Comparative cytogenetic study after treatment of mouse spermatogonia with mitomycin C

Male (101 × C₃H)F₁ hybrid mice, 10–12 weeks old, were injected i.p. with single doses of 2.5, 3.75 or 5.0 mg/kg of mitomycin C (MC). Spermatogonia were sample for mitotic chromosome analyses 6, 18 or 24 h after treatment. Spermatocytes were sample for meiotic chromosome analyses 50 or 60 days after treatment.The maximal yield of chromatid-type aberrations induced in spermatogonia was found 24 h after treatment with 5.0 mg/kg of MC. More than 50% of the cells carried at least one chromatid exchange. The majority (90%) of these were whole-arm exchanges derived from breaks in the centromeric heterochromatin.No translocation multivalents were found in spermatocytes analysed 50 or 60 days after treatment. The discrepancy between the presence of many symmetrical exchanges in spermatogonia and the absence of translocation multivalents in primary spermatocytes may be result of insensitivity of the stem cell spermatogonia against exchange induction by MC or of complete germinal selection against induced translocations before and/or during early meiosis. However, the possibility of missing translocations due to whole-arm exchanges in acrocentric chromosomes during the analysis of diakineses-metaphases I is also discussed.It is emphasized that comparisons of chromatid exchange frequencies in spermatogonia with the yield of translocation multivalents in spermatocytes descended from these spermatogonia as opposed to those from stem cells might provide an estimate of pre-diakinesis germinal selection against chromatid exchanges or the resulting translocations. This estimate is important for the quantitative evaluation of the genetic risk from environmental mutagens.     

A correlative study on the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice : I. CNU-ethanol

Cytogenetic damage induced by a wide range of concentrations of CNU-ethanol in mice was evaluated by determining the frequencies of (a) micronuclei nuclei in polychromatic erythrocytes of the bone marrow, (b) chromatid aberrations in bone marrow, (c) chromatid aberrations in spermatogonia, and (d) reciprocal translocations induced in spermatogonia and scored in spermatocytes.For CNU-ethanol the following order of sensitivity was found between the tests performed: micronuclei > aberrations in bone marrow > aberrations in spermatogonia > translocations in spermatocytes.Correlation coefficients were calculated for the first three parameters. Positive correlations existed (a) between micronuclei in polychromatic erythrocytes and chromatid aberrations in bone marrow on the first day after treatment, and (b) between chromatid aberrations in bone marrow and spermatogonia at the first day after treatment.Three reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes; all were of a rare type, namely between an X chromosome and an autosome.     

Bleomycin-induced structural chromosomal aberrations in spermatogonia and bone-marrow cells of mice

The induction of structural chromosomal aberrations by bleomycin (BLM) was studied in bone-marrow cells and spermatogonia of the mouse at doses of 10, 20, 40 and 80 mg/kg. BLM induced genetically important reciprocal translocations in stem-cell spermatogonia as measured with the spermatocyte test, and the response of bone-marrow cells to BLM was not markedly different from that of spermatogonia.     

A correlative study of the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice. III. 1,3-Bis (2-chloroethyl)-3-nitrosourea (BCNU)

Cytogenetic damage induced by various concentrations of BCNU was evaluated by determining the frequencies of (a) micronuclei in polychromatic erythrocytes of bone marrow, (b) chromatid aberrations in bone marrow, (c) chromatid aberrations in spermatogonia, and (d) reciprocal translocations induced in spermatogonia and scored in spermatocytes. The order of sensitivity for the four parameters tested was: micronuclei greater than chromatid aberrations in bone marrow greater than aberrations in spermatogonia greater than translocations in spermatocytes, a situation similar to that found in an earlier study with CNU-ethanol. When the effect of concentration of the chemical was taken into consideration there was no correlation among the four parameters tested, so that information on induced frequencies of one parameter does not have predictive value for the frequencies of the others. A comparison of the results obtained with the bifunctional BCNU and the mono-functional CNU-ethanol at equal concentrations indicated that BCNU had a similar or a lesser clastogenic effect than did CNU-ethanol. In an experiment in vitro the situation was different in that BCNU was more effective than CNU-ethanol.     

In vivo cytogenetics: mammalian germ cells

This chapter summarizes the most relevant methodologies available for evaluation of cytogenetic damage induced in vivo in mammalian germ cells. Protocols are provided for the following endpoints: numerical and structural chromosome aberrations in secondary oocytes or first-cleavage zygotes, reciprocal translocations in primary spermatocytes, chromosome counting in secondary spermatocytes, numerical and structural chromosome aberrations, and sister chromatid exchanges (SCE) in spermatogonia, micronuclei in early spermatids, aneuploidy in mature sperm. The significance of each methodology is discussed. The contribution of novel molecular cytogenetic approaches to the detection of chromosome damage in rodent germ cells is also considered.     

