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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 immunohistochemical 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 testicular 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 importance 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 weeks, concentration of radioactivity was found in the cytoplasm of eosinophils, while all other cell types remained unlabeled and neither nuclear nor cytoplasmic uptake in epithelial cells, fibroblasts and muscle cells was ever observed. In comparative in vivo experiments, no convincing evidence for eosinophil labeling could be obtained (32). HISTOCHEMISTRY OF STEROID HORMONE RECEPTORS 563

In vitro incubation of uterine tissue blocks in 10-? M [3H] estradiol-17p and subsequent fixation and plastic embedding for light and electron microscopic autoradiography (1), also showed radioactivity in cytoplasm of eosinophils in specific granules. In addition, radioactivity was found in cytoplasm of lumenal epithelium. Enhancement of this response was observed after pretreatment with estradiol and addition of hydrogen peroxide to the incubation medium, requiring only a few days exposure time for the demonstration of "[3H] estradiol". "Estradiol binding and peroxidase reactivity", demonstrated in the same specimen, correspond with each other in lumenal epithelium, being strongest in the apical region. Brokelmann (1) proposed that estradiol is covalently bound to peroxidase or hemoprotein, such as cytochrome P-450, for inactiviation in a fashion similarly to the biochemically determined covalent binding of the phenolic steroid estrone to liver microsomes. With the same procedure, after in vivoadministration of [3H] estradiol, Brokelmann was unable to obtain the radioactive labeling of cytoplasm of eosinophils and lumenal epithelium seen in vitro. There was no nuclear labeling in uterine tissues in all of his in vivoand in vitro experiments (1) . Therefore, the results of the work of Tchernitchin (34), as well as of Brokelmann (1) must be viewed with caution. Their results may be attributable to artifacts due to histological procedure, rather than to biological processes. No adequate precautions were taken, even though technical pitfalls had been demonstrated earlier in a comparative study for diffusible compounds with six different techniques (29) . Similarly, when immunohistochemical techniques were introduced in recent years and recommended for quick diagnosis of estrogen "receptors" in breast and prostatic tumors, diffusion and loss of hormone as well as of "receptor" were not controlled, even though strong evidence for possible pitfalls in the literature should have precluded disregard of such indispensable precautions. Autoradiography, if used with necessary care and following the guidelines developed more than 15 years ago (29, 30), is likely to be a useful histochemical technique for tumor diagnosis of steroid hormone receptors, using tritiated hormone with high specific activity. The potential with [125I]-labeled hormone needs to be explored, and appears to be attractive because of the high specific activity and short exposure time. Autoradiography can be used to distinguish between nontarget and target cells, and to determine differential binding capacity of heterogeneous target cells through quantification (33). Of special interest is the development of autoradiographic techniques for the electronmicroscopic localization of steroid hormones. Two avenues have been pursued, one involves freezing and cutting of unembedded ultrathin frozen sections, the other utilizes modifications of liquid fixation and embedding techniques. With the low temperature approach, to date no success has been obtained. With a modified fixation embedding procedure developed by Mizuhira et al. (16), ultrastructural localization of 3H estradiol was reported by Mizuhira (15) in nuclei of mouse uterine muscle and epithelial cells. This success appears promising and pro- vides an incentive for further development and application.

HISTOCHEMISTRYWITH CONJUGATED STEROID HORMONE Estradiol or estrone has been conjugated to fluorescein via albumin or 0- carboxy-methyl-hydroxylamine in position 6 (13, 23) or in position 17 (6, 22, 23, 564 STIJMPF AND SAR

35). Walker et al. (35) used peroxidase as a marker conjugated to position 17. There seems to be a consensus that the 17-conjugated estrogen is more suitable than the 6-conjugated estrogen (3, 6, 23). Rao et al. (23) are "convinced" that the 17-conjugated estradiol "can be used to detect estrogen receptors". These authors reported in 4 and 8µm frozen sections of human breast tumors fluorescence in ductal and glandular epithelium "in cytoplasm alone, in cytoplasm and nucleus, and in nucleus alone"; "uptake in nucleoli was observed in MCF-7 and in R3327-AT tumor cells" from cultures (23). According to Pertschuk et al. (22), "the majority of breast tumor cells exhibits cytoplasmic fluorescence". Lee (13), after incubating uterine sections with 2 x 10-6 M estradiol (albumin-fluorescein-conjugate), found various degrees of fluorescence in surface and glandular epithelium, eosinophilic leukocytes, myometrium and peritoneum.

