TRPM8 activation by sustained-release menthol mitigates exertional heatstroke-induced testicular injury and sperm ultrastructural damage in rats

AIMS: Exertional heatstroke (EHS) can induce abrupt scrotal hyperthermia and impair male reproductive integrity. This study tested whether sustained-release menthol protects against EHS-induced testicular and genitourinary injury through transient receptor potential melastatin 8 (TRPM8). MATERIALS AND Some rats received AMTB, a selective TRPM8 antagonist. Scrotal temperature change (ΔTscrotal) and circulating hormones were measured, and genitourinary tissues were examined on day 3. Blood-testis barrier proteins (claudin-11 and ZO-1), TRPM8, and apoptosis of Sertoli and Leydig cells were quantified by four-colour immunofluorescence, and sperm ultrastructure was assessed by scanning and transmission electron microscopy. KEY FINDINGS: Vehicle-treated EHS rats showed marked scrotal hyperthermia (Δ6.5 °C vs 0.75 °C in controls), reduced testosterone with elevated luteinizing hormone, sperm dysmorphology, and multisite genitourinary injury. EHS reduced claudin-11/ZO-1 and TRPM8 in seminiferous tubules and increased Sertoli/Leydig cell apoptosis. Electron microscopy revealed sperm head deformation, head-neck discontinuity, mitochondrial sheath disruption in the midpiece, and fissures/perforations along the principal piece. Menthol pretreatment attenuated scrotal hyperthermia (Δ2.6 °C), improved testosterone, reduced supporting-cell apoptosis, and lessened histological and ultrastructural injury. These benefits were blunted by AMTB, supporting TRPM8-dependent protection. SIGNIFICANCE: Sustained-release menthol mitigates EHS-induced male reproductive and genitourinary injury in rats, at least partly through TRPM8 activation. TRPM8 may represent a prophylactic target for reducing heat-related reproductive damage.