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Prenatal exposure to ethanol: A specific effect on the H19 gene in sperm
Reproductive Toxicology 31 (2011) 507–512
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Prenatal exposure to ethanol: A specific effect on the H19 gene in sperm Christelle Stouder a , Emmanuel Somm b , Ariane Paoloni-Giacobino a,c,∗ a
Department of Genetic and Laboratory Medicine, Geneva University Hospital, 1211 Geneva 14, Switzerland Department of Pediatrics, Geneva University Hospital, 1211 Geneva 14, Switzerland c Swiss Center for Applied Human Toxicology, University of Geneva Medical School, 1211 Geneva 4, Switzerland b
a r t i c l e
i n f o
Article history: Received 31 March 2010 Received in revised form 17 January 2011 Accepted 28 February 2011 Available online 5 March 2011 Keywords: Ethanol Epigenetics Methylation Fertility Sperm
a b s t r a c t 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. © 2011 Elsevier Inc. All rights reserved.
1. 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].
∗ Corresponding author at: Department of Genetic and Laboratory Medicine, CMU, 1 Michel-Servet, 1211 Geneva 4, Switzerland. Tel.: +41 22 379 59 47; fax: +41 22 379 52 28. E-mail address: [email protected] (A. Paoloni-Giacobino). 0890-6238/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2011.02.009
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
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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. 2. Materials and methods 2.1. 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 month old female mice were naturally mated with male mice of the same age. Finding of a copulation plug the next morning signified coitus and gestation day 1 (GD1). Plug-positive females were separated into 2 groups. One group, consisting in 8 pregnant dams, was treated by oral administration of alcohol at a dose of 0.5 g/kg/day, and another group of 11 pregnant dams was treated by an equal volume of water between GD10 and 18, to encompass major events during organogenesis and fetal development [6]. One offspring from each dam was terminated at the postnatal age of 8 weeks for DNA analysis. In some cases another offspring was terminated at 8 weeks for sperm count. One pup from each litter was sacrificed as a newborn for brain analysis. In order to study possible transgenerational effects of ethanol treatment, 7–8 F1 males were bred to 7–8 FVB/N females. F2 males were then analyzed at the age of 8 weeks. Only one offspring from each dam was analyzed. Animal protocols used in these studies were approved by the Commission d’ Ethique de l’Expérimentation Animale of the University of Geneva Medical School and by the Geneva Veterinarian Office. 2.2. Sperm collection and count The vas deferens and epididymis were dissected free of connective tissue, placed into a Petri dish, delicately scored with a razor blade in a droplet of Phosphate Buffer Saline (PBS), and left for 10 min at 37 ◦ C to allow sperm to swim out into the medium. The medium with sperm was then carefully transferred to another tube and centrifuged at 6000 × g for 3 min to pellet the sperm. In some experiments an aliquot of the supernatant’s sperm was used for sperm counting, using a hemocytometer. The corresponding sperm concentration was calculated using the formula: cells per ml = the average count per square × dilution factor × 104 (counts 10 squares). 2.3. Tissue collection Fragments of 5 mm × 5 mm size of tail, liver and tibialis anterior muscle of the 8 week-old offspring were collected, cut into small pieces in a Petri dish and transferred into DNA-extraction buffer. Brain hemispheres from these same animals were collected and hippocampus from both hemispheres were dissected and pooled into DNA-extraction buffer as a single tissue. Whole brains from newborn pups terminated immediately after birth were also collected, cut into small pieces in a Petri dish and transferred into DNA-extraction buffer. 2.4. DNA isolation Sperm DNA was extracted fusing the QIAampDNA microkit (Qiagen, CA, USA). Total genomic DNA was extracted from tails, livers, tibialis anterior muscle, hippocampus and whole brains by incubation for 3 h at 55 ◦ C in DNA-extraction buffer (50 mM Tris–HCl, pH 8, 100 mM EDTA, 0.5%
SDS) followed by phenol–chloroform–isoamyl alcohol extractions and ethanol precipitation. 2.5. Bisulfite treatment Using the EZ Methylation Gold-Kit (Zymo Research, Orange, CA, USA) the extracted DNA was treated with sodium bisulfite in order to convert unmethylated cytosine residues to uracil. The converted DNA was eluted in 10 l of TE buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 7.5). Two microlitres of the post bisulfite-treated DNA were used for subsequent PCR amplification. 