Juliette Salvaing
email: juliette.salvaing@jouy.inra.fr

Tiphaine Aguirre-Lavin

Claire Boulesteix

Gae ¨tan Lehmann

Pascale Debey

Nathalie Beaujean

5-Methylcytosine and 5-Hydroxymethylcytosine Spatiotemporal Profiles in the Mouse Zygote

Background: In the mouse zygote, DNA methylation patterns are heavily modified, and differ between the maternal and paternal pronucleus. Demethylation of the paternal genome has been described as an active and replication-independent process, although the mechanisms responsible for it remain elusive. Recently, 5-hydroxymethylcytosine has been suggested as an intermediate in this demethylation.

Methodology/Principal Findings: In this study, we quantified DNA methylation and hydroxymethylation in both pronuclei of the mouse zygote during the replication period and we examined their patterns on the pericentric heterochromatin using 3D immuno-FISH. Our results demonstrate that 5-methylcytosine and 5-hydroxymethylcytosine localizations on the pericentric sequences are not complementary; indeed we observe no enrichment of either marks on some regions and an enrichment of both on others. In addition, we show that DNA demethylation continues during DNA replication, and is inhibited by aphidicolin. Finally, we observe notable differences in the kinetics of demethylation and hydroxymethylation; in particular, a peak of 5-hydroxymethylcytosine, unrelated to any change in 5-methylcytosine level, is observed after completion of replication.

Conclusions/Significance: Together our results support the already proposed hypothesis that 5-hydroxymethylcytosine is not a simple intermediate in an active demethylation process and could play a role of its own during early development.

Introduction

DNA methylation, and epigenetic modifications in general, play a major role during early embryonic development, in particular embryonic genome activation, X inactivation, differentiation [START_REF] Corry | Epigenetic regulatory mechanisms during preimplantation development. Birth defects research Part C[END_REF][START_REF] Jeanblanc | Embryonic genome activation]. Gyne ´cologie, obste ´trique & fertilite[END_REF]… DNA methylation levels undergo major modifications that appear to be essential for early development [START_REF] Li | Targeted mutation of the DNA methyltransferase gene results in embryonic lethality[END_REF][START_REF] Tate | The methyl-CpG binding protein MeCP2 is essential for embryonic development in the mouse[END_REF] and necessary for the establishment of a pluripotent state by the demethylation of many pluripotency regulators [START_REF] Farthing | Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes[END_REF]. DNA methylation changes during early embryonic development have been studied in many species. One of the main features, observed primarily in mice, is the asymetric dynamics between the paternally and maternally inherited parts of the genome in 1-cell embryos just after fertilization [START_REF] Mayer | Demethylation of the zygotic paternal genome[END_REF][START_REF] Santos | Dynamic reprogramming of DNA methylation in the early mouse embryo[END_REF][START_REF] Oswald | Active demethylation of the paternal genome in the mouse zygote[END_REF][START_REF] Dean | Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos[END_REF][START_REF] Zaitseva | Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro[END_REF]. Indeed, in mouse embryos, using a 5-methylcytosine (5MeC) antibody, a very rapid demethylation was observed in the paternal genome (constituting the paternal pronucleus), prior to the onset of replication [START_REF] Mayer | Demethylation of the zygotic paternal genome[END_REF][START_REF] Santos | Dynamic reprogramming of DNA methylation in the early mouse embryo[END_REF][START_REF] Oswald | Active demethylation of the paternal genome in the mouse zygote[END_REF], while progressive demethylation in the maternal one occurs across the following cell-cycles until the morula stage [START_REF] Mayer | Demethylation of the zygotic paternal genome[END_REF][START_REF] Santos | Dynamic reprogramming of DNA methylation in the early mouse embryo[END_REF][START_REF] Oswald | Active demethylation of the paternal genome in the mouse zygote[END_REF][START_REF] Rougier | Chromosome methylation patterns during mammalian preimplantation development[END_REF]. Demethylation in the paternal pronucleus has thus been called ''active'' as opposed to the ''passive'' demethylation observed in the maternal pronucleus. Passive demethylation results from a dilution of the original methylation pool, due to the absence of methylation maintenance during replication [START_REF] Rougier | Chromosome methylation patterns during mammalian preimplantation development[END_REF][START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF]. Indeed, DNA methylation is ensured by enzymes called DNA methyl-transferases (Dnmt), which include the maintenance methyl-transferase Dnmt1, involved in copying DNA methylation patterns on the newly synthetised strand during replication, and the de novo methyltransferase Dnmt3A and 3B [START_REF] Jin | DNA Methylation: Superior or Subordinate in the Epigenetic Hierarchy?[END_REF], that establish newly methylated domains.

Active demethylation has been observed on a large scale only in embryos and in primordial germ cells [START_REF] Hajkova | Epigenetic reprogramming in mouse primordial germ cells[END_REF], and the mechanism sustaining it remains largely unknown, even if some advances in this domain have been published lately [START_REF] Okada | A role for the elongator complex in zygotic paternal genome demethylation[END_REF][START_REF] Wossidlo | Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes[END_REF][START_REF] Hajkova | Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway[END_REF]. Last year, the elongator complex has been suggested to play a role in DNA methylation, but it remains unclear whether that role is direct or indirect [START_REF] Okada | A role for the elongator complex in zygotic paternal genome demethylation[END_REF], and probably the most prominent mechanism proposed so far involve DNA repair pathways [START_REF] Wossidlo | Dynamic link of DNA demethylation, DNA strand breaks and repair in mouse zygotes[END_REF][START_REF] Hajkova | Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway[END_REF].

However, the recent rediscovery of 5-hydromethylcytosine (5hMeC) [START_REF] Tahiliani | Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1[END_REF][START_REF] Ito | Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification[END_REF] has led to new speculations about the function of this mark as an intermediate in the DNA methylation pathway. Recent studies in the mouse embryo have shown a good complementarity between 5MeC and 5hMeC levels, the latter increasing in the paternal pronucleus when 5MeC decreases [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF][START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF][START_REF] Ruzov | Lineagespecific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development[END_REF][START_REF] Gu | The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes[END_REF]. Additionally, while rings of 5MeC persist around the nucleolar precursor bodies (NPBs) in the paternal pronucleus [START_REF] Yamazaki | Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging[END_REF], similar rings were observed for 5hMeC in the maternal pronucleus [START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF]. Since the localization of those rings strongly reminds that of pericentric heterochromatin [START_REF] Martin | Genome restructuring in mouse embryos during reprogramming and early development[END_REF][START_REF] Probst | Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote[END_REF][START_REF] Maalouf | Three-dimensional fluorescence in situ hybridization in mouse embryos using repetitive probe sequences[END_REF], it has been proposed that 5MeC and 5hMeC complement each other, the first one marking the paternal heterochromatin, while the second one marks the maternal heterochromatin. 5hMeC can be further converted to 5carboxylcytosine (5caC) and to 5-formylcytosine (5fC) [START_REF] Ito | Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine[END_REF][START_REF] He | Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA[END_REF], both of which can be detected in the mouse zygote [START_REF] Inoue | Generation and replicationdependent dilution of 5fC and 5caC during mouse preimplantation development[END_REF]. In cells, 5caC can be excised by the thymine-DNA glycosylase [START_REF] He | Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA[END_REF] while in embryos 5caC and 5fC appear to be lost by progressive dilution [START_REF] Inoue | Generation and replicationdependent dilution of 5fC and 5caC during mouse preimplantation development[END_REF]. However, 5hMeC persists much longer than it would be expected for a simple intermediate in DNA demethylation [START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF] and seems to be removed during preimplantation development by a passive dilution mechanism similar to the one observed for 5MeC in the maternal pronucleus [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF]. In addition, as the levels of 5hMeC remain high in undifferentiated cells and are lost only upon differentiation [START_REF] Ruzov | Lineagespecific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development[END_REF][START_REF] Ficz | Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation[END_REF], it raises the possibility that 5hMeC might have an additional function in pluripotent stem cells as well as in the totipotent early embryo.

In order to investigate in more details the spatiotemporal relationship between 5MeC and 5hMeC patterns during the first cell cycle in the early mouse embryo, we examined the colocalization of 5MeC, 5hMeC and pericentric heterochromatin, using immunoFISH experiments. In a second part, we performed a quantitative analysis of 5MeC and 5hMeC dynamics during the replication phase, using labelled deoxyuridine incoporation, which allows precise timing of replication [START_REF] Bouniol-Baly | Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation[END_REF][START_REF] Adenot | Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos[END_REF]. All together, our results show that the complementarity between 5MeC and 5hMeC is not as perfect as it seems at first glance.

