Publications by authors named "Yutaka Takeuchi"

52 Publications

Characterization of the sex differentiation and gonadal development in small yellow croaker (Larimichthys polyactis) and its hybrid (L. polyactis ♀ × L. crocea ♂).

Fish Physiol Biochem 2021 Jul 29. Epub 2021 Jul 29.

Key Lab of Mariculture and Enhancement of Zhejiang Province, Zhejiang Marine Fisheries Research Institute, Zhoushan, 316021, China.

Interspecific hybridization has been considered as a possible approach to improve biological traits and has been applied in aquaculture practices. In the present study, artificial hybridization was carried out in the small yellow croaker (SYC; Larimichthys polyactis) ♀ × large yellow croaker (LYC; L. crocea) ♂ by artificial insemination, and the processes of sex differentiation and gonadal development in SYC and its hybrid were investigated under controlled conditions. Histological analysis of SYC larvae showed that migrating primordial germ cells (PGCs) were observed at 4 days post-hatching (dph), a genital ridge was formed on the dorsal side of the peritoneum at 6 dph, and a pair of primary gonads was first observed at 10 dph. Signs of the differentiated ovary and ovarian cavity were observed at 45 dph. However, some presumptive testes showed alterations in morphology, including an increase in the number of oocytes and an enhanced basophilia at 50 dph. These presumptive testes seemed to alter again, and numerous gonial cells were arranged in cyst-like groups with several degenerating oocytes that developed into residual body-like structures during 60-90 dph. Compared with SYC, the hybrid had a lower number of PGCs and showed retarded gonadal development at the early stage. Ovarian differentiation in the hybrid was observed at 50 dph, while testicular differentiation occurred at 60 dph. The presence of vitellogenic oocytes and spermatozoa at 360 dph in the hybrids suggested that hybrid individuals can undergo successful gametogenesis in females and males, respectively. Overall, the present results suggest that morphological sex differentiation occurred at 40 and 50 dph in SYC and its hybrid, respectively, both of which have normal gametogenesis. Moreover, some level of heterosis (hybrid vigor) occurred in the growth of the hybrid (total length and body weight) compared with that in the growth of SCY over time. Gonadal development of the hybrid was also found to be advanced at 360 dph. The present information will contribute to the potential use and management of these fish for aquaculture.
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http://dx.doi.org/10.1007/s10695-021-00975-0DOI Listing
July 2021

Production of functional sperm from cryopreserved testicular germ cells following intraperitoneal transplantation into allogeneic surrogate in yellowtail (Seriola quinqueradiata).

Cryobiology 2021 06 5;100:32-39. Epub 2021 Apr 5.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo, 108-8477, Japan.

The aim of this study was to establish a method for the cryopreservation of spermatogonia of the yellowtail (Seriola quinqueradiata), which is the most commonly farmed fish in Japan. Testicular cells were prepared by enzymatic dissociation of testicular fragments containing an abundance of type A spermatogonia and were added to cryomedium containing dimethyl sulfoxide (DMSO), ethylene glycol, glycerol, or propylene glycol at concentrations of 0.5-2.5 M. The cells were then frozen and stored in liquid nitrogen for 3 days. After thawing, their survival and transplantability were evaluated. Testicular cells were most successfully cryopreserved in 1.0 M DMSO as indicated by survival of 34% of cells. Furthermore, in situ hybridization using the yellowtail vasa probe showed that these recovered cells contained a similar proportion of germ cells to fresh testicular cells before freezing. Transplantation of the recovered cells into the peritoneal cavities of allogeneic larvae resulted in 94% of surviving recipients having donor-derived germ cells in their gonads after 28 days. Sperm were then collected from seven randomly selected recipients once they reached 2 years of age and used to fertilize wild-type eggs, which led to an average of 26% of the first filial (F1) offspring being derived from donor fish, as confirmed through the use of microsatellite markers. Thus, we successfully cryopreserved yellowtail spermatogonia and produced functional sperm via intraperitoneal transplantation into allogeneic recipients.
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http://dx.doi.org/10.1016/j.cryobiol.2021.04.001DOI Listing
June 2021

Assessment of Yangtze sturgeon as recipient for the production of American paddlefish gametes through spermatogonia transplantation.

Theriogenology 2020 Dec 15;158:168-179. Epub 2020 Aug 15.

Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture and Rural Affairs, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, 430223, China. Electronic address:

The Chinese paddlefish (Psephurus gladius), one of the world's largest freshwater fish, was last seen alive in 2003; they are presumed now to be extinct. In fish, germ cell transplantation is currently known as one of the most powerful assisted reproductive technologies for the conservation of endangered species. In the event that a Chinese paddlefish is unexpectedly caught in the near future, we aimed to develop an experimental strategy to produce paddlefish gametes in the gonads of surrogate sturgeon. Spermatogonia were collected from the testes of 2.5-year-old immature male American paddlefish (Polyodon spathula), the species most closely related to the Chinese paddlefish, by Percoll gradient centrifugation, and transplanted into the peritoneal cavity of Yangtze sturgeon (Acipenser dabryanus) larvae at 7-8 days post-hatch. At two months post-transplantation, donor-derived spermatogonia had efficiently colonized in the recipient gonads and proliferated. A PCR analysis developed to detect xenogenic donor-derived mtDNA sequences in recipient gonads revealed that American paddlefish germ cells survived for at least seven months after transplantation in the gonads of Yangtze sturgeon recipients. These results show that the somatic microenvironment of Yangtze sturgeon gonads was able to support the colonization, proliferation, and survival of xenogeneic germ cells from a different taxonomic family. This study provides key information that could lead to future restoration of Chinese paddlefish using germ cell transplantation.
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http://dx.doi.org/10.1016/j.theriogenology.2020.08.005DOI Listing
December 2020

Production of donor-derived eggs after ovarian germ cell transplantation into the gonads of adult, germ cell-less, triploid hybrid fish†.

Biol Reprod 2020 12;103(6):1289-1299

Noto Center for Fisheries Science and Technology, Faculty of Biological Science and Technology, Kanazawa University, Ishikawa, Japan.

