Researcher Profile and Settings

Affiliation

• Faculty of Fisheries Sciences　Marine Life Science　Aquaculture Genetics and Genomics

Job Title

• Professor

Degree

• Ph. D.（2005/03 Hokkaido University）

URL

http://kaken.nii.ac.jp/d/r/10400003.ja.html

Research funding number

• 10400003

J-Global ID

• 201201079026400200

Research Interests

• germ cell   hybrid   species identification   Salmonidae   hybridization   meiosis   polyploid   clone   chromosome set manipulation   cryopreservation   development and differentiation   biotechnology   loach   ICSI   

Research Areas

• Life sciences / Aquaculture

Educational Organization

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

Academic & Professional Experience

• 2024/04 - Today Hokkaido University Faculty of Fisheries Sciences, Division of Marine Life Science, Laboratory of Aquaculture Genetics and Genomics Professor
• 2012/04 - 2024/03 Hokkaido University Faculty of Fisheries Sciences, Division of Marine Life Science, Laboratory of Aquaculture Genetics and Genomics Associate Professor
• 2010/09 - 2012/03 Department of Fisheries and Oceans, Canada West Vancouver Lab. Postdoctoral fellow
• 2008/04 - 2010/08 Hokkaido University Field Sicence Center for Northern Biosphere Postdoctoral fellow
• 2005/06 - 2008/03 Hokkaido University Faculty of Fisheries Sciences Postdoctoral fellow
• 2005/04 - 2005/05 Hokkaido University Faculty of Fisheries Sciences Postdoctoral fellow

Education

• 2002/04 - 2005/03  Hokkaido University  Graduate School of Fisheries Sciences, Doctoral course  Division of Marine Life Science
• 2000/04 - 2002/03  Hokkaido University  Graduate School of Fisheries Sciences, Master course  Division of Marine Life Science
• 1996/04 - 2000/03  Hokkaido University  School of Fisheries Sciences  Department of Marine Biological Science

Association Memberships

• JAPANESE SOCIETY OF ANIMAL BREEDING AND GENETICS   JAPANESE SOCIETY FOR AQUACULTURE SCIENCE   THE JAPANESE SOCIETY OF FISH GENETICS AND BREEDING SCIENCE   THE ZOOLOGICAL SOCIETY OF JAPAN   THE JAPANESE SOCIETY OF FISHERIES SCIENCE   

Research Activities

Published Papers

• Development of a repetitive DNA marker for identification of the exotic large-scale loach introduced to Japan

  Masamichi Kuroda, Noriko Azuma, Takafumi Fujimoto, Katsutoshi Arai

  Fisheries Science 90 (1) 53 - 64 0919-9268 2023/11/02 [Refereed]
  
• Cryopreservation of masu salmon sperm using glucose-methanol extender and seminal plasma biomarkers related to post-thaw sperm motility

  Takafumi Fujimoto, Takahisa Kaneyasu, Mitsuru Endoh, Yuya Kogame, Joanna Nynca, Andrzej Ciereszko, Eisuke Takahashi, Etsuro Yamaha, Kiyoshi Naruse, Katsutoshi Arai

  Aquaculture 557 738305 - 738305 0044-8486 2022/08 [Refereed][Not invited]
  
• An assessment of hybridization potential between Atlantic and Pacific salmon

  Devlin, R, Biagi, C, Sakhrani, D, Fujimoto, T, Leggatt, R, Smith, J, Yesaki, T

  Canadian Journal of Fisheries and Aquatic Sciences 2022/02/03 [Refereed][Not invited]
  
• Nucleotide Sequence and Chromosome Mapping of 5S Ribosomal DNA from the Dojo Loach, Misgurnus anguillicaudatus

  Kiko Shibata, Masamichi Kuroda, Etsuro Yamaha, Katsutoshi Arai, Takafumi Fujimoto

  Cytogenetic and Genome Research 162 (10) 570 - 578 1424-8581 2022 [Refereed]
   

  There are 2 genetically divergent groups in the dojo loach Misgurnus anguillicaudatus: A and B. Although most wild-type diploids reproduce sexually, clonal diploids (clonal loach) reproduce gynogenetically in certain areas. Clonal loaches produce unreduced isogenic eggs by premeiotic endomitosis, and such diploid eggs develop gynogenetically following activation by the sperm of sympatric wild-type diploids. These clonal loaches have presumably arisen from past hybridization events between 2 different ancestors. The genomic differences between these 2 groups have not been completely elucidated. Thus, new genetic and cytogenetic markers are required to distinguish between these 2 groups. Here, we compared the 5S rDNA region to develop markers for the identification of different dojo loach groups. The nontranscribed sequence (NTS) of the 5S rDNA was highly polymorphic and group-specific. NTSs were found in clades of 2 different groups in clonal loaches. In contrast, we did not find any group-specific sequences in the coding region of the 5S rRNA gene. Sequences were located near the centromere of the short arm of the largest submetacentric chromosomes in groups A and B and clonal loaches. Thus, the 5S rDNA of the dojo loach is conserved at the chromosomal location. Whereas, the sequences of the NTS regions evolved group-specifically in the dojo loach, with the sequences of both groups being conserved in clonal loaches.
• Improvement in group identification of dojo loach, Misgurnus anguillicaudatus, using PCR-restriction fragment length polymorphism

  Masamichi Kuroda, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai

  Conservation Genetics Resources 13 (4) 457 - 463 1877-7252 2021/12 [Refereed]
  
• Skewed genetic sex ratio in reciprocal hybrids between pink salmon and masu salmon

  FUMIKA SHIMA, REI ASANUMA, TAKAHISA KANEYASU, MASAKI ICHIMURA, EISUKE TAKAHASHI, ETSURO YAMAHA, TAKAFUMI FUJIMOTO, KATSUTOSHI ARAI

  NIPPON SUISAN GAKKAISHI 87 (5) 473 - 482 0021-5392 2021/09/15 [Refereed][Not invited]
  
• Control of developmental speed in zebrafish embryos using different incubation temperatures

  Hirotaro Urushibata, Kazuaki Sasaki, Eisuke Takahashi, Toshikatsu Hanada, Takafumi Fujimoto, Katsutoshi Arai, Etsuro Yamaha

  Zebrafish 18 (5) 316 - 325 1545-8547 2021/09/03 [Refereed][Not invited]
  
• Chromosome set manipulation and genome manipulation in aquaculture

  Takafumi Fujimoto, Toshiya Nishimura

  Nippon Shokuhin Kagaku Kogaku Kaishi 68 (7) 277 - 289 1341-027X 2021/07/15 [Refereed]
  
• Diploid and aneuploid sperm in tetraploid ginbuna, Carassius auratus langsdorfii

  Fumi Yamaguchi, Takafumi Fujimoto, Hiroko Suzuki, Hideki Tanaka, Masaru Murakami, Etsuro Yamaha, Katsutoshi Arai

  Theriogenology 172 95 - 105 0093-691X 2021/06/08 [Refereed][Not invited]
   

  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.
• FISH identifies chromosome differentiation between contemporary genomes of wild types and the ancestral genome of unisexual clones of dojo loach, Misgurnus anguillicaudatus

  Masamichi Kuroda, Kiko Shibata, Takafumi Fujimoto, Masaru Murakami, Etsuro Yamaha, Katsutoshi Arai

  Cytogenetic and Genome Research 161 (3-4) 178 - 186 1424-8581 2021/05/10 [Refereed]
   

  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.
• Comparative analysis of mitochondrial genomes in genetically distinct groups of the dojo loach Misgurnus anguillicaudatus

  Kiko Shibata, Duong Thuy Yen, Takafumi Fujimoto, Katsutoshi Arai

  Mitochondrial DNA Part B 5 (4) 3810 - 3812 2020/10/01 [Refereed]
  
• Developmental potential of somatic and germ cells of hybrids between Carassius auratus females and Hemigrammocypris rasborella males

  Yuki Naya, Tomoka Matsunaga, Yu Shimizu, Eisuke Takahashi, Fumika Shima, Mitsuru Endoh, Takafumi Fujimoto, Katsutoshi Arai, Etsuro Yamaha

  Zygote 28 (6) 470 - 481 0967-1994 2020/08/10 [Refereed][Not invited]
   

  The 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.
• Ploidy manipulation using diploid sperm of a wild tetraploid ginbuna (Japanese silver crucian carp, Carassius auratus langsdorfii)

  Fujimoto, Takafumi, Fujimoto, Suzu, Murakami, Masaru, Yamaha, Etsuro, Arai, Katsutoshi

  Bulletin of Fisheries Sciences, Hokkaido University 70 (1) 103 - 111 2020/08 [Not refereed][Not invited]
  
• Isogenic lines in fish--a critical review

  Roman Franěk, Abdul Rasheed Baloch, Vojtěch Kašpar, Taiju Saito, Takafumi Fujimoto, Katsutoshi Arai, Martin Pšenička

  Reviews in Aquaculture 12 1412 - 1434 2020/08 [Refereed][Not invited]
  
• Hybrid between Danio rerio female and Danio nigrofasciatus male produces aneuploid sperm with limited fertilization capacity

  Mitsuru Endoh, Fumika Shima, Miloš Havelka, Rei Asanuma, Etsuro Yamaha, Takafumi Fujimoto, Katsutoshi Arai

  PLoS ONE 15 (5) e0233885 - e0233885 2020/05/29 [Refereed][Not invited]
  
• Morphological differences in embryos of goldfish (Carassius auratus auratus) under different incubation temperatures.

  Urushibata, H, Takahashi, E, Shimizu, Y, Miyazaki, T, Fujimoto, T, Arai, K, Yamaha, E

  Intenational Journal of Developmental Biolog 63 (11-12) 597 - 604 2019/12 [Refereed][Not invited]
   

  The goldfish (Carassius auratus auratus) is a useful species for embryonic micromanipulations because of its large egg size and wide temperature tolerance. Here, we describe in detail the rate of development and morphological characteristics of goldfish embryos incubated at temperatures between 10 °C and 30 °C. The cleavage speed increased rapidly as temperature increased. Synchronized cell divisions occurred at 131 min intervals at 10 °C, at 33 min intervals at 20 °C, and at 19 min intervals at 30 °C during the cleavage period. The rate of hatched abnormal embryos significantly increased at temperatures of 26 °C and above, while there was no change in the number of abnormal embryos at temperatures less than 24 °C. Moreover, the blastomeres around the center of the blastodisc rose in the direction of the animal pole at temperatures less than 14 °C. At the lower temperatures, clusters of maternally-supplied germplasm were visualized both at the ends of the first three cleavage furrows and at the border between the lower and upper tiers at the 16- to 32-cell stage, with injection of artificial mRNA and vasa in situ hybridization. This study showed that temperature affects not only developmental speed but also the shape of the blastodisc and the distribution of maternally-supplied materials in the blastodisc. By controlling the temperature, it is possible for researchers to prepare many stages of embryos and shapes of the blastodisc from a single batch of eggs.
• Aberrant meiotic configurations cause sterility in clone-origin triploid and Inter-group hybrid males of the dojo loach, Misgurnus anguillicaudatus

  Masamichi Kuroda, Takafumi Fujimoto, Masaru Murakami, Etsuro Yamaha, Katsutoshi Arai

  Cytogenetic and Genome Research 158 (1) 46 - 54 2019/06 [Refereed][Not invited]
   

  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.
• Preparing a fish embryo (Prochilodus lineatus) for staging, chorion removal and PGC traceabilityPreparation of a fish embryo for micromanipulation: staging of development, removal of chorions and traceability of PGC in Prochilodus lineatus

  Geovanna C.Z. Coelho, Isaac S. Yo, Tatiana M. Mira-López, Paulo S. Monzani, Dilberto R. Arashiro, Takafumi Fujimoto, José A. Senhorini, George S. Yasui

  International Journal of Developmental Biology 63 (1/2) 57 - 65 2019/01 [Refereed][Not invited]
  
• Intraooplasmic injection of a multiple number of sperm to induce androgenesis and polyploidy in the dojo loach Misgurnus anguillicaudatus (Teleostei: Cobitidae)

  George Shigueki Yasui, Taiju Saito, Yan Zhao, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai

  Zygote 26 408 - 416 2018/10 [Refereed][Not invited]
  
• Clonal reproduction assured by sister chromosome pairing in dojo loach, a teleost fish

  Masamichi Kuroda, Takafumi Fujimoto, Masaru Murakami, Etsuro Yamaha, Katsutoshi Arai

  Chromosome Research 26 (4) 1 - 11 1573-6849 2018/06/07 [Refereed][Not invited]
   

  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.
• Triploid or hybrid tetra: Which is the ideal sterile host for surrogate technology?

