Evolutionary pathway of pseudogenization of globin genes, α5 and ß5, in genus Oryzias.

Hemoglobin transports oxygen in many organisms and consists of α- and ß-globin chains. Previously, using molecular phylogenetic analysis, we proposed that both α- and ß-globins of teleost could be classified into four groups. We also showed that the Hd-rR strain of medaka (Oryzias latipes) inhabiting southern Japan had all four groups of globin genes but that the α- and ß-globin genes of group III were pseudogenized (α5(ψα), ß5(ψß)). Based on the small degree of nucleotide variations, the pseudogenization of ß5 was assumed to have occurred at a relatively late stage of evolution. Here, we compared the α5(ψα)-ß5(ψß) of two other strains of O. latipes and found that both α5(ψα) and ß5(ψß) of the northern Japanese and Korean strains were pseudogenized similar to those of Hd-rR. In a Philippine population (Oryzias luzonensis), α5(ψα) was also pseudogenized, but the structure was different from that of O. latipes, and ß5(ψß) was almost deleted. Interestingly, an Indonesian population (Oryzias celebensis) had α5 and ß5 genes that were deduced to be functional. Indeed, they were expressed from the young to adult development stages, and this expression pattern was consistent with the expression of α2 and ad.α1 in Hd-rR. Because α2 and ad.α1 in Hd-rR were assigned to groups I and II, respectively, we speculate that their expression patterns might be altered by pseudogenization of group III genes. These results provide a basis for further investigations of recruiting and changing expression patterns of one globin gene after pseudogenization of other globin genes during evolution.

Molecular co-evolution of a protease and its substrate elucidated by analysis of the activity of predicted ancestral hatching enzyme.

BACKGROUND: Hatching enzyme is a protease that digests the egg envelope, enabling hatching of the embryo. We have comprehensively studied the molecular mechanisms of the enzyme action to its substrate egg envelope, and determined the gene/protein structure and phylogenetic relationships. Because the hatching enzyme must have evolved while maintaining its ability to digest the egg envelope, the hatching enzyme-egg envelope protein pair is a good model for studying molecular co-evolution of a protease and its substrate. RESULTS: Hatching enzymes from medaka (Oryzias latipes) and killifish (Fundulus heteroclitus) showed species-specific egg envelope digestion. We found that by introducing four medaka-type residue amino acid substitutions into recombinant killifish hatching enzyme, the mutant killifish hatching enzyme could digest medaka egg envelope. Further, we studied the participation of the cleavage site of the substrate in the species-specificity of hatching enzyme. A P2-site single amino acid substitution was responsible for the species-specificity. Estimation of the activity of the predicted ancestral enzymes towards various types of cleavage sites along with prediction of the evolutionary timing of substitutions allowed prediction of a possible evolutionary pathway, as follows: ancestral hatching enzyme, which had relatively strict substrate specificity, developed broader specificity as a result of four amino acid substitutions in the active site cleft of the enzyme. Subsequently, a single substitution occurred within the cleavage site of the substrate, and the recent feature of species-specificity was established in the hatching enzyme-egg envelope system. CONCLUSIONS: The present study clearly provides an ideal model for protease-substrate co-evolution. The evolutionary process giving rise to species-specific egg envelope digestion of hatching enzyme was initiated by amino acid substitutions in the enzyme, resulting in altered substrate specificity, which later allowed an amino acid substitution in the substrate.

Adaptive evolution of fish hatching enzyme: one amino acid substitution results in differential salt dependency of the enzyme.

Embryos of medaka Oryzias latipes hatch in freshwater, while those of killifish Fundulus heteroclitus hatch in brackish water. Medaka and Fundulus possess two kinds of hatching enzymes, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), which cooperatively digest their egg envelope at the time of hatching. Optimal salinity of medaka HCE was found in 0 mol l(-1) NaCl, and activity decreased with increasing salt concentrations. One of the two Fundulus HCEs, FHCE1, showed the highest activity in 0 mol l(-1) NaCl, and the other, FHCE2, showed the highest activity in 0.125 mol l(-1) NaCl. The results suggest that the salt dependencies of HCEs are well adapted to each salinity at the time of hatching. Different from HCE, LCEs of both species maintained the activity sufficient for egg envelope digestion in various salinities. The difference in amino acid sequence between FHCE1 and FHCE2 was found at only a single site at position 36 (Gly/Arg), suggesting that this single substitution causes the different salt dependency between the two enzymes. Superimposition of FHCE1 and FHCE2 with the 3-D structure model of medaka HCE revealed that position 36 was located on the surface of HCE molecule, far from its active site cleft. The results suggest a hypothesis that position 36 influences salt-dependent activity of HCE, not with recognition of primary structure around the cleavage site, but with recognition of higher ordered structure of egg envelope protein.

Intron-loss evolution of hatching enzyme genes in Teleostei.

BACKGROUND: Hatching enzyme, belonging to the astacin metallo-protease family, digests egg envelope at embryo hatching. Orthologous genes of the enzyme are found in all vertebrate genomes. Recently, we found that exon-intron structures of the genes were conserved among tetrapods, while the genes of teleosts frequently lost their introns. Occurrence of such intron losses in teleostean hatching enzyme genes is an uncommon evolutionary event, as most eukaryotic genes are generally known to be interrupted by introns and the intron insertion sites are conserved from species to species. Here, we report on extensive studies of the exon-intron structures of teleostean hatching enzyme genes for insight into how and why introns were lost during evolution. RESULTS: We investigated the evolutionary pathway of intron-losses in hatching enzyme genes of 27 species of Teleostei. Hatching enzyme genes of basal teleosts are of only one type, which conserves the 9-exon-8-intron structure of an assumed ancestor. On the other hand, otocephalans and euteleosts possess two types of hatching enzyme genes, suggesting a gene duplication event in the common ancestor of otocephalans and euteleosts. The duplicated genes were classified into two clades, clades I and II, based on phylogenetic analysis. In otocephalans and euteleosts, clade I genes developed a phylogeny-specific structure, such as an 8-exon-7-intron, 5-exon-4-intron, 4-exon-3-intron or intron-less structure. In contrast to the clade I genes, the structures of clade II genes were relatively stable in their configuration, and were similar to that of the ancestral genes. Expression analyses revealed that hatching enzyme genes were high-expression genes, when compared to that of housekeeping genes. When expression levels were compared between clade I and II genes, clade I genes tends to be expressed more highly than clade II genes. CONCLUSIONS: Hatching enzyme genes evolved to lose their introns, and the intron-loss events occurred at the specific points of teleostean phylogeny. We propose that the high-expression hatching enzyme genes frequently lost their introns during the evolution of teleosts, while the low-expression genes maintained the exon-intron structure of the ancestral gene.

Different hatching strategies in embryos of two species, pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, that belong to the same order Clupeiformes, and their environmental adaptation.

Pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, which belong to the same order Clupeiformes, spawn different types of eggs: demersal adherent eggs and pelagic eggs, respectively. We cloned three cDNAs for Pacific herring hatching enzyme and five for Japanese anchovy. Each of them was divided into two groups (group A and B) by phylogenetic analysis. They were expressed specifically in hatching gland cells (HGCs), which differentiated from the pillow and migrated to the edge of the head in both species. HGCs of Japanese anchovy stopped migration at that place, whereas those of Pacific herring continued to migrate dorsally and distributed widely all over the head region. During evolution, the program for the HGC migration would be varied to adapt to different hatching timing. Analysis of the gene expression revealed that Pacific herring embryos synthesized a large amount of hatching enzyme when compared with Japanese anchovy. Chorion of Pacific herring embryo was about 7.5 times thicker than that of Japanese anchovy embryo. Thus, the difference in their gene expression levels between two species is correlated with the difference in the thickness of chorion. These results suggest that the hatching system of each fish adapted to its respective hatching environment. Finally, hatching enzyme genes were cloned from each genomic DNA. The exon-intron structure of group B genes basically conserved that of the ancestral gene, whereas group A genes lost one intron. Several gene-specific changes of the exon-intron structure owing to nucleotide insertion and/or duplication were found in Japanese anchovy genes.

