Preliminary results of blood growth differentiation factor-9 (GDF-9) measurement in cats: future aspects of GDF-9 on stage of the cycle and spaying history.

Growth differentiation factor-9 (GDF-9), an oocyte-derived member of the TGF-ß superfamily, plays an essential role in regulation of follicular development. This study aimed to determine the cyclic changes in serum GDF-9 concentration, compare its levels before and after ovariohysterectomy (OHE), and investigate its potential as a tool in ovarian remnant syndrome (ORS) diagnosis in cats. GDF-9 measurements were performed on 50 cats referred for routine OHE. The stage of the estrous cycle was determined by vaginal cytology and measurement of serum estradiol and progesterone levels was carried out to detect the cyclic changes in circulating GDF-9. One week after OHE, serum samples were collected again from 30 cats to reveal differences in GDF-9 levels. GDF-9 levels in the follicular phase were significantly higher than those in the interestrus (p⟨0.05). The postoperative analysis could be performed. GDF-9 levels slightly decreased one week after OHE (p=0.053). In conclusion, blood GDF-9 levels change during the estrous cycle, and may decrease with age in cats. However, further studies are needed to reveal the efficiency of GDF-9 in ORS diagnosis.

GDF9 affects the development and tight junction functions of immature bovine Sertoli cells.

Transforming growth factor-ß (TGFß) superfamily are critical regulators of germ cell development that act as extracellular ligands of the signal transduction pathways regulating proliferation, apoptosis and other aspects of cell behaviour. As a member of the TGF-ß superfamily, growth differentiation factor 9 (GDF9) plays a critical role in ovarian follicular development and the ovulation rate in females; however, its role in the testis has not been well elucidated. The aim of this study was to investigate the expression of GDF9 and its receptor genes BMPRII and ALK5 in prepuberal bovine Sertoli cells (SCs). In addition, we assessed the effects of GDF9 on immature SCs apoptosis, the cell cycle and tight junction functions. We found that GDF9 and its receptor genes BMPRII and ALK5 were expressed in immature SCs. Exogenous GDF9 significantly promoted SCs proliferation and inhibited the apoptosis of SCs by significantly upregulating Cyclin E (cell cycle) and bcl-2 (anti-apoptosis) mRNA expression and downregulating caspase-3 (pro-apoptosis) mRNA expression. Meanwhile, exogenous GDF9 significantly decreased the value of transepithelial electrical resistance by significantly downregulating claudin-11 mRNA expression and influencing the distribution of occludin. In conclusion, this study reveals that GDF9 is a key regulator of bovine SCs through the modulation of the cell cycle, apoptosis and tight junction functions.

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.

Androgen actions in the ovary: balance is key.

For many decades, elevated androgens in women have been associated with poor reproductive health. However, recent studies have shown that androgens play a crucial role in women's fertility. The following review provides an overall perspective about how androgens and androgen receptor-mediated actions regulate normal follicular development, as well as discuss emerging concepts, latest perceptions, and controversies regarding androgen actions and signaling in the ovary.

Growth differentiation factor 9 (GDF9) forms an incoherent feed-forward loop modulating follicle-stimulating hormone ß-subunit (FSHß) gene expression.

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses from the hypothalamus and regulates follicle-stimulating hormone ß-subunit (FSHß) gene expression in pituitary gonadotropes in a frequency-sensitive manner. The mechanisms underlying its preferential and paradoxical induction of FSHß by low frequency GnRH pulses are incompletely understood. Here, we identify growth differentiation factor 9 (GDF9) as a GnRH-suppressed autocrine inducer of FSHß gene expression. GDF9 gene transcription and expression were preferentially decreased by high frequency GnRH pulses. GnRH regulation of GDF9 was concentration-dependent and involved ERK and PKA. GDF9 knockdown or immunoneutralization reduced FSHß mRNA expression. Conversely, exogenous GDF9 induced FSHß expression in immortalized gonadotropes and in mouse primary pituitary cells. GDF9 exposure increased FSH secretion in rat primary pituitary cells. GDF9 induced Smad2/3 phosphorylation, which was impeded by ALK5 knockdown and by activin receptor-like kinase (ALK) receptor inhibitor SB-505124, which also suppressed FSHß expression. Smad2/3 knockdown indicated that FSHß induction by GDF9 involved Smad2 and Smad3. FSHß mRNA induction by GDF9 and GnRH was synergistic. We hypothesized that GDF9 contributes to a regulatory loop that tunes the GnRH frequency-response characteristics of the FSHß gene. To test this, we determined the effects of GDF9 knockdown on FSHß induction at different GnRH pulse frequencies using a parallel perifusion system. Reduction of GDF9 shifted the characteristic pattern of GnRH pulse frequency sensitivity. These results identify GDF9 as contributing to an incoherent feed-forward loop, comprising both intracellular and secreted components, that regulates FSHß expression in response to activation of cell surface GnRH receptors.

