Mutagenesis, Vol. 14, No. 2, 173-179,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Detection of aneuploidy by multicolor FISH in mouse sperm after in vivo treatment with acrylamide, colchicine, diazepam or thiabendazole
1 Institut für Säugetiergenetik, GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Neuherberg, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany and 2 Yunnan Normal University, Kumming, Peoples Republic of China
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Abstract |
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Multicolor fluorescence in situ hybridization (FISH) was used to investigate the induction of aneuploidy during meiosis in young adult male mice treated with chemicals chosen for the EU sponsored aneuploidy project (acrylamide, colchicine, diazepam and thiabendazole). The aim of the present study was to evaluate the frequency of aneuploid sperm induced by each of these chemicals by sperm FISH. Male (102/ElxC3H/El)F1 mice were treated with acrylamide (120 and 60 mg/kg single dose i.p.), colchicine (1.5 and 3 mg/kg single dose, i.p.), diazepam (300, 150 and 75 mg/kg single dose by oral intubation) or thiabendazole (100 and 300 mg/kg daily for 11 days by oral intubation). At 22 days after the last treatment, sperm were collected from the cauda epididymis. Three chromosome FISH was applied to determine hyperhaploid and diploid sperm with DNA probes specific for the chromosomes X, Y and 8. Five animals were treated per dose group and sperm aneuploidy was evaluated in 10 000 sperm per animal. We found significant increases in the frequency of total hyperhaploidy for the males treated with 3.0 mg/kg colchicine (0.092 versus 0.056%, P < 0.05) and with 1.5 mg/kg colchicine (0.082 versus 0.050%, P < 0.05), as well for the males treated with 300 mg/kg diazepam (0.081 versus 0.050%, P < 0.05), indicating that colchicine and diazepam each induced germ cell aneuploidy. We also found significant increases in the frequency of total diploidy for the males treated with 300 mg/kg diazepam (P < 0.05) and with 300 mg/kg thiabendazole (P < 0.05). No significant effects were found for 120 and 60 mg/kg acrylamide or for the other doses of diazepam and thiabendazole. These first results indicate that the multicolor FISH method is useful to determine aneuploidy induction in sperm of mice.
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Introduction |
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During the past 15 years, an important aim of the Aneuploidy Projects sponsored by the Environmental Research Programme of the European Union (EU) was to contribute to a better understanding of the mechanisms of aneuploidy induction by chemicals (Parry, 1996
). These coordinated research programs led to the development of new methods for the detection and assessment of aneugenic chemicals. Tests for aneuploidy in male mouse germ cells have been established to quantify the aneugenic potential of chemicals (Miller and Adler, 1992; Leopardi et al., 1993), to discriminate between aneugenic and clastogenic effects (Kallio and Lähdetie, 1993) or to identify a possible correlation between aneuploidy and meiotic delay (Miller and Adler, 1992; Adler et al., 1993). Although these methods provide some useful indirect information on aneugenic activity, they could not, in general, elucidate the mechanisms of aneuploid induction.
An important advancement in sperm cytogenetics for detecting aneuploidy was the adaptation of fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes (Wyrobek et al., 1990; Robbins et al., 1995). Using FISH to score the numbers of signals in sperm, large numbers of sperm can be scored quickly. Furthermore, the use of multicolor FISH techniques made it possible to study several chromosomes simultaneously for hyperhaploidy (disomy) and to distinquish between diploid and disomic sperm (Spriggs et al., 1995, 1996; Robbins et al., 1997a). The method can be applied to sperm of experimental rodents and humans. Using the FISH aneuploidy assay, Robbins et al. (1997a) were able to demonstrate increased hyperhaploidy in the sperm of men undergoing cancer chemotherapy, while Robbins et al. (1997b) reported data which indicate a significant association for caffeine and alcohol and suggestive evidence for a smoking effect. Given the difficulties in conducting sperm studies of exposed humans, Wyrobek et al. (1996) noted that complementary experimental approaches in animals are needed to identify aneugens, elucidate their modes of action and determine threshold concentrations.
