RAC1 belongs to the small G protein Rho subfamily and is implicated in regulating gene expression, cell proliferation and differentiation in mammals and humans; nevertheless, the function of RAC1 in growth and development of hen ovarian follicles is still unclear. This study sought to understand the biological effects of RAC1 on granulosa cell (GC) proliferation and differentiation of hen ovarian prehierarchical follicles. Firstly, our results showed expression levels of RAC1 mRNA in the follicles with diameters of 7.0–8.0 mm, 6.0–6.9 mm and 1.0–3.9 mm were greater than other follicles (p < 0.05). The RAC1 protein was mainly expressed in oocyte and its around GCs and stromal tissues of the prehierarchical follicles by immunohistochemistry. Further investigation revealed the RAC1 gene remarkably enhanced the mRNA and protein expression levels of FSHR (a marker of follicle selection), CCND2 (a marker of cell-cycle progression and GC differentiation), PCNA (a marker of GC proliferation), StAR and CYP11A1 (markers of GC differentiation and steroidogenesis) (p < 0.05). Furthermore, our data demonstrated siRNA interference of RAC1 significantly reduced GC proliferation (p < 0.05), while RAC1 gene overexpression enhanced GC proliferation in vitro (p < 0.05). Collectively, this study provided new evidence that the biological effects of RAC1 on GC proliferation, differentiation and steroidogenesis of chicken ovary follicles.

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: This study aims to investigate the regulation of small mothers against decapentaplegic 2 and 3 (Smad2/3) protein phosphorylation and nuclear exclusion in follicular granulosa cells (GCs) by chicken follicle-stimulating hormone (FSH) through the phosphatidylinositol 3-kinase (PI3K) signaling pathway, as well as the effect of Smad2/3 proteins on forkhead box O 3 and 4 (FoxO3/4). This lays the foundation for exploring the regulatory functions of signaling pathways closely related to follicular growth and development, as well as the molecular mechanisms of subcellular localization and nuclear exclusion of various effector factors (including transcription factors). Methods: In this study, we used granulosa cells from 6–8 mm prehierachical follicles of chickens and performed immunofluorescence, quantitative real-time PCR (RT-qPCR), and Western blotting analysis to detect the phosphorylation and nuclear exclusion of Smad2/3 induced by FSH, as well as the regulatory effect of Smad2/3 on FOXO3/4 proteins. Results: The results showed that 10 ng/mL FSH and 50 μg/mL PI3K activator significantly reduced the phosphorylation level of Smad2/3 (p < 0.05), while no nuclear exclusion was observed. On the other hand, 16 nM/mL PI3K inhibitor and 50 μg/mL alkaline phosphatase significantly increased the phosphorylation level of Smad2/3 (p < 0.05). Overexpression of Smad2/3 increased the phosphorylation level of FOXO3/4 (p < 0.05); Smad2/3 interference resulted in a decrease in FOXO3/4 phosphorylation levels (p < 0.05). Conclusions: FSH can inhibit Smad2/3 phosphorylation and retain it in the nucleus through the PI3K signaling pathway. Smad2/3 and FOXO3/4 act as downstream effectors of the PI3K signaling pathway, and Smad2/3 can promote the phosphorylation of FOXO3/4.

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.

Follicle-stimulating hormone (FSH) supports granulosa cell survival during follicular development, but the RNA networks involved remain incompletely understood. To investigate these networks, we integrated whole-transcriptome profiling and functional assays in cultured granulosa cells from chicken prehierarchical follicles. Transcriptomic comparisons included control, FSH and FSH plus leptomycin B (LMB), a nuclear export inhibitor. Integrated analysis of these profiles identified a candidate circARHGAP21/gga-miR-146a-5p/DEAF1 regulatory module. FSH increased circARHGAP21 and DEAF1 expression and decreased gga-miR-146a-5p abundance, whereas LMB opposed these changes in FSH-treated cells. Reporter assays showed that gga-miR-146a-5p suppressed reporter activity through the predicted sites in circARHGAP21 and the DEAF1 3′ untranslated region. Transfection with the oe-circARHGAP21 construct reduced apoptosis and increased follicle-stimulating hormone receptor (FSHR) expression. Construct transfection also increased proliferating cell nuclear antigen (PCNA) expression and decreased caspase-3 (CASP3) expression. Cotransfection with a gga-miR-146a-5p mimic attenuated these effects, whereas DEAF1 overexpression produced changes similar to those observed after oe-circARHGAP21 construct transfection. Compared with the oe-circARHGAP21 construct group, the corresponding LMB-treated group showed lower DEAF1, FSHR and PCNA expression and higher CASP3 expression and apoptosis. Together, these findings identify an FSH-responsive circARHGAP21/gga-miR-146a-5p/DEAF1 module associated with granulosa cell survival and follicle-selection-related gene expression.