During pre-implantation development, the embryonic genome undergoes extensive epigenetic modifications. This epigen-etic reprogramming allows gene expression reprogramming that accompanies crucial steps in early development, such as the loss of blastomere totipotency and gradual cell lineage specification, together with the onset of embryonic genome transcriptional activation. Among these epigenetic modifica-tions, DNA methylation reprogramming directly affects DNA; it involves the addition of a methyl group to the 5 position of the cytosine pyrimidine ring therefore called 5-metyl-cytidine (5-mC).1 During this period, a correct reprogramming of DNA methylation is necessary for long-term gene regulation during development2 and early aberrant DNA methylation patterns have been correlated with subsequent developmental failure.3,4 Furthermore, DNA methylation has been shown to be sensitive to environmental conditions, and especially to in vitro culture
alterations to DNa methylation have been attributed to in vitro culture and may affect normal embryo development. We chose to analyze DNa methylation reprogramming in the rabbit which, of the species with delayed transcriptional activation of the embryonic genome, allows easy comparisons between in vivo-developed (IVD) and in vitro-cultured (IVc) embryos. In this species, variations in DNa methylation had not previously been quantified, even in IVD embryos. IVD and IVc embryos were recovered at the 2-, 4-, 8- and 16-cell, morula and blastocyst stages. Immunostaining for 5-methyl-cytidine and normalization of the quantity of methylated DNa vs. the total DNa content were then performed. Our quantitative results evidenced DNa demethylation during pre-implantation development in both IVD and IVc embryos, but with different kinetics. Demethylation occurred earlier in vitro than in vivo between the 2- and 8-cell stages in IVc embryos, reaching its lowest level, while it only started at the 4-cell stage and ended at the 16-cell stage in IVD embryos. We also showed that an absence of serum from the culture medium significantly altered the degree of DNa demethylation. Finally, at the blastocyst stage, IcM was more methylated than the trophectoderm in all cases. Despite a morphological delay observed in in vitro cultured blastocysts, the difference in DNa methylation between IcM and trophectoderm cells appeared at the same time post-fertilization in IVD and IVc embryos, which may reflect another difference in the dynamics of DNa methylation during blastocyst formation. Our data thus clearly establish an effect of embryonic environment on DNa methylation reprogramming during pre-implantation development in a non-rodent species.
Alteration of DNA demethylation dynamics by in vitro culture conditions
in rabbit pre-implantation embryosadriana R. Reis e silva,1-3 céline Bruno,1,3,† Renaud Fleurot,1,3 Nathalie Daniel,1,3 catherine archilla,1,3 Nathalie peynot,1,3
carolina M. Lucci,2 Nathalie Beaujean1,3 and Véronique Duranthon1,3,*
1INRa; UMR1198 Biologie du Développement et Reproduction; Jouy-en-Josas, France; 2Faculty of Veterinary Medicine; University of Brasília; Brasília, Brazil; 3ENVa; Maisons alfort, France
†current affiliation: chU Montpellier; Département de Biologie de la Reproduction; hôpital arnaud de Villeneuve; Montpellier, France
Keywords: rabbit embryos, DNA methylation, in vivo development, in vitro culture, inner cell mass, trophectoderm
conditions, in many cell types. In the early embryo, modifi-cations of DNA methylation have been attributed to in vitro culture, which in turn may be linked to later defects in gene expression and development.5-7
During the first few hours after fertilization, a first wave of active DNA demethylation of the paternal genome has been reported in most mammal species during the first cell cycle.5,8-
11 Passive DNA demethylation occurs then gradually during the following cleavages. This was demonstrated first of all for the female genome in the mouse then in cattle12-14 between the 2- and 8-cell stages, in sheep15,16 and recently in pigs.6 At the blastocyst stage, when pluripotent cells from the inner cell mass segregate from the differentiated trophectoderm, differential methylation has been observed in most species, with ICM cells being more methylated than trophectoderm cells.14,15
Several DNA methyltransferases (DNMTs) that catalyze global genomic DNA methylation and modulate the dynam-ics of its pattern in mammals have been identified. The
monoclonal 5-mC antibody for methylated DNA, and EthD-2 for total DNA, in 2, 4, 8, 16-cell and morula stages are shown in Figure 1.
