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growth differentiation factor 9 OKDB#: 24
 Symbols: GDF9 Species: human
 Synonyms: POF14  Locus: 5q31.1 in Homo sapiens

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General Comment GDF-9 is an oocyte-derived paracine factor important for growth of follicles beyond the primary stage (Dong et al., 1996). It belongs to the TGF-beta superfamily having six or more cysteine residues which form a cysteine knot, characteristic for this family. GDF-9 exhibits only the six residues necessary for formation of the cysteine knot, however it lacks an additional cysteine which is used for homodimer formation in other members of this family (McPherron and Lee, 1993 ).//////Evolutionary Origin of Bone Morphogenetic Protein 15 and Growth and Differentiation Factor 9 and Differential Selective Pressure Between Mono- and Poly-Ovulating Species. Monestier O 2014 et al. Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are TGFbeta-like oocyte-derived growth factors involved in ovarian folliculogenesis as critical regulators of many granulosa cell processes and ovulation rate. Ovarian phenotypic effect caused by alterations in BMP15 and GDF9 genes appears to differ between species and may be relevant to their mono- or poly-ovulating status. Through phylogenetic analysis we recently showed that these two paralogous genes are strongly divergent and in rapid evolution as compared to other members of the TGFbeta superfamily. Here, we evaluate the amino-acid substitution rates of a set of proteins implicated in the ovarian function, including BMP15 and GDF9, with special attention to the mono- or poly-ovulating status of the species. Among a panel of mono- and poly-ovulating mammals, we demonstrate a better conservation of some areas in BMP15 and GDF9 within mono-ovulating species. Homology modeling of BMP15 and GDF9 homodimers and heterodimers 3D structures was suggestive that these areas may be involved in dimer formation and stability. A phylogenetic study of BMP15/GDF9 related proteins reveals that these two genes diverged from the same ancestral gene along with BMP3 and GDF10, two other paralogous genes. A substitution rate analysis based on this phylogenetic tree leads to the hypothesis of an acquisition of BMP15/GDF9 specific functions in ovarian folliculogenesis in mammals. We propose that high variations observed in specific areas of BMP15 and GDF9 in poly-ovulating species change the equilibrium between homodimers and heterodimers, modifying the biological activity and thus allowing poly-ovulation to occur. /////////////////////////
NCBI Summary: This gene encodes a secreted ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins. Ligands of this family bind various TGF-beta receptors leading to recruitment and activation of SMAD family transcription factors that regulate gene expression. The encoded preproprotein is proteolytically processed to generate each subunit of the disulfide-linked homodimer. This protein regulates ovarian function. Reduced expression of this gene may be associated with polycystic ovary syndrome and mutations in this gene may be more common in mothers of dizygotic twins. [provided by RefSeq, Jul 2016]
General function Ligand, Growth factor
Comment Cumulin, an oocyte-secreted heterodimer of the transforming growth factor-β family, is a potent activator of granulosa cells and improves oocyte quality. Mottershead DG et al. (2015) Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions, and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modelling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways, and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15 or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependant on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.////////////////// Modifications of human growth differentiation factor 9 to improve the generation of embryos from low competence oocytes. Li JJ et al. (2014) Growth differentiation factor 9 (GDF9) is an oocyte-derived growth factor that plays a critical role in ovarian folliculogenesis and oocyte developmental competence and belongs to the TGF-β family of proteins. Recombinant human GDF9 (hGDF9) is secreted in a latent form, which in the case of the fully processed protein, has the proregion noncovalently associated with the mature region. In this study, we investigated a number of amino acid residues in the mature region of hGDF9 that are different from the corresponding residues in the mouse protein, which is not latent. We designed, expressed, and purified 4 forms of chimeric hGDF9 (M1-M4) that we found to be active in a granulosa cell bioassay. Using a porcine in vitro maturation model with inherent low developmental competence (yielding 10%-20% blastocysts), we tested the ability of the chimeric hGDF9 proteins to improve oocyte maturation and developmental competence. Interestingly, one of the chimeric proteins, M3, was able to significantly increase the level of embryo production using such low competence oocytes. Our molecular modeling studies suggest that in the case of hGDF9 the Gly(391)Arg mutation probably increases receptor binding affinity, thereby creating an active protein for granulosa cells in vitro. However, for an improvement in oocyte developmental competence, a second mutation (Ser(412)Pro), which potentially decreases the affinity of the mature region for the proregion, is also required.////////////////// Amino Acid 72 of Mouse And Human GDF9 Mature Domain Is Responsible For Altered Homodimer Bioactivities But Has Subtle Effects On GDF9:BMP15 Heterodimer Activities. Peng J 2014 et al. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted paralogs of the transforming growth factor beta (TGFbeta) superfamily. In mammals, these two growth factors play critical roles in folliculogenesis. As previously reported, an arginine in the pre-helix loop of GDF5 defines the high binding specificity to its type 1 receptor. Interestingly, bioactive mouse GDF9 and human BMP15 share the conserved arginine in the pre-helix loop, but their low activity counterparts (mouse BMP15 and human GDF9) have a glycine or a proline instead. To address the question whether the arginine residue defines the different activities of GDF9 and BMP15 homodimers and their heterodimers in human and mouse, we used site-directed mutagenesis to change the species-specific residues in human and mouse proteins, and examined their activities in our in vitro assays. While amino acid 72 of mature GDF9 is responsible for altered homodimer bioactivities, neither the corresponding BMP15 amino acid 62 nor the intact pre-helix loop is indispensable for BMP15 homodimer activity. However, amino acid 72 in GDF9 only has only subtle effects on GDF9:BMP15 heterodimer activity. Based on previous studies and our recent findings, we provide hypothetical models to understand the molecular mechanism to define activities of the homodimeric and heterodimeric ligands. The arginine residue in the pre-helix loop of GDF9 homodimer may prevent the inhibition from its pro-domain or directly alter receptor binding, but this residue in GDF9 does not significantly affect the heterodimer activity due to suggested conformational changes during heterodimer formation. ///////////////////////// Activation of Latent Human GDF9 by a Single Residue Change (Gly391Arg) in the Mature Domain. Simpson CM et al. Growth differentiation factor 9 (GDF9) controls granulosa cell growth and differentiation during early ovarian folliculogenesis and regulates cumulus cell function and ovulation rate in the later stages of this process. Similar to other TGF-?superfamily ligands, GDF9 is secreted from the oocyte in a noncovalent complex with its prodomain. In this study, we show that prodomain interactions differentially regulate the activity of GDF9 across species, such that murine (m) GDF9 is secreted in an active form, whereas human (h) GDF9 is latent. To understand this distinction, we used site-directed mutagenesis to introduce nonconserved mGDF9 residues into the pro- and mature domains of hGDF9. Activity-based screens of the resultant mutants indicated that a single mature domain residue (Gly(391)) confers latency to hGDF9. Gly(391) forms part of the type I receptor binding site on hGDF9, and this residue is present in all species except mouse, rat, hamster, galago, and possum, in which it is substituted with an arginine. In an adrenocortical cell luciferase assay, hGDF9 (Gly(391)Arg) had similar activity to mGDF9 (EC(50) 55 ng/ml vs. 28 ng/ml, respectively), whereas wild-type hGDF9 was inactive. hGDF9 (Gly(391)Arg) was also a potent stimulator of murine granulosa cell proliferation (EC(50) 52 ng/ml). An arginine at position 391 increases the affinity of GDF9 for its signaling receptors, enabling it to be secreted in an active form. This important species difference in the activation status of GDF9 may contribute to the variation observed in follicular development, ovulation rate, and fecundity between mammals. Characterization of oocyte-expressed GDF9 gene in buffalo and mapping of its TSS and putative regulatory elements. Roy B et al. SummaryIn spite of emerging evidence about the vital role of GDF9 in determination of oocyte competence, there is insufficient information about its regulation of oocyte-specific expression, particularly in livestock animals. Because of the distinct prominence of buffalo as a dairy animal, the present study was undertaken to isolate and characterize GDF9 cDNA using orthologous primers based on the bovine GDF9 sequence. GDF9 transcripts were found to be expressed in oocytes irrespective of their follicular origin, and shared a single transcription start site (TSS) at -57 base pairs (bp) upstream of ATG. Assignment of the TSS is consistent with the presence of a TATA element at -23 of the TSS mapped in this study. Localization of a buffalo-specific minimal promoter within 320 bp upstream of ATG was consolidated by identification of an E-box element at -113bp. Presence of putative transcription factor binding sites and other cis regulatory elements were analyzed at ~5 kb upstream of TSS. Various germ cell-specific cis-acting regulatory elements (BNCF, BRNF, NR2F, SORY, Foxh1, OCT1, LHXF etc.) have been identified in the 5' flanking region of the buffalo GDF9 gene, including NOBOX DNA binding elements and consensuses E-boxes (CANNTG). Presence of two conserved E-boxes found on buffalo sequence at -520 and -718 positions deserves attention in view of its sequence deviation from other species. Two NOBOX binding elements (NBE) were detected at the -3471 and -203 positions. The fall of the NBE within the putative minimal promoter territory of buffalo GDF9 and its unique non-core binding sequence could have a possible role in the control of the core promoter activity. Functional and molecular characterization of naturally occurring mutations in the oocyte-secreted factors BMP-15 and GDF-9 Liao WX, et al . Immunohistochemical localization of advanced glycation end-products (AGEs) and their receptor (RAGE) in polycystic and normal ovaries. Diamanti-Kandarakis E et al. The aim of the present study was to investigate the localization/immunohistochemical distribution of AGEs and RAGE, as well as their putative signalling mediator NF-kappaB in ovaries of women with polycystic ovary syndrome (PCOS) compared to normal. Archival ovarian-tissue samples from biopsies of six women with PCOS and from six healthy of similar age women, were examined immunohistochemically with monoclonal anti-AGEs, anti-RAGE and anti-NF-kappaB(p50/p65) specific antibodies. In healthy women, AGE immunoreactivity was observed in follicular cell layers (granulosa and theca) and luteinized cells, but not in endothelial cells. PCOS specimens displayed AGE immunoexpression in theca interna and granulosa cells as well as in endothelial cells, but staining of granulosa cells was stronger than in that of normal ovaries. RAGE was highly expressed in normal and PCOS tissues. Normal tissue exhibited no staining differences between granulosa cell layer and theca interna. However, in PCOS ovaries, granulosa cells displayed stronger RAGE expression compared to theca interna cells in comparison to controls. NF-kappaB(p50/p65) was expressed in the cytoplasm of theca interna and granulosa cells of both normal and PCOS ovaries; whereas the NF-kappaB p65 subunit was only observed in granulosa cells nuclei in PCOS tissue. In conclusion, these findings demonstrate for the first time that RAGE and AGE-modified proteins with activated NF-kappaB are expressed in human ovarian tissue. Furthermore, a differential qualitative distribution of AGE, RAGE and NF-kappaB p65 subunit was observed in women with PCOS compared to healthy controls, where a stronger localization of both AGE and RAGE was observed in the granulosa cell layer of PCOS ovaries.
Cellular localization Secreted
Comment Serum concentrations of oocyte-secreted factors BMP15 and GDF9 during IVF and in women with reproductive pathologies. Riepsamen AH et al. (2019) Oocyte-secreted factors, bone morphogenetic protein-15 (BMP15) and growth differentiation factor-9 (GDF9) are critical for folliculogenesis and fertility. This study developed ELISAs for the measurement of BMP15 and GDF9 in serum and investigated their usefulness as biomarkers of female reproductive function. Serum samples were obtained from women undergoing infertility treatments (n=154), and from peri- and post-menopausal women (n=28). Serum concentrations of BMP15 and GDF9 in women relative to age, AMH, number of oocytes retrieved, and polycystic ovaries/syndrome (PCO(S)) after superovulation for IVF. BMP15 and GDF9 immunoassays were validated for specificity, sensitivity (24 and 26 pg/ml, respectively) and reproducibility. BMP15 and GDF9 were detectable in 61% and 29% of women, respectively. BMP15 and GDF9 varied 64- and 15-fold respectively between women, but did not change within subjects following ovarian stimulation with gonadotropins. Serum GDF9 concentration, but not BMP15, was associated with oocyte number retrieved in non-PCO(S) patients (p=0.018). GDF9 and BMP15 associations with oocyte number differed significantly (p<0.05) with PCO(S) status. GDF9 concentrations were lower in poor responders (women with <4 oocytes retrieved or cancelled cycles; p=0.020). Serum BMP15, but not GDF9, was lower in women over 55 years, compared to women of reproductive age (p<0.01). This study develops and validates immunoassays to quantitate BMP15 and GDF9 in human serum, and to correlate concentrations with female reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine.////////////////// Immunoneutralization of Growth Differentiation Factor 9 Reveals It Partially Accounts for Mouse Oocyte Mitogenic Activity. Gilchrist RB,et al . Paracrine factors secreted by oocytes play a pivotal role in promoting early ovarian follicle growth and in defining a morphogenic gradient in antral follicles, yet the exact identity of these oocyte factors remains unknown. This study was conducted to determine the extent to which the mitogenic activity of mouse oocytes can be attributed to growth differentiation factor 9 (GDF9). To do this, specific anti-human GDF9 monoclonal antibodies were generated. Based on epitope mapping and bioassays, a GDF9 neutralizing antibody, mAb-GDF9-53, was characterised with very low cross-reactivity with related TGF-beta superfamily members, including BMP15 (also called GDF9B). Pep-SPOT epitope mapping showed that mAb-GDF9-53 recognizes a short 4-aa sequence, and 3D-peptide modelling suggested that this binding motif lies at the C-terminal fingertip of mGDF9. As predicted by sequence alignments and modelling, the antibody detected recombinant GDF9, but not BMP15 in a Western blot, and GDF9 protein in oocyte extract and oocyte-conditioned medium. In a mouse mural granulosa cell (MGC) bioassay, mAb-GDF9-53 completely abolished the mitogenic effects of GDF9, but had no effect on TGF-beta1 or activin A-stimulated MGC proliferation. An unrelated IgG at the same dose had no effect on GDF9 activity. This GDF9 neutralizing antibody was then tested in an established oocyte-secreted mitogen bioassay, where denuded oocytes co-cultured with granulosa cells promote cell proliferation in a dose-dependent manner. mAb-GDF9-53 dose-dependently (0-160 micro g/ml) decreased the mitogenic activity of oocytes, but only by ~45% at the maximum dose of mAb. Just 5 micro g/ml of mAb-GDF9-53 neutralized 90% of recombinant mGDF9 mitogenic activity, but only 15% of oocyte activity. Unlike mAb-GDF9-53, a TGF-beta-pan-specific neutralizing antibody did not affect the mitogenic capacity of the oocyte, but completely neutralized TGF-beta1-induced DNA synthesis. This study has characterised a specific GDF9 neutralizing antibody. Our data provide the first direct evidence that the endogenous GDF9 protein is an important oocyte-secreted mitogen, but also shows that GDF9 accounts for only part of total oocyte bioactivity. PHOSPHORYLATION OF BMP-15 AND GDF-9 PLAYS A CRITICAL ROLE IN DETERMINING AGONISTIC OR ANTAGONISTIC FUNCTIONS. McMahon HE et al. Two highly homologous oocyte-secreted growth factors, bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are known to control folliculogenesis and ovulation through direct effects on granulosa cells in the developing follicles. Although much is known about the expression and biology of these proteins, the impact of posttranslational modifications of BMP-15 and GDF-9 is unknown. Here, we report that: i) recombinant human BMP-15 (rhBMP-15) and rhGDF-9 are phosphorylated; ii) the phosphorylation is essential for bioactivity and iii) the de-phosphorylated forms of rhBMP-15 and rhGDF-9 can abolish the bioactivity of rhBMP-15, rhGDF-9, and rhBMP-7 but not rhActivin A. These results indicate that the phosphorylation state of rhBMP-15 and rhGDF-9 is a determinant of their agonistic and antagonistic activities.
Ovarian function Follicle development, Initiation of primordial follicle growth, Primary follicle growth, Preantral follicle growth, Antral follicle growth, Cumulus cell differentiation, Cumulus expansion, Follicle atresia, Steroid metabolism, Oocyte maturation, Early embryo development
Comment A variant of human growth differentiation factor-9 that improves oocyte developmental competence. Stocker WA et al. (2020) Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are co-expressed exclusively in oocytes throughout most of folliculogenesis and play central roles in controlling ovarian physiology. Although both growth factors exist as homodimers, recent evidence indicates that GDF9 and BMP15 can also heterodimerize to form the potent growth factor cumulin. Within the cumulin complex, BMP15 "activates" latent GDF9, enabling potent signaling in granulosa cells via type I receptors (i.e., activin receptor-like kinase-4/5 ALK4/5]) and SMAD2/3 transcription factors. In the cumulin heterodimer, two distinct type I receptor interfaces are formed compared with homodimeric GDF9 and BMP15. Previous studies have highlighted the potential of cumulin to improve treatment of female infertility, but, as a non-covalent heterodimer, cumulin is difficult to produce and purify without contaminating GDF9 and BMP15 homodimers. In this study we addressed this challenge by focusing on the cumulin interface formed by the helix of the GDF9 chain and the fingers of the BMP15 chain. We demonstrate that unique BMP15 finger residues at this site (Arg-301, Gly-304, His-307, and Met-369) enable potent activation of the SMAD2/3 pathway. Incorporating these BMP15 residues into latent GDF9 generated a highly potent growth factor, called hereafter Super-GDF9. Super-GDF9 was >1000-fold more potent than wildtype human GDF9 and 4-fold more potent than cumulin in SMAD2/3-responsive transcriptional assays in granulosa cells. Our demonstration that Super-GDF9 can effectively promote mouse cumulus cell expansion and improve oocyte quality in vitro represents a potential solution to the current challenges of producing and purifying intact cumulin.////////////////// Growth and differentiation factor 9 promotes oocyte growth at the primary but not the early secondary stage in three-dimensional follicle culture. [Cook-Andersen H et al. (2016) Factors that differentially regulate oocyte and granulosa cell growth within the early preantral follicle and how these factors differ at each stage of follicle growth remain poorly understood. The aim of this study was to isolate and evaluate the effect of recombinant growth and differentiation factor 9 (GDF9) on oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle growth during in vitro culture. Primary stage follicles (diameters of 50-89 μm) and early secondary stage follicles (diameters of 90-120 μm) were isolated from immature mice, and individual, intact follicles were cultured in vitro in the presence and absence of recombinant GDF9. The effects of GDF9 on follicle growth were determined by the assessment of changes in the follicle volume during culture. The growth of the granulosa cell and oocyte compartments of the follicles was evaluated separately at each stage. GDF9 significantly increased the growth of isolated follicles at both the primary and early secondary follicle stages. Independent evaluation of the granulosa cell and oocyte compartments revealed that, while GDF9 promoted granulosa cell growth at both stages of folliculogenesis, oocyte growth was stage specific. GDF9 promoted growth of the oocyte at the primary, but not the early secondary, follicle stage. These findings demonstrate a stage-specific role for GDF9 in the regulation of oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle development.