Bone morphogenetic protein-9 downregulates StAR expression by inducing snail expression via SMAD1/5/8 signaling in human granulosa-lutein cells.

Ovarian steroidogenesis mediated by granulosa cells is pivotal in maintaining normal female reproductive function. The steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis. Bone morphogenetic protein-9 (BMP-9), also known as growth differentiation factor-2 (GDF-2), is a member of the transforming growth factor-beta (TGF-ß) superfamily. BMP-9 induces epithelial-mesenchymal transition (EMT) that contributes to cancer progression. However, the function of BMP-9 in the female reproductive system remains largely unknown. It has been recently shown that BMP-9 is expressed in human follicular fluid and can downregulate StAR expression in human ovarian granulosa cells. However, the underlying molecular mechanisms warrant investigation. Our results show that treatment of primary granulosa-lutein (hGL) cells with BMP-9 downregulates StAR expression. In addition, two EMT-related transcription factors, Snail and Slug, are upregulated by the treatment of BMP-9. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we show that BMP-9 upregulates Snail and Slug expression by activating SMAD1/5/8 signaling. We also examine the effects of BMP-9 on SMAD-independent signaling pathways, including ERK1/2, p38, JNK, AKT, and CREB. However, none of them is affected by the BMP-9. Moreover, we use gain- and loss-of-function approaches to reveal that only Snail, not Slug, is required for the BMP-9-induced downregulation of StAR expression in hGL cells. This study increases the understanding of the physiology function of BMP-9 in hGL cells and provides important insights into the regulation of StAR expression.

The transcription factor RUNX1 affects the maturation of porcine oocytes via the BMP15/TGF-ß signaling pathway.

Bone morphogenetic protein 15 (BMP15) is specifically expressed in oocytes in pigs at all stages from early stages to ovulation and has an important role in oocyte maturation. However, there are few reports on the molecular mechanisms by which BMP15 affects oocyte maturation. In this study, we identified the core promoter region of BMP15 using a dual luciferase activity assay and successfully predicted the DNA binding motif of the transcription factor RUNX1. The effect of BMP15 and RUNX1 on oocyte maturation was examined using the first polar body extrusion rate, a reactive oxygen species (ROS) assay and total glutathione (GSH) content at three time points of 12, 24 and 48 h of in vitro culture of porcine isolated oocytes. Subsequently, the effect of the transcription factor RUNX1 on the TGF-ß signaling pathway (BMPR1B and ALK5) was further verified using RT-qPCR and Western blotting. We found that the overexpression of BMP15 significantly increased the first polar body extrusion rate (P < 0.01) and total glutathione content of oocytes cultured in vitro for 24 h and decreased reactive oxygen levels (P < 0.01), whereas interference with BMP15 decreased the first polar body extrusion rate (P < 0.01), increased reactive oxygen levels in oocytes cultured in vitro for 24 h (P < 0.01), and decreased glutathione content (P < 0.01). The dual luciferase activity assay and online software prediction showed that RUNX1 is a potential transcription factor binding to the core promoter region (-1203/-1423 bp) of BMP15. Overexpression of RUNX1 significantly increased the expression of BMP15 and oocyte maturation rate, while inhibition of RUNX1 decreased the expression of BMP15 and the oocyte maturation rate. Moreover, the expression of BMPR1B and ALK5 in the TGF-ß signaling pathway increased significantly after overexpression of RUNX1, whereas their expression decreased after inhibition of RUNX1. Overall, our results suggest that the transcription factor RUNX1 positively regulates the expression of BMP15 and influences oocyte maturation through the TGF-ß signaling pathway. This study provides a theoretical basis for further complementing the BMP15/TGF-ß signaling pathway to regulate mammalian oocyte maturation.

Concentrations of oocyte secreted GDF9 and BMP15 decrease with MII transition during human IVM.

