Diploid Sperm Research Articles

Morphological Analysis of Giant Oocytes to Assess Their Relevance for the Origin of Triploid Zygote Formation

We studied the frequency, morphological characteristics, and clinical significance of giant oocytes. The union of gametes during normal fertilization is characterized by the formation of two pronuclei (2PN) at syngamy. Development of triploidy (3PN) zygotes is considered to be abnormal fertilization mechanisms. Main explanations for the occurrence of 3PN include the failure of the second polar body extrusion, dispermic penetration, or the entry of one diploid sperm. Here we report the observation of giant oocytes, which can offer an additional explanation for the origin of 3PN. Observational Study This study includes 10 giant oocytes obtained from 10 patients who underwent conventional IVF or ICSI. For IVF cases, giant oocytes were identified during the fertilization assessment. As for ICSI cases, unfertilized giant oocytes could be identified during a course of cumulus cell removal just before the ICSI procedure. They were examined for the number of the pronucleus and polar bodies(PB)18-20 hours after IVF/ICSI. Fertilized giant oocytes were further cultured to assess the developmental potential. Results: A total of 2564 oocytes were collected during the study period. 10 of them (10/2564 oocytes: 0.39%) were classified as giant oocytes. When considering only those patients having giant oocytes, the frequency of giant oocytes was found to be 9.6%(10/104 oocytes). Diameter of all giant oocytes was measured to be around 150 μm. The volume of giant oocytes was calculated to be about twice as large as that of a normal sized oocyte. For ICSI cases(n=4), all giant oocytes had apparently the two distinct first-PBs at the 9 o’clock and 12 o’clock position in perivitelline space in relation to the holding pipette. Afterward 3PN were confirmed and the second-PB extrusion was clearly recognized adjacent to the respective first-PB. All 3PN zygotes showed subsequent early cleavage to reach blastocyst stage. For IVF cases, half of giant oocytes(n=3) showed 3PN formation with extrusion of two second-PBs, and the rest of them(n=3) remained unfertilized. 1) An additional explanation for the occurrence of 3PN zygote after ICSI/IVF can be drawn from this observation. Most giant oocytes seems to have two sets of metaphase II(MII)/PB structures. It is possible that chromosomal content in these giant oocytes with two first-polar bodies is diploid, so that giant oocytes can cause digynic triploidy. 2) Considering the volume of a giant oocyte, one mechanism of its formation might be the fusion of two oocytes. 3) The early recognition of 3PN embryo is of considerable clinical significance. Majority of 3PN embryos arising from giant oocytes are thought to be composed of all triploid blastomeres. No risks should be taken and these embryos are always excluded from transfer.

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.

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.

A case of triploidy

To report a case of triploidy in a women who conceived after intracytoplasmic sperm injection (ICSI). Case report. Maternity unit of a university hospital. A 29-year-old woman who was pregnant following intracytoplasmic sperm injection (ICSI). Ultrasound and serum screening. Amniocentesis and postmortem findings. Amniocentesis confirmed the karyotype to be 69XXY, and the pregnancy was terminated. This case refers to diandric triploidy resulting from fertilization by diploid sperm.

A Novel Class of MYB Factors Controls Sperm-Cell Formation in Plants

In contrast to animals, the plant male germline is established after meiosis in distinctive haploid structures, termed pollen grains. The germline arises by a distinct asymmetric division of the meiotic products . The fates of the resulting vegetative and generative cells are distinct. In contrast to the larger vegetative cell, arrested in the G1 phase of the cell cycle, the smaller generative cell divides once to produce the two male gametes or sperm cells. Sperm cells are delivered to the female gametes by the pollen tube, which develops from the vegetative cell. In spite of recent efforts to understand pollen development , the molecular pathway controlling sperm-cell ontogenesis is unknown. Here, we present the isolation of DUO1, a novel R2R3 MYB gene of Arabidopsis, as the first gene shown to control male gamete formation in plants. DUO1 is specifically expressed in the male germline, and DUO1 protein accumulates in sperm-cell nuclei. Mutations in DUO1 produce a single larger diploid sperm cell unable to perform fertilization. DUO1 appears to be evolutionarily conserved in several plant species and defines a new subfamily of pollen-specific MYB genes.

