Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular leydig cells

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Abstract
The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor. Intense immunocytochemical staining was seen predominantly in the cytoplasm of the Leydig cells, whereas the cells of the seminiferous epithelium were weakly stained. Thus, the present data reveal a high glucocorticoid receptor content within Leydig cells and a lower level within cells of the seminiferous epithelium. Furthermore, the present experiments demonstrate for the first time the usefulness of dexamethasone 21-mesylate as an affinity label for the morphological localization of the glucocorticoid receptor. The localization of glucocorticoid receptors in Leydig cells suggest a role for this steroid in regulating the function of these cells.