α-SNAP, γ-SNAP, and NSF localization during meiotic maturation and MII oocytes activation.

α-SNAP (left panel), γ-SNAP (middle panel) and NSF (right panel) were immunodetected at different stages: GV-intact oocytes (GV), MII oocytes(MII), parthenogenetic activated MII oocytes with 10mM strontium chloride during 1 or 7 h post activation (SrCl₂ (1h) and (7h), respectively), and two pronucleus (2PN) embryos after in vitro fertilization. Green, positive staining for primary α-/β-SNAP, γ-SNAP or NSF antibody detected by secondary antibodies conjugated to DyLight 488; red, cortical granules stained with LCA-Rhodamine; blue, DNA labeled with Hoechst 3342.A-C, panels show the fluorescence intensity profiles for α-SNAP, γ-SNAP and NSF (green) and cortical granules stained with LCA-Rhodamine (red). Fluorescence intensities were measured along dashed lines traced in each panel. The intensities of α-SNAP, γ-SNAP and NSF are indicated by green lines, and the intensity of cortical granules is indicated by red lines. Scale bar: 20 μm. D-F, cortical and cytoplasmic patterns of protein distribution. Cortical region was defined as the region of 10 μm thickness from the oocyte plasma membrane towards the oocyte centre. Those cells in which fluorescence decay at 10 μm were considered to present a cortical staining, and those cells which present high fluorescence at 10um towards the center and beyond were considered to present cytoplasmic staining. Percentage analysis was assessed. Number of analyzed cells for α-SNAP, γ-SNAP and NSF were, respectively: GV = 85,51,32; MII = 138,37,35; MII SrCl₂ 1h = 101, 36, 30; MII SrCl₂ 7h = 35, 27, 33; IVF = 13, 6, 9.