Experimentally Induced Autotetraploidy and Allotetraploidy in Two Japanese Pond Frogs

We examined cytogenetic features and reproductive capacity in newly arisen tetraploid amphibians. Autotetraploids were produced by suppressing second polar body formation in haploid eggs of diploid females of Rana nigromaculata inseminated with diploid sperm of autotetraploid R. nigromaculata. Allotetraploids were also produced by suppressing second polar body formation in haploid eggs of diploid females of Rana porosa brevipoda inseminated with diploid sperm of autotetraploid R. nigromaculata. In male R. nigromaculata autotetraploid offspring, mostly quadri- and bivalents were found in Metaphase I, and 26 dyads were seen in Metaphase II. Autotetraploid males were then mated with autotetraploid females, and most of the resultant offspring were tetraploids possessing four chromosome sets of diploid R. nigromaculata. Male allotetraploid offspring showed 26 bivalents in Metaphase I and 26 dyads in Metaphase II. A large number of the offspring resulting from crosses between allotetraploid males and females were tetraploids having chromosome sets of diploid R. nigromaculata and diploid R. p. brevipoda. These results seem to indicate that, if such tetraploids develop a distinguishing premating mechanism, they should continue as a sexual fertile species in one generation and coexist with their diploid parental species.

Triploid females and diploid males: underreported phenomena in Polistes wasps?

In hymenopteran species, males are usually haploid and females diploid. However, in species that have complementary sex determination (CSD), diploid males arise when a female produces offspring that are homozygous at the sex-determining locus. Although diploid males are often sterile, in some species they have been shown to produce diploid sperm, thus producing triploid daughters if they mate successfully. Diploid males have been observed in very few species of social wasps, and we know of no published reports of triploid females. In this paper, we review the existing literature on diploid males and triploid females in the Hymenoptera, and report the observation of triploid females in three species of Polistes paper wasps. Although polyploid offspring may be produced parthenogenetically, the more likely scenario is that Polistes wasps have CSD and produce diploid males via homozygosity at the sex-determining locus. Therefore, female triploidy indicates that diploid males do exist in Polistes species where they are presumed to be absent, and are likely to be even more frequent among species that have experienced a genetic bottleneck. We conclude by cautioning against the assumption of a selective advantage to the production of early males, and by discussing the implications of male diploidy and female triploidy for measurement of sex ratio investment and assumptions of reproductive skew theory.

Sperm selection for ICSI: shape properties do not predict the absence or presence of numerical chromosomal aberrations.

We hypothesize that the potential relationship between abnormal sperm morphology and increased frequency of numerical chromosomal aberrations is based on two attributes of diminished sperm maturity: (i) cytoplasmic retention and consequential sperm shape abnormalities; and (ii) meiotic errors caused by low levels of the HspA2 chaperone, a component of the synaptonemal complex. Because sperm morphology and aneuploidies were assessed in semen, but not in the same spermatozoa, previous studies addressing this relationship were inconclusive. We recently demonstrated that sperm shape is preserved following fluorescence in situ hybridization (FISH). Thus, we examined the shape and chromosomal aberrations in the same sperm. We performed phase contrast microscopy and FISH, using centromeric probes for chromosomes X, Y, 10, 11 and 17 in 15 men. The fluorescence and respective phase contrast images were digitized using the Metamorph program. We studied 1286 sperm (256 disomic, 130 diploid and 900 haploid sperm) by three criteria: head and tail dimensions, head shape and Kruger strict morphology. Furthermore, in each analysis, we considered whether disomic or diploid sperm may be distinguished from haploid sperm. There was an overall, but not discriminative, relationship between abnormal sperm dimensions or shape and increased frequencies of numerical chromosomal aberrations. However, approximately 68 of the 256 disomic, and four of 130 diploid sperm showed head and tail dimensions comparable with the most normal, lowest tertile of the 900 haploid spermatozoa. Considering all 1286 sperm, among those with the most regular, symmetrical shape (n = 367), there were 63 and five with disomic and diploid nuclei, respectively. In line with these findings, among the 256 disomic sperm, 10% were Kruger normal. Sperm dimensions or shape are not reliable attributes in selection of haploid sperm for ICSI.

