Induction Of Acrosome Reaction And Preliminary Exploration On The Function Of Sperm BK Channel Of Eriocheir Sinensis

To explored the function of large-conductance calcium-activated potassium channel?known as BK channel?and its relationship with acrosome reaction in Chinese mitten crab?E.sinensis??known as crab?sperm.In the study,we acquired E.sinensis sperms,as experimental material,by accessory sex gland digestion.We carried out a series of studies,which were as follows,in vitro,the sperms were induced with calcium ionophore A23187.And the changes of sperm intracellular calcium concentration were detected before and after the acrosome by Fluo-4AM,which is a calcium fluorescence probe.Then,we detected and recorded the wholecell potassium current by whole-cell patch-clamp technique.Also,we analysised the electrophysiological properties and type by using the channel blockers.And we simulated the change of sperm intracellular calcium concentration by increasing the Ca²⁺ concentration of pipette solution,which had an effect on potassium current of crab sperms.We analysised and explored the effect of potassium channel in E.sinensis sperm acrosome reaction.The main results were as follows:1.With different concentration of calcium ionophore A23187,diluted by ASW?artificial sea water?and Ca²⁺-FASW?Calcium-free artificial sea water?respectively,to induce acrosome reaction of E.sinensis sperms.A23187 could significantly increase the acrosome reaction rate of sperm.And the sperms which had accomplished acrosome reaction were most in the III or IV period.So,the best concentration of A23187 was 80 ?g/m L.And the best time of induction was 30 min.Also,we found that Ca²⁺ played a leading role in sperm acrosome reaction of E.sinensis.2.Inducing acrosome reaction by A23187,while labelling Ca²⁺ of the crab sperms by calcium fluorescence probe Fluo-4AM,we tracked and detected the Ca²⁺ concentration change of a single E.sinensis sperm before and after acrosome reaction by confocal laser scanning technology.Studies found that the Ca²⁺ was mainl distributed in the cytoplasm and the acrosome tube of the acrosomal vesicle before acrosome reaction.The intracellular Ca²⁺ concentration had significantly increased in the moment of the acrosome reaction,which was inducing by A23187.And the Ca²⁺ was distributed in the cytoplasm and the front of acrosome tube that had be extended.In addition,the calcium fluorescence of sperm had no significant increase in the calcium-free buffer,which indicating that the sperm acrosome reaction mainly depends on the external calcium influx.3.In E.sinensis sperm,we used the patch-clamp technique to record the whole-cell potassium current,which included fast IA-like current and IK-like delay current.In calcium-free pipette solution,after adding BK blocker IbTX?Iberiotoxin 10 nmol/L?,the IA-like and IK-like potassium current was significantly inhibited,which the inhibition rate was 17.74 % and 21.51 %,which indicating that the sperm of E.sinensis possess BK channel,which was activated by membrance depolarization.4.We simulated the Ca²⁺ influx of E.sinensis sperm acrosome reaction by adding Ca²⁺ into pipette solution?50 ?mol/L?,found that the potassium currents of IA-like and IK-like had significantly increased,which increasing 61.49 % and 44.11 % respectively.So,the Ca²⁺ can enhance the whole-cell outward potassium current.After adding IbTX,the potassium currents of IA-like and IK-like were significantly inhibited,which the inhibition rate was 27.17 % and 22.65 % respectively.So,BK current was important in sperm outward potassium current and was dependent on Ca²⁺.These research results showed that the Ca²⁺ influx could activate BK channel,caused heavily outflow of K+,changed membrane potential,triggered sperm acrosome reaction,when the E.sinensis sperm was induced by A23187 in vitro.Showing that BK channel played an important role in sperm acrosome reaction of E.sinensis.