研究者データベース

山羽 悦郎(ヤマハ エツロウ)
北方生物圏フィールド科学センター 水圏ステーション 七飯淡水実験所
教授

基本情報

所属

  • 北方生物圏フィールド科学センター 水圏ステーション 七飯淡水実験所

職名

  • 教授

学位

  • 博士(水産学)(北海道大学)

J-Global ID

研究キーワード

  • 始原生殖細胞   PGC   借腹養殖   雄性発生   雌性発生   受精卵   発生工学   surrogate aquaculture   androgenesis   gynogenesis   genetic breeding   

研究分野

  • ライフサイエンス / 水圏生産科学

職歴

  • 2004年 - 北海道大学北方生物圏フィールド科学センター 教授
  • 1998年 - 2001年 北海道大学水産学部 助教授
  • 1998年 - 2001年 Hokkaido Univ. Faculty of Fisheries, Assistant Professor
  • 2001年 - 北海道大学北方生物圏フィールド科学センター 助教授
  • 2001年 - Hokkaido Univ. Field Science Center for Northern Biosphere, Assistant Professor
  • 1995年 - 1998年 北海道大学水産学部附属七飯養魚実習施設 助手
  • 1995年 - 1998年 Hokkaido Univ. Faculty of Fisheries Nanae Fish-Culture Exp. Stu. Associate Professor
  • 1985年 - 1995年 北海道大学水産学部 助手
  • 1985年 - 1995年 Hokkaido Univ. Faculty of Fisheries, Associate Professor

学歴

  •         - 1985年   北海道大学   水産学研究科   水産増殖学
  •         - 1985年   北海道大学
  •         - 1980年   北海道大学   水産学部   水産増殖学科
  •         - 1980年   北海道大学

所属学協会

  • 水産育種研究会   日本発生生物学会   日本動物学会   日本水産学会   

研究活動情報

論文

  • Takafumi Fujimoto, Takahisa Kaneyasu, Mitsuru Endoh, Yuya Kogame, Joanna Nynca, Andrzej Ciereszko, Eisuke Takahashi, Etsuro Yamaha, Kiyoshi Naruse, Katsutoshi Arai
    Aquaculture 557 738305 - 738305 2022年08月
  • 島 文華, 浅沼 黎, 兼安 敬久, 市村 政樹, 高橋 英佑, 山羽 悦郎, 藤本 貴史, 荒井 克俊
    NIPPON SUISAN GAKKAISHI 87 5 473 - 482 2021年09月15日 [査読有り]
  • Hirotaro Urushibata, Kazuaki Sasaki, Eisuke Takahashi, Toshikatsu Hanada, Takafumi Fujimoto, Katsutoshi Arai, Etsuro Yamaha
    Zebrafish 2021年09月03日 [査読有り]
  • Fumi Yamaguchi, Takafumi Fujimoto, Hiroko Suzuki, Hideki Tanaka, Masaru Murakami, Etsuro Yamaha, Katsutoshi Arai
    Theriogenology 172 95 - 105 2021年09月 [査読有り]
     
    Ginbuna (Carassius auratus langsdorfii (Teleostei: Cyprinidae)) occur in diploid, triploid, and tetraploid forms in wild populations. Diploid females reproduce bisexually, whereas polyploid (triploid and tetraploid) females reproduce gynogenetically with no contribution from sperm nuclei. However, tetraploid males produce diploid sperm. The mechanism responsible for the differences in egg and sperm ploidy has not been elucidated as tetraploid males are rare in wild populations. Here, we aimed to characterize the types of sperm and elucidate the mechanism of spermatogenesis in ginbuna. In the present study, we artificially produced tetraploid males by crossbreeding triploid ginbuna females with diploid goldfish (Carassius auratusauratus) males via accidental incorporation of sperm nuclei. We then examined spermatogenesis to reveal the process by which reduced diploid sperm are generated from tetraploid germ cells. DNA fingerprinting by random amplified polymorphic DNA (RAPD)-PCR indicated that the tetraploid progeny had a paternally derived genome. For the tetraploid male sperm, there were narrow (N-type) and broad (B-type) flow cytometrical histograms. The N-type were determined to be diploid with a low coefficient of variation (CV) by flow cytometry. The B-type were found to be aneuploid (hypodiploid to hexaploid) with a high CV. The head sizes of B-type sperm were variable, whereas those of the N-type sperm were uniform. Computer-assisted sperm analysis (CASA) revealed that both the haploid and diploid B-type sperm were weakly motile compared with the haploid sperm of goldfish and the diploid N-type sperm of tetraploid males. Bivalents and various multivalents were observed in the meiotic configurations of diploid spermatogenesis. In aneuploid spermatogenesis, most of the chromosomes were unpaired univalents and there were very few bivalents. Our findings provide empirical evidence for two different types of spermatogenesis in tetraploid C. a. langsdorfii males. Meiotic synapses might explain the observed differences in the ploidy status of the two sperm types.
  • Masamichi Kuroda, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai
    Conservation Genetics Resources 13 4 457 - 463 2021年08月24日 [査読有り]
  • Masamichi Kuroda, Kiko Shibata, Takafumi Fujimoto, Masaru Murakami, Etsuro Yamaha, Katsutoshi Arai
    Cytogenet Genome Res 161 3-4 178 - 186 2021年05月 [査読有り]
     
    In dojo loach (<i>Misgurnus anguillicaudatus</i>), although most wild types are gonochoristic diploids that are genetically differentiated into 2 groups, A and B, clonal lineages appear in certain localities. Clonal loaches have been considered to have hybrid origins between the 2 groups by a series of genetic studies. In this study, using FISH with a newly developed probe (ManDra-A), we identified 26 (1 pair of metacentric and 12 pairs of telocentric chromosomes) of 50 diploid chromosomes in contemporary wild-type group A loach. In contrast, ManDra-A signals were not detected on metacentric chromosomes derived from the ancestral group A of clonal loach. The FISH results clearly showed the presence of certain differentiations in metacentric chromosomes between ancestral and contemporary group A loach. Two-color FISH with ManDra-A and group B-specific ManDra (renamed ManDra-B) probes reconfirmed the hybrid origin of clones by identifying chromosomes from both groups A and B in metaphases. Our results showed the hybrid origin of clonally reproducing fish and the possibility that chromosomal differentiation between ancestral and contemporary fish can affect gametogenesis. In meiotic spermatocytes of sex-reversed clones, ManDra-A, and not ManDra-B, signals were detected in 12 out of 50 bivalents. Thus, the results further support the previous conclusion that clonal gametogenesis was assured by pairing between sister chromosomes duplicated from each ancestral chromosome from group A or B. Our study deepens the knowledge about the association between clonality and hybridity in unisexual vertebrates.
  • Yuki Naya, Tomoka Matsunaga, Yu Shimizu, Eisuke Takahashi, Fumika Shima, Mitsuru Endoh, Takafumi Fujimoto, Katsutoshi Arai, Etsuro Yamaha
    Zygote 28 6 470 - 481 2020年12月 [査読有り]
     
    SummaryThe cause of hybrid sterility and inviability has not been analyzed in the fin-fish hybrid, although large numbers of hybridizations have been carried out. In this study, we produced allo-diploid hybrids by cross-fertilization between female goldfish (Carassius auratus) and male golden venus chub (Hemigrammocypris rasborella). Inviability of these hybrids was due to breakage of the enveloping layer during epiboly or due to malformation with serious cardiac oedema around the hatching stage. Spontaneous allo-triploid hybrids with two sets of the goldfish genome and one set of the golden venus chub genome developed normally and survived beyond the feeding stage. This improved survival was confirmed by generating heat-shock-induced allo-triploid hybrids that possessed an extra goldfish genome. When inviable allo-diploid hybrid cells were transplanted into goldfish host embryos at the blastula stage, these embryos hatched normally, incorporating the allo-diploid cells. These allo-diploid hybrid cells persisted, and were genetically detected in a 6-month-old fish. In contrast, primordial germ cells taken from allo-diploid hybrids and transplanted into goldfish hosts at the blastula stage had disappeared by 10 days post-fertilization, even under chimeric conditions. In allo-triploid hybrid embryos, germ cells proliferated in the gonad, but had disappeared by 10 weeks post-fertilization. These results showed that while hybrid germ cells are inviable even in chimeric conditions, hybrid somatic cells remain viable.
  • Mitsuru Endoh, Fumika Shima, Miloš Havelka, Rei Asanuma, Etsuro Yamaha, Takafumi Fujimoto, Katsutoshi Arai
    PLOS ONE 15 5 e0233885 - e0233885 2020年05月29日 [査読有り]
  • Urushibata, H, Takahashi, E, Shimizu, Y, Miyazaki, T, Fujimoto, T, Arai, K, Yamaha, E
    Intenational Journal of Developmental Biolog 63 597 - 604 2019年12月 [査読有り][通常論文]
  • Goto R, Saito T, Matsubara T, Yamaha E
    Methods in molecular biology (Clifton, N.J.) 1874 475 - 487 2019年 [査読無し][通常論文]
  • Saito T, Goto R, Rivers N, Yamaha E
    Methods in molecular biology (Clifton, N.J.) 1920 327 - 341 2019年 [査読無し][通常論文]
  • Kuroda M, Fujimoto T, Murakami M, Yamaha E, Arai K
    Cytogenetic and genome research 158 1 46 - 54 2019年 [査読有り][通常論文]
     
    Gonochoristic wild-type dojo loaches (Misgurnus anguillicaudatus) are diploid (2n = 50) and reproduce bisexually. However, sympatric clonal diploids generate unreduced diploid isogenic eggs that develop gynogenetically. Clone-origin triploidy arises following the incorporation of a haploid wild-type sperm nucleus into the diploid egg. Triploid females produce fertile haploid eggs by meiotic hybridogenesis, while triploid males are sterile. Clonal loaches arose from past hybridization event(s) between genetically diverse groups, A and B. Artificial hybrid females between the 2 groups produce unreduced and/or aneuploid eggs, but the hybrid males are sterile. In this study using FISH, we analyzed chromosome pairing in meiotic cells of clone-origin triploid and inter-group hybrid males to clarify the cytogenetic mechanisms underlying the male-specific sterility. We used a repetitive sequence probe to identify group B-derived chromosomes and a 5.8S + 28S rDNA probe to identify pairs of homologous chromosomes. We found that asynapsis and irregular synapsis occur in triploid and hybrid males containing 2 different genomes and that this may cause the formation of sterile germ cells. These results will help us to understand hybrid sterility from the viewpoint of synapsis behavior.
  • Yasui GS, Saito T, Zhao Y, Fujimoto T, Yamaha E, Arai K
    Zygote (Cambridge, England) 26 5 408 - 416 2018年10月 [査読有り][通常論文]
  • Masamichi Kuroda, Takafumi Fujimoto, Masaru Murakami, Etsuro Yamaha, Katsutoshi Arai
    Chromosome Research 26 4 1 - 11 2018年06月07日 [査読有り][通常論文]
     
    Wild-type dojo loach (Misgurnus anguillicaudatus) commonly reproduces bisexually as a gonochoristic diploid (2n = 50), but gynogenetically reproducing clonal diploid lines (2n = 50) exist in certain districts in Japan. Clones have been considered to develop from past hybridization event(s) between two genetically diverse groups, A and B, within the species. Fluorescence in situ hybridization analyses using the repetitive sequence “ManDra” as a probe clearly distinguished 25 chromosomes derived from group B out of a total of 50 diploid chromosomes of the clone, providing strong molecular cytogenetic evidence of its hybrid origin. In meiosis, diploid wild-type showed 25 bivalents, while diploid clones revealed 50 bivalents, indicating the presence of 100 chromosomes. In meiotic chromosome spreads in sex-reversed clonal males, ManDra signals were detected in 25 out of 50 bivalents, and only one out of two bivalents possessing major ribosomal RNA coding regions exhibited two positive ManDra signals. In clonal females, ManDra signals were detected in approximately 25 out of 50 bivalents. Thus, unreduced gametes should be generated by the pairing between sister chromosomes doubled from each ancestral chromosome from the different groups by premeiotic endomitosis. Sister chromosome pairing should assure production of unreduced isogenic clonal gametes due to the absence of the influence of recombination or crossing over.
  • Hino H, Nakanishi A, Seki R, Aoki T, Yamaha E, Kawahara A, Shimizu T, Hibi M
    Developmental biology 434 1 96 - 107 2018年02月 [査読有り][通常論文]
     
    In early zebrafish development, the program for dorsal axis formation begins soon after fertilization. Previous studies suggested that dorsal determinants (DDs) localize to the vegetal pole, and are transported to the dorsal blastomeres in a microtubule-dependent manner. The DDs activate the canonical Wnt pathway and induce dorsal-specific genes that are required for dorsal axis formation. Among wnt-family genes, only the wnt8a mRNA is reported to localize to the vegetal pole in oocytes and to induce the dorsal axis, suggesting that Wnt8a is a candidate DD. Here, to reveal the roles of maternal wnt8a, we generated wnt8a mutants by transcription activator-like effector nucleases (TALENs), and established zygotic, maternal, and maternal zygotic wnt8a mutants by germ-line replacement. Zebrafish wnt8a has two open reading frames (ORF1 and ORF2) that are tandemly located in the genome. Although the zygotic ORF1 or ORF2 wnt8a mutants showed little or no axis-formation defects, the ORF1/2 compound mutants showed antero-dorsalized phenotypes, indicating that ORF1 and ORF2 have redundant roles in ventrolateral and posterior tissue formation. Unexpectedly, the maternal wnt8a ORF1/2 mutants showed no axis-formation defects. The maternal-zygotic wnt8a ORF1/2 mutants showed more severe antero-dorsalized phenotypes than the zygotic mutants. These results indicated that maternal wnt8a is dispensable for the initial dorsal determination, but cooperates with zygotic wnt8a for ventrolateral and posterior tissue formation. Finally, we re-examined the maternal wnt genes and found that Wnt6a is an alternative candidate DD.
  • Mitsuru Endoh, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai
    ZEBRAFISH 15 1 33 - 44 2018年02月 [査読有り][通常論文]
     
    Androgenesis is useful for induction of doubled haploids from male genetic resources and contributes to the restoration of individuals from cryopreserved sperm. Here, we determined the suitable conditions for egg in vitro preservation and the suitable dose of UV irradiation for genetic inactivation of the egg nucleus, and established an improved procedure for induction of androgenetic-doubled haploids in zebrafish. The suitable solution for egg preservation was evaluated by the fertilization rate using different types of solutions or conditions. Hank's solution with 0.5% bovine serum albumin (pH8.0) was suitable for the preservation of zebrafish eggs. In addition, we discovered an improvement of fertilization rates by temporal preservation of ovulated eggs in the suitable solution. UV irradiation of eggs at 50-75mJ/cm(2) induced haploid embryos. Microsatellite genotyping using eight loci revealed the paternity and homozygosity of the putative androgenetic doubled haploids. The yield rate of androgenetic doubled haploids, which were induced by UV irradiation and heat shock, ranged from 0.4% to 10.7%.
  • Milos Havelka, He Zhou, Seishi Hagihara, Masaki Ichimura, Takafumi Fujimoto, Etsuro Yamaha, Shinji Adachi, Katsutoshi Arai
    Fisheries Science 83 4 587 - 595 [Springer Japan] 2017年07月 [査読有り][通常論文]
  • Yanagimachi R, Harumi T, Matsubara H, Yan W, Yuan S, Hirohashi N, Iida T, Yamaha E, Arai K, Matsubara T, Andoh T, Vines C, Cherr GN
    Biology of reproduction 96 4 780 - 799 2017年04月 [査読有り][通常論文]
     
    Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical. Summary Sentence In fish, sperm entry into the egg through the micropyle is guided either chemically or physically depending on the species
  • Eisuke Takahashi, Yu Shimizu, Hirotaro Urushibata, Yutaka Kawakami, Katsutoshi Arai, Etsuro Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 61 6-7 397 - 405 2017年 [査読有り][通常論文]
     
    In teleost fish, the gonad originates from primordial germ cells (PGCs) and somatic cells. However, it is not clear whether the final gonadal position is determined by anteroposterior and dextrosinistral differentiation of endodermal organs or by the distribution of PGCs. The pond smelt has a transparent body even after hatching, enabling clear observation of PGC distribution and endodermal differentiation. Here, we first examined normal embryonic development to define the spatio-temporal characteristics of our developmental model. Second, the origin of PGCs was investigated by in situ hybridization. Third, the migration route of PGCs was tracked by microinjection of GFP-nos3 3' UTR mRNA and visualization of PGCs by green fluorescent protein. Lastly, differentiation of gonadal and endodermal organs was examined histologically. Maternal vasa transcripts were detected at the ends of cleavage furrows, indicating that PGCs differentiated by inheritance of germplasm as in other teleosts. During gastrulation, PGCs migrated following somatic cell movement and lined both sides of the embryonic body. During the segmentation period, PGCs moved posteriorly and were distributed in a line among dorsal mesentery cells around the posterior part of the intestinal bulb in the 16th to 24th somite region at 3 days post hatching. At 1 month post hatching, the gonad was formed at the 20th somite region. PGC distribution was biased to the left side of the body cavity, while the pancreas was formed on the right side. These results indicate that PGCs accumulate at the gonadal region by dorsal mesentery cells, and gonadal position is determined by the digestive system.
  • Eisuke Takahashi, Yutaka Kawakami, Katsutoshi Arai, Etsuro Yamaha
    Fisheries Science 82 4 639 - 652 [Springer Japan] 2016年07月 [査読有り][通常論文]
  • Jilun Hou, Takafumi Fujimoto, Taiju Saito, Etsuro Yamaha, Katsutoshi Arai
    SCIENTIFIC REPORTS 5 13346  2015年08月 [査読有り][通常論文]
     
    Generation of clonal zebrafish will facilitate large-scale genetic screening and help us to overcome other biological and biotechnological challenges due to their isogenecity. However, protocols for the development of clonal lines have not been optimized. Here, we sought to develop a novel method for generation of clonal zebrafish by androgenesis induced by cold shock. Androgenetic zebrafish doubled haploids (DHs) were induced by cold shock of just-fertilized eggs, and the eggs were then heat shocked to double the chromosome set. The yield rate of putative DHs relative to the total number of eggs used was 1.10% +/- 0.19%. Microsatellite genotyping of the putative DHs using 30 loci that covered all 25 linkage groups detected no heterozygous loci, confirming the homozygosity of the DHs. Thus, a clonal line was established from sperm of a DH through a second cycle of cold-shock androgenesis and heat-shock chromosome doubling, followed by genetic verification of the isogenic rate confirming the presence of identical DNA fingerprints by using amplified fragment length polymorphism markers. In addition, our data provided important insights into the cytological mechanisms of cold-shock-induced androgenesis.
  • Keh-Weei Tzung, Rie Goto, Jolly M. Saju, Rajini Sreenivasan, Taiju Saito, Katsutoshi Arai, Etsuro Yamaha, Mohammad Sorowar Hossain, Meredith E. K. Calvert, Laszlo Orban
    STEM CELL REPORTS 5 1 156 - 156 2015年07月 [査読有り][通常論文]
  • Rie Goto, Taiju Saito, Yutaka Kawakami, Tomoe Kitauchi, Misae Takagi, Takashi Todo, Katsutoshi Arai, Etsuro Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 59 10-12 465 - 470 2015年 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) appear during early embryogenesis and differentiate into gametes through oogenesis or spermatogenesis. Teleost PGCs can be visualized by injecting RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3). Although this method has been widely applied to teleost PGCs, the visualization of PGCs in pelagic species that have eggs with a hard chorion is more problematic due to the technical difficulty of microinjection into their eggs. In this study, we developed a reliable method for microinjection of fertilized eggs in a pelagic species, the barfin flounder. Using a microneedle with a constriction "brake", we were able to introduce gfp-nos3 3'UTR mRNA into embryos and to determine the migration route of PGCs. We also isolated the barfin flounder nos3 (bf-nos3) gene to compare its 3'UTR sequence with that of zebrafish. The 3'UTR of the bf-nos3 sequence was longer than that of zf-nos3. However, PGCs were also visualized after injection of gfp-bf-nos3 3'UTR mRNA both in zebrafish and barfin flounder. These results suggest that the function of nos3 is conserved between these species regardless of the sequence differences. The method developed here for labeling PGCs with gfp-nos3 mRNA will provide a means to study PGC development in the embryos of a wide range of marine fish species.
  • Keh-Weei Tzung, Rie Goto, Jolly M. Saju, Rajini Sreenivasan, Taiju Saito, Katsutoshi Arai, Etsuro Yamaha, Mohammad Sorowar Hossain, Meredith E. K. Calvert, Laszlo Orban
    STEM CELL REPORTS 4 1 61 - 73 2015年01月 [査読有り][通常論文]
     
