Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives.

For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1-5, 6-10, 11-15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or ß-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of ß-laminarin-treated granulocytes decreased significantly for zymosan and zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of ß-glucan receptors for phagocytosis.

Differences in integrin-dependent phagocytosis among three hemocyte subpopulations of the Pacific oyster "Crassostrea gigas".

Integrins play a key role in immunoresponses such as attachment, spreading, and phagocytosis in invertebrate hemocytes. This study was designed to identify integrin expression patterns at the hemocyte subpopulation level, and correlate the expression levels with phagocytic ability. First, we cloned a beta integrin from Crassostreagigas hemocytes and used real-time RT-PCR to analyze the quantitative expression level of its encoding mRNA. The expression level in hyalinocytes was significantly higher than that in granulocytes and agranulocytes. Subsequently, we investigated the phagocytic ability of each subpopulation using anti-alpha(5)beta(1) integrin antibody, and found that phagocytosis of hyalinocytes was inhibited by neutralization with the antibody but enhanced against the antibody-conjugated microspheres. In contrast, phagocytic abilities of granulocytes and agranulocytes showed high and zero levels, respectively, regardless of the antibody. These results suggest that phagocytosis of hyalinocytes is regulated by an integrin-dependent mechanism and that of granulocytes is elicited by other functional receptors.

Immunogen-dependent quantitative and qualitative differences in phagocytic responses of the circulating hemocytes of the lobster Homarus americanus.

Phagocytic responses in circulating hemocytes of the lobster Homarus americanus were measured before and after treatment of lobsters with 2 different immunogens: (1) lipolysaccharide (LPS) or endotoxin from a non-pathogenic Pseudomonas perolens, and (2) a vancomycin/live Gram-positive pathogen (Aerococcus viridans [var.] homari) combination, essentially attenuated cells, shown previously to induce a high degree of resistance to this pathogen. The responses elicited by each of the immunogens were markedly different. Hemocytes drawn from LPS-treated lobsters showed significant, largely non-specific, increases in phagocytic responses over baseline values against sheep red blood cells and an array of test bacteria, with the notable exception of the pathogen. In marked contrast, induction with the vancomycin/live pathogen combination resulted in highly significant and specific increases in phagocytic responses to the pathogen and to the related, (but avirulent) strains of the pathogen, as well as inducing in the lobsters the usual high degree of resistance to the pathogen. These results suggest that quantitative and qualitative variations in phagocytic and resistance levels induced in at least 1 crustacean genus are determined largely by the particular characteristics of the immunogen.

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.

Molecular characterization of a cDNA encoding putative vitellogenin from the Pacific oyster Crassostrea gigas.

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.

Estrogen synthesis in relation to gonadal development of Japanese scallop, Patinopecten yessoensis: gonadal profile and immunolocalization of P450 aromatase and estrogen.

Aromatase activities and estrogen contents in the gonad of Japanese scallop, Patinopecten yessoensis, were determined during gonadal development and estrogenic cells in the testis were identified immunohistochemically. Ovaries and testes developed rapidly during January and February to reach the mature stage in March and the spawning stage in April. Increases in aromatase activities of the ovary and testis preceded the onset of the ovarian and testicular development. Aromatase activities reached the highest level at the growing stage in February and the mature stage in March, and showed a striking decrease at the spawning stage in April. Contents of ovarian and testicular estradiol-17beta changed similarly to the profile of aromatase activities in the ovary and testis, although estrone showed no change. Immunoreactivities against P450 aromatase and estradiol-17beta were detected in the cells along the inside of the acinar wall of the testis, whereas in the previous reports, the cells are distributed along the outside of the acinar wall in the ovary. This study thus suggests that estrogen is synthesized in the estrogenic cells of the ovary and testis through aromatization by P450 aromatase and that testicular estrogen may play a physiological role in spermatogenesis.

Oyster estrogen receptor: cDNA cloning and immunolocalization.

A cDNA encoding the Pacific oyster, Crassostrea gigas, estrogen receptor (cgER) was cloned using degenerate PCR primers. The open reading frame predicted 485 amino acid residues. Comparisons of the amino acid sequence of cgER with other mollusk ERs show high similarities of the C domain (95-97%), and the E domain (56-66%). The amino acid sequence of the C domain of cgER shows 86 and 89% identity with the respective sequences of human ER-alpha and ER-beta. The amino acid sequence of the E domain of cgER shows 45% identity with those of human ER-alpha and ER-beta. The phylogenetic analysis indicated that the cgER is an ortholog of the other mollusk ERs. In the C domain, the positions of cysteine residues and other residues around them that constitute the two zinc finger motifs and the P-box are conserved. The cgER mRNA was expressed in various tissues including the ovary. Reporter gene assay revealed that cgER is unresponsive to estrogen. This result is similar to those of other mollusk ERs. ER immunoreactivity was localized mainly in the nuclei of follicle cells, the site of vitellogenin synthesis, and in oocytes. This result suggests that cgER could work as a nuclear receptor.

Pacific oyster hemocytes undergo apoptosis following cell-adhesion mediated by integrin-like molecules.

Previously, we demonstrated that in the Pacific oyster (Crassostrea gigas, a bivalve mollusc) apoptosis could be induced in hemocytes by treatment with Arg-Gly-Asp (RGD) peptides which are known to function as an integrin ligand. However, it is unclear where the RGD peptides are binding to the C. gigas hemocytes, or what mechanism or molecules are involved, e.g., integrin-ligand interactions. Therefore, this study was undertaken to investigate the binding interactions in C. gigas hemocytes. Initially, to confirm the presence of RGD-recognizing integrin-like molecule(s) on the hemocytes, we assessed the enhancement of spreading ability, and found that spreading ability was enhanced by immobilized human fibronectin, a fibronectin fragment containing the RGD motif, and C. gigas plasma in the presence of divalent cations. Interestingly, viability of the spreading hemocytes dramatically decreased 24 h later and DNA fragmentation with oligonucleosomal laddering of 180-200 bp in length was detected in the dead hemocytes by electrophoresis and TUNEL assay. These results indicated that hemocyte adhesion mediated by integrin-like molecules triggered apoptosis and suggested that integrin-activation contributes to the induction of apoptosis. This is the first report showing the possibility of an integrin functioning in the induction of apoptosis in invertebrate hemocytes.

Vitellogenin synthesis in the ovary of scallop, Patinopecten yessoensis: Control by estradiol-17 beta and the central nervous system.

Elucidation of a profile of scallop vitellin formation associated with oogenesis and its endocrine control, and identification of a vitellogenin synthesizing site were immunologically undertaken by using anti-scallop Vn serum. Vn content increased during ovarian growth and accounted for more than 80% of the water soluble protein of the ovary at the mature stage. In vivo injection of estradiol-17 beta (E(2)) resulted in an increase in Vn content in the ovary. In vitro accumulation of Vn in the ovarian tissue was promoted with E2 and a vitellogenesis promoting factor (VPF) from cerebral plus pedal ganglion which was heat stable, less than MW 10,000 and trypsin/chymotrypsin resistant. Estrogen receptor (ER)-like immunoreactivity was found in the growing oocyte and the auxiliary cell in close contact with growing oocytes, in which Vn immunoreactivity was also found. It is suggested that the vitellogenin synthesis occurred inside the ovary, especially in the auxiliary cell, and is controlled by E2 and VPF via ER.