PH REGULATION IN MOUSE SPERM - IDENTIFICATION OF NA-DEPENDENT, CL--DEPENDENT, AND HCO3--DEPENDENT AND ARYLAMINOBENZOATE-DEPENDENT REGULATORY MECHANISMS AND CHARACTERIZATION OF THEIR ROLES IN SPERM CAPACITATION()

Intracellular pH (pH(i)) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pHi in these cells are not understood. The pHi of mouse cauda epididymal sperm was determined from the fluorescence excitation ratio of 2,7-bis(carboxyet hyl)-5(6)carboxyfluorescein and calibrated with nigericin and elevated external [K+]. Two acid efflux mechanisms were identified following i mposition of acid loads. One pathway has many anticipated characterist ics of the somatic Na+-dependent Cl-HCO3- exchanger, although sperm an d somatic mechanisms can be distinguished by their ion selectivity and inhibitor sensitivity. Sperm may have an isoform of this exchange pat hway with novel functional characteristics. The second acid-export pat hway does not require extracellular anions or cations and is inhibited by aryl aminobenzoates (flufenamic acid, diphenylamine-2-carboxylate) . Mouse sperm also recover spontaneously from intracellular alkaliniza tion. Recovery rates in N-methyl-D-glucamine(+) Cl- or in 0.25 M sucro se are not significantly different from that in a complex culture medi um. Thus, recovery from alkalinization does not utilize specific, ion- dependent transport mechanisms. Other widely distributed acid-efflux m echanisms, such as the Na+-H+ antiport pathway and the Na+-independent Cl--HCO3- exchanger are not major regulators of mouse sperm pH(i). Sp erm capacitation results in pH(i) increases (from 6.54 +/- 0.08 to 6.7 3 +/- 0.09) that require a functional Na+-, Cl--, and HCO3--dependent acid-efflux pathway. Inhibition of this regulatory mechanism attenuate s alkaline shifts in pHi during capacitation as well as the ability of sperm to produce a secretory response to zona pellucida agonists. The se data suggest that one aspect of mouse sperm capacitation is the sel ective activation of one major pH(i) regulator. (C) 1996 Academic Pres s, Inc.