2.6. PCR amplification of bisulfite-treated DNAs for subsequent pyrosequencing The PCR amplifications aimed at pyrosequencing were performed starting from 100 to 140 ng of bisulfite-treated sperm, tail, liver and skeletal muscle DNA. The PCR conditions were the same for all the genes tested, i.e., 94 ◦ C for 15 min, followed by 40 cycles of 94 ◦ C, 30 s, 55 ◦ C, 30 s, 72 ◦ C, 30 s, and by a 72 ◦ C 10 min final extension step. The characteristics of the amplicons and of the oligonucleotides chosen have been described in a previous publication [26]. A diagram showing the locations of the DMDs, within the corresponding genes, and of the DNA fragments of the 5 genes amplified by PCR has been presented in a previous study [26]. For each imprinted gene between 5 and 23 CpGs were present in the PCR amplicon analyzed. All reactions were performed with a PCR mixtures (total volume 25 l) containing oligonucleotides at 0.5 mM concentration and 12.5 l of HotStarTaq Master Mix (Qiagen, CA, USA). The biotinylated PCR products were purified using streptavidin-sepharose beads (Amersham) and sequenced using the PSQ 96 Gold reagent kit (Biotage AB, Uppsala, Sweden) with the following primers: H19: 5 -GTGTAAAGATTAGGGTTGT-3 , Gtl2: 5 -GTTATGGATTGGTGTTAAG-3 , Peg1: 5 -TCAATATCAACTAAATAATC-3 , Snrpn: 5 -GAATTGGAGTTGTGTGG-3 , Peg3: 5 -AATTGATAAGGTTGTAGATT-3 . The degree of methylation at each CpG site was determined using Pyro Q-CpG Software (Biotage AB, Uppsala, Sweden). All samples were analyzed in duplicate. The average methylation value of each mouse was the average of the 2 duplicate values. The H19 amplicon, which was analyzed further in this study, contains only one CTCF binding site (i.e., site 2) (Fig. 4A). 2.7. PCR amplification of bisulfate-treated DNAs for subsequent amplicon subcloning and sequencing The PCR amplifications of individual clones were performed starting from 100 to 140 ng of bisulfite-treated sperm DNA. H19: amplification of a 182 bp fragment, encompassing 8 CpGs, was performed with and 5 oligonucleotides 5 -GGGGGTAGGATATATGTAAAAAAAAGG-3 AAAAAAACTCAATCAATTACAATCC-3 . PCR cycling was performed with a profile consisting of 94 ◦ C for 15 min, followed by 40 cycles of 94 ◦ C, 30 s, 55 ◦ C, 30 s, 72 ◦ C, 30 s, and by a 72 ◦ C 10 min final extension step. All reactions were performed with a PCR mixture (total volume 25 l) containing oligonucleotides at 0.5 mM concentrations and 12.5 l of HotStarTaq Master Mix (Qiagen, CA, USA). The amplified fragments were then gel-purified using the QIAEX II Gel Extraction kit (Qiagen, CA, USA). Gel-purified fragments were subcloned into pCR 2.1-TOPO by TOPO-TA cloning (Invitrogen, Carlsbad, CA) and individual clones were sequenced by an ABI 3130 XL sequencer using M13-Reverse oligonucleotide. Each result was obtained by sequencing 12 clonal colonies per mouse. The average methylation value of each mouse was the average of the 12 clone values. 2.8. Statistical analysis Significances were evaluated using the unpaired Student’s t test and set at p < 0.05. The non-parametric Mann–Whitney U test was also used.
3. 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 nonmethylated) CpGs. In the tail, which represents a heterogeneous sample of somatic cells, the amount of methylated CpGs in control 8 week-old male offspring was close to 50% of the total methylated + non methylated CpGs in all the imprinted genes tested. This value is in total agreement with the theoretical value expected for imprinted genes in which DNA methylation/unmethylation is present on one of the
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percent of o total num mber of CpG sites
B percent of total numb p ber of CpG G sites
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Newborn whole brain Fig. 1. Methylation status of the paternally methylated H19 and Gtl2 genes and of the maternally methylated Peg1, Snrpn and Peg3 genes A: in the tail, B: in the liver, C: in the skeletal muscle, D: in the hippocampus and E: in the newborn whole brain of F1 male offspring of control and of alcohol-fed mice. Control: water only. Alcohol: alcohol-fed. The results are the means ± sem of 8–10 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites.
two parental alleles only. No difference was observed in the methylation status of the 5 imprinted genes tested between control and alcohol-administered offspring (Fig. 1A). The methylation patterns of our 5 target genes were also analyzed in 8 week-old male offspring liver, skeletal muscle and hippocampus and in newborn
whole brain. As observed in the tail, the amount of methylated CpGs in the control offspring was close to the theoretical value of 50% of the total CpGs in all the imprinted genes tested. Again, no difference was observed between control and alcohol-administered offspring (Fig. 1B–E).
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percent of f total number of CpG G sites
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Sperm Fig. 2. (A) Methylation status of the paternally methylated H19 and Gtl2 genes and of the maternally methylated Peg1, Snrpn and Peg3 genes in the sperm of F1 offspring of control and of alcohol-fed mice. Results obtained using the pyrosequencing technique. The results are the means ± sem of 8–11 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites. ***p < 0.005 vs. controls. (B) Methylation status of the paternally methylated H19 gene in the sperm of F1 offspring of control and of alcohol-fed mice. Results obtained using the amplicon subcloning and sequencing technique. n = 6, **p < 0.02 vs. controls.
