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The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

Aims The impact of periodontal inflammation on lipid metabolism is controversial. This study aimed to investigate the association between full-mouth periodontal inflammation and serum lipid levels. Materials and methods In this cross-sectional study, we performed periodontal and bacteriological examinations during medical checkup on 131 subjects. The association between the periodontal inflamed surface area (PISA) and the lipid markers was analyzed by multiple linear regression, adjusting for age, sex, smoking, and body mass index. Results Overall, 118 medically healthy participants were analyzed. The proportions of none, mild, moderate, and severe periodontitis were 37.3%, 32.2%, 25.4%, and 5.1%, respectively. Multivariate analysis showed that high-density lipoprotein cholesterol was significantly higher in participants with the lowest tertile of PISA values (PISA low, coefficient: 7.94; 95% confidence interval [CI]: 1.63, 14.26, p = .01) compared to those in other tertiles (PISA high). Low-density/high-density lipoprotein cholesterol and total/high-density lipoprotein cholesterol ratios were significantly lower in the PISA-low group than the PISA-high group (coefficient: -0.26 and -0.30; 95% CI: -0.50, -0.02, and -0.59, -0.0002; p = .04 and .0498). Serum high-sensitivity C-reactive protein level, but not serum Porphyromonas gingivalis antibody titer, partly explained the association between PISA and high-density lipoprotein cholesterol. A significant interaction between female sex and PISA values toward high-density lipoprotein cholesterol level was detected. Conclusion Periodontal inflammation was inversely associated with higher high-density lipoprotein cholesterol, especially in females. Elevated serum C-reactive protein partly explained this association.

Hematopoietic tissues of vertebrates are highly radiation sensitive and the effects of ionizing radiation on the hematopoiesis have been studied in mammals and teleosts for decades. In this study, radiation responses in the kidney, the main hematopoietic organ in teleosts, were investigated in Japanese medaka (Kidney, the main hematopoietic tissue of adult medaka fish, was locally irradiated using proton and carbon ion beams irradiation system of Takasaki Ion Accelerator for Advanced Radiation Application (TIARA), QST, and the effects on peripheral blood cells and histology of the kidney were investigated.When only kidneys were locally irradiated with proton or carbon ion beam (15 Gy), the hematopoietic cells in the irradiated kidney and cell density in the peripheral blood decreased 7 days after the irradiation in the same manner as after the whole-body irradiation with γ-rays (15 Gy). These results demonstrate that direct irradiation of the hematopoietic cells in the kidney induced cell death and/or cell cycle arrest and stopped the supply of erythroid cells. Then, the cell density in the peripheral blood recovered to the control level within 4 days and 7 days after the γ-ray and proton beam irradiation (15 Gy), respectively, while the cell density in the peripheral blood did not recover after the carbon ion beam irradiation (15 Gy). The hematopoietic cells in the irradiated kidneys temporarily decreased and recovered to the control level within 21 days after the γ-ray or proton beam irradiation (15 Gy), while it did not recover after the carbon ion beam irradiation (15 Gy). In contrast, the recovery of the cell density in the peripheral blood delayed when anemic medaka were irradiated 1 day after the administration of phenylhydrazine. With and without γ-ray irradiation, a large number of hematopoietic cells was still proliferating in the kidney 7 days after the anemia induction.The results obtained strongly suggest that the hematopoietic stem cells in medaka kidney prioritize to proliferate and increase peripheral blood cells to eliminate anemia, even when they are damaged by high-dose irradiation.

In the oviparous medaka fish, Oryzias latipes, mature spermatozoa that were artificially introduced into the ovarian cavity retaining ovulated eggs could internally fertilize these eggs. This enabled us to examine the effect of ovarian gestation on the ovulation cycle. Most freshly ovulated eggs could be internally fertilized in the ovarian cavity. Yet eggs ovulated 24 h after single insemination remained unfertilized in the ovarian cavity. Artificially pregnant females persisted in a daily cycle of ovulation, which occurred shortly before the onset of light under the present reproductive conditions. Females continuously ovulated a certain number of eggs despite ovarian gestation, that is, the presence of embryos within the ovarian cavity. Repeated cycles of ovulation led to crowding in the ovarian cavity because the group of fertilized eggs, with their hardened egg envelope (chorion or zona radiata), plugged the genital orifice. The development of fertilized eggs was retarded and ceased around the initiation stage of blood circulation, but when they were transferred from the ovarian cavity into regular saline, they regained their ability to develop normally up to hatching. These results show that in oviparous female medaka, ovarian gestation exerted little effect on the time of ovulation and the number of ovulated eggs.

Hematopoietic tissues of vertebrates are highly radiation sensitive and the effects of ionizing radiation on the hematopoiesis have been studied in mammals and teleosts for decades. In this study, radiation responses in the kidney, the main hematopoietic organ in teleosts, were investigated in Japanese medaka (Oryzias latipes), which has been a model animal and a large body of knowledge has been accumulated in radiation biology. Kidney, the main hematopoietic tissue of adult medaka fish, was locally irradiated using proton and carbon ion beams irradiation system of Takasaki Ion Accelerator for Advanced Radiation Application (TIARA), QST, and the effects on peripheral blood cells and histology of the kidney were investigated. When only kidneys were locally irradiated with proton or carbon ion beam (15 Gy), the hematopoietic cells in the irradiated kidney and cell density in the peripheral blood decreased 7 days after the irradiation in the same manner as after the whole-body irradiation with γ-rays (15 Gy). These results demonstrate that direct irradiation of the hematopoietic cells in the kidney induced cell death and/or cell cycle arrest and stopped the supply of erythroid cells. Then, the cell density in the peripheral blood recovered to the control level within 4 days and 7 days after the γ-ray and proton beam irradiation (15 Gy), respectively, while the cell density in the peripheral blood did not recover after the carbon ion beam irradiation (15 Gy). The hematopoietic cells in the irradiated kidneys temporarily decreased and recovered to the control level within 21 days after the γ-ray or proton beam irradiation (15 Gy), while it did not recover after the carbon ion beam irradiation (15 Gy). In contrast, the recovery of the cell density in the peripheral blood delayed when anemic medaka were irradiated 1 day after the administration of phenylhydrazine. With and without γ-ray irradiation, a large number of hematopoietic cells was still proliferating in the kidney 7 days after the anemia induction. The results obtained strongly suggest that the hematopoietic stem cells in medaka kidney prioritize to proliferate and increase peripheral blood cells to eliminate anemia, even when they are damaged by high-dose irradiation.

