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研究者情報

マスター

アカウント（マスター）

  • 氏名

    東藤　孝(トウドウ　タカシ), トウドウ　タカシ

所属（マスター）

  • 水産科学研究院　海洋応用生命科学部門　増殖生物学分野

所属（マスター）

  • 水産科学研究院　海洋応用生命科学部門　増殖生物学分野

独自項目

syllabus

  • 2021, 増殖生物学特論Ⅰ, Advanced Aquaculture Biology Ⅰ, 修士課程, 水産科学院, 増殖、養殖、生理学、生化学、分子生物学、魚類、ウニ類
  • 2021, 増殖生物学特論Ⅱ, Advanced Aquaculture Biology Ⅱ, 修士課程, 水産科学院, 日本語：増殖、養殖、生理学、生化学、分子生物学、魚類、ウニ類
  • 2021, 水圏生化学実験, Laboratory Works on Aquatic Biochemistry, 学士課程, 水産学部, タンパク質、糖質、卵形成、生理学、生化学、免疫化学、筋肉
  • 2021, 環境と人間, Environment and People, 学士課程, 全学教育, 海、河川、生命、細胞、遺伝子、魚、海藻、微生物、代謝、進化
  • 2021, 水族生化学, Biochemistry of Aquatic Animals, 学士課程, 水産学部, 免疫生化学　抗原・抗体反応　蛋白質精製法　蛋白質同定法　蛋白質測定法　血清蛋白質　遺伝子工学
  • 2021, 比較生理学, Comparative Physiology, 学士課程, 水産学部, 繁殖，ホルモン，免疫系，生理機能，生体防御

researchmap

プロフィール情報

学位

  • 博士（水産学）（北海道大学）

プロフィール情報

  • 姓

    東藤, トウドウ

  • 名

    孝, タカシ

  • ID各種

    201301037244745948

対象リソース

業績リスト

研究キーワード

  • 卵形成   油球   環境ホルモン   水産学   脂質   エストロゲン   卵母細胞   リポタンパク   ウナギ   発生・分化   性ステロイドホルモン   生殖細胞   タンパク質   メダカ   サケ科魚類   卵黄   核内レセプター   性転換   器官培養   卵成長   ベラ科魚類   レセプター   卵巣   核内受容体   精子形成   生理学   アポトーシス   精巣   性ホルモン   アンドロゲン   

研究分野

  • ライフサイエンス / 形態、構造
  • ライフサイエンス / 水圏生命科学
  • ライフサイエンス / 水圏生産科学
  • 環境・農学 / 化学物質影響
  • 環境・農学 / 放射線影響
  • ライフサイエンス / 発生生物学

経歴

  • 2005年04月 - 現在 北海道大学 大学院水産科学研究院 准教授
  • 2003年04月 - 2005年03月 北海道大学 大学院水産科学研究科 助教授
  • 1998年02月 - 2003年03月 新潟大学 理学部附属臨海実験所 助手
  • 1997年04月 - 1998年01月 岡崎国立共同研究機構基礎生物学研究所 生殖研究部門 日本学術振興会特別研究員
  • 1996年11月 - 1997年03月 岡崎国立共同研究機構基礎生物学研究所 生殖研究部門 リサーチ・アソシエイト
  • 1995年04月 - 1996年10月 岡崎国立共同研究機構基礎生物学研究所 生殖研究部門 特別協力研究員

学歴

  • 1992年04月 - 1995年03月   北海道大学   水産学部   水産学研究科水産増殖学専攻博士後期課程
  • 1990年04月 - 1992年03月   北海道大学   水産学部   水産学研究科水産増殖学専攻修士課程
  • 1986年04月 - 1990年03月   北海道大学   水産学部   水産増殖学科

委員歴

  • 2020年03月 - 2022年03月   公益社団法人日本水産学会   水産増殖懇話会幹事
  • 2014年04月 - 2018年03月   公益社団法人日本水産学会   シンポジウム企画委員

論文

  • Shuichiro Watanabe, Ken Matsuzaki, Utano Shimizu, Ichiro Higuchi, Takashi Todo, Yasuaki Takagi, Kazuhiro Ura
    Fisheries Science 90 6 907 - 924 2024年06月10日
  • Yo Yamaguchi, Takuma Kawasaki, Akihiko Hara, Takashi Todo, Naoshi Hiramatsu
    Aquaculture 583 2024年03月30日 
    Viviparous rockfishes (Sebastes spp.) are ecologically and economically important fisheries and aquaculture species. Because the males lack obvious morphological characteristics related to reproductive status, it is challenging to select semen donors for artificial insemination. We previously identified lipocalin-type prostaglandin D2 synthase homolog protein (LPGDShp) in the urine of male black rockfish (Sebastes schlegelii). Subsequently, LPGDShp-like protein (hereafter, lipocalin-like protein) was immunologically detected in the serum of two Sebastes species, black rockfish and white-edged rockfish (S. taczanowskii). In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed to explore the potential of serum lipocalin-like protein as a biomarker for selecting mature males. The ELISA measured lipocalin-like protein in the sera of black rockfish and white-edged rockfish over an assay range between 0.391 and 12.5 ng/mL. In male black rockfish, serum levels of lipocalin-like protein were higher during the copulation season, and they were higher than levels in females during both the copulation and non-copulation seasons. In male white-edged rockfish sampled nearly monthly for a year, serum lipocalin-like protein levels showed synchronous dynamics with gonadosomatic index (GSI). Histological examination of testis revealed that the serum levels were elevated in the late/functional maturation stages, as compared to early/mid-maturation and resting stages. In both rockfish species, while serum levels of lipocalin-like protein were positively correlated with GSI in males, no such trend was observed in females. These results indicate, for the first time, that serum lipocalin-like protein can be employed as a hematological biomarker to detect the reproductive status of male rockfishes.
  • Yo Yamaguchi, Jin Namgung, Jun Nagata, Takuma Kawasaki, Akihiko Hara, Takashi Todo, Naoshi Hiramatsu
    Gene 147093 - 147093 2022年12月 [査読有り]
  • Yamaguchi, Y, Nagata, J, Nishimiya, O, Kawasaki, T, Hiramatsu, N, Todo, T
    Comparative Biochemistry and Physiology, Part A 261 111055 - 111055 2021年 [査読有り][通常論文]
  • Nagata, J, Wada, S, Nishimiya, O, Wu, M, Mushirobira, Y, Yamaguchi, Y, Yokono, T, Kawasaki, T, Matsubara, T, Todo, T, Hara, A, Hiramatsu, N
    Zoological Science 38 5 451 - 458 2021年 [査読有り][通常論文]
  • Nagata, J, Mushirobira, Y, Nishimiya, O, Yamaguchi, Y, Fujita, T, Hiramatsu, N, Hara, A, Todo, T
    General and Comparative Endocrinology 310 113812 - 113812 2021年 [査読有り][通常論文]
  • Namgung, J, Mizuta, H, Yamaguchi, Y, Nagata, J, Todo, T, Yilmaz, O, Hiramatsu, N
    Comparative Biochemistry and Physiology, Part A 257 110967 - 110967 2021年 [査読有り]
  • 莚平裕次, 川崎琢真, 中田訓彰, 竹中映美, 永田淳, 石田良太郎, 山口浩志, 佐藤充, 東藤孝, 平松尚志
    水産増殖 68 1 1 - 8 2020年 [査読有り][通常論文]
  • Haruna Amano, Seiichi Uno, Jiro Koyama, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara
    General and comparative endocrinology 281 67 - 72 2019年09月15日 [査読有り][通常論文]
     
    Non-competitive, enzyme-linked immunosorbent assays (ELISAs) for three distinct sole vitellogenins (VtgAa, VtgAb and VtgC) were designed using their purified lipovitellin (Lv) products and corresponding digoxigenin-labeled, anti-Lv polyclonal antibodies, primarily for employment in monitoring estrogenic pollution of the environment. The working range of the ELISAs was from 0.97 to 1,000 ng/mL for all Vtg subtypes. Each ELISA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed ELISAs were lower than 10%. Three Vtg subtypes were induced in serum of immature fish by estradiol-17β (E2) injection (0.5 mg/kg body weight). All Vtg subtypes were induced one day after the injection, reaching peak levels (Lv equivalents) within three days, as follows: 39.1 ± 28.9 μg/mL (VtgAa), 57.9 ± 30.7 μg/mL (VtgAb) and 12.6 ± 4.8 μg/mL (VtgC). In wild-caught males, VtgAa, VtgAb and VtgC were detected in ranges from 0.26 to 1.21, 0.19 to 8.69, and 0.17 to 53.50 μg/mL, respectively, over various sampling periods. In vitellogenic females sampled in January, the average level of VtgAb (8,744.43 ± 733.93 μg/mL) was significantly higher than for VtgAa (150.33 ± 22.35 μg/mL) or VtgC (57.08 ± 6.00 μg/mL); thus VtgAb appeared to be the most dominant Vtg subtype. The present study entails the first report on development of subtype-specific Vtg ELISAs in marbled sole, which empowers us to detect and monitor estrogenic contamination in aquatic environments inhabited by this species.
  • Georgia Thomson-Laing, Erin L Damsteegt, Jun Nagata, Shigeho Ijiri, Shinji Adachi, Takashi Todo, Naoshi Hiramatsu, P Mark Lokman
    Biology of reproduction 100 5 1319 - 1332 2019年05月01日 [査読有り][通常論文]
     
    Estradiol-17β (E2) and 11-ketotestosterone (11KT) have been implicated in vitellogenesis and in regulating expression of the follicle-stimulating hormone receptor (fshr), respectively. To override the captivity-induced reproductive block in shortfinned eel, Anguilla australis, we hypothesized that in combination, 11KT and E2 would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E2 (0, 0.2, 2, 5 mg) with or without 11KT (1 mg). Vtg levels were determined in plasma, liver, and ovarian tissues by histological examination, qPCR, immunoblotting, or single radial immunodiffusion. The expression of gonadotropin-beta subunits and gonadotropin receptors in the pituitary and ovary, respectively, were analyzed to determine mechanisms by which steroid effects may be exerted. When administered alone, E2 increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E2 could induce uptake of Vtg into oocytes, although E2 treatment appeared necessary for uptake to occur. This was the case despite 11KT dramatically increasing both oocyte size and fshr mRNA levels. Astonishingly, the uptake of Vtg was successfully induced by co-treatment with 11KT and E2, suggesting that 11KT might facilitate the incorporation of Vtg into the developing oocyte. These results highlight the potential of sex steroid co-treatment, an approach aimed at mimicking oogenesis in wild eels, to induce vitellogenesis, specifically ovarian yolk deposition, even in the absence of exogenous gonadotropin treatment.
  • Haruna Amano, Akihiro Kotake, Naoshi Hiramatsu, Toshiaki Fujita, Takashi Todo, Jun-Ya Aoki, Kiyoshi Soyano, Hirohiko Kagawa, Akihiko Hara
    General and comparative endocrinology 271 30 - 38 2019年01月15日 [査読有り][通常論文]
     
    Chemiluminescent immunoassays (CLIAs) were developed for each of three subtypes of vitellogenin (VtgAa, VtgAb and VtgC) in grey mullet, primarily for use in monitoring estrogenic pollution of the environment. The working range of VtgAa-CLIA and VtgAb-CLIA was from 0.975 to 1,000 ng/ml, while that of VtgC-CLIA was from 0.487 to 1,000 ng/ml. Each CLIA appeared to be specific to the targeted Vtg subtype. Intra- and inter-assay coefficients of variation in the developed CLIAs were lower than 10%. In male serum, VtgAa, VtgAb and VtgC were detected in ranges from 0.01 to 0.38, 0.02 to 1.01, and 0.01 to 3.12 μg/ml, respectively, during various sampling periods. In vitellogenic females (October), serum VtgAb levels (1,192.05 ± 237.81 μg/ml) were significantly higher than levels of the other two Vtg subtypes (120.82 ± 30.42 and 119.23 ± 16.95 μg/ml for VtgAa and VtgC, respectively). When immature mullet were fed diets containing 17α-ethinylestradiol (EE2) at three different doses (0.4, 40 and 4,000 ng/g body weight), all Vtg subtypes were induced by 40 ng/g and 4,000 ng/g EE2. The VtgC (610.30 ± 150.18 μg/ml) was most highly expressed among the three Vtgs in fish fed 40 ng/g EE2, while VtgAb (33.25 ± 13.58 mg/ml) was highest in expression in fish fed 4,000 ng/g EE2. The present study provided practical subtype-specific Vtg assays for the first time in grey mullet, providing the necessary means to evaluate estrogenic activities in aquatic environments.
  • Benjamin Reading, Linnea Andersen, Yong-Woon Ryu, Yuji Mushirobira, Takashi Todo, Naoshi Hiramatsu
    Fishes 3 4 45 - 45 2018年11月21日 [査読無し][通常論文]
     
    Egg quality in fishes has been a topic of research in aquaculture and fisheries for decades as it represents an important life history trait and is critical for captive propagation and successful recruitment. A major factor influencing egg quality is proper yolk formation, as most fishes are oviparous and the developing offspring are entirely dependent on stored egg yolk for nutritional sustenance. These maternally derived nutrients consist of proteins, carbohydrates, lipids, vitamins, minerals, and ions that are transported from the liver to the ovary by lipoprotein particles including vitellogenins. The yolk composition may be influenced by broodstock diet, husbandry, and other intrinsic and extrinsic conditions. In addition, a number of other maternal factors that may influence egg quality also are stored in eggs, such as gene transcripts, that direct early embryonic development. Dysfunctional regulation of gene or protein expression may lead to poor quality eggs and failure to thrive within hours of fertilization. These gene transcripts may provide important markers as their expression levels may be used to screen broodstock for potential spawning success. In addition to such intrinsic factors, stress may lead to ovarian atresia or reproductive failure and can impact fish behavior, fecundity, and ovulation rate. Finally, postovulatory aging may occur when eggs become overripe and the fish fails to spawn in a timely fashion, leading to low fertility, often encountered during manual strip spawning of fish.
  • Yuji Mushirobira, Osamu Nishimiya, Jun Nagata, Takashi Todo, Akihiko Hara, Benjamin J. Reading, Naoshi Hiramatsu
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 267 157 - 166 2018年10月 [査読有り][通常論文]
     