Ploidy status of progeny from the crosses between tetraploid males and diploid females in mud loach ( Misgurnus mizolepis)

For the first time, DNA content of the sperm of tetraploid mud loach ( Misgurnus mizolepis) males and the ploidy status of progenies generated by crossing tetraploid males with diploid females are described. Of 48 tetraploid males tested, 12 showed significantly retarded gonad development, but the other 36 produced functional sperm. Of these 36, 26 produced haploid sperm which, on fertilizing haploid eggs, generated diploid progenies. Seven tetraploid males were mosaics in their sperm, as indicated by the production of diploid, aneuploid and/or triploid offspring. Only three males produced diploid sperm rendering the production of all-triploid progenies. The DNA content of sperm of tetraploid males was consistent throughout three progeny tests, i.e., the haploid sperm-producing 4n males persisted to produce haploid sperm only; likewise, the diploid sperm producing 4n males consistently produced diploid sperm only, when progeny testing was extended to 3 successive but alternate years. Hence, a careful and direct examination of the DNA profile of sperm from tetraploid males is a pre-requisite for reproductive containment of genetically modified fish.

Variation in the membrane transport properties and predicted optimal rates of freezing for spermatozoa of diploid and tetraploid Pacific oyster, Crassostrea gigas.

In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 microm in diameter and a "stick" 41 microm in length and 0.14 microm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 microm in diameter and a "stick" 53 microm in length and 0.17 microm wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, Vb, of 0.6 Vo, where Vo is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: Lpg = 0.30 x 10(-15) m(3)/Ns (0.0017 microm/min-atm) and ELp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.27 x 10(-15) m(3)/Ns (0.0015 microm/min-atm) and ELp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20 degrees C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: Lpg = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: Lpg[cpa] = 0.34 x 10(-15) m(3)/Ns (0.0019 microm/min-atm) and ELp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70 degrees C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas.

Early male production is not linked to a reproductive strategy in the Japanese paper wasp, Polistes chinensis antennalis (Hymenoptera: Vespidae)

Diploid males in haplo-diploid insects are sterile, because they produce diploid sperm. Our previous report revealed that early males of the Japanese paper wasp Polistes chinensis antennalis are diploid but did not reveal how often this occurs. We analyzed the genotypes of early males using six microsatellite markers. Two of the 41 early males (5%) from six colonies were haploid, but the other males were diploid. This evidence suggests that we can ignore the reproductive success of the early males of P. chinensis antennalis.

Meiotic segregation of sex chromosomes in mosaic and non-mosaic XYY males: case reports and review of the literature.

The aim of this study was to determine the incidence of sex chromosome aneuploidy in spermatozoa of two males with a 47,XYY karyotype and one male with a 46,XY/47,XYY constitution. Spermatozoa obtained from two oligospermic patients and one volunteer semen donor were studied by multicolour fluorescence in situ hybridization. In the XY/XYY male, the frequencies of X-bearing to Y-bearing sperm were significantly different from the 1 : 1 expected ratio. Significantly increased frequencies were found in the mosaic and non-mosaic males for 24,XX and 24,YY sperm when compared with control donors. The number of 24,XY sperm was significantly different from the controls in the XYY males, but not in the mosaic male. The incidence of disomy 18 and the rate of diploidy also increased in the three patients. However, the mosaic male had the lowest cumulative rate of disomic and diploid spermatozoa when compared with the two XYY patients. Our data suggest that: (i) chromosome abnormalities observed in spermatozoa of the two XYY oligoasthenoteratospermic (OAT) males arise through segregation errors in XY germ cells rather than normal meiosis of XYY germ cells, (ii) mosaic XYY males with normal semen parameters have a lower risk of producing offspring with a sex chromosomal abnormality than OAT males with XYY karyotype.

Efficacy of the swim-up method in eliminating sperm with diminished maturity and aneuploidy.

