Figure 5
Extensive cross-linking of MSGb5Cer with 6E2 inhibits cytokinesis and development of mouse preimplantation embryos. (A) Deposition of BODIPY®FL-MSGb5Cer at the interfaces of 2-cell stage embryos cultured in the presence of 6E2. Embryos labeled with BODIPY®FL-MSGb5Cer (green) were cultured for 1 hour in M16 medium containing 100 μg/ml of 6E2 or 15B2 antibodies. A fluorescence 2D image of BODIPY®FL-MSGb5Cer and the overlay of a 2D image and DIC micrograph of cultured embryos are shown. These images shown are representative of twelve 2-cell embryos. (B) Effect of cross-linking of MSGb5Cer with 6E2 on cytokinesis and the development of mouse preimplantation embryos. Two-cell embryos were cultured for 60 hours in M16 medium containing 100 μg/ml of 15B2 (red squares), ECCD-2 (blue diamonds), and 6E2 (green triangles), and the living embryos were counted and their DIC images were taken at the times indicated. Arrows indicate compacted embryos. Twenty of 2-cell stage embryos were cultured for each experiment groups. (C) Localization of F-actin and E-cadherin in MSGb5Cer-cross-linked 2-cell embryos. Two-cell embryos treated for 2 hours with 6E2 (left column) or 15B2 (right column) were fixed and stained with Alexa Fluor® 633 phalloidin and anti E-cadherin/Alexa Fluor® 546-conjugated rabbit anti-rat IgG. These images shown are representative of twelve 2-cell embryos. (D) Inhibitory effect of cross-linking of MSGb5Cer with 6E2 on the localization of E-cadherin at interfaces. Embryos were immunostained with ECCD-2/Alexa Fluor® 546-conjugated Rabbit Anti-rat IgG (red) in the presence of various concentrations of 6E2. The upper row and the bottom row of each panel represent a 2D image and the overlay of a 2D image and DIC micrograph, respectively. These images shown are representative of thirty morula embryos.