Induction of aneuploidy in male mouse germ cells detected by the sperm-FISH assay: a review of the present data base

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.     

多色荧光原位杂交检测小鼠精子非整倍体

比多色荧光原位杂交技术评价了２－（４－噻唑）苯丙咪唑（ＴＢ）在雄性小鼠生殖形成过程中的非整倍体诱发效应。根据小鼠精子发育周期,以口饲法处理动物１１天,间隔２２天后取小鼠精子涂片,聚合应用８号,Ｘ及Ｙ染色体特异性ＤＮＡ探针进行多色ＦＩＳＨ,检测精子中出现的二倍体、双体、和缺本频率。结果发现：在２００ｍｇ／ｋｇ剂量组,上述３类异常精子频率均显著高于溶剂对照,其他２个剂量组的非整倍体精子频率与对照无显著     

Analysis of aneuploidy frequencies in sperm from patients with hereditary nonpolyposis colon cancer and an hMSH2 mutation

Hereditary nonpolyposis colon cancer (HNPCC) has been shown to be caused by mutations in the mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2. Recent evidence has demonstrated that mutations in mismatch repair genes disrupt meiosis in mice. A large HNPCC kindred in Newfoundland, Canada, has an hMSH2 mutation-an A-->T transversion at the +3 position of the splice-donor site of exon 5. We have studied sperm from men with this hMSH2 mutation, since it is possible that mismatch repair mutations in humans might also have an effect on meiosis and normal segregation of chromosomes. The frequencies of aneuploid and diploid sperm were determined in 10 men with the hMSH2 mutation, by use of multicolor FISH analysis for chromosomes 13, 21, X, and Y. A minimum of 10,000 sperm per man was studied per chromosome probe. Control individuals consisted of men in the same kindred with HNPCC who did not carry the mutation and of other normal men from Newfoundland. A total of 321,663 sperm were analyzed: 200,905 sperm were from men carrying the hMSH2 mutation and 120,758 sperm were from control men. There was a significantly increased frequency of disomy 13, disomy 21, XX, and diploidy in mutation carriers compared with control men. These results suggest that the hMSH2 mutation may affect meiosis in humans.     

Meiotic behaviour of sex chromosomes investigated by three-colour FISH on 35 142 sperm nuclei from two 47,XYY males

Meiotic segregation of sex chromosomes from two fertile 47,XYY men was analysed by a three-colour fluorescence in situ hybridisation procedure. This method allows the identification of hyperhaploidies (spermatozoa with 24 chromosomes) and diploidies (spermatozoa with 46 chromosomes), and their meiotic origin (meiosis I or II). Alpha-satellite probes specific for chromosomes X, Y and 1 were observed simultaneously in 35 142 sperm nuclei. For both 47,XYY men (24 315 sperm nuclei analysed from one male and 10 827 from the other one) the sex ratio differs from the expected 1:1 ratio (P < 0.001). The rates of disomic Y, diploid YY and diploid XY spermatozoa were increased for both 47,XYY men compared with control sperm (142 050 sperm nuclei analysed from five control men), whereas the rates of hyperhaploidy XY, disomy X and disomy 1 were not significantly different from those of control sperm. These results support the hypothesis that the extra Y chromosome is lost before meiosis with a proliferative advantage of the resulting 46,XY germ cells. Our observations also suggest that a few primary spermatocytes with two Y chromosomes are able to progress through meiosis and to produce Y-bearing sperm cells. A theoretical pairing of the three gonosomes in primary spermatocytes with an extra sex chromosome, compatible with active spermatogenesis, is proposed. Received: 12 April 1996 / Revised: 26 August 1996     

