Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions

Yano, Ayaka; Suzuki, Kensuke; Yoshizaki, Goro

doi:10.1095/biolreprod.107.064667

Cited by 86 publications

(72 citation statements)

References 36 publications

(38 reference statements)

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“…Rainbow trout (Oncorhynchus mykiss) used in the present study were maintained at the Oizumi Station of the Field Science Center of Tokyo University of Marine Science and Technology (Yamanashi, Japan). Immature whole testes [testis weight, 0.014 ± 0.001 g; gonad-somatic index (gonad weight divided by body weight × 100), 0.091 ± 0.004%] were obtained from 8-to 11-mo-old dominant orange-colored (heterozygous, OR/WT) (27) vasa-GFP transgenic (hemizygous, vasa-GFP/−) (28, 29) rainbow trout donors (standard length, 11.4 ± 0.2 cm), and ASGs were labeled by using the GFP gene (30). Collected testes were maintained in Eagle minimum essential medium (EMEM; Nissui) supplemented with 5% (vol/vol) FBS (Gibco), 25 mM Hepes (Sigma-Aldrich), and 2 mM L-glutamine (Sigma-Aldrich), and kept on ice before use.…”

Section: Methodsmentioning

confidence: 99%

Generation of functional eggs and sperm from cryopreserved whole testes

Lee

Iwasaki

Shikina

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The conservation of endangered fish is of critical importance. Cryobanking could provide an effective backup measure for use in conjunction with the conservation of natural populations; however, methodology for cryopreservation of fish eggs and embryos has not yet been developed. The present study established a methodology capable of deriving functional eggs and sperm from frozen type A spermatogonia (ASGs). Whole testes taken from rainbow trout were slowly frozen in a cryomedium, and the viability of ASGs within these testes did not decrease over a 728-d freezing period. Frozenthawed ASGs that were intraperitoneally transplanted into sterile triploid hatchlings migrated toward, and were incorporated into, recipient genital ridges. Transplantability of ASGs did not decrease after as much as 939 d of cryopreservation. Nearly half of triploid recipients produced functional eggs or sperm derived from the frozen ASGs and displayed high fecundity. Fertilization of resultant gametes resulted in the successful production of normal, frozen ASG-derived offspring. Feasibility and simplicity of this methodology will call for an immediate application for real conservation of endangered wild salmonids.fish genetic resources | testes cryopreservation | germ cell transplantation | stem cell | triploid trout B ecause of the environmental effects of human activities, a growing number of fish species have become threatened or are already extinct (1). Salmonid species are particularly vulnerable to habitat destruction and the effects of climate change as they occupy a unique ecological niche resulting from their longrange migration between freshwater and marine habitats (2, 3). Conservation strategies for these threatened fish must therefore be developed and implemented with all possible haste. Complicating the conservation of threatened salmonid populations are the risks associated with captive breeding and translocation of live fish, such as facility accidents, pathogen infections, genetic drift, and reduced fitness of individuals within natural habitats (4, 5).Cryobanking of fish gametes to semipermanently (6) store genetic resources could serve as an alternative approach to traditional conservation methods (7); however, past attempts at cryopreservation of fish embryos and mature oocytes have been unsuccessful (8), as a result of their large size, high yolk content, and low membrane permeability (9, 10). Although androgenesis performed by using frozen sperm and γ-ray-inactivated xenogenic eggs can regenerate live fish, their survival rate is extremely low, and resulting offspring become nuclear-cytoplasmic hybrids (11). The loss of maternally inherited materials including mitochondrial DNA makes this method impractical, as it also does for the transfer of nuclei from cryopreserved somatic cells into xenogenic oocytes (12, 13). In zebrafish, in vitro maturation of immature oocytes subsequent to cryopreservation is also impossible. Although techniques for cryopreservation of stage I and II oocytes (diameter, 90-350 μm) (14) and ...

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Section: Methodsmentioning

confidence: 99%

Generation of functional eggs and sperm from cryopreserved whole testes

Lee

Iwasaki

Shikina

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

132

118

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“…The availability of fish models expressing green fluorescent protein gene, under the promoter region of the vasa gene, will probably provide new possibilities of characterizing the different spermatogonial generations (Yoshizaki et al 2000;Krovel and Olsen 2002;Takeuchi et al 2002;Okutsu et al 2006a, b;Shikina et al 2008). This is possible because vasa is strongly expressed in early spermatogonia, and the association with FACS (fluorescent activated cell sorting) enables isolation of undifferentiated spermatogonia (Yano et al 2008).…”

Section: Testis Structure and Spermatogenesismentioning

confidence: 99%

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish

Nóbrega

Batlouni

França

2008

Fish Physiol Biochem

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Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology-transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species.

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“…The expression of scp3 was shown to be restricted to the gonads of rat (Lammers et al 1994) and rainbow trout (Yano et al 2008). Similarly, scp3 mRNA was predominantly expressed in the testis (at the recrudescent stage) and ovary (at the early vitellogenic stage) of ricefield eels, which was further confirmed by Western blot analysis using the antiserum against ricefield eel Scp3.…”

Section: Discussionmentioning

confidence: 60%

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel Monopterus albus

Shi

et al. 2016

Fish Physiol Biochem

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Synaptonemal complex protein 3 (Scp3), which is encoded by scp3, is a meiotic marker commonly used to trace the timing of gonadal differentiation in vertebrates. In the present study, the ricefield eel scp3 cDNA was cloned, and a fragment encoding amino acids 49 to 244 was overexpressed. The recombinant Scp3 polypeptide was purified and used to generate a rabbit anti-Scp3 polyclonal antiserum. In adult ricefield eels, scp3 mRNA was predominantly detected in the gonads and faintly detected in discrete brain areas. In the gonads, Scp3 immunoreactivities were shown to be localized to the germ cells, including meiotic primary growth oocytes, spermatocytes, and pre-meiotic spermatogonia. During early ovarian differentiation, immunoreactive Scp3 was not detected in the gonads of ricefield eels at 6 days post-hatching (dph) but was found to be abundantly localized in the cytoplasm of some oogonia at 7 dph, coinciding with the appearance of the ovarian cavity and ovarian differentiation. At 14 dph, strong Scp3 immunostaining was detected on one side of the nucleus with a distinct polarity in some germ cells, presumably at the leptotene stage. Consistent with these results, the expression of scp3 mRNA was faintly detected in the gonads of ricefield eels at 6 dph, increased at 8 dph, and then remained relatively high thereafter. Taken together, these results suggest that the appearance of immunoreactive Scp3 in oogonia could be a marker for early ovarian differentiation in ricefield eels. The translocation of the Scp3 protein from the cytoplasm to the nucleus in the oogonium of ricefield eels appears to be a controlled process that warrants further study.

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Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions

Cited by 86 publications

References 36 publications

Generation of functional eggs and sperm from cryopreserved whole testes

Generation of functional eggs and sperm from cryopreserved whole testes

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel Monopterus albus

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