X-ray-induced chromosome aberrations in mouse dictyate oocytes. I. Time and dose relationships.

Structural chromosome aberrations were analyzed in superovulated metaphase-I oocytes of the mouse, Mus musculus, at various times after a single acute dose of 200 R of X-rays. The aberrations seen were of the chromatid type, i.e., chromatid interchanges, isochromatid deletions and chromatid deletions. The aberration frequency was low during the interval 24 h to 5 days between irradiation and ovulation; peak frequency was reached when irradiation was given 14 days prior to ovulation. A dose-response study was made 14 days prior to ovulation at doses of 50, 100, 200, 300 and 400 R. A curve of these data indicated that a significant two-track component was present for both interchanges and deletions. Centromere staining revealed that symmetrical and asymmetrical interchanges occurred at approximately equal frequency and also that the asymmetrical equivalent of crossing-over was induced at a measurable frequency.     

The fate of X-ray-induced chromosome aberrations in blood lymphocyte culture

The BrdU-Giemsa method which facilitates an unequivocal identification of metaphases at different cycles has been utilized to investigate the fate of X-ray-induced chromosome aberrations in the blood lymphocyte culture system of the Indian muntjac which has the lowest diploid number (2n = 6 female/7 male) and easily distinguishable large-sized chromosomes. The results demonstrate that about 50% of dicentrics and only 12% of rings were transmitted from the first cycle to the second. There were as high as 73% abnormal cells in the second cycle as against 94% in the first cycle following 4.0 Gy. However, the frequencies of dicentrics, rings and of abnormal cells were greatly reduced in the third+ cycles. The frequencies of acentric fragments per post-irradiated first, second and third+ division cell were 2.21, 0.64 and 0.24, respectively. In sharp contrast to all earlier reports, about 75% of them were retained as a single acentric fragment in the second cycle. Analysis of fragment segregation during anaphase separation supports this finding. The survival probability of dicentrics and rings was found to be more than 60% in the second and only 18% in the third+ cycle.     

Induced chromosome aberrations in unfertilized oocytes of mice

Summary Just before ovulation we treated 10–12-week-old female mice of the strains C3H and (101×C3H)F₁ with 2.3.5.-triethyleneimonobenzoquinone-1.4 (T), cyclophosphamide (C) and methotrexat (M). The chromosome analysis took place shortly after ovulation at the time of metaphase II. The method described in detail seems to be appropriate to examine the induction of genetic defects during oogenesis by chemical agents.

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Zusammenfassung Wir behandelten 10–12 Wochen alte weibliche Mäuse der Stämme C3H und (101×C3H)F₁ kurz vor der Ovulation mit 2.3.5.-Triethyleniminobenzochinon-1.4 (T), Cyclophosphamid (C) und Methotrexat (M) Die Chromosomenanalyse erfolgte kurz nach der Ovulation zum Zeitpunkt der Metaphase II. Die im Detail beschriebene Methode erscheint geeignet, die Auslösung genetisch bedingter Eireifungsstörugen durch chemische Agentien zu untersuchen.

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This work was sponsored by the Deutsche Forschungsgemeinschaft.     

Cytogenetic analysis of X-ray-induced chromosome aberrations in spontaneously leukaemic AKR mice.

The increased frequency of numerical and structural chromosomal aberrations in spontaneously leukaemic AKR mice, compared with the values of healthy control CBA/H-T6T6 mice, induced by X-irradiation, migh be connected with the predisposition to malignant growth, probably indirectly helping the virus activation, or acting together with the immune deficiency, by creating a weaker system that is more sensitive to carcinogenic agents.     

High resistance of cultured Mongolian gerbil cells to X-ray-induced killing and chromosome aberrations.

