Researcher Profile and Settings

Affiliation

• Faculty of Fisheries Sciences　Marine Life Science　Aquaculture Biology

Job Title

• Professor

URL

http://kaken.nii.ac.jp/d/r/60303111.ja.html

J-Global ID

• 201301037244745948

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

Educational Organization

•  Bachelor's degree program　School of Fisheries Sciences
•  Master's degree program　Graduate School of Fisheries Sciences
•  Doctoral (PhD) degree program　Graduate School of Fisheries Sciences

Academic & Professional 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

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 Activities

Published Papers

• Identification and characterization of lipocalin-type prostaglandin D2 synthase homologs in the urine of male rockfish

  Yo Yamaguchi, Jin Namgung, Jun Nagata, Takuma Kawasaki, Akihiko Hara, Takashi Todo, Naoshi Hiramatsu

  Gene 147093 - 147093 0378-1119 2022/12 [Refereed]
  
• Molecular characterization of fshb and lhb subunits and their expression profiles in captive white-edged rockfish, Sebastes taczanowskii

  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]
  
• Changes in the hepatic three vitellogenin subtype genes during ovarian development in female white-edged rockfish (Sebastes taczanowskii)

  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]
  
• Hepatic estrogen-responsive genes relating to oogenesis in cutthroat trout (Oncorhynchus clarki): The transcriptional induction in primary cultured hepatocytes and the in vitro promoter transactivation in responses to estradiol-17

  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]
  
• Knock out of a major vitellogenin receptor gene with eight ligand binding repeats in medaka (Orizias latipes) using the CRISPER/Cas9 system

  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]
  
• Development of a species identification method for shishamo smelt larvae using whole-mount immunohistochemistry

  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]
  
• Development of specific enzyme-linked immunosorbent assays for multiple vitellogenins in marbled sole, Pleuronectes yokohamae.

  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.
• Synergistic effects of estradiol and 11-ketotestosterone on vitellogenin physiology in the shortfinned eel (Anguilla australis).

  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.
• Development of specific chemiluminescent immunoassays for three subtypes of vitellogenin in grey mullet (Mugil cephalus).

  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.
• Oogenesis and Egg Quality in Finfish: Yolk Formation and Other Factors Influencing Female Fertility

  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.
• Molecular cloning of vitellogenin gene promoters and in vitro and in vivo transcription profiles following estradiol-17 beta administration in the cutthroat trout

  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.
• Expression analysis of estradiol-17β responsive genes in the liver of female cutthroat trout (Oncorhynchus clarki) during a reproductive cycle

  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.
• Species-identification of eggs of three flounders by whole-mount immunostaining

  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.
• Ovarian expression and localization of clathrin (Cltc) components in cutthroat trout, Oncorhynchus clarki: Evidence for Cltc involvement in endocytosis of vitellogenin during oocyte growth

  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.
• Development of artificial insemination techniques for viviparous black rockfish (Sebastes schlegelii)

  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.
• Molecular cloning and characterization of hagfish estrogen receptors

  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.
• ヌタウナギ2種エストロジェン受容体（ER）のcDNAクローニングと機能解析

  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
• Molecular cloning of cDNAs encoding multiple vitellogenins in Amur sturgeon Acipenser schrenckii

  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.
• Molecular Cloning and Partial Characterization of a Low-Density Lipoprotein Receptor-Related Protein 13 (Lrp13) Involved in Vitellogenin Uptake in the Cutthroat Trout (Oncorhynchus clarki)

  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.
• Ovarian yolk formation in fishes: Molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins

  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.
• Visualization of primordial germ cells in the fertilized pelagic eggs of the barfin flounder Verasper moseri

  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.
• Lrp13 is a novel vertebrate lipoprotein receptor that binds vitellogenins in teleost fishes

  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.
• Development and partial characterisation of an antiserum against apolipoprotein B of the short-finned eel, Anguilla australis

  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.
• Molecular Cloning and Characterization of the Expression Profiles of Vitellogenin Transcripts in the Dojo Loach (Misgurnus anguillicaudatus) in Response to 17 alpha-ethinylestradioland 17 beta-estradiol Administration

  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.
• Biochemical and Immunochemical Characterization of Two Discrete Vitellogenin Proteins and Their Derived Lipovitellins in the Inshore Hagfish (Eptatretus burgeri)

  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.
• Molecular cloning and partial characterization of an ovarian receptor with seven ligand binding repeats, an orthologue of low-density lipoprotein receptor, in the cutthroat trout (Oncorhynchus clarki)

  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.
• Ovarian expression and localization of a vitellogenin receptor with eight ligand binding repeats in the cutthroat trout (Oncorhynchus clarki).

  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.
• Characterization of Alpha-fetoprotein Levels in Three Dolphin Species: Development of Sensitive Immunoassays for Analysis of the Pregnancy-associated Variations

  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.
• Characterization of vitellogenin and its derived yolk proteins in cloudy catshark (Scyliorhinus torazame)

  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.
• Molecular Cloning and Transcript Expression of Genes Encoding Two Types of Lipoprotein Lipase in the Ovary of Cutthroat Trout, Oncorhynchus clarki

  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.
• Changes in levels of dual vitellogenin transcripts and proteins in cutthroat trout Oncorhynchus clarki during ovarian development

  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.
• Multiple ovarian lipoprotein receptors in teleosts

  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.
• スジイルカにおけるpregnancy‐associated glycoprotein cDNAのクローニング

  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]
  
• Androgens and Very Low Density Lipoprotein Are Essential for the Growth of Previtellogenic Oocytes from Japanese Eel, Anguilla japonica, In Vitro

  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.
• Characterization of Alpha-fetoprotein in Fetal Striped Dolphin (Stenella coeruleoalba): Purification of Protein Product and Molecular Cloning of the Corresponding Transcript

  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.
• cDNA cloning of pregnancy-associated glycoprotein striped dolphin

  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。
• Effects of reproductive stage and 11-ketotestosterone on LPL mRNA levels in the ovary of the shortfinned eel

  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.
• Purification and characterization of a novel incomplete-type vitellogenin protein (VgC) in Sakhalin taimen (Hucho perryi)

  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.
• Expression of androgen receptor mRNA in the ovary of Japanese eel, Anguilla japonica, during artificially induced ovarian development

  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.
• Purification of Multiple Precursors for Egg Chorion Proteins in Atlantic Cod (Gadus morhua)

  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.
• Purification and Classification of Three Lipovitellin Subtypes in the Marbled Sole (Pleuronectes yokohamae)

  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.
• Purification of subtilase from short-spined sea urchin Strongylocentrotus intermedius

  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.
• Choriogenin and vitellogenin in red lip mullet (Chelon haematocheilus): Purification, characterization, and evaluation as potential biomarkers for detecting estrogenic activity

  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.
• Differentiation and maturation of sea urchin gonad

  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。
• Purification and characterization of lipovitellin from Pacific saury Cololabis saira

  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.
• In vitro induction of gonadal sex change in a protogynous fish, three-spotted wrasse (Halichoeres trimaculatus)

  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.
• In vitro induction of oil droplet accumulation into previtellogenic oocytes of Japanese eel, Anguilla japonica

  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.
• Changes in localization of cytochrome P450 cholesterol side-chain cleavage (P450scc) in Japanese eel testis and ovary during gonadal development

  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.
• Changes in serum thyroid hormone levels and thyroid gland activity in artificially maturing female Japanese eel (Anguilla japonica)

  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.
• Effects of rearing water quality on expression of brain corticotropin-releasing hormone and gonadal development in goldfish (Carassius auratus)

  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.
• Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.)

