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

Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, The Queen Elizabeth Hospital, University of Adelaide, Woodville, South Australia 5011, Australia

Address all correspondence and requests for reprints to: Robert B. Gilchrist, Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia. E-mail: robert.gilchrist{at}adelaide.edu.au.

	Abstract

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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, TGFß1, 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 TGFß antagonist neutralized TGFß-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 TGFß, GDF9 can enable FSH-induced cumulus expansion, but more importantly, demonstrates that neither GDF9 nor TGFß alone, nor the two in unison, account for the critical oocyte-secreted factors regulating mouse cumulus expansion.

	Introduction

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IN VIVO, THE MIDCYCLE LH surge initiates the ovulatory cascade resulting in expansion of the cumulus cell mass surrounding the oocyte. Cumulus expansion facilitates the release of the oocyte into the abdominal cavity, capture of the oocyte by the oviductal fimbria, sperm penetration, and subsequent fertilization. Correct cumulus expansion is a critical physiological process because impaired expansion leads to sterility (1). In vitro, cumulus expansion is not induced by LH but can be mimicked by FSH, epidermal growth factor (EGF) or EGF-like peptides (2, 3, 4). These hormones induce cumulus cells to secrete a number of extracellular matrix (ECM) molecules including hyaluronan, the production of which is primarily controlled by the enzyme hyaluronan synthase-2 (HAS2), resulting in an expanded mucified ECM surrounding the oocyte (5).

Development of the ovarian follicle is controlled by bidirectional communication between the germ cell and somatic cells (6). Endocrine, paracrine, autocrine, and gap-junctional signaling are responsible for the growth and development of the follicle (7). It is now well established that oocyte paracrine signaling to cumulus cells is essential for mouse cumulus expansion. Mouse oocytes secrete a soluble factor(s) that enables cumulus cells to produce matrix molecules in response to FSH (2, 8, 9, 10). Removal of the oocyte from the cumulus-oocyte complex (COC) by microsurgery (oocytectomy) eliminates cumulus expansion. However, by coculturing oocytectomized (OOX) complexes with fully grown denuded oocytes (DOs), cumulus expansion is restored (2, 8). This demonstrates that mouse oocytes produce a soluble cumulus expansion-enabling factor (CEEF) that is absolutely required for cumulus expansion (2, 8, 9).

Since these pioneering studies on the role of the oocyte in cumulus expansion, it is now widely recognized that oocyte paracrine factors regulate a multitude of other processes involved in folliculogenesis (reviewed in Refs. 11 and 12), including regulation of granulosa cell proliferation (13, 14, 15) and steroidogenesis (16); modulation of inhibin, activin, and follistatin synthesis (17, 18); regulation of expression of kit-ligand (19), LH receptor (20); and urokinase plasminogen activator (21, 22). Clearly oocyte paracrine signaling to follicular granulosa cells is essential for regulating normal folliculogenesis and fertility, yet to date the exact identities of these oocyte-secreted factors (OSFs) are unknown. This represents a clear deficiency in our current understanding of fundamental mechanisms regulating ovarian biology and fertility.

Members of the TGFß superfamily, in particular growth differentiation factor 9 (GDF9), bone morphogenetic protein (BMP) 15, and BMP6, are currently the prime candidate molecules for OSF due to their ability to mimic the actions of oocytes on granulosa cells in vitro (12, 23). Many studies have now demonstrated that both TGFß1 and TGFß2 are able to completely substitute for the oocyte and mimic many oocyte-regulated granulosa cell functions, including estradiol production (23), mural granulosa cell proliferation (23, 24, 25), and cumulus cell expansion (23, 26). However, in all these studies, the addition of a specific TGFß-neutralizing antibody could only attenuate the specific effects of recombinant TGFß1 and TGFß2 and not the effects of oocytes, demonstrating that TGFß1 and TGFß2 alone are not the critical OSF mediating these granulosa events.

