Melatonin Relieves Heat-induced Spermatocyte Apoptosis By Inhibiting ATF6 And PERK Signaling Pathways

The mammalian testis is located in the scrotum outside the body cavity.The normal spermatogenic process requires the temperature of the scrotum to be lower than the normal body temperature.Localized high temperature in the scrotum is currently recognized by the World Health Organization as an important factor that seriously damages male fertility.Excessive temperature can cause heat stress(HS),affect the spermatogenesis process of the testicles,reduce the quality and quantity of sperm,and even lead to infertility.At present,abnormal sperm occurrence and decreased reproductive performance of breeding animals caused by heat stress are important problems that are common in animal husbandry and need to be solved urgently.The endoplasmic reticulum plays an important role in the biogenesis of many cells,especially the secretion of proteins,the folding and transportation of membrane proteins,the metabolism of lipids and carbohydrates,and the storage and release of calcium ions.When cells are stimulated,a large number of misfolded or unfolded proteins accumulate in the endoplasmic reticulum,which makes the protein processing ability of the endoplasmic reticulum and leads to the disorder of the endoplasmic reticulum function,that is,endoplasmic reticulum stress occurs(Endoplasmic reticulum stress,ERS),ERS is considered to be a key factor leading to a large number of pathologies.At present,studies have reported heat stress can trigger ERS,but the relationship between heat stress-induced male reproductive system has not yet been reported.Melatonin is a neuroendocrine hormone with antioxidant and antiapoptotic capabilities.Melatonin plays an important role in reproductive regulation by regulating the secretion of gonadotropins and testosterone to affect testicular development.In addition,its antioxidant activity helps prevent testicular damage caused by stress and inflammation.The reproductive damage caused by heat stress has seriously affected animal production.How to use the anti-oxidation and anti-apoptotic functions of melatonin to prevent and treat the occurrence of male reproductive diseases,and reveal its specific regulatory mechanism to prevent and control the testis of male animals caused by heat stress dysfunction has important scientific significance and potential application value.In this study,through the establishment of a testicular heat stress model,histological and molecular tests found that: heat stress can lead to a decrease in the number of germ cells in the seminiferous tubules of the testes,a disorder of the seminiferous epithelium,and the production of ERS and apoptosis.The results of TUNEL staining further prove that apoptosis mainly occurs in testicular spermatocytes.In subsequent in vitro(GC2 cell line)experiments,we confirmed the above results from RNA and protein levels,and further found that the ATF6 and PERK signaling pathways were activated in cells after heat induction,while the IRE1 signaling pathway did not change significantly during this process.Interfering with the above genes by si-RNA can effectively reduce the damage of spermatocytes caused by heat stress.On this basis,we explored the effect of melatonin on spermatocyte apoptosis and ERS caused by heat stress.Through immunofluorescence,flow cytometry,Western blotting and other experiments,it was found that melatonin can pass through the melatonin receptor(MT1/MT2)inhibit the expression levels of Atf6,Perk and apoptosis-related genes Caspase12,Caspase3 and Chop.Finally,experiments confirmed that melatonin can alleviate the decrease in the number of seminiferous germ cells and spermatocyte apoptosis caused by heat stress in vivo.This study shows that heat stress can cause testicular damage and the occurrence of spermatocyte ERS and apoptosis.Melatonin can alleviate the apoptotic response of spermatocytes caused by heat stress through ATF6 and PERK signaling pathways,and affect the damaged testis.Play a repair role.This study suggests that melatonin may be used as a drug to prevent spermatocyte apoptosis caused by high temperature and protect the testes from heat stress.