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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.

The inner layer of egg envelope of the medaka, Oryzias latipes, comprises two major groups of glycoprotein subunits, ZI-1,2 and ZI-3. Their precursor proteins, choriogenin H (Chg H) and choriogenin L (Chg L), respectively, are synthesized in spawning female liver. In the present study, the primary structures of the precursors and the corresponding mature subunits were compared by peptide mapping and amino acid sequencing to find what difference in their molecular structures is relevant to the assembly of the soluble precursors into the insoluble inner layer. The primary structures of the solubilized subunits were mostly identical to those of the respective precursors, but they lacked C-terminal partial sequences that their precursors possessed, namely, ZI-1,2 subunit was shorter than Chg H by 34 amino acid residues and ZI-3 was shorter than Chg L by 27 residues. In addition, a consensus amino acid sequence, Arg-Lys-X-Arg, was found at the putative cleavage sites in the C-terminal region of the precursors. It is conjectured that the truncation of the precursor proteins is prerequisite for formation of mature chorion subunit proteins and their assembly into chorion.

The inner layer of the egg envelope of a teleost fish, the medaka, Oryzias latipes, consists of two major subunit groups, ZI-1,2 and ZI-3. On SDS-PAGE, the ZI-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). ZI-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 ZI-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.

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 time course and pattern of expression of the genes for choriogenin H and choriogenin L in the liver of the E2-treated male fish was investigated according to the methods of dot blotting analysis and in situ hybridization. Four hours after the beginning of estrogen treatment (final concentration in rearing water: 100 ng ml-1), expression of the choriogenins genes was induced in every parenchymal cell in the liver and the amounts of the gene transcripts were generally increased. As a primary step for analysis of the mechanism of the estrogen action, the amount of estrogen receptor and expression of its gene were examined in the liver of estrogen-treated male fish.

The hatching enzyme of the teleost, Oryzias latipes, is composed of two proteases, high choriolytic enzyme (choriolysin H, HCE) and low choriolytic enzyme (choriolysin L, LCE), which are similar in some enzymological characteristics and protein structure (55% identity in amino acid sequence) and belong to the astacin family. Two isoforms of HCE are detected. In the present study, the genes for HCE and LCE were isolated from the genomic library constructed from DNA of the inbred drR strain fish. In contrast to the close similarity of the enzymes, there was a marked difference in their gene organization. The LCE gene was a single copy gene and composed of eight exons interrupted by seven introns. The HCE genes were multicopy genes and lacked introns. In the haploid genome of the drR strain fish, there are eight HCE genes, seven of which were cloned. Each HCE gene was identified as that for either of the two isoforms of HCE. 5' flanking regions of the LCE gene and the HCE genes had consensus TATA box sequences, but not CAT box nor GC box sequences. The big difference in the exon-intron organization between the HCE genes and the LCE gene is discussed from an evolutionary viewpoint.

The inner layer of most teleostean egg envelopes, especially those after hardening, is almost insoluble in ordinary solvent, and therefore the inner layer of only the unhardened egg envelope has been subjected to solubilization with some potent solvents. We comparatively evaluated the methods of solubilization of the inner layer of egg envelope of medaka, Oryzias latipes, with SDS, urea and guanidium chloride (GuHCI). Analysis of the solubilized samples by SDS-polyacrylamide gel electrophoresis, comparison of their amino acid compositions or peptide maps using high-performance liquid chromatography and partial determination of their amino acid sequences showed that SDS and GuHCI were appropriate for solubilization and characterization of the envelope. Urea solubilization resulted in some artificial modifications of lysine and/or cysteine residues of envelope proteins. Partial determination of amino acid sequence of a subunit, ZI-3, isolated from the SDS-or GuHCI-solubilized envelope strongly suggested the identity of the envelope subunit, ZI-3, and its precursor, L-SF.

