Detection of aneuploidy in rodent and human sperm by multicolor FISH after chronic exposure to diazepam

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.     

Rotifers in aging research: use of rotifers to test various theories of aging

Four theories of aging are discussed to examine how effectively they might explain the aging process in rotifers. One of the early theories, the rate of living theory of aging can perhaps be discounted. Although the theory predicts that increased biological energy expenditure, in the form of increased activity or reproduction, would lead to a shorter lifespan, these predictions are not born out by experimental evidence. At the whole animal level, a case can be made for a theory of programmed aging, where the end of reproduction signals the end of the lifespan. Support for this view comes from the observation that lifespan is positively correlated with reproductive parameters, that treatments that extend lifespan usually act to extend the reproductive period, and that the end of reproduction is associated with high mortality and senescent biochemical changes. Two molecular theories of aging are also discussed; the free radical theory of aging and the calcium theory of aging. These theories point to the fact that molecular damage accumulates and that calcium influx increases in the course of aging. When free radical buildup or calcium homeostasis is reduced, lifespan is extended. A molecular explanation of aging does not necessarily exclude the idea of programmed aging. It is probable that an eventual understanding of the aging process will rest on both a physiological and molecular basis.     

Aneuploidy induction in mice: construction and use of a tester stock with 100% nondisjunction

A new murine tester stock for primary nondisjunction incorporates three genetically marked Robertsonian translocations with tribrachial homology (TBH): Rb(6.15)1Ald, Rb(4.6)2Bnr, and Rb(4.15)4Rma. The resultant tricentromeric meiotic configuration leads to 100% aneuploid gametes, but the TBH stock can be maintained by intercrossing, through the complementation of nullisomic and disomic gametes. The only neonatal survivors from tescrosses to wild type come from complementation of aneuploid gametes and genetic tests allow wild type gains or losses of Chromosomes 4, 6, and 15 to be distinguished. Alternatively, cytogenetic examination allows products of wild type chromosome gain, with one metacentric, to be separated from chromosome loss with two metacentrics. A pilot study, with 0-2 Gy X-irradiation of oocytes at diakinesis, revealed twelve examples of chromosome loss in wild type gametes but none of chromosome gain and thus provided no evidence for the induction of nondisjunction.     

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.     

The relationship between paternal age, sex ratios, and aneuploidy frequencies in human sperm, as assessed by multicolor FISH.

We studied the frequencies of X- and Y-chromosome-bearing sperm, diploidy and disomy for chromosomes 1, 12, X, and Y in sperm from 10 normal men aged 21-52 years, to determine whether there was any relationship between donor age and any of these variables. Multicolor FISH was used to control for lack of probe hybridization and to distinguish diploid sperm from disomic sperm. A minimum of 10,000 sperm per donor was evaluated for each chromosome, for a total of 225,846 sperm studied. Sperm were considered disomic if two fluorescent signals were separated by a minimal distance of one signal domain. The mean frequencies of X- and Y-bearing sperm were 50.1% and 49.0%, respectively; not significantly different from 50%. There was no correlation between paternal age and "sex ratio" in sperm. Similarly, there was no association between the frequency of diploid sperm (mean, .16%; range, .06-.42%) and donor age. For disomy frequencies, there was no relationship between donor age and disomy 12 (mean, .16%; range, .10%-.25%), XX (mean, .07%; range, .03%-.17%), and XY sperm (mean, .16%; range, .08%-.24%). There was a significant increase in the frequency of YY sperm (P = .04; mean, .18%; range, .10%-.43%) and disomy 1 sperm (P = .01; mean, .11%; range, .05%-.18%) with donor age. In summary, our results do not support a correlation between paternal age and sex ratio or diploidy.     

Aneuploidy detection in human sperm nuclei using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was accomplished with fluoresceinated avidin and antiavidin. For each of the chromosomes studied, chromosome number was determined by counting the fluorescent signals, representing hybridized regions, within the sperm nuclei. The frequencies for disomy, that is for nuclei containing two signals, for chromosomes 1, 12 and X were 0.06%, 0.04% and 0.03%, respectively. The congruence of these results with those determined by the cross-species hamster oocyte-human sperm assay, and the high efficiency of hybridization indicate that FISH is a sensitive and reliable tool for aneuploidy detection in human sperm.     

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.     

Molecular definition of high-resolution multicolor banding probes: first within the human DNA sequence anchored FISH banding probe set.

Fluorescence in situ hybridization (FISH) banding approaches are standard for the exact characterization of simple, complex, and even cryptic chromosomal aberrations within the human genome. The most frequently applied FISH banding technique is the multicolor banding approach, also abbreviated as m-band, MCB, or in its whole genomic variant multitude MCB (mMCB). MCB allows the differentiation of chromosome region-specific areas at the GTG band and sub-band level and is based on region-specific microdissection libraries, producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first bacterial artificial chromosome (BAC) array comparative genomic hybridization (aCGH) mapped, comprehensive, genome-wide human MCB probe set. All 169 region-specific microdissection libraries were characterized in detail for their size and the regions of overlap. In summary, the unique possibilities of the MCB technique to characterize chromosomal breakpoints in one FISH experiment are now complemented by the feature of being anchored within the human DNA sequence at the BAC level.     

