Investigation of Zona Pellucida Hardening with Atomic Force Microscopy and Nonlinear Optimization

The Zona Pellucida (ZP) is an extracellular membrane surrounding mammalian oocytes. The so called “zona hardening” has a key role in the fertilization process as it produces a block of polyspermy also through an increase of the stiffness of the membrane. In this study, mechanical properties of the ZP membrane extracted from mature and fertilized bovine oocytes were investigated with Atomic Force Microscopy (AFM) nanoindentation measurements. Both inner and outer sides of the ZP isolated from the fertilized oocyte were characterized to gain a deeper comprehension of the mechanisms involved in the structural re-organization of the ZP membrane leading to mechanical hardening. The full understanding of the “zona-hardening” mechanisms as well as a correct estimation of ZP elastic properties is of fundamental importance as alterations of ZP represent a potential cause of infertility. This work proposes the application of a hybrid procedure combining experimental measurements, Finite Element analysis and nonlinear optimization to analyze the indentation curves. Unlike the classical Hertz theory traditionally applied to analyze AFM data, the novel methodology presented here provides a reliable derivation of the elastic properties of the ZP as it accounts for hyperelastic mechanical behavior of the membrane and nonlinearity of the finite indentation process.