  Lucas Henrique Piva, Diógenes Henrique de Siqueira-Silva, Caio Augusto Gomes Goes, Takafumi Fujimoto, Taiju Saito, Letícia Veroni Dragone, José Augusto Senhorini, Fabio Porto-Foresti, José Bento Sterman Ferraz, George Shigueki Yasui

  Theriogenology 108 239 - 244 0093-691X 2018/03/01 [Refereed][Not invited]
   

  This work was aimed at developing an effective procedure to obtain sterile ideal host fish in mass scale with no endogenous germ cells in the germinal epithelium, owning permanent stem-cell niches able to be colonized by transplanted germ cells in surrogate technology experiments. Thus, triploids, diploid hybrids, and triploid hybrids were produced. To obtain hybrid offspring, oocytes from a single Astyanax altiparanae female were inseminated by sperm from five males (A. altiparanae, A. fasciatus, A. schubarti, Hyphessobrycon anisitsi, and Oligosarcus pintoi). Triploidization was conducted by inhibition of the second polar body release using heat shock treatment at 40 °C for 2 min. At 9-months of age, the offspring from each crossing was histologically evaluated to access the gonadal status of the fish. Variable morphological characteristics of the gonads were found in the different hybrids offspring: normal gametogenesis, gametogenesis without production of gametes, sterile specimens holding germ cells, and sterile specimens without germ cells, which were considered “ideal hosts”. However, only in the hybrid derived from crossing between A. altiparanae and A. fasciatus, 100% of the individuals were completely sterile. Among them 83.3% of the male did not present germ cells inside germinal epithelium, having only somatic cells in the gonad. The other 16.7% also presented spermatogonia inside the niches. Such a methodology allows the production of sterile host in mass scale, opening new insights for application of surrogate technologies.
• Short-term storage of the oocytes affects the ploidy status in the yellowtail tetra Astyanax altiparanae

  Matheus Pereira Dos Santos, Nivaldo Ferreira Do Nascimento, George Shigueki Yasui, Nycolas Levy Pereira, Takafumi Fujimoto, José Augusto Senhorini, Laura Satiko Okada Nakaghi

  Zygote 26 (1) 89 - 98 1469-8730 2018/02/01 [Refereed][Not invited]
   

  Summary In fish, many factors can affect reproduction during in vitro fertilization, therefore determination of the factors that affect affecting gamete quality is needed. However, few studies have focused on gamete quality and the ploidy status. This study was conducted to elucidate whether oocyte storage can affect ploidy status, survival, and embryo viability in the characid species Astyanax altiparanae. Oocytes were stored in Dulbecco's phosphate-buffered saline (PBS) at 26°C, then aliquots were fertilized immediately after extrusion (control) and also after 60, 120, 180, and 240 min of storage. Fertilization and hatching rates were measured, and the developmental stages were analyzed at each stage before describing the main abnormalities. Ploidy status was analyzed by flow cytometry and blood smear. In the control group, 100% of the samples were diploid. After treatment for 60 min, 95.56 ± 4.44% samples were diploid and 4.44 ± 4.44% were triploid. After 120 min, 94.44 ± 9.62% of the samples was diploid and 5.56 ± 5.56% were triploid 100% of the samples were diploid after 180 min and, after 240 min, there was no survival. In other treatments, the highest percentage of hatching was after 60 min (88.93 ± 5.15% P = 0.015), and treatment with 180 min storage resulted in the highest percentage of abnormal larvae (95.76 ± 12.67% P = 0.012). These results show that oocyte storage can affect ploidy status and may be an interesting parameter for analysis in studies on chromosome set manipulation and micromanipulation.
• Genome incompatibility between rainbow trout (Oncorhynchus mykiss) and sea trout (Salmo trutta) and induction of the interspecies gynogenesis

  Marcin Polonis, Takafumi Fujimoto, Stefan Dobosz, Tomasz Zalewski, Konrad Ocalewicz

  Journal of Applied Genetics 59 (1) 91 - 97 1234-1983 2018/02/01 [Refereed][Not invited]
   

  Rainbow trout (Oncorhynchus mykiss Walbaum) and sea trout (Salmo trutta Linnaeus, 1758) show large karyotypic differences and their hybrid offspring is not viable due to unstable karyotype and chromosome fragmentation. However, gametes from these two species were used to induce gynogenetic development. Rainbow trout eggs activated by UV-irradiated sea trout sperm were subjected to high hydrostatic pressure (HHP) shock to prevent release of the 2nd polar body (early shock) or to inhibit the first cleavage (late shock) in order to produce diploid meiotic gynogenotes and gynogenetic doubled haploids (DHs), respectively. Cytogenetic analysis proved fish that development was induced by the sea trout spermatozoa were rainbow trout. In turn, molecular examination confirmed homozygosity of the gynogenetic DHs. Presumed appearance of the recessive alleles resulted in lower survival of the gynogenetic DH larvae (~25%) when compared to survival of the heterozygous (meiotic) gynogenotes (c. 50%). Our results proved that genomic incompatibilities between studied trout species result in the hybrid unviability. However, artificial gynogenesis including activation of rainbow trout eggs with UV-irradiated sea trout spermatozoa was successfully induced. As both species are unable to cross, application of the UV-irradiated sea trout spermatozoa to activate rainbow trout development assures only maternal inheritance with no contamination by the residues of the paternal chromosomes.
• Improved Procedure for Induction of the Androgenetic Doubled Haploids in Zebrafish

  Mitsuru Endoh, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai

  ZEBRAFISH 15 (1) 33 - 44 1545-8547 2018/02 [Refereed][Not invited]
   

  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%.
• Grooves surrounding the micropyle decrease the inseminating dose in fish

  Matheus Pereira-Santos, Eduardo Shimoda, Andre Furugen Cesar de Andrade, Luciano Andrade Silva, Takafumi Fujimoto, Jose Augusto Senhorini, George Shigueki Yasui, Laura Satiko Okada Nakaghi

  ZYGOTE 25 (6) 731 - 739 0967-1994 2017/12 [Refereed][Not invited]
   

  In fish with external fertilization, sperm must reach the oocyte through the micropyle to enter the cytoplasm. Fertilization success is then influenced by characteristics of oocytes or sperm. In this study, we evaluated oocyte morphology and sperm motility parameters and their effects on the inseminating dose in a teleost fish Astyanax altiparanae. Interestingly, we found one of the lowest yet described inseminating doses in teleosts (2390 spermatozoa oocyte(-1) ml(-1)). Such a fertilization efficacy may be explained by the long duration of sperm motility (>75 s), the small oocyte diameter (695.119 mu m), large micropyle diameter (7.57 mu m), and the presence of grooves on the oocyte surface that guides spermatozoon to the fertilization area. Additionally, we have described for the first time a structure that combines grooves on the chorion surface and a ridge in the micropylar area.
• The effect of temperature on the initial development of Brycon amazonicus Spix & Agassiz, 1829 as tool for micromanipulation of embryos

  Regiane Cristina da Silva, Matheus Pereira dos Santos, Jose Augusto Senhorini, Maria do Carmo Faria Paes, Fernanda Nogueira Valentin, Takafumi Fujimoto, Nivaldo Ferreira do Nascimento, George Shigueki Yasui, Laura Satiko Okada Nakaghi

  ZYGOTE 25 (5) 637 - 651 0967-1994 2017/10 [Refereed][Not invited]
   

  Primordial germ cell (PGC) transplant is a promising tool in aquaculture; however, successful use of this technique requires in depth knowledge of the early stages of embryo and larval development. The aim of this study was to analyse the effect of different temperatures (22, 26, and 30 degrees C) on the early development of B. amazonicus. The newly fertilized eggs were distributed into tanks with controlled temperature and oxygenation. Samples were collected at pre-established times and analysed under light and fluorescence microscopy. Temperature influenced the speed and duration of each stage of early development, including hatching time. The highest pronuclei fusion rate was observed 8 min post-fertilization (mpf) at 22 and 26 degrees C, and 6 mpf at 30 degrees C. The duration of the 512-1000 blastomeres phase during in the blastocyst stage was 1 h 30 min at 22 degrees C, and 25 min at 26 and 30 degrees C. Hatching occurred at 24 h 30 mpf at 22 degrees C, 16 h post-fertilization (hpf) at 26 degrees C, and 11 h 30 mpf at 30 degrees C. The rate of morphologically normal larvae was 88.34% at 22 degrees C, 90.49% at 26 degrees C, and 73% at 30 degrees C. Malformations of the head, yolk sac, heart, and tail were observed in all temperatures. Nevertheless, B. amazonicus embryos were able to develop satisfactory in all three temperatures tested. These results enable embryo manipulation at different temperatures to optimize the micromanipulation time of embryos and larvae for biotechnological studies.
• Triploid Induction in the Yellowtail Tetra, Astyanax altiparanae, Using Temperature Shock: Tools for Conservation and Aquaculture

  Nadya Soares de Macedo Adamov, Nivaldo Ferreira do Nascimento, Elayna Cristina Silva Maciel, Matheus Pereira-Santos, Jose Augusto Senhorini, Leonardo Luiz Calado, Mariana Machado Evangelista, Laura Satiko Okada Nakaghi, Alan Hertz Marin Guerrero, Takafumi Fujimoto, George Shigueki Yasui

  JOURNAL OF THE WORLD AQUACULTURE SOCIETY 48 (5) 741 - 750 0893-8849 2017/10 [Refereed][Not invited]
   

  Triploidization is an interesting tool to produce sterile fish. In the yellowtail tetra, Astyanax altiparanae, this can be applied for aquaculture and surrogate technologies. In this study, we compared the efficacy of cold (2 C) or heat shock (38 C, 40 C, and 42 C) on triploid induction in the yellowtail tetra. The eggs were treated with cold or heat shock, 2 min postfertilization (30min in cold shock or 2min in heat shock). Intact embryos served as the control group. Ploidy status was confirmed by karyotyping, flow cytometry, and nuclear diameter of erythrocytes. The hatching rate decreased after cold shock (12.69 +/- 15.76%) and heat shock at 42 C(0.35 +/- 0.69%) in comparison with the control group (63.19 +/- 16.82%). At 38 C and 40 C, hatching rates (61.29 +/- 17.73% and 61.75 +/- 22.1%, respectively) were not decreased. Only one triploid arose at 38 C (1/80). At 40 C, a high number of triploids arose (72/78). At 42 C, very few embryos developed into the hatching stage. A large number of haploid individuals arose after cold shock (61/75), with only one triploid. Our results indicate that heat shocking of embryos at 40 C is optimum for triploid production in the yellowtail tetra.
• A Flow Cytometry Protocol to Estimate DNA Content in the Yellowtail Tetra Astyanax altiparanae

  Pedro L. P. Xavier, Jose A. Senhorini, Matheus Pereira-Santos, Takafumi Fujimoto, Eduardo Shimoda, Luciano A. Silva, Silvio A. dos Santos, George S. Yasui

  FRONTIERS IN GENETICS 8 131  1664-8021 2017/09 [Refereed][Not invited]
   

  The production of triploid yellowtail tetra Astyanax altiparanae is a key factor to obtain permanently sterile individuals by chromosome set manipulation. Flow cytometric analysis is the main tool for confirmation of the resultant triploids individuals, but very few protocols are specific for A. altiparanae species. The current study has developed a protocol to estimate DNA content in this species. Furthermore, a protocol for long-term storage of dorsal fins used for flow cytometry analysis was established. The combination of five solutions with three detergents (Nonidet P-40 Substitute, Tween 20, and Triton X-100) at 0.1, 0.2, and 0.4% concentration was evaluated. Using the best solution from this first experiment, the addition of trypsin (0.125, 0.25, and 0.5%) and sucrose (74 mM) and the effects of increased concentrations of the detergents at 0.6 and 1.2% concentration were also evaluated. After adjustment of the protocol for flow cytometry, preservation of somatic tissue or isolated nuclei was also evaluated by freezing (at -20 degrees C) and fixation in saturated NaCl solution, acetic methanol (1:3), ethanol, and formalin at 10% for 30 or 60 days of storage at 25 degrees C. Flow cytometry analysis in yellowtail tetra species was optimized using the following conditions: lysis solution: 9.53 mM MgCL2.7H(2)O; 47.67 mM KCl; 15 mM Tris; 74 mM sucrose, 0.6% Triton X-100, pH 8.0; staining solution: Dulbecco's PBS with DAPI 1 mu g mL(-1); preservation procedure: somatic cells (dorsal fin samples) frozen at -20 degrees C. Using this protocol, samples may be stored up to 60 days with good accuracy for flow cytometry analysis.
• Development of nuclear DNA markers to characterize genetically diverse groups of Misgurnus anguillicaudatus and its closely related species

  Takafumi Fujimoto, Aya Yamada, Yukihiro Kodo, Kohei Nakaya, Michiko Okubo-Murata, Taiju Saito, Kazuto Ninomiya, Michiko Inaba, Masamichi Kuroda, Katsutoshi Arai, Masaru Murakami

  FISHERIES SCIENCE 83 (5) 743 - 756 0919-9268 2017/09 [Refereed][Not invited]
   

  Repetitive DNA sequences, ManDra and ManBgl, were isolated from the DraI and BglII digests of the genomic DNA of Misgurnus anguillicaudatus, respectively. A primer set of ManDra distinguished two genetically different groups (A and B) of M. anguillicaudatus by specific electrophoretograms. A primer set of ManBgl amplified the DNA of M. anguillicaudatus and M. mizolepis. The individuals of M. anguillicaudatus were divided into two groups depending on the fragment sizes, in which the groups A and B (B-1 and B-2) showed 400 and 460 bp, respectively. M. mizolepis was distinguished by a different pattern (400-, 460-, and 510-bp fragments). PCR-RFLP analyses of recombination activating gene 1 gave a clear difference between A or B-2 (443-bp fragment) and B-1 groups (296- and 147-bp fragments). Clonal lineages and hybrids between B-1 and B-2 groups could be identified by appearance of three fragments (443, 296, and 147 bp). The combined analyses using the above three nuclear markers discriminated among nuclear genomes of genetic groups (A, B-1 and B-2) of M. anguillicaudatus and M. mizolepis. In several localities, natural hybridizations between the group B-1 and B-2 loaches and introgressions of clonal mitochondrial genomes into the group B-1 loaches were detected.
• Stereological analysis of gonads from diploid and triploid fish yellowtail tetra Astyanax altiparanae (Garutti & Britski) in laboratory conditions