    Transcription of vitellogenin (vtg) genes are initiated when estradiol-17 beta (E-2)-estrogen receptor (ER) complexes bind estrogen response elements (ERE) located in the gene promoter region. Transcriptional regulation of dual vtg subtypes (major salmonid A-type vtg: vtgAs; minor C-type vtg: vtgC) by E-2 was investigated under co-expression of a potential major transcriptional factor, era1 , in cutthroat trout. Two forms of trout vtgAs promoters (1 and 2) and one vtgC promoter were sequenced. These promoters structurally differ based on the number of EREs present. The vtgAs promoter 1 exhibited the highest maximal transcriptional activity by in vitro gene reporter assays. The concentration of E-2 that induces 50% of gene reporter activity (half-maximal effective concentrations, EC50) was similar among all vtg promoters and also to the EC50 of E-2 administered to induce vtg transcription in vivo. This study revealed a difference in transcriptional properties of multiple vtg promoters for the first time in a salmonid species, providing the basis to understand mechanisms underlying regulation of vitellogenesis via dual vtg gene expression.
  • 永田 淳, 莚平 裕次, 西宮 攻, 藤田 敏明, 平松 尚志, 原 彰彦, 東藤 孝
    水産増殖 66 2 91 - 101 水産増殖談話会 2018年 [査読有り][通常論文]
     
    サケ科魚類の雌の肝臓中では，エストラジオール-17β（E2）は卵膜蛋白前駆物質（コリオジェニン， Chg）や卵黄蛋白前駆物質（ビテロジェニン， Vtg）とエストロジェン受容体α1（Erα1）遺伝子の発現を誘導する。本研究では，カットスロートトラウト雌の生殖周期に伴う血中 E2 量，肝臓中の3種 chg（chgHα; chgHβ; chgL），2種 vtgs（vtgAs; vtgC），erα1の各mRNA 発現量の関係を調べた。血中 E2 量と肝臓 chg 発現量は，肝臓 vtg 発現量と同様に卵黄形成の進行とともに上昇した。しかし，chg 発現量は排卵後にも高値を維持し，血中 E2 量や vtg 発現量は低下した。肝臓 erα1 発現量は，血中 E2 量や chg，vtg 発現量の増加に先立つ卵黄形成開始時の8月にピークを示した。これらの結果から，chg，vtg，erα1 等の E2 応答性遺伝子の発現は異なる機構で制御されていることが示唆された。
  • 永田 淳, 東藤 孝, 原 彰彦, 平松 尚志, 笠井 慶, 峯野 博和, 藤崎 雄大, 莚平 裕次, 南宮 眞, 武田 康孝, 藤田 敏明, 川崎 琢真
    水産増殖 66 4 257 - 266 水産増殖談話会 2018年 [査読無し][通常論文]
     
    ホールマウント免疫染色法を用いて，マガレイ（Pseudopleuronectes herzensteini），スナガレイ（Limanda punctatissima）およびソウハチ（Cleisthenes pinetorum）の3種カレイ卵種判別を試みた。先ず，各カレイ排卵卵の卵膜を抗原としたポリクローナル抗体（a-マガレイ VE，a-スナガレイ VE，a-ソウハチ VE）を作製した。試料として，受精後24時間以内の各カレイ授精卵を用い，同抗体と標識二次抗体を用いた方法（2ステップ法）および，標識1次抗体を用いた方法（1ステップ法）でホールマウント免疫染色を行った。その結果，いずれの手法でも，対象種授精卵を特異的に染色することができた。以上，本研究で開発した免疫染色によるカレイ卵の種判別法は，カレイ類の生活史初期における資源量調査の簡易化に大きく役立つと考えられた。
  • Hiroko Mizuta, Yuji Mushirobira, Jun Nagata, Takashi Todo, Akihiko Hara, Benjamin J. Reading, Craig V. Sullivan, Naoshi Hiramatsu
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY 212 24 - 34 2017年10月 [査読有り][通常論文]
     
    To evaluate potential involvement of clathrin in endocytosis of vitellogenin (Vtg) by teleost oocytes, cDNAs encoding clathrin heavy chain (cltc) were cloned from ovaries of cutthroat trout. Quantitative PCR revealed three types of cltc (cltc-al, cltc-a2, cltc-b) to be expressed in 10 different tissues including the ovary. The cltc-al alone exhibited a significant decrease in ovarian expression during vitellogenesis; this was correlated with a corresponding decrease in transcripts encoding the major Vtg receptor (Vtgr). No development-related changes in ovarian cltc-a2 or cltc-b transcript levels were observed. In situ hybridization revealed a strong ctic signal in pre-vitellogenic oocytes, but not in vitellogenic oocytes. Western blotting using a rabbit antiserum (a-Cltc) raised against a recombinant Cltc preparation detected a polypeptide band with an apparent mass of similar to 170 kDa in vitellogenic ovary extracts. Immunohistochemistry using a-Cltc revealed Cltc to be uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, translocated to the periphery of lipid droplet stage oocytes, and localized to the oolemma during vitellogenesis. These patterns of citc/Cltc distribution and abundance during oogenesis, which are identical to those previously reported for vtgr/Vtgr in this species, constitute the first empirical evidence that cltc-al/Cltc-al is involved in Vtg endocytosis via the Vtgr in teleost fish.
  • 川崎 琢真, 清水 洋平, 森 立成, 平松 尚志, 東藤 孝
    水産増殖 65 1 73 - 82 水産増殖談話会 2017年 [査読有り][通常論文]
     
    クロソイは本邦における重要産業種である。胎生魚という特徴から，その種苗生産は自然交尾した親魚に依存しており，仔魚の安定生産が困難である。本研究ではクロソイ種苗生産の安定化を目的とし，人工授精技術の開発を試みた。成熟サイズの天然クロソイの雌20尾および雄15尾を用い，人工授精の時期，精子の希釈方法および精子を提供する雄の生死の影響を検討した。妊娠した雌の産仔数を調べ，マイクロサテライト解析による親子鑑定を行った。人工授精を行った結果，20尾中12尾の雌が妊娠し，平均産仔数は136,461尾であった。人工授精の時期，精子の希釈方法，雄親魚の生死の検討では，全ての試験条件で妊娠魚が得られた。親子鑑定の結果，全ての仔魚は人工授精由来であることが明らかになった。更に，複数の雄の精子を混合して人工授精した雌魚は，用いた全ての雄の子供を産仔した。本研究は，海産胎生魚での人工授精が可能であることを示した。
  • Osamu Nishimiya, Yoshinao Katsu, Hiroyuki Inagawa, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara
    JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 165 190 - 201 2017年01月 [査読有り][通常論文]
     
    One or more distinct forms of the nuclear estrogen receptor (ER) have been isolated from many vertebrates to date. To better understand the molecular evolution of ERs, we cloned and characterized er cDNAs from the inshore hagfish, Eptatretus burgeri, a modern representative of the most primitive vertebrates, the agnathans. Two er cDNAs, er1 and er2, were isolated from the liver of a reproductive female hagfish. A phylogenetic analysis placed hagfish ER1 into a position prior to the divergence of vertebrate ERs. Conversely, hagfish ER2 was placed at the base of the vertebrate ER beta Glade. The tissue distribution patterns of both ER subtype mRNAs appeared to be different, suggesting that each subtype has different physiological roles associated with estrogen actions. An estrogen responsive-luciferase reporter assay using mammalian HEK293 cells was used to functionally characterize these hagfish ERs. Both ER proteins displayed estrogen-dependent activation of transcription. These results clearly demonstrate that the hagfish has two functional ER subtypes. (C) 2016 Elsevier Ltd. All rights reserved.
  • 西宮 攻, 勝 義直, 平松 尚志, 東藤 孝
    比較内分泌学 43 160 21 - 23 日本比較内分泌学会 2017年
  • 前林 衛, 平松 尚志, 稲岡 雄平, 吉田 達哉, 萩原 聖士, 西宮 攻, 莚平 裕次, 足立 伸次, 原 彰彦, 東藤 孝
    水産増殖 64 1 63 - 76 水産増殖談話会 2016年 [査読有り][通常論文]
     
    Estradiol-17β処理したアムールチョウザメの肝臓から卵黄蛋白前駆物質であるビテロジェニン（Vtg）の cDNA クローニングを行った。得られた3種の cDNA は一時的に vtg1，vtg2 および vtg3 と名付けた。vtg1，vtg2 および vtg3（各々5,307，5,247，5,319 bp）は，それぞれアミノ酸1,769，1,749 および 1,773 aa からなる完全長の翻訳領域を含んでいた。演繹アミノ酸配列間の相同性は64.3%から47.9%の間であり，3種の Vtgs は全ての卵黄蛋白質ドメインを持つ完全型 Vtg であった。系統樹解析において，アムールチョウザメ Vtg1 は既知のシロチョウザメ Vtg と同じクレードを形成したが，Vtg2 および Vtg3 とは異なるクレードを形成した。アムールチョウザメ Vtg は3種とも VtgAB タイプに分類され，vtg1/Vtg1，vtg2/Vtg2 および vtg3/Vtg3 は，それぞれ vtgAB1/VtgAB1，vtgAB2a/VtgAB2a および vtgAB2b/VtgAB2b と名付けられた。本研究は，チョウザメ類の Vtg の多型性を理解する基礎を提供し，将来的にそれらを生殖関連バイオマーカーとして利用する第一歩となった。
  • Yuji Mushirobira, Hiroko Mizuta, Wenshu Luo, Takashi Todo, Akihiko Hara, Benjamin J. Reading, Craig V. Sullivan, Naoshi Hiramatsu
    MOLECULAR REPRODUCTION AND DEVELOPMENT 82 12 986 - 1000 2015年12月 [査読有り][通常論文]
     
    Multiple ovarian membrane proteins that bind vitellogenin (Vtg) have been detected in teleosts. One of these Vtg receptors was recently identified as low-density lipoprotein receptor-related protein 13 (lrp13/Lrp13) in perciform species, but little is known about this Vtg receptor in salmonid fish. In this study, a cDNA encoding a putative Vtg receptor with 13+1 ligand binding repeats (lr13+1) was cloned from the ovary, and identified as an lrp13 ortholog for cutthroat trout (Oncorhynchus clarki). This lrp13 was predominantly expressed in the pre-vitellogenic stage ovary, and its expression decreased during vitellogenesis. Ovarian localization of Lrp13 was observed by immunohistochemistry using specific antiserum against recombinant Lrp13. Lrp13 immunoreactivity was observed at the oolemma, throughout the zona radiata, and within the perivitelline space between the zona radiata and granulosa cells in ovarian follicles at both the lipid-droplet and vitellogenic stages of growth-an expression pattern that mimics that of a lr8/LR8-type Vtg receptor in this species and of lrp13/Lrp13 in Morone species. Six discrete Vtg-binding proteins were detected in cutthroat trout ovarian membrane proteins when probing with a digoxygenin-labeled salmonid A-type Vtg (VtgAs) followed by chemiluminescent ligand detection. Western blotting using the anti-Lrp13 serum revealed a broad signal consisting of two proteins with masses ranging from similar to 190 to similar to 210 kDa, which corresponded with some of the VtgA-binding proteins. These findings suggest that, in addition to lr8/LR8, lrp13/Lrp13 acts as a VtgA receptor in trout. (C) 2015 Wiley Periodicals, Inc.
  • Naoshi Hiramatsu, Takashi Todo, Craig V. Sullivan, Justin Schilling, Benjamin J. Reading, Takahiro Matsubara, Yong-Woon Ryu, Hiroko Mizuta, Wenshu Luo, Osamu Nishimiya, Meiqin Wu, Yuji Mushirobira, Ozlem Yilmaz, Akihiko Hara
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 221 9 - 15 2015年09月 [査読有り][通常論文]
     
    Fish egg yolk is largely derived from vitellogenins, which are synthesized in the liver, taken up from the maternal circulation by growing oocytes via receptor-mediated endocytosis and enzymatically processed into yolk proteins that are stored in the ooplasm. Lipid droplets are another major component of fish egg yolk, and these are mainly composed of neutral lipids that may originate from maternal plasma lipoproteins. This review aims to briefly summarize our current understanding of the molecular mechanisms underlying yolk formation in fishes. A hypothetical model of oocyte growth is proposed based on recent advances in our knowledge of fish yolk formation. (C) 2015 Elsevier Inc. All rights reserved.
  • Rie Goto, Taiju Saito, Yutaka Kawakami, Tomoe Kitauchi, Misae Takagi, Takashi Todo, Katsutoshi Arai, Etsuro Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 59 10-12 465 - 470 2015年 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) appear during early embryogenesis and differentiate into gametes through oogenesis or spermatogenesis. Teleost PGCs can be visualized by injecting RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3). Although this method has been widely applied to teleost PGCs, the visualization of PGCs in pelagic species that have eggs with a hard chorion is more problematic due to the technical difficulty of microinjection into their eggs. In this study, we developed a reliable method for microinjection of fertilized eggs in a pelagic species, the barfin flounder. Using a microneedle with a constriction "brake", we were able to introduce gfp-nos3 3'UTR mRNA into embryos and to determine the migration route of PGCs. We also isolated the barfin flounder nos3 (bf-nos3) gene to compare its 3'UTR sequence with that of zebrafish. The 3'UTR of the bf-nos3 sequence was longer than that of zf-nos3. However, PGCs were also visualized after injection of gfp-bf-nos3 3'UTR mRNA both in zebrafish and barfin flounder. These results suggest that the function of nos3 is conserved between these species regardless of the sequence differences. The method developed here for labeling PGCs with gfp-nos3 mRNA will provide a means to study PGC development in the embryos of a wide range of marine fish species.
  • Benjamin J. Reading, Naoshi Hiramatsu, Justin Schilling, Katelyn T. Molloy, Norm Glassbrook, Hiroko Mizuta, Wenshu Luo, David A. Baltzegar, Valerie N. Williams, Takashi Todo, Akihiko Hara, Craig V. Sullivan
    JOURNAL OF LIPID RESEARCH 55 11 2287 - 2295 2014年11月 [査読有り][通常論文]
     
    Transcripts encoding a novel member of the lipoprotein receptor superfamily, termed LDL receptor-related protein (Lrp)13, were sequenced from striped bass (Morone saxatilis) and white perch (Morone americana) ovaries. Receptor proteins were purified from perch ovary membranes by protein-affinity chromatography employing an immobilized mixture of vitellogenins Aa and Ab. RT-PCR revealed lrp13 to be predominantly expressed in striped bass ovary, and in situ hybridization detected lrp13 transcripts in the ooplasm of early secondary growth oocytes. Quantitative RT-PCR confirmed peak lrp13 expression in the ovary during early secondary growth. Quantitative mass spectrometry revealed peak Lrp13 protein levels in striped bass ovary during late-vitellogenesis, and immunohistochemistry localized Lrp13 to the oolemma and zona radiata of vitellogenic oocytes. Previously unreported orthologs of lrp13 were identified in genome sequences of fishes, chicken (Gallus gallus), mouse (Mus musculus), and dog (Canis lupus familiaris). Zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus) lrp13 loci are discrete and share genomic synteny. The Lrp13 appears to function as a vitellogenin receptor and may be an important mediator of yolk formation in fishes and other oviparous vertebrates.jlr The presence of lrp13 orthologs in mammals suggests that this lipoprotein receptor is widely distributed among vertebrates, where it may generally play a role in lipoprotein metabolism.
  • Erin L. Damsteegt, Hiroko Mizuta, Yuichi Ozaki, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara, Shigeho Ijiri, Shinji Adachi, P. Mark Lokman
    JOURNAL OF COMPARATIVE PHYSIOLOGY B-BIOCHEMICAL SYSTEMIC AND ENVIRONMENTAL PHYSIOLOGY 184 5 589 - 599 2014年07月 [査読有り][通常論文]
     
