Linares-Hernández L, Guzmán-Grenfell A M, Hicks-Gomez J J, González-Martínez M T
Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F.
Biochim Biophys Acta. 1998 Jun 24;1372(1):1-12. doi: 10.1016/s0005-2736(98)00035-2.
There are several physiological and pharmacological evidences indicating that opening of voltage dependent calcium channels play a crucial role in the induction of the acrosome reaction in mammalian sperm. In mature sperm, physiological inductors of the acrosome reaction such as ZP3, a zona pellucida protein, and the steroid hormone progesterone, induce depolarization and calcium influx, which are required for the acrosome reaction. In this paper, we describe a voltage-dependent calcium influx present in human sperm. We report an experimental procedure that allows measurement of intracellular calcium and membrane potential simultaneously using the fluorescent dyes DiSC3(5) and Fura-2. We found that in human uncapacitated sperm, depolarization induces a nifedipine-insensitive calcium influx that, in most cases, was transient. Calcium influx was observed in the range of -60 to -15 mV (the range tested). At resting membrane potential (around -40 mV), potassium addition depolarized and induced calcium influx, but when the depolarization was preceded by a hyperpolarization (induced with valinomycin), calcium influx was remarkably enhanced, suggesting that at -40 mV, channels are in a putative inactivated state. When sperm was incubated in medium without calcium, calcium restoration caused calcium influx that depended on voltage, and decayed between 1 and 2 min after depolarization. Unlike ram, mouse or bovine sperm, in which an alkalinization is required to induce calcium influx with potassium, the voltage-dependent calcium influx observed in human sperm did not require an increase in internal or external pH. However, we observed that ammonium, which increases intracellular pH, enhanced the voltage-dependent calcium influx about 90%. Furthermore, depolarization by itself caused a small increase in intracellular pH suggesting that pH can be regulated by membrane potential in human sperm.
有若干生理学和药理学证据表明，电压依赖性钙通道的开放在哺乳动物精子顶体反应的诱导过程中起关键作用。在成熟精子中，顶体反应的生理诱导剂，如透明带蛋白ZP3和甾体激素孕酮，会诱导去极化和钙内流，而这是顶体反应所必需的。在本文中，我们描述了人类精子中存在的电压依赖性钙内流。我们报告了一种实验方法，该方法允许使用荧光染料DiSC3(5)和Fura-2同时测量细胞内钙和膜电位。我们发现，在人类未获能精子中，去极化会诱导硝苯地平不敏感的钙内流，在大多数情况下，这种内流是短暂的。在-60至-15 mV（测试范围）内观察到了钙内流。在静息膜电位（约-40 mV）时，添加钾会使膜去极化并诱导钙内流，但当去极化之前先进行超极化（由缬氨霉素诱导）时，钙内流会显著增强，这表明在-40 mV时，通道处于一种假定的失活状态。当精子在无钙培养基中孵育时，钙的恢复会导致依赖电压的钙内流，并且在去极化后1至2分钟内衰减。与公羊、小鼠或牛的精子不同，在这些精子中需要碱化才能用钾诱导钙内流，而在人类精子中观察到的电压依赖性钙内流不需要细胞内或细胞外pH值升高。然而，我们观察到，增加细胞内pH值的铵会使电压依赖性钙内流增强约90%。此外，去极化本身会导致细胞内pH值略有升高，这表明在人类精子中pH值可由膜电位调节。
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