BMP15 regulates AMH expression via the p38 MAPK pathway in granulosa cells from goat.

Anti-Mullerian hormone (AMH), a member of the TGF-ß superfamily, is produced by granulosa cells (GCs) of preantral and small antral follicles and plays a role in regulating the recruitment of primordial follicles and the FSH-dependent development of follicles. However, the regulation of AMH expression in follicles remains poorly understood. The objectives of this study were to determine the following: 1. the association between bone morphogenetic protein 15 (BMP15) and AMH; 2. whether BMP15 can regulate the expression of AMH by inhibiting the p38 MAPK pathway; and 3. whether SRY-related HMG box 9 (SOX9), a transcription factor for AMH, is involved in the regulation of AMH expression by BMP15. In this study, an inhibitor of p38 MAPK and an siRNA specific for p38 MAPK were used to prevent the function of the p38 MAPK signaling pathway. Then, AMH mRNA expression and AMH secretion were detected in goat GCs using an RT-PCR assay and ELISA, respectively, after treatment with BMP15. The results indicated that BMP15 up-regulates the transcription of AMH and that the inhibition of p38 MAPK decreases the BMP15-induced expression of AMH and SOX9, suggesting that BMP15 up-regulates the expression of AMH via the p38 MAPK signaling pathway, and this process involves the SOX9 transcription factor.

[The effect of follicular fluid from patients with endometriosis, follicle stimulating hormone and bone morphogenetic protein 15 on the proliferation and progesterone secretion of granular cells].

Objective: The study was designed to evaluate the effect of follicular fluid from patients with endometriosis, follicle stimulating hormone (FSH), bone morphogenetic protein 15 (BMP-15) on the proliferation and progesterone secretion of human luteinized granular cells in vitro. Methods: Cumulus granulosa cells were collected from the patients who underwent in vitro fertilization and embryo transfer (IVF-ET) ovulation due to tubal or male factor infertility on the day of the retrieval. The cells in the control group were cultured with complete medium of DMEM/F-12, an extra of FSH at a dose of 12 µg/L was added in the FSH group, an extra of BMP-15 at a dose of 6 µg/L was added to the BMP-15 group, an extra of 5% of the follicular fluid from the patients with tubal or male factor infertility was added to the tubal group, an extra of 5% of the follicular fluid from the patients with endometriosis infertility was added to the endometriosis group, an extra of 5% of the follicular fluid from the patients with endometriosis infertility plus FSH at a dose of 12 µg/L were added to the endometriosis plus FSH group, and an extra of 5% of the follicular fluid from the patients with endometriosis infertility plus BMP-15 at a dose of 6 µg/L were added to the endometriosis plus BMP-15 group. Hemacytometer counting method was used to observe the growth of cells after 48 hours, and chemiluminescence method was utilized to measure the level of progesterone in culture supernatant. Results: The cell proliferation was enhanced in the FSH group, while the proliferation was inhibited in the endometriosis group and the endometriosis plus BMP-15 group, compared to the control group, both of which, were statistically significant. Compared to the control group, the progesterone levels from the culture supernatant of granular cells were significantly elevated in the FSH group, tubal group and endometriosis group. The secretion of progesterone in the endometriosis group was lower than that in the tubal group. After addition of FSH into the endometriosis group (the endometriosis plus FSH group), the secretion level of progesterone was significantly increased, compared to the control group and the endometriosis group. After adding BMP-15 into the endometriosis group (the endometriosis plus BMP-15 group), the secretion level of progesterone was increased, compared to the control group. Conclusions: FSH, but not BMP-15, was able to enhance the proliferation and progesterone secretion of granular cells. The proliferation of granular cells and secretion of progesterone were inhibited by the follicular fluid from patients with endometriosis, which was reversed by FSH. However, BMP-15 had no effect on the outcome of follicular fluid from patients with endometriosis.

The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders.

