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BOR - Papers in Press, published online ahead of print October 12, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.046284
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BIOLOGY OF REPRODUCTION 74, 307–313 (2006)
DOI: 10.1095/biolreprod.105.046284
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Effect of Zona Pellucida Removal on DNA Methylation in Early Mouse Embryos1

Ricardo C. Ribas 2 3 46 , Jane E. Taylor 3 4, Caroline McCorquodale 4, Ana C. Mauricio 56 , Mário Sousa 6, and Ian Wilmut 3 4

Roslin Institute,4 Roslin, Midlothian EH25 9PS, United Kingdom Centro de Estudos de Ciência Animal (CECA)-Instituto de Ciências e Tecnologicas Agrárias e Agro-Alimentares (ICETA),5 Campus Agrário de Vairão, 4485–661 Vairão, Portugal Instituto Ciências Biomédicas Abel Salazar (ICBAS),6 4099–003 Porto, Portugal

ABSTRACT

Removal of the zona pellucida is known to affect mouse development to term. Zygotes were recovered immediately after fertilization and their zona pellucida removed by exposure to pronase before culture and comparison with zona-intact embryos. The effect of removing the zona pellucida was assessed in terms of embryo development to blastocyst, DNA methylation, histone acetylation, and expression of three developmentally regulated genes. No significant differences were seen in percentage of embryos that developed to the blastocyst stage. However, zona-free embryos showed a significant reduction in the DNA methylation level at two-cell and four-cell stages, but no differences at pronuclear, morula, and blastocyst stages, as observed by immunofluorescence. Mechanical or enzymatic removal of the zona pellucida showed similar DNA methylation staining patterns at the two-cell stage. The time when the zona pellucida was removed appears to influence the levels of DNA methylation. When zona removal was delayed for 8 h, there was no difference in DNA methylation levels between zona-free and zona-intact two-cell embryos, indicating that the critical time is early on, between 1 and 8 h postfertilization. In contrast, when immunofluorescence analysis of histone acetylation was performed, no significant differences were seen between zona-free and zona-intact embryos at any of the developmental stage. Similarly, no differences were found regarding the onset of transcription of Dnmt1s, Nanog, and Fgf4 genes.

developmental biology, early development, embryo, gene regulation

INTRODUCTION

Oocytes of all vertebrates are surrounded by an extracellular matrix. This is known as chorion in fish [1], perivitelline membrane in birds [2], and zona pellucida (ZP) in mammals [3]. In the mouse, the ZP starts to be produce during folliculogenesis. This process starts just after birth and continues during the reproductive life of the female [4], to prepare the eggs for fertilization. After fertilization, the ZP surrounds the embryo during early development, until the blastocyst stage. At this point, embryos must hatch from the ZP to establish direct contact with endometrium, allowing implantation to take place [5]. Other suggested functions of the ZP include mechanical protection for the oocyte and embryo until blastocyst stage, facilitating the development of tight junctions between blastomeres during compaction [6, 7], protection of the early embryo from potential immunological rejection [8], and preventing embryo adherence. Zona-free (ZF) mouse embryos transferred to recipient mice at the one-cell, two-cell, and four-cell stages do not implant and seem to adhere to the oviduct wall and/or to each other in groups [9]. Fertilization is one of the main roles in which the ZP is involved: it provides binding sites to sperm receptors, promoting initiation of the acrosome reaction before fusion to the oocyte's oolemma and prevents polyspermy after spermatozoa entry [6].

During early embryo growth, development, and differentiation, gene expression is highly dependent on epigenetic modifications of the genome (reviewed in [10]). These modifications can occur either at the amino terminal tail of the core histones, as methylation, acetylation, phosphorylation, ubiquitination, but also in the DNA itself as methylation (reviewed in [11]). The most described modification in the DNA is its methylation, which is characterized by a covalent addition of a methyl group mainly in the cytosine residues at position 5 in CpG dinucleotides, becoming 5-methylcytosine. A relationship between DNA methylation and gene silencing has been shown [12], for example, when expression is not required or may be detrimental to genomic stability (reviewed in [13]). The enzymes responsible for this process are known as DNA methyltransferases (Dnmts). The most abundant is Dnmt1. Deletion of Dnmt1 in the mouse leads to global demethylation and embryonic death [14]. In somatic cells, DNA methylation is known to have other functions in X-chromosome inactivation, genomic imprinting, inactivation of retroviral sequences (reviewed in [10]; [15]), tumor formation (reviewed in [16]), and aging process (reviewed in [17]).

