purkinje neurons of the cerebellum, are now defined by rapid block by the spider toxin co-Aga IVA [Mintz et al., 1992].  1.5.3.4 Intercellular calcium waves Calcium waves are not only confined to single cells but can diffuse from one cell to another via two separate mechanisms.  One mode of transmission depends on the  diffusion of either calcium itself or IP3 through gap junctions [Boitano et al, 1992]. In 37  cells lacking gap junctions, waves spread by means of a secreted intermediate [Osipchuk and Cahalan, 1992].  38  1.5.4 Protein kinase C 1.5.4.1 Introduction The P K C family, a group of widely distributed serine/threonine kinases, mediates intracellular  signaling  of numerous  cellular  regulators  including  hormones,  neurotransmitters and growth factors [Nishizuka, 1984; Berridge, 1993; Dekker LV, 1997]. There are currently 13 known P K C isozymes that are divided into four groups based on their requirements for activation and structures.  Conventional PKCs (cPKC)  (a, p i , p i l and y) are regulated by diacylglycerol (DAG), phosphatidylserine and C a . 2 +  p i and p i l are alternative mRNA splicing products of the same gene, differing by fewer than 50 amino acids in the V5 region. Novel PKCs (8, e, 9 and r\) (nPKC) require diacylglycerol and phophatidylserine, but are calcium-independent. kinase Cs (c^, i and X) (aPKC) require neither calcium nor D A G .  Atypical protein  PKCu. and v, a fourth  subfamily, are activated by D A G but not structurally related to P K C .  P K C v has 77.3%  similarity to human PKCp. [Newton, 1997; Jaken, 1996; Hayashi et al, 1999].  1.5.4.2 Structure (Fig. 10) Protein kinase C, including four conserved (C1-C4) and five variable (V1-V5) regions, consists of a regulatory domain attached by a hinge region to a conserved kinase catalytic domain.  The regulatory domain contains two domains, C l and C2, and an  autoinhibitory pseudosubstrate sequence within the C l domain. The C l region of the cPKCs and nPKCs contains two copies of a Cys-rich zinc finger motif that constitutes the 39  DAG/phorbol ester binding site. The aPKCs have only one copy in C l region that does not bind D A G or phorbol ester. The C2 region of the cPKCs contains an Asp-rich calcium and phospholipid-binding site, which is absent in nPKCs and aPKCs.  The  catalytic domain contains the ATP-binding site, C3, and the substrate-binding site, C4 [Mollor and Parker, 1998].  PKCfi is also referred to as P K D in mouse, which is  characterized by lack of the typical pseudosubstrate site as well as the presence of unique amino-terminal hydrophobic domains together with its unusually large molecular size. Furthermore, a pleckstrin homology (PH) domain is identified in the regulatory region. Functional studies reveal that D A G and phorbol ester promote PKCja kinase activity [Johannes et al., 1994; Dieterich et al., 1996.  40  Catalytic domain  Regulatory domain  Subspecies  VI  Cl  V2  C2  V3 C3 V4  ,,,,,,  Conventional a,pT/(3II,y  PMA/DAG  Ca /PS 2+  C4  • ATP  Substrate  Novel 5,s,ri,e Cysteine rich sequences Atypical  t  t  Putative membrane spanning domain  PH  Fig. 10. P K C structure. The classical group (cPKC) contains all four conserved amino acid regions. The novel group (nPKC) lacks a calcium binding C2 region. The atypical group (aPKC) also lacks the C2 region as well as half of the C l region. P K C p contains a pleckstrin homology (PH) domain.  41  V5  1.5.4.3 Distribution of P K C isozymes P K C a , 8, and C, seem to be the most widely distributed isozymes of the P K C family. In contrast, PKCy is expressed mainly in the central nervous system, while PKC9 is expressed in skeletal muscle and haemopoetic cells. Further, PKCr) is highly expressed in skin and lung tissue, with low levels detected in the brain and spleen [Wetsel et al., 1992; Nishizuka, 1988; Osada et al.,1992].  Various P K C isoforms are present in the ovary of different species. In the rabbit corpus luteum, a, (3 and 8 isoforms of P K C are identified [Maizels et al., 1992], while porcine corpora lutea contain a and P [DeManno et al., 1992]. Western blot analysis reveals that bovine corpus luteum expresses a and 8 [Orwig et al., 1994].  1.5.4.4 Signal transduction After  binding  to  G-protein  coupled  receptors,  an  array  of  hormones,  neurotransmitters and growth factors activate phospholipase C (PLC) resulting in the 2+  generation of IP3 and D A G , which releases Ca  from stores in the endoplasmic  reticulum and subsequently activates P K C . Alternatively, phospholipase D, which can be activated by a receptor, by P K C or by kinases such as mitogen activated protein kinase (MAPK), cleaves phosphatidylcholine (PC) to phosphatidic acid (PA).  P A either  activates P K C directly or is hydrolysed to D A G for P K C activation [Nishizuka, 1995]. Atypical  PKCc^  is activated  via a different  phosphatidylinositol-3,4,5—triphosphate  (PIP3) 42  transduction generated  pathway by  -  through  activation  of  phosphatidylinositol-4,5-diphosphate-3-kinase  (PI-3-kinase).  It  is  believed that  phosphoinositide-dependent-kinase-1 (PDK-1) mediates the activation of P K C ^ [Zhou et al., 1994; Toker, 2000] (Fig. 11).  1.5.4.5 Localization of P K C isozymes Active P K C may translocate to specific cellular compartments and bind to certain proteins, which direct PKCs to second messenger activators and position PKCs in the proximity of appropriate substrate proteins. There are two general categories of proteins that are associated with active P K C binding. The first includes substrates that interact with C-kinases (STICKs) and the second includes nonsubstrate protein (receptors for activated C-kinase; R A C K s ) that place PKCs in close contact with substrate proteins [Jaken and Parker, 2000].  1.5.4.6 The role of P K C A wide range of cellular function is known to be regulated via the P K C signaling pathway such as cell differentiation and proliferation, secretion, cytoskeleton function, cell-cell contacts, gene expression, cell survival and the modulation of membrane ion channels or receptors.  In addition, there is evidence that P K C has a dual action by  providing positive forward actions as well as negative feedback, controlling various signaling steps [Nishizuka, 1989; Nishizuka, 1986].  43  Agonist  PKCc;  P K C : protein kinase C PI3-K: phosphoinositide-3-kinase PLC: phospholipase C PDK-1: phosphoinositide-dependent-kinase-1 PLD: phospholipase D P M E : phosphomonoesterase D A G : diacylglycerol PI4,5P2: phosphatidylinositol 4,5 bisphosphate PI3,4,5P3: phosphatidylinositol 3,4,5 Trisphosphate IP3: inositol triphosphate PC: phosphatidylcholine PA: phosphatidic acid  Fig. 11 Signal transduction pathway for the activation of P K C  44  1.5.5 Mitogen-activated protein kinase (MAPK)  1.5.5.1 Introduction The ubiquitous M A P kinases comprise a family of serine/threonine kinases that are involved in the transduction of externally derived signals regulating cellular proliferation, differentiation and division. Activated M A P K may mediate various cellular functions in the membrane, cytoplasm, nucleus, or cytoskeleton.  Upon activation, M A P kinases  translocate to the nucleus, resulting in the phosphorylation and activation of nuclear transcription factors involved in D N A synthesis and cell division [van Biesen et al., 1996; Blenis, 1993].  1.5.5.2 Classification and activation cascades M A P kinases have been classified into three subfamilies: extracellular-signal regulated kinases (ERKs), stress-activated protein kinases/ c-jun N-terminal kinases (SAPKs/JNKs) and p38 kinase [Lopez-Ilasaca, 1998]. Both tyrosine kinase receptors and G-protein-coupled receptors have been demonstrated to activate ERKs.  The J N K  and p38 M A P K are activated by environmental stresses (osmotic shock, heat shock, U V radiation) or cytokines (interleukin-1 and tumor necrosis factor-cc) [Raingeaud et al., 1995]. The discovery of the first mammalian M A P K was based on the identification of p42 and p44 M A P K s (ERK1 and ERK2, respectively) in 1987 [Ray and Sturgill, 1987]. These two M A P K  isoforms are activated by dual phosphorylation on a T E Y  (Threonine-Glutamic acid-Tyrosine) motif by the M E K I (MAPK/ERK-activating kinase,  45  M A P K kinases) and M E K 2 isoforms). Both M E K 1 and 2 are themselves activated by phosphorylation of two serine residues by the protein kinase Raf ( M A P K K kinase) [Ann et al, 1992; Brunet and Pouyssegur, 1997].  There are currently seven different M A P K kinases ( M A P K K ) identified. The first M A P K K s to be cloned were MEK1/2 which activate ERK1/2.  M K K 4 and M K K 7  phosphorylate and activate J N K . M K K 3 and M K K 6 specifically phosphorylate and activate p38, whereas M K K 5 activates ERK5 [Fanger GR, 1999] (Fig. 12).  Translocation of activated M A P K to the nucleus and subsequent phosphorylation of a variety of transcription factors including c-Myc, Elk-1 and ATF2 support the involvement of M A P K in transducing cytoplasmic signals to nucleus. ERK1 targets E1K-1, whereas ERK2 prefers c-Myc.  Specifically,  SAPK/JNK is able to effect c-jun,  ATF2 and Elk, and p38 targets ATF2, Elk and Max [Lopez-Ilasaca, 1998] (Fig. 13).  46  (Rafs, M E K K s )  MAPKKH  (MEKs, M K K s )  MAPKKJ  (ERK, JNK, p38)  MAPKJ  1 Transcription factors!  (Elk-1, c-Myc, jun, ATF2)  Fig. 12 Regulation of sequential kinase pathways that activate M A P K s .  MAPKs  such as E R K , JNK, p38 are activated by tyrosine and threonine phosphorylation by M A P K K , M E K s or M K K s .  M A P K K s are activated by serine and threonine  phosphorylation by the M A P K K K s such as the Rafs and M E K K s .  M A P K : mitogen-activated protein kinase  M A P K K : M A P K kinase  M A P K K K : M A P K K kinase  M E K : M A P K / E R K kinase  M E K K : M E K kinase E R K : extracellular-signal regulated kinase JNK: c-Jun N-terminal kinase  47  Growth factor, hormone, Neurotransmitter  Stress/cytokine  Heat shock Osmotic shock UV  TNFa ILla,P  1  r  M K K 3/6  Tyrosine  coupled  kinase  II PLC  I 1 J  G-protein-  MEKK  I  MKK4/7  PKC  Rafs  I  MEK1/2  MAPK/EriT)  Elk-1, Jun, ATF2  Cell proliferation & differentiation  Stress response  Fig. 13 Signal-transduction pathways of receptors or stress-activated M A P K s .  48  1.5.6 Cyclic AMP /PKA signal transduction  The c A M P signaling system is a second messenger-dependent pathway that converts extracellular signal into intracellular responses and regulates many vital functions such as gene transcription, proliferation, differentiation, reproductive function and secretion. A T P has been demonstrated to increase intracellular c A M P production by activating adenylate cyclase in several cell systems [Communi et al., 1997; Conigrave et al., 1998], indicating the stimulation of protein kinase A (PKA).  Many neurotransmitters and hormones transduce their signals into cells through the cAMP-dependent P K A pathway.  These agonists include neurotransmitters such as  acetylcholine, dopamine, norepinephrine, serotonin and histamine as well as peptide ligands such as somatostatin, corticotropin-releasing factor,  growth-hormone-releasing  hormone, follicle-stimulating hormone and luteinizing hormone [Brandon et al., 1997].  The c A M P signaling pathway is composed of a cascade of regulatory proteins.  The  cascade elements consist of a hormone or neurotransmitter (first messenger)-specific transmembrene receptor that is coupled to a heterotrimeric G-protein complex (Get and G(3y), resulting in the activation of an intracellular effector adenylate cyclase. Adenylate cyclase generates c A M P (second messenger) which activates P K A responsible for the phosphorylation of appropriate substrates [Spiegel et al., 1992; Tang and Gilman, 1991; Clapham and Neer, 1993].  The c A M P signal is terminated by cyclic nucleotide  phosphodiesterase, which hydrolyzes c A M P to 5'-AMP,  49  and by  phosphoprotein  phosphatases, which dephosphorylate the phosphoproteins [Ishikawa, 1998; Reifsnyder, 1990; Shenolikar and Nairn, 1991].  The elevation of c A M P leads to the activation of P K A , a tetrameric enzyme, which is composed of a dimeric regulatory (R) subunit and two monomeric catalytic (C) subunits.  The R-subunit, a pseudosubstrate to inhibit phosphotransferase  activity of  C-subunit, binds with c A M P resulting in the release of active C-subunit [Beebe, 1994]. Catalytic subunits migrate into the nucleus where they phosphorylate a single serine residue  on  CREB  protein  (cyclic  A M P response element-binding  protein,  a  transcriptional activator) and thereby activate it. The active C R E B then interact with the c A M P response element (CRE) found in the promoters of cAMP-responsive genes to initiate transcription [Sassone-Corsi, 1995] (Fig. 14).  50  Neurotransmitters: acetylcholine, dopamine, norepinephrine, serotonin and histamine Ligands  Extracellular  Peptide ligands: somatostatin, corticotropin-releasing factor, growth-hormone-releasing hormone, follicule-stimulating hormone, luteinizing hormone and human chorionic gonadotropin  CRE Binding protein (CREB)  cAMP Response Element (CRE)  Activate transcription of cAMP-responsive gene  Fig. 14 The cAMP signal transduction pathway.  51  HYPOTHESIS The ovary is a well-innervated endocrine organ, drawing our attention to investigate the role of neurotransmitters in modulating ovarian function. Considering that ATP is released from nerve endings and evokes cytosolic calcium oscillations in human granulosa cells (hGLCs), it is hypothesized that ATP plays a role in regulating ovarian function such as steroidogenesis.  To address this hypothesis, a series of studies are  performed to examine the expression of P2 purinergic receptor, signaling pathways and functional role of ATP in hGLCs.  SPECIFIC OBJECTIVES 1.  To examine the expression and regulation o f P2 purinergic receptors in hGLCs.  2.  To examine the role of protein kinase C in regulating ATP-induced cytosolic calcium oscillcitons in h G L C s .  3.  To examine the mechanism o f antigonadotropic action o f A T P i n h G L C s : the involvement o f protein kinase C .  4.  To examine the effect o f A T P on activation o f M A P K , its intracellular signaling pathway and functional role in h G L C s .  52  PART 2 GENERAL MATERIALS AND METHODS  2.1 Human granulosa-luteal cells culture  Human GLCs were collected from patients undergoing an In Vitro Fertilization Embryo Transfer program. The use of human GLCs was approved by U B C Clinical Screening Committee for Research and Other Studies Involving Human Subjects. The follicular fluid collected during oocyte retrieval was centrifuged at 1000 xg for 10 min. The pellet containing granulosa cells and red blood cells was resuspended with Dulbecco's Modified Eagle Medium (DMEM).  Granulosa cells were then separated  from red blood cells in follicular aspirates by centrifugation through equal volume of Ficoll Paque at 1000 xg for 15 min. Granulolsa cells sitting on interphase were collected and washed twice with D M E M .  After brief centrifugation, cell pellet of granulosa cells  were suspended in D M E M containing 100 U penicillin G sodium/ ml, 100 pg streptomycin/ml and 10% fetal bovine serum.  The cells were plated at a density of  approximately 200,000 cells per dish in 35-mm culture dishes. The dishes were incubated at 37 °C under a water-saturated atmosphere of 5% C 0 2 in air for 3 days.  Human GLCs were collected from patients undergoing in vitro fertilization treatment who ranged in age from 23 to 43 years. Forty-nine percent had severe male factor infertility while the remainder had various female factors or long-standing unexplained infertility. Ovarian stimulation entailed a long luteal phase down-regulation protocol for women under 40 years or a follicular phase flare protocol for women >40 53  years, as previously described (Yuzpe et al., 2000).  Human granulosa-luteal cells  obtained from 105 patients were uses in this study.  2.2 Isolation of total RNA  Total R N A was prepared from the cultured hGLCs by the phenol-chloroform method of Chromczynski and Sacchi [Chromczynski and Sacchi, 1987]. Human GLCs were washed twice with phosphate buffered saline (PBS; 137 m M NaCI, 2.7 m M KC1, 4.3 m M Na2HP04, 1.4 m M KH2P04, pH7.3), and cells were lysed in 500 pi of solution D (4 M guanidine thiocynate, 25 m M sodium citrate (pH 7.0), 0.5% N-lauroyl sarcosine, and 0.1 M (3-mercaptoethanol). microcentrifuge  tube,  The lysate was then transferred to a 1.7 ml  50 pi of sodium acetate (pH 4.0), 500  pi of DEPC  (diethylpyrocarbonate)-saturated phenol (pH 6.0) and 100 pi of chloroform: isoamyl alcohol mixture (24:1) was added, and the tube was vortexed briefly and cooled on ice for 20 min. After incubation, the tube was centrifuged at 4 °C at 10,000 xg for 20 min, and superantant was collected into a new tube without disturbing the interphase.  For the  second extraction, the supernatant was mixed with 400 pi of chloroform: isoamyl alcohol mixture, vortexed briefly and centrifuge for 5 min. The supernatant was collected and 500 pi of isopropanol was added. The R N A was then precipitated at -70 °C for 1 h.  Following precipitation, the tube was centrifuged at 10,000 xg for 20 min. The supernatant was removed and the pellet was washed twice with 500 pi of 70% ethanol. 54  Finally, the pellet containing some residual ethanol was dried by speedvac. The pellet was resuspended in 20 ul of DEPC-water.  The R N A concentration was determined based on absorbance at 260 nm. examine  the  integrity,  extracted  R N A was  checked  by  separation  in  To 1%  formaldehyde-agarose gel. R N A samples were loaded along with ethidium bromide containing gel loading buffer (6X loading buffer= 50% glycerol, 1 m M E D T A , 0.4% bromophenol blue, 0.4% xylene cyanol), and run at 70 V for 3 h. The demonstration of two R N A bands (18S and 28S) revealed the integrity of extracted R N A .  2.3 Reverse transcription of RNA to first-strand cDNA  R N A obtained from human granulosa-luteal cells was reverse transcribed into cDNA using the First Strand c D N A Synthesis Kit (Amersham Pharmacia Biotech, Oakville, Canada).  One microgram of total R N A dissolved in DEPC-water (8 ul in  total) was heated at 65 °C for 10 min and cooled on ice for 5 min. DTT (1 ul), oligo-dT (1 u.1) and bulk mixture (5 ul) was added to the sample, and the mixture was incubated at 37 °C for l h . After incubation, the sample was boiled for 10 min to inactivate reverse transcriptase and stored at -20 °C until use.  55  2.4 Southern blot analysis  Nine microliter of PCR products were mixed with 1 ul of 10X gel loading buffer and loaded on 1% agarose gel containing ethidium bromide (200 u.g/ 100 ml gel). The gel was run at 100 Volts for 1 h in I X T B E running buffer (90 m M Tris-borate, 2 m M E D T A , pH 8.0). The gel was then incubated in denaturing solution (1.5 M NaCI, 0.5 M NaOH) with agitation for 30 min and neutralized in neutraling solution (1.5 M NaCI, 1.0 M Tris, pH 8.0) for 15 min x 2. The samples in the gel were transferred overnight to a Nylon membrane (Hybond N , Amersham Pharmacia Biotech) using an apparatus (Fig. 15) containing 10X SSC ( I X SSC= 0.15 M NaCI, 0.015 Sodium citrate(2H20), pH 7.0). The membrane was wrapped in Saran Wrap and exposed to U V light for 5 min to crosslink D N A with the membrane.  