HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 72/1/195    most recent biolreprod.104.033357v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gui, L.-M. Articles by Joyce, I. M. Search for Related Content PubMed PubMed Citation Articles by Gui, L.-M. Articles by Joyce, I. M. Agricola Articles by Gui, L.-M. Articles by Joyce, I. M.

RNA Interference Evidence That Growth Differentiation Factor-9 Mediates Oocyte Regulation of Cumulus Expansion in Mice¹

School of Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In mouse ovaries, growth differentiation factor-9 (GDF9) is an oocyte-derived growth factor that plays an essential role during early follicular development. However, the role of GDF9 during later stages of follicular development is uncertain. In the present study, a long double-stranded (ds) RNA interference 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 reverse transcription-polymerase chain reaction (RT-PCR) and for measurement of GDF9 protein levels using Western blot analysis 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 of FSH, cumulus shells cocultured with buffer- and Bmp15 dsRNA-injected oocytes exhibited a high degree of expansion, whereas cumulus shells cocultured with Gdf9 dsRNA-injected oocytes exhibited only limited expansion. Supporting this observation, Has2 and Ptgs2 mRNA levels after 8 h of coculture were lower in cumulus cells cocultured with Gdf9 dsRNA-injected oocytes than in those cocultured with buffer-injected oocytes. The present results strongly support the concept that GDF9 is a key mediator of oocyte-enabled cumulus expansion in mice.

cumulus cells, fertilization, follicular development, growth factors, oocyte development

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In mammals, granulosa cells in preovulatory follicles are divided into two distinct populations: mural granulosa cells lining the follicle wall, and cumulus cells that surround and form an intimate association with the oocyte. In response to the preovulatory LH surge, the cumulus cell layer undergoes expansion (i.e., a process of cell separation and extracellular mucification). Cumulus expansion is associated with dramatic changes in gene expression in cumulus cells. For example, mRNA expression of prostaglandin synthase-2 (Ptgs2) and hyaluronan synthase-2 (Has2) is initiated in cumulus cells within 4 h of the LH surge [1, 2]. Functionally, in addition to facilitating efficient fertilization, the process of cumulus expansion is important for successful detachment of the cumulus-oocyte complex (COC) from the follicle wall and subsequent ovulation [3–5] as well as for transport of the COC through the oviduct and maintenance of oocyte viability in the oviduct [3]. Cumulus expansion therefore plays important roles during oocyte development, ovulation, and fertilization in higher mammals.

In 1990, Buccione et al. [6] presented evidence that cumulus expansion is dependent on the presence of one or more factors derived from the oocyte. They termed this substance cumulus expansion-enabling factor. Subsequently, the same group showed that production of cumulus expansion-enabling factor is developmentally regulated in mouse oocytes and that the factor likely is a secreted protein [7, 8]. In 1999, Elvin et al. [9], using a recombinant form of growth differentiation factor-9 (GDF9; an oocyte-derived growth factor), demonstrated that this protein promoted cumulus expansion in vitro. The authors therefore proposed that GDF9 was the cumulus expansion-enabling factor. Supporting this, Vanderhyden et al. [10] found that unlike wild-type oocytes, oocytes derived from Gdf9^null mice are unable to promote cumulus expansion in vitro.

Questions, however, remain. In the study by Elvin et al. [9], recombinant GDF9, unlike oocytes, promoted cumulus expansion in vitro in the absence of FSH or epidermal growth factor, suggesting that this recombinant form of GDF9 does not act identically to cumulus expansion-enabling factor. Similarly, the study by Vanderhyden et al. [10] left questions unanswered because of the difficulty of assessing the developmental status of oocytes derived from Gdf9^null mice: In these animals oogenesis is compromised, while folliculogenesis does not progress beyond the earliest stages of preantral follicular development. This is considerably earlier than the preovulatory follicles in which oocytes secreting cumulus expansion-enabling factor are found [11].

