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Embryo |
Department of Molecular Animal Breeding and Biotechnology,4 University of Munich, 85764 Oberschleissheim, Germany
Gene Center,5 University of Munich, 81377 Munich, Germany
Institute of Human Genetics,6 Mainz University School of Medicine, 55101 Mainz, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, cumulus cells, early development, embryo, fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is possible that the highly specialized gametic genomes require parent-specific reprogramming to establish biparental totipotency, which is a prerequisite for the generation of all cell lineages in the embryo [5]. The genetically inactive chromatin of the highly differentiated sperm and egg cells must be remodeled and the differential gametic marks be erased to achieve a totipotent state in the early embryo. Active demethylation of the paternal zygotic genome may make it easier for the cellular machinery of the fertilized egg to remodel the paternal chromosomes and to reestablish higher order chromatin structures to reprogram appropriate patterns of gene expression for early development [6, 7]. It is now well established that changes in DNA methylation are fundamental to mammalian development [7, 8]. Previously, we have shown that aberrant methylation patterns in mouse two-cell embryos correlate with subsequent developmental failure [9]. On the other hand, somatic cell nuclear transfer (SCNT) studies in different mammalian species demonstrated incomplete or abnormal methylation reprogramming of the donor genome in cloned embryos [3, 10, 11], which is associated with a variable but generally low cloning efficiency. However, the successful cloning of mammals from widely different orders [12, 13] implies that at least some highly methylated and differentiated somatic nuclei can be reprogrammed for embryonic development.
Normal mammalian development and viability do not only require correct genome reprogramming in the preimplantation embryo but also depend on euploid paternal and maternal chromosome sets. Proper meiotic and mitotic chromosome segregation is responsible for the stable inheritance of genetic information. Segregation errors during meiosis of the parental germ cells and/or the mitotic divisions of the early cleaving embryo are an important cause of spontaneous abortions and abnormal phenotypes in humans and other mammals [14, 15]. Aneuploidy that is a diploid chromosome complement with gain or loss of a particular chromosome or subset of chromosomes is the most common class of chromosome abnormality in humans, occurring in at least 0.3% of newborns, 5% of stillbirths, and 50% of recognized first-trimester spontaneous abortions [16, 17]. Chromosome abnormalities are also common in bovine embryos; however, the incidence, ranging from approximately 10% to >70%, varies significantly with the method of embryo production, stage of development, and also method of analysis [18]. In general, in vitro-produced embryos exhibit a higher rate of abnormalities than in vivo-produced embryos [19, 20]. Preimplantation bovine embryos frequently demonstrate ploidy errors affecting the whole chromosome complement. Haploidy, polyploidy, or mixoploidy (i.e., diploid-triploid or diploid-tetraploid) are not the result of meiotic or postzygotic nondisjunction but arise at or very soon after fertilization, i.e., through polyspermic fertilization or retention of the second polar body. The very few studies that have been carried out so far on the chromosomal constitution of cloned bovine embryos suggest an increased rate of ploidy errors and aneuploidy compared with in vivo- or in vitro-produced embryos [21, 22].
Rabbit is a common and important animal model in biological and medical research. Although there have been many attempts to clone this species [23–25], so far, only one group successfully produced live offspring from rabbit SCNT [26]. The difficulties with rabbit cloning may at least partially be due to insufficient remodeling of somatic cell nuclei in oocytes. To study possible species differences in epigenetic reprogramming during preimplantation development, we have performed methylcytosine (MeC) staining on in vivo-fertilized and cloned rabbit embryos. In addition, we have used fluorescence in situ hybridization (FISH) to compare the chromosomal aneuploidy rates of rabbit embryos derived from in vivo fertilization and SCNT.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal experiments were approved by the Ethical Committee for animal experimentation of the University of Munich and were performed in accordance with the European Union normative for care and use of experimental animals. Oocytes were obtained from sexually mature outbred Zika rabbits during the natural breeding season. Female rabbits were superovulated by injection of 100 IU eCG (Intergonan; Intervet, Unterschleissheim, Germany) intramuscularly and 100 IU human chorionic gonadotropin (hCG; Ovogest 1500; Intervet) intravenously 72 h later. Mature oocytes were flushed from the oviducts 16 h post-hCG injection in warm PBS supplemented with 4 mg/ml BSA and incubated in M199 medium (Sigma, St. Louis, MO) containing 5 µg/ml hyaluronidase (Sigma) for 15 min at 38°C. Cumulus cells were removed by gentle pipetting with a small-bore pipette.
