PurposeSpermiogenesis, the process of deformation of sperm head morphology and flagella formation, is a phenomenon unique to sperm. Axonemal dynein light chain proteins are localized to sperm flagella and are known to be involved in sperm... more
PurposeSpermiogenesis, the process of deformation of sperm head morphology and flagella formation, is a phenomenon unique to sperm. Axonemal dynein light chain proteins are localized to sperm flagella and are known to be involved in sperm motility. Here, we focused on the gene axonemal dynein light chain domain containing 1 (Axdnd1) with the aim to determine the function of its protein product AXDND1.MethodsTo elucidate the role of AXDND1 in spermatogenesis, we generated Axdnd1 knockout (KO) mice using the CRISPR/Cas9 system. The generated mice were subjected to fertility tests and analyzed by immunohistochemistry.ResultThe Axdnd1 KO mouse exhibited sterility caused by impaired spermiogenesis during the elongation step as well as abnormal nuclear shaping and manchette, which are essential for spermiogenesis. Moreover, AXDND1 showed enriched testicular expression and was localized from the mid‐pachytene spermatocytes to the early spermatids.ConclusionAxdnd1 is essential for spermatog...
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The present experiment was undertaken to investigate the seasonal variations of levels of prostaglandins (PGs) and regulation of these levels in the ovary and hemolymph of the scallop. The levels of prostaglandin F2 alpha (PGF2 alpha) and... more
The present experiment was undertaken to investigate the seasonal variations of levels of prostaglandins (PGs) and regulation of these levels in the ovary and hemolymph of the scallop. The levels of prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) in the hemolymph and ovary increased during sexual maturation, and these levels in the ovary showed a marked increase in the spawning season. Consecutive administration of antiestrogen inhibited the increase of the levels of PGF2 alpha and PGE2 during sexual maturation. These results indicate that the seasonal variations of the levels of PGF2 alpha and PGE2 are closely related to the reproductive cycle, suggesting that PGF2 alpha and PGE2 may be involved in the sexual maturation and spawning of the scallop. Furthermore, it was supposed that estrogen likely plays a role in the regulation of PGs production in female, well known in mammals.
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Research Interests: Aquaculture and Fishery
Previously, we isolated jacalin-related lectins termed PPL2, PPL3 (PPL3A, 3B and 3C) and PPL4 from the mantle secretory fluid of Pteria penguin (Mabe) pearl shell. They showed the sequence homology with the plant lectin family,... more
Previously, we isolated jacalin-related lectins termed PPL2, PPL3 (PPL3A, 3B and 3C) and PPL4 from the mantle secretory fluid of Pteria penguin (Mabe) pearl shell. They showed the sequence homology with the plant lectin family, jacalin-related β-prism fold lectins (JRLs). While PPL3s and PPL4 shared only 35%–50% homology to PPL2A, respectively, they exhibited unique carbohydrate binding properties based on the multiple glycan-binding profiling data sets from frontal affinity chromatography analysis. In this paper, we investigated biomineralization properties of these lectins and compared their biomineral functions. It was found that these lectins showed different effects on CaCO3 crystalization, respectively, although PPL3 and PPL2A showed similar carbohydrate binding specificities. PPL3 suppressed the crystal growth of CaCO3 calcite, while PPL2A increased the number of contact polycrystalline calcite composed of more than one crystal with various orientations. Furthermore, PPL4 alo...
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The diversity of protostomes is demonstrated by the classification of these animals into more than 20 phyla and 1,000,000 species. Many species of aquatic protostomes are considered valuable for basic studies of reproduction as well as... more
The diversity of protostomes is demonstrated by the classification of these animals into more than 20 phyla and 1,000,000 species. Many species of aquatic protostomes are considered valuable for basic studies of reproduction as well as for fishery resources. The aim of this chapter is to provide a brief overview of the process and mechanism of fertilization in three protostome groups, the mollusks, annelids, and arthropods, in which various modes of sexual reproduction have evolved. Regarding the series of interactions between oocytes and sperm at fertilization, we have described the structural changes in gametes and the regulatory mechanisms of polyspermy block and increases in intracellular Ca2+, which enable the successful fertilization of oocytes. Prior to fertilization, oocytes and sperm are produced and matured in gonads and released at the optimal time. The second half of the article focuses on gonial cell multiplication, oocyte growth, and spawning (oocyte release and sperm ...