To establish more fully the nature of cumulus expansion-enabling factor, in the present study an RNA interference (RNAi) technique was used to suppress Gdf9 levels in preovulatory-stage oocytes (hereafter termed Gdf9^knockdown oocytes). We then assessed the efficiency of Gdf9^knockdown oocytes in regulating cumulus expansion. The present results indicate that GDF9 is the cumulus expansion-enabling factor identified by Buccione et al. in 1990 [6].

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Synthesis of DNA Template and Long Double-Stranded RNAs

A 589-base pair (bp) Gdf9 template and a 528-bp Bmp15 template were synthesized by high-fidelity nested polymerase chain reaction (PCR) using a ProofStart PCR kit (Qiagen Ltd., Crawley, U.K.). The outer Gdf9 primer pairs were 5'-gtgagacccctaagctgca-3' and 5'-gatggctttctgccctcgacg-3', and the outer Bmp15 primer pairs were 5'-taccatcgttcggctgacc-3' and 5'-gaggagcaatgatccagtg-3'. Inside primers contained a T7 promoter attached to the 5'-end of both the forward and reverse primers. For Gdf9, the primer pairs were 5'-gcgtaatacgactcactatagggagaccagagcactctactacatg-3' and 5'-gcgtaatacgactcactatagggagactgtaaaggcctccaggtgg-3'. For Bmp15, the inner primer pairs were 5'-gcgtaatacgactcactatagggagagaggctggtaaagccgtcg-3' and 5'-gcgtaatacgactcactatagggagaaatgctgcatgcttggcgg-3'. All primers were produced by MWG Biotech (Ebersberg, Germany). The PCR amplification conditions for both Gdf9 and Bmp15 templates were as follows: initial denaturation at 94°C for 3 min; 40 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 45 sec; and a 7-min incubation at 72°C. The Gdf9 and Bmp15 templates were purified using a PCR purification kit (Qiagen), and 1–2 µg of each were transcribed in vitro using a MEGAscript RNAi kit (Ambion, Huntingdon, U.K.) to obtain the corresponding long double-stranded (ds) RNA. The in vitro transcription products were assessed for integrity on a 1.5% agarose gel, diluted in injection buffer (10 mM Tris and 0.1 mM EDTA, pH 7.5; Ambion), and stored at –80°C until used.

Oocyte Isolation, Microinjection, and Culture

Oocytes were obtained from 21- to 23-day-old (C57BL/6 x FVB/N) F1 mice 44–48 h after i.p. injection of 5 U of eCG (Sigma-Aldrich, Poole, U.K.). Ovaries from these mice were placed in M2 medium supplemented with 0.1 µM milrinone (Sigma-Aldrich) to maintain oocytes at the germinal vesicle stage. Large antral follicles were then punctured with sterile, 26-gauge needles to release COCs, and denuded oocytes were isolated following repeated pipetting of COCs. All experiments were conducted in accordance with established ethical guidelines from the Home Office for the care and use of laboratory animals.

Microinjection of dsRNA into the cytoplasm of oocytes was undertaken using a constant flow system (Femtojet; Eppendorf, Cambridge, U.K.). Oocytes to be microinjected were placed in a 30-µl drop of M2 medium containing 0.1 µM milrinone under mineral oil (Sigma-Aldrich) and maintained at 37°C. Each oocyte was injected with approximately 10 pl of dsRNA. As a control, Bmp15 dsRNA or injection buffer alone was injected into the cytoplasm of oocytes. After microinjection, oocytes (one per microliter) were cultured for 24 h in M16 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 0.1 µM milrinone in a modular incubation chamber infused with humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ and maintained at 37°C.

