PFOS-induced mitochondrial ROS impairs male reproductive function by suppressing FTO-dependent m6A RNA demethylation.

Perfluorooctanesulfonic acid (PFOS), a persistent per- and polyfluoroalkyl substance, has been associated with male reproductive toxicity, but the underlying molecular mechanisms remain incompletely defined. Here, we show that chronic exposure to environmentally relevant doses of PFOS caused dose-dependent testicular injury and spermatogenic dysfunction in mice, as indicated by reduced testis weight, disrupted seminiferous tubule architecture, and impaired sperm quality. Transcriptomic profiling revealed marked enrichment of pathways related to oxidative stress, mitochondrial dysfunction, and reproductive regulation. Mechanistically, PFOS increased mitochondrial reactive oxygen species (ROS) production in GC-2 spermatocyte cells, leading to mitochondrial membrane potential collapse, lipid peroxidation, and depletion of antioxidant defenses. Pharmacological scavenging of mitochondrial ROS with Mito-TEMPO attenuated PFOS-induced mitochondrial injury in vitro and improved testicular damage and sperm quality in vivo. We further found that PFOS suppressed the RNA demethylase fat mass and obesity-associated protein (FTO) and promoted its ROS-dependent redistribution from the nucleus to mitochondria, resulting in global N6-methyladenosine (m6A) hypermethylation. Integrated m6A epitranscriptomic analysis showed that PFOS remodeled m6A modification patterns on key transcripts involved in spermatogenesis, steroidogenesis, and redox homeostasis, including CYP11A1, SOX9, STRA8, and glutathione peroxidase (GPX4). FTO bound these transcripts, and restoration of FTO expression reversed PFOS-induced m6A hypermethylation and rescued reproductive gene expression. Together, these findings identify a mitochondrial ROS-FTO-m6A regulatory axis that links PFOS-induced mitochondrial oxidative stress to epitranscriptomic disruption and male reproductive dysfunction. Our study defines FTO-dependent RNA demethylation as a critical molecular mechanism and potential intervention target in PFOS-induced reproductive injury.