Alterations in Sperm DNA Methylation, Non-coding RNA and Histone Retention Associate with DDT Induced Epigenetic Transgenerational Inheritance of Disease

Environmental factors have been shown to induce the epigenetic transgenerational inheritance of disease through the germline. This study was designed to investigate DDT induced concurrent alterations of a number of different epigenetic processes including DNA methylation and histone retention in sperm. Gestating female rats were exposed transiently to DDT during fetal gonadal development and then F1, F2 and F3 generation sperm were analyzed. The experimental design involved a transient exposure of gestating female F0 generation rats during post-conception day 8-14 to 25 mg/kg body weight of DDT in DMSO daily using an intraperitoneal injection. A control generation lineage involved the exposure during day 8-14 of gestation to vehicle DMSO (dimethylsulfoxide) alone. Six different gestating females from different litters for each control and DDT lineage were used. The F1 generation offspring were obtained and aged to 90 day postnatal age and selected males and females bred within the control or DDT lineage. The F2 generation offspring were obtained and aged to 90 days and selected males and females from different litters bred to generate the F3 generation. No sibling or cousin breeding were used to avoid any inbreeding artifacts. All the males were sacrificed at 120 days of age for epididymal sperm collection. The differential DNA methylation regions (DMRs) between the control versus DDT lineages were determined using methylated DNA immunoprecipitation (MeDIP) followed by DNA sequencing (MeDIP-Seq) and bioinformatics analysis. The differential histone retention regions (DHRs) for the F1, F2 and F3 generation for the control versus DDT lineage sperm were analyzed. The procedure involved assessment of differential histone retention sites (DHRs) through a comparison of the control versus DDT lineage sperm using the edgeR analysis similar to that used for the DNA methylation analysis.