In Vivo Embryo Production
Female rabbits were superovulated as described above and mated immediately after hCG injection. For recovery of zygotes, females were slaughtered 24 h after mating and laparotomized. The oviducts were flushed and the fertilized oocytes transferred into 100-µl drops of MPM medium supplemented with 10% (v/v) fetal calf serum (FCS) (Biochrom, Berlin, Germany), covered with mineral oil, and cultured at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 [27]. Half of the embryos were cultured up to the blastocyst stage and the embryonic development was recorded. The other half of the embryos was cultured to the required stages for immunofluorescence or FISH analysis. Because only 4–6 slides (embryos) were processed at the same time, at least 10 independent MeC staining and FISH experiments each were performed on in vivo-fertilized and cloned embryos.
Donor Cells and Cell Culture
Rabbit cumulus cells and fetal skin fibroblasts from primary cultures (within six passages) were used as nuclear donors. Cells were cultured in Dulbecco modified Eagle medium (Gibco BRL, Grand Island, NY) supplemented with 10% (v/v) FCS, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 2 mM nonessential amino acids, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Gibco) at 37.5°C in a humidified atmosphere of 5% CO2 in air. Before using the cells as nuclear donors, they were grown in a medium containing only 0.5% FCS for 5 days. Cells with diameters of 10–15 µm were chosen for nuclear transfer (NT).
Enucleation and Nuclear Transfer
Cumulus-free meiosis II oocytes with first polar body were incubated for 10 min in 10 µg/ml Hoechst 33342 (Sigma) and then transferred into M199 medium (Sigma) containing 7.5 µg/ml cytochalasin B. The chromosomal DNA and a small part of the adjacent cytoplasm were aspirated. Enucleation was assessed by visualization of the metaphase plate under ultraviolet (UV) light. The time of exposure to UV light must not exceed 10 sec. All manipulations were done in M199 medium supplemented with 25 µM Hepes and 10% FCS. Transfer of donor nuclei into enucleated oocytes was carried out essentially as described previously [28]. Donor cells were transferred into the perivitelline space of the oocytes. Karyoplast-cytoplast complexes (KCCs) were exposed to a double electric pulse of 2.1 kV/cm for 10 µs 19–21 h post-hCG using a Zimmermann cell fusion instrument (Bachofer, Reutlingen, Germany). Fusion rate was determined 30 min after the fusion pulses. Fused KCCs were activated by an additional double electric pulse of 2.1 kV/cm for 10 µsec, followed by a 5-min culture in 7% ethanol and a then 5-h incubation in 10 µg/ml cycloheximide and 5 µg/ml cytochalasin B. KCCs were washed and transferred into culture medium as described previously.
Interspecies Nuclear Transfer
Cumulus-oocyte complexes were obtained by aspiration of 3- to 8-mm follicles from bovine ovaries collected from a local slaughterhouse. After 16–18 h of maturation, oocytes were incubated in PBS containing 3 mg/ ml FCS and 0.3 mg/ml hyaluronidase and vortexed for 2 min. Cumulus cells were removed by gentle pipetting. Oocytes showing a dense cytoplasm and a well-extruded polar body were selected for NT. Nuclei of rabbit fetal fibroblasts were transferred into the perivitelline space of oocytes that were enucleated with minimal cytoplasmic volume. The KCCs were fused and activated using a Zimmermann cell fusion instrument as described earlier. Then they were transferred into 100-µl drops of synthetic oviduct fluid supplemented with 5% (v/v) estrous cow serum, covered by paraffin oil, and cultured at 39°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2.
Embryo Fixation and Slide Preparation
Air-dried preparations were made according to the method of Tarkowski [29]. The rabbit embryos were placed in hypotonic solution (1% sodium citrate) at room temperature for 15 min. Each single embryo was then transferred in a 5-µl volume onto a well-cleaned glass slide and fixed immediately with several drops of freshly prepared 3:1 mixture of methanol and acetic acid. After air drying, the slides were stored in a covered box at 4°C.
