Development of a genetic sexing strain of Anopheles arabiensis for KwaZulu-Natal, South Africa.

An efficient sexing system is important for the release of sterile males for any control programme using the sterile insect technique. This study describes the development and characterization of a new genetic sexing strain from South Africa (GMK), needed for the planned implementation of such a programme in northern KwaZulu-Natal Province. The base colony used was a locally modified laboratory strain of Anopheles arabiensis containing a sex-linked gene conferring dieldrin resistance to male mosquitoes. Female A. arabiensis mosquitoes from northern KwaZulu-Natal were mated with these males and backcrossed to introduce the dieldrin resistance gene to the Y chromosome. The resulting strain therefore had an overall genotype representing the local population but with the Y chromosome containing the dieldrin resistance gene. Life-history characteristics, stability of the sex-linked resistance marker, and reduction in dieldrin waste were investigated. The strain showed semi-sterility exhibited by low egg hatch rates, faster development in the immature stages and longer adult survivorship compared with the parental strains. While the GMK strain carrying the dieldrin-resistant gene was successfully established, the stability of the gene is limited, requiring periodic purification. Dieldrin waste can be limited by treating many more eggs than currently recommended.

A new cause of male infertility after cisplatin exposure: the effect of cisplatin on Y chromosomes.

OBJECTIVES: To investigate the impact of cisplatin (CP) on the testes-specific protein, Y-linked (TSPY) gene situated on the Y chromosome. METHODS: The control group consisted of 10 rats. Group IIA consisted of 15 rats that underwent orchiectomy and received three cycles of 1 mg/kg, 2.5 mg/kg, or 5 mg/kg CP. Group IIB was exposed to the same doses of three cycles of chemotherapy but was examined after 3 months of chemotherapy. Group III was exposed to the same doses of chemotherapy without initial orchiectomy. Reverse transcriptase polymerase chain reaction for TSPY messenger ribonucleic acid (mRNA) and immunohistochemical staining for histone 2B were performed on the testes. Results were evaluated by one-way analysis of variance. RESULTS: Compared with the controls, the expression of TSPY mRNA in Group IIA after exposure to 1 mg/kg CP did not change; however, mRNA levels after exposure to 2.5 mg/kg and 5 mg/kg CP were decreased by 40% and 78%, respectively. In Group III after exposure to the same doses of CP, mRNA levels decreased by 30%, 87.5%, and 88%, respectively. The expression of TSPY was at normal levels except in rats that received 5 mg/kg CP in Group IIB. Immunohistochemical study revealed that histone 2B expression was decreased in a dose-dependent manner. None of the rats from any of the groups died during the study period. CONCLUSIONS: Decreased TSPY expression after CP exposure might be another mechanism for male infertility.

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.

Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome.

OBJECTIVE: To evaluate the incidence of sperm aneuploidy in men screened for infertility and identify any eventual relation with assisted reproductive outcome. DESIGN: Controlled prospective study. SETTING: University hospital-based IVF program. PATIENT(S): Infertile couples who were screened for sperm aneuploidy and evaluated for IVF treatment. INTERVENTION(S): Fluorescence in situ hybridization was used to identify chromosomes 18, 21, X, and Y. The assisted reproductive techniques of IVF and intracytoplasmic sperm injection were used for infertility treatment. MAIN OUTCOME MEASURE(S): The incidence of sperm aneuploidy, semen parameters, fertilization rate, pregnancy characteristics, and rate of neonatal malformations were determined. RESULT(S): Oligozoospermic and teratozoospermic men had a significantly higher incidence of chromosomal abnormalities than men with normal semen parameters (2.7% vs. 1.8%). The increased frequency of sperm aneuploidy did not appear to affect pregnancy losses or the occurrence of neonatal malformations. CONCLUSION(S): Suboptimal semen samples had a higher incidence of aneuploidy. In this study, the increased frequency of chromosomal abnormalities did not have a direct effect on the fertilization rate, pregnancy characteristics, or neonatal outcome.

Sperm aneuploidy among Chinese pesticide factory workers: scoring by the FISH method.

