Sperm chromosome segregation of rob(4;16) and rob(4;16)inv(4) in the brown brocket deer (Mazama gouazoubira).

The genus Mazama stands out among the Neotropical deer due to their wide intra and interspecific karyotypic diversification, which is associated with an accentuated chromosomal fragility. There are reports of heterozygous Robertsonian translocation (RT) carriers in a free-range population of Mazama gouazoubira (brown brocket deer), as well as in captive animals of this and other species of the genus. To analyze possible negative impacts of heterozygous chromosome rearrangements on reproductive fitness of the carriers, we performed an analysis of sperm meiotic segregation in four brown brocket bucks, carriers of a rob(4;16), and compared the results with those of a normal buck. We established a reliable FISH and sperm-FISH protocol for the brown brocket deer using bovine (Bos taurus; diploid number, 2n = 60) whole chromosome painting (WCP) and BAC probes. Using BAC probes, we revealed the presence of a paracentric inversion (PAI) of the fused chromosome 4 in two of the four analyzed RT carriers. The mean frequency of normal/balanced sperm in the translocation carriers was significantly lower than in the normal buck (94.78% vs 98.40%). The mean value of total unbalanced spermatozoa was almost doubled in the RT/PAI carriers (6.68%) when compared to RT carriers (3.76%), but the difference was not statistically significant. This study demonstrated the efficiency of FISH with bovine WCP and BAC probes in the characterization of chromosome rearrangements and gametic segregation patterns in brown brocket deer. Our results indicate a low to moderate increase in the rates of unbalanced meiotic segregation products in brown brocket bucks heterozygous for RT and RT/PAIs.

Different chromosome damage in lymphocytes of newly diagnosed gastrointestinal and breast cancer patients.

Structural chromosome aberrations are a predictive biomarker of cancer risk. Conventional chromosome analysis widely used for these purposes detects unstable chromosome aberrations that are eliminated during cell division. Stable aberrations that may persist in the body and tend to accumulate during a lifetime can be detected by fluorescence in situ hybridization (FISH). The aim of the study was to investigate the level of chromosome damage in newly diagnosed cancer patients and control subjects by FISH. Both groups of untreated cancer patients had increased frequency of aberrant cells. However, chromosome damage affected different cytogenetic endpoints. Stable translocations and cells with complex rearrangements were elevated in breast cancer patients whereas unstable chromosome aberrations (dicentric chromosomes and acentric fragments) were elevated in gastrointestinal cancer patients. These associations observed in nonsmokers were typically not pronounced in smokers (with the exception of dicentric chromosomes in gastrointestinal patients). Exposure to tobacco smoke increased aberrations in healthy controls but not in the cancer patients. Our study suggests an association between cancer and stable chromosomal rearrangements in breast cancer patients. Unstable aberrations elevated in gastrointestinal cancer patients may be at least partly ascribed to the exposure to diagnostic X-rays.

[Biomonitoring of Work with Genotoxic Substances and Factors in a Cancer Treatment Facility]. / Biomonitoring práce s genotoxickými látkami a faktory v onkologickém zdravotnickém zarízení.

BACKGROUND: A long-term occupational exposure of healthcare staff to cytostatics and ionizing radiation is associated with a possible manifestation of their genotoxic, carcinogenic and teratogenic effects. MATERIAL AND METHODS: A total number of 101 employees working with cytostatics or ionizing radiation were examined (some of them repeatedly) in a cancer treatment facility. The control group consisted of 119 persons excluded from the risk exposure. Fluorescence in situ hybridization with three pairs of whole-chromosomal probes and a pancrossomeric probe was used and the translocation frequency was determined. RESULTS: The total number of chromosomal rearrangements of healthcare professionals and control group correlates with age. Taking into account the age dependence, an increased level of chromosomal reconstruction was found in the case of 11 individuals, 10 of which were female, working on the positions of pharmacist, general nurse, physician. A total of 9 of those case involved the work with cytostatics. Five of these cases were re-examined two years later and the observed levels dropped to the control level. CONCLUSION: The results of biomonitoring should be evaluated on a group basis and individually, taking into account the personal history and possible non-professional effects on individuals - in particular those related to specific environmental measurement results.Key words: preventive medicine - occupational exposure - cytostatic agents - chromosome aberrations - in situ hybridization - fluorescence The authors declare they have no potential conflicts of interest concern ing drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This work was supported by project of Ministry of Health Czech Republic. reg. No. 15-33968A.Submitted: 12. 4. 2018Accepted: 16. 4. 2018.

