Validation of an assay for numerical chromosome changes
in male mouse germ cells

Ilse-Dore Adler1*, Adolf Baumgartner2, Thomas E. Schmid3

1Institut für Experimentelle Genetik
GSF-Forschungszentrum für Umwelt und Gesundheit, GmbH
Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany,
2Present address: Institut für Experimentelle Genetik
GSF-Forschungszentrum für Umwelt und Gesundheit, GmbH
Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany
3Present address: Department of Biomedical Sciences,
University of Bradford, Bradford BD7 1DP, UK

*Tel.: +49-89-3187-2302
*Fax: +49-89-3187-2210
*e-mail: adler@gsf.de

Abstract

The paper reviews studies on the effects of potential aneugenic chemicals undertaken over the period of 1994-2000. An aneuploidy assay in male mouse germ cells was developed by A. Wyrobek, LLNL, Livermore, USA. This assay uses molecular cytogenetic techniques, i.e. fluorescence in situ hybridization with three different chromosome-specific DNA probes on sperm to detect disomic or diploid cells. In our laboratory, the assay was refinded and validated. 
The refinement of the assay consisted in a determination of the optimum sperm-sampling time after chemical treatment. BrdU-labelled sperm were counted daily during days 20-24 after treatment of the mice. Only treatment with the chemicals colchicine and etoposide delayed the appearance of BrdU-labelled sperm in the epididymis for 48 and 24 h, respectively.
The validation of the assay consisted of testing 10 chemicals chosen on the basis of previous indications that these chemicals are known or suspect aneugens. Acrylamide, omeprazole and taxol gave negative results. Colchicine and trichlorfon induced disomic sperm. Carbendazim and thiabendazole induced only diploid sperm. Diazepam and griseofulvin induced disomic and diploid sperm. Vinblastine gave inconclusive results. In a comparative study between mice and humans, it could be shown that chronic Valium® abuse increased the rates of disomic sperm in men while prolonged treatment with diazepam, the active ingredient of Valium®, had no effect in mice. It is concluded that the reasonably simple sperm-FISH assay could be of general use to determine the aneugenic potential of chemicals in male germ cells of mammals.

I. Introduction

The goal of the present studies was to adapt and to validate an aneuploidy assay in male mouse germ cells. Over the period we had started to adapt a molecular cytogenetic assay to detect aneuploid or diploid mouse sperm employing fluorescence in situ hybridization (FISH) in collaboration with the group of A. Wyrobek at the Lawrence Livermore National Laboratory (LLNL) (Adler et al., 1996; Lowe et al., 1996; Wyrobek and Adler, 1996). The assay uses chromosome-specific DNA-probes labelled with different fluorochromes. These probes are hybridized to epididymal sperm obtained from  rodents or humans after chemical exposure. Fluorescent signals are then counted in individual sperm under the fluorescence microscope. From here on, the test will be called “the sperm-FISH assay”. 
The assay had to be refinded and validated  over the period 1997-200. In particular, the optimal timing of sperm sampling needed verification. It was originally based upon the timing of spermatogenesis reported by Oakberg (1965), i.e. 22 days after treatment was chosen. An assay was developed to test if the chemical treatment altered the duration of the meiotic divisions. In this assay, primary spermatocytes were labelled with BrdU and the appearance of BrdU-containing sperm in the epididymis was then detected at various intervals after treatment with the test chemicals. Scoring of BrdU-labelled sperm was performed automatically using a Laser Scanning Cytometer (LSC). It could  was shown that the positive control compound colchicine and the topoisomerase-II-inhibitor etoposide prolonged the duration of the meiotic divisions by one or two days. This observation is important in order to select the appropriate sampling times for sperm from the epididymis.
In the sperm-FISH assay, the scoring criteria for fluorescent signals were harmonized between our laboratory and the group of A. Wyrobek at LLNL. We also cross-checked the results in our laboratory between different scorers. Special attention was paid to the correct assessment of diploid sperm by using phase-contrast microscopy to determine the number of sperm flagellae. 
The sperm-FISH assay offers the possibility to study aneuploidy in any species for which chromosome-specific DNA-probes are available, including humans (Baumgartner et al., 1999; Lowe et al., 1998; Robbins et al., 1997). Therefore, we also performed the assay on human sperm of patients under chronic Valium® abuse in collaboration with the Department of Psychiatry of the Ludwig-Maximilians-University, Munich. 
The sperm-FISH assay was performed with 10 chemicals which were chosen by an EU  contact group  based on various indications of their aneugenic potential. Among these chemicals colchicine and vinblastine were selected as positive controls. The other chemicals were acrylamide, the monomer of polyacrylamide synthetics, carbendazim, the active component of the pesticide benomyl, diazepam, the active component of the sedative Valium®, the fungicide griseofulvin, the anti-ulcer drug omeprazole, the anthelminitic drug thiabendazole and the pesticide trichlorfon.

