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

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.

Introduction

Mammalian sex determination depends on the primary developmental decision of the gonad to differentiate as testis or ovary. The gonad develops as a bipotential organ with the capacity to respond to two different genetic stimuli: the activation of the SRY/SOX9 pathway that induces testicular development, or the expression of the R-spondin1 (RSPO1)/beta-catenin pathway that regulates ovarian differentiation [1]. Indeed in humans and mice, male sex determination is initiated by the expression of the Y-linked gene SRY [2], [3], [4]. Sry expression in turn activates the transcriptional regulator SOX9 [5]. Subsequently, SOX9 initiates Sertoli cell differentiation, the supporting cell of the testicular sex cords [6], [7]. Signaling pathways initiated in these cells contribute to the organization of the XY gonads [8], as well as to the differentiation of other testicular cell lineages such as the Leydig steroidogenic cells [9], [10] and the pro-spermatogonia [11], [12], ultimately leading to testis formation and, in turn, male development. In 46,XY patients, loss-of-function mutations in SRY and SOX9 promote male-to-female sex reversal [13], [14], whereas translocations of the SRY locus to another chromosome can yield 46,XX patients with female-to-male sex reversal [3]. Loss-of-function mutations [6], [7], [15], [16] and gain-of-function mutations [4], [17], [18] of Sry and Sox9 have been generated in mouse models, showing that Sry and Sox9 are necessary and sufficient to induce testis differentiation and the associated male development. As a consequence, these genes have been considered as the master inducers of testis differentiation and male development.

In the absence of SRY (XX individuals), up-regulation of RSPO1, an activator of the WNT/beta-catenin signaling pathway, promotes ovarian differentiation. Mutations in RSPO1 are responsible for skin disorders and female-to-male sex reversal in 46,XX patients [19]. Similarly, ablation of Rspo1 in mice yields female-to-male sex reversal and promotes Sox9 up-regulation correlated with differentiation of Sertoli cells and formation of testis cords at birth [20]. This gonadal dysgenesis yields development of an ovotestis, a gonad displaying both testicular and ovarian regions [20], [21]. Rspo1 expression in turn activates expression of Wnt4 [21], another activator of the WNT/beta-catenin signaling pathway involved in ovarian differentiation [22], [23]. When the canonical beta-catenin signaling pathway is activated in XY gonads, this induces male-to-female sex reversal indicating that this pathway acts on top of ovarian differentiation [23]. Indeed, activation of WNT/beta-catenin is required for expression of Foxl2 [24], a transcription factor involved in folliculogenesis [25], [26] and homeostasis of the ovary [27]. Thus Rspo1 appears to be the gene instructing the molecular network leading to ovarian development.

Since ablation of Rspo1 promotes SOX9 expression concomitantly with Sertoli cell differentiation [20], it was assumed that Sox9 is the sex reversal inducer in XX Rspo1 mutants. We now show that i) testicular differentiation occurs in XX Rspo1 mutants indicating that neither Sry nor Sox9 are required for female-to-male sex reversals; ii) testicular differentiation also occurs in XY Rspo1 mutants indicating that Rspo1 is required for male-to-female sex reversal in XY Sox9 mutants.

Results/Discussion

Rspo1 is required for ovarian development in XY Sox9 mice

Sox9 is required for Sertoli cell differentiation, testis formation and male development. Indeed, deletion of Sox9 in XY Sox9, (referred to as XY Sox9) triggers male-to-female sex reversal [16]. However the factor(s) inducing sex reversal in XY Sox9 remained to be identified. Given (i) the prominent role of RSPO1, an activator of beta-catenin signaling, in female sex determination [19], and (ii) the fact that ectopic activation of beta-catenin in XY gonads can induce male-to-female sex reversal [23], we hypothesized that Rspo1 expression induced male-to-female sex-reversal in XY Sox9 gonads. According to this scenario, neither testicular (which is Sox9-dependent) nor ovarian (which is Rspo1/beta-catenin-dependent) differentiation should occur in XY Sox9 gonads additionally lacking Rspo1. To test this hypothesis, we have generated and analyzed double loss-of-function mice (i.e. XY Rspo1, referred to as XY Rspo1).

