A covalently dimerized recombinant human bone morphogenetic protein-15 variant identifies bone morphogenetic protein receptor type 1B as a key cell surface receptor on ovarian granulosa cells.

Genetic studies have identified bone morphogenetic protein-15 (BMP15) as an essential regulator of female fertility in humans and in sheep. Oocyte-derived BMP15 is a noncovalently linked dimeric growth factor mediating its effects to ovarian somatic cells in a paracrine manner. Although receptor ectodomains capable of binding BMP15 have previously been reported, no cell surface receptor complex involved in BMP15 signaling has previously been characterized. Here we have expressed and purified recombinant human BMP15 noncovalent and covalent dimer variants. The biological effects of these BMP15 variants were assessed in cultured human granulosa-luteal cells or COV434 granulosa cell tumor cells using BMP-responsive transcriptional reporter assays and an inhibin B ELISA. Biochemical characterization of ligand-receptor interactions was performed with affinity-labeling experiments using [(125)I]iodinated BMP15 variants. Both ligand variants were shown to form homodimers and to stimulate Smad1/5/8 signaling and inhibin B production in human granulosa cells in a similar manner. [(125)I]Iodination of both ligands was achieved, but only the covalent dimer variant retained receptor binding capacity. The [(125)I]BMP15(S356C) variant bound preferentially to endogenous BMP receptor 1B (BMPR1B) and BMPR2 receptors on COV434 cells. Binding experiments in COS cells with overexpression of these receptors confirmed that the [(125)I]BMP15(S356C) variant binds to BMPR1B and BMPR2 forming the BMP15 signaling complex. The results provide the first direct evidence in any species on the identification of specific cell surface receptors for a member of the GDF9/BMP15 subfamily of oocyte growth factors. The fact that BMP15 uses preferentially BMPR1B as its type I receptor suggests an important role for the BMPR1B receptor in human female fertility. The result is well in line with the demonstration of ovarian failure in a recently reported human subject with a homozygous BMPR1B loss-of-function mutant.

The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: characterization of the purified untagged processed mature region.

Oocyte-derived bone morphogenetic protein-15 (BMP15) is critical for the regulation of mammalian fertility. Previously we have found that a C-terminal His(6)-tag destroys the bioactivity of growth differentiation-9 (GDF9, a homolog of BMP15). In this study we found that recombinant human BMP15 is produced by HEK-293T cells in an active form, but the bioactivity is lost by C-terminal modification, specifically, fusion to a Flag tag. After purification the mature BMP15 wt is active in transcriptional reporter assays specific for Smad1/5/8 in human granulosa-luteal (hGL) and COV434 granulosa tumor cells, whereas BMP15 with a carboxy-terminal Flag tag remains inactive. Using these same cell models we found that treatment with purified mature BMP15 wt causes a rapid phosphorylation of Smad1. The purified BMP15 wt is a potent stimulator of rat granulosa cell DNA synthesis, which could be antagonized by the BMPRII ectodomain-Fc fusion molecule, whereas the BMP15C-Flag was completely inactive. Further, the BMP15 wt form is a potent stimulator of inhibin B production in hGL cells. We found that the purified BMP15 wt consists of P16 and -17, both of which are post-translationally modified forms. This is the first characterization of a purified untagged human BMP15 mature region, which is stable and highly bioactive in human and rodent granulosa cells and as such is of importance for studies on human fertility.

Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice.

