Natrium Fluoride Influences Methylation Modifications and Induces

Aug 7, 2014 - ... and nutrient compositions in various culture media used during in vitro manipulations of early mouse embryos can change imprinted ge...
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Natrium Fluoride Influences Methylation Modifications and Induces Apoptosis in Mouse Early Embryos Mingzhe Fu, Xinying Wu, Jie He, Yong Zhang, and Song Hua* College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province The People’s Republic of China 712100 S Supporting Information *

ABSTRACT: This study epigenetically examined the effect of fluoride on early embryos of Kunming mice administered with 0, 20 (low), 60 (medium), and 120 mg/L (high) sodium fluoride (NaF). The results showed that NaF repressed oocyte maturation, fertilization and blastocyst formation in all NaF-treated groups. Meanwhile, TUNEL assay showed that embryo apoptosis was induced dramatically in blastocyst stage at either low or medium doses, and in 8-cell stage at high dose, compared to the control, suggesting a dose-dependent effect. Furthermore, the immunostaining displayed global increases of DNA methylation, H3K9m2 and H3K4m2 with increasing dose, which were consistent with gene expression results, exhibiting general increases of DNMT1, DNMT3a, G9a, LSD1, and MLL1 and a reduction of JHDM2a in transcription and protein levels. More closely, the differential methylation domain in parentally imprinted gene H19 showed low methylation, while materanlly imprinted gene IGF2 showed high methylaiton in NaF-treated groups compared to the control group, which corresponded with high expression of H19 and low expression of IGF2 confirmed by qPCR. Collectively, we demonstrated that fluoride epigenetically impaired mouse oocyte maturation and embryonic development, supplying a better knowledge of fluoride in toxicology and a deeper evaluation of its potential influence in physiological and clinical implications.

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As a potential environmental pollutant, fluoride comprises 0.065% of the Earth’s uppermost crust, and its concentration in superficial aquifer groundwater is 0.05−10 mg/L. Although at low dose, it is well documented that the concentration of sodium fluoride (NaF) between 0.8 and 1.0 mg/L has beneficial effect on tooth decay prevention (The national standard of the people’s Republic of China, GB5749-85). However, given the fact that since for many years it has been widely used in many industrial processes, it is considered a widespread industrial pollutant, which could result in its accumulation in the environment.8 In the previous studies, indeed, acute or chronic exposure to high fluoride dose caused great damage to our health.9−11 Also, a number of epidemiological investigations have demonstrated an apparent connection between excessive fluoride exposure to male infertility and low birth rates.12 Animal experimental studies further found a negative impact of fluoride ingestion on spermatogenesis13 and sperm fertilizing ability.14,15 Recently, a study reported that NaF may interact directly with the embryo to disrupt the maintenance of normal gene imprinting after male mice were exposed to NaF.16 Nevertheless, the study of its effect on the other epigenetic modifications during early embryo development is rare. As oral

INTRODUCTION

Dynamic epigenetical reprogramming is initiated from the fusion of oocyte and sperm to the formation of blastocyst in mammals, generally involving covalent modifications on both DNA and core histones without alterations in the DNA sequence.1 Among them, DNA methylation and histone H3 modifications have been well-studied, showing strong links to gene expression either in a separated manner or in a coordinated way. The former(MeC), regarded as a relatively stable mark and catalyzed by DNA (cytosine-5)-methyltransferases (DNMTs), always appears in the differential methylation domain (DMD) of 5′ region of genes, which is closely associated with gene expression activity.2 The representative examples are the two well-known imprinted genes, IGF2 and H19, in which the well-defined DMD regions play decisive roles in controling gene expressions.3,4 Disruption of DNA methylation in DMD regions can result in biallelic expression and abnormal embryo development.5 The latter (histone H3 modifications), including repressive mark, for example, H3K9m2 and active marks, for example, H3K9ac and H3K4m2, could have reciprocal effects on gene expression.6 However, these epigenetic marks are very sensitive to environmental factors, suggesting that animal life activities, food composition and bioactive components can change DNA methylation patterns and histone modifications, either globally or locally.7 © 2014 American Chemical Society

