Takezawa Y , Yoshida K , Miyado K , Sato M , Nakamura A , Kawano N , Sakakibara K , Kondo T , Harada Y , Ohnami N , Kanai S , Miyado M , Saito H , Takahashi Y , Akutsu H , Umezawa A .

	Figure 1. Expression of E-cadherin and β-catenin and localization of E-cadherin/β-catenin complex in oocytes.(a) Localization of E-cadherin, β-catenin and α-catenin in ovulated oocytes. Similar distribution pattern of E-cadherin and β-catenin on an oocyte suggests complex formation between these two proteins. IF, immunofluorescence; BF, bright field. Scale bars: 20 µm. (b) Localization of β- and γ-actin isoforms and β-catenin beneath the oocyte cell membrane and their fluorescent intensities. The route scanned on the membrane was indicated as a dotted line. Fluorescence intensities for each protein were measured and graphed based on the 3D image, as described in the Experimental Procedures. Red and green lines in the lower panel indicate intensities of β-catenin and actin, respectively. Scale bar: 5 µm. (c) Biochemical evidence for the presence of E-cadherin/β-catenin complex in oocytes. The extract from 905 oocytes immunoprecipitated (IP) by anti-β-catenin mAb and mouse IgG purified from preimmune serum (Control) was subjected to immunoblotting with anti-E-cadherin, anti-β-catenin or anti-β-tubulin mAb. Extracts from mouse embryonic carcinoma cell line P1935 were also subjected to immunoprecipitation with anti-β-catenin mAb and the resulting immunoprecipitates were reacted with each mAb as a positive control. Note that the extract (IP) immunoprecipitated by anti-anti-β-catenin mAb was reactive with both anti-β-catenin and anti-E-cadherin mAbs, but the extract (Unbound) that was not immunoprecipitated by anti-β-catenin mAb failed to bind to both antibodies. On the other hand, the β-tubulin was detectable in the Ab-unbound (but not Ab-IP) fractions. (d) Disassembly of β-catenin, E-cadherin and actin induced by latrunculin A (latA) treatment. Oocytes were doubly immunostained with anti-β- and γ-actin isoforms mAbs and DAPI (shown as ‘Actin DAPI') or with anti-β-catenin mAb or with anti-E-cadherin mAb and DAPI (shown as ‘β-catenin DAPI' or ‘E-cadherin DAPI'). In the lower panels, the fluorescence intensities measured after being traced along dotted lines in the figures of the upper panels are shown. The intensities of latA-treated oocytes are indicated by red lines, while those of the latA-untreated oocytes are shown by black lines. Scale bar: 20 µm.
	Figure 2. Expression of E-cadherin and β-catenin and their interaction in sperm.(a) Expression of E-cadherin and β-catenin in epididymal sperm. E-cadherin and β-catenin, but not N-cadherin, in the sperm were detected by immunoblotting (IB). Sperm (S1 and S2) were collected from the epididymis of two males and used for IB. Extracts from P19 cells were used as a positive control. IB was performed using anti-E-cadherin, anti-β-catenin or anti-N-cadherin mAbs. M, molecular weight markers. (b) Interaction between E-cadherin and β-catenin in sperm. Extracts from the sperm (used as input sample (IN)) were immunoprecipitated by anti-E-cadherin or anti-β-catenin mAb. The precipitates (IP) and unbound extracts (U) were immunoblotted (IB) with anti-E-cadherin or anti-β-catenin mAb. M, molecular weight markers. (c) Localization of E-cadherin and β-catenin in sperm. Unpermeabilized or permeabilized sperm were doubly immunostained with anti-E-cadherin (ECCD-2) and anti-β-catenin mAbs, and their nuclei were stained with DAPI. ECCD-2, which recognizes an epitope in the N-terminal extracellular region of E-cadherin, bound to E-cadherin without permeabilization pretreatment. Scale bar: 5 µm. (d) The fluorescence intensity profiles of E-cadherin and β-catenin in sperm shown in (c). Fluorescence intensities were measured after being traced on the sperm along dotted lines shown at the bottom of the panels in (c).
	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.
