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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?

School of Animal and Microbial Sciences, The University of Reading, Whiteknights, Reading RG6 6AJ, United Kingdom

Address all correspondence and requests for reprints to: Prof. P. G. Knight, School of Animal and Microbial Sciences, The University of Reading, Whiteknights, Reading RG6 6AJ, United Kingdom. E-mail: p.g.knight{at}reading.ac.uk.

	Abstract

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We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and -7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, -6, and -7 potently suppress both basal (P < 0.0001; respective IC₅₀ values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced (P < 0.0001; respective IC₅₀ values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3ß-hydroxy- steroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment (P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and -7, respectively, and the observation that both antagonists enhanced (P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP-4 and -7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.

	Introduction

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RECENT EVIDENCE INDICATES that bone morphogenetic proteins (BMPs) are expressed in a cell-specific manner in the mammalian ovary and have important roles in the regulation of folliculogenesis, ovulation, and corpus luteum function (1, 2). In several species, including rat and bovine, BMP-4 and -7 are expressed by theca cells (3, 4, 5), BMP-2 and -6 are expressed by granulosa cells (4, 5), and BMP-15 and -6 are expressed by oocytes (4, 5, 6, 7, 8). In common with other members of the TGF-ß superfamily, BMPs are dimeric proteins that interact with target cells by forming heterooligomeric complexes with two types of serine/threonine kinase receptor (type-I and type-II) located on the cell surface (9, 10, 11). Transphosphorylation of type-I receptor by complexed type-II receptor, leads to phosphorylation of intracellular signaling intermediaries called Smads 1, 5, or 8, which associate with co-Smad4 before translocating to the nucleus to alter gene transcription. Consistent with autocrine/paracrine BMP signaling within the ovary, theca cells, granulosa cells, oocytes, and luteal cells express a range of BMP-responsive type-I and -II receptors (3, 4, 5). These include three type-I receptors (also referred to as activin receptor-like kinases, ALK) called BMPRIA (ALK3), BMPRIB (ALK6), and ActRIA (ALK2) and three type-II receptors called BMPRII, ActRIIA, and ActRIIB.

Such evidence for a functional intraovarian BMP system is supported by the in vivo finding that sheep with naturally occurring mutations in either the BMPRIB or BMP-15 gene display aberrant ovarian follicle development (12, 13, 14). Moreover, in vitro experiments on isolated granulosa cells have revealed modulatory effects of BMPs on steroidogenesis (3, 5, 15, 16, 17), inhibin/activin secretion (5, 17), follistatin secretion (5), and cell proliferation (5, 16). For instance, with rat granulosa cells, BMP-4 and -7 enhanced FSH-induced estradiol secretion and cell proliferation while suppressing FSH-induced progesterone (P4) secretion (3, 15). With bovine granulosa cells, BMP-4, -6, and -7 enhanced both basal and IGF-stimulated secretion of estradiol, inhibin-A, activin-A, and follistatin, while suppressing P4 secretion (5).

The synthesis of androgens by thecal cells is a key aspect of follicle function because, in accordance with the two-cell, two-steroid model (18), the ability of granulosa cells in developing antral follicles to synthesize increasing amounts of estrogen requires an adequate supply of androgen substrate for P450 aromatase. Moreover, hypersecretion of thecal androgens is a characteristic of polycystic ovarian syndrome (PCOS), a widespread although poorly understood condition that compromises human fertility and has other pathophysiological consequences (19, 20). Despite this, there is a relative paucity of information on the local regulatory factors affecting theca cell function. Given the recent discovery of the intraovarian BMP system and the demonstration that theca cells express BMP receptors, the possibility that BMP ligands of thecal, granulosa, or oocyte origin exert autocrine/paracrine actions to modulate basal and gonadotropin-dependent theca cell function warrants detailed evaluation. An earlier study (21), involving a human ovarian theca-like tumor cell model, showed that BMP-4, like activin, reduced forskolin-induced androgen secretion and P450c17 expression. However, we are not aware of any studies to date investigating the actions of BMPs on primary theca cell cultures from any species.

