Abstract
MicroRNAs (miRNAs) have been established as important regulators of gene expression in the mammalian ovary. A previous screen of small RNA in the porcine ovary identified the downregulation of miR-574 during oocyte maturation, although its role during this process was not established. Here, we found that miR-574 directly targets the transcript for hyaluronan synthase 2 protein (HAS2), a key enzyme in the production of extracellular matrix by the surrounding cumulus cells. Inhibiting this miRNA during in vitro maturation (IVM) increased HAS2 levels along with several markers of oocyte quality. Furthermore, inhibiting miR-574 increased oocyte meiotic progression. We then stably overexpressed miR-574 using a lentiviral vector to transduce cumulus cells during IVM. This gain-of-function approach resulted in a 50% decrease in HAS2 expression and nearly 20% reduction in oocyte progression through meiosis. To confirm the specific targeting of HAS2 by miR-574, we constructed several luciferase vectors harboring the HAS2 3'-untranslated region. Cotransfection of the reporter and miR-574 attenuated luciferase activity. After mutating the putative miR-574 binding site, however, this effect was abolished and luciferase activity remained high. Our results show that the direct targeting of HAS2 by miR-574 negatively impacts oocyte quality during IVM and that inhibiting miR-574 derepresses HAS2 expression and subsequently improves oocyte maturation. Taken together, we help to elucidate a mechanism of posttranscriptional regulation by miRNA in the mammalian ovary.
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