Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

DOI： 10.3389/fgene.2021.739781

PubMed

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

DOI： 10.1007/s10695-018-0606-x

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Other Link： https://link.springer.com/article/10.1007/s10695-018-0606-x/fulltext.html

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

DOI： 10.1007/s10695-020-00892-8

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Other Link： https://link.springer.com/article/10.1007/s10695-020-00892-8/fulltext.html

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

DOI： 10.1007/s12562-020-01481-7

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Other Link： http://link.springer.com/article/10.1007/s12562-020-01481-7/fulltext.html

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

The original version of this Article contained a typographical error in the Abstract. “Up to 6.7% of the transplanted eggs underwent early development, and one feeding larva (0.5%) was successfully produced.” now reads: “Up to 12% of the transplanted eggs underwent early development, and one feeding larva (0.5%) was successfully produced.” In addition, the Acknowledgement section contained a typographical error. “The study was financially supported by the Ministry of Education, Youth, and Sports of the Czech Republic
projects “CENAKVA” (South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses
CZ.1.05/2.1.00/01.0024) and “CENAKVA II” (LO1205 under the NPU I program)
and the Czech Science Foundation (P502/13/26952 S). The study was also supported by the Grant Agency of the University of South Bohemia in Ceské Budejovice (GAJU Fatira 068/2017/Z) and was partially funded by the French CRB Animal project «Investissements d’Avenir», ANR-11-INBS-0003. The support of the EU COST Actions FA1205: AQUAGAMETE is gratefully acknowledged. We are in debt to Ian AE Butts for the English language editing and Martin Flajšhans for his scientific inputs.” now reads: “The study was financially supported by the Ministry of Education, Youth, and Sports of the Czech Republic
projects “CENAKVA” (South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses
CZ.1.05/2.1.00/01.0024) and “CENAKVA II” (LO1205 under the NPU I program)
and the Czech Science Foundation (17-19714Y). The study was also supported by the Grant Agency of the University of South Bohemia in Ceské Budejovice (GAJU Fatira 068/2017/Z) and was partially funded by the French CRB Animal project (Investissements d’Aveni), ANR-11-INBS-0003. The support of the EU COST Actions FA1205: AQUAGAMETE is gratefully acknowledged. We are in debt to Ian AE Butts for the English language editing and Martin Flajšhans for his scientific inputs.” These errors have now been corrected in the PDF and HTML versions of the Article.

DOI： 10.1038/s41598-020-58285-z

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

The surrogate broodstock technique produces donor-derived gametes via transplantation of germ cells into surrogate fish. The technique is a promising approach for improving the establishment and management of aquaculture strains with desirable genetic traits. The tiger puffer (Takifugu rubripes) is one of the most popular aquaculture fishes in Japan. However, brood stocks have large body sizes and require a long time to reach sexual maturity. The current study established a sterilization technique using gene knockdown in the grass puffer (T. alboplumbeus), which has a small body size and matures in half the time taken by the tiger puffer, and assessed the possibility of using germ cell-deficient grass puffers as recipients to efficiently produce donor-derived tiger puffer gametes. Dead end 1, which has two transcribed variants, was identified in the grass puffer. Morphants resulting from the microinjection of antisense morpholino oligonucleotides against the transcribed variants, into fertilized eggs, showed germ cell deficiency. Germ cell-deficient morphants exhibited sexual dimorphism with respect to their morphological and gene expression patterns. Testicular germ cells prepared from the testes of tiger puffer were intraperitoneally transplanted into morphant hatchlings. These recipients matured normally and produced functional donor-derived gametes separately from endogenous gametes. Thus, the use of germ cell-deficient recipients that produce only donor-derived gametes improves the efficiency of surrogate production and may accelerate the breeding process in tiger puffer aquaculture.