Results

Methylation and hydroxymethylation of pericentric heterochromatin

Previous studies described complementary patterns of 5methylcytidine (5MeC) and 5-hydroxymethylcytidine (5hMeC) both in terms of expression levels as in terms of localization [START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF]. In particular, a persisting ring of 5MeC was observed in the paternal pronucleus around the nucleolar precursor bodies (NPB) [START_REF] Yamazaki | Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging[END_REF], while a similar ring of 5hMeC was shown around the NPB in the maternal pronucleus [START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF]. The authors proposed that those rings correspond to the pericentric heterochromatin, which shows a very similar localization [START_REF] Martin | Genome restructuring in mouse embryos during reprogramming and early development[END_REF][START_REF] Probst | Structural differences in centromeric heterochromatin are spatially reconciled on fertilisation in the mouse zygote[END_REF], but they did not provide any direct evidence. We therefore performed immuno-FISH experiments on 1-cell stage embryos with a preserved 3D organization [START_REF] Maalouf | Three-dimensional fluorescence in situ hybridization in mouse embryos using repetitive probe sequences[END_REF], using an antibody against 5MeC or an antibody against 5hMeC, together with specific probes for the major satellites (pericentric DNA). Embryos were fixed at different time points [START_REF] Ito | Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF][START_REF] Gu | The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes[END_REF]25 and 29 hphCG), allowing us to observe them at all the stages of the first cell cycle, from fertilization up to the first mitosis. We used the PN classification from Adenot [START_REF] Adenot | Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos[END_REF] to sort the embryos.

As expected, 5MeC is found preferentially in the maternal pronucleus while 5hMeC is more strongly expressed in the paternal one (figure 1). It is however important to note that we could always detect a signal for 5MeC and 5hMeC respectively in the paternal and maternal pronuclei. It should also be mentioned that we did not observe any strong differences between the 5MeC and 5hMeC signals obtained with immunoFISH and those obtained with classical immunostainings (figure 1A vs 1B/C).

As shown on figures 1B and C, in the maternal pronucleus major satellites remain localized around the NPBs, with sometimes a few signals at the periphery, from PN1 until the end if the 1-cell stage (figure 1 B andC). We could not detect any accumulation of 5MeC on these, except for a very weak and partial accumulation sometimes observed at the PN5 stage (figure 1B; arrow). On the other hand, we observed accumulations of 5hMeC, in partial rings around the NPBs or sometimes as spots near the nuclear periphery, from PN2 until mitosis (figure 1C; arrows). However, only a small fraction of the major satellites signals clearly colocalizes with 5hMeC, suggesting that it accumulates on the pericentric heterochromatin of a subset of chromosomes only.

In the paternal pronucleus, we could not detect any strong accumulation of 5MeC on the major satellites during the early stages (until early PN3; figure 1B). During that time, the major satellites remain mostly in clusters, and organize around the NPBs at the PN3 stage only (figure 1 B andC). Once the rings are formed, a very strong 5MeC signal colocalizing with the major satellites signal is observed. Interestingly, at the PN3 and PN4 stages, 5MeC signal is very strong around the NPBs surrounded by major satellites, with an almost perfect colocalization; also, a very faint signal is sometimes observed around the other NPBs (arrow). Finally, at the PN5 stage, 5MeC rings are clearly observed around all NPBs, even in the absence of pericentric DNA (arrow). In contrast to 5MeC, there is no accumulation of 5hMeC signal on the pericentric heterochromatin regions at PN2 and 3 (figure 1C). However, faint and partial rings start to be visible around the NPBs at PN4, and at PN5 all the NPBs, including those not surrounded by pericentric heterochromatin, are surrounded with strong 5hMeC signal. There is therefore no exclusion of 5MeC and 5hMeC on the pericentric heterochromatin at the end of the first cell stage.

DNA methylation, hydroxymethylation and Replication

Since DNA methylation and hydroxymethylation patterns are not fully complementary on the spatial level, we wanted to adress their complementarity on the temporal level. The replication in particular is a very interesting period: while DNA methylation has been described as replication-independent and complete before the start of replication [START_REF] Santos | Dynamic reprogramming of DNA methylation in the early mouse embryo[END_REF], recent studies [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF][START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF] as well as our own observations (figure 1A) have shown changes in the methylation levels up to PN4 or PN5, that is after the beginning of replication [START_REF] Bouniol-Baly | Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation[END_REF][START_REF] Adenot | Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse embryos[END_REF]. Double stainings for replication and either 5MeC or 5hMeC were therefore performed. Embryos were sorted in 5 different classes (Table 1), based on the previous work from Bouniol-Baly and collaborators [START_REF] Bouniol-Baly | Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation[END_REF]. Those without any replication staining were called ''pre-replication'' or ''post-replication''. Embryos with homogeneous replication staining in both pronuclei were classified as ''early replication'', those with homogeneous replication staining in the maternal pronucleus but peri-NPB staining in the paternal one as ''mid replication'', and those with peri-NPB staining in the maternal pronucleus and peri-nuclear staining in the paternal one as ''late replication'' (figure 2).

Interestingly, all the ''early replication'' PN3 embryos show peri-NPBs rings of 5MeC, which are fainter or even absent in the ''pre-replication'' ones (figure 2A). This suggests that the formation of the peri-NPB rings precedes slightly the onset of replication. The 5MeC signal in the paternal pronucleus appears already weaker than in the maternal one prior to replication (figure 2A), while there is little difference for 5hMeC between both pronuclei at this stage (figure 2B). However, 5hMeC patterns change dramatically in the early replication population, with a much stronger signal detected in the paternal pronucleus (figure 2B). 5MeC dynamics are more difficult to assess: the passive demethylation occuring in the maternal pronucleus [START_REF] Rougier | Chromosome methylation patterns during mammalian preimplantation development[END_REF][START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF] as well as the increase in pronuclei size make any change difficult to visualize (figure 2A) without proper quantification.

For each embryo, we thus quantified the total DNA methylation content and calculated the paternal/maternal ratio. As can be observed on figure 3A, for 5MeC this ratio decreases the most between the pre-replication and the early replication stage (pvalue,10 24 ), with a slighter but constant decrease later on and goes up again after replication (p-value,0.05). The kinetics of the paternal/maternal ratio for 5hMeC is nearly opposite, with a sharp increase between pre-replication and early replication (pvalue,10 211 ), no change at mid-replication and a small decrease later on (p-value,0.01 between mid and post-replication).

In order to assess the dynamics of methylation and hydroxymethylation in both pronuclei independently, we used the DNA content as a reference. Since the DNA denaturation necessary for 5MeC and 5hMeC stainings strongly impaired DNA labelling using usual dyes, we used an anti-single stranded DNA antibody. Embryos were again sorted in 4 different replication classes related to the PN stage and hphCG as described above (Table 1). We also analyzed late 1-cell stage embryos at PN5 (28-29 hphCG).

As shown on figure 3B, the normalized DNA methylation content in the maternal pronucleus is divided by around 2 during replication. The most important changes are observed between pre-replication and early replication (p-value,10 23 ) and between mid/late replication and post-replication (p-value,10 24 ). Normalized 5hMeC levels in the maternal pronucleus decrease between pre-replication and early replication (p-value,10 23 ). There is then little change during replication but a sharp and surprising increase after, with a peak post-replication (pvalue,10 23 ) followed by an important decrease at the end of the cell cycle (p-value,0.01).

A similar peak is observed in the paternal pronucleus (figure 3C). Indeed normalized 5hMeC levels increase more importantly during replication (from1.9060.17 prereplication to 2.7060.15 in early replication; p-value,0.01; and 3.0760.19 in mid/late replication; p-value,10 24 ), and even more between mid/late replication and postreplication (p-value,10 23 ) before strongly decreasing at the end of the cell cycle (p-value,10 23 ). During the same time, the normalized DNA methylation constantly decreases Figure 1. DNA methylation and hydroxymethylation on pericentric heterochromatin. Z-stack images of 1-cell embryos (PN1 to Prophase) were acquired in 3D but only single representative z-sections for each PN are shown here. Images were rotated if necessary to have the maternal PN on the left and the paternal PN on the right. A) 3D immunostainings using either an anti-5-methylcytosine antibody (n = 148, upper row) or an anti-5hydroxymethylcytosine antibody (n = 226, lower row). B) 3D immunoFISH using an anti-5MeC antibody (upper row) and specific probes for the major satellites (middle row). Merged images are shown on the lower row (green: 5MeC; red: major satellites) ; a total of 75 embryos were analyzed C) 3D immunoFISH using an anti-5hMeC antibody (upper row) and specific probes for the major satellites (middle row). Merged images are shown on the lower row (green: 5hMeC; red: major satellites); a total of 61 embryos were analyzed. Scale Bar: 5 mm. doi:10.1371/journal.pone.0038156.g001 and is divided by 3, suggesting that passive demethylation cannot be the only mechanism involved in demethylating the paternal DNA, and that active demethylation still takes place during replication.

