A novel peptide identified from visceral ganglia induces oocyte maturation, spermatozoa active motility, and spawning in the pen shell Atrinapectinata.

The identification of novel peptides that regulate reproduction is essential for studying reproductive physiology in bivalves. Therefore, we aimed to identify peptides that affect the reproductive physiology of bivalves. We identified an oocyte maturation-, sperm motility-, and spawning-inducing peptide from the visceral ganglia of the pen shell, Atrina pectinata. The peptide consisted of 26 amino acid residues (GFDSINFPGTIDGFKDYSSNKKERLL). This peptide induced oocyte maturation and sperm motility activation at less than 1 nM upon the treatment of gonad fragments and induced spawning at 1 nmol when injected into mature individuals. Mature eggs and sperms artificially spawned by peptide administration were fertilized, and we confirmed that the development proceeded normally to veliger (D-shape) larvae. These results indicated that GFDSINFPGTIDGFKDYSSNKKERLL stimulated the gonads of pen shells and induced oocyte maturation, sperm motility activation, and spawning.

Reproduction-related genes in the pearl oyster genome.

Molluscan reproduction has been a target of biological research because of the various reproductive strategies that have evolved in this phylum. It has also been studied for the development of fisheries technologies, particularly aquaculture. Although fundamental processes of reproduction in other phyla, such as vertebrates and arthropods, have been well studied, information on the molecular mechanisms of molluscan reproduction remains limited. The recently released draft genome of the pearl oyster Pinctada fucata provides a novel and powerful platform for obtaining structural information on the genes and proteins involved in bivalve reproduction. In the present study, we analyzed the pearl oyster draft genome to screen reproduction-related genes. Analysis was mainly conducted for genes reported from other molluscs for encoding orthologs of reproduction-related proteins in other phyla. The gene search in the P. fucata gene models (version 1.1) and genome assembly (version 1.0) were performed using Genome Browser and BLAST software. The obtained gene models were then BLASTP searched against a public database to confirm the best-hit sequences. As a result, more than 40 gene models were identified with high accuracy to encode reproduction-related genes reported for P. fucata and other molluscs. These include vasa, nanos, doublesex- and mab-3-related transcription factor, 5-hydroxytryptamine (5-HT) receptors, vitellogenin, estrogen receptor, and others. The set of reproduction-related genes of P. fucata identified in the present study constitute a new tool for research on bivalve reproduction at the molecular level.

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.

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis.

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.

Tissue preferential expression of estrogen receptor gene in the marine snail, Thais clavigera.

Sex steroid hormones have been widely detected in molluscs, and experiments have shown the importance of sex steroids in sex determination, gonadal tissue maturation and gametogenesis. Nevertheless, the signaling pathways of sex steroids in invertebrates have not yet been elucidated. In order to gain insights into the mechanism of sex steroid signaling in molluscs, we have, therefore, tried to isolate molluscan estrogen receptors from the prosobranch mollusc Thais clavigera. Cerebral ganglia of T. clavigera (Mollusca, Gastropoda, Prosobranchia) were subjected to RNA extraction, and degenerate primers for amino acid sequences conserved in vertebrate estrogen receptors were designed. PCR amplification using cerebral RNA and degenerate primers followed by 5'- and 3'-RACE identified the cDNA encoding T. clavigera estrogen receptor 1 (tcER1). The deduced amino acid sequence showed 93% identity in the DNA-binding domain and 72% identity in the ligand binding domain when compared to Aplysia estrogen receptor. Reporter gene assay revealed that tcER1 is constitutively active and unresponsive to estrogen. Quantitative analysis of the tcER1 mRNA level demonstrated the preferential expression in the ovary. Furthermore, cerebral ganglia expressed tcER1 at a high level in the spring followed by subsequent enlargement of the ovary in later seasons. These results suggest importance of tcER1 in the seasonal development of reproductive organs in T. clavigera.