Total globozoospermia associated with increased frequency of immature spermatozoa with chromatin defects and aneuploidy: a case report.

Globozoospermia, characterised by the presence of round spermatozoa lacking acrosomes in an ejaculate, is a known cause of male infertility. Semen analysis, including sperm chromatin structure assay, toluidine blue, chromomycin A3 and aniline blue staining and fluorescence in situ hybridisation, was performed in an infertile globozoospermic patient to establish to which extent these genetic factors contributed to his infertility. No spermatozoa capable of hyaluronan (HA) binding were detected in the HA binding assay. Increased rates of immature spermatozoa with defective replacement of histones by protamines, DNA breaks and disturbed chromatin integrity and sperm aneuploid for the sex chromosomes were observed. Intracytoplasmic sperm injection (ICSI) was used in three in vitro fertilisation (IVF) cycles, and enough morphologically well-developing embryos were obtained in each cycle. However, no pregnancy was achieved. The infertility of our couple, resistant to IVF/ICSI treatment, was most probably caused by a combination of male and female factors.

A comparative study of meiotic recombination in cattle (Bos taurus) and three wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus).

The karyotypic evolution in the family Bovidae is based on centric fusions of ancestral acrocentric chromosomes. Here, the frequency and distribution of meiotic recombination was analyzed in pachytene spermatocytes from Bos taurus (2n = 60) and 3 wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus) (2n = 58) using immunofluorescence and fluorescence in situ hybridization. Significant differences in mean numbers of recombination events per cell were observed between B. taurus and members of the genus Connochaetes (47.2 vs. 43.7, p < 0.001). The number of MLH1 foci was significantly correlated with the length of the autosomal synaptonemal complexes. The average interfocus distance was influenced by interference. The male recombination maps of bovine chromosomes 2 and 25 and of their fused homologues in wildebeests were constructed. A significant reduction of recombination in the fused chromosome BTA25 was observed in wildebeests (p = 0.005). This was probably caused by interference acting across the centromere, which was significantly stronger than the intra-arm interference. This comparative meiotic study showed significant differences among the species from the family Bovidae with the same fundamental number of autosomal arms (FNa = 29) which differ by a single centric fusion.

Testicular sperm aneuploidy in non-obstructive azoospermic patients.

BACKGROUND: Non-obstructive azoospermic (NOA) men can father children after testicular sperm extraction (TESE). Previous studies suggest that they may be at risk of producing chromosomally abnormal spermatozoa, but the number of sperm analysed per patient was usually very low. METHODS: Multicolour fluorescence in situ hybridization was used for detection of chromosome 13, 15, 16, 18, 21, 22, X and Y disomy and diploidy in sperm obtained from NOA men (n = 17) and control donors (n = 10). At least 500 testicular sperm were scored in each patient to increase the precision of our study. RESULTS: The mean frequency of overall disomy (2.32%) and diploidy (0.80%) found in 13 689 testicular spermatozoa of NOA patients was significantly higher than in the ejaculated sperm of normospermic control donors, disomy (0.62%) and diploidy (0.29%). A highly significant increase in frequencies of chromosome 15, Y and overall disomy (P < 0.001), and a significant increase in disomy of chromosome 13 (P = 0.002), 16 (P = 0.031) and 21 (P = 0.018), overall diploidy (P = 0.031) and diploidy caused by errors in meiosis I (P = 0.011) were observed in the NOA group. CONCLUSIONS: Testicular sperm samples of NOA patients show a higher incidence of numerical chromosomal abnormalities compared with ejaculated sperm of control donors. Appropriate genetic counselling is necessary in NOA men undergoing TESE.

