Nanoscale characterization of the biomechanical hardening of bovine zona pellucida

Abstract

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.

A hybrid characterization framework to determine the visco-hyperelastic properties of a porcine zona pellucida

“…This ability represents a significant step further with respect to the current literature and may be a good start basis for future studies on the sperm-ZP interactions to assess the complex relationships between sperm motility, mechanical behaviour of ZP and fertility impairments. The next step of the research will be to analyse the visco-hyperelastic response of the ZP at different stages of the fertilization process to confirm the relationships between mechanical hardening and architecture of polymeric network [16]. Furthermore, the effect of the indenter shape on the visco-hyperelastic response will be studied in order to understand the relationship between sperm head shape and penetration [39].…”

“…(0) other mechanical characterization studies of nonlinear materials at the micro-and nanoscale [26][27][28]16]. The identification algorithm, coded in the Matlab (The Mathworks Inc., Austin, TX, USA) software environment, minimizes the difference between nanoindentation data and FE analyses via nonlinear optimization.…”

“…The bounds of the shear modulus m 8chain were chosen so as to cover the range of variation of the apparent Young's modulus of porcine oocytes reported in Papi et al [20] for the same indentation rate range (by scaling the Poisson ratio from the value of 0.33 used in Papi et al [20] to the value of 0.5 used in this study, the apparent Young's modulus would range from 25 to 135 kPa; because for the Arruda -Boyce model it holds E ¼ 3m 8chain , the shear modulus would be between 8.3 and 45 kPa), and to account for the fact that values of elastic moduli determined under the assumption of hyperelastic behaviour may be up to one or two orders of magnitude smaller than those determined with the classical Hertzian model, which assumes instead linear elasticity, 'infinite space', perfect spherical indenter and small contact area (e.g. [16,[29][30][31]). In general, material property bounds were set large enough to increase design freedom and rapidly converge to global optimum.…”

“…AFM was used to evaluate mechanical properties of immature, mature and fertilized bovine oocyte ZPs [13,14], also trying to assess the effect of ZP structure inhomogeneity across membrane thickness [15,16], and the role played by residual stresses that develop in the tested ZP membrane (which is originally spherical shaped) when specimens are spread out on a rigid substrate for nanoindentation measurements [17].…”

“…In a previous study conducted by some of the present authors [16], a hybrid mechanical characterization procedure combining AFM nanoindentation measurements, nonlinear finite-element (FE) analysis and nonlinear optimization was developed to determine the ZP hyperelastic parameters of immature, mature and fertilized bovine oocytes. This approach was successfully used to characterize both inner and outer sides of ZP specimens extracted from the fertilized oocyte.…”