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Arachidonic acid as a potential intracellular regulator of aromatase activity in human term trophoblast Lin, Show Whei 1992

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ARACHIDONIC ACID AS A POTENTIAL INTRACELLULARREGULATOR OF AROMATASE ACTIVITY INHUMAN TERM TROPHOBLASTbySHOW WHEI LINM.B., (Medicine), Chung Shan Medical College. Taiwan, 1975A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE MASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDepartment of Obstetrics and Gynaecology(Reproductive and Developmental Sciences)We accept this thesis as conformingto the required standardUNIVERSITY OF BRITISH COLUMBIAApril 14 1992© Show Whei Lin, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of Obstetrics and Gynaecology (Reproductive and Development Science)The University of British ColumbiaVancouver, CanadaDate Apr . 21 1992DE-6 (2/88)itABSTRACTThe potential role of arachidonic acid as an intracellular regulator ofaromatase activity of human term trophoblast was investigated in short term (3 h)incubations. Melittin, a known activator of phospholipase A2, suppressed thearomatase activity of physically dissociated trophoblast cells in a dose-dependentmanner. Indomethacin (10 -4M), a cyclooxygenase inhibitor of arachidonic acidmetabolism, suppressed aromatase activity and potentiated the inhibitory effect ofmelittin (P<0.05). Nordihydroguaiaretic acid (NDGA) alone, a lipoxygenaseinhibitor, also decreased aromatase activity but did not affect the inhibitory effectof melittin. The addition of exogenous arachidonic acid (10 4M) to trophoblast cellsdecreased aromatase activity in a dose-dependent manner (P<0.01). Arachidonicacid metabolites such as prostaglandin F2, (10-6M) and leukotriene B4 (10 -6M) hadno effect on aromatase activity. The presence of hypoxanthine (10 4 M) and xanthineoxidase (10 raU/m1), also attenuated aromatase activity in trophoblast cells (P<0.01),presumably via an elevation of intracellular free arachidonic acid concentration.Interestingly, melittin suppressed aromatase activity during short term (3 h)incubation but was ineffective on trophoblast cells from 24 h and 48 h incubations.The role of cAMP in the action of aromatase activity was investigated. CyclicAMP had no effect on 173-estradiol production in human trophoblast cells duringshort term (3 h) incubation. But cAMP enhanced 1713-estradiol production during 24h and 48 h incubations. These results suggested that aromatase activity of freshlyiiiobtained term trophoblast cells was at or near maximal capacity or cAMP may nothave been able to exert its steroidogenic action on human term trophoblast cellsrapidly enough to be detected during a 3 h incubation. Cyclic AMP stimulatedaromatase activity on human term trophoblast cells during 24 h and 48 hincubation, suggesting that cAMP achieves its actions by increasing protein andmRNA synthesis.Effects of arachidonic acid, cAMP, hCG and 25-OH-cholesterol onprogesterone production were investigated. None of them had effects onprogesterone production during a 3 h incubation. These observations suggest thatfresh term trophoblast cells may perform at or near opitmal capacity onprogesterone production.The results supported the potential intracellular regulatory role of arachidonicacid on aromtase activity in human term placenta.ivTABLE OF CONTENTSABSTRACT^TABLE OF CONTENTS ^  ivLIST OF ABBREVIATIONS  viiLIST OF FIGURES ^  viiiACKNOWLEDGEMENTS  ix1.0 LITERATURE REVIEW ^  11.1 The human placenta  11.1.1 Development, anatomy and histology of the placenta ^ 11.1.1.1 Development of the placenta ^  11.1.1.2 Anatomy and histology of the placenta ^ 31.1.2 Functions of the placenta ^  41.1.2.1 Transfer functions of the placenta  41.1.2.2 Immunologic functions of the placenta ^ 51.1.2.3 Endocrine function of the placenta  61.1.2.3.1 Protein hormones of the placenta^ 71.1.2.3.2 Steroid hormones of the placenta  101.2 Biosynthesis of sex steroid hormones by term placenta ^ 101.2.1 Feto-placental unit ^  101.2.2 Progesterone synthesis and secretion in the placenta ^ 111.2.2.1 Role of LDL and cholesterol in steroid hormonesynthesis  151.2.2.2 Cholesterol side chain cleavage (P-450scc) enzyme and3P-HSD/A" isomerase ^  171.2.3 Estrogen Synthesis and secretion in the placenta ^ 181.2.3.1 Estradiol and estrone synthesis and secretion ^ 181.2.3.2 Estriol synthesis and secretion in the placenta  191.2.3.3 Steroid sulfatase and sulfotransferase ^ 201.2.3.4 Placental aromatase^  211.2.3.5 Regulation of placental aromatase  211.3 Signal transduction and regulation of hormone production ^ 241.3.1 The second messenger cAMP^  241.3.2 The second messengers: Ca' and DG  25V1.3.3 The second messenger: cGMP ^  261.3.4 Phospholipase C ^  261.3.5 Phospholipase A2 and arachidonic acid ^ 281.4 Objectives ^  332.0 MATERIALS AND METHODS ^  342.1 Cell preparation ^  342.1.1 Preparation of human term trophoblasts ^ 342.1.2 Preparation of porcine granulosa cells  362.2 Enzyme assay: aromatase ^  372.3 Radioimmunoassay (RIA)  372.3.1 Estradiol RIA  382.3.2 Progesterone RIA ^  412.3 Statistical analysis  423.0 RESULTS^  433.1 Aromatase activity of human term trophoblasts ^ 433.1.1 Effects of melittin on aromatase activity  433.1.2 Effects of a cyclooxygenase inhibitor, indomethacin, onaromatase activity ^  433.1.3 Effects of a lipoxygenase inhibitor, NDGA, on aromataseactivity^  463.1.4 Effects of combined melittin, indomethacin and NDGA onaromatase activity ^  473.1.5 Effects of arachidonic acid with or without NDGA andindomethacin on aromatase activity ^ 483.1.6 Effects of arachidonic acid metabolites, prostaglandin F2a andleukotriene B4 ^  513.1.7 Effects of hypoxanthine and xanthine oxidase on aromataseactivity  513.1.8 Effects of hCG or 8-br-cAMP on aromatase activity ^ 543.1.9 Comparison of RIA and enzyme assay determinations ofaromatase activity ^  573.1.10 Effect of aromatase activity of melittin and cAMP after 24 hand 48 h incubation  573.2 Aromatase activity on porcine granulosa cells ^  613.2.1 Effects of treatment with indomethacin and melittin onviaromatase activity of porcine granulosa cells ^ 613.2.2 Dose-response effects of melittin and indomethacin on porcinegranulosa cells ^  613.3 The modulation of Progesterone production of human termtrophoblasts ^  643.3.1 Effects of melittin, hCG, cAMP on P4 production ^ 643.3.2 Effects of melittin and 25-OH-cholesterol on P4 production . . . ^ 644.0 DISCUSSION ^  674.1 Effects of arachidonic acid on aromatase activity of human termtrophoblasts  674.2 Effects of cAMP on aromatase activity in human term trophoblasts . . ^ 714.3 Short-term modulation of human placental progesterone production in vitrd734.4 Effects of indomethacin and melittin on porcine granulosa cells ^ 76CONCLUSION ^  78REFERENCES  79LIST OF ABBREVIATIONSAA^Arachidonic acidACTH Adrenocorticotropic hormoneATP^Adenosine trisphosphatecGMP cyclic guanosine-3',5'-monophosphateCL Corpus luteumDHA^DehydroepiandrosteroneDHAS Dehydroepiandrosterone sulfateDG DiacylgicerolE l^EstroneE2^EstradiolE3 EstriolEDTA^Ethylenediamine-tetraacetic acidFSH Follicle-stimulating hormoneGnRH^Gonadotropin releasing hormoneIGF-I Insulin growth factor IIndo^IndomethacinInsP3^Inositol 1,4,5-trisphosphateLDL Low density lipoproteinLH Luteinizing hormoneLTB4^Leukotriene B4M199 Medium 199NDGA^Nordihydroguaiaretic acidP4 ProgesteroneP450..^(pigment)-450 side chain cleavage enzymesP450 (pigment)-450 aromatasePKC^Protein kinase CPLA2^Phospholipase A2PLC Phospholipase CPGF2.^Prostaglandin FlaPtd-Ins PhosphatidylinositolPtd-InsP2^Phosphatidylinositol-4,5-bisphosphateRIA^RadioimmunoassayTRH Thyrotropin-releasing hormone33-HSD^3J3-hydroxysteroid dehydrogenase8-br-cAMP^8-bromo-cyclic adenosine monophosphateviiLIST OF FIGURESFig. 1 Estrogen biosynthesis in the feto-placental unit. ^ 12Fig. 2 The main sources of steroid hormone precursor.  13Fig. 3 A simple schematic structure of LDL and its transportationacross the membrane. ^  15Fig. 4 A schematic representation of aromatization of androgencatalyzed by aromatase  22Fig. 5 Molecular basis of PLA2 and PLC action. ^  29Fig. 6 The arachidonic acid cascade. ^  30Fig. 7 Proposed pathways for the mobilization of AA from Ptd-Ins andPtd-E in the cell. ^  31Fig. 8 Effects of melittin and AA on aromatase activity. ^ 44Fig. 9 Effects of a cyclooxygenase inhibitor, indomethacin  45Fig. 10 Effects of a lipoxygenase inhibitor, NDGA, on aromataseactivity. ^  47Fig. 11 Effects of melittin, indomethacin and NDGA on aromataseactivity  49Fig. 12 Effects of arachidonic acid with or without NDGA andindomethacin on aromatase activity ^  50Fig. 13 Effects of arachidonic acid metabolites, PGF 2a and leukotrieneB4 on aromatase activity^  52Fig. 14 Effects of hypoxanthine and xanthine oxidase on aromataseactivity ^  53Fig. 14a Effects of different doses of xanthine oxidase and hypoxanthineon aromatase activity^  54Fig. 15 Effects of hCG or 8-br-cAMP on aromatase activity ^ 56Fig. 16 The comparison between RIA and enzyme assay of aromataseactivity. ^  58Fig. 17 Effects of melittin and cAMP after 24 h incubation onaromatase activity ^  59Fig. 18 Effect of melittin and cAMP after 24 h and 48 h incubation. ^ 60Fig. 19 Effects of indomethacin and melittin on aromatase activity ofporcine granulosa cells.  62Fig. 20 Dose-response effects of melittin and indomethacin on porcinegranulosa cells. ^  63Fig. 21 Effects of melittin, hCG and cAMP on P4 production in humanterm trophoblasts.  