Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Fisheries Sciences　Marine Life Science　Aquaculture Biology

Affiliation (Master)

• Faculty of Fisheries Sciences　Marine Life Science　Aquaculture Biology

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Profile and Settings

Degree

• Fisheries science（Hokkaido University）

Profile and Settings

• Name (Japanese)

  Hiramatsu

• Name (Kana)

  Naoshi

• Name

  201001027884272437

Achievement

Research Interests

• Sebastes   breeding   bioreactor   gene modification   genome editing   seed production   aquaculture   ビテロジェニン   卵形成   魚類   ビテロジェニン受容体   受容体   リポ蛋白   魚類繁殖生理学   サケ科魚類   水産学   卵黄蛋白   バイオマーカー   エストロジェン   免疫測定法   卵黄タンパク質   環境毒性学   ビテロゲニン   Fish Vitellogenin   

Research Areas

• Life sciences / Aquaculture

Research Experience

• 2012/04 - Today Hokkaido University Faculty of Fisheries Sciences Associate Professor
• 2007/04 - 2012/03 Hokkaido University Faculty of Fisheries Sciences Assistant professor
• 2006/07 - 2007/03 Hokkaido University Faculty of Fisheries Sciences Assistant professor
• 2001/11 - 2006/06 ノースカロライナ州立大学 主任研究員
• 2001 - 2006 Senior research associate in North Carolina State University
• 2000/11 - 2001/11 ノースカロライナ州立大学 研究員
• 2000 - 2001 Research associate in North Carolina State University
• 1998/04 - 2000/10 日本学術振興会 特別研究員（PD）
• 1996 - 2000 Research fellowship for young scientist (JSPS)
• 1996/04 - 1998/03 日本学術振興会 特別研究員(DC2)

Education

• 1995/04 - 1998/03  Hokkaido University
•        - 1998  Hokkaido University  Graduate School, Division of Fisheries
• 1993/04 - 1995/03  Hokkaido University
• 1989/04 - 1993/03  Hokkaido University  School of Fisheries Sciences
•        - 1993  Hokkaido University  Faculty of Fisheries

Awards

• 2010/03 日本水産学会 水産学奨励賞
   魚類の卵形成に関与する卵黄前駆物質および その受容体に関する研究 

  受賞者: 平松 尚志

Published Papers

• Serum lipocalin-like protein in Sebastes rockfish: A novel biomarker for reproductive stage males

  Yo Yamaguchi, Takuma Kawasaki, Akihiko Hara, Takashi Todo, Naoshi Hiramatsu

  Aquaculture 583 0044-8486 2024/03/30 

  Viviparous rockfishes (Sebastes spp.) are ecologically and economically important fisheries and aquaculture species. Because the males lack obvious morphological characteristics related to reproductive status, it is challenging to select semen donors for artificial insemination. We previously identified lipocalin-type prostaglandin D2 synthase homolog protein (LPGDShp) in the urine of male black rockfish (Sebastes schlegelii). Subsequently, LPGDShp-like protein (hereafter, lipocalin-like protein) was immunologically detected in the serum of two Sebastes species, black rockfish and white-edged rockfish (S. taczanowskii). In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed to explore the potential of serum lipocalin-like protein as a biomarker for selecting mature males. The ELISA measured lipocalin-like protein in the sera of black rockfish and white-edged rockfish over an assay range between 0.391 and 12.5 ng/mL. In male black rockfish, serum levels of lipocalin-like protein were higher during the copulation season, and they were higher than levels in females during both the copulation and non-copulation seasons. In male white-edged rockfish sampled nearly monthly for a year, serum lipocalin-like protein levels showed synchronous dynamics with gonadosomatic index (GSI). Histological examination of testis revealed that the serum levels were elevated in the late/functional maturation stages, as compared to early/mid-maturation and resting stages. In both rockfish species, while serum levels of lipocalin-like protein were positively correlated with GSI in males, no such trend was observed in females. These results indicate, for the first time, that serum lipocalin-like protein can be employed as a hematological biomarker to detect the reproductive status of male rockfishes.
• Variation in responses to photoperiods and temperatures in Japanese medaka from different latitudes.

  Ai Shinomiya, Daisuke Adachi, Tsuyoshi Shimmura, Miki Tanikawa, Naoshi Hiramatsu, Shigeho Ijiri, Kiyoshi Naruse, Mitsuru Sakaizumi, Takashi Yoshimura

  Zoological letters 9 (1) 16 - 16 2023/07/22 

  Seasonal changes are more robust and dynamic at higher latitudes than at lower latitudes, and animals sense seasonal changes in the environment and alter their physiology and behavior to better adapt to harsh winter conditions. However, the genetic basis for sensing seasonal changes, including the photoperiod and temperature, remains unclear. Medaka (Oryzias latipes species complex), widely distributed from subtropical to cool-temperate regions throughout the Japanese archipelago, provides an excellent model to tackle this subject. In this study, we examined the critical photoperiods and critical temperatures required for seasonal gonadal development in female medaka from local populations at various latitudes. Intraspecific differences in critical photoperiods and temperatures were detected, demonstrating that these differences were genetically controlled. Most medaka populations could perceive the difference between photoperiods for at least 1 h. Populations in the Northern Japanese group required 14 h of light in a 24 h photoperiod to develop their ovaries, whereas ovaries from the Southern Japanese group developed under 13 h of light. Additionally, Miyazaki and Ginoza populations from lower latitudes were able to spawn under short-day conditions of 11 and 10 h of light, respectively. Investigation of the critical temperature demonstrated that the Higashidori population, the population from the northernmost region of medaka habitats, had a critical temperature of over 18 °C, which was the highest critical temperature among the populations examined. The Miyazaki and the Ginoza populations, in contrast, were found to have critical temperatures under 14 °C. When we conducted a transplant experiment in a high-latitudinal environment using medaka populations with different seasonal responses, the population from higher latitudes, which had a longer critical photoperiod and a higher critical temperature, showed a slower reproductive onset but quickly reached a peak of ovarian size. The current findings show that low latitudinal populations are less responsive to photoperiodic and temperature changes, implying that variations in this responsiveness can alter seasonal timing of reproduction and change fitness to natural environments with varying harshnesses of seasonal changes. Local medaka populations will contribute to elucidating the genetic basis of seasonal time perception and adaptation to environmental changes.
• 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.

  Yo Yamaguchi, Jun Nagata, Osamu Nishimiya, Takuma Kawasaki, Naoshi Hiramatsu, Takashi Todo

  Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 261 111055 - 111055 2021/11 [Refereed]
   

  Fundamental knowledge on the regulation of reproduction by gonadotropins (Gths) is quite limited in viviparous fishes. In the present study, we performed molecular cloning and characterization of cDNAs encoding two Gth subunits (fshb and lhb) from the pituitaries of viviparous white-edged rockfish, Sebastes taczanowskii; expression profiles of both gene transcripts were elucidated in the pituitaries of reproductive males and females which were kept in a captive environment. The cloned fshb and lhb fragments exhibited high sequence identities with corresponding β-subunit sequences from black rockfish, S. schlegelii. Notably, the fshb of white-edged rockfish appeared to lack a putative N-glycosylation site, whereas lhb conserved it. Expression of fshb and lhb transcripts in the rockfish pituitaries largely changed in synchrony but for minor exceptions. In males, levels of both transcripts increased with progression of spermatogenesis, although the peak for fshb (October) appeared slightly earlier than that for lhb (November). In females, both gene transcripts exhibited synchronous bimodal changes. High expression of fshb and lhb transcripts in the female pituitary during the gestation period, followed by the drastic decrease at parturition, suggest their possible involvement in regulation of gestation of this species. The knowledge gained for Sebastes in this study superimposes fundamental information necessary for further physiological understanding of viviparity in teleost fish.
• 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-17β.

  Jun Nagata, Yuji Mushirobira, Osamu Nishimiya, You Yamaguchi, Toshiaki Fujita, Naoshi Hiramatsu, Akihiko Hara, Takashi Todo

  General and comparative endocrinology 310 113812 - 113812 2021/09/01 [Refereed]
   

  Estradiol-17β (E2) regulates transcription of estrogen-responsive genes via estrogen receptors (Esr). In many teleost species, choriogenin (chg), vitellogenin (vtg) and esr genes are transactivated by E2 in the liver. This study aimed i) to compare expression properties of all subtypes of these genes (chg: chgHα, chgHβ, chgL; vtg: vtgAs, vtgC; esr: esr1a, esr1b, esr2a, esr2b) in response to estrogen stimulation, and ii) to confirm how each of four Esr subtypes is involved in the transcriptional regulation of these estrogen-responsive genes in cutthroat trout hepatocytes. In hepatocytes in primary culture, all chg and vtg subtype mRNA levels, and those of esr1a, were increased by E2 treatment (10-6 M) at 24 and 72 h post initiation (hpi), but esr1b, esr2a and esr2b mRNA levels were not. Treatment of hepatocytes with various concentrations of E2 (10-11-10-6 M) induced dose-dependent increases in the levels of all chg and vtg subtype mRNAs at 24 and 72 hpi. At both time points, the lowest dose that induced a significant increase in the expression levels of mRNAs (LOEC) for E2 differed among the genes; LOECs were estimated as 10-11 M for chgHα at 24 hpi, as 10-9 M for vtgC at 72 hpi, and as 10-10 M for other mRNAs at both 24 and 72 hpi. Meanwhile, the levels of esr1a mRNA exhibited a dose-dependent increase at 24 and 72 hpi, but the LOEC shifted from 10-9 M at 24 hpi to 10-7 M at 72 hpi because of a decrease in mRNA levels at treatment groups exposed to high concentrations of E2. All Esr subtypes transactivated chg, vtg and esr1a promoters in the presence of E2 in vitro. The activation levels indicated that promoter activity of chgHα ≥ vtgAs > chgHβ > chgL ≥ vtgC ≥ esr1a when mediated by Esr1a, chgHβ > chgHα > chgHL > vtgAs ≥ vtgC ≥ esr1a by Esr1b, chgHβ ≥ chgL > chgHα ≥ vtgAs > vtgC > esr1a by Esr2a, and chgHβ ≥ chgHα ≥ vtgAs > chgL ≥ vtgC > esr1a by Esr2b. Collectively, different Esr subtypes were distinctly different in their ability to transactivate estrogen-responsive target genes, resulting in differential expression of chg, vtg and esr1a genes in the estrogen-exposed hepatocytes.
• Changes in the Hepatic Expression of Three Vitellogenin Subtype Genes During Ovarian Development in Female White-Edged Rockfish (Sebastes taczanowskii)

  Jun Nagata, Satoru Wada, Osamu Nishimiya, Meiqin Wu, Yuji Mushirobira, Yo Yamaguchi, Takanori Yokono, Takuma Kawasaki, Takahiro Matsubara, Takashi Todo, Akihiko Hara, Naoshi Hiramatsu

  Zoological Science 38 (5) 0289-0003 2021/08/18
• Knock out of a major vitellogenin receptor gene with eight ligand binding repeats in medaka (Oryzias latipes) using the CRISPR/Cas9 system.

  Jin Namgung, Hiroko Mizuta, Yo Yamaguchi, Jun Nagata, Takashi Todo, Ozlem Yilmaz, Naoshi Hiramatsu

  Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 257 110967 - 110967 2021/07 [Refereed]
   

  Recent studies of vitellogenesis engendered a novel model of teleost yolk formation in which multiple yolk precursors, vitellogenins (Vtgs), and their receptors (Vtgrs) interact to ensure proper yolk composition for embryonic development and larval growth. As a step toward verification of this concept, we examined the role of one candidate Vtgr, termed low-density lipoprotein receptor relative with eight ligand-binding repeat (Lr8), in the medaka, a representative teleost and established laboratory model. A homozygous lr8 knock out (lr8-KO) medaka was produced to perform reverse-genetic functional analyses. In ovaries of wild type (WT) medaka, Western blotting detected a putative Lr8 protein band at ~130 kDa, while immunohistochemistry detected the putative Lr8 signal at the periphery of the oocyte underneath the zona radiata. These signals disappeared in ovaries of the lr8-KO group. Offspring of lr8-KO medaka exhibited decreased survival rate compared to WT fish, but KO of lr8 was not 100% lethal. There was no significant difference in total yolk protein content or size of eggs between WT and lr8-KO fish. However, LC-MS/MS analyses revealed a remarkable decrease in the relative abundance of yolk proteins derived from VtgAb in lr8-KO eggs, in conjunction with a compensatory increase in proteins derived from VtgAa1. These findings strongly support the conclusion that Lr8 is an important receptor for VtgAb in medaka. The disruption of proper yolk composition by lr8-KO is possibly one cause of the low offspring survival.
• Development and characterization of polyclonal antibodies against subtype specific vitellogenin of the dojo loach, Misgurnus anguillicaudatus

  Weilong Wang, Qingping Lian, Yuange Chen, Naoshi Hiramatsu, Meiqin Wu

  AQUACULTURE 532 0044-8486 2021/02 [Refereed]
   

  Fish vitellogenin (Vtg) has become an important biomarker for assessing the estrogenic potency of chemicals and the exposure of animals to estrogenic contaminants present in aquatic environments. In the current study, the polyclonal antibodies against subtype-specific Vtgs of the dojo loach (Misgurnus anguillicaudatus) were developed and identified by gene recombination and expression. Immuno-biochemical analyses revealed that VtgAo1 protein appeared to be the major Vtg type in this species. Enhanced chemiluminescent Western blotting was developed using the antiserum against VtgAo1. Exposure of male loach to 17 alpha-ethinylestradiol (EE2) and 17 beta-estradiol (E2) via water induced the VtgAo1 protein induction with a lowest-observed-effect concentration (LOEC) by 100 ng L-1 and 1000 ng L(-1)group at 7 day post-initiation (dpi), respectively. The results indicated that this method provided a valuable tool for detecting estrogenic activities in aquatic environments and excluded any difficulties expected in purification procedures to separate a highly pure Vtg subtype from circulating proteins, and the other Vtg subtypes.
• 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 日本水産増殖学会 68 (1) 1 - 8 0371-4217 2020/04 [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 0016-6480 2019/09 [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-17 beta (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 mu g/mL (VtgAa), 57.9 +/- 30.7 mu g/mL (VtgAb) and 12.6 +/- 4.8 mu g/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 mu g/mL, respectively, over various sampling periods. In vitellogenic females sampled in January, the average level of VtgAb (8,744.43 +/- 733.93 mu g/mL) was significantly higher than for VtgAa (150.33 +/- 22.35 mu g/mL) or VtgC (57.08 +/- 6.00 mu g/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 [Refereed][Not invited]
   

  Estradiol-17 beta (E-2) 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 E-2 would stimulate ovarian uptake of vitellogenin (Vtg). Early pubertal eels received hormone implants containing varying concentrations of E-2 (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, E-2 increased hepatic production and plasma levels of Vtg. In contrast, 11KT decreased plasma levels of Vtg, seemingly reducing its production. Neither 11KT nor E-2 could induce uptake of Vtg into oocytes, although E-2 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 E-2, 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.Combined treatment with estradiol-17 beta and 11-ketotestosterone results in evident yolk accumulation in the shortfinned eel ovary, showing the potential of steroid co-treatment to induce vitellogenesis, even in the absence of exogenous gonadotropins.
• 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 0016-6480 2019/01 [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 mu g/ml, respectively, during various sampling periods. In vitellogenic females (October), serum VtgAb levels (1,192.05 +/- 237.81 mu g/ml) were significantly higher than levels of the other two Vtg subtypes (120.82 +/- 30.42 and 119.23 +/- 16.95 mu g/ml for VtgAa and VtgC, respectively). When immature mullet were fed diets containing 17 alpha-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 mu g/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 [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.
• Production of recombinant salmon insulin-like growth factor binding protein-1 subtypes

  Hanae Tanaka, Gakuto Oishi, Yusuke Nakano, Hiroko Mizuta, Yuta Nagano, Naoshi Hiramatsu, Hironori Ando, Munetaka Shimizu

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 257 184 - 191 0016-6480 2018/02 [Refereed][Not invited]
   

  Insulin-like growth factor (IGF)-I is a growth promoting hormone that exerts its actions through endocrine, paracrine and autocrine modes. Local IGF-I is essential for normal growth, whereas circulating IGF-I plays a crucial role in regulating the production and secretion of growth hormone (GH) by the pituitary gland. These actions of IGF-I are modulated by six insulin-like growth factor binding proteins (IGFBPs). In teleosts, two subtypes of each IGFBP are present due to an extra round of whole-genome duplication. IGFBP-1 is generally inhibitory to IGF-I action under catabolic conditions such as fasting and stress. In salmon, IGFBP-1a and -1b are two of three major circulating IGFBPs and assumed to affect growth through modulating IGF-I action. However, exact functions of salmon IGFBP-1 subtypes on growth regulation are not known due to the lack of purified or recombinant protein. We expressed recombinant salmon (rs) IGFBP-1a and -1b with a fusion protein (thioredoxin, Trx) and a His-tag using the pET-32a(+) vector expression system in Escherichia coli. Trx.His.rsIGFBP-1s were isolated by Ni-affinity chromatography, enzymatically cleaved by enterokinase to remove the fusion partners and further purified by reversed-phase HPLC. We next examined effects of rsIGFBP-1a and -1b in combination with human IGF-I on GH release from cultured masu salmon (Oncorhynchus masou) pituitary cells. Unexpectedly, IGF-I increased GH release and an addition of rsIGFBP-1a, but not rsIGFBP-1b, restored GH levels. The results suggest that IGFBP-1a can inhibit IGF-I action on the pituitary in masu salmon. Availability of recombinant salmon IGFBP-1s should facilitate further functional analyses and assay development. (C) 2017 Elsevier Inc. All rights reserved.
• 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 [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.
• 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.
• 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.
• 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
• 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 (Pt B) 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.
• Vitellogenesis and choriogenesis in fishes

  Akihiko Hara, Naoshi Hiramatsu, Toshiaki Fujita

  FISHERIES SCIENCE 82 (2) 187 - 202 0919-9268 2016/03 [Refereed][Not invited]
   

  In studies of sex discrimination in fish in the early 1900s, a specific antigen in the blood of gravid females was identified using immunological methods. At present, this specific antigen is known as vitellogenin, the major precursor of egg yolk protein that is synthesized in the female liver and is secreted into the blood to be incorporated into the egg. Recently, protein and gene analyses have revealed the presence of several vitellogenin variants. In addition, in the 1980s, choriogenin was identified as a novel precursor of egg envelope proteins that is secreted into the blood in response to stimulation by estrogen, similarly to vitellogenin. These two proteins not only play key roles in the process of oogenesis, but they are also used as effective biomarkers for assessing the impact of estrogen-like endocrine-disrupting chemicals (environmental hormones) in aquatic ecosystems.
• 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.
• How do eggs get fat? Insights into ovarian fatty acid accumulation in the shortfinned eel, Anguilla australis

  Erin L. Damsteegt, Hiroko Mizuta, Naoshi Hiramatsu, P. Mark Lokman

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 221 94 - 100 0016-6480 2015/09 [Refereed][Not invited]
   

  Previous research using eels has shown that 11-ketotestosterone can induce ovarian triacylglyceride accumulation both in vivo and in vitro. Further, accumulation is dramatically enhanced in the presence of very-low density lipoprotein. This study examined the involvement of the low density lipoprotein receptor and vitellogenin receptor in oocyte lipid accumulation. Specific antisera were used in an attempt to block the vitellogenin receptor and/or the low density lipoprotein receptor. Accordingly, incubation with the low density lipoprotein receptor antiserum clearly reduced the oocyte diameter and the amount of oil present within the oocyte. In contrast, blocking the vitellogenin receptor had little effect on either oocyte surface area or the abundance of oil droplets in the cytosol. In keeping with birds, we conclude that the low density lipoprotein receptor is a major player involved in mediating ovarian fatty acid accumulation in the eel. However, lipoprotein lipase-mediated fatty acid accumulation also remains conceivable, for example through interactions between this enzyme and the low density lipoprotein receptor. (C) 2015 Elsevier Inc. All rights reserved.
• Machine learning reveals sex-specific 17-estradiol-responsive expression patterns in white perch (Morone americana) plasma proteins

  Justin Schilling, Angelito I. Nepomuceno, Antonio Planchart, Jeffrey A. Yoder, Robert M. Kelly, David C. Muddiman, Harry V. Daniels, Naoshi Hiramatsu, Benjamin J. Reading

  PROTEOMICS 15 (15) 2678 - 2690 1615-9853 2015/08 [Refereed][Not invited]
   

