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BOR - Papers in Press, published online ahead of print October 23, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.007187
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BIOLOGY OF REPRODUCTION 68, 922–928 (2003)
DOI: 10.1095/biolreprod.102.007187
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Numerical Chromosome Errors in Day 7 Somatic Nuclear Transfer Bovine Blastocysts

Paul J. Booth1,a, Dorthe Viuffb, Shijian Tana,c, Peter Holma, Torben Greveb, and Henrik Callesena

a Section of Reproductive Biology, Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark b Department of Clinical Studies, Reproduction, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark c Guangxi University, Institute of Animal Reproduction, Nanning 530005, China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulation method: half-cytoplasts were made from zona-free oocytes by bisection, after which two half-oocytes and one granulosa cell (serum-starved primary culture) were fused together and activated. The NT embryos were cultured in modified synthetic oviductal fluid containing essential and nonessential amino acids, myoinositol, sodium citrate, and 5% cattle serum in microwells for 7 days, at which time nuclei from all blastocysts were extracted and chromosome aberrations were evaluated using dual-color fluorescent in situ hybridization with bovine chromosome 6- and 7-specific probes. Five embryo clone families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 ± 3.7% (mean ± SEM) diploid, 2.6 ± 0.5% triploid, 10.0 ± 3.1% tetraploid, and 0.5 ± 0.2% pentaploid or greater; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 ± 4.5% and 73.7 ± 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing <=91 nuclei, were recorded. The ploidy distributions (classified as 2N, 3N, 4N, and >=5N chromosome complements, respectively) between two clone families were different (P < 0.01), as were blastocyst yields between other clone families (P < 0.01). Blastocyst yield was not correlated to % total ploidy error between clone families, but an inverse relationship (P < 0.01) between blastocyst total cell number and total % chromosome abnormality was observed within embryos. Categorization of the blastocysts into three quality grades (good, medium, and poor) and comparison of the distribution of ploidies when classified into 0%, 0.1–5.0%, 5.1–10.0%, 10.1–15.0%, and 15.1–100% errors within embryos indicated that medium- and poor-grade embryos were different (P < 0.05) from good-quality, in vitro-produced embryos. In a separate study, 11 different granulosa cell cultures (that did not correspond to those used for NT) were evaluated and found to possess only 0.23 ± 0.12% ploidy errors. These results demonstrate that 1) the percentage of ploidy errors in bovine NT blastocysts is inversely related to total blastocyst cell number, 2) the mixoploid condition is representative of the majority of embryos, 3) 100% polyploid NT blastocysts can exist, and 4) the ploidy errors seem not to be derived from the donor cells.

assisted reproductive technology, developmental biology, embryo


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ploidy constitution of nuclear transfer (NT) blastocysts is of fundamental interest because of the abnormally high mixoploid condition of in vitro-fertilized and -cultured (IVC) embryos [1]; the known high rate of embryonic, fetal, and perinatal mortality; and the incidence of both large and abnormal offspring derived from NT embryos after transfer [2–5], which generally exceeds that of in vitro-produced (IVP) embryos [6]. The timing and mechanism of induction of ploidy errors in NT embryos are largely speculative, but cell-cycle asynchrony between donor cell and cytoplast is one instigator [7]. In addition, the chemical treatments used to activate NT embryos produce a wide range of ploidy conditions in parthenogenetic embryos ([8, 9]; unpublished results). The question therefore arises whether more extensive ploidy errors could contribute to the greater embryonic and fetal wastage observed in NT embryos generated by this more manipulative technique compared to IVP embryos, or whether aberrant expression of either imprinted [10] or nonimprinted genes [11, 12] induced by in vitro culture conditions and/or manipulation is the primary cause. The observation that diploid/tetraploid mouse embryo chimeras developed larger placentae than normal fetuses is intriguing [13], especially because a frequent feature of the large-offspring syndrome is abnormal placentation [14, 15]. Any link between these parameters in domestic species, either suspected or real, is currently unknown.

