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557 Biphasic regulation of activin A by in cultured human ovarian granulosa-luteal cells leads to decreasing activin:inhibin ratios during continuing stimulation

T Vänttinen1, J Liu2, C Hydén-Granskog3 and R Voutilainen1,2 1Department of Pediatrics, Kuopio University and University Hospital, Kuopio, Finland 2Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland 3Department of and Gynecology, Helsinki University Central 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; Email: Raimo.Voutilainen@uku.fi)

Abstract Pituitary gonadotropins mediate part of their effects on experiments, FSH and LH increased inhibin A and ovarian function via local and growth factors secretion after both 24 and 48 h of treatment. produced by granulosa cells. Activins and inhibins are 8-BrcAMP (0·1–100 µM) increased activin A in 24- and among these factors, and they have often opposite effects 48-h experiments (to 206% and 148% of control respect- on various components of the reproductive system. The ively; P<0·01 for both). Inhibin A and progesterone purpose of this study was to investigate the regulation of secretion were stimulated by 8-BrcAMP time- and ovarian activin A secretion using cultured human ovarian dose-dependently. TPA increased activin A secretion granulosa-luteal cells as a model. The granulosa-luteal dose-dependently (0·1–100 ng/ml) in both 24- and 48-h cells, obtained from women taking part in an in vitro experiments. At 100 ng/ml concentration, it increased fertilization program, were cultured and treated with FSH, activin A up to 61-fold and inhibin A up to 16-fold of LH, 8-bromo cAMP (8-BrcAMP, a kinase A control in 24-h experiments. We conclude that gonado- activator) and 12-O-tetradecanoyl phorbol-13-acetate tropins regulate immunoreactive activin A secretion (TPA, a protein kinase C activator). Conditioned cell biphasically in cultured human granulosa-luteal cells: ini- culture media were analyzed for activin A, inhibin A and tial stimulation is followed by inhibition. In contrast, progesterone concentrations with specific enzyme immuno- gonadotropins increase inhibin A and progesterone secre- assays. FSH and LH (1–100 IU/l) increased activin A tion continuously. Consequently, continuing gonado- secretion with 24 h of treatment (to 132% and 253% tropin stimulation leads to a decreasing activin:inhibin of control respectively; P<0·05 for both), but their ratio, which may have a significant role in the local effects were inhibitory in 48-h treatments (26% and 16% fine-tuning of ovarian steroidogenesis. decreases respectively; P<0·05 for both). In the same Journal of Endocrinology (2002) 172, 557–563

Introduction preovulatory and early luteal granulosa cells, the - and A-subunits are highly expressed, whereas the expression Activins and inhibins are structurally related dimeric of the B-subunit is absent, which suggests that activin A consisting of subunits (, AorB), and inhibin A are the most important activin/inhibin each coded by a separate, differentially regulated gene forms during these stages. Gonadotropins up-regulate - (reviewed in Ying 1988, Hillier 1991, Knight & Glister and A-subunit expression (Erämaa et al. 1994) and 2001). Activins are dimers of -subunits (A:A, A:B activin A and inhibin A secretion in human and B:B for activin A, activin AB and activin B granulosa-luteal cells (Muttukrishna et al. 1997, Vänttinen respectively). Inhibins have a common -subunit con- et al. 2000). nected with either of the -subunits (:A and :Bfor Activins and inhibins have often opposite effects on inhibin A and inhibin B respectively). ovarian function. Hypotheses as to the para/autocrine role In the human , the synthesis of activins and of activins and inhibins in human are based on inhibins occurs mainly in the granulosa cells, where the in vitro studies. In cultured human follicular cells, activin genes for the subunits are expressed A modulates steroidogenesis by inhibiting progesterone throughout the (Roberts et al. 1993). In secretion and aromatase activity (Rabinovici et al. 1992).

