Xiu Lowe

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 fathers 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.

Etoposide induces heritable chromosomal aberrations and aneuploidy during male meiosis in the mouse

Etoposide, a topoisomerase II inhibitor widely used in cancer therapy, is suspected of inducing secondary tumors and affecting the genetic constitution of germ cells. A better understanding of the potential heritable risk of etoposide is needed to provide sound genetic counseling to cancer patients treated with this drug in their reproductive years. We used a mouse model to investigate the effects of clinical doses of etoposide on the induction of chromosomal abnormalities in spermatocytes and their transmission to zygotes by using a combination of chromosome painting and 4′,6-diamidino-2-phenylindole staining. High frequencies of chromosomal aberrations were detected in spermatocytes within 64 h after treatment when over 30% of the metaphases analyzed had structural aberrations (P < 0.01). Significant increases in the percentages of zygotic metaphases with structural aberrations were found only for matings that sampled treated pachytene (28-fold, P < 0.0001) and preleptotene spermatocytes (13-fold, P < 0.001). Etoposide induced mostly acentric fragments and deletions, types of aberrations expected to result in embryonic lethality, because they represent loss of genetic material. Chromosomal exchanges were rare. Etoposide treatment of pachytene cells induced aneuploidy in both spermatocytes (18-fold, P < 0.01) and zygotes (8-fold, P < 0.05). We know of no other report of an agent for which paternal exposure leads to an increased incidence of aneuploidy in the offspring. Thus, we found that therapeutic doses of etoposide affect primarily meiotic germ cells, producing unstable structural aberrations and aneuploidy, effects that are transmitted to the progeny. This finding suggests that individuals who undergo chemotherapy with etoposide may be at a higher risk for abnormal reproductive outcomes especially within the 2 months after chemotherapy.

Meiotic Segregation, Recombination, and Gamete Aneuploidy Assessed in a t(1;10)(p22.1;q22.3) Reciprocal Translocation Carrier by Three- and Four-Probe Multicolor FISH in Sperm

Meiotic segregation, recombination, and aneuploidy was assessed for sperm from a t(1;10)(p22.1;q22.3) reciprocal translocation carrier, by use of two multicolor FISH methods. The first method utilized three DNA probes (a telomeric and a centromeric probe on chromosome 1 plus a centromeric probe on chromosome 10) to analyze segregation patterns, in sperm, of the chromosomes involved in the translocation. The aggregate frequency of sperm products from alternate and adjacent I segregation was 90.5%, and the total frequency of normal and chromosomally balanced sperm was 48.1%. The frequencies of sperm products from adjacent II segregation and from 3:1 segregation were 4.9% and 3.9%, respectively. Reciprocal sperm products from adjacent I segregation deviated significantly from the expected 1:1 ratio (P < .0001). Our assay allowed us to evaluate recombination events in the interstitial segments at adjacent II segregation. The frequencies of sperm products resulting from interstitial recombination in chromosome 10 were significantly higher than those resulting from interstitial recombination in chromosome 1 (P < .006). No evidence of an interchromosomal effect on aneuploidy was found by use of a second FISH method that simultaneously utilized four chromosome-specific DNA probes to quantify the frequencies of aneuploid sperm for chromosomes X, Y, 18, and 21. However, a significant higher frequency of diploid sperm was detected in the translocation carrier than was detected in chromosomally normal and healthy controls. This study illustrates the advantages of multicolor FISH for assessment of the reproductive risk associated with translocation carriers and for investigation of the mechanisms of meiotic segregation of chromosomes.

Numerical and structural chromosomal abnormalities detected in human sperm with a combination of multicolor FISH assays.

