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289 Regulation of immunoreactive inhibin A and B in cultured human granulosa-luteal cells by , activin A and -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 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 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 (hCG), activin A, 8-bromo cyclic was insignificant. AMP (8-BrcAMP), staurosporine (a kinase C We conclude that gonadotropins via the protein kinase inhibitor) and an antagonist of IGF action (type-1 IGF A pathway are the main positive receptor 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 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 and testes but they are also have a role in gonadal tumorigenesis (Matzuk et al. 1992, synthesized in , 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 . originally thought to be the suppression of follicle- The expresses the A-subunit gene, while stimulating (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

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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 . 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 secretion by these assessed by measuring 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, (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. Follicular aspiration Whitney test. The level of significance was chosen as was performed 36–38 h after hCG (Pregnyl (Organon) P<0·05.

Journal of Endocrinology (2000) 167, 289–294 www.endocrinology.org

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Results

Each experiment was performed with cells derived from the follicles of a single woman, or in some cases with cells pooled from 2–6 women. Both inhibin A and inhibin B secretion was detectable in all conditioned media of cultured human ovarian granulosa-luteal cells. There was considerable variation in the absolute concentrations of both forms of inhibin in the conditioned media of different pools. The mean (...) secretion of inhibin A was 537·9(131·7) pg/ml/24 h (n=16; n means the number of experiments pooled together, each experiment consisted of a single culture with 2–3 parallel Figure 1 The effects of rhFSH (100 IU/l), rhLH (100 IU/l), hCG wells), and that of inhibin B 433·0(80·4) pg/ml/24 h (100 ng/ml) and 8-BrcAMP (cAMP, 100 M) on inhibin secretion (n=18) at the stage when the experiments were performed in cultured human ovarian granulosa-luteal cells during 24 h of treatment. MeansS.E.M. from 4–16 different experiments in each (7th–11th days of culture). The inhibin A/B ratio in the group are shown with the control mean adjusted to 100. N=the same cultures was 1·240·21 (mean...). As the number of experiments pooled in each treatment group. *P<0·05, absolute inhibin values do not have any particular signifi- **P<0·01 compared with the respective control. cance in cell culture experiments, and due to the above mentioned considerable variation in the absolute concen- trations of the secreted inhibins in different cell pools, the Activin A addition (40 ng/ml) into the culture medium  regulation data below are presented as changes compared increased basal inhibin B secretion up to 409·2 61·6% of    < with the control situation in each experiment. Progester- control (mean . . .; P 0·05; n=3) and it further < one was measured in selected conditioned media and its stimulated FSH-induced inhibin B secretion (P 0·05; ff secretion into the medium was about 1500 nmol/l/24 h at n=3) (Fig. 3). The stimulatory e ect of activin A was dose the time the experiments were performed. dependent, detectable from 3–10 ng/ml upwards, and Both rhFSH and rhLH increased dose dependently reached its maximum between 30 and 100 ng/ml (dose- ff inhibin A and B secretion into the culture medium; the response data not shown). Activin A addition had no e ect maximal effect was reached between 30 and 100 IU/l on inhibin A secretion (Fig. 3). (dose–response data not shown). In repeated experiments Addition of the type-1 IGF receptor antibody IR3 ff with cells from separate cell pools rhFSH (30 or 100 IU/l) (which blocks the e ect of IGFs through the type-1 increased inhibin A and B secretion up to 179·921·5% and 166·727·4% respectively of control (mean...; P<0·01, n=8 for both). rhLH (30 or 100 IU/l) increased inhibin A and B secretion up to 192·414·7% (P<0·05, n=4) and 139·020·3% (P<0·05, n=4) respectively of control, and hCG (100 ng/ml) up to 210·153·5% (P<0·01, n=6) and 127·432·1% (not significant (NS), n=6) respectively of control. 8-BrcAMP (100 µM) in- creased inhibin A secretion up to 243·324·5% (P<0·01, n=16) and inhibin B secretion up to 132·815·8% (NS, n=16) of control (presented graphically in Fig. 1). In time-course experiments no increase in inhibin secretion was detectable after four hours of hormonal manipulations but after 24 h rhFSH, rhLH, hCG and 8-BrcAMP had increased inhibin secretion (time-course data not shown). The protein kinase C inhibitor staurosporine decreased basal inhibin B secretion to 49·25·6% (mean...) < (P 0·01, n=7) of control, but it did not have any Figure 2 The effect of staurosporine (St, 30 nM) on basal and significant effect on inhibin A secretion. Staurosporine 8-BrcAMP-induced (cAMP, 100 M) inhibin secretion in cultured also decreased inhibin B (P<0·05, n=4) but not human granulosa-luteal cells during 48 h of treatment. Means inhibin A secretion in 8-BrcAMP-treated cultures (Fig. 2). S.E.M. from 4–7 different experiments in each group are shown FSH-induced inhibin B secretion was also inhibited with the control mean (both untreated and 8-BrcAMP-treated) adjusted to 100. N=the number of experiments pooled in each by staurosporine in two cultures performed (data not treatment group. *P<0·05; **P<0·01 compared with the respective shown). control. www.endocrinology.org Journal of Endocrinology (2000) 167, 289–294

