Analysis of Hardening of the Egg Envelope (Chorion) of the Fish, Oryzias latipes

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Abstract
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.