Preparation and Coculture of Cumulus Shells with Oocytes

Cumulus shells were microsurgically prepared from COCs isolated as detailed above following the protocol described by Buccione et al. [6]. The resulting oocytectomized COCs (i.e., cumulus shells) consisted of the spherical zona pellucida surrounded by the cumulus cell mass. Successful oocytectomy was assessed by the removal of the oocyte germinal vesicle along with the majority of ooplasm. Cumulus shells were then washed three times in M16 medium and cocultured with oocytes that had been injected with Gdf9 dsRNA, Bmp15 dsRNA, or buffer 24 h previously and cultured as described above. Following a protocol modified from Buccione et al. [6], oocytes (one per microliter) and cumulus shells (one per microliter) were cocultured in M16 medium supplemented with 10% FBS and 0.5 IU/ml of recombinant FSH (Puregon; Organon, Oss, The Netherlands). Cocultures were undertaken under paraffin oil and incubated for 8 h (for determination of Has2 and Ptgs2 mRNA levels) or for 24 h (for morphological assessment of expansion) in a modular incubation chamber infused with humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 37°C. Results of preliminary experiments confirmed that under these conditions, expansion of cumulus shells was fully evident when cocultured with noninjected oocytes. Differential interference contrast images were captured using 20x magnification on a Zeiss LSM510 inverted microscope (Carl Zeiss Ltd., Herts, U.K.).

Quantification of Gdf9, Bmp15, Has2, and Ptgs2 mRNA Levels

The mRNA levels of Gdf9, Bmp15, Has2, and Ptgs2 were quantified using real-time reverse transcription (RT)-PCR. Briefly, total RNA was extracted using a mini-total RNA isolation kit (Qiagen). An additional DNase step was used to reduce genomic DNA in the total RNA preparation to less than detectable levels. Complimentary DNA was then prepared from total RNA using oligo-dT (Promega, Southampton, U.K.) and Sensiscript reverse transcriptase (Qiagen). Real-time PCR was performed on a CycleriQ Real-Time Detection System using iQSYBR Green Supermix kit (Bio-Rad Laboratories Ltd., Hertfordshire, U.K.) in a final volume of 25 µl. Standard curves were constructed by serial dilution using cDNA from oocytes or cumulus cells as appropriate. The cDNA levels of Gdf9, Bmp15, Has2, and Ptgs2 in the standard curve spanned those in the samples. Ribosomal L19 (Rpl19) was used to normalize for RT-PCR efficiency [2, 12]. The Rpl19 primer pairs were 5'-tcatggagcacatccacaa-3' and 5'-gtgcttccttggtcttagac-3'. The Gdf9 primer pairs were 5'-gagtgtgttgaccattgaacc-3' and 5'-gcacctcgtagctatcatgtc-3'. The Bmp15 primer pairs were 5'-ttgctcctcgtctctatacc-3' and 5'-ctgaatgatggcatggttgg-3'. The Has2 primer pairs were 5'-atcttggctggtgctgtgtagg-3' and 5'-ccttggtgctctttttgcttcg-3', and the Ptgs2 primer pairs were 5'-aagccctctacagtgacatcga-3' and 5'-atggtctccccaaagatagcat-3'. Real-time PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 45 cycles of 94°C; 15 sec, 58°C for 15 sec, and 72°C for 30 sec. Fluorescence was measured at the end of each cycle with the temperature held at 85°C. Each sample was analyzed in triplicate. The relative expression level of each gene was calculated using the standard curve before normalization to Rpl19 mRNA levels. The specificity of primers was confirmed with gel electrophoresis.

Immunoblot and Immunostaining Analysis

Immunoblotting was performed as described by Matsudaira et al. [13]. One-hundred oocytes were suspended in 2x Laemmli loading sample buffer [14] with protease inhibitor cocktail (Sigma-Aldrich) and boiled for 3 min before storage at –80°C. Protein extract was separated by 10% SDS-PAGE and transferred to a Hybond polyvinylidene difluoride membrane (Amersham, Buckinghamshire, U.K.) with CAPS buffer (pH 11.0; Sigma-Aldrich). The protein extract was then probed using a goat anti-mouse GDF9 polyclonal antibody (R&D Systems Europe Ltd., Abingdon, U.K.) and horseradish peroxidase-conjugated donkey anti-goat immunoglobulin G (Sigma-Aldrich). The ECL Plus detection system (Amersham) was used to visualize the specific immunoreactive proteins by exposure to autoradiographic film.