II. Materials and Methods

II.1 Studies on the duration of meiosis:
To label male germ cells for the measurement of the duration of meiosis, male (102/E1xC3H/E1)F1 mice at the age of 10 - 12 weeks were intraperitoneally injected with 100 mg/kg bromodesoxyuridine (BrdU) at S-phase during preleptotene stage of meiosis. During meiosis I and II, 13 days later, the mice were treated with the test chemicals. The chemicals and concentrations are given in Tables 2-4. At daily intervals sperm were collected from the Caudae epididymes between 20 and 24 days after chemical treatment. BrdU-containing sperm were identified with a fluorescein-labeled anti-BrdU antibody (Diagnostic International, Germany). A minimum of 10,000 BrdU postive and BrdU negative cells (Figure 1) were scored automatically with a laser-scanning cytometer (LSC) (Compucyte, USA) on each slide (Figure 2). Photographic images were taken with Ektachrome Panther 400X film on a Zeiss Axioplan fluorescence photo-microscope (Zeiss, Germany) equipped with the  appropriate filters for the fluorochromes used.

Figure 1: Illustration of the BrdU positive and negative sperm. The sperm were labelled with a fluorescein anti-BrdU antibody (green fluorescence) and counterstained with propidiumiodide (red fluorescence).

Figure 2: Histogram of the green (FITC) fluorescent sperm scored with the Laser Scanning Cytometer (LSC)

II.2 Sperm-FISH studies:
Male (102/ElxC3H/El)F1 mice aged 10-14 weeks and weighing 25-30 g were employed. The chemicals chosen to be tested were acrylamide, carbendazim, colchicine diazepam, griseofulvin, omeprazole, taxol, thiabendazole, trichlorfon and vinblastine. The dose, routes of application and the solvents are given with the results in Tables 5-17. The choices of doses were based on previous studies or the determination of maximum tolerated doses. Mice were sacrified 22 days after treatment and sperm were collected from the Caudae epididymides. In preparation for the hybridization procedure, the sperm were decondensed by incubating the slides successiveley in a 10 mM dithiothreitol (DTT) solution in 0.1 M Tris-buffer at pH 8.0 (30 min) and a 3,5-diiodosalicylic acid solution (LIS) in 0.1 M Tris-buffer at pH 8.0 (60 min), dried on a slide warmer at 70°C and stored in -20°C.
Mouse DNA probes for chromosomes 8 and X were kindly supplied by A. Wyrobek (LLNL, California) and the Y-probe was supplied by U.-H. Weier (LBNL, California). Indirect labelling of the probes with the fluorescent dyes FITC and CY3 was performed by nick translation using the streptavidin or digoxygenin systems. The Y-probe was labelled with FITC (green signal), the X-probe was labelled with a combination of FITC and CY3 (yellow signal) and the probe for chromosome 8 was labelled with CY3 (red signal).  The sperm DNA was counterstained with DAPI. 
Scoring was performed on a Zeiss Axioplan fluorescence photo-microscope (Zeiss, Germany) equipped with the appropriate filters for the fluorochromes used. Records of every sperm with an abnormal number of hybridization domains were taken by digitizing the microscopic image with the computer program ISIS3 (MetaSystems, Germany). Data were computed with the software Cytoscore, Version 4.12, developed al the Lawrence Livermore National Laboratory, Livermore, CA, USA.
The sperm were assigned to the specific fluorochrome phenotypes as determined by the combinations of fluorescence signals in each nucleus: X8, Y8 as normal sperm, XX8, YY8, XY8, X88, or Y88 as disomic sperm (Figure 8), XY88 as diploid sperm resulting from the first meiotic division suppression, XX88 or YY88 as diploid sperm resulting from second meiotic division suppression. Cells were scored as having two domains of the same colour if the two signals were a) similar in size and intensity and b) separated by at least the distance of half the diameter of the domain. The frequencies of sperm with hypohaploid phenotypes (X0, Y0, 08) were scored but not included in the statistical anallysis. Sperm without signal did not occur.

Figure 8: Examples of 5 disomic and one diploid sperm (didgitized with the computer software Isis3, MetaSystem, Altlussheim, Germany)

III. Results

III.1 Influences of chemical treatments on the duration of meiotic division
There were no significant differences in the frequencies of BrdU-positve sperm between the first two control groups (Figure 3). The controls of the following three experiments showed the same time course of appearance of BrdU-labbeled sperm (Table 1). Of the 11 chemicals tested in the BrdU-assay, nine (acrylamide, carbendazim, diazepam, griseofulvin, omeprazole, taxol, thiabendazole, trichlorfon and vinblastine) showed no statistically significant changes in the duration of the meiotic divisions in mouse spermatocytes (Tables 2 to 4). Only after the treatment with 3 mg/kg colchicine and 50 mg/kg etoposide, was the appearance of BrdU-labelled sperm delayed by 48 and 24 hours, respectively (Tables 2 and 4, Figures 4 and 5). 