BACKGROUND: A large number of studies have contributed to understanding the general mechanisms driving ovarian folliculogenesis in humans and show a complex endocrine dialog between the central nervous system, the pituitary and the ovary, integrated by various intraovarian paracrine messages. The role of intraovarian paracrine regulation has acquired more relevance in the recent years owing to the discovery of previously unknown factors, such as the oocyte-derived bone morphogenetic protein (BMP)15. METHODS: A thorough literature search was carried out in order to summarize what has been reported so far on the role of BMP15, and the BMP15 paralog, growth and differentiation factor 9 (GDF9), in ovarian function and female fertility. Research articles published in English until March 2014 were included. RESULTS: The biological actions of BMP15 include: (i) the promotion of follicle growth and maturation starting from the primary gonadotrophin-independent phases of folliculogenesis; (ii) the regulation of follicular granulosa cell (GC) sensitivity to FSH action and the determination of ovulation quota; (iii) the prevention of GC apoptosis and (iv) the promotion of oocyte developmental competence. The existence of biologically active heterodimers with GDF9, and/or the synergistic co-operation of BMP15 and GDF9 homodimers are indeed relevant in this context. Experimental disruption of the bmp15 gene in mice resulted in a mild fertility defect limited to females, whereas natural missense mutations in ewes cause variable phenotypes (ranging from hyperprolificacy to complete sterility) depending on a fine gene dosage mechanism also involving GDF9. Strong evidence supports the concept that such a mechanism plays an important role in the regulation of ovulation rate across mammalian and non-mammalian species. Following the discovery of sheep fecundity genes, several research groups have focused on alterations in human BMP15 associated with primary ovarian insufficiency (POI) or polycystic ovary syndrome. Several variants of BMP15 are significantly associated with POI supporting their pathogenic role, but the underlying biological mechanism is still under investigation and of great interest in medicine. BMP15 maps to the Xp locus involved in the determination of the ovarian defect in Turner syndrome and significantly contributes to the determination of ovarian reserve. Pioneering studies in women undergoing controlled ovarian stimulation indicate that BMP15 may represent a marker of ovarian response or oocyte quality. CONCLUSIONS: BMP15, an oocyte-derived growth and differentiation factor, is a critical regulator of folliculogenesis and GC activities. Variations in BMP15 gene dosage have a relevant influence on ovarian function and can account for several defects of female fertility. The modulation of BMP15 action may have interesting pharmacological perspectives and the analysis of BMP15 may become a useful marker in IVF procedures. Recent outcomes indicate that the close interactions of BMP15/GDF9 have a critical biological impact that should be taken into account in future studies.

Role of oocyte-derived paracrine factors in follicular development.

Mammalian oocytes secrete transforming growth factor ß (TGF-ß) superfamily proteins, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 6 (BMP6) and BMP15, and fibroblast growth factors (FGFs). These oocyte-derived paracrine factors (ODPFs) play essential roles in regulating the differentiation and function of somatic granulosa cells as well as the development of ovarian follicles. In addition to the importance of individual ODPFs, emerging evidence suggests that the interaction of ODPF signals with other intra-follicular signals, such as estrogen, is critical for folliculogenesis. In this review, we will discuss the current understanding of the role of ODPFs in follicular development with an emphasis on their interaction with estrogen signaling in regulation of the differentiation and function of granulosa cells.

[Effects of phosphatidylinositol-3 kinase/protein kinase b/bone morphogenetic protein-15 pathway on the follicular development in the mammalian ovary].

In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.

Divergence of intracellular signaling pathways and early response genes of two closely related fibroblast growth factors, FGF8 and FGF18, in bovine ovarian granulosa cells.

Fibroblast growth factors (FGFs) modulate ovarian function, including FGF8 and FGF18. These FGFs activate the same receptors, although FGF18 is unusual in that it increases apoptosis in ovarian granulosa cells whereas the 'typical' response to FGF is increased proliferation. The objective of the present study was to determine which early response genes and pathways are activated by FGF8 and FGF18 in bovine granulosa cells. FGF8 increased abundance of mRNA encoding the FGF-responsive genes SPRY1, SPRY2, SPRY4, NR4A1 and NR4A3 whereas FGF18 did not. FGF8 increased but FGF18 decreased levels of mRNA encoding the growth arrest associated protein, GADD45B. FGF8 increased ERK1/2 phosphorylation but FGF18 did not. Microarray analysis identified EGR1, FOS, FOSL1, BAMBI, XIRP1 and PLK2 as other FGF8 immediate-early response genes, and FGF18 stimulated EGR1, FOSL1, BAMBI and PLK2, but not FOS or XIRP1. This study demonstrates that FGF8 and FGF18 signal through divergent pathways in ovarian granulosa cells, despite reportedly similar receptor activation patterns.

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes.

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus-oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.

Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions.

The TGF-ß superfamily is the largest family of secreted proteins in mammals, and members of the TGF-ß family are involved in most developmental and physiological processes. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), oocyte-secreted paralogs of the TGF-ß superfamily, have been shown genetically to control ovarian physiology. Although previous studies found that GDF9 and BMP15 homodimers can modulate ovarian pathways in vitro, the functional species-specific significance of GDF9:BMP15 heterodimers remained unresolved. Therefore, we engineered and produced purified recombinant mouse and human GDF9 and BMP15 homodimers and GDF9:BMP15 heterodimers to compare their molecular characteristics and physiological functions. In mouse granulosa cell and cumulus cell expansion assays, mouse GDF9 and human BMP15 homodimers can up-regulate cumulus expansion-related genes (Ptx3, Has2, and Ptgs2) and promote cumulus expansion in vitro, whereas mouse BMP15 and human GDF9 homodimers are essentially inactive. However, we discovered that mouse GDF9:BMP15 heterodimer is ∼10- to 30-fold more biopotent than mouse GDF9 homodimer, and human GDF9:BMP15 heterodimer is ∼1,000- to 3,000-fold more bioactive than human BMP15 homodimer. We also demonstrate that the heterodimers require the kinase activities of ALK4/5/7 and BMPR2 to activate SMAD2/3 but unexpectedly need ALK6 as a coreceptor in the signaling complex in granulosa cells. Our findings that GDF9:BMP15 heterodimers are the most bioactive ligands in mice and humans compared with homodimers explain many puzzling genetic and physiological data generated during the last two decades and have important implications for improving female fertility in mammals.

Species differences in the expression and activity of bone morphogenetic protein 15.

Oocyte-derived bone morphogenetic protein 15 (BMP15) regulates ovulation rate and female fertility in a species-specific manner, being important in humans and sheep and largely superfluous in mice. To understand these species differences, we have compared the expression and activity of human, murine, and ovine BMP15. In HEK293F cells, human BMP15 is highly expressed (120 ng/ml), ovine BMP15 is poorly expressed (15 ng/ml), and murine BMP15 is undetectable. Because BMP15 synthesis is dependent upon interactions between the N-terminal prodomain and the C-terminal mature domain, we used site-directed mutagenesis to identify four prodomain residues (Glu(46), Glu(47), Leu(49), and Glu(50)) that mediate the high expression of human BMP15. Substituting these residues into the prodomains of murine and ovine BMP15 led to significant increases in growth factor expression; however, maximal expression was achieved only when the entire human prodomain was linked to the mature domains of the other species. Using these chimeric constructs, we produced and purified murine and ovine BMP15 and showed that in a COV434 granulosa cell bioassay, these molecules displayed little activity relative to human BMP15 (EC(50) 0.2nM). Sequence analysis suggested that the disparity in activity could be due to species differences at the type I receptor binding interface. Indeed, murine BMP15 activity was restored when specific residues through this region (Pro(329)/Tyr(330)) were replaced with the corresponding residues (Arg(329)/Asp(330)) from human BMP15. The identified differences in the expression and activity of BMP15 likely underlie the relative importance of this growth factor between species.

Effects of species differences on oocyte regulation of granulosa cell function.

The aims were to investigate whether oocyte-secreted growth factors from a high (i.e. rat) and low (i.e. sheep) ovulation rate species could stimulate (3)H-thymidine incorporation in granulosa cells (GC) from antral follicles from the same or across species. Denuded oocytes (DO) were co-incubated with GC with or without specific antibodies to growth differentiating factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15). Co-incubations of DO-GC from the same or across species significantly increased thymidine incorporation in GC with increasing numbers of DO. GDF9 immuno-neutralisation reduced thymidine incorporation in rat GC co-incubated with either rat or ovine DO and in ovine GC co-incubated with ovine or rat DO. BMP15 immuno-neutralisation only reduced thymidine incorporation when ovine DO were co-incubated with either ovine or rat GC. Western blotting of oocytes co-incubated with GC identified GDF9 and BMP15 proteins for sheep and GDF9 protein for rats in oocyte lysates and incubation media. With respect to rat BMP15, a promature protein was identified in the oocyte lysate but not in media. Expression levels of GDF9 relative to BMP15 mRNA in DO co-incubated with GC were highly correlated (R (2)=0.99) within both species. However, the expression ratios were markedly different for the rat and sheep (4.3 vs 1.0 respectively). We conclude that during follicular development, rat oocytes secrete little, if any, BMP15 and that GDF9 without BMP15 can stimulate proliferation of rat and ovine GC. In contrast, ovine oocytes secrete both BMP15 and GDF9, and both were found to stimulate proliferation in ovine and rat GC.

