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• W2005444928 abstract "The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. Growth factors emitted by oocytes integrate and promote this process. Growth differentiation factor-9 (GDF-9), bone morphogenetic protein (BMP)-15, and BMP-6 are oocyte-derived members of the transforming growth factor-β superfamily. In contrast to the recent studies on GDF-9 and BMP-15, nothing is known about the biological function of BMP-6 in the ovary. Here we show that, unlike BMP-15 and GDF-9, BMP-6 lacks mitogenic activity on rat granulosa cells (GCs) and produces a marked decrease in follicle-stimulating hormone (FSH)-induced progesterone (P4) but not estradiol (E2) production, demonstrating not only the first identification of GCs as BMP-6 targets in the ovary but also its selective modulation of FSH action in steroidogenesis. This BMP-6 activity resembles BMP-15 but differs from GDF-9 activities. BMP-6 also exhibited similar action to BMP-15 by attenuating the steady state mRNA levels of FSH-induced steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage enzyme (P450scc), without affecting P450 aromatase mRNA level, supporting its differential function on FSH-regulated P4 and E2 production. However, unlike BMP-15, BMP-6 inhibited forskolin- but not 8-bromo-cAMP-induced P4production and StAR and P450scc mRNA expression. BMP-6 also decreased FSH- and forskolin-stimulated cAMP production, suggesting that the underlying mechanism by which BMP-6 inhibits FSH action most likely involves the down-regulation of adenylate cyclase activity. This is clearly distinct from the mechanism of BMP-15 action, which causes the suppression of basal FSH receptor (FSH-R) expression, without affecting adenylate cyclase activity. As assumed, BMP-6 did not alter basal FSH-R mRNA levels, whereas it inhibited FSH- and forskolin- but not 8-bromo-cAMP-induced FSH-R mRNA accumulation. These studies provide the first insight into the biological function of BMP-6 in the ovary and demonstrate its unique mechanism of regulating FSH action. The process of ovarian folliculogenesis is composed of proliferation and differentiation of the constitutive cells in developing follicles. Growth factors emitted by oocytes integrate and promote this process. Growth differentiation factor-9 (GDF-9), bone morphogenetic protein (BMP)-15, and BMP-6 are oocyte-derived members of the transforming growth factor-β superfamily. In contrast to the recent studies on GDF-9 and BMP-15, nothing is known about the biological function of BMP-6 in the ovary. Here we show that, unlike BMP-15 and GDF-9, BMP-6 lacks mitogenic activity on rat granulosa cells (GCs) and produces a marked decrease in follicle-stimulating hormone (FSH)-induced progesterone (P4) but not estradiol (E2) production, demonstrating not only the first identification of GCs as BMP-6 targets in the ovary but also its selective modulation of FSH action in steroidogenesis. This BMP-6 activity resembles BMP-15 but differs from GDF-9 activities. BMP-6 also exhibited similar action to BMP-15 by attenuating the steady state mRNA levels of FSH-induced steroidogenic acute regulatory protein (StAR) and P450 side-chain cleavage enzyme (P450scc), without affecting P450 aromatase mRNA level, supporting its differential function on FSH-regulated P4 and E2 production. However, unlike BMP-15, BMP-6 inhibited forskolin- but not 8-bromo-cAMP-induced P4production and StAR and P450scc mRNA expression. BMP-6 also decreased FSH- and forskolin-stimulated cAMP production, suggesting that the underlying mechanism by which BMP-6 inhibits FSH action most likely involves the down-regulation of adenylate cyclase activity. This is clearly distinct from the mechanism of BMP-15 action, which causes the suppression of basal FSH receptor (FSH-R) expression, without affecting adenylate cyclase activity. As assumed, BMP-6 did not alter basal FSH-R mRNA levels, whereas it