Introduction
The incomplete or aberrant reprogramming of donor nuclei by the oocyte is the main cause of the inefficient cloning (
Jouneau and Renard, 2003). The levels of epigenetic modification of donor cells may affect their reprogramming ability following nuclear transfer (NT). Histone acetylation and DNA methylation are heritable epigenetic modifications. In naturally fertilized zygotes, the paternal genome undergoes an “active demethylation” prior to DNA synthesis, but the maternal genome of the oocyte undergoes a “passive demethylation.” (
Morgan et al., 2005) During the embryo development, DNA is
de novo methylated by the blastocyst stage. Following nuclear transfer, the somatic nucleus carries the specific epigenetic modifications of its tissue type, which must be erased during nuclear reprogramming. Failure to erase and reestablish epigenetic marks will lead to a lack of embryo totipotency and affect further differentiation and development (
Kang et al., 2001). It has been reported that the differentiated somatic donor cells undergo a reduced or incomplete passive demethylation after injection into the oocytes and subsequently a
de novo methylation event occurs at an earlier stage (
Bourc’his et al., 2001;
Kang et al., 2001). The abnormal DNA methylation in NT embryos could lead to the abnormal expression of genes and result in the failed or disrupted reactivation of genes that are essential for proper embryonic development (
Bortvin et al., 2003). For example, the incomplete epigenetic reprogramming of somatic cell nuclear transfer (SCNT) embryos might cause the incomplete demethylation of OCT4 promoter, and consequently influence the normal expression of the OCT4 and other pluripotency genes (
Yamazaki et al., 2006).
It was reported that the methylation state of the donor cell affects the efficiency of genomic reprogramming. The Dnmt1 hypomorphic donor cells with a global hypomethylation state were more efficiently reprogrammed into pluripotent ES cells than their wild-type counterpart (
Blelloch et al., 2006). The
in vitro development of the SCNT embryos derived from 5-aza-2-deoxycytidine treated donor cells, which have a reduced DNA methylation level, does not improve because of the toxic effects of 5-aza-2-deoxycytidine. When the concentration of 5-aza-dC is much lower (0.01 μmol/L), there are no deleterious effects on the development of embryos cloned from treated cells, but these embryos failed to develop to term
in vivo (
Enright et al., 2003,
2005). S-adenoysl homocysteine (SAH), a DNA demethylation agent, can reduce the DNA methylation level of treated cells and cause a significant elevation in the frequency of cleaved embryos to reach hatched blastocyst stage (26.1%
vs 33.8%); however; the rate of developing to term
in vivo needs further study (
Jeon et al., 2008).
In the present study, we used ES cells, which were knocked out of the DNA methyltransferase Dnmt3a and Dnmt3b as donor cells, to examine the effect of genomic hypomethylation on reprogramming efficiency. We found that the percentage of blastocyst and E6.5 embryo formation was significantly elevated when using the DKO cell as donor cells in contrast to the wild type J1 cells. There was no significant difference in the OCT4 expression pattern between DKO and J1 cloned blastocysts or E6.5 embryos.
Materials and methods
Oocytes collection
Cumulus-oocyte complexes were collected from superovulated ICR mice (8-12 weeks old) 15 h post human chorionic gonadotrophin (hCG) injection and cumulus cells were removed with hyaluronidase (ICN Pharmaceuticals, Costa Mesa, CA, USA). Before micromanipulation, oocytes were cultured in CZB medium supplemented with 3 mg/mL bovine serum albumin (BSA) at 37°C and 5% CO2.
Animals were handled according to the Guidelines for the Care and Use of Laboratory Animals established by the Beijing Association for Laboratory Animal Science.
Nuclear transfer
The “one step micromanipulation” (OSM) method was used in the ES cell nuclear transfer process, and the method to prepare the ES donor cells was performed as previously described (
Zhou et al., 2001,
2003;
Jouneau et al., 2006). Reconstructed embryos were activated with 10 mmol/L SrCl
2 in calcium-free CZB medium for 3 .
Embryo transfer
Embryonic stem cell nuclear transfer (ECNT) blastocysts were transferred into the uterus of E2.5 pseudo-pregnant ICR surrogate mothers. All the pregnant recipient females were euthanized at E6.5, and the number of implantation site and embryo were counted and further analyzed.
Establishment of NT-ES cell lines and stem cell culture
The NT-ES cell lines derived from J1 (wt) and DKO ES cell cloned balstocysts were established, and ES cells were cultured as previously described (
Brook and Gardner, 1997;
Wakayama, 2003;
Zhao et al., 2007). DMEM/F12 (1∶1; Gibco No.11320-033) with leukaemia inhibitory factor (LIF, 2000 U; Chemicon, ESG1107) was used for NT-ES cell line establishment, whereas the concentration was decreased by half for ES cell culture.
