Mutagenesis, Vol. 16, No. 4, 339-343,
July 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Effect of chemicals on the duration of male meiosis in mice detected with laser scanning cytometry
Institut für Experimentelle Genetik and 1 Institut für Strahlenbiologie, 2 Arbeitsgruppe Durchflusszytometrie, GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, Ingolstädter Landstraße 1, 85764 Neuherberg, Germany
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Abstract |
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Aneuploidy studies in sperm such as the sperm-FISH assay require a precise knowledge of the duration of spermatogenesis, especially of the meiotic stages. This is important in order to sample sperm from the epididymis at appropriate intervals after animal treatment. However, aneugens may delay the cell cycle. The progression from meiotic divisions to epididymal sperm was determined by labelling the last S-phase before meiosis with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. In a time frame of 20–24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). We studied the effects of the chemicals acrylamide, colchicine, diazepam, griseofulvin, taxol, thiobendazole, trichlorfon and vinblastine on the duration of meiotic divisions in male mice. Colchicine treatment prolonged the duration of meiotic divisions by about 48 h. On days 21 and 22, the frequencies of BrdU-labelled sperm in the colchicine group were 11.7 and 9.4%, respectively, while they were 28.4 and 30.6%, respectively, in the concurrent controls (P > 0.01). On day 24 after treatment, the frequency of labelled sperm in the colchicine group reached the control level. Etoposide treatment resulted in an elevation of BrdU-labelled sperm at 23 rather than 22 days. The other chemicals showed no significant effect of prolonging meiotic cell cycle progression. On the basis of the colchicine and etoposide data, it is suggested that the effect of a chemical on the meiotic cell cycle progression is determined first in order to chose the appropriate sperm sampling time to detect aneuploidy induction.
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Introduction |
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Aneuploidy that is transmitted via germ cells is one of the most common forms of chromosome damage known to occur in humans. Whereas 35% of spontaneous abortions are aneuploid indicating that most aneuploid conceptions are lost prior to birth (Hassold, 1986
), approximately one of 300 newborns is still affected (Jacobs, 1992). Therefore, there is currently increasing interest in the study of rodent germ cells to determine the incidence and mechanisms of chemically induced aneuploidy in rodents. From such data estimates of the contribution of chemicals to the human effects may be made.
Fluorescence in situ hybridization (FISH) of chromosome-specific DNA probes to epididymal sperm (sperm-FISH assay) offers the possibility of detecting the consequences of chemically-induced missegregation during meiosis in spermatocytes. With multicolour FISH techniques, several chromosomes can be analysed simultaneously for hyperploidy (disomy) and diploid and disomic sperm can be distinguished (Spriggs et al., 1995; Wyrobek et al., 1995; Adler et al., 1996; Lave et al., 1996; Robbins et al., 1997a, b). The sperm-FISH assay has been applied to test the aneugenic properties of a number of chemicals selected in the Aneuploidy Project sponsored by the Environmental Research Programme of the European Union (Schmid et al., 1999a; Shi et al., 1999; Sun et al., 2000).
In the sperm-FISH assay, a crucial prerequisite for the timing of sperm sampling is a precise knowledge of both the duration of the meiotic stages and the possible temporal changes of meiotic divisions induced by aneugens. Such agents can induce cell-cycle delay in somatic cells (Chen et al., 1981). Miller and Adler (1992) discussed a possible correlation between induction of aneuploidy and meiotic delay in mouse spermatocytes. In recent studies with the sperm-FISH aneuploidy assay (Schmid et al., 1999a; Shi et al., 1999; Sun et al., 2000), a 22 day sperm-sampling time was used according to the timing of mouse spermatogenic stages (Adler, 1996). It was noted that relatively low disomy scores were obtained in sperm after treatment with griseofulvin, which was strongly aneugenic in female germ cells (Marchetti et al., 1992; Shi et al., 1999). One possible explanation could be that sperm were not sampled at the optimum time after treatment because of induced meiotic delay. To improve the sperm-FISH assay, it appeared necessary to test the effect of chemicals on the duration of male meiosis in mice.
We present here results from the analysis of the effect of chemicals on the duration of meiosis in male mice. The time of development from meiotic divisions in spermatocytes to epididymal sperm was determined by labelling cells with 5-bromo-2'-deoxyuridine (BrdU) during the last S-phase (preleptotene) and chemical treatment during the following meiotic divisions, i.e. after 13 days. The appearance of BrdU-labelled sperm in the epididymis was quantified at daily intervals between 20 and 24 days later using a laser scanning cytometer (LSC).