inhibited FSH- and forskolin- but not 8-bromo-cAMP-induced FSH-R mRNA accumulation. These studies provide the first insight into the biological function of BMP-6 in the ovary and demonstrate its unique mechanism of regulating FSH action. progesterone adenylate cyclase bone morphogenetic protein-6 bone morphogenetic protein-15 diethylstilbestrol estradiol follicle-stimulating hormone follicle-stimulating hormone receptor granulosa cell growth differentiation factor-9 luteinizing hormone receptor P450 aromatase P450 side-chain cleavage enzyme steroidogenic acute regulatory protein transforming growth factor-β 8-bromo-cAMP 3-isobutyl-1-methylxanthine reverse transcription-polymerase chain reaction One major direction of current research in the investigation of mechanisms controlling folliculogenesis is the identification of the biological functions of autocrine/paracrine factors that are produced in the ovary. Some of these molecules are synthesized and secreted by the oocyte (1Eppig J.J. Chesnel F. Hirao Y. O'Brien M.J. Pendola F.L. Watanabe S. Wigglesworth K. Hum. Reprod. 1997; 12: 127-132PubMed Google Scholar) and act as morphogens to control follicle growth as well as differentiation (2Erickson G.F. Shimasaki S. Trends Endocrinol. Metab. 2000; 11: 193-198Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). There is a large body of evidence indicating that oocyte-derived factors modulate ovarian function (1Eppig J.J. Chesnel F. Hirao Y. O'Brien M.J. Pendola F.L. Watanabe S. Wigglesworth K. Hum. Reprod. 1997; 12: 127-132PubMed Google Scholar, 2Erickson G.F. Shimasaki S. Trends Endocrinol. Metab. 2000; 11: 193-198Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 3Matzuk M. Mol. Cell. Endocrinol. 2000; 163: 61-66Crossref PubMed Scopus (108) Google Scholar). In vitro experiments have shown that these factors can act to inhibit progesterone (P4)1 production (4Goldschmit D. Kraicer P. Orly J. Endocrinology. 1989; 124: 369-378Crossref PubMed Scopus (30) Google Scholar), follicle-stimulating hormone (FSH)-induced expression of P450 side-chain cleavage enzyme (P450scc) (4Goldschmit D. Kraicer P. Orly J. Endocrinology. 1989; 124: 369-378Crossref PubMed Scopus (30) Google Scholar), and luteinizing-hormone receptor (LH-R) mRNA (5Eppig J.J. Wigglesworth K. Pendola F. Hirao Y. Biol. Reprod. 1997; 56: 976-984Crossref PubMed Scopus (242) Google Scholar), while acting to stimulate estradiol (E2) production (6Vanderhyden B.C. Tonary A.M. Biol. Reprod. 1995; 53: 1243-1250Crossref PubMed Scopus (117) Google Scholar) and granulosa cell (GC) mitosis (7Vanderhyden B.C. Telfer E.E. Eppig J.J. Biol. Reprod. 1992; 46: 1196-1204Crossref PubMed Scopus (204) Google Scholar). Three oocyte-derived members of the transforming growth factor-β (TGF-β) superfamily, namely growth differentiation factor-9 (GDF-9), bone morphogenetic protein-15 (BMP-15), and BMP-6 are, in particular, potentially involved in mediating these important biological consequences triggered by the putative oocyte factors.Recently, there have been a number of studies published on the role of GDF-9 and BMP-15 in the ovary. With regard to GDF-9, female mice lacking GDF-9 have resulted in an early block in folliculogenesis leading to infertility, suggesting that GDF-9 is obligatory for normal folliculogenesis and female fertility (8Dong J. Albertini D.F. Nishimori K. Kumar T.R. Lu N. Matzuk M. Nature. 1996; 383: 531-535Crossref PubMed Scopus (1297) Google Scholar, 9Carabatsos M.J. Elvin J. Matzuk M.M. Albertini D.F. Dev. Biol. 1998; 204: 373-384Crossref PubMed Scopus (260) Google Scholar, 10Elvin J.A. Yan C. Matzuk M.M. Mol. Cell. Endocrinol. 2000; 159: 1-5Crossref PubMed Scopus (239) Google Scholar). In vitrostudies using rat and mice GCs have demonstrated that GDF-9 regulates GC mitogenesis and steroidogenesis as well as cumulus expansion (11Hayashi M. McGee E.A. Min G. Klein C. Rose U.M. Van Duin M. Hsueh A.J.W. Endocrinology. 