643 BIOLOGY OF REPRODUCTION 71, 643–649 (2004) Published online before print 28 April 2004. DOI 10.1095/biolreprod.104.027466 -H2AX Expression Pattern in Non-Irradiated Neonatal Mouse Germ Cells and after Low-Dose -Radiation: Relationships Between Chromatid Breaks and DNA Double- Strand Breaks 1 Anne Forand, Bernard Dutrillaux, and Jacqueline Bernardino-Sgherri 2 INSERM U566-CEA-Paris 7, Laboratoire de radiosensibilite ´ des cellules germinales, De ´partement de Radiobiologie et Radiopathologie, Direction des sciences du vivant, Commissariat a ` l’E ´ nergie Atomique, BP 6, 92265 Fontenay-aux-roses Cedex, France ABSTRACT The DNA double-strand breaks (DSBs) are considered to be the most relevant lesions for the deleterious effects of ionizing radiation exposure. The discovery that the induction of DSBs is rapidly followed by the phosphorylation of H2AX histone at Ser- 139, favoring repair protein recruitment or access, opens the possibility for a wide range of research. This phosphorylated his- tone, named -H2AX, has been shown to form foci in interphase nuclei as well as megabase chromatin domains surrounding the DNA lesion on chromosomes. Using detection of -H2AX on germ cell mitotic chromosomes 2 h after -irradiation, we stud- ied radiation-induced DSBs during the G 2 /M phase of the cell cycle. We show that 1) non-irradiated neonatal germ cells ex- press -H2AX with variable patterns at metaphase, 2) -irradi- ation induces foci whose number increases in a dose-dependent manner, 3) some foci correspond to visible chromatid breaks or exchanges, 4) sticky chromosomes characterizing cell radiation exposure during mitosis are a consequence of DSBs, and 5) - H2AX remains localized at the sites of the lesions even after end-joining has taken place. This suggests that completion of DSB repair does not necessarily imply disappearance of - H2AX. developmental biology, gametogenesis, spermatogenesis, stress, testis INTRODUCTION The DNA double-strand breaks (DSBs) are considered to be the most biologically significant radiation-induced DNA lesions leading to mutations, which increase the risk of both cancer and hereditary diseases. It has been esti- mated that exposure of one cell to 1 Gy of low-linear-en- ergy-transfer radiation induces 25 to 40 DSBs [1–3]. The repair of DSBs is error-prone and frequently leads to mu- tations. In addition, multiple DSBs can lead to chromosom- al rearrangements by illegitimate joining of free extremities [4]. In the germline, this has great importance, because the clonal proliferation and differentiation of germ cells with genetic alterations can impair the genetic inheritance of fu- ture generations. The nature of radiation-induced chromo- 1 Supported by Electricite ´ de France (EDF) and the Program of Nuclear Toxicology of the Commissariat a ` l’Energie Atomique (CEA). 2 Correspondence: J. Bernardino-Sgherri, CEA/DSV/SEGG/LRCG, BP 6, 92265 Fontenay-aux-roses Cedex, France. FAX: 33 0 1 46 54 99 06; e-mail: jacqueline.bernardino@cea.fr Received: 15 January 2004. First decision: 5 February 2004. Accepted: 9 April 2004.  2004 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org some alterations at first metaphase following exposure de- pends on the phase of the cell cycle at the time of exposure: Translocations, inversions, and deletions are induced in the G 1 phase, whereas chromatid exchanges (radial figures) and breaks are induced in the G 2 phase. Detailed characteriza- tion of radiation-induced chromosome alterations in the late G 2 /M phase has revealed different types of alterations, such as gaps and sticky chromosomes, whose mechanisms of occurrence have not been elucidated [5]. The counting of these abnormalities helps to define the radiation sensitivity of cells. A highly conserved histone H2A variant, H2AX, ac- counts for 10–20% of total H2A proteins of the chromatin [6]. It is found in large amounts in adult germ cells [7–10]. Most importantly, it has been shown to be rapidly phos- phorylated after DSB induction. This modification is as- sumed to change local chromatin structure, enabling the recruitment of proteins involved in DNA repair [11]. Phos- phorylated H2AX (i.e., -H2AX) can be visualized by im- munocytochemistry of cell nuclei and chromosomes [12, 13]. However, accurate quantification of -H2AX foci on interphase nuclei is difficult, and to our knowledge, the only published data regarding chromosomes have been impaired by the poor quality of the metaphase spreads [13]. To our knowledge, no study has examined the effect of ionizing radiation on neonatal germ cell chromosomes, probably because of the difficulty in distinguishing germ cells from somatic cells at metaphase. We took advantage of our previous finding that the overall chromosome DNA methylation pattern distinguishes somatic cells from germ cells within testicular cell spreads [14] to study neonatal germ cell chromosome radiosensitivity during the G 2 /M phase of the cell cycle. This enabled us to quantify on meta- phase chromosomes the -H2AX foci induced by various doses of radiation and to examine the relationships among induced DSBs, chromatid breaks, and chromatid exchanges. MATERIALS AND METHODS Mice and Irradiation Eight-day-old NMRI mice bred in our animal facility were whole-body exposed to -rays from a 137 Cs source (IBL 637; CIS Bio International, France). Four doses were tested (0.2, 0.5, 1, and 2 Gy) at a dose rate of 0.652 Gy/min. Just after irradiation, i.p. injections of colcemid (Sigma, Saint Quentin Fallavier, France) were administered (1.2 mg/kg), and the animals were killed 2 h later. Two independent experiments were per- formed at each dose. The animals were used and maintained according to the French regulation (Ministry of Agriculture Decree 87-848). The animal installation is accredited by the Veterinary Inspectorate (A92-032-02). Downloaded from https://academic.oup.com/biolreprod/article/71/2/643/2667426 by guest on 13 July 2022