The hatching enzyme of the medaka, Oryzias latipes, consists of two proteases, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They are synthesized and accumulated in the same unicellular hatching glands and are secreted from them at the end of embryonic development to digest the egg envelope. Recently, these enzymes were purified, and their cDNA clones were isolated. In the present study, we examined temporal and spatial patterns of expression of the hatching enzyme genes during embryogenesis using cDNAs for HCE and LCE as probes. According to Northern blotting analysis, the expression of both genes started at the same time (stage 21-22 embryos: brain differentiation and lens formation) and the patterns of expression changed in parallel during development. In situ hybridization to whole embryo and the sections revealed that the expression of the HCE genes was detected first in the anterior end of the hypoblast layer in stage 16-17 (late gastrula) embryos. Distinct signals of the HCE gene expression were then detected in a group of cells located at the front of the head rudiment of embryos at stage 18-19 (1 somite). Treatment of the embryos with retinoic acid, which is known to affect the anterior differentiation of embryos, suppressed the hatching gland cell differentiation in accordance with the result of in situ hybridization. In stage 22 embryos, the HCE-positive cells dispersed in an ectodermal layer under the forebrain and optic vesicles. Thereafter, the hatching gland cells expressing the HCE mRNA were aligned along the branchial arches and finally rearranged to the inner wall of the pharyngeal cavity, following a marked elongation of the lower jaw. The results of in situ hybridization to whole embryos at consecutive developmental stages demonstrated that the hatching gland cells located at the most anterior portion of the hypoblast migrated posteriorward to endoderm (pharyngeal endoderm) by way of ectoderm, while they were expressing mRNA for the hatching enzyme. Retinoic acid treatment of embryos gave rise to aberrations in the final location of the hatching gland cells probably by disturbing their migration. Moreover, the number of hatching gland cells increased markedly during their migration. This fact strongly suggested a concurrence of gene expression and mitosis of a gland cell and/or a successive initiation of gene expression in maturing gland cells during migration.

cDNA clones for L-SF, the precursor of a low-molecular-weight subunit (ZI-3) of the inner layer of the Oryzias latipes egg envelope were isolated from Lambda ZAP cDNA libraries constructed from the poly(A)+ RNA of the liver of spawning female fish and estrogen-treated male fish. Among them, a clone, L-SF41, is 1473 bp long and contains an open reading frame encoding a signal peptide of 19 amino acids and L-SF protein of 420 amino acids. L-SF protein seems to be glycosylated, judging from the result of the glycanase digestion. L-SF protein contains a domain similar to ZP-domains in ZP3 of some mammalian species. Northern blot analysis employing XhoI-SmaI fragments of the cloned cDNA as probes revealed that expression of the L-SF gene occurred exclusively in the livers of spawning female fish and estrogen-treated male fish and that there was no mRNA encoding L-SF in the ovary of the spawning female fish.

Estrogen stimulation of production and accumulation of L-SF and H-SF, the low- and high-molecular-weight precursors of the egg envelope subunits in Oryzias latipes , was analyzed by immunohistochemical and immunoblotting methods. Both SF substances were detected in the liver of the male fish as quickly as 16-24 hr after estrogen treatment, and their production and accumulation in the liver and blood plasma increased as estrogen treatment continued. Immunohistochemical analysis revealed that the number of cells producing both SP substances was increased in the liver as estrogen treatment continued. These results strongly suggest the occurrence of synchronous production of L-SF and H-SF in response to estrogen. In liver extracts, but not in blood plasma, an additional L-SF-like immunoreactive protein was detected on immunoblots. Preliminary data indicate parallel appearance of vitellogenin and SF substances in the liver of estrogenized fish.

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.

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.

Hardening of the chorion of medaka eggs was quantitated in terms of the solubility of its constituent proteins. After activation of unfertilized eggs with the Ca2+-ionophore A23187, hardening of chorion (named ionophore-activation hardening) proceeded and 60 min after activation the solubility of the proteins in 1 N NaOH had decreased to 20% of that of proteins of unhardened chorions. On SDS-PAGE, the chorions of unfertilized eggs gave four major protein bands (150, 83, 78 and 51 K). After Ca2+-ionophore activation, new two protein bands (135 and 61 K) appeared, with concurrent disappearance of all the original bands except the 51 K band. Isolated chorions of unfertilized eggs were also hardened by Ca2+and 60 min after addition of Ca2+the solubility of their proteins in 1 N NaOH had decreased to about 45% of that originally. During this type of hardening (named ‘Ca2+-hardening), however, the SDS-PAGE pattern of the proteins remained unchanged. Therefore, there are two mechanisms of hardening. The ‘ionophore-activation hardening was inhibited by cadaverine. When chorions were isolated 20 min after Ca2+-ionophore activation and kept in Ca2+-free conditions, the ‘ionophore-activation hardening process was arrested: further hardening was resumed on addition of Ca2+to the medium. These results suggest the presence of some hardening machinery in isolated chorions.

