Essential functions of RNA helicase Vasa in maintaining germline stem cells and piRNA-guided silencing in spermatogenesis.

DEAD-box RNA helicase Vasa is required for gonad development and fertility in multiple animals. Vasa is implicated in many crucial aspects of oogenesis, including translation regulation, primordial germ cell specification, piRNA silencing of transposable elements, and maintenance of germline stem cells (GSCs). However, data about Vasa functions in spermatogenesis remain controversial. Here we showed that loss-of-function mutations led to failures of GSC maintenance in the testes, a severe loss of total germ cell content, and a cessation of male fertility over time. Defects in GSC maintenance in mutant testes were not associated with an increasing frequency of programmed cell death, indicating that a premature loss of GSCs occurred entering differentiation. We found that Vasa is implicated in the positive regulation of expression both in the testes and ovaries. The introduction of a transgene copy of , encoding a nuclear component of piRNA pathway machinery, in mutant background allowed us to restore premeiotic stages of spermatogenesis, including the maintenance of GSCs and the development of spermatogonia and spermatocytes. However, piRNA-guided repression of genes in spermatocytes of mutant testes with additional copy was not restored, and male fertility was disrupted. Our study uncovered a novel mechanistic link involving Vasa and Rhino in a regulatory network that mediates GSC maintenance but is dispensable for the perfect biogenesis of piRNAs in testes. Thus, we have shown that Vasa functions in spermatogenesis are essential at two distinct developmental stages: in GSCs for their maintenance and in spermatocytes for piRNA-mediated silencing of genes.