Thesis title: Functional studies of microRNA 17-92 cluster members in bovine granulosa cells and oocyte maturation
Name: Eryk Andreas
Public e-mail:
Institute address: Kampus IPB Darmaga
Bogor, 16680
West Java- Indonesia
Host institute: Institute of Animal Science
EndenicherAllee 15, 53115 Bonn
Germany
Dynamic transcript of genes expression are believed to occur during follicular development and oocyte maturation, which one way or the other is regulated by a post-transcriptional modifier, namely microRNA. In the previous work, among others, miR-17-92 cluster members were overexpressed in granulosa cell of subordinate follicle at day 19 of estrous cycle compared to the dominant ones. Thus, we hypothesized the potential involvement of miR-17-92 cluster members in follicular function at the late stage of the estrous cycle. Therefore, the aim of this thesis was to investigate the role of miR-17-92 cluster members in bovine granulosa cell and oocyte maturation. First, potential target gene of miR-17-92 cluster were predicted insilico followed by validation using luciferase assay. In order to investigate the role of miR-17-92 cluster member in granulosa cell function and oocyte maturation, we modulated the expression of those microRNAs in granulosa cells and cumulus-oocyte complexes (COCs) under in vitro condition. Target prediction and validation revealed that PTEN and BMPR2 are direct target genes of miR-17-92 cluster members. This result was confirmed by the alteration of PTEN and BMPR2 expression in granulosa cells transfected with miR-17-92 cluster members mimic and inhibitor. In this study, we observed that overexpression of miR-17-92 cluster increased proliferation and decreased differentiation rate of granulosa cells. On the other hand, inhibition of miR-17-92 cluster showed the opposite phenotypes. However, progesterone level in spent media of granulosa cells culture was not persistent with the cell differentiation rate. Further, cross-validation by target knockdown PTEN and BMPR2 genessimulated the results obtained from granulosa cells transfected miR-17-92 cluster member. In addition, the expression of one of miR-17-92 cluster members (miR-20a) in cumulus cells increased after in vitro maturation (IVM). Contrastively, it was decreased in oocytes after IVM. Moreover, the expression of miR-20a in cumulus cells and oocytes was affected by the presence or absence of their companion cells during culture. The expression of miR-20a in cumulus cells and oocytes from COCs cocultured with miR-20a mimic or inhibitor suggested that the transfection was restricted in the cumulus cells.In this study, miR-20a overexpression in COCs culture increased oocyte maturation rate and cumulus cell progesterone synthesis. On the other hand, inhibition of miR-20a did not affect the oocyte maturation rate, but decreased progesterone synthesis. In conclusion, the miR-17-92 cluster members involved in granulosa cell proliferation and differentiation, as well as oocyte maturation by targeting PTEN and BMPR2 genes.