Follicle selection is a pivotal process that determines which dominant prehierarchical follicle will enter the preovulatory hierarchy in the hen ovary and directly affects egg-laying productivity, in which granulosa cells (GCs) are characterized by active proliferation and significantly enhanced FSHR mRNA expression. Increasing evidence has shown that the PI3K/Akt signaling pathway and its important target and effector FOXO1, which promotes GC apoptosis, play crucial roles in ovarian follicular development in mammals. To investigate the molecular mechanism by which follicle-stimulating hormone (FSH)-mediated forkhead box O1 (FOXO1) participates in follicle selection, we treated granulosa cells from 6–8 mm prehierarchical follicles of chickens with FSH and leptomycin B (LMB). The results showed that under FSH and/or LMB treatment, the expression levels of FSHR, FOXO1, and its phosphorylated forms (p-FOXO1) at the predicted protein kinase B (PKB/Akt) phosphorylation sites Thr²⁴, Ser²⁴⁸, and Ser³¹¹ were differentially regulated. The subcellular localization of p-FOXO1 in hen ovarian GCs was determined by Western blotting and immunofluorescence staining (IF) analysis. And the expression of FOXO1 was significantly reduced, whereas the expression of p-FOXO1 corresponding to the PKB phosphorylation sites Ser²⁴⁸ and Ser³¹¹ was noticeably boosted in cultured GCs induced by FSH, accompanied by exclusion of FOXO1 from the nucleus to the cytoplasm. Subsequently, the effects of the PI3K/Akt signaling pathway on phosphorylation levels and nuclear exclusion of p-FOXO1 at the sites Ser²⁴⁸ and Ser³¹¹ were examined. The results indicate that the PI3K/Akt-dependent phosphorylation at these sites directly resulted in nuclear exclusion of FOXO1 in ovarian GCs, in which the Ser²⁴⁸ site is more essential than the Ser³¹¹ site. Subsequently, the FSH-induced acetylation of FOXO1 mediated by the cAMP/PKA pathway can enhance the phosphorylation level of FOXO1 at the Ser²⁴⁸ site. In summary, our findings demonstrate that FSH induces FOXO1 phosphorylation, nuclear exclusion, and functional inactivation by activating the PI3K/Akt signaling pathway. Moreover, during follicular development and selection, FOXO1 acts as a pivotal mediator linking the PI3K/Akt and P62/Keap1/Nrf2 signaling pathways to regulate granulosa cell proliferation and apoptosis, thereby exerting a central regulatory role.

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.

The tumor suppressor gene NF2 (Neurofibromatosis type 2) is involved in critical biological processes, including organ-size control, cell proliferation, differentiation, apoptosis, and cancer development by activating the SWH signaling pathway (also named Hippo pathway) in humans and Drosophila. However, the exact roles and molecular regulatory mechanisms of NF2 in chicken granulosa cells (GCs) proliferation, differentiation, apoptosis, and steroidogenesis are required for ovarian follicle growth, prehierarchical follicular development, and follicular selection remain poorly understood. This study demonstrated that the NF2 mRNA was expressed in various-sized prehierarchical follicles (PFs) and GCs. NF2 protein was localized in the GC cytoplasm and predominantly expressed in the undifferentiated GCs and oocytes of the PFs. Here, the effects of NF2 on the proliferation, differentiation, apoptosis, and mitochondrial function of GCs were detected. NF2 regulates GC proliferation, differentiation, and apoptosis via SWH signaling pathway downstream transcriptional co-activator YAP1 in vitro. Additionally, NF2/SWH significantly increased the phosphorylation level of p38 via transcriptional co-activator YAP1. To explore the role of the p38 signaling pathway in the process of NF2/SWH-induced apoptosis in GCs, an activator of p38 (U46619) was added to the si-NF2 treatment, and inhibitor of p38 (SB203580) added to the si-NF2 treatment. We found that NF2/SWH signaling pathway promoted GC apoptosis by affecting the function of GC mitochondria via p38 signaling via SWH signaling pathway downstream transcriptional co-activator YAP1. In conclusion, these results demonstrated that NF2/SWH inhibited proliferation, and differentiation of GCs via SWH signaling pathway downstream transcriptional co-activator YAP1, and promoted apoptosis of GCs by p38 MAPK signaling and mitochondrial function in chicken via SWH signaling downstream transcriptional co-activator YAP1.