Immunofluorescence was modified from the method described by Polanski et al. [15]. Before fixation, the zona pellucida was removed from oocytes using acid Tyrode solution (pH 2.5). Oocytes were fixed in 3.7% paraformaldehyde (Sigma-Aldrich) at 4°C overnight, followed by permeabilization. After preincubation in 1% fish gelatin in 0.1% Triton-100 PBS, oocytes were incubated with anti-mouse GDF9 polyclonal antibody (R&D Systems Europe). After four washings of 15 min each, oocytes were transferred to a 1:750 dilution of fluorescein-conjugated goat anti-mouse antibody (Jackson ImmunoResearch, West Grove, PA) and 2 µg/ml of 4',6'-diamidino-2-phenylindole (Tris-like) (Sigma-Aldrich) in blocking solution. In order to ensure membrane permeability, 0.5% saponin (Sigma-Aldrich) was added throughout the procedure. Finally, oocytes were mounted on a glass slide and examined using a 10x magnification on an Olympus IX70 inverted microscope (Olympus Ltd., London, U.K.) with a CoolSNAP HQ CCD camera (Roper Scientific, AZ) and SoftWorx image capturing software (Applied Precision, Inc., Issaquah, WA). Control experiments were performed with the second antibody alone to confirm the specificity of labeling.

Statistical Analysis

Each experiment was replicated between three and seven times and analyzed using one-way ANOVA. When a significant effect among treatment groups was detected, the individual groups were compared with the Tukey post-hoc test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Generation of Gdf9^knockdown Oocytes

Following 24 h of culture, Gdf9 and Bmp15 mRNA levels were quantified in Gdf9 dsRNA-, Bmp15 dsRNA-, and buffer-injected oocytes using real-time RT-PCR. Oocytes injected with Gdf9 dsRNA displayed an 89.2% reduction in Gdf9 mRNA levels, whereas oocytes injected with Bmp15 dsRNA showed a 78.8% reduction in Bmp15 mRNA (Fig. 1). Furthermore, this effect appeared to be specific, because Gdf9 dsRNA did not reduce the abundance of Bmp15 mRNA, and vice versa.

View larger version (19K): [in this window] [in a new window]   FIG. 1. Effect of Gdf9 and Bmp15 dsRNA injection on the relative abundance of Gdf9 and Bmp15 mRNA expression in fully grown mouse oocytes. White bars indicate Gdf9 mRNA levels, and patterned bars indicate Bmp15 mRNA levels. Data are presented with the experimental mean as one; individual bars show the treatment mean ± SEM. Within a series, bars with different letters are significantly different (P < 0.05)

To confirm that Gdf9^knockdown oocytes had been generated, GDF9 levels were compared in Gdf9 dsRNA- and buffer-injected oocytes using Western blot analysis. An immunoreactive band of 65.2 kDa (representing the glycosylated, unprocessed form of pro-GDF9) was prominent for the buffer-injected oocytes but barely detectable for the Gdf9 dsRNA-injected oocytes (Fig. 2A). Supporting this observation, confocal microscopic examination of oocytes immunostained with the anti-GDF9 antibody revealed intense spots of dense staining distributed as fine granules in the cytoplasm of buffer-injected oocytes. In comparison, immunostaining in Gdf9 dsRNA-injected oocytes was granular but considerably less intense (Fig. 2B). In the negative control (lacking the anti-GDF9 antibody), only limited and diffuse immunostaining of oocytes was observed. In sum, these results indicate that Gdf9^knockdown oocytes had been generated within 24 h of injection of Gdf9 dsRNA.

