219 METHYLATION STATUS OF A DIFFERENTIALLY METHYLATED REGION (DMR) WITHIN THE BOVINE Igf2 GENE IN PREIMPLANTATION EMBRYOS

Specific DNA regions within imprinted genes become differentially methylated on the maternal and paternal chromosomes during germ cell development. These DMRs play a crucial role in the regulation of imprinted gene expression. The murine insulin-like growth factor2 gene (Igf2) is imprinted and contains an intragenic DMR within the last exon. Recently it became known that the bovine Igf2 gene is also imprinted (Dindot et al. 2004 Biol. Reprod. 71, 470–478) where we have now identified an intragenic DMR in the last exon with the paternal allele being methylated. Aberrant methylation patterns within the bovine Igf2 gene could result in deregulated gene expression and could therefore be involved in the development of fetal abnormalities such as the large offspring syndrome (LOS) in cattle. We have studied the methylation status of 27 CG dinucleotides within this DMR in bovine pre-implantation embryos of different origin by bisulfite sequencing. DNA was isolated from expanded blastocysts collected in vivo and generated by in vitro fertilization (IVF), somatic nuclear transfer (NT), and parthenogenesis (PA). Additionally, DNA was obtained from fibroblasts derived from a female and a male adult animal and used as donor cells for NT and from zygotes and 4-cell embryos both produced by IVF. After PCR amplification of the bisulfite-treated DNA, PCR products were cloned and sequenced. Methylation percentages were calculated for each individual clone by division of the 27 CpGs with the number of methylated CpGs per sample. The methylation levels (%) from each sample were then used to obtain the global methylation levels of the analyzed region. Methylation decreased during the transition from the zygote (28.4% ± 3.8 SEM) to the 4-cell embryo (6.3% ± 2.2 SEM) indicating that the DMR is demethylated after fertilization. An increased methylation level was observed in expanded blastocysts (in vivo: 10.2% ± 1.2 SEM; IVF: 10.1% ± 0.7 SEM; female NT: 12.4% ± 1.4 SEM). Thus, remethylation starts before the blastocyst stage. The higher methylation level of male NT blastocysts (22% ± 1.9 SEM) in comparison to their in vivo and IVF counterparts could be due to an insufficient reprogramming of the donor cells after nuclear transfer. Female and male donor cells were both heavily methylated (77% ± 2.2 SEM; 72% ± 2.9 SEM, respectively). Parthenogenetic expanded blastocysts were less methylated (2.3% ± 1 SEM), probably due to their diploid maternal genome. Results show for the first time that the methylation status at this DMR is associated with the origin of the embryo. Analysis of methylation patterns in pre-implantation embryos could provide a diagnostic tool to unravel mechanisms involved in fetal malformations often observed after the use of in vitro fertilization and/or nuclear transfer.