Journal of Endocrinology (2002) 172, 557–563 Online version via http://www.endocrinology.org 0022–0795/02/0172–557  2002 Society for Endocrinology Printed in Great Britain

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Activins inhibit production in cultured human responsive in this culture system (Erämaa et al. 1994, theca cells, whereas inhibins stimulate it (Hillier et al. Voutilainen et al. 1986). At this stage the cultures were 1991a,b). The mechanisms of the antagonistic actions of subconfluent. The functional viability of the cells was inhibins and activins have not been understood until assessed by measuring progesterone secretion by the cells. recently, when inhibins were shown to be able to block Recombinant human FSH and luteinizing (LH) activin receptors via the transforming growth factor- type were gifts from Serono (Vantaa, Finland). 8-Bromo cAMP III receptor, betaglycan (Lewis et al. 2000, Matzuk 2000). (8-BrcAMP) and 12-O-tetradecanoyl phorbol-13-acetate The purpose of the present study was to examine the (TPA) were purchased from Sigma (St Louis, MO, USA). dynamics of gonadotropin-regulated activin A secretion and to compare it with that of inhibin A and progesterone Hormone assays secretion, to gain a better understanding of the endocrine The concentrations of total (free and -bound) regulation of the activin/inhibin system in the human activin A in culture media were measured by an ultra- ff ovary. We found that gonadotropins had a biphasic e ect sensitive ELISA intended for use with various human and on activin A secretion – initial stimulation was followed animal fluids, including culture media. The method has by inhibition – whereas they increased inhibin A and been described in detail previously (Knight et al. 1996). progesterone secretion continuously. The activin A assay kit (product code MCA 1426 KZZ) was purchased from Serotec (Oxford, UK). The detection limit of the assay was 50 pg/ml. The intra- and interassay Materials and Methods coefficients of variation were 6·1 and 6·5% respectively. All samples were assayed in duplicate. According to the Ethical considerations manufacturer, there is no detectable cross-reaction with The study was approved by the Research Ethics other forms of activins or inhibins. Committees of the Kuopio and Helsinki University Inhibin A was measured with an ultrasensitive two-site Hospitals, and the patients gave informed written consent immunosorbent assay by a technique similar to that used to participate. for activin A. The method has been described in detail previously (Groome et al. 1994, Muttukrishna et al. 1994). The inhibin A assay kit (product code MCA 950 KZZ) Cell cultures was purchased from Serotec. The limit of detection of the Human granulosa cells were obtained from women assay was 2 pg/ml. The intra- and interassay coefficients of undergoing an in vitro fertilization (IVF) program in variation were 2·4 and 7·6% respectively. There should Helsinki University Hospital as described previously not be any detectable cross-reaction with inhibin B or (Vänttinen et al. 2000, Liu et al. 2001). Before follicular activins. aspiration, the patients were treated with a gonado- Progesterone was measured by a competitive enzyme tropin-releasing hormone analog to suppress endogenous immunoassay purchased from Diagnostic Systems gonadotropin secretion. Follicle-stimulating hormone Laboratories, Inc. (Webster, Texas, USA; product code (FSH) was used to induce the development of several DSL-10-3900). The limit of detection of the assay was follicles. Follicular aspirations were performed after 36– 1 nmol/l. The intra- and interassay coefficients of variation 38 h of administration of a single dose of human chorionic were reported to be 7·5% and 9·4% respectively. gonadotropin (hCG). After follicular aspiration, the cumulus–oocyte complexes were separated for IVF and Statistical analyses the granulosa cells from all follicles of several women were Each experiment was repeated at least three times with pooled, pelleted and transported immediately to the different cell batches. Single experiments consisted of laboratory. several hormonal manipulations each performed in two or The granulosa-luteal cells were purified and cultured as three parallel wells. The data are presented as arithmetic described previously (Vänttinen et al. 2000). In brief, the meansS.E.M., with the control mean adjusted to 100%. cells were dispersed and separated from red blood cells, The statistical significances were estimated by the non- washed and plated with a medium containing DMEM– parametric Mann–Whitney test or the Kruskal–Wallis test Ham’s F12 (1:1) supplemented with 10% fetal calf serum in the multiple comparisons. (Gibco or Bioclear UK Ltd, Calne, Wilts, UK), 2 mM -glutamine, 100 IU/ml penicillin, and 100 µg/ml strep- tomycin sulfate (Gibco) at a density of 2–5105 cells/ Results well on 35-mm six-well dishes. The cells were grown at  Basal activin A, inhibin A and progesterone secretion by 37 C in a 95% air–5% CO2 humidified environment, and the culture media were changed every 2–3 days. All cultured granulosa-luteal cells hormonal and other treatments were performed during the Cultured granulosa-luteal cells secreted activin A, inhibin 7th to 10th days of culture, when the cells are the most A and progesterone with different patterns, depending on

