The zona pellucida (ZP) is an extracellular membrane surrounding mammalian oocytes. The so-called zona hardening plays a key role in fertilization process, as it blocks polyspermy, which may also be caused by an increase in the mechanical stiffness of the ZP membrane. However, structural reorganization mechanisms leading to ZP's biomechanical hardening are not fully understood yet. Furthermore, a correct estimate of the elastic properties of the ZP is still lacking. Therefore, the aim of the present study was to investigate the biomechanical behaviour of ZP membranes extracted from mature and fertilized bovine oocytes to better understand the mechanisms involved in the structural reorganization of the ZP that may lead to the biomechanical hardening of the ZP. For that purpose, a hybrid procedure is developed by combining atomic force microscopy nanoindentation measurements, nonlinear finite element analysis and nonlinear optimization. The proposed approach allows us to determine the biomechanical properties of the ZP more realistically than the classical analysis based on Hertz's contact theory, as it accounts for the nonlinearity of finite indentation process, hyperelastic behaviour and material heterogeneity. Experimental results show the presence of significant biomechanical hardening induced by the fertilization process. By comparing various hyperelastic constitutive models, it is found that the Arruda-Boyce eight-chain model best describes the biomechanical response of the ZP. Fertilization leads to an increase in the degree of heterogeneity of membrane elastic properties. The Young modulus changes sharply within a superficial layer whose thickness is related to the characteristic distance between cross-links in the ZP filamentous network. These findings support the hypothesis that biomechanical hardening of bovine ZP is caused by an increase in the number of inter-filaments cross-links whose density should be higher in the ZP inner side.
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