Telomere Damage Effect During MEHP-induced Germ Cell Damage And Preliminary Mechanism Exploration

BackgroundMale reproductive health has been identified by the World Health Organization（WHO）as a major health risks of the 21st century,and therefore the exploration of risk factors for male reproductive health has become a hot topic of research in this field.The reproductive health of men is influenced by a combination of genetic,environmental,social and behavioral factors;however,a growing body of research indicates that environmental pollution is emerging as a primary risk factor for male reproductive health.As typical endocrine disrupting chemicals（EDCs）,phthalic acid esters（PAEs）are used in a wide range of plastics due to their good plasticity and ductility.Various environmental media,such as soil,water and air,can detect a certain level of PAEs,which can enter human body via the gastrointestinal tract,respiration and skin,leading to a series of health damaging effects in the body.At present,it is well documented that PAEs are closely related to male reproductive toxicity and that the testis is their primary target organ.Studies have demonstrated that PAEs act like estrogen in humans and animals,interfering with the body’s normal endocrine function,reducing semen quality,affecting reproductive health and ultimately leading to male infertility.Despite the fact that PAEs have been well documented to cause definite androgenic toxicity,the molecular events,molecular mechanisms by which PAEs induce this damaging effect still remain to be further elucidated.Recently,studies have discovered that some EDCs can elicit structural and functional damage to telomeres,which is intimately associated with male infertility.However,very few studies have been conducted to explore the relationship between PAEs-induced male reproductive toxicity and telomere damage,as well as scarce mechanistic studies.Therefore,for this study,we chose to use Di-2-ethylhexyl phthalate（DEHP）with its primary metabolite,Mono-2-ethylhexyl phthalate（MEHP）,to construct an in vivo and in vitro model of toxicity to investigate the role of damage to telomere structure and function in PAEs-induced male reproductive damage,and to probe deeper into the underlying molecular mechanisms of MEHP-induced telomere dysfunction in germ cells.Methods1.Study of the role on telomere damage during spermatogenic cell cycle arrest and apoptosis caused by MEHP.（1）MEHP-induced spermatogenic cell cycle arrest and apoptosisEstablished an in vitro model of MEHP-stained GC-1 mouse spermatogonia.Changes in cell viability,cycle,apoptosis and reactive oxygen species（ROS）were detected by using Cell counting kit-8（CCK-8）,propidium iodide（PI）,Annexin V/FITC or DCFH-DA in combination with C6 flow cytometry.At the same time,Quantitative real-time PCR（RT-q PCR）was performed to analyze the expression of G₀/G₁-related genes and Western Blot（WB）to assay for G₀/G₁-related and apoptosis-related proteins for evaluating the toxic effects of MEHP on GC-1 cells.（2）Study on the structural and functional damage of telomeres in spermatogenic cells by MEHPThe telomere length（TL）,telomerase activity（TA）and telomerase reverse transcriptase（TERT）expression were assayed by RT-q PCR,Enzyme-linked immunosorbent assay（ELISA）and Western Blot to assess the telomere damage effect of MEHP on GC-1 cells.（3）Role of telomere damage in MEHP-induced spermatogenic cell cycle arrestBased on the above study,a TERT inhibitor（BIBR1532）was used to construct a TERT-interfering GC-1 cell model for the evaluation of the role of TERT-mediated telomere damage in MEHP-induced cell cycle arrest.2.The role and mechanism of c-Myc signaling of MEHP-mediated telomere damage in spermatogenic cells（1）c-Myc signaling pathway change detectionBased on an in vitro model of MEHP-stained GC-1 mouse spermatogonia,the expression of c-Myc,Max m RNA and protein in GC-1 cells was examined by RT-q PCR and Western Blot techniques,which evaluated the effect of MEHP on c-Myc,an upstream modulator of TERT.（2）Effect of c-Myc signaling pathway intervention on the effects of MEHP telomere damageThe c-Myc inhibitor（10058-F4）was used to construct a GC-1 cell model with disordered c-Myc expression for assessing the role of c-Myc in MEHP-induced cell cycle arrest and telomere damage.（3）Analysis of transcriptional regulatory molecules upstream of the c-Myc signaling pathwayThree public databases,Cistrome DB,TRRUST and Genecards,were used to screen for five c-Myc upstream target genes with overlap.The expression levels of m RNA and protein of these five target genes were inspected by RT-q PCR and Western Blot techniques as a means of assessing the role of c-Myc upstream transcription factors in MEHP-induced germ cell cycle arrest and telomere damage.