Normalized DNA methylation levels in IVD embryos (Fig. 2A) did not vary significantly between the 2- and 4-cell stages. They gradually and significantly (p < 0.05) decreased from the 4-cell to the 16-cell stage, then remained stable between the 16-cell stage and morula stage. Interestingly these DNA methylation dynamics appeared to be modified by the in vitro culture of embryos. In IVC embryos, the level of DNA meth-ylation decreased from the 2-cell stage onward, until the 8-cell stage. It remained stable between the 8- and 16-cell stages and then started to increase between the 16-cell and morula stages (Fig. 2B).
Differential methylation between the ICM and trophec-toderm at the blastocyst stage. In order to quantify the DNA methylation level in both the inner cell mass (ICM) and the trophectoderm cells of blastocysts, IVD embryos at 91 h post-coitum (hPC) (n = 15) and 97 hPC (n = 5) or IVC embryos (n = 17) at 98 hPC were hemi-sectioned (Fig. 3A). Our initial results indeed showed that ICM cells were less methylated than trophectoderm cells (data not shown). However, because these results contrasted with those observed in other species, we won-dered whether they could be due to a reduced penetration of labeled molecules into the blastocoele. For this reason, we fur-ther investigated hemi-blastocysts containing ICM and troph-ectoderm from the same embryo. Methylated DNA and total DNA contents were quantified in these hemi-embryos. In IVD and IVC blastocysts, the normalized DNA methylation level was significantly (p < 0.05) higher in the ICM than in the trophec-toderm (Fig. 3B–D). Moreover, in IVD blastocysts, this differ-ence rapidly increased in line with development, as evidenced by a comparison of 91 and 97 hPC blastocysts (Fig. 3B and C). In vitro development seemed to affect the kinetics of the establish-ment of this difference in DNA methylation levels between ICM and trophectoderm. While 98 hPC IVC blastocysts were mor-phologically (blastocyst diameter and relative volume of the blas-tocoele) close to 91 hPC IVD blastocysts, their difference in the level of DNA methylation between the ICM and trophectoderm was close to that of 97 hPC IVD blastocysts (Fig. 3C and D).
Variations in DNA demethylation kinetics in line with the composition of culture media. Because we observed some alter-ations to DNA demethylation kinetics during cleavage induced by the in vitro culture of embryos, we wondered whether the composition of the culture medium might affect this demeth-ylation. Because DNA methylation decreased between the 2- and 8-cell stages in vitro, we decided to compare the extent of this decrease under two different in vitro culture conditions. For this purpose 19 hPC recovered embryos were cultured in either B2 or B2 plus 2.5% fetal calf serum. The embryos were observed at the 2- and 8-cell stages (after 9 and 25 h of culture respectively). While DNA methylation levels did not differ at the 2-cell stage, the DNA methylation of 8-cell embryos was sig-nificantly lower in B2 than in B2 plus serum (Fig. 4), thus evi-dencing a sharper decrease in DNA methylation in the absence of serum.
maintenance enzyme called DNMT1 is the most abundant and is mostly involved in copying DNA methylation patterns on the newly synthesized DNA strand during replication.17 DNMT3A and DNMT3B are both de novo methyltransferases that estab-lish newly methyl groups to previously unmethylated genomic regions.18 The activity of both enzymes increases in the presence of DNMT3L, a DNT3A/B-like protein.19
Passive DNA demethylation is thought to take place in the absence of DNMT1 that cannot maintain methylation of newly synthesized DNA strands.20 On the other hand, no demethylat-ing enzyme has been identified so far that could explain active DNA demethylation. The elongator complex as well as DNA repair have been suggested to play a role in DNA demethylation; also, it remains unclear whether that role is direct or indirect.21,22 Recent studies in the mouse embryo have also shown a comple-mentarity between DNA methylation and hydroxymethylation, leading to new speculations regarding the function of this mark, 5-hydromethyl-cytidine (5-hmC), as an intermediate in the DNA demethylation pathway.23-25 Very interestingly, 5-hmC has been reported to be related to ICM specification.26
Despite inter-species variations regarding its extend, the pat-tern of demethylation during cleavages and the differential re-methylation of both cell lineages at the blastocyst stage have been reported in most mammalian embryos. However, the rabbit embryo has been reported to differ with respect to DNA meth-ylation dynamics during cleavages and segregation of the first cell lineages.27 In this species, qualitative data have shown an absence of demethylation during cleavages and a lower level of methyla-tion in ICM cells than in trophectoderm cells, a situation appar-ently shared with primate embryos.28,29 However, no quantitative data have yet been published on the rabbit.
