Functional characterization of IZUMO1 mutants. (A) Quantification of content mixing in BHK cells expressing vectors, WT IZUMO1, and its mutants (Ecto, ΔIg, W148A, and FWW; see Fig. 4). Bar chart showing means ± SEM. n = 1,000 nuclei per independent experiment. Comparisons by one-way ANOVA followed by Bonferroni’s test against the vector (black) and against WT (red). *P < 0.05, ***P < 0.001, ****P < 0.0001. (B and C) BHK-oocyte binding assay. BHK cells expressing IZUMO1 (WT and mutants). (B) Quantification of the binding of BHK cells to oocytes. Cells were transfected with pCI::GFPnes empty vector or encoding for IZUMO1, IZUMO1^W148A, or IZUMO1^FWW and incubated with WT oocytes. The number of BHK cells bound per oocyte was determined; n = total number of oocytes analyzed in parenthesis. Bar chart showing means ± SEM. Comparisons by one-way ANOVA followed by Dunnett’s test against the vector. *P < 0.05. (C) Representative images of oocytes from one experiment in B taken under a dissecting microscope (upper row) and a wide-field illumination microscope showing the merged differential interference contrast (DIC) and GFP channels (lower row). Scale bar, 20 µm.