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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.
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