Until very recently, experimental neutralization of OSF has not been possible for the more recently identified members of the TGFß superfamily, GDF9 and BMP15. Both GDF9 and BMP15 are homodimeric proteins, expressed primarily in gametes of which oocyte expression is essential for female fertility in a species-specific manner (27, 28, 29, 30). GDF9 uses the bone morphogenetic protein receptor type-II (BMPRII) and the TGFß type I receptor such that GDF9 elicits a TGFß-like intracellular response (31, 32, 33, 34). Importantly, GDF9 and BMP15, like TGFß1/ß2, are able to mimic most oocyte-regulated granulosa cell activities described so far (35, 36). However, it remains to be shown whether GDF9 and BMP15 are acting like TGFß1/ß2 and mimicking OSF or whether these molecules are in fact the key OSF. Until recently these questions have remained unanswered, in part due to the lack of GDF9 and BMP15 experimental reagents. Recently our laboratory characterized a GDF9 monoclonal-neutralizing antibody (mAb-GDF-53) specific within the TGFß superfamily to GDF9 (25). mAb-GDF9–53 is a potent GDF9 antagonist, eliminating all recombinant GDF9 and approximately 50% of oocyte granulosa cell mitogenic activity (25).

Currently, GDF9 is considered to be the most likely candidate molecule for the CEEF in the mouse. Cumulus expansion is induced when OOX complexes are treated with recombinant GDF9 (22, 37). Hence, to some extent, GDF9 acts like TGFß1, mimicking the expansion-stimulating properties of oocytes. However, it is puzzling that recombinant GDF9 promotes cumulus expansion in the absence of FSH, in contrast to oocyte-induced expansion, which requires FSH. Furthermore, oocytes obtained from GDF9 null mice were unable to induce cumulus expansion of OOX complexes in vitro (23). More recently an RNA interference approach was used to show that cumulus expansion was significantly reduced when oocytes were injected with GDF9 double-stranded RNA (38). The current inference from the literature is that GDF9 alone is the mouse CEEF (22, 35, 38), although based on these results and other evidence outlined above, this remains a controversial and open question.

This study examines the hypothesis that GDF9 is the key OSF responsible for enabling cumulus expansion in the mouse. To determine whether oocyte-secreted GDF9 is the CEEF in the mouse, we attempted to antagonize oocyte-induced cumulus expansion and HAS2 expression using a novel GDF9-neutralizing antibody and a previously described GDF9 antagonist. Findings from this study provide evidence against the hypothesis that GDF9 alone is the mouse CEEF.

	Materials and Methods

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Unless specified, all chemicals and reagents were purchased from Sigma (St. Louis, MO).

Isolation of COCs
Mice used in this study were maintained at the Queen Elizabeth Hospital animal house. The study was approved by local animal ethics committees and was conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Twenty-one- to 28-d-old 129/SV mice were injected with 5 IU equine chorionic gonadotropin (Folligon; Intervet, Castle Hill, Australia), and ovaries were collected 46 h later. Ovaries were cleaned free of adherent adipose and connective tissues and placed in HEPES-buffered tissue-cultured medium-199 (H-TCM-199; ICN Biomedicals Inc., Costa Mesa, CA) supplemented with 0.1% (wt/vol) BSA (H-TCM-199/BSA). COCs were isolated by puncturing antral follicles with 27-gauge needles and collected in H-TCM-199/BSA. Only COCs with a uniform covering of compacted cumulus cells were used in this study.

Culture of COCs and OOX complexes
Microsurgical removal of the oocyte from the COCs was performed using a micromanipulation apparatus on an inverted microscope as previously described by Buccione et al. (8). Approximately 200 OOX complexes were generated per hour. Treatment drops of 15 µl of Waymouth MB 752/1 medium (WAY) supplemented with penicillin G (100 U/ml), streptomycin sulfate (100 mg/ml), 5% (vol/vol) fetal calf serum (FCS) (Trace Biosciences, Castle Hill, New South Wales, Australia); 50 mIU/ml recombinant human FSH (Puregon; Organon, Oss, The Netherlands); and with or without treatment reagent were set up and overlaid with mineral oil in falcon petri dishes. COCs or OOX complexes were transferred in a 10-µl volume to the 15-µl drops to give a total volume of 25 µl. Ten COCs or OOX complexes were cultured per 25 µl drop. The complexes were cultured for 20 h at 37 C, 96% humidity in 5% CO₂ in air, before being assessed for degree of cumulus expansion.

Treatment of OOX complexes
Denuded oocytes.
Oocytes were denuded of their surrounding cumulus cells by rapidly agitating COCs using a vortex mixer for approximately 4 min in 2 ml H-TCM-199/BSA. Groups of 20 DOs and 10 OOX complexes were transferred together in a 10-µl volume to the 15-µl treatment drops. This resulted in a concentration of 0.8 DO/µl, which preliminary experiments revealed to generate maximal cumulus expansion.