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(132·311·3% of control; P<0·05), but in 48-h experi- ments the effect of FSH was inhibitory (74·013·5% of control; P<0·05). Inhibin A and progesterone secretion were increased in the presence of FSH after 24 h (177·724·0% and 248·229·6% of control respect- ively; P<0·01 for both) and 48 h (181·432·5% and 405·9121·2% of control respectively; P<0·01 for both) of incubation. LH (Fig. 2B) increased activin A secretion in 24-h experiments (253·165·7% of control; P<0·05), but inhibited it in 48-h experiments (83·97·7% of control; P<0·05). Inhibin A and progesterone secretion were increased in the presence of LH after 24 h (201·6 9·2% and 309·695·1% of control respectively; P<0·01 for both). After 48 h, the effect of LH on inhibin A and progesterone secretion was less pronounced: inhibin A was increased up to 132·716·8% of control (P=0·663) and progesterone up to 138·18·6% of control (P<0·01). The inhibitory effect of gonadotropins on activin A secretion was confirmed by performing five 48-h experi- ments with different doses of FSH (1–100 IU/l) and LH Figure 1 Concentrations (logarithmic scale) of activin A (black (1–100 IU/l) (Fig. 2C and D). FSH proved to have more bars), inhibin A (white bars) and progesterone (shaded bars) in significant effects than LH, but the pattern of their conditioned culture media of human granulosa-luteal cells in dose-dependent effects was similar. Low concentrations of relation to the period of culture. The cells were cultured for 12 days. Culture media were replaced every third day and analyzed FSH (1 IU/l) increased activin A secretion but higher for activin A, inhibin A and progesterone concentrations. The bars concentrations (10–100 IU/l) tended to inhibit it represent mean+S.E.M. hormone concentrations (ng/ml per 72 h) (P=0·08; Kruskal–Wallis test), whereas inhibin A secre- from four separate experiments. All hormone concentrations tion was increased dose-dependently (P<0·05). Low con- < changed significantly during the 12-day culture period (P 0·01; centrations of LH (1–10 IU/l) stimulated activin A Kruskal–Wallis test). secretion, but high concentrations (100 IU/l) were inhibitory (P<0·05). the culture age (Fig. 1). Activin A secretion followed a U-shaped pattern, increasing clearly between days 7–12 Effect of 8-BrcAMP and TPA on activin/inhibin A secretion in culture. The patterns of secretion of inhibin A and progesterone were similar to each other, decreasing In preliminary experiments, 8-BrcAMP (an activator of gradually after 6 days of culture. The activin A:inhibin A protein kinase A) increased the secretion of activin A, ratio increased 10-fold from days 7–9 to 10–12 of culture inhibin A and progesterone dose dependently (0·1– (P<0·05; Mann–Whitney test), whereas the sum of activin 1000 µM) in both 24 and 48 h treatments (dose–response A and inhibin A secretion did not change at this stage. data not shown). 8-BrcAMP (100 µM; Fig. 3) stimulated activin A secretion in the 24-h experiments (205·8 < ff 47·9% of control; P 0·01) and in the 48-h experiments, E ect of gonadotropins on activin A, inhibin A and but in the latter the stimulation was less (148·028·5% of progesterone secretion control; P<0·01). The secretion of inhibin A and proges- FSH and LH had similar effects on the secretion of activin terone was stimulated in both 24-h experiments (221·7 A, inhibin A and progesterone. In preliminary experi- 18·2% and 271·045·4% of control respectively; P<0·01 ments a dose-dependent stimulatory effect of these for both) and 48-h experiments (267·544·6% and gonadotropins (1–100 IU/l) on activin A secretion was 490·1116·3% of control respectively; P<0·01 for both). measurable after 6 h, reaching a maximum after approxi- The protein kinase C activator, TPA (0·1–100 ng/ml, mately 24 h, and diminishing rapidly after 36 h in culture dose–response data not shown), stimulated activin A (data not shown). On the basis of the preliminary experi- secretion dose dependently, with the maximal effect at the ments, gonadotropin concentrations of 100 IU/l were used concentration of 100 ng/ml in 24-h treatments. TPA in the following experiments. (100 ng/ml) increased activin A and inhibin A secretion in Incubation of granulosa-luteal cells with FSH (Fig. 24-h treatments (6097·61255·6% and 1554·9967·7% 2A) increased activin A secretion in 24-h experiments of control respectively; P<0·05) and 48-h treatments www.endocrinology.org Journal of Endocrinology (2002) 172, 557–563

