川口 眞理

Abstract During the fertilization of fish eggs, the hardening of the egg envelope is mediated by transglutaminase (hTGase). After fertilization, TGase undergoes processing. We isolated hTGase from extracts of unfertilized and water-activated rainbow trout eggs. Rainbow trout hTGase (Rt-hTGase) appeared as an 80 kDa protein, and its processed form was 55 kDa. Their N-terminal amino acid sequences were nearly identical, suggesting processing in the C-terminal region. The specific activities were not significantly different, indicating that C-terminal processing does not activate the enzyme itself. We cloned the cDNA by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers followed by RACE-PCR. The deduced amino acid sequence of the cDNA was similar to that of factor XIII subunit A (FXIIIA). Molecular phylogenetic and gene syntenic analyses clearly showed that hTGase was produced by duplication of FXIIIA during the evolution to Teleostei. The 55 kDa processed form of Rt-hTGase is predominantly composed of an enzyme domain predicted from the amino acid sequence of the cDNA. It is hypothesized that the C-terminal domain of Rt-hTGase binds to egg envelope proteins, and that processing allows the enzyme to move freely within the egg envelope, increasing substrate–enzyme interaction and thereby accelerating hardening.

INTRODUCTION: Fishes of the Syngnathidae family are rare in having male pregnancy: males receive eggs from females and egg development occurs in the male brood pouch that diverged during evolution. The family is divided into two subfamilies: Nerophinae and Syngnathinae. METHODS: We compared histologically five types of the brood pouch in Syngnathinae: an open pouch without skinfolds (alligator pipefish); an open pouch with skinfolds (messmate pipefish); a closed pouch with skinfolds (seaweed pipefish); and closed pouches with a sac-like pouch on the tail (pot-bellied seahorse) or within a body cavity (Japanese pygmy seahorse). RESULTS: Histological observations revealed that all the examined species possess vascular egg compartments during the brooding period. The present immunohistochemical study revealed that the pregnant egg compartment epithelium grows thin in both open and closed pouches. The placenta of open and closed pouches is composed of dermis and reticulin fibers, respectively. The closed pouch placenta is a flexible and moist tissue, suitable for substance transport between the father and embryos through the epithelium and blood vessels and responsible for supplying nutrition and removing waste. DISCUSSION: These results suggest that the basic egg incubation structures were established at an early stage of Syngnathinae evolution. On the other hand, it is likely that the innovation of tissue structure, where dermis was replaced with reticular fibers, occurred in closed brood pouches to regulate the pregnant pouch environment. The present study presents the morphological evolutionary pathway of the brood pouch in Syngnathinae, providing a basis for further molecular-level evolutionary studies.

The zona pellucida (ZP) protein constitutes the egg envelope, which surrounds the vertebrate embryo. We performed a comprehensive study on the molecular evolution of ZP genes in Teleostei by cloning and analyzing the expression of ZP genes in fish of Anguilliformes in Elopomorpha, Osteoglossiformes in Osteoglossomorpha, and Clupeiformes in Otocephala to cover unsurveyed fish groups in Teleostei. The present results confirmed findings from our previous reports that the principal organ of ZP gene expression changed from ovary to liver in the common ancestors of Clupeocephala. Even fish species that synthesize egg envelopes in the liver carry the ovary-expressed ZP proteins as minor egg envelope components that were produced by gene duplication during the early stage of Teleostei evolution. The amino acid repeat sequences located at the N-terminal region of ZP proteins are known to be the substrates of transglutaminase responsible for egg envelope hardening and hatching. A repeat sequence was found in zona pellucida Cs of phylogenetically early diverged fish. After changing the synthesis organ, its role is inherited by the N-terminal Pro-Gln-Xaa repeat sequence in liver-expressed zona pellucida B genes of Clupeocephala. These results suggest that teleost ZP genes have independently evolved to maintain fish-specific functions, such as egg envelope hardening and egg envelope digestion, at hatching.

BACKGROUND: Hatching is identified as one of the most important events in the reproduction of oviparous vertebrates. The genes for hatching enzymes, which are vital in the hatching process, are conserved among vertebrates. However, especially in teleost, it is difficult to trace their molecular evolution in detail due to the presence of other C6astacins, which are the subfamily to which the genes for hatching enzymes belong and are highly diverged. In particular, the hatching enzyme genes are diversified with frequent genome translocations due to retrocopy. RESULTS: In this study, we took advantage of the rapid expansion of whole-genome data in recent years to examine the molecular evolutionary process of these genes in vertebrates. The phylogenetic analysis and the genomic synteny analysis revealed C6astacin genes other than the hatching enzyme genes, which was previously considered to be retained only in teleosts, was also retained in the genomes of basal ray-finned fishes, coelacanths, and cartilaginous fishes. These results suggest that the common ancestor of these genes can be traced back to at least the common ancestor of the Gnathostomata. Moreover, we also found that many of the C6astacin genes underwent multiple gene duplications during vertebrate evolution, and the results of gene expression analysis in frogs implied that genes derived from hatching enzyme genes underwent neo-functionalization. CONCLUSIONS: In this study, we describe in detail the molecular evolution of the C6astacin gene in vertebrates, which has not been summarized previously. The results revealed the presence of the previously unknown C6astacin gene in the basal-lineage of jawed vertebrates and large-scale gene duplication of hatching enzyme genes in amphibians. The comprehensive investigation reported in this study will be an important basis for studying the molecular evolution of the vertebrate C6astacin genes, hatching enzyme, and its paralogous genes and for identifying these genes without the need for gene expression and functional analysis.

