2021/04/12 更新

写真a

ヤマシロ ヒデアキ
山城 秀昭
YAMASHIRO Hideaki
所属
教育研究院 自然科学系 農学系列 准教授
自然科学研究科 生命・食料科学専攻 准教授
農学部 農学科 准教授
職名
准教授
連絡先
メールアドレス
外部リンク

学位

  • 博士(農学) ( 2009年3月   東北大学 )

研究分野

  • ライフサイエンス / 動物生命科学

経歴(researchmap)

  • 新潟大学   農学部 農学科 准教授

    2018年6月 - 現在

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  • 新潟大学   農学部 農業生産科学科 助教

    2011年4月 - 2018年5月

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  • ハワイ大学 Institute for Biogenesis Research

    2009年5月 - 2011年3月

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    国名:アメリカ合衆国

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  • 日本学術振興会,日本学術振興会特別研究員

    2007年4月 - 2009年3月

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  • 国際協力機構 青年海外協力隊,ボツワナ国 家畜飼育

    2002年4月 - 2004年7月

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    国名:ボツワナ共和国

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経歴

  • 新潟大学   自然科学研究科 生命・食料科学専攻   准教授

    2018年6月 - 現在

  • 新潟大学   農学部 農学科   准教授

    2018年6月 - 現在

  • 新潟大学   農業生産科学科   助教

    2011年4月 - 2018年5月

学歴

  • 東北大学   Graduate School of Agricultural Science  

    2006年4月 - 2009年3月

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  • 広島大学    

    2004年10月 - 2006年3月

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  • 酪農学園大学   Faculty of Dairy Science  

    1994年4月 - 1999年3月

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論文

  • Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon ( Procyon lotor ) 査読

    Komatsu K, Iwasaki T, Murata K, Yamashiro H, Goh VST, Nakayama R, Fujishima Y, Ono T, Kino Y, Shimizu Y, Takahashi A, Shinoda H, Ariyoshi K, Kasai K, Suzuki M, Palmerini MG, Belli M, Macchiarelli G, Oka T, Fukumoto M, Yoshida MA, Nakata A. Miura T

    Reproduction in Domestic Animals   2020年12月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    DOI: 10.1111/rda.13887

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  • Contribution of light and electron microscopy in the identification of morphological alterations in large Japanese field mouse (Apodemus speciosus) testes exposed to low-dose rate-radiations 査読

    Gatti M, Palmerini MG, Belli M, Nottola SA, Fujishima Y, Yamashiro H, Nakata A, Miura T, Macchiarelli G

    Microscopie31   59 - 63   2020年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Environmental radiation on large Japanese field mice in Fukushima reduced colony forming potential in hematopoietic progenitor cells without inducing genomic instability 査読

    Kentaro Ariyoshi, Tomisato Miura, Kosuke Kasai, Valerie Swee Ting Goh, Yohei Fujishima, Akifumi Nakata, Atsushi Takahashi, Yoshinaka Shimizu, Hisashi Shinoda, Hideaki Yamashiro, Colin Seymour, Carmel Mothersill, Mitsuaki A. Yoshida

    International Journal of Radiation Biology   1 - 31   2020年8月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Informa UK Limited  

    DOI: 10.1080/09553002.2020.1807643

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  • Seasonal changes in the spermatogenesis of the large Japanese field mice (Apodemus speciosus) controlled by proliferation and apoptosis of germ cells 査読

    Jun Ito, Kanna Meguro, Kazuki Komatsu, Takuya Ohdaira, Rina Shoji, Takahisa Yamada, Satoshi Sugimura, Yohei Fujishima, Akifumi Nakata, Manabu Fukumoto, Tomisato Miura, Hideaki Yamashiro

    Animal Reproduction Science214   2020年3月

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    掲載種別:研究論文(学術雑誌)  

    © 2020 The aim of this study was to investigate the proliferation and apoptosis of male germ cells during the seasonal reproductive cycle of the large Japanese field mice (Apodemus speciosus). Male mice residing in their natural habitat were captured in Niigata, Japan. Testis sections were stained with haematoxylin and eosin, and mitotic male germ cells were identified using immunofluorescence staining for proliferating cell nuclear antigen (PCNA). Apoptosis was analysed using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay. The phases of spermatogenesis during the seasonal reproductive cycle were classified as active, transitional, and inactive based on the diameter of the seminiferous tubules. The number of PCNA-positive germ cells was less during the inactive than other phases. The percentage of TUNEL-positive germ cells per seminiferous tubule was greater during the inactive than active and transitional phases. Spermatogenesis during the seasonal reproductive cycle is controlled by proliferation and apoptosis in male germ cells. This species of undomesticated mice could be used as an animal model to study spermatogenesis as a valuable indicator of the effects of ecological and anthropogenic factors on animal reproduction.

    DOI: 10.1016/j.anireprosci.2020.106288

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  • Analysis of Radioactive Elements in Testes of Large Japanese Field Mice Using an Electron Probe Micro-Analyser after the Fukushima Accident 査読

    Takuya Ohdaira, Kanna Meguro, Kazuki Komatsu, Rina Syoji, Yohei Fujishima, Valerie Swee Ting Goh, Kosuke Kasai, Kentaro Ariyoshi, Akifumi Nakata, Yusuke Urushihara, Kazuma Koarai, Yasushi Kino, Tsutomu Sekine, Masatoshi Suzuki, Atsushi Takahasi, Yoshinaka Shimizu, Hisashi Shinoda, Mitsuaki A. Yoshida, Manabu Fukumoto, Hideaki Yamashiro, Tomisato Miura

    Ionizing and Non-ionizing Radiation   2020年1月

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    掲載種別:論文集(書籍)内論文   出版者・発行元:IntechOpen  

    DOI: 10.5772/intechopen.84634

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  • Effects of rice feeding and carnitine addition on growth performance and mRNA expression of protein metabolism-related genes in broiler grower chicks 査読

    Hiroya Fujimoto, Keisuke Matsumoto, Minori Koseki, Hideaki Yamashiro, Takahisa Yamada, Ryozo Takada

    Animal Science Journal91 ( 1 ) e13390   2020年1月

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    掲載種別:研究論文(学術雑誌)  

    © 2020 Japanese Society of Animal Science. This study was carried out to evaluate the nutritional effects of rice feeding and carnitine addition to a diet for broiler chicks. Thirty-six male 10-day-old broiler chicks were assigned to one of the following four treatment groups: corn-based diet (corn group), rice-based diet (rice group), and each diet with added carnitine (100 ppm). The experimental period was 2 weeks. Rice feeding resulted in significantly higher growth performance (body weight gain and feed efficiency) compared to corn feeding. Carnitine addition also resulted in higher growth performance. Breast muscle and thigh muscle weight (g) were significantly higher in broiler chicks fed rice and those fed diets with added carnitine. Liver mRNA expression of IGF-I was significantly higher in broiler chicks fed rice compared to those fed corn. There was no significant difference in mRNA expression of muscle atrogin-1 or liver CPT-I between broiler chicks fed rice and those fed corn, not between broilers chicks fed diets containing carnitine or not. Overall, these results show that rice feeding and carnitine addition improve the growth performance of broiler chicks by increasing mRNA expression of liver IGF-I. In addition, carnitine action is not affected by different cereals (corn and rice).

    DOI: 10.1111/asj.13390

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  • Induction of superovulation using inhibin antiserum and competence of embryo development in wild large Japanese field mice (Apodemus speciosus) 査読

    Kanna Meguro, Kazuki Komatsu, Takuya Ohdaira, Naomi Nakagata, Akifumi Nakata, Manabu Fukumoto, Tomisato Miura, Hideaki Yamashiro

    Reproduction in Domestic Animals54 ( 12 ) 1637 - 1642   2019年12月

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    掲載種別:研究論文(学術雑誌)  

    © 2019 Blackwell Verlag GmbH Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild-derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non-reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non-reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2- and/or 4-cell stage using both fresh and frozen-thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non-reproductive seasons. This study is the first to develop IVF–IVC wild large Japanese field mice beyond the 2- and/or 4-cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos.

    DOI: 10.1111/rda.13573

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  • Intestinal bacteria as powerful trapping lifeforms for the elimination of radioactive cesium 査読

    Kazuki Saito, Kengo Kuroda, Rie Suzuki, Yasushi Kino, Tsutomu Sekine, Hisashi Shinoda, Hideaki Yamashiro, Tomokazu Fukuda, Jin Kobayashi, Yasuyuki Abe, Junko Nishimura, Yusuke Urushihara, Hiroshi Yoneyama, Manabu Fukumoto, Emiko Isogai

    Frontiers in Veterinary Science6   70   2019年3月

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    掲載種別:研究論文(学術雑誌)  

    © 2019 Saito, Kuroda, Suzuki, Kino, Sekine, Shinoda, Yamashiro, Fukuda, Kobayashi, Abe, Nishimura, Urushihara, Yoneyama, Fukumoto and Isogai. In March 2011, an accident at the Fukushima Daiichi Nuclear Power Plant led to major problems, including the release of radionuclides such as Cesium (Cs)-137 into the environment. Ever since this accident, Cs-137 in foods has become a serious problem. In this study, we determined the concentration of Cs-137 in the feces, urine, and ruminal contents of cattle and demonstrated the possibility of its elimination from the body by intestinal bacteria. The results revealed a high Cs-137 concentration in the feces; in fact, this concentration was higher than that in skeletal muscles and other samples from several animals. Furthermore, intestinal bacteria were able to trap Cs-137, showing an uptake ratio within the range of 38-81% in vitro. This uptake appeared to be mediated through the sodium-potassium (Na+-K+) ion pump in the bacterial cell membrane. This inference was drawn based on the fact that the uptake ratio of Cs-137 was decreased in media with high potassium concentration. In addition, it was demonstrated that intestinal bacteria hindered the trapping of Cs-137 by the animal. Cattle feces showed high concentration of Cs-137 and intestinal bacteria trapped Cs-137. This study is the first report showing that intestinal bacteria contribute to the elimination of Cs-137 from the body.

    DOI: 10.3389/fvets.2019.00070

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  • Haematological analysis of Japanese macaques (Macaca fuscata) in the area affected by the Fukushima Daiichi Nuclear Power Plant accident 査読

    Yusuke Urushihara, Toshihiko Suzuki, Yoshinaka Shimizu, Megu Ohtaki, Yoshikazu Kuwahara, Masatoshi Suzuki, Takeharu Uno, Shiori Fujita, Akira Saito, Hideaki Yamashiro, Yasushi Kino, Tsutomu Sekine, Hisashi Shinoda, Manabu Fukumoto

    Scientific Reports8 ( 1 ) 16748   2018年12月

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    掲載種別:研究論文(学術雑誌)  

    © 2018, The Author(s). Several populations of wild Japanese macaques (Macaca fuscata) inhabit the area around Fukushima Daiichi Nuclear Power Plant (FNPP). To measure and control the size of these populations, macaques are captured annually. Between May 2013 and December 2014, we performed a haematological analysis of Japanese macaques captured within a 40-km radius of FNPP, the location of a nuclear disaster two years post-accident. The dose-rate of radiocaesium was estimated using the ERICA Tool. The median internal dose-rate was 7.6 μGy/day (ranging from 1.8 to 219 μGy/day) and the external dose-rate was 13.9 μGy/day (ranging from 6.7 to 35.1 μGy/day). We performed multiple regression analyses to estimate the dose-rate effects on haematological values in peripheral blood and bone marrow. The white blood cell and platelet counts showed an inverse correlation with the internal dose-rate in mature macaques. Furthermore, the myeloid cell, megakaryocyte, and haematopoietic cell counts were inversely correlated and the occupancy of adipose tissue was positively correlated with internal dose-rate in femoral bone marrow of mature macaques. These relationships suggest that persistent whole body exposure to low-dose-rate radiation affects haematopoiesis in Japanese macaques.