    Despite its key role in transportation of triacylglycerides in blood, the distribution, localisation and molecular weight variants of apolipoprotein B (Apob) in teleost fish have essentially escaped study. To address this, a specific short-finned eel (Anguilla australis) Apob antiserum was produced by an immunised rabbit, purified and partially characterised. Localisation of Apob at both the mRNA (in situ hybridisation) and protein (immunohistochemistry) levels mirrored that of mammals; thus immunostaining was confined to the interstitial spaces of the liver and the vascular core of the intestinal villi. Immunostaining of proteins by Western blotting, followed by high-resolution LC-MS, indicated that peptide sequence coverage of Apob in low-density lipoproteins spanned the full-length protein. We conclude that only full-length Apob is produced by eels and that both liver and intestine are key sites for its synthesis.
  • Meiqin Wu, Osamu Nishimiya, Misato Nakamori, Kiyoshi Soyano, Takashi Todo, Akihiko Hara, Naoshi Hiramatsu
    ZOOLOGICAL SCIENCE 31 4 202 - 212 2014年04月 [査読有り][通常論文]
     
    The gene, vitellogenin (vtg) was cloned and characterized in the dojo loach (Misgurnus anguillicaudatus), an indigenous freshwater species in East Asia, in order to develop tools for detecting the effects of estrogenic endocrine-disrupting chemicals (EEDCs). Full-length cDNAs encoding seven distinct vtg transcripts (vtg1-7) were obtained. The corresponding deduced amino acid sequences (Vtg1-7) were divided into two types; type I (Vtg1-6; 89-99% identical), which contained both lipovitellin (Lv) and phosvitin (Pv), and type II (Vtg7), which contained Lv alone. Phylogenetic analysis revealed that the type I and type II Vtgs in the loach could be classified as VtgAo1 and VtgC types, respectively. Immuno-biochemical analyses using type-specific Vtg antisera revealed that VtgAo1 proteins appeared to be the major Vtg type in this species. Males were administered (aqueous exposure) either 17 beta-estradiol (E2) or 17 alpha-ethinylestradiol (EE2), the results from which were used to determine that hepatic vtgAo1 expression was estrogen-sensitive. The precise classification of the loach vtg/Vtg products, as well as their induction profiles following the estrogenic stimulation, provide a basis for their use as sensitive biomarkers when EEDC activities are evaluated in the freshwater environments in East Asia.
  • Osamu Nishimiya, Yasuyuki Kunihiro, Naoshi Hiramatsu, Hiroyuki Inagawa, Takashi Todo, Akihiko Hara
    ZOOLOGICAL SCIENCE 31 4 251 - 257 2014年04月 [査読有り][通常論文]
     
    Vitellogenesis has been extensively studied in oviparous vertebrates, including teleost fishes, while not much is known with regard to jawless hagfishes, modern representatives of the most primitive vertebrate class. This study aimed to characterize vitellogenin (Vtg) and yolk protein (YP) in the inshore hagfish (Eptatretus burgeri) as an initial step to understand vitellogenesis in this species. A putative Vtg fraction was purified from the serum of female hagfish by combinations of hydroxylapatite and ion-exchange chromatography, followed by gel filtration. The purified fraction appeared to contain two distinct Vtgs (Vtg1 and Vtg2) and exhibited biochemical properties resembling those previously reported for teleost Vtgs; these appeared to be female-specific serum proteins and high-molecular-weight proteins in gel filtration (similar to 505 kDa as the mixture fraction of both Vtgs) and in SDS-PAGE (Vtg1 and Vtg2; similar to 210 kDa and similar to 195 kDa, respectively). A major YP was also purified from hagfish eggs by combinations of hydroxylapatite chromatography and gel filtration; the apparent native mass of the purified YP was unusually large (>669 kDa). The purified YP consisted of four polypeptides in SDS-PAGE; the peptide pattern indicated that it consisted of two lipovitellins (Lv1 and Lv2) giving rise to two sets of heavy chains (similar to 116 kDa and similar to 106 kDa, respectively) and two light chains (similar to 32 kDa and similar to 28 kDa, respectively). Additional immunological analysis, N-terminal amino acid sequencing and cDNA cloning firmly confirmed the precursor-product relationship between hagfish Vtgs and Lvs.
  • Wenshu Luo, Yuta Ito, Hiroko Mizuta, Kiyohiro Massaki, Naoshi Hiramatsu, Takashi Todo, Benjamin J. Reading, Craig V. Sullivan, Akihiko Hara
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY 166 2 263 - 271 2013年10月 [査読有り][通常論文]
     
    Teleost fish eggs contain a substantial yolk mass consisting of lipids and proteins that provides essential nutrients for embryonic and larval development. The polar lipid and protein components of the yolk are delivered to oocytes by circulating vitellogenins, however the source(s) of the neutral lipid remains unknown. We cloned a cDNA encoding an orthologue of low-density-lipoprotein receptor (LDLR) from the ovary of cutthroat trout, Oncorhynchus clarki (ct-Ldlr). Predominant expression of ct-ldlr mRNA was observed in the ovary and moderate expression was detected in intestine, gill and brain. The relative abundance of ct-ldlr transcripts was highest in early pre-vitellogenic ovaries and significantly decreased during vitellogenesis, followed by a slight increase during final maturation and in post-ovulatory follicles. In situ hybridization revealed an intense and evenly distributed localization of ct-ldlr transcripts in the ooplasm of pre-vitellogenic oocytes and these signals disappeared in vitellogenic follicles. Collectively, these results suggest that the ldlr is involved in deposition of yolk lipids in cutthroat trout oocytes. The ct-ldlr transcripts also were detected in theca and granulosa cells, suggesting that this receptor may be involved in cholesterol uptake for ovarian steroidogenesis. This is the first report on partial characterization of an ldlr orthologue in any fish species. (C) 2013 Elsevier Inc. All rights reserved.
  • Hiroko Mizuta, Wenshu Luo, Yuta Ito, Yuji Mushirobira, Takashi Todo, Akihiko Hara, Benjamin J Reading, Craig V Sullivan, Naoshi Hiramatsu
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 166 1 81 - 90 2013年09月 [査読有り][通常論文]
     
    A cDNA encoding a vitellogenin receptor with 8 ligand binding repeats (vtgr) was cloned from ovaries of the cutthroat trout, Oncorhynchus clarki. In situ hybridization and quantitative PCR analyses revealed that the main site of vtgr mRNA expression was the oocytes. Expression was strongly detected in perinucleous stage oocytes, gradually decreased as oocytes grew, and became hardly detectable in vitellogenic oocytes. A rabbit antibody (a-Vtgr) was raised against a recombinant Vtgr protein in order to immunologically detect and localize Vtgr within the ovarian follicles. Western blotting using a-Vtgr detected a bold band with an apparent mass of ~95-105kDa in an ovarian preparation that also bound Sakhalin taimen, Hucho perryi, vitellogenin in ligand blots. Immunohistochemistry using a-Vtgr revealed that the Vtgr was uniformly distributed throughout the ooplasm of perinucleolus stage oocytes, subsequently translocated to the periphery of lipid droplet stage oocytes, and became localized to the oolemma during vitellogenesis. We provide the first characterization of Vtgr at both the transcriptional and the translational levels in the cutthroat trout, and our results suggest that this receptor is involved in uptake of Vtg by oocytes of this species.
  • Yuka Morita, Naoshi Hiramatsu, Toshiaki Fujita, Haruna Amano, Etsuko Katsumata, Kazutoshi Arai, Toshihide Iwasaki, Takashi Todo, Akihiko Hara
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 59 4 368 - 377 2013年08月 [査読有り][通常論文]
     
    A single radial immunodiffusion (SRID) assay and a chemiluminescent immunoassay (CLIA) were initially developed for alpha-fetoprotein (AFP) of the striped dolphin. Utilizing these developed assays, we investigated pregnancy-associated changes in the levels of AFP in the sera of fetuses and pregnant females of three dolphin species; samples were either collected from captive individuals or obtained as fishery by-products. The concentrations of AFP in the fetal serum ranged from 419.0 to 2026.3 mu g/ml in the striped dolphin, 12.6 to 1218.7 mu g/ml (for an AFP equivalent; eqAFP) in the common bottlenose dolphin and 770.6 to 3129.1 mu g eqAFP/ml in the Risso's dolphin. AFP levels decreased with increased fetal size in fetuses over 20 cm in length. The concentrations of AFP in sera of pregnant females ranged from 7.18 to 8068.7 ng/ml in the striped dolphin, 6.6 to 1241.1 ng eqAFP/ml in the common bottlenose dolphin and 3.4 to 2868.7 ng eqAFP/ml in the Risso's dolphin. The levels in most pregnant females were equal to or lower than those found in males and nonpregnant individuals, although a few pregnant females exhibited extremely high levels (in the range of hundreds to thousands of nanograms per milliliter). Such high levels of AFP were not observed during pseudopregnancy. To our knowledge, this is the first report on basal profiles for serum AFP levels in small odontocetes. The profiles indicated that AFP may play a significant role during embryonic development, although maternal levels do not appear to be a diagnostic biomarker for monitoring pregnancy.
  • Kodai Yamane, Tomoki Yagai, Osamu Nishimiya, Rieko Sugawara, Haruna Amano, Toshiaki Fujita, Naoshi Hiramatsu, Takashi Todo, Takahiro Matsubara, Akihiko Hara
    FISH PHYSIOLOGY AND BIOCHEMISTRY 39 2 373 - 390 2013年04月 [査読有り][通常論文]
     
    Elasmobranchs (sharks and rays) exhibit unique reproductive characteristics and, in contrast to the situation in teleosts, very little is known about the identity, structure and physical characteristics of their egg yolk proteins. The aims of this study were to (1) detect and purify the vitellogenin (Vtg; egg yolk precursor) and yolk proteins (YPs) of the cloudy catshark (Scyliorhinus torazame), (2) examine the relationships between Vtg and YPs and (3) characterize and classify the deduced primary structure of the Vtg transcript (vtg). The apparent molecular weights of purified Vtg and putative Vtg-related YPs (lipovitellin: Lv, phosvitin: Pv) were determined by gel filtration and were similar to 560, > 669 and similar to 58 kDa, respectively. Following SDS-PAGE, these purified products (i.e., Vtg, Lv and Pv) appeared as bands of similar to 210, similar to 110 and similar to 22 kDa, respectively. On Western blots, antisera against purified Vtg, Lv and Pv recognized the similar to 210 kDa Vtg band. Catshark Pv, in contrast to teleost Pvs, had a very low serine content. The catshark Vtg cDNA sequence (vtg) appeared to contain an open-reading frame consisting of domains encoding Lv, Pv and beta'-component (beta'-c). A phylogenetic analysis, with a consideration of genome duplication events, placed catshark vtg into the 'vtgAB type.' It is concluded that at least a single major type of Vtg protein, which is transcribed and translated from catshark vtgAB gene, is the precursor of three egg yolk proteins (Lv, Pv and beta'-c) in catshark.
  • Yong-Woon Ryu, Ricako Tanaka, Ayumi Kasahara, Yuta Ito, Naoshi Hiramatsu, Takashi Todo, Craig V. Sullivan, Akihiko Hara
    ZOOLOGICAL SCIENCE 30 3 224 - 237 2013年03月 [査読有り][通常論文]
     
    Large amounts of neutral lipids (NLs) are stored as lipid droplets in the ooplasm of fish oocytes, providing an essential energy resource for developing embryos and larvae. However, little is known about the origin of such lipids or about mechanisms underlying their uptake and accumulation in oocytes. We have proposed a model for this lipidation of teleost oocytes, as follows: very low density lipoprotein (Vldl) is metabolized by lipoprotein lipase (Lpl) outside and/or inside of the oocyte and the resulting fatty acids (FAs) are then utilized for de novo biosynthesis of NLs. As a first step toward verification of this model, cDNAs for genes encoding two types of Lpl, lpl1 and lpl2, were cloned from the ovary of cutthroat trout, Oncorhynchus clarki. Examination of Lpl polypeptide sequences deduced from the cDNAs revealed features similar to LPLs/Lpls in other species, including several conserved structural and functional domains. Both types of lpl mRNA were highly expressed in lipid storage tissues (e. g., adipose tissue, muscle, and ovary) and were predominantly expressed in the granulosa cells of ovarian follicles. Ovarian lpl1 mRNA levels showed a remarkable peak in April (early oocyte lipid droplet stage) and then decreased to low values sustained until November (mid-vitellogenesis), after which time a small peak in lpl1 gene expression was observed in December (late vitellogenesis). The mRNA levels of lpl2 also were elevated in April and were highest in June (late lipid droplet stage), but did not show other pronounced changes. These results suggest that, in the cutthroat trout, Vldl is metabolized by the action of Lpls in the granulosa cell layer to generate free FAs for uptake and biosynthesis of neutral lipids by growing oocytes.
  • 莚平裕次, 水田紘子, 羅ウェンシュウ, 盛田祐加, 澤口小有美, 松原孝博, 平松尚志, 東藤孝, 原彰彦
    日本水産学会誌 79 2 175 - 189 2013年03月 [査読有り][通常論文]
     
    Two types of vitellogenin cDNA (vtgAs and vtgC) were cloned from the liver of cutthroat trout. Quantification of hepatic expression levels of vtg transcripts revealed that both vtg mRNA levels showed strong positive correlations with gonad somatic index (GSI). Quantification of serum levels of Vtg proteins revealed that these levels also showed positive, albeit weak, correlations with GSI; however, Vtg levels seemed to be maintained at high but constant levels in individuals with high GSI (i.e., greater than 2). This is the first report on changes associated with ovarian growth in levels of dual vtg/Vtg sub-types within single salmonidae species, providing a basis for a dual vtg/Vtg model in this group of fish.
  • N. Hiramatsu, W. Luo, B. J. Reading, C. V. Sullivan, H. Mizuta, Y. -W. Ryu, O. Nishimiya, T. Todo, A. Hara
    FISH PHYSIOLOGY AND BIOCHEMISTRY 39 1 29 - 32 2013年02月 [査読有り][通常論文]
     