  With growing abundance and awareness of endocrine disrupting compounds (EDCs) in the environment, there is a need for accurate and reliable detection of EDC exposure. Our objective in the present study was to observe differences within and between the global plasma proteomes of sexually mature male and female white perch (Morone americana) before (Initial Control, IC) and after 17-estradiol (E-2) induction. Semiquantitative nanoLC-MS/MS data were analyzed by machine learning support vector machines (SVMs) and by two-way ANOVA. By ANOVA, the expression levels of 44, 77, and 57 proteins varied significantly by gender, treatment, and the interaction of gender and treatment, respectively. SVMs perfectly classified male and female perch IC and E-2-induced plasma samples using the protein expression data. E-2-induced male and female perch plasma proteomes contained significantly higher levels of the yolk precursors vitellogenin Aa and Ab (VtgAa, VtgAb), as well as latrophilin and seven transmembrane domain-containing protein 1 (Eltd1) and kininogen 1 (Kng1). This is the first report that Eltd1 and Kng1 may be E-2-responsive proteins in fishes and therefore may be useful indicators of estrogen induction.
• 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.
• Proportional Accumulation of Yolk Proteins Derived From Multiple Vitellogenins is Precisely Regulated During Vitellogenesis in Striped Bass (Morone saxatilis)

  Valerie N. Williams, Benjamin J. Reading, Haruna Amano, Naoshi Hiramatsu, Justin Schilling, Scott A. Salger, Taufika Islam Williams, Kevin Gross, Craig V. Sullivan

  JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL AND INTEGRATIVE PHYSIOLOGY 321 (6) 301 - 315 2471-5638 2014/07 [Refereed][Not invited]
   

  We quantified three vitellogenins (VtgAa, VtgAb, VtgC) or their derived yolk proteins (YPs) in the liver, plasma, and ovary during pre-vitellogenic (PreVG), mid-vitellogenic (MVG), and late-vitellogenic (LVG) oocyte growth and during post-vitellogenesis (PostVG) in the striped bass (Morone saxatilis) using label-free quantitative mass spectrometry (MS). Western blotting of the samples using antisera raised against gray mullet (Mugil cephalus) lipovitellins derived from VtgAa, VtgAb, and VtgC confirmed the MS results. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed liver as the primary site of expression for all three Vtgs, with extrahepatic transcription weakly detected in ovary, foregut, adipose tissue, and brain. Quantitative real-time RT-PCR confirmed vtgAb to be primarily expressed in liver and VtgAb proteins were predominant in liver and plasma from MVG to PostVG. However, the primary period of deposition into oocytes of VtgAb occurred up until MVG, whereas VtgAa was primarily deposited from MVG to LVG. The VtgC was gradually taken up by oocytes throughout vitellogenesis and was detected at trace levels in plasma. The ratio of yolk proteins derived from VtgAa, VtgAb, VtgC (YPAa/YPAb/YPC) in PostVG ovary is 1.4:1.4:1, which differs from ratios previously reported for other fish species in that YPC comprises a greater proportion of the egg yolk. Our results indicate that proportional accumulation of multiple Vtgs in the yolk may depend both on the precise rates of their hepatic secretion and specific uptake by oocytes. Furthermore, composition of the Vtg-derived yolk may vary among Acanthomorph fishes, perhaps reflecting their different early life histories and reproductive strategies. (C) 2014 Wiley Periodicals, Inc.
• 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.
• Multiple vitellogenins and product yolk proteins in striped bass, Morone saxatilis: molecular characterization and processing during oocyte growth and maturation

  V. N. Williams, B. J. Reading, N. Hiramatsu, H. Amano, N. Glassbrook, A. Hara, C. V. Sullivan

  FISH PHYSIOLOGY AND BIOCHEMISTRY 40 (2) 395 - 415 0920-1742 2014/04 [Refereed][Not invited]
   

  The multiple vitellogenin (Vtg) system of striped bass, a perciform species spawning nearly neutrally buoyant eggs in freshwater, was investigated. Vitellogenin cDNA cloning, Western blotting of yolk proteins (YPs) using Vtg and YP type-specific antisera, and tandem mass spectrometry (MS/MS) of the YPs revealed the complex mechanisms of yolk formation and maturation in this species. It was discovered that striped bass possesses a tripartite Vtg system (VtgAa, VtgAb, and VtgC) in which all three forms of Vtg make a substantial contribution to the yolk. The production of Vtg-derived YPs is generally similar to that described for other perciforms. However, novel amino-terminal labeling of oocyte YPs prior to MS/MS identified multiple alternative sites for cleavage of these proteins from their parent Vtg, revealing a YP mixture far more complex than reported previously. This approach also revealed that the major YP product of each form of striped bass Vtg, lipovitellin heavy chain (LvH), undergoes limited degradation to smaller polypeptides during oocyte maturation, unlike the case in marine fishes spawning buoyant eggs in which LvHAa undergoes extensive proteolysis to osmotically active free amino acids. These differences likely reflect the lesser need for hydration of pelagic eggs spawned in freshwater. The detailed characterization of Vtgs and their proteolytic fate(s) during oocyte growth and maturation establishes striped bass as a freshwater model for investigating teleost multiple Vtg systems.
• 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 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 similar to 95-105 kDa 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. (C) 2013 Elsevier Inc. All rights reserved.
• 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.
• Relationships between gill Na+,K+-ATPase activity and endocrine and local insulin-like growth factor-I levels during smoltification of masu salmon (Oncorhynchus masou)

  Takahiro Shimomura, Takuro Nakajima, Moeri Horikoshi, Anai Iijima, Hirokazu Urabe, Shinya Mizuno, Naoshi Hiramatsu, Akihiko Hara, Munetaka Shimizu

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 178 (2) 427 - 435 0016-6480 2012/09 [Refereed][Not invited]
   

  We established profiles of insulin-like growth factor (IGF)-I mRNA in the liver, gill and white muscle and circulating IGF-I during smoltification of hatchery-reared masu salmon, and compared with that of gill Na+,K+-ATPase (NKA) activity. Gill NKA activity peaked in May and dropped in June. Liver igf1 mRNA was high in March and decreased to low levels thereafter. Gill igf1 increased from March, maintained its high levels during April and May and decreased in June. Muscle igf1 mRNA levels were relatively high during January and April when water temperature was low. Serum IGF-I continuously increased from March through June. Serum IGF-I during March and May showed a positive correlation with NKA activity, although both were also related to fish size. These parameters were standardized with fork length and re-analyzed. As a result, serum IGF-I and gill igf1 were correlated with NKA activity. On the other hand, samples from desmoltification period (June) that had high serum IGF-I levels and low NKA activity disrupted the relationship. Expression of two IGF-I receptor (igf1r) subtypes in the gill decreased in June, which could account for the disruption by preventing circulating IGF-I from acting on the gill and retaining it in the blood. The present study suggests that the increase in gill NKA activity in the course of smoltification of masu salmon was supported by both endocrine and local IGF-I, and the decrease during desmoltification in freshwater was due at least in part to the down-regulation of gill IGF-I receptors. (C) 2012 Elsevier Inc. All rights reserved.
• Effects of ethynylestradiol on vitellogenin synthesis and sex differentiation in juvenile grey mullet (Mugil cephalus) persist after long-term exposure to a clean environment

  Jun-ya Aoki, Ayaka Hatsuyama, Naoshi Hiramatsu, Kiyoshi Soyano

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY 154 (4) 346 - 352 1532-0456 2011/11 [Refereed][Not invited]
   

  We investigated the continuing effects of exposure to ethynylestradiol (EE2) in juvenile grey mullet after transfer to a clean environment. Eleven-month-old juvenile fish containing immature phenotype gonad were fed dry diets: the low and high EE2-treated groups were fed diets with 0.04 and 4 mu g EE2/g body weight for 4 weeks, respectively. After treatment, they were transferred to clean seawater, and reared with an EE2 free diet for 350 days. Vitellogenin (VTG) was not detected in the serum of the control group throughout the experimental period. However, in both treatment groups, abnormal values of serum VTG were detected until approximately 100 days after transfer to a clean environment. In the control group, sex differentiation was not confirmed until 206 days after transfer to a clean environment. However, some of the fish in the 0.04 mu g EE2-treated group had ovarian cavity and oocytes at 26 days. In most of the fish in the 4 mu g EE2-treated group, the ovarian cavity had already appeared at the end of EE2 treatment (0 day), and oocytes were observed at 26 days, suggesting that EE2 accelerates ovarian differentiation. These results suggest that previous exposure to EE2 has long-term effects on VTG synthesis and gonadal development. (C) 2011 Elsevier Inc. All rights reserved.
• 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 [Refereed][Not invited]
  
• 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.
• Disparate Binding of Three Types of Vitellogenin to Multiple Forms of Vitellogenin Receptor in White Perch

  Benjamin J. Reading, Naoshi Hiramatsu, Craig V. Sullivan

  BIOLOGY OF REPRODUCTION 84 (2) 392 - 399 0006-3363 2011/02 [Refereed][Not invited]
   

  Three types of white perch (Morone americana) vitellogenin (VtgAa, VtgAb, and VtgC) were purified, labeled with digoxigenin (DIG), and subjected to Vtg receptor (Vtgr) binding assays in 96-well plates coated with perch ovarian membrane proteins or to ligand blotting procedures. Binding specificity was evaluated by incubating membrane protein preparations with constant amounts of DIG-Vtg tracer (VtgAa, VtgAb, VtgC, or a mixture of VtgAa and VtgAb [VtgAa/b]) alone or in the presence of unlabeled Vtg ligands. At 250-fold excess molar concentration relative to the tracer, VtgAa and VtgAb were each able to displace only approximately 50% of bound DIG-VtgAa/b, but VtgAa/b could fully displace DIG-VtgAa and DIG-VtgAb under the same conditions. Over a broad range of excess molar ratios, unlabeled VtgAa and VtgAb each displaced their respective DIG-Vtg tracer much more effectively than each did the heterologous tracer (DIG-VtgAb and DIG-VtgAa, respectively). Ligand blotting revealed three forms of Vtgr, a large receptor (>212 kDa) that bound only to VtgAa and two smaller receptors (similar to 116 and similar to 110.5 kDa) that bound preferentially to VtgAb. The VtgC did not specifically bind to ovarian membrane proteins in either assay. Collectively, these results indicate the presence of a system of multiple ovarian Vtgrs with disparate binding to the three types of Vtg present in higher-order teleosts (Acanthomorpha). To our knowledge, this is the first report on binding of multiple types of Vtg to multiple forms of Vtgr in any vertebrate.
• Evaluation of environmental estrogenic activities in the Yangtze River estuary: utilization of vitellogenin production in red lip mullet.

  Mei-qin WU, Lei HONG, Wenshu LUO, Naoshi HIRAMATSU, Junya AOKI, Kiyoshi SOYANO, Jun-sheng ZHONG, Akihiko HARA

  Acuaculture Sci. 水産増殖談話会 59 (1) 65 - 73 0371-4217 2011 [Refereed][Not invited]
   

  本研究では、中国揚子江河口における3つの汽水域について、エストロジェン活性の有無を調査した。調査はメナダ（Chelon haematocheilus）を用い、本種のエストロジェン感受性蛋白であるビテロジェニンBサブタイプ（VgB）の血中レベルと肝臓でのmRNA発現をマーカーとし、化学発光免疫測定法（CLIA）とリアルタイムPCR測定系によりこれらを測定した。同調査域から捕獲されたメナダにおいて、VgB蛋白およびmRNAの発現は、測定系の検出限界以下もしくは極めて低く、人工海水環境で順致された個体から得たコントロール値とほぼ同一であった。また、同捕獲個体の生殖腺は、40個体中1個体に精巣卵が観察された以外、異常な形態を示すものは全く無かった。このことより調査域はエストロジェン活性に関しては殆ど危険性の無い状態にあると判断された。
• 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.
• Studies on yolk precursors and their receptors involved in fish oogenesis

  Naoshi Hiramatsu

  NIPPON SUISAN GAKKAISHI 公益社団法人 日本水産学会 76 (4) 613 - 616 0021-5392 2010/07 [Not refereed][Not invited]
  
• Development of chemiluminescent immunoassay for vitellogenin in red lip mullet, Liza haematocheila, Journal of fisheries of China

  Wu M, Wada T, Luo W, Morita Y, Hiramatsu N, Hara A, Zhong J

  Journal of fisheries of China 34 (4) 581 - 589 2010 [Refereed][Not invited]
  
• Purification of multiple precursors for egg chorion proteins in Atlantic cod (Gadus morhua).

  Toshiaki Fujita, Alexander P Scott, Loanna Katsiadaki, Haruna Amano, Lei Hong, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara

  Zoological science 26 (12) 870 - 7 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 microg/ml and 768.71+/-120.39 microg/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.
• Induction of vitellogenin production in male tilapia (Oreochromis mossambicus) by commercial fish diets.

  Lori K Davis, Bradley K Fox, Chhorn Lim, Naoshi Hiramatsu, Craig V Sullivan, Tetsuya Hirano, E Gordon Grau

  Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 154 (2) 249 - 54 1095-6433 2009/10 [Refereed][Not invited]
   

  Mozambique tilapia, (Oreochromis mossambicus), are a euryhaline teleost and an important biological model species. Captive male tilapia frequently have high levels of the estrogen-induced yolk precursor protein vitellogenin (Vg), a common indicator of exposure to estrogenic compounds. Sex steroids are found in commercial fish diets, but relatively few studies have examined the relationship between commercial diets and Vg production. In a fasting experiment to ascertain a dietary role in male Vg production, plasma Vg was reduced to negligible levels after 2 weeks of fasting, while no change in estrogen receptor (ER) expression was seen. When male tilapia were fed a squid-based diet that replaced the commercial trout diet, plasma Vg was reduced to undetectable levels over 40 days, concomitant with significant reductions in hepatic expression of Vgs A, B, and C, and ERbeta, compared with control fish fed commercial trout diet. Female tilapia fed the squid-based for 20 days had no change in these parameters. When male tilapia were fed a defined, soy-based diet, plasma Vg reduced to 20% of levels in fish given either commercial trout diet or a defined, fishmeal-based diet. Overall, results from these studies suggest that estrogens in a commercial trout diet induce vitellogenin production by increasing expression of Vg, but not ER genes in male tilapia.
• 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.
• Effects of o,p'-DDE, heptachlor, and 17beta-estradiol on vitellogenin gene expression and the growth hormone/insulin-like growth factor-I axis in the tilapia, Oreochromis mossambicus.

  Lori K Davis, Nancy Visitacion, Larry G Riley, Naoshi Hiramatsu, Craig V Sullivan, Tetsuya Hirano, E Gordon Grau

  Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 149 (4) 507 - 14 1532-0456 2009/05 [Refereed][Not invited]
   

  Effects of two endocrine disruptors, o,p'-DDE and heptachlor, and 17beta-estradiol (E(2)) on vitellogenin (Vg) and the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis were examined in male tilapia. In the first experiment, fish were given 5 weekly injections of either E(2), o,p'-DDE or heptachlor (5 microg/g). E(2) treatment increased plasma Vg and hepatic expression of three Vg genes (Vgs A, B, and C) and estrogen receptor alpha (ERalpha), while reducing plasma levels of IGF-I and suppressing the expression of IGF-I, the GH receptor (GHR2) and the putative somatolactin receptor (GHR1). Neither pesticide greatly affected the other parameters examined, except for a significant reduction in expression of GHR2 and increased plasma IGF-I. In the second experiment, fish were given a single injection of o,p'-DDE or heptachlor (100 microg/g), or E(2) (5 microg/g) and sacrificed 5 days post-injection. Treatment with E(2) stimulated expression of all three Vg genes. Both o,p'-DDE and heptachlor increased expression of VgB, whereas only o,p'-DDE increased VgA expression. There was no effect of o,p'-DDE or heptachlor on VgC expression or plasma Vg levels. Treatment with o,p'-DDE and heptachlor as well as E(2) increased ERalpha and ERbeta transcript levels. Similarly, both pesticides increased GHR1 and IGF-I expression, whereas no significant effect of E(2) was observed on GHR1, GHR2 or IGF-I expression. These results indicate that o,p'-DDE and heptachlor have varying temporal and dose effects on modulation of Vg and the GH/IGF-I axis that are distinct from E(2).
• Conserved and Variant Molecular and Functional Features of Multiple Egg Yolk Precursor Proteins (Vitellogenins) in White Perch (Morone americana) and other Teleosts

  Benjamin J. Reading, Naoshi Hiramatsu, Sayumi Sawaguchi, Takahiro Matsubara, Akihiko Hara, Mark O. Lively, Craig V. Sullivan

  MARINE BIOTECHNOLOGY 11 (2) 169 - 187 1436-2228 2009/04 [Refereed][Not invited]
   

  Three complete cDNAs encoding different forms of vitellogenin (Vtg) were isolated from a white perch (Morone americana) liver cDNA library and characterized with respect to immunobiochemical and functional features of the three Vtgs and their product yolk proteins (YPs) in this species and in the congeneric striped bass (Morone saxatilis). The two longest cDNAs encoded Vtgs with a complete suite of yolk protein domains that, based on comparisons with vtg sequences from other species, were categorized as VtgAa and VtgAb using the current nomenclature for multiple teleost Vtgs. The shorter cDNA encoded a Vtg that lacked a phosvitin domain, had a shortened C-terminus, and was categorized as VtgC. Mapping of peptide sequences from the purified Vtgs and their derived YPs to Vtg sequences deduced from the cDNAs definitively identified the white perch VtgAa, VtgAb, and VtgC proteins. Detailed comparisons of the primary structures of each Vtg with partial or complete sequences of Morone yolk proteins or of Vtgs from other fishes revealed conserved and variant structural elements of teleost Vtgs with functional significance, including, as examples, signal peptide cleavage sites, dimerization sites, cathepsin D protease recognition sites, and receptor-binding domains. These comparisons also yielded an interim revision of the classification scheme for multiple teleost Vtgs.
• 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. Toxicology & pharmacology : CBP 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 B: VgB) were purified from red lip mullet. The mass of intact Chg H and Chg L were estimated to be approximately 215 kDa and approximately 69 kDa, respectively. In SDS-PAGE, Chg H and Chg L separated to positions corresponding to approximately 51 kDa and approximately 44 kDa, respectively. The mass of intact VgB was approximately 530 kDa and resolved into a polypeptide of approximately 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 estradiol-17beta (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.
• Multiple vitellogenin-derived yolk proteins in gray mullet (Mugil cephalus): disparate proteolytic patterns associated with ovarian follicle maturation.

  Haruna Amano, Toshiaki Fujita, Naoshi Hiramatsu, Hirohiko Kagawa, Takahiro Matsubara, Craig V Sullivan, Akihiko Hara

  Molecular reproduction and development 75 (8) 1307 - 17 1040-452X 2008/08 [Refereed][Not invited]
   

  Disparate proteolytic patterns of yolk proteins, derived from three types of vitellogenin (VgA, VgB, and VgC), were observed in gray mullet. Immuno-biochemical analyses of extracts obtained from vitellogenic ovaries (VO) and ovulated eggs (OE) confirmed that a large proportion of VgA-derived lipovitellin (LvA) was degraded into free amino acids (FAAs) during ovarian follicle maturation. The maturation-associated alteration of VgB-derived Lv (LvB) involved only limited proteolysis; the heavy and light LvB chains were dissociated into at least three and one polypeptide fragments, respectively. The native mass of VgC-derived Lv (LvC) exhibited little difference between VO and OE, although it was apparent that the LvC was 'nicked' during maturation, resulting in the appearance of several bands in OE. Similar analyses confirmed that VgA-derived beta'-component (beta'-cA) and VgB-derived beta'-c (beta'-cB) decreased during maturation in both quantity and native mass, while phosvitin derived from either VgA (PvA) or VgB (PvB) appeared to be degraded into FAAs. The pattern of maturation-associated proteolysis of mullet yolk proteins is similar to that reported for other marine teleosts spawning pelagic eggs. However, the depository ratio of the three distinct types of Lv in the mullet VO appeared to be different from that estimated for another marine pelagophil, the barfin flounder. These results support a recent paradigm regarding the significance of Vg multiplicity upon successive physiological events in this group of fishes including the hydration of maturing oocytes, the acquisition of proper egg buoyancy, and the generation of requisite nutrient stocks for each stage of embryogenesis and larval development.
• Molecular cloning and characterization of three distinct choriogenins in masu salmon, Oncorhynchus masou.