Nevertheless, the rabbit embryo is an interesting model for the analysis of early epigenetic events and their modifications by the embryonic environment in non-mouse species, particularly because of the availability of embryos developed both in vivo and in vitro at all stages. We therefore decided to obtain precise quan-titative data on DNA methylation dynamics during pre-implan-tation development in this species. The analysis performed on embryos developed both in vivo and in vitro showed that the embryonic environment affected the kinetics and degree of DNA methylation reprogramming in the early embryo.
Results
DNA demethylation during embryo cleavages. DNA meth-ylation dynamics were analyzed in rabbit embryos fertilized in vivo that were either developed in vivo (IVD, n = 185) or cultured in vitro (IVC, n = 216) in B2 medium plus serum (B2S). DNA methylation was analyzed by quantifying 5-mC labeling in each blastomere. In order to take account of varia-tions in the DNA content due to S phase in each blastomere, the total DNA content of each blastomere was also determined by EthD-2 signal quantification. This made it possible to nor-malize the quantity of methylated DNA vs. the total DNA content during pre-implantation development. Representative pictures of pre-implantation rabbit embryos labeled with a mouse
in the embryonic cytoplasm.31 When comparing our results on rabbit DNA demethylation kinetics in vivo with those quantified in sheep15 and pig,6 the notable feature was the delay observed in the initiation of this demethylation in the rabbit where it only became significant from the 4-cell stage onwards. This delay in the initiation of significant passive demethylation implies that maintenance DNMT is functional at least during the S phase of the 2-cell stage. This is compatible with our previous dem-onstration of such activity during the first S phase in the rabbit zygote.11 Thereafter, progressive and significant demethylation is observed between the 4- and 8-cell stages in the three spe-cies, probably due to a reduction in maternally inherited DNMT. This reduction continues between the 8- and 16-cell stages in the rabbit, which contrasts with the sheep for which it remains constant until morula.15 No data are currently available in the
Discussion
The present study provides the first quantitative analysis of DNA methylation dynamics during both in vivo and in vitro pre-implantation devel-opment in the rabbit. To date, qualitative data had only been available on embryos developed in vitro.27 Moreover, this is the first study to have strictly isolated the effect of in vitro culture on DNA demethylation during pre-implantation development in a species with delayed embry-onic genome activation since studies in sheep15 and pig6 embryos analyzed in vitro cultured embryos obtained by the in vitro fertilization of in vitro matured oocytes. However, because the mucin coat deposited by the oviduct on the rab-bit embryo during in vivo development impairs antibody access to embryonic cell nuclei, it was necessary to treat the in vivo developed embryos with pronase in order to remove the mucin coat (and the zona pellucida). And because such a treatment could only be applied to embryos developed in vivo, it was not possible to compare the absolute values obtained for DNA or 5-mC staining on a stage-by-stage basis between in vivo treated and in vitro non-treated embryos. For this reason, we focused our comparisons on the dynamics of DNA demethylation both in vivo and in vitro.
Our quantitative data evidenced a gradual demethylation in IVD and IVC embryos during successive cell cleavages, as reported in cattle (in vitro produced embryos14), mice (in vitro produced embryos;9 IVD14), sheep (IVD embryos15,16) and described in pigs after quantitative immunofluorescence assess-ment (IVD embryos6). In mice, this loss of DNA methylation was associated with a reduction in DNA methyltransferase activity in pre-implantation embryos30 and the active retention of DNMT1
Figure 1. Dynamics of DNa methylation reprogramming in rabbit embryos. (a) Representative images of EthD-2 labeling (red) and 5-mc immunos-taining (green) during the pre-implantation development of in vivo-developed rabbit embryos.
