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Research Article |
Department of Bioscience, Tokyo University of Agriculture, Tokyo 156-8502, Japan
ABSTRACT
Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.
early development, embryo, oocyte development, placenta, trophoblast
INTRODUCTION
Embryo cloning technology is furthering the research in ontology, particularly in the investigation of genomic irreversibility and reprogramming of epigenetic modifications. In the process of embryo cloning, a donor nucleus is reprogrammed to acquire totipotency under the influence of unknown factors specific to MII oocytes [1, 2]. Occasionally, after nuclear transfer (NT), the genes necessary for early embryonic development are completely activated, and differentiation-associated genes that had been transcribed in the original donor cells are suppressed. However, the successful reprogramming of a donor nucleus in cloned embryos remains uncertain because it is always associated with some degree of error, inappropriate epigenetic modifications, and the expression of certain genes in somatic clones [3–5]. The cloned embryos may cease to develop at any stage because of such multiple failures that occur during reprogramming. Even when the embryos fortunately develop to term, some abnormalities always accompany embryo cloning (e.g., large-offspring syndrome, respiratory defects, immune defects, and placental enlargement) [6–11].
Among these, placental abnormalities were consistently observed in the somatic cloned embryos, regardless of the species and cell types [8, 12–15]. In studies on cattle, more than 50% of the clones were lost in the first trimester because of poor chorioallantoic development in the placentome [16]. In mice, the hypertrophic placentae of clones were often found to be 2–3-fold heavier than those of controls derived from natural mating [6, 8, 11, 17]. Many studies in the past focused on the molecular aspects of cloning in order to investigate the cause of such abnormalities in cloned embryos, for example, imprinted gene expression and DNA methylation [3, 4, 18, 19]. However, the underlying mechanism leading to such abnormalities is not yet fully understood.
To date, certain indirect evidences have helped in gaining some understanding of the irreversible changes occurring in donor nuclei during development and cell differentiation. It appears that the ability of the cloned embryos to develop to term is reduced as the donor embryos cleave after fertilization [16]. Cloned pups were produced from two- and four-cell embryos by standard NT in mice [2, 20, 21], whereas cloned embryos derived from eight-cell and morula embryos and lineages of blastocysts (i.e., both ICM and trophoblastic cells) can develop to term when pronucleus is transferred into an enucleated fertilized one-cell embryo (serial NT method) [21–23]. However, the birthrates from these cloned embryos were certainly low. From these results, it appears that the capability of donor nuclei to be reprogrammed is rapidly reduced during preimplantation development, and the cytoplasm derived from fertilized eggs possesses an activity that modifies nuclear reprogramming and assists in the development of the embryonic cloned embryos.
Thus, we focused on embryonic clones in order to obtain a better understanding of the irreversible changes that occur in the genome during development. Therefore, we conducted experiments to determine the point at which irreversible epigenetic changes occur, leading to developmental arrest and various abnormalities during preimplantation development. Thus, developmental competence and placental morphology were examined in cloned concepti, which were reconstructed by standard and serial NT methods using nuclear donors (four- and eight-cell embryos, morula embryos, ICM cells, and embryonic stem [ES] cells). Here, we have shown that advancing development of a donor embryo leads to loss of developmental competence in embryonic cloned embryos, and failure of cloning is definite, particularly if morula embryos are used. Subsequently, we have demonstrated that placental malformation first occurs in trophoblastic giant cells even when four-cell embryos were used as nuclear donors, and an excessive enlargement of placenta becomes evident in the clones derived from morula embryos, ICM cells, and ES cells.
MATERIALS AND METHODS
Animals
Adult female and male B6CBF1 (C57BL/6 x CBA) mice and ICR mice were obtained from Charles River Laboratories Japan, Inc. During the course of the experiments, food and water were provided ad libitum to all mice, and they were maintained at a controlled temperature (23 ± 2°C) under 12L:12D conditions. All the mice were maintained and used in accordance with the Guidelines for the Care and Use of Laboratory Animals established by the Japanese Association for Laboratory Animal Science.
