Effects And Mechanisms Of XAF1 And CHCHD10 In Bisphenol A Induced Spermatogenic Cell Damage

Background:The quality of human semen has been declining and the male infertility population has been increasing over the past 60 years.The continuous exposure of human to environmental pollutants is an important reason for the decline of male reproductive function.Bisphenol A(BPA)is one such typical environmental endocrine disruptors.It is widely used in food packaging,electronic products,construction,medical equipment and other industries as a chemical raw material.The population is generally exposed to BPA due to the residue in the production process and the dissolution during the use of it.It is important to study the toxic effects and explore the molecular mechanisms of BPA on male reproduction.Contents and methods:1.Evaluation of the toxic effects of BPA on male reproductive system in vivo study.32 mice were randomly assigned to 4 groups and administered with 0 mg/kg,3 mg/kg,30 mg/kg or 300 mg/kg BPA each day by gavage for 35 d to establish a male reproductive toxicity model in vivo.Epididymal sperm motility parameters were measured by computer assisted sperm analyzer;Sperm morphology was observed under optical microscope;H&E staining was used to observe the pathological morphology of mouse testis;TUNEL method was used to detect the apoptosis of mouse testis;Transmission electron microscopy was used to observe the ultrastructure of testis.2.Microarray detection and bioinformatics analysis.GC-2 cells were treated with 0 ?M,20 ?M,40 ?M and 80 ?M BPA for 48 h to establish male reproductive toxicity model in vitro.80 ?M BPA was chosen as the exposure concentration in Affymetrix GeneChip analysis due to its obvious toxic effects.GeneChip hybridization and analysis were conducted commercially to obtain the relative fold change of expression between two groups.Enrichment analysis was performed to obtain the biological functions and signaling pathways involved in these differentially expressed genes.qRT-PCR method was used to screen and verify the differentially expressed genes after BPA exposure.3.Researches on the role and mechanisms of XAF1 in BPA induced GC-2 cell apoptosis.The apoptosis of GC-2 cells after BPA treatment was evaluated by flow cytometry,ultrastructural observation and Western blot method respectively.The expressions of XAF1 in GC-2 cells and mouse testis after BPA exposure were detected by qRT-PCR,Western blot or Immunofluorescence staining method.XAF1 knockdown GC-2 cell model was established to detect the cell apoptosis rate and the levels of apoptosis-related proteins after BPA exposure by flow cytometry and Western blot method.Next,we detected the expressions of IFN? in BPA exposure models both in vitro and in vivo by ELISA,and then detected the expression of XAF1 and XIAP by Western blot method in IFN? knockdown GC-2 cell model after BPA exposure.4.Effects and mechanisms of CHCHD10 in BPA induced mitochondrial damage in GC-2 cells.The ultrastructure and morphology of mitochondria in GC-2 cells after BPA treatment were observed using transmission electron microscopy and confocal microscopy.Based the BPA exposure and CHCHD10 knockdown GC-2 cell model,the ATP and ROS levels were detected by ATP detection kit and ROS detection kit.Besides,the mitochondrial morphology were observed using confocal microscopy and the levels of oxidative phosphorylation and mitochondrial fusion/fission related genes and proteins were detected by qRT-PCR and Western blot method respectively.In addition,we analyzed the correlation between CHCHD10 expression and sperm quality parameters in a population study.Results:1.BPA showed potential male reproductive toxicity in in vivo study.Progressive degenerative lesions were observed in mouse testes exposed to different concentrations of BPA.The most serious pathological changes were found in 300 mg/kg/d BPA group,including the disordered and absent germ cells,the reduced number of spermatozoa in the lumen and even the deformed and atrophied of seminiferous tubules.Besides,the sperm motility and sperm density decreased significantly while the abnormal sperm deformity rate increased obviously in 300 mg/kg/d BPA group(P<0.05).Meanwhile,excessive apoptosis and abnormal mitochondrial structure of spermatocytes and sperm cells were also found in this group,which indicated the obvious male reproductive toxicity of 300 mg/kg/d BPA exposure.For 30 mg/kg/d BPA group,the rapid progressive sperm percentage decreased and the head abnormality of sperm increased significantly