////////////////// Increased GDF9 and BMP15 mRNA levels in cumulus granulosa cells correlate with oocyte maturation, fertilization, and embryo quality in humans. Li Y 2014 et al. BACKGROUND Oocyte secreted factors (OSFs), including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), play an important role in the process of follicular development and oocyte maturation. Since OSFs are expressed in oocytes and cumulus granulosa cells, the aim of the present study was to explore whether the expression levels of GDF9 and BMP15 mRNAs in cumulus granulosa cells can be used as molecular markers for predicting oocyte developmental potential. METHODS Cumulus cells of 2426 cumulus-oocyte complexes were collected from 196 female patients who underwent intracytoplasmic sperm injection (ICSI) and were used for mRNA detection on the egg retrieval day. Pearson correlation analysis was used to analyze the correlation between OSF expression and general physiological parameters. Partial correlation analysis was used to analyze the correlation between OSF expression and oocyte developmental potential. Covariance analysis was used to compare OSF expression among different groups. Receiver operating characteristic curves were used to examine the diagnostic value of GDF9 and BMP15 mRNA for predicting pregnancy. RESULTS The expression levels of GDF9 and BMP15 mRNAs were significantly associated with age, body mass index (BMI), oocyte maturation, normal fertilization, and cleavage rate (P < 0.05). The expression levels of GDF9 and BMP15 mRNAs in the group with high-quality embryos were significantly higher than those in the group without high-quality embryos (P < 0.05). The expression levels of GDF9 and BMP15 mRNAs in the pregnancy group were significantly higher than those in the nonpregnancy group (P < 0.05). The cut-off value of GDF9 mRNA for predicting pregnancy was 4.82, with a sensitivity of 82% and a specificity of 64%. The cut-off value of BMP15 mRNA for predicting pregnancy was 2.60, with a sensitivity of 78% and a specificity of 52%. CONCLUSIONS The expression levels of GDF9 and BMP15 mRNAs were closely associated with oocyte maturation, fertilization, embryo quality, and pregnancy outcome; therefore, GDF9 and BMP15 mRNAs in cumulus granulosa cells may be considered as new molecular markers for predicting oocyte developmental potential. ///////////////////////// Effects of Growth Differentiation Factor 9 and Bone Morphogenetic Protein 15 on the in vitro Maturation of Porcine Oocytes. Lin ZL 2013 et al. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-?(TGF-? family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGF?GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation. ///////////////////////// Growth differentiation factor 9:bone morphogenetic protein 15 heterodimers are potent regulators of ovarian functions. [Peng J et al. The TGF-?superfamily is the largest family of secreted proteins in mammals, and members of the TGF-?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-?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.///// Importance of the GDF9 signaling pathway on cumulus cell expansion and oocyte competency in sheep. Rouhollahi Varnosfaderani S et al. Acquisition of developmental competency in cumulus oocyte complexes (COCs) is derived from endocrine hormones and oocyte secreted factors. The contribution of these factors in oocyte maturation and development is an active area of research. The objective of this research was to investigate whether growth differentiation factor 9 (GDF9) that is secreted by oocyte affects cumulus expansion and oocyte development in sheep. Immature ovine COCs were cultured in the presence of recombinant human GDF9 (rhGDF9), denuded oocytes, SB-431542, a specific inhibitor of activin-like kinase 4/5/7; or a combination of these factors. Routine in?vitro maturation of COCs and denuded oocytes were used as external control samples. Cultured COCs were used for assessment of (1) cumulus expansion; (2) expression of cumulus-related transcripts including pentraxin 3, hyaluronan synthase 2 (HAS2), tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, B-cell lymphoma 2 (BCL2), and Bcl2-associated X (BAX); and (3) yield and quality of embryo development. It was observed that cumulus expansion was not affected by any of these treatments. HAS2 mRNA expression confirmed this observation. In the presence of exogenous GDF9, cleavage rate was reduced, blastocyst rate did not differ from other groups, and trophectoderm cell number significantly increased. This suggests that exogenous GDF9 could improve embryo quality. It was also observed that oocyte secreted factors reduced proapoptotic BAX mRNA, and BCL2 mRNA expression was not significantly different from other groups. This study provides evidence that GDF9 signaling might have a minor influence on ovine cumulus expansion and oocyte development and that other signaling pathway(s) might have a dominant role. In vivo treatment with GDF-9 stimulates primordial and primary follicle progression and theca cell marker CYP17 in ovaries of immature rats. Vitt UA et al. Growth differentiation factor (GDF)-9 is a cystine knot-containing hormone of the transforming growth factor-beta superfamily produced by the oocyte. In GDF-9 null mice, follicle development is arrested at the primary stage and GDF-9 treatment in vitro enhances preantral follicle growth. Immature female rats were treated with recombinant GDF-9 for 7 or 10 days. At 10 days, treatment with GDF-9 augmented ovarian weights, concomitant with an increase in the number of primary and small preantral follicles by 30 and 60%, respectively. Furthermore, the number of primordial follicles was decreased by 29%, but the number of large preantral follicles was not affected. In contrast, treatment with FSH increased the number of small and large preantral follicles by 36 and 177% but did not influence the number of primary and primordial follicles. Immunoblot analysis showed an increase of CYP17, a theca cell marker, in the ovarian homogenate after treatment with GDF-9 but not FSH. The present results indicate that in vivo treatment with GDF-9 enhances the progression of primordial and primary follicles into small preantral follicles. Thus, GDF-9 treatment could provide an alternative approach to stimulate early follicle development in addition to the widely used FSH that acts mainly on the development of more advanced follicles. Growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin B production in human granulosa cells. Shi F et al. Activin A or GDF9 alone can increase betaB mRNA level in human granulosa-lutein (hGL) cells from women undergoing in vitro fertilization but their potential interactions and related cell signaling pathways involved are unknown. We therefore compared inhibin subunit and inhibin levels, and activation of activin receptors (ACVRs) and Smad signaling pathway in these hGL cells with and without GDF9 and/or activin A treatment. Inhibin subunit (alpha, betaA, betaB), ACVR and Smad2/3/4/7 mRNA levels, inhibin A and B production, and Smad phosphorylation were assessed by RT-PCR, ELISA, and immunoblotting, respectively. Data were analyzed by ANOVA followed by Tukey's test. Activin A (1-50 ng/ml) or GDF9 (1-200 ng/ml) alone had only little stimulatory effects on alpha and betaA mRNA levels. In contrast, GDF9 could stimulate betaB subunit levels but to a lesser degree than the dose- and time-dependent effects of activin A. Compared to untreated cells, GDF9 pretreatment for 24 h significantly enhanced activin A-induced betaB mRNA levels, inhibin B secretion and Smad2/3 phosphorylation (effects attenuated by BMPR2 ECD, a GDF9 antagonist); and induced ACVR2B/1B and Smad2/3 but reduced Smad7 (an inhibitory Smad) mRNA levels. We report here for the first time that GDF9 enhances cell response to activin A by modulating key components of the activin signaling pathway in regulating inhibin subunits, and hence, inhibin B production in human granulosa-lutein cells. Effects of Growth Differentiation Factor 9 (GDF-9) on Cell Cycle Regulators and ERK 42/44 in Human Granulosa Cell Proliferation. Huang Q et al. Background: GDF-9 stimulates granulosa cell proliferation, and plays important roles during folliclogenesis. However, its molecular mechanisms are still far from clear, particularly its roles in human granulosa cells around the periovulatory stage. We therefore investigated the effects of GDF-9 on cell cycle distribution, regulatory molecules, and signaling pathways involved in human luteinized granulosa (hLG) cells in vitro. Methods: Primary cultures of hLG cells obtained from women undergoing IVF and treated with and without recombinant GDF-9 were evaluated with and without a specific inhibitor to ALK-5 (SB431542), ERK 42/44 (PD098059), or Smad 3 (SIS3). Cell proliferation, cell cycle distribution, and mRNA and protein expression of relevant cell cycle molecules were determined by (3)H-thymidine incorporation, flow cytometry, quantitative PCR, and immunoblotting, respectively. Results: GDF-9 stimulated (3)H-thymidine incorporation, enhanced cell transition from G0/G1 to S and G2/M phases (while both SB431542 and PD098059 attenuated these changes), increased mRNA and protein expression of cyclin D1 and E, and decreased those of the cyclin-dependent kinase (CDK) inhibitors, p15(INK4B) and p16(INK4A). GDF-9 also activated Rb protein (a critical G1 to S-phase regulator), ERK 42/44, and Smad 3. PD098059 blocked Rb protein phorsphorylation and the increase in cyclin D1 and E, but not the decrease in p15(INK4B) and p16(INK4A) induced by GDF-9. In contrast, SIS3 reversed the decrease in p15(INK4B) and p16(INK4A) but not the increase in cyclin D1 and E induced by GDF-9. Conclusions: GDF-9 stimulates hLG cells proliferation by stimulating cyclin D1 and E, and suppressing p15(INK4B) and p16(INK4A) via both Smad-dependent and Smad-independent pathways. Key words: cyclin D1, cyclin E, p15INK4B, p16INK4A. Growth and differentiation factor-9 stimulates activation of goat primordial follicles in vitro and their progression to secondary follicles. Martins FS et al. The aim of the present study was to investigate the effects of growth and differentiation factor-9 (GDF-9) on the survival and activation of preantral follicles, as well as their subsequent progression to secondary follicles, using goat ovarian cortical culture in vitro. Pieces of ovarian cortex were cultured for 1 and 7 days in minimum essential medium (MEM) with or without different concentrations of GDF-9 (1-200 ng mL(-1)). On Day 0 and after 1 and 7 days of culture, cortical pieces were fixed for histological and transmission electron microscopy evaluation. Preantral follicles were classified according to their development stage (primordial, intermediate, primary and secondary) and on the basis of morphological features (normal or degenerated). In addition, follicular and oocyte diameters were determined before and after culture. The results showed that, compared with non-cultured cortical tissue (Day 0), the culture of ovarian tissue significantly reduced (P < 0.05) the percentage of normal follicles in all media tested, except for tissue cultured in the presence of 200 ng mL(-1) GDF-9. Furthermore, in all media tested, the percentage of primordial follicles was significantly reduced (P < 0.05), with a concomitant increase in the percentage of developing follicles. The highest percentage of secondary follicles was observed after 7 days of culture in MEM plus 200 ng mL(-1) GDF-9. At all concentrations of GDF-9 tested, follicular diameter increased significantly after 7 days of culture compared with non-cultured cortical tissue. In conclusion, the results of the present study indicate that 200 ng mL(-1) GDF-9 maintains the survival of preantral follicles and promotes activation of primordial follicles. Furthermore, GDF-9 stimulates the transition from primary to secondary follicles, maintaining ultrastructural integrity of the follicles. Oocyte regulation of metabolic cooperativity between mouse cumulus cells and oocytes: BMP15 and GDF9 control cholesterol biosynthesis in cumulus cells. Su YQ et al. Oocyte-derived bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are key regulators of follicular development. Here we show that these factors control cumulus cell metabolism, particularly glycolysis and cholesterol biosynthesis before the preovulatory surge of luteinizing hormone. Transcripts encoding enzymes for cholesterol biosynthesis were downregulated in both Bmp15(-/-) and Bmp15(-/-) Gdf9(+/-) double mutant cumulus cells, and in wild-type cumulus cells after removal of oocytes from cumulus-cell-oocyte complexes. Similarly, cholesterol synthesized de novo was reduced in these cumulus cells. This indicates that oocytes regulate cumulus cell cholesterol biosynthesis by promoting the expression of relevant transcripts. Furthermore, in wild-type mice, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol and Ebp, which encode enzymes required for cholesterol synthesis, were highly expressed in cumulus cells compared with oocytes; and oocytes, in the absence of the surrounding cumulus cells, synthesized barely detectable levels of cholesterol. Furthermore, coincident with reduced cholesterol synthesis in double mutant cumulus cells, lower levels were also detected in cumulus-cell-enclosed double mutant oocytes compared with wild-type oocytes. Levels of cholesterol synthesis in double mutant cumulus cells and oocytes were partially restored by co-culturing with wild-type oocytes. Together, these results indicate that mouse oocytes are deficient in synthesizing cholesterol and require cumulus cells to provide products of the cholesterol biosynthetic pathway. Therefore, oocyte-derived paracrine factors, particularly, BMP15 and GDF9, promote cholesterol biosynthesis in cumulus cells, probably as compensation for oocyte deficiencies in cholesterol production. GDF-9 deficient mice show arrest of follicular development at the primary one-layer follicle stage (Dong et al., 1996). Furthermore recombinant GDF-9 induces preantral rat follicle growth in vitro (Hayashi et al., 1999). It was shown, that GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2) and steroidogenic acute regulator protein (StAR) mRNA synthesis and suppresses urokinase plasminogen activator (uPA) and LHR mRNA. Furthermore, GDF-9 in absence of FSH increases progesterone synthesis in vitro and induces cumulus expansion (Elvin et al., 1999). However, GDF-9 suppresses FSH-induced granulosa cell differentiation (steroidogenesis, cAMP production and LH-receptor expression) in vitro (Vitt et al., 2000).Using biologically active recombinant GDF-9 (rGDF-9) and an androgen-producing immortalized theca-interstitial cell (TIC) line or primary TIC, Solovyeva et al 2000 have examined the action of this paracrine hormone on theca cell steroidogenesis. The effect of GDF-9 on TIC progesterone synthesis was marginal and inconsistent in the primary cultures. In immortalized theca cells, GDF-9 attenuated the forskolin-stimulated progesterone accumulation. More significantly, this oocyte-derived growth factor enhanced both basal and stimulated androstenedione accumulation in the primary and transformed TIC cultures. The effects of GDF-9 on steroidogenesis by preovulatory follicles were relatively modest. Likewise, it did not affect the maturation of follicle-enclosed oocytes. The effect of GDF-9, an oocyte product, on TIC androgen production suggests a regulatory role of the oocyte on theca cell function and hence on follicle development and differentiation. This direct effect of GDF-9 on thecal steroidogenesis is consistent with its recently demonstrated actions on thecal cell recruitment and differentiation. Roh et al reported that growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells. Juengel JL, et al reported the effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes. Immunization of ewes against growth differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) can lead to an increased ovulation rate; however, it is not known whether normal pregnancies occur following such treatments. Short-term immunization against either BMP15 or GDF9 peptides resulted in an increase in ovulation rate with no apparent detrimental affects on fertilization of released oocytes, the ability of fertilized oocytes to undergo normal fetal development, or the ability of the immunized ewes to carry a pregnancy to term. Therefore, regulation of BMP15, GDF9, or both is potentially a new technique to enhance fecundity in some mammals. Gui LM, et al 2004 reported RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice. In mouse ovaries, growth differentiation factor-9 (GDF9) is an oocyte-derived growth factor that plays an essential role in early follicular development. However, the role of GDF9 at later stages of follicle development is uncertain. In this study, a long, double-stranded (ds) RNA interference (RNAi) approach was used to investigate the possible role of GDF9 in mediating oocyte regulation of cumulus expansion. Fully-grown mouse oocytes injected with Gdf9 dsRNA, Bmp15 dsRNA or injection buffer were cultured for 24 h and processed for measurement of Gdf9 and Bmp15 mRNA levels using real-time RT-PCR, and for measurement of GDF 9 protein levels using Western blotting and immunofluorescence. Injection with Gdf9 dsRNA knocked down Gdf9 but not Bmp15 mRNA expression in oocytes, and vice versa. Furthermore, GDF9 protein levels were reduced in the Gdf9 dsRNA injected oocytes. To investigate the role of GDF9 in cumulus expansion, two endpoints were used to evaluate cumulus expansion: Has2 and Ptgs2 mRNA levels were measured in cumulus cells using real-time RT-PCR; and assessment of cumulus expansion was undertaken morphologically. After 24 h of culture in the presence of 0.5 IU/ml FSH, cumulus shells co-cultured with buffer- and Bmp15 dsRNA-injected oocytes exhibited a high degree of expansion, while cumulus shells co-cultured with Gdf9 dsRNA-injected oocytes exhibited only limited expansion. Supporting this observation, after 8 h of co-culture Has2 and Ptgs2 mRNA levels were lower in cumulus cells co-cultured with Gdf9 dsRNA-injected oocytes than in those co-cultured with buffer-injected oocytes. These results strongly support the concept that GDF 9 is a key mediator of oocyte-enabled cumulus expansion in mice. Role of oocyte-secreted growth differentiation factor 9 in the regulation of mouse cumulus expansion Dragovic RA, et al . Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex (COC), which is necessary for ovulation. Oocyte-secreted growth differentiation factor 9 (GDF9) signals through the bone morphogenetic protein receptor II (BMPRII) and is currently the primary candidate molecule for the cumulus-expansion enabling factor (CEEF). This study was conducted to determine whether GDF9 is the mouse CEEF. COCs were collected from mice and the oocyte was microsurgically removed to generate an oocytectomized (OOX) complex. OOX complexes treated with FSH alone or recombinant mouse GDF9 alone failed to expand, whereas expansion was induced in the presence of FSH by either GDF9, TGFbeta1 or co-culture with oocytes. A specific GDF9 neutralizing antibody, mAb-GDF9-53, neutralized the expansion of OOX complexes in response to GDF9, but not the expansion of OOX complexes co-cultured with oocytes. Using real time RT-PCR, hyaluronan synthase 2 (HAS2) mRNA expression by OOXs was up regulated 4- to 6-fold by oocytes and GDF9. mAb-GDF9-53 attenuated GDF9-induced OOX HAS2 expression but not oocyte-induced HAS2 expression. A TGFbeta antagonist neutralized TGFbeta-induced, but not oocyte-induced, expansion of OOX complexes, and when combined with mAb-GDF9-53 also failed to neutralize oocyte-induced expansion. Furthermore, a soluble portion of the BMPRII ectodomain, which is a known GDF9 antagonist, completely antagonized GDF9-induced expansion, but only partially neutralized oocyte-induced expansion. This study provides further evidence that like TGFbeta, GDF9 can enable FSH-induced cumulus expansion, but more importantly, demonstrates that neither GDF9 nor TGFbeta alone, nor the two in unison, account for the critical oocyte-secreted factors regulating mouse cumulus expansion. Hickey et al reported androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells. Growth differentiation factor-9 has divergent effects on proliferation and steroidogenesis of bovine granulosa cells. Spicer LJ et al. In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1-5 mm) and large (8-22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150-600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P<0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P<0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P<0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P>0.10). Treatment with 600 ng/ml GDF-9 increased (P<0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P<0.05) progesterone production (by 28%), increased (P<0.05) cell numbers (by 60%), and had no effect (P>0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13-48%) and estradiol (by 20-51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P<0.05) steroidogenesis by 27-42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation. Growth Differentiation Factor-9 Is Anti-Apoptotic during Follicular Development from Preantral to Early Antral Stage. Orisaka M et al. Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the 'penultimate' stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined if and how oocyte-derived growth differentiation factor-9 (GDF-9) and FSH (FSH) regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intra-oocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, while addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the PI3K inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is anti-apoptotic in preantral follicles, and protects granulosa cells from undergoing apoptosis via activation of the PI3K/Akt pathway. Adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia. Growth Differentiation Factor 9 Is Antiapoptotic during Follicular Development from Preantral to Early Antral Stage. Orisaka M et al. Ovarian follicular atresia represents a selection process that ensures the release of only healthy and viable oocytes during ovulation. The transition from preantral to early antral stage is the penultimate stage of development in terms of gonadotropin dependence and follicle destiny (survival/growth vs. atresia). We have examined whether and how oocyte-derived growth differentiation factor 9 (GDF-9) and FSH regulate follicular development and atresia during the preantral to early antral transition, by a novel combination of in vitro gene manipulation (i.e. intraoocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Injection of GDF-9 antisense suppressed basal and FSH-induced preantral follicle growth in vitro, whereas addition of GDF-9 enhanced basal and FSH-induced follicular development. GDF-9 antisense activated caspase-3 and induced apoptosis in cultured preantral follicles, a response attenuated by exogenous GDF-9. GDF-9 increased phospho-Akt content in granulosa cells of early antral follicles. Although granulosa cell apoptosis induced by ceramide was attenuated by the presence of GDF-9, this protective effect of GDF-9 was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002 and a dominant negative form of Akt. Injection of GDF-9 antisense decreased FSH receptor mRNA levels in cultured follicles, a response preventable by the presence of exogenous GDF-9. The data suggest that GDF-9 is antiapoptotic in preantral follicles and protects granulosa cells from undergoing apoptosis via activation of the phosphatidylinositol 3-kinase/Akt pathway. An adequate level of GDF-9 is required for follicular FSH receptor mRNA expression. GDF-9 promotes follicular survival and growth during the preantral to early antral transition by suppressing granulosa cell apoptosis and follicular atresia. The Effects of Immunizing Sheep with Different BMP15 or GDF9 Peptide Sequences on Ovarian Follicular Activity and Ovulation Rate. McNatty KP et al. The aims of these studies were to determine the ability of antisera against different regions of ovine bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) to inhibit ovarian follicular activity, estrus (mating) and ovulation in sheep. The 9-15 mer peptides conjugated to keyhole limpet hemocyanin (KLH) were designed to generate antibodies against the flexible N-terminal regions of the mature protein as well as against regions where dimerisation of the protein or interaction with a type1 BMP or a type 2 TGFB or BMP receptor were predicted to occur. Ewes (10 per treatment group) were vaccinated with KLH or the KLH-BMP15 (N=9 different peptides) or KLH-GDF9 (N=10) peptides in Freund's adjuvant at 5 consecutive monthly intervals. Overall, the antisera generated against peptides corresponding to the amino acid residues 1-15 of the N-terminal end of the BMP15 or GDF9 mature protein or GDF9 amino acid residues 21-34 were the most potent at inhibiting ovulation following a primary and single booster vaccination. Several other BMP15 (8/9) or GDF9 (6/10) treatment groups but not KLH alone also led to significant reductions in the numbers of animals ovulating but either 2, 3 or 4 booster vaccinations were required. Anovulation was commonly associated with an inhibition of normal ovarian follicular development and anestrus. The in vitro neutralization studies with IgG from the BMP15 or GDF9 immunized ewes showed that the average inhibition of BMP15 plus GDF9 stimulation of (3)H-thymidine uptake by rat granulosa cells was around 70% for animals with no corpora lutea (CL) whereas for animals with 1-3 or > 3 CL, the mean inhibition was between 24-33% and 27-42% respectively. In summary these data suggest that reagents that block the biological actions of BMP15 or GDF9 at their N-terminal regions have potential as contraceptives or as sterilizing agents.Oocyte-Secreted Factor Activation of SMAD 2/3 Signaling Enables Initiation of Mouse Cumulus Cell Expansion. Dragovic RA et al. Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. TGFB superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR oocytes were found to express the two activin subunits, Inhba and Inhba, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin binding protein, neutralized activin-induced expansion, but had no effect on oocyte-induced expansion. The type-I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B- and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized EGF-stimulated oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A- and oocyte-induced OOX expression of hyaluronan synthase-2, tumour necrosis factor alpha-induced protein-6, prostaglandin synthase-2 and pentraxin-3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion. Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice. Yeo CX et al. BACKGROUND Successful oocyte in vitro maturation (IVM) would eliminate the need for hormonal stimulation used in assisted reproduction techniques. Unfortunately, oocytes matured in vitro have compromised developmental competence possibly due to disrupted oocyte-cumulus communication resulting from inappropriate levels of oocyte-secreted factors such as growth differentiation factor 9 (GDF9). Hence, the aim of this study was to investigate the effects of exogenous GDF9 during IVM of mouse oocytes on development and subsequent fetal viability. METHODS Cumulus-oocyte complexes from pregnant mare's serum gonadotrophin primed mice were cultured with or without 200 ng/ml exogenous recombinant GDF9, 50 mIU/ml FSH and 10 ng/ml epidermal growth factor (EGF). After 18 h, cumulus expansion was scored and oocytes were fertilized in vitro. Cleavage, blastocyst development, blastocyst total, inner cell mass (ICM) and trophectoderm cell numbers were assessed. Viability of embryos was assessed by transfer to recipient females and pregnancy outcome determined at day 15. RESULTS Oocytes matured with exogenous GDF9 in the presence of FSH and EGF had higher rates of development, percentage of hatching blastocyst and blastocyst total and ICM cell numbers (all P < 0.05). Although implantation rate and fetal and placental weights were not affected, the number of viable fetuses at day 15 was increased with exogenous GDF9. CONCLUSIONS Exogenous GDF9 during IVM improved embryo development and fetal viability and provides a promising approach for human IVM. Growth Differentiation Factor 9 (GDF9) Stimulates Proliferation and Inhibits Steroidogenesis by Bovine Theca Cells: Influence of Follicle Size on Responses to GDF9. Spicer LJ et al. Ovarian follicular development is controlled by numerous paracrine and endocrine regulators including oocyte-derived growth differentiation factor 9 (GDF9) and a localized increase in bioavailable IGF1. The effects of GDF9 on function of theca cells collected from small (3 to 6 mm) and large (8 to 22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis as measured by a (3)H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells but had no effect on IGF1R, STAR or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated Similar to Mothers Against Decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development. Growth Differentiation Factor 9 Promotes Rat Preantral Follicle Growth by Up-regulating Follicular Androgen Biosynthesis. Orisaka M et al. 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-9 (GDF-9) during preantral follicle growth is incomplete. We have 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. intra-oocyte injection of GDF-9 antisense oligos) and preantral follicle culture. Intra-oocyte 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 non-aromatizable androgen 5alpha-dihydrotestosterone (DHT) 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. Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells. Sasseville M et al. Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.
Expression regulated by FSH, NOBOX, thyroid hormone
Comment GDF9 is Transiently Expressed in Oocytes before Follicle Formation in the Human Fetal Ovary and is Regulated by a Novel NOBOX Transcript. Bayne RA et al. (2015) During human fetal ovary development, the process of primordial follicle formation is immediately preceded by a highly dynamic period of germ cell and somatic cell reorganisation. This is regulated by germ-cell specific transcription regulators, by the conserved RNA binding proteins DAZL and BOLL and by secreted growth factors of the TGFβ family, including activin βA: these all show changing patterns of expression preceding follicle formation. In mice, the transcription factor Nobox is essential for follicle formation and oocyte survival, and NOBOX regulates the expression of GDF9 in humans. We have therefore characterised the expression of GDF9 in relation to these known key factors during follicle formation in the human fetal ovary. mRNA levels of GDF9, BMP15 and NOBOX were quantified by qRT-PCR and showed dramatic increases across gestation. GDF9 protein expression was localised by immunohistochemistry to the same population of germ cells as those expressing activin βA prior to follicle formation but did not co-localise with either BOLL or DAZL. A novel NOBOX isoform was identified in fetal ovary that was shown to be capable of up-regulating the GDF9 promoter in reporter assays. Thus, during oogenesis in humans, oocytes go through a dynamic and very sharply demarcated sequence of changes in expression of these various proteins, even within individual germ cell nests, likely to be of major functional significance in determining selective germ cell survival at this key stage in ovarian development. Transcriptional variation may contribute to the range of age of onset of POI in women with NOBOX mutations.////////////////// A reporter promoter assay confirmed the role of a distal promoter NOBOX binding element in enhancing expression of GDF9 gene in buffalo oocytes. Roy B et al. 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 1.8kb 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. Growth Differentiation Factor-9 Mediates Follicle-Stimulating Hormone-Thyroid Hormone Interaction in the Regulation of Rat Preantral Follicular Development. Kobayashi N et al. FSH regulates follicular growth in a stage-development fashion. Although preantral follicle stage is gonadotropin responsive, FSH is not required for preantral follicular growth. With the antrum, the follicles continue growing under the influence of FSH and become gonadotropin dependent. Although thyroid hormone is important for normal female reproductive function, its role and interaction with FSH in the regulation of preantral ovarian follicular growth is yet to be defined. In the present study, we have examined the action and interaction of FSH and T3 in the regulation of the growth of preantral follicles, especially in their transition from preantral to early antral stage, using an established follicle culture system and evaluated the involvement of growth differentiation factor-9 (GDF-9) in this process in vitro. We have demonstrated that although T3 alone had no effect on follicular development, it markedly enhanced FSH-induced preantral follicular growth. Although FSH alone significantly down-regulated FSH receptor (FSHR) mRNA abundance in the preantral follicles and T3 alone was ineffective, expression of the message was significantly increased in the presence of both hormones. In addition, intra-oocyte injection of GDF-9 antisense oligonucleotides (GDF-9 morpholino) induced follicular cell apoptosis and suppressed follicular growth induced by FSH and T3. These responses were attenuated by exogenous GDF-9. Our findings support the concept that thyroid hormone regulates ovarian follicular development through its direct action on the ovary and that promotes FSH-induced preantral follicular growth through up-regulation of FSHR, a mechanism dependent on the expression and action of oocyte-derived GDF-9. Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function in ruminants McNatty KP, et al . Regulation of Growth Differentiation Factor 9 Expression in Oocytes In Vivo: A Key Role of the E-Box. Yan C et al. Growth differentiation factor 9 (GDF9) is preferentially expressed in oocytes and is essential for female fertility. To identify regulatory elements that confer high-level expression of GDF9 in the ovary but repression in other tissues, we generated transgenic mice in which regions of the Gdf9 locus were fused to reporter genes. Two transgenes (-10.7/+5.6mGdf9-GFP) and (-3.3/+5.6mGdf9-GFP) that contained sequences either 10.7kb or 3.3kb upstream, and 5.6kb downstream of the Gdf9 initiation codon demonstrated expression specifically in oocytes, thereby mimicking endogenous Gdf9 expression. In contrast, transgenes, -10.7mGdf9-Luc and - 3.3mGdf9-Luc, which lacked the downstream 5.6kb region, demonstrated reporter expression not only in oocytes but also high expression in male germ cells. This suggests that the downstream 5.6kb sequence contains a testis-specific repressor element, and 3.3kb of 5' flanking sequence contains all of the cis-acting elements for directing high expression of Gdf9 to female (and male) germ cells. To define sequences responsible for oocyte expression of Gdf9, we analyzed sequences of GDF9 genes from 16 mammalian species. The proximal ~400bp upstream of these GDF9 genes are highly conserved and contain a perfectly conserved E-box (CAGCTG) sequence. When this 400bp region was placed upstream of a luciferase reporter (- 0.4mGdf9-Luc), oocyte-specific expression was observed. However, a similar transgene construct (-0.4MUT-mGdf9-Luc) with a mutation in the E-box, abolished oocyte expression. Likewise, the presence of an E-box mutation in a longer construct (-3.3MUT-mGdf9-Luc) abolished expression in ovary but not in testis. These observations indicate that the E-box is a key regulatory sequence for Gdf9 expression in the ovary. Expression of GDF-9 in the oocytes is essential for the development of primordial follicles in the hamster ovary. Wang C et al. Postnatal GDF-9 expression in the hamster oocytes precedes the formation of primordial follicles. We examined the functional significance of GDF-9 in primordial folliculogenesis in the hamster ovary using RNAi knockdown of GDF-9 mRNA and protein expression. Fifteen-day-old fetal (E15) ovaries were cultured for 9 days with or without 1 ng FSH, 1 microl Metafectane, 100 nM control non-targeting siRNA, GDF-9 siRNA, or GDF-9 siRNA + FSH, and the development of primordial follicles examined. The efficiency of siRNA transfecting ovarian cells in the organ culture was tested by culturing ovaries with siGlo, a non-targeting control siRNA labeled with Cy3. More than 90% cells in the ovary were siGlo positive, and neither the Metafectane nor the siRNA induced cellular apoptosis. Control siRNA did not affect the basal levels of GDF-9 mRNA, but GDF-9 siRNA slightly but significantly reduced the level. FSH markedly up-regulated the levels of GDF-9 mRNA and protein, and the effect was completely suppressed by GDF-9 siRNA. However, GDF-9 siRNA did not affect the levels of BMPRIA or beta-actin mRNA. GDF-9 siRNA alone also reduced GDF-9 protein expression. Concurrent with GDF-9 expression, FSH significantly augmented primordial follicle formation, but the effect was abolished by GDF-9 siRNA. These results suggest that endogenous GDF-9 plays an important role in somatic cell differentiation and the formation of primordial follicles. Further, FSH, by virtue of regulating GDF-9 expression, modulates oocyte regulation of primordial follicles formation. Growth differentiation factor-9 inhibits 3'5'-adenosine monophosphate-stimulated steroidogenesis in human granulosa and theca cells. Yamamoto N et al. Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor superfamily, modulates the development and function of granulosa and theca cells. Targeted deletion of GDF-9 in the mouse revealed that GDF-9 was essential for the establishment of the thecal cell layer during early folliculogenesis. During later stages of follicular development, the roles of GDF-9 are less well understood, but it has been postulated that oocyte-derived GDF-9 may prevent premature luteinization of follicular cells, based on its ability to modulate steroidogenesis by rodent ovarian cells. In the rodent, GDF-9 is expressed solely by the oocyte from the early primary follicular stage through ovulation. Recent studies in the rhesus monkey demonstrated that granulosa cells express GDF-9, suggesting a broader role for this protein in ovarian function in primates. We examined the effect of recombinant GDF-9 on proliferating human granulosa and thecal cell steroidogenesis and the expression of steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage, and P450 aromatase. We also examined granulosa cell GDF-9 expression by quantitative RT-PCR and by Western analysis. GDF-9 inhibited 8-Br-cAMP-stimulated granulosa progesterone synthesis by approximately 40%, but did not affect basal progesterone production. Concordant with reduced steroid production, 8-Br-cAMP-stimulated StAR protein expression was reduced approximately 40% in granulosa cells, as were expression of StAR mRNA and StAR promoter activity. Additionally, GDF-9 inhibited 8-Br-cAMP-stimulated expression of P450 side-chain cleavage and P450 aromatase. Human granulosa cells expressed GDF-9, as determined by RT-PCR and Western analysis. Treatment of human thecal cells with GDF-9 blocked forskolin-stimulated progesterone, 17alpha-hydroxyprogesterone, and dehydroepiandrosterone synthesis. Thecal cells exhibited greater sensitivity to GDF-9, suggesting that this cell may be a primary target of GDF-9. Moreover, GDF-9 increased thecal cell numbers during culture, but had no effect on granulosa cell growth. Our findings implicate GDF-9 in the modulation of follicular steroidogenesis, especially theca cell function. Because GDF-9 mRNA and protein are detectable in granulosa-lutein cells after the LH surge, the concept of GDF-9 as a solely oocyte-derived luteinization inhibitor needs to be reevaluated. Characterization of nobox DNA binding specificity and its regulation of GDF9 and POU5F1 promoters. Choi Y et al. Nobox (newborn ovary homeobox gene) deficiency disrupts early folliculogenesis and the expression of oocyte-specific genes in mice. Here, we identified several cis-acting sites, TAATTG, TAGTTG and TAATTA as a NOBOX DNA binding elements (NBEs) using a library of randomly generated oligonucleotides by cyclic amplification of sequence target assay and mutation analyses. We show that NOBOX preferentially binds to the NOBOX binding elements with high affinity. In addition, we found that promoter regions of mouse Pou5f1 and Gdf9 contain one (-426) and three NOBOX binding elements (-786, -967, and -1259), respectively. NOBOX bound to these putative NOBOX binding elements with high affinity and increased transcriptional activity of luciferase reporter driven by mouse Pou5f1 and Gdf9 promoter containing the NOBOX binding elements. In chromatin immunoprecipitation assays, DNA sequences from Pou5f1 and Gdf9 promoters co-precipitated with anti-NOBOX antibody. These results suggest that NOBOX may directly regulate the transcription of Pou5f1 and Gdf9 in the oocyte during early folliculogenesis.
Ovarian localization Oocyte, Follicular Fluid
Comment Influence of follicular fluid GDF9 and BMP15 on embryo quality. Gode F et al. (2011) To evaluate the association between follicular fluid levels of propeptide and mature forms of growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 with subsequent oocyte and embryo quality. Prospective clinical study. University hospital. Eighty-one infertile patients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). The expression levels of the propeptide and mature forms of follicular fluid GDF9 and BMP15 were determined by western blot analysis. The levels of follicular fluid hormones (FSH, E2, and P) were measured with automated chemiluminescent enzyme immunoassays. The relationships between the levels of GDF9 and BMP15, hormones, oocyte maturation, and embryo quality. Mature GDF9 levels were significantly correlated with the nuclear maturation of oocytes. The mean mature GDF9 level was 4.87±0.60 in the high-embryo-quality group and 1.45±0.81 in the low-embryo-quality group. There were no statistically significant differences in embryo quality among the patients regarding propeptide GDF9 and BMP15 expression status. There was a negative correlation between follicular fluid levels of P and the mature form of GDF9. Higher mature GDF9 levels in the follicular fluid were significantly correlated with oocyte nuclear maturation and embryo quality.////////////////// GDF-9 mRNA was shown to be expressed exclusively in oocytes (McGrath et al., 1995). GDF-9 protein was shown to be present in rat oocytes using immunoblots with specific antibodies (Hayashi et al., 1999). Expression and Function of Growth Differentiation Factor-9 in an Oviparous Species, Gallus domesticus Johnson PA,et al .
Follicle stages Primary, Secondary, Antral, Preovulatory
Comment GDF-9 mRNA is found in oocytes of all follicular stages except primordials (McGrath et al., 1995).The protein was found in oocytes of primary, secondary and larger rat follicles (Hayashi et al., 1999). In contrast to studies in mice and rats in which GDF-9 was first detected beginning at the primary (type 2) follicle stage, in ovine and bovine ovaries GDF-9 mRNA was expressed beginning at the primordial (type 1) follicle stage (Bodensteiner et al., 1999). In human, both GDF-9 mRNA and protein are abundantly expressed in oocytes of primary follicles (Aaltonen et al., 1999). Expression of growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15) and anti-mullerian hormone (AMH) in cultured mouse primary follicles. Sadeu JC et al. Growth and differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15) and anti-mullerian hormone (AMH) play an important role in the primary-to-secondary follicle transition and follicle activation in vivo. In organ culture of neonatal mouse ovaries, it was observed that significantly less primary follicles develop to the secondary stage. The objectives of this study were: 1) to compare ovarian follicular populations between organ-cultured neonatal mouse ovaries and freshly isolated age-matched control ovaries; 2) to quantify RNA levels of GDF-9, BMP-15 and AMH, in cultured primary follicles; and 3) to immunolocalize GDF-9 and AMH in cultured ovaries. Ovaries from 3-day-old (PND 3) mice were cultured for 7 or 10 days in absence or presence of FSH. Follicular populations were counted in freshly isolated 13-day-old (PND 13) ovaries and organ-cultured ovaries. Transcripts were quantified in isolated primary follicles using real-time RT-PCR, and protein expressions were localized using immunohistochemistry. The number of secondary follicles in organ cultured ovaries was significantly lower than in vivo, controls. GDF-9 and BMP-15 mRNA expression levels were similar as in controls. AMH mRNA levels were significantly (P < 0.05) lower after day 10 of culture in absence of FSH. GDF-9 and AMH proteins were respectively detected in the oocytes and granulosa cells (GC) beginning at the primary and primordial stage onward. GDF-9 and BMP-15 production in cultured primary follicles are not different from in vivo controls; hence abnormal early follicle growth was not related to a deficient transcription of these factors.
Phenotypes POF (premature ovarian failure)
Mutations 16 mutations

