INTRODUCTION

Epigenetic modifications of the genome play a significant role in the elaboration of the genetic code and affect early growth and development through their influence on gene expression. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is a primary cause of low embryo production efficiency and high embryonic and fetal losses observed after somatic cell nuclear transfer (SCNT). It is assumed that aberrant epigenotypes of embryos and fetuses cause abnormalities that are frequently found in SCNT individuals, e.g., placental anomalies or fetal overgrowth phenotype^), large offspring syndrome (LOS). However, epigenetic perturbations have also been associated with disorders caused solely by in vitro culture of oocytes and embryos, and thus may occur independent of the manipulations associated with the nuclear transfer procedure. The phenotypic consequences are, to some extent, reminiscent of abnormalities observed after SCNT and include altered organ development, placental anomalies, fetal overgrowth/ LOS, and an increased frequency of rare congenital disorders associated with imprinting defects.

Epigenetic modifications of the genome include meth-ylation of DNA at cytosine residues, histone protein modifications such as acetylation and methylation, and the modification and assembly of regulatory protein complexes on DNA. These modifications can be functionally linked and are reversible. Methylation of cytosine residues at CpG dinucleotides is the most extensively characterized epigenetic mark in mammals, which is generally associated with transcriptional silencing and imprinting. During germ cell development, loss of DNA methylation is associated with the erasure of imprinting marks, which are subsequently reset in the developing gametes. After fertilization, during the second phase of large-scale epigenetic reprogramming, a general loss of DNA methylation is observed throughout the preimplantation period of development, but imprinted DNA methylation is maintained. The presence of DNA methylation brings about the deace-tylation of histone H4 and methylation of Lys9 of histone H3 and prevents methylation of Lys4 of histone H3. This highlights the importance of DNA methylation patterns established in early embryogenesis for setting up the structural profile of the genome.

Analysis of specific sequences by bisulfite sequencing and restriction enzymes and the investigation of chromosomal or global methylation patterns with antibodies against 5-methylcytosine (5mC) revealed highly aberrant methylation profiles in bovine preimplantation SCNT embryos. Compared with their normal counterparts, a large fraction of SCNT preimplantation embryos exhibited hypermethylation of DNA on a sequence-specific or global scale, which is presumably caused by inefficient passive demethylation during the initial embryonic cell divisions and/or precocious de novo methylation. Hypermethylation of DNA in SCNT preimplantation embryos is associated with histone H3 Lys9 hypermethylation and is negatively correlated with the developmental potential of embryos.

Epigenetic changes have been associated with altered gene expression and fetal overgrowth/LOS after sheep embryo culture, but this observation was based on a single locus, and data for the bovine system is lacking. In a more recent study, global hypomethylation was reported for DNA samples from tissues of aborted or viable bovine SCNT fetuses in comparison with in vitro fertilization (IVF) and artificial insemination (AI) controls. However, this investigation described extraordinarily high cytosine methylation levels of up to 36% even in samples from noncloned controls, although it has been demonstrated that less than 6% of cytosine residues are methylated in mammals, including cattle. We have, therefore, designed a standardized experiment to quantify global meth-ylation of cytosine in viable bovine Day 80 fetuses generated by in vitro (SCNT, IVF) or in vivo (AI) procedures with a highly accurate method and explored the relationships between the fetal overgrowth phenotype and 5mC content of liver and cotyledon tissue.