The Effects Of Different Treatments On The Expression And Distribution Of Protein Typrosine Phosphorylation Quality Of Pig Sperm

The purpose of this study was to evaluate the effects of capcitation freezing and treat time on the expression and distribution of protein tyrosine phosphorylation quality of pig sperm. Giemsa staining dye. Coomassie brilliant blue dye staining, immunofluorescence, SDS-PAGE and Western blotting analysis were used to detect the sperm motility, acrosomal integrity, distribution and expression of protein tyrosine phosphorylation sites Test can be divided into:Ⅰadopting different freezing methods,different thawing temperatures and incubation at different time to detect the quality of sperm;Ⅱdetecting the sperm capacitation efficiency which plced in mTBM processing different time.Ⅲadopting indirect immunofluorescence to treat sperm of different time capacitation and shows changes of sperm protein tyrosine phosphorylation sites under Fluorescence microscopy.ⅣAdopting SDS-PAGE and Western blotting analysis to treat sperm of different time capacitating.Results:1,there was no significant difference between slow freezing and rapid freezing (P>0.05);When using 0.5ml thin tube,there was significantly different between slow freezing and rapid freezing (P<0.05). After thawed. motility and the rate of acrosomal integrity of sperm were no significantly difference when adopted slow freezing with 0.25ml and 0.5ml thin tube (P >0.05);but adopted rapid freezing, there was a significantly difference, 0.25ml thin tube was better than 0.5 thin tube (P>0.05);thawed in 40℃and 45℃water,there was no significantly difference (P>0.05),but was better than 35℃and there was a significantly difference,but was better than 35℃and there was a significantly difference (P>0.05).when used 0.25ml thin tube,40℃was better than 45℃and there was a significantly difference (P<0.05)after thawed.Sperm motility was significantly difference between 0.25ml thin tube and 0.5ml thin tube thawed in the same time (P<0.05), 0.25ml thin tube was better than 0.5ml thin tube,incubated 20min,30min and 40min after thawed, motility and the rate of acrosomal integrity of sperm were significantly different,30min was best, after 40min incubation. sperm motility and acrosomal integrity were significantly decreased. 2,with the time of capacitation increased. sperm motility gradually reduced.2h was significantly different from others.time of capacitation had a great influence to the integrity of the sperm plasma membrane,in precess of capacitation, integrity of the sperm plasma membrane decreased.3,Capacitation had a significantly effect on levels of pig sperm protein tyrosine phosphorylation.Untreated sperm could only be seen in the neck of sperm where there were slightly sites of tyrosine phosphorylation. The treated sperm protein tyrosine phosphorylation was in a deeper level, color of fluorescent was evident, and sites of Tyrosine phosphorylation could be seen in acrosome and tail. with the time of capacitation increasing, fluorescent color became increasingly evident in acrosome and tail, proving that capacitating could cause internal tyrosine phosphorylation of sperm phosphorylated.Treated for 1h and 1.5h of capacitation,tyrosine phosphorylation of sperm focused on acrosome and tail.4,Alone with time of capacitation increasing, there were some new proteins in semen while part of them were lost at 2h. There was a 27 ku tyrosine phosphorylation protein which tyrosine phosphorylation level was increased when detected porcine sperm after 0 to 5 h in vitro capacitation. In vitro culture 1h,there was another 47 ku tyrosine phosphorylation protein which tyrosine phosphorylation level was highest after 1.5h in vitro cuiture.Then with the process of capacitating, tyrosine phosphorylation level was decreased.