Wyrobek et al. (1995) utilized late step testicular spermatids of mice to detect aneuploidy for one autosome and one sex chromosome. An improved method was applied to detect increased levels of aneuploidy in mice of advanced age by adding a DNA probe for the second sex chromosome (Lowe et al., 1995). However, to bridge the gap between experimental rodents and humans for comparative hazard evaluation, the FISH technology had to be adapted to rodent epididymal sperm, which involved the problem of localizing specific chromosomal subdomains within mature sperm. For this purpose, Lowe et al. (1996) developed a novel method to detect aneuploidy in epididymal sperm. Using this method with DNA probes specific for chromosomes X, Y and 8 and a three-color FISH technique, Adler et al. (1996b) determined the spontaneous frequencies of aneuploid sperm of young adult mice and compared inter-laboratory variation for the method. The results of this study suggested that the sperm FISH methodology was a promising procedure for the detection of aneuploidy in mouse sperm. Consequently, there was a practical need to examine its sensitivity to known or suspected aneugenic chemicals.
Therefore, in the present study the aneugenic effects of acrylamide (AA), colchicine (COL), diazepam (DZ) and thiabendazole (TB) during male mouse meiosis were examined by applying the three-color FISH method to mouse epididymal sperm. AA is an important industrial chemical used mainly in sewage and waste water treatment plants, in the paper industry, in the treatment of potable water and in research laboratories (Gutierrez-Espeleta et al., 1992). COL, DZ and TB are among the 10 model compounds selected for testing within the EU Aneuploidy Programmes. DZ is the active compound of the common tranquilizing drug valium, TB acts as a broad spectrum antihelmintic drug and COL was used as positive control.
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Materials and methods |
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Animals and chemical treatment
All experiments were performed with male (102/ElxC3H/El)F1 mice aged 10–14 weeks and weighing 25–29 g. Animals were bred in the GSF mouse colony and were maintained on a 12 h light/dark cycle with mouse pellet food and water ad libitum. The mice were treated with 60 or 120 mg/kg AA or with 1.5 or 3 mg/kg COL once by i.p. injection, with 75, 150 or 300 mg/kg DZ by oral intubation and with 100 or 300 mg/kg TB by oral intubation given daily for 11 consecutive days. AA and COL, obtained from Sigma (Deisenhofen, Germany), were dissolved in physiological saline; TB, obtained from Sigma, was dissolved in corn oil; DZ, obtained from Hoffmann-La Roche (Basel, Switzerland), was dissolved in ethanol/corn oil 1:10. Groups of five males were randomly assigned to treatment and concurrent solvent control groups. Concurrent controls were prepared with every dose group in order to code the slides and avoid scoring biases. The exception is DZ, where three dose groups were combined with one control group. The injected volume was 0.1 ml/10 g body wt. Males were killed 22 days after the last application of the test chemical or the solvent and sperm were collected from the cauda epididymis.
The techniques of preparation and decondensation of epididymal sperm, the preparation of the DNA probes and the hybridization conditions were followed as described earlier (Adler et al., 1996b, Lowe et al., 1996).
Preparation of epididymal sperm
The preparation technique for epididymal sperm was based on the newly developed procedure by Lowe et al. (1996). Both epididymides were dissected, placed in a Petri dish, prepared and incisions were made. Subsequently, they were placed individually into an Eppendorf cup filled with 300 µl fetal calf serum. The cups were placed in an Eppendorf incubator at 32°C for 30 min to allow the sperm to actively leave the epididymides. The epididymides were removed from the cups and the sperm suspensions were stored at –80°C. Fresh or thawed sperm suspensions (5 µl) were pipetted onto clean dry glass slides. Unfixed cells were smeared across the slide and allowed to dry overnight. The slides were stored at –20°C under nitrogen until use.