<title>Abstract</title>Generally, animals extract nutrients from food by degradation using digestive enzymes. Trypsin and chymotrypsin, one of the major digestive enzymes in vertebrates, are pancreatic proenzymes secreted into the intestines. In this investigation, we report the identification of a digestive teleost enzyme, a pancreatic astacin that we termed pactacin. Pactacin, which belongs to the astacin metalloprotease family, emerged during the evolution of teleosts through gene duplication of astacin family enzymes containing six cysteine residues (C6astacin, or C6AST). In this study, we first cloned C6AST genes from pot-bellied seahorse (<italic>Hippocampus abdominalis</italic>) and analyzed their phylogenetic relationships using over 100 C6AST genes. Nearly all these genes belong to one of three clades: pactacin, nephrosin, and patristacin. Genes of the pactacin clade were further divided into three subclades. To compare the localization and functions of the three pactacin subclades, we studied pactacin enzymes in pot-bellied seahorse and medaka (<italic>Oryzias latipes</italic>). In situ hybridization revealed that genes of all three subclades were commonly expressed in the pancreas. Western blot analysis indicated storage of pactacin pro-enzyme form in the pancreas, and conversion to the active forms in the intestine. Finally, we partially purified the pactacin from digestive fluid, and found that pactacin is novel digestive enzyme that is specific in teleosts.

In the present study, we aimed to evaluate the effects of hatching enzymes on the egg envelope digestion during the hatching period in the male brooding seahorse. The complementary DNAs encoding two hatching-enzyme genes, high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE), were cloned and functionally characterized from the lined seahorse (Hippocampus erectus). The genomic-synteny analysis confirmed that teleosts shared LCE gene synteny. In contrast, the genomic location of HCE was found to be conserved with pipefish, but not other teleosts, suggesting that translocation into a novel genomic location occurred. Whole-mount in situ hybridization showed that HCE and LCE mRNAs were expressed in hatching gland cells. To determine the digestion mechanisms of HCE and LCE in hatching, recombinant HCE and LCE were generated and their enzyme activities were examined using fertilized egg envelopes and synthetic peptides. Seahorse HCE and LCE independently digested and softened the egg envelopes of the lined seahorse. Although the egg envelope was digested more following HCE and LCE co-treatment, envelope solubilization was not observed. Indeed, both HCE and LCE showed similar substrate specificities toward four different synthetic peptides designed from the cleavage sites of egg envelope proteins. HCE and LCE proteins from other euteleostean fishes showed different specificities, and the egg envelope was solubilized by the cooperative action of HCE and LCE. These results suggest that the function of LCE was degenerated in the lined seahorse. Our results imply a digestion mechanism for evolutionary adaptation in ovoviviparous fish with male pregnancy.

The genus Oryzias consists of 35 medaka-fish species each exhibiting various ecological, morphological and physiological peculiarities and adaptations. Beyond of being a comprehensive phylogenetic group for studying intra-genus evolution of several traits like sex determination, behaviour, morphology or adaptation through comparative genomic approaches, all medaka species share many advantages of experimental model organisms including small size and short generation time, transparent embryos and genome editing tools for reverse and forward genetic studies. The Java medaka, Oryzias javanicus, is one of the two species of medaka perfectly adapted for living in brackish/sea-waters. Being an important component of the mangrove ecosystem, O. javanicus is also used as a valuable marine test-fish for ecotoxicology studies. Here, we sequenced and assembled the whole genome of O. javanicus, and anticipate this resource will be catalytic for a wide range of comparative genomic, phylogenetic and functional studies. Complementary sequencing approaches including long-read technology and data integration with a genetic map allowed the final assembly of 908 Mbp of the O. javanicus genome. Further analyses estimate that the O. javanicus genome contains 33% of repeat sequences and has a heterozygosity of 0.96%. The achieved draft assembly contains 525 scaffolds with a total length of 809.7 Mbp, a N50 of 6,3 Mbp and a L50 of 37 scaffolds. We identified 21454 predicted transcripts for a total transcriptome size of 57, 146, 583 bps. We provide here a high-quality chromosome scale draft genome assembly of the euryhaline Javafish medaka (321 scaffolds anchored on 24 chromosomes (representing 97.7% of the total bases)), and give emphasis on the evolutionary adaptation to salinity.