Documentation of the normality (or otherwise) regarding the cytogenetic status of NT embryos has been somewhat limited, and the results are undoubtedly confounded by differences between NT protocols, karyotyping methods, stages of development evaluated, types of donor cell, donor cell-cytoplast cell cycle synchronization, and perhaps, species. Of the few published studies, indications of karyokinetic abnormalities can be evident as early as the pronuclear stage [16]; likewise, ploidy irregularities are observed in cleavage-stage embryos [17, 18]. At later stages of development, analysis of cell lines derived from a limited number of NT bovine fetuses has shown normal karyotypes [19]. Adult cloned calves have also been examined for chromosome errors by Yazawa et al. [20] and by Sims and First [21], who recorded that overall, 50–91% of lymphocytes in such offspring were diploid/tetraploid mosaics in which tetraploid cells accounted for only 10% of the total. Here again, however, interpretation of the results should be cautious, because in the study of Yazawa et al. [20], the mosaic pattern of ploidy in the lymphocytes became resolved as the calves grew older and could have been the result of a viral infection.

These observations in NT offspring, albeit limited, may concur with the evidence in naturally conceived offspring that the birth of pure polyploid individuals, which invariably die perinatally, is exceptional [22], although several reports of apparently healthy diploid/polyploid mosaic cattle offspring have appeared [23, 24]. The vast majority of pure polyploid or diploid/polyploid mosaic conceptuses are undoubtedly eliminated embryonically or aborted within the first trimester of pregnancy [23, 25]. In cattle, evidence suggests that a proportion of polyploid embryos are eliminated before the blastocyst stage [1], and De La Fuente and King [26] speculate that this selection process may be largely complete during the blastocyst-elongation phase in domestic species. It must be stated, however, that the presence of polyploid cells in the trophectoderm of the elongating embryo/fetus is a normal phenomenon that, in cattle, arises after Day 12 ([27]; unpublished results) and that shunting of polyploid cells to the trophectoderm has been reported to occur in experimentally constructed diploid/tetraploid mouse chimeras [13]. One can only speculate at present whether such a mechanism could rescue IVP or NT embryos by the selective transfer of polyploid cells to the trophectoderm.

Clearly, the collection of articles cited above are insufficient to provide a detailed overview of the occurrence or, indeed, the mechanisms of induction of any ploidy errors in NT embryos. This encouraged us to perform a preliminary investigation into the ploidy of a limited number of bovine NT blastocysts reconstructed from blastomeres, in which we established that the proportion of completely normal NT embryos was no less than those produced by IVP but that the distribution of chromosome abnormalities was different, indicating that when chromosome errors were present, the NT embryos were more severely affected than those produced in vitro [28]. To investigate such differences more thoroughly, and now that somatic cell NT is a common technique, the present study examined ploidy in relation to the quality of NT blastocysts generated from granulosa cells. Although we have no evidence to suggest that ploidy errors are likely to be greater in somatic compared to blastomeric NT embryos, the negative relationship between the stage of development from which the donor cell is derived and the subsequent level of embryonic and fetal wastage observed [4, 5, 29] may not, perhaps, be entirely explained by epigenetic defects [30] (for review, see [6]). Furthermore, the different NT protocols used to coordinate cell-cycle synchronization between donor cell and cytoplast when using somatic or blastomere donor cells are worthy of investigation, especially because the theoretical ploidies of bovine embryos generated by parthenogenetic activation rarely match with reality ([8, 9]; unpublished results).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Chemicals were supplied by Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Cattle serum was derived from the Danish Veterinary Laboratory (Frederiksberg, Denmark). All embryo manipulations (including electrofusion) were performed at 37°C except for oocyte bisection, which was undertaken at room temperature (~20°C).