  Nivaldo Ferreira do Nascimento, Diogenes Henrique de Siqueira-Silva, Matheus Pereira-Santos, Takafumi Fujimoto, Jose Augusto Senhorini, Laura Satiko Okada Nakaghi, George Shigueki Yasui

  ZYGOTE 25 (4) 537 - 544 0967-1994 2017/08 [Refereed][Not invited]
   

  This study aimed to examine the gonadal morphology of diploid and triploid fish through stereological analysis. Triploid individuals were obtained after temperature shock (40 degrees C for 2 min) at 2 min post-fertilization and reared until 175 days post-fertilization (dpf). Intact eggs were used to obtain the diploids. Gonads were collected for histological analysis at 83, 114, 144 and 175 dpf. Diploid females and males presented normal oogenesis and spermatogenesis through all the experimental period. Conversely, stereological analysis revealed that triploid females were sterile and oogonia were the prevalent cell type in the ovaries. Triploid males presented increased amounts of spermatocyte cysts and a large area of lumen when compared with diploids and in addition the amount of spermatozoa was lower than that observed for diploids. However, some triploid males presented spermatogenesis similar to diploids. Therefore, we concluded that triploidization is an interesting alternative to produce sterile individuals in A. altiparanae.
• Spontaneous polyploidization in critically endangered Acipenser mikadoi

  Milos Havelka, He Zhou, Seishi Hagihara, Masaki Ichimura, Takafumi Fujimoto, Etsuro Yamaha, Shinji Adachi, Katsutoshi Arai

  FISHERIES SCIENCE 83 (4) 587 - 595 0919-9268 2017/07 [Refereed][Not invited]
   

  We investigated the source of spontaneous polyploidization in the critically endangered Acipenser mikadoi. Fourteen sib progeny of A. mikadoi and 11 hybrids between an A. mikadoi female and a Huso dauricus male, all showing atypically high ploidy, were analysed. Parent assignment based on five highly polymorphic microsatellite markers confirmed spontaneous duplication of maternal chromosome sets via retention of the second polar body to be the source of spontaneous polyploidization. To our knowledge, this provides the first evidence of the maternal origin of spontaneous polyploidization in A. mikadoi. Factorial correspondence analysis of the multilocus microsatellite genotypes placed the parent fish of spontaneous polyploids in clearly delineated clusters of A. mikadoi and H. dauricus, and parent fish had mitochondrial control region haplotypes corresponding to their presumed species. Thus, parent fish were confirmed to be of pure genetic origin, and hybridization did not promote the observed spontaneous polyploidization.
• Nuclear DNA markers for identification of Beluga and Sterlet sturgeons and their interspecific Bester hybrid

  Milos Havelka, Takafumi Fujimoto, Seishi Hagihara, Shinji Adachi, Katsutoshi Arai

  SCIENTIFIC REPORTS 7 1694  2045-2322 2017/05 [Refereed][Not invited]
   

  Sturgeons (Acipenseriformes) are among the most endangered species in the world due to fragmentation and destruction of their natural habitats and to overexploitation, mainly for highly priced caviar. This has led to the development of sturgeon culture, originally for reintroduction, but more recently for caviar production. In both cases, accurate species identification is essential. We report a new tool for accurate identification of Huso huso and Acipenser ruthenus based on nuclear DNA markers. We employed ddRAD sequencing to identify species- specific nucleotide variants, which served as specific binding sites for diagnostic primers. The primers allowed identification of Huso huso and Acipenser ruthenus as well as their discrimination from A. baerii, A. schrenckii, A. gueldenstaedtii, A. stellatus, A. persicus, A. mikadoi, A. transmontanus, and H. dauricus and identification of A. ruthenus and H. huso hybrids with these species, except hybrid between A. ruthenus and A. stellatus. The speciesspecific primers also allowed identification of bester (H. huso x A. ruthenus), the most commercially exploited sturgeon hybrid. The tool, based on simple PCR and gel electrophoresis, is rapid, inexpensive, and reproducible. It will contribute to conservation of remaining wild populations of A. ruthenus and H. huso, as well as to traceability of their products.
• Comparison of Egg Envelope Thickness in Teleosts and its Relationship to the Sites of ZP Protein Synthesis

  Kaori Sano, Mari Kawaguchi, Keita Katano, Kenji Tomita, Mayu Inokuchi, Tatsuki Nagasawa, Junya Hiroi, Toyoji Kaneko, Takashi Kitagawa, Takafumi Fujimoto, Katsutoshi Arai, Masaru Tanaka, Shigeki Yasumasu

  JOURNAL OF EXPERIMENTAL ZOOLOGY PART B-MOLECULAR AND DEVELOPMENTAL EVOLUTION 328 (3) 240 - 258 1552-5007 2017/05 [Refereed][Not invited]
   

  Teleost egg envelope generally consists of a thin outer layer and a thick inner layer. The inner layer of the Pacific herring egg envelope is further divided into distinct inner layers I and II. In our previous study, we cloned four zona pellucida (ZP) proteins (HgZPBa, HgZPBb, HgZPCa, and HgZPCb) from Pacific herring, two of which (HgZPBa and HgZPCa) were synthesized in the liver and two (HgZPBb and HgZPCb) in the ovary. In this study, we raised antibodies against these four proteins to identify their locations using immunohistochemistry. Our results suggest that inner layer I is constructed primarily of HgZPBa and Ca, whereas inner layer II consists primarily of HgZPBa. HgZPBb and Cb were minor components of the envelope. Therefore, the egg envelope of Pacific herring is primarily composed of liver-synthesized ZP proteins. A comparison of the thickness of the fertilized egg envelopes of 55 species suggested that egg envelopes derived from liver-synthesized ZP proteins tended to be thicker in demersal eggs than those in pelagic eggs, whereas egg envelopes derived from ovarian-synthesized ZP proteins had no such tendency. Our comparison suggests that the prehatching period of an egg with a thick egg envelope is longer than that of an egg with a thin egg envelope. We hypothesized that acquisition of liver-synthesized ZP proteins during evolution conferred the ability to develop a thick egg envelope, which allowed species with demersal eggs to adapt to mechanical stress in the prehatching environment by thickening the egg envelope, while pelagic egg envelopes have remained thin. (C) 2017 Wiley Periodicals, Inc.
• Novel decaplex PCR assay for simultaneous detection of scallop species with species-specific primers targeting highly variable 5 end of the 16S rRNA gene

  Alan Marin, Claudio Villegas-Llerena, Takafumi Fujimoto, Katsutoshi Arai

  AQUACULTURE RESEARCH 48 (3) 920 - 930 1355-557X 2017/03 [Refereed][Not invited]
   

  Scallops (family Pectinidae) comprise species of high commercial value, supporting both commercial fisheries and mariculture activities. Accurate and reliable molecular methods for the species level identification are of outstanding utility for taxonomic and food authentication surveys. The mitochondrial 16S rRNA gene has been used to design species-specific primers for identification of different bivalve species. However, the low interspecific variability at the 3 end of this gene has limited its utility and only few scallop species have been assessed. In this study, we used the high variable 5 end of the 16S gene to develop a novel decaplex PCR assay that enabled a fast and accurate identification of eight commercially important scallop species in a single PCR reaction. A total of 285 individuals including fresh and manufactured samples from eight different processed presentations from 11 different scallop species were collected representing diverse locations around the world. Our assay accurately identified all the analysed samples at the species level. Furthermore, to enhance the utility of our assay, the PCR product amplified by the family specific primer set that was utilized as positive control was also used for the identification of unknown (non-target) scallop species by DNA sequencing analysis. In its present form, our multiplex PCR method can be of great utility for different types of studies involving scallop species and for research institutes and governmental agencies that regulate seafood authentication around the world.
• Growth, fatty acid composition, and reproductive parameters of diploid and triploid yellowtail tetra Astyanax altiparanae

  Nivaldo Ferreira do Nascimento, Matheus Pereira-Santos, Lucas Henrique Piva, Breno Manzini, Takafumi Fujimoto, Jose Augusto Senhorini, George Shigueki Yasui, Laura Satiko Okada Nakaghi

  AQUACULTURE 471 163 - 171 0044-8486 2017/03 [Refereed][Not invited]
   

  The aim of this study was to evaluate the growth, carcass yield, fatty acid composition, and reproductive parameters of diploid and triploid Astyanax altiparanae. Triploidization was induced by heat shock (40 degrees C for 2 min) 2 min after fertilization. Fish were reared for 175 days from fertilization. Triploid females showed lower proportion of saturated and poly-unsaturated fatty acids and higher amount ofmono-unsaturated fatty acids than diploids. They were sterile, with immature gonads, and showed higher carcass yield than diploid females, which exhibited higher gonadosomatic indices. Triploid and diploid males showed similar gonad morphology, although diploid males produced greater numbers of spermatozoa. All males exhibited the secondary sex characteristic of spines in the pelvic and anal fins. Fatty acid composition and growth were similar in triploid and diploid males. Triploidy did not ensure sterility in males, but triploid females were sterile and had increased carcass yield, and may be used in aquaculture to increase the production of A. altiparanae. Statement of relevance: To our knowledge, this is the first study addressing the effect of triploid induction on growth parameters of a neotropical species. The important results for carcass yield and sterility in female triploid will improve the production of A. altiparane, as this species is being used as live-bait for sport fishing and direct consumption. (C) 2017 Elsevier B.V. All rights reserved.
• Morphology of gametes, post-fertilization events and the effect of temperature on the embryonic development of Astyanax altiparanae (Teleostei, Characidae)

  Matheus Pereira dos Santos, George Shigueki Yasui, Pedro Luiz Porfirio Xavier, Nadya Soares de Macedo Adamov, Nivaldo Ferreira do Nascimento, Takafumi Fujimoto, Jose Augusto Senhorini, Laura Satiko Okada Nakaghi

  ZYGOTE 24 (6) 795 - 807 0967-1994 2016/12 [Refereed][Not invited]
   

  The aim of this study was to describe the morphology of gametes, post-fertilization events and subsequent temperature effects on the early developmental stages of the neotropical species Astyanax altiparanae. The sperm of this species presents a typical morphology of teleost sperm with a spherical head (diameter = 1.88 mu m), midpiece (diameter = 0.75 mu m) and a single flagellum (length = 18.67 mu m). The extrusion of the second polar body and fusion of male and female pronucleus were reported for the first time in this species. Additionally, we observed the formation of the fertilization cone, which prevents polyspermic fertilization. Developmental stages at 22 degrees C, 26 degrees C and 30 degrees C gave rise to fertilization rates at 91.12, 91.42 and 93.04% respectively. Hatching occurred at 25 hpf at 22 degrees C, 16 hpf at 26 degrees C and 11 hpf at 30 degrees C and the hatching rates were 61.78%, 62.90% and 59.45%, respectively. At 22 degrees C, the second polar body was extruded at approximate to 6 mpf and the male and female pronucleus fused at approximate to 10 mpf. This fundamental information is important for the field and opens up new possibilities in fish biotechnology, including micromanipulation and chromosome-set manipulation.
• Utility of different sugar extenders for cryopreservation and post-thaw storage of sperm from Salmonidae species

  Joanna Nynca, Sylwia Judycka, Ewa Liszewska, Stefan Dobosz, Joanna Grudniewska, Katsutoshi Arai, Takafumi Fujimoto, Andrzej Ciereszko

  AQUACULTURE 464 340 - 348 0044-8486 2016/11 [Refereed][Not invited]
   

  The effect of replacement of glucose in glucose-methanol extender (GM) with either sucrose and trehalose on the sperm motility characteristics of cryopreserved semen and the post-thaw storage of salmon, brown trout, brook trout, sex-reversed female rainbow trout and whitefish was investigated. Semen samples were diluted 1:5 or 1:9 (for sex-reversed females) in 9% methanol containing 0.18 M glucose, sucrose or trehalose. After thawing (40 degrees C, 5 s), spermwas stored for 0, 30, 60, 120 and 240 min at 4 degrees C and assessed for sperm motility. The type of sugar had no effect on the percentage of sperm motility of brown trout (65-73%), brook trout (51-58%) or sex-reversed female rainbow trout (38-40%). Glucose and trehalose secured the highest post-thaw sperm motility after thawing (0 min) in salmon (64 +/- 7%) and whitefish (52 +/- 8%), respectively. The longest period (determined as the last time point when the percentage of motility did not significantly change) was recorded for cryopreserved semen of brook trout (60-120 min). The shortest was recorded for white fish semen (30 min). Our results suggest that glucose, sucrose or trehalose can be used interchangeably for the cryopreservation of brown trout, brook trout and sex-reversed semen. However, the best results in terms of post-thaw sperm motility were achieved for salmon with glucose and sucrose and for whitefish with trehalose and sucrose. Cryopreserved sperm can be stored for prolonged periods of at least 30 min without a loss in sperm motility, which can facilitate more organised hatchery work. Statement of relevance: The results of this study confirmed the high level of utility of cryopreservation employing a 0.18Mglucose and 9% methanol extender for freezing, among Salmonidae species. We have demonstrated for the first time the utility of this method for freezing Atlantic salmon semen. Furthermore it was shown that disaccharides are more efficient than glucose for preserving of whitefish post-thaw motility. (C) 2016 Elsevier B.V. All rights reserved.
• Microsatellite loci in the tetraploid spined loach, cobitis biwae, and cross-species amplification in four related species