IMMUNOHISTOCHEMISTRY WITH ANTIBODIESTO STEROID HORMONE Immunohistochemistry with antibodies to steroid hormone for the cellular localization of immunoreactive hormone and "receptors" has been employed first by Bubenik et al. (2) for testosterone in testis, using both fluorescein or peroxidase labeling. This was followed by Nenci et al. (19) and Pertschuk et al. (21) for estrogen in breast tumor and uterine tissues. In most applications, prior to the immunohistochemical treatment, the sections or cells are incubated with high doses of the antigen, e.g., estradiol or polyestradiol phosphate, in order to increase the response for optimal visualization. However, the hormone doses used of 10-g M or higher are in considerable excess to that necessary to saturate estrogen "receptors" (4, 14). Apparently the immunohistochemically detectable estrogen is not bound to the "receptor" or Clark's Type I, but rather to Type II or III steroid binding sites (4) . According to generally held concepts of steroid-"receptor" binding, steroid antibodies cannot recognize the steroid hormone in the "receptor"-cavity, since it is not accessible. When detached from the "receptor", the immune reaction could take place, but probably not at the original site, since the histochemical procedures are likely to translocate the hormone. Morrow et al. (18) noted a discrepancy between the results obtained from dextran-coated charcoal assay for cytosolic estrogen "receptor" and the immunohistochemical polyestradiol phosphate technique, not only in breast tumors, but also in follow-up studies with an animal model. These authors concluded that estradiol antibodies do not react with estradiol "receptor" complexes formed in vitro or in vivo, and "that polyestradiol phosphate cannot compete with estradiol for the receptor sites in vitro". It appears questionable that under in vitro conditions estradiol antibodies and conjugated steroid hormones can detect "receptors". Such claims should not have been made by the various investigators with the information at hand, even though under certain conditions data have been obtained, which seem to be specific for target cells. The latter can be derived from a comparison with available biochemical and especially autoradiographic data. For instance, there is some agreement between the identification of testosterone target sites in the testis as obtained by Bubenik et al. (2) and the results from our autoradiographic studies with [3H] testosterone (25). Limited agreement between our autoradiographic data and those HISTOCHEMISTRY OF STEROID HORMONE RECEPTORS 565 obtained by the other histochemical approaches exists for certain uterine tissues and in human breast tumor as reviewed above. Correspondence seems to exist in mouse thymus between the occurrence of binding of fluorescein-albumin-estradiol- 17,Q6-position conjugate in reticuloepithelial cells (10) and nuclear concentration of [3H] estradiol in the same cell type in rat thymus (32). In the report of Kalland and Forsberg (10), however, no subcellular distribution in thymic cells has been detailed and the uterine stroma of the same animals was negative for fluorescence.