The effect of the swim-up and hyaluronan-binding methods on the frequency of abnormal spermatozoa detected by FISH and SCSA in carriers of balanced chromosomal translocations.

BACKGROUND: The swim-up and hyaluronan (HA)-binding methods are used for the selection of good quality spermatozoa to improve pregnancy rates and embryo quality and to reduce the number of miscarriages after IVF. We evaluated whether the processing of sperm by these methods reduces the frequency of spermatozoa with abnormal karyotypes and altered chromatin quality in balanced translocation carriers. METHODS: Semen samples of 12 carriers of balanced chromosomal translocations were analysed for the frequency of spermatozoa, which are chromosomally unbalanced due to the segregation of balanced translocations, aneuploidies for chromosomes 7, 8, 13, 18, 21, X or Y, diploid sperm or sperm with fragmented DNA and poorly condensed chromatin. Results obtained by fluorescence in situ hybridization (FISH) and sperm chromatin structure assay were compared between ejaculated (n = 12), swim-up (n = 12) and HA-binding processed (n = 6) semen samples of the translocation carriers and with the control group (n = 10). RESULTS: The mean frequencies of unbalanced segregation products were 17.5 and 16.5% in neat and swim-up processed samples from Robertsonian translocation carriers, and 55.4, 54.5 and 50.9% in neat, swim-up and HA-bound sperm samples from reciprocal translocation carriers. Significant decreases in the frequency of sperm showing chromosome 18 and XY disomy and of diploidy, and in the rates of high-density staining sperm were observed in the motile swim-up fractions. There were significantly more sperm showing fragmented chromatin in the group of translocation carriers than in the control group, but no differences in the aneuploidy and diploidy rates were observed. CONCLUSIONS: The swim-up method is suitable for selection of sperm with condensed chromatin and a lower frequency of some aneuploidies and of diploidy. The frequency of spermatozoa chromosomally unbalanced due to the segregation of reciprocal (but not Robertsonian) translocations is significantly lower in HA-bound sperm. However, the advantages of either method for selecting normal sperm are limited.

Detection of translocation rob(1;29) in bull sperm using a specific DNA probe.

The Robertsonian translocation rob(1;29), connected with reduced fertility, is widespread in different cattle breeds all over the world. After laser microdissection, DOP-PCR, cloning and sequencing, a highly sensitive translocation-specific DNA probe, suitable for detection of rob(1;29) in cattle metaphase and interphase cells, including spermatozoa was designed. Sperm samples of five heterozygous translocation carriers were analyzed using this probe and a control probe for chromosome 6. One thousand decondensed spermatozoa from each bull were scored. Signals of the translocation-specific probe were detected in 48.8, 50.9, 50.1, 51.8, and 54.8% of spermatozoa, respectively. In contrast, semen samples from five chromosomally normal bulls showed only signals of the control probe for chromosome 6. Semen from a chimeric (XX/XY) bull, showing 57.5% of 59,XX,rob(1;29) and 42.5% of 60,XY cells in cultured peripheral lymphocytes, was also examined using this probe. No sperm head with signal of the translocation-specific probe was observed among 1,000 spermatozoa analyzed in this bull, demonstrating that female cells do not pass through the process of spermatogenesis.

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.

Frequency of aneuploidy in pig oocytes matured in vitro and of the corresponding first polar bodies detected by fluorescent in situ hybridization.