In the sperm of control male offspring the numbers of methylated CpGs were close to the theoretical values of 100% and 0% of the total CpGs in paternally and maternally methylated genes, respectively (Fig. 2A). Alcohol induced a 3% decrease in the number of methylated CpGs of H19. This effect, calculated using the unpaired Student’s t test had a p value <0.005 (Fig. 2A) and, using the nonparametric Mann–Whitney U test, a p value <0.005 (not shown). The effect of alcohol was validated on 6 of the 8–9 offspring by another method, consisting in PCR amplification of the bisulfate-treated DNAs and subsequent amplicon subcloning and sequencing. Alcohol was found, by this other approach, to induce a 5% decrease in the number of methylated CpGs of H19: unpaired Student’s t test: p < 0.02 (Fig. 2B) and Mann–Whitney U test: p < 0.006 (not shown). We analyzed separately the 6 CpGs of the H19 CTCF-binding site 2 PCR-amplicon. As shown in Fig. 3A, CpGs 1–5 belong to the CTCF-binding site 2 and CpG 6 is located downstream CTCF-binding site 2. The 3rd, 4th and 5th CpG methylation patterns were similar in alcohol-administered offspring and control mice. The 1st, 2nd and 6th CpGs exhibited significant methylation percentage losses in alcohol-exposed offspring as compared to the same positions in
Fig. 3. (A) Schematic representation of the H19 differentially methylated domain (DMD) analyzed. Gray circles: CpGs 1–5 of CCCTC-factor-binding site 2 (CTCF2). Gray bar: CTCF2 binding site. Empty circle: CpG 6 downstream CTCF2-binding site 2. GB: GenBank. (B) Methylation status H19 DMD CpGs 1–6 in the sperm of F1 offspring of control and of alcohol-fed mice. The results are the means ± sem of 8–11 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites. **p < 0.02, ***p < 0.005 vs. controls.
control mice (Fig. 3B). A Bonferroni correction for multiple testing was performed and only CpGs 1 and 2 remained significantly different from controls. The possibility that the administration of 0.5 g/kg/day of alcohol to pregnant female mice might also affect spermatogenesis was tested. A sperm count was performed in 8 control mice and in 7 alcohol-administered F1 offspring. As shown in Fig. 4, the mean sperm concentration of the alcohol-fed female offspring was decreased, amounting 76% of that of the controls, in the F1 and back to a normal value in the F2 offspring. The possibility that the demethylation of H19 observed in the sperm of the F1 offspring might affect the methylation pattern of somatic tissues in the F2 offspring was tested. There was no change in the litter size and sex ratio and birth weight in the F2 offspring of the affected F1 males. The H19 gene average amount of methylated CpGs was not changed in any of the F2 offspring tail, liver and skeletal muscle as compared to that of controls (52.1 ± 0.5, 52.1 ± 0.6 and 53.1 ± 0.7, respectively). The only exception was the newborn whole brain. As shown in Fig. 5A, the number of methylated CpGs of the H19 gene, that was not changed in the 8 week-old F1 offspring brain, was decreased by 4% as compared to that of controls in the F2 offspring brain. The 6 CpGs of the H19 CTCFbinding site 2 PCR-amplicon were analyzed separately. The 1st, 2nd, 3rd and 6th CpGs exhibited significant methylation percentage losses in alcohol-exposed offspring as compared to the same positions in control mice. A Bonferroni correction for multiple testing was performed and all the affected CpGs, i.e., the 1st, 2nd, 3rd and 6th remained significantly different from controls (Fig. 5B). The
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*
10
5
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Sperm concentration (mill/ml)
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Fig. 4. Sperm concentration, in millions of spermatozoa per ml of sperm, in F1 and F2 offspring of control and alcohol-fed mice. The results are the means ± sem of 7–8 mice. *p < 0.05.
4. 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 effects on fetal development. In mice exposed to alcohol, by gavage, at doses of 0.6 or 1.7 g/kg, the reported peak Blood Alcohol Concentration (BAC) ranged between 40 and 45 mg/dl and 140–145 mg/dl, respectively [29]. From these data, we estimate that in our experiments, the doses of 0.5 mg/kg should produce a BAC below 40 mg/dl. Our results show that alcohol exposure of the pregnant mice decreases the number of H19 gene methylated CpGs in the sperm of the offspring. It is the first time that, as a consequence of alcohol administration during pregnancy, a change in allele-specific DNA-methylation at an imprinted locus is reported in the offspring, which could possibly have resulted in disruption of imprinting control in the region. This phenomenon could be detected because the methylation pattern of H19 gene was analyzed in various tissues in parallel. To date, an exposure to alcohol of pregnant mice has been shown to induce changes in the methylation pattern of H19 gene in the placenta but not in the whole embryo [17]. Alcohol exposure of the pregnant mother, however, does not affect H19 DNA-methylation in somatic tissues of the offspring. Even in the hippocampus which has been reported to be a target tissue for the deleterious effects of alcohol during pregnancy [9], H19 DNAmethylation was unaltered. This suggests that, at the low doses of alcohol used in this study, the allele-specific methylation control mechanisms of H19 gene is more robust in the somatic than in the germ cells and/or that methylation changes occur during the reprogramming of germ cells. The analysis, in addition to H19, of 1 paternally (Gtl2) and 3 maternally (Peg1, Snrpn and Peg3) imprinted genes shows, interestingly, that of the genes tested and at the alcohol dosage used,
Newborn whole brain
B
Control Alcohol percent of total number of CpG sites
alcohol-induced demethylation pattern of H19 in the F2 offspring brain was essentially similar with that observed in the F1 offspring sperm with the exception of the 3rd CpG which was not affected in the sperm (Fig. 3B).
