AKT is required for FSH-stimulation of beta-catenin accumulation in bovine granulosa cells

Folliculogenesis is a multifaceted process in which follicles mature and produce estradiol (E2) and ovulate an oocyte. beta-catenin (CTNNB1) is required as a transcriptional co-factor for FSH-simulated E 2 production. In bovine large antral follicles, greater concentrations of intra-follicular E2 are associated with increased abundance of CTNNB1 protein. Likewise, bovine granulosa cells (GC) in culture, have increased CTNNB1 and protein kinase B (AKT) protein, and WNT2 mRNA expression following FSH stimulation. These data indicate that FSH regulates CTNNB1 through the canonical WNT or AKT signaling pathways. AKT and WNT signaling pathways are both capable of phosphorylating GSK3-beta, a component of the CTNNB1 degradation complex. Immunofluorescence FSH time course in bovine GCs from 0 to 48 h was conducted to evaluate localization of CTNNB1 from the cell junctions to the cytoplasm and nucleus. beta-catenin fluorescence into the cytoplasm and nucleus was greatest at 6 h and remained the same through 24 h, and was slightly reduced at 48h. The objective of following studies was to elucidate AKT's role in CTNNB1 accumulation. Preliminary studies for AKT inhibitor (LY294002) and (insulin-like growth factor 1; IGF-I) dose response were conducted to determine the optimal response. Inhibition of the AKT pathway with LY294002 was confirmed by the reduction in phosphorylated AKT (pAKT). Inhibition of AKT reduced FSH-mediated CTNNB1 accumulation and subsequent E2 production in bovine GCs. This suggests that FSH-stimulation of CTNNB1is mediated through the AKT signaling pathway. Another known AKT stimulator, IGF-I, was investigated for its ability to regulate CTNNB1. Insulin-like growth factor 1 increased pAKT compared to control and FSH, confirming AKT activation. Phosphorylated GSK3-beta was below control and FSH levels for the IGF-I treated GCs, and IGF-I did not stimulate CTNNB1 accumulation above control. However, GC production of P4 and E2 were increased as expected. Suggesting IGF-I activation of AKT stimulates activation of other pathways, and CTNNB1 may not be required for IGF-I stimulation of steroid production. Stimulation with FSH increased accumulation of CTNNB1 protein and E2, and requires AKT. AKT is required for steroid production, but is not sufficient to stimulate CTNNB1 accumulation.