BACKGROUND: A study of the prevalence of sperm aneuploidy among pesticide factory workers was conducted in Anhui, China. METHODS: We recruited 75 men: 32 subjects from a large pesticide-manufacturing plant and 43 subjects from a nearby textile factory free of pesticide exposure. Each subject met the following criteria: age of 20-40 years; continuous work in the plant for 3 months prior to the study, no congenital anomalies or acquired disease of the external genitalia and no history of recent febrile illness or mumps. Within one hour after collection from each subject, semen was evaluated in terms of several parameters and smear slides were prepared. RESULTS: Exposure assessment revealed that workers in the pesticide plant were exposed to ethyl parathion or methamidophos, each of which is a potent organophosphate pesticide, at a median level of 0.02 mg/m3 (8-hour time weighted average as measured by personal pump) while workers in the control plant had no such occupational exposure. Twenty-nine semen slides (13 from the exposed group and 16 from the unexposed group) were randomly chosen for aneuploidy scoring by the three-color fluorescence in situ hybridization (FISH) method with scorers being unaware of exposure status. Median semen parameters were as follows for exposed (and unexposed) men: abstinence period, 3 days (4 days); sperm concentration, 52.8x10(6)/ml (53.1x10(6)/ml); proportion of sperm with normal motility, 50.5% (61.3%); and proportion of sperm with normal morphology, 59% (61.5%). The specific chromosome abnormalities of interest were disomy for chromosome 18 and the three different types of sex chromosome disomy (i.e. XX, XY, YY disomy). The crude proportion of all aneuploidy combined was 0.30% and 0.19% for sperm from exposed and unexposed men, respectively. Poisson regression with overdispersion adjustment yielded significantly different crude risks of aneuploidy - 3.03 and 1.94 per 1,000 sperm from exposed and unexposed men, respectively - giving a rate ratio of 1.56 (95% CI, 1.06-2.31). The regression coefficients remained statistically significant after adjustment for inter-technician variability giving a rate ratio of 1.51 (95% CI, 1. 04-2.20). CONCLUSIONS: We conclude that occupational exposure to organophosphate pesticides moderately increases the prevalence of sperm aneuploidy.

[Chromosome damage in the course of laryngeal squamous cell carcinoma]. / Uszkodzenia chromosomów w przebiegu plaskonablonkowego raka krtani.

The aim of the article is a review of own cytogenic studies on laryngeal cancer confronted with the literature data. Spontaneous and bleomycin-induced chromosome instability was analysed in peripheral blood lymphocytes in relation to genetic risk of cancer incidence and progression. Comparative genome hybridization (CGH) was applied to demonstrate gains and losses of DNA copy number in tumour and non-tumour laryngeal mucosa. The profiles of imbalances of DNA copy number were shown to differ between metastazing and non-metastazing tumours. Preliminary data indicate a frequent loss of Y chromosome in tumour cells. The loss of heterozygosity at chromosome p53 locus (17p) has been shown to be more frequent than at chromosome locus coding 16 gene (9p). Altogether, the experiments have proven that a dynamics of chromosome aberrations is highest at the stage of metastasis.

Aneuploidy in male Indian muntjac cells is limited to the Y2 chromosome.

In untreated cell cultures of Indian muntjac (Munticus muntiacus vaginalis; 2n = 6 female, 7 male) we observed that spontaneous aneuploidy is limited primarily to the Y2 chromosome. We therefore treated the cells with aneugenic agents to determine if induced aneuploidy follows the same pattern and, hence, if there are limitations on the generation of aneuploidy or survival of cells lacking certain chromosomes. Exposing the cells to benomyl (8-100 micrograms/ml for 1 h), caffeine (5 x 10(-5)-2 x 10(-4) M for 2, 24 and 72 h) and colchicine (2 x 10(-4) and 5 x 10(-5) M for 1, 24, 48 and 72 h) resulted in cells primarily aneuploid for the Y2 chromosome. The frequency of cells lacking Y2 was far higher than those having an extra Y2 chromosome. The frequency of cells aneuploid for all other chromosomes combined was much lower than that for Y2. The data imply that the Indian muntjac genome can tolerate loss of the Y2 chromosome only and that aneuploidy for other chromosomes might cause lethality. This might be because the small number of chromosomes in the genome predisposes aneuploid cells to an imbalance of genes carrying out the basic activities required for cell division and cell survival. Because of the small chromosome number, the large size of the chromosomes and the ease of distinguishing every chromosome without banding or any other special treatment, e.g. FISH, this system could be useful and convenient in the study of induction of aneuploidy. This simple and inexpensive system can be utilized as a screening system for preliminary studies dealing with induced aneuploidy.

