Effects of melatonin on phosphorylation of memory-related proteins in the hippocampus and the perirhinal cortex in male mice.

We recently demonstrated that a single post-training administration of either melatonin, an MT1/MT2 melatonin receptor agonist ramelteon, or a brain melatonin metabolite N1-acetyl-5-methoxyquinuramine (AMK) enhanced object recognition memory. The present study aims to investigate the effects of melatonin, ramelteon, and AMK on relative phosphorylation levels of memory-related proteins in order to explore candidate signaling pathways associated with the receptor-mediated and nonreceptor-mediated memory-enhancing effects of melatonin. We first confirmed that post-training administration of either melatonin, ramelteon, or AMK at 1 mg/kg promoted long-term memory formation, using the novel object recognition task. Next, the effects of the same doses of these drugs on relative phosphorylation levels of the extracellular signal-regulated kinase (ERK) and calcium/calmodulin-dependent kinases (CaMKs) in the hippocampus and the perirhinal cortex (PRC) were examined by western blot analysis. In the hippocampus, treatment with ramelteon or AMK significantly increased and decreased phosphorylation levels of ERK and cAMP-response element binding protein (CREB) and those of CaMKIIα and ß, respectively. In the PRC, phosphorylation levels of ERK and those of CaMKIIß were significantly increased by both ramelteon and AMK and by ramelteon, respectively. Neither ramelteon nor AMK altered the phosphorylation levels of CaMKIV in either hippocampus or PRC. These results suggest that melatonin may be involved in promoting the formation of long-term object recognition memory in a similar, if not identical, manner by modulating the phosphorylation levels of memory-related proteins such as ERK, CaMKIIs, and CREB in both receptor-mediated and nonreceptor-mediated signaling pathways.

Orphan gene expressed in flame cone cells uniquely found in seahorse epithelium.

The seahorse is one of the most unique teleost fishes in its morphology. The body is surrounded by bony plates and spines, and the male fish possess a brooding organ, called the brood pouch, on their tail. The surfaces of the brood pouch and the spines are surrounded by characteristic so-called flame cone cells. Based on our histological observations, flame cone cells are present in the seahorse Hippocampus abdominalis, but not in the barbed pipefish Urocampus nanus or the seaweed pipefish Syngnathus schlegeli, both of which belong to the same family as the seahorse. In the flame cone cells, we observed expression of an "orphan gene" lacking homologs in other lineages. This gene, which we named the proline-glycine rich (pgrich) gene, codes for an amino acid sequence composed of repetitive units. In situ hybridization and immunohistochemical analyses detected pgrich-positive signals from the flame cone cells. Based on a survey of the genome sequences of 15 teleost species, the pgrich gene is only found from some species of Syngnathiformes (namely, the genera Syngnathus and Hippocampus). The amino acid sequence of the seahorse PGrich is somewhat similar to the sequence deduced from the antisense strand of elastin. Furthermore, there are many transposable elements around the pgrich gene. These results suggest that the pgrich gene may have originated from the elastin gene with the involvement of transposable elements and obtained its novel function in the flame cone cells during the evolution of the seahorse.

LC-MS/MS quantitation of elastin crosslinker desmosines and histological analysis of skin aging characteristics in mice.

Elastic fibers consist of an insoluble inner core of elastin, which confers elasticity and resilience to vertebral organs and tissues. Desmosine (DES) and isodesmosine (IDES) are potential biomarkers of pathologies that lead to decreased elastin turnover. Mice are commonly used in research to mimic humans because of their similar genetics, physiology, and organ systems. The present study thus used senescent accelerated prone (SAMP10) and senescent accelerated resistant (SAMR1) mice to examine the connection between aging and histological or biomolecular changes. Mice were divided into three groups: SAMP10 fed a control diet (CD), SAMP10 fed a high-fat diet (HFD), and SAMR1 fed a CD. The percent liver to total body weight ratio (%LW/BW), desmosines (DESs or DES/IDES) content, and histological alterations in skin samples were evaluated. DESs were quantified using an isotope-dilution liquid chromatography-tandem mass spectrometry method with isodesmosine-13C3,15N1 as the internal standard (ISTD). The assays were repeatable, reproducible, and accurate, with %CV values ≤ (1.90, 1.77, and 3.03), ISTD area %RSD of (1.54, 0.92, and 1.13), and %AC of (99.02 ± 1.86, 101.00 ± 2.30, and 101.30 ± 2.90) for the calibrations (equimolar DES/IDES, DES, and IDES, respectively). The average DESs content per dry-weight abdominal skin and %LW/BW were similar between the three groups. Histological analyses revealed elastin fibers in five randomly selected samples. The epidermis and dermal white adipose tissue layers were thicker in SAMP10 mice than SAMR1 mice. Thus, characteristic signs of aging in SAMP10 and SAMR1 mice could not be differentiated based on measurement of DESs content of the skin or %LW/BW, but aging could be differentiated based on microscopic analysis of histological changes in the skin components of SAMP10 and SAMR1 mice.

