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The increased number of cell divisions undergone by spermatogonia of older fathers cannot fully account for the observed increase in germline genetic damage. Studies have shown that the mechanisms induced in germ cells in response to oxidative damage varies with age, that DNA repair efficiency declines, and both sperm DNA damage and spontaneous mutations increase. However, it is not known whether the altered response with age is a cause, or consequence, of an age-associated change in cell susceptibility to genetic damage. Following a single 150 mg/kg dose of cyclophosphamide (CP), young (8-weeks old) and aged (17-month old) male mice were examined 24 h later for induced genetic damage in epididymal spermatozoa using the alkaline comet and sperm chromatin stability assays. Apoptosis among testicular cells was examined on tissue cross-sections using the TUNEL assay. Sperm showed no significant increase in DNA strand breaks with age (detected by the comet assay) and no change in sperm chromatin stability (detected by the SCSA assay). Following CP treatment, there was no effect on DNA-strand breakage but sperm chromatin instability was significantly higher. Furthermore, it was also significantly elevated in old treated, compared with young treated, animals suggesting that increased age affects the sensitivity of epididymal sperm to chromatin damage. There was no difference in apoptosis in testicular germ cells from either young or old control animals, while CP administration resulted in a significant increase in apoptosis among young animals but not old animals. Following genotoxin exposure, an increase in chromatin instability in the spermatozoa of old animals and a decrease in the ability of their testicular germ cells undergo apoptosis suggests an age-related decrease in genome protection mechanisms. Since those germ cells are only transiently present in the testis, it is likely that this age-related deterioration originates in the spermatogonial stem cells. The findings are also evidence that the safety evaluation of reproductive genotoxins should consider young and old individuals separately., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Male-mediated developmental toxicity has been of concern for many years. The public became aware of male-mediated developmental toxicity in the early 1990s when it was reported that men working at Sellafield might be causing leukemia in their children. Human and animal studies have contributed to our current understanding of male-mediated effects. Animal studies in the 1980s and 1990s suggested that genetic damage after radiation and chemical exposure might be transmitted to offspring. With the increasing understanding that there is histone retention and modification, protamine incorporation into the chromatin and DNA methylation in mature sperm and that spermatozoal RNA transcripts can play important roles in the epigenetic state of sperm, heritable studies began to be viewed differently. Recent reports using molecular approaches have demonstrated that DNA damage can be transmitted to babies from smoking fathers, and expanded simple tandem repeats minisatellite mutations were found in the germline of fathers who were exposed to radiation from the Chernobyl nuclear power plant disaster. In epidemiological studies, it is possible to clarify whether damage is transmitted to the sons after exposure of the fathers. Paternally transmitted damage to the offspring is now recognized as a complex issue with genetic as well as epigenetic components.

Background: Older men tend to have poorer semen quality and are generally at higher risks for infertility and abnormal reproductive outcomes., Methods: We employed proton-induced X-ray emission (PIXE, 3 MeV proton beam) to investigate the concentrations of zinc, copper, calcium, sulfur, chlorine, potassium, titanium, iron and nickel in washed sperm and seminal plasma from non-smoking groups of 10 older men (65-80 years old) and 10 younger men (22-28 years old) who were concurrently assayed for sperm function and genomicly defective sperm., Results: The older group showed elevated zinc, copper and calcium in sperm and elevated sulfur in seminal plasma compared with the younger men. The older group also showed reduced motility as well as increased sperm DNA fragmentation, achondroplasia mutations, DNA strand breaks and chromosomal aberrations. Sperm calcium and copper were positively associated with sperm DNA fragmentation (P < 0.03). Seminal sulfur was positively associated with sperm DNA fragmentation and chromosomal aberrations (P < 0.04), and negatively associated with sperm motility (P < 0.05). Sperm calcium was negatively associated with sperm motility, independent of male age (P = 0.01)., Conclusions: We identified major differences in elemental concentrations between sperm and seminal plasma and that higher sperm copper, sulfur and calcium are quantitatively associated with poorer semen quality and increased frequencies of genomic sperm defects.