65Fig. 22 Effects of melittin and 25-OH-cholesterol on P4 production. ^ 66ixACKNOWLEDGEMENTSFirst, I am deeply grateful to Dr. Leung, my supervisor, who guided my theresearch direction and provided information and advice. Next, Dr. Ledwitz-Rigbytaught me the detection of aromatase activity from porcine granulosa cells and gaveme continual support and suggestion. I want to thank for Dr. W. David Currie whogave me assistance from very tiny experimental techniques to very thoughtfultheory. And then David Boon, Wei Li and Gillian Steele also gave me lots ofsupport. Thanks to all of them.I also want to thank the nurses of the Labor and Delivery Unit in the O.R.and the physicians of Obstetrics and Gynaecology.1.0 LITERATURE REVIEW1.1 The human placentaThe human placenta is a transient organ that mediates physiological exchangebetween the mother and the developing fetus. The human placenta is geneticallyprogrammed to function for 9 months. The placenta provides a barrier betweenmaternal and fetal compartments and is a site of hormonal production andmetabolism. The barrier function of the placenta is similar to membranes active inphysiological exchange, such as the pulmonary alveolar lining and the nephron ofthe kidney. The endocrine function mimics certain functions of the adult pituitary,ovary and hypothalamus.1.1.1 Development, anatomy and histology of the placenta1.1.1.1 Development of the placentaThe ovulated human ovum is fertilized at the outer third of the fallopian tubeand transported to the uterus by ciliary action of tubal cilia approximately 4 daysafter ovulation. As the fertilized ovum passes through the fallopian tube, itundergoes cellular cleavage and forms a solid mass of cells called the morula. Themorula continues dividing to form the blastocyst which floats free in the1intrauterine fluid, from which it receives nutrients, for about 3 days.The human blastocyst, surrounded by the primitive trophoblast cells, consistsof the blastocyst cavity and inner cell mass. The inner cell mass differentiates intothe fetus. The primitive trophoblast underlying the inner cell mass penetrates thedecidua (pregnant endometrium) when the trophoblast cells contact the uterineepithelium. The trophoblast is the precursor of the fetal placenta.The penetrating embryo is totally covered by uterine epithelium by 11 days.Microscopic examination of the 11-day embryo reveals that rapid growth anddifferentiation of the trophoblast has occurred around the entire circumference. Thetrophoblast has differentiated into two cell layers: an inner layer of cytotrophoblastcomposed of individual cells and an outer layer of syncytiotrophoblast withoutdiscrete cell boundaries. The syncytiotrophoblast is formed by fusion ofcytotrophoblast which are mitotically active (Richart, 1961). The syncytium nowpossesses spaces called lacunae that contain maternal blood. Lacunae communicatewith each other and with maternal sinusoids and veins. These vascular connectionsallow the placental protein hormone, human chorionic gonadotropin (hCG), to entermaternal circulation at this time. This is evidence of a functional maternal vascularconnection. Proliferation of the cellular trophoblasts forms the tip-like villi whichbecome covered with syncytiotrophoblast. The villi enlarge and branch andprogressively assume the form of the fully developed human placenta.1.1.1.2 Anatomy and histology of the placenta2A full term placenta is discoid in shape with a diameter of 15-25 cm. It isapproximately 3 cm thick and has a weight of 500-600 gm. It consists of a fetal partformed by the chorion and a maternal part formed by the decidua basalis. The fetalside is bordered by the chorionic plate, the maternal side by decidual basalis.Between the chorionic plate and decidual basalis are the intervillous spaces whichare filled with maternal blood. After delivery, the maternal side of the placenta has15-20 slightly bulging areas, the cotyledons, covered by a thin layer of decidualbasalis. The fetal surface of the placenta is covered entirely by chorionic plate.The chorionic villi are the structures of the placenta central to function. Allplacental functions are centered in these areas. By the beginning of the third week,primary villi are formed. Primary villi are trophoblastic cords coated withsyncytiotrophoblast. During further development, mesodermal cells invade theprimary villous core and the secondary villi develop. By the end of the third week,blood cells and vessels are differentiated in the mesodermal core and maturechorionic villi are formed. The mature chorionic villus consists of 6 layers;syncytiotrophoblast, cytotrophoblast, basement membrane, interstitial cell layer,capillary endothelium and basement membrane. As pregnancy advances theinterstitial cell space is reduced and the villous vessels move from a central positionto a peripheral position (Fox, 1979). The thickness of the syncytiotrophoblastsdecreases from approximately 10 gm in early gestation to 1.7 um in late gestation(Robertson et al., 1975).31.1.2 Functions of the placentaThe placenta has several functions; exchange of metabolic and gaseousproducts between maternal and fetal circulation, immunological protection of thefetus from the maternal immune system and hormone production.1.1.2.1 Transfer functions of the placentaThe transport activities of the placenta are complex because numerousmaterials required for the synthesis of fetal tissues must be transferred and thewaste products of fetal metabolism removed. The transfer of substances across theplacenta occurs by several mechanisms including simple diffusion, facilitateddiffusion and active transport.Simple Diffusion: It is the most common and quantitatively significanttransport mechanism. 02, CO2, steroids and fatty acids are transferred by thismethod. Free fatty acids passively diffuse across the placenta and fetal levels arelower than maternal levels (Elphick et al., 1976).Facilitated diffusion: Transfer via a membrane bound carrier, usually atrates greater than achieved by simple diffusion. Some carbohydrates and glucoseare transported by facilitated diffusion, leading to rapid equilibrium with only a4small maternal-fetal gradient (Schneider et al. 1981; Hauguel et al., 1983). Aprotein (molecular weight 52,000) believed to bind glucose and act as a carrier infacilitated glucose diffusion has been isolated from placental membrane fractions(Johnson et al. 1982).Active transport: Transfer against a chemical gradient requires energy.Some amino acids and cations (Fe, Ca) are actively transported. Amino acid areactively transported across the placenta, making the concentrations in the fetalblood higher than in the maternal blood (Schneider et al., 1979).1.1.2.2 Immunologic functions of the placentaThe placenta prevents the fetus from attack by the maternal immune system.The mechanisms involved are poorly understood. The fetal trophoblast of thechorionic villi, which is in contact with maternal blood and immunocompetent cellsin the intervillous spaces over a large surface area, has been shown to lack antigensof the major histocompatibility complex (MHC) (ie. those antigens important inallograft rejection reactions) (Sunderland et al., 1981; Bulmer et al., 1985).Placental progesterone (P4) levels in humans are in the range of 2 x 103 ng/g wettissue to 6 x 103 ng/g. It has been demonstrated that P4 at these concentrations actsas an immunosuppressive agent in lymphocyte cultures stimulated by allogenicantigens (Mori et al., 1977; Stites et al., 1979). Estradiol-17(3 (E 2) and testosterone5are also able to block rIeljthymidine incorporation into human mixed lymphocrcultures (Clemens et al., 1979). In studies utilizing nonpregnant rats, an inversrelationship was demonstrated between thymic weight and circulating E2 atestosterone. In the absence of gonadal steroids (as by castration) thymic weightincreased while in the presence of elevated steroid levels thymic weight decreased(Chiodi et al., 1976; Grossman et al., 1979). Elevated E2 most certainly causes thechanges in thymic tissue structure observed in pregnancy.The action of these sex steroid hormones during pregnancy may play animportant role in the prevention of maternal-fetal allograft rejection and thusmaintains pregnancy to term.1.1.2.3 Endocrine function of the placentaIn 1905, Halban first suggested that the placenta was an endocrine organ(Halban, 1905). Since then, many investigators have contributed to a definition ofthe endocrine functions of the human placenta, including the formation of steroid,glyco-protein, and peptide hormones.The syncytiotrophoblast is thought to be the placental source of humanchorionic gonadotropin (hCG). Human CG is a gonadotropic glycoprotein hormonethat maintains the corpus luteum (CL) of pregnancy by mimicking luteotropicactions of luteinizing hormones (LH) during the first few months of pregnancy. Thesyncytiotrophoblast is also believed to be the site of production of human placental6lactogen (hPL). Human PL is a protein hormone that is primarily lactogenic inaction but also has detectable growth-promoting activity. In addition, P4 andestrogens (predominantly estriol(E3)) are produced by the placenta but placentalformation of estrogens requires cooperation of the fetal adrenal cortex and liver.1.1.2.3.1 Protein hormones of the placentaThe endocrine alterations that accompany human pregnancy increase theformation of sex steroid hormones and produce protein and polypeptide hormonessuch as hPL and hCG. The placenta also produces gonadotropin-releasing hormone(GnRH), human chorionic thyrotropin (hCT), corticotrophin-releasing factor (CRF),somatostatin and inhibin.Human chorionic gonadotropin--Human CG is a glycoprotein with a molecularweight of 36,700. The polypeptide portion accounts for 70% of the molecular weightand the carbohydrate portion, which appears to be essential for bioactivity, accountsfor approximately 30%. Human CG shares immunologic and biologic properties withLH (Hussa, 1980). Like LH, follicle-stimulating hormone (FSH) and TRH, hCGconsists of two subunits, a and f3. Human CG and its subunits have been localizedto syncytiotrophoblasts by immunocytochemical methods (Dreskin et al., 1970; Delkonicoff et al., 1973). Human CG can be detected in maternal blood by the tenthday after ovulation (Lenton et al., 1982). From day 10 onward, the hCG7concentration in pregnant women increases rapidly until week 8 of pregnancy.From weeks 8 to 12, hCG concentrations are fairly constant and then decrease untilabout week 18. After week 18 of pregnancy, the hCG concentration remainsrelatively constant for the remainder of pregnancy (Kletzky et al., 1985).The principal role of hCG is similar to that of LH, that is stimulation ofsteroidogenesis. Several lines of evidence suggest that hCG rescues the CL duringthe luteal phase of a fertile cycle by the action of extension of the life span of CLand increases the P4 and 17a-OH-P4 concentration in the maternal circulation(Caldwell et al., 1980).The effect of hCG in the CL is mediated by the action of adenyl cyclase, whichstimulates the conversion of adenosine trisphosphate (ATP) to cyclic adenosine-3%5%monophosphate (cAMP). Subsequently, cAMP augments the conversion ofcholesterol to pregnenolone and then increases P4 production (Hamberger et al.,1979; Dennefors et al., 1982). However, the role of hCG in the regulation of humanplacental steroidogenesis is still controversial. Human CG has been reported tohave no effect on the rate of conversion of either cholesterol or pregnenolone to P4(Macome et al., 1972; Talwar GP. 1979; Paul et al., 1981). The role of hCG in theregulation of estrogen synthesis in the human placenta is not clearly defined. It hasbeen reported that hCG either stimulates estrogen production (Cedard et al., 1970)or has no effect on estrogen synthesis (Laumas et al., 1968).Human placental lactogen —Human PL consists of a single polypeptide chain with8191 amino acid residues and two disulfide bonds. It has 96% sequence homologywith human growth hormone and 67% homology with human prolactin (Bewley etal., 1972; Cooke et al., 1981) Immunochemical location studies have demonstratedthat hPL is present in the syncytiotrophoblast by the second week after conception(Beck, 1970; Fujimoto et al., 1986).Human PL may stimulate basal insulin secretion (Nielsen, 1982). Human PLalso has growth-promoting activity, as demonstrated by hPL-induced body weightgain (Kaplan et al., 1964). In rats, the luteotropic activity