TGFß superfamily growth factors
Production of recombinant mouse GDF9 used in this study has previously been described (25, 39). In brief, recombinant mouse GDF9 was produced in-house using a transfected 293 human embryonic kidney cell line (293H), generously donated by Dr. Olli Ritvos (University of Helsinki, Helsinki, Finland). Control conditioned medium from untransfected 293H cells inhibited cumulus expansion and raw conditioned medium from GDF9-transfected cells did not promote cumulus expansion. Consequently, conditioned media were subjected to partial purification using hydrophobic interaction chromatography (Hickey, T. E., D. L. Marrocco, F. Amato, L. J. Ritter, R. J. Norman, R. B. Gilchrist, and D. T. Armstrong, manuscript submitted) which was effective at removing the inhibitory factors from the 293H parent cell line. These techniques generate a partially pure, mostly processed GDF9 of 17.5 kDa. Recombinant human TGFß1 and recombinant human BMP6 were obtained (R&D Systems, Minneapolis, MN) and were used at concentrations previously described (22, 23).

Growth factor antagonists
Attempts were made to antagonize recombinant and oocyte-secreted GDF9 bioactivity using a recently described GDF9 neutralizing monoclonal antibody, mAb-GDF9–53, which was generously donated by Prof. Nigel Groome (Oxford Brookes University, Oxford, UK) (25). This mouse mAb was raised against a 32-amino acid peptide at the C terminus of human GDF9. mAb-GDF9–53 has strong immunoaffinity for recombinant mouse GDF9 and has very weak cross-reactivity with other members of the TGFß superfamily, including BMP15. mAb-GDF9–53 specifically neutralizes the mitogenic activity of recombinant mouse GDF9 and partially antagonizes that of mouse OSF (25). A synthetic portion of the BMPRII extracellular domain (ECD) and the TGFß receptor II (TGFßRII) ECD fused to the human IgG-Fc region were both obtained from R&D Systems. These solubilized receptors act as antagonists by binding their respective ligands, thereby dramatically reducing ligand interaction with the native type II receptor. The BMPRII ECD presumably antagonizes the many ligands using this receptor, and it has been shown to neutralize the bioactivity of the recombinant forms of the key putative OSF, GDF9 (31), and BMP15 (32). Importantly, the BMPRII ECD completely neutralizes the granulosa cell growth-promoting bioactivity of mouse oocytes (40).

Assessment of cumulus expansion
Cumulus expansion of COCs and OOX complexes was recorded after a 20-h culture period. This was a blinded assessment to eliminate bias. The degree of cumulus expansion was assessed according to a subjective scoring system (0 to +4). In brief, score 0 indicates no expansion and score +4 indicates complete expansion of all cumulus cell layers. A cumulus expansion index (0.0–4.0) was calculated as previously described (9, 41).

Real-time RT-PCR
RNA isolation.
An experiment was conducted to examine the effect of neutralization of oocyte-secreted GDF9 on cumulus cell HAS2 mRNA levels after 6 h. OOX complexes were cultured in WAY supplemented with 5% (vol/vol) FCS + FSH (50 mIU/ml) and one of the following treatments: 1) 0 (control), 2) oocytes (0.8/µl), 3) oocytes + mAb-GDF9–53 (40 µg/ml), 4) GDF9 (250 ng/ml), or 5) GDF9 + mAb-GDF9–53. Ten OOX complexes were cultured per treatment group, each treatment group was in quadruplicate, and the experiment was replicated on five separate occasions. After the 6-h incubation, the DOs were removed and the OOX complexes were washed in H-TCM-199/BSA. The OOX complexes were transferred to Eppendorf tubes (40 OOX per tube) on ice, and RNA was isolated using a micro RNA isolation kit (QIAGEN, Victoria, Australia). This included addition of 20 ng of carrier RNA to each sample before homogenization, and all samples were DNase treated to eliminate any contaminating genomic DNA. RNA was quantified using a Ribogreen RNA quantification kit (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol.