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Discussion

Previous studies using cultured human granulosa-luteal cells have shown that inhibin - and A-subunit gene expression is induced by FSH, hCG and cAMP analogs. However, B-subunit mRNA accumulation is not increased by these agents (Erämaa et al. 1994, Tuuri et al. 1996). Gonadotropins mediate their effects on human granulosa-luteal cells via increased cAMP production and protein kinase A activation. In this study rhFSH, rhLH, hCG and 8-BrcAMP increased significantly inhibin A secretion while the stimulatory effect of these agents on inhibin B was less pronounced or insignificant. These data fit well with previous mRNA regulation data; induction of - and A-subunits leads to increased inhibin A produc- tion, and the slightly increased inhibin B production can be explained by increased -subunit expression assuming Figure 3 The effect of activin A (Act A, 40 ng/ml) on basal, and rhFSH-induced (100 IU/l) inhibin secretion in cultured human that enough B-subunits are available. Our inhibin A and granulosa-luteal cells during 48 h of treatment. MeansS.E.M. from B secretion data fit fairly well with those of Muttukrishna 3 different experiments in each group are shown with the control et al. (1997) who recently reported gonadotropin-induced mean (both untreated and FSH-treated) adjusted to 100. N=the inhibin A and B secretion in cultured human ovarian number of experiments pooled in each treatment group. *P<0·05 compared with the respective control. granulosa-luteal cells. Staurosporine is a relatively specific protein kinase C inhibitor. It reduced basal and gonadotropin-induced inhibin B secretion, suggesting that the protein kinase receptor) into the culture medium decreased inhibin A C-dependent regulatory pathway is important in the secretion to 69·53·3% and inhibin B secretion to regulation of inhibin B synthesis. To our knowledge there 49·77·9% of control (P<0·01 for both; n=5). In hCG- are no convincing data on the effects of protein kinase C treated cultures IR3 decreased inhibin A secretion to regulators on B-subunit gene expression to explain the 71·912·5% and inhibin B to 54·34·0% (P<0·05 for mechanism for the clear inhibition of inhibin B secretion both, n=3) of the secretion detected without IR3 caused by staurosporine in these cells. However, protein addition. IR3 decreased slightly inhibin B secretion in kinase C has been reported to be involved in the induction 8-BrcAMP-treated cultures (P<0·05; n=5), but it had no of A-subunit gene expression in human granulosa-luteal significant effect on inhibin A secretion (Fig. 4). cells (Tuuri et al. 1996). Activin A turned out to be a very potent inducer of inhibin B secretion. A similar effect of activin has been found in cultured rat granulosa cells (Lanuza et al. 1999). Increased inhibin B secretion can be explained by the activin A-induced B-subunit gene expression reported previously (Erämaa et al. 1995). Activin A is produced by human granulosa-luteal cells (Muttukrishna et al. 1997) enabling it to have a physiological autocrine/paracrine role in the regulation of ovarian inhibin production. In our experiments activin A did not increase inhibin A secretion, which is in contrast to rat granulosa cell data (Lanuza et al. 1999), but fits well with the human mRNA data showing no induction of -orA-subunit gene expression during activin treatment (Erämaa et al. 1995). IGFs are likely to upregulate inhibin secretion in human Figure 4 The effect of the type-1 IGF receptor antibody IR3 granulosa-luteal cells. This hypothesis is supported by the (1 g/ml) on basal, and hCG- (100 ng/ml) and 8-BrcAMP- (cAMP, clear inhibition of inhibin secretion by IR3 which blocks 100 M) regulated inhibin secretion in cultured human granulosa- the IGF-I- and -II-mediated signaling through the type 1 luteal cells during 48 h of treatment. MeansS.E.M. from 3–5 IGF receptor. Previous studies have shown that endogen- different experiments in each group are shown with the control ous IGF-I production is essential for inhibin -subunit mean (untreated, hCG- and 8-BrcAMP-treated) adjusted to 100.  N=the number of experiments pooled in each treatment group. expression in rat granulosa cells (Kubo et al. 1998, Li et al. *P<0·05; **P<0·01 compared with the respective control. 1998). In addition, IGF-I increased inhibin production by

