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a Department of Clinical Studies, Reproduction,
b Department of Anatomy and Physiology,
c Department of Mathematics and Physics, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark
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ABSTRACT |
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developmental biology, implantation/early development, IVF/ART
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro bovine embryos were produced as described earlier [10, 11], except that the maturation medium contained 1 mg/ml polyvinylalcohol (PVA; Sigma, St. Louis, MO) instead of serum. Our IVP system based on the same bulls generated cleavage rates (Day 2) of 84% ± 4.3% and a blastocyst rate (Day 8) of 32% ± 6.5% over the same period of time as the present experiment was performed. Embryos were collected from a randomized population obtained from five experiments, including a total of 2500 inseminated oocytes at Days 2, 3, 4, and 5 pi without any grading of the stage or the quality of the embryos; only the number of nuclei per embryo was noted.
Embryo Fixation
All embryos were spread using the method described previously for intact human embryos [12]. Briefly, individual embryos were quickly washed in a lysing buffer (0.01 N HCl, 0.1% Tween 20) and transferred in a small droplet to a Superfrost Plus slide (Menzel Gläser, Braunschweig, Germany). The embryos were constantly observed with an inverted phase-contrast microscope. After the lysis buffer had been added, the zona pellucida and the blastomere cytoplasm dissolved gradually and immediately before the nuclei dried out, and 3:1 methanol:glacial acetic acid was added dropwise to the slide. The specimens were then fixed in 3:1 methanol:glacial acetic acid at 4°C for at least 24 h, air-dried, and incubated at 60°C overnight. Slides that were not immediately hybridized were stored at -80°C.
Fluorescence In Situ Hybridization
A chromosome 6-specific probe (p33E39) and a chromosome 7-specific probe (cJAB8) were used [5]. DNA from p33E39 and from cJAB8 was isolated using the Qiagen DNA purification system (Diagen GmbH, Hilden, Germany). DNA from cJAB8 was labeled with digoxigenin (Boehringer Mannheim, Mannheim, Germany), and DNA from p33E39 was labeled with biotin (Life Technologies, Tåstrup, Denmark) by a standard nick-translation reaction. Fluorescence in situ hybridization was performed essentially as described by Viuff et al. [5]. Briefly, slides were washed for 2 min in PBS, fixed in 1% phosphate-buffered paraformaldehyde for 2 min and washed twice in PBS. Chromosomal DNA was denatured by immersing slides in 70% formamide, 2x SSC (pH 7) for 2 min at 70°C, and the slides were then immediately dehydrated in an ice-cold ascending ethanol series and air dried. The biotinylated p33E39 DNA and the digoxigenated cJAB8 DNA were added to the hybridization solution (50% deionized formamide, 10% dextran sulfate, 2x SSC, 10 µg salmon sperm DNA, and 12 µg genomic DNA) at a final concentration of 15–30 ng/µg, denatured at 75°C for 5 min, and then left to preanneal at 37°C for 30–60 min. Aliquots (5 µl) of this solution were placed on each slide, coverslipped, sealed, and after incubation overnight at 42°C, slides were washed three times in 0.05x SSC for 3 min at 42°C. Hybridization sites of biotinylated probes were visualized using Cy3-conjugated streptavidin (Jackson ImmunoResearch, West Grove, PA) after one round of amplification using biotinylated goat anti-avidin antibodies (Vector Laboratories, Burlingame, CA). Hybridization sites of digoxigenated probes were visualized using Anti-Dig-Fluorescein (Boehringer Mannheim). Nuclear DNA was counterstained with diamidino-phenyl-indole (DAPI, 1 µg/ml) in DABCO (Sigma) antifade solution. Images of FITC, Cy3-conjugated streptavidin and DAPI fluorescence were recorded separately using a Quantix CCD camera (Photometrix, Tucson, AZ) and subsequently merged using IPLab Spectrum software (Scanalytics, Fairfax, VA).