Real-time RT-PCR analysis
Ninety nanograms of RNA was reverse transcribed using random primers (Roche Molecular Biochemicals, Mannheim, Germany) and a Superscript II RT kit (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions. A negative reverse transcription control substituting water for RNA was included. Primer pairs were designed for mouse ribosomal protein L19 and HAS2 using Primer Express software (PE Applied Biosystems, Foster City, CA), and synthesized by Geneworks (Adelaide, Australia). The sequence for each primer pair was as follows; L19 sense, 5'-GAAAGTGCTTCCGATTCCA-3', and antisense primer, 5'-TGATCGCTTGATGCAAATCC-3', based on mouse L19 sequence (accession no. NM_009398) and HAS2 sense primer, 5'-CATTCCCAGAGGACCGCTTAT-3', and antisense primer, 5'-AAGACCCTATGGTTGGAGGTGTT-3', based on mouse HAS2 sequence (accession no. U52524).

To consider L19 as an appropriate housekeeping gene, the critical threshold (C_T) value for all samples should not vary significantly across treatment groups. Using an ABI GeneAmp 5700 machine (PE Applied Biosystems), L19 mRNA levels were measured in triplicate and were then normalized to total RNA measurements for each sample. There were no significant differences in L19 mRNA levels between treatment groups (P > 0.05). Primer amplification efficiencies were also examined to ensure that the housekeeping gene L19, and the target gene HAS2, primed with the same amplification efficiency. Each primer set was run with serially diluted cDNA and the slopes of each primer set were determined using the C_T values plotted against log dilutions of the cDNA. Slopes for each gene were determined and the L19 slope was statistically comparable with that of HAS2 (P > 0.05).

Each experimental sample was run in triplicate on an ABI GeneAmp 5700 sequence detection system. Each sample consisted of: 3 µl of diluted cDNA sample (1:9), 10 µl of 2 x SYBR green master mix (PE Applied Biosystems) and 10 pmol of each primer. Samples were treated at 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of amplification at 95 C for 15 sec and 60 C for 1 min. No template controls, substituting water for cDNA, and a negative reverse transcription were used in each run. HAS2 gene expression was calculated for each sample relative to the housekeeping gene, L19, using the 2^–C_T method as described in Ref. 42 . After RT-PCR amplification, a dissociation analysis was run on all products to ensure that a single product was produced during the PCR process. Products were then run on a 2% agarose gel for confirmation of single, correctly sized products. Finally, the identity of the each PCR product was verified by sequencing. The L19 amplicon was 97% homologous to mouse L19 (accession no. NM_009398), and the HAS2 amplicon was 98% homologous to mouse HAS2 (accession no. U52524).

Data analyses
Each experiment was replicated three to five times (see figure legends). Treatment effects on cumulus expansion were examined using a Kruskal-Wallis one-way ANOVA on ranks, and differences between means were detected using Dunn’s method post hoc comparisons or t tests. Real-time RT-PCR data underwent log transformation to satisfy ANOVA criteria and were then subjected to one-way ANOVA followed by Tukey comparisons. P < 0.05 was considered statistically significant. All statistical analyses were performed using the software package SigmaStat for Windows (version 2.03; Jandel Corp., San Ramon, CA).

	Results

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Oocyte regulation of cumulus expansion is mimicked by certain members of the TGFß superfamily
To examine the effect of OSF and TGFß superfamily OSF candidate molecules on cumulus expansion, COCs and OOX complexes were cultured alone or as positive and negative controls, or OOX complexes were treated with DO, GDF9, TGFß1, BMP6, or 293H (GDF9-negative control-conditioned media) in the presence of FCS and FSH. As expected, intact COCs expanded to a degree of 2.7 ± 0.1, and the negative control OOX complexes cultured alone failed to expand (Fig 1A). However, expansion of OOX complexes was significantly (P < 0.05) induced by coculture with oocytes (2.6 ± 0.2), GDF9 (3.4 ± 0.2), or TGFß1 (2.0 ± 0.2) (Fig. 1A). OOX complexes treated with 293H-conditioned medium or BMP6 failed to undergo expansion. GDF9 enabled OOX cumulus expansion in a dose-dependent manner, restoring expansion to COC levels at 250 ng/ml (Fig. 1B).