Preparation of Donor Embryos and Cell Cycle Arrest at Metaphase
Donor embryos to be used for NT were collected from mature B6CBF1 females that were mated with males of the same strain after superovulation by consecutive injections of 7.5 IU eCG and 7.5 IU hCG. Fertilized one-cell embryos that were retrieved 22 h after the hCG injection were cultured in modified CZB medium containing 5.56 mM glucose (mCZB). Following a period of 58–60, 66–68, and 78–80 h after the hCG injection, four-cell, eight-cell, and morula embryos were synchronized at metaphase by culturing them in the mCZB medium containing 1 µg/ml nocodazole for 4 h. The ICM cells were isolated from a blastocyst by immunosurgery 100 h after the hCG injection. In brief, the zonae pellucidae were removed by brief treatment of the blastocysts in acid Tyrode solution. The blastocysts were washed in M2 medium and incubated at 37°C for 30 min in M2 medium containing 10% rabbit anti-mouse serum. After washing three times in mCZB medium, the blastocysts were treated with 1% guinea pig serum complement in M2 medium at 37°C for an additional 30 min. Finally, the trophectodermal cells were completely removed by pipetting. The ICM cells were cultured individually for 10 h in 5-µl drops of knockout Dulbecco modified Eagle medium (DMEM; Invitrogen/Gibco) supplemented with 1 µg/ml nocodazole, 15% (v/v) knockout serum replacement (Invitrogen/Gibco), and the following reagents: 2 mM L-glutamine (Invitrogen/Gibco), 1% (v/v) nonessential amino acid solution (Invitrogen/Gibco), and 5.5 x 10–5 M 2-mercaptoethanol solution (Wako). All incubations were performed at 37°C under 5% CO2, 5% O2, and 90% N2.
The donor ES cells (TT2 G9a #36 cell line) were prepared as described previously [24]. ES cells were cultured in collagen-coated dishes without a feeder layer for 3 days in knockout DMEM (Invitrogen/Gibco) supplemented with 15% (v/v) fetal bovine serum (Invitrogen/Gibco), 103 U/ml leukemia inhibitory factor (ESGRO; CHMICON Int.), and the following regents: 2 mM L-glutamine (Invitrogen/Gibco), 1% (v/v) nonessential amino acid solution (Invitrogen/Gibco), and 5.5 x 10–5 M 2-mercaptoethanol solution (Wako). To synchronize at metaphase, the ES cells were cultured for 2 h in a medium containing 0.4 µg/ml nocodazole (Sigma-Aldrich), a microtubule polymerization inhibitor. Cells floating in the medium were collected. While being sucked into a transfer pipette, only the cells arrested at metaphase were selected and used as nuclear donors.
Single and Serial NT
Cloned embryos were constructed by the single NT method, which is the standard NT method, and the serial NT method using four- and eight-cell embryos, morula embryos, ICM cells, and ES cells arrested at metaphase, as shown in Figure 1. The recipient oocytes were collected from mature B6CBF1 female mice. Micromanipulations were performed in M2 medium containing 5 µg/ml cytochalasin B (Sigma) and 1 µg/ml nocodazole in a micromanipulation chamber.
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The zonae pellucidae of the four- and eight-cell embryos were slit, and a portion of the cytoplasm containing the metaphase-arrested nucleus, which lacked a nuclear membrane, was aspirated with a micropipette. When the morula embryos were used as donor nuclei, whole blastomeres were aspirated with a micropipette after cutting the zonae pellucidae. Individual ICM cells that were arrested at metaphase were easily isolated without chemical treatment to weaken adhesion of the metaphase cells.
A karyoplast or a whole cell was introduced into the perivitelline space of the enucleated oocyte by using an inactivated Sendai virus (HVJ; 2700 hemagglutinating activity units/ml). The oocytes that successfully fused with donor cells were incubated for 2 h in mCZB medium. Following a brief culture period, the oocytes were artificially activated with 10 mM strontium chloride for 6 h and were subsequently placed in mCZB medium. In serial NT, 8 h after activation, the nucleus of a reconstructed one-cell embryo was again transferred into a fertilized one-cell embryo, which was previously enucleated 12 h after insemination. Fertilized one-cell embryos were produced by in vitro fertilization using B6CBF1 females and males (8–10 wk old) as oocyte and sperm donors. Cloned embryos with ES cells were reconstructed as described previously [24]. To produce control pups, the female and male pronuclei of zygotes (B6CBF1 x B6CBF1) that were manipulated by NT were transferred into enucleated zygotes (pronuclear transfer [PNT]).
All types of reconstructed embryos were cultured in mCZB medium at 37°C under 5% CO2, 5% O2, and 90% N2. Blastocysts were selected on Day 4 of in vitro culture, and these were then transferred to the uterine horns of ICR female mice (8–10 wk old) on Day 2.5 of pseudopregnancy. The number of implantation sites on the uterus was calculated when live pups were recovered at E19.5. To calculate implantation rate, we record the number of live and dead pups, degenerated concepti, and vestige of concepti, which was clearly detected as a small dark spot suggesting that the conceptus ceased at early implantation stage.
