Uniformity of the porosity of the zona pellucida of unfertilized mouse eggs. (A) Unfertilized mouse eggs surrounded by the zona pellucida were soaked in KSOM medium containing 70 kD TRITC-dextran and imaged using confocal fluorescence microscopy. Scale bar, 50 µm. (B) An intensity scan was performed along the circumference of the zona pellucida (dotted line) to examine the uniformity of the structure. The 70-kD dextran has a hydrodynamic radius of ∼6.5 nm (Armstrong et al., 2004) and thus could penetrate through the zona pellucida, filling the space within its porous structure. The fluorescence signal at the zona pellucida was higher than that within the egg cell, whose membrane is not permeable to dextran, and it was lower than in the surrounding buffer due to the porosity. Colored plots in B indicate data from different samples (n = 5). The angle 0° is defined as the point of the protruding cortex of the egg. The relative fluorescence intensity, as normalized based on the mean intensity of each circular scan, varied by 8.6 ± 0.7% (n = 5) along the circumference of the zona pellucida, but the intensity minima and maxima did not appear at any specific location.