Methylcytosine Staining
Methylated DNA was visualized by a well-characterized monoclonal antibody against 5-methylcytosine [1–3, 6, 9, 10, 30]. After dehydration in an ethanol series (70%, 85%, 100%), the embryo preparations were denatured in 70% formamide, 2x saline sodium citrate (SSC) for 1 min at 70°C. The dehydrated and air-dried slides were then incubated with blocking solution (3% BSA, 0.1% Tween 20, 4x SSC) in a Coplin jar for 30 min at room temperature and with mouse anti-MeC antibody (hybridoma supernatant), diluted 1:50 with PBS in a humidified incubator at 37°C for 30 min. The slides were then washed in PBS three times for 10 min each and incubated for 30 min with fluorescein-isothiocyanate (FITC)-conjugated anti-mouse IgG (Dianova, Hamburg, Germany) appropriately diluted with PBS. After three further washes with PBS, the preparations were counterstained with 1 µg/ml 4',6'-diamidino-2-phenylindole (DAPI) in 2x SSC for 5 min. The slides were mounted in 90% glycerol, 0.1 M Tris-HCl, pH 8.0, and 2.3% 1,4-diazobicyclo-2,2,2-octane. An antibody staining control was performed for each immunofluorescence experiment. Omission of the primary or secondary antibody in the staining procedure resulted in complete absence of immunofluorescence staining.
DNA Probes
Twenty bacterial artificial chromosome (BAC) clones were randomly selected from the LBLN-1 rabbit BAC library and obtained from the Children's Hospital Oakland Research Institute (http://bacpac.chori.org). BAC DNA was prepared with the PhasePrep BAC DNA kit (Sigma) and labeled with either biotin-16-dUTP or digoxigenin-11-dUTP (Roche, Mannheim, Germany) by standard nick translation. All BACs were FISH mapped on metaphase chromosomes and interphase nuclei from rabbit fibroblasts. Only chromosome-specific BACs, LBLN1-94F4, 198D8, 219P16, 315E19, that produced two distinct hybridization signals in >95% of fibroblast nuclei were used as chromosome enumeration probes.
Fluorescence In Situ Hybridization
For FISH, the embryo preparations were treated with 100 µg/ml RNase A in 2x SSC, pH 7.0, at 37°C for 30 min and with 0.01% pepsin in 10 mM HCl at 37°C for 10 min, then rinsed two times in PBS and once in PBS containing 50 mM MgCl2. After fixing the preparations for 10 min in PBS, 50 mM MgCl2, 1% formaldehyde, they were dehydrated and denatured, as described earlier. For hybridization of one slide, 250 ng each of biotinylated and digoxigenated BAC DNA were coprecipitated with 10–20 µg of sheared rabbit genomic DNA (as competitor) and 10 µg salmon sperm DNA (as carrier) and redissolved in 50% formamide, 10% dextran sulphate, 2x SSC. The hybridization mixture was denatured at 80°C for 5 min and preannealed at 37°C for 30 min. Fifteen microliters of hybridization mix was applied to each slide and sealed under a coverslip. Hybridization was done for at least 18 h in a moist chamber at 37°C. For signal detection, slides were washed three times for 5 min in 2x SSC at 45°C and another three times for 5 min in 0.2x SSC at 65°C. Then they were blocked with 3% BSA, 4x SSC, 0.1% Tween 20 at 37°C for 30 min. Probes were detected with FITC-conjugated avidin and Cy3-conjugated antidigoxigenin antibody.
Fluorescence Microscopy
Images were taken with a Leica epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) equipped with a thermoelectronically cooled charge-coupled device camera, which was controlled by a personal computer. Leica CW FISH 400 imaging software was used to capture gray-scale images and to superimpose the source images into a color image.
Statistical Analysis
The proportion of euploid and aneuploid embryos from each experiment was compared by chi-square analysis. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vivo-fertilized rabbit embryos from the one-cell to the blastocyst stage (21 zygotes; 11, 2-cell embryos; 6, 4-cell embryos; 13, 8-cell embryos; 8, 16-cell embryos; 6, morulae; and 4 blastocysts) were processed for anti-MeC immunofluorescence. All 21 fertilized rabbit zygotes analyzed showed equivalently high methylation levels of both male and female pronuclei (Fig. 1A). The nuclei of 2-cell to 32-cell embryos displayed a relatively uniform strong immunofluorescence staining (Fig. 1, B–E), suggesting the absence of genome-wide passive demethylation at least up to the 16-cell stage. In the four blastocysts analyzed, a proportion (20–50%) of nuclei had lost most of their anti-MeC fluorescence (Fig. 2). Because the cells with weak immunofluorescence were usually located in the interior of the blastocyst and the cells with strong fluorescence more outward, it is plausible to assume that the demethylated cells represent embryonic cells from the inner cell mass. All embryos were hypotonically treated before fixation on the slide and then strongly denatured. Although this protocol inevitably destroys much of the embryo's three-dimensional morphology, we found that it largely improves accessibility of the nuclear target sites to the antibody macromolecules and increases DNA resolution. Collectively, our data suggest that the global methylation level of the diploid embryonic genome is largely maintained from the 1-cell up to the 16-cell stage during rabbit preimplantation development; however, some of the embryonic cells from the inner cell mass are demethylated at the blastocyst stage.
