Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s10228-019-00707-8

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Other Link： http://link.springer.com/article/10.1007/s10228-019-00707-8/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ARC Publications Pvt Ltd.  

DOI： 10.20431/2454-7670.0603003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.

DOI： 10.1038/s41598-019-50418-3

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Authorship：Lead author,　Corresponding author  

DOI： 10.1016/j.theriogenology.2019.03.032

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Language：English   Publishing type：Research paper (scientific journal)  

Bluefin tuna is one of the most important aquaculture species in several countries; however, information regarding the primordial germ cell (PGC) development and migration in this species is scarce. This information is vital for application in reproductive biotechnology, for example, induced sterility through targeted cell ablation or PGC manipulation. Teleost PGC can be visualized by injecting an RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3) into eggs. In this study, we identified the PGC and its migratory pathway during early embryogenesis and larvae development by injecting the GFP-zf-nos3 3'UTR mRNA into the Pacific bluefin tuna (PBT, Thunnus orientalis). PBT PGCs were initially found around the marginal and dorsal regions of the blastodisc at 50%-epiboly stage. The PGCs were aligned as two elongated lines at the posterior part of the embryonic body during the early segmentation period, and eventually formed a single tight cluster underneath somites 10 to 15 of the embryonic body until the late segmentation period. Although the aggregated PGCs stayed at the same position during hatching, they started migrating anteriorly and were split into two populations at 3 days after hatching (DAH). Until 15 DAH, these PGCs settled in two bilateral lines at the apex of the peritoneal cavity. Histological analysis of PBT larvae revealed that at 3 and 5 DAH, the PGCs were not enclosed by the somatic cells, whereas at 15 DAH, they were entirely covered by the somatic cells, indicating the development of the primordial gonads. These results are essential for future experiments in germ line control technologies for bluefin tuna.

DOI： 10.1016/j.theriogenology.2019.03.031

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Publishing type：Part of collection (book)   Publisher：Springer New York  

DOI： 10.1007/978-1-4939-8831-0_27

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DOI： 10.1007/978-1-4939-9009-2_20

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Aquaculture Research, Nishimura Toushadou Ltd.  

Japanese anchovy (Engraulis japonica) is an important marine species for commercial fisheries in addition to being a key part of coastal ecosystems around Japan. The species is a multiple spawner having oval-shaped pelagic eggs. Based on its morphological changes and observation of spawning, this study shows that Japanese anchovy completes final oocyte maturation (FOM) within eight hours, and the process can be classified into five stages: stage-I (St-I), post-vitellogenic oocyte (PVO)
stage-II (St-II), germinal vesicle migration (GVM)
stage-III (St-III), germinal vesicle breakdown (GVBD)
stage-IV (St-IV), late hydration
and stage-V (St-V), ovulation under photoperiod of 14L:10D with water temperature 19.5 to 20.9C. The average wet weight of St-I oocytes was 69 μg with a longitudinal diameter of 728 μm and 75% water content and then increased to 244 μg, 1,386 μm, and 94%, respectively, at St-V. Gel chromatographic analysis indicated a change in peak position of native lipovitellin from 430 to 170 kDa. Moreover, SDS-PAGE analysis indicated proteolytic degradation during the latter stages of FOM. The free amino acid (FAA) content in the St-I oocytes (2 nmol/individual) became approximately 11-fold greater in St-V eggs (22 nmol/individual), suggesting that the FAAs are produced from the proteolytic degradation of the yolk proteins and regulate the buoyancy of the eggs through osmotic effector generation in the Japanese anchovy.

DOI： 10.11233/aquaculturesci.65.29

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.stemcr.2015.07.001

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV BASQUE COUNTRY UPV-EHU PRESS  

Primordial germ cells (PGCs) appear during early embryogenesis and differentiate into gametes through oogenesis or spermatogenesis. Teleost PGCs can be visualized by injecting RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3). Although this method has been widely applied to teleost PGCs, the visualization of PGCs in pelagic species that have eggs with a hard chorion is more problematic due to the technical difficulty of microinjection into their eggs. In this study, we developed a reliable method for microinjection of fertilized eggs in a pelagic species, the barfin flounder. Using a microneedle with a constriction "brake", we were able to introduce gfp-nos3 3'UTR mRNA into embryos and to determine the migration route of PGCs. We also isolated the barfin flounder nos3 (bf-nos3) gene to compare its 3'UTR sequence with that of zebrafish. The 3'UTR of the bf-nos3 sequence was longer than that of zf-nos3. However, PGCs were also visualized after injection of gfp-bf-nos3 3'UTR mRNA both in zebrafish and barfin flounder. These results suggest that the function of nos3 is conserved between these species regardless of the sequence differences. The method developed here for labeling PGCs with gfp-nos3 mRNA will provide a means to study PGC development in the embryos of a wide range of marine fish species.