Objective: To investigate whether lifestyle factors such as increased dietary intake of micronutrients reduce the risks of sperm DNA damage, and whether older men benefit more than younger men., Design: Cross-sectional study design with equalized assignments into age groups., Setting: National laboratory and university., Patient(s): Nonclinical group of 22-80-year-old nonsmoking men (n = 80) who reported no fertility problems., Main Outcome Measure(s): Sperm DNA damage measured by alkaline and neutral DNA electrophoresis (i.e., sperm Comet assay)., Result(s): Sociodemographics, occupational exposures, medical and reproductive histories, and lifestyle habits were determined by questionnaire. The average daily dietary and supplement intake of micronutrients (vitamin C, vitamin E, b-carotene, zinc, and folate) was determined using the 100-item Modified Block Food Frequency Questionnaire (FFQ). Men with the highest intake of vitamin C had approximately 16% less sperm DNA damage (alkaline sperm Comet) than men with the lowest intake, with similar findings for vitamin E, folate, and zinc (but not β-carotene). Older men (>44 years) with the highest vitamin C intake had approximately 20% less sperm DNA damage compared with older men with the lowest intake, with similar findings for vitamin E and zinc. The older men with the highest intake of these micronutrients showed levels of sperm damage that were similar to those of the younger men. However, younger men (<44 years) did not benefit from higher intakes of the micronutrients surveyed., Conclusion(s): Men with higher dietary and supplement intake of certain micronutrients may produce sperm with less DNA damage, especially among older men. This raises the broader question of how lifestyle factors, including higher intakes of antioxidants and micronutrients, might protect somatic as well as germ cells against age-associated genomic damage., (Copyright © 2012. Published by Elsevier Inc.)

Benzene is a primary industrial chemical and a ubiquitous environmental pollutant that causes human leukemia and maybe other malignancies. Occupational exposure to benzene has been associated with increased chromosomal aneuploidies in blood lymphocytes and, in separate studies, in sperm. However, aneuploidy detection in somatic and germ cells within the same benzene-exposed individuals has never been reported. To compare aneuploidies in blood lymphocytes and sperm within the same individuals exposed to benzene, a cross-sectional study was conducted in 33 benzene-exposed male workers and 33 unexposed workers from Chinese factories. Air benzene concentrations in the exposed workers ranged from below the detection limit to 24 ppm (median, 2.9 ppm) and were undetectable in the unexposed subjects. Aneuploidies of chromosomes 21, X, and Y in blood lymphocytes were examined by multicolor fluorescence in situ hybridization and were compared to the previously reported aneuploidies in sperm. The results showed that benzene exposure was positively associated with the gain of chromosome 21 but not sex chromosomes in blood lymphocytes. This was in contrast to analysis of sperm, where the gain of sex chromosomes, but not chromosome 21, was significantly increased in the exposed workers. Furthermore, a significant correlation in the gain of sex chromosomes between blood lymphocytes and sperm was observed among the unexposed subjects, but not among the exposed workers. The findings suggest that benzene exposure induces aneuploidies in both blood cells and sperm within the same individuals, but selectively affects chromosome 21 in blood lymphocytes and the sex chromosomes in sperm., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Benzene is an industrial chemical that causes blood disorders, including acute myeloid leukemia. We previously reported that occupational exposures near the U.S. Occupational Safety and Health Administration permissible exposure limit (8 hr) of 1 ppm was associated with sperm aneuploidy., Objective: We investigated whether occupational exposures near 1 ppm increase the incidence of sperm carrying structural chromosomal aberrations., Methods: We applied a sperm fluorescence in situ hybridization assay to measure frequencies of sperm carrying partial chromosomal duplications or deletions of 1cen or 1p36.3 or breaks within 1cen-1q12 among 30 benzene-exposed and 11 unexposed workers in Tianjin, China, as part of the China Benzene and Sperm Study (C-BASS). Exposed workers were categorized into low-, moderate-, and high-exposure groups based on urinary benzene (medians: 2.9, 11.0, and 110.6 µg/L, respectively). Median air benzene concentrations in the three exposure groups were 1.2, 3.7, and 8.4 ppm, respectively., Results: Adjusted incidence rate ratios (IRRs) and 95% confidence intervals (CIs) for all structural aberrations combined were 1.42 (95% CI: 1.10, 1.83), 1.44 (95% CI: 1.12, 1.85), and 1.75 (95% CI: 1.36, 2.24) and for deletion of 1p36.3 alone were 4.31 (95% CI: 1.18, 15.78), 6.02 (95% CI: 1.69, 21.39), and 7.88 (95% CI: 2.21, 28.05) for men with low, moderate, and high exposure, respectively, compared with unexposed men. Chromosome breaks were significantly increased in the high-exposure group [IRR 1.49 (95% CI: 1.10, 2.02)]., Conclusions: Occupational exposures to benzene were associated with increased incidence of chromosomally defective sperm, raising concerns for worker infertility and spontaneous abortions as well as mental retardation and inherited defects in their children. Our sperm findings point to benzene as a possible risk factor for de novo 1p36 deletion syndrome. Because chromosomal aberrations in sperm can arise from defective stem cells/spermatogonia, our findings raise concerns that occupational exposure to benzene may have persistent reproductive effects in formerly exposed workers.