of hPL is established(Zumpe et al., 1974). However, several women are known to have maintained anormal duration pregnancy despite nearly undetectable hPL levels (Nielsen et al.,1979, Sideri et al., 1983).Gonadotropin -releasing hormone—Human placental tissues have been shown tocontain gonadotropin-releasing hormone-like activity (Khodr et al., 1978b). It wasreported that gonadotropin-releasing hormone-like factor (LRF) stimulates secretionof placental hCG, a-hCG, P4, E2, estrone (E i) and E3 release in vitro (Khodr et al.,1978a).1.12.3.2 Steroid hormones of the placentaThe placenta produces large amounts of P 4, El, E2 and E3 during pregnancyand distributes these steroid hormones to both maternal and fetal compartments(Tulchinsky et al., 1975). Normal pregnancy depends upon placental steroid actions9on the maternal reproductive organs and metabolic system. In pregenancy, estrogenstimulates growth of the uterine muscle mass, which will eventually supply thecontractile force needed to deliver the fetus. Progesterone may maintains the uterinestability during pregnancy.1.2 Biosynthesis of sex steroid hormones by term placenta1.2.1 Feto-placental unitThe biomolecular interactions between the fetus and mother during humanpregnancy are orchestrated by means of a fetal-maternal communication system.The human placenta lacks 17a-hydroxylase and C 17,93 lyase and is, therefore,unable to convert acetate into estrogen. The placenta is capable of converting C19steroids into estrogen (Ryan, 1959; Siiteri et al., 1963). It was suggested that thehuman fetal adrenal may provide precursors for estrogen biosynthesis (Frandsen etal., 1961). It has also been shown that DHAS of maternal and fetal adrenal origincontributed about equally to placental E 1 and E2 formation, while over 90% of theE3 as synthesized from 16-hydroxy-DHAS of fetal origin (Siiteri et al., 1963). (Fig.1)101.2.2 Progesterone synthesis and secretion in the placentaMost of the low density lipoprotein (LDL) in fetal plasma arises by de novosynthesis in the fetal liver (Carr et al., 1984). During pregnancy the placenta usesmaternal plasma cholesterol, primarily in the form of LDL, as its precursor for theformation of placental steroids, especially P4, rather than synthesizing cholesterolfrom acetate (Hellig et al., 1970) (Fig. 2).Progesterone is one of the major hormones synthesized during pregnancy.During the first trimester, the CL is primarily responsible for synthesis of thishormone, whereas during the second and third trimesters the main site of P4synthesis is the placenta. Conversion of cholesterol to P4 involves several steps.The conversion of cholesterol to pregnenolone in mitochondria is the first rate-limiting and hormonally regulated step in the synthesis of all steroid hormones(Golos et al., 1987; Ringler et al, 1989). Three enzymes are involved in this step;20a-hydroxylase, 22-hydroxylase and 20,22-lyase (Lieberman, et al., 1984). Mostpregnenolone is readily converted to P4 the action of 30-hydroxysteroiddehydrogenase (313-HSD) and 2, e4-isomerase. Approximately, 90% of P4 which isformed by the placenta appears in maternal circulation, where it is metabolized topregnanediol. The other 10% of P4 transported to the fetus as precursor for othersteroids (Tseng et al., 1978).11MOTHER FETUSPLACENTA50,11^ 50% DHAS ^eel fIDHA111,-HAndrostonedionoAROMA^ AddDHAS(fetal adrenal)id-hy dddd lameTDHAS113 - hydroxy lass 0AnOIATAlla2b-Hal)aillrateee90%10%1 8 -OH -DHAS(fetal liver)1 8 - OH - DHASFig. 1 Estrogen biosynthesis in the feto-placental unit.Fetal DHA is synthesized and sulfatized in adrenal. Around 70% of DHASproduced by fetal adrenal is converted to 16a-OH-DHAS in fetal liver and thentransported to placenta for E3 synthesis. Maternal and fetal compartmentcontributed the same amount of DHAS for the synthesis of E 1 and E2, while over90% of the estriol was synthesized from 16a-OH-DHAS obtained from the fetus.Estriol formation in the placenta is not from the conversion of E l , but directly from16a-OH-DHAS under a series of enzymatic action (sulfatase, 3f3-HSD, and t 1'5isomerase and aromatase).12Placenta FetusMotherCholesterolPregnenoloneProgesteroneFig. 2 The main sources of steroid hormone precursor.Placenta uses maternal cholesterol, mainly LDL, as a primary source for theP4 production. Ninety percent of P4 production are transported to the maternalcompartment and 10% to fetal compartment.10%CholesterolAcetate131.2.2.1 Role of LDL and cholesterol in steroid hormone synthesisCholesterol is the major substrate of steroid hormone biosynthesis in humanplacenta. The minimal capacity of the placenta to synthesize cholesterol isconfirmed by the lack of conversion of acetate to cholesterol demonstrated byplacental perfusion (Telegdy et al., 1970). A large proportion of cholesterol comesfrom LDL (Brown et al., 1979). A simplified model for the structure of LDL isshown in Fig. 3 (based on Brown et al., 1991). LDL contains a nonpolar core inwhich many molecules of hydrophobic cholesteryl esters are packed to form an oildroplet. Hydrophobic cholesteryl esters account for most of the mass of the LDLarticles. Uptake of lipoprotein from plasma is regulated by the serum concentrationof lipoprotein and the lipoprotein receptor-dependent uptake system. In humans70%-80% of LDL removed from the plasma each day by the LDL receptor pathway.Receptor-bound LDL enters the cells by receptor-mediated endocytosis andcholesterol is stored within cytoplasm in an esterified form.14Nonesterlfied• \ 10 6 ChoisterolPhospbolipidmembraneCell membraneEndocytosis^CrawlStructurs of Low Density of Lipoprotein (LDL)^ L receptorFig. 3 A simple schematic structure of LDL and its transportation acrossthe membrane.The core of the spherical lipoprotein particle is consisted of two nonpolarlipids which are triglyceride and cholesteryl ester. The nonpolar core is surroundedwith an outer coat composed chiefly of phospholipids. LDL carried by the plasmabinds to the LDL receptors in the cell membrane and is taken up by the cell withendocytosis. LDL is digested by lysosomes within the cells and cholesteryl esters arehydrolyzed into cholesterol by cholesteryl esterase.15Upon stimulation of the target organs by tropic hormones such as LH,adrenocorticotropic hormone (ACTH) or cAMP, a cholesterol esterase is activated,and the free cholesterol formed is transported into mitochondria, where acytochrome P450 side chain cleavage enzyme (P-450. x) converts cholesterol topregnenolone. Free cholesterol, which is insoluble in the aqueous cytosol, may betransported to mitochondria by a Sterol Carrier Protein 2 (SCP-2) (Noland et al.,1980, Tanaka et al., 1984). All mammalian sex steroid hormones are synthesizedfrom cholesterol via pregnenolone through a series of reactions that occur in eitherthe mitochondria or endoplasmic reticulum of sex steroid specific producing cells.1.2.2.2 Cholesterol side chain cleavage (P-450scc) enzyme and 313-HSD/A"isomeraseConversion of cholesterol to pregnenolone is the first rate-limiting andhormonally regulated step in the synthesis of steroid hormones (waterman et al.1985; Golos et a/.1987; Voutilainen et ala. 1987). It was suggested that threeseparate and distinct enzymes were involved: 20-hydroxylase, 22-hydroxylase anda 20,22-lyase (Lieberman et al. 1984). Carbon monoxide studies suggested that thesesteps were mediated by separate cytochrome P-450 enzyme. P450 functions as aterminal electron receiver in the mitochondrial electron transport system. Theelectron transport process begins with the accepting electrons (e) of adrenodoxinreductase from NADPH. These e• are passed to adrenodoxin, an iron-sulfur protein16within mitochondria (Picado-Leonard et a/. 1988). P-450scc accepted the e - fromadrenodoxin and catalyzed the cleavage of cholesterol to pregnenolone.Conversion from pregnenolone to P4, it involves two enzymes: 313-HSD, and5-4 isomerase. The enzymatic reactions of these two enzymes are poorlyunderstood. Some studies suggested the presence of two to three different isozymesof isomerase specific for the three steroidogenic pathways i.e mineralocorticoid,gluco-corticoidogenic and sex steroid (Gower et al. 1983).1.2.3 Estrogen Synthesis and secretion in the placentaThe placenta becomes the primary source of estrogen after approximatelyweek 9 of pregnancy (Oakey, 1970). Estrogens are formed in the placenta bymaternal and fetal 2 androgen precursors. The placenta is unable to produce theseprecursors de novo. Estrogen production rate increases continuously duringpregnancy and reaches 100-120 mg/24h. E3 comprises 60-70% of the total estrogens;300-500 times greater than in the non-pregnant state.1.2.3.1 Estradiol and estrone synthesis and secretionThe most important androgen precursor for E l and E2 synthesis is DHAS. Thematernal compartment contributes 40-45% of DHAS and DHA for the synthesis ofE l and E2 (Siiteri et al., 1966). The fetal compartment and particularly the fetal17adrenals provide 60% of the DHAS. The first step in estrogen production ishydrolyzation of DHAS by steroid sulfatase followed by transformation of e 6 DHAto A' androstenedione by 313-HSD and A" isomerase. Androstenedione isaromatized to E l by placental aromatase or is transformed to testosterone and thenaromatized to E2.1.2.3.2 Estriol synthesis and secretion in the placentaEstriol is synthesized by the placenta primarily from fetally derived 16a-OH-DHAS and estriol diffuses to the maternal and fetal circulations (Siiteri et al., 1966;Tulchinsky et al., 1973). It is estimated that 90% of the 16a-OH-DHAS precursorfor E3 synthesis is derived from the fetus and 10% form the mother, which is muchless than that for E 1 and E2 (Madden et al., 1978).Fetal adrenals use maternal LDL as substrate to produce DHA and sulfatizeDHA to DHAS. Approximately 70% of total fetal DHAS is transferred to the fetalliver where DHAS is hydroxylized to 16a-OH-DHAS. 16a-OH-DHAS isquantitatively the most important androgen circulating in the fetal compartment.16a-OH-DHAS reaches the placenta from both maternal and fetal compartments butthe maternal contribution is less than from the fetus (fetus 90%, mother 10%). Inthe placenta, E3 is formed after desulfation and aromatization of 16a-OH-DHAS byplacental sulfatase and placental aromatase respectively.181.2.3.3 Steroid sulfatase and sulfotransferaseSteroid sulfates may be synthesized directly from cholesterol sulfate or maybe formed by sulfation of steroids by cytosolic sulfotransferases (Miller et al., 1981).An important biochemical characteristic of the placenta is its capacity tohydrolyze steroid sulfates. Placental steroid sulfatase is a microsomal enzyme (Roseet al., 1982). It was detected in syncytiotrophoblasts by immunohistochemistry andhybridization studies. Neither steroid sulfatase immunoreactivity nor steroidsulfatase mRNA were detected in cytotrophoblast (Salido et al., 1990). In humanplacenta, P4 is synthesized from cholesterol, whereas estrogens are produced mainlyfrom maternal