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. Hardening occurred also in the chorion isolated from the unfertilized eggs. The conversion of the 64 K, 56 K and 50 K components into the higher molecular weight intermediates was Ca2+-dependent, accelerated by 2-mercaptoethanol and inhibited by a high temperature (60°C). Optimum pH of this hardening system was acidic, i.e., 5.0 to 6.0. Cadaverine inhibited the change in solubility of chorion in NaOH or Sample Buffer, while it could not inhibit the disappearance of the 64 K, 56 K and 50 K components and the new formation of the higher molecular weight intermediates. This observation indicates existence of two processes in chorion hardening, which are tentatively named cadaverine-insensitive and cadaverine-sensitive processes.

1. 1. Molecular weight estimation and subunit analysis of four yolk phosphoproteins (PP1-PP4) in medaka (Oryzias latipes) eggs were performed. 2. 2. PP1 (Mr ≈ 210,000) and PP2 (Mr ≈ 180,000) were found to be heterodimers composed of subunits of 113,000 and 94,000 and subunits of 84,000 and 72,000, respectively. 3. 3. PP3 and PP4 [phosvitins of medaka (Murakami et al., 1990, Devl. Growth Differ.32, 619–627)], were monomeric phosphoproteins having mol. wts of about 40,000 and about 20,000, respectively. 4. 4. Lipid composition of the mixture of PP1 and PP2, vitellogenin and yolk were found to be almost the same. PP1 and PP2 are probably lipovitellins of medaka.

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.

Publisher Summary This chapter discusses the activity, specificity and structural chemistry of choriolysin H. Choriolysin H hydrolyzes the inner layer of egg envelope and releases unique proline-rich polypeptides from it. As a result of hydrolysis by choriolysin H, the inner layer of egg envelope is swollen. The released polypeptides consist of repeats of Pro-Xaa-Yaa, mainly Pro-Glu-Yaa, which are present in ZI-1,2, one of the major subunit proteins of the egg envelope. The pH optimum is about 8.0 and 8.7 for caseinolytic activity and choriolytic activity, respectively. Both activities are inhibited by EDTA at a concentration of ImM, but are not inhibited by DFP, iodoacetamide or iodoacetic acid. The enzyme contains zinc, calcium and magnesium as revealed by metal analysis. Choriolysin H tends to bind tightly to the egg envelope. One of the antichoriolysin H monoclonal antibodies does not affect caseinolytic activity, but inhibits both choriolytic activity and binding to the egg envelope. The choriolysin H genes are multicopy genes and lack introns. Six of seven HCE genes cloned are situated in a cluster and arranged in tandem. A putative promoter region of each gene contains a TATA box sequence, but neither a CAT box nor GC box sequences.

Publisher Summary This chapter elaborates the activity, specificity and structural chemistry of choriolysin L. Choriolysin L displays very low choriolytic activity when examined by the turbidimetric method. It hardly digests the intact inner layer of egg envelope, but efficiently digests the swollen inner layer of egg envelope prepared by a limited digestion by choriolysin H. The specificity of choriolysin L is 8,000 and 100,000 times greater than those of trypsin and thermolysin, respectively. The hatching of medaka embryos results from a two-step digestion of the two enzymes; choriolysin H swells the inner layer of egg envelope by limited digestion, and choriolysin L solubilizes the swollen part of it completely. Choriolysin L also hydrolyzes casein and some synthetic substrates such as Suc-Leu-Leu-Val-Tyr-fNHMec . The pH optimum is about 8.6 for caseinolytic activity. Choriolysin L is a single-chain protein of about 25.5 kDa and pi 9.8. The enzyme is synthesized as a preproenzyme and the mature enzyme consists of 200 amino acids. Choriolysin L is colocalized with choriolysin H in the secretory granules of the hatching gland cells that are located in the inner wall of the pharyngeal cavity of prehatching medaka embryos.

