Background: R-Spondin1 (Rspo1) is a novel regulator of the Wnt/β-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway.

Sex determination in vertebrates, the process of forming an ovary or testis from a bipotential gonad, can be initiated by genetic or environmental factors. Elements of the downstream molecular pathways underlying these different sex-determining mechanisms have been evolutionarily conserved. We find the first evidence that Sox9 expression is preferentially organized in the testis early in the temperature-sensitive period in a species with temperature-dependent sex determination (Trachemys scripta). This pattern occurs before sexually dimorphic Mis expression and in a temporal hierarchy that is similar to mammals. Furthermore, we extend previous findings that Dmrt1 expression at early stages of sex determination has a dimorphic pattern consistent with a possible upstream role in determining the fate of the bipotential gonad.

Gonadogenesis, the process of forming an ovary or a testis from a bipotential gonad, is critical to the development of sexually reproducing adults. Although the molecular pathway underlying vertebrate gonadogenesis is well characterized in organisms exhibiting genotypic sex determination, it is less well understood in vertebrates whose sex is determined by environmental factors. We examine the response of six candidate sex-determining genes to sex-reversing temperature shifts in a species with temperature-dependent sex determination (TSD). For the first time, we report the regulation of FoxL2, Wnt4, Dmrt1, and Mis by temperature, confirming their involvement in the molecular pathway underlying TSD and placing them downstream of the action of temperature. We find evidence that FoxL2 plays an ovarian-specific role in development, whereas Wnt4 appears to be involved in both testis and ovary formation. Dmrt1 expression shows rapid activation in response to a shift to male-producing temperature, whereas Mis up-regulation is delayed. Furthermore, early repression of Mis appears critical to ovarian development. We also investigate Dax1 and Sox9 and reveal that at the level of gene expression, response to temperature is comparatively later in gonadogenesis. By examining the role of these genes in TSD, we can begin to elucidate elements of conservation and divergence between sex-determining mechanisms.

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