EPITHELIAL CELL PROFILING IN DEVELOPING EXCURRENT DUCT

Massachusetts General Hospital, Boston MA

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Abstract

The male reproductive tract and accessory glands comprise a complex but interrelated system of tissues that are composed of many distinct cell types, all of which contribute to the ability of spermatozoa to carry out their ultimate function of fertilizing an oocyte. Our laboratories are actively examining the role of specific epithelial cell types in modifying the tubular environment in the male reproductive system, and we have developed a battery of cell-specific markers and new animal models that will be used to achieve the aims of the present RFA, i. e., to study the development and the roles of different cell types in the genitourinary tract. The composition and pH of the luminal fluid in which sperm mature is defined by the secretion and absorption of ions and other molecules, including proteins, by distinct epithelial cells. Impairment of these functions, including the abnormal development of specific cell types, will result in deficient sperm maturation, lower fertility or other pathological conditions including cancer. However, our understanding of the development and regulation of these specific epithelial cell types and their associated transport processes is very limited. Many discussions and models addressing epididymal function, for example, fail even to acknowledge that considerable cellular heterogeneity exists in this organ. The present application is, therefore, aimed at developing new experimental tools to study gene expression in the pre- and postnatal male reproductive tract, which ultimately leads to the establishment and regulation of cell-type specific functions that may be important in the development of the reproductive tract, as well as for its mature function. Specific Aim 1: a) To develop transgenic mice that express cell-specific markers (EGFP, lacZ) driven by the H+ATPase 56 kD (B1 subunit) promoter to tag proton-pump (PP) rich cells in the epithelia of the reproductive tract (e. g., clear cells in the cauda epididymis, PP-rich cells in the prostate). b) To develop transgenic mice expressing cell specific transgenes driven by aquaporin promoters (AQP2 for vas deferens epithelial cells). Specific Aim 2: To develop cell-specific culture models that correspond to major epithelial cell types in the urogenital tract, particularly principal cells and PP-rich cells in the epididymis: a) using affinity-purification of distinct cell types with antibodies against extracellular domains of cell- specific proteins; b) using transgenic mice expressing EGFP constructs in the different epithelial cell types. Specific Aim 3: To determine cell-specific patterns of gene activation during pre- and postnatal epithelial development that leads to the heterogeneous cellular phenotype of the epididymis and the vas deferens. Pre- and postnatal tissues will be examined, because the heterogeneous epithelial phenotype in adults is acquired progressively after birth, during pre-pubertal maturation. For example, proton-pump rich cells (narrow and clear cells) are not detectable in neonatal epididymis. This will be achieved by the production of EST libraries from pre- and postnatal developing tissue using; a) laser capture microdissection to sample different cell types at different developmental stages; and b) using the purified PP-rich cell and principal cell preparations developed in Specific Aim 2.