Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa
博士 === 國立臺灣大學 === 生化科學研究所 === 94 === 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 k...
Main Authors: | , |
---|---|
Other Authors: | |
Format: | Others |
Language: | zh-TW |
Published: |
2006
|
Online Access: | http://ndltd.ncl.edu.tw/handle/54376424667220781625 |
id |
ndltd-TW-094NTU05103004 |
---|---|
record_format |
oai_dc |
spelling |
ndltd-TW-094NTU051030042015-12-16T04:38:20Z http://ndltd.ncl.edu.tw/handle/54376424667220781625 Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa 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 Jyh-Bing Tang 湯智斌 博士 國立臺灣大學 生化科學研究所 94 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 Yee-Hsiung Chen 陳義雄 2006 學位論文 ; thesis 99 zh-TW |
collection |
NDLTD |
language |
zh-TW |
format |
Others
|
sources |
NDLTD |
description |
博士 === 國立臺灣大學 === 生化科學研究所 === 94 === 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
|
author2 |
Yee-Hsiung Chen |
author_facet |
Yee-Hsiung Chen Jyh-Bing Tang 湯智斌 |
author |
Jyh-Bing Tang 湯智斌 |
spellingShingle |
Jyh-Bing Tang 湯智斌 Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa |
author_sort |
Jyh-Bing Tang |
title |
Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa |
title_short |
Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa |
title_full |
Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa |
title_fullStr |
Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa |
title_full_unstemmed |
Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa |
title_sort |
identification of a tyrosine-phosphorylated ccctc-binding nuclear factor in capacitated mouse spermatozoa |
publishDate |
2006 |
url |
http://ndltd.ncl.edu.tw/handle/54376424667220781625 |
work_keys_str_mv |
AT jyhbingtang identificationofatyrosinephosphorylatedccctcbindingnuclearfactorincapacitatedmousespermatozoa AT tāngzhìbīn identificationofatyrosinephosphorylatedccctcbindingnuclearfactorincapacitatedmousespermatozoa AT jyhbingtang xiǎoshǔjīngzihuònéngfǎnyīngyǐnfāccctcjiéhézhuǎnlùyīnzilàoànsuānlínsuānhuàzhīyánjiū AT tāngzhìbīn xiǎoshǔjīngzihuònéngfǎnyīngyǐnfāccctcjiéhézhuǎnlùyīnzilàoànsuānlínsuānhuàzhīyánjiū |
_version_ |
1718149558076178432 |