A cDNA for choriogenin H (Chg H; formerly high-molecular weight spawning female-specific substances, or H-SF), a precursor protein of the inner layer subunits of egg envelope (chorion) of the teleost fish, Oryzias latipes , was cloned and analyzed. The clone consisted of 1913 bp and contained an open reading frame encoding a signal peptide of 22 aa and Chg H protein of 569 aa. The Chg protein possessed three potential N-glycosylation sites and Pro-X-Y repeat sequences in the first two-fifths of the N terminus. There were amino acid sequence similarities between Chg H and a gene product expressed in the liver of female winter flounder during vitellogenesis. Moreover, the amino acid sequence of Chg H is similar to that of ZP2 rather than ZP3 of zona pellucida of some mammals. Northern blot analysis indicated that gene expression for Chg H occurred only in the livers of spawning female fish and 17β-estradiol-treated male fish, but not in the ovary of the spawning female fish. Gene expression for Chg H and Chg L (formerly low-molecular weight spawning female-specific substance, or L-SF) was induced and increased in parallel in the male fish liver after 17β-estradiol treatment.

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.

High- and low-molecular weight spawning-female-specific (H-SF and L-SF) substances are liver-derived putative precursors of the inner layer of egg envelope of the medaka Oryzias latipes, and are also found in the ascites which are accumulated in the male fish on prolonged treatment with oestrogen. In the present study, the H-SF substances purified from the ascites of the oestrogen-treated male medaka were found to consist of three proteins. Their molecular weights ranged from 74 000 to 76 000, which corresponds to those of ZI-1 and ZI-2, the major components of medaka egg envelope. The amino acid composition of the H-SF substances was characterised by high contents of proline (17.58 mol%) and glutamic acid/glutamine (13.34 mol%). The characteristics were almost identical to those of ZI-1 and ZI-2 but differed from those of L-SF substance, a precursor of ZI-3, another major component of the egg envelope. These results confirm our view that H-SF substances are the precursors of ZI-1 and ZI-2. The average amino acid compositions of H-SF substances and L-SF subsatance coincided with the amino acid composition of whole egg envelope. This result strongly suggests that an equal amount of H-SF substances and L-SF substance would construct the inner layer of egg envelope after a little modification.

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.

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.

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.

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.

“Probable vitellogenin” [Hamazaki et al. (1985) J. Exp. Zool., 235:269–279] of the medaka, Oryzias latipes, was purified from the ascites of estrogen-fed (EF) fish and was identified as the real vitellogenin by examining some of its biochemical characteristics. It has an amino acid composition and phosphorus content (0.8%) quite similar to those of the vitellogenin of rainbow trout and goldfish. The molecular weight (mw) in the native state was about 420,000, but sodium dodecyl sulfate denaturation produced a group of peptides that included a major peptide of about 200,000 mw with an isoelectric point of 3.8–4.0. Immunohistochemical observations with the use of the anti-vitello-genin antibody raised against the purified vitellogenin as a probe indicated that a considerable immunoreactivity appeared in some vesicles of the oocytes at the yolk-vesicle stage (ca, 150–250 μm in diameter). However, yolk deposition in the oocytes seemed to occur at the later stage (vitellogenic stage, over 300 μm in diameter) as judged from histological examination of the the hematoxylin-stained materials. Moreover, the immunofluorescence was observed in the intercellular spaces among the follicle cells of the vitellogenic oocyte. The observation indicates that the pathway of vitellogenin transportation through the follicle cell layer into the oocyte is present in the extracellular matrix of the follicular epithelium.

SYNOPSIS. Mechanisms of two constituent steps of the hatching process, i.e. , secretion of hatching enzyme from the gland cells and enzymatic choriolysis, in the Medaka, Oryzias latipes , are described. The ultrastructural changes of the hatching gland cells occurring at the initiation of electrically induced secretion as well as of natural secretion were the swelling of each gland cell and the separation of joints of the epithelial cells covering the gland cells, followed by a resultant exposure of the apical part of the gland cells. These changes, though their triggering mechanisms are not sufficiently clarified, suggest an intervention of some mechanical stimuli in the initiation of secretion. Decrease in electron density of the secretory granules also occurred immediately prior to the initiation of secretion. The secreted hatching enzyme was found to dissolve the inner layer of chorion by attacking the scleroprotein of the inner layer at some restricted sites and liberating a group of soluble glycoproteins of high molecular weights. This selective digestion appears to be the reason why choriolysis proceeds efficiently during a short period of time at hatching.

