Abstract
RNA interference (RNAi) is an effective tool for studying gene function in oocytes, but no studies have targeted somatic cells of primary cultured cumulus cell-oocyte complexes (COCs). This is probably due to difficulty in introducing RNAi-inducing molecules, such as a short-hairpin RNA (shRNA) gene, into COCs by commonly used transfection reagents. We therefore tested whether a developmental process of intact COCs could be suppressed by adenovirus-mediated shRNA expression. Has2, encoding hyaluronan synthase 2, was selected as the target transcript, because the process of cumulus expansion depends upon expression of Has2 mRNA and this process is easily evaluated in vitro. Intact COCs were infected with replication-incompetent adenoviruses containing an expression sequence of shRNA targeting either Has2 (Has2 shRNA) or a control transcript not expressed in cumulus cells, and the effects on epidermal growth factor (EGF)-stimulated cumulus expansion were determined. Has2 shRNA expression suppressed Has2 mRNA levels in COCs by more than 70%, without affecting expression levels of Ptgs2, Ptx3, Tnfaip6 mRNAs, which are also required for cumulus expansion, or other transcripts not related to expansion. Interestingly, levels of Areg and Ereg mRNAs were decreased in COCs expressing Has2 shRNA when compared with those in controls, while Btc mRNA levels remained unaffected. Furthermore, the degree of cumulus expansion by Has2 shRNA-expressing COCs was significantly less than that of controls. Thus adenovirus-mediated introduction of shRNA produces specific gene silencing and a phenotype in intact COCs, providing proof of principle that this method will be a helpful tool for understanding mechanisms of COC development.