Extracellular ATPs produced in seminal plasma exosomes regulate boar sperm motility and mitochondrial metabolism

Artificial insemination using diluted semen with reduced presence of seminal plasma has been applied worldwide. Sperm stored in seminal plasma rather than diluted or removed show improvement in survival and motility. However, the link between seminal plasma and sperm remains poorly understood. This study focuses on the effect of extracellular adenosine triphosphate (exATP) produced by boar seminal plasma exosomes on sperm motility, as well as the underlying molecular mechanisms. The seminal plasma exosomes had an average diameter of 86.6 nm and showed universal exosome markers, such as heat shock protein 70 (HSP70) and CD63. Production of net ATP increased when exosomes were incubated with glucose and partly inhibited by a glycolytic inhibitor such as iodoacetate. Fresh boar sperm incubated with exATP significantly increased sperm motility and reduced apoptotic rate. Ser21 phosphorylation of glycogen synthase kinase 3α (inactivation) also significantly increased, consistent with the increase in mitochondrial transmembrane potential in the exATP-treated sperm. Moreover, exATP treatment increased the intracellular ATP (inATP) concentration and decreased the ADP/ATP ratio in boar sperm. Lactate content in the incubation medium was decreased, whereas lactate dehydrogenase activity in sperm was increased. This finding suggested that exATP could prompt lactate to produce inATP in order to sustain motility. The combined results indicate that exATP produced in seminal plasma exosomes may finely modulate mitochondrial metabolism to control sperm motility. The results can provide insights into semen dilution and artificial insemination.