View larger version (19K): [in this window] [in a new window]   FIG. 1. Oocyte-secreted factors and TGFß superfamily members that enable cumulus cell expansion. A, OOX complexes were cultured alone, with denuded oocytes (0.8/µl), GDF9 (250 ng/ml), TGFß1 (10 ng/ml), BMP6 (50 ng/ml), or 293H (20% vol/vol; conditioned medium from the untransfected 293H parent cell line). OOX complexes and COCs were cultured in media supplemented with 50 mIU/ml FSH and 5% FCS. B, OOX complexes were cultured in the presence of 50 mIU/ml FSH with an increasing dose of recombinant GDF9 (16–500 ng/ml). The degree of cumulus expansion was determined after 20 h of culture using a scale of 0 (no expansion) to +4 (maximal expansion). Results represent the mean ± SEM from three experiments, each with a total of 10 complexes (OOX or COC) per treatment group. An asterisk represents a significant difference to OOX alone (P < 0.05).

View larger version (14K): [in this window] [in a new window]   FIG. 2. GDF9 enables FSH-stimulated cumulus cell expansion. OOX complexes were cultured with 50 mIU/ml FSH with or without denuded oocytes (0.8/µl) or with recombinant mouse GDF9 (250 ng/ml) with or without FSH. FSH is required for GDF9-induced cumulus expansion. The degree of cumulus expansion was determined after 20 h of culture using a scale of 0 (no expansion) to +4 (maximal expansion). Results represent the mean ± SEM from three experiments each using a total of 10 complexes (OOX or COC) per treatment group. Bars with different superscript letters are significantly different (P < 0.05).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Effect of anti-GDF9 mAb-GDF9–53 on cumulus expansion and cumulus cell HAS2 mRNA expression. A, OOX complexes were cocultured with oocytes (0.8/µl) or treated with GDF9 (250 ng/ml) to induce cumulus expansion and with an increasing dose (0–40 µg/ml) of mAb-GDF9–53. All control and treatment groups were cultured for 20 h in media supplemented with 50 mIU/ml FSH, and then cumulus expansion was measured according to the subjective scoring system: 0 (no expansion) to +4 (maximal expansion). The GDF9-neutralizing antibody antagonized GDF9-induced, but not oocyte-induced, cumulus expansion. Means within a line graph with different superscript letters are significantly different (P < 0.05). Asterisks indicate the two means at that dose of antagonist are significantly different (P < 0.05). B, Fold differences in cumulus cell expression of HAS2 mRNA after 6 h culture in the presence of 50 mIU/ml FSH. Real-time RT-PCR analysis was performed using primer sets for HAS2 and L19 using RNA from cumulus cells (OOX) cultured alone, with GDF9 (250 ng/ml), or with denuded oocytes (0.8/µl), either in the presence or absence of 40 µg/ml mAb-GDF9–53. Results are from five independent experiments and are expressed as a fold change from mRNA levels in OOX complexes cultured alone (control). All samples have been normalized to L19 mRNA content. An asterisk denotes a significant effect of mAb-GDF9–53 (P < 0.05).

A second and well-known GDF9 antagonist was examined for its capacity to neutralize oocyte-induced cumulus cell expansion. It has previously been documented that BMPRII is the type II receptor for GDF9 and that an ECD of BMPRII completely antagonizes recombinant GDF9 bioactivity (31) as well as the mitogenic effects of OSF on granulosa cells (40). OOX complexes cocultured with oocytes were treated with an increasing dose of BMPRII ECD. The BMPRII ECD caused a dose-dependent partial neutralization of oocyte-induced cumulus expansion. GDF9-induced cumulus expansion was notably antagonized by BMPRII ECD in a dose-dependent manner. At a dose of 10 µg/ml of BMPRII ECD, which completely abolished GDF9-induced expansion, oocyte-induced expansion was still comparable with the COC-positive control and was significantly higher than the GDF9 treatment at that dose (P < 0.05; Fig. 4).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Effect of BMPRII ECD on expansion of mouse OOX complexes. OOX complexes were cultured in the presence of FSH with either oocytes (0.8/µl) or recombinant mGDF9 (250 ng/ml) and treated with an increasing dose of BMPRII ECD (5–20 µg/ml). The degree of cumulus expansion was measured using a scale of 0 (no expansion) to +4 (maximal expansion). Means within a line graph with different superscript letters are significantly different (P < 0.05). Asterisks indicate the two means at that dose of antagonist are significantly different (P < 0.05).

