RHOB Regulates Apoptosis of Granulosa Cells in Muscovy Duck Follicles via Mitochondrial Pathway

Muscovy ducks (Cairina moschata) exhibit strong nesting tendencies, which result in reduced egg-laying performance. The research team previously identified differential expression of the Ras homolog family member B (RHOB) gene in the ovaries of Muscovy duck during the nesting and laying periods through RNA-seq and quantitative real-time polymerase chain reaction (qPCR) analysis. This finding suggested that RHOB may be associated with nesting behavior in Muscovy ducks. Previous studies have demonstrated that the nesting behavior of Muscovy ducks is closely associated with the proliferation and Apoptosis of their ovarian granulosa cells. It is speculated that RHOB may be involved in the proliferation and Apoptosis of Muscovy duck ovarian granulosa cells. This study employed qPCR, immunofluorescence staining, live-cell Caspase3 activity and mitochondrial membrane potential assays, Reactive Oxygen Species (ROS) staining, 5-Ethynyl-2'-deoxyuridine (EdU) staining, cell cycle analysis, cell Apoptosis detection and cell counting kit-8 (CCK-8) assays. Our results showed that RHOB inhibited granulosa cell Apoptosis and promoted granulosa cell proliferation. Similarly, in a granulosa cell Apoptosis model, RHOB was also found to inhibit Apoptosis in Muscovy duck granulosa cells. Further studies revealed that RHOB regulates mitochondrial function in granulosa cells. The combined experimental results indicate that RHOB regulates granulosa cell Apoptosis in Muscovy duck follicles via the mitochondrial Apoptosis pathway. These findings provide an experimental basis and theoretical foundation for the selective breeding of desirable traits in Muscovy ducks, such as low nesting behavior and high egg production.