MF研究者総覧

Researcher Database

Advance
Researchers' Page on researchmap
Last Updated :2024/11/17

Researcher Profile and Settings

Master

Affiliation (Master)

  • Faculty of Fisheries Sciences　Marine Life Science　Aquaculture Biology

Affiliation (Master)

  • Faculty of Fisheries Sciences　Marine Life Science　Aquaculture Biology

researchmap

Profile and Settings

Profile and Settings

  • Name (Japanese)

    Todo

  • Name (Kana)

    Takashi

  • Name

    201301037244745948

Alternate Names

Achievement

Research Interests

  • 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   

Research Areas

  • Life sciences / Morphology, anatomy
  • Life sciences / Marine/Aquatic life sciences
  • Life sciences / Aquaculture
  • Environmental science/Agricultural science / Environmental effects of chemicals
  • Environmental science/Agricultural science / Environmental effects of radiation
  • Life sciences / Developmental biology

Research Experience

  • 2005/04 - Today Hokkaido University Faculty of Fisheries Sciences Associate Professor
  • 2003/04 - 2005/03 Hokkaido University Graduate School of Fisheries Sciences Associate Professor
  • 1998/02 - 2003/03 Niigata University Sado Marine Biological Station, Faculty of Science Assistant Professor
  • 1997/04 - 1998/01 National Institute for Basic Biology Laboratory of Reproductive Biology Postdoctoral Researcher of JSPS
  • 1996/11 - 1997/03 National Institute for Basic Biology Laboratory of Reproductive Biology Reseach Associate
  • 1995/04 - 1996/10 National Institute for Basic Biology Laboratory of Reproductive Biology Research Fellow

Education

  • 1992/04 - 1995/03  Hokkaido University  Faculty of Fisheries  Graduate School of Fisheries, Doctor Course
  • 1990/04 - 1992/03  Hokkaido University  Faculty of Fisheries  Graduate School of Fisheries, Master Course
  • 1986/04 - 1990/03  Hokkaido University  Faculty of Fisheries

Published Papers

  • Shuichiro Watanabe, Ken Matsuzaki, Utano Shimizu, Ichiro Higuchi, Takashi Todo, Yasuaki Takagi, Kazuhiro Ura
    Fisheries Science 90 (6) 907 - 924 0919-9268 2024/06/10
  • Yo Yamaguchi, Takuma Kawasaki, Akihiko Hara, Takashi Todo, Naoshi Hiramatsu
    Aquaculture 583 0044-8486 2024/03/30 
    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 0378-1119 2022/12 [Refereed]
  • Yamaguchi, Y, Nagata, J, Nishimiya, O, Kawasaki, T, Hiramatsu, N, Todo, T
    Comparative Biochemistry and Physiology, Part A 261 111055 - 111055 1095-6433 2021 [Refereed][Not invited]
  • 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 0289-0003 2021 [Refereed][Not invited]
  • Nagata, J, Mushirobira, Y, Nishimiya, O, Yamaguchi, Y, Fujita, T, Hiramatsu, N, Hara, A, Todo, T
    General and Comparative Endocrinology 310 113812 - 113812 0016-6480 2021 [Refereed][Not invited]
  • Namgung, J, Mizuta, H, Yamaguchi, Y, Nagata, J, Todo, T, Yilmaz, O, Hiramatsu, N
    Comparative Biochemistry and Physiology, Part A 257 110967 - 110967 1095-6433 2021 [Refereed]
  • 莚平裕次, 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 0371-4217 2020 [Refereed][Not invited]
  • Haruna Amano, Seiichi Uno, Jiro Koyama, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara
    General and comparative endocrinology 281 67 - 72 2019/09/15 [Refereed][Not invited]
     
    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 0006-3363 2019/05/01 [Refereed][Not invited]
     
    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 2019/01/15 [Refereed][Not invited]
     
    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 2018/11/21 [Not refereed][Not invited]
     
    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 0016-6480 2018/10 [Refereed][Not invited]
     