Background: Diabetes mellitus (DM) is a well-known risk factor for periodontal disease, independent of age and smoking. However, the relationship between glycaemic control and tooth loss has not been well characterized. We aimed to determine whether the haemoglobin A1c (HbA1c) level is associated with the number of teeth remaining. Methods: This was a cross-sectional study of 233,567 individuals aged 20–74 years in a database comprising health insurance claim and health check-up data that was collected between April 1, 2015 and March 31, 2016. The participants were allocated to five groups according to their HbA1c, and the number of teeth was compared between age-groups. DM was defined by an HbA1c ≥6·5%. Findings: Fifty-two-point-seven percent were women and the mean (SD) age was 47·7 (9·6) years. Higher HbA1c was associated with fewer teeth if >30 years of age ( P for trend 30 years of age ( P

Transgenic expression in medaka of the Xiphophorus oncogene xmrk, under a pigment cell specific mitf promoter, induces hyperpigmentation and pigment cell tumors. In this study, we crossed the Hd-rR and HNI inbred strains because complete genome information is readily available for molecular and genetic analysis. We prepared an Hd-rR (p53+/−, p53−/−) and Hd-rR HNI hybrid (p53+/−) fish-based xmrk model system to study the progression of pigment cells from hyperpigmentation to malignant tumors on different genetic backgrounds. In all strains examined, most of the initial hyperpigmentation occurred in the posterior region. On the Hd-rR background, mitf:xmrk-induced tumorigenesis was less frequent in p53+/− fish than in p53−/− fish. The incidence of hyperpigmentation was more frequent in Hd-rR/HNI hybrids than in Hd-rR homozygotes; however, the frequency of malignant tumors was low, which suggested the presence of a tumor suppressor in HNI genetic background fish. The effects on tumorigenesis in xmrk-transgenic immature medaka of a single 1.3 Gy irradiation was assessed by quantifying tumor progression over 4 consecutive months. The results demonstrate that irradiation has a different level of suppressive effect on the frequency of hyperpigmentation in purebred Hd-rR compared with hybrids.

The tumor suppressor protein p53 is considered a guardian of genome integrity, regulating the induction of apoptosis and cell cycle arrest in response to irradiation to block the transmission of teratogenic mutations to progeny cells. We examined the function of p53 in highly radiosensitive tissues, the developing brain and mature testis, using a small fish model, medaka (Oryzias latipes). Medaka offer advantages as a vertebrate model system, as the transparency and small size of the embryos enables clear detection of apoptotic cells in the developing brain. In addition, the simple architecture of medaka testes enables more precise identification of the differentiating spermatogenic stages compared with mammals. We found that in irradiated p53-deficient embryonic brain, diminished induction of apoptosis facilitated tissue regeneration earlier compared to irradiated wild-type embryos, which remained structural abnormalities in the retina at hatching. Moreover, the prominent delay in apoptotic induction in irradiated p53-deficient testes could induce transient mis-differentiation during spermatogenesis, such as the formation of ovum-like cells (testis-ova). However, all testis-ova cells were eliminated via p53-independent apoptosis, and spermatogenesis was completely restored within 1 month after irradiation. Collectively, these data indicate that p53 is not indispensable for the restoration of irradiation-induced damaged tissues.

Induction of apoptosis in response to various genotoxic stresses could block transmission of teratogenic mutations to progeny cells. The severity of biological effects following irradiation depends on the stage at which embryos are irradiated during embryogenesis. We reported previously that irradiation of medaka embryos 3 days post fertilization (dpf) with 10 Gy of gamma rays induced high incidence of apoptotic cells in the mid-brain, however, the embryos hatched normally and developed without apparent malformations. To determine the severity of biological effects following irradiation during a later period of embryogenesis, embryos of various developmental stages were irradiated with 15 Gy of gamma rays and examined for apoptotic induction at 24 h after irradiation in the brain, eyes and pharyngeal epithelium tissues, which are actively proliferating and sensitive to irradiation. Embryos irradiated at 3 dpf exhibited many apoptotic cells in these tissues, and all of them died due to severe malformations. In contrast, embryos irradiated at 5 dpf showed no apoptotic cells and subsequently hatched without apparent malformations. Embryos irradiated at 4 dpf had relatively low numbers of apoptotic cells compared to those irradiated at 3 dpf, thereafter most of them died within 1 week of hatching. In adult medaka, apoptotic cells were not found in these tissues following irradiation, suggesting that apoptosis occurs during a restricted time period of medaka embryogenesis throughout the life. No apoptotic cells were found in irradiated intestinal tissue, which is known to be susceptible to radiation damage in mammals, whereas many apoptotic cells were found in proliferating spermatogonial cells in the mature testis following irradiation. Taken together, with the exception of testicular tissue, the results suggest a limited period during medaka embryogenesis in which irradiation-induced apoptosis can occur.

