Abstract
BackgroundAberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis.ResultsThe 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes.ConclusionIn vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.
Highlights
Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos
Aberrant hyper or hypomethylated genomic loci were increased in blastocysts derived from embryos that were exposed to in vitro conditions at 2- and 8-cell stages compared to the 16-cell stage indicating that the suboptimal culture condition induced aberrant DNA methylation pattern more strongly before and during the embryonic genome activation than later (Fig. 2)
After identification of genomic regions commonly differentially methylated in all blastocyst groups, we investigated the effects of aberrant DNA methylation patterns on the blastocyst gene expression by superimposing the DNA methylation profile with the transcriptome data [[22]], GSE33314 and GSE111990]]
Summary
Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. Alteration in the global DNA methylation patterns of human placenta derived from pregnancies established from embryos cultured under high oxygen tension [13], and alterations in the allelic methylation patterns of maternally or paternally expressed genes in mice embryonic stem cell fetuses [14] are additional evidences indicating how suboptimal culture condition could affect the genome methylation at the later stage of embryo development. Oocyte maturation media was confirmed to cause hypermethylation in the liver of fetuses at day 50 of the gestation period [16] indicating the long term effect of culture condition on the DNA methylome of the embryo or the fetal genome
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