BACKGROUND: The suggested effects of the oocyte secreted GDF9 and BMP15 growth factors on oocyte maturation are currently based on recombinant proteins, and little is known about native GDF9 and BMP15 in humans. METHODS: Human immature cumulus-oocyte complexes (COCs) obtained in connection with ovarian tissue cryopreservation (OTC) underwent in vitro maturation (IVM). Oocyte-produced GDF9 and BMP15 were detected in COCs using immunofluorescence, and in fresh GV oocytes and in GV and MII oocytes after IVM by western blot. Concentrations of GDF9, BMP15 homodimers, and GDF9/BMP15 heterodimer in spent media after IVM were measured by ELISA. The relative expression of seven genes from the GDF9 and BMP15 signaling pathways (BMPR2, ALK5, ALK6, SMAD1, SMAD2, SMAD3, and SMAD5) was evaluated in fresh cumulus cells (before IVM) and in cumulus cells from GV and MII oocytes after IVM by RT-qPCR. RESULTS: We detected native pro-mature GDF9 and BMP15 in human oocytes with molecular weights (Mw) of 47 kDa and 43 kDa, respectively. Concentrations of GDF9 and BMP15 in spent media after IVM were detected in 99% and 64% of the samples, respectively. The GDF9/BMP15 heterodimer was detected in 76% of the samples. Overall, the concentration of GDF9 was approximately 10-times higher than BMP15. The concentrations of both GDF9 and BMP15 were significantly lower in spent medium from MII oocytes than in media from oocytes that remained at the GV stage. Concentrations of the GDF9/BMP15 heterodimer did not differ between GV and MII oocytes. Furthermore, BMPR2, SMAD3, and SMAD5 were significantly upregulated in cumulus cells from MII oocytes, indicating that both GDF9 and BMP15 signaling were active during oocyte meiotic resumption in vitro. CONCLUSION: These data suggest that the driving mechanisms for oocyte nuclear maturation may involve both GDF9 and BMP15 homodimers, while the role of the GDF9/BMP15 heterodimer is questionable.

Oocyte-derived growth factors promote development of antrum-like structures by porcine cumulus granulosa cells in vitro.

Oocytes communicate with the surrounding somatic cells during follicular development. We examined the effects of two oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the development of porcine oocyte-cumulus cell complexes (OCCs) in vitro. We collected OCCs from early antral follicles (1.2-1.5 mm) and prepared oocytectomized cumulus cell complexes (OXCs), which were then cultured in a growth medium supplemented with 0-100 ng/ml GDF9 and/or BMP15 for 7 days. In the medium without GDF9 or BMP15, OCCs developed during culture, and approximately 30% of them formed antrum-like structures. GDF9 promoted OCC development and structure formation in a dose-dependent manner. However, OXCs did not form antrum-like structures without growth factors. GDF9 promoted the development of OXCs, and 50 and 100 ng/ml GDF9 promoted the formation of the structures by 8% and 26%, respectively; however, BMP15 did not promote the formation of these structures. OXCs were then cultured with 100 ng/ml GDF9 and various concentrations of BMP15 to investigate their cooperative effects on the formation of antrum-like structures. BMP15 promoted the formation of antrum-like structures in a dose-dependent manner. In conclusion, GDF9 derived from oocytes is probably important for the formation of antrum-like structures in porcine OXCs, and BMP15 cooperates with GDF9 to form these structures.

L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9.