Background and Objective The biochemical effects of an over-the-counter (OTC) medication were studied, which consists of a single-tuft brush containing cetylpyridinium chloride as a bactericidal agent, dipotassium glycyrrhizate as an anti-inflammatory drug and allantoin as a promoter of cell proliferation and wound healing, for delivery to hardly brushed sites. Material and Methods This randomized controlled double-blind study was performed in 61 subjects with chronic periodontitis in supportive periodontal therapy phase (test group: n = 27; placebo group: n = 28; dropout: n = 6). The OTC medication was self-applied twice a day for 12 wk to two molars with probing pocket depths of 4–6 mm. Biochemical indicators were evaluated at baseline and 12 wk using the suspension array system for eight cytokines and chemokines (interleukin [IL]-1β, IL-1ra, IL-4, IL-6, IL-8, IL-10, monocyte chemoattractant protein-1 and tumor necrosis factor [TNF]-α) in gingival crevicular fluid. Results The levels of IL-1β, IL-6, IL-8 and TNF-α remained significantly lower in the test group compared to the placebo group. In the placebo group, when the probing pocket depth at baseline was 4 mm, IL-1β increased, particularly in the second molar tooth, and the greatest increase was seen when PPD at baseline was 5–6 mm. In the test group, IL-1β decreased markedly in cases with furcation involvement and low bleeding on probing at baseline. In both groups, IL-1β, IL-6 and TNF-α were closely correlated with each other. Conclusion This OTC medication is biochemically effective for steady chronic periodontitis in the supportive periodontal therapy phase.

Gamma-ray irradiation promotes the formation of testis-ova in p53-deficient medaka (Oryzias latipes) and this finding suggests the possible involvement of p53 in sex differentiation in medaka [1]. Since administration of female hormone, estrogen, induces testis-ova in male fish, irradiation may disturb the hormonal balance and induce the testis-ova. To investigated whether the involvement of the hormonal imbalance by irradiation in the testis-ova induction, we put a 5 mm-thick acrylic plate for shield and separately irradiated the head and body of adult male fish using the carbon ion beam in Takasaki Ion Accelerators for Advanced Radiation Application (TIARA), QST[2]. Whereas testis-ova were not observed in the testis of the head-irradiated males 14 days after irradiation, testis-ova were observed in the body-irradiated males, demonstrating that direct irradiation of the testis is required to induce testis-ova. To confirm this finding, we used a collimated carbon ion microbeam irradiation (2 Gy and diameter of 250 µm) in TIARA to conduct targeted irradiation of p53-deficient male medaka testis. Seven days after irradiation, we found that testis-ova were induced only in the cyst of type A spermatogonia (Ga) that was expected to be located in the path of the microbeam. These results indicate that direct irradiation of p53-deficient testis induced abnormal differentiation of Ga to testis-ova and that microbeam is highly applicable to investigate the irradiation-induced cellular response in vivo., 第23回小型魚類研究会

The kidney is a hematopoetic tissue in medaka (Oryzias latipes) and the hematopietic tissue is sensitive to irradiation. We have been obsereved reduction of hematopietic cells in the kidney by carbon ion beam irradiation up to 2.2 mm in depth of medaka using Takasaki Ion Accelerators for Advanced Application (TIARA) of National institutes for Quantum and Radiological Science and Technology (Nagata.et.al., 2016). In this study, we made further analyses to clarify the radiation response of hematopoetic tissues by peripheral blood cell number and histologically evaluation of the kidney. When the 15 Gy of carbon ion beam was irradiated from the dorsal and ventral side of the kidney, the number of prripheral blood cell was decreased and increased from control 8 days after irradiation, respectively. This result confirmed the histological alterations of our preveous study. Neither the number of the peripheral blood cell nor the heamatopietic cells in the kidney recovered as the control to the 28 days after the irradiation of carbon ion beam from the dorsal side, whereas the histology of the kidney was recovered without increase of the number of the blood cell 28 days after the whole body irradiation by 15 Gy of gamma rays (137Cs). We then shielded medaka with 5-mm-thick acrylic plate with a hole on the position of the kidney to irradiate only kidney area, and irradiated 15 Gy one side or both sides on the kidney from the dorsal side. Seven days after the irradiation, the atrophy and reduction of hematopoietic cells were observed in the irradiated kidney and numbers of the blood cell were fewer in the kidney irradiated group than control. We are now establishing a precise local irradiation system that using heavy-ion microbeam of QST-Takasaki (diameter 250 μm) to analyze the local alterations by the irradiation. It is known that phenylhydrazine (PHZ) cause anemia and anemia may induce proliferation of hematopietic cells. So we used PHZ on medaka as an proliferation activator of the hematopoietic cells. Contrary to our expectation, the number of peripheral blood cells did not decrease 7 days after irradiation. This result suggests that hematopoietic cells become radiation resistant when proliferation was induced. In this study, we established a local irradiation system and clarified the radiation sensitivity of hematopoietic cells in different cell states. These results can contribute to reveal the mechanism of radiation response., 第23回小型魚類研究会

Radiotherapy is widely used in cancer treatment. In addition to inducing effects in the irradiated area, irradiation may induce effects on tissues close to and distant from the irradiated area. Japanese medaka, Oryzias latipes, is a small teleost fish and a model organism for evaluating the environmental effects of radiation. In this study, we applied low-energy carbon-ion (26.7 MeV/u) irradiation to adult medaka to a depth of approximately 2.2 mm from the body surface using an irradiation system at the National Institutes for Quantum and Radiological Science and Technology. We histologically evaluated the systemic alterations induced by irradiation using serial sections of the whole body and conducted a heart rate analysis. Tissues from the irradiated side showed signs of serious injury that corresponded with the radiation dose. A 3D reconstruction analysis of the kidney sections showed reductions in the kidney volume and blood cell mass along the irradiated area, reflecting the precise localization of the injuries caused by carbon-beam irradiation. Capillary aneurysms were observed in the gill in both ventrally and dorsally irradiated fish, suggesting systemic irradiation effects. The present study provides an in vivo model for further investigation of the effects of irradiation beyond the locally irradiated area.

