Aberrant methylation of donor genome in cloned bovine embryos

Abstract

Despite recent successes in cloning various animal species, the use of somatic cells as the source of donor nuclei has raised many practically relevant questions such as increased abortion rates, high birth weight and perinatal death. These anomalies may be caused by incomplete epigenetic reprogramming of donor DNA. Genome-wide demethylation occurs during early development, 'erasing' gamete-specific methylation patterns inherited from the parents. This process may be a prerequisite for the formation of pluripotent stem cells that are important for the later development. Here, we provide evidence that cloned bovine embryos may have impaired epigenetic reprogramming capabilities. We found highly aberrant methylation patterns in various genomic regions of cloned embryos. Cloned blastocysts closely resembled donor cells in their overall genomic methylation status, which was very different from that of normal blastocysts produced in vitro or in vivo. We found demethylation of the Bov-B long interspersed nuclear element sequence in normal embryos, but not in cloned embryos, in which the donor-type methylation was simply maintained during preimplantation development. There were also significant variations in the degree of methylation among individual cloned blastocysts. Our findings indicate that the developmental anomalies of cloned embryos could be due to incomplete epigenetic reprogramming of donor genomic DNA.

Altered imprinted gene expression and methylation patterns in mid-gestation aborted cloned porcine fetuses and placentas

“…Similar results were also found in mouse [20]. In cloned bovine embryos, these repeated regions of the donor genome exhibited aberrant methylation patterns [8]. In our study, the aborted cloned pigs showed aberrant high levels of methylation.…”

“…Previous studies demonstrated that the majority of imprinted genes play important roles in regulating the normal development of the fetus and placenta, and the expression of these genes is regulated by the methylation status of the promoters [5,6]. Numerous studies reported that aberrant DNA methylation occurs in cloned mammalian embryos [7,8]. PRE-1, a short interspersed nucleotide element (SINEs), has 10 5 copies in porcine genome, whereas centromeric satellite DNA is a highly repetitive sequence in porcine heterochromatic region.…”

Reprogramming of genome function through epigenetic inheritance

“…Such interactions may result in both de novo methylation and demethylation of specific loci of donor nuclei, which may occur stochastically, resulting subsequently in aberrant patterns of gene expression and failure of development. In one recent study, the methylation status of the donor nucleus at the blastocyst stage was found to be either unchanged or aberrant compared to the control 59 . Reprogramming of the X chromosome can occur, however, as the inactive X chromosome can be reactivated in the somatic nucleus, although the molecular memory of the inactive X chromosome is retained, resulting in its preferential inactivation in the trophectoderm 60 .…”

Mammalian nuclear transfer

“…Several groups have investigated DNA methylation patterns in NT embryos and reported abnormalities in DNA methylation (Dean et al, 1998BourcЈhis et al, 2001;Kang et al, 2001aKang et al, , 2002Ohgane et al, 2001). In cloned bovine embryos, satellite sequence methylation levels are more similar to the donor cells than to control embryos (Kang et al, 2001a).…”

“…These findings suggest that different chromosomal regions might respond differently to demethylation in the egg cytoplasm. Of interest, when satellite sequences that had been examined in bovine (Kang et al, 2001a) were analyzed in a different species (porcine) methylation levels at the blastocyst stage of cloned embryos were more comparable to those of fertilized control embryos (Kang et al, 2001b), suggesting also species-specific differences. A recent study that analyzed several imprinted genes in cloned murine blastocysts showed that most of the examined genes displayed aberrant methylation and expression patterns (Mann et al, 2003).…”