Background The reproductive strategies of vertebrates are diverse. Seahorses (Pisces: Syngnathidae) possess the unique characteristic of male pregnancy i.e., males, not females, incubate embryos in a specialized structure called a € brood pouch'. The brood pouch is formed along the ventral midline of the tail. The lumen of the brood pouch is surrounded by loose connective tissue, called pseudoplacenta, and dermis. Results We visualized and evaluated the morphology of brood pouch formation in Hippocampus abdominalis to gain generalizable insights into this process in seahorses. First, we employed several staining methods to characterize the pseudoplacenta and dermis of the brood pouch of mature male seahorses. The pseudoplacenta is composed mainly of reticular fibers, while the dermis is composed mainly of collagenous fibers. Further observations showed that pouch formation is initiated by linear projections of epithelia on both ventrolateral sides of the body. These projections elongated toward the ventral midline, eventually fused together, and then formed a baggy structure composed of a single dermis layer with neither smooth muscle nor pseudoplacenta. Finally, the pseudoplacenta was formed, together with two layers of dermis and smooth muscle. Thus, a fully developed brood pouch was established. The morphology of the luminal epithelium also changed during pouch formation. We analyzed the localization of C-type lectins as markers haCTL II was localized in both the outer and luminal epithelia of the brood pouch throughout development in the male seahorse, whereas haCTL IV, which was not detected in the early stage of seahorse development, became localized only in the luminal epithelium as development proceeded. Conclusions We categorized the processes of brood pouch formation during male seahorse development into three stages: (1) the early stage, characterized by formation of a baggy structure from the primordium (2) the middle stage, characterized by the differentiation and establishment of brood pouch-specific tissues and (3) the late stage, characterized by a fully formed pouch with developing blood vessels and a pouch fold ultimately capable of carrying and incubating embryos.

Teleost egg envelope generally consists of a thin outer layer and a thick inner layer. The inner layer of the Pacific herring egg envelope is further divided into distinct inner layers I and II. In our previous study, we cloned four zona pellucida (ZP) proteins (HgZPBa, HgZPBb, HgZPCa, and HgZPCb) from Pacific herring, two of which (HgZPBa and HgZPCa) were synthesized in the liver and two (HgZPBb and HgZPCb) in the ovary. In this study, we raised antibodies against these four proteins to identify their locations using immunohistochemistry. Our results suggest that inner layer I is constructed primarily of HgZPBa and Ca, whereas inner layer II consists primarily of HgZPBa. HgZPBb and Cb were minor components of the envelope. Therefore, the egg envelope of Pacific herring is primarily composed of liver-synthesized ZP proteins. A comparison of the thickness of the fertilized egg envelopes of 55 species suggested that egg envelopes derived from liver-synthesized ZP proteins tended to be thicker in demersal eggs than those in pelagic eggs, whereas egg envelopes derived from ovarian-synthesized ZP proteins had no such tendency. Our comparison suggests that the prehatching period of an egg with a thick egg envelope is longer than that of an egg with a thin egg envelope. We hypothesized that acquisition of liver-synthesized ZP proteins during evolution conferred the ability to develop a thick egg envelope, which allowed species with demersal eggs to adapt to mechanical stress in the prehatching environment by thickening the egg envelope, while pelagic egg envelopes have remained thin. (C) 2017 Wiley Periodicals, Inc.

Hatching gland cells (HGCs) originate from different germ layers between frogs and teleosts, although the hatching enzyme genes are orthologous. Teleostei HGCs differentiate in the meso-endodermal cells at the anterior end of the involved hypoblast layer (known as the polster) in late gastrula embryos. Conversely, frog HGCs differentiate in the epidermal cells at the neural plate border in early neurula embryos. To infer the transition in the developmental origin of HGCs, we studied two basal ray-finned fishes, bichir (Polypterus) and sturgeon. We observed expression patterns of their hatching enzyme (HE) and that of three transcription factors that are critical for HGC differentiation: KLF17 is common to both teleosts and frogs; whereas FoxA3 and Pax3 are specific to teleosts and frogs, respectively. We then inferred the transition in the developmental origin of HGCs. In sturgeon, the KLF17, FoxA3, and HE genes were expressed during the tailbud stage in the cell mass at the anterior region of the body axis, a region corresponding to the polster in teleost embryos. In contrast, the bichir was suggested to possess both teleost- and amphibian-type HGCs, i.e. the KLF17 and FoxA3 genes were expressed in the anterior cell mass corresponding to the polster, and the KLF17, Pax3 and HE genes were expressed in dorsal epidermal layer of the head. The change in developmental origin is thought to have occurred during the evolution of basal ray-finned fish, because bichir has two HGCs, while sturgeon only has the teleost-type.

The effect of microwave heating (MW) on the activity of a well-known enzyme (catalase) was elucidated by examining the catalase-assisted decomposition of hydrogen peroxide (H2O2 at various heating times (0 to 12 min)). For comparison, conventional water bath heating (WB) was also examined under identical temperature conditions. Microwave radiation had a positive effect on the activity of catalase only over a very short time (less than 3 min), presumably because of the possible disruption of the catalase structural integrity under microwave irradiation at longer times (a negative influence) as evidenced by Gel Permeation Chromatographic (GPC) and MALDI Time-Of-Flight-Mass-Spectrometric (MALDI-TOFMS) analyses. The effect of temperature on the catalase activity was also probed at 39, 37, and 25 degrees C. Results indicate that utilizing a hybrid heating approach with conventional heating (water bath) coupled to microwaves was more effective provided microwave irradiation was carried out for a short time (also less than 3 min). Moreover, it is demonstrated that microwave heating in degrading hydrogen peroxide was most effective when the enzymatic reaction was carried out at a lower temperature, particularly at 25 degrees C.