Species: ovine
Mutation name: None
type: naturally occurring
fertility: infertile - ovarian defect
Comment: Mutations in the Genes for Oocyte-Derived Growth Factors GDF9 and BMP15 Are Associated with Both Increased Ovulation Rate and Sterility in Cambridge and Belclare Sheep (Ovis aries) Hanrahan JP, et al . Belclare and Cambridge are prolific sheep breeds whose origins involved selecting ewes with exceptionally high litter size records from commercial flocks. The variation in ovulation rate in both breeds is consistent with segregation of a gene (or genes) with a large effect on this trait. Sterile ewes, due to a failure of normal ovarian follicle development, occur in both breeds. New naturally occurring mutations in genes for the oocyte derived growth factors GDF9 and BMP15 are described. These mutations are associated with increased ovulation rate in heterozygous carriers and sterility in homozygous carriers in both breeds. This is the first time that a mutation in the gene for GDF9 has been found that causes increased ovulation rate and infertility in a manner similar to inactivating mutations in BMP15, and shows that GDF9 is essential for normal folliculogenesis in sheep. Furthermore it is shown, for the first time in any species, that individuals with mutations in both GDF9 and BMP15 have a greater ovulation rate than sheep with either of the mutations separately.

Species: mouse
Mutation name: None
type: null mutation
fertility: None
Comment: Differential Oocyte-Specific Expression of Cre Recombinase Activity in GDF-9-iCre, Zp3cre, and Msx2Cre Transgenic Mice. Lan ZJ,et al . Oocyte-specific deletion of ovarian genes using Cre/loxP technology provides an excellent tool to understand their physiological roles during folliculogenesis, oogenesis, and preimplantation embryonic development. We have generated a transgenic mouse line expressing improved Cre recombinase (iCre) driven by the mouse growth differentiation factor-9 (GDF-9) promoter. The resulting transgenic mouse line was named GDF-9-iCre mice. Using the floxed ROSA reporter mice, we found that Cre recombinase was expressed in postnatal ovaries, but not in heart, liver, spleen, kidney, and brain. Within the ovary, the Cre recombinase was exclusively expressed in the oocytes of primordial follicles and follicles at later developmental stages. The expression of iCre of GDF-9-iCre mice was shown to be earlier than the Cre expression of Zp3Cre and Msx2Cre mice, in which the Cre gene is driven by zona pellucida protein 3 (Zp3) promoter and a homeobox gene Msx2 promoter, respectively, in the postnatal ovary. Breeding wild-type males with heterozygous floxed germ cell nuclear factor (GCNF) females carrying the GDF-9-iCre transgene did not produce any progeny having the floxed GCNF allele, indicating that complete deletion of the floxed GCNF allele can be achieved in the female germline by GDF-9-iCre mice. These results suggest that GDF-9-iCre mouse line provides an excellent genetic tool for understanding functions of oocyte-expressing genes involved in folliculogenesis, oogenesis, and early embryonic development. Comparison of the ontogeny of the Cre activities of GDF-9-iCre, Zp3Cre, and Msx2Cre transgenic mice shows there is sequential Cre activity of the three transgenes that will allow inactivation of a target gene at different points in folliculogenesis.