	Figure 4. Reduced levels of β-catenin localized beneath the cell membrane of both oocytes and sperm after membrane adhesion.(a) β-catenin disassembly induced by membrane adhesion in oocytes and sperm. In the ‘Non-adhered' group (upper panels), ZP-denuded C57BL/6N oocytes were stained with DAPI and subsequently reacted with anti-β-catenin mAb. Also, epididymal sperm were stained with DAPI and anti-β-catenin mAb. In the ‘Adhered' group (lower panels), ZP-denuded C57BL/6N oocytes were stained with DAPI and then subjected to IVF for 30 min prior to incubation with anti-β-catenin mAb. Arrows indicate areas where fluorescent intensities of β-catenin are reduced. In each panel, boxes in middle sets of panels were enlarged and shown on the right. Scale bars: 20 and 10 µm in left and middle panels, respectively. (b) Fluorescence intensities of oocytes before and after sperm-oocyte adhesion. Fluorescence intensities were measured after being traced along dotted lines in the left panels of (a). Arrows indicate both sides of the oocyte cell membranes. (c) Fluorescence intensities of sperm before and after sperm-oocyte adhesion. Fluorescence intensities were measured after being traced along dotted lines in the right panels of a. Red and blue lines indicate fluorescent intensities for β-catenin and DAPI, respectively. (d) β-Catenin disassembly induced by membrane adhesion in oocytes and sperm. In the ‘Non-adhered' group (upper panels), ZP-denuded CD9-/- oocytes were stained with DAPI and subsequently reacted with anti-β-catenin mAb. In the ‘Adhered' group (lower panels), ZP-denuded CD9-/- oocytes were preloaded with DAPI as depicted in Fig. 3c and then subjected to IVF for 30 min prior to incubation with anti-β-catenin mAb. Arrows indicate areas where fluorescent intensities of β-catenin are reduced. In each panel, Scale bars: 20 µm. (e) Fluorescence intensities of CD9-/- oocytes before and after sperm-oocyte adhesion. Fluorescence intensities were measured after being traced along dotted lines in the left panels of d. Arrows indicate both sides of the oocyte cell membranes.
	Figure 5. Reduced fusing ability of oocytes treated with UBE1-41, an inhibitor of ubiquitination.(a) Experimental flow. Cumulus cells attached to the oocytes collected from oviducts were removed. The oocytes were then treated with UBE1-41 for 30 min, followed by ZP removal. After 20-min incubation with DAPI-containing medium and subsequent washing, ‘zona-free' oocytes were mixed with the sperm and incubated for 30 min. Only oocytes having at least one fused sperm were counted as fused oocytes. (b) Sustained expression of β-catenin on the surface of “zona-free” wild-type oocytes treated with UBE1-41 after membrane adhesion with sperm. Arrows indicate oocyte cell membranes where β-catenin deposition is noted. One sperm fused with the untreated oocyte (upper panel), but not with the UBE1-41-treated oocyte (lower panel). To focus on the β-catenin expression, only fluorescent images are shown. Single slice, Image captured when the diameter of an oocyte was longest; 3D, 3-dimensional image reconstructed from serial scanned images. Bars: 20 µm. (c) Decreased number of fused sperm on “zona-free” wild-type oocytes after treatment with UBE1-41. The relative rate of oocytes carrying fused sperm was compared between UBE1-41-treated and -untreated wild-type oocytes. The comparative values relative to the control (set to 100.0) are displayed as the relative rate of fused oocytes. As described in the legend of Fig. 3c, oocytes fused with sperm are defined as those with at least one DAPI-positive sperm. (d) Comparison of the relative rate of oocytes carrying fused sperm between UBE1-41-treated and untreated oocytes (β-catenin-deficient (f/fcre) vs. β-catenin-intact (f/f) oocytes). Comparison was made as described in (c). NS, not significant. Parentheses indicate the total number of oocytes examined in triplicate experiments. Values are the mean ± SE. (e) A model of the possible role of β-catenin in transition from membrane adhesion to fusion. At fertilization (as described in (1)), adhesion of sperm to the surface of an oocyte is mediated by E-cadherin/β-catenin complex; however, subsequent fusion requires rapid degradation of β-catenin. On the other hand, in the presence of UBE1-41 (as described in (2)), adhesion occurs normally, but fusion is impaired since UBE1-41 inhibits degradation of β-catenin.

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