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When incubated in vitro, human sperm gradually become capable of acrosome-reacting in response to the agonist progesterone. Loss of unesterified cholesterol is required for sperm to become responsive to progesterone, but how cholesterol regulates acrosomal responsiveness is unknown. These experiments tested the hypothesis that loss of sperm cholesterol leads to a rise in the intracellular pH (pH(i)) that makes the sperm responsive to progesterone. pH(i) was measured using BCECF (2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein) in freshly ejaculated sperm (T0 sperm) and in sperm incubated in vitro overnight (T24 sperm). During incubation, pH(i) increased from 6.94 +/- 0.03 to 7.08 +/- 0.01 (mean +/- SEM, n = 4, p &lt; 0.01). Incubating sperm 24 h in medium supplemented with 1 microM cholesterol to prevent loss of sperm cholesterol suppressed the rise of pH(i) (T24C sperm, pH(i) = 6.96 +/- 0.03, n = 4, p = 0.64 compared to T0 sperm). To test whether their lower pH(i) prevents T24C sperm from reacting, we treated T24C sperm with the alkalinizing agents trimethylamine chloride (TMA) or NH4Cl. These agents did cause T24C sperm to respond to progesterone in a dose-dependent fashion, but they also caused a similar increase in the number of reacting T24 sperm. These agents probably do not reverse the inhibiting effects of high cholesterol but rather make responsive a subpopulation of sperm that is present regardless of the cholesterol content. NH4Cl and TMA did not make T0 sperm responsive to progesterone. The acidifying agent sodium propionate did not diminish the response of T24 sperm to progesterone. In summary, pH(i) increases during incubation in vitro in a cholesterol-dependent fashion. Elevated pH(i) alone is probably not sufficient to make sperm acrosomally responsive.

Control of Human Sperm Intracellular pH by Cholesterol and its Relationship to the Response of the Acrosome to Progesterone1