Flow-cytometric enrichment of Pacific bluefin tuna type A spermatogonia based on light-scattering properties

• Published in: Theriogenology 2017-10, Vol.101, p.91-98
• Main Authors: Ichida, Kensuke, Kise, Kazuyoshi, Morita, Tetsuro, Yazawa, Ryosuke, Takeuchi, Yutaka, Yoshizaki, Goro
• Format: Article
• Language: English
• Subjects: Animals; Cell Separation - methods; Cell Separation - veterinary; Flow cytometry; Flow Cytometry - methods; Flow Cytometry - veterinary; Light; Light scatter; Male; Pacific bluefin tuna; Scattering, Radiation; Spermatogonia - cytology; Testis - cytology; Tuna; Type A spermatogonia
• ISSN: 0093-691X; 1879-3231
• DOI: 10.1016/j.theriogenology.2017.06.022

We previously established surrogate broodstock in which the donor germ cells transplanted into the peritoneal cavities of xenogeneic recipients were capable of developing into functional eggs and sperm in teleost fish. In this transplantation system, only the undifferentiated germ cells such as type A spermatogonia (ASG) or a portion of the ASG population were capable of being incorporated into the genital ridges of the recipients and undergo gametogenesis. Therefore, the use of enriched ASGs can be expected to achieve efficient donor-cell incorporation. Here, we established a method of isolation and enrichment of the ASG of Pacific bluefin tuna using flow cytometry. Whole testicular cell suspensions were fractionated by forward and side scatter properties, following which ASGs were enriched in a fraction in which the forward scatter signal was relatively high and side scatter signal was relatively low. The diameter of sorted cells using the fraction was identical to the size of ASGs observed in histological analysis, and these cells also expressed the vasa gene. In addition, we succeeded in applying this method to several maturation stages of Pacific bluefin tuna. Since this method was based on light-scattering characteristics of ASGs, it can potentially be applied to various teleosts. We expect that this method can contribute to the production of seeds of Pacific bluefin tuna using surrogate broodstock. •Type A spermatogonia were enriched by light–scattering properties from whole testicular cells in Pacific bluefin tuna.•The sorted cells possessed both morphological and molecular characteristics of type A spermatogonia.•This method was successfully applied to several maturation stages of Pacific bluefin tuna.