and fetal steroid sulfate precursors, DHAS and 16a-OH-DHAS.These 2,C 19 androgen precursors are desulfated by steroid sulfatase in the placentabefore entering the pathway catalyzed by 3f3-HSD, 2' isomerase and aromataseto yield estrogens.The fetal adrenal and liver convert estrogen and P4 precursors, pregnenolone,17a-OH-pregnenolone, DHA and 16a-OH-DHA, into sulfated forms mostly at the 313position by sulfotransferase. The sulfated forms are transported to the placentawhere the sulfated group is cleaved off by sulfatase to produce estrogens and P4.1.2.3.4 Placental aromataseThe placenta is a major site of conversion of C a androgen precursors to19estrogens because of the amount and the high rate of activity of aromatase (Doodyet al., 1989). The enzyme was first purified and its amino acid sequencecharacterized from human placenta (Chen et al., 1986; Nakajin et al., 1986). Major(P2a) and minor (P3) forms of human placental aromatase have been solubilized andidentified by chromatography (Osawa et al., 1987).Most steroidogenic enzymes are members of the cytochrome P450 groups ofoxidases (Nebert et al., 1987). Aromatization of C18 estrogenic steroids from C19androgens is mediated by P450 , found in the endoplasmic reticulum. The systemconsists of an aromatase cytochrome P450 and a flavoprotein, NADPH-cytochromeP450 reductase (Thompson et al., 1974). The aromatization process involves lossof the angular C-19 methyl group and stereospecific elimination of the 1-13 and 2-Phydrogens from the androgens (Cole et al., 1990). This process includes 3 enzymatichydroxylations requiring 3 moles of 02 and NADPH per mole of estrogen formed.Furthermore, these irreversible reactions require the participation of bothcytochrome P-450 and NADPH cytochrome C-reductase enzymes (Seymour et al.,1984) (Fig. 2).1.2.3.5 Regulation of placental aromataseFollicle-stimulating hormone (FSH) and cAMP have been shown to stimulatearomatase activity in ovarian tissues (Dorrington et al., 1975, Erichson et al., 1978,Gore-Langton et al., 1981). Aromatase activity in human choriocarcinoma cells may20be stimulated by CAMP and theophylline (Bellino et al., 1978). Insulin, insulin-likegrowth factor I (IGF-I) and insulin-like growth factor II (IGF-II) may be potentinhibitors of aromatase activity in human placental cytotrophoblasts (Nestler et al.,1987 & 1990).210 20#H1HO I HNADPH02ENZYME ISLOWO .NADPHENZYME IIV. SLOWo#HONO ENZYMEV. FASTC,))HO-HO/Estrogen biosynthesis sequence0II+ HC-0-Fig. 4 A schematic representation of aromatization of androgen catalyzedby aromatase.Aromatization process requires a sequence of several microsomal enzymes,referred to as aromatase. The process involves three enzymatic hydroxylations.Enzyme I catalyzes two hydroxylations. Enzyme II catalyzes one hydroxylation andis the slowest step of the three. The third step is no enzyme involved.221.3 Signal transduction and regulation of hormone production:Cells respond to their environment through cell surface receptors that bindspecific ligands. These "signals" are transduced into cells and change cellularmetabolism and function. Many extracellular ligands act by increasing theintracellular concentrations of second messengers such as cAMP, Cap* or thephosphoinositides. Receptors generally associate with a transmembrane signalingsystem with three separate components. The first messenger (ligand) is recognizedby membrane-bound receptors specific to the ligand. The activated ligand-receptorcomplex triggers the activation of G-protein located inside the plasma membrane.The G-protein in turn changes the activity of effector elements, such as adenylatecyclase and PLC. The effector element for cAMP is adenylate cyclase, which is atransmembrane protein that converts intracellular ATP to cAMP. PLC andguanylate cyclase, respectively, are the effector elements of Cam, DG and cGMP.1.3.1 The second messenger: cAMPCyclic AMP, which is derived from ATP through the action of adenylatecyclase, plays an important role in the action of a number of hormones such as FSH,LH, angiotensin II, etc. Cyclic AMP binds to the regulatory subunits of a cAMP-dependent protein kinase and dissociates the catalytic subunits from the proteinkinase. The active catalytic subunits catalyze the phosphorylation of proteins.23Cyclic AMP is an important intracellular mediator in reproductiveendocrinology. Stimulation of P4 secretion from placenta by (Bu)2cAMP and ii-adrenergic agonists has been reported (Caritis et al., 1983). Cyclic AMP alsoincreases P4 production by human chorion (Tonkowicz et al., 1985). The regulationof P4 secretion by 8-br-cAMP was proposed as a result of 8-br-cAMP-provokedincreases in protein mRNA in placental cells (Nulsen et al., 1989). There are alsoconflicting reports regarding cAMP. It had been shown that cAMP had no effect onthe conversion of pregnenolone to P4 of human placenta (Ferre et al., 1975). It wasalso reported that 8-br-cAMP inhibited aromatase activity in cultured first trimesterhuman placenta (Rodway et al., 1990).1.3.2 The second messengers: cam and DGThis second messenger system involves hormonal stimulation of Ptd-Inshydrolysis. As soon as the extracellular signal (such as GnRH or angiotensin) bindswith its specific receptor in the plasma membrane, a series of chain reactions occurs.The binding of ligand and receptor activates GTP-binding regulatory proteins (G-proteins) and then triggers the enzyme, phosphoinositide-specific PLC. PLCcatalyzes plasma membrane Ptdlns-P 2 breakdown and generates two products: 1P3and DG.IP3 is water soluble and diffuses into cytoplasm where it triggers the releaseof sequestered Ca' from within the cell. The elevation of cytosolic ce enhances24the binding of Cap* with the calcium-binding protein calmodulin, which regulatesactivities of calcium-dependent protein kinase.DG has two potential signaling roles. DG is confined to the plasmamembrane where it activates the calcium-dependent PKC. DG can also be cleavedto release AA, which can be used as an intracellular regulator and/or continue tobreakdown into its eicosanoid metabolites.1.3.3 The second messenger: cGMPUnlike cAMP, the versatile intracellular second messenger, cyclic guanosine-3',5'-monophosphate (cGMP), is a more localized messenger in only a few cell types,such as intestinal mucosa and vascular smooth muscle. Increased intracellularcGMP concentrations cause vascular smooth muscle relaxation through a kinase-mediated mechanism. This seems to involve phosphorylation of myosin light chainkinase.Atrial natriuretic factor (ANF) and endothelial derived releasing factor(EDRF, probably nitric oxide) cause to guanylate cyclase activation. How ANF andEDRF regulate guanylate is unknown, but the system does not appear to use a G-protein.1.3.4 Phospholipase C25The activation of PLC is most likely mediated by a GPT-binding protein(Cockcroft S. 1987; Spiegel AM. 1987). Recent studies to purify the enzyme haveshown the existence of three immunologically distinct forms of PLC: type I, type II,type III (Suh et al. 1988). PLC-I was evenly distributed between the cytosol and theparticulate fraction. PLC-II is exclusively cytosolic. It was reported recently thatEGF-induced tyrosine phosphorylation of PLC-II, suggesting direct activation ofeffector by the receptor (Margolis et al. 1988).The activation of receptors stimulates PLC which degrades membrane-boundPtdlns-4,5-bisphosphate to produce two second messengers: IP 3, which can liberateCap* from intracellular stores and DG, which activates PKC (Berridge M.J., 1987,Nishizuka Y., 1984). The fatty acid in the sn-i position of DG is hydrolyzed by DGlipase followed by hydrolytic cleavage of the fatty acid from the sn-2 position bymono-acylglycerol lipase (Okazaki et al., 1981). This leads to the release of AA fromthe sn-2 position of 1,2-diacyl-sn-glycerols (Fig. 5).Upon the liberation of intracellular unesterified fatty acid from abovephospholipases, AA is rapidly oxidized in two separate pathways. One pathway iscatalyzed by an enzyme complex referred to as prostaglandin synthase orcydooxygenase and leads to the production of prostaglandins and thromboxanes.Another pathway of AA oxidation is catalyzed by lipoxygenase, an enzyme complexindependent of the cydooxygenase system, and leads to the formation of leukotrieneB4 leukotriene Co Do E4. (Fig. 6)261.3.5 Phospholipase As and arachidonic acid12:PLA2 enzyme is found in nearly all animal cell types examined. PLA2liberates AA from phospholipids in the membrane (Irvine, 1982). This enzyme maybe present in the plasma membrane or the cytosol (Balsinde et al., 1988). MostPLA2 enzymes need ce ions for full activity and also can be regulated by Gproteins linked to adrenergic or cholinergic receptors (Burch et al., 1987; Felder etal., 1990). In the presence of elevated cytosolic ce ions or phospholipase activators(e.g. melittin), phospholipase A2 is activated and splits membrane phospholipids intolysophospholipid and AA (Fig. 7). PLA2 activity is the major source of AA in the cell(Bell et al., 1979).Arachidonic acid: Several lines of evidence suggest that AA and its metabolites play animportant role as intracellular regulators in reproductive endocrinology. AA hasbeen shown to stimulate P4 production in rat ovarian cells (Wang and Leung 1988,1989). It was demonstrated that GnRH-stimulated LH release is mediated by AA(and/or its metabolites) and protein kinase C in pituitary cells (Chang et al., 1986).PKC activated by phorbol ester (TPA) was shown to stimulate AA mobilization andphospholipid hydrolysis in human uterine decidua cells (Schrey et al. 1987). Theyconcluded that PKC activation by TPA leads to AA mobilization from decidua-cellphospholipid by a mechanism involving phospholipase A-mediated Ptd-Inshydrolysis and phospholipase C-mediated phosphotidylcholine (Ptd-C) hydrolysis,27coupled with further hydrolysis of the DG product. It was reported that AAstimulated calcium and hPL release in isolated trophoblast cells (Zeitler et ai. 1986).The stimulation of hPL by AA may be due, at least in part, to the effects of the fattyacid on cellular calcium mobilization. AA also stimulated the rapid appearance ofinositol monophosphate in placental cells (Zeitler et al. 1985). The effect on AA wasspecific for hydrolysis of phosphoinositides and phosphatidylserine and did notinvolve other phospholipids. Arachidonate and unsaturated fatty acid have also beensuggested that they might activate directly and regulate protein kinase C as equallyimportant as DG in receptor function and cellular regulation in the neutrophil(McPhail et al., 1984). In neutrophils, AA directly activates a GTP-binding proteinand therefore may act as a role of second messenger in signal transduction(Abramson et al., 1991). PGF Ptd-Ins 4,5-bisphosphate hydrolysis andmobilizes intracellular Ca' in bovine luteal cells (Davis et al., 1987). It wassuggested that PGF its cellular responses in luteal cells by stimulating thephospholipase C-inositol 1,4,5-trisphosphate (InsP3) and DG second messengersystem. Recently, data had been reported suggesting that ovarian leukotriene B4(LTB4), one of the lipoxygenase pathway products, may induce ovulation bymechanical events within the ovary that are required for rupture of matureGraafian follicles in the rabbit (Yoshimura et al. 1991).2861MembtanePIE•PbodPidmoo— al 2IntiV^8 IcxV, 0-I imk 2 i 012-04--•\"/-\/-\,--\,----N, COOKaradzidottic acid (20 =Woe),I extended confonnetionCCIOH00.