  O. Jablonska, A. Mar�n, K. Kowalewska, T. Fujimoto, K. Arai

  Genetics and Molecular Research 15 (3) gmr.15039027  1676-5680 2016/09/23 [Refereed][Not invited]
   

  Fifteen microsatellite loci were identified in the tetraploid spined loach, Cobitis biwae (Teleostei: Cobitidae). Among these, 14 were polymorphic (5-31 alleles) and showed moderate to high cross-species amplification transferability in four related species, Cobitis matsubarai, Cobitis taenia, Misgurnus anguillicaudatus, and Misgurnus fossilis. The loci, described herein, will be useful for population genetics, phylogeny, parentage analysis, and detection of hybridization among Cobitis species.
• Non-motile tetraploid spermatozoa of Misgurnus loach hybrids

  Yan Zhao, Takafumi Fujimoto, Martin Psenicka, Taiju Saito, Katsutoshi Arai

  FISHERIES SCIENCE 82 (1) 127 - 135 0919-9268 2016/01 [Refereed][Not invited]
   

  We have compared various properties of spermatozoa from the wild diploid male pond loach Misgurnus anguillicaudatus to those from the interspecific male hybrid of the cross between a female M. anguillicaudatus and a male mud loach M. mizolepis. Our results show that spermatozoa from this interspecific hybrid had poor motility, low viability, abnormal morphology, a larger volume of mitochondrial mass per cell and higher ATP content of spermatozoa with tetraploid DNA content, and they were present at a low concentration. The interspecific hybrid males produced spermatozoa with a larger head, with either no flagellum (36.4 %), one flagellum (46.7 %) or two flagella (16.9 %). These flagella were shorter than those of the normal wild-type male M. anguillicaudatus and often presented with abnormalities in microtubule structure. An abnormally shorter flagellum has difficulty in propelling tetraploid spermatozoa with an increased head size in normal progressive motility, although they had higher energy, as shown by their larger volume of mitochondrial mass and higher ATP content. These tetraploid spermatozoa are likely produced by the arrest of the regular meiotic division after chromosomal replication, followed by abnormal spermiogenesis.
• Hybrid Origin of Gynogenetic Clones and the Introgression of Their Mitochondrial Genome Into Sexual Diploids Through Meiotic Hybridogenesis in the Loach, Misgurnus anguillicuadatus

  Aya Yamada, Yukihiro Kodo, Masaru Murakami, Masamichi Kuroda, Takao Aoki, Takafumi Fujimoto, Katsutoshi Arai

  JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY 323 (9) 593 - 606 1932-5223 2015/11 [Refereed][Not invited]
   

  In a few Japanese populations of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae), clonal diploid lineages produce unreduced diploid eggs that normally undergo gynogenetic reproduction; however the origin of these clones remains elusive. Here, we show the presence of two diverse clades, A and B, within this loach species from sequence analyses of two nuclear genes RAG1 (recombination activating gene 1) and IRBP2 (interphotoreceptor retinoid-binding protein, 2) and then demonstrate heterozygous genotypes fixed at the two loci as the evidence of the hybrid nature of clonal lineages. All the clonal individuals were identified by clone-specific mitochondrial DNA haplotypes, microsatellite genotypes, and random amplified polymorphic DNA fingerprints; they commonly showed two alleles, one from clade A and another from clade B, whereas other wild-type diploids possessed alleles from either clade A or B. However, we also found wild-type diploids with clone-specific mitochondrial DNA and nuclear genes from clade B. One possible explanation is an introgression of a clone-specific mitochondrial genome from clonal to these wild-type loaches. These individuals likely arose by a cross between haploid sperm from bisexual B clade males and haploid eggs with clone-specific mtDNA and clade B nuclear genome, produced by meiotic hybridogenesis (elimination of unmatched A genome followed by meiosis after preferential pairing between two matched B genomes) in clone-origin triploid individual (ABB). (C) 2015 Wiley Periodicals, Inc.
• The mitochondrial genomes of Pecten albicans and Pecten maximus (Bivalvia: Pectinidae) reveal a novel gene arrangement with low genetic differentiation

  Alan Marin, Takafumi Fujimoto, Katsutoshi Arai

  BIOCHEMICAL SYSTEMATICS AND ECOLOGY 61 208 - 217 0305-1978 2015/08 [Refereed][Not invited]
   

  Scallops (Bivalvia: Pectinidae) comprise more than 350 extant taxa including species of high economic importance. However, its phylogenetic classification remains unclear and so far only 7 scallop mitogenomes have been determined. In this study, the mitochondrial genomes of two congeneric scallop species Pecten albicans and Pecten maximus were determined. Both mitogenomes contain 12 protein-coding genes (atp8 is missing), two ribosomal genes, 20-22 transfer RNA genes, all encoded in the same strand. Overall, both Pecten mitogenomes are highly similar with high nucleotide (93%) and amino acid (98.7%) sequence identity. Both mitogenomes also contain the same gene order arrangement, which is a novel contribution within Pectinidae. The highly similar mitochondrial organization between both Pecten species suggested a recent speciation event. Phylogenetic analysis based on complete protein-coding gene information as well as large synteny gene blocks confirmed the sister group relationship between the genera Pecten (subfamily Pectininae) and Argopecten (tribe Aequipectini). (C) 2015 Elsevier Ltd. All rights reserved.
• Generation of clonal zebrafish line by androgenesis without egg irradiation

  Jilun Hou, Takafumi Fujimoto, Taiju Saito, Etsuro Yamaha, Katsutoshi Arai

  SCIENTIFIC REPORTS 5 13346  2045-2322 2015/08 [Refereed][Not invited]
   

  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.
• Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish

  G. S. Yasui, J. A. Senhorini, E. Shimoda, M. Pereira-Santos, L. S. O. Nakaghi, T. Fujimoto, L. Arias-Rodriguez, L. A. Silva

  ANIMAL 9 (3) 464 - 470 1751-7311 2015/03 [Refereed][Not invited]
   

  In fish, in vitro fertilization is an important reproductive tool used as first step for application of others biotechniques as chromosome and embryo manipulation. In this study, we aimed to optimize gamete quality and their short-term storage from the yellowtail tetra Astyanax altiparanae, for future application in laboratory studies. Working with sperm, we evaluated the effects of spawning inducers (carp pituitary gland and Ovopel (R) [(D-Ala6, Pro9-NEt) - mGnRH+metoclopramide]) and the presence of female on sperm motility. Additionally, we developed new procedures for short-term storage of sperm and oocytes. Briefly, sperm motility was higher when male fish were treated with carp pituitary gland (73.1 +/- 4.0%) or Ovopel (R) (79.5 +/- 5.5%) when compared with the control group treated with 0.9% NaCl (55.6 +/- 27.2%; P=0.1598). Maintenance of male fish with an ovulating female fish also improved sperm motility (74.4 +/- 7.4%) when compared with untreated male fish (42.1 +/- 26.1%; P=0.0018). Storage of sperm was optimized in modified Ringer solution, in which the sperm was kept motile for 18 days at 2.5 degrees C. The addition of antibiotics or oxygen decreased sperm motility, but partial change of supernatant and the combination of those conditions improve storage ability of sperm. Fertilization ability of oocytes decreased significantly after storage for 30, 60 90 and 120 min at 5, 10, 15 and 20 degrees C when compared with fresh oocytes (P=0.0471), but considering only the stored samples, the optimum temperature was 15 degrees C. Those data describe new approaches to improve semen quality and gametes short-term storage in yellowtail tetra A. altiparanae and open new possibilities in vitro fertilization.
• The variable 5' end of the 16S rRNA gene as a novel barcoding tool for scallops (Bivalvia, Pectinidae)

  Alan Marin, Takafumi Fujimoto, Katsutoshi Arai

  FISHERIES SCIENCE 81 (1) 73 - 81 0919-9268 2015/01 [Refereed][Not invited]
   

  Scallops (Bivalvia, Pectinidae) are among the most valuable source of marine food. With about 350 extant species distributed worldwide and a total global production comprising 18 species, the development of proper species-level identification assays is imperative. DNA barcoding has proven to be a useful tool in species identification. A partial region at the 5' end of the mitochondrial cytochrome c oxidase subunit I (COI) gene, known as the "Folmer region," was proposed as the most suitable DNA barcoding marker. However, Folmer primers have failed to amplify polymerase chain reaction (PCR) products in different organisms, including scallops. Searching for an alternative barcoding gene region, we analyzed the complete mitochondrial 16S rRNA gene in 15 scallop species. We found that the interspecific variation at the 5' end is twice as high as that at the 3' end. Based on that evidence, we designed a novel Pectinidae family-specific primer set, aiming to amplify a partial region at the 5' end of the 16S rRNA gene, and tested its suitability as a barcoding tool. A neighbor-joining analysis identified correctly 100 % of the scallop specimens analyzed, with high bootstrap support. Our new primers are well suited for DNA barcoding analysis and may contribute to scallop food industry surveys, as well as routine taxonomic surveys.
• Mitochondrial genome of the Peruvian scallop Argopecten purpuratus (Bivalvia: Pectinidae)

  Alan Marin, Ruben Alfaro, Takafumi Fujimoto, Katsutoshi Arai

  MITOCHONDRIAL DNA 26 (5) 726 - 727 1940-1736 2015 [Refereed][Not invited]
   

  The mitochondrial genome of the Peruvian scallop Argopecten purpuratus was determined. The length of the mitochondrial coding region is 15,608 bp. A typical bivalve mitochondrial composition was detected with 12 protein-coding genes, 2 ribosomal RNA genes and 21 transfer RNA genes, with the absence of the atp8 gene. Fifty percent of the protein-coding genes use typical ATG start codon, whereas five genes utilize ATA as their start codon. Only one gene was found to utilize TTG as its start codon. The A. purpuratus mitogenome shows a significant similarity to that of A. irradians irradians, in length as well as in gene composition.
• Comparison of spermatozoa parameters, fine structures, and energy-related factors among tetraploid, hyper-tetraploid, and hyper-triploid loaches (Misgurnus anguillicaudatus)

  Yan Zhao, Taiju Saito, Martin Psenicka, Takafumi Fujimoto, Katsutoshi Arai

  JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY 321 (4) 198 - 206 1932-5223 2014/04 [Refereed][Not invited]
   

  To evaluate the influence of ploidy elevation and aneuploidy on spermatozoa in the loach Misgurnus anguillicaudatus, we investigated some parameters (motility, concentration, and viability), fine structures (gross morphology, head size, and flagellum length), and energy-related biochemical factors (volume of mitochondrial mass per cell and ATP content) in diploid, hyper-diploid, and hexaploid-range spermatozoa produced in natural tetraploid, hyper-tetraploid, and hyper-triploid male loaches, respectively. Diploid spermatozoa exhibited vigorous movement and sufficient duration of motility similar to those in haploid spermatozoa. They had longer flagella, higher numbers and larger volume of mitochondria, and higher ATP content than haploid spermatozoa of wild-type diploids. No differences were observed in parameters and morphological characteristics between diploid and hyper-diploid spermatozoa. In contrast, the hexaploid-range spermatozoa of hyper-triploid males exhibited poor progressive motility in spite of a higher ATP content of spermatozoa. Spermatozoa with no flagella (36.0%) or multiple flagella (18.6%) were also observed in hyper-triploids. Ratios of head to flagellum length in hexaploid-range spermatozoa were significantly different from those of haploid spermatozoa. In addition to the normal 9 + 2 microtubule structure of the flagellum, an abnormal 9 + 1 microtubule structure was also observed in the spermatozoa of hyper-triploids. J. Exp. Zool. 321A: 198-206, 2014. (c) 2014 Wiley Periodicals, Inc.
• Androgenetic doubled haploids induced without irradiation of eggs in loach (Misgurnus anguillicaudatus)

  Jilun Hou, Taiju Saito, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai

  AQUACULTURE 420 S57 - S63 0044-8486 2014/01 [Refereed][Not invited]
   

  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 degrees C for 30 min, to eliminate the female nucleus. The eggs were then incubated in a water bath at 20 +/- 0.5 degrees C for 35 min. Finally, diploidy was restored (65 min after activation) by heat-shock treatment at 42 +/- 0.5 degrees 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. (C) 2013 Elsevier B.V. All rights reserved.
• Growth and endocrine effect of growth hormone transgene dosage in diploid and triploid coho salmon

  Robert H. Devlin, Dionne Sakhrani, Carlo A. Biagi, Jack L. Smith, Takafumi Fujimoto, Brian Beckman

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 196 112 - 122 0016-6480 2014/01 [Refereed][Not invited]
   

  Growth-hormone transgene dosage, polyploidy, and parental effects on growth and endocrine responses have been assessed in coho salmon. Diploid fry with one or two transgene doses grew equally, whereas later-stage juvenile homozygotes grew faster than hemizygotes. In contrast, homozygotes and hemizygotes grew equally after smoltification, both in sea water and fresh water. Triploid transgenic salmon showed impaired growth which could not be fully overcome with additional transgene copies. Levels of muscle GH mRNA were elevated in two vs. one transgene dose diploids, but in triploids, a dosage effect was observed in muscle but not for animals carrying three transgene doses. IGF-I mRNA levels were elevated in transgenic vs. non-transgenic animals, but a dosage effect was not observed. Diploids and tripbids with two transgenes had higher plasma GH levels than one-dose animals, but three-dose triploids showed no further elevation. Circulating IGF-I levels also showed a dosage effect in diploids, but not among any transgene doses in triploids. The present study reveals complex interactions among transgene dosage, maternal effects, developmental stage, and ploidy on growth and endocrine parameters in GH transgenic coho salmon. Specifically, GH transgenes do not always express nor have effects on growth that are directly correlated with the number of transgenes. Further, the reduced growth rate seen in tripbid transgenic animals could not be fully overcome by increasing transgene dosage. The findings have relevance for understanding growth physiology, transgene function, and for environmental risk assessments that require understanding phenotypes of hemizygous vs. homozygous transgenic animals in populations. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.
• Genetic characterization of the progeny of a pair of the tetraploid silver crucian carp Carassius auratus langsdorfii

