THE ANATOMICAL RECORD 205119-130 (1983) Uterine Simple and Complex Nuclear Bodies Are Separate Structural Entities HELEN A. PADYKULA AND SHTRWIN M. POCKWINSE Department of Anatomy, University of Massachusetts Medical School, Worcester, MA 01605 ABSTRACT In rat uterine luminal epithelial cells, nuclear bodies occur in the euchromatin in varying numbers in relation to the nuclear concentration of the estrogen receptor (Clark et al., 1978; Padykula et al., 1981, 1982). This functional responsiveness indicates that nuclear bodies may be useful indicators of the degree of cellular estrogenization. Because these filamentous bodies vary in size (200-1200 nm), shape, and composition, quantitative analysis of frequency of their occurrence has been difficult. A fundamental division into 2 categories can be made by the following criteria: 1)simple nuclear bodies (200500 nm) consisting of a protein mesh of microfilaments, and 2) complex nuclear bodies (200-1200 nm) composed of a n outer filamentous protein capsule enclosing a lucent core that may contain granules. Previous quantitative analyses at the electron microscopic level has excluded “simple bodies” because they might actually be ultrathin sections through the filamentous capsule of complex bodies (Le Goascogne and Baulieu, 1977; Clark et al., 1978). To resolve this sampling problem, we have performed serial ultrathin section analysis of nuclear bodies in hyperestrogenized luminal epithelial cells. Ultrastructural evidence presented here demonstrates that simple and complex nuclear bodies are anatomically separate entities. Ultrathin sections through the capsule of complex nuclear bodies will be misidentified as profiles of simple bodies during quantitative analysis. This anatomic distinctness of simple and complex nuclear bodies correlates with their differing responses to estrogenic stimulation and withdrawal (Fitzgerald and Padykula, pp. 131-141, this volume). Thus the existence of these two major categories should be taken into consideration during quantitative analyses. The frequency of observation of nuclear bodies have been recognized (Bouteille et al., bodies in rat uterine luminal epithelial cell 1974). Two principal populations of nuclear has been correlated with the degree of estro- body profiles are easily recognizable by size genic stimulation (LeGoascogne and Bau- and structural differences, i.e., nuclear bodlieu, 1977; Clark et al., 1978; Padykula et al., ies and complex nuclear bodies (Fig. 1). Sim1981).Because nuclear bodies are not readily ple bodies are small (diameter 200-500 nm) visible by light microscopy, quantitative and structurally quite homogeneous; they are analysis is performed by direct visualization composed of a protein (Krishnan et al., 1967; in ultrathin sections by electron microscopy Dupuy-Coin et al., 1972) mesh of microfila(Padykula and Clark, 1981). The diameter of ments and possibly microtubules. By conthese rat uterine nuclear bodies ranges from trast, the complex nuclear bodies are strucapproximately 200-1200 nm, and thus in turally heterogeneous and have a greater routine ultrathin sections (approximately 50 range in size (200-1200 nm), shape, and comnm) only slices through them are observed in position. They consist of a n electron dense electron micrographs. Such ultrathin sec- filamentous capsule that encloses a n electron tions are called “nuclear body profiles.” lucent core which may contain granules of a This sampling problem is further compli- varying size, density, and shape. Cytochemicated by the structural heterogeneity of nuclear bodies. Various subcategories of nuclear Received August 2, 1982; accepted October 25, 1982. 0003-276X/83/2052-0119$03.50 0 1983 ALAN R. LISS, INC. QUANTITATIVE ANALYSIS OF NUCLEAR BODIES Diagram illustrating sampling problem Nuclear Profile-Luminol Epithelial Cell Immature rat uterus 50 nm f 50nm 250nm 800nm L II 5$nm 7,000nm I Fig. 1. The difficulty of specific identification of simple nuclear bodies in routine ultrathin plastic sections is shown here. The two outermost sections of a spherical cal analysis indicates that the granules in complex nuclear bodies are composed of protein and that some may also contain RNA (Kierszenbaum, 1969; Dupuy-Coin et al., 1972). Since the protein capsule of the complex nuclear bodies resembles closely the substance of the simple bodies, the possibility has existed that so-called simple bodies may represent only a surface section through the capsule of a nuclear body (Fig. 1).Thus, the existence of simple bodies has not been established. Because of this limitation in interpretation, quantitative analyses of the frequency of occurrence of nuclear body profiles during different endocrine states have been