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標題: | 小鼠精子獲能反應引發CCCTC-結合轉錄因子酪胺酸磷酸化之研究 Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa |
作者: | Jyh-Bing Tang 湯智斌 |
指導教授: | 陳義雄(Yee-Hsiung Chen) |
關鍵字: | 精子獲能反應,酪胺酸磷酸化,電泳移動率遲緩試驗, sperm capacitation,tyrosine phosphorylation,electrophoretic mobility shift assay, |
出版年 : | 2006 |
學位: | 博士 |
摘要: | 雄性哺乳動物射精之後,精子可以迅速泳動,但是卻缺乏與卵子受精的能力;必須在雌性生殖道內經過一段時間的修飾,這個過程稱之為獲能反應(capacitation)。哺乳動物精子進行獲能反應的分子基礎,不論是在雌性生殖道裡的體內反應(in vivo)或是在試管中誘導的體外反應(in vitro),其機制到目前為止仍不甚清楚。然而根據先前的研究顯示,精子進行獲能反應時,會受到環狀腺核甘酸單磷酸-依賴性(cAMP-dependent)蛋白激酶(protein kinase)的調控,使得許多蛋白質分子的酪胺酸殘基被磷酸化(tyrosine phosphorylation)。利用二維膠體電泳搭配西方轉漬技術(Western blot)分離了小鼠精子獲能反應後細胞萃取物中的酪胺酸磷酸化蛋白,其中一個分子量為130 kDa的蛋白分子經過胰蛋白酶(trypsin)分解及質譜儀的分析,得到六個寡胜肽的序列片段,經過資料庫的比對,鑑定出這個蛋白分子是一個廣泛表現且高度保留的CCCTC-結合轉錄因子(CCCTC-binding nuclear factor;CTCF)。進一步利用免疫沉澱(immunoprecipitation)搭配西方轉漬技術,也發現獲能反應後的精子細胞萃取物經過與抗CTCF的抗體培養一段時間後,其結合沉澱物分別利用另一種抗CTCF的抗體以及抗磷酸酪胺酸抗體進行西方轉漬分析,均可以偵測出一個130 kDa的蛋白訊號;這些結果均證明在獲能反應後的小鼠精子內有酪胺酸磷酸化CTCF蛋白的存在。而間接免疫螢光染色(indirect immunofluorescent staining)的結果顯示,CTCF位於精子的頂體(acrosome)部位。進一步以乙型澱粉樣蛋白前驅物(amyloid The molecular basis of mammalian sperm capacitation, the process to acquire the ability to fertilize the oocyte in the female genital tract in a time-dependent manner, either in vivo in the female reproductive tract or in vitro, is poorly understood. It is well known that sperm capacitation is associated with a cyclic AMP-dependent increase in tyrosine phosphorylation of a subset of proteins. We resolved the phosphoproteins in the cell lysate of mouse sperm after capacitation by two-dimensional gel electrophoresis. Among the protein targets, one tyrosine-phosphorylated 130-kDa protein spot was trypsin-digested, and six oligopeptide sequences were further established from the trypsin digests by mass spectral analysis. These data were completely confirmed in a CCCTC-binding nuclear factor (CTCF), a widely expressed and highly conserved nuclear factor. Although we were unable to determine the exact site of phosphorylation of CTCF for the time being, we did demonstrate, using a cross-immunoprecipitation approach, that this protein is tyrosine phosphorylated during capacitation. Both an anti-phosphotyrosine antibody and an anti-CTCF antibody showed immunoreactivity to a 130-kDa component in the immunoprecipitates obtained after incubation of the cell lysate from the capacitated sperm using another anti-CTCF antibody. The data support the presence of a tyrosine-phosphorylated CTCF in the capacitated mouse sperm. Immunolocalization of the CTCF revealed fluorescent staining in the acrosome region in both capacitated and incapacitated sperm. The electrophoretic mobility shift assay, using a CTCF target sequence 5’-GGCGGCGCCGCTAGGGGTCTCTCT-3’ found in the promoter of the amyloid |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33528 |
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顯示於系所單位: | 生化科學研究所 |
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