Jiri Rubes

The use of laser microdissection for the preparation of chromosome-specific painting probes in farm animals.

Laser microbeam microdissection and laser pressure catapulting procedure were used for the construction of chromosome-specific painting probes, arm-specific probes and probes for chromosomal subfragments. We report on a method for generation of fluorescence in-situ hybridization probes from laser dissected chromosomes of farm animals. So far, using the described method, a set of chromosome-specific painting probes has been obtained for all porcine chromosomes, 17 chromosomes of cattle and selected equine chromosomes. It is concluded that the laser technology appears to be a useful and powerful tool for the construction of chromosome-specific painting probes. Its main advantage is the fast non-contact collection of chromosomes.

FAST-FISH with laser beam microdissected DOP-PCR probe distinguishes the sex chromosomes of Silene latifolia

We present an improved FISH strategy for differentiating the sex chromosomes of the dioecious model plant, Silene latifolia. Fixed mitotic protoplasts were dropped on a polyethylene naphthalate membrane, the X or Y chromosomes were isolated using nitrogen laser beam microdissection, catapulted by laser pressure, and amplified by DOP-PCR. A modified FAST-FISH protocol based on a short hybridization time combined with a low concentration of probe was used. The success of this approach is demonstrated by the differential labeling of the X and Y chromosomes and it could represent a quick method for comparing organization of plant genomes.

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.

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.

Genetic polymorphisms influence the susceptibility of men to sperm DNA damage associated with exposure to air pollution.

The purpose of the present study was to investigate the impact of carcinogenic polycyclic aromatic hydrocarbons and volatile organic compounds on sperm quality in a group of city policemen in Prague during a period of increased concentrations of ambient air-pollutants (winter season) compared to a period of low exposure (spring). Polymorphisms in metabolic genes (CYP1A1, EPHX1, GSTM1, GSTP1, GSTT1), folic acid metabolism genes (MTR, MTHFR) and DNA repair genes (XRCC1, XPD6, XPD23, hOGG1) were evaluated in these men as potential modifiers of associations between air pollution exposure and changes in sperm quality. The study population was a group of 47 policemen working in the center of the city. Seasonal differences in exposure were verified by ambient and personal monitoring. Markers of sperm injury included semen volume, sperm concentration, sperm morphology, sperm motility, and sperm DNA damage measured with the sperm chromatin structure assay The sperm chromatin structure assay (SCSA) includes a measure of DNA damage called DNA Fragmentation Index (DFI). The % of cells with detectable DFI (detDFI) by this assay includes sperm with either medium or high DNA damage; the term hDFI is used to define the % of sperm with only high DNA damage. The assay also detects immature sperm defined by high density staining (HDS). No significant differences were found in any of the standard semen parameters between the sampling periods except for vitality of sperms. Both DFI and HDS were significantly higher in winter than in spring samples for all men and for non-smokers. At the bivariate level, significant associations between hDFI or detDFI and polymorphisms of the repair genes XRCC1, XPD6 and XPD23 were observed. In multivariate models, polymorphisms of the genes XPD6, XPD23 and CYP1A1MspI were associated with hDFI and HDS. Moreover, HDS was significantly associated with polymorphisms in GSTM1 gene.

Phylogenomic study of spiral-horned antelope by cross-species chromosome painting

Chromosomal homologies have been established between cattle (Bos taurus, 2n = 60) and eight species of spiral-horned antelope, Tribe Tragelaphini: Nyala (Tragelaphus angasii, 2n = 55♂/56♀), Lesser kudu (T. imberbis, 2n = 38♂,♀), Bongo (T. eurycerus, 2n = 33♂/34♀), Bushbuck (T. scriptus, 2n = 33♂/34♀), Greater kudu (T. strepsiceros, 2n = 31♂/32♀), Sitatunga (T. spekei, 2n = 30♂,♀) Derby eland (Taurotragus derbianus 2n = 31♂/32♀) and Common eland (T. oryx 2n = 31♂/32♀). Chromosomes involved in centric fusions in these species were identified using a complete set of cattle painting probes generated by laser microdissection. Our data support the monophyly of Tragelaphini and a clade comprising T. scriptus, T. spekei, T. euryceros and the eland species T. oryx and T. derbianus, findings that are largely in agreement with sequence-based molecular phylogenies. In contrast, our study suggests that the arid adaptiveness of T. oryx and T. derbianus is recent. Finally, we have identified the presence of the rob(1;29) fusion as an evolutionary marker in most of the tragelaphid species investigated. This rearrangement is associated with reproductive impairment in cattle and raises questions whether subtle distinctions in breakpoint location or differential rescue during meiosis underpin the different outcomes detected among these lineages.

