Actions of seminal proteins in mated Drosophila females.

 描述(申请人提供)：精液蛋白(SFP)是从昆虫到哺乳动物的动物生育能力的重要组成部分，但往往被忽视。SFP对交配雌性的生殖生理、雌性体内精子的储存和释放以及最近在小鼠身上显示的后代表型都有重大影响。特定SFP异常的雄性动物是不育或不育的；这一点的证据延伸到人类。一些SFP与精子紧密结合；另一些留在精浆中，因此在一些辅助生殖技术(ART)中被移除。然而，尽管SFP在生殖中很重要，但人们对SFP是如何影响女性的知之甚少。这一建议结合了遗传学、生理学、进化和蛋白质的方法来解决关于SFP作用的两个紧迫问题：(1)SFP是如何与雌性相互作用而引起交配后效应的？(2)SFP是如何与精子紧密结合并对精子储存产生影响的？我们利用果蝇的实验优势来解决这些问题，果蝇是我们建立的一个主要的遗传模型系统，用来分析SFP功能，它的SFP与哺乳动物的SFP在分子和现象学上有许多相似之处。为了解决第一个问题，目标1专注于卵蛋白，一种刺激排卵的果蝇SFP。奥韦林诱导八胺能信号，进而调节生殖道的肌肉收缩，导致排卵率增加。我们将通过测试我们的候选GPCRs在基因敲除时的排卵表型以及在培养细胞中与卵蛋白的结合来鉴定女性的卵蛋白受体(OVR)。然后，使用有针对性的OVR基因敲除，我们将检验卵子蛋白直接作用于神经系统的假设。我们还将确定卵蛋白进入雌性时发生的蛋白质分解过程是否会增强卵蛋白的活性及其与OVR的结合。为了阐明精子结合SFP的机制和功能，目标2重点研究了果蝇的性肽，SP。我们最近定义了一个将SP与精子结合的SFP网络。我们将通过确定SP是否能与纯射精液中的精子结合来测试男性对SP精子结合的贡献是否足够，或者女性是否也有贡献因素。然后我们将确定SP结合所需的精子蛋白。SP操作 才能有效地将精子从储藏中释放出来。在目标2的最后部分，我们将检验这一假设，即SP是通过神经系统(和章鱼胺能信号)而不是通过精子储存器官内的作用来实现这一点的。在分子水平上阐明SFP如何与女性相互作用并影响女性是一个非常新的领域，直到现在才开始适合研究。拟议工作的结果与理解和诊断基于SFP的不育症和考虑ARTS的策略有关，ARTS可能受益于纳入关键的SFP。此外，了解双翅目昆虫中的生殖分子将有助于控制传播病毒的蚊子的策略，这些病毒会导致登革热和疟疾等严重的人类疾病。

 DESCRIPTION (provided by applicant): Seminal fluid proteins (Sfps) are an important, yet often overlooked, component of fertility in animals from insects to mammals. Sfps have significant effects on the reproductive physiology of mated females, on storage and release of sperm inside females and, as recently shown in mice, on the phenotype of progeny. Male animals abnormal for specific Sfps are sterile or subfertile; evidence for this extends to humans. Some Sfps bind tightly to sperm; others remain in the seminal plasma and therefore are removed during some Assisted Reproductive Technologies (ARTs). Yet despite their importance in reproduction, little is known about exactly how Sfps act to influence the female. This proposal combines genetic, physiological, evolutionary, and protein methodologies to address two pressing questions about Sfps' action: (1) How, precisely, do Sfps interact with the female to cause post- mating effects? (2) How do Sfps bind tightly to sperm and exert effects on sperm storage? We address these by using the experimental strengths of Drosophila, which we have established as a premier genetic model system with which to dissect Sfp function and whose Sfps have many molecular and phenomenological parallels to those of mammals. To address the first question, Aim 1 focuses on ovulin, a Drosophila Sfp that stimulates ovulation. Ovulin induces octopaminergic signaling, which in turn regulates muscle contraction in the reproductive tract, causing an increase in ovulation rate. We will identify the female's receptor for ovulin (OvR) by testing our candidate GPCRs for ovulation phenotypes upon knockdown, and for binding to ovulin in cultured cells. Then, using targeted knockdowns of OvR we will test the hypothesis that ovulin acts directly on the nervous system. We also will determine whether the proteolytic processing of ovulin that occurs when it enters the female enhances ovulin's activity and its binding to OvR. To address the mechanism and function of sperm-bound Sfps, Aim 2 focuses on the Drosophila "sex peptide", SP. We recently defined a network of Sfps that binds SP to sperm. We will test whether male contributions are sufficient for SP's sperm binding, or whether females also contribute factors, by determining whether SP can bind to sperm in pure ejaculates. We will then identify the sperm protein that is required for SP binding. SP action is required to efficiently release sperm from storage. In the final part of Aim 2 we will test the hypothesis that SP does so through the nervous system (and octopaminergic signaling) rather than by action within the sperm storage organs. Elucidating how Sfps interact with and affect the female at the molecular level is a very new area that is only now becoming amenable to study. Results of the proposed work are relevant to understanding and diagnosing Sfp-based infertilities and in considering strategies for ARTs, which may benefit from inclusion of critical Sfps. Additionally, understanding reproductive molecules in Dipteran insects will assist in strategies to control mosquitoes that transmit viruses that cause serious human diseases like dengue and malaria.