Histological Analysis of Placentae
The placentae that developed from embryo clones, clones derived from ES cells, and controls were collected from live pups at birth, fixed with Bouin solution, and processed for wax embedding. Serial sections were mounted on slides and stained with hematoxylin and eosin.
Statistical Analysis
The resulting embryos that developed to the two-cell and morula/blastocyst stages, underwent implantation, and reached term (shown in Table 1) were analyzed by chi-square analysis. The body weight and placental mass were analyzed by one-way ANOVA using the Prism software (http://www.graphpad.com/prism/Prism.htm). Post hoc analyses were carried out with the Bonferroni method for multiple comparisons. Differences were considered significant when P < 0.05.
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RESULTS
Development of Embryonic Cloned Embryos
We first examined the developmental competence of cloned embryos in vitro and in vivo (Table 1). In the first series of experiments, cloned embryos were constructed by standard NT. When four-cell embryos were used as donors, the proportion of embryos that developed to the morula-blastocyst stage and to term was 71.2% and 22.7%, respectively. In eight-cell cloned embryos, the ability to develop to term decreased dramatically (1.8%), although there was no significant difference in the in vitro ability to develop to the morula-blastocyst stage (76.3%). Furthermore, when morulae were used as nuclear donors, only 11.7% developed into morula-blastocysts, and none developed to term. Although the fact that no clones derived from morulae by the single NT method developed to term is likely to be a function of the small sample size, it is clear that dynamic developmental defects occurred during the preimplantation development. In contrast, 74.4% of the cloned embryos derived from ICM cells developed to the morula-blastocyst stage, and 5.4% gave rise to live pups. With regard to the cloned embryos derived from ES cells, 50.6% developed into morula-blastocysts, and live pups were produced from 3.6% of the reconstructed embryos.
When eight-cell and morula embryos were used as nuclear donors, serial NT enhanced the developmental ability of the cloned embryos (Table 1); 17.1% and 11.4% were developed to term, respectively. However, the deterioration of developmental competence of the cloned embryos was clear in the standard NT group; only 1.8% and 0% of the embryos were developed to term. On the contrary, the effect of serial nuclear transfer was not observed when ICM and ES cells were used as nuclear donors, although the proportion of embryos developed to term was quite limited in both methods.
In order to assess whether full development was a result of the unknown activities in the cytoplasm of the zygote that was used as recipient eggs in the second NT, we tested the competence of the cytoplasm from parthenogenetic eggs, which had been artificially activated with strontium chloride. The use of serial NT and parthenogenetic eggs was also found to be effective in accelerating development and improved the developmental ability of the cloned embryos that had received nuclei from morulae. Of the reconstructed embryos, 82.6% developed into morula-blastocysts, and pups were produced from 6.0% of the cloned embryos (Table 1). This result suggests that the acceleration of development among cloned embryos obtained by serial NT was due to either dilution of detrimental substance derived from the donor cytoplasm or supplementation of factors derived from one-cell eggs.
Phenotype of Cloned Pups and Placentae
All pups obtained from cloned embryos derived from four- and eight-cell embryos and morula embryos were healthy and showed no aberrant phenotypes. A well-developed placenta without a fetus and a dead fetus were recovered at Embryonic Day (E) 19.5 in series comprising embryo clones derived from ICM cells. Compared with the PNT controls, the cloned pups derived from ICM or ES cells showed slight overgrowth (Fig. 2A). Cloned placentae constructed by single and serial NT and those of controls were collected at E19.5, and their weights were compared (Fig. 2B). The placental weight of cloned mice derived from four- and eight-cell embryos was within the normal range of the PNT controls; no significant difference was observed between the single and serial NT groups. Although the data were obtained only from the serial NT group, a slightly heavy placenta was present in cloned embryos reconstructed using morula embryos. A remarkable enlargement of the placenta (weighing more than 300 mg) was observed in three of the seven clones from ICM cells (350, 352, and 404 mg) in the single NT group. Of the six clones produced by serial NT, two showed slight enlargement of the placenta (228 and 238 mg). With regard to the ES cell-derived clones, all placentae were enlarged (190–536 mg) in both the standard group and the serial NT group.