Since previous results have shown that sex reversal can appear quite late during fetal development [20], [22], we first analyzed adult stages when the sexual development is likely to be completed. At P60 (postnatal day 60), the anogenital distance in XY Rspo1 mice was equivalent to that of XX controls but the internal genitalia contained both male and female organs including oviducts, uterine horns and vaginal tissues, as well as epididymides, vasa deferensia, seminal vesicles and prostate (Figure S1). The XY Sox9 developed as ovaries (Figure 1b, 1g, 1l, 1q and Figure S1), as expected from a previous report [16]. Interestingly, XY Rspo1 gonads developed as hypoplastic testes containing well-defined seminiferous tubules as evidenced by histological analysis (Figure 1c, 1h and Figure S1). We next examined whether the supporting cells forming the seminiferous tubules differentiated as granulosa cells, the ovarian supporting cells expressing FOXL2 [25], [28] or as Sertoli cells expressing DMRT1 [29]. In P21 gonads, immunostaining experiments showed that the supporting cells forming the seminiferous tubules in XY Rspo1 gonads were DMRT1-expressing Sertoli cells (Figure 1r), even though SOX9 was clearly missing (Figure 1m). However, a few FOXL2-positive granulosa cells were found within the alignment of the Sertoli cells forming the seminiferous tubules (Figure 1m, r) and in a few XY Rspo1 mice (3 out of 18), rare and abnormal follicles were observed (Figure S2A). The mixed genetic background of Rspo1 mice is a likely factor causing the variation of this phenotype.

Figure 1

Testicular differentiation in XY and XX Rspo1 (Rspo1) mice.

Macroscopic views of gonads of 2 month-old mice show hypoplasic testis and ovotestis development in XY (c) and XX (d) Rspo1 mice, respectively. Seminiferous tubules are revealed by PAS histological analysis of XY (h) and XX (i) Rspo1 gonadal sections. They are less abundant than in XY controls (f). XY Sox9 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 (Sox9) eliminates SOX9 expression in Sertoli cells (l, m, n), and promotes male-to-female sex reversal in XY Sox9 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) Rspo1 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 controls, XY Sox9 gonads (b, g, l, q), XY (c, h, m, r) and XX (d, i, n, s) Sox9 respectively. XX Rspo1 and XX Sox9 gonads appeared similar (see Figure S2B).

Altogether this shows that a genetic pathway activated by RSPO1 is required for the male-to-female sex-reversal of XY Sox9 and indicates that Sertoli cell differentiation and seminiferous tubules formation can occur in the absence of SOX9.

Sertoli cell differentiation occurs without Sry and Sox9 in XX Rspo1 gonads

Our study also allowed us to evaluate the effect of Sox9 removal in a female-to-male sex reversal context (i.e. in XX Rspo1). Given that homozygous mutations of Rspo1 promote Sertoli cell differentiation around birth, a process that is associated with Sox9 up-regulation in these cells [20], we hypothesized that Sox9 is the inducing factor of testicular differentiation in XX Rspo1 mice. If Sox9 is indeed the main switch for female-to-male sex reversal in XX individuals, one expects an impaired differentiation of Sertoli cell and seminiferous tubules in the absence of both Rspo1 and Sox9 in XX Rspo1 gonads. Unexpectedly, at P60, these XX double mutants displayed hermaphroditism of the reproductive tracts (Figure S1). Histological analysis revealed that XX Rspo1 mice exhibited ovotestes with an extensive presence of sex cords (Figure 1d, 1i and in Figure S2B, f) as do XX Rspo1gonads (shown in Figure S2B, S2e and in previous analyses [20], [21]). Thus, the development of XX Rspo1 mouse genitalia is indistinguishable from that of XX Rspo1 mice indicating that the additional deletion of Sox9 in XX Rspo1 gonads does not change the fate of XX Rspo1 gonads. We next examined whether the supporting cells forming the sex cords differentiated as granulosa cells, the ovarian supporting cells expressing FOXL2 [25], [28], or as Sertoli cells expressing DMRT1 [29]. In three weeks old mice (P21), Sox9-depleted cells forming the seminiferous tubules generally lacked the follicular cell marker FOXL2 and instead expressed DMRT1 (Figure 1n, 1s). These data clearly indicate that Sertoli cell, seminiferous tubule and testis differentiation can occur in the absence of Sry and Sox9 in XX Rspo1 gonads.

Steroidogenic cells are present in Rspo1 embryonic gonads

Previous studies clearly show that the development of male genitalia depends on androgens secreted by the embryonic testis [30]. In XX Rspo1 gonads, steroidogenic cells appear before Sertoli cell differentiation [20], [31] and this was also observed in Wnt4 gonads [22], [32], Wnt4 being up-regulated upon Rspo1 expression in XX gonads [20], [21]. In addition, lack of Wnt4 expression was shown to allow ectopic migration of steroidogenic cells from the neighboring adrenals into gonads [32], [33] and subsequent androgen synthesis [34], which explains the development of male genitalia in these mutants. When investigating whether steroidogenic cells were present in XX and XY Rspo1 gonads, we found that P450Scc, a gene encoding for a precursor involved in androgen synthesis was expressed at 14.5 dpc in XY controls, XY and XX Rspo1 gonads and XX Rspo1 gonads, but not in XX controls (Figure S3A). However, Cyp21, a marker for adrenal cells [35], was not strongly expressed in Rspo1 gonads at 13.5 dpc (Figure S3A), suggesting that the steroidogenic cells in Rspo1 gonads, did not come from the arenals or, alternatively, have undergone reprogramming as Leydig cells. Whatever the situation, it is likely that male hormones synthesized in the developing mutant gonads can contribute to stimulate epididymides, vasa deferentia and seminal vesicles development.

Delayed testicular formation in Rspo1 mice

We next investigated the timing of testicular cord formation in XY and XX Rspo1 gonads. In wild-type embryos, the earliest morphological sign of testis development occurs at 12.0–12.5 dpc when testis cord are formed [36]. Accordingly at 13.5 dpc, the testis cords were highlighted in XY controls by the prominent expression of SOX9 and AMH, two markers of Sertoli cells (Figure 2a). In contrast, Rspo1 gonads did not show a clear testicular organization as they lack AMH at 13.5 dpc (Figure 2b, 2c, 2f, 2g). As AMH synthesis and secretion by Sertoli cells promotes the elimination of the female reproductive tract during embryogenesis [37], the absence of AMH in Rspo1 gonads provides an explanation for the maintenance of the Mullerian derivatives (oviducts, uterine horns and vaginal tissues) in these mutant mice. In addition, Sertoli cell differentiation is delayed in gonads lacking both Sox9 and Rspo1, as indicated by the maintenance of SRY expression, in the XY Rspo1 gonads at 13.5 dpc (Figure 2f), a stage at which SRY expression has already ceased for one day in the control situation [38], [39], [40]. Along these lines, the maintenance of SF1 expression in XX Rspo1 gonads at 13.5 dpc (Figure 2j, 2k), a factor whose expression is normally down-regulated between 13.5 and 16.5 dpc in the ovary [41] (Figure 2l), also suggests that the XX Rspo1 gonads are still undifferentiated or have differentiated as testis. However, with respect to the latter, the absence of AMH expression shows that no Sertoli cell differentiation has occurred (Figure 2c, 2g). Altogether these data indicate that the Rspo1 gonads are still undifferentiated at 13.5 dpc.

Figure 2

Non-differentiated XY and XX Rspo1 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) Rspo1 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 Rspo1 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 Rspo1 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 controls, XY (b, f, j) and XX (c, g, k) Sox9 respectively.

The first signs of Sertoli cell differentiation appeared at 16.5 dpc in Rspo1 gonads, with some rare DMRT1-positive cells in comparison to XY controls (Figure S3B). Then, few rudimentary testis cords were observed around 17.5 dpc (Figure S3B). At P0, Sertoli cells aligned to form sex cords as evidenced by the localization of DMRT1 positive-cells (Figure S4A c, d). Quantitative PCR experiments further confirmed that Dmrt1 expression was strongly expressed in XY Rspo1 gonads and weakly in XX Rspo1 gonads at P0, highlighting that more Sertoli cells were present in XY Rspo1 gonads in comparison to XX Rspo1 gonads (Figure S4C). In addition, some FOXL2-positive cells were also detected in Rspo1 gonads (Figure S4A c, d). However, quantitative PCR experiments showed that Foxl2 expression was significantly reduced in comparison to XX control or XY Sox9 gonads (Figure S4B) as expected for a gonad developing as ovotestis or testis.