Background: Benzene is a common industrial chemical known to induce leukemia and other blood disorders, as well as aneuploidy, in both human blood cells and sperm at exposures > 10 ppm. Recent reports have identified health effects at exposure levels < 1 ppm, the permissible exposure limit (PEL; 8 hr) set by the U.S. Occupational Safety and Health Administration., Objective: We investigated whether occupational exposures to benzene near 1 ppm induce aneuploidy in sperm., Methods: We used multicolor fluorescence in situ hybridization to measure the incidence of sperm with numerical abnormalities of chromosomes X, Y, and 21 among 33 benzene-exposed men and 33 unexposed men from Chinese factories. Individual exposures were assessed using personal air monitoring and urinary concentrations of benzene and trans,trans-muconic acid (E,E-MA). Air benzene concentrations were not detectable in unexposed men; in exposed men, concentrations ranged from below the detection limit to 24 ppm (median, 2.9 ppm), with 27% of exposed men (n = 9) having concentrations of

Cigarette smoking in men has been associated with increased chromosomal abnormalities in sperm and with increased risks for spontaneous abortions, birth defects and neonatal death. Little is known, however, about the reproductive consequences of paternal exposure to second-hand smoke. We used a mouse model to investigate the effects of paternal exposure to sidestream (SS) smoke, the main constituent of second-hand smoke, on the genetic integrity and function of sperm, and to determine whether male germ cells were equally sensitive to mainstream (MS) and SS smoke. A series of sperm DNA quality and reproductive endpoints were investigated after exposing male mice for two weeks to MS or SS smoke. Our results indicated that: (i) only SS smoke significantly affected sperm motility; (ii) only MS smoke induced DNA strand breaks in sperm; (iii) both MS and SS smoke increased sperm chromatin structure abnormalities; and (iv) MS smoke affected both fertilization and the rate of early embryonic development, while SS smoke affected fertilization only. These results show that MS and SS smoke have differential effects on the genetic integrity and function of sperm and provide further evidence that male exposure to second-hand smoke, as well as direct cigarette smoke, may diminish a couple's chance for a successful pregnancy and the birth of a healthy baby.

Background: The trend for men to have children at older age raises concerns that advancing age may increase the production of genetically defective sperm, increasing the risks of transmitting germ-line mutations., Methods: We investigated the associations between male age and sperm DNA damage and the influence of several lifestyle factors in a healthy non-clinical group of 80 non-smokers (mean age: 46.4 years, range: 22-80 years) with no known fertility problems using the sperm Comet analyses., Results: The average percentage of DNA that migrated out of the sperm nucleus under alkaline electrophoresis increased with age (0.18% per year, P = 0.006), but there was no age association for damage measured under neutral conditions (P = 0.7). Men who consumed >3 cups coffee per day had approximately 20% higher percentage tail DNA under neutral but not alkaline conditions compared with men who consumed no caffeine (P = 0.005)., Conclusions: Our findings indicate that (i) older men have increased sperm DNA damage associated with alkali-labile sites or single-strand DNA breaks and (ii) independent of age, men with substantial daily caffeine consumption have increased sperm DNA damage associated with double-strand DNA breaks. DNA damage in sperm can be converted to chromosomal aberrations and gene mutations after fertilization, increasing the risks of developmental defects and genetic diseases among offspring.