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Histological Analysis of the Placentae
As shown in previous studies [8, 24], placental enlargement in clones derived from somatic and ES cells is accompanied by an extensive proliferation of trophoblastic and glycogen cells in the spongiotrophoblast layer as well as the enlargement of trophoblastic giant cells. Histological analysis has produced several lines of evidence that indicate the formation of abnormal placentae even in the embryonic cloned concepti (Fig. 3). Trophoblastic giant cells in the placenta were already differentiated and showed prominent enlargement in all cloned embryos that were reconstructed using nuclei from cleavage-stage embryos. Among the placentae, the ratio of the area of spongiotrophoblast layer (which comprised glycogen cells and trophoblastic giant cells) to that of the labyrinthine layer increased with the progression of the developmental stage of the donor cells (Table 2). The hypertrophic placentae in the morula- and ICM-cloned concepti were also associated with typical histological abnormalities such as the extensive proliferation of trophoblastic and glycogen cells as well as distortion of the boundary between the spongiotrophoblast and labyrinthine layers.
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Although serial NT achieved better numerical success in the case of eight-cell clones, we did not observe any distinctive histological difference in the placenta between both the NT methods as well as those of clones derived from four-cell embryos and ES cells. However, distortion of the boundary between the spongiotrophoblast and labyrinthine layers in the hypertrophic placentae was reduced in the placentae reconstructed by serial NT using an ICM donor cell (Fig. 3).
DISCUSSION
When Do Decisive, Irreversible Epigenetic Changes Occur?
According to an emerging hypothesis, the differentiation state of donor cells is inversely correlated with their cloning efficiency [25–27]. However, little is known about the developmental competence and abnormalities in the concepti in embryonic cloning. One notable report described the developmental capability of reconstructed oocytes with nuclei from preimplantation-stage embryos and referred to the reprogramming ability of the donor nuclei. Recently, Hiiragi and Solter have compared the ability of G1-stage nuclei from zygote, two-, four-, and eight-cell embryos to support development [2]. They were able to produce cloned pups from two- and four-cell embryos; however, this was not possible with eight-cell embryos. These data suggest that the ability of reprogramming after NT reduces rapidly during the early preimplantation stage.
In the present study, we succeeded in producing cloned pups from eight-cell embryos (1.8%) by conventional NT. However, the results showed that the barrier to reprogramming of donor nuclei was clearly present from the eight-cell stage, and this was most evident in the morula-stage embryos; however, this was not observed in ICM or ES cell donors. The secure barrier that prevented the development of eight-cell and morula embryo nuclei was successfully overcome by the use of serial NT, and 17.1% and 11.4% of the respective cloned embryos developed to term. The result obtained from serial NT experiments showed a decline in the full-term developmental competence of cloned embryos derived from morula and ICM donor nuclei (11.4% and 6.6%, respectively). Additionally, when ICM cells were used as donor nuclei, the rate of development to term was more than double that of the ES cloned embryos; however, developmental regression and developmental disorder of placentae were recognized in ICM clones as well as ES clones. These results suggest that significant epigenetic changes, which result in inappropriate reprogramming of donor nuclei, occur in the ICM cells of blastocysts. The findings reported to date suggest that Sertoli cells from newborn mice could be the most competent donors for somatic cell cloning [28] with 5%–6% of the reconstructed embryos giving rise to live pups.
It is also possible to observe irreversible epigenetic modifications in the placentae of cloned concepti derived from preimplantation embryos. Although Tsunoda and Kato reported that live pups could be obtained from ICM and trophectodermal cells of blastocysts using serial NT [23], there was no mention of abnormalities in the cloned mice. Here, the first sign of development of a placental abnormality was the significant enlargement of giant cells, which was observed in all embryonic cloned placentae. However, no enlargement was observed in the giant cells that were produced by PNT. A distortion of the boundary between the spongiotrophoblast and labyrinth layers was first detected in placentae of clones derived from eight-cell embryos. This distortion became severe with the progression of donor embryonic stages. Subsequently, placental enlargement was found to progress starting from clones derived from morula. The typical placental enlargement was observed when ICM cells were used as nuclear donors. The placenta attained a weight of more than 300 mg with extensive proliferation of trophoblastic and glycogen cells in the spongiotrophoblast layer. These findings suggest that irreversible epigenetic changes occur in a stepwise manner during early development and become decisive at the morula stage.
Mitotic Donor Nucleus Is Beneficial for Embryo Cloning
We first succeeded in embryo cloning from eight-cell embryos (1.8%) and ICM cells (5.4%) by conventional NT. This improvement was the result of using metaphase nuclei as donors. The cell cycle is arrested at the M phase by nocodazole with high synchronicity and low toxicity [29]. The use of nocodazole treatment tended to synchronize the cells, thereby reducing the selection of S-phase cells. Contamination with S-phase nuclei has been shown to be incompatible with development because premature chromosome condensation causes pulverization of the replicating chromosome [30–32]. The use of G1-phase nuclei as donors decreased the efficiency of two-cell formation following nuclear transfer from preimplantation embryos [2, 22, 23]. The short duration of the G1 period was suggested as being a prominent factor that contributed to the poor cloning success with nuclei in progressively later stages of cleavage [2, 33]. These results suggest that a mitotic donor nucleus is beneficial for embryo cloning.