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We determined the primary structures of jacalin-related lectins termed PPL3s (PPL3A, 3B, and 3C, which are dimers consisting of sequence variants α + α, α + β, β + β, respectively) and PPL4, which is heterodimer consisting of α + β... more
We determined the primary structures of jacalin-related lectins termed PPL3s (PPL3A, 3B, and 3C, which are dimers consisting of sequence variants α + α, α + β, β + β, respectively) and PPL4, which is heterodimer consisting of α + β subunits, isolated from mantle secretory fluid of Pteria penguin (Mabe) pearl shell. Their carbohydrate-binding properties were analyzed, in addition to that of PPL2A, which was previously reported as a matrix protein. PPL3s and PPL4 shared only 35–50% homology to PPL2A, respectively; they exhibited significantly different carbohydrate-binding specificities based on the multiple glycan binding profiling data sets from frontal affinity chromatography analysis. The carbohydrate-binding specificity of PPL3s was similar to that of PPL2A, except only for Man3Fuc1Xyl1GlcNAc2 oligosaccharide, while PPL4 showed different carbohydrate-binding specificity compared with PPL2A and PPL3s. PPL2A and PPL3s mainly recognize agalactosylated- and galactosylated-type glycan...
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In the published article, “Phenotypic Stability of Sex and Expression of Sex Identification Markers in the Adult Yesso Scallop Mizuhopecten yessoensis throughout the Reproductive Cycle. [...]
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The objective of the present study was to analyze the phenotypic stability of sex after sex differentiation in the Yesso scallop, which is a gonochoristic species that has been described as protandrous. So far, no study has investigated... more
The objective of the present study was to analyze the phenotypic stability of sex after sex differentiation in the Yesso scallop, which is a gonochoristic species that has been described as protandrous. So far, no study has investigated in detail the sexual fate of the scallop after completion of sex differentiation, although bivalve species often show annual sex change. In the present study, we performed a tracking experiment to analyze the phenotypic stability of sex in scallops between one and two years of age. We also conducted molecular marker analyses to describe sex differentiation and gonad development. The results of the tracking experiment revealed that all scallops maintained their initial sex phenotype, as identified in the last reproductive period. Using molecular analyses, we characterized my-dmrt2 and my-foxl2 as sex identification markers for the testis and ovary, respectively. We conclude by proposing that the Yesso scallop is a sex-stable bivalve after its initial ...
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There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis. The objective of this paper is to characterize candidate invGnRH peptides... more
There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis. The objective of this paper is to characterize candidate invGnRH peptides of Yesso scallop Patinopecten yessoensis (i.e., peptide identification, immunohistochemical localization, and immunoquantification) in order to reveal their bioactive form in bivalves. Using mass spectrometry (MS), we identified two invGnRH (py-GnRH) peptides from the scallop nerve ganglia: a precursor form of py-GnRH peptide (a non-amidated dodecapeptide; py-GnRH12aa-OH) and a mature py-GnRH peptide (an amidated undecapeptide; py-GnRH11aa-NH2). Immunohistochemical staining allowed the localization of both py-GnRH peptides in the neuronal cell bodies and fibers of the cerebral and pedal ganglia (CPG) and the visceral ganglion (VG). We found that the peptides showed a dimorphic distribution pattern. Notably, the broad distribution of mature py-GnRH i...