    DOI: 10.1038/s41598-018-35104-0

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  • <sup>90</sup>Sr specific activity of teeth of abandoned cattle after the Fukushima accident – teeth as an indicator of environmental pollution 査読

    Kazuma Koarai, Yasushi Kino, Atsushi Takahashi, Toshihiko Suzuki, Yoshinaka Shimizu, Mirei Chiba, Ken Osaka, Keiichi Sasaki, Yusuke Urushihara, Tomokazu Fukuda, Emiko Isogai, Hideaki Yamashiro, Toshitaka Oka, Tsutomu Sekine, Manabu Fukumoto, Hisashi Shinoda

    Journal of Environmental Radioactivity183   1 - 6   2018年3月

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    掲載種別:研究論文(学術雑誌)  

    © 2017 Elsevier Ltd 90Sr specific activity in the teeth of young cattle that were abandoned in Kawauchi village and Okuma town located in the former evacuation areas of the Fukushima-Daiichi Nuclear Power Plant (FNPP) accident were measured. Additionally, specific activity in contaminated surface soils sampled from the same area was measured. (1) All cattle teeth examined were contaminated with 90Sr. The specific activity, however, varied depending on the developmental stage of the teeth during the FNPP accident; teeth that had started development before the accident exhibited comparatively lower values, while teeth developed mainly after the accident showed higher values. (2) Values of 90Sr-specific activity in teeth formed after the FNPP accident were higher than those of the bulk soil but similar to those in the exchangeable fraction (water and CH3COONH4 soluble fractions) of the soil. The findings suggest that 90Sr was incorporated into the teeth during the process of development, and that 90Sr in the soluble and/or leachable fractions of the soil might migrate into teeth and contribute to the amount of 90Sr in the teeth. Thus, the concentration of 90Sr in teeth formed after the FNPP accident might reflect the extent of 90Sr pollution in the environment.

    DOI: 10.1016/j.jenvrad.2017.12.005

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  • Effects on Glycemic Control in Impaired Wound Healing in Spontaneously Diabetic Torii (SDT) Fatty Rats 査読

    Miyajima Katsuhiro, Soon Hui Teoh, Hideaki Yamashiro, Masami Shinohara, Fatchiyah Fatchiyah, Takeshi Ohta, Takahisa Yamada

    Medical Archives72 ( 1 ) 4 - 8   2018年2月

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    掲載種別:研究論文(学術雑誌)  

    Introduction: Impaired diabetic wound healing is an important issue in diabetic complications. The present study aims to evaluate the protective effect on glycemic control against impaired diabetic wound healing using a diabetic rat model. We investigated the wound healing process and effect on the impaired wound repair by glycemic control in the Spontaneously Diabetic Torii (SDT) fatty rat, which is a new animal model of obese type 2 diabetes and may be a good model for study impaired wound healing.Material and methods: Male SDT fatty rats at 15 weeks of age were administered orally with sodium glucose co-transporter (SGLT) 2 inhibitor for 3 weeks. Wounds were induced at 2 weeks after SGLT 2 inhibitor treatment, and the wound areas were periodically examined in morphological and histological analyses.Results: The SDT fatty rats showed a delayed wound healing as compared with the normal rats, but a glycemic control improved the impaired wound healing. In histological analysis in the skin of SDT fatty rats showed severe infiltration of inflammatory cell, hemorrhage and many bacterial masses in the remaining and slight fibrosis of crust on skin tissue. Thought that this results skin performance to be a delay of crust formation and regeneration of epithelium; however, these findings were ameliorated in the SGLT 2 inhibitor treated group.Conclusion: Glycemic control is effective for treatment in diabetic wounds and the SDT fatty rat may be useful to investigate pathophysiological changes in impaired diabetic wound healing.

    DOI: 10.5455/medarh.2018.72.4-8

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  • Location and expression of Juno in mice oocytes during maturation 査読

    Banri Suzuki, Yukou Sugano, Jun Ito, Haruka Saito, Sueo Niimura, Hideaki Yamashiro

    JBRA Assisted Reproduction21 ( 4 ) 321 - 326   2017年12月

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    掲載種別:研究論文(学術雑誌)  

    © 2017, Sociedade Brasileira de Reproducao Assistida. All rights reserved. Objective: Oocyte-sperm interaction is the essential step in fertilization. Juno, which has been known as Folate receptor 4, is the Izumo1 receptor expressed on the oocyte membrane. This study aims to investigate the location and expression of Juno in mice oocytes during maturation. Methods: To confirm the stage at which Juno expression begins in the mice oocytes and its location pattern, we performed immunostaining methods. Next, we evaluated Juno mRNA expression by a half quantitative RT-PCR. Juno knockdown oocytes were generated by microinjecting siRNA into the germinal vesicle (GV) stage oocytes, and analyzed the maturation rate. Results: Our results showed that Juno was expressed on the surface of the oocyte cytoplasmic membrane at the GV stage and it continues to be expressed at similar levels in the metaphase II (MII) stages of oocytes maturation. Interestingly, Juno is also expressed on the first polar body membrane at the MII stage. Fluorescence showing Juno expression was decreased in the oolemma of siRNA injected oocytes, but it was not completely disappearing in knock down oocytes. MII stage-rates of siRNA injected oocytes were not significantly different from sham controls. Conclusion: Juno was expressed in oocytes at the GV stage and it continues to be expressed at similar levels in later stages of oocytes maturation. Juno accumulation in oolemma during oocyte maturation is essential for fertilization, such as membrane recognition of both gametes.

    DOI: 10.5935/1518-0557.20170065

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  • The Causal Relationship between DNA Damage Induction in Bovine Lymphocytes and the Fukushima Nuclear Power Plant Accident 査読

    Asako J. Nakamura, Masatoshi Suzuki, Christophe E. Redon, Yoshikazu Kuwahara, Hideaki Yamashiro, Yasuyuki Abe, Shintaro Takahashi, Tomokazu Fukuda, Emiko Isogai, William M. Bonner, Manabu Fukumoto

    Radiation Research187 ( 5 ) 630 - 636   2017年5月

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    掲載種別:研究論文(学術雑誌)  

    © 2017 by Radiation Research Society. The Fukushima Daiichi Nuclear Power Plant (FNPP) accident, the largest nuclear incident since the 1986 Chernobyl disaster, occurred when the plant was hit by a tsunami triggered by the Great East Japan Earthquake on March 11, 2011. The subsequent uncontrolled release of radioactive substances resulted in massive evacuations in a 20-km zone. To better understand the biological consequences of the FNPP accident, we have been measuring DNA damage levels in cattle in the evacuation zone. DNA damage was evaluated by assessing the levels of DNA double-strand breaks in peripheral blood lymphocytes by immunocytofluorescence-based quantification of γ-H2AX foci. A greater than two-fold increase in the fraction of damaged lymphocytes was observed in all animal cohorts within the evacuation zone, and the levels of DNA damage decreased slightly over the 700-day sample collection period. While the extent of damage appeared to be independent of the distance from the accident site and the estimated radiation dose from radiocesium, we observed age-dependent accumulation of DNA damage. Thus, this study, which was the first to evaluate the biological impact of the FNPP accident utilizing the γ-H2AX assays, indicated the causal relation between high levels of DNA damage in animals living in the evacuation zone and the FNPP accident.

    DOI: 10.1667/RR14630.1

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  • Analysis of the Effect of Chronic and Low-Dose Radiation Exposure on Spermatogenic Cells of Male Large Japanese Field Mice (Apodemus speciosus) after the Fukushima Daiichi Nuclear Power Plant Accident 査読

    Sachio Takino, Hideaki Yamashiro, Yukou Sugano, Yohei Fujishima, Akifumi Nakata, Kosuke Kasai, Gohei Hayashi, Yusuke Urushihara, Masatoshi Suzuki, Hisashi Shinoda, Tomisato Miura, Manabu Fukumoto

    Radiation Research187 ( 2 ) 161 - 168   2017年2月

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    掲載種別:研究論文(学術雑誌)  

    © 2017 by Radiation Research Society. In this study we analyzed the effect of chronic and low-dose-rate (LDR) radiation on spermatogenic cells of large Japanese field mice (Apodemus speciosus) after the Fukushima Daiichi Nuclear Power Plant (FNPP) accident. In March 2014, large Japanese field mice were collected from two sites located in, and one site adjacent to, the FNPP ex-evacuation zone: Tanashio, Murohara and Akogi, respectively. Testes from these animals were analyzed histologically. External dose rate from radiocesium (combined 134Cs and 137Cs) in these animals at the sampling sites exhibited 21 μGy/day in Tanashio, 304-365 μGy/day in Murohara and 407-447 μGy/day in Akogi. In the Akogi group, the numbers of spermatogenic cells and proliferating cell nuclear antigen (PCNA)-positive cells per seminiferous tubule were significantly higher compared to the Tanashio and Murohara groups, respectively. TUNEL-positive apoptotic cells tended to be detected at a lower level in the Murohara and Akogi groups compared to the Tanashio group. These results suggest that enhanced spermatogenesis occurred in large Japanese field mice living in and around the FNPP ex-evacuation zone. It remains to be elucidated whether this phenomenon, attributed to chronic exposure to LDR radiation, will benefit or adversely affect large Japanese field mice.

    DOI: 10.1667/RR14234.1

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  • Combination of spindle and first polar body chromosome images for the enhanced prediction of developmental potency of mouse metaphase II oocytes 査読

    Yukou Sugano, Manami Yazawa, Sachio Takino, Sueo Niimura, Hideaki Yamashiro

    Zygote24 ( 6 ) 900 - 908   2016年12月

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    掲載種別:研究論文(学術雑誌)  

    © Cambridge University Press 2016. The objective of this study was to classify spindle and first polar body (PB1) chromosome images in ovulated mouse oocytes over time to predict the developmental competence of metaphase II (MII) oocytes. Oocytes were collected at 12, 15, 20, and 25 h after human chorionic gonadotropin (hCG) injection, and stained for spindle tubulin, chromosomes, and PB1 chromosomes. MII spindle morphology was classified as tapered type or barrel type and PB1 chromosomes were categorized as aggregated, separated, dot, or collapsed. To determine whether differences in spindle and PB1 images in MII oocytes are associated with fertilization success, we performed in vitro fertilization (IVF) at various times after hCG injection. Barrel-type spindles and aggregate-type PB1 were dominant at 12 h after hCG injection. Oocyte spindles collected 1 h after injection were tapered, and PB1 chromosomes were separated. At 20 and 25 h after treatment, spindle and PB1 images were classified as collapsed. The rate of development to 2-cell embryos after IVF did not differ between the 12 h and 15 h treatments; however, it was significantly lower for the 25 h treatment than for other treatments. The rates of development to blastocysts at 12, 15, 20, and 25 h after hCG injection were 61, 46, 42, and 9%, respectively. MII oocytes with barrel-type spindles and aggregate-type PB1 had high rates of fertilization and blastocyst development, and spindle and PB1 characteristics were correlated with the outcomes of IVF and embryo culture. These results suggested that images of spindles combined with those of PB1 chromosomes enable the prediction of oocytic and/or embryonic quality.

    DOI: 10.1017/S096719941600023X

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  • Software development for estimating the concentration of radioactive cesium in the skeletal muscles of cattle from blood samples 査読

    Tomokazu Fukuda, Masahiro Hiji, Yasushi Kino, Yasuyuki Abe, Hideaki Yamashiro, Jin Kobayashi, Yoshinaka Shimizu, Atsushi Takahashi, Toshihiko Suzuki, Mirei Chiba, Kazuya Inoue, Yoshikazu Kuwahara, Motoko Morimoto, Masafumi Katayama, Kenichiro Donai, Hisashi Shinoda, Tsutomu Sekine, Manabu Fukumoto, Emiko Isogai

    Animal Science Journal87 ( 6 ) 842 - 847   2016年6月

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    掲載種別:研究論文(学術雑誌)  

    © 2015 Japanese Society of Animal Science The 2011 earthquake severely damaged the Fukushima Daiichi Nuclear Power Plant (FNPP), resulting in the release of large quantities of radioactive material into the environment. The deposition of these radionuclides in rice straw as livestock feed led to the circulation of contaminated beef in the market. Based on the safety concern of the consumers, a reliable method for estimating concentrations of radioactive cesium in muscle tissue is needed. In this study, we analyzed the concentrations of radioactive cesium in the blood and skeletal muscle of 88 cattle, and detected a linear correlation between them. We then developed software that can be used to estimate radioactive cesium concentrations in muscle tissue from blood samples. Distribution of this software to the livestock production field would allow us to easily identify high-risk cattle, which would be beyond the safety regulation, before shipping out to the market. This software is planned to be released as freeware. This software would contribute to food safety, and aid the recovery of the livestock industry from the damage creacted by the 2011 Tohoku earthquake and tsunami.