Species: mouse
Mutation name: None
type: null mutation
fertility: subfertile
Comment: Synergistic roles of BMP15 and GDF9 in the development and function of the oocyte-cumulus cell complex in mice: genetic evidence for an oocyte-granulosa cell regulatory loop. Su YQ, et al . Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-specific growth factors that appear to play key roles in granulosa cell development and fertility in most mammalian species. We have evaluated the role(s) of these paracrine factors in the development and function of both the cumulus cells and oocytes by assessing cumulus expansion, oocyte maturation, fertilization, and preimplantation embryogenesis in Gdf9(+/-)Bmp15(-/-) [hereafter, double mutant (DM)] mice. We found that cumulus expansion, as well as the expression of hyaluronon synthase 2 (Has2) mRNA was impaired in DM oocyte-cumulus cell complexes. This aberrant cumulus expansion was not remedied by coculture with normal wild-type (WT) oocytes, indicating that the development and/or differentiation of cumulus cells in the DM, up to the stage of the preovulatory luteinizing hormone (LH) surge, is impaired. In addition, DM oocytes failed to enable FSH to induce cumulus expansion in WT oocytectomized (OOX) cumulus. Moreover, LH-induced oocyte meiotic resumption was significantly delayed in vivo, and this delayed resumption of meiosis was correlated with the reduced activation of mitogen-activated protein kinase (MAPK) in the cumulus cells, thus suggesting that GDF9 and BMP15 also regulate the function of cumulus cells after the preovulatory LH surge. Although spontaneous in vitro oocyte maturation occurred normally, oocyte fertilization and preimplantation embryogenesis were significantly altered in the DM, suggesting that the full complement of both GDF9 and BMP15 are essential for the development and function of oocytes. Because receptors for GDF9 and BMP15 have not yet been identified in mouse oocytes, the effects of the mutations in the Bmp15 and Gdf9 genes on oocyte development and functions must be produced indirectly by first affecting the granulosa cells and then the oocyte. Therefore, this study provides further evidence for the existence and functioning of an oocyte-granulosa cell regulatory loop.