Inhibition of ADP-ribosylation increases X-ray-induced chromosomal damage in mouse testis and bone-marrow cells in vivo

The effect of 3-aminobenzamide (3AB) treatment on chromosomal radiosensitivity of mouse spermatogonial stem cells and bone-marrow cells was studied using various doses of X-rays. The results show that 3AB increases the induction of reciprocal translocations in slowly cycling spermatogonia as well as the frequency of chromosomal aberrations in actively dividing bone-marrow cells. The experiments indicate that both types of tissue are suitable to study the ability of inhibitors of ADP-ribosylation to modulate chromosome-breaking damage induced by ionizing radiation in vivo.     

Effect of follicle-stimulating hormone (FSH) on radiation-induced chromosomal aberrations in mouse germ cells and Chinese hamster cells in vitro

Earlier observations on the induction by X-rays of reciprocal translocations in stem-cell spermatogonia of the rhesus monkey have established a correlation between the level of follicle-stimulating hormone (FSH) in blood at the moment of irradiation and the final recovery of translocations (van Buul, 1980). In the present study, FSH treatment of mice did not induce chromosomal aberrations in bone-marrow cells or stem-cell spermatogonia, nor did it change the radiosensitivity of stem-cell spermatogonia for the induction of chromosomal translocations. Experiments in vitro with Chinese hamster ovary cells (CHO), however, showed a clear radiosensitizing effect of FSH on the induction of structural chromosomal aberrations.     

Dose—response relationship for radiation-induced translocations in somatic and germ cells of mice

Dose—response curves (0–600 rad X-rays) for induced reciprocal translocations in bone-marrow cells and in spermatogonia (scored in spermatocytes) of the mouse were constructed. The obtained results suggest that factors influencing aberration yields in somatic cells, are similar to those in germ cells and strengthen the premise for qualitative extrapolation from somatic cells to germ cells.     

Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells

The clastogenic activity of hydroquinone (HQ) in germ cells of male mice was evaluated by analysis of chromosomal aberrations in primary spermatocytes and differentiating spermatogonia. In the first experiment with treated spermatocytes the most sensitive stage of meiotic prophase to aberration induction by HQ was determined. Testicular material was sampled for microscopic analysis of cells in diakinesis-metaphase I at 1, 5, 9, 11, and 12 days after treatment with 80 mg/kg of HQ, corresponding to treated diplotene, pachytene, zygotene, leptotene and preleptotene. The frequencies of cells with structural chromosome aberrations peaked at 12 days after treatment (p less than 0.01). This indicates that the preleptotene when DNA synthesis occurred was the most sensitive stage of meiotic prophase. In the second experiment the dose response was determined 12 days post treatment by applying 2 additional doses of 40 mg/kg and 120 mg/kg. The clastogenic effects induced by 40 and 80 mg/kg were significantly different from the controls (p less than or equal to 0.01) and higher than the results obtained with 120 mg/kg of HQ. A humped dose-effect relationship was observed. In a third experiment the same doses were used to analyse chromosomal aberrations in dividing spermatogonia of mice 24 h after treatment with HQ. All the administered doses gave results statistically different from the control values (p less than or equal to 0.01) and the data were fitted to a linear equation. HQ was found to be clastogenic in male mouse germ cells. It is concluded that the clastogenic effect in male germ cells is of the same order of magnitude as in mouse bone marrow cells.     

Clastogenic effects of acrylamide in mouse bone marrow cells

Acrylamide, known to induce dominant-lethal mutations (Shelby et al., 1986; Smith et al., 1986) and heritable translocations (Shelby et al., 1987) in rodent germ cells, was hitherto a questionable clastogen in rodent bone marrow (Shiraishi, 1978). Therefore, it was tested for chromosomal aberrations in mouse bone marrow cells, spermatogonia and by the micronucleus test. The intraperitoneally injected doses ranged from 50 to 150 mg/kg. In the chromosomal bone marrow test and the micronucleus assay positive results were obtained with acrylamide, and in the latter test the effect increased linearly with dose. Chromosomal aberrations were not induced in differentiating spermatogonia by the acute acrylamide treatment. Cisplatin was used as a positive control and gave the expected positive response in all 3 tests. The present results demonstrate that acrylamide is no exception among clastogens. It breaks chromosomes not only in mammalian germ cells but also in somatic cells.     