Table1: Percentages of BrdU-labelled sperm at various intervals after chemical treatment of male mice: Control values
BrdU-positive sperm (%)						
	day 20 (mean±SD)	day 21 (mean±SD)	day 22 (mean±SD)	day 23 (mean±SD)	day 24 (mean±SD)	
Control 1	17.0  7.5	28.4  7.6	30.6  5.2	38.1  16.5	35.1  11.8	
Control 2	24.8  11.0	24.1  2.6	34.9  7.1	36.8  12.3	24.5  3.4	
Control 3	19.5  4.8	26.9  4.1	33.6  6.0	21.8  7.2	21.0  2.0	
Control 4	16.9  6.8	30.3  8.2	41.1  7.7	31.3  2.5	31.2  5.6	
Control 5	19.9  4.3	28.6  10.1	29.2  7.9	30.1  17.0	29.9  3.6	

Table 2: Percentages of BrdU-labelled sperm at various intervals after chemical treatment of male mice: Colchicine, Diazepam, Griseofulvin and Vinblastine
BrdU-positive sperm (%)						
Chemicals
	day 20 (mean±SD)	day 21 (mean±SD)	day 22 (mean±SD)	day 23 (mean±SD)	day 24 (mean±SD)	
Control 1	17.0  7.5	28.4  7.6	30.6  5.2	38.1  16.5	35.1  11.8	
Colchicine
3 mg/kg	8.3  3.7	11.7  3.7 *	9.4  2.5 **	21.2  9.7	35.5  10.5	
Diazepam
300 mg/kg	19.4  7.6	41.4  7.3	27.3  5.5	30.4  12.0	34.4  19.6	
Griseofulvin
2000 mg/kg	14.4  4.6	15.2  3.5 	26.4  7.5	24.0  7.5	16.6  6.6 	
Vinblastine
1 mg/kg	16.7  7.6	26.2  7.5	23.3  10.7	26.4  6.3	17.9  3.9 	
* p=0.0075 (t-test)
** p=0.0003 (t-test)

Table 3: Percentages of BrdU-labelled sperm at various intervals after chemical treatment of male mice: Acrylamide, Taxol, Thiabendazole and Trichlorfon
BrdU-positive sperm nuclei (%)						
	day 20 (mean±SD)	day 21 (mean±SD)	day 22 (mean±SD)	day 23 (mean±SD)	day 24 (mean±SD)	
Control 2	24.8  11.0	24.1  2.6	34.9  7.1	36.8  12.3	24.5  3.4	
Acrylamide
120 mg/kg	22.6  8.3	22.2  3.2	28.0  6.5	20.9  2.8	19.1  6.2	
Taxol
50 mg/kg	15.8  3.2 	32.3  8.4	35.4  5.5	27.6  6.8	25.5  4.1	
Thiabendazole
300 mg/kg	25.4  3.2	26.6  3.2	29.2  7.2	25.3  5.8	30.4  5.5 	
Trichlorfon
200 mg/kg	24.7  10.7	28.5  3.4 	30.8  3.2	24.4  3.5	27.3  3.7	

Table 4: Percentages of BrdU-labelled sperm at various intervals after chemical treatment of male mice: Carbendazim, Etoposide and Omeprazole
BrdU-positive sperm (%)						
	day 20 (mean±SD)	day 21 (mean±SD)	day 22 (mean±SD)	day 23 (mean±SD)	day 24 (mean±SD)	
Control 3	19.5  4.8	26.9  4.1	33.6  6.0	21.8  7.2	21.0  2.0	
Carbendazim
1000 mg/kg	12.5  2.7	29.2  7.9	29.7  8.1	25.2  2.4	23.1  3.3 	
Control 4	16.9  6.8	30.3  8.2	41.1  7.7	31.3  2.5	31.2  5.6	
Etoposide	11.7  3.4	17.4  9.2	14.2  5.6*	25.8  13.1	23.7  10.7 	
Control 5	19.9  4.3	28.6  10.1	29.2  7.9	30.1  17.0	29.9  3.6	
Omeprazole
300 mg/kg	22.1  6.2	29.9  13.5	32.1  12.1	26.9  11.3	24.4  12.6 	
* p=0.0012 (t-test)

Fig. 3: Time course of appearance of BrdU-labelled sperm in the epididymis. 
Control 1 vs control 2

Fig. 4: Time course of appearance of BrdU-labelled sperm in the epididymis.
Colchicine groups vs control 1 (*p<0.01)

Figure 5: Time course of appearance of BrdU-labelled sperm in epididymis.
Etoposide groups vs. control 1

III.2 Sperm-FISH studies:

Acrylamide
Two doses were tested, the higher one being the maximum tolerated dose. Acrylamide did not significantly increase the frequencies of disomic sperm or of diploid sperm (Table 5) (Schmid et al., 1999). 

