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Nagao Y.,Utsunomiya University | Nagao Y.,Tokyo University of Agriculture and Technology | Ohta Y.,Utsunomiya University | Ohta Y.,Science Service Inc. | And 2 more authors.
Zygote | Year: 2010

In this study, we investigated the effects of methyl-β-cyclodextrin (MBCD) on in vitro fertilization and the subsequent development of bovine oocytes. Bovine oocytes matured in serum-free medium were inseminated with frozen-thawed sperm pre-incubated in protein-free modified Brackett and Oliphant medium (BO) containing various concentrations of MBCD for various periods. MBCD decreased the frequency of live sperm, however enhanced the capacitation and acrosome reaction of the live sperm. Pre-incubation of sperm with 0.5, 1.0 and 1.5 mM MBCD for 2 and 4 h increased the frequency of normal fertilization. Embryos derived from oocytes fertilized with spermatozoa pre-incubated with MBCD developed normally to the blastocyst stage and term. There were individual differences and similar tendencies in four different sires in terms of the effects of MBCD upon fertilization. These results indicate that the pre-incubation of bovine sperm with MBCD affects viability and capacitation status of the sperm and promotes fertilization in vitro. Embryos derived from oocytes fertilized with sperm pre-incubated with MBCD developed normally to the blastocyst stage and term. © 2010 Cambridge University Press.


PubMed | Japan National Institute of Radiological Sciences, Utsunomiya University, Kyoto University and Science Service Inc.
Type: Journal Article | Journal: Zygote (Cambridge, England) | Year: 2016

Successful in vitro fertilization (IVF) of all inbred strains of laboratory mice has not yet been accomplished. We have previously shown that a high calcium concentration improved IVF in various inbred mice. However, we also found that in cumulus-free ova of C3H/He mice such IVF conditions significantly increased the deficiency of extrusion of the second polar body (PBII) in a dose-dependent manner (2% at 1.71 mM and 29% at 6.84 mM, P < 0.05) and that PBII extrusion was affected by high calcium levels at 2-3 h post-insemination. While developmental competence of ova without PBII extrusion to blastocysts after 96 h culture was not affected, a significant reduction in the nuclear number of the inner cell mass was observed in blastocyst fertilized under high calcium condition. We also examined how high calcium concentration during IVF affects PBII extrusion in C3H/He mice. Cumulus cells cultured under high calcium conditions showed a significantly alleviated deficient PBII extrusion. This phenomenon is likely to be specific to C3H/He ova because deficient PBII extrusion in reciprocal fertilization between C3H and BDF1 gametes was observed only in C3H/He ova. Sperm factor(s) was still involved in deficient PBII extrusion due to high calcium concentrations, as this phenomenon was not observed in ova activated by ethanol. The cytoskeletal organization of ova without PBII extrusion showed disturbed spindle rotation, incomplete formation of contractile ring and disturbed localization of actin, suggesting that high calcium levels affect the anchoring machinery of the meiotic spindle. These results indicate that in C3H/He mice high calcium levels induce abnormal fertilization, i.e. deficient PBII extrusion by affecting the cytoskeletal organization, resulting in disturbed cytokinesis during the second meiotic division. Thus, use of high calcium media for IVF should be avoided for this strain.


Ishida Y.,Japan National Institute of Radiological Sciences | Ishida Y.,Juntendo University | Takabatake T.,Japan National Institute of Radiological Sciences | Kakinuma S.,Japan National Institute of Radiological Sciences | And 14 more authors.
Carcinogenesis | Year: 2010

Accurate cancer risk assessment of low-dose radiation poses many challenges that are partly due to the inability to distinguish radiation-induced tumors from spontaneous ones. To elucidate characteristic features of radiation-induced tumors, we analyzed 163 medulloblastomas that developed either spontaneously or after X-ray irradiation at doses of 0.05-3 Gy in Ptch1 heterozygous mice. All spontaneous tumors showed loss of heterozygosity in broad regions on chromosome 13, with losses at all consecutive markers distal to Ptch1 locus (S-type). In contrast, all tumors that developed after 3 Gy irradiation exhibited interstitial losses around Ptch1 with distal markers retained (R-type). There was a clear dose-dependent increase in the proportion of R-type tumors within the intermediate dose range, indicating that the R-type change is a reliable radiation signature. Importantly, the incidence of R-type tumors increased significantly (P=0.007) at a dose as low as 50 mGy. Integrated array-comparative genomic hybridization and expression microarray analyses demonstrated that expression levels of many genes around the Ptch1 locus faithfully reflected the signature-associated reduction in genomic copy number. Furthermore, 573 genes on other chromosomes were also expressed differently between S-type and R-type tumors. They include genes whose expression changes during early cerebellar development such as Plagl1 and Tgfb2, suggesting a recapitulation of gene subsets functioning at distinct developmental stages. These findings provide, for the first time, solid experimental evidence for a significant increase in cancer risk by low-dose radiation at diagnostic levels and imply that radiation-induced carcinogenesis accompanies both genomic and gene expression signatures. © The Author 2010. Published by Oxford University Press. All rights reserved.