Synaptonemal complex aberrations in the pseudoautosomal region of X, Y chromosomes in irradiated hamsters.

The effects of X-radiation, bleomycin and amsacrine (m-AMSA) on the meiotic chromosomes of male Armenian hamsters were determined by electron microscopic analysis of synaptonemal complex (SC) damage. Pachytene stage cells were analyzed 5 or 6 days following their treatment at putative preleptotene-leptotene stages of meiosis. Of the multiple types of SC aberrations observed to be significantly increased over control levels, lateral element breakage and synaptic anomalies were most prevalent. The focus of these studies was on the sex chromosomes which, in the Armenian hamster, reveal an unusually well-defined pseudoautosomal region. In the XY pair, radiation and chemical treatments caused certain forms of structural and synaptic anomalies which appeared to be preferentially localized to telomeric and/or crossover regions. The nature of these specific aberrations, involving breakage, bridge formation and asynapsis, is not well understood; however, their distributions are suggestive of possible relationships with sites and processes of crossing over.

Fluorescence in situ hybridization to Y chromosomes in decondensed human sperm nuclei.

Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome-specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.

Inhibition of heterochromatin condensation of human Y chromosome by distamycin-A.

Distamycin-A, an oligopeptide antibiotic with a N-methylpyrrole ring system and propionamide side chain, preferentially forms stable bonds with AT rich double stranded DNA. When introduced to cell cultures, it inhibits condensation of the heterochromatic region of the Y chromosome. The frequency of metaphases showing inhibition of heterochromatin condensation of the Y chromosome was found to be dependent on the treatment time and concentration of distamycin-A in the culture medium. When distamycin-A was added to a concentration of 100 micrograms/ml at the start of the culture (72 hours), the frequency of Y heterochromatin decondensation was found to be 48%, 30% and 6% in amniotic fluid, lymphocyte and fibroblast cultures respectively. The highest frequency of metaphases with decondensed Y heterochromatin were observed when distamycin-A treatment was carried out for the last 24 hours prior to harvest, the frequencies being 94%, 72% and 59% in amniotic fluid, lymphocyte and fibroblast cultures respectively. Increase in the concentration of distamycin-A from 25 micrograms/ml to 50 micrograms/ml during the last 24 hours of culture increased the incidence of metaphases with Y heterochromatin decondensation from 51% to 69% in amniotic fluid, 40 to 49% in lymphocyte and 29% to 31% in fibroblast cultures. Highest frequency of metaphases with Y heterochromatin decondensation were observed when the cultures were exposed to distamycin-A at a concentration of 100 micrograms/ml for the last 24 hours of culture.

[The heterochromatin regions of the mitotic chromosomes in cattle]. / Geterokhromatinovye uchastki mitoticheskikh khromosom krupnogo rogatogo skota.

Cytochemical and molecular peculiarities of heterochromatic regions of bovine chromosomes have been studied, using specific fluorochrome staining induced decondensation, in situ hybridization, pretreatment of restriction enzymes. The heterochromatin of autosomes demonstrated a strong homogeneity. In chromosome Y two small specific heterochromatic regions were found lacking a long repeated tandem block of nucleotides enriched in GC base pairs and having no tandem block of Bkm repeats (10(4) b.p.). This class repeats are probably interspersed in the bovine genome. A rather seldom character of mammalian karyotypes is the absence of cytochemical heterochromatin in the X chromosome.

Precocious desynapsis of XY-bivalent in mouse germinal cells: influence of mitomycin C on tumour-bearing and normal mice.

A comparative study on the cytotoxic potential of anticancer-antibiotic mitomycin C has been made on tumour-bearing and normal mice considering precocious desynapsis of sex bivalent as parameter. The study indicates a strikingly differential effect of the drug on the phenomenon in two different types of mice. The administration of mitomycin C at therapeutic dose although enhances the frequency of precocious desynapsis of XY-bivalent in non-tumour (normal) mice to a significant extent (compared to control), the same drug at the same dose fails to produce a similar effect on tumour-bearing specimens. Discussions have been made on: (i) the probable cause for this differential effect, (ii) the mechanism of mitomycin action on precocious desynapsis of sex bivalent and, (iii) the possible significance of the findings in relation to cancer chemotherapy.