Here we report the use of a primary bovine theca interna cell culture model to investigate the effects of BMP-4, -6, and -7 on 1) basal and LH-induced secretion of androstenedione (A4) and P4 and cell proliferation/survival, 2) accumulation of phosphorylated (p)Smad1, and 3) expression of key regulatory proteins and enzymes involved in the steroidogenic pathway. Having demonstrated a potent suppressive effect of each BMP on A4 secretion, P450c17 expression and pSmad1 accumulation, we 4) evaluated the ability of a range of potential BMP antagonists to neutralize these BMP actions. This approach then allowed us to test the hypothesis that endogenous BMP-4 and/or -7 produced by theca cells serve to attenuate androgen secretion in an autocrine/paracrine fashion.

	Materials and Methods

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Isolation and culture of theca interna cells
Ovaries were obtained from cattle slaughtered at random stages of the estrous cycle at an abattoir. Follicles (4–6 mm diameter) were dissected from ovaries, aspirated, and hemisected, and a plastic inoculation loop was used to dislodge the granulosa cell layer as previously described (22, 23). Follicle halves were then shaken vigorously to remove any remaining granulosa cells, and the medium was changed three times. Follicle halves were examined under a dissecting microscope, and the theca interna layer was gently peeled away from the basement membrane. Pooled theca layers were incubated with collagenase (type IV, 1 mg/ml; Sigma Ltd., Poole, UK) and trypsin inhibitor (100 µg/ml; Sigma) in a shaking water bath at 37 C. After 30 min, the cell layers were triturated with a Pasteur pipette and returned to the water bath for another 15 min. Finally, any remaining undigested material was allowed to settle, and the resulting theca-cell-rich supernatant decanted. Cells were centrifuged (800 x g for 10 min) and the pellet resuspended in PBS before being subjected to an osmotic shock treatment (22) to lyse any red blood cells present. After washing again, cells were resuspended in culture medium [McCoy’s 5A modified medium supplemented with 1% (vol/vol) antibiotic-antimycotic solution, 10 ng/ml bovine insulin, 2 mM L-glutamine, 10 mM HEPES, 5 µg/ml apotransferrin, 5 ng/ml sodium selenite, and 0.1% BSA; all purchased from Sigma). Cells were counted using a hemocytometer, and viability, assessed using trypan blue, ranged from 80–90%. Cells (75 x 10³ viable cells/50 µl) were dispensed into wells of 96-well plates or 16-well chamber slides containing preequilibrated medium and incubated at 38.5 C in 5% CO₂ and 95% air for 48 h. Conditioned media were then removed (discarded), and cells were incubated for two successive 48-h periods with/without treatments. Conditioned media samples were stored at –20 C for steroid immunoassays. At the end of culture period, viable cell number was determined by neutral red dye uptake as described elsewhere (22, 24).

Steroid measurements
Concentrations of A4 were determined by RIA (25) with a detection limit of 50 pg/ml and intra- and interassay coefficients of variation of 8 and 10%, respectively. P4 was measured by ELISA (26) with a detection limit of 20 pg/ml and intra- and interassay coefficients of variation of 7 and 11%, respectively.

Experiment 1: dose-dependent effects of LH on secretion of A4 and P4
Theca cells were cultured for 4 d with a wide range of LH dose levels [0 and 25–3200 pg/ml; NIDDK-oLH-S26, National Hormone and Pituitary Program (NHPP), Torrance, CA]. Conditioned media were collected for measurement of A4 and P4, and viable cell number was determined at the end of the culture period.

Experiment 2: effect of BMPs on steroid production and cell number under basal and LH-stimulated conditions
Theca cells were cultured for 4 d with recombinant human BMP-4, -6, and -7 (R&D Systems, Abingdon, UK) at dose levels of 0, 0.08, 0.4, 2, 10, and 50 ng/ml both in the absence and presence of LH (0, 100, and 1600 pg/ml). As above, conditioned media were collected and viable cell number determined at the end of culture.