limited to counts of complex nuclear bodies (Le- A I complex nuclear body (800 nm) represent solely the capsular material which would appear as filamentous meshwork. (Taken from Padykula et al., 1981.) Goascogne and Baulieu, 1977; Clark et al., 1978) or to counts of total nuclear body profiles which combined complex and so-called simple nuclear bodies (Padykula et al., 1981). When we subsequently analyzed the data from this latter investigation, in which the observations a t the electron microscope had been recorded as the frequency of simple or complex nuclear bodies, it was evident that distinctly different functional responses by complex and simple nuclear bodies occur in response to a single injection of estradiol or a single injection of the nonsteroidal estrogen antagonist, nafoxidine (Fitzgerald and Padykula, pp. 131-141, this volume). Since the view has been advanced that simple nuclear bodies may be regularly present in nuclei UTERINE SIMPLE AND COMPLEX NUCLEAR BODIES (Bouteille et al., 19741, it became necessary to solve this structural problem. To confront this sampling problem, we have prepared serial ultrathin sections (80 nm) of hyperestrogenized uterine luminal epithelial cells to determine whether or not simple bodies are distinctly separate entities. Our analysis demonstrates that simple nuclear bodies are structurally different from complex nuclear bodies. In the accompanying report (Fitzgerald and Padykula, pp. 131-141, this volume), quantitative evidence is presented which indicates that simple and complex bodies respond differently to a single injection of the estrogen antagonist-agonist, nafoxidine, and to a single injection of estradiol. MATERIALS AND METHODS Hyperestrogenized immature rat uterine luminal cells were selected for serial section analysis because nuclear bodies occur in high frequency (Padykula et al., 1982). Estradiolcontaining silastic tubes were implanted subcutaneously in nape of 21 day old female rats and removed 72 hours later. During this period of steady estrogenic stimulation, the uterine luminal epithelial cells hypertrophy to twice (30 pm) the control height (15 pm), and a t least one profile of nuclear bodies is observed in 48% of the nuclear profiles observed in ultrathin sections. Procedure for obtaining serial ultrathin sections through nuclear bodies Blocks of uterine cross sections were chosen to include a n expanse of the luminal epithelium sectioned along the longitudinal axes of the cells. Ultrathin sections (beige, approximately 80 nm thick) were prepared on a Sorvall MT-1 ultramicrotome and placed on 0.5% formvar coated copper one-slot grids. A ribbon of serial sections was cut, and one or two sequential sections were placed on each coated grid. The grids were touched to a piece of filter paper to remove excess water and then sequentially placed into a n LKB grid storage box. The grids were then stained in 4% aqueous uranyl acetate for 3 minutes and, one by one, carefully rinsed by dipping into three beakers of distilled water. A grid was slowly immersed perpendicularly to the meniscus, raised and lowered ten times under the the meniscus, and slowly brought perpendicularly out of the distilled water. They were then stained in Reynolds lead citrate for 5 minutes and rinsed as before. After removal of excess water by touching the grid 121 to a piece of filtering paper, the grid was placed in a n LKB grid storage box. Mode o f study at the transmission electron microscope (JEOL lO@S) At 8000 times magnification, a grid from the middle of the series of sections was scanned for serial longitudinal sections of nuclei that contain nuclear bodies. Starting with the number 3 or number 4 grid of a partially serially sectioned nucleus, a small homogeneous profile of a nuclear body was located in a nucleus of a representative longitudinal section of uterine epithelial cell and photographed a t 8000 times magnification and then 5000 times magnification to record the entire nuclear profile. After scanning the same nucleus for other nuclear bodies, which usually were present, each location was noted and photographed. To aid in locating the same cell and nucleus in sequential.sections, the shapes of the surrounding cells around and below the chosen cell were noted. Proceeding to serial sections on either side of the starting point, the adjacent profiles of the chosen cell, its nucleus, and the nuclear bodies were photographed a t 8000 and 5000 times magnification. Several nuclear bodies were traced through five, six, seven, or eight serial sections in this manner. Fourteen different nuclei were thus analyzed photographically; some of these lacked one or two sections from the serial sequence. Another 15-20 were viewed