IMMUNOHISTOCHEMISTRYWITH ANTIBODIESTO "RECEPTOR" OR TARGET CELL SPECIFIC BINDINGPROTEINS Specific antibodies have been prepared with purified nuclear or cytosolic estradiol receptor proteins from calf uterus (7, 17). With these antibodies limited information is available on immunohistochemical localization of receptor in target tissues. Greene et al. (7, 8) produced specific antibodies to calf uterine nuclear estrogen receptor in rabbit and goat. These antibodies cross react with nuclear receptor of rat uterus and extranuclear receptor of calf, rat, mouse and guinea pig uterus, as well as of human breast cancer. Morel et al. used antibodies against calf uterine cytosol receptor to localize estradiol receptor in rat pituitary cells at the ultrastructural level (17) . These authors observed both cytoplasmic and nuclear localization in gonadotropes, lacto- tropes and somatotropes, but not in thyrotropes and corticotropes. Parikh et al. (20) demonstrated cytoplasmic immunostaining in rat uterine cells with antibodies to calf cytosolic estradiol receptor protein raised in guinea pig. Although immunostaining is highly sensitive and specific, these cytosolic and nuclear receptor antibodies may not be satisfactory for receptor localization, and further improvements are needed. For instance, the usefulness of rabbit, guinea pig, and goat antibodies is limited, due to their cross reactivity and heterogeneity (8). In order to obtain pure antibodies, recently monoclonal antibodies against nuclear estrogen receptor from calf uterus have been produced by hybridoma derived from mouse spleen (9). These antibodies crossreact specifically with receptor protein from calf uterus.

EXAMPLES Figures 1-5 show results with rodent uterine tissues from in vitro experiments, which are contrasted in Figs. 6-8 with results from in vivo studies. There is con- sistency in our autoradiographic results, when localization of radioactivity after 3H estradiol in vitro slice incubation (Fig. 1) and in vivo injection (Figs. 6, 7) are compared with each other. With both autoradiographic approaches, nuclear concentration is obtained in epithelial, stromal and muscle (not shown) cells, while nucleoli (Fig. 7) and eosinophils (Fig. 8) remain unlabeled. In contrast, in vitro section incubation with 3H estradiol or conjugated estradiol shows concentration of radioactivity or the conjugate in cytoplasm of eosinophils (Figs. 2, 3), without labeling of nuclei. Concentration of the conjugate, but not of the radioactivity, is also apparent in nucleoli and perinuclear cytoplasm (Fig. 3). In vitro section incubation with antibodies to estradiol cytosol "receptor" shows staining of cytoplasm of certain epithelial and stromal cells, while cell nuclei are unstained or only 566 STUMPF AND SAR HISTOCHEMISTRY OF STEROID HORMONE RECEPTORS 567 568 STUMPF AND SAR

FiGs. 1-5. In vitro rat uterine localization of 3H estradiol by autoradiography (Figs. 1 and 2), of estradiol conjugated with fluorescein (Fig. 3) and of estradiol cytosol-receptor antibody (Figs. 4 and 5). In Fig. 1 longitudinally slit uteri were incubated at 37°C for 1 hr in 10-9M 3H estradiol in Krebs-Ringer-Henseleit and dry-mount autoradiograms prepared from 4 µm freeze-dried. sections (27), showing nuclear concentration of radioactivity in epithelial and stromal cells. When 4µm frozen sections, thawed on a slide, are incubated in 10-12M 3H estradiol, radioactivity is found concentrated in cytoplasm of eosinophils only, but not elsewhere (Fig. 2, reproduced from 34). Four µm frozen section, when incubated with 10-3 M conjugated estradiol in phosphate- buffer solution, shows fluorescence in eosinophils (arrows) and in cytoplasm and nucleoli of stromall and epithelial cells, but little or no fluorescence in nuclei (Fig. 3). When 4 µm paraffin sections are incubated with antibodies to calf estradiol cytosol receptor protein, immunoreaction is seen in cytoplasm of lumenal (Fig. 4) and glandular (Fig. 5) epithelium, and in stromal cells (Figs. 4 and 5), but relatively weak or no staining of nuclei (Sar and Parikh, unpublished). Animals were immature intact (Fig. 4) or castrated adult (Figs. 1-3). Fig. 1 stained with methylgreen pyronin; exposure time: 95 days. x 540. FIGS. 6-8. In vivo mouse (Fig. 6) and rat (Figs. 7 and 8) uterine localization of 3H estradiol by thaw-mount autoradiography, showing nuclear concentration of radioactivity in epithelial and stromal cells (Fig. 6, x 620, and Fig. 7, x 1400), but not in nucleoli (Fig. 7) and not in eosinophils (Fig. 8, arrows; x 680), which is in contrast to the results obtained after section incubation (Figs. 2 and 3). Four µm, stained with methylgreen pyronin (Figs. 6 and 7) or hematoxylin eosin (Fig. 8). Exposure times: 170 days (Fig. 6), 35 days (Fig. 7), 158 days (Fig. 8). Fics. 9-12. Examples of thaw-mount (Figs. 9 and 10; 4 µm) and smear-mount (Figs. 1 I and 12) autoradiograms showing typical nuclear concentration of radioactivity after 3H estradiol in vitro incubation in dog prostate explant (Fig. 9), in vivo injection in rat DMBA-induced mammary tumor (Fig. 10) and in vitro incubation of human uterine tumor cells (Fig. 11), as well as after 3H dihydrotestosterone in vitro incubation of porcine granulosa cells (Fig . 12). Stained with methylgreen pyronin. Exposure times: 120 days (Fig. 9), 180 days (Fig. 10), 10 days (Fig. 11), 128 days (Fig. 12). HISTOCHEMISTRY OF STEROID HORMONE RECEPTORS 569 weakly stained. Nuclear radioactivity is also obtained with thaw-mount autoradiography after in vitro 3H estradiol incubation in prostate explants (Fig. 9; Ofner, Leav, Sar and Stumpf, unpublished), in vivo in rat mammary tumor cells (Fig. 10), as well as in smear-mount autoradiograms with human endometrial tumor cells (Fig. 11) and after 3H dihydrotestosterone incubation in porcine granulosa cells (Fig. 12 ; Nandedkar, Schomberg, Sar and Stumpf, unpublished).

