Mechanical analysis of chorion softening in prehatching stages of zebrafish embryos

Abstract—During early development, the chorion envelope of the zebrafish embryo undergoes a thinning process called “chorion softening, ” which has so far only been characterized chemically. In this study, a micromechanical force sensing system was used to characterize and quantitate mechanical modifications of the zebrafish embryo chorion during early development. Quantita-tive relationships between applied forces and chorion structural deformations were established at various embryonic stages. The measured penetration force into the chorion at the blastula stage was 1.3-fold greater than those at the prehatching stage. Fur-thermore, chorion elastic modulus values were determined by using a biomembrane elastic model. The elastic modulus of the chorion at the blastula stage was 1.66-fold greater than that at the prehatching stage, thus indicating that the chorion envelope become mechanically “softened ” at the prehatching stage. The experimental results quantitatively describe “chorion softening,” which is most likely due to proteolytic activities at the prehatching stage. Gradual chorion softening during embryonic development was also artificially achieved by treating blastula chorion with pronase, a proteolytic enzyme. The forces required to penetrate the pronase-treated chorion were similar to those at the prehatching stage. This similarity suggests that “chorion softening ” may be induced by the release of protease from the embryos, and the chemical nature of the process involves proteolytic fragmentation of the ZP2 protein. Index Terms—Cellular force sensing, chorion softening, elastic modulus, hatching, peptide fragmentation, protease, proteolytic activities, zebrafish embryo. I