ACTIONS OF SEMINAL PROTEINS IN MATED DROSOPHILA FEMALES

精液绝不是精子传递的被动媒介，精液中含有的蛋白质对交配后的雌性产生重大影响。这在昆虫中得到了最好的理解，在昆虫中，精液蛋白（“Acps”）刺激排卵和产卵，这是雌性储存精子所必需的，并缩短了她的寿命。尽管其重要性，精液蛋白的分子作用是未知的。我们将使用可处理的果蝇分子遗传学来确定Acps如何控制三个主要的生殖过程。在目标1中，我们讨论了Acp26Aa（一种男性来源的激素原）是如何刺激排卵的。使用Acp26Aa零突变，我们将识别转导Acp26Aa效应的信号。我们还将确定Acp26Aa起作用的受体，以及Acp26Aa是否需要加工，以及其类似于软体动物产卵激素的区域。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。在目的2中，我们研究精液蛋白如何引起精子储存。同样，我们将从单个分子Acp36DE中得出结论，我们已经证明，Acp36DE在基因上是这个过程的核心。我们将检查gfp标记的精子在没有Acp36DE的情况下的行为，测试Acp36DE是在精子储存器官内起作用还是使精子进入储存。我们将测试Acp36DE的功能是否需要与精子结合，并将识别Acp36DE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。Acp62F是果蝇精液中的一种胰蛋白酶抑制剂。有趣的是，Acp62F进入昆虫循环系统后是有毒的。我们提出了一种突变分析来确定Acp62F在精液中的功能，如控制关键生殖蛋白的蛋白水解。我们将测试Acp62F在交配期间进入雌性循环系统是否会缩短她的寿命。我们将识别结合Acp62F的分子；这些应该包括它抑制的蛋白酶。我们的研究结果将确立控制蛋白水解在生殖中的作用，并将测试精液中为什么存在有毒蛋白的进化假设。在目标4中，我们将继续筛选ems诱导的Acps突变集合。分析这些突变体和Acps的异位表达，将发现调节排卵、精子储存或蛋白水解的新Acps。

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.