The father, like the mother, can transmit genetic defects to his offspring that are detrimental for normal development and a healthy life. Epidemiological studies have identified associations between several paternal exposures and abnormal reproductive outcomes, but these types of studies are inherently complex and expensive, and the risk factors for the paternal contribution to abnormal reproductive outcomes remain poorly understood. Several sensitive methods have been developed for detecting mutations and chromosomal damage directly in sperm. These assays are potential bioindicators for paternal risk factors for infertility, spontaneous abortions, aneuploidy syndromes, and genetic diseases in children. Among these methods, fluorescence in situ hybridization (FISH) has been adapted for the detection of numerical and structural chromosomal abnormalities in the sperm of an expanding number of species, including humans and rodents. Sperm FISH has identified several potential paternal risk factors such as age, drugs, lifestyles, and various environmental/occupational exposures. Here, we summarize the status of the development and usage of these sperm-FISH assays and suggest strategies for prioritizing chemical agents for epidemiological investigations to assess paternal risk for abnormal reproductive outcome.

Some chemotherapy regimens include agents that are mutagenic or clastogenic in model systems. This raises concerns that cancer survivors who were treated before or during their reproductive years may be at increased risks for abnormal reproductive outcomes. However, the available data from offspring of cancer survivors are limited, representing diverse cancers, therapies, time to pregnancies, and reproductive outcomes. Rodent breeding data after paternal exposures to individual chemotherapeutic agents illustrate the complexity of factors that influence the risk for transmitted genetic damage including agent, dose, end point, and germ cell susceptibility profiles that vary across agents. Direct measurements of chromosomal abnormalities in sperm of mice and humans by sperm fluorescent in situ hybridization have corroborated the differences in germ cell susceptibilities. The available evidence indicates that the risk of producing chromosomally defective sperm is highest during the first few weeks after the end of chemotherapy and decays with time. Thus, sperm samples provided immediately after the initiation of cancer therapies may contain treatment-induced genetic defects that will jeopardize the genetic health of offspring.

In recent years, two techniques for detecting genetic damage in the whole genome have gained importance: the alkaline comet assay, to detect DNA damage such as strand breaks and alkali-labile sites, and a multicolour FISH method, spectral karyotyping (SKY), to identify chromosomal aberrations simultaneously in all metaphase chromosomes. In the present study, the induction of DNA damage in human sperm and lymphocytes in vitro has been studied employing an anticancer drug, doxorubicin (DX). An increase in DNA damage was observed with the comet assay as the median per cent head DNA of sperm significantly decreased from 82.07 and 85.14% in the untreated control groups to 63.48 and 72.52% at doses of 0.8 micro M DX. At 1.6 micro M the percentage declined to 60.96% (the corresponding tail moment increased from 4.42 to 12.19). In stimulated lymphocytes, a significant increase was observed in tail moment, from 0.72 and 0.53 in controls to 15.17 and 12.10 at 0.2 micro M DX, continuing at the same level to a final concentration of 1.6 micro M. Structural aberrations found in the parallel SKY study in stimulated lymphocytes at 0.2 micro M DX consisted of 14% chromatid-type and 2% chromosome-type aberrations; none were found in controls. The SKY results correlate very well with the findings of the comet assay in lymphocytes where DNA damage was observed at similar doses. This study is the first reporting use of the comet assay and SKY analysis in parallel after chemical treatment. The potential of the two techniques together is evident, as they represent a set of assays feasible for evaluating damage in human somatic and germ cells after chemical treatment (i) by direct observation of two different end-points, detecting general DNA damage and chromosomal aberrations and (ii) by extrapolation from lymphocytes to sperm, which provides a 'parallelogram' approach in human cells.

