Inhomogeneous Morphology and Elasticity of Mouse Oocyte Zona Pellucida Pre- And Post-Fertilization

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The mature mammalian oocyte is encapsulated within a thick membrane, the zona pellucida (ZP), which is composed of a small number of glycoproteins. The proteins are secreted by the oocyte during maturation. Sperm has to penetrate the ZP first before it reaches the oocyte. The ZP controls for species-specific fertilization and acts as the barrier to polyspermy, i.e. it prevents a multiplicity of sperms from reaching the oocyte. All ZP proteins contain a characteristic ZP domain but, otherwise, their 3-D structure is unknown as is the architecture of the zona membrane. Upon fertilization, one of the proteins (ZP2) is cleaved near its N-terminal. The cleavage is believed to alter the ZP structure which, henceforth, acts as a barrier to further sperm penetration.We used the atomic force microscope (AFM) to examine both the ZP structure and mechanical properties of the wildtype mouse ZP under physiological conditions. For that purpose, patches of isolated mouse ZP were immobilized on polylysine coated mica. Imaging revealed two predominant membrane surface morphologies consistent with previously reported electron microscopy images of the outer and inner membrane surfaces, respectively, from different species. One is a rough, ruffled surface and the other is a smoother surface with the appearance of a tighter construction. In addition to the surfaces, the structure across the wall thickness was visualized at high resolution revealing a layered, well organized architecture. Elastic modulus estimates from force-indentation data also showed systematic variability mirroring the morphological inhomogeneity. For example, significant differences in elasticity were measured between the rough regions, hypothesized to be outer surfaces, and the smoother regions. Interestingly, these properties also appear to undergo appreciable changes upon fertilization pointing to the structural change effected by the ZP2 cleavage.