    As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.
  • Jilun Hou, Taiju Saito, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai
    Aquaculture 420-421 1 s57 - s63 Elsevier 2014年04月 [査読有り][通常論文]
     
    Androgenetic doubled haploids (DHs) were induced in the loach Misgurnus anguillicaudatus (Cobitidae) without irradiation of the eggs. The eggs of wild-type females were activated with the intact sperm of an orange-phenotype male, and treated (within 10 s of activation) at 3 ± 0.5 °C for 30 min, to eliminate the female nucleus. The eggs were then incubated in a water bath at 20 ± 0.5 °C for 35 min. Finally, diploidy was restored (65 min after activation) by heat-shock treatment at 42 ± 0.5 °C for 2 min. Under these conditions, the yield rate (mean ± SD) of putative DHs relative to the total number of eggs used was 10.43 ± 1.69%, which was significantly higher (P < 0.05) than the yield rates obtained under the remaining heat-shock initiation conditions (55 min, 60 min, and 70 min after activation). We analyzed the ploidy status of the putative DH by using flow cytometry. All-male inheritance was confirmed by the expression of the recessive orange body color trait and microsatellite genotypes. We detected no maternally derived alleles or heterozygous genotypes at any of the 28 loci (covering 27 linkage groups) of loach, indicating the exclusively paternal inheritance and homozygosity of the obtained androgenetic DHs.
  • Taiju Saito, Martin Psenicka, Rie Goto, Shinji Adachi, Kunio Inoue, Katsutoshi Arai, Etsuro Yamaha
    PLOS ONE 9 2 e86861  2014年02月 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) arise elsewhere in the embryo and migrate into developing gonadal ridges during embryonic development. In several model animals, formation and migration patterns of PGCs have been studied, and it is known that these patterns vary. Sturgeons (genus Acipenser) have great potential for comparative and evolutionary studies of development. Sturgeons belong to the super class Actinoptergii, and their developmental pattern is similar to that of amphibians, although their phylogenetic position is an out-group to teleost fishes. Here, we reveal an injection technique for sturgeon eggs allowing visualization of germplasm and PGCs. Using this technique, we demonstrate that the PGCs are generated at the vegetal pole of the egg and they migrate on the yolky cell mass toward the gonadal ridge. We also provide evidence showing that PGCs are specified by inheritance of maternally supplied germplasm. Furthermore, we demonstrate that the migratory mechanism is well-conserved between sturgeon and other remotely related teleosts, such as goldfish, by a single PGCs transplantation (SPT) assay. The mode of PGCs specification in sturgeon is similar to that of anurans, but the migration pattern resembles that of teleosts.
  • Shogo Higaki, Yutaka Kawakami, Yoshiki Eto, Etsuro Yamaha, Masashi Nagano, Seiji Katagiri, Tatsuyuki Takada, Yoshiyuki Takahashi
    CRYOBIOLOGY 67 3 374 - 382 2013年12月 [査読有り][通常論文]
     
    The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me2SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0-2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me2SO and VS containing 3 M EG + 2 M Me2SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me2SO and VS containing 3 M EG + 2 M Me2SO, and that recovered PGCs retained ability to differentiate into functional gametes. (C) 2013 Elsevier Inc. All rights reserved.
  • Jie Dong, Masaru Murakami, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai
    Fisheries Science 79 6 935 - 941 The Japanese Society of Fisheries Science 2013年11月 [査読有り][通常論文]
     
    Silver crucian carp Carassius auratus langsdorfii comprises a diploid-polyploid complex in wild Japanese populations. Bisexually reproducing diploids are sympatrically distributed with gynogenetically developing triploids and tetraploids. Triploid and tetraploid males are very rare among Japanese silver crucian carp due to their gynogenetic reproduction. We examined the genetic characteristics of progeny that arose in a tank by natural spawning of a tetraploid silver crucian carp pair. The ploidy status of 120 samples randomly collected from these progeny was determined to be tetraploid by DNA content flow cytometry. DNA fingerprints from a random amplified polymorphic DNA assay indicated that almost all the progeny examined had genotypes identical to the maternal tetraploid female with no paternally derived fragments. Selected specimens' cytogenetic analyses revealed that the progeny examined had tetraploid chromosome numbers, categorized into 40 metacentric, 80 submetacentric, and 80 subtelocentric or telocentric chromosomes, which were arranged into quartets and six supernumerary microchromosomes. Fluorescence in situ hybridization signals were detected in four homologous chromosomes in all analyzed metaphases prepared from diploid goldfish specimens. Contrary, tetraploid silver crucian carp gave eight rDNA signals. These results suggest that gynogenetic development in eggs spawned by tetraploid females should be triggered by tetraploid males' homospecific sperm.四倍体ギンブナ雌雄の自然産卵により得た子孫のDNA 量を調べたところ、全個体が四倍体であった。さらにRAPD-PCR 法により分析したところ、子孫と母親の間で電気泳動像は一例を除き同一で、かつ父親の遺伝的影響は認められなかった。子孫は染色体数4n=206、核型40M+80SM+80ST/T+6m(微小染色体)を示し、5.8S+28SrDNA をプローブとしたFISH では、二倍体キンギョの4 シグナルに対して8 シグナルを示した。以上の結果は、四倍体の精子によっても、四倍体の卵の雌性発生が生じ、子孫は四倍体クローンとなったことを示す。
  • Takafumi Fujimoto, Suzu Sakao, Kouzou Oshima, Etsuro Yamaha, Katsutoshi Arai
    Aquaculture International 21 4 769 - 781 Springer 2013年08月 [査読有り][通常論文]
     
    Tetraploid fish, which are considered as key resources of diploid gametes for further breeding and ploidy manipulation, can be artificially induced by inhibition of the mitotic cell division with hydrostatic pressure or temperature treatments. Although many attempts have been made to induce artificial tetraploid strains, successful establishment of viable and fertile tetraploid strains are rare. In pond loach, Misgurnus anguillicaudatus, natural tetraploid individuals are distributed in wild populations and diploid gametes from the tetraploid fish have been used for the induction of polyploid individuals, but artificially induced tetraploid strains have not been established yet. In the present study, we optimised starting timing of the heat-shock treatment (41 °C for 2 min) to inhibit a mitotic cell division in fertilised eggs of the normal diploid pond loach between 21 and 51 min after insemination at 20 °C. After the treatment, we observed external appearance of hatching larvae and flow cytometrically determined ploidy status of the resultant larvae. Although tetraploid and diploid/tetraploid mosaic larvae were obtained, the optimum timings for induction of tetraploidy varied amongst crosses. Various kinds of ploidy such as haploidy, diploidy, triploidy, pentaploidy, hexaploidy, aneuploidy and mosaic were detected in non-optimum heat-shock timings for tetraploidisation. Survivors, a tetraploid and a diploid/tetraploid mosaic male, matured at the age of 1-year-old, but they produced functional haploid spermatozoa.
  • Jilun Hou, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai
    THERIOGENOLOGY 80 2 125 - 130 2013年07月 [査読有り][通常論文]
     
    Diploid androgenotes were produced without egg irradiation in the loach, Misgurnus anguillicaudatus. Eggs of wild-type diploid females were fertilized with diploid sperm of a neo-tetraploid male and then cold-shock treated at 3 degrees C (range, +/- 0.5 degrees C) for 30 minutes just after fertilization to eliminate the female nucleus. After hatching, ploidy status of the hatched larvae was analyzed by flow cytometry, which revealed putative diploid androgenotes as well as larvae possessing other ploidies. Five independent microsatellite DNA markers were genotyped to confirm all-male inheritance of the resultant diploid larvae. The mean +/- SD yield rate of diploid androgenetic larvae to total eggs used was 12.29 +/- 3.25% in the cold-shock group and 22.23 +/- 13.42% in the UV-irradiated group (P > 0.05). No diploid androgenetic larvae were detected in the intact control group. To our knowledge, this is the first report demonstrating successful induction of diploid androgenotes without egg irradiation in fish. (C) 2013 Elsevier Inc. All rights reserved.
  • H. Zhou, T. Fujimoto, S. Adachi, S. Abe, E. Yamaha, K. Arai
    JOURNAL OF APPLIED ICHTHYOLOGY 29 1 51 - 55 2013年02月 [査読有り][通常論文]
     
    The ploidy status of Acipenser mikadoi was examined using nuclear DNA contents, karyotypes and fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. In flow-cytometrically sorted specimens with 8.29.1 pg DNA content per somatic cell, i.e. genetic diploid, the best informative metaphase with 268 chromosomes had 80 biarmed meta- or submetacentric (M or SM) chromosomes, 48 monoarmed telocentric (T) chromosomes and 140 microchromosomes. In genetic triploid specimens with 12.613.0 pg DNA content, the best informative metaphase with 402 chromosomes showed 120 biarmed M or SM, 72 monoarmed T chromosomes and 210 microchromosomes. The rDNA FISH detected a maximum 18 and 27 signals in the diploid and triploid A. miakdoi, respectively. The obtained findings thus corroborated a clear parallel between nuclear DNA contents and karyological or FISH profiles in the genetic diploid and triploid specimens, suggesting 1.5 times chromosome complements of diploid counterparts or three sets of homologues in the triploid sturgeons. Moreover, the estimated genome size and the observed molecular cytogenetic features in the diploid A. mikadoi strongly suggest that this species is a member of a functional tetraploid group recently proposed in the literature.
  • Y. Kawakami, M. Ishihara, T. Saito, T. Fujimoto, S. Adachi, K. Arai, E. Yamaha
    JOURNAL OF ANIMAL SCIENCE 90 12 4256 - 4265 2012年12月 [査読有り][通常論文]
     
    Primordial germ cells (PGC) are the only cell type in developing embryos with the potential to transmit genetic information to the next generation. In this study, PGC of Japanese eel (Anguilla japonica) were visualized by injection of mRNA synthesized from a construct carrying the green fluorescent protein (GFP) gene fused to the 3' untranslated region of the Japanese eel nanos gene. We investigated the feasibility of cryopreserving Japanese eel PGC by vitrification of dechorionated whole somite stage embryos. The GFP-labeled PGC were rapidly cooled using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (methanol, dimethyl sulfoxide, and glycerol for 10 min and ethylene glycol for 10, 20, and 30 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose for 1, 5, and 10 min. Ethylene glycerol is an effective cryoprotectant for embryonic cells and shows no evidence of ice formation after thawing. Vitrified and thawed PGC were transplanted into blastula stage embryos from zebrafish (Danio rerio). The GFP-labeled PGC migrated toward the host gonadal ridge, suggesting maintenance of their normal migration motility. These techniques may assist in achieving inter- and intraspecies germ-line chimers using donor Japanese eel PGC.
  • D Inoue, T Fujimoto, Kawakami Y, GS Yasui, E Yamaha, K Arai
    Journal of Applied Ichthyology 28 6 919 - 924 2012年12月 [査読有り][通常論文]
  • T Kitauchi, T Saito, T Motomura, K Arai, E Yamaha
    Journal of Applied Ichthyology 28 6 998 - 1005 2012年12月 [査読有り][通常論文]
  • Y Zhao, M Psenicka, T Fujimoto, T Saito, GS Yasui, E Yamaha, K Arai
    Journal of Applied Ichthyology 28 6 1006 - 1012 2012年12月 [査読有り][通常論文]
  • Rie Goto, Taiju Saito, Takahiro Takeda, Takafumi Fujimoto, Misae Takagi, Katsutoshi Arai, Etsuro Yamaha
    DEVELOPMENTAL BIOLOGY 370 1 98 - 109 2012年10月 [査読有り][通常論文]
     
    The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells. (C) 2012 Elsevier Inc. All rights reserved.
  • T. Mueller, T. Molnar, A. Szabo, E. Yamaha, Eva Zs. Jarasi, M. Bercsenyi, A. Specziar, B. Urbanyi, R. Romvari
    ACTA BIOLOGICA HUNGARICA 63 2 180 - 188 2012年06月 [査読有り][通常論文]
     
    The present study aimed in vivo tracking of maturation of male eel by computed tomography (CT). Additionally, individually monitored testes sizes were correlated with the conventionally used external maturity indicators (i.e. eye and nose indexes) in order to test and improve their usefulness at individual level. Testes could be clearly identified with the CT from the end of the third week of hCG administration routinely used to induce maturation in fish. The volume of testes increased exponentially during hormone treatment, and by the end of the sixth week of maturation procedure all males produced motilable spermatozoa. Present results prove that testes size can noninvasively be monitored with CT from maturity level where testes size rich 3000 mm(3) volume. Eye and nose indexes are in close correlation with testes volume and thus can also be effectively used to monitor maturaty level of male eel, but preferably only at stock level. However, due to their high individual variability, these indexes can be applied only with caution at individual level and should be supplemented with other noninvasive techniques such as CT.
  • Y. Kawakami, T. Saito, T. Fujimoto, R. Goto-Kazeto, E. Takahashi, S. Adachi, K. Arai, E. Yamaha
    JOURNAL OF ANIMAL SCIENCE 90 2 495 - 500 2012年02月 [査読有り][通常論文]
     
    The feasibility of cryopreserving common carp (Cyprinus carpio) primordial germ cells (PGC) by vitrification of whole embryos at the 22- to 28-somite stage was investigated. Green fluorescent protein (GFP)-labeled PGC were cooled rapidly using liquid nitrogen after exposure to a pretreatment solution containing 1.5 M cryoprotectant (ethylene glycol or dimethyl sulfoxide, 30 or 50 min) and a vitrification solution containing 3 M cryoprotectant and 0.5 M sucrose (5, 10, 20, or 30 min). Embryonic cells that were pretreated for 30 min and vitrified for 20 min with ethylene glycol had the greatest rate of survival of embryonic cells (68.6%; P < 0.01), an optimal highest percentage of viable PGC (73.8 to 74.9%; P < 0.05), and no evidence of ice formation after thawing. The vitrified/ thawed PGC were transplanted into blastula-stage embryos from goldfish (Carassius auratus). The PGC maintained their motility and moved to the gonadal ridge of the host embryo. Thus, the combination of vitrification and transplantation to produce germline chimeras is a powerful tool for the artificial production of next-generation offspring.
  • T Muller, A Horvath, E Takahashi, B Kolics, K Bakos, K Decsi, B Kovacs, J Taller, B Urbanyi, M Bercsenyi, L Horvath, S Adachi, K Arai, E Yamaha
    Aquaculture 2012 350-353 130 - 133 Elsevier B.V. 2012年 [査読有り][通常論文]
     
    Eggs of Japanese eel (Anguilla anguilla) were fertilized with cryopreserved sperm of European eel (A. anguilla) obtained from fish reared and matured in freshwater conditions. A new genomic PCR-RFLP marker was developed to distinguish the two species and detect maternally and paternally derived DNA fragments to genetically verify the hybrid nature of the offspring.
  • Kagayaki Morishima, Takafumi Fujimoto, Mami Sato, Ayako Kawae, Yan Zhao, Etsuro Yamaha, Katsutoshi Arai
    BMC BIOTECHNOLOGY 11 116  2011年11月 [査読有り][通常論文]
     
    Background: Androgenesis (all-male inheritance) is generally induced by means of irradiating the eggs to inactivate the maternal genome, followed by fertilization with normal sperm. In fish, the conventional technique for induced androgenesis has been applied for rapid fixation to traits, recovery of cryopreserved genotypes, sex-control, etc. A new method of androgenesis that eliminates the need to irradiate the egg was proposed using the loach, Misgurnus anguillicaudatus (a teleost fish). Results: When the eggs of wild-type females were fertilized with sperm of albino or orange phenotype males and cold-shocked at 0 to 3 degrees C for 60 min duration just after fertilization, generally more than 30% (with a peak of 100%) of the hatched progeny were androgenotes. While a few of them were the normal diploid, most of them turned out to be abnormal haploid. All-male inheritance was verified by the expression of the recessive color trait (albino or orange) and microsatellite genotypes comprising only paternally derived alleles. Nuclear behavior after the cold-shock treatment was traced by microscopic observation of DAPI (4'6-diamidino-2-phenylindole)-stained samples and hematoxylin-eosin stained histological sections, and the extrusion of egg (maternal) nucleus was observed in eggs treated in the optimum timing. Conclusion: In this paper, we demonstrate that cold-shock treatment (at 0 and 3 degrees C) of loach eggs for 60 min just after fertilization successfully induces androgenetic haploid development. The most likely mechanism of cold-shock induced androgenesis is an elimination of the egg nucleus together along with the second polar body and subsequent development of a decondensed sperm nucleus or male pronucleus.
  • Taiju Saito, Rie Goto-Kazeto, Yutaka Kawakami, Kazuharu Nomura, Hideki Tanaka, Shinji Adachi, Katsutoshi Arai, Etsuro Yamaha
    PLOS ONE 6 9 e24460  2011年09月 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders).
  • G. S. Yasui, T. Fujimoto, S. Sakao, E. Yamaha, K. Arai
    JOURNAL OF ANIMAL SCIENCE 89 8 2380 - 2388 2011年08月 [査読有り][通常論文]
     
    An efficient procedure for the cryopreservation of fish blastomeres followed by restoration through germ-line chimera formation was established. Blastomeres of the loach (Misgurnus anguillicaudatus) were cryopreserved in 250-mu L straws in Eagle's minimum essential medium with various concentrations of dimethyl-sulfoxide (0, 5, 10, 15, and 20%), and the best concentration was combined with glycerol (1, 2, and 4%) and external cryoprotectants (1 or 2% sucrose; 2, 5, or 10% fetal bovine serum; 1 or 2% BSA). Post-thaw viability of the blastomeres was used to optimize cryopreservation conditions. Donor blastomeres were injected with zebrafish green fluorescence protein-nos1 3' untranslated region mRNA and biotin dextran before cryopreservation in the optimal freeze medium. Host embryos were injected with zebrafish DsRed-nos1 3' untranslated region mRNA and reared to the blastula stage. Donor blastomeres were thawed at 25 degrees C for 10 s and transplanted to the host embryos either immediately or after incubation for 16 h at 20 degrees C. Donor and host primordial germ cell migration was visualized with fluorescent imaging during the early stages of embryogenesis, and also by histology in 4-d-old embryos. Transplantation of blastomeres immediately after thawing gave decreased hatching rates (approximately 3%) and generated a smaller percentage of germ-line chimeras (approximately 1.1%). In contrast, incubation of a cryopreserved sample for 16 h followed by transplantation of the green fluorescence protein-positive blastomeres improved the hatching rate to 90%, and successfully produced presumable germ-line chimeras at a rate of 16.5%. The improved survival rates and germ-line chimerism may be an effective method for gene banking and subsequent reconstitution of endangered fish genotypes.
  • Yutaka Kawakami, Taiju Saito, Takafumi Fujimoto, Rie Goto-Kazeto, Eisuke Takahashi, Shinji Adachi, Katsutoshi Arai, Etsuro Yamaha
    Aquaculture 317 1-4 245 - 250 Elsevier 2011年07月 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. We previously visualized the PGCs of several teleostean embryos by injecting RNA synthesized from constructs encoding green fluorescent protein (GFP) fused to the 3'UTR of the zebrafish (Danio rerio) nanos1 gene (nos1). However, this technique was not always suitable for visualizing PGCs in embryos from all teleost species. In this study, we compared the visualization of PGCs in common carp (Cyprinus carpio) embryos using two artificial constructs containing GFP fused to the 3'UTR of nanos from either common carp or zebrafish. Visualization was better using GFP fused to the 3'UTR of the nanos gene from common carp, compared with that from zebrafish. The visualized PGCs successfully migrated toward the gonadal ridge after transplantation into goldfish host embryos, suggesting that they maintained normal migratory motility. These techniques could be useful for the production of inter-specific germline chimeras using common carp donor PGCs.
  • H Zhou, T Fujimoto, S Adachi, E Yamaha, K Arai
    Journal of Applied Ichthyology 27 2 484 - 491 2011年04月 [査読有り][通常論文]
  • Takafumi Fujimoto, Toshiya Nishimura, Rie Goto-Kazeto, Yutaka Kawakami, Etsuro Yamaha, Katsutoshi Arai
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 107 40 17211 - 17216 2010年10月 [査読有り][通常論文]
     
