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: The egg-laying performance of hens is primarily regulated by ovarian follicle growth and development; these follicles are susceptible to oxidative damage caused by excessive reactive oxygen species (ROS). Oxidative damage can lead to follicular atresia and impaired reproductive performance. Melatonin (MT), a known endogenous antioxidant, plays a role in regulating oxidative damage, but its precise mechanisms in mitigating H₂O₂-induced oxidative damage via mitophagy regulation in granulosa cells remain unclear. Methods: An in vitro oxidative damage model was established by determining the optimal H₂O₂ concentration using CCK-8 fluorescence quantification. The optimal MT concentration was identified through fluorescence quantification and catalase (CAT) activity assays. The protective effects of MT against H₂O₂-induced oxidative damage in follicular granulosa cells were investigated using flow cytometry, Western blotting, ELISA, and quantitative fluorescence analysis. Results: An in vitro oxidative damage model was established using H₂O₂-induced granulosa cells, characterized by P53 and LC3-II upregulation and LC3-I and BCL-2 downregulation. The optimal MT concentration for reducing cellular injury was determined. MT co-treatment enhanced CAT, GSH, and SOD activities, decreased LC3-II/LC3-I conversion, and increased P62 expression. Furthermore, MT reduced autophagic vesicle formation and restored mitochondrial membrane potential, demonstrating its protective effect against H₂O₂-induced oxidative damage. Conclusions: Melatonin alleviates H₂O₂-induced oxidative damage in chicken follicular granulosa cells by modulating antioxidant defense, autophagy, and mitochondrial function. These findings provide newer insights to our understanding of the regulatory mechanisms underlying the alleviation of the H₂O₂-induced oxidative damage in granulosa cells during ovarian follicle development in chickens.

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.

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.