The membrane was stored at 4 °C until the probing  steps.  Digoxigenin-labeled cDNA probes for a targeted molecule were prepared using DIG D N A labeling kit following manufacturer's protocol (Roche Molecular Biochemicals). Briefly, template cDNA (1 u.g) was denatured by boiling for 10 min and quickly chilled on ice. The c D N A was labeled in 20 ul reaction containing 2 u.1 of hexanucleotide mix, 2 Jul of dNTP mix, 1 ul of Klenow enzyme at 37 °C for 3 h. After incubation, the reaction was stopped by adding 2ul of 0.2 M E D T A (pH 8.0). Labeling efficiency was determined by comparing signal intensity with DIG-labeled control-DNA provided by the manufacturer. 56  The membrane was prehybridized for 2 h in hybridization buffer (50% deionized formamide, 5 X SSC, 0.1% w/v N-lauroylsarcosine, 0.02% SDS, 2% blocking reagent) and hybridized with denatured DIG-labeled cDNA probe (boiled for 10 min) at 42 °C for 20 h. The membrane was then washed twice with 2 X SSC, 0.1% SDS for 10 min at room temperature (RT), followed by twice high stringency washing with 0.1 X SSC, 0.1 % SDS at 65 °C for 15 min. After washing, the membrane was incubated for 30 min in buffer solution (Buffer 2, 1% blocking reagent in Buffer 1 [0.1 M Maleic acid, 0.15 M MaCl, pH 7.5]) and incubated with anti-DIG-AP connjugate (1: 10, 000) for 30 min. The membrane was washed twice in washing buffer ( 0.3% Tween 20 in Buffer 1 ) and equilibrated in Buffer 3 (0.1M Tris-HCl, 0.1 M NaCI, 50 m M MgC12, p H 9.5) for 5 min, followed by incubation with CSPD  (1:200) at room temperature (RT) for 10 min.  Finally, the membrane was wrapped with saran wrap and incubated at 37 °C for 15 min prior to exposure to Kodak Omat X-ray film (Eastman Kodak Co., Rochester, N Y ) .  57  Fig. 15 The apparatus for D N A transferring.  58  2.5 Subcloning and plasmid isolation  PCR products were fractioned in 1% agarose gel and eluted. Briefly, PCR products of correct size were cut from the gel and boiled in 500 pi solution containing I X T B E and 0.5 M NaCI for 3 min, followed by heated at 65 °C until completely dissolved. Samples were added with prewarmed (65 °C) TE-saturated phenol (pH7.5, 500 pi), vortexed and centrifuged at RT for 5 min.  The supernatant was collected and  centrifuged for another 5 min to remove residual gel. The collected supernatant was added with phenol/chloroform/isoamyl alcohol (25: 24:1), vortexed and centrifuged for 5 min.  The supernatnat was collected, added with equal volume of isopropanol and 0.1  volume of 3 M sodium acetate (pH 5.2), vortexed briefly and stored at -20 °C for 2 h, followed by centrifugation at 4 °C at 14,000 rpm for 20 min. The pellet was rinsed with 70% ethanol, centrifuged and dried with speedvac.  The pellet was then dissolved in 15  pi of TE and stored at -20 °C until use.  PCR products isolated from gel were cloned into pCR vector using TA Cloning Kit (Invitrogen, San Diego, CA). Briefly, purified PCR products (10 ng) was ligated in 10 pi ligation reaction containing 25 ng pCR II vector, 1 X ligation buffer, 20 IU T4 D N A ligase at 4 °C for 16 h. The ligated plasmid was incubated with competent E. Coli (One Shot ™ ) on ice for 30 min, transformed into E. Coli by heat shock at 42 °C for 1 min and incubated on ice for 5 min. Two hundred and fifty microliter of L B broth ( 10 g Bacto-tryptone, 5 g Bacto-yeast extract, 5 g NaCI in 1000 ml distilled H20) was added to the mixture and incubated at 37 °C for l h .  59  E. Coli mixture was then plated on  LB-ampicillin plates (50 mg/ ml L B ) pre-coated with X-Gal (20 ul of 20 mg/ml stock solution) and IPTG (10 |ug/ plate) and incubated at 37 °C for 18 h. White and blue colonies represent positive (cDNA-incorporated vector) and negative (vector only) results, respectively.  Positive colonies were selected and cultured 2 ml L B and plasmid was isolated using the alkaline lysis procedure.  Briefly, bacteria culture (1.5 ml of LB) was transferred to  microcentrifuge tube and centrifuged at 10,000 xg for 1 min.  The pellet was  resuspended with 200 ul of ice-cold Solution I (50 m M Tris-Cl, pH 8.0, 10 m M EDTA, 100 ug I ml RNase), lysed with 200 ul of Solution II (200 m M NaOH, 1% SDS) for 5 min and neutralized with 200 ul of ice-cold Solution III (3.0 M Potassium acetate, pH 5.5).  The mixture was centrifuged at 10, 000 xg for 20 min, and supernatant was  transferred to a new tube. The supernatant was added with equal volume of mixture of phenol: chloroform: isoamylalcohol (25: 24: 1) and centrifuged at 4 °C for 20 min. The plasmid-containing supernatant was added with equal volume of isopropanol for precipitation and stored at -20 °C for 1 h.  Plasmid pellet was obtained after  centrifugation at 4 °C for 20 min, washed with 70 % ethanol, dried with speedvac and dissolved in 20 ul of TE. The cDNA-containing plasmid was stored at -20 °C until use.  2.6 Sequence analysis  The cDNA-containing plasmid was sequenced by the dideoxy nucleotide chain 60  termination method using the T7 D N A polymerase sequencing kit (Amersham Pharmacia Biotech). Sequencing analysis was performed using universal M l 3 forward and reverse primers. Double-strain cDNA-containing plasmid (2 pg in 32 pi TE) was denatured by adding 8 pi of 2 M NaOH to a final volume of 40 pi. D N A was then precipitated with sodium acetate/ ethanol (7 pi of 3 M sodium acetate, pH 4.8, 4 pi of distilled water, and 120 pi of 100% ethanol ), collected by centrifugation at 10, 000 xg for 20 min, washed with 70% ethanol, and dried with speedvac.  The denatured D N A template was  resuspended in 10 pi of distilled water, folowed by adding 2 pi of primer (forward or reverse) and 2pl of annealing buffer (1 M Tris-HCl, pH 7.6, 100 m M MgC12, 160 m M DTT) to a final volume of 14 pi. The reaction was performed at 65 °C for 5 min and 37 °C for 10 min, followed by RT for 15 min to promote annealing. After the annealing reaction, 3 pi of Labeling Mix (including dCTP, dTTPand dGTP), l p l of [a- S]dATP 35  (10 pCi, Ammersham Pharmacia Biotech.), and 2 pi of diluted T7 D N A polymerase (3U) were added to a final volume of 20 pi and incubated for 5 min at RT. Meanwhile, four tubes of 2.5 pi dNTP mix (A,C,G or,T mix, A-mix including ddATP with dNTP) were prepared and prewarmed at 37 °C for 5 min prior to adding 4.5 pi (out of 20 p i ) of the prepared labeling reaction mixture. After incubation at 37 °C for 5 min, the reaction was terminated by adding 5 pi of Stop solution (0.3% bromphenol blue and xylene cyanol, 10 m M E D T A , pH 7.5, and 97.5 % deionized formamide) and stored at -20 °C until use.  Polyacrylamide 6%/ 7 M urea sequencing gels was prepared and prerun at 45 Walts for 1 h before loading the samples.  Samples (2.5 pi) were boiled for 5 min prior to  61  loading into the gel and then run for 3 to 4 h at 45 W constant power. Gel was dried at 80 °C for 2 h using a gel dryer ( Model 583, Biorad Laboratories, Richmond, CA) and exposed to Kodak Omat X-ray film at -70 °C for 24 h. The sequence of PCR product was sent to GeneBank at NCBI (National Center for Biotechnology Information) through the internet  (www.ncbi.nlm.gov) to compare the identity with published  sequences.  2.7 Northern blot analysis  Approximately 15 ug of total R N A was disolved in denaturing solution'.( 50 % formamide, 2.2 M formaldehyde) and incubated at 60 °C for 15 min. Samples were added with  I X gel loading buffer and separated by electrophoresis in a 1%  agarose-formaldehyde gel ( 20 m M MOPS, 2.2 M formaldehyde, 8 m M sodium acetate, 1 m M EDTA, p H 8.0) at 70 V for 3 h. R N A was transferred onto a nylon membrane (Amersham, Hybond-N) in 2 X SSPE (20X SSPE= 3 M NaCI, 0.2 M NaH2P04-H20, 0.025 M EDTA) for 18 h. Membrane was then incubated under U V light for 5 min to crosslink the R N A to the membrane. The membrane was pre-wetted in 5X SSPE for 30 min. They were then incubated in a prehybridization solution of 5X SSPE containing 50% deionized formamide, 5X Denhardt's, 1% SDS and 100 ug/ ml heat-denatured salmon sperm D N A at 42 °C for 3 hours.  Meanwhile, radiolabeled cDNA probe was prepared using the Random Labeling Kit 62  (Life Technologies, Inc.). According to manufacturer's protocol, D N A template (100 ng) in 23 ul was denatured at 100 °C for 5 min and placed on ice for 5 min. Two ul of each dNTP (dCTP, dGTP and dTTP) with 15 ul of random priming buffer, 5 ul of [a- P]dATP (50 uCi, Amersham Pharmacia Biotech) and 1 ul of Klenow fragment 32  were added into denatured D N A sapmle and incubated at RT for 3h. The labeled D N A was purified using G-50 Sephadex column (Amersham Pharmacia Biotech.).  The radiolabeled probe was denatured by boiling for 5 min and then added to the prehybridization solution. The blot was incubated in the presence of the radiolabeled probe at 42 °C for 16 hours, then washed twice with 2X SSPE at room temperature (5 min / wash), twice with 2 X SSPE containing 1% SDS at 55 °C (30 min / wash), twice with 0.2X SSPE at room temperature (30 min / wash) and finally exposed to Kodak Omat X-ray film.  2.8 Western blot analysis  The hGLCs were washed twice with ice-cold PBS and lysed with 100 uL of cell lysis buffer (RIPA, 150 m M NaCI, 50 m M Tris-HCl (pH 7.5), 1 % Nonidet P-40, 0.5 % deoxycholate, 0.1% SDS, 1.0 m M PMSF, 10 ug/mL leupeptin and 100 ug/mL aprotinin) at 4 °C for 30 min. The cell lysate was centrifuged at 10,000 x g for 5 min and the supernatant was collected for Western blot analysis. 63  The amount of protein was quantified using Bio-Rad DC Protein Assay kit (Bio-Rad Laboratories, Richmond, C A ) . Cellular extract (10 pi) was incubated with 25 pi of reagent A ( an alkaline copper tartrate solution) and 200 pi of reagent B ( a dilute Folin Reagent) for 15 min with gentle shaking at room temperature.  This assay is based on the  reaction of protein with an alkaline copper tartrate solution and Folin reagent. As with the Lowry assay, there are two steps which lead to color development: the reaction between protein and copper in an alkaline medium, and the subsequent reduction of Folin reagent by the copper-treated protein [Lowry et al., 1951]. Color development is mainly due to the amino acids tyrosine and tryptophan, and to a lesser extent, cystine, cysteine and histidine [Lowry et al., 1951; Peterson, 1979]. Proteins induce a reduction of the Folin reagent by loss of 1, 2, or 3 oxygen atoms so as to produce one or more of several possible reduced species that have a characteristic blue color with maximum absorbance at 750 nm and minimum absorbance at 450 nm [Peterson, 1979]. Absorbance of the sample was measured using microplate reader (Model EL311, Bio-Tek instruments, Vermont, C A ) at 630 nm. A standard curve for protein assay is constructed in each assay using standard solution of bovine serum albumin (Fig. 16).  Aliquots (30 pg) of protein were mixed with I X sample buffer (8X loading buffer= 100 m M Tris-HCl, pH 6.8, 4 % SDS, 0.2 % bromphenol blue, 20 % glycerol, 8 % 2-mecaptoethanol) and boiled for 10 min. The samples were then subjected to 10 % SDS-polyacrylamide gel electrophoresis [Laemmli, 1970] in I X gel running buffer (25 m M Tris, pH 8.0, 250 m M glycine, 0.1% SDS) at 30 miniampere for 3 h. The proteins  64  were then electrophoretically transferred from the gels onto nitrocellulose membranes (Amersham Pharmacia Biotech, Oakville, Canada) [Towbin et al., 1979] in transfer buffer (200 m M Glycin, 25 m M Trizma base, 20% methanol) at 100 Volts for 90 min. These nitrocellulose membranes were then pre-incubated with blocking solution (5% skim milk, 0.05% Tween 20 in TBS (Tris-buffered saline, p H 8.0, 5 m M Tris base, 100 m M sodium chloride)) for 2 h and incubated with primary antibody for 16 h at 4 °C with gently shaking in sample bag (VWR). After washing three times with TBS-T (0.1% Tween 20 in TBS) and two times with TBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1: 1000 dilution in blocking solution), and the signal was visualized using E C L system (enhanced chemiluminescent system, Amersham Pharmacia Biotech, Oakville, Canada) followed by autoradiogrphy.  The  autoradiograms were quantified using a laser densitometer (BIO-RAD, Model 620, Video Densitometer).  65  y = 3.1487x- 0.155  0  0.2  0.4 0.6 Optical Density  0.8  Fig. 16 Standard curve for protein assay. Absorbance of the samples was measured using ELISA reader at 630 nm.  66  1  2.9 Microspectrofluorimetry  Fluorescence ratio imaging is a widely used technique for the detection of calcium in living cells.  The Fura-2 is a calcium chelator that emits quantitatively different  fluorescence (510 nm) at different excitation wavelengths.  In the presence of high  concentrations of calcium, Fura-2 fluoresces brightly when excited at 340 nm and dimly when excited at 380 nm. In conditions of low calcium, the fluorescence intensity at 340 nm and 380 nm is reversed with bright at 380nm and dim at 340 nm. Thus, Fura-2 is a dual-excitation/ single-emission dye [Grynkiewicz et al., 1985].  The reversal of  fluorescence intensity in response to alterations of calcium level is the key for use of fluorescence ratio imaging to detect cytosolic calcium oscillations.  Human GLCs were seeded onto 25-mm circular glass cover slips (5,000 /slip) and incubated for 3 days at 37 °C in humidified air with 5% C 0 2 prior to microfluorimetric experiments. Cytosolic calcium concentrations were measured using the dual-excitation single-emission fluorimetric technique, as described previously [Squires et al., 1997]. Briefly, the cells were incubated with 5-10 p M fura-2 A M acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at 37 °C in humidified air with 5% C 0 2 . The cover slip was mounted onto the perifusion chamber and equilibrated for 10 min with balanced salt buffer (NaCI 137 m M , KC1 5.36 mM, CaC12 1.26 m M , MgS04-7H20 0.81 mM, Na2HP04-7H20 0.34 m M , KH2P04 0.44 mM, NaHC03 4.17 m M , HEPES 10 m M , glucose 2.02 m M , pH7.4) in humidified air with 5% C 0 2 .  The fura-2 ratio  measurements were performed using the Attoflour Digital Fluorescence Microscopy  67  System (Atto Instruments, Rockville, MD). The perifusion chamber was connected to a multiunit six-channel perifusion system with a flow rate of 1-2 ml/min. Fura-2 loaded cells were observed through a 40X fluorescent objective lens and were illuminated alternatively with light at 340 nm and 380 nm. Emitted light was filtered using a 510 nm long-pass filter and detected using a low light sensitive camera.  Measurements of  cytosolic calcium were performed at 1-2 sec intervals. A l l records were corrected for background fluorescence (determined from cell-free region of cover slip). Changes in the fluorescence ratio recorded at 340 and 380 nm correspond to changes in cytosolic calcium.  68  2.10 Radioimmunoassay for progesterone  Progesterone  levels in the culture medium were measured  radioimmunoassay [Vaananen et al, 1997].  by established  Anti-progesterone antibody was kindly  provided by Dr. D. T. Armstrong (University of Western Ontario). Briefly, samples were diluted 200 times with PBS-G (phosphate buffered saline-gelatin, 0.1 M PBS, 0.1% gelatin; I X PBS-G= 5.18 g/ L Na2HP04»7H20, 16.65 g/ L NaH2P04«H20, 9g/L NaCI, 0.1% gelatin, 0.1 g/L Thimerosal). Samples (100 ul) were incubated with antibody (100 ul) and tracer (100 ul), with a final concentration of 7,000 cpm/ml of [l,2,6,7,16,17-3H]Progesterone (Amersham Pharmacia Biotech). A standard curve was constructed in each assay with progesterone ranging between 0.39-25 ng/ ml (Fig. 17). After incubation at 4 °C for 16-24 hours, 500 ul charcoal/dextran solution (0.5g/ 0.05g in 200 ml of PBS-G) was added to each tube to remove unbound progesterone or tracer. After centrifugation at 4 °C at 4500 rpm for 30 min, 700 ul of the supernatant was collected and added with 3 ml of scintillation cocktail (Amersham Pharmacia Biotech), and the vials were counted with a (3-counter ( L K B Wallac, Turku, Finland). The cells in each dish were harvested for quantifying protein amount using Bio-Rad Protein Assay kit.  Samples were assayed in triplicate and  progesterone concentrations were standardized against total protein content.  69  A.  0  10  20  30  Progesterone (rg/rri)  B.  -g § m  y = -13.283Ln(x) + 55.681  80 70 60 50 40 an 20 10  o 0.1  1  io  ioo  logtProgesterone (ng/tnl)]  Fig. 17 Standard curves for radioimmunoassay. A. Progesterone v.s. % Bound. B. Log[Progesterone] v.s. % Bound.  70  2.11 Radioimmunoassay for cAMP  Prior to scheduled treatments, hGLCs were incubated in serum-free medium containing 0.1% B S A and 0.5 m M 3-isobutyl-lmethylxanthine (IBMX, Sigma-Aldrich Corp., phosphodiesterase inhibitor) for 30 min.  After treatment, the medium was  removed and ice-cold 100 % ethanol was added, followed by incubation on ice for 20 min.  Cyclic AMP-containing ethanol was collected and centrifuged at 4 °C at 10, 000 xg  for 20 min. The supernatant was collected and dried using a speedvac. The pellet was dissolved in 120 u l of Reagent 1 (0.05 M Tris, pH 7.5, 4 m M EDTA).  Intracellular  c A M P levels were measured using the [ -H]-cAMP assay system, following the protocol 3  provided by manufacturer (Amersham Pharmacia Biotech).  A standard curve was  constructed in each assay with c A M P concentrations between 1- 16 pmole (Fig. 18). The sample (50 ul) was incubated with 100 ul of binding protein (purified from bovine muscle) and 50 jul of [8- H]adenosine 3',5'-cyclic phosphate. 3  After incubation at 4 °C  for 16 h, 100 jul of charcoal was added to remove unbound antigen. After centrifugation at 4500 rpm for 30 min, 200 ul of the supernatant was collected and added with 3 ml of scintillation cocktail, and the vials were counted with a (3-counter. A l l samples were assayed in duplicate.  