Radiosensitivity of somatic cells and imaginal disks of male and female Drosohila]

Primary chromosome damages as well as the frequency of spontaneous and X-rays induced chromosome aberrations in Drosophila melanogaster males and females are studied. It is found using cytofluorimetric method that primary chromosome damages in ganglia cells of females and males are the same. In these cells as well as in cells of imaginal discs the frequency of induced chromosome aberrations, except gaps, is considerably higher for females. Ganglia cells of females and males of Drosophila are found not to differ from each other in their proliferation activity. The frequency of morphoses for both sexes is also the same. The assumption is made concerning the role of the non-identical repair in the increased mutability of female somatic cells, which is more intensive in this sex, as well as concerning more intensive identical repair in imaginal discs of females.     

Chromosome aberrations induced by gamma radiation in the somatic cells of a radiosensitive mutant line of Drosophila melanogaster

Chromosome aberrations induced by gamma-rays in ganglia cells of Drosophila melanogaster larvae have been studied. Two strains of Drosophila were used: radiosensitive mutant rad (2) 201G1 and normal strain. It has been shown that the frequency of cells with chromosome aberrations in radiosensitive larvae is much more than in normal larvae after gamma-irradiation. The ratio of chromosome and chromatid deletions number to the number of exchange type aberrations is the same for both strains. The kinetics of chromosome aberrations induced in rad-larvae is similar to the normal one. The conclusion has been made that the realization of rad (2) 201G1 mutation takes place on the cell level.     

Delayed Formation of Chromosome Aberrations in Mouse Pachytene Spermatocytes Treated with Triethylenemelamine (Tem)

Induction of chromosome aberrations in pachytene spermatocytes of mice by 2 mg/kg TEM was compared with induction by 400 R X rays. These doses induced comparably high dominant lethal effects in pachytene spermatocytes of mice. Cytological analysis at diakinesis–metaphase I stage showed that whereas 76.4% of the cells treated with X rays at pachytene stage had aberrations, the frequencies observed in two TEM experiments were only 0.8 and 2.2%. On the other hand, 5% of the progeny from TEM-treated pachytene spermatocytes were found to be translocation heterozygotes. This is the first report on the recovery of heritable translocations from treated spermatocytes of mice. The aberration frequencies observed for TEM in diakinesis–metaphase I were much too low to account for all the lethal mutations and heritable translocations. Thus, the formation of the bulk of aberrations induced by TEM in pachytene spermatocytes was delayed—a marked contrast to the more immediate formation of X-ray-induced aberrations. It is postulated that the formation of the bulk of TEM-induced aberrations in pachytene spermatocytes and in certain postmeiotic stages occurs sometime during spermiogenesis, and not through the operation of postfertilization pronuclear DNA synthesis.     

Cell-stage-specific enhancement by caffeine of the frequency of chromatid aberrations induced by X-rays in neural ganglia of Drosophila melanogaster

Caffeine (10(-2) M) induced a high level of chromatid aberrations in neural ganglia of third-instar larvae of Drosophila melanogaster only when it was added to cells in late G2 and mitotic prophase. No aberrations were observed after treatment in late S--middle G2 or C-mitosis. We observed that, in these stages, caffeine strongly increased X-ray-induced damage (500 R). This potentiation was quantitatively similar. But it involved all types of aberration after treatment in C-mitosis, and essentially isochromatid deletions and chromatid exchanges after treatment in S--G2. Some hypotheses are put forth to explain the possible mechanism of action of caffeine in the potentiation of X-ray-induced damage.     

An autoradiographic study of unscheduled DNA synthesis in the germ cells of male mice treated with X-rays and methyl methanesulfonate

Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.     

Action of mitomycin C on mouse spermatogonia

The induction of chromosome aberrations in mouse spermatogonia and bone marrow cells by treatment with Mitomycin (MC) was tested. The following dosages were used: 3.5; 1.75; 0.35; and 0.035 mg/kg body weight. Chromatid interchanges and terminal deletions were induced in both tissues. Regarding the chromosome damage, spermatogonia seemed to be more sensitive to the test substance than bone marrow cells.The aberrations observed were considered to represent the cause of dominant lethals induced in spermatocytes after treatment with MC by others. The squash technique adapted for examination of mitoses of mouse spermatogonia proved to be a useful tool in testing potential chemical mutagens.     