1999; 140: 1236-1244Crossref PubMed Google Scholar, 12Elvin J.A. Clark A.T. Wang P. Wolfman N.M. Matzuk M.M. Mol. Endocrinol. 1999; 13: 1035-1048Crossref PubMed Google Scholar, 13Elvin J.A. Yan C. Matzuk M.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10288-10293Crossref PubMed Scopus (158) Google Scholar, 14Vitt U.A. Hayashi M. Klein C. Hsueh A.J.W. Biol. Reprod. 2000; 62: 370-377Crossref PubMed Scopus (279) Google Scholar). As for BMP-15, which is most closely related in structure to GDF-9, an intriguing finding has been reported recently by Gallowayet al. (15Galloway S.M. McNatty K.P. Cambridge L.M. Laitinen M.P.E. Juengel J.L. Jokiranta T.S. McLaren R.J. Luiro K. Dodds K.G. Montgomery G.W. Beattie A.E. Davis G.H. Ritvos O. Nat. Genet. 2000; 25: 279-283Crossref PubMed Scopus (832) Google Scholar), in which they identified a causative point mutation in the bmp-15 gene in Inverdale sheep that has been known to exhibit aberrant follicle development and ovulation rate. Of interest, the homozygous Inverdale female sheep are infertile because follicle growth is arrested at the primary preantral stage, whereas the heterozygous females exhibit increased ovulation rate. Thus this mutation causes increased ovulation and infertility in a dosage-sensitive manner, suggesting an important role for BMP-15 in folliculogenesis and ovulation. By in vitro studies with rat GCs, we have shown that BMP-15 stimulates mitosis independent of FSH and regulates steroidogenesis by inhibiting FSH receptor (FSH-R) expression (16Otsuka F. Yao Z. Lee T.H. Yamamoto S. Erickson G.F. Shimasaki S. J. Biol. Chem. 2000; 275: 39523-39528Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 17Otsuka F. Yamamoto S. Erickson G.F. Shimasaki S. J. Biol. Chem. 2001; 276: 11387-11392Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar).In contrast, although BMP-6 mRNA is highly expressed in both immature and mature oocytes of mice (10Elvin J.A. Yan C. Matzuk M.M. Mol. Cell. Endocrinol. 2000; 159: 1-5Crossref PubMed Scopus (239) Google Scholar, 18Lyons K.M. Pelton R.W. Hogan B.L.M. Genes Dev. 1989; 3: 1657-1668Crossref PubMed Scopus (398) Google Scholar), there have been few studies designed to investigate the biological functions and target cells of BMP-6 in the ovary. Mice with homozygous loss-of-function mutations in the bmp-6 gene by targeted deletion are fertile with normal sized litters (19Solloway M.J. Dudley A.T. Bikoff E.K. Lyons K.M. Hogan B.L. Robertson E.J. Dev. Genet. 1998; 22: 321-339Crossref PubMed Scopus (303) Google Scholar), which may suggest thatbmp-6 is dispensable for fertility. The lack of perturbative reproductive phenotype seen in these mutant mice could be the reflection of compensation by related BMP(s) expressed in the ovary by virtue of a redundant safety system to maintain homeostasis. Alternatively, it is possible that BMP-6 evokes no biological effects in the ovary.Because no information is available at present from any species about the biological function of BMP-6 in the ovary, its role in the ovary remains beyond speculation. In the present study, we report the identification of GCs as a target cell type for BMP-6 in the rat ovary. Furthermore, we report that BMP-6 evokes biological activities that are distinct from other TGF-β superfamily members and that it utilizes novel cellular mechanism in GCs.RESULTSWe have first assumed that GCs are target cells for BMP-6 because rat GCs express BMP receptor type IB (BMPR-IB, also known as ALK-6) and type II (BMPR-II) and at lesser levels BMPR-IA (ALK-3) (28Shimasaki S. Zachow R.J. Li D. Kim H. Iemura S.-I. Ueno N. Sampath K. Chang R.J. Erickson G.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7282-7287Crossref PubMed Scopus (278) Google Scholar) as well as activin type II and type IIB receptors (29Cameron V.A. Nishimura E. Mathews L.S. Lewis K.A. Sawchenko P.E. Vale W.W. Endocrinology. 