Analytical Criteria and Controls
Embryo nuclei were scored only if they were intact and nonoverlapping. The specific signals detected in a given blastomere were considered to reflect a true chromosome constitution if the signals were of similar size, shape, and intensity and were more than the diameter of a single signal apart. A nucleus was considered diploid if it presented either 2 + 2 (Fig. 1A), 2 + 1, or 2 + 0 signals; triploid if 3 + 3 (Fig. 1B), 3 + 2, 3 + 1, or 3 + 0 signals were found; and tetraploid if 4 + 4, 4 + 3, 4 + 2, 4 + 1, or 4 + 0 signals were observed. Nuclei with higher ploidy were classified accordingly. Nuclei with potential monosomy of either chromosome 6 or 7 were disregarded in this study unless all nuclei within an embryo displayed the same pattern. In addition, nuclei lacking signals such as 0 + 0, 0 + 1, 1 + 1, 1 + diffuse, or 0 + diffuse signals were considered to be false negative, and only embryos having less than 20% false-negative nuclei were included in the analysis. Furthermore, only embryos with at least 50% successfully analyzed nuclei were included. Those embryos for which not all nuclei could be analyzed and where all analyzed nuclei were diploid, were scored as diploid. Embryos in which some nuclei displayed one type of chromosome complement and other nuclei another were considered to be mixoploid, that is, mosaic embryos, whereas only embryos in which all nuclei were analyzed and shown to be polyploid were considered to be true polyploid embryos. Thus, our estimates of mixoploidy and polyploidy frequencies are conservative.
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Metaphase spreads of male bovine cells were prepared from lymphocyte cultures using standard cytogenetic methods and used as control for the FISH staining. At standard stringency, p33E39 and cJAB8 produce a strong signal on chromosome 6 and 7, respectively, in metaphase spreads. At interphase, nuclei p33E39 and cJAB8 produced different signals: p33E39 produced a large and rather diffuse signal containing a number of small spots, whereas cJAB8 showed small and more well-defined signals. At low stringency, we observed a high background in the euchromatic regions of all bovine chromosomes. In a few cells, we observed two distinct labeling sites close together. We interpret these double signals as originating from nuclei in which the DNA has been replicated, for instance, in the case of cells in late S-phase, in G2, or in early mitosis. According to the analytical criteria given above, such double signals would, however, be scored as one.
Statistical Analysis
The relative frequencies of chromosome abnormalities were analyzed by Fisher's Exact Test and logistic regression, in which the probability of chromosome abnormality was modeled as a function of the stage. For the latter analysis, the embryonic stage was quantified for each embryo by the midpoint of the stage interval, that is, 3.5 for cells in the 3- to 4-cell stage. The validity of the logistic regression model was checked by a statistical test that compared the description of the data by the regression model with a description of the data without any assumptions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 3805 nuclei were scored in 426 embryos collected during Days 2–5 pi. Not all nuclei of the embryos could be analyzed because of loss of nuclei during preparation. However, an average of 1.9 nuclei could be analyzed per embryo for the 2-cell embryos, 3.6 nuclei for the 3- to 4-cell embryos, 6.4 nuclei for the 5- to 8-cell embryos, 10.0 nuclei for the 9- to 16-cell embryo, 17.0 nuclei for the 17- to 25-cell embryos, 23.1 nuclei for the 26- to 35-cell embryo, and finally, 46.7 nuclei per embryo for the embryos containing more than 36 cells. Further, the analytical criteria given in the previous sections were designed to provide conservative estimates of polyploid and mixoploid embryos in those cases in which not all nuclei could be analyzed. The percentage of nuclei with false negative scores at Days 2, 3, 4, and 5 pi were 2.2%, 1.1%, 5%, and 3.5%, respectively.
Results of the chromosome analysis of embryos collected during Days 2, 3, 4, and 5 pi are given in Table 1. The frequency of normal diploid embryos was 73%, 72%, 81%, and 58% at Days 2, 3, 4, and 5, respectively. Of the embryos containing abnormal cells, mixoploidy was more frequent than polyploidy. The percentages of mixoploidy at Days 2, 3, 4, and 5 pi were 22%, 15%, 16%, and 42%, respectively, whereas the percentages of true polyploidy, that is, cases in which all nuclei in the embryo were analyzed and were polyploid, were 5%, 13%, 3%, and 0%, respectively. Although no embryos were categorized as polyploid, there were three embryos collected at Day 5 in which all the nuclei that could be analyzed were polyploid (triploid) but not all nuclei that were observed before preparation were found on the slide. Consequently, the embryos were categorized as mixoploid, although we realize that they may in fact have been true triploid embryos.