Kokubo T.,Japan National Institute of Radiological Sciences | Kokubo T.,Juntendo University | Kakinuma S.,Japan National Institute of Radiological Sciences | Kobayashi T.,Juntendo University | And 7 more authors.
Cancer Science | Year: 2010

Exposure to carcinogens early in life may contribute to cancer development later in life. The amount of radiation exposure children experience during medical procedures has been increasing, so it is important to evaluate the radiation risk of cancer in developing organs. Toward this goal, we assessed the risk of developing renal cell carcinoma using Eker rats as a kidney tumor model. F1 hybrids of male Eker (Tsc2 mutant) and female F344 rats were irradiated with 0.5 or 2 Gy gamma radiation on gestation days 15 and 19, and on postnatal days 5, 20, and 49. At 27 weeks of age, kidneys were examined for proliferative lesions. Preneoplastic lesions such as phenotypically altered tubules increased after postnatal irradiation as a function of age-at-irradiation, and hyperplasia were greatly increased after perinatal and postnatal irradiation. In contrast, development of adenoma and adenocarcinoma were evident in animals irradiated at perinatal ages, being maximal at gestational day 19. The frequency of LOH at the Tsc2 locus was unexpectedly low - 0% (0 of 4) for the unirradiated control, and 17% (6 of 35) for the irradiated group. Irrespective of LOH, the mTOR (mammalian target of rapamycin) pathway, which is negatively regulated by the Tsc1/2 complex, was activated in both benign and malignant lesions, as evidenced by phosphorylation of S6 ribosomal protein and 4E-BP1. This suggests that the wild-type Tsc2 allele may be functionally inactivated. In conclusion, actively growing kidneys in perinatal-aged (F344 × Eker) F1 rats (Tsc2+/-) are at risk for radiation-induced malignant transformation of the renal epithelium associated with mTOR activation. © 2010 Japanese Cancer Association.


Tsutsumi S.,Japan Aerospace Exploration Agency | Ishii T.,Japan Aerospace Exploration Agency | Ui K.,Japan Aerospace Exploration Agency | Tokudome S.,Japan Aerospace Exploration Agency | Wada K.,Science Service Inc.
Journal of Spacecraft and Rockets | Year: 2015

Subscale model tests and a numerical investigation are performed to predict and attenuate the acoustic level of the Epsilon launch vehicle at liftoff. Requirements for the subscale model test to predict a full-scale acoustic environment are investigated, and then the scale size of the model test is set at 1/42. The launch pads employed herein are designed to attenuate reflection of the Mach wave radiated from the free jet and the acoustic wave due to the jet's impinging on the flame deflector. When comparing the acoustic result taken at the fairing location with that at three different altitudes, 9De, 14De, and 22De, whereDe represents the nozzle exit diameter, the acoustic level observed at 14De is the highest and exceeds the design requirement at all frequencies. It is revealed from the numerical analysis that the reflection of Mach waves radiating from the free jet within the flame path is the dominant acoustic source.Afront cover is attached to the vertical flame path to shield the Mach wave radiation, and 3 dBattenuation in the overall sound pressure level is achieved. The design methods and the knowledge obtained in this study are valid for the design of the launch pad to attenuate liftoff acoustics. Copyright © 2014 by the American Institute of Aeronautics and Astronautics, Inc.


Tsutsumi S.,Japan Aerospace Exploration Agency | Ishii T.,Japan Aerospace Exploration Agency | Ui K.,Japan Aerospace Exploration Agency | Tokudome S.,Japan Aerospace Exploration Agency | Wada K.,Science Service Inc.
53rd AIAA Aerospace Sciences Meeting | Year: 2015

Acoustic design of launch pad and prediction of full-scale acoustic environment are carried out by the numerical simulation and 1/42-scale model test for the Epsilon launch vehicle. Post-flight acoustic analysis of the Epsilon’s first flight is conducted. It is found that the acoustic level inside and outside the payload fairing satisfies the design requirement. The overall sound pressure level inside the payload fairing is achieved to be 132.3 dB without water injection system. Acoustic level outside the payload faring is reduced by 10 dB in compared with the former M-V launcher through the modification of the launch pad. The flight result reveals the effectiveness of the acoustic design methodology developed by the authors for acoustic attenuation at lift-off. Accuracy for predicting full-scale launch environment based on the results of the subscale model test and the numerical simulation is also assessed in this study. © 2015 by the American Institute of Aeronautics and Astronautics, Inc. All rights reserved.

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