Although stable in somatic cells, DNA methylation changes during early development [18]. During mouse embryo development, an active demethylation wave and DNA decondensation, seen as an increase in pronuclei size, occurs in the male pronucleus within hours after fertilization, but in the female, pronucleus demethylation occurs passively in subsequent cleavage divisions. Santos et al., in 2002, showed that, in mouse, de novo methylation occurs in the blastocyst in the inner cell mass but not in the trophectoderm [19]. Enzymes responsible for de novo DNA methylation are Dnmt3a and Dnmt3b [20]. However, this pattern varies within different species [21].

Histone acetylation is a regulatory histone modification that occurs predominantly at the level of the lysines in the amino-terminal tail of histones 3 and 4. It plays an important role in gene expression by altering the accessibility of DNA to proteins, such as transcription factors. In the majority of cases, genes that are undergoing transcription are hyperacetylated, and those that are repressed are deacetylated [22, 23].

In view of previous results showing that removal of the ZP affects mouse development to term [24], and because normal regulation of DNA methylation is essential for normal development, the effect of the ZP removal on DNA methylation of preimplantation mouse embryos was performed by 5-methylcytosine antibody. Histone 4 lysine 5 (H4K5) acetylation antibody was also used to study the effect of zona removal on histone acetylation.

MATERIALS AND METHODS

All chemicals were supplied by Sigma-Aldrich (Dorset, U.K.) and all embryo manipulation was performed at 37°C unless indicated otherwise.

Production and Collection of Murine In Vitro Fertilized Embryos

All animal procedures were under strict accordance with U.K. Home Office regulations and within a project license issued under the Animal (Scientific Procedures) Act of 1986. All mice were bred and raised at the Roslin Institute. Eight- to 10-wk-old B6D2F1 (C57BL/6JxDBA/2) mouse females were superovulated by intraperitoneal injections of 5 IU eCG (Folligon; Intervet) followed by 5 IU hCG (Chorulon; Intervet) 48 h later and mated with B6D2F1 mouse males. Zygotes were recovered approximately 16 h after hCG injection. At this point, a majority of the oocytes should have been fertilized for 1 h [25]. Immediately after recovery, cumulus cells were removed by incubation with hyaluronidase (300 U/ml) in 4-(2-hydroxyethil-1-piperazinethane sulfonic acid) (Hepes)-buffered CZB media-[hCZB] [26] and ZP digested from half of the embryos, using 5 mg/ml pronase in hCZB. In later studies, the ZP was removed using pronase 8 h after recovery from the oviduct. In some cases, the ZP was removed mechanically using a Nikon Eclipse TE300 microscope with a holding pipette and a sharp tool, which is used to remove the ZP. All embryos were cultured individually in vitro in 5-µl droplets of CZB [26] under paraffin oil (Fluka) at 37°C and 5% CO2 in air for 4 days (Day 0 = recovery). Embryo development was assessed daily.

The ZP was removed from control zona-intact (ZI) embryos before fixation using the methods described above to prevent differences in immunostaining techniques resulting from the presence of a ZP.

Number of Cells per Blastocyst

Some ZF and ZI embryos that reached the blastocyst stage were stained using Hoechst (5 µl/ml). The mean number of cells per blastocyst was determined after three consecutive counts for each blastocyst.

5-Methylcytosine and H4 Lysine 5 Acetylation Immunodetection in EmbryosM

Embryos were washed in phosphate buffer solution (PBS) and fixed overnight at 4°C in 4% paraformaldehyde. To compare several development stages in the same immunostaining experiment, some embryos were kept in 4% paraformaldehyde at 4°C for 1–6 days (control experiments showed no differences in staining within embryo from the same stage when processed within 7-day intervals [13]).