In animals, spermatogonial transplantation in sterile adult males is widely developed; however, despite its utility, ovarian germ cell transplantation is not well developed. We previously showed that the interspecific hybrid offspring of sciaenid was a suitable model for germ cell transplantation studies as they have germ cell-less gonads. However, all these gonads have testis-like characteristics. Here, we tested whether triploidization in hybrid embryos could result in germ cell-less ovary development. Gonadal structure dimorphism and sex-specific gene expression patterns were examined in 6-month-old triploid hybrids (3nHybs). Thirty-one percent of 3nHybs had germ cell-less gonads with an ovarian cavity. cyp19a1a and foxl2, ovarian differentiation-related genes, were expressed in these gonads, whereas dmrt1 and vasa were not expressed, suggesting ovary-like germ cell-less gonad development. Some (26%) 3nHybs had testis-like germ cell-less gonads. Ovarian germ cells collected from homozygous green fluorescent protein (GFP) transgenic blue drum (BD) (Nibea mitsukurii) were transplanted into 6-month-old 3nHybs gonads via the urogenital papilla or oviduct. After 9 months, the recipients were crossed with wild type BD. Among the six 3nHyb recipients that survived, one female and one male produced fertile eggs and motile sperm carrying gfp-specific DNA sequences. Progeny tests revealed that all F1 offspring possessed gfp-specific DNA sequences, suggesting that these recipients produced only donor-derived eggs or sperm. Histological observation confirmed donor-derived gametogenesis in the 3nHyb recipients' gonads. Overall, triploidization reduces male-biased sex differentiation in germ cell-less gonads. We report, for the first time, donor-derived egg production in an animal via direct ovarian germ cell transplantation into a germ cell-less ovary.
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http://dx.doi.org/10.1093/biolre/ioaa168DOI Listing
December 2020

Suitability of hybrid mackerel (Scomber australasicus × S. japonicus) with germ cell-less sterile gonads as a recipient for transplantation of bluefin tuna germ cells.

Gen Comp Endocrinol 2020 09 2;295:113525. Epub 2020 Jun 2.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. Electronic address:

We aim to establish a small-bodied surrogate broodstock, such as mackerel, which produces functional bluefin tuna gametes by spermatogonial transplantation. When reproductively fertile fish are used as recipients, endogenous gametogenesis outcompetes donor-derived gametogenesis, and recipient fish predominantly produce their gametes. In this study, we assessed fertility of hybrid mackerel, Scomber australasicus × S. japonicus, and its suitability as a recipient for transplantation of bluefin tuna germ cells. Hybrid mackerel were produced by artificially inseminating S. australasicus eggs with S. japonicus spermatozoa. Cellular DNA content and PCR analyses revealed that F1 offspring were diploid carrying both paternal and maternal genomes. Surprisingly, histological observations found no germ cells in hybrid mackerel gonads at 120 days post-hatch (dph), although they were present in the gonad of 30- and 60-dph hybrid mackerel. The frequency of germ cell-less fish was 100% at 120-dph, 63.1% at 1-year-old, and 81.8% at 2-year-old. We also confirmed a lack of expression of germ cell marker (DEAD-box helicase 4, ddx4) in the germ cell-less gonads of hybrid mackerel. By contrast, expression of Sertoli cell marker (gonadal soma-derived growth factor, gsdf) and of Leydig cell marker (steroid 11-beta-hydroxlase, cyp11b1) were clearly detected in hybrid mackerel gonads. Together these results showed that most of the hybrid gonads were germ cell-less sterile, but still possessed supporting cells and steroidogenic cells, both of which are indispensable for nursing donor-derived germ cells. To determine whether hybrid gonads could attract and incorporate donor bluefin tuna germ cells, testicular cells labeled with PKH26 fluorescent dye were intraperitoneally transplanted. Fluorescence observation of hybrid recipients at 14 days post-transplantation revealed that donor cells had been incorporated into the recipient's gonads. This suggests that hybrid mackerel show significant promise for use as a recipient to produce bluefin tuna gametes.
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http://dx.doi.org/10.1016/j.ygcen.2020.113525DOI Listing
September 2020

Influence of Benz[]anthracene on Bone Metabolism and on Liver Metabolism in Nibbler Fish, .

Int J Environ Res Public Health 2020 02 21;17(4). Epub 2020 Feb 21.

Noto Marine Laboratory, Institute of Nature and Environmental Technology, Division of Marine Environmental Studies, Kanazawa University, Noto-cho, Ishikawa 927-0553, Japan.

It has been reported that spinal deformity was induced in developing fish by the addition of polycyclic aromatic hydrocarbons (PAHs). To examine the mechanism of the disruption of fish bone metabolism, the effect of benz[]anthracene (BaA), a kind of PAH, on plasma calcium, inorganic phosphorus, osteoblasts, and osteoclasts was investigated in this study. We also measured several plasma components to analyze the toxicity of BaA on other metabolisms. BaA (1 or 10 ng/g body weight) was intraperitoneally injected (four times) into nibbler fish during breeding, for 10 days, and it was indicated, for the first time, that injecting high doses of BaA to nibbler fish induced both hypocalcemia and hypophosphatemia. Furthermore, in the scales of nibbler fish treated with high doses of BaA, both osteoclastic and osteoblastic marker messengerRNA (mRNA) expressions decreased. These results are a cause of disruption of bone metabolism and, perhaps, the induction of spinal deformities. In addition, we found that total protein, metabolic enzymes in the liver, total cholesterol, free cholesterol, and high-density lipoprotein cholesterol levels significantly decreased in BaA-injected fish. These results indicate that BaA may affect liver diseases and emphasize the importance of prevention of aquatic PAH pollution.
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http://dx.doi.org/10.3390/ijerph17041391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068328PMC
February 2020

Germ cell-less hybrid fish: ideal recipient for spermatogonial transplantation for the rapid production of donor-derived sperm†.

Biol Reprod 2019 08;101(2):492-500

Division of Fisheries Resource Sciences, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima City, Japan.