Slide processing and analysis
Prior to in situ hybridization the slides were heated for 5 min at 70°C on a hotplate. Thereafter, the slides were incubated in a Coplin jar in 10 mM dithiotreitol (Sigma) for 30 min on ice followed by incubation in 4 mM lithium-3,5-diiodosalicylic acid (Sigma) for 30 min on ice. The slides were dried by heating for 5 min at 70°C on a hotplate.
Preparation of the DNA probes
Chromosome-specific probes for chromosome 8 (Boyle and Ward, 1992), chromosome X (Disteche et al., 1985) and chromosome Y (Bishop and Hatat, 1987; Weier et al., 1994) were used for multicolor FISH. Plasmid DNA for chromosomes X (clone DXWas70) and 8 (clones 84 and 85e) were transformed in Escherichia coli XL1-blue and DNA was isolated using the Qiagen Plasmid Maxi Kit (Qiagen, Chatsworth, MD).
The chromosome 8 probe was labeled using the Gibco Nick Translation System with biotin-dUTP (Boehringer, Mannheim, Germany). The chromosome X probe was labeled with a combination of biotin-16-dUTP and digoxigenin-11-dUTP (Boehringer, Mannheim, Germany). The chromosome Y probe was prepared from flow sorted murine chromosomes by primer-directed DNA amplification using PCR with the JUN1 and UW4B primers (Weier et al., 1994). Alternatively, the clone pY353/B (Bishop and Hatat, 1987) was transformed in E.coli XL1-blue and the DNA was isolated using the Qiagen Plasmid Maxi Kit (Qiagen). The chromosome Y probe was labeled using the Gibco Nick Translation System with digoxigenin-dUTP (Boehringer, Mannheim, Germany).
Three chromosome FISH method
Hybridizations were performed according to a modified technique of Pinkel et al. (1986, 1988). Labeled probes were mixed with Master Mix 2.1 (55% formamide, 10% dextran in 1x SSC) and denatured at 78°C for 8 min. The sperm slides were denatured in 70% formamide (in 2x SSC, pH 7.0) at 78°C for 5 min, dehydrated in an alcohol series (70, 90 and 100%, 2 min each) and dried on a slide warmer at 37°C for 3 min before application of the denatured hybridization mix.
Hybridization was carried out for 24–48 h in a moist chamber at 37°C. Post-hybridization washing consisted of two steps: 15 min in 50% formamide (2x SSC, pH 7.0) at 45°C and 30 min in PN buffer at 37°C. The probes were detected with streptavidin-Cy3 (chromosome 8) and anti-digoxigenin-FITC (Y chromosome) or a combination of both (X chromosome). The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (0.1 µg/ml PBS) for 10 min at room temperature and cover-slipped in Vectashield (Vector Laboratories, CA). Slides were stored at 4°C in the dark.
Scoring
For this study at least 10 000 cells/animal and a total of ~870 000 sperm were microscopically examined for aneuploidy using a Zeiss Axiopan Fluorescence Photo Microscope (Zeiss, Germany). The microscope was equipped with the following filters: triple filter (triple band-pass filter set no. 61000; Chroma Technology) for simultaneous visualization of green (FITC), yellow (FITC + Cy3), red (Cy3) fluorescence hybridization domains and the blue (DAPI) fluorescence of the sperm nuclei; individual filters for each of the fluorochromes [FITC, HQ 480/40 and HQ 535/50; Cy3, HQ 535/50 and HQ 610/75 (Chroma Technology); DAPI, BP 365 and LP 397 (Zeiss)] to digitize the image. Records of every sperm with an abnormal number of hybridization domains were taken by digitizing the microscopic image with the computer program ISIS3 (MetaSystems, Altlussheim, Germany). Photographic images were taken with Ektachrome Panther 400 X film.
