| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
research-article |
Department of Reproduction and Development,4 Kunming Institute of Zoology & Kunming Primate Research Center, the Chinese Academy of Sciences, Kunming, Yunnan 650223, China
Graduate University of Chinese Academy of Sciences,5 Beijing 100049, China
INRA,6 UMR 1198; ENVA; CNRS, FRE 2857, Biologie du Développement et Reproduction, Jouy en Josas, F-78350, France
The State Key Laboratory of Reproductive Biology,7 Institute of Zoology, the Chinese Academy of Sciences, Beijing 100860, China
ABSTRACT
Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy. During preimplantation development, 67% of two-cell- and 50% of eight-cell-cloned embryos showed higher DNA methylation levels than their in vitro fertilization (IVF) counterparts, which undergo gradual demethylation until the early morula stage. Moreover, whereas an asymmetric distribution of DNA methylation was established in an IVF blastocysts with a lower methylation level in the inner cell mass (ICM) than in the trophectoderm, in most cloned blastocysts, ICM cells maintained a high degree of methylation. Finally, two donor cell lines (S11 and S1–04) that showed a higher level of H3K9 acetylation supported more blastocyst formation after nuclear transfer than the other cell line (S1–03), with a relatively low level of acetylation staining. In conclusion, we propose that abnormal DNA methylation patterns contribute to the poor quality of cloned preimplantation embryos and may be one of the obstacles to successful cloning in primates.
DNA methylation, donor cells, early development, embryo, embryonic development, epigenetic reprogramming, histone acetylation, in vitro fertilization, rhesus monkey, somatic cell nuclear transfer
Although many mammals have been successfully cloned, including rodents, carnivores, lagomorphs, and ungulates, primate cloning remains to be mastered [1–10]. When combined with gene-targeting technologies, nonhuman primate cloning by somatic cell nuclear transfer (SCNT) procedures would support the production of loss-of-function monkey models for the study of human disease in which mouse models have been irrelevant and reduce the number of animals required for biomedical research.
Over the past years, investigations to optimize nonhuman primate SCNT cloning have revealed major problems concerning meiotic spindle extraction and mitotic spindle defects during the first cell cycles of embryonic development [11–13]. Besides this, we found that about 30% of SCNT two-cell embryos could develop into blastocysts [14], a rate consistent with the results published recently by Simerly et al. [13]. However, none of the SCNT-reconstructed embryos transferred into surrogate mothers resulted in pregnancies to term [11, 13], though one cloned monkey offspring has been achieved by embryonic cell nuclear transfer [15].
To explore the factors that could actually affect the early or late development of these reconstructed embryos, we focused on the epigenetic reprogramming of the donor cells. It is believed that after SCNT procedures, epigenetic reprogramming of the donor nuclei transferred into the enucleated oocytes is likely to have a crucial role in establishing nuclear totipotency and normal development of the cloned animals. Indeed, whereas somatic nuclei acquire highly specialized epigenetic modifications and remain largely constant, there are at least two developmental periods in germ cells and preimplantation embryos during which genome-wide reprogramming of the epigenetic patterns takes place, generating cells with a broad developmental potential [16]. After fertilization, the gametes undergo a drastic reprogramming that includes changes in DNA methylation and histone modifications. However, abnormal DNA methylation reprogramming has been found in most SCNT-cloned embryos or animals in several species, such as the mouse [17], bovine [18–21], rabbit [22], and sheep [23]. In cloned mouse embryos, previous studies have found that the regulation of DNA methyltransferase expression was defective [24], the methylation pattern and expression of imprinted genes were disrupted in most blastocysts [25], and the demethylation of the Pou5f1 (previously known as Oct4) promoter was inefficient [26].
