| Ion channels are deem to play a critical role on the regulation of physiological function in living cells.Recent researches demonstrate that sperm-specific K+ ion channel(KSper)and Ca2+ ion channel(Cat Sper)are expressed in mammalian sperm.KSper is responsible for mediating the membrane potential and subsequently,affects the opening probability of voltage-dependent ion channels and transporters in sperm.Cat Sper is found to involve in the modulation of intracellular calcium([Ca2+]i).As the important second message,Ca2+ is required for a series of physiological functions in sperm.Benefiting to the established knockout model of mouse,the deficiency of functional Cat Sper and KSper channel results in male infertility.More importantly,the patient whose Cat Sper gene was mutated suffers from the infertile syndrome.The abnormal membrane potential controlled by h KSper may cause the severe fertilizing malfunction.Therefore,the physiological regulation of KSper and Cat Sper are indispensable to the completion of fertilization process in sperm.With the extensive application of sperm patch-clamping technology,the regulatory mechanism of KSper and Cat Sper are well elucidated.Previous studies showed that KSper and Cat Sper channel were potentiated by the elevation of intracellular p H(p Hi).When sperm are ejaculated from the epididymis to female reproductive tract and guided to swim toward the location of oocyte,KSper and Cat Sper are activated by responding to extracellular alkalization and initialed a variety of important physiological functions to accomplish the fusion with oocyte.Kinds of regulatory mechanisms,such as Na+/H+ exchangers(NHEs)and voltage-dependent proton channel(Hv1),are reported to take participate in mediating the intracellular alkalization.However,whether these molecular mechanisms of p H regulation are functional coupled to the activation of KSper and Cat Sper remain to be revealed.Focusing on the above problem,by performing the patch-clamp techniques and sperm functional experiments,we examined the effect of NHEs and Hv1 inhibitors on KSper and Cat Sper channel and put forward these findings: 1)As a broad-spectrum antagonist of NHEs,DMA dramatically inhibited the activation of m KSper and m Cat Sper,and subsequently,impaired the sperm motility and the occurrence of progesterone-induced acrosome reaction via decreasing the p Hi.2)Cariporide that aims to inhibit NHE1 and NHE5 failed to produce the inhibition of m Cat Sper channel.In this case,we supposed that the biological activity of sperm-specific NHE(s NHE)was the key prerequisite for the alkalify-activated KSper and Cat Sper,although there was no suitable antibody of s NHE to confirm the effect of s NHE on m KSper and m Cat Sper channel.3)Both NHEs and Hv1 participated in the regulation of alkalization-activated h KSper and h Cat Sper channel.NHEs were suggested to be the main factor on the regulation of h Cat Sper activation.As a consequence,the amplitude of [Ca2+]i signals and sperm motility were partially reduced by the incubation with DMA.Except for the property of p H-sensitivity,KSper channel in human sperm was found to be mediated by [Ca2+]i.Early researches showed that about 50 ?M [Ca2+]i could evoke the potentiation of h KSper.However,whether the elevation of [Ca2+]i signaling under the physiological condition is sufficient to regulate the h KSper channel needed more crucial evidences.In order to elucidate the physiological mechanism how h KSper was modulated by [Ca2+]i,we investigated the effect of progesterone-stimulated [Ca2+]i increase on h KSper channel.Our results manifested that Ca2+ influx induced by progesterone(P4)at 500 n M failed to mediate the membrane potential.Whilst,Ca2+ ionophore(ionomycin)or artificial injection of 1 m M Ca2+ into the sperm cytoplasm was capable to bring about the hyperpolarization of membrane potential.By comparing the alteration of membrane potential in the presence or absence of Ca2+ chelator(BAPTA),we found and verified that membrane potential recorded from the same samples was depolarized due to the reduction of basal [Ca2+]i.Hence,we conjectured that the failure of h KSper activation caused by P4 was resulted from the effect of basal [Ca2+]i on h KSper channel.To confirm this hypothesis,we tested whether P4 mediated the h KSper channel in the absence of basal [Ca2+]i.Although [Ca2+]i elevation elicited by P4 was partially chelated by BAPTA,a slight hyperpolarization of membrane potential was observed.According to our results,we speculated that the maintenance of basal [Ca2+]i was of importance for the opening of h KSper channel.The physiological regulations of sperm ion channels play a decisive role on the sperm capacitation,hyperactivity and acrosome reaction.However,the involvement of ion channels in sperm chemotaxis still obscure.P4 was proved to induce the activation of h Cat Sper and sperm chemotaxis,while the concentration of P4 that enhances the h Cat Sper current was much higher than the effective concentration of P4 on sperm chemotaxis.Therefore,in order to clarify the function of h Cat Sper on sperm chemotaxis,more convincing evidences need to be proposed.Recently,a kind of ?-defensins named DEFB119 and its mouse ortholog DEFB19 were reported as the potent chemoattractant for human and mouse sperm.Thus,we explored the effect of recombinant DEFB119/19(r DEFB119/19)on Cat Sper channel.Our results showed that r DEFB119/19 had the capability to potentiate the Cat Sper channel and resulted in the elevation of [Ca2+]i.To further confirm that the enhanced current was derived from the activation of Cat Sper,we verified that mibefradil and RU1968,two Cat Sper inhibitors,abolished the amplitude of Cat Sper currents induced by r DEFB119/19.More importantly,r DEFB19 failed to evoke the observable augment of current recorded from the sperm in a Cat Sper-deficient mouse.Besides,we also found that C-C chemokine receptor 6(CCR6),the physiological receptor of r DEFB119/19,was required for the r DEFB119/19-mediated Cat Sper activation.Therefore,all of the results indicated that r DEFB119/19 activated Cat Sper channel and modulated sperm chemotaxis via binding to their receptor CCR6.Meanwhile,C-type natriuretic peptide(NPPC),another possible chemoattractant,also increased the amplitude of current under the recording condition of Cat Sper.However,NPPC had no effect on the [Ca2+]i signals.As a result,we surmised that NPPC-mediated sperm chemotaxis was Cat Sper-independent.The potentiation of current was derived from other unknown ion channel.Based on these results,we proposed that different chemoattractants possessed their own regulatory mechanism and Cat Sper was not required for all of the chemotactic effects.In conclusion,our study illuminated that intracellular alkalization mediated by NHEs and Hv1 was of significance on the activation of KSper and Cat Sper.We also presented a new insight that basal [Ca2+]i was necessary to the opening of h KSper channel.At last,we investigated the importance of Cat Sper channel on sperm chemotaxis.Our research will be beneficial to have a better understanding of the vital role of KSper and Cat Sper on sperm functions and achieve the targeted settlement on the fertilizing problems. |
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