View larger version (42K): [in this window] [in a new window]   FIG. 2. Effect of Gdf9 dsRNA injection on GDF9 levels in fully grown mouse oocytes. A) Immunoblot assay for GDF9. Molecular weights are indicated in kilodaltons. A strong band of 62.5 kDa was detected in buffer-injected oocytes in comparison with Gdf9 dsRNA-injected oocytes. B) Immunofluorescence assay for GDF9. Representative selected images were captured using an Olympus IX70 inverted microscope with a CoolSNAP HQ CCD camera and SoftWorx image capturing software. Shown are a negative control (a; buffer-injected oocyte stained with fluorescein isothiocyanate-conjugated antibody to goat immunoglobulin G alone), a buffer-injected oocyte (b), and a Gdf9 dsRNA-injected oocyte (c). The inset shows representative lower-magnification images of multiple oocytes. Bar = 50 µm

Effect of Gdf9^knockdown Oocytes on Cumulus Expansion

Morphological analysis To examine the effect of Gdf9^knockdown oocytes on cumulus expansion, Gdf9 dsRNA-, Bmp15 dsRNA-, and buffer-injected oocytes were cultured alone for 24 h. These oocytes were then cocultured with freshly isolated cumulus shells for a further 24 h. In the buffer-injected and Bmp15 dsRNA-injected groups, cumulus expansion was clearly evident. In these groups, cumulus cells had separated, and evidence of mucification of the extracellular matrix was seen (Fig. 3, a and b). On the other hand, cumulus shells cocultured with Gdf9^knockdown oocytes exhibited only limited evidence of expansion: Nearly all the cumulus cells remained in close association, and little evidence of extracellular mucification was seen (Fig. 3c).

View larger version (68K): [in this window] [in a new window]   FIG. 3. Representative photomicrographs showing the effect of GDF9 knockdown on cumulus expansion. a and b) Expanded cumulus shells following 24-h coculture with buffer-injected oocytes (a) and Bmp15 dsRNA-injected oocytes (b) the presence of 0.5 IU/ml of FSH. c) Cumulus shells showing little evidence of expansion following 24-h coculture with Gdf9knockdown oocytes in the presence of 0.5 IU/ml of FSH. Differential interference contrast images were captured using 20x magnification on a Zeiss LSM510 inverted microscope. Bar = 50 µm

Analysis of Has2 and Ptgs2 mRNA expression To examine the effect of Gdf9^knockdown oocytes on cumulus cell mRNA expression of Has2 and Ptgs2 (i.e., two cumulus cell-expressed genes associated with cumulus expansion), Gdf9 dsRNA- and buffer-injected oocytes were cultured alone for 24 h. These oocytes were then cocultured with freshly isolated cumulus shells for a further 8 h. Following this period, Has2 and Ptgs2 mRNA levels in the cumulus cells were assayed by real-time RT-PCR. Both Has2 and Ptgs2 mRNA levels were significantly higher in cumulus cells cocultured with buffer-injected oocytes than in cumulus cells cocultured with Gdf9^knockdown oocytes (Fig. 4).

View larger version (22K): [in this window] [in a new window]   FIG. 4. Effect of GDF9 knockdown on Has2 and Ptgs2 mRNA expression in cumulus cells. Cumulus shells were cocultured for 8 h with fully grown oocytes that had been injected with either Gdf9 dsRNA or buffer 24 h earlier. Culture medium was supplemented with 0.5 IU/ml of FSH. White bars indicate Ptgs2 mRNA levels, and patterned bars indicate Has2 mRNA levels. Data are presented with the experimental mean as one; individual bars show the treatment mean ± SEM. Within a series, bars with different letters are significantly different (P < 0.05)

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The RNAi technique is a posttranscriptional gene-silencing mechanism initiated by dsRNA homologous in sequence to that of the targeted gene. RNAi has been used extensively to determine gene function in a number of organisms, including mammalian cells [16–19]. Unlike in mammalian somatic cells, long dsRNA does not elicit an apoptotic response in mouse oocytes and preimplantation embryos, possibly because these cells lack an interferon response. For this reason, mouse oocytes and preimplantation embryos are a suitable system in which to undertake RNAi with long dsRNA [16, 20–22].

