Delayed and Progressive Post Exposure Testicular Injury in Rats Due to Fine Particulate Matter (PM2.5)

Fine particulate matter (PM2.5) is increasingly implicated in male reproductive impairment. However, because most research remains acute-focused, delayed, persistent, or progressive post-exposure testicular damage-critical for long-term risk assessment-remains poorly understood. We established a time-resolved rat model to determine whether injury persists or worsens after exposure. Seventy-two male Sprague-Dawley rats were randomized into nine groups (control; vehicle at 24 h, 1, 2, and 4 months; PM2.5 at the same time points) and received intranasal PM2.5 at 5 mg/kg/day for 7 days. We quantified sperm quality and reproductive organ indices, examined testicular histology and ultrastructure, measured serum testosterone, FSH, LH, and E2, profiled inflammatory cytokine mRNAs (IL-1β, IL-6, TNF-α, IFN-γ), assessed germ-cell apoptosis by TUNEL, and evaluated junctional and stress-related proteins (Connexin-43, Occludin, JNK). PM2.5 exposure resulted in sustained declines in sperm concentration and motility, along with increased malformations and testicular and epididymal atrophy that did not recover over the 4-month post-exposure period. Histology showed progressive thinning of the seminiferous epithelium and ultrastructural degeneration. Endocrine disruption was also evident, with reduced testosterone and altered gonadotropins. Mechanistically, PM2.5 exposure maintained elevated IL-1β and IL-6 expression, promoted germ cell apoptosis, and downregulated Connexin-43 and Occludin while exhibiting patterns consistent with activation of the JNK pathway. Together, these findings demonstrate delayed and progressive testicular toxicity after PM2.5 exposure ends and suggest that preserving junctional integrity and targeting JNK may mitigate long-term reproductive harm.