Actions of Seminal Proteins in Mated Drosophila Females

精液蛋白在交配果蝇雌性中的作用

• 批准号: 7002329
• 负责人: Mariana Federica Wolfner
• 金额: $ 26.65万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7002329
• 关键词: Drosophilidae; RNA interference; arthropod genetics; biological signal transduction; female reproductive system; fertilization; gene expression; gene mutation; glycoproteins; green fluorescent proteins; immunoprecipitation; molecular cloning; ovulation; peptide hormone biosynthesis; polymerase chain reaction; protease inhibitor; protein structure function; proteolysis; semen; sperm; tissue /cell culture; trypsin inhibitors; western blottings; yeast two hybrid system

In animals with internal fertilization, seminal proteins transferred with sperm play crucial roles in the fertility of both males and females. This is best understood in the model system Drosophila, whose seminal proteins ("Acps") induce females to increase egg production, store sperm, and modify their behavior, but also to die younger than unmated females. We have identified 90% of all Acps in the fly genome (52 genes). They include members of conserved seminal-protein classes such as peptides, sperm-binding proteins and protease inhibitors; 29% show signs of having evolved rapidly, a trait seen for reproductive proteins in many organisms. We have also identified effectors and genes regulated by Acps in mated females. Our goal is to determine the Acps that mediate specific reproductive changes in females, and how they carry out their functions. In Aim 1 we will screen for the reproductive function of individual Acps. We will assess the targeting, biochemical activity and structure of these molecules. We will determine their in vivo functions by ectopic expression assays in females, and by the consequences of removing individual Acps from males. We have adapted the Gateway cloning system to carry out these assays with high efficiency. In Aim 2 we will analyze in detail the functions of individual Acps that regulate egg production, sperm storage or lifespan effects/protease inhibition. Initially we will extend our analysis of three Acps, each representing one of these areas. We will determine molecular requirements for their activity, the downstream genes/effectors they regulate, and their binding proteins. We will then similarly analyze Acps identified in Aim 1 to be important in egg production, sperm storage or proteolysis regulation. Through this revised research plan we will obtain a comprehensive functional view of the fly tissue analogous to the mammalian prostate gland, and a specific view of the actions of individual seminal proteins and the responses they trigger in females. This information will help us understand the molecular interactions between males and females, and to evaluate hypotheses about the functions and evolution of reproductive proteins. In addition to their relevance to reproductive mechanisms in a range of organisms including mammals, we anticipate that our findings will have practical applications in the control of insect vectors of human disease.

在具有内部受精的动物中，具有精子转移的精液蛋白在男性和女性的生育中都起着至关重要的作用。最好在模型系统果蝇中理解这一点，他们的精液蛋白（“ ACP”）诱导女性增加卵的产量，存储精子并改变其行为，但也比未经未来的女性更年轻。我们已经确定了苍蝇基因组中所有ACP的90％（52个基因）。它们包括保守的Seminal蛋白质类别的成员，例如肽，结合蛋白和蛋白酶抑制剂； 29％的人表现出迅速发展的迹象，这是许多生物体中生殖蛋白的特征。我们还确定了由ACP调节的效应子和基因。我们的目标是确定介导女性特定生殖变化的ACP，以及它们如何执行其功能。 在AIM 1中，我们将筛选单个ACP的生殖功能。我们将评估这些分子的靶向，生化活性和结构。我们将通过女性的异位表达测定以及从男性中去除单个ACP的后果来确定其体内功能。我们已经调整了网关克隆系统，以高效进行这些测定。 在AIM 2中，我们将详细分析调节卵产生，精子储存或寿命效应/蛋白酶抑制的单个ACP的功能。最初，我们将扩展对三个ACP的分析，每个ACP代表这些领域之一。我们将确定其活性的分子需求，它们调节的下游基因/效应子和结合蛋白。然后，我们将类似地分析AIM 1中确定的ACP，以在产生卵，精子储存或蛋白水解调节中很重要。 通过这个修订后的研究计划，我们将获得类似于哺乳动物前列腺类似的蝇组织的全面功能视图，以及对单个精液蛋白的作用及其在女性中触发的反应的特定视图。这些信息将有助于我们了解雄性与女性之间的分子相互作用，并评估有关生殖蛋白功能和进化的假设。除了它们与包括哺乳动物在内的一系列生物中的生殖机制相关，我们预计我们的发现将在控制人类疾病的昆虫媒介中具有实际应用。