Six aspartic proteinase precursors, a pro-cathepsin E (ProCatE) and five pepsinogens (Pgs), were purified from the stomach of adult newts (Cynops pyrrhogaster). On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the molecular weights of the Pgs and active enzymes were 37-38 kDa and 31-34 kDa, respectively. The purified ProCatE was a dimer whose subunits were connected by a disulphide bond. cDNA cloning by polymerase chain reaction and subsequent phylogenetic analysis revealed that three of the purified Pgs were classified as PgA and the remaining two were classified as PgBC belonging to C-type Pg. Our results suggest that PgBC is one of the major constituents of acid protease in the urodele stomach. We hypothesize that PgBC is an amphibian-specific Pg that diverged during its evolutional lineage. PgBC was purified and characterized for the first time. The purified urodele pepsin A was completely inhibited by equal molar units of pepstatin A. Conversely, the urodele pepsin BC had low sensitivity to pepstatin A. In acidic condition, the activation rates of newt pepsin A and BC were similar to those of mammalian pepsin A and C1, respectively. Our results suggest that the enzymological characters that distinguish A-and C-type pepsins appear to be conserved in mammals and amphibians.

Syngnathiform fishes carry their eggs in a brood structure found in males. The brood structure differs from species to species: seahorses carry eggs within enclosed brood pouch, messmate pipefish carry eggs in the semi-brood pouch, and alligator pipefish carry eggs in the egg compartment on abdomen. These egg protection strategies were established during syngnathiform evolution. In the present study, we compared the hatching mode of protected embryos of three species. Electron microscopic observations revealed that alligator pipefish and messmate pipefish egg envelopes were thicker than those of seahorses, suggesting that the seahorse produces a weaker envelope. Furthermore, molecular genetic analysis revealed that these two pipefishes possessed the egg envelope-digesting enzymes, high choriolytic enzyme (HCE), and low choriolytic enzyme (LCE), as do many euteleosts. In seahorses, however, only HCE gene expression was detected. When searching the entire seahorse genome by high-throughput DNA sequencing, we did not find a functional LCE gene and only a trace of the LCE gene exon was found, confirming that the seahorse LCE gene was pseudogenized during evolution. Finally, we estimated the size and number of hatching gland cells expressing hatching enzyme genes by whole-mount in situ hybridization. The seahorse cells were the smallest of the three species, while they had the greatest number. These results suggest that the isolation of eggs from the external environment by paternal bearing might bring the egg envelope thin, and then, the hatching enzyme genes became pseudogenized. (C) 2016 Wiley Periodicals, Inc.

We investigated the evolution of the hatching enzyme gene using bester sturgeon (hybrid of Acipencer ruthenus and Huso huso), a basal member of ray-finned fishes. We purified the bester hatching enzyme from hatching liquid, yielding a single band on SDS-PAGE, then isolated its cDNA from embryos by PCR. The sturgeon hatching enzyme consists of an astacin family protease domain and a CUB domain. The CUB domains are present in frog and bird hatching enzymes, but not in teleostei, suggesting that the domain structure of sturgeon hatching enzyme is the tetrapod type. The purified hatching enzyme swelled the egg envelope, and selectively cleaved one of five egg envelope proteins, ZPAX. Xenopus hatching enzyme preferentially digests ZPAX, thus, the egg envelope digestion process is conserved between amphibians and basal ray-finned fish. Teleostei hatching enzymes cleave the repeat sequences at the N-terminal region of ZPB and ZPC, suggesting that the targets of the teleostei hatching enzymes differ from those of amphibians and sturgeons. Such repeat sequences were not found in the N-terminal region of ZPB and ZPC of amphibians and sturgeons. Our results suggest that the change in substrates of the hatching enzymes was accompanied by the mutation of the amino acid sequence of N-terminal regions of ZPB and ZPC. We conclude that the changes in the mechanism of egg envelope digestion, including the change in the domain structure of the hatching enzymes and the switch in substrate, occurred during the evolution of teleostei, likely triggered by the teleost-specific third whole genome duplication. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 720-732, 2015.

The present article examined the advantages of 5.8-GHz microwaves versus the more commonly used 2.45-GHz microwaves using a modular non-commercial apparatus on the proteolysis of the Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly peptide at the amino side of the aspartic acid (Asp) using the Asp-N metallo-endoproteinase enzyme. The microscale sample was subjected to microwaves' electric field (E-field) and magnetic field (H-field) radiation: good temperature control for samples at the mu L scale was achieved using an apparatus that emitted microwaves at a precise frequency (5.800000 GHz). Temperature-dependent experiments with the Asp-N metallo-enzyme confirmed the activity of this enzyme to be greatest at 37 degrees C reached within 15 s on irradiation with the 5.8-GHz microwaves' H-field component with an input power of 1.7W under air cooling conditions. Enzymatic activity decreased significantly above and below this temperature by a slight temperature change of 1 degrees C. Proteolysis yields of the peptide by the Asp-N enzyme at 30-42 degrees C under microwave E-field and H-field heating and under conventional heating revealed that enhancement of the proteolysis of the peptide at 37 degrees C by E-field irradiation (42.6%) was 1.5 times greater than by conventional heating (27.5%), whereas under microwave H-field irradiation (63.5%) it was 2.3 times greater than conventional heating. The relative dielectric loss factors (epsilon(r)'') of the sample solution (peptide + enzyme) were also determined: 22.19 (5.8 GHz) and 12.78 (2.45 GHz) at 22 degrees C, which decreased with increasing temperature (faster for the 5.8-GHz microwaves) so that the initial heating efficiency of 5.8-GHz microwaves was nearly twofold greater than for 2.45-GHz microwaves. Results indicated that in addition to a thermal factor, microwave non-thermal factors also had a significant influence as the microwaves considerably enhanced the proteolytic process relative to traditional heating. (C) 2015 Elsevier B.V. All rights reserved.

