Study Of The Regulation And Function Of Spermatozoa Potassium Ion Channel

Background and objective:With the change of the modern lifestyle and environment, the rate of infertility is rising gradually. At present, about fifteen percent of couples have fertility problems at different levels and around fifty percent of which is associated with men. Therefore it is the basis of accurate diagnosis and treatment of male infertility to illuminate the regulating mechanism of spermatozoa's function. Sperm must undergo capacitation, hyperactivation and Acrosome Reaction before sperm fertilize an egg in the female tract. And the misfunction of the processes caused by physiological or environmental factors may affect the fertilization. K+ ion channel, KSper and Ca2+ ion channel, CatSper are the most important specific ion channels on the spermatozoa. KSper consists of the main subunit Slo3 and auxiliary subunit LRRC52. It is reported that Slo3-/- male mice are infertile, and LRRC52-/- male mice have severely impaired fertility, which suggests that KSper is the key factor of regulating the spermatozoa function. It has been known well that under physiological conditions, increased intracellular pH is critical for activating the KSper. However, it hasn't been elucidated that the regulating mechanisms of intracellular pH of spermatozoa and that which molecules can activate the KSper through regulating the intracellular pH. Furthermore, whether some environmental factors affect the function of spermatozoa by the KSper have not been reported.We aim to clarify that whether the NHE is the important molecule to regulate the KSper in the physiological conditions on the basis of published reports that NHE is the pH regulator on the spermatozoa. In addition, this study tries to illustrate that whether the damage effect of BPA, an endocrine-disrupting chemical, on spermatozoa function is induced by KSper. Taken together, we want to illuminate the regulating mechanism and function of KSper in the spermatozoa and provide some new information for the regulation of sperm function and the sperm damage by environmental factors. Methods:1. Studing the role of NHE inhibitors in the activation of KSper. we should filter a broad spectrum NHE inhibitor that didn't inhibit the KSper channel. That K+ flow out induced by KSper induce membrane hypopolarization. The change of membrane potential was directly response to the activation of KSper, and the change of pH. In different buffer pipette solution, we detected the influence of NHE inhibitor on spermatozoa membrane potential by using the patch clamp technology. This study is to elucidate whether the NHE regulated the KSper through intracellular pH. In addition, we examined the effect of inhibitors on the spermatozoa motility and AR using CASA and AR technology.2. Examining the effect of BPA in different concentrations on the expression and function of the KSper in vivo and vitro. In the vivo, the mice were fed with BPA in different concentrations for three months. Then, detected the expression and the current of main subunit Slo3 by using Western blot and patch clamp technique. Also the spermatozoa motility and AR were examined. In vitro, we examined the effect of BPA in various concentrations on the current of KSper, sperm motility and AR, Results:1. we filtered a broad spectrum NHE inhibitor that doesn't inhibit the KSper channel, DMA.2. When the pipette solution pH was low buffer(without Hepes), 20 ?M DMA inhibited the sperm membrane potential significantly under the physiological extracellular solution, suggesting that NHE is very important for increasing intracellular pH and activate KSper in the physiological conditions.3. When the pipette solution pH was strong buffer(containing 20 mM Hepes), DMA didn't inhibit the sperm membrane potential in the physiological extracellular solution. This further indicated that that effect was induced by pH rather than other factors.4. Intracellular pH only affected the initial membrane potential, but didn't affect the effect of DMA on the depolarization.5. Fluorescence measurement indicated that DMA decreased the intracellular pH of spermatozoa. This rusults were consisted with the above results.6. In addition, DMA had a significant inhibition on P4-induced AR. But DMA had no significant effect on spermatozoa motility and spontaneous AR.7. In vivo experiment, Sperm that mice were feed with different concentration BPA had low motility, and the AR was lower. The expression of Slo3 and the KSper current had no effect.8. In vitro experiment, BPA could damage sperm motility and AR directly when BPA was incubated with spermatozoa. But BPA had no effect on KSper current rather than CatSper. Conclutions:1. NHE is the important molecule to regulate the intracellular pH and KSper on spermatozoa in the physiological conditions. This result provides direct evidence that NHE regulate the KSper channel by regulating the intracellular pH. DMA, an inhibitor of NHE, decreased the intracellular pH of mice spermatozoa, and then induced depolarization of mice spermatozoa membrane potential induced by KSper. DMA had a significant inhibition to P4-induced AR, which is likely related to the inhibiton of DMA on AR induced by P4.2. BPA, a kind of environmental disrupting chemical, obviously suppressed the sperm motility and AR, but BPA had no significant effect on expression and function of KSper channel. This effect of BPA on spermatozoa function may affect the CatSper channel, a kind of sperm-specific proteins.