Although germline cells can form multipotential embryonic stem (ES)/embryonic germ (EG) cells, these cells can be derived only from embryonic tissues, and such multipotent cells have not been available from neonatal gonads. Here we report the successful establishment of ES-like cells from neonatal mouse testis. These ES-like cells were phenotypically similar to ES/EG cells except in their genomic imprinting pattern. They differentiated into various types of somatic cells in vitro under conditions used to induce the differentiation of ES cells and produced teratomas after inoculation into mice. Furthermore, these ES-like cells formed germline chimeras when injected into blastocysts. Thus, the capacity to form multipotent cells persists in neonatal testis. The ability to derive multipotential stem cells from the neonatal testis has important implications for germ cell biology and opens the possibility of using these cells for biotechnology and medicine.

No 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

Mice were exposed at various ages to 1 Gy or 2 Gy of X rays, and translocation frequencies in peripheral blood T cells, spleen cells, and bone marrow cells were determined with FISH painting of chromosomes 1 and 3 when the animals were 20 weeks old. It was found that the mean translocation frequencies were very low (< or =0.8%) in mice exposed in the fetal or early postnatal stages. However, with the increase in animal age at the time of irradiation, the frequency observed at 20 weeks old became progressively higher then reached a plateau (about 5%) when mice were irradiated when > or =6 weeks old. A major role of p53 (Trp53)-dependent apoptosis for elimination of aberrant cells was not suggested because irradiated fetuses, regardless of the p53 gene status, showed low translocation frequencies (1.8% in p53(-/-) mice and 1.4% in p53(+/-) mice) compared to the frequency in the p53(-/-) mother (7.4%). In contrast, various types of aberrations were seen in spleen and liver cells when neonates were examined shortly after irradiation, similar to what was observed in bone marrow cells after irradiation in adults. We interpreted the results as indicating that fetal cells are generally sensitive to induction of chromosome aberrations but that the aberrant cells do not persist because fetal stem cells tend to be free of aberrations and their progeny replace the pre-existing cell populations during the postnatal growth of the animals.

Ionizing radiation activates a series of DNA damage response, cell cycle checkpoints to arrest cells at G1/S, S and G2/M, DNA repair, and apoptosis. The DNA damage response is thought to be the major determinant of cellular radiosensitivity and thought to operate in all higher eukaryotic cells. However, the radiosensitivity is known to differ considerably during ontogeny of mammals and early embryos of mouse for example are much more sensitive to radiation than adults. We have focused on the radiation-induced damage response during pre-implantation stage of mouse embryo. Our study demonstrates a hierarchy of damage responses to assure the genomic integrity in early embryonic development. In the sperm-irradiated zygotes, p53 dependent S-phase checkpoint functions to suppress erroneous replication of damaged DNA. The transcription-dependent function is not required and the DNA-binging domain of the protein is essential for this p53 dependent S-phase checkpoint. p21 mediated cleavage arrest comes next during early embryogenesis to prevent delayed chromosome damage at morula/ blastocyst stages. Apoptosis operates even later only in the cells of ICM at the blastocyst stage to eliminate deleterious cells. Thus, early development of sperm-irradiated embryos is protected at least by three mechanisms regulated by p53 and by p21.

The S-phase DNA damage checkpoint is activated by DNA damage to delay DNA synthesis allowing time to resolve the replication block. We previously discovered the p53-dependent S-phase DNA damage checkpoint in mouse zygotes fertilized with irradiated sperm. Here, we report that the same p53 dependency holds in mouse embryonic fibroblasts (MEFs) at low doses of irradiation. DNA synthesis in p53 wild-type (WT) MEFs was suppressed in a biphasic manner in which a sharp decrease below 2.5 Gy was followed by a more moderate decrease up to 10 Gy. In contrast, p53À/À MEFs exhibited radioresistant DNA synthesis below 2.5 Gy whereas the cells retained the moderate suppression above 5 Gy. DNA fiber analysis revealed that 1 Gy irradiation suppressed replication fork progression in p53 WT MEFs, but not in p53À/À MEFs. Proliferating cell nuclear antigen (PCNA), clamp loader of DNA polymerase, was phosphorylated in WT MEFs after 1 Gy irradiation and redistributed to form foci in the nuclei. In contrast, PCNA was not phosphorylated and dissociated from chromatin in 1 Gy-irradiated p53À/À MEFs. These results demonstrate that the novel low-dosespecific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA.

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.

Perfluorinated compounds (PFCs) have been employed as surface treatment agents in a variety of products. Perfluorooctanoic acid (PFOA), a PFC that is found globally in the environment and in human tissues, has been increasing significantly in serum levels over the past 50 years. Here, we demonstrated that PFOA inhibits feeding behavior as potently as the endogenous peroxisome proliferator-activated receptor (PPAR)-α ligand, oleoylethanolamide (OEA), via the activation of PPAR-α, the vagal nerve and hypothalamic neuropeptides. Peripherally administered PFOA decreased food intake as potently as OEA. PFOA decreased gastric emptying and increased the expression level of the gene encoding urocortin 1 in the hypothalamus and the immunoreaction for urocortin 1 in the paraventricular nucleus. Vagotomy attenuated the inhibitory effects of PFOA on feeding. The inhibition of food intake and bodyweight gain by PFOA was completely mitigated in PPAR-α-/mice. Our studies demonstrated that the ubiquitous environmental pollutant PFOA works as an imitator of OEA mimicking its action in the feeding regulatory system, providing a new mode of action as represented by environmental 'anorexigens'.