View larger version (26K): [in this window] [in a new window]   FIG. 5. Effect of TGFß ECD and/or mAb-GDF9–53 on cumulus expansion. All treatments consisted of 10 OOX/COC complexes cultured in media supplemented with 50 mIU/ml FSH. A, OOX complexes were cultured with oocytes (0.8/µl) or 10 ng/ml TGFß1 in the presence or absence of 200 ng/ml TGFßRII ECD. COCs were also cultured with or without TGFßRII ECD. B, OOX complexes were cultured with denuded oocytes (0.8/µl) or 250 ng/ml GDF9 in the presence or absence of 40 µg/ml mAb-GDF9–53. COCs were also cultured either with or without mAb-GDF9–53. C, OOX complexes were cultured alone, with GDF9, or with TGFß1 in the presence or absence of mAb-GDF9–53 or TGFßRII ECD, respectively. OOX complexes were cocultured with oocytes and treated with mAb-GDF9–53, TGFßRII ECD, or the two antagonists together. After 20 h of culture, the degree of cumulus expansion was assessed using the subjective scoring system: 0 (no expansion) to +4 (maximal expansion). Results show the mean ± SEM of three to four individual experiments, and bars within a graph with different superscript letters are significantly different (P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In vitro, cumulus expansion requires both the oocyte and stimulation, either by FSH or EGF (2, 3). Microsurgical removal of the oocyte from the COC ablates FSH-stimulated cumulus expansion, which is restored by coculture of OOXs with oocytes but only in the presence of FSH (8). Our results show that OOX complexes treated with GDF9 in the absence of FSH do not undergo cumulus expansion but do in the presence of FSH, demonstrating that our GDF9 is behaving in the same manner as the oocyte in enabling cumulus expansion. Curiously, other groups (22, 43) recently reported that GDF9 in the absence of FSH stimulates cumulus expansion in vitro. Reasons for this discrepancy are unclear and warrant further study. One possible explanation is that the chemical nature of the GDF9 used in these two studies somehow differs. GDF9 is not commercially available, and the production conditions in different laboratories will ultimately lead to different molecular forms of GDF9. The GDF9 from our laboratory is partially purified using hydrophobic interaction chromatography, a process that is required to demonstrate GDF9 bioactivity using the cumulus expansion bioassay. Moreover, the proregion of the GDF9 that we produce has been proteolytically cleaved to generate mostly mature processed GDF9 (25, 39). It has previously been hypothesized that cumulus expansion requires a FSH-stimulated cumulus cell product that alters the structure of native GDF9, e.g. processes the proregion, making it biologically active and thereby enabling expansion (43). In this case, recombinant GDF9, which is produced in this modified processed form, would therefore not require the FSH-dependent factor produced by cumulus cells (43). Our results do not support this hypothesis because they illustrate that fully processed recombinant GDF9, just like OSF, requires FSH to stimulate cumulus expansion.

Although GDF9 specifically induces cumulus expansion, this does not mean that GDF9 is the key OSF that normally mediates this process. This is most clearly illustrated by the fact that just like GDF9, TGFß1 and TGFß2 can also behave in this manner and mimic a diverse range of oocyte-regulated granulosa/cumulus cell processes, including cumulus expansion, proliferation, and steroidogenesis. However, TGFß1/ß2 are not the OSF regulating these processes (23, 24, 26). This has now been demonstrated in many studies using TGFß neutralizing antibodies, which are unable to inhibit oocyte-paracrine effects on granulosa cells (23, 24, 25, 26). Until recently this kind of experimental approach has not been possible with regard to GDF9 because GDF9 is a newly described molecule, and specific reagents and experimental tools are still limited.

This study is the first to use a GDF9-neutralizing antibody in the cumulus expansion assay. The antagonist mAb-GDF9–53 is a highly specific monoclonal neutralizing antibody recently characterized in detail in our laboratory (25). In the current study, mAb-GDF9–53 effectively inhibited recombinant GDF9-induced cumulus expansion but did not antagonize oocyte-induced cumulus expansion. COCs treated with the GDF9-neutralizing antibody also underwent cumulus expansion. Failure of mAb-GDF9–53 to antagonize expansion of an intact complex is perhaps not surprising because the IgG may not effectively penetrate the cumulus mass to reach the source of the OSF. However, there can be little doubt the antibody has access to OSF in the OOX + DO cultures. This is the first example of antibody-mediated neutralization of GDF9-induced cumulus expansion, although this was anticipated based on the characteristics of the antibody. mAb-GDF9–53 is a mouse monoclonal raised against a 32-amino acid peptide at the C terminus of human GDF9 (25). Epitope mapping and alignment of the binding sequence indicate that the motif is highly conserved across species and so the neutralizing activity is unlikely to be species specific. Alignment of the epitope with related members of the TGFß superfamily illustrates low homology with BMP15 and no homology with the next closest member of the superfamily, and as such, mAb-GDF9–53 has low immunoaffinity for BMP15 and does not antagonize TGFß1 or activin A bioactivity on granulosa cells (25). Importantly, mAb-GDF9–53 does recognize the molecular form of GDF9 secreted by mouse oocytes and furthermore does partially antagonize oocyte-stimulated granulosa cell proliferation (25), even though in the current study this antibody failed to inhibit oocyte-induced cumulus expansion.

