Human Molecular Genetics, 2001, Vol. 10, No. 9 963-972
© 2001 Oxford University Press
Analysis of a malsegregating mouse Y chromosome: evidence that the earliest cleavage divisions of the mammalian embryo are non-disjunction-prone
Department of Genetics and the Center for Human Genetics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland OH, USA
Received 5 January 2001; Revised and Accepted 20 January 2001.
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ABSTRACT |
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Despite the clinical importance of human aneuploidy, we know little of the causes of mammalian non-disjunction. In part, this reflects the fact that, unlike lower organisms, segregation ‘impaired’ chromosomes are virtually non-existent in mammals. To address this issue, we have studied the mouse Y chromosome on the BALB/cWt (‘Wt’) inbred background, a system in which loss of the Y chromosome in gonadal tissue has been linked to hermaphroditism. Our results indicate that the Wt Y chromosome is stably transmitted during meiotic cell divisions, but non-disjoins at an extremely high frequency in mitosis. Surprisingly, the non-disjunction events are largely restricted to the earliest cleavage divisions, indicating that there is a temporal ‘window’ during which the Wt Y chromosome is susceptible to non-disjunction. The non-disjunction phenotype has both cis and trans components: the Wt Y chromosome malsegregates on a variety of genetic backgrounds, demonstrating an intrinsic defect; however, the incidence of non-disjunction is significantly influenced by strain background, indicating the existence of modifying loci and thus providing evidence for a genetic effect on mammalian non-disjunction. These studies suggest that the earliest cell divisions in mammals are non-disjunction-prone, an interpretation which provides an explanation for the high rate of chromosome mosaicism observed in studies of in vitro fertilization (IVF)-derived human preimplantation embryos. Further, our observations raise the possibility that the IVF setting adversely affects chromosome segregation and suggest that genetic quality be an important consideration in any attempt to improve or modify in vitro procedures for use on human eggs and embryos.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Since the initial description of trisomy 21 as the basis of Down’s syndrome (1,2), the causes of human aneuploidy have been exhaustively investigated. Nevertheless, we still know surprisingly little about the molecular etiology of aneuploidy in humans or, indeed, in any other mammalian species. In lower eukaryotes, both non-disjunctional mutants and non-disjunction-prone chromosomes are well known, aiding in the understanding of aneuploidy in these organisms (3,4). In contrast, few such reagents are available for mammalian studies. For example, many mutations are known to disrupt mammalian meiotic chromosome synapsis (5) or recombination (6), or other components of the meiotic/mitotic cell cycle machinery; however, none is known to yield a non-disjunction ‘phenotype’.
Furthermore, chromosomes that are genetically susceptible to segregation errors are virtually non-existent in mammals. One rare exception—and the subject of the present report—is the segregation-deficient mouse Y chromosome, the BALB/cWt (‘Wt’) Y chromosome. In studies conducted over 20 years ago, Whitten (7,8) reported an unusual abnormality of sex differentiation among the progeny of Wt mice. Specifically, he observed a high incidence (3%) of hermaphroditism and noted a skewed sex ratio among the remaining mice, with nearly two-thirds being phenotypic females. Both effects appeared to be mediated through the male, as crosses of Wt males with females of other strains produced hermaphroditism and disturbed sex ratio levels, whereas reciprocal crosses did not (9).
Subsequent cytogenetic analyses of Wt animals (8,10) revealed the reason for the abnormalities—invariably, the hermaphrodites were sex chromosome mosaics. That is, XO/XY, XO/XYY or XO/XY/XYY mosaicism was identified in bone marrow or fetal liver preparations from hermaphrodites. Thus, it seemed likely that the Wt Y chromosome was mitotically unstable, that the instability occasionally gave rise to gonads which were partially or totally populated by XO cells and that, depending on the level of XO cells, ovotestes or ovaries formed in animals that had begun development as genetic males. Consistent with this interpretation, Eicher et al. (10) found that, in Wt fetal hermaphrodites, there was a direct correlation between the proportion of XO cells in fetal liver and the amount of ovarian tissue in ovotestes.
These early studies provided indirect evidence that the Wt Y chromosome was segregation-deficient. However, as the focus of these studies was on hermaphroditism and not chromosome segregation, no attempt was made to characterize the non-disjunction phenotype of the Wt Y chromosome. Here, as part of our efforts to develop mouse models of human non-disjunction, we have re-examined the Wt Y chromosome to determine whether it is, indeed, non-disjunction-prone, and whether this liability extends to meiosis.
