GSE137963
Developmental Origins of Transgenerational Sperm Histone Retention Following Ancestral Exposures
This study was designed to investigate the developmental origins of the transgenerational differential histone retention sites (called DHRs) during gametogenesis of the sperm. Vinclozolin and DDT were independently used to promote the epigenetic transgenerational inheritance of these DHRs. Male control, DDT lineage and vinclozolin lineage F3 generation rats were used to isolate round spermatids, caput epididymal spermatozoa, and caudal sperm. The DHRs between the control versus DDT lineage or vinclozolin lineage were determined in these three developmental stages. DHRs and a reproducible core of histone H3 retention sites were observed using an H3 chromatin immunoprecipitation (ChIP-Seq) analysis in the germ cell populations. The chromosomal locations and genomic features of the DHRs were also analyzed. A cascade of epigenetic histone retention site alterations was found to be initiated in or prior to the round spermatids that is further altered during epididymal sperm maturation. Observations show that in addition to alterations in sperm DNA methylation previously identified, the induction of differential histone retention sites (DHRs) in the later stages of spermatogenesis also occurs. Overall design: Three different germ cell stages from transgenerational great-grand offspring F3 generation control, DDT and vinclozolin lineage male rats were used in this experimental design for epigenetic analysis. The F0 generation gestating female rats at 90 days of age were transiently exposed to DDT, vinclozolin or a vehicle control during gestational days 8-14.  At 90 days of age, the F1 generation offspring were bred within the control, DDT and vinclozolin lineages to generate the F2 generation (grand offspring) which were then bred at 90 days of age to obtain the transgenerational F3 generation for each control, DDT and vinclozolin lineage. No cousin or sibling breeding was used to avoid any inbreeding artifacts. Only the F0 generation gestating females were exposed to DDT or 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 F3 generation is the first transgenerational generation not directly exposed, so was used to study the developmental origins of the transgenerational histone retention. The F3 generation control, DDT and vinclozolin lineage male rats were aged to 120 days for isolation of round spermatid, caput spermatozoa, and cauda epididymal sperm. Three different pools each with different individual sets of animals from different litters were obtained with 3 adult males per pool. The H3 histones and methylation H3K27me3 histones were isolated from each purified round spermatid stage pool and from individual animals for epididymal caput spermatozoa and cauda sperm and then three pools generated for subsequent epigenetic analysis. The analysis of differential histone retention regions (DHRs) was performed with a chromatin immunoprecipitation (ChIP) procedure followed by next generation sequencing for a ChIP-Seq protocol.