The sperm proteome of the Pacific oyster <i>Crassostrea gigas</i> and immunolocalization of heat shock proteins

<div><p>The Pacific oyster <i>Crassostrea gigas</i> 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.</p></div

Statistics of protein identification.

<p>More details were provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135008#pone.0135008.s002" target="_blank">S1 Table</a>.</p><p>^a: include one hit from the decoy database.</p><p>Statistics of protein identification.</p

The primers of 30 interested genes and reference genes.

<p>The primers of 30 interested genes and reference genes.</p

A part of differentially expressed genes/proteins between CL and JU.

<p>We present the major differentially expressed proteins of curiosity in this table. An expanded table is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135008#pone.0135008.s004" target="_blank">S3 Table</a>. The fold changes (FCs) were calculated using the formula: FC = Log₂ (JU/CL). “↑” and “↓” indicate higher expression in JU and CL, respectively. “–” indicates no significant difference between the two stages (no variation, n.v.).</p><p>^a: The Student’s <i>t</i>-test was used to compare the mRNA expressions between the two stages and the difference was considered significant when <i>p</i> <0.05.</p><p>A part of differentially expressed genes/proteins between CL and JU.</p

Quantitative analysis between the two developmental stages.

<p>a. The scatter plot distributions of the FCs of the 240 proteins that were detectable in both stages. The Y axis represent FC and the X axis represents the log₂ transformation of total spectra from the four runs. The range of one SD of the mean is indicated by dashed lines. Those proteins fell out of the range are candidate differentially expressed proteins. b. The distributions of the 117 differentially expressed proteins. All proteins have the H-sc of at least ten.</p

A part of differentially expressed genes/proteins between CL and JU.

<p>We present the major differentially expressed proteins of curiosity in this table. An expanded table is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135008#pone.0135008.s004" target="_blank">S3 Table</a>. The fold changes (FCs) were calculated using the formula: FC = Log₂ (JU/CL). “↑” and “↓” indicate higher expression in JU and CL, respectively. “–” indicates no significant difference between the two stages (no variation, n.v.).</p><p>^a: The Student’s <i>t</i>-test was used to compare the mRNA expressions between the two stages and the difference was considered significant when <i>p</i> <0.05.</p><p>A part of differentially expressed genes/proteins between CL and JU.</p

A Label-Free Proteomic Analysis on Competent Larvae and Juveniles of the Pacific Oyster <i>Crassostrea gigas</i>

<div><p>Current understandings on the molecular mechanisms underlying bivalve metamorphosis are still fragmentary, and a comprehensive description is required. In this study, using a large-scale label-free proteomic approach, we described and compared the proteomes of competent larvae (CL) and juveniles (JU) of the Pacific oyster, <i>Crassostrea gigas</i>. A total of 788 proteins were identified: 392 in the CL proteome and 636 in the JU proteome. Gene Ontology analysis of the proteome from each sample revealed active metabolic processes in both stages. Further quantitative analyses revealed 117 proteins that were differentially expressed between the two samples. These proteins were divided into eight groups: cytoskeleton and cell adhesion, protein synthesis and degradation, immunity and stress response, development of particular tissues, signal regulation, metabolism and energy supply, transport, and other proteins. A certification experiment using real-time PCR assay confirmed 20 of 30 examined genes exhibited the same trends at the mRNA and protein levels. The differentially expressed proteins may play roles in tissue remodeling, signal transduction, and organ development during and after metamorphosis. Novel roles were proposed for some differentially expressed proteins, such as chymotrypsin. The results of this work provide an overview of metamorphosis and post-metamorphosis development of <i>C</i>. <i>gigas</i> at the protein level. Future studies on the functions of the differentially expressed proteins will help to obtain a more in-depth understanding of bivalve metamorphosis.</p></div

Protein identification and GO analysis.

<p>a. A Venn diagram showing the numbers of proteins identified from each sample. The number in the bracket indicates a hit from the decoy database. b. Results of GO analysis (the "biological process" category). More information of GO analysis is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135008#pone.0135008.s003" target="_blank">S2 Table</a>.</p

The temporal expression pattern of MmeLR in the primary cells from the binding assay.

<p>(A) Cells of the matrigel-coated group and the control group were harvested at 0 h, 12 h, 24 h, 48 h, 72 h, 6 days and 12 days. At 6 days and 12 days, the MmeLR mRNA expression levels of both groups were approximately 2 – 2.5-fold higher than at earlier time points (<i>P</i><0.05). At 12 h and 24 h, the MmeLR mRNA expression levels of the control group were approximately 2.5-fold higher than that of the matrigel-coated group, and the difference was significant. *indicates significant difference (<i>P</i><0.05). (B) Cells of the two groups were harvested at 0 h, 24 h, 6 days and 12 days for western blots. The β-actin protein (42 kDa) was used as the endogenous reference. Lane m: Cells of the matrigel-coated group. Lane c: Cells of the control group. At 24 h, cells of the matrigel-coated group expressed less MmeLR than the control group. At 6 days and 12 days, in both groups, MmeLR was expressed at higher levels than at 24 h.</p

Immunofluorescence analysis of laminin.

<p>White arrows show the location of laminin detected by FITC (green) signals. Red signals indicate the outline of the mantle tissue slices. The laminin is primarily localized in the ECM of the clam, <i>M. meretrix</i>.</p