71  Fig. 18 Standard curve for cAMP assay. Co: the cpm bound in the absence of unlabelled cAMP. Cx: the cpm bound in the presence of standard or unknown unlabelled cAMP.  72  PART 3 EXPRESSION AND REGULATION OF P2U-PURINERGIC RECEPTOR IN H U M A N GRANULOSA-LUTEAL C E L L S  3.1 Abstract The P2U purinoceptor (P2UR) has been identified pharmacologically in the ovary. However, the expression and regulation of the P2UR messenger ribonucleic acid (mRNA) in human ovarian cells are still poorly characterized. The present study was designed to examine the expression and regulation of the P2UR in human granulosa-luteal cells (hGLCs) by reverse transcription - polymerase chain reaction (RT-PCR) and Northern blot analysis. A P C R product corresponding to the expected 599 bp P2UR c D N A was obtained from hGLCs. Molecular cloning and sequencing of the P C R product revealed an identical sequence to the reported P2UR cDNA. Two mRNA transcripts of 2.0 kb and 4.6 kb were identified in hGLCs using Northern blot analysis. The expression ofthe P2UR mRNA was down-regulated by h C G in a dose- and time-dependent manner. Treatment with 8-bromo-cAMP and forskolin also attenuated P2UR mRNA levels. Calcium signaling following the activation of the P2UR in single hGLCs was studied using microspectrofluorimetry. uridine triphosphate  It revealed that, like adenosine triphosphate (ATP),  (UTP) also induced cytosolic calcium mobilization in a  dose-dependent manner.  These results demonstrate for the first time that the P2UR  mRNA is expressed in hGLCs, and that P2UR mRNA is regulated by hCG, c A M P and forskolin. The P2UR expressed in hGLCs is functional, since activation of the P2UR by 73  A T P or UTP resulted in rapid and transient mobilization of cytosolic calcium at the single cell level.  These findings further support a potential role of this neurotransmitter  receptor in the human ovary.  3.2 Introduction  Adenosine triphosphate (ATP) is released from cells such as platelets and co-released with neurotransmitter granules from autonomic nerves by exocytosis [Gordon, 1986]. Extracellular ATP binds to a G protein-coupled P2 purinoceptor that activates phospholipase C and phosphatidylinositol hydrolysis, generating diacylglycerol and inositol 1,4,5-triphosphate, which stimulate protein kinase C and cytosolic calcium ([Ca2+]i) mobilization, respectively [Berridge, 1984; el-Moatassim et al., 1992]. Thereafter, ATP may participate in various types of physiological responses, including secretion, membrane potential, cell proliferation, platelet aggregation, neurotransmission, cardiac function, and muscle contraction [el-Moatassim et al., 1992; Burnstock, 1990].  Purinergic receptors have been classified as PI receptors and P2 receptors. Pharmacologically, the PI receptors have a high affinity for extracellular adenosine and A M P (Adenosine > A M P > A D P > ATP), whereas P2 receptors have high affinity for ATP and A D P (ATP > A D P > A M P > adenosine) [Gordon, 1986; el-Moatassim et al., 1992; Burnstock, 1990]. Six subtypes of P2 purinergic receptors, P2X, P2Y, P2D, P2T, P2Z, and P2U, have been identified in pharmacological and molecular cloning studies 74  [Fredholm e t a l , 1994].  Functionally, a P2U purinoceptor  (P2UR) has been detected in human  granulosa-luteal cells (hGLCs) using microspectrofluorimetry [Kamada et al, 1994]. Autonomic nerves have been shown to innervate the ovary and may be involved in regulating steroidogenesis [Owman et al., 1967; Bodis et al., 1993a; Bodis et al., 1993b]. It is tempting to speculate that the co-released A T P from autonomic nerve endings in the ovary may play a role in regulating ovarian function. A T P has been shown to regulate the production of progesterone and estradiol in hGLCs [Kamada et al., 1994].  These  findings provide further evidence that A T P is able to regulate ovarian function through binding to A T P receptors.  Although P2UR has been identified pharmacologically in human ovary [Kamada et al., 1994, Lee P S N et al., 1996], its expression and regulation at the messenger R N A (mRNA) levels have not as yet been characterized.  To understand further the potential  role of ATP and the receptor of this neurotransmitter in the ovary, the present study was designed to detect the expression of the P2UR in hGLCs and to examine the regulation and signaling of this receptor in vitro.  75  3.3 Materials and Methods  Reagents and Materials  Prostaglandin F2a, gonadotropin-releasing hormone (GnRH), human chorionic gonadotropin  (hCG),  estradiol,  progesterone,  8-bromo-adenosine-3',5'-cyclic  monophosphate (8-bromo-cAMP), forskolin, A T P and uridine triphosphate (UTP) were obtained from Sigma Chemical Co. (St. Louis, MO) Dulbecco's Modified Eagle Medium ( D M E M , phenol red free), penicillin-streptomycin, were obtained from GIBCO-BRL (Burlington, Ontario, Canada).  Fura-2 A M was purchased from Molecular Probes  (Eugene, OR).  Human granulosa-luteal cells culture and treatments  Human GLCs were collected from patients undergoing an In Vitro Fertilization Embryo Transfer program and processed as mentioned in P A R T 2. Human GLCs were then cultured in phenol-red free D M E M containing 100 U penicillin G sodium/ ml, 100 u.g streptomycin/ml and 10% fetal bovine serum at a density of approximately 200,000 cells per dish in 35-mm culture dishes. The dishes were incubated at 37 °C under a water-saturated atmosphere of 5% C 0 2 in air for 3 days. To examine the regulation of the P2UR mRNA, hGLCs were incubated in serum-free medium for 24 hours prior to 76  treatment with estradiol (10" M), progesterone (10" M ) , prostaglandin F2a (10" M ) , 7  7  7  GnRH, (lO" M ) , hCG, (5 IU/ml) or A T P (10 pM) for 24 hours. For dose-response 7  experiments, hGLCs were treated with different concentrations of hCG (0.1,1,5,10 IU/ml) for 24 hours. For time-course analysis, hGLCs were treated with 5 IU/ml of hCG for 0,3,6,12,24 or 48 hours. To further delineate the underlying mechanism, by which the expression of P2UR mRNA was regulated, cells were treated with 8-bromo-cAMP (ImM) or forskolin (10 pM) for 24 hours prior to the determination of P2UR mRNA levels.  Total RNA isolation and RT-PCR  Total R N A was prepared from the cultured hGLCs by the phenol-chloroform method of Chromczynski and Sacchi [Chromczynski and Sacchi, 1987] as mentioned in PART 2. One microgram of total R N A obtained from human granulosa-luteal cells was reverse transcribed into cDNA using the First Strand cDNA Synthesis Kit (Pharmacia Biotech,  Morgan,  Canada).  One  CCTGGAATGCGTCCACCACATAT-3  set and  of  oligonucleotide  primers  (5-  5-  GACGTGGAATGGCAGGAAG  C A G A -3) based on the published human P2U receptor sequence [Parr et al., 1994] was designed for polymerase chain reaction (PCR) to amplify the P2UR from hGLCs. P C R reactions were performed in the presence of 10 m M Tris-HCl (pH 8.3), 50 m M KC1, 1.5 m M MgCl2, 400 p M dNTPs, 0.25 U Taq D N A polymerase, 2 p M primers, and l p l c D N A template per 25 pi reaction. Amplification was carried out for 33 cycles with a condition of denaturation at 94°C for 60 seconds, annealing at 64°C for 35 seconds and extension at  77  72°C for 90 seconds, and a final extension at 72°C for 15 minutes. The same amount of cDNA of each sample was used for amplification of glyceraldehyde 3-phosphate dehydrogenase (  (GAPDH).  Primers  for  5'-ATGTTCGTCATGGGTGTGAACCA-3'  GAPDH and  5 ' - T G G C A G G T T T T T C T A G A C G G C A G - 3 ' ) were designed based on published sequence [Tokunaga et al., 1987]. Amplification was carried out for 18 cycles with a condition of denaturation at 94°C for 60 seconds, annealing at55°C for 35 seconds and extension at 72 °C for 90 seconds, and a final extension at 72°C for 15 minutes.  Cloning and sequencing of RT-PCR product  Ten ul of PCR products of P2UR were fractionated in a 1% agarose gel stained with ethidium bromide. The expected P C R products (599 bp) were isolated from gel, cloned using the T A cloning kit (Invitrogen) and sequenced by the dideoxy chain termination method using a T7 D N A polymerase sequencing kit (Pharmacia Biotech, Morgan, Canada). The sequence of the cDNA was sent to GenBank at N C B I (National Center for Biotechnology Information) through internet (www.ncbi.nlm.nih.gov) to compare the identity with published human P2UR. This cDNA was then used as the template for making probes for Northern and Southern blot analysis.  Northern blot analysis 78  Approximately 15 pg total R N A was separated by electrophoresis in a 1% agarose-formaldehyde gel and transferred onto a charged nylon membrane (Amersham, Hybond-N) as described in P A R T 2. The Northern blots were incubated in 5X SSPE for 30 min.  They were then transferred to a prehybridization solution of 5X SSPE  containing 50% deionized formamide, 5X Denhardt's, 1% SDS, water and heat-denatured salmon sperm D N A (final concentration, 0.2 mg/ ml). The blots were prehybridized at 42 °C for 3 hours. The radiolabeled P2UR probe was then added to the prehybridization solution. The blots were incubated in the presence of the radiolabeled probe at 42 °C for 16 hours, then washed twice with 2 X SSPE at room temperature (5 min / wash), twice with 2X SSPE containing 1% SDS at 55 °C (30 min / wash), twice with 0.2X SSPE at room temperature (30 min / wash) and finally exposed to Kodak Omat X-ray film.  Southern blot analysis  After sequencing, the cloned cDNA of P2U receptor was used as the template to make digoxigenin (DIG)-labeled probe using a DIG D N A Labeling Kit following the protocol provided by manufacturer (Boehringer Mannheim Laval, Canada). The PCR products in 1 % agarose gel were transferred to Hybond-N nylon membranes (Amersham Inc., Canada) and hybridized with DIG-labeled P2U receptor cDNA. were processed as per the manufacturer's protocol. exposed for 10 min at room temperature to X-ray  79  The membranes  Finally, the membranes were film.  The autoradiograms were  scanned with a laser densitometer (BIO-RAD, Model 620, Video Densitometer) and P2UR mRNA levels were standardized against G A P D H .  Quantification ofP2UR mRNA To compare the expression and regulation of P2UR mRNA, semiquantitative P C R was am  performed.  For validation, various cycles of (27-38) P C R were performed to  plify P2UR mRNA.  G A P D H was used in the present study to normalize the P C R  product of P2UR mRNA.  For G A P D H , 15-27 cycles of P C R were performed for  validation. Southern blot analysis was carried out using equal amount of amplified PCR products (10 pg), and the results were quantified using a laser densitometer.  Microspectrofluorimetry  Human GLCs were seeded onto 25-mm circular glass cover slips (5,000 /slip) and incubated for 3 days at 37 °C in humidified air with 5% C02 prior to microfluorimetric experiments. Cytosolic calcium concentrations were measured using the dual-excitation single-emission fluorimetric technique, as described in PART 2.  To confirm the  presence of functional P2UR in hGLCs, cells were treated with 100 p M of ATP or UTP (Sigma Chemical Co.).  For dose-response experiments, cells were treated with various  concentrations of A T P or UTP (1, 10, and 100 pM), prior to cytosolic calcium determinations.  80  Data analysis  Relative P2UR mRNA levels were expressed as the ratio of P2UR to G A P D H . For each patient, the data are represented as the percent change relative to the control. Data of the same treatment groups are represented as means ±  standard errors (SE).  Statistical analysis was performed by one-way analysis of variance followed by Tukey test. Differences were considered significant at p < 0.05.  3.4 Results  Expression of the P2U receptor mRNA in human granulosa-luteal cells  The expression of P2U receptor mRNA in hGLCs was examined by RT-PCR using one set of primers designed on the basis of the published human P2UR expressed in airway epithelium. The positions and sequences of primers were shown as Fig. 19A. A n expected 599-bp D N A fragment was observed in ethidium bromide-stained gel from hGLCs isolated from 3 different patients (Fig. 19B). No product was obtained from the negative control (without first strain c D N A template in P C R reaction). products from hGLCs were subcloned and sequenced.  81  The P C R  Sequence analysis revealed that  the cloned c D N A is identical to nucleotide position 436-1034 of the published human P2U receptor [Parr, et al., 1994]. This cDNA was then used as a template for making probes for Northern and Southern blot analyses.  Using Northern blot analysis, two  P2UR transcripts of 2.0 kb and 4.6 kb were detected in hGLCs as shown in Fig. 19C.  82  A.  Forward 436  458 1380  250 1012  1034 Reverse  Forward primer: 5 - C C T G G A A T G C G T C C A C C A C A T AT-3 Reverse primer: 5-GACGTGGAATGGCAGGAAGCAGA-3  Fig. 19A. Expression of P2UR mRNA in human granulosa-luteal cells (hGLCs). Demonstration of positions and sequences of primers designed on the basis of published human P2U purinoceptor (P2UR).  This set of primers was used in  polymerase chain reaction (PCR) to amplify P2UR mRNA isolated from hGLCs.  83  Control  Fig  patient 1  patient 2  patient 3  1 9 B . E t h i d i u m b r o m i d e - s t a i n e d D N A gel s h o w i n g the P C R products o f three  patients.  O n e p g o f total m R N A o f h G L C s f r o m each patient was reverse transcribed  into c D N A . a n d aliquots were a m p l i f i e d u s i n g P C R w i t h p r i m e r s s h o w n i n F i g . 1 A . 5 9 9 bp product was obtained i n P C R f r o m three patients. c D N A in P C R .  84  A  C o n t r o l represented w i t h o u t  Patient 1  Patient 2  Patient 3  Fig. 19C. Demonstration of P2UR mRNA in hGLCs by Northern blot analysis. Fifteen ug of total mRNA was loaded and separated by electrophoresis in 1% agarose-formaldehyde gel and transferred onto a charged nylon membrane. Radio-labeled c D N A of P2UR was used as the probe to detect the presence of P2UR mRNA in hGLCs.  Finally, the blot was subjected to radioautography.  Two transcripts of 2.0 kb and 4.6 kb mRNA were detected in this study.  85  Validation of PCR for P2UR transcript  To determine the condition under which the amplification of P2UR was in the logarithmic phase, various cycles of P C R were performed.  Equal aliquots (lpl) of  cDNA were amplified by different P C R cycles. Ten pi of the P C R products were fractionated, transferred onto a charged nylon membrane, detected by DIG-labeled c D N A and finally subjected to radioautography. densitometer.  The results were quantified using a laser  A linear relationship between PCR products and amplification cycles was  observed in both P2UR (Fig. 20) and G A P D H (data not shown). Thirty-three cycles for P2UR and 18 cycles for G A P D H were employed for quantification in subsequent regulation studies.  86  Cycle Number 27  30  33  35  38  -599 bp  d  a  3 2 1  0  1  24  1  27  30  33  36  39  Cycle Number  Fig. 20 Validation of semiquantitative P C R for P2UR in hGLCs. Total R N A of hGLCs was isolated and reverse transcribed in the first strain cDNA.  Equal  aliquots (lul) of cDNA were amplified by different PCR cycles (27-38) as described in Materials and Methods. Ten pi of the PCR products were fractionated by electrophoresis in 1% agarose gel and transferred onto a charged nylon membrane.  DIG-labeled cDNA of P2UR was used as the probe to detect the  expression of P2UR in hGLCs.  After washing, the membrane was exposed to  Kodak Omat X-ray film, and the result was quantified using a laser densitometer. A linear relationship was observed between PCR products and amplification cycles.  87  Regulation of the P2UR mRNA in hGLCs  To examine the regulation of P2UR mRNA, hGLCs were treated with hCG (5 IU/ml), estradiol (IO" M), progesterone (IO" M), ATP(lOuM), prostaglandin F2 a (IO" 7  7  7  M), or GnRH (IO M ) , respectively. As shown in Fig. 21, no significant change of -7  P2UR m R N A levels was observed in the groups treated with estradiol, progesterone, ATP, prostaglandin F2a, or GnRH. In contrast, about 30% decrease of P2UR mRNA (P<0.05) was noted in the hCG-treated group. To further examine the effect of hCG on P2UR expression, hGLCs were treated with increasing concentrations of hCG for 24 hours.  As shown in Fig. 22, hCG down-regulated the level of P2UR mRNA in a  dose-dependent manner.  Further,  a time-course  analysis revealed  that h C G  down-regulated P2UR mRNA in a time-dependent manner (Fig. 23).  It is well  established that hCG activates adenylate cyclase and increases the production of c A M P in ovarian cells. To examine the possible mechanism by which P2UR m R N A is regulated by hCG, hGLCs were treated with 8-bromo-cAMP and forskolin, an activator of adenylate cyclase.  As shown in Fig. 24, both c A M P and forskolin significantly  down-regulated the expression of P2UR mRNA.  88  Control  hCG  PGF  GnRH  P4  E2  ATP  ^mm^m%^mWk$mWlflBmm% mm%  }APDH  1.2  Control  hCG  PGF  GnRH  P4  E2  ATP  Fig. 21 The effect of different reagents on the regulation of P2UR mRNA in cultured hGLCs.  Cells were treated with human chorionic gonadotropin (hCG, 5 IU/ml),  prostaglandin F2a (PGF, IO" M), GnRH (IO" M), progesterone (P4, IO" M), estradiol 7  7  7  (E2, 10" M) or ATP (10 uM) for 24 hours in serum-free condition as described in 7  Materials and Methods. The PCR products (upper panel) were normalized by G A P D H (middle panel). The size of PCR product is shown on the right hand side of upper panel. Data represent the means ± standard error for four separate experiments with samples from four patients.  *, Significantly different from control (p<0.05)  89  Control  0.1  1  5  10  (IU/ml)  -599 bp  control  0.1  1  5  10  H C G (IU/ml) Fig. 22 The dose effect of human chorionic gonadotropin (hCG) on the regulation of P2UR mRNA in cultured hGLCs.  Cells were treated with various concentrations of  hCG (0.1-10 IU/ml) for 24 hours in serum-free condition as described in Materials and Methods.  The P C R products (upper panel) were normalized by G A P D H  (middle panel). The size of PCR product is shown on the right hand side of upper panel. Data represent the means ± standard error for four separate experiments with samples from four patients.  *, Significantly different from control (p<0.05)  90  bp  3  6  12  24  48  Fig. 23 The time effect of human chorionic gonadotropin (hCG) on the regulation of P2UR mRNA in cultured hGLCs.  Cells were treated with 5 IU/ml of hCG for 0-48  hours in serum-free condition as described in Materials and Methods.  The PCR  products (upper panel) were normalized by G A P D H (middle panel). The size of PCR product is shown on the right hand side of upper panel. Data represent the means ± standard error for three separate experiments with samples from four patients. Significantly different from control (p<0.05)  91  *,  Control  cAMP  forskolin  -599 bp  P2UR  GAPDH  1.2 <  1  pi  0.8 0.6  2 0.4 c3  £  0.2 0  Control  cAMP  forskolin  Fig. 24 The effects of 8-bromo-cAMP (cAMP) and forskolin on the regulation of P2UR mRNA in cultured hGLCs.  