The Role And Mechanism Of Telomere And Mitochondrial Compromise In BaP-Induced Male Reproductive Damage

Background:Benzo[a]pyrene（BaP）is a representative compound of Polycyclic Aromatic Hydrocarbons（PAHs）,and studies have shown that BaP and its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide（Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide,BPDE）have a clear male（male）reproductive impairment effect,but the exact molecular mechanism remains unclear.Telomeres are DNA repetitive sequences located at the ends of chromosomes that protect chromosome structure and are also involved in maintaining genomic stability during germ cell meiosis,thus playing an important role in male reproduction.However,the guanine-rich nature of telomeric DNA sequences makes them vulnerable to cellular oxidative stress induced by environmental toxicants such as BaP.Meanwhile,mitochondria are sensitive targets in oxidative stress damage,and they have also been shown to be sensitive organelles in BaP/BPDE-induced male reproductive damage.In recent years,it has been increasingly shown that the telomeric reverse transcriptase（TERT）gene is involved in mediating telomere and mitochondrial compromise in cells.On the one hand,TERT is an important factor regul ating telomere lengthening in the nucleus,and inhibition of TERT expression may lead to impaired maintenance of telomere structure and subsequent.On the other hand,it is related to the non-telomere function of the TERT gene,i.e.,TERT may play a different role than promoting telomere lengthening by localizing to organelles outside the nucleus,including a possible role in resisting oxidative stress damage by localizing to mitochondria.Previous studies have shown that BaP and its active metabolite BPDE can cause damage to spermatogenic cells by inducing telomere and mitochondrial compromise,so what is the molecular mechanism of TERT regulation in this process?Are they involved in germ cell damage through"non-telomere function"?It is worth exploring in depth.In addition,telomeres are important structures for maintaining chromosome integrity,but the association between sperm telomere length（STL）and traditional DNA damage indicators is unclear.The group has previously established a reproductive health cohort of university students in Chongqing,and has examined the relevant indicators in the sperm samples of the cohort.The correlation between STL and traditional DNA damage indicators,including nuclear DNA and mitochondrial DNA damage,needs to be investigated to explore whether STL may be a potential indicator of sperm quality.Objectives:1.To investigating the molecular mechanism of TERT regulation in telomere damage induced by BaP and BPDE in spermatogenic cells.2.To investigating the role of mitochondrial TERT expression in BaP/BPDE-induced mitochondrial compromise.3.To investigate the correlation between sperm telomere length and sperm nuclear DNA integrity and mitochondrial DNA abnormalities,and to explore the potential application of sperm telomere length as a biological indicator for predicting male sperm function.Methods:1.To constructed animal model,BaP was continuously treated by gavage for one spermatogenic cycle（35 days）which can be used to clarify the effects of male reproductive damage and target cells induced by BaP.Male ICR mice were treated with different doses of BaP（1,10 mg/kg BW）by gavage for 35 days,while the telomerase agonist ABG was used for intervention.Semen quality was measured by CASA,apoptosis of spermatogenic tubules by TUNEL,histopathological changes in testis by HE staining,ultrastructure of testicular tissue by transmission electron microscopy,and target cells of spermatogenic disorders by immunofluorescence staining.After extracting testicular tiss ue proteins,Western blotting was used to detect the expression of germ cell markers,TERT transcriptional regulation-related proteins SIRT1.2.SIRT1,FOXO3a,c-MYC,and mitochondrial biosynthesis-related proteins PGC-1αand COX IV.The mRNA levels of TERT gene and its transcriptional regulation-related molecules were detected by qPCR after RNA extraction from testis tissues.Telomere length was detected by qPCR after extraction of testicular tissue and sperm DNA.3.Based on the clarification of spermatocyte damage,BPDE-treated GC-2 cells were used as an in vitro cellular experimental model to further investigate the molecular mechanism of TERT expression inhibition by BPDE.The cell cycle was detected by propidium iodide staining combined with flow cytometry（FCM）,the levels of Senescence Associated Secretory Phenotype（SASP）and Telomerase（TE）were detected by ELISA,Annexin V-APC/PI combined with FCM,and apoptosis was detected by Western blotting.FCM was used to detect apoptosis,Western blotting and qPCR to detect the protein and mRNA expression levels of SIRT1,FOXO3a and c-MYC,respectively,and qPCR to detect telomere length.After pretreatment with ABG in BPDE-treated cells,the above indices were then examined to evaluate the role of TERT expression regulation in BPDE-induced telomere damage.In addition,the expression of SIRT1,FOXO3a and c-MYC was upregulated by transfecting cells with pLV-EGFP-SIRT1,pLV-EGFP-FOXO3a and pLV-EGFP-c-MYC vectors,respectively,to verify that BPDE inhibits TERT transcriptional expression via the SIRT1/FOXO3a/c-MYC pathway,which in turn causes GC-2 Molecular mechanism of telomere damage in cells.4.The role of mitochondrial TERT expression in BPDE-induced mitochondrial compromise was investigated using BPDE-tainted GC-2 cells as a model.Cellular NAD⁺/NADH ratio was detected by NAD⁺/NADH assay kit,cellular complexⅠactivity was detected by mitochondrial respiratory chain complexⅠassay kit,cellular complexⅠexpression was detected by Western blotting,and cellular MOTS-c content was detected by ELISA kit.Immunofluorescence staining combined with laser confocal assay was used to detect the localization changes of TERT protein,and Western blotting was used to detect the expression levels of TERT in cytoplasm,nucleus and mitochondria,respectively.Further,GC-2 cells stably overexpressing mitochondria-targeted TERT(OE-TERT^mst)and