Furthermore, NT using metaphase nuclei is beneficial for culturing cells such as ES cells in which it is difficult to synchronize the cell cycle to the G0/G1 phase. ES cells are fast-dividing cells with short G1 and G2 phases [34]. Previous studies have demonstrated that compared to interphase nuclei, metaphase nuclei from ES cells have a significantly higher rate of development to the morula or blastocyst stage [35, 36]. Metaphase donor chromatin may be more susceptible to reprogramming than G1 phase chromatin. The reasons for this beneficial effect remain unclear. Chromatin that is already condensed and organized into chromosomes may offer increased mechanical resistance to the forces of the mitotic spindle. In contrast, the structural changes that accompany the rapid condensation of donor chromosomes after nuclear transfer of interphase cells may be potentially harmful. Chromatin condensation after introduction into the recipient MII cytoplasm often leads to an abnormal metaphase with chromosomes dispersed along the spindle [30]. In addition, it also leads to a decrease in the number of asters, probably because some of the asters have participated in the formation of the spindle that anchors the donor chromosomes in mice [37].
Furthermore, transferring a nucleus as condensed chromosomes is a convenient means of ensuring that transcription is silenced globally. This has been considered particularly important in mice where the onset of zygotic activities is required early after the first cleavage [38, 39]. Although a substantial amount of attention has been focused on the mechanism by which the chromosomes condense and segregate, much less is known regarding the mechanism leading to the transcription silencing during mitosis [40, 41].
Advantages of Serial NT
The advantages of serial NT have been reported primarily by our group as well as Tsunoda's group [21, 23]. The effect of this approach can be observed when fertilized one- and two-cell embryos are used for obtaining cells with the recipient cytoplasm in the second NT. However, the evidence obtained from previous reports is not necessarily sufficient to illustrate the advantages of serial NT or to clearly demonstrate the potential effect of this method. Here, we compared the effect of standard NT and serial NT to clarify this issue. Interestingly, serial NT was clearly more effective not only in terms of achieving a greater degree of developmental competence of reconstructed embryos but also with respect to the inhibition of abnormalities in embryonic cloning. When eight-cell embryos and morula embryos were used as nuclear donors, the maximum effect in terms of embryo cloning was achieved. Serial NT did not lead to significant amelioration of the placental abnormalities observed in the hypertrophic placentae, but distortion of the boundary between the spongiotrophoblast and labyrinthine layers in the hypertrophic placentae was reduced when an ICM donor cell were used.
The mechanism(s) and the cause of these effects are not entirely understood; however, the present results may have been due to either the supplementation of molecules produced by the embryonic genome after oocyte activation or the reduction in donor cell-derived molecules. The observed effect was not reduced when parthenogenetic one-cell embryos were used, suggesting that the molecules involved were not specific to the origin of fertilization. The effect of serial NT was clearly observed in the cloned embryos derived from eight-cell embryos and morula embryos. In such preimplantation-stage embryos, the molecules produced during a period extending for several hours following fertilization may be insufficient for supporting normal development.
Here, we demonstrated that the extent of placental malformation caused by embryo cloning is related to the origin of the donor nuclei, and this type of malformation progresses with embryo development. Placental malformation was observed not only in cloned concepti derived from ES cells but also in those derived from embryos. These findings suggest that faulty reprogramming was the result of irreversible epigenetic changes that had occurred during the preimplantation stage. Thus, we have shown here that developmental abnormalities such as placentomegaly and embryo loss during development occurred even in cloned embryos reconstructed with preimplantation-stage embryos. In addition, it was demonstrated that such effects are not specific to somatic cloning.
FOOTNOTES
1 Supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan and from the Ministry of Agriculture, Forestry and Fisheries of Japan (Development of stable production technology of clone animals by somatic cell nuclear transfer) to T.K. and from the Japan Society for the Promotion of Science (JSPS) for Young Scientists to Y.O. 
2 Correspondence: Department of Bioscience, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan. FAX: 81 3 5477–2543; tomohiro{at}nodai.ac.jp
3 Current address: Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.
Received: 8 November 2005.
First decision: 26 November 2005.
Accepted: 3 May 2006.
REFERENCES
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