We then studied SDMG1 expression, a cytoplasmic marker of Sertoli cells and of granulosa cells when follicles form (Best et al. 2008). Using this marker, sex cords were evident at P0 (Figure 3c, 3d and Figure S5c, S5d) and, at puberty (P12), development of the seminiferous tubules appeared complete in Rspo1 gonads (Figure 3h, 3i and Figure S5h, S5i). At puberty, androgen receptor (AR) immunostaining indicated that, in addition to Sertoli cells, peritubular myoid and Leydig cells were also present both in XY Rspo1 testes (Figure 4i) and in testicular parts of the XX Rspo1 ovotestes (data not shown). In addition, follicle development appeared at P12 in XX Rspo1 ovotestes and XX control ovaries (Figure S2B d, f). Together our results indicate that seminiferous tubule development is delayed in the absence of Sox9 and Rspo1, thereby explaining the small size of the XY Rspo1 testes (Figure 1c).

Figure 3

Post-natal development of sex cords in XY and XX Rspo1 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 Rspo1 gonads, with more developing sex cords in XY Rspo1 testis (c, h, m, r) in comparison to XX Rspo1 ovotestis (d, i, n, s). At P12, the sex cords are fully developed in both XY (h) and XX (i) Rspo1 mice. In XY Sox9 (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 controls, XY Sox9 gonads (b, g, l, q), XY (c, h, m, r) and XX (d, i, n, s) Rspo1 gonads respectively.

Figure 4

Sertoli cells support germ cell differentiation in XY Rspo1 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 Rspo1 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 Sox9 mutant gonads normally expressed AR (h). (scale bars: 50 µm). XY (a, d, g, j) Rspo1 controls, XY Sox9 gonads (b, e, h, k) and XY Rspo1 (c, f, i, l) gonads.

SOX9-negative Sertoli cells can support germ cell differentiation until initiation of meiosis

We next investigated whether the Sertoli cells that differentiate in the Rspo1 gonads can support germ cell differentiation. Since XX germ cells cannot survive in a testicular environment [42], [43], the analysis was only carried out in XY Rspo1 gonads. In the normal fetal testis, following Sertoli cell differentiation, prospermatogonia become quiescent from 14.5 dpc and express the multipotency marker Oct4 until 17.5 dpc [44]. At that time, Cyp26b1, a protein involved in retinoic acid degradation, contributes to prevent germ cells from entering meiosis [45], [46]. As expected, the majority of prospermatogonia in XY Rspo1 gonads expressed Oct4 at 14.5 dpc (Figure S6o). Nonetheless, some cells had already committed to meiosis (Figure S6k) and expressed the meiotic marker Stra8 [47], possibly because of the low level of Cyp26b1 expression in XY Rspo1 gonads (Figure S6g). The reduced level of Cyp26b1 expression is however not sufficient to allow all germ cells to enter meiosis in XY Rspo1 gonads.

At P10, GATA1, Androgen Receptors (AR) and Clusterin (Clu) were normally expressed in Sertoli cells of XY Rspo1 gonads (Figure 4c, 4i, 4l), suggesting that these cells have acquired their identity and may be capable to support spermatogenesis. Accordingly, XY germ cells had committed to meiosis at P10, as assessed by immunodetection of the pre-meiotic and meiotic markers STRA8 and γH2AX, respectively (Figure 4c, 4f, 4i). However, later stages of spermatogenesis cannot however be analyzed, as hypoplasia of germ cells occurred within the seminiferous tubules of adult XY Rspo1 gonads (Figure 1h and Figure S1m), most likely because of cryptorchidism.