F2
50
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**
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40 30 20 10
0 1
2
3
4
5
6
CpGs of H19 DMD CTCF2
Newborn whole brain F2 Fig. 5. (A) Methylation status of the paternally methylated H19 gene in the newborn whole brain of F1 and F2 male offspring of control and of alcohol-fed mice. Control: water only. Alcohol: alcohol-fed. The results are the means ± sem of 7–8 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites. ***p < 0.005 vs. controls. (B) Methylation status of H19 DMD CpGs 1–6 in the newborn whole brain of F2 male offspring of control and of alcohol-fed mice. The results are the means ± sem of 7–8 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites. **p < 0.01, ***p < 0.005 vs. controls.
only H19 methylation was affected. This is in sharp contrast with the effects on the offspring of an exposure of the pregnant mother to the endocrine-disrupting chemical vinclozolin. The latter, indeed affected all the imprinted genes tested (H19, Gtl2, Peg1, Snrpn and Peg3) in the sperm and 1 imprinted gene (Peg3) in the somatic cells [26]. The results of our study, showing the sensitivity to alcohol of H19 imprinting, are in agreement with the finding that chronic alcohol consumption in men is correlated with a demethylation of H19 and IG genes in the sperm [16] and that an exposure to alcohol of pregnant mice induced changes in the methylation pattern of the H19 gene in the placenta [17]. However, in the latter study no other imprinted genes were tested and therefore our study is the first to identify H19 as a specific target for the effects of alcohol exposure during pregnancy.
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The DMD of the H19 gene analyzed by Haycock and Ramsay [17] is a putative imprinting control region (ICR), that encompasses 4 CCCTC-factor (CTCF) DNA-binding sites that bind their ligand preferentially under their unmethylated form. The authors analyzed the first 2 CTCF DNA-binding sites of the H19 gene DMD, CTCF binding sites 1 and 2. In the placenta of alcohol treated pregnant mouse embryos they identified a demethylation on the paternal allele of the H19 CTCF-binding site 1 that was partly correlated with a decrease in placental weight. Our data show a demethylation of the H19 CTCF-binding site 2 in the sperm of alcohol-exposed offspring. The observation that, in this site, only a fraction of the CpGs exhibited an abnormal methylation pattern is in agreement with Ouko et al. [16] who showed that alcohol use may affect specific CpG positions in CTCF-binding sites of H19 and IG genes in human male gametes. The mechanism by which a single site is preferentially demethylated remains unclear. It is known that the methylation of the CTCF binding sites of the H19 gene paternal allele prevents the binding of the CTCFs thus increasing Igf2 promoter–enhancer interaction and expression of paternal Igf2 [30]. Igf2 is the precursor peptide of the mitogen factor insulin-like growth factor 2 that plays an important role in fetal growth [31]. Nevertheless, the existence of a link between the demethylation of the H19 CTCF-binding site 2 observed in this study and the spermatogenic defect remains speculative. First, it is not known if the magnitude of the demethylation is sufficient to modify the imprinting status of H19 and Igf2 expression and if the latter is involved in the control of spermatogenesis. It would be interesting to test the effect of an alcohol treatment like that of this study on Igf2 expression. Second, the possibility that the spermatogenic phenotype is due to epigenetic/expression changes at loci elsewhere in the genome cannot be excluded. Third, the loss of DNA methylation might be a result and not a cause of reduced spermatogenesis. The observation of a H19 demethylation in the F2 offspring whole brain suggests that the H19 demethylation observed in the F1 offspring sperm may have been inherited in the F2 offspring. The already small effect of alcohol on the H19 gene in the F1 offspring sperm (3%) should become even smaller upon transmission to the next generation and therefore undetectable in most of the somatic tissues tested. Another reason why the effect of alcohol was not observed in tail, muscle, liver and hippocampus of the F2 offspring may be that the demethylation was erased/corrected during the epigenetic reprogramming by chance fluctuation in the allelespecific DNA methylation. The demethylation of H19 observed in the brain of the F2 offspring and the good concordance of the CpG demethylation patterns of the F1 offspring sperm and of the F2 offspring brain suggest that the methylation change in the sperm of the F1 offspring might have functional consequences. The effects of alcohol exposure on both the number of H19 gene methylated CpGs in the sperm and on the sperm count were not observed in the F2 offspring. The only effect observed consistent with transgenerational transmission was a demethylation of H19 in the F2 offspring whole brain. Our study supports the idea that imprinting defects can be involved in the deleterious effects of alcohol exposure during pregnancy. The findings of this study warrant additional studies to evaluate their potential biological relevance. 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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A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome. Proc Natl Acad Sci USA 1999;96:8064–9. [24] Reinhart B, Paoloni-Giacobino A, Chaillet JR. Specific differentially methylated domain sequences direct the maintenance of methylation at imprinted genes. Mol Cell Biol 2006;26:8347–56. [25] Chaillet JR, Bader DS, Leder P. Regulation of genomic imprinting by gametic and embryonic processes. Genes Dev 1995;9:1177–87. [26] Stouder C, Deutsch S, Paoloni-Giacobino A. Superovulation in mice alters the methylation pattern of imprinted genes in the sperm of the offspring. Reprod Toxicol 2009;28:536–41. [27] Datta S, Turner D, Singh R, Ruest LB, Pierce WMJ, Knudsen TB. Fetal alcohol syndrome (FAS) in C57BL/6 mice detected through proteomics screening of the amniotic fluid. Birth Defects Res A Clin Mol Teratol 2008;82:177–86. [28] Amini SA, Dunkley PR, Murdoch RN. Teratogenic effects of ethanol in the Quackenbush special mouse. Drug Alcohol Depend 1996;41:61–9. [29] Fish EW, Riday TT, McGuigan MM, Faccidomo S, Hodge CW, Malanga CJ. Alcohol, cocaine, and brain stimulation-reward in C57Bl6/J and DBA2/J mice. Alcohol Clin Exp Res 2010;34:81–9. [30] Kurukuti S, Tiwari VK, Tavoosidana G, Pugacheva E, Murrell A, Zhao Z, et al. CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2. Proc Natl Acad Sci USA 2006;103:10684–9. [31] Tycko B, Morison IM. Physiological functions of imprinted genes. J Cell Physiol 2002;192:245–58.