(/anchidonic add ,folded defamationMenixesePlicePholiPid1n^csV^8 I 2C- 0— CHPhoepho►ipese CIDOL"""e■A".W ci 0_ 26 7Arschidcnic AcidPhoepholipsee A2PLClipase.Fig. 5 Molecular basis ofPLA, and PLC action.PLA2 cleaves phospholipidon sn-2 position and yieldsAA and lysophospholipid(upper panel).The lower panelshows PLC action onmembrane phospho-lipids.The PLC splits the phospho-lipids into IP3 and DG. DGmay further break-down intoAA.DGL:diacylglycerol lipase.—121C^MGL:monoacylglycerol29N D G A --^ast- )-■ ep–00isc _LTA 4\N HEPETELTB4 LTC4Soo^44-i— Indomethacintit.ei,PGG2 4/ \ ey„, 416%4-,14.14.•PGF2 TXA2PGF2 PGD2The arachidonic acid cascadePHOSPHOLIPIDCl4010IIIMelittin --e . ).2-Calcium^o=lonophores^o.•0=a.4."..... GlucocorticoidsLocal anesthetics •ARACHIDONIC ACIDFig. 6 The arachidonic acid cascade.This is one pathway through which AA is generated. Release of AA isfacilitated by the hydrolytic action of PLA 2 on membrane phospholipid. Theliberation of AA is followed rapidly by the actions of cyclooxygenase andlipoxygenase, producing eicosanoid metabolites.30Phosphatidyl-ethanolamine IMOPhosPhatklY1-inositolPhosphandicacid N PLC  I^>Diacylglycerol(1)Monoacylglycerol(2)IArachidonic Acidi____J I 1^ILT-S^PG-S TX-SFig. 7^Proposed pathways for the mobilization of AA from Ptd-Ins andPtd-E in the celLPhospholipids are catalyzed by PLA 2 and PLC. DG produced from Ptd-I hasat least two pathways: phosphorylation to phosphatidic acid and hydrolysis tomonoacylglycerol (MG) by DG-lipase (1). MG is further degraded to AA and glycerolby MG-lipase (2). Mobilization of AA from phosphatidylethanolamine (Ptd-E) seemsto involve a single reaction catalyzed by PLA2.31AA also enhanced P4 production in rat granulosa cells and AA may act as astimulatory mediator of LH-releasing hormone action in the rat ovary (Wang &leung., 1988 & 1989).1.4 ObjectivesThis study was intended to obtain information relative to understanding AAand cAMP effects on human term placenta. The study examined AA and cAMPregulation of aromatase activity and P-450. ce in short term (3 h) human termplacental cell cultures. Human term placental cells cultured for 3 to 4 days wereused to examine differences in response of aromatase activity to AA and cAMP inshort term versus long-term cell cultures.322.0 MATERIALS AND METHODS2.1 Cell preparation2.1.1 Preparation of human term trophoblastsGeneral materials:* Supplemented M199 (Gibco, Mississauga, Ontario):1% (v/v) heat inactivated, defined fetal calf serum (FCS, Hyclone, Logan, UT).Penicillin G sodium (100 U/ml, Gibco).Streptomycin (100 pg/ml, Gibco).Hepes (15 mM, Gibco).NaHCO3 (26 mM, Sigma, St. Louis, MO).* Hank's balanced salt solution (HBSS, Gibco).* Arachidonic acid (Sigma).* Cyclic adenosine-3',5'-monophosphate (Sigma)* Human chorionic gonadotropin (Sigma)* Hypoxanthine (Sigma).* Indomethacin (Sigma)* Leukotriene B4 (Sigma)* Melittin (Sigma)* Nordihydroguaiaretic acid (Sigma).33* Prostaglandins F2a (Sigma)* Scintiverse (Fisher, Vancouver, B.C).* [10,213,-3M-testosterone (40.4 mCiJmmol, Dupont, Boston, MA).* Xanthine oxidase (Sigma).Methods--Normal full term placentae (36-42 weeks gestation) were obtainedimmediately after elective cesarean section. Four cotyledons dissected fromunderlying fibrous tissues were rinsed with supplemented M199 and dissociatedthrough a stainless steel sieve (150 pm screen, Sigma, St. Louis, MO). Aggregatedcells were removed by filtration through 48 pm nylon mesh (B&SH Thompson,Scarborough, ONT). Dissociated cells were centrifuged (1000g x 6 min), supernatantdecanted and cells resuspended in M199. Hematocytes were removed bycentrifuging (1700g x 20 min) the suspension on 40% (v/v) percoll (Sigma) in HBSS.Cell viability as determined by 0.05% trypan blue (Gibco) exclusion staining wasconsistently greater than 90%.Cells were pipetted into borosilicate glass tubes (12 x 75 mm, Canlab,Burnaby, BC) at a density of 2 x 10 6 cells/ml M199. Each tube was treated with amixture of [18,213-3M-testosterone (30,000 cpm) and unlabelled testosterone (2 x 10 -7M). Tubes containing this testosterone mixture were then treated with additionalfactors such as indomethacin (1 x 104M), melittin (1.5 x 104M), NDGA (1 x 10-5M),AA (1 x 10-5M), PGF2,,, (1 x 104M), LTB4 (1 x 104M) or hypoxanthine (1 mM) plusxanthine oxidase (10 mU/m1). Cells were cultured in a shaking incubator (60 rpm,345% CO2, 37C) for 3 h. After 3 h incubation, 500 pl of a charcoal (BDH, Vancouver,BC, 20 mg/m1 distilled water) and dextran (BDH, 2 mg/ml distilled water) mixture(4C) was added at 4C. The tubes were vortexed (30 cycles/sec) for 1 minute with amultiple-tube vortexer and let them stand for 15 minutes. The tubes were thencentrifuged (2000g x 15 min) at 4C. The supernatant was decanted to scintillationvials (Packard, Canberra, Australia) and 3 ml of scintillation cocktail added.Radiation was counted (1 min/vial) using a beta-counter (LKB-Wallac C1217RackBeta, Turku, Finland).All experiments were performed in hexaplicate.2.1.2 Preparation of porcine granulosa cellsGeneral Materials—As outlined in section 2.1.1.1Methods--Porcine ovaries were obtained from the abattoir in the morning. Pigswere electrically stunned (400 volts) and exsanguinated. Ovaries were immediatelyremoved, submerged in 0.154 M saline in an insulated container and transportedto the laboratory.Disposable plastic syringes (5 ml) with 23 gauge needles were used topuncture and aspirate follicular fluid. The mixture of follicular fluid and granulosacells was centrifuged (1000g x 6 min) The supernatant was decanted and the pelletresuspended in M199. The cells were pelleted and resuspended a second time.35Cells were counted using an Improved Neubauer Hemocytometer (Canlab) and areverse phase microscope (10X, Nikon). Cell viability was determined using 0.05%trypan blue exclusion staining. Cell viability was approximately 30-40%.Cells were pipetted into borosilicate glass tubes (12 x 75 mm) at a density of1 x 106 cells/ml. Each tube received a mixture of [113,2f3, 3H]testosterone (30,000cpm) and unlabelled testosterone ( 2 x 10-7M) and subsequently treated withcombinations of indomethacin (1 x 10 4M), melittin (3 x 10-7M) and NDGA (1 x 10 -5m) .2.2 Enzyme assay: aromataseHuman term trophoblast cells (2 x 106 cells/nil) incubated with [113,41-3H]testosterone (30,000 cpm) and a corresponding amount of unlabelled testosteroneto give a final concentration of 200 nM. The formation of total estrogen by thearomatase is equimolar to the tritiated water produced. Detection of tracer waswith a liquid scintillation counter (LKB-Wallac C1217 RackBeta) detecting 3Hdecay with 60% efficiency. Scintillation vials were counted for 60 sec each.Aromatase activity was measured by the recovery of tritiated water from theconversion of [113,213-311]-testosterone to estrogen.The following equations were use for calculation of conversion:(200 nM was the testosterone concentration added in the media.)36Rate of convert - the recovery rate of tritiated water-Backgroundtotal countEstrogen=rate of convert x 200 nti2.3 Radioimmunoassay (RIA)2.3.1 Estradiol RIAReagents1. Buffer was 50 mM phosphate buffered saline (pH 7.4, 12 mM EDTA) with0.1% w/v gelatin. All buffer reagents were from BDH.Mix and stir until dissolved in 900 ml distilled water:1.0 g gelatin, dissolved in 100 ml hot distilled water9.0 g sodium chloride, NaC15.3 g monobasic sodium phosphate, NaH 2PO4 crystalline.8.8 g dibasic sodium phosphate, Na 2HPO4 andydrous.0.1 g sodium azide, NaN22. The standard (Sigma E1132, Sigma) was prepared and stored in polystyreneat -20C. Standards were diluted in M199 media without serum3.^^The antiserum was rabbit #3, bled 12/10/82, raised against 1,3,5(10)-estratriene-3,17p-dio1-6-one-6-carboxymethyl-oxide:BSA conjugate (Steraloids,37Wilton, New Hamphire). The antiserum was used at a final dilution of1:750000 and gave 71% binding of label (12.000 cpm).4. The labelled hormone was 2,4,6,7,-31I-E2 (Amersham, Oakville, Ontario) witha specific activity of approximately 114 Ci/mmol. Initial stock was 1 pCi/p1in toluene:ethanol (9:1 v:v). Solvents were evaporated and stock was dilutedin buffer (2000 a- 15,000 cpm).5. The charcoal suspension (0.25% w/v Norit A + 0.025% Dextran w/v, BDH) inbuffer was mixed in glass for 30 min at 20C, stored at 4C and mixed for 30min at 4C prior to use.6. The extraction solvent was ethyl ether (Fisher). Extraction efficiency wasapproximately 90%.7. The scintillation cocktail was Scintiverse (Fisher).ProcedureStandards (range from 1pg-lng/m1) and samples (diluated 5X) were assayedin quadruplicate. Extraction tubes were 16 x 150 mm borosilicate glass (Fisher); RIAtubes were 12 x 75 mm borosilicate glass (Fisher). Scintillation vials werepolyethylene (Packard).Day 1: Extraction Efficiency100 ul tracer added to 3 scintillation vials and 3 extraction tubes.0.5 ml M199 added.38M199 extracted, ether phase evaporated and 3 ml Scintiverse added.Extraction500 pl sample added to extraction tubes.4 ml ethyl ether added to extraction tubes.Extraction tubes vortexed twice for 30 s at a 1 h interval at 21C and incubated foran additional 1 h at 21C.Extraction tubes cooled at -70C for 12 minEther phase decanted to assay tubes and evaporated.RIA200 pl standard added to appropriate tubes.200 pl buffer added to total counts assayed tubes (TCA) and non-specific bindingtubes (NSB).200 pl antiserum in buffer added to standards and samples.Vortexed.Incubated for 1 h at 20C.200 pl tracer added to all tubes.Vortexed.Incubated for 24 h at 4C.Day 2:39500 1.11 charcoal suspension added in less than 2 min to all but TCA tubes.500 jil buffer added to TCA tubes.Vortexed.Tubes loaded in centrifuge precooled to 4C.Assay tubes centrifuged at 2000g for 15 min, beginning 10 min after addition ofcharcoal to first tube.Supernatant decanted to scintillation vials.3 ml Scintiverse added.Votexed and incubated for 1 h at 21C.Counted for 1 min per vial (LKB-Wallac).2.3.2 Progesterone RIAReagents and procedure:Tracer: (1,23H(N)lprogesterone with a specific activity of approximately 115Ci/mmol (Amersham)Procedures: The P4 concentration in the culture medium was determined by themethod of Leung et al., 1979, and antiserum was kindly provided by Dr. D.T.Armstrong of the University of Western Ontario (Leung and Armstrong, 1979).Standards (range from 80pg-lOng/m1) were used triplicate and samples wereassayed in duplicate. RIA tubes were 10 x 75 mm borosilicate glass (Fisher).Scintillation vials were polyethylene (Packard).40Day 1:100 Al std added to appropriate tubes.300 ill buffer