Actions of Seminal Proteins in Mated Drosophila Females

在体内受精的动物中，通过精子转移的精液蛋白对雄性和雌性的生育能力都起着至关重要的作用。这一点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，并改变它们的行为，但也比未交配的雌性更早死亡。我们已经确定了苍蝇基因组(52个基因)中90%的ACPs。它们包括保守的精液蛋白类成员，如多肽、精子结合蛋白和蛋白酶抑制剂；29%的人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。我们还在交配的雌性中发现了由ACP调控的效应器和基因。我们的目标是确定调节雌性特定生殖变化的ACP，以及它们如何执行其功能。 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性、生化活性和结构。我们将通过在雌性体内的异位表达分析以及从雄性体内移除单个ACP的后果来确定它们在体内的功能。我们已经采用了Gateway克隆系统来高效地进行这些检测。 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，我们将扩展我们对三个ACP的分析，每个ACP代表这些领域中的一个。我们将确定它们活性的分子需求，它们调节的下游基因/效应器，以及它们的结合蛋白。然后，我们将类似地分析目标1中确定的ACPs在卵子产生、精子储存或蛋白质分解调节中的重要作用。 通过这项修订的研究计划，我们将获得类似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活动及其在雌性体内引发的反应的具体视图。这些信息将有助于我们理解雄性和雌性之间的分子相互作用，并评估关于生殖蛋白功能和进化的假说。除了它们与包括哺乳动物在内的一系列生物的生殖机制有关外，我们预计我们的发现将在控制人类疾病的昆虫媒介方面具有实际应用。

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.