Fenvalerate Inhibits T-type Ca～(2+) Currents In Mouse Spermatogenic Cells By Calmodulin

Part One: Properties of T-type Ca2+ currents in mouse pachytene spermatocytes using patch clampCa2+ is an essential element in all ions influenced physiological processes for mammal sperm. Intracellular Ca2+ is the key to the aspects including spermatogenesis, mature, capacitation and acrosome reaction. The voltage dependent calcium channels is one of main paths to controlling Ca2+ influx and T-type Ca2+ channel is the main Ca2+ current carrier in mouse spermatocyte and sperm. Previous study on T-type Ca2+ channel in mouse spermatogenic cells adopted the method of enzyme digestion which we have known could affect the channel function in cells and it would take too much time for the test to meet the requirement of quickness in pharmacology and toxicology. So properties of Ca2+ channels in acutely dissociated mouse spermatogenic cells devoid of enzyme digestion were studied using whole-cell patch clamp technique. Ca2+ currents were obtained by stepping membrane potential to voltage between -80 and +10mV, in 10mV increments from a holding potential of -90mV. The threshold for Ca2+ current activation was at about -60mV, thepeak amplitude occurred at -30mV and the reversal potential about was 48mV. The constant of both the peak time and the time of inactivation is voltage dependent, kinetics changing with e-fold and the voltage change is 13.11 and 9.72mV, respectively. Half-activation value of -49.43+0.62 mV and steepness parameter of 6.05+055mV were obtained from the data, and activation occurs between -65 and -10mV. Half-inactivation value and steepness parameter averaged -60.79+0.24mV, 4.71+0.22mV, and inactivation occurs from -90 to -40mV. Kinetics of activation and inactivation show that Ca2+ channels presented in mouse spermatogenic cells belong to T-type channels. Accordingly, NiCl2(100umol/L and CdCl2(100umol/L) reduced current amplitude by 62.48% and 20.03%, respectively. Compared with the results reported by Darszon, time constant of inactivation was significant elongation resulted from its good behavior.Part Two Potent mechanism of fenvalerate on T-type Ca2+ channels in mouse pachytene spermatocytesSperm must undergo some crucial physiological courses before fertilization, including capacitation and acrosome reaction. Only acrosome-reacted sperm can penetrate the egg and fertilization. During the course of increase of intracellular Ca2+ plays an important role in acrosome reaction and the main path of Ca2+ influx is through T-type Ca2+ channels. In the study, the effect of fenvalerate on T-type Ca2+ channel in mouse pachytene spermatocytes, was observed using patch clamp technique. The results showed that fenvalerate had a significant inhibitory effect on the currents presented in mouse pachytene spermatocytes in a dose-dependent manner. The maximum inhibitory rate of the currents was reached in the presence of 1 micromolar fenvalerate. It took 3 min to appear any sign of effects and 9-1 Omin reached maximum inhibition after addition of fenvalerate to the bath, which suggests that the slow rate offenvalerate action was controlled by the rate at which fenvalerate moves and unbinds from membrane lipid phase. And the current was irreversible after the drug removal. Analysis of the kinetics of Ca2+ channels indicated that fenvalerate could affect both the activation and inactivation of Ca2+ channel. The curves of activation and inactivation were shifted in hyperpolarizing direction for about 14 mV, suggesting the open time of Ca2+ channel was unchanged. It was indicated the change of permeability of the channel to ions accounts for the inhibition of fenvalerate on Ca2+ current. Fenvalerate may block the course of spermatogensis through inhibiting T-type Ca2+ current, which could mediate differentiation and maturation. These Ca2+ channels are also likely to be present in these cells, where they may contribute to the Ca2+ influx required to trigger the acrosome reaction. Thus the reduction of Ca2+ influx by fenvalerate resulted in the suppression of acrosome reaction.Part Three The eff...