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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.

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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.

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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.

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Most teleostean embryos develop and hatch without parental assistance, though some receive parental care. We focused on a paternal brood-care species, the barred-chin blenny (Rhabdoblennius nitidus [Günther, 1861]). As hatching approached, fanning behavior by the male parent drastically increased and then embryos hatch. In the absence of the male parent, most embryos failed to hatch. However, the hatching rate was greatly assisted by introducing an artificial water current, suggesting that paternal assistance other than for aeration is required for successful embryo hatching. Next, we analyzed genes for the hatching enzyme and egg-envelope protein, which were successfully cloned from barred-chin blenny, and found the expression patterns differed from those of other euteleosts. Generally, high choriolytic enzyme swells the intact egg envelope, and then low choriolytic enzyme solubilizes the swollen envelope. The expression levels of both the enzymes, but especially the latter, were much lower in barred-chin blenny that is known in most other oviparous species. In addition, the main component of the egg envelope was changed into ChgHm and choriogenin L (ChgL) in barred-chin blenny, whereas ChgH and ChgL for other euteleosts. These in barred-chin blenny would result in ineffective egg-envelope digestion because the posthatching egg envelopes were observed to be swollen but not solubilized. Male parental assistance by fanning until hatching may compensate for this insufficiency. Our study illustrates an example of the evolution of parent-embryo interaction built on a novel relationship: Degradation of the hatching enzyme/egg-envelope digestion system, accompanied by male parental hatching assistance.

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Environmentally cued hatching is well documented in anurans, enabling embryos to escape diverse threats. However, knowledge of anuran hatching mechanisms is limited and based largely on aquatic-breeding species without known plasticity in hatching timing. Generally, hatching gland cells produce a hatching enzyme that degrades the vitelline membrane. We investigated hatching and its regulation in terrestrial embryos of hourglass treefrogs, Dendropsophus ebraccatus, which accelerate hatching to escape dehydration. We specifically tested if changes in hatching gland cell development or hatching enzyme gene expression are associated with accelerated hatching. We measured perivitelline chamber size of well-hydrated eggs over development as an indicator of breakdown of the vitelline membrane and found that the size of the perivitelline chamber increased steadily until hatching, suggesting gradual hatching enzyme release and vitelline membrane degradation. Hatching gland cells peaked in abundance and began regression substantially prior to hatching, but we found no developmental differences in the abundance or surface area of hatching gland cells between dry and well-hydrated embryos. Hatching enzyme gene expression also peaked early in development then declined, with no difference between hydration treatments. In D. ebraccatus breakdown of the vitelline membrane appears gradual, mediated by hatching enzyme release starting long before hatching. However, hatching acceleration is not associated with ontogenetic changes in hatching gland cell development or hatching enzyme gene expression. This hatching process contrasts with that of red-eyed treefrogs, Agalychnis callidryas, which appear to release enzyme acutely at hatching, yet both species are capable of hatching to escape acute threats.

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In animals, hatching represents the transition point from a developing embryo to a free-living individual, the larva. This process is finely regulated by many endogenous and environmental factors and has been shown to be sensitive to a variety of chemical agents. It is commonly evaluated in bioassays in order to establish the effects of different agents on early development and reproductive capabilities in fish and other aquatic animals. In fish, the breakdown of the chorion is achieved by the secretion of choriolysin by hatching gland cells (HGCs) into the perivitelline space (PVS), coupled with spontaneous movements of the developing larva. In this work, we used zebrafish to assay the effects of a family of widely used agrochemicals-triazoles Triadimefon (FON), Triadimenol (NOL) and free triazole (1,2,4-T)-on hatching success. We found a strong inhibition of hatching by triazole exposure which was correlated with morphological changes and a reduction in the secretory function of the HGCs. As a consequence, the release of choriolytic enzymes by HGCs was reduced. We also found that HGC secretion reduction after exposure to FON can be rescued by co-incubation with a dopamine D2 receptor antagonist but not by antagonists of the D1-like receptors. This suggests a specific pathway through which this family of fungicides may be impairing a critical event in the fish life cycle.

