Role of Estrogen Receptor Binding and Transcriptional Activity in the Stimulation of Hyperestrogenism and Nuclear Bodies

Proc. Natl. Acad. Sci. USA Vol. 75, No. 6, pp. 2781-2784, June 1978 Cell Biology

Role of estrogen receptor binding and transcriptional activity in the stimulation of hyperestrogenism and nuclear bodies (nuclear morphology/nafoxidine/estradiol) JAMES H. CLARK*, JAMES W. HARDIN*, HELEN A. PADYKULAt, AND CONSTANCE A. CARDASISt eDepartment of Cell Biology, Baylor College of Medicine, Houston, Texas 77030; and t Laboratory of Electron Microscopy, Wellesley College, Wellesley, Maschusetts 02181 Communicated by Elwood V. Jensen, March 15,1978

ABSTRACT The effects of estradiol and nafoxidine on nu- MATERIALS AND METHODS clear estrogen receptor binding, RNA polymerase activities, and Animals. Immature (21 days old) female rats of the uterine ultrastructure were studied. Animals were either injected with estradiol, implanted with estradiol/paraffin pellets, Sprague-Dawley strain were obtained from Texas Inbred or injected with nafoxidine. Animals treated with nafoxidine Mouse Co. (Houston, TX). Animals were kept in a controlled or estradiol implants showed sustained long-term nuclear re- environment (22°; constant relative humidity; light/dark cycle tention of estrogen receptor and increased nuclear RNA poly- of 12 hr each with the light cycle starting at 0700), and food and merase activities for up to 72 hr. A single injection of estradiol water were provided ad lib. caused initial increases in these variables which returned to Estradiol was dissolved in 0.9% NaCl/1% ethanol, and control levels by 24 hr after hormone treatment. Uterine tissue nafoxidine was dissolved 1% ethanol in distilled water. Alter- was examined by light and electron microscopy 72 hr after hormone treatments. Uteri from either estradiol-implanted or natively, 50-mg paraffin implants were prepared containing nafoxidine-treated animals showed markedly increased hy- 10% (wt/wt) estradiol by liquifying a known quantity of par- pertrophy of the luminal epithelial cells. Nuclei in sections of affin, mixing the appropriate quantity of hormone with it, and the uteri of these hyperestrogenized animals displayed a large dropping a uniform volume of the liquid mixture onto a pre- number and wide array of nuclear bodies composed of a fila- cooled glass surface (9). mentous capsule and granular cores. We conclude that hyp Vehicle (0.25 ml), estradiol (1.0 Ag), or nafoxidine (50,ug) was erestrogenization, a condition that eventually results in ab- administered subcutaneously at various times prior to sacrifice. normal cell growth, is correlated with increased and sustained nuclear binding of the estrogen receptor, increased and sus- In experiments involving implants, animals were placed under tained RNA polymerase activity, and the appearance of nuclear light ether anesthesia, a small incision was made in the nape of bodies. the neck, a paraffin implant was placed subcutaneously, and the incision was closed with one or two sutures. The nonsteroidal estrogen antagonist nafoxidine is an atypical Animals were sacrificed by cervical dislocation. The uteri long-acting estrogen in the immature rat. An injection of were rapidly removed, stripped of adhering fat, weighed, and nafoxidine causes the translocation of the estrogen receptor placed in either ice-cold 0.9% NaCl/5 mM ethylene glycol- from the cytoplasm to the nucleus of uterine cells with a sub- bis(fi-aminoethyl ether)-N,N'-tetraacetic acid for nuclear iso- sequent long-term nuclear retention of the receptor, presum- lation or in the appropriate fixative for morphological exami- ably as a receptor-nafoxidine complex. This long-term nuclear nation (see below). retention of the receptor is accompanied by sustained stimu- Isolation of Nuclei. Nuclei were isolated by a modification lation of uterine size and long-term increase of nuclear RNA of the hexylene glycol procedure as described (5). polymerase activities (1-5). A single injection of the physio- Assay of Endogenous RNA Polymerase Activities. Total logical hormone 17,B-estradiol also results in nuclear retention endogenous nuclear RNA polymerase activities were measured of the receptor, stimulation of uterine hypertrophy, and in- by the addition of an aliquot of nuclei (usually 5-15 ,g of DNA) crease of nuclear RNA polymerase activities but these effects to a tube that contained 2.5 Mmol of Tris-HCl (pH 8.0), 0.05 are of a much shorter duration than those observed after Mumol of MnC12, 0.1 Mmol of MgCl2, 3 ,mol of (NH4)2SO4, 0.1 nafoxidine