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.

Hemocyte migration and expression of four Sox genes during wound healing in Pacific abalone, Haliotis discus hannai.

In molluscs, migration of hemocytes and epithelial cells is believed to play central roles in wound healing. Here, we assessed cellular and molecular mechanisms of wound healing in Pacific abalone, a marine gastropod. Light and electron microscopy in the wounds showed early accumulation of putative hemocytes, collagen deposition by fibroblasts, and further coverage of this tissue by migration of adjacent epithelial cells. Cell labelling technique allowed us to track hemocytes, which migrated to wound surface within 24 h. The migrated cells first expressed PCNA and SoxF weakly, and then the epithelial cells expressed abundant PCNA and SoxB1, SoxB2, and SoxC. These findings imply that abalone SoxF is involved in hemocyte migration or their differentiation into fibroblasts, and suggest that the migrated epithelia acquire stem cell-like property and undergo active proliferation. This study is the first to show direct evidence of hemocyte migration to wounds and expression of Sox genes in molluscan wound healing.

Gluconeogenesis and glycogen metabolism during development of Pacific abalone, Haliotis discus hannai.

In lecithotrophic larvae, egg yolk nutrients are essential for development. Although yolk proteins and lipids are the major nutrient sources for most animal embryos and larvae, the contribution of carbohydrates to development has been less understood. In this study, we assessed glucose and glycogen metabolism in developing Pacific abalone, a marine gastropod mollusc caught and cultured in east Asia. We found that glucose and glycogen content gradually elevated in developing abalone larvae, and coincident expression increases of gluconeogenic genes and glycogen synthase suggested abalone larvae had activated gluconeogenesis and glycogenesis during this stage. At settling, however, glycogen sharply decreased, with concomitant increases in glucose content and expression of Pyg and G6pc, suggesting the settling larvae had enhanced glycogen conversion to glucose. A liquid chromatography-mass spectrometry (LC/MS)-based metabolomic approach that detected intermediates of these pathways further supported active metabolism of glycogen. Immunofluorescence staining and in situ hybridization suggested the digestive gland has an important role as glycogen storage tissue during settlement, while many other tissues also showed a capacity to metabolize glycogen. Finally, inhibition of glycolysis affected survival of the settling veliger larvae, revealing that glucose is, indeed, an important nutrient source in settling larvae. Our results suggest glucose and glycogen are required for proper energy balance in developing abalone and especially impact survival during settling.

Identification and Function of GnRH-like Peptide in the Pacific Abalone, Haliotis discus hannai.

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproductive function in various vertebrates and invertebrates. In the present study, we have identified the GnRH-like peptide cDNA and peptide from the cerebral ganglion (CG) of the Pacific abalone, Haliotis discus hannai. Pacific abalone GnRH-like peptide (hdhGnRH-like peptide) cDNA encodes precursor, which possesses the typical organization of the known mollusk GnRH-like peptide precursors, including a hydrophobic signal peptide, GnRH-like peptide, and a cleavage site followed by a GAP-like peptide region. Three hdhGnRH-like peptides, pQNYHFSNGWHAamide (hdhGnRH-11amide), pQNYHFSNGWHA (hdhGnRH-11OH), and pQNYHFSNGWHAG (hdhGnRH-12OH), were determined from the acid/acetone extract of the CG by mass spectrometry (LC-MS/MS) analysis. The hdhGnRH-like peptide mRNA expression was detected not only in the CG but also in gonads, and hdhGnRH-11amide was also detected in the extract of gonads. The mRNA expression of hdhGnRH-like peptide in the CG was lower in spawned males than in non-spawned animals, while no change in hdhGnRH-like peptide mRNA expression was shown in both ovulated and non-ovulated abalone. The hdhGnRH-11amide induces spawning and ovulation of both mature males and females in a concentration-dependent fashion following intramuscular injection. These results indicate that three hdhGnRH-like peptides are yielded from a single hdhGnRH-like peptide precursor, and that at least hdhGnRH-11amide is involved in the control of reproduction of the Pacific abalone.