Background: Modern reproductive technologies are enabling the treatment of infertile men with severe disturbances of spermatogenesis. The possibility of elevated frequencies of genetically and chromosomally defective sperm has become an issue of concern with the increased usage of ICSI, which can enable men with severely impaired sperm production to father children. Several papers have been published reporting aneuploidy in oligozoospermic patients, but relatively little is known about chromosome structural aberrations in the sperm of these patients., Methods: We examined sperm from infertile, oligozoospermic individuals for structural and numerical chromosomal abnormalities using a multicolour ACM fluorescence in situ hybridization (FISH) assay that utilizes DNA probes specific for three regions of chromosome 1 to detect human sperm that carry numerical chromosomal abnormalities plus two categories of structural aberrations: duplications and deletions of 1pter and 1cen, and chromosomal breaks within the 1cen-1q12 region., Results: There was a significant increase in the average frequencies of sperm with duplications and deletions in the infertility patients compared with the healthy concurrent controls. There was also a significantly elevated level of breaks within the 1cen-1q12 region. There was no evidence for an increase in chromosome 1 disomy, or in diploidy., Conclusions: Our data reveal that oligozoospermia is associated with chromosomal structural abnormalities, suggesting that oligozoospermic men carry a higher burden of transmissible, chromosome damage. The findings raise the possibility of elevated levels of transmissible chromosomal defects following ICSI treatment.

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.

Background: The possibility that oligozoospermic men may have elevated levels of genetic damage in their sperm is of particular concern as they could transmit defects to their offspring., Methods: Sperm samples were obtained from 12 infertile, oligozoospermic patients and 12 healthy normozoospermic volunteers. Fluorescence in-situ hybridization (FISH) was used to determine aneuploidy rates in sperm and inverse restriction site mutation (iRSM) assay to determine gene mutations; defective chromatin packaging was quantified by sperm chromatin structure assay (SCSA) and DNA strand breaks by the Comet assay., Results: FISH analysis showed a significant increase in gonosomal X,Y,18 (P < 0.01) disomy and diploid sperm with X,Y,18,18 (P < 0.05) in the infertility patients compared with the controls. A significant increase (P < 0.01) in disturbed sperm chromatin was found in the infertility patients compared with the control group using the SCSA assay. In the Comet assay, a significant increase (P < 0.01) in the tail moment was found in the infertility patients compared with the control group, indicating significantly high levels of DNA strand breaks. There was no significant increase in point mutations detected by iRSM assay., Conclusions: The data indicate that infertile oligozoospermic men have an elevated level of XY aneuploidy and XY diploidy in the germ-line, as well as elevated levels of sperm chromatin disturbances and sperm DNA strand breaks. These data demonstrate that oligozoospermic infertility patients show several different types of genetic damage in their sperm. Thus, such men appear to have defects at a variety of levels of spermatogenesis and their infertility may not just be a result of the oligozoospermia.

The spontaneous mutation rate in the male germ-line increases with age. The reason for this is unknown, but presumably involves an age-related degeneration in the efficacy of cellular processes. To investigate the possibility that rates of apoptosis and genetic damage (represented by aneuploidy) might vary with age in mice, the testes and sperm of 2- and 12-month-old male MF-1 mice were examined by a modified TUNEL technique and 3-colour sperm-FISH assay, respectively. Sperm were labeled with probes to chromosomes 8, X and Y and 20,000 sperm scored from each of 5 animals per group. A significant increase in gonosomal disomy was found in the aged mice, especially X-X-8. This suggests that advanced paternal age is associated primarily with meiosis II rather than meiosis I disjunction errors. Neither diploidy nor autosomal disomy was affected in the older group. The rate of germ cell apoptosis (apoptotic cells per seminiferous tubule cross-section per animal per group) was higher in the old mice than controls, but not significantly. Considerable inter-animal variability was observed in the older group. The finding of an increase in levels of sperm aneuploidy is novel for 1-year-old mice and confirms the genotoxic effect of ageing in mice. Since apoptosis is assumed to eliminate cells with unrepaired damage, it may be that the apoptotic response in older mice is compromised, resulting in the higher levels of aneuploidy in sperm. However, given the inter-animal variability in testicular germ cell apoptosis, this awaits confirmation., (Copyright 2003 Wiley-Liss, Inc.)