DOI： 10.1016/j.aquaculture.2020.735385

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Singapore  

Sturgeons are one of the oldest, biggest, most valuable and today also most endangered group of fish species. Germ stem cells (GSCs), such us embryonic primordial germ cells (PGCs) or spermatogonial/oogonial stem cells, can be a key for an effective conservation and possible restoration of these unique and astonishing fishes. In this chapter, labeling, development, isolation, and transplantation of GSCs were studied in sturgeons. It was shown that the maternally supplied germ plasm, which determines the PGC origin, is localized in vegetal pole of ovulated egg and remains there throughout the cleavage period, and therefore, the PGC specification pattern is similar to that of anuran amphibians rather than teleostean fishes. This knowledge enabled to develop an original PGC labeling method using common cell tracer dye injection into the vegetal pole of two- to eight-cell stage embryo. Next inhibition of maternally supplied dead end RNA resulted in PGC mismigration and general sterilization of individuals. This method enables preparation of recipients for germ cell transplantation. Isolation and transplantation of spermatogonia and oogonia were developed as well. It was tested that one sturgeon juvenile (Siberian sturgeon) can provide approximately one million germ cells suitable for transplantation. Moreover, it was shown that these cells are capable of propagation via an in vitro culture system and of cryopreservation. After freezing/thawing of sturgeon gonadal tissue followed by enzymatic dissociation, above 90% of viable cells were obtained and used for transplantation. The technique of surrogate production can be applied for conservation and possibly restoration of critically endangered sturgeon species with a long term of maturation and a big body size (e.g., beluga), whereas a more common species with shorter term of maturation and smaller body size (e.g., sterlet) can be used as a recipient (surrogate parent).

DOI： 10.1007/978-981-15-2290-1_17

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

DOI： 10.20431/2454-7670.0603003

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

DOI： 10.1038/s41598-019-46892-4

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

DOI： 10.1111/raq.12389

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

DOI： 10.1016/j.theriogenology.2019.03.032

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DOI： 10.1007/978-1-4939-9009-2_20

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

Somatic cell nuclear transfer (SCNT) is a very promising cloning technique for reconstruction of endangered animals. The aim of the present research is to implement the interspecific SCNT (iSCNT) technique to sturgeon
one fish family bearing some of the most critically endangered species. We transplanted single cells enzymatically isolated from a dissociated fin-fragment of the Russian sturgeon (Acipenser gueldenstaedtii) into non-enucleated eggs of the sterlet (Acipenser ruthenus), two species bearing different ploidy (4n and 2n, respectively). Up to 6.7% of the transplanted eggs underwent early development, and one feeding larva (0.5%) was successfully produced. Interestingly, although this transplant displayed tetraploidism (4n) as the donor species, the microsatellite and species-specific analysis showed recipient-exclusive homozygosis without any donor markers. Namely, with regards to this viable larva, host genome duplication occurred twice to form tetraploidism during its early development, probably due to iSCNT manipulation. The importance of this first attempt is to apply iSCNT in sturgeon species, establishing the crucial first steps by adjusting the cloning-methodology in sturgeon's biology. Future improvements in sturgeon's cloning are necessary for providing with great hope in sturgeon's reproduction.

DOI： 10.1038/s41598-018-24376-1

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

DOI： 10.1007/s10695-018-0553-6

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

DOI： 10.1017/s0967199418000448

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

DOI： 10.1093/biolre/ioy076

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：John Wiley and Sons Inc.  

In oocytes, RNA localization has critical implications, as assembly of proteins in particular subcellular domains is crucial to embryo development. The distribution of mRNA molecules can identify and characterize localized transcripts. The goal of this study was to clarify the origin of primordial germ cells in the oocyte body plan and to reveal the generation of cell lineages by localized RNAs. The distribution of 12 selected mRNAs in sterlet Acipenser ruthenus oocytes was investigated by qPCR tomography and compared with known patterns of mRNA localization in Xenopus laevis. We investigated the distribution of two gene clusters in the ooplasm along the animal–vegetal axis of the sturgeon oocyte, both of which showed clearly defined intracellular gradient pattern remarkably similar to their distribution in the frog oocyte. We elucidated the localization of sturgeon egg germplasm markers belonging to the vegetal group of mRNAs. The mRNAs coding otx1, wnt11, and veg1 found to be localized in the sturgeon animal hemisphere are, in contrast, distributed in the vegetal hemisphere in amphibian. Actinopterygii and Sarcopterygii, two major lineages of osteichthyan vertebrates, split about 476 Ma (Blair &amp
Hedges,), albeit basal lineages share conserved biological features. Acipenseriformes is one the most basal living lineages of Actinopterygii, having evolved about 200 Ma (Bemis, Birstein, &amp
Waldman,), contemporaneous with modern amphibians (Roelants et al.,).

DOI： 10.1002/jez.b.22802

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

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.

DOI： 10.1007/s10695-018-0506-0

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

DOI： 10.1093/biolre/ioy092

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

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.

DOI： 10.1016/j.theriogenology.2017.12.013

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

Knowledge of embryo development is essential to the application of reproductive biotechnology in aquaculture, including for pikeperch Sander lucioperca. We describe pikeperch embryo development and demonstrated effects of temperature on the duration of embryogenesis. Developmental stages in embryos incubated at 15 degrees C were identified as zygote, 0-1.5 h post-fertilization (hpf); cleavage, 2.5-7.5 hpf; blastula, 9-18.75 hpf; gastrula, 21-39, hpf; segmentation, 45-105 hpf; and hatching, 125-197 hpf. Additional groups of eggs were fertilized and incubated at 10, 15, 20, and 25 degrees C to document stages of development, development rate, and survival. The optimal fertilization and incubation temperature was shown to be 15 degrees C, with the highest fertilization, survival, and hatching rates. Embryo development was slower at 10 degrees C, with 45% of fertilized embryos surviving to hatching. Development was accelerated at 20 degrees C, and resulted in a 56% survival rate of fertilized embryos. At 25 degrees C, embryos did not develop to the blastula stage. Pikeperch could be a valuable percid model for research in which flexible incubation temperatures is required. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2017.07.050

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

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.

DOI： 10.1007/s12562-017-1108-y

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

This review provides a general view on teleost germline development using primordial germ cells (PGCs) or spermatogonia (SG), highlighting recent progress in research on these two cell types in teleost fishes. Due to the interest of these cells for gene banking purposes, this chapter also reviews the available protocols for cryopreservation and the techniques that have been employed to assess cellular and molecular status after the process. Protocols for in vitro culture and the possibility of generating PGCs in vitro from non-committed embryonic cells are also discussed. Furthermore, the potential of these cells in surrogate production is presented, analyzing the transplantation and xenotransplantation experiments performed in fish. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.aquaculture.2016.03.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CZECH ACADEMY AGRICULTURAL SCIENCES  

Pikeperch Sander lucioperca is a valuable fish in Europe, and basic information about its embryonic development, especially primordial germ cell (PGC) migration, is important for use in biotechnology. We categorized pikeperch embryonic development into six stages as in other fish species: zygote, cleavage, blastula, gastrula, segmentation, and hatching and described PGC migration. PGCs were visualized by injection of synthesized green fluorescent protein (GFP) within the 3' untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to location at the gonadal ridge. We conducted blastomere transplantation (BT) at the blastula stage. Donor embryos were labelled with GFP-nos3 3' UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve BT chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that BT can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.

DOI： 10.17221/40/2016-CJAS

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

Several sturgeon species are near extinction; therefore an efficient conservation strategy is required. Germ stem cells can be used for long-term storage and restoration of genetic information using surrogate reproduction. This study compared cryopreservation procedures of early stages of Siberian sturgeon Acipenser baerii testicular and ovarian cells. Whole gonad tissue or dissociated cells were frozen at a cooling rate of 1 degrees C/min in phosphate buffered saline with 0.5% bovine serum albumin, 50 mM glucose, and one of four different 1.5 M cryoprotectants: dimethyl sulfoxide, glycerol, ethylene glycol, or dimethyl sulfoxide with propanediol. The number of living cells obtained from 0.1 g of gonadal tissue after freeze/thaw of both whole tissue and dissociated cells was higher using ethylene glycol than with other cryoprotectants. Although there were no differences in the number of living cells in cryopreserved whole tissue vs. dissociated cells, the number of dead cells was lower with whole tissue cryopreservation, indicating that cells that died during freeze/thaw were digested during subsequent enzymatic dissociation. This resulted in more than 90% live cells after freeze/thaw and dissociation. The thawed tissue cryopreserved using ethylene glycol as protectant as well as fresh gonadal tissue were dissociated, and the cells were labelled by PKH26 and transplanted into larvae of sterlet Acipenser ruthenus. Ninety days post-transplant of both fresh and cryopreserved cells, introduced cells proliferated in more than half of the recipients. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cryobiol.2016.02.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

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.