    Germ cell-deficient fish usually develop as phenotypic males. Thus, the presence of germ cells is generally considered to be essential for female gonadal differentiation or the maintenance of ovarian structure. However, little is known of the role of germ cells in the determination of the sexual fate of gonadal somatic cells. We have established an inducible germ cell deficiency system in the loach ( Misgurnus anguillicaudatus, Cypriniformes: Cobitidae), a small freshwater fish, using knockdown of the dead end gene with a morpholino antisense oligonucleotide. Interestingly, loach lacking germ cells could develop as either phenotypic males or females, as characterized morphologically by the presence or absence of bony plates in the pectoral fins, respectively. The phenotypic males and females had testicular and ovarian structures, respectively, but lacked germ cells. Gene expression patterns in these male and female germ cell-deficient gonads were essentially the same as those in gonads of normal fish. Our observations indicate that sexually dimorphic gonads can develop in germ cell-deficient loach. In contrast to the situation in other model fish species, the gonadal somatic cells in phenotypic females autonomously differentiated into ovarian tissues and also played a role in the maintenance of gonadal structure. On the basis of our observations, we propose two possible models to explain the role of germ cells in sex determination in fish.
  • Gaku Kanno, Takahito Yamaguchi, Hideki Kishimura, Etsuro Yamaha, Hiroki Saeki
    FISH PHYSIOLOGY AND BIOCHEMISTRY 36 3 637 - 645 2010年09月 [査読有り][通常論文]
     
    Trypsin from the pyloric ceca of masu salmon (Oncorhynchus masou) cultured in fresh water was purified by a series of chromatographies including Sephacryl S-200, Sephadex G-50 and diethylaminoethyl cellulose to obtain a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. The molecular mass of the purified trypsin was estimated to be approximately 24,000 Da by SDS-PAGE. The enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and N (alpha) -p-tosyl-l-lysine chloromethyl ketone. Masu salmon trypsin was stabilized by calcium ion. The optimum pH of the masu salmon trypsin was around pH 8.5, and the trypsin was unstable below pH 5.0. The optimum temperature of the masu salmon trypsin was around 60A degrees C, and the trypsin was stable below 50A degrees C, like temperate-zone and tropical-zone fish trypsins. The N-terminal 20 amino acid sequence of the masu salmon trypsin was IVGGYECKAYSQPHQVSLNS, and its charged amino acid content was lower than those of trypsins from frigid-zone fish and similar to those of trypsins from temperate-zone and tropical-zone fish. In the phylogenetic tree, the masu salmon trypsin was classified into the group of the temperate-zone fish trypsin.
  • Shogo Higaki, Kentaro Mochizuki, Yuichiro Akashi, Etsuro Yamaha, Seiji Katagiri, Yoshiyuki Takahashi
    JOURNAL OF REPRODUCTION AND DEVELOPMENT 56 2 212 - 218 2010年04月 [査読有り][通常論文]
     
    The feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells (PGCs) by rapid cooling (i.e., vitrification) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EC), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG) and 1,3-butylene glycol (1,3-BG). According to the results of glass-forming and embryo toxicity tests, pretreatment solution (PS) containing 2 or 3 M cryoprotectant and vitrification solution (VS) containing 5 M cryoprotectant and 0.5 M sucrose were prepared using each cryoprotectant. Dechorionated embryos, the PGCs of which were visualized by injection of green fluorescence protein-nos1 3'UTR mRNA, were cooled rapidly by plunging into liquid nitrogen after serial exposure to PS and VS. All embryos cooled with MeOH, PG and 1,3-BG showed ice formation during cooling, and few embryos had live PGCs after warming. Most embryos cooled with GC did not show ice formation; however, few embryos had live PGCs. All embryos cooled with EC and most embryos cooled with DMSO had live PGCs when the embryos did not show ice formation during cooling. Based on the number of live PGCs in fresh embryos, the maximum survival rates of PGCs recovered from embryos cooled with EC, and DMSO were estimated to be about 40 and 20%, respectively. The present study indicates that rapid cooling of dechorionated whole embryos, especially using EG-based solutions, could be utilized as a simple and promising tool for cryopreservation of PGCs.
  • Shogo Higaki, Yoshiki Eto, Yutaka Kawakami, Etsuro Yamaha, Noriko Kagawa, Masashige Kuwayama, Masashi Nagano, Seiji Katagiri, Yoshiyuki Takahashi
    REPRODUCTION 139 4 733 - 740 2010年04月 [査読有り][通常論文]
     
    This study aimed to produce fertile zebrafish (Danio rerio) possessing germ cells (gametes) that originated from cryopreserved primordial germ cells (PGCs). First, to improve the vitrification procedure of PGCs in segmentation stage embryos, dechorionated yolk-intact and yolk-removed embryos, the PGCs of which were labeled with green fluorescent protein, were cooled rapidly after serial exposures to equilibration solution (ES) and vitrification solution (VS), which contained ethylene glycol, DMSO, and sucrose. Yolk removal well prevented ice formation in the embryos during cooling and improved the viability of cryopreserved PGCs. The maximum recovery rate of live PGCs in the yolk-removed embryos vitrified after optimum exposure to ES and VS was estimated to be about 90%, and about 50% of the live PGCs showed pseudopodial movement. Next, to elucidate the ability of cryopreserved PGCs to differentiate into functional gametes, PGCs recovered from the yolk-removed embryos (striped-type) that were vitrified under the optimum exposure to ES and VS were transplanted individually into 218 sterilized recipient blastulae (golden-type). Two days after the transplantation, 7.5% (14/187) of morphologically normal embryos had PGC(s) in the genital ridges. Six (5 males and 1 female) of the 14 recipient embryos developed into mature fish and generated progeny with characteristics inherited from PGC donors. In conclusion, we demonstrated the successful cryopreservation of PGCs by vitrification of yolk-removed embryos and the production of fertile zebrafish possessing germ cells that originated from the PGCs in vitrified embryos. Reproduction (2010) 139 733-740
  • Takafumi Fujimoto, Taiju Saito, Suzu Sakao, Katsutoshi Arai, Etsuro Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 54 5 827 - 835 2010年 [査読有り][通常論文]
     
    In teleosts, viable nucleocytoplasmic hybrids, formed by combining a nucleus from one species with the egg cytoplasm of another, have been used as one of the methods for breed improvement in aquaculture, but have been little exploited for developmental biology studies. Here, we used an artificial androgenesis technique to form nucleocytoplasmic hybrids comprising a goldfish haploid nucleus and loach egg cytoplasm. These hybrids were used to investigate interactions between the nucleus and cytoplasm during embryonic development. Additionally, the developmental characteristics of embryonic cells of nucleocytoplasmic hybrids were examined in chimeras produced by transplantation of blastomeres into recipient loach or goldfish embryos. We found that the nucleocytoplasmic hybrids arrested at the dome stage of embryonic development and did not form any gastrula structures. The goosecoid (gsc) and no tail (nil) genes were expressed normally before gastrulation in nucleocytoplasmic hybrids, similar to diploid loach. However, expression of the gsc and ntl genes was not maintained in nucleocytoplasmic hybrids. In chimeric embryos, blastomeres derived from nucleocytoplasmic hybrids were found to mix with the cells of recipient loach embryos at the gastrula stage. The transplanted blastomeres formed small clusters at the somitogenesis stage and, finally, small spots at the hatching stage. In contrast, when the blastomeres were transplanted into goldfish embryos, the transplanted blastomeres aggregated in the chimeric embryos. Thus, embryonic cells from nucleocytoplasmic hybrids that arrest before gastrulation could survive beyond the somitogenesis stage depending on the cytoplasmic environment in the recipient embryos.
  • Taiju Saito, Rie Goto-Kazeto, Takafumi Fujimoto, Yutaka Kawakami, Katsutoshi Arai, Etsuro Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 54 10 1481 - 1486 2010年 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.
  • Rie Goto-Kazeto, Taiju Saito, Misae Takagi, Katsutoshi Arai, Etsuro Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 54 10 1487 - 1492 2010年 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3'UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos 1 3'UTR mRNA into 1-4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.
  • Yutaka Kawakami, Rie Goto-Kazeto, Taiju Saito, Takafumi Fujimoto, Shogo Higaki, Yoshiyuki Takahashi, Katsutoshi Arai, Etsuro Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 54 10 1493 - 1501 2010年 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. In our previous study, a single PGC transplanted into a host differentiated into fertile gametes and produced germ-line chimeras of cyprinid fish, including zebrafish. In this study, we aimed to induce germ-line chimeras by transplanting donor PGCs from various sources (normal embryos at different stages, dissociated blastomeres, embryoids, or embryoids cryopreserved by vitrification) into host blastulae, and compare the migration rates of the PGCs towards the gonadal ridge. Isolated, cultured blastomeres not subject to mesodermal induction were able to differentiate into PGCs that retained their motility. Moreover, these PGCs successfully migrated towards the gonadal ridge of the host and formed viable gametes. Motility depended on developmental stage and culture duration: PGCs obtained at earlier developmental stages and with shorter cultivation periods showed an increased rate of migration to the gonadal ridge. Offspring were obtained from natural spawning between normal females and chimeric males. These results provide the basis for new methods of gene preservation in zebrafish.
  • E Yamaha, R Goto-Kazeto, T Saito, Y Kawakami, T Fujimoto, S adachi, K Arai
    Journal of Applied Ichthyology 26 5 816 - 822 2010年 [査読有り][招待有り]
  • Takafumi Fujimoto, George Shigueki Yasui, Mayumi Hayakawa, Suzu Sakao, Etsuro Yamaha, Katsutoshi Arai
    Aquaculture 308 Suppl.1 S133 - S139 Elsevier 2010年 [査読有り][通常論文]
     
    Tetraploid loaches have been discovered amongst specimens recovered from fish markets in Japan. These tetraploids can be used as a source of diploid gametes to assist the further expansion of ploidy manipulation. Here, we produced a first generation neo-tetraploid strain by fertilizing eggs from a normal diploid female with diploid spermatozoa from a natural tetraploid male, followed five minutes later by heat shock (42°C, 2 min duration) to inhibit release of the second polar body. Diploid spermatozoa from the neo-tetraploid males produced were then used to create androgenetic diploid progeny by fertilizing UV-irradiated eggs from a normal diploid female. Triploid progeny were produced by crossing a normal diploid female with a neo-tetraploid male. Tetraploid progeny were produced by cold shock (1°C, 40 min duration), beginning 5 min after fertilizing normal eggs with diploid spermatozoa from first generation neo-tetraploid males. Reproductive performance of second generation progeny was also examined. Androgenetic diploid males generated fertile haploid spermatozoa. Triploid males were sterile, but a triploid female laid fertile haploid eggs. Second generation neo-tetraploid males were considered sterile.
  • Shogo Higaki, Kentaro Mochizuki, Hiroko Baba, Yuichiro Akashi, Etsuro Yamaha, Seiji Katagiri, Yoshiyuki Takahashi
    JAPANESE JOURNAL OF VETERINARY RESEARCH 57 2 119 - 128 2009年08月 [査読有り][通常論文]
     
    We investigated the feasibility of cryopreservation of zebrafish (Danio rerio) blastomeres and primordial germ cells (PGCs) by rapid freezing of dechorionated whole embryos at the blastula, gastrula and segmentation stages. Initially we examined the glass-forming properties and embryo toxicities of 5 cryoprotectants: methanol (MeOH), ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (1,3-BG). Embryos at the blastula and gastrula stages had high sensitivities to cryoprotectant toxicities and were fragile against mechanical damage. Thus the segmentation stage embryos, the PGCs of which were visualized by injecting green fluorescence protein-nos1 3'UTR mRNA, were frozen using solutions containing each cryoprotectant at 6 M (first trial) and 2 types of cryoprotectants at 3 M each (second trial). In the first trial, live PGCs were recovered from most of the embryos frozen with EG (about 2 cells/embryo); however, a few embryos had live PGCs when embryos were frozen with other cryoprotectants. In the second trial, a mixture of EG + PG better preserved the viability of PGCs in frozen embryos. Live PGCs were recovered from all embryos frozen with EG + PG (about 3 cells/embryo), and the survival rate of PGCs was estimated to be about 25% based on the number of live PGCs in fresh embryos (about 12 cells/embryo). The present study indicates that we can utilize rapid freezing of dechorionated whole embryos at the segmentation stage for the cryopreservation of PGCs.
  • Rie Goto-Kazeto, Takahiro Takeda, Taiju Saito, Misae Takagi, Katsutoshi Arai, Etsuro Yamaha
    Biology of Reproduction 81 Suppl{\_}1 39  Oxford University Press ({OUP}) 2009年07月 [査読有り][通常論文]
  • Sakao Suzu, Fujimoto Takafumi, Kobayashi Terumasa, Yoshizaki Goro, Yamaha Etsuro, Arai Katsutoshi
    FISHERIES SCIENCE 75 4 993 - 1000 Springer Japan 2009年07月 [査読有り][通常論文]
     
    Diploid gametes generated with tetraploid animals are a stepping stone to improving techniques of chromosome manipulation. But artificially induced tetraploid individuals generally die soon after hatching. Diploid gametes could have been induced by in vivo cultures of tetraploid primordial germ cells (PGCs) through germ-line chimera. In the present study, characteristics of PGCs were studied in inviable tetraploid masu salmon, Oncorhynchus masou. Histological observation of tetraploid embryos revealed that the same or smaller numbers of PGCs were observed and they migrate into the genital ridges as did diploid PGCs during gonadogenesis. By whole mount in situ hybridization using vasa mRNA, 4-35 vasa-positive signals were detected in a pair of genital ridges of tetraploids. By cytological observation of genital ridge cell suspensions, several large round cells were observed, some of which extended pseudopodia. They also contained large nuclei and round granules in their cytoplasm, characteristics of PGCs. As the results suggest that inviable artificial tetraploids have PGCs, we expect to achieve diploid gamete production through surrogate propagation and tetraploid fish production.
  • Hiroyuki Yoshikawa, Kagayaki Morishima, Takafumi Fujimoto, Taiju Saito, Tohru Kobayashi, Etsuro Yamaha, Katsutoshi Arai
    BIOLOGY OF REPRODUCTION 80 5 973 - 979 2009年05月 [査読有り][通常論文]
     
    The natural clonal loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) is diploid (2n = 50) and produces genetically identical unreduced eggs, which develop into diploid individuals without any genetic contribution from sperm. Artificially sex-reversed clones created by the administration of 17alpha-methyltestosterone produce clonal diploid sperm. In metaphase spreads from testicular cells of the sex-reversed clones, spermatocytes had twice the normal number of chromosomes (50 bivalents) compared with those of normal diploids (25 bivalents). Thus, the production of unreduced diploid spermatozoa is initiated by premeiotic endomitosis (or endoreduplication), chromosome doubling before meiosis, and is followed by two quasinormal divisions. Larger nuclei in the germ cells were observed in all stages of type B spermatogonia in the testes of the sex-reversed clones. In contrast, besides having larger type A spermatogonia, the sex-reversed clones also had the type A spermatogonia that were the same size as those of normal diploids. It follows that chromosome duplication causing unreduced spermatogenesis occurred in the type A spermatogonia. The presence of tetraploid type A and early type B spermatogonia, identified by labeling with antispermatogonia-specific antigen 1, was verified using DNA content flow cytometry. These results support the conclusion that chromosome doubling occurs at the type A spermatogonial stage in diploid spermatogenesis in the clonal fish.
  • Yoshikawa, H, Morishima, K, Fujimoto, T, Arias-Rodriguez, L, Yamaha, E, Arai, K
    Journal of Applied Ichthyology 24 4 410 - 414 Blackwell Publishing 2008年08月 [査読有り][通常論文]
     
    In the loach, Misgurnus anguillicaudatus (Teleoste: Cobitidae), diploid sperm can be obtained from natural tetraploid individuals with four sets of homologous chromosomes. Using diploid sperm, various kinds of polyploids and androgenetic diploids have been produced. Cryptic clonal lineages are also recognized in wild populations of the loach. They produce unreduced diploideggs genetically identical to somatic cells of the mother fish and most diploid eggs develop gynogenetically as a member of the clone. However, some eggs develop to triploid and/or diploid-triploid mosaic individuals by incorporation of sperm nucleus. Diploid-triploid mosaic males exclusively generate fertile diploid sperm with clonal genotypes. Such diploid sperm can also be obtained from artificially sex-reversed clonal individuals. Recent population studies suggested that Japanese M. anguillicaudatus might not be a single species, but a complexinvolving cryptic species, because wild populations exhibited genetic differentiation at interspecific level. This implies possible relationship between atypical reproduction and natural hybridization in the loach.
  • Taiju Saito, Rie Goto-Kazeto, Katsutoshi Arai, Etsuro Yamaha
    BIOLOGY OF REPRODUCTION 78 1 159 - 166 2008年01月 [査読有り][通常論文]
     
    Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. in many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.
  • T Fujimoto, GS Yasui, H Yoshikawa, E Yamaha, K Arai
    Journal of Applied Ichthyology 24 4 430 - 437 Blackwell Publishing 2008年 [査読有り][通常論文]
     
    Diploid and triploid interspecific hybrid male progeny obtained from mating Misgurnus anguillicaudatus with M. mizoleis were reported to have histologically fertile and sterile testes, respectively. However, their reproductive capacity is still unclear because mating tests have not been examined using mature hybrids. Here, we examined physiological and genetic characteristics of spermatozoa of diploid and triploid hybrids. In diploid hybrid males, 1n, 2n and 4n spermatozoa showing low motility were detected. However, spermatozoa of three diploid hybrid males could generate 2n larvae. Therefore, only 1n spermatozoa of diploid hybrid males was fertile to produce larva. The chromosomes of diploid hybrid males were transmitted to spermatozoa by random segregation between the homologous chromosomes because most larvae had one allele derived from both M. anguillicaudatus and M. mizolepis at all loci examined. In triploid hybrid males, spermatozoa could be categorized to three different types based on their ploidy status. Type 1: In the first and second males, sperm samples mainly comprised 6n spermatozoa. Motility and fertility were not recorded. Type 2: The third male gave a large proportion of 6n spermatozoa as well as a small proportion of 1n spermatozoa. Although no motility was observed, larvae arose from eggs inseminated with such spermatozoa. Type 3: In the fourth male, only 1n spermatozoa were detected and their motility was vigorous. When eggs were fertilized with such 1n spermatozoa, normal larvae hatched. 1n spermatozoa of the triploid hybrid male only included the M. anguillicaudatus genome. In Misgurnus fishes, diploid hybrid males exhibited semi-sterility or slight fertility. On the contrary, triploid hybrid males were sometime fertile due to the production of 1n spermatozoa by a kind of transformation of meiosis like meiotic hybridogenesis.
  • Takafumi Fujimoto, Suzu Sakao, Etsuro Yamaha, Katsutoshi Arai
    JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY 307A 8 449 - 462 2007年08月 [査読有り][通常論文]
     
    Genetic inactivation of the egg nucleus is an indispensable step in the production of androgenetic embryos in teleosts. However, few experimental studies have focused on determining the most effective means of achieving complete inactivation of the maternal genome. Here, we sought to identify the optimum conditions of ultraviolet (UV) irradiation for complete inactivation of the loach egg nucleus. Unfertilized eggs were UV irradiated from above with a dose in the range 0-200 mJ/cm(2). Successful inactivation of the maternal genome was evaluated by the exclusive expression of a paternally inherited color phenotype. The presence or absence of putative maternal chromosome fragments was screened by flow cytometry of DNA content and by cytogenetic analysis. The majority of the larvae derived from irradiated eggs had an abnormal appearance. Haploid individuals were detected by measurement of DNA content flow cytometry and by chromosome counting in the groups that received more than 75 mJ/cm(2) groups. Although the coefficient of variation of DNA content was apparently reduced in the 125-200 mJ/cm(2) groups, chromosome fragments were still detected in all the groups from irradiated eggs. Inactivation of the egg nucleus was also histologically elucidated by the presence or absence of residual nuclear material in anuclear embryos that developed from UV-irradiated eggs fertilized with UV-irradiated sperm. Embryos that were completely or near-completely anuclear were found in the 150 and 200 mJ/cm(2) groups. We conclude that the optimum UV dose for complete genetic inactivation of the egg nucleus is more than 150 mJ/cm(2).
  • Hiroyuki Yoshikawa, Kagayaki Morishima, Satoshi Kusuda, Etsuro Yamaha, Katsutoshi Arai
    JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY 307A 2 75 - 83 2007年02月 [査読有り][通常論文]
     