Figure 2. Normalized DNa methylation levels (5-mc/EthD-2) in the nuclei of (a) in vivo-developed or (B) in vitro-developed (B2s) rabbit embryos. On these box plots, values between the inner and outer fences are plotted with asterisks. n = number of blastomeres analyzed at each stage. The arrows indicate a significant difference (p < 0.05) between two consecutive developmental stages.
of homocysteine have been shown to induce a hypermethylation of genomic DNA as well as developmental retardation, giving a new percep-tion of nutrient-sensitive epigenetic regulation.32 Whatever the case, the earlier re-methylation that we observed in vitro may have reflected a kind of adaptive response by the embryo to the situation generated by the in vitro environment.
Because we compared the kinetics of demeth-ylation rather than absolute values, our results cannot be compared directly with those pre-viously published on other species, especially rodents5 and pigs6 using quantitative immu-nofluorescence. In these latter two studies, the authors concluded as to a hypermethylation of in vitro cultured zygotes and in vitro fertilized and cultured embryos respectively obtained from in vitro matured oocytes. These results have recently been confirmed, although at the blastocyst stage, using alternative methods such as the microarray assessment of DNA methyla-
tion in in vitro cultured mouse embryos.33 Alterations to global DNA methylation caused by the in vitro environment thus seems to have been confirmed in several species and using dif-ferent methods. Moreover, because epigenetic differences have also been evidenced between blastocysts obtained in vitro and in vivo33 and between ES cells derived from either in vitro or in vivo developed blastocysts,34 we can conclude that the early alterations observed during the demethylation phase are not compensated by the subsequent re-methylation phase that accompanies blastocyst formation.9,14 Our analysis also points to a finely tuned effect of in vitro culture conditions, insofar as the degree of demethylation between the 2- and 8-cell stages increased when fetal calf serum was absent from the culture medium. Such differential effects of culture conditions have been observed with respect to imprint maintenance at specific loci in the mouse model.35 Moreover, direct epigenetic effects of adding serum to culture media have
pig. As complementarities between 5-mC and 5-hmC seem con-served in rabbit zygotes,25 it would be interesting to examine the levels of 5-hmC during rabbit pre-implantation development to see whether 5-hmC is linked with epigenetic reprogramming in other mammals than mouse.
Interestingly, however, we observed different DNA demeth-ylation kinetics in vivo and in vitro. While DNA demethylation only became significant as from the 4-cell stage in vivo, it started as early as the 2-cell stage in vitro. At later stages normalized DNA methylation levels also increased earlier in vitro (as from the 16-cell stage) than in vivo (not observed at the morula stage). Whether this earlier in vitro demethylation is due to a deregulation of maternal DNMT through maternal mRNA or protein degra-dation, or to a difference in the kinetics of methyl group provid-ers available for DNA methylation reactions in the cell remains unknown. In cattle embryos, for example, high concentrations
Figure 3. Dynamics of DNa methylation in rabbit blastocysts. (a) Representative example of an in vivo-developed blastocyst after hemi-section. (B–D) Normalized DNa methylation levels (5-mc/EthD-2) in blastocysts that were either in vivo-developed (IVD) (B and c) or in vitro-cultured (IVc) in B2 serum (D). n, number of blastomeres analyzed. On these box plots, values between the inner and outer fences are plotted with asterisks. The letters indicates a significant difference (p < 0.05) between the inner cell mass (IcM) and trophectoderm (Tp) cells of blastocysts within each development time for each condition of development (IVD at 91 hpc, IVD at 97 hpc or IVc at 98 hpc).
Figure 4. Normalized DNa methylation levels (5-mc/EthD-2) in rabbit embryos cultured in B2 or B2s media. n, number of blastomeres analyzed. On these box plots, values between the inner and outer fences are plotted with asterisks and values outside the outer fence with empty circles. The arrows indicate a significant difference (p < 0.05) between two consecutive developmental stages. The letters indicate a significant difference (p < 0.05) between treatments.
animals, as promulgated by the Society for the Study of Reproduction, and with the European Convention on Animal Experimentation. The researchers involved in work with the ani-mals were all licensed for animal experimentation by the French veterinary services.
Animals. New Zealand White female rabbits (20–22 week-old) were superovulated by means of 5 subcutaneous adminis-trations of pFSH (Stimufol®, Merial) for 3 d before mating: two doses of 5 μg on day 1 at 12-h intervals, two doses of 10 μg on day 2 at 12-h intervals, and one dose of 5 μg on day 3, followed 12 h later by an intravenous administration of 30 IU HCG (Chorulon, Intervet) at the time of mating (natural mating).
