In the epididymis spermatozoa mature, gain the ability to fertilize in vivo and are stored prior to ejaculation. Vitamin A is important in epididymal function since it is required to maintain epithelial structures. Targeted mutation of the retinoic acid receptor alpha (RARalpha) results in squamous metaplasia of the epididymal epithelium with resulting infertility (1). The retinoids being hydrophobic labile compounds are transported in a aqueous environments bound to specific carrier proteins. We have found that the epithelium of a restricted region of the epididymis (distal caput) secretes an androgen dependent retinoic acid binding protein (E-RABP) in the lumen of the epididymal duct. We have cloned, sequenced and localized on chromosome 2 the murine E-RABP gene. We have shown that 5 kb of the 5'flanking region ligated in front of the CAT reporter gene targets a surprisingly high level of gene expression in the caput. Our hypothesis is that mE-RABP is essential for the homeostasis and trafficking of retinoids within the epididymis and that its region-specific expression is determined by epididymal transcription factors. Our goals are to establish transgenic mouse models 1) to identify and characterize the minimal sequences conferring region specificity to mE-RABP gene expression and 2) to disrupt mE-RABP gene expression to determine the function of the mE-RABP protein.
The specific aims of this study are: 1) To determine the role of the androgen receptor in the region-specific expression of the mE- RABP gene within the epididymis. 2) To identify the regulatory elements and their associated binding proteins involved in the epididymis-and region-specific expression of the mE-RABP gene. 3) To determine the function of mE-RABP using deletion and substitution variants of the mE- RABP gene in transgenic mouse models. Completion of the project will provide valuable tools for future studies designed to probe epididymal function. The mE-RABP promoter can be used to disrupt in vivo other epididymal genes allowing one to analyze the functional role of these genes in the epididymis. The mE-RABP knock-out mice can be used to re- express mE-RABP in an ectopic site (cauda or vas deferens), allowing one to determine the functional importance of region-specific gene expression in the epididymis. This proposal is part of our long-term goal to understand the mechanisms by which the gene expression in the epididymis. This proposal is part of our long-term goal to understand the mechanisms by which the epididymis provides the optimal milieu for male gametes and contributes to the production of a fertile ejaculate. The ultimate goal is to provide the basic knowledge for the rational treatment of some forms of male infertility.
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