    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.
  • Nagata Jun, Mushirobira Yuji, Nishimiya Osamu, Fujita Toshiaki, Hiramatsu Naoshi, Hara Akihiko, Todo Takashi
    Aquaculture Science 水産増殖談話会 66 (2) 91 - 101 0371-4217 2018 [Refereed][Not invited]
     
    Choriogenin (Chg) and vitellogenin (Vtg) subtypes and estrogen receptor α1 (Erα1) are upregulated by estradiol-17β (E2) in the liver of female salmonids. The aim of this study was to examine the relationships among hepatic mRNA levels for chgs (chgHα; chgHβ; chgL), vtgs (vtgAs; vtgC), erα1 and serum E2 levels in female cutthroat trout (Oncorhynchus clarki) during a reproductive cycle. Levels of serum E2 and hepatic chg mRNAs, as well as hepatic vtg mRNAs, increased in correlation with the progress of vitellogenesis. In the ovulated fish, chg mRNA remained at high levels while serum E2 and vtg mRNA levels decreased. Hepatic erα1 mRNA levels exhibited a peak in August (at the beginning of vitellogenesis) before levels of E2, chg and vtg mRNAs start to increase. These results suggest that expression levels of chg, vtg and erα1 genes are potentially regulated through E2 stimulation by different mechanisms.
  • Nagata Jun, Todo Takashi, Hara Akihiko, Hiramatsu Naoshi, Kasai Kei, Mineno Kazuhiro, Fujisaki Yudai, Mushirobira Yuji, Namgung Jin, Takeda Yasutaka, Fujita Toshiaki, Kawasaki Takuma
    Aquaculture Science 水産増殖談話会 66 (4) 257 - 266 0371-4217 2018 [Not refereed][Not invited]
     
    Whole-mount immunostaining (WI) was used for identification of eggs of brown sole (Pseudopleuronectes herzensteini), sand flounder (Limanda punctatissima) and pointhead flounder (Cleisthenes pinetorum). Polyclonal antibodies were raised in rabbit against vitelline envelope (VE) of ovulated eggs of each flounder (a-brown sole VE, a-sand flounder VE and a-pointhead flounder VE). A WI method was first developed using the non-labeled primary a-VE antibodies in conjunction with a labeled-secondary antibody (2-step method). Another WI method using the labeled-primary a-VE anitbodies alone was developed (1-step method) in order to omit the use of the secondary antibody. When fertilized eggs of each species (2-24 hrs post-fertilization) were examined, both WI methods effectively stained the eggs in a species-specific manner. Immunological tools developed for identification of flounder eggs in this study will contribute to simplify fishery surveys of flounder species during their early life stages.
  • 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 1095-6433 2017/10 [Refereed][Not invited]
     
    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.
  • Kawasaki Takuma, Shimizu Yohei, Mori Tatsunari, Hiramatsu Naoshi, Todo Takashi
    Aquaculture Science 水産増殖談話会 65 (1) 73 - 82 0371-4217 2017 [Refereed][Not invited]
     
    Black rockfish (Sebastes schlegelii) is an economically important aquaculture species in Japan. Because it is a viviparous species, seedling production is typically performed using natural mating and the numbers of larva that are produced tend to be unstable. In the present study, we aimed to develop artificial insemination techniques for black rockfish to ensure stable seedling production. We used 20 female and 15 male mature individuals and examined when artificial insemination is best performed, in which diluent the sperm is best diluted and whether sperm collected from live or dead males both have fertilizing capability. The number of larvae produced by each pregnant female was counted. In addition, microsatellite markers were used to determine the relationship between larvae and parents. Following artificial insemination, 12 of 20 females became pregnant. The average litter size per pregnant female was about 130,000 fry. Pregnant females were obtained under all experimental conditions, regardless of season, sperm diluent and whether the male was live or dead. Paternity testing revealed that all larvae were produced by artificial insemination. Furthermore, females artificially inseminated by several males appeared to produce offspring derived from all males. The present study demonstrates that artificial insemination is possible in marine viviparous fish.
  • Osamu Nishimiya, Yoshinao Katsu, Hiroyuki Inagawa, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara
    JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 165 190 - 201 0960-0760 2017/01 [Refereed][Not invited]
     