In mammals, DNA methylation and histone modifications are major epigenetic marks. DNA methylation occurs predominantly at CpG dinucleotides and is involved in several key genome functions, such as imprinting, X chromosome inactivation, genome stability, silencing of retrotransposons, and inactivation of genes in cancers [27–29]. Most of all, DNA methylation suppresses gene expression by recruiting methyl-CpG-binding proteins, such as MECP2 (methyl-CpG-binding protein 2), MBD1 (methyl-CpG-binding domain protein 1), MBD2, and MBD3, as well as associated histone deacetylases, corepressor proteins, and chromatin remodeling machineries at the promoter regions of specific genes [30]. It is recognized that histones play important roles in maintaining the dynamic equilibrium of chromatin through which the regulation of gene expression is attained during all stages of multicellular organism development. It has become evident that changes within the chromatin structure brought about by covalent modifications of histones are of crucial importance in determining many biological processes, including development [31]. Acetylation of histone H3 on lysine 9 (H3K9ac), for example, is often found within active chromatin configurations [32] and inversely correlates with DNA methylation levels in several genes, especially in cancers [33, 34]. More interestingly, the different patterns of lysine acetylation observed in mouse preimplantation embryos suggest that acetylation is also important in epigenetic reprogramming [35]. Histone H3 and H4 lysines (with the exception of H4 Lys 5) are deacetylated after fertilization in the mouse, as well as in the somatic nuclei that have been transferred into enucleated oocytes [36].
In the present study, we investigated the epigenetic reprogramming of both global DNA methylation and histone H3K9 acetylation in SCNT and in vitro fertilization (IVF) rhesus monkey embryos during preimplantation development. We also compared the epigenetic characteristics of three donor cell lines with different capacities in terms of development after SCNT.
Mature rhesus macaque males and females were supplied by Kunming Primate Research Center, and housed in individual cages when used for the present study. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Kunming Primate Research Center, the Chinese Academy of Sciences.
All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise stated.
Establishment of Rhesus Monkey Fibroblast Cell Lines and Culture
Fibroblast cell lines (S1 and S11) were derived from the ear skin tissue of healthy neonatal rhesus monkeys. The skin tissue was washed five times in Ca2+- and Mg2+-free PBS that contained penicillin G and streptomycin (100 IU/ml), washed one time in Dulbecco modified Eagle medium (DMEM, no. 23700–024; Gibco) supplemented with 10% fetal calf serum (FCS; Invitrogen), and then minced into 1-mm3 pieces. The tissue pieces were evenly plated in 50-ml culture flasks containing a small amount of DMEM with 10% FCS. Once the tissue pieces had stably adhered to the flask surface, 5 ml of culture medium was added. Fibroblasts were harvested with PBS containing 0.25% (w/v) trypsin and 0.02% (w/v) EDTA when the culture had grown to full confluency. The fibroblast cells were passaged two more times before being frozen in DMEM with 10% FCS and 10% dimethyl sulfoxide and stored in liquid nitrogen. S1–03 and S1–04 donor cells were subcloned from the S1 fibroblast cell line.
Before nuclear transfer, all donor cells were serum starved for 4 days in DMEM supplemented with 0.5% FCS. For indirect immunofluorescence detection, cycling cells that had reached 70%–80% confluency either were fixed with 4% paraformaldehyde, as for the controls, or were serum starved for 4 days before fixation.
Ovarian Stimulation, Recovery of Oocytes, IVF, and Embryo Culture
Beginning on Days 1–3 of the menstrual cycle (Day 1 = first day of menses), 18 IU of recombinant human FSH (r-hFSH, Gonal F; Laboratories Serono SA, Switzerland) was injected i.m. twice daily for 8 days. On Day 9 of treatment, 2000 IU of hCG (Lizhu Groups, Shenzhen, China) was injected to induce oocyte maturation. Thirty to thirty-four hours post-hCG injection, animals that responded to exogenous gonadotropins were anesthetized with ketamine (10–12 mg/kg) for oocyte retrieval with the laparoscopic follicular aspiration system as described previously [11]. Briefly, the contents of enlarged follicles (>2 mm in diameter) were aspirated with a single-lumen needle (20 gauge; ECHO-TIP, Cook, Australia). The collected follicular contents were diluted immediately with Tyrode lactate (TL)-Hepes medium containing heparin (5 IU/ml). Oocytes were stripped of cumulus cells by pipetting after a brief exposure (<1 min) to hyaluronidase (0.5 mg/ml) to allow the classification of nuclear maturity as prophase I, metaphase I (MI), metaphase II (MII), and atretic (presence of fragmentation or vacuoles in ooplasms).