Sperm fluorescence in situ hybridization study of meiotic segregation and an interchromosomal effect in carriers of t(11;18)

BACKGROUND Alanced translocations are associated with infertility, spontaneous abortions and birth defects. METHODS We report the analysis, by multicolour fluorescence in situ hybridization (FISH), of meiotic segregation and aneuploidy of chromosomes X, Y, 7, 8 and 21 in sperm from three men who are carriers of two different translocations involving chromosomes 11 and 18. A control group comprised ten young, healthy normospermic men. RESULTS The higher prevalence of alternate segregation followed by adjacent 1, adjacent 2 and 3:1, and other segregants was observed in all three patients. Two carriers of the same translocation differed only in the frequency of adjacent 2 segregation (P < 0.01). The carrier of the other translocation showed significantly higher frequency of alternate (P < 0.01) and less adjacent 1 and 3:1 segregation products (P < 0.01). An increased frequency of XY (P < 0.01), YY (P < 0.05) and diploid (P < 0.01) sperm was also detected in the group of translocation carriers compared with the control group. This difference was caused by elevated frequencies of disomy and diploidy in two of our carriers. CONCLUSIONS The incidence of chromosomally unbalanced or aneuploid gametes varies in the individual translocation carriers even if the same chromosomes are included in the translocation. FISH analysis provides information useful for genetic counseling and assisted reproduction.

Aneuploidy detection in porcine embryos using fluorescence in situ hybridization

In contrast to human embryos, there are very few studies published on the frequency of chromosomal aneuploidy in farm animals. The objectives of this study were to apply a three-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in porcine embryos using chromosome-specific DNA probes, establish baseline frequencies of aneuploidy in embryos and compare the results with our previous findings of aneuploidy in spermatozoa and oocytes. The embryos were collected from superovulated gilts, which were slaughtered 48 h after insemination. FISH was performed using probes specific for the centromeric regions of porcine chromosomes 1, 10 and Y. Altogether 403 blastomeres from 114 porcine embryos were successfully investigated. Diploidy was observed in 101 (88.6%) embryos, triploidy in 2 (1.8%) embryos, mosaicism/mixoploidy in 9 (7.9%) embryos, and trisomy for chromosomes 1 or 10 in 2 (1.8%) embryos. No blastomere showed aneuploidy for chromosome Y. These findings correspond with the frequencies of aneuploidy we have found previously in porcine germ cells.

Chromosome evolution in the subtribe Bovina (Mammalia, Bovidae): The karyotype of the Cambodian banteng (Bos javanicus birmanicus) suggests that Robertsonian translocations are related to interspecific hybridization

Three subspecies of banteng (Bos javanicus) have been described: B. j. javanicus in Java, B. j. lowi in Borneo, and B. j. birmanicus in Cambodia, Lao PDR, Myanmar, Thailand and Vietnam. In this paper we provide the first description of the karyotype of the Cambodian banteng. The chromosomal complement of B. j. birmanicus differs from that of B. j. javanicus, which was previously found to be similar to that of cattle, Bos taurus (2n = 60). The Cambodian banteng karyotype has a diploid number of 2n = 56 (FN = 62) and the karyotype consists of 26 pairs of acrocentric chromosomes and two pairs of submetacentric chromosomes. Comparisons with other species of the subtribe Bovina show that the two pairs of bi-armed chromosomes resulted from two centric fusions involving the equivalent of cattle chromosomes 1 and 29, and 2 and 28, respectively. Cross-species fluorescence in-situ hybridization (FISH) with B. taurus whole chromosome paints and satellite DNA I probes was used to identify the chromosomes involved in the translocations, and their orientation. We suggest that Robertsonian translocations (1;29) and (2;28) have been fixed in the common ancestor of Cambodian banteng as a consequence of hybridization with the kouprey (Bos sauveli) during the Pleistocene epoch.

Frequency of Aneuploidy Related to Age in Porcine Oocytes

It is generally accepted that mammalian oocytes are frequently suffering from chromosome segregation errors during meiosis I, which have severe consequences, including pregnancy loss, developmental disorders and mental retardation. In a search for physiologically more relevant model than rodent oocytes to study this phenomenon, we have employed comparative genomic hybridization (CGH), combined with whole genome amplification (WGA), to study the frequency of aneuploidy in porcine oocytes, including rare cells obtained from aged animals. Using this method, we were able to analyze segregation pattern of each individual chromosome during meiosis I. In contrast to the previous reports where conventional methods, such as chromosome spreads or FISH, were used to estimate frequency of aneuploidy, our results presented here show, that the frequency of this phenomenon was overestimated in porcine oocytes. Surprisingly, despite the results from human and mouse showing an increase in the frequency of aneuploidy with advanced maternal age, our results obtained by the most accurate method currently available for scoring the aneuploidy in oocytes indicated no increase in the frequency of aneuploidy even in oocytes from animals, whose age was close to the life expectancy of the breed.