compared to the control(P<0.05).Meanwhile,some pathological changes including the vacuoles,exfoliated and increased apoptotic germ cells as well as some abnormal structure of mitochondria were found in seminiferous tubules or germ cells.Although no significant difference were found between 3 mg/kg/d BPA group and the control group,the downward trend of sperm motility and slight pathological changes were observed.These results suggested that BPA may have potential male reproductive toxicity under lower dose exposure conditions.Spermatocytes and sperm cells were the mainly target cells for BPA induced apoptosis and mitochondria is the important target organelles for BPA-induced spermatogenic cell damage.2.BPA resulted in spermatogenic cell damage by regulating multiple biological functions and signaling pathways.Affymetrix GeneChip results showed that 492 genes were up-regulated and 931 genes were down-regulated after BPA exposure in GC-2 cells.The results of bioinformatics analysis showed that BPA exposure could lead to abnormal cholesterol biosynthesis,type I interferon receptor binding,cell apoptosis,mitochondrial cytochrome C release and ATP complex enzyme transport in GC-2 cells and the signal pathways such as Toll-like receptor signaling pathway,RIG-like receptor signaling pathway,oxidative phosphorylation related signaling pathway were also involved in.Besides,we screened out some differential genes,such as XAF1,CHCHD10,Mterf and Irf7,which may play important roles in BPA-induced spermatogenic cell damage.3.XAF1 induced spermatogenic cell apoptosis via IFN?-XAF1-XIAP pathway.Exposure to BPA increased the apoptosis rate,altered the expression of apoptosis-related proteins,and showed obvious morphological characteristics of apoptosis in GC-2 cells.XAF1 was up-regulated in GC-2 cells and mice testes after BPA exposure.The expression levels of XIAP and XAF1 were inversely correlated after BPA exposure.Knockdown of XAF1 expression partially inhibited the apoptosis,suppressed the activation of caspase 3 and improved the BPA-induced XIAP expression in GC-2 cells.On the other hand,IFN? levels were significantly up-regulated after BPA exposure both in vitro and in vivo,and these levels were positively related to the expression of XAF1.Knockdown of IFN? reduced the expression of XAF1 and increased the expression of XIAP in BPA-treated GC-2 cells.4.CHCHD10 participated in BPA-induced mitochondrial dysfunction in GC-2 cells.The abnormal mitochondrial ultrastructure,decreased ATP level,elevated ROS level and abnormal expressions of mitochondrial respiratory chain-related genes and proteins after BPA exposure were founded in our study.Meanwhile,we found that the number of long tubular mitochondria decreased significantly while the punctate or fragmented mitochondria increased in BPA treated GC-2 cells.Besides,Western blot results showed that the expression of mitochondrial fission proteins Fis1 and Drp1 increased while the mitochondrial fusion proteins Mfn2 and Opa1 decreased significantly in GC-2 cells.These results suggested that BPA can result in the imbalance of mitochondrial fusion and fission.The mRNA and protein levels of CHCHD10 increased significantly in GC-2 cells after BPA treatment.Based on the CHCHD10 knockdown cell model,we found that specific knockdown the expression of CHCHD10 could significantly reverse the expression of mitochondrial respiratory chain and fusion/fission related genes and proteins,increase the number of long tubular mitochondria and decrease the number of punctate mitochondria after BPA exposure.In addition,the negative correlation between CHCHD10 level and rapid progressive sperm percentage,sperm concentration and total sperm count were also demonstrated by population studies.Conclusion:1.BPA has potential male reproductive toxicity in vivo study.Excessive apoptosis and mitochondrial damage in spermatogenic cells might be important reasons in BPA-induced male reproductive toxicity.2.BPA exerted its reproductive toxicity by regulating the signaling pathways such as Toll-like receptor signaling pathway,RIG-like receptor signaling pathway,oxidative phosphorylation and cytokine-cytokine receptor interaction related signaling pathway as well as the expressions of the related genes in GC-2 cells.3.BPA could trigger male germ cell apoptosis in mice via IFN?-XAF1-XIAP pathway.4.CHCHD10 participated in BPA-induced mitochondrial damage by mediating the expressions of mitochondrial respiratory chain related genes and mitochondrial fusion/fission related proteins in GC-2 cells.