Polyvinyl alcohol (PVA) at a concentration of 0.1 mg/ml was added to the media to prevent ZF embryos from sticking to the dishes during the staining procedure. After fixation, embryos were washed extensively in 0.05% Tween 20 (VWR) and permeabilized in 0.2% Triton X-100 (VWR) for 30 min at room temperature. After extensive washes in 0.05% Tween 20, embryos were subjected to 2 M hydrochloric acid for 1 h at 37°C to denature the DNA and then washed in 0.05% Tween 20. After blocking for 1 h in 2% bovine serum albumin (BSA) at room temperature, methylated DNA was visualized with a mouse monoclonal antibody against 5-methylcytosine (Eurogentec, Belgium). Incubation with this antibody was performed at 37°C for 1 h (1:400 dilution in block solution [BS]: 1% BSA, 0.05% Tween 20 in PBS). Embryos were washed four times in BS for 15 min each and incubated in donkey anti-mouse IgG conjugated to FITC (Jackson ImmunoResearch) at a concentration of 1:200 for 1 h at room temperature. After extensive washes in BS, embryos were postfixed in 4% paraformaldehyde for 30 min and mounted on multiwell slides (VWR) with Vectashields containing 4,6-diamino-2-phenylindole (DAPI) (Vector Laboratories Ltd.).

The staining protocol for acetylation of H4 lysine 5 was as described above for 5-methylcytosine, without the denaturation step. Acetylation at histone 4 at lysine 5 was visualized with an anti-H4Ac5 antibody (R40; kind gift from Bryan Turner). Incubation in primary antibody (1:200 dilution in BS) as well as in secondary antibody fluorescein coupling (Jackson ImmunoResearch) was performed at room temperature for 1 h. Comparisons were made between ZF and ZI embryos for each stage of development. Control embryos were prepared by omitting the primary antibody or both the primary and secondary antibodies.

Whole-Mount Fluorescence Microscopy and Quantitative Analysis of Nuclei Fluorescence

Fluorescence observation was performed on a Nikon upright microscope (Microphot), using a filter wheel equipped with standard filters for FITC and DAPI emissions. Images were captured through a Nikon Fluar 40x objective (NA 0.75) by a cooled CCD Digital Pixel camera with a KAF 1600 sensor coupled to the IPLab Digital Pixel software. The same software was used for quantification of total embryonic fluorescence intensities.

Nuclear fluorescence intensities of nuclei in cleavage stage embryos were measured by manually outlining all nuclei. For blastocysts, at least 15 nuclei were randomly selected from inner cell mass and trophoectoderm cell populations based on cellular morphology (small and compact cells in inner cell mass compared with elongated cells in trophectoderm). A total fluorescence intensity emitted by each individual nucleus was measured using IPLab Digital Pixel software and averaged per each embryo.

Assessment of Gene Expression

Total RNA was extracted from groups of six preimplantation embryos using the Qiagen QIAshredder kit and RNeasy kit, following manufacturer's instructions. Reverse transcription was performed immediately after RNA extraction using a random hexamer (pd(N)6) primer and the First-Strand cDNA Synthesis kit (Amersham Biosciences). Polymerase chain reaction (PCR) was carried out using Thermo-Start PCR master mix (ABgene, U.K.) in a total reaction volume of 25 µl containing PCR master mix (1.5 mM magnesium; 0.8 mM dNTP, 1U Thermo-Start Taq polymerase), 250 nM each of forward and reverse primers, and 2 µl template cDNA. After a 5-min initial incubation at 95°C, reactions were subjected to 40 cycles of denaturation (94°C for 30 sec), annealing (58°C: Dnmt1s, Ezh2, Nanog; and 60°C: Fgf4 for 30 sec), and extension (72°C for 45 sec), with a final extension time of 10 min at 72°C. All PCR products were separated on a 2% (w/v) agarose gel, visualized after staining with ethidium bromide, cloned into the pCR4-TOPO vector (TA cloning kit; Invitrogen, The Netherlands) and sequenced by MWG Biotech, UK. Primers used were 5'-AAGCACAATGCAACACCAAA-3' and 5'-AGACGGTGCCAGCAGTAAGT-3' for Ezh2; 5'-GACTACCTGCTGGGCCTCAAAAG-3' and 5'-TTGGTCCGCCCGTTCTTACTGAG-3' for Fgf4; 5'-CTTACAAGGGTCTGCTAC-3' and 5'-CTGAAACCTGTCCTTGAG-3' for Nanog and Dnmt1s [27]. Each experiment was repeated at least four times for each gene, and Figure 5 shows a representative PCR.