Cells were treated with c A M P (1 mM) and  forskolin (10 pM) for 24 hours in serum free condition as described in Materials and Methods. The P C R products (upper panel) were normalized by G A P D H (middle panel). The size of PCR product is shown on the right hand side of upper panel. Data represent the means ± standard error for four separate experiments with samples from four patients.  *, Significantly different from control (p<0.05).  92  Effects of ATP and UTP on intracellular calcium mobilization in single cells  The P2UR expressed in hGLCs was tested functionally and pharmacologically using microspectrofluorimetry in single cell studies. As shown in Fig. 25, hGLCs responded equally well to 100 p M A T P and UTP, indicating the expression of a functional P2UR in these cells at the level of calcium signaling. The cytosolic calcium mobilization was characterized by a spike and a marked increase in cytosolic calcium, followed by numerous  oscillations  with  decreasing  amplitudes.  To  examine  further  the  dose-response relationship, hGLCs were treated with increasing concentrations of ATP or UTP (1 - 100 pM). It has been demonstrated that submicromolar concentrations of ATP were incapable of mobilizing cytosolic calcium [Lee PSN et al., 1996]. As shown in Fig. 26, both A T P and U T P were able to induce cytosolic mobilization in micromolar levels with maximal responses reached when treated with lOpM of A T P or UTP, and no difference was noted between cells treated with 10 p M and 100 p M .  93  Fig. 25 Effects of ATP and UTP on inducing cytosolic calcium mobilization in cultured hGLCs using microspectrofluorimetry.  Fura-2 loaded human  granulosa-luteal cells were treated with 100 p M of ATP and UTP. Data of calcium oscillations were presented as ratio (340:380 nm). Both ATP and UTP were able to induce calcium oscillations, and no significant difference was noted between these two treatments in hGLCs.  94  3  1  25  00 en  2  o  o  cn  "—'  o  1.5  1  n _>  0.5  0  (ATP) B.  1 pM  100 p M  10 u M  35  3  1  o  25  ©  2  oo cn  \  cn  ^—^  _o  Is _>  'M "5  1.5  if  —  '--Tk  1 180  ^  0.5  0  (UTP)  1 uM  10 p M  100 p M  Fig. 26 Dose effects of ATP and UTP on inducing cytosolic calcium mobilization in cultured  hGLCs  using microspectrofluorimetry.  A . Fura-2  loaded human  granulosa-luteal cells were treated with various concentrations of ATP (1-100 pM). B.  Fura-2 loaded human granulosa-luteal cells  were treated  with various  concentrations of UTP (1-100 pM). Data of calcium oscillations were presented as ratio (340:380 nm). The results demonstrated that both ATP and UTP were able to induce calcium oscillations in a dose-dependent manner.  95  3.5 Discussion  Sources of extracellular ATP are mainly neuronal in origin. ATP is either released from purinergic nerve endings, or co-released with other neurotransmitter granules such as acetylcholine and noradenaline during neurotransmission [Morel and Meunier, 1981; Winkler and Carmichael, 1982]. The concentration of ATP in adrenergic granules of sympathetic nerves and in acetylcholine-containing granules of parasympathetic nerves can be as high as 150mM [Winkler and Carmichael, 1982]. Exocytotic release of ATP has also been found in non-neuronal cells, including platelets [Born and Kratzer, 1984], adrenal chromaffin cells [Cena and Rojas, 1990], mast cells, and basophilic leukocytes [Osipchuk and Cahalan, 1992]. Although ATP is present in millimolar concentrations in the cytosol, extracellular levels of the nucleotide will normally be maintained at very low levels by the ubiquitous ecto-ATPase and ecto-ATP diphosphohydrolase [Dombrowski et al., 1998; Dubyak and el-Moatassim, 1993; Zimmermann et al., 1998].  Adrenergic and cholinergic nerves have been shown to innervate the ovary and may be involved in the regulation of steroidogenesis [Mohsin and Pennefather, 1979; Burden and Lawrence, 1978; Stefenson et al., 1981].  In human granulosa cells,  epinephrine and norepinephrine have been shown to stimulate progesterone secretion via interaction with the P-adrenergic receptor [Webley, 1988]. In other reproductive tissues, extracellular A T P has been shown to activate contraction in the intact myometrium [Osa and Maruta, 1987].  Amnion cells isolated from the human placenta express A T P  receptors that are coupled to inositol phospholipid breakdown and Ca2+ mobilization  96  [Vander Kooy et al., 1989]. A T P can trigger the acrosome reaction in human sperm in vitro [Foresta et al., 1992].  Following binding to P2-purinergic receptors, A T P can  increase the secretion of testosterone in rat Leydig cell [Foresta et al., 1996].  The human P2UR gene has been mapped to chromosome l l q 13.5-14.1 [Dasari et al., 1996]. The human P2UR cDNA was cloned and sequenced from airway epithelium [Parr et al., 1994]. The P2UR in the human ovary has not as yet been characterized. The present study demonstrates for the first time the expression of P2UR in human ovarian cells. Northern blot analysis revealed that two species of mRNA, 2.0kb and 4.6 kb, were expressed in hGLCs. Interestingly, human uterine cervical cells express at least four distinct transcripts, 2.0, 2.2, 3.0 and 4.6 kb [Gorodeski et al., 1998], while human nasal and proximal-tubule epithelia and liver express only a single 2.1 kb mRNA [Parr et al., 1994]. The expression of P2UR in hGLCs supports the hypothesis that extracellular ATP might play a role in the regulation of ovarian function.  Relatively few studies have focused on the regulation of P2UR mRNA in response to hormone treatments.  For example, retinoids have been shown to regulate the  expression of P2UR mRNA in human uterine cervical cells [Gorodeski et al., 1998]. In the present study, hGLCs were treated with estradiol, progesterone, PGF2oc, GnRH, A T P and hCG. The result shows that only hCG attenuated the expression of P2UR mRNA in these cells, suggesting that L H / h C G may play a role in regulating the expression of P2UR in the human ovary. It is well established that activation of L H / C G receptor activates adenylate cyclase and P K A [Lustbader et a l , 1998]. To further elaborate the mechanism  97  by which h C G regulates the expression of P2UR mRNA, hGLCs were treated with exogenous 8-bromo-cAMP and forskolin, an activator of adenylate cyclase. Our results show that both 8-bromo-cAMP and forskolin markedly down-regulated the expression of P2UR mRNA levels, supporting the notion that hCG down-regulation of the expression of P2UR mRNA may be mediated by adenylate cyclase and c A M P . Recently, we and others have shown that h C G can alter the mRNA levels of GnRH receptor and P G F 2 a receptor [Peng et al., 1994; Ristmaki et al., 1997; Vaananen et al., 1998] in hGLCs.  There appears to be a complex interaction of GnRH and P G F  2 a  on steroid  hormone production in hGLCs [Vaananen et al., 1997]. In the present study, hCG, but not GnRH or PGF2cc, has been demonstrated to down-regulate the expression of P2UR in dose- and time-dependent manners.  The physiological significance of the hCG effect on  P2UR remains to be determined. It has been reported that A T P may act as a trigger for apoptosis or programmed cell death [Zheng et al. 1991], and that A T P at a concentration of 2.0 m M causes cell necrosis and death in the ovary [Charming, 1970].  It is  conceivable that hCG is capable of minimizing the detrimental effect of A T P , at least in part, by down-regulation of P2UR expression in hGLCs.  ATP has been shown to induce cytosolic calcium oscillations in human granulosa-luteal cells [Fredholm et al., 1994; Lee PSN et al., 1996; Squires et al., 1997]. Pharmacologically,  the  order  of  agonist  potency  for  P2UR  ATP=UTP>ATPyS»2MeSATP [Kamada et al., 1994; Harden et al., 1995].  is  It also  has been demonstrated that the cytosolic calcium oscillations evoked by A T P are initiated by the release of calcium from cytosolic stores and maintained by extracellular calcium  98  influx [Lee P S N et al., 1996]. In the present study, we confirmed the presence of a functional P2UR in hGLCs. Further, our results clearly indicate that, like ATP, UTP is also capable of evoking cytosolic calcium mobilization in a dose-dependent manner. These data provide further evidence that the P2UR expressed in hGLCs is functional following receptor activation by the ligand, at the level of signal transduction.  In summary, our results demonstrate for the first time the expression of P2UR in the human ovary at the mRNA level.  We have determined that the level of P2UR  mRNA is down-regulated by hCG, presumably via a cAMP-mediated mechanism. The P2UR expressed in hGLCs is functional, in terms of calcium signaling. Taken together, these findings further support a role for ATP and the P2UR in the regulation of human ovarian function.  99  PART 4 ADENOSINE CALCIUM  TRIPHOSPHATE-EVOKED OSCILLATIONS  GRANULOSA-LUTEAL  CELLS:  CYTOSOLIC  IN ROLE  HUMAN OF  PROTEIN  KINASE C  4.1 Abstract  Adenosine triphosphate (ATP) has been shown to modulate progesterone production in human granulosa-luteal cells (hGLCs) in vitro. After binding to a G-protein coupled P2 purinergic receptor, ATP stimulates phospholipase C. The resultant production of diacylglycerol and inositol triphosphate  activates protein kinase C (PKC) and  intracellular calcium [Ca^+Ji mobilization, respectively.  In the present study, we  examined the potential cross-talk between the P K C and C a 2 pathway in ATP signal +  transduction.  Specifically, the effect of P K C on regulating ATP-evoked [Ca^ ]i +  oscillations were examined in hGLCs.  Using microspectrofluorimetry, [Ca2 ]i +  oscillations were detected in Fura-2 loaded hGLCs in primary culture. The amplitudes of the ATP-triggered [Ca^+Ji oscillations were reduced in a dose-dependent manner by pretreating the cells with various concentrations (1 n M to 10 pM) of the P K C activator, phorbol-12-myristate-13-acetate (PMA).  Ten p M of P M A completely suppressed 10 100  u M ATP-induced oscillations. The inhibitory effect occurred even when P M A was given during the plateau phase of ATP evoked [ C a ] i oscillations, suggesting that 2+  extracellular calcium influx was inhibited. The role of P K C was further substantiated by the observation that, in the presence of a P K C inhibitor, Bisindolylmaleimide I, ATP-induced  [Ca2+]i  oscillations were  not  completely  suppressed  by P M A .  Furthermore, homologous desensitization of ATP-induced calcium oscillations was partially reversed by Bisindolylmaleimide I, suggesting that activated P K C may be involved in the mechanism of desensitization.  These results demonstrate that P K C  negatively regulates the ATP-evoked [Ca2+]i mobilization from both intracellular stores and extracellular influx in hGLCs and further support a modulatory role of ATP and P2 purinoceptor in ovarian steroidogenesis.  101  4.2 Introduction  Adenosine triphosphate (ATP), released from autocrine nerves by exocytosis, activates  phospholipase  C (PLC) through binding to a G protein-coupled P2  purinoceptors. This activation leads to the production of diacylglycerol and inositol 1,4,5-triphosphate,  which in turn activates protein kinase C (PKC) and mobilizes  intracellular calcium ([Ca ]i), respectively [Berridge, 1984; Gordon 1986]. Through 2+  this signaling pathway, ATP may participate in various types of physiological responses, including secretion, membrane  potential, cell proliferation, platelet  aggregation,  neurotransmission, cardiac function and muscle contraction [el-Moatassim et al., 1992; Burnstock, 1990].  Protein kinase C, a serine-threonine  kinase which can be activated by  tumor-promoting phorbol esters, has been shown to play a key role in intracellular signaling and regulate a wide range of cell functions [Hug and Sarre, 1993; Nishizuka, 1992]. In many systems, P K C has been shown to regulate calcium channel activity and modulate calcium signaling pathway [Nishizuka, 1992; Berridge, 1991; Tsien and Tsien, 1990]. In the ovary, activated P K C has been reported to alter ATP-triggered intracellular calcium oscillations in chicken granulosa cells [Morley et al., 1996] and inhibit steroidogenesis in swine granulosa cells [Veldhuis and Demers, 1986]. Recently, ATP has been shown to evoke calcium oscillations and regulate steroidogenesis in human granulosa cells [Kamada et al., 1994; Lee et al., 1996; Squires et al., 1997]. However, the cross-talk between the ATP-triggered P K C and C a  102  2 +  signaling pathways in the  human ovary is not understood. The present study was designed to examine the potential effect of P K C in the regulation of ATP-trigger calcium oscillations in human granulosa-luteal cells (hGLCs).  As well, the role of P K C in the homologous  desensitization of ATP-triggered calcium oscillations was investigated.  4.3 Materials and Methods  Reagents and Materials  ATP and phorbol- 12-myristate-13-acetate (PMA) were obtained from Sigma Chemical Co. (St. Louis, MO).  Dulbecco's Modified Eagle Medium (DMEM),  penicillin-streptomycin and fetal bovine serum (FBS) were purchased from GIBCO-BRL (Burlington, Ontario, Canada). Fura-2 A M was purchased from Molecular Probes (Eugene,  OR).  Bisindolylmaleimide I, a P K C inhibitor, was  obtained  from  C A L B I O C H E M (Cedarlane, Ontario, Canada).  Human granulosa-luteal cells (hGLCs) in culture  Human GLCs were collected from patients undergoing In Vitro Fertilization Embryo Transfer (IVF-ET) program and processed as mentioned in PART 2. Human  103  GLCs were seeded onto 25-mm circular glass cover slips (5,000 cells /slip) and incubated for 3 days at 37 °C in humidified air with 5% C02 prior to microfluorimetric experiments [Lee PS etal., 1996].  Microspectrofluorimetry  Cytosolic calcium concentrations were measured  using the dual-excitation  single-emission fluorimetric technique, as described previously.  Treatments  To examine the effect of ATP on inducing intracellular calcium oscillations, hGLCs were treated with various concentrations of ATP (1, 10, or 100 uM) prior to cytosolic calcium determinations on day 3. Further, hGLCs were cultured for various days (3,5 or 7 days) prior to 10 u M ATP treatment.  To investigate the effect of P K C on regulating ATP-triggered calcium oscillations, hGLCs  were  treated  with  various  concentrations  of  PKC  activator,  phorbol-12-myristate-13 -acetate (PMA) (1 , 10, 100 n M , 1 or 10 uM) for 5 min, followed by treatment with 10 u M ATP for 3 min.  104  To further investigate the role of P K C in the regulation of ATP-induced calcium oscillations, hGLCs were pretreated with 1 p M Bisindolylmaleimide I, a P K C inhibitor [Toullec et al., 1991], for 2 minutes prior to P M A and ATP stimulation as performed in the previous experiments.  It has been demonstrated that the intracellular calcium changes are initiated by the release of calcium from cytosolic stores and followed by extracellular calcium influx. To examine whether P K C affects the calcium influx in ATP-evoked calcium mobilization, cells were treated with P M A during the plateau phase of calcium oscillations.  To examine the role of P K C in homologous desensitization of ATP-evoked calcium oscillations, P M A was administered between two A T P treatments. In addition, hGLCs were treated repeatedly with A T P in the absence or presence of Bisindolylmaleimide I.  Data analysis  Data were shown as means of three individual experiments and presented as the mean ±SD. The data were analyzed by one way A N O V A followed by Tukey test. Data were considered significant when P< 0.05.  105  4.4 Results  Induction of cytosolic calcium oscillation by ATP in hGLCs  Human GLCs were treated with various concentrations of ATP (1, 10, or 100 uM) for 3 min. Our results showed that ATP triggered calcium oscillations in these cells (Fig. 27A).  The response to ATP was characterized by a spike and a marked increase in  cytosolic calcium, followed by numerous oscillations with decreasing amplitudes to pre-activated levels. As shown in Fig. 27A, ATP induced cytosolic mobilization in a dose-dependent manner, with maximal response reached when treated with lOpM of ATP, and no difference was noted between cells treated with 10 p M and 100 p M . Fig. 27B demonstrated the effects of 10 p M ATP on inducing calcium mobilization in hGLCs with various culturing days.  There were no significant difference in both of the patterns and  amplitudes of ATP-evoked calcium oscillations.  106  A.  (ATP)  1 uM  10 u M  100 u M  Fig. 27A Effects of ATP on inducing cytosolic calcium oscillations in cultured hGLCs.  Fura-2 loaded hGLCs were treated with various concentrations of ATP  (1-100 uM). Data of calcium oscillations were presented as ratio (340:380 nm).  107  B.  Day 3  Day 3  Day 5  25  2-  s  1.5 -  o 'to  1 -  ativ  c  j  0.5 -  cr  Day 7 ATP  "33  cr  o •!•  Day 7  Fig. 27B. Effects of 10 u M ATP on hGLCs cultured for various days (Day 3-Day 7). Data of calcium oscillations were presented as ratio (340:380 nm).  108  The role of PKC in ATP-triggered calcium oscillations in hGLCs  To determine the role of activated P K C in ATP-triggered calcium oscillations, hGLCs were pre-treated with increasing concentrations of P K C activator, P M A (1 n M , lOnM, lOOnM, 1 u M or 10 uM) for 5 min, and then stimulated with 10 u M ATP. As shown in Fig. 28, P M A pretreatment reduced the amplitudes of ATP-induced calcium oscillations in a dose-dependent manner. Complete inhibition of initial [Ca^+Ji spike was noted when cells were pretreated with 10 u M P M A .  To further examine the role of P K C in the regulation of ATP-triggered calcium oscillations,  Fura-2  loaded  hGLCs  were pretreated  in sequence with  1 uM  Bisindolylmaleimide I for 2 min, and Bisindolylmaleimide I plus 10 u M P M A for 5 min, prior to treatment of 10 u M ATP. The results revealed that, in contrast to pretreatment with P M A alone (Fig. 28F), ATP induced calcium oscillations when the cells were pretreated with both P M A and the P K C inhibitor (Fig. 29).  To examine if P M A affects the calcium influx in ATP-evoked calcium mobilization, cells were treated with 10 u M P M A during the plateau phase of ATP-triggered calcium oscillations. The result, when compared with control (Fig. 3 OA), demonstrated that the amplitudes of calcium oscillations of PMA-treated cells declined to baseline level abruptly (Fig. 30B), suggesting that calcium influx was inhibited by activated P K C .  B. 2.5 2  1.5  1 0.5 0  P M A (1 nM) I  D. .15  I  2  •I 0.5  I  ATP PMAXJ,00 nM) ii  Di  E.  T ii  25  1.5 ATP 0.5  Di  P M A (1 \M)  Fig. 28 Dose-dependent effects of P M A on ATP-evoked cytosolic calcium oscillations in  cultured hGLCs.  Fura-2 loaded hGLCs  were pre-treated  with various  concentrations of P M A (1 n M - 10 p M , B-F) for 5 min prior to treatment with 10 p M ATP. Data of calcium oscillations were presented as ratio (340:380 nm). 