Normal spermatogenic processes require the scrotal temperature to be lower than that of the body as excessive heat affects spermatogenesis in the testes,reduces sperm quality and quantity,and even causes infertility.Endoplasmic reticulum stress(ERS)is a crucial factor in many pathologies.Although several studies have linked ERS to heat stress,researchers have not yet determined which ERS signaling pathways contribute to heat-induced testicular damage.Melatonin activates antioxidant enzymes,scavenges free radicals,and protects the testes from inflammation;however,few studies have reported on the influence of melatonin on heat-induced testicular damage.Using a murine model of testicular hyperthermia,we observed that heat stress causes both ERS and apoptosis in the testes,especially in the spermatocytes.These observations were confirmed using the mouse spermatocyte cell line GC2,where the Atf6 and Perk signaling pathways were activated during heat stress.Knockout of the above genes effectively reduced spermatocyte damage caused by heat stress.Pretreatment with melatonin alleviated heat-induced apoptosis by inhibiting the Atf6 and Perk signaling pathways.This mitigation was dependent on the melatonin receptors.In vivo experiments verified that melatonin treatment relieved heat-induced testicular damage.In conclusion,our results demonstrated that ATF6 and PERK are important mediators for heat-induced apoptosis,which can be prevented by melatonin treatment.Thus,our study highlights melatonin as a potential therapeutic agent in mammals for subfertility/infertility induced by testicular hyperthermia.