Fig. 3. Wnt pathways in dormant and activated mouse blastocysts. (A) Differential patterns of phosphorylated (inactive) and dephosphorylated (active) β-catenin during blastocyst activation. Whereas total β-catenin distribution was comparable in dormant (Dor) and activated (Act) blastocysts, phosphorylated β-catenin was primarily detected in the inner cell mass (ICM) and active β-catenin mostly in the trophectoderm (Tr) of activated blastocysts. (B) Wnt3a was induced in activated blastocyst Tr, whereas Wnt4 and Wnt5a were detected at high levels in the same cell-types in both dormant and activated blastocysts. (C) Dynamic expression of Wnt antagonists Dkk1, Dkk2 and Sfrp1 in delayed-implanting blastocysts. Whereas Dkk1 was downregulated, Dkk2 was induced in the Tr with blastocyst activation. By contrast, Sfrp1 expression was restricted to the ICM of activated blastocysts. (D) Expression of Wnt receptor subtypes Fzd2, Fzd4, Lrp5, Lrp6, Kremen1 and Kremen2 in dormant and activated blastocysts. An intriguing observation is the internalization and nuclear import of Wnt receptors in activated blastocysts. (E) Expression of Dvl1-3 proteins in delayed-implanting blastocysts. It is notable that Dvl1 and Dvl3 increasingly accumulated in the cytoplasm with visible nuclear localization of Dvl1 in the Tr during blastocyst activation. (F) c-Myc was induced in Tr cells of activated blastocysts. (G) Downregulation of total and GTP-bound (active) RhoA GTPase in the Tr during blastocyst activation. Representative immunofluorescence staining images depict Cy3-labeled antigens in red, SYTO-13-labeled nuclei in green, and merge in yellow. Scale bars: 50 µm.