Sox8 and Sox10 are expressed in the absence of Sox9

Interestingly, we found that AMH was expressed in Sertoli cells of both XX and XY Rspo1 gonads at P12 (Figure 5A). Given that (i) Amh is a target-gene of SOX9 [48], [49], (ii) Amh expression can be induced by SOX8 [50] and SOX10 [51], and (iii) Sox10 ectopic up-regulation in XX gonads can induce testis differentiation [51], we hypothesized that a Sox factor distinct from Sox9 could have induced late AMH expression in Rspo1 gonads and delayed testicular differentiation. In agreement with this possibility, expression of both Sox8 and Sox10 was activated in Rspo1 mutants at P12 and P0 respectively (Figure 5B, 5C). Previous data have shown that Sox8 becomes crucial from 14.5 dpc onwards, for the maintenance of testis development [52], suggesting that Sertoli cell differentiation can be induced by Sox genes other than Sox9 during late embryogenesis. However, the function of these Sox genes during late development is likely not sufficient to replace the role of Sox9 in early Sertoli cells development, thus leading to the formation of an hypoplastic testis, as is the case in the XY Rspo1 mice. To date, the only factors that have been shown to be able to induce Sertoli cell differentiation are Sox genes [51], [53], while other factors such as Dmrt1 are required after birth (P7) for the maintenance of Sertoli cell identity [54]. Further studies are required to address whether DMRT1 is able to allow Sertoli cell differentiation from undifferentiated supporting cells. Given that Sox9 expression is controlled by Wt1 when Sry expression has ceased [55], we can speculate that Wt1 might also be involved in Sox8 and Sox10 expression in these mutants. Furthermore, FGF9 or PGD2, two secreted factors synthesized in the undifferentiated gonads, [56], [57] can also contribute to Sertoli cell differentiation [58], [59], [60]. Whether Wt1, PGD2 or FGF9 signaling also regulate Sox8 and Sox10 remains to be investigated.

Figure 5

AMH and SOX genes are expressed in XY and XX Rspo1 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) Rspo1 gonads and theca cells of the ovarian region of XX Rspo1 gonads (d) and XY Sox9 gonads (b). AMH expression is observed in Sertoli cells of the XY (c, h) and XX (d, i) Rspo1 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 Rspo1 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 controls, XY Sox9 gonads (b, g), XY (c, h) and XX (d, i) Rspo1 gonads respectively. B- Sox8 is expressed in XY and XX Rspo1 gonads. In situ hybridization of Sox8 transcripts. Sox8 is expressed in Sertoli cells at P5 in XY control (a), XY (c) and XX (d) Rspo1 gonads, but not in XY Sox9 ovaries (b). (a) XY Rspo1 controls, (b) XY Sox9 gonads, XY (c) and XX (d) Rspo1 gonads respectively. C- Sox10 is expressed in XY and XX Rspo1 gonads. QPCR analysis shows that Sox10 is significantly up-regulated both in XY and XX Rspo1 gonads, when compared to XY controls. The differences between XY controls and XY Sox9 are not significant.

In addition, when XX and XY Rspo1 gonads are compared at the same stage, XY gonads always appear more masculinized than XX gonads (Figure 1, Figure 3, Figure S1, Figure S3, Figure S5), because they contain more sex cords/seminiferous tubules. At a molecular level, the main difference between XX and XY Rspo1 gonads is the expression of SRY in XY gonads (Figure 2). Indeed, SRY expression is maintained in XY Rspo1 gonads at 13.5 dpc, while at this time point its expression has ceased in XY control gonads. This suggests that SRY participates in the masculinization of the XY Rspo1 gonads by inducing the expression of genes other than Sox9 to promote sex cord formation.

In summary, here we have shown that (i) both SRY and SOX9 are dispensable for female-to-male sex reversal in Rspo1, (ii) RSPO1 signaling is required for male-to-female sex reversal in Sox9, (iii) Sertoli cell differentiation and seminiferous tubule formation can occur in the absence of SOX9, possibly because of a functional redundancy with other SOX proteins such as SOX8 and SOX10. Indeed, ectopic expression of Sox10 in XX gonads has been shown to promote testicular differentiation [51]. Altogether these data show that SRY and SOX9 are not the only masculinizing factors, since other SOX proteins can induce female-to-male sex reversal in pathophysiological conditions (Figure 6). Following Sertoli cell differentiation, DMRT1 expression becomes required for the maintenance of the Sertoli cells and the testicular tissue [54]. Furthermore, our data suggest that the feminizing factors remaining in Rspo1 mice can be overtaken by SOX proteins, when they are activated in XX gonads. Testicular differentiation in the absence of Rspo1 expression in XY Sox9 gonads was unexpected since female development is thought to be a default pathway [61], [62]. Our results imply that instead the female pathway needs to be activated. Therefore our genetic study suggests that mammalian sex determination is regulated by a finely tuned balance between two main factors [56], [63], which are the SOX genes on the one hand and the RSPO1/WNT/beta-catenin signaling pathway on the other hand.

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., Rspo1) 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 Rspo1 gonads restores testis development. This is associated with the expression of other SOX genes like SOX 8 and SOX10, other masculinising factors.

