Germ Cell and Dose-Dependent DNA Damage Measured by the Comet Assay in Murine Spermatozoaa after Testicular X-Irradiation1

Abstract

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 X-rays 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.

Recovery of a low mutant frequency after ionizing radiation-induced mutagenesis during spermatogenesis

“…Neonatal [47] or pubertal exposure to a single dose of γ-ray did not result in significantly reduced testis weight, except for testes collected from 60 d/o mice irradiated at 25 day old (Table 2). Other studies have reported reduced testes weights after irradiation of fetal [48,49], pubertal [50] and adult mice [51,52], with IR doses ranging from 0.25 Gy to 6.0 Gy. The different mouse stains (C57BL/6 in the present study vs. CBA/P [52]), animal ages (prepuberty vs. fetal [48,49] or adult [51,52]) could partly explain the discrepancy.…”

Direct and delayed X‐ray‐induced DNA damage in male mouse germ cells

“…This phenomenon has also been observed in male germ cells [de Boer et al, 2010]. Spermatogonial irradiation causes delayed genomic instability in descendant sperm [Sailer et al, 1995;Haines et al, 2001Haines et al, , 2002Cordelli et al, 2003] as well as in the progeny of irradiated male parents in rodents [Baulch and Raabe, 2005;Barber and Dubrova, 2006;Barber et al, 2009] and humans [Aghajanyan et al, 2011]. Remodeling of chromatin domains and nuclear matrix during spermiogenesis and in the zygote has been suggested to play a role in these phenomena [de Boer et al, 2010], but how and when they operate is still unclear.…”

“…Mechanistic understanding of non-targeted effects on the germ line is necessary to determine whether it is relevant for genetic risk assessment of radiation exposure. Mouse spermatozoa derived from irradiated premeiotic germ cells carry DNA strand breaks [Sailer et al, 1995;Haines et al, 2001Haines et al, , 2002Cordelli et al, 2003]. The persistence of these strand breaks is incompatible with the multiple mitotic cycles and checkpoints, meiosis and postmeiotic differentiation processes that take place in cells from the time of exposure to the generation of spermatozoa.…”

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

“…12,13 Although sperm DNA is packed in a highly compacted and stable form, 90 chromatin abnormalities and DNA damage exist, derived from either premeiotic testicular insults, during spermiogenesis, when DNA is packed or during further chromatin building (protamine dominance) during epididymal maturation. [91][92][93] Alternatively, it could be the result of free radical-induced damage 94 or a consequence of apoptosis, in some species. 95 Sperm DNA fragmentation can be assessed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, which can identify DNA strand breaks with modified nucleotides.…”