  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.
• Transgenic cell lines which stably express progestogen receptors (PRS) alpha and beta and the PR-responsive reporter genes

  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.
• 11-ketotestosterone potentiates estrogen-induced vitellogenin production in liver of Japanese eel (Anguilla japonica)

  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.
• Molecular cloning and expression during spermatogenesis of a cDNA encoding testicular 11 beta-hydroxylase (P450(11 beta)) in rainbow trout (Oncorhynchus mykiss)

  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.
• Characterization and expression of steroidogenic acute regulatory protein and MLN64 cDNAs in trout

  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.
• Teleost ovarian carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase: Potential role in the production of maturation-inducing hormone during final oocyte maturation

  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.
• Changes in hepatic vitellogenin mRNA levels during oocyte development in the Japanese eel, Anguilla japonica

  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).
• A novel progestogen receptor subtype in the Japanese eel, Anguilla japonica

  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.
• Two subtypes of androgen and progestogen receptors in fish testes

  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.
• Molecular cloning and characterization of Japanese eel ovarian P450c17 (CYP17) cDNA

  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.
• Isoleucine-15 of rainbow trout carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase is critical for coenzyme (NADPH) binding

  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.
• ホルモン受容体 (特集 魚類の配偶子形成機構--水産における基礎と応用)

  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]
  
• Characterization of a testicular 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (a spermiation-inducing steroid in fish) receptor from a teleost, Japanese eel (Anguilla japonica)

  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.
• Molecular cloning of estrogen receptors alpha and beta in the ovary of a teleost fish, the Tilapia (Oreochromis niloticus) (vol 16, pg 653, 1999)

  XT Change

  ZOOLOGICAL SCIENCE 16 (5) 853 - 853 0289-0003 1999/10 [Refereed][Not invited]
  
• Estradiol-17 beta stimulates the renewal of spermatogonial stem cells in males

  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.
• cDNA cloning of a novel androgen receptor subtype

  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).
• Molecular cloning of estrogen receptors alpha and beta in the ovary of a teleost fish, the Tilapia (Oreochromis niloticus)

  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.
• Cloning and expression of two carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase cDNAs in ovarian follicles of rainbow trout (Oncorhynchus mykiss)

  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.
• Fish androgen receptor: cDNA cloning, steroid activation of transcription in transfected mammalian cells, and tissue mRNA levels

  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.
• The establishment of a quantitative RT-PCR assay for estrogen receptor mRNA in Japanese eel, Anguilla japonica

  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.
• 魚類のステロイドホルモンレセプタ-の構造と機能 (総特集 魚類の生育・適応機能と遺伝子)

  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]
  
• RT-PCR analysis of estrogen receptor expression in various tissues in the Japanese eel, Anguilla japonica

  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.
• cDNA cloning and protein expression of the gonadotropin receptor from the tilapia, Oreochromis niloticus

  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.
• Steroidogenesis by the salmonid ovarian follicle: the two-cell type model revisited

  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.
• Molecular cloning and characterization of Japanese eel estrogen receptor cDNA

  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]
  
• Molecular cloning of the cDNAs encoding pituitary glycoprotein hormone α- And gonadotropin IIβ-subunits of the Japanese eel, Anguilla japonica, and increase in their mRNAs during ovarian development induced by injection of chum salmon pituitary homogenate

  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.
• Hepatic estrogen receptors in the Japanese eel, Anguilla japonica: Characterization and changes in binding capacity during artificially-induced sexual maturation

  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.
• Molecular basis of vitellogenesis in the Japanese eel, Anguilla japonica, during artificially-induced sexual maturation

  T Todo, S Adachi, K Yamauchi

  AQUACULTURE 135 (1-3) 77 - 77 0044-8486 1995/10 [Refereed][Not invited]
  
• DEVELOPMENT OF A SENSITIVE SANDWICH ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR VITELLOGENIN IN THE JAPANESE EEL ANGUILLA-JAPONICA

  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.

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

Conference Activities & Talks

• カットスロートトラウト初代培養肝細胞を用いたエストラジオール‐17β応答性遺伝子の発現解析  [Not invited]

  永田淳, 莚平裕次, 西宮攻, 藤田敏明, 平松尚志, 原彰彦, 東藤孝

  日本水産学会大会講演要旨集  2018/03
• カットスロートトラウト雌の肝臓におけるエストラジオール‐17β応答性遺伝子の生殖周期に伴う発現変化  [Not invited]

  永田淳, 莚平裕次, 西宮攻, 平松尚志, 原彰彦, 東藤孝

  日本水産学会大会講演要旨集  2017/03
• メダカにおけるLR8型ビテロジェニン受容体遺伝子のノックアウト:CRISPR/Cas9による変異導入初世代の作出  [Not invited]

  JIN Namgung, 小山海斗, 勘林優樹, 水田紘子, 廣島美枝, YEO In‐Kyu, 東藤孝, 平松尚志

  日本水産学会北海道支部大会講演要旨集  2017
• ホールマウント免疫染色法による3種カレイ受精卵の種判別  [Not invited]

  笠井慶, 永田淳, 峯野博和, 藤崎雄大, 莚平裕次, 莚平裕次, 武田康孝, 藤田敏明, 東藤孝, 原彰彦, 平松尚志

  日本水産学会北海道支部大会講演要旨集  2017
• 遺伝子導入メダカ初世代における組換え蛋白質の発現:pT2/TransposaseシステムとpCAGGS/I‐SceIシステムの比較  [Not invited]

  塚原杏奈, 吉田達哉, 莚平裕次, 東藤孝, 平松尚志

  日本水産学会北海道支部大会講演要旨集  2017
• メダカにおける低密度リポ蛋白質受容体遺伝子ノックアウト:CRISPR/Cas9による欠損導入初世代の作出  [Not invited]

  小山海斗, JIN Namgung, 勘林優樹, 永田淳, 莚平裕次, 吉田達也, 東藤孝, 平松尚志

  日本水産学会北海道支部大会講演要旨集  2017
• GnRH投与によるエゾメバル雌の性成熟促進  [Not invited]

  紫藤勇磨, 川崎琢真, 莚平裕次, 平松尚志, 東藤孝

  日本水産学会北海道支部大会講演要旨集  2016/10
• カットスロートトラウト(Oncorhynchus clarki)における2型ビテロジェニンプロモーターの性状解析  [Not invited]

  莚平裕次, 西宮攻, 永田淳, 東藤孝, 原彰彦, 平松尚志

  日本水産学会大会講演要旨集  2016/03
• Molecular cloning of cDNAs encoding multiple vitellogenins in Amur sturgeon Acipenser schrenckii  [Not invited]

  前林衛, 稲岡雄平, 吉田達哉, 萩原聖士, 西宮攻, 莚平裕次, 足立伸次, 原彰彦, 東藤孝, 平松尚志

  水産増殖  2016/03
• Molecular mechanisms underlying yolk formation in fish: how to make the tailor-made yolk?  [Invited]

  Naoshi Hiramatsu, Takashi Todo

  The 2nd Joint Symposium of HU/NTOU & 1st International Conference on Quaternary and Future Earth  2016/01
• エゾメバルの卵巣発達に伴う3型ビテロジェニン遺伝子発現量の変化  [Not invited]

  和田怜, 西宮攻, 横野孝典, 莚平裕次, 東藤孝, 川崎琢真, 松原孝博, 澤口小有美, 原彰彦, 平松尚志

  日本水産学会大会講演要旨集  2015/03
• ウニの生殖腺刺激ホルモン様物質の探索  [Not invited]

  相澤俊介, 佐藤卓史, 東典子, 東藤孝, 浦和寛, 都木靖彰

  日本水産学会大会講演要旨集  2015/03
• エゾメバル(Sebastes taczanowski)における生殖腺刺激ホルモン各サブユニットのcDNAクローニングと発現解析  [Not invited]

  横野孝典, 西宮攻, 川崎琢真, 平松尚志, 東藤孝

  日本水産学会北海道支部大会講演要旨集  2015
• 免疫染色法によるシシャモ仔稚魚の判別技術開発  [Not invited]

  川崎琢真, 清水洋平, 石田良太郎, 中田訓彰, 平松尚志, 東藤孝

  日本水産学会北海道支部大会講演要旨集  2015
• Ovarian yolk formation in fishes: Molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins  [Not invited]

  Naoshi Hiramatsu, Takashi Todo

  10th International Symposium on Reproductive Physiology of Fish  2014/05
• ホールマウント免疫染色法を用いた3種カレイ卵の同定  [Not invited]

  峯野博和, 南宮眞, 文惠拏, 呂寅圭, 武田康孝, 西宮攻, 鳴海一侑, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2014/03
• カットスロートトラウト(Oncorhynchus clarki)における血漿リポ蛋白質の分離とアポリポ蛋白質Eの検出  [Not invited]

  莚平裕次, 竹下眞広, 東藤孝, 原彰彦, 平松尚志

  日本水産学会大会講演要旨集  2014/03
• クロソイおよびキツネメバル人工授精技術の現状と課題  [Not invited]

  川崎琢真, 清水洋平, 高畠信一, 森立成, 小渡賢太, 横野孝典, 和田怜, 平松尚志, 東藤孝

  日本水産学会北海道支部大会講演要旨集  2014
• Molecular cloning and characterization of estrogen receptors from the most primitive vertebrate, the inshore hagfish Epatretus burgeri  [Not invited]

  Osamu Nishimiya, Yoshinao Katsu, Hiroyuki Inagawa, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara

  17th International Congress of Comparative Endocrinology  2013/07
• サケ科魚類のビテロジェニンに結合するリポ蛋白質受容体の探索と性状解析  [Not invited]

  莚平裕次, 水田紘子, 羅ぶん妹, 東藤孝, 原彰彦, 平松尚志

  日本水産学会大会講演要旨集  2013/03
• カットスロートトラウト卵巣におけるクラスリン重鎖転写産物および同翻訳産物の局在に関する組織学的解析  [Not invited]

  水田紘子, 東藤孝, 原彰彦, 平松尚志

  日本水産学会大会講演要旨集  2013/03
• ヌタウナギの2種エストロジェン受容体の機能解析  [Not invited]

  西宮攻, 勝義直, 稲川裕之, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2013/03
• カットスロートトラウトにおける常染色体劣性高コレステロール血症原因蛋白質(ARH)のcDNAクローニング及び遺伝子発現解析  [Not invited]

  水田紘子, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2012/03
• ヌタウナギの2種ビテロジェニン遺伝子および2種エストロジェン受容体遺伝子の発現解析  [Not invited]

  西宮攻, 國弘康之, 稲川裕之, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2012/03
• 低濃度エストロジェン投与によるマコガレイ3タイプビテロジェニンの血中動態  [Not invited]

  天野春菜, 細見靖道, 森山俊介, 藤井一則, 平松尚志, 東藤孝, 原彰彦

  日本比較内分泌学会大会及びシンポジウムプログラム・講演要旨  2012
• イトヨ腎臓での男性ホルモン刺激によるスピギン合成にはARαが必須である  [Not invited]

  長江真樹, 松尾昴, 瀬川卓也, 東藤孝, 勝義直, 井口泰泉, SCOTT Alexander P, KATSIADAKI Ioanna

  日本内分泌かく乱化学物質学会研究発表会要旨集  2011/12
• スジイルカにおけるpregnancy‐associated glycoprotein cDNAのクローニング  [Not invited]

  盛田祐加, 平松尚志, 岩崎俊秀, 東藤孝, 原彰彦

  J Reprod Dev  2011/08
• マコガレイの3型ビテロジェニンに対する酵素免疫測定法の確立  [Not invited]

  天野春菜, 森山俊介, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2011/03
• イトヨ腎臓におけるスピギンとアンドロジェン受容体の発現解析  [Not invited]

  瀬川卓也, 松尾昴, 長江真樹, 平松尚志, 東藤孝, 原彰彦

  日本水産学会北海道支部大会講演要旨集  2011
• カットスロートトラウト(Oncorhynchus clarki)における2型ビテロジェニン遺伝子の発現解析  [Not invited]

  莚平裕次, 水田紘子, 羅ぶんしょ, 澤口小有美, 松原孝博, 平松尚志, 東藤孝, 原彰彦

  日本水産学会北海道支部大会講演要旨集  2011
• カットスロートトラウト卵巣におけるリポ蛋白質受容体の局在解析  [Not invited]

  水田紘子, 伊東優太, 平松尚志, 東藤孝, 原彰彦

  日本水産学会北海道支部大会講演要旨集  2011
• ヌタウナギにおける2種エストロジェン受容体のcDNAクローニングおよび発現解析  [Not invited]

  西宮攻, 國弘康之, 稲川裕之, 平松尚志, 東藤孝, 原彰彦

  日本水産学会北海道支部大会講演要旨集  2011
• カットスロートトラウト卵巣におけるビテロジェニン受容体蛋白質の性状解析  [Not invited]

  水田紘子, 伊東優太, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2010/03
• イルカ類の胎児および妊娠雌血清中におけるalpha‐fetoproteinの動態  [Not invited]

  盛田祐加, 平松尚志, 藤田敏明, 天野春菜, 岩崎俊秀, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2010/03
• ヌタウナギの2型ビテロジェニン遺伝子のプロモーター領域解析  [Not invited]

  西宮攻, 國弘康之, 天野春菜, 藤田敏明, 稲川裕之, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2010/03
• カットスロートトラウト卵巣におけるリポタンパクリパーゼファミリー遺伝子の発現解析  [Not invited]

  柳蓉法, 田中莉夏子, 伊東優太, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2010/03
• ヌタウナギのエストロジェン受容体遺伝子のクローニング  [Not invited]

  西宮攻, 國弘康之, 稲川裕之, 平松尚志, 東藤孝, 原彰彦

  日本水産学会北海道支部大会講演要旨集  2010
• ヌタウナギの2型ビテロジェニン遺伝子のクローニング  [Not invited]

  國弘康之, 天野春菜, 藤田敏明, 稲川裕之, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2009/03
• カットスロートトラウト卵巣におけるリポタンパクリパーゼのcDNAクローニングと発現解析  [Not invited]

  東藤孝, 笠原あゆみ, 田中莉夏子, 伊東優太, 全先清博, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  2009/03
• マコガレイの卵黄蛋白リポビテリンの検索と精製  [Not invited]

  天野春菜, 藤田敏明, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2009/03
• スジイルカalpha‐fetoproteinの精製と定性  [Not invited]

  盛田祐加, 平松尚志, 藤田敏明, 天野春菜, 岩崎俊秀, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2009/03
• カットスロートトラウト低密度リポ蛋白質受容体(LDLR)遺伝子の発現解析  [Not invited]

  伊東優太, 全先清博, 伊藤貴洋, 平松尚志, 東藤孝, 藤田敏明, 原彰彦

  日本水産学会大会講演要旨集  2009/03
• Purification of Subtilase from Short-Spined Sea Urchin Strongylocentrotus intermedius  [Not invited]

  浦和寛, 田中恵梨, 東藤孝, 後藤孝弘, 清水幹博, 尾島孝男, 都木靖彰

  北海道大学水産科学研究い報  2009/02 

  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.
• イトヨの多型ビテロジェニンの精製  [Not invited]

  高橋美咲, 天野春菜, 藤田敏明, 平松尚志, 東藤孝, 松原孝博, 原彰彦

  日本水産学会大会講演要旨集  2008/03
• クロマグロ卵膜蛋白遺伝子の検出  [Not invited]

  久田剛輝, 藤田敏明, 玄浩一郎, 二階堂英城, 升間主計, 松原孝博, 東藤孝, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  2008/03
• カットスロートトラウト低密度リポ蛋白質受容体のcDNAクローニング  [Not invited]

  全先清博, 平松尚志, 東藤孝, 藤田敏明, 原彰彦

  日本水産学会大会講演要旨集  2008/03
• 雌化養成ウナギの卵巣および生殖関連ホルモンの周年変化  [Not invited]

  登坂亮太, 風藤行紀, 井尻成保, 東藤孝, 足立伸次, 山内晧平

  日本水産学会大会講演要旨集  2008/03
• ヌタウナギのビテロジェニンならびにその関連卵黄蛋白の検索および精製  [Not invited]

  國弘康之, 天野春菜, 藤田敏明, 稲川裕之, 平松尚志, 東藤孝, 原彰彦

  日本水産学会大会講演要旨集  2008/03
• ウニの生殖細胞マーカーとしての抗Vasa抗体の作製  [Not invited]

  東藤孝, 森川遥, 武智静子, 大澤秀一, 須川祐, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  2008/03
• カットスロートトラウト卵巣における脂肪酸輸送膜蛋白(CD36)のcDNAクローニング  [Not invited]

  伊藤貴洋, 平松尚志, 東藤孝, 藤田敏明, 千葉仁志, 原彰彦

  日本水産学会大会講演要旨集  2008/03
• クロマグロ高度化―奄美栽培漁業センターにおける養成3歳魚雌クロマグロの繁殖特性  [Not invited]

  玄浩一郎, 二階堂英城, 香川浩彦, 松原孝博, 澤口小有美, 東藤孝, 平松尚志, 原彰彦, 武部文行, 井手健太郎, 西明文, 塩澤聡, 升間主計

  日本水産学会大会講演要旨集  2008/03
• エゾバフンウニにおけるnanos遺伝子のクローニングと発現解析  [Not invited]

  東藤孝, 久保田陽子, 浦和寛, 都木靖彰, 足立伸次, 山内皓平

  日本水産学会大会講演要旨集  2007/03
• 環境操作によるウナギの前卵黄形成期の卵成長および初期卵黄形成の誘導  [Not invited]

  登坂亮太, 宮崎智史, 風藤行紀, 井尻成保, 東藤孝, 足立伸次, 山内皓平

  日本水産学会大会講演要旨集  2007/03
• ニホンウナギ組換え生殖腺刺激ホルモンおよびその受容体の機能解析  [Not invited]

  風藤行紀, 小原真由子, 三浦猛, 三浦智恵美, TRANT JM, 東藤孝, 足立伸次, 山内皓平

  日本水産学会大会講演要旨集  2007/03
• ニホンウナギ卵巣におけるプロゲスチンレセプター遺伝子の発現解析  [Not invited]

  飯塚慧, 風藤行紀, 東藤孝, 足立伸次, 山内皓平

  日本水産学会大会講演要旨集  2007/03
• ミツボシキュウセンの生殖腺器官培養系を用いた性転換機構の解析  [Not invited]

  佐藤茉菜, 東藤孝, 蘆田美沙, 山口直宏, 小林靖尚, 足立伸次, 山内皓平, 中村將

  日本水産学会大会講演要旨集  2007/03
• ニホンウナギ卵巣器官培養系による油球蓄積機構の解析  [Not invited]

  遠藤琢, 東藤孝, 井尻成保, 足立伸次, 山内皓平

  日本水産学会大会講演要旨集  2007/03
• ウナギの試験管内卵生産に用いる卵巣の保存条件の検討  [Not invited]

  安部智貴, 井尻成保, 東藤孝, 足立伸次, 山内皓平

  日本水産学会大会講演要旨集  2007/03
• 海藻を用いた魚類循環飼育水の浄化および魚類の生理状態に及ぼす水質悪化の影響  [Not invited]

  中山大樹, 川崎琢真, 安井肇, 東藤孝, 足立伸次, 山内皓平

  水産増殖  2007/03
• ニホンウナギの生体外卵成熟,排卵および発生誘導  [Not invited]

  安部智貴, 東藤孝, 足立伸次, 山内皓平

  水産増殖  2007/03
• Changes in Serum Thyroid Hormone Levels and Thyroid Gland Activity in Artificially Maturing Female Japanese Eel (Anguilla japonica)  [Not invited]

  QU Xian-Cheng, SHIN Dong-Hwan, NAGAE Masaki, TODO Takashi, ADACHI Shinji, YAMAUCHI Kohei

  水産増殖  2006/09 

  ニホンウナギAnguilla japonicaの生殖における甲状腺の役割を調べることを目的として、サケ脳下垂体磨砕物（SPH）投与により人為催熟された雌個体の甲状腺活性を測定した。甲状腺活性（濾胞上皮細胞高）は、SPH投与前（前卵黄形成期）に高く、卵黄形成初期（約8.3μm）でさらに増加し、卵黄形成後期と核移動期（約4.2μm）には低下した。甲状腺ホルモンであるチロキシン（T4）およびトリヨードチロニン（T3）の血中量は卵黄形成初期（T4；約3.5ng／ml、T3；約4.1ng／ml）で高く、その後減少（T4；約0.4ng／ml、T3；約0.03ng／ml）し、甲状腺活性を反映した。以上の結果から、人為催熟されたニホンウナギにおいては、甲状腺活性は卵巣発達と負の関係にあることが示唆された。排卵前のニホンウナギにおける血中甲状腺ホルモン量は他魚種における値と比較して低く、正常値ではないかもしれない。
• ニホンウナギの卵質と核成熟との関係  [Not invited]

  佐合洋祐, 申東かん, 鵜沼辰哉, 黒川忠英, 野村和晴, 松原創, 田中秀樹, 小林亨, 長浜嘉孝, 小林弘子, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  2006/03
• エゾバフンウニにおけるvasa関連遺伝子のクローニングと発現解析  [Not invited]

  久保田陽子, 浜岡未知子, 東藤孝, 酒井勇一, 近田靖子, 浦和寛, 都木靖彰, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  2006/03
• ニホンウナギにおける2種アンドロゲンレセプターの発現解析  [Not invited]

  登坂亮太, 風藤行紀, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  2006/03
• アンドロゲン処理された雌ウナギの人為催熟  [Not invited]

  清水研二, 瀬名波絢子, 石上博紹, 申東かん, 原口泉, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  2005/04
• 魚類のステロイド合成酵素の免疫組織化学的検出  [Not invited]

  山口直宏, 市川真幸, 中村将, 武井則雄, 風藤行紀, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  2005/04
• アメリカ産ナマズの脳型アロマテースの機能検索  [Not invited]

  風藤行紀, 東藤孝, 足立伸次, 山内こう平, JM Trant

  日本水産学会大会講演要旨集  2005/04
• ウナギおよびキンギョの卵質解析  [Not invited]

  申東かん, 石上博紹, 原口泉, 山下正兼, 小林亨, 小林弘子, 長浜嘉孝, 東藤孝, 足立伸次

  日本水産学会大会講演要旨集  2005/04
• ニホンウナギの初期卵成長に及ぼす性ステロイドホルモン経口投与の影響  [Not invited]

  瀬名波絢子, 木村郷, 松原創, 高橋隆行, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  2004/03
• 魚類におけるステロイド合成酵素の免疫組織化学  [Not invited]

  武井則雄, 山口直宏, 井尻成保, 東藤孝, 足立伸次, 中村将, 山内こう平

  日本水産学会大会講演要旨集  2004/03
• キンギョの循環飼育による水質悪化とCRH,POMCmRNAの発現変化  [Not invited]

  川崎琢真, 永野恭子, 宮田愛, 井尻成保, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  2004/03
• アンドロゲン経口投与によるニホンウナギ初期卵成長の促進  [Not invited]

  清水研二, 瀬名波絢子, 松原創, 東藤孝, 足立伸次, 山内こう平

  日本水産学会北海道支部大会講演要旨集  2004
• コレステロール側鎖切断酵素抗体を用いた魚類のステロイド産生細胞の検出  [Not invited]

  山口直宏, 市川真幸, 中村将, 武井則雄, 東藤孝, 足立伸次, 山内こう平

  日本水産学会北海道支部大会講演要旨集  2004
• 月周期と関連したカンモンハタの性成熟と産卵  [Not invited]

  征矢野清, 増本貴士, 田中英洋, 東藤孝, 中村将

  日本水産学会大会講演要旨集  2003/04
• アルキルフェノール類のメダカ及びヒトエストロゲン受容体に対する活性の比較  [Not invited]

  西川智浩, 白石不二雄, 東藤孝, 西川淳一, 白石寛明, 森田昌敏

  日本内分泌かく乱化学物質学会研究発表会要旨集  2002/11
• マダイのアンドロゲンレセプターcDNAのクローニング  [Not invited]

  玄浩一郎, 奥沢公一, 東藤孝, 香川浩彦

  日本水産学会大会講演要旨集  2002/03
• カンモンハタの成熟・産卵と月周期との関係  [Not invited]

  増本貴士, 中村将, 比嘉幹彦, 竹村明洋, 東藤孝, 征矢野清

  日本水産学会大会講演要旨集  2002/03
• メダカ(Oryzias latipes)における3種類のエストロジェンレセプター  [Not invited]

  東藤孝, 酒泉満, 浜口哲

  日本内分泌かく乱化学物質学会研究発表会要旨集  2001/12
• ニジマスのsteroidogenic acute regulatory protein(StAR) cDNAのクローニング  [Not invited]

  東藤孝, 日下部誠, MCQUILLAN H J, YOUNG G

  日本比較内分泌学会大会及びシンポジウムプログラム・講演要旨  2000/11
• HCG投与による精子形成に伴う精巣での2種類のアンドロゲン受容体の動態  [Not invited]

  池内俊貴, 東藤孝, 小林亨, 長浜嘉孝

  日本水産学会大会講演要旨集  2000/04
• ウナギ精巣における2種類の最終成熟誘起ステロイド受容体  [Not invited]

  東藤孝, 池内俊貴, 小林亨, 長浜嘉孝

  日本水産学会大会講演要旨集  2000/04
• ウナギ精巣における2種類のプロゲストゲン受容体  [Not invited]

  池内俊貴, 東藤孝, 小林亨, 長浜嘉孝

  日本動物学会大会予稿集  1999/09
• 魚類の配偶子形成における内分泌機構 II‐1. ホルモン受容体  [Not invited]

  東藤孝

  日本水産学会大会講演要旨集  1999/09
• ウグイの産卵に及ぼす産卵場環境の作用  [Not invited]

  伊藤文成, 長江真樹, 東藤孝, 山崎継子, 吉田大作, 征矢野清

  日本水産学会大会講演要旨集  1999/04
• 種々のホルモン投与によるウグイ雌の最終成熟および排卵誘起  [Not invited]

  東藤孝, 伊藤文成, 長江真樹, 吉田大作, 征矢野清

  日本水産学会大会講演要旨集  1999/04
• ウナギ精巣における11‐ケトテストステロン(精子形成誘起ホルモン)受容体  [Not invited]

  池内俊貴, 東藤孝, 小林亨, 長浜嘉孝

  日本水産学会大会講演要旨集  1999/04
• 11 - keto testosterone (spermatogenesis inducement hormone ) acceptor in eel testiculus.  [Not invited]

  池内俊貴, 東藤孝, 小林亨, 長浜嘉孝

  日本動物学会大会予稿集  1998/09
• cDNA cloning of two types of 20.BETA.-hydroxysteroid dehydrogenase(20.BETA.-HSD) from rainbow trout ovaries.  [Not invited]

  GUAN G, 田中実, 東藤孝, YOUNG G, 長浜嘉孝

  日本分子生物学会年会プログラム・講演要旨集  1997/12
• Expression of aromatase in egg follicular cell of rainbow trout .  [Not invited]

  東藤孝, YOUNG G, 関桂君, 田中実, 小林亨, 長浜嘉孝

  日本水産学会大会講演要旨集  1997/04
• Change of expression of estrogen receptor gene with artificial maturation acceleration of female Japan eel.  [Not invited]

  中村郁美, 長江真樹, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  1997/04
• Cloning of vitellogenin gene of Japan eel and expression in liver.  [Not invited]

  奥村浩美, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  1997/04
• cDNA cloning and expression of the progesterone receptors of eel.  [Not invited]

  東藤孝, 小林亨, 小林弘子, 鈴木邦昌, 吉国通庸, 山内こう平, 長浜嘉孝

  日本動物学会大会予稿集  1996/09
• Altered expression of vitellogenin with artificial maturation of Japanese female eel.  [Not invited]

  奥村浩美, 佐伯文博, 東藤孝, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  1996/03
• Cloning of progesterone receptor cDNA of Japanese eel.  [Not invited]

  東藤孝, 小林亨, 小林弘子, 鈴木邦昌, 吉国通庸, 山内こう平, 長浜嘉孝

  日本水産学会大会講演要旨集  1996/03
• ニホンウナギ培養肝細胞を用いたビテロゲニン合成,分泌能の解析  [Not invited]

  佐伯文博, 東藤孝, 奥村浩美, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  1995/04

MISC

• 不妊体ニジマスの魚卵アレルギーフリー食材としての有用性評価

  渡辺彩希, 石田慎太郎, 笹岡友季穂, 清水裕, 東藤孝, 平松尚志, 趙佳賢, 佐伯宏樹  日本水産学会大会講演要旨集(CD-ROM)  2022-  2022
• クロソイ養殖の効率化に向けた補償成長に関する給餌試験

  芝竜太郎, 山口燿, 竹内真論, 木村和世, 平井輝孝, 川崎琢真, 清水宗敬, 東藤孝, 平松尚志  水産増殖  69-  (4)  2021
• メダカ卵への物質輸送システム開発における同種主要ビテロジェニンAa1プロモーターの利用

  小島哲也, 南宮眞, 永井優里, 永田淳, 莚平裕次, 川上浩一, 東藤孝, 平松尚志  日本水産学会大会講演要旨集(CD-ROM)  2021-  2021
• オミクス解析並びに免疫生化学的手法を用いたクロソイSebastes schlegelii交尾期雄尿タンパク質の同定

  山口燿, 芝竜太郎, 川崎琢真, 原彰彦, 東藤孝, 平松尚志  日本水産学会大会講演要旨集(CD-ROM)  2021-  2021
• Development of a species identification method for shishamo smelt larvae using whole-mount immunohistochemistry

  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 = Aquaculture science  68-  (1)  1  -8  2020/04  [Not refereed][Not invited]
  
• カットスロートトラウト肝臓で発現するエストロジェン応答性遺伝子のプロモーター解析

  永田淳, 莚平裕次, 西宮攻, 藤田敏明, 平松尚志, 原彰彦, 東藤孝  日本水産学会大会講演要旨集  2019-  30  2019/03/26  [Not refereed][Not invited]
  
• カットスロートトラウト初代培養肝細胞を用いたエストラジオール‐17β応答性遺伝子の発現解析

  永田淳, 莚平裕次, 西宮攻, 藤田敏明, 平松尚志, 原彰彦, 東藤孝  日本水産学会大会講演要旨集  2018-  13  2018/03/26  [Not refereed][Not invited]
  
• カットスロートトラウト雌の肝臓におけるエストラジオール‐17β応答性遺伝子の生殖周期に伴う発現変化

  永田淳, 莚平裕次, 西宮攻, 平松尚志, 原彰彦, 東藤孝  日本水産学会大会講演要旨集  2017-  16  2017/03/26  [Not refereed][Not invited]
  
• メダカにおける低密度リポ蛋白質受容体遺伝子ノックアウト:CRISPR/Cas9による欠損導入初世代の作出

  小山海斗, JIN Namgung, 勘林優樹, 永田淳, 莚平裕次, 吉田達也, 東藤孝, 平松尚志  日本水産学会北海道支部大会講演要旨集  2017-  25  2017  [Not refereed][Not invited]
  
• 遺伝子導入メダカ初世代における組換え蛋白質の発現:pT2/TransposaseシステムとpCAGGS/I‐SceIシステムの比較

  塚原杏奈, 吉田達哉, 莚平裕次, 東藤孝, 平松尚志  日本水産学会北海道支部大会講演要旨集  2017-  26  2017  [Not refereed][Not invited]
  
• ホールマウント免疫染色法による3種カレイ受精卵の種判別

  笠井慶, 永田淳, 峯野博和, 藤崎雄大, 莚平裕次, 莚平裕次, 武田康孝, 藤田敏明, 東藤孝, 原彰彦, 平松尚志  日本水産学会北海道支部大会講演要旨集  2017-  23  2017  [Not refereed][Not invited]
  
• GnRH投与によるエゾメバル雌の性成熟促進

  紫藤勇磨, 川崎琢真, 莚平裕次, 平松尚志, 東藤孝  日本水産学会北海道支部大会講演要旨集  2016-  51  2016/10/22  [Not refereed][Not invited]
  
• カットスロートトラウト(Oncorhynchus clarki)における2型ビテロジェニンプロモーターの性状解析

  莚平裕次, 西宮攻, 永田淳, 東藤孝, 原彰彦, 平松尚志  日本水産学会大会講演要旨集  2016-  14  2016/03/26  [Not refereed][Not invited]
  
• エゾメバルの卵巣発達に伴う3型ビテロジェニン遺伝子発現量の変化

  和田怜, 西宮攻, 横野孝典, 莚平裕次, 東藤孝, 川崎琢真, 松原孝博, 澤口小有美, 原彰彦, 平松尚志  日本水産学会大会講演要旨集  2015-  215  2015/03/27  [Not refereed][Not invited]
  
• カットスロートトラウト(Oncorhynchus clarki)における血漿リポ蛋白質の分離とアポリポ蛋白質Eの検出

  莚平裕次, 竹下眞広, 東藤孝, 原彰彦, 平松尚志  日本水産学会大会講演要旨集  2014-  178  2014/03/27  [Not refereed][Not invited]
  
• サケ科魚類のビテロジェニンに結合するリポ蛋白質受容体の探索と性状解析

  莚平裕次, 水田紘子, 羅ぶん妹, 東藤孝, 原彰彦, 平松尚志  日本水産学会大会講演要旨集  2013-  39  2013/03/26  [Not refereed][Not invited]
  
• カットスロートトラウト(Oncorhynchus clarki)における2型ビテロジェニン遺伝子の発現解析

  莚平裕次, 水田紘子, 羅ぶんしょ, 澤口小有美, 松原孝博, 平松尚志, 東藤孝, 原彰彦  日本水産学会北海道支部大会講演要旨集  2011-  28  2011  [Not refereed][Not invited]
  
• Development of evaluation systems for the detection of estrogenic endocrine disrupting chemicals (EDCs) in aquatic environments using estrogen-inducible biomarkers.

  Hiramatsu N, Luo W, Hong L, Soyano K, Aoki J, Amano H, Fujita T, Todo T, Matsubara T, Hara A  Journal of The National Taiwan Museum  14-  61  -69  2010/06  [Not refereed][Not invited]
  
• クロマグロ高度化―奄美栽培漁業センターにおける養成3歳魚雌クロマグロの繁殖特性

  玄浩一郎, 二階堂英城, 香川浩彦, 松原孝博, 澤口小有美, 東藤孝, 平松尚志, 原彰彦, 武部文行, 井手健太郎, 西明文, 塩澤聡, 升間主計  日本水産学会大会講演要旨集  2008-  238  2008/03/27  [Not refereed][Not invited]
  
• クロマグロ卵膜蛋白遺伝子の検出

  久田剛輝, 藤田敏明, 玄浩一郎, 二階堂英城, 升間主計, 松原孝博, 東藤孝, 平松尚志, 原彰彦  日本水産学会大会講演要旨集  2008-  224  2008/03/27  [Not refereed][Not invited]
  
• 海藻を用いた魚類循環飼育水の浄化および魚類の生理状態に及ぼす水質悪化の影響

  中山大樹, 川崎琢真, 安井肇, 東藤孝, 足立伸次, 山内皓平  水産増殖  55-  (1)  2007
• ニホンウナギの卵質と核成熟との関係

  佐合洋祐, 申東かん, 鵜沼辰哉, 黒川忠英, 野村和晴, 松原創, 田中秀樹, 小林亨, 長浜嘉孝, 小林弘子, 東藤孝, 足立伸次, 山内こう平  日本水産学会大会講演要旨集  2006-  2006
• The cause of deterioration in egg quality of the Japanese eel and goldfish.

  Sago Y, Shin D H, Haraguchi I, Unuma T, Kurokawa T, Nomura K, Matsubara H, Tanaka H, Kobayashi T, Kobayashi H, Nagahama Y, Yamashita M, Todo T, Adachi S, Yamauchi K  The 4th International Symposium “Potential and Perspective of Marine Bio-Manipulation”  2005/10  [Not refereed][Not invited]
   

  ニホンウナギと金魚の卵質について調べた。（Sago Y, Shin D H, Haraguchi I, Unuma T, Kurokawa T, Nomura K, Matsubara H, Tanaka H, Kobayashi T, Kobayashi H, Nagahama Y, Yamashita M, Todo T, Adachi S, Yamauchi K.）The 4th International Symposium “Potential and Perspective of Marine Bio-Manipulation”Proceedings, , 2005:p89 ポスター発表
• Hatching rate and thyroid hormone levels in eggs from artificially matured Japanese eel.

  Ishigami H, Shin D H, Haraguchi I, Unuma T, Kurokawa T, Nomura K, Matsubara H, Tanaka H, Todo T, Adachi S, Yamauchi K  The 4th International Symposium “Potential and Perspective of Marine Bio-Manipulation”  2005/10  [Not refereed][Not invited]
   

  人為催熟ニホンウナギの排卵卵中の甲状腺ホルモン量と孵化率の関係を調べた。（Ishigami H, Shin D H, Haraguchi I, Unuma T, Kurokawa T, Nomura K, Matsubara H, Tanaka H, Todo T, Adachi S, Yamauchi K. ）Potential and Perspective of Marine Bio-Manipulation”Proceedings, Japan, 2005:p88掲載.ポスター発表
• Effects of androgen on the expression of lipidation related genes in female Japanese eel (Anguilla japonica)

  Senaha A, Matsubara H, Todo T, Adachi S, Yamauchi K  The 1st International Symposium “Potential and Perspective of Marine Bio-Manipulation”  2005/02  [Not refereed][Not invited]
   

  ニホンウナギ雌の脂肪酸合成関連因子の発現にアンドロゲンが及ぼす影響を調べた。（Senaha A, Matsubara H, Todo T, Adachi S, Yamauchi K.）ポスター発表 Good poster award
• ウナギおよびキンギョの卵質解析

  申東かん, 石上博紹, 原口泉, 山下正兼, 小林亨, 小林弘子, 長浜嘉孝, 東藤孝, 足立伸次  日本水産学会大会講演要旨集  2005-  2005
• Maturity of ovulated egg in artificially matured Japanese eel Anguilla japonica

  Shin D H, Ishihara M, Park C W, Matsubara H, Kaneko S, Kotani T, Yamashita M, Todo T, Adachi S, Yamauchi K  Suisanzoshoku  (52)  434  -435  2004/12  [Not refereed][Not invited]
   

  ニホンウナギの排卵卵の卵質を調べた。(Shin D H, Ishihara M, Park C W, Matsubara H, Kaneko S, Kotani T, Yamashita M, Todo T, Adachi S, Yamauchi K.) Suisanzoshoku 2004; 52: p434-435.掲載
• Relationship between egg quality and maturity of ovulated egg in Japanese eel Anguilla japonica

  Shin D H, Ishihara M, Park C W, Matsubara H, Kaneko S, Kotani T, Yamashita M, Todo T, Adachi S, Yamauchi K  The Seventh Asian Fisheries Forum  2004/11  [Not refereed][Not invited]
   

  ニホンウナギの排卵卵の成熟度と卵質の関係を調べた。(Shin D H, Ishihara M, Park C W, Matsubara H, Kaneko S, Kotani T, Yamashita M, Todo T, Adachi S, Yamauchi K.)
• The role of androgen on early oogenesis in eel

  Senaha A, Matsubara H, Takahashi T, Todo T, Adachi S, Yamauchi K  Zoological Science  1503  -1503  2003/12  [Not refereed][Not invited]
   

  前卵黄形期のニホンウナギ雌に11-ケトテストステロンを経口投与したところ、卵母細胞の成長が促進した。（Senaha A, Matsubara H, Takahashi T, Todo T, Adachi S, Yamauchi K.）Zoological Science 2003; 20: p1603.ポスター発表
• Induction of oogenesis by injections of salmon pituitary homogenates in hypophysectomized immature Japanese eel

  Matsubara H, Senaha A, Takei N, Shin D H, Ijiri S, Todo T, Adachi S, Yamauchi K  Zoological Science  1603  -1603  2003/12  [Not refereed][Not invited]
   

  脳下垂体を摘除したニホンウナギ雌に人為催熟を施したところ卵成長が進行した。（Matsubara H, Senaha A, Takei N, Shin D H, Ijiri S, Todo T, Adachi S, Yamauchi K.）Zoological Science 2003; 20: p1603.ポスター発表
• SEARCH OF THE COELOMIC FLUID SPECIFIC PROTEINS IN FEMALE FISH(Endocrinology,Abstracts of papers presented at the 74^ Annual Meeting of the Zoological Society of Japan) :

  Miyashita Tadashi, Matsubara Takahiro, Hara Akihiko, Todo Takashi, Adachi Shinji, Yamauchi Kohei  Zoological science  20-  (12)  1603  -1603  2003
• THE ROLE OF ANDROGEN IN EARLY OOGENESIS IN THE JAPANESE EEL (ANGUILLA JAPONICA)(Endocrinology,Abstracts of papers presented at the 74^ Annual Meeting of the Zoological Society of Japan) :

  Senaha Ayako, Matsubara Hajime, Takahashi Takayuki, Todo Takashi, Adachi Shinji, Yamauchi Kohei  Zoological science  20-  (12)  1603  -1603  2003
• Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.) (vol 28, pg 353, 2003)

  H 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  29-  (3)  253  -253  2003  [Not refereed][Not invited]
  
• COMPARISON OF THE LIGAND SPECIFICITY OF PROGESTOGEN RECEPTORS α AND β

  IKEUCHI Toshitaka, KOBAYASHI Tohru, TODO Takashi, NAGAHAMA Yoshitaka  (17)  68  2002/12/01
• ニジマスsteroidogenic acute regulatory protein(StAR)のcDNAクローニング

  東藤孝  新潟大学理学部附属臨海実験所研究概要・年次報告  (31)  8  -10  2001/07  [Not refereed][Not invited]
  
• ウナギ精巣における2種類の最終成熟誘起ステロイド受容体

  東藤孝  新潟大学理学部附属臨海実験所研究概要・年次報告  (30)  10  -11  2000/05  [Not refereed][Not invited]
  
• 第52回Yamada Conference International Symposium on Molecular Steroidogenesisに参加して

  東藤 孝  日本比較内分泌学会ニュース = Newsletter of Japan Society for Comparative Endocrinology  97-  25  -26  2000/05  [Not refereed][Not invited]
  
• Molecular cloning of two androgen receptor cDNAs from eeltestis.

  東藤孝  新潟大学理学部附属臨海実験所研究概要・年次報告  (29)  8  -9  1999/07  [Not refereed][Not invited]
  
• Two progestogen receptor subtypes from testes of Japanese eel : Anguilla japonica(Endocrinology)(Proceedings of the Seventieth Annual Meeting of the Zoological Society of Japan) :

  Ikeuchi T., Todo T., Kobayashi T., Nagahama Y.  Zoological science  16-  6  -6  1999
• Functional organization of steroid hormone receptors in eel.

  東藤孝  新潟大学理学部附属臨海実験所研究概要・年次報告  (28)  6  -7  1998/04  [Not refereed][Not invited]
  
• 11-KETOTESTOSTERONE (SPERMATOGENESIS-INDUCING HORMONE) RECEPTOR IN EEL TESTES(Developmental Biology)(Proceedings of the Sixty-Ninth Annual Meeting of the Zoological Society of Japan) :

  Ikeuchi T., Todo T., Kobayashi T., Nagahama Y.  Zoological science  15-  83  -83  1998
• EXPRESSION OF STEROIDOGENIC ENZYME mRNAS IN OVARIAN FOLLICLE OF RAINBOW TROUT, ONCORHYNCHUS MYKISS

  TODO Takashi, YOUNG Graham, GUAN Guijun, TANAKA Minoru, KOBAYASHI Tohru, NAGAHAMA Yoshitaka  Proceedings of the Japan Society for Comparative Endocrinology  12-  43  -43  1997/07

Research Grants & Projects

• Analysis of vitellogenesis in fish using genome genome editing technology

  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
• Studies to find a mechanism of gonadal growth stimulation in sea urchin

  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
• Is there gonadal stimulation hormone in sea urchins?

  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.
• Studies on molecluar mechanism of lipid droplet formation in telesot oocytes

  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
• Study of the role of two AR subtypes on spiggin synthesis in stickleback kidney

  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.
• Studies on mechanism of lipid uptake into teleost oocytes

  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.
• Mechanism of previtellogenic oocyte growth in fish

  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.
• Studies of fish vitellogenesis based on the multiple vitellogenin model

  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.
• Analysis of mechanisms controlling gonadal sex change in fish using an in vitro culture system

  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.
• 催熟技術改善のためのウナギ卵形成機構の解析

  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。
• ウナギ雌の性成熟過程を制御する性ステロイドホルモンの作用機構に関する研究

  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。
• The behavioral and physiological study on lunar-related maturation and spawning in honeycomb grouper.

  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.
• 魚類の生殖に及ぼす内分泌撹乱化学物質のエストロゲン受容体を介した作用機構の解析

  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。
• INFLOUENCE OF ENDOCREINE DISRUPTORS ON EMBRYO AND FRY OF VIVIPAROUS FISH.

  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.

Educational Activities

Teaching Experience