  Jie Dong, Masaru Murakami, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai

  FISHERIES SCIENCE 79 (6) 935 - 941 0919-9268 2013/11 [Refereed][Not invited]
   

  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.
• Rapid species identification of fresh and processed scallops by multiplex PCR

  Alan Marin, Takafumi Fujimoto, Katsutoshi Arai

  FOOD CONTROL 32 (2) 472 - 476 0956-7135 2013/08 [Refereed][Not invited]
   

  Food control policies regarding to seafood label authenticity have become a global issue due to increased incidence of species substitution or mislabelling. Proper species-level identification in processed scallop products is hindered by the lack of morphological characters such as their valves. In order to identify four commercially important scallop species (Argopecten purpuratus, Argopecten irradians, Mizuhopecten yessoensis, Pecten albicans) a species-specific multiplex PCR reaction is described herein. Novel reverse species-specific primers in combination with one universal forward primer designed to amplify a partial region of the mitochondrial 16S rRNA gene were assayed in fresh as well as in manufactured scallop samples. All PCR reactions showed a high specificity allowing an unambiguous species authentication. (C) 2013 Elsevier Ltd. All rights reserved.
• Heat-shock-induced tetraploid and diploid/tetraploid mosaic in pond loach, Misgurnus anguillicaudatus

  Takafumi Fujimoto, Suzu Sakao, Kouzou Oshima, Etsuro Yamaha, Katsutoshi Arai

  AQUACULTURE INTERNATIONAL 21 (4) 769 - 781 0967-6120 2013/08 [Refereed][Not invited]
   

  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 A degrees 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 A degrees 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.
• Production of androgenetic diploid loach by cold-shock of eggs fertilized with diploid sperm

  Jilun Hou, Takafumi Fujimoto, Etsuro Yamaha, Katsutoshi Arai

  THERIOGENOLOGY 80 (2) 125 - 130 0093-691X 2013/07 [Refereed][Not invited]
   

  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.
• Genetic structure of the Peruvian scallop Argopecten purpuratus inferred from mitochondrial and nuclear DNA variation

  Alan Marin, Takafumi Fujimoto, Katsutoshi Arai

  MARINE GENOMICS 9 1 - 8 1874-7787 2013/03 [Refereed][Not invited]
   

  The population genetic structure of the Peruvian scallop Argopecten purpuratus from three different wild populations along the Peruvian coast was analyzed using nine microsatellite loci and a partial region (530 bp) of the mitochondrial 16S rRNA gene. A total of 19 polymorphic sites in the 16S rRNA gene defined 18 unique haplotypes. High genetic diversity was presented in all populations. Statistical analysis of mitochondrial DNA revealed no significant genetic structure (Phi(ST)=0.00511, P = 0.32149) among the three localities. However, microsatellite analysis showed low (2.86%) but highly significant (P = 0.0001) genetic differentiation among populations, most of the variation was found in Independencia Bay population, which is located in the Peruvian National Reserve of Paracas. Neutrality tests based on mitochondrial haplotypes were performed to assess signatures of recent historical demographic events. Overall results from Tajima's D and Fu's F-S tests indicated significant deviations from neutrality. To our knowledge, this study constitutes the first investigation based on mitochondrial and microsatellite markers on the genetic structure of A. purpuratus. (c) 2012 Elsevier B.V. All rights reserved.
• Molecular cytogenetic study on the ploidy status in Acipenser mikadoi

  H. Zhou, T. Fujimoto, S. Adachi, S. Abe, E. Yamaha, K. Arai

  JOURNAL OF APPLIED ICHTHYOLOGY 29 (1) 51 - 55 0175-8659 2013/02 [Refereed][Not invited]
   

  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.
• Genomic Constitution and Atypical Reproduction in Polyploid and Unisexual Lineages of the Misgurnus Loach, a Teleost Fish

  K. Arai, T. Fujimoto

  CYTOGENETIC AND GENOME RESEARCH 140 (2-4) 226 - 240 1424-8581 2013 [Refereed][Invited]
   

  The loach (Misgurnus anguillicaudatus) is an excellent animal model to elucidate biological origin and evolutionary significance of genome duplication and unisexual reproduction because artificially induced and naturally occurring polyploids and parthenogenetic (gynogenetic, androgenetic) animals can be compared. First, we summarize the chromosome manipulation techniques to induce triploids and tetraploids by inhibiting meiotic or mitotic divisions of inseminated eggs, respectively, as well as parthenogenetic animals, obtained after fertilization with genetically inactivated gametes. Then, we review the knowledge on natural polyploid and unisexual lineages found in Misgurnus loaches. A natural diploid-tetraploid complex occurs in wild populations in central China, and these diploid and tetraploid loaches reproduce bisexually. Chinese tetraploids are considered autotetraploid, which may have arisen by doubling of the entire genome of an ancestral diploid, based on cytogenetic results from FISH (fluorescence in situ hybridization) karyotypes and meiotic configurations. In contrast, gynogenetically reproducing clonal diploid lineages have been discovered in a few wild populations in Japan, although most wild-type individuals are bisexually reproducing diploids. Such clonal diploid loaches sometimes produce triploid progeny by accidental incorporation of a sperm nucleus into an unreduced diploid egg, and the resulting triploid generates haploid eggs by meiotic hybridogenesis. Unreduced diploid gametes of clonal loaches are generated by a cytological mechanism, premeiotic endomitosis, which likely occurs in the early (gonium stage) germ cells. Initiation of gynogenetic development is related to a failure of decondensation of the male (sperm) pronucleus in unreduced diploid eggs of a clonal loach. Clonal lineages may have arisen from a past hybrid event between genetically divergent groups, but their exact origins are unknown at present. See also the sister article focusing on plants by Hegarty et al. in this themed issue. (C) 2013 S. Karger AG, Basel
• Vitrification of primordial germ cells using whole embryos for gene-banking in loach, Misgurnus anguillicaudatus

  D. Inoue, T. Fujimoto, Y. Kawakami, G. S. Yasui, E. Yamaha, K. Arai

  JOURNAL OF APPLIED ICHTHYOLOGY 28 (6) 919 - 924 0175-8659 2012/12 [Refereed][Not invited]
   

  In gene-banking, primordial germ cells (PGCs), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGCs have a potential to differentiate into both eggs and sperm via germ-line chimera. Here, we have established vitrification methods for PGCs cryopreservation using 12- to 17-somite stage embryos in loach, Misgurnus anguillicaudatus, which were dechorionated, removed their yolk and injected with green fluorescent protein (GFP) -nos1 3'UTR mRNA to visualize their PGCs. In order to optimize cryopreservation medium for vitrification, the toxicity of cryoprotectants was analyzed. Different concentrations (2, 3, 4, 5 m) of dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and propylene glycol (PG) as cryoprotectants were tested. Then, 5 m DMSO showed significantly-high toxicity. Based on this information, combinations called DMP (2 m (14.2% [v/v]) DMSO, 2 m (8.1% [v/v]) MeOH and 2 m (14.4% [v/v]) PG), DP (2 m (14.2% [v/v]) DMSO and 4 m (28.7% [v/v]) PG) and DE (2.1 m (15% [v/v]) DMSO and 2.7 m (15% [v/v]) EG) were evaluated for their toxicities and efficacy of PGCs cryopreservation using two types of equilibration step: direct immersion of cryopreservation media (one-step) and serial exposure to half and full concentration of cryopreservation media (two-step). Viable PGCs were obtained from post-thaw embryos which were cryopreserved by DP and DE with both 1- and 2-step equilibrations. Despite DP showing the highest toxicity, it gave the highest survival rate of embryonic cells after cryopreservation. When PGCs recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGCs were able to migrate to a host genital ridge similarly as endogenous PGCs. It suggests that our methods could be useful to create a germ-line chimera for the production of gametes from PGCs of cryopreserved embryos.
• Motility, morphology, mitochondria and ATP content of diploid spermatozoa from sex-reversed clonal diploid and neo-tetraploid loach, Misgurnus anguillicaudatus

  Y. Zhao, M. Psenicka, T. Fujimoto, T. Saito, G. S. Yasui, E. Yamaha, K. Arai

  JOURNAL OF APPLIED ICHTHYOLOGY 28 (6) 1006 - 1012 0175-8659 2012/12 [Refereed][Not invited]
   

  Diploid spermatozoa were obtained from sex-reversed clonal diploid males and neo-tetraploid which derived from normal diploid x natural tetraploid cross followed by the 2nd polar body release inhibition. The spermatozoa from sex-reversed clonal diploid males exhibited a significant reduction in motility when compared with those from normal diploid and neo-tetraploid males. Electron microscopy revealed that diploid spermatozoa from sex-reversed clonal diploid and neo-tetraploid males exhibited normal shape similar to haploid spermatozoa from normal diploid males, except for larger sperm-head size and longer flagellum. Considering flow-cytometric results, volume of mitochondrial mass per spermatozoon was also increased in proportion to the elevation of ploidy status from haploid to diploid sperm. Diploid spermatozoa from neo-tetraploid increased the number of mitochondria and the ATP content, but those from sex-reversed clonal diploid males did not show such results.
• Cryopreservation of green fluorescent protein (GFP)-labeled primordial germ cells with GFP fused to the 3 ' untranslated region of the nanos gene by vitrification of Japanese eel (Anguilla japonica) somite stage embryos

  Y. Kawakami, M. Ishihara, T. Saito, T. Fujimoto, S. Adachi, K. Arai, E. Yamaha

  JOURNAL OF ANIMAL SCIENCE 90 (12) 4256 - 4265 0021-8812 2012/12 [Refereed][Not invited]
   

  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.
• Germ cells are not the primary factor for sexual fate determination in goldfish

  Rie Goto, Taiju Saito, Takahiro Takeda, Takafumi Fujimoto, Misae Takagi, Katsutoshi Arai, Etsuto Yamaha

  DEVELOPMENTAL BIOLOGY 370 (1) 98 - 109 0012-1606 2012/10 [Refereed][Not invited]
   

  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.
• The effect of ions and cryoprotectants upon sperm motility and fertilization success in the loach Misgurnus anguillicaudatus

  George Shigueki Yasui, Takafumi Fujimoto, Lenin Arias-Rodriguez, Yasuaki Takagi, Katsutoshi Arai

  AQUACULTURE 344 147 - 152 0044-8486 2012/05 [Refereed][Not invited]
   

  The solutions commonly used to dilute or cryopreserve sperm are commonly composed of salts, buffers and cryoprotectants, which may affect gametes and subsequent fertilization success. Here, we have evaluated the effects of several cryoprotectants (methanol; MeOH, dimethyl sulfoxide; DMSO and dimethyl acetamide; DMA at concentrations of 0.25, 0.5 and 1%) and different ions (potassium, calcium and magnesium at concentrations of 1.25, 2.5, 5.0 and 10 mM) as sperm diluents upon sperm motility and fertilization success in the loach Misgurnus anguillicaudatus sperm. Our results demonstrated that DMSO (at 1%) decreased sperm motility while calcium and magnesium ions (from 2.5 mM) induced sperm aggregation and reduced sperm motility. Reduced fertilization rates were observed with potassium (from 1.25 mM), calcium (at 10 mM), magnesium (at 10 mM), DMA (at 1%), and DMSO (at 1%). We conclude that specific ions and cryoprotectants, and their relative concentrations caused effect upon loach gametes. These data are important to consider for the preparation of sperm diluents and activating solutions in order to manage gamete quality for artificial propagation. (C) 2012 Elsevier B.V. All rights reserved.
• Fish Breeding and Chromosome Set Manipulation

  Fujimoto, T, Arai, K

  水産育種 水産育種研究会 41 (2) 111 - 123 1343-7917 2012/03 [Refereed][Not invited]
  
• Isolation and characterization of 12 microsatellite loci in the Peruvian scallop Argopecten purpuratus and cross-species amplification in other scallop species (family Pectinidae)

  Alan Marin, Takafumi Fujimoto, Katsutoshi Arai

  CONSERVATION GENETICS RESOURCES 4 (1) 179 - 182 1877-7252 2012/03 [Refereed][Not invited]
   

  Argopecten purpuratus is an economically important marine bivalve. Twelve microsatellite loci were isolated and characterized in 20 individuals of A. purpuratus collected in Isla Independencia (Pisco, Peru) and tested for cross-species amplification in other 4 scallops (family Pectinidae). Ten loci were polymorphic. The number of alleles per locus ranged from 2 to 13. The observed (H (O)) and expected (H (E)) heterozygosities ranged from 0.100 to 0.900 and from 0.185 to 0.903, respectively. Two loci showed significant deviation from Hardy-Weinberg equilibrium. A low rate of cross-species amplification was found.
• Technical note: Viability and motility of vitrified/thawed primordial germ cell isolated from common carp (Cyprinus carpio) somite embryos