administration (1-7). ,4mol of dithiothreitol, 0.03 umol each of ATP, CTP and GTP, It has recently been shown that a single injection of nafoxi- 3 nmol of unlabeled UTP, and 2.5 ,Ci of [3H]UTP (12-16 Ci/ dine or Clomid, a closely related compound, in neonatal rats mmol) in a final volume of 50 ,l. a-Amanitin was used to de- causes hyperestrogenization of the reproductive tract which termine the amount of activity due to RNA polymerase II. RNA results in the appearance of multiple abnormalities and tumors polymerase I activity was determined by subtraction of the in the adult animal (8). a-amanitin-inhibited activity from the total (5). Reactions were To relate and extend the above biochemical and histo- incubated at 300 for 20 min and terminated by removing a pathological observations to morphological changes that occur 25-Ml aliquot from each reaction and spotting it on a 2.5-cm in the normal immature rat uterus in response to nafoxidine and DE-81 filter paper disc. The disc was immediately transferred estradiol treatment, we examined the ultrastructure of uterine to a wash solution of 0.5 M Na2HPO4. Filters were washed and luminal epithelial cells as well as changes in nuclear RNA assayed for radioactivity as described by Roeder (10). polymerase activities after long-term exposure to these com- DNA Determination. DNA was estimated by the diphe- pounds. nylamine method of Burton (11). Measurement of Nuclear Estrogen Receptors. The level The costs of publication of this article were defrayed in part by the of nuclear estrogen receptors was measured by the [3H]estradiol payment of page charges. This article must therefore be hereby marked exchange assay as described (12). "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate Fixation and Sectioning Procedures for Light and Electron this fact. Microscopy. Uterine cross sections (1-2 mm thick) were ini- 2781 Downloaded by guest on October 1, 2021 2782 Cell Biology: Clark et al. Proc. Natl. Acad. Sci. USA 75 (1978)

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Time, hr FIG. 1. Relationship of nuclear binding of receptor-estrogen complex to endogenous RNA polymerase activities. Immature rats were either injected with 1 gg of estradiol (A), implanted with an estradiol/paraffm pellet (B), or injected with 50 ug of nafoxidine (C). At the indicated times, animals were sacrificed, uteri were removed, and nuclei were isolated. Each point represents the mean of six determinations. 0, Recep- tor-estrogen complex; ,, RNA polymerase I; 0, RNA polymerase II. tially fixed for 2 hr at room temperature in one of two aldehydic the nuclear retention of the estrogen receptor. To define further mixtures: (i) 2% glutaraldehyde 2% formaldehyde/picric acid the relationship of nuclear retention of estrogen receptor and (13) or (ii) 4% glutaraldehyde/1% formaldehyde (14, 15). After the stimulation of uterine endogenous RNA polymerase ac- rinsing in 0.1 M sodium cacodylate/0.25 M sucrose, pH 7.4, tivities, animals were either injected with estradiol or nafoxidine they were treated with 1% OS04 in barbital/acetate buffer, or implanted with estradiol and sacrificed at 24-hr intervals. dehydrated, and embedded in Epon. Semithin (0.75 Mim) and Nafoxidine-treated and estradiol-implanted animals showed ultrathin sections were prepared on a Sorvall MT-2 ultrami- increased and sustained levels of RNA polymerase activities at sections were stained with 0.25% toluidine all times examined (Fig. 1). In contrast, estradiol injection crotome. Semithin caused increased levels of polymerase activities by 12 hr with blue in 0.25% borax. Ultrathin sections were stained with uranyl a return to control levels by 24 hr. These alterations in poly- acetate and lead hydroxide and were examined in a Siemens merase activities closely parallel levels of nuclear estrogen re- 1A electron microscope. ceptors. To relate the above biochemical observations to morpho- RESULTS logical changes that occur in the normal immature rat uterus Previous results (5) from this laboratory had demonstrated that in response to nafoxidine and estradiol, samples of uteri treated a single injection of either nafoxidine or estradiol to immature as above were prepared for light and electron microscopy. The rats stimulated endogenous nuclear RNA polymerase activities. ultrastructure of the uterine luminal epithelial cells was ex- The extent and the degree of stimulation were correlated with amined in these samples. The height of the luminal epithelial

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FIG. 2. Imnmature rat uterine luminal epithelium and stroma 72 hr after treatment with estradiol or nafoxidine. (A) Injected with saline; (B) injected with 1 ug of estradiol; (C) implanted with estradiol/paraffi pellets; (D) injected withsg50 of nafoxidine. (Epon sections, 1 Mm; toluidine blue; X500.) Downloaded by guest on October 1, 2021 Cell Biology: Clark et al. Proc. Natl. Acad. Sci. USA 75 (1978) 2783

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FIG. 3. Nuclear bodies in the uterine luminal epithelial cells of immature rats 72 hr after a single injection of 50 gsg of nafoxidine. In these two profiles ofluminal epithelial nuclei, three nuclear bodies are apparent in the left nucleus and two in the right (arrows). (Insets A-D) Principal forms of the nuclear bodies observed after nafoxidine injection or estradiol implantation: (A) singlet (filamentous capsule and less-dense core); (B) granular nuclear body (small granules in core); (C) doublet (double core); (D) granular nuclear body with large electron-opaque inclusions. (Main picture, X14,000; Insets, X50,000.) cells was greatly increased (over the height of control cells) by rough cisternal endoplasmic reticulum as well as enlarged Golgi either nafoxidine treatment or estradiol implantation (Fig. 2). systems, features that are indicative of stimulated cytoplasmic After a single injection of estradiol the height of luminal epi- protein synthesis. thelial cells was not significantly different from that of control Several types of nuclear bodies were seen in tissue from either tissue. Thise~~~~ a state that follows nafoxidine-treated or animals probably represents regressed estradiol-implanted (Fig. 3). the hypertrophy and hyperplasia and occurs 24-48 hr after a Nuclear bodies were quantitated in cells that were judged to single injection of estradiol. At the ultrastructural level, the be sectioned in the longitudinal plane. Electron microscopic hypertrophied cells resulting from nafoxidine treatment or counts were made on ultrathin sections of nuclei that are re- estradiol implants had an abundance of free polysomes and ferred to as nuclear "profiles." Only 22% of control nuclear Downloaded by guest on October 1, 2021 2784 Cell Biology: Clark et al. Proc. Nati. Acad. Sci. USA 75 (1978) profiles (n = 188) contained these inclusions; 19% of the nuclear The specific function and composition of nuclear bodies profiles (n = 88) from estradiol-injected animals contained present in hyperestrogenized uteri are not known. In this paper nuclear bodies. In estradiol-implanted animals 37% (n = 65) we have shown that they are one of the visible manifestations contained inclusions and 53% of those (n = 129) in nafoxid- of hyperestrogenization. Their presence and complexity are ine-treated animals contained nuclear bodies. Interestingly, only closely related to the transcriptional activity of the uterus-i.e., 5% of control profiles contained more than one nuclear body nuclei of quiescent cells have fewer and morphologically sim- whereas in estradiol-implanted animas 28% of positive profiles pler nuclear bodies whereas nuclei of hyperactive cells contain contained more than one body and 38% of the profiles in many bodies of varying complexity. Increased transcriptional nafoxidine-treated animals had multiple nuclear bodies. No activity and the appearance of nuclear bodies is correlated with instances of multiple nuclear bodies were seen 72 hr after a long-term retention of the estrogen receptor in uterine nuclei. single estradiol injection. Thus, nuclear bodies may represent nuclear organelles that are Four distinct morphological classes of nuclear bodies were involved in increasing transcriptional activity or may be by- observed during the course of this study (Fig. 3). These included products of such singlets with a filamentous capsule and less-dense core (size activity. Recently, it was demonstrated that range, 280-305 nm; Fig. 3A), nuclear bodies containing gran- single injections of nafoxidine or Clomid given to neonatal rats ules in the core (size range, 700-1000 nm; Fig. 3B), doublets result in multiple reproductive tract abnormalities in the adult with double cores (Fig. 3C), and granular bodies with large animal (8). whether there is any relationship to nuclear bodies electron-opaque inclusions (Fig. 3D). The last three classes were and reproductive tract abnormalities is unknown, although it in control tissue. is known that many neoplastic cells contain nuclear bodies (15, never observed 16, 20). Because we have shown that nuclear bodies are induced DISCUSSION by compounds that