Using this approach, we have demonstrated that the expression of Gdf9 and Bmp15 mRNA is inhibited by microinjection of Gdf9 long dsRNA and Bmp15 long dsRNA, respectively, into the cytoplasm of mouse oocytes. Quantification of this effect using real-time RT-PCR indicated that Gdf9 and Bmp15 mRNA levels were reduced by 89.2% and 78.8%, respectively, in comparison with buffer-injected oocytes. Moreover, this effect was specific: Injection of Gdf9 dsRNA did not reduce the abundance of Bmp15 mRNA, and vice versa, despite the fact that these genes share a high level of sequence homology. Elvin et al. [9] showed that GDF9 protein is synthesized in oocytes coincident with the expression of Gdf9 mRNA. In the present study, evidence from both Western blot analysis and immunofluorescence assays indicates that injection of oocytes with Gdf9 dsRNA efficiently inhibited the translation of Gdf9 mRNA. These results support and extend those of previous studies indicating that long dsRNA directed toward a specific target mRNA effectively results in sequence-specific gene silencing in mouse oocytes [16, 20, 22].

In the present study, the Gdf9^knockdown oocytes generated by RNAi were valuable in investigating the role of GDF9 in cumulus expansion. Whereas cumulus expansion was clearly evident in cumulus shells cocultured with buffer-injected and Bmp15 dsRNA-injected oocytes, little morphological evidence of cumulus expansion was observed when Gdf9^knockdown oocytes were cocultured with cumulus shells, suggesting that oocyte-secreted GDF9 is, indeed, required for cumulus cell expansion.

Supporting this observation, mRNA levels of Has2 and Ptgs2 were lower in cumulus cells cocultured with Gdf9^knockdown oocytes than in cumulus cells cocultured with buffer-injected oocytes. Expression of the Has2 gene in cumulus cells is critical to the formation of hyaluronan (i.e., the predominant matrix component surrounding expanded cumulus cells), with a strictly temporal regulation of hyaluronan synthesis in cumulus cells appearing to occur at the level of transcription of Has2 [1]. Recombinant GDF9 can induce the expression of Has2 mRNA in mural granulosa cells in vitro [9], and the present study confirms a role for GDF9 in promoting cumulus cell expression of Has2 during cumulus expansion.

The PTGS2 is a key enzyme in the prostaglandin synthesis pathway. A role for this enzyme in cumulus expansion has been demonstrated by the Ptgs2^null mouse, which exhibits reduced ovulatory rates and severely impaired cumulus expansion [4, 23]. Oocyte regulation of Ptgs2 in cumulus cells has been demonstrated [2]. Evidence suggests that GDF9 may be important in this effect, because recombinant GDF9 up-regulated Ptgs2 mRNA levels in cultured mural granulosa cells [9]. The present results therefore confirm a role for oocyte-derived GDF9 in promoting cumulus cell expression of Ptgs2 during cumulus expansion.

In summary, the present results provide convincing evidence that GDF9 is a critical mediator of oocyte regulation of cumulus expansion in mice and that GDF9 is, indeed, the elusive cumulus expansion-enabling factor. Using a highly transferable experimental paradigm, the present study also underscores the power of RNAi as a tool for investigating the mechanisms underlying oocyte-granulosa cell interaction.

	ACKNOWLEDGMENTS

 
The authors would like to thank Prof. Alan Handyside and Dr. David Brooke for technical advice concerning micromanipulation and Dr. Gareth Howell for image capture. We are grateful to Drs. John Eppig and Rob van den Hurk and to Wanzi Muruvi and Nick Salmon for reviewing the manuscript and for helpful comments. We are also grateful to Dr. Peter Pimpl for editorial assistance and to Organon for generously supplying the recombinant FSH.

	FOOTNOTES

 
¹ Supported by BBSRC project grant S16550 and a grant from the Royal Society.