GC-2 cells without mitochondria-targeted sequence TERT(OE-TERT^wt)were constructed,and on this basis,BPDE treatment was performed,and cellular mitochondrial membrane potential was detected by TMRE probe and enzyme marker,Mito SOX staining combined with FCM was used to detect mitochondrial ROS level,and qPCR for mitochondrial DNA copy number（mt DNAcn）,and the aforementioned methods for MOTS-c content,cellular complexⅠactivity and NAD⁺/NADH ratio,respectively,to clarify the role of mitochondrial TERT expression in BPDE-induced mitochondrial compromise.5.Based on the reproductive health cohort population of college students in Chongqing established by the group in the early stage,444 volunteers who met the criteria were included and questionnaires were used to collect demographic information.Semen samples were collected from individuals and analyzed by CASA for routine semen parameters,qPCR for sperm telomere length（STL）and mitochondrial DNA copy number（mt DNAcn）,sperm chromatin structure assay（SCSA）and comet assay for sperm nuclear DNA integrity,respectively.The sperm chromatin structure assay（SCSA）and comet assay were used to detect the integrity of sperm nuclear DNA and Long-PCR to detect the integrity of sperm mitochondrial DNA,respectively.The correlation between individual STL and sperm nuclear DNA integrity and mt DNA abnormalities was analyzed by linear regression,and the correlation between STL and age,parental reproductive age and BMI of the study subjects was analyzed by Spearman’s correlation analysis,to initially explore the potential application of sperm telomere length as a biological indicator of sperm function.Results:1.BaP exposure also caused a significant decrease in the expression of the meiotic and spermatocyte markers HSPA2 and SYCP3,suggesting that BaP caused impaired maintenance of the spermatocyte population in mice.The spermatocyte population maintenance was impaired by BaP.Subsequently,using a mouse spermatocyte-derived GC-2 cell line as an in vitro experimental model,BPDE treatment was found to cause senescence damage in GC-2 cells,including cellular S-phase arrest and increased levels of SASP（IL-6,IL-8 and MMP9）.Meanwhile,both BaP and BPDE-induced telomere damage in germ cells（including telomere length shortening and reduced TERT expression）were significantly alleviated by ABG pretreatment,and ABG also ameliorated BPDE-induced cellular senescence,cycle block and apoptosis.The mRNA assay of TERT gene showed that BPDE inhibited TERT transcriptional expression,and ABG was able to revert the BPDE-induced TERT transcriptional repression,so this study further explored the molecular regulatory mechanism of BPDE-induced TERT transcriptional repression.MYC protein and mRNA expression levels were significantly reduced in BaP-treated mouse testis and BPDE-exposed GC-2 cells.Up-regulation of SIRT1 expression alleviated BPDE-induced cell cycle arrest,apoptosis and increased SASP secretion,and reduced FOXO3a acetylation,increased c-MYC and TERT protein expression and up-regulated TERT mRNA levels,and BPDE-induced telomere length shortening and telomerase content reduction were also significantly restored.Similarly,upregulation of FOXO3a or c-MYC expression improved BPDE-induced cellular senescence and telomere damage,including shortened telomere length,reduced telomerase content and decreased TERT expression were significantly alleviated,and c-MYC overexpression significantly improved BPDE-induced TERT transcriptional repression.2.Mitochondrial structural and functional damage was observed in BaP-treated mouse testicular spermatocytes.The results of cellular assays showed that BPDE inhibited the NAD⁺/NADH ratio,complexⅠactivity,complexⅠprotein expression and mitochondria-derived peptide MOTS-c levels in GC-2 cells,indicating that BPDE induced mitochondrial compromise in GC-2 cells.Protein expression and localization analysis showed that BPDE significantly inhibited mitochondrial TERT expression in GC-2 cells.Experiments using the constructed TERT stable overexpression cells showed that both OE-TERT^mst and OE-TERT^wt ameliorated BPDE-induced cellular senescence,apoptosis,decreased telomerase levels and mitochondrial damage（including mitochondrial membrane potential,Mito SOX,mt DNAcn,MOTS-c,NAD⁺/NADH and complexⅠactivity were reverted）,and mitochondria-targeted TERT overexpression was more effective in alleviating BPDE-induced mitochondrial compromise and apoptosis compared with OE-TERT^wt.3.The results of the correlation study in the population showed that sperm telomere length（STL）was not correlated with each semen quality parameter,and STL was significantly and positively correlated with sperm DNA fragments index（DFI）and with DNA damage indicators analyzed by the comet assay.correlation analysis of STL with sperm mitochondrial DNA indicators showed that STL was positively correlated with sperm mt DNAcn and negatively correlated with sperm mt DNA integrity was negatively correlated.The correlation analysis of STL with individual’s age,parental childbearing age and BMI showed that STL was positively correlated with paternal childbearing age.These results suggest that STL is associated with sperm nuclear DNA integrity and sperm mitochondrial DNA abnormalities,suggesting that sperm telomere length may be used as an indicator of human sperm quality.Conclusion:1.BaP and BPDE may induce spermatocyte senescence,cycle arrest and apoptosis by inducing telomere damage,leading to spermatogenic disorders.Meanwhile,BPDE may inhibit the transcriptional expression of TERT through SIRT1/FOXO3a/c-MYC pathway,which leads to telomere damage.2.BaP and BPDE could cause mitochondrial damage in spermatocytes,while BPDE could inhibit mitochondrial TERT expression.In vitro experiments showed that inhibition of TERT mitochondrial expression mediated BDPE-induced mitochondrial damage and cell senescence and apoptosis in spermatocytes.3.STL is significantly associated with sperm nuclear DNA integrity and sperm mitochondrial DNA abnormalities,providing a reference for the possible application of sperm telomeres as a biological indicator of sperm function.