Individual variation in the frequency of sperm aneuploidy in humans

To examine interindividual differences in sperm chromosome aneuploidy, repeated semen specimens were obtained from a group of ten healthy men, aged 20-21 at the start of the study, and analyzed by multi-color fluorescence in situ hybridization (FISH) analysis to determine the frequencies of sperm aneuploidy for chromosomes X, Y, 8, 18 and 21 and of diploidy. Semen samples were obtained three times over a five-year period. Statistical analysis examining the stability of sperm aneuploidy over time by type and chromosome identified two men who consistently exhibited elevated frequencies of sperm aneuploidy (stable variants): one with elevated disomy 18 and one with elevated MII diploidy. Differences among frequencies of aneuploidy by chromosome were also seen. Overall, disomy frequencies were lower for chromosome X, 8 and 18 than for chromosomes 21 or Y and for XY aneuploidy. The frequency of chromosome Y disomy did not differ from XY sperm frequency. Also, the frequency of meiosis I (XY) and II (YY + XX) sex chromosome errors did not differ in haploid sperm, but the frequency of MII errors was lower than MI errors in diploid sperm. Frequencies of sperm aneuploidy were similar between the first sampling period and the second, two years later. However, the frequency of some types of aneuploidy (XY, disomy Y, disomy 8, total autosomal disomies, total diploidy, and subcategories of diploidy) increased significantly between the first sampling period and the last, five years later, while others remained unchanged (disomy X, 21 and 18). These findings confirm inter-chromosome differences in the frequencies of disomy and suggest that some apparently healthy men exhibit consistently elevated frequencies of specific sperm aneuplodies. Furthermore, time/age-related changes in sperm aneuploidy may be detected over as short a period as five years in a repeated-measures study.     

Estimates of sperm sex chromosome disomy and diploidy rates in a 47,XXY/46,XY mosaic Klinefelter patient

A 47,XXY/46,XY male was investigated for the incidence of aneuploidy in sperm sex chromosomes using a three-colour X/Y/18 fluorescence in situ hybridisation (FISH) protocol. A total of 1701 sperm nuclei were analysed. The ratio of X-bearing to Y-bearing sperm did not differ from the expected 1 : 1 ratio although there were more 23,Y sperm than 23,X sperm (844 vs 795). There was a significantly increased proportion of disomy XY and XX sperm compared with normal controls (0.41% vs 0.10%, P < 0.001 and 0.29% vs 0.04%, P < 0.01). However, the incidence of YY sperm was similar to the controls (0.06% vs 0.02%). The diploidy rate was also significantly increased (1.7% vs 0.13%, P < 0.0001), as was disomy 18 (0.71% vs 0.01%) and 25,XXY (0.47% vs 0%). The results support the hypothesis that some 47,XXY cells are able to undergo meiosis and produce mature spermatozoa. Patients with mosaic Klinefelter syndrome with severe oligozoospermia have significantly elevated incidences of disomy XY and XX sperm and may be at a slightly increased risk of producing 47,XXX and 47,XXY offspring. Additionally, they may be at risk of producing offspring with autosomal trisomies. Hence, patients with Klinefelter mosaicism scheduled for intracytoplasmic sperm injection intervention should first undergo FISH analysis of their sperm to determine their risk. Received: 16 November 1998 / Accepted: 16 February 1999     

Orthovanadate increased the frequency of aneuploid mouse sperm without micronucleus induction in mouse bone marrow erythrocytes at the same dose level

The objective of the current study was to investigate the ability of orthovanadate to induce aneuploidy in mouse sperm and micronuclei in mouse bone marrow cells at the same dose levels. The BrdU-incorporation assay was performed to test if the chemical treatment altered the duration of the meiotic divisions. It was found that orthovanadate (25mg/kg bw) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated by intraperitoneal (i.p.) injection with 5, 15 or 25mg/kg bw orthovanadate and sperm were sampled from the Caudae epididymes 22 days later. Fluorescence in situ hybridization (FISH) was performed with DNA-probes for chromosomes 8, X or Y. Significant increases in the frequencies of total hyperhaploid sperm (p<0.01) were found with 15 and 25mg/kg bw orthovanadate, indicating induced non-disjunction during male meiosis. The dose-response was described best by a linear equation. Orthovanadate did not significantly increase the frequencies of diploid sperm at any of the three doses tested, indicating that no complete meiotic arrest occurred. Orthovanadate was investigated also by the micronucleus test at i.p. doses of 1, 5, 15 or 25mg/kg bw, followed by bone marrow sampling 24h after treatment. None of the orthovanadate doses caused a significant increase in the rates of micronuclei (MN). Since the results show that orthovanadate induced non-disjunction during male meiosis without an accompanying induction of MN in bone marrow erythrocytes under the present experimental conditions and doses, it is concluded that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis). However, induction of micronuclei was reported in the literature with orthovanadate, vanadylsulfate and ammonium metavanadate, which contradicts the notion that vanadium compounds might be unique germ cell aneugens.     

Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) with single-color chromosome-specific probes was used to study the rates of disomy for chromosome 1, 16, X, and Y in sperm of fertile and infertile subjects. Diploidy rates were studied using a two-color cocktail of probes for chromosomes 17 and 18 in the same sperm samples. Two-color methodology was not available at the outset of the study. A total of 450,580 spermatozoa were studied from 21 subjects (9 fertile, 12 infertile). Significant differences were observed in the disomy rates between chromosomes with the highest frequency observed for chromosome 16 (0.17%) and the lowest for the Y chromosome (0.10%). No differences were observed between fertile and infertile subjects for either diploidy or disomy. Total disomy rates for chromosomes 1, 16, X and Y ranged from 0.34% to 0.84% among infertile subjects, and 0.32% to 0.61% among fertile subjects. Our data suggest that generalized aneuploidy in sperm is not a major contributor to unexplained infertility.     

Reliability of aneuploidy estimates in human sperm: Results of fluorescence in situ hybridization studies using two different scoring criteria

Aneuploidy estimates for chromosomes 1, 12, X, and Y were obtained in human sperm from five donors using multicolor fluorescence in situ hybridization (FISH) analysis. Disomy frequencies were obtained by scoring a minimum of 10,000 sperm for each chromosome probe per donor. This analysis was replicated for two scoring criteria: one used one half of a signal domain as the minimum distance between two signals to be counted as two and thus disomic; the other set one signal domain as the minimum distance between two signals. A total of 120,870 sperm were assessed using one half of a domain as the criterion, and 113,478 were scored using one domain as the criterion. The percentage of disomy for chromosomes 1, 12, X, Y, and XY was 0.18, 0.16, 0.15, 0.19, and 0.25, respectively, using the one-half-domain criterion, and 0.08, 0.17, 0.07, 0.12, and 0.16, respectively, using the one-domain criterion. The percentage of disomy decreased significantly with use of one domain as the minimum distance for signal separation for all chromosomes except for chromosome number 12. These lower disomy frequencies correlated well with frequencies derived from human sperm karyotypes analyzed in our laboratory. This suggests that the fluorescent signals for chromosomes 1, X, and Y split into more than one domain in decondensed interphase sperm, and that the use of the one-half-domain criterion would lead to an overestimate of aneuploidy frequencies. The factors known to affect aneuploidy estimates derived from FISH studies are discussed, and recommendations for stringent scoring criteria are proposed. © 1995 wiley-Liss, Inc.     

Increased incidence of hyperhaploid 24,XY spermatozoa detected by three-colour FISH in a 46,XY/47,XXY male

Meiotic segregation of gonosomes from a 46,XY/47,XXY male was analysed by a three-colour fluorescence in situ hybridisation (FISH) procedure. This method allows the identification of hyperhaploid spermatozoa (with 24 chromosomes), diploid spermatozoa (with 46 chromosomes) and their meiotic origin (meiosis I or 11). Alpha satellite DNA probes specific for chromosomes X, Y and 1 were observed on 27,097 sperm nuclei. The proportions of X-and Y -bearing sperm were estimated to 52.78% and 43.88%, respectively. Disomy (24,XX, 24,YY, 24,X or Y,+1) and diploidy (46,XX, 46,YY, 46,XY) frequencies were close to those obtained from control sperm, whereas the frequency of hyperhaploid 24,XY spermatozoa (2.09%) was significantly increased compared with controls (0.36%). These results support the hypothesis that a few 47,XXY germ cells would be able to complete meiosis and to produce mature spermatozoa.     

Analysis of aneuploidy for chromosomes 13, 21, X and Y by multicolour fluorescence in situ hybridisation (FISH) in a 47,XYY male.