Mature oocytes (MII) were fertilized in vitro with capacitated and stimulated sperm (2 x 107/ml) diluted in Tyrode albumin lactate pyruvate culture medium, according to Bavister et al. [37]. Fertilized oocytes were then cultured in hamster embryo culture medium 9 (HECM-9) containing 10% FCS at 37°C in 5% CO2.
A one-step micromanipulation technique was used for nuclear transfer, as described previously [10]. In brief, a monkey fibroblast cell was aspirated into a 10-µm (inner diameter) pipette to break the cell membrane and then injected into an oocyte at the opposite side of the MII plate after zona and oolemma drilling with a piezo actuator. The injection pipette was then slowly withdrawn in such a way until the tip was in the vicinity of the oocyte metaphase chromosomes, visible with a differential interference contrast (DIC) microscope. The MII chromosomes were sucked into the pipette, which was then removed, allowing closure of the oocyte oolemma.
Two hours after injection, activation was induced by incubation in Ca2+- and Mg2+-free TL-Hepes buffer that contained 5 µM ionomycin for 4 min and then in 2 mM DMAP [N, N-dimethylamino)phenol] for 5 h at 37°C in 5% CO2. Activated embryos were cultured in HECM-9 containing 10% FCS at 37°C in 5% CO2.
The method used for double indirect immunofluorescence was adapted from Santos et al. [20], with some modifications. All steps were performed at room temperature, unless otherwise mentioned. Early embryos and donor cells were first washed in PBS and then fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.2% Triton X-100 in PBS (30 min for embryos and 1 h for cells). After several washes with 0.05% Tween-20 in PBS, all samples were treated with 4 M HCl for 10 min and fully rinsed again with 0.05% Tween-20 in PBS. They were then left in blocking solution (1% BSA and 0.05% Tween-20 in PBS) overnight at 4°C. Embryos were incubated for 1 h with a mouse monoclonal antibody to 5-methyl cytosine (NA81, dilution 1 µg/ml; Ococgene) and for 1 h with a secondary goat anti-mouse Texas red antibody (dilution 1:200; Jackson ImmunoResearch). After further washes with the blocking solution, they were stained for 1 h with a rabbit polyclonal antibody to histone H3 acetylated at position lysine 9 that cannot distinguish different subfamilies of H3 (no. 06–942, dilution 1:250; Upstate Biotechnology) or to POU5F1, previously known as OCT4 (sc-9081, dilution 1:50; Santa Cruz) and then incubated for 1 h with a fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (dilution 1:200; Jackson ImmunoResearch). Finally, samples were mounted on slides with antifading medium. For donor cells, antibodies to 5-methyl cytosine and H3K9ac were applied on separate samples, and experiments were replicated at least three times.
Stained embryos and cultured cells mounted on slides were observed on a laser scanning microscopy (LSM) 510 META microscope (Zeiss) with a Plan Neofluar 40x/1.3 Oil DIC objective having excitation wavelengths of 488 and 543 nm. Collection of each channel signal was done sequentially. For each wavelength, serial optical sections (Z-stack) were collected at 2-µm intervals through the specimens, with scan averaging and a slow scan speed. For each experiment, the same detector gain, amplifier offset, and pinhole parameters were used. These Z-stacks were later merged with the Zeiss LSM Image Examiner and ImageJ software, v.1.34s (National Institutes of Health, Bethesda, MD) to produce a two-dimensional image depicting the staining patterns and total intensities of all the nuclei. All processed images were assembled by Adobe Photoshop v6.0 (Adobe Systems, San Jose, CA).