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Similar to the situation in in vivo-fertilized rabbit embryos, we did not observe genome-wide methylation changes in 73 rabbit embryos (1 zygote; 14, 2-cell embryos; 4, 4-cell embryos; 15, 8-cell embryos; 12, 16-cell embryos; 18 morulae; and 9 blastocysts) derived from cumulus cell and fetal fibroblast NT (Fig. 3). At the cytological level, there was no detectable difference in the MeC staining patterns between in vivo-fertilized and NT rabbit embryos. The nuclei of cloned 1- to 32-cell embryos were uniformly and highly methylated, whereas blastocysts displayed both highly and weakly methylated nuclei. Rabbit fibroblast cells, which were processed along with the embryo preparations, showed a medium overall nuclear immunofluorescence with brightly fluorescing foci in regions of more highly condensed and/or more highly methylated chromatin (Fig. 4A). In contrast, cumulus cells showed a relatively homogeneous MeC staining of their nuclei (Fig. 4B), comparable with that of in vivo-fertilized and NT embryos. Interestingly, neither fibroblast nor cumulus cell NT embryos from two-cell to morula stage displayed nuclear foci. This implies that the punctate MeC staining pattern of fibroblast nuclei disappears at or soon after NT. Unfortunately, fibroblast cell NT zygotes were not available for analysis.
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Chromosomal Aneuploidy in In Vivo-Fertilized and Cloned Rabbit Embryos
During preimplantation development, we observed a significantly higher rate of developmental failure in NT embryos in all stages analyzed compared with in vivo-fertilized embryos (Table 1). To study whether the reduction of viability in cloned embryos is associated with chromosomal aneuploidies, two-color FISH with chromosome enumeration probes was performed on in vivo-fertilized rabbit embryos, cumulus cell NT rabbit embryos, and interspecies embryos derived from NT of rabbit fibroblast nuclei into enucleated bovine oocytes. Embryonic nuclei were cohybridized with two differently fluorescence-labeled BACs, either LBNL1-198D8 and LBNL1-315E19 or LBLN1-94F4 and LBNL1-219P16. Each probe combination delineated two different chromosomes (see metaphase plates in Fig. 5, B–D). Although the hybridization efficiency of these BAC probes on fibroblast nuclei was >95%, scoring individual embryos, in particular one- and two-cell stages, for aneuploidy was still difficult. Even after hypotonic, RNase, and pepsin treatment of a preimplantation embryo, its nuclei were often covered by excessive cytoplasm, protein masses, and/or RNA from the egg that produced a relatively high background staining and/or reduced accessibility of the probe DNA to its nuclear target sequences. In all FISH experiments, a percentage of embryonic nuclei had to be discarded from analysis because of nonspecific background hybridization. In addition, there were only very few cells available for analysis.
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To avoid gross overestimation of the number of aneuploid embryos, only cells showing at least one hybridization signal for each probe were evaluated. The unreplicated chromosomes during G1 phase produced singlet hybridization signals, whereas the replicated chromosomes of G2 phase cells appeared as doublets. Therefore, nuclei showing two discrete (single-dot or double-dot) hybridization signals for each probe were considered euploid. Nuclei with only one or three and more signals for at least one probe were considered aneuploid. One- to four-cell embryos that had at least one aneuploid cell were scored as aneuploid, whereas in 5- to 16-cell embryos, one aneuploid cell was considered a single-cell hybridization artifact. In morula and blastocyst stages, up to two aneuploid cells were considered technical artifacts. Similar grading systems for the exclusion of false-positive results are routinely used for the prenatal diagnosis of human chromosomal aneuploidies by FISH on uncultured fetal cells [31]. In addition, a very low level of chromosomally abnormal cells is not thought to have detrimental effects because these cells can be eliminated during early development or diverted to the extraembryonic structures [32, 33].