All samples in these experiments were coded. The sperm were assigned to the specific fluorochrome phenotypes as determined by the combination of fluorescence signals in each nucleus: X-8, Y-8 as normal sperm. Strict scoring criteria were employed for assigning cells to one of five hyperhaploidy classes (X-X-8, Y-Y-8, X-Y-8, X-8-8, Y-8-8), to X-Y-8-8 as diploid sperm resulting from first meiotic division suppression and to X-X-8-8 or Y-Y-8-8 as autodiploid sperm resulting from second meiotic division suppression (Lowe et al., 1995).
Cells were scored as having two domains of the same color if both signals were of similar size and intensities and were separated by a distance of more than half the diameter of one domain. The frequencies of sperm with hypohaploid phenotypes (X-0, Y-0, 0-8) were scored but not included in the statistical analysis. This procedure is justified because loss of a chromosome domain could be due to a technical artifact.
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Results |
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The appearance of sperm painted by multicolor FISH and examples of hyperhaploid (disomic) or diploid sperm are shown in Figure 1
. Alterations of the chromosome numbers in sperm involving painted target chromosomes can be easily detected as colored domains. The DNA probes are identified by a yellow domain for chromosome X (Cy3 and FITC), a large green domain for chromosome Y (FITC) and a red domain for chromosome 8 (Cy3).
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The results of the analysis of aneugenic effects in mice after exposure to AA, COL, DZ and TB are presented in Tables 1–4
, together with the concurrent control data for each chemical dose in all cases where treatment with different doses were not performed at the same time. For a statistical overall comparison of the sperm frequencies the 
2 test with Yate's correction was used and comparisons on an individual animal basis were carried out with the Mann–Whitney U-test (Siegel, 1956).
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Independent of the treatment with the four chemicals, no significant differences from an expected sex ratio of 1:1 could be observed between sperm carrying X-8 and Y-8 (Tables 1–4
). Furthermore, there were no significant differences in the frequencies of disomic sperm among the eight control groups.
As shown in Table 1, treatment with 60 and 120 mg/kg AA did not induce significant increases in diploid (0 and 0.002%) or disomic sperm (0.052 and 0.074%) compared with the corresponding control values of 0.004 and 0.002% or 0.054 and 0.070%, respectively.
As seen in Tables 2 and 3, treatment with COL and DZ induced disomic sperm. Compared with the corresponding control value the frequency of disomic sperm induced by 1.5 and 3 mg/kg COL was significantly increased by a factor of ~2 (P < 0.05), i.e. 0.082 and 0.092% compared with the corresponding control values of 0.050 and 0.056%, respectively. Similarly, a significant increase in the frequency of disomic sperm by a factor of ~2 (P < 0.05) was caused by treatment with 300 mg/kg DZ, 0.081% compared with the corresponding control value of 0.046%. Treatment with 150 and 75 mg/kg DZ increased the disomic frequency slightly but not significantly (P < 0.10), i.e. 0.066 and 0.058% compared with the corresponding control value of 0.046%. Furthermore, treatment with 300 mg/kg DZ induced diploid sperm, i.e. 0.018% compared with the corresponding control value of 0.002% (P < 0.05). Figure 2 indicates a uniform dose dependence for diploid and disomic sperm frequencies (± SE) induced by the different doses of DZ. The standard errors were calculated on the basis of the Poisson distribution. This was justified because the 2 test for Poisson homogeneity among 50 untreated animals analyzed in our laboratory (unpublished data) showed no significant variability (2 = 20.3, d.f. 49, P > 0.05).