Ovoviviparous fish, whose embryonic development and hatching take place in the maternal body, is one of the good model organisms for studying adaptive evolution. Using genome database of the ovoviviparous platy Xiphophorus maculatus, we tried to search hatching enzyme genes (high choriolytic enzyme HCE and low choriolytic enzyme LCE) and egg envelope protein genes (choriogenin H, Hm, and L). Analysis of genes co-localized with them confirmed that shared synteny was found between platy and medaka genome. Both hatching enzyme genes HCE and LCE were pseudogenized in platy. In addition, one of the three choriogenin genes Hm was completely lost from the genome, the other two genes H and L encoded functional proteins. On the other hand, the expression of H and L was very low as compared to oviparous fishes, and the platy egg envelope was extremely thinner. Considering that ovoviviparous fish embryos are protected in the maternal body, an importance of egg envelope for protection of egg/embryo would be reduced in the ovoviviparous fishes. Platy embryos would escape from their thin egg envelope without help of hatching enzymes. In another ovoviviparous fish, black rockfish belonging to different order from the platy, one of the hatching enzyme genes has been reported to be pseudogenized, that is, the embryo of black rockfish can escape from egg envelope by only one hatching enzyme HCE. Adaptive evolution of the hatching strategy of ovoviviparous teleosts may be established by pseudogenization of hatching enzyme genes and/or lowering of expression and/or pseudogenization of hatching enzyme and egg envelope genes. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 41-50, 2015. (c) 2014 Wiley Periodicals, Inc.

We compared several characteristics of the pelagic eggs of Verasper variegatus with those of demersal eggs of Pseudopleuronectes yokohamae, both in the order Pleuronectiformes (halibuts or flatfishes). V. variegatus eggs had about twice the diameter of P. yokohamae eggs. However, the total egg protein weight of P. yokohamae was similar to that of V. variegatus. The specific gravity of P. yokohamae eggs was calculated to be 7-fold that of V. variegatus. The difference in size is the main feature distinguishing the two types of egg. The thickness of the egg envelope of P. yokohamae-more than twice that of V. variegatus-must affect the manner of hatching. The amount of hatching enzyme synthesized in pre-hatching embryo was estimated to be larger in P. yokohamae than V. variegatus. The distribution of hatching gland cells differed between the species. In V. variegates embryos, these were located on the yolk sac as a narrow ring-shaped belt, resulting in cleavage of the egg envelope into two parts by digesting a limited region of the egg envelope, called "rim-hatching". The hatching gland cells of P. yokohamae embryos were distributed all over the surface of the yolk sac, forming a hole through which the embryo could escape. Thus, the location of the hatching gland cells in pre-hatching embryos varied during the evolution of the Pleuronectiformes, depending on the egg type and manner of hatching.

Background: Duplication and subsequent neofunctionalization of the teleostean hatching enzyme gene occurred in the common ancestor of Euteleostei and Otocephala, producing two genes belonging to different phylogenetic clades (clade I and II). In euteleosts, the clade I enzyme inherited the activity of the ancestral enzyme of swelling the egg envelope by cleavage of the N-terminal region of egg envelope proteins. The clade II enzyme gained two specific cleavage sites, N-ZPd and mid-ZPd but lost the ancestral activity. Thus, euteleostean clade II enzymes assumed a new function; solubilization of the egg envelope by the cooperative action with clade I enzyme. However, in Otocephala, the clade II gene was lost during evolution. Consequently, in a late group of Otocephala, only the clade I enzyme is present to swell the egg envelope. We evaluated the egg envelope digestion properties of clade I and II enzymes in Gonorynchiformes, an early diverging group of Otocephala, using milkfish, and compared their digestion with those of other fishes. Finally, we propose a hypothesis of the neofunctionalization process. Results: The milkfish clade II enzyme cleaved N-ZPd but not mid-ZPd, and did not cause solubilization of the egg envelope. We conclude that neofunctionalization is incomplete in the otocephalan clade II enzymes. Comparison of clade I and clade II enzyme characteristics implies that the specificity of the clade II enzymes gradually changed during evolution after the duplication event, and that a change in substrate was required for the addition of the mid-ZPd site and loss of activity at the N-terminal region. Conclusions: We infer the process of neofunctionalization of the clade II enzyme after duplication of the gene. The ancestral clade II gene gained N-ZPd cleavage activity in the common ancestral lineage of the Euteleostei and Otocephala. Subsequently, acquisition of cleavage activity at the mid-ZPd site and loss of cleavage activity in the N-terminal region occurred during the evolution of Euteleostei, but not of Otocephala. The clade II enzyme provides an example of the development of a neofunctional gene for which the substrate, the egg envelope protein, has adapted to a gradual change in the specificity of the corresponding enzyme.

