Yasumasu Shigeki

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.

ABSTRACT False clownfish (Amphiprion ocellaris) employ a hatching strategy regulated by environmental cues, wherein parents provide water flow to encourage embryos to hatch after sunset on the hatching day. Despite previous studies demonstrating the necessity of complete darkness and water agitation for hatching, the regulatory mechanisms underlying these environmental cues remain elusive. This study aimed to investigate how darkness and water agitation affect the secretion of hatching enzymes and the hatching movements of embryos in false clownfish. Assessment of chorion digestion and live imaging of Ca²⁺ in hatching glands using GCaMP6s, a Ca²⁺ indicator, revealed that darkness stimulation triggers the secretion of hatching enzymes by increasing Ca²⁺ levels in hatching gland cells. On the other hand, water agitation primarily stimulated hatching movements in embryos, which led to the rupture of their egg envelopes. These results suggest that changes in light environments following sunset induce embryos to secrete hatching enzymes and that water agitation provided by parents stimulates hatching movements. These responses to environmental cues, light and water agitation, contribute to the rapid and synchronous hatching in false clownfish.

Teleost oocytes are surrounded by a structure, called chorion or egg envelopes, which is composed of zona pellucida (ZP) proteins. As a result of the gene duplication in teleost, the expression site of the zp genes, coding the major component protein of egg envelopes, changed from the ovary to the maternal liver. In Euteleostei, there are three liver-expressed zp genes, named choriogenin (chg) h, chg hm, and chg l, and the composition of the egg envelope is mostly made up of these Chgs. In addition, ovary-expressed zp genes are also conserved in the medaka genomes, and their proteins have also been found to be minor components of the egg envelopes. However, the specific role of liver-expressed versus ovary-expressed zp genes was unclear. In the present study, we showed that ovary-synthesized ZP proteins first form the base layer of the egg envelope, and then Chgs polymerize inwardly to thicken the egg envelope. To analyze the effects of dysfunction of the chg gene, we generated some chg knockout medaka. All knockout females failed to produce normally fertilized eggs by the natural spawning. The egg envelopes lacking Chgs were significantly thinner, but layers formed by ZP proteins synthesized in the ovary were found in the thin egg envelope of knockout as well as wild-type eggs. These results suggest that the ovary-expressed zp gene is well conserved in all teleosts, including those species in which liver-derived ZP proteins are the major component, because it is essential for the initiation of egg envelope formation.

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.

Steroid hormones play very important roles in gonadal differentiation in many vertebrate species. Previously, we have determined a threshold dosage of testosterone (T) to induce female-to-male sex reversal in Glandirana rugosa frogs. Genetic females formed a mixture of testis and ovary, the so-called ovotestis, when tadpoles of G. rugosa were reared in water containing the dosage of T, which enabled us to detect primary changes in the histology of the masculinizing gonads. In this study, we determined a threshold dosage of estradiol-17β (E2) to cause male-to-female sex reversal in this frog. We observed first signs of histological changes in the ovotestes, when tadpoles were reared in water containing the dosage of E2. Ovotestes were significantly larger than wild-type testes in size. By E2 treatment, male germ cells degenerated in the feminizing testis leading to their final disappearance. In parallel, oocytes appeared in the medulla of the ovotestis and later in the cortex as well. Quantitative polymerase chain reaction analysis revealed that the expression of sex-related genes involved in testis formation was significantly decreased in the ovotestis. In addition, immuno-positive signals of CYP17 that is involved in testis differentiation in this frog disappeared in the medulla first and then in the cortex. These results suggested that oocytes expanded in the feminizing gonad (ovary) contemporaneously with male germ cell disappearance. Primary changes in the histology of the gonads during male-to-female sex reversal occurred in the medulla and later in the cortex. This direction was opposite to that observed during female-to-male sex reversal in the G. rugosa frog.

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.

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.