Materials and Methods

Mouse strains and genotyping

The experiments here described were carried out in compliance with the relevant institutional and French animal welfare laws, guidelines and policies. They have been approved by the French ethics committee (Comité Institutionnel d'Ethique Pour l'Animal de Laboratoire; number NCE/2011-12). All mouse lines were kept on a mixed 129SV/C57BL6/J background. Rspo1, Sox9 mice and Sf1:cre transgenic mice (a kind gift from Keith Parker) were described previously [64]. Rspo1 male were mated with Sox9; Sf1:cre female [16] to obtain Rspo1 females and Rspo1 males. Matings between these littermates allowed us to generate Rspo1 mice, referred to as Rspo1 mice, and controls. Gonad samples were collected from timed pups (day of birth = P0). Genotyping was performed as described [7], [20], [64] using DNA extracted from tail tip or ear biopsies of mice. The presence of the Y chromosome was determined as described previously [65]. Pax6 primer set (5′-GCAACAGGAAGGAGGGGGAGA-3′; 5′-CTTTCTCCAGAGCCTCAATCTG-3′) was included in each PCR reaction as an internal control.

Histological analysis

Urogenital organs were dissected, fixed in Bouin's solution overnight, and then embedded in paraffin. Microtome sections of 5 µm thickness were stained with periodic acid Schiff (PAS) or hematoxylin and eosin (H&E) according to standard procedures. Pictures were taken with an Axiocam mrm camera (Zeiss) and processed with Adobe Photoshop.

Immunological analyses

Gonad samples were fixed with 4% (w/v) paraformaldehyde overnight and then processed for paraffin embedding. Gonad samples for cryosections were successively fixed for 2 hours in 4% (w/v) paraformaldehyde, washed in cold phosphate-buffered saline (PBS), equilibrated in 10% (w/v) sucrose solution during 3 hours, then in 30% (w/v) sucrose solution overnight at 4°C, embedded in Cryomount (Histolab) and stored at −80°C. For paraffin-embedded and Cryomount-embedded samples, sections of 5 and 8 µm thickness were processed, respectively. The following dilutions of primary antibodies were used: AMH/MIS (C-20, sc-6886, Santa Cruz), 1∶200; AR (sc-816, SantaCruz), 1∶100; DMRT1 (kindly provided by David Zarkower), 1∶500; FOXL2 (ab5096, Abcam), 1∶250; γH2AX (U5-636, Upstate), 1∶500; GATA1 (sc-265, SantaCruz), 1∶50; SDMG1 (a kind gift from Ian Adams) 1∶2000; SF1 (kindly provided by Ken Morohashi) 1∶1500; SOX9 (HPA-001758, Sigma) 1∶250 and SRY [59], [66] 1∶100, STRA8 (ab49602, Abcam), 1∶100. Counterstain with 4′,6-diamidino-2-phenylindole (DAPI) was used to detect nuclei (in blue). Fluorescent studies were performed with a motorized Axio ImagerZ1 microscope (Zeiss) and pictures were taken with an Axiocam mrm camera (Zeiss) and processed with Axiovision LE.

In situ hybridization

Embryos were fixed with 4% paraformaldehyde (PFA) in 1×PBS at 4°C overnight. Further processing of embryos and in situ hybridization were carried out essentially as described [67]. Sox9 riboprobe was synthesized according to [68] and Sox8 to [69], P450scc, Stra8 and Oct4 riboprobes synthesis were carried out as described previously [20]. In situ hybridisation (ISH) with digoxigenin–labelled probes was performed as described [70], using 10 µm–thick cryosections. Each experiment was repeated on at least two gonads. Post–hybridization washes were done in 100 mM maleic acid pH7.5, 150 mM NaCl, 0.1% (v/v) tween–20 (MABT). To increase the sensitivity, 5% (v/v) polyvinyl alcohol (Sigma) was added to the staining solution [71]. Nuclei were counterstained with DAPI diluted in the mounting medium at 10 µg/ml (Vectashield, Vector laboratories). ISH signals corresponding to Clu-positive cells were converted into a red false color on the merged pictures. The plasmids containing Lgals1 (366 bp–long; exons 2–4; MGI:96777) or Clu (942 bp–long; exons 5–9; MGI:88423) cDNA fragments were linearized and used as a templates for the synthesis of the sense or antisense riboprobes.