Molecular evolution of patristacin genes in teleosts based on the genome survey.

During the evolution of astacin metalloprotease family genes, gene duplication occurred, especially in the lineage of teleosts, in which several types of astacins containing six conserved cysteines (c6ast) emerged. One of them is patristacin, originally found in syngnathid fishes, such as pipefishes and seahorses. Patristacin is expressed in the brood pouch and is present on the same chromosome as other c6ast (pactacin and nephrosin) genes. We first surveyed all the genes from 33 teleost species using a genome database, and characterized the genes by phylogenetic analysis. Pactacin and nephrosin gene homologs were found from all the examined species with only few exceptions, while patristacin gene homologs were found from only several lineages. The patristacin gene homologs were found as multicopy genes in most species of Percomorpha, one of the diverged groups in teleosts. Further diversification of the gene occurred during the evolution of Atherinomorphae, one of the groups in Percomorpha. Fishes of Atherinomorphae possess two types of patristacin, belonging to subclades 1 and 2. Among the Atherinomorpha, we chose the southern platyfish to examine the patristacin gene expression. Platyfish possess eight patristacin gene homologs, called XmPastn1, 2, 3, 4, 5, 7, 10, and 11. Of these genes, only XmPastn2 belongs to subclade 1, while the other seven belong to subclade 2. Only XmPastn2 showed strong expression in several organs of adult platyfish, as observed in reverse-transcription polymerase chain reaction of RNA extracts. Cells expressing XmPastn2 were predominantly mucus-secreting cells found in epidermis around the jaw, as revealed by in-situ hybridization. This result suggests that XmPastn2 is secreted and may contribute to mucus formation or secretion.

N-acetyl-5-methoxykynuramine enhance object location and working memory performances via modulating CaMKII, ERK and CREB phosphorylation.

OBJECTIVES: Melatonin (MEL) has been reported to enhance cognitive performance. Recently, we have demonstrated that a MEL metabolite N-acetyl-5-methoxykynuramine (AMK) promoted the formation of long-term object recognition memory more potently than MEL. Here, we examined the effects of 1 mg/kg MEL and AMK on both object location memory and spatial working memory. We also investigated the effects of the same dose of these drugs on relative phosphorylation/activation levels of memory-related proteins in the hippocampus (HP), the perirhinal cortex (PRC) and the medial prefrontal cortex (mPFC). METHODS: Object location memory and spatial working memory were assessed using the object location task and the Y-maze spontaneous alternation task, respectively. Relative phosphorylation/activation levels of memory-related proteins were assessed using western blot analysis. RESULTS: AMK, as well as MEL, enhanced object location memory and spatial working memory. AMK increased the phosphorylation of cAMP-response element-binding protein (CREB) in both the HP and the mPFC 2 h after the treatment. AMK also increased the phosphorylation of extracellular signal-regulated kinases (ERKs) but decreased that of Ca2+/calmodulin-dependent protein kinases II (CaMKIIs) in the PRC and the mPFC 30 min after the treatment. MEL increased CREB phosphorylation in the HP 2 h after the treatment, whereas no detectable changes in the other proteins examined were observed. CONCLUSION: These results suggested the possibility that AMK exerts stronger memory-enhancing effects than MEL by more remarkably altering the activation of memory-related proteins such as ERKs, CaMKIIs and CREB in broader brain regions, including the HP, mPFC and PRC, compared to MEL.

LC-MS/MS analysis of elastin crosslinker desmosines and microscopic evaluation in clinical samples of patients with hypertrophy of ligamentum flavum.

Ligamentum flavum (LF) pathologies often lead to severe myelopathy or radiculopathy characterized by reduced elasticity, obvious thickening, or worsened ossification. Elastin endows critical mechanical properties to tissues and organs such as vertebrae and ligaments. Desmosine (DES) and isodesmosine (IDES) are crosslinkers of elastin monomers called tropoelastin. These crosslinkers are potential biomarkers of chronic obstructive pulmonary disease. As a biological diagnostic tool that supplements existing symptomatic, magnetic resonance imaging scanning or radiological imaging diagnostic measures for LF hypertrophy and associated pathologies, an isotope-dilution liquid chromatography-tandem mass spectrometry method with selected reaction monitoring mode for the quantitation of DESs in human plasma, urine, cerebrospinal fluid (CSF), and yellow ligamentum was investigated. Isotopically labeled IDES-13C3,15N1 was used as an internal standard (ISTD) for DES quantitation for the first time. The samples plus ISTD were hydrolyzed with 6 N hydrochloric acid. Analytes and ISTD were extracted using a solid phase extraction cellulose cartridge column. The assays were repeatable, reproducible, and accurate with % CV ≤ 7.7, ISTD area % RSD of 7.6, and % AC ≤ (101.2 ± 3.90) of the calibrations. The ligamentum samples gave the highest average DES/IDES content (2.38 µg/mg) on a dry-weight basis. A high percentage of the CSF samples showed almost no DESs. Urine and plasma samples of patients showed no significant difference from the control (p-value = 0.0519 and 0.5707, respectively). Microscopy of the yellow ligamentum samples revealed dark or blue-colored zones of elastin fibers that retained the hematoxylin dye and highly red-colored zones of collagen after counterstaining with van Gieson solution. Thus, we successfully developed a method for DES/IDES quantitation in clinical samples.