We report here that the Wt Y chromosome is stably transmitted in meiosis, but non-disjoins at extremely high frequency in mitosis. Surprisingly, the non-disjunction events are largely restricted to the earliest cleavage divisions, indicating that there is a temporal ‘window’ during which the Wt Y chromosome is susceptible to non-disjunction. Further, the incidence of non-disjunction is dependent on strain background, providing evidence for genetic effects on Wt Y chromosome non-disjunction. Thus, the Wt Y chromosome represents an important reagent for modeling aspects of human non-disjunction.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Wt Y chromosome segregates normally in meiosis
To determine whether the Wt Y chromosome is prone to malsegregation during meiosis, we used three-color fluorescence in situ hybridization (FISH) to screen for aneuploidy in mouse epididymal sperm from Wt males. For controls, we performed a similar analysis with males carrying a non-Wt Y chromosome; for these studies, we used C57BL/6 (‘B6’) males. We found no evidence for an increase in either XY or YY disomy among the Wt males, nor was there any obvious increase in other categories of disomy or in diploidy (Table 1). Further, there was no obvious deviation from a 1:1 ratio for Y- and X-bearing sperm (data not shown). Thus, our data provide no evidence that the Wt Y chromosome is susceptible to non-disjunction in meiosis.
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The Wt Y chromosome is unstable in mitosis
To assess mitotic stability of the Wt Y chromosome, we first examined metaphase preparations from 13.5 days post-coitum (d.p.c.) case and control fetuses. For these analyses, cases were defined as fetuses derived from matings of Wt males to either Wt females or females from a closely related strain, BALB/cBy (‘By’); controls were defined as fetuses from matings of By males to Wt or By females. Wt mothers were routinely karyotyped to ensure that none was XO. In total, we analyzed 74 case fetuses from nine litters and 43 control fetuses from eight litters; as no among-litter effects were identified in any of our analyses (data not shown), the results were pooled for the cases and controls.
In our initial cytogenetic analyses, 40 of 74 (54%) case fetuses and 19 of 43 (44%) controls were scored as having an XX sex chromosome complement; in each of these, results of PCR analysis were consistent with this interpretation. Thus, these fetuses were assumed to originate from XX zygotes, and were eliminated from further consideration. Subsequent analyses involved the non-XX case and control fetuses, which were assumed to derive from XY zygotes. For these animals, we scored approximately 50 cells from each of two embryologically distinct tissue types, namely fetal fibroblasts and liver, with representative metaphases provided in Figure 1 and the results summarized in Figure 2.
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We identified a remarkable level of aneuploidy among the 34 case fetuses, with only one (2.9%) having a normal XY chromosome complement. Of the others, four were scored as non-mosaic XOs and 29 as mosaics for sex chromosome aneuploidy. Among the mosaics, most had XO, XY and XYY cell lines and three even had XYYY cells (Fig. 2). The proportion of abnormal cells varied widely among the aneuploid animals; the majority of animals had
10% aneuploid cells, but in over one-third of the animals 
25% of the cells were aneuploid. In six animals—one an XO/XYY/XYYY mosaic, one an XO/XY mosaic and four non-mosaic XOs—virtually all cells were aneuploid.
These results were in striking contrast to those involving the 24 control fetuses. Although aneuploid cells were occasionally observed in controls, they were almost always monosomic rather than trisomic, suggesting that they originated from artifactual loss of the Y chromosome in slide preparation. Further, only one of the 24 controls fit the scoring criteria for mosaicism, a highly significant reduction from that observed for the case fetuses (
2 = 46.3; P < 0.001).
Thus, our results provide direct evidence that the Wt Y chromosome is non-disjunction-prone, at least by comparison with the By Y chromosome. Further, these data strongly suggest that the non-disjunctional events in Wt fetuses occur early in development. That is, although the level of aneuploidy varied widely among the 34 Wt fetuses, the distribution of aneuploid cell types was virtually identical in the two cell lineages from the same animal (Fig. 2); indeed, only one of the 34 fetuses exhibited significant between-tissue differences in aneuploidy. Thus, these initial results suggest that Wt Y chromosome malsegregation is temporally restricted, with the vast majority of non-disjunction occurring before the divergence of fetal fibroblasts from liver cells.