Growth differentiating factor 9 (GDF9) and bone morphogenetic protein 15 both activate development of human primordial follicles in vitro, with seemingly more beneficial effects of GDF9.

CONTEXT: The signals initiating growth of primordial follicles are unknown. Bone morphogenetic protein 15 (BMP15) and growth differentiating factor 9 (GDF9) are promising candidates. OBJECTIVE: The objective of the study was to evaluate for the first time the effects of human recombinant BMP15 and human recombinant GDF9 on the in vitro development of human primordial follicles. DESIGN AND SETTING: This was a controlled culture study performed in a major tertiary university-affiliated medical center. MATERIALS: Materials included ovarian tissue from 17 girls/women and three aborted human fetuses. INTERVENTION: There were no interventions. MAIN OUTCOME MEASURE: Histological and immunohistochemical (proliferating cell nuclear antigen, BMP15, and GDF9) studies and an endocrine assay of 17ß-estradiol were conducted. RESULTS: In the samples from girls/women, the number of developing follicles was greater with GDF9 or BMP15 alone than with no BMP15 or GDF9. Higher 17ß-estradiol secretion was noted after treatment with GDF9 than with BMP15 or with GDF9+anti-GDF9. The number of atretic follicles was greater with BMP15 than with GDF9. Proliferating cell nuclear antigen expression was greater with the higher dose of both growth factors than the lower dose. Expression of BMP15 and GDF9 was identified in samples cultured without BMP15 or GDF9. Results for the fetal follicles yielded no distinguishable pattern. CONCLUSIONS: Although both BMP15 and GDF9 promoted activation of human primordial follicles from girls/women (but not human fetuses) in a dose-dependent manner, GDF9 seems more beneficial.

A single nucleotide polymorphism in BMP15 is associated with high response to ovarian stimulation.

There is substantial variability in ovarian response to exogenous gonadotrophins in women undergoing ovarian stimulation for IVF. Genetic variation in signalling pathways of the ovary may influence ovarian stimulation outcome. One previous study showed an association between single nucleotide polymorphisms (SNP) in the gene for bone morphogenetic protein 15 (BMP15) and ovarian hyperstimulation syndrome (OHSS). This article presents a retrospective case-controlled genetic-association study designed to test the association between SNP in the BMP15 gene and two clinically important outcomes of ovarian stimulation: low and high response. Blood samples from 53 high responders, 38 low responders and 100 controls were analysed for five SNP of interest. Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated by a multivariate logistic regression model. We found an association between the BMP15 -9G allele and high response to ovarian stimulation (OR=2.7, 95% CI=1.3-5.7). This association confirms previous findings in a different population and strengthens the case for an association between this SNP and ovarian stimulation outcome.

Polymorphisms in GDF9 and BMP15 associated with fertility and ovulation rate in Moghani and Ghezel sheep in Iran.

The genetic base of fertility and ovulation rate in Moghani and Ghezel sheep in northwestern Iran and northeastern Turkey is important because of their fat-tailed meat and carpet quality wool. The genes encoding bone morphogenetic (BM) protein 15 and growth differentiation (GD) factor 9, respectively BMP15 and GDF9 have been shown to affect female productivity in domesticated sheep. Recently, numerous investigations have been performed on a variety of breeds to determine the association between mutations in these genes and fertility. Thus, in this study, we assessed such mutations in the Moghani and Ghezel breeds using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) with appropriate enzymes. Our data were similar to those of the previous studies showing that the genotypes were heterozygous for GD (G -->A) and BM (C -->T) mutations. These heterozygous genotypes resulted in higher ovulation rates, illustrating that one copy of each of the BMP15 and GDF9 mutations had equivalent effects on the ovulation rate. We demonstrate for the first time that the BM variant may not be sufficient on its own for infertility. In addition, although the previous studies have shown no notable relationship between the GD variant, known as the non-effecting mutation and sterility, we report that this mutation has an important role in the Moghani and Ghezel breeds.