Protein Kinase A anchoring is critical for sperm capacitation

STIVA C; BARO GRAF C; BUFFONE MG; LUQUE GM; KRAPF D; RITAGLIATI C; VISCONTI PE; KRAPF D

Holderness, NH

Congreso; Gordon Conference on Fertilization and the Activation of Development.; 2017

Gordon Conference

Capacitation is the process by which mammalian sperm acquire the ability to fertilize and can be mimic in vitro. Once completed, sperm obtain acrosomal responsiveness and a hyperactivated flagellar movement.When sperm are exposed to HCO3-, there is rapid intracellular increase in cAMP, that activates PKA, which acts as a key player during capacitation. Its regulation has been mainly studied focusing on its catalytic activity. However, biological activity is also dependent on its subcellular localization, which is mediated by interaction with scaffolding AKAP proteins.Here, we study the role of PKA anchoring during capacitation murine sperm. The binding and anchoring of PKA to AKAP was affected by the peptide st-HT31, which disrupts PKA-AKAP interaction without affecting catalytic activity.When PKA anchoring was impaired throughout capacitation, sperm neither acquired acrosome responsiveness (assessed by PNA-staining of the acrosome), nor hyperactivated motility. Phosphorylation of PKA substrates as well as tyr phosphorylation were also blocked, as evaluated by western-blot.Conversely, when PKA de-anchoring was induced in capacitated sperm, AR was triggered by a mechanism that involved 1) membrane depolarization, and 2) Ca2+ influx, seemingly through CatSper channel. These events were assessed by single-cell microscopy and population fluorimetric measures, using specific-sensitive dyes.Addition of the inhibiting peptide st-HT31 induced AR only in capacitated sperm but not in non-capacitated cells, showing there are capacitation-associated events that prime the sperm for this response to PKA de-anchoring. In addition, AR needed active PKA activity, since inhibition of PKA right before st-HT31 exposure impaired AR. Moreover, an inactive analogue of st-HT31, named st-HT31p, did not show any effect.Overall, we show that PKA anchoring is essential for its biological activity in sperm processes.