Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa
Date Issued
2006
Date
2006
Author(s)
Tang, Jyh-Bing
DOI
zh-TW
Abstract
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
Subjects
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
Type
other
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