| 摘要: | 本研究的目的為探討精子的半乳醣、乙醯半乳醣胺以及乙醯葡萄醣胺醣蛋白於成熟前後、獲能反應以及頂體反應後在精子中的分佈與表現量。利用凝集素PNA、SBA及WGA染色結果顯示半乳醣、乙醯半乳醣胺以及乙醯葡萄醣胺醣化物質的分佈及表達量會隨著精子發展成熟而改變。藉由PNA親和性層析法分離出15個精子半乳醣蛋白，而質譜儀鑑定法偵測出精子含有蛋白質p38。p38多株抗體可於未成熟精子中辨認出~38kDa 及~40kDa之蛋白質條帶，然而在精子成熟後此抗體只能觀察到~38kDa的條帶。p38的表現量於獲能反應及頂體反應之精子並無改變。本研究亦觀察到p38蛋白於精子頭部之分佈會隨精子發展成熟而改變。
Testicular sperm are not motile and have to undergo extratesticular maturation in epididymis. Nevertheless, freshly ejaculated sperm cannot fertilize oocytes. As sperm reside in female reproductive tract, a final step of maturation, termed capacitation, takes place and sperm become fully fertilizable. As sperm approach oocytes, the extracellular matrix, zona pellucida glycoproteins, surrounding the oocytes induce sperm to undergo acrosome reaction. This exocytotic event allows sperm to release enzymes that facilitate sperm to move forward prior to gamete fusion. Recent studies, using carbohydrates-binding lectins, have demonstrated that sperm glycocomponents participate in sperm maturation, capacitation, acrosome reaction, and fertilization. The purpose of this study is to investigate the presence and the distribution of sperm specific galactosylated, N-acetylgalactosamylated and N-acetylglucosamylated glycocomponents during
epididymal maturation, prior to and following capacitation and acrosome reaction. Fluorescent staining study using three lectins, PNA, SBA, WGA, showed that the presence of galactosylated, N-acetylgalactosamylated and N-acetylglucosamylated glycocomponents and these glycosylated materials distributed in sperm head region. Following extratesticular maturation and development, sperm glycocomponents underwent relocalization and they were differentially expressed. Using PNA affinity chromatography, we isolated fifteen sperm galactosylated glycoproteins for LC/MS MS protein identification and our spectrometric results showed that a sperm specific protein, p38, was present. In epididymal sperm, two major protein bands with molecular weight close to 38kDa and 40kDa were identified by antibody specific to p38. Following capacitation and acrosome reaction, p38 was only identified as a single protein band and there was no significant change in p38 protein expression, compare to that of in epididymal sperm. Finally, p38 was localized to the sperm head region and underwent redistribution following acrosome reaction. |
