Transient development of ovotestes in XX Sox9 transgenic mice

AbstractThe sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9Tg/+ gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads and is able to induce the expression of EGFP, knocked into the 3′ UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9–EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX fetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth

XY Sox9 embryonic loss-of-function mouse mutants show complete sex reversal and produce partially fertile XY oocytes.

International audienceGonadal differentiation is the first step of mammalian sex determination. The expression of the Y chromosomal testis determining factor Sry leads to up-regulation of the transcription factor Sox9 which promotes testis differentiation. Previous studies showed that Sox9 deficiency induces expression of ovarian markers in XY mutant fetal gonads before they die. To better understand the genome-wide transcriptional profile underlying this process we compared samples from XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads in which Sox9 is ablated in Sertoli-precursor cells during early stages of gonad development to XX Sox9(flox/flox) ovaries and XY Sox9(flox/flox) testes at E13.5. We found a complex mRNA signature that indicates wide-spread transcriptional de-regulation and revealed for XY mutants at E13.5 an intermediate transcript profile between male and female gonads. However, XY Sf1:Cre(Tg/+); Sox9(flox/flox) mutant gonads develop as ovaries containing XY developing follicles at P0 but less frequently so than in XX control ovaries. Furthermore, we studied the extent to which developing XY mutant ovaries are able to mediate adult fertility and observed that XY oocytes from XY mutant ovaries are competent for fertilization; however, two thirds of them fail to develop beyond two-cell stage embryos. Taken together, we found that XY Sf1:Cre(Tg/+); Sox9(flox/flox) females are capable of producing viable offspring albeit at a reduced level

Conserved genomic signature defines fetal sertoli cell fate in mammals

International audienc

Testicular Differentiation Occurs in Absence of R-spondin1 and Sox9 in Mouse Sex Reversals

<div><p>In mammals, male sex determination is governed by SRY-dependent activation of <em>Sox9</em>, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated <em>Sry</em> and <em>Sox9</em> 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 <em>Rspo1</em> mutant mice correlates with <em>Sox9</em> 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 <em>Rspo1</em> and <em>Sox9</em> (referred to as XX <em>Rspo1^KOSox9^cKO</em>⁾, indicating that <em>Sry</em> and <em>Sox9</em> are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both <em>Sox8</em> and <em>Sox10</em>, suggesting that activation of <em>Sox</em> genes other than <em>Sox9</em> can induce male differentiation in <em>Rspo1^KOSox9^cKO</em> mice. Moreover, since testis development occurs in XY <em>Rspo1^KOSox9^cKO</em> mice, our data show that <em>Rspo1</em> is the main effector for male-to-female sex reversal in XY <em>Sox9^cKO</em> mice. Thus, <em>Rspo1</em> 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.</p> </div

Non-differentiated XY and XX <i>Rspo1^KOSox9^cKO</i> gonads at 13.5 d<i>pc</i>.

<p>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) <i>Rspo1^KOSox9^cKO</i> gonads shows that Sertoli cell differentiation did not occur at 13.5 d<i>pc</i>. Note that the kidneys (K) are positive for SOX9. This is accompagnied with the maintenance of SRY expression in the XY <i>Rspo1^KOSox9^cKO</i> 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 <i>Rspo1^KOSox9^cKO</i> 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) <i>Rspo1^+/−; Sox9^flox/flox</i> controls, XY (b, f, j) and XX (c, g, k) <i>Sox9^cKO Rspo1^KO</i> respectively.</p

Opposing function of SOX and RSPO1 signaling in the fate of the gonad.

<p>A- In XX gonads, RSPO1 activates WNT/beta-catenin signaling to promote ovarian differentiation. Ablation of <i>Rspo1</i> results in partial sex reversal with ovotestis development, which coincides with <i>Sox9</i> expression. However additional deletion of <i>Sox9</i> in the XX <i>Rspo1</i>^KO (i.e., <i>Rspo1^KOSox9^cKO</i>) still allows ovotestis formation, implying that <i>Sry</i> and <i>Sox9</i> are not required for testicular differentiation in female-to-male sex reversal. B- In XY gonads, whereas <i>Sox9</i> deletion triggers ovarian development, additional deletion of <i>Rspo1</i> in XY <i>Rspo1^KOSox9^cKO</i> gonads restores testis development. This is associated with the expression of other SOX genes like SOX 8 and SOX10, other masculinising factors.</p

Testicular differentiation in XY and XX <i>Rspo1^−/−; Sox9^flox/flox; Sf1;cre^Tg/+</i> (<i>Rspo1^KOSox9^cKO</i>) mice.