Berenil-induced undercondensation in human heterochromatin.

The aromatic diamidine berenil specifically inhibits the condensation of a subset of constitutive heterochromatin in human lymphocyte cultures. In the normal male chromosome complement, only the quinacrine-brilliant Y heterochromatin exhibits distinct undercondensation. The optimal culture conditions for inhibiting heterochromatin condensation are achieved when berenil is added at a final concentration of 150 micrograms/ml 24 h before cell harvest. Various examples of the use of berenil in the analysis of chromosome rearrangements involving quinacrine-brilliant heterochromatin are presented. A variant, giant-satellited chromosome 22 was found to respond to berenil treatment, although its enlarged and quinacrine-bright short-arm region did not contain Y heterochromatin. Southern blot analysis and chromosome in situ hybridization suggested that most chromosome 22 variants do not stem from Y; acrocentric translocations. The experimentally undercondensed Y heterochromatin is characterized by moderate C-band labeling, bright quinacrine fluorescence, and specific silver staining. At the ultrastructural level, undercondensation is associated with loosely packed, mutliply folded chromatin fibers with a diameter of approximately 250 A and organized probably as loops.

A genotoxic study of metal workers exposed to trichloroethylene. Sperm parameters and chromosome aberrations in lymphocytes.

Metal workers exposed to trichloroethylene for the degreasing of metals were studied to evaluate the genotoxicity of this exposure. For 15 workers presently exposed to high doses of trichloroethylene there was no difference from unexposed persons with respect to sperm count and morphology, and a small increase of two fluorescent bodies (YFF%) in spermatozoa. In contrast, there was a highly significant increase in frequency of structural aberrations (breaks, gaps, translocation, deletions, inversions) and hyperdiploid cells in cultured lymphocytes from trichloroethylene degreasers. As control groups, physicians from chemically non-exposed surroundings and a concurrently sampled reference from cytogenetic investigations were used. This study indicates positive correlations between exposure to trichloroethylene and somatic chromosome aberrations, whereas no effect on male germ cells could be demonstrated.

Insect sex chromosomes. VI. A presumptive hyperactivation of the male X chromosome in Acheta domesticus (L.).

The functional status of the X chromosome in Acheta domesticus has been analysed at the whole chromosome level on the basis of (1) 3H-thymidine autoradiography, (2) 5-BrdU/AO fluorescence microscopy (3) in vivo 5-BrdU incorporation and (4) 3H-UdR induced aberrations. The rationale of these techniques in relation to the functional aspect of the X chromosome is that the inactive X chromosome would (1) show asynchrony in DNA synthesis, (2) show differential fluorescence, (3) respond differentially to in vivo 5-BrdU treatment and (4) the active X chromosome would show aberrations when treated with 3H-Uridine. From the results, it appears that the X chromosomes in both male (XO) and female (XX) somatic cells of Acheta are euchromatic (active). Further, the single X in the male is transcriptionally as active as the two X chromosomes in the female. In other words, the single X in the male is hyperactive when compared with the single X in the female. From this it is inferred that the male X chromosome is differentially regulated in order to bring about an equalization of it's gene product(s) to that produced by both Xs in the female. Drosophila melanogaster has a comparable system of dosage compensation. Thus, Acheta is yet another insect showing evidence for an X chromosome regulatory mechanism of dosage compensation. Additionally, it is surmised that sex determination in Acheta is based on an autosomes/X chromosome balance mechanism.

Evaluation of the genetic activity of industrially produced carbon black.

Commercially produced oil furnace carbon black (Chemical Abstract Service Registry No. 1333-86-4) has been evaluated by five different assay for genetic activity. These were the Ames Salmonella typhimurium reverse mutation test, sister chromatid exchange test in CHO cells, mouse lymphoma test, cell transformation assay in C3H/10T1/2 cells, and assay for genetic effects in Drosophila melanogaster. Limited cellular toxicity was exhibited but no significant genetic activity was noted.