Secretory granules of hatching gland were isolated from a 0.3 M sucrose homogenate of whole medaka embryos at prehatching stage by differential centrifugation, followed by a Percoll density gradient centrifugation. The obtained preparation was almost free of melanosomes and composed exclusively of the secretory granules of hatching gland (hatching enzyme granules), as judged by morphological as well as enzymological criteria. The aqueous extracts of the purified secretory granules showed a specific choriolytic activity as high as about 40 times that of a partially purified secretory granule preparation, P1,000, and represented a single protein band with molecular weight of about 21,000 on SDS-polyacrylamide gel electrophoresis. It was also revealed that a major component of the hatching enzyme preparation (P II–0.3 enzyme, 13) purified from the hatching liquid was identical with the 21,000 molecular weight band. These results suggest that the hatching enzyme is present in the secretory granules of prehatching embryos in an active molecular form.

A large number of spherical bodies, a major insoluble component of cortical alveolus, were isolated from blastulae of medaka, Oryzias latipes. Spherical body material was found to be abundant in carbohydrates, especially in galactose and hexosamine, and total carbohydrate content amounted to about 1.3 times that of protein. Immunohistochemical studies using anti-spherical body antiserum as a probe revealed that a substance crossreacting with spherical body material was localized only in “yolk vesicles” of growing oocytes and later in cortical alveoli of full-grown oocytes. Thus the present results identified immunochemically yolk vesicle material with cortical alveolar material, supporting the view that the yolk vesicle is a precursory structure of cortical alveolus.

The relationship between the hatching enzymes I and II of the medaka, Oryzias latipes, was studied by examining their behavior on gel filtration column chromatography and their responsiveness to added reagents including certain inhibitors. Enzyme I, of higher molecular weight, could be converted to the lower molecular weight enzyme II during purification. The results support the conclusion that enzyme I is a complex form of enzyme II combined with another substance.

A “spawning female-specific substance” (SF substance) [Hamazaki et al. (1985) J. Exp. Zool., 235:269–279] was isolated from the ascites fluid of the estrogen-fed (EF) male or nonspawning female fish, Oryzias latipes, by gel filtration chromatography, followed by high-performance ion-exchange liquid chromatography (HPLC). Biochemical analyses revealed that the SF substance was a glycoprotein that contained 3.01% carbohydrates and showed 49,000 molecular weight (mw) on SDS-PAGE. Amino acid composition and the carbohydrate content of the SF substance were similar to those of the glycoproteins, F1 and F2, which were purified from the hatching enzyme digest of the inner layer (zona interna) of egg chorion (egg envelope). The inner layer of chorion from ovarian egg (oocyte) was separable into three different glycoproteins, ZI-1 (mw 76,000), Z1-2 (mw 74,000), and ZI-3 (mw 49,000) on SDS-PAGE. The major glycoprotein, ZI-3, was found to show the mw and amino acid composition that were almost identical to those of the purified SF substance. These results indicate that a substance that is almost the same as the major component glycoprotein of the inner layer of chorion, in terms of the biochemical as well as immunological properties, is present out-side the ovary and is produced in the liver of the spawning female fish as well as the EF fish.

Morphological changes in the chorion of the Medaka, Oryzias latipes, brought about by the hatching enzyme were examined by transmission as well as scanning electron microscopy. The structure of the intact chorion, especially its thick multilamellar inner layer, does not change during development until about 1 hr before the onset of hatching. As choriolysis proceeds, the inner layer of the chorion is digested to yield soluble proteins of relatively high molecular weight. During this process it appears that each lamella is successively solubilized from the inner surface of the chorion. Finally, a thin outer layer with accompanying villi and attaching filaments remains. Under the experimental conditions used, the enzyme was in direct contact with both the inner and outer layers of the chorion. Because of this, the enzyme could penetrate the outer layer and act on some peripheral parts of the underlying inner layer. Based on these morphological changes, a mechanism is proposed to account for the solubilization of the chorion by the hatching enzyme.

