Shigeki Yasumasu

Molecular and cellular basis of formation, hardening, and breakdown of the egg envelope in fish

Publisher Summary This chapter highlights some of the processes from formation to breakdown of the egg envelope (egg membrane) in fish from the perspective of cell and developmental biology. The chapter describes the structure and functions of the egg envelope, compares the egg envelopes of various species, and deduces a biological concept of the egg envelope. The chapter explores the egg envelope as a probe that can be used in the analysis of molecular, cellular, and developmental phenomena in living systems. The egg envelope is an acellular structure enclosing the egg and embryo of all multicellular animals except sponges and some coelenterates. The number of egg envelopes varies from one to several in different animal species. Most of the egg envelopes in fish consist of two or three layers. These layers are different in morphology, ultrastructure, stainability, and chemical properties. The outer one or two layers are thin, while the innermost layer is usually the thickest. The egg envelope of fish has been considered to be synthesized in oocytes or follicle cells and is classified as the primary or secondary egg envelope. The envelopes of fertilized eggs of many fish are hard and tough structures with strong elasticity and are also insoluble in water. The constituent proteins of the egg envelope are, therefore, inconvenient as immunogens to raise antibodies. The hatching enzyme does not break down the egg envelope completely into free amino acids or small peptides but, by limited proteolysis, produces a mixture of water-soluble, high-molecular-weight glycoproteins.

SPECIES-DEPENDENT MIGRATION OF FISH HATCHING GLAND CELLS THAT COMMONLY EXPRESS ASTACIN-LIKE PROTEASES IN COMMON

Two constituent proteases of the hatching enzyme of the medaka (Oryzias latipes), choriolysin H (HCE) and choriolysin L (LCE), belong to the astacin protease family. Astacin family proteases have a consensus amino acid sequence of HExxHxxGFxHExxRxDR motif in their active site region. In addition, HCE and LCE have a consensus sequence, SIMHYGR, in the downstream of the active site. Oligonucleotide primers were constructed that corresponded to the above‐mentioned amino acid sequences and polymerase chain reactions were performed in zebrafish (Brachydanio rerio) and masu salmon (Oncorynchus masou) embryos. Using the amplified fragments as probes, two full‐length cDNA were isolated from each cDNA library of the zebrafish and the masu salmon. The predicted amino acid sequences of the cDNA were similar to that of the medaka enzymes, more similar to HCE than to LCE, and it was conjectured that hatching enzymes of zebrafish and masu salmon also belonged to the astacin protease family. The final location of hatching gland cells in the three fish species: medaka, zebrafish and masu salmon, is different. The hatching gland cells of medaka are finally located in the epithelium of the pharyngeal cavity, those of zebrafish are in the epidermis of the yolk sac, and those of masu salmon are both in the epithelium of the pharyngeal cavity and the lateral epidermis of the head. However, in the present study, it was found that the hatching gland cells of zebrafish and masu salmon originated from the anterior end of the hypoblast, the Polster, as did those of medaka by in situ hybridization. It was clarified, therefore, that such difference in the final location of hatching gland cells among these species resulted from the difference in the migratory route of the hatching gland cells after the Polster region.

Purification and characterization of zebrafish hatching enzyme – an evolutionary aspect of the mechanism of egg envelope digestion

There are two hatching enzyme homologues in the zebrafish genome: zebrafish hatching enzyme ZHE1 and ZHE2. Northern blot and RT‐PCR analysis revealed that ZHE1 was mainly expressed in pre‐hatching embryos, whereas ZHE2 was rarely expressed. This was consistent with the results obtained in an experiment conducted at the protein level, which demonstrated that one kind of hatching enzyme, ZHE1, was able to be purified from the hatching liquid. Therefore, the hatching of zebrafish embryo is performed by a single enzyme, different from the finding that the medaka hatching enzyme is an enzyme system composed of two enzymes, medaka high choriolytic enzyme (MHCE) and medaka low choriolytic enzyme (MLCE), which cooperatively digest the egg envelope. The six ZHE1‐cleaving sites were located in the N‐terminal regions of egg envelope subunit proteins, ZP2 and ZP3, but not in the internal regions, such as the ZP domains. The digestion manner of ZHE1 appears to be highly analogous to that of MHCE, which partially digests the egg envelope and swells the envelope. The cross‐species digestion using enzymes and substrates of zebrafish and medaka revealed that both ZHE1 and MHCE cleaved the same sites of the egg envelope proteins of two species, suggesting that the substrate specificity of ZHE1 is quite similar to that of MHCE. However, MLCE did not show such similarity. Because HCE and LCE are the result of gene duplication in the evolutionary pathway of Teleostei, the present study suggests that ZHE1 and MHCE maintain the character of an ancestral hatching enzyme, and that MLCE acquires a new function, such as promoting the complete digestion of the egg envelope swollen by MHCE.

The third egg envelope subunit in fish: cDNA cloning and analysis, and gene expression.