    Clone loaches reproduce unisexually in a wild population of Hokkaido Island, Japan. These clone loaches produce genetically identical unreduced eggs which develop to diploid individuals without any genetic contribution of sperm donors. In the present study, sex reversal of clone loaches was attempted and the reproductive potential of resultant clone males was examined. Clone loaches administered 0.5 ppm of 17-alpha methyltestosterone (MT) for 30 days from 1 month after hatching differentiated into physiological males. These sex-reversed clone males produced fertile spermatozoa with a diploid DNA content. Diploid spermatozoa had significantly larger heads than normal haploid sperm, but had a normal shape showing a head, mid-piece, and tail. The motility of diploid spermatozoa was low after ambient water was added. Concentration of diploid spermatozoa per unit of sperm was lower than that of control haploid spermatozoa. Microsatellite genotyping revealed that triploid progeny from the cross between a normal diploid female and a sex-reversed clone male had two alleles specific to the diploid clone male and one allele of the mother loach. These results indicated that the sex-reversed clone males produced fertile diploid spermatozoa genetically identical to the clone lineage.
  • Masaki Itono, Naoki Okabayashi, Kagayaki Morishima, Takafumi Fujimoto, Hiroyuki Yoshikawa, Etsuro Yamaha, Katsutoshi Arai
    JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY 307A 1 35 - 50 2007年01月 [査読有り][通常論文]
     
    The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.
  • Etsuro Yamaha, Taiju Saito, Rie Goto-Kazeto, Katsutoshi Arai
    Journal of Sea Research 58 1 8 - 22 Elsevier B.V. 2007年 [査読有り][招待有り]
     
    This review introduces surrogate production as a new technique for fish-seed production in aquaculture. Surrogate production in fish is a technique used to obtain the gametes of a certain genotype through the gonad of another genotype. It is achieved by inducing germ-line chimerism between different species during early development. Primordial germ cells (PGCs) are the key material of this technique to induce germ-line chimera. In several species, it has been reported that PGCs differentiated from the blastomeres inherited some maternally supplied mRNA located in the terminal regions of the early cleavage furrows. PGCs from donor species (or strains) are isolated and transplanted into host species to induce the germ-line chimera. Four methods for inducing germ-line chimera are described: blastomere transplantation, blastoderm-graft transplantation, transplantation of PGC from the genital ridge, and transplantation visualised PGC with GFP fluorescence. Several problems preventing the successful induction of germ-line chimera in various fish species are discussed. Surrogate production, however, opens the possibility of efficient fish-seed production and effective breeding and transfer of biodiversity to an aquaculture strain. Conservation and efficient utilisation of genetic resources will be achieved through surrogate production combined with the cryopreservation of PGCs.
  • H Yoshikawa, K Morishima, T Fujimoto, E Yamaha, K Arai
    Journal of Fish Biology 71 Suppl B 250 - 263 Fisheries Society of the British Isles 2007年 [査読有り][通常論文]
     
    In the loach Misgurnus anguillicaudatus, very few diploid-triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with UV-irradiated goldfish sperm indicated that diploid-triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid-triploid mosaics revealed that triploid genotypes of mosaic mothers consisted of two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.
  • Takafumi Fujimoto, Takashi Kataoka, Suzu Sakao, Taiju Saito, Etsuro Yamaha, Katsutoshi Arai
    ZOOLOGICAL SCIENCE 23 11 977 - 989 2006年11月 [査読有り][通常論文]
     
    The staging of embryonic and larval development, and the germ cell lineage of the loach, Misgurnus anguillicaudatus, are described. Fertilized eggs were obtained by artificial insemination. For the convenience of detailed observation and photography of the external appearance, we use dechorionated embryos. Through a series of operations, these embryos were cultured at 20 degrees C in an incubator. Embryonic and larval development of the loach was divided into five periods: cleavage, blastula, gastrula, segmentation, and hatching. Stages were assigned within each of these periods. Developmental stages were determined and named by morphological features and somite number. The staging series were photographed and tabulated. The germ cell lineage was then elucidated by whole mount in situ hybridization of mRNA expression of the germ-cell-specific marker vasa and histological analysis. Primordial germ cells (PGCs) of the loach derived from the cleavage furrows of 8-cell stage embryos began proliferation in the late blastula period and migrated to the gonadal anlagen through a migration pathway similar to that of the zebrafish. However, it is characteristic of the loach that PGCs migrate a long distance and stay in the posterior part of the yolk-extension region.
  • Etsuro Yamaha
    NIPPON SUISAN GAKKAISHI 72 5 960 - 961 2006年09月 [査読有り][通常論文]
  • Arai Katsutoshi, Yoshizaki Goro, Yamaha Etsuro
    NIPPON SUISAN GAKKAISHI 72 5 951 - 951 The Japanese Society of Fisheries Science 2006年09月 [査読有り][通常論文]
  • M Itono, K Morishima, T Fujimoto, E Bando, E Yamaha, K Arai
    JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-COMPARATIVE EXPERIMENTAL BIOLOGY 305A 6 513 - 523 2006年06月 [査読有り][通常論文]
     
    The natural clone loach produces unreduced eggs genetically identical to somatic cells of the mother fish and such diploid eggs normally develop as a clone without genetic contribution of sperm. Following the identification of clonal nature and diploidy of eggs, we conducted cytological studies to determine the mechanisms responsible for this unusual oogenesis. Cytolological observation of full-grown oocytes cultured in vitro revealed that oocytes of both the clone and the control loach underwent two successive meiotic divisions: formation of a bipolar spindle and metaphase in meiosis I and equal segregation of chromosomes, extrusion of the first polar body and the appearance of metaphase of meiosis II. However, spindle size of the clone was larger than that of the control. Bivalent chromosome number of germinal vesicle of oocytes was 25 in the control diploid, whereas 50 in the clone. The results suggest that chromosomes are duplicated by mitosis without cytokinesis before meiosis, i.e. premeiotic endomitosis and then oocytes differentiated from tetraploid oogonia undergo a quasinormal meiosis followed by two successive divisions to produce diploid eggs.
  • R Goto-Kazeto, Y Abe, K Masai, E Yamaha, S Adachi, K Yamauchi
    AQUACULTURE 254 1-4 617 - 624 2006年04月 [査読無し][通常論文]
     
    The present study analyzes the temperature-dependent sex differentiation in goldfish, which have an XX-XY sex determining system. Hatchlings from an XX male-XX female cross were reared at 23 +/- 0.5 degrees C until Day 12 (days after fertilization), and divided into experimental groups reared at 15, 17, 20, 23, 25, 28 or 30 +/- 0.5 degrees C. The proportions of females were 94.6% at 15 degrees C, 100% at 17 degrees C, 94.6% at 20 degrees C 90.5% at 23 degrees C 46.6% at 25 degrees C, 15.4% at 28 degrees C, and 7.7% at 30 0.5 degrees C. This result confirms that genotypic females can be changed to phenotypic males by high temperature treatment. To determine the temperature sensitive period (TSP) of sex differentiation, the sex ratio was measured in groups of larvae shifted reciprocally between 23 and 30 degrees C as development progressed. The percentage of females increased in test groups shifted from 23 to 30 degrees C at Days 17, 22 and 27 and decreased in groups shifted from 30 to 23 degrees C at Day 17 and 22. The end point of the TSP occurs on Day 17 at 30 degrees C and on Day 22 at 23 degrees C, implying that the TSP is shortened at higher temperatures. To investigate the effect of fluctuating temperatures on sex differentiation, several one-day temperature pulses from 23 to 30 degrees C or 30 to 23 degrees C were applied, during the TSP. The pulse to 23 degrees C, from the basal temperature of 30 degrees C, resulted in a decreased percentage of males, however, the pulse to 30 degrees C, from the basal temperature of 23 degrees C, had no effect on the sex ratio. Theses results show that high temperature must be maintained during the TSP in order to produce a high proportion of masculinized genetic female goldfish. In conclusion, water temperature can play an important role in the process of sex differentiation in goldfish, as has been seen in other teleosts and reptiles, and be applied to aquaculture as a tool for control of sex. (c) 2005 Elsevier B.V. All rights reserved.
  • Taiju Saito, Takafumi Fujimoto, Shingo Maegawa, Kunio Inoue, Minoru Tanaka, Katsutoshi Arai, Etsuro Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 50 8 691 - 700 2006年 [査読有り][通常論文]
     
    In some teleost fish, primordial germ cells (PGCs) inherit specific maternal cytoplasmic factors such as vasat and nanos 1 (nos1) mRNA. It has been shown that the 3' untranslated regions (UTRs) of vasa and nos1 have critical roles for stabilization of these RNAs in zebrafish PGCs. In this study, to determine whether this role of the nos1 3'UTR isconserved between teleost species,we injected artificially synthesized mRNA, combining green fluorescent protein (GFP) and the zebrafish nos1 3'UTR (GFP-nos1 3'UTR mRNA), into the fertilized eggs of various fish species. The 3'UTR of the Oryzias latipes vasa homologue (olvas) mRNA was assayed in the same manner. We demonstrate that the PGCs of seven teleost species could be visualized using GFP-nos1 3'UTR mRNA. GFP-olvas 3'UTR mRNA did not identify PGCs in herring or loach embryos, but did enable visualization of the PGCs in medaka embryos. Our results indicate that the 3'UTR of the zebrafish nos1 mRNA can promote maintenance of RNAs in the PGCs of different fish species. Finally, we describe and compare the migration routes of PGCs in seven teleost species.
  • Saito Taiju, Goto-Kazeto Rie, Arai Katsutoshi, Yamaha Etsuro
    Zoological Science 23 12 1170  2006年 [査読有り][通常論文]
  • Suzu Sakao, Takafumi Fujimoto, Shizuo Kimura, Etsuro Yamaha, Katsutoshi Arai
    Aquaculture 252 2 147 - 160 Elsevier 2006年 [査読有り][通常論文]
     
    Although tetraploidy is considered to be important and useful for the production of sterile triploids and fertile allotetraploids in aquaculture, in most cases the resultant embryos exhibit extremely low survival. In this study, we aimed to clarify the cause of the inviability of tetraploids in masu salmon. Firstly, we compared developmental rates of the first cell cycle among 9 single pairs produced by all possible matings between 3 females and 3 males. The results showed that the fertilized eggs developed almost synchronized among individuals from each pair but asynchronous among pairs and this asynchronous development was more apparent among pairs from different female than those from different male. Secondly, we induced both tetraploidy (4N) and gynogenetic diploidy (G2N) by the same hydrostatic pressure shock (PS) conditions (700 kg/cm2, 7 min duration) for the first cleavage inhibition using single-pair mating. This experiment was designed to verify whether the cause of mortality of induced tetraploidy was the elevated ploidy itself. Eggs from one female were divided into 2 groups and fertilized with normal or UV-irradiated sperm from one male, respectively. Then each group of eggs was subdivided into 6 groups: one was an intact control and the other 5 groups were treated with PS at every 30 min from 5 to 7 hpf at 10 °C. As a result, the treated eggs of 4N and G2N showed the highest survival (90.8% and 60.6%, respectively) and embryogenesis rate (both 100% relative to surviving eggs) at 33 dpf, only when PS treatment was performed at late prometaphase (6 hpf and 6.5 hpf, respectively). The other PS groups showed extremely low survival (1.5-52.8% and 3.1-14.8%), ceased morphogenetic development and developed into only undifferentiated cell masses. This experiment was repeated 2 more times using other single-pair mating and the results showed the same tendency. The embryonic bodies were confirmed by flow cytometry to have approximately objective ploidy, tetraploid, hypo- or hyper- tetraploid in 4N and diploid or hypo diploid in G2N. However, all tetraploid embryos, even those with normal morphology, began to die simultaneously around the hatching period (34 dpf), while G2N embryos showed normal morphology and survived beyond 50 dpf (55.6%). Most tetraploid embryos showed malformation and had very poor vascular systems even in normal-looking ones. These results suggest that the mortality of induced tetraploids depends not only on the side effects of the PS treatment but also strongly depends on the induced tetraploidy itself.
  • Satoshi Kusuda, Nobuhisa Koide, Hiloshi Kawamura, Tetsuo Teranishi, Jun-Ichiro Nakajima, Etsuro Yamaha, Katsutoshi Arai, Hiromi Ohta
    Fisheries Science 71 2 293 - 298 Japanese Society of Fisheries Science 2005年03月 [査読有り][通常論文]
  • S Otani, T Kitauchi, T Saito, S Sakao, S Maegawa, K Inoue, K Arai, E Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 49 7 843 - 850 2005年 [査読有り][通常論文]
     
    The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nosl 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.
  • Fujimoto Takafumi, Saito Taiju, Sakao Suzu, Yamaha Etsuro, Arai Katsutoshi
    Zoological Science 22 12 1459  2005年 [査読有り][通常論文]
  • Kouzou Oshima, Kagayaki Morishima, Etsuro Yamaha, Katsutoshi Arai
    Ichthyological Research 52 1 1 - 8 Ichthyological Society of Japan 2005年 [査読有り][通常論文]
  • 長井輝美, 大谷哲, 斎藤大樹, 前川真吾, 井上邦夫, 荒井克俊, 山羽悦郎
    日本水産学会誌 71 1 1 - 9 The Japanese Society of Fisheries Science 2005年01月 [査読有り][通常論文]
     
    ゼブラフィッシュを用い,胚盤移植を試み,同法の発生への影響および生殖系列キメラの作出への有効性を検討した。胞胚期の胚盤全体,胚盤上部,または胚盤下部を他胚の動物極へ移植した。全ての移植群で,正常な頭部.背方構造を持つ個体が得られたが,胚盤下部を移植した胚では頭部構造を欠く個体の出現頻度が高かった。胚盤全体または胚盤下部を移植した胚から発生した多くの個体(10/11)の生殖隆起にドナー胚盤に由来するPGCsが認められ,本種での生殖系列キメラ個体の作出に,胚盤移植法が有効であることが明らかとなった。To elucidate the effects of blastoderm graft for chimeric-fish production on further development, transplantations of upper, lower or entire part of blastoderm were performed onto the animal part of host embryos during the blastula stage in zebrafish. In the case of upper, all resultant chimeric embryos developed normally, while in the lower part, many acephalic embryos appeared. When the entire blastoderm was transplanted, many resultant embryos showed normal phenotype with extra cell-mass, while a few had double axis. vas mRNA, one of the markers of PGCs, also kept the expressions in both donor and recipient blastomeres in transplantation of the entire blastoderm. Donor PGCs were frequently detected at germinal anlagen in histological sections of 6-day larvae developed from transplantations of lower and entire blastoderm, but seldom in those of the upper part. These results might suggest that graft transplantation was effective for production of germ-line chimera, but sometimes caused un-regulaory differentiation of graft cells under the community effect.
  • T Saito, S Otani, T Fujimoto, T Suzuki, T Nakatsuji, K Arai, E Yamaha
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 48 10 1079 - 1085 2004年12月 [査読有り][通常論文]
     
    In order to determine the origin and migration of ukigori primordial germ cells (PGCs), we observed the aggregation of vasa mRNA by whole mount in situ hybridization. To observe PGC migration in the germ layers, we analyzed HE-stained paraffin sections. The germ line lineages were derived from the edge of the first, second and third cleavage furrows. During subsequent cleavages, vasa mRNA aggregations were respectively taken into four to eight cells in each embryo and vasa expressing cells proliferated from the sphere stage. At the bud to early somitogenesis period, PGCs aligned from head to tail bud regions on both sides of the embryonic body. During the late somitogenesis period, PGCs mainly aggregated just underneath the body axis. After gut formation, PGCs aligned along both sides of the gut at the 4th- to 8th- somite regions. Finally, PGCs reached the genital ridge via the inside of the lateral plate mesoderm and dorsal peritoneum. These results suggest that localized patterns of vasa transcripts and the migration routes of PGCs are different among fish (Teleost) species, perhaps depending on the amount of germinal cytoplasm derived maternally and the timing of endoderm differentiation.
  • T Fujimoto, T Kataoka, S Otani, T Saito, T Aita, E Yamaha, K Arai
    ZOOLOGICAL SCIENCE 21 7 747 - 755 2004年07月 [査読有り][通常論文]
     
    Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20degreesC after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.
  • Tanaka M, Yamaha E, Arai K
    Journal of experimental zoology. Part A, Comparative experimental biology 301 491 - 501 6 2004年06月 [査読有り][通常論文]
  • Morishima K, Oshima K, Horie S, Fujimoto T, Yamaha E, Arai K
    Journal of experimental zoology. Part A, Comparative experimental biology 301 502 - 511 6 2004年06月 [査読有り][通常論文]
  • 斎藤大樹, 荒井克俊, 山羽悦郎
    水産増殖 52 2 177 - 184 日本水産増殖学会 2004年06月 [査読有り][通常論文]
     
    20℃におけるシマウキゴリの胚発生過程を詳細に観察した。さらに, 卵割期における単一割球の標識実験により, 胚盤における分化の方向性が決定される時期を明らかにした。その結果, 未受精卵の段階から細胞質が卵黄から分離していること, 体節形成期に至るまで胚細胞中には卵黄顆粒が分布しているが体節形成期以降消失し胚体が透明化すること, 第3卵割以降, 卵割の様式が不規則になることが明らかとなった。また, 16細胞期から32細胞期に標識された細胞追跡の結果, 胞胚期から初期嚢胚期まで, 標識細胞は未標識の細胞と胚盤内で大規模な混合を起こすことが明らかとなった。この結果は, 初期嚢胚期まで胚盤の細胞は分化多能性を有していることを示唆している。
  • KUSUDA Satoshi, KOIDE Nobuhisa, KAWAMULA Hiloshi, TERANISHI Tetsuo, YAMAHA Etsuro, ARAI Katsutoshi
    Suisanzoshoku 52 2 171 - 175 水産増殖談話会 2004年06月 [査読有り][通常論文]
     
    イトウ精子の凍結保存における適切な保存液を開発するため、受精率を指標に耐凍剤の種類を検討した。300mMグルコースを90%、耐凍剤(DMSO、メタノール、グリセリン)を10%含む保存液で精液を4倍に希釈し、予めドライアイスに開けた小孔に100μl ずつ滴下し凍結した。滴下3分後に各ペレットを液体窒素に投入し、2時間以上浸漬した。各種ペレット4粒を30℃の120mM NaHCO3 16ml 中で解凍し、20gの卵に媒精した。卵は8℃の水中に14時間以上静置した後、ブアン氏液で固定し、卵割の有無を指標に受精率を算出した。受精率は耐凍剤がメタノールの場合に最も高く、DMSO、グリセリンの順に有意に低下した。メタノールを含む保存液で凍結した精液は、DMSOを含む保存液で凍結した精液より約2倍高い受精率を示した。以上の結果は、メタノールがイトウ精子の耐凍剤として適していることを示唆している。
  • Y Hashimoto, S Maegawa, T Nagai, E Yamaha, H Suzuki, K Yasuda, K Inoue
    DEVELOPMENTAL BIOLOGY 268 1 152 - 161 2004年04月 [査読有り][通常論文]
     
    Maternally supplied factors in fertilized eggs play essential roles in the establishment of primordial germ cells. In zebrafish, cytoplasm at the distal ends of the first and second cleavage furrows has been assumed to contain germ lineage determinants, since maternal transcripts of genri lineage-specific genes are localized to ends of the cleavage furrows. To investigate whether these parts of cytoplasm are required for germ cell formation, we removed all four regions of the cytoplasm by glass capillary at the 4-cell stage. Histological analysis revealed that the ablation of cytoplasm at the ends of the cleavage planes resulted in a severe reduction in the number of germ cells. In addition, the expression of germ lineage markers was eliminated by cytoplasmic ablation. These results demonstrated that cytoplasm at the distal ends of cleavage furrows is essential for germ cell formation. We also found novel localization patterns for zDazl and brul mRNAs along the cleavage planes. Our findings provide the first direct evidence that localized cytoplasmic factors are indispensable for germ cell establishment in zebrafish. (C) 2004 Elsevier Inc. All rights reserved.
  • S Kusuda, T Teranishi, N Koide, T Nagai, K Arai, E Yamaha
    JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY 301A 2 131 - 138 2004年02月 [査読有り][通常論文]
     
    To examine the pluripotency of cryopreserved blastomeres, we transplanted them into blastula. Donor blastomeres were prepared from blastula of goldfish (Carassius auratus) and cryopreserved in liquid nitrogen for two months. Fifty-five percent and 44% of blastomeres survived after thawing. Cryopreserved blastomeres were transplanted to the blastula of triploid crucian carp (C. a. longsdorfii), which reproduces gynogenetically in nature. At four days after the operation, resultant chimeric embryos transplanted with cryopreserved blastomeres showed a survival rate (41.6%) lower than that of embryos transplanted with unfrozen blastomeres (57.1%). Transplanted blastomeres were histologically identified in various organs derived from all three germ layers. A primordial germ cell differentiated from a cryopreserved blastomere was detected in one of the 32 chimeric fish examined. These results suggest blastomeres that survive after cryopreservation retain their pluripotency and are able to differentiate into both somatic and germ cell lines. (C) 2004 Wiley-Liss, Inc.
  • Taiju SAITO, Etsuro YAMAHA
    The journal of animal genetics 31 2 47 - 55 Japanese Society of Animal Breeding and Genetics 2004年 [査読有り][通常論文]
  • Fujimoto Takafumi, Saito Taiju, Sakao Suzu, Yamaha Etsuro, Arai Katsutoshi
    Zoological Science 21 12 1294 - 1295 2004年 [査読有り][通常論文]
  • E Yamaha, M Murakami, K Hada, S Otani, T Fujimoto, M Tanaka, S Sakao, S Kimura, S Sato, K Arai
    GENETICA 119 2 121 - 131 2003年10月 [査読有り][通常論文]
     
