Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas.

Knowledge of sperm maturation process is limited in the Pacific oyster Crassostrea gigas and major factors of fertilization success of this free spawning animal are unknown. We investigated proteins associated to spermatozoa by analyzing two cellular fractions obtained from a 40-80% Percoll gradient fractioning of germ cell of mature male gonads. Mature spermatozoa were enriched in the lower Percoll fraction while the upper fraction contained less mature or earlier germ cells. A 2-DE proteomic approach was used to identify differentially expressed proteins in both fractions. We screened out 31 differential proteins (P<0.05) which included 14 up-regulated and 17 down-regulated proteins. Using MALDI-TOF/TOF MS and bioinformatics search against a C. gigas database, 13 and 8 proteins were identified for the up-regulated and down-regulated groups, respectively. In the spermatozoa enriched fraction, proteins regarding flagellum formation and control, energy production and Proteosome subunit beta were increased. In less mature germ cell fraction, proteins regarding developmental processes and chaperon molecules were mainly increased. Our results improve current knowledge of proteins associated with spermatozoa maturation related to zootechnical practices used in mollusk hatcheries. BIOLOGICAL SIGNIFICANCE: This is the revised version of the manuscript "Proteomic identification of protein associated to mature spermatozoa in the Pacific oyster Crassostrea gigas" by Kingtong et al. to the Journal of Proteomics. The corrections have been done by the team carefully. This work highlight the enrichment method of spermatozoa of Pacific oyster from stripped complex sample using Percoll gradient. The results reflexed developmental stages of germ cells in gonadal tubules of this species. We have used proteomic approach to identify differentially expressed proteins in mature spermatozoa fraction compared to less mature spermatozoa fraction which provided candidates of protein associated to mature spermatozoa in the Pacific oyster. This work improves the current knowledge in marine bivalve hatchery.