Background: Hatching enzyme is a protease that digests the egg envelope, enabling hatching of the embryo. We have comprehensively studied the molecular mechanisms of the enzyme action to its substrate egg envelope, and determined the gene/protein structure and phylogenetic relationships. Because the hatching enzyme must have evolved while maintaining its ability to digest the egg envelope, the hatching enzyme-egg envelope protein pair is a good model for studying molecular co-evolution of a protease and its substrate. Results: Hatching enzymes from medaka (Oryzias latipes) and killifish (Fundulus heteroclitus) showed species-specific egg envelope digestion. We found that by introducing four medaka-type residue amino acid substitutions into recombinant killifish hatching enzyme, the mutant killifish hatching enzyme could digest medaka egg envelope. Further, we studied the participation of the cleavage site of the substrate in the species-specificity of hatching enzyme. A P2-site single amino acid substitution was responsible for the species-specificity. Estimation of the activity of the predicted ancestral enzymes towards various types of cleavage sites along with prediction of the evolutionary timing of substitutions allowed prediction of a possible evolutionary pathway, as follows: ancestral hatching enzyme, which had relatively strict substrate specificity, developed broader specificity as a result of four amino acid substitutions in the active site cleft of the enzyme. Subsequently, a single substitution occurred within the cleavage site of the substrate, and the recent feature of species-specificity was established in the hatching enzyme-egg envelope system. Conclusions: The present study clearly provides an ideal model for protease-substrate co-evolution. The evolutionary process giving rise to species-specific egg envelope digestion of hatching enzyme was initiated by amino acid substitutions in the enzyme, resulting in altered substrate specificity, which later allowed an amino acid substitution in the substrate.

Fish egg envelopes consist of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among chordates. Euteleosts synthesize ZP proteins in the liver, while elopomorphs synthesize them in the ovaries. In Cypriniformes, zp genes are expressed in the ovaries. We investigated the zp genes of two Otocephalan orders: Clupeiformes (Pacific herring and Japanese anchovy) and Gonorynchiformes (milkfish), which diverged earlier than Cypriniformes. cDNA cloning of zp gene homologs revealed that Pacific herring and Japanese anchovy possessed both ovary- and liver-expressed zp genes however, the zp genes of milkfish were only expressed in the ovaries. Molecular phylogenetic analysis showed that ovary- and liver-expressed zpc genes of two the Clupeiformes formed independent clades. Based on this, we hypothesized the evolution of teleostean zp genes, focusing on the organ expressing zp gene. As in other chordates, the original site of expression of zp genes was likely the ovary. In the early stage of teleostean evolution, the ancestral zp genes acquired the ability to express in the liver. Later, one of the two expression sites became dominant. The liver-expressed zp genes are component proteins of the egg envelope in the Euteleostei. In Otocephala, Clupeiformes possess both ovary- and liver-expressed genes that presumably participate in egg envelope formation, whereas the Gonorynchiformes and Cypriniformes have primarily preserved ovary expressed zp genes. © 2013 Wiley Periodicals, Inc.

We purified eel hatching enzyme (EHE) from the hatching liquid of Japanese eel Anguilla japonica belonging to Elopomorpha to a single band on SDS/PAGE. TOF-MS analysis revealed that the purified EHE contained several isozymes with similar molecular masses. Comparison of the egg envelope digestion specificities of the purified EHE and of recombinant EHE4, one of the EHE isozymes, suggested that the isozymes contained in the purified EHE were functionally the same in terms of egg envelope digestion. By electron microscopy, the egg envelope became swollen after treatment with the purified EHE. The EHE cleavage sites on the zona pellucida (ZP) protein of the egg envelope were located in the N-terminal repeat regions. In previous phylogenetic analysis, we suggested that fishes included in Elopomorpha, as basal teleosts, possess a single type of hatching enzyme genes, and that fishes in Otocephala and Euteleostei gain two types of hatching enzyme genes called clade I and II genes by duplication. Further, the clade I enzymes, zebrafish hatching enzyme (ZHE1) and medaka high choriolytic enzyme (HCE), swell the egg envelope by cleaving the N-terminal regions of ZP proteins, while the clade II enzyme, medaka low choriolytic enzyme (LCE), solubilizes the swollen envelope by cleaving the site at the middle region on the ZP domain. In this evolutionary scenario, our findings support that hatching of Japanese eel conserves the ancestral mechanism of fish egg envelope digestion. The clade I enzymes inherit the ancestral enzyme function, and the clade II enzymes gain a new function during evolution to Otocephala and Euteleostei. Embryo of Japanese eel, an early species in Teleostei, has only a single type of hatching enzyme (EHE). At hatching, a part of egg envelope is softened by EHE. The cleavage sites of EHE are located in the N-terminal repeat regions, not in ZP domain. This manner of EHE is the ancestral form of egg envelope digestion in teleostean evolution. © 2011 FEBS.

