DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development

In mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation.

To this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.

The findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.