The nuclear intensities of integrated fluorescence were measured by manually outlining all nuclei, except in late morula and blastocysts. The total fluorescence intensity emitted by each individual nucleus was measured on merged images, after background subtraction, by the ImageJ software, and averaged per embryo. By means of SPSS software, a one-way ANOVA was used to compare the values of different embryo stages and treatments.
Epigenetic Reprogramming in IVF Preimplantation Monkey Embryos
Prior to studying cloned SCNT monkey embryos, we first examined embryos produced by IVF (control group). As several studies had previously suggested that there were species differences in DNA methylation dynamics during mammalian preimplantation development and as no report regarding analysis of epigenetic marks in nonhuman primate embryos was available, we analyzed global changes that took place with respect to DNA methylation and histone H3K9 acetylation during preimplantation development in the rhesus monkey.
After fertilization, at the single-cell stage, we observed that most zygotes were presenting two pronuclei as the result of parental genome decondensation and that only one of them was highly methylated (n = 19, Fig. 1A, left). On the other hand, both pronuclei presented a strong staining for histone H3 acetylated on position 9 (H3K9ac) (Fig. 1A, middle). Interestingly, in polyspermic zygotes, only one of the pronuclei had a high level of methylation, whereas the other pronuclei (two or three) were less methylated (data not shown).
|
At the two-cell stage, the nuclei of both blastomeres were uniformly labeled by the anti-H3K9ac antibody (Fig. 1B, middle) but asymmetrically labeled by the 5-methyl cytosine antibody, with only half of each nuclei stained (n = 15, Fig. 1B, left). This asymmetry in the 5-methyl cytosine labeling is consistent with the differential methylation pattern observed between the male and female pronuclei during the first cell cycle.
Subsequently, from the two-cell stage to the early morula stage, DNA methylation staining seemed to decrease progressively and significantly (Fig. 2, B–E). To examine whether this could be due to a visual artifact, we performed quantification analysis of the overall levels of methylation on merged images. As shown in Figure 4, it confirmed a significant decrease in the DNA methylation intensity from the two-cell to the early morula stage (an 81% decrease, P < 0.05). By the late morula stage, the process of demethylation had reverted, and an increase in nuclear methylation levels was observed, which could have been the result of de novo methylation events (Figs. 2F and 4). However, this type of methylation did not happen in all cells and resulted in an asymmetric distribution of the 5-methyl cytosine labeling in the blastocyst: although the inner cell mass (ICM) cells were faintly labeled, the trophectoderm (TE) cells were more highly methylated (Figs. 2G and 3). In fact, the TE cells presented 65% more methylation than the ICM that maintained the same level of methylation as the early morula cells (Fig. 4). To verify this asymmetric pattern, we performed POU5F1 immunostaining, as it was previously shown that POU5F1 expression characterizes the ICM of expanded blastocysts [38]. As shown on Figure 3, the POU5F1 signal within the ICM colocalizes with more weakly stained cells in terms of DNA methylation, thus demonstrating the lower methylation signal in ICM versus TE cells.
|
|
|
Similarly, H3K9 acetylation decreased progressively during the preimplantation period (Fig. 2, a–g). The global level of acetylation was reduced by almost 90% from the two-cell to the morula stage (data not shown). At the blastocyst stage, nearly all nuclei were faintly and homogeneously labeled. It should be underlined that no statistical difference was found between the ICM and TE cells for H3K9ac (P < 0.05), which suggests that the observed undermethylation of the ICM is not due to antibody penetration problems.
Abnormalities in the Reprogramming of SCNT-Cloned Embryos
We next examined and quantified IVF versus SCNT (donor line S11) preimplantation monkey embryos after immunostaining with the same antibodies as above. Note that, because of monkey oocyte restrictions, not all stages were analyzed. We selected key stages according to previous studies of other species, i.e., two-cell, eight-cell, and blastocyst stages.
