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Reproductive Technology |
RIKEN Bioresource Center,3 Tsukuba, Ibaraki 305-0074, Japan
CREST,4 JST, Saitama 332-0012, Japan
Department of Veterinary Science,5 National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan
Gene Research Center,6 Tokyo Institute of Technology, Yokohama 226-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, cumulus cells, embryo, implantation, Sertoli cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since the first successful somatic cell cloning in the mouse in 1998 [3], cumulus cells with the hybrid F1 genotype (BDF1 or B6C3HF1) have become the standard donors in mouse cloning experiments. Generally, normal cloned mice are not born alive from donor cells from inbred strains, with the exception of the 129 strains (cumulus cells [4] and tail-tip cells [5]). The 129 group represents inbred strains of mice that are excellent sources of embryonic stem (ES) cell lines [6]. The underlying mechanism of this unique feature of the 129 mouse strain is not fully understood, but it is possible that the epigenetic status of the 129 genome is less stable and more easily modified in comparison with those of other strains of mice. If this is true, it may be possible to reprogram the genome of the 129 donor cells and thereby improve the subsequent development of reconstructed embryos.
The donor cell type is another factor that is generally thought to affect cloning efficiency in mammals. Precise assessment of the effects of cell type on somatic cell cloning may be performed using the mouse as a model because different donor cells with the same genetic background are readily available in this species. Previously we reported that the nuclei of immature Sertoli cells from neonatal BDF1 mice supported embryonic development at a relatively higher rate than did cumulus cells with the BDF1 background [7]. More recently it was reported that fetal neural BDF1 cells further increased the efficiency of cloning [8]. In contrast, fetal fibroblasts, which are the most commonly used donor cells in domestic species, are not necessarily good donors in the mouse [9], irrespective of their genotype [2, 5].
The present study was undertaken to examine whether donor cell type, donor genotype, or a combination thereof increased the efficiency of mouse cloning. In the first series of experiments, we used cumulus cells and immature Sertoli cells from 129/Sv-ter mice, their F1 hybrids, and BDF1 mice as donors in a 2 x 6 factorial analysis. In the second series of experiments, we examined whether the presence of the 129 genome enhanced the development of hybrid F1 clones that had the wild-type genome (JF1, Mus musculus molossinus; [10]). The production of clones with these hybrid genotypes is of practical significance because these genomes are amenable to allele-specific analysis of polymorphisms in laboratory and wild-type mice. In this study, we analyzed the parental expression pattern of the imprinted Xist gene, which is involved in the initiation of X-chromosome inactivation [11], in cloned placentas with the JF1 hybrid genotype. In both experiments, statistical analyses were performed on results that pertained to the developmental ability of the reconstructed embryos (i.e., the rates of cleavage, development into morulae/blastocysts, implantation, and development into term offspring).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The donor cells were collected from inbred and F1 hybrid mice, as shown in Table 1. Inbred strains, such as C57BL/6, DBA/2, and JF1, with the exception of strain 129, were not used as donors because their reconstructed embryos show extremely poor developmental ability in vitro and in vivo [4, 5, 7]. BDF1 (Japan SLC, Shizuoka, Japan) and ICR (Japan Crea, Tokyo, Japan) female mice were used for the collection of recipient oocytes and as the embryo transfer recipients, respectively. The mice were maintained under specific-pathogen-free conditions at the National Institute of Infectious Diseases (Japan). They were provided with water and commercial laboratory mouse chow ad libitum and housed under controlled lighting conditions (daily light period of 0700–2100 h). All animals were maintained in accordance with the guidelines of the National Institute of Infectious Diseases.