    Recent investigations have revealed multiplicity in maternal yolk precursors and their corresponding ovarian lipoprotein receptors (LRs) in diverse oviparous vertebrates, including fishes. This mini-review describes further evidence for the system of fish egg yolk formation mediated by multiple ovarian LRs, which have been obtained by studies utilizing a combination of conventional molecular and biochemical analyses, and modern proteome and transcriptome technologies. A hypothetical "multiple ovarian LR" model is proposed based on our current and previous knowledge of fish yolk formation.
  • 盛田祐加, 平松尚志, 岩崎俊秀, 東藤孝, 原彰彦
    J Reprod Dev 57 Suppl Japanese Issue J174  2011年08月20日 [査読無し][通常論文]
  • Taku Endo, Takashi Todo, P. Mark Lokman, Hideaki Kudo, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi
    BIOLOGY OF REPRODUCTION 84 4 816 - 825 2011年04月 [査読有り][通常論文]
     
    To investigate the regulation of lipid uptake into the eel oocyte in more detail, effects of 11-ketotestosterone (11-KT) and lipid transporters (lipoproteins) were determined in vitro. Ovarian explants from previtellogenic Japanese eels (Anguilla japonica) were incubated for 28 days with 11-KT and/or with very low density lipoproteins (Vldl), low density lipoproteins (Ldl), or high density lipoproteins (Hdl) purified from eel plasma. The androgen 11-KT induced notable increases in oocyte diameter, which were accompanied by the appearance of vacuoles rather than lipid. Ldl and Hdl increased oocyte diameters, whereas Vldl did not. However, coincubation of 11-KT and Vldl, but not of Ldl or Hdl, resulted in dramatic increases in oocyte size and lipid droplet surface area. Effects of both 11-KT (oocyte size) and Vldl (lipid droplet surface area) were dose-dependent between 1 and 100 ng/ml and between 0.5 and 5 mg/ml, respectively. Interestingly, abnormal oocyte cytology under conditions of coculture with 11-KT and Vldl could essentially be prevented if Vldl concentrations were high enough (>= 5 mg Vldl/ml medium). Unlike 11-KT, estradiol-17beta had no effect on oocyte diameter or lipid droplet surface area. We conclude that Vldl is a key transporter of neutral lipids that accumulate into the eel oocyte during oogenesis and that Vldl-dependent lipid uptake is stimulated by the androgen 11-KT.
  • Yuka Morita, Naoshi Hiramatsu, Toshiaki Fujita, Haruna Amano, Takashi Todo, Akihiko Hara
    ZOOLOGICAL SCIENCE 28 3 215 - 224 2011年03月 [査読有り][通常論文]
     
    Alpha-fetoprotein (AFP) is a fetal glycoprotein that is known as a biomarker for monitoring pregnancy in many mammalian species. However, characterization of AFP has not yet been undertaken in any cetacean species. Here, we purified AFP from the serum of fetal striped dolphin by chemical precipitation followed by a combination of immunoadsorbent column chromatography and gel filtration. The molecular masses of native and denatured dolphin AFP were estimated to be similar to 78,000 Da by gel filtration and similar to 68,000 Da by SDS-PAGE, respectively, representing typical masses reported for mammalian AFPs. In fetal serum, only the AFP band (similar to 68,000 Da) appeared to be immunoreactive to an antiserum against purified dolphin AFP, indicating sufficient specificity for the development of an AFP immunoassay. Full-length cDNA encoding for the dolphin AFP was cloned from fetal liver and revealed an open reading frame comprising 610 amino acid residues, which included a putative signal peptide of 18 amino acid residues. This was followed by a sequence identical to the N-terminus of purified AFP. The deduced amino acid sequence of dolphin AFP showed more than 80% identity to those of other mammalian AFPs. To our knowledge, the present report represents the first identification and characterization of AFP from any cetacean species.
  • 盛田 祐加, 平松 尚志, 岩崎 俊秀, 東藤 孝, 原 彰彦
    日本繁殖生物学会 講演要旨集 104 0 1066 - 1066 日本繁殖生物学会 2011年 [査読有り][通常論文]
     
    【目的】Pregnancy-associated glycoprotein (PAG)は胎盤で発現する妊娠関連タンパク質であり，妊娠母体の血中に出現することから，その検出や動態は数種の陸生哺乳類において妊娠の診断やモニタリング等に利用されている。本研究は鯨類における繁殖生理機構の一端を解明し，得られた知見を繁殖管理へ応用することを目的としてスジイルカPAG cDNAのクローニングを行った。【方法】 試料には和歌山県太地町のイルカ漁にて捕獲されたスジイルカを用いた。妊娠初期から後期のスジイルカ4個体から得た胎盤をプールしRNAを抽出後，cDNAを合成した。他の陸上哺乳類PAGの配列を元に設計したdegenerateプライマーと胎盤cDNAをPCRに供し，得られたPAG様PCR産物の配列を決定した。さらに5'および3'RACE法を用いてPAGのcDNAクローニングを行い，全一次構造の解析を試みた。【結果】スジイルカ胎盤組織より PAG のクローニングを行った結果，6 種類のPAG cDNA (pag1～6) が確認され，それらは397個（pag1,2）および404個（pag3～6）のアミノ酸翻訳領域から構成されていた。同アミノ酸配列は他の陸上哺乳類 pag と高い相同性を示し，本種の PAG をコードしていると考えられた。スジイルカpag配列には3～5個のN-glycosylationサイトおよびアスパラギン酸プロテアーゼファミリーに特徴的なドメインが含まれていた。またアスパラギン酸プロテアーゼ活性に必要なcatalytic モチーフを含んでいたことより，構造的には同活性を持ち得る可能性が示唆された。各pag cDNAの塩基配列および演繹アミノ酸配列は約90%以上の相同性を示したが，系統樹を作成したところ，2つのクラスターに分類された。さらに本種pag配列は同モチーフを有するウマおよびブタpag配列に最も近いクレードを形成した。以上本研究によりスジイルカのPAGが遺伝子レベルで同定され，鯨類で初めて詳細な性状が明らかとなった。
  • Sean L. Divers, H. James McQuillan, Hajime Matsubara, Takashi Todo, P. Mark Lokman
    JOURNAL OF LIPID RESEARCH 51 11 3250 - 3258 2010年11月 [査読有り][通常論文]
     
    To understand the dynamics of lipid uptake into the ovary and the potential role that lipoprotein lipase plays in this event, changes in LPL transcript abundance during oogenesis were measured in both wild-caught and pituitary homogenate-induced artificially maturing eels. Also, the effects of 11-ketotestosterone (11-KT) on LPL mRNA levels were investigated in vivo and in vitro. Normalized ovarian LPL transcript abundance increased as oogenesis advanced, and it rose particularly rapidly during midvitellogenesis, corresponding to pronounced increases in ovarian lipid deposits and LPL activity. Furthermore, LPL mRNA levels were dramatically increased following 11-KT treatment in vivo, findings that were reinforced as trends in ovarian tissue incubated in vitro. Ovarian LPL appears to be directly involved in the uptake of lipids into the eel ovary, an involvement that appears to be controlled, at least in part, by the androgen 11-KT.-Divers, S. L., H. J. McQuillan, H. Matsubara, T. Todo, and P. M. Lokman. Effects of reproductive stage and 11-ketotestosterone on LPL mRNA levels in the ovary of the shortfinned eel. J. Lipid Res. 2010. 51: 3250-3258.
  • Haruna Amano, Machiko Mochizuki, Toshiaki Fujita, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY 157 1 41 - 48 2010年09月 [査読有り][通常論文]
     
    A novel, incomplete-type vitellogenin (VgC) and its derived yolk lipovitellin (LvC) were immunologically detected in female serum and egg extracts, respectively, of Sakhalin taimen (Hucho perryi) using a subtype-specific antiserum against LvC of grey mullet (Mugil cephalus). The taimen VgC was purified from the sera of vitellogenic females by a combination of gel filtration, anion exchange, and immunoadsorbent column chromatography. Gel filtration of the purified VgC revealed that it had an apparent native mass of similar to 380 kDa, while the mass of the VgC polypeptide that appeared following SOS-PAGE was estimated to be similar to 140 kDa. An antiserum was raised against the purified VgC and utilized for the development of a subtype-specific immunoassay for VgC. Levels of VgC in the serum of female taimen increased from 25 mu g/mL to similar to 1 mg/mL, with an increase of GSI. Levels of complete-type Vg and estradiol-17 beta (E-2) in the serum of E-2-administered juvenile taimen increased and reached peak levels similar to those found in vitellogenic females. Although VgC could be induced in the serum of E-2-administered taimen, it stayed at levels (35.5-73 mu g/mL) lower than those obtained in females. This is the first report on the presence of serum VgC and yolk LvC in a salmonid species: these findings indicate that for Sakhalin taimen, like other highly-evolved teleost species, this minor subtype of Vg is significant in the formation of egg yolk. (C) 2010 Elsevier Inc. All rights reserved.
  • Ryota Tosaka, Takashi Todo, Yukinori Kazeto, P. Mark Lokman, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 168 3 424 - 430 2010年09月 [査読有り][通常論文]
     
    In order to elucidate how androgens may mediate their effects on ovarian growth, we investigated the mRNA levels of two subtypes of androgen receptor (ara and arb) in the ovary of feminized Japanese eel (Anguilla japonica) during artificially induced ovarian development by quantitative real-time reverse transcriptase polymerase chain reaction and in situ hybridization. Ara mRNA levels were high from the late oil droplet stage to the late vitellogenic stage, whereas arb mRNA levels were high from the late oil droplet stage to the midvitellogenic stage. Both or mRNAs were predominantly observed in the follicle cells and the epithelial cells of the ovigerous lamellae in all stages. In the oil droplet stage, oogonia exhibited intense signals for or mRNAs. There was no obvious difference in localization pattern between ara and orb in all ovaries examined, irrespective of maturational stage. It was difficult to identify the follicle cell types that were positive for or mRNA during ovarian development. Only in post-ovulatory follicles could theca and granulosa cells be clearly identified, and ar signals were observed in both layers. The predominant localization of ar mRNA in the follicle cells suggests that androgens play important roles in oocyte growth by acting on these cells in this species. We have shown the expression profile and localization of or mRNA during ovarian development for the first time in an oviparous vertebrate. (C) 2010 Elsevier Inc. All rights reserved.
  • Toshiaki Fujita, Alexander P. Scott, Ioanna Katsiadaki, Haruna Amano, Lei Hong, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara
    ZOOLOGICAL SCIENCE 26 12 870 - 877 2009年12月 [査読有り][通常論文]
     
    Egg chorion precursors (zona radiata proteins; Zrps) were purified from the blood plasma of female Atlantic cod (Gadus morhua) by salting-out and column chromatography. The salting-out procedure employed a relatively low (30%) concentration of saturated ammonium sulfate. This was a critical step that separated Zrps from approximately 89% of other plasma proteins. Subsequently, three subtypes of Zrp (Zrp-alpha, -beta and -gamma) were purified by four (Zrps-alpha, -gamma) or five (Zrp-beta) serial column chromatography steps. The intact masses of purified Zrp-alpha, -beta and -gamma. were 290 kDa, 134 kDa, and 73 kDa, while masses estimated by SDS-PAGE were 78 kDa, 54 kDa, and 47 kDa, respectively. Antibodies were prepared against Zrp-beta and -gamma and utilized to develop specific immunoassays. The plasma levels of Zrp-beta and -gamma in reproductive female cod were estimated to be 591.42+/-77.59 mu g/ml and 768.71+/-120.39 mu g/ml, respectively. Thus, practical procedures for the separation of Zrp subtypes were developed in cod, which resulted in the development of subtype-specific Zrp immunoassays in this species; a similar method could be adopted for the separation, detection, and quantification of Zrp subtypes in other teleosts.
  • Haruna Amano, Toshiaki Fujita, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara
    ZOOLOGICAL SCIENCE 26 7 510 - 516 2009年07月 [査読有り][通常論文]
     
    An immunological analysis using subtype-specific antisera of the major yolk protein lipovitellin (Lv) of the grey mullet (Mugil cephalus) confirmed the presence of the three corresponding Lv subtypes (LvA, LvB, and LvC) in vitellogenic ovaries of the marbled sole (Pleuronectes yokohamae). These three Lv subtypes were purified from sole ovaries by using various combinations of anion exchange, hydroxylapatite, immunoadsorbent, and gel-filtration chromatography. Purified LvA, LvB, and LvC had an apparent native mass of similar to 482, similar to 380, and similar to 372 kDa, respectively, estimated by gel filtration. Analysis of their tertiary structures by SDS-PAGE indicated that LvA, LvB, and LvC were typical of teleost Lvs in having a heavy (H) chain (similar to 105, similar to 102, and similar to 107 kDa, respectively) and a light (L) chain (similar to 22, similar to 19.5, and similar to 25 kDa, respectively). The N-terminal amino acid (AA) sequences were obtained for the LvA H chain, the LvB H and L chains, and the LvC L chain and compared to the deduced AA sequences of their precursors, vitellogenins (Vgs), in several species. This comparison of LvA, LvB, and LvC with various teleost VgA, VgB, and VgC sequences, respectively, revealed high identities (60-100%). The purified Lv subtypes were subjected to double immunodiffusion using an antiserum against an unclassified Lv of the sole (Hashimoto et al., 1998); only the LvB subtype exhibited immunoreactivity with this antiserum. This result indicates that the previously developed immunoassay using this anti-Lv for the detection of sole Vg is effectively a VgB-specific assay.
  • 浦 和寛, 田中 恵梨, 東藤 孝, 後藤 孝弘, 清水 幹博, 尾島 孝男, 都木 靖彰
    北海道大学水産科学研究彙報 58 3 21 - 28 北海道大学大学院水産科学研究院 = Research Faculty of Fisheries Sciences, Hokkaido University 2009年02月 [査読無し][通常論文]
     
    We purified a subtilase from digestive system of short spined sea urchin Strongylocentortus intermedius by a combination of ion-exchange chromatography and gel filtration. The molecular weight of purified subtilase on SDS-PAGE under both reducing and nonreducing conditions was 35,000. Antiserum against subtilase specifically immunostained its antigen. By Western blot analysis, immunoreactive with this antibody were observed in stomach and intestine.
  • Lei Hong, Toshiaki Fujita, Tatsunori Wada, Haruna Amano, Naoshi Hiramatsu, Xiumei Zhang, Takashi Todo, Akihiko Hara
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY 149 1 9 - 17 2009年01月 [査読有り][通常論文]
     