Whereas the Galβ1-4Gal epitope is rarely found in mammalian glycans, it has been found in glycans of various species of non-mammalian vertebrates, such as fish, amphibians and birds. Although glycans containing Galβ1-4Gal in these vertebrates were detected by precise structural analysis of the glycans using mass spectrometry and/or NMR spectrometry, there are no convenient methods to detect Galβ1-4Gal from various samples. To analyze systematically the distribution of Galβ1-4Gal in nature, we generated mouse monoclonal antibodies (mAbs) specific for Galβ1-4Gal using extracts of medaka eggs as an immunogen. Four mAbs (two immunoglobulin (Ig)Ms and two IgG1s) were obtained by enzyme-linked immunosorbent assay-based screening. The specificities of these mAbs were evaluated by frontal affinity chromatography using 142 kinds of 2-aminopyridine (PA)-derivatized oligosaccharides. While all mAbs interacted with (Galβ1-4Gal)-containing oligosaccharides at their non-reducing termini with dissociation constants (K(d)) ranging from 1.0 x 10⁻⁵ to 2.8 x 10⁻⁴ M, no apparent interaction was observed with any other glycans. The number of branches containing Galβ1-4Gal on N-glycans did not significantly affect K(d) of mAbs of IgG1 subclasses, but those of IgM mAbs were decreased by ∼1 order of magnitude, in increments of the number of branches present. Using the mAbs, we established that Galβ1-4Gal is also expressed on glycoproteins in various tissues from the African clawed frog. Immunohistochemical staining of medaka sections revealed that Galβ1-4Gal epitopes were expressed in the endothelium, epithelium and epidermis, which directly contact the external environment or invading organisms. Thus, these mAbs are useful for systematically investigating the species-specific expression of glycans, which may act as a barrier against infection.

Purpose: Exposure to heavy-ion radiation is considered a critical health risk on long-term space missions. The developing central nervous system (CNS) is a highly radiosensitive tissue; however, the biological effects of heavy-ion radiation, which are greater than those of low-linear energy transfer (LET) radiation, are not well studied, especially in vivo in intact organisms. Here, we examined the effects of iron-ions on the developing CNS using vertebrate organism, fish embryos of medaka (Oryzias latipes). Materials and methods: Medaka embryos at developmental stage 28 were irradiated with iron-ions at various doses of 0-1.5 Gy. At 24 h after irradiation, radiation-induced apoptosis was examined using an acridine orange (AO) assay and histo-logically. To estimate the relative biological effectiveness (RBE), we quantified only characteristic AO-stained rosette-shaped apoptosis in the developing optic tectum (OT). At the time of hatching, morphological abnormalities in the irradiated brain were examined histologically. Results: The dose-response curve utilizing an apoptotic index for the iron-ion irradiated embryos was much steeper than that for X-ray irradiated embryos, with RBE values of 3.7-4.2. Histological examinations of irradiated medaka brain at 24 h after irradiation showed AO-positive rosette-shaped clusters as aggregates of condensed nuclei, exhibiting a circular hole, mainly in the marginal area of the OT and in the retina. However, all of the irradiated embryos hatched normally without apparent histological abnormalities in their brains. Conclusion: Our present study indicates that the medaka embryo is a useful model for evaluating neurocytotoxic effects on the developing CNS induced by exposure to heavy iron-ions relevant to the aerospace radiation environment.

Some studies for radiological protection of the environment have been made at the National Institute of Radiological Sciences (NIRS). Transfer of radionuclides and related elements has been investigated for dose estimation of non-human biota. A parameter database and radionuclide transfer models have been also developed for the Japanese environments. Dose (rate)-effect relationships for survival, growth and reproduction have been investigated in conifers, Arabidopsis, fungi, earthworms, springtails, algae, duckweeds, daphnia and medaka. Also genome-wide gene expression analysis has been carried out by high coverage expression profiling (HiCEP). Effects on aquatic microbial communities have been studied in experimental ecosystem models, i.e., microcosms. Some effects were detected at a dose rate of 1 Gy day(-1) and were likely to arise from interspecies interactions. The results obtained at NIRS have been used in development of frameworks for environmental protection by some international bodies, and will contribute to environmental protection in Japan and other Asian countries.

We have examined cerebellar morphogenesis after neural tube stage in medaka (Oryzias latipes), a ray-finned fish, by conventional histology and immunohistochemistry using anti-proliferating cell nuclear antigen (PCNA) and anti-acetylated tubulin antibodies. Our results indicate that the medaka cerebellum is formed in 4 successive stages: (1) formation and enlargement of the cerebellar primordia; (2) rostral midline fusion of the left/right halves of the cerebellar primordia; (3) formation of the cerebellar matrix zones in the midline and caudalmost regions of the primitive cerebellum, and (4) growth and differentiation of the cerebellum. Our results also show that cerebellar morphogenesis is different from that in mammals in 3 important points: the developmental origins of the primordia, directions along which cerebellar fusion proceeds, and number, locations and duration of the cerebellar matrix zones. During the course of this study, an alar-derived membranous structure between the cerebellum and the midbrain in the adult medaka brain was identified as the structure homologous to the rostrolateral part of the mammalian anterior medullary velum. We have named this structure in the adult teleostean brains as the ‘mesencephalic sheet’. The present study indicates that there exists both conserved and divergent patterns in cerebellar morphogenesis in vertebrates.

To observe the sequential radiation-induced apoptosis in a living embryo, we injected Acridine Orange (AO) solution into the yolk of embryo and visualized radiation-induced apoptosis in developing optic tectum (OT). Medaka embryos at stage 28, when neural cells proliferate rapidly in the OT, were irradiated with 5 Gy X-rays which is a non-lethal dose for irradiated embryos at hatching. The irradiated embryos hatched normally without morphological abnormalities in their brains, even though a large number of apoptotic cells were induced transiently in OT. By yolk injection, apoptotic cells in OT were distinguished as AO-positive small nuclei at 3 h after irradiation. At 8-10 h after irradiation, AO-positive rosette-shaped clusters were obviously distinguished in marginal tectal regions of OT where cells are proliferating intensely. The AO-positive clusters became bigger and more obvious, but the number did not increase up to 24 h after irradiation and completely disappeared up to 49 h after irradiation. This characteristic appearance of the AO-positive nuclei/clusters is in good agreement with our previous results, based on the examination of fixed specimens stained with AO by injection into the peri-vitelline space, suggesting that the AO-yolk injection method is highly reliable for detecting apoptotic cells in living embryos. The live imaging of apoptotic cells in developing Medaka embryos by AO-yolk injection method is expected to reveal more of the details of the dynamics of apoptotic responses in the irradiated brain and other tissues.