When applying these criteria, 56% of in vivo-fertilized rabbit embryos (24 of 43 analyzed), 83% of cumulus cell NT rabbit embryos (35 of 42), and 79% of interspecies NT embryos (11 of 14) were aneuploid (Table 2). The aneuploidy rate in cumulus cell NT and interspecies NT embryos was significantly higher (P < 0.05) than that in in vivo-fertilized embryos, whereas intra- and interspecific NT embryos showed comparable aneuploidy rates. Altogether, 239 cells from in vivo-fertilized 2- to 16-cell embryos, 202 cells from cumulus cell NT 2- to 16-cell embryos, and 91 cells from interspecies NT 2- to 16-cell embryos were evaluated by FISH. We found 42% (98) aneuploid cells in in vivo-fertilized embryos, 56% (112) in rabbit NT embryos, and 63% (57) in interspecies NT embryos. Again, there was a significant difference (P < 0.05) between NT embryos and in vivo-fertilized embryos, but not between intra- and interspecific NT embryos. Morula and blastocyst stages were excluded from this analysis to avoid that single embryos with a higher-than-average-number of euploid or aneuploid cells strongly influence the statistics.
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It is noteworthy that only one in vivo-fertilized embryo was the result of meiotic nondisjunction, displaying only monosomic cells. All other aneuploid embryos, including 23 in vivo-fertilized, 35 rabbit NT, and 11 interspecies NT embryos were mixtures (mosaics) of euploid and aneuploid cells, consistent with postzygotic chromosomal nondisjunction. In general, there was a good correlation between chromosomal aneuploidy frequency and the rate of developmental arrest during the preimplantation stage: 48% of in vivo-fertilized and 79% of rabbit NT embryos failed to develop to the blastocyst stage, respectively (Table 1). Using our grading system, 56% of in vivo-fertilized and 83% of NT embryos were classified as aneuploid (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although these immunofluorescence results are unexpected, they are consistent with an earlier study using methylation-sensitive restriction enzymes. Molecular analyses of digested genomic DNA of early cleaving rabbit embryos suggested that the rabbit embryonic genome remains highly methylated at least up to the 8-cell stage [34]. In this context, it is important to emphasize that most MeCs in mammalian DNA reside in repeated DNA sequences that represent 30–40% of the genome, whereas CpG islands make up only 1% [35, 36]. Therefore, immunofluorescence staining and restriction analysis of genomic DNA mainly reflect the density of MeCs in various repeat DNA families. We cannot rule out the possibility that dynamic methylation changes occur at gene loci and/or some sequence classes, whereas most of the genome remains relatively undisturbed throughout rabbit preimplantation development. In addition, immunofluorescence staining cannot detect simultaneous demethylation and remethylation waves, which do not dramatically change the methylation percentage of CpGs in the genome.
Although active zygotic demethylation appears to be conserved in different mammalian orders, rabbit may not be the only species that maintains high methylation levels during preimplantation development. Similar to the situation in rabbit, sheep male pronuclei did not show dramatic loss of methylation in normally fertilized sheep zygotes [13]. These species differences in methylation reprogramming promote the idea that active zygotic demethylation and genome-wide methylation changes are not a prerequisite for normal mammalian development. This is consistent with the observation that, despite incomplete and delayed demethylation of somatic cell nuclei that have been introduced into activated bovine eggs during cloning [3, 10, 11], at least a small percentage of the cloned embryos develop apparently normally [13]. Although the requirement of DNA methylation for early development is well documented [4, 11], it may not be the primary mechanism for epigenetic reprogramming.