    Two vitelline envelope precursors (choriogenin H: Chg H; choriogenin L: Chg L) and an egg yolk precursor (vitellogenin 13: VgB) were purified from red lip mullet. The mass of intact Chg H and Chg L were estimated to be similar to 215 kDa and similar to 69 kDa, respectively. In SDS-PAGE, Chg H and Chg L separated to positions corresponding to similar to 51 kDa and similar to 44 kDa, respectively. The mass of intact VgB was similar to 530 kDa and resolved into a polypeptide of similar to 185 kDa in SDS-PAGE. Specific antisera were raised against each purified protein and specific immunoassays were developed. When Chg H, Chg L and VgB were induced in the serum of immature mullet by injection with various doses of estracliol-17 beta (E-2), VgB exhibited the most sensitive response exhibiting high variation in its induced levels. The variation in induced levels of Chg H and L was relatively minimal although induction required higher doses of E-2 than with VgB. Serum samples obtained from immature mullet populations collected from their natural habitat exhibited similar profiles in the levels of these proteins. The present study suggests that the utilization of multiple biomarkers holds great importance for the reliable and accurate evaluation of estrogenic activity in aquatic environments. (C) 2008 Published by Elsevier Inc.
  • 山野 恵祐, 東藤 孝, 浦 和寛
    水産総合研究センター研究報告 26 129 - 134 水産総合研究センター 2008年12月 [査読無し][通常論文]
     
    近年のウニ類の漁獲量は一万数千トンを推移しており、北海道を中心とした北日本ではエゾバフンウニ、キタムラサキウニ、九州を中心とした西日本ではアカウニが主要な漁獲対象となっている。これらのウニの資源増大を図るため、種苗生産・放流も長年に渡って実施されている。ウニ類の漁業生産はもっぱら漁獲に依っており、一部、養殖が行われているケースもあるが、養殖による生産量は極めて少なく漁業統計には現れない程度に過ぎない。食品としてのウニを見た場合、ウニは水産物の中でも特異な性質を有している。すなわちウニ類では生殖巣（卵巣及び精巣）だけが食される。また食用に適した生殖巣は、栄養物を生殖巣内に十分に蓄積しているが成熟期に入る前の未成熟な状態のものである。このため食品としての品質や歩留まりは成熟状態と密接に関連しており、良質の生殖巣を得られる時期、いわゆる旬は、種毎に限られた季節となる。本課題では、ウニの生殖巣の分化及び生殖巣の発達・成熟に関する研究を実施した。
  • Haruna Amano, Makiko Kitamura, Toshiaki Fujita, Naoshi Hiramatsu, Takashi Todo, Satoshi Suyama, Akihiko Hara
    FISHERIES SCIENCE 74 4 830 - 836 2008年08月 [査読有り][通常論文]
     
    Lipovitellin (Lv), the major yolk protein derived from vitellogenin (Vg), was purified from vitellogenic ovaries of Pacific saury Cololabis saira using hydroxylapatite column chromatography followed by gel filtration. The apparent native mass of purified Lv was approximately 420 kDa, while the tertiary structure of Lv revealed by sodium dodecylsulfate-polyacrylamide gel electrophoresis was typical of teleost Lvs, consisting of a heavy chain (similar to 99 kDa) and a light chain (similar to 34 kDa). Western blot analysis using rabbit antiserum raised against Pacific saury Lv revealed a specific reaction with a polypeptide (similar to 194 kDa) that is present in serum from female Pacific saury but not in male serum, suggesting the approximately 194-kDa polypeptide to be the Vg monomer. This study describes the first step toward the development of specific immunoassays for Pacific saury Vg, which will be an effective tool for monitoring the reproductive development of this species.
  • Takashi Todo, Mana Sato, Misa Ashida, Naohiro Yamaguchi, Yasuhisa Kobayashi, Shinji Adachi, Kohei Yamauchi, Masaru Nakamura
    CYBIUM 32 2 106 - 106 2008年07月 [査読無し][通常論文]
     
    Recently, we succeeded in inducing in vitro spermatogenesis in a protogynous hermaphrodite fish, three-spotted wrasse (Halichoeres trimaculatus), during organ culture of ovary with a serum-supplemented medium. In the present study, we further optimized and used this culture system to determine effective steroid doses to induce sex change, and to investigate the importance of apoptosis during sex change in vitro. The results show that the restructuring from ovary to testis in the wrasse could be induced in vitro with a serum-free medium. Gonadal sex change can be triggered during basic in vitro culture due to a lack of endogenous factors (especially, estrogens) for female sexuality, and can be accelerated by androgens. Furthermore, oocyte apoptosis may be an important mechanism effecting gonadal sex change.
  • Taku Endo, Takashi Todo, P. Mark Lokman, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi
    CYBIUM 32 2 239 - 240 2008年07月 [査読無し][通常論文]
     
    Accumulation of oil droplets in previtellogenic oocytes of Japanese eel, Anguilla japonica, could be induced in vitro. Co-treatment with very low-density lipoprotein (VLDL) and 11-ketotestosterone (11-KT) resulted in significant oil droplet accumulation. It is therefore suggested that lipids in oil droplets originate, at least in part, from VLDL, and that 11-KT plays an important role in their transfer and/or accumulation into oocytes.
  • S Ijiri, N Takei, Y Kazeto, T Todo, S Adachi, K Yamauchi
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 145 1 75 - 83 2006年01月 [査読有り][通常論文]
     
    In this study, we generated and characterized a polyclonal antiserum against eel P450 cholesterol side-chain cleavage (P450scc) using a recombinant protein as the antigen. We examined the localization and abundance of P450scc by immunohistochemistry in Japanese eel testes and ovaries during artificially induced gonadal development. P450scc mRNA localization was also examined by in situ hybridization. In male eels, testicular development was induced by a single injection of human chorionic gonadotropin (HCG). In females, ovarian development was induced by weekly injections of salmon pituitary homogenate (SPH). Before HCG injection, the testis contained germ cells that were primarily type A spermatogonia. Additionally, several clusters of immunoreactive cells for P450scc were localized in the interstitial Leydig cells, but no P450scc mRNA signals were detected. This suggests that P450scc is either a relatively stable protein or it is produced by a mRNA that is present at too low a level to detect. Shortly after a single injection of HCG, expression of P450scc mRNA was stimulated and the number of immunoreactive clusters and their staining intensity were both increased. P450scc mRNA fell to an undetectable level 3 days after hormonal stimulation. Although the P450scc protein also decreased at the same time as the mRNA, it remained at a detectable level throughout this period. P450scc mRNA, but not the P450scc protein, was also detected in the spermatids and spermatozoa. The biological significance of P450scc mRNA expression at this stage is unknown. Prior to experimentation, the ovary contained oocytes that were developed to the oil-droplet stage, with several clusters of immuno reactive cells localized in the thecal layer and ovigerous lamella epithelium. Expression of P450scc, mRNA was also stimulated by SPH injections in the ovary. In contrast to the testis, P450scc mRNA was continuously detected in the thecal cell layer throughout artificially induced maturation, possibly due to a repeated stimulus by the SPH injection every week. Clusters of immunoreactive cells in the thecal cell layer increased in number as ovarian development progressed. This increase ill P450scc mRNA and protein may explain, at least in part, the increase in serum steroid hormones in female eels. The P450scc antiserum clearly immunostained interrenal steroidogenic cells in the head kidney of not only eel but also goldfish, indicating that this antibody could also be used in other teleost species. (c) 2005 Elsevier Inc. All rights reserved.
  • Qu XC, Shin DH, Nagae M, Todo T, Adachi S, Yamauchi K
    Aquaculture Science 54 3 283 - 292 日本水産増殖学会 2006年 [査読有り][通常論文]
     
    ニホンウナギAnguilla japonicaの生殖における甲状腺の役割を調べることを目的として, サケ脳下垂体磨砕物 (SPH) 投与により人為催熟された雌個体の甲状腺活性を測定した。甲状腺活性 (濾胞上皮細胞高) は, SPH投与前 (前卵黄形成期) に高く, 卵黄形成初期 (約8.3μm) でさらに増加し, 卵黄形成後期と核移動期 (約4.2μm) には低下した。甲状腺ホルモンであるチロキシン (T4) およびトリヨードチロニン (T3) の血中量は卵黄形成初期 (T4; 約3.5ng/ml, T3; 約4.1ng/ml) で高く, その後減少 (T4; 約0.4ng/ml, T3; 約0.03ng/ml) し, 甲状腺活性を反映した。以上の結果から, 人為催熟されたニホンウナギにおいては, 甲状腺活性は卵巣発達と負の関係にあることが示唆された。排卵前のニホンウナギにおける血中甲状腺ホルモン量は他魚種における値と比較して低く, 正常値ではないかもしれない。
  • K Nagano, T Kawasaki, S Ijiri, T Todo, S Adachi, K Yamauchi
    FISH PHYSIOLOGY AND BIOCHEMISTRY 28 1-4 437 - 438 2003年 [査読有り][通常論文]
     
    The effects of changes in water quality in a recirculating system on brain corticotropin-releasing hormone (CRH) gene expression in goldfish were examined. In the experimental group, fish were reared without water change, while in the control group, 50% of water was changed everyday. In the experimental group, total ammonia-nitrogen, nitrate and phosphate were increased over the experimental period. Brain CRH mRNA levels in the control group seemed higher than those in the experimental group, but differences were confounded by variation among individuals. There were no differences in gonadal development between both groups.
  • M Matsubara, PM Lokman, A Senaha, Y Kazeto, S Ijiri, A Kambegawa, T Hirai, G Young, T Todo, S Adachi, K Yamauchi
    FISH PHYSIOLOGY AND BIOCHEMISTRY 28 1-4 353 - 354 2003年 [査読有り][通常論文]
     
    The control of 11-ketotestosterone (11-KT) biosynthesis and its physiological roles were examined in female Japanese eel (Anguilla japonica) and New Zealand longfinned eel (Anguilla dieffenbachii). 11-KT was detected in serum of female eels of both species. Among various tissues from Japanese eel, the ovary had the greatest capacity to synthesize 11-KT in vitro. In addition, the oocyte diameters of eels treated with 11-KT had increased significantly. Furthermore, these oocytes were found to have an increased number of oil droplets. These findings suggest that 11-KT in female eels may be mostly of ovarian origin and that this androgen appears to play an important role in controlling pre-vitellogenic oocyte growth.
  • T Ikeuchi, T Kobayashi, T Todo, Y Nagahama
    FISH PHYSIOLOGY AND BIOCHEMISTRY 28 1-4 151 - 151 2003年 [査読有り][通常論文]
     
    Transgenic cell lines which stably express progestogen receptors (PRs) and the PR-responsive reporter genes were developed. They are a good system for rapid, sensitive and reproducible screening of various ligands.
  • H Asanuma, H Ohashi, H Matsubara, S Ijiri, T Matsubara, T Todo, S Adachi, K Yamauchi
    FISH PHYSIOLOGY AND BIOCHEMISTRY 28 1-4 383 - 384 2003年 [査読有り][通常論文]
     
    Isolated hepatocytes from male Japanese eel at various stages of human chorionic gonadotropin (HCG)-induced spermatogenesis were cultured in the presence of estradiol-17beta (E2). Hepatic vitellogenin (VTG) production in response to E2 increased during testicular development. This indicates that VTG production is influenced by gonadal maturity not only in females but also in males, where serum levels of 11-ketotestosterone (11KT) increase during testicular development. Recently, it has been confirmed that significant levels of 11KT are detected in maturing female eel. Therefore, in a next experiment, effect of 11KT on E2-induced VTG production was examined in vitro. As a result, in both male and female hepatocytes, 11KT enhanced E2-induced VTG production, although 11KT alone failed to induce VTG production.
  • M Kusakabe, T Kobayashi, T Todo, PM Lokman, Y Nagahama, G Young
    MOLECULAR REPRODUCTION AND DEVELOPMENT 62 4 456 - 469 2002年08月 [査読有り][通常論文]
     
    Cytochrome P450 1beta-hydroxylase (P450(11beta)) is an important steroidogenic enzyme for glucocorticoid and mineralocorticoid production in vertebrates. In teleosts, P450(11beta) also plays a role in the production of 11-ketotestosterone (11-KT), the predominant androgen in male fish. In this study, we cloned a cDNA encoding rainbow trout (Oncorhynchus mykiss) testicular P450(11beta). The cDNA contains 1,740 nucleotides that encode a protein of 551 amino acids which shares 65.2% homology with testicular P450(11beta) from Japanese eel, and 33-45% homology with adrenal P450(11beta) from rat, human, and frog. HEK293 cells transfected with an expression vector containing the rainbow trout P450(11beta) cDNA open reading frame showed 11beta-hydroxylating activity in the presence of exogenous testosterone. Analysis of tissue distribution by RT-PCR showed great abundance of P450(11beta) mRNA in testis and head kidney. In order to clarify the sites of P450(11beta) gene expression, cRNA in situ hybridization analysis was performed. Hybridization signals were detected in Leydig cells and head kidney inter-renal cells. The results of Northern blot analysis indicated a single 1.8-kb transcript encoding P450(11beta) in testis and in head kidney, suggesting that the testicular form of P450(11beta) may also be involved in cortisol production by inter-renal cells. Seasonal changes in total P450(11beta) mRNA levels in testes during spermatogenesis showed a pattern similar to that of plasma androgens. These results suggest that androgen production in male rainbow trout is partially regulated by changes in abundance of P450(11beta) mRNA. (C) 2002 Wiley-Liss, Inc.
  • M Kusakabe, T Todo, HJ McQuillan, FW Goetz, G Young
    ENDOCRINOLOGY 143 6 2062 - 2070 2002年06月 [査読有り][通常論文]
     
    Complementary DNA-encoding proteins with high homology to steroidogenic acute regulatory proteins (StAR) of mammals were cloned from rainbow trout head kidney and a mixture of several brook trout tissues. A cDNA encoding an MLN64 homolog was also cloned from brook trout. The C-terminal domains of rainbow trout StAR and brook trout StAR were very highly conserved compared with StAR of mammals. In rainbow trout, Northern and RT-PCR analyses showed abundant StAR transcripts in head kidney, ovary, and testis, and weaker signals were found in intestine, pyloric caeca, spleen, and kidney. Brief acute stress resulted in elevated plasma cortisol levels and a 2-fold increase in rainbow trout StAR transcripts in head kidneys sampled 3 h after exposure to the stressor. In brook trout, StAR transcripts were detected only in known steroidogenic tissues. Ovarian brook trout StAR mRNA was not seen until the onset of final maturation. Its abundance increased during germinal vesicle breakdown, peaked during and just following ovulation, and decreased by 2 wk post ovulation. Brook trout MLN64 transcripts were found in all tissues tested, and transcript abundance in ovarian samples did not vary during final oocyte maturation and ovulation. Both StAR structure and function appear to be highly conserved throughout the vertebrates.
  • M Tanaka, S Nakajin, D Kobayashi, S Fukada, GJ Guan, T Todo, B Senthilkumaran, Y Nagahama
    BIOLOGY OF REPRODUCTION 66 5 1498 - 1504 2002年05月 [査読有り][通常論文]
     