A correlative study of the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice. III. 1,3-Bis (2-chloroethyl)-3-nitrosourea (BCNU)

Cytogenetic damage induced by various concentrations of BCNU was evaluated by determining the frequencies of (a) micronuclei in polychromatic erythrocytes of bone marrow, (b) chromatid aberrations in bone marrow, (c) chromatid aberrations in spermatogonia, and (d) reciprocal translocations induced in spermatogonia and scored in spermatocytes. The order of sensitivity for the four parameters tested was: micronuclei greater than chromatid aberrations in bone marrow greater than aberrations in spermatogonia greater than translocations in spermatocytes, a situation similar to that found in an earlier study with CNU-ethanol. When the effect of concentration of the chemical was taken into consideration there was no correlation among the four parameters tested, so that information on induced frequencies of one parameter does not have predictive value for the frequencies of the others. A comparison of the results obtained with the bifunctional BCNU and the mono-functional CNU-ethanol at equal concentrations indicated that BCNU had a similar or a lesser clastogenic effect than did CNU-ethanol. In an experiment in vitro the situation was different in that BCNU was more effective than CNU-ethanol.     

Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium

Cytogenetic damage induced by a wide range of concentrations of uranyl fluoride injected into mouse testes was evaluated by determining the frequencies of chromosomal aberrations in spermatogonia and primary spermatocytes. Breaks, gaps and polyploids were observed in spermatogonia. The frequencies of the significant type of aberration, breaks, were induced according to the injected doses of uranyl fluoride. Primary spermatocytes were examined for fragments, univalents and multivalents. The multivalents observed in this study resulted either from chromatid interchanges or from reciprocal translocations. The reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes. For primary spermatocytes the incidence of aberrant cells largely depended on the administered dose. Sampling time after treatment could affect the frequencies of chromosomal aberrations in male mouse germ cells.     

Analysis of the Chromosome Aberrations Induced by X-Rays in Somatic Cells of DROSOPHILA MELANOGASTER

A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.-The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chromatid aberrations. They also display a greater frequency of spontaneous aberrations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G(2) and G(1). These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferentially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translocations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level.     

Protoplasmic Incompatibility and Cell Lysis in PODOSPORA ANSERINA. I. Genetic Investigations on Mutations of a Novel Modifier Gene That Suppresses Cell Destruction

The distribution of chromosomal aberrations between and within chromosomes of male D, melanogaster somatic cells after treatment with UV has been analyzed. -- Distribution of the breaks between chromosomes was largely nonrandom since we found a higher aberration frequency than that expected on the Y chromosome. Moreover, within the chromosomes the aberrations are clustered in the pericentromeric heterochromatic regions. The above distribution is compared with that of the breaks induced by X rays and methyl-methane-sulphonate (MMS) which were distributed in a different pattern.     

Micronuclei and chromosome aberrations in Xenopus laevis spermatocytes and spermatids exposed to adriamycin and colcemid

Cultured testes and spermatocytes from the frog Xenopus laevis have been incubated (40-42 h) with adriamycin or colcemid followed by quantitation of chromosome aberrations in secondary spermatocytes and quantitation of micronuclei in secondary spermatocytes, early round spermatids, and round spermatids with acrosomal vacuoles (AV) at 18-162 h of culture. Micronucleus frequencies were consistently higher in secondary spermatocytes relative to round spermatids after exposure to either adriamycin or colcemid due to a higher rate of micronucleus formation during meiosis I compared to meiosis II. Also, some of the micronuclei formed during meiosis I did not survive meiosis II to form micronucleated spermatids. Micronucleus formation occurred in 3-7% of secondary spermatocytes with detectable chromosome aberrations, depending upon drug treatment. Thus, the ratio of micronuclei to total chromosome aberrations in secondary spermatocytes was always higher in colcemid-treated cells compared to adriamycin-treated cells following 18- and 42-h treatment periods. Adriamycin induced significant increases in micronuclei in both secondary spermatocytes and spermatids after 162 h of culture, the time for initial pachytene stages to develop into secondary spermatocytes and spermatids. The data show that cultured testes and spermatocytes from Xenopus may be used to quantify specific meiotic chromosome aberrations induced by both clastogens and spindle poisons using either a rapid secondary spermatocyte micronucleus assay or meiotic chromosome analysis.     

In vivo cytogenetics: mammalian germ cells

This chapter summarizes the most relevant methodologies available for evaluation of cytogenetic damage induced in vivo in mammalian germ cells. Protocols are provided for the following endpoints: numerical and structural chromosome aberrations in secondary oocytes or first-cleavage zygotes, reciprocal translocations in primary spermatocytes, chromosome counting in secondary spermatocytes, numerical and structural chromosome aberrations, and sister chromatid exchanges (SCE) in spermatogonia, micronuclei in early spermatids, aneuploidy in mature sperm. The significance of each methodology is discussed. The contribution of novel molecular cytogenetic approaches to the detection of chromosome damage in rodent germ cells is also considered.     

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.     

