Study On The Roles And Mechanisms Of GSTK1 In Testis Leydig Cell Autophagy Induced By Oxidative Stress

Background and aimsGenerally speaking,women who cohabit for more than two years after marriage without taking any contraceptive measures and are not pregnant are called infertility.Male infertility refers to infertility caused by male factors.According to statistics,around 10%-15%of married couples worldwide suffer from infertility,of which male infertility accounts for about 50%.Male infertility is one of the most important diseases in human reproductive health.Nearly 75%of these diseases are caused by oligoasthenospermia,i.e.too little sperm and low sperm motility.It is estimated that male factors account for half of infertility patients,and 25%to 87%of male infertility is thought to be caused by oxidative stress.Statistical data showed that oxidative stress was one of the main causes of the decrease in sperm number and motility.Oxidative stress refers to the state in which oxides(including reactive oxygen species,ROS and reactive nitrogen species,RNS)are produced more than they can be removed,leading to excessive accumulation of oxides.Excessive oxides are important factors leading to DNA oxidative damage(including base damage and DNA strand breakage)and protein denaturation.Oxidative stress can damage sperm plasma membrane,protein and DNA,leading to structural damage and functional decline of sperm.Testosterone is the most important reproductive hormone in men.It directly acts on spermatogenic cells and plays an important role in regulating spermatogenesis and maintaining normal sperm count.Testosterone is mainly secreted by Leydig cells.Leydig cells distribute between seminiferous tubules,accounting for only 2%of testicular weight,but they secrete 95%of total testosterone.Under the action of luteinizing hormone,mesenchymal cells activate the related adenylate cyclase and accelerate the production of cyclic adenosine phosphate in cells,as well as a series of cascade reactions,thus inducing the conversion of cholesterol to testosterone.However,oxidative stress can damage mesenchymal cells,resulting in a decline in cell viability and function,resulting in a significant decrease in testosterone levels,and ultimately lead to spermatogenesis disorders.Glutathione S-transferase(GST)is a group of antioxidant enzymes involved in the scavenging of oxygen free radicals in cells.It can catalyze conjugation reaction between reduced glutathione and electrophiles to reduce the activity of electrophiles,including binding with xenobiotic substances and oxidative intermediates,so as to reduce their toxicity and facilitate their removal from cells,thereby protecting membrane lipids,DNA and proteins.Quality is not damaged.In physiological state,there are high levels of GSTs in testis and semen.Under oxidative stress,various forms of GSTs occur and accumulate in mitochondria to protect mitochondria from ROS,LPO and other oxidants.Studies have shown that GST gene families such as GSTM1 and GSTT1 subtypes are deleted in male infertility patients,accompanied by poor sperm quality and high levels of oxidative stress.Our previous results suggest that GSTK1 is mainly expressed in testicular Leddish cells.When mice were treated with tert-butyl hydroperoxide(TBHP),Leddish cells did not apoptosis,but autophagy was significantly enhanced.Whether GSTK1 is involved in the autophagy of mesenchymal cells induced by oxidative stress,and its role and regulatory mechanism are unclear.In this study,Cre-loxP system was used to construct Gstk1-specific knockout mice.Combined with tert-butyl hydrogen peroxide TBHP-induced oxidative stress model,the role of GSTK1 in oxidative stress-induced autophagy of testicular Leydig cells and its regulatory mechanism were explored.Materials and methods1.Immunohistochemistry(IHC)/immunofluorescence staining(IF)was used to determine the expression and localization of GSTK1 in testis of postnatal and adult mice.2.TBHP was injected intraperitoneally into adult wild type B6 mice to establish oxidative stress model.Malondialdehyde(MDA)was used to detect oxidative stress.IF and western blot(WB)were used to detect autophagy and GSTK1 expression,TUNEL was used to detect apoptotic level,and real-time quantitative PCR(qPCR)was used to detect the expression of steroid-producing genes in Lydich cells.3.Leydig cells of adult mice were isolated and cultured in primary culture.After treatment with TBHP and H2O2,WB and flow cytometry(FC)were used to detect autophagy,apoptosis and GSTK1 expression.4.Leydig cell-specific Gstkl knockout mice were constructed by Cre-loxP system.The knockout mice(Gstkl-cKO)and control mice were identified by genotype identification,IF and WB.5.Hematoxylin-eosin(H&E)staining was used to observe the effect of Gstkl knockout on the morphology and structure of testis and epididymis,and sperm counting was also evaluated.6.Control and Gstkl-cKO mice were injected intraperitoneally with TBHP,and WB and IF were used to detect the autophagy of Leydig cells after Gstkl deletion under oxidative stress.Results1.IHC and IF results showed that GSTK1 was mainly expressed in Leydig cells of mouse testis.2.After TBHP treatment,cell apoptosis increased significantly,but specifically occurred in seminiferous epithelium.Cell autophagy,rather than cell apoptosis,was enchanced in Leydig cells.Besides,the expression level of genes involved in steroid production decreased significantly,but the expression and localization of GSTK1 did not change significantly.3.In primary cultured Leydig cells,both TBHP and H2O2 treatment promoted cell autophagy,but there was no significant change in cell apoptosis,nor in GSTK1 protein level.4.Genotyping,WB and IF showed that Gstk1-cKO mice were successfully generated.H&E staining showed that the morphology and structure of testis and epididymis did not change significantly after Gstk1 knockout,and the number of sperm in epididymis was normal.5.After TBHP treatment,the level of autophagy of Leydig cells in Gstk1-cKO mice testis increased significantly.Conclusion1.GSTK1 was predominantly expressed in mouse testicular Leydig cells.2.Under in vivo and in vitro oxidative stress,there was no significant change in the apoptosis of Leydig cells,but autophagy was significantly increased,which may lead to the decrease in steroid production of Leydig cells.3.The testis and epididymis of the mice whose Gstk1 gene was specifically knocked out in Leydig cells had no obvious phenotype and,the cKO mice had normal sperm count,suggesting that Gstk1 is dispensable for testicular development and spermatogenesis under physiological conditions.4.Under oxidative stress,Gstk1-cKO mice had a higher level of autophagy,suggesting that GSTK1 might be involved in the regulation of autophagy and function of Leydig cells under oxidative stress.