We purified two hatching enzymes, namely high choriolytic enzyme (HCE EC 3.4.24.67) and low choriolytic enzyme (LCE EC 3.4.24.66), from the hatching liquid of Fundulus heteroclitus, which were named Fundulus HCE (FHCE) and Fundulus LCE (FLCE). FHCE swelled the inner layer of egg envelope, and FLCE completely digested the FHCE-swollen envelope. In addition, we cloned three Fundulus cDNAs orthologous to cDNAs for the medaka precursors of egg envelope subunit proteins (i.e. choriogenins H, H minor and L) from the female liver. Cleavage sites of FHCE and FLCE on egg envelope subunit proteins were determined by comparing the N-terminal amino acid sequences of digests with the sequences deduced from the cDNAs for egg envelope subunit proteins. FHCE and FLCE cleaved different sites of the subunit proteins. FHCE efficiently cleaved the Pro-X-Y repeat regions into tripeptides to dodecapeptides to swell the envelope, whereas FLCE cleaved the inside of the zona pellucida domain, the core structure of egg envelope subunit protein, to completely digest the FHCE-swollen envelope. A comparison showed that the positions of hatching enzyme cleavage sites on egg envelope subunit proteins were strictly conserved between Fundulus and medaka. Finally, we extended such a comparison to three other euteleosts (i.e. three-spined stickleback, spotted halibut and rainbow trout) and found that the egg envelope digestion mechanism was well conserved among them. During evolution, the egg envelope digestion by HCE and LCE orthologs was established in the lineage of euteleosts, and the mechanism is suggested to be conserved. © 2010 The Authors Journal compilation © 2010 FEBS.

A fish egg envelope is composed of several glycoproteins, called zona pellucida (ZP) proteins, which are conserved among vertebrate species. Euteleost fishes synthesize ZP proteins in the liver, while otocephalans synthesize them in the growing oocyte. We investigated ZP proteins of the Japanese eel, Anguilla japonica, belonging to Elopomorpha, which diverged earlier than Euteleostei and Otocephala. Five major components of the egg envelope were purified and their partial amino acid sequences were determined by sequencing. cDNA cloning revealed that the eel egg envelope was composed of four ZPC homologues and one ZPB homologue. Four of the five eel ZP (eZP) proteins possessed a transmembrane domain, which is not found in the ZP proteins of Euteleostei and Otocephala that diverged later, but is found in most other vertebrate ZP proteins. This result suggests that fish ZP proteins originally possessed a transmembrane domain and lost it during evolution. Northern blotting and RT-PCR revealed that all of the eZP transcripts were present in the ovary, but not in the liver. Phylogenetic analyses of fish zp genes showed that ezps formed a group with other fish zp genes that are expressed in the ovary, and which are distinct from the group of genes expressed in the liver. Our results support the hypothesis that fish ZP proteins were originally synthesized in the ovary, and then the site of synthesis was switched to the liver during the evolutionary pathway to Euteleostei. © 2010 The Authors Journal compilation © 2010 FEBS.

Hatching of medaka embryos from the fertilized egg envelope involves two enzymes, HCE and LCE. HCE swells the envelope and then LCE completely dissolves it. We determined HCE and LCE cleavage sites on the egg envelope that are primarily constructed of two groups of subunit proteins, ZI-1,2 and ZI-3. HCE and LCE cleaved different target sequences on the egg envelope proteins but shared one common cleavage site. HCE cleaved the N-terminal region of ZI-1,2 and ZI-3, mainly the Pro-Xaa-Yaa repeat sequence of ZI-1,2 into hexapeptides, but not the site within a zona pellucida (ZP) domain that is considered to be the core structure of the egg envelope. The cleavage of these N-terminal regions results in swelling and softening of the envelope. LCE cleaved the middle of the ZP domain of ZI-1,2, in addition to the upstream of the trefoil domain of ZI-1,2 and the ZP domain of ZI-3. This middle site is in the intervening sequence connecting two subdomains of the ZP domain. Cleaving this site would result in the solubilization of the swollen egg envelope by the disruption of the filamentous structure that is thought to be formed by the non-covalent polymerization of ZP domains.

