Ovary

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

Abstract
In mouse ovaries, growth differentiation factor-9 (GDF9) is an oocyte-derived growth factor that plays an essential role in early follicular development. However, the role of GDF9 at later stages of follicle development is uncertain. In this study, a long, double-stranded (ds) RNA interference (RNAi) approach was used to investigate the possible role of GDF9 in mediating oocyte regulation of cumulus expansion. Fully-grown mouse oocytes injected with Gdf9 dsRNA, Bmp15 dsRNA or injection buffer were cultured for 24 h and processed for measurement of Gdf9 and Bmp15 mRNA levels using real-time RT-PCR, and for measurement of GDF 9 protein levels using Western blotting and immunofluorescence. Injection with Gdf9 dsRNA knocked down Gdf9 but not Bmp15 mRNA expression in oocytes, and vice versa. Furthermore, GDF9 protein levels were reduced in the Gdf9 dsRNA injected oocytes. To investigate the role of GDF9 in cumulus expansion, two endpoints were used to evaluate cumulus expansion: Has2 and Ptgs2 mRNA levels were measured in cumulus cells using real-time RT-PCR; and assessment of cumulus expansion was undertaken morphologically. After 24 h of culture in the presence of 0.5 IU/ml FSH, cumulus shells co-cultured with buffer- and Bmp15 dsRNA-injected oocytes exhibited a high degree of expansion, while cumulus shells co-cultured with Gdf9 dsRNA-injected oocytes exhibited only limited expansion. Supporting this observation, after 8 h of co-culture Has2 and Ptgs2 mRNA levels were lower in cumulus cells co-cultured with Gdf9 dsRNA-injected oocytes than in those co-cultured with buffer-injected oocytes. These results strongly support the concept that GDF 9 is a key mediator of oocyte-enabled cumulus expansion in mice.

Key words: Cumulus cells • Fertilization • Follicular development • Growth factors • Oocyte development

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