Figure 4: JUNO antibody fails to stain the contact site of an egg and Izumo1-expressing cells.

(a) Distribution of JUNO on oocyte in cell–oocyte assay. Localization of JUNO (TH6, red), CD9 (MZ3, green) and IZUMO1 (Mab125, magenta) in the cell–oocyte assay was observed by each specific antibody. Final concentrations of antibodies were 0.05, 0.5 and 0.5 μg ml⁻¹, respectively. Two hours after incubation, images were captured. (b) The state of dimer IZUMO1 and JUNO on the egg. In combination with the BiFC analysis, cell–oocyte assays were performed. The asterisks and the arrow indicate adhered cells and the early stage of binding, respectively. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. (c) The scanning electron microscope image of cell–oocyte. Cell–oocyte assays were observed with a scanning electron microscope (left; scale bar, 20 μm). A magnified image of the region surrounded by a black square is shown on the right (scale bar, 2 μm). (d,e) cfSGFP2-JUNO is excluded from the contact site of the egg and Izumo1-expressing cells. Experimental diagram of germinal vesicle (GV) stage oocyte injection. Western blot analysis of the lysate of 15 eggs expressing cfSGFP2-JUNO with α-JUNO (12A5) and α-GFP. Arrow and arrowhead indicate cfSGFP2-JUNO and endogenous JUNO, respectively (d). After mRNA microinjection, GV-stage oocytes were incubated in 10% fetal bovine serum/TYH medium for 21 h at 37 °C up to maturation at metaphase II. Then, they were subjected to cell–oocyte assay with Mab125-Alexa546 (magenta), α-GFP polyclonal antibody labelled with α-rabbit IgG antibodies-Alexa405 (blue) and α-JUNO-Alexa647 (red) (e). The images were simultaneously taken with autofluorescence (cfSGFP2-JUNO, green). Scale bar, 20 μm. (f–h) The effects of IASD in a cell–oocyte assay. This membrane-impermeable thiol-reactive reagent drastically decreased the number of attached COS-7 cells expressing Izumo1. Cell–oocyte assays were performed with 10 mM IASD. Bright field images were taken 2 h after incubation. Scale bar, 100 μm (f), and attached cells were counted (excluding aggregated cells), values are presented as mean±s.e.m., *P<0.0001 (Student’s t-test; g). The oocytes were stained with α-JUNO-Alexa647 (red), Mab18-Alexa488 (green) and Mab125-Alexa546 (magenta) for 30 min after treatment with IASD. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm (h).