Visualization and tracking of live type a spermatogonia using a fluorescence-conjugated antibody in Salmo species

Abstract

Combining germ cell cryopreservation with transplantation is a powerful tool for preserving the genetic resources of various fish strains. In this germ cell transplantation system, since only type A spermatogonia (ASGs) among the various testicular cells have the potency to colonize recipient gonads and resume gametogenesis, techniques for visualization, isolation, and tracing of ASGs facilitate ease of manipulation. Here we established an ASG visualization technique in two Salmo species: brown trout and Atlantic salmon. We applied a monoclonal antibody (antibody No. 95), which specifically recognizes a cell surface antigen of ASGs obtained from rainbow trout, against Salmo species. Immunohistochemistry studies showed that antibody No. 95 also specifically detected the ASGs of brown trout and Atlantic salmon. Furthermore, marker-gene analyses of brown trout testicular cells sorted by flow cytometry using Alexa flour 488-conjugated antibody No. 95 revealed that fluorescent-positive cells possessed molecular characteristics of ASGs. Finally, to investigate the transplantability of antibody-positive cells, ASGs pre-stained with antibody No. 95 were transplanted into the peritoneal cavity of rainbow trout larvae. As a result, fluorescent cells incorporated into the recipient gonads, suggesting that the visualized ASGs possess transplantability. Thus, it is expected that the ASG visualization methods developed in this study will facilitate the development of more efficiency of germ cell transplantation techniques in Salmo species.