Reproduction Abstracts

Introduction: Genome-wide analysis of DNA methylation in mammalian preimplantation embryos, particularly at the single embryo level, will be of particular use for assessing the epigenetic status and health of in vitro-derived embryos, including humans. To date, single embryo DNA methylation analysis has been limited to a small number of genes (<5). In this study we used an adaptation of reduced representation bisulfite sequencing (RRBS) to i) perform genome-wide analysis of the DNA methylome of the bovine blastocyst using pooled blastocysts and ii) to assess whether the same aim could be achieved in single blastocysts.

Materials and methods: Embryonic genomic DNA was isolated from bovine in vitro-derived blastocysts. Two pooled blastocyst samples were generated (pool 1, n=17 blastocysts; pool 2, n=5 blastocysts). In addition, DNA from three single bovine blastocysts was isolated. RRBS reads were mapped using Bismark and the Bioconductor package BiSeq for methylation calling, clustering, and analysis.

Results and discussion: Assessment of RRBS read clusters revealed that greater than 34 000 CpG clusters were commonly methylated between the two pooled blastocyst samples and >13 000 CpG clusters were common between the three single blastocyst samples (clusters contain >20 CpGs). Genome-wide plots for methylated CpG clusters and methylated CpG islands will be presented. Using DAVID analysis, the highly methylated loci include members of the MAPK signalling pathway, members of the pathways in cancer and several miRNAs as examples. This report is the first genome-wide analysis of DNA methylation in the bovine blastocyst and the first to show that comprehensive genome-wide methylation analysis in single mammalian blastocysts is possible.