The biological significance of species-specific methylation reprogramming is still unclear. Zygotic gene activation (ZGA) is essential for the transition from maternal to embryonic control of development. It is interesting to note that ZGA also differs between mammalian species. In mouse [37] and most likely in cattle [38], ZGA is already seen at the 2-cell stage, whereas in rabbit, it does not occur until the 8- to 16-cell stage [39]. In contrast with the early mouse embryo, in which most poly(A)-containing mRNA is degraded [40], the amounts of both total and polyadenylated RNAs remain high until ZGA in rabbit [41, 42]. The total protein content decreases from mouse zygote to morula stage [43] and the pattern of protein synthesis changes abruptly around ZGA [44]. In rabbit embryos, the protein content remains relatively stable until the late morula stage [45]. Then the total amount of proteins increases rapidly between the late morula and early blastocyst stage [46]. Thus, maternal factors seem to control nearly the entire cleavage period in rabbit embryos. It is plausible to assume that the overall high methylation level throughout rabbit preimplantation development is interrelated with delayed activation of the embryonic genome. Both the first DNA replication and ZGA may be involved sequentially in establishing a repressive chromatin state in rabbit embryos [47]. Considering that many preimplantation events are not identical in mouse and rabbit, it is not surprising that there are also marked differences in methylation reprogramming. The mouse embryonic genome undergoes dramatic demethylation and is activated very early during preimplantation development. The rabbit does not require ZGA until the 16-cell stage and, therefore, may not need this dramatic erasure of methylation after fertilization. There is, however, still little knowledge on the factors controlling early mammalian development.
Species differences in the molecular and cellular events that occur shortly after fertilization may also have important implications for reprogramming of transplanted somatic cell nuclei in cloned embryos. A highly variable cloning efficiency, with 0–4% of NT embryos developing to term, has been observed in mammalian species [12, 13]. The absence of active demethylation mechanisms correlates well with the very low success rate of rabbit embryo cloning experiments. Nuclei of rabbit NT embryos showed relatively high methylation levels that were indistinguishable from those in in vivo-fertilized rabbit embryos. Evidently, active zygotic demethylation is not an obligate requirement for normal mammalian development. However, it may help the oocyte to revive the highly condensed sperm nucleus in normally fertilized eggs for somatic development and, by extrapolation, to restore totipotency of the transplanted somatic cell nucleus in cloned embryos. The fact that sheep oocytes, which also do not show active zygotic demethylation, have a higher capability of reprogramming somatic cell nuclei for full-term development indicate that other cellular factors influence outcome. For example, in this study, we have shown that the failure to segregate chromosomes properly and to maintain ploidy is one important cause of early embryo loss.
Chromosomal aneuploidy can be the result of meiotic and postzygotic (mitotic) nondisjunction events. Accumulating experimental evidence suggests that in vitro manipulation and culture of early embryos are associated with alterations in the expression of the embryonic genome and a great reduction in viability [48–51]. By FISH, chromosomal abnormalities (ploidy errors) were detected in 25% of morphologically normal bovine blastocysts from superovulated heifers [19]. In another study, the incidence of the chromosomally abnormal bovine embryos was estimated to be 20% and 40% after in vivo and in vitro fertilization, respectively [20]. Evidently, embryo culture alone can already double the chromosomal abnormality frequency. The incidence of chromosome abnormalities appears to be even higher in bovine NT embryos [18, 21, 22]. Considering the manifold effects of in vitro culture on embryo quality, it is not surprising that in our study >50% of in vivo-fertilized but cultured rabbit embryos displayed postzygotic nondisjunction. However, using the same culture conditions, a significantly higher aneuploidy frequency of approximately 80% was found in rabbit and interspecies NT embryos. This can only be explained by additive detrimental effects of the NT procedure itself. Although we used serum-starved donor cells, it is possible that asynchronous cell cycles of donor nucleus and recipient oocyte [13] and/or mechanically disturbed mitotic spindle assembly [52] lead to chromosome segregation errors. On the other hand, NT and/or embryo culture conditions may affect the expression of genes that are crucial for cell cycle control and the mechanism of mitosis. Because gain or loss of an entire chromosome in a significant proportion of blastomeres can cause embryonic, fetal, and perinatal loss or produce abnormal offspring, chromosomal abnormalities may contribute significantly to the low overall success rate of mammalian embryo cloning. We propose that the cloning procedure interferes with chromosome stability and segregation mechanisms during the first mitotic divisions after NT.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Thomas Haaf, Johannes Gutenberg-Universität Mainz, Langenbeckstrasse 1, Bau 601, 55131 Mainz, Germany. FAX: 49 6131 175690; haaf{at}humgen.klinik.uni-mainz.de
3 Current address: Department of Development and Genetics, Evolutionary Biology Center, Uppsala University, Norbyvägen 18A, 75236 Uppsala, Sweden
Received: 22 October 2003.
First decision: 18 November 2003.
Accepted: 12 March 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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