This study was designed to investigate the effect of different concentrations of L-cysteine supplementation into the maturation medium on the oocyte nuclear maturation, cumulus cell expansion, ultrastructure of the oocytes and the expression of oocyte-derived growth factors BMP-15, GDF-9 and CB-1 genes. Cumulus oocyte complexes (COCs) were collected from cow's ovaries obtained from abattoir and incubated at 38.5°C in maturation media supplemented with 0, 0.6, 0.8 or 1 mM L-cysteine in 5% CO2 under humidified air for 24 hr. We found that a significantly higher percentage of oocytes progressed to metaphase II stage in the in vitro maturation (IVM) medium supplemented with L-cysteine, particularly 0.8 mM group, compared with untreated control oocytes. Additionally, L-cysteine treatment significantly increased the number of expanded COCs and the degree of expansion of individual COCs. Results of RT-qPCR showed significant increase in expression levels of BMP-15 and GDF-9 in L-cysteine-treated groups compared with control one. Electron microgram showed improvement of cytoplasmic maturation regarding ultrastructure of the oocytes and oocyte-cumulus cell gap junction communication in all L-cysteine-treated groups especially 0.8 mM L-cysteine-treated one. In conclusion, supplementation of IVM medium with a potential anti-oxidant, L-cysteine can effectively improve in vitro oocytes cytoplasmic and nuclear maturation via activation of oocyte maturation related BMP-15 and GDF-9 genes in bovine oocytes, benefiting the extended researches about the potential applications of L-cysteine in mammalian breeding technologies.

Roles of NR5A1 and NR5A2 in the regulation of steroidogenesis by Clock gene and bone morphogenetic proteins by human granulosa cells.

The functional role of the transcription factors NR5A1 and NR5A2 and their interaction with Clock gene and bone morphogenetic proteins (BMPs) were investigated in human granulosa KGN cells. Treatment with BMP-15 and GDF-9 suppressed forskolin (FSK)-induced steroidogenesis as shown by the mRNA expression levels of StAR and P450scc but not the mRNA expression level of P450arom. Of interest, treatment with BMP-15 and GDF-9 also suppressed FSK-induced NR5A2 mRNA expression. Treatment with BMP-15 suppressed NR5A2 mRNA and protein expression but increased Clock mRNA and protein expression levels by granulosa cells. The mRNA expression levels of NR5A1, but not those of NR5A2, were positively correlated with the levels of Clock mRNA, while the mRNA levels of Id-1, the target gene of BMP signaling, were positively correlated with those of NR5A1 but not with those of NR5A2. It was also demonstrated that the mRNA expression levels of NR5A1 were positively correlated with those of P450arom and 3ßHSD, whereas the mRNA expression level of NR5A2 was correlated with those of StAR and P450scc. Furthermore, inhibition of Clock gene expression by siRNA attenuated the expression of NR5A1, and the mRNA levels of Clock gene were significantly correlated with those of NR5A1. Collectively, the results suggested a novel mechanism by which Clock gene expression induced by BMP-15 is functionally linked to the expression of NR5A1, whereas NR5A2 expression is suppressed by BMP-15 in granulosa cells. The interaction between Clock NR5A1/NR5A2 and BMP-15 is likely to be involved in the fine-tuning of steroidogenesis by ovarian granulosa cells.

Oocyte-secreted factors strongly stimulate sFRP4 expression in human cumulus cells.

Secreted frizzled-related protein-4 (SFRP4) belongs to a family of soluble ovarian-expressed proteins that participate in female reproduction, particularly in rodents. In humans, SFRP4 is highly expressed in cumulus cells (CCs). However, the mechanisms that stimulate SFRP4 in CCs have not been examined. We hypothesise that oocyte-secreted factors such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are involved in the regulation of SFRP4. Human CCs were collected from patients undergoing fertility treatments and treated with GDF9 or BMP15 or their combination in the presence of FSH or vehicle. FSH treatment significantly decreased SFRP4 mRNA levels when compared with nontreated cells. However, SFRP4 mRNA levels were increased significantly by GDF9 plus BMP15 in a concentration-dependent manner in the presence or absence of FSH. The combination of GDF9 plus BMP15 also increased SFRP4 protein levels and decreased the activity of the ß-catenin/T cell factor-responsive promoter significantly. GDF9 plus BMP15 inhibited steroidogenic acute regulatory protein and LH/hCG receptor stimulation by FSH, while treatment with SFRP4 blocked the stimulatory effect of FSH on these genes. The evidence demonstrates that GDF9 and BMP15 act in coordination to stimulate SFRP4 expression and suggests that SFRP4 mediates the anti-luteinising effects of the oocyte in human CCs.

