The Properties Of Human Sperm Membrane CI~- Channel Reconstituted Into Giant Liposome And The Inhibition Of Male Antifertility Drugs On Mouse Spermatogenic Cell Ca~2+channels

Ion channel plays a key role in the maturation, capacitation and acrosome reaction (AR) of mammalian sperm. It was reported that AR was inhibited by Cl- channel blockers or in Cl--free medium. But due to the small size, complex geometry and high differentiation of sperm, the types and properties of mature sperm Cl- channels are difficult to be studied by the patch-clamp technique directly. In this experiment, the membrane proteins extracted from the human sperm were reassembled into liposome bilayer, and then the liposomes were fused into giant liposomes with a diameter more than 10 (m by dehydration-rehydration procedure. The giant liposomes were used to study the Cl- channel activities by patch-clamp technique. Three kinds of single-channel events with different unit conductance weredetected in NMDG-Cl solution system. Their activities were voltage-dependent and all of them were blocked by SITS in a concentration-dependent manner. By constructing the open and close dwell time distribution histograms and then fitting them with exponential function, two time constants were obtained in both the open and the close states. The burst activity and conductance substate of the channels were observed.Many of traditional Chinese medicines including gossypol and Tripterygium wilfordi were found to have male antifertility effects and the interest on these as potential contraceptive drugs arose. Up to now, several monomers were isolated from GTW (multi-glycosides of Tripterygium wilfordii, GTW) and their antifertility effects were demonstrated. However, the effects of these drugs on ion channels have not been directly studied by electrophysiological techniques. The spermatogenic cell, a developmental precursor of mature sperm, is often used as a model system to study the effects of the agents on the channel events in mature sperm. In the present study, by using whole-cell recording, the effects of gossypol, GTWand three isolated monomers from GTW, demethylzeylasteral (TZ), L-epicatechin and celastrol, on the T-type Ca2+ channels in mouse spermatogenic cells were investigated. Meanwhile, the effect of TZ on sperm AR was evaluated by chlortetracycline fluorescence staining. It was observed that all of these agents significantly inhibited Ca2+ currents. The inhibition of the progesterone-evoked AR by TZ at the concentration to block the Ca2+ channels was also observed. These data suggest that the inhibition of T-type Ca2+ currents could be responsible for the antifertility activity of these compounds.