Mouse Growth Differentiation Factor 9 Research Articles

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.

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 of 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. Although 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, because of suggested conformational changes during heterodimer formation.

Mouse GDF9 decreases KITL gene expression in human granulosa cells.

Kit ligand (KITL) is an important granulosa cell-derived growth factor in ovarian folliculogenesis, but its expression and function in human granulosa cells are currently poorly understood. Based on studies performed in animal models, it was hypothesised that KITL gene expression in human granulosa cells is regulated by androgens and/or growth differentiation factor 9 (GDF9). We utilised two models of human granulosa cells, the KGN granulosa tumour cell line and cumulus granulosa cells obtained from preovulatory follicles of women undergoing assisted reproduction. Cells were treated with combinations of 5α-dihydrotestosterone (DHT), recombinant mouse GDF9, and the ALK4/5/7 inhibitor SB431542. KITL mRNA levels were measured by quantitative real-time PCR. No change in KITL mRNA expression was observed after DHT treatment under any experimental conditions, but GDF9 treatment resulted in a significant decrease in KITL mRNA levels in both KGN and cumulus cells. The effect of GDF9 was abolished by the addition of SB431542. These results indicate that KITL is not directly regulated by androgen signalling in human granulosa cells. Moreover, this study provides the first evidence that GDF9 negatively regulates KITL gene expression in human granulosa cells providing new information on the regulation of these important growth factors in the human ovary.

Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice

The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

Extra-ovarian expression and activity of growth differentiation factor 9

Growth differentiation factor 9 (GDF9) produced within the ovary plays an essential role during follicle maturation through actions on granulosa cells, but extra-ovarian expression, signalling and actions of GDF9 are less well characterised. The present studies confirm GDF9 expression in the mouse testis, pituitary gland and adrenocortical cancer (AC) cells, and establish its expression in L beta T2 gonadotrophs, and in mouse adrenal glands, particularly foetal and neonatal cortical cells. AC, L beta T2, TM3 Leydig and TM4 Sertoli cells express the requisite GDF9 binding signalling components, particularly activin receptor-like kinase (ALK) 5 and the bone morphogenetic protein (BMP)/GDF type II receptor, BMPRII (BMPR2). We therefore compared GDF9 activation of these potential extra-ovarian target cell types with its activation of granulosa cells. Recombinant mouse GDF9 stimulated expression of activin/transforming growth factor-beta-responsive reporters, pGRAS-luc or pAR3-lux, in TM4 and AC cells (IC50=145 ng/ml in the latter case), and two granulosa cell lines, KGN and COV434. The ALK4/5/7 inhibitor, SB431542, blocked GDF9 activity in each case. By contrast, GDF9 lacked specific effects on TM3 cells and rat primary pituitary and mouse L beta T2 gonadotrophs. Our findings show that GDF9 regulates the expression of R-SMAD2/3-responsive reporter genes through ALK4, 5 or 7 in extra-ovarian (adrenocortical and Sertoli) cells with similar potency and signalling pathway to its actions on granulosa cells, but suggest that expression of BMPRII, ALK5 (TGFBR1) and R-SMADs 2 and 3 may not be sufficient for a cell to respond to GDF9.

121. EXTRACELLULAR ROLES FOR PROREGIONS OF MOUSE BMP15 AND GDF9 IN VIVO

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are essential for normal follicular development and ovulation in mammalian species. As TGF-beta family proteins, BMP15 and GDF9 are expressed as pre-pro-mature proteins, with a signal region prompting extracellular secretion, a proregion that is post-translationally cleaved, and a mature protein that is known to be biologically active as a non-covalently interacting dimer. Until recently, the proregion was thought primarily to facilitate correct folding and dimerization of the mature proteins within the oocyte prior to processing and cleavage. However, our in vitro work with recombinant mouse BMP15 and GDF9 has shown that each of the proregion, mature protein and the non-processed promature protein are secreted from transfected 293H cells. We showed non-covalent interactions existing between the proregion and mature protein of each of BMP15 and GDF9, and between the BMP15 proregion and GDF9 mature protein. Importantly, a mouse BMP15 proregion antibody was able to abolish cooperative BMP15 and GDF9 bioactivity measured using a granulosa cell thymidine incorporation bioassay, providing strong evidence for an extracellular role for the mouse BMP15 proregion. Currently, to find out whether BMP15 and GDF9 proregions have extracellular roles in vivo, our investigation has utilised knock-down of BMP15 and GDF9 proregion proteins by mouse immunisation. Ovaries of mice immunised with the GDF9 proregion had significantly increased numbers of corpora lutea (p&lt;0.005), while ovaries from mice immunised with BMP15 proregion peptides had significantly fewer corpora lutea (p&lt;0.005). These findings provide the first evidence that the proregions of mouse GDF9 and BMP15 have different physiological roles outside the oocyte. Our future aim is to elucidate the function of these proregions, how these may differ between BMP15 and GDF9, and whether these aspects differ between species with different ovulation quota.

