ACROSOMAL ENZYME BIOSYNTHESIS BY SPERMATIDS

这项研究计划的长远目标是： 了解参与生物发生和成熟的过程， 哺乳动物精子顶体，受精所必需的细胞器。 第一个具体的目标是确定两种顶体蛋白何时被 首先合成，并将其合成与 编码多肽的信使RNA。 本实验室曾 以前证明，顶体的合成之一， 顶粒蛋白在减数分裂期间开始，并持续到更早的时期。 精子发生 第二个具体目标是考察 顶粒蛋白通过测试假设，它是类似于蛋白质 在其他分泌颗粒中发现，可能与包装有关 其他成分进入顶体 第三个具体目标是 检查附睾相关的顶体变化的影响， 组件的功能。 第一，修改 蛋白酶酶原顶体酶原的寡糖侧链将被 检查以确定这些变化是否影响活化的酶的 活动 此外，精子顶体基质的稳定性 将检查来自附睾不同区域的精子， 这种基质的性质可能会影响精子的能力， 进行完全的顶体反应。 最后的具体目标是 检查顶体蛋白原和顶粒蛋白对分泌颗粒的靶向作用 垂体细胞系AtT-20的细胞已经转染了 编码顶体蛋白原和顶粒蛋白的cDNA。 这些实验将 利用细胞生物学、生物化学和分子生物学的方法 来观察顶体的生物起源。 这些蛋白质将 定位于睾丸切片中特定细胞的细胞器， 光镜和电镜免疫细胞化学。 功能测定 将研究顶体酶原/顶体酶的酶活性和 顶粒蛋白的聚集特性。 过程中 项目，编码顶体蛋白原和顶粒蛋白的cDNA将被 分离并表征。 这些探测器将使我们能够看到 精子发生过程中顶体蛋白原和顶粒蛋白mRNA的表达， 以确定蛋白质的推导氨基酸序列。 将制备重组构建体，使得这些生殖细胞蛋白质 可以在体细胞中表达。 稳定转染细胞的产生 表达这些蛋白质的细胞系将允许更容易的检查 这些蛋白质的生物合成运输。 结果从这些 实验将有助于解释顶体蛋白是如何被运输的 从细胞质到顶体。 对这一过程的了解可能 为男性特发性不育症提供新的检查途径 并可能为避孕开发开辟新的目标。

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.