<p>Macroscopic views of gonads of 2 month-old mice show hypoplasic testis and ovotestis development in XY (c) and XX (d) <i>Rspo1^KOSox9^cKO</i> mice, respectively. Seminiferous tubules are revealed by PAS histological analysis of XY (h) and XX (i) <i>Rspo1^KOSox9^cKO</i> gonadal sections. They are less abundant than in XY controls (f). XY <i>Sox9^cKO</i> 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 <i>Sox9</i> with <i>Sf1:cre</i> (<i>Sox9^cKO</i>) eliminates SOX9 expression in Sertoli cells (l, m, n), and promotes male-to-female sex reversal in XY <i>Sox9^cKO</i> gonads as highlighted by robust FOXL2 expression (l, q). However, <i>Sox9</i> deletion no longer allows ovarian cells differentiation when <i>Rspo1</i> is deleted in the XY (m, r) and XX (n, s) <i>Rspo1^KOSox9^cKO</i> 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) <i>Rspo1^+/−; Sox9^flox/flox</i> controls, XY <i>Sox9^cKO</i> gonads (b, g, l, q), XY (c, h, m, r) and XX (d, i, n, s) <i>Sox9^cKO Rspo1^KO</i> respectively. XX <i>Rspo1^KO</i> and XX <i>Sox9^cKO Rspo1^KO</i> gonads appeared similar (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003170#pgen.1003170.s002" target="_blank">Figure S2B</a>).</p

Sertoli cells support germ cell differentiation in XY <i>Rspo1^KOSox9^cKO</i> gonads.

<p>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 <i>Clu</i> 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 <i>Clu</i> expression show that the Sertoli cells mature in XY controls (a, g, j) and XY <i>Rspo1^KOSox9^cKO</i> 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 <i>Sox9^cKO</i> mutant gonads normally expressed AR (h). (scale bars: 50 µm). XY (a, d, g, j) <i>Rspo1^+/−; Sox9^flox/flox</i> controls, XY <i>Sox9^cKO</i> gonads (b, e, h, k) and XY <i>Rspo1^KOSox9^cKO</i> (c, f, i, l) gonads.</p

AMH and SOX genes are expressed in XY and XX <i>Rspo1^KO Sox9^cKO</i> gonads.

<p>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 <i>Sox9</i> with <i>Sf1:cre</i> eliminates SOX9 expression in <i>Sf1:cre</i> positive cells of the gonads, which are Sertoli cells in XY (c) and XX (d) <i>Rspo1^KOSox9^cKO</i> gonads and theca cells of the ovarian region of XX <i>Rspo1^KOSox9^cKO</i> gonads (d) and XY <i>Sox9^cKO</i> gonads (b). AMH expression is observed in Sertoli cells of the XY (c, h) and XX (d, i) <i>Rspo1^KOSox9^cKO</i> 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 <i>Rspo1^KOSox9^cKO</i> 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) <i>Rspo1^+/−;Sox9^flox/flox</i> controls, XY <i>Sox9^cKO</i> gonads (b, g), XY (c, h) and XX (d, i) <i>Rspo1^KO Sox9^cKO</i> gonads respectively. B- <i>Sox8</i> is expressed in XY and XX <i>Rspo1^KO Sox9^cKO</i> gonads. <i>In situ</i> hybridization of <i>Sox8</i> transcripts. <i>Sox8</i> is expressed in Sertoli cells at P5 in XY control (a), XY (c) and XX (d) <i>Rspo1^KOSox9^cKO</i> gonads, but not in XY <i>Sox9^cKO</i> ovaries (b). (a) XY <i>Rspo1^+/−; Sox9^flox/flox</i> controls, (b) XY <i>Sox9^cKO</i> gonads, XY (c) and XX (d) <i>Rspo1^KO Sox9^cKO</i> gonads respectively. C- <i>Sox10</i> is expressed in XY and XX <i>Rspo1^KO Sox9^cKO</i> gonads. QPCR analysis shows that <i>Sox10</i> is significantly up-regulated both in XY and XX <i>Rspo1^KOSox9^cKO</i> gonads, when compared to XY controls. The differences between XY controls and XY <i>Sox9^cKO</i> are not significant.</p

Post-natal development of sex cords in XY and XX <i>Rspo1^KOSox9^cKO</i> mice.

<p>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 <i>Rspo1^KOSox9^cKO</i> gonads, with more developing sex cords in XY <i>Rspo1^KOSox9^cKO</i> testis (c, h, m, r) in comparison to XX <i>Rspo1^KOSox9^cKO</i> ovotestis (d, i, n, s). At P12, the sex cords are fully developed in both XY (h) and XX (i) <i>Rspo1^KOSox9^cKO</i> mice. In XY <i>Sox9^cKO</i> (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) <i>Rspo1^+/−; Sox9^flox/flox</i> controls, XY <i>Sox9^cKO</i> gonads (b, g, l, q), XY (c, h, m, r) and XX (d, i, n, s) <i>Rspo1^KO Sox9^cKO</i> gonads respectively.</p