A functional bone morphogenetic protein system in the ovary

Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development.     

Establishment of a human nonluteinized granulosa cell line that transitions from the gonadotropin-independent to the gonadotropin-dependent status

The ovary is a complex endocrine organ responsible for steroidogenesis and folliculogenesis. Follicles consist of oocytes and two primary steroidogenic cell types, the granulosa cells, and the theca cells. Immortalized human granulosa cells are essential for researching the mechanism of steroidogenesis and folliculogenesis. We obtained granulosa cells from a 35-yr-old female and immortalized them by lentivirus-mediated transfer of several genes so as to establish a human nonluteinized granulosa cell line (HGrC1). We subsequently characterized HGrC1 and investigated its steroidogenic performance. HGrC1 expressed enzymes related to steroidogenesis, such as steroidogenic acute regulatory protein, CYP11A, aromatase, and gonadotropin receptors. Stimulation with FSH increased the mRNA levels of aromatase, which consequently induced the aromatization of androstenedione to estradiol. Activin A increased the mRNA levels of the FSH receptor, which were synergistically up-regulated with FSH stimulation. HGrC1 also expressed a series of ligands and receptors belonging to the TGF-β superfamily. A Western blot analysis showed that bone morphogenetic protein (BMP)-4, BMP-6, and BMP-7 phosphorylated small mother against decapentaplegic (Smad)1/5/8, whereas growth differentiation factor-9 phosphorylated Smad2/3. BMP-15 and anti-Müllerian hormone phosphorylated Smad1/5/8 while also weakly phosphorylating Smad2/3. These results indicate that HGrC1 may possess the characteristics of granulosa cells belonging to follicles in the early stage. HGrC1 might also be capable of displaying the growth transition from a gonadotropin-independent status to gonadotropin-dependent one.     

Regulatory role of kit ligand-c-kit interaction and oocyte factors in steroidogenesis by rat granulosa cells

Although kit ligand (KL)-c-kit interaction is known to be critical for oogenesis and folliculogenesis, its role in ovarian steroidogenesis has yet to be elucidated. We studied the impact of KL-c-kit interaction in regulation of steroidogenesis using rat oocyte/granulosa cell co-culture. In the presence of oocytes, soluble KL suppressed FSH-induced estradiol production and aromatase mRNA expression without affecting FSH-induced progesterone production. The KL effect on steroidogenesis was interrupted by an anti-c-kit neutralizing antibody, suggesting that KL-c-kit interaction is involved in suppression of estrogen by granulosa cells through oocyte c-kit action. The cAMP-PKA pathway activity was not directly involved in the estrogen regulation by KL-c-kit action. It was of note that KL treatment increased the expression levels of oocyte-derived FGF-8, GDF-9 and BMP-6, while it reduced the expression levels of oocyte-derived BMP-15 in the oocyte-granulosa cell co-culture. Given the findings that FGF-8, but not GDF-9, BMP-6 or -15, suppressed FSH-induced estrogen production by granulosa cells, oocyte-derived FGF-8 is linked to suppression of FSH-induced estrogen production through the KL-c-kit interaction. Furthermore, the suppression of FSH-induced estrogen production by KL in the co-culture was reversed by a FGF receptor kinase inhibitor and the effect of the inhibitor was enhanced in combination with extracellular-domain protein of BMPRII, which interferes with BMP-15 and GDF-9 activities. Thus, the actions of endogenous oocyte factors including FGF-8 and BMP-15/GDF-9 were involved in the KL activity that inhibited FSH-induced estradiol production. Collectively, the results indicate that KL-c-kit interaction plays a role in estrogenic regulation through oocyte-granulosa cell communication.     

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?

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 IC(50) values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced (P < 0.0001; respective IC(50) 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 3beta-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.     

Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells

In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P < 0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P < 0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P < 0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P > 0.10). Treatment with 600 ng/ml GDF-9 increased (P < 0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P < 0.05) progesterone production (by 28%), increased (P < 0.05) cell numbers (by 60%), and had no effect (P > 0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P < 0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.     

Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells.

Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.     