Figure 5
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  		FIG. 5. Gene expression of Nanog, Dnmt1s, and Fgf4 during mouse preimplantation development of in vivo-fertilized embryos, cultured with and without ZP. Gene expression was assayed by RT-PCR of a pool of six embryos from two-cell ZF, two-cell ZI, four-cell ZF, four-cell ZI, eight-cell ZF, eight-cell ZI, morula ZF, morula ZI, blastocysts ZF and blastocyst ZI. Ezh2 served as a ubiquitously expressed positive control

Statistical Analysis

Percentages. Percentages of embryos reaching the two-cell, four-cell, morula, and blastocyst stages were analyzed using a generalized linear mixed model (GLMM) and the method of Breslow and Clayton [28]. This method took account of a binomial distribution of the numbers of embryos reaching the various stages of development and any extra variation between repeat runs of the experiment and between treatment groups of embryos within runs. Treatment means and standard errors of differences were estimated in the logistic scale by this method, and statistical significance of differences between these treatment means was determined approximately using Student t-tests with degrees of freedom equal to those associated with random interaction of runs and treatments.

Blastocyst cell number. Residual maximum likelihood (REML) was used to estimate the treatment effects of zona type on blastocyst cell number taking into account random variation between experimental runs, between treatment groups within runs, and between blastocysts within groups. Cell numbers were left untransformed, as there was little evidence of the variance changing with the mean. Statistical significance was determined approximately as described earlier.

Quantification. REML [29] was also used to estimate the effects of ZP removal, development stage, and blastocyst cell type taking into account random variation between experimental runs, between treatment groups within runs, between embryos within groups, and between cells within embryos. Quantification was transformed to the logarithmic scale before analysis, as there was evidence of variation increasing with the mean. In addition, the variance between cells within embryos appeared to change with development stage, and this was also accounted for in the REML analysis.

Statistical significance of differences between means in the logarithmic scale was established approximately using Student t-tests as above. REML is an extension of the analysis of variance for observations with unequal replication and a nested structure as described above. Exact significance tests are not generally available with REML or GLMM.

Units for area and total nuclear fluorescence are arbitrary units given by the software.

Deviations to the means or back-transformed means where the logistic or logarithmic scale was used were represented by standard errors (SEM).

RESULTS

In Vivo Fertilized Control Development Until Blastocyst and Blastocyst Quality

There was no significant difference in development to blastocyst for in vivo fertilized embryos cultured with and without ZP (70.3% ± 8.0% vs. 61.0% ± 9.1%, respectively; P > 0.2 Fig. 1), although previous results from our group have shown a significant reduction in development to term after removal of the ZP [24].


Figure 1
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  		FIG. 1. Development of zona-free (ZF; n = 410) and zona-intact (ZI; n = 449) in vivo-fertilized embryos. Bar graphs represent mean ± SEM

Blastocyst quality was assessed by number of cells per blastocyst. Morphologically, ZF blastocysts look smaller, less expanded, and more irregular than ZI (Fig. 2); however, the number of cells per blastocyst was not significantly different (37.5 ± 2.21, n = 118; 37.8 ± 2.36, n = 73; P > 0.2, for blastocysts cultured with and without ZP, respectively).


Figure 2
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  		FIG. 2. Blastocyst morphology of ZF and ZI embryos. Blastocysts were fertilized in vivo and then cultured without (a) and with (b) ZP. ZF blastocysts look smaller, less expanded, and more irregular than ZI ones. Bar = 90 µm

Effect of Zona Removal on DNA Methylation in Two-Cell-, Four-Cell-, Morula-, and Blastocyst-Stage Embryos

After ZP removal and culture of in vivo fertilized embryos, 5-methylcytosine fluorescence was quantified in two-cell, four-cell, morula, and inner cell mass and trophectoderm in the blastocyst. Interestingly, ZF embryos showed a 30% reduction in the levels of DNA methylation compared with ZI embryos at two-cell stage (2.04 ± 0.36 vs. 2.90 ± 0.51, respectively; P < 0.05) and 43% reduction at four-cell stage (1.32 ± 0.23 vs. 2.33 ± 0.41, respectively; P < 0.01; Fig. 3, a and b). At the morula stage however, the reduction in DNA methylation of ZF embryos had lessened compared with ZI embryos and was not statistically different (1.46 ± 0.26 vs. 1.81 ± 0.32, respectively; P > 0.1). At the blastocyst stage, the levels of fluorescence were very similar for ZF and ZI embryos in both inner cell mass (1.35 ± 0.24 vs. 1.43 ± 0.25, respectively; P > 0.2) and trophectoderm (0.90 ± 0.16 vs. 0.92 ± 0.16, respectively; P > 0.2; Fig. 3, a and b).


