The purpose of this proposal is to study the molecular mechanism of androgen action on testes at the chromatin and/or nuclear level by determining specificity, saturability, localization (distribution), chemical nature and the hormonal regulation of chromatin acceptor sites (CAS) for androgen-receptor complexes (ARC) in specific cell types of seminiferous epithelium ad attempting to correlate the relationship between acceptor sites and gene transcription. Some general properties of germ cell CAS will also be investigated. Nuclei and chromatin will be prepared from cultured Sertoli cells and isolated germ cells. ARC and other steroid-receptor complexes will be purified by chromatography on DEAE cellulose, phosphocellulose, etc. and by ammonium sulfate precipitation to deplete inhibitor(s) or stimulator(s) of the binding interaction. ARC from different target tissues will be compared to probe their similarity. The specificity of the binding interaction of CAS isolated from purified testicular cell types with ARC will be investigated by competition with other steroid-receptor complexes. The saturability of CAS of Sertoli cells and fractionated germ cells will be assessed. Furthermore, the effect of androgen on the number of available (unmasked) Sertoli cell CAS, nuclear transcriptional activity and chromatin initiation sites will be explored. Further effort will be made to localize Sertoli cell CAS in chromatin subfractions, i.e., transcriptional active or inactive, nucleolar or extranucleolar as well as the nuclear matrix. The fractionation of chromatin subfractions will be achieved by controlled enzymatic digestion or sonication followed by separation based on solubility and sedimentation properties. The Sertoli cell CAS will be isolated by systematic partial chromatin deproteinization. Attempts will be made to study the properties, kinetics and nature of the interaction of purified ARC with isolated CAS fraction covalently bound to activated Sepharose, as well as with partially deproteinized or reconstituted chromatin. The knowledge gained is crucial to an understanding of the molecular mechanisms by which androgen regulate testicular function.
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