Gdnf, a germ cell-derived factor, regulates zebrafish germ cell stemness through the creation of new spermatogonial niches (germ and Sertoli cells) and inhibition of spermatogonial differentiation in an autocrine and paracrine manners

Glial cell line-derived neurotrophic factor (GDNF) and its receptor (GDNF Family Receptor 1 - GFR1) are well known to mediate spermatogonial stem cell (SSC) proliferation and survival in the mammalian testes. In nonmammalian species, Gdnf and Gfr1 orthologs have been found but their functions remain poorly investigated in the testis. Considering this background, this study aimed to understand the roles of Gdnf-Gfr1 signaling pathway in the zebrafish testis by combining in vivo, in silico and ex vivo approaches. Our analysis showed that zebrafish exhibited two paralogs of Gndf (gdnfa and gdnfb) and its receptor, Gfr1 (gfr1a and gfr1b), in agreement with the teleost-specific third round (3R) of whole genome duplication. Expression analysis further revealed that gdnfa and gfr1a were the most expressed copies in the zebrafish adult testes. Subsequently, we demonstrated that gdnfa is expressed in the germ cells, while Gfr1a was detected in early spermatogonia (mainly in types Aund and Adiff) and Sertoli cells. Functional ex vivo analysis showed that Gdnf promoted the creation of new available niches by stimulating proliferation of both type Aund spermatogonia and their surrounding Sertoli cells, but without changing pou5f3 mRNA levels. Strikingly, Gdnf also inhibited late spermatogonial differentiation as shown by the decrease of type B spermatogonia and down-regulation of dazl in the co-treatment with Fsh. Altogether, our data revealed for the first time that a germ cell-derived factor is associated with maintaining germ cell stemness through the creation of new available niches, supporting development of differentiating spermatogonial cysts and inhibiting late spermatogonial differentiation in autocrine and paracrine manners.