Departments of Anatomy and Pharmacology University of North

ACTA HISTOCHEM. CYTOCHEM, Vol. 15, No. 4, 1982 HISTOCHEMICAL APPROACHES FOR THE LOCALIZATION OF STEROID HORMONE RECEPTORS WALTER E. STUMPF AND MADHABANANDA SAR Departmentsof Anatomyand PharmacologyUniversity of North Carolina, Chapel Hill, NC 27514 Histochemical identification and visualization of steroid hormone receptors remains a goal, despite earlier success with dry- and thaw-mount autoradiography. Applications of various histochemical and immunohisto- chemical procedures for the identification of steroid hormone target cells and subcellular binding sites are reviewed. Results obtained with dry- and thaw- mount autoradiography after in vivo administration of [3H] estradiol are contrasted with those obtained by in vitro liquid emulsion autoradiography, incubation with conjugated estradiol, antibodies to estradiol, or antibodies to estradiol "receptor". Dry- and thaw-mount autoradiography in vivo after single injection of [3H] estradiol, and in vitro after tissue slice incubation with [3H] estradiol consistently show preferential nuclear concentration of radioactivity, little in cytoplasm, and none in nucleoli. In contrast, liquid emulsion autoradiography after in vitro uterine section incubation with [3H] estradiol shows no nuclear uptake, but only labeling of cytoplasm of eosinophils. Eo- sinophils are also labeled with conjugated estradiol. Histochemical studies with conjugated estradiol or estradiol antibodies show variable results with preferential cytoplasmic and distinct nucleolar labeling of uterine epithelial and stromal cells; however, there is only occasional nuclear labeling, and some lack of identification of target cells, when compared to results obtained with our autoradiographic techniques. The specificity and utility of certain histochemical techniques need to be established, since some of the results are probably related to technique and do not reflect biological events. Develop- ment and use of refined histochemical techniques are needed to further contri- bute to the clarification of the mechanism of steroid hormone action and to provide a tool for the clinical diagnosis of steroid hormone dependent tumors. Evidence for a nuclear concentration and retention of steroid hormones has kcen obtained during the early 1960's. Both biochemical and histochemical approaches helped to establish the concept of nuclear or genomic sites of action of stcroid hormones. Biochemical techniques are disruptive of tissue and cell structure and, therefore, generally deficient regarding information about cell and cell type specific events. Histoch.emical techniques leave the tissue relatively intact and provide information which is difficult or impossible to be obtained otherwise. During the last two decades, hundreds of biochemical laboratories have focused on the study of steroid hormone mechanisms of action, without having been able to clarify the gap between cytoplasmic-nuclear uptake and nuclear-cytoplasmic events. 560 HISTOCHEMISTRY OF STEROID HORMONE RECEPTORS 561 Related histochemical studies, although not less important , were left to a few meagerly funded laboratories. A receptor is, by generally accepted definition, the molecular site where the action or chain of actions is initiated. For steroid hormones, a nuclear receptor can be postulated, because of "high affinity and limited capacity" of nuclear uptake after exogenous administration and the subsequent occurrence of nuclear events, including stimulation of RNA, protein and DNA synthesis. Certain cytoplasmic and nuclear binding proteins have been isolated by biochemists and termed "receptor", although it is not demonstrated yet that binding of the hormone to them initiates action or the chain of actions, a requirement for the designation as receptor. What is currently called by biochemists "acceptor", probably corresponds to the receptor, and what is called "cytosol receptor" and "nuclear receptor", probably designates specific transport and binding proteins. These binding proteins seem to be essential for a sustained action at the genomic receptor, which apparently is locat- ed at certain chromatin or DNA sites. Specific steroid binding proteins are also found outside of target cells, for instance, in the blood and in certain secretions of target tissues. Steroid binding proteins exist in prostatic alveolar lumen, in uterine lumen, in ovarian follicular fluid, in mammary gland milk and cystic fluid, in testi- cular seminiferous tubules and epididymal lumen, in pituitary follicles and cysts, and in brain cerebrospinal fluid. In all of these compartments accumulation of radioactivity has been observed in autoradiograms after injection of tritium labeled steroid hormone, and corresponding binding proteins with "high capacity and low