This paper summarizes research activities in National Institute of Radiological Sciences (NIRS) for evaluation of the radiation effects on selected terrestrial and aquatic organisms as well as the ecosystems. Seven organisms, conifers, fungi, earthworms, springtails, algae, daphnia and Medaka are presently selected to study. For the estimation of possible radiation dose, transfers of radionuclides and related elements from medium to organisms are evaluated. Dose-effect relationships of acute gamma radiation on the survival, growth, and reproduction of selected organisms have been studied. Studies on the effect of chronic gamma radiation at low dose rate were also started. In order to understand the mechanism of radiation effects and to find possible indicators of the effects, information of genome- and metagenome-wide gene expression has been collected. Evaluation of ecological effects of radiation is more challenging task. Study methods by using three-species microcosm were established, and an index for the holistic evaluation of effects on various ecological parameters was proposed. The microcosm has been simulated as a computer simulation code. Developments of more complicated and practical model ecosystems have been started. The Denaturant Gradient Gel Electrophoresis (DGGE) has been applied on soil bacterial community in order to evaluate the radiation effects on soil ecosystems., International Conference on Radioecology & Environmental Radioactivity

Developing neural tubes are bilaterally symmetric in all vertebrate embryos, irrespective of the presence of gene networks that generate left-right asymmetry. To explore the mechanisms that underlie the bilaterally symmetric formation of the neural tube, we examined a medaka (Oryzias latipes) dominant mutant, Oot, the neural tube of which transiently lacks normal symmetry in the optic tectum. We found that spatial changes in isthmic fgf8 expression do not occur on one side of the mutant, resulting in a transient desynchronized expression that correlates with tectal asymmetry. The application of exogenous FGF8 on one side of a wild-type embryo mimics the Oot phenotype, indicating that the bilaterally equivalent expression of isthmic fgf8 is crucial for the bilaterally symmetric development of the tectum. These results suggest that tectal symmetry is not a “default” state, but rather is maintained actively by a bilaterally coupled and synchronized regulation of isthmic fgf8 expression. genesis 46:537–545, 2008. © 2008 Wiley-Liss, Inc.

The cerebellar structures of teleosts are markedly different from those of other vertebrates. The cerebellum continues rostrally into the midbrain ventricle, forming the valvula cerebelli, only in ray-finned fishes among vertebrates. To analyze the ontogenetic processes that underlie this morphological difference, we examined the early development of the cerebellar regions, including the isthmus (mid/hindbrain boundary, MHB), of the medaka (Oryzias latipes), by histology and in-situ hybridization using two gene (wnt1 and fgf8) probes. Isthmic wnt1 was expressed stably in the caudalmost mesencephalic region in the neural tube at all developmental stages examined, defining molecularly the caudal limit of the mesencephalon. The wnt1-positive mesencephalic cells became located rostrally to the isthmic constriction at Iwamatsu's stages 25-26. Isthmic fgf8 expression changed dynamically and became restricted to the rostralmost metencephalic region at stage 24. The rostralmost part (prospective valvula cerebelli) of the fgf8-positive rostral metencephalon protruded rostrally into the midbrain ventricle, bypassing the isthmic constriction, at stages 25-26. Thus, the isthmic constriction shifted caudally with respect to the molecularly defined MHB at stages 25-26. Paired cerebellar primordia were formed from the alar plates of the fgf8-positive rostral metencephalon and the fgf8-negative caudal metencephalon in the medaka neural tube. Our results show that cerebellar development differs between teleosts and murines: both the rostral and caudal metencephalic alar plates develop into the cerebellum in medaka, whereas in the murines only the caudal metencephalic alar plate develops into the cerebellum, and the rostral plate is reduced to a thin membrane.

We describe a novel method for rapid and quantitative evaluation of the degree of radiation-induced apoptosis in the developing brain of medaka (Oryzias latipes). Embryos at stage 28 were irradiated with 1, 2, 3.5, and 5 Gy x-ray. Living embryos were stained with a vital dye, acridine orange (AO), for 1-2 h, and whole-mount brains were examined under an epifluorescence microscope. From 7 to 10 h after irradiation with 5 Gy x-ray, we found two morphologically different types of AO-stained structures, namely, small single nuclei and rosette-shaped nuclear clusters. Electron microscopy revealed that these two distinct types of structures were single apoptotic cells with condensed nuclei and aggregates of apoptotic cells, respectively. From 10 to 30 h after irradiation, a similar AO-staining pattern was observed. The numbers of AO-stained rosette-shaped nuclear clusters and AO-stained single nuclei increased in a dose-dependent manner in the optic tectum. We used the number of AO-stained rosette-shaped nuclear clusters/optic tectum as an index of the degree of radiation-induced brain cell death at 20-24 h after irradiation. The results showed that the number of rosette-shaped nuclear clusters/optic tectum in irradiated embryos exposed to 2 Gy or higher doses was highly significant compared to the number in nonirradiated control embryos, whereas no difference was detected at 1 Gy. Thus, the threshold dose for brain cell death in medaka embryos was taken as being between 1-2 Gy, which may not be so extraordinarily large compared to those for rodents and humans. The results show that medaka embryos are useful for quantitative evaluation of developmental neurocytotoxic effects of radiation.