² Correspondence: Ieuan M. Joyce, School of Biology, University of Leeds, Leeds LS2 9JT, UK. FAX: 44 0 113 343 2835; ochrfawr{at}yahoo.co.uk

Received: 25 June 2004.

First decision: 8 July 2004.

Accepted: 27 August 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Fulop C, Salustri A, Hascall VC. Coding sequence of a hyaluronan synthase homologue expressed during expansion of the mouse cumulus-oocyte complex. Arch Biochem Biophys 1997 337:261-266[CrossRef][Medline]
2. Joyce IM, Pendola FL, O'Brien M, Eppig JJ. Regulation of prostaglandin-endoperoxide synthase-2 messenger ribonucleic acid expression in mouse granulosa cells during ovulation. Endocrinology 2001 142:3187-3197[Abstract/Free Full Text]
3. Hess KA, Chen L, Larsen WJ. Inter--inhibitor binding to hyaluronan in the cumulus extracellular matrix is required for optimal ovulation and development of mouse oocytes. Biol Reprod 1999 61:436-443[Abstract/Free Full Text]
4. Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC, Langenbach R. Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E₂ and interleukin-1ß. Endocrinology 1999 140:2685-2695[Abstract/Free Full Text]
5. Chen L, Russell PT, Larsen WJ. Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass. Mol Reprod Dev 1993 34:87-93[CrossRef][Medline]
6. Buccione R, Schroeder AC, Eppig JJ. Interactions between somatic cells and germ cells throughout mammalian oogenesis. Biol Reprod 1990 43:543-547[Abstract]
7. Vanderhyden BC, Caron PJ, Buccione R, Eppig JJ. Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol 1990 140:307-317[CrossRef][Medline]
8. Eppig JJ, Peters AH, Telfer EE, Wigglesworth K. Production of cumulus expansion enabling factor by mouse oocytes grown in vitro: preliminary characterization of the factor. Mol Reprod Dev 1993 34:450-456[CrossRef][Medline]
9. Elvin JA, Clark AT, Wang P, Wolfman NM, Matzuk MM. Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endocrinol 1999 13:1035-1048[Abstract/Free Full Text]
10. Vanderhyden BC, Macdonald EA, Nagyova E, Dhawan A. Evaluation of members of the TGFß superfamily as candidates for the oocyte factors that control mouse cumulus expansion and steroidogenesis. Reprod Suppl 2003 61:55-70[Medline]
11. Vanderhyden BC. Species differences in the regulation of cumulus expansion by an oocyte-secreted factor(s). J Reprod Fertil 1993 98:219-227
12. Salmon NA, Handyside AH, Joyce IM. Oocyte regulation of anti-mullerian hormone expression in granulosa cells during ovarian follicle development in mice. Dev Biol 2004 266:201-208[CrossRef][Medline]
13. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 1987 262:10035-10038[Abstract/Free Full Text]
14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970 227:680-685[CrossRef][Medline]
15. Polanski Z, Ledan E, Brunet S, Louvet S, Verlhac MH, Kubiak JZ, Maro B. Cyclin synthesis controls the progression of meiotic maturation in mouse oocytes. Development 1998 125:4989-4997[Abstract]
16. Svoboda P, Stein P, Hayashi H, Schultz RM. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development 2000 127:4147-4156[Abstract]
17. Sharp PA. RNA interference–2001. Genes Dev 2001 15:485-490[Free Full Text]
18. Sharp PA, Zamore PD. Molecular biology: RNA interference. Science 2000 287:2431-2433[Free Full Text]
19. Vaucheret H, Beclin C, Fagard M. Posttranscriptional gene silencing in plants. J Cell Sci 2001 114:3083-3091[Abstract/Free Full Text]
20. Yu J, Deng M, Medvedev S, Yang J, Hecht NB, Schultz RM. Transgenic RNAi-mediated reduction of MSY2 in mouse oocytes results in reduced fertility. Dev Biol 2004 268:195-206[CrossRef][Medline]
21. Fedoriw AM, Stein P, Svoboda P, Schultz RM, Bartolomei MS. Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 2004 303:238-240[Abstract/Free Full Text]
22. Wianny F, Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol 2000 2:70-75[CrossRef][Medline]
23. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997 91:197-208[CrossRef][Medline]

