The zona pellucida porosity: three-dimensional reconstruction of four types of mouse oocyte zona pellucida using a dual beam microscope.

In the last decade, the applicability of focus ion beam-field emission scanning electron microscopy (FIB-FESEM) in the biological field has begun to get relevance. Among the possibilities offered by FIB-FESEM, high-resolution three-dimensional (3D) reconstruction of biological structures is one of the most interesting. Using this tool, the 3D porosity of four different types of mouse oocyte zona pellucida (ZP) was analyzed. A surface analysis of the mouse oocyte ZP was first performed by SEM. Next, one oocyte per ZP type was selected, and an area of its ZP was completely milled, using the cut and view mode, in the FIB-FESEM. Through a 3D reconstruction of the milled area, a map of the distribution of the pores across the ZP was established and the number and volume of pores were quantified, thus enabling for the first time the study of the inner porosity of the mouse ZP. Differences in ZP porosity observed among the four types analyzed allowed us to outline a model to explain the changes that the ZP undergoes through immature, mature, predegenerative, and degenerative stages.