Figure 5: JUNO disappears from the contact site of not only cell–cell but also sperm–egg adhesion.

(a,b) The reconstitution approach in COS-7 cells. COS-7 cells transfected with a mammalian expression vector inserted with Juno and Izumo1 cDNA were mixed and incubated for 24 h before observation using confocal microscopy. Lower magnification images are shown in the left panels. The region of the contact site is shown by a dashed box. (a) COS-7 cells were stained with α-JUNO-Alexa647 (red), Mab18-Alexa488 (green) and Mab125-Alexa546 (magenta). (b) BiFC analysis (green) was performed with α-JUNO-Alexa647 (red) and Mab125-Alexa546 (magenta). Magnification of the orange or blue dashed box is in the right panels. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (c,d) BiFC in Izumo1-VNC155-expressing cells. COS-7 cells were transiently co-transfected with Izumo1-VN155 and VC155. After 48-h incubation, they were stained with Mab125-Alexa647 (magenta) and JUNO-FC (c, red) or Mab18-Alexa546 (d, red). After Z-sectioning and 3D reconstitution, magnified images (right panel) were taken from the direction of the arrow. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification. (e,f) The characteristics of each antibody before and after sperm–egg fusion. Zona-free oocytes and spermatozoa were co-incubated with Mab18-Alexa488 (green), Mab125-Alexa546 (magenta) and JUNO (red) antibodies (e,f). Sperm–egg fusion was visualized with Hoechst transfer (blue). Images of before (e) and after (f) fusion were taken after fixation. Highly magnified photographs correspond to the dashed box in the left pictures. Scale bar, 20 μm in lower magnification, 10 μm in higher magnification.