Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo

The H19 gene is imprinted with preferential expression from the maternal al lele. The putative imprinting control region for this locus is hypermethyla ted on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic ex pression has been documented in embryos and embryonic stem cells experiment ally manipulated by in vitro culture conditions. In this study the effect o f 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 litt le paternal expression was observed following culture in KSOM containing am ino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleoti de located in the upstream imprinting control region revealed a loss in met hylation in embryos cultured in Whitten's medium but not in embryos culture d 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 KSO M+AA approximated more closely the in vivo situation. It is unlikely that b iallelic expression of H19 following culture in Whitten's medium is a gener alized effect of lower methylation levels, since the amount of DNA methyltr ansferase activity and the spatial distribution of Dnmt1 protein were simil ar 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, affe ct the expression of imprinted genes provides a model system for further st udy of the linkage between DNA methylation and gene expression.