DOI： 10.1007/s12562-015-0939-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CZECH ACADEMY AGRICULTURAL SCIENCES  

Recently, transplantation of germ cells has attracted attention as a potential technique for efficient reproduction of fish. One of the well-proven techniques to deliver donor germ cells into a recipient is the transplantation of primordial germ cells (PGCs) during the blastula stage. Nevertheless, the application of such techniques so far has been limited to model fish species such as zebrafish, due to the lack of information about early development in many fish species. We propose that pikeperch (Sander lucioperca) can be a useful model species for establishing this technique in the order Perciformes, which includes commercially and ecologically important marine species. In this study, we described the important events, namely, embryonic staging, yolk syncytial layer (YSL) formation, and midblastula transition (MBT) during the blastula stage in pikeperch to obtain basic information about early embryonic development. The chorion was softened by treating with 0.2% trypsin and 0.4% urea in Ringer's solution so as to remove it easily by forceps. Although the first cleavage occurred at about 2.5 h post fertilization, blastomeres divided approximately every one hour after this at 15 degrees C. The YSL was formed after the breakdown of marginal cells during the 512- to 1k-cell stage. Cell division analysis by 4'-6-diaminido-2-phenylindole (DAPI) staining revealed that transition from synchronous to asynchronous division occurred after the 10th cleavage (1k-cell stage). Our results indicate that zygotic gene expression (MBT) starts after this stage. Next, we performed blastodisc isolation assay to find the competent stage for embryonic manipulation. Embryos were manipulated by using a microneedle every hour from the 512-cell to the sphere stage, and then developmental rates were evaluated at the hatching stage. The highest survival rate was obtained when we performed this manipulation at the 1k-cell stage. These results clearly showed that the MBT is the best stage for transplantation of PGCs or any cells in pikeperch.

DOI： 10.17221/35/2015-CJAS

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

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production: application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (drid) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-mu M MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization. (C) 2015 The Authors. Published by Elsevier Inc.

DOI： 10.1016/j.theriogenology.2015.07.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Primordial germ cells (PGCs) are the origin of all germ cells in developing embryos. In the sturgeon embryo, PGCs develop from the vegetal hemisphere, which mainly acts as an extraembryonic source of nutrition. Current methods for studying sturgeon PGCs require either killing the fish or using costly and time-consuming histological procedures. Here, we demonstrate that visualization of sterlet (Acipenser ruthenus) PGCs in vivo is feasible by simply labeling the vegetal hemisphere with fluorescein isothiocyanate (FITC)-dextran. We injected FITC-dextrans, with molecular weights varying between 10 000 and 2 000 000, into the vegetal pole of 1- to 4-cell stage embryos. At the neurula to tail-bud developmental stages, FITC-positive PGC-like cells appeared ventrally around the developing tail bud in the experimental group that received a high-molecular-weight FITC-dextran. The highest average number of FITC-positive PGC-like cells was observed in embryos injected with FITC-dextran having a molecular weight of 500 000 (FD-500). The pattern of migration of the labeled cells was identical to that of PGCs, clearly indicating that the FITC-positive PGC-like cells were PGCs. Labeled vegetal cells, except for the PGCs, were digested and excreted before the embryos starting feeding. FITC-labeled PGCs were observed in the developing gonads of fish for at least 3 mo after injection. We also found that FD-500 could be used to visualize PGCs in other sturgeon species. To the best of our knowledge, this report is the first to demonstrate in any animal species that PGCs can be visualized in vivo for a long period by the injection of a simple reagent.

DOI： 10.1095/biolreprod.115.128314

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

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.

DOI： 10.1038/srep13346

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Language：English   Publisher：CELL PRESS  

DOI： 10.1016/j.stemcr.2015.07.001

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

We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii, using 0.3% trypsin (2 hours, 23 degrees C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell-specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate-labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P < 0.05). The efficiency of transplantation was 60% (counted 90 dpt). (C) 2015 The Authors. Published by Elsevier Inc.

DOI： 10.1016/j.theriogenology.2014.12.010

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

We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii, using 0.3% trypsin (2 hours, 23 degrees C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell-specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate-labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P < 0.05). The efficiency of transplantation was 60% (counted 90 dpt). (C) 2015 The Authors. Published by Elsevier Inc.

DOI： 10.1016/j.theriogenology.2014.12.010

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

As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.

DOI： 10.1016/j.stemcr.2014.10.011

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

As complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.