A pair of multicolor FISH assays (X‐Y‐21 and A‐M‐16) was developed for human sperm to simultaneously measure sex ratios; aneuploidies involving chromosomes 1, 16, 21, X, and Y; meiotic diploidies; and structural aberrations involving chromosome 1p. Sex ratios in sperm were not significantly different from unity among healthy men. Baseline frequencies of disomic sperm for chromosomes 1, 8, and 21 were similar (6.7 per 104 sperm, 95% CI of 5.6–8.1), suggesting that among these three chromosomes, chromosome 21 was not especially prone to nondisjunction. Frequencies of disomy 16 sperm were significantly lower, however (3.5 per 104 sperm, 95% CI of 2.0–6.2; P < 0.02). The baseline frequencies of sperm disomy by FISH for chromosomes 16 and 21 were validated against aneuploidy data obtained by the hamster‐egg technique for human sperm cytogenetics. The frequencies of X‐X, Y‐Y, X‐Y (“Klinefelter”) sperm and sex‐null (“Turner”) sperm were 5.5, 5.1, 5.5, and 7.8 per 104 sperm, respectively. For chromosomes 16 and 21, the frequencies of nullisomic and disomic sperm were similar, suggesting that gain and loss events occurred symmetrically. However, more gain than loss was reported for chromosomes 1, X, and Y. The frequency of MI and MII diploid sperm (with flagella) was ∼12 per 104 (range 8.3–16.7 per 104 sperm). Based on flagella data, the frequency of somatic cells in the semen was estimated to be ∼1.8 per 104 sperm. Loss or gain of a portion of chromosome‐arm 1p occurred in 5.5 per 104 sperm, and the percentage of sperm carrying structural aberrations within the haploid genome as calculated from FISH (1.4%), was similar to that obtained with the hamster‐egg technique. These complementary sperm FISH assays have promising applications in studies of chromosomally abnormal sperm after exposure to occupational, medical, and environmental toxicants. Environ. Mol. Mutagen. 33:49–58, 1999 Published 1999 Wiley‐Liss, Inc.

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

Abstract.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.

Induction of chromosomal aberrations in mouse zygotes by acrylamide treatment of male germ cells and their correlation with dominant lethality and heritable translocations

The objectives of this research were: 1) to investigate the time course of the cytogenetic defects induced by acrylamide (AA) treatment (5 × 50 mg/kg) of male germ cells in first‐cleavage zygote metaphases using PAINT/DAPI analysis, and 2) to characterize the correlation between chromosomal aberrations at first cleavage, dominant lethality, and heritable translocations. PAINT/DAPI analysis employs multicolor fluorescence in situ hybridization painting plus DAPI staining to detect both stable and unstable chromosomal aberrations at first‐cleavage metaphase of the zygote. High levels of chromosomally defective zygotes were detected after mating at all postmeiotic stages (20–190‐fold, P < 0.001). Early spermatozoa (6.5 d post‐treatment) were the most sensitive, with 76% of the zygotes carrying cytogenetic defects. A significant 10‐fold increase was also detected 27.5 d post‐treatment, indicating that AA had a cytogenetic effect on meiotic stages. PAINT/DAPI analysis revealed that: 1) AA‐induced chromosomal breaks occurred at random, and 2) the frequencies of symmetrical and asymmetrical exchanges were similar at all mating days, except 9.5 d after AA treatment, where significantly (P < 0.02) more asymmetrical aberrations were found. Furthermore, the proportions of zygotes carrying unstable and stable chromosomal aberrations followed a similar post‐treatment time course as the proportions of dominant lethality among embryos and heritable translocations among offspring. These findings indicate that PAINT/DAPI analysis of zygotic metaphases is a promising method for detecting male germ cell mutagens capable of inducing chromosomal aberrations and for evaluating the associated risks for embryonic loss and balanced translocations at birth. Environ. Mol. Mutagen. 30:410–417, 1997

Spontaneous rates of sex chromosomal aneuploidies in sperm and offspring of mice : a validation of the detection of aneuploid sperm by fluorescence in situ hybridization

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.

Micronuclei and developmental abnormalities in 4‐day mouse embryos after paternal treatment with acrylamide