Received: Revised: Accepted: Published: 10398

January 22, 2014 August 6, 2014 August 7, 2014 August 7, 2014 dx.doi.org/10.1021/es503026e | Environ. Sci. Technol. 2014, 48, 10398−10405

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ingestion of NaF in water is usually the major source of fluoride toxicosis, in the present study, the toxicity of NaF was evaluated epigenetically by treating female mice with different concentrations of NaF in water based on dectecions of changes in global DNA methylaiton, H3K9 acetylaiton and dimethylation, H3K4 dimethylation as well as local DNA methylation in differential methylation domains (DMD) of IGF2 and H19.

instructions. Briefly, embryos collected at 96 h post IVF were fixed with 4% paraformaldehyde, and permeabilized in 0.5% Triton X-100. Samples in treatment group were placed in 5 μL TdT+45 μL FITC-labeled dUTP mixture; while embryos in negative control group were placed in 50 μL FITC-labeled dUTP. Next, treatment group samples were supplemented with 50 μL TUNEL reaction mixture, negative control samples were just supplemented with 50 μL FITC-labeled dUTP, and they were covered with coverslip and incubated for 60 min at 37 °C in a humidified atmosphere in the dark. Following three washes, the nuclei were stained with propidium iodide (PI, 5 mg/mL). Finally, embryos were mounted on slides and analyzed by fluorescence microscopy (Nikon). Only blastomere nuclei that showed fragmented or condensed morphology by PI staining and positive for TUNEL staining were considered apoptotic. Immunofluorescence Staining. Immunofluorescence staining of genome-wide detection (on 5-methylcytosine), H3K4m2, H3K9m2, and H3K9ac was performed according to the report.20 Briefly, embryos were fixed at 96 h post culture in Immunol Staining Fix Solution (Beyotime, Jiangsu, China) for 1 h, and then they were permeabilized with 0.2% Triton X-100 in PBS-PVA for 30 min. After three washes, they were blocked in the Immunol Staining Blocking Solution (Beyotime) for 1 h at room temperature and then incubated with the primary antibody (rabbit monoclonal anti-5-methylcytosine/antiH3K9ac/anti-H3K4m2/H3K9m2) diluted (1:500) in blocking solution (2% BSA in PBS) overnight at 4 °C. After washing for three times, they were incubated with the Alexa Fluor 488labeled secondary antibody (goat antirabbit IgG) diluted (1:500) in blocking solution for 1 h at 37 °C, again followed by washing for three times. At last nuclear staining was done with 10 μg/mL DAPI for 5 min, and then they were mounted on microscope slides using a drop of mounting medium (PBS) and imaged under a Zeiss LSM 510 Axiovert 100 M confocal microscope equipped with a Plan-Apochromat ×63/1.4 oil DIC objective. All the images were converted to grayscales and then all the nuclei areas were outlined for measurement. The intensity of 5-methylcytosine, H3K4m2, H3K9m2, and H3K9ac (green fluorescence) was analyzed using the microcomputer imaging device MCID 7.0 (Imaging Research, St. Catherine’s, Ontario, Canada) software and validated by a DAPI signal (blue fluorescence) based on the reported method.21 Images were converted to grayscale and inverted, and average cytoplasmic intensity was measured for normalization to background. After calibration of optical density, all individual nuclei of embryos were outlined, and integrated optical density (IOD) and area were measured. The average normalized fluorescence intensity for a single embryo was represented by “sum IOD/sum area”. Genome-wide DNA methylation, H3K4m2, H3K9m2, or H3K9ac fluorescent intensities were divided by total fluorescence intensities stained by DAPI to calculate normalized genome-wide DNA methylation, H3K4m2, H3K9m2, or H3K9ac levels, respectively. All images were obtained using the same exposure times, adjustments and settings of the microscope to maximally minimize the difference among embryos. Furthermore, all parameters were kept same to perform the quantification of the intensity of immunofluorescence using Image-Pro plus software. The experiments were replicated three times, and 6−8 embryos per group were processed in each replication. Expression Analysis of Developmentally Important Genes. The total RNA from embryos at 96 h post culture was extracted using the High Pure Viral RNA kit (Roche,

