Combination treatment of recombinant growth differentiation factor-9 and Cetrorelix improves gestational origin of the polycystic ovarian syndrome in female rats.

AIMS: Polycystic ovary syndrome (PCOS) is a hyper-androgenic endocrinopathy prevalent in premenopausal women with no cure available. The current study aimed to investigate the therapeutic effect of recombinant GDF-9 and Cetrorelix on the gestational origin of dehydroepiandrosterone (DHEA) induced PCOS in postnatal pups' delivered to rat dams. MAIN METHODS: The body weight measurement, blood and serum analysis for glucose tolerance, lipid profile, liver enzymes, sex hormones (Testosterone, Estradiol, and Progesterone), estrus cyclicity assessment, histological staining of ovary and liver, molecular markers expressions of pro-inflammatory by qRT-PCR and immuno-histochemistry technique for folliculogenesis genes and histological staining studies of liver and ovary were done. KEY FINDINGS: The combinational treatment was found to normalize the biochemical parameters and reduction in the estrus irregularity by altering the sex hormones as well as the glucose metabolism and insulin resistance via HOMA-IR value. Further, molecular markers expression confirmed the pro-inflammatory (IL-1ß, TNF-α, and IL-6) and folliculogenesis (GDF-9, BMPR2, and TGF-ßR1) genes associated with PCOS were improved by combinational therapy. SIGNIFICANCE: In conclusion, rGDF-9 could be a potential therapeutic agent in combination with Cetrorelix as a better treatment regime for metabolic and reproductive phenotypes in PCOS. However, the effect of rGDF-9 on infertility-associated phenotypes in PCOS needs further evaluation.

High concentration of growth differentiation factor-9 (GDF-9) to the ram semen had a negative effect on the sperm positive rheotaxis.

Herein, the microfluidic device technique was used to investigate the effects of GDF-9 concentrations and exposure time on the ram sperm positive rheotaxis (PR). Semen was collected from six rams and utilized for PR, motility and sperm kinetic parameter analysis using a computer-assisted sperm analysis program with controlled flow velocity following 0, 10, 20 or 30 min of incubation at 37°C with GDF-9 (200 , 400 or 600 ng/ml; semen sample without GDF-9 was used as a control). Results revealed that there was not an interaction between effects of GDF-9 concentrations and incubation duration on PR% (p = .457) and TM% (p = .099). A simple main effects analysis showed that GDF-9 concentrations had an effect on PR% (p = .003). However, the incubation duration did not have an effect on PR% (p = .101). GDF-9 concentrations had not an effect on TM% (p = .817). By contrast, the incubation duration affected on TM% (p = .026). A higher PR% was found (p < .05) at 200 ng GDF-9 after 10 min (46.7 ± 10.3) and 20 min (45.5 ± 11.5) of incubation. After 30 min of incubation, the PR% was found lowest (p < .05) at 400 ng of GDF-9 (30.6 ± 14.1) and 600 ng of GDF-9 (32.2 ± 9.6). There was no difference (p > .05) in the sperm kinetic parameters between the four treatment groups. In conclusion, the ram sperms had the best rheotaxis properties after 10 and 20 min of incubation with 200 ng of GDF-9 and were sensitive to high concretions.

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.

Effect of growth differentiation factor 9 (GDF-9) on in vitro development of collared peccary preantral follicles in ovarian tissues.

The aims were to identify the effects of growth differentiation factor 9 (GDF-9) on the in vitro development of ovarian preantral follicles (PAFs) of collared peccaries. Ovarian fragments were in vitro cultured for 1 or 7 days without or with inclusion of GDF-9 in the medium (0, 50, 100, or 200 ng/mL). The non-cultured (control) and cultured fragments were evaluated for PAF viability, activation, and cell proliferation. Although there were no differences in the percentage of morphologically normal follicles, the percentage of growing follicles was greater compared to the control in all treatment groups, especially those cultured with 200 ng/mL GDF-9 for 7 days (P < 0.05). The inclusion of GDF-9 in the medium did not interfere with PAF viability (P> 0.05); however, treatment with 200 ng/mL GDF-9 resulted in greater (P < 0.05) cell proliferation in PAFs cultured for 1 or 7 days (∼2.5 nucleolar organizing regions - NORs) compared to the follicles of the control group (2.0 NORs). In addition, peccary ovarian cortexes were subjected to PCR analysis and there was detection of the mRNA GDF-9 receptor transcripts of the BMPR2 (type I receptor) and ALK-5 (type II receptor) types. In conclusion, GDF-9, especially at a 200 ng/mL inclusion in the culture medium, was actively involved in the in vitro development of collared peccary PAFs.

