摘要Background Heat stress(HS)poses a significant threat to male goat reproduction.Sertoli cells(SCs)provide both structural and nutritional support necessary for germ cells.HS induces physiological and biochemical changes in SCs.Nevertheless,the molecular mechanisms involved are still not fully understood.Melatonin is a classic anti-oxidant that can alleviate HS-induced male reproductive damage.However,the underlying molecular mechanisms by which melatonin mitigates damage to goat testicular SCs remain unclear and require further investigation.Results In this study,an in vivo heat stress model was established in goats.The results showed that HS exposure led to testicular injury,abnormal spermatogenesis and apoptosis of SCs.To elucidate the mechanism of HS-induced SC apoptosis,primary SCs were isolated and cultured from goat testes,then exposed to HS.HS exposure increased the production of reactive oxygen species(ROS),decreased adenosine triphosphate(ATP)synthesis,and reduced mitochondrial membrane potential in SCs.Additionally,HS increased the expression of mitochondrial fission pro-teins 1(FIS1)and dynamin-related protein 1(DRP1)while decreasing the expression of mitochondrial fusion proteins Mitofusin 1(MFN1),Mitofusin 2(MFN2),and optic atrophy 1(OPA1).This resulted in excessive mitochondrial fis-sion and mitochondria-dependent apoptosis.Mdivi-1(DRP1 inhibitor)reduces mitochondria-dependent apoptosis by inhibiting excessive mitochondrial fission.Mitochondrial fission is closely related to mitophagy.HS activated upstream mitophagy but inhibited autophagic flux,disrupting mitophagy and exacerbating mitochondria-depend-ent apoptosis.Finally,the classical antioxidant melatonin was shown to reduce mitochondria-dependent apoptosis in SCs exposed to HS by decreasing ROS levels,restoring mitochondrial homeostasis,and normalizing mitophagy.Conclusions In summary,these findings indicated that the mechanism of HS-induced mitochondria-dependent apoptosis in SCs is mediated by hyperactivation of the ROS-DRP1-mitochondrial fission axis and inhibition of mito-chondrial autophagy.Melatonin inhibited HS-induced mitochondria-dependent apoptosis in SCs by restoring mito-chondrial homeostasis.This study enhances the understanding of the mechanisms through which heat stress triggers apoptosis and provides a vision for the development of drugs against HS by targeting mitochondria in goats.