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MATERIALS AND METHODS Main Reagents. Sodium fluoride (NaF, Sigma Chemical Company), reproductive hormones PMSG and hCG (Ningbo Sansheng Pharmaceutical Co., Ltd., China), anti-5-methylcytosine/anti-H3K9ac/anti-H3K4m2/H3K9m2 (Abcam, Cambridge UK), Apoptosis detection kit (Roch, Applied Science, Germany). Animal Treatment. One hundred and 60 adult female (4 weeks old, average weight 22 ± 0.8 g) and 20 male (4 weeks old, average weight 25 ± 0.5 g) Kunming mice were purchased from the experimental animal center of the fourth military medical university. The female mice were allotted randomly into four groups: a control group, which was provided deionized water, and other three experimental groups supplied with 20, 60, and 120 mg/L NaF in deionized water, respectively. The values were according to the toxicity dose of NaF dissolved in deionized water tested in mice.15,17,18 They were kept for 30 days (reached sexual maturity), and had free access to food (the total content of NaF is 0.67 ± 0.08 mg/kg, fluoride-ion selective electrode method) and water under optimum temperature (22−25 °C), 12 h light every day, ventilation, and hygienic conditions. The present study was carried out in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Research Council, and all experimental protocols associated with animals were approved by the Animal Care and Use Committee of Northwest A&F University (Approval number was NWAFSX-20130121). Oocyte Maturation and in Vitro Fertilization (IVF). Female mice were superovulated by intraperitoneal injection with 10 IU of pregnant mare serum gonadotrophin (PMSG), after 46 h of hCG injection, the females were deeply anesthetized with inhaled isofluorane (about 2.5%), and then killed by cervical dislocation and ovarian was isolated under sterile conditions. Under the stereomicroscope the cumulusenclosed oocyte complexes (COC) with a homogeneous cytoplasm and more than three compact layers of cumulus cells were aspirated from the follicles; COC was selected for in vitro maturation. Oocytes maturation was carried out by placing the COC in sterile Petri dishes each containing 50−60 COC and incubated in an incubator at 37 °C with 5% CO2 in humidified air for 18 h. Male mice were deeply anesthetized using isofluorane and killed by cervical dislocation, then sperm were obtained by flushing of both vas deferens. A concentration of about 2 × 105 sperm/ml was capacitated in potassium simplex optimized medium (KSOM) with 4 mg/mL BSA for 1 h, and were coincubated with COC at 37 °C in humidified CO2 incubator for 4 h. After this, presumptive zygotes were washed with fresh KSOM and finally incubated in KSOM droplets (50 μL) under oil.19 Fertilization was determined at the two-cell stage to assess cleavage and embryos at 96 h after IVF were used to further experiments. TUNEL Assay. Embryo cells apoptosis was analyzed using in situ cell death detection kit (Roch) according to the 10399

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Figure 1. Regions analyzed within the H19 and IGF2 upstream sequence. The coordinates and sequences of the amplified regions and positions in relation to their transcription start site were shown. CpG dinucleotides are italic and underlined. DMD referred to differential methylation region, the grids on the DMD upstream IGF2 were the sequence tested for IGF2 gene; the grids (Region 1 and Region 2) on the DMD upstream H19 were the sequences tested for H19 gene.