DOI： 10.1387/ijdb.150008rg

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Primordial germ cells (PGCs) arise elsewhere in the embryo and migrate into developing gonadal ridges during embryonic development. In several model animals, formation and migration patterns of PGCs have been studied, and it is known that these patterns vary. Sturgeons (genus Acipenser) have great potential for comparative and evolutionary studies of development. Sturgeons belong to the super class Actinoptergii, and their developmental pattern is similar to that of amphibians, although their phylogenetic position is an out-group to teleost fishes. Here, we reveal an injection technique for sturgeon eggs allowing visualization of germplasm and PGCs. Using this technique, we demonstrate that the PGCs are generated at the vegetal pole of the egg and they migrate on the yolky cell mass toward the gonadal ridge. We also provide evidence showing that PGCs are specified by inheritance of maternally supplied germplasm. Furthermore, we demonstrate that the migratory mechanism is well-conserved between sturgeon and other remotely related teleosts, such as goldfish, by a single PGCs transplantation (SPT) assay. The mode of PGCs specification in sturgeon is similar to that of anurans, but the migration pattern resembles that of teleosts.

DOI： 10.1371/journal.pone.0086861

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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.

DOI： 10.1016/j.ydbio.2012.07.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders).

DOI： 10.1371/journal.pone.0024460

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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.

DOI： 10.1016/j.aquaculture.2011.04.019

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/j.1439-0426.2010.01548.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：40  

DOI： 10.1073/pnas.1007032107

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：10  

DOI： 10.1387/ijdb.103111ts

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：10  

DOI： 10.1387/ijdb.093059yk

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：10  

DOI： 10.1387/ijdb.092914rg

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press ({OUP})  

DOI： 10.1093/biolreprod/81.s1.39

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Thyroid-stimulating hormone receptors (TSHRs) are primarily expressed in the thyroid of vertebrates, however recently, transcripts encoding TSHR have been found abundantly in the gonads in a variety of fish species. The purpose of this study is to characterize the channel catfish TSHR and to examine whether the transcript are translated into protein in the gonad or store the transcript as maternal RNA for later use. The cDNA encoding the TSHR was isolated from the channel catfish thyroid but the transcript was determined to be expressed in a number of tissues, including the gonads. In fact, the ovarian expression of TSHR changed significantly during the reproductive season and peaked after the vitellogenic growth phase. Furthermore, the TSHR transcript was also detected in unfertilized eggs but not in fertilized egg of catfish. LM-PAT analysis demonstrated that catfish TSHR transcripts were fully polyadenylated in thyroidal follicles, gonads and unfertilized eggs suggesting that they were translated into protein opposed to being "stored mRNA". Western blot analysis using polyclonal antibodies against the catfish TSHR verified this assumption by visualizing immunoreactive protein in the thyroid, testis, and the post-vitellogenic ovary in abundance. A functional assay clearly showed that the recombinant catfish TSHR was specifically activated by bovine TSH but not by recombinant catfish follicle-stimulating hormone (FSH) and luteinizing hormone (LH). As in other species, the heterologous gonadotropin, hCG, partially activated the receptor. These results suggested that TSHR plays important roles for gametogenesis rather than embryogenesis.

DOI： 10.1016/j.ygcen.2009.01.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：1  

DOI： 10.1095/biolreprod.107.060038

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

DOI： 10.1016/j.seares.2007.02.003

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The present study analyzes the temperature-dependent sex differentiation in goldfish, which have an XX-XY sex determining system. Hatchlings from an XX male-XX female cross were reared at 23 ± 0.5 °C until Day 12 (days after fertilization), and divided into experimental groups reared at 15, 17, 20, 23, 25, 28 or 30 ± 0.5 °C. The proportions of females were 94.6% at 15 °C, 100% at 17 °C, 94.6% at 20 °C, 90.5% at 23 °C, 46.6% at 25 °C, 15.4% at 28 °C, and 7.7% at 30 ± 0.5 °C. This result confirms that genotypic females can be changed to phenotypic males by high temperature treatment. To determine the temperature sensitive period (TSP) of sex differentiation, the sex ratio was measured in groups of larvae shifted reciprocally between 23 and 30 °C as development progressed. The percentage of females increased in test groups shifted from 23 to 30 °C at Days 17, 22 and 27 and decreased in groups shifted from 30 to 23 °C at Day 17 and 22. The end point of the TSP occurs on Day 17 at 30 °C and on Day 22 at 23 °C, implying that the TSP is shortened at higher temperatures. To investigate the effect of fluctuating temperatures on sex differentiation, several one-day temperature pulses from 23 to 30 °C or 30 to 23 °C were applied, during the TSP. The pulse to 23 °C, from the basal temperature of 30 °C, resulted in a decreased percentage of males, however, the pulse to 30 °C, from the basal temperature of 23 °C, had no effect on the sex ratio. These results show that high temperature must be maintained during the TSP in order to produce a high proportion of masculinized genetic female goldfish. In conclusion, water temperature can play an important role in the process of sex differentiation in goldfish, as has been seen in other teleosts and reptiles, and be applied to aquaculture as a tool for control of sex. © 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquaculture.2005.10.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.aquatox.2005.10.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioscientifica  