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Hatching enzyme is a protease which can degrade the membrane of egg. In this study, a hatching enzyme was purified from starfish (Asterina pectinifera) with 6.34 fold of purification rate, 5.04 % of yield, and 73.87 U/mg of specific activity. The molecular weight of starfish hatching enzyme was 86 kDa, which was reduced to 62 kDa after removal of N-linked oligosaccharides. The optimal pH and temperature of the hatching enzyme activity were pH 7.0 and 40 °C, respectively, while those of stability were pH 8 and 20 °C. The kinetic parameters, Vmax , Km , K cat and Kcat/Km values were 0.197 U/ml, 0.289 mg/ml, 112.57 s-1, and 389.52 ml/mg s, respectively. Zn2+ increased the enzyme activity by 167.28 %, while EDTA, TPCK, TGCK, leupeptin, PMSF, and TLCK decreased. In addition, Ca2+, Mg2+, and Cu2+ did not affect the enzyme activity. The starfish hatching enzyme activity pretreated with EDTA was recovered by Zn2+. Therefore, the starfish hatching enzyme was classified as a serine-zinc protease.

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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 mesoendodermal 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.

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Zebrafish embryos are increasingly used for developmental toxicity screening of candidate drugs and are occasionally co-incubated with a metabolic activation system at 32°C for 1, 2 or 4h, depending on their developmental stage. As this temperature is higher than the optimal temperature for zebrafish embryonic development (26-28.5°C), we investigated whether continuous incubation of zebrafish embryos from 2.5 until 96h post fertilization (hpf) at high temperatures (30.5-36.5°C) causes malformations. At 32.5°C tail malformations were observed as early as 24hpf, and these became even more prominent at 34.5 and 36.5°C. Cardiovascular and head malformations, edema and blood accumulations throughout the body were present at 36.5°C. Finally, temperatures higher than 28.5°C accelerated embryonic development except for 36.5°C, at which a lower hatching rate and hatching enzyme activity were observed. In conclusion, incubation of zebrafish embryos at 32.5°C and above from 2.5 until 96hpf causes malformations as early as 24hpf.

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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.

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We have analyzed a gene, designated VEB4, that is expressed transiently in very early blastulae of the sea urchin, Strongylocentrotus purpuratus. Sequence analysis of the complete open reading frame shows that VEB4 encodes an unusual, highly charged protein with a pl of 9.55. We show here that VEB4 mRNA accumulate in a spatial pattern that is indistinguishable from that of two other recently described genes encoding metallo-endoproteases, SpAN, related to astacin and SpHE, the hatching enzyme (Reynolds et al. 1992). VEB4 and other members of this gene set encode the earliest strictly zygotic gene products that have been identified. The asymmetric accumulation of VEB4 mRNA in non-vegetal blastomeres of the 16 cell embryo and their descendants reflects the animal-vegetal maternal developmental axis.

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To explore the substrate or subsite specificity of a mouse hatching enzyme, effects of leupeptin [acetyl(P4 )-Leu(P3 )-Leu(P2 )-argininal(P1 )] and its analogs (peptidyl argininals) on mouse blastocyst hatching were investigated. The compounds containing benzyloxycarbonyl group (Z) in the P4 position inhibited the hatching more strongly than those containing acetyl group or unprotected N-terminal amino acid. Among five Z-Leu-P2 -argininals, a derivative containing a P2 Ser residue was the most potent inhibitor, and the derivatives containing Leu, Thr, Pro, and Gly in the P2 position followed in this order. Then, we synthesized four Z-P3 -Ser-argininals and tested their effects on hatching. The result indicated that the compound with Phe residue in the P3 position was the strongest inhibitor, and the Leu-, Pro-, and Ala-containing derivatives were ranked in this order. Thus, among Z-dipeptidyl-argininals tested, Z-Phe-Ser-argininal most potently inhibited the mouse embryonic hatching, suggesting the preference of the mouse hatching enzyme for Phe(P3 )-Ser(P2 )-Arg(P1 ) sequence as a substrate.