The hatching enzyme is an embryo-secreted enzyme(s) which digests the egg envelope, allowing the embryo to emerge at the time of hatching. The hatching enzyme of the fish, Oryzias latipes, has recently been found to consist of two kinds of proteases which may digest the inner layer of chorion (egg envelope) cooperatively [Yasumasu, S. et al. (1988) Zool. Sci. 5, 191-195]. In the present study, one of them, high choriolytic (egg envelope digesting) enzyme (HCE) was purified and some biochemical and enzymological properties were examined. The enzyme was a basic protein with a molecular weight of about 24 kDa, and exhibited choriolytic activity as well as proteolytic (caseinolytic) activity. The results of inhibitor studies and metal analyses strongly suggested that it was a zinc-protease. The purified HCE consisted of two probable isomers, HCE-1 and HCE-2. Both of them were markedly similar in amino acid composition, specific activities of choriolysis and proteolysis, and substrate specificity as determined using MCA-peptides. Moreover, they were not separable on SDS-PAGE, electrofocusing PAGE, or ultracentrifugal analysis, but were discriminated only on HPLC with a CM-300 column. Thus, the mixture of HCE-1 and HCE-2 could be regarded as almost a single enzyme, HCE. When it acted on an intact chorion, the purified HCE caused a remarkable swelling of its inner layer with concomitant release of peptides from it. Once the inner layer of chorion was swollen, the enzyme hardly digested it.

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.

High choriolytic enzyme (HCE), a constituent protease of the hatching enzyme of the teleost, Oryzias latipes, swells its natural substrate, egg envelope (chorion) by hydrolyzing it partially. This enzyme was found to be bound tightly to the chorion when it exerted catalytic action. This was evidenced by the experimental results showing (i) that the turnover of this enzyme seemed to be hindered by the chorion, (ii) that the enzyme bound to the chorion could be recovered by washing with an alkaline medium, and (iii) that the bound enzyme could be quantified by radioimmunological estimation. The bound enzyme sustained its original activity and the binding between the enzyme and the chorion seems to be stoichiometric.

A “spawning female-specific substance” cross-reactive with an anti-chorion glycoprotein antibody is present not only in the ovary but also in the liver and blood plasma of the teleost, Oryzias latipes [Hamazaki et al. (19851, J. Exp. Zool., 235269-279]. Upon the administration of estrogen, this substance was produced in hepatic cells of both a nonspawning adult female and an adult male. Moreover, a large amount of the substance was accumulated in the ascites fluid that was accumulated in the abdominal cavity of the estrogen fed (EF) fish. The ascites fluid as well as blood plasma of these fish contained also a considerable amount of “probable vitellogenin” (recently identified as real vitellogenin) that showed no cross-reactivity with the anti-body. Thus these two substances were major components of the ascites fluid. The antigenic substance, as well as vitellogenin, seems to remain in its native molecular state in the ascites fluid without undergoing enzymatic digestion. On electron microscopic observation of the hepatic cells of the estrogenized male and nonspawning female fish, an unusual formation of electron dense droplets was found among well developed rough-surfaced endoplasmic reticuli (r-ER). Immunocytochemical examination revealed that the antigenic substance was localized in the lumen of the r-ER, Golgi apparatus, some transport vesicles. These results strongly suggest that this “spawning female-specific substance” is normally produced in the liver under the influence of an estrogenic hormone.

A substance with high immunoreactivity to rabbit anti-chorion (egg envelope) glycoprotein antibody was found in liver and blood plasma as well as ovary of the spawning female medaka, while it was absent in the male and the non-spawning female fish. This substance disappeared first from the hepatic cells of the female fish when it ceased spawning, whereas the immunoreactivity still remained in ovary and blood plasma. The relative electrophoretic mobility of the antigenic substance derived from these tissues was identical. The molecular weight and isoelectric point (pI) of the substance, as determined by polyacrylamide electrophoresis with varied gel concentrations and isoelectric focusing method, were about 60,000 and about 4.5, respectively. The substance was evidently distinct from the unreactive band of probable vitellogenin (ca 420,000 and pI, ca 3.8–4.0) which was found in blood plasma of the spawning female. In ovary sections, cytoplasm of the oocyte at the initial stage of chorion formation, containing neither vitellogenin nor egg yolk, showed a strong immunoreactivity. Cytoplasm of the oocytes, later than the vitellogenic stage, exhibited little immunoreactivity. Changes of this substance in connection with the spawning cycle strongly suggest that this substance is related to oogenesis.