Experiment 3: effect of BMPs on expression of mRNAs encoding LH receptor (LHR), steroidogenic acute regulatory protein (StAR), P450scc, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and P450c17.
For mRNA expression studies, theca cells were cultured with/without BMPs (2 ng/ml) at the same density as above but in 25-cm² flasks (5 x 10⁶ cells/10 ml per flask). At the end of the culture period, cells were dislodged using a cell scraper and transferred with conditioned medium to a 20-ml tube. After centrifugation (10 min at 800 x g), medium was removed for steroid assay, the cell pellet was washed with culture medium, and total RNA was extracted using 1 ml of Trizol reagent (Sigma) according to the manufacturer’s recommendations. To exclude DNA contamination, isolated RNA samples were treated with RNase-free DNase (Promega, Southampton, UK) and the Trizol reagent extraction process was repeated; integrity and purity of final RNA extracts were assessed by agarose gel electrophoresis and spectrophotometry (A₂₆₀/A₂₈₀ ratio). First-strand cDNA was synthesized from total RNA using a PowerScript reverse transcriptase kit (Clontech, Palo Alto, CA) according to the manufacturer’s recommendations. The cDNA product was diluted 1:10 with nuclease-free water and stored at –20 C. PCR primer pairs (see Table 1) were designed to amplify partial cDNAs for bovine LHR, StAR, P450scc, 3ß-HSD, P450c17, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCRs were carried out in a total volume of 25 µl using Extensor Hi Fidelity ReddyMix PCR Master Mix (Abgene, Epsom, UK). All PCR included a denaturing step (94 C for 60 sec), annealing step (62–72 C for 30 sec; see Table 1) and extension step (68 C for 60 sec). For negative controls, cDNA template was substituted with nuclease-free water. PCR products were analyzed by agarose gel electrophoresis. Gels were stained with ethidium bromide and photographed under UV illumination, and images were subsequently analyzed using an imaging densitometer with Quantity One software (Bio-Rad, Hemel Hempstead, UK). Expression values for each mRNA were normalized to GAPDH expression.

Experiment 5: effects of BMP-4, -6, and -7 and activin-A on cellular accumulation of pSmad1 and -2
After culture with/without LH (100 pg/ml) for 4 d, cells were exposed to BMP-4, -6, or -7 or activin-A (50 ng/ml) for 4 h before fixing (30 min in 4% paraformaldehyde) and immunostaining with affinity-purified rabbit polyclonal antibodies against pSmad1 and pSmad2 (5 µg/ml; Upstate, Botolph Claydon, UK) as described previously for granulosa cells (5). Normal rabbit IgG (5 µg/ml) was used as a control. The second antibody used was Alexa 488-conjugated goat antirabbit (10 µg/ml; Molecular Probes). Images were captured and fluorescence intensities quantified as described above for experiment 4.

Experiment 6: effects of putative BMP antagonists on steroid production in the presence and absence of exogenous BMPs
Theca cells were cultured under both basal and LH-stimulated conditions as described for experiments 1 and 2. Cells were exposed to different combinations of BMP-4, -6, or -7 (at 0 and 2 ng/ml) with/without three putative BMP antagonists, recombinant mouse chordin (0 and 1.25 µg/ml; R&D Systems), recombinant gremlin (0 and 1.25 µg/ml; R&D Systems), and recombinant human follistatin (0 and 0.14 µg/ml; NHPP). The concentrations of chordin and gremlin used were selected on the basis of potency estimates provided by the supplier.

Statistical analysis
Unless stated otherwise, values are presented as arithmetic means ± SEM and P values < 0.05 were considered to be statistically significant. ANOVA of log-transformed data from four or more independent cultures were used to evaluate the effect of LH, BMPs, and BMP antagonists on A4 or P4 secretion and on viable cell number. Post hoc Fisher’s protected least significant difference tests were used for pair-wise comparisons providing ANOVA yielded a significant F ratio. Comparison of the relative fluorescence intensities of cells immunostained with P450c17 or pSmad1/2 antibodies were made by ANOVA and Fisher’s protected least significant difference test with results based on three independent cell cultures. To facilitate evaluation of semiquantitative RT-PCR data, mRNA expression values (normalized to GAPDH expression) from three independent cultures were plotted as means ± 95% confidence intervals.

