A r c h i v e o f S I D Cytogenetic Alterations in Preimplantation Mice Embryos Following Male Mouse Gonadal Gamma-irradiation: Comparison of Two Methods for Reproductive Toxicity Screening (original) (raw)
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Genome instability is a main cause of chromosomal alterations in both somatic and germ cells when exposed to environmental, physical and chemical genotoxicants. Germ cells especially spermatozoa are more vulnerable to suffering from DNA damaging agents during spermatogenesis and also more potent in transmitting genome instability to next generation. To investigate the effects of γ-rays on inducing abnormalities manifested as numerical Chromosome Aberrations (CA) and Micronucleus (MN) in preimplantation embryos, adult male NMRI mice were irradiated with 4 Gy of γ-rays. They were then mated at weekly intervals with superovulated, non-irradiated female mice in 6 successive weeks. About 68 hr post coitous, four to eight cell embryos were retrieved and fixed on slides using standard methods in order to screen for CA and MN. In embryos generated from irradiated mice, the frequency of aneuploidy and MN increased dramatically at all post-irradiation sampling times as compared to the control...
Mutagenesis, 2005
Mice conceived 6 weeks after paternal exposure to ionizing radiation were fathered by sperm that were Type B spermatogonia at the time of irradiation. Previous studies of these offspring showed that this paternal F 0 germ cell irradiation led to decreased embryonic cell proliferation rates, altered enzyme activities, protein levels and whole-body weights. In the present study, we examined four generations of CD1 mice following paternal F 0 irradiation of the Type B spermatogonia (1.0 Gy, 137 Cs g rays) to determine the stability of the heritable effects. Offspring were evaluated for changes in protein kinase C and mitogen-activated protein kinase enzyme activities and Trp53 and p21 waf1 protein levels. Two or more endpoints were significantly altered in all four generations of offspring from the irradiated F 0 sire (P 0.05). To test the hypothesis that these heritable biochemical effects are random stochastic responses rather than some predictable uniform response, each endpoint was also evaluated in terms of a variability index (VI). Results of VI analyses show that the observed heritable phenotype is unpredictable in magnitude and direction of change for an endpoint between generations and within generations. These results indicate that irradiated spermatogonia develop a capacity to transmit a type of heritable genomic instability to four generations of offspring.
2008
Cytogenetic damage expressed as micronuclei (MN) in 4-8 cells embryos generated after irradiation of male or male and female mice in the absence and presence of vitamin C was investigated. Male NMRI mice were whole body exposed to 4 Gy gamma-rays and mated with non irradiated super-ovulated female mice in 6 successive weeks after irradiation in a weekly interval. In experiments involving irradiation of both male and female mice, irradiated male mice for 6 weeks post-irradiation were mated with female mice irradiated after induction of super-ovulation. Effect of 100 mg/kg vitamin C (ascorbic acid) on the frequency of MN was also studied. Pregnant animals were euthanized and embryos flushed from the oviducts and fixed on slides. The rate of MN observed in embryos generated from irradiated male compared to control group dramatically increased (P<0.01). Frequency of MN in this group decreased dramatically after vitamin C treatment (P<0.01). Frequency of MN in embryos generated by mating both male and female irradiated mice was higher than that observed for those embryos generated by irradiated male mice alone. However, a considerable modifying effect of vitamin C was observed for this group too (P<0.05).
Gamma-irradiation increased meiotic crossovers in mouse spermatocytes
Mutagenesis, 2011
In mice, the occurrence of immunofluorescent foci for mismatch repair protein MLH1 correlates closely with the occurrence of crossovers, as detected genetically, and MLH1 foci represent virtually all prospective crossover positions. To examine the effects of g-irradiation on meiotic crossovers in mouse spermatocytes, male mice were subjected to wholebody g-irradiation at different sub-stages of meiotic prophase and crossovers on synaptonemal complexes (SCs) were analysed by visualising and quantifying the immunofluorescent MLH1 foci. At both 24 and 48 h after exposure, significant dose-dependent increases in the number of total MLH1 foci per spermatocyte were observed at late zygotene-early pachytene with the gradient increase of radiation dose from 0, 1.5, 3-6 Gy. Furthermore, irradiation at preleptoteneleptotene still led to significant dose-dependent increased meiotic crossovers in the spermatocytes analysed 120 h after exposure. In further analysis, these dose-dependent increases in the number of total MLH1 foci per cell were attributed to significant dose-dependent decreases in autosomal SCs with 0 MLH1 focus, and the dose-dependent increases in autosomal SCs with 2 MLH1 foci and the percentage of cells with MLH1 focus on XY bivalent. The increased number of cells with an MLH1 focus on the pseudoautosomal regions (PARs) may indicate that there is a delay in meiotic progression in the irradiated cells. Although significant dose-dependent increases in the number of total MLH1 foci per cell were examined 24, 48 or 120 h after exposure with the gradient increase of radiation doses, these increases were mild compared to the control groups. This suggests that there is tight control of crossover formation (at least with respect to MLH1 foci number). The mechanisms underlying irradiationinduced DNA lesion repair, cellular responses independent of DNA damage and meiotic crossover homeostasis in mammals will be the subjects of future study.
