Reactive Oxygen Species-evoked Genotoxic Stress Mediates Arsenic-induced Suppression Of Male Germ Cell Proliferation And Decline In Sperm Quality

Objective This study focused on the effects of arsenic exposure on sperm quality in male mice,and explored the role and mechanism of genotoxic stress in As-induced suppression of male germ cell proliferation and decline in sperm quality.Methods This study included in vivo and in vitro experiments.The in vivo experiment consisted of three independent experiments.Experiment one,forty male mice were randomly separated into two groups:control and As groups.In As group,mice drank ultrapure water containing Na As O₂（15 mg/L）for 70 d,while mice drank ultrapure water in the control group.All mice were sacrificed 70 d after As exposure.The testes were collected for subsequent analysis.The apoptosis of testicular germ cells was detected by TUNEL assay.Ki67 and PCNA were used to detect the proliferation of testicular germ cells.Experiment two,we performed an acute As exposure.Forty-eight mice were randomly separated into four groups.In control group,mice were injected with normal saline.As-exposed mice were injected with Na As O₂（4 mg/kg）.Testes were collected at different time points（6,24 or 72 h）after As injection.The protein levels of HO-1 and p-ATR were detected by Western blotting.Experiment three,we explored the protective effect of NAC pretreatment on As-induced testicular toxicity.Forty-eight mice were randomly separated into four groups.Mice in control group drank ultrapure water.In As and As+NAC groups,mice were given with ultrapure water containing Na As O₂（15 mg/L）.In NAC and As+NAC groups,mice were orally administered with NAC（200 mg/kg）.Preliminary results showed that NAC（200mg/kg）had no testicular toxicity.Testes were collected 35 d after As exposure.PCNA levels were detected by immunohistochemistry.In vitro experiments:RTCA and Ki67immunostaining were used to detect cell proliferation.Cell cycle was detected by flow cytometry.The protein levels of CDK1,Cyclin B1,p-ATR,p53 and p21 were detected by Western blotting.Comet assay andγH2AX immunostaining were used to detect DNA damage.ROS level was detected by DCFH-DA.NAC intervention test was used to detect HO-1,p21,Ki67 and other indicators.Results The mature seminiferous tubules and epididymal sperm count were reduced in As-exposed mice.Cell proliferation,determined by immunostaining with Ki67,was suppressed in As-exposed seminiferous tubules and GC-1 cells.PCNA,a proliferation marker,was reduced in As-exposed mouse testes.Cell growth index was decreased in As-exposed GC-1 cells.Flow analysis showed that As-exposed GC-1 cells were retarded at G2/M phase.CDK1 and cyclin B1 were reduced in As-exposed GC-1 cells and mouse testes.Additional experiment revealed that p-ATR,a marker of genotoxic stress,was elevated in As-exposed mouse testes and GC-1 cells.Accordingly,p-p53and p21,two downstream molecules of ATR,were increased in As-exposed GC-1 cells.Excess reactive oxygen species（ROS）,measured by immunofluorescence,and DNA-strand break,determined by Comet assay,were observed in As-exposed GC-1cells.γH2AX,a marker of DNA-strand break,was elevated in As-exposed seminiferous tubules and GC-1 cells.NAC alleviated As-evoked DNA damage,genotoxic stress,cell proliferation inhibition and sperm count reduction.Conclusion In conclusion,ROS-evoked genotoxic stress mediates As-induced germ cell proliferation inhibition and decline in sperm quality.