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The type of chromosome abnormality in mixoploid embryos is illustrated in Table 2. In total, diploidy-triploidy was the most frequent abnormality (65%), whereas diploid-tetraploid and diploid-triploid-tetraploid mosaics were observed in 11% and 24% of embryos, respectively. Among the polyploid embryos, triploidy was the most common aberration: at Day 2, there were three triploid and three tetraploid embryos; at Day 3, there were nine triploid and six tetraploid embryos; and at Day 4, there were one triploid, one tetraploid, and one pentaploid embryo.
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There is general decline in the percentage of polyploidy from Days 2 to 5. Statistical analysis by comparing each day with any of the other days showed that there was a significant difference between Day 3 and 4 (P = 0.006) and between Day 2 and Day 3 versus Day 5 (P = 0.031 and <0.001). This is also reflected by a significant difference between Days 2, 3, and 4 (pooled together) to Day 5 (P = 0.002), but this difference is largely due to the high percentage of polyploidy at Day 3 compared with the percentage at Days 2 and 4. In contrast, the mixoploidy rate increased from an average of 17% at Days 2, 3, and 4 to 42% at Day 5. Statistical analysis revealed that there were no significant differences among the mixoploidy frequencies of Days 2, 3, and 4, whereas the difference between the frequencies on these days and Day 5 was statistically significant (P < 0.001).
Ploidy of Embryos in Relation to the Day of Collection and the Stage of Embryonic Development
The distribution of the chromosome constitution in relation to the stage of embryonic development and the day of collection is presented in Table 1. According to our statistical test, the logistic regression model was an acceptable representation of the data for the frequency of polyploidy at Days 2 and 3 (P = 0.998) and for the mixoploidy data (P = 0.179). Analysis of the data using this model showed that there are significantly negative regression coefficients for Day 2 and Day 3, meaning that there are more chromosomally aberrant embryos in the slow-developing embryos compared with the case of the fast-developing embryos on Day 2 (P = 0.0156) and Day 3 (P < 0.0001). This pattern was not identified at Days 4 and 5. There were some indications that the stage-dependent decreasing pattern was steeper for Day 2 than for Day 3 (P = 0.0795). For mixoploid embryos, there were no significant relations to the stage of embryonic development, indicating that there is no overrepresentation of the mixoploid embryos in the slowly developing group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The basis of polyploidy could be found during gametogenesis, at in vitro fertilization, or induced by in vitro culture. Earlier studies have shown that the frequencies of chromosomally abnormal germ cells vary according to the species examined, but in general, chromosomally unbalanced gametes have been noticed more frequently among oocytes (2.4–14.6%) than among spermatocytes (0–7%) [15]. Unfortunately, we have not yet obtained data on the chromosome aberration frequency of gametes from the in vitro production system used.
The relatively high incidence (37%) of polyploid embryos at the 2-cell stage might be due to the fact that our first collection was performed at Day 2 (44–52 h pi), when the 2-cell embryos already were judged as being developmentally retarded. Actually, Slimane et al. [16] reported preliminary data indicating that IVP bovine 2-cell embryos collected at 24 h pi have a lower incidence of chromosome abnormalities compared with the incidence for 2-cell embryos collected at 48 h pi. In addition, Benadiva et al. [17] found that developmentally normal Day 4 human embryos have a slightly lower percentage of abnormalities (45.4%) than slow/fragmented (51.3%) or arrested embryos (59.9%). Both these reports are in agreement with previous studies that have shown that the time of first cleavage post-insemination has major, long-lasting effects on the rate of blastocyst development and cell number of the resultant embryos [18–20]. Thus, we consider it likely that polyploidy is one of the main reasons for that slowly developing cattle embryos have a low blastocyst rate as shown by Holm et al. [21].