We propose here a novel interpretation of the embryonic origin of cells of diencephalic sensory relay nuclei in teleosts based on our recent studies of gene expression patterns in the medaka (Oryzias latipes) embryonic brain and comparative hodological studies. It has been proposed that the diencephalic sensory relay system in teleosts is unique among vertebrates. Teleost relay nuclei, the preglomerular complex (PG), have been assumed to originate from the basal plate (the posterior tuberculum) of the diencephalon, whereas relay nuclei in mammals are derived from the alar plate (dorsal thalamus) of the diencephalon. Our results using in situ hybridization show, however, that many pax6- or dlx2-positive cells migrate laterally and ventrocaudally from the diencephalic alar plate to the basal plate during development. Massive clusters of the migrated alar cells become localized in the mantle layer lateral to the posterior tubercular neuroepithelium, from which main nuclei of the PG appear to differentiate. We therefore consider most if not all neurons in the PG to be of alar, not basal, origin. Thus, the teleost PG, at least in part, can be regarded as migrated alar nuclei. Developmental and hodological data strongly suggest that the teleost PG is homologous to a part of the mammalian dorsal thalamus. The organization and origin of the diencephalic sensory relay system might have been conserved across vertebrates.

To understand how humans adapt to the space environment, many experiments can be conducted on astronauts as they work aboard the Space Shuttle or the International Space Station (ISS). We also need animal experiments that can apply to human models and help prevent or solve the health issues we face in space travel. The Japanese medaka (Oryzias latipes) is a suitable model fish for studying space adaptation as evidenced by adults of the species having mated successfully in space during 15 days of flight during the second International Microgravity Laboratory mission in 1994. The eggs laid by the fish developed normally and hatched as juveniles in space. In 2012, another space experiment (Medaka Osteoclast) was conducted. Six-week-old male and female Japanese medaka (Cab strain osteoblast transgenic fish) were maintained in the Aquatic Habitat system for two months in the ISS. Fish of the same strain and age were used as the ground controls. Six fish were fixed with paraformaldehyde or kept in RNA stabilization reagent (n = 4) and dissected for tissue sampling after being returned to the ground, so that several principal investigators working on the project could share samples. Histology indicated no significant changes except in the ovary. However, the RNA-seq analysis of 5345 genes from six tissues revealed highly tissue-specific space responsiveness after a two-month stay in the ISS. Similar responsiveness was observed among the brain and eye, ovary and testis, and the liver and intestine. Among these six tissues, the intestine showed the highest space response with 10 genes categorized as oxidation-reduction processes (gene ontogeny term GO:0055114), and the expression levels of choriogenin precursor genes were suppressed in the ovary. Eleven genes including klf9, klf13, odc1, hsp70 and hif3a were upregulated in more than four of the tissues examined, thus suggesting common immunoregulatory and stress responses during space adaptation., 形態: カラー図版あり, Physical characteristics: Original contains color illustrations, 資料番号: PA1610027000

The tumor suppressor protein, p53, plays pivotal roles in regulating apoptosis and proliferation in the embryonic and adult central nervous system (CNS) following neuronal injuries such as those induced by ionizing radiation. There is increasing evidence that p53 negatively regulates the self-renewal of neural stem cells in the adult murine brain; however, it is still unknown whether p53 is essential for self-renewal in the injured developing CNS. Previously, we demonstrated that the numbers of apoptotic cells in medaka (Oryzias latipes) embryos decreased in the absence of p53 at 12-24 h after irradiation with 10-Gy gamma rays. Here, we used histology to examine the later morphological development of the irradiated medaka brain. In p53-deficient larvae, the embryonic brain possessed similar vacuoles in the brain and retina, although the vacuoles were much smaller and fewer than those found in wild-type embryos. At the time of hatching (6 days after irradiation), no brain abnormality was observed. In contrast, severe disorganized neuronal arrangements were still present in the brain of irradiated wild-type embryos. Our present results demonstrated that self-renewal of the brain tissue completed faster in the absence of p53 than wild type at the time of hatching because p53 reduces the acute severe neural apoptosis induced by irradiation, suggesting that p53 is not essential for tissue self-renewal in developing brain.

Developmental neurotoxicity/Brain/X-rays/Cell death/Medaka. Medaka ( Oryzias latipes ) embryos at 25‐26 and 28‐30 stages were irradiated with a single acute dose of 10 Gy of X-ray, which is lower than the LD 50 of the embryos. The effects on developing brains were examined under a stereomicroscope in living embryos until hatching. All the irradiated embryos survived; however, from 6 to 35 h after X-ray irradiation, massive clusters of optically opaque and round cells were observed either in the entire brain region (when irradiated at 25‐26 stages) or mainly in the optic tectum (when irradiated at 28‐30 stages). Histological examination and TUNEL showed that these cells are clusters of dead cells. These dead cell clusters disappeared thereafter, and the irradiated embryos continued to develop apparently normally. The grown irradiated embryos, however, had smaller brains and eyes than the nonirradiated control embryos. At hatching, the irradiated embryos exhibited histological abnormalities in the brain, particularly in the torus longitudinalis, and in the retina, although most of them hatched normally and survived. The results indicate that brain cell death and a reduced brain size can be observed in living irradiated embryos, and suggest that the medaka embryo is useful for screening the developmental neurotoxicity effects of various hazardous factors.