Uptake of nanoparticles from sunscreen physical filters into cells arising from increased environmental microwave radiation: increased potential risk of the use of sunscreens to human health.

This study examines the microwave chemical risks posed by photocatalysts present in sunscreens (physical filters) against the increasing use of microwaves (radio waves) in the environment, sometimes referred to as electronic smog. Specifically, the study assesses the damage caused by silica-coated physical filters (photocatalysts, TiO2⋅ and/or ZnO) contained in commercially available sunscreens and fresh silica-coated ZnO for sunscreens to mouse skin fibroblasts cells (NIH/3T3) evaluated in vitro by the life/death of cells using two types of electromagnetic waves: UV light and microwave radiation, and under simultaneous irradiation with both UV light and microwaves. Conditions of the electromagnetic waves were such as to be of lower light irradiance than that of UVA/UVB radiation from incident sunlight, and with microwaves near the threshold power levels that affect human health. The photocatalytic activity of the physical filters was investigated by examining the degradation of the rhodamine B (RhB) dye in aqueous media and by the damage caused to DNA plasmids from E. coli. Compared to the photocatalytic activity of ZnO and TiO2 when irradiated with UV light alone, a clear enhanced photocatalytic activity was confirmed upon irradiating these physical filters concurrently with UV and microwaves. Moreover, the uptake of these metal oxides into the NIH/3T3 cells led to the death of these cells as a result of the enhanced photocatalytic activity of the metal oxides on exposure to microwave radiation.

Brood pouch evolution in pipefish and seahorse based on histological observation.

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.

Lineage-specific evolution of zona pellucida genes in fish.

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.

Molecular evolution of hatching enzymes and their paralogous genes in vertebrates.

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.

Effects of hatching enzymes on egg envelope digestion in the male-brooding seahorse.

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.

Pactacin is a novel digestive enzyme in teleosts.

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 (Hippocampus abdominalis) 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 (Oryzias latipes). 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.

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.

Genome Sequence of the Euryhaline Javafish Medaka, Oryzias javanicus: A Small Aquarium Fish Model for Studies on Adaptation to Salinity.

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, behavior, 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.

Male parental assistance in embryo hatching of barred-chin blenny Rhabdoblennius nitidus.

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.

Translocation of promoter-conserved hatching enzyme genes with intron-loss provides a new insight in the role of retrocopy during teleostean evolution.

The hatcing enzyme gene (HE) encodes a protease that is indispensable for the hatching process and is conserved during vertebrate evolution. During teleostean evolution, it is known that HE experienced a drastic transfiguration of gene structure, namely, losing all of its introns. However, these facts are contradiction with each other, since intron-less genes typically lose their original promoter because of duplication via mature mRNA, called retrocopy. Here, using a comparative genomic assay, we showed that HEs have changed their genomic location several times, with the evolutionary timings of these translocations being identical to those of intron-loss. We further showed that HEs maintain the promoter sequence upstream of them after translocation. Therefore, teleostean HEs are unique genes which have changed intra- (exon-intron) and extra-genomic structure (genomic loci) several times, although their indispensability for the reproductive process of hatching implies that HE genes are translocated by retrocopy with their promoter sequence.

IFPA meeting 2018 workshop report I: Reproduction and placentation among ocean-living species; placental imaging; epigenetics and extracellular vesicles in pregnancy.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2018 there were nine themed workshops, four of which are summarized in this report. These workshops discussed new knowledge and technological innovations in the following areas of research: 1) viviparity in ocean-living species; 2) placental imaging; 3) epigenetics; and 4) extracellular vesicles in pregnancy.

Morphology of brood pouch formation in the pot-bellied seahorse Hippocampus abdominalis.

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.

Comparison of Egg Envelope Thickness in Teleosts and its Relationship to the Sites of ZP Protein Synthesis.

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.

Evolutionary Changes in the Developmental Origin of Hatching Gland Cells in Basal Ray-Finned Fishes.

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.