Katsuyoshi Mori

Changes in vitellin during oogenesis and effect of estradiol-17β on vitellogenesis in the Pacific oyster Crassostrea gigas

Summary 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 oys...

GONADAL ESTROGEN PROFILE AND IMMUNOHISTOCHEMICAL LOCALIZATION OF STEROIDOGENIC ENZYMES IN THE OYSTER AND SCALLOP DURING SEXUAL MATURATION

Abstract Estrogen levels in the gonads of marine bivalves, the Pacific oyster Crassostrea gigas and scallop Patinopecten yessoensis were determined by high performance liquid chromatography (HPLC) using an electrochemical detector. Estrone (E 1 ), estradiol-17 β (E 2 ), and a small amount of estriol (E 3 ) were identified in the ovary, while only E 2 was found in the testis. The level of E 2 in the ovary was consistently higher than E 1 and it increased with sexual maturation. These results indicate that E 2 may play a role in the reproductive events of the oyster and scallop. In vitro experiments demonstrated the presence of 17 β -hydroxysteroid dehydrogenase (17 β -HSD) in the ovaries of both bivalves. The activity of 17 β -HSD in the ovary was lower in the postspawning stage than in the early differentiating stage. The evidence for the presence of aromatase activity in the scallop ovary was obtained by 3 H-water assay. The immunoreactivity against 3 β -hydroxysteroid dehydrogenase (3 β -HSD), P450 aromatase and E 2 was detected in the cells along the outside of germinal acini of the scallop ovary. It is concluded that estrogens can be synthesized in the gonad, that their levels vary with the reproductive cycle, and that they have a role in the development of gametes.

In vitro production of hydrogen peroxide by the amoebocytes of the scallop, patinopecten yessoensis (JAY)

In vitro production of hydrogen peroxide (H2O2) by the amoebocytes of the scallop, Patinopecten yessoensis, was studied. The authors first confirmed the availability of the direct quantitative method using homovanillic acid, and found that both resting and stimulated amoebocytes produced H2O2. However, the latter showed higher capacity for H2O2 production. The amoebocytes stimulated by concanavalin A released a large amount of H2O2 compared with the cells challenged with three species of bacteria.

Molecular Characterization of a cDNA Encoding Putative Vitellogenin from the Pacific Oyster Crassostrea gigas

Abstract To elucidate the molecular mechanisms involved in oogenesis, we applied a differential display method to identify genes whose expression was detected only in ovaries containing oocytes. One of the cDNA fragments isolated by mRNA differential display was similar in structure to vitellogenin. Using this fragment, a full-length cDNA encoding putative vitellogenin in the Pacific oyster Crassostrea gigas was cloned by RACE (rapid amplification of cDNA ends), and its amino acid sequence was deduced. The open reading frame predicted 1583 amino acid residues. The deduced primary structure of putative vitellogenin in C. gigas was shown to be similar to vitellogenins of various other mollusk, fish, crustacean and nematode species, especially in the N-terminal region. Reverse transcription-mediated PCR revealed that mRNA encoding putative vitellogenin was expressed only in the ovary. In situ hybridization analysis revealed that putative vitellogenin mRNA was expressed strongly in the follicle cells in the ovary. It is concluded that the follicle cells are the site of putative vitellogenin synthesis.

Pharmacological characterization of serotonin receptor in the oocyte membrane of bivalve molluscs and its formation during oogenesis

Serotonin (5-hydroxytryptamine, 5-HT) is a major neurotransmitter that triggers the spawning of bivalve molluscs and oocyte germinal vesicle breakdown (GVBD) of oocytes. The present study employed [3H] 5-HT binding assay to characterize a putative receptor on oocyte membranes prepared from scallops and oysters. Results demonstrated a profile of hormone control of 5-HT receptor formation during oocyte growth. The dissociation constants for 5-HT on the surfaces of scallop and oyster oocytes were estimated to be 2.64 μM and 2.01 μM, respectively, and the maximal binding capacity of the oocyte surface was 152 pmol/mg protein in scallops and 529 pmol/mg protein in oysters. The inhibition of [3H] 5-HT bound by various agonists and antagonists provided pharmacological evidence for a 5-HT receptor in each oocyte, a mixed profile of 5-HT1/5-HT2 in scallops and a single profile of 5-HT1 in oyster. Formation of 5-HT receptor on the surface of the scallop oocyte was induced by estradiol-17β(E2), which was suggested as a primary promoter of the effect of E2 on sensitivity to 5-HT, resulting in the induction of spawning. This formation occurred during oocyte growth controlled by E2. J. Exp. Zool. 281:124–131, 1998.