An interspecific hybrid marine fish that developed a testis-like gonad without any germ cells, i.e., a germ cell-less gonad, was produced by hybridizing a female blue drum Nibea mitsukurii with a male white croaker Pennahia argentata. In this study, we evaluated the suitability of the germ cell-less fish as a recipient by transplanting donor testicular cells directly into the gonads through the urogenital papilla. The donor testicular cells were collected from hemizygous transgenic, green fluorescent protein (gfp) (+/-) blue drum, and transplanted into the germ cell-less gonads of the 6-month-old adult hybrid croakers. Fluorescent and histological observations showed the colonization, proliferation, and differentiation of transplanted spermatogonial cells in the gonads of hybrid croakers. The earliest production of spermatozoa in a hybrid recipient was observed at 7 weeks post-transplantation (pt), and 10% of the transplanted recipients produced donor-derived gfp-positive spermatozoa by 25 weeks pt. Sperm from the hybrid recipients were used to fertilize eggs from wild-type blue drums, and approximately 50% of the resulting offspring were gfp-positive, suggesting that all offspring originated from donor-derived sperm that were produced in the transplanted gfp (+/-) germ cells. To the best of our knowledge, this is the first report of successful spermatogonial transplantation using a germ cell-less adult fish as a recipient. This transplantation system has considerable advantages, such as the use of comparatively simple equipment and procedures, and rapid generation of donor-derived spermatogenesis and offspring, and presents numerous applications in commercial aquaculture.
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http://dx.doi.org/10.1093/biolre/ioz045DOI Listing
August 2019

Green light irradiation during sex differentiation induces female-to-male sex reversal in the medaka Oryzias latipes.

Sci Rep 2019 02 20;9(1):2383. Epub 2019 Feb 20.

The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, 890-0056, Japan.

This study investigated whether irradiation of a specific light wavelength could affect the sex differentiation of fish. We first found that the photoreceptor genes responsible for receiving red, green, and ultraviolet light were expressed in the eyes of medaka during the sex differentiation period. Second, we revealed that testes developed in 15.9% of genotypic females reared under green light irradiation. These female-to-male sex-reversed fish (i.e. neo-males) showed male-specific secondary sexual characteristics and produced motile sperm. Finally, progeny tests using the sperm of neo-males (XX) and eggs of normal females (XX) revealed that all F1 offspring were female, indicating for the first time in animals that irradiation with light of a specific wavelength can trigger sex reversal.
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http://dx.doi.org/10.1038/s41598-019-38908-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382872PMC
February 2019

Transcriptional response to low temperature in the yellow drum (Nibea albiflora) and identification of genes related to cold stress.

Comp Biochem Physiol Part D Genomics Proteomics 2018 12 7;28:80-89. Epub 2018 Jul 7.

Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan.

The yellow drum (Nibea albiflora) is an economically important maricultured fish in China, but the aquaculture of this species is severely affected by overwinter mortality associated with cold stress. Characterization of the molecular mechanisms underlying the susceptibility of the yellow drum to cold might increase our understanding of how this fish adapts to environmental challenges. Here, the transcriptional response of the yellow drum to cold stress (7.5 °C) was investigated with RNA-Seq analysis. We compared brain and muscle transcriptomes among cold-tolerant (Tol) fish that survived the cold treatment, cold-sensitive (Sen) fish that were killed by the cold treatment, and control (Con) fish that were not subjected to cold. Our analysis recovered 233,245 unigenes. The genes (DEGs) differentially expressed in the brain and muscle of the Tol versus Con group, the Sen versus Con group, and the Tol versus Sen group had tissue-specific expression patterns. Gene ontology, enrichment, and pathway analyses indicated the most highly enriched pathways in the DEGs were signaling molecules and interaction, signal transduction, carbohydrate metabolism, lipid metabolism, digestive system, and endocrine system pathways. These pathways were all associated with biological functions relevant to cold adaptation in the yellow drum, including transduction of stress signals, energy metabolism, and stress-induced cell membrane changes. We identified genes likely to be involved in cold-susceptibility and -tolerance as those differentially expressed in the Tol group as compared to the Sen group. Further investigation and characterization of these candidate genes might improve our understanding of the mechanisms underlying cold adaptation in the yellow drum.
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http://dx.doi.org/10.1016/j.cbd.2018.07.003DOI Listing
December 2018

Adjuvant effect in aquaculture fish of cell-wall glycolipids isolated from acid-fast bacteria.

Dev Comp Immunol 2018 08 14;85:142-149. Epub 2018 Apr 14.

The United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-8580, Japan; Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima, 890-0056, Japan. Electronic address:

Mycobacteriosis and nocardiosis in cultured fish caused by infections with acid-fast bacteria, are responsible for large economic losses globally. In this study, we suggest a novel adjuvant using glycolipids that activates host immune systems. The immune response to glycolipids stimulation was investigated using ginbuna crucian carp. Ginbuna vaccinated with FKC (formalin-killed cells) + glycolipids isolated from Mycobacterium sp., upregulated inflammatory- and Th1-related cytokines, and a DTH (delayed-type hypersensitivity) response was confirmed only in ginbuna vaccinated with FKC + glycolipids. These observations suggest that glycolipids activated host innate and cell-mediated immunity. Subsequently, we evaluated the adjuvant effect of glycolipids against amberjack nocardiosis. In a challenge test, a higher survival rate was observed in amberjack vaccinated with FKC + glycolipids emulsified with conventional oil adjuvant than in fish vaccinated with FKC + oil adjuvant without glycolipids. Therefore, glycolipids potentially could be used as a practical, economical and safe adjuvant for aquaculture fish.
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http://dx.doi.org/10.1016/j.dci.2018.04.012DOI Listing
August 2018

Gonadal Transcriptome Analysis of Pacific Abalone Haliotis discus discus: Identification of Genes Involved in Germ Cell Development.

Mar Biotechnol (NY) 2018 Aug 3;20(4):467-480. Epub 2018 Apr 3.

Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima, 890-0056, Japan.