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–––) and diploid (—As shown in Table 4
, treatment with 100 and 300 mg/kg TB did not induce disomic sperm, i.e. 0.067 and 0.072% compared with the corresponding control values of 0.049 and 0.069%. However, treatment with 300 mg/kg TB induced diploid sperm (0.047%) compared with the corresponding control value of 0.023% (P < 0.05). |
Discussion |
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Since FISH has greatly improved the possibility for a precise and rapid detection of aneuploidy in a large number of various human and mammalian cells, this method can be used efficiently to analyse the occurrence of sex chromosomal aneuploidy in mice. Whereas Wyrobek et al. (1995), Lowe et al. (1995) and Baulch et al. (1996) performed such studies in late step spermatids of mice using multicolor FISH, Lowe et al. (1996) modified the procedure for epididymal sperm. This method was employed by Adler et al. (1996b) to analyze the spontaneous rates of sex chromosomal aneuploidy in sperm of young adult mice.
Using this method the present study was performed to investigate chemically induced aneuploid sperm. Similar to the study of Adler et al. (1996b), a three-color FISH was applied to determine the frequencies of disomic and diploid sperm with DNA probes specific for chromosomes X, Y and 8. Initially, the spontaneous frequencies of aneuploidy in epididymal sperm of young adult mice were determined. The present data of the eight control groups resulting from scoring of >400 000 sperm confirm in essence the findings of our laboratory in an earlier study (Adler et al., 1996b), where three groups of controls treated with different common solvents had been analyzed. In fact, all the present findings, including those of the earlier reported solvent controls, fall within a relatively small range of variation in disomic frequencies. Therefore, the spontaneous disomic frequencies seem to be very similar, provided only young adult male mice are used. This finding is an important prerequisite for the evaluation of the frequency of chemically induced aneuploid effects by sperm FISH in mice, because age-dependent increases in aneuploid sperm have been reported in mice (Lowe et al., 1995) and in humans (Griffin et al., 1995, Robbins et al., 1995; Kinakin et al., 1997).
The known spindle poison COL, which inhibits polymerization of tubulins (Margolis and Wilson, 1977; Margolis et al., 1980; Liang and Brinkley, 1985) was used in the present study as the positive control. Whereas the induced frequencies of disomic sperm were significantly higher than the corresponding controls, there was no similar tendency for the induction of diploid sperm.
The evidence that AA is neurotoxic, genotoxic or carcinogenic was extensively reviewed by Dearfield et al. (1988). It appeared that the major genotoxic effect of AA was due to its clastogenic activity (Moore et al., 1987). Results obtained with classical germ cell mutagenicity tests indicate that AA is not only positive in differentiating germ cells but also in stem cell spermatogonia (an overview is given in Adler et al., 1996a). Using a differential staining of spindle and chromatin or an immunofluorescent spindle staining technique, Gassner and Adler (1995) mainly observed multipolar spindles in male mouse germ cells induced in vivo by AA. Since it can be assumed that cells bearing multipolar spindles are filtered out by meiotic selection, it can be suggested that AA may not be a strong aneugen. This suggestion is in line with the results of the present study, which did not indicate an aneugenic potential of AA under the applied experimental conditions.
Previous studies reported that DZ is capable of inducing mitotic or meiotic arrest and of increasing the frequency of aneuploidy in mammalian cells. Firstly, Andersson et al. (1981) found that DZ (dose range 40–80 µg/ml) induced mitotic arrest in human fibroblasts. Staining for tubulin of mitotic cells by indirect tubulin-specific fluorescence, it was observed that DZ did not affect microtubule integrity but inhibited the separation of centrioles in prometaphase. This observation was confirmed by Hsu et al. (1983) and Lafi et al. (1987), who reported on increased incidences of mitotic arrest in Chinese hamster cell lines after treatment with different doses of DZ (dose range 10–600 µg/ml). Due to the observation of monopolar spindles in these three studies, it was concluded that DZ induced mitotic arrest resulting from abnormal centrioles which cannot initiate anaphase or telophase. However, these findings differ in some respects from those of Wallin and Hartley-Asp (1993), who tested DZ on the assembly of isolated bovine microtubules and studied their morphology by electron microscopy. They found that at the highest dose tested (1 mM) DZ produced microtubules with some aberrant structures. Therefore, these authors suggested that in the previous experiments the changes in morphology may have been overlooked and, additionally, as centrioles are complex structures of microtubules (microtubule organizing centers), an effect via microtubules cannot be excluded.
