Early prenatal alcohol exposure alters imprinted gene expression in placenta and embryo in a mouse model

Abstract

Prenatal alcohol exposure (PAE) can harm the embryonic development and cause life-long consequences in offspring's health. To clarify the molecular mechanisms of PAE we have used a mouse model of early alcohol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first eight days of gestation (GD 0.5-8.5). Owing to the detected postnatal growth-restricted phenotype in the offspring of this mouse model and both prenatal and postnatal growth restriction in alcohol-exposed humans, we focused on imprinted genes Insulin-like growth factor 2 (Igf2), H19, Small Nuclear Ribonucleoprotein Polypeptide N (Snrpn) and Paternally expressed gene 3 (Peg3), which all are known to be involved in embryonic and placental growth and development. We studied the effects of alcohol on DNA methylation level at the Igf2/H19 imprinting control region (ICR), Igf2 differentially methylated region 1, Snrpn ICR and Peg3 ICR in 9.5 embryonic days old (E9.5) embryos and placentas by using MassARRAY EpiTYPER. To determine alcohol-induced alterations globally, we also examined methylation in long interspersed nuclear elements (Line-1) in E9.5 placentas. We did not observe any significant alcohol-induced changes in DNA methylation levels. We explored effects of PAE on gene expression of E9.5 embryos as well as E9.5 and E16.5 placentas by using quantitative PCR. The expression of growth promoter gene Igf2 was decreased in the alcohol-exposed E9.5 and E16.5 placentas. The expression of negative growth controller H19 was significantly increased in the alcohol-exposed E9.5 embryos compared to controls, and conversely, a trend of decreased expression in alcohol-exposed E9.5 and E16.5 placentas were observed. Furthermore, increased Snrpn expression in alcohol-exposed E9.5 embryos was also detected. Our study indicates that albeit no alterations in the DNA methylation levels of studied sequences were detected by EpiTYPER, early PAE can affect the expression of imprinted genes in both developing embryo and placenta.

Fig 1. Schematic structure of studied imprinted genes on mouse chromosome 7q.

Proximally, near the centromere, are Paternally expressed gene 3 (Peg3) and Small Nuclear Ribonucleoprotein Polypeptide N (Snrpn), and distally Insulin-like growth factor 2/H19 locus (Igf2/H19). Gray boxes illustrate maternally or paternally expressed active alleles and black boxes inactive alleles. Imprinting control regions (ICR) or differentially methylated regions (DMR) are marked with methylated (black) or unmethylated (white) balls. Arrows and sequences below the ICRs or DMR represent the regions of interest. Not drawn into scale.

Fig 2

DNA methylation levels of CpG sites at the Igf2/H19 ICR (A), Igf2 DMR1 (B), Snrpn ICR (C), Peg3 ICR (D), and Line-1 (E) in 9.5 embryonic days old (E9.5) control and alcohol-exposed embryos and placentas. Control samples are colored in black and alcohol-exposed samples in gray. The numbers of samples are presented in columns. CpG sites 3 and 5 at Igf2 DMR as well as CpG site 13 at Peg3 ICR in E9.5 placentas have been excluded since no methylation value could be detected by EpiTYPER. Error bars denote the SD.

Fig 3. Expression levels of imprinted genes in control and alcohol-exposed 9.5 embryonic day old (E9.5) embryos and placentas as well as E16.5 placentas.

Igf2, H19, Snrpn and Peg3 expressions in control and alcohol-exposed E9.5 embryos (A), E9.5 placentas (B) and in E16.5 placentas (C). Control samples are colored in black and alcohol-exposed samples in gray. The numbers of samples are presented in columns (expression of Snrpn in one embryo and one E9.5 placenta samples was not detected). Error bars denote the SD. P-value; (A) two-way Student’s t-test: *p<0.05, **p<0.001, (C) Two-way ANOVA: *p<0.05.