（4）In vivo reproductive toxicity evaluation and validationBased on the results of the cellular assay,an animal model of DEHP（0,250 and 500mg/kg）gavage stained C57BL/6J mice for 35 days was established in this study to further validate the results of the in vitro GC-1 cellular assay.Results1.MEHP induces G₀/G₁ phase block and apoptosis in germ cellsThe results showed that MEHP could induce significant cytotoxic effect damage to GC-1cells.Gradually,the survival of GC-1 cells declined in response to increasing concentrations of MEHP treatment and presented a definite dose-effect relationship.MEHP additionally enhanced the G₀/G₁ phase ratio,apoptotic cell percentage as well as ROS levels in GC-1 cells when treated with 100,200 and 400μM for 48 h.Meanwhile,levels of m RNA expression of Cdk4,Cdk6,Ccnd1 and Rb1,critical genes that modulate cell cycle progression across the G₁to S phases,decreased with increasing doses of MEHP staining,CDK4,Cyclin D1 and the anti-apoptosis-related protein Bcl-2 exhibited progressively decreasing protein expression levels,in contrast to the incrementally increasing protein expression levels of the apoptosis-related protein Bax.2.MEHP causes telomere length shortening,telomerase activity suppression and TERT expression depression in spermatogenic cellsIt was shown that in MEHP-treated GC-1 cells,cells had shorter telomere length and a lower level of telomerase activity expression,as well as significantly reduced m RNA and protein expression of TERT,a rate-limiting determinant of telomerase activity.3.MEHP-induced TERT-mediated telomere damage may be associated with G₀/G₁phase arrest in germ cellsThe above findings suggest that telomere disruption may be tightly linked to MEHP-induced spermatogenic cell damage.The TERT inhibitor BIBR1532 was used to establish a cell model for TERT inhibition and it was found that the combined treatment of TERT inhibitor with MEHP resulted in more severe telomere shortening and G₀/G₁ phase arrest in GC-1 cells.This implies that TERT may in some sense be involved in and mediate the MEHP-induced G₀/G₁ phase arrest in spermatogonia associated with telomere damage.4.c-Myc signaling mediates MEHP-induced telomere damage and G₀/G₁ phase block in germ cellsThe results displayed that MEHP treatment reduced the expression of c-Myc and Max m RNA and protein in GC-1 cells in a dose-dependent manner.Moreover,study of GC-1spermatogonia treated with c-Myc inhibitor revealed that the treatment with c-Myc inhibitor10058-F4 exacerbated to some extent the MEHP-induced reduction in TERT expression and proliferation inhibition in GC-1 cells and gave further rise to a more severe G₀/G₁ phase arrest of the cells.This suggests that a reduction in c-Myc probably participates in the MEHP-induced G₀/G₁ phase arrest and a reduction in TERT expression in germ cells.5.MEHP may suppress c-Myc signalling by specific upstream transcriptional regulatorsBy examining transcription factors upstream of c-Myc,we found that MEHP-induced repression of c-Myc expression may be related to a decrease in the expression levels of CTCF,ESR1 and C-JUN m RNA and protein,which are upstream target genes of c-Myc.6.DEHP exposure induces testicular telomere damage and male reproductive toxicity in male C57BL/6J miceIn vivo animal studies showed that DEHP staining shortened telomere length,reduced TERT protein expression,damaged c-Myc signalling pathway and induced testicular toxicity in male C57BL/6J mice,including apoptosis,reduced sperm density,reduced sperm viability and altered testicular histomorphology,which further validated the results of the cytological experiments.ConclusionIn this study,based on a mouse spermatogenic GC-1 cell model of in vitro MEHP staining,we discovered that MEHP exposure induced cell cycle dysregulation and apoptosis through mediating abnormal telomere structure and function in spermatogenic cells.The telomerase reverse transcriptase TERT plays an invaluable role in this damage process,and the reduction in TERT expression regulates telomere dysfunction mediating MEHP-induced G₀/G₁ phase arrest in male germ cells.Further studies revealed that telomere damage may be correlated with reduced expression of c-Myc,an transcription factor that regulates TERT expression,and that the expression levels of CTCF,ESR1 and C-JUN,the upstream transcriptional regulators of c-Myc signalling,were reduced distinctly.These findings implicate that MEHP may induce spermatogenic cell block and apoptosis through specific transcriptional regulators that inhibit c-Myc signaling,resulting in reduced TERT activity,telomere function and structural damage.The results obtained from the cellular model study were validated in an in vivo DEHP mouse staining model.This discovery will contribute to a deeper understanding of the molecular mechanisms of male reproductive damage induced by PAEs and provide new intervention clues for better prevention of MEHP-induced male reproductive toxicity.