Vacquier V D, Moy G W
Scripps Institution of Oceanography, University of California, San Diego, California 92093-0202, USA.
Dev Biol. 1997 Dec 1;192(1):125-35. doi: 10.1006/dbio.1997.8729.
The sea urchin sperm cell is an advantageous model for studying ligand-mediated exocytosis. Sperm can be obtained in vast quantities and induced to undergo exocytosis of the acrosomal vesicle with great synchrony. During sea urchin fertilization, egg jelly (EJ) triggers the sperm acrosome reaction (AR) which is required for sperm binding and fusion with the egg. Uncertainty exists as to the exact biochemical nature of the AR inducer. The following study was performed in an attempt to clarify the nature of the inducer. EJ from individual females (Strongylocentrotus purpuratus) was analyzed on SDS-PAGE gels. Each female had a unique composition of EJ macromolecules, but all females possessed the previously described fucose sulfate polymer (FSP). Two electrophoretic isotypes of FSP were discovered; 87% of females had only one isotype and 13% had both. Both FSP isotypes bound to the REJ protein (receptor for egg jelly) purified from sperm. The two FSP isotypes had almost equal potency in inducing the AR. EJ was fractionated by DEAE chromatography in 6 M urea/4% beta-mercaptoethanol. All AR-inducing activity coeluted with FSP. FSP, purified by trypsin digestion followed by dialysis, was twice as active as the non-trypsin-digested control at inducing the AR. EJ was digested with proteinase K, boiled in detergent and beta-mercaptoethanol, and subjected to sucrose density gradient sedimentation. The FSP and AR activity had superimposable sedimentation patterns. Purified FSP had no associated peptide component. Sperm from individual males differed in the concentration dependency of purified FSP to induce the AR. The data indicate that the 138/82 kDa EJ glycoproteins, previously thought to act as AR inducers, do not appear to be involved in triggering the AR. The data are consistent with the hypothesis that FSP is the only inducer of the AR of this sea urchin species.
海胆精子细胞是研究配体介导的胞吐作用的一个有利模型。可以大量获取精子，并诱导其顶体小泡高度同步地发生胞吐作用。在海胆受精过程中，卵胶（EJ）触发精子顶体反应（AR），这是精子与卵子结合和融合所必需的。关于AR诱导剂的确切生化性质尚不确定。进行以下研究以试图阐明诱导剂的性质。对来自单个雌性（紫海胆）的EJ在SDS-PAGE凝胶上进行分析。每个雌性的EJ大分子组成独特，但所有雌性都拥有先前描述的硫酸岩藻糖聚合物（FSP）。发现了FSP的两种电泳同型物；87%的雌性只有一种同型物，13%的雌性同时拥有两种。两种FSP同型物都与从精子中纯化的REJ蛋白（卵胶受体）结合。两种FSP同型物在诱导AR方面的效力几乎相等。EJ在6 M尿素/4%β-巯基乙醇中通过DEAE色谱法进行分级分离。所有诱导AR的活性都与FSP共洗脱。通过胰蛋白酶消化然后透析纯化的FSP在诱导AR方面的活性是非胰蛋白酶消化对照的两倍。用蛋白酶K消化EJ，在去污剂和β-巯基乙醇中煮沸，然后进行蔗糖密度梯度沉降。FSP和AR活性具有可叠加的沉降模式。纯化的FSP没有相关的肽成分。来自单个雄性的精子在纯化的FSP诱导AR的浓度依赖性方面存在差异。数据表明，先前被认为作为AR诱导剂的138/82 kDa EJ糖蛋白似乎不参与触发AR。这些数据与FSP是这种海胆物种AR的唯一诱导剂这一假设一致。
