RNA interference evidence that growth differentiation factor-9 mediates clocyte regulation of cumulus expansion in mice

In mouse ovaries, growth differentiation factor-9 (GDF9) is an oocyte-derived growth factor that plays an essential role 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.