The developmental consequences of paternal exposure to acrylamide (50 mg/kg i.p. for 5 days) were assessed in preimplantation embryos. There was a significant increase in the proportion of morphologically abnormal embryos after postmeiotic treatment during spermatogenesis (88.7% vs. 14.8% in control). Abnormal embryos had an average of 1.8 ± 3.5 cells and >80% had at least one fragmented nucleus. In addition, morphologically normal embryos were significantly delayed (34.3 ± 12.8 cells per embryo vs. 57.6 ± 15.7 in control, P < 0.001). Acrylamide caused 10‐ and 20‐fold increases in frequencies of cells with micronuclei (MN) in morphologically normal and abnormal embryos, respectively (41 and 93 MN per 1,000 cells). Both centromere‐negative (M−) and centromere‐positive (M+) were induced. Nuclei of abnormal embryos were significantly larger (900 μm2 vs. 250 μm2) than controls. In addition, MN of abnormal embryos were larger than those of normal embryos (21.2 μm2 vs. 6.5 μm2, P < 0.01). Among control embryos, M+ were significantly larger than M− (P < 0.05). These findings suggest that the preimplantation embryo is a sensitive indicator of paternally transmitted effects on early development. Multiple mechanisms appear to be involved, including cytogenetic damage, proliferation arrest/delay, and fertilization failure. Future studies are needed to establish how induced cytological defects in preimplantation embryos contribute to birth defects and other postimplantation abnormalities. Environ. Mol. Mutagen. 31:206–217, 1998

Epididymal sperm aneuploidies in three strains of rats detected by multicolor fluorescence in situ hybridization.

A multicolor fluorescence in situ hybridization (FISH) method was developed to detect aneuploidy and diploidy in epididymal sperm of rats using DNA probes specific for chromosomes 4 and Y. Fourteen healthy young‐adult rats from three strains were evaluated: inbred Fisher 344/N/ehs, outbred Sprague‐Dawley, and outbred WU Wistar/CPB. The hybridization efficiency of the FISH procedure was >99.9%, the sex‐ratio in sperm was ∼1 as expected, and there was no significant variation among two independent scorers. No significant variations were detected within or among strains in the frequencies of sperm disomy for chromosome 4 (1–6.5 per 10,000 cells per animal) or the Y chromosome (0–2.5 per 10,000 cells per animal). There was a trend toward increased variation among Wistar rats. The frequencies of sperm‐carrying hyper‐ and hypohaploidy for chromosome 4 were similar, suggesting a symmetrical mechanism of chromosome gain and loss during meiosis. The frequencies of Y‐Y‐4‐4 sperm, which represent genomic meiosis II errors, did not differ significantly across strains (0.1–0.7 per 10,000 cells per strain). This FISH method for detecting aneuploidy in rat epididymal sperm provides a promising interspecies biomarker of male germ cell aneuploidy and introduces the rat as an animal model for investigating the heritable risk to offspring associated with paternal genotype, physiology, and exposure to environmental mutagens. There appear to be no significant differences among young healthy rats, mice, and men in the baseline frequencies of sperm with Y chromosomal disomy, the only chromosome for which data currently exists for all three species. Environ. Mol. Mutagen. 31:125–132, 1998

Paternally inherited chromosomal structural aberrations detected in mouse first-cleavage zygote metaphases by multicolour fluorescencein situ hybridization painting

We describe a fluorescencein situ hybridization (FISH) procedure for assessing zygotic risk of paternal exposure to endogenous or exogenous agents. The procedure employs multicolour FISH with chromosome-specific DNA painting probes plus DAPI staining for detecting both balanced and unbalanced chromosomal aberrations in mouse first-cleavage (1-Cl) zygote metaphases. Four composite probes specific for chromosomes 1, 2, 3 or X, each labelled with biotin, plus a composite probe specific for chromosome Y labelled with digoxigenin, were used. We applied this method to evaluate the effects of paternal exposure to acrylamide, a model germ cell clastogen. First-cleavage zygote metaphases, collected from untreated females mated to males whose sperm or late spermatids were treated with acrylamide, were scored for the induction of structural aberrations using bothchromosome painting (PAINT analysis) andDAPI analysis. Structural chromosomal aberrations were observed in the sperm-deried, but not in the eggderived, pronuclei. While 59.4% of the zygotes had structural aberrations by DAPI analysis, 94.1% of the same zygotes had structural aberrations by PAINT analysis (P<0.001), illustrating the increased sensitivity for detecting translocations and insertions obtained by adding chromosome painting. These findings show that FISH painting of mouse 1-Cl zygotes when used in conjunction with DAPI analysis is a powerful model for investigating the cytogenetic defects transmitted from father to offspring.