title: Enrichment of spermatogonial stem cells using side population in teleost.
abstract: Spermatogenesis originates from a small population of spermatogonial stem cells; this population can maintain continuous sperm production throughout the life of fish via self-renewal and differentiation. Despite their biological importance, spermatogonial stem cells are not thoroughly characterized because they are difficult to distinguish from their progeny cells that become committed to differentiation. We previously established a novel technique for germ cell transplantation to identify spermatogonial stem cells based on their colonizing activity and their ability to initiate donor-derived gametogenesis in the rainbow trout (Oncorhynchus mykiss). Although spermatogonial stem cells can be retrospectively identified after transplantation, there is currently no technique to prospectively enrich for or purify spermatogonial stem cells. Here, we describe a method for spermatogonial stem cell enrichment using a side population. With optimized Hoechst 33342 staining conditions, we successfully identified side-population cells among type A spermatogonia. Side-population cells were transcriptomically and morphologically distinct from non-side-population cells. To functionally determine whether the transplantable spermatogonial stem cells were enriched in the side-population fraction, we compared the colonization activity of side-population cells with that of non-side-population cells. Colonization efficiency was significantly higher with side-population cells than with non-side-population cells or with total type A spermatogonia. In addition, side-population cells could produce billions of sperm in recipients. These results indicated that transplantable spermatogonial stem cells were enriched in the side-population fraction. This method will provide biological information that may advance our understanding of spermatogonial stem cells in teleosts. Additionally, this technique will increase the efficiency of germ cell transplantation used in surrogate broodstock technology.
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Aichi, Japan.
authors.forename: Masanao
authors.initials: M
authors.lastname: Sato
authors.affiliation: Beckman Coulter Japan, Tokyo, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Nagasaka
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan.
authors.forename: Sakiko
authors.initials: S
authors.lastname: Sadaie
authors.affiliation: Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Aichi, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Kobayashi
authors.affiliation: Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan goro@kaiyodai.ac.jp.
authors.forename: Goro
authors.initials: G
authors.lastname: Yoshizaki
mesh_terms: D053687:Adult Stem Cells / Q000637:transplantation*; D000818:Animals; D005398:Fisheries; D008297:Male; D017686:Oncorhynchus mykiss; D013091:Spermatogenesis / Q000502:physiology*; D013093:Spermatogonia / Q000166:cytology*; D013234:Stem Cells / Q000166:cytology*; D013737:Testis / Q000166:cytology*
keywords: Hoechst 33342 dye; fish; flow cytometry; germ cell transplantation; side population; spermatogonial stem cells