Studying The Mechanism Of Extracellular ADP Actions On Human Mature Sperm

Before successful fertilization,human spermatozoa need to undergo a series of physiological and biochemical processes,including capacitation,hyperactivation,chemotaxis and acrosome reaction.Physiological factors in the female reproductive tract play an important regulatory role during the physiological and biochemical processes.Studies have shown that extracellular ATP?ATPe?has a significant effect on the sperm motility,hyperactivation and acrosome reaction.ADP is one of the natural metabolites of ATP.However,whether extracellular ADP?ADPe?affects the physiological functions of human sperm and the underlying mechanisms has not been explored.Ca²⁺,as a second messenger,plays a very important role in regulating sperm functions.The intracellular Ca²⁺concentration([Ca²⁺]_i)was detected by a multi-mode plate reader.The study first examined the effect of ADPe on human sperm[Ca²⁺]_i.The result showed that ADPe?50,100,200,500,1000,2500?M?dose-dependently increased[Ca²⁺]_i and reached to saturation at 1000?M.By application of Ca²⁺chelator BAPTA to keep sperm in the Ca²⁺-free environment,ADPe did not elicit[Ca²⁺]_i increase in human sperm,suggesting that the ADPe increased[Ca²⁺]_i is due to extracellular Ca²⁺influx.The P2Y receptors mediate ADP-induced[Ca²⁺]_i increase in other systems.In this study,we found the expression of P2Y?1,2,13,14?receptors in human sperm.Meanwhile the borad-spectrum P2Y receptor inhibitors PPADs and suramin significantly suppressed ADP-induced[Ca²⁺]_i increase.Further investigation of the effect of P2Y?1,2,13,14?antibodies on the increase of[Ca²⁺]_i induced by ADPe,the result revealed that only P2Y13 antibody showed significant inhibition,indicating that P2Y13 receptor mediates ADPe-induced[Ca²⁺]_i increase.As a G-protein-coupled receptor,P2Y13 does not directly mediates Ca²⁺influx.Based on CatSper,a sperm-specific calcium channel,this study further verified whether CatSper channel mediates ADPe-elicited[Ca²⁺]_i increase.Patch clamping results showed that ADPe rapidly enhanced CatSper current,while P2Y receptor inhibitors PPADs and suramin,as well as P2Y13 antibody significantly inhibited ADPe-induced[Ca²⁺]_i increase.These results manifest that ADPe binds to P2Y13receptor to activate CatSper channel,which result in Ca²⁺influx.However,the meachnism for how to activate CatSper channel after P2Y13 receptor activation remains obscure.This study also examined the effect of ADPe on human sperm functions.The results revealed that the above-mentioned concentration of ADPe could promote human sperm hyperactivation,which is consistent with the molecular meachnism that CatSper channel regulates human sperm hyperactivation..In summary,ADPe activates human CatSper channel via P2Y13,which results in extracellular Ca²⁺entry.In addition,ADPe also increased the human sperm hyperactivation.This project certainly elucidates molecular mechanism of ATPe/ADPe actions on human spermatozoa and the relevant physiological functions.