1994; 134: 799-808Crossref PubMed Scopus (192) Google Scholar), all to which BMP-6 can bind (30Ebisawa T. Tada K. Kitajima I. Tojo K. Sampath T.K. Kawabata M. Miyazono K. Imamura T. J. Cell Sci. 1999; 112: 3519-3527Crossref PubMed Google Scholar). We have, therefore, tested whether GCs would respond to BMP-6. Similar to our previous studies on BMP-4, BMP-7, and BMP-15 (16Otsuka F. Yao Z. Lee T.H. Yamamoto S. Erickson G.F. Shimasaki S. J. Biol. Chem. 2000; 275: 39523-39528Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar,28Shimasaki S. Zachow R.J. Li D. Kim H. Iemura S.-I. Ueno N. Sampath K. Chang R.J. Erickson G.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7282-7287Crossref PubMed Scopus (278) Google Scholar), we examined the effect of BMP-6 on P4 and E2 production induced by FSH using primary rat GCs cultured in serum-free medium. As observed earlier (16Otsuka F. Yao Z. Lee T.H. Yamamoto S. Erickson G.F. Shimasaki S. J. Biol. Chem. 2000; 275: 39523-39528Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 28Shimasaki S. Zachow R.J. Li D. Kim H. Iemura S.-I. Ueno N. Sampath K. Chang R.J. Erickson G.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7282-7287Crossref PubMed Scopus (278) Google Scholar), FSH alone increased P4 and E2 production in a dose-dependent manner (Fig.1). By comparison, BMP-6 alone (up to 300 ng/ml) did not affect basal levels of P4 and E2production. However, co-treatment of GCs with a saturated dose of FSH (10 ng/ml) and increasing doses of BMP-6 (0–300 ng/ml) caused a marked inhibition (80%) of the FSH-induced P4 production (ED50 = 10 ng/ml). On the contrary, BMP-6 had no effect on the FSH-induced E2 production. These findings indicate that rat GCs express a functional type I and type II receptor set for BMP-6 and that P4 synthesis induced by FSH is selectively down-regulated by virtue of BMP-6.Based on these findings, we hypothesized that BMP-6 may be specifically involved in modulating the expression of steroidogenic genes in response to FSH stimulation. To elucidate this hypothesis, we analyzed mRNA levels for three key regulators of GC steroidogenesis StAR, P450scc, and P450arom in the GCs treated with FSH and/or BMP-6 by a quantitative competitive RT-PCR. We have carefully validated and established this technique in our laboratory as an accurate technique for the quantification of RNA expression as reported in our previous studies (17Otsuka F. Yamamoto S. Erickson G.F. Shimasaki S. J. Biol. Chem. 2001; 276: 11387-11392Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). As shown in Fig. 2, treatment of GCs for 48 h with a saturating dose (100 ng/ml) of BMP-6 alone had no effect on the steady state mRNA levels for StAR, P450scc, P450arom and a housekeeping gene L19. Treatment with FSH, in contrast, markedly increased their mRNA levels except for L19. Interestingly, BMP-6 reduced FSH-stimulated StAR and P450scc mRNA levels to the basal levels but had no effect on P450arom mRNA level. L19 mRNA level showed no difference at all among these four different treatments. Given the fact that StAR and P450scc are major rate-limiting factors of P4synthesis in GCs (31Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar), the suppression of their mRNA expressions are likely to reflect the selective inhibition of FSH-induced P4 production. On the other hand, the failure of BMP-6 to inhibit FSH-stimulated P450arom (catalytic enzyme to convert androstenedione to E2) mRNA expression is consistent with that to inhibit FSH-induced E2 production.Figure 2Effects of FSH and BMP-6 on the expression of the mRNAs for StAR , P450scc , P450arom , and L19. GCs were cultured either alone or together with FSH (10 ng/ml) and/or BMP-6 (100 ng/ml) in the presence of androstenedione (100 nm) for 48 h after which total RNA was extracted and then subjected to quantitative competitive RT-PCR analysis as described under “Experimental Procedures.” The PCR products are shown in theupper panel, and the ratios of PCR products (target/internal control) are graphed. Bars