  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 0021-8812 2012/02 [Refereed][Not invited]
   

  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.
• Cold-shock eliminates female nucleus in fertilized eggs to induce androgenesis in the loach (Misgurnus anguillicaudatus), a teleost fish

  Kagayaki Morishima, Takafumi Fujimoto, Mami Sato, Ayako Kawae, Yan Zhao, Etsuro Yamaha, Katsutoshi Arai

  BMC BIOTECHNOLOGY 11 116  1472-6750 2011/11 [Refereed][Not invited]
   

  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.
• Production of loach (Misgurnus anguillicaudatus) germ-line chimera using transplantation of primordial germ cells isolated from cryopreserved blastomeres

  G. S. Yasui, T. Fujimoto, S. Sakao, E. Yamaha, K. Arai

  JOURNAL OF ANIMAL SCIENCE 89 (8) 2380 - 2388 0021-8812 2011/08 [Refereed][Not invited]
   

  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.
• Visualization and motility of primordial germ cells using green fluorescent protein fused to 3'UTR of common carp nanos-related gene

  Yutaka Kawakami, Taiju Saito, Takafumi Fujimoto, Rie Goto-Kazeto, Eisuke Takahashi, Shinji Adachi, Katsutoshi Arai, Etsuro Yamaha

  AQUACULTURE 317 (1-4) 245 - 250 0044-8486 2011/07 [Refereed][Not invited]
   

  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. (C) 2011 Elsevier B.V. All rights reserved.
• Genome size variation estimated by flow cytometry in Acipenser mikadoi, Huso dauricus in relation to other species of Acipenseriformes

  H. Zhou, T. Fujimoto, S. Adachi, E. Yamaha, K. Arai

  JOURNAL OF APPLIED ICHTHYOLOGY 27 (2) 484 - 491 0175-8659 2011/04 [Refereed][Not invited]
   

  In Acipenseriformes, extant species can be categorized into three ploidy groups based on their genome size inferred from chromosome numbers and / or nuclear DNA content. However, genome size is still inconclusive in some sturgeon species. Here, we reported genome size of Acipenser mikadoi, Huso dauricus, other seven sturgeon and one paddlefish species quantified by nuclear DNA content flow cytometry. We also measured DNA content of artificially propagated progenies of A. mikadoi and H. dauricus, and progenies from several hybrids using eggs of the two sturgeon species and the hybrid bester. Nuclear DNA content of A. mikadoi was 8.2 pg and thus this species was categorized into functional tetraploid or evolutionary octaploid species like closely related A. medirostris. Although H. dauricus has been considered to be functional diploid or evolutionary tetraploid species based on previous results, this species had 8.3 pg DNA content and was classified into the functional tetraploid or evolutionary octaploid group which includes A. mikadoi. Among artificially propagated A. mikadoi, high frequencies of triploid individuals (45%) and a little portion of tetraploid individuals (1.5%) were detected. The occurrence of polyploid larvae was also detected in hybrid progenies using eggs of A. mikadoi. All 1-year-old survivors (n = 3) of artificially propagated H. dauricus were triploid. The spontaneous occurrence of such genetic triploid and tetraploid progenies suggests that polyploidization event presumably occurs even at present and may cause intraspecific variation of genome size in Acipenseridae.
• Sexual dimorphism of gonadal structure and gene expression in germ cell-deficient loach, a teleost fish

  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 0027-8424 2010/10 [Refereed][Not invited]
   

  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.
• Primordial germ cell in teleost fish with special references to its specification and migration

  E. Yamaha, R. Goto-Kazeto, T. Saito, Y. Kawakami, T. Fujimoto, S. Adachi, K. Arai

  JOURNAL OF APPLIED ICHTHYOLOGY 26 (5) 816 - 822 0175-8659 2010/10 [Refereed][Not invited]
   

  P>Germ-line chimeras provide a valuable approach to surrogate propagation in poultry farming and aquaculture. The appropriate combinations of donor germ cells and host individuals enable the production of eggs and sperm and of offspring from strains with a high commercial value or valuable genotype via an easily reared species or strain. Primordial germ cells (PGCs) are one of the candidate cell types that can be used in the production of germ-line chimeras. The model teleost species, zebrafish, has been used to study specification, differentiation and migration of PGCs. However, it is important that the properties of PGCs are also determined for fish species used in aquaculture. Recent technical developments together with improvements in embryo manipulation techniques have enabled analyses of the differentiation of PGCs and their migration to the genital ridge. Here, we report the characteristic properties of PGCs from several teleost species and investigate their behavior in germ-line chimeras using fluorescently-tagged PGCs.
• Generation of germ-line chimera zebrafish using primordial germ cells isolated from cultured blastomeres and cryopreserved embryoids

  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 0214-6282 2010 [Refereed][Not invited]
   

  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.
• Inter-species transplantation and migration of primordial germ cells in cyprinid fish

  Taiju Saito, Rie Goto-Kazeto, Takafumi Fujimoto, Yutaka Kawakami, Katsutoshi Arai, Etsuro Yamaha

  INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 54 (10) 1481 - 1486 0214-6282 2010 [Refereed][Not invited]
   

  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.
• Developmental potential of embryonic cells in a nucleocytoplasmic hybrid formed using a goldfish haploid nucleus and loach egg cytoplasm

  Takafumi Fujimoto, Taiju Saito, Suzu Sakao, Katsutoshi Arai, Etsuro Yamaha

  INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 54 (5) 827 - 835 0214-6282 2010 [Refereed][Not invited]
   

  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.
• Reproductive capacity of neo-tetraploid loaches produced using diploid spermatozoa from a natural tetraploid male

  Takafumi Fujimoto, George Shigueki Yasui, Mayumi Hayakawa, Suzu Sakao, Etsuro Yamaha, Katsutoshi Arai

  AQUACULTURE 308 (Suppl.1) S133 - S139 0044-8486 2010 [Refereed][Not invited]
   

  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 5 min later by heat shock (42 degrees 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 degrees 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. (C) 2010 Elsevier B.V. All rights reserved.
• Restoration of the loach, Misgurnus anguillicaudatus, from cryopreserved diploid sperm and induced androgenesis

  George Shigueki Yasui, Takafumi Fujimoto, Katsutoshi Arai

  AQUACULTURE 308 (Suppl.1) S140 - S144 0044-8486 2010 [Refereed][Not invited]
   

  In the present study, we evaluated the feasibility of using cryopreserved diploid sperm as a repository genebank for the loach, Misgurnus anguillicaudatus, along with a rederivation strategy utilizing induced-androgenesis. Firstly, we evaluated three types of media for egg inactivation: Hank's saline solution + 0.5% bovine serum albumin (BSA). Ringer's solution + 0.5% BSA, and masu salmon seminal plasma. Haploid and diploid sperm were taken from diploid and tetraploid loaches, respectively. Fresh and cryopreserved haploid or diploid sperm were then used to fertilize intact or UV-irradiated eggs from wild diploid females. The irradiation media evaluated here successfully maintained the egg quality over 2 h. Fertilization and hatching rates of eggs fertilized with cryopreserved diploid sperm were 11.68 +/- 6.74% and 7.14 +/- 6.29% respectively, compared to 63.51 +/- 10.68% and 45.19 +/- 16.2% for intact eggs fertilized by fresh haploid sperm. All-male inheritance was confirmed by determination of larval morphology, ploidy status and microsatellite genotypes of putative androgenetic progeny. (C) 2010 Elsevier B.V. All rights reserved.
• Cutting edge of chromosome manipulation for aquaculture and conservation of Salmonids.

  Arai, K, Sakao, S, Fujimoto, T, Yamaha, E

  Journal of the National Taiwan Museum 14 49 - 59 2010 [Not refereed][Not invited]
  
• A sperm cryopreservation protocol for the loach Misgurnus anguillicaudatus and its applicability for other related species

  George Shigueki Yasui, Lenin Arias-Rodriguez, Takafumi Fujimoto, Katsutoshi Arai

  ANIMAL REPRODUCTION SCIENCE 116 (3-4) 335 - 345 0378-4320 2009/12 [Refereed][Not invited]
   

  The aim of the present study was to establish a protocol of sperm cryopreservation in Misgurnus anguillicaudatus and verify the applicability of the obtained protocol in other loach species. We evaluated the following parameters: inseminating dose, thawing temperatures (20, 25 and 30 degrees C for 10s), extenders (loach or cyprinid extenders), internal cryoprotectants (dimethyl sulfoxide (DMSO), dimethylacetamide (DMA), glycerol (Gly), ethylene glycol (EG),and methanol (MeOH)at 0, 5, 10 and 15%), external cryoprotectants (bovine serum albumin 1 and 2%; sucrose 0.5 and 1%; glucose 0.5 and 1%: glycine 0.5 and 1%), activating solutions (distilled water, dechlorinated tap water, 25 mM NaCl and 50 mM NaCl), and hatchability of the eggs when fertilized with fresh or cryopreserved sperm. After the evaluation of these parameters, we optimized the cryopreservation using the following procedure: thawing temperature at 25 degrees C for 10 s; loach or cyprinid extenders; methanol at 10 or 15% as internal cryoprotectants; glycine 0.5% or bovine serum albumin 1% as external cryoprotectants and 50 mM NaCl for sperm activation. Using this procedure, the fertilizability of the post-thawed sperm was 47% in comparison to the fresh sperm, at the minimum inseminating dose (687.65 spermatozoa egg(-1) mL(-1)). Based on this protocol, sperm from other loach species Lefua nikkonis, Misgurnus mizolepis and Barbatula toni were cryopreserved successfully. (C) 2009 Elsevier B.V. All rights reserved.
• Artificially induced tetraploid masu salmon have the ability to form primordial germ cells

  Suzu Sakao, Takafumi Fujimoto, Terumasa Kobayashi, Goro Yoshizaki, Etsuro Yamaha, Katsutoshi Arai

  FISHERIES SCIENCE 75 (4) 993 - 1000 0919-9268 2009/07 [Refereed][Not invited]
   

  Diploid gametes generated with tetraploid animals are a stepping stone to improving chromosome manipulation techniques. However, artificially induced tetraploid individuals generally die soon after hatching. Diploid gametes could be 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 messenger RNA (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.
• Chromosome Doubling in Early Spermatogonia Produces Diploid Spermatozoa in a Natural Clonal Fish

  Hiroyuki Yoshikawa, Kagayaki Morishima, Takafumi Fujimoto, Taiju Saito, Tohru Kobayashi, Etsuro Yamaha, Katsutoshi Arai

  BIOLOGY OF REPRODUCTION 80 (5) 973 - 979 0006-3363 2009/05 [Refereed][Not invited]
   

  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.
• SIMPLE AND INEXPENSIVE METHOD FOR CRYOPRESERVATION OF FISH SPERM COMBINING STRAW AND POWDERED DRY ICE

  George Shigueki Yasui, Lenin Arias-Rodriguez, Takafumi Fujimoto, Katsutoshi Arai

  CRYOLETTERS 29 (5) 383 - 390 0143-2044 2008/09 [Refereed][Not invited]
   

  Here, we propose a simple and inexpensive method for fish sperm cryopreservation. Sperm samples of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) were diluted 7-fold by an extender containing 63.5mM NaCl, 114mM KCl, 20mM Tris and 10% methanol. The cryogenic straws were placed in three kinds of self-made tubes which diameter was changed by commercially available materials and then immersed into powdered dry ice for 2 min and plunged into liquid nitrogen. This procedure resulted in a cooling rate at -421.4 +/- 119.84 (control), -55.8 +/- 4.32 (tube 1), -40.2 +/- 3.43 (tube 2) and -33.3 +/- 2.09 degrees C/min (tube 3). In the slowest cooling rate by the tube 3, total motility (72 +/- 3%), duration (146 +/- 12s) and hatching rates (29 +/- 04%) were higher than those by other rates. Progressive motility (83 +/- 5%) did not differ significantly from fresh samples.
• Ploidy manipulation using diploid sperm in the loach, Misgurnus anguillicaudatus: a review

  H. Yoshikawa, K. Morishima, T. Fujimoto, L. Arias-Rodriguez, E. Yamaha, K. Arai

  JOURNAL OF APPLIED ICHTHYOLOGY 24 (4) 410 - 414 0175-8659 2008/08 [Refereed][Not invited]
   

  This paper assesses the present state of the art of ploidy manipulation in the loach, Misgurnus anguillicaudatits (Teleoste: Cobiticlac). Diploid sperm can be obtained from natural tetraploid individuals with four sets of homologous chromosomes. Using diploid sperm, various polyploids and androgenetic diploids have been produced. Cryptic clonal lineages are also recognized in wild populations of the loach. They produce unreduced diploid eggs genetically identical to somatic cells of the mother fish and most diploid eggs develop gynogenctically 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. anglui-ficaudatus might not be a single species, but a complex involving cryptic species, because wild populations exhibited genetic differentiation at interspecific level. This implies possible relationship between atypical reproduction and natural hybridization in the loach.
• Genetic and reproductive potential of spermatozoa of diploid and triploid males obtained from interspecific hybridization of Misgurnus anguillicaudatus female with M. mizolepis male

  T. Fujimoto, G. S. Yasui, H. Yoshikawa, E. Yamaha, K. Arai

  JOURNAL OF APPLIED ICHTHYOLOGY 24 (4) 430 - 437 0175-8659 2008/08 [Refereed][Not invited]
   

