Isolation and proteomic analysis of mouse sperm detergent-resistant membrane fractions: Evidence for dissociation of lipid rafts during capacitation
Isolation and proteomic analysis of mouse sperm detergent-resistant membrane fractions: Evidence for dissociation of lipid rafts during capacitation
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DOI：
10.1095/biolreprod.105.041533
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发表时间：
2005-10-01
影响因子：
3.6
通讯作者：
Visconti, PE
中科院分区：
文献类型：
--
作者：
Sleight, SB;Miranda, PV;Visconti, PE
哺乳动物的精子在雌性生殖道中获得受精能力，这一过程被称为获能。本研究探讨是否在获能过程中胆固醇流出改变精子质膜的生物物理特性，通过潜在地减少脂筏结构域的程度分析的洗涤剂抗性膜馏分使用蔗糖梯度的隔离。此外，这项工作调查是否在获能过程中耐洗涤剂的膜部分的解离改变常驻精子筏蛋白。通过银染、串联质谱和Western印迹分析研究了与这些组分相关的小鼠精子蛋白。小窝蛋白1被确定在精子脂筏中的多聚体状态，包括一个高分子量的低聚物，在高pH值的还原条件下降解敏感。获能导致减少光澄清剂密度，洗涤剂抗性膜馏分和减少阵列的蛋白质分离在这部分，包括损失的高分子量的小窝蛋白1低聚物。蛋白质组学分析的精子蛋白质分离的光澄清剂密度部分确定了几种蛋白质，包括己糖激酶1，睾丸丝氨酸蛋白酶1和2，TEX101，透明质酸酶（PH20，SPAM1），促进葡萄糖转运蛋白3，乳酸脱氢酶A，碳酸酐酶IV，IZUMO，泛泡素，basigin，和富含半胱氨酸的抑制性分泌蛋白1。获能也导致精子标记的荧光脂质类似物DilC16显着减少，表明获能改变了精子质膜中的液体有序结构域。获能改变抗洗涤剂膜组分的蛋白质组成的观察结果与获能过程中胆固醇流出解离脂筏成分，引发导致精子获能的信号事件的假设一致。
Mammalian sperm acquire fertilization capacity after residing in the female tract during a process known as capacitation. The present study examined whether cholesterol efflux during capacitation alters the biophysical properties of the sperm plasma membrane by potentially reducing the extent of lipid raft domains as analyzed by the isolation of detergent-resistant membrane fractions using sucrose gradients. In addition, this work investigated whether dissociation of the detergent-resistant membrane fraction during capacitation alters resident sperm raft proteins. Mouse sperm proteins associated with such fractions were studied by silver staining, tandem mass spectrometry, and Western blot analysis. Caveolin 1 was identified in sperm lipid rafts in multimeric states, including a high-molecular-weight oligomer that is sensitive to degradation under reducing conditions at high pH. Capacitation resulted in reduction of the light buoyant-density, detergent-resistant membrane fraction and decreased the array of proteins isolated within this fraction, including loss of the high-molecular-weight caveolin 1 oligomers. Proteomic analysis of sperm proteins isolated in the light buoyant-density fraction identified several proteins, including hexokinase 1, testis serine proteases 1 and 2, TEX101, hyaluronidase (PH20, SPAM1), facilitated glucose transporter 3, lactate dehydrogenase A, carbonic anhydrase IV, IZUMO, pantophysin, basigin, and cysteine-rich inhibitory secretory protein 1. Capacitation also resulted in a significant reduction of sperm labeling by the fluorescent lipid-analog DilC16, indicating that capacitation alters the liquid-ordered domains in the sperm plasma membrane. The observations that capacitation alters the protein composition of the detergent-resistant membrane fractions is consistent with the hypothesis that cholesterol efflux during capacitation dissociates lipid raft constituents, initiating signaling events that lead to sperm capacitation.