The first obvious observation was that most cloned embryos presented a higher level of DNA methylation than their IVF counterparts (Figs. 4 and 5). The most striking difference was at the two-cell stage, when 67% of the cloned embryos (n = 9) were more intensively labeled than the IVF embryos (P < 0.01, Fig. 5d). Moreover, no particular polarity was observed within the nuclei: the 5-methyl cytosine staining covered the entire surface of the nuclei, suggesting that the somatic genome did not undergo the same reprogramming changes as in the IVF embryos.
|
At the eight-cell stage, half of the cloned embryos (n = 16) showed higher DNA methylation but lower H3K9 acetylation than the IVF controls (representative example on Fig. 5, e and E). Intriguingly, the levels of these two epigenetic marks were very heterogeneous within and between the cloned eight-cell embryos. Some cloned embryos that slightly stained for DNA methylation presented either low or high levels of H3K9 acetylation. We then investigated if this variability could be linked with embryo development rates in culture. Cleavage rates of cloned and IVF embryos were compared during in vitro culture (Table 1). As expected, the first mitosis after the nuclear transfer and activation of the cloned embryo is the first checkpoint of embryonic development (63%–70% cleavage versus 81% in the IVF group) and illustrates successful reconstructions. The developmental rate to the eight-cell stage was not significantly different between the IVF embryos and the three SCNT groups; however, the morula and blastocyst formation rates for the IVF embryos were obviously higher than for the others (P < 0.05). This suggests that another developmental barrier takes place in cloned embryos between the 8- and 16-cell stages, with cleavage rates from the 8-cell to the morula stage ranging from 51% to 68% in clones derived from the three different donor cell lines versus 78% in the IVF group.
|
Finally, at the blastocyst stage, although nearly all nuclei in some cloned embryos of good morphology were quite homogenously labeled for H3K9ac, as were the IVF embryos, some cells (especially the TE cells) were more intensively labeled in the cloned blastocysts with fewer cells and a smaller size (Fig. 5F). Moreover, compared with their IVF counterparts, cloned blastocysts had a stronger overall level of staining for DNA methylation in the ICM cells (n = 7, P < 0.01, Fig. 4). Although the difference between the TE and ICM cells was also significant in most clones (5 of 7, P < 0.05), it was not as obvious as in the IVF controls (Fig. 4). Again, this suggests that monkey SCNT embryos are not fully reprogrammed, unlike their IVF counterparts. Because of monkey oocyte restrictions, we could not analyze the cloned morula. It is therefore impossible to determine if hypermethylation of the ICM cells in cloned blastocysts is due to an insufficient demethylation of the genome between the reconstruction and the morula stage or to an unspecific de novo methylation of all cells at the blastocyst stage.
Epigenetic Characteristics of Donor Cell Lines
As the first step of the SCNT procedure is the preparation of the donor cells, we then analyzed the effect of serum starvation on three fetal fibroblast cell lines in terms of DNA methylation levels and H3K9 acetylation.
The analysis showed that the global DNA methylation level was obviously reduced after serum starvation in all three of the cell lines in comparison to serum-fed cells (Fig. 6, upper panel). We noticed that the global distribution pattern was quite heterogeneous in the serum-fed cells, whereas in the serum-starved cells, the staining was more homogeneous. One explanation could be that serum-fed cells progress through the cell cycle nonsynchronously, whereas the serum-starved cells were synchronized at the G0/G1 stage.
|
On the other hand, in both S11 and S1–03 cultures, serum starvation had no effect on the level of H3K9 acetylation, with S11 cells more acetylated than S1–03 cells (Fig. 6, lower panel). Most intriguing was that the two subcloned cell lines (S1–04 and S1–03) were quite hypoacetylated in cycling cultures and that only the S1–04 cell line reacted to serum starvation with an increase in the global level of H3K9ac, thereby reaching a level similar to that of S11 cells.
Interestingly, the two cell lines (S11 and S1–04) that were hyperacetylated after serum starvation gave good blastocyst formation rates, around 30% and 28%, respectively, versus 13% with the S1–03 cell line (Table 1). As only S11-derived embryos were stained for epigenetic marks in the present study, we cannot say if the S1–03/S1–04-derived embryos would follow similar or differential reprogramming in comparison to S11-derived embryos.
