陳義雄臺灣大學:生化科學研究所謝孟純HSIEH, MENG-TSUNMENG-TSUNHSIEH2010-05-042018-07-062010-05-042018-07-062009U0001-2307200916000600http://ntur.lib.ntu.edu.tw//handle/246246/178886成熟的精子必需經過許多的修飾才具備與卵受精的能力，其中最主要的變化為獲能效應 (capacitation)。目前獲能效應的相關機制尚不是完全清楚，因此尋找與獲能效應相關的蛋白質並進一步探討其功能便是目前重要方向之一。已被證實跟獲能效應相關的現象有：精子細胞膜上脂質之改變、精子的超活動力、精子內鈣離子的增加及tyrosine磷酸化的增加。 類胰島素生長因子 (insulin-like growth factor-1 , IGF-1) 可藉由細胞中各種訊息傳遞路徑調控細胞功能，其中包含tyrosine phosphorylation pathway，而酪胺酸磷酸化對於精子的獲能效應為一重要因素。因此本研究以小白鼠精子為材料，探討IGF-1的功能和精子獲能效應間是否有關聯性。利用間接螢光染色技術 (indirect fluorescence technique) 證實精子頭部含IGF-IR。也於任何雄性和雌性的生殖腺檢測到IGF-1的mRNA。利用酵素免疫分析法，也證實IGF-1蛋白存於貯精囊液。進一步的實驗結果提示IGF-1可能促進獲能效應伴隨的精子蛋白酪胺酸磷酸化，加入AG538 (IGF-1受體抑制劑)，則此生化反應的程度會下降。另一方面BSA引發的獲能效應，就會使得精子上IGF-IR的酪胺酸磷酸化，這個生化反應也會被AG538抑制。推測獲能效應引發IGF-IR的磷酸化而不必經由IGF-1的結合就能加強其激酶活性。Ejaculated spermatozoon of mammalians should undergo a serious of modification in female to production tract before they acquire the ability to fuse with the oocytes in order to have a successful fertilization. This process is generally termed “capacitation”. The molecular mechanism of capacitation is still far from understanding, despite of the reports about the capacitation-related events, including the rearrangement of plasma membrane lipid, hyperactivated motility, intracellular Ca2+ elevation and protein tyrosine phosphorylation.nsulin-like growth factor-1 (IGF-1) is well known to stimulate the tyrosine kinase of its receptor (IGF-IR) in the regulation of cellular activity. In the seminal vesicle fluid, I could demonstrate IGF-1 by ELISA. Moreover, I could detect IGF-1 mRNA in any sexual glands of adult mice, and examine also IGF-IR on the mouse sperm head by indirect fluorescence technique. These data together prompted me to assess whether IGF-IR is involved in the capacitation-related protein tyrosine phosphorylation. I found the ability of IGF-1 to elicit the capacitation-related protein phosphorylation. The biochemical event could be suppressed by AG538, an inhibitor of IGF-IR. Meanwhile, the sperm capacitation induced by BSA caused the tyrosine phosphorylation of IGF-IR, and the protein tyrosine phosphorylation associated the capacitation could be greatly reduced by AG538. These data suffer ligand-independent IGF-IR on sperm head in the capacitation-related protein phosphorylation.目次文摘要………………………………………………………………………...…….1文摘要………………………………………………………………………………2寫表…………………………………………………………………………………3一章 概論殖學概論……………………………………………………………………..……..4乳類雄性生殖系統………………………………………………………..………..4精囊發育與功能…………………………………………………………..………..7子的形成與成熟………………………………………………………..…………..7能效應……………………………………………………………………………..10胰島素生長因子…………………………………………………………………..11二章 材料與方法驗材料……………………………………………………………………………..13驗方法……………………………………………………………………………..13. Total RNA 萃取…………………………………………………...……………13. RNA品質檢定………………………………………………………………….14. 逆轉錄-聚合酶連鎖反應……………………………………………………….14. 聚合酶連鎖反應……………………………………………………...………...15. SDS-聚丙烯醯胺膠體電泳………………………………………………….....17. 西方墨點法……………………………………………………………………..18. Sperm preparation………………………………………………………………20. AG538抑制精子獲能作用及IGF-IR磷酸化…………………………………20. In vitro fertilization………………………………………………………..……210. 包夾式酵素免疫分析…………………………………………………………..221. Indirected immunofluorescene………………………………………………….23三章 實驗結果. IGF-1在各個生殖系統中之表現………………………………………………24. 儲精囊液中IGF-1的表現量…………………………………………………..24. Localization of the IGF-1 receptor on mouse sperm……………………………25. IGF-1 對精子獲能效應引發蛋白酪胺酸磷酸化的影響……………………..25. 獲能效應誘發精子上IGF-IR的激酶活性…………………………………….26次ig III-1 IGF-1在各個生殖系統中之表現………………………………………….27ig III-2以酵素免疫分析法量測IGF-1在儲精囊液中的含量……………………29ig III-3利用免疫螢光染色觀察精子上之IGF-IR………………………………...31ig III-4利用西方點墨法 (western blot analysis) 觀察精子之IGF-1 receptor..….33ig III-5 IGF-1對精子獲能效應所引發蛋白酪胺酸磷酸化的影響…………….....35ig III-6 IGF-IR的抑制物AG538降低精子獲能效應所引發蛋白酪胺酸磷酸化..37ig III-7. AG538對精子獲能反應之影響…………………………………………..39四章 實驗討論…………………………………………..………………….…41五章 參考文獻………………………………………………………………..42application/pdf17494231 bytesapplication/pdfen-US類胰島素生長因子類胰島素生長因子受體IGF-1IGF-I receptorhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178886/1/ntu-98-R96b46019-1.pdf