The Influence Of Heat Shock On Mouse Sertoli Cell Metabolism And Reproductive Function

Male infertility is a worldwide medical problem,and many factors affect it,such as genetic factors,immune factors,environmental factors(ionizing radiation,ultrasound,microwave,temperature)and so on.Among them,the temperature is essential for the occurrence of normal sperm.Studies have shown that cryptorchidism or testicular long-term high temperature environment will lead to heat shock,heat shock induced apoptosis of spermatogenic cells,and often accompanied by Sertoli cell morphology and function changes.However,the research on heat shock-induced Sertoli cell dysfunction and its mechanism is not very clear.Sertoli cell metabolism regulation is closely related to spermatogenesis,and the lactic acid produced by the support of cells through the "Weberger effect" is an important energy substrate for spermatogenic cells.Studies have shown that lactic acid can inhibit spermatogenic cell apoptosis,and exogenous injection of lactic acid can promote spermatogenesis in cryptorchidism rats.Therefore,the study of heat shock on the Sertoli cell metabolism and reproductive function,has important implications for improving male reproductive health.Based on the above research,the testicular biopsy tissue of male patients with cryptorchidism infertility was used as the tissue model,and the mouse Sertoli cell line(TM4)was used as an in vitro cell model to explore the effect of heat shock on testicular tissue and Sertoli cell in this paper.This article mainly studies in the following three aspects:1.Screening metabolism-associated genes in cryptorchidism infertility and bio-informatics analysis:The expression of 119 metabolism-associated genes in testicular tissue of patients with cryptorchidism infertility and normal fertility was detected by qPCR array.Five differentitally expressed genes were identified:LDHA,IGF1,MCAT,CRAT,ARG2.And the relationship between the five differentially expressed genes,azoospermia and abnormal spermatozoa was analyzed by GEO database.By analyzing the expression differences of these five metabolic genes in three kinds of sterile pathological types,we found that the expression level of LDHA was the most significant,and it was significantly down-regulated in patients with cryptorchidism and infertility.2.Effects of heat shock on metabolic function of TM4 cells:(1)Establishment of TM4 cell heat shock model:Heat shock activates the expression of HSFs and HSPs in cells,and the expression of HSP70/90 is closely related to heat shock.The effects of heat treatment on TM4 cells were detected by apoptotic analysis and HSP70/90 mRNA expression.The TM4 cells were treated with TM4 cells for 3 min at 37 ℃,followed by gradient time repair for 6 h,12 h and 24 h.(2)Effects of heat shock on metabolic function of TM4 cells:Based on the successful construction of TM4 model,the response of LDHA to mRNA and protein was detected by qPCR and Western Blot.It was found that the expression of LDHA mRNA was down-regulated and the protein level was not changed after TM4 cells were heat shock.And the lactic acid production and glucose uptake were significantly down-regulated through metabolic biochemical assay.These two groups of results suggest that the metabolic level of TM4 cells was slowed down after heat shock.3.The effect of interfering with LDHA expression on TM4 cell metabolism and reproductive function:Given that the high expression of LDHA in TM4 cells,RNA interference was used to reduce LDHA expression.The results showed that lactate dehydrogenase activity,lactic acid production and glucose uptake in TM4 cells were significantly decreased,while the production of ATP and ROS increased significantly,indicating that interference with LDHA expression promoted mitochondrial respiration.MTT and scratch test results showed that interfering with LDHA expression could inhibits TM4 cell proliferation and migration ability.The mRNA expression of ZO-1,ZO-2,β-catenin,Occludin,N-cadherin in the TM4 cells were down-regulated,among these ZO-1 and ZO-2 were significantly decreased,suggesting that interference with LDHA expression could destroy cell junction and BTB in TM4 cells.In summary,this study found that heat shock could alter the metabolic levels of Sertoli cell significantly,and LDHA participating in lactic acid production is an important response factor.Reducing LDHA expression may change Sertoli cell metabolism and reproductive function,suggesting that LDHA may act as an candidate indicator molecule in heat shock-induced spermatogenic dysfunction.