Little is known about the molecular mechanisms governing gonadal developmental processes in abalones. Here, we conducted transcriptome analysis of Pacific abalone Haliotis discus discus for gene discovery in the brain, ovary, testis, and unfertilized eggs. Among the annotated unigenes, 48.6% of unigenes were identified by Venn diagram analysis as having universal or tissue-specific expression. Twenty-three genes with gonad-biased gene ontology (GO) terms were first obtained. Secondly, 36 genes were found by screening known gene names related to germ cell development. Finally, 17 genes were obtained by querying the annotated unigene database for zygotically expressed gonadal genes (ovary and testis) and maternally expressed gonadal genes (ovary, testis, and unfertilized eggs) using keywords related to reproduction. To further verify tissue distribution pattern and subcellular localization of these genes, RT-PCR and in situ hybridization were performed using a unigene encoding a germ cell marker, vasa, as control. The results showed that vasa was expressed mainly in the early developmental stages of germ cells in both sexes. One of the candidate genes, vitelline envelope zona pellucida domain protein 12 (ZP12), was expressed in the primordial germ cells of immature gonad and early developmental stages of germ cells of the adult female. The results obtained from the present study suggest that vasa and ZP12 are involved in germ cell development of Pacific abalone and that ZP12 is an especially useful germ cell-specific marker in immature adults. The current gonadal transcriptome profile is an extensive resource for future reproductive molecular biology studies of this species.
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http://dx.doi.org/10.1007/s10126-018-9809-5DOI Listing
August 2018

Hybrid Sterility in Fish Caused by Mitotic Arrest of Primordial Germ Cells.

Genetics 2018 06 2;209(2):507-521. Epub 2018 Apr 2.

Research Center for Advanced Science and Technology, Tokyo University of Marine Science and Technology, Tateyama, Chiba 294-0308, Japan

Sterility in hybrid animals is widely known to be due to a cytological mechanism of aberrant homologous chromosome pairing during meiosis in hybrid germ cells. In this study, the gametes of four marine fish species belonging to the Sciaenid family were artificially fertilized, and germ cell development was examined at the cellular and molecular levels. One of the intergeneric hybrids had gonads that were testis-like in structure, small in size, and lacked germ cells. Specification of primordial germ cells (PGCs) and their migration toward genital ridges occurred normally in hybrid embryos, but these PGCs did not proliferate in the hybrid gonads. By germ cell transplantation assay, we showed that the gonadal microenvironment in hybrid recipients produced functional donor-derived gametes, suggesting that the germ cell-less phenotype was caused by cell autonomous proliferative defects of hybrid PGCs. This is the first evidence of mitotic arrest of germ cells causing hybrid sterility in animals.
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http://dx.doi.org/10.1534/genetics.118.300777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972423PMC
June 2018

Production of Tiger Puffer Takifugu rubripes Offspring from Triploid Grass Puffer Takifugu niphobles Parents.

Mar Biotechnol (NY) 2017 Dec 23;19(6):579-591. Epub 2017 Sep 23.

Department Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo, 108-8477, Japan.

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2-5 kg) much larger than average market size (0.6-1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.
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http://dx.doi.org/10.1007/s10126-017-9777-1DOI Listing
December 2017

Genomic DNA variation confirmed Seriola lalandi comprises three different populations in the Pacific, but with recent divergence.

Sci Rep 2017 08 24;7(1):9386. Epub 2017 Aug 24.

Genecology Research Centre, Faculty of Science, Health, Education, and Engineering, University of the Sunshine Coast, Maroochydore, DC, QLD 4558, Australia.

Captive breeding programs and aquaculture production have commenced worldwide for the globally distributed yellowtail kingfish (Seriola lalandi), and captive bred fingerlings are being shipped from the Southern Hemisphere to be farmed in the Northern Hemisphere. It was recently proposed that Pacific S. lalandi comprise at least three distinct species that diverged more than 2 million years ago. Here, we tested the hypothesis of different "species" in the Pacific using novel genomic data (namely single nucleotide polymorphisms and diversity array technology markers), as well as mtDNA and DNA microsatellite variation. These new data support the hypothesis of population subdivision between the Northeast Pacific, Northwest Pacific and South Pacific, and genetic divergence indicates restriction to the gene flow between hemispheres. However, our estimates of maximum mtDNA and nuclear DNA divergences of 2.43% and 0.67%, respectively, were within the ranges more commonly observed for populations within species than species within genera. Accordingly our data support the more traditional view that S. lalandi in the Pacific comprises three distinct populations rather than the subdivisions into several species.
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http://dx.doi.org/10.1038/s41598-017-07419-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571200PMC
August 2017

Flow-cytometric enrichment of Pacific bluefin tuna type A spermatogonia based on light-scattering properties.

Theriogenology 2017 Oct 22;101:91-98. Epub 2017 Jun 22.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan Minato-ku, Tokyo 108-8477, Japan.

We previously established surrogate broodstock in which the donor germ cells transplanted into the peritoneal cavities of xenogeneic recipients were capable of developing into functional eggs and sperm in teleost fish. In this transplantation system, only the undifferentiated germ cells such as type A spermatogonia (ASG) or a portion of the ASG population were capable of being incorporated into the genital ridges of the recipients and undergo gametogenesis. Therefore, the use of enriched ASGs can be expected to achieve efficient donor-cell incorporation. Here, we established a method of isolation and enrichment of the ASG of Pacific bluefin tuna using flow cytometry. Whole testicular cell suspensions were fractionated by forward and side scatter properties, following which ASGs were enriched in a fraction in which the forward scatter signal was relatively high and side scatter signal was relatively low. The diameter of sorted cells using the fraction was identical to the size of ASGs observed in histological analysis, and these cells also expressed the vasa gene. In addition, we succeeded in applying this method to several maturation stages of Pacific bluefin tuna. Since this method was based on light-scattering characteristics of ASGs, it can potentially be applied to various teleosts. We expect that this method can contribute to the production of seeds of Pacific bluefin tuna using surrogate broodstock.
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http://dx.doi.org/10.1016/j.theriogenology.2017.06.022DOI Listing
October 2017

Adjuvant effect of recombinant interleukin-12 in the Nocardiosis formalin-killed vaccine of the amberjack Seriola dumerili.

Fish Shellfish Immunol 2017 Aug 7;67:263-269. Epub 2017 Jun 7.

