| Part one: Role of potassium channel on sperm in vitro capaictaion in miceThe primary aim of this study was to observe the correlation between the mouse sperm capacitaion state and K+ channel, and then to observe the effect of valinomycin (Val), a potassium ionophore, in sperm in vitro capacitation. Mouse in vitro sperm capacitation was assessed by the B pattern of staining by chlortetracycline (CTC), a Ca2+-dependent fluorescence microscopictechnique, and its is currently the assay of choice because it distinguishes five different stages of sperm activation: noncapacitated, capacitated acrosome-intact , capacitated acrosome-reacted, in an early stage of the AR and nonidentified sperm.When spermatozoa were cultured in valinomycin (Val) at theconcentration of 50nmol.L-1, the capacitation was increased significantly (n=10,p<0.05).As we all know, valinomycin induces potassium efflux in cell, suggesting that potassium efflux is important in capatitation.Then we recorded the delayed rectifier K+ currents(IDRK) in mouse pachytene spermatocyte using whole-cell patch clamp technique so as to determine their characteristics. Meanwhile we observed the effects of valinomycin (Val) on delayed rectified K+ currents presents in mouse pachytene spermatocyte. A outward current activated at -40mV was obtained when inward Ca2+ current and outward Ca2+-dependent potassium current were blocked by Cd2+and EGTA separated. The current increased dramatically when depolarization in creased from -40mV to +70 mV, without any deactivation in 400MS. And it had an outwardly rectifying current-voltage relationship. According to the characteristics of the current, we primarily inferred that it was delayed rectifier K+ current. Furthermore , 4-AP, a know transient inactivation K+ current blocker, did not significantly affect these K+ currents. The current were blocked by 20mmol.L-1 TEAC1, a know voltage-dependent K+ currents. These observations suggest that the outward K+ currents are generated by activate relayed rectifier K+ channels.We found that 50nmol.L-1 Val increased the amplitude of IDRK currents significantly, up-regulated the I-V curve of IDRK current. These results suggest that valinomy promotes potassium ion efflux by activation of delayed rectifier potassium channels in mouse pachytene spermatocyte, and then mediated hyperpolarization itself appears capable of inducing sperm capacitation.Part two: Effect of fenvalerate on sperm in vitro capaciation in mice and its mechanismWhen spermatozoa were cultured in various fenvalerateconcentrations(umol .L-1, 0, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0) for 2 h in an atmosphere of 5%co2 in air at 37C, the percentage of spermatozoa showing pattern B was 55.05 + 0.43, 52.02+1.29, 47.13 + 0.87, 40.08+0.32, 35.79 + 0.88,28.66 + 0.96,27.89+1.56 separately, the ED50 is 5 u mol.L/-1.This suggest that fenvalerate can inhibit capacitation was in a concentration-dependent manner. In the same time we also observed that fenvalerate can inhibit capacitation in a time-dependent manner.To study the mechanism of fenvalerate's action, we observe the effect of fenvalerate on IDRK channels in moue pachytene spermatocyte, was observed using patch clamp technique. The results showed that fenvalerate had a slightly inhibitory effect on the currents presented in moue pachytene spermatocyte, even though at higher fenvalerate concentration. These results indicated that 1DRK channel was not the primary regulation for fenvalerate to inhibit sperm capacitation.Insecticides have been shown to partition into membrane and cause change in membrane fluidity. So at the same time we monitored the fluidity parameters to reveal the dynamic changes in sperm membrane due to fenvalerate interaction by measuring the fluorescence polarization of l,6-diphlnyl-1,3,5-hextriene DPH incorporated into the membrane. Fenvalerate was found to increase the DPH fluorescence polarization value in a concentration-dependent manner, implication that it makes the membrane less fluid. These results suggest that...
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