110  Effect of PKQ 1.6 1  "~~  Fig. 29 The role of P K C in ATP induced-calcium oscillations in cultured hGLCs.  Fura-2 loaded hGLCs were pretreated in sequence with 1 u M  Bisindolylmaleimide I (PKCI) for 2 min, and Bisindolylmaleimide I plus 10 u M P M A for 5 min, prior to treatment with 10 u.M ATP. Data of calcium oscillations were presented as ratio (340:380 nm).  ill  A. ATP evoked calcium oscillations 2.5 O CO OO  <Z5 T OO  1.5  Of  •3  0.5  180 sec  •ATP(lOulvl)  0  Fig. 30 The effect of P M A on ATP-evoked cytosolic calcium oscillations in cultured hGLCs.  A . The biphasic pattern of ATP-induced cytosolic calcium  oscillations in cultured hGLCs, which was initiated by the release of calcium from cytosolic store and followed by extracellular calcium influx.  B . Fura-2 loaded  hGLCs were treated with 10 u M P M A during the plateau phase of calcium oscillations. Data of calcium oscillations were presented as ratio (340:380 nm). 112  The role of PKC in homologous desensitization of ATP-triggered calcium oscillations in hGLCs  Calcium replacement is required to maintain cytosolic calcium oscillations during repeated ATP treatments [Squires et al., 1997]. In the present study, treatment of hGLCs with P M A completely suppressed the subsequent ATP-induced calcium oscillations (Fig. 31 A), suggesting that activated P K C may play a role in mediating homologous desensitization.  In addition, calcium oscillations were partially reversed during  subsequent exposures of hGLCs to ATP in the presence of Bisindolylmaleimide I (Fig. 31C),  when  compared  with  repeated  ATP exposures  in  the  absence  of  Bisindolylmaleimide I (Fig, 31B), supporting the proposal that P K C may be involved in homologous desensitization of ATP-triggered calcium oscillations.  113  A.  2.5  Fig. 31 A The role of P K C in homologous desensitization of ATP induced-calcium oscillations in cultured hGLCs. Fura-2 loaded hGLCs were treated with 10 p M P M A for 5 min following exposure to ATP. No cytosolic calcium oscillations were induced by subsequent ATP treatment.  114  B.  c.  Fig. 31B The role of P K C in homologous desensitization of ATP induced-calcium oscillations in cultured hGLCs.  B . and C. The effect of P K C was observed in the  absence or presence of P K C inhibitor, Bisindolylmaleimide (PKCI) during repeated treatment of ATP in hGLCs. Data of calcium oscillations were presented as ratio (340:380 nm).  Data represent the means ± standard error.  different from control (p<0.05)  115  *, Significantly  4.5 Discussion  ATP, released from nerve endings, has been shown to participate in various types of physiological responses [el-Moatassim et al., 1992; Burnstock, 1990; Owman et al., 1967; Bodis etal., 1993a; Bodis etal., 1993b].  It  is  tempting  to  speculate that  the  co-released ATP from autonomic nerve endings in the ovary may play a role in regulating ovarian function.  A T P has been shown to regulate the production of progesterone and  estradiol in hGLCs [Kamada et al., 1994]. We have reported previously that the P2U purinoceptor is expressed in hGLCs [Tai et al., 2000], further supporting a physiological role of A T P in the human ovary.  Calcium, a second messenger, has been shown to mediate several physiological activities including fertilization, embryo development, cell proliferation and cell death [Berridge et al., 1998]. As demonstrated in this study, A T P is able to mobilize cytosolic calcium, implicating a role of ATP in the control of ovarian function. This finding leads us  to  postulate  that  several  calcium  dependent  kinases  such  as  P K C or  Ca /calmodulin-dependent protein kinase [James and Putney, 1998] may be involved in 2+  regulating cellular function.  However, the precise role of calcium oscillations is not  clear yet [Tsien and Tsien, 1990]. P K C has been reported to modulate the activities of ion channels including calcium channels and potassium channels [Shearman et al., 1989]. In addition, P K C has been shown to modulate cytosolic calcium and c A M P levels induced by activation of P2U-purinergic receptor on rat glioma cells [Munshi et al., 1993].  In the ovary, P K C has been reported to modulate ATP-evoked calcium  116  oscillation in chicken granulosa cells, supporting the notion that the calcium oscillations were reduced by either activation or inhibition of P K C activity [Morley et al., 1996]. In the present study, a role of P K C in regulating ATP-induced calcium oscillations was revealed in hGLCs. negatively regulated  Our results demonstrate that the activation of P K C activity the ATP-evoked cytosolic calcium mobilization from  both  intracellular stores and extracellular influx in cultured hGLCs. Pretreatment with a P K C inhibitor reversed the inhibitory effect of activated P K C , further supporting the role of PKC in ATP-evoked calcium oscillations in the human ovary.  ATP has been shown to effect a homologous desensitization of ATP-receptors [Dickenson and Hill, 1993]. Homologous desensitization is characterized by a reduced response to an agonist due to repeated treatments with the same agonist.  It has been  suggested that P K C activated by agonists may be involved in the mechanism of desensitization in several studies [Dickenson and Hill, 1993; Brown et al., 1987; Jones et al., 1990]. ATP-evoked calcium oscillations are dependent upon calcium mobilization from both cytosolic stores and extracellular influx. Calcium replacement is required to maintain cytosolic calcium oscillations during repeated A T P treatments [Squires et al., 1997]. However, the calcium replacement still cannot prevent the down-regulation of the amplitudes of oscillations during repeated A T P treatments, implying that another regulator exists.  Several other studies have linked this type of desensitization with  activated P K C [Munshi et a l , 1993; Wilkinson et a l , 1994]. In many systems, P K C has been shown to regulate calcium channel activity and modulate calcium signaling pathway [Nishizuka, 1992; Berridge, 1991; Tsien and Tsien, 1990]. In the present study, repeated treatment of ATP decreased the amplitude of the initial spike of calcium oscillations, 117  which can be partially reversed by pretreatment with P K C inhibitor.  This result  indicates that ATP induced homologous desensitization in calcium oscillations in hGLCs.  The mechanism of P K C in regulating calcium oscillations is not clear.  Several  proteins in the A T P signal transduction pathway can be proposed to act as potential targets of activated P K C .  Considering several potential phosphorylation sites in P2U  purinoceptor [Lustig et al., 1996], the P2U purinoceptor function may be affected by activated P K C (Fig. 32-®). This proposal is supported by the finding that phorbol ester, a P K C activator, can inhibit the function of G-protein-coupled receptor [Leeb-Lundberg et al., 1985; Thomopoulos et al., 1982]]. With respect to receptor-coupled G-proteins, several studies have shown that phorbol ester can regulate G-protein-mediated responses Orellana et al., 1987; Sagi-Eisenberg, 1989; Krishnamurthi et al., 1989], indicating that P2UR-coupled G-protein may be inhibited by activated P K C in hGLCs (Fig. 32-©). In addition, activated P K C has been identified to attenuate agonist-induced inositol phospholipid hydrolysis [Zavoico et al., 1985; Dubyak, 1986; Ryu et al., 1990], suggesting that agonist-stimulated phospholipase C may be desensitized through a negative feedback involving the activation of P K C (Fig. 32-®). Inositol triphosphate (IP3), a product of inositol phospholipid hydrolysis, binds to IP3 receptors on endoplasmic reticulum and induces the release of calcium from the intracellular stores. P K C has been shown to phosphorylate a serine site on IP3 receptors [Ferris et al., 1991; Ferris et al., 1992], implying that activated P K C may shut down cytosolic calcium mobilization through inactivation of the function of IP3 receptors (Fig. 32-®).  Calcium  influx from the extracellular environment plays a critical role in maintaining the plateau phase following an initial peak of cytosolic calcium oscillations [Lee et al., 1996]. 118  Protein kinase C has been demonstrated to down-regulate or alter calcium influx in agonist-induced calcium mobilization in different systems [Shearman et al., 1989; D i Virgilio et al., 1986; Rane and Dunlap, 1986; Clunes and Kemp, 1996] (Fig. 32-©). Based on above findings, it can be proposed that the ATP-activated P K C may feedback at different levels intracellularly, including the P2U purinoceptor, G-protein, phospholipase C, IP3 receptor or calcium channel, culminating in a shutdown of the calcium signaling pathway in hGLCs.  119  Fig. 32 A proposed model of the potential cross-talk between ATP-activated protein kinase C (PKC) and cytosolic calcium oscillations in hGLCs. P2UR = P2U purinoceptor on cell membrane; G = G-protein; P L C = phospholipase C; PIP2 = phosphatidyl-inositol 4,5-bisphosphate; D A G = diacylglycerol; IP3 = inositol 1,4,5-triphosphate; IP3R = IP3 receptor.  120  In conclusion, our results demonstrated that (1) A T P was capable of inducing calcium oscillation in human granulosa-luteal cells in a dose-dependent manner,  (2)  P K C negatively regulated the ATP-evoked [ C a ] i mobilization from both intracellular 2+  stores and extracellular influx in cultured hGLCs, and ATP-induced homologous desensitization in hGLCs.  (3) P K C was involved in  Taken together, these results  indicate that A T P may exert a feedback regulation on its own signaling pathway through activation of P K C in the human ovary.  121  PART 5  ANTIGONADOTROPIC GRANULOSA-LUTEAL  ACTION OF A T P IN H U M A N CELLS:  INVOLVEMENT  OF  PKCa  5.1 Abstract The presence of P2U purinoceptor in human granulosa-luteal cells (hGLCs) indicates the potential role of ATP in regulating ovarian function.  Human chorionic  gonadotropin (hCG) exerts its action via increasing the accumulation of intracellular cAMP. In this study, the inhibitory effect of ATP on hCG-induced c A M P production was observed.  Extracellular ATP has been shown to activate protein kinase C (PKC)  after binding to a purinoceptor.  To understand the role of P K C in mediating ATP action,  hCG-stimulated c A M P level was examined in the presence of P K C activator, 1 u M phorbol- 12-myristate-13-acetate (PMA), or P K C inhibitor, 1 u M staurosporin or 1 u M bisindolylmaleimide I. c A M P production.  P M A , like 10 u M ATP, significantly reduced hCG-evoked  In addition, the inhibitory effect of ATP was reversed by  staurosporin and bisindolylmaleimide I. To further investigate the involvement of P K C isoforms in mediating inhibitory effect of ATP, the presence of P K C isoforms in cultured hGLCs was examined by Western blot using monoclonal antibodies against specific isoforms. As well, translocation of P K C isoform from cytosolic fraction to membrane  122  fraction was studied to identify the active P K C isozyme subsequent to ATP treatment, and the change of P K C isoform in PKC-depleted cells (achieved by exposure to P M A for 18 h) was examined.  Our results demonstrated the presence of P K C a , 8, i and X  isoforms in hGLCs and the translocation of P K C a subsequent to ATP treatment.  In •  PKC-depleted cells, P K C a level was reduced, and no significant effect of ATP on hCG-stimulated c A M P production was noted.  To our knowledge, this is the first  demonstration of P K C isoforms in hGLCs and the involvement of activated P K C in mediating the antigonadotropic effect of extracellular ATP. Taken together, these results further support a role of this neurotransmitter in regulating ovarian function.  123  5.2 Introduction  Binding of human chorionic gonadotropin (hCG) with the L H / C G receptor activates adenylate cyclase, leading to the production of c A M P which is the intracellular messenger mediating several cellular functions [Lustbader et al., 1998; Marsh, 1976; Marsh, 1975; Furger et al., 1996].  Adenosine triphosphate (ATP) is released from cells such as platelets or co-released with neurotransmitter granules from autocrine nerves by exocytosis [Gordon, 1986]. ATP has been shown to participate in various types of physiological responses, including secretion, membrane potential, cell proliferation, platelet aggregation, neurotransmission, cardiac function, and muscle contraction [el-Moatassim et al., 1992; Burnstock, 1990; Owman et al., 1967; Bodis et al., 1993a; Bodis et al., 1993b]. It is tempting to speculate that the co-released ATP from autonomic nerve endings in the ovary may play a role in regulating ovarian function. We have reported previously that the P2U purinoceptor is expressed in hGLCs [Tai et al, 2000a], further supporting a physiological role of A T P in the human ovary.  After binding to a G protein-coupled P2 purinoceptor, extracellular A T P activates phospholipase C and phosphatidylinositol hydrolysis, generating diacylglycerol and inositol 1,4,5-triphosphate,  which stimulate protein kinase C (PKC) and cytosolic  calcium mobilization, respectively [el-Moatassim et al., 1992; Tai et al., 2000a; Berridge, 1984].  The P K C family, a group of widely distributed serine/threonine kinases,  124  mediates intracellular signaling of numerous cellular regulators including hormones, neurotransmitters  and growth factors [Nishizuka, 1984; Berridge, 1993].  Thirteen  isozymes have been identified and categorized into four subclasses: (a) conventional protein kinase Cs (a,  [31, [311 and y) which are regulated  by diacylglycerol,  phosphatidylserine and C a , (b) novel protein kinase Cs (8, s, 0 and n) which are 2 +  regulated by diacylglycerol and phophatidylserine, (c) atypical protein kinase Cs (£, i and A.) whose regulation have not been clearly established, and (d) a fourth subfamily, u. and v [Newton, 1997; Jaken, 1996; Hayashi et al., 1999]. It is noteworthy to mention that multiple and various P K C isoforms are present in the ovary of different species. In the rabbit corpus luteum, a, p and 8 isoforms of P K C are identified [Maizels et al., 1992], while porcine corpora lutea contain a and p [DeManno et al., 1992].  Western blot  analysis reveals that bovine corpus luteum expresses a and 8 [Orwig et al., 1994].  In the present study, we demonstrated that A T P reduced hCG-induced c A M P accumulation in human granulosa-luteal cells (hGLCs). To establish the mechanism, we examined the effect of P K C on hCG-induced c A M P production, the expression of P K C isozymes and the translocation of P K C isozyme subsequent to A T P treatment.  125  5.3 Materials and Methods  Reagents and Materials  ATP,  staurosporin,  human  chorionic  gonadotropin  (hCG)  and  phorbol-12-myristate-13-acetate (PMA) were obtained from Sigma Chemical Co. (St. Louis, MO).  Dulbecco's Modified Eagle Medium (DMEM), penicillin-streptomycin and  fetal bovine serum (FBS) were purchased from GIBCO-BRL (Burlington, Ontario, Canada), bisindolylmaleimide I, a P K C inhibitor, was obtained from C A L B I O C H E M (Cedarlane, Ontario, Canada).  Human granulosa-luteal cells culture  Human GLCs were collected from patients undergoing an In Vitro Fertilization Embryo Transfer program and processed as mentioned in PART 2.  Radioimmunoassay for intracellular cAMP  To determine the effect of ATP on hCG-induced intracellular c A M P accumulation, hGLCs were incubated in serum-free medium containing 0.1% B S A and 0.5 m M 3-isobutyl-lmethylxanthine (IBMX, Sigma-Aldrich Corp.) for 30 min. Human GLCs  126  were min  then treated with hCG (1 IU/mL) in the presence or absence of ATP (10 uM) for 20  . Human GLCs were lysed with 100% ethanol. Intracellular c A M P levels were  measured using the [ H]-cAMP assay system, following the protocol provided by 3  manufacturer (Amersham Pharmacia Biotech).  Treatment for cyclic AMP assay  To investigate the role of P K C in hCG-evoked c A M P accumulation, hGLCs were treated with 1 IU/ml hCG in the presence or absence of 1 u M P M A , a P K C activator. To understand the involvement of P K C in the effect of ATP on hCG-induced c A M P production, hGLCs were treated with ATP plus hCG in the presence or absence of P K C inhibitor (1 u M staurosporin or 1 u M bisindolylmaleimide I). In this study, hGLCs were treated with staurosporin or bisindolylmaleimide I for 15 min followed by the administration of ATP.  Western blot analysis  To establish the expression of P K C isozymes, hGLCs were processed as described in PART 2.  Aliquots (30 ug) were subjected to  10% SDS-polyacrylamide gel  electrophoresis under reducing condition, as previously described [Laemmli, 1970]. The proteins were then electrophoretically transferred from the gels onto nitrocellulose  127  membranes (Amersham Pharmacia Biotech, Oakville, Canada) according to the procedures of Towbin et al. [Towbin et al., 1979]. These nitrocellulose membranes were probed with a mouse monoclonal antibody directed against the P K C isozymes (Transduction Laboratories, Lexington, K Y ) at 4 C for 16 h.  After washing, the  membranes were incubated with HRP-conjugated goat-anti mouse secondary antibody, and the signal was visualized using E C L system (Amersham Pharmacia Biotech, Oakville, Canada) followed by autoradiogrphy.  Reverse Transcription-Polymerase Chain Reaction (RT-PCR)  In view of PKCy being expressed mainly in the nervous system [Nishizuka, 1988], and the monoclonal antibody for P K C a may cross-react with PKCy, a set of primers, as reported previously, was used to examine the existence of PKCy in hGLCs [Moore et al, 1999]. Total R N A was isolated from hGLCs as mentioned before [Tai et al., 2000a]. As a positive control, the mRNA from human antral gastrin cells was kindly provided by Dr. Buchan [Moore et al., 1999].  Translocation experiment of PKC isozymes  Human GLCs were incubated in serum-free medium for 4 h prior to treatment. To examine the translocation of activated P K C , hGLCs were treated with 10 u M ATP for 1  128  or 5 min. Fractionation of cytosolic and membrane proteins was performed as described previously [Tippmer et al., 1994]. In brief, cells were harvested in test buffer (10 m M Tris/HCl pH 7.4, 250 m M sucrose, 2 m M EDTA, 10 m M E G T A , 2mM dithiothreitol, 1000 U/ml aprotinin, 0.8 pg/ml leupeptin, 2mM PMSF), disrupted by three freeze/ thaw steps, and centrifuged at 17,000 xg for 30 min.  The supernatant was collected as  cytosolic fraction. The pellet was redissolved in lysis buffer (20 m M Hepes/ NaOH pH 7.4, 150 m M NaCI, 1% Triton X-100, 10% glycerol, 8 m M E G T A , 15 m M MgCl2, 2 m M PMSF) and centrifuged at 17,000 xg at 4 °C for 30 min. The supernatant was collected as membrane fraction. Equal amounts of cytosolic and membrane proteins (20 pg) were loaded for Western blot analysis. The translocation of P K C isoforms was detected using monoclonal antibodies against P K C a , 5, i or A,.  