Megumi Toyoshima

Generation of Pluripotent Stem Cells from Neonatal Mouse Testis

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.

p21 provides stage specific DNA damage control to preimplantation embryos

The early stage embryogenesis of higher eukaryotes lacks some of the damage response pathways such as G1/S checkpoint, G2/M checkpoint and apoptosis. We examined here the damage response of preimplantation stage embryos after fertilization with 6 Gy irradiated sperm. Sperm-irradiated embryos developed normally for the first 2.5 days, but started to exhibit a developmental delay at day 3.5. p21 was activated in the delayed embryos, which carried numerous micronuclei owing to delayed chromosome instability. Apoptosis was observed predominantly in the inner cell mass of the day 4.0 embryos. Sperm-irradiated p21−/− embryos lacked the delay, but chromosome instability and apoptosis were more pronounced than the corresponding p21 wild-type embryos. We conclude from the result that damage responses come in a stage-specific manner during preimplantation stage development; p53-dependent S checkpoint at the zygote stage, p21-mediated cell cycle arrest at the morula/blastocyst stages and apoptosis after the blastocyst stage in the inner cell mass.

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

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 gamma-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 gamma-H2AX in mouse embryos gamma-irradiated in utero. Our analysis revealed that although H2AX is present in early preimplantation embryos, its phosphorylation after 3 Gy gamma-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.

The Novel Surveillance Mechanism of the Trp53-Dependent S-Phase Checkpoint Ensures Chromosome Damage Repair and Preimplantation-Stage Development of Mouse Embryos Fertilized with X-Irradiated Sperm

Abstract Shimura, T., Toyoshima, M., Taga, M., Shiraishi, K., Uematsu, N., Inoue, M. and Niwa, O. The Novel Surveillance Mechanism of the Trp53-Dependent S-Phase Checkpoint Ensures Chromosome Damage Repair and Preimplantation-Stage Development of Mouse Embryos Fertilized with X-Irradiated Sperm. Radiat. Res. 158, 735–742 (2002). 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.

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint.

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, p53−/− 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 p53−/− 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 p53−/− MEFs. These results demonstrate that the novel low-dose-specific p53-dependent S-phase DNA damage checkpoint is likely to regulate the replication fork movement through phosphorylation of PCNA.

Chromosome Aberrations do not Persist in the Lymphocytes or Bone Marrow Cells of Mice Irradiated In Utero or Soon after Birth

Abstract Nakano, M., Kodama, Y., Ohtaki, K., Nakashima, E., Niwa, O., Toyoshima, M. and Nakamura, N. Chromosome Aberrations do not Persist in the Lymphocytes or Bone Marrow Cells of Mice Irradiated In Utero or Soon after Birth. Radiat. Res. 167, 693–702 (2007). 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 (≤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 ≥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.

Transcription-Independent Suppression of DNA Synthesis by p53 in Sperm-Irradiated Mouse zygotes

Cell cycle arrest in response to DNA damage is important for the maintenance of genomic integrity in higher eukaryotes. We have previously reported the novel p53-dependent S-phase checkpoint operating in mouse zygotes fertilized with irradiated sperm. In the present study, we analysed the detail of the p53 function required for this S-phase checkpoint in mouse zygotes. The results indicate that ATM kinase is likely to be indispensable for the p53-dependent S-phase checkpoint since the suppression was abrogated by inhibitors such as caffeine and wortmannin. However, ATM phosphorylation site mutant proteins were still capable of suppressing DNA synthesis when microinjected into sperm-irradiated zygotes lacking the functional p53, suggesting that the target of the phosphorylation is not p53. In addition, the suppression was not affected by α-amanitin, and p53 protein mutated at the transcriptional activation domain was also functional in the suppression of DNA synthesis. However, p53 proteins mutated at the DNA-binding domain were devoid of the suppressing activity. Taken together, the transcription-independent function of p53 associated with the DNA-binding domain is involved in the S-phase checkpoint in collaboration with yet another unidentified target protein(s).

