Résumé
The internal pH (pHi) of human spermatozoa was measured by the fluorescent indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and the distribution of the radioactive [C-14]-methylamine under different external ionic conditions. The effect of the addition of progesterone and human follicular fluid (HFF) on the spermatozoa pHi was also analysed. The pHi values obtained were almost identical with the two probes used. In sodium (NaM) and potassium (KM) media, a linear relationship between the internal and external pH was observed. In NaM, the pHi values were similar to 0.4 pH unit less than the external pH. In KM, the pHi measured was higher than in NaM and only slightly inferior to the external pH (0.1-0.2 pH unit). Addition of 10 mu M progesterone, oestradiol 17 beta or 20% HFF to spermatozoa incubated at pH 7.2 in NaM did not induce any rapid variation of the BCECF fluorescence or change in the accumulation of methylamine. A slight change in pH (similar to 0.5 units) occurred with progesterone after 15 min. As a control, addition of 10 mM of NH4Cl induced a rapid alkalinization (0.4 pH unit) of the cell interior while 10 mM lactate produced only a slight acidification (similar to 0.2 pH unit). Under the same conditions (NaM, pH 7.2), the pHi of the spermatozoa prepared by Percoll gradient was found more acidic by 0.2 pH unit than washed unfractionated spermatozoa. Progesterone, oestradiol 17 beta and HFF had no effect on the pHi of these spermatozoa. The results obtained in this study show that it is possible to measure accurately the internal pH of human spermatozoa. Internal pH was found to be dependent upon the pH of the external medium and a quasi-linear relationship exists between the internal and external pH, suggesting no specific pH regulatory mechanisms. Our data suggest instead that the protons, under our experimental conditions, are passively distributed. Progesterone, oestradiol 17 beta and HFF, known to promote both capacitation and the acrosome reaction, do not act through a rapid pHi change.