Table 5: Result of the three-colour FISH with epididymal sperm of mice treated 
ip with acrylamide (AA)
	Control I
(saline)	AA
60 mg/kg	Control II
(saline)	AA
120 mg/kg	
No. of animal	5	5	5	5	
Sperm scored	50,077	50,093	60,060	50,057	
X/Y	0.999	0.999	0.999	1.002	
XY88, XX88, YY88	2	2  	1	1	
% Diploidies	0.004 	0.004	0.002 	0.002 	
XX8	6	7	14	17	
YY8	3	6  	  3	  3	
XY8	3	3	  6	  1	
X88	8	5	  8	  9	
Y88	7	5	  4	  7	
% Disomies	0.054 	0.052 	0.070 	0.074 	

Carbendazim
Carbendazim was tested at two dose levels simultaneously with one concurrent solvent control. The doses were chosen according to published data on micronucleus induction (Seiler, 19776). While the frequencies of disomic sperm were not significantly increased at either dose, the lower dose of 500 mg/kg significantly increased the frequency of diploid sperm tenfold, from 0.002% in the concurrent control to 0.020% (Table 6). The higher dose of 1000 mg/kg increased the diploidy level to 0.012%, however the difference to the concurrent control was not significant. 

Table 6: Result of the three-colour FISH with epididymal sperm of mice treated 
po with carbendazim (CARB)
	Control 
(corn oil)	CARB
500 mg/kg	CARB 
1000 mg/kg	
No. of animal	5	5	5	
Sperm scored	50,082	50,070	50,084	
X/Y	1.001	0.998	0.995	
XY88, XX88, YY88	1	10	6	
% Diploidies	0.002 	0.020*	0.012 	
XX8	5	6	6	
YY8	6	6  	4	
XY8	1	2	2	
X88	9	4	4	
Y88	6	6	6	
% Disomies	0.054 	0.050 	0.044 	
*p<0.01 (2-test)

Colchicine
Colchicine gave the expected positive response at both doses tested, however, the effect did not increase with dose (Table 7) (Schmid et al., 1999).

Table 7: Result of the three-colour FISH with epididymal sperm of mice treated 
ip with colchicine (COL)
	Control I
(saline)	COL
1.5 mg/kg 	Control II
(saline)	COL
3 mg/kg 	
No. of animal	5	5	5	5	
Sperm scored	50,093	50,062	50,074	50,069	
X/Y	1.003	0.989	0.999	1.003	
XY88, XX88, YY88	0	2  	1	2  	
% Diploidies	0.0	0.004	0.002	0.004 	
XX8	9	14 	9  	14 	
YY8	4	  5  	2  	  6  	
XY8	2	  4  	4  	  4  	
X88	6	10 	8  	  9  	
Y88	4	  8  	5  	13 	
% Disomies	0.050	0.082*	0.056 	0.092*	
*p<0.05 (2-test)

Diazepam
Diazepam was dissolved in a 10:1 mixture of corn oil and ethanol. The doses were chosen based on our previous studies (Gassner and Adler, 1995). Since ethanol may have aneugenic properties, two concurrent control were included, corn oil alone and the corn oil-ethanol mixture. The two controls gave similarly negative results (Table 8). Single acute doses of 75, 150, and 300 mg/kg of diazepam were tested. Diazepam significantly increased the frequency of disomic sperm at the highest dose tested and a linear dose-response could be observed (Table 8). Similarly, the frequencies of diploid sperm were increased in a dose-related way (Schmid et al., 1999). 

Table 8: Result of the three-colour FISH with epididymal sperm of mice treated 
po with diazepam (DZ)
	Control I (corn oil) 	Conrol II (corn oil + ethanol) 	DZ
75 mg/kg	DZ
150 mg/kg	DZ
300 mg/kg	
No. of animal	5	5	5	5	5	
Sperm scored	50,256	52,076	50,097	50,075	50,876	
X/Y	1.004	1.008	0.999	0.995	0.998	
XY88	1	1	3	2	2	
XX88,YY88	0	0	1	4	7	
% Diploidies	0.002	0.002	0.008	0.012	0.018*	
XX8	6	7	11	9	13	
YY8	5	6	  4	8	  7	
XY8	3	2	  4	4	  5	
X88	4	7	  8	7	10	
Y88	5	4	  2	5	  6	
% Disomies	0.046	0.050	0.058	0.066	0.081*	
*p<0.05 (2-test)