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Figure 2 Time-dependent (A, B) and dose-dependent (C, D) effects of FSH and LH on activin A, inhibin A and progesterone secretion by cultured human granulosa-luteal cells. The cells were incubated for 24 or 48 h in the presence of FSH (100 IU/l) (A) or LH (100 IU/l) (B). The bars represent the relative activin A, inhibin A and progesterone per 24 and 48 h (mean+S.E.M.) when the respective control is adjusted to 100%. N, number of experiments performed. In dose–response experiments (N=5), FSH (1–100 IU/l) (C) decreased activin A secretion at high concentrations, whereas it increased inhibin A secretion dose dependently during a 48-h treatment period. LH treatment (D) caused a similar trend in activin A and inhibin A secretion. *P<0·05, **P<0·01 (Mann–Whitney test).

(1264·7189·0% and 380·9124·8% of control respect- biphasic manner. Activin A secretion is increased when ively; P<0·05), but it had no significant effect on proges- the treatment period is short or the gonadotropin dose terone secretion in either 24-h (134·719·5% of control; is low, but long-term treatment or high gonadotropin P=0·48) or 48-h treatments (163·844·1% of control; concentrations lead to its inhibition. As the secretion P=0·25) (Fig. 4). of inhibin A is meanwhile stimulated by increasing gonadotropin concentrations and incubation time, the result is a clear decrease in the activin A:inhibin A Discussion secretion ratio. In addition, without gonadotropin stimu- lation, cultured granulosa-luteal cells exhibit the opposite Our data show that activin A secretion in cultured tendency, to increase the activin:inhibin secretion ratio granulosa-luteal cells is regulated by gonadotropins in a spontaneously.

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expression of -subunit for at least 48 h. Our data suggest that the rapid stimulation of A-subunit expression leads to increased activin A secretion, whereas the later domi- nance of the -subunit leads to increased inhibin A secretion during continuing gonadotropin stimulation. 8-BrcAMP also induced A-subunit mRNA expression at 48 h (Tuuri et al. 1996), which explains the increase in activin A secretion after both 24 and 48 h of incubation. Tuuri and co-workers (1996) showed that TPA increases the expression of the A-subunit gene without any effect on the -subunit. Our data suggest that most of this increased A-subunit mRNA is translated and pro- cessed to activin A. The increase in inhibin A after TPA treatment shows that granulosa-luteal cells form sufficient -subunit for inhibin production. However, we do not Figure 3 Time-dependent effect of 8-BrcAMP (protein kinase A know the significance of these regulatory mechanisms activator) on activin A, inhibin A and progesterone secretion by in vivo, as the physiological activators of the protein kinase cultured human granulosa-luteal cells. The cells were incubated for C pathway in granulosa cells are poorly 24 or 48 h in a culture medium containing 8-BrcAMP (100 M) or known. The observation that the ratio of A- to -subunit culture medium only (control). The bars represent the relative mRNAs increases during the period of culture (Erämaa secretion of activin A, inhibin A and progesterone per 24 and 48 h (mean+S.E.M.) by the cells when the respective control is et al. 1994) is also in agreement with our data, as the adjusted to 100%. N, number of experiments performed. **P<0·01 activin A:inhibin A ratio increased in relation to the time (Mann–Whitney test). in culture. On the basis of the findings of the present and previous studies, it seems that the activin A:inhibin A secretion ratio can be predicted well from the mRNA expression data of the activin:inhibin subunits. Muttukrishna and co-workers (1997) were the first to show that the secretion of activin A in cultured human granulosa-luteal cells is stimulated by gonadotropins. Their finding that LH stimulates activin A secretion only after 24 h, but not after 48 h, is in accordance with our data. However, their report that FSH stimulates activin A secretion weakly after 48 h but not after 24 h of incubation is in contrast to our results. A possible reason for this difference is that they used a markedly different model, with granulosa-luteal cells cultured for only 24 or 48 h, in contrast to our cells, which were cultured for 1 week or more before hormonal treatments. It is possible that their granulosa-luteal cells were desensitized to FSH at the experimental period as a result of in vivo ovarian hyper- Figure 4 Time-dependent effect of 12-O-tetradecanoyl phorbol- stimulation. This is supported by the fact that FSH did 13-acetate (TPA, protein kinase C activator) on activin A, inhibin A not affect the steroid secretion of their cells, in contrast to and progesterone secretion (logarithmic scale) by cultured human ours, which exhibited significantly increased progesterone granulosa-luteal cells. The cells were incubated for 24 or 48 h in a culture medium containing TPA (100 ng/ml) or no TPA (control). secretion in the presence of FSH. FSH and LH did not The bars represent the relative secretion of activin A, inhibin A have completely similar effects in our study either. In and progesterone per 24 and 48 h (mean+S.E.M.) when the particular, the sensitivity to LH-treatment varied, indicat- respective control is adjusted to 100%. N, number of experiments < ing that granulosa-luteal cells harvested from a hetero- performed. *P 0·05 (Mann–Whitney test). genous group of patients do not always behave exactly similarly. Nevertheless, the inhibin:activin secretion ratio increased uniformly during prolonged stimulation with The biphasic gonadotropin regulation of activin A both FSH and LH. secretion can be explained by previous mRNA It has been hypothesized that the follicular environment expression data from similar granulosa-luteal cell models. changes from activin- to inhibin-dominant during follicu- Gonadotropin (Erämaa et al. 1994) and 8-BrcAMP (Tuuri logenesis, because granulosa cells exhibit an increase in et al. 1996) stimulation increased the expression of A- gene expression of the -subunit towards the end of the subunit rapidly and transiently, whereas they increased the (Hillier 1991, Knight & Glister 2001, www.endocrinology.org Journal of Endocrinology (2002) 172, 557–563