In Vitro Maturation and Culture

The in vitro maturation (IVM) and IVC systems were identical to methods previously described [31, 32]. In brief, oocytes were aspirated from 2- to 6-mm follicles from slaughterhouse ovaries and selected for the presence of an even ooplasm and an intact compact cumulus investment several layers deep. Twenty-five cumulus-oocyte complexes were matured in 400 µl of modified TCM-199 supplemented with 10 IU/ml of eCG and 5 IU/ml of hCG (Suigonan Vet; Intervet, Skovlunde, Denmark) and 15% (v/v) cattle serum. The IVM was performed at 38.6°C in 5% CO2 in humidified air. Reconstructed embryos were cultured in 400 µl of modified synthetic oviductal fluid containing essential and nonessential amino acids, myoinositol, sodium citrate, and 5% (v/v) cattle serum (mSOFaacis) in 5% CO2, 5% O2, and 90% N2 until Day 7. Both IVM and IVC were performed under 400 µl of paraffin oil (Uvasol; Merck, Darmstadt, Germany)

Oocyte Preparation and Bisection

At 18 h after the start of IVM, the granulosa cells were removed from the oocytes by vortexing in calcium- and magnesium-free BCMF medium (composed of 95.0 mM NaCl, 6.0 mM KCl, 0.56 mM glucose, 5.0 mM Na-lactate, 0.33 mM Na-pyruvate, 0.2 mM L-glutamine, 10.0 mM MEM [minimum essential medium] amino acids, 20.0 mM BME [basal medium Eagle] amino acids, 25.0 mM Hepes, 2.0 mM NaHCO3, and 24.0 mM sorbitol). The following zona-free NT technique was performed as described by Booth et al. [33]: Oocytes were placed in 5 mg/ml of pronase until the zonae were digested, at which time they were transferred to TCM-Hepes containing 5% (v/v) serum to neutralize the enzyme. Subsequently, oocytes were placed in TCM-Hepes containing 5 µg/ml of cytochalasin B, 5 µg/ml of Hoechst 33342 DNA stain, and 5% (v/v) cattle serum in a plastic Petri dish. Bisection was performed with a microsurgical knife mounted on a manipulator, whereby the knife was gradually lowered through the oocyte. Each bisected oocyte was exposed to ultraviolet light for no more than 1 sec to select the half containing the metaphase plate, which was then immediately discarded. Half-cytoplasts were then returned to the maturation medium to await NT.

Embryo Reconstruction, Activation, and Microwell Culture

Half-cytoplasts were placed in 300 µg/ml of phytohemagglutinin-P (prepared in TCM-Hepes) together with granulosa cells that were of medium size (actual size not determined) and round with smooth membranes. One half-cytoplast was adhered to one granulosa cell by manipulation using a fine glass rod and observed through a stereo microscope. These couplets, together with other half-cytoplasts, were equilibrated in electrofusion medium comprising 0.3 M mannose, 0.1 mM MgSO4, and 1% (v/v) serum. In the fusion chamber, which comprised two parallel wires 0.5 mm apart, each couplet (one half-cytoplast adhered to a granulosa cell) was placed next to another half-cytoplast so that both adhering membranes were parallel to the electrofusion wires. An alternating current (3.3 V, 600 kHz) was applied for 15 sec, followed by a single fusion pulse (0.6 kV/cm, 30 µsec). Constructs were examined within 1 h after the fusion pulse. Those that had not fused received a second fusion pulse using a higher voltage (1.2 kV/cm, 30 µsec). No attempt was made to differentiate those embryos reconstructed with the first fusion pulse from those successfully refused at the second attempt. The constructs were transferred to mSOFaacis and remained there for 0.5–4 h before activation. The effect of the duration between reconstruction and activation on ploidy was not investigated. Activation comprised calcium ionophore A23187 (10 µM, 1.5–5 min) followed by 2 mM DMAP (6-dimethylaminopurine) for 4 h. The NT embryos were then washed and cultured in microwells in mSOFaacis until Day 7. Microwells were prepared in four-well dishes and were created by making indentations in the bottom of each dish with an unheated darning needle [33].

Granulosa Cell Primary Cultures

Ovaries not possessing dominant follicles were selected from five different cows to generate five primary cultures of granulosa cells for NT, resulting in five embryo clone families. Granulosa cells were cultured in TCM-199 supplemented with 15% (v/v) cattle serum. After 3–4 days, the concentration of serum was reduced to 0.5% (v/v) to divert the cell cycle into a presumptive, predominantly G0/G1 status [34, 35]. After 5–6 days in low serum, the cells were disaggregated at 38.5°C in 2.5 mg/ml of trypsin (type XII-S) in calcium- and magnesium-free BCMF medium (see above) before neutralization of the enzyme with serum (final concentration, 5% v/v). A 45-min period of trypsinization was necessary to generate granulosa cells that were completely round with a smooth cell membrane.