are known to induce reproductive tract A single injection of nafoxidine or an estradiol implant causes abnormalities and neoplasia, the continued study of nuclear extensive cellular hypertrophy of the immature rat uterus This bodies as a possible precursor to the neoplastic state should be hypertrophy is characterized by long-term nuclear retention of interest. of the estrogen receptor and the prolonged stimulation of endogenous nuclear RNA polymerase activities (Fig. 1). This very The authors acknowledge the excellent technical assistance of K. active state of uterine cells appears to be one manifestation of O'Connor, J. Kovar, and L. Swain. These studies were supported by the hyperestrogenized uterus. Hyperestrogenization is also grants from the National Institutes of Health (HD-09209) and the indicated by the high level of nuclear receptor in the nafoxid- American Cancer Society (BC-92). ine-treated and estrogen-implanted rats. In both groups, ap- proximately 80-90% of the total receptor was translocated to 1. Clark, J. H., Anderson, J. N. & Peck, E. J., Jr. (1973) Steroids 22, the nucleus, and this level greatly exceeds the 10-20% that is 707-718. necessary to maximize uterine growth (6, 7). 2. Clark, J. H., Peck, E. J., Jr. & Anderson, J. N. (1974) Nature 251, At an ultrastructural level, we observed the presence of a 446-448. large number of complex nuclear bodies in hyperestrogenized 3. Capony, F. & Rochefort, H. (1975) Mol. Cell. Endocrlnol. 3, uteri. These nuclear bodies closely resemble some of the pro- 233-251. teinaceous nuclear bodies previously described cytochemically 4. Katzenellenbogen, B. S. & Ferguson, E. R. (1975) Endocrinology by Bouteille and coworkers (15, 16) who showed that the nu- 97, 1-12. clear bodies in their studies consisted of a filamentous protein 5. Hardin, J. W., Clark, J. H., Glasser, S. R. & Peck, E. J., Jr. (1976) capsule and a core that probably contained either RNA or DNA. Biochemristry 15,1370-1374. Such bodies have often been identified in neoplastic cells (15, 6. Anderson, J. N., Clark, J. H. & Peck, E. J., Jr. (1972) Blochem. 16). It is not known whether the different forms of nuclear Biophys. Res. Commun. 48,1460-1468. 7. Anderson, J. N., Peck, E. J., Jr. & Clark, J. H. (1975) Endocri- bodies shown in Fig. 3 represent unique types of inclusions or nology 96,160-167. whether they might simply represent sections taken at different 8. Clark, J. H. & McCormack, S. (1977) Science 197, 164-165. angles through segments of a single class of nuclear bodies. 9. Rudali, G., Apiou, F. & Mael, B. (1975) Eur. J. Cancer 11, 39- Nuclear bodies have also been demonstrated in the adrenal zona 47. fasciculata of the calf after corticotropin treatment, and a nu- 10. Roeder, R. G. (1974) J. Biol. Chem. 249,241-248. cleolar channel system has been described in endometrial gland 11. Burton, K. (1956) Biochem. J. 62,315-323. cells of the human uterus (17, 18). Neither of these types of 12. Anderson, J. N., Clark, J. H. & Peck, E. J., Jr. (1972) Biochem. nuclear bodies appears to be identical to those described in this J. 126,561-567. paper and the relationship, if any, to them is unknown. 13. Ito, S. & Karnovsky, M. J. (1968) J. Cell Biol. 39, 168a. Two interesting observations concerning nuclear bodies were 14. Karnovsky, M. J. (1965) J. Cell Biol. 27, 137a. made in the course of this study. The number and complexity 15. Bouteille, M., Kalifat, S. R. & Delarue, J. (1967) J. Ultrastruct. of nuclear bodies were increased in a hyperestrogenized animal Res. 19,474-486. and nuclear bodies appeared to be present predominantly in 16. Dupuy-Coin, A. M., Kalifat, S. R. & Bouteille, M. (1972) J. Ul- luminal epithelial cells. This latter observation appears to relate trastruct. Res. 38, 174-183. 17. Weber, A., Whipp, S., Usenik, E. & Frommes, S. (1964) J. Ul- to other recent results from this laboratory (unpublished data) trastruct. Res. 11,564-576. that nafoxidine preferentially stimulates growth of luminal 18. More, I. A. R., Armstrong, E. M., McSeveney, D. & Chatfield, epithelial cells while having little effect on growth of myome- W. R. (1974) J. Ultrastruct. Res. 47,74-85. trium and stromal cells. Kang et al. (19) have also shown that 19. Kang, Y.-H., Anderson, W. A. & DeSombre, E. R. (1975) J. Cell CI-628, a compound similar to nafoxidine, causes extensive Biol. 64,682-691. epithelial hypertrophy but does not induce mitotic activity in 20. Krishan, A., Uzman, B. G. & Hedley-Whyte, E. T. (1967) J. Ul- the mucosa as does estradiol. trastruct. Res. 19,536-547. Downloaded by guest on October 1, 2021