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.

在体内受精的动物中，随精子转移的精液蛋白在雄性和雌性的生育能力中起着至关重要的作用。这在果蝇的模型系统中得到了最好的理解，果蝇的精液蛋白（“Acps”）诱导雌性增加卵子产量，储存精子，改变它们的行为，但也比未交配的雌性更早死亡。我们已经鉴定了果蝇基因组中90%的Acps（52个基因）。它们包括保守的生殖蛋白类的成员，如肽，精子结合蛋白和蛋白酶抑制剂; 29%显示出快速进化的迹象，这是许多生物体中生殖蛋白的特征。我们还确定了在交配的女性Acps调控的效应子和基因。我们的目标是确定介导女性特定生殖变化的Acps，以及它们如何发挥其功能。 在目标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。我们将确定其在体内的功能，异位表达测定在女性中，并从男性中删除个别Acps的后果。我们已经调整了Gateway克隆系统，以高效地进行这些测定。 在目标2中，我们将详细分析单个Acp调节卵子产生，精子储存或寿命效应/蛋白酶抑制的功能。首先，我们将扩展我们的分析三个ACP，每个代表这些领域之一。我们将确定其活性的分子要求，它们调节的下游基因/效应子，以及它们的结合蛋白。然后，我们将类似地分析目标1中鉴定的Acps在卵子产生、精子储存或蛋白水解调节中的重要性。 通过这项修订后的研究计划，我们将获得类似于哺乳动物前列腺的苍蝇组织的全面功能视图，以及单个精液蛋白的作用及其在女性中引发的反应的具体视图。这些信息将帮助我们了解雄性和雌性之间的分子相互作用，并评估有关生殖蛋白质功能和进化的假设。除了它们与包括哺乳动物在内的一系列生物体的生殖机制的相关性之外，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。