Oleanolic acid prevents ferroptosis and enhances oocyte competence during in vitro maturation in a porcine model

Abstract

The in vitro production of porcine embryos is a valuable model for livestock biotechnology and human reproductive research. However, oocyte quality is often compromised during culture by ferroptosis, which is an iron-dependent form of cell death driven by lipid peroxidation and redox imbalance. This study induced ferroptosis using RSL3 during porcine oocyte maturation to evaluate the protective potential of oleanolic acid (OA), an antioxidant triterpenoid compound. Exposure to RSL3 increased Fe²⁺ accumulation (as detected by FerroOrange staining), lipid peroxidation (as determined by the MDA assay), apoptosis, and mitochondrial dysfunction. These changes were consistent with ferroptotic stress and were accompanied by impaired cumulus expansion, meiotic progression, and blastocyst formation. Supplementing with 1 mg/L OA significantly improved oocyte competence, partially restored blastocyst development, enhanced cumulus expansion, and reduced apoptosis. At the cellular level, OA reduced ROS, MDA, and abnormal mitochondrial distribution while maintaining glutathione levels and mitochondrial membrane potential. Molecular analysis revealed that OA modulated ferroptosis-related gene expression, including partial restoration of GPX4 and SLC7A11 expression and suppression of ACSL4 and TFRC upregulation. Collectively, these findings indicate that OA partially alleviated RSL3-induced ferroptotic damage in porcine oocytes, as reflected by coordinated improvements in iron homeostasis, redox status, mitochondrial function, and the expression of ferroptosis- and autophagy-associated markers. OA could therefore represent a promising technique to improve oocyte quality and embryonic development, with potential application value in reproductive medicine and livestock biotechnology.