猪精子获能前后细胞亚组分蛋白分离及酪氨酸磷酸化蛋白的鉴定

Separating Subcellular Proteins in Uncapacitation and Capacitation of Boar Sperm Cells and Identifying the Tyrosine Phosphoprotein

本研究对猪精子获能前后细胞亚组分蛋白进行分离以及对酪氨酸磷酸化蛋白进行鉴定,旨在为哺乳动物精子受精生物学研究奠定理论基础。利用动物精子体外获能培养、细胞亚组分分离技术及蛋白免疫印迹的方法,分离猪精子细胞亚组分蛋白及酪氨酸磷酸化蛋白鉴定。结果表明,猪精子经过获能培养后各项活力指标均得到显著提高,且与精子蛋白发生酪氨酸磷酸化修饰密切相关;获能精子中126、108、79ku的高分子量蛋白磷酸化程度明显高于未获能精子;分子质量约为25、47、50ku的膜蛋白及47ku胞浆蛋白发生酪氨酸磷酸化,其中25、47ku的膜蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05);分子量约为23、37、42～50ku的核蛋白发生酪氨酸磷酸化,获能精子中23ku的核蛋白酪氨酸磷酸化程度显著高于未获能精子(P<0.05)。结果提示,猪精子细胞不同亚组分中,发生酪氨酸磷酸化修饰的蛋白以膜蛋白及核蛋白为主,同时有少量的胞浆蛋白。

The aim of this study was to separate subsets proteins in uncapacitation and capacitation of boar sperm cells and to identify the protein tyrosine phosphorylation,and to establish the theoretical foundation for the mammalian sperm fertilization biology research.In vitro,the boar sperms were cultivated for capacitation and separated subsets proteins.The tyrosine phosphorylated proteins in uncapacitation and capacitation sperms were identified by using SDS-PAGE and Western blot.In capacitation boar sperm,the degree of tyrosine phosphorylation on 126,108,79 ku proteins were significantly higher than them on uncapacition boar sperm.The vigor indexes and the results of SDS-PAGE of uncapacitation and capacitation sperms were different,all the vigor indexes of capacitation sperms were significantly improved and these changes were related with tyrosine phosphorylation.The molecular weight of tyrosine phosphorylated proteins on membrane were approximately 25,47 and 50 ku and on cytoplasm is about 47 ku.The degree of tyrosine phosphoproteins of 25 and 47 ku in capacitation sperm were significantly higher than them in the uncapacitation sperm（P〈0.05）.This phenomenon also existed in cell nucleus.In sperm nucleus,these proteins’ molecular weight were 23,37 and 42-50 ku.The degree of tyrosine phosphrylated 23 ku protein in nucleus of capacitation sperms are higher.The results suggests that the main tyrosine phosphorylated proteins were on the nucleus and membrane proteins,and a few of them are plasma proteins.