Abstract

Oxidative stress (OS) within the follicular microenvironment compromises bovine fertility largely by damaging granulosa cells (GCs), thereby impairing oocyte developmental competence. Betaine has been implicated in redox regulation, yet its direct actions and molecular mechanisms in bovine GCs remain insufficiently defined. In this study, primary bovine GCs were exposed to an H 2 O 2 -induced OS model after betaine pretreatment, and cell viability, intracellular ROS, GSH/GSSG homeostasis, and apoptosis were evaluated using CCK-8, CM-H2DCFDA fluorescence imaging, glutathione assays, and Annexin V-APC/PI flow cytometry. RNA-seq, qPCR, western blotting, siRNA-mediated GSTA5 knockdown, STAT3 activator/inhibitor assays, promoter prediction, structural modeling, and ChIP-qPCR were further used to characterize the associated transcriptional mechanism. Betaine pretreatment significantly attenuated H 2 O 2 -induced loss of GC viability and improved cellular redox status. Transcriptome profiling identified GSTA5 as a core betaine-responsive gene under oxidative challenge. Consistently, GSTA5 knockdown weakened betaine-mediated restoration of the GSH/GSSG balance, promoted renewed ROS accumulation, and reduced anti-apoptotic protection. Mechanistically, STAT3 activation was associated with GSTA5 expression, and ChIP-qPCR supported STAT3 occupancy at the GSTA5 promoter. Collectively, these findings indicate that betaine protects bovine GCs from oxidative stress mainly through GSTA5-dependent antioxidant responses and suggest that STAT3-associated transcriptional regulation contributes to this process.