specificity" have been found in most of them. For the histochemical identification of target cells, of special interest and im- portance is the presence of specific and heterogeneous intracellular steroid binding proteins as observed in uterine cytosol for estrogen (5). The questions arise, to which degree can results from the various histochemical techniques be utilized for the distinction of heterogeneous binding proteins, the detection of steroid hormone target cells, the identification of receptor sites, as well as the delineation of artifactual binding. Upon in vitro incubation, artifactual binding, which does not exist in vivo, may occur due to tissue damage. Because of the tendency of steroids to chemically interact with various proteins, artifactual binding, which simulates "receptor"- binding, must be considered, when results from in vitro studies are interpreted. Potential histochemical approaches for the localization of cellular and subcellular hormone receptor sites include: (1) autoradiography with radioactively labeled hormone-in vivo labeled and in vitro; (2) histochemistry with autofluorescent hormone or analogue-in vivo and in vitro; (3) immunohistochemistry with antibodies to the hormone or analogue which remain immobilized and assayable during histochemical processing-in vitro; (4) histochemistry with conjugated (fluorescent compound or enzyme) hormone-in vitro; (5) immunohistochemistry with antibodies to receptor protein or target cell specific binding protein-in vitro. 562 STUMPF AND SAR AUTORADIOGRAPHY Hormones or their analogs are utilized for autoradiography and, perhaps, autofluorescence, without substitution or conjugation of the ligand. This is of advantage, since with such compounds in vitro as well as in vivo applications are possible and results from the different applications can be compared. Autofluores- cence of steroid hormones is, however, weak and inadequate at dilutions of 10-9 M. Coumarin, a plant estrogen, was used by Pertschuk (22) for in vitro autofluorescence at 10-4 to 10_5 M solution. Autoradiography, which was adapted to the study of non-covalently bound substances by introducing the dry-mount and thaw-mount techniques (29, 31), made the first authentic histochemical contributions for nuclear receptor localization at the light microscopic level. Accordingly, after in vivo application of a single pulse of tritium labeled steroid hormone, a nuclear concentration and retention of radioactivity ensues in target cells. This nuclear concentration occurs rapidly and is seen in perivascular uterine cells already at one min after intravenous injection of 0.1 µg [3H] estradiol-17~. In the case of [3H] estradiol, at one to two hr after the injection, the cytoplasm of cells with nuclear concentration contains very little radioactivity with a ratio of nuclear-cytoplasmic radioactivity of 10 : 1 or more. The nuclear half-life or radioactivity from [3H] estradiol is 2-3 hr, as estimated from autoradiograms, if one assumes that there is termination of action by degrada- tion and no reuptake after nuclear occupation. Nucleoli stand out by being free of radioactivity. No evidence exists, under the conditions of the experiments, for preferential plasma membrane or nuclear membrane binding (28, 32). Under in vitro conditions with slices of longitudinally slit uteri and [3H] estradiol-179 incubation at 10_9 M in Krebs-Ringer-Henseleit solution for 1 hr at 37°C, results comparable to the in vivo studies are obtained in our autoradiograms. However, in contrast to the in vivo results, the in vitro autoradiograms may show a gradient of penetration of radioactivity and strongest nuclear uptake in peripheral uterine structures, with decreasing or lack of nuclear concentration in more central portions of the tissue (27). Prostatic explants, incubated for 2 hr with [3H] estradiol or [3H] dihydrotestosterone and then processed for thaw-mount autoradiography, show nuclear concentration of radioactivity in epithelial and stromal cells (12). Similarly, Nandedkar, Schomberg, Sar and Stumpf have shown nuclear concentration of radioactivity in porcine granulosa cells in culture after incubation with 3H estradiol or 3H-dihydrotestosterone (unpublished). Recently, Sar has observed nuclear concentration of radioactivity in human endometrial cells in culture when incubated with 10_9 M 3H estradiol for 30 minutes (unpublished). Utilizing a different autoradiographic approach, Tchernitchin (34) prepared autoradiograms of 4 um frozen sections of rat uterus melted on glass slide. After several washings in isotonic saline, the sections were incubated in 0.2 ml of 1.6><10-12 M [3H] estradiol solution for 10 min at room temperature, followed by liquid emulsion dipping. After a short exposure time of one or two

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