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Members of the vertebrate CYP3A subfamily are involved in the metabolism of steroids and a wide range of xenobiotics. In this study two CYP3A-like mRNAs have been isolated from the mussel (Mytilus edulis), and their seasonal expression... more
Members of the vertebrate CYP3A subfamily are involved in the metabolism of steroids and a wide range of xenobiotics. In this study two CYP3A-like mRNAs have been isolated from the mussel (Mytilus edulis), and their seasonal expression profile and modulation by estrogens examined. Sexual dimorphism of CYP3A-like mRNA expression was not observed in mussel gonads of individuals collected throughout a year. Nevertheless, natural variation in gonadal CYP3A-like mRNA expression was observed, with highest levels of CYP3A isoform1 and lowest levels of CYP3A isoform2 mRNA during the maturation and spawning season. Exposure to a 10% sewage treatment works extract did not result in any significant changes in mRNA expression of CYP3A-like. In contrast, exposure to E2 (200 ng/L) and TBT (100 ng/L) significantly down-regulated the expression of CYP3A-like isoform1 but not CYP3A-like isoform2 suggesting differential regulation.
Research Interests: Marine And Environmental Pollution, International Marine Environmental Law, Biological Sciences, GE, Environmental Sciences, and 12 moreEndocrine disruptors, Animals, CHEMICAL SCIENCES, Sewage, Isoenzymes, Mytilus edulis, Endocrine Disruptors, Amino Acid Sequence, Elsevier, Gonads, Down-Regulation, and Molecular Sequence Data
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Research Interests: Biology, Environmental Pollution, Medicine, Multidisciplinary, Estrogen Receptor, and 15 moreEndocrine disruptors, Female, Animals, Male, Statistical Significance, mRna expression levels, Reproductive cycle, Estrogens, Natural Variation, Mytilus edulis, Endocrine Disruptors, Elsevier, Environmental, Gametogenesis, and Estrogen Receptor beta
ABSTRACT mantle > adductor muscle. Bioconcentration factors for TBTO in the oyster ranged from 2400 to 7800. Accumulated TBTO in the gill was readily eliminated up to the control level, while the gonad retained the highest level of... more
ABSTRACT mantle > adductor muscle. Bioconcentration factors for TBTO in the oyster ranged from 2400 to 7800. Accumulated TBTO in the gill was readily eliminated up to the control level, while the gonad retained the highest level of TBTO. The influence of accumulated TBTO in the ovary and testis on fertilization and embryonic development was investigated. The rates of fertilization and development declined with increasing concentration of TBTO in the ovary, whereas no relationship between these rates and TBTO in the testis was observed. These results lead to the tentative conclusion that TBTO accumulated and retained in the eggs would interfere with embryonic development in the oyster.
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Research Interests: Aquatic Toxicology, Biology, Medicine, Biological Sciences, Environmental Sciences, and 15 moreHumans, Endocrine disruptors, Female, Animals, Male, CHEMICAL SCIENCES, mRna expression levels, Estrogens, Endocrine Disruptors, Amino Acid Sequence, Estradiol, Gonads, Gametogenesis, Gene Expression Regulation, and Molecular Sequence Data
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ABSTRACT The kinetic profiles of vitellin accumulation in the oyster ovary during oocyte growth and the effects in vivo and in vitro of estradiol-17β (E2) on vitellin formation were examined in this study. The relative vitellin content... more
ABSTRACT The kinetic profiles of vitellin accumulation in the oyster ovary during oocyte growth and the effects in vivo and in vitro of estradiol-17β (E2) on vitellin formation were examined in this study. The relative vitellin content measured by an enzyme-linked immunosorbent assay (ELISA) shows an apparent increase as the oocyte develops. Immunoblotting of the vitellin using anti-vitellin indicated that two main bands (179 and 110 kD), which begin to accumulate at an early stage of maturation, become pronounced during oocyte growth. Meanwhile, the major peak of the intact form of vitellin (530 kD) in gel filtration also enlarges with oocyte growth, supporting the results of immunoblot analysis and vitellin determination. E2 treatment in vivo causes significant increases in oocyte diameter and vitellin content in the female oyster. A similar trend was observed in ovarian tissues cultured in the presence of E2. It is concluded that E2 is one of the major factors which control the vitellogenesis in the oyster and that the ovary is undoubtedly the site of synthesis of vitellin.