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Preparation of Donor Cells
Cumulus cells and immature Sertoli cells were collected from mature females (2–4 mo old) and immature males (0–5 days old), respectively. The cells were prepared for nuclear transfer as described previously [3, 7]. Briefly, cumulus cells were isolated from the cumulus-oocyte complexes of mice that were superovulated, as described below. The cumulus cells were allowed to disperse in KSOM medium [12] that contained 0.1% bovine testicular hyaluronidase (Sigma Chemical Co., St. Louis, MO) for a few minutes and used for injection. Immature Sertoli cells were collected as a testicular cell suspension, which was prepared by treatment with 0.1 mg/ml collagenase (Sigma) and 0.01 mg/ml deoxyribonuclease (DNase; Sigma) for 30 min at 37°C, followed by 0.2 mg/ml trypsin (Sigma) for 5 min at 37°C. The testicular cell suspension was washed with PBS(-) that contained 4 mg/ml BSA and used for injection. Immature Sertoli cells were identified on the basis of size and morphology under Nomarski optics [7].
Collection of Recipient Oocytes
The BDF1 females were induced to superovulate at 8 to 10 weeks of age by injections of 7.5 IU of eCG (Sankyo, Tokyo, Japan) and 7.5 IU of hCG (Sankyo) at a 48-h interval. Mature MII oocytes were collected from the oviducts 15 h after hCG injection, released from the cumulus cells by treatment with 0.1% bovine testicular hyaluronidase (Sigma) in KSOM, washed three times, and incubated in fresh medium at 37.5°C in an atmosphere of 6.2% CO2 in air.
Nuclear Transfer
Nuclear transfer to enucleated oocytes was carried out as described previously for the nuclei of cumulus cells [3] and immature Sertoli cells [7]. Using a warmed manipulation stage (37°C), the recipient MII oocytes were enucleated together with a small amount of the surrounding cytoplasm in Hepes-buffered KSOM that contained 5 µg/ml cytochalasin B (Sigma). The chromosomes of the BDF1 oocytes were easily discernible under Nomarski optics and could be removed using a Piezo-driven micromanipulator (Prime Tech, Ibaraki, Japan), within 10 sec of puncturing the zona pellucida. After the cytochalasin B was removed by repeated washes, the enucleated oocytes were incubated in KSOM for 30 min to 2 h to allow complete restoration of the membranes. The donor nuclei were injected into enucleated oocytes in Hepes-buffered KSOM at room temperature using the Piezo-driven micromanipulator. It was not necessary to isolate the donor nuclei from the cytoplasm completely because cumulus and immature Sertoli cells are nonrigid and their nuclei are readily exposed to the ooplasm on injection. The injected oocytes were cultured in KSOM for 1 to 2 h until the donor chromosomes condensed in the MII cytoplasm [3]. These reconstructed oocytes were activated for 1 h with Ca2+-free KSOM that contained 3 mM SrCl2 and 5 µg/ml cytochalasin B and then cultured for 5 h in KSOM that contained 5 µg/ml cytochalasin B. The cytochalasin B was removed by washing with fresh KSOM, and the oocytes were cultured in KSOM for 72 h.
Embryo Transfer
Reconstructed embryos that reached the morula/blastocyst stage by 72 h in culture were transferred into pseudopregnant ICR females (8 to 12 wk old) on Day 3 (Day 1 was designated as the day following sterile mating). Eight to 10 embryos were transferred into each uterine horn (16 to 20 per recipient). On Day 20, the recipient females were examined for the presence of fetuses, and live pups were nursed by lactating ICR females.
Experimental Design and Statistical Analysis
In Experiment 1, the effects of donor cell type and genotype on the development of cloned embryos were examined using laboratory mouse strains. In Experiment 2, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. It is critically important for valid statistical analysis that day-dependent variations in the experimental condition be minimized. The duration of the experiments was therefore limited to 1 yr (from December 2001 to December 2002); during this period, the basal level of cloning efficiency was monitored at least twice a month using BDF1 cumulus cells or immature Sertoli cells, which are the standard donors for mouse cloning. Each group consisted of at least 200 reconstructed embryos, with a minimum of two replicates. At least 40 reconstructed embryos were cultured in each experiment per day. The percentages of embryos that developed to the two-cell and morula/blastocyst stages underwent implantation and reached term were analyzed using arcsine transformation, followed by two-way ANOVA analysis. A post hoc procedure using Scheffe's F test was adopted for multiple comparisons between the groups where appropriate. For the statistical analysis, we used a computer program (Stat123 for Windows, http://www.vector.co.jp, in Japanese), which is capable of performing factorial ANOVA with unequal replication because the number of replicates for the BDF1 groups was larger than those for the other groups.
