Involvement of cystic fibrosis transmembrane conductance regulator in mouse sperm capacitation
Involvement of cystic fibrosis transmembrane conductance regulator in mouse sperm capacitation
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DOI：
10.1074/jbc.m701603200
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发表时间：
2007-08-17
影响因子：
4.8
通讯作者：
Darszon, Alberto
中科院分区：
文献类型：
--
作者：
Hernandez-Gonzalez, Enrique O.;Trevino, Claudia L.;Darszon, Alberto
哺乳动物精子在一个被称为获能的过程中在雌性生殖道中获得可受精性。在小鼠精子中，这一过程与蛋白酪氨酸磷酸化增加、膜电位超极化、细胞内pH和Ca 2+增加以及超活化运动性有关。参与这些变化的分子机制尚不完全清楚。目前的证据表明，在小鼠精子中，与获能相关的膜超极化是由cAMP/蛋白激酶A依赖性途径调节的，该途径涉及激活内向整流K+通道和抑制上皮钠通道（ENaCs）。囊性纤维化跨膜传导调节因子（CFTR）是一种控制包括ENaC在内的几种转运蛋白活性的Cl-通道。在这里，我们探讨了CFTR是否参与调节精子中的ENaC抑制，因此对于获能相关的超极化是必不可少的。使用逆转录-PCR，蛋白质印迹和免疫细胞化学，我们的文件CFTR在小鼠和人类精子的存在。有趣的是，CFTR抑制剂（二苯胺-2-羧酸; 250 μ M）的加入抑制了容量相关的超极化，防止ENaC关闭，并减少了透明质酸诱导的顶体反应，而不影响酪氨酸磷酸化的增加。精子在无Cl-培养液中培养也可消除获能相关的超极化。另一方面，CFTR激活剂（染料木黄酮; 5-10 μ M）促进超极化小鼠精子孵育条件下，不支持获能。二丁酰环磷酸腺苷的非获能小鼠精子增加细胞内氯离子。这些结果表明，cAMP依赖的Cl-通量通过CFTR参与在获能过程中的ENaC的调节，从而有助于观察到的超极化与此过程相关。
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 mu M) inhibited the capacitation-associated hyperpolarization, prevented ENaC closure, and decreased the zona pellucida-induced acrosome reaction without affecting the increase in tyrosine phosphorylation. In cubation of sperm in Cl-- free medium also eliminated the capacitation associated hyperpolarization. On the other hand, a CFTR activator (genistein; 5-10 mu M) 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.