Nematode ageing: Putting metabolic theories to the test.

The increased life span caused by certain mutations in the nematode Caenorhabditis elegans has been interpreted in terms of two metabolic theories of ageing: the oxidative damage theory and the rate of living theory. New findings support the former, but not the latter interpretation.     

Cloned DNA probes regionally mapped to human chromosome 21 and their use in determining the origin of nondisjunction.

A number of unique sequence recombinant DNA clones were isolated from a recombinant DNA library constructed from DNA enriched for chromosome 21 by flow sorting. Of these, five were mapped to chromosome 21 using a somatic cell hybrid. Regional mapping of these probes and of a probe previously assigned to chromosome 21, was carried out with the aid of chromosome 21 rearrangements using both chromosome sorting and a somatic cell hybrid. Three probes were shown to be located on either side of the breakpoint 21q21.2. Two of the probes were shown to identify restriction fragment length polymorphisms (RFLPs) with high rare-allele frequencies (0.46 and 0.43). A Bgl II RFLP revealed the parental origin of non-disjunction in three of ten families with Down's syndrome.     

Aneuploidy in the human blastocyst

Studies of human cleavage stage embryos, 3 days after fertilization of the oocyte, have revealed remarkably high levels of chromosome abnormality. In addition to meiotic errors derived from the gametes, principally the oocyte, mitotic errors occurring after fertilization are also common, leading to widespread chromosomal mosaicism. The prevalence of chromosome anomalies in embryos may explain the relatively poor fertility and fecundity in humans and the low success rates of assisted reproductive treatments (e.g., IVF). While much is known concerning the incidence of aneuploidy during the first 3 days following fertilization, it is only in the last couple of years that large numbers of embryos at the final stage of preimplantation development, the blastocyst stage, 5 days after fertilization, have been subjected to detailed analysis. Here we discuss the latest data from the comprehensive cytogenetic analysis of blastocysts. These findings indicate that the majority of selection against chromosome abnormalities does not occur until the time of implantation or shortly after, with aneuploidy typically affecting more than 50% of blastocysts. Additionally, clinical results presented suggest that screening of blastocyst stage embryos for chromosome abnormality, with preferential transfer to the uterus of those found to be euploid, may help to improve the success rates of assisted reproductive treatments.     

Visualization of a self-incompatibility gene in Brassica campestris L. by multicolor FISH

 The physical localization of the S-glycoprotein (SLG) locus in the chromosome of Brassica campestris L. ‘pekinensis’ cv ‘Kukai’ was visualized by multi-color fluorescent in situ hybridization (McFISH). ‘Kukai’, which is an F₁ hybrid between two parental lines, T-17 and T-18, has two SLG genes from both T-17 and T-18. In this study, a 1.3-kb DNA fragment was amplified from the genomic DNA of T-17 by PCR using a set of primers specific to the class-I SLG. From the genomic DNA of T-18, no DNA fragment was amplified using these primers. In the genomic Southern hybridization, a cloned PCR product hybridized with the genomic DNA of T-17 or F₁ but not with that of T-18. The PCR product had a sequence homology of approximately, 85% to another class-I SLG gene, SLG-9. Therefore, the PCR product from T-17 was named SLG-17, as it is thought to be a member of the class-I SLG. Using SLG-17 as the probe, FISH was carried out to visualize the position of the SLG locus. McFISH was also carried out simultaneously using the SLG-17 and SLG-9 genes as probes. The SLG-17 gene was detected as a doublet signal at the interstitial region close to the end of a small chromosome, with the signal site being identical to that of SLG-9. Therefore, it is concluded that the SLG-17 gene is localized at the interstitial region close to the end of the chromosome derived from T-17 in Brassica campestris L. ‘pekinensis’ cv ‘Kukai’. Received: 18 September 1997 / Accepted: 6 October 1997     

Mechanisms of nondisjunction in human spermatogenesis

A reduction in recombination in the pseudoautosomal region is associated with an increased frequency of aneuploid 24,XY human sperm. Similarly, individuals with paternally derived Klinefelter syndrome (47,XXY) also have a paucity of recombination in the chromosomes that have undergone nondisjunction. Meiotic studies using newly developed immunocytogenetic techniques have demonstrated errors of chromosome synapsis and significantly reduced recombination in infertile men with nonobstructive azoospermia. These men have an increased risk of aneuploidy in sperm that have been surgically removed from the testes. Thus, evidence is starting to accumulate that reduced recombination has a marked effect on the generation of aneuploid sperm.     

