The Effects Of Acrylamide On Sperm In Mice And The Mechanisms Of Transgenerational Inheritance

Objective:The toxic effects of acrylamide(AA)on spermsfrom F0,F1 and F2 mice were evaluated by detecting sperm number,activity and deformity.DNA methylation and gene expression patternswere examined in spermand hippocampal to understand the mechanisms of transgeneration inheritance.Methods:C57male mice were randomly distributed into fivetreatment groups with six mice in each group.After being exposed to AA at 0,0.01,1,10 ppm and its metabolite glycidamide(GA)at 10 ppmfor six weeks,mice were mated to produce F0 and F1 genreations.The number and activity of sperms were examined by an automatic sperm biochemical detector andverifiedunder microscope.The structure of testis wereexaminedby histological section.The testosterone and ROS were detected by ELISA.DNA damage was detected by alkaline comet assay.Morris water maze was used to evaluate the learning and memory ability of mice.Gene expression changes were screened by RNA-seq,and validated by qPCR.MethylRAD andnext generationsequencing(NGS)were used to detect the whole genome DNA methylation of spermatozoa in parental male mice,which were further validated by COBRA.Results:Compared with control group,nosignificantchange in sperm numberwas found in miceexposedto AA and GA,but sperm activity and malformationratewere decreased and increased respectively in a dose-dependent manner.For F1 and F2 offspring,neither sperm number nor motility nor malformation rate show significant change.High dose of acrylamide(10ppm)and glycidamide(10ppm)also increasedsperm ROS and decreased serum testosterone in AA exposed mice andtheir offspring.Meanwhile,the arrangement of epithelial cells of seminiferous tubules was disturbed in the testes of exposed mice andtheir offspring,with some spermatogenic cells falling off into the seminiferous tubules.No DNA damage was detected.In water maze experiment,the escape time of the mice exposed to AA(10 ppm)and GA was significantly delayed at test day 1 and day 2 compared to control mice,which turn to normal at test day 3 and day 4.For F1 and F2 male mice,the escape time of AA 10 ppm and GA groups at test day 3 was significantly lower compared to that of control group,and at test day 4,the time delay was still exist for F2 male mice.Expression changes of genes related to signal pathways such as complement systems were found in the hippocampus of the mice exposed to AA at 10 ppm.The down-regulation of Kcne2 and S100 a and the up-regulation of Ttrwerevalidated by qPCR.The overexpression of Ttr was also found in F2 male mice.For DNA methylation changes,1004 CCGG sites and 106 CCWGG sites were found in mice exposed to high dose of AA(10 ppm).The hypermethylation ofUshbp1 and the hypomethylation G19 were not only confirmed in AA exposed mice but also found in the second generation(F2 mice).No DNA methylationchange was found in repeat sequences IAP and LINE1 in AA/GA exposed mice and their offspring.Conclusion:Acrylamide and its main metabolite GA decreased sperm number and motility,increased malformationrate,sperm ROS and serum testosterone.and other parental changes can be inherited to the F2 male mice.Moreover,the changes in sper malformation rate,sperm ROS and serum testosterone was inherited to F2 male mice.Acrylamide also cause neurotoxicity in exposed mice and their offspring,and could induce gene expression changes in hippocampus.The transgenerational effects may be due to the aberrent sperm DNA methylation patterns induced by acrylamide.