	Results

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Effect of LH on steroid secretion and cell number
Treatment of theca cells with LH had a marked dose-dependent stimulatory effect on steroid secretion (Fig. 1). LH-induced A4 secretion showed a biphasic response (P < 0.0001) with a maximum 6-fold increase over basal levels seen in cells treated with 100 pg/ml LH; higher dose levels of LH gave suboptimal A4 secretory responses. In contrast to A4 secretion, the P4 secretory response (P < 0.0001) was monophasic, and the maximal response was elicited by the highest dose level of LH tested (3200 pg/ml). Treatment with LH promoted a modest although significant decrease in viable cell number (2-fold; P < 0.0001) determined at the end of the 144-h culture period.

View larger version (13K): [in this window] [in a new window]   FIG. 1. Dose-dependent effect of LH on secretion of androstenedione (A) and progesterone (B) by bovine theca interna cells in primary culture. C, Viable cell number at the end of the culture period. Values are means ± SEM (n = 8 independent cultures), and ANOVA results are indicated.

View larger version (36K): [in this window] [in a new window]   FIG. 2. Effects of BMP-4, -6, and -7 alone (A) and in the presence of 100 pg/ml LH (B) or 1600 pg/ml LH (C) on the secretion of androstenedione and progesterone and on viable cell number at the end of the culture period. Values are means ± SEM (n = 4 independent cultures), and results of two-way ANOVA are summarized.

Viable cell number was modestly (2-fold; P < 0.0001) increased by all three BMPs both in the absence and presence of LH at 100 or 1600 pg/ml.

Effect of BMP-4, -6, and -7 on the expression of mRNAs encoding LHR, StAR, P450scc, 3ß-HSD, and P450c17
As shown in Fig. 3, semiquantitative RT-PCR revealed that all three BMPs dramatically reduced steady-state levels of mRNA for P450c17 relative to GAPDH expression. The three BMPs also reduced levels of mRNA encoding P450scc, 3ß-HSD, and StAR but to a lesser degree.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Semiquantitative RT-PCR analysis of RNA isolated from bovine theca cells cultured for 4 d in the absence (control) or presence of BMP-4, -6, or -7 (each at 2 ng/ml). Bars show relative mRNA expression (normalized to GAPDH expression) for P450c17 (A), P450scc (B), 3ß-HSD (C), StAR (D), and LHR (E). Insets show representative amplicon bands for each target mRNA (upper band) and the corresponding GAPDH mRNA (lower band). Values are means ± 95% confidence intervals based on three independent cell culture experiments.

View larger version (10K): [in this window] [in a new window]   FIG. 4. Representative confocal micrographs of bovine theca cells immunostained (green) with rabbit antiserum against P450c17 after culturing in the presence and absence of BMP-4, -6, and -7. Controls were stained with normal rabbit serum in place of rabbit anti-P450c17. Nuclei are counterstained (red) with propidium iodide. The histograms show the results of quantitative assessment of relative fluorescence intensity of cells stained with anti-P450c17. Values are means ± SD (n = 3 independent cultures), and letters denote significant differences (P < 0.01). Scale bar, 10 µm.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Representative confocal micrographs showing that 4-h exposure to BMP-4, -6, and -7 selectively induces nuclear accumulation of pSmad1 in bovine theca cells whereas activin-A selectively induces accumulation of pSmad2. Controls were stained with normal rabbit IgG in place of rabbit anti-pSmad1 IgG. The histograms aligned to each row show the results of quantitative assessment of relative fluorescence. Values are means ± SD (n = 3 independent cultures), and means without a common letter are significantly different (P < 0.05). Scale bar, 10 µm.

View larger version (34K): [in this window] [in a new window]   FIG. 6. Interactions between BMP-4, -6, and -7 and three potential BMP antagonists (chordin, gremlin, and follistatin) in modulating thecal androstenedione secretion by bovine theca cells cultured under basal (A) and LH-stimulated (B) conditions. Values are means ± SEM based on data from four independent culture experiments. Within each set of bars, means without a common letter are significantly different (P < 0.05).

