Structure and developmental expression of hatching enzyme genes of the Japanese eel<Emphasis Type="Italic"> Anguilla japonica</Emphasis>: an aspect of the evolution of fish hatching enzyme gene

We isolated seven cDNA clones from embryos of the Japanese eel Anguilla japonica. Each deduced amino acid sequence consisted of a signal peptide, a propeptide and a mature enzyme portion belonging to the astacin protease family. A phylogenetic analysis showed that the eel enzymes resembled the high choriolytic enzyme (HCE) of medaka Oryzias latipes, and the hatching enzymes of the zebra fish Danio rerio and masu salmon Oncorhynchus masou. Hatching enzymes of these teleosts belonged to the group of the medaka HCE, and not the medaka low choriolytic enzyme (LCE), another hatching enzyme of medaka. Southern blot analysis showed that the genes of the eel hatching enzymes were multicopy genes like the medaka HCE genes. However, one of the eel hatching enzyme genes comprised eight exons and seven introns, and the exon-intron organization was similar to the medaka LCE gene, which is a single-copy gene. The molecular evolution of the fish hatching enzyme genes is discussed. In addition, whole-mount in situ hybridization and immunocytochemistry showed that the eel hatching enzyme was first expressed in the pillow anterior to the forebrain of early neurula, and finally in the cell mass on the yolk sac of later stage embryos. The early differentiation profile of eel hatching gland cells was similar to that of medaka, masu salmon and zebrafish, whereas the final location of the gland cells was different among fishes.Edited by N. Satoh     

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.     

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.     

Two constituent proteases of a teleostean hatching enzyme: concurrent syntheses and packaging in the same secretory granules in discrete arrangement.

Formation, accumulation, and storage of two components of the Oryzias latipes hatching enzyme, high and low choriolytic enzymes (HCE and LCE), were examined by immunocytochemical and immunoblotting methods. Both of the enzymes were found to be formed specifically in the hatching gland cells at the stages of lens formation to eye pigmentation and their accumulation proceeded markedly and concurrently up to Day 5.5 embryos (the stage just before hatching). The amount of HCE formed was more abundant than that of LCE. In the hatching gland cells, HCE and LCE were found to be packaged in the same secretory granules but in distinct arrangement; HCE is localized to the inside of granules whereas LCE is situated at the periphery of the same granules. Their segregated arrangement is compatible with their relative quantities formed per embryo. The results provide not only the cellular and developmental basis for a view that this hatching enzyme is an enzyme system composed of HCE and LCE but also a clue to the regulatory mechanism of concurrent syntheses of two different specific proteins in the same embryonic cell.     

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.     

Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching enzyme of the teleost, Oryzias latipes

One of the two component proteases of the hatching enzyme of the fish, Oryzias latipes, low choriolytic enzyme (LCE), was isolated from the hatching liquid and partly characterized. The enzyme was a basic protein with molecular weight of about 25.5 kDa. Like high choriolytic enzyme (HCE), the other component of the O. latipes hatching enzyme [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], LCE was considered to be a zinc-protease from the results of inhibitor studies and metal analyses. However, LCE was found to be distinct from HCE not only in some biochemical characteristics such as molecular weight, amino acid composition, and isoelectric point, but also in some enzymological properties such as substrate specificity, heat stability, and mode of action toward their natural substrate, chorion (egg envelope). Although LCE was almost incapable of digesting the inner layer of intact chorion, it very efficiently digested the inner layer of chorion that had been swollen previously by the action of HCE. Taking account of the fact that HCE swells the inner layer of intact chorion by partial proteolysis but does not efficiently digest the swollen chorion any more [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], the present results demonstrated an essential role of LCE in choriolysis, in cooperation with HCE.     

Conservation of the egg envelope digestion mechanism of hatching enzyme in euteleostean fishes

We purified two hatching enzymes, namely high choriolytic enzyme (HCE; EC 3.4.24.67) and low choriolytic enzyme (LCE; EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved.     

