Trophic hormones acutely stimulate steroid production by acting on the rate-determining step of steroidogenesis, the transport of the substrate, cholesterol, from intracellular stores to the inner mitochondrial membrane. There, cholesterol will be metabolished to pregnenolone. Using biochemical, morphological and molecular approaches, we demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) mediates the transport of cholesterol from the outer to the inner mitochondrial membrane and the subsequent steroid biosynthesis. It is our hypothesis that PBR functions as a channel for cholesterol thus allowing this steroid precursor to get into the outer membrane and cross from the outer to the inner mitochondrial membrane through the intermembrane contact sites. Therefore, regulation of the expression of this PBR channel protein will control the amount of cholesterol available for testosterone synthesis and consequently testicular function and fertility. In the first aim, we will examine the structure/function relationship of the PBR receptor by deletion mutations and site-directed mutagenesis followed by reconstitution of the receptor in bacteria and PBR-negative R2C Leydig tumor cells, generated by PBR gene targeting. The information obtained, together with molecular modeling studies of PBR, will help define the structure of the receptor and its function as a channel for cholesterol, identify the ligand binding domains and the domains important for cholesterol uptake and transfer. In the second aim, we will examine the regulation of the expression of PBR by the endocrine disruptors, peroxisome proliferators (PPs), some of which are extensively used in humans and are shown to exert anti- androgenic activity and testicular atrophy in rodents. In preliminary studies, we observed that PPs, including hypolipidemic drugs and native and substituted long chain fatty acids, inhibit PBR expression and the hormone-stimulated Leydig cell steroidogenesis. These results will be confirmed and extended. Transcriptional suppression, mediated by the peroxisome proliferators-activated receptors transduction pathway, may be responsible for the biological effect of PPs on PBR expression. It is our goal to elucidate the mode of action and define the health hazard risk of hypolipidemic drugs and perfluorinated carboxylic acids PPs, putative suppressors of Leydig cell PBR, cholesterol transport, steroidogenesis, and testicular function.
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