Hernández-González Enrique O, Treviño Claudia L, Castellano Laura E, de la Vega-Beltrán José L, Ocampo Ana Y, Wertheimer Eva, Visconti Pablo E, Darszon Alberto
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca Morelos 62210, México.
J Biol Chem. 2007 Aug 17;282(33):24397-406. doi: 10.1074/jbc.M701603200. Epub 2007 Jun 22.
Mammalian sperm acquire fertilizing ability in the female tract during a process known as capacitation. In mouse sperm, this process is associated with increases in protein tyrosine phosphorylation, membrane potential hyperpolarization, increase in intracellular pH and Ca2+, and hyperactivated motility. The molecular mechanisms involved in these changes are not fully known. Present evidence suggests that in mouse sperm the capacitation-associated membrane hyperpolarization is regulated by a cAMP/protein kinase A-dependent pathway involving activation of inwardly rectifying K+ channels and inhibition of epithelial sodium channels (ENaCs). The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that controls the activity of several transport proteins, including ENaCs. Here we explored whether CFTR is involved in the regulation of ENaC inhibition in sperm and therefore is essential for the capacitation-associated hyperpolarization. Using reverse transcription-PCR, Western blot, and immunocytochemistry, we document the presence of CFTR in mouse and human sperm. Interestingly, the addition of a CFTR inhibitor (diphenylamine-2-carboxylic acid; 250 microM) inhibited the capacitation-associated hyperpolarization, prevented ENaC closure, and decreased the zona pellucida-induced acrosome reaction without affecting the increase in tyrosine phosphorylation. Incubation of sperm in Cl- -free medium also eliminated the capacitation-associated hyperpolarization. On the other hand, a CFTR activator (genistein; 5-10 microM) promoted hyperpolarization in mouse sperm incubated under conditions that do not support capacitation. The addition of dibutyryl cyclic AMP to noncapacitated mouse sperm elevated intracellular Cl-. These results suggest that cAMP-dependent Cl- fluxes through CFTR are involved in the regulation of ENaC during capacitation and thus contribute to the observed hyperpolarization associated with this process.
哺乳动物的精子在称为获能的过程中于雌性生殖道内获得受精能力。在小鼠精子中，这一过程与蛋白质酪氨酸磷酸化增加、膜电位超极化、细胞内pH值和Ca2+升高以及超活化运动性有关。这些变化所涉及的分子机制尚未完全明确。目前的证据表明，在小鼠精子中，与获能相关的膜超极化由一种cAMP/蛋白激酶A依赖性途径调控，该途径涉及内向整流钾通道的激活和上皮钠通道（ENaCs）的抑制。囊性纤维化跨膜传导调节因子（CFTR）是一种氯离子通道，可控制包括ENaCs在内的多种转运蛋白的活性。在此，我们探讨了CFTR是否参与精子中ENaC抑制的调节，因此对于与获能相关的超极化是否至关重要。通过逆转录PCR、蛋白质印迹和免疫细胞化学，我们证明了CFTR在小鼠和人类精子中的存在。有趣的是，添加CFTR抑制剂（二苯胺-2-羧酸；250微摩尔）可抑制与获能相关的超极化，阻止ENaC关闭，并减少透明带诱导的顶体反应，而不影响酪氨酸磷酸化的增加。将精子在无氯离子培养基中孵育也消除了与获能相关的超极化。另一方面，CFTR激活剂（染料木黄酮；5 - 10微摩尔）在不支持获能的条件下孵育的小鼠精子中促进超极化。向未获能的小鼠精子中添加二丁酰环磷酸腺苷可提高细胞内氯离子浓度。这些结果表明，在获能过程中，通过CFTR的cAMP依赖性氯离子通量参与了ENaC的调节，从而导致了与该过程相关的超极化。
