| Flow-cytometric isolation of testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions. | |
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MedLine Citation:
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PMID: 17901070 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay. |
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Authors:
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Ayaka Yano; Kensuke Suzuki; Goro Yoshizaki |
Publication Detail:
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Type: Journal Article Date: 2007-09-26 |
Journal Detail:
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Title: Biology of reproduction Volume: 78 ISSN: 0006-3363 ISO Abbreviation: Biol. Reprod. Publication Date: 2008 Jan |
Date Detail:
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Created Date: 2007-12-21 Completed Date: 2008-03-25 Revised Date: 2008-11-21 |
Medline Journal Info:
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Nlm Unique ID: 0207224 Medline TA: Biol Reprod Country: United States |
Other Details:
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Languages: eng Pagination: 151-8 Citation Subset: IM |
Affiliation:
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Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Cell Separation / methods Cloning, Molecular Fish Proteins / genetics, metabolism* Flow Cytometry Green Fluorescent Proteins / genetics* Male Oncorhynchus mykiss / genetics, metabolism* Phylogeny Promoter Regions, Genetic Spermatozoa / cytology* Testis / cytology* |
| Chemical | |
Reg. No./Substance:
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0/Fish Proteins; 147336-22-9/Green Fluorescent Proteins |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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