Repeated daily treatments with a low dose of 3 mg/kg during the entire meiotic prophase (11 days) significantly increased the frequency of autosomal disomies (X88 and Y88), but not the total disomy rate (Table 9). Also the frequency of diploid sperm was elevated, however, the difference was only significant against the corn oil control and not against the corn oil-ethanol control (Baumgartner et al., 2000). 
In a parallel human study, two men were evaluated who had chronically ingested >0,3 mg/kg/d DZ for more than 6 months. Multicolor FISH was applied to human sperm probing for chromosomes X, Y, and 13. Frequencies for sperm with disomy 13 (2.2-fold; p<0.01), disomy X (2.5-fold; p<0.01), and total sex-chromosomal disomies (2.0-fold; p<0.01) were found to be significantly elevated after chronic DZ-exposure  compared to control subjects (Table 10) (Baumgartner et al., 2000).

Table 9: Result of the three-colour FISH with epididymal sperm of mice after 
subchronic po diazepam exposure (DZ) 
(treatment during entire meiotic prophase, 11 days)
	Controls 	DZ	
	Oil	Oil-ethanol	(3 mg/kg/d)	
No. of animals	5	5	5	
Scored sperm cells	50,409	50,562	50,545	
	(%)	(%)	(%)	
Autosomal disomy 88	0.016	0.020	0.044*§	
Gonosomal disomy XX	0.016	0.012	0.008	
	disomy YY	0.002	0.006	0.002	
	disomy XY	0.002	-	-	
All gonosomal disomies	0.020	0.018	0.010	
Total Disomies	0.036	0.038	0.054	
Diploidies	-	0.004	0.008*	
*Significantly different from oil-control group, p < 0.01 (Mann-Whitney test).
§Significantly different from oil-ethanol-control group, p < 0.05 (Mann-Whitney test).

Table 10: Result of the three-colour FISH in human sperm after chronic diazepam (DZ)-exposure 
	Control group	DZ
>0.3 mg/kg/d	
No. of individuals	2	2	
Sperm scored 	20,278	20,398	
	(%)	(%)	
Autosomal disomy 13-13	0.049	0.108*	
Gonosomal disomy XX	0.044	0.103*	
	disomy YY	0.010	0.015	
	disomy XY	0.025	0.039	
All gonosomal disomies	0.079	0.157*	
Total Disomies 	0.128	0.265**	
Diploidies a 	0.104	0.162	
Tetraploidiesa 	-	0.005	
*p < 0.05 (2 test):**p < 0.01 (2 test);aOnly sperm with one or more flagellae.

Griseofulvin
Three experiments were performed with griseofulvin doses of 500, 1000 and 2000 mg/kg, each with a concurrent corn-oil control-group. The frequencies of diploidies were 0.01-0.02% in sperm of the solvent control animals and increased dose-dependently to 0.03%, 0.068% and 0.091%, respectively, for 500, 1,000 and 2,000 mg/kg of griseofulvin (Table 11). The frequencies of disomic sperm were increased significantly above the controls in all griseofulvin-treated  groups but showed no dose response (Shi et al., 1999).

Table 11: Result of the three-colour FISH with epididymal sperm of mice treated 
po with griseofulvin (GF)
	Control I
(corn oil)	GF 
500 
mg/kg	Control II
(corn oil)	GF 
1000 
mg/kg 	Control III (corn oil)	GF 
2000 mg/kg	
No. of animals	5	5	5	5	5	5	
Sperm scored	50,643	50,488	50,269	50,121	50,533	50,687	
X/Y sperm ratio	1.0162	1.0009	1.0013	 0.9984	1.0141	1.0039	
XY8-8	  0 	  6 	  0 	  9	  2 	  8	
XX8-8	  8	  9	  4	21	  3	27 	
Y-Y-8-8	  0 	  0 	  1	  4	  3	11	
% Diploidies	 0.016	0.03	0.01	0.068**	0.02	0.091**	
XX8	  7	15	  8	12	10	12	
YY8	  6	13 	  9	14	  8	11	
XY8	  7	  8	  2	  6 	  5	  5 	
X88	  6	  8	  5 	  9 	  5	11	
Y88	10	22 	  9	11	12	34	
% Disomies	0.071	0.131**	0.066	0.104*	0.079	0.144**	
p< 0.05, ** p < 0.01 compared with the concurrent solvent control (2-test)

Omeprazole
Omeprazole was tested at two doses, 150 and 300 mg/kg. Neither disomic nor diploid sperm were induced (Table 12).