Evolution of teleostean hatching enzyme genes and their paralogous genes

We isolated genes for hatching enzymes and their paralogs having two cysteine residues at their N-terminal regions in addition to four cysteines conserved in all the astacin family proteases. Genes for such six-cysteine-containing astacin proteases (C6AST) were searched out in the medaka genome database. Five genes for MC6AST1 to 5 were found in addition to embryo-specific hatching enzyme genes. RT-PCR and whole-mount in situ hybridization evidenced that MC6AST1 was expressed in embryos and epidermis of almost all adult tissues examined, while MC6AST2 and 3 were in mesenterium, intestine, and testis. MC6AST4 and 5 were specifically expressed in jaw. In addition, we cloned C6AST cDNA homologs from zebrafish, ayu, and fugu. The MC6AST1 to 5 genes were classified into three groups in the phylogenetic positions, and the expression patterns and hatching enzymes were clearly discriminated from other C6ASTs. Analysis of the exon–intron structures clarified that genes for hatching enzymes MHCE and MAHCE were intron-less, while other MC6AST genes were basically the same as the gene for another hatching enzyme MLCE. In the basal Teleost, the C6AST genes having the ancestral exon–intron structure (nine exon/eight intron structure) first appeared by duplication and chromosomal translocation. Thereafter, maintaining such ancestral exon–intron structure, the LCE gene was newly diversified in Euteleostei, and the MC6AST1 to 5 gene orthologs were duplicated and diversified independently in respective fish lineages. The HCE gene lost all introns in Euteleostei, whereas in the lineage to zebrafish, it was translocated from chromosome to chromosome and lost some of its introns.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.The nucleotide sequence data reported in the present paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers from AB256940 to AB256952.     

非洲爪蟾孵化酶的cDNA结构及其部分生物化学特性的研究

以UVS．2为探针从第25期非洲爪蟾胚胎头背部的cDNA文库中筛选出了一个1．8kb的孵化酶基因(xhe),其转录产物最早出现于第17期胚胎的头背部,在第30期转录量达到高峰,随后便逐渐减少。该基因含有编码514个氨基酸的一个开框阅读框架,含有信号肽和原酶序列。所推测出的成熟蛋白有425个氨基酸,包括位于N一端的含有200个氨基酸的金属蛋白酶序列和位于C端的两个各110个氨基酸的CUB重复区。而UVS．2只代表该基因C端大约3／4的部分。同时还发现该酶分子量为60kDa,是一种胰蛋白酶类型的金属蛋白酶。它很不稳定,在纯化过程中极易降解为40kDa分子。60kDa分子具有很强的卵黄膜溶解活性和蛋白酶活性。其中CUB重复区很可能在介导卵黄膜和40kDa分子中起着重要作用,而40kDa分子很可能是在纯化操作过程中,由60kDa分子发生降解或自身降解丢失了两个CUB重复区而形成的,它只是60kDa分子中的一个金属蛋白酶主功能区,所以它没有卵黄膜溶解活性,尽管仍具有很强的蛋白酶活性。     

A new member to the astasin family of metalloendopeptidases: A novel 1,25-dihydroxyvitamin D-3-stimulated mRNA from chorioallantoic membrane of quail

1,25-Dihydroxyvitamin D-3 is essential for the utilization of eggshell calcium by avian embryo through the chorioallantoic membrane (CAM). A cDNA library was constructed from poly(A)⁺ RNA extracted from vitamin D-deficient CAMs given 1,25-dihydroxyvitamin D-3. Screening this library by differential hybridization yielded a full-length (∼ 1.8 kb) cDNA, whose corresponding mRNA is increased 3-fold 2.5 h after a single injection of 1,25-(OH)₂D₃. The complete nucleotide sequence for the full-length cDNA has been determined. An open-reading frame, corresponding to a 310 amino acid, 41 kDa protein was found. Searching protein sequence data bases revealed a strong similarity to the following proteases: astacin, a crayfish digestive protease, Oryzias latipes hatching enzyme constituent protease (Orz), Xenopus laevis developmentally regulated UVS.2 protein secreted by the hatching gland of embryos, the NH₂-terminal domain of human bone morphogenetic protein (BMP-1) and Drosophila dorsal-ventral patterning tolloid. The cDNA has approximately 36% overall identity with astacin and BMP-1, and is more than 60% identical to either Orz or UVS.2. Moreover, multiple alignment analysis indicates that 37 residues, including 3 cysteine residues, are strictly conserved in the complete 200-amino acid astacin sequence. All 6 proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases. This motif is found within an extended sequence of HEXXHXXGFXHE that is unique to this subgroup of metalloendopeptidases. In addition, the 6 proteins have 50% identity (including the present cDNA) and 79% are conserved in 4 of these proteins in a 24-amino acid sequence that includes the putative active site. The level of mRNA for the new protein reaches a maximum at day 12 of embryonic life and declines thereafter. It is suggested that this clone corresponds to an mRNA encoding for a protease that may play a role in the degradation of eggshell matrix.     

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.     

