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

Abstract

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