Dataset features


Dataset type: Methylation profiling by high throughput sequencing
Number of samples: 36
Release date: Jan 19 2019
Last update date: Jan 19 2019
Access: Public
Dataset link Developmental Origins of Transgenerational Sperm DNA Methylation Epimutations Following Ancestral DDT Exposure

Experimental Protocol

F3 generation control and DDT lineage male rats were used in this experimental design to isolate various germ cell stages for epigenetic analysis. The F0 generation gestating female rats at 90 days of age were transiently exposed to DDT or a vehicle control during embryonic days 8-14 when the PGCs were migrating to colonize the indifferent gonad during early stage of gonadal sex determination. The F1 generation offspring were obtained and when they reached 90 days of age they were bred within the control and DDT 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 and DDT lineage. No cousin or sibling breeding were used to avoid any inbreeding artifacts. Only the F0 generation gestating females were exposed to DDT, 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 and was used to study the developmental origins of the sperm epimutations. The F3 generation control and DDT lineage male rats were aged to the embryonic day 16 (E16) fetal stage for prospermatogonia cell isolation, to postnatal day 10 (P10) for spermatogonial cell isolation, and to 120 days of age for pachytene spermatocyte isolation, round spermatid isolation, caput spermatozoa isolation, and cauda epididymal sperm isolation. Three different pools each with different individual sets of animals were obtained from 5-6 males per pool for the E16 stage, 6-7 males per pool for the P10 stage, 3 adult males per pool for the adult stage. Therefore, 3-7 different males were used in the analysis within the three different pools analyzed for both the control and DDT lineages. Genomic DNA was isolated from each pool and from individual animals for caput and cauda sperm for subsequent epigenetic analysis. The analysis of differential DNA methylation regions (DMRs) was performed with a methylated DNA immunoprecipitation (MeDIP) procedure followed by next generation sequencing for an MeDIP-Seq protocol.










Michael Skinner