The inner layer of the egg envelope of a teleost fish, the medaka, Oryzias latipes, consists of two major subunit groups, Zl‐1,2 and Zl‐3. On SDS‐PAGE, the Zl‐1,2 group presents three glycoprotein bands that were considered to be composed of a common polypeptide moiety derived from their precursor, choriogenin H (Chg H). Zl‐3 is a single glycoprotein derived from the precursor, choriogenin L (Chg L). In the present study, a fraction of a novel subunit protein was found in the V8 protease digest of Zl‐1,2 that was partially purified from oocyte envelopes. This protein fraction was not present in the purified precursor, Chg H. By RT‐PCR employing the primers based on the amino acid sequence of this fraction, a cDNA for the novel subunit was amplified, and a full‐length clone of the cDNA was obtained by screening a cDNA library constructed from the spawning female liver. The clone consisted of 2025 b.p. and contained an open reading frame encoding the novel protein of 634 amino acids. This protein included Pro‐X‐Y repeat sequences in two‐fifths of the whole length from its N‐terminus. Northern blot analysis revealed that the gene expression for this protein occurred in the liver but not in the ovary of spawning female fish. This protein is considered as the third major subunit of the inner layer of the egg envelope of medaka.

Morphogenesis and regionalization of the medaka embryonic brain

We examined the morphogenesis and regionalization of the embryonic brain of an acanthopterygian teleost, medaka (Oryzias latipes), by in situ hybridization using 14 gene probes. We compared our results with previous studies in other vertebrates, particularly zebrafish, an ostariophysan teleost. During the early development of the medaka neural rod, three initial brain vesicles arose: the anterior brain vesicle, which later developed into the telencephalon and rostral diencephalon; the intermediate brain vesicle, which later developed into the caudal diencephalon, mesencephalon, and metencephalon; and the posterior brain vesicle, which later developed into the myelencephalon. In the late neural rod, the rostral brain bent ventrally and the axis of the brain had a marked curvature at the diencephalon. In the final stage of the neural rod, ventricles began to develop, transforming the neural rod into the neural tube. In situ hybridization revealed that the brain can be divided into three longitudinal zones (dorsal, intermediate, and ventral) and many transverse subdivisions, on the basis of molecular expression patterns. The telencephalon was subdivided into two transverse domains. Our results support the basic concept of neuromeric models, including the prosomeric model, which suggests the existence of a conserved organization of all vertebrate neural tubes. Our results also show that brain development in medaka differs from that reported in other vertebrates, including zebrafish, in gene‐expression patterns in the telencephalon, in brain vesicle formation, and in developmental speed. Developmental and genetic programs for brain development may be somewhat different even among teleosts. J. Comp. Neurol. 476:219–239, 2004.

HCE, a constituent of the hatching enzymes of Oryzias latipes embryos, releases unique proline‐rich polypeptides from its natural substrate, the hardened chorion

HCE, a constituent protease of the hatching enzymes of Oryzias latipes embryos [1,2], releases unique proline‐rich polypeptides from its natural substrate, the hardened chorion. The polypeptides consist of repeats of Pro‐X‐Y, mainly Pro‐Glx‐X. In addition, the polypeptides contain abundant γ‐glutamyi ϵ‐lysine isopeptides which are regarded to be responsible for chorion hardening. These findings suggest that HCE recognizes specific site(s) of the chorion, releases the proline‐rich polypeptides from it, and makes the substrate accessible to LCE, another protease of the hatching enzymes.

Structure and developmental expression of hatching enzyme genes of the Japanese eel Anguilla japonica: 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.

cDNAs and the Genes of HCE and LCE, Two Constituents of the Medaka Hatching Enzyme

The hatching enzyme of animals is present in only developing embryos. It is synthesized in a definite group of embryonic cells at a definite period of development, i . e., its synthesis is strictly controlled spatio-temporally in the embryonic body. Thus the hatching enzyme has long been regarded as a candidate of appropriate probes to analyze the mechanism of synthesis of a specific protein(s) in the developmental system in connection with differentiation of embryonic cells (9, 33). However, uncertainties about the molecular properties of the hatching enzyme of any animal species has long hindered progress in molecular biological exploitation of this enzyme. Recently, the hatching enzymes of the teleost, medaka, Oryzias latipes, and the sea urchins, Paracentrotus lividus, and Hemicentrotus pulcherrimus, were highly purified and information was obtained on their physical chemical characteristics and the enzymological properties (14, 23, 41, 42). Unexpectedly, the results on the hatching enzyme of the medaka showed that it was not a single enzyme, but an enzyme system composed of two similar but distinct enzymes (35, 40), whereas the hatching enzyme in the sea urchin was single enzyme (14, 23). Within the next few years, cDNAs for the Paracentrotus enzyme (1 5) and the two constituent proteases of the teleostean enzyme (45) were cloned and so these embryo-specific enzymes are now exploitable as molecular biological probes. No information is available at present on the gene(s) and gene expression of the hatching enzyme(s) in other animals, but it seems likely that the nature of the hatching enzymes of the zebrafish and amphibians will be clarified at a molecular level in the near future. The present article surveys the results of some recent studies on the cDNAs and the genes for the

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.

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.