Effect Of AC220 And ZK 93426 On Sperm Capacitation And Acrosome Reaction

Spermatozoa produced in the testis don't have the ability to fertilize the egg.It needs to gain forward movement ability in the epididymis,undergoes capacitation and acrosome reaction in the female reproductive tract,passes through the zona pellucida,and integrates with the egg to complete the fertilization.If capacitation or acrosome reaction is abnormal,male fertility will decrease.At present,there have been extensive studies on capacitation and acrosome reaction,but the specific mechanism has not been completely resolved.Previously AC220 and ZK 93426 were found during the screening of small molecular compounds that affect capacitation and acrosome reaction.The aim of this study was to explore the effect of these two compounds on sperm capacitation and acrosome reaction and to further explore the underlying mechanism.AC220 is a potent,selective and efficacious FMS-like tyrosine kinase-3(FLT3)inhibitor while ZK93426 is a selective benzodiazepine receptor antagonist,it belongs to ?-carboline derivatite.We first detected the effect of AC220 on the acrosome reaction of mouse and human sperm and the result showed that AC220 could inhibit the acrosome reaction significantly.Later experiment showed that AC220 had no significant effect on mouse sperm viability and motility.Further examination revealed that AC220 could significantly inhibit capacitation-related protein tyrosine phosphorylation.Furthermore,PKA pathway agonist dbc AMP can reverse the inhibitory effect of AC220 on capacitation.In addition,we analyzed FLT3,which is the target protein of AC220 and found FLT3 is present in mouse spermatogenic cells,and FLT3 is located in the head of mouse sperm and in the neck of human sperm.On the other hand,we tested the effect of ZK 93426 on mouse and human sperm capacitation.The results showed that ZK 93426 significantly promoted capacitation-related protein tyrosine phosphorylation.The promoting effect was more intensive in HMB in the absence of calcium and immunofluorescence confirmed this conclusion.ZK 93426 was subsequently found to have no significant effect on mouse sperm motility.We selected the Cat Sper channel inhibitors Mibefradil and NNC 55-0396 and the PKA pathway inhibitor H89 to further explore their mechanism and found that Mibefradil and NNC 55-0396 couldn't inhibit the promoting effect of ZK 93426 on capacitation-related tyrosine phosphorylation while H89 could inhibit its effect.In summary,AC220 inhibits the acrosome reaction by down-regulating protein tyrosine phosphorylation.ZK 93426 promotes capacitation-related protein tyrosine phosphorylation.AC220 and ZK 93426 may play a corresponding role by acting upstream of the PKA pathway but the promoting effect of ZK 93426 is independent of the Cat Sper channel.This study could help reveal the signal pathways and molecular mechanism in sperm capacitation,and provide new ideas for the diagnosis and treatment of male infertility.