The objectives of this study were to develop a two-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in gilt oocytes using chromosome-specific DNA probes, and to establish baseline frequencies of aneuploidy in pig oocytes matured in vitro. The ovaries were collected from gilts at the local slaughterhouse. Immature oocytes were isolated by slicing the cortex of the ovaries. The oocytes were matured in microplate wells using TCM-199 medium supplemented with 10% estrous cow serum, sodium pyruvate, antibiotics, and gonadotrophins. After 44 h of maturation the oocytes were incubated with hyaluronidase and the cumulus cells were removed by vortexing. Single oocytes were transferred into 1 microL drops of a lysing buffer (0.01 N HCl/0.1% Tween 20) on clean microscopic slides. Two-color FISH was performed using probes specific for Chromosomes 1 and 10. The probe for Chromosome 1 was labeled with Cy3-dUTP and a probe labeled with fluorescein-11-dUTP was used for Chromosome 10. Only oocytes in which a complementary first polar body was found were confirmed as aneuploid. The final assessment of aneuploidy was based on results of 1189 haploid oocytes. Thirty-four (3%) of the examined oocytes were aneuploid. Disomy of Chromosome 1 and Chromosome 10 was found in 12 of 34 and 8 of 34 of the aneuploid oocytes, respectively. Nullisomy of Chromosome 1 and Chromosome 10 was found in 8 of 34 and 6 of 34 of the aneuploid oocytes. No significant differences were found in the frequencies of disomies and nullisomies of oocytes or in the frequencies of aneuploidies of Chromosomes 1 and 10. The frequency of aneuploid oocytes determined by FISH seems to be higher than that determined by conventional methods in other laboratories.

[Air pollution and sperm quality]. / Znecistení ovzdusí a kvalita spermií.

BACKGROUND: It has been described that an exposition of males to chemical substances may significantly impoverish quality and quantity of produced spermatozoa. The aim of our study was to test whether the polluted air in the Teplice district has negative effects on the quality of sperm of males living in this district. METHODS AND RESULTS: 325 males 18-year-old living in the Teplice district and in the control district of Prachatice were tested. Samples were taken in 1992 and 1994, always at the end of winter and in autumn. According to WHO laboratory manual for investigation of the human sperm, basic parameters were determined: volume of the semen, pH, motility, number and morphology of spermatozoa. In selected groups of males the frequency of aneuploidia of spermatozoa was also examined. Examination of aneuploidia was done using three color fluorescence in situ hybridisation with satellite DNA proves specific for X, Z and 8 chromosomes. Logistic regression was used for the data analysis and Odd's Ratio was estimated (OR's). OR's was found for the morphology of spermatozoa (4.1 and 10.1 for medium and high exposition respectively), for the head morphology (6.1 and 4.1) and in the percentage of motile spermatozoa (9.8 and 3.5). More intensively exposed males had higher frequency of disomy in chromosomes X (p = 0.012), XY (p = 0.01), and Y (p < 0.001). CONCLUSIONS: Bio-indicators of toxic and genetic impairment have shown lower quality of sperm in males in Teplice district.

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.

Cytogenetic analysis of peripheral lymphocytes in medical personnel by means of FISH.

Genotoxic effects of occupational exposure to cytostatics were investigated in 20 nurses and physicians working in various departments of one hospital. The group was divided into two equal subgroups one of which was involved in the administration of cytostatics (exposed subgroup) and the other was not (unexposed subgroup). The whole group and the two subgroups were compared with a control group of 11 healthy blood donors. Two differently labeled whole chromosome painting (WCP) probes specific for the chromosomes 1 and 4 were used simultaneously. Chromosome aberrations were classified in terms of the Protocol for Aberration Identification and Nomenclature (PAINT) nomenclature. The results obtained by the painting method were compared with findings of conventional unbanded chromosome analysis. Significant differences in the numbers of translocations (FG/100 = 2.25 +/- 1.50 vs. 0.66 +/- 0.21, p < 0.01) and unstable chromosome aberrations determined by the conventional method (AB.C/100 = 2.70 +/- 2.31 vs. 1.63 +/- 1.59, p < 0.05) were found between the exposed subgroup and controls. The unexposed subgroup differed from the controls only in the number of translocations (FG/100 = 2.93 +/- 2.79 vs. 0.66 +/- 0.51, p < 0.05). No significant differences in the number of stable and unstable aberrations were found between the exposed and the unexposed subgroups. On the other hand, highly significant differences (p < 0.01) were demonstrated by the two methods between the whole group (all medical personnel) and the controls. All differences which were found to be significant when translocations were compared were also found to be significant when total stable chromosome exchanges, i.e., the sum of translocations and insertions, were considered. Multicolour chromosome painting is apparently a more sensitive method than the conventional metaphase-based analysis.