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. 2012;8(12):e1003170.
doi: 10.1371/journal.pgen.1003170. Epub 2012 Dec 27.

Testicular differentiation occurs in absence of R-spondin1 and Sox9 in mouse sex reversals

Rowena Lavery  1 , Anne-Amandine Chassot, Eva Pauper, Elodie P Gregoire, Muriel Klopfenstein, Dirk G de Rooij, Manuel Mark, Andreas Schedl, Norbert B Ghyselinck, Marie-Christine Chaboissier
Affiliations

Testicular differentiation occurs in absence of R-spondin1 and Sox9 in mouse sex reversals

Rowena Lavery et al. PLoS Genet. 2012.

Abstract

In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1(KO)Sox9(cKO) ()), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1(KO)Sox9(cKO) mice. Moreover, since testis development occurs in XY Rspo1(KO)Sox9(cKO) mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9(cKO) mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Testicular differentiation in XY and XX Rspo1−/−; Sox9flox/flox; Sf1;creTg/+ (Rspo1KOSox9cKO) mice.
Macroscopic views of gonads of 2 month-old mice show hypoplasic testis and ovotestis development in XY (c) and XX (d) Rspo1KOSox9cKO mice, respectively. Seminiferous tubules are revealed by PAS histological analysis of XY (h) and XX (i) Rspo1KOSox9cKO gonadal sections. They are less abundant than in XY controls (f). XY Sox9cKO gonads (g) develop as ovaries (j). (T: testicular region, O: ovarian region, scale bar: 200 µm). Immunofluorescence of SOX9 (k–o) or DMRT1 (p–t) (a Sertoli cell marker, in red), FOXL2 (k–t) (a follicular cell marker, in green) and DAPI (a nuclear marker in blue) (scale bar, 50 µm). Deletion of Sox9 with Sf1:cre (Sox9cKO) eliminates SOX9 expression in Sertoli cells (l, m, n), and promotes male-to-female sex reversal in XY Sox9cKO gonads as highlighted by robust FOXL2 expression (l, q). However, Sox9 deletion no longer allows ovarian cells differentiation when Rspo1 is deleted in the XY (m, r) and XX (n, s) Rspo1KOSox9cKO mice. This is evidenced by the robust expression of DMRT1 in 3 week-old XY (s) and XX (r) mutant gonads and XY controls (p), and the low or absent expression of FOXL2 in these gonads (k, m, n, p, r, s). XY (a, f, k, p) and XX (e, j, o, t) Rspo1+/−; Sox9flox/flox controls, XY Sox9cKO gonads (b, g, l, q), XY (c, h, m, r) and XX (d, i, n, s) Sox9cKO Rspo1KO respectively. XX Rspo1KO and XX Sox9cKO Rspo1KO gonads appeared similar (see Figure S2B).
Figure 2
Figure 2. Non-differentiated XY and XX Rspo1KOSox9cKO gonads at 13.5 dpc.
Immunofluorescence of SOX9 (Sertoli cell marker, in red) and AMH (Sertoli cell marker green) (a–d), AMH (Sertoli cell marker, in green) and SRY (pre-Sertoli and Sertoli cell marker in red) (e–h) and SF1 (undifferentiated supporting cell, Sertoli and Leydig cell marker) (i–l). Counterstain is DAPI (in blue). Lack of SOX9 and AMH expression in XY (b) and XX (c) Rspo1KOSox9cKO gonads shows that Sertoli cell differentiation did not occur at 13.5 dpc. Note that the kidneys (K) are positive for SOX9. This is accompagnied with the maintenance of SRY expression in the XY Rspo1KOSox9cKO gonads (f) whereas SRY expression has ceased in XY controls (e). SF1 expression is maintained in absence of Sertoli cells differentiation in XY and XX Rspo1KOSox9cKO gonads (j and k respectively) (scale bar: 100 µm). Note that SF1 is also expressed in steroidogenic cells of the adrenals (A). XY (a, e, i) and XX (d, h, l) Rspo1+/−; Sox9flox/flox controls, XY (b, f, j) and XX (c, g, k) Sox9cKO Rspo1KO respectively.
Figure 3
Figure 3. Post-natal development of sex cords in XY and XX Rspo1KOSox9cKO mice.
Immunofluorescence of SDMG1 (in red). Counterstain is DAPI (in blue). SDMG1 is expressed in Sertoli cells (XY controls a, f, k, p) and in follicular cells of growing ovaries as evidenced at P12 onwards (j, o, t). Sertoli cells are present and formed sex cords in both XY and XX Rspo1KOSox9cKO gonads, with more developing sex cords in XY Rspo1KOSox9cKO testis (c, h, m, r) in comparison to XX Rspo1KOSox9cKO ovotestis (d, i, n, s). At P12, the sex cords are fully developed in both XY (h) and XX (i) Rspo1KOSox9cKO mice. In XY Sox9cKO (b, g, l, q) and XX control (e, j, o, t) gonads, ovarian follicles express SDMG1 at P12, P21 and P60. At these stages, SDMG1 is also expressed in the follicles of the XX double mutant ovotestes (see n) and in XY double mutant follicles when they develop (scale bars: 100 µm). XY (a, f, k, p) and XX (e, j, o, t) Rspo1+/−; Sox9flox/flox controls, XY Sox9cKO gonads (b, g, l, q), XY (c, h, m, r) and XX (d, i, n, s) Rspo1KO Sox9cKO gonads respectively.