    DOI: 10.1111/asj.12490

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  • Analysis of plasma protein concentrations and enzyme activities in cattle within the ex-evacuation zone of the Fukushima Daiichi Nuclear Plant Accident 査読

    Yusuke Urushihara, Koh Kawasumi, Satoru Endo, Kenichi Tanaka, Yasuko Hirakawa, Gohei Hayashi, Tsutomu Sekine, Yasushi Kino, Yoshikazu Kuwahara, Masatoshi Suzuki, Motoi Fukumoto, Hideaki Yamashiro, Yasuyuki Abe, Tomokazu Fukuda, Hisashi Shinoda, Emiko Isogai, Toshiro Arai, Manabu Fukumoto

    PLoS ONE11 ( 5 ) 1 - 13   2016年5月

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    掲載種別:研究論文(学術雑誌)  

    © 2016 Urushihara et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The effect of the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on humans and the environment is a global concern. We performed biochemical analyses of plasma from 49 Japanese Black cattle that were euthanized in the ex-evacuation zone set within a 20-km radius of FNPP. Among radionuclides attributable to the FNPP accident, germanium gamma-ray spectrometry detected photopeaks only from 134Cs and 137Cs (radiocesium) commonly in the organs and in soil examined. Radioactivity concentration of radiocesium was the highest in skeletal muscles. Assuming that the animal body was composed of only skeletal muscles, the median of internal dose rate from radiocesium was 12.5 μGy/day (ranging from 1.6 to 33.9 μGy/day). The median of external dose rate calculating from the place the cattle were caught was 18.8 μGy/day (6.0-133.4 μGy/day). The median of internal and external (total) dose rate of the individual cattle was 26.9 μGy/day (9.1-155.1 μGy/day). Plasma levels of malondialdehyde and superoxide dismutase activity were positively and glutathione peroxidase activity was negatively correlated with internal dose rate. Plasma alanine transaminase activity and percent activity of lactate dehydrogenase (LDH)-2, LDH-3 and LDH-4 were positively and LDH-1 was negatively correlated with both internal and total dose rate. These suggest that chronic exposure to low-dose rate of ionizing radiation induces slight stress resulting in modified plasma protein and enzyme levels.

    DOI: 10.1371/journal.pone.0155069

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  • <sup>90</sup>Sr in teeth of cattle abandoned in evacuation zone: Record of pollution from the Fukushima-Daiichi Nuclear Power Plant accident 査読

    Kazuma Koarai, Yasushi Kino, Atsushi Takahashi, Toshihiko Suzuki, Yoshinaka Shimizu, Mirei Chiba, Ken Osaka, Keiichi Sasaki, Tomokazu Fukuda, Emiko Isogai, Hideaki Yamashiro, Toshitaka Oka, Tsutomu Sekine, Manabu Fukumoto, Hisashi Shinoda

    Scientific Reports6   24077   2016年4月

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    掲載種別:研究論文(学術雑誌)  

    Here we determined the 90Sr concentrations in the teeth of cattle abandoned in the evacuation area of the Fukushima-Daiichi Nuclear Power Plant (FNPP) accident. 90Sr activity concentrations in the teeth varied from 6-831 mBq (g Ca)-1 and exhibited a positive relationship with the degree of radioactive contamination that the cattle experienced. Even within an individual animal, the specific activity of 90Sr (Bq (g Sr)-1) varied depending on the development stage of the teeth during the FNPP accident: teeth that were early in development exhibited high 90Sr specific activities, while teeth that were late in development exhibited low specific activities. These findings demonstrate that 90Sr is incorporated into the teeth during tooth development; thus, tooth 90Sr activity concentrations reflect environmental 90Sr levels during tooth formation. Assessment of 90Sr in teeth could provide useful information about internal exposure to 90Sr radiation and allow for the measurement of time-course changes in the degree of environmental 90Sr pollution.

    DOI: 10.1038/srep24077

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  • A comprehensive dose evaluation project concerning animals affected by the Fukushima Daiichi Nuclear Power Plant accident: Its set-up and progress 査読

    Shintaro Takahashi, Kazuya Inoue, Masatoshi Suzuki, Yusuke Urushihara, Yoshikazu Kuwahara, Gohei Hayashi, Soichiro Shiga, Motoi Fukumoto, Yasushi Kino, Tsutomu Sekine, Yasuyuki Abe, Tomokazu Fukuda, Emiko Isogai, Hideaki Yamashiro, Manabu Fukumoto

    Journal of Radiation Research56   36 - 41   2015年12月

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    掲載種別:研究論文(学術雑誌)  

    © 2015 The Author. It is not an exaggeration to say that, without nuclear accidents or the analysis of radiation therapy, there is no way in which we are able to quantify radiation effects on humans. Therefore, the livestock abandoned in the ex-evacuation zone and euthanized due to the Fukushima Daiichi Nuclear Power Plant (FNPP) accident are extremely valuable for analyzing the environmental pollution, its biodistribution, the metabolism of radionuclides, dose evaluation and the influence of internal exposure. We, therefore, sought to establish an archive system and to open it to researchers for increasing our understanding of radiation biology and improving protection against radiation. The sample bank of animals affected by the FNPP accident consists of frozen tissue samples, formalin-fixed paraffin-embedded specimens, dose of radionuclides deposited, etc., with individual sampling data.

    DOI: 10.1093/jrr/rrv069

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  • Electron probe X-ray microanalysis of boar and inobuta testes after the Fukushima accident 査読

    Hideaki Yamashiro, Yasuyuki Abe, Gohei Hayashi, Yusuke Urushihara, Yoshikazu Kuwahara, Masatoshi Suzuki, Jin Kobayashi, Yasuyuki Kino, Tomokazu Fukuda, Bin Tong, Sachio Takino, Yukou Sugano, Satoshi Sugimura, Takahisa Yamada, Emiko Isogai, Manabu Fukumoto

    Journal of Radiation Research56   42 - 47   2015年12月

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    掲載種別:研究論文(学術雑誌)  

    © 2016 The Author. We aimed to investigate the effect of chronic radiation exposure associated with the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on the testes of boar and inobuta (a hybrid of Sus scrofa and Sus scrofa domestica). This study examined the contamination levels of radioactive caesium (Cs), especially 134Cs and 137Cs, in the testis of both boar and inobuta during 2012, after the Fukushima accident. Morphological analysis and electron-probe X-ray microanalysis (EPMA) were also undertaken on the testes. The 134Cs and 137Cs levels were 6430 ± 23 and 6820 ± 32 Bq/kg in the boar testes, and 755 ± 13 and 747 ± 17 Bq/kg in the inobuta testes, respectively. The internal and external exposure of total 134Cs and 137Cs in the boar testes were 47.1 mGy and 176.2 mGy, respectively, whereas in the inobuta testes, these levels were 6.09 mGy and 59.8 mGy, respectively. Defective spermatogenesis was not detected by the histochemical analysis of radiation-exposed testes for either animal. In neither animal were Cs molecules detected, using EPMA. In conclusion, we showed that adverse radiation-induced effects were not detected in the examined boar and inobuta testes following the chronic radiation exposure associated with the FNPP accident.

    DOI: 10.1093/jrr/rrv070

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  • Effect of seasonal changes on testicular morphology and the expression of circadian clock genes in Japanese wood mice (Apodemus speciosus) 査読

    Akiyama M, Takino S, Sugano Y, Yamada T, Nakata A, Miura T, Fukumoto M, Yamashiro H

    Journal of Biological Regulators and Homeostatic Agents29 ( 3 ) 589 - 600   2015年7月

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    掲載種別:研究論文(学術雑誌)  

    This study aimed to determine the seasonality of reproduction throughout the year in Japanese wood mice (Apodemus speciosus). The effect of seasonal changes on testicular morphology and the periodic expression of circadian clock genes in the hypothalamus and testes of male individuals was evaluated. We also examined the morphology of the testes and caudae epididymides of male mice. In addition, RT-PCR analysis was carried out with mRNA extracted from the hypothalamus and testes to evaluate the expression of the circadian clock genes Clock, Bmal1, Per1, and Cry1. The complete induction of testicular activity was detected from February to April and from August to October, with testes weight increasing with the completion of spermatogenesis (reproductive season). From May to early June and from November to early January, testicular weight declined, the seminiferous tubules reduced in size, spermatogenesis was arrested, and sperm were not produced (non-reproductive season). From mid- June to July and mid-January, the re-induction of testicular activity for spermatogenesis was observed in the seminiferous tubules (transitional season). Out of the four examined genes, Cry1 had the highest expression level in both the hypothalamus and testes throughout the year, followed by Bmal1, Per1, and Clock. The expression of Bmal1 was significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive and non-reproductive seasons. Cry1 transcript levels were also significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive season. In conclusion, the results indicating changes in testicular morphology revealed annual reproductive, non-reproductive, and transmission periods in Japanese wood mice. When an increase in testicular activity was observed indicating the onset of the reproductive season, the mean day length was approximately 11–13 h. The expression of the circadian clock genes Bmal1 and Cry1 in the hypothalamus and testes during the reproductive season was significantly higher than that of the same genes during the transitional season. Consequently, completion of spermatogenesis occurred in the seminiferous tubules of Japanese wood mice testes during the reproductive period.

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  • Pathophysiological changes by short-term food restriction in obese diabetic rats 査読

    Miyajima K, Toriniwa Y, Motohashi Y, Otohashi Y, Ishii M, Shinohara M, Yamashiro H, Yamada T, Ohta T

    British Journal of Medicine and Medical Research9 ( 6 ) 1 - 7   2015年6月

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  • Association of the expression level of the MYBPC1 gene in skeletal muscle with marbling trait in Japanese Black beef cattle 査読

    Bin Tong, Youji Muramatsu, Takeshi Ohta, Hiroyuki Kose, Hideaki Yamashiro, Toshie Sugiyama, Takashisa Yamada

    Annals of Animal Science15 ( 2 ) 349 - 358   2015年4月

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    掲載種別:研究論文(学術雑誌)  

    © 2015 by Bin Tong. Marbling characterized by the amount and distribution of intramuscular fat (IMF) in longissimus muscle (LM) and measured as beef marbling score (BMS), is an economically important trait of beef cattle in Japan. The myosin binding protein C, slow type (MYBPC1) gene, involved in efficient energy metabolism and homeostasis during muscle contraction in slow skeletal muscle, has been previously shown to be expressed at different levels in the LM between high-marbled and lowmarbled steer groups using differential-display PCR (ddPCR). In this study, we found that IMF area (%) in the sacrococcygeus muscle (SM) was positively correlated with BMS in the LM in Japanese Black steers (n=22, r=0.941, P<0.0001). This suggested that the IMF area (%) in the SM tends to equate marbling level in the LM. Furthermore, we showed that the MYBPC1 expression level in SM was significantly higher in the Japanese Black steers (n=5) with high BMS than in the Japanese Black steers (n=5) with low BMS (P<0.001). Moreover, correlation analyses showed that the expression level of the MYBPC1 gene was positively correlated with IMF area (%) (n=22, r=0.858, P<0.0001) and BMS (n=22, r=0.769, P<0.0001), indicating the association of MYBPC1 expression level with marbling trait. These results, together with the previous ddPCR result, suggested that high level of MYBPC1 expression may be associated with the development of marbling in Japanese Black beef cattle.

    DOI: 10.1515/aoas-2015-0014

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  • Effects on fat distribution by food restriction in zucker diabetic fatty rats 査読

    Ohta T, Ishii Y, Shinohara M, Yamashiro H, Yamada T

    British Journal of Medicine and Medical Research7   699 - 704   2015年3月

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  • Effect of Isolation Stress on Glucose/Lipid Metabolism in Spontaneously Diabetic Torii (SDT) Fatty Rats 査読

    K. Miyajima, Y. Toriniwa, Y. Motohashi, Y. Ishii, M. Shinohara, H. Yamashiro, T. Yamada, T. Ohta

    British Journal of Medicine and Medical Research8 ( 7 ) 588 - 594   2015年1月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Sciencedomain International  

    DOI: 10.9734/bjmmr/2015/17988

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  • Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: Analyses of cattle within the evacuation zone of the Fukushima Daiichi Nuclear Power Plant 査読

    Tomokazu Fukuda, Yasushi Kino, Yasuyuki Abe, Hideaki Yamashiro, Jin Kobayashi, Yoshinaka Shimizu, Atsushi Takahashi, Toshihiko Suzuki, Mirei Chiba, Shintaro Takahashi, Kazuya Inoue, Yoshikazu Kuwahara, Motoko Morimoto, Hisashi Shinoda, Masahiro Hiji, Tsutomu Sekine, Manabu Fukumoto, Emiko Isogai

    Animal Science Journal86 ( 1 ) 120 - 124   2015年1月

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    掲載種別:研究論文(学術雑誌)  

    © 2014 Japanese Society of Animal Science. The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium (137Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a 137Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood 137Cs and muscle 137Cs (Y=28.0X, R2=0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high-risk cattle before they enter the market, and will contribute to food safety.

