Ichiro Iuchi

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.

Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development.

The hatching enzyme of medaka consists of two types of proteases (HCE, LCE). cDNA clones for LCE and HCE were isolated from a lambda gt11 cDNA library constructed with poly(A)+ RNA of Day 3 embryos. LCE cDNA is 936 bp long and contains an 813-bp open reading frame encoding a preproenzyme with a 20-amino-acid signal sequence, a 51-amino-acid propeptide, and a 200-amino-acid mature enzyme. For HCE, two distinct cDNAs (HCE21, HCE23) having nucleotide sequences with 92.8% similarity were obtained. These cDNAs contain open reading frames encoding preproenzymes of 279 and 270 amino acids, respectively. The mature enzyme forms of both consist of 200 amino acids, the similarity between them being 95.5%. On Northern blotting analysis, the transcripts of LCE and HCE genes were first detected coincidentally in Day 2 embryos shortly before the production of LCE and HCE, accumulated thereafter in parallel, and dramatically decreased after hatching. The amino acid sequence, the HExxH motif, which is known to constitute an active site in some Zn proteases, is also found in LCE and HCE. However, the sequence analyses strongly suggest that both the enzymes belong to the astacin (protease) family, being distinct from sea urchin hatching enzyme, which is reportedly similar to collagenase.

A glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida interna) of the fish, Oryzias latipes☆

A glycoprotein from the liver, which shares epitopes with chorion (egg envelope or zona pellucida) glycoproteins, is present only in the spawning female fish, Oryzias latipes, under natural conditions. This spawning female-specific (SF) substance is distinct from vitellogenin but closely resembles a major glycoprotein component, ZI-3, of the inner layer (zona radiata interna) of the ovarian egg envelope with respect to some biochemical and immunochemical characteristics. Here we report that the [125I]SF substance, injected into the abdominal cavity of the spawning female fish, was rapidly transported by the blood circulation into the ovary and incorporated into the inner layer of egg envelope of the growing oocytes. The result strongly suggests that the SF substance from the liver is a precursor substance of the major component, ZI-3, of the inner layer of egg envelope in the fish.

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.

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.

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.

Spawning Female-Specific Egg Envelope Glycoprotein-Like Substances in 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.

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

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.

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.