Allelic Expression Analysis of the XistGene
In Experiment 2, we generated clones with the JF1 hybrid genotypes, which enabled us to identify the active alleles of the imprinted genes. To see whether the X-inactivation memory of the donor somatic cells was inherited by the trophectoderm lineage, we analyzed the parental expression pattern of the Xist gene in the cloned placentas. Total RNA was extracted from the placental tissue using ISOGEN (Nippon Gene, Tokyo, Japan) and treated with RNase-free DNase I (Nippon Gene) to eliminate residual contamination of genomic DNA. First-strand cDNA was synthesized with the Xist-specific primer F1063AS [13] using 1 mg total RNA at 50°C and Superscript II (Life Technologies Inc., Rockville, MD), as recommended by the manufacturer. A 1/1000 dilution of the cDNA was then subjected to PCR using ExTaq (TaKaRa, Kyoto, Japan) for 30 cycles of 96°C for 15 sec, 65°C for 30 sec, and 72°C for 30 sec, followed by a 1-min extension step at 72°C. The following primer sequences were used: for Xist-specific cDNA synthesis (F1063AS), 5'-GCACAACCCCGCAAATGCTA-3'; for PCR (700P2), 5'-CGGGGCTTGGTGGATGGAAAT-3'; AS1634F, 5'-GCGTAACTGGCTCGAGAATA-3'. The amplified PCR products were subjected to direct sequencing using the BigDye cycle sequencing kit (Applied Biosystems, Foster City, CA). The expressed allele was determined with a single nucleotide polymorphism between B6 and JF1, as described by Sado et al. [13].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The in vitro and in vivo development profiles of the embryos that were reconstructed from cumulus or Sertoli cell nuclei are summarized in Table 2. In the control BDF1 groups, the percentages of embryos that developed into the morula/blastocyst stage (per cleaved embryo) and underwent implantation were always higher than 45% and 35%, respectively, throughout the experimental period. Their SDs were not statistically different from those of our routine microinsemination experiments (P > 0.05, F-test) (data not shown). Thus, we considered that our experimental protocol was stable enough to perform statistical analysis.
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The rates of development to the two-cell and morula/blastocyst stages per cultured embryo were higher when cumulus cells were used as the donors (P < 0.005 and P < 0.001, respectively) (Table 3). The cell types and genotypes had no effects on the rate of development into morulae/blastocysts per cleaved embryos or on the implantation rate (Table 3). There was a significant effect of cell type on the birth rate per transfer, with Sertoli cells performing better (P < 0.005). Normal pups were obtained in all of the experimental groups, except for the (B6 x 129) cumulus clones. The highest mean birth rate was obtained with (B6 x 129) Sertoli cells (10.8 ± 2.1%) (Table 2). The highest birth rate in the one embryo transfer experiment was 15.0% (12 of 80), which was with the (DBA x 129) Sertoli cells (Fig. 1). Because there was a significant interaction on the birth rate between the cell type and genotype (P < 0.005; Table 3), the analysis was simplified to examine the effect of all combinations of the two factors using one-way ANOVA analysis. We found that there was a significant difference between the groups (P < 0.005); the multiple comparison test revealed that the (B6 x 129) and (DBA x 129) immature Sertoli cells developed into offspring with higher efficiencies (P < 0.05) than did the BDF1 cumulus cells or BDF1 Sertoli cells, which are the standard donors for mouse-cloning experiments (Table 2, Fig. 2). As shown in Table 2, more pups were born from Sertoli cells than from cumulus cells in each genotype group, and there were statistically significant differences in the 129 and B6 x 129 groups (P < 0.005 and P < 0.001, respectively) (Fig. 2). Significant interactions were also observed for the rate of development into morulae/blastocysts per cultured embryo and per cleaved embryo (P < 0.01 and P < 0.05, respectively; Table 3).