  Toshiaki Fujita, Haruhisa Fukada, Munetaka Shimizu, Naoshi Hiramatsu, Akihiko Hara

  Molecular reproduction and development 75 (7) 1217 - 28 1040-452X 2008/07 [Refereed][Not invited]
   

  Three cDNAs, each encoding a different choriogenin (Chg), were isolated from a female masu salmon (Oncorhynchus masou) liver cDNA library. Two of the cDNA clones, Chg Halpha and Chg Hbeta, showed a close relationship and contained the typical domains of zona pellucida (ZP) B genes in fish, namely proline and glutamine rich repeats, a trefoil factor family domain, and a ZP domain. Specific antibodies against recombinant Chg H products (rmHalpha and rmHbeta) were generated to elucidate the relationship between the Chg H cDNAs and two types of serum Chg H protein, which were previously purified and characterized, and designated as very-high-molecular-weight vitelline envelope-related protein (vhVERP) and Chg H of masu salmon. The immunobiochemical analyses revealed that the Chg Halpha and Chg Hbeta clones encoded vhVERP and Chg H proteins, respectively. The third cDNA clone (Chg L) appeared to be a ZPC gene and, by mapping the N-terminal sequence of purified Chg L, was shown to encode serum Chg L protein. Various types of heteromultimer of the three Chgs were identified immunologically as high molecular weight chorion components, indicating the involvement of complex heterodimerization of multiple Chgs in the construction of chorion architecture in masu salmon.
• Molecular alteration of three forms of vitellogenins and their product yolk proteins during oocyte growth and maturation in grey mullet (Mugil cephalus)

  Haruna Amano, Toshiaki Fujita, Naoshi Hiramatsu, Hirohiko Kagawa, Sayumi Sawaguchi, Takahiro Matsubara, Craig V. Sullivan, Akihiko Hara

  CYBIUM 32 (2) 156 - 158 0399-0974 2008/07 [Refereed][Not invited]
   

  Seven yolk proteins (YPs) derived from three distinct types of vitellogenin (VgA, VgB, and VgC) were purified from the vitellogenic ovaries of grey mullet (Mugil cephalus). Three YPs appeared to be VgA derivatives and were designated as lipovitellin A (LvA), beta'-component A (beta'-cA), and LvA-phosvitin A complex (LvA-PvA). In ovulated eggs, these VgA derivatives were mostly degraded. Three other YPs (LvB, beta'-cB, and PvB) appeared to be VgB derivatives. In eggs, beta'-cB, and PvB were hardly detected, while LvB remained as a large peptide with a slight decrease in mass. One YP component (LvC) appeared to be a VgC derivative that underwent no alteration in native mass but was proteolytically nicked during oocyte maturation. The maturation-associated proteolysis of mullet YPs is similar to that which we have reported for other marine teleosts spawning pelagic eggs, supporting the significance of Vg multiplicity to reproductive physiology in this group of fishes.
• Deduced primary structures of three vitellogenins and specific binding to putative multiple ovarian receptors in white perch (Morone americana)

  Benjamin J. Reading, Naoshi Hiramatsu, Takahiro Matsubara, Akihiko Hara, Craig V. Sullivan

  CYBIUM 32 (2) 159 - 161 0399-0974 2008/07 [Refereed][Not invited]
   

  Three complete cDNAs encoding different forms of vitellogenin (vtg) were cloned from a white perch (Morone americana) liver cDNA library. The two longest cDNAs encoded Vtgs with a complete suite of yolk protein domains that, based on comparisons with vtg sequences from other species, were categorized as VtgAa and VtgAb. The shorter cDNA encoded a Vtg that lacked a phosvitin domain, had a shortened C-terminus, and was categorized as VtgC. Ligand blots and binding assays revealed multiple distinct Vtg receptors in the ovary of perch with disparate affinities for VtgAa and VtgAb. Furthermore, in this species, VtgC does not bind to the ovarian membrane.
• Estrogen regulation of multiple vitellogenin and estrogen receptor genes and of the growth hormone-insulin like growth factor axis in tilapia

  Lori Davis, Naoshi Hiramatsu, Craig Sullivan, Tetsuya Hirano, E. Gordon Grau

  CYBIUM 32 (2) 242 - 243 0399-0974 2008/07 [Refereed][Not invited]
   

  We studied the regulation of multiple vitellogenin (Vg) and estrogen receptor (ER) genes, and the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis by estradiol (E2) in liver and gonads of male, female, and E2-treated male tilapia. Plasma Vg and liver expression of three Vg genes and ER alpha increased in females and E2-treated males, while plasma IGF-I and liver expression of ERB, IGF-I and GH receptor (GHR) were reduced. Expression of Vgs, ERs, IGF-I and GHR was higher in ovary than in testis. These results suggest that E2 acts on the liver to shift energy away from somatic growth towards Vg production.
• Differential production and uptake of dual vitellogenins in Japanese medaka (Oryzias latipes)

  Naoshi Hiramatsu, Mihoka Inoue, Hiroki Ideuchi, Toshiaki Fujita, Haruna Amano, Takahiro Matsubara, Craig V. Sullivan, Akihiko Hara

  CYBIUM 32 (2) 260 - 260 0399-0974 2008/07 [Refereed][Not invited]
   

  Differential production and uptake of two forms of vitellogenin (VgA/B and VgC) was verified in serum and oocytes, respectively, of Japanese medaka (Oryzias latipes). The ratio of the two types of Vg (VgA/B:VgC) in the serum of females varied with reproductive stage (range similar to 2:1-8:1). In males exposed to waterborne E2 for various durations or at various dosages, the induced Va ratios were fairly constant (similar to 2.4:1-3.4:1)except that VgC alone was detected after a very short (2 h) exposure to E2. An antiserum against VgC (a-VgC) immunostained yolk granules in pre-vitello 'genic oocytes when no immunoreactivity was found by staining with a-VgA/B. Both antisera stained yolk globules in vitellogenic oocytes. The reproductive phase-specific differences between plasma levels of VgA/B and V-C likely reflect different rates of their hepatic synthesis and uptake by oocytes.
• Controlled accumulation of multiple vitellogenins into oocytes during vitellogenesis in the barfin flounder, Verasper moseri

  Sayumi Sawaguchi, Nobuyuki Ohkuio, Haruna Amano, Naoshi Hiramatsu, Akihiko Hara, Craig V. Sullivan, Takahiro Matsubara

  CYBIUM 32 (2) 262 - 262 0399-0974 2008/07 [Refereed][Not invited]
   

  For a quantitative examination of the accumulation by growing oocytes of the multiple yolk proteins which are derived from three forms of vitellogenins in barfin flounder, Verasper moseri, six enzyme-linked immunosorbent assays (ELISAs) were developed using antisera against vitellogenin (Vg) A, VgB, VgC as phosvitinless vitellogenin, lipovitellin (Lv) A, LvB and LvC, respectively. Concentrations of the Vgs in serum and liver, and contents of the Lvs in ovarian follicles were measured by the developed ELISAs in female fish sampled throughout the year. Results of the measurements show that this controlled accumulation of multiple Vgs is regulated primarily by rates of hepatic Vg synthesis and secretion and secondarily by mechanisms for receptor-mediated uptake of Vgs into oocytes.
• Gender-specific expression of multiple estrogen receptors, growth hormone receptors, insulin-like growth factors and vitellogenins, and effects of 17 beta-estradiol in the male tilapia (Oreochromis mossambicus).

  Lori K Davis, Andrew L Pierce, Naoshi Hiramatsu, Craig V Sullivan, Tetsuya Hirano, E Gordon Grau

  General and comparative endocrinology 156 (3) 544 - 51 0016-6480 2008/05/01 [Refereed][Not invited]
   

  Gender-specific expression of estrogen receptors (ER alpha and ER beta), growth hormone receptors (GHR1 and GHR2), insulin-like growth factors (IGF-I and IGF-II) and three vitellogenins (Vgs A-C) was examined in the liver, gonad, pituitary, and brain of sexually mature male, female, and 17 beta-estradiol (E2)-treated male tilapia (Oreochromis mossambicus). Reflecting greater growth rate in male tilapia, hepatic expression of GHR1, GHR2, IGF-I and IGF-II as well as plasma IGF-I levels were higher in males than in females, whereas the expression of Vgs A-C and ER alpha was higher in females. On the other hand, expression of all genes measured was higher in the ovary than in testis. Forty eight hours after E2 injection (5 microg/g) into male fish, hepatic expression of most transcripts measured were altered to levels that were similar to those seen in females. The changes included decreased expression of GHR1, GHR2, IGF-I, and IGF-II, and increased expression of ER alpha and Vgs A-C. E2 treatment also increased Vg and decreased IGF-I in the plasma. Brain expression of ER alpha, ER beta, GHR1, and IGF-I was higher in females than in males, whereas pituitary expression of GHR2 and IGF-I was lower in females; only brain expression of GHR1 was increased by E2 treatment. These findings suggest that E2 stimulates Vg production primarily through activation of ER alpha and down-regulation of the GH/IGF-I axis, thus shifting energy from somatic growth towards vitellogenesis at the level of the liver.
• Purification of multiple vitellogenins in grey mullet (Mugil cephalus)

  Haruna Amano, Toshiaki Fujita, Naoshi Hiramatsu, Sayumi Sawaguchi, Takahiro Matsubara, Craig V. Sullivan, Akihiko Hara

  MARINE BIOLOGY 152 (6) 1215 - 1225 0025-3162 2007/11 [Refereed][Not invited]
   

  Three female specific serum proteins were detected immunologically in the sera of grey mullet (Mugil cephalus) which were named vitellogenin A (VgA), VgB, and VgC, based upon their distinct antigenicity against specific antisera raised against three types of mullet lipovitellins (Lvs). These Vgs were subsequently purified from the serum of estradiol-treated mullet by combining several types of chromatography columns (anion exchanger, hydroxylapatite, immunoadsorbent column, and gel filtration). Purified native VgA, VgB, and VgC exhibited molecular masses of 570, 580, and 335 kDa, respectively. Following, SDS-PAGE, the estimated mass of polypeptide bands evident for VgA and VgB were similar to 179 and similar to 175 kDa, respectively; VgC appeared to be similar to 132 kDa. The two larger Vgs (VgA and VgB) appeared to be phosphorylated, suggesting that these Vgs contain a highly phosphorylated, serine-rich phosvitin (Pv) domain. Furthermore, two discrete Vg-type specific antisera, anti-VgA and anti-VgB, were developed and each generated two precipitin lines against ovary extracts in immunoelectrophoresis, indicating that these Vgs contain additional antigenic yolk protein domains: Lv and beta'-component. The small Vg (VgC) appeared to lack a Pv domain because of its low serine content (5.35%) and failure to show positive results in phospho-staining experiments. In conjunction with N-terminal amino acid sequencing analyses of the purified Vgs, our present results have conclusively identified the purified Vg products in grey mullet as typical A-type (VgA), B-type (VgB), and C-type (VgC) Vgs.
• Induction of three vitellogenins by 17beta-estradiol with concurrent inhibition of the growth hormone-insulin-like growth factor 1 axis in a euryhaline teleost, the tilapia (Oreochromis mossambicus).

  Lori K Davis, Naoshi Hiramatsu, Kaori Hiramatsu, Benjamin J Reading, Takahiro Matsubara, Akihiko Hara, Craig V Sullivan, Andrew L Pierce, Tetsuya Hirano, E Gordon Grau

  Biology of reproduction 77 (4) 614 - 25 0006-3363 2007/10 [Refereed][Not invited]
   

  The objective of the present study was to utilize the male Mozambique tilapia (Oreochromis mossambicus) as a model for examining the molecular mechanisms that mediate the physiological transition between somatic and gonadal growth in female teleost fish, and in vertebrates in general. Partial cDNAs that encode multiple forms of vitellogenin (Vtg), which is the major precursor of yolk proteins, were cloned from estrogen-treated males and utilized to develop real-time quantitative RT-PCR assays, which were supplemented by an assay for Vtg immunoreactivity in the plasma. Alignment analyses of the amino acid sequences deduced from the vtg cDNAs revealed three distinct tilapia Vtgs, which were categorized as Aa-, Ab-, and C-type Vtgs. A single injection of male tilapias with 17beta-estradiol (E(2)) at 5 microg/g body weight significantly increased the plasma E(2) and hepatic levels of all three vtg transcripts within 1 day. Plasma E(2) levels declined after 3 days, whereas the plasma Vtg immunoreactivity and hepatic levels of the three vtg transcripts continued to increase. Hepatic expression of the estrogen receptor (esr) 1 gene, but not the esr2 gene, also increased markedly 1 day after E(2) injection and remained elevated for 5 days. While plasma growth hormone (Gh) levels were unaffected, hepatic expression of transcripts that encoded the Gh receptor and insulin-like growth factor 1 (Igf1) was suppressed by E(2), as were the plasma Igf1 levels. These results clearly suggest a distinct negative interplay between the growth and reproductive axes at the molecular level of key hepatic regulatory pathways involved in the control of energy utilization by gonadal and somatic growth processes.
• Egg yolk proteins in gray mullet (Mugil cephalus): Purification and classification of multiple lipovitellins and other vitellogenin-derived yolk proteins and molecular cloning of the parent vitellogenin genes

  Haruna Amano, Toshiaki Fujita, Naoshi Hiramatsu, Munetaka Shimizu, Sayumi Sawaguch, Takahiro Iatsubara, Hirohiko Kagawa, Masaki Nagae, Craig V. Sullivan, Akihiko Hara

  JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL AND INTEGRATIVE PHYSIOLOGY 307A (6) 324 - 341 2471-5646 2007/06 [Refereed][Not invited]
   

  Seven yolk proteins (YPs), four large lipoproteins (YPs1-4) and three minor yolk components (YPs5-7) including one phosphoprotein (YP7), were purified from extracts of vitellogenic ovaries of gray mullet (Mugil cephalus) by combinations of hydroxylapatite, ion exchange, immunoadsorbent, and gel filtration chromatography. The molecular masses of native Y-P1, YP2, YP3, and YP4 were estimated to be 330, 325, 335, and 570 kDa, respectively. The tertiary structures of YP1, YP2, and YP3 revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis were typical of teleost lipovitellins (Lvs), consisting of a heavy chain (similar to 110, similar to 99, and similar to 97 kDa, respectively) and a light chain (similar to 30, similar to 29, and similar to 21.5 kDa, respectively), while YP4 exhibited a heavy chain (similar to 110 kDa) and two more polypeptide bands (similar to 70 and similar to 54kDa). Mapping of N-terminal peptide sequences of the purified YPs to the primary structure of multiple mullet vitellogenins (Vgs) deduced from their respective complete cDNAs, which were cloned and sequenced, conclusively identified YP1, YP2, and YP3 as Lvs derived from mullet VgA, VgB, and VgC, respectively. The fourth YP (YP4) appeared to be a proteolytic variant consisting of Lv and phosvitin components of VgA. Two other YPs (YP5 and YP6) were identified as beta'-components derived from VgA and VgB based on their structures and common, but not identical, antigenicity to salmonid beta'-component, while purified YP7, a phosphoprotein with a high content of serine residues, was identified as a phosvitin derived from VgB. This is the first report, of which we are aware, on purification and molecular classification of three distinct forms of Lv from any oviparous vertebrate.
• Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs

  S Sawaguchi, H Kagawa, N Ohkubo, N Hiramatsu, CV Sullivan, T Matsubara

  MOLECULAR REPRODUCTION AND DEVELOPMENT 73 (6) 719 - 736 1040-452X 2006/06 [Refereed][Not invited]
   

  Full-length cDNAs encoding three forms of vitellogenin (Vg) were obtained from a liver cDNA library of estrogen-treated red seabream, Pagrus major. Two of the three Vg sequences had high homology with type-A and -B Vgs (VgA and VgB) of other teleosts. The third red seabream Vg was classified as a type-C or phosvitinless (Pvl) Vg due to its lack of a phosvitin (Pv) domain. Two Vg preparations (610 and 340 kDa) from blood serum of estradiol-treated fish were biochemically characterized. Analyses of precursor-product relationships by examination of N-terminal amino acid sequences verified cleavage of the 610 kDa Vg into a 540 kDa lipovitellin (Lv) and a 32 kDa PI-component. Each of these yolk preparations comprising both VgA- and VgB-derived polypeptides. The 340 kDa Vg, which was immunologically verified to be a PvlVg, was accumulated by vitellogenic oocytes with no alterations to its native molecular mass. During oocyte maturation, the VgA- and VgB-derived yolk proteins were differentially processed, presumably to generate a pool of free amino acids for oocyte hydration or for allocation of specific types of nutrients, amino acids, and proteins, to the developing embryo. Conversely, the 340 kDa Vg-derived yolk protein is unlikely to contribute to oocyte hydration or diffusible nutrients since the molecule underwent only minor proteolytic nicking during oogenesis. The present study elucidates for the first time specific functions of three different forms of Vg and their product yolk proteins in a higher taxonomic group of marine teleosts that spawn pelagic eggs.
• Multiple piscine vitellogenins: biomarkers of fish exposure to estrogenic endocrine disruptors in aquatic environments

  N Hiramatsu, T Matsubara, T Fujita, CV Sullivan, A Hara

  MARINE BIOLOGY 149 (1) 35 - 47 0025-3162 2006/04 [Refereed][Not invited]
   

  Vitellogenin (Vg), a major estrogen-inducible yolk precursor protein, has become an important biomarker for assessing the estrogenic potency of chemicals and the exposure of animals to estrogenic contaminants present in aquatic environments. These contaminants, which can disrupt functioning of the vertebrate neuroendocrine system, are known as endocrine disrupting chemicals (EDCs). In general, investigations of the significance of estrogenic EDCs have failed to keep pace with recent developments in our understanding of vitellogenesis in fishes. Recent gene cloning and immunobiochemical analyses have verified the general multiplicity of piscine Vg and led to exploration of the unique roles of yolk proteins derived from different forms of Vg in the processes of oogenesis and embryogenesis. The levels of circulating Vg proteins (or Vg gene transcripts) during oogenesis and their degree of induction by estrogens appear to vary among species and among different types of Vg within species. The kinetics of induction of distinct types of Vg by estrogens in fishes appears to depend on environmental factors (e.g., water temperature and photoperiod), life history stage, and the concentration and type of estrogenic compound. Consideration of these findings will contribute to development of Vg-based bioassays superior to those currently based on the outdated "single Vg" model.
• Annual changes in serum levels of two choriogenins and vitellogenin in masu salmon, Oncorhynchus masou.

  Toshiaki Fujita, Haruhisa Fukada, Munetaka Shimizu, Naoshi Hiramatsu, Akihiko Hara

  Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 141 (2) 211 - 7 1096-4959 2005/06 [Refereed][Not invited]
   

  Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17beta (E2) were quantified in masu salmon, Oncorhynchus masou, using specific immunoassays. Serum Chg levels were higher than Vg during the previtellogenic growth phase when circulating E2 levels were low ( approximately 0.1 ng/mL), suggesting higher sensitivity of Chg to E2. When oocyte growth shifted to the vitellogenic phase, Vg levels increased and became the most abundant in serum coincident with elevations of E2 and GSI. Chg H, Chg L and Vg peaked 1 month prior to ovulation at 0.61+/-0.08, 0.98+/-0.18 and 10.93+/-3.24 mg/mL, respectively. These results suggest that chorion formation by Chgs occurs prior to vitellogenesis and that the sensitivity of Chgs to low circulating E2 is closely related to the sequential events of oocyte growth.
• Multiple vitellogenins (Vgs) in mosquitofish (Gambusia affinis): identification and characterization of three functional Vg genes and their circulating and yolk protein products.

  Sayumi Sawaguchi, Yasunori Koya, Norio Yoshizaki, Nobuyuki Ohkubo, Tadashi Andoh, Naoshi Hiramatsu, Craig V Sullivan, Akihiko Hara, Takahiro Matsubara

  Biology of reproduction 72 (4) 1045 - 60 0006-3363 2005/04 [Refereed][Not invited]
   

  The objectives of this study were to characterize multiple forms of vitellogenin (Vg) in mosquitofish (Gambusia affinis) and to discover the fate of each Vg during its processing into product yolk proteins. Two Vg preparations, with apparent masses of 600 kDa (600 Vg) and 400 kDa (400 Vg), were isolated from the plasma of fish treated with estradiol-17beta (E(2)) by various chromatographic procedures. Immunological analyses verified the presence of two different Vg proteins (600 VgA and 600 VgB) in the 600 Vg preparation and of a single protein in the 400 Vg preparation. Three major yolk proteins (Yps) with apparent masses of 560, 400, and 28 kDa were observed in extracts of ovarian follicles from vitellogenic females. Immunological analyses demonstrated that the 400 Vg underwent no change in native mass after being incorporated into oocytes. The 600 Vgs gave rise to a 28 kDa beta'-component and a native 560 kDa Yp, which was heterodimeric in structure, consisting of two types of complexes between phosvitin (Pv) and lipovitellin (Lv) heavy- and light-chains. Full-length cDNAs encoding the 600 VgA, 600 VgB, and 400 Vg were isolated from a liver cDNA library of E(2) treated fish. Similar to the zebrafish vg3 gene, the 400 Vg cDNA lacked a Pv domain and was classified as an incomplete or phosvitinless (C-type) Vg. The deduced primary structures of 600 VgA and 600 VgB were complete, and these were categorized as type A and type B Vgs, respectively, according to our recent classification scheme. This is the first report on the characterization of three functional Vg genes and their circulating and yolk protein products in any vertebrate species.
• Chapter 16 Vitellogenesis and endocrine disruption

  Naoshi Hiramatsu, Ann O. Cheek, Craig V. Sullivan, Takahiro Matsubara, Akihiko Hara

  Biochemistry and Molecular Biology of Fishes 6 (C) 431 - 471 1873-0140 2005 [Refereed][Not invited]
   

  Over the last decade, much progress has been made with regard to identification of EDCs and evaluation of their estrogenic potency. These developments have included establishment of accurate and reliable assay systems for measuring circulating Vgs and Chgs, as well as identification of other new biomarkers with the potential to detect and evaluate the potency of estrogenic EDCs. Future investigations need to focus on development of specific and sensitive immunoassays for individual Vg and Chg molecules and, in general, far more consideration needs to be given to the constitutive multiplicity of teleost Vgs and Chgs. On the other hand, substantial knowledge of the extent of estrogenic endocrine disruption in fish has accumulated from field surveys. It is becoming apparent that exposure of fishes to these compounds is widespread in marine, brackish, and flesh waters around the world. Combined with results from several laboratory studies of fishes, these observations send the obvious warning that many contemporary aquatic environments possess an estrogenic potency and have the potential to disrupt reproduction of fishes, or even wildlife and humans consuming water or aquatic species from these areas. The extent of estrogenic EDC impact on reproduction of wild fishes, and the proximal causes of such effects, have been and remain largely unknown. Basic research on reproductive processes in fish will reveal critical mechanisms subject to impairment by EDCs. Especially important in this regard will be complete elucidation of the roles of dual or multiple Vgs, cathepsins, and Chgs in ovarian follicle growth and maturation, egg quality, and embryonic and larval development. Such basic studies of the functional mechanisms of oogenesis and embryogenesis will, in turn, provide the next generation of biomarkers for assessing the potential impacts of aquatic EDCs on fishes, wildlife, and human beings. © 2005 Elsevier B.V. All rights reserved.
• Identification, characterization and cDNA cloning of three forms of vitellogenin, two general forms and a phosvitinless form, in a viviparous mosquitofish, Gambusia affinis.