DOI： 10.1016/j.stemcr.2014.10.011

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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：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Sturgeon spermatozoa are immotile in the testis and acquire the potential for motility after contact with urine in Wolffian duct. The present study tested if in vitro incubation of testicular sperm in seminal fluid from Wolffian duct sperm leads to the acquisition of sperm fertilization ability. Sterlet sperm was taken from the testes, matured in vitro and cryopreserved. The fertility and motility of cryopreserved semen were tested. Matured testicular sperm showed freeze-thaw survival rates similar to Wolffian duct sperm, which is commonly used in sturgeon artificial propagation. Matured testicular sperm and Wolffian duct sperm post-thaw motility rate and curvilinear velocity were not significantly different, while duration of matured testicular sperm motility was significantly shorter than that of Wolffian duct sperm. Development rates of embryos obtained with post-thaw matured testicular sperm and Wolffian duct sperm were not significantly different. In vitro maturation of sterlet testicular sperm can potentially be useful in sperm cryobanking. (C) 2014 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cryobiol.2014.07.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Embryonic and larval development of the tench, Tinca tinca, is described, along with the origin and migration routes of germ cell lineage. Primordial germ cells (PGCs) potentially transmit genetic information to the next generation, and represent a powerful tool for creating a germ-line chimera within fish species. PGCs were identified by injecting synthesized mRNA, combining green fluorescent protein and the zebrafish nos1 3UTR, under the blastodisc of embryos at the 1-4 cell stage. Developmental stages were divided into five periods defined by morphological features: cleavage (40min-5h post-fertilization (hpf), blastula (3.6-12hpf), gastrula (12-20hpf), segmentation (20-65hpf), and hatching (65-188hpf). The migration pathways of fluorescent PGCs were detected from 100% epiboly (18hpf) to the end of the hatching period (184hpf) in 69.3% of injected embryos, which migrated to the site of future gonads. Each hatching larva possessed 3-10 labeled PGCs with 95.1% of these cells localized at the genital ridge. These data may have use in practical aquaculture as well as in research to investigate germ-line chimerism in tench.

DOI： 10.1111/jai.12429

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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.

DOI： 10.1002/jez.1851

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

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.

DOI： 10.1016/j.aquaculture.2013.05.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CZECH ACADEMY AGRICULTURAL SCIENCES  

A practical technique for isolation and cryopreservation of tench (Tinca tinca) (Cyprinidae, Teleostei) early stages of germ cells (GC), including spermatogonia and spermatocytes, is reported for the first time. The germ-line cells possess the ability to differentiate into functional gametes of both sexes. These early stages of germ cells are small enough to be well-suited to cryopreservation, which, together with their high level of plasticity, makes their preservation a promising tool for maintaining genetic resources. Testicular cells were distinguished and separated by Percoll gradient, with the highest proportion of GC (62.2%) obtained from the 30% layer. The concentration and viability of GC were determined, and specific staining (DDX4) for germ cells was used to distinguish GC from somatic cells. Early stages of germ cells were cryopreserved in an extender composed of phosphate buffered saline (pH 8) with 0.5% BSA, 50mM D-glucose, and containing 1.5M cryoprotectant in the pre-programmed PLANER Kryo10 series III using a cooling protocol from +10 degrees C to -80 degrees C at a rate of 1 degrees C/min. The effect of six cryoprotectants - methanol, dimethyl sulfoxide, dimethyl sulfoxide + propanediol (1 : 1), glycerol, ethylene glycol, and dimethylacetamid was assessed, and the results were evaluated by comparing the percentage of viable frozen/thawed GC by ANOVA, Tukey's HSD test (P < 0.05). Almost the same viability rates were obtained with no significant differences among tested cryoprotectants, indicating high stability of GC in cryoprotectants. Nevertheless, glycerol at a concentration of 1.5M was associated with the highest survival rate of thawed tench GC (57.69 +/- 16.85%).