The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-8580, Japan; Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, Japan. Electronic address:

Nocardiosis causes serious economic damage in the fish farming of Japanese yellowtail fish. Nocardia seriolae identified as pathogenic bacterium is an intracellular-pathogen. In general, induction of cell-mediated immunity (CMI) is effective in infection defense against intracellular-pathogen. However, the conventional formalin-killed N. seriolae (FKC) vaccine induces humoral immunity. Interleukin-12 (IL-12) is Th1 type heterodimeric cytokine and induces cell differentiation in mammals. Our previous study showed that recombinant amberjack IL-12 has a role in CMI induction in vitro and could be a possible CMI inducing adjuvant. However, its adjuvant effect of fish IL-12 was not studied. In the present study, six types of amberjack recombinant IL-12 (rIL-12) were mixed and injected into amberjack with FKC. Firstly, we analyzed Th1- and Th2- related gene expression and monitored Th1/Th2 status followed by investigation of antibody titer. As a result, Th1-type immunity was induced in FKC + rIL-12 vaccinated fish. Secondly, we checked Th1/Th2 status of vaccinated fish after 10 days of N. seriolae infection using the expression of related genes. High T-bet/GATA-3 ratio was observed in FKC + rIL-12 vaccinated fish, suggesting that Th1 cells possesing antigen memory were induced against N. seriolae infection. Finally, the survival rate in challenge test showed that 88% of FKC + rIL-12 vaccinated fish was survived at 34 days after N. seriolae injection whereas PBS (control) and FKC only were exterminated. These result suggest that i) rIL-12 is viable CMI inducible adjuvant and ii) production of Th1 cells having antigen memory resulting from activation of IL-12 signaling pathway is important for defense against N. seriolae infection.
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http://dx.doi.org/10.1016/j.fsi.2017.06.025DOI Listing
August 2017

Establishment of intraperitoneal germ cell transplantation for critically endangered Chinese sturgeon Acipenser sinensis.

Theriogenology 2017 May 13;94:37-47. Epub 2017 Feb 13.

Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan, 430223, China; Institute of Hydrobiology, Chinese Academy of Sciences, University of the Chinese Academy of Sciences, Wuhan, 430072, China; Freshwater Fisheries Research Center, Chinese Academy of Fisheries Science, Wuxi, 214081, China. Electronic address:

Recent progress in germ cell transplantation techniques in fish has paved the way for the conservation of endangered species. Here, we developed an intraperitoneal germ cell transplantation procedure using Chinese and Dabry's sturgeon as donor and recipient species, respectively. Histological analysis revealed that primordial germ cells migrated on the peritoneal wall at 16 days post-hatch (dph) in Dabry's sturgeon. The genital ridges of Dabry's sturgeon (recipient) first formed at 28 dph, suggesting that for successful colonization of donor germ cells in the recipient gonads, the transplantation should be performed earlier than this age. Sexual dimorphism of gonadal structure was first observed at 78 dph. Gonadal germ cell proliferation was not seen in either sex during this period. Immunohistochemistry using the anti-Vasa antibody found that donor testes from 2-year-old Dabry's sturgeon mainly consisted of single- or paired-type A spermatogonia, while donor ovaries from 11.5-year-old Chinese sturgeon had perinucleolus stage oocytes and clusters of oogonia. Donor cells isolated from Dabry's sturgeon testes or Chinese sturgeon ovary labeled with PKH26 fluorescent dye were transplanted into the peritoneal cavity of the 7- or 8-dph Dabry's sturgeon larvae. More than 90% and 70% of transplanted larvae survived after 2 days post-transplantation (dpt) and 51 dpt, respectively. At 51 dpt, PKH26-labeled cells exhibiting germ cell-specific nuclear morphology and diameter were observed in excised recipient gonads by fluorescent and confocal microscopy. The colonization rate of allogeneic testicular germ cell transplantation (Group 1) was 70%, while that of two batches of xenogeneic ovarian germ cell transplantation (Group 2 and Group 3) were 6.7% and 40%, respectively. The ratio of colonized germ cells to endogenous germ cells was 11.96%, 5.35% and 3.56% for Group 1, Group 2 and Group 3, respectively. Thus, we established a germ cell transplantation technique for the critically endangered Chinese sturgeon using the most closely related species as a recipient and demonstrated the successful preparation of transplantable female germ cells from aged adult Chinese sturgeon.
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http://dx.doi.org/10.1016/j.theriogenology.2017.02.009DOI Listing
May 2017

The predictive value of endobronchial ultrasonography with a guide sheath in the diagnosis of the histologic subtypes of lung cancer.

Respir Investig 2016 Nov 1;54(6):473-478. Epub 2016 Jul 1.

First Department of Medicine, Hokkaido University School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan. Electronic address:

Background: Recent studies have shown differential response to chemotherapy among the subtypes of non-small cell lung carcinoma (NSCLC). Therefore, to accurately differentiate between the types of lung cancer is of paramount importance. Transbronchial biopsy using endobronchial ultrasonography with a guide sheath (EBUS-GS) is a promising method for the diagnosis of NSCLC. The purpose of this study was to evaluate the consistency between the types of lung cancer histologically diagnosed by bronchial biopsy or cytologically by EBUS-GS, and the final diagnosis of the resected specimen.

Methods: A retrospective analysis was performed on 203 patients having primary lung cancers diagnosed by EBUS-GS, who subsequently underwent curative pulmonary resection at the Hokkaido University Hospital between July 2003 and December 2011. In the present study, non-small cell carcinoma was defined as non-squamous cell carcinoma, and squamous cell carcinoma (Sq) was excluded.

Results: Of the 40 cases diagnosed as Sq by EBUS-GS, 37 cases were diagnosed as Sq, and 3 cases were diagnosed as non-Sq after surgical resection. Of the 159 cases diagnosed as non-Sq by EBUS-GS, 151 cases were diagnosed as non-Sq, 6 as Sq, and 2 as small cell carcinoma after surgical resection. These results showed that the positive predictive value of EBUS-GS in the diagnosis of Sq was 93%, and its positive predictive value in diagnosing non-Sq was 95%.

Conclusions: The pathological subtyping of NSCLC using small tissue and cytology samples obtained by EBUS-GS appears to effectively distinguish between Sq and non-Sq and is therefore considered useful in making a treatment decision.
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http://dx.doi.org/10.1016/j.resinv.2016.05.003DOI Listing
November 2016

Small-scale capture, transport and tank adaptation of live, medium-sized Scombrids using "Tuna Tubes".

