Experimental Research Of Human Sperm KCNQ1/KCNE1 Channel In Signal Transduction During Hyposmotic Stress In Vitro

Aim:After ejaculating into the female reproductive tract,the mammalian sperm need to get through the uterus,fallopian tubes and find the egg to complete fertilization.In this process,the osmotic pressure of the surrounding environment gradually decline,sperm subsequently launched a series of signal transduction.Some studies noticed the lower osmotic pressure at different positions in the fertilization process,they all focused on the capacity of regulatory volume decrease.No scholars systematic study the signal transduction pathway of sperm under hyposmotic stress.In this study,we explore human sperm signal transduction pathway under hypotonic condition and the role of the potassium channel KCNQ1/KCNE1 hypotonic changes to explain physiological significance of osmotic pressure on human sperm and the role in male infertility.Methods and Results:This study can be divided into five parts.There is no research confirmed KCNQ1/KCNE1 channel on human sperm membrane.So in the first part,we identified KCNQ1/KCNE1 at RNA and protein levels in human sperm and,confirmed KCNQ1/KCNE1 on human sperm membrane;Second,osmotic pressure is an important factor to affect the water balance inside and outside cells,when the cells in hypotonic environment,the intracellular water increases result cell swelling.At the same time,the cells start capacity adjusting mechanism,compensatory mechanism of activation of the ion transport the ions within the cell outflow,so that the osmotic pressure within the cell,the outflow of water within the cell,the cell body is gradually restored,the process known as regulatory volume decrease(RVD).Potassium channels,chloride ion channels,water channels involved in sperm RVD.We detected sperm volume using flow cytometry(FCM)and sperm intracellular potassium concentration by fluorescent probe of potassium.The use of a specific inhibitor of the potassium ion channel KCNQ1/KCNE1,alcohol 293B,confirmed KCNQ1/KCNE1 involved in human sperm RVD,the channel of human sperm in hypotonic state mediated potassium efflux.In the third part,in order to clarify the physiological osmotic pressure on the significance of the sperm from sperm movement,shape intracellular calcium ion concentration change the angle of the impact of the low permeability of sperm.The experimental results confirm the linear motion of the sperm rate of decline,increased rate of capuchin confirmed hypotonic screening role of sperm into the female reproductive tract.We found that when KCNQ1/KCNE1 channel blocked,the more obvious changes in sperm parameters,which means that in hypotonic condition,the potassium channel KCNQ1/KCNE1 channel open to help sperm caused confrontation hypotonic the adverse impact on the vitality.The results of calcium imaging display intracellular calcium ion concentration is increased in hypotonic and further through the role of extracellular calcium chelating agents.We found that the increase in intracellular calcium ions resulted from calcium stores within the sperm.In the fourth part,after identified potassium ion channels in human sperm hypotonic change,we try to find the upstream regulatory mechanisms.We detected the sperm membrane phosphatidylserine muscle-acyl 4,5-diphosphate(PIP2)in hypotonic when the osmotic pressure changed.PIP2 in hypotonic condition decreased and increased after a few minutes,this suggesting that hypotonic stress resulted PIP2 hydrolysis,which is related to the activation of phospholipase PLC.The sperm intracellular calcium ion and potassium ion concentration does not change in U73122,a PLC blockers pretreated sperm.These results suggest that the PLC is activated in hypotonic stress,hydrolysis of PIP2 to generate inositol trisphosphate(IP3)and diacyl glycerol(DAG).IP3 binded IP3 receptor and activated calcium store the RNE release within the sperm calcium,causing the increase of intracellular calcium concentration.Generated DAG may cause KCNQ1/KCNE1 channels open through the activation of protein kinase PKC-mediated hypotonic intracellular potassium ion outflow.In the fifth part,in order to confirmed in hypotonic potassium of ion channel KCNQ1/KCNE1 the under electrophysiological level open with PLC-PIP2-IP3/DAG of pathways related to building potassium channel KCNQ1/KCNE1 model in HEK cells,the use of patch clamp technique confirmed the KCNQ1/KCNE1 open in hypotonic condition.PLC inhibitor U73122 hypotonic induced potassium ion channel KCNQ1/KCNE1 of open disincentive.Conclusion:Phospholipid signal transduction system will be activated in hypotonic state on the cell membrane of sperm,the potassium channel KCNQ1/KCNE1,related to the activation of PLC,play an important role in the outflow of potassium.KCNQ1/KCNE1 have an adversely affect on sperm motility decrease resulted by the hyposmotic stress.Calcium release from calcium stores in this process can provide the ionic basis for human sperm.