272. Signalling pathways involved in mouse GDF9 and BMP15 stimulated thymidine uptake by rat granulosa cells

The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are essential for ovarian follicular growth and development. Understanding the molecular mechanisms of these factors could assist with the development of future products for fertility control. Thymidine uptake by rat granulosa cells is stimulated cooperatively by GDF9 and BMP15. Inhibitors of the activin receptor-like kinase (ALK) 4,5,7 and the nuclear factor kappaB (NFKB) second messenger pathways block ovine GDF9 and BMP15 stimulated thymidine incorporation. The ALK 4,5,7 receptor pathway is known to be essential for the cooperative effects of mouse (m)GDF9 and mBMP15 on thymidine incorporation but the role of other pathways has yet to be determined, which was the focus of this study. Inhibitors of NFKB (Sn50; 10µg/mL), ALK 2,3,6 receptor (Dorsomorphin; 1µM), p38 mitogen-activated protein kinase (p38 MAPK; SB239063; 5 µM) and c-Jun-N-terminal kinase (JNK; TAT-TI-JIP153–163; 5 µM) pathways were each cultured with recombinant mGDF9 (25 ng/mL) and mBMP15 (6 ng/mL) in a rat granulosa cell [3H]-thymidine bioassay. The p38 MAPK inhibitor caused partial inhibition of thymidine uptake but this appeared to be non-specific as a similar level of suppression was observed in the control cultures. Neither the ALK 2,3,6 receptor nor the NFKB pathway inhibitors had any effect on mGDF9 and mBMP15 stimulated thymidine uptake. The JNK inhibitor showed a 1.7-fold increase in stimulation above the mGDF9 and mBMP15 effect (P &lt; 0.01) but a similar stimulation was also observed in some controls. This differs from the results observed with ovine GDF9 and BMP15 where thymidine uptake was completely blocked by the NFKB inhibitor and the JNK inhibitor had no effect. In conclusion, the molecular mechanisms of GDF9 and BMP15 function are dependent on the species of origin of the growth factor and therefore caution is needed when extrapolating findings from one species to another.

439. GDF9 and BMP15 are germ-free regulators of Sertoli cell function

Oocyte-secreted Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are critical regulatory factors in female reproduction. Together they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study, we show that the expression and stage-specific localisation of both factors are limited to the germ cells of the rat seminiferous epithelium; with GDF9 being principally localised in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of the GDF9 and BMP15 signalling receptors (ALK5, ALK6, and BMPRII). Individual receptor types were expressed throughout the seminiferous epithelium, but co-expression of type I and type II receptors was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFβ ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction formation and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localisation of the junctional proteins claudin-11, occludin and ZO-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 upregulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together these results demonstrate that GDF9 and BMP15 are germ cell specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.

Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells

Growth differentiation factor-9 (GDF9) is an oocyte secreted paracrine factor essential for mammalian ovarian folliculogenesis. Like other members of the transforming growth factor-beta (TGFbeta) superfamily, GDF9 is synthesized as a prepropeptide which needs processing by furin-like proteases to result in an active mature protein. We have previously characterized a preparation of unpurified recombinant mouse GDF9 which is bioactive as produced by human embryonic kidney 293T (HEK-293T) cells. However, we find that unpurified recombinant human GDF9 (hGDF9) produced by HEK-293T cells is not bioactive. Purified recombinant hGDF9 is bioactive and here we report the characterization of this protein. We find that the purified untagged mature region of hGDF9 is active in transcriptional reporter assays specific for Smad3/4 in human granulosa-luteal (hGL) cells. We also demonstrate the use of a BMP (Smad1/5) responsive (BRE-luciferase) adenovirus in primary cultures of hGL cells to detect BMP responses. Using this adenovirus we find that purified human GDF9 does not activate the Smad1/5 pathway. Purified hGDF9 mature region activated the Smad3 pathway also in the FSH responsive human granulosa tumor cell line KGN. Primary cultures of rat granulosa cells responded to purified hGDF9 with an increase in DNA synthesis as measured by [3H]-thymidine uptake. Here we also report that the inclusion of a C-terminal affinity purification tag destroys GDF9 bioactivity. This study is the first characterization of purified biologically active human GDF9 and as such is of importance for studies on human fertility, and efforts aimed at treating infertility conditions.

