Study On The Mechanism Of Melatonin Partially Alleviates The Damage Induced By Heat Stress In Mice Testis And Human Spermatozoa

Sperm requires the temperature of the scrotum to be lower than body temperature.Studies have shown that elevated scrotal temperature will lead to reduced male fertility and even infertility.Previous studies have shown that elevated testicular temperature induces heat stress and ultimately leads to spermatogenesis disorders.The detailed regulation of spermatogenic abnormalities caused by heat stress and how to improve testicular damage caused by heat stress are not very clear.Heat stress is often accompanied by the occurrence of oxidative stress,and melatonin acts as a neuroendocrine hormone,and itself and its metabolites have better anti-oxidative stress and anti-apoptosis effects.However,there is no research on the protective effect and mechanism of melatonin on male germ cell heat stress injury.In view of this,this study will systematically explain the possible mechanisms of melatonin in alleviating spermatogenic disorders caused by heat stress from the following three aspects:1.Identification of melatonin to alleviate the abnormality of mouse testicular spermatogenesis caused by heat stress;2.Analyze the regulation mechanism of melatonin on the protective effect of mouse spermatocyte damaged by heat stress;3.Explore the protective effect and mechanism of melatonin on human sperm heat stress injury.Part1 Protective effect of melatonin on mouse testicular spermatogensis arrest caused by heat stressObjective:To investigate the effects of heat stress on sperm spermatogenesis in mice testicular and sperm quality in mouse epididymis,and study the protective effect of melatonin on mice testicular and epididymis damage induced by heat stress.Methods:The mice were divided into four groups:control group?Con?,melatonin group?Mel?,heat stress group?HS?,heat stress+melatonin group?HS+Mel?.Melatonin was administered by intraperitoneal injection,and a heat stress model was established by placing the mouse testes in a water bath at 42°C for 30 min.The oxidative stress parameters of mouse epididymis spermatozoa were analyzed by flow cytometry.The DNA fragment rate of epididymal spermatozoa was detected by SCD assay.The oxidative stress level of mouse testis tissue was detected by ELLISA.The mRNA and protein expressions of endoplasmic reticulum stress and apoptosis related molecules in mouse testis tissue were detected by RT-PCR and Western Blot respectively,HE staining and TUNEL staining were used for detection of spermatogenesis and apoptosis in seminiferous tubules.Results:Compared with the Con group,the motility and viability of the mouse epididymis sperm were decreased,the sperm oxidative stress level and DNA fragmentation rate were increased.The oxidative stress level of mouse testis tissue was increased;The mRNA and protein expressions of endoplasmic reticulum stress and apoptosis related molecules GRP78,p-eIF2?,CHOP,Caspase3 increased,and the expression of Bcl-2/Bax decreased;spermatogenic cells in seminiferous tubules were lost and apoptosis.However,intraperitoneal injection of melatonin can partially alleviate the above damage caused by heat stress on the epididymis and testis of mice.Conclusion:Melatonin relieves testicular spermatogenesis disorder caused by heat stress,reduces oxidative stress,endoplasmic reticulum stress and apoptosis in mouse testicular cells;reduces oxidative stress and DNA fragmentation rate of sperm in epididymis,and improves spermatogenesis quality.Part2 Protective mechanism of melatonin on mouse spermatocyte damage induced by heat stressObjective:To inverstigate the regulation mechanism of the protective effect of melatonin on heat stress injury of mouse spermatocytes.Methods:The pachytene spermatocytes of the testicular tissues of each group were isolated by BSA gradient method.And H₂O₂,endoplasmic reticulum stress inducer/inhibitor?Tunicamycin/4-PBA?were added to the culture medium to induce/inhibite oxidative stress and endoplasmic reticulum stress in GC2 cells,and knock out the expression of endogenous CHOP in GC2 cell line by using CHOP siRNA.The oxidative stress level,and the expression of endoplasmic reticulum stress and apoptosis-related molecules were detected by immunofluorescence,ELISA,RT-PCR and Western Blot.Results:1.In the isolated mouse pachytene spermatocytes,compared with Con group the 4-HNE content the HS group was higher;the expression of endoplasmic reticulum stress and apoptosis related molecules in spermatocytes increased at mRNA and protein levels,Compared with HS group,the content of 4-HNE in the pachytene spermatocytes of the HS+Mel group was decreased,and the mRNA and protein levels of the endoplasmic reticulum stress and apoptosis related molecules were significantly decreased too.The lipids peroxidation product 4-HNE is co-localized with the CHOP,as well as CHOP and c-Caspase3 in the spermatocytes after heat stress.In the GC-2 cell line,the addition of H₂O₂ and melatonin,we found that melatonin can alleviate the upregulation of CHOP and Caspase3 induced by H₂O₂.2.The number of sites of DSB marker?-H2AX in HS group was significantly higher than that in Con group investigated by chromosome micro-spreading technique.The number of sites in HS+Mel group was significantly lower than that in HS group.In the GC-2 cell line,the expression of CHOP was increased after the addition of tunicamycin,and the expression of DNA damage repair protein Rad51was decreased.And it was found that 4-PBA inhibite the reduce of the expression of Rad51 the addition of tunicamycin and 4-PBA together.In addition,the DSB repair ability of GC-2 cells at different time points after X-ray irradiation was detected by immunofluorescence,and the endoplasmic reticulum inducer tunicamycin was found to reduce DSB repair ability of GC-2 cells.3.The addition of the endoplasmic reticulum stress inducer tunicamycin to the GC-2 cell line increased the expression of CHOP and Caspase3.However,after knocked out the endogenous CHOP of the GC-2 cell line,the amount of expression of Caspase3 induced by tunicamycin was significantly lower than that of the unknocked out group.Conclusion:Melatonin can reduce oxidative stress and endoplasmic reticulum stress in mouse pachytene spermatocytes caused by heat stress,promotes DSB repair of spermaocytes,and thus reduce apoptosis of pachytene spermatocytes.Part3 Protective mechanism of melatonin on human spermatozoa damage induced by heat stressObjective:To explore the protective role of melatonin on human spermatozoa from heat stress and its mechanismMethods:Divide each sample into four aliquots as four groups:control group?Con?,melatonin group?Mel?,heat stress group?HS?,heat stress+melatonin group?HS+Mel?.The Mel group and the HS+Mel group were incubated with 1 mM melatonin at 37°C for 30 min,and then the HS group and the HS+Mel group were placed in a 42°C incubator for 3h,and then transferred to a 37°C incubator.After24h,the difference of sperm motility and viability between the samples was tested.The differences of sperm oxidative stress and apoptosis markers between each group were detected by flow cytometry.The sperm DNA oxidative damage and DNA fragmentation were detected by immunofluorescence and SCD assay separately.Results:Compared with Con group,in HS group,sperm motility and viability were decreased,sperm mitochondrial ROS,lipid peroxidation product 4-HNE,sperm apoptosis were increased,and sperm mitochondrial membrane potential lost,Compared with the HS group,the sperm motility and viability of the HS+Mel group were increased,sperm mitochondrial oxidative stress,sperm mitochondrial membrane potential loss,sperm lipid peroxidation product 4-HNE,sperm DNA oxidative damage,DNA fragmentation and sperm apoptosis were decreased.Conclusion:It is found that melatonin can effectively alleviate heat-induced spermatozoa oxidative damage,improve the sperm quality.