289 Regulation of immunoreactive inhibin A and B secretion in cultured human granulosa-luteal cells by gonadotropins, activin A and insulin-like growth factor type-1 receptor T Vänttinen1, J Liu1,3, C Hydén-Granskog4, M Parviainen2, I Penttilä2 and R Voutilainen1,3 1Department of Pediatrics, Kuopio University Hospital, Kuopio, Finland 2Department of Clinical Chemistry, Kuopio University Hospital, Kuopio, Finland 3Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland 4Department of Obstetrics and Gynecology, Helsinki University Hospital, Helsinki, Finland (Requests for offprints should be addressed to R Voutilainen, Department of Pediatrics, Kuopio University Hospital, PO Box 1777, FIN-70211 Kuopio, Finland) Abstract Inhibins are gonadal glycoproteins with endocrine effects P<0·01; 139%, P<0·05; 127%, P>0·05; 133%, P>0·05 of on pituitary FSH secretion and para/autocrine effects on the controls respectively). IR3 decreased inhibin A and B ovarian and testicular function. The purpose of this study secretion down to 70% (P<0·01) and 50% (P<0·01) was to investigate the endocrine and para/autocrine respectively of the control. Staurosporine decreased regulation of inhibin A and inhibin B secretion in human inhibin B secretion down to 49% (P<0·01) of the control; ovarian granulosa-luteal cells. The cells were obtained its effect on inhibin A secretion was not significant. from women undergoing in vitro fertilization, and the Activin A increased inhibin B secretion up to fourfold of primary cultures were treated with FSH, LH, human the control (P<0·05) while its effect on inhibin A secretion chorionic gonadotropin (hCG), activin A, 8-bromo cyclic was insignificant. AMP (8-BrcAMP), staurosporine (a protein kinase C We conclude that gonadotropins via the protein kinase inhibitor) and an antagonist of IGF action (type-1 IGF A signal transduction pathway are the main positive receptor antibody IR3). The secretion of inhibins was regulators of inhibin A and B secretion in human measured by ELISA assays capable of reliably distinguish- granulosa-luteal cells. The protein kinase C signal trans- ing between inhibin A and B. duction pathway seems to be important especially for FSH, LH, hCG and 8-BrcAMP increased inhibin A inhibin B secretion. Locally produced IGFs are probably secretion on average up to 180% (P<0·01), 192% important inducers of the production of both forms (P<0·05), 210% (P<0·01) and 243% (P<0·01) respectively of inhibin in human ovaries while activins seem to of the control level, while their stimulatory effect on upregulate inhibin B secretion. inhibin B secretion was less pronounced (up to 167%, Journal of Endocrinology (2000) 167, 289–294 Introduction (Vale et al. 1988, Ying 1988). In addition to their endocrine effects on pituitary FSH secretion, inhibin and Inhibins are dimeric glycoproteins consisting of an activin peptides have important autocrine and paracrine -subunit and either a A- (inhibin A) or B- (inhibin functions in the organs in which they are produced B) subunit. Activins are composed of -subunits. The (Mather et al. 1992, Findlay 1993, Woodruff 1998). homodimer of A-subunits forms activin A, the Inhibins and activins regulate ovarian steroidogenesis in homodimer of B-subunits activin B, and the heterodimer both rodents and primates (Hsueh et al. 1987, Miró & of A- and B-subunits activin AB. Circulating inhibins Hillier 1992, Rabinovici et al. 1992). Inhibins may also are secreted mainly by ovaries and testes but they are also have a role in gonadal tumorigenesis (Matzuk et al. 1992, synthesized in placenta, adrenals and some gonadal tumors 1996). (reviewed in Vale et al. 1988, Ying 1988, Woodruff 1998). All inhibin subunit genes (Mason et al. 1986) are The main biological function of the inhibins was expressed in human ovaries through the menstrual cycle. originally thought to be the suppression of follicle- The corpus luteum expresses the A-subunit gene, while stimulating hormone (FSH) secretion by the pituitary B-subunit gene expression may be minimal or absent gland, whereas activins increase pituitary FSH secretion (Schwall et al. 1990, Roberts et al. 1993). Inhibin subunit Journal of Endocrinology (2000) 167, 289–294 Online version via http://www.endocrinology.org 0022–0795/00/0167–289 2000 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/23/2021 02:13:14PM via free access 290 TVA}NTTINEN and others · Inhibin A and B in granulosa cells peptides have been localized by immunohistochemistry in or Profasi (Serono)) administration. After removal of the human ovaries (Yamoto et al. 1992, Arora et al. 1997). On cumulus–oocyte complex, the granulosa cells from all the basis of in situ hybridization histochemistry it was follicles of each woman were pooled and pelleted. The assumed that inhibin B is the dominant form at the cells were then dispersed in 0·1% hyaluronidase (Sigma beginning of follicular development and that inhibin A Chemicals Co., St Louis, MO, USA) in Dulbecco’s becomes more