with different letters indicate that group means are significantly different atp < 0.05. i.c., internal control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To elucidate further the mechanism by which BMP-6 suppresses FSH-induced expression of StAR and P450scc, we utilized forskolin and 8-Br-cAMP that mimic FSH action in steroid synthesis by bypassing FSH-R and G proteins. Forskolin is a direct activator of adenylate cyclase (AC) and 8-Br-cAMP is a stable analog of cAMP. As shown in Fig.3, forskolin (10 µm) stimulated the expression of StAR and P450scc mRNA, whereas BMP-6 suppressed the forskolin-induced mRNA levels of StAR and P450scc, similar to those had seen with the FSH treatment. However, in striking contrast to the FSH and forskolin results, BMP-6 failed to change the 8-Br-cAMP-induced mRNA levels of these steroidogenic factors. The mRNA level of the control housekeeping gene, L19, did not change in response to treatment with any of the indicated reagents and their combinations.Figure 3Effects of forskolin (10 µm), 8-Br-cAMP (0.2 and 1 mm), and BMP-6 (100 ng/ml) on the expression of the mRNAs for StAR , P450scc , and L19. The experimental design is as described in Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We further compared the effects of BMP-6 on FSH-, forskolin-, and 8-Br-cAMP-induced P4 production by GCs (Fig.4). Consistent with the results shown in Fig. 1, FSH-induced P4 was significantly suppressed by BMP-6 up to ∼80%. As expected, BMP-6 also suppressed forskolin-induced P4 by ∼80% but did not significantly inhibit 8-Br-cAMP-induced P4 production. These data reinforce our findings that BMP-6 exerts its biological activity by inhibiting FSH signaling at a site downstream of the FSH-R and upstream of cAMP signaling.Figure 4Effect of BMP-6 on progesterone production induced by FSH , forskolin, and 8-Br-cAMP. GCs (105viable cells) were cultured in serum-free media with 100 nmandrostenedione in the presence or absence of FSH (10 ng/ml), forskolin (10 µm), and 8-Br-cAMP (1 mm) with the combination of BMP-6 (100 ng/ml). After 48 h of culture, the levels of progesterone in the medium were measured by radioimmunoassay. Data are shown as mean ± S.E. of percent change. *,p < 0.05 compared with the control level.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Therefore, we next examined the possible effect of BMP-6 on AC activity with which cellular AMP is converted to cAMP, leading to the activation of protein kinase A. For this analysis, GCs were treated with FSH (10 ng/ml) or forskolin (10 µm) in the presence or absence of BMP-6 (100 ng/ml). In order to prevent the metabolic effect of phosphodiesterase on cAMP, IBMX (specific inhibitor of phosphodiesterase activity) was added to the culture medium at 0.1 mm (32Gitay-Goren H. Lindenbaum E.S. Kraiem Z. Mol. Cell. Endocrinol. 1989; 61: 69-76Crossref PubMed Scopus (22) Google Scholar, 33Rice V.M. Limback S.D. Roby K.F. Terranova P.F. Endocrine. 1999; 10: 19-23Crossref PubMed Scopus (24) Google Scholar). After 48 h of culture, cAMP concentrations in the culture media were measured. As shown in Fig.5, the level of cAMP in the medium from the cells without FSH or forskolin treatment was low, and BMP-6 did not appear to alter basal cAMP production. However, cAMP levels stimulated by either FSH or forskolin were significantly suppressed by the co-treatment with BMP-6. The ability of BMP-6 to reduce the cAMP levels in the presence of IBMX should be in response to decreased synthesis of cAMP by AC rather than increased metabolic activity of phosphodiesterase. These data suggest that BMP-6 inhibits AC activity enhanced by FSH and forskolin, and the attenuation of FSH- and forskolin-induced StAR and P450scc mRNA expression by BMP-6 seems to be, at least in part, attributed to the decreased activity of AC.Figure 5Effects of FSH , forskolin, and BMP-6 on cAMP production. GCs (105 viable cells) were cultured in serum-free media and in the presence or absence of FSH (10 ng/ml) and forskolin (10 µm) with the combination of BMP-6 (100 ng/ml). After 48 h culture, the extracellular concentrations of cAMP were measured by enzyme immunoassay after the acetylation of samples. Bars with different letters indicate that group means are significantly different at p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since FSH-R expression in GCs is known to be stimulated by FSH, forskolin (34Tilly J.L. LaPolt P.S. Hsueh A.J.W. Endocrinology. 