View larger version (15K): [in this window] [in a new window]   FIG. 7. Stimulatory effect of three potential BMP antagonists (chordin, gremlin, and follistatin) on androstenedione secretion by bovine theca cells cultured without exogenous BMPs. Values are means ± SEM based on data from four independent culture experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present serum-free culture model, the theca interna cells proliferate some 4-fold over 6 d and aggregate into small cellular clumps that attach firmly to the culture surface; they secrete readily measurable amounts of A4 and P4 under both nonstimulated basal conditions and in response to LH stimulation. Although responsiveness to LH in terms of A4 secretion was biphasic (optimal LH dose level of 100 pg/ml), P4 secretion increased monophasically and was maximal at the highest LH dose level tested (3200 pg/ml). This differential A4/P4 responsiveness to LH in vitro appears to reflect functional luteinization of the cells in response to high LH exposure, as occurs in vivo after the preovulatory LH surge. However, it should be noted that LH had a small although highly consistent dose-dependent inhibitory effect on cell proliferation/survival, whereas luteinization is characterized by enhanced cell proliferation.

Our results clearly show that the three BMPs tested (BMP-4, -6, and -7) exerted a similar effect on each aspect of theca cell function examined, although BMP-6 was significantly more potent than BMP-4 and -7 in suppressing basal and LH-induced A4 secretion. Whether these differences reflect true potency differences among the BMPs or arise from differential association with endogenous binding proteins produced by the cultured cells is unknown at this stage.

It is now well established that the interaction of BMPs with type-I and -II receptors on the cell surface leads to the activation of an intracellular signaling pathway involving Smad1, Smad5, and Smad8. In contrast, activin signaling involves activation of a Smad2/3 pathway (9, 10, 11). The present observations that exposure of bovine theca cells to BMP-4, -6, or -7 led to nuclear accumulation of pSmad1 (but not pSmad2) whereas, conversely, exposure to activin-A promoted nuclear accumulation of pSmad2 (but not pSmad1) are consistent with this and confirm that functional BMP ligand-receptor interactions were occurring. Despite this, it is not known which particular combinations of type-I and -II receptors are recruited by each BMP to elicit a biological response in terms of Smad activation, altered steroidogenesis, and cell proliferation/survival. As we reported recently (5), bovine theca cells express at least two type-I receptors (BMPRIB and ActRIA) and three type-II receptors (BMPRII, ActRIIA, and ActRIIB) that can potentially bind BMPs. Unexpectedly, we noted that pSmad2 accumulation in BMP-treated cells was significantly lower than in untreated control cells. One possible explanation for this is that the exogenous BMP is competing with an endogenous ligand (activin or perhaps TGF-ß) for binding to ActRIIA or -IIB leading to reduced activin/TGF-ß signaling via the Smad2/3 pathway.

In an effort to determine the mechanism(s) through which BMP-4, -6, and -7 inhibit androgen production and raise P4 production, semiquantitative RT-PCR was used to compare steady-state levels of mRNA expression for key regulatory proteins and enzymes involved in the steroidogenic pathway including LHR, StAR, P450scc, 3ß-HSD, and P450c17. The observation that all three BMPs promoted a profound suppression in P450c17 mRNA expression, coupled with our immunocytochemical finding of a parallel BMP-induced fall in cellular content of P450c17 protein, identifies this enzymatic step in the steroidogenic pathway as a key target of BMP action. Down-regulation of P450c17 expression by BMPs would block the conversion of C21 steroids to C19 steroids, an action consistent with the inverse changes in A4 and P4 secretion observed here. A previous study using a human ovarian theca-like tumor cell culture model also revealed a suppressive effect of BMP-4 on P450c17 expression and androgen secretion (21). Likewise, activin, another TGF-ß superfamily member, was shown to inhibit P450c17 expression and androgen production in this model, whereas inhibin enhanced P450c17 expression and androgen production (27). Similar opposing effects of activin and inhibin on thecal androgen production have been reported for primary cultures of rat (28), human (29), and bovine (25) thecal cells and have been confirmed using the present bovine theca interna culture model (data not shown).