Crystallization and preliminary X-ray analysis of ZHE1, a hatching enzyme from the zebrafish Danio rerio.

The hatching enzyme of the zebrafish, ZHE1 (29.3 kDa), is a zinc metalloprotease that catalyzes digestion of the egg envelope (chorion). ZHE1 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. Two diffraction data sets with resolution ranges 50.0-1.80 and 50.0-1.14 A were independently collected from two crystals and were merged to give a highly complete data set over the full resolution range 50.0-1.14 A. The space group was assigned as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 32.9, b = 62.5, c = 87.4 A. The crystal contained one ZHE1 molecule in the asymmetric unit.

Hatching enzyme of the ovoviviparous black rockfish Sebastes schlegelii- environmental adaptation of the hatching enzyme and evolutionary aspects of formation of the pseudogene.

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned; however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing.

Analysis of the exon-intron structures of fish, amphibian, bird and mammalian hatching enzyme genes, with special reference to the intron loss evolution of hatching enzyme genes in Teleostei.

Using gene cloning and in silico cloning, we analyzed the structures of hatching enzyme gene orthologs of vertebrates. Comparison led to a hypothesis that hatching enzyme genes of Japanese eel conserve an ancestral structure of the genes of fishes, amphibians, birds and mammals. However, the exon-intron structure of the genes was different from species to species in Teleostei: Japanese eel hatching enzyme genes were 9-exon-8-intron genes, and zebrafish genes were 5-exon-4-intron genes. In the present study, we further analyzed the gene structures of fishes belonging to Acanthopterygii. In the species of Teleostei we examined, diversification of hatching enzyme gene into two paralogous genes for HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) was found only in the acanthopterygian fishes such as medaka Oryzias latipes, Fundulus heteroclitus, Takifugu rubripes and Tetraodon nigroviridis. In addition, the HCE gene had no intron, while the LCE gene consisted of 8 exons and 7 introns. Phylogenetic analysis revealed that HCE and LCE genes were paralogous to each other, and diverged during the evolutionary lineage to Acanthopterygii. Analysis of gene synteny and cluster structure showed that the syntenic genes around the HCE and LCE genes were highly conserved between medaka and Teraodon, but such synteny was not found around the zebrafish hatching enzyme genes. We hypothesize that the zebrafish hatching enzyme genes were translocated from chromosome to chromosome, and lost some of their introns during evolution.

Cloning and expression of xP1-L, a new marker gene for larval surface mucous cells of tadpole stomach in Xenopus laevis.

The amphibian gastrointestinal tract is remodeled from a larval-type to an adult-type during metamorphosis. In the present study, we examined the products of subtractive hybridization between tadpole and frog stomach cDNAs of Xenopus laevis in order to identify genes expressed specifically in the larval stomach epithelium. A new gene homologous to xP1 was obtained and named xP1-L. In the genome database of Silurana tropicalis, we found a homologue of xP1-L and named it stP1-L. RT-PCR showed that the expression of xP1-L was detected in stage 41/42 tadpoles. In addition, in situ hybridization showed that xP1-L was localized to surface mucous cells of the larval stomach. The H(+)/K(+)-ATPase beta subunit, a marker gene for manicotto gland cells in the tadpole stomach, was also detected at the same time. However, adult marker genes such as xP1 for surface mucous cells and pepsinogen C (PgC) for oxynticopeptic cells were not expressed in the tadpole stages. The expression of xP1-L gradually decreased towards the metamorphic climax and disappeared after stage 61 when larval-type gastric epithelium is replaced by adult-type. We found that xP1-L was never expressed in surface mucous cells of the adult-type stomach, and xP1, instead of xP1-L, was expressed. During T3-induced metamorphosis, xP1-L expression decreased in the same manner as during natural metamorphosis. Thus, xP1-L is a useful marker for larval surface mucous cells in tadpole stomach. This is the first demonstration of a marker gene specific for the surface mucous cells of the larval stomach.

