Water and solute transport in the epididymis

产品说明：男性生殖道流出道上皮细胞的液体重吸收导致精子浓度增加，这对生育力至关重要。对这一过程的调控机制知之甚少。AQP 9被认为是附睾顶端的主要水通道。然而，其在附睾经上皮转运中的作用尚未研究。AQP 9是一种水甘油孔蛋白，除了水之外还可以运输中性溶质。该应用程序旨在 表征附睾中AQP 9相关的水和溶质转运的功能和调节。具体目的1将检验两个假设：1）AQP 9参与顶端水和/或中性溶质转运，并负责建立最佳管腔渗透压和精子浓度; 2）附睾上皮的基底外侧膜含有尚未鉴定的水通道蛋白。我们将表征顶端和基底侧水和/或溶质运输的途径，使用多学科的方法，包括在体内和体外灌注的附睾小管，定量数字成像（细胞体积测量和免疫荧光），和顶膜囊泡中的停流光散射。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性筛选，确定必须参与水和/或溶质流过附睾上皮的基底外侧水通道蛋白。有趣的是，自上次提交以来，我们已经表明AQP 11 mRNA在附睾上皮中表达。我们现在将研究AQP 11的mRNA定位原位杂交，蛋白质表达蛋白质印迹和免疫荧光。SPECIFIC AIM 2将测试AQP 9是否通过以下方式调节：1）直接磷酸化;或2）与伴侣蛋白相互作用。我们将研究急性调节AQP 9通过磷酸化使用纯化的AQP 9。蛋白质间相互作用 将使用下拉、免疫共沉淀和凝胶覆盖测定来检查“PDZ”结构域。 将研究与CFTR、NHERF 1或PDZK 1的相互作用。拟议的研究将提供独特的和新颖的信息，这是重要的，我们的理解如何水和溶质运输的流出道上皮细胞有助于产生一个管腔环境中的精子成熟，储存，并集中。

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.