Sperm DNA methylation alterations induced by gestational arsenic exposure are established stepwise during spermatogenesis.

Abstract

Background: Gestational exposure to environmental toxicants has been reported to be associated with epigenetic alterations in the male germ line. We have previously demonstrated that gestational arsenic exposure induces characteristic DNA methylation alterations in F1 sperm, namely the global DNA hypomethylation and the retrotransposon-associated alterations, including an increased frequency of differentially methylated cytosines (DMCs) and a predominance of hypomethylated DMCs (hypoDMCs) within long terminal repeats (LTRs) and long interspersed nuclear elements (LINEs). Hypomethylation of retrotransposon promoters is associated with increased detrimental retrotransposition and may contribute to disease development. Determining when and how these methylation alterations occur during spermatogenesis is important for elucidating the underlying molecular mechanisms.

Methods: To determine the developmental stage at which these alterations are established, prospermatogonia and type A spermatogonia were isolated from late-gestation and early postnatal testes of F1 male mice exposed to arsenic during gestation. Genome-wide DNA methylation profiles were analyzed in each cell population using reduced representation bisulfite sequencing (RRBS). Global methylation levels and the genomic distribution of DMCs, with particular focus on retrotransposon-associated regions, were examined. In addition, the expression of genes encoding DNA methylation-related enzymes was measured.

Results: Global DNA hypomethylation was observed in prospermatogonia derived from arsenic-exposed mice and was also detected in type A spermatogonia. In contrast, an increased frequency of DMCs and a predominance of hypoDMCs within retrotransposon-associated regions were not observed in prospermatogonia or type A spermatogonia, suggesting that these locus-specific alterations may be established during later stages of spermatogenic differentiation subsequent to the type A spermatogonial stage. Furthermore, decreased expression of Dnmt and Tet family genes was observed in prospermatogonia from the arsenic-exposed group.

Conclusions: These findings demonstrate that sperm DNA methylation alterations induced by gestational arsenic exposure are established in a stepwise manner during spermatogenesis. Global DNA hypomethylation associated with alterations in epigenetic remodeling processes is established at the prospermatogonial stage. Subsequently, more locus-specific alterations, characterized by an increased frequency of DMCs and a predominance of hypoDMCs primarily within LTR and LINE retrotransposon regions, emerge during later stages of spermatogenic differentiation subsequent to the type A spermatogonial stage.