Thrombin induces assembly in vitro of vitelline envelope components of common carp eggs

Assembly in vitro of vitelline envelope (VE) components of VE extracts from fish eggs was induced by thrombin. The VE components and VEs themselves contained a chorionic transglutaminase, which was visualized on slab gels and in unfixed and frozen sections stained with monodansylcadaverine. Adding thrombin to the reaction system containing the sample followed by vortex mixing resulted in formation of fibrous material in vitro. Electron microscopy revealed that this fibrous material consisted of aggregates of network-like sheets or sheets of filamentous substances of various thicknesses arranged in parallel with several filamentous interconnections and the filamentous substances were sometimes decorated with variable numbers of oval particles (24-40 x 82-130 nm). The main scaffold of individual sheets was organized by branching and anastomosing of thin (ca. 25-40 nm) and thick (ca. 140-350 nm) filamentous substances. The networks varied in size and consisted of a varying numbers of fine networks. VE component assembly in vitro was inhibited by the presence of p-chloromercuribenzoate, iodoacetamide, L-cysteine, diisopropylfluorophosphate and hirudin-pretreated thrombin and did not occur in the absence of thrombin. Thus, VE component assembly in vitro appears at least to some extent to reproduce the phenomena that occur during the process of fertilization envelope (FE) formation. Moreover, these results suggest a new approach to investigating the FE assembly process in vitro. (C) 1998 Wiley-Liss,Inc.