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Downloaded from Bioscientifica.com at 09/23/2021 02:13:14PM via free access Inhibin A and B in granulosa cells · TVA}NTTINEN and others 293 cultured rat granulosa cells (Lanuza et al. 1999). We can Hsueh AJW, Dahl KD, Vaughan J, Tucker E, Rivier J, Bardin CW & assume that IGF-II is the main autocrine/paracrine IGF Vale W 1987 Heterodimers and homodimers of inhibin subunits have different paracrine action in the modulation of luteinizing upregulating inhibin synthesis in human ovaries. This hormone-stimulated . PNAS 84 5082–5086. assumption is based on the high and gonadotropin- Kubo T, Shimasaki S, Kim H, LiD&Erickson GF 1998 dependent expression of the IGF-II gene in human ovaries Activin-induced inhibin -subunit production by rat granulosa cells (Voutilainen & Miller 1987, Voutilainen et al. 1996). requires endogenous insulin-like growth factor-I. Biology of In summary, our data show that gonadotropins via the Reproduction 58 712–718. Lanuza GM, Groome NP, Baranao JL & Campo S 1999 Dimeric protein kinase A signal transduction pathway are the main inhibin A and B production are differentially regulated by positive regulators of inhibin A and B secretion in human and local factors in rat granulosa cells. Endocrinology 140 granulosa-luteal cells. The protein kinase C signal trans- 2549–2554. duction pathway seems to be important especially for Li D, Kubo T, Kim H, ShimasakiS&Erickson GF 1998 Endogenous insulin-like growth factor-I is obligatory for stimulation of rat inhibin B secretion. Locally produced activins are probably inhibin -subunit expression by follicle-stimulating hormone. important inducers of inhibin B production, and IGFs Biology of Reproduction 58 219–225. seem to upregulate both forms of inhibin in human Mason AJ, Niall HD & Seeburg PH 1986 Structure of two human ovaries. ovarian inhibins. Biochemical and Biophysical Research Communications 28 957–964. Mather JP, Woodruff TK & Krummen LA 1992 Paracrine regulation of reproductive function by inhibin and activin. Proceedings of the Acknowledgements Society for Experimental Biology and Medicine 201 1–15. Matzuk MM, Finegold MJ, Su J-GJ, Hsueh AJW & Bradley A 1992 -Inhibin is a tumour suppressor gene with gonadal specificity in Ms Merja Haukka and Ms Irma Janhunen are thanked for mice. Nature 360 313–319. their technical assistance. Recombinant human FSH and Matzuk MM, Kumar TR, Shou W, Coerver KA, Lau AL, Behringer LH for in vitro experiments were generously provided by RR & Finegold MJ 1996 Transgenic models to study the roles of inhibins and activins in reproduction, oncogenesis, and Serono-Nordic (Vantaa, Finland), and activin A peptide development. Recent Progress in Hormone Research 51 123–154. (Dr A F Parlow) and hCG (CR-127) by the National Miró F & Hillier SG 1992 Relative effects of on Hormone and Pituitary Program, NIDDK, NIH. This synthesis in primate granulosa cells. Journal of study was financially supported by the Academy of Clinical Endocrinology and 75 1556–1561. Finland, the Finnish Foundation for Pediatric Research, Muttukrishna S, GroomeN&Ledger W 1997 Gonadotropic control of secretion of dimeric inhibins and activin A by human granulosa- and the Kuopio University Hospital (Research Contract luteal cells in vitro. Journal of Assisted Reproduction and Genetics 14 No. 5107 to R V). 