    17alpha,20beta-Dihydroxy-4-pregnen-3-one is the major oocyte maturation-inducing hormone of several teleost species. Gonadotropin-induced increase in ovarian 20beta-hydroxysteroid dehydrogenase activity is essential for the synthesis of maturation-inducing hormone. Cloning and expression studies suggest that ayu (Plecoglossus altivelis) ovarian carbonyl reductase can function as 20beta-hydroxysteroid dehydrogenase. The amino acid sequence deduced from the isolated cDNA had 276 amino acid residues and shared approximately 60% homology with mammalian and teleostean carbonyl reductases. The sequence data search showed that the ayu cDNA clone belongs to the short-chain dehydrogenase/reductase family. The clear lysate prepared from Escherichia coli harboring the cDNA catalyzed the production of maturation-inducing hormone. Its identification was confirmed by two-dimensional, thin-layer chromatography followed by recrystallization. Purification of the E. coli-expressed cDNA product revealed that it possessed both carbonyl reductase and steroid dehydrogenase activities, and 17alpha-hydroxyprogesterone, the endogenous immediate precursor of maturation-inducing hormone, was one of the preferred substrates. Furthermore, Northern blot analysis denoted that the transcripts are present both in fully grown, immature ovarian follicles and at higher levels in mature ovarian follicles. These results demonstrate that the carbonyl reductase of ayu ovary is involved in the production of maturation-inducing hormone, and they provide evidence for a novel physiological role of this enzyme in the final maturation of oocytes. Based on its functional properties, the enzyme can be referred to as carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase.
  • H Okumura, T Todo, S Adachi, K Yamauchi
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 125 1 9 - 16 2002年01月 [査読有り][通常論文]
     
    The induction of vitellogenesis is a complex process requiring coordinated control and expression of many hepatic gene products, such as vitellogenin (VTG). To investigate the regulation of VTG synthesis, knowledge of the molecular genetics of VTG is required. Here, the authors have isolated a partial cDNA encoding Japanese eel VTG using an immunoscreening technique. This cDNA contained an open reading frame of 1629 bp, predicted to encode 543 amino acid residues, the sequence of which showed high homology with the VTG of other fishes. Northern blot analysis yielded a VTG transcript of approximately 5.8 kb from eel hepatic tissue. Experimentally, VTG synthesis could be induced by treatment with salmon pituitary homogenate. The levels of VTG mRNA in the liver during the artificial maturation of female Japanese eels correspond well to levels of E-2 and VTG in the serum. (C) 2002 Elsevier Science (USA).
  • T Ikeuchi, T Todo, T Kobayashi, Y Nagahama
    FEBS LETTERS 510 1-2 77 - 82 2002年01月 [査読有り][通常論文]
     
    A cDNA encoding a second type of a progestogen receptor (ePR2) was isolated from the same library as we had previously cloned a functional PR (ePR1) in eel testis. The amino acid sequence of the ePR2 shows low homology with ePR1 (34%), but both PRs showed progestogen-dependent transactivation in transfection experiment. Tissue distribution of ePR2 mRNA was clearly different from that of ePR1. Protein interaction between two PRs was demonstrated in vitro by a glutathione S-transferase pull-down assay. These results indicate that ePR2 is also a functional PR. This is the first isolation of two different functional PR molecules from a vertebrate. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
  • T Ikeuchi, T Todo, T Kobayashi, Y Nagahama
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 129 2-3 449 - 455 2001年06月 [査読有り][通常論文]
     
    Two subtypes (a and P) of androgen (AR) and progestogen receptors (PR) are present in the testis of Japanese eel (Anguilla japonica). Amino acid homology of the open reading frames between alpha and beta in AR or PR is approximately 40%, but the DNA- and ligand-binding domains show high homology between subtypes. Judging from these structures, alpha and beta are not isoforms derived from translational initiation at two in-phase ATG codons, alternative splicing, or tetraploidy. In transient transfection assays using a reporter construct containing a steroid-responsive promoter, each subtype showed its corresponding hormone-dependent transactivation. The ligand affinity for transactivation between AR and PR subtypes was similar for physiological ligands. Tissue distribution of both subtype mRNAs was different. Protein interaction between subtypes was demonstrated in vitro by GST pull-down assays. These results clearly indicate that two functional subtypes of AR and PR exist in eel. These findings will advance our understanding of the mechanisms underlying sex steroid signaling. (C) 2001 Elsevier Science Inc. All rights reserved.
  • Y Kazeto, S Ijiri, T Todo, S Adachi, K Yamauchi
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 118 1 123 - 133 2000年04月 [査読有り][通常論文]
     
    As a first step in investigating the mechanism underlying the steroidogenic shift from the production of ovarian androgens (vitellogenic stage) to that of 17 alpha-hydroxylated progestins (maturational stage) in Japanese eel during induced oogenesis, a cDNA encoding Japanese eel (Anguilla japonica) ovarian P450c17 (CYP17: steroid 17 alpha-hydroxylase/C17-20 lyase) was cloned and sequenced. This cDNA contained the complete coding region representing 510 amino acid residues, which showed high sequence homology to those of rainbow trout (74%) and mammals (45-55%). The protein encoded by this cDNA possessed high enzymatic activities of 17 alpha-hydroxylase and C17-20 lyase, thus quickly converting pregnenolone and progesterone to their respective delta(4) and delta(5) C19 products. P450c17 produced a single transcript of 2.4 kb in length, as assessed by Northern blot. Transcript levels of this enzyme significantly increased throughout artificially induced ovarian development. Considering this together with the previous data showing that C17-20 lyase activity decreased from the vitellogenic to the maturational stage, whereas 17 alpha-hydroxylase activity increased, the present data suggest that changes in C17-20 lyase activity (the production of androgens) do not depend on transcriptional changes of the P450c17gene. (C) 2000 Academic Press.
  • GJ Guan, T Todo, M Tanaka, G Young, Y Nagahama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 97 7 3079 - 3083 2000年03月 [査読有り][通常論文]
     
    Carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/ 20 beta-HSD) is an enzyme that converts 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (the maturation-inducing hormone of salmonid fish). We have previously isolated two types of CR/20 beta-HSD cDNAs from ovarian follicle of rainbow trout (Oncorhynchus mykiss), Recombinant proteins produced by expression in Escherichia coli in vitro showed that one (type A) had CR and 20 beta-HSD activity but that the other (type B) did not. Among the three distinct residues between the protein products encoded by the two cDNAs, two residues (positions 15 and 27) are located in the N-terminal Rossmann fold, the coenzyme binding site. To investigate the structure/function relationships of CR/20 beta-HSDs, we generated mutants by site-directed mutagenesis at the following positions: MutA/l15T, MutB/T15I, and MutB/Q27K. Enzyme activity of wild-type A was abolished by substitution of Ile-15 by Thr (MutA/I15T). Conversely, enzyme activity was acquired by the replacement of Thr-15 with lie in type B (MutB/T15I), MutB/T15I mutant showed properties similar to the wild-type A in every aspect tested, Mutation MutB/Q27K had only partial enzyme activity, indicating that Ile-15 plays an important role in enzyme binding of cofactor NADPH.
  • 東藤 孝, 池内 俊貴, 大場 裕一
    海洋 32 2 95 - 101 海洋出版 2000年02月 [査読無し][通常論文]
  • T Todo, T Ikeuchi, T Kobayashi, H Kajiura-Kobayashi, K Suzuki, M Yoshikuni, K Yamauchi, Y Nagahama
    FEBS LETTERS 465 1 12 - 17 2000年01月 [査読有り][通常論文]
     
    A cDNA encoding a nuclear 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP, spermiation-inducing hormone in fish) receptor (DPR) aas, for the first time, isolated from an eel testis cDNA library. The amino acid sequence of DPR shows high homology with those of human and chicken progesterone receptors, The affinity of the bacterial recombinant DPR ligand binding domain protein for 17 alpha,20 beta-DP is higher than that of progesterone. In transfection experiments using COS7 cells, the DPR showed progestin-dependent activation of transcription. 17 alpha,20 beta-DP was the most effective activator of transcription. These results indicate that the cDNA encodes a functional eel DPR, and show that 17 alpha,20 beta-DP has a nuclear receptor-mediated action in eel testes. (C) 2000 Federation of European Biochemical Societies.
  • XT Change
    ZOOLOGICAL SCIENCE 16 5 853 - 853 1999年10月 [査読有り][通常論文]
  • T Miura, C Miura, T Ohta, MR Nader, T Todo, K Yamauchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 264 1 230 - 234 1999年10月 [査読有り][通常論文]
     
    In this work, we examined the functions of the female hormone "estrogen" on spermatogenesis of the Japanese eel (Anguilla japonica). Estradiol-17 beta (E-2), a natural estrogen in vertebrates, was present in the serum and its receptor was expressed in the testis during the whole process of spermatogenesis, Spermatogonial stem cell renewal was promoted by E-2 implantation but was suppressed by tamoxifen (an antagonist of estrogen), In vitro, 10 pg/ml of E-2 was sufficient to induce spermatogonial stem cell division in cultured testicular tissue, therefore confirming the in vivo observations. These results clearly show that estrogen is an indispensable "male hormone" in the early spermatogenetic cycle. (C) 1999 Academic Press.
  • T Ikeuchi, T Todo, T Kobayashi, Y Nagahama
    JOURNAL OF BIOLOGICAL CHEMISTRY 274 36 25205 - 25209 1999年09月 [査読有り][通常論文]
     
    There has been general acceptance that only one type of androgen receptor (AR) exists in an individual. This contrasts with other members of the nuclear receptor superfamily where multiple forms have been reported (e.g. estrogen receptor alpha/beta, thyroid hormone receptor alpha/beta, etc.). We have previously identified 11-ketotestosterone (a potent androgen in teleosts) as the spermatogenesis-inducing hormone of the Japanese eel and have cloned its receptor (eAR1) cDNA from eel testis. Here we report on the cloning of a cDNA encoding a second type of AR (eAR2) from the eel testis and the functional characterization of the encoded protein. This cDNA contains a complete open reading frame encoding 797 amino acid residues. The amino acid sequence of eAR2 shows high homology with other ARs, including eAR1, in the DNA-binding (98-88%) and ligand-binding (59-85%) domains, whereas the other domains show low homology (<35%). In transient transfection assays of mammalian cells, the eAR2 protein displayed androgen-dependent activation of transcription from the androgen-responsive murine mammary tumor virus promoter. Tissue distribution of its mRNA was different from that of eAR1, We conclude that eAR2 is a novel AR in the eel, which we suggest should be named eel ARP to distinguish it from eAR1 (eAR alpha).
  • XT Chang, T Kobayashi, T Todo, T Ikeuchi, M Yoshiura, H Kajiura-Kobayashi, C Morrey, Y Nagahama
    ZOOLOGICAL SCIENCE 16 4 653 - 658 1999年08月 [査読有り][通常論文]
     
    Estrogen receptors (ER) in mammals have recently been shown to be encoded by two distinct genes, ER alpha and ER beta. In this study, cDNAs encoding two tilapia ER subtypes, tER5.1 and tER4.3, were cloned from an ovarian cDNA library of a teleost fish, the tilapia Oreochromis niloticus. The tER5.1 and tER4.3 contain complete open reading frames encoding 585 and 557 amino acid residues, respectively. The two receptors share about 12% homology in the A/B domain, 96% in the DNA binding domain (C domain), 12% in the D domain, 57% in the ligand binding domain (E damain), and 20% in the F domain. Phylogenetic analysis of ER proteins from various vertebrate species indicated that vertebrate ERs consist of two major groups (ER alpha and ER beta); tER5.1 and tER4.3 belong to ER alpha and ER beta subtypes, respectively. Thus, we consider tER5.1 and tER4.3 to be the tilapia homologs of ER alpha (tER alpha) and ER beta (tER beta), respectively. In transient transfection assays using mammalian COS-7 cells, both tER alpha and tER beta showed estradiol-17 beta dependent activation of transcription from the estrogen-responsive ERE-Luc promoter. This is the first report of the presence of ER alpha and ER beta within a single non-mammalian vertebrate species.
  • GJ Guan, M Tanaka, T Todo, G Young, M Yoshikuni, Y Nagahama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 255 1 123 - 128 1999年02月 [査読有り][通常論文]
     
    In salmonid fish, 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) is a key enzyme involved in the production of oocyte maturation-inducing hormone (MIH), 17 alpha,20 beta-dihydroxy-4-pregnen-3-one. Here we report the isolation of two cDNAs which encode proteins with high homology to carbonyl reductase-like BOP-HSD (CR/20 beta-HSD) from rainbow trout (Oncorhynchus mykiss) ovarian follicles. Genomic DNA analysis showed that the two CR/20 beta-HSD cDNAs are derived from two different genes. Northern blot and RT PCR analysis demonstrated that trout CR/20 beta-HSDs are broadly expressed in various tissues. Enzymatic characterization using recombinant CR/20 beta-HSD proteins produced in E. coli showed that the product of one of the two cDNAs had both SOP-HSD and CR activity, but the other had neither activity. Although the functional significance of the two genes remains unresolved, these results clearly demonstrate the presence of two distinct CR/20 beta-HSD transcripts in the trout ovary. (C) 1999 Academic Press.
  • T Todo, T Ikeuchi, T Kobayashi, Y Nagahama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 254 2 378 - 383 1999年01月 [査読有り][通常論文]
     
    We have previously identified 11-ketotestosterone (11KT, a portent androgen in teleosts) as the spermatogenesis-inducing hormone of the Japanese eel. In this study, a cDNA encoding the eel androgen receptor (AR) was isolated from the Japanese eel testis, This cDNA contains a complete open reading frame encoding 848 amino residues. The amino acid sequence of the eel AR shows high homology with other ARs. In transient transfection assays of mammalian cells, the eel AR showed androgen-dependent activation of transcription from the androgen-responsive MMTV promoter. Of the endogenous androgens found in the Japanese eel, 11KT was the most effective activator of transcription. These results indicate that the cloned cDNA encodes a functional eel AR, and its major native ligand is 11KT. This is the first isolation of an AR molecule from fish, and is the first evidence that 11KT acts via a nuclear receptor. (C) 1999 Academic Press.
  • Nakamura, I, T Todo, M Nagae, Y Kazeto, S Adachi, K Yamauchi
    ZOOLOGICAL SCIENCE 15 6 923 - 930 1998年12月 [査読有り][通常論文]
     
