Proteomics: a subcellular look at spermatozoa

<p>Abstract</p> <p>Background</p> <p>Male-factor infertility presents a vexing problem for many reproductively active couples. Many studies have focused on abnormal sperm parameters. Recent advances in proteomic techniques, especially in mass spectrometry, have aided in the study of sperm and more specifically, sperm proteins. The aim of this study was to review the current literature on the various proteomic techniques, and their usefulness in diagnosing sperm dysfunction and potential applications in the clinical setting.</p> <p>Methods</p> <p>Review of PubMed database. Key words: spermatozoa, proteomics, protein, proteome, 2D-PAGE, mass spectrometry.</p> <p>Results</p> <p>Recently employed proteomic methods, such as two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential in gel electrophoresis, have identified numerous sperm-specific proteins. They also have provided a further understanding of protein function involved in sperm processes and for the differentiation between normal and abnormal states. In addition, studies on the sperm proteome have demonstrated the importance of post-translational modifications, and their ability to bring about physiological changes in sperm function. No longer do researchers believe that in order for them to elucidate the biochemical functions of genes, mere knowledge of the human genome sequence is sufficient. Moreover, a greater understanding of the physiological function of every protein in the tissue-specific proteome is essential in order to unravel the biological display of the human genome.</p> <p>Conclusion</p> <p>Recent advances in proteomic techniques have provided insight into sperm function and dysfunction. Several multidimensional separation techniques can be utilized to identify and characterize spermatozoa. Future developments in bioinformatics can further assist researchers in understanding the vast amount of data collected in proteomic studies. Moreover, such advances in proteomics may help to decipher metabolites which can act as biomarkers in the detection of sperm impairments and to potentially develop treatment for infertile couples.</p> <p>Further comprehensive studies on sperm-specific proteome, mechanisms of protein function and its proteolytic regulation, biomarkers and functional pathways, such as oxidative-stress induced mechanisms, will provide better insight into physiological functions of the spermatozoa. Large-scale proteomic studies using purified protein assays will eventually lead to the development of novel biomarkers that may allow for detection of disease states, genetic abnormalities, and risk factors for male infertility. Ultimately, these biomarkers will allow for a better diagnosis of sperm dysfunction and aid in drug development.</p

Two-dimensional differential in-gel electrophoresis-based proteomics of male gametes in relation to oxidative stress

Objective: To identify the relative abundance of proteins in pooled reactive oxygen species (ROS)-positive (ROSþ) and ROS-negative (ROSÀ) semen samples with the use of two-dimensional differential in-gel electrophoresis (2D-DIGE). Design: Spermatozoa suspensions from ROSþ and ROSÀ groups by 2D-DIGE analysis. Setting: Tertiary hospital. Patient(s): 20 donors and 32 infertile men. Intervention(s): Seminal ejaculates evaluated for semen and proteomic analysis. Main Outcome Measure(s): Semen samples from 20 donors and 32 infertile men were pooled, divided into ROSþ and ROSÀ groups based on the cutoff value of &lt;20 relative light units/s/10 6 sperm and frozen. From each pooled group, spermatozoa were labeled with Cy3/Cy5 fluorescent dye. Duplicate 2D-DIGE gels were run. Image analysis was performed with the use of Decider software. Protein spots exhibiting R1.5-fold difference in intensity were excised from the preparatory gel and identified by liquid chromatographymass spectrometry. Data were analyzed with the use of Sequest and Blast programs. Result(s): A total of 1,343 protein spots in gel 1 (ROSÀ) and 1,265 spots in gel 2 (ROSþ) were detected. The majority of protein spots had similar expression, with 31 spots were differentially expressed. Six spots were significantly decreased and 25 increased in the ROSÀ sample compared with the ROSþ sample. Conclusion(s): Significantly different expression of protective proteins against oxidative stress was found in ROSÀcompared with ROSþ samples. These differences may explain the role of oxidation species in the pathology of male infertility. (Fertil Steril Ò 2013;99:1216-26. Ó2013 by American Society for Reproductive Medicine.

Integrin α(D)β(2), an adhesion receptor up-regulated on macrophage foam cells, exhibits multiligand-binding properties

Integrin α(D)β(2), the most recently discovered member of the β(2) subfamily of integrin adhesion receptors, is up-regulated on macrophage foam cells. Although other members of the subfamily have been subjects of extensive research, the recognition specificity and the molecular basis for α(D)β(2) ligand binding remain unknown. Based on the high extent of structural homology between α(D)β(2) and the major myeloid-cell-specific integrin α(M)β(2) (Mac-1), noted for its capacity to bind multiple ligands, we considered that the 2 integrins have similar recognition specificity. In this study, using recombinant and natural α(D)β(2)-expressing cells, we demonstrate that α(D)β(2) supports adhesion and migration to many extracellular matrix proteins in a fashion similar to α(M)β(2). Consistent with these data, the recombinant α(D)I-domain of the receptor bound selected ligands. The binding was activation-dependent because the α(D)I-domain with its C-terminal α7 helix truncated, but not the form with the C-terminal part extended, bound ligands. When the α(D)I-domain segment Lys(244)-Lys(260) (highly homologous to its α(M)I-domain counterpart Lys(245)-Arg(261) responsible for α(M)β(2) multiligand-binding properties) was inserted into the mono-specific α(L)I-domain, the chimeric protein bound many ligands with affinities similar to those of wild-type α(D)I-domain. These results establish integrin α(D)β(2) as a multiligand receptor and indicate that the mechanism whereby α(D)β(2) exhibits broad ligand specificity resembles that used by α(M)β(2), the most promiscuous member of the integrin family