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Because it is possible that the large multiplicity of replicates for the BDF1 groups may compromise the optimum power of the statistical analysis, we also performed the ANOVA analysis without the BDF1 groups. The data obtained using this supplemental analysis were essentially the same as those obtained when the BDF1 groups were included, with the exception that no significant interaction was observed for development into morulae/blastocysts per cultured or cleaved embryo (Table 3). Thus, it is very likely that the cell type effects and interaction between the two factors observed in Experiment 1 are statistically valid.
Experiment 2
On the basis of the results obtained in the multiple comparison test in Experiment 1, we speculated that the presence of the 129 genome increased cloning efficiency. In the second series of experiments, we examined whether the developmental ability of clones with the wild-type genotype (JF1) was improved when combined with the 129 genotype. The results are summarized in Table 4. There was no effect of cell type or genotype on in vitro development or implantation (Table 5). Normal cloned pups were obtained from cumulus and immature Sertoli cells of the (129 x JF1) and (JF1 x 129) genotypes, whereas no pups were born from cells with the (B6 x JF1) genotype (Table 4), which indicates a highly significant effect of genotype on birth rate (P < 5 x 10-8) (Table 5). Furthermore, the Sertoli cell clones in this experiment produced significantly higher birth rates (P < 0.005; Table 5). The interaction between the two factors was statistically significant (P < 0.005; Table 5), as found in Experiment 1. The result of the multiple comparison test between groups is shown in Table 4.
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Allelic Expression Analysis of the XistGene
The allelic expression of the Xist gene in cloned (129 x JF1) placentas was analyzed by direct sequencing of the transcripts. As shown in Figure 3, the expressed parental allele was clearly identified by a single nucleotide polymorphism. Six of the eight cloned placentas (including two placenta-only conceptuses [2]) expressed the 129 maternal allele, and two expressed the JF1 paternal allele; mixed patterns of expression were not observed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We demonstrate that the effect of cell type on cloning appears as early as the first cleavage, as shown in Experiment 1, in which significantly more cumulus cell embryos than immature Sertoli cell embryos developed into the two-cell stage. Although not statistically significant, the same tendency was observed in Experiment 2, in which the hybrid JF1 genotypes were used (Table 4). The improved cleavage of cumulus cell-derived embryos probably reflects their larger G0/G1 population (about 90% of the total; [21]), as compared with that of the immature Sertoli cells (about 70%) [22]. It is interesting to note that once cleavage into the two-cell form occurred, cell type and genotype had no significant effects on subsequent development into morulae/blastocysts (the rate per cleaved embryo) or progression to the implantation stage (Tables 3 and 5). Therefore, when the nuclei of fresh donor cells are transferred into recipient oocytes, we can roughly estimate that about 70% of the cleaved embryos will develop into morulae/blastocysts, and about 50% of the transferred embryos will undergo implantation, as indicated in Tables 2 and 4.
In contrast to late embryonic development through implantation, postimplantation embryonic development was affected significantly by the cell type. Two-way ANOVA analysis revealed that immature Sertoli cells were significantly better donors than cumulus cells in terms of their ability to support fetal development to term (Tables 3 and 5). Assuming that the levels of sensitivity to physical damage are similar among these two cell types, it seems reasonable to conclude that the genomes of immature Sertoli cells are more easily reprogrammed by nuclear transfer and are more successful at activating the genes necessary for normal fetal development. Although the exact mechanisms that determine nuclear reprogrammability are unclear, we know empirically that smaller donor cells often provide better clone development than larger cells. Immature Sertoli cells (8 µm diameter) are slightly smaller than cumulus cells (10–12 µm) and are much smaller than fetal fibroblast cells (15–20 µm), which are very difficult to clone in the mouse [2, 9]. Interestingly, highly confluent (80%–90%) donor ES cell cultures contained a high proportion of smaller cells and showed significantly improved development of reconstructed embryos in vitro and in vivo, compared with subconfluent (60%–70%) ES cell cultures, whereas the percentage of G1 cells was similar between the two groups [20]. Chromatin modifications that are associated with the compacted nucleus may enhance the genomic reprogramming of MII oocytes, at least in the mouse.