We have previously shown that after 80% Percoll centrifugation there is an overall 2.7-fold reduction of sperm with chromosomal disomies and diploidies (3.2-fold and 2.0-fold respectively), and of sperm with diminished maturity as detected by cytoplasmic retention. The relationship between disomies and immature sperm was r = 0.7, suggesting that disomy primarily originates in immature sperm. In the present work we studied the efficacy of the swim-up method in elimination of sperm with diminished maturity and with chromosomal aberrations in the swim-up sperm fractions of 10 patients (sperm concentration: 20 +/- 3.9 x 10(6)/ml, range 8.9-45.5; sperm motility: 45.2 +/- 2.4, all mean +/- SEM). The validity of the study was enhanced by assessing each sperm fraction with three-colour (X, Y and 17; 5000 sperm) and two-colour (10 and 11; 5000 sperm) chromosome probes using fluorescence in-situ hybridization (FISH). Thus, in each sample 10 000 sperm were evaluated. The incidence of diminished maturity sperm was assessed with creatine kinase immunocytochemistry. In the swim-up fractions there was a reduction in the frequencies of disomic sperm, whether considering the sex chromosomes (1.4-fold) or the three autosomal chromosomes (1.5-fold based on the aggregate frequencies of disomy 10, 11 and 17). There was also a 1.5-fold reduction in diminished maturity sperm, indicating a relationship between the proportion of immature sperm and chromosomal aneuploidies (r = 0.46, P < 0.05, n = 20). Diploid sperm were reduced at a 2.7-fold rate, whether assessed with two- or three-colour FISH. There was a slight increase in the X/Y ratios. Swim-up reduces the proportion of sperm with chromosomal aberrations and of sperm with diminished maturity. When compared with the results of the previous study with gradient centrifugation performed on semen samples with similar quality, the efficacy after swim-up is lower for disomies and higher for diploidies than that of gradient centrifugation.

Genetic damage in oligozoospermic patients detected by fluorescence in-situ hybridization, inverse restriction site mutation assay, sperm chromatin structure assay and the Comet assay.

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. 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. 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. 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.

An increased incidence of autosomal aneuploidies in spermatozoa from a patient with Klinefelter’s syndrome

Objective To determine whether there is an increased incidence of autosomal aneuploidies in spermatozoa from a subject with nonmosaic Klinefelter’s syndrome. Design Analysis of sperm nuclei by fluorescence in situ hybridization. Setting Hospital-based laboratory for reproductive biology. Patient(s) A patient with Klinefelter’s syndrome and a 46,XY fertile man were analyzed. Intervention(s) The sperm samples were prepared for fluorescence in situ hybridization. Main outcome measure(s) The disomy frequencies for chromosomes 7, 9, 13, 18, and 21 and sex chromosomes were determined using fluorescence in situ hybridization. Result(s) Significant differences were found in the frequency of disomy for chromosomes 13, 18, and 21 between the patient and a normospermic control. No significant differences were observed for chromosomes 7 and 9. The frequencies of gonosomal abnormalities and diploid spermatozoa were also significantly increased in the patient. Conclusion(s) Our results indicate that there is an increased incidence of autosomal aneuploidies for chromosomes 13, 18, and 21 in the spermatozoa from the patient with Klinefelter’s syndrome than in the general population. Furthermore, most of the studies have also shown an increased frequency of autosomal aneuploidy in the spermatozoa from 46,XY males with oligozoospermia or oligoasthenoteratozoospermia. Thus, the offspring of the patients with Klinefelter’s born by intracytoplasmic sperm injection may be at higher risk of autosomal aneuploidy due to oligoasthenoteratozoospermia.

Numerical chromosomal abnormalities in rat epididymal spermatozoa following chronic cyclophosphamide exposure.

Chronic, low-dose treatment of male rats with cyclophosphamide, a chemotherapeutic agent, is known to affect progeny outcome adversely in a dose-dependent and time-specific manner, resulting in increased pre- and postimplantation loss as well as malformations. Concern exists regarding the genetic quality of mature gametes exposed to cyclophosphamide during mitosis and meiosis. The goal of the present study was to determine the effect of chronic cyclophosphamide treatment during spermatogenesis on the frequency of numerical chromosomal anomalies in epididymal spermatozoa. Male rats were treated with either saline or cyclophosphamide (6 mg kg-1 day-1) for 6 or 9 wk, and cauda epididymal spermatozoa were collected. The rat sperm Y-4 fluorescence in situ hybridization assay was used to assess the induction of spermatozoal disomy, nullisomy, and diploidy involving chromosomes Y and 4. The overall frequency of numerically abnormal spermatozoa was elevated approximately 2-fold (P < 0.001) after 9 wk of cyclophosphamide treatment. Exposure for 9 wk, but not for 6 wk, significantly increased the frequency of spermatozoa with chromosome 4 disomy (P < 0.02) and nullisomy (P < 0.05), but disomy Y and diploidy were not significantly increased with treatment compared to corresponding controls. Independent of treatment, only 27% of aneuploid spermatozoa presented with morphological abnormalities, but all diploid spermatozoa were approximately twice the size of normal cells. Thus, cyclophosphamide disrupts meiotic events before pachynema during spermatogenesis, emphasizing the potential for adverse progeny outcomes following genotoxic damage.