added to TCA and NSB tubes.100 pl antiserum in buffer added to stds and samples.100 Al tracer added to all tubes.Vortexed.Incubated for 24 h at 4C.Day 2:500 pil charcoal suspension added in less than 2 min to all but TCA tubes.Vortexed.Tubes loaded in centrifuge precooled 4C.Assay tubes centrifuged at 2000g for 15 min, beginning 10 min after addition ofcharcoal to first tube.Supernatant decanted to scintillation vials.3 ml Scintiverse added.Incubated for 4 h at 4C.Counted for 1 min per vial in beta-counter (LKB-Wallac).2.3 Statistical analysisControl and treatment groups were compared by one-way or multiple analysisof variance (ANOVA) where applicable. Briefly, a variance ratio (the variance of41between treatment groups^the variance of within treatment groups) of approximately1 indicates no true treatment differences. A variance ratio much greater than 1suggests a true treatment effect and was followed by Sheffe's multiple comparisontest (Thomas, 1991).423.0 RESULTSAromatase activity was measured by the recovery of tritiated water from theconversion of [10,2P-3M-testosterone to estrogen.3.1 Aromatase activity of human term trophoblasts:3.1.1 Effects of melittin on aromatase activity (Fig. 8):Human term trophoblasts dissociated from full term placenta were incubatedfor 3 h with tritiated testosterone (30,000 cpm, 2 x 10 -7M) and in the presence orabsence of increasing melittin or exogenous AL Following a 3 h incubation, therewas a dose-dependent decrease in aromatase activity of the cells in the presence ofmelittin. Whereas melittin at the concentration of 10 -7M was ineffective, thearomatase activity was attenuated (P<0.01) in the presence of 10 -6M and 10 -5Mmelittin (Fig. 8A). Treatment of the cells with 104M AA also resulted in 80%suppression of aromatase activity (Fig. 8B).3.1.2 Effects of a cyclooxygenase inhibitor, indomethacin, on aromataseactivity (Fig. 9):The possible involvement of the cydooxygenase pathway in43,,,,Fig. 8 Effects of melittin and AA on aromatase activity.Melittin suppressed aromatase activity in a dose-dependent manner (104-10-6M) during a 3 h incubation (P4.01). Melittin (10 -7M) was ineffective. AA (104M)also suppressed aromatase activity of trophoblast cells (P4.01).44Control^I ndo^Melittin^Melittin^(10-4 M)^(1 .5x10-4 M) Ind°**P<0.01Fig. 9 Effects of a cyclooxygenase inhibitor, indomethacin.A cyclooxygenase pathway inhibitor, indomethacin (10M), suppressedaromatase activity of human term trophoblast cells (P<0.01). Indomethacin +melittin further suppressed aromatase activity relative to melittin alone (P<0.05).45the inhibitory effect of melittin on aromatase activity wasinvestigated. Human trophoblast cells were incubated in the presence or absenceof melittin (1.5 x 10 -5M), indomethacin (104M) or melittin + indomethacin. Thepresence of this dose of indomethacin suppressed aromatase activity following a 3h incubation period (Fig. 9), whereas lower concentrations had no effect (data notshown). As expected, melittin suppressed aromatase activity (P<0.01) withoutaffecting cell viability. The combined presence of melittin + indomethacin furtherattenuated aromatase activity in the cells (2.4 ± 0.2 vs 3.4 ± 0.1 nM; indomethacin+ melittin vs melittin, P<0.05).3.1.3 Effects of a lipoxygenase inhibitor, NDGA, on aromatase activity (Fig.10) :The effect of a lipoxygenase inhibitor, NDGA, on the action of melittin wasinvestigated in parallel experiments. NDGA (10 -5M) suppressed aromatase activityfollowing a 3 h incubation period (Fig. 10). Melittin (1.5 x 10 4M) decreasedaromatase activity (P<0.01). The combined treatment of cells with melittin + NDGAresulted in similar suppression of aromatase activity as in the melittin group alone(3.3 ± 0.3 nM vs 3.4 ± 0.1 nM).46Control^NDGA^Melittin NDGA+Melittin^(10-5 M)^(1 .5x1 0 -5 H)Fig. 10 Effects of a lipoxygenase inhibitor, NDGA, on aromatase activity.NDGA (10M) suppressed the aromatase activity of trophoblast cells duringa 3 h incubation (P<0.01). Melittin (1.5 a 104M) inhibited aromatase activity(P<0.01). Treatment of the cells with NDGA + melittin had an effect similar tomelittin alone.473.1.4 Effects of combined melittin, indomethacin and NDGA on aromataseactivity (Fig. 11):The effect melittin on aromatase activity of trophoblast cells was investigatedin the presence or absence of NDGA and/or indomethacin. Melittin (1.5 x 10 -6M)suppressed aromatase activity following a 3 h incubation (Fig. 11, P<0.01). Theeffect of melittin was not affected by the presence of NDGA (10 -6M). As previouslyobserved, indomethacin (104M) + melittin suppressed aromatase activity furtherthan melittin alone (P<0.01). Combined treatment with melittin + NDGA +indomethacin suppressed aromatase activity further than NDGA + melittin (P<0.05)but not further than melittin + indomethacin.3.1.5 Effects of arachidonic acid with or without NDGA and indomethacinon aromatase activity (Fig. 12):The presence of AA (104M) decreased aromatase activity in trophoblast cellsduring a 3 h incubation period (P<0.01). treatment with AA + NDGA (10-5m) did notsuppress aromatase activity further than AA alone (Fig. 12). Concomitanttreatment with AA and indomethacin further suppressed aromatase48Control^Melittin^Melittin(1 .5x1 0-5 M) NDGAi*P<0.01 *N0.05 (10 -5 m)MelittinIndo(10-' hi)MelittinNDGAInd°Fig. 11 Effects of melittin, indomethacin and NDGA on aromatase activity.Trophoblast cells were treated with indomethacin and/or NDGA in thepresence of melittin. Melittin suppressed aromatase activity (P<0.01). There wasno difference in aromatase activity of the cells treated with melittin and thosetreated with melittin + NDGA. Melittin + indomethacin decreased aromataseactivity further than melittin alone (P<0.05). The aromatase activity of cells treatedwith melittin + indomethacin did not differ from cells treated with melittin +indomethacin + NDGA.49Fig. 12 Effects of arachidonic acid with or without NDGA andindomethacin on aromatase activity.AA (10 -4M) inhibited aromatase activity during a 3 h incubation (P<0.01).Treatment with AA and NDGA did not affect aromatase activity. AA +indomethacin suppressed aromatase activity further than AA alone (P<0.01). Again,there was no difference between aromatase activity in cells treated with AA +indomethacin or AA + NDGA + indomethacin.50activity when compared with AA alone (P<0.01). Finally, combined treatment withAA + indomethacin + NDGA did not suppress aromatase activity further than AA+ indomethacin.3.1.6 Effects of arachidonic acid metabolites, prostaglandin F2a andleukotriene B4(Fig. 13):The possible effects of two products of AA metabolism were investigated.Neither PGF (a cyclooxygenase pathway metabolite) nor LTB 4 (lipoxygenasepathway metabolite) affected aromatase activity, whereas melittin suppressedaromatase activity in trophoblast cells (Fig. 13, P<0.01).3.1.7 Effects of hypoxanthine and xanthine oxidase on aromatase activity(Fig. 14):Trophoblast cells were incubated with either melittin or hypoxanthine +xanthine oxidase. Both treatments suppressed aromatase activity in the trophoblastcells (Fig. 14, P<0.01). Treatment of cells with hypoxanthine + xanthine oxidaseresulted in 35% suppression of aromatase activity, which was less than thatobserved with 1.5 x 10 -6M melittin. Increasing the concentrations of xanthineoxidase to 20 mU/m1 did not further suppress aromatase activity (Fig. 14a).51Fig. 13 Effects of arachidonic acid metabolites, PGF2 ^lenkotriene B4on aromatase activity.Trophoblast cells were treated with two AA metabolites, PGF 2cyclooxygenase inhibitor) and LT13, (lipoxygenase metabolite). Neither affectedaromatase activity of trophoblast cells during a 3 h incubation period. Melittinsuppressed aromatase activity (P<0.01).52Fig. 14 Effects of hypoxanthine and xanthine oxidase on aromataseactivity.Hypoxanthine combined with xanthine oxidase had been shown to generateAA production. Hypoxanthine (1 mM) + xanthine oxidase (10 mM) suppressedaromatase activity of human term trophoblast cells (35% suppression). Melittin (1.5x 10-6M) suppressed aromatase activity more potently than hypoxanthine + xanthineoxidase.53Fig. 14a Effects of different doses of xanthine oxidase and hypoxanthine onaromatase activity.Xanthine oxidase (5 mU/m1 to 20 mU/m1) + hypoxanthine (1 mM) hadinhibitory effects on aromatase activity of trophoblast cells during a 3 h incubationperiod. Increasing xanthine oxidase concentrations did not further suppressaromatase activity.543.1.8 Effects of hCG or 8-br-cAMP on aromatase activity (Fig. 15):The presence of hCG (1 IU/m1) or 8-br-cAMP (2 mM) had no effect ontrophoblast cells during the 3 h incubation period. Melittin (1.5 x 10 -8M) and AA(10-4M) decreased (P<0.01) aromatase activity. Human CG and 8-br-cAMP did notaffect aromatase activity (Fig. 15).55**P<0.01Control Mellitin^hCG 8-br-cAMP AA(1 .5x10-SA) (1 iu/ml)^(2x1 0-3m)^(1 0-41A)Human term trophoblasts(3h, enzyme assay)1 1 -****10 -Mai 8-6 -.çi5 _4 -0a) 3 -C2 -O-0Fig. 15 Effects of hCG or 8-br-cAMP on aromatase activity.Human CG (1 IU/m1) and 8-bromo-cAMP (2 x 10 -3M) had no effect onaromatase activity during a 3 h incubation period. Melittin and AA inhibitedaromatase activity of trophoblast cells (P<0.01).563.1.9 Comparison of RIA and enzyme assay determinations of aromataseactivity (Fig. 16):The RIA and enzyme assay provided similar data. Melittin suppressedaromatase activity as determined by both RIA and enzyme assay (Fig. 16). HumanCG and 8-br-cAMP had no effect on aromatase activity in 3 h trophoblast incubationas determined by both methods. Data suggesting that AA inhibited aromataseactivity (P<0.01) was obtained using the enzyme assay but was not evaluated usingRIA .3.1.10 Effect of aromatase activity of melittin and cAMP after 24 h and 48h incubation (Fig. 18):Dispersed trophoblast cells were precultured for 48 h with 10% FCS in M199on the first day and 1% FCS in M199 on the second day. During preculture thesecells were exposed to melittin (1.5 x 10 -6M) or cAMP (2 x 10 -3M) in the presence oftestosterone (2 x 10 -7M). Media samples were collected after 24 h and 48 hincubation. During 24 h incubation, cAMP increased aromatase activity (Fig. 17).During 48 h incubation, cAMP enhanced aromatase activity (Fig. 18). Aromataseactivity was higher in cells incubated for 48 h than for 24 h. Melittin had no effecton aromatase activity of cells cultured for either 24 or 48 h.57Fig. 16 Thecomparison betweenRIA and enzyme assayof aromatase activity.The upper panel showsaromatase activity asdetermined by enzymeassay. The lower panelshows E2 values asdetermined by RIA. Bothexperiments used cellsfrom the same placenta.AA and melittinattenuated aromataseactivity. Neither 8-br-cAMP nor hCG affectedaromatase activity ofhuman term trophoblastsin short term incubation(3 h).580.0Control**P<0.01^Me littin^8—br—cAMP.5x1 o-S.0 (2xi D-31)24h incubation**3.6 -3.0 -E 2.4rnc 1.8 -c5-oID 1.2U)1.1.10.6 -Fig. 17 Effects of melittin and cAMP after 24 h incubation on aromataseactivity.Dispersed