CONCLUSIONS The varied experimental conditions among the different histochemical techniques discussed do not permit a strict comparison. The reasons for the differences of the results obtained with the individual techniques need to be studied and ex- plained. Especially it needs to be clarified, which data are authentic and represent events which occur in vivo, and what is attributable to procedure. According to our autoradiographic data obtained with the dry-mount, thaw-mount and smear- mount techniques in vivo and in vitro, the localization of estradiol in uterine eosinophils and apical cytoplasm of lumenal epithelium reported by others, is probably introduced by in vitro conditions of the sectioned tissue and not related to in vivo steroid binding protein. It is likely that some of the "results" obtained with conjugated steroid hormone and antibodies to steroid hormone are relevant and useful for the identification of target tissues, after experimental in vitro artifacts have been identified. Clarifi- cations between artifacts and results are necessary before techniques can be recommended. With the present techniques, using conjugated steroid hormone or antibodies to steroid hormone, it is unjustified to apply the term "receptor". Clari- fication about specificity is needed. Here, immunohistochemistry and combined autoradiography-immunohistochemistry (11, 24) can be expected to make important contributions. It is further expected that estradiol "receptor" protein can be visualized with immunohistochemical methods if specific antibodies can be obtained that can bind selectively to steroid hormone receptor. Immunohistochemistry with receptor protein antibodies will provide new dimensions in the understanding and detection of target and non-target tissue as well as of steroid hormone dependent and in- dependent tumors. A comparison of results obtained with the different histochemical techniques under comparable conditions will be revealing, not only with respect to in vitro findings, which do not correspond to in vivo observations, but also regarding the subcellular events related to uptake, transport and true receptor interaction. Further advancements of techniques are needed. An extension of the histochemical techniques to the ultrastructural level is necessary. The combined use of autoradiography with labeled steroid hormone and of immunohistochemistry with antibodies to "receptor"-protein can be expected to clarify the relationships of unoccupied or occupied "receptors" (26). The significance of steroid hormones for the regulation of life and the importance of related research may be characterized in this haiku: View of Spring and Fall Steroid And of Steroid Receptor Inochi no himitsu Show Secret of Life me ni utsuri 570 STUMPF AND SAR

ACKNOWLEDGEMENTS This work was supported by PHS grant NS09914. We thank Drs. Vinci Mizuhira, Yoshihiro Hamashima, Paul Nakane, Yutaka Futaesaku, and Tetsushi Nagata for providing the Japanese version of the haiku.

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