Contents lists available at ScienceDirect
Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Prenatal exposure to ethanol: A specific effect on the H19 gene in sperm Christelle Stouder a , Emmanuel Somm b , Ariane Paoloni-Giacobino a,c,∗ a
Department of Genetic and Laboratory Medicine, Geneva University Hospital, 1211 Geneva 14, Switzerland Department of Pediatrics, Geneva University Hospital, 1211 Geneva 14, Switzerland c Swiss Center for Applied Human Toxicology, University of Geneva Medical School, 1211 Geneva 4, Switzerland b
a r t i c l e
i n f o
Article history: Received 31 March 2010 Received in revised form 17 January 2011 Accepted 28 February 2011 Available online 5 March 2011 Keywords: Ethanol Epigenetics Methylation Fertility Sperm
a b s t r a c t 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. © 2011 Elsevier Inc. All rights reserved.
1. 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].
∗ Corresponding author at: Department of Genetic and Laboratory Medicine, CMU, 1 Michel-Servet, 1211 Geneva 4, Switzerland. Tel.: +41 22 379 59 47; fax: +41 22 379 52 28. E-mail address: [email protected] (A. Paoloni-Giacobino). 0890-6238/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2011.02.009
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
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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. 2. Materials and methods 2.1. 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 month old female mice were naturally mated with male mice of the same age. Finding of a copulation plug the next morning signified coitus and gestation day 1 (GD1). Plug-positive females were separated into 2 groups. One group, consisting in 8 pregnant dams, was treated by oral administration of alcohol at a dose of 0.5 g/kg/day, and another group of 11 pregnant dams was treated by an equal volume of water between GD10 and 18, to encompass major events during organogenesis and fetal development [6]. One offspring from each dam was terminated at the postnatal age of 8 weeks for DNA analysis. In some cases another offspring was terminated at 8 weeks for sperm count. One pup from each litter was sacrificed as a newborn for brain analysis. In order to study possible transgenerational effects of ethanol treatment, 7–8 F1 males were bred to 7–8 FVB/N females. F2 males were then analyzed at the age of 8 weeks. Only one offspring from each dam was analyzed. Animal protocols used in these studies were approved by the Commission d’ Ethique de l’Expérimentation Animale of the University of Geneva Medical School and by the Geneva Veterinarian Office. 2.2. Sperm collection and count The vas deferens and epididymis were dissected free of connective tissue, placed into a Petri dish, delicately scored with a razor blade in a droplet of Phosphate Buffer Saline (PBS), and left for 10 min at 37 ◦ C to allow sperm to swim out into the medium. The medium with sperm was then carefully transferred to another tube and centrifuged at 6000 × g for 3 min to pellet the sperm. In some experiments an aliquot of the supernatant’s sperm was used for sperm counting, using a hemocytometer. The corresponding sperm concentration was calculated using the formula: cells per ml = the average count per square × dilution factor × 104 (counts 10 squares). 2.3. Tissue collection Fragments of 5 mm × 5 mm size of tail, liver and tibialis anterior muscle of the 8 week-old offspring were collected, cut into small pieces in a Petri dish and transferred into DNA-extraction buffer. Brain hemispheres from these same animals were collected and hippocampus from both hemispheres were dissected and pooled into DNA-extraction buffer as a single tissue. Whole brains from newborn pups terminated immediately after birth were also collected, cut into small pieces in a Petri dish and transferred into DNA-extraction buffer. 2.4. DNA isolation Sperm DNA was extracted fusing the QIAampDNA microkit (Qiagen, CA, USA). Total genomic DNA was extracted from tails, livers, tibialis anterior muscle, hippocampus and whole brains by incubation for 3 h at 55 ◦ C in DNA-extraction buffer (50 mM Tris–HCl, pH 8, 100 mM EDTA, 0.5%
SDS) followed by phenol–chloroform–isoamyl alcohol extractions and ethanol precipitation. 2.5. Bisulfite treatment Using the EZ Methylation Gold-Kit (Zymo Research, Orange, CA, USA) the extracted DNA was treated with sodium bisulfite in order to convert unmethylated cytosine residues to uracil. The converted DNA was eluted in 10 l of TE buffer (10 mM Tris–HCl, 0.1 mM EDTA, pH 7.5). Two microlitres of the post bisulfite-treated DNA were used for subsequent PCR amplification. 