In order to further investigate the relationship between replication and this demethylation, we blocked replication using aphidicolin. As shown on figure 4A, the paternal/maternal ratio at 25 hphCG after aphidicolin treatment is significantly different from that of methanol control embryos (0.4560.07 vs 0.3460.08; p-value,10 23 ) but not significantly different from that of prereplication embryos (0.4960.13; p-value.0.3). We then looked at DNA methylation levels independantly. As shown on figure 4B, an effect of the aphidicolin treatment can be observed in both pronuclei, but is much milder in the maternal pronucleus. It thus appears that in the paternal pronucleus the passive dilution mechanism as well as the active mechanism are blocked following aphidicolin treatment.

Discussion

In this study, we have analyzed the comparative dynamics of DNA methylation and hydroxymethylation during the first-cell stage in the mouse embryo, both qualitatively, using pericentric heterochromatin and quantitatively, over the replication period.

5hMeC as an intermediate of DNA demethylation

Previous studies about the kinetics of hydroxymethylation during the first cell stage have shown that hydroxymethylation remains very low in the maternal pronucleus while it increases in the paternal one, in parallel to demethylation [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF][START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF][START_REF] Ruzov | Lineagespecific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development[END_REF][START_REF] Gu | The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes[END_REF]. While our results regarding the dynamics of 5hMeC in the paternal pronucleus are mostly in agreement with those data, it is not the case in the maternal pronucleus, where we always observe a non negligible signal. Looking at the protocols used, it appears that in several studies [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF][START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF][START_REF] Gu | The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes[END_REF], the blocking time was increased while the incubation time with the primary antibodies was reduced when compared with standard protocols usually used for 5MeC 3D immunostainings in embryos [START_REF] Zaitseva | Dynamics of DNA-demethylation in early mouse and rat embryos developed in vivo and in vitro[END_REF][START_REF] Beaujean | Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer[END_REF][START_REF] Park | Dynamic DNA methylation reprogramming: active demethylation and immediate remethylation in the male pronucleus of bovine zygotes. Developmental dynamics: an official publication of the American Association of Anatomists[END_REF][START_REF] Fulka | DNA methylation pattern in human zygotes and developing embryos[END_REF]. In another study [START_REF] Ruzov | Lineagespecific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development[END_REF], the authors used a protocol similar to ours but with a much lower dilution of the antibody (1:5000 vs 1:500) and a signal amplification. In addition, in all of the above mentioned papers, data were obtained with double immunostainings for 5MeC and 5hMeC together.

When we compared these protocols, we found that the modified protocols affect the results: 5hMeC signal is of lower quality and thus appears more prominent in the paternal pronucleus (Figure S1). We observed that the signal ratio between the maternal and paternal pronuclei was modified when the antibody concentration went too low (1:2000 dilution; data not shown), because some of the signal was lost especially in the maternal pronucleus. It is therefore not surprising that, with a higher antibody dilution (1:5000) Ruzov and collaborators only observed paternal pronucleus staining: even if they used an amplification system, it cannot restore lost signal [START_REF] Ruzov | Lineagespecific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development[END_REF]. On the other hand, using lower antibody dilutions (1:100; [START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF]) might saturate the signal in the paternal pronucleus and alter the paternal/maternal ratio. To minimize these problems, we chose an intermediate dilution (1:500; as in [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF]) that provided reproducible results in terms of signal distribution and intensity. Finally, the use of double immunostainings also increased the difference in intensity between both pronuclei, in comparison to single immunostaining (Figure S2). We think this might be due to antibody competition, caused by steric hindrance when both marks are found in the vicinity of each other. It therefore appears that 5hMeC staining is very sensitive to the protocol used and this could explain the differences between our study and previous ones.

Following its rediscovery, 5hMeC has rapidly been proposed as an intermediate in the active demethylation process [START_REF] Tahiliani | Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1[END_REF]. It also fits with the patterns of 5hMeC observed in previous studies [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF][START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF][START_REF] Ruzov | Lineagespecific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development[END_REF][START_REF] Gu | The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes[END_REF][START_REF] Inoue | Generation and replicationdependent dilution of 5fC and 5caC during mouse preimplantation development[END_REF]. Indeed, in the paternal pronucleus, all studies, including ours, show an increase of 5hMeC concomitant with the decrease of 5MeC, which is in agreement with the hypothesis that 5MeC is converted to 5hMeC. In the maternal pronucleus, it has been suggested that maternal demethylation happens by dilution, i-e through a passive replication-dependent mechanism. This hypothesis is supported by data from Rougier [START_REF] Rougier | Chromosome methylation patterns during mammalian preimplantation development[END_REF] and Inoue [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF] showing the progressive disappearance of 5MeC from metaphase chromosomes during the first embryonic division, as well as by our own data showing that the 5MeC/DNA ratio is divided by 2 during replication in the maternal pronucleus. The absence of 5hMeC in the maternal pronucleus would be in agreement with this hypothesis. Some data however do not fit in this perfect picture. First, our results do not only show the presence of 5hMeC in the maternal pronucleus all along the cell cycle, but also its strong increase during the replication phase. Second, a closer look at 5MeC and 5hMeC kinetics in the paternal pronucleus shows that the increase in 5hMeC observed between late and post-replication is much more important than we would expect from the 5MeC decrease at the same time. Third, at the end of the cell cycle, we observe an important enrichment of 5hMeC on the major satellites in the paternal pronucleus, unrelated to any decrease of the 5MeC signal. Finally, we show that at least part of the active demethylation in the paternal pronucleus is dependent on replication, while Wossidlo and collaborators have shown that 5hMeC does not depend on replication [START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF].

Several non exclusive hypotheses could explain these results. First, 5hMeC could participate in passive demethylation too. This has already been proposed by Tahiliani and collaborators [START_REF] Tahiliani | Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1[END_REF], based on the fact that Dnmt1 recognizes poorly 5hMeC [START_REF] Valinluck | Endogenous cytosine damage products alter the site selectivity of human DNA maintenance methyltransferase DNMT1[END_REF]. However, while this would explain in part the 5hMeC increase in the maternal pronucleus it does not explain the other observations. Second, the demethylation process could be more complex than it appears at first glance, with the coexistence of passive and active demethylation as well as of methylation in both pronuclei. This would require the presence of an active Dnmt. Several studies have shown that the maintenance Dnmt1 [START_REF] Vassena | Species-dependent expression patterns of DNA methyltransferase genes in mammalian oocytes and preimplantation embryos[END_REF][START_REF] Cirio | Preimplantation expression of the somatic form of Dnmt1 suggests a role in the inheritance of genomic imprints[END_REF][START_REF] Kurihara | Maintenance of genomic methylation patterns during preimplantation development requires the somatic form of DNA methyltransferase 1[END_REF] and the de novo Dnmt3A [START_REF] Hirasawa | Maternal and zygotic Dnmt1 are necessary and sufficient for the maintenance of DNA methylation imprints during preimplantation development[END_REF], are expressed in both pronuclei of mouse zygotes. Some methylation could therefore take place in the maternal pronucleus as well as in the paternal pronucleus. This hypothesis is supported by the reinforcement of 5MeC, as well as of 5hMeC, that we observe on peri-NPB sequences in the paternal pronucleus between PN4 and PN5. Moreover, methylation of the paternal genome has already been shown in 1-cell embryos of rabbit [START_REF] Reis | Dynamics of DNA methylation levels in maternal and paternal rabbit genomes after fertilization[END_REF] and bovine [START_REF] Park | Dynamic DNA methylation reprogramming: active demethylation and immediate remethylation in the male pronucleus of bovine zygotes. Developmental dynamics: an official publication of the American Association of Anatomists[END_REF] using 5-Azacytidine, a DNA methylation inhibitor. Preliminary experiments we performed in the mouse embryos suggest that methylation at 29 hphCG, but not at 25 hphCG, is indeed reduced in both pronuclei after treatment with 5-Azacytidine (data not shown), but this would need further examination.

