MAMMALIAN SPERM SURFACE ORGANIZATION AND FUNCTION

我们研究的长期目标是了解 精子表面结构与精子功能的关系 在 在先前的工作中，我们鉴定了两种精子表面蛋白，PH-20和PH-30， 在受精过程中起作用。 在这里，我们建议证明他们的 在受精中的精确功能，并将此功能与其领域联系起来 精子表面的定位、表面密度和侧向移动性。 在目的I中，我们提出确定负责形成的机制 精子发生过程中精子表面结构域的变化。 我们将区分 两种可能的机制之间：1）表面表达的时间 精子发生过程中的蛋白质决定了它们的结构域定位; 2） 表面蛋白含有分选信号， 域. (The排序信号可用于初始插入 正确域或从“不正确”域中删除）。 我们还将 分析创建前后头的后续步骤 附睾中精子成熟过程中的结构域。 在目标II中，我们提出了实验来获得PH-20是 一种透明体的精子受体 我们也会测试效果 PH-20的表面密度和侧向流动性对其粘附活性的影响， 确定其粘合伙伴在涂料和测试，如果PH-20在另一个 物种（小鼠）在精子结合中起作用。 我们在目标III中提议研究PH-30的功能，PH-30由 α和β亚基的结合 我们将测试两个假设：PH-30 α在精卵融合中起作用，其推定的融合肽是 一个必需的活性位点; PH-30 β在将精子结合到卵子中起作用 质膜通过其N末端去整合素结构域。 此外，本发明还提供了一种方法， 我们建议确定PH-30 β的粘附伙伴（可能是一种 整合素），并测试PH-30是否被激活 由于其在附睾通过、获能和/或 顶体反应 这项工作的重要性在于，我们将在界定 研究步骤的细胞和分子基础， 受精 此外，PH-20和PH-30均处于活跃状态 作为避孕疫苗抗原的研究和扩大 了解它们的基本生物学将有助于这一领域的工作。

The long term goal of our research has been to understand the relationship of sperm surface organization and sperm function. In previous work we identified two sperm surface proteins, PH-20 and PH-30, that have a role in fertilization. Here we propose to demonstrate their precise function in fertilization nd relate this function to their domain localization, surface density and lateral mobility on the sperm surface. In Aim I we propose to determine the mechanisms responsible for formation of sperm surface domains during spermatogenesis. We will distinguish between two possible mechanisms: 1) the time of expression of surface proteins during spermatogenesis determines their domain localization; 2) surface proteins contain sorting signals that target them to specific domains. (The sorting signal may be used for initial insertion into the correct domain or removal from the "incorrect" domain). We will also analyze the later steps in creation of the anterior and posterior head domains during sperm maturation in the epididymis. In Aim II we present experiments to obtain direct evidence that PH-20 is a sperm receptor for the zona pellucida. We will also test the effect of PH-20's surface density and lateral mobility on its adhesion activity, define its adhesion partner in the zona and test if PH-20 in another species (mouse) functions in sperm-zona binding. We propose in Aim III to study the function of PH-30 which is comprised of the subunits alpha and beta. We will test two hypotheses: PH-30 alpha functions in sperm-egg fusion and its putative fusion peptide is a required active site; PH-30 beta functions in binding sperm to the egg plasma membrane through its N-terminal disintegrin domain. In addition, we propose to identify PH-30 beta's adhesion partner (probably an integrin) on the egg plasma membrane and to test if PH-30 is activated for its role in fusion during epididymal passage, capacitation and/or the acrosome reaction. The importance of this work is that we will make progress in defining both the cellular and molecular basis of the studied steps in fertilization. Furthermore, PH-20 and PH-30 are both under active investigation as antigens for a birth control vaccine and expanded understanding of their basic biology will be useful in that area.