The frequency of aneuploid sperm was assessed by fluorescence in situ hybridisation (FISH) in a 47,XYY male previously studied by sperm karyotyping. A total of 20,021 sperm were studied: 10,017 by two-colour FISH for chromosomes 13 and 21 and 10,002 by three-colour FISH for the sex chromosomes using chromosome 1 as an autosomal control for diploidy and lack of hybridisation. Results were compared with more than 500,000 sperm from 18 normal men. The frequencies of X-bearing (49.4%) and Y-bearing sperm (49.8%) were not significantly different from 50% as shown in our sperm karyotyping study. There was no significant increase in the frequency of diploid sperm compared with control donors. There was a significant increase in the frequency of disomy for chromosome 13 (p < 0.0001) and XY disomy (p = 0.0008) compared with control donors. However, since the frequency of disomy was 0.40% for chromosome 13 and 0.55% for XY disomy, it is not surprising that these increases were not discovered previously in our analysis of 75 sperm karyotypes. Our results suggest that the extra Y chromosome is eliminated during spermatogenesis in the majority of cells but that there may be a small but significant increase in the frequency of aneuploid sperm in these men.     

CENP-B is not critical for meiotic chromosome segregation in male mice.

Centromere protein B (CENP-B) is a constitutive protein that binds to a highly conserved 17bp motif located at most mammalian centromeres. To determine whether disruption of this gene affects chromosome segregation in male germ cells, we evaluated the frequencies of disomic and diploid sperm in CENP-B heterozygous and homozygous null mice using the mouse epididymal sperm aneuploidy (m-ESA) assay, a multicolor FISH method with probes for chromosomes X, Y and 8. The specificity and sensitivity of the m-ESA assay was demonstrated using Robertsonian (2.8) translocation heterozygotes as positive controls for sperm aneuploidy. Our results show that the frequencies of disomic and diploid sperm did not differ significantly between CENP-B heterozygous and homozygous null mice (P> or = 0.5) or from 129/Swiss isogenic mice (P> or = 0.5) and B6C3F1 mice (P> or = 0.2). These findings indicate that CENP-B does not have an essential role during chromosome segregation in male meiosis.     

Cigarette smoking and aneuploidy in human sperm

Cigarette smoke contains chemicals which are capable of inducing aneuploidy in experimental systems. These chemicals have been shown to reach the male reproductive system, increasing oxidative DNA damage in human sperm and lowering semen quality. We have examined the association between smoking and aneuploid sperm by studying 31 Chinese men with similar demographic characteristics and lifestyle factors except for cigarette smoking. None of the men drank alcohol. These men were divided into three groups: nonsmokers (10 men), light smokers (< 20 cigarettes/day, 11 men), and heavy smokers (> or = 20 cigarettes/day, 10 men). There were no significant differences in semen parameters or in age across groups. Two multi-color fluorescence in situ hybridizations (FISH) were performed: two-color FISH for chromosomes 13 and 21, and three-color FISH for the sex chromosomes using chromosome 1 as an internal autosomal control for diploidy and lack of hybridization. The mean hybridization efficiency was 99.78%. The frequency of disomy 13 was significantly higher in light and heavy smokers than in non-smokers, while no significant differences in the frequency of disomy 21, X or Y were observed across groups. Significant inter-donor heterogeneity in every category of disomic sperm examined was found in both light and heavy smokers, while in nonsmokers only XY disomy showed significant inter-donor differences. Thus, we conclude that cigarette smoking may increase the risk of aneuploidy only for certain chromosomes and that men may have different susceptibilities to aneuploidy in germ cells induced by cigarette smoking. Mol. Reprod. Dev. 59: 417-421, 2001.     

Frequency of XY sperm increases with age in fathers of boys with Klinefelter syndrome

