Some Properties of the Hardening Process in Chorions Isolated from Unfertilized Eggs of Medaka, Oryzias latipes: (Fish Egg Envelope/Chorion/Chorion Proteins/Chorion Hardening/In Vitro Ca -Hardening).

Chorions isolated from unfertilized eggs of medaka, Oryzias latipes, harden during incubation with Ca ions (Masuda et al., 1991). In this process, i.e. in vitro Ca -hardening, the amounts of the major proteins of unfertilized egg chorions (83 K, 78 K and 51 K, corresponding to ZI-1, 2 and 3 of oocyte chorions reported by Hamazaki et al, 1987) decreased and new proteins having molecular weights of 148 K or more appeared. Immunoblotting analysis using anti-ZI-1, 2 antisera and anti-ZI-3 antisera showed that the 148 K protein was an intermediate formed during polymerization of the original proteins. The mechanism of in vitro Ca -hardening was studied by examining the decrease in ZI-1, 2, and 3, the formation of 148 K protein, and the change in solubility of chorions in 6% sodium dodecylsulfate-1% 2-mercaptoethanol-15% glycerol-0.2 M Tris-HCl (pH 6.8). In vitro Ca -hardening was inhibited at temperatures higher than 70°C and its optimum pH was about 5.5. It was inhibited by neither aminotriazole nor cadaverine. The results suggested that in vitro Ca -hardening was generated by some factor(s) other than ovoperoxidase and transglutaminase.