Figure 7

DNA methylation establishment is undisturbed by EDs in prospermatogonia at paternally methylated imprinted DMRs. DNA methylation was mapped in purified G1R and G2R prospermatogonia and spermatozoa using MIRA-chip and custom Nimblegen imprinting arrays. The mouse matings were conducted as depicted in Additional file 4. (A) Summary of MIRA-chip results at imprinted DMRs in custom imprinting arrays (groups 5 to 8). The average MIRA/input log2 ratios (n = 3 for MGCs and n = 2 for sperm) were calculated for known imprinted DMRs and are depicted with red (maternal and blue (paternal) flags in the range of -1.9 to +1.9. The full calculations are provided in Additional file 6. Note that paternally methylated DMRs have positive MIRA/input log2 ratios and maternally (MAT) methylated DMRs have negative MIRA/input log2 ratios, as expected. No MAT DMR exhibited increased methylation and no PAT DMR had decreased DNA methylation, using the cutoff values of ±5% and P <0.05 (Student’s t-test). (B) The MIRA profile is depicted at paternally methylated imprinted DMRs (black rectangles) in biological duplicate samples for VZ treatment or control. The DNA methylation signals of MIRA versus input DNA were plotted as -log10 P values in the range of 0 to 8.4. The average % DNA methylation levels at each CpG as determined by whole genome bisulfite sequencing (WGBS) are shown compared to that of normal MGCs at 16.5 dpc [18] and normal sperm [20]. Note, that DNA methylation at paternal DMRs is undisturbed by ED treatment in the exposed prospermatogonia and in the prospermatogonia of the next generation.