Semiquantitative RT-PCR analysis also indicated that expression of StAR, P450scc, and 3ß-HSD were also reduced after BMP treatment but to a lesser extent than P450c17 expression. Thus, it appears likely that activation of the BMP signaling pathway(s) can affect thecal steroidogenesis at multiple levels. Indeed, BMP-6 and -7 have been shown to inhibit expression of StAR by cultured rat granulosa cells (15, 16), whereas BMP-7 has been shown to inhibit P450scc expression (16). Despite this, the finding that basal P4 secretion increased in response to BMP treatment suggests that, under the present culture conditions, these other potential inhibitory actions are not rate limiting with respect to thecal synthesis of C21 steroids, at least in the absence of LH stimulation. Thus, the BMP-induced rise in progesterone can be accounted for by a reduced rate of conversion to androgen that more than compensates for any decrease in the rate of progesterone synthesis arising from reduced StAR expression. It is also possible that steady-state levels of StAR mRNA may not reflect the amount of functionally active StAR protein that is known to be regulated at the posttranslational level (30, 31).

To further substantiate our findings, we evaluated the abilities of three potential BMP antagonists, chordin (32), gremlin (33), and follistatin (5, 34, 35), to reverse the suppression of androgen secretion induced by BMP-4, -6, and -7 under basal and LH-induced conditions. It is well established that follistatin is expressed in the ovary, principally by granulosa cells (1, 2). More recently, it has been reported that granulosa cells also express gremlin (36) and a related BMP binding protein called protein related to DAN and cerebrus (PRDC) (37). The present demonstration that chordin and gremlin effectively reversed the suppressive effects of BMP-7 and BMP-4, respectively, but did not influence the response to BMP-6 offers further evidence that the actions of these exogenous treatment preparations on androgen production in our culture model are indeed specific. The apparently selective ability of chordin and gremlin to reverse the actions of exogenous BMP-7 and BMP-4, respectively, was unexpected because both are considered antagonists of these two BMPs. Differential neutralizing ability could simply reflect the heterologous nature of the preparations used; the chordin and gremlin preparations were recombinant mouse proteins, whereas the BMP preparations were recombinant human proteins. However, using recombinant human gremlin Sudo et al. (37) also observed a selective reversal of BMP-4 action, but not BMP-6 or BMP-7 action in a luciferase-reporter gene bioassay.

Unexpectedly, follistatin did not reverse the suppressive effects of either BMP-4, 6, or -7 on androgen secretion despite being added at an approximately 60-fold molar excess. The explanation for this is unknown at present because in a previous study (5) we showed that a 25-fold molar excess of follistatin completely reversed the effect of BMP-4 and partially reversed the effect of BMP-6 on pSmad1 accumulation in bovine granulosa cells. The present observation that follistatin actually enhanced rather than antagonized the suppressive effect of BMP-4 and -7 on LH-induced androgen secretion is notable given our previous finding that follistatin enhanced the effect of BMP-7 on pSmad1 accumulation in granulosa cells (5). Similarly, Amthor et al. (35) presented evidence that follistatin may facilitate rather than inhibit the presentation of BMP-7 to BMP receptors on embryonic myoblasts. These paradoxical findings are most likely attributable to the reversible, relatively low-affinity binding between follistatin and BMPs (1, 5, 35) and the association of follistatin to cell surface proteoglycans potentially forming a BMP reservoir at the cell surface (38). A similar mechanism might explain the seemingly paradoxical observation that chordin enhanced, rather than reversed, the suppressive effect of BMP-4 on LH-induced androgen secretion. In this regard, chordin has recently been shown to associate with cell surface proteoglycans (39), and thus, like follistatin, it could potentially act as a BMP reservoir near the cell surface. Subsequent dissociation of sequestered BMP, perhaps aided by the action of specific metalloproteinase(s) on chordin (40, 41) could result in enhanced interaction of BMP with its cognate receptors. To resolve such issues, more information is needed on the intrafollicular expression patterns of the various extracellular binding proteins implicated in the regulation of BMPs and other TGF-ß superfamily members.

