| Mouse spermatozoa express a pH-dependent K+current (Ksper) thought to induce hyperpolarization to enhance Ca2+influx via an alkaline-activated calcium channel (Catsper) to initial a so-called sperm capacitation while staying in female genital tract for fertilization. However, the regulating mechanism of the Ksper and Catsper channels by membrane potential and pHi remains uncertain because the complexities of two channel kinetics in sperms are hardly to overcome at this stage. Using confocal calcium imaging, here showed differences of the intracellular [Ca2+]i between the wild-type (WT) and knockout (KO) Ksper (or Slo3-/-) mice in the application of the Slo3blockers, Quinidine (QD), Clofilium, and NH4Cl indicating that Ksper channels encoded by Slo3gene, dominates the membrane potential of mouse sperms to increase the intracellular [Ca2+]i and [pH]j during the capacitation process to play a vital role in fertility. Furthermore, model sperm built directly with the native Ksper and Catsper currents in sperms reveals two functions of membrane potential and intracellular pHi, allowing us to calculate the intracellular pHi by NH4Cl, based on membrane potentials recording from current-clamp experiments. Obviously, the profound work is necessary to explore the details of the channel gating and the physiological role of sperm in the future. Since data from both our studies and those of others laboratories have shown that [Ca2+]i plays a central and fundamental roles, and the interaction with the female reproductive tract is essential for the "normal" Ca2+signaling and fertilizing ability of sperm. We report that quinidine at different concentration in μM(0.1,1,10,100, and1000) induced a completely reversible reduction in the intracellular Ca2+of sperms at the lower concentration (0.1and1μM), but a partially reversible reduction at the higher concentrations (10,100and1000μM). Furthermore, clofilium at a concentration in uM (50,100and500) decreased [Ca2+]i by inhibiting extracellular Ca2+entry to sperm by blocking ksper channel. This indicates that the blockade of Clofilium is irreversible in contrast its reversible block with the lower concentration of quinidine through which the Ca2+concentration sustained to normal state after washout with HS containing2mM Ca2+at pH7.4. These blockers effects are dependent on K+flux and membrane potential. Moreover, the application of10μM QD+10mM NH4Cl and50μM Clofilium+10mM NH4Cl reduced the Ca2+with a reversible recovery in WT sperm. Showed that the role of the three drugs in WT, are stronger than Slo3-/-sperm cells. The Slo3-KO mouse used to demonstrate turther that all of the above phenomena are whether derived from Catsper or KSper (Slo3) channels. As a comparison, we re-examined the changes of Ca2+signal in the Slo3-null mouse sperms under the similar conditions, the Slo3-/-sperms showed a complete recovery of Ca2+signal from the Clofilium inhibition due to absent of Slo3irreversible factor. Concluded that the reductions in Ca2+signal caused by QD and Clofilium in Slo3-/-sperms were not from the blocking of KSper (Slo3), but possibly from the direct blocking of Catsper channel or others. Additionally, the application of10mM NH4Cl also enhanced the Ca2+signal in Slo3-/-sperms, similar to that in wt sperms suggesting that the Catsper channel should be pH-dependent too. The simulations results of sperm membrane potential dependence on pH by using current clamp technique, the membrane potential recorded at different pH in WT and Slo3-/-mutant mice sperm. The experimental results showed the Slo3membrane potential increase with pH elevation produced hyperpolarization. In contrast, in the mice Slo3gene knockout, the increase of sperm cell membrane potential opposite to the pH change.We concluded from that, the current model not only explains the capacitation mechanism induced by Ksper channels and introduces two functions of the pHj and membrane potential Vm for quickly calculating the values of pHj in wt and Slo3-/-sperms according to the values of Vm measured by Current-Clamp experiments. Recently, there is developing of a new approach of male contraception, based on targeting specific processes in sperm development, maturation, and function by using quinidine as a one of calcium channel blocker. Short-Term uses of this reagent, to treat malaria have a significant antispermatogenic and antifertility or contraceptive effect on mice sperms and testicular tissue in vitro, and our findings showed the toxic effect of QD on sperms parameters concluded that QD had harmful effects on seminiferous tubules and sperm formation in male mice and probably caused disorder in spermatogenesis and male fertility... |
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