RESUMO
Chorions isolated from unfertilized eggs of medaka, Oryzias latipes, harden during incubation with Ca2+ ions (Masuda et al., 1991). In this process, i.e. in vitro Ca2+ -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 Ca2+ -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 Ca2+ -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 Ca2+ -hardening was generated by some factor(s) other than ovoperoxidase and transglutaminase.
RESUMO
In addition to the spawning female-specific (SF) substance (3, 6), a group of new higher molecular weight proteins cross-reacting with anti-egg envelope (chorion) glycoprotein (F1) antibody (original antibody) were found in the liver, blood plasma and the ovary of spawning female fish and in the ascites of the estrogenized fish of Oryzias latipes. Exploiting the antibodies specific for the SF substance and the new proteins, which were made of the original antibody by absorbing with the new proteins or the SF substance, the new proteins were found to behave very similarly to the SF substance concerning their localization in the inner layer of the oocyte envelope, intrahepatic formation in response to estrogen etc. They include the protein bands corresponding to Zl-1 and -2, two major constituent glycoproteins of the oocyte envelope, while the SF substance corresponds to ZI-3, the third major constituent of the envelope. Thus the three major constituent proteins of the inner layer of oocyte envelope are probably formed in the liver under the influence of estrogen in this fish.
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.
RESUMO
Changes in yolk phosphoproteins of Oryzias latipes embryos during early development were examined by electrophoresis. Four phosphoproteins were identified in the yolk of blastulae by polyacrylamide gel electrophoresis. Two of them were high molecular weight phosphoproteins containing 0.7% (w/w) phosphorus and with similar amino acid compositions to that of vitellogenin. The other two were low molecular weight phosphoproteins, characterized by high contents of phosphorus [12.2% (w/w)] and serine (44.8 mole%) and low contents of aromatic amino acid residues. From these characteristics, together with their behaviors on DEAE-cellulose chromatography and electrophoresis and low stainability with dyes, the latter two were concluded to be phosvitins. These phosvitins were isolated and partially characterized. The yolk phosphoproteins, especially the phosvitins, were degraded, their amounts in embryos decreased throughout early development. Studies on the mechanism of endogenous phosphoprotein degradation strongly suggested the participation of some protease(s) that was precipitated on centrifugation of the egg homogenate at 14,000 × g for 10 min.
RESUMO
Mode of action of two stimulants of the hatching enzyme secretion, electric current (AC) and potassium cyanide, was analyzed by applying them to Medaka embryos in the presence or absence of suppressants of nervous system-mediated secretion, tetrodotoxin or MS-222. Electric current (AC) stimulated the secretion of the hatching gland of the embryos that had been treated with these suppressants, while potassium cyanide did not. These results strongly suggest that electric current acts as a stimulant of hatching enzyme secretion directly on the gland cell itself, while potassium cyanide stimulates the secretion indirectly, probably through nervous system of the embryo. In the present experiments, it was also shown that Ca2+ and ionophore, X-537A, when applied directly to the hatching gland extracellularly, induced a marked secretion-associated morphological change of the gland cells instantaneously. However, it was found that chum salmon prolactin did not induce the secretion-associated morphological changes in the hatching gland cells when it was applied directly to the gland cells in situ or indirectly through embryonic circulation.
