Reproduction Abstracts

Introduction: Proliferation of granulosa cells (GCs) is essential for oocyte growth toward ovulation. Extreme proliferation of GCs is taken place under hypoxic condition due to lack of vascularization in the follicles. However, the mechanisms underlying proliferation of GCs under hypoxia have not been elucidated. In the present study, we examined the effect of low oxygen level on molecular background of cellular proliferation in bovine GCs.

Methods: Bovine GCs obtained from 2 to 5 mm follicles were incubated under 5% (low) and 21% (high) oxygen level for 24 h. To understand comprehensive gene expression, RNA-seq was conducted using next generation sequence technique. From the results of RNA-seq analysis, we compared the expression level of HIF-1α and VEGF using western blotting. Proliferative activity was examined by BrdU intake, and amount of phosphorylated mTOR, AKT, and S6RP were compared under low and high oxygen level. To examine the role of VEGF in the hypoxia-induced cellular proliferation, proliferation activity and phosphorylation of AKT and mTOR was examined using anti-VEGF neutralizing antibody. We examined the effect of oxygen level on expression level of SIRT1 and effects of SIRT1 activation by resveratrol (Res) under low oxygen on proliferative activity and the amount of phosphorylated proliferation related proteins.

Results and discussion: Gene expression analysis revealed upregulation of genes associated with HIF-1α. Low oxygen level increased proliferation activity of GCs, expression level of VEGF, amount of phosphorylated mTOR, AKT, and S6RP with high HIF-1α expression, whereas, anti-VEGF antibody treatment reversed activation of AKT- mTOR pathway and reduced cellular proliferative activity. On the other hand, low oxygen level reduced SIRT1, and activation of SIRT1 by Res decreased cellular proliferation with reduction of phosphorylated mTOR. These results suggest that low oxygen level stimulates the VEGF-AKT-mTOR pathway, which contributes to GC proliferation, and SIRT1 is major regulator of the hypoxia associated cellular proliferation.