  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, In, 2n and 4n spermatozoa showing low motility were detected. However, spermatozoa of three diploid hybrid males could generate 2n larvae. Therefore, only In 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 In sperinatozoa. Although no motility was observed, larvae arose from eggs inseminated with such spermatozoa. Type 3: In the fourth male, only In spermatozoa were detected and their motility was vigorous. When eggs were fertilized with such In spermatozoa, normal larvae hatched. In 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 In spermatozoa by a kind of transformation of meiosis like meiotic hybridogenesis.
• Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

  Ekaterina Bubenshchikova, Elena Kaftanovskaya, Nami Motosugi, Takafumi Fujimoto, Katsutoshi Arai, Masato Kinoshita, Hisashi Hashimoto, Kenjiro Ozato, Yuko Wakamatsu

  DEVELOPMENT GROWTH & DIFFERENTIATION 49 (9) 699 - 709 0012-1592 2007/12 [Refereed][Not invited]
   

  Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.
• Simultaneous formation of haploid, diploid and triploid eggs in diploid-triploid mosaic Roaches

  H. Yoshikawa, K. Morishima, T. Fujimoto, E. Yamaha, K. Araj

  JOURNAL OF FISH BIOLOGY 71 250 - 263 0022-1112 2007/10 [Refereed][Not invited]
   

  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 ultraviolet-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 possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the 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. (c) 2007 The Authors Journal compilation (c) 2007 The Fisheries Society of the British Isles.
• Evaluation of different doses of UV irradiation to loach eggs for genetic inactivation of the maternal genome

  Takafumi Fujimoto, Suzu Sakao, Etsuro Yamaha, Katsutoshi Arai

  JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL GENETICS AND PHYSIOLOGY 307A (8) 449 - 462 1932-5223 2007/08 [Refereed][Not invited]
   

  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).
• Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei : Cobitidae)

  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 1932-5223 2007/01 [Refereed][Not invited]
   

  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.
• Developmental stages and germ cell lineage of the loach (Misgurnus anguillicaudatus)

  Takafumi Fujimoto, Takashi Kataoka, Suzu Sakao, Taiju Saito, Etsuro Yamaha, Katsutoshi Arai

  ZOOLOGICAL SCIENCE 23 (11) 977 - 989 0289-0003 2006/11 [Refereed][Not invited]
   

  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.
• Premeiotic endomitosis produces diploid eggs in the natural clone loach, Misgurnus anguillicaudatus (Teleostei : Cobitidae)

  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 1548-8969 2006/06 [Refereed][Not invited]
   

  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.
• Drastic mortality in tetraploid induction results from the elevation of ploidy in masu salmon Oncorhynchus masou

  S Sakao, T Fujimoto, S Kimura, E Yamaha, K Arai

  AQUACULTURE 252 (2-4) 147 - 160 0044-8486 2006/03 [Refereed][Not invited]
   

  Although tetraploidy is considered to be important and useful for the production of sterile triploids and fertile allo-tetraploids 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/cm(2), 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 degrees 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 hypertetraploid in 4N and diploid or hypodiploid 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. (c) 2005 Elsevier B.V. All rights reserved.
• Visualization of primordial germ cells in vivo using GFP-nos1 3 ' UTR mRNA

  Taiju Saito, Takafumi Fujimoto, Shingo Maegawa, Kunio Inoue, Minoru Tanaka, Katsutoshi Arai, Etsuro Yamaha

  INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 50 (8) 691 - 700 0214-6282 2006 [Refereed][Not invited]
   

  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.
• The germ line lineage in ukigori, Gymnogobius species (Teleostei : Gobiidae) during embryonic development

  T Saito, S Otani, T Fujimoto, T Suzuki, T Nakatsuji, K Arai, E Yamaha

  INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY 48 (10) 1079 - 1085 0214-6282 2004/12 [Refereed][Not invited]
   

  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.
• Embryonic stages from cleavage to gastrula in the loach Misgumus anguillicaudatus

  T Fujimoto, T Kataoka, S Otani, T Saito, T Aita, E Yamaha, K Arai

  ZOOLOGICAL SCIENCE 21 (7) 747 - 755 0289-0003 2004/07 [Refereed][Not invited]
   

  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.
• Clonal diploid sperm of the Diplcoid-Triploid mosaic loach, Misgurnus anguillicaudatus (Teleostei : Cobitidae)

  K Morishima, K Oshima, S Horie, T Fujimoto, E Yamaha, K Arai

  JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-COMPARATIVE EXPERIMENTAL BIOLOGY 301A (6) 502 - 511 0022-104X 2004/06 [Refereed][Not invited]
   

  The loach Misgurnus anguillicaudatus comprises diploid, triploid and diploid-triploid mosaic individuals in a wild population of the Hokkaido island, Japan. Previous studies revealed the presence of a cryptic clonal lineage among diploid loaches, which is maintained by uniparental reproduction of genetically identical diploid eggs. In the present study, we analyzed distribution and genetic status of diploid and triploid cells in infrequent mosaic males. Flow cytometry, microsatellite genotyping and DNA fingerprinting verified that mosaic males consisted of diploid cells with genotypes identical to the natural clone and triploid cells with diploid genomes of the clonal lineage plus haploid genome from sperm nucleus of the father. Thus, the occurrence of diploid-triploid mosaicism might be caused by accidental fertilization of a diploid blastomere nucleus with haploid sperm after the initiation of clonal development of unreduced eggs. Such mosaic males produced fertile sperm with diploid DNA content. The experimental cross between normal diploid female and diploid-triploid mosaic male gave rise to the appearance of triploid progeny which exhibited two microsatellite alleles identical to the clonal genotype and one allele derived from the normal female. In DNA fingerprinting, such triploid progeny gave not only all the DNA fragments from the clone, but also other fragments from the normal female. Induced androgenesis using UV irradiated eggs and sperm of the mosaic male gave rise to the occurrence of diploid individuals with paternally derived microsatellite genotypes and DNA fingerprints, absolutely identical to the natural clonal lineage. These results conclude that the diploid-triploid mosaic male produced unreduced diploid sperm with genetically identical genotypes. The spermatogenesis in the clonal diploid cells under the mosaic condition suggests that triploid male somatic cells might transform genetically all female germ cells to differentiate into functionally male gametes. The discovery of the mosaic male producing unreduced sperm suggests the theoretical occurrence of triploids and other polyploids by the syngamy of such paternally derived diploid gametes. (C) 2004 Wiley-Liss, Inc.
• Clonal diploid sperm of the diploid-triploid mosaic loach, Misgurnus anguillicaudatus (Teleostei:Cobitidae).

  Morishima, K, Oshima, K, Horie, S, Fujimoto, T, Yamaha, E, Arai, K

  Journal of experimental zoology. Part A, Comparative experimental biology 301 502 - 511 1548-8969 2004/06 [Refereed][Not invited]
  
• Recovery of fertility in male hybrids of a cross between goldfish and common carp by transplantation of PGC (primordial germ cell)-containing graft

  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 0016-6707 2003/10 [Refereed][Not invited]
   

  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.
• Aberrant and arrested embryos from masu salmon eggs treated for tetraploidization by inhibition of the first cleavage

  S Sakao, T Fujimoto, M Tanaka, E Yamaha, K Arai

  NIPPON SUISAN GAKKAISHI 69 (5) 738 - 748 0021-5392 2003/09 [Refereed][Not invited]
   

  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 C) 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.
• Spontaneous mosaicism occurred in normally fertilized and gynogenetically induced progeny of the kokanee salmon Oncorhynchus nerka

  M Tanaka, S Kimura, T Fujimoto, S Sakao, E Yamaha, K Arai

  FISHERIES SCIENCE 69 (1) 176 - 180 0919-9268 2003/02 [Refereed][Not invited]
   

  In normally fertilized progeny of the kokanee salmon Oncorhynchus nerka, DNA content flow cytometry revealed that all the externally normal embryos were diploid, whereas abnormal embryos exhibited haplo-diploid, diplo-tetraploid and haplo-diplo-tetraploid mosaicisms, together with a few haploid and diploid individuals. When gynogenetic development was artificially induced by fertilization of eggs obtained from a female of the same kokanee brood stock with UV-irradiated sperm, haplo-diploid mosaics appeared most frequently. These mosaics were likely to happen by certain cytological events, such as meiotic or mitotic errors during the process of maturation, fertilization or early cleavage.
• Haplo-diploid mosaic embryos in cutthroat trout Oncorhynchus clarki

  Yamaha, E, Kimura, S, Tanaka, M, Sakao, S, Fujimoto, T, Arai, K

  Fish Genetics and Breeding Science 32 121 - 126 2002 [Refereed][Not invited]
  
• Polyploid and mosaic crucian carp found in Tokotan-numa Swamp, Akkeshi, Hokkaido

  Yamaha, E, Murakami, M, Takeyama, S, Morishima, K, Otani, S, Horie, S, Tanaka, M, Fujimoto, T, Oshima, K, Sakao, S, Aida, T, Arai, K

  Fish Genetics and Breeding Science 32 19 - 26 2002 [Refereed][Not invited]
  
• Germ-line chimera by lower-part blastoderm transplantation between diploid goldfish and triploid crucian carp

  E Yamaha, M Kazama-Wakabayashi, S Otani, T Fujimoto, K Arai

  GENETICA 111 (1-3) 227 - 236 0016-6707 2001 [Refereed][Not invited]
   

  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.

Books etc

• Sex Control in Aquaculture Volume I

  Katsutoshi ARAI, Takafumi FUJIMOTO (Joint workChapter 6 Chromosome Manipulation Techniques and Applications to Aquaculture)
  Wiley and Sons, Ltd. 2019 (ISBN: 9781119127291)
• Fish Genetics and Breeding Science

  Takafumi Fujimoto (ContributorChapter 6 and 10)
  Tohoku University Press, Sendai 2017/03 (ISBN: 9784861632709) 243 99-118、171-186

Industrial Property Rights

• WO2011-099528:生殖系列キメラを介して致死的魚類半数体に由来する生殖細胞から遺伝的に同一な配偶子を得る方法  2011/08/18

  山羽 悦郎, 荒井 克俊, 藤本 貴史, 斎藤 大樹  国立大学法人北海道大学  201303084993461420

Awards & Honors

• 2017/03 The Japanese Society of Fisheries Science Progress Award
   Loach as a model fish for developmental engineering and chromosome manipulation 

  受賞者: Takafumi Fujimoto
• 2010/03 The Japanese Society of Fisheries Science Award of Excellence for a Scientific Paper (2009)
   

  受賞者: Sakao S;Fujimoto T;Kobayashi T;Yoshizaki G;Yamaha E;Arai K

Research Grants & Projects

• 半クローン雑種の総括研究-永続性の実証（究極要因）とゲノム削除機構（至近要因）

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/04 -2024/03 

  Author : 宗原 弘幸, 藤本 貴史

   

  半クローンやクローンは、雌だけを産むため個体群増殖速度が速いが、遺伝的な多様性を作り出せないなどの欠点から、系統寿命は短いと考えられてきた。しかし、アイナメ属半クローン雑種を用いた研究から、一世代で父親ゲノムを「置換」できる半クローンの特性により、母種の雄と交配することで組換え可能な子を作り出せる。本研究では、アイナメ属の系統進化の過程で、ホスト種を換えることによって半クローンと組換え世代を繰り返し、系統寿命を伸ばしてきたことを実証する。さらに半クローン配偶子が形成過程を細胞学的に解明する。 本年度は以下のことを実施した。 1. 父種ゲノムの削除機構の細胞学的観察と遺伝マーカーの作成 半クローンは、体細胞では父種と母種のゲノムが協働して雑種成体となるが、生殖細胞は母種ゲノムだけを伝える配偶子を作る。父種ゲノムが排除される仕組みを明らかにするため、卵原細胞から卵母細胞になるまでの染色体動態を蛍光観察するためのプローブ作成を行った。 2. 半クローン遺伝子のホストスイッチを経由した永続性の実証 スジアイナメゲノムの半クローン雑種には、アイナメを宿主にするアイナメ系とクジメを宿主にするクジメ系が北海道南部とロシア沿海州で見つかっている。スジアイナメは北太平洋東岸で起源し極東まで分布を広げた。その過程には、多様な環境があったが、そこを近縁種のゲノムを宿主にして乗り越えて極東にたどり着いたと考えられる。これを『ホストスイッチ仮定』とよんでいる。これを実証するために、もっとも寒冷域にまで分布するエゾアイナメを厚岸町から入手した。エゾアイナメの精子で半クローンの卵を人工受精した。その結果、受精し、孵化仔魚も他の純粋種と比較して順調に育つことがわかった。本年度は得られた稚魚のDNA量、両親種のゲノムが遺伝したことを確認した。次年度はこの雑種が半クローンかどうかを確認する。
• Mechanism of diploid gamete formation and its application for genetic breeding

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

  Date (from‐to) : 2021/04 -2024/03 

  Author : 藤本 貴史, 西村 俊哉, 田中 啓介, 黒田 真道

   

