Direct male development in chromosomally ZZ zebrafish

Abstract

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 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.

A maternal-to-zygotic-transition gene block on the zebrafish sex chromosome

“…2014 ), likely because it was derived by several generations of gynogenesis ( Walker-Durchanek 1980 ); thus, AB is likely chromosomally WW, a genotype that usually becomes females but can also develop into neomales ( Wilson et al . 2014 , 2024 ; Valdivieso et al . 2022 ).…”

“…In the NA strain, all ZZ fish develop directly into males and ZW fish usually become females but some ZW fish develop as neomales, showing that the W is necessary, but not sufficient, for female development ( Wilson et al . 2014 , 2024 ; Valdivieso et al . 2022 ).…”

“…We doubt that eggshell proteins are primary sex-determining factors because these genes are not expressed in ZW gonads until oocytes develop to Selman Stage IB ( Selman et al . 1993 ) and gonads in ZZ individuals develop directly into testes without even forming Stage IA oocytes ( Wilson et al . 2024 ).…”

“…Region-4 near the right telomere of Chr4 contains 3 groups of genetic elements important for egg development: (1) a factor that is necessary but not sufficient for the bipotential gonad to develop into an ovary in natural zebrafish strains ( Wilson et al . 2014 , 2024 ); (2) mucin genes encoding egg chorion proteins ( Fig. 1 ); and (3) several 45S ribosomal RNA genes that are expressed specifically in oocytes but not in somatic cells, which express a different set of 45S rRNA genes ( Locati, Pagano, Girard et al .…”

“…Here, we describe the discovery of strong testis-biased gene expression in hundreds of protein-coding genes in a heterochromatic region of the long arm of the homomorphic sex chromosome Chr4 near the sex-determining locus in a zebrafish strain that retains the wild ZW female/ZZ male sex-determining system ( Wilson et al . 2014 ) and in which ZZ fish develop directly into males, avoiding the juvenile ovary stage observed in laboratory strains ( Wilson et al . 2024 ).…”