Figure 3
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  		FIG. 3. Methylation analysis in preimplantation ZF and ZI mouse embryos. A) 5-Methylcytosine Immunofluorescence of in vivo-fertilized two-cell (a, b), four-cell (c, d), morula (e, f), and blastocyst (g, h) embryos, cultured with (ZI) and without (ZF) ZP. Bar = 20 µm. B) Quantification of total nuclear DNA methylation intensities in in vivo-fertilized two-cell, four-cell, morula, and blastocyst embryos (ICM, inner cell mass; TE, trophoectoderm) cultured with (ZI) and without (ZF) ZP. Each column represents the back-transformed mean value of these intensities per developmental stage except for blastocysts, where we distinguished between ICM and TE. Samples sizes (n) are indicated above the corresponding column. Bar graphs represent mean ± SEM. Significant differences are shown by * P < 0.05 and ** P < 0.01

Effect of Mechanical Zona Removal on DNA Methylation in Two-Cell-Stage Embryos

To exclude the possibility that enzymatic digestion with pronase was in some way responsible for the changes observed in DNA methylation levels, the ZP was removed by an alternative method. Mechanical removal of the ZP resulted in a similar reduction in DNA methylation levels in two-cell embryos, as seen after removal with pronase digestion compared with ZI embryos (Fig. 4). However, there was no difference between the two methods of ZP removal (2.10 ± 0.44 vs. 2.06 ± 0.44; P > 0.2 for pronase and mechanical removal, respectively).


Figure 4
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  		FIG. 4. 5-Methylcytosine total nuclear fluorescence of in vivo-fertilized two-cell embryos, cultured with and without ZP from the zygotes stages. ZP was removed either with pronase (Pr; n = 87) OR mechanically (MR; n =38). ZI represents control ZI embryos (n = 62). Bar graphs represent mean ± SEM. a,b, P < 0.02; a,c, P > 0.2; b,c, P < 0.01

Effect of Delaying ZP Removal on DNA Methylation

To determine if the time of ZP removal was critical for normal development, zygotes were collected from mothers and cultured for 8 h before ZP removal with pronase incubation. Eight hours culture time was chosen to ensure that the zygotes were still in the first cell cycle, before DNA replication. Two-cell ZF embryos showed a nonsignificant reduction in 5-methylcytosine fluorescence intensity compared with ZI (2.23 ± 0.28 vs. 2.68 ± 0.34; P > 0.2, respectively), indicating that the critical period is between 1 and 8 h postfertilization.

Effect of Zona Removal on DNA Methylation in Male and Female Pronuclei

Seven hours after zona removal, male and female pronuclei were analyzed for DNA fluorescence intensity and size. Determination of pronulear area is necessary because the male pronucleus increases in size in the first hours after fertilization, due to DNA decondensation. The aim was to investigate the effect of the ZP removal on DNA methylation at an earlier stage of development. The male pronuclei of ZF embryos were smaller and more methylated than those of ZI embryos, although not significantly different. Similarly, female pronuclei size and DNA methylation levels were similar for ZF and ZI groups (Table 1).


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  		TABLE 1. 5-Methylcytosine total nuclear fluorescence and size of in vivo-fertilized pronuclear stage embryos approximately 7 h after fertilization, cultured with (ZI) and without (ZF) zona pellucida

Effect of Zona Removal on H4K5 Acetylation in Two-Cell-, Four-Cell-, Morula-, and Blastocyst-Stage Embryos

Histone acetylation plays an important role in gene expression by altering the accessibility of DNA to proteins such as transcription factors. In the majority of cases, genes that are undergoing transcription are hyperacetylated, and those that are repressed are deacetylated. Comparisons were made between ZF and ZI embryos at each stage of development (two-cell, four-cell, morula, and blastocyst inner cell mass and trophectoderm) to assess the acetylation of lysine 5 on histone 4. Similar levels of fluorescence were observed for ZF and ZI embryos for each stage of development: two-cell (5.27 ± 0.53, n = 21 for ZF; and 4.69 ± 0.47, n = 21 for ZI; P > 0.2); four-cell (2.05 ± 0.21, n= 20 for ZF; and 2.40 ± 0.24, n = 21 for ZI; P > 0.1); morula (1.7 ± 0.17, n = 20 for ZF; and 2.02 ± 0.2, n = 22 for ZI; P > 0.1); and blastocyst, including inner cell mass (1.38 ± 0.15, n = 15 for ZF; and 1.23 ± 0.13, n = 16 for ZI; P > 0.2) and trophectoderm (0.93 ± 0.10, n = 15 for ZF; and 0.93 ± 0.10, n = 16 for ZI; P > 0.2).