Comparative investigations on cytogenetic effects of X-irradiation on the germinal epithelium of male mice and Chinese hamsters

Summary In one short-term-experiment and one long-term-experiment spermatogonia of mice and Chinese hamsters were compared for their sensitivity of X-ray induced chromosome aberrations.Short-term-experiment: Six hours after varying doses of X-rays the spermatogonia of both species were analysed and the number of induced chromatid breaks determined. At the dose range from 25–125 R the number of induced chromatid breaks per cell per roentgen is 0.01 in mice. In Chinese hamsters this value is 0.0072.The frequencies of chromatid breaks were studied in both species after a single dose of 100 R until 48 h p.i. The frequency in mice decreased more slowly than in hamster spermatogonia. After 12 h p.i. the ratio breaks in mice cells: breaks in hamster cells was 3.5:1, after 24 h this ratio was 5.2:1 after 48 h both frequencies were on the same level.Long-term-experiment: Analysis of spermatogonia and primary spermatocytes has been done 5 weeks after irradiation of the mice and 2, and 4 months after irradiation of the Chinese hamsters. The number of observed reciprocal translocations turned out to be higher in spermatogonial mitoses than in diakinesis-metaphases I in each animal.The conclusion is drawn for mice that a selection against abnormal cells is taking place already during pre-meiosis. In hamster pre-meiosis, the results are only indicative for a similar effect.These investigations were sponsored by the Deutsche Forschungsgemeinschaft within the SFB 35 (Klinishe Genetik).     

Clastogenic effects of cis-diamminedichloroplatinum. II. Induction of chromosomal aberrations in primary spermatocytes and spermatogonial stem cells of mice

The clastogenic effect of the anticancer drug cis-diamminedichloroplatinum (II) (cisplatin) on meiotic prophase in primary spermatocytes and on spermatogonial stem cells of male (101/E1 x C3H/E1)F1 mice was studied. The intraperitoneal doses of cisplatin tested were 5.0, 7.5 and 10.0 mg/kg. Chromosomal aberrations were examined at diakinesis-metaphase 1 of meiosis 1-13 days after treatment, representing cells treated at diplotene, pachytene, zygotene, leptotene an preleptotene. Reciprocal translocations were evaluated 63-70 days after treatment, representing treated stem-cell spermatogonia. Cisplatin had a toxic effect in zygotene to preleptotene of meiosis, as indicated by the significant reduction in testicular weight. At diplotene, pachytene and zygotene no enhancement of aberrations was found. An increase in aberrant cells was observed during leptotene with preleptotene being the most sensitive stage. The dose-response relationship for aberrant cells was linear on day 13 after treatment. It is concluded that, like mitomycin C (Adler, 1976), cisplatin primarily caused aberrations during the premeiotic phase of DNA synthesis. No significant increase of translocation multivalents was found after treatment of stem-cell spermatogonia.     

Chromosomal abnormalities induced by iodine-125 in mouse germ cells

Swiss albino male mice were injected intraperitoneally with 0, 185, 370 or 555 kBq (0, 5, 10 or 15 microCi) of iodine-125 (125I). All the animals were killed on the sixtieth day and chromosomal aberrations were screened in spermatocytes at meiotic metaphase I. A significant increase in the percentage of chromosomal aberrations including translocations (0, 1.2, 1.8 and 2.3 per cent translocations in controls, 185, 370 and 555 kBq groups respectively) was recorded at all dose levels indicating the clastogenic effects of 125I in mouse spermatocytes.     

Comparison of frequencies of radiation-induced stable chromosomal aberrations in somatic and germ tissues of the mouse

Radiation-induced stable chromosome aberrations have been studied in the testes and bone-marrow of the mouse (Mus musculus). 60 days after whole-body irradiation with a dose of 400 rad X-rays, the frequency of visible chromosome aberrations in bone-marrow cells was 19.8%. The frequency of chromosome aberrations in spermatogonia of the same mice, scored as multivalents in spermatocytes, was considerably lower — only 4.7%. The possible mechanisms underlying this marked difference in translocation yield are discussed.     

Clastogenic effects of cis-diamminedichloroplatinum II. Induction of chromosomal aberrations in primary spermatocytes and spermatogonial stem cells of mice

The clastogenic effect of the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) on meiotic prophase in primary spermatocytes and on spermatologonial stem cells of male (101/E1 × C3H/E1)F₁ mice was studied. The intraperitoneal doses of cisplatin tested were 5.0, 7.5 and 10.0 mg/kg. Chromosomal aberrations were examined at diakinesis-metaphase 1 of meiosis 1–13 days after treatment, representing cells treated at diplotene, pachytene, zygotene, leptotene an preleptotene. Reciprocal translocations were evaluated 63–70 days after treatment, representing treated stem-cell spermatogonia.Cisplatin had a toxic effect in zygotene to preleptotene of meiosis, as indicated by the significant reduction in testicular weight. At diplotene, pachytene and zygotene no enhancement of aberrations was found. An increase in aberrant cells was observed during leptotene with preleptotene being the most sensitive stage. The dose-response relationship for aberrant cells was linear on day 13 after treatment. It is concluded that, like mitomycin C (Alder, 1976), cisplatin primarily caused aberrations during the premeiotic phase of DNA synthesis. No significant increase of translocation multivalents was found after treatment of stem-cell spermatogonia.     

