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.

Background: Follicle stimulating hormone (FSH) is key regulator for follicular development, dif-ferentiation and maturation, the effects involve various intra follicular factors, such as members of 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 6-8mm chicken follicles to detect FSH induced phosphorylation and nuclear rejection of FOXO3/4, and its regulatory effects on follicular GC pro-liferation, differentiation and apoptosis, using immunofluorescence, Western blotting, RT-qPCR and flow cytometry methods. Results: This study found that the expression level of FSHR mRNA was highest in GC treated with 10 ng/ml FSH for 8 and 12 hours. The level of p-FOXO3/4 protein sig-nificantly increased in cells treated with FSH for 12 hours, while the expression level of non phosphorylated FOXO3/4 significantly decreased. After co-treatment with 10ng/ml LMB, FOXO3/4 phosphorylation was effectively prevented. The immunofluorescence results showed that FOXO3 and FOXO4 were originally distributed in GC nucleus and cytoplasm, whereas they were almost accumulated in cytoplasm as 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 cell, but significantly reduced GCs apoptosis. However, LMB also eliminated these stimulating or inhibitory effects on cell proliferation. Conclusion: These findings provided new evidence that FSH induced FOXO3/4 nuclear rejection promoted GCs proliferation and reduced GCs apoptosis during hen follicular development.