The Mongolian gerbil (Meriones unguiculatus) is known to be one of the most radioresistant animals. We have examined the X-ray sensitivity of normal diploid fibroblasts from Mongolian gerbil embryos compared with those of cultured embryo cells obtained from various laboratory animals and a normal human. There was a wide difference in X-ray sensitivity for cell killing among different mammalian species. The D0 values for Mongolian gerbil cells ranged from 2.08 to 2.28 Gy, values which are twice as high as those for human cells. The mean D0 value for human cells was 1.06 Gy. Mouse, rat, Chinese hamster, and Syrian/golden hamster cells showed similar D0 values ranging from 1.30 to 1.56 Gy. When cells were irradiated with X rays, ten times more chromosome aberrations were detected in human cells than in Mongolian gerbil cells. The frequencies of chromosome aberrations in other rodent cells were between the values for cells from humans and those from gerbils. These data indicate that the Mongolian gerbil cells are resistant to X-ray-induced cell killing and chromosome aberrations, and that the radiation sensitivity of mammalian cells in primary culture may be reflected by their radioresistance in vivo.     

The influence of maternal age on radiation-induced chromosome aberrations in mouse oocytes

The relative sensitivities of dictyate oocytes from young and old female mice to radiation-induced chromosome damage were examined in 2 separate experiments. Firstly, females were given either 2 or 4 Gy of X-rays and metaphase I stage oocytes collected 16.5 days later. Analysis of these cells showed dose-related increases in chromosome aberrations in both age groups. The response was significantly greater in oocytes of older females. In the second experiment, females were given 4 Gy of X-rays and metaphase I stage oocytes collected 3.5 days later. Again, a significantly larger frequency of aberrations was present in cells from older animals. Overall, these 2 experiments provide unambiguous evidence that the radiosensitivity of mouse dictyate oocytes increases with advancing maternal age.     

RNA synthesis in preovulatory mouse oocytes

RNA synthesis, previously shown to take place during oocyte growth, has been demonstrated throughout the growth-quiescent period preceding ovulation of the mouse oocyte. In the final 7-day preovulatory period, the level of incorporation of (5,6-3H)uridine into ovulated oocytes decreased as the interval between exposure to precursor and ovulation decreased; significant incorporation was detectable within 2 days before ovulation. Analysis of the frequency and density of label in ovarian oocytes at successive stages of meiosis in relation to the interval between adminstration of labeled precursor and collection of oocytes revealed that RNA synthesis continues up to within 2 h before GVBD.     

An explanation of interspecific differences in sensitivity to X-ray-induced chromosome aberrations and a consideration of dose-response curves

Using 1-β- -arabinofuranosylcytosine (AraC) which is an inhibitor of DNA-repair resynthesis, previous studies have shown that the frequency of chromosome-type aberrations is influenced by the rate of repair of araC-inhibitable DNA damage. The experiments described here are a further test of this hypothesis and also an attempt to determine if the different sensitivities of lymphocytes of different species to X-ray-induced aberrations are related to the rate of endonucleolytic incision during repair of DNA damage. Unstimulated lymphocytes from 4 species were exposed to an X-ray dose of 200 rad, and then incubated with araC for 0, 1, 2, 3 or 4 h. The aberration frequencies increased in all species up to 3–4 h. It was also clear that the rate of increase was different between species and was approximately proportional to the ratios of X-ray-induced aberrations observed in the absence of araC. For example, human lymphocytes are approximately twice as sensitive as rabbit lymphocytes to the induction of aberrations by X-rays and the rate of increase of aberrations in the presence of araC was about twice as great in human as rabbit lymphocytes. In addition, using 50, 100, 200 or 300 rad of X-rays and treating human lymphocytes for 0, 1, 2 or 3 h in araC post-irradiation, we have shown that the rate of increase in aberrations is proportional to the amount of araC-inhibitable DNA damage; with a limiting dose at about 50 rad. These results appear to provide a basis for interpreting differences in sensitivities to aberration induction among mammalian species.     

Blood lymphocyte culture system: quantitative analysis of X-ray-induced chromosome aberrations in man, muntjac and cattle

