Screening Differentially Expressed Proteins Of X And Y Sperms Of Dairy Cows

The study of specific expression proteins from X and Y-sperm is a theoretical basis for sex identification and sex control research in dairy cows.The sperm of Simmental Cow were sorted by flow cytometry,and the sperm were tested by artificial insemination,the best sperm protein cleavage effects were obtained by comparison of trizol,TCA-acetone,urea guanidine hydrochloride three protein cleavage methods;The two-dimensional electrophoresis system of X and Y sperm-specific proteins was optimizated,and the specific expression proteins of X and Y-sperm were screened.The qualitative and functional analysis proteins were identified by protein Q Exactive mass spectrometer and bioinformatics prediction.The results were as follows:(1)The purity of 96% X and Y sperm were obtained by flow cytometry,sperm motility(43.81%,41.49%,46.16%)and sperm acrosome integrity(56.54%,58.21%,58.10%)were not significantly different from sex control sperms and conventional sperms.Thirteen female calves were obtained from nineteen young cows by artificially inseminate with X-sperm,and fourteen female calves were obtained from 20 young cows by artificially inseminate with Y-sperm,the ratio of sex control was 100%,respectively.(2)In the two-dimensional electrophoresis system,the method of trizol cleavage can be obtained 16 protein bands form 3.0×107 sperms which was higher than the TCA-acetone(10 bands)and the urea guanidine hydrochloride(7 bands),respectively.It obtained more independent protein spots by the optimization of programs(IEF,isoelectric focusing;pH4-7 IPG stripssilver stains)than by the programs of pre-IEF programs,p H3-10 IPG strips,blue-siliver stains,respectively.(3)The specific expression of twenty-five protein spots form X and Y sperms were detected,of which ten protein spots form X sperms and fifteen protein spots form Y sperms.Three X-sperm and three Y-sperm specific protein spots were identified by protein Q Exactive mass spectrometer.Among them,two proteins werepresumed histone H2 B,and the putative 14-3-3Z protein,they were located in the X sperms,nucleus and no transmembrane structure,the apolipoprotein A-I was located Y sperms,and outside the cell membrane,but no transmembrane structure.The other three unnamed protein sequences were named protein sequence one and uncharacterized protein sequence two no found in the transmembrane structure,the protein sequence three had a cross-nuclear membrane.Conclusions: The validity of X and Y-sperm were confirmed for sex control in cows;Optimization of the two-dimensional electrophoresis system of X and Y sperm-specific proteins was established;X sperm-specific proteins ten and Ysperm-specific fifteen proteins were obtained by 2DE.The six X and Y sperm-specific protein spots were identified by mass spectrometry.The six proteins were hydrophilic and antigenic determinants,which are related to sperm production,metabolism,metabolism and signal transduction.It was presumed that the presumed histone H2 B and apolipoprotein A-I were related to male sterility of dairy cattle,but the six proteins were not satisfied at the conditions of vaccine.