DNA Methylation In The Disturbance Of Spermatogenesis Caused By Exposure To Chemical In Neonatal SD Rats

During germ cell development,the building of DNA methylation patterns plays an important role in the maintaince of male reproduction.The change of DNMT activity and expression may induce abnormal DNA methylation and further interfere with normal spermatogenesis.It has been noticed for a long time that many germ cells in testis were apoptosis during the fetal and early postnatal ages.Cell cycles and apoptosis were regulated by gene.However,change of the DNA methylation level in gene promoter region could influence the expression of these genes and might induce apoptosis.Although DNA methylation and apoptosis could regulate the spermatogenesis,the molecular events governing these facts have not been clearly characterized.5-Aza-2 -deoxycytidine(5-Aza-CdR) is a cytidine analogue which can incorporate into replicating DNA,and indirectly caused genomic hypomethylation It was also reported that 5-Aza-CdR can cause reproductive dysfunction in laboratory animals.Cd and PCB153,persistent bioaccumulative pollutants in environment,can be absorbed into the bodies through food chain.Cd and PCB have been demonstrated a variety of adverse effects including reproductive development,and they also have been reported changing DNA metyltransferase in vitro.Gonocytes resume into mitosis at early neonatal stage,and many of them differentiate into spermatogonia,while others degenerate at this time.The maintance and de novo methylation are important for spermatogenesis.Therefore neonatal exposure to 5-Aza-Cd,Cd and PCB153 models were used to investigate global DNA methylation,DNMT activity and expression of Dnmts and P53 promoter methylation,which helps to explore the role of DNA methylation in chemical induced spermatogenesis dysfunction.Neonatal male rats were randomly divided into 10 groups,24 rats per group and received oral administration of 5-Aza-CdR(0.025,0.25mg/kg),PCB153(0.025,0.25, 2.5mg/kg) and Cd(1.0,2.0,4.0mg/kg) from PND3 for five days.12 rats were killed 24h after the last administration.The remains were fed until 12 weeks.Testes were taken for histological study,immunohistochemistry detection and DNA methylation assay. After exposure for 5d,5-Aza-CdR significantly reduced body weight of rats, which compared with control group,while in adult rats of 12-week age,only 0.25mg/kg dose group lost weight.But there was no significant difference between different dose groups as far as the organ coefficient and anogenital distance were concerned.Although there was no significant abnormal in adult histology,the exposure to 5-Aza-CdR did repress the spermatogenesis and reduced the rate of daily sperm production.The event that 5-Aza-CdR exposure decreased global DNA methylation may happen via repressing the expression of Dnmt3a and lowering DNMT activity.5-Aza-CdR also decreased P53 promoter region methylation level while increased P53 expression therefore it could induce apoptosis.5-Aza-CdR exposure significantly increased apoptosis compare with control group further confirm the thesis.Exposure to Cd resulted in losing weight of rats at PND8 and then it presented no significant change till12-week.No obvious abnormal events happened in histology of PND8 testis.While there was obvious abnormal in 12-week testis,Histologic study showed that there was less muration sperm in tubules and the arrangement of cell was disorder.And sperm heads count from testis homogenates showed significantly decrease in Cd exposure groups compared with control.Cd exposure decreased global DNA methylation may via de novo methylation represse the expression of Dnmt3a, Dnmt3b and therefore lowered DNMT activity.Cd exposure significantly increased apoptosis in testis compared with control group.But it was different with 5-Aza-CdR,because P53 independent may explain why Cd induced apoptosis.As far as PCB153 was concerned,there was no obvious change in body weight, histology of testis both at PND8 and 12 week.And there was no significant change in DNA methylation and apoptosis and related gene expression.But this study did find the daily sperm production decrease at adulthood.This suggested there may be other pathway such as endocrine that can affect spermatogenesis.This study indicated that DNA methylation could be affected by chemical during neonatal exposure via multiple pathways.The abnormal pattern of DNA methylation could cause reproduction dysfunction and disturb the spermatogenesis.Further studies are warranted to increase our understanding of the effect ofDNA methylation in relation to the underlying mechanisms of chemical induced disturbance of spermatogenesis.