Wt Y non-disjunction is restricted to the early cleavage divisions
To determine whether there is, indeed, a temporal ‘window’ during which Wt non-disjunction is maximized, we conducted FISH analyses on 2-, 4-, 8-, 16- and 32-cell preimplantation embryos (Fig. 3). For these analyses, we compared the sex chromosome constitutions of cells from 361 Wt Y chromosome-bearing preimplantation embryos with those of 136 control By Y chromosome-bearing embryos (Table 2).
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We identified a remarkable level of Y chromosome mosaicism in Wt Y chromosome-bearing embryos. Overall, 150 of 361 (41%) Wt Y-bearing early embryos were found to be sex chromosome mosaics, with X0/XY and X0/XY/XYY chromosome constitutions predominating. These results were in sharp contrast to the 3% level of mosaicism identified in By Y-bearing embryos (
2 = 68.9; P < 0.001), demonstrating that the Wt Y chromosome is, indeed, prone to malsegregation in the earliest embryonic cell divisions. The 3% level of mosaicism detected among By fetuses may reflect a propensity to early mitotic non-disjunction, even among control chromosomes; however, as interphase FISH analysis is subjective (11), we think it more likely that this represents the ‘background’ level of mis-scoring of FISH signals.
By analyzing mosaicism at several different cell divisions, we were able to examine the cumulative frequency of mosaicism over the first few cleavage divisions and to assess possible division-specific differences in the probability of Wt Y chromosome malsegregation (Fig. 4). Our results indicate that, by the 32-cell stage, approximately one-half of all Wt males are already mosaics for sex chromosome aneuploidy. Further, they suggest extraordinary variation in malsegregation among the earliest cell divisions. That is, we calculated the probability that an individual cell malsegregates at a given cell division, d (e.g. 2
4 cells), as:
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Pd = [(
d+1 – d) / (1 – d)] / N
where d is the observed incidence of sex chromosome aneuploidy among all embryos at the initiating cell stage (e.g. the 2-cell stage) and N is the number of cells at the initiating cell stage.
Using this approach to examine the data from Table 2, we estimated the cell-specific probability of Wt Y chromosome malsegregation at the first, second, third, fourth and fifth cell divisions as 0.12, 0.165, 0.025, 0.005, and 0.001, respectively. Thus, these data indicate that the first two divisions are more vulnerable to non-disjunction than are the later divisions. Clearly, these estimates rely on a number of uncertain assumptions, e.g. that XO, XY and XYY cells divide at relatively similar rates in the early embryo, that the efficiency of our FISH analysis is not dependent on cell stage and that our analysis correctly identifies all aneuploid embryos. Nevertheless, the magnitude of the differences between the first and second divisions and the third to fifth divisions provides strong evidence that the Wt Y chromosome is especially prone to non-disjunction during the first two cleavage divisions.
Non-disjunction of the Wt Y chromosome is dependent on genetic background
Early studies of Wt Y chromosome-carrying animals demonstrated significant variation in the level of hermaphroditism on different maternal backgrounds. That is, crosses of Wt males with females of several different inbred lines (e.g. B6) yielded no hermaphrodites, whereas crosses with other strains (e.g. A/HeJ) yielded ‘intermediate’ levels of hermaphroditism of 
1%, and still other crosses (e.g. Wt or By) produced ‘high’ levels of hermaphroditism of 3% (9). Conceptually, there are at least two possible explanations for these strain differences: firstly, the Wt Y chromosome might non-disjoin at similar levels on the different genetic backgrounds, with the variation in hermaphroditism being attributable to strain-specific differences in testis formation; or alternatively, the Wt Y chromosome might non-disjoin at different levels on the backgrounds, resulting in varying proportions of XO cells in the fetal gonad.
To directly examine the ‘non-disjunction’ hypothesis, we analyzed metaphases from 13.5 d.p.c. fetuses resulting from crosses of Wt males with either B6 females (‘no’ hermaphroditism) or A/J (AJ) females (a substrain of A, as is A/HeJ, associated with ‘intermediate’ hermaphroditism) and compared these data with those from Wt males crossed with Wt (or By) females (‘high’ hermaphroditism; Fig. 2).
