Loss of Nobox prevents ovarian differentiation from juvenile ovaries in zebrafish

As a species without master sex-determining genes, zebrafish displays high plasticity in sex differentiation, making it an excellent model for studying the regulatory mechanisms underlying gonadal differentiation and gametogenesis. Despite being a gonochorist, zebrafish is a juvenile hermaphrodite that undergoes a special phase of juvenile ovary before further differentiation into functional testis and ovary. The mechanisms underlying juvenile ovary formation and subsequent gonadal differentiation remain largely unknown. In this study, we explored the role of Nobox/nobox (new born ovary homeobox protein), another oocyte-specific transcription factor in females, in early zebrafish gonadogenesis using CRISPR/Cas9 technology. As in mammals, nobox is specifically expressed in zebrafish gonads with a dimorphic pattern at juvenile stage. In contrast to the mutant of figla (factor in the germline alpha, another oocyte-specific transcription factor), the nobox mutants showed formation of typical perinucleolar (PN) follicles at primary growth (PG) stage in juvenile gonads, suggesting occurrence of follicle assembly from cystic oocytes (chromatin nucleolar stage, CN). These follicles, however, failed to develop further to form functional ovaries, resulting in all-male phenotype. Despite its expression in adult testis, the loss of nobox did not seem to affect testis development, spermatogenesis and male spawning. In summary, our results indicate an important role for Nobox in zebrafish ovarian differentiation and early folliculogenesis. Graphical Abstract Using genome-editing method, we demonstrated that Nobox, an oocyte-specific transcriptional factor, plays an essential role in ovarian formation during zebrafish gonadal differentiation and folliculogenesis in females. Although follicles could be seen to some extent in the absence of Nobox, their formation and further development are both retarded.