Global epigenetic reprogramming has been reported as a major factor that is required to take place following SCNT for normal development and successful cloning. Previous studies on cloned embryos have demonstrated defects in global methylation changes, at specific imprinted genes and other loci, as well as defective regulation of Dnmt1 expression (reviewed by Latham [39]). As a matter of fact, somatic cell nuclei, unlike the gamete genome, usually possess stable epigenetic marks that probably make it difficult to recapitulate the reprogramming that occurs soon after fertilization in normal embryos.
In our rhesus monkey SCNT experiments, we observed differential blastocyst formation rates with three donor cell lines. The cell line that showed low levels of methylation and hypoacetylation after the serum starvation treatment was the worst cell line for SCNT. One explanation could be that the original global methylation level of the donor nuclei may be not crucial to the following initial reprogramming after nuclear transfer, but chromatin remodeling may be important for the reprogramming process. In the early cloned embryo, H3K9 acetylation would then represent a measure of chromatic remodeling and accessibility to factors present in the oocyte cytoplasm that are crucial for early development. Along with this hypothesis, Enright et al. [40] showed that the treatment of donor cells by trichostatin A (TSA), a histone deacetylase inhibitor, can increase the blastocyst formation, but not the development to term, in the bovine model. More intriguingly, it was recently reported that TSA treatment following oocyte activation resulted in a more efficient in vitro development of somatic cloned mouse embryos to the blastocyst stage [41] and a more efficient derivation of nuclear transfer-embryonic stem cells and even an increased development to term [42]. Further studies are needed that will elicit how TSA treatment influences the reprogramming of donor nuclei and the role of histone acetylation/deacetylation in epigenetic reprogramming during preimplantation development.
Although many other epigenetic marks may well play a role in the reprogramming of the somatic genome, we focused on two major epigenetic marks, H3K9 acetylation and DNA methylation, and compared the developmental dynamics of the cloned embryos to their IVF counterparts. First, diametrically opposed methylation levels were uniquely observed between the two pronuclei in zygotes, as previously described as in the mouse, rat, bovine, pig [19], and human [43]. However, it is unknown whether the undermethylated pronucleus would be the paternal one and whether this could be due to active demethylation, as in mouse zygotes [44, 45]. More studies are needed to understand the epigenetic similarities and differences between mammalian species [46]. As in the mouse, H3K9ac staining in the male pronuclei, in our opinion, results from the rapid replacement of the protamines by histones after fertilization [47].
As a consequence of this differential methylation between the two pronuclei, in two-cell IVF embryos, the methylated staining pattern presented a polarity within each nucleus, demonstrating that the maternal and paternal genomes were distributed in two different territories. To our knowledge, this is the first report of spatial separation in the parental genome at the two-cell stage in a species other than the mouse [44, 45]. On the contrary, in cloned embryos at the two-cell stage, DNA methylation labeling covered the entire nuclei, and quantification showed that the staining intensity was significantly higher than in the IVF embryos. These results in clones could indicate inefficient or delayed demethylation and insufficient chromatin remodeling of the differentiated donor nuclei, as suggested by a study of sheep SCNT embryos [23].
Upon further development, H3K9 acetylation and DNA methylation decreased progressively, as in the bovine preimplantation embryos [20]. Passive demethylation, due to semiconservative replication at each cell cycle, was clearly shown in previous studies on chromosome spreads with mouse and bovine embryos [18, 48]. In monkey embryos, progressive demethylation after the single-cell stage probably results from the same passive process as in the mouse and bovine; however, the global methylation level does not reach a minimum by the eight-cell stage, as in the bovine, but reaches it at the early morula stage. Recently, Vassena et al. [49] investigated the expression of DNA methyltransferase genes in the monkey in comparison with the mouse and found a significant decrease of DNMT3A mRNA from the two-cell stage to the morula stage, whereas DNMT1 mRNA declined gradually and became significantly reduced by the eight-cell stage. Our results are consistent with these expression patterns.
