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 ...

In vitro folliculogenesis of cryopreserved ovarian tissue could be an effective method for insuring fertility for patients who receive gonadotoxic treatment. Although several culture systems have been described for growing female gametes in vitro, the production of competent oocytes for further development remains a considerable challenge. The purpose of our study was to determine whether maternal primary imprinting progresses normally during mouse oocyte growth in vitro. We analysed the DNA methylation status of differentially methylated regions of the imprinted genes H19, Mest/Peg1 and Igf2R using fully grown germinal vesicle-stage oocytes (fg oocytes) produced by in vitro folliculogenesis from early preantral follicles. When compared to fg oocytes removal from control females, we observed after in vitro development, a loss of methylation at the Igf2R locus in six out of seven independent experiments and Mest/Peg1 locus (one out of seven), and a gain of methylation at the H19 locus (one out of seven). These results provide insight into the dysregulation of the process of primary imprinting during oocyte growth in vitro and highlight the need for effective new biomarkers to identify complete nuclear reprogramming competence after in vitro folliculogenesis.