566–574. Rabinovici J, Spencer SJ, Doldi N, Goldsmith PC, SchwallR&Jaffe RB 1992 Activin-A as an intraovarian modulator: actions, localization, and regulation of the intact dimer in human ovarian References cells. Journal of Clinical Investigation 89 1528–1536. 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Journal of Clinical Tuuri T, Erämaa M, Van Schaik RHN & Ritvos O 1996 Differential Endocrinology and Metabolism 79 1670–1677. regulation of inhibin/activin -andA-subunit and follistatin Erämaa M, Hilden K, TuuriT&Ritvos O 1995 Regulation of mRNAs by cyclic AMP and phorbol ester in cultured human inhibin/activin subunit messenger ribonucleic acids (mRNAs) by granulosa-luteal cells. Molecular and Cellular Endocrinology 121 1–10. activin A and expression of mRNAs in cultured Vale W, Rivier C, Hsueh A, Campen C, Meunier H, Bicsak T, human granulosa-luteal cells. Endocrinology 136 4382–4389. Vaughan J, Corrigan A, Bardin W, Sawchenko P, Petraglia F, Findlay JK 1993 An update on the roles of inhibin, activin, and Yu J, Plotsky P, SpiessJ&Rivier J 1988 Chemical and biological follistatin as local regulators of . Biology of Reproduction characterization of the inhibin family of protein hormones. Recent 48 15–23. Progress in Hormone Research 44 1–34. Groome NP & Lawrence M 1991 Preparation of monoclonal VoutilainenR&Miller WL 1987 Coordinate to the beta A subunit of ovarian inhibin using a synthetic regulation of mRNAs for insulin-like growth factor II and peptide immunogen. Hybridoma 10 309–310. cholesterol side-chain cleavage enzyme, P450 scc, in human Groome NP, Hancock J, Betteridge A, Lawrence M & Crawen R steroidogenic tissues. PNAS 84 1590–1594. 1990 Monoclonal and polyclonal antibodies reactive with the 1–32 Voutilainen R, Tapanainen J, Chung B-C, Matteson KJ & Miller WL amino terminal sequence of the alpha subunit of human 32 K 1986 Hormonal regulation of P450 scc (20,22-desmolase) and inhibin. Hybridoma 9 31–42. P450c17 (17-hydroxylase/17,20-lyase) in cultured human granulosa Groome NP, Illingworth PJ, O’Brien M, Pai R, Rodger FE, Mather cells. Journal of Clinical Endocrinology and Metabolism 63 202–207. JP & McNeilly AS 1996 Measurement of dimeric inhibin B Voutilainen R, Franks S, Mason HD & Martikainen H 1996 throughout the human menstrual cycle. Journal of Clinical Expression of insulin-like growth factor (IGF), IGF-binding protein, Endocrinology and Metabolism 81 1401–1405. and IGF receptor messenger ribonucleic acids in normal and www.endocrinology.org Journal of Endocrinology (2000) 167, 289–294

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polycystic ovaries. Journal of Clinical Endocrinology and Metabolism 81 Ying SY 1988 Inhibins, activins, and follistatins: gonadal proteins 1003–1008. modulating the secretion of follicle-stimulating hormone. Endocrine Woodruff TK 1998 Regulation of cellular and system function by Reviews 9 267–293. activin. Biochemical Pharmacology 55 953–963. YamotoM,MinamiS,NakanoR&Kobayashi M 1992 Immunohistochemical localization of inhibin/activin subunits in Received 25 October 1999 human ovarian follicles during the menstrual cycle. Journal of Revised manuscript received 27 April 2000 Clinical Endocrinology and Metabolism 74 989–993. Accepted 30 June 2000

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