Protein Profile of Seminal Plasma and Serine/Threonine Phosphoproteome Related to Capacitation of Boar Spermatozoa
Date Issued
2008
Date
2008
Author(s)
Lin, Yun
Abstract
Sperm capacitation refers to the process of ejaculated sperm developing the ability to fertilize eggs in female reproductive tracts. Capacitation can be inhibited by decapacitation factors existed in the seminal plasma. However, the molecular mechanisms regarding the capacitation and decapacitation process are still needs to be resolved. Thus the purpose of this study was to understand the capacitation-related proteins in boar semen by proteomic study. The advancement of high sensitivity mass spectrometry and bioinformatics software bring us the better chances analyzing the proteomic composition of boar semen. In order to evaluate the sperm capacitation profile, porcine sperm and seminal plasma were analyzed separately. First, porcine sperm was assorted into capacitated and uncapacitated sperm to reveal the phosphoproteome involved in sperm capacitation. Second, the proteome of the seminal plasma was analyzed to unveil the decapacitation factors existed in the seminal plasma.rotein extracts of the capacitated and uncapacitated sperm were resolved in the SDS-PAGE, digested in gel by trypsin, and isolated the Serine/Threonine (Ser/Thr) containing phosphopeptides. The phosphoproteome was analyzed by liquid chromatography combined with linear ion trap–fourier transform ion cyclotron resonance mass spectrometry (LC LTQ-FT ICR MS). The preliminary data of Ser/Thr phosphoproteome showed that 9 proteins were phosphorylated only in uncapacitated sperm such as tubulin α and β subunit, which is the constituent of microtubles and may related to sperm hypermotility. There are 4 proteins were phosphorylated only in capacitated sperm. Moreover, distinct phosphorylation sites were found in 6 proteins when compared the phosphoproteome between capacitated and uncapacitated sperm. It includes cAMP-dependent protein kinase and outer dense fiber protein. The cAMP-depent protein kinase type I and type II can regulate signal transduction chains mediating membrane association. In addition, outer dense fiber protein located on outside of the axoneme in the sperm tail may help maintain the passive elastic structure of sperm tail. Hence, it is reasonable to assume that the process of sperm capacitation is regulated by altering phosphorylation status of Ser/Thr residuals. he proteins extracted from seminal plasma were separated on a SDS-PAGE gel, digested in gel by trypsin, and subjected to LC LTQ-FT ICR MS analyses. There were 107 proteins identified from porcine seminal plasma. The proteome data was further analyzed using Gene Ontology program and the results were classified into 8 categories base upon molecular functions. Among them, the phosphorylation-related are phospholipid binding, transferase activity (transferring phosphorus-containing groups), phospholipase inhibitor activity and pyridoxal phosphate binding. These proteins possess the ability to bind the phospholipids, to inhibit the phospholipase activity, and to catalyze the transfer of phosphorus-containing groups, which may cause the change of protein phosphoylation sites on sperm. The category of ion binding and transporting will involve metal ion binding, anion binding, anion transmembrane transporter activity, and calcium-dependent protein binding which could regulate ion efflux and influx especially the calcium that holds an important role in sperm capacitation. Enzyme inhibitors equipped activity of protease and phospholipase inhibitor may reduce or terminate the activity of phospholipase involved in sperm capacitation. inally, using the proteome of seminal plasma as backbone and subtracting the proteins presented in the capacitated sperm which left 6 proteins only present in uncapacitated sperm and seminal plasma. It could be the potential candidates of sperm decapacitation factors existed in seminal plasma. n conclusion, it is the first time that the phosphoproteome of capacitated and uncapacitated sperm was compared, and the proteome of porcine seminal plasma was unveiled. These results will be helpful for our further understanding the complex mechanism of mammalian sperm capacitation.
Subjects
boar sperm
seminal plasma
proteomics
Type
thesis
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