Other functions for 5hMeC?

Results from several studies [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF][START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Ruzov | Lineagespecific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development[END_REF] have shown a persistence of 5hMeC during preimplantation development, while other studies have emphasized the relatively strong presence of 5hMeC in pluripotent cells [START_REF] Ruzov | Lineagespecific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development[END_REF][START_REF] Ficz | Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation[END_REF]. Inoue and collaborators have shown that 5hMeC but also its derivates (5-formylcytosine and 5carboxylcytosine) are lost by dilution through the cell cycles [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF][START_REF] Inoue | Generation and replicationdependent dilution of 5fC and 5caC during mouse preimplantation development[END_REF]. This 5hMeC persistence suggests that it plays a role on its own. In agreement with this hypothesis, we show that the patterns of 5MeC and 5hMeC on the pericentric heterochromatin are not exactly complementary, unlike what had been suggested by other authors [START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF][START_REF] Wossidlo | 5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming[END_REF]. 5hMeC appears enriched only on a fraction of the pericentric heterochromatin in the maternal pronucleus, which probably represents a subset of the chromosomes (in mouse all chromosomes, except for the Y, carry pericentric heterochromatin). This in accordance with images from Inoue and collaborators [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF], on which only 6 out of 20 maternally inherited chromosomes show a strong 5hMeC staining. Interestingly, while 5MeC does not show any enrichment on the maternal heterochromatin during most of the cell cycle, a partial enrichment is detected on those sequences at the end of the cell cycle (PN5 and beyond), probably also on a subset of chromosomes. This had already been noted on metaphase chromosomes by Rougier and collaborators [START_REF] Rougier | Chromosome methylation patterns during mammalian preimplantation development[END_REF], and is also visible on images from Inoue [START_REF] Inoue | Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos[END_REF], where a colocalization of Embryos were first sorted according to their PN stage and the timing of their fixation. Then, for each category, embryos were sorted again according to their replication staining. Early PN3 embryos are exclusively in pre-replication, PN3 are in majority in early replication, early PN4 are mostly in mid/late replication and most of the late PN4 have completed replication. doi:10.1371/journal.pone.0038156.t001

5MeC and 5hMeC can be observed on some chromosomes. It would be interesting to determine which chromosomes show enrichment for either one or both marks on its pericentric heterochromatin.

Coexistence of both marks is also observed at the end of the cell cycle in the paternal pronucleus, not only on pericentric heterochromatin but also on other sequences localizing around the NPBs. Those could correspond to other repetitive sequences such as LINEs, in agreement with results from Iqbal [START_REF] Iqbal | Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine[END_REF], or the ribosomal DNA [START_REF] Romanova | High resolution mapping of ribosomal DNA in early mouse embryos by fluorescence in situ hybridization[END_REF]. Moreover, as this coexistence of 5MeC and 5hMeC can also be observed in ES cells [START_REF] Ficz | Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation[END_REF][START_REF] Williams | TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity[END_REF] but not in somatic adult cells where 5hMeC is mostly absent from repeat sequences [START_REF] Nestor | Tissue-type is a major modifier of the 5-hydroxymethylcytosine content of human genes[END_REF], we can hypothesize that it may represent a specific epigenetic state of embryos or embryonic cells.

Finally, the dynamics of 5hMeC lead to a peak of this mark in both pronuclei after the completion of replication. Since 5hMeC has been related to the regulation of gene expression in ES cells [START_REF] Williams | TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity[END_REF][START_REF] Xu | Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells[END_REF][START_REF] Wu | Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells[END_REF], it is possible that 5hMeC plays a role in the minor activation of the embryonic genome, which occurs simultaneously with this peak [START_REF] Bouniol-Baly | Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation[END_REF][START_REF] Aoki | Regulation of transcriptional activity during the first and second cell cycles in the preimplantation mouse embryo[END_REF]. Further studies are necessary to assess a possible role of 5hMeC in this process, as well as in the major activation which occurs at the 2-cell stage in mouse.

In conclusion, the conversion of 5MeC to 5hMeC could indeed be an intermediate step in demethylation during early development, but increasing evidences indicate that it could be as well a conversion from a repressive epigenetic mark to another, more permissive for transcription.

Materials and Methods

Ethics Statement

Animal care and handling were carried out according to European regulations on animal welfare. NB and JS both have the authorization to work with laboratory animals from the departmental veterinary regulatory services (Nu 78-95 and Nu 78-137, respectively). This work has been approved by the local ethics committee (agreement 11/048 from the Comethea Jouy-en-Josas/ AgroParisTech).

Embryo collection and culture

All products are from Sigma-Aldrich, France unless otherwise stated.

Embryos were produced by natural fertilization of C57/CBA F1 mice. Superovulation was induced by injection of pregnant mare serum gonadotropin (PMSG, Intervert, 5 UI) followed by injection of human chorionic gonadotropin (hCG, Intervert, 5 UI) 48 hours later. Female mice were then mated with C57/CBA F1 males. Fertilization occurred at about 12 hours after hCG injection which was used as reference point for embryonic development (hours post-hCG i.e., hphCG). Fertilized eggs were collected at 18-19 hphCG from the ampulla in M2 medium after a brief treatment with 1 mg/ml of hyaluronidase in phosphate buffered saline (PBS, pH7.5, AMRESCO, Solon, OH) to separate them from the surrounding follicular cells.

After collection, embryos were transferred to M16 medium and kept in an incubator at 37uC under 5% CO 2 . Depending on the experiment set, various compounds could be added to the M16 medium.

All experimental sets contained embryos from six to ten different mice. All experiments were repeated at least twice.

Assessment of replication

To avoid cross-reactivity with anti 5hMeC or 5MeC antibodies, labeling of replication was performed either by BrdU incorporation or by DIG-dUTP micro-injection.

In the first case, embryos were incubated in the presence of BrdU (100 mM in M16 medium) for 30 minutes in an incubator at 37uC under 5% CO 2 and subsequently fixed with 4% paraformaldehyde (PFA, EMS, Hatfield, PA) in PBS overnight at 4uC. These embryos incubated with BrdU were later used for anti-5hMeC and anti-BrdU double immunostainings. In the second case, DIG-dUTP (40 mM in Pipes/KCl, Roche, Switzerland) was injected in embryos using a Nikon inverted microscope with Narishige micromanipulators and an Eppendorf microinjector. Injections were performed between 22 and 25 hphCG. After injection embryos were placed in M16 in the incubator at 37uC under 5% CO 2 for 30 minutes and subsequently fixed with 4% paraformaldehyde (PFA) in PBS overnight at 4uC.

Embryos micro-injected with DIG-dUTP were later used for anti-5MeC and anti-DIG double immunostainings.

Aphidicolin experiments and controls

Embryos were collected at 18 hphCG. As a first control, some embryos were incubated in the presence of BrdU for 30 minutes at 19 hphCG and fixed in PFA 4% overnight at 4uC to check that replication had not started. Immunostaining was therefore performed on those control embryos with an anti-BrdU antibody. The rest of the embryos were incubated in M16 supplemented with either Aphidicolin (10 mg/ml, 1/400 from a 4 mg/ml stock solution diluted in methanol) or methanol (1/400 v/v) as a second control, starting from 19 hphCG. The medium was changed every 2 hours to avoid temperature-linked degradation of the drug. At 23 hphCG, a group of embryos from both batches were incubated with BrdU, fixed in PFA 4% overnight at 4uC and later stained with an anti-BrdU antibody to verify the absence of replication in the aphidicolin treated ones. The remainder of the embryos (aphidicolin treated and methanol controls) were fixed at 25 hphCG, in PFA 4% overnight at 4uC and processed for 5MeC immunostaining.

Immunofluorescence and Mounting

Embryos were fixed with 4% PFA in PBS overnight at 4uC and permeabilized with 0.5% Triton X-100 (30 min, Room Temperature (RT)) after several washes with 0.05% Tween-20 in PBS. They were blocked with 2% bovine serum albumin (BSA) in PBS for 1 h. Embryos were then incubated in 2N HCl solution at 37uC for 1 hour. Incubation with various combinations of the following primary antibodies, was performed overnight at 4uC: anti-5methylcytosine (5MeC, mouse monoclonal Eurogentec, BI-MECY1000), anti-5-hydroxymethylcytosine (5hMeC, rabbit polyclonal Active Motif 39769), anti-BrdU (mouse monoclonal Becton Dickinson 347580) and anti-digoxigenin (DIG, sheep polyclonal, Roche 11333089001) diluted in 2% BSA-PBS at 1:500, 1:500, 1:100 and 1:200, respectively.