This article has been cited by other articles:

				J. E. Swain and T. B. Pool ART failure: oocyte contributions to unsuccessful fertilization Hum. Reprod. Update, July 5, 2008; (2008) dmn025v1. [Abstract] [Full Text] [PDF]

				R. B. Gilchrist, M. Lane, and J. G. Thompson Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality Hum. Reprod. Update, March 1, 2008; 14(2): 159 - 177. [Abstract] [Full Text] [PDF]

				D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]

				F. J. Diaz, K. Wigglesworth, and J. J. Eppig Oocytes determine cumulus cell lineage in mouse ovarian follicles J. Cell Sci., April 15, 2007; 120(8): 1330 - 1340. [Abstract] [Full Text] [PDF]

				X. Gueripel, V. Brun, and A. Gougeon Oocyte Bone Morphogenetic Protein 15, but not Growth Differentiation Factor 9, Is Increased During Gonadotropin-Induced Follicular Development in the Immature Mouse and Is Associated with Cumulus Oophorus Expansion Biol Reprod, December 1, 2006; 75(6): 836 - 843. [Abstract] [Full Text] [PDF]

				O. Yoshino, H. E. McMahon, S. Sharma, and S. Shimasaki A unique preovulatory expression pattern plays a key role in the physiological functions of BMP-15 in the mouse PNAS, July 11, 2006; 103(28): 10678 - 10683. [Abstract] [Full Text] [PDF]

				S. Mazerbourg and A. J.W. Hsueh Genomic analyses facilitate identification of receptors and signalling pathways for growth differentiation factor 9 and related orphan bone morphogenetic protein/growth differentiation factor ligands Hum. Reprod. Update, July 1, 2006; 12(4): 373 - 383. [Abstract] [Full Text] [PDF]

				S. A. Pangas, X. Li, E. J. Robertson, and M. M. Matzuk Premature Luteinization and Cumulus Cell Defects in Ovarian-Specific Smad4 Knockout Mice Mol. Endocrinol., June 1, 2006; 20(6): 1406 - 1422. [Abstract] [Full Text] [PDF]

				K. Nganvongpanit, H. Muller, F. Rings, M. Hoelker, D. Jennen, E. Tholen, V. Havlicek, U. Besenfelder, K. Schellander, and D. Tesfaye Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA. Reproduction, May 1, 2006; 131(5): 861 - 874. [Abstract] [Full Text] [PDF]

				E. Clelland, G. Kohli, R. K. Campbell, S. Sharma, S. Shimasaki, and C. Peng Bone Morphogenetic Protein-15 in the Zebrafish Ovary: Complementary Deoxyribonucleic Acid Cloning, Genomic Organization, Tissue Distribution, and Role in Oocyte Maturation Endocrinology, January 1, 2006; 147(1): 201 - 209. [Abstract] [Full Text] [PDF]

				S. A. Pangas and M. M. Matzuk The Art and Artifact of GDF9 Activity: Cumulus Expansion and the Cumulus Expansion-Enabling Factor Biol Reprod, October 1, 2005; 73(4): 582 - 585. [Abstract] [Full Text] [PDF]

				R. A. Dragovic, L. J. Ritter, S. J. Schulz, F. Amato, D. T. Armstrong, and R. B. Gilchrist Role of Oocyte-Secreted Growth Differentiation Factor 9 in the Regulation of Mouse Cumulus Expansion Endocrinology, June 1, 2005; 146(6): 2798 - 2806. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 72/1/195    most recent biolreprod.104.033357v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gui, L.-M. Articles by Joyce, I. M. Search for Related Content PubMed PubMed Citation Articles by Gui, L.-M. Articles by Joyce, I. M. Agricola Articles by Gui, L.-M. Articles by Joyce, I. M.