Investigation Of The Regulatory Effects Of SNHE On The Sperm-specific Ion Channels

After entering the 21st century,an increasing number of individuals are disturbed by infertility with the change of environment and life style.So far,about ten to fifteen percent of childbearing age couples are with reproductive problems throughout the world.Among them,half of patients are relevant to male reproductive factors.Sperm dysfunction is the main reason in the male infertility.Therefore,it is significant to study the regulatory mechanism of sperm function for solving the male reproductive troubles.When sperm enter the female genital tract,sperm undergo a series of physiological changes such as capacitation,hyperactivation and acrosome reaction,which is the fatal premise to achieve fertilization with an egg.KSper and CatSper,the sperm specific channels,play important roles in these processes.The study of mouse model indicated that KSper is composed by main subunit SLO3 and at least one accessory subunit LRRC52.Sperm exhibited an impaired reproductive function in the mutated SLO3 or LRRC52 mice.In human sperm,the molecular component of KSper still remains to be elucidated,and its characteristics are also different from mice.However,the abnormal current of KSper related to male infertility had been reported,which implied that KSper is also a vital factor in regulating human sperm function.KSper regulates sperm function via regulating the sperm membrane potential.In addition,CatSper,which is consisted of CatSper1-4 main subunits and many accessory subunits,is a dominated ion channel to mediate Ca²⁺influx on sperm membrane.No matter which subunits were mutated,the infertility of male mice would be caused.Meanwhile,many reports showed that the mutation of CatSper in human sperm was able to cause the male infertility.It is worth to be emphasized that KSper and CatSper are both pH-dependent ion channels.Hence,identifying the main factor of regulating sperm pH and exploring its coupled relationship with the above mentioned two specific ion channels have the practical significance and theoretical basis for clarifying the underlying mechanism of sperm function and elucidating the reason of male infertility precisely.Many p H-regulated transporters were discovered in spermatozoa,and NHEs were one kind of Na⁺-H⁺exchanger.Primary experiments in our lab indicated that NHEs inhibitor decreased intracellular pH and induced the depolarization in mouse sperm caused by the decline of KSper activation,suggesting the coupling relation between NHEs and KSper in mouse sperm.However,it's not clear yet whether there is a coupling relation between NHEs and CatSper and whether NHEs affect sperm function by acting on pH-sensitive channels in mouse sperm.More importantly,it's still unclear that whether there is coupling relationship between NHEs and KSper or CatSper in human sperm.Based on these questions,this project tries to search the regulatory and functional effects of s NHE on KSper and CatSper.Methods:1.We study the role of KSper in mice and human sperm using the sodium hydrogen exchanger?sNHE?inhibitor named DMA.It had been known that the activation of KSper can lead to the efflux of potassium ions,resulting in the hyperpolarization on sperm membrane.Besides,CatSper is a pH-sensitive channel,which is significantly affected by intracellular pH.In order to clarify the effect of pH on relevant channels,the whole-cell patch clamp technique was used to detect the activation or inhibition of the KSper and CatSper in the sperm.2.Sperm functional and molecular experiments are effective methods to test sperm quality and drug effects.Based on the results of patch clamp,sNHE inhibitors are also used to test sperm function.Detecting sperm function need to use computer-assisted sperm analysis?CASA?,calcium signal,single cell calcium signal measurement,acrosome reaction,penetration assay,immunofluorescence and other experiments in vitro.Results:1.In mouse sperm,when the intracellular pH buffer was low,adding sNHE inhibitor into extracellular environment could lead to the depolarization significantly.When the intracellular pH buffer was high?even if s NHE was inhibited,the intracellular pH would not be affected?,the addition of DMA in extracellular environment did not lead to the depolarization of the sperm membrane potential.Because the results in the above was only related to the intracellular pH change,it was indicated that the inhibitory effect of DMA on KSper channel was due to the suppression of sNHE rather than other non-specific reasons.The results further confirmed that sNHE and KSper have the functional coupling.2.The effects of DMA on intracellular Ca²⁺were measured by fluorescence staining and enzyme labeling in mouse sperm.In functional experiments,the acrosome reaction induced by progesterone was significantly reduced after adding DMA.3.In human sperm,sNHE and KSper have co-located expressions.Fluorescence method was used to measure the influence of DMA on intracellular pH.DMA also reduced the intracellular pH,which was further confirmed in the single-cell experiment.Next,based on the findings that DMA had no effect on KSper by the patch clamp recordings,we explored the effect of DMA on human membrane potential.The results indicated that the sensitivity of the sperm KSper to pH changes was significantly lower than the mice.We hypothesized that the effect of DMA on the intracellular pH might not affected the KSper but influenced the activation of CatSper whose pH-sensitivity is higher than KSper.4.The sNHE inhibitor DMA did reduce intracellular Ca²⁺concentration and progesterone-induced intracellular Ca²⁺influx significantly through the fluorescence measurement.Similarly,in single-cell fluorescence measurement,intracellular Ca²⁺fluorescence intensity was significantly inhibited after adding DMA.These results further verified that human sperm CatSper mediated intracellular Ca²⁺influx due to pH changes.5.In human sperm functional experiments,the sperm motility parameters,penetrated mucus ability and acrosome reaction had the remarkable impairment after adding DMA.We hypothesized that the DMA could affect sNHE function to change the intracellular Ca²⁺indirectly,which affect sperm function significantly.In the patch clamp experiment,the addition of DMA could reduce the current of CatSper and progestorene-induced CatSper current,which also fully confirmed the above conjecture.