DOI： 10.17221/7589-cjas

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In teleosts, primordial germ cells (PGC)s are formed by the inheritance of cytoplasmic determinants called germ plasm during early embryonic development. Although several molecular components of the germ plasm have been identified, it is still unclear if cytoplasmic regions that show expression of germ cell specific marker genes such as vasa and nanos1 are necessary and sufficient for inducing PGCs in the embryo. In this study, the relationship between PGC formation and yolk granules was examined by reducing numbers of granules in the embryo shortly after fertilization. Ten minutes after fertilization, goldfish eggs were centrifuged at 700g. This treatment resulted in the formation of a transparent blastodisc with apparently no yolk granules. When this segment was removed from the centrifuged egg and placed in culture, it exhibited a holoblastic mode of cleavage and developed into a spherical embryoid body. Maternal vasa mRNA was detected at the connections between blastomeres that developed from the separated fragment. Electron dense structures could be detected along the cleavage furrow in a position seemingly identical to vasa mRNA localization. When the spherical embryoid body from the centrifuged eggs was transplanted onto the blastodisc of a normal blastula stage embryo, PGCs from the donor embryo were detected around the genital ridge in the resulting chimeric larva at 10days post-fertilization. These results suggest that cytoplasmic fragments from centrifuged eggs contain factors sufficient for PGC differentiation. However, transplantation of the putative cytoplasmic germ plasm into host blastodiscs did not result in the formation of PGCs.

DOI： 10.1111/jai.12068

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

DOI： 10.1111/jai.12069

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC ANIMAL SCIENCE  

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.

DOI： 10.2527/jas.2011-4884

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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：AMER SOC ANIMAL SCIENCE  

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.

DOI： 10.2527/jas.2011-4329

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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)   Publisher：SOC STUDY REPRODUCTION  

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa); Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 +/- 108 DsRed-positive germ cells was recovered from each female. For transplantations, a total of approximately 620 +/- 242 EGFP-positive cells of which 12 +/- 4.7 were DsRed-positive germ cells were introduced into the abdominal cavity under the swim bladder of 2-wk-old sterile hybrid larvae. Six weeks after transplantation, a total of 10 recipients, obtained from 2 different transplantations, were examined, and 2 individuals (20%) were identified that possessed a large number of DsRed-and EGFP-positive cells in the gonadal region. The transplanted ovarian germ cells successfully colonized the gonads and differentiated into sperm in the male hybrid recipients. Of 67 adult recipients, 12 (18%) male chimeric fish reproduced and generated normal offspring when paired with wild-type zebrafish females. The fertilization efficiency ranged from 23% to 56%. Although the fertile male chimeras were generated by transplantation of ovarian germ cells, the F1 generation produced by the male chimeras contained both male and female progeny, indicating that male sex determination in zebrafish is not controlled by sex chromosome heterogamy. Our findings indicate that a population of ovarian germ cells that are present in the ovary of adult zebrafish can function as germline stem cells, able to proliferate and differentiate into testicular germ cells and functional sperm in male recipients. The high frequency of germline chimera formation achieved with the ovarian germ cells and the convenience of identifying the chimeras in the sterile host background should make this transplantation system useful for performing genetic manipulations in zebrafish.

DOI： 10.1095/biolreprod.110.088427

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

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.

DOI： 10.1111/j.1439-0426.2010.01548.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3'UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos 1 3'UTR mRNA into 1-4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.

DOI： 10.1387/ijdb.092914rg

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

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.

DOI： 10.1387/ijdb.092896tf

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

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.

DOI： 10.1387/ijdb.093059yk

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

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.

DOI： 10.1387/ijdb.103111ts

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

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.

DOI： 10.1095/biolreprod.108.075150

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Language：English   Publisher：SOC STUDY REPRODUCTION  

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Language：English   Publisher：SOC STUDY REPRODUCTION  

DOI： 10.1093/biolreprod/81.s1.39

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. in many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.

DOI： 10.1095/biolreprod.107.060038

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

This review introduces surrogate production as a new technique for fish-seed production in aquaculture. Surrogate production in fish is a technique used to obtain the gametes of a certain genotype through the gonad of another genotype. It is achieved by inducing germ-line chimerism between different species during early development. Primordial germ cells (PGCs) are the key material of this technique to induce germ-line chimera. In several species, it has been reported that PGCs differentiated from the blastomeres inherited some maternally supplied mRNA located in the terminal regions of the early cleavage furrows. PGCs from donor species (or strains) are isolated and transplanted into host species to induce the germ-line chimera. Four methods for inducing germ-line chimera are described: blastomere transplantation, blastoderm-graft transplantation, transplantation of PGC from the genital ridge, and transplantation visualised PGC with GFP fluorescence. Several problems preventing the successful induction of germ-line chimera in various fish species are discussed. Surrogate production, however, opens the possibility of efficient fish-seed production and effective breeding and transfer of biodiversity to an aquaculture strain. Conservation and efficient utilisation of genetic resources will be achieved through surrogate production combined with the cryopreservation of PGCs. (c) 2007 Elsevier B.V All rights reserved.

