title: Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation.
abstract: 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 (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse 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 of 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 mouse sperm, and undetectable in Slo3 knockout mouse 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 of a cAMP-dependent pathway and is mediated by the activation of SLO3 K(+) channels.
authors: Department of Veterinary and Animal Science, Integrated Sciences Building, University of Massachusetts, Amherst, Massachusetts.
authors: Jessica
authors: J
authors: Escoffier
authors: Department of Veterinary and Animal Science, Integrated Sciences Building, University of Massachusetts, Amherst, Massachusetts.
authors: Felipe
authors: F
authors: Navarrete
authors: Department of Veterinary and Animal Science, Integrated Sciences Building, University of Massachusetts, Amherst, Massachusetts.
authors: Doug
authors: D
authors: Haddad
authors: Department of Anatomy and Neurobiology. Washington University School of Medicine, St. Louis, Missouri.
authors: Celia M
authors: CM
authors: Santi
authors: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnologia-Universidad Nacional Autónoma de México, Cuernavaca, México darszon@ibt.unam.mx.
authors: Alberto
authors: A
authors: Darszon
authors: Department of Veterinary and Animal Science, Integrated Sciences Building, University of Massachusetts, Amherst, Massachusetts pvisconti@vasci.umass.edu.
authors: Pablo E
authors: PE
authors: Visconti
mesh_terms: D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology; D015640:Ion Channel Gating; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D010539:Permeability; D011188:Potassium; D015398:Signal Transduction / Q000502:physiology; D012964:Sodium; D013075:Sperm Capacitation / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology*; D013194:Staining and Labeling
chemical_list: C110627:Kcnu1 protein, mouse; D051036:Large-Conductance Calcium-Activated Potassium Channels; D012964:Sodium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011188:Potassium
keywords: ENac; SLO3; capacitation; flow cytometry; membrane potential