Studying The Mechanism Of Extracellular ATP Actions On Human Mature Sperm

After human spermatozoa are ejaculated into women reproductive tract,they must undergo a series of physiological and biochemical processes including capacitation,hyperactivation and acrosome reaction that enable them to fertilize the oocyte.During this transit course,sperm functions are regulated by many physiological factors within the women reproductive tract.Among these,extracellular ATP?ATPe?,an important physiological factor,has been reported that it exists in the women reproductive tract and plays a significant role in promoting human sperm hyperactivation,acrosome reaction and in-vitro fertilizing capacity.However,the mechanism of ATPe actions on human sperm function is not elucidate.Based on the central role of intracellular Ca²⁺ signaling in controlling mature sperm function,we therefore firstly examined the effect of ATPe on human sperm intracellular Ca²⁺ concentration([Ca²⁺]_i)using a multi-mode plate reader.The results showed that ATPe?0.05,0.1,0.2,0.5,1,2.5,5 mM?dose-dependently increased [Ca²⁺]_i and reached to saturation at 2.5 mM.After depleting the Ca²⁺ in the extracellular medium with the Ca²⁺ chelator BAPTA,ATPe did not induce an increase of sperm [Ca²⁺]_i,suggesting that ATPe increased human sperm [Ca²⁺]_i is due to extracellular Ca²⁺ influx.It is well known that the action of ATPe is primarily mediated by cell-surface P2 receptors.Thus,we choose two broad-spectrum P2 receptor inhibitors PPADs and suramin,and found they significantly inhibited ATPe induced human sperm [Ca²⁺]_i.To identify which P2 receptor mediates ATPe actions on human spermatozoa,we used western-blot to detect the expression of P2 receptors and revealed that P2X?2,3,4,7?and P2Y?1,2?were expressed in human spermatozoa.Since the action of ATPe is mediated by P2X2 receptor channel in mouse spermatozoa,whereas we could not record ATPe activated P2X2 currents in human spermatozoa with patch-clamp technique.In addition,by testing the effect of P2 receptors antibodies on ATPe induced [Ca²⁺]_i,among P2X?2,3,4,7?and P2Y?1,2?antibodies,only P2Y1 antibody significantly inhibited ATPe increased [Ca²⁺]_i,indicating that ATPe actions on human spermatozoa by targeting P2Y1 receptor.Because P2Y1 belongs to G protein-coupled receptor and ATPe induced human sperm [Ca²⁺]_i is due to extracellular Ca²⁺ influx,hence,we supposed that P2Y1 activated by ATPe may act on other Ca²⁺ channel in the plasma membrane.Spermspecific CatSper channel is the most likely candidate because of its vital role in regulating intracellular Ca²⁺ signaling and sperm functions.Indeed,we found ATPe could enhance CatSper currents,and CatSper blockers mibefradil and AR720 could inhibit ATPe increased [Ca²⁺]_i and CatSper currents,demonstrating that ATPe induced human sperm Ca²⁺ influx is mediated by CatSper channel.Moreover,P2Y1 antibody could repress ATPe enhanced CatSper currents;P2Y1 and CatSper have similar localization in human spermatozoa,which mainly located in the flagellum;and increasing sperm intracellular GTP concentration could strengthen the elevation effect of ATPe on CatSper currents;suggesting that the activation of ATPe on CatSper is an indirect effect which is mediated by P2Y1 receptor.In this study,we further analyzed the relation between P2Y1 and human sperm motility,and the results showed that the expression level of P2Y1 protein were significantly higher in spermatozoa from normozoospermic compared with those from asthenozoospermic men.Additionally,ATPe induced [Ca²⁺]_i were also significantly higher in spermatozoa from normozoospermic compared with those from asthenozoospermic men.The accordance between P2Y1 expression and ATPe induced [Ca²⁺]_i in human spermatozoa suggests that P2Y1 expression and function are positive correlated with human sperm motility.Taken together,ATPe targets human sperm P2Y1 receptor,then leads to CatSper activation,and thereby resulting in [Ca²⁺]_i elevation.The present study has a certain degree elucidating the molecular mechanism of ATPe actions on human spermatozoa;firstly reveals the function of P2Y1 receptor in human spermatozoa;and provides a novel pattern in the regulation of human sperm CatSper activation.