Globin gene enhancer activity of a DNase-I hypersensitive site-40 homolog in medaka, Oryzias latipes.

In medaka, we found a C16orf35-like gene in the region within 1 Kbp 3' downstream of the psibeta end of the 36-Kbp embryonic globin gene cluster ((5')alpha0(3')-(3')beta1(5')-(5')alpha1(3')-(5')beta2(3')-(5')alpha2(3')-(3')alpha3(5')-(5')beta3(3')-(3')beta4(5')-(5')alpha4(3')-(3')psialpha(5')-(5')psibeta(3')). Intron 5 of the gene contained a region having NF-E2 binding sites located between GATA boxes. The region was homologous to human HS-40 in terms of the existence and structure of characteristic transcription-factor binding sites and was named Ol-HS-40. Injection of the fusion gene construct Ol-HS-40-alpha0(up-2)GFP, consisting of Ol-HS-40, a 5' upstream 200-bp minimum promoter for alpha0, and green fluorescent protein (GFP), showed that Ol-HS-40, as in human HS-40, had the ability to strongly enhance GFP expression in erythroid cells of embryos. Further analysis using transgenic technology revealed that Ol-HS-40 had the ability to change the type of the GFP expression from embryo-to-young fish to embryo-to-adult. In addition, the results suggest that Ol-HS-40, although its natural function remains unclear, has strong enhancer activity for the expression of not only the alpha-globin gene but also the beta-globin gene.

Purification and gene cloning of Fundulus heteroclitus hatching enzyme. A hatching enzyme system composed of high choriolytic enzyme and low choriolytic enzyme is conserved between two different teleosts, Fundulus heteroclitus and medaka Oryzias latipes.

Two cDNA homologues of medaka hatching enzyme -- high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE) -- were cloned from Fundulus heteroclitus embryos. Amino acid sequences of the mature forms of Fundulus HCE (FHCE) and LCE (FLCE) were 77.9% and 63.3% identical to those of medaka HCE and LCE, respectively. In addition, phylogenetic analysis clearly showed that FHCE and FLCE belonged to the clades of HCE and LCE, respectively. Exon-intron structures of FHCE and FLCE genes were similar to those of medaka HCE (intronless) and LCE (8-exon-7-intron) genes, respectively. Northern blotting and whole-mount in situ hybridization showed that both genes were concurrently expressed in hatching gland cells. Their spatio-temporal expression pattern was basically similar to that of medaka hatching enzyme genes. We separately purified two isoforms of FHCE, FHCE1 and FHCE2, from hatching liquid through gel filtration and cation exchange column chromatography in the HPLC system. The two isoforms, slightly different in molecular weight and in MCA-peptide-cleaving activity, swelled the inner layer of chorion by their limited proteolysis, like the medaka HCE isoforms. In addition, we identified FLCE by TOF-MS. Similar to the medaka LCE, FLCE hardly digested intact chorion. FHCE and FLCE together, when incubated with chorion, rapidly and completely digested the chorion, suggesting their synergistic effect in chorion digestion. Such a cooperative digestion was confirmed by electron microscopic observation. The results suggest that a hatching enzyme system composed of HCE and LCE is conserved between two different teleosts Fundulus and medaka.

Evolution of globin genes of the medaka Oryzias latipes (Euteleostei; Beloniformes; Oryziinae).