    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 日本比較内分泌学会 43 (160) 21 - 23 1882-6636 2017
  • Maebayashi Mamoru, Hiramatsu Naoshi, Inaoka Yuhei, Yoshida Tatsuya, Hagihara Seishi, Nishimiya Osamu, Mushirobira Yuji, Adachi Shinji, Hara Akihiko, Todo Takashi
    Aquaculture Science 水産増殖談話会 64 (1) 63 - 76 0371-4217 2016 [Refereed][Not invited]
     
    Molecular cloning of cDNAs encoding an egg yolk precursor, vitellogenin (Vtg), was performed using the liver of estrogen-treated Amur sturgeon. Three kinds of vtg cDNA were cloned and temporarily named as vtg1, vtg2 and vtg3. These vtg cDNAs were obtained as contiguous sequences; each of vtg1, vtg2 and vtg3 sequences contained a complete open reading frame (5,307, 5,247 and 5,319 bp, respectively), each encoding 1,769, 1,749 and 1,773 aa residues, respectively. Similarity among amino acid sequences deduced from the three vtg cDNAs were relatively low, ranging from 64.3% to 47.9%. Three Vtgs appeared to be a complete-type, consisting of all representative yolk protein domains. Phylogenetic analysis revealed that Amur sturgeon Vtg1 formed a clade together with a published white sturgeon Vtg, while Amur sturgeon Vtg2 and Vtg3 formed another distinct clade. The results of phylogenetic analysis confirmed all three Vtgs belong to VtgAB type; vtg1/Vtg1, vtg2/Vtg2 and vtg3/Vtg3 were hereby designated as vtgAB1/VtgAB1, vtgAB2a/VtgAB2a and vtgAB2b/VtgAB2b, respectively. This study provided a basis to understand multiplicity in sturgeon vtg/Vtg and set a stage for their future application as reproductive biomarkers in sturgeon aquaculture.
  • 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 1040-452X 2015/12 [Refereed][Not invited]
     
    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 0016-6480 2015/09 [Refereed][Not invited]
     
    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 0214-6282 2015 [Refereed][Not invited]
     
    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 0022-2275 2014/11 [Refereed][Not invited]
     
    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 0174-1578 2014/07 [Refereed][Not invited]
     
    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 0289-0003 2014/04 [Refereed][Not invited]
     
    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 0289-0003 2014/04 [Refereed][Not invited]
     
    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 1095-6433 2013/10 [Refereed][Not invited]
     
    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 1096-4959 2013/09 [Refereed][Not invited]
     
    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 0916-8818 2013/08 [Refereed][Not invited]
     
    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 0920-1742 2013/04 [Refereed][Not invited]
     
    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 0289-0003 2013/03 [Refereed][Not invited]
     
    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.
  • Yuji Mushirobira, Hiroko Mizuta, Wenshu Luo, Yuka Morita, Sayumi Sawaguchi, Takahiro Matsubara, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara
    NIPPON SUISAN GAKKAISHI 79 (2) 175 - 189 0021-5392 2013/03 [Refereed][Not invited]
     
    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 0920-1742 2013/02 [Refereed][Not invited]
     
    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  0916-8818 2011/08/20 [Not refereed][Not invited]
  • Taku Endo, Takashi Todo, P. Mark Lokman, Hideaki Kudo, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi
    BIOLOGY OF REPRODUCTION 84 (4) 816 - 825 0006-3363 2011/04 [Refereed][Not invited]
     
    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 0289-0003 2011/03 [Refereed][Not invited]
     
    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.
  • MORITA Yuka, HIRAMATSU Naoshi, IWASAKI Toshihide, TODO Takashi, HARA Akihiko
    The Journal of Reproduction and Development Supplement 日本繁殖生物学会 104 (0) 1066 - 1066 2011 [Refereed][Not invited]
     
