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First published online August 2, 2004
doi: 10.1242/10.1242/dev.01266

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Disrupted gonadogenesis and male-to-female sex reversal in Pod1 knockout mice

Shiying Cui^1,*, Andrea Ross^2,*, Nancy Stallings³, Keith L. Parker³, Blanche Capel² and Susan E. Quaggin^1,4,

¹ Department of Maternal and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Ontario M5G 1X5, Canada
² Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
³ Department of Internal Medicine and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
⁴ Division of Nephrology, St Michael's Hospital, University of Toronto, Toronto, Ontario M5B 1W8, Canada

View larger version (107K): [in a new window]   Fig. 1. Both male and female Pod1 KO mice have abnormal reproductive tracts. (A-F) Urogenital tracts of 18.5 dpc mice: (A,C,D) XY; (B,E,F) XX. In wild-type XY embryos, the testes (t) are rounded and have descended to the level of the bladder (b). In Pod1 KO XY embryos, the gonads are much smaller than controls and are adjacent to the kidneys (A). The gonads in Pod1 KO mice are irregular in shape with an uneven surface (D). In addition, the reproductive duct (green arrowheads) in the XY mutant has not developed into a normal epididymis (e), compared with in the wild-type mouse (C), and the internal genitalia rather resemble those of wild-type and Pod1 KO females. The ovaries (ov) in a wild-type embryo at 18.5 dpc have descended to just below the kidney (B). In XX Pod1 KO mice, the right gonad is sometimes observed in this location, but the left gonad never descends properly (B). Pod1 KO ovaries (F) are similar in size to the wild-type structures (E), but are irregularly shaped and display a morphology similar to the Pod1 KO testes. Of note, kidneys and bladders in both XY and XX Pod1 KO mice are also greatly reduced in size relative to controls. t, testis; ov, ovary; a, adrenal gland; k, kidney; b, bladder; e, epididymis. Semi-thin sections demonstrate that the wild-type testis at 18.5 dpc (G) exhibits well-organized testicular cords with Sertoli cells (sc) surrounding germ cells (g), peritubular myoid cells (pm), and extensive interstitial tissue, including Leydig cells (le) and vessels. The Pod1 KO testis (H) appears to have degenerated with a number of apoptotic cells (arrows, in H). At 18.5 dpc, most germ cells from control ovaries are in meiotic prophase (g, in I), but in the mutant XX gonad, they still appear as primordial germ cells similar to those in the testes (g in J). TUNEL labeling is shown in XY (K,L) and XX (M,N) gonads from 16.5 dpc embryos. In control gonads (K,M), no apoptotic cells are found in the sections, but in the gonads of Pod1 XY (L) and XX (N) mutants, a marked increase in the number of apoptotic cells (arrows) is observed.

View larger version (71K): [in a new window]   Fig. 2. Pod1 expression during gonad development. Illustrated are wholemounts (A,B,E,F) and longitudinal sections (C,D,G,H) of Pod1+/– gonads (go) stained for expression of the lacZ reporter gene. At 11.5 dpc, lacZ staining was observed in gonads from both XY (A) and XX (B) genital ridges. Strong expression was observed in the coelomic epithelium (ce) in male embryos and in mesonephric stromal cells at the boundary between the gonad and the mesonephros (m) (arrowheads) in both XY (C) and XX embryos (D). At 12.5 dpc, lacZ again was expressed in both XY (E) and XX (F) gonads, but higher levels of expression were observed in the male. Again, expression was primarily limited to the coelomic epithelium and some underlying cells (G,H). At 18.5 dpc, high levels of lacZ expression were observed in the interstitial cells of the testis (I,K) both in fetal Leydig cells (le) and in the peritubular myoid cells (pm) surrounding the testicular cords (tc). lacZ was also expressed throughout the developing ovary (J,L). Expression was concentrated particularly in the ovarian medulla (arrowhead), and in cells surrounding the developing follicles, which may represent theca cell progenitors. The inset (L) shows Pod1 expression in pericytes (pe) associated with a capillary. go, gonad; ce, coelomic epithelium; m, mesonephros; le, Leydig cells; pm, peritubular myoid cells; tc, testicular cords; pe, pericyte; en, endothelial cell.

View larger version (123K): [in a new window]   Fig. 3. Abnormal gonadal morphology in both male and female Pod1 mutants. (A) At 12.5 dpc, gonads from XY Pod1 KO mice (bottom) were smaller than in the wild-type testes (top). (B) Gonads from a Pod1 KO XX embryo at 12.5 dpc also were reduced in size compared with the control. In both XY and XX Pod1 KO embryos, an abnormal invagination of the surface was observed at the anterior region of the gonad (g) (arrowheads). g, gonad; m, mesonephros. (C-F) Semi-thin sections from 13.5 dpc gonads were stained with Toluidine Blue. At 13.5 dpc, the wild-type XY gonad (C) showed organized testicular cords (tc), peritubular myoid cells (pm) and extensive mesenchyme (m). However, no organized testicular cords, peritubular myoid cells or mesenchyme were seen in Pod1 KO XY gonads (E). At 13.5 dpc, Pod1 KO XX gonads (F) showed a similar arrangement of germ (g) and somatic (s) cells as the control (D), but lacked the small but distinct mesenchymal zone near the coelomic epithelium. tc, testicular cord; pm, peritubular myoid cell; m, mesenchyme; ce, coelomic epithelium; g, germ cell; s, somatic cell.