Springerplus 2015 13;4:604. Epub 2015 Oct 13.

Faculty of Science, Health, Education and Engineering, Genecology Research Centre, University of the Sunshine Coast, Maroochydore DC, QLD 4558 Australia.

The transport of live fish is a crucial step to establish fish culture in captivity, and is especially challenging for species that have not been commonly cultured before, therefore transport and handling methods need to be optimized and tailored. This study describes the use of tuna tubes for small-scale transport of medium-sized pelagic fish from the Scombridae family. Tuna tubes are an array of vertical tubes that hold the fish, while fresh seawater is pumped up the tubes and through the fish mouth and gills, providing oxygen and removing wastes. In this study, 19 fish were captured using rod and line and 42 % of the captured fish were transported alive in the custom-designed tuna tubes to an on-shore holding tank: five mackerel tuna (Euthynnus affinis) and three leaping bonito (Cybiosarda elegans). Out of these, just three (15.8 % of total fish) acclimatized to the tank's condition. Based on these results, we discuss an improved design of the tuna tubes that has the potential to increase survival rates and enable a simple and low cost method of transporting of live pelagic fish.
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http://dx.doi.org/10.1186/s40064-015-1391-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4627978PMC
November 2015

Functional Sperm of the Yellowtail (Seriola quinqueradiata) Were Produced in the Small-Bodied Surrogate, Jack Mackerel (Trachurus japonicus).

Mar Biotechnol (NY) 2015 Oct 4;17(5):644-54. Epub 2015 Aug 4.

Central Research Laboratory, Nippon Suisan Kaisha, Ltd., 1-32-3 Nanakuni, Hachioji-shi, Tokyo, 192-0991, Japan,

Production of xenogeneic gametes from large-bodied, commercially important marine species in closely related smaller surrogates with short generation times may enable rapid domestication of the targeted species. In this study, we aimed to produce gametes of Japanese yellowtail (Seriola quinqueradiata) using jack mackerel (Trachurus japonicus) as a surrogate with a smaller body size and shorter maturation period. Donor spermatogonia were collected from the testes of yellowtail males and transferred into the peritoneal cavity of 10- and 12-day-old jack mackerel larvae. Twenty days later, 59.5% of the recipients survived of which 88.2% had donor-derived germ cells in their gonads. One year later, genomic DNA templates were prepared from the semen of 96 male recipients and subjected to polymerase chain reaction (PCR) analyses using primers specific for the yellowtail vasa sequence, resulting in the detection of positive signals in semen from two recipients. The milt collected from the recipients was used for fertilization with yellowtail eggs. Of eight hatchlings obtained from the crosses, two were confirmed to be derived from donor yellowtail by DNA markers, although the others were gynogenetic diploids. These findings indicate that it is possible to produce donor-derived sperm in xenogeneic recipients with a smaller body size and shorter generation time by transplanting spermatogonia. Thus, the xenogeneic transplantation of spermatogonia might be a potential tool to produce gametes of large-bodied, commercially important fish, although the efficiency of the method requires further improvement. This is the first report demonstrating that donor-derived sperm could be produced in xenogeneic recipient via spermatogonial transplantation in carangid fishes.
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http://dx.doi.org/10.1007/s10126-015-9657-5DOI Listing
October 2015

Assessment of yellowtail kingfish (Seriola lalandi) as a surrogate host for the production of southern bluefin tuna (Thunnus maccoyii) seed via spermatogonial germ cell transplantation.

Reprod Fertil Dev 2016 Oct;28(12):2051-2064

Genecology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Qld 4558, Australia.

Germ cell transplantation is an innovative technology for the production of interspecies surrogates, capable of facilitating easier and more economical management of large-bodied broodstock, such as the bluefin tuna. The present study explored the suitability of yellowtail kingfish (Seriola lalandi) as a surrogate host for transplanted southern bluefin tuna (Thunnus maccoyii) spermatogonial cells to produce tuna donor-derived gametes upon sexual maturity. Germ cell populations in testes of donor T. maccoyii males were described using basic histology and the molecular markers vasa and dead-end genes. The peripheral area of the testis was found to contain the highest proportions of dead-end-expressing transplantable Type A spermatogonia. T. maccoyii Type A spermatogonia-enriched preparations were transplanted into the coelomic cavity of 6-10-day-old post-hatch S. lalandi larvae. Fluorescence microscopy and polymerase chain reaction analysis detected the presence of tuna cells in the gonads of the transplanted kingfish fingerlings at 18, 28, 39 and 75 days after transplantation, indicating that the transplanted cells migrated to the genital ridge and had colonised the developing gonad. T. maccoyii germ cell-derived DNA or RNA was not detected at later stages, suggesting that the donor cells were not maintained in the hosts' gonads.
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http://dx.doi.org/10.1071/RD15136DOI Listing
October 2016

Polyunsaturated fatty acid metabolism in a marine teleost, Nibe croaker Nibea mitsukurii: Functional characterization of Fads2 desaturase and Elovl5 and Elovl4 elongases.

Comp Biochem Physiol B Biochem Mol Biol 2015 Oct 23;188:37-45. Epub 2015 Jun 23.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. Electronic address:

To reduce the requirement for fish oil in marine aquaculture, it would be advantageous to endow marine fish species with the capability for the endogenous biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). For this purpose, we have previously produced transgenic Nibe croaker (Nibea mitsukurii) carrying an elongase of very-long-chain fatty acids 2 (elovl2) gene isolated from Masu salmon (Oncorhynchus masou). However, fatty acid analysis revealed that 24:5n-3 accumulated in the liver of the transgenic fish, whereas the DHA level did not differ between non-transgenic and transgenic fish. Therefore, to select more effective enzymes for successful transgenic synthesis of DHA, understanding the endogenous DHA biosynthetic pathway in the Nibe croaker is considered to be important. The present study aimed to investigate the biochemical functions of the Elovl5, Elovl4 and Fads2 enzymes involved in the DHA biosynthetic pathway in the Nibe croaker. The results showed that both Elovl5 and Elovl4 were able to elongate C18 fatty acids to C22 fatty acids and that Fads2 had Δ6 desaturase activity toward C18 fatty acids and weak Δ8 desaturase activity toward C20 fatty acids. On the other hand, Fads2 was found to lack the ability to convert 24:5n-3 to 24:6n-3, a fatty acid that can directly be converted to DHA via β-oxidation.
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http://dx.doi.org/10.1016/j.cbpb.2015.06.005DOI Listing
October 2015

A phase I study of farletuzumab, a humanized anti-folate receptor α monoclonal antibody, in patients with solid tumors.