PKC depletion  Long term treatment of P M A (16 h) is associated with P K C depletion [Abayasekara, 1993a]. In the down-regulation experiment, hGLCs were pretreated with 1 p M P M A for 18 h prior to the treatment. Separate studies were performed to examine the expression of P K C a and the effect of ATP on hCG-induced c A M P accumulation.  129  Statistical Analysis  Intracellular c A M P levels were shown as pmole per 2 x 10 cells/dish. Data were 5  represented as means ± standard errors (SE). Statistical analysis was performed by one-way  analysis of variance followed  by Tukey's multiple comparison test.  Differences were considered significant at p < 0.05.  130  5.4 Results  Effect of ATP on hCG-induced cAMP production  ATP has been demonstrated to increase intracellular c A M P production by activating adenylyl cyclase in several cell systems [Communi et al, 1997; Conigrave et al., 1998]. To examine the effect of ATP on intracellular c A M P production, hGLCs were treated with 10 u M ATP, ATP plus hCG or 1 IU/mL hCG alone for 20 min. As demonstrated in Fig. 33, h C G markedly increased intracellular c A M P level.  In contrast, ATP did not  increase intracelluar c A M P accumulation in hGLCs, when compared with control group. This result indicates that the P2U purinoceptor expressed in hGLCs is not coupled to adenylyl cyclase. Instead, ATP reduced hCG-evoked c A M P production by 40 %, when compared to hCG treatment alone.  131  Control  Fig. 33 The effect  ATP  hCG  ATP+hCG  of ATP on hCG-stimulated intracellular c A M P  production in human granulosa-luteal cells (hGLCs). Human GLCs were treated with hCG (1 IU/ml) in the presence or absence of ATP (10 pM) for 20 min as described in the Materials and Methods. Samples were assayed in triplicates following manufacturer's protocol. Values were presented as the Mean ± SE of three individual experiments.  Differences were  considered significant at p < 0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. hCG.  132  The role of PKC in hCG-induced intracellular cAMP accumulation  Phorbol ester has been shown to activate protein kinase C in human ovarian tissue [Kawai et al., 1985].  When hGLCs were treated with 1 u M P M A , hCG-stimulated  c A M P production was reduced by 30 % (Fig. 34).  To further investigate the role of P K C , hGLCs were treated with ATP plus hCG in the  presence  or  absence of P K C inhibitor (1  u M staurosporin  or  1 uM  Bisindolylmaleimide I). As shown in Fig. 35 & 36, the inhibitory effect of ATP on hCG-evoked c A M P production was reversed by P K C inhibitors, supporting the active involvement of P K C in the regulation of c A M P production.  133  Fig. 34 The effect of P M A on hCG-stimulated intracellular c A M P production in human granulosa-luteal cells (hGLCs). Human GLCs were treated with hCG (1 IU/ml) in the presence or absence of P M A (1 uM) for 20 min as described in the Materials and Methods. Samples were assayed in triplicates following the manufacturer's protocol. presented as the Mean±SE of three individual experiments.  Values were Differences  were considered significant at p < 0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. hCG.  134  600  Control  ATP  hCG  ATP+hCG  ST+ATP+hCG  Fig. 35 The role of staurosporin (ST) in the inhibitory effect of A T P on hCG-stimulated c A M P production. Human GLCs were treated with h C G plus A T P in the presence or absence of staurosporin (1 pM). Values were presented as the M e a n ± S E of three individual experiments.  Differences were considered  significant at p < 0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. h C G  135  Fig. 36 The role of bisindolylmaleimide I (Bis) in the inhibitory effect of A T P on hCG-stimulated c A M P production.  Human GLCs were treated with hCG plus A T P  in the presence or absence of bisindolylmaleimide I (1 uM). Values were presented as the Mean±SE of three individual experiments.  Differences were considered  significant at p < 0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. hCG  136  Expression of PKC isozymes in hGLCs  Eight antibodies against various P K C isozymes were used for the Western blot analysis. When compared with the positive control, five (a, y, 8, t and X) isoforms were identified by showing bands of the expected sizes (Fig. 37). PKCp, s and 9 were absent in hGLCs.  In the present study, the positive controls were Jurkat cells for PKC9 and rat  brain for the rest of P K C isoforms.  Considering that the monoclonal antibody against PKCy may crossreact with P K C a , and PKCy is found mainly in the nervous system [Nishizuka, 1988], RT-PCR was performed to examine the presence of PKCy in hGLCs. absence of PKCy in hGLCs.  Our results demonstrated the  Primers specific to PKCy amplified a band of the expected  size from the positive control, human antral gastrin cells, but not from hGLCs (Fig. 38). This observation ruled out the existence of PKCy in hGLCs.  137  PKC isoforms in human granulosa-luteal cells  a  GLC  Cnt  P  GLC  X  y  Cnt  GLC  Cnt  GLC  Cnt  S  6  G L C Cnt  G L C Cnt  GLC  Cnt  Fig. 37 The presence of PKC isoforms in human granulosa-luteal cells.  G L C Cnt  Monoclonal  antibodies against eight different PKC isoforms were used in western blot analysis as  described in the Materials and Methods. G L C , human granulosa-luteal cells; Cnt, positive  control.  138  Control  Antral Cells  GLC1  GLC2  GLC3  Fig. 38 PCR product showing the absence of PKCy in hGLCs in three different patients.  139  Translocation of PKCa from cytosolic to membrane fraction  Activation of P K C is associated with a translocation of the enzyme from the cytosolic fraction to the plasma membrane [Tippmer et al., 1994]. In the present study, hGLCs were treated with 10 u M ATP for 1 or 5 min. Of the 4 P K C isoforms, only P K C a , mainly in cytoplasm, was noted with increased expression in membrane fraction and reduced expression in the cytosolic fraction after treatment (Fig. 39).  The  translocation of P K C a isoform to the plasma membrane was accompanied by a decrease in the amount of P K C a in the cytosolic fraction.  Effect of PKC  depletion  After treatment with P M A for 18 h, the P K C a isozyme in hGLCs was significantly down-regulated, when compared with control (Fig. 40A).  As well, there was no  significant effect of ATP on hCG-stimulated c A M P production after P M A pretreatment (Fig. 40B).  140  f  \  flflgP* C  5 min  1 min  Control  f  \  mmm M  '  m  C  M  *  mmm C  M  Fig. 39 Translocation of P K C a from cytosolic to membrane fraction after ATP treatment in hGLCs.  Human GLCs was treated with ATP for 1 or 5 min. The  P K C a levels in different fractions were detected by Western blot analysis. C, cytosolic fraction; M , membrane fraction.  141  Control  PMA  B. 200 ag  150 | §  100 50  u  0 Control  PMA18h  P M A 18h  +hCG  ATP+hCG  Fig. 40 A . Down-regulation of P K C a in hGLCs achieved by prolonged treatment with 1 u M P M A for 18 hours.  B. The effect of ATP on  hCG-induced c A M P accumulation in hGLCs after treatment with P M A for 18 hours.  Values were presented as the Mean ± SE of three individual  experiments.  Differences were considered significant at p < 0.05. a, p < 0.05  vs. control 142  5.5 Discussion  The presence of P2U purinoceptor in hGLCs highlights a role of extracellular ATP in the human ovary [Tai et al., 2000a].  In this study, we demonstrated that P K C was  associated with the inhibitory effect of ATP on hCG-stimulated c A M P accumulation. The presence of P K C a in hGLCs and its translocation from cytosolic fraction to membrane fraction suggest a role of P K C a in mediating ATP action on hCG-induced c A M P production.  Cyclic A M P is well established in transducing hCG actions such as progesterone production in the ovary.  Prostaglandin F2a, an antigonadotropic agent, inhibits  gonadotropin-induced progesterone production via reducing gonadotropin-stimulated c A M P accumulation [Abayasekara et al., 1993b]. In this study, we demonstrated that ATP reduced hCG-induced c A M P production, further supporting a role of extracellular ATP in regulating ovarian function.  Evidence reveals that extracellular ATP can regulate cellular function through activation of P K C [Gordon, 1986; el-Moatassim et al., 1992; Burnstock, 1990]. Studies also indicate that P K C has dual actions by providing positive forward actions as well as negative feedback in controlling various signaling steps [Nishizuka, 1989; Nishizuka, 1986]. We reported recently that ATP was able to induce cytosolic calcium oscillations, and activated P K C negatively regulated ATP-evoked calcium mobilization from both intracellular stores and extracellular influx in hGLCs [Tai et al., 2000b]. In this study, 143  the forward action of P K C in mediating the effect of ATP on hCG-induced c A M P accumulation was demonstrated using a P K C activator and P K C inhibitors. kinase  C isozymes  consist  of single polypeptide  chains,  each  Protein  containing  an  amino-terminal regulatory region and a carboxy-terminal kinase domain [Nishizuka, 1992]. Phorbol esters cause activation of conventional and novel P K C isozymes through binding to the regulatory region [Nishizuka, 1989]. In the present study, P M A mimicked the effect of ATP by reducing the hCG-induced c A M P production (Fig. 34). In addition, staurosporin, a potent P K C inhibitor [Watson et al., 1988], and bisindolylmaleimide I [Toullec et al., 1991], a selective P K C inhibitor of PKCa,p,8 and s, effectively reversed the inhibitory action of ATP in hCG-evoked c A M P production, supporting the notion that P K C plays a role in mediating ATP action in the human ovary.  Multiple and various P K C isoforms have been demonstrated in the ovary of different species [Maizels et al., 1992; DeManno et al., 1992; Orwig et al., 1994]. In the present study, we identified the presence of P K C a , 5, i and X isoforms in hGLCs.  PKC  subspecies are expressed specifically in certain tissues [Nishizuka, 1988]. PKCy appears to be present predominantly in the nervous system such as the brain and spinal cord [Coussens et al, 1986]. Outside of the nervous system, PKCy is identified in human antral gastrin cells [Moore et a l , 1999]. Based on RT-PCR results, we ruled out the presence of this isoform in hGLCs.  We reported previously that 10 u M ATP induced  cytosolic calcium oscillations in hGLCs [Tai, et al., 2000a], implying the activation of a calcium-dependent P K C isoform subsequent to ATP exposure.  According to our  observations, the P K C a , a calcium-dependent P K C isoform, was translocated from the  144  cytosolic fraction to membrane fraction after ATP treatment, specifying the P K C isoform involved.  Long term exposure to phorbol esters cause down-regulation of P K C activity that is associated with proteolysis of P K C occurring in the hinge region between regulatory and catalytic domains by proteases such as calpain or serine protease [Solanki et al., 1983; Pontremoli et al., 1990; Chida et al, 1986].  Prolonged exposure of P M A (16 h)  down-regulates P K C activity in hGLCs [Abayasekara et al., 1993a]. In this study, long term treatment of hGLCs with P M A downregulated the expression of P K C a (Fig. 40A), which was shown to be activated by ATP (Fig. 39).  ATP lost its effect on  hCG-stimulation c A M P accumulation in PKC-depleted cells, indicating the involvement of P K C a in reducing hCG-induced c A M P production a (Fig. 40B).  The observation that ATP inhibited intracellular c A M P responses to h C G through activation of P K C leads us to speculate several potential action sites of activated P K C . Considering the presence of potential P K C phosphorylation sites in the third intracellular loop and at the C-terminal of the LH/hCG receptor, this receptor may be affected by activated P K C following ATP treatment (Fig. 41-©).  These intracellular regions of  LH/hCG receptor are related to the coupling of the Gs protein, indicating the possibility of dissociation between LH/hCG receptor and Gs protein by active P K C [Loosfelt et al, 1989; Macfarland et al., 1989]. With respect to receptor-coupled G-proteins, several studies have shown that phorbol ester can regulate G-protein-mediated responses [Orellana et al., 1987; Sagi-Eisenber, 1989; Krishnamurthi et al, 1989], indicating that  145  LH/hCG-coupled Gs-protein may be inhibited by activated P K C in hGLCs (Fig. 41-©). Adenylyl cyclase activity is closely related with c A M P accumulation. P K C has been demonstrated to alter the responses of adenylyl cyclase to G-protein [Zimmermann and Tausig, 1996], pointing out another action site for activated P K C subsequent to ATP treatment in hGLCs (Fig. 41-®). cAMP  and  affects  Cyclic A M P phosphodiesterase causes degradation of  intracellular c A M P  accumulation.  Stimulation of  cAMP  phosphodiesterase via P K C is reported in cultured hGLCs [Michael and Webley, 1991], suggesting one more factor associated with regulating cytosolic c A M P level (Fig. 41-®).  In conclusion, our results demonstrated  that (1) extracellular ATP reduced  hCG-stimulated c A M P accumulation, (2) the P K C a , 8, i and X isoforms were present in hGLCs, (3) P K C a was translocated subsequent to ATP treatment, and (4) P K C was involved in mediating the antigonadotropic action of extracellular ATP. Taken together, these results further support a role of this neurotransmitter in ovarian steroidogenesis.  146  Fig. 41 A proposed model of the potential cross-talk between ATP-activated protein kinase C a (PKCa) and hCG-induced c A M P production in hGLCs. P2UR = P2U purinoceptor on cell membrane; G = G-protein; P L C = phospholipase C; PIP2 = phosphatidyl-inositol 4,5-bisphosphate; D A G = diacylglycerol; IP3 = inositol 1,4,5-triphosphate; LH/hCG R = LH/hCG receptor.  147  PART 6 ATP  ACTIVATES  MITOGEN-ACTIVATED  PROTEIN  KINASE IN H U M A N GRANULOSA-LUTEAL C E L L S  6.1 Abstract  ATP has been shown to activate the phospholipase C (PLC)/ diacylglycerol/ protein kinase C (PKC) pathway.  However, little is known about the downstream signaling  events. The present study was designed to examine the effect of ATP on activation of mitogen-activated protein kinase (MAPK) signaling pathway and its physiological role in human granulosa-luteal cells (hGLCs).  Western blot analysis, using a monoclonal  antibody which detected the phosphorylated forms of ERK1 and ERK2 ( p 4 2 P k and ma  p44 niapk respectively), demonstrated that A T P activated M A P K in a dose- and ;  time-dependent manner.  Treatment of the cells with suramin (a P2-purinoceptor  antagonist), neomycin (a P L C inhibitor), staurosporin (a P K C inhibitor) or PD98059 (a M E K , M A P K / E R K kinase, inhibitor) significantly attenuated the ATP-induced activation of M A P K .  In contrast, ATP-induced M A P K activation was not significantly affected by  pertussis toxin (a G i inhibitor). To examine the role of Gs protein, intracellular c A M P level was determined after treatment with ATP or human chorionic gonadotropin (hCG). No significant elevation of intracellular c A M P level was noted after ATP treatment. To determine the role of M A P K in steroidogenesis, hGLCs were treated with ATP, hCG, or ATP plus hCG in the presence or absence of PD98059. Radioimmunoassay revealed 148  that ATP alone did not significantly affect basal progesterone concentration. However, hCG-induced progesterone production was reduced by ATP treatment.  PD98059  reversed the inhibitory effect of ATP on hCG-induced progesterone production. To our knowledge, this is the first demonstration of ATP-induced activation of M A P K signaling pathway in the human ovary. These results support the notion that the M A P K signaling pathway is involved in mediating ATP actions in the human ovary.  149  6.2 Introduction  Extracellular adenosine triphosphate (ATP) is co-released with neurotransmitter granules from nerve endings by exocytosis [Gordon, 1986].  After binding to a G  protein-coupled P2 purinoceptor, ATP activates phosphoinositide hydrolysis, generating diacylglycerol and inositol 1,4,5-trisphosphate, which stimulate protein kinase C and cytosolic calcium mobilization, respectively [Berridge, 1984; el-Moatassim et al., 1992]. Thereafter, ATP may participate in various types of physiological responses, including secretion, membrane potential, cell proliferation, platelet aggregation, neurotransmission, cardiac function and muscle contraction [el-Moatassim et al., 1992; Burnstock, 1990]. Considering that the ovary is a well-innervated organ, it is tempting to speculate that the co-released ATP from nerve endings may play a role in regulating ovarian function. We reported previously the expression of P2U purinoceptor in hGLCs [Tai et al., 2000a], further supporting a physiological role of ATP in the human ovary.  Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine kinases involved in converting extracellular stimulus into intracellular signals. Extracellular signal regulated kinases (ERKs), one of M A P K s subfamilies, have been shown to be activated  by  extracellular  agonists  such  as  cytokines,  growth  factors  and  neurotransmitters [Cobb and Goldsmith, 1995; Fanger, 1999]. It is believed that two classes of cell surface receptors, G-protein-coupled receptor and receptor tyrosine kinases are associated with the activation of M A P K s [Fanti et al., 1993; Lopez-Ilasaca, 1998; Chabre, 1995]. When activated, ERK1 and E R K 2 (also known as p 4 2 P m a  150  k  and p44  mapk respectively), phosphorylate a variety of substrates, including transcription factors, 5  which have been implicated in the control of cell proliferation and differentiation [Post and Brown, 1996; Cano and Mahadevan, 1995; Blenis, 1993].  The demonstration of P2U purinoceptor in hGLCs highlights the significance of ATP in regulating ovarian function, but little is known about the signaling events and cellular responses subsequent to the binding of ATP to its receptor in the human ovary. Activation of P2 purinoceptor has been shown to increase M A P K activity [Dickenson et al., 1998]. However, the role of M A P K in ovarian cells is poorly understood.  In the  present study, the signaling cascade proximal to M A P K activation subsequent to ATP exposure was determined in hGLCs.  In addition, the functional role of activated M A P K  following ATP treatment was studied.  6.3 Materials and Methods  Reagents and Materials  ATP, suramin, pertussis toxin (PTX), neomycin, staurosporin and human chorionic gonadotropin (hCG) were obtained from Sigma Chemical Co. (St. Louis, MO). PD98059, a M E K inhibitor, was purchased from New England Biolabs Inc., Berverly, MA.  