Exposure of mouse embryos to ethanol during preimplantation development: effect on DNA-methylation in the H19 imprinting control region
Date
2009-02-23T13:15:28Z
Authors
Haycock, Philip Charles
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Abstract
ABSTRACT
Ethanol is a classic teratogen capable of inducing a wide range of developmental
abnormalities that vary in severity, from the barely perceptible to spontaneous abortion.
These defects are collectively referred to as foetal alcohol spectrum disorders (FASD).
Foetal alcohol syndrome (FAS) lies at the extreme end of this spectrum and is associated
with three broad domains: prenatal and/or postnatal growth retardation, distinctive facial
features and brain damage. Epidemiological and animal studies clearly indicate that the
clinical variability of FASD is related to four distinct window periods: preconception,
preimplantation, gastrulation and postorganogenesis. These developmental windows are
correlated with peak periods of epigenetic reprogramming, suggesting a common
mechanism of ethanol teratogenesis. Together with experimental evidence that ethanol
inhibits DNA-methyltransferase, as well as folate metabolism, this suggests an
‘epigenetic model of FASD’.
The aim of the present study was to explore the validity of this model by
investigating the relationship between ethanol-induced growth retardation and imprinting,
following ethanol exposure during the preimplantation period. Employing an
experimental study design, together with a hybrid mouse model, embryos and placentae
were harvested at 10.5 days post coitus (dpc). The weights of embryos and placentae, as
well as methylation profiles at the H19 imprinting control region (ICR) – an important
regulator of growth - were measured.
It was found that ethanol-treated embryos and placentae were severely growth
retarded in comparison to controls: r=-0.760 (p<0.01, one-tailed) and r=-0.816 (p<0.05,
two-tailed), respectively. Bisulphite genomic sequencing revealed that the methylation
profile at the H19 ICR was unaffected in ethanol-treated embryos, in comparison to
saline-treated controls. Conversely, methylation at the paternal and maternal alleles in
placentae was found to be reduced and increased, respectively, in comparison to
embryos. These results imply that mechanisms for the maintenance of imprinting in the
embryo are more robust than in the placenta. This is consistent with the relatively longlived
nature of the embryo, which must maintain imprinting for a considerably longer
period of time than the placenta.
Bisulphite sequencing also revealed that the paternal allele of the H19 ICR had
significantly decreased levels of methylation, while the maternal allele had increased
levels of methylation, in ethanol treated-placentae, in comparison to saline controls. The
changes observed at the paternal allele were localized to the CTCF1 DNA-binding site,
while a trend for increased methylation at the maternal allele was observed at the CTCF2
site. A partial correlation further revealed that demethylation at the paternal allele in
placentae partly mediated the effect of ethanol on placental weight. An ‘epigenetic switch
model’, whereby paternal Igf2 is downregulated by the epigenetic switching of the
paternal allele to the maternal epigenotype, is proposed to explain this relationship.
However, partial correlations also indicated that demethylation at the paternal allele of
the H19 ICR, as well as placental growth retardation, did not mediate the effect of
ethanol on embryo growth.
Collectively, these data suggest that imprinting at the H19 ICR is not a
mechanism of embryo growth retardation prior to 10.5 dpc. In explaining these results, it
is proposed that the growth retarded placenta was able to meet the nutritional demands of
the similarly growth retarded embryo up until 10.5 dpc. However, an important question
for future research would be to examine the relationship between ethanol-induced growth
retardation and imprinting during late gestation. During the final growth spurt (>14.5
dpc) the growth retarded placenta may become unable to meet the increased demands for
nutrition, which would exacerbate foetal growth restriction.
In sum, the present study revealed a novel mechanism of ethanol-induced growth
retardation in the placenta but indicated that imprinting at the H19 ICR does not mediate
the effect of ethanol on the early embryo. Further research is required to resolve the
relationship between imprinting and ethanol-induced growth retardation.
Description
Keywords
mouse embryos, ethanol-induced growth retardation, imprinting, H19 imprinting control region