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

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 Ca*+-ionophore A231 87, 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 Ca'+-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.

Identification and cDNA Cloning of Alveolin, an Extracellular Metalloproteinase, Which Induces Chorion Hardening of Medaka (Oryzias latipes) Eggs upon Fertilization

“…Therefore, the progress of chorion hardening is easily analyzed as decreased proteinaceous bands by SDS-PAGE 1 (13,14). Three isolated chorions were * This work was supported in part by Grant-in-aid for Research for the Future (JSPS-RFTF 96L00401) (to Y. N.) and for Scientific Research priority areas 07283104 (to Y. N.) and 09640798 (to M.…”

“…In medaka (12)(13)(14), cod (15), and rainbow trout (16 -18), the chorion of unfertilized eggs is composed of a few major glycoproteins. In medaka, ZI-1,2 and ZI-3 were shown to be the major chorion proteins.…”

“…In medaka, ZI-1,2 and ZI-3 were shown to be the major chorion proteins. After limited hydrolysis to produce 61-and 62-kDa proteins early in the process of chorion hardening, ZI-1,2 polymerizes with ZI-3 to form insoluble higher molecular weight complexes (13,14). Chorion hardening is accomplished by limited hydrolysis and polymerization of chorion proteins.…”

“…Chorion hardening is accomplished by limited hydrolysis and polymerization of chorion proteins. In rainbow trout (19,20), cod (15), and medaka (13,21), it is reported that the polymerization is catalyzed by transglutaminase via ⑀-(␥-glutamyl)lysine isopeptide bonds between chorion proteins. The transglutaminase is located within the unfertilized egg chorion (22,23).…”

“…The transglutaminase is located within the unfertilized egg chorion (22,23). Increases in mechanical strength and decreases in solubility are due to the polymerization of chorion proteins (13)(14)(15). However, the molecular mechanism initiating of chorion hardening remains largely unknown.…”