The results for 33 B6Wt F1 fetuses and 23 AJWt F1 fetuses are summarized in Figure 5. We observed significant variation in sex chromosome aneuploidy, with the results paralleling those reported in the previous studies of hermaphroditism (9). Specifically, B6Wt F1 males had the lowest level of sex chromosome aneuploidy, with only two of 33 animals having 10% abnormal cells; AJWt F1 males exhibited an intermediate level, with seven of 23 animals having 10% aneuploid cells; and the inbred Wt males exhibited the highest level, with 18 of 34 animals having 10% aneuploid cells. Comparison of population means using Mann–Whitney two-sample tests indicated a highly significant difference between the B6Wt and Wt animals (P < 0.001), a marginal difference between the B6Wt and AJWt F1 animals (P = 0.035) and a non-significant difference between the AJWt F1 and Wt animals.
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To verify that these observations reflected differences in non-disjunction and not strain-specific differences in the viability of aneuploid conceptuses, we examined preimplantation embryos from crosses of Wt males with B6 females. Results of these analyses are summarized in Table 2 and Figure 4. On this F1 background, the Wt Y chromosome was still susceptible to malsegregation, as 18 of 143 (13%) of the 2-, 4-, 8- or 16-cell embryos were scored as mosaics; however, the level of malsegregation was markedly reduced from that observed for Wt males (Table 2). Further, at each cell stage, the level of mosaicism was significantly lower among the B6Wt F1 animals (Fig. 4). Thus, our data provide strong evidence of an effect of genetic background on the ability of the Wt Y chromosome to properly disjoin during the earliest cleavage divisions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Wt Y chromosome is prone to malsegregation during the earliest cleavage divisions
The purpose of the present study was two-fold: to determine whether the Wt Y chromosome is, indeed, non-disjunction-prone and, if so, to investigate possible factors influencing the likelihood of Wt Y non-disjunction. Our results demonstrate that the Wt Y is susceptible to mitotic, although not meiotic non-disjunction, and provide strong evidence for both temporal and genetic background effects on the likelihood of Wt Y malsegregation.
The demonstration of an apparently structurally normal, non-disjunction-prone chromosome is, to our knowledge, unprecedented in mammals. To be sure, there are many examples of mammalian chromosomes that are subject to malsegregation. Translocations or other structural rearrangements can disrupt the normal pairing and disjunction of homologous chromosomes during meiosis, or separation of sister chromatids during mitosis. Additionally, there are rare examples of ‘programmed’ non-disjunction; e.g. the X chromosome of the creeping vole, Microtus oregoni, is normally eliminated prior to the meiotic divisions in the male (12,13). However, such examples typically involve specialized meiotic or pre-meiotic adaptations to sex determination. In contrast, the Wt Y appears to segregate faithfully in meiosis, but is prone to high rates of malsegregation in the earliest mitotic cell divisions.
Although the underlying mechanism of Wt Y malsegregation is not yet clear, our observations indicate that the process more often results in chromosome loss than gain. That is, while we observed both hypo- and hyperdiploid cells among mid-gestation fetuses, the most common abnormality was an XO cell. Further, a well known correlate of mitotic chromosome loss, namely micronucleus formation, was a common feature of Wt preimplantation embryos (Fig. 3); indeed, Wt Y chromosome-containing micronuclei were observed in approximately one-half (71/150) of all mosaic preimplantation embryos.
From these observations, it seems reasonable to conclude that the movement of the Wt Y chromosome is compromised during early cleavage divisions; either it is deficient in forming microtubular attachments and congressing to the metaphase plate or, if a stable bipolar attachment is achieved, it is defective in sister chromatid separation and segregation at anaphase. These two errors suggest different types of centromeric defects; a failure to form bipolar attachments and align at the spindle equator implies a defect in formation of a functional kinetochore, while anaphase lagging suggests a centromere-specific defect in the binding of sister chromatid cohesion proteins (for reviews see refs 14 and 15). Analysis of prometaphase and anaphase stages of first and second cleavage division embryos may distinguish between these possibilities.