Species: human
Mutation name: None
type: naturally occurring
fertility: subfertile
Comment: Mutations and sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. Laissue P et al. Background and objective: Mutations in bone morphogenic protein 15 (BMP15) and growth/differentiation factor 9 (GDF9) lead to altered fertility in animal models. In the human, a heterozygous point mutation of BMP15 has been associated with premature ovarian failure (POF). Subject and methods: We have directly sequenced both genes in a cohort of 203 POF patients presenting with primary or secondary amenorrhea and high FSH levels and in a control population including 54 women with regular menstrual cycles who had at least one child. RESULTS: We have identified several heterozygous variants. One alteration in GDF9 (S186Y) and one in BMP15 (L148P) may have pathogenic effects as both positions are conserved in vertebrate species, ranging from the chicken to mammals. These variants were absent in the control samples. We also found synonymous and neutral substitutions. CONCLUSIONS: We propose that although mutations in BMP15 and GDF9 are not a major cause of ovarian insufficiency, they may be involved in POF. Identification of novel missense mutations of GDF9 in Chinese women with polycystic ovary syndrome. Wang B et al. The gene for growth differentiation factor 9 (GDF9) is expressed in human oocytes and has an important function in regulating early follicle growth and fertility. Polycystic ovary syndrome (PCOS) is one of the common defects that causes ovary dysfunction and is linked to aberrant processes in folliculogenesis. Previous studies have discovered several mutations in the screening of GDF9 in premature ovarian failure but none in PCOS. This current study focused on the mutational analysis of the coding region of GDF9 among 216 Chinese PCOS patients. Of the 10 different variants found in this study, five novel missense mutations in GDF9 were discovered namely c.15C>G, c.118T>G, c.133A>G, c.1025A>T and c.1275C>A. The above-mentioned mutations indicate GDF9 may be potentially associated with PCOS patients. As far as is known, this study is the first to provide evidence for such an association.

