Membrane potential hyperpolarization induces cytoplasmic alkalinization of mouse sperm

Abstract

To gain fertilization ability sperm must undergo a series of physiological modification inthe female tract called capacitation, which leads to the acquisition of hyperactivatedmotility (HA) and the ability to undergo acrosome reaction (AR). Membrane potential(Em) hyperpolarization and alkalinization of the intracellular pH are necessary for HAand AR to take place. It has been reported either pharmacologically or genetically(potassium channel Slo3 knockout mice) that hyperpolarization is necessary andsufficient for sperm acrosomal responsiveness. In addition, intracellular alkalinizationallows the opening of CatSper channels which lead to an increase in intracellular calciumthat is fundamental for the HA of sperm. The aim of this work was to elucidate theinterplay between Em and pH in mouse sperm. We used BCECF-AM and DISC3(5), probesthat measures changes in cytoplasmic pH and Em, respectively. First, we studied thedependence of pH and Em, by co-incubating sperm with both probes followed by FlowCytometry 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 determinedthat sperm incubated under non-capacitating conditions with the addition of 1 μMvalinomycin displayed a robust increase in pH suggesting that changes in Em arenecessary for the rise in pH observed during capacitation. This increase was abrogatedin the presence of high K+ in the extracellular space. In addition, we studied the kineticsof this changes by assessing Em and pH at different time points of capacitation (0 to 60min). Our results showed that there is a rapid pH alkalinization during the first 15 min ofincubation 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 resultssuggested that changes in the Em induces modifications in intracellular pH during mousesperm capacitation.