  ①ゲノム倍加を生じる系統間ゲノムの違い、②ゲノム倍加の分子機構の解明、③雑種由来の二倍性配偶子の育種応用に向けた実証研究の3項目の概要を下記に示す。 ①ドジョウB系統の倍加半数体の遺伝子型が完全同型接合であることを確認し、倍加半数体の高分子DNAをロングリードシーケンサーによるゲノム解析に供した。得られたリードデータを用いて、ゲノム構造解析の基盤整備を行った。また、B系統特異的なFISHプローブを開発するとともに、人為的に作出した二系統間雑種でGISHを行った結果、A系統をプローブDNAに用いた場合はA系統由来と推定される染色体の全域が染色されたのに対し、B系統をプローブDNAに用いた場合はB系統と推定される染色体のセントロメア領域が強く染色され、系統間におけるゲノム構造の変異が示唆された。 ②クローンドジョウの卵巣をトリプシンやコラゲナーゼ等の消化酵素を用いて細胞を開始した後、メッシュフィルターを通して大型卵母細胞を除去することにより、小型の細胞のみを回収した。これらの細胞を固定後、Vasa抗体を用いて免疫蛍光染色に供した結果、比較的大型の核を有する卵原細胞が確認された。また、FACSによる分析では、Vasa陽性の細胞がDAPI蛍光強度で3種類に分類できることが明らかとなった。一方、生殖細胞の可視化にむけた遺伝子組換えクローンドジョウでは、piwiあるいはvasaのプロモーター領域とEGFPからなるコンストラクトを作製し、これらを顕微注入した遺伝子組換え候補個体の作出を行った。 ③サケ科ではイワナ属の種間雑種では受精能を有する半数性の配偶子形成が確認された。他のサケ科雑種に関しては成熟に至らず、現在も継続して飼育している。ドジョウ雑種ではカラドジョウ雌と各系統のドジョウ雄の雑種雄は不妊性を示すことが確認された。メダカ属雑種では、雑種個体を誘起し成熟に向けて飼育を継続している。
• 育種・染色体操作を用いたサケ科魚類の魚卵アレルゲン性低減化の試み

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2020/04 -2023/03 

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

   

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

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

  Date (from‐to) : 2016/04 -2021/03 

  Author : YAMAHA Etsuro

   

  It takes a long time for genetic breeding in commercially important fish species, because of their low survival rate in their early stage of development and long generation time. When germline chimeras, in which primordial germ cells (PGCs) with genetic diversity are transplanted into host blastula of different species are induced, only PGCs adapted under different gonadal environment are expected to differentiate into normal gametes. As these chimera individuals will select such adapted PGCs in vivo, they shorten the breeding times. In this application, we established such techniques, namely cell sorting of visualized PGCs with GFP fluorescence at the blastula stage, loading into glass needle as cell mass, and transplantation into the host blastula. When a large number of sorted PGCs were transplanted, low number of them migrated to host gonadal region, while many distributed various regions of the host embryo and formed cell mass in some cases. Other methods will be required.
• Origin and evolution of hemiclonal Hexagrammos hybrids elongating the lineage lifetime by host-switching

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

  Date (from‐to) : 2017/04 -2020/03 

  Author : MUNEHARA Hiroyuki

   

  Unisexual vertebrates (i.e., those produced through clonal or hemiclonal reproduction) are typically incapable of purging deleterious mutations, and as a result, are considered short-lived in evolutionary terms although high increasing rate. However, most of clonal and hemiclonal vertebrates have existed far longer than expected theoretical generation longevities. This study clarified that hemiclonal Hexagrammos hybrids use two-way backcrossing (clonal genomes are returned to the gene pool where they can undergo recombination plays an important role in increasing the genetic variability of the hemiclonal genome and reducing the extinction risk). In this way, hemiclonal lineages may have survived longer than predicted through occasional recombinant generation. In addition, genes inducing hemiclonal reproduction by discarding of paternal genome during oogenesis have been identified by RNA-seq although not yet being completely analyzed.
• Control of fish spermatogenesis by genome editing

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

  Date (from‐to) : 2016/04 -2019/03 

  Author : FUJIMOTO TAKAFUMI, YAMAZAKI kyo, NISHIHARA Hiroki

   

  Artificial induction of infertile fish is necessary for application of transgenic and genome edited fish for aquaculture. In this study, mutation of genes associated with sperm flagellum movement and spermiogenesis were induced by genome editing using CRISPR/Cas9 in order to develop novel male specific sterilization in zebrafish Danio rerio and dojo loach Misgurnus anguillicaudatus. We successfully induced frame shift mutations in the both genes in germ cells of F0 individuals, because the F0 males produced sperm with the mutations. The sperm showed motility as well as wild type and could fertilize to induce F1 generation. F2 generation with homozygous deficiency of spermiogenesis related gene was successfully produced by mating within F1 individuals of heterozygous mutation. But production of functional sperm was observed in the F2 generation. The unexpected functional sperm production might be caused by illegitimate translation and/or expression of orthologue genes.
• Molecular mechanisms of atypical development and reproduction caused by hybridization and the application to breeding in fishes

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

  Date (from‐to) : 2015/04 -2019/03 

  Author : ARAI KATSUTOSHI, FUJIMOTO Takafumi, YAMAHA Etsuro, MUNEHAEA Hiroyuki

   

  Hybridization between different fish species produced viable or inviable progeny. Recovery of survival potential by allotriploidization was often recorded in certain inviable hybrids. In viable hybrids, germ cells were not normally differentiated and hermaphroditic gonads were frequently detected. Natural clonal loach has the hybrid origin between two genetically diverse strains. Each chromosome of the clone cannot find its counterpart for pairing in the course of meiosis. Thus, each chromosome is duplicated by premeiotic endomitosis and then sister chromosome pairing assures the formation of isogenic gametes. Natural Hexagrammos hybrids have large metacentic chromosomes closely linking to hemi-clonal reproduction (hybridogenesis). Thus, these chromosomes were used as marker of hemi-clone to detect presence or absence of back-crossed progeny between hemi-clonal hybrid female and paternal pure species.
• Studies on reduced 2n sperm and unreduced 4n egg formation in tetraploid crucian carp

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

  Date (from‐to) : 2015/04 -2017/03 

  Author : ARAI Katsutoshi, FUJIMOTO Takafumi, YAMAHA Etsuro, MURAKAMI Masaru, SUZUKI Hiroko, TANAKA Hideki

   

  Although triploid (3n) silver crucian carp reproduces by gynogenesis as all-female clonal line, both female and male appear in tetraploiod (4n). 4n females generate unreduced 4n eggs which reproduce by gynogenesis, while 4n males produce reduced 2n sperm. Here, the mechanism responsible for such a different meiotic manner between the two sexes is attempted to disclose and obtained results are as follows:(1) some males produced aneuploid sperm, (2) 3n progeny, 4n progeny, or both 3n and 4n progeny occurred in cross-fertilization between 3n female and diploid goldfish male, (3) Samples analyzed here included unusually high rates of Chinese silver crucian carp. Thus, irregular meiosis and gametogenesis in 4n crucian carp may relate to the genomic consititution of each 4n individual.
• Effects of genomic constitution in germ cell on gametogenesis in fish

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

  Date (from‐to) : 2013/04 -2016/03 

  Author : Fujimoto Takafumi

   

  The effects of genomic constitution in germ cell on gametogenesis was studied using progeny, which were produced by artificial hybridization using loach derived from two genetically different groups and clonal line. Allodiploid males produced a small amount of haploid sperm and female suggested a fertility because of oocyte growth in the ovary. In allotriploids, males showed infertility due to the defect in meiosis, although females showed fertility as well as the allodiploid female. In the infertile allotriploid male, abnormal pairing in meiotic chromosomes were detected by Fluorescence in situ hybridization. Genomic in situ hybridization revealed genetic differences between chromosomes of the two groups. Transcriptome analysis based on RNA-seq to compare gene expressions between gonads with unreduced or reduced gametogenesis showed different expressing genes associated with cell division and cell cycle etc.
• Revision and application of polyspermy in fish

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

  Date (from‐to) : 2013/04 -2015/03 

  Author : ARAI KATSUTOSHI, FUJIMOTO Takafumi

   

  About 1, 10, 100 or 1000 spermatozoa of albino male were microinjected into an activated and then dechorionated egg of wild-type female loach Misgurnus anguillicaudatus. The group injected with about 1000 spermatozoa gave 87% cleavage rate, almost equivalent to control and 6% hatched. When dechorionated eggs were inseminated with sperm to induce polyspermy, 2% hatched. Expression of albino trait and all-male inheritance of microsatellite DNA markers concluded that most resultant haploid, mosaic including haploid cells, and tetraploid embryos were androgenetically developed progeny. While diploid, triploid, aneuploid and mosaic embryos also occurred by the fusion of egg nucleus and single to multiple numbers of sperm nuclei. Similar development was observed in microinjected and polyspermy eggs of zebrafish.
• 魚類の倍数性配偶子誘導にむけた自然クローン魚におけるゲノム倍加機構の解明

  日本学術振興会：科学研究費助成事業 研究活動スタート支援

  Date (from‐to) : 2012/08 -2014/03 

  Author : 藤本 貴史

   

  一部のドジョウでは天然でクローン生殖や倍数性配偶子の産出が認められ、その特殊な配偶子形成を人為的に制御できれば非常に有用な技術となりうるが、その分子メカニズムは未だ不明である。このメカニズム解明において、ゲノム倍加が起こるタイミングの特定とその時期の生殖細胞を単離し解析しなければならず、そのためにはドジョウの生殖腺発達段階のステージングと生殖細胞を解析するための新たな実験系を構築する必要がある。そこで、本年度はサンプルとして入手が容易な通常両性生殖個体を用いて実験家系を作出し、継時的に体長測定を行うとともに生殖腺を採取し、組織学的に解析に供した。そして、本年度の研究によって仔魚期から性分化期までの生殖腺の発達段階のステージングをおおむね行うことができ、生殖細胞の増殖期と減数分裂開始時期の特定ができた。その結果、成長初期の生殖細胞の活発な増殖はメスで顕著に観察され、ある一定の体サイズに成長した段階で、雌の生殖腺では減数分裂に移行し卵形成が開始することが明らかとなった。本知見はクローンドジョウの生殖腺における生殖細胞のゲノム倍加が生殖腺発達段階のどの時期で起こっているかを調査するためには必須の基礎的知見であり、希少なクローン個体を用いた生殖腺サンプリングにおいて、調査に必要なサンプリング時期を定めるためには重要である。しかし、生殖腺ステージングで明らかとなった減数分裂開始時期は、卵巣発達過程においてかなり早い段階で起きており、この発達段階の卵巣を実体顕微鏡下で単離することはかなりの困難を要する。この対策として密度勾配遠心による卵巣細胞の分離が考えられ、本年度はパーコールを用いた生殖細胞の分離・純化技術の開発を遂行し、特定の密度分画における生殖細胞の分離が可能となった。
• サケ科交雑種の生存性と、異種ゲノムの組合せが遺伝子発現に与える影響

  北海道大学：北海道大学総長室事業推進経費「若手研究者自立支援」

  Date (from‐to) : 2012 -2012 

  Author : 藤本 貴史
• 魚類の精子を用いた遺伝資源の保存と個体再生に関する研究

  日本学術振興会：科学研究費助成事業 若手研究(B)

  Date (from‐to) : 2006 -2007 

  Author : 藤本 貴史

   

  本年度は、初年度に開発した技術の応用と個体の解析に主眼をおいて研究を行った。凍結保存精子からの個体再生実験では、ドジョウ凍結精子と通常卵との受精からは孵化仔魚を得ることができたが、凍結精子を用いた人為雄性発生では生存性の孵化仔魚を得ることができず、精子ゲノムのみでの個体再生には至らなかった。しかしながら、ドジョウ精子の凍結保存方法を他のドジョウ属魚類(カラドジョウ、フクドジョウ、エゾホトケドジョウ)へ応用したところ、本実験で用いたドジョウ属魚類の凍結精子は解凍後にも運動性を示したことから、本研究で開発した凍結保存方法が広範囲のドジョウ属魚類に応用可能であることが示された。二倍性精子に関しては、まず、初年度に作製した新四倍体系統が産する二倍性精子に由来する雄性発生個体の雄の妊性を確認した。これらの成熟した個体からは受精能を有する半数性精子が産出され、正常個体が生じたことから、新四倍体系統の産する二倍性精子は再生産可能な二倍体個体を作出できることが明らかとなった。次に、本年度ではポリエチレングリコール溶液(PEG)と高カルシウム溶液(高Ca)を用いた化学処理を施し、通常二倍体が産する半数性精子の接着・融合による倍加法を検討した。核とミトコンドリアを蛍光染色した精子の形態観察では、処理群においてのみ接着・融合している精子が観察された。また、各化学処理を比較したところ、PEG処理では僅かに接着・融合精子が見受けられるものの、PEG濃度の増加とともに精子の運動性が顕著に低下し、多数の精子凝集塊が生じた。一方、高Ca処理では、精子の運動性は濃度や処理時間によって大きな影響は受けず、PEG処理よりも多数の接着・融合精子が生じた。このことから、高Ca処理は精子の接着・融合に有効な処理方法であると考えられた。
• ドジョウを用いた宿主不妊化技術の研究

  北海道大学：21世紀COEプログラム若手研究者研究活動経費

  Date (from‐to) : 2005 -2005 

  Author : 藤本 貴史

Educational Activities

Teaching Experience

Social Contribution

Social Contribution

Social Contribution

• 道南おさかな図鑑「どじょう」

  Date (from-to) : 2021/09/28

  Role : Contribution

  Sponser, Organizer, Publisher  : 北海道新聞社

  Event, Program, Title : 北海道新聞

Academic Contribution

• Expert investigator

  Date (from-to) :2013/08/01-Today

  Role: Academic research planning

  Type: Academic research

  Organizer, responsible person: National Institute of Science and Technology Policy (NISTEP)