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BOR - Papers in Press, published online ahead of print September 26, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.064667
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BIOLOGY OF REPRODUCTION 78, 151–158 (2008)
DOI: 10.1095/biolreprod.107.064667
© 2008 by the Society for the Study of Reproduction, Inc.

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

Ayaka Yano 2, Kensuke Suzuki 2, and Goro Yoshizaki 1 2 3

Department of Marine Biosciences,2 Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan Solution Oriented Research for Science and Technology (SORST),3 Japan Science and Technology Agency, Saitama 332-0012, Japan

ABSTRACT

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.

flow cytometry, GFP, spermatogenesis, spermatogonial transplantation, vasa

Correspondence: 1Goro Yoshizaki, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. FAX: 81 3 5463 0558; e-mail: goro{at}kaiyodai.ac.jp






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Copyright © 2008 by the Society for the Study of Reproduction.