Abstract

High-temperature cooking processes generate oxidized lipids such as 9,10-epoxy stearic acid (ESA), yet its reproductive toxicity remains poorly characterized. Here, we demonstrated that ESA exposure in mice induces hepatotoxicity, reduced serum testosterone, and male reproductive dysfunction, manifested by testicular injury, disrupted spermatogenesis, and impaired sperm quality. Integrated metabolomics and proteomics analysis revealed that ESA disrupts amino acid and carbohydrate metabolic homeostasis, and analysis of these two datasets suggests that mitochondrial dysfunction is likely a key factor in its toxicity. ESA activated the Bax/Bcl-2-mediated mitochondrial apoptotic pathway, leading to increased germ cell apoptosis. In vitro validation in GC-2spd(ts) confirmed that ESA downregulated key mitochondrial-related genes, including Ndufa4l2 and Mettl20 , reduced ATP production, suppressed mitochondrial Complex I activity, and induced oxidative stress. ESA also significantly downregulated essential spermatogenesis-related genes, including Cxcr4 , Bbof1 , Spata25 , Sdc1 , Rhox5, and Foxj3 . These findings establish that ESA impairs male reproductive function through mitochondrial dysfunction, manifesting as impaired energy metabolism, elevated oxidative stress, and activation of the intrinsic apoptotic pathway, alongside suppression of spermatogenesis-regulatory gene networks. This study identifies ESA as a key toxic monomer in oxidized frying oil and underscores the need to incorporate specific lipid oxidation products into food safety assessments.