Bone morphogenetic protein 15 supplementation enhances cumulus expansion, nuclear maturation and progesterone production of in vitro-matured bovine cumulus-oocyte complexes.

In vitro embryo production (IVP) efficiency is reduced when compared to in vivo. The basic knowledge of bovine in vitro oocyte maturation (IVM) mechanisms provides support to improve in vitro embryo production yields. The present study assessed the effects of bone morphogenetic protein 15 (BMP15), fibroblast growth factor 16 (FGF16) and their combined action on cumulus cells (CC) expansion, oocyte and CC DNA fragmentation, oocyte nuclear maturation, energetic metabolism and progesterone production in bovine IVM. Cumulus-oocyte complexes (COC) were matured in control or supplemented media containing BMP15 (100 ng/ml), FGF16 (10 ng/ml) or BMP15 combined with FGF16; and assessed at 0 and 22 hr of IVM. BMP15 alone or its association with FGF16 enhanced cumulus expansion. BMP15 decreased DNA fragmentation in both CC and oocytes, and improved oocyte nuclear maturation rate. In addition, BMP15 increased CC progesterone production, an effect not previously reported. The present study reinforces previous data pointing to a beneficial influence of BMP15 during IVM, while providing novel evidence that the underlying mechanisms involve increased progesterone production.

Bone morphogenetic protein 15 induces differentiation of mesenchymal stem cells derived from human follicular fluid to oocyte-like cell.

Follicular fluid (FF) is essential for developing ovarian follicles. Besides the oocytes, FF has abundant undifferentiated somatic cells containing stem cell properties, which are discarded in daily medical procedures. Earlier studies have shown that FF cells could differentiate into primordial germ cells via forming embryoid bodies, which produced oocyte-like cells (OLC). This study aimed at isolating mesenchymal stem cells (MSC) from FF and evaluating the impacts of bone morphogenetic protein 15 (BMP15) on the differentiation of these cells into OLCs. Human FF-derived cells were collected from 78 women in the assisted fertilization program and cultured in human recombinant BMP15 medium for 21 days. Real-time polymerase chain reaction and immunocytochemistry staining characterized MSCs and OLCs. MSCs expressed germline stem cell (GSC) markers, such as OCT4 and Nanog. In the control group, after 15 days, OLCs were formed and expressed zona pellucida markers (ZP2 and ZP3), and reached 20-30 µm in diameter. Ten days after induction with BMP15, round cells developed, and the size of OLCs reached 115 µm. A decrease ranged from 0.04 to 4.5 in the expression of pluripotency and oocyte-specific markers observed in the cells cultured in a BMP15-supplemented medium. FF-derived MSCs have an innate potency to differentiate into OLCs, and BMP15 is effective in promoting the differentiation of these cells, which may give an in vitro model to examine germ cell development.

BMP-15 activity on in vitro development of collared peccary (Pecari tajacu Linnaeus, 1758) preantral follicles.

This study investigated the effects of BMP-15 on the in vitro development of preantral follicles of collared peccaries. Ovarian fragments were cultured for 1 or 6 days in Tissue Culture Medium 199 (TCM199+ ) supplemented with BMP-15 at rates of 0, 1, 25 or 50 ng/ml. The fragments were analysed histologically by evaluating follicular morphology, activation and growth as well as the potential for proliferation of granulosa cells. Our results show the addition of 25 ng/ml BMP-15 in the medium provided the greatest percentage of normal follicles (79.67% ± 0.69) when compared to other treatments (p < .05); however, this result is similar to 1 ng/ml BMP-15 (74.00% ± 1.90, p > .05). Moreover, 25 and 50 ng/ml of BMP-15 promoted follicular activation. BMP-15 supplements did not affect oocyte and follicular growth. All concentrations of BMP-15 increased the number of nucleolus organizer regions (NORs) after 1 day of culture when compared to fresh fragments or the control samples (p < .05). However, at the end of the experiment, the number of NORs in follicles cultured in all treatments was higher than that observed in the fresh control (sample taken prior to culturing) (p > .05). In summary, the addition of 25 ng/ml BMP-15 to the culture medium of collared peccary preantral follicles maintained a high number of morphologically healthy follicles and stimulated the activation of primordial follicles after 6 days in culture.

Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells.

CONTEXT: Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2 (IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown. OBJECTIVE: To determine the role of oocyte-secreted factors (OSFs) such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production by hGCs. DESIGN: Primary human cumulus GCs in culture. SETTING: University infertility center. PATIENTS OR OTHER PARTICIPANTS: GCs of women undergoing in vitro fertilization. INTERVENTION(S): Cells treated with GDF9 and BMP15 in the presence of vehicle, follicle-stimulating hormone (FSH), dibutyryl cyclic-AMP (dbcAMP), or mothers against decapentaplegic homolog (SMAD) inhibitors. MAIN OUTCOME MEASURE(S): Quantification of mRNA, protein, promoter activity, and DNA methylation. RESULTS: FSH stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9 and BMP15 (G+B) combination (P < 0.0001) in a concentration-dependent manner showing a maximal effect at 5 ng/mL each. However, GDF9 or BMP15 alone or in combination (G+B) have no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors inhibited G+B enhancement of IGF2 stimulation by FSH (P < 0.05) but had no effect on FSH induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B potentiation of FSH actions (P < 0.009). CONCLUSIONS: For the first time, we show that the oocyte actively participates in the regulation of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via SMAD2/3, which in turn target mechanisms downstream of the FSH receptor.

Molecular mechanism of FSHR expression induced by BMP15 in human granulosa cells.

PURPOSE: Follicle-stimulating hormone receptor (FSHR) expression in granulosa cells is critical in enabling follicles to achieve accelerated growth. Although FSHR expression has been reported to be epigenetically regulated, the mechanism is unclear. Cooperation between oocytes and granulosa cells is also essential for normal follicular growth. Among oocyte-derived factors, bone morphogenetic protein 15 (BMP15) promotes follicular growth and is suggested to have epigenetic effects. We examined the role of BMP15 in the acquirement of FSHR in human granulosa cells. METHODS: Immortalized non-luteinized human granulosa (HGrC1) cells were stimulated with trichostatin A (TSA) or BMP15 to analyze FSHR expression, histone modifications, and USF1/2 binding at the FSHR promoter region. Histone acetyl transferase (HAT) activity and phosphorylation of Smad 1/5/8 and p38 MAPK were examined with or without BMP15, SB203580, and LDN193189. CYP19A1 expression and estradiol production were also studied. RESULTS: TSA and BMP15 induced FSHR mRNA expression in a dose-dependent manner and histone modifications were observed with increased binding of USF1/2. BMP15 increased FSHR protein expression, which was suppressed by LDN193189. BMP15 increased phosphorylation of Smad 1/5/8 and significantly increased HAT activity, which was inhibited by LDN193189, but not by SB203580. BMP15 increased phosphorylation of p38 MAPK and USF1. LDN193189 suppressed BMP15-induced phosphorylation of both p38 MAPK and USF1, whereas SB203580 suppressed the phosphorylation of USF1. BMP15 increased CYP19A1 mRNA expression and estradiol production. CONCLUSION: BMP15 induced FSHR expression in human granulosa cells through Smad and non-Smad pathways. This mechanism of FSHR induction by BMP15 may be utilized for controlling follicular growth.

Cumulin and FSH Cooperate to Regulate Inhibin B and Activin B Production by Human Granulosa-Lutein Cells In Vitro.

The oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) interact functionally, and it is hypothesized that this interaction may be mediated by formation of a GDF9:BMP15 heterodimer termed cumulin. GDF9 and BMP15 regulate folliculogenesis and ovulation rate and have been shown to regulate inhibin and activin, local regulators of folliculogenesis. The objective of this study was to determine whether cumulin regulates granulosa cell inhibin and activin production and whether this requires cooperation with FSH. Human granulosa-lutein (hGL) cells collected from patients undergoing in vitro fertilization were cultured with or without FSH with various forms of recombinant cumulin (native and cysteine mutants, with or without the prodomains), and cysteine mutant GDF9 or BMP15. Messenger RNA expression of the subunits of inhibins/activins (INHA, INHBA, INHBB) and secretion of inhibin A, inhibin B, and activin B were measured. Mature forms and proforms of cumulin stimulated comparable INHBB mRNA expression and secretion of inhibin B and activin B, whereas GDF9 or BMP15 exhibited no effect. Cumulin, but not GDF9 or BMP15, interacted synergistically with FSH to increase INHBB mRNA and inhibin B expression. FSH markedly stimulated INHA, which encodes the α subunit of inhibin A/B, and suppressed activin B. Cumulin with or without FSH did not significantly alter inhibin A. Together these data demonstrate that cumulin, but not GDF9 or BMP15, exerts paracrine control of FSH-induced regulation of inhibin B and activin B. The prodomains of cumulin may have a minimal role in its actions on granulosa cells.

Influence of growth differentiation factor 9 and bone morphogenetic protein 15 on in vitro maturation of canine oocytes.

Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) have pivotal roles in oocyte development in many species, therefore the aim was to investigate these factors during in vitro maturation (IVM) of canine oocytes. Canine cumulus oocytes complexes (COCs) were cultured in six groups for 72 hr in a supplemented TCM199-Hepes medium as (a) Control group; (b) GDF-9 antibody (Ab); (c) BMP-15 Ab; (d) recombinant human (rh) GDF-9; (e) rh BMP-15 or (f) rh BMP-15 and GDF-9. Data were evaluated by ANOVA. The Abs against GDF-9 or BMP-15 had a negative impact on meiotic development. Higher (p < 0.05) number of oocytes was arrested at GVBD stage when they were incubated with either GDF-9 Ab (64.4 ± 2.1%) or BMP-15 Ab (67.2%± 4.9%) in comparison to those in control group (32.4 ± 7.8%). In contrast, more (p < 0.05) oocytes in control group reached MI (37.4 ± 1.3%) and MII stages (10.2 ± 2.1%) comparing to those groups with GDF-9 Ab (23.1 ± 4.7% MI; 0.0% MII) or BMP-15 Ab (16.4 ± 2.4%MI; 5.9% ± 2.1 MII). Higher rates (p < 0.05) of oocytes in control group stayed still arrested at GV (19.9 ± 8.6%) in comparison to those cultured with either rhGDF-9 (3.7 ± 0.4%) or rhBMP-15 (10.9 ± 0.7%). However, there were no differences in MII rates between oocytes cultured with GDF-9 (14.7 ± 3.1) and BMP-15 (7.8 ± 2.5) separately. But, more oocytes (p < 0.05) reached the MII stage (20.5 ± 3.8%) compared to those exposed to each protein separately and to the control group. These results suggest that these proteins likely contribute to the meiotic development in dogs.

Anti-Müllerian hormone type II receptor in avian follicle development.

Anti-Müllerian hormone (AMH) helps maintain the ovarian reserve by regulating primordial follicle activation and follicular selection in mammals, although its role within the avian ovary is unknown. In mammals, AMH is primarily produced in granulosa cells of preantral and early antral follicles. Similarly, in the hen, the granulosa cells of smaller follicles are the predominant source of AMH. The importance of AMH in mammalian ovarian dynamics suggests the protein and its specific Type II receptor, AMHRII, may have conserved functions in the hen. AMHRII mRNA expression is highest (P < 0.01) in small follicles of the hen and decreases as follicle size increases. Similarly, expression of AMHRII and AMH is highest in granulosa cells from small follicles as compared to larger follicles. Dissection of 3-5 mm follicles into ooplasm and granulosa components shows that AMHRII mRNA levels are greater in ooplasm than granulosa cells. Furthermore, immunohistochemistry also revealed AMHRII staining in the oocyte and granulosa cells. AMH expression in mammals is elevated during periods of reproductive dormancy, possibly protecting the ovarian reserve. AMHRII and AMH mRNA were significantly higher (P < 0.05) in nonlaying ovaries of broiler hens. In molting layer hens, AMHRII mRNA was significantly greater (P < 0.05) compared to nonmolting hen ovaries. These results suggest that AMH may have a direct effect on the oocyte and, thereby, contribute to bidirectional communication between oocyte and granulosa cells. Enhanced expression of AMHRII and AMH during reproductive quiescence supports a potential role of AMH in protecting the ovarian reserve in hens.