1. 1. The muscle LDH-A 4 and -B 4 isozymes of the inbred and outbred fish of a Japanese cyprinodont, Oryzias latipes , were purified and some of the protein chemical as well as enzymological characteristics were examined. 2. 2. The molecular weights of the A and B subunits were about 36.0 kD and 36.5 kD, respectively. There was a marked similarity in amino acid composition between the corresponding subunits of this fish and the salmon, Oncorhynchus tshawytscha . 3. 3. The optimal pHs of both A 4 and B 4 isozymes were almost the same, being about 6.5 for pyruvate reduction and about 10.0 for lactate oxidation. 4. 4. The B 4 enzyme was markedly more heat-stable and of higher affinity to the substrates in the reciprocal reactions than the A 4 enzyme.

Ultrastructural changes of the hatching gland during electrically induced precocious secretion were compared with those during natural secretion in the medaka, Oryzias latipes . The gland cells are covered by a layer of epithelial cells, which adjoin one another just on the apical center of each gland cell. When the natural as well as the precocious secretion occurred, each gland cell was swollen upward and rounded, and separation of the epithelial joints occurred, giving rise to an exposure of the apical portion of the gland cells. There were marked differences between these two kinds of secretion process in the behavior of the secretory granules prior to secretion and in the mode of discharge of the secretory substances. The changes which occurred during both types of secretion and which, therefore, seemed to be essential to the secretory processes of this gland cell were the swelling up of the gland cells in the initiation of secretion and the reduction of the electron density of the zymogen granules. These secretion-associated ultrastructural changes are discussed in view of the difference in the maturation of the gland cells.

Ontogeny of the hemoglobins of Honmasu (Oncorhynchus rhodurus, HXH), brook trout (Salvelinus fontinalis, BXB) and their reciprocal hybrids (HXB and BXH) was examined electrophoretically as well as spectrophotometrically. All of these fish have electrophoretically distinct larval and adult type hemoglobins and the transition from one type to the other occurred early during post-hatching development. In the case of larval hemoglobins, the electrophoretic pattern of a hybrid, BXH, resembled that of the maternal parent, brook trout (BXB), while the hemoglobin of the reciprocal hybrid, HXB, differed markedly from that of the maternal parent, Honmasu (HXH), in that it possesses a diffuse pattern. Spectrophotometric examinations indicated that this was mostly methemoglobin. Adult type hemoglobins at more advanced stages of both hybrids were electrophoretically resolved into the parental hemoglobins. However, no “hybrid” hemoglobins were detected. Because there was abnormal hemoglobin formation in the larval erythrocytes (erythrocytes L) of the HXB hybrid, and then normal hemoglobin synthesis in the adult erythrocytes (erythrocytes A) of the same fish at later developmental stages, it appears that the paternal effects upon the hemoglobin differed in these two erythroid cell lines of the developing trout.

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.

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 [ 125 I]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 evelope in the fish.

A choriolytic enzyme (hatching enzyme) of the teleost Oryzias latipes was isolated from the culture medium in which many larvae hatched out synchronously by successive fractionation with (NH4)2SO4 precipitation, Sephadex gel filtration and CM-cellulose column chromatography. The enzyme protein is found to represent a single band upon starch gel electrophoresis at pH 8.6 and 5.2, respectively. The specific activity of the isolated enzyme preparation is approximately 180 to 210 times that of the original culture medium as determined by the assay of proteolytic activity toward casein and of clearing (choriolytic) activity toward natural substrate, chorion. The molecular weight of the enzyme as estimated roughly by gel filtration is approximately 8000. Although it seems probable that there are more than one choriolytic and/or proteolytic enzymes in the original medium, the isolated enzyme is found to have a powerful clearing activity as well as proteolytic activity and to be responsible for an effective choriolysis by itself.

1. 1. Some properties of an isolated hatching enzyme of the medaka, Oryzias latipes , were studied. 2. 2. Under the assay conditions, the enzyme was inhibited by high concentrations of Na + , K + , Ca 2+ and Mg 2+ , while it was slightly activated by them at low concentrations. The optimum pH of this enzyme was around 7·9. 3. 3. This enzyme was more stable in neutral or alkaline medium than in acid medium and was inhibited by ethylenediaminetetracetic acid but not by diisopropylfluorophosphate or monoiodoacetamide. 4. 4. A substrate specificity test using some synthetic substrates revealed that this enzyme was medially specific.