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.

BACKGROUND: The absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored.
RESULTS: By differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells.
CONCLUSIONS: Our findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

Zebrafish are routinely used to model reproductive development, function, and disease, yet we still lack a clear understanding of the fundamental steps that occur during early bipotential gonad development, including when endothelial cells, pericytes, and macrophage arrive at the bipotential gonad to support gonad growth and differentiation. Here, we use a combination of transgenic reporters and single-cell sequencing analyses to define the arrival of different critical cell types to the larval zebrafish gonad. We determined that blood initially reaches the gonad via a vessel formed from the swim bladder artery, which we have termed the gonadal artery. We find that vascular and lymphatic development occurs concurrently in the bipotential zebrafish gonad and our data suggest that similar to what has been observed in developing zebrafish embryos, lymphatic endothelial cells in the gonad may be derived from vascular endothelial cells. We mined preexisting sequencing datasets to determine whether ovarian pericytes had unique gene expression signatures. We identified 215 genes that were uniquely expressed in ovarian pericytes, but not expressed in larval pericytes. Similar to what has been shown in the mouse ovary, our data suggest that pdgfrb+ pericytes may support the migration of endothelial tip cells during ovarian angiogenesis. Using a macrophage-driven photoconvertible protein, we found that macrophage established a nascent resident population as early as 12 dpf and can be observed removing cellular material during gonadal differentiation. This foundational information demonstrates that the early bipotential gonad contains complex cellular interactions, which likely shape the health and function of the mature gonad.

The fish reproductive system is a complex biological system. Nonetheless, reproductive organ development is conserved, which starts with sex determination and then sex differentiation. The sex of a teleost is determined and differentiated from bipotential primordium by genetics, environmental factors, or both. These two processes are species-specific. There are several prominent genes and environmental factors involved during sex determination and differentiation. At the cellular level, most of the sex-determining genes suppress the female pathway. For environmental factors, there are temperature, density, hypoxia, pH, and social interaction. Once the sexual fate is determined, sex differentiation takes over the gonadal developmental process. Environmental factors involve activation and suppression of various male and female pathways depending on the sexual fate. Alongside these factors, the role of the brain during sex determination and differentiation remains elusive. Nonetheless, GnRH III knockout has promoted a male sex-biased population, which shows brain involvement during sex determination. During sex differentiation, LH and FSH might not affect the gonadal differentiation, but are required for regulating sex differentiation. This review discusses the role of prominent genes, environmental factors, and the brain in sex determination and differentiation across a few teleost species.

In the early fetal stage, the gonads are bipotent and only later become the ovary or testis, depending on the genetic sex. Despite many studies examining how sex determination occurs from biopotential gonads, the spatial and temporal organization of bipotential gonads and their progenitors is poorly understood. Here, using lineage tracing in mice, we find that the gonads originate from a T+ primitive streak through WT1+ posterior intermediate mesoderm and appear to share origins anteriorly with the adrenal glands and posteriorly with the metanephric mesenchyme. Comparative single-cell transcriptomic analyses in mouse and cynomolgus monkey embryos reveal the convergence of the lineage trajectory and genetic programs accompanying the specification of biopotential gonadal progenitor cells. This process involves sustained expression of epithelial genes and upregulation of mesenchymal genes, thereby conferring an epithelial-mesenchymal hybrid state. Our study provides key resources for understanding early gonadogenesis in mice and primates.

Disorders of sexual development (DSD) are conditions with an atypical chromosomal, gonadal or phenotypic sex, which leads to differences in the development of the urogenital tract and different clinical phenotypes. Some genes have been implicated in the sex development during gonadal and functional differentiation where the maintenance of the somatic sex of the gonad as either male or female is achieved by suppression of the alternate route. The diagnosis of DSD requires a structured approach, involving a multidisciplinary team and different molecular techniques. We discuss the dimorphic genes and the specific pathways involved in gonadal differentiation, as well as new techniques for genetic analysis and their diagnostic value including epigenetic mechanisms, expanding the evidence in the diagnostic approach of individuals with DSD to increase knowledge of the etiology.

Sex-determining factors are usually assumed to be either genetic or environmental. The present paper aims at drawing attention to the potential contribution of developmental noise, an important but often-neglected component of phenotypic variance. Mutual inhibitions between male and female pathways make sex a bistable equilibrium, such that random fluctuations in the expression of genes at the top of the cascade are sufficient to drive individual development toward one or the other stable state. Evolutionary modeling shows that stochastic sex determinants should resist elimination by genetic or environmental sex determinants under ecologically meaningful settings. On the empirical side, many sex-determination systems traditionally considered as environmental or polygenic actually provide evidence for large components of stochasticity. In reviewing the field, I argue that sex-determination systems should be considered within a three-ends continuum, rather than the classical two-ends continuum.