A further 11 primary cultures of granulosa cells were generated from a further 11 cows to evaluate numerical chromosome composition after serum starvation. The granulosa cells were prepared and treated identically to those destined for NT. After serum starvation, the nuclei of the granulosa cells were extracted and subjected to in situ hybridization (see below). The ploidies of 200 nuclei in each of the 11 granulosa cell cultures were evaluated by fluorescent in situ hybridization (FISH).

Nuclear Extraction and FISH

The nuclei of all Day 7 blastocysts were extracted by lysing the embryos in 0.1% (v/v) Tween 20 and 0.01 N HCl. The total number of nuclei in each blastocyst were counted under phase-contrast microscopy before they were fixed on slides using methanol:glacial acetic acid (3:1 [v/v]). No attempt was made to isolate and stain the ICM and trophectoderm cells separately. The FISH technique was essentially as described by Viuff et al. [36]. Briefly, a chromosome 6-specific probe (p33E39) and a chromosome 7-specific probe (cJAB8) were labeled with biotin (Life Technologies, Tåstrup, Denmark) and digoxigenin (Boehringer Mannheim, Mannheim, Germany), respectively. Hybridization sites of the biotinylated probe were visualized using Cy3-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) after one round of amplification using biotinylated goat antiavidin antibodies (Vector Laboratories, Burlingame, CA), whereas digoxygeninated probes were visualized using anti-digoxigenin-fluorescein.

FISH Analytical Criteria

The FISH analytical criteria were those described by Viuff et al. [36]. Briefly, the nuclei of blastocysts that were overlapping or nonintact were excluded from analysis. The number of fluorescent signals per nucleus generated from the chromosome 6 and chromosome 7 probes were counted. Consequently, each nucleus was registered with two scores: a score of 2+2, 2+1, or 2+0 would thereby present a diploid nucleus possessing at least two signals from either chromosome 6 or 7. Triploid nuclei were defined as possessing 3+3, 3+2, 3+1, or 3+0 signals. Nuclei containing either 4+4, 4+3, 4+2, 4+1, or 4+0 signals were classified as tetraploid. A similar pattern of criteria was used to define nuclei of higher ploidy. Nuclei were not classified as monosomic for chromosome 6 or 7 in the present study. Furthermore, nuclei possessing 0+0, 0+1, 1+1, 1+diffuse, or 0+diffuse were categorized as false negatives. Blastocysts possessing false negatives of more than 20% were rejected from the analysis.

Statistical Analysis

The relative frequency distribution of chromosome abnormalities between embryo types was analyzed by the chi-square test. Correlation analysis was performed by a generalized linear model technique [37]. Probability values of <=0.05 were regarded as significant.

The NT embryo quality, in terms of the distribution of total percentage chromosome abnormalities, was compared to previously published data of such distributions in morphologically excellent- and good-quality, Days 7–8 IVP and Day 7 in vivo-flushed blastocysts [36]. The classes in the distribution analysis were categorized as 0%, 0.1–5.0%, 5.1–10.0%, 10.1–15.0%, and 15.1–100% chromosome abnormalities. The NT blastocysts were classified into three groups in both a subjective and an objective manner. First, the subjective method performed under a stereomicroscope comprised allocation of blastocysts into three quality grades and was performed as a guide to assist future practical selection of NT blastocysts destined for embryo transfer. The three grades were labeled as good, medium, and poor blastocysts. Good blastocysts were defined as large, expanded blastocysts containing a good inner cell mass (ICM). Medium blastocysts were classified as those blastocysts of medium size containing a good ICM, whereas poor embryos comprised all small blastocysts plus blastocysts of any size either possessing a poor ICM or no recognizable ICM at all. Second, the objective method comprised allocation of blastocysts according to total cell (nuclei) number, which was measured in conjunction with the FISH analysis. The range of total nuclei (24–292) observed in the NT blastocysts was divided into three categories: low (24–113), medium (114–203), and high (204–292).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The blastocyst rate, blastocyst total cell number, mean number of nuclei examined, and hybridization error for clone families 1–5 are shown in Table 1. Of the 112 blastocysts that were analyzed, 26 (23.2%) possessed completely diploid nuclei (Fig. 1). The number of completely diploid blastocysts (and the percentages of the total number of such blastocysts within each clone family) were 1 (7.1%), 5 (20.8%), 2 (22.2%), 6 (35.3%), and 12 (25.0%) for clone families 1–5, respectively. The remaining 80 blastocysts (71.4% of total) were either pure polyploid (not possessing any diploid nuclei) or were diploid/polyploid mixoploids. Of the latter category, 49% were either diploid/tetraploid (2N and 4N) mosaics or diploid/triploid/tetraploid (2N, 3N, and 4N) mosaics (Fig. 1).