    In germ-line chimera, gametes originate from both the donor and recipient. In order to increase the proportion of gametes from the donor, the elimination or reduction of primordial germ cells (PGCs) from the recipient is required. In the present study, histological and genetic analyses were performed in the chimeric fish obtained when sterile goldfish x common carp hybrid and fertile goldfish embryos were used as a recipient and donor, respectively. Chimerism was induced by transplantation of the lower part of the goldfish blastoderm into the hybrid blastoderm at the blastula stage. Neither spermatid nor spermatozoa were observed in the testis of the male hybrid. Motile sperm were obtained from 15 chimeric males by human chorionic gonadotropin (HCG) injection. When the sperm of chimeric fish were genetically analyzed, only goldfish-specific repetitive DNA sequences were detected. These results revealed that chimeric fish of the cross between a sterile male hybrid and fertile goldfish produced sperm exclusively derived from the donor goldfish.
  • 坂尾寿々, 藤本貴史, 田中稔, 山羽悦郎, 荒井克俊
    日本水産学会誌 69 5 738 - 748, 852 The Japanese Society of Fisheries Science 2003年09月 [査読有り][通常論文]
     
    染色体操作において第一卵割阻止処理胚の生残率は極端に低いことが知られている。本研究では四倍体誘起個体が死亡する原因を細胞学的に解明することを目的とした。通常受精後10℃の水温条件下で培養し,第一卵割を阻止する目的で受精後5時間から7時間に,700kg/cm^2,7分間の圧力処理を施した。処理胚では,初期卵割期に分裂異常,胞胚期に無核細胞,発眼期に異数体およびモザイク個体の出現が認められた。これらの異常胚出現が四倍体誘起胚の生残率を低下させる原因であると考えられた。Although tetraploid is important for the mass production of sterile triploids by hybridizing with diploid, its survival capacity is extremely low. In this study, we aimed to identify the cause of such a high mortality of embryos treated with hydrostatic pressure shock (700 kg/cm^2, 7 min duration, 5-7 h after fertilization at 10℃ in masu salmon. In treated eggs, cleavage rates were delayed and aberrant cell divisions were observed during the early cleavage stage. Histological observation in the blastula stage revealed frequent occurrence of aberrant blastoderms including anuclear and/or small blastomeres. Ploidy analysis of eyed and hatching embryos revealed successful production of pure tetraploid individuals. However tetraploid survivors exhibited abnormal appearances. In the groups treated at 6.5 and 7 h, no embryonic body was observed at hatching stage. Judging from the results of each treated group, the optimum timing for the inhibition of first cleavage was 6 h after fertilization, when the eggs were cytologically staged as prometaphase to metaphase of the first cleavage . Aneuploids and mosaics were also detected in treated embryos by flow cytometry. These results suggest that abnormal cleavage and blastomeres mosaicism caused by the treatment at the first cleavage give rise to high mortality.
  • M Tanaka, S Kimura, T Fujimoto, S Sakao, E Yamaha, K Arai
    Fisheries Science 69 1 176 - 180 Japanese Society of Fisheries Science 2003年 [査読有り][通常論文]
  • T Saito, S Otani, T Nagai, T Nakatsuji, K Arai, E Yamaha
    ZOOLOGICAL SCIENCE 19 9 1027 - 1032 2002年09月 [査読有り][通常論文]
     
    Shiro-uo (ice goby; teleost fish), Leucopsarion petersii, shows a unique cleavage pattern characterized by two tires of blastomeres at 8-cell stage, like that of echinoderm and amphibian embryo. Such a pattern is suitable to isolation and cell lineage experiments. In this study, cell lineage of germ-line was traced by histological observation and cell labelling experiment at the 8-cell stage. Primordial germ cells (PGCs) were first detected histologically at the 10-somite stage, and migrated to gonadal anlage at 10 days post-fertilization, through usual way described in other teleost species. When a single blastomere was labelled with tracer dye at 8-cell stage, both upper and lower tires generated labelled PGCs at gonadal anlage although upper tires occasionally. This result suggests that all blastomeres at the 8-cell stage have potential to produce PGCs in shiro-uo.
  • S Otani, S Maegawa, K Inoue, K Arai, E Yamaha
    ZOOLOGICAL SCIENCE 19 5 519 - 526 2002年05月 [査読有り][通常論文]
     
    vas RNA has been identified in germ-line cells and its precursors in zebrafish, with the result that the germ-line lineage can be traced throughout embryogenesis. In the present study, we described vas localization and the migration of vas-positive cells in goldfish, using whole mount in situ hybridization. The signals of vas mRNA localization appeared at the marginal part of the first to third cleavage planes. The eight signals were detected during the period from the 8- cells to the 512-cell stage. At the late-blastula stage, additional numbers of vas-positive cells were observed, suggesting the proliferation of these cells. At the segmentation period, vas-positive cells showed a long extended distribution along the embryonic axis, but did not form any clusters. vas-positive cells were occasionally distributed at the head region, especially around the future otic vesicle. These signals were inherited to the primordial germ cells, suggesting that vas-positive cells were primordial germ cells (PGCs) in goldfish.
  • K Morishima, S Horie, E Yamaha, K Arai
    ZOOLOGICAL SCIENCE 19 5 565 - 575 2002年05月 [査読有り][通常論文]
     
    In Memanbetsu town, Hokkaido island, Japan, a high frequency of natural triploid loaches Misgurnus anguillicaudatus (7.4% on average) was detected by flow cytometry for relative DNA content. Among sympatric diploid females (n=6) from a single population, we found two unique females that laid unreduced diploid eggs. They gave normal diploid progeny even after induction of gynogenesis with genetically inert UV-irradiated sperm. When fertilized with normal loach sperm, some unreduced eggs developed into triploids, but the rest into diploids. Hybridization using goldfish Carassius auratus sperm gave both normal diploid loaches and inviable allotriploid hybrids possessing the diploid loach genome and the haploid goldfish genome. Microsatellite genotyping and DNA fingerprinting demonstrated that the diploid progeny developing from the unreduced eggs were genetically identical to the mother, while the triploids had some of the paternal DNA. These results indicate that the diploid eggs reproduced unisexually as a diploid clone and in other cases developed into triploids after accidental incorporation of sperm nucleus. The presence of at least one clonal line in this area was shown by the identical DNA fingerprint detected in five out of 17 diploid loaches examined.
  • カットスロートトラウトSalmo clarkiにみられた半数体-二倍体モザイク胚.
    山羽悦郎, 木村志津雄, 田中稔, 阪尾寿々, 藤本貴史, 荒井克俊
    水産育種 32 121 - 126 2002年 [査読有り][通常論文]
  • 山羽悦郎, 村上賢, 武山悟, 森島輝, 大谷哲, 堀江晋, 田中稔, 藤本貴史, 大嶋耕造, 阪尾寿々, 相田貴紀, 荒井克俊
    水産育種 32 1 19 - 26 水産育種研究会 2002年 [査読有り][通常論文]
  • Yamaha E, Otani S, Minami A, Arai K
    Fisheries Science 68 2 313 - 319 Japanese Society of Fisheries Science 2002年 [査読有り][通常論文]
  • T Saito, M Kamimoto, A Miyake, E Yamaha, T Suzuki, N Nakatsuji, T Nakatsuji
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 45 4 661 - 668 2001年06月 [査読有り][通常論文]
     
    Isolation of cleavage-stage blastomeres and the study of their developmental potential has been used extensively for analyzing the mechanisms of embryogenesis in vertebrates, including amphibians and echinoderms. We devised a method to isolate 8-cell stage blastomeres in the teleost, shiro-uo, by utilizing its unique cleavage pattern of the horizontal 3rd cleavage plane. Removal of all the upper blastomeres at the 8-cell stage allowed almost normal embryogenesis from the remaining lower blastomeres and yolk cell mass. Isolated upper or lower blastomeres formed vesicles and spherical bodies, which later showed morphological changes during cultivation. Mesoderm formation was detected not only in the cultivated lower blastomeres or whole blastomeres but also in the upper blastomeres isolated from the yolk cell mass at the 8-cell stage, although at a lower frequency than the lower blastomeres. These results indicated the presence of very early signaling for mesoderm induction, which is independent from the currently postulated signals from the yolk syncytial layer at later stages. This also indicated non-equivalence or differentiation of the blastomeres from the very early cleavage stage in teleost embryos.
  • E Yamaha, M Kazama-Wakabayashi, S Otani, T Fujimoto, K Arai
    GENETICA 111 1-3 227 - 236 2001年 [査読有り][通常論文]
     
    Germ-line chimerism was successfully induced by blastoderm transplantation from donor triploid crucian carp, which reproduces gynogenetically, to recipient diploid goldfish, which reproduces bisexually. Lower part of donor blastoderm including primordial germ cells (PGCs) was sandwiched between recipient blastoderm at the mid- to late-blastula stage. When donor grafts were prepared from intact embryos or ventralized ones by removing vegetal yolk hemisphere at the 1- to 2-cell stage, malformations including double axes were observed in the resultant chimeras transplanted with grafts from intact embryos at the hatching stage, while a few malformations in those from ventralized embryos. PGCs originated from donor grafts were observed around the gonadal anlage at 10 days post-fertilization in chimeras. When ploidy of erythrocytes and epidermal cells in chimeric fish was examined by flow-cytometry, no triploid cells were detected at 1- and 5-year-old chimeras. Three-year-old chimeric fish (n = 5) laid eggs originated from the donor together with those from the recipient. The frequency of eggs from the donor crucian carp blastoderm varied from 3.1 to 89.3% between chimeras.
  • 水野俊朗, 山羽悦郎
    タンパク質/核酸/酵素 45 2712 - 2719 17 Suppl 2000年12月 [査読無し][通常論文]
  • E Yamaha, T Mizuno, K Matsushita, Y Hasebe
    NIPPON SUISAN GAKKAISHI 65 4 709 - 717 1999年07月 [査読有り][通常論文]
     
    In order to contribute developmental technology, the early embryonic stages during the pre-gastrula were studied by cytological, histological or biochemical viewpoints in goldfish Carassius auratus. Synchronous cleavages occur 9 times every about 30 min from 50 min to 6 h post-fertilization at 20 degrees C, and, thereafter, transpose asynchronous phase, suggesting mid-blastula transition (MBT). This timing is staged as the mid-blastula. Genomic expression of mesodermal markers, goosecoid and no fail, are detected with in situ hybridization assay from 8 h post-fertilization. This timing is staged as the late-blastula. Cytoplasmic protrusions as cell movement are ovserved after MET, and autonomous mixing of blastomeres from the late-blastula stage. Deep cells were produced from the surface part of blastomeres and central part of yolk cell during the late-blastula stage. Marginal yolk syncytial layer forms during the mid-blastula stage, but central yolk syncytial layer forms after the late-blastula stage. This result suggests that each yolk syncytial layer has a different origin of nuclei.
  • T Mizuno, E Yamaha, A Kuroiwa, H Takeda
    MECHANISMS OF DEVELOPMENT 81 1-2 51 - 63 1999年03月 [査読有り][通常論文]
     
    To examine the nature of cytoplasm determinants for dorsal specification in zebrafish, we have developed a method in which we remove the vegetal yolk hemisphere of early fertilized eggs (vegetal removed embryos). When the vegetal yolk mass was removed at the I-cell stage, the embryos frequently exhibited typical ventralized phenotypes: no axial structures developed. The frequency of dorsal defects decreased when the operation was performed at later stages. Furthermore, the yolk cell obtained from the vegetal-removed embryos lost the ability to induce goosecoid in normal blastomeres while the normal yolk cell frequently did so in normal and vegetal-removed embryos. These results suggested that the vegetal yolk cell mass contains the dorsal determinants, and that the dorsal-inducing ability of the yolk cell is dependent on the determinants. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
  • M Alonso, A Fujiwara, E Yamaha, S Kimura, S Abe
    HEREDITAS 131 3 221 - 225 1999年 [査読有り][通常論文]
     
    Nucleolus-forming 5.8S, 18S and 28S ribosomal RNA gene (rDNA) loci were assigned by fluorescence in situ hybridization (FISH) to the distal half of the short arms of a large-sized submetacentric pair in the Alaskan char (Salvelinus malma) and to the distal region of the long arms of a medium-sized submetacentric pair in the chum salmon (Oncorhynchus keta), respectively. In each species, heteromorphic FISH signals, spanning whole satellite region and secondary constriction, imply an intraspecific variation in the size of rDNA loci. Size variation of silver-stained nucleolar organizer regions (AgNORs) was also apparent between or within the assigned rDNA loci in each species, suggesting a possible inter- or intralocus inactivation of rDNAs. C-band positivity of assigned rDNA loci and AgNORs unequivocally showed their association with heterochromatin in these species.
  • A Fujiwara, S Abe, E Yamaha, F Yamazaki, MC Yoshida
    CHROMOSOME RESEARCH 6 6 463 - 471 1998年09月 [査読有り][通常論文]
     
    Ribosomal RNA gene (rDNA) loci, including those of nucleolus-forming 18S, 5.8S and 28S (major) and non-nucleolus-forming 5S (minor) rDNA, were assigned using fluorescence in situ hybridization (FISH) to the embryonic chromosomes of rainbow trout (Oncorhynchus mykiss), masu salmon (O. masou), brook trout (Salvelinus fontinalis) and Japanese huchen (Hucho perryi). In these species, the minor rDNA loci were located basically on 2-4 chromosome pairs, whereas the major rDNA loci were found essentially on one chromosome pair, except for the brook trout. Its major rDNA loci were dispersed on about half of the chromosome complement, showing a considerable interindividual variation in the number and location. The major and minor rDNA loci were separated onto different chromosomes in the examined species, except for the rainbow trout, in which one chromosome pair had tandemly aligned minor and major rDNA loci. Chromosome regions containing both kinds of rDNA loci in each species were found to be stained with C-banding, showing an association of these loci with heterochromatin. Comparison of the assigned major rDNA loci and sequentially detected silver (Ag)-stained nucleolar organizer regions (AgNORs) in all the species revealed a considerable polymorphism in the number and size of AgNORs among or within those loci, suggesting a possible inter- or intralocus inactivation of the major rDNAs.
  • E Yamaha, T Mizuno, Y Hasebe, H Takeda, F Yamazaki
    DEVELOPMENT GROWTH & DIFFERENTIATION 40 3 267 - 275 1998年06月 [査読有り][通常論文]
     
    The teleost dorsoventral axis cannot be morphologically distinguished before gastrulation. Previous studies by the current authors have shown that localized dorsalizing activity in the yolk cell (YC) induces the dorsal tissues in the overlying blastoderm. In order to examine whether or not dorsal blastomeres are committed to their dorsal fate before the gastrula stage, a variety of transplant operations were performed in goldfish blastoderms at the mid- to late-blastula stages. When the blastoderm was cut from the YC, rotated horizontally at 180 degrees, and recombined with the YC, the blastoderm frequently developed two axes, indicating that dorsal blastomeres of the blastula had already acquired the ability to differentiate into the organizer in the absence of dorsalizing signals from the YC. This result was further confirmed by experiments using ventralized embryos in which no dorsal structures formed. the axis formation was frequently observed in the normal blastoderm combined with the ventralized YC at the blastula stage. However, the axes formed in the absence of dorsal information from the YC exhibited a lower dorso-anterior index. Furthermore the dorsal specification was not stably maintained when the dorsal cells were located far from the YC. These results suggest that the inductive and permissive influence of the YC may be required for the blastoderm to undergo full dorsal differentiation.
  • E Yamaha, T Mizuno, Y Hasebe, F Yamazaki
    FISHERIES SCIENCE 63 4 514 - 519 1997年08月 [査読有り][通常論文]
     
    Chimeric fish were produced by exchanging the upper halves of blastoderms at the blastula stage between goldfish-common carp hybrids with pigmented scales and goldfish with clear scales, or between goldfish with clear scales and triploid crucian carp with pigmented scales. Blastoderm transplantation was carried out in Ringer's solution containing 1.6% albumen within one minute after isolation of the blastoderm. In the absence of albumen, the transplantation was not successful. The larvae and young fish were confirmed to be chimeric fish, as transplanted blastoderm cells could be detected histologically and as the fish exhibited phenotypes typical of both fish used to produce the chimeras. Blastomere mixing between the upper exchanged and lower halves of blastoderms occurred but not to a great extent, and transplanted cells from the upper halves of blastoderms labeled with biotin were mainly distributed in the head region.
  • A Fujiwara, S Abe, E Yamaha, F Yamazaki, MC Yoshida
    CHROMOSOMA 106 1 44 - 52 1997年06月 [査読有り][通常論文]
     
    Chromosome elimination through chromosome loss and partial deletion is known to be one of the causes of embryonic inviability in some salmonid interspecific hybrids. Using fluorescence in situ hybridization and related techniques, including whole chromosome painting and comparative genomic hybridization, parental origin of eliminated chromosomes was identified in the inviable hybrids between masu salmon (Ms, Oncorhynchus masou) female and rainbow trout (Rb, O. mykiss) male at the early embryonic stage prior to death. In these hybrids, the haploid Rb chromosome number decreased to nearly half, whereas the Ms chromosomes were retained as one or occasionally two full haploid complements. The Rb chromosomes were also involved in the frequently observed fragments and micronuclei. Whereas the occurrence of fragments was constant throughout the observed period, chromosome loss occurred mainly from just after fertilization to the blastulae stage. In tissue sections and cell spreads of late blastula, some Rb chromosomes were trapped in the midzone from ana- to telophase, resulting in micronuclei at the subsequent interphase. Micronuclei and mitotic abnormalities were also observed in the androgenetic haploid hybrids. However, such abnormalities were seldom or never observed in the viable reciprocal hybrids. The present findings suggest that the paternal Rb chromosomes in the inviable hybrids are preferentially eliminated through mitotic abnormalities during early embryogenesis, owing to a possible incompatibility between the maternal Ms cytoplasm and paternal Rb genome.
  • M Suzuki, E Yamaha, F Yamazaki
    FISHERIES SCIENCE 63 3 474 - 475 1997年06月 [査読有り][通常論文]
  • K. Inoue, K. Inoue, S. Odo, T. Noda, S. Nakao, S. Takeyama, E. Yamaha, F. Yamazaki, S. Harayama
    Marine Biology 128 1 91 - 95 1997年04月01日 [査読無し][通常論文]
     
    The adhesive protein allele of mussels collected at 13 points in Japan from Hokkaido to Kyushu was analyzed by the polymerase chain reaction using a set of primers which amplifies a part of the nonrepetitive region of the adhesive protein gene. While most mussels exhibited a 126 bp fragment, characteristic of the pure Mytilus galloprovincialis, 55 of 64 mussels sampled at Hiura and 1 of 14 mussels at Hakodate Port exhibited 168 and 126 bp fragments. Sequence analysis of the two fragments indicated that the 168 and 126 bp fragments are almost identical to previously reported sequences in M. trossulus and M. galloprovincialis, respectively. Since the frequency of heterozygous individuals in Hiura is very high, it is unlikely that they are simple hybrids. However, it is evident that mixing of genes occurred between the two species off Hokkaido.
  • K Inoue, Y Takeuchi, S Takeyama, E Yamaha, F Yamazaki, S Odo, S Harayama
    JOURNAL OF MOLECULAR EVOLUTION 43 4 348 - 356 1996年10月 [査読有り][通常論文]
     
    cDNA encoding the adhesive protein of the mussel Mytilus coruscus (Mcfp1) was isolated. The coding region encoded 848 amino acids (a.a.) comprising the 20-a.a. signal peptide, the 21-a.a. nonrepetitive linker, and the 805-a.a. repetitive domain. Although the first 204 nucleotides and the 3'-untranslated region of Mcfp1 cDNA were homologous to corresponding parts of M. galloprovincialis adhesive protein (Mgfp1) cDNA, the other parts diverged. The representative repeat motif of the repetitive domain, YKPK(I/P)(S/T)YPP(T/S), was similar but slightly different from the repeat motif of Mgfp1. The codon usage patterns for the same amino acids were different in different positions of the decapeptide motif. Almost identical nucleotide sequences encoding the two to 13 repeats appeared several times in the repetitive region, which suggests that the adhesive protein genes of mussels have evolved through the duplication of these repeat units.
  • Toshiro Mizuno, Etsuro Yamaha, Masami Wakahara, Atsushi Kuroiwa, Hiroyuki Takeda
    Nature 383 6596 131 - 132 1996年09月 [査読有り]
  • T INOKUCHI, S ABE, E YAMAHA, F YAMAZAKI, MC YOSHIDA
    HEREDITAS 121 3 255 - 265 1994年 [査読有り][通常論文]
     
