Belmonte S A, López C I, Roggero C M, De Blas G A, Tomes C N, Mayorga L S
Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Casilla de Correo 56, 5500 Mendoza, Argentina.
Dev Biol. 2005 Sep 15;285(2):393-408. doi: 10.1016/j.ydbio.2005.07.001.
The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.
顶体是覆盖在精子细胞核上的一种分泌性颗粒。在不同刺激下，它会经历钙调节的胞吐作用。刚射出的哺乳动物精子不能立即发生顶体反应。获得这种能力被称为获能，涉及精子生理过程中一系列尚未完全明确的变化。质膜胆固醇的去除是与获能相关的精子修饰之一。然而，甾醇如何影响顶体胞吐作用尚不清楚。在这里，我们表明在刺激前用环糊精（一种胆固醇去除剂）进行短时间孵育可促进顶体胞吐作用。此外，在用钙刺激的通透细胞中也观察到了这种效应，表明胆固醇在与顶体反应相关的钙依赖性胞吐作用中起直接作用。使用光抑制性钙螯合剂，我们表明胆固醇影响胞吐级联反应的早期事件，而不是脂质双层混合。功能数据表明胆固醇作用的一个靶点是Rab3A。甾醇含量不影响Rab3A的激活 - 失活循环，但调节其膜锚定。蛋白质印迹分析和免疫电子显微镜证实胆固醇外流促进Rab3A与精子质膜的结合。我们的数据表明，获能过程中发生的胆固醇外流优化了顶体反应所需融合机制有效组装的条件。