Cleft palate caused by perfluorooctane sulfonate is caused mainly by extrinsic factors

Perfluorooctane sulfonate (PFOS) is found ubiquitously in the environment, and is known to cause developmental toxicity, including cleft plate (CP). The aim of the present study was to elucidate the mechanism of CP associated with in utero exposure to PFOS in mice. We first examined whether the concentration of PFOS in fetal serum was related to susceptibility to CP. We compared palatogenesis following the administration of various concentrations of PFOS to dams. We conducted histological examination on gestational day (GD) 15 and 18, and alizarin red/alcian blue staining of fetal heads on GD18. Finally, we cultured palatal shelves (PSs) of GD14 fetuses, which had not yet made contact with each other, for 48h, to examine whether the shelves maintained the ability to fuse. The incidence of CP increased from 7.3% with a fetal serum concentration of PFOS of 110.7+/-13.4microg/ml (13mg/kg) to 78.3% with 138.6+/-0.9microg/ml (20mg/kg). PFOS at 50mg/kg on GD11-15 caused CP at a rate of 6.1%, meanwhile PFOS at 20mg/kg on GD1-17 caused a CP rate of 89.3%. Failure of palatal shelf elevation was observed with 20mg/kg PFOS. PFOS at 20mg/kg on GD1-17 and 50mg/kg on GD11-15 inhibited mandibular growth to the same extent, even though the rate of CP was different. Explants exposed to PFOS 20mg/kg and Tween 20 showed 94% (34/36) and 100% (31/31) fusion, respectively. We demonstrated that increasing the oral dose of PFOS from 13 to 20mg/kg resulted in a significant increase in CP even though there was only a small increase in serum concentration of PFOS. PFOS prevented elevation of the PSs above the tongue because their growth/fusion potential was maintained. Mandibular hypoplasia did not seem to play a critical role in the pathogenesis of CP.

Roles of neuropeptides in O,O,S -trimethylphosphorothioate (OOS-TMP)-induced anorexia in mice

O,O,S-Trimethylphosphorothioate (OOS-TMP), an impurity present in various organophosphorus insecticides, has previously been shown to induce hypophagia. The major goal of this study was to investigate its mechanism of action. Both intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) injection transiently induced hypophagia at a dose of 5mg/kg within 6h, without causing lung injury. Hypophagia was accompanied by up-regulation of corticotropin releasing factor (CRF) (2.92+/-0.45 vs. 1.7+/-0.5, at 2h after i.c.v., 3.40+/-1.38 vs. 1.76+/-0.41 at 6h after i.p., P<0.05) in the hypothalamus. After i.c.v. injection, hypophagia recovered by 6h after dosing. At doses higher than 5mg/kg, i.c.v. injection induced continuous hypophagia from 20min to 72h after dosing, accompanied by hypothermia and lung injury. OOS-TMP was considered to induce hypophagia through enhancing expression of CRF.

Levels of N-acylethanolamines in O,O,S-trimethylphosphorothioate (OOS-TMP)-treated C57BL/6J mice and potential anti-obesity, anti-diabetic effects of OOS-TMP in hyperphagia and hyperglycemia mouse models.

O,O,S-Trimethylphosphorothioate (OOS-TMP) has been shown to induce hypophagia and hypopraxia. Recent studies suggest that OOS-TMP-induced anorexia is partly mediated by its effect on the central nervous system. In this study, we examined the profiles of N-acylethanolamines (NEAs), including five amide-linked compounds, in the gastrointestinal system in C57BL/6J (B6) mice. The present results shown an orexigenic profile of the levels of NEAs with downregulation of the anorectic lipid, N-stearoylethanolamine (SEA), upregulation of the orexigenic lipid, 2-arachidonoyl glycerol (2-AG), at 2 h and upregulation of 2-AG at 24 h albeit with significant anorexia. However, the data indicated that the high level of 2-AG may be responsible for the hypopraxia. We next explored whether OOS-TMP may affect two models of hyperphagia and hyperglycemia, ins2(+/Akita) B6 (Akita) and B6-lepr(db)/lepr(db) mice (db/db). We identified potential anorexigenic effects in B6, Akita and db/db mice. Moreover, OOS-TMP was found to reduce blood glucose in Akita mice but not in db/db mice. Collectively, these findings suggest that N-acylethanolamines are not involved in the hypophagia but rather hypopraxia, and may play multiple physiological roles in this process. OOS-TMP might be a promising candidate for anti-obesity and anti-diabetic drug development.