Relationships Between Mitochondrial Function And Boar Sperm Motility And The Role Of Extracellular ATP

Sperm motility is one of the most important conditions during fertilization.Sperm movement relied on large amounts of adenosine triphosphate(ATP).It was reported that glycolysis was the main pathway of glucose utilization in boar sperm,whether mitochondria works as energy production in boar sperm is unclear.Moreover,microenvironment where sperm were contained amounts of extracellular ATP.However,the relationship between extracellular ATP and boar sperm motility is unknown.In the present study,we use mature boar sperm as model to gain insight into the relationships between mitochondria DNA content,mitochondrial activity,and sperm motility,and the regulation of extracellular ATP on mitochondrial function,intracellular ATP content and boar sperm motility.1 Relationships between mitochondrial DNA content,mitochondrial activity and boar sperm motilityMitochondria owned its DNA,namely mitochondrial DNA(mtDNA).The mtDNA encoded 13 proteins are subunits of mitochondrial respiratory chain,which participate in oxidative phosphorylation(OXPHOS)to produce ATP.However,there were less mitochondria and mtDNA contents in motile sperm than less motile sperm in human and mouse,whether there was a similar phenomenon in boar sperm was yet unclear.We separated motile and less motile sperm by discontinuous density-gradient centrifugation and compared the differences in mtDNA content,and mitochondrial activity.Results showed that motile sperm had less mitochondria(P<0.01)and mtDNA(P<0.05)contents compared to less motile sperm.Transmission electron microscopy was employed to evaluate mitochondrial ultrastructure in boar sperm,motile sperm mitochondria contained more distinct inner membrane than less motile sperm.Higher mitochondrial transmembrane potential(P<0.01)and mitochondrial respiratory chain complex I activity were found in motile sperm.Motile sperm contained more mitochondrial reactive oxygen species(mROS)(P<0.01)than less motile sperm.Although owned less mtDNA contents,motile sperm had higher mitochondrial activity than less motile sperm.When mitochondrial respiratory chain complex I activity and OXPHOS were inhibited,sperm motility was significantly decreased(P<0.01),which indicated that OXPHOS played an important role in sperm motility.The level of mtDNA-encoded protein cytochrome c oxidase subunit 1(COX1)(P<0.01)and peroxisome proliferator coactivated receptor gamma co-activator 1-alpha(PGC-1 a)(P<0.01),mitochondrial transcription factors A(TFAM)(P<0.01)were higher in motile sperm than less motile sperm,which futher indicated that motile sperm owned higher mitochondrial activity.The results are helpful to interpret why mtDNA-less sperm have higher mitochondrial activity and better motility.2 Extracellular ATP regulates sperm motility and ATP contentStudies had shown that sperm,epithelial cell and exosome secrete extracellular ATP.However,the modulatory function of extracellular ATP in sperm motility is unclear.After treated with extracellular ATP 3 h,sperm motility increased(P<0.05)and apoptotic sperm significantly decreased(P<0.01).Further,a significant increase in the serine phosphorylation of GSK3a was observed in extracellular ATP group(P<0.01).Extracellular ATP significantly improved the content of intracellular ATP(P<0.001)in sperm and decreased the ADP:ATP ratio(P<0.001).Higher mitochondrial transmembrane potential was observed in ATP group,but there were no differences in ATP synthetase and complex ? activity between control and ATP group.Creatine kinase(CK)and pyruvate kinase(PK)play important roles in producing ATP,but extracellular ATP affects neither CK nor PK activity.Extracellular ATP improved mitochondrial function but had no effects on mitochondrial activity.Interestingly,lactate content in ATP group significantly decreased(P<0.001)and lactate dehydrogenase activity in seminal plasma and sperm also significantly decreased(P<0.001),which indicated that extracellular ATP affected glycolysis in sperm.Extracellular ATP could improve ATP content and sperm motility which may be associated with GSK3? phosphorylation level.Extracellular ATP improve intracellular ATP content by neither mitochondrial function nor the adenosine salvage,but by glycolysis.