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.

Genome-wide analysis of circular RNAs in bovine cumulus cells treated with BMP15 and GDF9.

Circular RNAs (circRNAs) are important members of the non-coding RNA family, and those relating to animal physiologies have been widely studied in recent years. This study aimed to explore the roles of circRNAs in the regulation of follicular development. We constructed four bovine cumulus cell cDNA libraries, including a negative control group (NC) and groups treated with BMP15, GDF9 and BMP15 + GDF9, and we sequenced the libraries on the Illumina HiSeq Xten platform. We identified 1706 circRNAs and screened for differential circRNA expression. We conducted a bioinformatics analysis of these circRNAs and screened for differential circRNAs. Functional annotation and enrichment analysis of the host genes showed that the differential circRNAs were related to locomotion, reproduction, biological adhesion, growth, rhythmic processes, biological phases and hormone secretion. According to the differential expression of circRNA between groups, there were 3 up-regulated and 6 down-regulated circRNAs in the BMP15 group as well as 12 up-regulated and 24 down-regulated circRNAs in the GDF9 group. Co-addition of both BMP15 and GDF9 resulted in 15 up-regulated and 13 down-regulated circRNAs. circ_n/a_75,circ_12691_1 and circ_n/a_303 were altered in both the BMP15 and GDF9 groups as well as in the BMP15 + GDF9 combination group. We focused on these three circRNAs because they were potentially associated with the additive effect of BMP15 and GDF9. Quantitative PCR analysis showed that the expression levels of these three circRNAs were consistent with the sequencing results. In addition, the target miRNAs of circ_n/a_75 and circ_n/a_303, miR-339a, miR-2400 and miR-30c, were down-regulated in the experimental group, which was in contrast to the circRNAs trend. These findings demonstrated that BMP15 and GDF9 may regulate the target gene through circRNA, as a miRNA sponge, in order to regulate the status of bovine cumulus cells and affect follicular development.

Effects of MiR-375-BMPR2 as a Key Factor Downstream of BMP15/GDF9 on the Smad1/5/8 and Smad2/3 Signaling Pathways.

BACKGROUND/AIMS: Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are secreted by oocytes, are important regulators of follicular growth and development and ovarian function. These two factors can regulate the proliferation and apoptosis of cumulus cells via modulation of the Smad signaling pathway. Studies have shown that BMP15 and GDF9 can affect the level of miR-375, whereas the target gene of miR-375 is BMPR2, the type II receptor of BMP15 and GDF9. However, whether or how the BMP15/ GDF9-miR-375-BMPR2 pathway affects the proliferation and apoptosis of bovine cumulus cells through regulation of the Smad signaling pathway remains unclear. METHODS: In this study, cumulus cells were first obtained from cumulus-oocyte complexes (COCs). Appropriate concentrations of BMP15 and GDF9 were added during the in vitro culture process. Cell Counting Kit-8 (CCK-8) analyses and flow cytometry were used to determine the effects of BMP15/GDF9 on bovine cumulus cells proliferation and apoptosis. Subsequently, miR-375 mimics, miR-375 inhibitor and BMPR2 siRNA were synthesized and used for transfection experiments. Western Blot analysis was used to detect changes before and after transfection in the expression levels of the BMP15/GDF9 type I receptors ALK4, ALK5 and ALK6; the phosphorylation levels of Smad2/3 and Smad1/5/8, which are key signaling pathway proteins downstream of BMP15/GDF9; the expression levels of PTX3, HAS2 and PTGS2, which are key genes involved in cumulus cells proliferation; and Bcl2/Bax, which are genes involved in apoptosis. RESULTS: The addition of 100 ng/mL BMP15 or 200 ng/mL GDF9 or the combined addition of 50 ng/mL BMP15 and 100 ng/mL GDF9 effectively inhibited bovine cumulus cell apoptosis and promoted cell proliferation. BMP15/GDF9 negatively regulated miR-375 expression and positively regulated BMPR2 expression. High levels of miR-375 and inhibition of BMPR2 resulted in increased expression of ALK4 and decreased expression of PTX3, HAS2 and PTGS2, whereas miR-375 inhibition resulted in the opposite results. BMP15 and GDF9 significantly activated the levels of p-Smad2/3 and p-Smad1/5/8, whereas miR-375 inhibited the levels of p-Smad2/3 and p-Smad1/5/8 by negatively regulating BMPR2 and also led to apoptosis. CONCLUSION: BMP15 and GDF9 have synergistic effects and can act through miR-375 to affect the expression levels of type I receptor ALK4 and type II receptor BMPR2 and the activation of Smad signaling pathway, which subsequently affected the proliferation, spread and apoptosis of cumulus cells.