Recently we cloned two globin gene clusters from the genome of medaka (Oryzias latipes): one designated the embryonic globin gene cluster (E1; (5')alpha0(3')-(3')beta1(5')-(5')alpha1(3')-(5')beta2(3')-(5')alpha2(3')-(3')alpha3(5')-(5')beta3(3')-(3')beta4(5')-(5')alpha4(3')-(3')psialpha(5')-(5')psibeta(3')) and the other the adult globin gene cluster (A1; (3')ad.alpha1(5')-(5')ad.beta1(3')-(3')ad.alpha2(5')). The E1 and A1 clusters map to linkage groups 8 and 19, respectively. The genes beta1/alpha1, alpha3/beta3, beta4/alpha4, psialpha/psibeta and ad.alpha1/ad.beta1 are organized in head-to-head orientation with respect to transcriptional polarity. The genes alpha0, alpha1 and alpha2 are arranged in tandem with the same orientation. The results suggest that a variety of events occurred in globin gene evolution such as chromosomal translocation, duplication of alpha/beta-paired genes, tandem duplication of single alpha genes and the transformation of one pair of alpha/beta-paired genes into pseudogenes (psialpha/psibeta). Amino acid sequences predicted from the genes were compared with those of 42 alpha and 55 beta teleostean globins using the neighbor-joining or maximum likelihood methods. The phylogenetic trees that were generated classified the teleostean globins into at least four groups, tentatively named 'Embryonic Hb Group (I)', 'Notothenioid Major Adult Hb Group (II)', 'Anodic Adult Hb Group (III)' and 'Cathodic Adult Hb Group (IV)'. The medaka genes alpha0, beta1, alpha1, alpha2, alpha3, beta3, beta4 and alpha4 belong to group I, and ad.alpha1 and ad.beta1 to group II. Further analysis suggests that psialpha/psibeta and beta2/ad.alpha2 belong to groups III and IV, respectively. Thus, globin genes in the medaka probably were diversified from four ancestral genes, one for each group. On the basis of the gene comparisons, we present a hypothetical pathway for globin gene evolution in the medaka.

Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka.

Choriogenins (chg-H, chg-L) are precursor proteins of egg envelope of medaka and synthesized in the spawning female liver in response to estrogen. We linked a gene construct chg-L1.5 kb/GFP (a 1.5 kb 5'-upstream region of the chg-L gene fused with a green fluorescence protein (GFP) gene) to another construct emgb/RFP (a cis-regulatory region of embryonic globin gene fused with an RFP gene), injected the double fusion gene construct into 1- or 2-cell-stage embryos, and selected embryos expressing the RFP in erythroid cells. From the embryos, we established two lines of chg-L1.5 kb/GFP-emgb/RFP-transgenic medaka. The 3-month-old spawning females and estradiol-17beta (E2)-exposed males displayed the liver-specific GFP expression. The E2-dependent GFP expression was detected in the differentiating liver of the stage 37-38 embryos. In addition, RT-PCR and whole-mount in situ hybridization showed that the E2-dependent chg expression was found in the liver of the stage 34 embryos of wild medaka, suggesting that such E2-dependency is achieved shortly after differentiation of the liver. Analysis using serial deletion mutants fused with GFP showed that the region -426 to -284 of the chg-L gene or the region -364 to -265 of the chg-H gene had the ability to promote the E2-dependent liver-specific GFP expression of its downstream gene. Further analyses suggested that an estrogen response element (ERE) at -309, an ERE half-site at -330 and a binding site for C/EBP at -363 of the chg-L gene played important roles in its downstream chg-L gene expression. In addition, this transgenic medaka may be useful as one of the test animals for detecting environmental estrogenic steroids.

Expression of CCAAT/enhancer binding protein delta is closely associated with degeneration of surface mucous cells of larval stomach during the metamorphosis of Xenopus laevis.

CCAAT/enhancer binding protein delta (C/EBP delta) is one of the transcription factors that have a basic-leucine zipper domain. In mammals, it has been suggested that this transcription factor plays a role in differentiation of adipocytes or in apoptosis of mammary gland epithelial cells. The factor also plays a role in acute-phase response in injury, infection and inflammation. We cloned Xenopus homologues of the C/EBP delta gene from metamorphosing stomach by subtractive hybridization and analyzed spatio-temporal expression pattern of the homologues. Two isoforms of C/EBP delta were isolated and named C/EBP delta-1 and -2. Their deduced amino acid sequences were highly similar to each other (identity, 91.2%). Expression of the C/EBP delta mRNAs in the stomach transiently increased during its metamorphosis-associated remodeling, and the transient up-regulation was also found in thyroid hormone-induced metamorphosis. The C/EBP delta mRNAs were exclusively localized in degenerating larval surface mucous cells, not in newly proliferating and differentiating adult-type epithelial cells. The result suggests a possibility that Xenopus C/EBP delta plays a role in apoptotic cell death of larval-type epithelium during the stomach remodeling.