Dispermy--origin of diandric triploidy: brief communication.

Triploidy may arise from either digynic or diandric fertilizations. Errors in the second meiotic division account for most digynic triploidy while most studies have found that approximately 2/3 of diandric triploids arise as the result of dispermy and 1/3 as the result of meiotic errors giving rise to diploid sperm. Using molecular markers very close to the centromere, all 14 cases of diandric triploidy were shown to be the result of dispermy with no evidence to support a meiotic error as the origin of diandric triploids.

Evaluation of segregation patterns of 21;21 Robertsonian translocation along with sex chromosomes and interchromosomal effects in sperm nuclei of carrier by FISH technique.

Meiotic segregation patterns of carriers of Robertsonian translocations (RT) are important for assessing the risk of unbalanced forms. We investigated the ratio of sperm with t(21;21) to sperm with nullisomy for chromosome 21; the segregation of the t(21;21) along with sex chromosomes, and also interchromosomal effects on chromosome 10 by using three color fluorescence in situ hybridization (FISH) with telomere specific (Tel 21q) and centromere-specific alpha satellite probes for chromosomes X, Y, and 10. The percentage of cosegregation of t(21;21) with sex chromosomes (49.50%) and without sex chromosomes (46.98%) was not significant. There are no significant differences between the percentages of cosegregation of t(21;21) with chromosome X (23.36%) and with chromosome Y (26.16%). No evidence of an interchromosomal effect on chromosome 10 was detected, the percentage of chromosome 10 aneuploidy being similar to that in controls. In addition, the frequency of diploid sperm nuclei was not significantly higher in the carrier (0.32%) than in the controls (0.44%) (P > 0.05). The sex ratio was similar within the carrier and the controls and between the carrier and the control. Three color-FISH analysis, using different probe combinations, seems a rapid and accurate tool for direct analysis of meiotic segregation product.

Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: studies with topoisomerase II inhibitors

The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2′-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 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). It was found that VP-16 (50 mg/kg) treatment induced a meiotic delay of about 24 h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50 mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50 mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.

Stable Variants of Sperm Aneuploidy among Healthy Men Show Associations between Germinal and Somatic Aneuploidy

Repeated semen specimens from healthy men were analyzed by sperm fluorescence in situ hybridization (FISH), to identify men who consistently produced elevated frequencies of aneuploid sperm and to determine whether men who were identified as stable variants of sperm aneuploidy also exhibited higher frequencies of aneuploidy in their peripheral blood lymphocytes. Seven semen specimens were provided by each of 15 men over a 2-year period and were evaluated by the X-Y-8 multicolor sperm FISH method (i.e., approximately 1,050,000 sperm were analyzed from 105 specimens). Three men were identified as stable aneuploidy variants producing significantly higher frequencies of XY, disomy X, disomy Y, disomy 8, and/or diploid sperm over time. In addition, one man and three men were identified as sperm-morphology and sperm-motility variants, respectively. Strong correlations were found between the frequencies of sperm with autosomal and sex-chromosome aneuploidies and between the two types of meiosis II diploidy; but not between sperm aneuploidy and semen quality. A significant association was found between the frequencies of sex-chromosome aneuploidies in sperm and lymphocytes in a subset of 10 men (r2=0.67, P=.004), especially between XY sperm and sex-chromosome aneuploidy in lymphocytes (r2=0.70, P=.003). These findings suggest that certain apparently healthy men can produce significantly higher frequencies of both aneuploid sperm and lymphocytes. Serious long-term somatic and reproductive health consequences may include increased risks of aneuploidy-related somatic diseases and of having children with paternally transmitted aneuploidies, such as Klinefelter, Turner, triple-X, and XYY syndromes.