Species: human
Mutation name: None
type: naturally occurring
fertility: fertile
Comment: Novel variants in growth differentiation factor 9 in mothers of dizygotic twins Palmer JS,et al . CONTEXT: Genes from the ovarian bone morphogenetic signaling pathway (GDF9 and BMP15) are critical for normal human fertility. We previously identified a deletion mutation in GDF9 in sisters with spontaneous dizygotic (DZ) twins, but the prevalence of rare GDF9 variants in twinning families is unknown. OBJECTIVE: The objective was to evaluate the frequency of rare variants in GDF9 in families with a history of DZ twinning. DESIGN AND SUBJECTS: We recruited 3450 individuals from 915 DZ twinning families (1693 mothers of twins) and 1512 controls of Caucasian origin. One mother of DZ twins was selected from 279 of the 915 families, and a DNA sample was screened for rare variants in GDF9 using denaturant HPLC. Variants were confirmed by DNA sequencing and genotyped in the entire sample by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. RESULTS: We found two novel insertion/deletions (c.392-393insT, c.1268-1269delAA) and four missense alterations in the GDF9 sequence in mothers of twins. Two of the missense variants (c.307C > T, p.Pro103Ser and c.362C > T, p.Thr121Leu) were located in the pro-region of GDF9 and two (c.1121C > T, p.Pro374Leu and c.1360C > T, p.Arg454Cys) in the mature protein region. For each variant, the frequencies were higher in cases compared with controls. The proportion of mothers of DZ twins carrying any variant (4.12%) was significantly higher (P < 0.0001) than the proportion of carriers in controls (2.29%). CONCLUSION: We describe new variants in the GDF9 gene that are significantly more common in mothers of DZ twins than controls, suggesting that rare GDF9 variants contribute to the likelihood of DZ twinning.

Species: ovine
Mutation name: None
type: naturally occurring
fertility: infertile - ovarian defect
Comment: Homozygosity for a single base pair mutation in the oocyte-specific GDF9 gene results in sterility in Thoka sheep. Nicol L et al. The control of fecundity is critical in determining mammalian offspring survival. It is regulated principally by the ovulation rate, so that primates and large farm species commonly have single offspring. Previously, several mutations have been identified in sheep which increase the naturally low ovulation rate, although in some cases homozygous ewes are infertile. In the present study we present a detailed characterisation of a novel mutation in growth differentiation factor 9 (GDF9), found in Icelandic Thoka sheep. This mutation is a single base change (A1279C) resulting in a non-conservative amino acid change (S109R) in the C-terminus of the mature GDF9 protein, which is normally expressed in oocytes at all stages of development. Genotyping all animals for which reproductive records were available confirmed this mutation to be associated with increased fecundity in heterozygous ewes and infertility in homozygotes. Analysis of homozygote ovarian morphology and a number of genes normally activated in growing follicles showed that GDF9 was not involved in oocyte activation, but in subsequent development of the follicle. This study highlights the importance of oocyte factors in regulating fertility and provides new information for structural analysis and investigation of the potentially important sites of dimerization or translational modifications required to produce biologically active GDF9. It also provides the basis for the utilisation of these animals to enhance sheep production.

Species: human
Mutation name: None
type: naturally occurring
fertility: subfertile
Comment: G546A polymorphism of growth differentiation factor-9 contributes to the poor outcome of ovarian stimulation in women with diminished ovarian reserve. Wang TT et al. The growth differential factor-9 (GDF-9) gene, an oocyte-specific factor, was screened in 106 Chinese women with diminished ovarian reserve (DOR), and three single-nucleotide polymorphisms, c.G169A, c.C447T and c.G546A, were detected. We found GDF-9 c.G546A, but not c.G169A or c.C447T, to be correlated with the poor ovarian stimulation and in vitro fertilization outcomes in women with DOR.

Species: None
Mutation name: None
type: naturally occurring
fertility: fertile
Comment: A new polymorphism in the Growth and Differentiation Factor 9 (GDF9) gene is associated with increased ovulation rate and prolificacy in homozygous sheep. Silva BD et al. Summary Brazilian Santa In?(SI) sheep are very well-adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are the first two genes expressed by the oocyte to be associated with an increased ovulation rate in sheep. All GDF9 and BMP15 variants characterized, until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study, we have found a new allele of GDF9, named FecG(E) (Embrapa), which leads to a substitution of a phenylalanine with a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecG(E) allele have shown an increase in their ovulation rate (82%) and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.

