The sperm proteome of the Pacific oysterCrassostrea gigasand immunolocalization of heat shock proteins

Abstract

The Pacific oyster Crassostrea gigas is a potential model organism of bivalve mollusks. Comprehensive studies on the proteome of its sperm are necessary to expand our understanding on its reproduction and development, which however are still poor currently. In this study, to improve the situation, we conducted a proteomic analysis on the sperm based on two-dimensional electrophoresis combined with protein identification through mass spectra data. Fifty-six protein spots with constant and relatively high expression levels were selected for protein identification. Among them, 36 were identified (corresponding to 31 proteins), including cytoskeletal proteins, proteins involved in energy supply, protein modifiers, signal regulators, antioxidant proteins, and others. We proposed that these proteins might play important roles in sperm motility, gamete interaction, and oxidation resistance. In particular, we observed several heat shock proteins that were proved to play essential roles in animal sperms. A further immunofluorescence experiment revealed a mitochondria localization of Hsp60s and wide distributions of Hsp70s, indicating these proteins might function in various processes such as mitochondrial function, gamete interaction, and regulation of receptor activity. Our data will provide fundamental supports for the studies on the mechanisms of fertilization and contribute to expand the understandings on reproduction and development of bivalve mollusks.

A comparative proteomic analysis reveals important proteins for the fertilization and early embryonic development of the oyster Crassostrea gigas

“…The localization of five differentially regulated proteins, namely eukaryotic translation elongation factor 1 (EEF1, Spot08), HSP70 (Spot10), an apextrin‐like protein (Spot13), proteasome subunit alpha type 6 (PMSA6, Spot14), and S‐adenosylhomocysteine hydrolase (SAHH, Spot15), during fertilization was investigated by IHC. The specificity of the anti‐HSP70 antibody in C. gigas was demonstrated previously and was therefore not tested again in the present study. The specificity of the other four antibodies was examined by Western blotting, and three of these antibodies generated a single band (Fig.…”

“…Nevertheless, few studies have focused on the whole proteomes of gametes and the changes that occur during fertilization. In 2015, we reported a proteomic analysis of the sperm of C. gigas, and the results indicate that some HSPs might play important roles in homeostasis, gamete recognition, and fertilization [27]. In the present study, we further analyzed the proteomes of C. gigas oocytes and early embryos (2/4-cell stage) to provide an overview of the oocyte proteome and to reveal the proteomic changes that occur during fertilization.…”

The Sperm Proteome of the Oyster Crassostrea hongkongensis

“…Proteomics has been shown to be a promising tool to rapidly profile sperm proteome, but its application in free-spawning invertebrates has been limited to only few species: the Biwa pearly mussel Hyriopsis schlegelii, [10] the red abalone Haliotis rufescens, [11] the blue mussel M. edulis, [12] the Mediterranean mussel Mytilus galloprovincialis, [13] the echiuran Urechis unicinctus, [14] and the Pacific oyster C. gigas. [15,16] The two previous studies of oyster sperm proteome have both used C. gigas as the study animal, and applied 2D gel electrophoresis-based techniques. Kingtong et al (2013) [15] conducted 2D gel analysis of the C. gigas sperm in two Percoll fractions-one fraction enriched with spermatozoa and another fraction with both spermatozoa and previous stages of germ cells.…”

The Sperm Proteome of the Echiuran Urechis unicinctus (Annelida, Echiura)

“…Nevertheless, proteomic studies of sperm cells have been conducted mainly in model organisms with a sequenced genome . In recent years, application of the transcriptome‐coupled LC‐MS/MS approach has allowed profiling of sperm proteome in a few non‐model animals, such as 975 proteins in the abalone Haliotis rufescens , 77 and 550 proteins in the mussel Mytilus edulis , and Mytilus galloprovincialis , respectively, 31 proteins in the Pacific oyster Crassostrea gigas , and 206 proteins in the rainbow trout Oncorhynchus mykiss . Abalones, mussels, and oysters belong to Mollusca, one of the most diverse phyla of Lophotrochozoa, which is characterized by having a swimming trochophore stage during the larval development.…”

“…The samples were shipped to Hong Kong Baptist University on dry ice, and kept in a deep refrigerator at −80 °C before proteomic analysis. Sperm proteome was analyzed as described previously for the mussel M. galloprovincialis . In brief, the samples were thawed, sonicated for 5 min, centrifuged to remove insoluble pellet, and protein quantity was determined using an RC DC Protein Assay Kit (BIO‐RAD, CA).…”