Dulbecco's Modified Eagle Medium (DMEM), penicillin-streptomycin and fetal  151  bovine serum (FBS) were obtained from GIBCO-BRL (Burlington, Ontario, Canada). Staurosporin and PD98059 were dissolved in dimethyl sulfoxide (DMSO) as suggested by manufacturers.  Human granulosa-luteal cells culture  Human GLCs were collected from patients undergoing an In Vitro Fertilization Embryo Transfer program and processed as described in PART 2.  Treatments  Human GLCs were incubated in serum-free medium for 4 h prior to treatment. To examine  the dose-response relationship,  hGLCs  were  treated  with  increasing  concentrations of A T P (100 n M , 1 p M , 10 p M or 100 pM) for 5 min. For time-course experiments, hGLCs were treated with 10 p M ATP for 1, 5, 10 or 20 min.  To determine the intracellular signaling pathway, hGLCs were treated with suramin (300 p M , an inhibitor of P2 purinergic receptor), P T X (200 ng/mL; a G i inhibitor), neomycin (10 m M ; a P L C inhibitor), staurosporin (1 p M ; a P K C inhibitor) or PD98059 (50 p M ; a M E K inhibitor) in the presence or absence of 10 p M ATP. Human GLCs were pretreated with suramin for 15 min, P T X for 1 h, neomycin for 15 min, staurosporin  152  for 15 min and PD98059 for 1 h prior to ATP treatment. The cells were collected 5 min after ATP exposure.  Western blot analysis  The hGLCs were processed as mentioned in PART 2.  Aliquots (30 ug) were  subjected to 10 % SDS-polyacrylamide gel electrophoresis under reducing condition, as previously described [Laemmli, 1970].  The proteins were then electrophoretically  transferred from the gels onto nitrocellulose membranes (Amersham Pharmacia Biotech, Oakville, Canada) according to the procedures of Towbin et al. [Towbin et al., 1979]. These nitrocellulose membranes were probed with a mouse monoclonal antibody (New England Biolabs Inc., Beverly, M A ) directed against the phosphorylated forms of ERK1 and ERK2 (P-MAPK, p 4 2 p k and p44 ma  m a  P , respectively) at 4 °C for 16 h. k  Alternatively, the membranes were probed with a rabbit polyclonal antibody for p42/p44 M A P K , which detected total M A P K (T-MAPK) levels (New England Biolabs Inc., Beverly, M A ) .  After washing, the membranes were incubated with HRP-conjugated  goat-anti mouse secondary antibody for P - M A P K and sheep-anti rabbit secondary antibody for T - M A P K , and the signal was visualized using E C L system (Amersham Pharmacia Biotech, Oakville, Canada) followed by autoradiogrphy. The autoradiograms were quantified using a laser densitometer (BIO-RAD, Model 620, Video Densitometer).  153  MAP Kinase Assay  To measure M A P kinase activity, a nonradioactive method was utilized (p44/42 M A P Kinase Assay Kit, New England Biolabs Inc.).  The kit contains two  phospho-antibodies, one to selectively precipitate active M A P kinase and a second to detect MAPK-induced phosphorylation of Elk-1.  Briefly, active M A P kinase from  hGLCs lysate (200 pg) treated with 10 p M ATP for 5 min was selectively immunoprecipitated with an immobilized monoclonal antibody to phospho-p44/42 M A P (Thr202 and Tyr204) kinase. For a positive control, active M A P Kinase (provided by the manufacturer) was added to the control cell extract.  The resulting precipitate was  incubated with an Elk-1 fusion protein in the presence of ATP which allowed immunoprecipitated active M A P K to phosphorylate Elk-1 [Marais et al., 1993; Janknecht et al., 1993; Gille et al., 1995].  Phosphorylation of Elk-1 at Ser383 was measured by  western blotting using a phospho-Elk-1 (Ser383) antibody. Ser383 of Elk-1 is a major phosphorylation site by M A P kinase and is required for Elk-1-dependent transcriptional activity [Marais et al., 1993; Janknecht et al., 1993; Gille et al., 1995].  154  Overview of p44/42 M A P Kinase Assay Step 1: Prepare cell extracts. (a) Treat cells. (b) Add cell lysis buffer. (c) Collect cell lysates Step 2: Selective immunoprecipitation of active (phosphorylated) M A P K using immobilized phospho-antibody. (a) Add immobilized phospho M A P K Ab (b) Immunoprecipitation of cell extracts using immobolized phospho-MAPK Ab.  Active (phosphorylated Thr202 and Tyr204) Step 3: Incubate immunoprecipitated pellets in buffer containing Elk-1 fusion protein and ATP.  Step 4: Analyze Elk-1 phosphorylation using phospho-antibodies by western blotting.  155  Radioimmunoassay for intracellular cAMP  Human GLCs (2 x IO cells) were plated onto 35 mm culture dishes and cultured for 5  4 days. The cells were then incubated in serum-free medium containing 0.1% B S A and 0.5 m M 3-isobutyl-lmethylxanthine (IBMX, Sigma-Aldrich Corp.) for 30 min. To determine ATP or hCG-induced intracellular c A M P accumulation, hGLCs were treated with ATP (10 uM) or hCG (1 IU/mL, a positive control) for 20 min. Intracellular c A M P levels were measured using the [ -H]-cAMP assay system, following the protocol 3  provided by manufacturer (Amersham Pharmacia Biotech).  Radioimmunoassay for progesterone  After culture in D M E M with 10% FBS for 3 days, hGLCs were incubated in D M E M for 4 h prior to treatment for steroidogenesis experiments. To determine the role of M A P K in steroidogenesis, hGLCs were treated with ATP (10 uM), h C G (1 IU/ml), or ATP plus hCG in the presence or absence of PD98059 for 6 h.  Progesterone levels in the culture medium were measured by an established radioimmunoassay [Vaananen et al, 1997].  Anti-progesterone antibody was kindly  provided by Dr. D. T. Armstrong (University of Western Ontario). Briefly, samples were incubated with antibody and tracer, with final concentration of 7,000 cpm/ml of [l,2,6,7,16,17-3H]Progesterone (Amersham Pharmacia Biotech). After incubation for  156  16-24 hours, a charcoal/dextran solution was added to remove unbound progesterone or tracer.  Scintillation cocktail (Amersham Pharmacia Biotech) was added to each sample,  and the vials were counted with a p-counter ( L K B Wallac, Turku, Finland). The cells in each dish were harvested for quantifying protein amount using Bio-Rad Protein Assay kit. Samples were assayed in triplicates and progesterone concentrations were standardized against total protein contents.  Human chorionic gonadotropin and MAPK in hGLCs  Gonadotropins have been demonstrated to activate M A P K in porcine granulosa cells [Cameron et al., 1996]. To examine the effect of hCG on M A P K activation, hGLCs were treated with 1 IU/ml hCG for 1, 5, 10 or 20 min and cell lysates were collected for western blot analysis. The effect of M A P K on hCG-stimulated progesterone production was studied by treating cells with 1 IU/ml hCG in the presence or absence of PD98059 for 24 h.  MAPK and antigonadotropic effect ofATP on hCG-induced cAMP production  To examine the activated M A P K and inhibitory effect of A T P on hCG-induced c A M P accumulation, hGLCs were treated with hCG, A T P plus hCG in the presence or absence of PD98059 for 20 min. Intracellular c A M P accumulation was measured as  157  mentioned previously.  Statistical Analysis  M A P K and progesterone levels were expressed as a relative ratio of basal levels. Intracellular c A M P levels were shown as pmole per 2 x 10 cells. Data were represented 5  as means ± standard errors (SE).  Statistical analysis was performed by one-way  analysis of variance followed by Tukey's multiple comparison test. Differences were considered significant at p < 0.05.  6.4 Results  Effect ofATP on MAPK activation To demonstrate the ability of ATP in activating M A P K , hGLCs were treated with increasing concentrations (100 nM-100 pM) of ATP for 5 min. For time-course analysis, the cells were treated with 10 p M ATP for varying time intervals (1-20 min). As shown in Fig. 42, ATP activated M A P K in hGLCs in a dose-dependent manner.  A significant  effect was observed at 1 p M with a maximum effect noted at 10 p M , and there is no statistically significant difference between cells treated with 10 p M and 100 p M ATP.  158  ATP was capable of rapidly inducing M A P K activity.  A significant effect was seen  within 5 min after treatment, and the activation of M A P K was sustained for at least 15 min (Fig. 43).  MAP kinase activity  In vitro M A P kinase activity was measured using a p44/42 M A P kinase assay As shown in Fig. 44, ATP significantly increased M A P K activity.  159  Control  0.1  1  10  100  uM P-MAPK  T-MAPK  Control  0.1  1 ATP(uM)  10  Fig. 42 The dose-response of ATP on M A P K activation in human granulosa-luteal cells (hGLCs).  Human GLCs were treated with increasing concentrations of  ATP (0, 100 nM, 1 p M , 10 p M or 100 pM) for 5 min as described in the Materials and Methods. The M A P K were detected by Western blot analysis. The data were shown as relative ratio to basal levels. Values were presented as the Mean±SE of three individual experiments.  Statistical analysis was performed by one-way  analysis of variance followed by Tukey test. significant at p < 0.05, *.  160  Differences were considered  5  1  Control  1  10  5  20 min  10  Time (riin)  Fig. 43 The time course of ATP on M A P K activation in human granulosa-luteal cells (hGLCs). Human GLCs were treated with 10 u M ATP for 0,1,5,10 or 20 min as described in the Materials and Methods. The M A P K were detected by Western blot analysis. The data were shown as relative ratio to basal levels. Values were presented as the Mean ± SE of three individual experiments. Differences were considered significant at p < 0.05, *.  161  Control  ATP  Positive Control  Phospho-Elk-  Control  ATP  Positive control  Fig. 44 M A P kinase activity in hGLCs detected using a M A P kinase assay kit.  Human GLCs were treated with 10 u M ATP for 5 min as described  in the Materials and Methods.  162  P2-purinergic receptor and ATP-induced MAPK activation  P2U purinergic receptor has been demonstrated in hGLCs [Tai et al., 2000a; Lee et a l , 1996]. To investigate the involvement of P2 purinoceptor in ATP-induced M A P K activation, hGLCs were pretreated with 300 u M suramin, a P2 purinoceptor antagonist [Hertog et al., 1992], for 15 min prior to the administration of ATP.  As demonstrated in  Fig. 45, ATP activated M A P K to about 230 % of basal (control) level. The co-treatment with suramin and ATP significantly reduced M A P K activity by 85 %, when compared to ATP treatment alone.  Pertussis toxin and ATP-induced MAPK activation  A PTX-insensitive G-protein, Gctq/11, is known to be expressed in hGLCs [Carrasco et al., 1997; Lopez Bernal et al., 1995]. To identify the subclass of G-protein involved in the ATP-induced activation of M A P K , human GLCs were pretreated with PTX for 1 h prior to exposure to ATP.  Pretreatment of PTX did not alter ATP-induced  M A P K activity, indicating that ATP acts through a PTX-insensitive G-protein (Fig. 46).  163  Control  ATP  Suramin  Suramin  + ATP P-MAPK  T-MAPK  +ATP  Fig. 45 The effect of suramin, a P2 purinoceptor inhibitor, on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs). Human GLCs were treated with 10 u.M ATP in the presence or absence of suramin (300 uM) as described in the Materials and Methods. The activated M A P K were detected by Western blot analysis.  The data were shown as relative ratio to basal levels.  presented as the Mean±SE of three individual experiments.  Values were  Differences were  considered significant at p < 0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. ATP.  164  Control  ATP  PTX+ATP  PTX  p44  P-MAPK  p42 p44  T-MAPK  p42  3.5  § 2.5 < 2 03  1.5 0.5 0  Control  ATP  PTX+ATP  PTX  Fig. 46 The effect of pertussis toxin (PTX), a Gi protein inhibitor, on ATP-induced M A P K  activation in human granulosa-luteal cells (hGLCs).  Human GLCs were treated with 10 u M ATP in the presence or absence of PTX (200ng/mL) as described in the Materials and Methods. The activated M A P K were detected by Western blot analysis. The data were shown as relative ratio to basal levels. Values were presented as the Mean±SE of three individual experiments.  Differences were considered significant at p < 0.05. a, p < 0.05 vs.  control; b, p < 0.05 vs. ATP.  165  PLC and ATP-induced MAPK activation  Neomycin, an aminoglycoside antibiotic, has been demonstrated to inhibit P L C [Spath et al., 1991]. In this study, hGLCs were pretreated with 10 m M neomycin for 15 min prior to the stimulation of ATP. As shown in Fig. 47, treatment of hGLCs with neomycin significantly inhibited the ATP-induced activation of M A P K .  The combined  treatment with neomycin and ATP significantly attenuated M A P K activity by 90 %, when compared to ATP treatment alone.  PKC and ATP-induced MAPK activation  Staurosporin, a potent inhibitor of protein kinase C [Watson et al., 1998], significantly attenuated the ATP-induced activation of M A P K  (Fig. 48).  The  concomitant treatment with the P K C inhibitor and ATP attenuated M A P K activation by 70 %, when compared to the level stimulated by ATP alone.  MEK and ATP-induced MAPK activation  In M A P K activation cascade, M E K is the immediate activator of M A P K .  M E K is  also known as M A P K Kinase [Fanger, 1999]. M E K inhibitor, PD98059, significantly decreased the ATP-induced activation of M A P K in hGLCs.  166  Simultaneous treatment  with PD98059 and ATP reduced M A P K activity to about 50 % ofthe level stimulated by ATP alone (Fig. 49).  167  Control  ATP  Neomycin  Neomycin  + ATP P-MAPK  T-MAPK  Control  ATP  I^omycin  Nfeomydn  +ATP  Fig. 47 The effect of neomycin, a PLC inhibitor, on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs). Human GLCs were treated with 10 p M ATP in the presence or absence of neomycin (10 mM) as described in the Materials and Methods. The activated M A P K were detected by Western blot analysis. The data were shown as relative ratio to basal levels. Values were presented as the Mean ±SE of three individual experiments.  Differences were considered significant at p <  0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. ATP.  168  Control  ATP  Staurosporin  Staurosporin  DMSO  + ATP  P-MAPK  T-MAPK  Control  ATP  Staurosporin Staurosporin  DMSO  +ATP  Fig. 48 The effect of staurosporin, a PKC inhibitor (PKCI), on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs).  Human GLCs were treated  with 10 u M ATP in the presence or absence of staurosporin (1 uM) as described in the Materials and Methods. The activated M A P K were detected by Western blot analysis.  The data were shown as relative ratio to basal levels.  presented as the Mean±SE of three individual experiments.  Values were  Differences were  considered significant at p < 0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. ATP.  169  Control  ATP  MEKI +ATP  MEKI  DMSO  P-MAPK  T-MAPK  +ATP  Fig. 49 The effect of PD98059, a M E K inhibitor (MEKI), on ATP-induced M A P K activation in human granulosa-luteal cells (hGLCs).  Human GLCs were treated  with 10 u M ATP in the presence or absence of PD98059 (50 u,M) as described in the Materials and Methods. analysis.  The activated M A P K were detected by Western blot  The data were shown as relative ratio to basal levels.  presented as the Mean±SE of three individual experiments.  Values were  Differences were  considered significant at p < 0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. ATP.  170  Effect ofATP on intracellular cAMP accumulation  ATP has been demonstrated to increase intracellular c A M P production by activating adenylyl cyclase in several cell systems [Communi et al., 1997; Conigrave et al., 1998]. To examine the effect of ATP on intracellular c A M P production, hGLCs were treated with 10 p M ATP for 20 min, while 1 IU/mL hCG was used as a positive control. Human C G markedly increased intracellular c A M P level.  In contrast, ATP was not able to  increase intracelluar c A M P accumulation in hGLCs, when compared with control group (Fig. 50). This result indicates that the P2U purinoceptor expressed in hGLCs is not coupled to adenylyl cyclase.  Effect ofATP-evoked MAPK activation on hCG-induced progesterone production  To determine the role of M A P K in ovarian steroidogenesis, hGLCs were treated with ATP (10 pM), h C G (1 IU/ml), or ATP plus h C G in the presence or absence of PD98059.  As shown in Fig. 51, 10 p M ATP had no effect on the basal level of  progesterone production, while hCG increased progesterone production to 250 % of control in hGLCs. Co-treatment of hGLCs with ATP and hCG significantly inhibited the progesterone production to 50 % of the level induced by hCG alone.  Further, the  presence of M E K inhibitor (PD98059) reversed the inhibitory effect of ATP on hCG-induced progesterone production.  171  Effect of A T P on cA M P production  Fig. 50 The effect  of ATP on intracellular c A M P production in human  granulosa-luteal cells (hGLCs). Human GLCs were treated with ATP (10 pM) or hCG (1 IU/ml) for 20 min as described in the Materials and Methods. Samples were assayed in triplicates following manifacturer's  protocol.  presented as the Mean±SE of three individual experiments. considered significant at p < 0.05. a, p < 0.05 vs. control.  172  Values were  Differences were  Control  ATP  hCG  ATP+hCG MEKI +ATP+hCG  MEKI  DMSO  Fig. 51 The effect of M A P K on progesterone production in human granulosa-luteal cells (hGLCs). Human GLCs were treated with ATP (10 uM), hCG (1 IU/ml), or ATP plus hCG in the presence or absence of PD98059 for 6 hours as described in the Materials and Methods. Samples were assayed in triplicates and progesterone concentrations were standardized against total protein content. presented as the Mean±SE of three individual experiments.  Values were  Differences were  considered significant at p < 0.05. a, p < 0.05 vs. control; b, p < 0.05 vs. ATP plus hCG.  173  Human chorionic gonadotropin activates MAPK in hGLCs  As shown in Fig. 52A, h C G was capable of activating M A P K in hGLCs in a time-dependent manner.  Phosphorylated M A P K increased significantly in 1 min, when  compared with the control, and reached a maximum response after treatment with 1 IU/ml hCG for 5 min. • To investigate the role of hCG-stimulated M A P K in steroidogenesis, hGLCs were treated with hCG in the presence or absence of M E K inhibitor. Radioimmunoassay demonstrated that there was no significant effect of M E K I on hCG-induced progesterone production (Fig. 52B).  MAPK and antigonadotropic effect of ATP on hCG-induced cAMP production  As shown in Fig. 53, the inhibitory effect of A T P on hCG-stimulated c A M P production was not altered significantly in the presence of M E K I , suggesting that the action site of active M A P K is secondary to c A M P production.  174  B.  