Wt Y malsegregation is subject to cis and trans effects
Regardless of the exact mechanism of Wt Y chromosome malsegregation, it is clear that it involves both cis (Wt Y chromosome-specific) and trans (genetic background) effects. The former effect is deduced from the fact that errors in the mitotic segregation of the Wt Y chromosome were observed on all backgrounds that were tested, i.e. in cells derived from mid-gestation Wt fetuses and two types of F1 hybrid fetuses, in cells of preimplantation Wt and B6Wt F1 embryos, and in cells of mid-gestation fetuses generated by repeated backcrosses of Wt Y-carrying males with B6 females (data not shown). From this we conclude that, even under optimal cellular conditions, the Wt Y is susceptible to chromosome breakage or non-disjunction, i.e. it is a ‘vulnerable’ chromosome.
The non-disjunctional events in the preimplantation embryos suggest that this cis effect is due to a ‘sub-optimal’ centromere, and cytogenetic observations of Wt Y-carrying mid-gestation fetuses provide further evidence to support this interpretation. That is, in a number of cells the normal Wt Y chromosome was replaced by an isochromosome for the long arm; indeed, in some cells 3–4 Y isochromosomes were present (Fig. 1). Such cells were never identified in control animals, nor were isochromosomes involving the X chromosome or autosomes identified in Wt animals; thus, isochromosome formation appears to be a characteristic, if infrequently occurring, property of the Wt Y chromosome. Recent molecular evidence suggests that, as originally proposed by Darlington (16), isochromosome formation can occur by centromere misdivision (17). That is, instead of a normal ‘longitudinal’ division in which sister chromatids segregate from one another, the chromosome divides ‘horizontally’ through the centromere, yielding one daughter cell containing an isochromosome for the short arm and the other cell an isochromosome for the long arm. Depending on the amount and content of the centromeric sequences retained by the isochromosome, malsegregation of the isochromosome might be a feature of subsequent divisions; we suggest this as the basis for those cells observed to contain multiple Wt Y isochromosomes.
Although we consider the Wt Y to be a ‘vulnerable’ chromosome, it is also clear that this vulnerability depends on trans-acting factors. In both mid-gestation fetuses and preimplantation embryos, we observed significant differences in aneuploidy levels between the inbred and F1 animals, indicating that genetic background affects the non-disjunction phenotype. The basis of this variation is unclear but may involve strain-specific differences in the first two cell divisions since, on the inbred BALB/cWt background, this is the stage at which non-disjunction is maximized. There is ample evidence that the first two mitotic cell divisions are unusual in comparison with subsequent ones. For example, during the first few cleavage divisions development depends largely on stored maternal mRNA and protein, since there is little transcription from the embryonic genome (18). Additionally, the morphology of the early cleavage division spindles is distinctive; at the first division the spindle is anastral and barrel-shaped and, similarly, the second division also has broad poles; in contrast, the third and later divisions are characterized by fusiform-shaped spindles with narrow poles (19). Also, the first two cell cycles are protracted in comparison with later ones, with the first cycle lasting 14–20 h, the second 18–22 h and subsequent cycles 10 h (20). Further, at least for the first cell cycle, the timing of cleavage is influenced by strain background, with both maternal and paternal effects (21,22) and, in studies utilizing Wt males, cleavage was delayed markedly in the resulting progeny (21).
Thus, it is likely that chromosome segregation at the earliest cleavage divisions is subject to different rules from subsequent somatic divisions. Consequently, it seems reasonable to suggest that the genetic effect on Wt Y segregation is, indeed, due to strain-specific differences in the first two cleavage divisions. If so, the effect could derive from variation in maternally expressed products, variation in the embryonic genome, or both. It should be relatively straightforward to distinguish between these alternatives. For example, by generating Wt Y chromosome-carrying consomic strains (e.g. B6Wt Y), and utilizing the consomic males and inbred Wt males in reciprocal crosses (e.g. Wt x B6Wt Y and B6 x WtWt Y), it will be possible to derive and examine F1 embryos with identical genotypes which nevertheless differ in maternally expressed transcripts/proteins. Such studies will guide subsequent mapping efforts to localize the genetic determinants of Wt Y chromosome malsegregation.
