DNA double-strand damage and repair following γ-irradiation in isolated spermatogenic cells (original) (raw)
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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...
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.
Direct and delayed X-ray-induced DNA damage in male mouse germ cells
Environmental and Molecular Mutagenesis, 2012
Sperm DNA integrity is essential for the accurate transmission of paternal genetic information. Various stages of spermatogenesis are characterized by large differences in radiosensitivity. Differentiating spermatogonia are susceptible to radiationinduced cell killing, but some of them can repair DNA damage and progress through differentiation. In this study, we applied the neutral comet assay, immunodetection of phosphorylated H2AX (g-H2AX) and the Sperm Chromatin Structure Assay (SCSA) to detect DNA strand breaks in testicular cells and spermatozoa at different times following in vivo X-ray irradiation. Radiation produced DNA strand breaks in testicular cells that were repaired within the first few hours after expo-sure. Spermatozoa were resistant to the induction of DNA damage, but non-targeted DNA lesions were detected in spermatozoa derived from surviving irradiated spermatogonia. These lesions formed while round spermatids started to elongate within the testicular seminiferous tubules. The transcription of pro-apoptotic genes at this time was also enhanced, suggesting that an apoptotic-like process was involved in DNA break production. Our results suggest that proliferating spermatogonia retain a memory of the radiation insult that is recognized at a later developmental stage and activates a process leading to DNA fragmentation. Environ. Mol. Mutagen. 00:000-000, 2012. V V C 2012 Wiley Periodicals, Inc.
Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2001
To investigate whether DNA damage arising in spermatogenic germ cells can be detected in resultant sperm, we have irradiated murine testis and collected spermatozoa from the vas deferens 45 days later. These cells were derived from spermatogonia present at the time of irradiation. Two forms of irradiation were used, external X-rays (4 Gy) and internal auger electrons from contamination of the male mouse with the isotope Indium-114m (1.85 MBq), which was localised in the testis. Both forms of irradiation produced a profound fall in vas deferens sperm count and testis weight, Indium-114m being more effective. Using the neutral Comet assay for double strand break detection, significant increases in sperm comet tail length and moment were observed. The levels of damage were similar for both treatments. Care had to be taken during the assay to distinguish between sperm and somatic cells as the proportion of the latter increased after irradiation. We conclude that the comet assay can detect DNA damage in spermatozoa after the in vivo exposure of male germ cells to a known testicular genotoxic agent. The assay may be useful for the assessment of sperm DNA damage (double stranded) associated with male infertility and post-fertilization developmental abnormalities in the offspring.
Fertilizing ability of DNA‐damaged spermatozoa
Journal of Experimental Zoology, 1999
In order to investigate the fertilizing ability of DNA-damaged sperm, they were exposed to gamma radiation prior to insemination. The presence of DNA-strand breaks were detected by the TUNEL test. Fertilization rates of 64.3, 59.9, 58.5, and 61.1% were achieved when sperm were subjected to 5, 10, 50, and 100 GY, respectively. This rate was 53.2% in the control group with no significant difference (P > 0.01). The blastocyst development was decreased from 49.8% in the control group to 20.3, 7.8, 3.4, and 2.3% with sperm exposed to doses of 5, 10, 50, and 100 GY, respectively. Of the transferred blastocyst in the control group, 69.8% were implanted and 33.9% developed into live fetuses. These rates were 57.1 and 21.4%, 20 and 0% when sperm were exposed to doses of 5 and 10 GY with a significant difference (P < 0.01). The present study clearly shows that DNA-damaged sperm (regardless of degree of damage) have the ability to fertilize the oocyte, but that embryonic development is very much related to the degree of DNA damage. However, the oocyte has the capacity to repair DNA damage of sperm when it is damaged less than 8%. Damage beyond this level will result in low rate of embryonic development and high early pregnancy loss.
Mutagenesis, 2011
Methods for analysing oxidised DNA lesions [formamidopyrimidine glycosylase (Fpg)-sensitive sites] in coelomocytes and spermatogenic cells from the earthworm Eisenia fetida using the Fpg-modified comet assay were established. The DNA integrity (SSBs 5 strand breaks plus alkali labile sites and Fpg-sensitive sites) in cells from E. fetida continuously exposed to 60 Co gamma-radiation (dose rates 0.18-43 mGy/h) during two subsequent generations (F0 and F1) were measured and related to effects on reproduction end points which have already been reported. The data suggest a slight increase of Fpg-sensitive sites in spermatogenic cells from worms exposed at 11 mGy/h in the F0 generation but not in F1, whereas reduced reproduction had been observed at dose rates at or >4 mGy/h in F0 and at 11 mGy/h in F1. Using acute X-rays (41.9 Gy/h), dose-response relationships were established for SSBs in coelomocytes and spermatogenic cells exposed in vitro. In vivo DNA repair was studied by measuring the decrease in damage (SSBs and Fpg-sensitive sites) in coelomocytes and spermatogenic cells isolated from worms at different times (0-6 h) after acute X-ray exposure (4 Gy). SSBs were repaired in coelomocytes following biphasic kinetics, i.e. with a fast and a slow half-life (t 1/2 ) of 36 min (95%) and 6.7 h (5%), respectively. Fpgsensitive sites were repaired at considerably lower rates (t 1/2 5 4-5 h). In spermatogenic cells, SSB repair during the first hour was observed but a half-life could not be estimated. Repair of Fpg-sensitive sites could not be determined. In general, a reduced repair of Fpg-sensitive sites suggests a higher potential for accumulation of oxidised lesions, compared to SSBs, in earthworms exposed to radiation and other environmental contaminants. This is the first study comparing DNA damage with reproduction in earthworms exposed to ionising radiation.
