公豬精子獲能相關之絲胺酸/酥胺酸磷酸化之蛋白質圖譜

Abstract

本研究旨在利用最新穎精確的蛋白質體學研究方法，來研究公豬精子獲能相關之蛋白質圖譜。為達此目的，吾人將公豬精液分為精子與精漿兩部分來分析探討。獲能前之精子表面結合有精漿蛋白質，這些蛋白質能抑制精子之獲能，稱去獲能因子。因此精子須先脫去其表面之精漿物質，方能發生獲能作用而達到受精之目的；該過程為一系列反應，包括精子中酪胺酸（Tyrosine，Tyr）的磷酸化。由於精子獲能時絲胺酸/酥胺酸（Serinr/ Threonine，Ser/Thr）磷酸化對獲能之影響所知甚少，因此本研究冀望利用蛋白質體學之方法，分別探討精子獲能時蛋白質中之Ser/Thr磷酸化的變化與精漿中去獲能因子的多樣性，探討公豬精子獲能相關之蛋白質圖譜。 首先，在精子蛋白質磷酸化之部分，為比較獲能精子與未獲能精子之Ser/Thr磷酸化蛋白質圖譜。首先利用chlortetracycline（CTC）染色、peanut agglutinin （PNA）染色與西方點墨法來確認精子的獲能狀態。在CTC染色與PNA染色結果，均顯示精子之獲能處理與未獲能處理有極顯著的差異（p < 0.01），同樣在西方點墨法也證實了精子獲能處理組之精子確實有獲能。接著利用串聯式質譜分析儀（LC-MS/MS）來偵測獲能精子與未獲能精子中的蛋白質體，並純化出個別的Ser/Thr磷酸化胜肽，同時由LC-MS/MS比對其Ser/Thr磷酸化蛋白質圖譜。結果顯示精子在獲能前後，在獲能精子中共有18個Ser/Thr位置發生磷酸化，而未獲能之精子則有33個Ser/Thr磷酸化位置。其中6個磷酸化蛋白質在獲能精子與未獲能精子中，具有不同的Ser/Thr磷酸化位置；4個蛋白質僅在獲能精子中發生Ser/Thr磷酸化；9個蛋白質僅在未獲能精子中發生Ser/Thr磷酸化。 其次，為分析公豬精漿之蛋白質體，利用一維電泳膠分離公豬精漿蛋白質後，配合LTQ-FT ICR MS質譜儀，共分析到107個蛋白質出現在公豬精漿中。利用人類及小鼠之資料庫，經基因功能體系分類後，以level 4做基準，將蛋白質之分子功能粗略分為蛋白質磷酸化、離子結合與運送、電子傳遞鍊、催化反應、核酸結合、酵素抑制劑、蛋白質結合功能以及其他，共八大類分子功能。依據文獻推測，蛋白質磷酸化、離子結合與運送、酵素抑制劑以及其他豬特異表現之蛋白質中，最有可能出現與精子獲能相關之因子。再則比較公豬精漿、獲能精子、未獲能精子此三個蛋白質體間之差異，分析獲得於公豬精漿中之major seminal plasma glycoprotein PSP-I、glutathione S-transferase P、malate dehydrogenase, cytoplasmic、aldose reductase、junction plakoglobin以及neutral α-glucosidase AB等，共六種蛋白質在精漿中為極具潛力之去獲能因子。

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. Protein 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. The 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. Finally, 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. In 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.