Dynamic CpG and Non-CpG Methylation of the Peg1/Mest Gene in the Mouse Oocyte and Preimplantation Embryo

Abstract

In somatic tissues, the CpG island of the imprinted Peg1/Mest gene is methylated on the maternal allele. We have examined the methylation of CpG and non-CpG sites of this differentially methylated CpG island in freshly ovulated oocytes, in vitro aged oocytes, and preimplantation embryos. The CpG methylation pattern was heterogeneous in freshly ovulated oocytes, despite the fact that they all were arrested in metaphase II. After short in vitro culture, Peg1/Mest became hypermethylated, whereas prolonged in vitro culture resulted in demethylation in a fraction of oocytes. Non-CpG methylation also occurred in a stage-specific manner. On alleles that were fully methylated at CpG sites, this modification was found, and it became reduced in twocell stage embryos and blastocysts. These observations suggest that the process of establishment of the methylation imprint at this locus is more dynamic than previously thought.Establishment of the mature epigenetic configuration of the genome is part of the maturation process of the gametes and is essential for normal development after fertilization. DNA methylation of CpG sites is one of the epigenetic modifications that regulates gene expression (for review, see (1)). The genome undergoes widespread changes in CpG methylation during germ cell maturation. Imprinted genes are of particular interest, because they are frequently associated with CpG-rich regions that are methylated differentially on the paternal and maternal chromosomes (for a review on imprinting, see Ref. 2). These differentially methylated regions (DMRs) 1 are believed to play an important role in the parental origin-dependent regulation of individual imprinted genes and of entire genomic regions during embryonic development. The methylation profile typical for the paternal or maternal chromosome is believed to be established during maturation of the male and female germ cells, but the exact kinetics of this process is still unclear. Some observations have even suggested that some imprinted genes reach their mature methylation profile only after fertilization (3).Peg1/Mest is a typical imprinted gene that is predominantly expressed from the paternal allele in the mesoderm and its derivatives (4). The methylation analysis of Peg1/Mest revealed that the CpG island in the promoter region was completely methylated on the maternal and unmethylated on the paternal chromosomes (5). It has been shown that the human PEG1/ MEST is already unmethylated in spermatogonia (6). In the maternal germ line, Peg1/Mest is fully methylated in ovulated oocytes that are arrested in metaphase of the second meiotic division (MII) (7).In a previous study (8), we have detected methylation heterogeneity in growing oocytes at several imprinted loci, including Peg1/Mest. The heterogeneity was substantially increased in the oocytes matured in vitro, suggesting that the methylation imprint, at this stage, is unstable and can be influenced by the cellular environment. In the present study, we investigated whether changes in the methylation ...

Unaltered imprinting establishment of key imprinted genes in mouse oocytes after in vitro follicle culture under variable follicle-stimulating hormone exposure

“…A study in human suggested aberrant DNA methylation at the imprinted H19 gene in oocytes following in vitro maturation (IVM) (Borghol et al, 2006) and prolonged IVM of mouse oocytes was associated with a loss of DNA methylation at Peg1 (Imamura et al, 2005). Another study reported aberrant DNA methylation at imprinted genes in oocytes after superovulation with high doses of gonadotrophins in mouse and human (Sato et al, 2007).…”

Epigenetic changes associated with oocyte aging

“…The hypermethylated pattern was maintained when time progressed to 21 and 29 h post hCG injection. By contrast, Imamura et al [46] reported that Peg1 lost methylation in oocytes during postovulatory aging. For Peg1, our result differed from that of Imamura et al [46].…”

“…By contrast, Imamura et al [46] reported that Peg1 lost methylation in oocytes during postovulatory aging. For Peg1, our result differed from that of Imamura et al [46]. Such discrepancy may be due to different oocyte aging times, or different domains of the same gene examined.…”

Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites, including expanded (CAG)n/(CTG)n repeats

“…12 This preference supports previous gene-specific observations. [13][14][15][16][17][18][19][20][21] Interestingly, the CAG/CTG site preference of non-CpG methylation matches the sequence preference of some mammalian DNA methyltransferases. 22,23 Non-CpG methylation has typically been assessed using bisulfite sequencing, a method with which most labs are not familiar.…”