Immunofluorescence staining
The blastocysts, E6.5 embryos and ES cells were fixed with 4% paraformaldehyde for 30 min at room temperature. After three times of washing, they were permeabilized with 0.5% Triton X-100 in PBS for 30 min. (For the detection of 5-methyl-cytosine (5-MeC), ES cells were treated with 4 mol/L HCl for 1 h at 37°C after permeabilization). Then all samples were blocked in 2% BSA in phosphate buffered saline (PBS) for one hour. Blastocysts were incubated with Oct4 (1∶200; Santa cruz) and Cdx2 antibodies (1∶200; Biogenex), E6.5 embryos were incubated with Oct4 antibody, J1 and DKO were treated by anti-5-methyl-cytosine antibody (1∶4000; a gift from Nathalie BEAUJEAN) and NT-ES cells were incubated with Dnmt3a and Dnmt3b antibodies (1∶1000; a gift from Guoliang XU) overnight at 4°C. After three washings, the embryos and ES cells were incubated with FITC or TRITC conjugated secondary antibodies at 37°C for 1 h. Finally, the DNA was stained with propidium iodide (PI, 10 μg/mL; Molecular Probes, OR, USA) for 15 min at 37°C and after three times washing, all the samples were mounted and observed under a Zeiss LSM 510 META confocal microscope (ZEISS, Germany).
Statistical analysis
Statistical analysis was performed by SPSS 13.0 statistical software. One-way ANOVA and Fisher's exact test were used for statistical analysis. For all statistical analyses, a value of P<0.05 was considered to be statistically significant.
Results
DNA methylation levels of J1, DKO, 3aKO and 3bKO ES cells
The level of m
5C in the DNA of ES cells homozygous for the DNA methyltransferase mutation was reduced to about one-third of that found in heterozygous or wild-type cells (
Li et al., 1992). Immunofluorescence confirmed that there was a distinct difference in the intensity of DNA methylation of J1 compared to that of DKO, 3aKO and 3bKO ES cells. However, the difference in global DNA methylation between DKO, 3aKO and 3bKO ES cells was nearly the same (Fig. 1).
Pre-implantation development of ECNT embryos
The cleavage rate and blastocyst formation of ECNT embryos cloned from J1, DKO, 3aKO and 3bKO were evaluated. As shown in Table 1, there was no significant difference in the number of 2-cell and 4-cell stage embryos between all four kinds of ES cell cloned embryos. However, few embryos cloned from J1 and 3aKO reached morula stage compared to DKO and 3bKO ES cell cloned embryos. The rate of blastocyst formation cloned from DKO and 3bKO ES cells was significantly higher than that of reconstructed embryos with J1 cells (Table 1; P<0.05). Yet, there was no significant difference in the rate of morula and blastocyst formation between the J1 and 3aKO cloned embryos.
Post-implantation development of ES-NT embryos
We then decided to investigate the effect of the DNA methylation on post-implantation development. ECNT blastocysts were transferred into pseudo-pregnant recipients, and embryos were collected at E6.5 (Table 2). Remarkably, the rate of E6.5 embryo was nearly two times higher for the blastocysts derived from DKO than that of J1 ES cells (P<0.01), although the rate of implantation was nearly the same between DKO and J1 ES cell cloned blastocysts.
Establishment of NT-ES cells from DKO and J1 ES cell cloned blastocysts
The DKO and J1 ES cell cloned blastocysts were explanted onto MEF-coated plates and cultured to derive NT-ES cells (Table 3). To our surprise, the efficiency of NT-ES cell derivation of DKO cloned blastocysts was 50% of the J1 cloned blastocysts based on the blastocysts, although the efficiency was nearly the same based on the 2-cell. The expression of Dmnt3a and Dnmt3b in NT-ES cells derived from J1 and DKO cloned blastocysts were tested by immunofluorescence staining, which showed that the NT-ES cells derived from DKO cloned blastocysts were negative, but the NT-ES cells derived from J1 cloned blastocysts were positive for Dnmt3a and Dnmt3b (Fig. 2).
Comparasion of Oct4 and CDX2 protein distribution in ECNT cloned blastocysts and fetus
Oct4 expression pattern of cloned blastocyst and postimplantation embryos was examined by immunofluorescence staining, and no distinct difference in OCT4 protein distribution between the blastocysts and the E6.5 embryo was found (Fig. 3, red). All the OCT4 positive cells were arranged in inner cell mass (ICM), and Cdx2 was specifically expressed in TE cells (Fig. 3, green) and no OCT4 and CDX2 double positive cell was detected in the blastocysts. In the E6.5 NT embryos, OCT4 positive cells were limited to the embryonic tissue (Fig. 3E and F). These results indicated that the DNA methylation state does not impact the Oct4 and CDX2 expression pattern in NT-ES blastocysts and E6.5 mouse embryos.