Refining insulin concentrations in culture medium containing growth factors BMP15 and GDF9: An in vitro study of the effects on follicle development of goats.

The aim of the present study was to investigate the influence of two insulin concentrations (10ng/mL and 10µg/mL) combined or in the absence of BMP15 and/or GDF9, on the in vitro survival and development of preantral follicles of goat ovarian tissue. Ovarian slices from the same goat ovary pair were randomly assigned to a non-cultured control treatment or to be in vitro cultured for 1 or 7days in α-MEM containing 10ng/mL (Low) or 10µg/mL (High) of insulin in the absence or presence of BMP15 and/or GDF9. With the low insulin treatment, there was a greater percentage of normal follicles than with the high insulin treatment. The addition of BMP15 alone or in association with GDF9 to the medium containing low insulin resulted in a lesser percentage of normal follicles (P<0.05). The addition of BMP15 and GDF9 separately or in combination with the high insulin concentration enhanced the percentage of normal follicles. On day 7 of culture, the use of medium containing low insulin alone or high insulin supplemented with BMP15 and BMP15+GDF9 resulted in a greater percentage of secondary follicles than the non-cultured control, although follicles cultured with low insulin were smaller than those from the control group and had greater rates of oxidative stress. In conclusion, in the presence of physiological concentrations of insulin (10ng/mL), medium supplementation with GDF9 and BMP15 alone or in combination is unnecessary for normal follicle development in vitro.

Regulation of AMH by oocyte-specific growth factors in human primary cumulus cells.

The regulation of AMH production by follicular cells is poorly understood. The purpose of this study was to determine the role of the oocyte-secreted factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on AMH production in primary human cumulus cells. Cumulus cells from IVF patients were cultured with a combination of GDF9, BMP15, recombinant FSH and specific signaling inhibitors. Stimulation with GDF9 or BMP15 separately had no significant effect on AMH mRNA levels. In contrast, simultaneous stimulation with GDF9 and BMP15 (G + B) resulted in a significant increase in AMH mRNA expression. Increasing concentration of G + B (0.6, 2.5, 5 and 10 ng/mL) stimulated AMH in a dose-dependent manner, showing a maximal effect at 5 ng/mL. Western blot analyses revealed an average 16-fold increase in AMH protein levels in cells treated with G + B when compared to controls. FSH co-treatment decreased the stimulation of AMH expression by G + B. The stimulatory effect of G + B on the expression of AMH was significantly decreased by inhibitors of the SMAD2/3 signaling pathway. These findings show for the first time that AMH production is regulated by oocyte-secreted factors in primary human cumulus cells. Moreover, our novel findings establish that the combination of GDF9 + BMP15 potently stimulates AMH expression.