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  		TABLE 1. Blastocyst rates, blastocyst cell number, number of nuclei examinable by FISH per blastocyst, and percentage hybridization error in nuclear transfer clone families 1–5



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  		FIG. 1. Classification of nuclear transfer blastocysts according to ploidy. The polyploid embryos were defined as those possessing no diploid nuclei, whereas mixoploid embryos were categorized as being diploid/polyploid mosaics. In mixoploid embryos, the diploid, triploid, tetraploid, and pentaploid conditions are represented by the abbreviations 2N, 3N, 4N, and 5N, respectively. The number of embryos within each class are given above each histogram bar

The percentages of nuclei in each blastocyst possessing diploid, triploid, tetraploid, or pentaploid or greater chromosome constitutions were calculated. The means of these values in clone families 1–5 are presented in Figure 2. Overall, the mean percentage of nuclei that were 2N, 3N, 4N, or >=5N were 86.9 ± 3.7%, 2.6 ± 0.5%, 10.0 ± 3.1%, and 0.5 ± 0.2%, respectively, indicating that the majority of nuclei in NT blastocysts were of normal ploidy. The vast majority (>75%) of polyploid nuclei were present in the tetraploid state, with triploid nuclei representing only 20% of abnormal nuclei.



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  		FIG. 2. Mean distributions of the ploidies of nuclei within nuclear transfer blastocysts between the five clone families. Total ploidy error is the sum of the 3N, 4N, and >=5N chromosome complements (see Fig. 1 for definitions). Ploidy distributions are different (P < 0.01) between clone families 2 and 3

Remarkably, 6 of the 112 blastocysts (5.4%) analyzed contained only pure polyploid nuclei (Fig. 1). These embryos were derived from clone families 2 (n = 4) and 5 (n = 2) and represented 16.7% and 4.2%, respectively, of embryos within each clone family. Notably, these polyploid embryos possessed lower-than-average total cell numbers (clone family: 2, 82, 68, 91, and 26; clone family 5: 88 and 24). Of these embryos, four were 3N/4N/>=5N mosaics, two were 3N/4N mosaics, one was a 4N/>=5N mosaic, and one was completely tetraploid. Expressed on a percentage basis, the tetraploid state was the predominant condition; the mean percentages of 3N, 4N, and >=5N nuclei were 12.3%, 85.5%, and 2.2%, respectively.

Analysis of the data according to clone family established an effect (P < 0.01) on ploidy distribution between clone families 2 and 3 (Fig. 2). This effect appeared to result from a shift in distribution of the percentage nuclei present in the diploid condition to the tetraploid state in clone family 2.

Blastocyst yield was not correlated to percentage total ploidy error. An inverse relationship (P < 0.01) between blastocyst total cell number and total percentage chromosome abnormality was observed (Fig. 3).