Discussion
In this study, we investigated the effect of DNA methylation status of donor cells on the pre- and post-implantation development of ECNT embryos. These results demonstrate that global hypomethylation of donor cell nucleus can strongly increase the pre- and post-implantation development of cloned embryos, although it did not affect the Oct4 expression in NT blastocysts and postimplantation embryos.
Abnormal SCNT embryo and developmental defects are thought to result from incomplete or aberrant reprogramming of donor nuclei by the oocyte (
Jouneau and Renard, 2003). The nuclear transfer-derived embryos typically show abnormal patterns of DNA methylation compared with non-SCNT embryos such as
in vitro or
in vivo fertilized embryos (
Bourc’his et al., 2001;
Kang et al., 2001;
Santos et al., 2003). In SCNT bovine embryos active demethylation occurs at the 1-cell stage with no further demethylation occurring subsequently (
Kang et al., 2001;
Santos et al., 2003). These abnormal patterns could result in the failure of normal reactivation and expression of early embryonic genes (
Bortvin et al., 2003). Previous attempts to improve reprogramming efficiency by changing the DNA methylation level using drugs such as the demethylation drug 5-aza-2-deoxycytidine were unsuccessful, but treating donor cell with S-adenosylhomocysteine hydrolase (SAH) can reduce the DNA methylation level and increase the
in vitro development rate of the NT embryos from the treated cells (
Jones et al., 2001;
Enright et al., 2003;
Jeon et al., 2008). It is consistent with our result that the rate of blastocysts and postimplantation embryos cloned from DKO was significantly higher than that of J1 cells, yet the efficiency of NT-ES cell line derivation was decreased significantly. Alternately, it is inconsistent with what was reported previously that global hypomethylation of donor cell nucleus can strongly increase the efficiency of deriving ES cell lines, but did not affect the cleavage rate of cloned embryo (
Blelloch et al., 2006). Some possible reasons may contribute to the contradiction, including (1) difference in mutation of DNMTs: we mutated the Dnmt3a and Dnmt3b, which are two
de novo methyltransferases and responsible for the DNA
de novo methylation after blastocyst stage. It will take a longer time to derive the NT-ES cells after the blastocysts are seeded on feeder and the lack of Dnmt3a and Dnmt3b may cause the cells to reestablish their DNA methylation stage. However, Robert Blelloch could avoid this problem by mutation of Dnmt1, a DNA methyltransferase that maintains CpG methylation. (2) Difference of donor cells: J1 cells are a type of ES cell that has a higher efficiency of cloning and derivation of NT-ES cell lines than that of somatic cells, such as tail tip fibroblasts which R. Blelloch used. Genome hypomethylation correlates with the active gene expression of pluripotency genes and makes donor cell nuclei more sensitive to reprogram by oocytes, which could increase the efficiency of cloned embryos subsequently. Up-regulation of telomerase has been shown to increase proliferation, cell survival and possibly alter cell state to a more undifferentiated, progenitor-like condition (
Perrault et al., 2005). Increase of telomerase activity levels in hypomethylated donor somatic cell cloned embryos could enhance the nuclear reprogramming efficiency (
Jeon et al., 2008).
The Oct4 promoter is inefficiently demethylated following nuclear transfer (
Yamazaki et al., 2006), consistent with incomplete epigenetic reprogramming causing the abnormal expression of Oct4 and other essential pluripotency genes (
Blelloch et al., 2006). Inadequate methylation caused by ablating Dnmt3a and Dnmt3b is associated with dysregulated expression of
Oct4 and
Nanog during the differentiation of pluripotent cells and mouse embryonic development (
Li et al., 2007). No discernible phenotype was apparent in homozygous ES cells in culture, homozygous mutant embryos display severe stunting, developmental delay, and death at mid-gestation at a time when organogenesis and rapid growth proceed in normal embryos (
Li et al., 1992). Yet in our results, there is no apparent difference in Oct4 expression distribution in the four ES cell line cloned blastocysts or E6.5 embryos by indirect immunofluoscence staining. In conclusion, changes in DNA methylation levels caused by mutation of Dnmt3a or Dnmt3b do not affect the Oct4 expression in cloned blastocysts and postimplantation E6.5 embryos.
In this study, we have demonstrated that the methylation state of the donor cell strongly influences the efficiency of NT embryos pre- and post-implantation development. This partly suggests that the epigenetic state of the donor genome is a very important factor affecting the reprogramming efficiency.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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