Chromosome modifications induced in mouse spermatogonia through exposure to high-energy neutrons]

The incidence of reciprocal translocations induced in mouse spermatogonia has been studied in CBA mice given X-ray or neutron exposure. Analysis of dividing spermatocytes at diakinesis-first metaphase stage of meiosis shows that in X-irradiated mice there is a linear dose-response relationship. After exposure to fast neutrons the yield of translocations follows a humped curve with a maximum of chromosome exchanges after exposure to 100 rad.     

Evaluation and re-evaluation of genetic radiation hazards in man: II. The arm number hypothesis and the induction of reciprocal translocation in man

The arm number hypothesis proposed by Brewen and colleagues in 1973 has been examined in the light of information thus far available from mammalian studies. In experiments with peripheral blood lymphocytes (radiation in vitro), a linear relationship between dicentric yield and the effective chromosome arm number of the species was obtained in the mouse, Chinese hamster, goat, sheep, pig, wallaby and man. However, the data are not consistent with such a relationship in several primate species (marmoset, rhesus monkey, cynomolgus monkey, squirrel monkey and the slow loris), the cat and the dog. In the rabbit, the data are conflicting.In the mouse and Chinese hamster the frequencies of reciprocal translocations recorded in spermatocytes descended from irradiated spermatogonia are in line with the expectation based on the arm number hupothesis, whereas in the golden hamster, rabbit and the rhesus monkey they are not. In man and the marmoset, the limited data are not inconsistent with a 2-fold higher sensitivity of these species relative to the mouse although they do not rule out a difference as high as 4-fold. In the guinea-pig, the situation is unclear.New data on the transmission of reciprocal translocations in mice suggest that the frequency in the F₁ progeny may be close to one-quarter of that recorded in the spermatocytes of the irradiated fathers (spermatogonial irradiation) at an exposure level of 150 R, whereas at higher exposures, the reduction factor is about one-eighth, the latter being in line with the earlier finding.All these results taken together suggest that inter-specific extrapolation from the radiosensitivity of somatic cells (to dicentric induction) to that of germ cells (to translocation induction) is fraught with uncertainty at present. Certain aspects that need to be studied in more detail in the context of induced chromosome aberrations are discussed.     

Delayed Formation of Chromosome Aberrations in Mouse Pachytene Spermatocytes Treated with Triethylenemelamine (Tem)

Induction of chromosome aberrations in pachytene spermatocytes of mice by 2 mg/kg TEM was compared with induction by 400 R X rays. These doses induced comparably high dominant lethal effects in pachytene spermatocytes of mice. Cytological analysis at diakinesis–metaphase I stage showed that whereas 76.4% of the cells treated with X rays at pachytene stage had aberrations, the frequencies observed in two TEM experiments were only 0.8 and 2.2%. On the other hand, 5% of the progeny from TEM-treated pachytene spermatocytes were found to be translocation heterozygotes. This is the first report on the recovery of heritable translocations from treated spermatocytes of mice. The aberration frequencies observed for TEM in diakinesis–metaphase I were much too low to account for all the lethal mutations and heritable translocations. Thus, the formation of the bulk of aberrations induced by TEM in pachytene spermatocytes was delayed—a marked contrast to the more immediate formation of X-ray-induced aberrations. It is postulated that the formation of the bulk of TEM-induced aberrations in pachytene spermatocytes and in certain postmeiotic stages occurs sometime during spermiogenesis, and not through the operation of postfertilization pronuclear DNA synthesis.     

Action of mitomycin C on mouse spermatogonia

The induction of chromosome aberrations in mouse spermatogonia and bone marrow cells by treatment with Mitomycin (MC) was tested. The following dosages were used: 3.5; 1.75; 0.35; and 0.035 mg/kg body weight. Chromatid interchanges and terminal deletions were induced in both tissues. Regarding the chromosome damage, spermatogonia seemed to be more sensitive to the test substance than bone marrow cells.The aberrations observed were considered to represent the cause of dominant lethals induced in spermatocytes after treatment with MC by others. The squash technique adapted for examination of mitoses of mouse spermatogonia proved to be a useful tool in testing potential chemical mutagens.     

Comparative sensitivities of meiotic phophase stages in male mice to chromosome damage by acute X- and chronic gamma-irradiation

Radiation-induced multivalents, fragments and bivalent separation were studied at metaphase I in mouse spermatocytes. These cells had been irradiated with 200 rad X-rays as spermatogonia or in different stages of prophase. Radiation sensitivity increased towards the latter end of prophase with respect to multivalents and fragments. These results were compared with protracted gamma-irradiation throughout prophase.