Biochemical studies of estrogen and androgen receptors, cell and tissue cultures of mouse and rat hypothalamus and use of mutants are combined to study mechanisms of sexual differentiation of mammalian brain, especially of hypothalamus. The approach is designed to define the causes and the duration fo the """"""""critical period"""""""" of brain sexual differentiation and to ascertain the relative roles of testosterone and the androgen metabolites, dihydrotestosterone and estradiol. We characterize estrogen and androgen receptors and their ontogeny in embryonic and neonatal hypothalami of mice and rats; these results lead to hypotheses to be tested in receptor biochemistry, in developing brain cultures and in the analysis of genetic lesions that effect steroid action or neuroendocrine function. Using ligand exchange during DNA-cellulose chromatography we shall examine occupied nuclear receptors for both estrogens and androgens in perinatal brain tissues. This method is very sensitive and results in an elution profile that defines both the binding macromolecule and the ligand. Eluted receptors will be analyzed further by isoelectric focusing. To determine when in development steroids act on hypothalamus and what they do, cultures of dissociated cells and of tissue explants of hypothalamus will be assayed for effects of steroids upon morphology, steroid metabolism, neurotransmitter production and the content and release of peptide hormones. Development of simple, defined media will permit comparative analyses of different steroids upon hypothalamic cells. Cultures will be prepared from wild-type mice and rats and from three mutations with the androgen-resistance syndrome, testicular feminization, Tfm mouse, TfmL mouse and Tfm rat. The biological lesions in these mutations will be defined further as they are used as probes of androgen action in the biochemistry and culture experiments. Other neuroendocrine mutations, such as the little (lit) mouse, will be analyzed in this system.