Quantitative PCR analysis

Individual gonads were dissected in PBS from P0 animals (day of birth) and immediately frozen at −80°C. RNA was extracted using the RNeasy Qiagen kit, and reverse transcribed using the RNA RT–PCR kit (Stratagene). Primers and probes were designed at Roche Assay design center (https://www.rocheappliedscience.com/sis/rtpcr/upl/adc.jsp). Primers are 5′-TCCTCCTCAGACCGCTTTT-3′ and 5′-CCTGGTTCATCATCGCTAATC-3′ (probe 95) for Hprt1, and 5′-ATGTCAGATGGGAACCCAGA-3′ and 5′-GTCTTTGGGGTGGTTGGAG-3′ (probe 21) for Sox10, 5′-aagaagtgcagcctgattgc-3′ and 5′-ggtggctgatacccagttct-3′ (probe 40) for Dmrt1, and 5′-ggcgtcgtgaactcctaca-3′ and 5′-tgcagatgatgtgcgtgag-3′ (probe 51) for Foxl2. All real-time, quantitative, PCR assays (QPCR) were carried out using the LC-Faststart DNA Master kit Roche, according to the manufacturer's instructions. QPCR was performed on cDNA from one gonad and compared to a standard curve. QPCR were repeated at least twice. Relative expression levels of each sample were quantified in the same run, and normalized by measuring the amount of Hprt1 cDNA (which represents the total amount of gonadal cells).

Statistical analysis

For each genotype (n = 6 individuals), the fold-change was the mean normalized expression levels divided by the mean normalized expression levels of the XY samples considered as the reference. Graphs illustrate fold-changes +1 s.e.m. The results were analyzed using Graphpad for statistical significance that was assessed using one-way ANOVA followed by Tukey-Kramer post-test for selected pairs of genotypes. Asterisks indicate : * p<0.05, ** p<0.01 and *** p<0.001.

Testicular differentiation in XY and XX Rspo1 mice. External genitalia of XX control mice (e) is similar to XY Sox9 (b), XY and XX Rspo1 mice (c and d respectively) at 2 months of age. The internal genitalia of XY and XX Rspo1 mice (h, i) show epididymides (E), vasa deferentia (VD) and seminal vesicles (SV), as in XY males (f) but also uterine horns (U) and oviducts (Ovi) as in XX controls (j) or XY Sox9mice (g). PAS stained histological sections of XY and XX Rspo1 gonads (m, n) show seminiferous tubules lacking germ cells because hypoplasia of germ cells occurred in these tubules. XY Sox9 gonads (l) are similar to ovaries (o) (scale bar, 50 µm). XY (a, f, k) and XX (e, j, o) Rspo1controls, XY Sox9 gonads (b, g, l), XY (c, h, m) and XX (d, i, n) Rspo1 gonads respectively.

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A- XY Rspo1 gonad containing a single follicle. XY Rspo1 gonad with a single grossly large follicle was located near the entrance of the oviduct (a). This follicle contained three oocytes and was observed in XY Rspo1 gonads on rare occasions (b). B- Comparison of XX Rspo1and XX Rspo1 gonads. Immunofluorescence detection of SDMG1 in Sertoli cells (cytoplasmic) of XX Rspo1 (b) and XX Rspo1 (c) gonads at P0. Some sex cords are clearly visible in XX Rspo1 and XX Rspo1 in contrast to XX control gonads (a). Histological analysis of XX Rspo1 and XX Rspo1 gonads at P12 and P21 (e, h and f, i respectively) show the presence of seminiferous tubules and follicles in comparison to XX controls containing only follicles (d, g). Empty and filled arrowheads indicate testis cords and follicles, respectively.