When no DNA immunostaining was performed, after two washes with 0.05% Tween-20 in PBS (15 min each, RT), embryos were incubated with the secondary antibodies, coupled with Fluorescein (FITC), Rhodamine (TRITC) or Cyanine 5 (Cy5) (Jackson Immunoresearch, West Grove, PA) and diluted in 2% BSA-PBS at 1:200, during 1 hr (RT). They were extensively rinsed again to remove excess of antibodies and briefly postfixed (2% PFA-PBS, 20 min, RT).

When DNA immunostaining was performed, after two washes with 0.05% Tween-20 in PBS (15 min each, RT), embryos were postfixed (2% PFA-PBS, 20 min, RT) and subsequently incubated in 4% non fat milk-0.1% Tween-PBS for 3 hours. Incubation with the primary anti-DNA single stranded specific antibody (ssDNA, mouse IgM monoclonal, Millipore clone F7-26) diluted in 4% non fat milk-0.1% Tween-PBS at 1:10 was performed for 36 hours at 4uC. After two washes with 0.05% Tween-20 in PBS (15 min each, RT), embryos were incubated with the secondary antibodies, coupled with FITC, TRITC or Cy5 (Jackson Immunoresearch, West Grove, PA) and diluted in 2% BSA-PBS at 1:100, during 1 hr (RT). They were extensively rinsed again to remove excess of antibodies and briefly postfixed (2% PFA-PBS, 20 min, RT).

The embryos were finally deposited on slides and mounted under a coverslip with citifluor (Citifluor Products, Canterbury, UK).

Examples of co-immunostainings obtained with the ssDNA and the 5hMeC antibodies are shown on Figure S3.

Protocol controls

To ensure that the signals detected for 5hMeC and 5MeC using our protocol were specific, antibodies against anti-5-hydroxymethylcytosine (5hMeC, rabbit polyclonal Active Motif 39769) or anti-5-methylcytosine (5MeC, mouse monoclonal Eurogentec, BI-MECY1000) were preincubated with either 1 mM 29-deoxy-59methylcytidine-59-triphosphate (dm 5 CTP, Fermentas) or 1 mM 29deoxy-59-hydroxymethylcytidine-59-triphosphate (Hydroxymethyl-dCTP, BIOLINE). Immunostaining experiments were then performed as described above using the preincubated anti-5-hydroxymethylcytosine or anti-5-methylcytosine antibodies. Results are presented in Figure S4.

3D ImmunoFISH

Embryos were fixed with 2% PFA in PBS 20 minutes at room temperature (RT) and permeabilized with 0.5% Triton X-100 (30 min, RT) after several washes with 0.05% Tween-20 in PBS. They were blocked with 2% bovine serum albumin (BSA) in PBS for 1 h. Embryos were then incubated in 4N HCl solution at RT for 20 minutes. Incubation with primary antibody either anti-5methylcytosine (5MeC, mouse monoclonal Eurogentec, BI-MECY1000), or anti-5-hydroxymethylcytosine (5hMeC, rabbit polyclonal Active Motif 39769) diluted in 2% BSA-PBS at 1:500 was performed overnight at 4uC. After two washes with 0.05% Tween-20 in PBS (15 min each, RT), embryos were incubated with the secondary antibodies, coupled with FITC (Jackson Immunoresearch, West Grove, PA) and diluted in 2% BSA-PBS at 1:200, during 1 hr (RT). They were extensively rinsed again to remove excess of antibodies and briefly postfixed (2% PFA-PBS, 20 min, RT). The remainder of the zona pellucida was removed using acidic tyrode (40 seconds, RT) and embryos were mounted on Superfrost slides after a brief rinse in PBS, and postfixed again in PFA 4% 30 minutes at RT. A new permeabilization was performed (Triton X100 0.5%, 30 minutes, RT), followed by a brief wash in 26 SSC pH6.3 and a RNAse A treatment (200 mg/ ml in 26 SSC pH6.3; 30 minutes, 37uC). Slides were then equilibrated in hybridization buffer (50% formamide, SCC 26, Denhardt 16, 40 mM NaH2PO4, 10% dextran sulfate) for 1 h at RT. Both probes (diluted in hybridization buffer) and slides were separately denatured by heating 10 minutes at 85uC, then the major satellites probes were applied on the embryos for incubation at 37uC overnight. After two 5 minutes washes in 26 SSC pH6.3 at 42uC, a last postfixation was performed (PFA 2%, 15 minutes, RT) and the slides were mounted with citifluor (Citifluor Products, Canterbury, UK).

Major satellites probes were prepared by PCR on genomic mouse DNA using the 2 following primers 59-CATATTC-CAGGTCCTTCAGTGTGC-39 and 59-CACTTTAGGACGT-GAAATATGGCG-39 and Cy5-labeling by random priming (Invitogen Kit, Ref 18095-011).

Fluorescence Microscopy and Image Analysis

For immunostainings experiments, FITC, TRITC and Cy5 signals were recorded with a Zeiss ApoTome structured illumination system using an oil-immersion objective (Plan Apochromatic 636, n.a.1.4) and 470 nm, 530 nm or 625 nm LEDs. The distance between two consecutive optical sections was 0.27 mm.

For ImmunoFISH experiments, FITC and Cy5 signals were recorded with a Zeiss LSM 510 confocal laser scanning microscope equipped with an oil-immersion objective (Plan Apochromatic 636, n.a.1.4) and lasers at 488-and 633-nm wavelengths. Entire embryos were scanned with 0.37 mm distance between light optical sections.

Quantitative analysis was performed using the ImageJ software. Projection summing intensities of all selected slices was performed independently on both pronuclei. A region of interest was then drawn around each pronucleus and the total fluorescence intensity was measured for 5MeC or 5hMeC and ssDNA stainings. Background correction was applied for all signals. ssDNA staining was used for normalization of paternal/maternal ratios and to obtain an estimation of 5MeC or 5hMeC kinetics in both pronuclei independently.

Graphs were obtained with the Gnuplot software and a specific macro, developed by Claude Monteil (INP/ENSAT Toulouse; http://www.inp-toulouse.fr/fr/espace-tice/excel-interactif. html#affichage) was used to generate boxplots with the Excel software (Microsoft). Figures were built-up using Adobe Photoshop CS4 software.

Statistical Analysis

The staining intensity ratios at the different stages were compared with a Mann-Whitney-Wilcoxon test at level 5% using the R statistical software package [START_REF]R: A Language and Environment for Statistical Computing[END_REF].

Figure S4 Specificity of 5hMeC staining. Z-stack projections of embryos stained with either an anti-5MeC (A,B) or an anti-5hMeC antibody (C,D) that were preincubated with methyl-dCTP (A,C) or with hydroxymethyl-dCTP (B,D), before the immunostaining procedure. Around 10 embryos were analyzed per group. Scale Bar: 10 mm. (TIF)

Figure 2 .

 2 Figure 2. DNA methylation and hydroxymethylation patterns during replication. Representative z-section images of maternal PN (left) and paternal PN (right) in 1-cell embryos with various replication statuses: from pre-replication to post-replication. A) Double immunostaining of 5MeC (upper row) and DIG dUTP (middle row). Merged images are shown on the lower row (green: 5MeC; red: replication); a total of 89 embryos were analyzed. B) Double immunostaining of 5hMeC (upper row) and BrdU (middle row). Merged images are shown on the lower row (green: 5MeC; red: replication); a total of 226 embryos were analyzed. Scale Bar: 5 mm. doi:10.1371/journal.pone.0038156.g002

Figure 3 .

 3 Figure 3. Quantification of 5MeC and 5hMeC over the replication period. Quantification of the global 5MeC and 5hMeC signal was performed and corrected for background and DNA content as described in Material and Methods. A) Comparison of the paternal/ maternal ratios obtained for 5MeC and 5hMeC from pre-replication to post-replication. 10 to 27 embryos were quantified for each stage. B) Comparison of 5MeC and 5hMeC dynamics in the maternal pronucleus. Respectively 21 to 46 embryos and 10 to 27 embryos were quantified for each stage. C) Comparison of 5MeC and 5hMeC dynamics in the paternal pronucleus. Respectively 21 to 46 embryos and 10 to 27

Figure 4 .