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.

Epidermal growth factor (EGF) sustains in vitro primordial follicle viability by enhancing stromal cell proliferation via MAPK and PI3K pathways in the prepubertal, but not adult, cat ovary.

This study examined the influences of epidermal growth factor (EGF) and growth differentiation factor 9 (GDF9) on in vitro viability and activation of primordial follicles in the ovarian tissue of prepubertal (age, <6 mo) versus adult (age, >8 mo) cats. Ovarian cortical slices were cultured in medium containing EGF and/or GDF9 for 14 days. EGF, but not GDF9, improved (P < 0.05) follicle viability in prepubertal donors in a dose-dependent fashion. Neither EGF nor GDF9 enhanced follicle viability in ovarian tissue from adults, and neither factor activated primordial follicles regardless of age group. We then explored how EGF influenced primordial follicles in the prepubertal donors by coincubation with an inhibitor of EGF receptor (AG1478), mitogen-activated protein kinase (MAPK; U0126), or phosphoinositide 3-kinase (PI3K; LY294002). EGF enhanced (P < 0.05) MAPK and AKT phosphorylation, follicle viability, and stromal cell proliferation. These effects were suppressed (P < 0.05) when the tissue was cultured with this growth factor combined with each inhibitor. To identify the underlying influence of age in response to EGF, we assessed cell proliferation and discovered a greater thriving stromal cell population in prepubertal compared to adult tissue. We conclude that EGF plays a significant role in maintaining intraovarian primordial follicle viability (but without promoting activation) in the prepubertal cat. The mechanism of action is via stimulation of MAPK and PI3K signaling pathways that, in turn, promote ovarian cell proliferation. Particularly intriguing is that the ability of cat ovarian cells to multiply in reaction to EGF is age-dependent and highly responsive in prepubertal females.

Oocyte-secreted growth differentiation factor 9 inhibits BCL-2-interacting mediator of cell death-extra long expression in porcine cumulus cell.

Oocyte-secreted factors (OSFs) maintain the low incidence of cumulus cell apoptosis. In this report, we described that the presence of oocytes suppressed the expression of proapoptotic protein BCL-2-interacting mediator of cell death-extra long (BIMEL) in porcine cumulus cells. Atretic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cumulus cells strongly expressed BIMEL protein. The healthy cumulus- oocyte complex exhibited a low BIMEL expression in cumulus cell while the removal of oocyte led to an about 2.5-fold (P < 0.5) increased expression in oocytectomized complex (OOX). Coculturing OOXs with denuded oocytes decreased BIMEL expression to the normal level. The similar expression pattern could also be achieved in OOXs treated with exogenous recombinant mouse growth differentiation factor 9 (GDF9), a well-characterized OSF. This inhibitory action of GDF9 was prevented by the addition of a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Luciferase assay further demonstrated that BIM gene expression was forkhead box O3a (FOXO3a)-dependent because mutation of FOXO3a-binding site on the BIM promoter inhibited luciferase activities. Moreover, the activity of BIM promoter encompassing the FOXO3a-binding site could be regulated by GDF9. Additionally, we found that GDF9 elevated the levels of phosphorylated AKT and FOXO3a, and this process was independent of the SMAD signal pathway. Taken together, we concluded that OSFs, particularly GDF9, maintained the low level of BIMEL expression in cumulus cell through activation of the PI3K/FOXO3a pathway.