Early cleavage divisions may be non-disjunction-prone in both mice and humans
Our studies imply that that the earliest cell divisions in mammals are non-disjunction-prone, an interpretation that provides an explanation for results of cytogenetic studies of human preimplantation embryos derived from in vitro fertilization (IVF). In multi-color FISH analyses of ‘spare’ preimplantation embryos, Delhanty et al. (23) concluded that 30% were mosaics; similarly, Munné et al. (24) reported a 29% level of mosaicism in an analysis of arrested or morphologically abnormal preimplantation embryos. Further, Delhanty et al. (23) and others have observed occasional ‘chaotic’ embryos, characterized by highly abnormal, but varying, chromosome constitutions in different blastomeres of the same conceptus. The biological significance of these observations has been uncertain, for at least two reasons: firstly, they involve analyses of in vitro fertilized embryos derived from infertile couples, and may not reflect the situation in naturally-occurring pregnancies; and secondly, they suggest an extraordinary level of mosaicism among human preimplantation embryos, much higher than that observed among clinically recognized human fetuses (25). However, given our observations on the Wt Y chromosome, it seems likely that the first cell divisions in humans are, indeed, particularly vulnerable to errors in chromosome segregation. The much higher level of mosaicism observed among IVF-derived preimplantation embryos than clinically recognized pregnancies may reflect early selection or, alternatively, the fact that the IVF procedure itself increases the risk of segregation errors during this vulnerable window of development. By studying the segregation of the Wt Y chromosome in IVF-derived mouse embryos, it should be possible to determine whether the frequency of Wt Y chromosome non-disjunction is, in fact, increased in an in vitro setting. Thus, the Wt Y chromosome provides a potential model for human IVF, as well as an important reagent for understanding factors that influence chromosome segregation in mammals.
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MATERIALS AND METHODS |
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Mice
Breeding stock of BALB/cWt (‘Wt’), BALB/cBy (‘By’), C57BL/6 (‘B6’) and A/J (‘AJ’) inbred strains was obtained from The Jackson Laboratory. Mice were housed in Thoren ventilated rack caging in a pathogen-free facility.
Sperm FISH analysis
To obtain mature sperm for FISH analysis, adult Wt and By males were killed and the testes removed and placed in PBS. The epididymis was dissected from each testis, placed in 2.2% sodium citrate, bisected longitudinally and incubated for 10 min at 37°C. Following incubation, the sperm-containing supernatant was collected and individual drops of sperm suspension were carefully spread onto clean microscope slides and allowed to air dry (26).
Three color FISH was performed using the Y-specific probe, pErs5 (27), the X-linked probe, DXWas70 (28), and a chromosome 8 probe consisting of four pooled subclones of a chromosome 8-specific repeat (29). Slides were processed for FISH according to the procedure of Lowe et al. (30). The hybridization solution consisted of digoxigenin-labeled (Boehringer Mannheim) pErs5, biotin-labeled (Boehringer Mannheim) DXWas70, and both digoxigenin- and biotin-labeled chromosome 8 subclones in 55% formamide/10% dextran sulfate/1x SSC. Hybridized slides were detected with fluorescein isothiocyanate-avidin and Rhodamine-labeled anti-digoxigenin (Boehringer Mannheim) and were counterstained with 200 ng/ml DAPI (4’-6-diamidino-2-phenylindole; Sigma) so that Y-, X- and 8-specific signals were visualized as red, green and yellow, respectively, on a blue total DNA background.
Sperm were analyzed by at least two independent observers on a Zeiss Axiophot epifluorescence microscope using dual or triple band-pass filters. Stringent scoring criteria (31) were applied before a sperm head was classified as disomic; i.e. the two signals had to be of equal intensity, separated from one another by at least one signal domain, regular in appearance, not diffuse and clearly positioned within the sperm head. We scored for disomy but not nullisomy, since failure to detect a signal could be due to technical difficulties as well as to non-disjunction.
Analysis of mid-gestation fetuses
To obtain fetal fibroblasts and liver cells for karyotypic analysis, pregnant female mice were killed at 13.5 d.p.c. and fetuses dissected from the uterine horns. A small tissue sample from each fetus was frozen for subsequent PCR analysis (see below) and gonads were removed, examined under a dissecting microscope and classified as ovary, testis, or ovotestis on the basis of gonadal architecture (32). Fetal livers were disaggregated and air dried chromosome preparations were made as described previously (33). Short-term fibroblast cultures were established by mincing tail and limb buds, plating the fragments on 60 mm tissue-culture dishes and culturing the cells in RPMI medium 1640 (Gibco BRL) supplemented with 15% fetal bovine serum (Gibco BRL). After 48 h in culture, 0.25 µg colcemid (Gibco BRL) was added for 1–2 h and slides for cytogenetic analysis were prepared using standard methodology (33).