important during the later stages of follicu- minimal essential medium (DMEM)–Ham’s F-12 logenesis (Schwall et al. 1990, Roberts et al. 1993). In- medium (GIBCO Laboratories, Grand Island, NY, hibin A was recently found to be the dominant form of USA) (1:1) for 30 min at 37 C with intermittent inhibin secreted by cultured human granulosa-luteal cells stirring. The granulosa cells were separated from red (Muttukrishna et al. 1997). blood cells by centrifugation in Ficoll-Paque (Pharmacia During the human menstrual cycle, serum immuno- Biotech AB, Uppsala, Sweden) for 15 min at 1000g. reactive inhibin concentrations follow the growth of the The cells were then washed and plated in DMEM– developing follicles, which are the main source of circu- Ham’s F-12 medium (1:1) supplemented with 10% fetal lating inhibins in the follicular phase. At this stage inhibin calf serum (GIBCO or Bioclear UK Ltd, Calne, Wilts, B is dominant but its concentration decreases rapidly UK), 2 mM -glutamine, 100 IU/ml penicillin, and during the luteal phase. Inhibin A concentration is highest 100 µg/ml streptomycin sulfate (GIBCO) at a density of in the luteal phase, and it also decreases rapidly before the 2–5105 cells/well on 35-mm six-well Cellstar dishes end of the cycle (Groome et al. 1996). Serum inhibin (Greiner Labortechnik Gmbh, Frickenhausen, Germany). concentrations during the menstrual cycle correlate well The cells were grown at 37 C in a 95% air–5% CO2 with inhibin subunit gene expression in the corpus luteum humidified environment and the cell culture media were (Schwall et al. 1990, Roberts et al. 1993). changed every 2–3 days. Gonadotropin- and protein kinase C-dependent regu- In vitro hormonal and other treatments were performed lation of the expression of inhibin and A subunit genes during the 7th to the 11th days of culture when the cells has previously been described in ovarian granulosa cells at are the most responsive in this culture system (Voutilainen mRNA level (Erämaa et al. 1994, Tuuri et al. 1996) but et al. 1986). The functional viability of the cells was information about the inhibin peptide secretion by these assessed by measuring progesterone concentrations in cells is scant (Muttukrishna et al. 1997). The aim of the selected culture media after different treatments. present work was to shed more light on the endocrine Recombinant human (rh) FSH (Gonal-F) and rhLH were (FSH, luteinizing hormone (LH), human chorionic gifts from Serono, and purified hCG (CR-127) was a gift gonadotropin (hCG)) and local (activins, insulin-like from the National Hormone and Pituitary Programme, growth factor (IGF) system) regulation of inhibin A and B NIDDK, NIH, Bethesda, MA, USA. Recombinant secretion in human ovaries by using cultured granulosa- human activin A peptide was generously provided by luteal cells with highly sensitive and specific inhibin assays. Dr A F Parlow (NIDDK’s National Hormone and The roles of protein kinase A- and C-dependent signal Pituitary Programme). 8-BrcAMP was purchased from transduction pathways in inhibin secretion were tested by Sigma, staurosporine from Boehringer Mannheim adding 8-bromo cyclic AMP (8-BrcAMP) (which acti- (Mannheim, Germany), and IGF type-1 receptor antibody vates protein kinase A) and staurosporine (which inhibits IR3 from Oncogene Sciences (Uniondale, NY, USA). protein kinase C) to the cell cultures. Inhibin A and B were measured by specific enzyme- linked immunosorbent assay (ELISA) kits (product codes MCA950 KZZ and MCA1312 KZZ respectively; Serotec Materials and Methods Ltd, Oxford, Oxon, UK) as described previously (Groome et al. 1990, 1996, Groome & Lawrence 1991). The Human ovarian granulosa cells were obtained by follicular detection limit for the assay was reported to be 2 pg/ml aspiration from women taking part in in vitro fertilization for inhibin A and 15 pg/ml for inhibin B. Both intra- programs. The study was approved by the Research Ethics and interassay coefficients of variation were below 10% Committees of Kuopio and Helsinki University Hospitals, and 7% for inhibin A and B respectively. The samples and the women gave informed written consent. The were assayed in duplicate. According to the manufacturer, women were treated with a gonadotropin-releasing there is minimal cross reaction with inhibin B or activins hormone (GnRH) analog (Synarela (Searle, Bretigny-Sur- in the inhibin A assay, and about 1% cross reaction Orge, France), Suprecur (Hoechst Marion Roussel, with inhibin A in the inhibin B assay. When activin added Frankfurt am Main, Germany) or Zoladex (Zeneca, into the cell culture medium (up to 40 ng/ml) was Alderley Park Macclesfield, Cheshire, UK)) and an FSH measured with the inhibin A and B assays, no cross reaction preparation (Gonal-F (Serono, Bari, Italy) or Puregon could be detected. Differences in the inhibin concentrations (Organon, Oss, The Netherlands)) to induce the between treatment groups were assessed by the Mann- development of multiple follicles.