Sexually dimorphic regulation of inhibin beta B in establishing gonadal vasculature in mice

Abstract

Sexually dimorphic differentiation of gonads is accomplished through balanced interactions between positive and negative regulators. One of the earliest features of gonadal differentiation is the divergent patterning of the vasculature. A male-specific coelomic vessel develops on the anterior to posterior of the XY gonad, whereas this vessel is absent in XX gonads. It is postulated that the testis-determining gene Sry controls formation of the coelomic vessel, but the exact molecular mechanism remains unknown. Here we reveal a novel role for inhibin beta B in establishing sex-specific gonad vasculature. In the testis, inhibin beta B contributes to proper formation of the coelomic vessel, a male-specific artery critical for testis development and, later in development, hormone transportation. On the other hand, in the ovary, inhibin beta B is repressed by WNT4 and its downstream target follistatin, leading to the absence of the coelomic vessel. When either Wnt4 or follistatin was inactivated, the coelomic vessel appeared ectopically in the XX ovary. However, when inhibin beta B was also removed in either the Wnt4-null or follistatin-null background, normal ovarian development was restored and no coelomic vessel was found. Our results indicate that the sex-specific formation of the coelomic vessel is established by positive components in the testis as well as an antagonizing pathway from the ovary. Inhibin beta B is strategically positioned at the intersection of these opposing pathways.

FIG. 1

Section in situ hybridization of Inhba in XY (A) and XX (C) gonads and Inhbb in XY (B) and XX (D) gonads at E12.5. Positive staining appears as purple deposits and is indicated by black arrows. Negative controls using sense Inhba or Inhbb probes are shown in E and F. Magnifications ×10 and ×40 (two magnifications are shown for each sample). Real-time PCR analysis of Inhbb expression in XY and XX gonads is shown in G. The Y axis represents the relative fold differences between XY and XX gonads. The star indicates a statistical difference (P < 0.001). The red dotted outlines indicate testis cords. G, Gonad; M, mesonephros.

FIG. 2

Whole mount in situ hybridization for Inhbb in gonads from E11.5 and 12.5 wild-type XY (A and E), wild-type XX (B and F), Wnt4−/− XX (C and G), and Fst−/− XX (D and H). G, Gonad; M, mesonephros; red arrow, gonad at E11.5. Original magnification ×6.

FIG. 3

Inhbb is responsible for ectopic formation of the coelomic vessel in Wnt4 and Fst−/− XX gonads. The coelomic vessel (white arrows) and vasculature was delineated by an anti-PECAM1 antibody (red), which also marks primordial germ cells (red cells in gonads). Sertoli cells were also highlighted by an anti-AMH antibody (green). A) Wild-type XY. B) Wnt4−/−; Inhbb+/+ XX. C) Fst−/−;Inhbb+/+ XX. D) Wild-type XX. E) Wnt4−/−;Inhbb−/− XX. F) Fst−/−;Inhbb−/− XX. G, Gonad; M, mesonephros. n = 4. Original magnification ×25.

FIG. 4

Inhbb, or its product activin B, contributes to formation of the coelomic vessel. A and B) When XX gonads were cultured with human activin B, the coelomic vessel was induced as marked by an anti-PECAM antibody (white arrows). Branching of the coelomic vessel similar to that in the XY gonad was also observed (white arrowheads). Treatment of activin B to the XX gonad did not induce Sertoli cell differentiation, because no AMH immuno-staining (green) was found. C and D) The coelomic vessel in E12.5 Inhbb−/− XY gonads had a smaller caliber and occasional discontinuity compared to the wild type (white arrows). Sertoli cell differentiation (green AMH staining) appeared normal in the Inhbb−/− XY gonad. E) The proposed model: Inhbb (or its product activin B) is strategically positioned where the WNT4/FST signaling cascade and the SRY pathway intersect during mammalian gonad differentiation. Our results also suggest that activin B contributes to coelomic vessel formation independent of the SRY pathway. Original magnification A and B, ×10; C and D, ×25.