Primordial Germ Cell Transgenerational Sperm DNA Methylation Epimutations Following Ancestral Vinclozolin Exposure

Previously the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis. Male control and vinclozolin lineage F3 generation rats were used as a source of embryonic day 13 (E13) primordial germ cells. The DMRs between the control versus vinclozolin lineage samples were determined for this stage. The experimental design involved the development of F3 generation control and vinclozolin lineage male rats to isolate the primordial germ cell (PGC) stage for epigenetic analysis. The F0 generation gestating female rats at 90 days of age were transiently exposed to vinclozolin or a vehicle control during embryonic days 8–14 when the PGCs were migrating to colonize the indifferent gonad and during the early stages of gonadal sex determination. The F1 generation offspring were obtained and at 90 days of age were bred within the control and vinclozolin lineages, respectively, to generate the F2 generation (grand offspring) which were then bred at 90 days of age to obtain the F3 generation for each control and vinclozolin lineage. No cousin or sibling breedings were used to avoid any inbreeding artifacts. Only the F0 generation gestating females were transiently exposed to vinclozolin, which also directly exposed the F1 generation fetus and the germline within the F1 generation fetus that will generate the F2 generation. Therefore, the transgenerational F3 generation is the first generation not directly exposed and was used to study the developmental origins of the sperm epimutations. The F3 generation control and vinclozolin lineage male rats were aged to the embryonic day 13 (E13) fetal stage for PGC cell isolation. Two pools each from different individual sets of animals were obtained from 5-6 males per pool for the E16 stage. Therefore, 3-7 different males were used in each of the three different pools analyzed for both the control and vinclozolin lineages. The DNA was isolated from each pool for subsequent epigenetic analysis.