Table 12: Result of the three-colour FISH with epididymal sperm of mice treated 
ip with omeprazole (OM)
	Control 
(saline)	OM
150 mg/kg	OM
300 mg/kg	
No. of animal	5	5	5	
Sperm scored	50,071	50,089	50,0794	
X/Y	1.009	1.007	0.993	
XY88, XX88, YY88	1	0	0	
% Diploidies	0.002 	0.0	0.0 	
XX8	4	7	5	
YY8	5	9	6	
XY8	2	4	0	
X88	6	5	6	
Y88	1	4	2	
% DisomiesD	0.036 	0.058 	0.038 	

Taxol
Taxol was only tested at the maximum tolerated dose of 50 mg/kg (Table 13). The increase of disomic sperm was at the borderline of significance, i.e. 0.064% vrs 0.038 of the corn oil control (p=0.06).

Table 13: Result of the three-colour FISH with epididymal sperm of mice
ip treated with taxol (TX)
	Control
(corn oil)	TX
50 mg/kg	
No. of animals	5	5	
Sperm scored	50,086	50,088	
X/Y sperm ratio	0.999	1.001	
XY88, XX88,YY88	1	 0	
% Diploidies	0.002	 0.0	
XX8	8	14	
YY8	2	  3	
XY8	4	  2	
X88	3	  2	
Y88	2	11	
% Disomies	0.038	  0.064§	
*p=0.06 (2-test)

Thiabendazole
Thiabendazole induced a significant increase in diploid sperm at 300 mg/kg when compared to the concurrent control. However, the first control value for diploidies was in the same range. Both control values are exceptionally high. Disomic sperm frequencies were elevated in both treatment groups, however, the increases were not statistically significant.

Table 14: Result of the three-colour FISH with epididymal sperm of mice treated 
po with thiabendazole (TB)
	Control I
(corn oil)	TB
100 mg/kg	Control II
(corn oil)	TB
300 mg/kg	
No. of animal	5	5	5	5	
Sperm scored	51,325	51,914	52,047	51,605	
X/Y	1.023	0.978	1.005	1.002	
XY88	4	6	5	6	
XX88,YY88	18	13	7	18	
% Diploidies	0.043	0.037	0.023	0.047*	
XX8	6	11	8	6	
YY8	1	5	3	4	
XY8	2	4	1	3	
X88	9	6	7	9	
Y88	6	9	17	15	
% Disomies	0.049	0.067	0.069	0.072	
*p<0.05 (2-test)

Trichlorfon
After treatment with trichlorfon at single doses of 200, 300 and 405 mg/kg, the frequencies of disomic sperm were 0.068%, 0.074% and 0.134%, respectively (Table 14). Compared to the corresponding control values of 0.046%, 0.042% and 0.056%, 
respectively, the disomy frequencies of the treated groups were significantly increased (p < 0.05 and  p < 0.01, respectively). The dose-response relationship is shown in Figure 6 (Sun et al., 2000). 

Table 15: Result of the three-colour FISH with epididymal sperm of mice treated 
ip with trichlorfon (TCF)
	Control I
(water)a	TCF
200 mg/kg	Control II
(water)a	TCF
300 
mg/kg	Control III
(water)a	TCF
405 
mg/kg	
No. Of animal	5	5	5	5	5	5	
Sperm scored	50,254	50,258	50,150	50,283	50,014	50,018	
X/Y	1.006	1.000	1.003	1.000 	1.004	0.9940	
XY8-8	0	1	0	0	0	0	
XX8-8, 
YY8-8	1	0	0	0	1	0	
%Diploidies 	0.002	0.002	0.0	0.0	0.002	0.0	
XX8	5	11	4	14	5	18	
YY8	1	4	1	5	5	11	
XY8	0	3	2	2	2	1	
X88	9	11	6	3	8	14	
Y88	8	5	8	13	8	23	
%Disomies	0.046	0.068*	0.042	0.074**	0.056	0.134**	
*p < 0.05 (Mann-Whitney-Test), **p < 0.01 (Mann-Whitney-Test). aBidistilled water at pH 5.5

Figure 6: Dose-response relationship for the induction of disomic sperm by trichlorfon

Frequency of disomic sperm (% ± SD)

Closed circles treated, open circles control group means.

Vinblastine
Vinblastine was tested at three dose levels in the first experiments. Each dose group was accompanied by a concurrent solvent control group. The two higher doses induced disomic sperm, however, there was no dose-related increase (Table 15). Some of the slides from the middle dose (1.0 mg/kg) were also scored by a collegue at the Lawrence Livermore National Laboratory (LLNL), Livermore, USA, who could not confirm our positive results due to higher rates of disomic sperm they scored in the contol group. Therefore, we decided to repeat the experiment and take the slides to the Livermore laboratory to adjust the scoring criteria. Even though the control rates of the LLNL-scorer became more in line with our scores, the result for 1 mg/kg of vinblastine was negative by both scorers (Table 16).

