Studies On The Role Of Slo3Channel In Mouse Sperm Ca²⁺ Signaling

Background and Objective:Infertility is a significant clinical disease which adversely impacts human physical and mental health and even human population. About half of the infertility cases are related to male factors. One of the most important male factors to affect fertility is sperm, which is generated in the testes, then matured and stored in the epididymis. Sperm from epididymis must undergo a complicated process called capacitation before they obtain the ability to fertilize the egg. When sperm enter into the female reproductive tract, they encounter an environment with varying physical and chemical factors, such as pH, osmotic pressure and hormone level. By responding to the environmental changes, sperm will experience many physiological processes including capacitation, hyperactivation and acrosome reaction. These physiological processes are essential parts of the sperm function, and they are very important to fertilization.As in other cells, Ca2+plays an important role as a second messenger in sperm, directly or indirectly regulating the physiological processes mentioned above. In sperm, the major resource of Ca2+is the Ca2+influx through Ca2+channels located on plasmic membrane. So far, there are only two kinds of ion currents in sperm have been substantially studied with electrophysiological techniques:one is Ca2+current mediated by CatSper channel; the other is K+current mediated by Slo3channel. Studies have shown that Slo3channel, the primary K+channel in mouse sperm, plays an important role in sperm capacitation. Along with sperm intracellular alkalization, the pH-dependent Slo3channels are activated, resulting in membrane potential hyperpolarization. Membrane potential hyperpolarization is speculated to affect Ca2+signaling by two ways:on one hand, it increases the driving force for Ca2+influx; on the other hand, it may decrease the open probability of voltage-dependent CatSper channel, subsequently inhibiting Ca2+influx. Therefore, the net contribution of Slo3channel on sperm Ca2+signaling is uncertain although it is speculated that it should be an important regulator. However, so far no Slo3-specific blockers have been identified. Among the Slo1-specific and general potassium channel blockers, such as CTX, IbTX, TEA,4-AP and quinidine, quinidine is the only blocker which strongly inhibits Slo3channels.In addition, reactive oxygen species have been shown effects on mouse sperm function. Appropriate amount of ROS is closely related to the sperm normal physiological function. Nevertheless, excess of ROS will cause sperm oxidation-antioxidant imbalance, which may result in sperm damage, including reduced sperm activity, inhibited acrosome reaction and reduced sperm fertilization ability. Interestingly, the sperm damages caused by excess ROS show some similarity to the phenotypes of Slo3-/-sperm. Furthermore, ROS has been shown to inhibit Slo1channel, whose intrinsic structure is speculated to be similar to that of Slo3channels. These similarities infer that ROS might modulate sperm functions through Slo3channels.Methods:1. Single sperm confocal Ca2+imaging:sperm obtained from3months old male mouse are divided into three groups randomly:control, quinidine and H2O2. Procedures:1. Sperm from caudal epididymides are placed into centrifuge tube, and then centrifuged.2. The centrifuged sperm are put into confocal chamber which have painted with Cell-Tak and Poly-L-lysine hydrobromide, intending to make sperm completely attached to the chamber bottom.3. Fluo-4AM is added into chamber, incubating for30minutes at room temperature and in dark.4. Single sperm Ca2+imaging is acquired in confocal system, in which60x Oil immersion objective is used. The excitation wavelength is488nm, and the emission between535-565nm is collected.2. Patch-clamp techniques:the inhibition of quinidine on KSper is substantially studied under whole-cell configuration, intending to find the dose-response relation-ship of quinidine block on native sperm potassium currents.Results:1. Quinidine decreases intracellular Ca2+concentration in sperm, and the reduction level is concentration dependent:the extent of reduction is increased with the increase of quinidine concentration. At all of the tested concentrations (0.1μM、1μM、1μM、10μM、100μM and1000μM), the averaged reduction levels are about15%,20%,40%,70%and90%at0.1μM,1μM,10μM,100μM and1000μM quinidine, respectively. The concentration of half reduction (IC50) is about11μM. Interestingly, the results showed that the IC50for quinidine to block KSper currents is also around10μM.2. Our preliminary data showed that H2O2may also affect sperm Ca2+signaling. Different from quinidine, at low concentrations H2O2seems to increase intracellular Ca2+concentration in sperm, for example,100μM of H2O2increases sperm Ca2+signal about30%. However, at high concentrations H2O2decreases Ca2+signal:the Ca2+signal was decreased by60%and90%at1000μM and1×106μM, respectively. Whether the effect of H2O2on sperm Ca2+signaling is related to Slo3channels will need further investigation.Conclusion:Inhibition of Slo3by quinidine decreases intracellular Ca2+concentration in sperm, suggesting that the activation of Slo3channels under alkalization may increase Ca2+influx.