  Sawaguchi S, Koya Y, Yoshizaki N, Ohkubo N, Andoh T, Sullivan C.V, Hiramatsu N, Hara A, Matsubara T

  Biology of Reproduction 72 1045 - 1060 2005 [Refereed][Not invited]
  
• Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana)

  N Hiramatsu, RW Chapman, JK Lindzey, MR Haynes, CV Sullivan

  BIOLOGY OF REPRODUCTION 70 (6) 1720 - 1730 0006-3363 2004/06 [Refereed][Not invited]
   

  A full-length (4021 base pair [bp]) cDNA encoding a polypeptide (844 amino acids) with a predicted mass of 93 kDa and other characteristic structural features of a vertebrate vitellogenin receptor (VgR) was isolated from a white perch (Morone americana) ovarian cDNA library. Northern blotting performed using a specific digoxygenin-labeled VgR cDNA probe revealed a distinct similar to4.1 kilobase (kb) hybridization signal in an mRNA preparation obtained from previtellogenic perch ovaries. The deduced amino acid sequence of the perch VgR was 89% and 82% identical, respectively, to that of the tilapia and rainbow trout. Because it possessed an eight-repeat ligand-binding domain (LR8) but lacked an O-linked sugar domain (-), the perch VgR was identified as a non-O-linked form of VgR (LR8-). Unlike the case in other vertebrates investigated, including tilapia and trout, no species of mRNA encoding an O-linked form of VgR (LR8+) could be detected when perch ovarian or liver mRNA reverse transcripts or cDNA libraries were screened by PCR using primer sets flanking the putative O-linked sugar domain. These novel findings call into question the assumptions that an LR8+ splice variant of the VgR always is dominantly present in somatic tissues and exists at lower levels in ovarian tissues to sequester lipoproteins distinct from Vg. A SYBR-green-based real-time reverse transcription-polymerase chain reaction assay was developed and used to quantitatively measure VgR expression in gonadal and somatic tissues, for the first time in any vertebrate. The main site of perch VgR mRNA expression was the ovary and the highest level of VgR mRNA expression was in ovaries whose largest follicles contained previtellogenic oocytes. Expression of VgR mRNA decreased with oocyte growth during vitellogenesis and was very limited in ovulated eggs. These quantitative results verify the concept that growing oocytes must extensively recycle LR8- forms of the VgR.
• Quantification of serum levels of precursors to vitelline envelope proteins (choriogenins) and vitellogenin in estrogen treated masu salmon, Oncorhynchus masou

  T Fujita, H Fukada, M Shimizu, N Hiramatsu, A Hara

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 136 (1) 49 - 57 0016-6480 2004/03 [Refereed][Not invited]
   

  Previously two precursors to vitelline envelope proteins, choriogenin H (Chg H) and choriogenin L (Chg L), were identified in masu salmon, Oncorhynchus masou, and specific antisera against these two proteins were generated in rabbits. In this study, two methods of immunoassay have been developed using these specific antibodies: single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA). Non-competitive sandwich ELISAs for Chg H and Chg L were designed using digoxigenin-labeled antibodies and purified Chgs as assay components. The working range of the ELISAs was 1-128 and 2-256 ng/ml for Chg H and Chg L, respectively. Using these immunoassays and a chemiluminescent immunoassay for vitellogenin (Vg), the changes in these three estrogen-responsive proteins were measured in the serum of masu salmon after treatment with various doses of estradiol-17beta (E-2). The changes in serum levels of Chgs and Vg in male fish differed according to the E-2 dose. When fish were given a 5 mg/kg body weight (BW) of E-2, Vg was induced to a greater extent than Chgs. By contrast, Chg levels were higher than that of Vg after a 10 tg/ kg BW of E-2 injection. A similar trend was seen in the response time to exogenous E-2. Serum Chgs were induced from 8 h after E-2 injection and reached a peak of about 5 mug/ml at 24 h. Although Vg was not detected until 8 h after E-2 injection, its levels remained considerably low at around 0.03 mug/ml, even after 24 h. Chg H was more sensitive than was Chg L to I mug/kg BW of estrogen: the long-term exposure of fish to E-2 showed that Chg H could be induced from a lower dose of E-2 than could Chg L. Taken together, these results suggest that the serum levels of Chg H, Chg L, and Vg in masu salmon are regulated by circulating levels of E-2. (C) 2003 Elsevier Inc. All rights reserved.
• Carp (Cyprinus carpio) vitellogenin: purification and development of a simultaneous chemiluminescent immunoassay

  H Fukada, Y Fujiwara, T Takahashi, N Hiramatsu, CV Sullivan, A Hara

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY 134 (3) 615 - 623 1095-6433 2003/03 [Refereed][Not invited]
   

  Vitellogenin (Vg) was purified from the serum of vitellogenic female carp (Cyprinus carpio) by hydroxylapatite column chromatography and gel filtration. Vg had an apparent molecular mass of 490 kDa and appeared as two bands corresponding to 190 and 156 kDa after SDS-PAGE under reducing conditions. These bands were immunoreacted in Western blotting using antiserum against carp lipovitellin (anti-Lv) which is an egg yolk protein derived from Vg. The amino acid composition of carp Vg was similar to previous reports of cyprinids. The chemiluminescent immunoassay (CLIA) for carp Vg was developed to quantify serum Vg using purified carp Vg and anti-Lv. Its measurable range was from 1.95 to- 1000 ng/ml. The dilution curve in the CLIA of vitellogenic female serum was parallel to the standard curve of purified Vg. The coefficient variations of intra- and inter-assay were less than 5%, respectively. Furthermore, the assay had cross-reactivity with the sera of other female cyprinids (crucian carp and Japanese dace). In fish diets-experiments, Vg was detected in all fish in the fish meal containing soybean (20%) group, but was not detected in almost all of the fish in the fish meal-group. This suggests that a soybean based-diet may induce Vg production in the serum of cultivated carp. (C) 2002 Elsevier Science Inc. All rights reserved.
• Atresia in temperate basses: cloning of hatching enzyme (choriolysin) homologues from atretic ovaries

  DM Donato, N Hiramatsu, KM Arey, K Hiramatsu, AM Kennedy, CL Morton, A Hara, CV Sullivan

  FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 329 - 330 0920-1742 2003 [Refereed][Not invited]
   

  Partial cDNAs encoding homologues of choriolysin were cloned from striped bass embryos and from atretic ovaries of white perch. Among these clones, one of the ovarian homologues may be a candidate bio-marker for detecting onset of atresia. Early detection of atresia may allow affected broodfish to be reproduced before they undergo complete reproductive failure.
• Multiple vitellogenins in white perch (Morone americana)

  K. Hiramatsu, N. Hiramatsu, A. Hara, T. Matsubara, C. V. Sullivan

  Fish Physiology and Biochemistry 28 (1-4) 347 - 348 0920-1742 2003 [Refereed][Not invited]
   

  Three forms of female-specific plasma protein were purified from blood plasma of estrogen-treated white perch, characterized, and classified as three distinct vitellogenins (VgA, VgB, and VgC). This study describes the first purification of three classes of native Vg from any vertebrate and sets the stage for discovery of the different functions of each type of Vg. © 2004 Kluwer Academic Publishers.
• Induced maturation and spawning: opportunities and applications for research on oogenesis

  CV Sullivan, N Hiramatsu, AM Kennedy, RW Clark, GM Weber, T Matsubara, A Hara

  FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 481 - 486 0920-1742 2003 [Refereed][Not invited]
   

  Broodstock management requires the ability to detect and regulate oocyte growth, acquisition of maturational competence, maturation of oocytes, and onset of ovarian atresia. Our research on temperate basses ( genus Morone) has supported development of these capabilities. These investigations have revealed that accumulation of neutral lipid droplets and deposition of vitellogenin-derived yolk proteins in growing oocytes are independent processes with different sensitivities to changing day length and water temperature. In these fishes, completion of oocyte growth is marked by disappearance of vitellogenin from ovarian biopsy samples. Competence of females for induced spawning is predicted by the ability of biopsied follicles to initiate oocyte meiosis in vitro in response to insulin-like growth factor I. Cytoplasmic maturation of the oocytes is triggered by the maturation-inducing steroid hormone and can be monitored by evaluating degradation of the yolk proteins. Onset of ovarian atresia is indicated by the appearance of edema in the granulosa cell layer of biopsied follicles, and can be delayed for months by holding gravid females at abnormally low temperature ('cold banking'). These novel findings hold strong promise for application to other farmed fishes.
• Oocyte growth in temperate basses: multiple forms of vitellogenin and their receptor

  N Hiramatsu, A Hara, T Matsubara, K Hiramatsu, CV Sullivan

  FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 301 - 303 0920-1742 2003 [Refereed][Not invited]
   

  A full-length cDNA encoding a vitellogenin (Vg) receptor (VgR) was isolated and sequenced from a white perch, Morone americana, ovarian cDNA library. The perch expresses a single VgR that is structurally classified as an LR8 type (without an 0-linked sugar domain) and appears to be the only species observed to possess no splice variant of the receptor. We proposed a hypothetical model for ovarian lipid transfer in Morone species, one in which the 'single VgR' transports Vg and other lipoproteins via a selective endocytotic pathway based on ligand affinity, in conjunction with a lipase-mediated pathway of fatty acid transport.
• Multiple vitellogenins and their unique roles in marine teleosts

  T Matsubara, M Nagae, N Ohkubo, T Andoh, S Sawaguchi, N Hiramatsu, CV Sullivan, A Hara

  FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 295 - 299 0920-1742 2003 [Refereed][Not invited]
   

  Vitellogenin (Vg) is the precursor to egg yolk proteins of teleost fishes, including lipovitellin (Lv), phosvitin (Pv), and beta'-component (beta'-c). Complete Vg molecules contain the yolk protein domains arranged in linear fashion: NH2-Lv heavy chain, Pv, Lv light chain, beta'-c, C-terminal coding region (C-t)-COOH. Western blots employing an antiserum raised against a recombinant C-t polypeptide revealed that the C-t domain of Vg gives rise to a fourth yolk protein in barfin flounder oocytes. Three classes of Vg appear to exist in teleosts, including two types of complete Vg (A-type and B-type) and a smaller, incomplete Vg lacking a Pv domain (Pv-less type). By sequencing cDNA amplified from distinctive regions of each type of Vg transcript from several teleosts, we discovered that members of higher taxa (e.g. Paracanthopterygii and Acanthopterygii) express both Vg A and Vg B, and that Pv-less Vg is widely present among teleosts. Vitellogenins A and B play distinct roles in regulation of egg buoyancy in barfin flounder through selective proteolysis of their product yolk proteins in oocytes undergoing final maturation. Protease specification procedures confirmed involvement of a cathepsin B-like enzyme in this maturation-associated proteolysis. Measurement of changing pH revealed that drastic acidification of the ooplasm accompanies hydrolysis of the yolk proteins. Thus, with respect to yolk protein hydrolysis and oocyte hydration, cytoplasmic maturation appears to be controlled by changing pH in maturing oocytes.
• Vitellogenin-derived yolk proteins of white perch, Morone americana: Purification, characterization, and vitellogenin-receptor binding

  N Hiramatsu, A Hara, K Hiramatsu, H Fukada, GM Weber, ND Denslow, CV Sullivan

  BIOLOGY OF REPRODUCTION 67 (2) 655 - 667 0006-3363 2002/08 [Refereed][Not invited]
   

  The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a similar to20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.
• Purification of serum precursor proteins to vitelline envelope (choriogenins) in masu salmon, Oncorhynchus masou.

  Toshiaki Fujita, Munetaka Shimizu, Naoshi Hiramatsu, Haruhisa Fukada, Akihiko Hara

  Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 132 (3) 599 - 610 1096-4959 2002/07 [Refereed][Not invited]
   

  Three vitelline envelope-related proteins (VERPs), very-high-molecular-weight VERP (vhVERP), high-molecular-weight VERP (hVERP) and low-molecular-weight VERP (lVERP) were purified from female masu salmon serum. The apparent molecular weights of vhVERP, hVERP and lVERP, in their native state, were 520, 88 and 54 kDa, respectively, by gel-filtration chromatography. Very-high-molecular-weight VERP comprises two subunits, corresponding to 175 and 126 kDa. On SDS-PAGE, hVERP and lVERP migrate at 53 and 47 kDa, respectively. Amino acid analysis of vhVERP and hVERP showed that they share a high content of glutamic acid and proline. By contrast, lVERP is rich in glutamic acid and asparatic acid. These features are in good agreement with the amino acid composition of the vitelline envelope. Immuno-biochemical analysis suggested that vhVERP is derived from hVERP by polymerization and/or aggregation. Antibodies against hVERP and lVERP specifically immunostained the vitelline envelope and liver of female masu salmon. In addition, both hVERP and lVERP were induced in the serum of estrogen-treated male fish. Taken together, it is suggested that hVERP and lVERP are homologous molecules with choriogenin H and choriogenin L in medaka, respectively. These results indicate that hVERP and lVERP are precursor proteins to the vitelline envelope (choriogenins) in masu salmon.
• Vitellogenin-derived yolk proteins in a hybrid sturgeon, bester (Huso huso x Acipencer ruthenus): identification, characterization and course of proteolysis during embryogenesis.

  Naoshi Hiramatsu, Kaori Hiramatsu, Kaori Hirano, Akihiko Hara

  Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 131 (2) 429 - 41 1095-6433 2002/02 [Refereed][Not invited]
   

  Vitellogenin (Vg) and its corresponding yolk protein (YP) products, YP1, YP2 and YP3, were isolated from serum of estrogen-treated hybrid sturgeon (bester; Huso huso X Acipencer ruthenus) and eggs from untreated fish, respectively. Vitellogenin had an apparent molecular mass of 580 kDa and appeared as two major bands corresponding to 180 kDa and 120 kDa after SDS-PAGE. Apparent molecular weights of YP1, YP2 and YP3 were 370 kDa, 88 kDa and 19 kDa, respectively. After SDS-PAGE, YP1 appeared as a main band of 110 kDa, while YP2 was resolved as a single band of 94 kDa and 29 kDa band under non-reducing and reducing conditions, respectively. Yolk protein 3 appeared as a diffuse band corresponding to 16 kDa and two faint bands below 14.4 kDa after SDS-PAGE. However, the 16 kDa band alone was observed after dephosphorylation with alkaline phosphatase. The course of cleavage of yolk proteins in bester embryos and alevins was observed by SDS-PAGE and Western blotting from fertilization onward. After hatching, the main 110 kDa band of YP1 was degraded into smaller peptides during development, while YP2 hardly showed any such structural changes. The amino acid compositions of purified yolk proteins indicated that YP1, YP2 and YP3 were bester lipovitellin, beta-component, and phosvitin, respectively.
• Identification and characterization of proteases involved in specific proteolysis of vitellogenin and yolk proteins in salmonids

  N Hiramatsu, N Ichikawa, H Fukada, T Fujita, GV Sullivan, A Hara

  JOURNAL OF EXPERIMENTAL ZOOLOGY 292 (1) 11 - 25 0022-104X 2002/01 [Refereed][Not invited]
   

  A pepstatin A-sensitive enzyme involved in yolk formation was purified from the masu salmon (Oncorhynchus masou) ovary using in vitro generation of yolk proteins from purified vitellogenin to assay enzymatic activity. Purification of the enzyme involved precipitation of ovarian extracts by water and ammonium sulfate followed by five steps of column chromatography. After SDS-PAGE and Western blotting, the purified enzyme appeared as a single similar to 42 kDa band that was immunoreactive to anti-human cathepsin D. The course of proteolytic cleavage of the three major yolk proteins (lipovitellin, beta'-component, and phosvitin) in Fertilized masu salmon and Sakhalin taimen (Hucho perryi) eggs and embryos was visualized by SDS-PAGE and Western blotting using specific antisera. Major yolk protein bands appeared in positions corresponding to 92 kDa, 68 kDa, and 22 kDa (lipovitellin-derived peptides), as well as 17 kDa (beta'-component). During embryo development, the 92 kDa and 22 kDa bands gradually decreased in intensity, becoming undetectable in alevins. The 68 kDa band and a minor 24 kDa band became more intense after the eyed stage. Two additional peptides, corresponding to 40 and 28 kDa, newly appeared in alevins. During embryonic growth, the beta'-component band (17 kDa) persisted and phosvitin appeared to be progressively dephosphorylated. In vitro analysis of lipovitellin proteolysis indicated that the enzyme involved is a Pefabloc SC-sensitive serine protease. These results demonstrate, for the first time, that a cathepsin D-like protease and serine proteases play key roles in yolk formation and degradation, respectively, in salmonid fishes. (C) 2002 Wiley-Liss, Inc.
• Identification, purification and classification of multiple forms of vitellogenin from white perch (Morone americana)

  N Hiramatsu, T Matsubara, A Hara, DM Donato, K Hiramatsu, ND Denslow, CV Sullivan

  FISH PHYSIOLOGY AND BIOCHEMISTRY 26 (4) 355 - 370 0920-1742 2002 [Refereed][Not invited]
   

  Three forms of female-specific plasma protein (FSPP 1-3) were purified from blood plasma of estrogen-treated white perch ( Morone americana) by combining several types of ion-exchange chromatography including a novel, fast flow, strong anion exchanger (POROS media), followed by gel filtration. Native FSPP 1, FSPP 2 and FSPP 3 had molecular masses of 532 kDa, 532 kDa and 426 kDa, respectively. The apparent mass of purified FSPP 1 and FSPP 2 after SDS-PAGE under reducing conditions was similar to 180 kDa, while FSPP 3 appeared as a major similar to 148 kDa band. All of the FSPPs resembled one another with respect to amino acid composition but each appeared to be immunologically distinct. In double immunodiffusion using anti-total FSPP ( antiserum raised against vitellogenic female plasma pre-absorbed by male plasma), each FSPP formed one precipitin line that crossed those produced by both others. A rabbit antiserum was raised against each FSPP and absorbed with combinations of the other two FSPPs to ensure specificity. Using the antisera, each FSPP was detected by immuno-electrophoresis in plasma from vitellogenic females or estrogen-treated male or immature fish, but no FSPP was detected in normal male plasma. Endoprotease (Asp-N) digests of the FSPPs were subjected to HPLC separation for N-terminal sequencing and mapping of isolated peptides to published vitellogenin (Vg) sequences. Results of these analyses indicate that white perch FSPP 1, FSPP 2, and FSPP 3 can be classified into three Vg groups identified in previous studies: VgA, VgB, and VgC-like protein, respectively. This is the first report, of which we are aware, on isolation of more than two Vg proteins from any species of vertebrate except the chicken.
• Cloning of cDNA for choriogenin H and choriogenin L of masu salmon

  Toshiaki Fujita, Haruhisa Fukada, Munetaka Shimizu, Naoshi Hiramatsu, Akihiko Hara

  Fisheries Science 68 1273 - 1274 1444-2906 2002 [Refereed][Not invited]
  
• Developmental changes in egg yolk proteins of walleye pollock, Theragra chalcogramma, and a comparative study of immunoreactivity of other north pacific teleosts and invertebrate eggs.

