Establishment of an In Vitro Assay for Analysis of the Mechanism Underlying Chorion Elevation in Zebrafish

Chorions, also known as egg membranes, form quickly after fertilization and play a role in subsequent embryonic development in fish. While the enzymatic hardening of the chorion due to the release of cortical alveoli (CA) components by the exocytosis of CA has been well-demonstrated, the initiation mechanism of this process has remained unresolved. Knockout lines with the prss59.1 trypsin paralog gene exhibited abnormalities in chorion elevation. Prss59.1 has been shown to be expressed on the chorion's surface. Therefore, we hypothesized that a trypsin-like enzyme expressed on the chorion could trigger chorion elevation. In this study, we attempted to improve the effectiveness of Hank's solution at preventing chorion elevation. By adjusting the concentration of the solution's contents, we developed a modified Hank's solution that can stop chorion elevation almost completely. Using this solution, we demonstrated that trypsin can induce chorion elevation. These results support our hypothesis that a trypsin-like enzyme initiates chorion elevation. This assay method can be used for the further analysis of the chorion elevation mechanism.