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. 2008 Jul 24:8:72.
doi: 10.1186/1471-213X-8-72.

Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development

Craig A Smith  1 , Christina M Shoemaker, Kelly N Roeszler, Joanna Queen, David Crews, Andrew H Sinclair
Affiliations

Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development

Craig A Smith et al. BMC Dev Biol. .

Abstract

Background: R-Spondin1 (Rspo1) is a novel regulator of the Wnt/beta-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.

Results: Gonadal 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.

Conclusion: These 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.

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Figures

Figure 1
Figure 1
A. Alignment of predicted R-SPONDIN1 proteins in human, mouse, chicken and turtle (T. scripta). B. Percentage amino acid similarities (identities) for RSPO1 orthologues.
Figure 2
Figure 2
Expression of mouse Rspo1 mRNA in FACS-sorted gonadal somatic and germ cells, over E12.5 – 15.5. Mean +/- SEM. ■ = female; □ = male.
Figure 3
Figure 3
A) Expression of RSPO1 mRNA in embryonic chicken gonads over development (Embryonic days (E) (stages), assayed by qRT-PCR. Values represent mean +/- SEM. ● = female; ○ = male. B) Effect of fadrozole aromatase inhibitor on gonadal RSPO1 expression in the chicken embryo. Embryos were treated at E3.5 with 0.1 ml PBS control or 0.1 ml (1 mg) fadrozole in PBS, and examined at E8.5 and E12.5. Quantitative RT-PCR. Mean +/- SEM. ■ = female; □ = male.
Figure 4
Figure 4
Gonadal expression of chicken WNT4, assessed by in situ hybridization. A) cWNT4 is expressed in the gonads of both sexes at E4.5 (prior to sexual differentiation) (arrows). Expression is similar between the sexes at E6.5 (near the beginning of sexual differentiation), but is down-regulated in males (ZZ) by E8.5. B) Transverse sections of over-stained E8.5 whole mounts showing cWNT4 expression in the outer region of the female gonad. Weak staining in the male gonad represents background.
Figure 5
Figure 5
Expression of RSPO1 mRNA in the gonads of turtle embryos (Trachemys scripta) during the temperature-sensitive period. Expression levels were examined by quantitative real-time PCR in three samples per sex/stage. Mean +/- SEM. ● = female; ○ = male. A) Time course of T. scripta Rspo1 expression, showing higher expression in developing ovaries from stage 17 (p = 0.0318). B) Turtle Rspo1 expression following temperature shifts. Expression data at MPT and FPT at three early stages during the temperature-sensitive period is expanded and compared to the response of gene expression to a shift in temperature. Embryos were shifted at stage 16 from either MPT to FPT (MPT → FPT) or FPT to MPT (FPT → MPT) and gonads dissected for analysis at two subsequent timepoints, stage 16.5 and stage 17. Following a FPT → MPT shift, expression is significantly decreased within one stage from female-typical levels (p = 0.0253) and is not different from male-typical levels (p = 1.0000). Following a MPT → FPT shift, expression has not yet changed from MPT-typical levels. Asterisks indicate statistically significant difference in expression between sex within stage at the p = 0.05 significance level, after correction for multiple pair-wise comparison tests by Tukey's HSD.
Figure 6
Figure 6
Immunofluorescent localization of Rspo1 protein in E14.5 embryonic mouse gonads. A) Rspo1 immunofluorescence in a female gonad. Expression is detectable throughout the gonad, in cell cytoplasm and at the cell surface (e.g.; arrow). Transverse section. B) Background staining in male gonad. Transverse section. C) Double staining for Rspo1 (green) and Wt1 (red) in a female gonad. Most cells that are Rspo1+ and also Wt1+ (e.g., arrow). Some cells stain for Rspo1 but not Wt1 (e.g;. arrowhead). Transverse section. D) Wt1 (red) but not Rspo1 expression in the male gonad (arrows show Wt1 protein in the seminiferous cords). E) Double-staining for Rspo1 (green) and the meiotic germ cell marker, Scp3 (red), showing Rspo1 protein at the germ cell surface (arrow), and also in Scp3- somatic cells (arrowhead). Transverse section. F) Double-staining for Rspo1 (green) and Scp3 (red) shows no expression in the male.
Figure 7
Figure 7
Immunofluorescent localization of RSPO1 protein in E8.5 and E12.5 embryonic chicken gonads (ten micron transverse sections). A) E8.5 left ovary. Double-staining for aromatase (red; medulla) and RSPO1 (green; cortex). B) E12. 5 left ovary. Double-staining for aromatase (red; medulla) and RSPO1 (green; cortex). C) E8.5 right female gonad, showing aromatase expression in the medulla, but no RSPO1 expression due to the reduced cortex. D) E8.5 testis, showing no expression of aromatase or RSPO1. E) High magnification view of the left ovarian cortex, showing RSPO1 expression (green) in the extracellular matrix and at the cell surface (e.g., arrows) and in cell cytoplasm (e.g., arrowhead). F) Loss of RSPO1 protein (green) in an E12.5 female left gonad treated with aromatase inhibitor at E3.5. Aromatase protein is weakly expressed (red), due to fadrozole inhibition, and the cortex is absent.
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