    The availability of chromosome replication banding for fish karyotype analysis was investigated in early embryos of three salmonid species: rainbow trout (Oncorhynchus mykiss), masu salmon (O. masou), and chum salmon (O. keta), at around 100 to 110 thermal units, after exposure to 5-bromodeoxyuridine (BrdU). The optimum conditions for obtaining reproducible replication bands were BrdU treatment of three to five embryos at the final concentration of 400 to 700 mu g/ml in 10 ml physiological saline, in the presence of 10(-5) M fluorodeoxyuridine and 10(-4) M deoxycytidine, for 2 to 2.5 days at 5 degrees C in the dark. Under these conditions, distinct banding patterns were obtained in the chromosomes of all three species. The karyotype of each species was subsequently determined. In addition, chromosome number variation, observed in one diploid embryo with 61 chromosomes and two haploid ones with 31 chromosomes of rainbow trout, was shown to be due to a Robertsonian centric fission of one biarmed chromosome, although the affected chromosome was not the same in each embryo. The present findings suggest that the BrdU replication banding in the chromosomes of early embryos is a promising technique in fish karyotype analysis, in terms of its technical simplicity and a high reproducibility to induce distinct chromosome bands.
  • E YAMAHA, F YAMAZAKI
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 37 2 291 - 298 1993年06月 [査読有り][通常論文]
     
    In this study, we report the induction of cell fusion between two fertilized eggs and viable chimeric fish from the fused egg in goldfish. Chemical fusion by polyethylene glycol between denuded fertilized egg induced egg adhesion but rarely egg fusion. This treatment seemed not suitable for egg fusion, because of tight adhesion between vitelline membrane and surrounding matrix. Electrically fertilized egg fusion was successfully induced at the rate of almost 100% under conditions of 400V/cm, 1 MHz dielectrophoresis pulse for 3 seconds and a successive, 750V/cm, 10 musec fusion pulse. The treatment had to be applied to the egg in the calcium-free low electrolyte medium within 20 min of fertilization at 20-degrees-C. Small numbers of fused eggs developed into young chimeric fish, and the different cells originating from two eggs survived together as an individual.
  • K YAMANO, E YAMAHA, F YAMAZAKI
    NIPPON SUISAN GAKKAISHI 54 9 1477 - 1481 1988年09月 [査読有り][通常論文]
  • E YAMAHA, K USUI, H ONOZATO, K HAMADA
    BULLETIN OF THE JAPANESE SOCIETY OF SCIENTIFIC FISHERIES 52 11 1929 - 1934 1986年11月 [査読有り][通常論文]
  • H ONOZATO, E YAMAHA
    BULLETIN OF THE JAPANESE SOCIETY OF SCIENTIFIC FISHERIES 49 5 693 - & 1983年 [査読有り][通常論文]

書籍

  • 稲葉, 一男, Hall-Spencer, Jason M. (担当:分担執筆範囲:Chapter 11 Development of Marine Fish: Several Preocedures for the Observation of Embryonic Development p.125-148)
    Springer Nature Singapore Pte Ltd 2020年04月 (ISBN: 9789811513251) xvi, 367 p.
  • 魚類学の百科事典
    山羽 悦郎 (担当:分担執筆範囲:第8章4−5 胚 - 受精から三胚葉形成, - エピボリー運動から胚体形成)
    丸善出版 2018年
  • 魚類発生学の基礎
    山羽悦郎, 荒井克俊 (担当:分担執筆範囲:第4章 初期胚発生)
    恒星社厚生閣 2018年
  • 水産遺伝育種学
    荒井克俊, 藤本貴史, 山羽悦郎 (担当:分担執筆範囲:第6章 染色体操作と育種)
    東北大学出版会 2017年
  • 帰山 雅秀, 永田 光博, 中川 大介 
    北海道大学出版会 2013年 (ISBN: 9784832982109)
  • 阿部 周一, 矢部 衞, 永田 光博, 帰山 雅秀, 桜井 泰憲, 上田 宏, 伴 真俊, 安東 宏徳, 浦野 明央, 佐藤 俊平, 荒井 克俊, 山羽 悦郎, 吉水 守, 笠井 久会, 高橋 是太郎, 天野 哲也, 鍛治 明香 
    北海道大学出版会 2009年 (ISBN: 9784832981935)
  • 初期胚の細胞質の機能
    水産育種に関わる形質の発現と評価(藤尾・谷口編)水産学シリーズ117.恒星社厚生閣 1998年

その他活動・業績

  • 黒田真道, 柴田季子, 村上賢, 山羽悦郎, 藤本貴史, 荒井克俊 日本水産学会大会講演要旨集 2019 95 2019年03月26日 [査読無し][通常論文]
  • 黒田真道, 柴田季子, 村上賢, 山羽悦郎, 藤本貴史, 荒井克俊 日本水産学会大会講演要旨集 2019 94 2019年03月26日 [査読無し][通常論文]
  • 遠藤 充, 山羽 悦郎, 藤本 貴史 日本繁殖生物学会 講演要旨集 111 (0) AW1 -4-AW1-4 2018年 [査読無し][通常論文]
     
    <p>【目的】魚類では異種間交雑や倍数性操作により<u>核ゲノム構成を人為的に制御</u>し,不妊個体が作出されてきた。一方,哺乳類ではミトコンドリアヘテロプラスミーの顕在化による雄性不妊が報告されている。魚類では<u>ヘテロプラスミーと生殖特性の関係</u>についての知見が少なく,細胞質に起因する妊性の変化を解明できれば,新たな不妊化技術となる可能性がある。本研究では,ヘテロプラスミーが配偶子形成に与える影響の調査に向け,ゼブラフィッシュ受精卵へキンギョミトコンドリアを移植し,人為的ヘテロプラスミー誘起を試みた。【方法】キンギョ未受精卵から単離したミトコンドリアをMitoTracker Green FMで蛍光染色した。このキンギョ由来ミトコンドリアを,人工授精により作出したゼブラフィッシュの1細胞期胚に顕微注入し,ミトコンドリア移植を行った。32個体の移植胚を個別培養し,生残率,奇形率を調べるとともに,蛍光顕微鏡によりキンギョ由来ミトコンドリアの動態を観察した。また,8細胞期から孵化期までの各胚発生段階におけるキンギョmtDNAの存否を,種判別プライマーを用いたPCRにより調査した。プライマーはゼブラフィッシュとキンギョのミトコンドリア遺伝子<i>atp6</i>に対して種特異的に増幅するように設計した。【結果】受精72時間後の孵化期における移植胚の生残率と奇形率は81.3%,11.1%であり,無処理胚(83.3%,5.7%)と比較して有意差はなかった。移植胚では,キンギョ由来ミトコンドリアを示す蛍光シグナルが胚発生の進行とともに胚体全体へ拡散した。また,移植胚のすべての胚発生段階でゼブラフィッシュとキンギョのmtDNAが検出された。以上のように,<u>移植したキンギョmtDNAは胚発生を通して存在</u>したため,本研究のミトコンドリア移植は,<u>魚類のヘテロプラスミー研究の良いモデル</u>であると考えられた。今後,ヘテロプラスミー個体での始原生殖細胞の分化や配偶子形成の解析により,ヘテロプラスミーが生殖特性へ与える影響を明らかにできると考えられる。</p>
  • 萩原聖士, 須山喜市, 東典子, 堤尚信, 西尾朋高, 市村政樹, 三坂尚行, 鈴木渉太, 宮城大助, 古市明文, 市川卓, 松原創, 川崎琢真, 宗原弘幸, 高橋英祐, 山羽悦郎, 井尻成保, 足立伸次 日本水産学会大会講演要旨集 2016 16 2016年03月26日 [査読無し][通常論文]
  • Japanese eel propagation and hybridisation of eel species by using cryopreserved sperm (Anguilla japonica ♀×A. anguilla♂)
    Müller T, Matsubara H, Kubara Y, Horváth Á, Urbányi B, Horváth L, Asturiano J F, Peñaranda D, Adachi S, Arai K, Yamaha E Aquaculture 2012 abstract 378 -378 2012年09月 [査読無し][通常論文]
     
    ヨーロッパウナギ精子とニホンウナギ卵による雑種を作製し、その方法を紹介した。(Müller T*, Matsubara H*, Kubara Y, Horváth Á, Urbányi B, Horváth L, Asturiano J F, Peñaranda D, Adachi S, Arai K, Yamaha E )Japanese
    eel propagation and hybridisation of eel species by using cryopreserved sperm (<I>Anguilla japonica </I>♀×<I>A. anguilla</I>♂). Aquaculture 2012, Prague, Czech Republic. abstract no: 378.ポスター発表
  • Eel hybridisation: cryopreserved sperm as tool for ex-situ conservation of Anguilla anguilla .
    Müller T, Matsubara H, Kubara Y, Horváth Á, Urbányi B, Horváth L, Asturiano J F, Peñaranda D, Adachi S, Arai K, Yamaha E Annual meeting of IUCN-SSC/Wetlands International Freshwater Fish Specialist Group 12 -13 2012年05月 [査読無し][通常論文]
     
    ヨーロッパウナギ精子とニホンウナギ卵による雑種を作製し、その方法を紹介した。(Müller T*, Matsubara H*, Kubara Y, Horváth Á, Urbányi B, Horváth L, Asturiano J F, Peñaranda D, Adachi S, Arai K, Yamaha E )Annual meeting of IUCN-SSC/Wetlands International Freshwater Fish Specialist Group. 1-3 May 2012,Chester, UK., abstract book pp. 12-13.ポスター発表
  • Artificial hybridisation betwen Japanese eel females (Anguilla japonica) and European eel males (Anguilla anguilla ) by using cryopreserved sperm. .
    Müller T, Matsubara H, Kubara Y, Horváth Á, Urbányi B, Horváth L, Asturiano J F, Peñaranda D, Adachi S, Arai K, Yamaha E 1st PRO-EEL Collaborative Workshop 2012年03月 [査読無し][通常論文]
     
    ヨーロッパウナギの凍結精子をニホンウナギ卵と交雑させ、雑種を作製、その形態を調べた。(Müller T*, Matsubara H*, Kubara Y, Horváth Á, Urbányi B, Horváth L, Asturiano J F, Peñaranda D, Adachi S, Arai K, Yamaha E )1st PRO-EEL Collaborative Workshop 27th March 2012, Valencia, Spain ポスター発表
  • 阪尾 寿々, 藤本 貴史, 小林 輝正, 吉崎 悟朗, 山羽 悦郎, 荒井 克俊 日本水産學會誌 = Bulletin of the Japanese Society of Scientific Fisheries 76 (4) 581 -581 2010年07月15日
  • Cutting edge of chromosome manipulation for aquaculture and conservation of salmonids
    Katsutoshi Arai, Suzu Sakao, Takafumi Fujimoto, Etsuro Yamaha Journal of the National Taiwan Museum Special Publication (14) 49 -59 2010年03月 [査読有り][通常論文]
  • 藤本貴史, 吉川廣幸, 荒井克俊, 山羽悦郎 日本動物学会大会予稿集 79th 127 2008年08月20日 [査読無し][通常論文]
  • 藤本貴史, YASUI George Shigueki, 吉川廣幸, 山羽悦郎, 荒井克俊 日本水産学会大会講演要旨集 2008 245 2008年03月27日 [査読無し][通常論文]
  • 藤本貴史, YASUI George Shigeki, 吉川廣幸, 山羽悦郎, 荒井克俊 日本水産学会大会講演要旨集 2007 5 2007年09月25日 [査読無し][通常論文]
  • 藤本貴史, 吉川廣幸, 森島輝, 山羽悦郎, 荒井克俊 日本水産学会大会講演要旨集 2007 108 2007年03月28日 [査読無し][通常論文]
  • 長井輝美, 斎藤大樹, 吉川智仁, 大谷哲, 前川真吾, 井上邦夫, 荒井克俊, 山羽悦郎 日本水産学会大会講演要旨集 2003 130 2003年04月01日 [査読無し][通常論文]
  • 斎藤大樹, 吉川智仁, 長井輝美, 大谷哲, 藤本貴史, 相田貴紀, 井上邦夫, 前川真吾, 山羽悦郎 日本水産学会大会講演要旨集 2003 130 2003年04月01日 [査読無し][通常論文]
  • T Nagai, E Yamaha, K Arai ZOOLOGICAL SCIENCE 18 (2) 215 -223 2001年03月 [査読無し][通常論文]
     
    In this study, primordial germ cells (PGCs) in zebrafish were described histologically using eosinophilic granules as a marker. PGC-like cells (PL-cells) with eosinophilic granules were identified initially at the sphere stage (4-hr postfertilization), and were observed until the bud stage, the earliest stage to which PGCs with proper morphology could be traced. The morphology and distribution of eosinophilic granules in PL-cells changed during epiboly. Mitoses of PL-cells were observed only from the shield to bud stage, after eosinophilic granules aggregated to the perinuclear region in PGCs. These shifts of eosinophilic granules corresponded histologically to those of germ plasm described in Xenopus. These results suggest that eosinophilic granules represent germ plasm in fish and that PL-cells with these granules correspond to the PGCs or presumptive PGCs (pPGCs).
  • 大谷哲, 前川真吾, 井上邦夫, 荒井克俊, 山羽悦郎 日本動物学会大会予稿集 71st 57 2000年09月 [査読無し][通常論文]
  • 長井輝美, 大谷哲, 前川真吾, 井上邦夫, 荒井克俊, 山羽悦郎 日本動物学会大会予稿集 70th 37 1999年09月27日 [査読無し][通常論文]
  • M Kazama-Wakabayashi, E Yamaha, F Yamazaki FISHERIES SCIENCE 65 (4) 577 -582 1999年08月 [査読無し][通常論文]
     
    The origin and translocation of primordial germ cells (PGCs) in goldfish Carassius auratus were studied, using both histological observations during embryonic development and by following the effects of partial elimination and duplication of blastoderm on the numbers of PGCs at the mid-blastula stage. Goldfish PGCs are distinguished from somatic cells by their large size, distinct outline and large nucleus, because they are morphologically identical with those of the other fish species. They were located in the gonadal anlage at ten days post-fertilization, and were traced back to 30-hour post-fertilization (hpf) embryos with seven to nine somites. They were distributed widely in an embryo at 30 hpf, except for brain, spinal cord, notochord and the enveloping layer. The numbers of PGCs did not increase from 30 hours to ten days post-fertilization. When the lower part of the blastoderm was eliminated at the mid-blastula stage, the number of PGCs decreased at the gonadal anlage. But, elimination of the upper part at same stage did not effect the number of PGCs. Moreover, the number of PGCs increased in the duplicated embryos, in which a normal blastoderm was transplanted onto a host embryo from which the upper part of the blastoderm had been removed. These results suggest that the PGCs-bearing-cells could be predetermined by cytoplasmic factor and were already distributed in the lower part of blastoderm at the mid-blastula stage, and that they can not regenerate after that.
  • 山羽悦郎, 風間真紀, 大谷哲, 長井輝美 月刊海洋 31 (5) 266 -271 1999年05月01日 [査読無し][通常論文]
  • 山羽 悦郎, Etsuro Yamaha, 北海道大学水産学部, Faculty of Fisheries Hokkaido University 日本水産学会誌 = Bulletin of the Japanese Society of Scientific Fisheries 65 (2) 315 -315 1999年03月15日
  • T Mizuno, E Yamaha, A Kuroiwa, H Takeda MECHANISMS OF DEVELOPMENT 81 (1-2) 51 -63 1999年03月 [査読無し][通常論文]
     
    To examine the nature of cytoplasm determinants for dorsal specification in zebrafish, we have developed a method in which we remove the vegetal yolk hemisphere of early fertilized eggs (vegetal removed embryos). When the vegetal yolk mass was removed at the I-cell stage, the embryos frequently exhibited typical ventralized phenotypes: no axial structures developed. The frequency of dorsal defects decreased when the operation was performed at later stages. Furthermore, the yolk cell obtained from the vegetal-removed embryos lost the ability to induce goosecoid in normal blastomeres while the normal yolk cell frequently did so in normal and vegetal-removed embryos. These results suggested that the vegetal yolk cell mass contains the dorsal determinants, and that the dorsal-inducing ability of the yolk cell is dependent on the determinants. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
  • 山羽悦郎, 水野寿朗, 松下健, 長谷部優 日本水産学会誌 65 (4) 709 -717 1999年 [査読無し][通常論文]
     
    In order to contribute developmental technology, the early embryonic stages during the pre-gastrula were studied by cytological, histological or biochemical viewpoints in goldfish Carassius auratus. Synchronous cleavages occur 9 times every about 30 min from 50 min to 6 h post-fertilization at 20°C, and, thereafter, transpose asynchronous phase, suggesting mid-blastula transition (MBT). This timing is staged as the mid-blastula. Genomic expression of mesodermal markers, goosecoid and no tail, are detected with in situ hybridization assay from 8 h post-fertilization. This timing is staged as the late-blastula. Cytoplasmic protrusions as cell movement are ovserved after MBT, and autonomous mixing of blastomeres from the late-blastula stage. Deep cells were produced from the surface part of blastomeres and central part of yolk cell during the late-blastula stage. Marginal yolk syncytial layer forms during the mid-blastula stage, but central yolk syncytial layer forms after the late-blastula stage. This result suggests that each yolk syncytial layer has a different origin of nuclei.
  • M Alonso, A Fujiwara, E Yamaha, S Kimura, S Abe HEREDITAS 131 (3) 221 -225 1999年 [査読無し][通常論文]
     
    Nucleolus-forming 5.8S, 18S and 28S ribosomal RNA gene (rDNA) loci were assigned by fluorescence in situ hybridization (FISH) to the distal half of the short arms of a large-sized submetacentric pair in the Alaskan char (Salvelinus malma) and to the distal region of the long arms of a medium-sized submetacentric pair in the chum salmon (Oncorhynchus keta), respectively. In each species, heteromorphic FISH signals, spanning whole satellite region and secondary constriction, imply an intraspecific variation in the size of rDNA loci. Size variation of silver-stained nucleolar organizer regions (AgNORs) was also apparent between or within the assigned rDNA loci in each species, suggesting a possible inter- or intralocus inactivation of rDNAs. C-band positivity of assigned rDNA loci and AgNORs unequivocally showed their association with heterochromatin in these species.
  • A Fujiwara, S Abe, E Yamaha, F Yamazaki, MC Yoshida CHROMOSOME RESEARCH 6 (6) 463 -471 1998年09月 [査読無し][通常論文]
     
    Ribosomal RNA gene (rDNA) loci, including those of nucleolus-forming 18S, 5.8S and 28S (major) and non-nucleolus-forming 5S (minor) rDNA, were assigned using fluorescence in situ hybridization (FISH) to the embryonic chromosomes of rainbow trout (Oncorhynchus mykiss), masu salmon (O. masou), brook trout (Salvelinus fontinalis) and Japanese huchen (Hucho perryi). In these species, the minor rDNA loci were located basically on 2-4 chromosome pairs, whereas the major rDNA loci were found essentially on one chromosome pair, except for the brook trout. Its major rDNA loci were dispersed on about half of the chromosome complement, showing a considerable interindividual variation in the number and location. The major and minor rDNA loci were separated onto different chromosomes in the examined species, except for the rainbow trout, in which one chromosome pair had tandemly aligned minor and major rDNA loci. Chromosome regions containing both kinds of rDNA loci in each species were found to be stained with C-banding, showing an association of these loci with heterochromatin. Comparison of the assigned major rDNA loci and sequentially detected silver (Ag)-stained nucleolar organizer regions (AgNORs) in all the species revealed a considerable polymorphism in the number and size of AgNORs among or within those loci, suggesting a possible inter- or intralocus inactivation of the major rDNAs.
  • E Yamaha, T Mizuno, Y Hasebe, H Takeda, F Yamazaki DEVELOPMENT GROWTH & DIFFERENTIATION 40 (3) 267 -275 1998年06月 [査読無し][通常論文]
     