Role of Oocyte-Secreted Growth Differentiation Factor 9 in the Regulation of Mouse Cumulus Expansion

Oocyte-secreted factors are required for expansion of the mouse cumulus-oocyte complex, which is necessary for ovulation. Oocyte-secreted growth differentiation factor 9 (GDF9) signals through the bone morphogenetic protein receptor II and is currently the primary candidate molecule for the cumulus-expansion enabling factor. This study was conducted to determine whether GDF9 is the mouse cumulus-expansion enabling factor. Cumulus-oocyte complexes 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 GDF9, TGFbeta1, or coculture 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 cocultured 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. Monoclonal neutralizing antibody-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 monoclonal neutralizing antibody-GDF9-53 also failed to neutralize oocyte-induced expansion. Furthermore, a soluble portion of the bone morphogenetic protein receptor II extracellular domain, 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.

Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice.

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.

251.Regulation of mouse cumulus expansion by oocyte-secreted growth differentiation factor-9 (GDF-9)

Oocyte paracrine signalling is necessary for mouse cumulus cell expansion, an important preovulatory process. The oocyte-secreted factor growth differentiation factor-9 (GDF-9) signals through the bone morphogenetic protein receptor-II (BMPR-II) and is currently the primary candidate molecule for the cumulus expansion enabling factor (CEEF). The present study was conducted to determine whether in the mouse GDF-9 is the CEEF. Cumulus oocyte complexes (COC) were collected from eCG-primed mice and the oocyte was microsurgically removed to generate an oocytectomised complex (OOX). An established scoring system was used to measure FSH-induced cumulus expansion; 0 (no expansion) to +4 (maximum expansion). OOX complexes treated with FSH alone failed to expand (score: 0), whereas expansion was significantly (P�&lt;�0.05) induced by either recombinant mouse GDF-9 (score; mean +/– SEM: 2.7 +/– 0.1), recombinant TGF-μ1 (score: 2.6 +/– 0.2) or co-culture with oocytes (score: 2.3 +/– 0.2). A GDF-9 neutralising antibody mAb-53, raised against hGDF-9, was effective in neutralising the response of OOX complexes to GDF-9 (score: 0.1 +/– 0.1), but had no significant effect on the expansion of OOX complexes co-cultured with oocytes (score: 2.3 +/– 0.2). Likewise, a TGF-μ antagonist neutralised (P�&lt;�0.05) TGF-μ-induced, but not oocyte-induced, expansion of OOX complexes. A soluble portion of the BMPR-II ectodomain, a known GDF-9 antagonist, failed to neutralise oocyte-induced cumulus expansion (P�&gt;�0.05) at the highest dose implying that BMPR-II is not a critical receptor involved in regulating cumulus expansion. Using real-time RT-PCR, hyaluronan synthase-2 (HAS2) mRNA expression by OOXs was upregulated 6- to 7-fold by oocytes and GDF-9. The GDF-9 neutralising antibody mAb-53, partially neutralised GDF-9-induced OOX HAS2 expression, but not oocyte-induced HAS2 expression. This study provides evidence that like TGF-μ1, GDF-9 can enable FSH-induced cumulus expansion, however more importantly demonstrates that neither GDF-9 nor TGF-μ1 alone account for the crucial oocyte-secreted factor regulating cumulus expansion in the mouse.

GCNF-dependent repression of BMP-15 and GDF-9 mediates gamete regulation of female fertility.

To determine the function of germ cell nuclear factor (GCNF) in female reproduction, we generated an oocyte-specific GCNF knockout mouse model (GCNF(fl/fl)Zp3Cre(+)). These mice displayed hypofertility due to prolonged diestrus phase of the estrous cycle and aberrant steroidogenesis. These reproductive defects were secondary to a primary defect in the oocytes, in which expression of the paracrine transforming growth factor-beta signaling molecules, bone morphogenetic protein 15 (BMP-15) and growth differentiation factor 9 (GDF-9), were up-regulated in GCNF(fl/fl)Zp3Cre(+) females at diestrus. This was a direct effect of GCNF, as molecular studies showed that GCNF bound to DR0 elements within the BMP-15 and GDF-9 gene promoters and repressed their reporter activities. Consistent with these findings, abnormal double-oocyte follicles, indicative of aberrant BMP-15/GDF-9 expression, were observed in GCNF(fl/fl)Zp3Cre(+) females. The Cre/loxP knockout of GCNF in the oocyte has uncovered a new regulatory pathway in ovarian function. Our results show that GCNF directly regulates paracrine communication between the oocyte and somatic cells by regulating the expression of BMP-15 and GDF-9, to affect female fertility.

Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B

Although targeted gene disruption of GDF-9, an oocyte derived growth factor, leads to an arrest of folliculogenesis and causes infertility in female mice, little is known on the expression of GDF-9 protein in the ovary. We show that GDF-9 protein is expressed in rat oocytes during folliculogenesis from the early primary follicle stage onwards but the most intensive immunostaining was seen in primary and preantral follicles. Northern blot analyses of the ontogeny of GDF-9 gene expression in postnatal rat ovaries showed that the GDF-9 transcript levels are clearly increased on the second postnatal day concomitant with the appearance of primary follicles. Interestingly, Northern blot and in situ hybridization analyses indicate a similar expression pattern for GDF-9B, the rat ortholog of a mouse GDF-9 like factor for which we recently reported the partial amino acid sequence. The polypeptide sequences deduced from isolated ovarian cDNAs indicate that the rat GDF-9 prepropeptide is 440 amino acids (aa) in length and the putative mature peptide is 135 aa whereas rat GDF-9B is 391 aa long and the mature region is 125 aa. We conclude that (1) the GDF-9 protein is highly expressed in the oocytes of primary follicles of rat ovaries suggesting that it plays a role mainly in early folliculogenesis and that (2) the full-length polypeptide sequence of GDF-9B suggests that this novel TGF-β family member is likely to be a secreted growth factor that may regulate folliculogenesis at similar developmental stages as GDF-9.

Paracrine actions of growth differentiation factor-9 in the mammalian ovary.

Although the transforming growth factor-beta (TGF-beta) superfamily is the largest family of secreted growth factors, surprisingly few downstream target genes in their signaling pathways have been identified. Likewise, the identities of oocyte-derived secreted factors, which regulate important oocyte-somatic cell interactions, remain largely unknown. For example, oocytes are known to secrete paracrine growth factor(s) which are necessary for cumulus expansion, induction of hyaluronic acid synthesis, and suppression of LH receptor (LHR) mRNA synthesis. Our previous studies demonstrated that absence of the TGF-beta family member, growth differentiation factor-9 (GDF-9), blocks ovarian folliculogenesis at the primary follicle stage leading to infertility. In the present study, we demonstrate that mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles. To explore the biological functions of GDF-9 in the later stages of folliculogenesis and cumulus expansion, we produced mature, glycosylated, recombinant mouse GDF-9 using a Chinese hamster ovary cell expression system. A granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semiquantitative RT-PCR. We find that recombinant GDF-9 induces hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppresses urokinase plasminogen activator (uPA) and LHR mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increases granulosa cell progesterone synthesis in the absence of FSH. Since induction of HAS2 and suppression of the protease uPA in cumulus cells are key events in the production of the hyaluronic acid-rich extracellular matrix which is produced during cumulus expansion, we determined whether GDF-9 could mimic this process. Using oocytectomized cumulus cell-oocyte complexes, we show that recombinant GDF-9 induces cumulus expansion in vitro. These studies demonstrate that GDF-9 can bind to receptors on granulosa cells to regulate the expression of a number of gene products. Thus, in addition to playing a critical function as a growth and differentiation factor during early folliculogenesis, GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte microenvironment, processes which are essential for normal ovulation, fertilization, and female reproduction.

A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis

Growth differentiation factor-9 (GDF-9) is a transforming growth factor-β (TGF-β) family member which is expressed in the oocytes in mouse ovaries (McGrath, S.A., Esquela, A.F., Lee, S.J., 1995. Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, 131–136). GDF-9 is indispensable for normal folliculogenesis since female mice deficient for the GDF-9 gene are infertile due to an arrest of follicular growth at the primary follicle stage (Dong, J., Albertini, D.F., Nishimori, K., Kumar, T.R., Lu, N., Matzuk, M.M., 1996. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531–535). We searched the GenBank Expressed Sequence Tag (EST) database with the mouse GDF-9 cDNA sequence, and identified from a mouse 2-cell embryo library an EST cDNA that encodes a putative member of the TGF-β superfamily, and named it as GDF-9B. Northern blot hybridization analyses of mouse ovaries revealed a single transcript of approximately 4.0 kilobases (kb) for GDF-9B and of 2.0 kb for GDF-9. We cloned by reverse transcription-polymerase chain reaction from mouse ovarian RNA a partial 821-base pair GDF-9B cDNA that spans the sequence encoding the putative mature region of GDF-9B. The COOH-terminal region of GDF-9B appears to be 53% homologous to GDF-9. Moreover, like GDF-9, GDF-9B lacks the cysteine residue needed for the covalent dimerization of several TGF-β family members. Using in situ hybridization analysis, we demonstrate that GDF-9B and GDF-9 mRNAs are co-localized in the oocyte. We also show that GDF-9B and GDF-9 genes are co-ordinately expressed during follicular development.