Multicolour fluorescence in situ hybridization (FISH) with chromosome-specific DNA-probes can be used to assess aneuploidy (disomy) and diploidy in sperm of any species provided the DNA-probes are available. In the present EU research project, DNA-probes for mouse chromosomes 8, X and Y were employed each labelled with different colours. Male mice were treated with the test chemicals and sperm were sampled from the Caudae epididymes 22-24 days later to allow spermatocytes exposed during meiosis to develop into mature sperm. At present, the data base comprises 10 chemicals: acrylamide (AA), carbendazim (CB), colchicine (COL), diazepam (DZ), griseofulvin (GF), omeprazole (OM), taxol (TX), thiobendazole (TB), trichlorfon (TF) and vinblastine (VBL). Of these, COL and TF induced disomic sperm only. DZ and GF induced disomic and diploid sperm, while CB and TB induced diploid sperm only. VBL gave contradictory results in repeated experiments in an inter-laboratory comparison. AA, OM and TX did not induce an increase in disomic or diploid sperm at the doses used. The induction of aneuploidy by DZ was also tested in humans. Sperm samples from patients after attempted suicide and from patients with chronic Valium((R)) abuse were evaluated using human DNA-probes specific for chromosomes 1,16, 21, X and Y. A quantitative comparison between mouse and man indicates that male meiosis in humans is 10-100 times more sensitive than in mice to aneuploidy induction by DZ. The positive response of mice to TF supports the hypothesis by Czeizel et al. [Lancet 341 (1993) 539] that TF may be causally related to the occurrence of congenital abnormality clusters in a Hungarian village.

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.

Background: Laser-scanning cytometry (LSC) allows fast automated scoring of fluorescence signals directly on microscopic slides. Frequencies of spontaneous aneuploidies in murine and human sperm were evaluated by using this new LSC technique. Rapid detection may be of great interest in reproductive toxicology, as certain chemicals act as aneugens during meiosis, increasing the production of aneuploid germ cells. Materials and Methods Selected chromosomes were detected by using fluorescence in situ hybridization (FISH) and fluorochrome-labeled DNA-probes. Sperm chromatin was counterstained with propidium iodide. By scanning across the slide, fluorescence signals within sperm nuclei were detected and counted., Results: In murine sperm, the frequencies of disomies for chromosomes 8 and X were 0.019% and 0.021%, respectively. The automated assessment in human sperm resulted in disomy frequencies of 0.061% and 0.090% for chromosomes 13 and X, respectively. These results were comparable to data obtained from the same samples by manual microscopic scoring and to literature data., Conclusions: Frequencies of genotypically abnormal sperm were not significantly different between automated and manual scoring. In conclusion, sperm aneuploidy was reliably determined and disomic sperm were successfully relocated by LSC. By virtue of rapid and reliable analyses, LSC has the powerful potential to replace manual microscopic FISH analysis in molecular cytogenetics., (Copyright 2001 Wiley-Liss, Inc.)

The mouse epididymal sperm aneuploidy (mESA) assay using 3-chromosome fluorescence in situ hybridization (FISH) was recently developed for assessing the aneugenic potential of chemicals on male germ cells. This study was designed to identify the major technical factors that affect inter-scorer and inter-laboratory variability of the mESA assay. Two laboratories participated in this study (GSF and Lawrence Livermore National Laboratory, LLNL). Mice (102/ElxC3H/El) F(1) were exposed in one laboratory (GSF) to vinblastine (VBL; single intraperitoneal injection of 0, 0.5, 1.0 or 2.0 mg/kg), one of the 10 priority compounds of the Commission of the European Communities (CEC) Aneuploidy Program. Twenty-two days later the mESA assay was applied to analyze sperm aneuploidy. In the initial evaluation, small but statistically significant differences were found between the two laboratories in baseline frequencies and there was also disagreement in the determination of a VBL aneuploid effect. Therefore, experiments were conducted to identify the sources of the inter-laboratory differences and technical factors that affected assay reliability and the VBL study was repeated. A harmonization experiment was conducted by bringing the microscope scorers from both laboratories to the same site (LLNL) for a cross-training exercise. Following this exercise, a second group of VBL-treated and control mice were evaluated, and we concluded that VBL is not a sperm aneugen. Our research has identified scoring criteria as the major source of inter-laboratory variation and emphasizes the importance of strict technical controls for the mESA assay, including controlling slide preparations for treatment-induced reductions in sperm count, coding of slides and selection of statistical tests. These considerations are particularly important for the interpretation of small effects (< or =2-fold) on sperm aneuploidy. Our findings suggest that 2-fold differences in frequencies can result from differences among scorers, samples and treatment groups, and are readily within the normal variation for the mESA assay. Such small differences should be viewed with caution until independently confirmed.