    The teleost dorsoventral axis cannot be morphologically distinguished before gastrulation. Previous studies by the current authors have shown that localized dorsalizing activity in the yolk cell (YC) induces the dorsal tissues in the overlying blastoderm. In order to examine whether or not dorsal blastomeres are committed to their dorsal fate before the gastrula stage, a variety of transplant operations were performed in goldfish blastoderms at the mid- to late-blastula stages. When the blastoderm was cut from the YC, rotated horizontally at 180 degrees, and recombined with the YC, the blastoderm frequently developed two axes, indicating that dorsal blastomeres of the blastula had already acquired the ability to differentiate into the organizer in the absence of dorsalizing signals from the YC. This result was further confirmed by experiments using ventralized embryos in which no dorsal structures formed. the axis formation was frequently observed in the normal blastoderm combined with the ventralized YC at the blastula stage. However, the axes formed in the absence of dorsal information from the YC exhibited a lower dorso-anterior index. Furthermore the dorsal specification was not stably maintained when the dorsal cells were located far from the YC. These results suggest that the inductive and permissive influence of the YC may be required for the blastoderm to undergo full dorsal differentiation.
  • A Fujiwara, S Abe, E Yamaha, F Yamazaki, MC Yoshida CHROMOSOMA 106 (1) 44 -52 1997年06月 [査読無し][通常論文]
     
    Chromosome elimination through chromosome loss and partial deletion is known to be one of the causes of embryonic inviability in some salmonid interspecific hybrids. Using fluorescence in situ hybridization and related techniques, including whole chromosome painting and comparative genomic hybridization, parental origin of eliminated chromosomes was identified in the inviable hybrids between masu salmon (Ms, Oncorhynchus masou) female and rainbow trout (Rb, O. mykiss) male at the early embryonic stage prior to death. In these hybrids, the haploid Rb chromosome number decreased to nearly half, whereas the Ms chromosomes were retained as one or occasionally two full haploid complements. The Rb chromosomes were also involved in the frequently observed fragments and micronuclei. Whereas the occurrence of fragments was constant throughout the observed period, chromosome loss occurred mainly from just after fertilization to the blastulae stage. In tissue sections and cell spreads of late blastula, some Rb chromosomes were trapped in the midzone from ana- to telophase, resulting in micronuclei at the subsequent interphase. Micronuclei and mitotic abnormalities were also observed in the androgenetic haploid hybrids. However, such abnormalities were seldom or never observed in the viable reciprocal hybrids. The present findings suggest that the paternal Rb chromosomes in the inviable hybrids are preferentially eliminated through mitotic abnormalities during early embryogenesis, owing to a possible incompatibility between the maternal Ms cytoplasm and paternal Rb genome.
  • Chimeric fish produced by blastoderm exchange in goldfish, ┣DBCarassius(/)-┫DB ┣DBauratus(/)-┫DB. (共著)
    Fisheries Science 63 514 -519 1997年 [査読無し][通常論文]
  • T Mizuno, E Yamaha, F Yamazaki DEVELOPMENT GENES AND EVOLUTION 206 (6) 389 -396 1997年01月 [査読無し][通常論文]
     
    The teleost dorsoventral axis cannot be distinguished morphologically before gastrulation. In order to examine whether the yolk cell affects axis determination, we bisect early cleavage embryos of the goldfish, Carassius auratus. When the vegetal yolk hemisphere is removed by bisection along the equatorial plane at the 2-cell stage, the embryos develop abnormally and exhibit a symmetrical morphology. No dorsal structures, such as notochord, somites and neural tube, differentiate and no embryonic shield is formed during gastrulation. In addition, no goosecoid mRNA is expressed before gastrulation. The frequency of abnormality decreases as the age at which the vegetal yolk hemisphere is removed increases. Most embryos removed at the 32-cell stage develop normally. Their morphological phenotype is similar to that of a Xenopus ventralized embryo generated by ultraviolet irradiation on the vegetal hemisphere soon after fertilization. We also observed that, when the embryos were bisected along the first cleavage plane at the 2-cell stage, the proportion of pairs of embryos of which one embryo developed normally was 44.8%. These results indicate that the vegetal yolk hemisphere of the early cleavage embryo of the goldfish contains axis determination factor(s), which are necessary for generation of dorsal structures. Furthermore, it is suggested that these determinant(s) are distributed asymmetrically within the vegetal yolk hemisphere.
  • 山羽 悦郎 水産育種 (23) 13 -20 1996年10月
  • E YAMAHA, F YAMAZAKI INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 37 (2) 291 -298 1993年06月 [査読無し][通常論文]
     
    In this study, we report the induction of cell fusion between two fertilized eggs and viable chimeric fish from the fused egg in goldfish. Chemical fusion by polyethylene glycol between denuded fertilized egg induced egg adhesion but rarely egg fusion. This treatment seemed not suitable for egg fusion, because of tight adhesion between vitelline membrane and surrounding matrix. Electrically fertilized egg fusion was successfully induced at the rate of almost 100% under conditions of 400V/cm, 1 MHz dielectrophoresis pulse for 3 seconds and a successive, 750V/cm, 10 musec fusion pulse. The treatment had to be applied to the egg in the calcium-free low electrolyte medium within 20 min of fertilization at 20-degrees-C. Small numbers of fused eggs developed into young chimeric fish, and the different cells originating from two eggs survived together as an individual.
  • A preliminary study on Brd U reprication banding in the chromosomes of Chum salmon, ┣DBOncorhynchus(/)-┫DB ┣DBkata(/)-┫DB. 共著
    Chromosome Information Service 54,30-32 1993年 [査読無し][通常論文]
  • E YAMAHA, M MATSUOKA, F YAMAZAKI NIPPON SUISAN GAKKAISHI 54 (11) 2043 -2043 1988年11月 [査読無し][通常論文]
  • E YAMAHA, H ONOZATO, F YAMAZAKI NIPPON SUISAN GAKKAISHI 54 (3) 537 -537 1988年03月 [査読無し][通常論文]

共同研究・競争的資金等の研究課題

  • 魚類致死性雑種の発生工学的利用
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2021年07月 -2023年03月 
    代表者 : 山羽 悦郎
  • in vivo 選抜育種による魚類育種の加速化実現
    日本学術振興会:科学研究費(基盤研究(A))
    研究期間 : 2016年 -2020年 
    代表者 : 山羽 悦郎
  • 魚類の交雑に起因する特異な発生・生殖の分子機構解明と育種応用
    日本学術振興会:科学研究費(基盤研究(A))
    研究期間 : 2015年 -2018年 
    代表者 : 荒井克俊
  • 雌/非還元型、雄/還元型の配偶子形成をする四倍体フナの減数分裂機構
    日本学術振興会:科学研究費(挑戦的萌芽研究)
    研究期間 : 2015年 -2016年 
    代表者 : 荒井克俊
  • 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
    日本学術振興会:科学研究費(基盤研究(A))
    研究期間 : 2012年 -2016年 
    代表者 : 足立伸次
  • UV照射等の卵核遺伝的不活性化を用いない新規雄性発生誘起法の開発と応用
    日本学術振興会:科学研究費(基盤研究(B))
    研究期間 : 2012年 -2014年 
    代表者 : 荒井克俊
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2012年 -2012年 
    代表者 : 山羽 悦郎
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2012年 -2012年 
    代表者 : 山羽 悦郎
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2009年 -2011年 
    代表者 : 荒井 克俊, 足立 伸次, 山羽 悦郎
     
    ミカドチョウザメはゲノムサイズ(8.2pg/N)から見て、進化的な四倍体種と推定された。そして、人工受精に従来する子孫に上記のDNA量を持つ遺伝的な二倍体(2n)のみならず、三倍体(3n)が高頻度で、また、四倍体(4n)が少数生じた。従って、現在もゲノム重複をもたらす染色体倍加が自然に生じている。そこで、倍加機構解明に資するため、予めフローサイトメトリーで倍数性を判定した2n、3n仔魚について細胞遺伝学的な解析を実施した。その結果、2nは250-280の、3nは360-402の染色体数を示し、2n=268細胞の核型は80M/SM(中部/次中部着糸型染色体)+48T(端部着糸型染色体)+140m(微小染色体)、3n=402細胞の核型は120M/SM+72T+210mであった。5.8S+28SrDNAをプローブとしたFISHを行ったところ、2nでは最大18、3nでは最大27のシグナルが検出された。以上の結果より、三倍体は1組過剰のゲノム(染色体セット)を有することが判明した。シロチョウザメ二倍体(8.4pg/N)と自然三倍体(13.2pg/N)の14歳魚卵巣について組織学的観察を行ったところ、周辺仁期卵母細胞が見られ、三倍体の妊性が示唆された。倍加原因を探るためミカドチョウザメの精子について透過型電子顕微鏡観察を行ったところ、他のチョウザメと同様に先体(acrosome)を有し、...
  • 文部科学省:科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2009年 -2010年 
    代表者 : 山羽 悦郎, 本村 泰三
     
    本申請では、発生工学の材料であるPGCを産業的に重要な魚種でも使えるようにするために、生殖細胞質を利用し人為的にPGCを誘導することを目的とし魚類の生殖細胞質に関する研究を進めている。生殖細胞質の因子と考えられているゼブラフィッシュBucky BallとGFPを結合したコンストラクトのmRNA(Buc-GFP)を異種キンギョ、タナゴ、ウナギ、マツカワの4魚種の受精卵へ顕微注入し、その発現を明らかにした。(1)キンギョでは、第1~3卵割期の卵割面にGFPのシグナルが確認された。128~胞胚期に胚盤周囲で蛍光顆粒をもった割球が確認された。(2)タナゴでは、8~16細胞期から卵割面の境界に蛍光シグナルが認められた。64~128細胞期の一部の胚で、胚盤の周囲には分布しない割球間での蛍光の顆粒の局在を観察した。(3)ウナギでは、胞胚期での顆粒の局在を確認した。良い卵が採れていないので、初期卵割期での確認は行えていない。(4)マツカワでは、4-8細胞期から蛍光顆粒の局在が確認される。個体によって、割球間に複数の顆粒の局在が認められた。胞胚期では、胚盤の周辺あるいは中央部でと、個体により異なる局在が認められた。シグナルは、胚環期から胚盾期には弱くなった。これらの結果より、このコンストラクトを用いることで、様々な種の生殖細胞質を顕在化させることが出来るようになった。Buc-GF mRNAを注...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2006年 -2008年 
    代表者 : 荒井 克俊, 阿部 周一, 山羽 悦郎, 村上 賢
     
    ドジョウは両性生殖で繁殖するのが普通であるが、通常の減数分裂によらず非還元二倍体の卵を産み、かつ、これらの卵が雌性発生により父親の遺伝的関与なく発生するクローン系統、および偶然の精子核取り込みにより生じる自然三倍体系統が野生集団に見られる。本研究は、このような特殊な生殖の機構と起源を明らかにするとともに、このような変異体を遺伝資源(育種素材)として、交雑・染色体操作と組み合わせることにより育種技術開発を行った。
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2006年 -2007年 
    代表者 : 荒井 克俊, 足立 伸次, 原 彰彦, 山羽 悦郎
     
    ミカドチョウザメの沿岸捕獲個体は得られなかったが、昨年度広尾水族館より入手した1個体(性別不明)を加えた合計6尾を北大七飯淡水実験所で飼育している。本年度は雄個体(体重11Kg)からLH-Rha注射により国内では初めて精子を得ることに成功した。そこで、同実験所において飼育中のダウリアチョウザメの雌個体(体重80Kg)からLH-Rha注射により得た成熟卵と人工授精を行なった。水温調節の問題から、受精卵の多くが死亡したが、国内で初めて交雑種の孵化仔魚(約50尾)を得ることができ、飼育を継続している。得られたミカドチョウザメの精液の凍結保存も試みたが、解凍後の精子運動比率は低く、さらに検討が必要と思われた。また、昨年度生検(biopsy)により成熟度判定を行なった雌個体(体重38Kg)について、本年度も再度成熟度判定を行なった。その結果、卵巣中の卵母細胞は約3.9mmに達し、黒色素の沈着が十分進行していたことから、本研究終了直後の採卵が見込まれた。さらに、他の雌個体(体重18Kg)についても成熟度判定を行なった結果、卵巣卵は約1.5mmに達していたことから、平成21-22年度に採卵可能となると思われた。引き続き、これら個体の繁殖に向けた飼育を継続する。上記のチョウザメ各種からは、DNAを採取、精製しており、これらから種判別、親子鑑定、性判別を行なうための分子マーカー開発するための準...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2004年 -2006年 
    代表者 : 山羽 悦郎, 足立 伸次, 平井 俊朗, 吉崎 吾朗
     
    1.ゼブラフィッシュのnos1遺伝子転写産物の3'UTRとGFPの構造遺伝子を結合した人工のGFP-nos1 3'UTR mRNAを合成し、これを注入したニシン、キンギョ、ドジョウ、パールダニオ、シロウオ、ウキゴリ、メダカ、マコガレイ、マツカワガレイ、ウナギにおいて始原生殖細胞(PGC)が可視化された。さらに、これら各魚種で可視化されたPGCの生殖隆起への動態を明らかにした。2.GFP-nos1 3'UTR mRNAの注入により可視化したPGCを、モルフォリノオリゴヌクレオチドにより不妊化した同種、他種の胞胚期へ移植する単一PGC移植法(SPT法)を開発した。SPT法により移植されたPGCは約50%の確率で宿主胚の生殖隆起へ移動した。3.パールダニオのPGCを不妊化したゼブラフィッシュへ移植した異種間の生殖系列キメラの誘導を行ない、成熟した個体の自然交配よりドナーのみの次世代を得た。また、キンギョ、ドジョウの可視化したPGCを不妊化したゼブラフィッシュへ移植した異属、異科間の生殖系列キメラでは、ドナーの機能的な精子が分化した。4.卵核を紫外線で不活化したドジョウ未受精卵にキンギョ精子を受精して誘起したキンギョ核-ドジョウ細胞質雑種胚は致死であるにもかかわらず、この胚細胞をドジョウ胚またはキンギョ胚へ移植すると宿主胚内で生残し、PGCも宿主の生殖隆起へ移動することを明らかにした...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2003年 -2005年 
    代表者 : 都木 靖彰, 山羽 悦郎, 阿部 周一, 新井 崇臣
     
    欧米諸国における魚類の耳石微量元素研究では、誘導結合プラズマ質量分析装置(ICPMS)が用いられているが、我が国では感度の低い電子線プローブマイクロアナライザー(EPMA)が用いられている。当該研究は従来のEPMA法による耳石微量元素測定と生活履歴解析、DNAマーカーを用いた集団判別研に加え、我が国で初めてのICPMSを用いて魚類の生活履歴解析および集団判別をおこない、以下の成果を得た。1.ICPMSよる耳石微量元素測定技術開発およびそれを利用した魚類の生活履歴解明と集団判別:ICPMSを用いた耳石および貝殻中の微量元素含有量測定法を開発し、微量元素組成による集団判別が養殖魚介類の産地判別に有効であることを示した。2.EPMA法による耳石微量元素測定を利用した魚類の生活履歴解明:EPMAによる耳石SrおよびCa含有量測定と、Sr : Ca比を用いて、サケ科魚類やウナギ属魚類の個体の生息水域の変化(回遊経路)を詳細に明らかにした。3.遺伝子を用いた集団判別:シロサケの系群解析を可能とするDNAマイクロアレイ方を開発した。本法は正確で時間もかからず、DNAシーケンサーなどの特別な機器を必要としないため、フィールドや調査船上における系群解析に極めて有効である。4.耳石の形成と石灰化機構および微量元素取り込み機構の解明と魚類の他硬組織との比較:耳石有機基質OMP-1およびotolin...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2002年 -2003年 
    代表者 : 山羽 悦郎, 吉崎 悟朗, 平井 俊朗, 荒井 克俊
     
    1)スズキ目魚類シロウオとウキゴリのPGCsの起源と動態を、vasa mRNAに対するwhole mount in situ hybridizationと組織学的観察により明らかにした。2)PGCsを含む胞胚期の胚盤断片の移植により生殖系列キメラの誘導を図った。その結果、同種間ではこの移植により生殖系列キメラが誘導されたが、異種間では、供与体胚細胞がホスト内で分散した場合のみ誘導された。3)生殖系列に特異的な遺伝子vasaあるいはnos1の3'末端非翻訳領域と緑色蛍光蛋白遺伝子を接続したキメラRNAを注入することで、ニシン、サケ科魚種、コイ目魚種、スズキ目魚種のPGCsがGFP蛍光で可視化された。4)胚盤断片の移植では誘起できなかった生殖系列キメラを誘導するため、可視化したPGCsを異種間での異時的な移植を行なった。その結果、少なくともコイ目の魚種間では、生殖系列キメラが誘導された。5)ニジマス孵化稚魚から単離したPGC、を異系統のニジマス宿主(孵化稚魚)に移植することで、移植PGC由来の卵・精子を得ることに成功した。なお、これらの配偶子は機能的であり、正常な次世代の作出も可能であった。これと同様の方法で、ニジマスPGCをヤマメに移植することで、ヤマメ精巣内で機能的なニジマス精子を得ることに成功し、次世代の作出にも成功した。6)Vasa-GFP遺伝子導入ニジマスから単離した生...
  • 文部科学省:科学研究費補助金(萌芽研究)
    研究期間 : 2002年 -2003年 
    代表者 : 荒井 克俊, 村上 賢, 山羽 悦郎
     
    1.北海道女満別町産のクローン1系統ドジョウ二倍体について、HCG注射による排卵誘起と人口受精の手法により、材料の維持・増殖を試み、現在三世代目集団を飼育している。しかしながら、クローン二倍体オランダ産フナについては親魚死亡により維持・繁殖ができなかった。2.十分な数のクローン供試魚を得られなかったため、生殖腺の組織学的・細胞遺伝学的な観察にいたらなかったが、染色体の核型において通常の二倍体(2n=50)との違いは認められなかった。3.クローン成魚数が少なかったため、in vitroでの卵巣培養による成熟過程の卵母細胞について細胞学的観察を行うことができなかったが、通常精子で受精したクローン由来卵において、進入した精子の凝縮、雌性前核のみの形成と第一卵割の進行が観察され、クローンドジョウの生殖は「雌性発生」によることが確認できた。4.ドジョウ野生集団について、自然三倍体のミトコンドリア(mt) DNAのRFLPハプロタイプを基にクローン候補二倍体を選別し、マイクロサテライトDNA、RAPD、DNAフィンガープリント分析を行ったところ、新たなクローンを北海道において一つ、石川県において二つ見出した。mtDNA調節領域の塩基配列では北海道のクローン間、石川県のクローン間で差はなく、両者間の差はわずかであった。5.北海道で得た二倍体-三倍体モザイクドジョウ雄は、DNAフローサイトメ...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2001年 -2003年 
    代表者 : 荒井 克俊, 奥村 誠一, 山羽 悦郎
     
    1.サクラマス受精卵の卵割阻止処理に由来する胚において、分裂の遅滞、割球サイズの不均一、染色体の断片化、多極分裂等の細胞学的異常を観察し、これらが致死的なモザイク胚の出現原因と推定した。2.第一卵割阻止処理由来のアマゴについて、生殖腺を含む様々器官・組織におけるモザイク性を検討し、他の器官に四倍性細胞が含まれない場合でも、生殖腺に四倍体由来の二倍性生殖細胞が含まれる場合があることを示すとともに、このような四倍体由来の二倍性配偶子(卵、精子)を用いて実際に三倍体を作出し、モザイクの利用による倍数体育種の可能性を示した。3.サケマス類の野生集団および人為雌性発生集団について、半数体-二倍体モザイク個体の生存性を示し、さらに、致死的な半数体個体であっても二倍体細胞とモザイク化することにより生存性回復が可能なことを、半数体と二倍体間の胚盤移植操作による半数体-二倍体キメラキンギョの作出により実験的に確認した。4.北海道内の野生フナ集団とドジョウ集団について、異なる倍数体細胞からなるモザイク個体の出現を認め、これらのモザイクにおける特殊な生殖様式(クローン卵あるいは精子の産生)の解明を行うとともに、カットスロートトラウト、ヒメマス養殖種苗に半数体-二倍体、二倍体-四倍体モザイクが出現することを見出し、卵質とモザイク出現の関係を考察した。5.第二極体放出により誘起したエゾアワビ三倍体集団...
  • 文部科学省:科学研究費補助金(基盤研究(B))
    研究期間 : 2000年 -2001年 
    代表者 : 山羽 悦郎, 木下 政人, 平井 俊朗, 荒井 克俊, 吉崎 悟朗
     
