The Relationship Between Protein Phosphorylation And Cryocapacitation As Well As Studies Of CLC To Improve Quality Of Frozen-thawed Gametes

The objective of this study was to elucidate the differences between cryocapacitation and capacitation generation mechanisms and to explore the effect of CLC in semen extender on the quality of frozen thawed pig gametes.The experiment was mainly divided into three parts.The first part mainly studies the effects of capacitation,acrosome reaction and cryocapacitation on expression and tyrosine phosphorylation of porcine sperm protein 32(sp32).The second part mainly studies effects of CLC on capacitation status,mitochondrial membrane potential and quality characteristics of frozen-thawed porcine sperm.The third part mainly studies effects of CLC on mitochondrial membrane potential,apoptotic gene expression,ZP ubiquitination and sperm-ZP binding ability of frozen-thawed porcine GV oocytes.The results are as follows:1,The results of capacitation,acrosome reaction and cryocapacitation showed that the expression of SP32 in capacitation and acrosome reaction groups was significantly higher than that in fresh and cryocapacitation group(P<0.05);2,The tyrosine phosphorylation level of sp32 in capacitation and acrosome reaction groups was significantly higher than that in fresh and cryocapacitation groups(P<0.05);3,After adding CLC(0,0.5,1.0 and 2.0 mg/mL)to semen extender,the results showed that motility,viability and acrosome integrity of 0.5 mg/mL CLC group was higher than those of other three groups(P<0.05),and the percentage of Capacitated and acrosome-reacted sperm was lower than those of the other three groups(P<0.05);4,The mitochondrial smembrane potential of sperm treated with CLC when boar semen stored at 4 0C and cryopreservation was significantly increased;5,After adding CLC(0,0.5,1.0 and 2.0 mg/mL)to semen extender,the results showed that the CLC fluorescence intensity of 10 mg/mL treatment group was significantly higher than that of the other three groups(P<0.05);6,5 mg/mL and 10 mg/mL CLC groups significantly increased the maturation rate of frozen-thawed porcine GV oocytes(P<0.05)7,The mitochondrial membrane potential of frozen-thawed porcine GV oocytes after sperm treated with 5 mg/mL CLC was higher than that of the other two groups,but significantly lower than that of the untreated control group(P<0.05);8,The expression of Caspase 3,Caspase 8 and Caspase 9 of frozen-thawed porcine GV oocytes after sperm treated with 5 mg/mL and 10 mg/mL CLC was significantly lower than that of control group(P<0.05);9,The sperm-ZP binding ability of frozen-thawed porcine GV oocytes after sperm treated with CLC was significantly higher than that of control group(P<0.05).In conclusion:1,sp32 or sperm protein tyrosine phosphorylation level can be used as a biomarker for porcine sperm capacitation.2,CLC improves the quality of frozen-thawed porcine sperm.CLC increases the motility,viability and acrosome integrity of frozen thawed porcine sperm,reduces the percentage of sperm capacitation and acrosome reaction after freezing and thawing,increases the mitochondrial membrane potential of frozen-thawed porcine sperm.3,CLC improves the quality of frozen-thawed porcine GV oocytes.CLC increases the maturation rate,mitochondrial membrane potential,ZP ubiquitination and ZP3 proteins,decreases the expression of apoptotic genes and improves the sperm-ZP binding ability of frozen-thawed porcine GV oocytes.