Because BMP-4 and -7 are expressed by theca cells (1, 2), we hypothesized that these BMPs might function in an autoregulatory capacity to modulate theca cell function. In direct support of this, when chordin, gremlin, or follistatin were added to theca cells cultured in the absence of exogenous BMPs, all three putative antagonists promoted a significant increase in basal androgen secretion; gremlin also promoted a significant increase in LH-induced androgen secretion. The capacities of the heterologous chordin (mouse), gremlin (mouse), and follistatin (human) preparations used in this study to bind and block the actions of endogenous bovine BMPs are unknown at this stage. Although they were added to cells at high concentrations, it is quite possible that other more effective antagonists would promote even greater increases in androgen output. Nonetheless, these observations are consistent with an autocrine/paracrine role of endogenous thecal-derived BMP-4 and/or -7 in suppressing androgen production, particularly under basal conditions.

PCOS is a major cause of infertility affecting approximately 5% of women. Despite intensive research, its etiology is poorly understood (19, 20). PCOS is associated with a variety of symptoms including menstrual irregularities, hirsutism, infertility, insulin resistance, and obesity. Ovarian hyperandrogenism is a pivotal feature and reflects increased thecal androgen production and enhanced expression of P450c17 and 3ß-HSD (19, 42). In addition, follicle development arrests at the small/mid-antral stage, and granulosa cells have the characteristics of prematurely luteinized cells (20, 43). We suggest that these changes in thecal and granulosa function associated with PCOS could be caused, at least in part, by a defect in an intraovarian BMP signaling system(s). Several lines of evidence support this hypothesis: 1) BMPs act on theca cells to suppress basal and LH/forskolin-induced androgen production and P450c17 expression (this study and Ref. 21); 2) BMP antagonists can promote thecal androgen production (this study); 3) BMPs enhance granulosa cell proliferation/survival and have an antiluteinization effect on these cells (3, 5); and 4) treatment of human granulosa-lutein cells with BMPs up-regulates production of inhibin-B, a marker of healthy growing follicles but not preovulatory or luteinized follicles (44). Thus, the notion of a functional deficit in BMP secretion and/or action within ovarian follicles would be compatible with both overproduction of thecal androgens and arrested follicle development caused by diminished proliferation and/or premature luteinization of granulosa cells. In addition, our evidence that BMPs reduce expression of other enzymes and proteins involved in steroidogenesis (P450scc, 3ß-HSD, and StAR) in addition to P450c17 is intriguing given the finding of multiple alterations in the thecal steroidogenic pathway in PCOS, including increased expression of P450scc, 3ß-HSD, and P450c17 (45).

In summary, the present findings extend our earlier observations (5) relating to the expression of BMP ligands and receptors in bovine ovarian cell types and indicate that an intrafollicular BMP signaling pathway may contribute to the negative regulation of thecal androgen production by down-regulating expression of P450c17, the key enzyme in the conversion of C21 steroids to C19 steroids. BMP-6 of granulosa and/or oocyte origin and BMP-4 and -7 of thecal origin are implicated in this regulatory process. The possibility that a defect in intrafollicular BMP signaling could be a contributory factor in the etiology of ovarian hyperandrogenism and PCOS warrants further investigation. Such a defect could arise from aberrant expression of BMP ligands, BMP binding proteins, BMP receptors, or components of the intracellular signaling pathway. Additional information regarding both local intrafollicular BMP concentrations and cell-specific pattern of expression of BMPs, their receptors, and extracellular binding proteins throughout the different stages of follicle development is required to fully evaluate the physiological relevance of these observations.

	Acknowledgments

 
We are grateful to Dr. A. F. Parlow (NHPP, Torrance, CA) for providing ovine LH and recombinant human follistatin and Dr. S. Kominami (Hiroshima University, Japan) for providing the P450c17 antibody.

	Footnotes

 
This work was supported by the Biotechnology and Biological Sciences Research Council of Great Britain.

First Published Online December 29, 2004

Abbreviations: A4, Androstenedione; ALK, activin receptor-like kinase; BMP, bone morphogenetic protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSD, hydroxysteroid dehydrogenase; LHR, LH receptor; p, phosphorylated; P4, progesterone; PCOS, polycystic ovary syndrome; StAR, steroidogenic acute regulatory protein.

Received October 5, 2004.

Accepted for publication December 21, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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