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Link to original content: https://pubmed.ncbi.nlm.nih.gov/21297984
Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling - PubMed Skip to main page content
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. 2011 Jan 28;6(1):e16366.
doi: 10.1371/journal.pone.0016366.

Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling

Sara Tomaselli  1 , Francesca Megiorni, Lin Lin, Maria Cristina Mazzilli, Dianne Gerrelli, Silvia Majore, Paola Grammatico, John C Achermann
Affiliations

Human RSPO1/R-spondin1 is expressed during early ovary development and augments β-catenin signaling

Sara Tomaselli et al. PLoS One. .

Abstract

Human testis development starts from around 42 days post conception with a transient wave of SRY expression followed by up-regulation of testis specific genes and a distinct set of morphological, paracrine and endocrine events. Although anatomical changes in the ovary are less marked, a distinct sub-set of ovary specific genes are also expressed during this time. The furin-domain containing peptide R-spondin1 (RSPO1) has recently emerged as an important regulator of ovary development through up-regulation of the WNT/β-catenin pathway to oppose testis formation. Here, we show that RSPO1 is upregulated in the ovary but not in the testis during critical early stages of gonad development in humans (between 6-9 weeks post conception), whereas the expression of the related genes WNT4 and CTNNB1 (encoding β catenin) is not significantly different between these tissues. Furthermore, reduced R-spondin1 function in the ovotestis of an individual (46,XX) with a RSPO1 mutation leads to reduced β-catenin protein and WNT4 mRNA levels, consistent with down regulation of ovarian pathways. Transfection of wild-type RSPO1 cDNA resulted in weak dose-dependent activation of a β-catenin responsive TOPFLASH reporter (1.8 fold maximum), whereas co-transfection of CTNNB1 (encoding β-catenin) with RSPO1 resulted in dose-dependent synergistic augmentation of this reporter (approximately 10 fold). Furthermore, R-spondin1 showed strong nuclear localization in several different cell lines. Taken together, these data show that R-spondin1 is upregulated during critical stages of early human ovary development and may function as a tissue-specific amplifier of β-catenin signaling to oppose testis determination.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. RSPO1 expression increases at key stages of early human ovary development.
(A) Analysis of CTNNB1, WNT4, and RSPO1 in human fetal gonads between 6–9 weeks post-conception. Testis and ovary samples showed higher expression of CTNNB1 (ANOVA, p<0.001), WNT4 (ANOVA, p<0.01) and RSPO1 (ANOVA, p<0.001) compared to control. Significantly higher expression of RSPO1 was detected in the ovary compared to testis (**p<0.01). (B) Analysis of RSPO1 expression levels in the testis and ovary during this period of development. A significant increase in RSPO1 was found in the ovary across this time course (ANOVA, P<0.0001; 8w >6–7w and 9w >6–7w, both ***p<0.001) (control, 8 wpc heart).
Figure 2
Figure 2. Expression analysis of factors crucial in gonadal development following disruption of RSPO1.
(A) CTNNB1 mRNA levels, determined by RT-PCR and compared to GAPDH mRNA levels, seem to have no difference between normal ovary (Ov) and patient's ovotestis (OT). (B) β-catenin protein expression appeared decreased in ovotestis tissue compared to control ovary and HEK293T cell line, in an immunoblot assay (as shown in Tomaselli et al, 2008 [21]). The weak residual band may represent extracellular β-catenin, which is implicated in cell-cell adhesion. (C) WNT4 and SOX9 mRNA levels were tested by RT-PCR and compared to GAPDH transcript levels. WNT4 was dramatically reduced in the patient's ovotestis (OT) in comparison to control ovary (Ov). SOX9 signal was detected in control testis (T), but did not appear upregulated in the ovotestis (OT) sample compared to the normal ovary (Ov).
Figure 3
Figure 3. R-spondin1 weakly activates a β-catenin-responsive promoter.
(A) The effect of WT RSPO1 on TCF-dependent transcriptional activation (left panel) was compared with that of a naturally-occurring RSPO1 mutant (MT) vector generating a protein lacking the first furin domain (right panel), using embryonic kidney tsa201 cells and the TOPFLASH reporter construct. Transient gene expression assays of increasing concentrations of plasmids encoding wild-type (WT) or mutant (MT) RSPO1 (0–100 ng/well) showed a mild activation (maximum 1.8-fold) of the TOPFLASH reporter with WT RSPO1 vector (ANOVA, p<0.001) and a complete lack of activity for the mutant. (B) Dose-dependent activation of the TOPFLASH reporter with increasing doses of beta-catenin (0–10 ng/well) (ANOVA, p<0.001). Luciferase data are reported as a mean ± SEM of at least three triplicate experiments, standardized for Renilla co-expression (compared to basal value, **p<0.01; ***p<0.001).
Figure 4
Figure 4. R-spondin1 augments β-catenin signaling.
(A) Co-transfection of increasing doses of wild-type (WT) RSPO1 (0–100 ng/well) with β-catenin (0–5 ng/well) showed dose-dependent augmentation of β-catenin signaling (left panel) (ANOVA: CTNNB1 0 ng, p<0.05; CTNNB1 2 ng, p<0.01; CTNNB1 5 ng, p = 0.07). A statistically significant synergistic effect was seen when doses of 10 ng RSPO1 and 50 ng RSPO1 were transfected on the background of increasing doses of CTNNB1 (ANOVA: RSPO1 10 ng, p<0.05; RSPO1 50 ng, p<0.05). No activity was seen after co-transfection of mutant (MT) RSPO1 (0–100 ng/well) (right panel). (B) Relative luciferase activity after stimulation of β-catenin transfected cells with Rspo1 peptide (0–3000 ng/ml) (ANOVA: CTNNB1 -, p<0.01; CTNNB1 +, p<0.001). Luciferase data are reported as a mean ± SEM of at least three triplicate experiments, standardized for Renilla co-expression (*p<0.05; **p<0.01; ***p<0.001 compared to basal value without RSPO1 vector or peptide for that study).
Figure 5
Figure 5. Effect of DKK1 treatment on R-spondin1 augmentation of β-catenin signaling.
(A) RSPO1/β-catenin co-transfected cells were treated 2 hours before transfection with different doses of DKK1 (0–400 ng/ml). After 24 h, cells were lysed and assayed for luciferase activity. No significant reduction in stimulation was seen (ANOVA: RSPO1 50 ng, p = 0.15). (B) Cells were treated with DKK1 (0–1000 ng/ml) and stimulated with Rspo1 peptide (0–2000 ng/ml). Luciferase activity was measured 24 h later. Luciferase data are reported as a mean ± SEM of at least three triplicate experiments, standardized for Renilla co-expression. (N.S., not significant; *p<0.05).
Figure 6
Figure 6. R-spondin1 can show nuclear and nucleolar localization.
(A) Immunofluorescent microscopy was used to detect the cellular distribution of a WT pRSPO1-GFP vector in H295R cells (left panel). DAPI stained nuclei and merged images are shown (center and right panels, respectively) (magnification 20X). (B) A similar pattern of cellular distribution was obtained in a CHO cell line (left panel). Fluorescent labeling performed with an antibody against C23-nucleolar protein (red) revealed that the GFP-tagged WT RSPO1 protein shows strong nucleolar localization in some of these cells (center and right panels) (magnification 20X). (C) Immunofluorescent analysis of MT pRSPO1-GFP showed similar cellular distribution. (D) The strong nuclear localization was confirmed following Western blot analysis of nuclear and cytosolic extracts prepared from HEK293T cells that had been transfected with either WT or MT pRSPO1-HA constructs.
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