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  		FIG. 3. Percentage of total chromosome abnormalities in individual, Day 7 bovine somatic nuclear transfer blastocysts according to total cell number and embryo grade

Embryos were also classified subjectively (on the basis of good, medium, and poor grades) or objectively (on the basis of high, medium, and low total nuclei numbers) and then further subdivided into the percentage of chromosome abnormality that each embryo exhibited. The numbers and percentages of embryos falling within these categories are presented in Figure 4. The distributions of ploidy errors were not different between blastocysts categorized according to grade (Fig. 4, top). However, classification according to total cell number (Fig. 4, bottom) indicated that chromosome abnormality distributions were different between blastocysts possessing high and low cell numbers (P < 0.01) and between those possessing medium and low cell numbers (P = 0.01). Figure 4 suggests that (irrespective of the method of classification), although the proportion of completely diploid embryos appears to be variable between classes of embryo, when abnormalities are present a shift in the distributions occurs such that lower-quality embryos tend to possess a higher proportion of more abnormal nuclei (i.e., the distribution profile shifts to the right). The number (and percentage) of embryos categorized as possessing high, medium, and low numbers of nuclei were 42 (37.5%), 22 (19.6%), and 48 (42.9%), respectively; the number (and percentage) of embryos grouped according to good, medium, and poor grades based on morphology were 28 (25%), 27 (24.1%), and 57 (50.9%), respectively.



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  		FIG. 4. The distribution of nuclear transfer Day 7 blastocysts classified according to the percentage of polyploid nuclei per embryo and according to embryo grade (top) and blastocyst total cell number (bottom). In the bottom panel, distributions were different between blastocysts possessing high and low cell numbers (P < 0.01) and between those with medium and low cell numbers (P = 0.01)

The distributions in the percentages of polyploid nuclei per embryo in NT embryos classified according to blastocyst grade were compared to data derived from IVP and in vivo-flushed blastocysts as published by Viuff et al. [36]. Such analysis established that the distributions of medium- and poor-grade blastocysts were different (both P < 0.05) compared to IVP embryos, whereas all grades of blastocyst were different (P < 0.001) compared to in vivo embryos.

Of 11 granulosa cell primary cultures that were evaluated for ploidy errors, eight possessed 100% diploid cells in the 200 nuclei assessed from each cell culture. Of the remaining three cell cultures, one possessed one triploid nucleus (representing a 0.5% ploidy error), and two other cell cultures each contained two tetraploid nuclei (1% ploidy error). Therefore, the overall rate of ploidy error was 0.23 ± 0.12% in the 11 cell cultures.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These data indicate that ploidy errors are correlated to blastocyst quality and that NT blastocysts that were graded as good morphologically possessed no more ploidy errors than morphologically excellent- and good-quality embryos as produced by in vitro fertilization by Viuff et al. [36]. In the present study, such good-quality embryos were defined as large, expanded blastocysts possessing a good ICM. Notably, morphologically poorer grades of blastocyst (medium and low grades) did differ in their proportions of ploidy errors compared, again, to morphologically good and excellent in vitro-fertilized embryos [36]. Consequently, from these data, one may speculate (in the absence of embryo transfer results) that selection of suitable embryos for transfer based on morphology would appear to be equally applicable to NT embryos as it has been traditionally for in vitro-fertilized and in vivo-flushed embryos [38]. Indeed, apart from other inherent defects, slower-developing embryos that are generally of a correspondingly poorer grade because of lower blastocyst cell numbers (i.e., buffalo [39]) have a tendency to exhibit greater chromosomal errors [40]. Such a relationship was clearly observed in the present study, in which the total blastocyst cell number was negatively correlated to the percentage of chromosome abnormalities present. Furthermore, although the percentage of chromosome abnormalities in embryos was variable between blastocyst grades when classified according to total cell number, a greater proportion of the nuclei in embryos defined as low quality possessed chromosome errors. In this respect, the 6 NT blastocysts that were totally polyploid (albeit that a minimum of 58.3% of nuclei were evaluated in these embryos) lay within this same category, being of low total cell number indicating delayed and/or abnormal development. Indeed, speed of development was reported to be reduced for in vitro-fertilized bovine embryos that were polyploid [41], perhaps because of delays in replicating the more abundant amounts of DNA.