In mouse spermatocytes DZ induced aneuploidy and a concomitant meiotic delay (Miller and Adler, 1992). At 6 h after treatment with a dose of 150 mg/kg DZ the frequency of hyperploidy in cells at meiotic metaphase II was increased, whereas the frequency of hypoploidy was unchanged. In mouse oocytes, however, Marchetti et al. (1994) reported that treatment with doses between 50 and 150 mg/kg DZ failed to induce either meiotic arrest or aneuploidy. Although several chemicals have been reported to induce aneuploidy in male germ cells but not in female germ cells (for a review see Mailhes and Marchetti, 1994), it remains unclear which factors may be responsible for these differences. Using their different staining procedures, Gassner and Adler (1995) found that in male mouse germ cells at 24 h after treatment with 150 mg/kg DZ the level of monopolar spindles was elevated and a loss of single chromosomes could be observed, suggesting an effect on motor proteins. The misplaced chromosomes may lead to aneuploidy in daughter cells. This assumption is confirmed by the findings of the present study. In the analyzed dose range between 75 and 300 mg/kg DZ the frequencies of both disomic and diploid sperm indicate a uniform dose dependence, though only at the dose of 300 mg/kg DZ was the observed increase significant compared with the corresponding controls. In general, using multicolor FISH in epididymal sperm of young adult mice, it can be demonstrated that DZ acts as an aneugenic chemical in germ cells.
TB is an antihelmintic drug used in human medicine and in veterinary practice (Campbell and Cukler, 1969). It induced non-disjunction in Aspergillus nidulans (Kappas et al., 1974). TB did not induce micronuclei in human lymphocytes (van Hummelen et al., 1995). In mouse bone marrow, TB did not induce c-mitotic effects and was negative in the micronucleus assay (Miller and Adler, 1989; Adler et al., 1991). In mouse spermatocytes, a single injection of TB did not induce hyperhaploid second metaphase cells (Miller and Adler, 1992), but when fed to mice over 22 days a positive effect was reported (Pacchierotti et al., 1984). This latter report led us to design the TB treatment over 11 days in order to cover the entire meiotic prophase. For the dose of 300 mg/kg we also counted chromosomes in second meiotic metaphase cells and found no difference in hyperhaploidy to the concurrent control (data not shown). Thus, we did not confirm the report of Pacchierotti et al. (1984).
In conclusion, the results in the present study indicate that the development of the multicolor FISH technique in epididymal sperm has obvious benefits for detection of aneuploidy in mouse sperm. However, since only a 2-fold increase in the chemically induced frequency of disomic sperm was found, further studies are needed to verify these results and to evaluate the sensitivity of this new approach for aneuploidy detection. Therefore, additional studies with the three-color FISH sperm aneuploidy assay should be untertaken to clarify whether a variety of other suspect aneugens cause sperm aneuploidy. Due to obvious difficulties in performing sperm studies on chemically exposed humans, there is a need to have such a complementary experimental approach in animals to identify aneugenic chemicals. Provided that such an evaluation is based on significant data which have been determined under strict scoring criteria, threshold concentrations with respect to risk assessment could be determined.
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Acknowledgments |
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Special thanks are due to Andy Wyrobek, Colin Bishop and Ulli Weier for providing the mouse chromosome-specific DNA probes. We also thank Helga Gonda and Martin Skerhut for assistance in the treatment of the mice. The mouse studies were supported by EU contract EN5V5-CT94-0403. Prof. Wang Xu was a guest of the GSF-Institut für Säugetiergenetik with a stipend from the BMBF.
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Notes |
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3 To whom correspondence should be addressed. Tel: +49 89 3187 2302; Fax: +49 89 3187 2210; Email: adler{at}gsf.de
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Received on August 21, 1998; accepted on October 7, 1998.
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