Effects Of Genistein On The Quality Of Frozen-Thawed Boar Sperm And Its Mechanism

Studies have shown that low temperature can induce tyrosine phosphorylation,passive capacitation and short survival time of frozen-thawed sperm.Genistein is a natural isoflavone compound,which plays an anticancer role by inhibiting the activity of tyrosine protein kinase and intracellular protein tyrosine phosphorylation.However,its use as a blocking agent of sperm capacitation has been rarely reported.The objective of the experiment was to decrease the protein tyrosine phosphorylation and capacitation–like changes of frozen-thawed sperm by adding genistein to the frozen diluent of boar semen,thereby further improving the quality of frozen-thawed sperm and laying a certain theoretical foundation and scientific basis for breaking through the technical bottleneck of unsatisfactory reproduction performance of frozen-thawed boar sperm.The specific research contents and results showed as followed:1.Effects of genistein on routine indicators of frozen-thawed boar sperm.Different concentrations?0,25,50,75,and 100?mol/L?genistein were added to the frozen diluent of boar semen.Semen was frozen after being incubated for the different time?30,60 and 90 min?.The routine indicators?motility,normal acrosome rate and plasma membrane integrity?of frozen-thawed boar spermatozoa were detected.After the semen was added 100?mol/L genistein and incubated for 60 min,the motility of frozen-thawed sperm was significantly increased?P<0.05?,and the acrosome integrity and plasma membrane integrity were slightly higher?P>0.05?than that of the control group.Conclusion:Genistein improved the quality of frozen-thawed boar sperm,and incubating for60 min in 100?mol/L genistein resulted in a better effect.2.Effects of genistein on capacitation status of frozen-thawed boar sperm.Ca²⁺concentration in frozen-thawed sperm was determined using fluorescence spectrophotometer.The capacitation state of frozen-thawed sperm treated with100?mol/L genistein for 60 min was detected using aureomycin staining.Compared with the control group,Ca²⁺concentration of frozen-thawed sperm in each group decreased?P>0.05?.Capacitated sperm count decreased in the treatment group?P<0.05?.Conclusion:Genistein inhibited the capacitation of boar sperm in freezing-thawing process.3.Effects of genistein on the level of protein tyrosine phosphorylation of frozen-thawed/capacitated boar sperm.The experiment was designed to determine the effects of genistein on tyrosine phosphorylation levels/sites in frozen-thawed?incubated with 100?mol/L genistein for 60 min?/fresh-capacitated?incubated with 100?mol/L genistein for 60 min?sperm using western blotting and indirect immunofluorescence.Results:?1?The tyrosine phosphorylation levels of 32 KDa and 69 KDa proteins in frozen-thawed sperm treated with genistein were significantly decreased compared with the control group?P<0.05?.?2?The tyrosine phosphorylation level of 58 KD protein in capacitated spermatozoa of the capacitated group and genistein+capacitated group was significantly increased compared with the fresh group?P<0.05?.The parameter of genistein+capacitated group was slightly lower than the capacitated group?P>0.05?.?3?The protein tyrosine phosphorylation sites of frozen-thawed and capacitated spermatozoa were mainly distributed in the acrosome?a?zone and acrosome+posterior acrosome?a+b?zone which had strong fluorescence.The phosphorylation sites in the?a+b?region of frozen-thawed spermatozoa were significantly lower than those of the control group?P<0.05?.?4?The phosphorylation sites in?a?/?a+b?zone of the genistein+capacitated sperm were not significantly reduced compared with the capacitated group?P>0.05?.Conclusion:genistein reduced protein tyrosine phosphorylation in the sperm?a+b?zone during freezing-thawing or capacitating.4.Effects of genistein on PKA and PKG activity of frozen-thawed/capacitated boar sperm.The experiment contained two groups:the treatment group?100?mol/L,60 min?and the fresh-capacitated?100?mol/L,60 min?group.And the activity of PKA/PKG in frozen-thawed and capacitated sperm was determined using enzyme-linked immunosorbent assay.Results:?1?The PKA activity of frozen-thawed sperm was significantly reduced compared with the control group?P<0.05?.?2?PKA/PKG activity of genistein+capacitated spermatozoa was not significantly reduced compared with the capacitated group?P>0.05?.Conclusion:genistein may inhibit the PKA activity in the c AMP/PKA signaling pathway,thereby inhibiting the capacitation of boar sperm during freezing and thawing.5.Effects of genistein on the spermadhesins family?AQN1,AQN3,AWN,PSP-I,and PSP-II?and SPMI m RNA expression of frozen-thawed/capacitated boar sperm.The collected semen was divided into two parts.The first was treated with 100?mol/L genistein for 60 min before being frozen.The second was incubated with 100?mol/L genistein during capacitating.The spermadhesins/SPMI m RNA expression of frozen-thawed and capacitated sperm was detected using q PCR.Results:?1?AWN m RNA expression of frozen-thawed sperm was significantly increased?P<0.01?,and the parameters of PSP-I?P<0.05?,PSP-II and SPMI?P<0.01?were significantly decreased in the treatment group compared with the control group.?2?AQN1 and AWN?P<0.01?/AQN3 and SPMI?P<0.05?m RNA expression of capacitated spermatozoa was significantly increased,and the parameter of PSP-I and PSP-II?P<0.01?were significantly decreased in genistein+capacitated group compared with the capacitated group.Conclusion:m RNA expression of spermadhesins and SPMI in the genistein treatment group was better than those in the control group,and close to those in the fresh group.In conclusion,the addition of genistein to boar sperm freezing solution had a positive effect on the quality of frozen-thawed sperm.Its mechanism may be the inhibition of genistein to sperm capacitation by decreasing tyrosine phosphorylation of acrosome/acrosome+posterior acrosome regions of frozen-thawed sperm and increasing m RNA expression of spermadhesins?AQN1,AQN3,and AWN?,or the increase of genistein to sperm motility by reducing the m RNA expression of spermadhesins?PSP-I and PSP-II?and SPMI.The experiments provided a theoretical basis for further research on the protection mechanism of genistein on boar sperm.