 4 Figure 4. Relationship between DNA demethylation and replication. A) Quantification of the 5MeC paternal/maternal ratio in control (methanol) and aphidicolin treated embryos with representative images (z-projections of 3D-stacks). Images were rotated if necessary to have the maternal PN on the left and the paternal PN on the right. Scale Bar: 10 mm. B) Separate quantification of 5MeC for both pronuclei in control and aphidicolin treated embryos. 4 experiments were performed and a total of 59 (5+17+25+12) control embryos versus 61 (7+18+22+14) aphidicolin treated ones were quantified. doi:10.1371/journal.pone.0038156.g004

Table 1 .

 1 Status of replication according to the PN stage and time of fixation.

		Pre	Early	Mid	Late	Post
	Early PN3 (20-21 hphCG)	23 (100%)
	PN3 (21-23 hphCG)	10 (16.9%)	43 (72.9%)	4 (6.8%)	1 (1.7%)	1 (1.7%)
	Early PN4 (23-24 hphCG)		2 (6.9%)	11 (37.9%)	14 (48.3%)	2 (6.9%)
	Late PN4 (25-26 hphCG)			3 (13%)	5 (21.7%)	15 (65.2%)

PLoS ONE | www.plosone.org

May 2012 | Volume 7 | Issue 5 | e38156

Acknowledgments

The authors are indebted to members of Unite ´Expe ´rimentale d'Infectiologie Expe ´rimentale des Rongeurs et Poissons (UEIERP) in charge of the mice. The authors wish to thank Justine Auge ´and Anne Jeandin for their help in initiating this study. They also thank the MIMA2 facility for access to confocal and ApoTome microscopes and the Re ´gion Ile-de-France for funding this last system.

This work was supported by an INRA (Institut National de la Recherche Agronomique) ''Young Team'' research grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supporting Information

Figure S1 Comparison of two immunostaining protocols used to detect 5hMeC. Images were acquired as 3D stacks and single representative sections (A/B) as well as z-stack projections (A9/B9) are shown. A and A9) Images obtained using the protocol from Iqbal et al., 2011 [20] (n = 13). B and B9) Images obtained using the procedure commonly used for 5MeC immunostaining (n = 25). Scale Bar: 10 mm. (TIF)


J Peydecastaing 
email: jerome.peydecastaing@ensiacet.fr

C Vaca-Garcia

E Borredon

Interactions with water of mixed acetic-fatty cellulose esters

Keywords: mixed cellulose esters, contact angle, differential vapor sorption, hydrophobicity, water repellency, low-substituted cellulose esters

Cellulose powder was acylated with mixtures containing acetic, fatty and acetic-fatty anhydrides to form acetic-fatty cellulose esters. The total degree of substitution (DS) of the mixed cellulose esters (MCE) ranged from 2x10 -2 to 2.92. MCE were characterized by their interactions with water. Static contact angles with water were measured on a regular smooth surface. The values found were dependent on the fatty acyl content and independent of the acetyl content. In the case of acetic-oleic cellulose esters, the minimum DS of the oleoyl moiety required to obtain permanent water repellency was 3x10 -4 . The microporosity of the samples may account for this exceptional hydrophobic character. Nevertheless, water vapor adsorption measurements on powder samples revealed only a limited increase in hydrophobicity of the MCE compared to cellulose acetate with the same acetyl content. It was thus demonstrated that water repellency and vapor water adsorption are not correlated.

Introduction

Mixed cellulose esters (MCE), i.e. bearing at least two types of substituents, are renewable-based multifunctional polymers. The main representative examples of this family are the cellulose acetate-butyrates and acetate-propionates, industrially produced and used for their thermoplastic properties which can be modulated according to the type and number of side chains. For instance, long chain cellulose esters have better water barrier properties than cellophane [START_REF] Bras | Oxygen and water vapor permeability of fully substituted long chain cellulose esters (LCCE)[END_REF]. Some properties change noticeably with the introduction of a small amount of substituents. It has been reported that long chain cellulose esters with a low degree of substitution (DS ≤ 0.15, i.e. acylation of only 5% of the hydroxyl groups) appear to present interesting properties such as water repellency for cotton yarns and fabrics, and an ability for selective adsorption of oily compounds, useful for the decontamination of polluted water [START_REF] Deschamps | Oil removal from water by selective sorption on hydrophobic cotton fibers. 1. Study of sorption properties and comparison with other cotton fiber-based sorbents[END_REF]. In addition, applied to chemical wood modification, these esters have recently achieved success industrially due to their excellent weathering properties [START_REF] Magne | Method for treating lignocellulosic materials, in particular wood and material obtained by said method[END_REF].

The properties obtained in the works cited above are difficult to correlate with the extent of esterification, because the DS-values are low and their determination is subject to high standard deviation using present techniques. However, thanks to the TMSH (trimethyl sulfonium hydroxide) GC analytical technique recently developed [START_REF] Peydecastaing | Accurate determination of the degree of substitution of long chain cellulose esters[END_REF], we are now able to precisely characterize cellulose esters with DS < 0.1.

In this article we report on the interactions of MCE with liquid water and water vapor, and establish correlations with the DS. Although the whole DS-range was explored, the main contribution of this paper is the study of very low DS-values (10 -4 to 0.1).

Static contact angles and equilibrium moisture contents were used as characterization tools.

Experimental

Chemicals

Pure water (18.2 MΩ.cm at 25°C) was obtained from a Milli-Q (A10) device from Millipore. Alpha-cellulose from Sigma-Aldrich France (degree of polymerization = 960, 4% pentosans and conditioned at 7% moisture content), was used here as reference for the water interactions study.

2.2. Mixed cellulose esters with DS total < 0.1.

Synthesis

The synthesis of MCE has already been fully described [START_REF] Peydecastaing | Reactivity of mixed acetic-fatty anhydride mixtures toward cellulose[END_REF]. In short, cellulose was reacted with an acylation mixture obtained from reaction of acetic anhydride and fatty acids without external catalyst. MCE were thoroughly washed with ethanol then purified by Soxhlet extraction with ethanol for 8 hr. The absence of residual reagents was assessed by FT-IR (Jasco 460 device used with KBr pellet method, spectral width 4000-400 cm -1 , 32 times accumulation, 4 cm -1 resolution). The purification method was also validated by impregnating a sample of cellulose with the acylation mixture at 20°C, followed by the same purification steps. The DS of this sample was null. The purified product was then vacuum dried to constant weight at 70°C for at least 24 hr. The samples were manipulated in such a way as to avoid contact with contaminants that might modify the surface energy.

Determination of the degree of polymerization

Degree of polymerization (DP). The cellulose esters were saponified under 'mild' conditions to regenerate cellulose. 0.5 g of purified sample was stirred for 30 min in 40 ml of aqueous ethanol (70%). After the addition of 20 mL of a 0.5 N NaOH aqueous solution, the stirring was continued for 48 h at 50°C. The unreacted NaOH was back-titrated with 0.5 N aqueous HCl. The solid was recovered by filtration and thoroughly washed with deionized water and ethanol, and dried under vacuum at 50°C for 48 h. The absence of ester functions in the saponified solid was confirmed by FTIR spectroscopy. The intrinsic viscosity (η) of diluted solutions of cellulose in cupriethylendiamine, was measured at 25°C with a capillary viscometer, according to the French AFNOR standard NF T 12-005. The DP was calculated using the Mark-Houwink-Sakurada equation (Brandrup and Immergut, 1975) which correlates η and the viscosimetric molecular weight (Mv) of cellulose:

where 0.0133 and 0.905 are the coefficients corresponding to the cupriethylendiamine system and η is expressed in ml/g. Blanks were also run to evaluate the reduction in DP due to the saponification.

Contact angle measurements

Pellets (10 mm diam.) of the MCE were obtained using a laboratory press (ICL, 10 t) and a conventional pellet mold. Metal surfaces in contact with the sample were carefully cleaned to avoid contaminant sources. A drop of Milli-Q water (3 µL) was placed on the surface of the pellet and the static contact angle was measured with a goniometer DGD-R&D (GBX Instruments, France) equipped with an automatic camera recording still images every 0.1 seconds for the whole analysis period (around 5 min). Contact angles were measured automatically using the triple point calculation method. Three specimens were used for each sample. Two contact angle measurements were made per specimen.

