The Effect of FSH-Induced Nuclear Exclusion of FOXO3/4 on Granulosa Cell Proliferation and Apoptosis of Hen Ovarian Follicles

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.