    1)キンギョの受精直後からPGCsが生殖隆起に至るまでの正常発生過程におけるvasa遺伝子産物の分布を明らかにし、ゼブラフィッシュと異なるいくつかの知見を得た。2)機能的なPGCsの形成には、中胚葉誘導を必要としないことを実験発生学的に明らかにした。3)キンギョの胞胚から嚢胚にかけてのPGCsの移動は、形態形成運動に依存することを実験発生学的手法を用いて明らかにした。4)胚盤の移植により胞胚期にPGCsの分布を動物極側に変更した胚で、その後のPGCsの分布を解析することで、PGCsは胞胚期での初期分布により移動経路が異なることを示した。5)胞胚期のPGCsを含む領域の移植により生殖系列キメラを確実に生み出す方法を確立した。この生殖系列キメラ個体から宿主とドナー両方の子孫が得られた。6)不妊となるキンギョ雌×コイ雄の雑種胚を宿主にすることで、ドナー由来の精子のみが作られることを明らかにした。7)vasa-GFP遺伝子導入によりPGCsを可視化したニジマスの系統化に成功した。8)このニジマス系統からフローサイトメーターを用いて大量にPGCsを精製する技法を開発した。9)ニジマス孵化稚魚の生殖隆起と他の組織間でのサブトラクション法により、PGCsを含む生殖系列に特異的に発現する遺伝子群を単離した。10)単離したニジマスPGCsをニジマス或いはヤマメ孵化稚魚に移植することで、宿主生殖...
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 1999年 -2000年 
    代表者 : 足立 伸次, 山羽 悦郎, 山口 晧平
     
    1 温度感受期の特定キンギョを用いて、仔魚期の様々な時期に適水温(23℃)から高水温(30℃)への昇温、あるいは、その逆の降温処理を行なった結果、23℃では27日、30℃では22日以前が温度感受期であることを特定した。マツカワについても同様の実験を行ない、全長約3cm以前が温度感受期であることを特定した。さらに、マコガレイについても同様の実験を行ない、全長約2cm以前が温度感受期であることをほぼ特定した。次に、キンギョの感受期内で短時間の温度変化が与える影響について調べた結果、温度感受期内の1日のみ適温から高温へ移行した場合は遺伝的雌の雄化は誘導されなかった。しかし、その逆に、温度感受期内の1日のみ高温から適温へ移行した場合は、高温による雄化の割合が大きく減少した。2 様々な飼育水温での性分化過程キンギョを用い、15、19、23および30℃での性分化過程を組織学的に観察した結果、30℃ではほとんどが雄になったため卵巣の観察ができなかったが、23℃以下の水温では明らかに低温ほど卵巣形成が遅れた。3 性決定に及ぼす飼育水温以外の環境要因遺伝的全雌のキンギョを用いて、飼育水のpH、密度および日照等の条件を変えた場合での性比を調べた結果、低pH(5.5)で有意に雄の割合が増加した。しかし、飼育密度および日照については、今回行なった条件では影響はみられなかった。また、マツカワについても...
  • 文部科学省:科学研究費補助金(一般研究(C), 基盤研究(C))
    研究期間 : 1995年 -1997年 
    代表者 : 山羽 悦郎
     
    (1)異種間での胚盤再構築を可能にするために必要な嚢胚期以前のキンギョの発生段階を、組織学的、細胞学的、発生遺伝学的な視点から検討を加えた。20℃の培養下で同調卵割から非同調卵割への移行(中期胞胚期遷移)は、9回の同調卵割の後受精約6時間に起こり、この時期以降を中期胞胚期と定めた。中胚葉分化の指標となるgoosecoidとno tailの発現は受精後8時間に観察され、この時期以降を後期胞胚期と定めた。(2)キンギョおよびゼブラフィッシュの、初期胚発生過程におけるPGCsの形態及びその動態を組織学的に明らかにした。キンギョでは20℃の培養下で受精後30時間に組織学的に区別されるPGCsが胚全体に広く分布した。この時期のPGCsを生体外部から細胞の大きさによって区別できる可能性が示された。しかしながら、これ以前の発生段階では区別できなかった。ゼブラフィッシュでは、組織学的なPGCsを6体節期まで遡ることができた。(3)PGCsの起源を実験発生学的に検討した結果、キンギョのPGCsは中期胞胚期には分化する割球が決まっており、その多くは胚盤の下部に位置するという結果が示された。(4)ゼブラフィッシュの2系統間、あるいはキンギョと3倍体のフナより作成された胚盤の下部を重複されたキメラ個体からは、両系統あるいは両亜種の生殖細胞由来の子孫が生まれることが明らかとなった。しかしながらその頻度...
  • 魚類の生殖細胞分化
    研究期間 : 1997年
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1994年 -1994年 
    代表者 : 山羽 悦郎
     
    1、同調分裂期にある胚細胞を胚から外科的に分離し、2つの胚細胞を電気融合させた場合、殆どの融合胚は1回目の分裂で4つの胚細胞となった。このため2種類のゲノムをあわせ一つの細胞を得るためには一方の細胞の中心粒を取り除く必要があると考えられた。2、ゲノム構造の変革の材料となる胚細胞の発生学的な特性の検討を行った結果、(1)キンギョの20℃の培養条件下で9回の同調分裂を行い、その後(受精後6時間)非同調的な分裂となった。この時期が中期胞胚期遷移(MBT)と考えられた。均一な同調胚細胞を使うにはMBT以前の胚細胞を材料とするのが最良であることが示唆された。人為的に多くの同調細胞を得るためには、同調期のM期での4μg/mlのアフィディコリンと1μg/mlのサイトカラシンBを含む生理的塩類溶液での処理が適当と考えられた。(2)ゼブラフィッシュで背方化のシグナルとして知られているgoosecoid遺伝子の発現が受精後8時間に確認され、さらに、受精後6、8時間の胚盤を周縁質上で切断し180°水平に回転を行った胚では2軸が形成されるなど、少なくともMBT以降の胚盤の一部の胚細胞は分化の方向性が決定されており、胚細胞の変革には不適である可能性が示唆された。(3)MBT直後の胚盤の下部を外科的に除去し、胚盤の上部のみからなる胞胚は形態的には正常に発生するものの、その始原生殖細胞の数は、対照胚(16...
  • 文部科学省:科学研究費補助金(奨励研究(A))
    研究期間 : 1993年 -1993年 
    代表者 : 山羽 悦郎
     
    卵黄の植物極側を初期卵割期に除去した受精卵より発生してきた胚は、形態的に両生類における無体軸胚と酷似しており、除去されたのは両生類で知られている背方決定の因子と考えられた。卵片発生の実験より、キンギョの背方決定の因子は、受精直後に胚盤とは反対側の卵黄内に局在し、32細胞期には動物半球胚盤側まで移動すること、2細胞期の第1卵割面を境にした片側にこの因子が局在している卵の割合が非常に高いことが明らかになった。また、受精卵を遠心処理する実験の結果より、背方決定の因子は、1細胞期の0.7の時期までに細胞内骨格に固定されるか、次のシグナルに変換される可能性が示唆された。この様なキンギョの背方決定の因子の動態は、両生類の背方化の過程と類似していた。背方化因子を取り除かれた胚は、リチウム処理により背方化された胚の胚盤周囲域の細胞を移植することにより、軸の回復がなされた。しかしながら軸回復胚には脊索の形成はなされていなかった。このことより、キンギョの体軸構造の形成には、リチウム処理で誘導されるgoosecoid遺伝子産物以外のいくつかの因子の発現が必要であり、これらの因子の発現に背方決定の因子が関わっていると考えられた。末受精卵より抽出されたtotalRNAには、軸回復能力は確認されず、背方化因子の実体はその他の(例えば蛋白性の)因子である可能性が残された。キンギョ胚の始原生殖細胞(PGCs...
  • 文部科学省:科学研究費補助金(国際学術研究)
    研究期間 : 1992年 -1992年 
    代表者 : 山崎 文雄, 朴 弘陽, 斉藤 節雄, 鳥居 茂樹, 山羽 悦郎, 清水 幹博, 麦谷 泰雄, 山田 寿郎
     
    本研究の主要目的はこれまで我国の科学研究の一般的傾向として情報の交換・人的交流が欧米に偏り,隣国韓国とは充分な交流が行われてこなかったとの認識に立って,今後両国がアジアの先進国として,アジア地域の繁殖のために水産増養殖研究,特にその基礎となる魚類の遺伝・育種・繁殖に関する研究分野において,科学研究協力の基礎作りとすることにあった。現在韓国から水産研究の分野で多数の留学生が我国で学んでいるが,韓国における水産増養殖の研究,特に今回の研究テーマである硬骨魚類の遺伝・育種・繁殖に関する研究の実体が十分に把握されていなかった。今回の国際学術研究において建国大学,畜産学科,家畜遺伝育種研究室の朴 弘陽教授の協力を得て,建国大学において魚類の遺伝育種に関するセミナーを開催し,建国大学における研究の実体を把握した。現在ソウル地域には水産培養殖研究にかかわる大学,研究機関は存在せず,建国大学に将来,水産に関する学科を新設する計画があり,我々との研究を強く希望していた。また韓国のアジアに誇るベき規模を有する,韓国海洋研究において魚類の遺伝・育種に関する研究報告会を開催し,関連の研究者と情報交換を行った。済州島では韓国国立水産振興院,水産種苗培養場を訪れ,ヒラメの種苗生産と親魚の繁殖,受精卵の管理と稚魚の飼育,養成に関する問題点について情報交換を行った。釜山地域では,韓国唯一の釜山水産大学校と水...
  • 文部科学省:科学研究費補助金(国際学術研究)
    研究期間 : 1991年 -1991年 
    代表者 : 山田 寿郎, 藤田 忠, 朴 弘陽, 鳥居 茂樹, 山羽 悦郎, 清水 幹博, 麦谷 泰雄, 山崎 文雄, ジェフレイ J.Z, 佐野 陽子, フレモンド J.F, 宮永 国子, 岩崎 はる子, 御手洗 昭治, ホセ デベラ, ソリ シメラネ, 中内 恒夫, 土居 弘元, 須貝 栄, 福永 美津子, 恩田 久雄, 大槻 繁雄, 安積 仰也
     
    本研究実績概要を対象別・方法論別でなく,地域別にまとめる。理由は各研究者が地域別に分散して国際交流をしているからである。アメリカー宮永は日米間の文化摩擦と交渉過程をテ-マに米国内に研究集団をつくる。特に,米国の代表的倫理学者グッドパスタ-博士の倫理的アプロ-チによる交渉研究の展開が期待される。須貝はハ-バ-ド・交渉研究所長のコルブ教授と討論する。それにより米国における行動科学的アプロ-チによる研究動向が明らかになる。特に,Negotiations in Organization Vol.I.II.III JA1 pressが最良書である。また,米国経営学会ヒンフリクト・マネジメント部会が交渉研究に活発である情況が明らかになる。土居が交渉過程の決定分析的アプロ-チによる米国の研究動向を調査する。特に,南加大学のW.エドワ-ド教授の多属性効用の計測法のSMARTの交渉への応用は重要である。また,スタンフォ-ド大学のR.ハワ-ド教授らによる決定分析の教育機関「決定教育センタ-」は交渉教育の点からも注目に値いする。斉藤はハ-バ-ド法学大学院の「調停者」セミナ-に参加する。これは最近脚光をあびているADR(Alternative Dispute Resolution)の一つである。この調停は中立的立場から紛争・解決を友好的に進展させるものである。この教育方法は理論学習だけでなく,シミュ...
  • 文部科学省:科学研究費補助金(一般研究(C))
    研究期間 : 1989年 -1990年 
    代表者 : 山羽 悦郎
     
    (1)細胞核を移植するための宿主胚の作成: Hoechst33342で核を顕在化し、卵膜を除去した卵より、蛍光顕微鏡下でマイクロインジェクタ-を用い核の除去を行った。針先を細胞質に挿入せず、卵黄膜表面に密着させ細胞質ごと核を吸引することで無核胚が得られた。受精卵の第1卵割に中期に核除去を行うと胚発生が進行しないが、前期と後期に除核をした卵は卵割し、胞胚程度まで発生が進行した。しかしながら、正常胚では上皮層、深層細胞、周縁質と3層に分化する胞胚が無核胚では3層に分化しなかった。(2)卵核除去卵への外来核導入法としての電気細胞融合法の検討: 卵同士の接着を誘起する高周波パルスは、今回購入した装置の設定範囲では非常に弱く融合の前段階の処理には用いることが出来なかった。融合へ導くパルスは、0.18Mショ糖、1.8mMCaCl_2溶液中では、電圧を高めるかパルス幅を広げるほど、卵の急激な収縮を引き起こすことが示された。この収縮は電気融合法の適用の阻害要因であった。胚盤が形成されるまでの卵の収縮は、融合パルスにより培養液中のCa^<2+>イオンの卵内への流入により収縮が引き起こされることが判明した。受精後30分以内の卵ならば50〜100μM程度の低濃度のキレ-ト剤存在下で、750V/cm,10μsのパルス処理により卵同士の融合は誘起された。胚盤の形成された卵ではこの条件下でも収縮が起こり...
  • 文部科学省:科学研究費補助金(海外学術研究)
    研究期間 : 1988年 -1988年 
    代表者 : 山崎 文雄, 井田 斉, 山羽 悦郎, 前川 光司, 後藤 晃, 実吉 峯郎
     
    集団の遺伝解析アラスカ州に生息するオショロコマは降海型を基本とする個体群から構成されているため、陸封を基本とする我国のオショロコマと比較した場合、集団間の遺伝的分化がどの程度になっているのか、また、コデアック島のカーラック川水系に古くからオショロコマと北極イワナ(S.alpinus)が共存するとされているが、この集団がオショロコマの変異型であるのか、極周辺に広く分布する北極イワナのレリック集団か、まだ一致した見解がない。本調査において以上の2点を考慮し、ジュノー市に本拠をおいて、ジュノー市周辺で5集団、ケチカン1集団、アンカレッジ1集団、コデアック島2集団の合計9集団およびチャンドラー湖で北極イワナ7個体を採集した。アイソザイム電気泳動法によって集団の遺伝子組成の解析を行った。用いた臓器は眼、肝臓、筋肉であり、15酵素から26遺伝子産を推定して解析した。ジュノー市周辺の5集団で遺伝子頻度を基に、集団の異質性を検定した結果、有意差はなく、各集団間で遺伝的類似性が極めて高いことを示しており、この事実はオショロコマの母川回帰性が厳密ではなく、各河川間で遺伝的交流のあることを示唆している。また、地理的に離れた集団間での遺伝的類似性はコディアック島Thumb湖の集団を除いて、極めて高いことが示され、各集団間の遺伝的分化の程度は、北海道のオショロコマ集団に比べて、著しく低い事が明らかにさ...
  • 文部科学省:科学研究費補助金(海外学術研究)
    研究期間 : 1987年 -1987年 
    代表者 : 山崎 文雄, 井田 斉, 山羽 悦郎, 前川 光司, 後藤 晃, 実吉 峯郎
     
    オショロコマは, サケ科イワナ属に属し, 我国を含め, 北方園に広く分布する魚種で最も寒冷地に適応した原始的魚種とされている. 変異の中が広く, 生活環には相分化がみられ, 河川残留型と降海型の2型に分かれる. 我国では, 河川残留型が, 一般的であるが, アラスカでは, 降海型が多く, この相分化の遺伝的な維持機構が問題となっている. 本研究では, アラスカのオショロコマについて繁殖生物学的な解析を目的として調査を行い, 降海型と残留型の繁殖の関与, 繁殖行動の解析, 地方集団の遺伝的解析, アイリザイム多型を調べ, 我国のオショロコマと比較検討を行うことを目的とする.本調査は, アラスカ州におけるサケ科魚類の繁殖時期に合わせて, 8月〜10月の期間に行った. この間, ジュノー市近郊では, 5河川, ケチカン市近郊では2河川, アナトヴィク近在のチャンドラ湖, コディアック島2河川, アンカレッヂ市近郊のチーケル川/河川を中心に採集を行った. 筋肉資料は電気泳動によってアイソザイムの解析を行った. その結果, Ldh,LL,Aat,Pgm,Lgg,6Pg,Gpi,Me,Aco,Agp,Sdhの各酵素蛋白に電気泳動的に分離される多型が観察された. この多型を利用した集団の解析が可能となり, 河川間で明瞭な差のあることが明らかとなった. また, コデアック島のカーラック川には...
  • 文部科学省:科学研究費補助金(一般研究(C))
    研究期間 : 1987年 -1987年 
    代表者 : 山崎 文雄, 山羽 悦郎, 後藤 晃
     
    本研究は既存の染色体または染色体断片を発生卵内に導入して, 新しい形質を有する個体の作出が可能か否かを知る事を目的として行った. 卵内への染色体断片の導入は受精による方法を用いた. 染色体の断片化には精子に^<60>Coγ線を照射する方法を用いた. 2倍性の回復には受精後第2極体放出を阻止する方法を用いた. 材料としてサクラマスの卵の染色体2セット(2n)にサクラマス, カラフトマスまたはサケの精子由来の染色体を受精によって卵内に導入して2n+α, (α:染色体断片)の染色体を持つ個体を作出した.精子に照射するγ線量は染色体の断片化に有効な5×10^3, 7×10^3, 10^4, 2×10^4Rとし, 完全に染色体が消失する10^5R照射精子も併せて用いた. これらの線量を照射した精子でサクラマス卵を媒精するといずれの線量においても胚の発生は正常に進行せず, 孵化までに全て全死する. しかし, 受精後7-10分後に水圧650Kg/cm^2を処理して卵由来の染色体2セットを確保するとサクラマス精子断片の場合, 7×10^3Rで28%が孵化し, 10^4Rでは, 1.3%が孵化した. 一方, カラフトマス精子の場合, 5×10^3Rで0.3%, 7×10^3Rで1.3%, 10^4Rでは0.5%, 2×10^4Rで1.1%が孵化した. これらの線量では, 染色体は断片化し, 孵...
  • 魚類の発生工学に関わる研究
  • 魚類の胚操作
  • Joint Research on Developmental Technology in Fish
  • Embryonic manipulation of teleost fish

教育活動情報

主要な担当授業

  • 生物圏科学特別講義Ⅱ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : Aquatic Biology, Marine Ecology, Oceanography
  • フィールド科学特別実習Ⅰ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : フィールド科学、森林圏、水圏、耕地圏、生物学、生態学、資源・生態系管理 field science, forest ecosystem, aquatic ecosystem, agricultural ecosystem, biology, ecology, resource and ecosystem management
  • フィールド科学特別実習Ⅱ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : フィールド科学、森林圏、水圏、耕地圏、生物学、生態学、資源・生態系管理 field science, forest ecosystem, aquatic ecosystem, agricultural ecosystem, biology, ecology, resource and ecosystem management
  • 生物生産学基礎論
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : 生物生産、植物栽培、畜産業、林業、養殖、餌、生態系、持続的生産、バイオテクノロジー、フィールド管理 animal products, aquaculture, biotechnology, crop production, ecosystem, field management, fish-seed, forestry, livestock, stockbreeding, sustainable production
  • 水圏科学特論Ⅱ
    開講年度 : 2021年
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : 水圏動物、頭足類、スルメイカ、バイオロギング、バイオテレメトリー、海棲哺乳類、高次捕食者、生物多様性、生物地理要素 Aquatic animals, bio-logging, bio-telemetry, cephalopod, Japanese flying squid, marine mammals, top predators, selective breeding, biodiversity, biogeological elements
  • 水圏生物学実験
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 受精、細胞分裂、胚発生、形態形成、ストレス応答、性決定、分子生物学、組織学、海藻、魚類
  • 環境と人間
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : サケ、回遊、人工ふ化放流、分類、気候変化、資源変動、生活史、個体群密度効果、生物多様性、バイオロギング、母川回帰、嗅覚、物質循環、海水適応、ゲノム、性統御、始原生殖細胞、流通、食品,機能性素材、アスタキサンチン,考古学、先住民族、市民運動
  • 水産増養殖実習
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 海藻,プランクトン,魚類,生育環境,生殖、人工授精、増養殖技術、水産関連施設
  • 環境と人間
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 海、河川、生命、細胞、遺伝子、魚、海藻、微生物、代謝、進化
  • 一般教育演習(フレッシュマンセミナー)
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : フィールド、体験型、環境科学、自然、産業
  • 北方生物圏機能生物学
    開講年度 : 2021年
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 水圏環境、水圏生物機能、水圏生物資源、養殖生産、生物改変手法

大学運営

学内役職歴

  • 2014年4月1日 - 2016年3月31日 北方生物圏フィールド科学センター 副センター長
  • 2016年4月1日 - 2018年3月31日 北方生物圏フィールド科学センター 副センター長
  • 2018年4月1日 - 2020年3月31日 北方生物圏フィールド科学センター 副センター長

委員歴

  • 2002年   水産育種研究会   会計幹事   水産育種研究会


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