Kinetics of γ-H2AX induction and removal in bone marrow and testicular cells of mice after X-ray irradiation

Abstract

Male germ cells have been shown to differ in their DNA damage response (DDR) with respect to somatic cells. In addition, DDR pathways are modulated along spermatogenesis, accompanying profound chromatin modifications. Histone H2AX phosphorylation is a fundamental step of DDR. Few data are available on the long-term kinetics of phosphorylated H2AX (γ-H2AX) after in vivo irradiation. We have investigated, by microscopic and flow cytometric immunochemistry, γ-H2AX induction and removal in testicular cells of irradiated mice, in comparison with bone marrow cells. In unirradiated testicular cells, much higher levels of γ-H2AX were measured by flow cytometry with respect to bone marrow cells. Irradiation induced a redistribution of γ-H2AX into discrete foci detectable by microscopy. In irradiated bone marrow, the percentage of labelled cells peaked at 1 h and rapidly declined, in agreement with data on in vitro cell lines. In contrast, spermatocytes and round spermatids showed persistent labelling until 48 h. During this time, in spermatids, topological changes were observed in γ-H2AX foci from a pattern of many uncountable dots to a pattern of few large spots. Observations of testicular sections confirmed this trend in the reduction of foci number in spite of substantially invariable percentages of labelled cells in the analysed timeframe. To assess whether γ-H2AX persistence in testicular cells was due to unrepaired DNA breaks, we performed comet assay and immunofluorescence analysis of Mdc1, a marker of DDR different from γ-H2AX. Comet assay showed that most breaks were repaired within 2 h. Forty-eight hours after irradiation, contrary to γ-H2AX foci that remained detectable in 80% of initially labelled cells, Mdc1 foci were observed in only 20-30% of cells. These data suggest that, at long times after irradiation, mechanisms additional to impairment of DNA break repair may account for the long persistence of γ-H2AX foci in male germ cells.

Persistent γH2AX: A promising molecular marker of DNA damage and aging

“…The persistent gH2AX foci may be present in the form of large foci. For example, in spermatids, the persistent gH2AX foci appeared as larger foci at 48 h after IR exposure [72]. Large persistent gH2AX foci were also observed in normal human fibroblasts (VH-10) and in HeLa cells after exposure to IR [73].…”

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

“…Phosphorylated H2AX (g-H2AX) foci are markers of DNA double strand breaks [Rogakou et al, 1998]. g-H2AX content in testicular cells is heterogeneous, higher than in cells from other organs and increases after irradiation [Hamer et al, 2003;Ahmed et al, 2007Ahmed et al, , 2010aBlanco-Rodriguez, 2009;Paris et al, 2011]. The presence of H2AX has been documented in human [Gatewood et al, 1990] and in mouse [Meyer-Ficca et al, 2009] sperm chromatin.…”

“…Together with the neutral comet data, the g-H2AX fluorescence results indicate that double strand breaks represent an important component of delayed DNA damage. However, the specificity of these assays for DNA double strand breaks has been questioned [Calini et al, 2002;Collins et al, 2008;de Boer et al, 2010;Costes et al, 2010;Paris et al, 2011] and chromatin structural alterations and other types of DNA breaks may be an additional component of delayed radiation damage in spermatozoa.…”

Limited Repair of Critical DNA Damage in Cells Exposed to Low Dose Radiation

“…To achieve this level of DNA compaction, sperm cells have replaced most of their histones by protamines (Oliva, 2006;Marchetti et al, 2007). So in the male germ cells DDR development is very slow, and formed RIF (γH2AX) are stored for longer periods of time than those in somatic cells (Paris et al, 2011). Studies in mice show that many radiation-induced DNA lesions in mature spermatozoa are not subject to repair and persist for at least 7 days (Marchetti and Wyrobek, 2008;Ahmed et al, 2010).…”

“…DNA repair in the spermatozoon occurs, to a large extent, after their penetration into the ovum, the repair systems of the latter being involved in the process. Prior to DNA repair, reconstruction of chromatin is, obviously, required, accompanied by substitution of oocyte histones for protamines (Marchetti and Wyrobek, 2008;Ahmed et al, 2010;Paris et al, 2011).…”