ACTIONS OF SEMINAL PROTEINS IN MATED DROSOPHILA FEMALES

精液远不是精子传递的被动媒介，它含有对交配雌性产生重大影响的蛋白质。这在昆虫中是最好理解的，其中精液蛋白质（“Acps”）刺激排卵和产卵，是雌性储存精子所需的，并减少她的寿命。 尽管其重要性，精液蛋白的分子作用是未知的。 我们将使用果蝇的易处理的分子遗传学来确定Acps如何控制三个主要的生殖过程。 在目标1中，我们讨论了Acp 26 Aa是如何刺激排卵的，Acp 26 Aa是一种男性来源的激素原。 使用Acp 26 Aa无效突变，我们将识别出抑制Acp 26 Aa效应的信号。我们还将确定Acp 26 Aa作用的受体，以及Acp 26 Aa是否需要加工，以及其区域是否类似于软体动物产卵激素。 我们的研究结果对生殖、激素作用和生殖隔离机制的进化具有重要意义。 在目标2中，我们研究了精液蛋白质如何引起精子储存。我们将再次从一个单一的分子，Acp 36 DE，我们发现基因是这个过程的核心。 我们将检查GFP标记的精子在缺乏Acp 36 DE的情况下的行为，测试Acp 36 DE是否在精子储存器官内起作用或使精子进入储存。 我们将测试Acp 36 DE的功能是否需要它与精子结合，并将确定Acp 36 DE结合的分子。 精子储存在动物中很普遍，我们的研究将确定其分子基础。 在目标3中，我们讨论了蛋白酶抑制剂在精液中的作用。 许多动物的精液中含有蛋白酶抑制剂，但其生殖功能尚不清楚。 Acp 62 F是果蝇精液中的胰蛋白酶抑制剂。有趣的是，Acp 62 F在引入昆虫循环系统时是有毒的。我们提出了一个突变分析，以确定Acp 62 F的功能，在精液中，如控制关键生殖蛋白的蛋白水解。 我们将测试Acp 62 F在交配期间进入雌性的循环系统是否会缩短她的寿命。 我们将鉴定结合Acp 62 F的分子;这些分子应该包括它抑制的蛋白酶。 我们的研究结果将建立受控蛋白水解的生殖作用，并将测试为什么有毒蛋白质存在于精液中的进化假说。在目标4中，我们将继续筛选EMS诱导的Acps突变。 分析这些突变体和Acps的异位表达，将确定新的Acps，调节排卵，精子储存或蛋白水解。

Far from being a passive medium for sperm delivery, seminal fluid contains proteins that exert major influences on mated females. This is best understood in insects, where seminal fluid proteins ("Acps") stimulate ovulation and egg-laying, are required for a female to store sperm, and decrease her lifespan. Despite their importance, the molecular actions of seminal fluid proteins are unknown. We will use the tractable molecular genetics of Drosophila to determine how three major reproductive processes are controlled by Acps. In aim 1, we address how ovulation is stimulated by Acp26Aa, a male-derived prohormone. Using an Acp26Aa null mutation, we'll identify signals that transduce the effect of Acp26Aa. We will also identify the receptor through which Acp26Aa acts, and whether Acp26Aa needs processing, and its region similar to mollusk egg-laying hormones, to act. Our results are significant to reproduction, hormone action, and the evolution of reproductive isolation mechanisms. In aim 2, we investigate how seminal fluid proteins cause sperm storage. Again we'll work out from a single molecule, Acp36DE, that we showed genetically is central to this process. We will examine the behavior of GFP-labeled sperm in the absence of Acp36DE, testing whether Acp36DE acts within the sperm storage organs or to get sperm into storage. We will test whether Acp36DE's function requires its binding to sperm, and will identify molecules to which Acp36DE binds. Sperm storage is widespread among animals; our study will determine its molecular basis. In aim 3, we address the role of protease inhibitors in seminal fluid. Seminal fluid of many animals contains protease inhibitors, but their reproductive function is unknown. Acp62F is a trypsin inhibitor in Drosophila seminal fluid. Intriguingly, Acp62F is toxic when introduced into insect circulatory systems. We propose a mutational analysis to determine Acp62F's function in the seminal fluid, such as controlling proteolysis of crucial reproductive proteins. We will test if entry of Acp62F into the female's circulatory system during mating shortens her life. We will identify molecules which bind Acp62F; these should include the proteases it inhibits. Our results will establish reproductive roles for controlled proteolysis, and will test evolutionary hypotheses for why toxic proteins exist in semen. In aim 4, we'll continue screening a collection of EMS-induced mutations for ones in Acps. Analyzing these mutants, and ectopic expression of Acps, will identify new Acps that regulate ovulation, sperm storage or proteolysis.