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

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.