Isolation and molecular characterization of sertoli and undifferentiated spermatogonial cell populations using GFP-expressing transgenic zebrafish lines

The preservation of the genetic resources (mitochondrial and nuclear genomes) and the regeneration of individuals are important economic and ecological issues for the sustainability of the aquaculture sector and conservation of the biodiversity, respectively. In fish, the cryopreservation of the ovocytes and embryos is impossible but the transplantation of cryopreserved diploid spermatogonial stem cells (SSC) in pre-hatching recipient embryos is a promising approach fulfilling the requirements. However, the rareness, and our incapacity to efficiently purify and amplify in vitro the SSCs limit the use and the dissemination of this procedure. In order to unravel mechanisms involved in the control of SSC self-renewal, we have undertaken the identification of paracrine factors that are released from the Sertoli cells within the germinal niche and could act on their cognate membrane bound receptors expressed on SSC. In the present study, we have produced transgenic zebrafish lines that express GFP specifically in the Sertoli cells (Dr_Gsdf:eGFP) or in spermatogonia (Dr_Vasa:eGFP) using promoter fragments of cell-specific genes (gsdf and vasa respectively). The expression pattern of the transgene was validated using whole mount RNA in situ hybridization and immunocytochemistry techniques. Note that transgene expression was not observed in the primordial germ cells (PGCs) of the developing Dr_vasa:eGFP embryos but was mainly detected in isolated or doublets of undifferentiated spermatogonial cells in the testis of pubertal males. The fluorescent cell populations were isolated from adult testes using a differential plating approach followed by a fluorescent assisted cell sorting. The enrichment of the candidate cell types was determined using real time quantitative PCR and a panel of 15 genes representative of different testicular cell types and germ cell stemness. We demonstrate that the transgenic zebrafish lines are valuable models to purify highly enriched populations of Sertoli cells or germ cells expressing high levels of certain but not all germ stem cell markers previously proposed in mammals and in fish.