As expected from previous studies of bovine- and sheep-cloned embryos [18–20, 23], many cloned eight-cell rhesus embryos were hypermethylated in comparison to their IVF counterparts. Most striking was the discovery that some eight-cell-cloned embryos were very heterogeneous in terms of DNA methylation and H3K9 acetylation. This variability may result from insufficient chromatin remolding, mitotic deficiencies, or DNA methyltransferase misexpression. It was suggested that in mouse eight-cell-cloned embryos, aberrant DNMT1 localization and expression contribute to defects in DNA methylation [24]. Abnormal DNA methylation and histone acetylation may subsequently prevent the correct expression of crucial genes at the eight-cell stage, which might explain the developmental loss observed in the present study at around the 8- and 16-cell stages and in a previous report by Mitalipov et al. [50]. Indeed, in rhesus preimplantation development, many chromatin regulatory factor-encoding genes, such as BAZ1A (previously known as ACF1), CHRAC1, and POLE3 (previously known as CHRAC17), are specifically activated at the eight-cell stage to support the transition from maternal to embryonic genome expression [51].
In the mouse and bovine, de novo methylation is specifically observed in the ICM but not in the TE cells of the blastocyst [19]. This asymmetric pattern associated with embryonic and extraembryonic lineages is believed to be essential to further development. However, in monkey blastocysts, we found that the ICM is undermethylated in comparison with TE cells, as in humans [43]. We suggest that it reflects differences that occur in the later stages of implantation and placenta formation between primates and other mammals [52]. More studies are needed to investigate this peculiar asymmetric pattern, especially as most cloned blastocysts showed a more highly methylated ICM than their IVF counterparts. Interestingly, this observation is consistent with the problems of fetal development that we and other researchers have experienced after the transfer of SCNT monkey embryos into surrogate mothers [11, 13]. Besides the hypermethylated ICM, the H3K9ac appeared more intensely labeled in some small cloned blastocysts of bad morphology. These abnormal and positively correlated H3K9 acetylation levels and DNA methylation levels were also found in cloned bovine blastocysts [20, 53].
For primate cloning, though the blastocyst formation rates are similar to other cloned species that exhibit successful births of offspring, no cloned primate animal has yet been obtained, to our knowledge. The effect of the abnormal reprogramming that we demonstrated in the present study on cloned rhesus monkey embryos with respect to imprinted gene expression and other epigenetic marks and the consequences for postimplantation development should be further investigated to address the challenge of bringing cloned rhesus monkey embryos to term. To establish the foundations for biomedical medical research and therapeutic cloning on human diseases, it also clearly appears that more detailed studies of the epigenetic environment of cloned embryos are needed.
ACKNOWLEDGMENTS
We are grateful to Dr. Fatima Santos from the Babraham Institute, Cambridge, for help with protocols and advice in the early stage of the present study.
FOOTNOTES
1Supported by Major Projects of Knowledge Innovation Program from the Chinese Academy of Sciences (KSCX1-05), National Natural Science Foundation of China (30370166), Joint Scholar Plan for the Development of Western China, National Basic Research Program of China (973 program, 2004CCA01300), and the PRA (Programme de Recherches Avancées Franco—Chinois). 
Correspondence: 2 FAX: 86 871 513 9413; e-mail: wji{at}mail.kiz.ac.cn
Correspondence: 3 FAX: 86 10 626 50042; e-mail: qzhou{at}ioz.ac.cn
Received: 2 February 2006.
First decision: 2 March 2006.
Accepted: 19 September 2006.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  L. H. Shi, J. S. Ai, Y. C. OuYang, J. C. Huang, Z. L. Lei, Q. Wang, S. Yin, Z. M. Han, Q. Y. Sun, and D. Y. Chen Trichostatin A and nuclear reprogramming of cloned rabbit embryos J Anim Sci, May 1, 2008; 86(5): 1106 - 1113. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
