In adult mammals, the urogenital system is comprised of the kidneys, urinary tracts, gonads, and reproductive ducts. Before GW7–GW8, the human gonadal ridges are undifferentiated. Genital ducts appear morphologically similar in both sexes with two paired structures: the Wolffian ducts (WDs), which are derived from the mesonephros, and the Müllerian ducts (MDs), whose differentiation is induced and guided by the WD. A major reorganization of the genital tracts accompanies sex determination (). In males, MDs regress under the influence of an anti-Müllerian hormone produced by the testes. In females, the WDs degenerate and the MDs transform into the female reproductive ducts ().

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Figure 6 3D Analysis of the Urogenital System Development in Male Embryos Show full caption All panels are LSFM images of solvent-cleared embryos and fetuses. (A and B) GW8 embryo stained for Pax2. Raw LSFM image (left) and 3D rendering image (right). The middle panel shows the segmentation and pseudocolorization of the kidney and ureters (yellow), Müllerian ducts (magenta, MD), and Wolffian ducts (cyan, WD; the mesonephric tubules are indicated by an arrowhead). (B) High magnification of the MD/WD junction (arrow). (C and D) Segmented (left) and 3D (right) images of the GW9.5 urogenital system labeled with Pax2. (D) The apical tip has enlarged (arrow). The distal tips of the MD (arrowheads in C and D) have extended along the WD (compare with A) but have not yet fused. (E) Segmented (left) and 3D (right) images of the GW10 urogenital system labeled with Pax2. The MD have fused distally (arrow) and began to fragment (arrowheads). (F) More advanced stage of MD regression and fragmentation (arrowhead) in a second GW10 fetus. (G) 3D image of the GW14 urogenital system labeled with Pax2. A short fragment of the Müllerian duct persists (arrowhead) and the vas deferens (VD) appears more developed (arrow). (H) GW10 testis (Te) labeled with Pax2 (red) and Sox9 (green). Sox9+ Sertoli cells are seen in the developing testis cords (arrowheads). (I) Single optical section (2 μm z projection) through a GW10 fetus testis labeled for Pax2, Sox9, and Plvap (endothelial cells). The arrow indicates the rete testis. (J) GW14 testis labeled for Sox9 and Pax2 segmented. The Müllerian (magenta) and Wolffian (cyan) ducts (Pax2+) have been segmented and pseudocolored. Abbreviations: Kid, kidney; MD, Müllerian duct; PU, prostatic utricle; WD, Wolffian duct; ♂, male. Scale bars, 400 μm in (A), (B), 500 μm in (C) and (E), 100 μm in (D) and (H, right panel), 300 μm in (F), (G), (H, left and middle panels) and (I), and 350 μm in (J). See also Figure S5 and Figure S6

Figure 7 Comparative 3D Analysis of the Urogenital System Development in Female and Male Embryos Show full caption All panels are LSFM images of solvent-cleared embryos and fetuses. (A) Original LSFM image (left), segmented/colorized (middle) and 3D (right) images of the urogenital system in a GW10.5 female fetus labeled with Pax2. The Wolffian ducts (cyan) are continuous and the Müllerian (magenta) ducts have fused. (B) Genital system of a GW11.5 female fetus stained with Pax2 after segmentation and 3D rendering. The arrow indicates the developing uterus and upper vagina. The Wolffian ducts start to regress (arrowheads). (C) Genital system of a GW13 female fetus stained with Pax2 after segmentation and 3D rendering. The size of the future uterus has increased and the Wolffian ducts have significantly regressed (arrowheads). The right panel is a high magnification at the level of the apical part of the Müllerian duct showing the developing fimbriae of the oviduct (arrow). (D and E) Vasculature of the developing gonads. (D) GW8 testis labeled for Pax2 and Plvap. A dense Plvap+ capillary network covers all the testis (arrow on the left) and the Wolffian duct (WD). By contrast, the Müllerian duct (MD) is not vascularized (arrowheads). The right panel is an optical section (1.2 μm) through the right testis and MD. (E) At GW10, the MD is still devoid of capillaries in male. (F, G) GW10.5 and GW13 ovaries labeled for Pax2 and Plvap. A dense vascular network covers the ovary (Ov). Unlike in males, both the Müllerian and Wolffian ducts are densely vascularized. Abbreviations: ♀, female; ♂, male; MD, Müllerian ducts; WD, Wolffian ducts; OV, ovary. Scale bars, 500 μm in (A), (B), and (C, left panel), 270 μm in (C, right panel), 100 μm in (D, middle and right panels), 160 μm in (D, left panel), 200 μm in (E)–(G). See also Figure S5 and Figure S6

