Delayed and stage specific phosphorylation of H2AX during preimplantation development of γ-irradiated mouse embryos

Abstract

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated in the serine 139 residue at the damage site. The phosphorylated H2AX, designated as g-H2AX, is visible as nuclear foci in the irradiated cells which are thought to serve as a platform for the assembly of proteins involved in checkpoint response and DNA repair. It is known that early stage mammalian embryos are highly sensitive to radiation but the mechanism of radiosensitivity is not well understood. Thus, we investigated the damage response of the preimplantation stage development by analyzing focus formation of g-H2AX in mouse embryos g-irradiated in utero. Our analysis revealed that although H2AX is present in early preimplantation embryos, its phosphorylation after 3 Gy g-irradiation is hindered up to the two cell stage of development. When left in utero for another 24-64 h, however, these irradiated embryos showed delayed phosphorylation of H2AX. In contrast, phosphorylation of H2AX was readily induced by radiation in post-compaction stage embryos. It is possible that phosphorylation of H2AX is inefficient in early stage embryos. It is also possible that the phosphorylated H2AX exists in the dispersed chromatin structure of early stage embryonic pronuclei, so that it cannot readily be detected by conventional immunostaining method. In either case, this phenomenon is likely to correlate with the lack of cell cycle arrest, apoptosis and high radiosensitivity of these developmental stages. Reproduction (2007) 133 415-422

Distribution of p53 binding protein 1 (53BP1) and phosphorylated H2A.X during mouse preimplantation development in the absence of DNA damage

“…Interestingly, they document a different degree of H2A.X phosphorylation responsiveness to DNA damage between male and female chromatin, in line with our observations. Our work contrasts to a report published earlier by Adiga et al, where basal levels of γH2A.X are undetectable in normal embryos (Adiga et al, 2007). This is very likely due to different immunostaining protocols and techniques (e.g.…”

Transgenerational changes in somatic and germ line genetic integrity of first-generation offspring derived from the DNA damaged sperm

“…It has been shown that radiation-induced chromosome instability in vitro could be attributed to the long-term delay in chromosome replication (13). It is also suggested that one of the initial steps of damage response is inefficient in early preimplantation stages of mouse embryos because of altered chromatin conformation at these stages (7), and these could be the possible reasons for delayed onset of genetic instability in preimplantation embryos derived from the DNA damaged sperm.…”

“…Although embryos do show some DNA repair capacity (7), the level of DNA damage that embryos tolerate during their development is not clear (8). It has been shown that the zygotic stage of mouse embryogenesis lacks G1/S arrest because fertilization of DNA-damaged sperm does not delay the entry of the zygotes to S-phase, although DNA synthesis in such embryos was severely suppressed (2).…”

Association between the extent of DNA damage in the spermatozoa, fertilization and developmental competence in preimplantation stage embryos

“…In the present study, while developmental abnormalities were observed on day 2.0 onwards, it was marked after day 3.0, which corresponds to the morula blastocyst transition stage. Hence, the embryonic response to genetic insult introduced by the sperm is stage specific, which is possibly associated with changes in chromatin conformation or activation of the embryonic genome (22). Further studies are required to elucidate the mechanism associated with sperm DNA damage response during embryonic genome activation.…”