Genomic organization and developmental expression of globin genes in the teleost Oryzias latipes.

We isolated globin genes from a genomic DNA library of the drR strain of medaka Oryzias latipes, and walked on chromosome. The present study is the first demonstration of the full-length structure of globin gene locus in the teleosts. Two gene clusters were found. One cluster of 36 kbp consisted of nine globin genes and two pseudogenes. Based on structural and phylogenetic similarity of amino acid sequences, the cluster was named embryonic globin gene cluster (E1). The orientation of the genes was in (5')alpha0(3')-(3')beta1(5')-(5')alpha1(3')-(5')beta2(3')-(5')alpha2(3')-(3')alpha3(5')-(5')beta3(3')-(3')beta4(5')-(5')alpha4(3')-(3')psialpha(5')-(5')psibeta(3'). The other cluster of 20 kbp contained three globin genes ((3')ad.alpha1(5')-(5')ad.beta1(3')-(3')ad.alpha2(5')), and was named adult globin gene cluster (A1). Genetic linkage analysis clarified that E1 and A1 were mapped on linkage groups 8 and 19, respectively. The E1 cluster included other genes homologous to human EST clone KIAA0172, Sushi-1 retrotransposon, and protein 14 gene-like gene, while the A1 cluster linked to aquaporin-8 gene-like gene. The expression patterns of the genes were classified into four types: embryo-specific expression (alpha3, beta3, alpha4 and beta4), expression in embryo to young fish (alpha0, beta1, alpha1 and ad.alpha2), expression in young to adult fish (alpha2 and ad.alpha1) and successive expression in embryo to adult (ad.beta1).

Mode of Action of some Stimulants of the Hatching Enzyme Secretion in Fish Embryos*: (hatching/secretion/fish embryo/Medaka).

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.

Yolk Phosphoprotein Metabolism during Early Development of the Fish, Oryzias latipes: (Fish egg/Yolk/Phosphoprotein/Oryzias latipes/Fish development).

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.

Participation of a metalloprotease in the fertilization-associated conversion of the egg envelope (chorion) of the fish, Oryzias latipes.

The unfertilized egg envelope of medaka (Oryzias latipes) consists of two major groups of subunits, ZI-1,2 (74-76 kDa) and ZI-3 (49kDa). During egg envelope hardening after egg activation, both subunit groups decreased in amount, new protein bands of 57-65, 110 and 125 kDa appeared and, finally, no bands were detectable on sodium dodecylsulfate-polyacrylamide gel electrophoresis. The 110 and 125 kDa bands are intermediates formed by polymerization of such subunit groups. In contrast, treatment with iodoacetamide, an inhibitor of polymerization, revealed that the 57-65 kDa intermediates originated from ZI-1,2 by limited hydrolysis. ZI-1,2 comprises at least three distinct proteins of quite similar structure with their N-termini undetectable by Edman degradation, while the 57-65 kDa intermediates also consist of at least three proteins with the same N-terminal amino acid sequence: DGKPSNPQQPQVPQYPSK-. This fact strongly suggests a participation of a protease in the conversion of ZI-1,2 into 57-65 kDa proteins. EDTA and 1,10-phenanthrolinium inhibited the conversion and both Ca2+ and Zn2+ recovered the inhibition. These results suggest that the assumed protease is a metalloprotease.

Some Properties of the Hardening Process in Chorions Isolated from Unfertilized Eggs of Medaka, Oryzias latipes: (Fish Egg Envelope/Chorion/Chorion Proteins/Chorion Hardening/In Vitro Ca2+ -Hardening).

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.

Spawning Female-Specific Egg Envelope Glycoprotein-Like Substances in Oryzias latipes: (egg envelope/spawning female fish/oogenesis/oocyte growth/Oryzias latipes).

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.