ACROSOMAL ENZYME BIOSYNTHESIS BY SPERMATIDS

这项研究提案的广泛、长期目标是 了解生物发生和成熟所涉及的过程 哺乳动物精子顶体，受精所必需的细胞器。 第一个特定的目标是确定两种顶体蛋白何时 首先合成它们，并将它们的合成与 编码其多肽的信使RNA。这个实验室有 先前证明了顶体的合成之一 顶体颗粒素从减数分裂开始，一直持续到早期。 精子发生。第二个具体目的是考察 通过检验与蛋白质相似的假说来检验顶端颗粒素 存在于其他分泌颗粒中，可能与包装有关 其他成分进入顶体。第三个具体目标是 观察附睾性改变对顶体的影响 组件对其功能的影响。首先，修改 蛋白水解酶原顶体酶原的寡糖侧链 检查以确定这些变化是否会影响激活的酶 活动。此外，精子顶体基质的稳定性 从附睾部的不同部位会检查到变化以来 这种基质的性质可能会影响精子 经历一个完全的顶体反应。最终的具体目标是 顶体酶原和顶体颗粒素对分泌颗粒的靶向性研究 已转染腺病毒的脑垂体细胞株ATT-20 编码顶体酶原和顶体颗粒素的cDNA。这些实验将 运用细胞生物学、生物化学和分子生物学的方法 来观察顶体的生物发生。这些蛋白质将是 定位于睾丸切片中的特定细胞细胞器 光镜和电子显微镜下的免疫细胞化学。功能分析 将观察原顶体酶/顶体酶的酶活性和 肢端颗粒素的聚集特性。在这个过程中， 项目中，编码原顶体酶和顶体颗粒蛋白的cDNA将是 与世隔绝的，有特点的。这些探测器将使我们能够看到 精子发生和发育过程中顶体酶原和顶体颗粒素的表达 以确定蛋白质的推导氨基酸序列。 将准备重组构建物，以便这些生殖细胞蛋白 可以在体细胞中表达。稳定表达细胞的建立 表达这些蛋白质的线条将允许更容易的检查 这些蛋白质的生物合成运输。这些研究的结果 实验将有助于解释顶体蛋白是如何运输的 从细胞质到顶体。了解这一过程可能会 为检查男性特发性不育病例提供新途径 并可能为避孕措施的发展开辟新的目标。

The broad, long-term objective of this research proposal is to understand the processes involved in the biogenesis and maturation of the mammalian sperm acrosome, an organelle essential for fertilization. The first specific aim is to determine when two acrosomal proteins are first synthesized and correlate their synthesis with that of the messenger RNA coding for their polypeptides. This laboratory has previously demonstrated that the synthesis one of the acrosomal components, acrogranin, begins during meiosis and continues into earlier spermogenesis. The second specific aim is examine the function of acrogranin by testing the hypothesis that it is similar to proteins found in other secretory granules and may be involved in the packaging of other components into the acrosome. The third specific aim is to examine the effects of epididymis-associated changes of acrosomal components on their function. First, the modification of the oligosaccharide side-chains of the protease zymogen proacrosin will be examined to determine if these changes affect the activated enzyme's activity. In addition, the stability of the acrosomal matrix of sperm from different regions of the epididymis will be examined since changes in the properties of this matrix may affect the ability of sperm to undergo a complete acrosome reaction. The final specific aim is to examine the targeting of proacrosin and acrogranin to secretory granules of cells of the pituitary line AtT-20 that have been transfected with cDNAs coding for proacrosin and acrogranin. These experiments will utilize the methods of cell biology, biochemistry, and molecular biology to look at the biogenesis of the acrosome. The proteins will be localized to specific cells organelles of cells in sections of testes by immunocytochemistry by light and electron microscopy. Functional assays will look at the enzymatic activity of proacrosin/acrosin and the aggregation characteristics of acrogranin. During the course of the project, cDNAs coding for both proacrosin and acrogranin will be isolated and characterized. These probes will allow us to look at the expression of proacrosin and acrogranin mRNA during spermatogenesis and to determine the deduced amino acid sequences for the proteins. Recombinant constructs will be prepared so that these germ cell proteins can be expressed in somatic cells. Creation of stably transfected cell lines expressing these proteins will allow the more facile examination of the biosynthetic transport of these proteins. The results from these experiments will help explain how acrosomal proteins are transported from the cell cytoplasm to the acrosome. Knowledge of this process may provide new avenues for examining cases of male idiopathic infertility and may open new targets for contraceptive development.