Effect of Zona Removal on Expression of Dnmt1s, Nanog, and Fgf4

To address the possibility that reduced levels of DNA methylation in ZF embryos at the time of genome activation may result in altered gene expression, onset of transcription was observed for three genes essential for early mouse development. Ezh2 was used as a positive control, as it is ubiquitously expressed during preimplantation development from oocytes to blastocyst. Onset of transcription of the somatic form of Dnmt1 (Dnmt1s), Nanog, and Fgf4 occurs after embryonic gene activation between the four-cell and morula stages, and no maternal mRNA is carried from the oocyte [27, 30, 31]. Figure 5 shows RT-PCR results for the three different genes. Transcription for Nanog started to occur for the majority of the embryos at the morula stage, although the Nanog transcript was observed in some embryos at the eight-cell stage. Similar results were seen in ZF and ZI embryos. Dnmt1s and Fgf4 mRNA expression began at the four-cell stage and continued through to the blastocyst stage. Despite changes in methylation of DNA, the onset of genome activation for these three genes was similar for embryos with or without ZP (Fig. 5). However, small but meaningful differences in gene expression between ZF and ZI embryos may have been missed by using a nonquantitative RT-PCR assay employing 40 amplification cycles.

DISCUSSION

In the present study, removal of the ZP immediately after recovery of fertilized eggs caused a reduction in DNA methylation at the two- and four-cell stages. As the same effect was seen when the zona was removed mechanically or by exposure to pronase, it does not reflect effects of an enzyme on the cells. There was no reduction if the zona was removed 8 h after zygote recovery, suggesting that the effect can only occur during a very brief period. DNA methylation levels were restored to normal in the morula and blastocyst stages. It is important that confirmation of these effects is sought by molecular analysis. Despite this transient reduction in DNA methylation, ZP removal had no effect on development to the blastocyst stage or cell number in blastocysts, although, in an earlier study, was observed to reduce development to term [24].

The time when the ZP was removed appears to influence the levels of DNA methylation. When zona removal was delayed for 8 h, there was no difference in DNA methylation levels between ZF and ZI two-cell embryos, indicating that the critical time is early on, between 1 and 8 h postfertilization. This suggests that the period immediately after the fertilization is the most sensitive. At this time, the male DNA is very close to the oolemma, which may make it more vulnerable to changes during ZP removal and possibly affecting DNA methylation as well. ZF and ZI embryos were observed at the pronucleus stage, approximately 7 h after zona removal. Zona removal had no effect on male or female pronuclear area or pronuclear DNA methylation level. Hence, visible changes arise between 7 and 24 h (two-cell stage) postfertilization.

During early stages of development in the mouse, demethylation of the DNA begins at fertilization and continues until the blastocyst stage. This occurs, first, in an active way in the paternal genome [32–34] and, second, in a passive way during the first cleavage divisions from two-cell to morula stage [32, 35]. Our ZI DNA methylation staining pattern confirms these results, showing passive demethylation until the blastocyst stage and de novo methylation in the inner cell mass of the blastocyst as previously shown [19]. By contrast, a reduction in the ZF fluorescence emission was observed when compared with ZI controls. Substantial differences in DNA methylation occurred at two-cell and four-cell stages, contrasting with no differences at morula or blastocyst stages (either in inner cell mass or trophectoderm). However, small differences may not have been detected because these may be masked by differences in access of antibody to the interphase nuclei. There is also a possibility at the later stages of development that nuclei may overlie one another, causing misleading measurements. As removal of the zona did not affect development to the blastocyst stage or the number of cells in the blastocyst, ZF embryos are able to recover the normal level of DNA methylation. It is interesting to note that DNA methylation in ZF embryos remained relatively constant for four-cell, morula, and blastocyst inner cell mass stages.