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are among the key regulators transmitting the signaling between the oocyte and the surrounding granulosa cells. Previously, it has been shown that a recombinant BMP type II receptor ectodomain-Fc fusion protein (BMPR2ecd-Fc) is able to inhibit the actions of GDF9 and BMP15 in vitro. Here, we have produced bioactive BMPR2ecd-Fc, which was injected i.p. into neonatal mice. Early folliculogenesis was first studied by injecting mice five times with various doses of BMPR2ecd-Fc during the postnatal days 4-12. Folliculogenesis was affected dose dependently, as evidenced by a decreased mitogenesis of granulosa cells of the growing follicles. Furthermore, we also noticed a decrease in the number of secondary and tertiary follicles as well as an increase in the oocyte size. Electron microscopic analysis revealed that the ultrastructure of the granulosa cells of the primary follicles was not affected by the BMPR2ecd-Fc treatment. A second study was conducted to investigate whether a longer treatment with 12 injections during postnatal days 4-28 would inhibit folliculogenesis. Similar effects were observed in the two studies on the early follicular developmental stages. However, in the long-term study, later stages of folliculogenesis were not blocked but rather increased numbers of antral follicles, preovulatory follicles, and corpora lutea were found. We conclude that BMPR2ecd-Fc is a potent modulator of ovarian folliculogenesis in vivo, and thus, is a valuable tool for studying the physiology and downstream effects of oocyte-derived growth factors in vivo.

Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells.

Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-beta (TGFbeta) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BRE-luciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.

Molecular basis of oocyte-paracrine signalling that promotes granulosa cell proliferation.

Oocytes regulate follicle growth by secreting paracrine growth factors that act on neighbouring granulosa cells (GCs). Those factors identified to date are mainly members of the transforming growth factor-beta (TGFbeta) superfamily, but little is known about which specific receptor/signalling system(s) they employ. This study was conducted to determine the requisite pathways utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded mouse oocytes are co-cultured with mural GCs. Oocytes, growth differentiation factor-9 (GDF9), TGFbeta1 and activin-A all promoted GC DNA synthesis, but bone-morphogenetic protein 6 (BMP6) did not. Subsequently, we tested the capacity of various TGFbeta superfamily receptor ectodomains (ECD) to neutralise oocyte- or specific growth factor-stimulated GC proliferation. The BMP type-II receptor (BMPR-II) ECD antagonised oocyte and GDF9 bioactivity dose-dependently, but had no or minimal effect on TGFbeta1 and activin-A bioactivity, demonstrating its specificity. The TGFbetaR-II, activinR-IIA and activinR-IIB ECDs all failed to neutralise oocyte- or GDF9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective native ligands. An activin receptor-like kinase (ALK) 4/5/7 inhibitor, SB431542, also antagonised both oocyte and GDF9 bioactivity in a dose-dependent manner. Consistent with these findings, oocytes, GDF9 and TGFbeta1 all activated SMAD2/3 reporter constructs in transfected GC, and led to phosphorylation of SMAD2 proteins in treated cells. Surprisingly, oocytes did not activate the SMAD1/5/8 pathway in transfected GCs although exogenous BMP6 did. This study indicates that oocyte paracrine factors primarily utilise a similar signalling pathway first identified for GDF9 that employs an unusual combination of TGFbeta superfamily receptors, the BMPR-II and a SMAD2/3 stimulatory ALK (4, 5 or 7), for transmitting their mitogenic actions in GC. This cell-signalling pathway may also have relevance in the hypothalamic-pituitary axis and in germ-somatic cell interactions in the testis.

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function.

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular growth and function. The effects of murine (m) and ovine (o) GDF9 as well as oBMP15, alone or together, on 3H-thymidine uptake and progesterone and inhibin production by granulosa cells from rats were determined. Murine GDF9 stimulated thymidine incorporation by granulosa cells whereas oGDF9 and oBMP15 alone had no effect. However, oBMP15 given together with mGDF9 or oGDF9 was very potent in stimulating 3H-thymidine incorporation by granulosa cells with a greater than 3-fold stimulation compared with any growth factor alone. The synergistic effect of oBMP15 and oGDF9 was almost completely blocked by antibodies generated against these growth factors when administered either alone or in combination. While neither GDF9 (murine or ovine) nor oBMP15 were able to modulate FSH-stimulated progesterone production on their own, FSH-stimulated progesterone production by granulosa cells was potently inhibited when BMP15 and GDF9 were administered together. Immunoreactive alpha-inhibin levels increased more than 15-fold from granulosa cells when BMP15 and GDF9 were given together whereas consistent stimulatory effects of either growth factor alone were not observed. The effects of GDF9 and BMP15, when added together, were different than those observed for the growth factors alone. Therefore, we hypothesize that within the ovary, these oocyte-secreted growth factors co-operate to regulate proliferation and gonadotropin-induced differentiation of granulosa cells in mammals.

Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants.

The oocyte-secreted polypeptide growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, also known as GDF9B) have both been shown to be essential for ovarian follicular development and ovulation rate. In addition, it is known from both in vivo and in vitro studies that these factors co-operate in some manner. To date, most studies examining the in vitro effects of these growth factors have used the rodent model. However, the evidence suggests that these growth factors have somewhat different roles between rodents and ruminants. Therefore, the objectives of these studies were to examine the effects of GDF9 and BMP15, alone and together, on the functions of ovine and bovine granulosa cells under in vitro conditions. Ovine (o)BMP15 given together with murine (m)GDF9 or oGDF9 was more potent in stimulating (3)H-thymidine incorporation by ovine granulosa cells compared with each growth factor alone. For bovine granulosa cells, there appeared to be little or no co-operativity between oBMP15 and oGDF9 as oBMP15 alone was as potent as any combination of the two growth factors in stimulating (3)H-thymidine uptake. The species of origin of GDF9 affected the progesterone response in ovine granulosa cells with mGDF9 stimulating and oGDF9 inhibiting progesterone production. Ovine BMP15 alone had no effect on progesterone production by ovine granulosa cells and these growth factors did not appear to co-operate. FSH-stimulated progesterone production by bovine granulosa cells was most potently inhibited when oBMP15 and murine or ovine GDF9 were administered together. As was observed for progesterone, the species of origin of GDF9 affected inhibin production by ovine granulosa cells where mGDF9 inhibited while oGDF9 stimulated production. Murine GDF9 also inhibited inhibin production from bovine granulosa cells. For both ovine and bovine granulosa cells, BMP15 alone had no effect on inhibin production and there did not appear to be any co-operation between GDF9 and BMP15. These results indicate that the effects of BMP15 and GDF9 varied with respect to the species of origin of the growth factor. Moreover, the effects of GDF9 and BMP15 together were often co-operative and not always the same as those observed for these growth factors alone.

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.

In the human ovary, cell growth and differentiation are regulated by members of the TGF-beta superfamily, including growth differentiation factor-9 (GDF9), TGF-beta, and activin. TGF-beta and activin are known to signal via Smad3 activation, and we have recently shown the involvement of Smad3 in cellular responses to GDF9. Recent studies with Smad3-deficient mice have also indicated a key role for this signaling mediator in ovarian folliculogenesis. We now demonstrate the use of a Smad3 reporter (CAGA-luciferase) adenovirus in primary cultures of human granulosa-luteal (hGL) cells to detect GDF9, TGF-beta, and activin responses. In rodent granulosa cells, TGF-beta and GDF9 signal through the TGF-beta type I receptor or activin receptor-like kinase 5 (Alk5), whereas the effect of activin is mediated though the activin type IB receptor, also known as Alk4. We now show that the GDF9 response in hGL cells is markedly potentiated upon overexpression of Alk5 by adenoviral gene transduction, as measured by the CAGA-luciferase reporter activity. A similar response to Alk5 overexpression was observed for TGF-beta, but not for activin. Adenoviral overexpression of the activin type IB receptor Alk4 in hGL cells specifically potentiated activin signaling, but not GDF9 or TGF-beta signaling. Alk5 overexpression in hGL cells also potentiated the GDF9 response when inhibin B production was used as the read-out. These results indicate that the CAGA-luciferase adenovirus can be used to study Smad3 signaling in primary cultures of human cells, and that adenoviral overexpression of wild-type receptors of the TGF-beta superfamily can be used to amplify the cellular response to ligands such as GDF9, TGF-beta, and activin. Furthermore, these studies indicate the involvement of Alk5 in GDF9 signaling in human cells and therefore, along with other recent studies, highlight how a limited number of type I and II receptors cooperate to generate specificity of action within the TGF-beta superfamily.