Recovery and culture of rabbit zygotes. In vivo-developed (IVD) embryos were collected from oviducts perfused with PBS at 24, 32, 39, 55, 67 and 91 h post coitum (hPC) to obtain embryos at 2-, 4-, 8-, 16-cell, morula and blastocyst stages respec-tively. At each stage embryos were recovered from at least 6 dif-ferent females. They were then incubated in 5 mg/ml Pronase (Sigma) at room temperature (RT) to remove zona pellucida and mucin coat, except the blastocysts. Blastocysts were hemi-sectioned with a scalpel in order to avoid biases due to reduced penetration of labeled molecules into the blastocoele. Analyses were performed on hemi-embryos containing both ICM and trophectoderm cells. In vitro-cultured (IVC) embryos were collected at 19 h PC and then cultured in 500 μl B2 medium (Laboratoire C.C.D.) with or without 2.5% (V/V) fetal calf serum (FCS, Gibco) for 9, 15, 25, 39, 53 and 79 h at 38.5°C in 5% CO
2 in air to recover 2, 4, 8 and 16 cells and morula and
blastocysts respectively.Immunostaining. Embryos were fixed in 4% paraformalde-
hyde (PFA, Euromedex) in PBS for 48 h at 4°C, that was changed every 24 h. Some embryos were kept in 4% PAF for 2–5 d to allow simultaneous immunostaining of embryos at all stages (2-cell to morula stages). During immunostaining, all steps were performed at room temperature, unless otherwise specified. The embryos were first washed in PBS for 1 h and permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 1 hr. The embryos were then treated with 2 N HCl for 1 hr followed by washes with 0.05% Tween-20 in PBS (10 min). After washing, the embryos were then incubated for 1 h in PBS containing 2% BSA and then with a monoclonal mouse anti-5-methylcytidine antibody (5-mC, 1:1,000 dilution in 2% BSA-PBS; Eurogentec) known to be specific for 5-mC (Beaujean et al. 2004) overnight at 4°C. After incubation they were washed with 0.05% Tween-20 in PBS for 30 min and incubated for 1 hr with a fluorescein isothiocya-nate (FITC)-conjugated anti-mouse secondary antibody (1:200 dilution; Jackson ImmunoResearch). After washing with 0.05% Tween-20 in PBS, the embryos were post-fixed in 2% PAF for 20 min, washed in PBS for 5 min and the DNA was counterstained for 30 min at 37°C with 0.002 mM Ethidium Homodimer-2 (EthD-2, Invitrogen). The embryos were finally mounted on slides with Vectashield (Vector). Negative control: no signal was observed for 5-mC in rabbit embryos when the mouse monoclonal anti-5-methylcytidine antibody was omitted (data not shown).
Quantitative analysis. The embryos were observed using a Carl Zeiss AxioObserver Zl fluorescence microscope equipped
been reported previously: in sheep, serum-starved fetal fibro-blasts used in somatic cell nuclear transfer procedures displayed levels of DNA methylation that were 27% lower than those seen in fibroblasts cultured in a serum-fed medium.36 In the mouse, pre-implantation cultures in the presence of serum affected the control of multiple growth-related imprinting genes, leading to abnormal fetal growth.37
Taken together, these results evidencing the differential effects of different culture conditions stress the need for further research aimed at optimizing culture media and conditions, particularly in the context of the development of Assisted Reproductive Technologies.
At the blastocyst stage, we examined the relative methyla-tion levels in ICM and trophectoderm cells. Our initial results in vivo seemed in agreement with previous data obtained in vitro,27 showing that ICM cells were less methylated than trophectoderm cells (data not shown). However, because these results contrasted to those observed in cattle,14 humans,38 mice,14 pigs6,39,40 and sheep,15,16 we wondered whether they could be caused by an arti-factual reduction in antibody access to ICM cells. For this reason, we decided to focus more closely on hemi-blastocysts. Under these conditions, and contrary to previous data in reference 27, we found that rabbit blastocysts behaved in the same way as cattle, mice, pigs, sheep and humans, insofar as their ICM cells were more methylated than their trophectoderm cells. Very interestingly, this result agreed with that reported by Manes and Menzel,41 in rabbit blastocysts using an alternative methodology based on the use of methylation-sensitive restriction enzymes. We therefore hypoth-esize that the reduced penetration of antibody that we observed when working with whole blastocysts might have affected results previously obtained in rabbit,27 primate28,29 and bovine42 embryos produced in vitro.