We also note that the frequency of an extra/missing, in other words, trisomy/monosomy, of 6 or 7 is low in the 426 bovine embryos investigated: only one trisomy/monosomy mosaicism was found. This is most likely generated by a somatic nondisjunction event in the first mitosis and not during gametogenesis. Thus, trisomy/monosomy 6 or 7 is in the per millions rather than in the percentage level in cattle embryos. Chromosomes 6 and 7 were chosen in this study because suitable probes were available, and it is unknown whether these chromosomes have an unusual high susceptibility to aneuploidy. However, if we assume that chromosomes 6 and 7 have aberration frequencies similar to those of the other chromosomes of the karyotype, then the total proportion of unbalanced gametes is not higher than a few percentage points and could in fact be significantly lower. Exceptions would be, for example, carriers of the 1/29 Robertsonian translocation that is relatively frequent in some cattle breeds and that reduces fertility by 5–7% [22]. Further, it should be remembered that FISH using two chromosome-specific DNA probes will not reveal all aneuploidies. We will only detect a trisomy/monosomy 6 or 7 if all cells of the embryo can be analyzed and if no hybridization errors or superimposed signals occur. In the latter case, the appearance of the signals is important: p33E39 produced a large and rather diffuse signal containing a number of small spots, which makes it more difficult to distinguish two distinctly separated signals. According to the analytical criteria, two signals that are closer than the diameter of a single signal are counted as one. In some cases, it is therefore difficult to distinguish diploid but superimposed signals from monosomy or a hybridizing error. We did chose to score this observation as a hybridizing error unless, as described above, all nuclei showed the same pattern or it was possible to count two or three signals for cJAB8. If the latter was the case, the cell was judged to be either diploid or triploid, respectively. Thus, our estimates of trisomy and monosomy 6 or 7 are conservative.
The basis of mixoploidy is most likely established either in abnormal fertilization or abnormalities imposed during in vitro culture of the embryos. It has recently been reported that abnormal fertilization, as in the case of polyspermy and parthenogenetically activated oocytes, can result in chromosomal mosaicism during subsequent cleavage divisions [23]. This could be a significant factor because 10–15% of the embryos are polyspermic and 3% are parthenogenetically activated within our IVP system [24]. In addition, abnormalities imposed during in vitro culture of the embryo may cause abnormal chromosome segregation leading to mosaicism from the first cleavage division. Studies of human IVP embryos, which have been characterized as normal by routine examination for pronuclei, have revealed an unexpected high development of postzygotic chromosomal abnormalities [25–28]. These include mitotic nondisjunction, ploidy mosaicism, and chaotic chromosome complement in a majority of nuclei. The high incidence of postzygotic chromosome abnormalities might be induced by a lack of cell cycle checkpoints during the cycles prior to the major activation of the embryonic genome. Thus, studies in Xenopus and Drosophila have demonstrated that embryos lack activated cell cycle checkpoints before the embryonic genome is activated [29, 30]. However, this effect will in our study only explain the initial mixoploidy level of 15–25% but not the major increase in the mixoploidy frequency that occurs after activation of the embryonic genome. We speculate on whether the culture system plays a role in increasing the rate of mixoploidy because our previous study revealed a relatively low frequency of mixoploidy frequency in Day 7–8 blastocysts developed in vivo. We are aware, however, that Schumacher et al. [31] concluded that physical factors associated with in vitro culture do not increase DNA ploidy abnormalities in rabbit embryos.
The consequences of chromosomal mosaicism for bovine embryonic development are still unknown. The fact that mixoploidy increased from 16% at Day 4 to 42% and up to 72% of Day 7/8 embryos [5] in an IVP system that gave rise to pregnancy at a rate of 64% [32] signaled that mixoploidy is of minor importance for at least the establishment of pregnancy. There are several theories that may explain how an embryo with some chromosomal abnormal blastomeres can give rise to a normal fetus. Firstly, selection against or apoptosis in one cell line need not be lethal to an embryo if a chromosomally normal core persists [33, 34]. Secondly, tetraploid blastomeres are known to be diverted to the trophectoderm in mice [35]. We are still intrigued by our initial data showing a significantly lower proportion of mixoploidy in bovine blastocysts developed in vivo. However, together with the present data, it seems likely that the mixoploidy frequency increases after activation of the embryonic genome. It will be interesting to learn whether it is a general phenomenon of IVP systems that polyploidy and mixoploidy occur in higher proportion compared with in vivo-developed embryos. We need to extend our studies of in vivo developed embryos to the earlier stages of development to learn more about when the difference during development is induced.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Danish Agricultural and Veterinary Research Council and Danish Biotechnology Program. Presented in part at the 32nd annual meeting of the Society for the Study of Reproduction, August 1999, Pullman, Washington. 
2 Correspondence: Dorthe Viuff, Department of Clinical Studies, Reproduction, Royal Veterinary and Agricultural University, Dyrlægevej 68, DK-1870 Frederiksberg C, Denmark. FAX: 45 35 28 29 72; dv{at}kvl.dk
Accepted: May 26, 2000.
Received: February 15, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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