Restraint Stress Of Male Mice Induces Apoptosis In Spermatozoa And Spermatogenic Cells:the Fas/fasL System Triggers Sperm Apoptosis By Facilitating Epididymal Secretion Of TNF-α And TRAIL

Modern society is full of pressure,which is a kind of serious psychological stress that can cause infertility.Thus,people’s reproductive health is facing enormous challenges.Stress is reported to affect male reproduction by disrupting reproductive endocrine and causing apoptosis.Studies have shown that stress can affect the process of spermatozoa production,impair spermatozoa quality,and thus damage the quality of semen,meanwhile,there are few researchs on the effects of stress on mature spermatozoa.The mechanisms by which psychological stress impairs semen quality are largely unknown.The restraint stress model can better simulate the psychological stress caused by the stress of daily life in human beings.So by using a restraint-stressed mouse model to mimic psychological stress,we studied the specific conditions of apoptosis in various types of spermatogenic cells and spermatozoa induced by male mice restraint stress and its relationship with Fas/FasL system,and further studied how the Fas/FasL system triggers apoptosis of spermatozoa in the cauda epididymis during restraint stress in this study.Kun Ming male mice were restrained for 48 h before examination for spermatozoa fertilizing potential and for apoptosis and expression of active caspase-3 and Fas in spermatozoa and spermatogenetic cells,and for expression of active caspase-3 and Fas/FasL in caudae epididymides.Meanwhile,in order to further confirm the role of Fas/FasL system in the apoptosis of spermatozoa and spermatogenetic cells caused by restraint stress,we used gld strain mice,which have a loss-function mutation in FasL,and wild C57BL/6J strain mice,the control mice of gld,to do further research,and we also assessed the motility and apoptosis of spermatozoa after preservation supplemented with soluble FasL in vitro.The results showed that:(1)Restraint stress caused a significant increase in serum cortisol levels in male mice.At1 h,24 h and 48 h after the initiation of restraint stress,the level of cortisol in mice was always significantly higher than that in the control group,suggesting that our stress model can cause stress in male mice effectively;(2)There was no significant difference in spermatozoa concentration between the restraint stress group and the control group;(3)There was no significant difference in fresh spermatozoa motility between the restraint stress group and the control group,but the spermatozoa motility of the restraint group was significantly lower than that of the control group at 90 minutes and later on of in vitro preservation,indicating that restraint stress accelerates the decline of spermatozoa motility in vitro;(4)The restraint stress caused a significant increase in the proportion of Annexin-V FITC positive spermatozoa,and the content of activated Caspase-3 and Fas protein in spermatozoa increased significantly,indicating that 48h restraint stress promoted spermatozoa apoptosis,and the pro-apoptotic effect of restraint stress on spermatozoa may be related to the Fas/FasL system;(5)The expression levels of active Caspase-3 and Fas/FasL protein in the epididymis were significantly increased after 48 hours of restraint stress,indicating that restraint stress promoted epididymal apoptosis;(6)After 48 hours of restraint stress,the proportion of Annexin-positive spermatogenic cells was significantly increased,and the expression of active Caspase-3 protein in spermatogenic cells was significantly increased,indicating that restraint stress promoted spermatogenic cell apoptosis for 48 hours;On the 35~thh day after restraint stress,the sperm motility of the stressed mice was significantly lower than that of the control,and the apoptosis rate of spermatozoa was significantly higher than that of the control,indicating that the restraint stress leads to the apoptosis of spermatogenic cells and further affects the quality of the spermatozoa differentiated from these cells;(7)The in vitro fertilization rate,apoptosis rate,the expression level of active Caspase-3in the spermatogenic tubules and the percentage of apoptotic spermatogenic cells in the control gld and C57BL/6J mice were not significantly different.After 48 hours of restraint stress,gld and C57BL/6J mice showed significant adverse changes in above indexes,but the gld mice were significantly less affected than C57BL/6J mice,indicating that the Fas/FasL system is involved in the damage of spermatozoa and spermatogenic cells caused by restraint stress.But there may also be other pathways that play a role in this process;(8)Incubation with sFasL did not affect spermatozoa motility and apoptosis,while incubation with sTNF-αdecreased the motility and promoted the apoptosis of spermatozoa,48h restraint stress caused a significantly high expression level of FasL in epididymis,the epididymis of the gld mice produced significantly less TNF-αand TRAIL than that of wild-type mice did after male restraint,indicating that the Fas/FasL system in the epididymis triggered spermatozoa apoptosis dependently through increasing secretion of sTNF-alpha and TRAIL during male restraint stress,meanwhile,there were Fas/FasL-independent pathways(TNF-alpha or TRAIL)in the epididymis,which induce spermatozoa apoptosis much stronger than the FasL/Fas pathway.Therefore,our results indicate that the restraint stress model can cause stress in male mice effectively.Restraint stress promotes apoptosis of mature spermatozoa by activating the Fas/FasL system and produces low-quality sperm by causing apoptosis of spermatogenic cells.FasL triggered spermatozoa apoptosis in epididymis dependently through promoting TNF-αand TRAIL secretion.