-2289bp) sense 5′- GATTT GTGTT GTTTG TAGTT TTGTT T-3′, antisense 5′- CCTCT CCAAC TCCTT CTATT TTACT AC-3′; H19-Region 2(−4097bp ∼ -3838bp) sense 5′- GTGGT AAGAT GTGTG TATTT TTGGA-3′, antisense 5′- ATTAT ATCCA CCCCA TAACC CTTAT A −3′; IGF(−159bp ∼ +86bp) sense 5′- TATTT TGTGT GTATT TTGTA AATAA −3′, antisense 5′- AAACC TCTAA AAAAC CACTA CTAAC-3′. Three independent PCR reactions were performed, and the products were purified using QIAquick Gel Extraction kit (Qiagen). All purified products were mixed together, and cloned into a pMD18-T vector (TaKaRa, China), followed by confirmation using PCR. Finally, one hundred colonies for each sample were sequenced. Bisulfite sequencing data and C/T conversion rates were analyzed by BIQ Analyzer software.24 Statistical Analysis. The DNA methylation levels of DMD and apoptotic rate were expressed as a percentage; all other values were expressed as means ± SEM and were statistically analyzed using the SPSS software 10.0. The mathematical model Y = μ + τi + βj + εij was used to analyze the original data about oocytes and embryos. One way ANOVA was used to compare among groups, and values of P < 0.05 were considered as significant difference. In addition, significance analysis of gene expression was compared with false discovery rate (FDR) controlled at 0.05.

Germany), and cDNA was prepared using a High Fidelity PrimeScript RT-PCR Kit (Takara Biotech, Dalian, China) according to the manufacturer’s instructions. A RT-PCR reaction mixture without reverse transcriptase was prepared as a negative control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was served as a reference gene. All the primers were designed using Primer Premier 6.0 according to sequences published in the National Center for Biotechnology Information database (shown in Supporting Information Table SI-1) Each reaction was run in a 25 μL reaction mixtures with 12.5 μL SYBR Premix Ex Taq (Takara Biotech, China), 0.5 μL sense primer, 0.5 μL antisense primer, 1.5 μL distilled water, and 10 μL of cDNA. Reaction conditions were as follows: 94 °C for 40 s, followed by 45 cycles at 94 °C for 30 s, 45 °C for 30 s, and 72 °C for 40 s. The specificity of amplification during PCR was monitored by evaluating the melting curve and detecting the PCR products of quantity PCR by agarose gel electrophoresis. Transcript levels presented for each gene was normalized to GAPDH levels, and the 2−ΔΔCt method was used to calculated expression levels as reported by Livak and Schmittgen.22 DNA Methylation Analysis by Sodium Bisulfite Genomic Sequencing. Methylated CpG sites on DMD of the imprinted gene IGF2 and H19 (Figure 1) were analyzed by sodium bisulfite genomic sequencing.23 Briefly, 15 embryos were randomly collected from each group and genomic DNA was extracted, using Wizard Genomic DNA purification kit (Promega) and quantified using NanoDropTM ND-1000 spectrophotometer (Thermo Finnigan, America). The extracted sample was treated according to the manufacture’s instructions of EpiTect Bisulfite Kit (Qiagen). The amplification of bisulfite-treated DNA was performed as an end-point PCR reaction with Zymo TaqTM DNA Polymerase (ZYMO RESEARCH). Cycling conditions were 95 °C for 6 min followed by 40 cycles of 94 °C for 30 s, 72 °C for 40 s and a final extension of 8 min at 72 °C. The primer design of bisulfate treated-DNA templates for each gene was done using the online software (http://www.urogene.org/methprimer/). Primer sequences were as follows, H19-Region 1(−2454bp ∼

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RESULTS In Vitro Maturation of Mouse Oocytes and Early Development. After 30 days exposure to NaF, oocytes were recovered and cultured in vitro. The percentages of mature oocytes and fertilization rates per mouse in the control group were significantly higher than that in other three treated groups (P < 0.05), and they decreased with increasing NaF concentration (Table 1). In addition, the number of embryos reaching the blastocyst stage was also significantly higher in the control group than in both lower dose and medium dose groups (P < 0.05); yet we just observed five embryos at 4−8cell stage in the higher dose group. 10400