Phagocytosis and hydrogen peroxide production by phagocytes of the sea urchin Strongylocentrotus nudus

Phagocytosis of erythrocytes by phagocytes from the sea urchin Strongylocentrotus nudus can occur in vitro, and is enhanced by opsonization with the coelomic fluid. This opsonic activity of coelomic fluid can be elevated over a 5-day period by injecting erythrocytes into the coelom. Phagocytes produce hydrogen peroxide during both resting and stimulated states. This result on hydrogen peroxide production is the first to be observed in echinoderms. During the stimulated state, phagocytes produce more hydrogen peroxide than resting phagocytes. However, hydrogen peroxide production by phagocytes is not affected by opsonic activity of the coelomic fluid. Phagocytes share similar functional properties with vertebrate macrophages and granulocytes.

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.

Cloning of cDNA encoding vitellogenin and its expression in red sea urchin, Pseudocentrotus depressus

Abstract Both male and female red sea urchins, Pseudocentrotus depressus, accumulate a large quantity of the major yolk protein (MYP) in the nutritive phagocytes of immature gonads before the initiation of game-togenesis. To examine the accumulation mechanism of this protein in the gonad, we cloned full-length cDNA encoding vitellogenin (Vg; the MYP precursor in the coelomic fluid), and investigated its expression in various tissues of immature adults. The nucleotide sequence of Vg contains an open reading frame of 4050 bp encoding 1349 amino acids. The deduced amino acid sequence near the N-terminal showed 25% homology to the vertebrate transferrin family. Vitellogenin mRNA was detected in the ovary, testis, stomach, intestine and rectum by Northern blot analysis, with the highest level of mRNA expression in the gonad. Weak expression was also detected in the esophagus and coelomocytes by RT-PCR. In situ hybridization demonstrated that nutritive phagocytes, which exclusively fill the lumina of the immature gonad, contained Vg mRNA. These results suggested that the MYP stored in the immature gonads is synthesized and accumulated mainly within the nutritive phagocytes.

Hemolymph lectin of the pearl oyster, Pinctada fucata martensii: A possible non-self recognition system

To elucidate a mutual correlation between the hemolymph lectin and hemocytes of the pearl oyster, Pinctada fucata martensii, we searched for common epitopes and ligands. Neither the hemocyte plasma membrane nor cytoplasm was immunoreactive to anti-hemolymph lectin antibody. The hemolymph lectin strongly bound to D-galactose and N-acetyl-D-galactosamine, and the plasma membrane of both granulocytes and agranulocytes had affinity only for D-mannose and N-acetyl-D-glucosamine-binding plant lectins. In the gonad, the hemolymph lectin selectively adsorbed injected horse red blood cells (HRBC), and its hemagglutinating activity probably prevented them from dispersing. Pinctada sp. may possess system of recognition of non-self by the hemolymph lectin.

Apoptosis by RGD-containing peptides observed in hemocytes of the Pacific oyster, Crassostrea gigas.

We observed in vitro that after treatment with the Arg-Gly-Asp (RGD) peptide, non-spreading Crassostrea gigas hemocytes underwent cell death. Utilizing a combination of a Hoechst staining method and a DNA fragmentation assay, the typical features of apoptosis were shown, i.e. cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. The hemocyte cell death caused by the RGD peptide appears to be sequence-specific, since no induction was shown in the alanine-substituted control peptide (RAD) treatment. Interestingly, the glutamic acid-substituted control peptide (RGE) also induced hemocytic cell death, but a different type of the death to that induced by the RGD peptide. This is the first report that specific peptides induce cell death in molluscan hemocytes.