Species: human
Mutation name: None
type: naturally occurring
fertility: subfertile
Comment: Identification of a mutation in GDF9 as a novel cause of diminished ovarian reserve in young women. Wang TT 2013 et al. STUDY QUESTION Do any mutations in growth differentiation factor 9 (GDF9) have a role in diminished ovarian reserve (DOR) in young women? SUMMARY ANSWER The GDF9 p.R146C mutation may be a source of DOR in some young women. WHAT IS KNOWN ALREADY DOR affects 10% of women under 37 years of age and is associated with accelerated expenditure of follicles. GDF9 is an oocyte-secreted factor that plays a critical role in follicular development and female fertility. Several GDF9 variants have been linked to ovarian dysfunction. STUDY DESIGN, SIZE, DURATION This case-control study included 139 women with DOR and 152 controls aged under 37 years. PARTICIPANTS/MATERIALS, SETTING, METHODS All women were recruited in a Chinese tertiary center and underwent DNA sequencing of GDF9 gene. We then determined the molecular and biological properties of mutant GDF9 proteins using protein expression, structural prediction and functional analyses. MAIN RESULTS AND THE ROLE OF CHANCE We identified two mutations in the proregion of GDF9 gene: c.169T > G (p.D57Y) and c.436T > C (p.R146C). The p.R146C mutation was found in three women with DOR but was absent in the control population. This mutation was also associated with significant reductions in GDF9 mature protein secretion in cultured cells. Functional studies with human granulosa cells (GCs) showed that the p.R146C mutation reduced the abilities of GDF9 to stimulate GC proliferation and to activate the Smad2 pathway. Protein structure modeling predicted that p.R146C disrupted an a-helix in GDF9 protein. In contrast with p.R146C, the p.D57Y mutation, found in both the DOR and control groups (6 versus 2), had no obvious deleterious effects. LIMITATIONS, REASONS FOR CAUTION Larger studies in varying populations may validate the role of GDF9 mutation in young women with DOR. WIDER IMPLICATIONS OF THE FINDINGS These results may provide new insights into the pathophysiological mechanisms of early-onset DOR. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China (30900517 and 81100468), the National Basic Research Program of China (2012CB944901 and 2011CB944502), the Fundamental Research Funds for the Central Universities, Ministry of Education, China (2011KYJD008), Program for Changjiang Scholars and Innovative Research Team in University (IRT1184) and Program for Key Subjects Group of Reproductive Medicine at Zhejiang Province (XKQ-009-002). The authors have no competing interests to declare. /////////////////////////

Species: human
Mutation name: None
type: naturally occurring
fertility: subfertile
Comment: Aberrant GDF9 expression and activation is associated with common human ovarian disorders. Simpson CM 2014 et al. Context. Growth differentiation factor 9 (GDF9) is a central regulator of folliculogenesis and ovulation rate. Fourteen mutations in human (h)GDF9 have been reported in women with premature ovarian failure (POF) or polycystic ovarian syndrome (PCOS), as well as in mothers of dizygotic (DZ) twins, implicating GDF9 in the aetiology of these conditions. We sought to determine how these mutations alter the biological activity of hGDF9. Objective. To determine whether aberrant GDF9 expression or activation is associated with common ovarian disorders. Design. Homology modelling was used to predict the location of individual mutations within structurally important regions of the pro- and mature- domains of hGDF9. Each hGDF9 variant was generated by site-directed mutagenesis, expressed from human embryonic kidney (HEK293T) cells and assessed as to whether it resulted in defective production or enhanced activation of mature hGDF9 in an in vitro granulosa cell proliferation bioassay. Results. Mutations observed in mothers of DZ twins (P103S and P374L) completely abrogated GDF9 expression, suggesting that women heterozygous for these mutations would have a 50% reduction in GDF9 levels. Comparable declines in GDF9 in ewes result in a 2-fold increase in ovulation rate and fecundity. Remarkably, three prodomain mutations associated with POF (S186Y, V216M and T238A) all resulted in activation of hGDF9. Mechanistically, these mutations reduced the affinity of the prodomain for mature hGDF9, allowing the growth factor to more readily access its signalling receptors. Conclusions. Together, these findings indicate that alterations to hGDF9 synthesis and activity can contribute to the most common ovarian pathologies. /////////////////////////

Species: None
Mutation name: None
type: naturally occurring
fertility: infertile - ovarian defect
Comment: Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes. Souza CJ 2014 et al. Litter size (LS) in sheep is determined mainly by ovulation rate (OR). Several polymorphisms have been identified in the growth differentiation factor 9 (GDF9) gene that result in an increase in OR and prolificacy of sheep. Screening the databank of the Brazilian Sheep Breeders Association for triplet delivery, we identified flocks of prolific Ile de France ewes. After resequencing of GDF9, a point mutation (c.943C>T) was identified, resulting in a non-conservative amino acid change (p.Arg315Cys) in the cleavage site of the propeptide. This new allele was called Vacaria (FecG(v) ). A flock of half-sib ewes was evaluated for OR in the first three breeding seasons, and Vacaria heterozygotes had higher OR (P?

Species: None
Mutation name:
type: naturally occurring
fertility: fertile
Comment: Maximum-likelihood approaches reveal signatures of positive selection in BMP15 and GDF9 genes modulating ovarian function in mammalian female fertility. Ahmad HI et al. (2017) Bone morphogenetic proteins (BMPs) and the growth factors (GDFs) play an important role in ovarian folliculogenesis and essential regulator of processes of numerous granulosa cells. BMP15 gene variations linked to various ovarian phenotypic consequences subject to the species, from infertility to improved prolificacy in sheep, primary ovarian insufficiency in women or associated with minor subfertility in mouse. To study the evolving role of BMP15 and GDF9, a phylogenetic analysis was performed. To find out the candidate gene associated with prolificacy in mammals, the nucleotide sequence of BMP15 and GDF9 genes was recognized under positive selection in various mammalian species. Maximum-likelihood approaches used on BMP15 and GDF9 genes exhibited a robust divergence and a prompted evolution as compared to other TGFβ family members. Furthermore, among 32 mammalian species, we identified positive selection signals in the hominidae clade resulting to 132D, 147E, 163Y, 191W, and 236P codon sites of BMP15 and 162F, 188K, 206R, 240A, 244L, 246H, 248S, 251D, 253L, 254F and other codon sites of GDF9. The positively selected amino acid sites such as Alanine, Lucien, Arginine, and lysine are important for signaling. In conclusion, this study evidences that GDF9 and BMP15 genes have rapid evolution than other TGFß family members and was subjected to positive selection in the mammalian clade. Selected sites under the positive selection are of remarkable significance for the particular functioning of the protein and consequently for female fertility.//////////////////

Species: ovine
Mutation name:
type: naturally occurring
fertility: fertile
Comment: Three Complete Linkage SNPs of GDF9 Gene Affect the Litter Size Probably Mediated by OCT1 in Hu Sheep. Li Y et al. (2020) Growth differentiation factor (GDF) 9 gene is involved in regulating reproductive traits in animals, but little is known about the promoter, single-nucleotide polymorphisms (SNPs), transcription factor binding sites, and regulating mechanism of GDF9 gene. In this study, the SNPs in the GDF9 promoter region were explored and their transcription mechanisms in regulating GDF9 expression were analyzed. Ear tissues of 267 Hu ewes were collected, and genomic DNA was extracted. GDF9 promoter region was amplified by PCRs, and identified SNPs genotyped by sequencing. SPSS16.0 software was used to analyze the association between genotypes and litter sizes. Flow cytometry assay was used to detect cell apoptosis, and dual-luciferase reporter assay was used to discover the promoter activity. A length of 1789 bp promoter region of GDF9 in Hu sheep was obtained by PCR amplification, and luciferase activity assay showed that there was a negative regulatory element in the region within -725 to -309 bp and a positive regulatory element in the region within -309 to +43 bp. Three complete linkage SNPs at -534A/G, -407T/G, and -332C/T were detected, resulting in three genotypes (namely, AA, AB, and BB). The association analysis indicated that the AA genotype ewes had larger litter size at average parity than those with the BB genotype. The -534A/G mutation created a novel binding site for the octamer transcription factor 1 (OCT1), and the Annexin V FITC/PI flow cytometry assay showed OCT1 promoted cell apoptosis in sheep ovarian granulosa cells. Overexpression of OCT1 considerably inhibited the luciferase activity of both genotypes and the inhibition effect of pGL3-BB was higher than that of pGL3-AA. Three complete linkage SNPs of the GDF9 gene regulate the litter size in Hu sheep probably via inhibition of the promoter activity by binding with OCT1 at -534 GG genotype and forming a complex between OCT1 and CCAAT/enhancer-binding protein (C/EBP).//////////////////

Species: human
Mutation name:
type: naturally occurring
fertility: fertile
Comment: GDF9 polymorphisms: influence on ovarian response in women undergoing controlled ovarian hyperstimulation. Bilibio JP et al. (2020) The study looked into the possible influence of GDF9 polymorphisms on ovarian response in women with a normal ovarian reserve undergoing controlled ovarian hyperstimulation for in vitro fertilization (IVF). This cross-sectional study included 67 women with normal ovarian reserve aged 30-39 years submitted to controlled ovarian hyperstimulation for IVF. We sequenced four polymorphisms in the GDF9 gene (C398G, C447T, G546A, and G646A) and analyzed their influence on follicular and oocyte outcomes. The mutant allele C398G decreased the total number of follicles >17mm (6.49 vs. 4.33, p=0.001), total number of follicles (10.11 vs. 7.33, p=0.032), number of MII oocytes retrieved, and serum progesterone levels on trigger day. The C447T polymorphism was associated with a greater number of follicles between 12 and 14 mm on the day of r-hCG, while the G546A polymorphism was associated with lower serum progesterone levels on trigger day. GDF9 gene polymorphisms C398G and C447T adversely affected ovarian response in women undergoing controlled ovarian hyperstimulation. These findings show that in addition to playing a role in the early stages of folliculogenesis, GDF9 polymorphisms have an important impact on the final stage of oocyte development.//////////////////

Species: human
Mutation name:
type: naturally occurring
fertility: infertile - ovarian defect
Comment: A homozygous truncating variant in GDF9 in siblings with primary ovarian insufficiency. Verma KP et al. (2021) Premature or primary ovarian insufficiency (POI) affects approximately 1% of women and can be due to a variety of causes. Genetic causes include syndromic and non-syndromic POI. There are several promising candidate genes for whom a clear Mendelian association with non-syndromic POI has not yet been conclusively established, including GDF9. GDF9 is an oocyte-secreted factor and is part of the TGF-beta superfamily of morphogens. It has an important role in follicular development and granulosa cell maturation. We report the case of two siblings with primary ovarian insufficiency (POI) and a homozygous truncating variant in GDF9 (c.604C>T; p.(Gln202*). This report helps establish a clear gene-disease association between GDF9 and POI and argues for routine evaluation for GDF9 variants in patients undergoing genomic investigation for POI.//////////////////

Species: mouse
Mutation name: GDF-9 knockout mouse
type: null mutation
fertility: infertile - ovarian defect
Comment: Ovaries from GDF-9-deficient female mice showed that primordial and primary one-layer follicles can be formed, but there is a block in follicular development beyond the primary one-layer follicle stage which leads to complete infertility. Oocyte growth and zona pellucida formation proceed normally, but other aspects of oocyte differentiation are compromised (Dong et al., 1996).
Genomic Region show genomic region
Phenotypes and GWAS show phenotypes and GWAS
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created: July 22, 1999, midnight by: Uschi   email:
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last update: April 12, 2021, 10:41 p.m. by: hsueh    email:



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