Molecular Mechanism Of The Proliferation Of Granulosa Cells Regulated By ActivinA In Mouse

Activin A (ActA) a member of TGF-P superfamily, plays an important role in the development of mammalian follicles, and regulates related genes expression at different stages of follicular development. Recent researches mainly focused on functions of ActA on follicular maturation and luteal, but there is little known about its effects on the proliferation of granulosa cells. This research focuses on the signal pathway of ActA regulating the proliferation of granulosa cells and its mechanism.Granulosa cells of12-14dpp mouse preantral follicles was cultured in vitro and the relationship of ActA and the proliferation of granulosa cells was investigated. The proliferation of cells was measured by BrdU staining experiments. The results indicated that:the number of BrdU positive cells was significantly increased incubated with ActA (100ng/ml) than in control after4days’ culture (P<0.001). The results of fluorescence immunohistochemistry showed that SMAD3mainly expressed in granulosa cells. ActA promoted granulosa cells proliferation and at the same condition increased the expression of Smad3both in mRNA and protein levels. The Smad3over-expression has the similar effects on granulosa cells proliferation. We found that the expression of NOTCH2and HEY2also increased in the presence of ActA. Western blot analysis indicated that ActA promoted expression of two protein in granulosa cells (P<0.01). Fouthermore, Notch2and Hey2expression levels are significantly increased when Smad3overexperssed (P<0.01). Granulosa cells exposured to Notch signal pathway inhibitor DAPT for3d or5d, and the results showed that DAPT repressed proliferation of granulosa cell significantly (P<0.01). And adding ActA or Smad3overexpression can relieve the inhibition of Notch signaling pathway. CoIP and ChIP were performed to determine the exact converging points between TGF-P and NOTCH signaling pathways. SMAD3protein integrated with ICN to form complex and act as a transcriptional factor binding to the specific DNA motifs of Notch target genes such as Hey1and Hey2to participate in transcriptional regulation and affect the proliferation of granulosa cells.In summary, ActA regulates granulosa cells proliferation by TGF-P signaling pathway, and which has a direct crosstalk with Notch signaling pathway.