    DOI: 10.1111/asj.12301

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  • Use of in ovo chorioallantoic membrane engraftment to culture testes from neonatal mice 査読

    Emi Uematsu, Sachio Takino, Hidemi Okajima, Bin Tong, Toshie Sugiyama, Takahisa Yamada, Sueo Niimura, Hideaki Yamashiro

    Comparative Medicine64 ( 4 ) 264 - 269   2014年8月

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    掲載種別:研究論文(学術雑誌)  

    Copyright © 2014 by the American Association for Laboratory Animal Science. Many attempts have been made to culture germ cells in vitro by mimicking their development in vivo. The objective of this study was to establish an alternative method of xenotransplantation by developing a new approach for the rapid induction of spermatogenesis by using the chorioallantoic membrane of developing chicken embryos. Fertilized chicken eggs were incubated for 7 d, after which a small window was cut into the shell of the egg. We then transplanted testes from 7- to 8-d-old B6D2F1 mice onto the vessels of the chorioallantoic membrane and incubated them at 35.0 °C for 14 d or 37.5 °C for 12 d. After this in ovo CAM (iCAM) culture, the survival rates of the eggs and testes were assessed histologically and immunohistologically. The transplanted testes in the chicken embryos that survived were supported by the CAM, with an associated chronic vascularization response. The testes cultured at 35.0 °C had lower rates of generation and higher rates of death than did those cultured at 37.5 °C. Histologic examination of the testes cultured at 37.5 °C revealed the presence of spermatogonia and primary spermatocyte-like germ cells in the seminiferous tubules. The number of cells positive for synaptonemal complex protein 3 in the seminiferous tubules was significantly higher than that in the noniCAM-cultured testes from control mice. These results suggest that iCAM culturing of neonatal donor testis induces androcyte development. This method could be the foundation for a method that would enable in vitro spermatogenesis.

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  • Association of a single-nucleotide polymorphism in myosin-binding protein C, slow-type (MYBPC1) gene with marbling in Japanese Black beef cattle

    Bin Tong, Seiki Sasaki, Youji Muramatsu, Takeshi Ohta, Hiroyuki Kose, Hideaki Yamashiro, Tatsuo Fujita, Takahisa Yamada

    Animal Genetics45 ( 4 ) 611 - 612   2014年8月

  • The nucleotide sequences for the dopamine receptor D4 gene in the Japanese crested ibis 査読

    Yuuta KOMATSU, Mai YAMASHITA, Takahisa YAMADA, Bin TONG, Takeshi OHTA, Toshie SUGIYAMA, Hideaki YAMASHIRO, Yoshinori KANEKO, Hisashi NAGATA, Hiroaki IWAISAKI

      42 ( 1 ) 3 - 9   2014年

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    掲載種別:研究論文(学術雑誌)  

    DOI: 10.5924/abgri.42.3

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  • Semen collection and polymerase chain reaction-based sex determination of black-headed and straw-necked ibis 査読

    K. Kaneko, E. Uematsu, Y. Takahashi, B. Tong, S. Takino, Y. Wajiki, T. Kimura, H. Yamashiro, Y. Kaneko, H. Iwaisaki, T. Sugiyama, T. Yamada, S. Yamagishi

    Reproduction in Domestic Animals48 ( 6 ) 1001 - 1005   2013年12月

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    掲載種別:研究論文(学術雑誌)  

    Contents: This study aimed to develop a polymerase chain reaction (PCR)-based sexing and effective semen collection methods for black-headed and straw-necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black-headed and 4 straw-necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W-chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro-ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw-necked ibis using the massage method. Limited motility, viability and concentration of straw-necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR-based sexing proved to be an accurate molecular sexing method for black-headed and straw-necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw-necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required. © 2013 Blackwell Verlag GmbH.

    DOI: 10.1111/rda.12200

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  • Effects of radioactive caesium on bull testes after the Fukushima nuclear plant accident 査読

    Hideaki Yamashiro, Yasuyuki Abe, Tomokazu Fukuda, Yasushi Kino, Isao Kawaguchi, Yoshikazu Kuwahara, Motoi Fukumoto, Shintaro Takahashi, Masatoshi Suzuki, Jin Kobayashi, Emi Uematsu, Bin Tong, Takahisa Yamada, Satoshi Yoshida, Eimei Sato, Hisashi Shinoda, Tsutomu Sekine, Emiko Isogai, Manabu Fukumoto

    Scientific Reports3   2850   2013年10月

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    掲載種別:研究論文(学術雑誌)  

    We aimed to investigate the effect of chronic radiation exposure associated with the Fukushima Daiichi Nuclear Plant accident on the testis from 2 bulls. Estimated dose of internal exposure in one bull was 0.7-1.2â.mGy (134 Cs) and 0.4-0.6â.mGy (137 Cs) and external exposure was 2.0â.mGy (134 Cs) and 0.8â.mGy (137 Cs) (196 days). Internal dose in the other was 3.2-6.1â.mGy (134 Cs) and 1.8-3.4â.mGy (137 Cs) and external dose was 1.3â.mGy (134 Cs) and 0.6â.mGy (137 Cs) (315 days). Sperm morphology and spermatogenesis were within normal ranges. 134, 137 Cs radioactivity was detected but Cs was not detectable in the testis by electron probe microanalysis. Thus, adverse radiation-induced effects were not observed in bull testes following chronic exposure to the above levels of radiation for up to 10 months. Since we could analyse a limited number of testes, further investigation on the effects of ionizing radiation on spermatogenesis should be extended to more animals.

    DOI: 10.1038/srep02850

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  • Distribution of radioactive cesium in ostrich (struthio camelus) after the Fukushima Daiichi Nuclear Power Plant accident 査読

    Isogai E, Kino Y, Abe Y, Yamashiro H, Shinoda H, Fukuda T, Fukumoto M, Kuroda K, Yoneyama H, Isogai H, Sekine T

    Radiation Emergency Medicine2 ( 2 ) 68 - 71   2013年9月

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  • Distribution of Artificial Radionuclides in Abandoned Cattle in the Evacuation Zone of the Fukushima Daiichi Nuclear Power Plant 査読

    Tomokazu Fukuda, Yasushi Kino, Yasuyuki Abe, Hideaki Yamashiro, Yoshikazu Kuwahara, Hidekazu Nihei, Yosuke Sano, Ayumi Irisawa, Tsutomu Shimura, Motoi Fukumoto, Hisashi Shinoda, Yuichi Obata, Shin Saigusa, Tsutomu Sekine, Emiko Isogai, Manabu Fukumoto

    PLoS ONE8 ( 1 ) e54312   2013年1月

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    掲載種別:研究論文(学術雑誌)  

    The Fukushima Daiichi Nuclear Power Plant (FNPP) accident released large amounts of radioactive substances into the environment. In order to provide basic information for biokinetics of radionuclides and for dose assessment of internal exposure brought by the FNPP accident, we determined the activity concentration of radionuclides in the organs of 79 cattle within a 20-km radius around the FNPP. In all the specimens examined, deposition of Cesium-134 (134Cs, half-life: 2.065 y) and 137Cs (30.07 y) was observed. Furthermore, organ-specific deposition of radionuclides with relatively short half-lives was detected, such as silver-110m (110mAg, 249.8 d) in the liver and tellurium-129m (129mTe, 33.6 d) in the kidney. Regression analysis showed a linear correlation between the radiocesium activity concentration in whole peripheral blood (PB) and that in each organ. The resulting slopes were organ dependent with the maximum value of 21.3 being obtained for skeletal muscles (R2 = 0.83, standard error (SE) = 0.76). Thus, the activity concentration of 134 Cs and 137Cs in an organ can be estimated from that in PB. The level of radioactive cesium in the organs of fetus and infants were 1.19-fold (R2 = 0.62, SE = 0.12), and 1.51-fold (R2 = 0.70, SE = 0.09) higher than that of the corresponding maternal organ, respectively. Furthermore, radiocesium activity concentration in organs was found to be dependent on the feeding conditions and the geographic location of the cattle. This study is the first to reveal the detailed systemic distribution of radionuclides in cattle attributed to the FNPP accident. © 2013 Fukuda et al.

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  • Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis 査読

    Joel Marh, Zoia Stoytcheva, Johann Urschitz, Atsushi Sugawara, Hideaki Yamashiro, Jesse B. Owens, Ilko Stoytchev, Pawel Pelczar, Ryuzo Yanagimachi, Stefan Moisyadi

    Proceedings of the National Academy of Sciences of the United States of America109 ( 47 ) 19184 - 19189   2012年11月

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    掲載種別:研究論文(学術雑誌)  

    We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community.

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  • Useful g.70014208G&gt;A SNP marker in MYBPC1 gene showing marbling-associated expression changes 査読

    Bin Tong, Seiki Sasaki, Youji Muramatsu, Takeshi Ohta, Hiroyuki Kose, Hideaki Yamashiro, Takahisa Yamada

    Journal of Animal and Veterinary Advances11 ( 23 ) 4446 - 4450   2012年

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    掲載種別:研究論文(学術雑誌)  

    Marbling, defined by the am eract with muscle protein including myosin, actin and titin to regulate muscle contraction and to express in slow-twitch oxidative fiber that is observed in high-marbled muscle and involved in development and maintenance of the fiber, possesses expression differences in musculus longissimus muscle between low-marbled Holstein and high-marbled Japanese Black steer groups. In the present study, researchers found that a marker (DIK4787) close to the MYBPC1 was polymorphic between low-marbled Holstein and high-marbled Japanese Black steer groups and exhibited significantly different allelic distribution between Japanese Black sires with extremely high predicted breeding value for marbling and with extremely low one. Further, researchers detected 3 Single Nucleotide Polymorphisms (SNPs) in the promoter region of the MYBPC1 gene between low-marbled Holstein and high-marbled Japanese Black steer groups. A SNP in the upstream of the promoter of the MYBPC1, referred to as g.70014208G>A, exhibited significantly different allelic distribution between animals with extremely high predicted breeding value for marbling and with extremely low one (p = 0.046). The other 2 SNPs seemed not to segregate in Japanese Black beef cattle. The findings suggest that the g.70014208G>A SNP may be related to changes in MYBPC1 gene expression and/or marbling. The g.70014208G>A SNP marker may be useful for effective marker-assisted selection to increase the levels of marbling. © Medwell Journals, 2012.

    DOI: 10.3923/javaa.2012.4446.4450

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  • Preliminary association study of single nucleotide polymorphism in MYHl and TRDN genes for growth-related traits hi japanese black beef cattle 査読

    Kaifeng Wu, Bin Tong, Seiki Sasaki, Youji Muramatsu, Takeshi Ohta, Hiroyuki Kose, Hideaki Yamashiro, Takahisa Yamada, Yanru Zhang

    Journal of Animal and Veterinary Advances11 ( 24 ) 4540 - 4543   2012年

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    掲載種別:研究論文(学術雑誌)  

    Growth performance as well as marbling is the main breeding objectives in Japanese Black cattle, the major beef breed in Japan. Researchers have recently reported the existence of Single Nucleotide Polymorphisms (SNPs), g.29850738G>A and g.3834941C>T in promoter regions of the Myosin Heavy chain 1 (MYHl) and the Triadin (TRDN) genes, respectively, in Japanese Black beef cattle population. The MYHl encodes an isoform of myosin heavy chain in type I fiber of skeletal muscle and the TRDN is known to be involved in muscle contraction and the 2 genes have been earlier shown to be located within genomic regions of quantitative trait loci for growth-related carcass traits. Thus, the MYHl and TRDN genes were considered as positional functional candidates for the gene responsible for growth performance. In this study, we analyzed the possible effects of the 2 SNPs on the growth-related carcass traits: Carcass Weight (CWT), Rib Thickness (RT), Rib Eye Area (REA) and Subcutaneous Fat Thickness (SFT) in Japanese Black beef cattle. The g.29850738G>A SNP in the MYHl, exhibited significantly different allelic distribution between Japanese Black sires with extremely high predicted breeding value and the sires with extremely low one for CWT and RT but not for the others. The allelic distribution of the g.3834941C>T SNP in the TRDN was indistinguishable between the sires with extremely high one and with extremely low one for all the traits. These findings suggest possible effect of the g.29850738G>A SNP on the growth-related trait in Japanese Black beef cattle. The MYHl SNP may be useful for effective marker-assisted selection to increase the beef productivity in Japanese Black beef cattle. © Medwell Journals, 2012.