  Bailey K. M, Merati N, Helser M, Hiramatsu N, Hara A

  Bull. Fac. Fish. Sci. Hokkaido Univ 北海道大学 53 (3) 95 - 105 1346-1842 2002 [Refereed][Not invited]
  
• Vitellogenesis in Aquatic Animals

  Naoshi Hiramatsu, Takahiro Matsubara, Gregory M. Weber, Craig V. Sullivan, Akihiko Hara

  FISHERIES SCIENCE 68 694 - 699 0919-9268 2002 [Refereed][Not invited]
   

  Vitellogenin (Vg) is the main precursor to egg yolk proteins (YPs) accumulated as nutrients for developing embryos of oviparous aquatic species. Recent gene cloning and immuno-biochemical analyses verified the presence of multiple Vgs in teleost fishes, similar to the case in chickens and Xenopus. These findings lead us to abandon the classical "single Vg model" and explore different functions of individual Vgs and their YP derivatives during teleost oocyte maturation and embryogenesis. The course of proteolysis of Vgs and their YP products appears to differ among species. Detailed characterization of the relevant proteolytic enzymes has been partly accomplished only for salmonid fishes. Investigations of the endocrine regulation of teleost Vg and YP proteolysis have only just begun. Over the past decade, much attention has been paid to Vg due to its promise as a biomarker of contaminants that mimic estrogen, which are present in the aquatic environment. Sensitive and specific assays for measuring Vg in male and juvenile fish have been and will be valuable tools for identifying environmental estrogens to which humans and wildlife are potentially exposed.
• Serum Immunoglobulin M (IgM) in Sakhalin Taimen (Hucho perryi): Purification, Caracterization, Circulating Levels, and Specific IgM Production by the Parasitic Salmincola stellatus

  HIRAMATSU Naoshi, FUKADA Haruhisa, KITAMURA Makiko, SHIMIZU Munetaka, FUDA Hirotoshi, KOBAYASHI Kunihiko, HARA Akihiko

  水産増殖 日本水産増殖学会 49 (3) 347 - 355 0371-4217 2001/09/20 [Refereed][Not invited]
   

  Immunoglobulin M (IgM) was purified from the serum of Sakhalin taimen (Hucho perryi) by salting-out, ion-exchange chromatography on DEAF-cellulose, and gel filtration on Sepharose 6B. The intact, tetrameric taimen IgM has a mass of 750 kDa with molecular architecture typical of IgM from other salmonids. The molecular weights of heavy (μ) chain and light chains (L) of the IgM monomer were estimated to be 68 kDa and 23 kDa, respectively. Purified taimen IgM was used to raise a specific rabbit antiserum and to develop a single radial immunodiffusion assay for measuring circulating IgM levels. The serum IgM levels in captive, immature or maturing female taimen varied between 1 and 5 mg/ml, showing seasonal changes regardless of fish age, with relatively low levels in spring and conversely high levels in autumn. Production of specific serum IgM to a parasite, Salmincola stellatus, was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The parasitized taimen serum could react with protein components of aqueous extracts from the parasite that were blotted on a nitrocellulose membrane after SDS-PAGE, but normal taimen serum did not, indicating that the parasitized fish produced the specific IgM to S. stellatus in the serum.
• Development and validation of chemiluminescent immunoassay for vitellogenin in five salmonid species

  H Fukada, A Haga, T Fujita, N Hiramatsu, CV Sullivan, A Hara

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR AND INTEGRATIVE PHYSIOLOGY 130 (1) 163 - 170 1095-6433 2001/08 [Refereed][Not invited]
   

  A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of vitellogenin (Vg) in five salmonids. The CLIA for salmon Vg was performed using the two-site method, with anti-masu salmon beta ' -component as primary antibody and chemiluminescent acridinium-labeled anti-rainbow trout lipovitellin F(ab)' (2) as the second antibody. Using cutthroat trout Vg as the standard, the working range of the CLIA was from 60 pg to 500 ng Vg/ml. Intra- and inter-assay coefficients of variation ranged from 3.04 to 6.67% and 3.23 to 5.86%, respectively. For the various salmonid species, serially diluted samples of serum from vitellogenic fish ran parallel to their purified Vg standard curve in the CLIA. In male cutthroat trout maturing during the 4 months before spawning, serum Vg levels ranged from 1.56 to 8000 ng/ml. High levels of Vg in some individuals may have resulted from temporary elevation of estradiol-17 beta levels in the same fish during December or January (1-2 months before spawning). This is the first report on changes in serum Vg levels in maturing male trout using CLIA, the most sensitive assay for Vg yet developed. (C) 2001 Elsevier Science Inc. All rights reserved.
• Estimation of baseline vitellogenin level in male salmonid serum.

  Haga A, Fukada H, Fujita T, Hiramatsu N, Hara A

  Enviromental Science 8 (2) 173  2001 [Refereed][Not invited]
  
• Simple and sensitive detection of vitellogenin receptor(s) in Sakhalin taimen (Hucho perryi).

  Naoshi Hiramatsu, Haruhisa Fukada, Craig V. Sullivan, Akihiko Hara

  Bull. Fac. Fish. Hokkaido Univ. 52 (1) 5 - 9 2001 [Refereed][Not invited]
  
• Changes of serum growth hormone and virellogenin levels during vitellogenesis in female masu salmon Oncorhynchus masou

  H Fukada, N Hiramatsu, M Kitamura, M Shimizu, A Hara

  FISHERIES SCIENCE 66 (4) 789 - 791 0919-9268 2000/08 [Refereed][Not invited]
  
• Development of a sensitive luminometric immunoassay for determining baseline seasonal changes in serum vitellogenin levels in male flounder (Pleuronectes yokohamae)

  Hiroki Bessho, Sunao Iwakami, Naoshi Hiramatsu, Akihiko Hara, Shinya Hashimoto

  International Journal of Environmental Analytical Chemistry 76 (3) 155 - 166 0306-7319 2000 [Refereed][Not invited]
   

  Vitellogenin is a sensitive biomarker used to study the effects of artificial estrogens in aquatic environments. We developed and optimized a luminometric immunoassay that was able to detect trace amounts of vitellogenin in the serum of male flounder collected from an uncontaminated reference site. The lowest measurable concentration of vitellogenin in the serum was approximately 0.08 ng/ml with purified protein diluted in buffer. The reproducibility of the vitellogenin measurements was determined by the analysis of triplicate samples and found to be about 5.3%, based on serum samples containing vitellogenin at 2.0 ng/ml. This method was successfully applied to samples collected from an uncontaminated reference site for the monitoring of baseline levels of serum vitellogenin in male flounder.
• Serum levels of precursors to vitelline envelop proteins (choriogenins) in Sakhalin taimen after treatment with estrogen and during oocyte growth.

  Munetaka Shimizu, Haruhisa Fukada, Toshiaki Fujita, Naoshi Hiramatsu, Akihiko Hara

  Journal of Fish Biology 57 (170) 181  2000 [Refereed][Not invited]
  
• Changes in serum choriogenin and vitellogenin levels in masu salmon (Oncorhynchus masou) during sexual maturation and after estrogen treatment.

  Fujita T, Hiramatsu N, Shimizu M, Sullivan CV, Hara A

  Proceedings of the 6th International Symposium on the Reproductive Physiology of Fish 314  1999 [Refereed][Not invited]
  
• Changes of serum growth hormone and vitellogenin levels in chtthroat trout during sexual maturation.

  Fukada H, Hiramatsu N, Shimizu M, Sullivan CV, Hara A

  Proceedings of the 6th International Symposium on the Reproductive Physiology of Fish 314  1999 [Refereed][Not invited]
  
• Purification of Immunoglobulin M (IgM) in Serum of Japanese Flounder (Paralichthys olivaceus)

  NISHIDA Hiroko, ENOKIDA Tsuyoshi, HIRAMATUS Naoshi, HARA Akihiko, YOSHIMIZU Mamoru

  Bulletin of the Faculty of Fisheries, Hokkaido University 北海道大学 49 (3) 157 - 164 0018-3458 1998/12 [Refereed][Not invited]
  
• Immunochemical detection, estrogen induction and occurrence in serum of vitelline envelope-related proteins of Sakhalin Taimen Hucho perryi

  M Shimizu, T Fujita, N Hiramatsu, A Hara

  FISHERIES SCIENCE 64 (4) 600 - 605 0919-9268 1998/08 [Refereed][Not invited]
   

  Vitelline envelope-Related proteins (VERPs) were immunologically detected in serum and liver of female Sakhalin taimen Hucho perryi, but not in males, using an antiserum against vitelline envelope. Two VERPs were observed in serum as female-specific proteins with molecular weights of 48 kDa and 42 kDa on SDS-PAGE under non-reduced conditions. VERPs were induced in serum and the liver of immature fish by injection of 10 mu g estradiol-17 beta (E-2) per kg of body weight. These results suggest that the origin of VERPs is the liver. During vitellogenesis, one VERP band first appeared in July in the serum of five-year-old fish. Two VERP bands were observed during winter and became undetectable in June after ovulation. The changes in VERPs were parallel to those in vitellogenin.
• Chemiluminescent Immunoassay (CLIA) for Salmon Growth Hormone (GH)

  Haruhisa Fukada, Naoshi Hiramatsu, Makiko Kitamura, Hitoshi Chiba, Akihiko Hara

  Luminescence 12 (6) 271 - 275 1522-7235 1997/11 [Refereed][Not invited]
   

  A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of growth hormone (GH) in salmonid species. The CLIA for salmon GH was performed using the sandwich method with anti-GH IgG as the first antibody and chemiluminescent acridinium ester-labelled specific anti-GH F(ab′)2 as the second antibody. The measurable range of salmon GH in the CLIA was 39-1250 pg/mL using a short assay (1 day) protocol and 3.9-125 pg/mL in a longer (2-day) assay. The dilution curve in the CLIA of serum from masu salmon (Oncorhynchus masou) was parallel to the standard curve of recombinant chum salmon (Oncorhynchus keta) GH. Seasonal changes of serum GH levels were measured in 1 year-old masu salmon cultivated in a pond from March to November. Their serum GH levels increased during smoltification from March to April, achieved a maximum level of 21 ng/mL in August, and then declined gradually to 11 ng/mL in October. © 1997 John Wiley & Sons, Ltd.
• Development of an ELISA for chum salmon (Oncorhynchus keta) growth hormone

  H Fukada, N Hiramatsu, K Gen, A Hara

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 117 (3) 387 - 392 0305-0491 1997/07 [Refereed][Not invited]
   

  A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of Low levels of serum growth hormone (GH) in chum salmon (Oncorhynchus keta). The antiserum to GH (a-rsGH) was obtained from a rabbit immunized with recombinant chum salmon GH. The noncompetitive ELISA was performed by a sandwich method using a-rsGH rabbit IgG as the first antibody, its biotinylated Fab' fragment as the second antibody, and the avidin-biotin reaction for signal amplification. This assay could he run in 3 days and routinely detected GH at concentrations as low as 0.5 ng/ml. The development of an ELISA for Chi made possible quantification of serum GH levels. In this assay system, parallel dilution curves were obtained using purified chum salmon GH and GH's from several species of the genus Oncorhynchus. (C) 1997 Elsevier Science Inc.
• アメマスSalvelinus leucomaenisビテロゲニンの卵黄蛋白質への特異的分解

  平松尚志, 原彰彦

  日本水産学会誌 63 (5) 701 - 708 0021-5392 1997 [Refereed][Not invited]
  
• Simple Purification of Growth Hormone from Chum Salmon(Oncorhynchus keta).

  深田陽久, 玄浩一郎, 平松尚志, 浦和寛, 原彰彦

  北海道大学水産学部研究い報 北海道大学 47 (2/3) 31 - 39 0018-3458 1996/12 [Not refereed][Not invited]
  

MISC

• クロソイ(Sebastes schlegelii)雄尿に含まれるリポカリンタンパク質のアンドロゲンによる産生制御について

  山口燿, 永田淳, 川崎琢真, 東藤孝, 平松尚志  日本水産学会大会講演要旨集(CD-ROM)  2023-  2023
• クロソイ(Sebastes schlegelii)雄尿タンパク質の同定:Three-finger proteinファミリーに属する新規タンパク質

  山口燿, 足達凛太郎, 南宮眞, 川崎琢真, 東藤孝, 平松尚志  日本水産学会大会講演要旨集(CD-ROM)  2023-  2023
• 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
• 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
• ２つの海に面する八雲：日本海と太平洋の海の生物・環境を比べてみよう 報告書

  平松尚志  海の宝アカデミックコンテスト 2019 ・ブルーオーシャン  2019/07  [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]
  
• 生物情報科学によるメバル属魚類の増養殖関連有用マーカーの探索

  平松 尚志  H30年度 南北海道学術振興財団助成事業報告書  2019  [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]
  
• 魚の卵を科学する！魚の血液検査でオス・メスが分かる？

  平松 尚志  H29年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング サイエンスカフェ  2017/07/29  [Not refereed][Not invited]
  
• サクラマスインスリン様成長因子結合蛋白‐1の機能的組換え蛋白の作製

  田中英絵, 中野裕介, 水田紘子, 平松尚志, 清水宗敬  日本水産学会大会講演要旨集  2017-  170  2017/03/26  [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]
  
• カレイ類受精卵及びシシャモ仔魚に対する種特異抗体の作製

  平松 尚志  H28年度北水協会 水産研究助成事業報告  2017  [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]
  
• カレイ類受精卵及びシシャモ仔魚に対する種特異抗体の作製

  平松 尚志  H27年度 北水協会水産研究助成事業報告  15  -23  2016  [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]
  
• 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  104-  (0)  1066  -1066  2011  [Not 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。
• 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

  Naoshi Hiramatsu, Wenshu Luo, Lei Hong, Kiyoshi Soyano, Junya Aoki, Haruna Amano, Toshiaki Fujita, Takashi Todo, Takahiro Matsubara, Akihiko Hara  National Taiwan Museum Special Publication  14-  (49)  56  2010  [Not refereed][Not invited]
  
• Purification and characterization of lipovitellin from Pacific saury Cololabis saira

  AMANO HARUNA, KITAMURA MAKIKO, FUJITA TOSHIAKI, HIRAMATSU NAOSHI, TODO TAKASHI, SUYAMA SATOSHI, HARA AKIHIKO  Fisheries science : FS  74-  (4)  830  -836  2008/08/01
• 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]
  
• 魚類ビテロジェニンの生体指標蛋白質としての利用

  平松 尚志, 天野春菜, 藤田敏明, 松原孝博, Craig V. Sullivan, 征矢野清, 原彰彦  拠点来額方式による日韓水産学術交流 平成18年度事業報告書  199  -202  2006/03  [Not refereed][Not invited]
  
• North Carolina State University, College of Agriculture and Life Science, Department of Zoology

  平松 尚志  日本比較内分泌学会ニュース = Newsletter of Japan Society for Comparative Endocrinology  (117)  28  -29  2005/05/01  [Not refereed][Not invited]
  
• Vitellogenesis in aquatic animals (Proceedings of International Commemorative Symposium--70th Anniversary of The Japanese Society of Fisheries Science)

  HIRAMATSU N.  Fisheries Science  68-  694  -699  2002/11
• 卵黄蛋白前駆物質（ビテロジェニン）を指標にした成熟度判定手法の開発

  原彰彦, 平松尚志  平成8年度新品種作出基礎技術開発事業研究成果の概要  274  -297  1998  [Not refereed][Not invited]
  
• Limited proteolysis of vitellogenin and its derived yolk proteins in salmonids

  N Hiramatsu, N Ichikawa, K Hosokawa, A Hara  ADVANCES IN COMPARATIVE ENDOCRINOLOGY, TOMES 1 AND 2  1481  -1486  1997  [Not refereed][Not invited]
   

  An enzyme, which specifically converts vitellogenin (Vg) into yolk proteins during vitellogenesis, was purified from vitellogenic ovaries of masu salmon. Purification was conducted with water and ammonium sulfate precipitation, followed by 5 steps of chromatography. Purified enzyme appeared as a single band of 42 kDa in SDS-PAGE, and reacted with. antiserum to human cathepsin-D in western blots. As a result of catabolism of yolk proteins during embryonic development, subunit band of lipovitellin (92 kDa) in SDS-PAGE degraded into smaller polypeptide. beta'-component showed no change in its band patterns, while phosvitin seemed to be dephosphorylated during embryogenesis. The present study is the first to suggest that the catalyst for the intraoocytic processing of Vg is cathepsin-D like proteinase in salmonids. furthermore, the degradation of yolk proteins occurred and appeared to be protein-specific during their embryonic growth.
• 卵黄蛋白前駆物質（ビテロジェニン）を指標にした成熟度判定手法の開発

  原彰彦, 平松尚志, 北村真紀子  平成7年度新品種作出基礎技術開発事業研究成果の概要  180  -192  1996  [Not refereed][Not invited]
  
• 卵黄蛋白前駆物質（ビテロジェニン）を指標にした成熟度判定手法の開発

  原彰彦, 平松尚志, 清水宗敬  平成6年度新品種作出基礎技術開発事業研究成果の概要  192  -203  1995  [Not refereed][Not invited]
  
• Three egg yolk proteins are derived from salmonid vitellogenin

  In proceedings of the fifth International Symposium on the reproductive physiology  364  1995  [Not refereed][Not invited]
  
• 卵黄蛋白前駆物質（ビテロジェニン）を指標にした成熟度判定手法の開発

  原彰彦, 平松尚志  平成5年度新品種作出基礎技術開発事業研究成果の概要  205  -213  1994  [Not refereed][Not invited]
  
• 卵黄蛋白前駆物質（ビテロジェニン）を指標にした成熟度判定手法の開発

  原彰彦, 平松尚志, 稲場琴美  平成4年度新品種作出基礎技術開発事業研究成果の概要  164  -178  1993  [Not refereed][Not invited]
  

Books etc

• 道南おさかな図鑑

  魚に魅せられた北大の研究者たち (Joint workイトウ―ペリー提督も見た幻の巨大魚)
  海文堂出版 2023/10 (ISBN: 9784303800116) 97p
• Vitellogenin as a biomarker for endocrine disruption: molecular and biochemical considerations. In: Biochemical and Molecular Biology of Fishes, pp.431-472, Vol. 6 Environmental Toxicology (T.W. Moon and T.P. Mommsen, editors).

  Elsevier Science BV, Amsterdam, 2005
• Vitellogenin as a biomarker for endocrine disruption: molecular and biochemical considerations. In: Biochemical and Molecular Biology of Fishes

  Elsevier Science BV, Amsterdam 2005

Presentations

• Introduction of a possible new local species for salmon aquaculture “Itou”=“Japanese huchen”  [Not invited]

  Naoshi Hiramatsu

  Hokkaido Innovation Week  2024/01
• Identification of urinary proteins of Sebastes rockfish: novel proteins in lipocalin family and three-finger protein family  [Not invited]

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

  The 17th International Meeting on Reproductive Biology of Aquatic Animals  2023/10
• Androgen signaling is responsible for the renal synthesis of the urinary lipocalin protein in rockfish  [Not invited]

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

  The 17th International Meeting on Reproductive Biology of Aquatic Animals  2023/10
• Increasing efficiency of aquaculture production by producing an all-female mono-sex population in black rockfish (Sebastes schlegelii)  [Not invited]

  Mutea Fridah Gacheri, Yo Yamaguchi, Takuma, Kawasaki, Takashi Todo, Naoshi Hiramatsu

  海洋研究センター成果報告会  2023/06
• メバル類のオスの成熟度を推定できる新たなバイオマーカー:リポカリン様タンパク質の測定系確立とオス成熟度指標としての利用可能性  [Not invited]

  山口 燿, 川崎 琢真, 原 彰彦, 東藤 孝, 平松 尚志

  海洋センター成果報告会  2023/06
• Development of enzyme-linked immunosorbent assay (ELISA) for serum lipocalin-like protein, a potential novel biomarker relating to the male gonadal development, in Sebastes rockfish  [Not invited]

  Yo Yamaguchi, Takuma Kawasaki, Akihiro Hara, Takashi Todo, Naoshi Hiramatsu

  12th International Symposium on Reproductive Physiology of Fish  2023/05
• クロソイ(Sebastes schlegelii)雄尿に含まれるリポカリンタンパク質のアンドロゲンによる産生制御について

  山口燿, 永田淳, 川崎琢真, 東藤孝, 平松尚志

  日本水産学会大会講演要旨集(CD-ROM)  2023
• クロソイ(Sebastes schlegelii)雄尿タンパク質の同定:Three-finger proteinファミリーに属する新規タンパク質

  山口燿, 足達凛太郎, 南宮眞, 川崎琢真, 東藤孝, 平松尚志

  日本水産学会大会講演要旨集(CD-ROM)  2023
• 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
• 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
• 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
• Multiple vitellogenins in Medaka (Oryzias latipes): Characterization of the promoters and yolk derivatives  [Not invited]

  Kojima T., Namgung J, Nagata T., Mushirobira Y., Todo T., Hara A., Reading B.J., Hiramatsu N.

  The 16th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2019/10
• Impact of environmental estrogens on vitellogenin and hybrid striped bass reproduction (Vitellogenesis in Fishes)  [Not invited]

  Reading, B.J., Kowalchyk, C., Andersen, L.K., Fischer, J., Mushirobira, Y., Todo, T., and Hiramatsu, N.

  EDC-NC: Endocrine Disruption Special Interest Group of the Center for Human Health and the Environment  2019/04
• 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  [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  2019/03
• VITELLOGENESIS IN FISHES  [Not invited]

  Benjamin Reading, Linnea Andersen, Justin Schilling, Yuji Mushirobira, Takashi Todo, Naoshi Hiramatsu

  AQUACULTURE 2019  2019/03
• The in vivo effects of estradiol and 11-ketotestosterone on vitellogenin physiology in shortfinned eel, Anguilla australis  [Not invited]

  Thomson-Laing, G, Samsteegt, E.L, Nagata, J, Ijiri, S, Adachi, S, Todo, T, Hiramatsu, N, Lokman, P.M

  11th International Symposium on Reproductive Physiology of Fish  2018/10
• Mutagenesis of a major vitellogenin receptor gene with eight ligand binding repeats in Medaka using CRISPR/Cas9 system  [Not invited]

  Namgung J, Oyama K, Mizuta H, Todo T, Hiramatsu N

  The 15th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2018/08
• Comparison of growth and muscle properties among black rockfish, fox jacopever, and the hybrid  [Not invited]

  Takeuchi M, Kawasaki T, Todo T, Hiramatsu N

  The 15th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2018/08
• Determination of efficient microsatellite markers for parentage diagnosis in viviparous rockfish  [Not invited]

  Numayama A, Kawasaki T, Todo T, Hiramastu N

  The 15th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2018/08
• Measurement of multiple vitellogenins in marbled sole, Pleunectes yokohamae, using type-specific enzyme-linked immunosorbent assays  [Not invited]

  Amano H, Uno S, Koyama J, Hiramatsu N, Todo T, Hara A

  The 15th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2018/08
• Expression of estrogen responsive genes in primary cultured hepatocyte of cutthroat trout, Oncorhynchus clarki  [Not invited]

  Nagata J, Mushirobira Y, Nishimiya O, Fujita T, Hiramastsu N, Hara A, Todo T

  The 15th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2018/08
• 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
• 北方性ソイ・メバル類の増養殖に向けた基礎および応用研究  [Not invited]

  柴田侑人, 平松尚志, 東藤孝

  H29年度函館市国際水産・海洋総合研究センター・マリンサロン  2018/01
• 函館市国際水産・海洋総合研究センターにおける海洋教育研究活動の実践  [Not invited]

  高原英生, 安部智貴, 平松尚志, 宮下和士

  2017年度日本海洋学学会秋季大会、海洋教育特別ポスターイベント：海洋教育・アウトリーチ活動の実践と課題  2017/10
• Changes in mRNA levels of estrogen responsive genes in the liver of cutthroat trout following estradiol-17beta injection.  [Not invited]

  Nagata J, Mushirobira Y, Hiramatsu N, Hara A, Todo T

  The 14th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea,  2017/09
• 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
• サクラマスインスリン様成長因子結合蛋白‐1の機能的組換え蛋白の作製  [Not invited]

  田中英絵, 中野裕介, 水田紘子, 平松尚志, 清水宗敬

  日本水産学会大会講演要旨集  2017/03
• クロソイとキツネメバルの天然妊娠魚における雄の交尾尾数の解析  [Not invited]

  村山大樹, 川崎琢磨, 東藤孝, 平松尚志, 原彰彦

  平成28年度函館国際水産・海洋都市構想シンポジウム・海洋研究センター研究発表会  2017/03
• GnRH投与によるエゾメバル雌の性成熟促進  [Not invited]

  紫藤勇磨, 川崎琢磨, 村山大樹, 猪股勇斗, 石毛友里絵, 平松尚志, 東藤孝

  平成28年度函館国際水産・海洋都市構想シンポジウム・海洋研究センター研究発表会  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
• Differential responses of oogenesis-related genes to estradiol-17β in the liver of cutthroat trout, (Onchorhychus clarki).  [Not invited]

  Nagata, J, Mushirobira, Y, Hiramatsu, N, Hara, A, Todo, T

  The 13th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2016/11
• Expression profiles of dual vitellogenin genes in the cutthroat trout (Onchorhychus clarki) following estradiol-17β administration.  [Not invited]

  Mushirobira,Y, Nagata, J, Todo, T, Hara, A, Hiramatsu, N

  The 13th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2016/11
• 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
• 魚の卵を科学する～基礎から応用への共同研究の可能性  [Not invited]

  平松 尚志

  第一回食科学プラットフォーム「水産ブロック」意見交換会  2016/09
• 魚の卵を科学する  [Not invited]

  平松 尚志

  平成28年度（第30回）北海道大学水産学部公開講座「弁天町発！！水産・海洋研究の最前線」  2016/07
• 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 mechanisms underlying yolk formation in fish: how to make the tailor-made yolk?  [Not invited]

  Hiramatsu, N, Todo, T

  The 2nd Joint Symposium of HU/NTOU & 1st International Conference on Quaternary and Future Earth  2016/01
• Oogenesis in fish: what makes fish eggs ? Current understanding of yolk formation in fish  [Not invited]

  HIRAMATSU Naoshi

  Hokkaido University (HU) Learning Satellite “Fish ‘n Czech” 2015  2015/09
• 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
• Analysis of lipid droplet formation in oocytes of the cutthroat trout, Oncorhynchus clarki in vitro  [Not invited]

  Nagata, J, Mushirobira, Y, Hiramatsu, N, Hara, A, Todo, T

  The 12th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2015
• Vitellogenesis in white perch (Morone americana): Multiple vitellogenins and their receptors  [Not invited]

  Schilling, J, Hiramatsu, N, Daniels, H.V, Reading, B.J

  In: abstract of Aquaculture America 2015 (https://www.was.org/meetings/ShowAbstract.aspx?Id=34659)  2015
• Molecular characterization of dual vitellogenin gene promoters in the cutthroat trout (Oncorhynchus clarki)  [Not invited]

  Mushirobira, Y, Nishimiya, O, Nagata, J, Todo, T, Hara, A, Hiramatsu, N

  The 12th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2015
• 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
• Detection and identification of plasma apolipoproteins using liquid chromatography-tandem mass spectrometry in cutthroat trout, Oncorhynchus clarki  [Not invited]

  Mushirobira, Y, Todo, T, Hara, A, Hiramatsu N

  The 11th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2014/12
• Sexual maturation of the white-edged rockfish (Sebastes taczanowskii): effects of photothermal manipulation on the gonadal growth and maturation  [Not invited]

  和田怜, 水田紘子, 川崎琢磨真, 高畠信一, 原彰彦, 東藤孝, 平松尚志

  食とバイオ国際交流シンポジウム  2014/06
• エゾメバル雄における性成熟誘導と人工授精に関する基礎研究  [Not invited]

  小渡賢太, 川崎琢真, 高畠信一, 和田怜, 西宮攻, 莚平裕次, 東藤孝, 原彰彦, 平松尚志

  食とバイオ国際交流シンポジウム  2014/06
• Ligand binding properties of ovarian lipoprotein receptors in the cutthroat trout  [Not invited]

  Mushirobira, Y, Mizuta, H, Luo, W, Todo, T, Hara, A, Reading, B.J, Sullivan, C.V, Hara, A

  10th International Symposium on Reproductive Physiology of Fish  2014/05
• How do eggs fat? Insights into triacylglyceride uptake in the oocytes of the shortfinned eel, Anguilla australis  [Not invited]

  Damsteegt, E.L, Mizuta, H, Hiramatsu, N, Lokman, P.M

  10th International Symposium on Reproductive Physiology of Fish  2014/05
• Very low-density lipoprotein is primary carrier of neutral lipids to ooplasm lipid droplets in teleosts  [Not invited]

  Ryu, Y.W, Todo, T, Hiramatsu, N, Matsubara, T, Sullivan, C.V, Hara, A

  10th International Symposium on Reproductive Physiology of Fish  2014/05
• Ovarian yolk formation in fishes: molecular mechanisms underlying formation of lipid droplets and vitellogenin-derived yolk proteins  [Invited]

  Hiramastu, N, Todo, T, Sullivan, C.V, Reading, B.J, Matsubara, T, Ryu, Y.W, Mizuta, H, Luo, W, Nishimiya, O, Wu, M, Mushirobira, Y, Hara, A

  10th International Symposium on Reproductive Physiology of Fish  2014/05
• 魚卵アレルゲンのIgE反応性変化を調べるツールとしての抗ペプチド抗体の有用性  [Not invited]

  清水裕, 平松尚志, 佐伯宏樹

  日本栄養・食糧学会大会講演要旨集  2014/04
• 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
• スケトウダラ卵に含まれる食物アレルゲンの精製と性状解析  [Not invited]

  田原大貴, 清水裕, 平松尚志, 渡辺一彦, 佐伯宏樹

  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
• Sexual maturation of the white-edged rockfish (Sebastes taczanowskii): effects of photothermal manipulation on the gonadal growth and maturation  [Not invited]

  Wada, S, Mizuta, H, Kawasaki, T, Takabatake, S, Hara, A, Todo, T, Hiramatsu, N

  The 10th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2013/11
• Ligand binding properties of ovarian lipoprotein receptors in the cutthroat trout (Oncorhynchus clarki)  [Not invited]

  Mushirobira, Y, Mizuta, H, Luo, W, Todo, T, Hara, A, Hiramatsu, N

  The 10th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2013/11
• Yolk formation in fish: Multiple vitellogenins and their receptors.  [Not invited]

  HIRAMATSU, NaoshiHiramatsu, N, Mizuta, H, Luo, W, Nishimiya, O, Wu, Meiqin, Mushirobira, Y, Reading, B.J, Sullivan, C.V, Todo, T, Hara, A

  Diversification in Inland Finfish Aquaculture II (DIFAII).  2013/09
• Molecular cloning and characterization of estrogen receptors from the most primitive vertebrate, the inshore hagfish /Eptatretus burgeri/.  [Not invited]

  Nishimiya, O, Katsu, Y, Inagawa, H, Hiramatsu,N, Todo T, Hara, A

  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
• ペプチド抗体を用いた魚卵アレルギーの魚種間交差性に関する検討  [Not invited]

  清水裕, 岸村栄毅, 平松尚志, 原彰彦, 佐伯宏樹

  日本水産学会大会講演要旨集  2013/03
• 卵生脊椎動物におけるビテロジェニン分子の成立と進化の解明  [Not invited]

  西宮攻, 国弘康之, 山根広大, 平松尚志, 東藤孝, 原明彦

  生命情報科学若手の会 第4回研究会  2013/03
• Multiplicity of vitellogenin and their receptors in genus Morone.  [Not invited]

  Sullivan CV, Reading BJ, Schlling JD, Williams VN, Glassbrook N, Hiramatsu N, Luo W, Mizuta H, Todo T, Hara A

  Aquaculture 2013  2013/02
• Dual vitellogenins in cutthroat trout (Oncorhynchus clarki): purification and changes in serum proten level during reproductive cycle.  [Not invited]

  Mushirobira Y, Mizuta H, Luo W, Morita Y, Sawaguchi S, Matsubara T, Hiramatsu N, Todo T, Hara A

  The 9th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2012/12
• Molecular biological and immunochemical studies of vitellogenin in loach (Misgurnus anguillicaudatus).  [Not invited]

  Wu M, Nishimiya O, Mizuta H, Hiramatsu N, Todo T, Nakamori M, Suzuki A, Soyano K, Hara A

  The 9th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2012/12
• Molecular cloning and characterization of two estrogen receptor subtypes from a hagfish Eptairetus burgeri.  [Not invited]

  Nishimiya O, Inagawa H, Katsu Y, Hiramatsu N, Todo T, Hara A

  The 9th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2012/12
• 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
• サクラマスの銀化に伴うインスリン様成長因子‐Iの変化と鰓Na+,K+‐ATPaseとの関係  [Not invited]

  下村考弘, 中嶋拓郎, 堀越萌李, 飯嶋亜内, 卜部浩一, 水野伸也, 平松尚志, 原彰彦, 清水宗敬

  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
• カットスロートトラウト卵黄におけるスカベンジャー受容体クラスＢタイプＩ遺伝子の発現解析  [Not invited]

  斎藤恭一, 柳ヨンウン, 伊東優太, 平松尚志, 東藤孝, 原彰彦

  第5回サケ学研究会  2011/12
• 卵黄蛋白前駆物質ビテロジェニンの異種間投与とその運搬過程：イトウとゼブラフィッシュを用いたモデルについて  [Not invited]

  櫻井秀之, 川北奈央子, 平松尚志, 東藤孝, 原彰彦

  第5回サケ学研究会  2011/12
• サクラマスにおけるインスリン様成長因子結合蛋白―1の発現パターンと成長の関係  [Not invited]

  川口航平, 下村孝弘, 中野祐介, 木村志津雄, 原彰彦, 清水宗敬

  第5回サケ学研究会  2011/12
• サクラマスの銀化変態期における鰓Na+/K+-ATPase 活性とインスリン様成長因子-1  [Not invited]

  下村孝弘, 中嶋拓郎, 堀越萌李, 飯嶋亜内, 卜部浩一, 水野伸也, 平松尚志, 原彰彦, 清水宗敬

  第5回サケ学研究会  2011/12
• Dual vitellogenins in cutthroat trout (Oncorhynchus clarki): molecular cloning, structural characterization, and changes in hepatic expression during reproductive cycle.  [Not invited]

  Mushirobira Y, Mizuta H, Luo W, Sawaguchi S, Matsubara T, Hiramatsu N, Todo T, Hara A

  The 8th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2011/10
• 抗シジミビテリン抗体を用いたヤマトシジミの雌雄判別  [Not invited]

  寺西哲夫, 水野伸也, 小出展久, 田中浩, 桑田博, 平松尚志, 足立伸次

  日本水産学会大会講演要旨集  2011/09
• 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

  平成23年度繁殖生物学会  2011/09
• Novel class of ovarian liporotein receptor in cutthroat trout: Molecular cloning and expression analysis  [Not invited]

  Hiramatsu N, Luo W, Mizuta H, Todo T, Reading BJ, SUllivan CV, Hara A

  9th international symposium on reproductive physiology of fish  2011/08
• マコガレイの3タイプの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

  第24回北里大学バイオサイエンスフォーラム  2011/08
• Expression of genes involved in oocyte lipidation in cutthroat trout, Oncorhynchus clarki.  [Not invited]

  Ryu Y, Tanaka R, Kasai A, Saito K, Kanno Y, Ito Y, Hiramatsu N, Todo T, Sullivan CV, Hara A

  9th international symposium on reproductive physiology of fish  2011/08
• Molecular characterization and expression analysis of estrogen receptor and vitellogenins in inshore hagfish (Eptatretus burgeri)  [Not invited]

  Nishimiya O, Kunihiro Y, Hiramatsu N, Inagawa H, Todo T, Matsubara T, Reading BJ, Sullivan CV, Hara A

  9th international symposium on reproductive physiology of fish  2011/08
• Molecular cloning and localization of two classical ovarian lipoprotein receptors in cutthroat trout (Oncorhynchus clarki)  [Not invited]

  Mizuta H, Hiramatsu N, Todo T, Ito Y, Gen K, Kazeto Y, Sullivan CV, Reading BJ, Hara A

  9th international symposium on reproductive physiology of fish  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
• スジイルカalpha‐fetoprotein遺伝子の発現動態  [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  2011/03
• 人工飼育条件下におけるヤマトシジミの成熟促進  [Not invited]

  寺西哲夫, 水野伸也, 小出展久, 守山義昭, 平松尚志, 足立伸次

  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
• サクラマスにおける海水適応能の発達とインスリン様成長因子ー１の発現パターン  [Not invited]

  下村孝弘, 堀越萌李, 中嶋拓郎, 飯嶋亜内, 卜部浩一, 水野伸也, 平松尚志, 原彰彦, 清水宗敬

  第4回サケ学研究会  2010/12
• 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]

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

  第4回サケ学研究会  2010/12
• Molecular cloning and structural characterization of a putative estrogen receptor in inshore hagfish (Eptatretus burgeri)  [Not invited]

  Nishimiya O, Kunihiro Y, Hiramatsu N, Inagawa H, Todo T, Hara A

  JSPS Core University Program Seminar: Proposal to Sustainable Fisheries  2010/12
• Expression analysis of lipoprotein lipase family genes: involvement in processes of lipid accumulation into oocyte of cutthroat trout, Oncorhynchus clarkii  [Not invited]

  Ryu YW, Tanaka R, Kasahara A, Massaki K, Ito Y, Hiramatsu N, Todo T, Hara A

  JSPS Core University Program Seminar: Proposal to Sustainable Fisheries  2010/12
• A novel ovarian membrane receptor (LRX+1) belonging to low density lipoprotein receptor gene family: characterization of the primary structure and expression in cutthroat trout  [Not invited]

  Luo W, Hiramatsu N, Todo T, Reading BJ, Sullivan CV, Hara A

  JSPS Core University Program Seminar: Proposal to Sustainable Fisheries  2010/12
• Expression and localization of two ovarian lipoprotein receptor proteins in cutthroat trout (Oncorhynchus clarkii)  [Not invited]

  Mizuta H, Hiramastu N, Todo T, Kudo H, Hara A

  JSPS Core University Program Seminar: Proposal to Sustainable Fisheries  2010/12
• Molecular characterization of hagfish (Eptatretus burgeri) estrogen receptor (ER)  [Not invited]

  Nishimiya O, Kunihiro Y, Hiramatsu N, Inagawa H, Todo T, Hara A

  The 7th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2010/11
• Characterization of ovarian lipoprotein receptor proteins in cutthroat trout: vitellogenin receptor and low-density lipoprotein rceptor  [Not invited]

  Mizuta H, Hiramatsu N, Todo T, Kudo H, Hara A

  The 7th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2010/11
• Molecular cloning and expression analysis of a novel ovarian lipoprotein receptor in cutthroat trout (Oncorhynchus clarkii)  [Not invited]

  Luo W, Hiramatsu N, Todo T, Reading BJ, Sullivan CV, Hara A

  The 7th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2010/11
• Studies on molecular mechanisms of lipid uptake into fish oocytes: identification and expression analysis of lipoproteins lipase family genes in the ovary of cutthroat trout, Oncorhynchus clarkii  [Not invited]

  Ryu YW, Tanaka R, Kasahara A, Massaki K, Ito Y, Hiramatsu N, Todo T, Hara A

  The 7th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2010/11
• ボラを調査対象生物とした東シナ海沿岸域における環境ホルモン汚染の実態調査  [Not invited]

  板敷祥昌, WU Meiqin, ZHONG Junsheng, PARK Chang‐Beom, 青木純哉, 平松尚志, 原彰彦, 征矢野清

  日本水産学会大会講演要旨集  2010/09
• ヤマトシジミビテリン量の測定  [Not invited]

  寺西哲夫, 水野伸也, 小出展久, 大川和之, 守山義昭, 平松尚志, 足立伸次

  日本水産学会大会講演要旨集  2010/09
• ヤマトシジミビテリンの精製と免疫組織化学的局在  [Not invited]

  寺西哲夫, 水野伸也, 小出展久, 吉田豊, 大川和之, 守山義昭, 平松尚志, 原彰彦, 足立伸次

  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
• 魚類の卵形成に関与する卵黄前駆物質およびその受容体に関する研究  [Not invited]

  平松尚志

  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
• Development of evaluation systems for the detection of estrogenic endocrine disrupting chemicals (EDCs) in aquatic environments using estrogen-inducible biomarkers.  [Not invited]

  Hiramatsu N, Luo W, Hong L, Soyano K, Aoki J, Amano H, Fujita T, Todo T, Matsubara T, Hara A

  International Symposium on Formosa Landlocked salmon and Masu Salomon  2009/10
• Purification and molecular cloning of alpha-fetoprotein in fetal striped dolphin, Stenella coeruleoalba  [Not invited]

  Morita Y, Hiramatsu N, Fujita T, Amano H, Iwasaki T, Todo T, Hara A

  18th Biennial confference on the Biology of Marine Mammals  2009/10
• Molecular cloning and expression analysis of lipoprotein lipase family genes from ovary of cutthroat trout, Oncorhynchus clarkii.  [Not invited]

  Ryu YW, Tanaka R, Kasahara A, Massaki K, Ito Y, Hiramatsu N, Todo T

  The 6th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2009/09
• Low-density-lipoprotein receptor in cutthroat trout (Oncorhynchus clarkii): molecular cloning and experssion analysis.  [Not invited]

  Ito Y, Massaki K, Ito T, Hiramatsu N, Todo T, Fujita T, Hara A

  The 6th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2009/09
• Estrogenic activities of coastal aquatic environments in China: evaluation using a model species, the red lip mullet (Chelon haematochelilus).  [Not invited]