Involvement of Phosphorylated Akt and FOXO3a in the Effects of Growth and Differentiation Factor-9 (GDF-9) on Inhibition of Follicular Apoptosis and Induction of Granulosa Cell Proliferation After In Vitro Culture of Sheep Ovarian Tissue.

This study aimed to evaluate the effects of growth and differentiation factor-9 (GDF-9) on the morphology, activation, apoptosis, and granulosa cell proliferation of ovine preantral follicles cultured within ovarian tissue slices and to verify whether GDF-9 could influence follicular activation through the phosphatidylinositol 3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway. Ovine ovarian fragments were cultured in α-MEM+ or α-MEM+ with GDF-9 (1, 50, 100, 200, or 400 ng/ml) for 7 days. Apoptosis and cell proliferation were analyzed. Next, the activation of the PI3K was inhibited with LY294002, and immunostaining for p-Akt and p-FOXO3a proteins was assessed. The concentration of 50 ng/ml GDF-9 had (P < 0.05) more morphologically normal follicles compared to all treatments, except 1 ng/ml GDF-9. Moreover, 50 ng/ml GDF-9 increased primordial follicle activation compared to all treatments, except α-MEM+ and 1 ng/ml GDF-9. However, the concentration of 50 ng/ml GDF-9 showed higher cell proliferation and lower apoptosis than α-MEM+ and 1 ng/ml GDF-9 treatments. Culture of the ovarian tissue with LY294002 inhibited the activation of primordial follicles and reduced p-Akt immunostaining in both α-MEM+ and 50 ng/ml GDF-9 treatments. In addition, after culture with LY294002, the percentage of oocytes with nuclear p-FOXO3 was higher in 50 ng/ml GDF-9 than in the control medium (α-MEM+). In conclusion, after culture of ovine ovarian cortical slices, the addition of 50 ng/ml GDF-9 reduces follicular apoptosis and promotes granulosa cell proliferation likely through the involvement of phosphorylated Akt and FOXO3a.

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.

Growth differentiation factor-9 improves development, mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles.

This study analysed the effect of growth differentiation factor-9 (GDF-9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α-MEM+ -control medium) or α-MEM+ supplemented with 1, 10, 50 or 100 ng/ml GDF-9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF-9 than other treatments, except for 10 ng/ml of GDF-9 (p > 0.05). Treatment containing 100 ng/ml GDF-9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF-9 showed more oocytes in MI than α-MEM+ , 1 or 50 ng/ml GDF-9 (p < 0.05). In conclusion, 100 ng/ml GDF-9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.

Growth differentiation factor 9 promotes follicle-stimulating hormone-induced progesterone production in chicken follicular granulosa cells.

The function of oocyte-derived growth differentiation factor 9 (GDF9) in ovarian follicles has thus far been poorly defined in avian species compared with the defined function in mammals. Our aim here is to investigate the effects of GDF9 on steroidogenesis and on chicken ovarian granulosa cell (GC) mitosis. Primary GCs from both prehierarchical (6-8 mm in diameter, phGCs) and preovulatory follicles (F1-F5, poGCs) were cultured in the presence or absence of the GDF9 protein. The progesterone (P4) levels in the culture medium were then measured by radioimmunoassay (RIA), and the expression levels of steroidogenesis genes were detected by quantitative PCR. We found that GDF9 alone showed no significant effect on the P4 levels by regulating the expression of steroidogenesis genes, such as STAR, CYP11A1 and HSD3B. Further experiments indicated that GDF9 promoted follicle-stimulating hormone (FSH)-induced P4 production and STAR expression. GDF9 also rescued the FSH-induced decrease of FSH receptor (FSHR) expression but had no effect on the forskolin-induced P4, STAR and forskolin-inhibited FSHR expression levels, suggesting that GDF9 might achieve its regulatory role of P4 by enhancing FSHR and STAR expression. In addition, GDF9 also promoted GC cell cycle progression, regulated the gene transcription of related genes, potentiated DNA replication and inhibited apoptosis. Interestingly, these effects differed between the phGCs and the poGCs. To our knowledge, this is the first report that illustrates the function of GDF9 on chicken GCs and the effects on ovarian steroidogenesis. Our findings highlight the regulation of central oocytes on the surrounding granulosa cells and emphasize the interaction between paracrine signals and endocrine hormones on ovarian progesterone production; these findings contribute to the understanding of the development of avian ovarian follicles.