Invest New Drugs 2015 Apr 9;33(2):332-40. Epub 2014 Nov 9.

Department of Medical Oncology, Saitama International Medical Center-Comprehensive Cancer Center, Saitama Medical University, Saitama, Japan,

Farletuzumab is a humanized monoclonal antibody against folate receptor α (FRA). The purpose of the study is to assess safety and tolerability, the pharmacokinetic (PK) profile, and preliminary antitumor effect. Patients with ovarian cancer (OC) or FRA-expressing solid tumors who are resistant to standard treatments were eligible for the study. After single-dose administration for PK assessment, farletuzumab was administered by intravenous injection, repeating every week until disease progression. Dose-limiting toxicities (DLTs) were defined as grade 4 hematological and grade 3/4 nonhematological toxicities. Dose escalation was planned in 4 cohorts (50, 100, 200, and 400 mg/m(2)). Fourteen patients with OC and two patients with gastric cancer (GC) received farletuzumab infusion. Neither DLTs nor grade 3/4 toxicities were reported in all cohorts. Major adverse events, including grade 1/2 infusion related reaction (15 patients, 93.8%), headache (seven patients, 43.8%), and nausea and decreased appetite (five patients each, 31.3%), were observed and medically managed. AUC and Cmax increased dose-dependently and linear PK profiles were observed. No tumor shrinkage was recorded, but long-term disease stabilization for 25 and 20 months was observed in one patient with clear cell OC (100 mg/m(2)) and one patient with GC (400 mg/m(2)), respectively. No cumulative toxicity occurred in any patient. Farletuzumab was well tolerated in Japanese patients with a similar PK profile as compared with the US population. Long-term disease stabilization was observed in a subpopulation of clear cell OC and GC; both of them were resistant and progressive after standard chemotherapies (ClinicalTrials.gov Identifier: NCT01049061).
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http://dx.doi.org/10.1007/s10637-014-0180-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4387250PMC
April 2015

Modification of the n-3 HUFA biosynthetic pathway by transgenesis in a marine teleost, nibe croaker.

J Biotechnol 2014 Feb 31;172:46-54. Epub 2013 Dec 31.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. Electronic address:

Marine fishes are generally unable to produce sufficient quantities of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) for their normal growth and survival, as the key fatty acid-metabolizing enzymes in the EPA and DHA biosynthetic pathway are limited. It is therefore necessary to supplement cultured marine fish species diets with fish oils in order to supply EPA and DHA. Given that freshwater fishes are capable of synthesizing both EPA and DHA, they presumably express all of the enzymes required for this biosynthetic pathway. Thus, we hypothesize that transgenic marine species carrying these fatty acid-metabolizing enzymes could be reared without the dietary supplementation of fish oil. As the first step toward this goal, we used marine fish, nibe croaker to produce a transgenic line carrying the elongase gene isolated from masu salmon. Fatty acid analysis revealed that the liver EPA (20:5n-3) content in the transgenic fish was lower (3.3% vs. 7.7%). However, docosapentaenoic acid (22:5n-3) content in the transgenic fish was 2.28-fold (4.1% vs. 1.8%) higher than in non-transgenic fish. Further, tetracosapentaenoic acid (24:5n-3) was specifically detected in the transgenic fish. We therefore conclude that the development of transgenic fish lines with these fatty acid-metabolizing enzymes could be a powerful tool for manipulating fatty acid metabolic pathways in fish.
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http://dx.doi.org/10.1016/j.jbiotec.2013.12.004DOI Listing
February 2014

The Pacific bluefin tuna (Thunnus orientalis) dead end gene is suitable as a specific molecular marker of type A spermatogonia.

Mol Reprod Dev 2013 Oct 26;80(10):871-80. Epub 2013 Aug 26.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.

We developed a spermatogonial transplantation technique to produce donor-derived gametes in surrogate fish. Our ultimate aim is to establish surrogate broodstock that can produce bluefin tuna. We previously determined that only type A spermatogonia (ASG) could colonize recipient gonads in salmonids. Therefore, it is necessary to develop a precise molecular marker that can distinguish ASG in order to develop efficient spermatogonial transplantation methods. In this study, the Pacific bluefin tuna (Thunnus orientalis) dead end (BFTdnd) gene was identified as a specific marker for ASG. In situ hybridization and RT-PCR analysis with various types of spermatogenic cell populations captured by laser microdissection revealed that localization of BFTdnd mRNA was restricted to ASG, and not detected in other differentiated spermatogenic cells. In order to determine if BFTdnd can be used as a molecular marker to identify germ cells with high transplantability, transplantation of dissociated testicular cells isolated from juvenile, immature, and mature Pacific bluefin tuna, which have different proportions of dnd-positive ASG, were performed using chub mackerel as the surrogate recipient species. Colonization of transplanted donor germ cells was only successful with testicular cells from immature Pacific Bluefin tuna, which contained higher proportions of dnd-positive ASG than juvenile and mature fish. Thus, BFTdnd is a useful tool for identifying highly transplantable ASG for spermatogonial transplantation.
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http://dx.doi.org/10.1002/mrd.22224DOI Listing
October 2013

Gonadal development and fertility of triploid grass puffer Takifugu niphobles induced by cold shock treatment.

Mar Biotechnol (NY) 2013 Apr 28;15(2):133-44. Epub 2012 Jul 28.

Nagasaki Prefectural Institute of Fisheries, 1551-4 Taira, Nagasaki-shi, Nagasaki 851-2213, Japan.