Background: Hatching enzyme, belonging to the astacin metallo-protease family, digests egg envelope at embryo hatching. Orthologous genes of the enzyme are found in all vertebrate genomes. Recently, we found that exon intron structures of the genes were conserved among tetrapods, while the genes of teleosts frequently lost their introns. Occurrence of such intron losses in teleostean hatching enzyme genes is an uncommon evolutionary event, as most eukaryotic genes are generally known to be interrupted by introns and the intron insertion sites are conserved from species to species. Here, we report on extensive studies of the exon-intron structures of teleostean hatching enzyme genes for insight into how and why introns were lost during evolution. Results: We investigated the evolutionary pathway of intron-losses in hatching enzyme genes of 27 species of Teleostei. Hatching enzyme genes of basal teleosts are of only one type, which conserves the 9-exon-8-intron structure of an assumed ancestor. On the other hand, otocephalans and euteleosts possess two types of hatching enzyme genes, suggesting a gene duplication event in the common ancestor of otocephalans and euteleosts. The duplicated genes were classified into two clades, clades I and II, based on phylogenetic analysis. In otocephalans and euteleosts, clade I genes developed a phylogeny-specific structure, such as an 8-exon-7-intron, 5-exon-4-intron, 4-exon-3-intron or intron-less structure. In contrast to the clade I genes, the structures of clade II genes were relatively stable in their configuration, and were similar to that of the ancestral genes. Expression analyses revealed that hatching enzyme genes were high-expression genes, when compared to that of housekeeping genes. When expression levels were compared between clade I and II genes, clade I genes tends to be expressed more highly than clade II genes. Conclusions: Hatching enzyme genes evolved to lose their introns, and the intron-loss events occurred at the specific points of teleostean phylogeny. We propose that the high-expression hatching enzyme genes frequently lost their introns during the evolution of teleosts, while the low-expression genes maintained the exon-intron structure of the ancestral gene.

Pacific herring Clupea pallasii and Japanese anchovy Engraulis japonicus, which belong to the same order Clupeiformes, spawn different types of eggs: demersal adherent eggs and pelagic eggs, respectively. We cloned three cDNAs for Pacific herring hatching enzyme and five for Japanese anchovy. Each of them was divided into two groups (group A and B) by phylogenetic analysis. They were expressed specifically in hatching gland cells (HGCs), which differentiated from the pillow and migrated to the edge of the head in both species. HGCs of Japanese anchovy stopped migration at that place, whereas those of Pacific herring continued to migrate dorsally and distributed widely all over the head region. During evolution, the program for the HGC migration would be varied to adapt to different hatching timing. Analysis of the gene expression revealed that Pacific herring embryos synthesized a large amount of hatching enzyme when compared with Japanese anchovy. Chorion of Pacific herring embryo was about 7.5 times thicker than that of Japanese anchovy embryo. Thus, the difference in their gene expression levels between two species is correlated with the difference in the thickness of chorion. These results suggest that the hatching system of each fish adapted to its respective hatching environment. Finally, hatching enzyme genes were cloned from each genomic DNA. The exon-intron structure of group B genes basically conserved that of the ancestral gene, whereas group A genes lost one intron. Several gene-specific changes of the exon-intron structure owing to nucleotide insertion and/or duplication were found in Japanese anchovy genes. © 2008 Wiley-Liss, Inc.

The hatching enzyme of oviparous euteleostean fishes consists of two metalloproteases: high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE). They cooperatively digest the egg envelope (chorion) at the time of embryo hatching. In the present study, we investigated the hatching of embryos of the ovoviviparous black rockfish Sebastes schlegelii. The chorion-swelling activity, HCE-like activity, was found in the ovarian fluid carrying the embryos immediately before the hatching stage. Two kinds of HCE were partially purified from the fluid, and the relative molecular masses of them matched well with those deduced from two HCE cDNAs, respectively, by MALDI-TOF MS analysis. On the other hand, LCE cDNAs were cloned however, the ORF was not complete. These results suggest that the hatching enzyme is also present in ovoviviparous fish, but is composed of only HCE, which is different from the situation in other oviparous euteleostean fishes. The expression of the HCE gene was quite weak when compared with that of the other teleostean fishes. Considering that the black rockfish chorion is thin and fragile, such a small amount of enzyme would be enough to digest the chorion. The black rockfish hatching enzyme is considered to be well adapted to the natural hatching environment of black rockfish embryos. In addition, five aberrant spliced LCE cDNAs were cloned. Several nucleotide substitutions were found in the splice site consensus sequences of the LCE gene, suggesting that the products alternatively spliced from the LCE gene are generated by the mutations in intronic regions responsible for splicing. © 2008 The Authors.

Using gene cloning and in silico cloning, we analyzed the structures of hatching enzyme gene orthologs of vertebrates. Comparison led to a hypothesis that hatching enzyme genes of Japanese eel conserve an ancestral structure of the genes of fishes, amphibians, birds and mammals. However, the exon-intron structure of the genes was different from species to species in Teleostei: Japanese eel hatching enzyme genes were 9-exon-8-intron genes, and zebrafish genes were 5-exon-4-intron genes. In the present study, we further analyzed the gene structures of fishes belonging to Acanthopterygii. In the species of Teleostei we examined, diversification of hatching enzyme gene into two paralogous genes for HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) was found only in the acanthopterygian fishes such as medaka Oryzias latipes, Fundulus heteroclitus, Takifugu rubripes and Tetraodon nigroviridis. In addition, the HCE gene had no intron, while the LCE gene consisted of 8 exons and 7 introns. Phylogenetic analysis revealed that HCE and LCE genes were paralogous to each other, and diverged during the evolutionary lineage to Acanthopterygii. Analysis of gene synteny and cluster structure showed that the syntenic genes around the HCE and LCE genes were highly conserved between medaka and Teraodon, but such synteny was not found around the zebrafish hatching enzyme genes. We hypothesize that the zebrafish hatching enzyme genes were translocated from chromosome to chromosome, and lost some of their introns during evolution. (c) 2006 Elsevier B.V. All rights reserved.