The chemicals examined in this study were acrylamide, colchicine, diazepam, etoposide, griseofulvin, taxol, thiobendazole, trichlorfon and vinblastine. Acrylamide 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). Diazepam is the active compound of the tranquilizer Valium®. Etoposide is a topoisomerase-II inhibitor. Griseofulvin is an orally applied fungicide for the treatment of dermatophytosis. Thiabendazole is an antihelmintic drug with a broad spectrum. Vinblastine and taxol are used in chemotherapy for the treatment of cancer. Trichlorfon, an inhibitor of acetylcholine esterase, is widely used not only as an insecticide but also as human medicine against Alzheimer's disease (Tariot et al., 1997). Colchicine was used as positive control. All these chemicals except for etoposide have already been tested with the sperm-FISH assay in the frame of the Aneuploidy Project sponsored by the Environmental Research Programme of the European Union. Acrylamide and taxol (unpublished data) have been found negative while colchicine, diazepam, griseofulvin, thiabendazole, trichlorfon have been found positive (Schmid et al., 1999a; Shi et al., 1999; Sun et al., 2000). Vinblastine gave equivocal results (unpublished data). Sperm-FISH experiments with etoposide are in progress.
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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 mouse colony of the GSF, and were maintained on a 12 h light/dark cycle with mouse pellet food and water ad libitum.
Mice were intraperitoneally injected with 100 mg/kg BrdU in order to label spermatocytes at S-phase during preleptotene of meiosis. During meiosis I and II, 13 days later, the mice were treated with the test chemicals. The chemical doses applied in the present study were the highest ones used in earlier studies with the sperm-FISH assay (Shi et al., 1999; Schmid et al., 1999a; Sun et al., 2000).
The chemical doses were 120 mg/kg acrylamide (AA), 3 mg/kg colchicine (COL), 300 mg/kg diazepam (DZ), 50 mg/kg etoposide (ET, maximum tolerated dose), 2000 mg/kg griseofulvin (GF), 50 mg/kg taxol (TX, maximum tolerated dose), 300 mg/kg thiobendazole (TB), 200 mg/kg trichlorfon (TF) and 1 mg/kg vinblastine (VBL). AA, COL, ET, GF, TX, TF and VBL were obtained from Sigma (Deisenhofen, Germany) and dissolved in physiological saline, whereas 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 25 males each were randomly assigned to the various treatment and the two concurrent solvent control groups (1 and 2). AA, COL, ET, TX, TF and VBL were intraperitoneally injected (ip), DZ, GF and TB were applied by oral intubation (po). The injected volume was 0.1 ml/10 g body weight. Groups of five males were killed daily on days 20–24 after the application of the test chemical or the solvent and sperm were collected from the caudae epididymes.
A separate experiment to test the source of variability in controls was performed using sperm samples from control group 1 on day 23.
Preparation of epididymal sperm
The preparation technique for epididymal sperm was based on the procedure described by Schmid et al. (1999a). 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 (FCS). The cups were placed on 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 spread across the slide.
Slide processing and immunofluorescence staining
The slides were air-dried for at least 1 week. Then, after heating the slides for 5 min at 70°C on a hot plate, they were incubated in a Coplin jar in 10 mM dithiothreitol (DTT; Sigma) for 30 min on ice followed by incubation in 4 mM lithium-3,5-diiodosalicylic acid (LIS; Sigma) for 30 min on ice. The slides were dried by heating for 5 min at 70°C on a hot plate.
Immunofluorescence staining with BrdU antibodies was performed according to a modified technique of Zink et al. (1998). The sperm smears were denatured in a mixture of 50 ml 0.1 N NaOH and 22 ml ethanol at room temperature for 1 min and neutralized with PBS buffer. Immunodetection was carried out in two steps in a humidified chamber: slides were incubated with a rat anti-BrdU (Seralab, 1/100) antibody for 30 min at room temperature. After washing for 3x5 min with PBS the slides were incubated with a FITC-conjugated anti-rat IgG antibody (Dianova, 1/100) for 30 min at room temperature and washed again (3x5 min PBS). The nuclei were counterstained with propidium iodide (1 µg/ml in PBS) for 10 min at room temperature and cover-slipped in Vectashield (Vector Laboratories, CA, USA). Slides were stored at –20°C in the dark.
Scoring
All slides were coded. The laser scanning cytometer (LSC; Compucyte, Cambridge, MA, USA) was equipped with an argon and a helium-neon laser and the fluorescence signals emitted by the cells were collected by the objective lens partially directed to the scanning mirror. The reflected fluorescence was detected via photomultipliers. Photographic images were taken with Ektachrome Panther 400 X film on a Zeiss Axioplan fluorescence photo-microscope (Zeiss, Germany) equipped with the following filters: triple filter (triple band-pass filter set No. 61000; Chroma Technology, Brattleboro, USA) for simultaneous visualization of green (FITC) and red (propidium iodide) fluorescence hybridization; individual filters for each of the fluorochromes (FITC: HQ 480/40, HQ 535/50; PI: HQ 535/50, HQ 610/75; Chroma Technology, Brattleboro, USA and DAPI: BP 365, LP 397; Zeiss, Germany) were used to digitize the image.
