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Fig. 9. Phosphorylation levels of AKT (a downstream effector of PI3K) in developing genital ridges in vitro and in vivo. (A) Sagittal sections of genital ridges (10-11 ts) cultured in the absence (cont) or presence of PI3K inhibitor (15 µM; LY294002; +LY) for 48 hours, and stained with anti-phospho-AKT. Positive signals for anti-phospho-AKT staining are detected in gonadal somatic cells located near germ cells, with a strong signal detected in the XY explant (XY cont) but only a weak signal in the XX explant (XX cont). Anti-phospho-AKT signals are clearly reduced in XY explants cultured in the presence of the PI3K inhibitor (XY + LY). (B) Immunoblot analyses of phosphorylation levels of AKT in XY and XX genital ridges (10-11 ts) cultured for 24 hours in the presence or absence of LY294002. In control XY explants, the AKT phosphorylation level is higher than that in XX control explants, and the level is clearly reduced by addition of LY294002 both in XY and XX explants. Repeat experiments were performed three times, and similar results were obtained each time. (C) Transverse sections of 11.5-dpc embryos (18 ts) and stained with anti-phospho-AKT. Anti-phospho-AKT reactions are found in the ventral region of neural tubes and dorsal root ganglion at similar levels in both sexes, but the reactions in the gonadal region are higher in XY than in XX embryos in vivo. These positive reactions in XY gonads are found in somatic cells near germ cells (asterisks). In plates A and C, right panels show higher magnification images, indicated by the boxed area in the corresponding left panel. asterisk, germ cell; drg, dorsal root ganglion; ms, mesonephros; nt, neural tube. Bar, 50 µm.