2.6. PCR amplification of bisulfite-treated DNAs for subsequent pyrosequencing The PCR amplifications aimed at pyrosequencing were performed starting from 100 to 140 ng of bisulfite-treated sperm, tail, liver and skeletal muscle DNA. The PCR conditions were the same for all the genes tested, i.e., 94 ◦ C for 15 min, followed by 40 cycles of 94 ◦ C, 30 s, 55 ◦ C, 30 s, 72 ◦ C, 30 s, and by a 72 ◦ C 10 min final extension step. The characteristics of the amplicons and of the oligonucleotides chosen have been described in a previous publication [26]. A diagram showing the locations of the DMDs, within the corresponding genes, and of the DNA fragments of the 5 genes amplified by PCR has been presented in a previous study [26]. For each imprinted gene between 5 and 23 CpGs were present in the PCR amplicon analyzed. All reactions were performed with a PCR mixtures (total volume 25 l) containing oligonucleotides at 0.5 mM concentration and 12.5 l of HotStarTaq Master Mix (Qiagen, CA, USA). The biotinylated PCR products were purified using streptavidin-sepharose beads (Amersham) and sequenced using the PSQ 96 Gold reagent kit (Biotage AB, Uppsala, Sweden) with the following primers: H19: 5 -GTGTAAAGATTAGGGTTGT-3 , Gtl2: 5 -GTTATGGATTGGTGTTAAG-3 , Peg1: 5 -TCAATATCAACTAAATAATC-3 , Snrpn: 5 -GAATTGGAGTTGTGTGG-3 , Peg3: 5 -AATTGATAAGGTTGTAGATT-3 . The degree of methylation at each CpG site was determined using Pyro Q-CpG Software (Biotage AB, Uppsala, Sweden). All samples were analyzed in duplicate. The average methylation value of each mouse was the average of the 2 duplicate values. The H19 amplicon, which was analyzed further in this study, contains only one CTCF binding site (i.e., site 2) (Fig. 4A). 2.7. PCR amplification of bisulfate-treated DNAs for subsequent amplicon subcloning and sequencing The PCR amplifications of individual clones were performed starting from 100 to 140 ng of bisulfite-treated sperm DNA. H19: amplification of a 182 bp fragment, encompassing 8 CpGs, was performed with and 5 oligonucleotides 5 -GGGGGTAGGATATATGTAAAAAAAAGG-3 AAAAAAACTCAATCAATTACAATCC-3 . PCR cycling was performed with a profile consisting of 94 ◦ C for 15 min, followed by 40 cycles of 94 ◦ C, 30 s, 55 ◦ C, 30 s, 72 ◦ C, 30 s, and by a 72 ◦ C 10 min final extension step. All reactions were performed with a PCR mixture (total volume 25 l) containing oligonucleotides at 0.5 mM concentrations and 12.5 l of HotStarTaq Master Mix (Qiagen, CA, USA). The amplified fragments were then gel-purified using the QIAEX II Gel Extraction kit (Qiagen, CA, USA). Gel-purified fragments were subcloned into pCR 2.1-TOPO by TOPO-TA cloning (Invitrogen, Carlsbad, CA) and individual clones were sequenced by an ABI 3130 XL sequencer using M13-Reverse oligonucleotide. Each result was obtained by sequencing 12 clonal colonies per mouse. The average methylation value of each mouse was the average of the 12 clone values. 2.8. Statistical analysis Significances were evaluated using the unpaired Student’s t test and set at p < 0.05. The non-parametric Mann–Whitney U test was also used.
3. 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 nonmethylated) CpGs. In the tail, which represents a heterogeneous sample of somatic cells, the amount of methylated CpGs in control 8 week-old male offspring was close to 50% of the total methylated + non methylated CpGs in all the imprinted genes tested. This value is in total agreement with the theoretical value expected for imprinted genes in which DNA methylation/unmethylation is present on one of the
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Newborn whole brain Fig. 1. Methylation status of the paternally methylated H19 and Gtl2 genes and of the maternally methylated Peg1, Snrpn and Peg3 genes A: in the tail, B: in the liver, C: in the skeletal muscle, D: in the hippocampus and E: in the newborn whole brain of F1 male offspring of control and of alcohol-fed mice. Control: water only. Alcohol: alcohol-fed. The results are the means ± sem of 8–10 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites.
two parental alleles only. No difference was observed in the methylation status of the 5 imprinted genes tested between control and alcohol-administered offspring (Fig. 1A). The methylation patterns of our 5 target genes were also analyzed in 8 week-old male offspring liver, skeletal muscle and hippocampus and in newborn
whole brain. As observed in the tail, the amount of methylated CpGs in the control offspring was close to the theoretical value of 50% of the total CpGs in all the imprinted genes tested. Again, no difference was observed between control and alcohol-administered offspring (Fig. 1B–E).
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Sperm Fig. 2. (A) Methylation status of the paternally methylated H19 and Gtl2 genes and of the maternally methylated Peg1, Snrpn and Peg3 genes in the sperm of F1 offspring of control and of alcohol-fed mice. Results obtained using the pyrosequencing technique. The results are the means ± sem of 8–11 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites. ***p < 0.005 vs. controls. (B) Methylation status of the paternally methylated H19 gene in the sperm of F1 offspring of control and of alcohol-fed mice. Results obtained using the amplicon subcloning and sequencing technique. n = 6, **p < 0.02 vs. controls.
