Kenjiro Yamagami

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.

7 Mechanisms of Hatching in Fish

Publisher Summary This chapter describes the mechanisms of hatching in fish. Hatching is a process by which an animal changes its life from an intracapsular to a free-living type and is, therefore, of great significance in animal ontogeny. Changes in histochemical stainability of hatching enzyme granules during the development of fish have been reported. The hatching-gland cells of medaka can be distinguished from other endodermal cells early in development by their relatively large size, the abundance of cisternae of endoplasmic reticulum, and a large electron-dense nucleus with a large nucleolus. At stages somewhat earlier than eye pigmentation, secretory granules appear first in the cytoplasmic matrix. In salmonid fishes, secretory granules become electron-lucent and fused together just before secretion. The gland cells discharge the granules together with some other cytoplasmic structures differently from ordinary exocytosis. The appearance of multiple hatching enzyme peaks on gel filtration chromatography has been reported also in some other fish species, such as rainbow trout and pike.

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.

Studies on the Hatching Enzyme (Choriolysin) and Its Substrate, Egg Envelope, Constructed of the Precursors (Choriogenins) in Oryzias latipes: A Sequel to the Information in 1991/1992

Abstract Enzymatic hatching of fish embryos is caused by a sequential occurrence of many elementary processes from the commitment of the hatching gland cells to the emergence of the embryos. Molecular biological approaches to the formation, properties and function of the hatching enzyme should be required for elucidation of the enzymatic hatching, since this enzyme is a key molecule to analyze these processes. Besides them, there are some other processes indirectly related to hatching, e.g., formation and hardening of the egg envelope. The present article describes the results of our studies on some of the above-mentioned problems in the fish, Oryzias latipes, which have been obtained mostly in the early 1990s.

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.

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

Change in Component Proteins of the Egg Envelope (Chorion) of Rainbow Trout during Hardening

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.