Hatching enzyme of Japanese eel Anguilla japonica and the possible evolution of the egg envelope digestion mechanism

We purified eel hatching enzyme (EHE) from the hatching liquid of Japanese eel Anguilla japonica belonging to Elopomorpha to a single band on SDS/PAGE. TOF-MS analysis revealed that the purified EHE contained several isozymes with similar molecular masses. Comparison of the egg envelope digestion specificities of the purified EHE and of recombinant EHE4, one of the EHE isozymes, suggested that the isozymes contained in the purified EHE were functionally the same in terms of egg envelope digestion. By electron microscopy, the egg envelope became swollen after treatment with the purified EHE. The EHE cleavage sites on the zona pellucida (ZP) protein of the egg envelope were located in the N-terminal repeat regions. In previous phylogenetic analysis, we suggested that fishes included in Elopomorpha, as basal teleosts, possess a single type of hatching enzyme genes, and that fishes in Otocephala and Euteleostei gain two types of hatching enzyme genes called clade I and II genes by duplication. Further, the clade I enzymes, zebrafish hatching enzyme (ZHE1) and medaka high choriolytic enzyme (HCE), swell the egg envelope by cleaving the N-terminal regions of ZP proteins, while the clade II enzyme, medaka low choriolytic enzyme (LCE), solubilizes the swollen envelope by cleaving the site at the middle region on the ZP domain. In this evolutionary scenario, our findings support that hatching of Japanese eel conserves the ancestral mechanism of fish egg envelope digestion. The clade I enzymes inherit the ancestral enzyme function, and the clade II enzymes gain a new function during evolution to Otocephala and Euteleostei.     

Cloning of a quail homologue of hatching enzyme: its conserved function and additional function in egg envelope digestion

The aim of the present study was to reveal molecular entities participating in the digestion of the egg envelope in the Japanese quail, Coturnix japonica. We isolated a 1,510-bp cDNA from extraembryonic tissues of developing embryos and designated it quail hatching enzyme (QHE) cDNA. The QHE cDNA was found to code a protein molecule comprising an astacin protease domain in the N-terminal half and a complement subcomponents C1r/C1s, Uegf, Bmp1 (CUB) domain in the C-terminal half. A phylogenetic analysis showed that QHE belonged to the hatching enzyme group and was distinct from other proteases in the astacin family. Northern blotting and in situ hybridization demonstrated that expression of the QHE mRNA occurred twice during the development: first in ectodermal cells of the yolk sac on days 0–5, then in those of the albumen sac on days 8–13. Zymography revealed that proteolytic activity in extracts of days 3–4 and 9–12 embryos appeared at the position of 40 kDa. Immunoblotting tests showed that anti-QHE antiserum stained a 40-kDa molecule in extracts of day 3 area vitellina. Anti-QHE antibody stained the ectodermal cells of the area opaca on days 0–1, those of the area vitellina of the yolk sac on days 2–5, and those of the albumen sac on days 9–12. The temporal and spatial expression pattern of QHE mRNA was closely associated with digestion of the vitelline membrane occurring on days 1–4, and with that of the egg white on days 9–12.     

Hatching mechanism of the Chinese soft-shelled turtle Pelodiscus sinensis

The mechanism by which the embryo hatches out of the egg envelope, the vitelline membrane and egg white, was studied in the Chinese soft-shelled turtle Pelodiscus sinensis. The cDNA of the turtle hatching enzyme (HE) was 1555 bp-long and a mature enzyme of 321 amino acids. The mature HE was composed of an astacin protease domain of 200 amino acids and a CUB domain of 121 amino acids, and the estimated molecular size was 35,311. The protease domain contained two active site consensus sequences, HExxHxxGFxHExxRxDR and MHY. An immunoblotting test of an extract of allanto-chorions revealed a 40-kDa band by cross-reaction with the anti-Xenopus HE antiserum. The first change in the envelopes was the appearance of a hole, 1 mm in diameter, at the location around the animal pole of day 8 incubation eggs. A cluster of tall cells, forming a circle in the avascular chorion of day 8 embryos and facing the edge of the hole, had various sizes of inclusion bodies and secretory granules that were labeled by immuno-electron microscopic staining with the antiserum. The egg envelopes were degraded gradually from the animal pole side towards the vegetal pole side in accordance with translocation of the avascular site of the chorion in the same direction. Labeled cells degenerated, presumably when the chorion was underlain by allantois in succeeding developmental stages. The vitelline membrane and egg white were totally digested, presumably by secreted HE, during the hatching period and were consumed for embryonic growth.