Cross N L, Razy-Faulkner P
Department of Anatomy, Pathology, and Pharmacology, Oklahoma State University, Stillwater 74078, USA.
Biol Reprod. 1997 May;56(5):1169-74. doi: 10.1095/biolreprod56.5.1169.
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 < 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.
在体外培养时，人类精子会逐渐能够响应激动剂孕酮而发生顶体反应。精子要对孕酮产生反应，需要消耗未酯化胆固醇，但胆固醇如何调节顶体反应性尚不清楚。这些实验检验了以下假设：精子胆固醇的消耗会导致细胞内pH值（pH(i)）升高，从而使精子对孕酮产生反应。使用BCECF（2',7'-双（2-羧乙基）-5（6）-羧基荧光素）测量新鲜射出精子（T0精子）和体外过夜培养精子（T24精子）的pH(i)。在培养过程中，pH(i)从6.94±0.03升高到7.08±0.01（平均值±标准误，n = 4，p < 0.01）。在补充有1 microM胆固醇的培养基中培养精子24小时以防止精子胆固醇消耗，可抑制pH(i)的升高（T24C精子，pH(i) = 6.96±0.03，n = 4，与T0精子相比p = 0.64）。为了测试较低的pH(i)是否会阻止T24C精子发生反应，我们用碱化剂氯化三甲胺（TMA）或NH4Cl处理T24C精子。这些试剂确实使T24C精子以剂量依赖方式对孕酮产生反应，但它们也使发生反应的T24精子数量有类似增加。这些试剂可能不会逆转高胆固醇的抑制作用，而是使无论胆固醇含量如何都存在的一部分精子产生反应。NH¬4Cl和TMA没有使T0精子对孕酮产生反应。酸化剂丙酸钠没有降低T24精子对孕酮的反应。总之，体外培养期间pH(i)以胆固醇依赖方式升高。单独升高pH(i)可能不足以使精子发生顶体反应。