Peripheral blood lymphocytes of 3 mammalian species, man, muntjac and cattle, which have various amounts of DNA and divergent karyotypes, were exposed to 100-400 rad of X-rays, and frequencies of dicentrics and other aberrations were analysed at first post-irradiation metaphases. During experiments, various preparative or physical and biological factors that could influence the yield of chromosome aberrations were taken into account. The frequency of dicentrics scored at first post-irradiation metaphases showed best fit to both linear and quadratic dose-response curves, y = a + bD and y = bD + cD2 with a high correlation coefficient of 0.98 (P less than 0.001). The frequency of dicentrics obtained at different post-irradiation fixation times did not show significant variation, indicating a homogeneous sensitivity of peripheral lymphocytes to X-irradiation. BrdU incorporation following X-irradiation showed no increase in the frequency of chromosome aberrations. The frequency of dicentrics in man, muntjac and cattle showed a close correlation with their DNA content, but no meaningful correlation was found between the yield of dicentrics and the chromosome arm number or the nuclear volume. The ratio of dicentric yields, 1.00:0.67:1.04 obtained in man, muntjac and cattle were comparable to the ratio of their DNA contents, 1.00: 0.65: 1.07. The base-line frequency of SCEs was similar in the 3 species and no significant variation in SCE frequency was noticed even after administration of 400 rad of X-rays.     

Radiation-induced chromosome aberrations in guinea-pig growing oocytes, and their relation to follicular atresia

The female guinea-pig has been shown to represent a good model to investigate the genetic hazard of ionizing radiation in humans. The sensitivity of the guinea-pig oocytes to radiation-induced chromosome aberrations was, therefore, studied at different stages of oocyte and follicular growth. The sensitivity of oocytes enclosed in small follicles (15 weeks before ovulation) was found to be low and comparable to that of immature oocytes present at birth. The sensitivity of growing oocytes remained low and almost constant until 3 weeks before ovulation, from which time it began to increase. The most dramatic increase of sensitivity occurred during the last week preceding ovulation: about 90% of oocytes X-irradiated with 4Gy, 2 days before ovulation showed one or more chromatid interchanges, as compared to 20% for those irradiated with the same dose 1 week earlier. A comparison of our results with those found by others in the mouse shows that considerable differences of sensitivity exist between oocytes of these two species irradiated at similar stages of development. The possible reasons for these differences are discussed.     

Dose-response relationship for induction of structural chromosome aberrations in Chinese hamster oocytes after X-irradiation

Recently, we determined the precise timing of changes of meiotic chromosomal radiosensitivity in oocytes during the spontaneous estrous cycle in the Chinese hamster, by assessing MII chromosomal lesions induced after 200 rad X-irradiation at various stages prior to ovulation (Mikamo et al., 1981). It was demonstrated that early diakinesis was the most radiosensitive phase, the incidence of oocytes with structural chromosome aberrations being 15 times higher than during dictyotene stages of diestrum. A high radiosensitivity was also found in mouse oocytes at a transitional stage from late dictyotene to diakinesis, although X-irradiation was restricted only to this single stage (Reichert et al., 1975).In the present work, we investigated the dose-response relationship for induction of chromosome aberrations in Chinese hamster oocytes at the most radiosensitive stage, and compared our results with those obtained previously in the mouse.     

Higher incidence of spontaneous sister-chromatid exchanges (SCEs) and X-ray-induced chromosome aberrations in peripheral blood lymphocytes during pregnancy

In vitro cultures of peripheral blood lymphocytes from human and muntjac (barking deer) females who were at an advanced stage of pregnancy (32-37 weeks pregnant women and 20-24 weeks pregnant muntjacs) showed an enhanced frequency of SCEs and X-ray-induced chromosome aberrations when compared with those of nonpregnant females. Lymphocyte cultures of nonpregnant females to which sex hormones progesterone, oestrogen and human chorionic gonadotropin (HCG) were added together exogenously also showed higher frequency of SCEs. The plausible reason(s) for such high incidence of SCEs during pregnancy is discussed.     

The meiotic stage of preovulatory oocytes in mares

Confusion exists as to whether the oocytes of the domestic horse are ovulated at the first meiotic metaphase (MI) or the second (MII). In this study eight oocytes were collected from the preovulatory follicles of 16 mares 36 h after human chorionic gonadotropin CG treatment. Six of the eight oocytes were judged to be at MII by the presence of the first polar body and this judgement was confirmed by semithin sectioning in one. Of the two that had no polar body, one was found to be at MII after fixation for chromosomal analysis and the meiotic stage of the other remained undetermined. Since all seven oocytes yielding conclusive evidence were at MII, it was concluded that horse oocytes, like those of most mammals studied, are ovulated after completion of the first meiotic division and formation of the first polar body.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.