Proteomic Analysis Of Gametes And Fertilization Of The Pacific Oyster Crassostrea Sisas

The Pacific oyster Crassotrea gigas is a worldwide distributed aquaculture speciesadapted in the intertidal zone. Additionally, its particular position in evolution and therelationship between the calcification of its shells and ocean acidification make C.gigas receive increasing attention. Fundamental studies into the fertilization biologyof C. giags would not only expand our understanding of the reproduction anddevelopment of mollusks but also benefits the marine culture industry.Fertilization biology involves three major fields: gametes, fertilization, and earlyembryonic development. In this study，we analyzed the proteomes of gametes and theproteins differentially expressed during fertilization process of C. gigas based on thetwo-dimensional electrophoresis (2-DE). Many proteins associated with gametemature, fertilization, and early embryonic development were indentified and theirpotential functions during these processes were discussed.Firstly, the abundant and steady expressed protein spots were screened out andidentified by mass spectra-based protein identification. Two reference maps of spermsand eggs were then constructed. In the proteomic analysis of sperms,56protein spotswere identified by peptide mass fingerprinting data (PMF). Thirty-six of them furtherwere certified by MS/MS data and they were divided into six groups includingcytoskeletal proteins, mitochondrial proteins, protein modifiers, signal regulators,antioxidant proteins and others. Seventy-three protein spots in the reference map of C.gigas eggs were divided into six groups as well including cytoskeletal proteins,mitochondrial proteins, protein synthesis and degradation associated proteins,development regulation and nutrition associated proteins, antioxidant proteins andothers. A significant amount of antioxidant proteins were identified in both sperm andegg proteomes, which suggested that oxidation resistance was crucial in gametes and fertilization. This result is consistent with a previous report.Then we compare the protein expression profiles of eggs and embryos (2-cell stage) inorder to screen differentially expressed proteins during fertilization. Seventeendifferently expressed protein spots were screened out and identified successfully.They were divided into six categories including vitellogenin, mitochondrial proteins,protein synthesis and degradation associated proteins, development associatedproteins, antioxidant proteins and so on. These proteins were suggested to playmultiple roles in fertilization and early embryonic development. For instance,vitellogenin may provide nutrition for fertilization and development; activities ofmitochondrial proteins may ensure sufficient energy supplement. In particular, anamount of protein synthesis and degradation associated proteins were suggested to beinvolved in multiple processes such as egg activation, sperm-egg recognition andfusion, and early development. Several development associated proteins wereproposed to participate in particular processes of early embryonic development, suchas the decision of apical-basal polarity.We further investigated the localization of some important proteins in the gametes ofC. gigas using immunofluorescence to predict their potential roles. The wideexpression of HSP70s in sperms suggests their multiple functions in spermatogenesis,sperm motility, sperm-egg recognition and so on. Apextin was revealed to beexpressed mainly on the extrocytopasmic surface of eggs, which may suggest animportant role in cell-to-cell connection and the decision of apical-basal polarity ofcleavages. The dispersed distributions of PSMA6in eggs may suggest its importantroles in egg maturation and fertilization.Conclusively, we analyze the proteomes of gametes of C. gigas as well as theirchanges during the fertilization. A number of important proteins in gamete maturation, fertilization and early embryonic development were revealed, some of which were notreported before. We further revealed the distributions of some proteins in gametes anddiscussed their potential roles.