Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger

Abstract

We previously demonstrated that the progesterone-(P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na+ (Na(-)0). Moreover, Na(-)0 depletion resulted in a decreased cytosolic pH (pHi), suggesting involvement of a Na(+)-dependent pHi regulatory mechanism during the P-initiated AR. We now report that the decreased pHi resulting from Na(+)0 depletion is reversible and mediated by a Na+/H+ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pHi, capacitated spermatozoa were incubated in Na(+)-deficient, bicarbonate/CO2-buffered (ONaB) medium for 15-30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na(+)-containing, bicarbonate/CO2-buffered (NaB) medium; Na(+)-containing, Hepes-buffered (NaH) medium; or maintained in the ONaB medium. Included in the NaH medium was the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA). The steady-state pHi was then determined by spectrofluorometric measurement of bis(carboxyethyl)5(6)-carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 microM) significantly (P < 0.05) inhibited the pHi recovery produced by NaH medium. Moreover, the pHi in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na(+)-dependent, bicarbonate-independent pHi regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE-1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P-initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE-dependent pHi regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility.