Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation 1 Running title: Flow cytometry analysis of sperm membrane potential.

To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential. In the present work, we used flow cytometry to analyze changes in sperm membrane potential (Em) during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mice sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed by sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K + , by PKA inhibitors and by SLO3 inhibitors in CD1 mice sperm, and undetectable in Slo3 knock-out mice sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3 and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream a