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Program for Developmental and Reproductive Biology, Biomedicum Helsinki (J.B., L.D., O.R.), and Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki (R.J., J.B., K.H., O.R.), and Hospital of Children and Adolescents, Helsinki University Central Hospital (T.R., L.D.), 00014 Helsinki, Finland; and School of Biological and Molecular Sciences (N.G.), Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom
Address all correspondence and requests for reprints to: Dr. Olli Ritvos, Biomedicum Helsinki, Room C502b, P.O. Box 63, Haartmaninkatu 8, 00014 University of Helsinki, Finland. E-mail: . olli.ritvos{at}helsinki.fi
Abstract
During the human menstrual cycle the circulating levels of inhibin B, a dimer of inhibin 
- and ßB-subunits, fluctuate in a fashion distinct from that of inhibin A, the -ßA-subunit dimer. This suggests that human inhibin subunits are each regulated in a distinct manner in human ovarian granulosa cells by endocrine and local factors. We have previously shown using cultures of human granulosa-luteal (hGL) cells that gonadotropins stimulate the steady state mRNA levels of inhibin - and ßA-subunits, but not those of the ßB-subunit, which, on the other hand, are up-regulated by, for instance, activin and TGFß. We recently identified the TGFß gene family member bone morphogenetic protein-3 (BMP-3) as a granulosa cell-derived growth factor, but whether BMP-3 or other structurally related BMPs regulate human granulosa cell inhibin production is not known. We show here that hGL cells express mRNAs for distinct serine/threonine kinase receptors (BMP-RIA and BMP-RII) and Smad signaling proteins (Smad1, Smad4, and Smad5) involved in the mediation of cellular effects of BMPs. Subsequently, we determined in hGL cell cultures the effects of distinct members of the BMP family previously found to be expressed in mammalian ovaries. Recombinant BMP-2 induces potently in a time- and concentration-dependent manner the expression of the inhibin ßB-subunit mRNAs in hGL cells without affecting the levels of - or ßA-subunit mRNAs. BMP-6 has a similar, but weaker, effect than BMP-2, whereas BMP-3 and its close homolog, BMP-3b (also known as growth differentiation factor-10) had no effect on inhibin subunit mRNA expression. hCG treatment of hGL cells was previously shown to abolish the stimulatory effect of activin on ßB-subunit mRNA levels, and here hCG is also shown to suppress the effect of BMP-2. Furthermore, BMP-2 stimulates hGL cell secreted dimeric inhibin B levels in a concentration-dependent manner. Depending on the experiment, maximal increases in inhibin B levels of 6- to 28-fold above basal levels were detected during a 72-h culture period. We conclude that activation of the BMP-signaling pathway in hGL cells stimulates inhibin ßB-subunit mRNA levels and leads at the protein level to a dramatic stimulation of secreted inhibin B dimers. Our results are consistent with the suggestion that in addition to the distinct activin- and TGFß-activated signaling pathways, the BMP-activated pathway is likely to be implicated in the complex regulation of inhibins in the human ovary.
INHIBINS AND ACTIVINS are dimeric polypeptides originally purified from ovarian follicular fluid as nonsteroidal factors able to inhibit or stimulate, respectively, pituitary FSH production (1). In addition to their endocrine role as regulators of FSH secretion, they have been implicated in the local regulation of ovarian folliculogenesis, exerting autocrine and paracrine effects on follicular granulosa and thecal cells (2). Inhibin A and inhibin B are dimers of a common -subunit covalently coupled by a disulfide bond to similar, but structurally distinct, ß-subunits, the ßA- or ßB-subunits, respectively. By contrast, activins are homo- or heterodimers of the ßA- or ßB-subunits. Inhibins and activins belong to the TGFß superfamily of over 40 known distinct growth factors that are involved in the regulation of a wide array of biological processes, such as early embryonic mesoderm induction and regulation of cell growth and differentiation in diverse tissues (3, 4).
The three inhibin/activin subunits are all expressed in human ovarian granulosa cells, and each of the different subunits seems to be regulated in a distinct manner by endocrine and local factors (5, 6, 7, 8, 9, 10). During the human menstrual cycle the dimeric inhibin B levels fluctuate in a typical cyclical manner, but with a different profile from that seen for dimeric inhibin A (11, 12). The levels of circulating inhibin B are elevated in the midfollicular phase, and they are significantly lower in the luteal phase (12). By contrast, dimeric inhibin A levels increase in the late follicular phase and are highest during the midluteal phase (11, 12). Immunohistochemical and in situ hybridization studies of the spatiotemporal expression of the inhibin subunit proteins and mRNAs in human and primate ovarian follicles are in line with the findings observed for the circulating inhibin dimers (6, 7, 13, 14). The inhibin ßB-subunit mRNAs are abundantly expressed in granulosa cells of small antral follicles when ßA-subunit mRNA levels are relatively low (6, 13, 14). In preovulatory follicles ßB-subunit levels fall dramatically, accompanied by an increase in inhibin - and ßA-subunit levels. We have previously shown, using cultures of human granulosa-luteal (hGL) cells, that gonadotropins stimulate the steady state mRNA levels of inhibin - and ßA-subunits (8), but not those of the ßB-subunit, which, on the other hand, are up-regulated by activin A and TGFß (9, 10). Also, recent studies performed with human granulosa cells of small antral follicles showed that inhibin A and inhibin B are differentially regulated (15, 16).
At the cellular level activins mediate their effects by first binding to specific type II serine/threonine (Ser/Thr) kinase receptors. The ligand-type II receptor complex then allows specific type I Ser/Thr kinase receptors to be recruited as activated kinases, which, in turn, activate distinct cytoplasmic signal transducer proteins of the Smad family (3, 4, 17). Recent evidence suggests that the cellular effects of inhibins are based on their ability to form high affinity complexes with activin type II receptors and betaglycan, a cell surface-anchored proteoglycan, and thereby inhibins restrict the number of the activin type II receptors available for activin signaling (18, 19). Another cell surface inhibin-binding protein, p120, was also recently discovered, and it is thought to influence the effect of inhibin on target cells (18, 20). Although a relatively large number of TGFß family ligands are known to regulate various biological processes, a significantly smaller number of type I and II Ser/Thr receptors and Smad proteins have been identified despite extensive efforts (3, 4). This suggests that many of the ligands share the same receptors, which then activate an even more restricted number of Smad signaling pathways. Two distinct Smad signaling pathways, the activin/TGFß-activated pathway and the bone morphogenetic protein (BMP)-activated pathway, are thought to mediate the cellular effects of most TGFß family members (4). The receptor-activated Smads (R-Smads), Smad2 and Smad3, are activated by TGFß or activin receptors, whereas Smad1, Smad5, and Smad8 are activated by BMP receptors. R-Smads then form complexes with the common partner Smad, Smad4. The Smad complexes translocate into the nucleus, where they interact with various other transcriptional regulators and control the transcription of target genes. In the ovary, activin and TGFß have been reported to regulate granulosa cell proliferation, steroidogenesis, and inhibin production, but little is known of the possible role of the BMP signaling pathway in ovarian physiology.
A number of TGFß family members belonging to the BMP subfamily have recently been reported to be expressed in the mammalian ovary. For example, BMP-3 mRNAs are highly expressed and hormonally regulated by gonadotropins in hGL cells (21). BMP-2 and BMP-3 mRNAs as well as growth differentiation factor-10 (GDF-10/BMP-3b) mRNAs have been detected in whole human and/or rat ovaries (22, 23), and BMP-4 and BMP-7 mRNAs have been identified in thecal cells of rat preovulatory follicles (24). Furthermore, BMP-6 (25) and GDF-9B/BMP-15 have been shown to be expressed in rodent, sheep, and human oocytes (26, 27, 28, 29, 30, 31). Collectively, these findings suggest that many members of the BMP family regulate mammalian ovarian function. Specifically in this study we determined which components of the BMP signaling pathway are expressed in hGL cells and tested whether distinct members of the BMP family found in the mammalian ovary regulate the inhibin system in these cells.
Materials and Methods
hGL cell cultures
hGL cells were obtained in conjunction with oocyte aspiration from women undergoing hormone treatment for in vitro fertilization. For each experiment, the cells of two to five patients were pooled, enzymatically dispersed, and separated from red blood cells by centrifugation through Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) as previously described (7). Thereafter, the cells were directly recovered for RNA extraction or plated at a density of 2–5 x 105 cells/well on six-well dishes or at a density of 5–10 x 104 cells/well on 24-well dishes (Greiner Labortechnik, Frickenhausen, Germany) for mRNA or protein detection, respectively. hGL cells were cultured in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS (Life Technologies, Inc.), 2 mmol/liter L-glutamine, and antibiotics at 37 C in a 95% air-5% CO2 humidified environment. Cell culture media were changed every other day, and treatments were performed between d 4 and 7 of culture for different time periods, as indicated in the text and figure legends.
Treatment of hGL cells with BMPs, activin A, follistatin, and hCG
Before adding the stimulants, the cells were transferred to DMEM supplemented with 2.5% FCS. Recombinant BMP-2, BMP-3, BMP-3b/(GDF-10), BMP-4, and BMP-6 were gifts from Dr. Vicki Rosen (Genetics Institute, Cambridge, MA). Recombinant activin A (gift from Dr. Eto, Aijnomoto, Inc, Kawasaki, Japan), recombinant human follistatin (288-amino acid follistatin; from National Hormone and Pituitary Program, Baltimore, MD) and purified hCG (CR-127, National Hormone and Pituitary Program) were used in the experiments as described previously (8, 9). The effects of BMPs on inhibin/activin subunit and Smad6 mRNA expression in hGL cells were first studied in 24- to 48-h treatments between d 4–6 of culture. In the concentration dependence studies hGL cells received 1–100 ng/ml BMP-2 for 48 h on d 4–6 of culture for mRNA expression studies or for 72 h on d 3 of culture for dimeric inhibin B measurement, respectively. For time-course experiments, hGL cells were treated with 50 ng/ml BMP-2 for 2, 8, 24, 48, 72, and 96 h on culture d 4 or 5. To study the protein synthesis dependence of BMP-2-stimulated inhibin ßB-subunit mRNA expression the cells were treated with 20 µg/ml cycloheximide (CHX; Sigma, St. Louis, MO) 30 min before BMP-2 (50 ng/ml) supplementation. The effects of hCG (100 ng/ml) on BMP-2 (50–100 ng/ml)- and activin A (50 ng/ml)-induced ßB-subunit mRNA were studied by adding both factors simultaneously. To determine whether the activin-binding protein, follistatin (250 ng/ml), is able to block the effect of BMP-2 (50 ng/ml) and activin A (50 ng/ml), follistatin was preincubated with BMP-2 or activin A at 37 C for 1 h before their addition to cultures. Each experiment was performed at least three times with triplicate cultures.
RNA isolation and Northern and dot blotting
Cytoplasmic RNAs from cultured hGL cells or total RNAs from freshly isolated granulosa cells were extracted with the modified Nonidet P-40 lysis procedure (8) or the guanidium isothiocyanate-cesium chloride method (32), respectively. RNA was quantitated by absorbance measurement at 260 nm. For Northern blots, 10–20 µg total RNA were size-fractionated in 1.5% agarose gels. Before transfers even loading of the gels was checked based on RNA fluorescence at 254 nm, after which they were transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). Dot-blot samples of 1–2 µg cytoplasmic RNA were spotted onto nylon membranes and UV cross-linked as previously described (8).
Preparation and labeling of cDNA probes and filter hybridizations
For the detection of inhibin -, ßA- and ßB-subunit mRNAs by Northern or dot-blot hybridization, double- or single-stranded cDNA probes were prepared as previously described (7). The BMP-RIA receptor cDNA was obtained as a full-length clone in pcDNA-1 vector (33), and a 783-bp proportion of BMP-RIA cDNA was PCR-amplified with primers 5'-ATGACTCAGCTATACATTTACAT-3' and 5'-CTAGGAAT-TCGG(A/G)AA(G/T)AT(C/T)TT(G/C)AC(T/A/G)GC(A/C)AC-3', subcloned into the pGEM-T vector (Promega Corp., Madison, WI), whereafter the hybridization probe insert was cut with SacII and PstI. A 1914-bp BMP-RII receptor cDNA probe was generated from the truncated form of the receptor (2030 bp) in vector pJT6 (34) by digesting with SacII. Smad1 and Smad2 cDNA inserts were obtained by SalI and BamHI digestion of pCMV5B-hSmad1-F (35) and EcoRI and XhoI digestion of pcDNA-3-Smad2-F (36), respectively. A 1007-bp Smad3 cDNA probe was cut with BglI from pRK-5-Smad3-M (37). Smad4 and Smad5 cDNA probes were cut with SalI and BglII from vector pCMV5B-Smad4-F (38) and with BamHI from vector pCMV5B-Flag (39), respectively. A human cyclophilin cDNA was used as a loading control for the experiments (40). Northern and dot-blot hybridizations were performed for 16 h at 42 C, and the filters were washed three times for 20 min each time with 0.1–1x SSC/0.1% SDS at 50 C. Thereafter, filters were exposed to a Fujifilm Ip-Reader Bio-Imaging Analyzer BAS 1500 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The relative densities of dot-blot hybridization signals were analyzed using MacBas software supplied by the manufacturer (Fuji).
Measurement of dimeric inhibin B by ELISA
The amounts of dimeric inhibin B secreted by hGL cells in the spent culture medium were quantified using an ELISA kit for dimeric inhibin B (Serotec, Oxford, UK). The signal amplification kit (Life Technologies, Inc.) was used in connection with the dimeric inhibin B kit. For determination of intraassay coefficient of variation (CV) supernatants from several stimulated cultures were pooled, followed by repeated measurements of inhibin B levels. The CVs at mean concentrations of 90 and 459 pg/ml were less than 7%. To achieve concentrations within the measuring range of the assay (15.6–1000 pg/ml), the spent culture medium from each experiment was diluted in fresh medium. The final concentrations were corrected accordingly. Serial dilutions of spent culture medium were linear with the human inhibin B standard. The interassay CV was less than 10%. Each experiment was repeated three times, and each experiment included three parallel wells, which were stimulated identically. Fresh cell culture medium did not contain any inhibin B immunoreactivity. The basal inhibin B levels in medium of cells cultured for 72 h varied between 200–800 pg/ml. Secreted inhibin B levels were normalized to total cellular protein contents. The cells were lysed in 250 ml 0.1 M NaOH at room temperature for 1 h, whereafter the suspension was neutralized with 250 ml 0.1 M HCl, and the protein concentrations were analyzed by a protein measurement kit based on the Bradford method (protein assay, Bio-Rad Laboratories, Inc., Hercules, CA).
Analysis of RNA and protein data
Statistical significance for single comparisons was analyzed by t test. For multiple comparisons, the data were first analyzed by one-way ANOVA, and statistical significance was determined by Scheffé’s multiple comparison test. The results of inhibin ßB-subunit mRNA measurements are represented as the mean ± SEM of values from triplicate cultures expressed in arbitrary densitometric units and adjusted to a value of 1.0 for the mean of the first control culture. The results of the protein experiments are presented relative to the mean of the basal inhibin B levels.
Results
Expression of BMP receptor and Smad signal transductor mRNAs in hGL cells
We first determined whether BMP receptor type IA (BMP-RIA; also called ALK-3), BMP-RII, and Smad1–6 are expressed in hGL cells. Northern blot analysis showed that BMP-RIA and BMP-RII as well as the common partner Smad4 mRNAs were present in freshly isolated hGL cells and in cells cultured for 6 d in in vitro (Fig. 1
, B–D). The mRNAs for BMP-activated R-Smads, Smad1 and Smad5, as well as activin/TGFß-activated Smad2 and Smad3 were also expressed in hGL cells (Fig. 1, E–H), indicating that the signaling components for both Smad pathways are found in these cells. A 3.1-kb Smad6 transcript was detected in hGL cells, and BMP-2 (50 ng/ml) clearly stimulated its expression in 48-h culture experiments (Fig. 2B).
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We determined whether a number of recombinant BMPs representing different BMP subgroups were able to regulate inhibin subunit mRNA expression in hGL cells. Recombinant BMP-2 (50–100 ng/ml) induced expression of the inhibin ßB-subunit transcripts (Fig. 2A
), but not those of the -subunits (Fig. 2A) or ßA-subunits (data not shown). On the other hand, BMP-3 and its close homolog, BMP-3b/GDF-10, were not able to stimulate the levels of mRNAs of any of the three inhibin subunits. BMP-6 stimulated inhibin ßB-subunit mRNAs, but to a somewhat lesser extent than BMP-2 (Fig. 2A). Also, consistent with our previous findings (8, 9), hCG stimulated inhibin -subunit mRNAs, but did not affect basal inhibin ßB-subunit transcript levels (Fig. 2A).
Northern and dot-blot experiments shown in Fig. 3, A–D, indicate that BMP-2 induced consistently a concentration-dependent stimulatory effect on inhibin ßB-subunit mRNA levels in 48-h cultures, with statistically significant stimulation obtained with 10–100 ng/ml BMP-2 (P < 0.05, by Scheffé’s test). In time-course experiments, the levels of inhibin ßB-subunit mRNAs increased significantly between 2 and 8 h after stimulation by BMP-2, rising gradually thereafter to a maximum level at 72–96 h (Fig. 4A). The effect of the protein synthesis inhibitor CHX on basal and BMP-2-stimulated inhibin ßB-subunit mRNA levels is shown in Fig. 4B. CHX treatment for 24 h markedly blocked BMP-2-stimulated inhibin ßB-subunit mRNA levels compared with control values.
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Figure 5 shows that BMP-2-induced ßB-subunit mRNA levels were suppressed by hCG in a 48-h culture (P < 0.05, by t test). Follistatin (250 ng/ml) itself had no marked effect on ßB-subunit mRNA levels, and it blocked the effect of activin A in hGL cells (Fig. 6, A and B) as previously reported (9). The activin-binding protein, follistatin, antagonizes the biological effects of activins, and recent reports indicated that BMP subfamily members (41, 42, 43) are also blocked by follistatin in different biological models. Follistatin blocked the effect of activin A in hGL cells; however, BMP-2-stimulated inhibin ßB-subunit mRNA levels were not affected by even a 7.3-fold molar excess of follistatin (Fig. 6, A and B).
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BMP-2 strongly stimulated the synthesis and release of dimeric inhibin B into culture medium in a concentration-dependent manner in a 72-h treatment (Fig. 7). Although the maximal stimulation levels differed in distinct experiments between 6- to 28-fold above basal levels, the stimulatory trend and concentration dependence were similar in these experiments. The lowest BMP-2 concentration capable of inducing significantly increased dimeric inhibin B secretion compared with controls was 15 ng/ml (P < 0.05; Fig. 7). In time-course experiments, a significant increase in dimeric inhibin B protein levels was first detected at 24 h of culture (data not shown).
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The synthesis and secretion of inhibin and activin dimers in the human ovary are dependent on the regulation of the three known inhibin/activin subunits that are controlled not only by endocrine hormones, but also by local factors. This study identifies the BMP signaling pathway as a possible regulatory system controlling inhibin production in the ovary. We provide evidence that human ovarian granulosa cells express mRNAs for BMP receptors and BMP-activated Smad signaling proteins. Furthermore, among the recombinant BMP proteins tested in this study, BMP-2 was found to most potently stimulate inhibin ßB-subunit mRNA levels and dimeric inhibin B production in human hGL cells.
This study together with our previous work (9, 10) indicate that hGL cells express the Ser/Thr kinase receptors and Smad signaling proteins needed for BMP, activin, and TGFß signaling. BMP-RIA (ALK3) and BMP-RII transcripts were here shown to be abundantly expressed in freshly isolated hGL cells, and the transcripts were also detectable in primary in vitro cultures. BMP-RIA is a well documented receptor for the closely structurally related BMP-2 and BMP-4 (33, 34, 44, 45). BMP-6 and BMP-7, which belong to the BMP5–8 subfamily, do not bind well to BMP-RIA, but they use ActRI (ALK2) or BMP-RIB (ALK6) for their signaling (33, 46). On the other hand, BMP-R II binds BMP-2, -4, -6, and -7 with the above-mentioned distinct type I receptors (34, 47, 48). The receptors for BMP-3 and BMP-3b/GDF-10, which form yet another BMP subgroup, have not been characterized. We found that BMP-2 had more profound effects on inhibin ßB-subunit mRNA expression than BMP-6, whereas BMP-3 and BMP-3b were without effect. Our results indicate that inhibin ßB-subunit mRNAs are differentially regulated by distinct representatives of various BMP subgroups. In addition to our present data on human granulosa cells other laboratories have recently shown the presence of BMP-RIA, BMP-IB, and BMP-RII in rodent, human, and ruminant ovaries (24, 34, 47, 48, 49, 50, 51), supporting an important role for the BMP signaling system in the ovaries of various mammalian species. In this study we demonstrate for the first time that the mRNAs for most of the known Smad signaling proteins are expressed in human granulosa cells. We further observed that BMP-2 stimulates in hGL cells the expression of Smad6, a BMP signaling pathway inhibitor (52, 53, 54). The inhibitory Smad6 is up-regulated by BMPs in various cell other types, and it prevents the phosphorylation of Smad1 by the activated BMP type I receptor (55, 56). Taken together, the results indicate that hGL cells possess a BMP signaling pathway functionally similar to that of other cell types. In preliminary experiments we have obtained evidence that BMP-2 and activin activate the phosphorylation of Smad1 and Smad2 proteins, respectively, in hGL cells, suggesting that these two distinct Smad pathways are activated by these ligands in a similar way as in other tissues (unpublished observation). However, further studies are needed to dissect the relative functional roles of distinct Ser/Thr kinase receptors and Smad proteins during different follicular stages.
We studied the effects of BMP-2 on inhibin ßB-subunit mRNA expression more extensively than those of other BMPs and compared them to the effects of activin A. Concentration dependence studies indicated that BMP-2 stimulates ßB-subunit mRNA levels at similar concentrations as was shown for activin A in a previous study (9). The maximal stimulatory effects of BMP-2 were similar or even stronger than those of activin A in many experiments. Time dependence studies indicated that BMP-2 causes a sustained, protein synthesis-dependent stimulation of ßB-subunit transcripts. Interestingly, hCG on its own does not affect ßB-subunit levels, but it suppresses both BMP-2- and activin-stimulated ßB-subunit mRNA induction. It is noteworthy that the previous studies have established that ßB-subunit transcripts are strongly expressed in small antral follicles of humans and primates (6, 13, 14). It is possible during early primate folliculogenesis that the ßB-subunit mRNAs are heavily stimulated by a concerted activation of both the activin/TGFß and the BMP signaling pathways. Cultured hGL cells used in this study do not represent the stages of granulosa cell differentiation at which ßB-subunit mRNAs levels would be typically elevated. Much caution is therefore needed in attempts to draw conclusions about the physiological relevance of the in vitro regulation of ßB-subunit mRNAs in hGLs cells. However, it can be concluded that the inhibin ßB-subunit mRNA levels are clearly up-regulated by multiple TGFß family ligands employing different signaling pathways, and this opens new insights into our understanding of inhibin mRNA regulation in the human ovary.
Although circulating inhibin B dimer profiles (12) and inhibin B concentrations in human follicular fluid (57) have been determined, little is known about the regulation of inhibin B production by human granulosa cells. In a previous study inhibin B was albeit modestly affected by gonadotropins in hGL cultures (58), which is well in line with our results at the mRNA level (8, 9). In another more recent study cultured granulosa cells from human small antral follicles were found to produce about 20 times more inhibin B than inhibin A, but gonadotropins were not found to much affect inhibin B levels (15). It is likely that local ovarian factors effectively maintain inhibin B synthesis in these cells, but to date the identity of such factors has remained elusive. The present study identifies the BMP pathway as one possible control mechanism driving inhibin B production in the human ovary. We expected on the basis of ßB-subunit mRNA up-regulation that inhibin B levels would be affected by BMP-2. The inhibin -subunit mRNAs are highly expressed in hGL cells (7, 8), and the inhibin -subunit polypeptide is likely to be available for dimerization with newly synthesized ßB-subunit. The present study shows clearly that inhibin B dimer production is greatly increased in BMP-2-treated hGL cells in a concentration-dependent manner. Preliminary experiments indicate that activin and TGFß also stimulate dimeric inhibin B production in cultured hGL cells, and the effect is abolished by hCG cotreatment (our unpublished results). It is possible that activin B dimer levels would also increase after BMP-2 treatment, but this could not be determined here because no suitable activin B assay is available as yet. In the rat, activin and TGFß were recently shown to increase inhibin B production in cultures of immature granulosa cells (59, 60) or follicles (60). The present studies with hGL cells taken together with those reported previously provide evidence that inhibin B production is influenced by several TGFß family ligands in the mammalian ovary.
Our studies have suggested that the BMP signaling pathway could be involved in the regulation of inhibin production, but a number of issues need to be addressed before the physiological role of BMPs in the ovary is well understood. Especially intriguing is the role of BMP-3 in the ovary. We and others have previously shown that BMP-3 is highly expressed in the ovary, but its biological roles in this tissue are still unclear (21, 22, 23). In the present study we did not observe any effect of BMP-3 or BMP-3b on inhibin production in cultured hGLs cells. Whatever phenomenon BMP-3 might regulate in the human ovary, it is worth noting that very recent evidence suggests that in the bone BMP-3 is not acting in a similar way as BMP-2 (61), and it may even antagonize the effects of BMP-2. This would suggest that these factors are acting in a different way than BMP-2 also in the ovary. Clearly, further experimentation is needed to unveil the role of BMP-3 in mammalian ovaries, including studies determining whether BMP-3 interferes with the effect of other BMPs or TGFß family members.
Recent studies have suggested that ovarian granulosa and thecal cell function is regulated by a complex network of factors belonging to different BMP subgroups. In the rat ovary, BMP-4 and BMP-7 of thecal cell origin appear to modulate gonadotropin-stimulated steroidogenesis in granulosa cells in vitro (24). In cultured human thecal tumor cells, BMP-4 regulates androgen production, suggesting that these BMPs may play a role as direct regulators of thecal cell differentiation (48). BMP-6, which here was shown to stimulate inhibin ßB-subunit mRNAs in hGL cells, is derived from the oocyte (25) and may thus exert paracrine effects on somatic follicular cells. Thus, members of the BMP-2/4 and BMP-5–8 subfamilies seem to regulate ovarian function in several mammalian species. Yet a further level of complexity to the ovarian BMP system is provided by the recently identified oocyte-derived TGFß family members GDF-9 and GDF-9B/BMP-15. Genetic evidence from GDF-9 knockout mice (62) and mutant GDF-9B/BMP-15 sheep families Inverdale and Hanna (30) have indicated that these factors are essential for early folliculogenesis in mice and sheep, respectively. In mice, GDF-9B/BMP-15 does not seem to be as important as it is in sheep, but it is likely to be needed for optimal fertilization rates and normal cumulus cell function in later stages of folliculogenesis (63). Both of these factors appear to have direct effects on granulosa cells (31, 64), but whether they interact with known BMP or activin/TGFß receptors and activate their respective downstream signaling components is still not known. Finally, yet another granulosa cell-derived TGFß family member, anti-Müllerian hormone (AMH) or Müllerian inhibiting substance, has been recently shown to signal through the BMP signaling pathway via the ALK-2 type I receptor (65, 66) or BMP-RIB (ALK6) (67). In contrast to GDF-9 and GDF-9B/BMP-15, AMH/Müllerian inhibiting substance naturally inhibits folliculogenesis, because in mice lacking AMH more primordial follicles are recruited than in wild-type mice (68). Finally, direct evidence for a role of BMP receptors in mammalian ovarian function was recently demonstrated by the increased fertility rates of sheep with a point mutation in the BMP-RIB gene (50, 51) and by the infertility phenotype of female BMP-RIB knockout mice, which ovulate very poorly fertilizable oocytes due to defective cumulus expansion (49). In conclusion, we have shown that the inhibin system in human granulosa cells is regulated by the BMP signaling pathway, which in light of our present understanding could be activated physiologically by various BMP family members emanating from the granulosa cell itself, the thecal cells, or the oocyte. All of these alternative options need closer experimental observation before the physiological roles of these multiple BMP family members in the ovary can be clearly established.
Acknowledgments
Ms. Anita Saarinen and Ms. Ritva Javanainen are warmly thanked for their skillful technical assistance. The personnel of the Family Federation of Finland and the Felicitas IVF Clinic are also kindly acknowledged for their cooperation. We thank Drs. Vicki Rosen and Yusuru Eto for their kind gifts of recombinant BMPs and activins, respectively. Drs. Jan Rosenbaum, Peter ten Dijke, Carl-Henrik Heldin, Rik Derynck, and X. F. Wang are thanked for BMP receptor and Smad cDNAs. Drs. Johanna Aaltonen and Ken McNatty are thanked for having critically read the manuscript.
Footnotes
This work was supported by the Emil Aaltonen Foundation, the Finnish Cultural Foundation, the Jalmari and Rauha Ahokas Foundation, the Sigrid Juselius Foundation, the Finnish Cancer Societies, the Urho Känkänen Foundation, the Medical Society of Finland, the Academy of Finland, and the Helsinki University Central Hospital Funds.
Abbreviations: AMH, Anti-Müllerian hormone; BMP-3, bone morphogenetic protein-3; BMP-RIA, BMP receptor type IA; CHX, cycloheximide; CV, coefficient of variation; GDF-10, growth differentiation factor-10; hGL, human granulosa-luteal; R-Smad, receptor-activated Smad; Ser/Thr, serine/threonine.
Received August 16, 2001.
Accepted December 6, 2001.
References
- and ßA-subunit messenger ribonucleic acid levels by chorionic gonadotropin and recombinant follicle-stimulating hormone in cultured human granulosa-luteal cells. J Clin Endocrinol Metab 79:1670–1677[Abstract]
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 S. L Al-Musawi, R. T Gladwell, and P. G Knight Bone morphogenetic protein-6 enhances gonadotrophin-dependent progesterone and inhibin secretion and expression of mRNA transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells Reproduction, August 1, 2007; 134(2): 293 - 306. [Abstract] [Full Text] [PDF]
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 K. B Lee, V. Khivansara, M. M Santos, P. Lamba, T. Yuen, S. C Sealfon, and D. J Bernard Bone morphogenetic protein 2 and activin A synergistically stimulate follicle-stimulating hormone {beta} subunit transcription J. Mol. Endocrinol., February 1, 2007; 38(2): 315 - 330. [Abstract] [Full Text] [PDF]
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L. Bodin, E. Di Pasquale, S. Fabre, M. Bontoux, P. Monget, L. Persani, and P. Mulsant A Novel Mutation in the Bone Morphogenetic Protein 15 Gene Causing Defective Protein Secretion Is Associated with Both Increased Ovulation Rate and Sterility in Lacaune Sheep Endocrinology, January 1, 2007; 148(1): 393 - 400. [Abstract] [Full Text] [PDF]
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P. G Knight and C. Glister TGF-{beta} superfamily members and ovarian follicle development. Reproduction, August 1, 2006; 132(2): 191 - 206. [Abstract] [Full Text] [PDF]
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C. Glister, S. L. Richards, and P. G. Knight Bone Morphogenetic Proteins (BMP) -4, -6, and -7 Potently Suppress Basal and Luteinizing Hormone-Induced Androgen Production by Bovine Theca Interna Cells in Primary Culture: Could Ovarian Hyperandrogenic Dysfunction Be Caused by a Defect in Thecal BMP Signaling? Endocrinology, April 1, 2005; 146(4): 1883 - 1892. [Abstract] [Full Text] [PDF]
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 J.L. Juengel and K.P. McNatty The role of proteins of the transforming growth factor-{beta} superfamily in the intraovarian regulation of follicular development Hum. Reprod. Update, March 1, 2005; 11(2): 144 - 161. [Abstract] [Full Text] [PDF]
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 N. Kaivo-Oja, D. G. Mottershead, S. Mazerbourg, S. Myllymaa, S. Duprat, R. B. Gilchrist, N. P. Groome, A. J. Hsueh, and O. Ritvos Adenoviral Gene Transfer Allows Smad-Responsive Gene Promoter Analyses and Delineation of Type I Receptor Usage of Transforming Growth Factor-{beta} Family Ligands in Cultured Human Granulosa Luteal Cells J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 271 - 278. [Abstract] [Full Text] [PDF]
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 S. Shimasaki, R. K. Moore, F. Otsuka, and G. F. Erickson The Bone Morphogenetic Protein System In Mammalian Reproduction Endocr. Rev., February 1, 2004; 25(1): 72 - 101. [Abstract] [Full Text] [PDF]
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C. Glister, C F. Kemp, and P. G Knight Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin Reproduction, February 1, 2004; 127(2): 239 - 254. [Abstract] [Full Text] [PDF]
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 E. E. Nilsson and M. K. Skinner Bone Morphogenetic Protein-4 Acts as an Ovarian Follicle Survival Factor and Promotes Primordial Follicle Development Biol Reprod, October 1, 2003; 69(4): 1265 - 1272. [Abstract] [Full Text] [PDF]
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E. Wiater and W. Vale Inhibin Is an Antagonist of Bone Morphogenetic Protein Signaling J. Biol. Chem., February 28, 2003; 278(10): 7934 - 7941. [Abstract] [Full Text] [PDF]
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N. Kaivo-Oja, J. Bondestam, M. Kamarainen, J. Koskimies, U. Vitt, M. Cranfield, K. Vuojolainen, J. P. Kallio, V. M. Olkkonen, M. Hayashi, et al. Growth Differentiation Factor-9 Induces Smad2 Activation and Inhibin B Production in Cultured Human Granulosa-Luteal Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 755 - 762. [Abstract] [Full Text] [PDF]
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R. K. Moore, F. Otsuka, and S. Shimasaki Molecular Basis of Bone Morphogenetic Protein-15 Signaling in Granulosa Cells J. Biol. Chem., January 3, 2003; 278(1): 304 - 310. [Abstract] [Full Text] [PDF]
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J.-S. Roh, J. Bondestam, S. Mazerbourg, N. Kaivo-Oja, N. Groome, O. Ritvos, and A. J. W. Hsueh Growth Differentiation Factor-9 Stimulates Inhibin Production and Activates Smad2 in Cultured Rat Granulosa Cells Endocrinology, January 1, 2003; 144(1): 172 - 178. [Abstract] [Full Text] [PDF]
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 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [Abstract] [Full Text] [PDF]
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