Figure 1. Testicular differentiation in XY and XX Rspo1^−/−; Sox9^flox/flox; Sf1;cre^Tg/+ (Rspo1^KOSox9^cKO) mice.

Macroscopic views of gonads of 2 month-old mice show hypoplasic testis and ovotestis development in XY (c) and XX (d) Rspo1^KOSox9^cKO mice, respectively. Seminiferous tubules are revealed by PAS histological analysis of XY (h) and XX (i) Rspo1^KOSox9^cKO gonadal sections. They are less abundant than in XY controls (f). XY Sox9^cKO 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^cKO) eliminates SOX9 expression in Sertoli cells (l, m, n), and promotes male-to-female sex reversal in XY Sox9^cKO 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^KOSox9^cKO 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^+/−; Sox9^flox/flox controls, XY Sox9^cKO gonads (b, g, l, q), XY (c, h, m, r) and XX (d, i, n, s) Sox9^cKO Rspo1^KO respectively. XX Rspo1^KO and XX Sox9^cKO Rspo1^KO gonads appeared similar (see Figure S2B).