An average of 10 000 BrdU-positive (green fluorescence) and BrdU-negative (red fluorescence) cells per animal (Figure 1) were scored automatically with LSC.
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Statistical methods
The differences between the numbers of BrdU-positive sperm in treated animals and the concurrent solvent controls on each day were tested for significance using Student's t-test.
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Results |
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Figure 2
illustrates a dotplot of murine sperm (a typical control) with FITC-tagged BrdU (green peak) and sperm without BrdU (red peak). For validating the LSC-based method, a series of control experiments were performed. As shown in Table I, the highest variability was observed between a cohort of five animals (36.8 ± 12.3). Variability between five slides from one animal was somewhat lower (33.7 ± 3.8) and even lower between different scoring areas on a single slide (28.4 ± 3.2). These findings indicate that interindividual variability rather than scoring variability is responsible for the large standard deviations of the results in these experiments.
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Figure 3
shows the comparison between the two control groups. No significant difference can be observed. The frequencies of BrdU-labelled sperm form a plateau between 21 and 23 days confirming the original choice of the sperm-sampling time of 22 days after treatment in the sperm-FISH assay.
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Of the nine chemicals tested in the BrdU assay, seven did not cause significant changes in the duration of the meiotic divisions in mouse spermatocytes (Tables II and III
). With DZ, a non-significant peak of BrdU-labelled sperm occurred on day 21; however, the results of the other days were in the control range (Table II). With GF and VBL, the frequencies of BrdU-labelled sperm on days 22 to 24 were lower than in the concurrent control groups, but the difference was not statistically significant. A similar observation was made with AA (Table III). With TX, TB and TF, the frequencies of BrdU-labelled sperm were similar to the concurrent controls.
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For the treatment with colchicine, a prolongation of the duration of the meiotic divisions was observed. On days 21 and 22, the frequencies of BrdU-labelled sperm in the colchicine groups were 11.7 and 9.4%, respectively (Table II
, Figure 4), the corresponding control frequencies were 28.4 and 30.6%, respectively (P = 0.007 and 0.0003, respectively). The values came up to control level on day 24. For the treatment with ET, the frequency of BrdU-labelled sperm was significantly below the control value on day 22 and came up to control levels only on day 23 (Table IV, Figure 5).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The goal of the present study was to determine the time interval of the development from meiotic divisions in spermatocytes to epididymal sperm in controls and a possible alteration of this time in chemically treated animals.
In these studies, BrdU-labelled sperm were automatically counted with the aid of a new instrument, the LSC. Quantitation of sperm with fluorescent signals by the LSC saves time, e.g. manual scoring of 10 000 sperm takes about 5 h while LSC scoring of 10 000 sperm takes only 20 min. Experiments to apply LSC scoring also to the sperm-FISH assay where multiple colour signals have to be detected are in progress (Schmid et al., 1999b).
From the control experiment (Table I), it can be concluded that the main source of the high standard deviations in Tables II and III is the variability between animals and not an inaccuracy of the LSC measurements.
The present data indicate that the treatment with 3 mg/kg COL prolonged the duration of the meiotic divisions in mouse spermatocytes for about 48 h (Figure 4). Therefore, the optimum time for sperm sampling in a sperm-FISH assay with COL would be 24 instead of 22 days. For ET, the optimum sampling time is 23 days after treatment (Figure 5). Respective experiments with ET and COL are in progress. The chemicals AA, DZ, GF, TX, TB, TF and VBL did not prolong the meiotic cell cycle. Thus, the 22 day sampling time used in recent studies was confirmed for these chemicals. Therefore, the difference in aneuploidy induction by DZ between male and female mice (Marchetti et al., 1994) cannot be attributed to the choice of an inappropriate sampling time for sperm but may be a real sex difference. It is concluded that in future experiments, the effect of a test chemical on meiotic division progression should be determined before designing the sampling time of sperm in aneuploidy studies with the sperm-FISH assay in order to optimize the test protocol and to avoid missing an effect by inappropriate timing of the sperm samples.
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Acknowledgments |
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We thank Helga Gonda, Isa Otten and Martin Skerhut for their technical assistance. We also thank Herbert Braselmann for his help with the statistical analysis. The studies were supported by the EU contract EN5V5-CT94-0403 and EN5V5-CT97-0471. S.Attia acknowledges the support from the Egyptian Government to perform his PhD thesis studies in Germany.
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Notes |
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3 To whom correspondence should be addressed. Email: adler{at}gsf.de
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References |
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Received on November 20, 2000; accepted on March 14, 2001.
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