The method used here to evaluate the ploidy of nuclei is based on the hybridization of chromosome-specific probes and is independent of the requirement that the blastomeres should be synchronized into metaphase, as is obligatory in (and a severe limitation of) traditional karyotyping methods. Thereby, FISH theoretically permits every nucleus in an embryo to be evaluated. Such contrasting techniques may account for the greater percentage of totally diploid (40% and 60%) DMAP-activated bovine NT blastocysts reconstructed from primordial germ cells and fetal fibroblasts as recently recorded by Yoo et al. [42] and Hill et al. [43], respectively, compared to the present study. Other differences in the NT technique, such as choice of somatic cells, culture medium, type of protocol, or additional factors, could also have contributed to these contrasting results.

The general mechanism by which ploidy errors are generated is still largely speculative, although our observed ploidy similarities between IVP and NT blastocysts suggest that polyspermic fertilization in vitro (representing 10–15% of embryos [1]) is not the sole source in IVP embryos. The formation of mixoploid embryos is assumed to result from cytokinetic failure during the first cell cycle, blastomere fusion, endoreduplication, or combinations of these events [23]. The artificial activation regimens that inadequately mimic the activational events consequent to fertilization [44] are themselves not innocuous, because they always possess nonspecific actions. Prolonged exposure to DMAP during activation has been directly implicated in reducing the percentage of the resulting parthenogenetic embryos that are diploid [9]. Theoretically, DMAP generates diploid embryos [45]. In practice, however, diploid embryos are a minor product of this treatment, because the tetraploid and mixoploid states represent more than 80% of embryos [8, 46], suggesting that a more complex series of karyokinetic events probably occurs, including spindle abnormalities [47], DNA replication abnormalities, pronuclei duplication, and at a later stage, binucleation of blastomeres [46].

Karyokinetic abnormalities are also seen in NT embryos where variations in pronuclear number are reported [16, 35] together with the unusual exclusion of patches of chromatin that subsequently coalesce to form pronuclei [48]. Cell-cycle incompatibility between the donor cell and cytoplast can also contribute to disturb numerical chromosome constitution [7]. However, this mechanism is perhaps unlikely to be of major significance in the present study, because the majority of donor cells would be expected to be in G0/G1 of the cell cycle and the larger donor cells (that are more likely to be in G2/M phase) were rejected from use [34].

Variations in the efficiency of NT blastocyst production have frequently been observed between cell lines [49]. Because each cell line was used only once in the present study, any differences between clones may simply be caused by daily variations in the efficiency of the NT procedure, the methylation pattern of the donor cells [6], or perhaps, any nucleocytoplasmic interactions between donor cells and cytoplasts that may possibly occur between breeds of cattle (as observed between strains of mice [14]) that were not controlled in this experiment. Contrasting states of ploidy of the donor cells could have generated the differences in ploidy distributions observed between clones, because alteration in karyotype is not an uncommon phenomenon in cultured cells [50]. However, our retrospective analysis of a different set of primary cell cultures indicated virtually no ploidy errors, suggesting that the origin of the alterations in ploidy distribution between clones was probably independent of the donor cells.

In conclusion, it appears that NT blastocysts of good morphological quality possess the least proportion of ploidy errors and, in this respect, are probably equivalent to good-quality IVP embryos. This suggests that the greater embryonic and fetal wastage recorded in NT compared to IVP embryos could be independent of ploidy defects and concurs with the accumulating evidence that alterations in both imprinted [10] and nonimprinted [11, 12] gene expression are a cause of these defects. However, this should not exclude karyotyping as an important criteron of fetal viability and the normality of NT offspring. It is also evident that clarification and improvement of the mechanisms of reprogramming and karyokinetic events occurring during the first cell cycle of NT embryos will be invaluable for the application of this technology.


   ACKNOWLEDGMENTS
 
The authors wish to thank Zaida Rasmussen, Anette Pedersen, Susanne Bøhrk, and Ruth Kristensen for their excellent technical assistance.


   FOOTNOTES
 
1 Correspondence: Paul Booth, Fertility Clinic, Holbaek Hospital, Smedelundsgade 60, 4300 Holbaek, Denmark. FAX: 45 59 48 42 69; e-mail: chpabo{at}vestamt.dk Back

Received: 30 May 2002.

First decision: 25 June 2002.

Accepted: 20 September 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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