Biology of Reproduction, 2002
The single-cell gel electrophoresis (Comet) assay has been widely used to measure DNA damage in human sperm in a variety of physiological and pathological conditions. We investigated the effects of in vivo radiation, a known genotoxin, on spermatogenic cells of the mouse testis and examined sperm collected from the vas deferens using the neutral Comet assay. Irradiation of differentiating spermatogonia with 0.25-4 Gy Xrays produced a dose-related increase in DNA damage in sperm collected 45 days later. Increases were found when measuring Comet tail length and percentage of tail DNA, but the greatest changes were in tail moment (a product of tail length and tail DNA). Spermatids, spermatocytes, differentiating spermatogonia, and stem cell spermatogonia were also irradiated in vivo with 4 Gy X-rays. DNA damage was indirectly deduced to occur at all stages. The maximum increase was seen in differentiating spermatogonia. DNA damaged cells were, surprisingly, still detected 120 days after stem cell spermatogonia had been irradiated. The distribution of DNA damage among individual sperm cells after irradiation was heterogeneous. This was seen most clearly when changes in the Comet tail length were measured when there were discrete undamaged and damaged populations. After increasing doses of irradiation, an increasing proportion of cells were found in the damaged population. Because a proportion of undamaged sperm cells remains after all but the highest dose, the possibility of normal fertility remains. However, fertilization with a spermatozoa carrying high amounts of DNA damage could lead to effects as diverse as embryonic death and cancer susceptibility in the offspring.
Iranian Journal of Reproductive Medicine
Background: Irradiation is one of the major causes of induced sperm DNA damage. Various studies suggested a relation between sperm DNA damage and fertilization rate after intra-cytoplasmic sperm injection (ICSI). Objective: In this study, fertilization rate and premature chromosome condensation (PCC) formation after ICSI of hamster oocytes with irradiated sperms from normal and oligosperm individuals was investigated. Materials and Methods: Human sperms were classified according to counts to normal and oligosperm. Ten samples were used for each group. Golden hamster oocytes were retrieved after super ovulation by PMSG and HCG injection. From retrieved oocytes, 468 were in metaphase II. Control and 4 Gy gamma irradiated sperms were then injected into oocytes. After pronuclei formation in injected oocytes and formation of 8 cells embryos, slides were prepared using Tarkowskie's standard air-drying technique. The frequency of embryos and PCC were analyzed using 1000× microscope aft...
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1995
Radiation-induced perturbations in the steady-state spermatogenesis of mouse exposed to 0.05 to 2 Gy of 6"Co gamma-radiation were studied at 7 to 70 days post-irradiation flow cytometrically. Five quantifiable populations viz: elongated spermatids (HC), round spermatids (lC), spermatogonia and other diploid cells (2C), spermatogonial cells synthesizing DNA (S-phase) and primary spermatocytes (4C) were identified in the sham-irradiated controls. Exposure of mice to different doses of radiation resulted in a significant decline in the total germ-cell transformation ratio (I C:2C) at 2 1 and 28 days post-irradiation as compared to the control group, except for the animals exposed to 0.05 Gy. The lC:2C ratio is subdivided into two components viz. 4C:2C and lC:4C. The 4C:2C ratio decreased significantly on day 14 post-irradiation, except for 0.05 Gy where it was non-significant. Consequently, meiotic transformation (lC:4C) showed a significant increase on day 14 post-irradiation compared to the sham-irradiated control barring 0.05 Gy where the difference between the two groups was non-significant. The ratio of HC: IC (cell transformation during spermiogenesis) increased significantly at day 21 post-irradiation 0.2 to 2 Gy and between day 7 and 14 for 0.05 Gy as compared to the control group. It appears that a dose as low as 0.05 Gy radiation was able to cause the perturbations in the steady-state spermatogenesis of mouse and normalcy was not restored even up to 70 days post-irradiation at all exposure doses.
Environmental and Molecular Mutagenesis, 2012
Large tandem repeat DNA loci such as expanded simple tandem repeats and minisatellites are efficient markers for detecting germline mutations; however, mutation detection using these loci can be imprecise and difficult to standardize across labs. Short-tandem repeats, such as microsatellites, offer more precise and high-throughput mutation detection, but germline mutation induction at these loci has not yet been studied in model organisms such as mice. In this study, we used microsatellite enrichment and large-scale DNA sequencing of several closely related inbred mouse lines to identify a panel of 19 polymorphic microsatellites with potentially high spontaneous mutation frequencies. We used this panel and four additional loci from other sources to quantify spontaneous mutation frequency in pedigrees of outbred Swiss-Webster mice. In addition, we also examined mutation induction in families in which sires were treated with acute doses of either 0.5 Gy or 1.0 Gy gamma-irradiation to spermatogonial stem cells. Per locus mutation frequencies ranged from 0 to 5.03 3 10 23 . Considering only the 11 loci with mutations, the mutation frequencies were: control 2.78 3 10 23 , 0.5 Gy 4.09 3 10 23 , and 1.0 Gy 1.82 3 10 23 . There were no statistically significant changes in mutation frequencies among treatment groups. Our study provides the first direct quantification of microsatellite mutation frequency in the mouse germline, but shows no evidence for mutation induction at pre-meiotic male germ cells following acute gamma-irradiation. Further work using the panel is needed to examine mutation induction at different doses of radiation, exposure durations, and stages during spermatogenesis. Environ. Mol. Mutagen. 00:000-000, 2012. V V C 2012 Wiley Periodicals, Inc.
Toxicology in vitro : an international journal published in association with BIBRA, 2007
In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and gamma-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of g...
Proceedings of the National Academy of Sciences, 1998
Germ-line mutation induction at mouse minisatellite loci by acute irradiation with x-rays was studied at premeiotic and postmeiotic stages of spermatogenesis. An elevated paternal mutation rate was found after irradiation of premeiotic spermatogonia and stem cells, whereas the frequency of minisatellite mutation after postmeiotic irradiation of spermatids was similar to that in control litters. In contrast, paternal irradiation did not affect the maternal mutation rate. A linear dose-response curve for paternal mutation induced at premeiotic stages was found, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. High frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be evaluated at low doses of exposure in very small population samples, which currently makes minisatellite DNA the most powerful tool for monitoring radiation-induced germ-line mutation.