DOI： 10.1016/j.seares.2007.02.003

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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.

DOI： 10.2108/zsj.23.977

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

Plasminogen activator inhibitor-1 (PAI-1) is a key molecule that regulates turnover of the extracellular matrix. In the present study, we characterized PAI-1 gene expression in mast cells and melanocytes. In bone marrow-derived cultured mast cells, up-regulation of the PAI-1 gene was observed upon treatment with TGF-beta(1), and was regulated at the transcriptional level. Microphthalmia-associated transcription factor (MITF), a member of the basic helix-loop-helix leucine zipper family of tissue-specific transcription factors predominantly expressed in mast cells, melanocytes and osteoclasts, also stimulated PAI-1 gene transcription, and TGF-beta(1) did not increase PAI-1 mRNA levels in mast cells from mi/mi mice expressing dominant-negative MITF. MITF isoforms regulated TGF-beta(1)-induced transcription of PAI-1 differently; MITF-E-mediated transcription was further increased by TGF-beta(1), whereas transcriptional activation by TGF-beta(1) was blocked by MITF-M or MITF-mc expression. In contrast, activin A, another member of the TGF-beta family, enhanced transcription induced by MlTF-M, as well as by MITF-E, although MITF-mc blocked activin A-induced transcription of PAI-1. Different regulation of PAI-1 gene expression upon TGF-beta(1) and activin A treatment was also detected in B16 melanocytes; TGF-beta(1) transiently increased the PAI-1 mRNA level, whereas activin A had prolonged effects on up-regulation of PAI-1. Our results on the control of PAI-1 gene expression by MITF isoforms, TGF-beta(1) and activin A suggest that discrete regulation of the plasminogen activator system occurs in a cell type-specific manner. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cellsig.2005.04.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

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.

DOI： 10.1387/ijdb.062143ts

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

The vasa genes are expressed in the germ cell lineage in many organisms, but their expression patterns show large variations. Recent studies suggest that vasa transcripts are involved in germ cell lineage development. In this paper, we isolated the vasa cDNA clone from a teleost, shiro-uo, Leucopsarion petersii and examined its expression pattern during embryogenesis. Then, we examined the functional significance of vasa mRNA during the formation of primordial germ cells (PGCs). The amino acid sequence of shiro-uo VASA is 61.1% identical to that of zebrafish. In whole-mount in situ hybridization, vasa transcripts appeared at the 4- and 8-cell stages as four spots at both ends of two cleavage planes between the lower tier of blastomeres and the yolk cell mass. At the 16-cell stage, eight spots were observed. After the blastula stage, shiro-uo vasa transcripts showed similar localization as in the zeblafish. Ultrastructural analysis of 4-cell stage embryos revealed the presence of a subcellular organelle that resembled "nuage" in the germ cell lineage observed in the embryos of various organisms. We carried out micromanipulation of 4- or 8-cell stage embryos to remove the vasa mRNA-containig spots and then measured the number of the vasa-expressing PGCs in the genital ridge of the manipulated embryos. The numbers decreased when all of the four spots were removed, indicating that the vasa-containing spots at early cleavage stages have important functions in the development of PGCs.

DOI： 10.1387/ijdb.062172am

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC FISHERIES SCIENCE  

To elucidate the effects of blastoderm graft for chimeric-fish production on further development, transplantations of upper, lower or entire part of blastoderm were performed onto the animal part of host embryos during the blastula stage in zebrafish. In the case of upper, all resultant chimeric embryos developed normally, while in the lower part, many acephalic embryos appeared. When the entire blastoderm was transplanted, many resultant embryos showed normal phenotype with extra cell-mass, while a few had double axis. vas mRNA, one of the markers of PGCs, also kept the expressions in both donor and recipient blastomeres in transplantation of the entire blastoderm. Donor PGCs were frequently detected at germinal anlagen in histological sections of 6-day larvae developed from transplantations of lower and entire blastoderm, but seldom in those of the upper part. These results might suggest that graft transplantation was effective for production of germ-line chimera, but sometimes caused un-regulaory differentiation of graft cells under the community effect.