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Table 1. In Vitro Maturation of Oocyte and Early Development Post Fertilization for Every Mouse groups (mg NaF/ L) 0 20 60 120

no. of oocytes cultured in vitro 49.3 40.7 44.7 44.1

± ± ± ±

3.4a 2.7b 2.9b 3.2b

no. of matured oocytes 43.6 35.2 31.7 27.4

± ± ± ±

3.1a 2.4b 1.9c 2.5d

no. of fertilization oocytes

no. of blastocyst

± ± ± ±

31.1 ± 2.6a 20.7 ± 1.4b 13.2 ± 2.1c 0

40.6 33.2 26.6 18.3

2.2a 1.7b 2.8c 2.1d

a

The different lowercase letters within the same column indicated significantly different (P < 0.05). The experiment was repeated 4 times with 10 mice in each group. The mathematical model Y = μ + τi + βj + εij (μ refers to average, τ refers to treatment effect, β refers to block effect, ε refers to random error) was used to analyze the original data. Since block effect was not significantly different, the mathematical model was changed into Y = μ + τi + εij and one-way ANOVA were used to analyze these data.

Apoptosis Detection of Blastomeres. We just tested the apoptosis rates of blastomeres in the control, lower dose and medium dose NaF groups as no any blastocyst was obtained in the higher dose group. The experiment was performed three times with a total of 45 embryos in each group. As shown in Table 2, the lower and medium dose NaF groups showed a

Figure 2. Apoptosis figures analyzed with TUNEL kit. Blastocysts were fixed and tested using In Situ Cell Death Detection Kit (Roch). The nuclei were stained with propidium iodide. Blastomere nuclei that showed fragmented (arrowhead) or condensed morphology and positive for TUNEL staining (upper panel) were considered apoptotic, and lower panel showed living cells. A. negative control; B. embryo derived from the 0 mg/L NaF; C. embryo derived from the 20 mg/L NaF; D. embryo derived from the 60 mg/L NaF. Scale bar indicates 80 μm.

Table 2. Percentages of Apoptotic Cell in Blastocysts Collected at 96 h after Culturea 0(mg/L) total blastomere number in 45 embryos TUNEL positive nuclei morphologically apoptotic

20(mg/L)

60(mg/L)

1991

1809

1717

121 (6.1%)c 57 (2.9%)c

361 (20.0%)b 244 (13.5%)b

496 (28.9%)a 348 (20.3%)a

Table 3. Percentage of Methylated CpG Sites on DMD of H19 and IGF2

a

Nuclei morphologically apoptotic refers to fragmented or condensed embryonic blastomere cell nuclei visualized by PI staining. The different lowercase letters within the same row indicated significantly different (P < 0.05). The experiment was repeated 3 times with 15 embryos in each group. The mathematical model Y = μ + τi + βj + εij was used to analyze the original data. One-way ANOVA were used to analyze these values.

DMD

0 mg/L

20 mg/L

60 mg/L

H19-Region I H19-Region II IGF2

45.76% 47.29% 46.94%

37.56% 26.88% 61.39%

28.63% 17.71% 87.59%

a The experiment was repeated three times with five embryos per replicate. Altogether 15 embryos were mixed together in each group and were used to methylation test. In total 100 clones in each group were random selected for sequencing.

