Actions of seminal proteins in mated Drosophila females.

 描述（由申请人提供）：精液蛋白（Sfps）是从昆虫到哺乳动物的重要但经常被忽视的生育力组分。SFPs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中显示的后代表型有显著影响。特定Sfps异常的雄性动物是不育或生育力低下的;这一点的证据延伸到人类。一些SFPs与精子紧密结合;另一些则留在精浆中，因此在某些辅助生殖技术（ART）中被去除。然而，尽管它们在繁殖中很重要，但人们对Sfps如何影响雌性却知之甚少。该建议结合遗传学、生理学、进化和蛋白质方法来解决关于Sfps作用的两个紧迫问题：（1）Sfps如何准确地与雌性相互作用以引起交配后效应？(2)SFPs如何与精子紧密结合并对精子储存产生影响？我们通过使用果蝇的实验优势来解决这些问题，我们已经建立了一个首要的遗传模型系统来解剖SFP功能，其SFP与哺乳动物的SFP有许多分子和现象学上的相似之处。为了解决第一个问题，Aim 1将重点放在ovulin上，这是一种刺激排卵的果蝇Sfp。排卵素诱导章鱼胺能信号传导，章鱼胺能信号传导反过来调节生殖道中的肌肉收缩，导致排卵率增加。我们将通过检测我们的候选GPCR在敲低后的排卵表型以及在培养细胞中与排卵蛋白的结合来鉴定女性的排卵蛋白受体（OvR）。然后，使用OvR的靶向敲除，我们将测试Ovulin直接作用于神经系统的假设。我们还将确定当卵蛋白进入雌性时发生的蛋白水解加工是否增强了卵蛋白的活性及其与OvR的结合。为了阐明精子结合Sfps的机制和功能，本研究的第二个目标是研究果蝇的性肽SP，我们最近定义了一个与精子结合的Sfps网络。我们将测试是否男性的贡献是足够的SP的精子结合，或女性是否也有助于因素，通过确定是否SP可以结合到精子在纯射精。然后，我们将确定SP结合所需的精子蛋白。SP作用 才能有效地将精子从储存中释放出来在目标2的最后一部分，我们将检验SP通过神经系统（和章鱼胺能信号传导）而不是通过精子储存器官内的作用来实现这一假设。阐明Sfps如何在分子水平上与女性相互作用并影响女性是一个非常新的领域，现在才开始适合研究。拟议的工作的结果是相关的理解和诊断SFP为基础的不孕症，并在考虑战略的ART，这可能会受益于列入关键SFP。此外，了解双翅目昆虫的生殖分子将有助于控制蚊子传播导致登革热和疟疾等严重人类疾病的病毒的策略。

 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.