88 VARIABLE DNA METHYLATION PROFILES AT IMPRINTED LOCI IN BOVINE EARLY PRE-IMPLANTATION EMBRYOS

Abstract

The DNA methylation imprints, at maternally imprinted gene differentially methylated regions, are established during the postnatal growth stage of oogenesis, with paternal imprints being acquired in the perinatal prospermatagonia. Murine DNA methylation marks, at imprinted loci, are widely regarded to be resistant to post-fertilization demethylation events that occur in the paternal pronucleus of the zygote and to passive demethylation of the maternally derived genomic content from cleavage to the 16-cell stage. However, the DNA methylation profile of bovine imprinted genes following fertilization remains unknown. The objective of the current study was to analyze the methylation dynamics at several imprinted gene differentially methylated regions during bovine embryo development. In addition, a previously published RNA-seq database (Mamo et al. 2011 Biol. Reprod.) was mined for transcript abundance of genes associated with establishing and maintaining genomic imprints. Single in vivo blastocysts (Day 7), hatched ovoid embryos (Day 14), filamentous embryos (Day 17), and implanting conceptii (Day 25) were collected (n = 4–9, per time point) from beef heifers. Genomic DNA was isolated and bisulfite modified, using the EZ DNA methylation direct kit (Zymo, Irvine, CA, USA), and used as template in bisulfite PCR reactions. The PCR products were verified by agarose gel electrophoresis and subsequently pyrosequenced. Observed methylation values were most highly variable in Day 7 blastocysts, with values ranging between 13 and 44% (IGF2R), 5 and 63% (PEG10), 7 and 59% (MEST), 3 and 61% (SNRPN), 12 and 64% (PLAGL1), and 20 and 32% (H19). There was a marked reduction in variability as embryonic development progressed, with values at Day 25 ranging from 37 to 41% (IGF2R), 34 to 38% (PEG10), 31 to 37% (MEST), 36 to 40% (SNRPN), 17 to 26% (PLAGL1), and 25 to 30% (H19). Statistical analysis (Levene’s test for equal variance) of methylation values for each gene at each time point confirmed that the methylation values observed in Day 7 embryos were significantly variable (P < 0.05) when compared with later developmental stages. Concordant with this finding, RNA transcript levels of associated methylation machinery genes DNMT3A, DNMT3B, and TRIM28 progressively increased from Day 7 to 13 and subsequently decreased from Day 13 to 16. Taken together our results demonstrate that in cattle DNA methylation marks, at imprinted loci, are highly variable at the blastocyst stage and are progressively stabilized with increasing days post-fertilization. This stabilization of imprint is coordinated with a window of increased levels of associated methylation machinery transcripts. Work presented here provides evidence of a novel mechanism for bovine embryonic DNA methylation imprint maintenance. This work was funded by SFI grant number 07/SRC/B1156.