Furthermore, we observed in IVD blastocysts that the dif-ference in methylation levels between ICM and trophectoderm cells increased in line with development, as described in pigs.39 Interestingly, despite the fact that 98 hPC IVC blastocysts were morphologically closer to 91 hPC than to 97 hPC IVD blasto-cysts, their ICM/trophectoderm differential methylation was more similar to that observed in 97 hPC IVD blastocysts. This may have been due to the earlier increase in DNA methylation observed in IVC morulae and might reflect another effect of the in vitro environment on the dynamics of DNA methylation during blastocyst formation. Indeed, the de novo DNA methyltransferase DNMT3B is expressed in most mouse morulae and blastocysts, preferentially in the trophectoderm of mouse blastocysts.43 One could suspect that in vitro culture affects DNMT3 expression thereby leading to the abnormal DNA methylation levels observed in IVC embryos. Because trophectoderm cells contribute to the chorionic component of the placenta, change made to their DNA methylation pattern by in vitro culture may be linked to an altered expression of the genes involved in placental and fetal growth.44,45
Materials and Methods
These experiments were performed in accordance with the International Guidelines on Biomedical Research involving
staining intensities, data from each replicate were normalized by the 4-cell stage median of the replicate. After log2 transforma-tion, data on the DNA methylation levels (methylated DNA/total DNA content) were analyzed by ANOVA using the Systat 13 (Systat Software Inc.). p values < 0.05 were considered to be significant.
Disclosure of Potential Conflicts of Interest
The authors declare that they have no conflict of interest.
Acknowledgments
The authors are indebted to members of Unité Commune d’Expérimentation Animale (UCEA) responsible for the rabbit colonies. They also thank the MIMA2 facility for access to the Apotome microscopy and the Region Ile-de-France for fund-ing this system and Dr. Pierre Adenot for his assistance with the confocal microscope on the platform. The authors would like to thank Luc Jouneau for his assistance with statistical analyses.
Financial Disclosures
This work was supported by grants awarded to A.R.R.eS. from the CAPES and Brazilian Ministry of Agriculture, Livestock and Food Supply. Experimental work was supported by an “AMP diagnostic prénatal et diagnostic génétique” 2009 grant from the French Agence de la Biomedecine and an INRA “Young Team” research grant.
with the ApoTome slider (MIMA2 Platform, INRA). The samples were observed for the area containing the nucleus with a 40x Plan-Neofluar oil objective (NA 1.3) and digital optical sections were collected using a Z-series acquisition feature every 1.0 μm. Quantitative analyses of DNA methylation levels and total DNA contents were estimated by quantifying fluorescence signals as follows, using Image-J software (National Institutes of Health): (1) the area of each nucleus was outlined manually and the mean fluorescence intensity was measured for both 5-mC and EthD-2 images, (2) the mean fluorescence intensities were divided by the acquisition times of the corresponding signal and (3) these corrected mean fluorescence intensities were multiplied by the nuclear areas to obtain the total fluorescence intensities for both 5-mC and EthD-2. Finally, DNA methylation levels (5-mC total fluorescence intensities) were divided by total DNA contents (EthD2 total fluorescence intensities) to calculate nor-malized methylated DNA quantities. The total number of nuclei analyzed at each stage and in each condition are indicated on the figures. These were obtained by analyzing 205 IVD embryos (20, 92, 24, 30, 19 and 20 at the 2-, 4-, 8- and 16-cell, morula and blastocyst stage respectively) and 233 IVC embryos (46, 93, 48, 17, 12 and 17 at the 2-, 4-, 8- and 16-cell, morula and blas-tocyst stage, respectively). For the comparison of IVC embryos obtained in B2 vs. B2 plus serum, a mean of 20 embryos were analyzed at each stage in each condition.
Statistical analysis. Most experimental groups were rep-licated three times. To reduce inter-replicate variability in
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