1992; 130: 1296-1302Crossref PubMed Scopus (74) Google Scholar), or 8-Br-cAMP (35Minegishi T. Kishi H. Tano M. Kameda T. Hirakawa T. Miyamoto K. Mol. Cell. Endocrinol. 1999; 149: 71-77Crossref PubMed Scopus (29) Google Scholar), the effect of BMP-6 on FSH-R mRNA expression was also examined (Fig.6). Control untreated cells spontaneously expressed basal level of FSH-R mRNA, and BMP-6 did not alter the level by itself. FSH treatment increased FSH-R mRNA level up to 2-fold, and BMP-6 abolished its effect. Stimulation of FSH-R mRNA expression by forskolin was significantly suppressed by BMP-6 but that by 8-Br-cAMP at two different concentrations was unchanged. Furthermore, virtually identical activity of BMP-6 was obtained from the experiments in which we tested the effect of BMP-6 on the expression of several other genes (Fig.7). Namely, BMP-6 inhibited the stimulatory action of FSH and forskolin on the steady state mRNA levels of inhibin/activin subunits (α, βA, and βB) and LH-R, but not that of 8-Br-cAMP.Figure 6Effects of FSH (10 ng/ml), forskolin (10 µm), 8-Br-cAMP (0.2 and 1 mm), and BMP-6 (100 ng/ml) on the expression of FSH-R mRNA. FSH-R mRNA levels in GCs with the indicated treatments were analyzed by a quantitative competitive RT-PCR and are presented together with those of L19 mRNA. Bars withdifferent letters indicate that group means are significantly different at p < 0.05. i.c.,internal control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Effects of FSH (10 ng/ml), forskolin (10 µm), 8-Br-cAMP (1 mm), and BMP-6 (100 ng/ml) on the expression of inhibin/activin subunits and LH-R mRNA. Target mRNA levels in GCs with the indicated treatments were analyzed by a semi-quantitative RT-PCR analysis using specific primer pairs. Two distinct bands of LH-R are due to alternative spliced transcripts. The representative data are shown from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since oocyte factors have been shown to stimulate GC mitosis, we also examined whether BMP-6 is capable of regulating GC proliferation (Fig.8). Treatment of primary cultured rat GCs with BMP-6 (0–300 ng/ml) produced no significant change in thymidine incorporation as well as numbers of GCs, in contrast to the positive effect by BMP-15.Figure 8Effect of BMP-6 on GC mitosis. GCs (2 × 105 viable cells) were cultured in serum-free media and treated for 24 h with [methyl-3H]thymidine plus increasing doses of BMP-6 (0–300 ng/ml) or BMP-15 (100 ng/ml) after 24 h of preculture. The labeled thymidine incorporated into the cells was counted. Actual GC number was also counted after 24 h of culture with or without BMP-6 (300 ng/ml) or BMP-15 (100 ng/ml). Data are mean ± S.E. *, p < 0.05 compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn the present study, we first provided evidence for the biological activity of BMP-6 in GCs. Experiments in which BMP-6 was added to cultured GCs indicated that BMP-6 is potent in suppressing FSH-induced P4 production without affecting FSH-induced E2 production and that BMP-6 does not exhibit any mitogenic activity in GCs. Our data demonstrate that the biological effects of BMP-6 on GCs are distinct from those of other TGF-β superfamily members, including inhibin (36Hutchinson L.A. Findlay J.K. de Vos F.L. Robertson D.M. Biochem. Biophys. Res. Commun. 1987; 146: 1405-1412Crossref PubMed Scopus (187) Google Scholar, 37Sugino H. Nakamura T. Hasegawa Y. Miyamoto K. Abe Y. Igarashi M. Eto Y. Shibai H. Titani K. Biochem. Biophys. Res. Commun. 