Environmentally induced epigenetic transgenerational inheritance of phenotypic variation and disease susceptibility requires the germ cell (sperm or egg) transmission of integrated epigenetic mechanisms involving DNA methylation, histone modifications, and non-coding RNA (ncRNA) actions. Previous studies have demonstrated that transgenerational exposure and disease-specific differential DNA methylation regions (DMRs) in sperm are observed and that ncRNA-mediated DNA methylation occurs. The current study was designed to determine if transgenerational exposure-specific ncRNAs exist in sperm. Specifically, toxicants with distinct mechanisms of action including the fungicide vinclozolin (anti-androgenic), pesticide dichlorodiphenyltrichloroethane (estrogenic), herbicide atrazine (endocrine disruptor at cyclic adenosine monophosphate level), and hydrocarbon mixture jet fuel (JP8) (aryl hydrocarbon receptor disruptor) were used to promote transgenerational disease phenotypes in F3 generation outbred rats. New aliquots of sperm, previously collected and used for DNA methylation analyses, were used in the current study for ncRNA sequencing analyses of nuclear RNA. Significant changes in transgenerational sperm ncRNA were observed for each transgenerational exposure lineage. The majority of ncRNA was small noncoding RNAs including piwi-interacting RNA, tRNA-derived small RNAs, microRNAs, rRNA-derived small RNA, as well as long ncRNAs. Although there was some overlap among the different classes of ncRNA across the different exposures, the majority of differentially expressed ncRNAs were exposure-specific with no overlapping ncRNA between the four different exposure lineages in the transgenerational F3 generation sperm nuclear ncRNAs. The ncRNA chromosomal locations and gene associations were identified for a small number of differential expressed ncRNA. Interestingly, an overlap analysis between the transgenerational sperm DMRs and ncRNA chromosomal locations demonstrated small populations of overlapping ncRNA, but a large population of non-overlapping ncRNAs. Observations suggest that transgenerational sperm ncRNAs have both exposure-specific populations within the different classes of ncRNA, as well as some common populations of ncRNAs among the different exposures. The lack of co-localization of many of the ncRNAs with previously identified transgenerational DMRs suggests a distal integration of the different epigenetic mechanisms. The potential use of ncRNA analyses for transgenerational toxicant exposure assessment appears feasible.
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