Membrane-bound progestin receptors (mPR) were recently cloned and characterized as a new class of steroid receptors that transduce cell-signals through alteration of MAP kinase- and cAMP-dependent pathways. To further develop our understanding of this new class of steroid receptors, we characterized the cDNAs and genes of thealpha, beta and gamma forms of the channel catfish mPRs (IpmPR). The predicted alpha and beta proteins have 49% sequence identity, whereas they only have 30% and 27% identities, respectively, with the gamma form. Furthermore, IpmPRalpha and IpmPRbeta genes have similar structures featuring intronless coding regions, while IpmPRgamma gene is composed of 8 exons and 7 introns. The 5'-flanking region of each IpmPR gene differs, but each contains putative transcriptional regulatory elements of factors known to influence reproductive physiology and endocrine disruption, for example, responsive elements for cAMP and steroids and the recognition sites for steroidogenic factor-1 and for the aryl hydrocarbon receptor. The IpmPRalpha gene was detected in all the tissues tested with relatively greater expression in brain, pituitary, muscle and testis. The expression of IpmPRbeta was much lower than that of IpmPRalpha and the transcript was predominantly observed in brain, pituitary, ovary and testis. In contrast, the IpmPRgamma transcript was mainly detected in gill, ventral aorta, intestine, and trunk kidney. In conclusion, all the structural features of the IpmPRs strongly suggest that the closely related alpha and beta forms are distantly related to the gamma form. Additionally, regulatory features of the 5'-flanking regions and the differences in tissue-specific expression of each IpmPR gene suggest that they are involved in different endocrine functions in catfish.

DOI： 10.1677/jme.1.01721

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Membrane-bound progestin receptors (mPRs) are potential intermediaries in meiotic maturation of fish oocytes and other physiological processes. In this study, gene expression of the mPRs in the ovary of catfish and zebrafish during the reproductive cycle and the hormonal regulation of the expression were investigated. The transcript abundance of catfish mPRalpha gradually increased in conjunction with ovarian growth and then decreased prior to spawning period whereas the ovarian gene expression of mPRbeta varied little throughout the reproductive cycle. In contrast, mPRgamma gene expression peaked early in the mid-vitellogenic stage. The transcript abundance of zebrafish mPRalpha and beta was low in ovarian follicles at early stages of oogenesis and gradually increased after the onset of vitellogenic growth and, thereafter, the gene expression did not vary. Gonadotropic treatment did not modulate the ovarian expression of mPRalpha and beta genes in either catfish or zebrafish. On the other hand, exposure to 17,20beta-dihydroxy-4-pregenen-3-one (the maturation-inducing steroid in this species) resulted in the down-regulation of mPRalpha in catfish ovary whereas gene expression was significantly induced by estradiol-17beta. Taken together, these findings suggest that gonadotropin-induced final oocyte maturation may not require an induction of mPR(s) expression or that the gonadotropin stimulates mPR protein production at the post-transcriptional level, presuming these receptors are indispensable for oocyte maturation.

DOI： 10.1016/j.ygcen.2005.01.017

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ygcen.2004.07.003

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Authorship：Lead author   Publishing type：Research paper (international conference proceedings)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1023/b:fish.0000030578.25321.8f

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1023/b:fish.0000030466.23085.94

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Fisheries Science  

DOI： 10.1046/j.1444-2906.2000.00062.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Fisheries Science  

Temperature-dependent sex determination has been demonstrated in several species of fish. This study analyzes this phenomenon in barfin flounder <i>Verasper moseri</i>. The sex ratio was 1:1 when fish were maintained at 14&deg;C from Day 34 to Day 95. However, acclimation to 18&deg;C for 62 days resulted in an all male population. The proportion of males from 10.1mm up to 29.7mm in total length was affected by high temperature treatments. Survival rates were not affected by these treatments. Morphological differentiation of the gonad into either ovary or testis became distinguishable at 35mm in total length. This time corresponds to the end of the temperature-sensitive period. These results suggest that gonadal sex was determined by temperature prior to the onset of gonadal sex differentiation in this fish.

DOI： 10.2331/fishsci.65.884

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