Interestingly, no differences were seen when acetylation at lysine 5 in histone 4 was analyzed in ZF or ZI embryos at any of the stages of the development, but there is a possibility that small differences may not have been detected. The lysine 5 is the final lysine to be acetylated in histone H4, after lysine 16, 8, and 12, reflecting the hyperacetylated state of H4, and is strongly correlated with the active states of the genes (reviewed in [36]; [37]). This could explain our data showing that no differences were found regarding onset of transcription for Dnmt1s, Nanog, and Fgf4, although, small differences in expression would not have been detected by the assay method used in this study. The present observations provide no indication of the sites at which DNA methylation was lost. This information could be obtained by molecular analyses of DNA methylation within regulatory regions of critical genes and in repeat sequences. Disturbance of DNA methylation at both regulatory regions and repeat sequences has been shown after embryo culture and manipulation [38, 39].

There are earlier observations showing epigenetic effects of zona removal or culture environment on embryo development and, in some cases, this has been shown to be associated with changes in DNA methylation. Removal of the ZP from mouse zygotes influences development to term. This effect was associated with differences in cell contact at the four-cell stage, and the authors suggested that cell association influences formation of the inner cell mass [40]. The health of offspring of several species is prejudiced by culture in inappropriate conditions, such as use of serum in the media [38, 39]. Gross abnormalities in gene expression have been observed in embryos, fetuses, and offspring produced by nuclear transfer, and in some cases, has been associated with abnormal DNA methylation [41]. In some species, the inefficiency of nuclear transfer is associated with epigenetic effects on expression of imprinted genes [42]. The mechanisms that bring about these epigenetic effects are not known. Several possible causes of the transient reduction in DNA methylation level in the present studies can be considered.

The mechanisms that regulate DNA methylation in the preimplantation embryos are not yet fully established in any species, although one important gene is Dmnt1o, which is synthesized in the oocyte, but resides in the cytoplasm and is functional in preimplantation embryos to maintain genomic imprinting [43]. It is also possible that the pronase, as a protease enzyme, could in some way affect some of the cytoplasmic enzymes of the zygote/embryo, which may have a downstream effect on DNA methylation, altering patterns of the preimplantation embryos. However, the fact that mechanical removal of the ZP had a similar effect suggests that it is not the pronase that is responsible for the altered levels of DNA methylation, but the absence of the zona itself.

Fertilization causes a release of cortical granules to the perivitelline space by exocytosis. Some of the proteins expelled by the cortical granules help the ZP to prevent polyspermy, but others remain in the perivitelline space, forming the cortical granule envelope [44, 45]. The premature loss of the proteins from the cortical granule envelope by the time of the zona removal may be responsible for the alterations of DNA methylation levels. A variety of proteins have been shown to be present in the cortical granules, including p62/p56, p32, p75, n-acetylglucosaminidase, ovoperoxidase, calreticulin, tissue plasminogen activator, heparin binding placental protein, and other proteinases (reviewed in [46]). Little is known about the proteins present in the extracellular matrix. However, Hoodbhoy et al., in 2001 [47], identified two proteins in the extracellular matrix with molecular weights of 62 kDa (p62) and 56 kDa (p56) using Western blots. ZF fertilized oocytes expressed lower levels of p62 and p56 proteins compared with ZI ones, showing that loss of these proteins occurs after zona removal [47]. It was also shown in mice that new synthesis of some of these proteins, such as p62 or p56, occurs at the two-cell stage, with greatest production at the eight-cell stage, suggesting that they are replenished after the first cleavage [47, 48].

Premature loss of proteins from the cortical granule envelope may account for the difference in DNA methylation seen in this study. This hypothesis could be tested by assessing the effect on DNA methylation of using RNA interference to reduce the level of these candidate proteins in ZI embryos.

ACKNOWLEDGMENTS

The authors would like to thank to all the members of the Ian Wilmut group at Roslin Institute, especially William Ritchie, for the help with mechanical removal of ZPs, all the staff from the small animal unit at Roslin Institute, and Bryan Turner for the H4K5 acetylation antibody gift.

FOOTNOTES

1 Supported by Fundação para a Ciência e Tecnologia (FCT), Ministério da Ciência e do Ensino Superior (MCES), Portugal. Back

2 Correspondence: FAX: 0044 131 242 6629; ricardo.ribas{at}icr.ac.uk Back

3 Current address: The Queen's Medical Research Institute, Centre for Reproductive Biology, Reproductive and Developmental Sciences, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom. Back

Received: 2 August 2005.

First decision: 22 August 2005.

Accepted: 12 October 2005.

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