Direct Male Development in Chromosomally ZZ Zebrafish

The genetics of sex determination varies across taxa, sometimes even within a species. Major domesticated strains of zebrafish ( Danio rerio ), including AB and TU, lack a strong genetic sex determining locus, but strains more recently derived from nature, like Nadia (NA), possess a ZZ male/ZW female chromosomal sex-determination system. AB strain fish pass through a juvenile ovary stage, forming oocytes that survive in fish that become females but die in fish that become males. To understand mechanisms of gonad development in NA zebrafish, we studied histology and single cell transcriptomics in developing ZZ and ZW fish. ZW fish developed oocytes by 22 days post-fertilization (dpf) but ZZ fish directly formed testes, avoiding a juvenile ovary phase. Gonads of some ZW and WW fish, however, developed oocytes that died as the gonad became a testis, mimicking AB fish, suggesting that the gynogenetically derived AB strain is chromosomally WW. Single-cell RNA-seq of 19dpf gonads showed similar cell types in ZZ and ZW fish, including germ cells, precursors of gonadal support cells, steroidogenic cells, interstitial/stromal cells, and immune cells, consistent with a bipotential juvenile gonad. In contrast, scRNA-seq of 30dpf gonads revealed that cells in ZZ gonads had transcriptomes characteristic of testicular Sertoli, Leydig, and germ cells while ZW gonads had granulosa cells, theca cells, and developing oocytes. Hematopoietic and vascular cells were similar in both sex genotypes. These results show that juvenile NA zebrafish initially develop a bipotential gonad; that a factor on the NA W chromosome or fewer than two Z chromosomes is essential to initiate oocyte development; and without the W factor or with two Z doses, NA gonads develop directly into testes without passing through the juvenile ovary stage. Sex determination in AB and TU strains mimics NA ZW and WW zebrafish, suggesting loss of the Z chromosome during domestication. Genetic analysis of the NA strain will facilitate our understanding of the evolution of sex determination mechanisms.