pH Regulation in Mouse Sperm: Identification of Na+-, Cl−-, and[formula]Dependent and Arylaminobenzoate-Dependent Regulatory Mechanisms and Characterization of Their Roles in Sperm Capacitation

AbstractIntracellular pH (pHi) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pHiin these cells are not understood. The pHiof mouse cauda epididymal sperm was determined from the fluorescence excitation ratio of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein and calibrated with nigericin and elevated external [K+]. Two acid efflux mechanisms were identified following imposition of acid loads. One pathway has many anticipated characteristics of the somatic Na+-dependent Cl−–[formula]exchanger, although sperm and somatic mechanisms can be distinguished by their ion selectivity and inhibitor sensitivity. Sperm may have an isoform of this exchange pathway with novel functional characteristics. The second acid-export pathway does not require extracellular anions or cations and is inhibited by arylaminobenzoates (flufenamic acid, diphenylamine-2-carboxylate). Mouse sperm also recover spontaneously from intracellular alkalinization. Recovery rates inN-methyl-D-glucamine+Cl−or in 0.25Msucrose are not significantly different from that in a complex culture medium. Thus, recovery from alkalinization does not utilize specific, ion-dependent transport mechanisms. Other widely distributed acid-efflux mechanisms, such as the Na+–H+antiport pathway and the Na+-independent Cl−–[formula]exchanger are not major regulators of mouse sperm pHi. Sperm capacitation results in pHiincreases (from 6.54 ± 0.08 to 6.73 ± 0.09) that require a functional Na+-, Cl−-, and[formula]-dependent acid-efflux pathway. Inhibition of this regulatory mechanism attenuates alkaline shifts in pHiduring capacitation as well as the ability of sperm to produce a secretory response to zona pellucida agonists. These data suggest that one aspect of mouse sperm capacitation is the selective activation of one major pHiregulator