ERK signaling is a central regulator for BMP-4 dependent capillary sprouting

OBJECTIVE: Bone Morphogenetic Protein-4 (BMP-4) and Extracellular-Signal Regulated Kinases (ERK) play crucial roles in vascular diseases. Here, we demonstrate that BMP-4 not only signals through the classical Smad cascade but also activates ERK phosphorylation as an alternative pathway in human umbilical vein endothelial cells (HUVEC) and that Smad and ERK pathways communicate through signal crosstalk. METHODS: HUVECs were treated with BMP-4 and/or MEK inhibitors. Smad 6 and constitutively active (ca) MEK1 were overexpressed. Loss of function of Smad 4 and Smad 6 was achieved by specific siRNA transfection. Cell lysates were analyzed by western blotting for Smad and ERK phosphorylation. HUVEC spheroids were generated for angiogenesis quantification. RESULTS: Treatment with BMP-4 results in a dose- and time-dependent activation of the MEK-ERK 1/2 pathway in addition to activation of the Smad pathway and is blocked by MEK inhibitors. Quantitative in-gel angiogenesis assays in the presence or absence of MEK inhibitors demonstrate that ERK signals are necessary for BMP-4 induced capillary sprouting. Furthermore sprouting is not blocked by inhibition of the Smad signaling pathway. Overexpression of the inhibitory Smad 6 inhibits ERK phosphorylation and ERK-induced capillary sprouting, whereas loss of function of Smad 4 has no effect. CONCLUSIONS: We demonstrate that ERK1/2 functions as an alternative pathway in BMP-4 signaling in HUVECs. Capillary sprouting induced by BMP-4 is dependent on ERK phosphorylation. ERK is essential for efficient transduction of BMP signals and serves as a positive feedback mechanism. On the other hand, stimulation of Smad 6 inhibits ERK activation and thus results in a negative feedback loop to fine-tune BMP signaling in HUVECs.     

Low molecular weight substance from rat ovary induces steroidogenesis in cultured granulosa cells

Ovaries from immature intact rats contain an apparently low molecular weight substance which mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induced cell-shape changes followed by intensive progestin production. Like FSH action, OS-induced steroidogenesis reversibly ceased upon washing the factor from the cultured cells, and could be blocked in the presence of cycloheximide or alpha-amanitin. Although OS stimulated aromatase activity in granulosa cells, it failed to elicit LH responsiveness in the cultured cells. Androstenedione synergistically augmented OS-induced progestin production and aromatase activity. OS itself synergistically augmented FSH-induced progestin but did not have any effect on FSH-induced aromatase activity. In contrast to FSH action which is mediated via cAMP formation, OS doses which evoked extensive synthesis of progestin products failed to stimulate significant increases in intracellular cAMP accumulation. These results suggest the existence of a putative intraovarian hormone-like substance which can mimic some effects of the gonadotropins on the follicular granulosa cell differentiation and may facilitate FSH action at yet unknown stages of the follicular development.     

Engagement of activin and bone morphogenetic protein signaling pathway Smad proteins in the induction of inhibin B production in ovarian granulosa cells

In the mammalian ovary cell growth and differentiation is regulated by several members of the transforming growth factor beta (TGF beta) superfamily including activins, inhibins, growth differentiation factors and bone morphogenetic proteins (BMPs). The effects of TGF beta family members are mediated to the target cells via heteromeric complexes of type I and II serine/threonine kinase receptors which activate Smad signaling protein pathways in various cell types. We have previously shown that inhibin B, a hormonally important product from human granulosa cells, is up regulated by activin and BMPs. Here, we report the use of adenoviral gene transfer methodology to manipulate the TGF beta growth factor signaling system in primary cultures of human granulosa cells. These cells are exceedingly difficult to transfect by conventional transfection methods, but were virtually 100% infected with recombinant adenoviruses expressing green fluorescent protein (GFP). Adenoviruses expressing constitutively active forms of the seven known mammalian type I activin receptor-like kinase receptors (Ad-caALK1 through Ad-caALK7) cause activation of endogenous and adenovirally transferred Smad signaling proteins so that Ad-caALK1/2/3/6 and Ad-caALK4/5/7 induced phosphorylation of the Smad1 and Smad2 pathways, respectively. Activin A and BMP-2 activated the Smad1 and Smad2 pathways as well as inhibin B production as did all the Ad-caALKs. Furthermore, overexpression of adenoviral Smad1 and Smad2 proteins without exogenously added ligands induced inhibin B production. The inhibitory Smad7 protein suppressed BMP-2 and activin induced inhibin B production. Collectively, the present data demonstrate that adenoviral gene transfer provides an effective approach for dissecting the TGF beta signaling pathways in primary ovarian cells in vitro and more specifically indicate that the Smad1 and Smad2 pathways are involved in the regulation of inhibin B production by TGF beta family ligands in the ovary.