Water and solute transport in the epididymis

描述：通过男性生殖道流出管上皮细胞的液体重吸收导致精子密度的增加，这对生育至关重要。人们对这一过程的调控机制知之甚少。AQP9已被确定为附睾尖的主要水通道。然而，它在附睾跨上皮运输中的作用还没有被研究。AQP9是一种水甘油疏松素，除了水外，还可以运输中性溶质。此应用程序的目标是 研究AQP9相关的水和溶质在附睾中运输的功能和调节。特异的AIM 1将检验两个假设：1)AQP9参与顶端水和/或中性溶质的运输，并负责建立最佳的管腔渗透压和精子浓度；2)附睾上皮基外侧膜含有一种未知的水通道蛋白(S)。我们将使用多学科方法，包括体内和体外的附睾管灌流，定量数字成像(细胞体积测量和免疫荧光)，以及顶膜囊泡中的停流光散射，来表征顶端和基底侧水和/或溶质运输的途径。我们将通过从激光捕获显微切割分离的上皮细胞中进行同源筛选，确定必须参与水和/或溶质通过附睾上皮流动的基底外侧水通道蛋白(S)。有趣的是，自从上次提交以来，我们已经表明AQP11mRNA在附睾上皮细胞中表达。我们现在将通过原位杂交来研究AQP11的mRNA定位，并通过蛋白质印迹和免疫荧光来研究AQP11的蛋白表达。特异性AIM 2将测试AQP9是否通过以下方式调节：1)直接磷酸化；或2)与伙伴蛋白相互作用。我们将使用纯化的AQP9来研究AQP9通过磷酸化的急性调节。蛋白质-蛋白质相互作用涉及 “PDZ”结构域将使用下拉、免疫共沉淀和凝胶覆盖分析进行检测。将研究与CFTR、NHERF1或PDZK1的相互作用。所提出的研究将提供独特和新颖的信息，对于我们理解水和溶质在流出管上皮细胞中的运输如何有助于精子成熟、储存和浓缩的管腔环境的产生具有重要意义。

DESCRIPTION: Fluid reabsorption across epithelial cells of the excurrent ducts of the male reproductive tract results in an increase in sperm concentration that is crucial for fertility. The mechanisms underlying the regulation of this process are poorly understood. AQP9 has been identified as the major apical water channel in the epididymis. However its role in epididymal transepithelial transport has not been investigated. AQP9 is an aquaglyceroporin that can transport neutral solutes in addition to water. This application is aimed at characterizing the function and regulation of AQP9-related water and solute transport in the epididymis. SPECIFIC AIM 1 will test two hypotheses: 1) AQP9 participates in apical water and/or neutral solute transport and is responsible for the establishment of an optimum luminal osmolarity as well as spermatozoa concentration; and 2) The basolateral membrane of the epididymal epithelium contains an as yet unidentified aquaporin(s). We will characterize the pathways of apical and basolateral water and/or solute transport, using a multidisciplinary approach including epididymal tubules perfused in vivo and in vitro, quantitative digital imaging (cell volume measurement and immunofluorescence), and stopped-flow light scattering in apical membrane vesicles. We will identify the basolateral aquaporin(s) that must participate in water and/or solute flow across the epididymal epithelium by homology screening from epithelial cells isolated by laser capture microdissection. Interestingly, since the previous submission, we have shown that AQP11 mRNA is expressed in the epididymal epithelium. We will now study AQP11 mRNA localization by in situ hybridization, and protein expression by western blotting and immunofluorescence. SPECIFIC AIM 2 will test whether AQP9 is regulated via: 1) direct phosphorylation; or 2) interaction with partner proteins. We will study the acute regulation of AQP9 via phosphorylation using purified AQP9. Protein-protein interaction involving "PDZ" domains will be examined using pull-down, co-immunoprecipitation and gel overlay assays. Interactions with CFTR, NHERF1 or PDZK1 will be studied. The proposed studies will provide unique and novel information that is important to our understanding of how water and solute transport across the excurrent duct epithelium contribute to the generation of a luminal environment in which spermatozoa mature, are stored, and are concentrated.