The Effects And Molecular Mechanisms Of Chronic Stress On Testicular Cells And Oocytes And The Involving Protective Role Of Melatonin In Mice

Background and Objective:Today,10?15%of couples are suffering from infertility problems which is increasing around the world,possibly due to a variety of causes such as social and behavioral factors as well as environmental exposures.Reproductive health research,including mechanisms for declining fertility and new strategies for protection,has become an important support of national key programs in China.With the rapid development of society and economy,stress is present at every level of society in the form of physical,social and psychological.Studies suggest that chronic stress can be harmful to reproduction,which can lead to abnormal spermatogenesis and decrease in the quality of oocytes,but the underlying mechanism remains to be further elucidated.Melatonin is mainly secreted by the pineal gland and is one of the most well-investigated antioxidants,and could defend against oxidants-induced damage in many tissues.Whether melatonin can relieve abnormal spermatogenesis and oocyte quality caused by chronic stress has not been reported yet.The aim of this study is to investigate the effects and molecular mechanisms of chronic stress on testicular cells and oocytes and the involving protective role of melatoninMaterials and Methods:Male and female 6-week-old mice were randomly divided into 4 groups:control(treated with vehicle without restraint stress),control+M(treated with melatonin without restraint stress),stress(treated with vehicle and restraint stress),and stress+M(treated with melatonin and restraint stress).The male mice were stressed for 35 days.First,the serum corticosterone,melatonin and sperm quality of mice were detected.Second,TUNEL,WB and ELISA were used to detect the apoptosis of testicular cells,signal molecules of apoptosis and oxidative stress.Finally,the effects of melatonin and H2O2 on testicular cells in vivo and in vitro were detected by TUNEL and WB.For female mice,after 28 days of restraint stress,first,the corticosterone in serum was detected by ELISA.Then,the morphology of spindle,chromosome arrangement,copy number of mtDNA,ATP content,mitochondrial membrane potential,mitochondrial distribution,autophagy and expression of key signaling protein PGC-l?and SIRT1 were detected to explore the effects and mechanism of melatonin and chronic stress on the oocyte in miceResults:Compared to control male mice,the sperm density was notably decreased in stressed mice;the number of TUNEL+testicular cells was significantly increased in the stressed group;the ratio of pro-apoptotic protein BAX over anti-apoptotic protein Bcl-2 and the activity of cleaved caspase-3 were significantly augmented in stressed male testes;the level of ROS increased approximately 1.5-fold in the stressed group as compared to that in control group;the anti-oxidant molecules,such as SOD and GSH were markedly decreased in the stressed group;stressed testes exhibited significantly elevated levels of the pro-oxidant enzyme,iNOS;stress notably increased the phosphorylation of I?B? and the nuclear translocation of p65 and significantly downregulated the expression of Nrf-2 and HO-1 in mice testes.H2O2 treatment increased the number of TUNEL+testicular cells,the ratio of pro-apoptotic protein BAX over anti-apoptotic protein Bcl-2,the level of cleaved caspase-3,the expression of iNOS,the phosphorylation of I?B? and the nuclear translocation of p65,and downregulated the antioxidant proteins Nrf2 and HO-1.But melatonin notably normalized all the effects of restraint stress and H2O2 on testicular cellsRestraint stress induced meiotic defects in mice MII oocytes,showing abnormal meiotic spindle and chromatin misalignment,resulted in mitochondria dysfunction including reduced ATP production,mtDNA copy number and mitochondrial membrane potential and mitochondria aggregation,down-regulated the biogenesis factor of mitochondria,PGC-1?,and the process of maintaining mitochondrial homeostasis,autophagy,as well as the expression of related proteins including Atg5 and LC3B,reduced the expression of SIRT1,and elevated intra-oocyte ROS level,all of which could be rescued by melatonin treatmentConclusion:Chronic stress could induce oxidative apoptosis of testicular cells and oocytes meiosis defects and mitochondria dysfunction which can be significantly alleviated by melatonin,in which NF-?B and Nrf2/HO-1 signaling pathways are involved in the testicular cells injury induced by chronic stress and the protective role of melatonin and SIRT1,PGC-1 ?,and autophagy-related signaling pathways are involved in the negative effects of chonic stress as well as the protective role of melatonin on oocytes.This study provides new evidence for that chronic stress could lead to fertility decline which can be rescued by melatonin.