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  • Polymorphisms in promoter regions of MYH1 and IRS1 genes showing marbling-associated expression changes 査読

    Bin Tong, Seiki Sasaki, Youji Muramatsu, Takeshi Ohta, Hiroyuki Kose, Hideaki Yamashiro, Takahisa Yamada

    Journal of Animal and Veterinary Advances11 ( 23 ) 4441 - 4445   2012年

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    掲載種別:研究論文(学術雑誌)  

    Marbling, defined by the amount and distribution of intramuscular fat is an economically important trait of beef cattle in Japan. Previously, researchers have found that the Myosin Heavy Chain 1 (MYH1) gene coding for an isoform of myosin heavy chain a major contractile protein which converts chemical energy into mechanical energy in muscle which is expressed in slow-twitch oxidative fiber and involved in development of the fiber and Insulin Receptor Substrate 1 (IRS1) gene encoding a signaling adaptor protein which is phosphorylated by insulin receptor and associated with insulin resistance and birth weight, possess expression differences in musculus longissimus muscle between low-marbled and high-marbled steer groups. In the present study, researchers detected Single Nucleotide Polymorphisms (SNPs), g.29850738G>A and g.l 20947716T>C in the promoter regions of the MYH1 and IRS1 genes, respectively between the 2 steer groups. However, the allelic distributions of the 2 SNPs were indistinguishable between Japanese Black sires with extremely high predicted breeding value for marbling and with extremely low one. An unknown SNP in the distal region upstream of ~4kb proximal promoter regions of the MYH1 and IRS 1 genes might be related to changes in gene expression and/or marbling. © Medwell Journals, 2012.

    DOI: 10.3923/javaa.2012.4441.4445

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  • Possible effects of single nucleotide polymorphism in CDC10, IRS1 and MFN2 Genes on growth-related traits in japanese black beef cattle 査読

    Bin Tong, Kaifeng Wu, Seiki Sasaki, Youji Muramatsu, Takeshi Ohta, Hiroyuki Kose, Hideaki Yamashiro, Yanru Zhang, Takahisa Yamada

    Journal of Animal and Veterinary Advances11 ( 24 ) 4536 - 4539   2012年

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    掲載種別:研究論文(学術雑誌)  

    Growth performance as well as marbling is the main breeding objectives in Japanese Black cattle, the major beef breed in Japan. Researchers have recently reported that existence of Single Nucleotide Polymorphisms (SNPs), g.63629097G>C, g.120947716T>C and g.38437771C>T in the promoter regions of the septin 7 (CDC10), the insulin receptor substrate 1 (IRS1) and the mitofusin 2 (MFN2) genes, respectively in Japanese Black beef cattle population. The CDC10 is known to be involved in cellular proliferation and the IRS1 is reported to be associated with insulin resistance and birth weight. The 2 genes have been earlier shown to be located within genomic regions of quantitative trait loci for growth-related traits. Thus, the CDC10 and IRS1 genes were considered as positional functional candidates for the gene responsible for growth performance. In addition, the MFN2 is known to play a role in energy balance through mitochondrial fusion, so the MFN2 gene was considered as functional candidate. In this study, researchers analyzed the possible effects of the 3 SNPs on the growth-related carcass traits: Carcass Weight (CWT), Rib Thickness (RT), Rib Eye Area (REA) and Subcutaneous Fat Thickness (SFT) in Japanese Black beef cattle. The g.63629097G>C and g. 120947716T>C SNPs in the CDC10 and IRS1, respectively, exhibited significantly different allelic distribution between Japanese Black sires with extremely high predicted breeding value and the sires with extremely low one for CWT and RT but not for the others. The g.38437771C>T SNP in the MFN2 had significantly different allelic distribution for RT. These findings suggest possible effects of the g.63629097G>C, g.l 20947716T>C and g.38437771C>T SNP on the growth-related trait in Japanese Black beef cattle. The SNPs in the CDC10, IRS1 and MFN2 genes may be useful for effective marker-assisted selection to increase the beef productivity in Japanese Black beef cattle. © Medwell Journals, 2012.

    DOI: 10.3923/javaa.2012.4536.4539

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  • A case study on cryopreservation of African sheep semen for the Red Maasai, dorper breeds and their crosses 査読

    Hideaki Yamashiro, John R. Wando, Edward Okoth, Satoshi Sugimura, Stefan Moisyadi, Eimei Sato, Edward O. Rege, Mwai Okeyo

    African Journal of Agricultural Research6 ( 4 ) 844 - 848   2011年2月

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    掲載種別:研究論文(学術雑誌)  

    The objective of this study was to examine the effect of different concentrations of trehalose and raffinose in Tris-citric acid-glucose (TCG) egg yolk extender for the sperm freezability of Red Maasai sheep, another African breed- the Dorper and their crosses. The results showed that Red Maasai, Dorper and their crosses sperm could be cryopreserved by extending with 0.075 to 0.01 M trehalose or raffinose-TCG egg yolk extender. We conclude that the cryopreservation of currently under threat Red Maasai sheep due to uncontrolled exotic breeds sperm makes it possible to establish a gene bank for the storage of animal genetic resources for endangered species, under the management of the International Livestock Research Institute for future genetic options in an uncertain world. © 2011 Academic Journals.

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  • Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects 査読

    Johann Urschitz, Miyuri Kawasumi, Jesse Owens, Kazuto Morozumi, Hideaki Yamashiro, Ilko Stoytchev, Joel Marh, James A. Dee, Kris Kawamoto, Craig J. Coates, Joseph M. Kaminski, Pawel Pelczar, Ryuzo Yanagimachi, Stefan Moisyadi

    Proceedings of the National Academy of Sciences of the United States of America107 ( 18 ) 8117 - 8122   2010年5月

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    掲載種別:研究論文(学術雑誌)  

    Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flankedby terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

    DOI: 10.1073/pnas.1003674107

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  • Lactate and adenosine triphosphate in the extender enhance the cryosurvival of rat epididymal sperm 査読

    Hideaki Yamashiro, Masaaki Toyomizu, Motoi Kikusato, Natsuki Toyama, Satoshi Sugimura, Yumi Hoshino, Hiroyuki Abe, Stefan Moisyadi, Eimei Sato

    Journal of the American Association for Laboratory Animal Science49 ( 2 ) 160 - 166   2010年

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    掲載種別:研究論文(学術雑誌)  

    We evaluated the cryosurvival of rat epididymal sperm preserved in raffinose-modified Krebs-Ringer bicarbonate-egg yolk extender supplemented with various energy-yielding substrates (glucose, pyruvate, lactate, and ATP) and assessed the effect on sperm oxygen consumption. The incubation of sperm at 37°C for 10 min in lactate-free extender decreased sperm motility and oxygen consumption before and after thawing compared with those of sperm in glucose- and pyruvate-free mediums. We then focused on the effect of supplementing the extender with lactate (0, 10.79, 21.58, 32.37, and 43.16 mM) and found that sperm frozen and thawed in extender supplemented with 32.37 mM lactate exhibited the highest motility. When we supplemented extender containing 32.37 mM lactate with ATP (0, 0.92, 1.85, 3.70, and 5.55 mM), sperm frozen and thawed in the extender supplemented with 1.85 mM ATP exhibited considerably higher motility and viability than those of sperm frozen and thawed in ATP-free extender. These results provide the first evidence that supplementation of the raffinose-modified Krebs-Ringer bicarbonate-egg yolk extender with 32.37 mM lactate and 1.85 mM ATP increases of number of motile sperm before freezing and enhances the cryosurvival of rat sperm. These supplements to the extender may enhance sperm cryosurvival by improving the metabolic capacity of sperm before freezing. Copyright 2010 by the American Association for Laboratory Animal Science.

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  • Extracellular ATP and dibutyryl cAMP enhance the freezability of rat epididymal sperm 査読

    Hideaki Yamashiro, Masaaki Toyomizu, Natsuki Toyama, Nobuya Aono, Masahiro Sakurai, Yuuki Hiradate, Masaki Yokoo, Stefan Moisyadi, Eimei Sato

    Journal of the American Association for Laboratory Animal Science49 ( 2 ) 167 - 172   2010年

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    掲載種別:研究論文(学術雑誌)  

    We studied the effects of ATP, ionomycin, and dibutyryl cAMP (dbcAMP) on the motility, freezability, and oxygen consumption of rat epididymal sperm. In vitro fertilization and intrauterine insemination were performed by using frozen-thawed rat sperm. Frozen-thawed sperm diluted in raffinose-modified Krebs-Ringer bicarbonate solution-egg yolk extender containing 1.85 mM ATP and 100 μM dbcAMP exhibited considerably higher motility and viability than sperm diluted in dbcAMP-free extender. Addition of ionomycin and dbcAMP to ATP-containing extenders did not alter the oxygen consumption rate of sperm, suggesting that extracellular ionomycin and dbcAMP are not involved in the mobilization of mitochondrial energy substrates in sperm. Further, high rates of pronucleus formation and progression to the blastocyst stage were observed in embryos produced by the fertilization of oocytes with fresh sperm in an in vitro fertilization medium supplemented with ATP and dbcAMP. Oocytes were not penetrated by frozen-thawed sperm when cocultured with cumulus-oocyte complexes in a medium without ATP and dbcAMP. In contrast, cryopreserved sperm penetrated oocytes when the gametes were cultured in an ATP- and dbcAMP-containing medium, and the resultant embryos formed blastocysts. Our results show that the dilution of rat sperm in raffinose-modified Krebs-Ringer bicarbonate solution-egg yolk extender supplemented with ATP and dbcAMP prior to sperm cryopreservation enhances the freezability of the cryopreserved sperm. Furthermore, the in vitro fertilization medium we developed effectively supports the production of embryos from both fresh and cryopreserved rat sperm. Copyright 2010 by the American Association for Laboratory Animal Science.

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  • Oxidation of exogenous lactate by lactate dehydrogenase C in the midpiece of rat epididymal sperm is essential for motility and oxidative activity 査読

    Yamashiro H, Toyomizu M, Kadowaki A, Takeda Z, Nakazato F, Toyama N, Kobayashi J, Sato E

    American Journal of Applied Sciences6 ( 10 ) 1854 - 1859   2009年10月

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  • Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos 査読

    S. Sugimura, K. Narita, H. Yamashiro, A. Sugawara, T. Shoji, Y. Terashita, K. Nishimori, T. Konno, M. Yoshida, E. Sato

    Theriogenology72 ( 4 ) 549 - 559   2009年9月

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    Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P < 0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells. © 2009 Elsevier Inc.

    DOI: 10.1016/j.theriogenology.2009.04.011

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  • Influence of the prostatic fluid from the first and third fractions of the ejaculates on the cryosurvival of poodle dog sperm 査読

    Hideaki Yamashiro, Kaori Narita, Satoshi Sugimura, Atsushi Sugawara, Yumi Hoshino, Masahiro Sakurai, Masaki Yokoo, Tsutomu Konno, Muneyoshi Yoshida, Eimei Sato

    American Journal of Animal and Veterinary Sciences4 ( 1 ) 14 - 20   2009年

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    掲載種別:研究論文(学術雑誌)  

    Problem statement: Two experiments were conducted to determine the effect of the prostatic fluid from the first and third fractions of the ejaculates on the freezability of poodle dog sperm. Approach: The ejaculates from 2 trained poodle dogs were collected. Effect of prostatic fluid from the first and third fractions of ejaculates on cryosurvival of dog sperm was examined. SDS-PAGE analyses were performed to verify whether there were any modifications in the protein patterns of the first and third fractions. Results: When second fractions of Dog A sperm were combined with the prostatic fluid from the first and third fractions of the ejaculates, sperm motility index was decreased compared to second fractions were collected alone. The post-thaw motility of Dog A sperm was significantly reduced when prostatic fluid from the first and third fractions of the ejaculates was added. The motility of frozen-thawed Dog B sperm did not appear to be altered when it was diluted with either of the prostatic fluids. The major bands present in both prostatic fluids corresponded to proteins with molecular masses of 30 and 75 kDa. The amount of proteins in the prostatic fluid from the third fraction was twice-three times as much protein present compared to the first fraction. Conclusion: Collection of the sperm-rich fraction along with either the prostatic fluid from the 1st and 3rd fractions of the ejaculates during sperm processing has varying detrimental effects on the motility characteristics of frozen-thawed sperm. © 2009 Science Publications.

