Abstract
BACKGROUND: Follicle stimulating hormone (FSH) is key regulator for follicular development, differentiation, and maturation, and the effects involve various intra follicular factors, such as members of the forkhead box O (FOXO) subfamily. However, the specific role and mechanism of FOXO3 and FOXO4 in growth and development of hen follicles by affecting granulosa cell (GC) division and FSH response function are still unclear. METHOD: This study selected GCs from 6-8 mm chicken follicles, and immunofluorescence and Western blot methods were used to detect FSH-induced FOXO3/4 phosphorylation and nuclear exclusion. Quantitative real-time PCR and flow cytometry were used to investigate the regulatory effects of FSH-induced FOXO3/4 phosphorylation and nuclear exclusion on follicular GC proliferation, differentiation, and apoptosis. RESULTS: This study found that the level of p-FOXO3/4 protein significantly increased in cells treated with FSH for 12 h, while the expression level of non-phosphorylated FOXO3/4 significantly decreased. After co-treatment with 10 ng/mL Leptomycin B (LMB), FOXO3/4 phosphorylation was effectively prevented. The immunofluorescence results showed that FOXO3 and FOXO4 were originally distributed in the GC nucleus and cytoplasm, whereas they were almost accumulated in cytoplasm when treated with FSH for 12 h. Conversely, FOXO3/4 nuclear translocation was blocked by LMB. Moreover, RT-qPCR and flow cytometry results showed that FSH treatment significantly increased proliferation and differentiation of cells but significantly reduced GCs apoptosis. However, LMB also eliminated these stimulating or inhibitory effects on cell proliferation. CONCLUSION: These findings provide new evidence that FSH-induced FOXO3/4 nuclear exclusion promotes GCs proliferation and reduces GCs apoptosis during hen follicular development.