Mechanisms Of Oviductal ATP Secretion And Effects Of Extracellular ATP On Sperm Capacitation

Adenosine triphosphate(ATP)is the most direct source of energy in the living organism,and extracellular ATP can exert physiological functions as a signal molecule.Previous studies have shown that extracellular ATP could induce acrosomal reaction of sperm and increase the fertilization rate.but the source of extracellular ATP is unknown.The oviduct is the site where mammalian sperm and egg make fertilization.Whether extracellular ATP involved in the regulation of sperm fertilization is released from the oviduct is still unknown.In this study,female mouse at different stages of estrous cycle were used as experimental models to investigate whether and how ATP is secreted in the oviduct of female mouse during estrous cycle.Besides,boar sperm were treated with ATP to investigate its effect on sperm fertilization.1 Mechanisms underlying oviductal ATP secretion and effects of ATP on sperm acrosomal reactionIn order to investigate the secretion of ATP and its mechanism in the oviduct of mouse.this study used female mouse at different stages of estrous cycle as experimental models to study ATP synthesis in oviduct epithelial cells and ATP secretion process mediated by cystic fibrosis transmembrane regulator(CFTR).The results showed that the ATP content of mouse oviduct fluid changed during estrous cycle.The ATP content in the Estrus group was significantly higher than that in the Proestrus group and the Diestrus group(P<0.05).Treatment of mouse sperm with ATP in vitro show that ATP significantly increased acrosomal reaction rate of sperm compared with the CON group(P<0.05).RT-PCR detection found that compared with Proestrus group and Diestrus group,the mRNA expression of CFTR in oviduct of Estrus group was significantly decreased(P<0.05).However,the protein level of CFTR in Estrus group was significantly higher than the other two groups(P<0.05).Compared with the Proestrus group and Diestrus group,the miR-381-3p abundance targeting the CFTR3’UTR region of individuals during estrus significantly decreased(P<0.05).In vivo experiments showed that inhibition of CFTR in female mouse induced a decrease in the number of maternal pregnancy and litters while ATP could alleviate the effects of this process.Electron microscopic observation of the oviduct showed that the structure of mitochondria in the Estrus group was clearer than that of the Proestrus group and Diestrus group.In addition,mitochondrial complex I activity in the Estrus group was significantly higher than that in the Proestrus group(P<0.05)which indicate mitochondrial activity elevated in the oviduct during estrus period.Compared with the Proestrus group,the mRNA expression of mitochondrial-related genes in the Estrus group and the Diestrus group were significantly upregulated(P<0.05).Compared with the Proestrus group and the Diestrus group,the protein expression of ERa in the Estrus group was significantly increased(P<0.05).ChIP analysis demonstrate that The enrichment of ERa and mtDNA D-loop in the Estrous group was significantly higher than that in the Proestrus group,which showed ERa in oviduct was involved in the transcriptional regulation of mtDNA during estrus period.In conclusion,this experiment found that ERa mediated mtDNA transcription during estrus period in the oviduct of mouse,made mitochondrial activity upregulate and ATP production increase,which then was released into lumen by CFTR to regulate sperrm acrosomal reaction.2 Effects of extracellular ATP on boar sperm capacitationIn order to investigate the effect of extracellular ATP on the capacitation of boar sperm,an in vitro capacitation model of boar sperm was established.It was found that ATP could significantly increase the acrosomal reaction rate of boar sperm(P<0.05),and the addition of mitochondrial ribosomal translation inhibitors(CP)did not affect the effect of ATP,indicating that the promotion of ATP in boar sperm capacitation was not related to protein translation;ATP treatment could significantly improve the mitochondrial membrane potential of sperm(P<0.05),which could be blocked by CP.In order to detect whether Ca2+is involved in the process of ATP promoting acrosomal reaction of boar sperm,blocking Ca2+transport by adding CatSper channel inhibitor MB in the capacitation fluid could inhibit the acrosomal reaction of boar sperm,suggesting that Ca2+ transport is involved in the process of ATP promoting acrosomal reaction of boar sperm.In addition,inhibition of Ca2+ transport could prevent ATP induced acetylation of sperm protein to some extent.In conclusion,extracellular ATP could increase the mitochondrial activity of boar sperm and promote its acrosomal reaction.Inhibition of CatSper channel and decrease of Ca2+ concentration could block the acrosomal reaction induced by ATP,and protein acetylation may be involved in this process.To sum up,this study found that the expression of CFTR protein in the oviduct of the mouse increased during estrus period,which mediated the release of ATP,enhanced the mitochondrial activity of oviduct during estrus period,and increased the production of ATP,which provided the raw material for the release of ATP.Finally the content of ATP in oviduct cavity during estrus period was significantly higher than that in other periods.Extracellular ATP can improve the mitochondrial activity of boar sperm and promote its acrosomal reaction.Ca2+ transport is involved in the process of extracellular ATP promoting boar acrosomal reaction and protein acetylation may be involved in this process.