Dynamic vapor sorption (DVS) analysis

All the experiments were performed on a DVS Advantage, automated gravimetric vapor sorption analyzer (Surface Measurement Systems Ltd., London, UK). DVS measures the uptake and loss of vapor gravimetrically using a Cahn D200 recording ultra-microbalance with a mass resolution of ±0.1 µg. The relative humidity around the sample, was controlled by mixing saturated and dry carrier gas streams using mass flow controllers. The temperature was maintained constant (±0.1°C) by enclosing the entire system in a temperature-controlled incubator. Samples were stored in a desiccator. For each experiment, cellulose ester was placed immediately into the DVS analyzer under a continuous stream of dry (< 0.1% relative humidity, RH) air. A sample size between 5 and 10 mg was used. Prior to exposure to any water vapor, the samples were dried at 0% RH to remove superficial water present and establish a dry baseline mass. The samples were exposed to the following relative humidity profile: 0%, 10%, 20%, … 90%, decreasing to 80%, 70%, … 0% RH. At each stage, the sample mass was allowed to reach equilibrium before the relative humidity was increased or decreased. An isotherm was calculated from the complete moisture sorption and desorption profile, using the DVS Advanced Analysis Suite v3.6 software. All experiments were performed at 25.0°C.

2.3. Mixed cellulose esters with DS total > 0.1.

These samples have been prepared and characterized (DS and contact angle)

previously (Vaca-Garcia and Borredon 1999), however the contact angle measurements have never been published. The synthesis protocol was as follows: a mixture composed of octanoic acid (5.6 eq/cellulose OH), acetic anhydride (2 eq/OH)

and H2SO4 catalyst (3-7 meq/OH) was heated at 90°C for 1 h. Solvent-exchanged cellulose (2.0 g dry basis) was added to the reaction medium. The whole mixture was stirred at 110-130°C for 1-3 h. At the end of the reaction, 150 mL of ethanol was added to precipitate the soluble fraction. The solid was separated by filtration over fritted glass and purified by Soxhlet extraction with ethanol for 8 h. The purified product was then dried at 105°C to constant weight. Static contact angles were measured as described above.

Results and discussion

3.1. MCE with total DS values greater than 0.1 Mixed acetic-octanoic cellulose esters obtained with sulfuric acid as catalyst showed total DS-values comprised between 0.26 and 2.9 (Table 1).

The water repellency of these MCE was evaluated by measuring the contact angle (Ѳ)

for 60 seconds. In our case, all the samples showed significant water repellency with contact angle-values of at least 89°. By convention, a material is qualified as hydrophobic if Ѳ is greater than 90°.

The upper line in Fig. 1 represents the plot of the contact angle values obtained at 60 s.

The contact angle decreases with the total degree of substitution. This fact is surprising for three reasons:

i) an increase of the total DS means less hydrophilic hydroxyl groups;

ii) the OH groups are substituted by fatty moieties supposed to increase the hydrophobicity;

iii) the proportion of octanoyl groups increases with the total DS as shown by the plot of DS 2 /DS 8 as a function of the total DS (Fig. 2). Where DS 2 is the DS of the acetyl groups and DS 8 the DS of octanoyl groups.

This disagreement can be explained by the change in porosity of the samples. In the synthesis of MCE in heterogeneous medium (solid cellulose, liquid acylating agent), the samples with low DS retain their fibrous structure and chemical modification takes place on the external molecules of the microfibrils. Conversely, a highly substituted sample loses its fibrous aspect and has a "plastic like" appearance. Consequently, when making pellets at room temperature from all these powders, the ones with lower DS-values should have a higher porosity and a more irregular surface than highly substituted ones. According to Cassie's law (eq.1), the apparent contact angle of a porous material is greater compared to a non porous version of the same material [START_REF] Cassie | Wettability of porous surfaces[END_REF].

cos Ѳ = x (cos Ѳ0 +1) -1 (eq.1)
where Ѳ 0 is the apparent contact angle of a perfect surface with no air absorbing pores and x represents the contact surface fraction of the drop with the material. Superhydrophobic materials are constructed on this principle, with nanoscale protuberances (nanopins) artificially created on the surface of a metal or other non-hydrophobic material, creating voids filled with air under the drop [START_REF] Kim | Superhydrophobic nanostructures based on porous alumina[END_REF]). Here, the x value in Cassie's equation tends to zero and Ѳ to 180°.

In the case of MCE with low DS, isolated fatty chains protuberate when fixed on the cellulose surface because this state requires less energy than being forced to lie on the surface of a hydrophilic substrate. These protuberances reinforce the Cassie effect.

Indeed, we observed a correlation between the degree of polymerization (DP) and the total DS (lower line of Fig. 1). The trend of this curve is quite similar to that of the contact angles. Indeed, the samples showing the highest DS were synthesized under more extreme conditions of temperature, catalyst and time (Vaca-Garcia and Borredon 1999) leading to lower DP values (Table 1). The decrease in DP must therefore diminish the entanglement of the cellulose chains leading to more compact configurations with less voids, and lower micro-porosity, all decreasing the contact angle values.

However, because MCE with DS values as low as 0.5 displayed water repellency with very high contact angles, we investigated their characteristics when prepared with limited chemical modification, i.e. synthesized without a strong acid catalyst (maximum preservation of the biopolymer). The fibrillar structure will thus be maintained and be closer to that of the original cellulose.

Low-substituted mixed cellulose esters

Acetic-fatty cellulose esters, with fatty chains from C8 to C18:1 (i.e. octanoyl to oleoyl), were prepared under protective conditions (i.e. treatment limiting degradation of cellulose: 1 h, 140°C, without catalyst). Their DS-values are shown in Table 2. A sample of cellulose acetate (Sample n°1) was also prepared by reacting cellulose with acetic anhydride under the same conditions.

Contact angle measurements with water were measured for 5 min as previously. The MCE samples showed high contact angle values even after this period and remained fairly constant. However, from Fig. 3 we can see that there is a slight decrease of Ѳ, and because this could correspond to a slow absorption of the water drop by the porous material or a slight evaporation phenomenon occurring, we experimented with a non-porous tablet of Teflon. As the slope of this line was identical to those of cellulose esters, we concluded that this decrease was effectively induced by the measurement conditions. All the MCE samples in Fig. 3 can be defined as exhibiting 5 min water repellency as their trends are parallel to the one observed for Teflon. Such a behavior will be qualified in this paper as "permanent", to distinguish it from many other works in the literature reporting 'instant' contact angles. It should also be noted that the slope of the line for the contact angle vs. time of cellulose acetate octanoate closely parallels that of Teflon.

These interesting results demonstrate that mixed cellulose esters with low DS of acetyl groups (DS 2 less than 0.1) and with extremely low DS of fatty acyl groups (DS f ), between 1.83x10 -3 and 6.82x10 -4 inclusive, nonetheless displayed marked water repellency (Samples 2 to 6 plus 14).

The total degree of substitution (DS total ) and DS 2 of these samples is practically the same, since DS f << DS 2 . Moreover, their values are almost constant whereas DS f varies over one order of magnitude (Table 2). The ratio DS 2 /DS f ranges from 27 (Sample 2)

to 203 (Sample 14). Consequently, the lack of correlation between contact angle and length of the fatty chain shown in Fig. 4 is not surprising.

Contrary to MCE, cellulose acetate with a DS 2 of 0.072 (Sample 1) showed high hydrophilicity, with the drop of water absorbed into the pellet in less than 2 seconds, despite the fact that the cellulose acetate sample, and indeed any of the MCE samples, have practically the same DS 2 . This fact illustrates the decisive impact of the very few fatty chains on the hydrophobicity of MCE, which, with the lowest DS f , corresponds to about one fatty chain per 1500 anhydroglucose units.

The control cellulose sample and the cellulose acetate sample behaved similarly in terms of water repellency. The control had an initial/instant contact angle value (at t 0 , just after the deposition of the drop) of 59.7° followed by complete absorption of the drop in less than 3 s, and the cellulose acetate sample a Ѳ of 68.6° with complete absorption in less than 5 s.

If we compare acetic-octanoic cellulose esters obtained with and without catalyst (Tables 1 and2), we can see that they give contact angle values of the same order even if their DS total values are very different. For example, sample 2 has Ѳ = 107° with DS 8 = 1.83x10 -3 while sample A, prepared with a sulfuric acid catalyst, has Ѳ = 103° with DS 8 40 times higher (7x10 -2 ).

In addition, all the MCE presented above exhibited the same behavior regardless of the type of fatty chain. The observations made on acetic-oleic MCE and on aceticoctanoic MCE can be generalized to include all acetic-fatty cellulose esters.