In order to know the general pattern of axonogenesis in vertebrates, we examined axonogenesis in the embryonic brain of a teleost fish, medaka (Oryzias latipes), and the results were compared with previous studies in zebrafish and mouse. The axons and somata were stained immunocytochemically using antibodies to a cell surface marker (HNK-1) and acetylated tubulin and visualized by retrograde and anterograde labeling with a lipophilic dye. The fiber systems developed correlating with the organization of the longitudinal and transverse subdivisions of the embryonic brain. The first axons extended from the synencephalic tegmentum, forming the first fiber tract (fasciculus longitudinalis medialis) in the ventral longitudinal zone of the neural rod, 38 hours after fertilization. In the neural tube, throughout the entire brain two pairs of longitudinal fiber systems, one ventral series and one dorsal or intermediate series, and four pairs of transverse fiber tracts in the rostral brain were formed sequentially during the first 16 hours of axon production. In one of the dorsal longitudinal tracts, its branch retracted and disappeared at later stages. One of the transverse tracts was found to course in the telencephalon and hypothalamus. The overall pattern of the longitudinal fiber systems in medaka brain is similar to that in mouse, but apparently different from that in zebrafish. We propose that a ventral tract reported in zebrafish partially belongs to the dorsal fiber system, and that the longitudinal fiber systems in all vertebrate brains pass through a common layout defined by conserved genetic and developmental programs.

Fungus and bacterium, which could grow under acidic conditions, were isolated, and their characteristics of growth and neutralization of acids were studied. They could grow in acidic media (pH 4.0) containing several kinds of acids at the same rate in the medium of pH 7.2 and neutralize acids. For bioremediation of pollutants in soil, we proposed a self-immobilization method of microorganisms forming aggregates in a shallow layer of soil. These isolated strains, which showed weak aggregation and were different in size, were successfully co-immobilized with Bacillus subtilis secreting viscous polymer in a shallow layer of model soil packed in a column and could neutralize acidic solutions flowing through packed soil.

We proposed a novel bioremediation method, bioremediation by the self-immobilization system (BSIS), for application to treat pollution of soil spreading over a wide area. In this method, cells are immobilized by spontaneous formation of aggregates and retained in soil. We selected B. subtilis IFO3335 strain, which secretes viscous filaments and forms strong aggregates, as one of strains suitable for this bioremediation method based on self-immobilization, and studied their behavior in soil. These cells could be retained at a shallow layer under soil for a long period.

Candida rugosa ATCC14830 cells have high tendency to make flocculent masses by entanglement of the mycelium-like shaped cells and can be well immobilized in porous polyvinyl formal (PVF) resin. Moreover, Candida lipolytica cells, which became to show a mycelium-like shape by mutation, were also immobilized in PVF resin.These C. rugosa and mutant C. lipolytica cells were immobilized in PVF resins and cultured by the draw-fill method 2–3 times. These C. rugosa and C. lipolytica cells could decompose L-lactic acid and produce bio-surfactant liposan, respectively. Because high cell densities could be attained in immobilized systems, the total productivity in repeated-batch operations with immobilized cells was higher than those in batch operations with free cells. Moreover, the decomposed amount of L-lactic acid did not decrease during the repeated-batch operations, because the number of immobilized cells in PVF resin was kept almost constant.

Microglia remove apoptotic cells by phagocytosis when the central nervous system is injured in vertebrates. Ionizing irradiation (IR) induces apoptosis and microglial activation in embryonic midbrain of medaka (Oryzias latipes), where apolipoprotein E (ApoE) is upregulated in the later phase of activation of microglia In this study, we found that another microglial marker, l-plastin (lymphocyte cytosolic protein 1), was upregulated at the initial phase of the IR-induced phagocytosis when activated microglia changed their morphology and increased motility to migrate. We further conducted targeted irradiation to the embryonic midbrain using a collimated microbeam of carbon ions (250 μm diameter) and found that the l-plastin upregulation was induced only in the microglia located in the irradiated area. Then, the activated microglia might migrate outside of the irradiated area and spread through over the embryonic brain, expressing ApoE and with activated morphology, for longer than 3 days after the irradiation. These findings suggest that l-plastin and ApoE can be the biomarkers of the activated microglia in the initial and later phase, respectively, in the medaka embryonic brain and that the abscopal and persisted activation of microglia by IR irradiation could be a cause of the abscopal and/or adverse effects following irradiation.

Radiation therapy (RT) is pivotal in the treatment of many central nervous system (CNS) pathologies; however, exposure to RT in children is associated with a higher risk of secondary CNS tumors. Although recent research interest has focused on the reparative and therapeutic role of microglia, their recruitment following RT has not been elucidated, especially in the developing CNS. Here, we investigated the spatiotemporal dynamics of microglia during tissue repair in the irradiated embryonic medaka brain by whole-mount in situ hybridization using a probe for Apolipoprotein E (ApoE), a marker for activated microglia in teleosts. Three-dimensional imaging of the distribution of ApoE-expressing microglia in the irradiated embryonic brain clearly showed that ApoE-expressing microglia were abundant only in the late phase of phagocytosis during tissue repair induced by irradiation, while few microglia expressed ApoE in the initial phase of phagocytosis. This strongly suggests that ApoE has a significant function in the late phase of phagocytosis by microglia in the medaka brain. In addition, the distribution of microglia in p53-deficient embryos at the late phase of phagocytosis was almost the same as in wild-type embryos, despite the low numbers of irradiation-induced apoptotic neurons, suggesting that constant numbers of activated microglia were recruited at the late phase of phagocytosis irrespective of the extent of neuronal injury. This medaka model of microglia demonstrated specific recruitment after irradiation in the developing CNS and could provide a useful potential therapeutic strategy to counteract the detrimental effects of RT.

Eye development and brain structures of a mutant teleost fish were investigated. The el(eyeless) mutation in medaka (Oryzias latipes) is recessive and affects eye formation; in the most severe cases, it results in the absence of eyes. Developmental studies revealed that normal eyeballs are not formed in the el mutant embryos, but small optic cup-like structures differentiate in situ in the walls of the prosencephalon without evagination. The anophthalmic el homozygous fish hatched normally, although they did not respond behaviorally to visual stimuli. A small fraction of these fish grew to adulthood. In the adult anophthalmic el homozygous fish, the brain exhibited abnormalities in several subdivisions. A pair of small abnormal protrusions was observed on the surface of the ventral telencephalon and preoptic area. Immunocytochemistry using a rhodopsin monoclonal antibody showed that opsin-positive cells were present in the abnormal structures. Bodian staining showed that the optic nerves were present near the abnormal structures, although the number of optic nerve fibers was extremely small. The optic tectum was extremely small, and the thickness of the stratum opticum and stratum fibrosum et griseum superficiale was reduced. These behavioral and morphological observations suggest that the adult anophthalmic el homozygous fish are functionally blind, although small retina-like structures were partially differentiated and persisted in the adult fish brain. Moreover, the adult anophthalmic el homozygous fish were infertile, and the sizes of the hypophysis and the hypothalamus were reduced. Thus, the el mutation affects not only the brain structures that are related to the visual system but also those related to the reproductive system.