Figure 4
Figure 4. Sertoli cells support germ cell differentiation in XY Rspo1KOSox9cKO gonads.
Immunofluorescence (a–i) of GATA1 (Sertoli cell marker, in green), AR (Androgen Receptor) (Sertoli, peritubular myoid and Leydig cell marker, in red), STRA8 (a premeiotic marker, in red), and γH2AX (a meiotic marker, in green) at P10. Counterstain is DAPI (in blue). In situ hybridization (j–l) using a probe for Clu transcripts, another marker for mature Sertoli cells, illustrated as computer–generated bright field superimpositions of the blue counterstain (DAPI) with the hybridization signal (red false color). GATA1, AR and Clu expression show that the Sertoli cells mature in XY controls (a, g, j) and XY Rspo1KOSox9cKO testes (c, i, l), and are able to support germ cell differentiation until meiosis initiation as revealed by STRA8 (a, c, d, f) and γH2AX (d, f, g, i) expression. Note that both Sertoli, peritubular myoid and Leydig cells of XY Sox9cKO mutant gonads normally expressed AR (h). (scale bars: 50 µm). XY (a, d, g, j) Rspo1+/−; Sox9flox/flox controls, XY Sox9cKO gonads (b, e, h, k) and XY Rspo1KOSox9cKO (c, f, i, l) gonads.
Figure 5
Figure 5. AMH and SOX genes are expressed in XY and XX Rspo1KO Sox9cKO gonads.
A- AMH expression in absence of SOX9. Immunofluorescence of SOX9 (in red) and AMH (in green). Counterstain is DAPI (in blue). SOX9 and AMH are synthetised in Sertoli cells of the testis (a, f). SOX9 is expressed in theca cells (white star in e) and AMH in follicular cells of the ovary at P12 (e, j). Deletion of Sox9 with Sf1:cre eliminates SOX9 expression in Sf1:cre positive cells of the gonads, which are Sertoli cells in XY (c) and XX (d) Rspo1KOSox9cKO gonads and theca cells of the ovarian region of XX Rspo1KOSox9cKO gonads (d) and XY Sox9cKO gonads (b). AMH expression is observed in Sertoli cells of the XY (c, h) and XX (d, i) Rspo1KOSox9cKO gonads even the absence of SOX9. (scale bar: 50 µm). Immunofluorescence of FOXL2 (in red) and AMH (in green). Counterstain is DAPI (in blue). Most of the AMH positive cells in the testicular cords of Rspo1KOSox9cKO gonads (h, i) are negative for FOXL2 indicating that they are not granulosa cells, some AMH/FOXL2 positive cells were observed outside of these cords indicating that they are granulosa cells (h, i). (scale bar: 100 µm). XY (a, f) and XX (e, j) Rspo1+/−;Sox9flox/flox controls, XY Sox9cKO gonads (b, g), XY (c, h) and XX (d, i) Rspo1KO Sox9cKO gonads respectively. B- Sox8 is expressed in XY and XX Rspo1KO Sox9cKO gonads. In situ hybridization of Sox8 transcripts. Sox8 is expressed in Sertoli cells at P5 in XY control (a), XY (c) and XX (d) Rspo1KOSox9cKO gonads, but not in XY Sox9cKO ovaries (b). (a) XY Rspo1+/−; Sox9flox/flox controls, (b) XY Sox9cKO gonads, XY (c) and XX (d) Rspo1KO Sox9cKO gonads respectively. C- Sox10 is expressed in XY and XX Rspo1KO Sox9cKO gonads. QPCR analysis shows that Sox10 is significantly up-regulated both in XY and XX Rspo1KOSox9cKO gonads, when compared to XY controls. The differences between XY controls and XY Sox9cKO are not significant.
Figure 6
Figure 6. Opposing function of SOX and RSPO1 signaling in the fate of the gonad.
A- In XX gonads, RSPO1 activates WNT/beta-catenin signaling to promote ovarian differentiation. Ablation of Rspo1 results in partial sex reversal with ovotestis development, which coincides with Sox9 expression. However additional deletion of Sox9 in the XX Rspo1 KO (i.e., Rspo1KOSox9cKO) still allows ovotestis formation, implying that Sry and Sox9 are not required for testicular differentiation in female-to-male sex reversal. B- In XY gonads, whereas Sox9 deletion triggers ovarian development, additional deletion of Rspo1 in XY Rspo1KOSox9cKO gonads restores testis development. This is associated with the expression of other SOX genes like SOX 8 and SOX10, other masculinising factors.
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This work was supported by Agence Nationale pour la Recherche (ANR-10-BLAN-1239-molmechmeiosis, ANR-09-GENM-009-03 GENIDOV) and Association pour la Recherche sur le Cancer (SFI20101201408). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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