Melatonin attenuates heat stress-induced oxidative stress and apoptosis in mouse testicular Leydig cells via the KEAP1-NRF2 pathway

Heat stress represents a critical environmental and occupational hazard that impairs male reproductive capacity across mammalian species, including humans. It primarily exerts its deleterious effects by inducing oxidative stress and apoptosis in testicular Leydig cells, ultimately disrupting testosterone biosynthesis. In this study, primary Leydig cells (PLCs) were subjected to heat stress, which significantly elevated intracellular ROS, MDA, and XO levels while reducing TAC and SOD activities, leading to increased apoptosis. Pretreatment with 0.1 μM melatonin effectively restored redox homeostasis, lowering ROS, MDA, and XO and enhancing TAC and SOD, thereby markedly decreasing apoptotic cell death. Mechanistic studies showed that melatonin increased NRF2 expression, and NQO1 levels, while decreasing KEAP1, and reversed heat-induced upregulation of Bax and γH2AX and downregulation of Bcl-2. In vivo, melatonin-treated mice exhibited preservation of 3β-HSD-positive Leydig cell number, reduced seminiferous tubule vacuolization, normalized serum testosterone levels, and restoration of STAR, CYP11A1, and 3β-HSD protein expression. These findings demonstrate that melatonin alleviates heat stress-induced oxidative injury and apoptosis via activation of the KEAP1-NRF2 pathway and thereby protects Leydig cell function and testosterone production.