    DOI: 10.3844/ajavsp.2009.14.20

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  • Noninvasive measurement of fecal progesterone concentration in toy poodles by time resolved fluoroimmunoassay (TR-FIA) 査読

    Satoshi Sugimura, Kaori Narita, Hideaki Yamashiro, Atsushi Sugawara, Katsuhiko Nishimori, Tsutomu Konno, Muneyoshi Yoshida, Eimei Sato

    American Journal of Animal and Veterinary Sciences3 ( 1 ) 43 - 46   2008年

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    掲載種別:研究論文(学術雑誌)  

    Progesterone is an important reproductive hormone and measurement of its level by repeated blood samplings is beneficial to monitoring of estrus cycle. However, since toy poodles have a small body size and thin-walled blood vessels, repeated blood samplings cause stress and affect their preparation for mating or artificial insemination (AI). Therefore, a noninvasive method for monitoring progesterone concentration should be developed. Here, we show that time-resolved fluoroimmunoassay (TF-RIA) is a useful noninvasive method for determining the progesterone concentration in serum and fecal samples obtained from toy poodles. Present results demonstrate that progesterone concentrations in the fecal correlated with the serum collected in same time and the sequential changes in progesterone concentrations in the feces are paralleled in the serum. Therefore, this technique may be suitable for monitoring the estrus cycle in toy poodles. © 2008 Science Publications.

    DOI: 10.3844/ajavsp.2008.43.46

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  • Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution 査読

    Hideaki Yamashiro, Young Joon Han, Atsushi Sugawara, Ikuo Tomioka, Yumi Hoshino, Eimei Sato

    Cryobiology55 ( 3 ) 285 - 294   2007年12月

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    掲載種別:研究論文(学術雑誌)  

    The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs-Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm. © 2007 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.cryobiol.2007.08.011

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  • Trehalose enhanced the freezability of Poodle dog sperm collected by an artificial vagina (AV) 査読

    Hideaki Yamashiro, Kaori Narita, Satoshi Sugimura, Young Joon Han, Atsushi Sugawara, Kanako Morohaku, Fumiaki Nakazato, Tsutomu Konno, Muneyoshi Yoshida, Eimei Sato

    Animal Reproduction Science102 ( 1-2 ) 165 - 171   2007年11月

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    掲載種別:研究論文(学術雑誌)  

    In an attempt to develop a suitable freezing method for Poodle dog sperm, an experiment was conducted to investigate semen collection methods of digital stimulation and an artificial vagina (AV), using Tris and trehalose-egg yolk extender, on the characteristics and cryopreservation of sperm. Two dogs (dogs A and B) were subjected to semen collection by digital stimulation and AV. The volume, sperm concentration, sperm motility index (SMI) and acrosome status of ejaculates were determined immediately after collection. The remainder was frozen as pellets in Tris and trehalose-egg yolk extender. Sperm motility index was evaluated after thawing and during a thermal resistance test, and acrosome integrity was also assessed. No significant differences regarding sperm concentrations, SMI and acrosome integrity were observed between semen collected by AV and digital stimulation. However, when dog sperm were collected by an AV and frozen in trehalose-egg yolk extender, the motility index of frozen-thawed sperm was significantly improved compared to sperm frozen in Tris-egg yolk extender which were collected by digital stimulation. In conclusion, semen collected by an AV and frozen in trehalose-egg yolk extender was effective in enhancing the freezability of Poodle dog sperm. © 2007 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.anireprosci.2007.02.026

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  • Studies of the role of steroid hormone in the regulation of oocyte maturation in cattle 査読

    Hai Feng Wang, Naoki Isobe, Kanako Kumamoto, Hideaki Yamashiro, Yasuhisa Yamashita, Takato Terada

    Reproductive Biology and Endocrinology4   4   2006年2月

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    掲載種別:研究論文(学術雑誌)  

    Background: The objective of this study was to investigate whether the steroid hormone(s) secreted from cumulus-oocyte complexes (COCs) is a prerequisite for bovine oocyte maturation and cumulus expansion using aminoglutethimide (AGT), a P450 cholesterol side-chain cleavage inhibitor. Methods: In experiment 1, COCs were cultured in maturation medium with various concentrations of AGT for 22 h to determine the effective concentration of AGT to inhibit steroid hormone secretion, meiotic maturation and cumulus expansion. In experiment 2, COCs were cultured in conditioned medium (CM) and TCM-199 medium with or without 10 mM AGT to check whether steroid hormones secreted from COCs were responsible for oocyte maturation and cumulus expansion. Experiments 3 and 4 were carried out to determine whether exogenous progesterone or estradiol-17beta was able to overcome the inhibitory effects of AGT on oocytes maturation and cumulus expansion. COCs cultured in 10 mM AGT-containing medium supplemented with various concentrations of progesterone or estradiol-17beta for 22 h were examined for oocyte maturation and cumulus expansion. Results: Experiment 1 showed that a concentration of 10 mM AGT in medium was sufficient to block steroid hormone secretion, oocyte maturation and cumulus expansion, and that these inhibitory effects were fully reversible. In experiment 2, the addition of 10 mM AGT to CM did not significantly prevent oocyte maturation and cumulus expansion, implying that CM contains the steroid hormone(s) secreted from COCs, which are closely associated with oocyte maturation and cumulus expansion. The results in experiments 3 and 4 demonstrated that the addition of any concentration of progesterone or estradiol-17beta in the medium did not reduce the inhibitory effects of AGT on oocyte maturation and cumulus expansion. Conclusion: Our results indicate that bovine oocytes surrounded by cumulus cells are prevented from maturation and cumulus expansion through the inhibition of steroid secretion due to AGT, and that these inhibitory effects of AGT on oocyte maturation and cumulus expansions can not be overcome by the addition of either progesterone or estradiol-17beta in the medium. These observations suggest that some steroid hormone(s) other than P4 and E2 secreted from bovine COCs is essential for their meiotic maturation and cumulus expansion. © 2006 Wang et al; licensee BioMed Central Ltd.

    DOI: 10.1186/1477-7827-4-4

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  • Enhanced freezability of goat spermatozoa collected into tubes containing extender supplemented with bovine serum albumin (BSA) 査読

    Hideaki Yamashiro, Haifeng Wang, Yasuhisa Yamashita, Kanako Kumamoto, Takato Terada

    Journal of Reproduction and Development52 ( 3 ) 407 - 414   2006年

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    掲載種別:研究論文(学術雑誌)  

    The purpose of this study was to investigate the effects of semen collection into tubes containing extender supplemented with BSA on the cryosurvival of goat spermatozoa. Semen was collected from two goats into empty tubes or tubes containing 10 ml extender supplemented with 0, 0.1, 1, or 5% BSA, and the washed spermatozoa were frozen as pellets in egg yolk-trehalose extender with the addition of 0.04% SDS and 4% glycerol. Sperm motion parameters were evaluated after post-thawing and during a thermal resistance test. The acrosome status of frozen-thawed spermatozoa was also observed using FITC-PNA staining. In frozen semen that was collected into tubes containing extender supplemented with 5% BSA, the post-thawed spermatozoa exhibited a significant improvement in motion parameters and maintained high motility throughout incubation and acrosome integrity, as compared with semen collected into tubes containing extender supplemented with lower concentrations of BSA. In conclusion, semen collection into tubes with a large volume of extender containing high concentrations of BSA dramatically improves the motility and acrosome integrity of frozen-thawed spermatozoa. This suggests that the in vitro functional freezability of spermatozoa is abruptly modified by reducing contact with seminal plasma and by flash contact with BSA at ejaculation.

    DOI: 10.1262/jrd.17105

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  • Effect of semen collection in extender solution on the characteristics of goat spermatozoa 査読

    Hideaki Yamashiro, Kanako Kumamoto, Hai Feng Wang, Yasuhisa Yamashita, Takato Terada

    Journal of Reproduction and Development52 ( 3 ) 397 - 406   2006年

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    掲載種別:研究論文(学術雑誌)  

    The objective of this experiment was to investigate whether the motility parameters and acrosome integrity of goat ejaculated spermatozoa are affected by collecting semen into tubes containing an extender, and thereby determine the significance of reducing contact between seminal plasma and the sperm membrane at ejaculation. Semen were collected from three goats into tubes containing 0, 1 or 10 ml extender, or collected into tubes containing 10 ml extender supplemented with 0.1, 1 or 5% BSA. Sperm motion parameters were evaluated immediately after collection, after washing, and during a 3-h thermal resistance test. Acrosome integrity was assessed using FITC-PNA staining. Semen collection into tubes containing 10 ml extender produced higher sperm motility, progressive motility, and acrosome integrity than that using a smaller volume of extender. Furthermore, collection into 5% BSA-containing extender exhibited higher sperm characteristics and maintained high sperm motility and progressive motility throughout incubation. In conclusion, semen collection into tubes with a large volume of extender, especially extender containing higher concentrations of BSA, improved the quality of ejaculated spermatozoa, strongly suggesting that the in vitro functional characteristics of the spermatozoa were abruptly modified by flash sperm contact with accessory sex gland fluid at ejaculation.

    DOI: 10.1262/jrd.17104

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  • Easy and rapid method for the determination of gene expression in cumulus cells incubated for oocyte maturation 査読

    Kanako Kumamoto, Haifeng Wang, Hideaki Yamashiro, Takato Terada

    Reproductive Biology and Endocrinology3   59   2005年10月

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    掲載種別:研究論文(学術雑誌)  

    Background: The objectives of this study were to develop an easy and rapid method for measuring gene expression in a small number of cells by real-time PCR without RNA extraction and purification, and to use this method to determine more precisely IGF-I gene expression in the cumulus cells surrounding oocytes. Methods: First, after small numbers of cumulus cells were lysed in cell lysis buffer, they were digested with various concentrations of DNase I for different periods at 37 to determine the optimal conditions for digestion of genomic DNA in the lysate. Since nonspecific amplification was liable to occur when the non-purified RT product of the cell lysate was used for real-time PCR with the given primers, the optimal conditions for Mg2+ and annealing temperature were well investigated. Further, to create the same conditions as in the actual sample reaction for measurement by real-time PCR, RT-minus product was added to the reaction mixture of the standard curve, and then the amplification efficiency was assessed. Next, IGF-I gene expression in cumulus cells collected from cumulus oocyte complexes (COCs) every 4h during maturation was determined using the developed method. Results: The optimal conditions for measuring gene expression using the cell lysate from a small number of cells were as follows: incubation of the cell lysate with 0.16 U/ microL DNase I with 10 U/ microL for 30 min, an Mg concentration of 1.5mM for amplification of target gene by real-time PCR using RT-product of the cell lysate. When the RT-minus products added to the reaction mixture for the standard curve, which was prepared for purified 18SrRNA plasmid, the PCR efficiency was similar between the sample and the standard. The IGF-I gene expression in the cumulus cells was elevated up through the first 8h of the culture and then declined gradually by the end of maturation, with the maximal gene expression (778-fold) seen at 8 h. Conclusions: It can be concluded that the method developed here, in which equivalent to cumulus cells collected from 0.03-0.075 COCs were employed per reaction, permits rapid and easy determination of target gene expression in a limited number of cells using real-time PCR without RNA extraction. © 2005 Kumamoto et al., licensee BioMed Central Ltd.