Tiger puffer Takifugu rubripes is one of the most valuable fish species in Japan; however, there has not been much progress in their selective breeding until recently despite their potential in aquaculture. Their long generation time and the large body size of their broodstock make breeding difficult. Recently, we made a surrogate broodstock, which produced gametes of different species in salmonids. Therefore, by using closely related recipients, which have small body sizes and short generation times, it is possible to accelerate breeding of the tiger puffer. Thus, we considered the grass puffer Takifugu niphobles, which has a short generation time and a small maturation size, as a potential recipient for gamete production of the tiger puffer. Furthermore, if sterile triploid individuals are used as recipients, the resulting surrogate broodstock would produce only donor-derived gametes. Therefore, we examined conditions for inducing triploidy by suppressing meiosis II to retain the second polar body in grass puffer. We found that cold shock treatment, which is 5°C for 30 min starting from 5 min after fertilization, is optimal to obtain high triploidization and hatching rates. Although the resulting triploid grass puffers produced small amounts of gametes in both sexes, the offspring derived from the gametes could not live for over 3 days. Furthermore, we found that triploid grass puffer showed normal plasma sex steroid levels compared with diploids. These are important characteristics of triploid grass puffer as surrogate recipients used for germ cell transplantation.
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http://dx.doi.org/10.1007/s10126-012-9470-3DOI Listing
April 2013

Production of donor-derived offspring by allogeneic transplantation of spermatogonia in the yellowtail (Seriola quinqueradiata).

Biol Reprod 2012 Jun 14;86(6):176. Epub 2012 Jun 14.

Central Research Laboratory, Nippon Suisan Kaisha, Ltd, Tokyo, Japan.

Although the yellowtail (Seriola quinqueradiata) is the fish most commonly farmed in Japan, breeding of this species has not yet started. This is primarily due to the lack of sufficiently sophisticated methods for manipulating gametogenesis, which makes it difficult to collect gametes from specific dams and sires. If it were possible to produce large numbers of surrogate fish by transplanting germ cells isolated from donor individuals harboring desirable genetic traits, then the probability of acquiring gametes carrying the donor-derived haplotype would increase, and breeding programs involving this species might increase as a result. As a first step, we established a method for the allogeneic transplantation of yellowtail spermatogonia and the production of donor-derived offspring. Donor cells were collected from immature (10-month-old) yellowtail males with testes containing abundant type A spermatogonia, labeled with PKH26 fluorescent dye, and transferred into the peritoneal cavities of 8-day-old larvae. Fluorescence observation at 28 days post-transplantation revealed that PKH26-labeled cells were incorporated into recipients' gonads. To assess whether donor-derived spermatogonia could differentiate into functional gametes in the allogeneic recipient gonads, gametes collected from nine male and four female adult recipients were fertilized with wild-type eggs and milt. Analysis of microsatellite DNA markers confirmed that some of the first filial (F(1)) offspring were derived from donor fish, with the average contribution of donor-derived F(1) offspring being 66% and the maximum reaching 99%. These findings confirmed that our method was effective for transplanting yellowtail spermatogonia into allogeneic larvae to produce donor-derived offspring.
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http://dx.doi.org/10.1095/biolreprod.111.097873DOI Listing
June 2012

Flow-cytometric isolation and enrichment of teleost type A spermatogonia based on light-scattering properties.

Biol Reprod 2012 Apr 12;86(4):107. Epub 2012 Apr 12.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.

The transplantation of germ cells is a powerful tool both for studying their development and for reproductive biotechnology. An intraperitoneal germ cell transplantation system was recently developed for use in several teleost species. Donor germ cells transplanted into the peritoneal cavity of hatchlings migrated toward and were incorporated into the recipient's genital ridges, where they underwent gametogenesis. Among male germ cells, only type A spermatogonia were capable of colonizing the recipient gonads, unlike those at more advanced stages. The enrichment of type A spermatogonia is therefore important to achieve efficient donor-cell incorporation and subsequent donor-derived gametogenesis. Here we established a simple and rapid system of isolation and enrichment for fish type A spermatogonia, using flow cytometry. Type A spermatogonia were found to have distinctive forward and side light scatter properties compared to that with other types of testicular cell. Based on these characteristics, we were able to isolate and enrich type A spermatogonia by using flow cytometry. After intraperitoneal transplantation, the enriched type A spermatogonia could be successfully incorporated into the recipient genital ridges. This flow cytometry approach using forward and side light scatter was also found to be applicable to other salmonid and sciaenid species, suggesting that it could be a powerful tool for isolating and enriching transplantable type A spermatogonia in a wide range of teleosts. We expect this method to contribute significantly to germ cell biology and biotechnology.
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http://dx.doi.org/10.1095/biolreprod.111.093161DOI Listing
April 2012

Lymphocyte antigen 75 (Ly75/CD205) is a surface marker on mitotic germ cells in rainbow trout.

Biol Reprod 2010 Oct 10;83(4):597-606. Epub 2010 Jun 10.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.

In mammals, several cell surface molecular markers have been characterized in order to identify the mitotic germ cells. However, little is known in fish about their cell surface antigen. In this study, we identified lymphocyte antigen 75 (Ly75/CD205) as a germ cell-specific cell surface marker by combination expressed sequence tag analysis of purified type A spermatogonia (A-SG) from immature testis, in silico prediction of membrane proteins, and expression studies. The ly75 transcripts were abundant in the testis and gills, and weak signals were detected in the head kidney and brain. In addition, ly75 mRNA was predominantly localized in the primordial germ cells of newly hatched embryos, A-SG in testis, oogonia, and chromatin nucleolus-stage oocytes in the ovary. In contrast, ly75 mRNA was not detected in spermatocytes, spermatids, spermatozoa, vitellogenic oocytes, or gonadal somatic cells from either males or females. The expression profile of Ly75 protein was similar to that of the mRNA. Furthermore, identification of various fish homologs of ly75 confirmed that their amino acid sequences are well conserved. Therefore, Ly75 may be appropriate for use as a versatile cell surface marker for mitotic germ cells in fish.
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http://dx.doi.org/10.1095/biolreprod.109.082081DOI Listing
October 2010

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources.

Comp Biochem Physiol Part D Genomics Proteomics 2011 Mar 15;6(1):55-61. Epub 2010 May 15.

Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Japan.

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.
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http://dx.doi.org/10.1016/j.cbd.2010.05.003DOI Listing
March 2011
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