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Roberts et al. 1993). This shift was demonstrated at the Groome NP, Illingworth PJ, O’Brien M, Cooke I, Ganesan TS, Baird protein level when Schneyer and co-workers (2000) DT & McNeilly AS 1994 Detection of dimeric inhibin throughout the human menstrual cycle by two-site enzyme immunoassay. showed a decrease in follicular fluid activin:inhibin ratio in Clinical Endocrinology 40 717–723. relation to follicular growth. The findings of our study Hillier SG 1991 Regulatory functions for inhibin and activin in suggest that the decrease in activin:inhibin ratio could human ovaries. Journal of Endocrinology 131 171–175. result from increasing gonadotropin stimulation during Hillier SG, Young EL, Illingworth PJ, Baird DT, Schwall RH & ff as the granulosa cells start to express first MasonAJ1991a E ect of recombinant activin on androgen synthesis in cultured human thecal cells. Journal of Clinical FSH and then LH/hCG receptors at the preovulatory Endocrinology and 72 1206–1211. stage (Minegishi et al. 1997a,b). We speculate that increas- Hillier SG, Young EL, Illingworth PJ, Baird DT, Schwall RH & ing gonadotropin stimulation would then allow granulosa MasonAJ1991b Effect of recombinant inhibin on androgen cells to produce increasing amounts of inhibin, whereas synthesis in cultured human thecal cells. Molecular and Cellular Endocrinology 75 R1–R6. the production of activin A would be less than during the Knight PG & Glister C 2001 Potential local regulatory functions of earlier stages of the menstrual cycle. It is also possible that inhibins, activins and follistatin in the ovary. Reproduction 121 the increase in secretion of progesterone is 503–512. partly a consequence of a diminution of the para/autocrine Knight PG, MuttukrishnaS&GroomeNP1996Developmentand inhibitory effect exerted on it by activin A towards the application of a two-site enzyme immunoassay for the determination of ‘total’ activin A concentrations in serum and luteal phase. This hypothesis is supported by the finding of follicular fluid. Journal of Endocrinology 148 267–279. increased serum activin A concentrations in patients with Lewis KA, Gray PC, Blount AL, MacConell LA, Wiater E, hypothalamic hypogonadotropism (Petraglia et al. 1998). Bilezikjian LM & Vale W 2000 Betaglycan binds inhibin and can mediate functional antagonism of activin signaling. Nature 404 411–414. Liu J, Hydén-GranskogC&Voutilainen R 2001 Gonadotrophins Conclusion inhibit and activin induces expression of inhibin/activin B subunit mRNA in cultured human granulosa-luteal cells. Molecular Human Reproduction 7 319–323. The secretion of immunoreactive activin A is regulated by Matzuk MM 2000 In search of binding – identification of inhibin gonadotropins via the protein kinase A signal transduction receptors [Editorial]. Endocrinology 141 2281–2284. pathway in human granulosa-luteal cells. Activin A secre- Minegishi T, Tano M, Igarashi M, Rokukawa S, Abe Y, Ibuki Y & tion is initially increased by gonadotropin stimulation, but Miyamoto K 1997a Expression of follicle-stimulating hormone later diminishes rapidly, whereas inhibin A and progester- receptor in human ovary. European Journal of Clinical Investigation 27 469–474. one secretion are increased time- and dose-dependently. Minegishi T, Tano M, Abe Y, Nakamura K, IbukiY&Miyamoto K This differential regulation of activin A and inhibin A 1997b Expression of /human chorionic secretion, leading to a decreasing activin:inhibin ratio gonadotropin (LH/HCG) receptor mRNA in the human ovary. during increasing