Membrane potential hyperpolarization induces cytoplasmic alkalinization of mouse sperm

JABLOÑSKI M; KRAPF D; BALESTRINI PA; LUQUE GM; GILIO N; BUFFONE MG

Mar del Plata

Congreso; Reunión Anual de Sociedades de Biociencia. SAIC, SAFE, SAB, SAP, AACYTAL.; 2019

To gain fertilization ability sperm must undergo a series of physiological modification in the female tract called capacitation, which leads to the acquisition of hyperactivated motility (HA) and the ability to undergo acrosome reaction (AR). Membrane potential (Em) hyperpolarization and alkalinization of the intracellular pH are necessary for HA and AR to take place. It has been reported either pharmacologically or genetically (potassium channel Slo3 knockout mice) that hyperpolarization is necessary and sufficient for sperm acrosomal responsiveness. In addition, intracellular alkalinization allows the opening of CatSper channels which lead to an increase in intracellular calcium that is fundamental for the HA of sperm. The aim of this work was to elucidate the interplay between Em and pH in mouse sperm. We used BCECF-AM and DISC3(5), probes that measures changes in cytoplasmic pH and Em, respectively. First, we studied the dependence of pH and Em, by co-incubating sperm with both probes followed by Flow Cytometry analysis. We observed that all the cells were grouped into two populations: 1) low pH and depolarized Em or 2) high pH and hyperpolarized Em. We also determined that sperm incubated under non-capacitating conditions with the addition of 1 μM valinomycin displayed a robust increase in pH suggesting that changes in Em are necessary for the rise in pH observed during capacitation. This increase was abrogated in the presence of high K+ in the extracellular space. In addition, we studied the kinetics of this changes by assessing Em and pH at different time points of capacitation (0 to 60 min). Our results showed that there is a rapid pH alkalinization during the first 15 min of incubation in capacitating medium that is followed by a transient acidification at 30 min. Alkalinization is then restored at 45 min and remain unchanged. Thus, our results suggested that changes in the Em induces modifications in intracellular pH during mouse sperm capacitation.