trophoblast cells were precultured for 48 h and incubated withmelittin (1.5 x 10-6M) or cAMP (2 x 10-3M) in the presence of testosterone (2 x 10 -7M)for 24 h. Melittin did not affect aromatase activity following 24 h incubation. cAMPincreased aromatase activity (P<0.01).59Fig. 18 Effect of melittin and cAMP after 24 h and 48 h incubation.Cells were precultured for 48 h and incubated with melittin (1.5 x 10 -6M) orcAMP (2 x 10 -3M) in the presence of testosterone (2 x 10 -7M) for 24 h and 48 h.Melittin (24 h & 48 h) did not affect aromatase activity. However, cAMP (24 h &48 h) stimulated aromatase activity (P<0.01).603.2 Aromatase activity on porcine granulosa cells:3.2.1 Effects of treatment with indomethacin and melittin on aromataseactivity of porcine granulosa cells (Fig. 19):Indomethacin and melittin were stimulatory to aromatase activity comparedto controls (3.55 ± 0.27 pg E 2/ml in the melittin treated cells, 4.37 ± 0.28 pg/ml inindomethacin treated cells and 2.12 ± 0.21 pg/ml in the control group, both p<0.01).Indomethacin + melittin increased aromatase activity further than indomethacin ormelittin alone (8.01 ± 0.07 pg EIml in indomethacin + melittin treated cellsp<0.01).3.2.2 Dose-response effects of melittin and indomethacin on porcinegranulosa cells (Fig. 20):Indomethacin stimulated aromatase activity in a dose-dependent manner overthe range of 104 to 104 M. The highest concentration of indomethacin doubledaromatase activity over control levels. Melittin (3 x 10-7M) had no effect onaromatase activity. Combined indomethacin (10 to 10 -4M) and melittin treatment.61** **Porcine Granulosa CellsControl^Melittin^Indo^Indo+Melittin** P<O, 011 09* *Fig. 19 Effects of indomethacin and melittin on aromatase activity ofporcine granulosa cells.Unlike their effects in human term trophoblast cells, indomethacin (10 4M)and melittin (3 x 10-7M) stimulated aromatase activity of porcine granulosa cells(P<0.01). Indomethacin + melittin further stimulated aromatase activity of porcinegranulosa cells (P<0.01).62Fig. 20 Dose-response effects of melittin and indomethacin on porcinegranulosa cells.This figure illustrates the effect of increasing concentrations of indomethacin(10' to 10 -4M), in the presence or absence of melittin (3 x 10-7M), on aromataseactivity during 3 h incubation. Indomethncin increased aromatase activity in adose-dependent manner and melittin enhanced the effect of indomethacin.633.3 The modulation of Progesterone production of human termtrophoblasts:The P-450 cholesterol side-chain cleavage step is the rate-limiting step insteroidogenesis and is hormonally regulated. LH stimulates the adenyl cyclasesystem and increases cAMP production (Marsh, 1975). Activation of LH receptorsis followed by cAMP formation and P4 synthesis.3.3.1 Effects of melittin, hCG, cAMP on P4 production by humantrophoblast cells (Fig. 21):The possible involvement of melittin (1.5 x 10 -6M), hCG (1 111/m1) and cAMP(2 x 10-3M) in P4 production were investigated using a 3 h incubation. None affectedP4 production in human term trophoblasts during short term incubation (Fig. 21).3.3.2 Effects of melittin and 25-OH-cholesterol on P4 production (Fig. 22):The universal steroid hormone precursor, cholesterol, was tested for progestinproduction in human term trophoblasts during short term incubation. 25-OH-cholesterol (10-5M), a water-soluble cholesterol, was not stimulatory to the P4production in this study. Melittin (1.5 x 10 -6M) + 25-OH-cholesterol also failed toaffect P4 production during short term incubation of human term trophoblasts.64Fig. 21 Effects of melittin, hCG and cAMP on P4 production in human termtrophoblasts.Human CG, AA and cAMP are known to enhance intracellular P4 synthesisin the ovary. This study examined the effects of hCG, melittin and cAMP on P4production by human term trophoblasts. Human term trophoblasts were exposedto melittin (1.5 x 10 -6M), hCG (1 rU/m1) and cAMP (2 x 10 -3M) during 3 h•incubations and P4 production was examined. There were no differences betweencontrol and treatment values observed.650 Control121 14eIttln(1.5d o-soOE 25-0H-Cholsaboo101741025-43H-Cholortoroi+Moittin25-0H-Cholesteml 25-0H-Ctiokoterol1441161NoffttinControl181513119 -Fig. 22 Effects of melittin and 25-OH-cholesterol on P4 production.The steroid precursor, 25-OH-cholesterol, and melittin were administered tohuman term trophoblasts to measure P-450. function during the third trimester ofpregnancy. The effects of melittin (1.5 x 10 4M) and 25-OH-cholesterol (10-5M) onP4 production by human term trophoblasts was examined using a 3 h incubation.There were no differences between control and treatment values observed.664.0 DISCUSSION4.1 Effects of arachidonic acid on aromatase activity of human termtrophoblasts:The trophoblast, through complex endocrine mechanisms, plays an essentialrole in the initiation and maintenance of pregnancy. However, the mechanismsregulating hormonogenesis within this tissue appear to remain poorly understood.The role of intracellular regulators, including second messengers, enzyme functionand the molecular events resulting in synthesis of sex steroids and peptidehormones require further elucidation for an improved understanding of themaintenance of pregnancy.Results of this study suggested that melittin, an activator of PLA 2, inhibitedaromatase activity in human term trophoblast cells in vitro. The effect of melittinwas dose-dependent and could not be blocked by the concomitant presence of acyclooxygenase inhibitor, indomethacin, or a lipoxygenase inhibitor, NDGA. Indeed,the presence of high concentrations of indomethacin or NDGA alone had asuppressive effect on aromatase activity. This may have resulted from anaccumulation of intracellular AA due to the enzymatic blockade of AA metabolism.Treatment of trophoblast cells with exogenous AA also reduced aromatase activity.Again, the inhibitory effect of exogenous AA was not reversible by the concomitant67presence of indomethacin or NDGA. Thus, inhibition of aromatase activity bymelittin could result from a direct action of intracellular unesterified AA onaromatase, rather than via conversion of AA to its metabolites of the cydooxygenaseor lipoxygenase pathways. Preliminary evidence has shown that neither PGF2c, norLTB4 could mimic the inhibitory action of melittin or AA on aromatase activity introphoblast cells.Melittin activates PLA2, a membrane associated, calcium-dependent enzymewhich liberates AA from membrane phospholipids. There is increasing evidencethat PLA2 activation and AA liberation could be an important link inhormonogenesis in reproduction. As an example, it has been reported that GnRH-stimulated gonadotropin release from anterior pituitary cells is closely associatedwith the dynamic action of AA and PKC (Chang et al., 1987). A similar situationmay be found to occur in the placenta. One or more of the lipoxygenase metabolitesof AA might be a component of a cascade of reactions initiated by interleukin-1. Thecascade ultimately results in gonadotropin release (Spangelo et al., 1991). AA itselfmay be an intracellular regulator of prolactin release from GH 3 cells (Kolesnick etal., 1984). It has been demonstrated that AA stimulates P4 production in ratovarian cells (Wang et al., 1988 & 1989). It has been reported that both melittinand AA stimulated hPL release but not hCG release from human trophoblast(Handwerger et al., 1981; Zeitler et al., 1983). There is preliminary evidence thatAA stimulates aromatase activity in porcine granulosa cells (Nickerson et al., 1990,Ledwitz-Rigby et al., 1992). Together with the observation above, the present68finding of an inhibitory action of AA in human trophoblast cells suggests that effectsof melittin or AA on aromatase could depend upon species and/or cell type underinvestigation.The mechanism by which activation of PLA 2 suppresses aromatase activityin human trophoblast remains unknown. The present data suggest that AA, itself,rather than the cyclooxygenase or lipoxygenase metabolic products may mediate thisaction. The lack of effect of PGF 2„, or LTB4 supports this notion of a direct actionof AA. Interestingly, treatment of the trophoblast cells with hypoxanthine andxanthine oxidase also suppressed aromatase activity. The combination ofhypoxanthine and xanthine oxidase is known to generate intracellular hyperoxideanions as well as increased intracellular AA concentrations (Ikebuchi et al., 1991).Although a possible direct effect of hyperoxide anions on aromatase activity cannotbe excluded, these data are consistent with an inhibitory action of AA.The majority of the cells used in this study are presumably cytotrophoblasts.Filtration to remove cellular clumps excluded syncytiotrophoblasts greater than 48iim in diameter. The present data on aromatase activity are in agreement withthose of Taman et al., (1986) and Nestler (1987), showing that freshly isolatedcytotrophoblasts aromatized androgens to estrogens. However, recent immuno-histochemical data have suggested that the cytochrome P450 aromatase is localizedin the syncytiotrophoblasts of first and second trimester placentae (Inkster et al.,1989). It is difficult to explain the apparent discrepancy between theseimmunohistochemical and biochemical data. Nevertheless, the possibility exists69that there might be different forms of aromatase that are recognized by theantibodies in the immunohistochemical studies (Harada 1988).The present results suggest that activation of PLA2 and AA may have aninhibitory effect on aromatase activity in human trophoblast cells. Thus, in additionto cAMP (Bellino et al., 1978; Hochberg et al., 1982; Rodway et al., 1990), AA couldbe another intracellular regulator of aromatase activity in human trophoblasts. Theinhibition caused by melittin could result from a direct action of intracellularunesterified AA rather than via its conversion to metabolites of the cyclooxygenaseor lipoxygenase pathways. The possible involvement of epoxygenated products ofAA metabolism remains unknown.Further, the physiological role of AA in the regulation of placental aromataseactivity remains to be determined Modulators of human placental hormonogenesis,such as IGF-I and insulin (Nestler et al., 1987), have been reported to stimulatephosphatidylinositol turnover and release AA (Michell, 1975). IGF-1, insulin andIGF-II have been reported to decrease aromatase activity in human trophoblast(Hochberg et al., 1983; Nestler, 1987 & 1990). The role of AA in the action of IGF-1,insulin or other regulators of placental hormonogenesis warrants furtherinvestigation. Interestingly, melittin suppressed aromatase activity during shortterm incubation but was ineffective on cells from 24 h and 48 h incubations.4.2 Effects of cAMP on aromatase activity in human term trophoblasts:70Cyclic AMP has been shown to be a second messenger in the release of manyhormones. Acute release (10 min) of hPL by cAMP was reported (Harman et al.,1987). FSH and LH act via cAMP to exert