  Wu M, Aoki J, Hiramatsu N, Hara A, Soyano K, Zhong J

  The 6th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2009/09
• 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
• 魚類のリポタンパク質と受容体に関する研究  [Not invited]

  平松 尚志

  平成20年度北海道海洋生物科学研究会シンポジウム  2008/11
• Involvement of multiple vitellogenin-system in control and adaptation mechanism of egg buoyancy in marine teleosts.  [Not invited]

  Matsubara T, Ohkubo N, Nagae M, Hiramatsu N, Amano H, Reading BJ, Sullivan CV, Hara A

  World Fisheries Congress  2008/10
• Molecular characterization of hagfish vitellogenin  [Not invited]

  Kunihiro Y, Hiramatsu N, Fujita T, Amano H, Inagawa H, Todo T, Hara A

  5th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2008/09
• Purification of putative alpha-fetoprotein in fetal serum of striped dolphin, Stenella coeruleoalba  [Not invited]

  Morita Y, Hiramatsu N, Fujita T, Amano H, Iwasaki T, Todo T, Hara A

  5th International Meeting on Reproductive Biology of Aquatic Animals of the East China Sea  2008/09
• Multiple biomarkers for detecting estrogenic endocrine disruption  [Not invited]

  Hiramatsu N, Hara A

  International Universities Exchange Seminar Sustainability and Risk Management of Seafood and Ocean Ecosystem Conservarion  2008/08
• Molecular cloning and characterization of low density lipoprotein receptor in cutthroat trout (Oncorhynchus clarki)  [Not invited]

  Massaki K, Hiramatsu N, Todo T, Fujita T, Sullivan CV, Hara A

  Sex Determination and Gametogenesis in Fish: CUrrent status and Future Directions  2008/05
• Expression of lipoprotein lipase gene in ovarian follicles of cutthroat trout (Oncorhynhus clarki) during gonadal development.  [Not invited]

  Todo T, Kasahara A, Massaki K, Hiramatsu N, Sullivan CV, Hara A

  Sex Determination and Gametogenesis in Fish: CUrrent status and Future Directions  2008/05
• Teleost oocyte growth, vitellogenesis, and cytoplasmic maturation  [Not invited]

  Sullivan CV, Reading BJ, Hiramatsu N, Todo T, Matsubara T, Sawaguchi S, Amano H, Hara A

  Sex Determination and Gametogenesis in Fish: CUrrent status and Future Directions  2008/05
• Yolk assembly in teleost: recent findings on the deposition of ovarian lipids and proteins  [Not invited]

  Hiramatsu N, Todo T, Ito T, Massaki K, Kasahara A, Amano H, Reading BJ, Matsubara T, Sawaguchi S, Sullivan CV, Hara A

  World Aquaculture 2008  2008/05
• 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
• 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
• Purification of multiple vitellogenins in three-spined stickleback (Gasterosteus aculeatus)  [Not invited]

  Takahashi M, Hiramatsu N, Fujita T, Todo T, Matsubara T, Hara A

  4th Japan-Korea, Korea-Japan Joint Meeting on Reproductive Biology of Aquatic Animals.  2007/11
• Molecular cloning of CD36 in cuttroat trout (Oncorhynchus clarkii)  [Not invited]

  Ito T, Hiramatsu N, Fujita T, Amano H, Todo T, Hara A

  4th Japan-Korea, Korea-Japan Joint Meeting on Reproductive Biology of Aquatic Animals.  2007/11
• Purification of vitellogenin and lipovitellin-like yolk protein in hagfish (Eptatretus burgeri)  [Not invited]

  Kunihiro Y, Amano H, Fujita T, Inagawa H, Hiramatsu N, Todo T, Hara A

  4th Japan-Korea, Korea-Japan Joint Meeting on Reproductive Biology of Aquatic Animals.  2007/11
• Multiple vitellogenins and their derived yolk proteins in grey mullet (Mugil cephalus): differential proteolytic patterns during oocyte growth and maturation  [Not invited]

  Amano H, Fujita T, Hiramatsu N, Matsubara T, Sullivan CV, Hara A

  4th Japan-Korea, Korea-Japan Joint Meeting on Reproductive Biology of Aquatic Animals.  2007/11
• Monitoring of endocrine disruptant usig serum protein as bio-markers  [Not invited]

  Hiramatsu N, Hara A

  International Workshop on the Evaluation of the 21st COE Program, Marine Bio-manipulation Frontier for Food Production.  2007/11
• サケ科魚類の卵黄形成―多型ビテロジェニンとコリオジェニン  [Not invited]

  藤田敏明, 望月麻智子, 天野春菜, 平松尚志, 東藤孝, 原彰彦

  第1回サケ学研究会  2007/09
• Differential synthesis and uptake of dual vitellogenins in Japanese medaka (Oryzias latipes).  [Not invited]

  Hiramastu N, Inoue M, Ideuchi H, Fujita T, Amano H, Matsubara T, Sullivan CV, Hara A

  8th International Symposium on Reproductive Physiology of Fish  2007/06
• Controlled accumulation of multiple vitellogenins into ocytes during vitellogenesis in the barfin flounder, Verasper moseri.  [Not invited]

  Sawaguchi S, Ohkubo N, Amano H, Hiramatsu N, Hara A, Sullivan CV, Matsubara T

  8th International Symposium on Reproductive Physiology of Fish  2007/06
• Yolk precursors in white perch (Morone americana): deduced primary structures of three types of vitellogenin (vg) proteins and disparate binding of the different Vgs to multiple ovarian receptors  [Not invited]

  Reading BJ, Hiramatsu N, Matsubara T, Hara A, Sullivan CV

  8th International Symposium on Reproductive Physiology of Fish  2007/06
• Purification and classification of egg yolk proteins derived from multiple vitellogenins in grey mullet (Mugil cephalus)  [Not invited]

  Amano H, Fujita T, Hiramatsu N, Matsubara T, Sullivan CV, Hara A

  8th International Symposium on Reproductive Physiology of Fish  2007/06
• トラザメのリポビテリンおよびホスビチンの精製  [Not invited]

  山根広大, 菅原理恵子, 天野春菜, 藤田敏明, 杉下孝治, 高谷広行, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  2007/03
• サケ科魚類雄魚の血中ビテロジェニンおよびコリオジェニン―回遊期と遡上期での比較―  [Not invited]

  西川友典, 望月麻智子, 藤田敏明, 遊佐清明, 山本潤, 亀井佳彦, 高木省吾, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  2007/03
• 大西洋タラの卵膜蛋白前駆物質(コリオジェニン)の精製  [Not invited]

  藤田敏明, 天野春菜, SCOTT A. P, KATSIADAKI I, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  2007/03
• ボラ卵黄蛋白の催熟前後における比較  [Not invited]

  天野春菜, 藤田敏明, 平松尚志, 香川浩彦, 原彰彦

  日本水産学会大会講演要旨集  2007/03
• Multiple egg yolk precursors (vitellogenins) and egg quality in teleost fishes.  [Not invited]

  Reading BJ, Hiramatsu N, Williams VN, Sullivan CV, Sawaguchi S, Matsubara T, Amano H, Hara A

  Aquaculture America 2007  2007/02
• Development of monitoring system on endocrine disruption.  [Not invited]

  Hiramatsu N, Amano H, Fujita T, Hara A

  Symposium on Developing Fisheries Science in Asia.  2006/12
• Fish vitellogenin: single versus multiple vitellogenin models.  [Not invited]

  Hiramatsu N, Amano H, Fujita T, Matsubara T, Todo T, Nagae M, Soyano K, Hara A

  3rd Japan-Korea, Korea-Japan Joint Meeting on Reproductive Biology of Aquatic Animals.  2006/11
• The multiple vitellogenin system of Xenotoca eiseni  [Not invited]

  Sullivan CV, Williams VN, Reading BJ, Hiramatsu N, Sawaguchi S, Matsubara T, Amano H, Hara A

  The III international symposium on viviparous fishes  2006/11
• Purification of C-type vitellogenin (VgC) in Sakhalin taimen.  [Not invited]

  Mochizuki M, Amano H, Fujita T, Hiramtasu N, Hara A

  The 6th Joint Seminar between Japan and Korea by Core University Program on Fisheries Sciences- Sustainability of Fisheries in Japan and Korea-  2006/08
• Multiple forms of vitellogenin and choriogenin in red lip mullet (Chelon haematocheilus): Immunological detection using type-specific antisera.  [Not invited]

  Hong L, Hiramatsu N, Amano H, Fujita T, Hara A

  The 6th Joint Seminar between Japan and Korea by Core University Program on Fisheries Sciences- Sustainability of Fisheries in Japan and Korea-  2006/08
• Purification and classification of egg yolk proteins in grey mullet (Mugil cephalus)  [Not invited]

  Amano H, Fujita T, Hiramatsu N, Shimizu M, Sawaguchi S, Matsubara T, Kagawa H, Sullivan CV, Hara A

  The 6th Joint Seminar between Japan and Korea by Core University Program on Fisheries Sciences- Sustainability of Fisheries in Japan and Korea-  2006/08
• 魚類ビテロジェニンの生体指標蛋白質としての利用  [Not invited]

  平松尚志, 天野春菜, 藤田敏明, 松原孝博, Sullivan CV, 征矢野清, 原彰彦

  The 6th Joint Seminar between Japan and Korea by Core University Program on Fisheries Sciences-Sustainability of Fisheries in Japan and Korea-  2006/08
• Purification of multiple vitellogenins from grey mullet.  [Not invited]

  Amano H, FUjita T, Hiramatsu N, Hara A

  The 5th International Symposium of Industrial-Academia-Govermental Collaboration for the Establishment of Marine Production in China and Japan  2006/07
• ボラの3型ビテロジェニンの精製  [Not invited]

  天野春菜, 藤田敏明, 平松尚志, 沢口小有美, 松原孝博, 原彰彦

  日本水産学会大会講演要旨集  2006/03
• ボラを用いた環境ホルモンのモニタリングー3型ビテロジェニンの精製ー  [Not invited]

  天野春菜, 藤田敏明, 平松尚志, 原彰彦

  21世紀COEプログラム「海洋生命統御による食糧生産の革新」都市エリア産学官連携促進事業「水産・海洋に特化したライフサイエンス領域」合同成果発表会「函館エリアにおけるライフサイエンスの最前線」  2006/03
• Molecular Characterization of three forms of vitellogenin and their yolk protein products during oovyte growth and maturation in red seabream, pagrus major  [Not invited]

  Sawaguchi S, Kagawa H, Ohkubo N, Hiramtasu N, Sullivan CV, Hara A, Matsubara T

  4th International Symposium of Reproductive, Genetica dn Desease Management in Aquaculture and Ocean Ranching  2005/08
• Multiple lipovitellins in grey mullet: N-terminal amino acid sequencing and mapping of their constituent polypeptides.  [Not invited]

  Amano H, Fujita T, Hiramatsu N, Sawaguchi S, Matsubara T, Kagawa H, Sullivan CV, Hara A

  4th International Symposium of Reproductive, Genetica dn Desease Management in Aquaculture and Ocean Ranching  2005/08
• ボラ血清中の複数ビテロジェニンの検索および精製  [Not invited]

  天野春菜, 藤田敏明, 平松尚志, YEO In‐Kyu, 香川浩彦, 原彰彦

  日本水産学会大会講演要旨集  2005/04
• ボラビテロジェニンの性状解析  [Not invited]

  天野春菜, 藤田敏明, 平松尚志, Yeo IK, 香川浩彦, 原彰彦

  北海道大学市民オープンフォーラム「海・函館・北海道発わたしたちの健康と水産物の安全・安心  2005/03
• Detection of three vitellogenins in serum of grey mullet.  [Not invited]

  Amano H, Fujita T, Hiramatsu N, Yeo IK, Kagawa H, Sullivan CV, Hara A

  1st International Symposium of "potential and perspective of Marine Bio-Manipulation".  2005/02
• Hybrid striped bass farming in the United States: Research and industry development. 4th International Symposium of "Reproductive, Genetic and Disease Management in Aquaculture and Ocean Ranching"  [Not invited]

  21st COE symposium  2005
• Hybrid striped bass farming in the United States: Research and industry development. 4th International Symposium of "Reproductive, Genetic and Disease Management in Aquaculture and Ocean Ranching"  [Not invited]

  21st COE symposium  2005
• Vitellogenin multiplicity and egg quality in teleost fishes  [Not invited]

  Reading BJ, Hiramatsu N, Matsubara T, Hara A, Sullivan CV

  134th Annual meeting of the American Fisheries Society  2004/08
• Immunoassays of fish vitellogenin  [Not invited]

  Hara A, Fukada H, Hiramatsu N

  World Aquaculture Society  2004/03
• Fish vitellogenin as a biomarker for environmental estrogens.  [Not invited]

  Bilateral Seminar Italy and Japan  2004
• Multiple vitellogenins in tilapia  [Not invited]

  ハワイ大学 夏期プログラム （Edwin W. Pauley Summer Program in Marine Biology)  2004
• Oogenesis in teleost: Myths and Miracles: Opportunities and Applications for Basic Research  [Not invited]

  ハワイ大学 夏期プログラム （Edwin W. Pauley Summer Program in Marine Biology)  2004
• Oogenesis in teleost: Myths and Miracles: Opportunities and Applications for Basic Research  [Not invited]

  Edwin W. Pauley Summer Program in Marine Biology  2004
• Multiple vitellogenins in tilapia  [Not invited]

  Edwin W. Pauley Summer Program in Marine Biology  2004
• Fish vitellogenin as a biomarker for environmental estrogens.  [Not invited]

  Bilateral Seminar Italy and Japan  2004
• Ovarian atresia in temperate basses: detection of hatching enzymes (choriolysins) in atretic ovaries  [Not invited]

  Donato D, Hiramatsu N, Arey KA, Hiramatsu K, Kennedy AM, Morton CL, Hara A, Sulivan CV

  7th International Symposium on Reproductive Physiology of Fish.  2003/05
• Oocyte growth and cytoplasmic maturation of temperate basses: multiple vitellogenins and their receptors.  [Not invited]

  Hiramatsu N, Hara A, Matsubara T, Hiramatsu K, Sullivan CV

  7th International Symposium on Reproductive Physiology of Fish.  2003/05
• Vitellogenin in male fish as a bio-marker for estrogenic contamination of the aquatic environment.  [Not invited]

  Hara A, Hiramatsu N, Soyano K, Matsubara T

  Inauguration conference and first academic symposium, CLARINET  2001/11
• Cloning of cDNA for choriogenin H and choriogenin L of masu salmon  [Not invited]

  Fujita T, FUkada H, Shimizu M, Hiramtasu N, Hara A

  70th Anniversary of JSFS, International Commemorative Symposium  2001/10
• マコガレイ血清の雌特異蛋白の動態 ビテロジェニンおよびコリオジェニン  [Not invited]

  小寺智志, 平松尚志, 藤田敏明, 深田陽久, 藤井一則, 原彰彦

  日本水産学会大会講演要旨集  2001/04
• Estimatio of baseline vitellogenin levels in salmonid male serum  [Not invited]

  Haga A, Fukada H, Fujita T, Hiramatsu N, Hara A

  環境ホルモン学会第3回研究発表会  2000/12
• Vitellogenin-derived yolk proteins of white perch: purification, characterization, and vitellogenin-receptor binding.  [Not invited]

  Hiramatsu N, Hiramatsu K, Fukada H, Weber GM, Hara A, Sullivan CV

  4th International Symposium on FIsh Endocrinnology  2000/07
• Development of chemiluminescent immunoassay for salmonid vitellogenin: serum vitellogenin and estradiol 17beta levels in male cutthroat trout  [Not invited]

  Fukada H, Haga A, Fujita T, Hiramatsu N, Sullivan CV, Hara A

  4th International Symposium on FIsh Endocrinnology  2000/07
• Estrogen induction of choriogenin H and choriogenin L in masu salmon Oncorhynchus masau.  [Not invited]

  Fujita T, Fukada H, Hiramatsu N, Shimizu M, Haga A, Hara A

  4th International Symposium on FIsh Endocrinnology  2000/07
• 化学発光イムノアッセイを用いたカットスロートトラウト血中Vg量の測定  [Not invited]

  芳賀歩, 深田陽久, 藤田敏明, 平松尚志, 木村志津雄, 原彰彦

  日本水産学会大会講演要旨集  2000/04
• マコガレイ血中ビテロジェニンの周年変化  [Not invited]

  小寺智志, 平松尚志, 藤田敏明, 原彰彦

  平成11年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1999/12
• Changes of serum choriogenin and vitellogenin levels in masu salmon (Oncorhynchus masou) during sexual maturation and after estrogen treatment.  [Not invited]

  Fujita T, Hiramatsu N, Shimizu M, Sullivan CV, Hara A

  6th International Symposium on the Reproductive Physiology of Fish.  1999/07
• Changes of serum growth hormone and vitellogenin levels in chtthroat trout  [Not invited]

  Fukada H, Hiramatsu N, Shimizu M, Sullivan CV, Hara A

  6th International Symposium on the Reproductive Physiology of Fish.  1999/07
• サクラマス卵膜蛋白前駆物質(コリオジェニン)の血中量の変化  [Not invited]

  藤田敏明, 清水宗敬, 平松尚志, 木村志津雄, 原彰彦

  日本水産学会大会講演要旨集  1999/04
• コイのビテロジェニン酵素免疫測定法(ELISA)の確立  [Not invited]

  平野香織, 深田陽久, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  1999/04
• カットスロートの卵黄形成初期におけるビテロジェニンとコリオジェニンについて  [Not invited]

  芳賀歩, 藤田敏明, 平松尚志, 木村志津雄, 原彰彦

  平成10年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1998/11
• ビテロジェニンのcross reaction  [Not invited]

  高橋みのり, 平松尚志, 原彰彦

  平成10年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1998/11
• Fatty acid composition of bony fishe egg and its change in early development.  [Not invited]

  尾崎雄一, 古賀英裕, 犬飼信子, 宮下和夫, 萱場隆昭, 平松尚志, 原彰彦, 足立伸次, 山内こう平

  日本水産学会大会講演要旨集  1998/04
• Limited degradation enzyme for vitellogenin and vitellus protein in Salmonidae fish.  [Not invited]

  平松尚志, 市川典正, 木村志津雄, 原彰彦

  日本水産学会大会講演要旨集  1998/04
• サケ科魚類の胚発生に伴う卵黄蛋白および酵素の性状解析  [Not invited]

  市川典正, 平松尚志, 原彰彦

  平成9年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1997/12
• Limited proteolysis of vitellogenin and its derived yolk proteins in salmonids  [Not invited]

  Hiramatsu N, Ichikawa N, Hosokawa K, Hara A

  XIII International Congress of Comparative Endocrinology  1997/11
• Cathepsin-D like proteinase involved in yolk formation in salmonid ovary  [Not invited]

  Hiramatsu N, Hara A

  American Society of Ichthyologists and Herpetologists, 77th Annual meeting  1997/06
• .BETA. component in serum and egg yolk in Salmonidae fishes.  [Not invited]

  北村真紀子, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  1997/04
• Change of egg yolk protein accompanied with embryonic development of Salmonidae fishes.  [Not invited]

  市川典正, 北村真紀子, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  1997/04
• Purification of Vg degrading enzyme of Salmonidae fishes.  [Not invited]

  平松尚志, 木村志津雄, 原彰彦

  日本水産学会大会講演要旨集  1997/04
• サクラマス卵の胚発生に伴う卵黄蛋白の変化  [Not invited]

  市川典正, 北村真紀子, 平松尚志, 木村志津男, 原彰彦

  平成8年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1996/12
• サケ科魚類ビテロゲニンの卵黄蛋白質成分への特異的分解  [Not invited]

  平松尚志, 原彰彦

  平成8年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1996/12
• Changes in egg yolk protein accompanied by the embryonic development of Oncorhyncus masou eggs.  [Not invited]

  市川典正, 北村真紀子, 平松尚志, 木村志津雄, 原彰彦

  日本水産学会北海道支部大会講演要旨集  1996/12
• Peculiarities of serum vitellogenin profiles in Sakhalin taimen  [Not invited]

  Hiramatsu N, Kitamura M, Hara A

  3rd International Symposium on Fish Endocrinology  1996/05
• A sensitive non-competitive avidin-biotin ELISA for chum salmon growth hormone  [Not invited]

  Fukada H, Hiramatsu N, Hara A

  3rd International Symposium on Fish Endocrinology  1996/05
• .BETA.' - component-like protein appearing in serum of Salmonidae fishes.  [Not invited]

  北村真紀子, 平松尚志, 原彰彦

  日本水産学会大会講演要旨集  1996/03
• Search of enzyme-like protein related to molecular disintegration of vitellogenin of Salmonidae fishes.  [Not invited]

  平松尚志, 北村真紀子, 木村志津雄, 原彰彦

  日本水産学会大会講演要旨集  1996/03
• Change of serum GH quantity with maturation process of cherry salmon.  [Not invited]