gonadotropin stimulation, may be an Molecular Human Reproduction 3 101–107. Muttukrishna S, Fowler PA, Groome NP, Mitchell GG, Robertson important component in the local fine-tuning of ovarian WR & Knight PG 1994 Serum concentrations of dimeric inhibin function. during the spontaneous human menstrual cycle and after treatment with exogenous gonadotrophin. Human Reproduction 9 1634–1642. Muttukrishna S, GroomeN&Ledger W 1997 Gonadotropic control of secretion of dimeric inhibins and activin A by human Acknowledgements granulosa-luteal cells in vitro. Journal of Assisted Reproduction and Genetics 14 566–574. The laboratory technicians Mss Merja Haukka and Petraglia F, Hartmann B, Luisi S, Florio P, Kirchengast S, Santuz M, Minna Heiskanen are thanked for their skillful assistance. Genazzani AD & Genazzani AR 1998 Low levels of serum inhibin A and inhibin B in women with hypergonadotropic amenorrhea Recombinant human FSH and LH for in vitro experi- and evidence of high levels of activin A in women with ments were generously provided by Serono-Nordic hypothalamic amenorrhea. Fertility and Sterility 70 907–912. (Vantaa, Finland). This study was financially supported by Rabinovici J, Spencer SJ, Doldi N, Goldsmith PC, SchwallR&Jaffe Academy of Finland, the Emil Aaltonen Foundation, the RB 1992 Activin-A as an intraovarian modulator: actions, Foundation for Pediatric Research, the Jalmari and Rauha localization, and regulation of the intact dimer in human ovarian cells. Journal of Clinical Investigation 89 1528–1536. Ahokas Foundation, Novo Nordisk Foundation, Sigrid Roberts VJ, Barth S, el-RoeiyA&YenSSC1993Expression of Juselius Foundation, and Kuopio University Hospital. inhibin/activin subunits and follistatin messenger ribonucleic acids and in ovarian follicles and the during the human menstrual cycle. Journal of Clinical Endocrinology and Metabolism 77 1402–1410. References Schneyer AL, Fujiwara T, Fox J, Welt CK, Adams J, Messerlian GM & Taylor AE 2000 Dynamic changes in the intrafollicular Erämaa M, Tuuri T, HildénK&Ritvos O 1994 Regulation of inhibin/activin/follistatin axis during human follicular development: inhibin -andA-subunit messenger ribonucleic acid levels by relationship to circulating hormone concentrations. Journal of Clinical chorionic gonadotropin and recombinant follicle stimulating Endocrinology and Metabolism 85 3319–3330. hormone in cultured human granulosa-luteal cells. Journal of Clinical Tuuri T, Erämaa M, Van Schaik RHN & Ritvos O 1996 Differential Endocrinology and Metabolism 79 1670–1677. regulation of inhibin/activin - and A-subunit and follistatin

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mRNAs by cyclic AMP and phorbol ester in cultured human granulosa cells. Journal of Clinical Endocrinology and Metabolism 63 granulosa-luteal cells. Molecular and Cellular Endocrinology 121 1–10. 202–207. Vänttinen T, Liu J, Hydén-Granskog C, Parviainen M, Penttilä I & Ying SY 1988 Inhibins, activins, and follistatins: gonadal proteins Voutilainen R 2000 Regulation of immunoreactive inhibin A and modulating the secretion of follicle-stimulating hormone. Endocrine B secretion in cultured human granulosa-luteal cells by Reviews 9 267–293. gonadotropins, activin A, and -like growth factor type-1 receptor. Journal of Endocrinology 167 289–294. Voutilainen R, Tapanainen J, Chung B-C, Matteson KJ & Miller WL 1986 Hormonal regulation of P450 scc (20,22-desmolase) and Received 25 September 2001 P450c17 (17-hydroxylase/17,20-lyase) in cultured human Accepted 23 November 2001

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