their steroidogenic actions in the ovary.However, the steroidogenic effects of cAMP on several other tissues (like placenta)are still controversial. Data obtained by RIA and enzyme assay in this study foundthat cAMP had no effect on estrogen production in human trophoblasts during shortterm (3 h) incubation (Fig. 16). The results suggested that cAMP may not havebeen able to exert its steroidogenic action on human term trophoblasts rapidlyenough to be detected during only a 3 h incubation. Alternately, aromatase activityof freshly obtained term trophoblasts was at or near maximal The possibility of thelatter explanation is less likely. It had been reported that aromatase specificactivity of trophoblast cells increased 10- to 15-fold 24 h after plating (Lobo et al.,1989). Cyclic AMP increased cytochrome P450 mRNA within 24 h of the initiationof culture (Ringler et al., 1989).After 24 h and 48 h incubation with cAMP in this experiment, the aromataseactivity of trophoblast cells was significantly increased (P<0.01). Moreover, cellsfrom 48 h incubation produced approximately 3 times more E2 than cells from 24 hincubation (11 ng/106 cells/ml vs 3 ng/106 cells/nil). The results suggested that thefirst 24 h incubation with cAMP primed the trophoblast cells to produce more E2 inthe following 24 h. The mechanism behind this event may have involved earlycAMP activation of the mRNA and protein synthetic machinery of the trophoblast.The results were in agreement with the report that cAMP-induced aromatase71specific activity was approximately 1.8-fold higher at 48 h than at 24 h of incubation(Lobo et al., 1989).Previous studies have demonstrated that 8-br-cAMP decreased basalproduction of estradiol in cultured term trophoblast cells (Benoit et al., 1988;Rodway et al., 1988). This discrepancy may be due to the different methods of cellpreparation use in the previous versus the present study. Placental cells werephysically dissociated (see "2.1.1 preparation of human term trophoblasts" fordetails) in this study but were enzymatically dissociated in the previous studies. Ithas been suggested that enzymatic dissociation has the inherent advantage ofstripping trophoblast tissues while releasing few fibroblasts from the villous core(Truman et al., 1989). Even small numbers of fibroblasts may be subject toovergrowth in placental culture (Truman et al., 1989; Veger et a!., 1989). However,trypsin and DAase used in the enzymatic dissociation of cells is suspected to extractmembrane bound receptor proteins from the cell surface, making the cellsunresponsive to physiological ligands for the first several days in culture. Theadvantages of physical dissociation for freshly harvested tissues are no enzymaticinterference of cell membrane recepotrs. The viabilities achieved are consistentlybetween 95 and 100%. The disadvantage is that some large syncytiotrophoblastcells (>45 pm) may be excluded. It has been reported, for example, that aromataseregulation by cAMP is critically dependent upon the methods used to isolate andculture the trophoblast cells (Lobo et al., 1989).One other thing should be considered when comparing the short term (3 h)72incubation to the long term (24 h or 48 h) incubation. The majority of freshlyprepared cells are trophoblasts. After 48 h of preculture, the cells are transformedto syncytiotrophoblasts (Kliman et al., 1986). The different cell populations mayrespond differently to cAMP.It was concluded that the effects of cAMP on aromatase activity of humantrophoblast cells were not significant during short term incubation (3h). Thestimulatory effects of cAMP on aromatase activity during 24 h and 48 h incubationcould be due to the enhanced activity of mRNA and protein synthetic machinery ofthe trophoblast cells.4.3 Short-term modulation of human placental progesterone production invitro:Cyclic AMP, hCG and melittin:It is documented that acute stimulation of steroidogenesis in the gonads isLH-dependent and mediated by the activation of cAMP-dependent protein kinaseA (PKA) (Dufau et al., 1982). Cyclic AMP induced P4 synthesis has been reportedin term trophoblast cells (Tonkowicz et al., 1985: Feinman et al., 1986: Rodway etal., 1988). Most of the experiments above involved incubation periods that wererelatively long-term (24 h or 48 h). However, data presented in this studydemonstrated that P4 production is not stimulated by 8-br-cAMP, hCG or melittinduring short term (3 h) incubation of human term trophoblasts (Fig. 21). Lack of73short term (3 h) modulation of in vitro ovine placental P4 secretion by LH, 8-br-cAMP and 25-OH-cholesterol has also been observed (De La Llosa-hermier et al.,1988). These results suggest that hCG, CAMP and possibly PLA 2 activation andincreased free AA substrate are not implicated in the short-term regulation of P4synthesis by human term trophoblasts.25-OH-cholesterol: Cholesterol is the most important substrate for the production of P4 by humanterm trophoblasts. The side chain cleavage enzymes are rate-limiting in theconversion of cholesterol to P4 (Toaff et al., 1982; Bagavandoss et al., 1987). 25-OH-cholesterol is a water-soluble steroid which readily enters cells and is metabolizedto pregnenolone in mitochondria (Toaff et al., 1982; Lino et al., 1985). 25-OH-cholesterol (2 pg/m1) was not observed to stimulate P4 production during short termincubation (3 h) in human term trophoblasts in this study (Fig. 22). It was reportedthat LDL cholesterol had no effect on basal output of 170-estradiol, P4 or hCG(Haning, et al., 1982). It was reported that 25-OH-cholesterol (2.5 pg=10 -5M) did notincrease P4 production over a 4 h incubation period of human chorion (Tonkowiczet al., 1985). However, 25-OH-cholesterol (20 pg/n21) increased P4 production in termtrophoblast cells during a 3 h incubation (Kliman et al., 1986). The concentrationof 25-OH-cholesterol (20 pg/m1=10 4M) is 10x higher than in this experiment (2pg/m1=10 -5M). Increase incubation period (5 h) and/or dosage of 25-OH-cholesterolalso increased P4 production (Tonkowicz et al. 1985).7425-OH-cholesterol (10-6M) did not affect P4 production by trophoblast cellsduring a 3 h incubation in this study.Melittin: This study used melittin to examine the involvement of PLA 2 activation andcytochrome P450scc enzyme in P4 production by trophoblast cells. Melittin hadsuppressive effects on aromatase activity (been discussed) above during short termincubation. In the process of P4 production, several steps in the cholesterol SCCreaction, such as uptake of cholesterol by mitochondria, the intramitochondrialaccess of cholesterol to the SCC enzyme complexes, and the modulation of themitochondrial cytochrome P-450 levels, are suspected to be under hormonal control(Leaven et al., 1981). It was found that melittin had no effect on P4 production byhuman term trophoblast cells during a 3 h incubation in this experiment. Melittindid increase P4 production by rat granulosa cells during a 5 h culture period (Wanget al., 1987). Melittin + 25-OH-cholesterol also had no effect on P4 production in thisstudy. This suggested that AA had no regulatory role in this regard. As mentionedabove, full term placenta may make optimal use of its resources, performing at ornear maximal capacity. Consequently, AA stimulated P4 production in other tissuesbut was unable to further increase the near maximal rate of P4 production byhuman term trophoblasts.754.4 Effects of indomethacin and melittin on porcine granulosa cellsContrasting the effects on human trophoblast cells, indomethacin or melittinstimulated aromatase activity of porcine granulosa cells. The stimulatory effect ofindomethacin on aromatase activity seems more effective than melittin in porcinegranulosa cells. This suggests that AA generated by melittin was rapidlyhydrolyzed to further eicosanoid metabolite. Whereas AA generated byindomethacin with blockade of the cyclooxygenase pathway can maintain higherconcentration to exert its stimulatory effect on aromatase activity. Indomethacinenhanced aromatase activity in a dose-dependent manner over a range of 10'M to10-4M. Indomethacin synergized with melittin to further stimulate aromataseactivity. The synergistic effect result from the accumulation of AA via differentpathways. One is from increasing AA production, yielded by the stimulatory actionof melittin on PLA2 and PLA2 enhanced membrane phospholipid hydrolysis andsubsequently release of AA. The other is from the blockade of AA metabolism byindomethacin. It was shown that PLA 2 + indomethacin had the same synergisticeffect as melittin + indomethacin (Ledwitz-Rigby et al., 1992). Granulosa cells fromdifferent size of follicles (large, medium, and small) had varying levels of aromataseactivity responding to indomethacin treatment (Ledwitz-Rigby, et al., 1992).Melittin and indomethacin stimulate aromatase activity of porcine granulosa cellsby blocking the cydooxygenase and lipoxygenase pathways of the AA cascade andthen increase intracellular AA concentration. The regulatory action of AA on76aromatase is still not clearly understood. However, increased intracellular Ca 2 ',PKC activation, and oxidation of AA via lipoxygenase (see 4.1) are all possible routesthrough which AA may mediate P4 production by placental trophoblast cells.Observed differences in the effects of indomethacin and melittin on aromataseactivity between porcine granulosa cells and human term trophoblast cells could bedue to variation in cell types and/or species examined.77CONCLUSIONAA inhibition of aromatase activity of human term trophoblasts during shortterm (3 h) incubation is a direct action of AA rather than of its eicosanoidmetabolites. AA had no attenuated effect on aromatase activity of human termtrophoblasts after 24 h and 48 h incubation.Human CG or 8-br-cAMP did not show any effect on aromatase activityduring short term (3 h) incubation with both RIA and enzyme assay method.However, 8-br-cAMP was significantly stimulatory of aromatase activity in humantrophoblasts after 24 h and 48 h incubation. Moreover, the estradiol productionduring 48 h incubations was 3 times more than that of 24 h incubation (11 ng/10 6cells/ml vs 3 ng/10 6 cells/nil).Human CG or 8-br-cAMP do not mediate short term regulation ofprogesterone production in human term trophoblasts. 25-OH-cholesterol had nostimulatory action on progesterone production during short term (3 h) incubation,suggesting that the rate of progesterone synthesis in human term trophoblasts ismaximal Melittin had no effect on progesterone production of human termtrophoblasts during short term incubation period. It can be concluded thatregulatory role is not implicated for arachidonic acid in the progesterone productionof human term trophoblasts in short term (3 h) incubation.78REFERENCESAbramson, S.B., Leszczynska-piziak, J. and Weissmann, G. 1991 Arachidonic acidas a second messenger, interaction with a GTP-binding protein of humanneutrophils. 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