GDF9 modulates the reproductive and tumor phenotype of female inha-null mice.

Intraovarian factors play important roles in coordinating germ cell and somatic cell growth in the ovary. Prior to the onset of gonadotropin stimulation and reproductive cyclicity, follicle development is dependent upon locally produced growth factors, such as the transforming growth factor beta family members inhibin, activin, and GDF9. In the absence of inhibin in prepubertal mice (Inha(-/-)), there are marked alterations in preantral follicle growth, but no evidence of ovarian tumors characteristic of adult Inha-null mice. To ascertain the contribution of GDF9 to the Inha-null phenotype, we analyzed folliculogenesis in postnatal Inha Gdf9 double knockout mice. Deletion of Gdf9 from Inha(-/-) rescues the initial growth defects found at early follicle stages in Inha(-/-) ovaries, but surprisingly enhances the onset of pretumor lesions. The normalization of growth dynamics between granulosa cells and oocytes of Inha Gdf9 double knockout mice is also accompanied by a reduction in levels of the activin/inhibin beta B subunit, Inhbb, which is upregulated in Inha(-/-) ovaries. However, at later ages, Inha Gdf9 double knockout ovaries are similar to Inha(-/-) ovaries, and show upregulation of the activin/inhibin subunits and downregulation of the growth factor, kit ligand, thus resulting in a local environment that is growth-promoting for granulosa cells but growth-inhibitory for oocytes. These data suggest a sequential mechanism of action initiated by GDF9 in the Inha knockout mouse that promotes defective folliculogenesis. These studies thus provide a novel role for GDF9 in causing reproductive defects and suppressing tumor initiation in the Inha(-/-) mouse model.

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.

A reporter promoter assay confirmed the role of a distal promoter NOBOX binding element in enhancing expression of GDF9 gene in buffalo oocytes.

Growth differentiation factor 9 is primarily expressed in oocytes and plays a vital role in oocyte cumulus crosstalk. Earlier studies with buffalo oocytes revealed differential expression of this gene under different media stimulation conditions which, in turn, are correlated with the blastocyst yield. In this study, different germ cell specific cis elements including a NOBOX binding elements (NBE) and several E-boxes were identified at the 5' upstream region of buffalo GDF9 gene and their potential role in GDF9 expression was investigated. Transfecting oocytes with GDF9 promoter deletion constructs harbouring the NBE reporter gene revealed a 33% increase in GFP as well as the luciferase signal signifying its role in stimulating the minimal promoter activity of GDF9 in buffalo oocytes. Site directed mutation of core binding nucleotides at NBE at 1.8 kb upstream to TSS further confirmed its role for enhancing the basal transcriptional activity of GDF9 promoter in buffalo oocytes. Current work will provide important leads for understanding the role of GDF9 in oocytes competence and designing a more physiological IVF protocol in case of buffalo.

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 differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells.

BACKGROUND: We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin ß(B)-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A. METHODOLOGY: hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin ß(B)-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey's test. PRINCIPAL FINDINGS: GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin ß(B)-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin ß(B)-subunit mRNA and inhibin B production were attenuated by adding FST. CONCLUSION: GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin ß(B)-subunit mRNA expression and inhibin B production in hGL cells.

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.

Growth and differentiation factor-9 promotes adhesive and motile capacity of prostate cancer cells by up-regulating FAK and Paxillin via Smad dependent pathway.