  深田陽久, 平松尚志, 島崎健二, 原彰彦

  日本水産学会大会講演要旨集  1996/03
• イトウ卵母細胞中の卵膜蛋白成分の検索  [Not invited]

  藤田敏明, 清水宗敬, 平松尚志, 原彰彦

  平成7年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1995/10
• イトウにおけるIgMの精製・定量および寄生性コペポーダsalmincola stellatusに対するspecific IgMの産生  [Not invited]

  平松尚志, 清水宗敬, 深田陽久, 北村真紀子, 木村志津男, 原彰彦

  平成7年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1995/10
• エストラジオール17βによるイトウの卵膜関連蛋白及びビテロジェニンの誘導  [Not invited]

  清水宗敬, 平松尚志, 原彰彦

  平成7年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1995/10
• サクラマスの成熟過程に伴う成長ホルモン及びビテロジェニンの変化  [Not invited]

  深田陽久, 清水宗敬, 平松尚志, 北村真紀子, 原彰彦

  平成7年度道立水産孵化場と北大水産学部との合同セミナー 魚の研究  1995/10
• Three egg yolk proteins are derived from salmonid vitellogenin.  [Not invited]

  HIRAMATSU Naoshi, HARA Akihiko

  The 5th International Symposium on the Reproductive Physiology of Fish  1995/07
• イトウ血清中の卵膜関連蛋白とその誘導  [Not invited]

  清水宗敬, 平松尚志, 島崎健二, 原彰彦

  日本水産学会大会講演要旨集  1995/04
• サケ成長ホルモンのサンドウイッチ法による酵素免疫測定法  [Not invited]

  深田陽久, 平松尚志, 玄浩一郎, 原彰彦

  日本水産学会大会講演要旨集  1995/04
• イトウのビテロゲニンは3つの卵黄蛋白へ分子解裂する  [Not invited]

  平松尚志, 稲場琴美, 木村志津雄, 原彰彦

  日本水産学会大会講演要旨集  1995/04
• 回遊魚の増養殖  [Not invited]

  原彰彦, 木村志津男, 平松尚志, 深田陽久, 清水宗敬

  第1回水産学部プロジェクト研究シンポジウム「回遊のメカニズム」  1994/04

Teaching Experience

Aquaculture production of salmonAquaculture production of salmon Hokkaido University

Association Memberships

• 水産増殖学会   日本水産学会   

Research Projects

• 養殖サーモンのブランド化と環境負荷低減に貢献する餌料開発および生産物の 健康機能性成分の定量分析

  地方大学・地域産業創生交付金事業キングサーモン完全養殖技術研究業務委託

  Date (from‐to) : 2022/04 -2027/03 

  Author : 平松尚志, 細川雅史, 東藤孝, 高橋勇樹, 藤田雅紀, 深田陽久, 大石岳人
• 魚卵アレルゲン低減化魚類の提案と最適な飼育環境の確立

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2023/04 -2026/03 

  Author : 清水 裕, 笹岡 友季穂, 平松 尚志, 佐伯 宏樹, 清水 宗敬
• フィールドサイエンスを基盤とした地球環境を再生する新たな持続的食料生産システムの構築と展開

  地域中核・特色ある研究大学強化促進事業（J-PEAKS）

  Date (from‐to) : 2024/04
• Analysis of vitellogenesis in fish using genome genome editing technology

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  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。
• 大型トラウトサーモン（ニジマス）の量産化に向けた 高効率配合飼料技術体系の研究開発

  水産庁マリノフォーラム21：養殖業成長産業化提案公募型実証事業

  Date (from‐to) : 2023 -2024
• 育種・染色体操作を用いたサケ科魚類の魚卵アレルゲン性低減化の試み

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  Date (from‐to) : 2020/04 -2023/03 

  Author : 清水 裕, 笹岡 友季穂, 藤本 貴史, 平松 尚志, 石田 晃彦, 渡慶次 学, 佐伯 宏樹

   

  進捗状況を3項目に分けて述べる。 1.不妊魚の作出：ニジマスの卵と凍結保存したブラウントラウトあるいはサクラマスの精子を受精し、第二極体放出阻止処理により作出された雑種三倍体候補（A：ニジマス×ブラウントラウト、B：ニジマス×サクラマス）の倍数性を調査した。その結果、候補Aでは全25個体で、候補Bでは1個体を除く30個体が三倍体であった。これらの個体はPITタグで標識し、現在も継続して飼育している。（藤本） 2.魚卵アレルゲン検知系の構築：引き続き、ニジマス卵アレルゲンであるβ’-component（BC）のペーパー免疫分析デバイスの構築に取り組んだ。デバイスは濾紙にインクを印刷して加熱する常法により作製し、インクで囲まれた領域を分析反応ゾーンとした。分析は、反応ゾーンに新規作製した抗BC抗体を固定化して試料、酵素標識抗BC抗体、発色試薬を順に加えて行う手順とした。本手法では、従来並みの感度（検出感度：約1 ng/mL）の分析が従来の1/100の時間（約20分）で可能となった。（渡慶次、石田） 加えて、交雑種に対応した検知系に使用する抗体の作成のため、サクラマス排卵からBCを精製し、これを家兎に免疫し抗血清を作製した。（平松） 3.不妊化魚の魚卵アレルゲン性の調査：全23個体の不妊化三倍体ニジマスの内臓組織に含まれるBCを測定したが、全個体の筋肉には魚卵アレルギー発症リスクが認められなかった。しかし、5個体において生殖線から少量のBCが検出されたが、その内3個体は目視にて明確な生殖腺の発達が観られた。目視で生殖線の発達が確認できなかった20個体について、生殖腺の組織切片を作製・観察し、その発達状況を確認した。その結果、３個体で発達中の卵母細胞が散在しているのが確認され、生殖腺の外観だけでアレルギー発症リスクの有無を見分けるのは困難であることが判明した。（清水、佐伯、笹岡、平松）
• 養殖サケマス身肉への色素蓄積制御に関する生物情報科学的研究

  南北海道学術振興財団：令和4年度学術研究支援事業

  Date (from‐to) : 2022/04 -2023/03 

  Author : 平松尚志
• 雄尿関連蛋白質を利用した北方性メバル類の養殖・育種効率化

  寿原記念財団：令和3年度研究助成

  Date (from‐to) : 2021/04 -2022/03 

  Author : 平松尚志
• 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
• Basic studies toward smart cell industry of salmon species

  Hokusui Foundation：令和2年度助成事業

  Date (from‐to) : 2020/04 -2021/03 

  Author : 平松 尚志
• 幻の魚イトウの海中養殖法確立のための有用遺伝子マーカーの探索

  南北海道学術振興財団：助成金

  Date (from‐to) : 2019/04 -2020/03 

  Author : 東藤 孝, 者, 平松
• 生物情報科学によるメバル属魚類の増養殖関連有用マーカーの探索

  南北海道学術振興財団：助成金

  Date (from‐to) : 2018/04 

  Author : 平松 尚志
• フサカサゴ科メバル属魚類の性成熟度判定および繁殖制御技術の開発

  北水協会：研究助成事業

  Date (from‐to) : 2017/04 

  Author : 川崎琢真
• Studies on mechanisms underlying formation of lipid droplets in oocytes of teleost fishes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2014/04 -2017/03 

  Author : Todo Takashi, HARA Akihiko, HIRAMATSU Naoshi

   

  We have previously shown about the mechanisms underlying formation of lipid droplets in teleost oocytes as follows; plasma very low density lipoprotein is the major carrier of neutral lipids to oocyte lipid droplets, and Vldl is metabolized at the outside of oocyte, then free fatty acids derived from the Vldl metabolism are incorporated into oocyte. In this study, we focused on the role of lipoprotein lipase (Lpl), a key enzyme of Vldl metabolism, and analyzed its expression pattern in ovarian follicle using salmonids, medaka, pipefish and zebrafish. As the results, it was suggested that the presence of Lpl expressed in ovarian follicle cells is indispensable for the formation of lipid droplets in oocytes.
• カレイ類受精卵及びシシャモ仔稚魚に対する種特異抗体の作製

  北水協会：研究助成事業

  Date (from‐to) : 2015/04 -2017/03 

  Author : 平松 尚志
• 魚類の卵母細胞を標的とする分子輸送体を産生するトランスジェニックメダカの作製

  文部科学省：科学研究費補助金 挑戦的萌芽研究

  Date (from‐to) : 2015/04 -2017/03 

  Author : 平松 尚志
• Hepatoprotective effects of novel antioxidant isolated from the Pacific oyster

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2016/03 

  Author : Fuda Hirotoshi, HIRAMATSU Naoshi, JIN Shigeki

   

  This study was undertaken to characterize the hepatoprotective potency of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a novel phenolic antioxidant, isolated from the Pacific oyster, using hepatoma-derived cells (C3A) cultured in the presence of pro-oxidant, as well as well-known antioxidants. We found that only DHMBA significantly protected C3A cells against cell death in a dose-dependent manner, whereas other antioxidants did not. Also our study assessed the effects of DHMBA-rich oyster extracts (DOE) in a novel non-alcoholic steatohepatitis (NASH) model mouse established by combination of a high-fat diet and oxidized low-density lipoprotein. The NASH model mouse exhibited obesity, insulin resistance, hepatic steatosis, inflammation, fibrosis, and apoptosis. These changes were significantly moderated by supplementation of DOE. From these results, DHMBA might serve as a functional food for people at elevated risk for ROS-related diseases, such as NASH.
• チョウザメの生殖統 御技術開発のための 性分化、卵成長およ び卵成熟の分子機構 解析

  文部科学省：科学研究費補助金 基盤研究Ａ

  Date (from‐to) : 2012/04 -2015/03 

  Author : 足立 伸次
• マガキで発見された 新規の抗酸化物質に よる非アルコール性 脂肪肝炎の予防効果

  文部科学省：科学研究費補助金 基盤研究Ｃ

  Date (from‐to) : 2012/04 -2014/03 

  Author : 布田博敏
• エピトープ情報を活 用した魚卵アレルゲ ン検知系の開発

  文部科学省：科学研究費補助金 基盤研究Ｃ

  Date (from‐to) : 2012/04 -2014/03 

  Author : 佐伯 宏樹
• Basic studies on development of a novel delivery system of materials into fish oocytes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2011/04 -2013/03 

  Author : HIRAMATSU Naoshi, GEN Koichiro

   

  A yolk precursor vitellogenin (Vg) is selectively incorporated from the maternal blood into oocyte following its binding to ovarian Vg receptor. This study demonstrated that Vg obtained from heterologous species, as well as Vg coupled with voluntary lipids, can be delivered to fish eggs and offsprings by injecting them into broodstocks. This indicated a potential use of fish Vg as a transporter of bioactive materials; the system can be universal to any fish species. This study also confirmed that such system was found to be applicable for the delivery of bioactive lipids and/or hydrophobic materials to eggs and offsprings across generations.
• 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) : 2010/04 -2013/03 

  Author : 原 彰彦
• 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.
• 卵巣特異リポ蛋白質受容体を標的とした物質輸送:光る魚の大量生産

  産学が連携した研究開発成果の展開 研究成果展開事業 研究成果最適展開支援プログラム(A-STEP) 探索タイプ

  Date (from‐to) : 2011 -2011 

  Author : 平松 尚志

   

  本研究は蛍光性物質を親魚から卵・稚仔魚へ輸送する技術の開発を目指し、1)卵母細胞に特異的に発現する2種類の受容体を標的とする抗体の作製、および2)蛍光標識抗体の生体投与試験を行った。実施においては計画した全ての実験を完了した。結果においては、これら抗体によるゼブラフィッシュの卵母細胞や受精卵・孵化仔魚への蛍光物質の輸送は殆ど検出できなかった。一方、本研究と並行した実験により、受容体に対する抗体ではなくリガンドを輸送体に使用した場合、卵母細胞および受精卵・孵化仔魚への蛍光物質の輸送が確認された。以上の結果より、輸送体にはリガンドを使用するべきであり、またその場合、蛍光物質の輸送が技術的に可能であることが示された。
• Studies on vitellogenin and its receptor in salmonid using interactome techniques

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2010 

  Author : HIRAMATSU Naoshi

   

  When fish grows ovaries, a yolk precursor vitellogenin (Vg) is incorporated from the blood into oocyte following its binding to ovarian Vg receptor (VgR). This study first identified a portion of VgR-binding region on salmon Vg (sVg) using cutting-edged interactome techniques. Further, a novel C-type Vg (VgC) was cloned and its recombinant protein was synthesized with a limited amount; this provided a basis for discovering the unrevealed VgC receptor.
• Studies on mechanism of lipid uptake into teleost oocytes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2010 

  Author : TODO Takashi, HARA Akihiko, HIRAMATSU Naoshi

   

  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.
• Hybrid striped bass farming: Applications of genomics and functional proteomics to solve problems in growth and reproduction

  国際研究協力

  Date (from‐to) : 2006 -2010
• Studies of fish vitellogenesis based on the multiple vitellogenin model

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2007 -2009 

  Author : Akihiko HARA, 松原孝博, 征矢野清, 東藤孝, 平松尚志, Takahiro MATSUBARA, Kiyoshi SOYANO, Takashi TODO, Naoshi HIRAMATSU

   

  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.
• Studies on the specific binding of vitellogenin and very-low-density lipoprotein to ovarian lipoprotein receptor(s) in teleosts

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(若手研究(B))

  Date (from‐to) : 2007 -2008 

  Author : Naoshi HIRAMATSU

   

  本研究は、魚卵中の卵黄についての研究であり、特に卵黄の素となる血液中の前駆体(ビテロジェニンと超低密度リポ蛋白質)が、どの様に卵へ取り込まれるかを調べた。スズキ類とボラ類の魚を使いこれらの蛋白質や遺伝子を解析した結果、まずビテロジェニンには3種類のサブタイプがあることが明らかとなった。また各種ビテロジェニンと超低密度リポ蛋白質は、それぞれ異なる受容体と結合してから卵の中に取り込まれる可能性が初めて示された。
• サケ科魚類の多型ビテロジェニンおよびコリオジェニンに対する免疫測定系の確立

  第8期日本化学工業協会：長期自主研究

  Date (from‐to) : 2007 -2008
• 魚類の新たな生体指標蛋白を利用した環境エストロジェン評価系の構築

  科学技術振興調整費

  Date (from‐to) : 2007 -2007 

  Author : 平松 尚志
• 魚類ビテロゲニンの卵母細胞への蓄積ならびに稚仔魚への吸収機構の解明

  日本学術振興会：特別研究員研究費（PD）

  Date (from‐to) : 1998/04 

  Author : 平松 尚志
• サケ科魚類におけるビテロゲニンの卵黄蛋白への分子解裂機構の解明

  日本学術振興会：DC2

  Date (from‐to) : 1996 -1997 

  Author : 平松 尚志
• Deposition and utilization of vitellogenin and yolk proteins in teleost fish.

Social Contribution

• 函館朝市ミニ水族館

  Date (from-to) : 2019/07/15-2019/12/30

  Role : Advisor

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : はこだて海の教室～海と日本PROJECT~
• 魚の卵を科学しよう！

  Date (from-to) : 2019/08/05

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道大学

  Event, Program, Title : 令和元年度オープンキャンパス【高校生限定プログラム（実験）】
• ２つの海に面する八雲：日本海と太平洋の海の生物・環境を比べてみよう –海と日本PROJECTー

  Date (from-to) : 2019/07/15

  Role : Lecturer

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : 海の宝アカデミックコンテスト2019・ブルーオーシャン活動に向けて（海と日本2019）
• 魚の卵を科学する

  Date (from-to) : 2019/06/27

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道高等学校理科研究会

  Event, Program, Title : 北海道高等学校理科研究会支部総会一般講演
• 海の宝アカデミックコンテスト2018

  Date (from-to) : 2018/11/10

  Role : Planner

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H30年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• 北の海の七福神（キラリス）ミニ水族館

  Date (from-to) : 2018/07-2018/09

  Role : Advisor

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H30年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• 北の海の七福神（函館空港）ミニ水族館

  Date (from-to) : 2018/08/01-2018/08/31

  Role : Advisor

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H30年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• 海って結構面白い 海の写真展とワークショップ

  Date (from-to) : 2018/08/06-2018/08/12

  Role : Organizing member

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H30年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• マリン・ラーニング 魚拓づくり

  Date (from-to) : 2018/07/21

  Role : Presenter

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H30年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• 海の宝アカデミックコンテスト2017

  Date (from-to) : 2017/11

  Role : Planner

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H29年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• 魚の卵を科学しよう！

  Date (from-to) : 2017/08/07

  Role : Appearance

  Sponser, Organizer, Publisher  : 北海道大学

  Event, Program, Title : 平成29年度オープンキャンパス【高校生限定プログラム（実験）】
• 実験に参加！！函館海洋センターバックヤード研修

  Date (from-to) : 2017/07/29-2017/07/30

  Role : Presenter

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H29年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• マリン・ラーニング 魚拓づくり

  Date (from-to) : 2017/07/16

  Role : Presenter

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H29年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• 海の宝アカデミックコンテスト2016

  Date (from-to) : 2016/11

  Role : Planner

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H28年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング：函館マリンフェスティバル
• パドルボードで海の宝ものを探そう

  Date (from-to) : 2016/08/06-2016/08/07

  Role : Planner

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H28年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング
• 魚の卵を科学する

  Date (from-to) : 2016/07/30

  Role : Lecturer

  Sponser, Organizer, Publisher  : 北海道大学

  Event, Program, Title : 平成28年度（第30回）北海道大学水産学部公開講座「弁天町発！！水産・海洋研究の最前線」
• 北の魚の赤ちゃんと海藻の世界

  Date (from-to) : 2016/07/23-2016/07/24

  Role : Appearance

  Sponser, Organizer, Publisher  : 日本財団

  Event, Program, Title : H28年度 日本財団「海と日本プロジェクト」：海の宝を巡る学びと体験マリンラーニング：函館マリンフェスティバル
• 魚の卵を科学しよう！

  Date (from-to) : 2015/08/03

  Role : Appearance

  Sponser, Organizer, Publisher  : 北海道大学

  Event, Program, Title : 平成27年度オープンキャンパス【高校生限定プログラム（実験）】
• 魚の卵を科学しよう！

  Date (from-to) : 2013

  Role : Appearance

  Sponser, Organizer, Publisher  : 北海道大学

  Event, Program, Title : 平成25年度オープンキャンパス【高校生限定プログラム（実験）】
• 魚の増える仕組み

  Date (from-to) : 2012/05/23

  Role : Presenter

  Sponser, Organizer, Publisher  : 北海道大学

  Event, Program, Title : 新水産・海洋都市はこだてを支える人材育成、H24年度海のものづくりプログラム
• 科学論文雑誌審査員

  Role : Others

  Event, Program, Title : Biochimica Biophysica Acta, Biology of Reproduction, Comparative Biochemistry and Physiology, Fish Physiology and Biochemistry, General Comparative Endocrinology, Journal of Experimental Biology, Journal of Histochemistry and C・・・

Media Coverage

• メバル類の成熟度をタンパク質で判別 北大水産院の山口さんら 人工授精効率化に期待

  Date : 2024/02/28

  Publisher, broadcasting station: 北海道新聞

  Program, newspaper magazine: 北海道新聞

  Internet
• 胎生メバル類の雄成熟度マーカーの開発に初めて成功～メバル類増養殖における種苗生産の効率化への貢献に期待～

  Date : 2024/02/14

  Writer: Myself

  Publisher, broadcasting station: 北海道大学

  Program, newspaper magazine: プレスリリース

  Others
• 釣りびと万歳トークショー～釣りびとたちと考える道南の海～

  Date : 2022/10/09

  Publisher, broadcasting station: NHK

  Program, newspaper magazine: NHK函館放送局 開局90周年 見ささるFes.

  Media report
• 「メバル」

  Date : 2022/09/30

  Publisher, broadcasting station: NHK Eテレ

  Program, newspaper magazine: ギョギョッとサカナ★スター

Academic Contribution

• パネルディスカッション

  Date (from-to) :2017/09/17

  Role: Panel chair etc

  Organizer, responsible person: 北海道大学

Others

• 2023 ソイ・メバル (Rockfish)

  水産学部HP上にFish of the Monthコンテンツとして記事掲載
• 2014 -2014 日本水産学会春季大会

  会場担当
• 2010 -2010 日本水産学会北海道支部大会

  評議委員会・総会担当
• 2009 -2009 日本水産学会北海道支部大会

  本部・会場担当
• 2007 -2007 日本水産学会秋季大会

  会場担当
• 2004 -2004 ハワイ大学サマープログラム

  米国ハワイ大学における夏期プログラム（Edwin W. Pauley Summer Program in Marine Biology)に出席し、講義および研究指導（Real-time PCRによるティラピアビテロジェニン遺伝子発現定量法の確立）を行った。