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MISC

  • CRISPR遺伝子編集系を電気穿孔法で受精卵に導入して作製したNanos3ノックアウトマウス生殖巣の形態解析

    大平 拓也, 中務 胞, 阿部 学, 夏目 理恵, 杉村 智史, 崎村 建司, 山城 秀昭

    The Journal of Reproduction and Development65 ( Suppl. ) j146 - j146   2019年9月

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    記述言語:日本語   出版者・発行元:(公社)日本繁殖生物学会  

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  • 野生アカネズミのIVFC技術を用いた染色体数の異なる生殖能の評価

    目黒 栞奈, 小松 一樹, 近能 真優, 坂 美幸, 藤嶋 洋平, 中潟 直己, 中田 章史, 三浦 富智, 山城 秀昭

    Journal of Mammalian Ova Research36 ( 1 ) S42 - S42   2019年4月

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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  • 透明帯除去マウス卵子を用いた被災アライグマ精子の卵子侵入能の評価

    庄司 莉那, 目黒 栞奈, 小松 一樹, 大平 拓也, 山城 秀昭, 藤嶋 洋平, ヴァレリー・ティング, 葛西 宏介, 中田 章史, 有吉 健太郎, 吉田 光明, 福本 学, 三浦 富智

    日本放射線影響学会大会講演要旨集61回   99 - 99   2018年11月

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    記述言語:日本語   出版者・発行元:(一社)日本放射線影響学会  

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  • 被災アライグマにおける卵巣の卵胞発育能の評価

    小松 一樹, 大平 拓也, 目黒 栞奈, 山城 秀昭, 藤嶋 洋平, ヴァレリー・ティング, 葛西 宏介, 中田 章史, 有吉 健太郎, 吉田 光明, 福本 学, 三浦 富智

    日本放射線影響学会大会講演要旨集61回   66 - 66   2018年11月

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    記述言語:日本語   出版者・発行元:(一社)日本放射線影響学会  

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  • 雄性SDT fattyラットにおけるアンジオテンシンⅡ受容体拮抗薬ロサルタンの薬理作用に関する検討

    美谷島 克宏, 剣持 佑介, 本橋 雄, 鳥庭 靖文, 村井 康高, 篠原 雅巳, 山城 秀昭, 山田 宜永, 太田 毅

    北信越畜産学会報 = Hokushinetsu journal of animal science ( 116 ) 1 - 8   2018年3月

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    記述言語:日本語   出版者・発行元:北信越畜産学会  

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  • 2型糖尿病モデル動物における血液凝固能の変化の検討

    剣持 佑介, 村井 康高, 竹内 里美, 山城 秀昭, 篠原 雅巳, 山田 宜永, 太田 毅

    北信越畜産学会報 = Hokushinetsu journal of animal science ( 116 ) 37 - 42   2018年3月

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    記述言語:日本語   出版者・発行元:北信越畜産学会  

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  • 肥満2型糖尿病モデル雌雄SDT fattyラットにおける摂餌制限による内臓脂肪および皮下脂肪の量的変動

    美谷島 克宏, 村井 康高, 石井 幸仁, 笹瀬 智彦, 篠原 雅巳, 山城 秀昭, 山田 宜永, 太田 毅

    北信越畜産学会報 = Hokushinetsu journal of animal science ( 116 ) 15 - 22   2018年3月

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    記述言語:日本語   出版者・発行元:北信越畜産学会  

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  • 培養したブタ卵母細胞の囲卵腔の大きさ、ヒアルロン酸の含有量および媒精後の多精子侵入の頻度

    神保 幸弘, 山城 秀昭, 新村 末雄

    北信越畜産学会報 ( 114 ) 9 - 19   2017年3月

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    記述言語:日本語   出版者・発行元:北信越畜産学会  

    低NaCl濃度のNorth Carolina State University37(NCSU37)(低NaCl-NCSU37)およびヒアルロン酸合成阻害剤である4-methylumbeliferone(MU)を0.25mM含むNCSU37(MU-NCSU7)で培養したブタ卵母細胞について、囲卵腔の大きさ、ヒアルロン酸含有量および媒精後の多精子侵入の頻度をそれぞれ観察して対象の卵母細胞と比較し、囲卵腔の大きさとヒアルロン酸含有量との関係および囲卵腔の大きさと多精子侵入の頻度との関係を検討した。また、多精子侵入防御に役割を果たす表層粒の分布状態についても観察した。胞状卵胞から採取直後の卵母細胞では、囲卵腔は0.63μmであったが、卵母細胞の囲卵腔は、通常のNaCl濃度のNCSU37で培養32時間以降有意に拡大し、培養後44時間には4.58μmになった。一方、低NaCl-NCSU37で44時間培養した卵母細胞の囲卵腔は6.25μmあり、対象のNCSU37で培養した卵母細胞の4.93μmに比べて有意に大きかった。また、MU処置した卵母細胞の囲卵腔は4.02μmであり、対照のMU処置していない卵母細胞の4.69μmに比べて有意に小さかった。ヒアルロン酸は、採取直後の卵母細胞では検出できなかったが、NCSU37で44時間培養後の卵母細胞では1個あたり227.76pg検出された。一方、低NaCl-NCSU37で44時間培養した卵母細胞では370.01pgのヒアルロン酸が検出され、この値はNCSU37で培養した卵母細胞の含有量に比べて有意に多かった。また、MU処置した卵母細胞のヒアルロン酸含有量は349.18pgであり、対照のMU処置していない卵母細胞の485.52pgに比べて有意に少なかった。受精率は、低NaCl-NCSU37で培養した卵母細胞と対照のNCSU37で培養した卵母細胞では80.9および82.5%、MU処置した卵母細胞と対照のMU処置していない卵母細胞では88.1および85.7%であり、それぞれの処置を施した卵母細胞と対照の卵母細胞との間で相違なかった。一方、多精子侵入の頻度は、低NaCl-NCSU37で培養した卵母細胞の32.7%に比べ、対照のNCSU37で培養した卵母細胞では有意に高く、51.9%であった。また、MU処置していない対照の卵母細胞の多精子侵入の頻度は47.6%であり、MU処置した卵母細胞の70.3%に比べて有意に低かった。表層粒のほとんどが細胞膜直下の細胞質に分布している卵母細胞の割合は、低NaCl-NCSU37で44時間培養した卵母細胞と対照のNCSU37で培養した卵母細胞の間、MU処置した卵母細胞と対照のMU処置していない卵母細胞の間でそれぞれ相違なかった。以上の結果から、囲卵腔の大きさとヒアルロン酸含有量との間には密接な関係のあることが推察されるとともに、囲卵腔の大きさと多精子侵入の頻度との間にも密接な関係があることが確かめられた。また、小型の囲卵腔を有する卵母細胞での媒精後の多精子侵入の高頻度は、これらの卵母細胞における表層粒の不備が原因ではないことが推察された。(著者抄録)

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  • 福島原発事故のその後、再生を目指して 被災雄牛の継世代影響解析

    山城 秀昭, 高橋 秀和, 藤 晋一, 漆原 佑介, 鈴木 正敏, 桑原 義和, 阿部 靖之, 杉村 智史, 福田 智一, 木野 康志, 篠田 壽, 磯貝 惠美子, 福本 学

    無菌生物46 ( 1 ) 22 - 23   2016年9月

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    記述言語:日本語   出版者・発行元:日本無菌生物ノートバイオロジー学会  

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  • 第一極体におけるJunoの局在と受精能の評価

    鈴木 万理, 菅野 有晃, 伊藤 洵, 斎藤 晴香, 及川 和聡, 新村 末雄, 山城 秀昭

    日本卵子学会誌1 ( 1 ) S41 - S41   2016年4月

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    記述言語:日本語   出版者・発行元:(一社)日本卵子学会  

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  • 野生アカネズミにおける体外繁殖実験系の検討

    瀧野 祥生, 藤宮 冴, 菅野 有晃, 鈴木 万里, 新村 末雄, 山城 秀昭

    日本畜産学会大会講演要旨集120回   73 - 73   2015年9月

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    記述言語:日本語   出版者・発行元:(公社)日本畜産学会  

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  • ニワトリ胚漿尿膜を用いた新生子マウス卵巣の器官培養

    岩島 玲奈, 瀧野 祥生, 菅野 有晃, 柳沼 日佳里, 杉山 稔恵, 新村 末雄, 山城 秀昭

    北信越畜産学会報 ( 110 ) 27 - 35   2015年3月

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    記述言語:日本語   出版者・発行元:北信越畜産学会  

    血管新生が盛んであるニワトリ胚漿尿膜(in ovo chorioallantoic membrane;iCAM)を用いて、新生子マウス卵巣の器官培養が可能か否か試みた。7-8日齢のBDF1雌マウスから採取した卵巣を、38.5度で7-9日間孵卵したニワトリ胚の漿尿膜上に静置し、孵卵器で10-12日間培養した。培養後、卵巣のパラフィン切片を作製して、HE染色を施し、卵巣組織および卵胞の発育を観察した。iCAM器官培養後の卵巣には、ニワトリ胚由来の血管が新生されて血液が全体に浸潤したもの、部分的に浸潤したもの、血液が全く浸潤していないものがあった。卵胞の形態を維持したiCAM器官培養後のHE染色した卵巣では、原始卵胞、1次卵胞、2次卵胞および胞状卵胞が観察され、ニワトリ胚由来の血液浸潤による赤血球も見られた。iCAM器官培養後の卵巣の切片1枚あたりの卵胞数は7.29±2.2個であり7-8日齢及び19日齢のBDF1雌マウスの卵巣のそれらと比較して有意に少なかった。また、直径が30μm未満の原始卵胞の割合は、58.9%であり、1次卵胞の21.9%、2次卵胞の17.8%、初期胞状卵胞を含む2次卵胞の2.7%、胞状卵胞の1.4%に比べて高かった。一方、卵胞の形態を維持できなかったiCAM器官培養卵巣では、ニワトリ胚由来の血液浸潤による赤血球が観察されるのみであった。以上、iCAM器官培養した31個の新生子マウス卵巣において、ニワトリ胚由来の血管新生による血液が全体に浸潤したものと部分的に浸潤した24サンプル中、7サンプルの卵巣において卵胞が維持されることが確かめられた。今後は、さらに卵胞を維持できるiCAM器官培養の方法を検討する必要がある。(著者抄録)

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  • 季節がアカネズミ精巣における精子形成と時計遺伝子発現に及ぼす影響

    秋山 将, 瀧野 祥生, 菅野 有晃, 岩島 玲奈, 高橋 実希, 矢澤 愛美, 福本 学, 山城 秀昭

    日本畜産学会大会講演要旨集119回   181 - 181   2015年3月

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    記述言語:日本語   出版者・発行元:(公社)日本畜産学会  

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  • 被災アカネズミの雄性生殖細胞における増殖能の評価

    瀧野 祥生, トウ・ビン, 菅野 有晃, 岩島 玲奈, 秋山 将, 中田 章史, 葛西 宏介, 三浦 富智, 福本 学, 山城 秀昭

    日本放射線影響学会大会講演要旨集57回   119 - 119   2014年9月

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    記述言語:日本語   出版者・発行元:(一社)日本放射線影響学会  

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  • マウス桑実胚におけるブリリアントクレシル青を利用した性判別の可能性

    小林 万優, 佟 彬, 山城 秀昭

    北信越畜産学会報 ( 108 ) 15 - 22   2014年3月

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    記述言語:日本語   出版者・発行元:北信越畜産学会  

    マウスの桑実胚について、X染色体の数とブリリアントクレシル青(BCB)に対する着色性との関係の有無を調べ、BCBに対する着色性から胚の性判別の可能性を検討した。BCBで染色した桑実胚の50%が還元促進液に浸漬後に陰性になる時間は、体内受精胚では22.6±0.21分であり、体外受精胚の22.4±0.30分、2倍体の単為発生胚の21.9±0.32分とは相違なかったが、半数体の単為発生胚の25.6±0.36分に比べて有意に短かった。なお、受精胚における時間は、有意差はないものの2倍体の単為発生胚よりも長い傾向にあった。これらのことより、マウス桑実胚において、BCBの還元速度とX染色体の数との間には関係があり、性判別にBCB染色を使用できる可能性が示唆された。一方、桑実胚期の体内受精胚の50%がBCBに陰性になる時間で、BCBに陽性と陰性それぞれの胚について雄特異的遺伝子であるSryの検出をPCR法で行ったところ、BCBに陰性で雌と推定された胚で、PCRで雌と判定された胚の割合は61.3%、BCBに陽性で雄と推定された胚で、PCRで雄と判定された胚の割合は71.9%であり、判別率の平均値は66.6%となった。以上のことから、胚のBCBに対する着色性と雌雄との間には関係のあることが示唆された。(著者抄録)

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  • 次世代シークエンサーを用いた被災ウシY染色体における突然変異の解析

    Tong Bin, 瀧野 祥生, 阿部 靖之, 桑原 義和, 山城 秀昭, 山田 宜永, 磯貝 恵美子, 福本 学

    日本放射線影響学会大会講演要旨集56回   178 - 178   2013年10月

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    記述言語:日本語   出版者・発行元:(一社)日本放射線影響学会  

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  • 原発事故が与える動植物への影響 福島第一原子力発電所事故に伴う家畜における放射性セシウムの臓器分布

    磯貝 恵美子, 福田 智一, 木野 康志, 福本 学, 篠田 壽, 関根 勉, 山城 秀昭, 黒田 健吾, 桑原 義和, 高橋 慎太郎, 井上 和也, 阿部 靖之, 小林 仁

