RESUMO
Hardening of the chorion of medaka eggs was quantitated in terms of the solubility of its constituent proteins. After activation of unfertilized eggs with the Ca2+ -ionophore A23187, hardening of chorion (named ionophore-activation hardening) proceeded and 60 min after activation the solubility of the proteins in 1 N NaOH had decreased to 20% of that of proteins of unhardened chorions. On SDS-PAGE, the chorions of unfertilized eggs gave four major protein bands (150, 83, 78 and 51 K). After Ca2+ -ionophore activation, new two protein bands (135 and 61 K) appeared, with concurrent disappearance of all the original bands except the 51 K band. Isolated chorions of unfertilized eggs were also hardened by Ca2+ and 60 min after addition of Ca2+ the solubility of their proteins in 1 N NaOH had decreased to about 45% of that originally. During this type of hardening (named 'Ca2+ -hardening), however, the SDS-PAGE pattern of the proteins remained unchanged. Therefore, there are two mechanisms of hardening. The 'ionophore-activation hardening was inhibited by cadaverine. When chorions were isolated 20 min after Ca2+ -ionophore activation and kept in Ca2+ -free conditions, the 'ionophore-activation hardening process was arrested: further hardening was resumed on addition of Ca2+ to the medium. These results suggest the presence of some hardening machinery in isolated chorions.
RESUMO
Egg envelope (chorion) of unfertilized eggs of rainbow trout, Oncorhynchus mykiss, consists of 4 major protein components whose molecular weights are approximately 110 K, 64 K, 56 K and 50 K. On hardening of the chorion after activation, 64 K, 56 K and 50 K components disappeared, some components higher than 160 K were newly formed and the solubility of the chorion in 1 N NaOH or 12 M urea-2% SDS-1% 2-mercaptoethanol (Sample Buffer) decreased. This implies that probable formation of covalent crosslinks between the constituents occurs during the hardening. Hardening occurred also in the chorion isolated from the unfertilized eggs. The conversion of the 64 K, 56 K and 50 K components into the higher molecular weight intermediates was Ca2+ -dependent, accelerated by 2-mercaptoethanol and inhibited by a high temperature (60°C). Optimum pH of this hardening system was acidic, i.e., 5.0 to 6.0. Cadaverine inhibited the change in solubility of chorion in NaOH or Sample Buffer, while it could not inhibit the disappearance of the 64 K, 56 K and 50 K components and the new formation of the higher molecular weight intermediates. This observation indicates existence of two processes in chorion hardening, which are tentatively named cadaverine-insensitive and cadaverine-sensitive processes.