Table 3 shows the characteristics for synthesized acetic-oleic cellulose esters. The DS of the fatty acyl groups (DS 18:1 ) are as low as 7.25x10 -5 but DS total were similar to those of samples presented above (samples A to P in Table 1 and 1 to 6 in Table 2).

The DP-values of the samples given in Tables 2 and3 were practically constant at 745 ± 30.

The contact angle values as a function of time for all the Table 3 powder samples, are given in Fig. 5. Even though all the samples show high contact angle values (> 75°)

initially, we can see that some of these are not, "permanent". Sample 19 can be considered as the crossover point between "permanent" and "temporary" water repellency. Conversely, the group of samples 8, 11-18, and 21-24 can be clearly defined as hydrophobic materials (same slope as Teflon and Ѳ > 90°).

From these graphs, we identified the contact angle values obtained at 60 s and attempted to correlate them with the DS total values (Fig. 6). No correlation was found between these parameters. Furthermore, as was explained before, DS 2 >> DS 18:1 (i.e.

DS total ≈ DS 2 ), confirming that there is no correlation between Ѳ and DS 2 . In other words, the acetate content has no influence on the water repellency of the samples considered.

However, there was a significant correlation between Ѳ and DS 18:1 (Fig. 7). Ѳ increases regularly and levels off at the DS 18:1 value of 3x10 -4 . The contact angle value reached at the plateau is about 95°, which is generally considered as, "hydrophobic".

What is noteworthy is that this high hydrophobicity is reached, with a substitution, equivalent to one fatty chain per 3,000 anhydroglucose units.

However, we must keep in mind that the 3x10 -4 DS threshold is for acetic-oleic cellulose esters and this value may vary with the length of the grafted fatty chain.

Contact angle values of the other samples (regardless of the fatty chain) are also plotted as a function of the fatty acyl content in Fig. 7. We can see that the contact angle depends only on the DS f for fatty chains with from 8 to 18 carbon atoms, and it is possible that a relationship exists between the increased apolarity of the aliphatic chain and the hydrophobic character. The number of carbon atoms grafted on the biopolymer would therefore be more relevant than the number of hydroxyl functions substituted. This is the case for many properties such as water vapor permeability [START_REF] Bras | Oxygen and water vapor permeability of fully substituted long chain cellulose esters (LCCE)[END_REF], glass transition [START_REF] Sealey | Novel cellulose derivatives .4. Preparation and thermal analysis of waxy esters of cellulose[END_REF]) and wood dimensional stability [START_REF] Hill | The dimensional stabilisation of Corsican pine sapwood by reaction with carboxylic acid anhydrides -The effect of chain length[END_REF].

As a final point, we characterized four samples by dynamic vapor sorption with water:

untreated cellulose (blank), cellulose acetate (sample 1), cellulose acetate-octanoate (sample 2) and cellulose acetate-oleate (sample 14). As the first two samples had not shown water repellency whereas samples 2 and 14 had shown 5 min water repellency, it was surprising therefore to find that the sorption isotherms of the cellulose derivatives showed practically the same trend regardless of the DS or the type of substituent (Fig. 8). Moreover, the reduction of the equilibrium moisture content with regard to cellulose was quite small; only one percentage point less than the blank. A sample of Teflon, a material recognized as being perfectly hydrophobic, has a water sorption of 0% over the whole range of relative humidity. Therefore, the MCE samples that had contact angles almost as high as Teflon now display water sorption behavior almost akin to hydrophilic cellulose.

These experiments demonstrate clearly that the hydrophobicity of a cellulosic material is shown in several ways that are not necessarily correlated. MCE with very low DS can be considered as hydrophobic (with regard to their water repellency). But from the standpoint of affinity to water vapor, they are only a little less hydrophilic than cellulose.

A possible explanation for these differences of behavior towards water vapor and liquid comes from their physical states. Vapor is constituted of individual molecules able to reach the numerous hydrophilic hydroxyl groups remaining in the cellulose esters. Conversely, liquid water is a cluster of hydrogen bonded molecules. A drop of water displays high surface energy and low wettability on hydrophobic surfaces, especially if the latter also have fatty nanopins.

Conclusions

Concerning the mixed acetic-fatty cellulose esters, formed into pellets, we have found that:

• Water repellency is obtained at very low DS total values (< 0.06). Contact angles are comprised between 90° and 100°.

• Water repellency depends essentially on the fatty acyl content. The minimum value for 5 min water repellency in the case of acetic-oleic cellulose esters is DS 18:1 = 3x10 -4 , which is extremely low.

• The contact angle of cellulose acetate (without fatty acyl groups) with DS 2 ≤ 0.072 is null.

Concerning the mixed acetic-fatty cellulose ester powders, we have found that:

• Equilibrium moisture content of MCE with very low DS is almost as high as cellulose acetate with very low DS.

• Neither of the samples can be considered as hydrophobic, Because of their affinity for water vapor they are just slightly less hydrophilic than cellulose.

As a general conclusion, the so-called hydrophobic cellulose esters can display completely different affinities for water depending on its physical state: liquid or vapor. Individual vapor molecules can reach the hydroxyl sites whereas a cluster of molecules making up a drop of water is confronted with a highly hydrophobic barrier, with the fatty chains probably protuberating on the surface of a porous material, and Cassie's effect may reinforce this hydrophobicity. 1.83x10 --3 8 5.19x10 --2 27 3 4.88x10 --2 1.02x10 --3 10 4.98x10 --2 48 4 5.03x10 --2 6.82x10 --4 12 5.10x10 --2 74 5 6.10x10 --2 6.78x10 --4 14 6.17x10 --2 90 6 6.37x10 --2 5.25x10 --4 16 6.43x10 --2 121 14 5.74x10 --2 2.83x10 --4 18:1 5.77x10 --2 203 2.30x10 --2 7.25x10 --5 2.31x10 --2 317 8 2.60x10 --2 3.51x10 --4 2.64x10 --2 74 9

Sample

3.78x10 --2 1.70x10 --4 3.80x10 --2 222 10 3.80x10 --2 1.64x10 --4 3.82x10 --2 231 11 5.20x10 --2 2.27x10 --4 5.22x10 --2 229 12 5.70x10 --2 2.83x10 --4 5.73x10 --2 202 13 5.70x10 --2 3.12x10 --4 5.73x10 --2 183 14 5.74x10 --2 2.83x10 --4 5.77x10 --2 203 15 6.10x10 --2 3.31x10 --4 6.14x10 --2 184 16

6.20x10 --2 2.54x10 --4 6.23x10 --2 244 17 6.30x10 --2 3.37x10 --4 6.33x10 --2 187 18 6.60x10 --2 2.20x10 --4 6.62x10 --2 300 19 6.70x10 --2 1.84x10 --4 6.72x10 --2 365 20 7.00x10 --2 1.06x10

Fig. 1 Fig. 3 Fig. 4 Fig. 6 Fig. 7

 13467 Fig. 1 Water contact angles and degree of polymerization of mixed acetic--octanoic cellulose esters with DS total > 0.1

Table 1 Acetic--octanoic cellulose esters with DS total comprised between 0.1 and 3

 1

		DS 2	DS 8	DS total	DS 2 /DS 8	Ѳ°	DP
	A	0.19	0.07	0.26	2.71	103	404
	B	0.21	0.08	0.29	2.63	106	450
	C	0.22	0.1	0.32	2.20	104	330
	D	0.25	0.1	0.35	2.50	104	299
	E	0.27	0.1	0.37	2.70	104	287
	F	0.66	0.25	0.91	2.64	97	190
	G	0.66	0.27	0.93	2.44	96	138
	H	0.67	0.29	0.96	2.31	96	183
	I	0.76	0.26	1.02	2.92	95	215
	J	0.75	0.34	1.09	2.21	94	174
	K	0.83	0.31	1.14	2.68	92	238
	L	0.80	0.41	1.21	1.95	95	136
	M	0.94	0.41	1.35	2.29	91	138
	N	1.31	0.69	2.00	1.9	92	100
	O	1.42	0.76	2.18	1.87	93	100
	P	1.86	1.04	2.90	1.80	89	100

Table 2 Acetic--fatty cellulose esters with DS total < 0.1. n = number of carbon atoms of the saturated acyl chain Sample DS 2 DS 18:1 DS total DS 2 /DS 18:1 7

 2

Table 3 Acetic--oleic cellulose esters with DS total < 0.1

 3

Acknowledgments: We thank LAPEYRE for financial support and Mr. Michael Charton for his precious technical help.