Figure 3. In vitro fertilizing ability of β-catenin-deficient oocytes.

(a) Experimental flow for testing sperm-oocyte membrane ‘adhesion', and comparison of the number of wild-type sperm adhered to an ‘zona-free' oocyte between f/fcre and f/f oocytes. After ZP removal, ‘zona-free' oocytes were mixed with sperm for 1 h. (b) The number of sperm adhered to an oocyte was counted by DAPI-derived fluorescence in sperm heads on the surface of an oocyte. Parentheses indicate the number of oocytes examined. Values are the mean ± standard error (SE). (c) Experimental flow for testing sperm-oocyte membrane ‘fusion' and fused sperm (shown as DAPI-positive sperm) in ‘zona-free' β-catenin-deficient (f/fcre) and control (f/f) oocytes. After ZP removal, subsequent preincubation for 20 min in the presence of DAPI and washing, ‘zona-free' oocytes were mixed with the wild-type sperm for 1 h. (d) Comparison of oocytes fused with sperm between f/fcre and f/f oocytes. BF, bright field. Bars: 20 µm. (e) Comparison of the relative rate of oocytes fused with sperm between f/fcre and f/f oocytes. Only oocytes having at least one fused sperm were counted. The comparative values relative to the control (f/f oocytes; set to 100.0) were displayed as the relative rate of fused oocytes. Parentheses indicate the number of oocytes examined in triplicate experiments. Values are the mean ± SE. (f) Experimental flow for testing sperm-oocyte membrane interaction and fused sperm in two-cell embryos developing from ‘cumulus-intact' β-catenin-deficient (f/fcre) and control (f/f) oocytes. DAPI staining was performed to detect fused sperm on the developing two-cell embryos. (g) Comparison of oocytes fused with sperm between “cumulus-intact” f/fcre and f/f oocytes. Bars: 20 µm. (h) Comparison of the relative rate of oocytes fused with sperm between “cumulus-intact” f/fcre and f/f oocytes. Parentheses indicate the number of oocytes examined in triplicate experiments. Values are the mean ± SE. (i) Comparison of the ratio of oocytes developing to two-cell stage 24 h after fertilization between “cumulus-intact” f/fcre and f/f oocytes, according to the procedure described in (f). Parentheses indicate the total number of oocytes examined in triplicate experiments. NS, not significant. Values are the mean ± SE.