To confirm that the measured changes in cumulus morphology are associated with quantitative changes in cumulus cell gene expression, HAS2 was assessed, which is the major hyaluronan synthase enzyme involved in regulating cumulus expansion. Cumulus expansion involves the formation of a mucoid ECM surrounding the oocyte of which hyaluronan is a major structural component (22). The present study demonstrates that both oocytes and GDF9 can up-regulate HAS2 expression. Confirming the cumulus morphological observations, the GDF9-neutralizing antibody antagonized GDF9-induced HAS2 expression but did not neutralize oocyte-induced HAS2 expression. This suggests there is likely redundant regulation of HAS2 by other OSFs as well as GDF9, further supported by the observation that COCs from BMP15 null mice exhibit reduced HAS2 expression (44). Together, these provide additional evidence against the hypothesis that GDF9 is the sole oocyte factor enabling cumulus expansion.

To provide an additional line of evidence, an alternative GDF9 antagonist was tested for its capacity to antagonize oocyte-induced cumulus expansion. Previously it has been shown that GDF9 binds the BMPRII and that a solubilized portion of the receptor ECD (BMPRII ECD) neutralizes GDF9 bioactivity (31) and, importantly, also completely eliminates oocyte growth-promoting activity (40). In the current study, BMPRII ECD completely antagonized GDF9-induced cumulus expansion but only partially neutralized oocyte-induced cumulus expansion. The latter suggests that signaling through BMPRII may be an important, but not an exclusive, feature of the cumulus expansion process and hence suggests that other receptors and their ligands are likely to be involved.

If neither GDF9 nor TGFß alone is the CEEF and oocyte factors other than those using BMPRII are involved in cumulus expansion, we further considered whether GDF9 and TGFß operate in a redundant manner to enable cumulus expansion. GDF9 and TGFß use different type II receptors, but they use a common type I receptor, activin receptor-like kinase 5, and hence a common intracellular signaling pathway, both activating mothers against decapentaplegic-2 and -3 (Smad 2/3) (34, 39). Here we demonstrate that simultaneous antagonism of GDF9 and TGFß using mAb-GDF9–53 and TGFßRII ECD, fail to neutralize oocyte-induced cumulus expansion. Together these results suggest that the mouse CEEF is composed of multiple OSFs, which may include GDF9 and TGFß among others.

The conclusion that GDF9 is not the sole constituent of the CEEF may appear to contradict some very recent studies. Vanderhyden et al. (23) showed that oocytes from GDF9-deficient mice are unable to promote expansion of OOX complexes. However, as acknowledged by the authors, results obtained from the GDF9 knockout mice should be interpreted with caution due to the likelihood that GDF9 is not the only factor missing from these oocytes. It is highly likely these oocytes are deficient in a multitude of developmentally regulated transcripts as a consequence of their abnormal growth and development (45). Gui and Joyce (38) used GDF9 double-stranded RNA interference to successfully knock down oocyte-GDF9 expression and thereby eliminate cumulus expansion and as a result concluded that GDF9 alone is the mouse CEEF. A key experimental approach in the current study was to neutralize oocyte-GDF9 using mAb-GDF9–53, although it is conceivable that this antibody may be less effective against the form of GDF9 secreted by oocytes as it is against recombinant GDF9. Recombinant GDF9 is mostly produced in a mature processed state, whereas preliminary data suggest native GDF9 may be secreted (in vitro at least) with its proregion intact (25). In addition, glycosylation status may differ between native and recombinant GDF9. mAb-GDF9–53 may have a lower affinity for oocyte-secreted GDF9; nonetheless, this antibody does partially antagonize the growth-promoting effects of oocyte-secreted GDF9 (25). Furthermore, the lack of complete neutralization of oocyte-induced cumulus expansion by the GDF9 antagonist, BMPRII ECD, provides additional evidence that GDF9 alone is not the CEEF. Despite 15 yr of research in this area, the elusive CEEF in the mouse still remains controversial.