Table 16: Result of the three-colour FISH with epididymal sperm of mice treated 
ip with vinblastine (VBL) (Scorer TES)
	Control I
(saline)	VBL
0.5 mg/kg	Control II
(saline)	VBL
1.0 mg/kg	Control III
(saline)	VBL
2.0 mg/kg	
No. Of animal	5	5	5	5	5	5	
Sperm scored	50,806	50,476	51,047	51,638	50,088	50,070	
X/Y	0.998	1.002	0.999	1.008 	1.002	1.004	
XY88	0	3	0	3	0	1	
XX88, 
YY88	3	5	2	3	4	0	
%Diploidies 	0.006	0.016	0.004	0.012	0.008	0.002	
XX8	8	8	7	11	7	15	
YY8	5	8	6	7	5	16	
XY8	3	4	2	8	2	6	
X88	7	81	12	13	5	8	
Y88	7	9	4	10	6	8	
%Disomies	0.059	0.073	0.057	0.097*	0.050	0.086*	

Table 17: Frequencies of aneuploid sperm after treatment with 1mg/kg vinblastine 
(Scorer CS, LLNL and TES, GSF, scored at Livermore)
	Scorer: CS	Scorer: CS	Scorer: TES	Scorer: TES	
	Control
(saline)	VBL
1.0 mg/kg	Control
(saline)	VBL
1.0 mg/kg	
No. of animal	5	5	5	5	
Sperm scored	47,365	50,448	50,154	50,172	
X/Y	0.999	0.980	0.994	0.990	
XY88	0	0	0	0	
XX88,YY88	0	1	1	1	
% Diploidies	0.000	0.002	0.002	0.002	
XX8	  6	  4	13	18	
YY8	  1	  0	 7	  6	
XY8	  1	  2	 2	  3	
X88	13	10	 5	10	
Y88	  5	  6	 5	  7	
% Disomies	0.055	0.044	0.064	0.088	

IV. Discussion

IV.1 Influences of chemical treatment on the duration of meiotic division
The sampling of epididymal sperm in the first experiments with the sperm-FISH assay were designed according to the timing of spermatogenesis described Oakberg (1956), i.e. 22 days after treatment with the test chemical. It became obvious that positive results obtained with this sampling interval never exceeded a doubling or tripling of the control rates for disomic sperm. Therefore, we decided to test, whether or not the chemical treatment prolonged the meiotic divisions. It has been often discussed that cell cycle delay and aneuploidy induction are closely if not causally related (Miller and Adler, 1992; Chen et al., 1981). Therefore, we developed the BrdU-assay to determine the time course for the appearance of BrdU-labelled sperm. In the controls, the BrdU-labelled sperm appeared in the epididymis 22-24 hours after meiosis confirming that our original timing was correct. Of the chemicals tested, only colchicine and etoposide delayed the appearance of BrdU-Labelled sperm by 48 and 24 hours, respectively. All other 9 chemicals tested failed to show any difference to the controls. The sperm-FISH assay with colchicine will be repeated with a 24 day sampling interval, the assay with etoposide using a sampling interval of 23 days is currently under way. For all other chemicals the 22 day sampling interval proved to be appropriate.

IV.2 Sperm-FISH studies:
A total of 10 chemicals have been tested with the sperm-FISH assay (Table 17). Three of these chemicals gave negative results: the monomer of synthetics acrylamide, the anti-ulcer drug omeprazol and the anti-cancer drug taxol. Two chemicals induced only disomic sperm, the spindle inhibitor colchicine and the pesticide trichlorfon. The prevalence of XX8 and YY8 sperm suggests that trichlorfon affected chromosome segregation predominantly during the second meiotic division (Table 14). Another two chemicals induced only diploid sperm which indicates that they suppressed the first or second meiotic division completely. These compounds were the pesticide carbendazim and the anthelmintic drug thiabendazole. 

Table 18: Summary of Results with the Sperm-FISH Assay
Chemical	Doses	Aneuploidy	Diploidy	
Acrylamide	    60 mg/kg	-	-	
	  120 mg/kg	-	-	
Omeprazole	   150 mg/kg	-	-	
	   300 mg/kg	-	-	
Taxol	    50 mg/kg	-	-	
Colchicine	      1.5 mg/kg	-	-	
	      3 mg/kg	+	-	
Trichlorfon	  200 mg/kg	+	-	
	  300 mg/kg	+	-	
	  405 mg/kg	+	-	
Carbendazim	  500 mg/kg	-	+	
	1000 mg/kg	-	-	
Thiabendazole	  100 mg/kg	-	-	
	  300 mg/kg	-	+	
Diazepam	      3 mg/kg	-	-	
	    75 mg/kg	-	-	
	  150 mg/kg	-	+	
	  300 mg/kg	+	+	
Griseofulvin	  500 mg/kg	+	-	
	1000 mg/kg	+	+	
	2000 mg/kg	+	+	
Vinblastine	      0.5 mg/kg	-	-	
	      1 mg/kg	+/-	-	
	      2 mg/kg	+	-	

Disomies and diploidies were induced by the sedative diazepam and the fungicide griseofulvin. The results with diazepam indicate that it acts as an aneugen during meiosis in male spermatogenesis, both in mice and humans. The quantitative comparison shows that after chronic exposure humans are at least 10 times more sensitive than mice for aneuploidy induction by DZ during male meiosis (Figure 7, Baumgartner et al., 2001). In another unpublissshed study with self poisoning patients which had taken an overdose of valium® the difference in diazepam effectiveness between humans and mice was 100 fold (publication submitted).

