MOLECULAR EVENTS INVOLVED IN THE HYPERPOLARIZATION OF THE PLASMA MEMBRANE OF MURINE SPERMATOZOA

NOVERO, ANALIA G.; CARRUBA, MICAELA; SCHIAVI-EHRENHAUS, LIZA J.; RITAGLIATI, CARLA; BARO GRAF, CAROLINA; STIVAL, CINTIA; GENTILE, IÑAKI; SANTI, CELIA M.; DE LA VEGA BELTRÁN, JOSÉ L.; TORRES RODRÍGUEZ, PAULINA; NISHIGAKI, TAKUYA; TREVIÑO, CLAUDIA L.; BUFFONE, MARIANO G.; KRAPF, DARIO

Congreso; Fertilization and Activation of Development Gordon Research Conference; 2023

Successful fertilization in mammals requires sperm physiological changes that occur during its passage through the female reproductive tract, collectively known as capacitation. At the molecular level, capacitation involves reorganization of the plasma membrane, post-translational modifications of proteins, and changes in ion membrane permeability, including hyperpolarization of the plasma membrane potential (Em), which is necessary and sufficient for the acrosome reaction to occur. Although the regulation of this mechanism is not fully understood, previous research suggested that PKA plays a role in this process. Understanding the molecular mechanisms underlying Em hyperpolarization is needed for to proper development of new contraceptive methods and diagnostic tools for male infertility.Using population fluorimetry measurements of Em, we investigated the role of PKA in hyperpolarization during capacitation. We found that the specific permeable PKA inhibitor sPKI did not block Em hyperpolarization. However, inhibition of cAMP synthesis by the specific inhibitor of adenylate cyclase (ADCY10) TDI-10229 prevented Em hyperpolarization, which was rescued by adding the permeable cAMP analog 8Br-cAMP. As previously shown, permeable cAMP analogs induced Em hyperpolarization when added to non-capacitating media. Em hyperpolarization observed in the presence of sPKI did not occur in sperm from Slo3 KO mice or under conditions of pharmacological inhibition of this channel, confirming that PKA blockade did not trigger Em changes through a pathway other than Slo3 opening. In this regard, direct catalytic stimulation of PKA by 6-Bnz-cAMP did not induce Em hyperpolarization. However, inhibition of PKA-AKAP binding by sHT-31 did inhibit Em hyperpolarization.Our results suggest that cAMP is required for Em hyperpolarization, independently of PKA catalytic activity. The permeable cAMP analog 8-pCPT-2'-O-Me-cAMP, which specifically activates EPAC, did not promote Em hyperpolarization. Based on the dependency of Slo3 channels on intracelular pH, we analyzed the effect of inhibition of Na+/H+ exchangers (NHE) present in sperm by dimethyl amiloride (DMA). This inhibitor prevented hyperpolarization of the plasma membrane, which was recovered by direct stimulation of Slo3 by the alkalizing agent NH4Cl.In summary, our data suggest that although PKA catalytic activity is not required for Em hyperpolarization, cAMP is needed in this signaling pathway, probably as alkalizing agent. Our results shed light on the different signaling modules involved in this process and contribute to the understanding of the molecular mechanisms guiding capacitation.