Aneuploidy induction in male germ cells of mice and men after chronic exposure to diazepam (DZ; CAS 439-14-5; Valium was assessed by multicolor fluorescence in situ hybridization (FISH). DZ, a widely administered sedative and muscle relaxant, was proposed to act as an aneugen by disturbing spindle function in various assay systems. Male mice were treated by oral intubation with 3mg/kg DZ once or daily for 14 consecutive days. At 22 days after the last treatment, epididymal sperm were collected from the caudae epididymes. Evaluation of aneuploid and diploid sperm (10,000 sperm per animal) was performed by multicolor FISH employing DNA probes specific for chromosomes X, Y, and 8 simultaneously. We found a significant increase in the frequency of disomy 8 in subchronically DZ-treated mice when compared to the concurrent solvent control group (2.4-fold; P<0.01), while no increase was detected for sex-chromosome hyperhaploidies. No effect was seen when mice were treated with a single dose (3mg/kg DZ). In a parallel human approach, two men were evaluated who chronically ingested >0.3mg/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, disomy X, and total sex-chromosomal disomies were found to be elevated among the two subjects after chronic DZ-exposure compared to control subjects. In conclusion, the results indicate that diazepam acts as an aneugen during meiosis in male spermatogenesis, both in mice and humans. The quantitative comparison indicates that humans may be at least 10 times more sensitive than mice for aneuploidy induction by DZ during male meiosis.

Griseofulvin (GF) was tested in male mouse germ cells for the induction of meiotic delay and aneuploidy. Starved mice were orally treated with 500, 1000 and 2000 mg/kg of GF in corn oil and testes were sampled 22 h later for meiotic delay analysis and chromosome counting in spermatocytes at the second meiotic metaphase (MMII). A dose-related increase in meiotic delay by dose-dependently arresting spermatocytes in first meiotic metaphase (MMI) or/and prolonging interkinesis was observed. Hyperhaploid MMII cells were not significantly increased. Sperm were sampled from the Caudae epididymes 22 days after GF-treatment of the males for three-color fluorescence in situ hybridization (FISH). 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, 1000 and 2000 mg/kg of GF. The frequencies of disomic sperm were increased significantly above the controls in all GF-treated groups but showed no dose response. The data for individual classes of disomic sperm indicated that MII was more sensitive than MI to GF-induced non-disjunction in male mice. 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., (Copyright 1999 Elsevier Science B.V.)

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.

This study was designed to evaluate the frequency of aneuploid sperm in young adult mice of the genotype (102/E1 x C3H/E1)F1 determined by the fluorescence in situ hybridization (FISH) procedure and to evaluate the frequencies of aneuploid sperm observed by FISH compared with the frequencies of aneuploid offspring. Three-chromosome FISH was applied to determine the fractions of hyperhaploid and diploid sperm with DNA probes specific for chromosomes X, Y and 8. The animals were treated with three common solvents. Sperm smears were prepared for FISH by two similar protocols and were scored by different persons and in two different laboratories. There were no significant differences between scorers or laboratories. The frequencies of the sex chromosome aneuploidies in sperm (Y-Y and X-Y) were compared to the frequencies of mice carrying sex chromosome aneuploidy among controls of the heritable translocation assay in studies conducted from 1975-1995. To identify aneuploid individuals, untreated males and females of the genotype (102/E1 x C3H/E1)F1 were mated to assess their fertility by observing three consecutive litters. Semisterile and sterile animals were further analysed by meiotic cytogenetics and by karyotyping to determine the incidence of reciprocal translocations and sex chromosome aneuploidies (XXY and XYY). Based on the analysis of 175247 sperm and 9840 progeny, the frequency of Y-Y sperm was 0.01% while 0.03% of the offspring were XYY. The frequency of X-Y sperm was 0.005% while 0.02% of the offspring were XXY. The frequencies of aneuploid sex chromosomes were not significantly different between sperm and offspring. This allows two conclusions. First, there was no detectable prenatal selection against these sex-chromosomal aneuploid offspring, and second, germ cell aneuploidy can be reliably determined in mice by sperm FISH analyses.