Locus-specific analysis of DNA methylation patterns in cloned and in vitro fertilized porcine embryos

Abstract

Porcine somatic cell nuclear transfer (SCNT) is currently inefficient, as 1-3.95% of reconstructed embryos survive to term; inadequate or erroneous epigenetic reprogramming of the specialized donor somatic nucleus could be a primary reason. Therefore, a locus-specific analysis of DNA methylation dynamics in embryogenesis and the DNA methylation status of gametes and donor cells used for SCNT were conducted in the following developmentally important gene loci: POU5F1, NANOG, SOX2, H19, IGF2, IGF2R, XIST; and the retrotransposon LINE-1. There were significant epigenetic differences between the gametes and the somatic donor cells. Three gamete-specific differentially methylated regions (DMRs) in POU5F1, XIST, and LINE-1 were identified. A delayed demethylation process at POU5F1 and LINE-1 loci occurred after three successive cleavages, compared to the in vitro fertilized (IVF) embryos. Although cloned embryos could undergo de-methylation and re-methylation dynamics at the DMRs of imprinted genes (H19, IGF2R, and XIST), the re-methylation process was compromised, unlike in fertilized embryos. LINE-1 loci are widely dispersed across the whole genome, and LINE-1 DMR might be a potential porcine nuclear reprogramming epi-marker. Data from observations in our present and previous studies, and two published articles were pooled to produce a schematic diagram of locus-specific, DNA methylation dynamics of cloned and IVF embryos during porcine early embryogenesis. This also indicated aberrant DNA methylation reprogramming events, including inadequate DNA demethylation and insufficient re-methylation in cloned embryos. Further research should focus on mechanisms underlying demethylation during the early cleavage of embryos and de novo DNA methylation at the blastocyst stage.

Keywords: Cloned embryos; DNA methylation; Donor cells; In vitro fertilized embryos; Porcine.

Fig. 1.

Gamete-specific DNA methylation patterns of the POU5F1 gene. The sequence of the POU5F1 gene (Genbank: CT737281.12) was analyzed for CpG islands (CGIs), using CpG Island Searcher (http://cpgislands.usc.edu/), and a typical island across the 5' upstream region, and the first exon was identified (blue bar in Fig. 1A). A CGI shore region (–88 bp to –367 bp, 280 bp, red bar in Fig. 1A) was subsequently identified as a gamete-specific differentially methylated region (DMR) (Fig. 1B). The use of “_|” in Fig. 1B represents an absent CpG.

Fig. 2.

Gamete-specific DNA methylation patterns of the XIST gene, and LINE-1 retrotransposon. The use of “_|” represents an absent CpG due to mutation.

Fig. 3.

Sequence analysis for DNA methylation and genome-wide distribution of porcine LINE-1 retrotransposon. The CpG island prediction tool (http://cpgislands.usc.edu/) indicates a CpG island (blue bar) in the 5'-untranslated region (Fig. 3A). A CpG shore containing six CpGs (red bar) within the antisense promoter (ASP; based on GenBank: ABR01162 annotation) of the LINE-1 retrotransposon was defined as a gamete-specific DMR and was hypermethylated in donor somatic cells with 65.5% methylation levels (Fig. 3A). The LINE-1 DMR sequence was BLASTed against Pig Sscrofa11.1 (genomic sequence) and 202 matched sequences (red box, red triangle) with, at least, 98.41% of sequence similarity (E-value < 1e^-1) were obtained (Fig. 3B).

Fig. 4.

DNA methylation levels at nine loci of porcine donor cells. DNA methylation patterns of male somatic donor cells at nine loci in eight genes, POU5F1, NANOG, SOX2, IGF2, H19, IGF2R, XIST, and LINE-1.

Fig. 5.

DNA Methylation reprogramming dynamics of POU5F1 during early embryogenesis. Cloned embryos undergo a delayed demethylation process from two-cell to the blastocyst-stage, compared with their in vitro fertilization (IVF) counterparts, which rapidly demethylate after the first cleavage.

Fig. 6.

DNA Methylation reprogramming dynamics of LINE-1 during early embryogenesis. Cloned embryos undergo a delayed demethylation process after three cleavages, compared with their in vitro fertilization (IVF) counterparts, which reach a lower methylation level (57.5% of cloned embryos vs. 37.5% of IVF embryos).

Fig. 7.

DNA Methylation reprogramming dynamics of IGF2R during early embryogenesis. Cloned and in vitro fertilization (IVF) embryos completely demethylate after the first cleavage but differ in the re-methylation process when the cloned embryos cannot establish imprinted hemi-methylation patterns, compared to the IVF embryos, that reach semi-methylation at the blastocyst stage.

Fig. 8.

Summary of locus-specific DNA methylation dynamics of cloned and in vitro fertilization (IVF) embryos during early porcine embryogenesis. (A) Locus-specific DNA methylation patterns of six gene loci at the level of sperms, oocytes, and somatic donor cells (B–E). DNA methylation reprogramming dynamics of four gene loci during early embryogenesis of cloned embryos (dark red lines) and IVF embryos (blue lines).