Normal spermatogenesis is dependent upon adequate levels of intratesticular androgens, primarily testosterone, and the androgen receptor (AR) is essential for mediating the actions of these steroids. We propose that the intracellular level and the transcriptional activity of the AR are likely to play key roles in determining androgen action in Sertoli cells and hence, in spermatogenesis.
In aim 1, we will examine the hypothesis that stage-specific regulation of AR gene expression in Sertoli cells is affected via the differential expression of endogenous transcription factors that bind to the AR promoter and enhance or repress transcription of the AR gene. The functional regulatory regions within the rat AR promoter will be determined in transfected Sertoli cells. The stage-specificity of expression of putative transcription factors will be determined by DNA-protein binding on gel shift and DNase footprinting assays using Sertoli cell nuclear extracts from stage-synchronized seminiferous tubules. Norther and Western Blots will be used to determine the stage-specific Sertoli cell expression of transcription factors that bind to the AR promoter. Transcription factors will be overexpressed in transient transfection assays to determine their effects on AR gene promoter activity. We will determine whether androgens, FSH, or vitamin A affect AR promoter activity in cultured rat Sertoli cells.
Aim 2 will examine the hypothesis that the AR is able to recruit endogenous co- regulatory proteins in Sertoli cells, and that these protein-protein interactions activate or repress AR transcriptional activity of genes required during spermatogenesis. The AR will be used in the yeast two- hybrid protein interaction assays to screen a rat testis cDNA library and to identify a cohort of co-regulatory proteins that interact with AR to activate or press androgen-regulated transcription. In vivo transfection and expression of cDNAs for putative co-regulators will verify that specific proteins interact with AR to alter transactivation of androgen- responsive reporter genes. Direct protein interactions will be verified by in vitro binding assays. cDNAs encoding putative interacting proteins will be used as probes on Northern blots to determine the expression of mRNAs in various tissues and testicular cells (Sertoli cells, Leydig cells, germ cells) and by in situ hybridization as a means to correlate the expression of co-regulators with AR. Our goal is to understand the control of AR expression and transcriptional activity in Sertoli cells as a primary mechanism by which androgen action is regulated during the spermatogenic cycle.
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