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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 10(4) 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 10(4) 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 10(4) 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 approximately 12 per 10(4) (range 8.3-16.7 per 10(4) sperm). Based on flagella data, the frequency of somatic cells in the semen was estimated to be approximately 1.8 per 10(4) sperm. Loss or gain of a portion of chromosome-arm 1p occurred in 5.5 per 10(4) 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.

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 approximately 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 cell 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.

The chromosomal effects of chloral hydrate (CH) on germ cells of male mice were investigated using two methods to detect and characterize spermatid micronuclei (SMN); (a) anti-kinetochore immunofluorescence (SMN-CREST) and (b) multicolor fluorescence in situ hybridization with DNA probes for centromeric DNA and repetitive sequences on chromosome X (SMN-FISH). B6C3F1 mice received single intraperitoneal (i.p.) injections of 82.7, 165.4, or 413.5 mg/kg and round spermatids were sampled at three time intervals representing cells treated in late meiosis, early meiosis, or as spermatogonial stem cells. No increases in the frequencies of SMN were detected for cells treated during meiosis using either SMN-CREST or SMN-FISH methods. After spermatogonial stem-cell treatment, however, elevated frequencies of SMN were detected by both methods. With SMN-FISH, dose trends were observed both in the frequencies of spermatids containing micronuclei and in the frequency of spermatids carrying centromeric label. These findings corroborate the recent report by Allen and colleagues [Allen JW et al.(1994): Mutat. Res. 323:81-88] that CH treatment of spermatogenic stem cells induced SMN. Furthermore, our findings suggest that chromosomal malsegregation or loss may occur in spermatids long after CH treatment of stem cells. Further studies are needed to understand the mechanism of action of the CH effect on stem cells and to determine whether similar effects are induced in human males treated with CH.