Cloning and expression of R-Spondin1in different vertebrates suggests a conserved role in ovarian development

Craig A Smith,Kelly N Roeszler,Andrew H Sinclair,Christina M Shoemaker,Joanna Queen,David Crews

doi:10.1186/1471-213x-8-72

Abstract

BackgroundR-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.ResultsGonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5–E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures.ConclusionThese results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.

Highlights

R-Spondin1 (Rspo1) is a novel regulator of the Wnt/β-catenin signalling pathway
The predicted sequence derived from the chicken genome database lacks 14 residues at the amino terminal relative to human RSPO1
RSPO1 has a conserved, female-specific expression profile that implies an important role in vertebrate ovarian development

Summary

Introduction

R-Spondin (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. A number of transcription factors and signalling molecules are known to play a role in testicular differentiation upstream and downstream of SRY. Wnt has been shown to play a role in both testis and ovary formation, since loss of Wnt signalling interferes with proper Sertoli cell development in males [12]. It has been shown that compound mutants lacking both Wnt and the transcription factor Foxl develop as XX males, with patent testes [13]. These observations point to an important additive role for these two genes in ovarian differentiation

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