Production of zebrafish offspring from cultured spermatogonial stem cells

Germ-line stem cells have the potential to be a very powerful tool for modifying the genetic information of individual animals. As a first step to use spermatogonial stem cells (SSCs) to enable genetic modification, we here describe effective long-term culture conditions for propagating zebrafish SSCs and for the production of offspring from these cultured SSCs after their differentiation into sperm in transplanted testicular cell aggregates. Dissociated testicular cells were cultured in specific medium with some modified supplements, including several mammalian growth factors. The spermatogonia actively proliferated and retained the expression of exogenous green fluorescent protein under the control of vas and sox17 promoters and also of promyelocytic leukemia zinc finger (Plzf), a marker of undifferentiated spermatogonia, after 1 month in culture. This is a longer period than the entire natural spermatogenic cycle (from SSCs to sperm). The use of subcutaneously grafted aggregates of these cultured spermatogonia and freshly dissociated testicular cells showed that these SSCs could undergo self-renewal and differentiation into sperm. Artificial insemination of these grafted aggregates successfully produced offspring. This culture method will facilitate the identification of new factors for the maintenance of SSCs and enable the future enrichment of genetically modified SSCs that will produce offspring in zebrafish.