Fig. 6. Wnt11-LRP6 signaling regulates axin levels in the stage-6 oocyte. (A) Western blots of wild-type control and LRP6-depleted (LRP6^-) stage-6 oocytes frozen after 3 days in culture and probed with an affinity purified axin polyclonal antibody. Axin levels were increased in LRP6-depleted oocytes compared with controls (^*36% and 46% increases in experiment 1 and 2, respectively). (B) Western blots of wild-type, manually defolliculated oocytes (Control) and collagenase-treated oocytes frozen after 3 days in culture and probed with an axin antibody. Axin levels were unchanged as a result of culture without the follicle cell layer. Two repeats of the experiment are shown. (C) Western blots of wild-type and Wnt11-depleted (Wnt11^-) stage-6 oocytes frozen after 3 days in culture and probed with an axin antibody. Axin levels were increased in Wnt11-depleted oocytes compared with controls (increased by 128%; range 10-128% in three experiments). (D) Western blots of wild-type control and LRP6-depleted (LRP6^-) stage-6 oocytes that were frozen after 3 days in culture and probed with a ß-catenin antibody. Oocytes were non-matured or stimulated with progesterone and cultured for 8 hours before freezing. ß-catenin was expressed at low levels in oocytes before maturation, compared with the levels after maturation. LRP6 depletion reduced the amount of ß-catenin protein in matured oocytes (23% less ß-catenin compared with controls). (E) Western blots of wild-type and axin-depleted (Axin^-) stage-6 oocytes that were frozen after 3 days in culture and probed with a ß-catenin antibody. ß-catenin levels were increased in axin-depleted oocytes compared with controls (99% increase compared with controls). (F) A model for steady-state canonical Wnt signaling. Axin is continually synthesized in full-grown, non-matured oocytes and degrades cytoplasmic ß-catenin. Wnt11 is also continually synthesized and secreted, binds to LRP6, and degrades some axin protein. Thus, a steady-state level of axin is maintained by Wnt signaling, which, in turn, keeps ß-catenin at a low level in the cytoplasm. In LRP6-depleted oocytes, Wnt11 is secreted, but signals cannot be transduced. Thus, axin levels increase, and ß-catenin is further reduced.