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.