用FISH技术研究人类体外未受精卵的21号染色体非整倍体

采用荧光原位杂交技术,选用人类21号染色体端粒探针（21qter）,检测人类体外未受精卵的21号染色体非整倍体发生率,并比较非整倍体率与25－30岁和31－35岁这两个女性年龄组、IVF指征、超排方案之间的关系,在54个未受精卵中,正常21号单体30枚,二体16枚,三体4枚,缺体4枚,非整倍体率为44.4％（24／54）;25－30岁和31－35岁这两个年龄组、IVF指征、超排方案的患者的21号染色体非整倍体率之间的差异无显著性,卵母细胞21号染色体的非整倍性是造成体外受精失败的重要原因之一。     

Aneuploidy in the human cleavage stage embryo

The cleavage stage embryo (days 1-3) stands out due to the high level of chromosomal anomalies, especially mosaicism that arises prior to global embryonic genome activation. Molecular cytogenetic studies show that an average of 60% of in vitro derived embryos have at least one aneuploid cell by the time they are 3 days old. However, comprehensive studies of the chromosome content of individual cells have revealed that 25% of these embryos have no aneuploid cells, a fact that sits well with the knowledge that at most 1 in 5 have the capacity to implant. The evidence is that extensive mosaicism, affecting several chromosomes, interferes with development to a greater extent than does uniform aneuploidy. Follow-up studies on embryos after pre-implantation genetic aneuploidy screening indicate that the frequency of meiotic errors varies according to the referral reason, with the highest frequency being recorded for the recurrent miscarriage category and the lowest in the repeated implantation failure group where younger women have a good response to ovarian stimulation. The exceptionally high incidence of pre- and post-zygotic chromosomal anomalies seen in early human embryos is thus the product of several mechanisms. Firstly, the error-prone cell cycle during the embryonic cleavage stage and secondly, parental susceptibility to meiotic and mitotic chromosomal instability together with their general genetic background.     

Declarative versus episodic: two theories put to the test

The question of whether the hippocampus plays a selective role in episodic memory or a more general role in both episodic and semantic memory (together termed declarative memory) is an unresolved and much-debated topic in the current literature. In two back-to-back articles in this issue of Neuron, Squire and his colleagues describe findings from a group of six patients with damage thought to be limited to the hippocampus. The reported findings provide new evidence toward resolving this much-debated controversy.     

Aneuploidy in human sperm: a review of the frequency and distribution of aneuploidy, effects of donor age and lifestyle factors

Application of fluorescence in situ hybridization (FISH) analysis has opened the way for comprehensive studies on numerical chromosome abnormalities in human sperm. During the last decade, more than five million sperm from approximately 500 normal men were analyzed by a number of laboratories from around the world by this approach. Except for chromosome 19 which has been analyzed in only one study, all other chromosomes have been examined by two or more studies with considerable differences in disomy frequency for an individual chromosome among studies. The mean disomy frequency is 0.15% for each of the autosomes and 0.26% for the sex chromosomes. Most chromosomes analyzed have an equal distribution of disomy with the exception of chromosomes 14, 21, 22 and the sex chromosomes, which display significantly higher disomy frequencies. Slight but significant increases in disomy frequency with advancing paternal age were observed for some chromosomes, in particular for the sex chromosomes. Some lifestyle factors such as smoking, alcohol drinking and caffeine consumption have been investigated and no consistent association between disomy frequency and any type of lifestyle factors has been established. The question of whether different geographic and ethnic groups of men have inherent differences in frequency of disomic sperm has been investigated by two studies with conflicting results.     

Association between nondisjunction and maternal age in meiosis-II human oocytes.

The relationship between advanced maternal age and increased risk of trisomic offspring is well known clinically but not clearly understood at the level of the oocyte. A total of 383 oocytes that failed fertilization from 107 patients undergoing in vitro fertilization were analyzed by FISH using X-, 18-, and 13/21-chromosome probes simultaneously. The corresponding polar bodies were also analyzed in 188 of these oocytes. The chromosomes in the oocyte and first polar body complement each other and provide an internal control to differentiate between aneuploidy and technical errors. Two mechanisms of nondisjunction were determined. First, nondisjunction of bivalent chromosomes resulting in two univalents going to the same pole and, second, nondisjunction by premature chromatid separation (predivision) of univalent chromosomes producing either a balanced (2 + 2) or unbalanced (3 + 1) distribution of chromatids into the first polar body and M-II oocytes. Balanced predivision of chromatids, previously proposed as a major mechanism of aneuploidy, was found to increase significantly with time in culture (P < .005), which suggests that this phenomenon should be interpreted carefully. Unbalanced predivision and classical nondisjunction were unaffected by oocyte aging. In comparing oocytes from women <35 years of age with oocytes from women > or = 40 years of age, a significant increase (P < .001) in nondisjunction of full dyads was found in the oocytes with analyzable polar bodies and no FISH errors. Premature predivision of chromatids was also found to cause nondisjunction, but it did not increase with maternal age.