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ABSTRACT 1. The present study was undertaken to determine the levels of endogenous catecholamines in various tissues of Patinopecten yessoensis, Crassostrea gigas and Mytilus edulis using high performance liquid chromatography (HPLC).2.... more
ABSTRACT 1. The present study was undertaken to determine the levels of endogenous catecholamines in various tissues of Patinopecten yessoensis, Crassostrea gigas and Mytilus edulis using high performance liquid chromatography (HPLC).2. Dopamine and noradrenaline were identified in the ganglia, gills and gonads. Dopamine levels in all tissue were from three to one hundred thirty times as much as noradrenaline levels. In the scallop, dopamine and noradrenaline levels decreased in the order of ganglia, gill and gonad. In the oyster, dopamine levels decreased in the order of gill and gonad, however both tissues showed the same amount of noradrenaline. Dopa and adrenaline were not detected in any of the tissues under the present experimental conditions.3. Dopamine levels of the scallops decreased during spawning induced by U.V. ray-irradiated seawater, whereas almost no change of noradrenaline levels were observed. These results suggest that the release of dopamine may be especially implicated in the spawning of scallops.
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ABSTRACT 1. The present study was undertaken to determine the levels of endogenous catecholamines in various tissues of Patinopecten yessoensis, Crassostrea gigas and Mytilus edulis using high performance liquid chromatography (HPLC).2.... more
ABSTRACT 1. The present study was undertaken to determine the levels of endogenous catecholamines in various tissues of Patinopecten yessoensis, Crassostrea gigas and Mytilus edulis using high performance liquid chromatography (HPLC).2. Dopamine and noradrenaline were identified in the ganglia, gills and gonads. Dopamine levels in all tissue were from three to one hundred thirty times as much as noradrenaline levels. In the scallop, dopamine and noradrenaline levels decreased in the order of ganglia, gill and gonad. In the oyster, dopamine levels decreased in the order of gill and gonad, however both tissues showed the same amount of noradrenaline. Dopa and adrenaline were not detected in any of the tissues under the present experimental conditions.3. Dopamine levels of the scallops decreased during spawning induced by U.V. ray-irradiated seawater, whereas almost no change of noradrenaline levels were observed. These results suggest that the release of dopamine may be especially implicated in the spawning of scallops.
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Vertebrate-like sex steroid hormones have been widely detected in mollusks, and numerous experiments have shown the importance of steroids in gonad development. Nevertheless, their signaling pathways in invertebrates have not been... more
Vertebrate-like sex steroid hormones have been widely detected in mollusks, and numerous experiments have shown the importance of steroids in gonad development. Nevertheless, their signaling pathways in invertebrates have not been uncovered yet. Steroid receptors are an ancient class of transcription factors with multiple roles in not only vertebrates but also invertebrates. Estrogen signaling is thought to have major roles in mollusk physiology, but the full repertoire of estrogen receptors is unknown. We presented the successful cloning of two novel forms of estrogen receptor-like genes. These receptors are present in two closely related species of Mytilus: Mytilus edulis and Mytilus galloprovincialis, commonly known and widely distributed sentinel species. Our phylogenetic analysis revealed that one of these receptors is an estrogen receptor (ER) and the other one is an estrogen-related receptor (ERR). Studies of expression analysis showed that both receptor mRNAs were localized in the oocytes and follicle cells in contact with developing oocytes in the ovary and Sertoli cells in the testis, and in the ciliated cells of the gill. In addition, we have evidence that one (ER) of these may have a capacity to autoregulate its own expression in the gonadal cells by estrogen (E2) and that this gene is responsive to estrogenic compounds.
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This study was undertaken to describe and quantitatively analyze the pattern of development of gametes in the Japanese scallop Patinopecten yessoensis. Incorporation of 5-bromo-2′-deoxyuridine (BrdU) into gonial cells was used to... more
This study was undertaken to describe and quantitatively analyze the pattern of development of gametes in the Japanese scallop Patinopecten yessoensis. Incorporation of 5-bromo-2′-deoxyuridine (BrdU) into gonial cells was used to quantitatively detect mitotically active gonial cells. Oogonia increased in number from November to December and decreased rapidly to March. A small number of oocytes was detected in November. Oocytes steadily increased in number and size up to March. The number of spermatogonia slightly increased from November to December, and increased markedly from January to March. Both ratios of BrdU-immunopositive gonial cells in the ovary and testis to gonial cells moderately increased from September to December. The ratio of BrdU-immunopositive spermatogonia to gonial cells drastically increased from January to February and kept an elevated level in March, whereas the oogonia started to disappear in January. The results suggest that the pattern of proliferation of g...