On the basis of previous studies on the genetic effects of donor cells [4, 5], we expected that the 129 genome would increase the reprogramming efficiency of the donor genome during nuclear transfer. The flexibility of the 129 genome has been demonstrated by the relatively easy establishment of ES cell lines from 129 strains [6, 23], the high incidence of spontaneous testicular teratoma [24], and the unstable expression of transgenes in the 129 genetic background [25]. For Sertoli cell cloning, but not for cumulus cell cloning, donors with the 129 genome showed improved efficiency of clone production, with B6 x 129 being significantly more efficient than BDF1. The superiority of the 129 genome for cloning was more apparent in Experiment 2. Normal pups were born after nuclear transfer with either 129 x JF1 or JF1 x 129, using cumulus and Sertoli cells as nuclear donors, whereas no pups were born from B6 x JF1 donor cells. We also confirmed that the development of embryos that were cloned from fetal germ cells was significantly improved when 129 strains were used as donors (K.I., unpublished data).
Another interesting finding in the present study is the significant interaction between donor cell type and genotype for the development to term of reconstructed embryos (Tables 3 and 5). This implies that the effect strength of one factor (e.g., cell type) on fetal development may vary according to the other factor (e.g., genotype) with which it is combined. In other words, the birth rate after transfer of cloned embryos is determined by the combination of donor cell types and genotypes. Genomic reprogramming during nuclear transfer apparently involves large-scale modification of the epigenetic status of the donor genome. This epigenetic status of the somatic donor genome, which encompasses DNA methylation and chromatin structure, may vary according to the cell type and genotype. This explains why combinations of cell type and genotype affect the fetal development of clones, which involves the normal activation of a large number of genes. In this study, cloning using four types of Sertoli cells (B6 x 129, DBA x 129, JF1 x 129, 129 x JF1) resulted in birth rates of approximately 10% per transfer. This efficiency is low, compared with the efficiency achieved using in vivo- or in vitro-fertilized embryos. However, it is possible that a 10% birth rate is maximal for mouse somatic cell cloning because several technical factors, such as physical damage to the donor cells and oocytes during handling in vitro, decrease the viability of the cloned embryos. Embryo transfer also decreases the yield of viable offspring. In our routine microinsemination experiments using round spermatids, only 20%–40% of the transferred embryos developed to term [5], although the genetic constitution and imprinting status of round spermatids are essentially the same as those of mature spermatozoa [26]. Because cloned embryos experience harsh treatments, such as enucleation, microinjection, and long-term exposure to cytochalasin, their viability is decreased considerably. Even when optimal ES cell lines, the genomes of which can be readily reprogrammed [27], are used for nuclear transfer, the rate of development to term of the reconstructed embryos is 10%–23% per transfer [19].
In Experiment 2, the 129 genotype was combined with the JF1 genotype to generate an F1 hybrid genotype, which facilitated polymorphic analysis for the identification of the expression allele. We examined the parental expression of the Xist gene in placentas that were cloned from 129 x JF1 cumulus cells by taking advantage of the single nucleotide polymorphism. Six of the eight cloned placentas (including two placenta-only conceptuses [2]) expressed the 129 allele, and two expressed the JF1 allele; mixed patterns of expression were not observed. This clearly indicates that X-chromosome inactivation in the trophectoderms of the clones is nonrandom (i.e., the X-inactivation memory of the donor somatic cells is inherited by the trophectoderm lineage), as reported previously [28]. Analysis of placentas that were cloned from cumulus and Sertoli cells with the JF1 hybrid genotypes confirmed our previous finding [29] that imprinted gene memories on the donor genome are not modified by somatic cell cloning (data not shown).
Future strategies for increasing cloning efficiency should include the establishment of novel nuclear transfer techniques that are based on information at the molecular level. The present study indicates that cloning efficiency would also be improved by choosing appropriate combinations of the donor cell type and genotype. Analysis of the embryos and animals that are cloned from donor cells with defined cell types and genotypes may provide us with a better understanding of the mechanisms of genome reprogramming following nuclear transfer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: A. Ogura, RIKEN Bioresource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan. FAX: 81 29 836 9172; ogura{at}rtc.riken.go.jp
Received: 28 March 2003.
First decision: 30 April 2003.
Accepted: 4 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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