In the sperm of control male offspring the numbers of methylated CpGs were close to the theoretical values of 100% and 0% of the total CpGs in paternally and maternally methylated genes, respectively (Fig. 2A). Alcohol induced a 3% decrease in the number of methylated CpGs of H19. This effect, calculated using the unpaired Student’s t test had a p value <0.005 (Fig. 2A) and, using the nonparametric Mann–Whitney U test, a p value <0.005 (not shown). The effect of alcohol was validated on 6 of the 8–9 offspring by another method, consisting in PCR amplification of the bisulfate-treated DNAs and subsequent amplicon subcloning and sequencing. Alcohol was found, by this other approach, to induce a 5% decrease in the number of methylated CpGs of H19: unpaired Student’s t test: p < 0.02 (Fig. 2B) and Mann–Whitney U test: p < 0.006 (not shown). We analyzed separately the 6 CpGs of the H19 CTCF-binding site 2 PCR-amplicon. As shown in Fig. 3A, CpGs 1–5 belong to the CTCF-binding site 2 and CpG 6 is located downstream CTCF-binding site 2. The 3rd, 4th and 5th CpG methylation patterns were similar in alcohol-administered offspring and control mice. The 1st, 2nd and 6th CpGs exhibited significant methylation percentage losses in alcohol-exposed offspring as compared to the same positions in
Fig. 3. (A) Schematic representation of the H19 differentially methylated domain (DMD) analyzed. Gray circles: CpGs 1–5 of CCCTC-factor-binding site 2 (CTCF2). Gray bar: CTCF2 binding site. Empty circle: CpG 6 downstream CTCF2-binding site 2. GB: GenBank. (B) Methylation status H19 DMD CpGs 1–6 in the sperm of F1 offspring of control and of alcohol-fed mice. The results are the means ± sem of 8–11 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites. **p < 0.02, ***p < 0.005 vs. controls.
control mice (Fig. 3B). A Bonferroni correction for multiple testing was performed and only CpGs 1 and 2 remained significantly different from controls. The possibility that the administration of 0.5 g/kg/day of alcohol to pregnant female mice might also affect spermatogenesis was tested. A sperm count was performed in 8 control mice and in 7 alcohol-administered F1 offspring. As shown in Fig. 4, the mean sperm concentration of the alcohol-fed female offspring was decreased, amounting 76% of that of the controls, in the F1 and back to a normal value in the F2 offspring. The possibility that the demethylation of H19 observed in the sperm of the F1 offspring might affect the methylation pattern of somatic tissues in the F2 offspring was tested. There was no change in the litter size and sex ratio and birth weight in the F2 offspring of the affected F1 males. The H19 gene average amount of methylated CpGs was not changed in any of the F2 offspring tail, liver and skeletal muscle as compared to that of controls (52.1 ± 0.5, 52.1 ± 0.6 and 53.1 ± 0.7, respectively). The only exception was the newborn whole brain. As shown in Fig. 5A, the number of methylated CpGs of the H19 gene, that was not changed in the 8 week-old F1 offspring brain, was decreased by 4% as compared to that of controls in the F2 offspring brain. The 6 CpGs of the H19 CTCFbinding site 2 PCR-amplicon were analyzed separately. The 1st, 2nd, 3rd and 6th CpGs exhibited significant methylation percentage losses in alcohol-exposed offspring as compared to the same positions in control mice. A Bonferroni correction for multiple testing was performed and all the affected CpGs, i.e., the 1st, 2nd, 3rd and 6th remained significantly different from controls (Fig. 5B). The
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Fig. 4. Sperm concentration, in millions of spermatozoa per ml of sperm, in F1 and F2 offspring of control and alcohol-fed mice. The results are the means ± sem of 7–8 mice. *p < 0.05.
4. 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 effects on fetal development. In mice exposed to alcohol, by gavage, at doses of 0.6 or 1.7 g/kg, the reported peak Blood Alcohol Concentration (BAC) ranged between 40 and 45 mg/dl and 140–145 mg/dl, respectively [29]. From these data, we estimate that in our experiments, the doses of 0.5 mg/kg should produce a BAC below 40 mg/dl. Our results show that alcohol exposure of the pregnant mice decreases the number of H19 gene methylated CpGs in the sperm of the offspring. It is the first time that, as a consequence of alcohol administration during pregnancy, a change in allele-specific DNA-methylation at an imprinted locus is reported in the offspring, which could possibly have resulted in disruption of imprinting control in the region. This phenomenon could be detected because the methylation pattern of H19 gene was analyzed in various tissues in parallel. To date, an exposure to alcohol of pregnant mice has been shown to induce changes in the methylation pattern of H19 gene in the placenta but not in the whole embryo [17]. Alcohol exposure of the pregnant mother, however, does not affect H19 DNA-methylation in somatic tissues of the offspring. Even in the hippocampus which has been reported to be a target tissue for the deleterious effects of alcohol during pregnancy [9], H19 DNAmethylation was unaltered. This suggests that, at the low doses of alcohol used in this study, the allele-specific methylation control mechanisms of H19 gene is more robust in the somatic than in the germ cells and/or that methylation changes occur during the reprogramming of germ cells. The analysis, in addition to H19, of 1 paternally (Gtl2) and 3 maternally (Peg1, Snrpn and Peg3) imprinted genes shows, interestingly, that of the genes tested and at the alcohol dosage used,
Newborn whole brain
B
Control Alcohol percent of total number of CpG sites
alcohol-induced demethylation pattern of H19 in the F2 offspring brain was essentially similar with that observed in the F1 offspring sperm with the exception of the 3rd CpG which was not affected in the sperm (Fig. 3B).