The majority of advanced prostate cancers metastasis to the bone. Mediators of bone remodelling, the bone morphogenetic proteins have extensively been implicated in the progression and metastasis of prostate cancer. The present study investigated the function of BMP member GDF-9, in prostate cancer. We overexpressed GDF9 in PC-3 cells using a mammalian expression construct, and knocked-down with the use of ribozyme transgenes. These cells were further used in in vitro adhesion and motility assays, in order to determine the effect of GDF-9 on these properties. Recombinant GDF-9 was generated to treat PC-3 WT cells before further analysing the effect on adhesion. The GDF-9 overexpressing PC-3 cells demonstrated a significantly enhanced adhesive and motile capacity compared to their controls. The opposite effect was seen in the GDF-9 knock-down cells. In addition, treating PC-3 cells with rh-GDF-9 resulted in them becoming more adhesive. Both endogenous and exogenous GDF-9 was demonstrated to up-regulate focal adhesion associated proteins FAK and paxillin which contribute to promoted cell adhesion and motility. With the use of a Smad3 inhibitor, this effect was inhibited suggesting that GDF-9 signals via Smad3 to up-regulate expression of these proteins. This study shows that GDF-9 can promote the motile and adhesive capacity of PC-3 prostate cancer cells by up-regulating expression of FAK and paxillin in a Smad dependent manner, suggesting a pro-tumourigenic role for GDF-9 in prostate cancer.

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.

Effects of endogenous growth differentiation factor 9 on activin A-induced inhibin B production in human granulosa-lutein cells.

BACKGROUND: We recently reported on the effects of exogenous growth differentiation factor 9 (GDF9) in enhancing activin A-induced inhibin beta(B)-subunit mRNA and inhibin B levels in human granulosa-lutein (hGL) cells by modulating key components of the activin signaling pathway. We undertook the following study to characterize the role of endogenous GDF9 in this regard. METHODS: We compared inhibin subunit (alpha, beta(A), and beta(B)) mRNA and inhibin B levels and activation of activin receptors (ACVRs) and Smad signaling pathway in hGL cells obtained from women undergoing in vitro fertilization and cultured with and without activin A treatment after GDF9-targeting small interfering RNA transfection. GDF9, inhibin subunits, ACVR2B/1B and Smad2/3/4/7 mRNA and/or protein levels, Smad phosphorylation, and inhibin B were assessed with RT-PCR, immunoblotting, and ELISA. Data were analyzed by ANOVA followed by Tukey's test. RESULTS: GDF9 was detected as mRNA and protein in hGL cells and protein in follicular fluid from all 11 patients tested. Reduced endogenous GDF9 expression after targeting small interfering RNA transfection was associated with decreased ACVR2B/1B and Smad2/3/4 but increased inhibitory Smad7 mRNA and protein levels and, consequently, reduced activin A-induced beta(B)-subunit mRNA and inhibin B levels. CONCLUSIONS: We report here for the first time autocrine roles for endogenous GDF9 in hGL cells in enhancing activin A-induced beta(B)-subunit mRNA and inhibin B levels via key components of the activin signaling pathway. However, the relative contributions of GDF9 in granulosa cells vs. oocyte as autocrine/paracrine regulators of beta(B)-subunit and inhibin B production in normal and abnormal human ovarian functions remain to be determined.

Growth differentiation factor 9 promotes rat preantral follicle growth by up-regulating follicular androgen biosynthesis.

The transition from preantral to early antral stage is the penultimate stage of ovarian follicular development in terms of gonadotropin dependence and follicle destiny. Although oocyte-somatic cell communication is important in early follicular development, our knowledge of the precise role of the oocyte-derived growth differentiation factor (GDF)-9 during preantral follicle growth is incomplete. We examined whether and by what means oocyte-derived GDF-9 controls follicular development and steroidogenesis during the preantral to early antral transition, by a combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Intraoocyte injection of GDF-9 antisense suppressed rat preantral follicle growth in vitro, whereas GDF-9 enhanced follicular development. GDF-9 augmented testosterone production in preantral follicles. GDF-9 antisense suppressed androgen production and CYP17A1 mRNA expression in cultured follicles, a response attenuated by exogenous GDF-9. The nonaromatizable androgen 5alpha-dihydrotestosterone rescued the follicular growth arrest caused by GDF-9 down-regulation. The specific androgen receptor antagonist flutamide suppressed GDF-9-induced preantral follicle growth in vitro. The data suggest that GDF-9 plays an important role in promoting preantral follicle growth by up-regulating follicular androgen biosynthesis. GDF-9 is essential for CYP17A1 expression during follicular development from the preantral to the early antral stage.