Effects of NaF on Histone Modifications. The levels of H3K4m2, H3K9m2, and H3K9ac were showed in Figure 3b−d and Figure 4. The intensity of H3K4m2 in the control group embryos was markedly lower than that in the lower dose group embryos (P < 0.05), and the level in the lower dose group was significantly lower than that in the medium dose group embryos (P < 0.01). Similarly, the level of H3K9m2 was the lowest in the control group embryos, and was the highest in the medium group embryos (P < 0.05). But no significant difference in H3K9ac intensity was observed among the three groups (P > 0.05). We also tested levels of H3K4m2, H3K9m2, and H3K9ac using Western blotting, which were consistent with the immunostaining results (shown in Supporting Information Figure SI-1). Effects of NaF on the Expression of Developmentally Important Related Genes. Imprinted genes H19 and IGF2 were selected for study because of their important biological functions and characteristic phenotypes during embryo development; And the other selected genes like LSD1, which is a specific methyltransferase to form methylated H3K4 (H3K4m1/2) and (H3K9m1/2). As for G9a and MLL1, they specially catalyze the formation of H3K9m2 and H3K4m2, respectively. And JHDM2a is a specific demethylase to remove

significantly higher percentages of apoptotic cell than the control group (P < 0.05) in terms of both morphological apoptosis and TUNEL detection positive, and they were concentration dependent (Figure 2 and Table 2). Effects of NaF on the DNA Methylation Level. The percentage of methylated CpG sites on Region 1 of H19 was significantly higher in the control group than NaF groups, and showed negative correlation (Table 3). Similarly, the level of methylated CpG sites on Region 2 showed the same changing patterns as the Region 1. In contrast, the profile of methylated CpG sites on DMD of IGF2 was significantly lower in the control group compared to the NaF groups, and NaF affected the methylation level in a dosage dependent manner. At the same time, the effect of NaF on gobal DNA methylation was analyzed by immunostaining, and the intensity of fluorescent images was compared (Figure 3a and Figure 4). The global DNA methylation level in the control groups was significantly lower than that in the treated groups, and in general they expressed a dosage dependent change. Furthermore, these results were confirmed by ELISA-based colorimetric assay, which were shown in Supporting Information Table SI-2. 10401

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Figure 3. Global DNA methylation and histone acetylation analyzed with immunofluorescence staining. Embryos were fixed and immunostained with antibodies anti-5-methylcytosine (green) or anti-H3K9ac (green) or anti-H3K4m2 (green) or H3K9m2 (green). Each sample was counterstained with DAPI to visualize DNA (Blue). Intensity levels of acetylated H3K4m2, H3K9m2, H4K9ac, and global DNA methylation on 5methylcytosine were compared to that of DAPI signal. Scale bar indicates 120 μm. Representative figures of (a) global DNA methylation, (b) H3K4m2, (c) H3K9m2, and (d) H4K9ac.

the methyls of H3K9m1/2. Additionally, GCN5 is one of histone acetyltransferases and HDAC1 is one of deacetylases; DNMT1 is thought to be responsible for maintenance of genomic methylation patterns, and DNMT3a can methylate DNA de novo methylation. Significance analysis of gene expression identified 11 (false discovery rate (FDR) = 0.05) transcripts differentially expressed among three groups embryos with different concentrations of NaF, of which 9 showed significantly different (P value = 0.0013, 0.0016, 0.0019, 0.0020, 0.00203, 0.0022, 0.0031, 0.0034, 0.0037, 0.047, 0.054). The imprinted gene IGF2 showed the highest expression level in the control group embryos, followed by the lower dose group, then the medium dose group. On the contrary, the expression level of H19 was the lowest in the control group and the highest in

the medium group. The expression levels of GCN5 and HDAC1 were not significantly different among the three groups (P > 0.05); yet the transcript levels of DNMT1 and DNMT3a increased significantly with the increasing concentration of NaF (P < 0.05). As shown in Figure 5. LSD1, G9a and MLL1 presented a similar expression pattern, and their expression levels increased markedly (P < 0.05); yet JHDM2a showed an opposite expression pattern, and its expression level decreased significantly with the increasing concentration of NaF (P < 0.05). Finally, apoptotic related gene BAX also showed significantly different expression levels among the three groups, in which the medium dose group was roughly 10 times as high as the control and 2-fold higher than the lower dose 10402

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Figure 4. Semiquantitative determination of global DNA methylation and histone acetylation levels. Values with different lowercase letters differ significantly (P < 0.05).