With increasing availability of drugs for impotence and advanced reproductive technologies for the treatment of subfertility, more men are fathering children at advanced ages. We conducted a study of the chromosomal content of sperm of healthy men aged 24-57 years to (a) determine whether father's age was associated with increasing frequencies of aneuploid sperm including XY, disomy X, disomy Y, disomy 21, and sperm diploidy, and (b) examine the association between the frequencies of disomy 21 and sex-chromosomal aneuploidies. The study group consisted of 38 fathers of boys with Klinefelter syndrome (47, XXY) recruited nationwide, and sperm aneuploidy was assessed using multicolor X-Y-21 sperm FISH ( approximately 10,000 sperm per donor). Paternal age was significantly correlated with the sex ratio of sperm (Y/X; P=.006) and with the frequency of XY sperm (P=.02), with a clear trend with age by decades (P<.006). Compared with fathers in their 20s (who had an average frequency of 7.5 XY sperm per 10,000), the frequencies of XY sperm were 10% higher among fathers in their 30s, 31% higher among those in their 40s, and 160% higher among those in their 50s (95% CI 69%-300%). However, there was no evidence for age effects on frequencies of sperm carrying nullisomy sex; disomies X, Y, or 21; or meiosis I or II diploidies. The frequencies of disomy 21 sperm were significantly associated with sex-chromosomal aneuploidy (P=.04)-in particular, with disomy X (P=.004), but disomy 21 sperm did not preferentially carry either sex chromosome. These findings suggest that older fathers produce higher frequencies of XY sperm, which may place them at higher risk of fathering boys with Klinefelter syndrome, and that age effects on sperm aneuploidy are chromosome specific.     

Aneuploidy in pig sperm: multicolor fluorescence in situ hybridization using probes for chromosomes 1, 10, and Y.

The objective of this research was to develop chromosome-specific probes for use in evaluating aneuploidy in boar spermatozoa through the application of fluorescence in situ hybridization (FISH) technology. A multicolor FISH method was developed to detect aneuploidy in the sperm of boars using DNA probes specific for small regions of chromosomes 1, 10, and Y. The average frequencies of sperm with disomy for chromosomes 1, 10, and Y were 0.075%, 0.067%, and 0.094%, respectively. The incidence of disomy did not differ significantly by chromosome. The average frequencies of diploidy were 0.177% for 1-1-10-10 and 0.022% for Y-Y-10-10. Thus, the incidence of overall diploidy (1-1-10-10) was significantly higher than that of disomy for the chromosomes examined (P < 0.01 for disomy of the autosomes and P < 0.05 for disomy of the Y chromosome). No significant age or breed effects on disomy and diploidy rates and no significant interindividual variations in disomy or diploidy were found. The observed level of numerical chromosome aberrations in pig sperm appear to be within the range of the baseline frequencies reported so far in men.     

CENP-B is not critical for meiotic chromosome segregation in male mice

Centromere protein B (CENP-B) is a constitutive protein that binds to a highly conserved 17 bp motif located at most mammalian centromeres. To determine whether disruption of this gene affects chromosome segregation in male germ cells, we evaluated the frequencies of disomic and diploid sperm in CENP-B heterozygous and homozygous null mice using the mouse epididymal sperm aneuploidy (m-ESA) assay, a multicolor FISH method with probes for chromosomes X, Y and 8. The specificity and sensitivity of the m-ESA assay was demonstrated using Robertsonian (2.8) translocation heterozygotes as positive controls for sperm aneuploidy. Our results show that the frequencies of disomic and diploid sperm did not differ significantly between CENP-B heterozygous and homozygous null mice (P≥0.5) or from 129/Swiss isogenic mice (P≥0.5) and B6C3F1 mice (P≥0.2). These findings indicate that CENP-B does not have an essential role during chromosome segregation in male meiosis.     

Risk assessment and segregation analysis in a pericentric inversion inv6p23q25 carrier using FISH on decondensed sperm nuclei

Fluorescent in situ hybridization (FISH) in decondensed sperm nuclei has been used to determine the percentage of normal/balanced or unbalanced spermatozoa produced by an inv(6)(p23q25) carrier, and the possible interchromosomal effect (ICE) of the reorganized chromosomes on other chromosome pairs. A dual color FISH with specific subtelomeric probes for the 6p and 6q regions was performed to determine the segregation pattern of the inverted chromosome. ICE on chromosomes 18, X and Y was assessed using a triple color FISH assay. In the segregation analysis 10,049 spermatozoa were analyzed, and only 45.7% of them were normal/balanced. The high number of unbalanced gametes in our carrier could be the consequence of the large size of the inverted segment. This situation could facilitate the formation of an inversion loop, where formation of an odd number of chiasmata (usually one) result in the production of 50% normal and 50% unbalanced sperm. Furthermore, an increase in the disomy rate for chromosome 6 was also observed. In the screening for ICE, 10,007 spermatozoa were analyzed. The disomy rate for the sex chromosomes and chromosome 18 were not significantly different from those found in our controls, suggesting no evidence of interchromosomal effects in this patient. The use of FISH in decondensed sperm nuclei has proved once more to be an accurate approach to determine the chromosome anomalies in sperm and could help to better establish a reproductive prognosis.     

