SMAD3 regulates the diverse functions of rat granulosa cells relating to the FSHR/PKA signaling pathway

Abstract

The function of Smad3, a downstream signaling protein of the transforming growth factor β (TGFβ) pathway, in ovarian follicle development remains to be elucidated. The effects of Smad3 on ovarian granulosa cells (GCs) in rat were studied. Female rats (21 days of age Sprague–Dawley) received i.p. injections of pregnant mare serum gonadotropin, and GCs were harvested for primary culture 48 h later. These cells were engineered to overexpress or knockdown Smad3, which were validated by immunohistochemistry and western blot. The expression of proliferating cell nuclear antigen (PCNA), cyclin D2, TGFβ receptor II (TGFβRII), protein kinase A (PKA), and FSH receptor (FSHR) was also detected by western blotting. Cell cycle and apoptosis of GCs were assayed by flow cytometry. The level of estrogen secreted by GCs was detected by ELISA. Smad3 overexpression promoted estrogen production and proliferation while inhibiting apoptosis of GCs. Reduction in Smad3 by RNAi resulted in reduced estrogen production and proliferation and increased apoptosis of GCs. Manipulation of Smad3 expression also resulted in changes in FSHR and PKA expression, suggesting that the effects of Smad3 on follicle development are related to FSHR-mediated cAMP signaling.