Gcrtrol  KJG  MEKT+H3G  Fig 52. A . The effect of hCG on M A P K activation in human granulosa-luteal cells (hGLCs). Human GLCs were treated with 1 IU/ml hCG for various time (1-20 min) as described in the Materials and Methods. The activated M A P K were detected by Western blot analysis.  B. The effect of PD98059, a M E K inhibitor (MEKI), on  hCG-induced progesterone production in human granulosa-luteal cells (hGLCs). Samples  were  assayed  in triplicates and  standardized against total protein content.  progesterone  concentrations  were  Values were presented as the Mean±SE of  three individual experiments. Differences were considered significant at p < 0.05. a, p < 0.05 vs. control. 175  Effect of MAPK on cAMP production 800  g  600  a  400  a,b  CD  Q_  "5  a  200 0 Control  A  T  hCG  P  ATP+hCG  MEKI+ATP+hCG  Fig. 53 The effect of M E K I in inhibitory effect of ATP on hCG-stimulated c A M P production.  Human GLCs were treated with hCG plus ATP in the presence or  absence of M E K I (50 pM). Values were presented as the Mean±SE of three individual experiments.  Differences were considered significant at p < 0.05. a, p  < 0.05 vs. control; b, p < 0.05 vs. ATP+hCG  176  6.5 Discussion  The M A P kinases have been implicated in the regulation of cell growth and differentiation [Brunet and Pouyssegur, 1997]. M A P kinases are classified into three subfamilies: (I) ERKs (extracellular signal-regulated kinases), including E R K 1 and ERK2, (II) SAPKs (stress-activated protein kinase), also called c-jun N-terminus kinases (JNKs), and (III) p38 kinase [Lopez-Ilasaca, 1998]. The M E K s , also known as M A P K kinases (MAPKKs), activate the M A P K s by dual phosphorylation on threonine and tyrosine residues of a T E Y (Thr-Glu-Tyr) motif [Ann et al., 1992]. The first M A P K s to be cloned are M A P K / E R K 1 and 2, which are phosphorylated and activated by M E K s [Boulton et al., 1990; Boulton et al., 1991].  M A P Kinases have been identified in  several steroidogenic cells [Chabre et al., 1995; McNeill et al., 1998], but little is known about their role(s) in steroidogenesis. PGF2a, an anti-gonadotropic hormone, has been demonstrated to stimulate the M E K I / M A P K signaling cascade in bovine luteal cells [Chen et al., 1998]. Recently, Kang et al. reported that M A P K s mediate the inhibitory effect of gonadotropin-releasing hormone in progesterone production in hGLCs [Kang et al., 2000], indicating the role of M A P K s in steroidogenesis. In the present study, the phospho-specific M A P K antibody, which detected phosphorylated T h r  2 0 2  and T y r  ERK1/2, was used to measure activated M A P K s by Western blot analysis. concentration  of ATP in adrenergic  granules  of sympathetic  nerves  2 0 4  on  The  and in  acetylcholine-containing granules of parasympathetic nerves can be as high as 150 m M [Winkler and Carmichael, 1982]. Our results demonstrated that M A P K s were activated by 10 p M ATP, and furthermore, M A P K s mediated the anti-gonadotropic action of ATP  177  in steroidogenesis in hGLCs.  The P2U purinoceptor has been identified in hGLCs [Tai et al., 2000a] and may be coupled to PTX-sensitive or insensitive G-proteins [Dubyak and el-Moatassim, 1993; Sternweis and Smrcka, 1992]. It has been reported previously that P2U purinoceptors are coupled to a PTX-insensitive G-protein in hGLCs, using microspectrofluorimetry [Lee et al., 1996]. In the present study, ATP-induced phosphorylation of M A P K was not affected by 200 ng/mL PTX, indicating the involvement of PTX-insensitive G-proteins such as Gaq/11 [Carrasco et al., 1997; Lopez Bernal et al., 1995]. P2 purinoceptors have been reported to be coupled to adenylyl cyclase in several systems [Communi et al., 1997; Conigrave et al., 1998; Post et al., 1998]. In this study, ATP failed to increase intracellular c A M P accumulation, indicating that the P2U purinoceptor expressed in hGLCs is not coupled to adenylyl cyclase.  After binding to the G-protein-coupled receptor, ATP has been reported to activate phospholipase C [Dubyak and el-Moatassim, 1993; Dubyak, 1991], resulting in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG), which in turn induces calcium mobilization and activates P K C , respectively.  PLC-p and PLC-y  isoforms have been identified in hGLCs [Carrasco et al, 1997]. Neomycin has been demonstrated to inhibit all three isoforms of PLCs [Spath et al., 1991]. In the present study, 10 m M Neomycin significantly reduced the level of the phosphorylated form of M A P K , indicating the role of P L C in ATP-induced M A P K activation. P K C has been shown to exert its effects in the ovary [Morley et al., 1996; Abayasekara, 1993a;  178  Abayasekara, 1993b; Michael and Webley, 1991]. In this study, ATP-induced M A P K activation was significantly attenuated in hGLCs pretreated with staurosporin, a potent P K C inhibitor [Watson et al., 1988], indicating the involvement of P K C in the M A P K activation cascade. M E K is an immediate activator of M A P K .  Our data demonstrated  that the M E K inhibitor, PD98059, significantly decreased the ATP-induced activation of MAPK.  Taken together, this study delineated the ATP signaling pathway in hGLCs from  PTX-insensitive G-protein, PLC, P K C , with a M E K to M A P K activation.  ATP has been demonstrated to induce the production of steroid hormones in steroidogenic cells [Foresta et al, 1996; Niitsu, 1992]. In the ovary, 100 p M ATP, A D P and A M P have been shown to regulate the basal levels of progesterone and estrogen in hGLCs, indicating the effects of ATP metabolites on steroidogenesis.  However, UTP  has no effect on basal progesterone level in hGLCs, implying that the stimulatory effects of purine nucleotides on progesterone production are not through P2U-purinoceptors, but via A2-adenosine receptors [Kamada et al, 1994]. As shown in the present study, a lower concentration of ATP (10 pM) had no effect on the basal level of progesterone production in hGLCs.  However, co-treatment of hGLCs with ATP significantly  inhibited the progesterone production induced by hCG, indicating an anti-gonadotropic action of ATP in hGLCs.  Furthermore, pretreatment of hGLCs with M E K inhibitor  reversed the inhibitory effect of ATP on hCG-induced progesterone production.  Luteinizing hormone has been demonstrated to increase M A P K activity in porcine granulosa cells [Cameron et al., 1996]. In the present study, hCG activated both ERK1  179  and ERK2 in a time-dependent manner.  However, the hCG-induced M A P K did not alter  hCG-stimulated progesterone production (Fig. 52). Taken together, these observations support the notion that a diverse array of ligands, including hormones, neurotransmitters and growth factors, are able to activate M A P K and cells may contain several M A P K signaling cascades, potentially regulated independently [Van Biesen et al., 1996].  The precise mechanism by which M A P K s affect ovarian steroidogenesis is not clear. As demonstrated in the present study, activated M A P K did not alter the antigonadotropic action of ATP in hCG-stimulated c A M P production, suggesting that the potential action site of M A P K is distal to c A M P production. Several steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage  (P450scc) and 3(3-hydroxysteroid dehydrogenase  (3p-HSD) have  been  demonstrated in the human ovary [Duncan et al., 1999; Kiriakidou et al, 1996]. Considering the nuclear translocation of activated M A P K s [Fanger, 1999; Post and Brown, 1996; Cano and Mahadevan, 1995; Blenis, 1993], it can be postulated that M A P K is involved in steroidogenesis through altering the production of steroidogenic enzymes (Fig. 54).  To our knowledge, this is the first demonstration of ATP-induced activation of M A P K signaling pathway in the human ovary. Through a PTX-insensitive G-protein and without affecting intracellular c A M P production, ATP activated M A P K subsequent to P L C and P K C activation in hGLCs.  These findings support a role of the M A P K  signaling pathway in mediating the ATP modulation of steroidogenesis in the human  180  ovary.  181  ATP P2U Receptor hCG  StAR  p42mapk  P450scc  p^^mapk  MEK  r 3(3-HSD  Fig. 54 Proposed intracellular signaling cascades of ATP in hGLCs.  ATP binds to a  PTX-insensitive G protein-coupled receptor that activates phospholipase  C and  phosphatidylinositol 4,5-biphosphate (PIP2) hydrolysis, generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3), which stimulate P K C and evoke intracellular calcium (Ca ) mobilization, respectively. 2+  and M A P K (p42  mapk  and p44  m a p k  P K C , in turn, activates M E K  ) , culminating in inhibition of hCG-induced  progesterone production.  182  PART 7 SUMMARY Adenosine triphosphate (ATP) is co-released with neurotransmitter granules from autonomic nerves by exocytosis. Extracellular A T P binds to a G protein-coupled P2 purinoceptor that activates phospholipase C and phosphatidylinositol hydrolysis, generating diacylglycerol and inositol 1,4,5-triphosphate, which stimulate protein kinase C (PKC) and cytosolic calcium ([Ca2+]i) mobilization, respectively. Thereafter, A T P may participate in various types of physiological responses, including secretion, membrane potential, cell proliferation, platelet aggregation, neurotransmission, cardiac function, and muscle contraction [el-Moatassim et al., 1992; Burnstock, 1990].  The reproductive tract including ovary, oviduct, uterus, cervix and vagina is innervated by autonomic nerves.  However, the effect of neurotransmitters  reproductive function is still poorly understood.  on  Considering that the ovary is a  well-innervated organ, it is tempting to speculate that the co-released A T P from autonomic nerve endings in the ovary may play a role in regulating ovarian function. In this study, a series of experiments were performed to examine (1) the expression and regulation of P2U purinergic receptor in human granulosa-luteal cells (hGLCs), (2) the functional role of extracellular A T P in the human ovary, (3) the signaling pathway subsequent to the binding of ATP to purinergic receptor in hGLCs, (4) the action and mechanism of antigonadotropic effect of ATP on hGLCs, (5) the roles of protein kinases including protine kinase C (PKC) and mitogen-activated protein kinase ( M A P K ) in mediating A T P action in hGLCs.  183  The present study demonstrates for the first time the expression of P2UR in human ovarian cells. The demonstration of P2UR mRNA in hGLCs provides the basis to explore the signaling pathway and functional role of extracellular A T P in the human ovary.  Northern blot analysis revealed that two species of mRNA, 2.0kb and 4.6 kb,  were expressed in hGLCs. Interestingly, human uterine cervical cells express at least four distinct transcripts, 2.0, 2.2, 3.0 and 4.6 kb [Gorodeski et al., 1998], while human nasal and proximal-tubule epithelia and liver express only a single 2.1 kb mRNA [Parr et al., 1994]. The P2UR was expressed functionally in hGLCs, since activation of the P2UR by both A T P and uridine triphosphate (UTP) resulted in rapid and transient mobilization of cytosolic calcium at the single cell level.  ATP has been demonstrated to regulate the production of steroid hormones in steroidogenic cells [Foresta et al, 1996; Niitsu, 1992]. In the present study, a lower concentration of ATP (10 pM) had no effect on the basal level of progesterone production in hGLCs.  However, co-treatment of hGLCs with A T P significantly  inhibited the progesterone production induced by hCG, indicating an anti-gonadotropic action of A T P in hGLCs.  Human C G was shown in our study to reduce the expression of P2U purinergic receptor in a dose- and time-dependent manner.  It is well established that activation of  L H / C G receptor activates adenylate cyclase and P K A [Lustbader et al., 1998].  To  further elaborate the mechanism by which hCG regulates the expression of P2UR mRNA, hGLCs were treated with exogenous 8-bromo-cAMP and forskolin, an activator of  184  adenylate cyclase. Our results show that both 8-bromo-cAMP and forskolin markedly down-regulated the expression of P2UR mRNA levels, supporting the notion that hCG down-regulation of the expression of P2UR mRNA may be mediated by adenylate cyclase and c A M P .  It has been reported that ATP may act as a trigger for apoptosis or  programmed cell death [Zheng et al. 1991], and that ATP at a concentration of 2.0 m M causes cell necrosis and death in the ovary [Charming, 1970]. It is conceivable that hCG is capable of minimizing the detrimental effect  of A T P , at least in part, by  down-regulation of P2UR expression in hGLCs.  The P K C family, a group of widely distributed serine/threonine kinases, mediates intracellular  signaling  of  numerous  cellular  regulators  including  hormones,  neurotransmitters and growth factors [Nishizuka, 1984; Berridge, 1993]. Multiple and various P K C isoforms are present in the ovary of different species. We demonstrated the presence of P K C a , 5, i and X isoforms in hGLCs.  It appears that P K C may have' dual  actions by providing forward actions as well as negative feedback in controlling various signaling steps. In this study, the forward action of active PKC-induced by A T P was demonstrated by reducing hCG-stimulated c A M P production. The inhibitory effect of ATP was reversed by P K C inhibitors, staurosporin and bisindolylmaleimide I, indicating the involvement of P K C in mediating antigonadotropic action of A T P in hGLCs. On the other hand, A T P induced calcium mobilization was negatively regulated by activated P K C from both intracellular stores and extracellular influx in cultured hGLCs, indicating the cross-talk between the P K C and Ca2+ pathway in A T P signal transduction.  185  Our data also demonstrated the potential cross-talk between protein kinase A (PKA) and P K C signaling pathway in hGLCs.  Humman C G downregulated the  expression of P2U purinergic receptor by activation of c A M P / P K A pathway, culminating in a reduction of purinergic receptor transcription. On the other hand, A T P significantly attenuated hCG-stimulated c A M P and progesterone production through P K C signaling pathway. These observations indicated the complex interactions between gonadotropin and neurotransmitter-related signaling pathway in the ovary.  Extracellular signal regulated kinases (ERK), one of the M A P K subfamilies, have been shown to be activated by extracellular agonists such as cytokines, growth factors and neurotransmitters [Cobb and Goldsmith, 1995; Fanger, 1999].  When activated,  ERK1 and E R K 2 (also known as p 4 2 P k and p44 rnapk^ respectively) phosphorylate a ma  variety of substrates, including transcription factors, which have been implicated in controlling cellular proliferation and differentiation [Post and Brown, 1996; Cano and Mahadevan, 1995; Blenis, 1993]. Our data demonstrated that ATP was able to activate E R K 1/2 in hGLCs in a dose- and time-dependent manner.  After binding to the  P2-purinoceptor, ATP activated M A P K subsequent to P L C and P K C activation through PTX-insensitive G-protein in hGLCs.  M A P K mediated the anti-gonadotropic action of  ATP in steroidogenesis by reducing hCG-stimulated progesterone production.  Physiologically, the ovarian cycle can be divided into three phases: the follicular phase, ovulation and the luteal phase. The cross-talk between extracellular ATP and the gonadotropin-related c A M P signaling pathway shown in this study encourages us to  186  explore in the future the potential action of A T P in the follicular phase, which is dominated by FSH.  In the present study, we demonstrated the role of A T P in regulating  hCG-stimulated progesterone secretion through the protein kinase Cl mitogen-activated protein kinase pathway in hGLCs obtained from women undergoing the IVF-ET program, indicating that A T P plays a role in the early luteal phase. Since the uterus is a target organ of progesterone, A T P may exert some effects on the regularity of the menstrual cycle and blastocyst implantation. It was observed that prostaglandin F2a exerted a similar effect in regulating gonadotropin-stimulated progesterone production [Tai et al, 2001b; Vaananen et al, 1997]. In view of the fact that prostaglandin F2ot is a luteolytic agent, it leads us to speculate that there is a possible luteolytic effect of extracellular A T P , which remains to be determined in the future.  Our data demonstrated the effect of extracellular A T P on regulating human ovarian function.  Furthermore, the demonstration of active involvement of multiple signaling  molecules such as protein kinase C, cytosolic calcium and mitogen-activated protein kinase delineates the pathway mediating the functional role of ATP in the ovary. These findings support the notion that extracellular A T P is a potent regulator of human ovarian function.  187  PART 8 FUTURE STUDIES  1. To examine the role of ATP in regulating steroidogenic protein and enzymes in hGLCs.  Steroidogenic acute regulatory protein, P450scc and 3(3-HSD control major steps in progesterone production. In a previous study, it was observed that ATP reduced hCG-stimulation by attenuating c A M P accumulation induced by hCG.  To explore  where other molecules are involved in mediating antigonadotropic effect of ATP, we will examine the effect of ATP on these steroidogenic protein and enzymes.  To examine the role of active P K C in regulating steroidogenic protein and enzymes in hGLCs.  P K C has been shown to mediate the antigonadotropic action of ATP in hGLCs by reducing hCG-stimulated c A M P production.  Considering the multiple potential  action sites of activated P K C , we will examine the role of this serine/threonine kinase in regulating the expression of steroidogenic protein and enzymes.  188  To examine the role of activated M A P K in regulating steroidogenic protein and enzymes in hGLCs.  Our results demonstrated that ATP activated M A P K in a dose- and time-dependent manner. Fufhermore, M A P K was shown to mediate the antigonadotropic action of ATP in hGLCs by reducing hCG-stimulated progesterone production but not c A M P accumulation. Considering the nuclear translocation of activated M A P K s [Fanger, 1999; Post and Brown, 1996; Cano and Mahadevan, 1995; Blenis, 1993], it can be postulated that M A P K s are involved in steroidogenesis through altering the synthesis of steroidogenic enzymes.  To examine the effect of ATP on inducing apoptosis (programmed cell death) in hGLCs.  ATP has been reported to induce death of hGLCs at a concentration of 2 m M [Charming, 1970]. However, there is no experiment to elucidate the underlying mechanism. Extracellular ATP may act as a trigger for apoptosis [Zheng et al., 1991].  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