Figure 7: Comparison of sensitivity to Diazepam 
between humans and mice (Baumgartner et al., 2001)

The griseofulvin data for individual classes of disomic and diploid sperm indicates that MII was more sensitive than MI to griseofulvin-induced non-disjunction in male mice (Table 11). A comparison of the present data from male mice and literature data from female mice suggests that mouse oocytes are more sensitive than mouse spermatocytes to GF-induced meiotic delay and aneuploidy (Marchetti and Mailhes, 1992).
The other positive control substance, vinblastine, gave a similar response as colchicine in that the disomic sperm were significantly more frequent than in the controls at the two higher doses tested (Table 15) but without showing a dose-response. Some of the vinblastine slides from the 1.0 mg/kg group were rescored at another laboratory (LLNL, USA) whereby the positive result was not confirmed. Therefore, the experiment was repeated with that dose and jointly scored under observation of harmonized scoring criteria (Schmid et al., 2001). Again, the result showed no increase in disomic sperm after vinblastine treatment (Table 16). Therefore, we conclude that the vinblastine response was inconclusive. The inconclusive vinblastine results can be attributed to the high variability in animal response which has been previously observed (Gassner 1995). The results with vinblastine raised the question of reproducibility of results. We have now 20 independent control groups of 5 males/50,000 sperm each. The mean control value for disomic sperm is 0.049 with a standard deviation of 0.016. Thus, our historical control is quite stable and reproducible so that we believe that scoring criteria within our group have been harmonized adequately. It has to be concluded that concurrent controls are mandatory and that historical controls need to be accumulated and any new scorer has to assertain if his concurrent controls are within the range of the historical controls. This is a common rule for mutagenicity tests. 

V. Conclusions

We have adapted and validated the sperm-FISH assay to detect the induction of numerical chromosome changes in male mammalian germ cells by chemical mutagens. The data base now contains results for 10 known or suspected aneugens. We also demonstrated with semen samples of diazepam patients that the assay is suitable to detect aneuploidy induced in human spermatocytes. This provided the first data set to compare human-mouse sensitivity differences and suggested that human spermatocytes were 10-100 fold more sensititve than those of mice. It also showed that the spontaneous aneuploidy rate in human sperm was about 4fold higher than in mice. 
As we demonstrated, some chemicals induced either only diploidies or only disomies and some induced both. Disomies are the result of non-disjunction during either first or second meiotic division and may be due to effects on different targets that control segregation fidelity such as centromeres with their kinetochores, telomeres, spindle tubuli, centrioles, or motor proteins. Diploidies are the result of suppressed cell division. The prevalence of YY- or XX-bearing diploid sperm indicates the supression of the second meiotic division. Similarly, a prevalence of XX- or YY-bearing hyperhaploid sperm indicates non-disjunction during the second meiotic division. Examples are colchicine, carbendazim, diazepam, griseofulvin and trichlorfon. So far, none of the aneugens exhibited their predominant effect during the first meiotic division. This suggests that either the spermatocytes are more sensitive during their second meiotic divison or that non-disjunction during or inhibition of the first meiotic division causes predominantly cell death. The latter conclusion is supported by the fact that the ratio between XY- versus XX- plus YY-bearing hyperhaploid sperm is 1:5 in the controls. It should be 1:1 unless one cell would undergo non-disjunction during first and second meiotic division which is rather unlikely. 
We will expand the data base, however, it seems timely to suggest that this reasonably simple assay could be of general use to determine the aneugenic potential of chemicals in male germ cells of mammals. It is mandatory for the reliability of the assay that strict scoring criteria are observed and that concurrent control values stay within the range of historical controls. We also advise that before starting a sperm-FISH assay, to perform a BrdU-assay to determine a possible delay in meiotic cell division induced by a given test chemical in order to chose an adequate sampling time for epididymal sperm.

VI. Acknowledgements

We appreciate the technical assistance by Helga Gonda, Isa S. Otten and Martin Skerhut. We are grateful to Dr. C.G. Schuetz of the Department of Psychiatry at the LMU, Munich, for providing semen samples of patients. The work was supported by the EU-Contracts EV5V-CT94-0403 and ENV4-CT97-0471.

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