All of the major putative OSFs, GDF9, BMP15, and BMP6, as well as other members of the TGFß superfamily, use BMPRII as their primary type II receptor, and because the BMPRII ECD partially antagonized oocyte-induced expansion, this suggests that one or a number of these growth factors contribute to the CEEF. Results from the current study suggest that neither GDF9 alone nor BMP6 alone regulate oocyte-induced cumulus expansion. Other possible molecules contributing to the mouse CEEF may include BMP15 and other BMPs. Like GDF9, oocyte-secreted BMP15 regulates a wide range of differentiation processes of granulosa cells attributed to OSF (46, 47). BMP15 null mice display decreased ovulation and fertilization rates (48); however, it is unclear whether the BMP15 null mice are subfertile due to assembly failure of their cumulus ECM or for other reasons. RNA interference of oocyte BMP15 failed to prevent cumulus expansion (38), although BMP15 null mice exhibit lower cumulus cell expression of HAS2 (44). A recent study by Su et al. (44) examined COCs from BMP15^–/– GDF9^+/– double-mutant mice, suggesting that these molecules may act in a synergistic manner. Whereas cumulus expansion was not overtly impaired in COCs from BMP15 null mice, it was in the double-mutant COCs, suggesting that these two OSFs may be working synergistically to promote expansion, either as independent homodimers or alternatively as a GDF9/BMP15 heterodimer (49). Even though some type of GDF9-BMP15 interaction appears necessary for cumulus expansion because both these factors require BMPRII and cumulus expansion was not prevented by the BMPRII ECD in the current study, oocyte factors in addition to these molecules must be involved.

In conclusion, the present study, together with other studies, demonstrated that it is common for several members of the TGFß superfamily to mimic the paracrine actions of oocytes on granulosa or cumulus cells in vitro. It is apparent that recombinant TGFß1 and GDF9 can mimic the oocyte and promote cumulus expansion, yet in both cases specific and more generalized antagonists to TGFß and GDF9 fail to prevent the expansion-inducing action of oocytes. The findings from this study provide strong evidence against the hypothesis that GDF9 is the sole oocyte-secreted factor regulating cumulus expansion in the mouse. This study supports the argument that the mouse CEEF is composed of multiple TGFß superfamily molecules, including at least one of which is a BMPRII ligand(s) and one or more that are not. The results reported here provide a better understanding of the process of cumulus expansion.

	Acknowledgments

 
The authors thank Dr. Olli Ritvos (University of Helsinki) for generously donating the GDF9-expressing cell line and Professor Nigel Groome (Oxford Brookes University) for kindly providing the mAb-GDF9–53. We also thank Jim Wang, Jeremy Thompson, Darryl Russell, and Theresa Hickey for helpful discussions.

	Footnotes

 
This work was supported by a National Health and Medical Research Council of Australia Program Grant and the Research Centre for Reproductive Health. R.A.D. was supported by Faculty of Health Sciences, Adelaide University, and The Queen Elizabeth Hospital Research Foundation. R.B.G. was the recipient of the F. T. T. Fricker Medical Research Associateship, Adelaide University.

First Published Online March 10, 2005

Abbreviations: BMP, Bone morphogenetic protein; BMPRII, BMP receptor type II; CEEF, cumulus expansion-enabling factor; COC, cumulus-oocyte complex; C_T, critical threshold; DO, denuded oocyte; ECD, extracellular domain; ECM, extracellular matrix; EGF, epidermal growth factor; FCS, fetal calf serum; GDF9, growth differentiation factor 9; 293H, 293 human embryonic kidney cell line; HAS2, hyaluronan synthase-2; H-TCM, HEPES-buffered tissue-cultured medium-199; mAb, monoclonal antibody; mAb-GDF-53, GDF9 monoclonal-neutralizing antibody; OOX, oocytectomized; OSF, oocyte-secreted factor; TGFßRII, TGFß receptor II; WAY, Waymouth MB 752/1 medium.

Received January 25, 2005.

Accepted for publication March 2, 2005.

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