    A sensitive quantitation system using reverse transcription-polymerase chain reaction (RT-PCR) was developed to measure the low estrogen receptor (ER) mRNA levels in Japanese eel (Anguilla japonica). Two types of cytoskeletal actins, beta- and gamma-actins, wee distinguished in Japanese eel and used as the internal control for exact quantitation. Actin and ER primers of this study annealed to different exons, allowing for the skipping of DNase treatment. Accordingly, ER and actin RT-PCR products showed a single band and were amplified with the same efficiency during PCR. The ER mRNA amount was calculated as a relative value, normalized over actin (beta and gamma) mRNA. The results thus obtained by RT-PCR agreed with the results from Northern blot analysis of liver from pre-vitellogenic and hormone-treated early vitellogenic eel. Using this system, the ER mRNA levels were further measured in coelomic epithelium, pituitary, brain and ovary. In the liver, ER mRNA levels of the early vitellogenic eel increased by about 2.7 folds compared to those of the immature eel. in contrast, changes in levels of ER transcripts were not observed in other tissues. This system can be used to detect relative ER mRNA levels around 100-fold lower than those of actin mRNA in all tissues in which it has been difficult to measure ER mRNA by Northern blot analysis.
  • 東藤 孝, 長浜 嘉孝
    海洋 30 2 96 - 102 海洋出版 1998年02月 [査読無し][通常論文]
  • Nakamura, I, M Nagae, T Todo, S Adachi, K Yamauchi
    ADVANCES IN COMPARATIVE ENDOCRINOLOGY, TOMES 1 AND 2 1375 - 1379 1997年 [査読有り][通常論文]
     
    A sensitive quantitation system by reverse transcription polymerase chain reaction (RT-PCR) was developed to measure the lon estrogen receptor (ER) mRNA levels in Japanese eel (Auguilla Japonica). This system was able to detect the relative expression of ER mRNA in all tissues that have been hard to measure ER mRNA by Northern blot. The level of the ER gene expression in the liver er increased at early vitellogenic and migratory nucleus stages. In contract, ER gene expression gradually decreased in the coelomic epithelium. In the pituitary, the basal ER gene expression was relatively high but obvious changes were not observed. In the brain and ovary, changes in gene expression levels of ER were observed. The results of RT-PCR analysis in various tissues suggested a tissue specific regulation of ER.
  • Z Yao, XT Chang, T Hirai, T Todo, M Yoshikuni, T Kobayashi, Y Nagahama
    ADVANCES IN COMPARATIVE ENDOCRINOLOGY, TOMES 1 AND 2 853 - 856 1997年 [査読有り][通常論文]
     
    A cDNA clone encoding a GTH receptor was isolated from the fish tilapia (Oreochromis niloticus). This clone is considered a fish GTH receptor clone based on the evidence of the homology comparison of the deduced amino acid sequence to its mammalian counterparts, the Northern blot analysis and the receptor-ligand binding studies on the membrane preparation of COS7 cells transfected by the isolated cDNA.
  • G Young, T Todo, T Kobayashi, G Guan, Y Nagahama
    ADVANCES IN COMPARATIVE ENDOCRINOLOGY, TOMES 1 AND 2 1443 - 1449 1997年 [査読有り][通常論文]
     
    Two models have been proposed for estradiol-17 beta (E2) and 17, 20 beta-dihydroxy-4-pregnen-3-one (DHP) production, with the thecal layer providing precursor steroids to the granulosa layer. cDNAs encoding cholesterol side-chain cleavage enzyme (P450(SCC)), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17-hydroxyrase/C17-C20 lyase (P450(c17)), and aromatase (P450(arom)) have been cloned, antisera against predicted proteins produced and used to study gene expression during the ovarian cycle. P450(arom) gene expression was restricted to granulosa cells, and peaked during vitellogenesis and then declined. 3 beta-HSD, P450(c17), and P450(SCC) gene expression in thecal cells was relatively low during vitellogenesis but subsequently increased. The 3 beta-HSD gene was also expressed in granulosa cells during vitellogenesis. Gonadotropin and forskolin increased 3 beta-HSD gene expression, and also induced immunoreactive P450(SCC) in theca and granulosa cells.
  • T Todo, S Adachi, K Yamauchi
    MOLECULAR AND CELLULAR ENDOCRINOLOGY 119 1 37 - 45 1996年05月 [査読有り][通常論文]
     
    A cDNA encoding the Japanese eel, Anguilla japonica. estrogen receptor (ER) was isolated and sequenced. This cDNA contains a complete open reading frame encoding 573 amino acid residues, and the molecular weight of this protein is calculated to be 63417. The amino acid sequence of the eel ER shows high homology of the DNA binding (80%) and ligand binding (55%,) domains with those of other species. However, the other domains show greatly reduced homology (10-20%). When the cDNA was ligated to the pSVL vector and transfected into COS7 cells, a protein was produced that had high affinity for estradiol-17 beta (E(2)) and specifically bound estrogens. Northern analysis showed that three ER mRNAs with lengths of 5.6, 3.8 and 1.2 kb were expressed in eel liver. Their expression was E(2) inducible, with the 5.6 kb mRNA being strongly dependent on E(2) stimulation.
  • Molecular cloning and sequence analysis of the cDNAs encoding the glycoprotein hormone α - and gonadotropin IⅠβ -subunits, and their expression in the pituitary of the Japanese eel,Angullia joponica
    Nagae.M, Todo.T, Gen.K, Kato.Y, Young.G, Adachi.S, Yamauchi.K
    J. Mol. Endocrinol 16 171-181  1996年 [査読無し][通常論文]
  • M. Nagae, T. Todo, K. Gen, Y. Kato, G. Young, S. Adachi, K. Yamauchi
    Journal of Molecular Endocrinology 16 2 171 - 181 1996年 [査読無し][通常論文]
     
    cDNAs encoding the glycoprotein hormone α- and gonadotropin (GTH) IIβ-subunits of Japanese eel (Anguilla japonica) pituitary were cloned using the polymerase chain reaction. The nucleotide sequence of the glycoprotein hormone α-subunit cDNA was 364 base pairs (bp) long, encoding 117 amino acids, and that of the GTH IIβ-subunit cDNA was 433 bp long, encoding 140 amino acids. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts, indicating that the structure of GTH subunits has been conserved during the evolution of teleosts. Changes in the expression of these subunit genes during ovarian development induced artificially by the injection of chum salmon pituitary homogenate were examined using Northern blot analysis. Glycoprotein hormone α-subunit mRNA increased almost linearly during ovarian development, whereas GTH IIβ-subunit mRNA was detected only at the late vitellogenic and migratory nucleus stages. These data indicate that eel GTH II is synthesized mainly at the late vitellogenic and migratory nucleus stages, and suggest that GTH II plays an important role in final oocyte maturation of Japanese eel. Changes in the expression of glycoprotein hormone α- and GTH IIβ-subunits mRNA correlate with the serum estradiol-17β (E2) and testosterone profile during ovarian development. The increase in mRNA of both subunits is probably due to positive feedback of E2and testosterone produced by ovarian follicles in response to the GTH contained in chum salmon pituitary homogenate.
  • T Todo, S Adachi, F Saeki, K Yamauchi
    ZOOLOGICAL SCIENCE 12 6 789 - 794 1995年12月 [査読有り][通常論文]
     
    Estrogen receptors were identified in cytosolic and nuclear extracts of livers of female Japanese eel, Anguilla japonica. A single class of high affinity binding sites was found, with a Kd=0.97 nM for the cytosolic estrogen receptor (cER) and Kd=0.85 nM for the nuclear estrogen receptor (nER). Binding of both the cER and the nER was specific for estrogens (diethylstilbestrol: DES >estradiol-17 beta E(2) > > estriol: E(3) >estrone: E(1)). These binding character istics of ERs were quite different from those of the serum estrogen-binding component; [H-3]E(2) binding to serum was not saturable, and was displaced by testosterone but not by DES, E(1) or E(3). The relationships between the levels of hepatic ERs, circulating E(2) and vitellogenin (VTG) during artificial maturation of cultivated female eels were examined, using eels injected weekly with chum salmon pituitary homogenate at a dose of 20 mu g/g-body weight. Serum E(2) levels were constantly low during pre- to midvitellogenesis, and dramatically increased in the migratory nucleus stage. However, VTG levels gradually increased from early to midvitellogenesis, and were greatly elevated in the migratory nucleus stage. Hepatic cER levels slightly increased in early vitellogenesis, and then increased significantly from midvitellogenesis to the migratory nucleus stage. In contrast, nER levels did not change significantly, although nER levels in the migratory nucleus stage were higher than those at other stages. The changes in cER levels represent increased hepatic responsiveness to estrogenic stimuli during artificial maturation. Lack of change in nER levels may be a feature of artificial maturation compared to sexual maturation in nature.
  • T Todo, S Adachi, K Yamauchi
    AQUACULTURE 135 1-3 77 - 77 1995年10月 [査読有り][通常論文]
  • H OKUMURA, A HARA, F SAEKI, T TODO, S ADACHI, K YAMAUCHI
    FISHERIES SCIENCE 61 2 283 - 289 1995年04月 [査読有り][通常論文]
     
    A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of low levels of serum vitellogenin (VTG) of the Japanese eel Anguilla japonica. The non-competitive avidin-biotm interaction was performed by a sandwich method using the specificity of the specific antiserum to eel yolk protein (a-lipovitellin) and biotinylated F(ab')(2) fragment of a-lipovitellin. The assay could be run in 2 days and routinely detect VTG in a concentration as low as 0.8 ng/ml. The appearance of low levels of serum VTG significantly increased 9-12 h after a single injection of estradiol-17 beta into immature fish. The development of an ELISA for VTG made possible the quantification of serum VTG and thereby in vitro analysis of the mechanism of the onset and course of vitellogenesis.

MISC

書籍等出版物

  • 魚類のDNA 分子遺伝学的アプローチ
    青木宙, 隆島史夫, 平野哲也 (担当:分担執筆範囲:17. 配偶子形成に関わる遺伝子)
    恒星社厚生閣 1997年06月

講演・口頭発表等

担当経験のある科目（授業）

  • 系統動物学新潟大学
  • 水産増殖学北海道大学
  • 比較生理学北海道大学
  • 水族生化学北海道大学

所属学協会

  • 日本比較内分泌学会   環境ホルモン学会 [正式名称;日本内分泌撹乱化学物質学会]   日本動物学会   日本水産学会   

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 東藤 孝, 平松 尚志
     
    本研究は、魚類の卵母細胞における広義の卵黄物質、すなわち中性脂質（油球）と卵黄タンパク質（卵黄球）の蓄積機構について、それらに関わる重要な分子群の機能を最先端のゲノム編集技術を用いることにより解析し、明らかにすることを目的としている。メダカを研究モデルとし、「課題1：油球形成機構の解析」と「課題2：卵黄球形成機構の解析」の2課題を設定して研究を進めている。 課題1：先ず、油球を構成する中性脂質の主要な供給源である超低密度リポタンパク質の代謝酵素（リポタンパクリパーゼ：Lpl）に着目し、メダカ卵濾胞組織におけるlpl遺伝子の発現を解析した。その結果、2種類のlpl遺伝子（lpl1とlpl2）のうち、lpl1のみが発現していることが示されたことから、lpl1の遺伝子欠損（KO）メダカを作出することとした。CRISPR/Cas9システムによる効率的な変異導入を達成するため、lpl１遺伝子に対する複数のガイドRNA（gRNA）候補を設計して検討した結果、フレームシフトを伴う変異導入に有効なgRNAの組み合わせが見出された。 課題2：卵黄タンパク前駆物質（ビテロジェニン：Vtg）の卵内への取り込みに必要な3種のリポタンパク質受容体（Lr7、Lr8、Lrp13）のそれぞれについて、メダカ卵濾胞での発現解析ならびに、KO魚の作出とそれらの表現型解析を進めている。これまでに、Lr8のKOメダカ系統（lr8-KO）の作出に成功し、その表現型解析を行った。その結果、lr8-KO系統メダカの孵化仔魚の生残率が野生型のそれよりも低下することが示された。さらにlr8-KO系統メダカにおいては、4種のVtg（VtgAa1、VtgAa2、VtgAb、VtgC）のうち、VtgAb由来の卵黄タンパク質成分が野生型と比べて相対的に減少しており、Lr8がVtgAbタイプの主要な受容体であることが明らかとなった。
  • 魚卵を標的とした物質輸送システムの開発：バイオリアクターによる輸送体の大量生 産
    文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2018年 -2022年 
    代表者 : 平松尚志
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 都木 靖彰, 浦 和寛, 東藤 孝
     
    ウニ生殖巣の肥大化メカニズムを研究するツールとして，生殖巣器官培養系を確立し，これを用いて生殖巣の肥大に重要な主要卵黄タンパク質（MYP）の合成を誘起する因子を探索した。仮説としてウニ体腔液中の因子と核内受容体に結合する脂質を設定し，ウニ生殖巣が肥大した時期の体腔液および肥大したウニ生殖巣から抽出した総脂質を添加して生殖巣を培養し，MYP mRNA量を定量ＰＣＲにより測定したが，どちらも優位な変動は認められなかった。本研究期間において，ウニ生殖巣中のMYP mRNAの発現を誘起する因子の決定はできなかった。今後，MYPの合成に関与する核内受容体を特定しそのリガンドを添加する実験が必要である。
  • 魚類の卵母細胞における油球形成機構の解明
    文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2014年 -2016年 
    代表者 : 東藤孝
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2011年 -2013年 
    代表者 : 都木 靖彰, 浦 和寛, 東藤 孝
     
    これまで、ウニの生殖巣の発達につれて生殖巣で合成・蓄積されるタンパク質（主要卵黄タンパク質 Major Yolk Protein :MYP）の遺伝子構造・発現部位が明らかにされていたが、生殖巣の発達を統御機構に関しては不明なまま残されていた。本研究では、MYPおよびGAPDH mRNA発現定量系の確立をおこなうとともに、MYPの合成を誘起する生殖腺刺激ホルモンの探索を行うための生殖巣器官培養の確立を試み、短期間の器官培養系を確立した。これにより、今後 MYP mRNA 発現量を指標に生殖の内分泌統御に関する研究が可能になる。
  • 文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2011年 -2013年 
    代表者 : 東藤 孝
     
    本研究は、サケ科魚類のカットスロートトラウトを主要なモデルに用い、魚類の卵母細胞における油球形成機構を分子レベルで明らかにすることを目的としており、本年度は以下の項目について解析した。1. リポタンパクの蛍光標識法の確立：先ず、カットスロートトラウトの血漿から段階的超遠心法を用いて各種リポタンパク（超低密度リポタンパク、VLDL；低密度リポタンパク、LDL；高密度リポタンパク、HDL）を精製した。次に、各精製リポタンパクの脂質部分をグラスビーズ法により緑色蛍光標識脂肪酸（BODIPY FL C16）で標識する方法を確立した。また、各リポタンパクのアポタンパク部分は、市販のキットにより赤色蛍光物質（Alexa 594）で標識できることを確認した。さらに、こられ脂質とタンパクの標識を同時に行って、二重標識されたリポタンパクを作製できることも確かめられた。2. カットスロートトラウト卵濾胞の生体外培養系の確立：前卵黄形成期（油球期）の卵巣を摘出し、ピンセットを用いて卵濾胞を単離した。単離された卵濾胞を、市販のL-15を基本とした培養液（0.5%ウシ血清アルブミン、各種抗生物質含有、pH 7.4）中で15°Cで培養したところ、少なくとも48時間までは形態と機能を維持できることが確認された。この培養系を用いて、1で作製した蛍光標識リポタンパクの卵濾胞への取り込みを調べた結果、脂質とタンパクの双方とも、時間経過とともに卵濾胞への取り込み量が増加した。さらに、組織切片による観察から、各種リポタンパクのうちVLDL由来の蛍光標識脂質のみが卵母細胞内の油球に取り込まれることが明らかとなった。
  • 魚類の卵黄球および油球形成機構の解明
    文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2009年 -2013年 
    代表者 : 原彰彦
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2009年 -2011年 
    代表者 : 長江 真樹, 征矢野 清, 東藤 孝
     
