Phenotypic characterization and in vitro propagation and transplantation of the Nile tilapia (Oreochromis niloticus) spermatogonial stem cells

In association with in vitro culture and transplantation, isolation of spermatogonial stem cells (SSCs) is an excellent approach for investigating spermatogonial physiology in vertebrates. However, in fish, the lack of SSC molecular markers represents a great limitation to identify/purify these cells, rendering it difficult to apply several valuable biotechnologies in fish-farming. Herein, we describe potential molecular markers, which served to phenotypically characterize, cultivate and transplant Nile tilapia SSCs. Immunolocalization revealed that Gfra1 is expressed exclusively in single type A undifferentiated spermatogonia (Aund, presumptive SSCs). Likewise, the expression of Nanos2 protein was observed in Aund cells. However, Nanos2-positive spermatogonia have also been identified in cysts with two to eight germ cells that encompass type A differentiated spermatogonia (Adiff). Moreover, we also established effective primary culture conditions that allowed the Nile tilapia spermatogonia to expand their population for at least one month while conserving their original undifferentiated (stemness) characteristics. The maintenance of Aund spermatogonial phenotype was demonstrated by the expression of early germ cell specific markers and, more convincingly, by their ability to colonize and develop in the busulfan-treated adult Nile tilapia recipient testes after germ cell transplantation. In addition to advancing our knowledge on the identity and physiology of fish SSCs, these findings provide the first step in establishing a system that will allow fish SSCs expansion in vitro, representing an important progress towards the development of new biotechnologies in aquaculture, including the possibility of producing transgenic fish.