Figure 5. Quantification of developmentally important gene transcript abundance. Bars with different superscripts (a, b, c) within the same gene differ significantly (P < 0.05).

reduced chromatin accessibility, therefore causing the transcript activation. As such, the repressed maternal allele of IGF2 was hypermethylated.29 Growing research indicated that early nutrition might alter imprinting regulation in rodents, and nutrient compositions in various culture media used during in vitro manipulations of early mouse embryos can change imprinted gene methylation and expression.5,7 Doherty et al. (2000) found that Whitten’s medium caused biallelic expression of H19, which correlated with hypomethylation in the 5′ DMD.5 Conversely, medium KSOM+AA resulted in embryos with monoallelic expression and the methylation patterns were similar to that observed in embryos in vivo. Khosla et al. (2001) found that preimplantation mouse embryos in a chemically defined culture medium (M16) without fetal calf serum (FCS) developed similarly to control embryos produced in vivo and that the addition of FCS to the M16 medium resulted in decreased viability and lower body weight, as well as reduced expression of H19 and IGF2, and increased DNA methylation at their DMDs compared with controls.3 We found that the two DMDs tested on H19 and the DMD on IGF2 showed significantly different methylation levels in the three groups of embryos and that the gobal DNA methylation levels in the control groups were significantly lower than that in NaF groups. These results suggested that NaF disturbed the DNA methylation patterns not only in the DMDs of imprinted genes, but also on a global scale. Latest study showed NaF treatment of male mice before copulation may disrupt the maintenance of normal gene imprinting during pregnancy, but may not directly affect the global DNA methylation in the sperm and liver.16 This may be in some part due to difference impacts after male and female mice were treated with NaF, respectively. In the present study, two key DMDs were detected based on the IGF2/H19 regulation model. In the

group (Figure 5). In addition, all results were verified by Western blotting (shown in SI Figure SI-2).

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DISCUSSION In the present study, we showed that oocyte maturation, in vitro fertilization and blastocyst formation were significantly impaired at either lower or medium dose of NaF in the treated groups compared to the control group and that all the embryos were blocked either before or at 4−8-cell stage in the high dose group. To our knowledge, this was the direct evidence to show that NaF damage animal embryo development, and this result is consistent with published data.17,25,26 The developmental failure could be linked to apoptosis based on the severe DNA damage we observed. DNA damage might be especially detrimental to preimplantation embryos through abnormal epigenetic modification.27 DNA methylation is a prominent epigenetic mechanism. The effect of NaF on embryo development were associated with DNA methylation or demethylation, because mouse embryo undergoes extensive DNA demethylation after fertilization and de novo methylation at the blastocyst stage.4 Moreover, two cycles of DNA demethylation and remethylation are closely linked with the DNA repair. The results will provide a basis to further explore molecular mechanism that NaF damage organs and issues. Since NaF resulted in embryo apoptosis, namely DNA damage, in a dose-dependent manner, we comprehensively analyzed the epigenetic changes in the next step. First of all, we had a look at imprinted genes. The representatives are IGF2 and H19 genes which are reciprocally imprinted and closely linked.28 Parental imprinting is an important genetic mechanism by which certain genes are can be expressed in a parent-of-origin-specific manner in regulating mammalian development. The paternally imprinted H19 gene showed hypermethylation on the DMD of repressed allele, and 10403

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concentration of NaF. But the expression of JHDM2a,39 a specific demethylase to remove the methyls (me1/2) from K9 site on histone 3 was decreased significantly with the increasing concentration of NaF. These changing patterns were consistent with those of H3K9m2 and H3K4m2. Surprisingly, the levels of acetylated H3K9 were constant among the three groups of embryo. Based on our results, we speculate that NaF may affect these epigenetic modification-related genes to disturb normal methylation modification and expression profiles of developmental related genes during mouse early embryo development. Finally, the expression amount of apoptotic related gene BAX was also significantly different in a dose-dependent manner, which was consistent with the results of apoptotic analysis. All in all, we demonstrated that fluoride epigenetically impaired mouse oocyte maturation and embryonic development, supplying a better knowledge of fluoride in toxicology and a deeper evaluation of its potential influence in physiological and clinical implications.

