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.

Background: The egg-laying performance of hens is primarily regulated by ovarian follicle growth and development, which 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 (ΔΨm), 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 mecha-nisms underlying the alleviation of the H₂O₂-induced oxidative damage in the granulosa cells during ovarian follicle development in chicken.