DOI： 10.2331/suisan.71.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

The property of primordial germ cells (PGCs) in fragmented goldfish embryos was investigated. When 1- and 2- cell embryos were cut at several perpendicular levels at the animal-vegetal axis, cells expressing vas mRNA were observed in the resultant embryos derived from all kinds of animal fragments. Blastodisc fragments from the 1- to 2-cell stage developed to spherical embryos containing yolk body with a yolk syncytial layer (YSL). Germ ring and no tail expression were not observed in the spherical embryo. When the spherical embryo labeled with tracer dye or GFP-nosl 3'UTR mRNA was transplanted onto the animal part of the blastoderm in a host embryo at the blastula stage, PGCs of spherical embryo origin were detected around the gonadal ridges in the resultant embryos which developed normally. These results suggest that small animal fragments should contain factors sufficient for PGC differentiation and that PGCs differentiate without mesoderm induction, since mesoderm is not induced in a spherical embryo.

DOI： 10.1387/ijdb.052027so

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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

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.

DOI： 10.1387/ijdb.041912ts

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：Japanese   Publisher：水産育種研究会  

CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

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.

DOI： 10.2108/zsj.21.747

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Language：Japanese   Publisher：Japanese Society for Aquaculture Science  

The embryonic development of shima-ukigori, <I>Gymnogobius opperiens</I> (Family; gobiidae) at 20°C was staged by morphological and histological criteria, and analyzed by cell-lineage tracing, with special reference to the embryonic patterning mechanism during pre-gastrula stage. Egg cytoplasm was separately distributed from yolk even in unfertilized egg, but included many yolk granules within it. The yolk granules were maintained until mid-somitogenesis stage, and disappeared thereafter. The third cleavage sometimes occurred horizontally. Yolk cell kept the spherical shape without yolk extension throughout the embryonic development. These properties are similar to those in gobiidae fish, but different from those in cyprinidae fish, such as zebrafish and goldfish. Cell-labeling at the 16- to 32-cell stage showed that the blastomeres mingled with each other during blastula to early gastrula stages, suggesting developmental fates are not destined in these stages. These results suggest that the fundamental mechanisms of embryonic patterning are conserved in varied fish species, although shapes of embryo and yolk cell are different.

DOI： 10.11233/aquaculturesci1953.52.177

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Language：Japanese   Publisher：Japanese Society of Animal Breeding and Genetics  

DOI： 10.5924/abgri2000.31.2_47

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Shiro-uo (ice goby; teleost fish), Leucopsarion petersii, shows a unique cleavage pattern characterized by two tires of blastomeres at 8-cell stage, like that of echinoderm and amphibian embryo. Such a pattern is suitable to isolation and cell lineage experiments. In this study, cell lineage of germ-line was traced by histological observation and cell labelling experiment at the 8-cell stage. Primordial germ cells (PGCs) were first detected histologically at the 10-somite stage, and migrated to gonadal anlage at 10 days post-fertilization, through usual way described in other teleost species. When a single blastomere was labelled with tracer dye at 8-cell stage, both upper and lower tires generated labelled PGCs at gonadal anlage although upper tires occasionally. This result suggests that all blastomeres at the 8-cell stage have potential to produce PGCs in shiro-uo.

DOI： 10.2108/zsj.19.1027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

Isolation of cleavage-stage blastomeres and the study of their developmental potential has been used extensively for analyzing the mechanisms of embryogenesis in vertebrates, including amphibians and echinoderms. We devised a method to isolate 8-cell stage blastomeres in the teleost, shiro-uo, by utilizing its unique cleavage pattern of the horizontal 3rd cleavage plane. Removal of all the upper blastomeres at the 8-cell stage allowed almost normal embryogenesis from the remaining lower blastomeres and yolk cell mass. Isolated upper or lower blastomeres formed vesicles and spherical bodies, which later showed morphological changes during cultivation. Mesoderm formation was detected not only in the cultivated lower blastomeres or whole blastomeres but also in the upper blastomeres isolated from the yolk cell mass at the 8-cell stage, although at a lower frequency than the lower blastomeres. These results indicated the presence of very early signaling for mesoderm induction, which is independent from the currently postulated signals from the yolk syncytial layer at later stages. This also indicated non-equivalence or differentiation of the blastomeres from the very early cleavage stage in teleost embryos.

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