    無菌生物43 ( 1 ) 46 - 50   2013年9月

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    記述言語:日本語   出版者・発行元:日本無菌生物ノートバイオロジー学会  

    2011年3月に起きた東京電力福島第一原子力発電所の爆発事故によって大気中に放出された放射性物質は福島県を中心に広範な地域に環境汚染をもたらした。本研究では福島原発20km圏内(警戒区域)で被災家畜の調査を行った。警戒区域内ではガンマースペクトルメーター測定において採取された家畜の筋肉などで134Cs、137Cs、110m Ag、129m Teのピークを認めた。放射性Csは血液などの体液、全身の臓器・組織から検出され、筋肉は最も高いレベルであった。また、血液と筋肉をはじめとする諸臓器との相関を認めた。福島原発の影響を知るためにも被災家畜を通して、人や動物への内部被ばくの影響を明らかにしていく必要がある。(著者抄録)

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  • in ovoでのマウス精巣内精原細胞の分化誘導

    瀧野 祥生, 植松 恵美, 岡嶋 秀美, 杉山 稔恵, 新村 末雄, 山城 秀昭

    日本畜産学会大会講演要旨集117回   100 - 100   2013年9月

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    記述言語:日本語   出版者・発行元:(公社)日本畜産学会  

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  • マウス胚盤胞の収縮運動に及ぼす透明帯の影響

    寺澤 瞳, 山下 優香, 山城 秀昭

    北信越畜産学会報 ( 106 ) 27 - 33   2013年3月

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    記述言語:日本語   出版者・発行元:北信越畜産学会  

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  • 電子線マイクロアナライザによる被災牛の精巣切片の元素分析

    山城 秀昭, 阿部 靖之, 福田 智一, 木野 康志, 福本 基, 桑原 義和, 高橋 慎太郎, 植松 恵美, 小林 仁, 磯貝 恵美子, 福本 学

    日本放射線影響学会大会講演要旨集55回   106 - 106   2012年9月

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    記述言語:日本語   出版者・発行元:(一社)日本放射線影響学会  

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  • ニワトリ胚漿尿膜を用いた新生子マウス精巣内精原細胞の器官培養

    植松 恵美, 岡嶋 秀美, 金子 小雪, 唐澤 春菜, 長島 愛美, 高橋 陽平, 杉山 稔恵, 山城 秀昭, 新村 末雄

    The Journal of Reproduction and Development58 ( Suppl. ) j130 - j130   2012年8月

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    記述言語:日本語   出版者・発行元:(公社)日本繁殖生物学会  

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  • 福島第一原子力発電所事故に伴う家畜の内部被ばく

    磯貝 恵美子, 阿部 靖之, 福田 智一, 木野 康志, 福本 学, 篠田 壽, 関根 勉, 山城 秀昭, 黒田 健吾, 米山 裕

    産業動物臨床医学雑誌3 ( 2 ) 102 - 104   2012年

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    記述言語:日本語   出版者・発行元:日本家畜臨床学会 ・ 大動物臨床研究会  

    DOI: 10.4190/jjlac.3.102

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  • イヌにおける再生医療の実現とその戦略

    中里 文昭, 杉村 智史, 山城 秀昭, ほか7名

    畜産の研究61 ( 6 ) 675 - 678   2007年6月

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    記述言語:日本語   出版者・発行元:養賢堂  

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    その他リンク: https://agriknowledge.affrc.go.jp/RN/2010741220

  • イヌ遺伝性疾患の診断技術と今後の展望

    菅原 淳史, 杉村 智史, 山城 秀昭, ほか5名

    畜産の研究61 ( 5 ) 548 - 554   2007年5月

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    記述言語:日本語   出版者・発行元:養賢堂  

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    その他リンク: https://agriknowledge.affrc.go.jp/RN/2010740797

  • イヌにおける再生医療の現状と可能性--ヒトと比較して考える

    中里 文昭, 杉村 智史, 山城 秀昭, ほか6名

    畜産の研究60 ( 11 ) 1172 - 1178   2006年11月

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    記述言語:日本語   出版者・発行元:養賢堂  

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    その他リンク: https://agriknowledge.affrc.go.jp/RN/2010732400

▶ 全件表示

共同研究・競争的資金等の研究

  • インドの高自然放射線地域における被ばく線量測定と生殖機能評価

    研究課題/領域番号:19KK0176  2019年10月 - 2023年03月

    日本学術振興会  科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))  国際共同研究加速基金(国際共同研究強化(B))

    山城 秀昭, 三浦 富智, 織部 祐介, 中田 章史, 篠田 壽

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    担当区分:研究代表者 

    配分額:18200000円 ( 直接経費:14000000円 、 間接経費:4200000円 )

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  • 放射線被ばくと糖尿病の二重ストレスの生体影響解析

    研究課題/領域番号:19K08142  2019年04月 - 2022年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    三浦 富智, 葛西 宏介, 有吉 健太郎, 山城 秀昭

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    担当区分:研究分担者 

    配分額:4290000円 ( 直接経費:3300000円 、 間接経費:990000円 )

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  • 異種の生殖器官および生殖細胞をマウスで再生する技術の開発

    研究課題/領域番号:18K05938  2018年04月 - 2021年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    山城 秀昭, 杉村 智史, 笹岡 俊邦

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    担当区分:研究代表者 

    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    【目的】生殖細胞の形成に不可欠であるNanos3をKOしたマウスの生殖巣を解析することを目的に、CRISPR/Cas9システムを電気穿孔法で受精卵に導入するTAKE法とGONAD法での変異導入効率の違いを胚盤胞および産仔の生殖巣の解析を行った。【方法】TAKE法:交配後に採取した前核期卵子は、Cas9mRNAとgRNAを満たしたチャンバー内に並べ、電気穿孔を行った。GONAD法:交配後のマウスの卵管に、Cas9とgRNAを卵管内に注入し、卵管全体に対し電気穿孔法を施した。TAKE法およびGONAD法にて得られた産仔の尾または胚盤胞からDNAを抽出し、PCRおよびsquenceにてhomo、hetero、wildに分類した。得られた産仔は10週齢で生殖巣を採取し、生殖巣表現型の差異と組織形態学的解析を行った。【結果】TAKE法の胚盤胞区では、17.6%(homo:2個/ 5.9%, hetero:4個/11.7%)、移植区では18.5%(homo:6匹/ 17.2%, hetero:1匹/ 1.3%)の効率で変異を確認できた。GONAD法では、18.6%の効率で変異が確認でき、全てhomo欠損個体であった。精巣および卵巣は、wild個体とhomo欠損個体でサイズに著しく差が生じていた。H&E染色像を観察した結果、homo欠損個体では、精子を含む生殖細胞は確認されず、セルトリ細胞のみが確認された。卵巣では、H&E染色像を観察した結果、homo欠損個体において卵子および卵胞は確認されなかった。以上、電気穿孔法で受精卵での遺伝子編集により、TAKE法とGONAD法では同程度の効率でNanos3遺伝子への変異を導入することが可能であり、かつ作製したNanos3ノックアウトマウスの生殖巣は、精巣の精細管内には精細胞が形成されず、卵巣内にも卵胞、卵母細胞が形成されないことを明らかにした。

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  • 被災雄牛における継世代影響評価系の構築と解析

    研究課題/領域番号:15K11952  2015年04月 - 2018年03月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    山城 秀昭, 杉村 智史

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    担当区分:研究代表者 

    配分額:5070000円 ( 直接経費:3900000円 、 間接経費:1170000円 )

    本研究では、旧警戒区域内で6ヶ月および2年間被ばくした雄ウシと8頭の産子において全ゲノムエクソン領域の網羅的な変異を抽出した。その結果、NLRP9など9遺伝子への変異が認められたが、生命の維持に重要な役割を果たす遺伝子への変異は認められなかった。さらに、被災雄牛の精子を用いて人工受精を実施し、10ヶ月飼育した後に全代謝産物のメタボローム解析を行った。その結果、対照の子牛のそれらの値に比較して差は認められなかった。本研究の成果は、福島県産の優良種雄牛の精子を用いて子牛を生産した場合においても、その子牛への放射線の影響が認められる可能性は極めて低いという一つの科学的な根拠を示すことができた。

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  • 福島第一原発事故に伴う被災動物における体内放射性物質の動態および影響解析研究

    研究課題/領域番号:26253022  2014年04月 - 2018年03月

    日本学術振興会  科学研究費助成事業 基盤研究(A)  基盤研究(A)

    福本 学, 木野 康志, 鈴木 正敏, 山城 秀昭

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    配分額:35230000円 ( 直接経費:27100000円 、 間接経費:8130000円 )

    福島第一原発事故の被災動物臓器アーカイブを構築し、以下の結果を得た。事故後2年経過し、ほぼ放射性セシウムのみが検出され、全臓器であった。ウシ血中の酸化ストレス関連因子は内部被ばく線量率と相関を示した。末梢血リンパ球のDNA二本鎖切断数は多かったが、線量とは無関係であった。ウシ精子に形成異常を認めなかった。地表を這い、γ、β線両方の被ばくが大きいアカネズミでは、精子形成の細胞回転が亢進しているが成熟精子に異常を認めなかった。野生ニホンザル骨髄は低形成傾向だが、抹消血球に異常を認めなかった。著変は認めないもののサルの内部被ばく線量率は現在も高く、生物影響を知るための詳細な観察継続が必要である。

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  • piggyBacトランスポゾンを用いた次世代型遺伝子改変ニワトリ作成法の開発

    研究課題/領域番号:24780269  2012年04月 - 2014年03月

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    山城 秀昭

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    担当区分:研究代表者 

    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    外来遺伝子導入により新しいニワトリ品種の作製は、有用タンパク質、鶏卵抗体やバイオ医薬品を卵黄や卵白で生産する技術に繋がり、次世代の新たな切り口を見出す基盤生産技術として期待されている。本研究は、ニワトリ生体の組織や器官とその生産物である鶏卵中に効率的に安定して、外来遺伝子であるEGFPが導入されたpiggyBacトランスポゾンを用いたプラスミドDNAと個体作成法を開発することを目的とした。 その結果、濃度2.5 ng/μlのpiggyBacトランスポゾンプラスミドを胚盤葉に注入した場合、生存率は低いが、孵化直前の鶏胎児に外来遺伝子を導入することが可能であることを明らかにした。

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  • 受精可能なラット精子の大量保存技術の確立

    研究課題/領域番号:07J11360  2007年 - 2008年

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    山城 秀昭

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    担当区分:研究代表者 

    配分額:1800000円 ( 直接経費:1800000円 )

    ラットは、生体機能モデルや疾患モデルとして利用される重要な実験動物である。しかし、多種多様なラット遺伝資源の効率的確保、安定した供給や保存において極めて重要な技術である精子の凍結保存技術は未だ実用化に至っていない。それ故、ラット精子の凍結保存技術の開発は急務となっており、その技術が実用可能になれば、開発の意義は計り知れない。そこで本研究では、極めて困難であるとされるラット精子凍結保存法の開発と凍結精子による体外受精技術の確立を目的として実験を行った。その結果、1)概知のラクトース卵黄保存液に代わる新たな精子の凍結保存液として基盤となる、ラフィノース・卵黄-修正クレブス-リンガー重炭酸液(modified Krebs-Ringer Bicarbonate;mKRB)保存液を開発した。2)ラット精子のエネルギー利用と供給に着目した、乳酸、およびエネルギー源であるATPを添加したラフィノース・卵黄-mKRB凍結保存液を改良した。3)精子運動活性化作用を有する物質に着目し、精子内のcAMP濃度を上昇させる効果のあるdbcAMPをさらに添加したdbcAMP・ATP・乳酸・ラフィノース・卵黄-mKRB保存液を開発した。4)新鮮精子を用いた体外受精および培養法において、ATPおよびdbcAMPを添加した受精培地を改良・開発し、従来の方法に比較して、高い受精率と胚の発生率を得ることが可能になった。さらに、凍結保存した精子においても、開発した受精培地を用いることにより、受精可能であることを証明した。以上、本研究において開発されたラット精子凍結保存法は、精子の凍結保存に有効な技術であるはかりではなく、保存精子を用いた体外受精においても有効な技術であることを示した。これらのことから、本研究の新たな知見と技術は、重要な実験動物であるラット遺伝資源の効率的維持、保存や安定した供給のため貢献するものである。

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