treated group, the reduction of DNA methylation in the primary DMD, namely H19-Region 1 and 2, suggested that these two subregions in the paternal allele underwent DNA demethylation, which was supported by the upregulation of H19 on the mRNA level. As for the secondary DMD (IGF2), it showed hypermethylation in the treated group, which was resulted from the gains of DNA methylation in the paternal allele. Because of the role of secondary DMD in controlling the promoter activity, as expected, the mRNA expression of IGF2 reduced as mentioned above. All the results above were consistent with the regulation model in the literature.28 The possibility of DNA methylation changes from the maternal side was ruled out based on the fact that the CpG sites in the primary and secondary DMDs of the maternal allele are free of methylation and fully methylated in the normal case, respectively. Collectively, NaF may globally result in the fluctuations of DNA methylation in DMD regions; further disrupt expressions of imprinted genes, examplified by IGF2/ H19 in our study, although only female mice were treated with NaF in a increasing concentration. The result was consistent with the study that NaF may interact with the oocyte to disrupt the maintenance of the imprinted genes in the subsequent embryo.16 As mouse H19 and IGF2 DMD is a methylation state-sensitive insulator that regulates transcriptional activation of both genes,30,31 the transcript analysis further confirmed that NaF adversely affected the normal expression of IGF2 and H19 in the early embryos. DNA methylation can directly prevent transcription factor binding and repress transcription, and its level is negatively related to the gene expression. In the present study, the methylation levels of DMD located on the H19 were decreased significantly, while its transcript levels were increased markedly with the increasing concentration of NaF. On the contrary, significantly increased methylation level of IGF2 resulted in decreased expression level. Abnormal patterns of DNA methylation are correlated with DNA instability and could ultimately damage embryonic development.32,33 It is worthwhile to note that the inconsistency between expression results of methyltransferases DNMT1 and DNMT3a and the methylated levels on DMDs tested in H19 gene might attribute to the common effect of many methyltransferases. Next, we came to the major histone modifications. A large number of epigenetic marks cooperatively act on promoters to regulate gene expression. H3K4m2 regulates chromatin assembly on promoters and transcript activation, while H3K9m2 plays opposite roles in control gene expression.34 A previous evidence suggests that there is a relationship between dietary constituents and histone acetylation modification,35,36 yet the effects of nutrient components on histone methylation were less studied. The most important finding is that abnormal histone methylation modification resulted from irrational nutritional diet at the early embryo development stages was associated with diet related chronic diseases including cardiovascular disease, type 2 diabetes, obesity, and cancer.37,38 In the present study, the levels of H3K4m2 and H3K9m2 analyzed by immunostaining were significantly different, but no significant difference in H3K9ac was observed among the three group embryos, suggesting that NaF destroyed the histone methylation modifications. LSD1 is a specific methyltransferase to form methylated H3K4 (H3K4me1/2) to activate transcription and H3K9 (H3K9m1/2) to repress transcription; G9a and MLL1 specially catalyze the formation of H3K9m2 and H3K4m2, respectively.3 Transcript levels of LSD1, G9a and MLL1 were increased significantly with the increasing

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Information (SI-Table 1, 2 and SI-Figure 1, 2). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 8629 87080092; fax: 8629 87080092; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the editor and the reviewers for their constructive suggestions to the manuscript. We also thank Dr. Jie Lan (University des Saarlandes) and Dr. Jeff Gale (an American teacher working in China) for extensive correction of the revised manuscript. The work was supported by National Natural Science Foundation of China (31001008).

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