To determine the postpartum smoking relapse rate among women in Japan who quit smoking during pregnancy and to clarify factors related to smoking relapse.A self-administered questionnaire survey was conducted as a cross-sectional study of all mothers of children who underwent health checkups after birth in randomly selected municipalities in Japan from May to July 2009. Using valid data of 20,601 mothers, smoking rate was calculated. In addition, χ(2) -test and multiple logistic regression analysis were used to clarify related factors to the smoking relapse.The smoking rates among women were 15.8% at the time when they became pregnant, 5.1% during pregnancy and 11.3% after giving birth. Among women who smoked at the time they became pregnant, the smoking rate during pregnancy was 31.1%. Among women who quit smoking during pregnancy, the postpartum smoking relapse rate was 41.0%. The odds ratios (95% confidence interval) for factors in smoking relapse were 0.72 (0.60-0.88) for women spending time with their child in a relaxed mood, 0.67 (0.47-0.94) for women having someone to talk to on the Internet about childrearing, 1.94 (1.60-2.35) for women who worked and 3.37 (2.61-4.35) for women whose partner smoked after they gave birth.It is hoped that future research will establish methods to further support smoking cessation and the continuation of smoking cessation after childbirth, and develop mechanisms to spread knowledge about the harm of smoking in society and encourage women not to start.

Fatty acid-binding proteins (FABPs) belong to a multigene family of small intracellular proteins that bind hydrophobic ligands. Recent studies have indicated that FABP7 plays important roles in neurogenesis or neuronal migration in vertebrates. In this study, we isolated cDNA and the genomic fragment containing the fabp7 gene for medaka fish and examined the expression of the medaka fabp7 gene through the development of their central nervous system (CNS). The medaka fabp7 gene consists of four exons in approximately 1 kb of the genomic region. Its deduced amino acid sequence exhibits over 80% identity with those of other higher vertebrates. In situ hybridization analysis demonstrated that fabp7-positive cells first appear at stage 22 in a small dorsal domain of the retina, dorsal diencephalon, and rhombencephalon, then expand to the entire CNS including the retina and the spinal cord. In addition, we generated two lines of transgenic medaka with 1.7 kb upstream of the fabp7 gene combined with the enhanced-green fluorescence protein (EGFP) gene. The spatio-temporal expression patterns of EGFP in these animals were consistent with the results of in situ hybridization analysis. The result of our reporter assays with a series of truncated fabp7 promoters suggested that POU elements play a role in fabp7 expression in medaka as well as in other vertebrates. Our transgenic animal will contribute to clarifying the role of FABP7 in the development of CNS. J. Exp. Zool. (Mol. Dev. Evol.) 310B:577–587, 2008. © 2008 Wiley-Liss, Inc.

We examined the morphogenesis and regionalization of the embryonic brain of an acanthopterygian teleost, medaka (Oryzias latipes), by in situ hybridization using 14 gene probes. We compared our results with previous studies in other vertebrates, particularly zebrafish, an ostariophysan teleost. During the early development of the medaka neural rod, three initial brain vesicles arose: the anterior brain vesicle, which later developed into the telencephalon and rostral diencephalon; the intermediate brain vesicle, which later developed into the caudal diencephalon, mesencephalon, and metencephalon; and the posterior brain vesicle, which later developed into the myelencephalon. In the late neural rod, the rostral brain bent ventrally and the axis of the brain had a marked curvature at the diencephalon. In the final stage of the neural rod, ventricles began to develop, transforming the neural rod into the neural tube. In situ hybridization revealed that the brain can be divided into three longitudinal zones (dorsal, intermediate, and ventral) and many transverse subdivisions, on the basis of molecular expression patterns. The telencephalon was subdivided into two transverse domains. Our results support the basic concept of neuromeric models, including the prosomeric model, which suggests the existence of a conserved organization of all vertebrate neural tubes. Our results also show that brain development in medaka differs from that reported in other vertebrates, including zebrafish, in gene-expression patterns in the telencephalon, in brain vesicle formation, and in developmental speed. Developmental and genetic programs for brain development may be somewhat different even among teleosts.

In this study, the roles of p53 in impaired spermatogenic male germ cells of p53-deficient medaka were investigated by analyzing histological changes, and gene expressions of 42Sp50, Oct 4 and vitellogenin (VTG2) by RT-PCR or in situ hybridization in the testes. We found that a small number of oocyte-like cells (testis–ova) differentiated spontaneously in the cysts of type A and early type B spermatogonia in the p53-deficient testes, in contrast to the wild-type (wt) testes in which testis–ova were never found. Furthermore, ionizing radiation (IR) irradiation increased the number of testis–ova in p53-deficient testes, increased testis–ova size and proceeded up to the zygotene or pachytene stages of premature meiosis within 14 days after irradiation. However, 28 days after irradiation, almost all the testis–ova were eliminated presumably by p53-independent apoptosis, and spermatogenesis was restored completely. In the wt testis, IR never induced testis–ova differentiation. This is the first study to demonstrate the pivotal role of the p53 gene in the elimination of spontaneous testis–ova in testes, and that p53 is not indispensable for the restoration of spermatogenesis in the impaired testes in which cell cycle regulation is disturbed by IR irradiation.