Figure S5 Development of the Müllerian and Wolffian Ducts in Male and Female Human Embryos, Related to Figure 6 and Figure 7 Show full caption All panels are LSFM images of the genital tracts of solvent-cleared embryos and fetuses labeled with Pax2 antibody, segmented and pseudocolored. All images are at the same magnification. (A) In males the Müllerian duct (MD) extends ventrally along the Wolffian duct (WD) and fuses with the opposite MD around GW9.5. The MDs then degenerate except the fused domain that will become the prostatic utricle. (B) In females, the MDs fuse to form the uterus and vagina and the WDs regress. Scale bars, 400 μm in A and B.

Knowledge of urogenital system development is primarily based on histological and electron microscopy analysis () and has not yet been studied at a cellular and molecular level in humans, unlike in mice (). We studied the development of the human urogenital system by using antibodies against Pax2 TF. Pax2 mRNA was previously detected in the mesonephros and WD at GW6–GW7 (). We used embryos and fetuses of either sex, including 11 males (GW8–GW14) and 9 females (GW7–GW14). All major components of the urogenital system were visualized in 3D ( Figures 6 7 , and S5 and Movie S6 ) and individually segmented. In the youngest male embryo (GW8), the caudal tip of the MD was in close contact with the WD but had not yet fully elongated ( Figures 6 A and 6B), and mesonephric tubules stemming from the WD covered the testis primordium. Kidneys still occupied a ventral position adjacent to the genital ridges ( Figure 6 A). At GW9.5 (n = 1), the MD further elongated along the WD but did not yet join ( Figures 6 C and 6D). In GW10 male fetuses (n = 2), the two MD were fused and extended medially between the two WD up to the urogenital sinus. However, in both cases, the MD started to fragment dorsally ( Figures 6 E and 6F and Figure S5 A), and this remnant of the fused MD might give rise to the prostatic utricle. At older ages, such as GW14 (n = 1), the mesonephric nephrons of the WD have regressed while the epididymis ducts and vas deferens have emerged ( Figure 6 G). As previously reported (), an apical MD remnant is still present next to the rete testis by GW14 ( Figures 6 G and 6J and Movie S6 ). Sox9 is essential to testes differentiation in males, and its mRNA was first detected around CS19–CS21 (GW7–GW7.5) (). Using whole-mount immunostaining for Sox9 on GW10, GW11, GW13.5, and GW14 fetuses (n = 1 for each), we visualized the 3D organization of the testes cords, containing Sox9+ Sertoli cells ( Figure 6 H and 6J, Movie S6, and data not shown). We also stained GW10.5–GW13 female fetuses with Pax2 (n = 6) and followed in 3D the reorganization of the urogenital tract ( Figures 7 and S5 ). In our youngest female fetus (GW10.5) the MD have already fused to form the uterovaginal canal ( Figure 7 A). The WD were still continuous but mesonephric tubules began to regress. At GW11.5, the regression of the mesonephros and WD was more pronounced and the length of the two MD increased ( Figure 7 B). At GW13, the distal part of the WD had dissolved whereas its cranial part transformed into the Fallopian tubes ( Figures 7 C and S5 B and Movie S6 ). The vascularization of the developing gonads is thought to play a role in their maturation (). We performed double staining for Pax2 and Plvap to study the interaction between the vasculature and the genital tracts. Interestingly, at GW8 and GW10, the developing testes and WD were embedded in a dense capillary meshwork but the MD was avascular ( Figures 7 D and 7E). In contrast, in a GW10.5 female fetus, both the MD and the WD were vascularized and a dense vascular network ensheathed the MD by GW13 ( Figures 7 F and 7G and Movie S6 ). While we could not study younger female embryos, these results suggest that MD angiogenesis may be sex dependent. This differential vascularization of the developing MD in males and females at GW10 suggest that their regression in males, which starts around this stage, could be linked to or facilitated by the lack of vascularization. By contrast, the WD are vascularized in both sexes. Interestingly, the distal fused part of the Müllerian ducts does not seem to regress and likely gives rise to the prostatic utricle, as previously proposed ().