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

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.