    【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 0022-2275 2010/11 [Refereed][Not invited]
     
    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 1095-6433 2010/09 [Refereed][Not invited]
     
    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 0016-6480 2010/09 [Refereed][Not invited]
     
    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 0289-0003 2009/12 [Refereed][Not invited]
     
    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 0289-0003 2009/07 [Refereed][Not invited]
     
    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
    Bulletin of fisheries sciences, Hokkaido University 北海道大学大学院水産科学研究院 = Research Faculty of Fisheries Sciences, Hokkaido University 58 (3) 21 - 28 1346-1842 2009/02 [Not refereed][Not invited]
     
    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 1532-0456 2009/01 [Refereed][Not invited]
     
    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
    Bulletin of Fisheries Research Agency 水産総合研究センター (26) 129 - 134 1346-9894 2008/12 [Not refereed][Not invited]
     
    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 0919-9268 2008/08 [Refereed][Not invited]
     
    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 0399-0974 2008/07 [Not refereed][Not invited]
     
    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 0399-0974 2008/07 [Not refereed][Not invited]
     
    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 0016-6480 2006/01 [Refereed][Not invited]
     
    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.
  • Xian-Cheng QU, Dong-Hwan SHIN, Masaki NAGAE, Takashi TODO, Shinji ADACHI, Kohei YAMAUCHI
    Aquaculture Science 日本水産増殖学会 54 (3) 283 - 292 0371-4217 2006 [Refereed][Not invited]
     
    In order to evaluate the role of the thyroid gland during reproduction in the female cultivated Japanese eel (Anguilla japonica), we investigated thyroid activity during sexual maturation induced by chum salmon pituitary homogenate (SPH) treatment. Thyroid gland activity (epithelial cell height) was high before SPH injection (approximately 6.3μm; i.e. during previtellogenic stages of ovarian development), further increasing at the early vitellogenic stage (approximately 8.3μm) and decreasing thereafter to the late vitellogenic and migratory nucleus stages (approximately 4.2μm) . The serum profiles of the thyroid hormones thyroxine (T4) and triiodothyronine (T3) changed notably during vitellogenesis. Levels of these hormones were high during previtellogenic and early vitellogenic stages (T4; approximately 3.5 ng/ml, T3; approximately 4.1ng/ml) and subsequently declined (T4; approximately 0.4ng/ml, T3; approximately 0.03ng/ml), thereby reflecting activity levels in the thyroid gland. Our results suggest that leves of thyroid activity inversely correlate with ovarian development during artificial maturation of the Japanese eel. The low levels of thyroid hormones observed in the serum of the Japanese eel prior to ovulation may not be normal as compared with that seen in other teleost species.
  • K Nagano, T Kawasaki, S Ijiri, T Todo, S Adachi, K Yamauchi
    FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 437 - 438 0920-1742 2003 [Refereed][Not invited]
     
    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 0920-1742 2003 [Refereed][Not invited]
     
    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 0920-1742 2003 [Refereed][Not invited]
     
    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 0920-1742 2003 [Refereed][Not invited]
     
    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 1040-452X 2002/08 [Refereed][Not invited]
     
    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 0013-7227 2002/06 [Refereed][Not invited]
     
    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 0006-3363 2002/05 [Refereed][Not invited]
     
    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 0016-6480 2002/01 [Refereed][Not invited]
     
    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 0014-5793 2002/01 [Refereed][Not invited]
     
    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 1096-4959 2001/06 [Refereed][Not invited]
     
    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 0016-6480 2000/04 [Refereed][Not invited]
     
    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 0027-8424 2000/03 [Refereed][Not invited]
     
    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 海洋出版 32 (2) 95 - 101 0916-2011 2000/02 [Not refereed][Not invited]
  • T Todo, T Ikeuchi, T Kobayashi, H Kajiura-Kobayashi, K Suzuki, M Yoshikuni, K Yamauchi, Y Nagahama
    FEBS LETTERS 465 (1) 12 - 17 0014-5793 2000/01 [Refereed][Not invited]
     
    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 0289-0003 1999/10 [Refereed][Not invited]
  • T Miura, C Miura, T Ohta, MR Nader, T Todo, K Yamauchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 264 (1) 230 - 234 0006-291X 1999/10 [Refereed][Not invited]
     
    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 0021-9258 1999/09 [Refereed][Not invited]
     
    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 0289-0003 1999/08 [Refereed][Not invited]
     
    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 0006-291X 1999/02 [Refereed][Not invited]
     
    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 0006-291X 1999/01 [Refereed][Not invited]
     
    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 0289-0003 1998/12 [Refereed][Not invited]
     
    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 海洋出版 30 (2) 96 - 102 0916-2011 1998/02 [Not refereed][Not invited]
  • Nakamura, I, M Nagae, T Todo, S Adachi, K Yamauchi
    ADVANCES IN COMPARATIVE ENDOCRINOLOGY, TOMES 1 AND 2 1375 - 1379 1997 [Refereed][Not invited]
     
    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 [Refereed][Not invited]
     
    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 [Refereed][Not invited]
     
    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 0303-7207 1996/05 [Refereed][Not invited]
     
    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 [Not refereed][Not invited]
  • M. Nagae, T. Todo, K. Gen, Y. Kato, G. Young, S. Adachi, K. Yamauchi
    Journal of Molecular Endocrinology 16 (2) 171 - 181 0952-5041 1996 [Not refereed][Not invited]
     
    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 E2 and 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 0289-0003 1995/12 [Refereed][Not invited]
     
    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 0044-8486 1995/10 [Refereed][Not invited]
  • H OKUMURA, A HARA, F SAEKI, T TODO, S ADACHI, K YAMAUCHI
    FISHERIES SCIENCE 61 (2) 283 - 289 0919-9268 1995/04 [Refereed][Not invited]
     
    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

Books etc

  • 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 (Contributor17. 配偶子形成に関わる遺伝子)
    恒星社厚生閣 1997/06

Presentations

Association Memberships

  • JAPAN SOCIETY FOR COMPARATIVE ENDOCRINOLOGY   Japan Society of Endocrine Disrupter Research   THE ZOOLOGICAL SOCIETY OF JAPAN   THE JAPANESE SOCIETY OF FISHERIES SCIENCE   

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2021/04 -2024/03 
    Author : 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))
    Date (from‐to) : 2018 -2022 
    Author : 平松尚志
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2017/03 
    Author : Takagi Yasuaki, URA Kazuhiro, TODO Takashi
     
    We developed organ culture system using the sea urchin gonads for analysis of endocrine system during the gonadal growth. To date, it is well known that growth of gonad by synthesis and stored of MYP. The aim of this present study is identification of hormonal chemicals to induce the gonadal growth in sea urchin. We performed organ culture using the sea urchin gonads in the coelomic fluids of sea urchin, we measured MYP mRNA levels in the gonads by qPCR after incubation for 3days. The levels of MYP mRNA were no changing after incubation. It is suggested that MYP synthesis by nuclear receptor in gonads. Moreover, we extracted total lipids fraction form the gonads of sea urchin, and then gonads were incubated in the medium containing total lipids. However, NYP mRNA was no induced in this experiment. In the present study, we can’t identify the gonadal stimulation hormone in sea urchin. In future, we will try the comparison of colemic fluids compounds during gonadl growth.
  • 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))
    Date (from‐to) : 2014 -2016 
    Author : 東藤孝
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2013 
    Author : TAKAGI Yasuaki, URA Kazuhiro, TODO Takashi
     
    Sea urchin gonadal growth is characterized by intragonadal nutrient storage and the use of the stored nutrient for gametogenesis. Before gametogenesis initiation, gonads increase in size by accumulating nutrients such as proteins, lipids and carbohydrates in nutritive phagocytes that fill the gonadal lumina of both sexes. The major protein is termed major yolk glycoprotein or major yolk protein (MYP), which is a glycoprotein having molecular weight of 160-180 kDa, and has significant roles in gametogenesis. However, we have no information about gonadal development mechanisms, as well as the regulation mechanism on the synthesis of MYP in gonads. Therefore, to understand the mechanisms of gonadal development, we developed quantitative PCR system of MYP mRNA expression as well as an organ culture systems using sea urchin gonads. These techniques will enable a endocrinological research on gonadal developments in the sea urchin.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2013 
    Author : TODO TAKASHI, HARA Akihiko, HIRAMATSU Naoshi
     
    In teleost fishes, high amount of lipids and proteins are accumulated in oocytes during their growth phase; they are later utilized as energy and nutrient resources by developing embryos and larvae. However, little is known about the origin of such lipids and the mechanisms underlying their accumulation into oocytes. In the present study, we aimed to clarify the mechanisms at the molecular levels. As the results, we confirmed that, for the first time, plasma very low density lipoprotein is the major carrier of neutral lipid to oocyte lipid droplets, and updated the model for the oocyte lipidation.
  • 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))
    Date (from‐to) : 2009 -2013 
    Author : 原彰彦
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2009 -2011 
    Author : NAGAE Masaki, SOYANO Kiyoshi, TODO Takashi
     
    Full length of ARαand ARβcDNAs were isolated from mature male stickleback kidney. It was showed by reporter gene assay that both were functional AR, and had almost same potency of transcriptional activation. The changes in the mRNA levels of spiggin and both ARs of immature female stickleback treated with androgen were measured and detected by quantitative RT-PCR and in situ hybridization. Spiggin mRNA levels increased in androgen dose and exposure duration-dependent manner. In contrast, the mRNA levels of both ARs were significantly decreased by androgen exposure. In addition, the levels of ARαwere about 10 times higher than that of ARβduring androgen exposure. These results suggested ARαwas essential for the spiggin synthesis in stickleback kidney.
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2008 -2010 
    Author : Takashi TODO, Akihiko HARA, Naoshi HIRAMATSU
     
    In teleost fishes, high amounts of lipids and proteins are accumulated in oocytes during their growth phase ; they are later utilized as energy and nutrient resources by developing embryos and larvae. However, little is known about the origin of such lipids and the mechanisms underlying their accumulation into oocytes. In the present study, we aimed to clarify the mechanisms at the molecular levels. As the results, we identified various factors involved in the lipid uptake into fish oocytes, and proposed a new model for the oocyte lipidation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2007 -2009 
    Author : ADACHI Shinji, IJIRI Shigeho, TODO Takashi
     
    Estrogen treatment may promote oocyte growth during ovarian differentiation in eel and sturgeon. Androgen treatment accelerates previtellogenic oocyte growth in eel. Somatic growth may promote oocyte growth between estrogen and androgen-dependent phase in fish.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2007 -2009 
    Author : HARA Akihiko, MATSUBARA Takahiro, SOYANO Kiyoshi, TODO Takashi, HIRAMATSU Naoshi
     
    The objective of this study is to elucidate mechanisms involved in the yolk formation in fish oocytes. Results included development of novel technologies for separation and quantification of multiple subtypes of a yolk precursor (vitellogenin : Vg) ; such techniques were initially developed for the grey mullet, and thereafter applied for several other teleosts. Findings endow the better understandings on fish oogenesis, as well as improvement of fish egg quality in aquaculture and development of technologies for the surveys on the impacts of environmental xenoestrogenic chemicals.
  • Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2006 -2007 
    Author : Takashi TODO, Masaru NAKAMURA
     
    Recently, we succeeded to induce in vitro spermatogenesis in a protogynous hermaphrodite fish, the three-spotted wrasse (Halichoeres trimaculatus) during organ culture of ovary with a serum-supplemented medium. This culture system appears to be a good model for further study of the mechanisms controlling gonadal sex change. In the present study, we re-examined this culture system for the three-spotted wrasse.Ovarian tissue from sexually immature three-spotted wrasse was cultured in Leibovitz L-15 medium supplemented with 0.5% bovine serum albumin (BSA) or 10% fetal bovine serum (FBS) using a floating tissue culture method. Tissue was exposed to androgens for a few, weeks at 26℃. The effects of various concentrations of androgens and estrogen were also examined. Furthermore, the detection of apoptotic cells during the culture was performed using the TUNEL method.In all experimental groups, including the control (no steroid), degeneration of oocytes and initiation of spermatogenesis was observed. Androgens enhanced the complete transition of ovarian tissue via spermatogonia to formation of spermatogenic crypts. There was no difference between supplementation with BSA and FBS. The effects of androgens on inducing spermatogenesis showed a dose dependent manner. Estrogen treatments reduced the degeneration of oocytes, and did not lead the occurrence of spermatogenesis. The TUNEL-positive oocytes were observed and increased during culture period.These results show that the restructuring from ovary to testis in the wrasse could be induced in vitro with a serum-free medium. The results also suggest that the gonadal sex change can be triggered by basic in vitro culture due to a lack of endogenous factors (especially, estrogens) for female sexuality, and accelerated by androgens. Furthermore, oocyte apoptosis is might be an important mechanism of the gonadal sex change.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2005 -2007 
    Author : 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))
    Date (from‐to) : 2004 -2005 
    Author : 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。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2003 -2004 
    Author : SOYANO Kiyoshi, ISHIMATSU Atsushi, NAKAMURA Masaru, TODO Takashi
     
    Honeycomb grouper, which is distributed in the sub-tropical and tropical area in Pacific Ocean, is known as a lunar-related spawner. However, details of gonadal development and spawning in this species are not well understood. In this study, we investigated the gonadal development and hormonal changes in female collected from the fringing reef area around Sesoko Island during spawning season and observed the spawning in artificial conditions. Moreover, we investigated the spawning migration to the outside of fringing reef for spawning using biotelemetry method. Gonadosomatic index increased rapidly and reached the maximum level around the full moon in late May or early June, when ovaries were occupied with oocytes of the tertiary yolk stage. The fish of maturational size disappeared from the fringing reefs for a few days after the full moon. When the fish appeared again in the same reef area, there was no vitellogenic oocyte in the ovary. These results indicate that the fish spawned in the area outside the fringing reefs after the full moon. In artificial conditions, the final oocyte maturation and spawning were observed for a few days after the full moon, while the maturated fish migrated outside the fringing reefs in natural conditions. The spawning of individuals was carried out for three or four days. This results elucidated clearly that honeycomb grouper is a lunar-related spawner.
  • 文部科学省：科学研究費補助金(奨励研究(A))
    Date (from‐to) : 2000 -2001 
    Author : 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。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1999 -2000 
    Author : SOYANO Kiyoshi, ISHIBASHI Yasuhiro, KOHRA Shinya, ISHIMATSU Atushi, TODO Takashi
     
    There is little information of endocrine disruptors in viviparous fish. The purpose of the present study was to investigate the influence of environmental estrogens on embryo and fry of mosquitofish, viviparous fish, using bisphenol A(BPA) which is found to exert estrogenic effects in fish. Females with ovulated eggs and pregnant females were exposed to BPA at concentration of 20ppb and reared until the parturition of fry. The mortality and the abnormality of newborn fry which was bred by BPA exposed pregnant females were examined. The stillbirth(92.1%) was induced by BPA exposure. The deformation of head, the curvature of vertebra and the hypertrophy of yolk sac were observed as abnormalities in all the stillborn fry. Newborn fry bred by normal females were exposed to BPA at the same concentration for 10, 20 or 30 days after the parturition. In this experiment, the mortality of exposed fry was not significantly different from control fry and the abnormality was not observed. These results suggest that environmental estrogens may influence the embryogenesis in viviparous fish.

Copyright © MEDIA FUSION Co.,Ltd. All rights reserved.