Quantitative analysis of radiation-induced changes in sperm morphology
PubMed, 1982
When developing spermatogenic cells are exposed to radiation, chemical carcinogens or mutagens, the transformation in the morphology of the mature sperm can be used to determine the severity of the exposure. In this study five groups of mice with three mice per group received testicular doses of X irradiation at dosage levels ranging from 0 rad to 120 rad. A random sample of 100 mature sperm per mouse was analyzed five weeks later for the quantitative morphologic transformation as a function of dosage level. The cells were stained with gallocyanin chrome alum (GCA) so that only the DNA in the sperm head was visible. The ACUity quantitative microscopy system at Lawrence Livermore National Laboratory was used to scan the sperm at a sampling density of 16 points per linear micrometer and with 256 brightness levels per point. The contour of each cell was extracted using conventional thresholding techniques on the high-contrast images. For each contour a variety of shape features was then computed to characterize the morphology of that cell. Using the control group and the distribution of their shape features to establish the variability of a normal sperm population, the 95% limits on normal morphology were established. Using only four shape features, a doubling dose of approximately 39 rad was determined. That is, at 39 rad exposure the percentage of abnormal cells was twice that occurring in the control population. This compared to a doubling dose of approximately 70 rad obtained from a concurrent visual procedure.
Mutation Research-fundamental and Molecular Mechanisms of Mutagenesis, 2010
This study was designed to obtain a better insight into the relative contribution of homologous recombination (HR) and non-homologous end joining (NHEJ) to the repair of radiation-induced DNA double-strand breaks (DSBs) at first meiotic prophase. Early and late pachytene and early diplotene spermatocytes that had completed crossing over were sampled. We studied the kinetics of ␥-H2AX chromatin foci removal after irradiation of mice deficient for HR and mice deficient for NHEJ. Analyzing ␥-H2AX signals in unirradiated RAD54/RAD54B deficient spermatocytes indicated incomplete meiotic recombination repair due to the pronounced increase of ␥-H2AX foci in late prophase primary spermatocytes. In these mice, 8 h after irradiation, early pachytene spermatocytes showed a reduction of the numbers of ␥-H2AX foci by 52% compared to 82% in the wild type, the difference being significant. However, after crossing over (in late pachytene and early diplotene), no effect of RAD54/RAD54B deficiency on the reduction of irradiationinduced foci was observed. In NHEJ deficient SCID mice, repair kinetics in early spermatocytes were similar to those in wild type mice. However, 1 h after irradiation in late pachytene and early diplotene spermatocytes 1.7 times more foci were found than in wild type mice. This difference might be related to the absence of a DNA-PKcs dependent fast repair component in SCID mice. As subsequent repair is normal, HR likely is taking over. Taken together, the results obtained in RAD54/RAD54B deficient mice and in SCID mice indicate that DSB repair in early pachytene spermatocytes is mainly carried out through HR. In late spermatocytes (late pachytenes and early diplotenes) NHEJ is active. However, probably there is an interplay between these repair pathways and when in late spermatocytes the NHEJ pathway is compromised HR may take over.
Differences in Radiation Sensitivity of Recovery of Spermatogenesis Between Rat Strains
Toxicological Sciences, 2012
Previous studies with Lewis/Brown-Norway (BN) F1 hybrid rats indicated that spermatogenesis was much more sensitive to ionizing radiation than in the widely studied outbred Sprague Dawley stock, suggesting that there were genetically based differences; however, the relative sensitivities of various inbred strains had not been established. As a first step to defining the genes responsible for these differences, we compared the sensitivities of seven rat strains to radiation damage of spermatogenesis. Recovery of spermatogenesis was examined 10 weeks after 5-Gy irradiation of seven strains (BN, Lewis, Long-Evans, Wistar Kyoto, spontaneously hypertensive [SHR], Fischer 344, and Sprague Dawley). The percentages of tubules containing differentiated cells and testicular sperm counts showed that BN and Lewis were most sensitive to radiation (< 2% of tubules recovered, < 2 3 10 5 late spermatids per testis), Long-Evans, Wistar Kyoto, Fischer, and SHR were more resistant, and Sprague Dawley was the most resistant (98% of tubules recovered, 2 3 10 7 late spermatids per testis). Although increases in intratesticular testosterone levels and interstitial fluid volume after irradiation had been suggested as factors inhibiting recovery of spermatogenesis, neither appeared to correlate with the radiation sensitivity of spermatogenesis in these strains. In all strains, the atrophic tubules without differentiated germ cells nevertheless showed the presence of type A spermatogonia, indicating that their differentiation was blocked. Thus, we conclude that the differences in radiation sensitivity of recovery of spermatogenesis between rat strains of different genetic backgrounds can be accounted for by differences in the extent of the radiation-induced block of spermatogonial differentiation.