    成熟したイトヨ雄腎臓からARαおよびARβcDNAを単離した。これらcDNAを導入したレポータージーンアッセイ系により、何れのARも機能的であり、両者の転写活性化能には違いがないことも明らかにされた。次に種々の濃度の男性ホルモンを未成熟のイトヨ雌に曝露し、腎臓でのスピギンおよび両ARmRNAの発現変化をリアルタイム定量RT-PCR法およびin situ hybridization法によりそれぞれ明らかにした。スピギンmRNA濃度は、男性ホルモン濃度および曝露期間に依存した増加が認められた。一方、AR mRNA濃度は、何れのタイプにおいても、曝露により顕著な発現量の低下が認められた。このことから、ARは腎臓でのスピギン合成を仲介すると同時に、男性ホルモン受容に応じて自身の発現量を調節(ダウンレギュレート)することが示唆された。また、両ARのmRNA濃度は大きく異なっており、ARαmRNA発現量はβのそれに比べて少なくとも10倍高値を示した。このことから、腎臓でのスピギン合成には、主にARαが関与するものと示唆された。
  • 文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2008年 -2010年 
    代表者 : 東藤 孝, 原 彰彦, 平松 尚志
     
    魚類の卵には、その成長過程で脂質やタンパク質が卵外から多量に取り込まれて蓄えられ、これらの物質は胚や稚仔魚の重要な栄養・エネルギー源として利用される。本研究は、これまで殆ど不明であった、魚類の卵における脂質の取り込み機構について分子レベルで解明することを目的に行われた。その結果、脂質の取り込みに関わる様々な因子の存在が遺伝子レベルで明らかにされ、この機構についての新しいモデルが提出された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2007年 -2009年 
    代表者 : 足立 伸次, 井尻 成保, 東藤 孝
     
    ウナギでは、性分化以前からのエストロゲン処理は雌化および卵巣形成ばかりではなく、初期卵成長促進効果があることが示唆された。一方、前卵黄形成期の卵母細胞の成長はアンドロゲン処理により促進されることが確認された。チョウザメでは、卵径約100μmから約400μmまでの卵成長には数年あるいは10数年を要するが、この時期の卵成長はエストロゲンあるいはアンドロゲン依存ではなく、体成長依存であることが示唆された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2007年 -2009年 
    代表者 : 原 彰彦, 松原 孝博, 征矢野 清, 東藤 孝, 平松 尚志
     
    本研究は魚の卵黄の蓄積メカニズムを詳細に解明しようとするものである。本研究の成果として、複数の卵黄前駆物質(卵黄の由来となる血清蛋白質)を各々分離する技術や個別に検出または測定する技術を、モデル魚(ボラ)を始めに、複数の魚類において初めて開発したことは特筆される。同技術の開発に成功したことにより魚類の卵形成に関する理解が深まったことに加え、増養殖の課題である卵質の向上や女性ホルモン様化学物質汚染の調査技術の確立を行うために必要不可欠な基礎的知見と技術を提供した。
  • 文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2006年 -2007年 
    代表者 : 東藤 孝, 中村 將
     
    本研究では、雌性先熟型の性転換を行うミツボシキュウセン(Halichoeres trimaculatus)の卵巣器官培養系を用いて、卵巣から精巣への転換を調節する分子機構について解析した。先ず、培養条件を再検討し、無血清条件下でも培養組織が良好に維持され、卵巣から精巣への転換も誘導できることを確認した。次に、様々な濃度のアンドロゲンの効果を調べるとともに、エストロゲンの影響についても検討した。その結果、ホルモン未添加の対照群でも性転換がみられたが、アンドロゲン処理により精子形成の進行が促進され、そのアンドロゲンの効果は濃度依存的に認められた。また、様々な濃度のエストロゲンをアンドロゲンとともに卵巣器官培養系に添加したところ、精子形成の進行はほとんどみられず、卵巣組織も維持されていた。この結果から、エストロゲンは卵巣機能を維持するとともに、アンドロゲンによる精子形成促進効果も抑制することが示唆された。また、培養下での卵巣から精巣への転換過程に伴う生殖細胞の増殖を観察した結果、先ず卵巣薄板の上皮において生殖細胞の増加が起こり、さらにそれらが生殖腺の内部に向かって増殖していく様子が認められた。さらに、卵巣から精巣への転換におけるアポトーシスの関わりについて調べたところ、卵母細胞の退行はアポトーシスによるものであることが示唆された。以上の結果から、魚類の性転換において性ステロイドホルモンが非常に重要な役割を担っていることがあらためて示された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2007年 
    代表者 : 山内 晧平, 足立 伸次, 東藤 孝
     
    本研究では、サケ脳下垂体投与により人為催熟された雌ニホンウナギを材料として、生殖関連分子の作用機構を解析し、良質卵大量生産技術の確立に寄与することを目的とした。初期卵成長(油球蓄積)機構解析のため、アンドロゲン(11-ケトテストステロン)合成に必須の11β-水酸化酵素(P45011β)を含む複数のステロイド合成酵素の特異抗体を作製した。また、卵巣の器官培養系を用いて、卵母細胞の油球蓄積に及ぼすアンドロゲンおよびリポタンパク質の影響を調べ、油球はリポタンパク質、特に超低密度リポタンパク質に由来し、アンドロゲンがその取り込みに作用していることを示した。次に、アンドロゲンで前処理された未熟雌ウナギを催熟することにより、前処理がその後の人為催熟に及ぼす影響を調べた結果、アンドロゲン前処理により卵径は有意に増大し、油球蓄積が顕著に進行した。魚類の卵母細胞の成熟は、最終成熟誘起ステロイド(MIS)により誘起される。MISは特異的受容体(MIS-R)を介してその作用を発現する。これまで、核型プロゲスチン受容体(PR)および膜結合型プロゲスチン受容体(mPR)が、MIS-Rであることが示唆されている。そこで、2種PR(PRαおよびPRβ)および膜型PRγ(mPRγ)の卵巣の発達に伴う発現変化およびその局在性を調べた。しかし、いずれのPRあるいはmPRもMIS-Rであると断定するには至らなかった。さらに、ニホンウナギの卵質悪化機構を明らかにすることを目的として、卵質を評価することのできるマーカータンパク質を探索し、卵質悪化には複数の要因が関与することを示した。
  • 文部科学省：科学研究費補助金(若手研究(B))
    研究期間 : 2004年 -2005年 
    代表者 : 東藤 孝
     
    ウナギの人工種苗生産技術を確立するためには、先ず何よりも良質な卵や精子の安定した供給が不可欠である。そのためには、ウナギにおける性成熟の生理機構を理解し、その知見をもとにウナギの性成熟過程を人為的に統御する必要がある。そこで本研究では、魚類の性成熟の重要な制御因子である性ステロイドホルモンに着目し、ウナギ雌の性成熟過程における性ステロイド作用を分子レベルで明らかにすることを目的として、各性ステロイド(エストロゲン、アンドロゲン、プロゲスチン)の核内受容体(ER、AR、PR)や膜型受容体の構造と機能について解析した。本年度は、先ずウナギの2種サブタイプAR(ARαとARβ)および2種サブタイプPR(PRαとPRβ)のそれぞれのmRNA量を測定するためのリアルタイム定量PCR系を確立した。この測定系を用いて、ウナギ雌雄の生殖腺におけるARとPR mRNA量の性成熟過程に伴う変化を調べた。その結果、卵巣では成熟の進行とともにARとPRの双方とも増加したが、ARではARαが、PRではPRβがそれぞれ他のタイプよりも発現量が高い傾向を示した。一方、精巣ではARとPRの双方とも精子形成の進行とともに発現量が減少し、ARでは2種間で差は見られなかったが、PRではPRαがPRβよりも総じて高い発現量を示す傾向にあった。この様に、ARとPRともに雌雄の生殖腺や成熟のステージによりそれぞれのサブタイプで発現量に相違が見られたことから、ARとPRのサブタイプは卵巣や精巣の発達において異なる役割を担っていることが示唆された。さらにin situハイブリダイゼーション法による解析から、ARとPRのmRNAは卵巣では双方とも主に濾胞細胞や間質細胞で発現しており、精巣では生殖細胞を含めた様々な細胞で発現していることが初めて明らかとなった。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2003年 -2004年 
    代表者 : 征矢野 清, 石松 惇, 中村 將, 東藤 孝
     
    ハタ科魚類は雌性先熟型の性転換を行うなど生物学的に興味深い特徴を有する魚種である。また、世界中の熱帯・温帯域に広く分布し、極めて美味であることから次世代の種苗生産対象魚として世界的に注目を集めていおり、水産的価値の高い魚種でもある。しかし、その成熟・産卵過程はほとんど明らかにされていなかった。我々は本研究を実施する前にカンモンハタの生殖腺発達に関する予備的研究を行い、沖縄周辺海域に生息するカンモンハタが月周期と完全に同調して成熟し、満月大潮直後に生息場であるサンゴ礁の礁池を離れ産卵することを見いだした。しかし、満月直後に起こることが予想される卵巣での劇的な生理変化を観察するには至らなかった。そこで、本種の成熟および産卵過程を月周期と関連づけて詳細に調べるとともに、産卵関連行動とそれに伴う生殖腺の生理変化を、組織学的・内分泌学的に解明する事を目的として、本研究を実施した。 本研究により得られた主な成果は以下の通りである。 1.カンモンハタは満月大潮後に生息地である珊瑚礁池から外洋に移動して産卵することを、目視及びバイオテレメトリー手法により裏付けた。 2.生殖腺は月周期と同調して発達し、飼育環境下でも満月大潮から数日後に産卵することが確認された。 3.生殖腺発達様式は他のハタ科魚類と類似しているが、北方系のマハタなどとは異なり、明瞭な月周性を持つことが分かった。 4.産卵は水温が上昇する5月以降に起こり7月下旬まで続づくことが分かった。 5.一尾の雌個体は満月大潮後の一産卵時に数日に分けて成熟卵を放出することが分かった。 本研究の成果はカンモンハタの生殖腺発達を理解するだけでなく、種苗生産対象魚として注目されている他のハタ科魚類の生殖腺発達を理解する上でも貴重な情報となる。また、南方系海産魚に多く見られる月周産卵の機構解明にも役立つことが期待される。
  • 文部科学省：科学研究費補助金(奨励研究(A))
    研究期間 : 2000年 -2001年 
    代表者 : 東藤 孝
     
    魚類における内分泌撹乱化学物質のエストロゲン受容体(ER)を介した作用機構を明らかにする目的で、メダカをモデルとして以下の研究を行った。近年、魚類のERにはαとβ、γの3種類が存在することが明らかとなったが、メダカではまだαタイプしか知られていない。そこで、先ずメダカERβとERγのcDNAクローニングを行うことにより、メダカにおける3種類のERの存在を確認し、3種類のERの構造や機能について比較・検討した。魚類ERβの保存配列より設計されたプライマーを用いたPCRにより、メダカ卵巣由来のcDNAから2種類のER cDNA断片が得られた。これらのcDNAは両者とも約500個のアミノ酸残基をコードするものであり、そのアミノ酸配列はERのタンパク翻訳領域の大部分を含んでいた。分子系統樹による解析から、得られた2種のcDNAはそれぞれメダカERβとERγであることが確認された。次に、メダカ雌における3種のER mRNAの発現組織をRT-PCR法により調べた。ERα mRNAは肝臓と卵巣にのみ検出されたが、ERβmRNAは脳や肝臓、卵巣、筋肉、鰭で発現しており、さらにERγ mRNAは調べた組織のほとんどで発現が認められた。以上のように、メダカにおいても3種類のERが存在することが初めて明らかにされた。また、3種類のER mRNAの発現組織に違いがみられたことから、エストロゲンや内分泌撹乱化学物質の作用において3種のERは異なる役割を担っている可能性が高いことが示された。しかし、3種類のERの機能的相違の詳細については、今後の課題として残された。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1999年 -2000年 
    代表者 : 征矢野 清, 石橋 康弘, 高良 真也, 石松 惇, 東藤 孝
     
    内分泌攪乱化学物質が魚類の生殖現象及び生残に与える影響を胎生魚のカダヤシを用いて調べた。本研究では、カダヤシの親魚をビスフェノールA(BPA)に暴露させ、親魚体内の胎児における形態異常の有無と生殖線の形態変化を観察した。また、正常に産仔された仔魚をBPAに暴露させ、生殖線の形態変化と生残率を調べた。本研究により得られた成果は以下の通りである。 1)受精卵及び胎児を持つ親魚を産仔までの期間(約23日間)20ppb及び2000ppbのBPAに暴露させ、産仔された仔魚の生残率と奇形率を調べたところ、20ppb暴露群ではほとんど生残仔魚は得られず92.1%が死産仔魚であった。これらの死産仔魚は全て頭部変形、脊椎湾曲、卵黄嚢肥大などの奇形形態を呈していた。2000ppb暴露群では産仔が行われなかった。 2)発生後期の胚を持つ親魚をBPAに暴露すると、産仔された仔魚の36.5%は死産であり、そのうち91.3%は頭部変形、脊椎湾曲などの奇形形態を呈していた。 3)受精期の欄を持つ親魚をBPAに暴露しても同様の奇形を持つ死産仔魚が認められた。 4)正常に産仔された仔魚を20ppbのBPAに30日間暴露したところ、形態異常は全く引き起こされず、生残率もBPAを暴露していない対照区の仔魚と変わらなかった。 5)BPA暴露した親魚より産仔された仔魚及び産仔後にPBA暴露した仔魚のいずれにおいても生殖腺形態の異常は観察されなかった。ただし、高濃度のPBAを暴露した親魚の生殖腺では、退行卵が観察された。 本研究の結果、親魚体内で発生中の胚は親魚を通してBPAの影響を強く受けることが明かとなった。また、産仔後の仔魚では同濃度のBPAにさらしても異常が認められないことから、化学物質の影響は胚発生過程にある胎児への影響が大きいと考えられる。

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