View larger version (79K): [in a new window]   Fig. 4. Structural and vascular defects in Pod1 KO mice. Confocal images are shown of 12.5 dpc gonads double-labeled with antibodies to laminin (green) and CD31/PECAM (red), which labels both germ cells (round) and vascular cells (elongated). In wild-type males at 12.5 dpc (A,C), the green laminin staining delineated numerous testicular cords (tc, arrows). The wild-type testes also displayed the characteristic coelomic vessel, which forms right beneath the coelomic epithelium (C, white arrowheads). In the Pod1 KO testes (B,D), no testicular cords were present and the coelomic vessel was also absent. The coelomic epithelium (blue arrowheads) was highly disorganized and invaginated in many locations. In wild-type females (E,G), no clear morphological organization was observed at this stage of development. However, compared with the control, the Pod1 KO XX gonad (F,H) exhibited a very disorganized surface epithelium, similar to that in gonads from the XY mutant. Of note, germ cell numbers did not appear to be affected in either XY or XX Pod1 KO mice.

View larger version (64K): [in a new window]   Fig. 5. Vascular defects in Pod1 KO gonads. Migration assays with GFP-expressing (green) wild-type mesonephros, and wild-type (A,C,E) or mutant (B,D,F) gonads at 72 hours, demonstrated a marked decrease in migration of endothelial cells (red) into Pod1 KO gonads. Yellow denotes double-staining of GFP and CD31/PECAM-positive cells, confirming the presence of mesonephric-derived vascular networks in wild-type but not Pod1 KO gonads. At 13.5 dpc, pericytes (per) were observed in close association with endothelial (en) cells of wild-type gonadal capillaries (G,I), but were never seen around Pod1 KO capillaries (H,J).

View larger version (65K): [in a new window]   Fig. 6. Disruption of male-specific markers in Pod1 KO gonads. Whole-mount in situ hybridization for Sox9 (A,B) and Dhh (C,D) was performed with gonads from 12.5 dpc embryos. Sox9 and Dhh were expressed by Sertoli cells in wild-type testes (A,C, top), but were absent in wild-type XX gonads (B,D, top). In Pod1 XY mutants (A,C, bottom), expression of both Sox9 and Dhh was reduced relative to controls, especially in the anterior domain of the gonad (arrowheads). Neither Sox9 nor Dhh was expressed in gonads from XX mutants (B,D, bottom). Scc, a marker of Leydig and adrenocortical cells, was expressed in the wild-type XY gonad (E), and in the adrenal primordial cells (ap) of both XY (E) and XX (F) wild-type embryos. Pod1 mutant XY and XX gonads (E,F) had greatly increased Scc expression, particularly at the posterior end of the gonad. Of note, the anterior ends of the Pod1 KO gonads did not separate from the adrenal primordium (E,F; arrowheads), although a distinct boundary between the gonads and adrenal glands was present in the controls (E,F, arrowheads). (G,H) At 11.5 dpc, Scc expression was restricted to adrenal primordia (ap) of wild-type XX and XY embryos, but was seen throughout the gonads (go) within the urogenital ridges of Pod1 mutant XX and XY gonads. (I,J) The adrenal-specific steroidogenic marker 11ß-hydroxylase was expressed appropriately in the adrenal primordia (ap) of both wild-type and Pod1 KO mice at 12.5 dpc. ap, adrenal primordia; go, gonad.

View larger version (68K): [in a new window]   Fig. 7. Early female-specific markers are expressed in Pod1 KO XX gonads. Whole-mount in situ hybridization for Wnt4 (A) and follistatin (B) is shown in XY and XX gonads from 12.5 dpc embryos. Expression of Dmc1, a marker of meiotic germ cells (C), is shown in 14.5 dpc embryos. Wnt4 is expressed in control XX gonads and mesonephroi, and was increased in mesonephroi of XX mutants (–/–). Follistatin is also expressed in XX mutant gonads, although the expression is somewhat decreased as compared with controls. Dmc1 expression is similar in XX mutants and controls.

View larger version (81K): [in a new window]   Fig. 8. Expansion of Sf1 expression in gonads and mesonephroi of Pod1 KO mice. (A) In situ hybridization analyses and immunohistochemistry of transverse sections and whole-mount genital ridges of XY gonads (go) at 11.5 dpc are shown. Gonads were labeled with a riboprobe against Pod1, or double-labeled with antibodies to Sf1 (green) and PECAM (red). Pod1 expression was concentrated in the coelomic epithelium (arrows) and the boundary of the gonad and mesonephros (arrowheads). Sf1-positive cells were evenly scattered in the interior of the control XY gonads. In the Pod1 KO XY gonads, Sf1 expression was increased in both the coelomic epithelium (arrow) and the interior of the gonad. A large population of Sf1-positive cells was seen at the boundary region between the gonad and the mesonephros (arrowhead). Note that the coelomic epithelium and the boundary between the gonad and the mesonephros are domains where Pod1 is expressed. (B) Confocal images of 13 dpc gonads double-labeled with antibodies to Sf1 (green) and ß-gal (red). In 13 dpc XX and XY control gonads, Sf1 is not expressed in cells that express the Pod1/lacZ reporter gene. By contrast, Sf1 is co-expressed in mutant Pod1/lacZ-expressing cells (arrowheads, yellow). (C) Model for Pod1 function in XY gonad development. Pod1 is proposed to repress Sf1 expression in a pluripotent interstitial cell precursor (in the mesonephros and/or coelomic epithelium, ce), thereby permitting differentiation of several interstitial cell lineages, including fetal Leydig cells (Le), peritubular myoid cells (PMC) and pericytes (Pe). Loss of Pod1 leads to ectopic expression of Sf1, and prematurely commits the progenitor cells to a steroidogenic cell lineage.

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