Prenatal exposure to ethanol: A specific effect on the H19 gene in sperm

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Abstract

Alcohol exposure during pregnancy induces a range of disorders in the offspring. Methylation changes in imprinted genes may play a role in the teratogenic effects of alcohol. We evaluated the possible effects of alcohol administration in pregnant mice on the methylation pattern of 5 imprinted genes (H19, Gtl2, Peg1, Snrpn and Peg3) in somatic and sperm cell DNAs of the male offspring. The effects observed were a 3% (p < 0.005) decrease in the number of methylated CpGs of H19 in the F1 offspring sperm, a 4% (p < 0.005) decrease in the number of methylated CpGs of H19 in the F2 offspring brain and a 26% (p < 0.05) decrease in the mean sperm concentration. CpGs 1 and 2 of the H19 CTCF-binding site 2 exhibited significant methylation percentage losses. H19 CTCF-binding sites are important for the regulation of Igf2 gene expression. The hypomethylation of H19 may contribute to the decreased spermatogenesis in the offspring.

Introduction

The teratogenic effects of alcohol consumption during pregnancy are well documented. They consist in a wide range of disorders referred to collectively as the fetal alcohol syndrome (FAS) [1] or as the fetal alcohol spectrum disorders (FASDs) [2]. They include physical, behavioral and cognitive abnormalities, like growth deficiency, central nervous system dysfunction, organ/skeletal pathology and craniofacial anomalies [3]. Maternal alcohol consumption might also be related cryptorchidism and the testicular dysgenesis syndrome which includes hypospadias, testis cancer and decreased semen quality [4].

All the features of FAS have been reproduced in mice after acute or chronic alcohol prenatal exposure. Their severity depended on the dose and timing of exposure [3], [5]. A critical period was that of organogenesis and neuronal growth [6]. The effects of alcohol also depended on the mice genetic background [5]. Prenatal chronic alcohol exposure was found to induce a dose-dependent alteration of hippocampal electrophysiological activity [7] and alcohol exposure in the adult inhibited neural progenitor cell proliferation and survival [8]. Many of the fetal alcohol neurobehavioral effects, like deficits in spatial learning or memory tasks could be attributed to an abnormal hippocampal development and function [9].

Epigenetic refers to changes in gene expression that do not involve DNA sequence but modifications of DNA, such as DNA methylation and post-translational modifications of histones such as acetylation, methylation and phosphorylation. Epigenetic changes may play a role in the effects of chronic alcohol exposure on NR2B (a subunit of the NMDA receptor) gene expression in the central nervous system [10] and in the teratogenic effects of alcohol during pregnancy [11]. Indeed, alcohol treatment of pregnant mice induced global DNA hypomethylation in the fetuses [12] and, recently, maternal ingestion of alcohol was found to induce an hypermethylation of the Agouti viable yellow (Avy) gene associated with an agouti colored coat [13]. Also aberrant changes in DNA methylation associated with changes in gene expression were induced in whole embryo culture by alcohol exposure [14].

Imprinting is an epigenetic form of gene regulation that mediates parent-of-origin-dependent expression of the alleles of a number of genes. It occurs at specific sites within or surrounding the gene, called differentially methylated domains (DMDs). Within a DMD, one parental allele is methylated on all or the majority of its CpG dinucleotides, and the opposite parental allele is methylated on none or a small percentage of its CpG dinucleotides. The detrimental effects of alcohol consumption on male reproductive hormones and on semen quality are well documented [15]. Indeed, chronic alcohol consumption in men was found to be correlated with a demethylation of the imprinted paternally methylated H19 and IG genes in the sperm [16]. Recently, a study showed that an oral exposure to alcohol of pregnant mice during the embryo preimplantation period induced changes in the methylation pattern of the H19 gene. This effect was observed in the placenta but not in the embryo, suggesting that the imprinting control mechanisms were more robust in the latter [17]. Altogether, the data of the studies of Ouko et al. [16] and of Haycock and Ramsay [17] suggest that alcohol affects the methylation of some imprinted genes, at least in certain, possibly more susceptible, tissues.

A few mouse and human DMDs have been well characterized. They are, in particular, the DMDs of the maternally expressed paternally methylated H19 [18] and Gtl2 [19] genes and the paternally expressed maternally methylated small nuclear ribonucleoprotein polypeptide N (Snrpn) [20], Peg1 [21], Peg3 [22] and the potassium channel 1 (Kcnq1) [23] genes.

The aim of the present study was to evaluate more systematically than ever before the possible effects of low dose alcohol administration in pregnant mice, from gestation day (GD) 10 to 18, on DNA methylation at imprinted genes. This was done by testing possible methylation changes induced by alcohol exposure of pregnant mice in a selection of target imprinted genes and tissues of the offspring. The DMD methylation patterns of 2 paternally (H19 and Gtl2) and 3 maternally (Peg1, Snrpn and Peg3) imprinted genes were analyzed in the tail, liver, skeletal muscle, hippocampus and sperm DNAs of the male offspring over 2 generations.

Section snippets

Mice

Normal FVB/N mice purchased from Charles River (Arbresle, France) were used. For toxicology studies focusing on epigenetic and DNA methylation changes, the FVB/N strain is well-characterized and widely used [24]. Furthermore, it has been well studied with regards to the regulation of imprinting reprogramming during fetal development [25]. How FVB/N strain aligns to the conventional C57BL/6J model used for the study of alcohol effects on the methylation of imprinted genes [17] is not known.

Two

Results

Alcohol administration to pregnant female mice during the gestational period corresponding to fetal organogenesis and gonadal sex determination did not affect litter size, sex ratio and birth weight among the two groups (not shown).

The DNA bisulfite treatment technique allows to measure the amount of methylated as compared to total (methylated and non-methylated) CpGs.

In the tail, which represents a heterogeneous sample of somatic cells, the amount of methylated CpGs in control 8 week-old male

Discussion

The goal of this study was to examine whether alcohol exposure during pregnancy could lead to methylation changes in imprinted genes of the progeny.

Most animal studies use one of 2 alcohol dosage paradigms: 2.9–6 g/kg on one or 2 occasions [27] or <3 g/kg daily exposure [11] during the developmental period of interest. A short term regimen of alcohol at a dose of 1.5 g/kg from GD7 to 10, did not adversely affect embryonic growth [28]. We chose a low dose of alcohol (0.5 g/kg) in order to avoid

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the Swiss Academy of Medical Sciences and the Swiss Center for Applied Human Toxicology.

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