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Factors that inhibit and stimulate the initiation of sperm motility were determined for Manila clam (Ruditapes philippinarum), Pacific oyster (Crassostrea gigas), and Japanese scallop (Patinopecten yessoensis). Compared with artificial... more
Factors that inhibit and stimulate the initiation of sperm motility were determined for Manila clam (Ruditapes philippinarum), Pacific oyster (Crassostrea gigas), and Japanese scallop (Patinopecten yessoensis). Compared with artificial seawater (ASW), serotonin (5-hydroxytryptamine creatinine sulfate, 5-HT) could fully trigger sperm motility and increase sperm velocity and motility duration. Sperm motility was decreased in ASW at pH 6.5–7.0 and suppressed at pH 4.0. In Manila clam and Pacific oyster, 5-HT could overcome the inhibitory effects of acidic pH on sperm motility. In the presence of nigericin (a K+/H+ exchanger), sperm motility was only triggered at pH 8.3. Testicular fluid K+ concentrations were two- to fourfold higher than that in ASW. Sperm motility and velocity were decreased in ASW or 5-HT containing ≥40 mM K+ or ≥2.5 mM 4-aminopyridine, suggesting K+ efflux requirement to initiate motility. Sperm motility and velocity were reduced in ASW or 5-HT containing EGTA or W-...
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ABSTRACT The kinetic profiles of vitellin degradation, protease activity and free amino acids in the embryo and larvae of the Pacific oyster, Crassostrea gigas, have been investigated during the period from the unfertilized egg through... more
ABSTRACT The kinetic profiles of vitellin degradation, protease activity and free amino acids in the embryo and larvae of the Pacific oyster, Crassostrea gigas, have been investigated during the period from the unfertilized egg through the 48 h straight-hinge larva. Immunoblotting using anti-vitellin showed that two major bands (179 and 110 kD) and several faint bands detected in the unfertilized egg become weak at the trochophore stage and disappear 48 h postfertilization. In gel filtration the main peak of the intact molecule of vitellin, estimated to be 530 kD, tended to become low from the blastula stage onwards. The relative vitellin content determined by enzyme-linked immunosorbent assay showed the same decreasing pattern as in gel filtration. The increase in the protease activity during larval development agreed well with the timing of the vitellin degradation and this protease directly degraded larval vitellin protein. The total free amino acids drastically increased at the same time as the increase in protease activity and were reduced at the 24 h straight-hinge larva stage. The results obtained here suggest that oyster vitellin is degraded during larval development and free amino acids, generated by hydrolysis of vitellin protein, may play a role in embryonic and larval development.
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Serotonin (5-hydroxytriptamin, 5-HT) triggers germinal vesicle breakdown (GVBD) of oocytes and the transporting of the mature oocyte through the gonoduct via cilia motility in bivalves. The 5-HT receptor in the oocyte membrane of the... more
Serotonin (5-hydroxytriptamin, 5-HT) triggers germinal vesicle breakdown (GVBD) of oocytes and the transporting of the mature oocyte through the gonoduct via cilia motility in bivalves. The 5-HT receptor in the oocyte membrane of the Japanese scallop, Patinopecten yessoensis, has been pharmacologically characterized as a mixed profile of 5-HT(1)/5-HT(2) and is induced by estradiol-17beta (E(2)). Here we report the isolation, cloning, and tissue expression of the 5-HT receptor from the gonad of the Japanese scallop. A full-length cDNA (1818 bp) encoding a putative 5-HT receptor (5-HT(py)) of 454 amino acid residues was isolated from the ovary and shared 53.3% and 40.2% homology with the Aplysia 5-HT(1ap) and mouse 5-HT(1A), respectively. The 5-HT(py) sequence possessed typical characteristics of 5-HT(1), including seven transmembrane domains, a long third inner loop, and a short fourth inner terminal. Phylogenetic analysis suggested that 5-HT(py) was classified into the 5-HT(1) subtype as well as other invertebrate 5-HT(1) receptors. Semi-quantitative RT-PCR showed the expression of the 5-HT(py) gene in both the nervous system and peripheral tissues and the induction of expression by E(2) in the ovarian tissue. In situ hybridization revealed a strong 5-HT(py) signal in the oocytes, spermatids, and ciliary epithelium of the gonoducts in the ovary and testis. These results suggest that the effects of 5-HT on the induction of oocyte maturation, sperm motility, and transport of mature oocytes and sperm through the ciliated epithelium of the gonoducts are mediated by 5-HT(py).