The presence or absence of Y chromosome material in tissues from mid-gestation animals was assessed karyotypically and by PCR analysis. For the latter, PCR was performed on genomic DNA from frozen tissue samples. The primer pair, 5'-TGAAGCTTTTGGCTTTGAG-3' and 5'-CCGCTGCCAAATTCTTTGG-3', which amplifies sequences from the Smcx gene on the X chromosome and from the Smcy gene on the Y chromosome, was used. Because these primers span an intron that is larger for Smcx than Smcy, Y chromosome-carrying mice can be distinguished from XO or XX mice by the presence of two bands (34,35).
For cytogenetic analysis, liver and fibroblast metaphase preparations were dehydrated in a cold ethanol series (70, 80 and 100%), air dried, denatured for 2 min in 70% formamide/2x SSC pH 7.0 at 72°C, and immediately dehydrated in a cold ethanol series (70, 80 and 100%). Hybridization solution containing X-, Y- and chromosome 8-specific probes (labeled as described for sperm FISH studies) was denatured for 5 min and applied to the slide for overnight hybridization at 37°C. Slides were washed in 2x SSC pH 7.0 at 72°C for 2 min and detected, washed and counter-stained as described above for sperm FISH studies.
Slides were scored by at least two independent observers who were blinded to the strain backgrounds of the fetuses. Initially, 3–5 metaphases were scored to identify fetuses with an XX sex chromosome constitution; no further analyses were conducted on these animals. For all remaining fetuses, we attempted to score at least 100 metaphases per mouse, i.e. 50 from liver and 50 from fibroblasts. Only cells with 39 or more chromosomes were scored, as those with 38 or fewer chromosomes were assumed to represent artifactual chromosome loss and were excluded from consideration. Cells were scored as 39,XO, 40,XY, 41,XYY, 42,XYYY or as an autosomal aneuploidy, depending on the number of chromosomes and the number and type of FISH signals. For each animal, the presence of one or more non-modal hyperploid metaphase or two or more identical non-modal hypoploid metaphases was interpreted as evidence of mosaicism. However, the presence of a single hypoploid cell in an otherwise euploid or euploid/trisomic animal was considered to represent artifactual chromosome loss and not mosaicism.
Analysis of preimplantation embryos
To obtain preimplantation embryos, 4- to 6-week-old females were superovulated with intraperitoneal injections of 2.5 I.U. pregnant mare serum gonadotropin (Sigma) followed 44–48 h later by 5 I.U. of human chorionic gonadotropin (Sigma) and mated with adult Wt or By males. Females were killed and embryos were flushed from the reproductive tract 24–72 h after mating. Preimplantation embryos were collected in M-16 modified medium (Specialty Media) containing 2 x 10–7 M colchicine (to enrich for metaphases) and incubated for 1 h. Following incubation, the cells were swollen slightly in 0.03 M citric acid (Sigma). Individual embryos were then placed into microdrops of water on clean microscope slides and fixed with 5:2 methanol:glacial acetic acid. Slides were processed for three-color FISH as described above and each slide was analyzed by at least two independent and blinded observers. For 2-, 4-, 8-, 16- and 32-cell stage embryos all analyzable metaphase and interphase cells were scored. Metaphase cells were counted when possible and scored as described above, depending on the number of chromosomes and the number and type of FISH signals present. Interphase cells were scored for the number of chromosome 8, X and Y signals. We defined mosaicism as the presence of two or more different cell types; thus, in this situation we considered the presence of a single cell with a missing or additional Y chromosome sufficient evidence for mosaicism.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Drs Aravinda Chakravarti and Eleanor Feingold for their statistical advice, Dr Huntington Willard for his helpful suggestions on the manuscript and Dr Eva Eicher for supplying the BALB/cWt mice and for her helpful review of the manuscript. This research was supported by research grants HD21341 (T.H.) and HD31866 (P.H.); C.B. was supported by training grant GM08056.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Department of Genetics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland OH 44106, USA; Tel: +1 216 368 6225; Fax: +1 216 368 0491; Email: tjh6@po.cwru.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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