F2
50
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0 1
2
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CpGs of H19 DMD CTCF2
Newborn whole brain F2 Fig. 5. (A) Methylation status of the paternally methylated H19 gene in the newborn whole brain of F1 and F2 male offspring of control and of alcohol-fed mice. Control: water only. Alcohol: alcohol-fed. The results are the means ± sem of 7–8 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites. ***p < 0.005 vs. controls. (B) Methylation status of H19 DMD CpGs 1–6 in the newborn whole brain of F2 male offspring of control and of alcohol-fed mice. The results are the means ± sem of 7–8 mice. They represent the number of methylated CpG sites and are expressed in percent of the total number of CpG sites. **p < 0.01, ***p < 0.005 vs. controls.
only H19 methylation was affected. This is in sharp contrast with the effects on the offspring of an exposure of the pregnant mother to the endocrine-disrupting chemical vinclozolin. The latter, indeed affected all the imprinted genes tested (H19, Gtl2, Peg1, Snrpn and Peg3) in the sperm and 1 imprinted gene (Peg3) in the somatic cells [26]. The results of our study, showing the sensitivity to alcohol of H19 imprinting, are in agreement with the finding that chronic alcohol consumption in men is correlated with a demethylation of H19 and IG genes in the sperm [16] and that an exposure to alcohol of pregnant mice induced changes in the methylation pattern of the H19 gene in the placenta [17]. However, in the latter study no other imprinted genes were tested and therefore our study is the first to identify H19 as a specific target for the effects of alcohol exposure during pregnancy.
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The DMD of the H19 gene analyzed by Haycock and Ramsay [17] is a putative imprinting control region (ICR), that encompasses 4 CCCTC-factor (CTCF) DNA-binding sites that bind their ligand preferentially under their unmethylated form. The authors analyzed the first 2 CTCF DNA-binding sites of the H19 gene DMD, CTCF binding sites 1 and 2. In the placenta of alcohol treated pregnant mouse embryos they identified a demethylation on the paternal allele of the H19 CTCF-binding site 1 that was partly correlated with a decrease in placental weight. Our data show a demethylation of the H19 CTCF-binding site 2 in the sperm of alcohol-exposed offspring. The observation that, in this site, only a fraction of the CpGs exhibited an abnormal methylation pattern is in agreement with Ouko et al. [16] who showed that alcohol use may affect specific CpG positions in CTCF-binding sites of H19 and IG genes in human male gametes. The mechanism by which a single site is preferentially demethylated remains unclear. It is known that the methylation of the CTCF binding sites of the H19 gene paternal allele prevents the binding of the CTCFs thus increasing Igf2 promoter–enhancer interaction and expression of paternal Igf2 [30]. Igf2 is the precursor peptide of the mitogen factor insulin-like growth factor 2 that plays an important role in fetal growth [31]. Nevertheless, the existence of a link between the demethylation of the H19 CTCF-binding site 2 observed in this study and the spermatogenic defect remains speculative. First, it is not known if the magnitude of the demethylation is sufficient to modify the imprinting status of H19 and Igf2 expression and if the latter is involved in the control of spermatogenesis. It would be interesting to test the effect of an alcohol treatment like that of this study on Igf2 expression. Second, the possibility that the spermatogenic phenotype is due to epigenetic/expression changes at loci elsewhere in the genome cannot be excluded. Third, the loss of DNA methylation might be a result and not a cause of reduced spermatogenesis. The observation of a H19 demethylation in the F2 offspring whole brain suggests that the H19 demethylation observed in the F1 offspring sperm may have been inherited in the F2 offspring. The already small effect of alcohol on the H19 gene in the F1 offspring sperm (3%) should become even smaller upon transmission to the next generation and therefore undetectable in most of the somatic tissues tested. Another reason why the effect of alcohol was not observed in tail, muscle, liver and hippocampus of the F2 offspring may be that the demethylation was erased/corrected during the epigenetic reprogramming by chance fluctuation in the allelespecific DNA methylation. The demethylation of H19 observed in the brain of the F2 offspring and the good concordance of the CpG demethylation patterns of the F1 offspring sperm and of the F2 offspring brain suggest that the methylation change in the sperm of the F1 offspring might have functional consequences. The effects of alcohol exposure on both the number of H19 gene methylated CpGs in the sperm and on the sperm count were not observed in the F2 offspring. The only effect observed consistent with transgenerational transmission was a demethylation of H19 in the F2 offspring whole brain. Our study supports the idea that imprinting defects can be involved in the deleterious effects of alcohol exposure during pregnancy. The findings of this study warrant additional studies to evaluate their potential biological relevance. 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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