Research Interests: Physiology, Zoology, In Situ Hybridization, Serotonin, Female, and 13 moreAnimals, Male, Serotonin Receptors, Spawning, Pectinidae, Receptor, Veterinary Sciences, Amino Acid Sequence, Base Sequence, Gonads, Molecular Sequence Data, Serotonin receptor, and reverse transcriptase polymerase chain reaction
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In bivalves, serotonin (5-hydroxytriptamine, 5-HT) acts as a major promotional factor in oocyte maturation, sperm motility, and sequential spawning. The previously reported novel neuronal protein, oocyte maturation arresting factor (OMAF)... more
In bivalves, serotonin (5-hydroxytriptamine, 5-HT) acts as a major promotional factor in oocyte maturation, sperm motility, and sequential spawning. The previously reported novel neuronal protein, oocyte maturation arresting factor (OMAF) that was found in the central nervous system and hemolymph of the Japanese scallop, Patinopecten yessoensis, has an inhibitory activity in the 5-HT-induced oocyte maturation via a receptor-mediated mechanism, resulting in an arrest of spawning [30]. In this study, OMAF protein was isolated from the supernatant of hemolymph of the scallop using gel and anion-exchange chromatography, and SDS-PAGE. Three digested partial peptides with 4, 11, and 16 amino acid residues were determined through reversed-phase HPLC and amino acid sequencing. The anti-OMAF antibodies generated against the obtained peptides with 11 and 16 amino acid residues were applied to immunohistochemistry and 5-HT-induced spawning and oocyte maturation assays. Fusiform OMAF neurons were localized in the external area of the anterior lobe of the cerebral ganglion, supporting our presumption that OMAF was secreted from the cerebral and pedal ganglia (CPG). Pretreatment with anti-OMAF antibody on three kinds of bivalve species showed a strong in vivo amplification of 5-HT-induced release of egg and sperm, and an in vitro restoration of 5-HT-induced germinal vesicle breakdown (GVBD) from inhibition by the CPG extract, suggesting the release from suppressive activity of OMAF due to the absorption with antibody. These results confirm that the isolated peptides are from OMAF and OMAF acts as an inhibitor of 5-HT-induced oocyte maturation and sperm motility as previously reported.
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Mantle tissue extracts from the Pacific oyster, Crassostrea gigas, exhibited anti-Gram-positive bacterial and lysozyme activities over a wide pH range, suggesting that multiple defensive mantle lysozymes were present. Degenerated... more
Mantle tissue extracts from the Pacific oyster, Crassostrea gigas, exhibited anti-Gram-positive bacterial and lysozyme activities over a wide pH range, suggesting that multiple defensive mantle lysozymes were present. Degenerated reverse-transcription PCR detected the expression of two mantle lysozymes, CGL-1 and a novel lysozyme CGL-3, confirming the presence of multiple lysozymes in the mantle. Since CGL-3 is a cognate protein of the digestive lysozyme CGL-2, it is assumed that CGL-3 has evolved specifically a defensive function. Functional assays using recombinant CGL-1 and CGL-3 suggested that CGL-1 and CGL-3 play a major defensive role in the mantle tissue, and that they are responsible for lysozyme activity under different pH, ionic strength and temperature conditions. Based on these observations, we conclude that multiple mantle lysozymes in the Pacific oyster are better for host-defense under broader conditions than a single lysozyme.