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A-Expression of the steroidogenic marker P450Scc and Cyp21 in XY Rspo1 gonads. Whole-mount in situ hydridization of gonads at 14.5 dpc and 13.5 dpc. P450Scc is expressed in XY Rspo1 gonads (b), and in XY controls (a) but not in XX controls (e). P450Scc, was expressed in cells at the anterior part of the XX Rspo1 and XX Rspo1 gonads (c and d respectively) at 14.5 dpc. Cyp21 was strongly expressed in the adrenals (f, g, h, i, j), whereas no signal was detected in the gonads at 13.5 dpc (f, g, h, i, j). Ad: adrenal, G: gonad, K: kidney. B-Delayed testicular cords formation in XY and XX Rspo1 gonads. Haematoxylin and eosin stained histological sections of XY and XX Rspo1 gonads at 17.5 dpc show that some sex cords are forming in the XY Rspo1 (b) gonads in contrast to the XY controls (a) containing already formed sex cords. In the littermates XX Rspo1 and Rspo1 gonads (c and d respectively), no sex cords were observed at this stage (scale bar, 10 µm). Insets in a and b show AMH (Red) and DMRT1 (green), two markers of Sertoli cells highlighting sex cords at 16.5 dpc. Sex cords were rare in XY Rspo1 gonads and absent from XX Rspo1 gonads at this stage.

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Detection of DMRT1 and FOXL2 in XY and XX Rspo1 gonads at P0. A- Immunofluorescence analysis of DMRT1 and FOXL2 in XY and XX Rspo1 gonads at P0. DMRT1 (red), a marker of postnatal Sertoli cells, was detected in the XY control (a), XY Rspo1 and XX Rspo1 gonads (c and d respectively) while in XY Sox9 (b) and XX (e) control gonads DMRT1 was not detected. The ovarian protein FOXL2 (green) was detected in XY Sox9 and XX control gonads, and few FOXL2-positive cells (granulosa cells) were found within the XY Rspo1 (c) and XX Rspo1 (d) gonads. B- qPCR anaylsis of Foxl2 in XY and XX Rspo1 gonads at P0. Foxl2 is significantly up-regulated in the XY Sox9 compared to XY and XX Rspo1 indicating that ablation of Rspo1 leads to reduced Foxl2 expression at a transcriptional level. C- qPCR anaylsis of Dmrt1 in XY and XX Rspo1 gonads at P0. Dmrt1 is significantly up-regulated in the XY Rspo1 gonads, indicating that ablation of both Rspo1 and Sox9 promotes Sertoli cell differentiation. Dmrt1 is not up-regulated in XX Rspo1 gonads. Indeed, at P0 few Sertoli cells have differentiated in XX Rspo1 gonads in comparison to XY Rspo1 gonads (see Figure 3). In addition it is noteworthy that Dmrt1 transcription occurs in the XY Sox9cKO but the protein is not detected (see same figure panel A). This suggests that the Rspo1 signaling pathway antagonizes the male pathway, at least partly at the post-transcriptional level, as shown for two other genes Wnt4 and Fgf9 [56], [72].

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Histological analysis of XY and XX Rspo1 gonads at P0, P12 and P21. Differentiation of seminiferous tubules containing germ cells in XY Rspo1hypoplasic testis (c, h, m) and XY controls (a, f, k). In XX Rspo1 (d, i, n), the gonads develop as ovotestes containing both seminiferous tubules and follicles (white star indicates a section of a follicle and white arrowhead shows a section (n) of a seminiferous tubule). However, as previously shown [73], the XX germ cells did not survive in post-natal seminiferous cords. In contrast, germ cells survived until P12 and some until P21 in the XY Rspo1 seminiferous tubules (h and m respectively) (scale bar: 50 µm). Controls XY (a, f, k) and XX (e, j, o), XY Sox9 (b, g, I) and XY Rspo1 (c, h, m), XX Rspo1 (d, i, n).

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Mixed germ cell differentiation in XY Rspo1 gonads at 14.5 dpc. In situ hybridization using a riboprobe for Sox9 (a–d), Cyp26b1 (e–h), Stra8 (i–l) and Oct4 transcripts (m–p) shows lack of Sox9 expression in the XY Sox9 (b) XY Rspo1 (c) and the XX control (d) gonads. Germ cells in XY Sox9 (j) and XX (i) control gonads have entered meiosis in as evidenced by robust Stra8 expression and weak expression of primordial germ cell marker Oct4 (n and p respectively). XY Rspo1 mutants (k) showed Stra8 expression at the periphery of the E14.5 gonad indicating these few cells have undergone meiosis (k) while the remaining germ cells were quiescent, thus, express Oct4 (o). Few Cyp26b1 expressing cells were detected in XY Rspo1 gonads (g). G: gonad, K: Kidney. XY (a, e, i, m) and XX (d, h, l, p) Rspo1 +/−; Sox9 controls, XY Sox9 (b, f, j, n) and XY Rspo1 (c, g, k, o).

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