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.

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.

Impact of oocyte-secreted factors on its developmental competence in buffalo.

Oocyte-secreted factors (OSFs) play an important role in the acquisition of oocyte developmental competence through bidirectional cross-talk between oocyte and cumulus cells via gap junctions. Thus, the present study was designed to investigate the effect of two OSFs, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the developmental competence of buffalo oocytes derived from two different follicle sizes. Cumulus-oocyte complexes (COCs) from large follicles (LF, >6 mm) or small follicles (SF, 0.05) between DOs and combination groups. Relative mRNA analysis revealed significantly higher (P > 0.05) expression of the cumulus cell marker genes EGFR, HAS2, and CD44 in LF-derived than SF-derived oocyte; the expression of these markers was significantly higher (P > 0.05) in DOs and combination groups, irrespective of the follicle size. These results suggested that LF-derived oocytes have a higher developmental competence than SF-derived oocytes and that supplementation of GDF9 and BMP15 modulates the developmental competence of buffalo oocytes by increasing the relative abundance of cumulus-enabling factors and thereby increasing cleavage and the quality of blastocyst production.

MicroRNA-21 plays a pivotal role in the oocyte-secreted factor-induced suppression of cumulus cell apoptosis.

It is known that oocytes and cumulus cells (CCs) are more resistant to apoptosis than other compartments of the antral follicle. However, although oocyte-secreted factors (OSFs) have been found to be involved in suppressing bovine CC apoptosis, little is known about the intracellular mechanisms by which OSFs render CCs resistant to apoptosis. Here, we show that coculture with mouse or pig cumulus-denuded oocytes, culture with recombinant mouse growth differentiation factor-9 (GDF-9), or culture in pig oocyte-conditioned medium (POCM) significantly inhibited CC apoptosis of mouse oocytectomized cumulus oophorus complexes (OOXs). The POCM contained both GDF-9 and bone morphogenetic protein-15, and their levels remained constant during culture of OOXs. The level of microRNA-21 (miR-21) was significantly lower in OOXs than in COCs after culture in a simplified α-MEM medium, but increased significantly when OOXs were cultured with GDF-9 or in POCM. The level of miR-21 in OSF-treated CCs was correlated with that of Dicer1 but not that of Drosha mRNA. Inhibiting activin receptor-like kinase 5 or SMAD3 completely abolished the beneficial effects of GDF-9 or POCM on CC apoptosis and miR-21 levels. Up- and downregulating miR-21 expression significantly reduced and increased CC apoptosis, respectively. The OSF-upregulated miR-21 expression suppressed CC apoptosis with activation of the PI3K/Akt signaling. In conclusion, miR-21 plays a pivotal role in the OSF suppression of CC apoptosis. OSFs upregulated miR-21 expression through the TGF-ß superfamily signaling, which worked through DICER. MicroRNA-21 prevented apoptosis via the PI3K/Akt signaling.

Effect of growth differentiation factor-9 (GDF-9) on the progression of buffalo follicles in vitrified-warmed ovarian tissues.

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor-9 (GDF-9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF-9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen-thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14 days in the presence or absence of GDF-9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF-9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF-9 was detected in mural granulosa cells and theca cells of pre-antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF-9. In corpora lutea, GDF-9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF-9 and cultured control that indicated the GDF-9 treatment has no effect on the primordial to primary follicle transition. GDF-9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre-antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF-9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14 days, on the expression of GDF-9. In conclusion, treatment with GDF-9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).