Aneuploidy frequency in sperm of fertile men

Analysis of sperm aneuploidy in 11 healthy men using two-or three-color FISH permitted to determine the average frequency of disomy for chromosomes 13 and 21 (0.11% and 0.2%, respectively), disomy for chromosome 18 (0.05%) and to reveal gonosomal aneuploidy variants and their frequency. The frequency of XX disomy was 0.04%; XY, 0.17%; YY, 0.06%; and gonosomal nullisomy, 0.29%. We assessed the frequency of meiotic nondisjunction of 13, 21, 18, X, and Y chromosomes and the frequency of XX, XY, and YY diploid spermatozoa. The XY variant prevailed in gonosomal aneuploidy and diploidy and was associated with abnormal chromosomal segregation in meiotic anaphase I. The contribution of human sperm chromosomal imbalance to early embryonic lethality and to some forms of chromosomal abnormalities in the off-spring is discussed.     

Sequence analysis of long FMR1 arrays in the Japanese population: insights into the generation of long (CGG) n tracts

Sperm chromosome abnormalities were assessed in testicular cancer patients before and after treatment with BEP (bleomycin, etoposide, cisplatin). The frequencies of disomy for chromosomes 1, 12, X, Y and XY were assessed along with diploid frequencies and sex ratios by multicolour fluorescence in situ hybridization (FISH). For each cancer patient, a minimum of 10 000 sperm was assessed for each chromosome probe before and after chemotherapy (CT). Data was analysed “blindly” by coding the slides. A total of 161 097 sperm were analyzed, 80 445 before and 80 642 after treatment. The mean disomy frequencies were 0.11% pre-CT vs 0.06% post-CT for chromosome 1, 0.18% vs 0.15% for chromosome 12, 0.10% vs 0.9% for the X chromosome, 0.13% vs 0.10% for the Y chromosome and 0.25% vs 0.20% for XY sperm. There was no significant difference in the frequency of disomy pre-CT vs post-CT for any chromosome except that chromosome 1 demonstrated a significant decrease after CT. The “sex ratios” and frequency of diploid sperm were also not significantly different in pre- and post-CT samples with 50.2% X-bearing sperm pre-CT and 50.5% X post-CT and 0.14% diploid sperm pre-CT vs 0.15% diploid sperm post-CT. There was no significant donor heterogeneity among the cancer patients. None of the values in the cancer patients differed significantly from 10 normal control donors. Thus our study suggests that BEP chemotherapy does not increase the risk of numerical chromosomal abnormalities in human sperm. Received: 11 June 1996 / Revised: 8 August 1996     

Aneuploid epididymal sperm detected in chromosomally normal and Robertsonian translocation-bearing mice using a new three-chromosome FISH method

We present a new method to detect epididymal sperm aneuploidy (ESA) in mice using simultaneous fluorescence in situ hybridization (FISH) with DNA probes specific for mouse chromosomes X, Y and 8. The method was applied to Robertsonian (Rb) translocation (8.14) heterozygotes and homozygotes as well as the chromosomally normal B6C3F1. The sex ratios of sperm did not differ from the expected 1∶1 and the hybridization efficiencies were ≈99.7% for over 60 000 sperm analyzed. Mice heterozygous for Rb (8.14) produced about tenfold higher rates of sperm with chromosome 8 hyperhaploidy than did Rb (8.14) homozygotes or chromosomally normal mice, while frequencies of sperm with hyperhaploidies for chromosomes X and Y were unaffected in all three lines of mice. Hyperhaploid frequencies obtained with the ESA method were consistent with those of the previous testicular FISH method and were validated by published data obtained by conventional cytogenetic analyses (meiotic metaphase II and first cleavage). Thus, the mouse three-chromosome ESA assay together with the previously developed aneuploidy assay for human sperm constitute a promising pair of interspecific biomarkers for comparative studies of the genetic and physiologic mechanisms of the induction and persistence of aneuploidy in male germ cells. Edited by: T. Hassold