1988; 153: 281-288Crossref PubMed Scopus (82) Google Scholar), activin (37Sugino H. Nakamura T. Hasegawa Y. Miyamoto K. Abe Y. Igarashi M. Eto Y. Shibai H. Titani K. Biochem. Biophys. Res. Commun. 1988; 153: 281-288Crossref PubMed Scopus (82) Google Scholar, 38Xiao S. Findlay J.K. Robertson D.M. Mol. Cell. 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A. 1999; 96: 7282-7287Crossref PubMed Scopus (278) Google Scholar), BMP-7 (28Shimasaki S. Zachow R.J. Li D. Kim H. Iemura S.-I. Ueno N. Sampath K. Chang R.J. Erickson G.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7282-7287Crossref PubMed Scopus (278) Google Scholar), and GDF-9 (14Vitt U.A. Hayashi M. Klein C. Hsueh A.J.W. Biol. Reprod. 2000; 62: 370-377Crossref PubMed Scopus (279) Google Scholar). However, it is intriguing that the function of BMP-6 with respect to GC steroidogenesis has a strong resemblance to that of BMP-15, another oocyte-secreted factor (16Otsuka F. Yao Z. Lee T.H. Yamamoto S. Erickson G.F. Shimasaki S. J. Biol. Chem. 2000; 275: 39523-39528Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Both BMP-6 and BMP-15 selectively modulate the biological effects of FSH on GCs by inhibiting FSH-induced P4 production without affecting FSH-induced E2synthesis. The maximal efficacy of BMP-6 inhibition of FSH-induced P4 was ∼30% more than that of BMP-15, but the ED50 of both factors were similar, ∼10−10m, which is within a physiological range. The biological effects of GDF-9, a closely related factor also secreted by oocytes, are clearly different from BMP-6. GDF-9 stimulates E2production by cultured primary rat GCs in the absence of FSH yet suppresses FSH-induced both E2 and P4production (14Vitt U.A. Hayashi M. Klein C. Hsueh A.J.W. Biol. Reprod. 2000; 62: 370-377Crossref PubMed Scopus (279) Google Scholar).The ability of BMP-6 to regulate specifically FSH-induced P4 biosynthesis without affecting FSH-induced E2 synthesis indicates that BMP-6 must preferentially inhibit the biochemical pathways that lead to P4production. The present studies revealed that BMP-6 reduced the steady state levels of FSH-induced mRNAs involved in P4synthesis, including StAR and P450scc. However, when BMP-6 was administered without FSH, no changes in the expression of these molecules were observed, indicating that BMP-6 must be acting only by regulating FSH activity. In this regard, our previous studies showed that BMP-15 regulated the sensitivity of GCs to FSH by inhibiting the expression of FSH-R (16Otsuka F. Yao Z. Lee T.H. Yamamoto S. Erickson G.F. Shimasaki S. J. Biol. Chem. 2000; 275: 39523-39528Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). The present finding that BMP-6 alone had no effect on the steady state levels of FSH-R mRNA indicates that BMP-6 must work by a mechanism that is different from BMP-15 and is downstream of the FSH-R.To elucidate the mechanism of BMP-6 regulation of FSH signaling, we investigated whether BMP-6 would also suppress the effects of forskolin and 8-Br-cAMP on StAR and P450scc mRNA levels in GCs. When added alone, both forskolin and 8-Br-cAMP exhibited similar activities to FSH in GCs. BMP-6 did inhibit forskolin-induced responses, which further supports the hypothesis that BMP-6 is acting downstream of the FSH-R, but did not inhibit 8-Br-cAMP-induced responses, which indicates that BMP-6 must be acting upstream of cAMP signaling. This action of BMP-6 was broadly observed in a battery of other genes includingFSH-R , LH-R , and the inhibin/activin subunits. Moreover, direct measurement of cAMP in GCs revealed that, when added with FSH or forskolin, BMP-6 caused a significant decrease in the levels of cAMP. Collectively, these data sug" @default.
• W2005444928 created "2016-06-24" @default.
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• W2005444928 date "2001-08-01" @default.
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