Differential Effects of Culture on Imprinted H19 Expression in the Preimplantation Mouse Embryo1

Abstract

The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.

Aberrant DNA methylation in porcine in vitro‐, parthenogenetic‐, and somatic cell nuclear transfer‐produced blastocysts

“…In this study, the in vivo-produced blastocysts were different from the other blastocysts in that there was no time in culture. For example, previous studies have shown that epigenetic changes caused by in vitro preimplantation culture results in aberrant methylation and expression of a number of imprinted genes (Sasaki et al, 1995;Doherty et al, 2000;Mann et al, 2004). Further, the authors suggest that the epigenetic remodeling and reprogramming of the trophoblast may be more sensitive to the disruptive effect of the culture media than the ICM.…”

Genomic imprinting as an adaptative model of developmental plasticity

“…It has been proposed that nutritional availability around conception may affect the post-fertilisation wave of epigenetic reprogramming [36,37]. Multiple studies of embryos fertilised and cultured in vitro have suggested that imprinting control elements may be more susceptible to the environment during this period than previously thought [38,39]. However, these studies are potentially confounded by the effects of superovulation, which has been shown to alter the epigenetic status of maternal ICRs [40].…”

The Epigenetics of Adult (Somatic) Stem Cells

“…112 Of greater interest is that although culture of embryos to the blastocyst stage in KSOM maintains the appropriate maternal monoallelic expression of the imprinted H19 gene, H19 expression is essentially biallelic following culture in Whitten's medium. 113 Moreover, the loss of imprinting was associated with the loss of methylation on the paternal, repressed allele in the differentially methylated region (DMR); 113 DNA methylation of the paternal allele in this region is strongly linked with the repression of the paternal H19 allele. 114 The loss of imprinting that occurs during culture under certain conditions, as well as global changes in gene expression, could have long-term consequences on development.…”