Unveiling the Mechanisms of Ozone-Induced Male Reproductive Toxicity: Insights from Testicular Proteomics and CTD...

Ambient ozone (O₃) is a major air pollutant, yet its mechanistic impact on male reproduction remains poorly understood, despite links to reduced sperm quality. We developed a short-term O₃ exposure mouse model and utilized testicular proteomics, Comparative Toxicology Database (CTD) mining, and in vitro investigations to identify mediators. Results showed that O₃ exposure dose-dependently impaired sperm quality, disrupted histology, and altered testosterone and luteinizing hormone levels. Proteomics revealed disturbances in cholesterol, steroid hormone, and retinol metabolism in mice testicular tissue in response to O₃ exposure, alongside impaired spermatogenesis. More importantly, HIF-1α/mTOR signaling was suggested as a crucial mediator to O₃-elicited testicular toxicity based on proteomic and CTD analysis. Further mechanistic validation using an in vitro air-liquid interface (ALI) coculture of MLE-12 alveolar epithelial and TM3 Leydig cells demonstrated that O₃ exposure triggered reactive oxygen species (ROS)-mediated oxidative stress, subsequently inactivated HIF-1 signaling, and impaired Leydig cell steroidogenesis, thereby contributing to testicular injury and declined sperm quality. Overall, our findings identify HIF-1 signaling as a pivotal regulator linking the exposure of O₃ to steroidogenic dysfunction and spermatogenic disruption in male reproduction, offering novel mechanistic insights for male reproductive toxicology.