De Geyter, Ch

De Geyter, M.

Holzgreve, W.

Miny, P.

Vollmer, M.

Zhang, H.

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Abstracts of the 15th Annual Meeting of the ESHRE, Tours, France 1999P-258. Effect of IGFI and IGFII on PGE2 and PGF2arelease by human luteal cellsApa R., Miceli F., Pierro E., Minici F., Fracasso A.M.,Tropea A., Mancuso S. and Lanzone A.Department of Obstetrics and Gynecology, UniversitaCattolica S. Cuoro, L.go A. Gemelli 8, Roma, ItalyIntroduction: The relevance of the insulin-like growth factor(IGFs) system in ovarian physiology has been extensivelydemonstrated. Moreover, IGFs have been shown to enhancesteroidogenesis both in human granulosa and luteal cells.Taking into account the reports that prostaglandin E2 (PGE2)and prostaglandin F2a (PGF2a) have opposite (i.e. luteotropicand luteolytic) effects on the human corpus luteum, we aimedto study whether a correlation exists between IGFs effect onprogesterone secretion and the activation of the prosta-glandin system.Materials and methods: Isolated human luteal cells werecultured for different times (12, 24 and 48 h) with IGFI, IGFII(10-100 ng/ml) and GH (100 ng/ml); PGE2 and PGF2a releasein culture media was assayed by radioimmunoassay.Results: Both IGFI and II were able to induce PGE2 synthesisin a time-and dose-dependent way while they both inhibitedPGF2a production. GH was also found able to significantlyreduce PGF2a synthesis; this effect was IGFI-mediated sinceit was reverted by increasing concentrations of anti-IGFIantibody. Conversely, GH had no effect on PGE2 production.Discussion: In a recent paper of ours, we showed that IGFIas well as II are able to stimulate progesterone secretion byhuman luteal cells, suggesting a direct luteotropic actionexerted by these substances. In light of the evidence of a well-defined correlation between IGFs and prostaglandin release byluteal cells, it is possible to suggest that IGFs could influenceluteal steroidogenesis by regulating the complex balance ofluteotropic and luteolytic effects exerted by the two differentprostanoids.P-259. Characterization of an immortalized humangranulosa cell line (COV434) for the study of thedevelopment of ovarian follicles in vitro*De Geyter Ch.1, Zhang H.1, Vollmer M.1, De Geyter M.1,Miny P.2 and Holzgreve W.11University Women's Hospital of Basel, Switzerland and2Division of Human Genetics at the Children's Hospital ofthe University of Basel, SwitzerlandIntroduction: The biological properties of an immortalizedhuman granulosa cell line (COV434) are presented. Thefollowing characteristics were considered essential for normalfunctionality of granulosa cells: (i) increased synthesis andsecretion of 17p-oestradiol after stimulation of cytochromeP450 aromatase with follicle stimulating hormone (FSH);(ii) establishment of intercellular connections between theimmortalized granulosa cells and cells of a cumulus oophorus;270and (iii) the potential response to similar inducers of apoptosisas compared to natural granulosa cells.Material and methods: The cell line of immortalized granu-losa cells was established from a primary human granulosacell tumour. Control granulosa cells were collected frompatients undergoing in-vitro fertilization (IVF) or intracyto-plasmic sperm injection (ICSI). The cells were studied underlight microscopy , with transmission electron microscopy andwith scanning electron microscopy under various conditions.To assess the aromatase activity the cells were cultured inDulbecco's minimal essential medium (DMEM), supplementedwith 10% fetal calf serum (FCS) and 4-androstene-3, 17-dionein presence of recombinant FSH, luteinizing hormone (LH)or urinary human chorionic gonadotrophin (HCG) and theconcentrations of 17p-oestradiol and progesterone were meas-ured in the supernatant. mRNA of apoptosis-associated geneswere analysed by reverse transcription-polymerase chain reac-tion (RT-PCR) and various apoptotic protein expressions wereexamined by Western blotting.Results: Aromatase activity in the COY434-granulosa cellswas demonstrated by the rapid rise of the 17p-oestradiolconcentration in presence of FSH. No rise of progesteroneconcentration was observed after the addition LH or HCGneither could the LH-receptor be induced with FSH and l7P-oestradiol. Expression of various apoptosis-associated geneswas similar in both granulosa cell types. Multiple intercellularconnections were formed during co-culture of COy434-granu-losa cells with cumulus cells containing a viable oocyte butnot with cumulus cells devoid of an oocyte. Co-cultured withCOy434-granulosa cells, immature human oocytes remainedviable for 8 days, but degenerated thereafter.Conclusions: The immortalized human granulosa cell lineCOY434 contains many of the properties of natural granulosacells and may be instrumentalized for experimental studies onfollicular development.*Project nr. 31-49834.96, funded by the Swiss NationalFoundation.P-260. Pattern of cycle-oxygenase expression in thehuman endometrium during the menstrual cycleKwon H.C., Yang H.W., Hwang K.J., Kim S.K.l, Cho D.J.1and Oh K.S.Department of Obstetrics and Gynaecology, Ajou UniversitySchool of Medicine, Suwon and 1Department of Obstetricsand Gynaecologyn Yonsei University College of Medicine,Seoul, KoreaIntroduction: Recent attention has been focused on theimplantation stage endometrial changes at the initiation ofdecidual reaction induced by prostaglandins (PGs). PGs arederived from arachidonic acid by the action of cyclo-oxygenase(COX), and are comprised of two isoforms, COX-l and COX-2. COX-1 is a constitutional enzyme, and is present in manyhuman tissues, whereas COX-2 is induced by endocrinologicalstimulation. The expression of COX-l is relatively uniform inthe oestrus cycle of mice, but the expression of COX-2 has

P-259. Characterization of an immortalized human granulosa cell line (COV434) for the study of the development of ovarian follicles in vitro

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