Studies on mutagen-sensitive strains of Drosophila melanogaster. VII. Effects of repair deficiency in males on X-ray-induced sex-linked recessive lethals in spermatozoa

The response of mature spermatozoa to the X-ray induction (500 R and 3000 R) of sex-linked recessive lethals was studied in Drosophila melanogaster males known to be deficient in excision- or post-replication repair of UV damage in somatic cells. The results show that the induced frequencies of recessive lethals in the excision-repair-deficient males (mei-9a and mei-9L1) are similar to those in the appropriate repair-proficient males (mei+ and Berlin-K). However, in the post-replication-repair-deficient males (w mus(1)101D1), these frequencies are significantly lower than in the comparable repair-proficient males (w) after 500 R, but not after 3000 R.     

Antimutagenic effect of chemosignals from isolated female house mouse on male germ cells (Mus musculus L.)

The influence of chemosignals from isolated mature females of the CBA strain on level of spontaneous and radiation-induced meiotic disturbances in spermatocytes I of males of the same strain was studied. Using an ana-telophase method, 24-hour exposure of males to soiled bedding containing isolated females’ chemosignals was shown to lead to a significantly lower frequency of chromosomal aberrations and other meiotic disturbances in spermatocytes I as compared to males kept on clean bedding. The same effect of female chemosignals was found in the germ cells of irradiated males (4 Gr). The mechanisms and importance of the revealed antimutagenic effect of mouse female chemosignals on the male reproductive cells in the reproduction process are discussed.     

Spermatocyte responses in vitro to induced DNA damage

Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gammaH2AX (which modifies chromatin associated with DSBs). Both gammaH2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etoposide treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.     

Synaptonemal complex karyotype of zebrafish

Meiotic cells of zebrafish have been prepared to show synaptonemal complexes (SCs) by light and electron microscopy. Completely paired SCs from both spermatocytes and oocytes were measured to produce an SC karyotype. The SC karyotype resembles the somatic karyotype of zebrafish and has no recognisable sex bivalent. Measurements of total SC length indicate that SCs grow longer and develop centromeres during pachytene. Oocytes consistently have longer SCs than spermatocytes, presumably correlated with the reported higher recombination frequency in females than in males.     

The induction by X-rays of chromosome aberrations in male guinea-pigs, golden hamsters and rabbits. II. Properties of translocations induced in post-meiotic stages.

Translocations induced by X-rays in post-meiotic germ cells of male guinea-pigs, golden hamsters and rabbits were studied cytologically in the F1 sons of the irradiated males. The percentage of spermatocytes displaying multivalent configurations varied with the translocation, but the average percentage appeared to depend on the species: fewer quadrivalents were observed in hamster than in guinea-pig heterozygotes and most were recorded for rabbit heterozygotes. Chain quadrivalents were more abundant than ring quadrivalents at meiosis for the guinea-pig and hamster, in contrast to the mouse. Too few translocation heterozygotes were examined to determine which meiotic configuration was the more prevalent in the rabbit. In all three species, as in the mouse, translocations were found which caused male sterility, due to partial or complete failure of spermatogenesis, although most translocations caused semi-sterility. For these semi-sterile males both the frequency and time of embryonic death in the progeny appeared to be the same as in the mouse. It is concluded that similar types of chromosome aberrations are induced by X-rays in post-meiotic germ cells of male guinea-pigs, rabbits, golden hamsters and mice.     

Cytogenetic studies on natural populations of grasshoppers with special reference to B-chromosomes

In Atractomorpha bedeli the frequencies of males with B-chromosomes in the males from the Tofuro-ato population were 21, 30, 32, 40, and 26%, respectively, over five years. Numbers of B's (0 to 7) in the primary spermatocytes were usually constant within the follicles but were variable among the follicles within the individuals. In males from the Okazyo-ato population, 35% contained B's in their primary spermatocytes. The cells of the gastric caeca were observed in 8 males with B's in the primary spermatocytes but no cells of the gastric caeca contained B's. Cells of ovariole walls and those of gastric caeca were analysed in 80 females from the same population; none of them contained B's. These facts indicate that B's are present in the germ line but absent in the somatic line. The difference between the germ line and the somatic one was explained on the basis of elimination of B's from the somatic cells. The inter-follicular variation in number of B's was ascribed to mitotic non-disjunction in the germ line prior to differentiation of the follicles.Contribution No. 86 from the Institute of Biology, Oita University.     

Comparative study of the somatic conjugation of chromosomes in the neural ganglia of male and female Drosophila melanogaster D-32

The frequency of somatic conjugation of the 2nd and 3d chromosome pairs has been analysed in male and female neural ganglia from Drosophila melanogaster larvae D-32 line. The frequency of conjugation found for males was 1.5 times lower than for females. On this basis a supposition concerning less duration of homologous conjugation in males has been put forward. It is suggested that it is due to the differences in space arrangement of homologous chromosomes holding their conjugation.