a t the electron microscope, but were not suitable for photographic record because of various technical limitations. RESULTS Structural and functional characteristics of hyperestrogenized uterine luminal epithelial cells A physiologic state in which nuclear bodies are numerous was selected for the serial section analysis. In a recent investigation, we observed that, during 72 hours of sustained estrogenic stimulation (subcutaneous silastic-estradiol implant) of the immature rat uterus, the luminal epithelial cells double in height (from 15 to 30 pm), and that twice as many nuclear profiles contain a t least one complex nuclear body profile (Padykula et al., 1982).In control cells, approximately 25% of the nuclear profiles contain a t least one complex nuclear body profile, whereas after 72 hours of steady estrogenic stimulation approximately twice as many nuclear profiles 122 H.A. PADYKULA AND S.M. POCKWINSE Figure 2 represents section 4 in a series of six serial sections that are illustrated in Fig. 3a, b. Note the four profiles of nuclear bodies which are numbered. Of these NB1, 2, and 4 are complex bodies composed of capsules and central cores. However, NB3 is smaller, without a distinct core, and composed of filamentous and tubular components. To determine whether or not this profile represents a simple nuclear body, study of the six serial sections (Fig. 3a, b) indicates that NB3 is different from NB1, 2, and 4, and that NB3 is a simple body rather than a component of a complex nuclear body. Its entire extent is most likely demonstrated in S1 through S5 (diameter 400 nm). Figure 4 represents section 3 in a series of five nuclear profiles. It contains a nuclear profile with four nuclear bodies which might be classified as follows: NB5 and 6, simple; NB8, small complex; NB7, ?. Serial section analysis in Figure 5 confirms that NB5 and 6 are simple nuclear bodies. NB7 appears to be a simple body also, although for determination of its complete structure a 6th section would be necessary. NB8 is a small complex body with a distinct core which is evident in section 3; it appears to be more angular in Serial section analysis of nuclear bodies its shape than NB5,6, and 7. in hyperestrogenized uterine luminal The problem of distinguishing between epithelial cells simple and complex nuclear bodies in ultraThe goal of this analysis was to determine thin sections is illustrated in Figure 3a, b. whether simple nuclear bodies are: 1) sepa- The following profiles of complex nuclear rate structural entities, and/or 2) tangential body profiles observed in isolation might be sections through the capsule of complex nu- classified erroneously as simple nuclear clear bodies.Using ultrathin sections that in- bodies: NB1 in S1, S2, S5; NB2 in S5; and clude luminal epithelial cells cut in the NB4, S3. From Figure 1, it is evident that longitudinal plane, nuclear profiles that con- the two sections tangential to the surface of tained a t least one small homogeneous nu- a complex nuclear body would usually inclear body were selected for serial section clude primarily filamentous capsular matestudy. The nuclear diameter (transverse axis) is approximately 4000 nm (Fig. 1). Hence, the six serial sections through the first nucleus (Figs. 2 and 3a,b) represent approximately 12% of its volume (six sections x 80 Figs. 2-5. Serial ultrathin sections (approximately nm = 480 nm). Within this region, four nu- 80 nm) through uterine luminal epithelial cells from clear bodies occur as conspicuous components immature rats which had received steady estrogenic of the euchromatin (Fig. 2). Heterochromatin stimulation for 72 hours from a subcutaneous implanted is sparse and localized primarily along the silastic tube filled with estradiol. inner surface of the nuclear envelope. PeriFig. 2. (~17,500) The nuclear profile illustrated here chromatin and interchromatin granules is one of six nuclear serial profiles. Regions containing (Monneron and Bernhard, 1969) contribute profiles of these four nuclear bodies are enlarged in to the overall granularity of the largely eu- Figure Za, b. Here in section 4 (S4),four nuclear bodies are evident. NBl, 2, and 4 are complex nuclear chromatic nucleus. Note the halo, a rela- (NB) bodies as identified by a n electron lucent core surtively clear area, that immediately surrounds rounded by a more opaque capsule. Note that NB 1 and 2 are bipartite. NB3 is small and lacks a distinct core. nuclear bodies. (48%) contain a t least one complex nuclear body profile. Profiles of simple nuclear bodies occur a t a relatively low frequency which varies around 25%. This cellular growth and differentiation occurs during a period when the cytoplasmic estrogen receptor complexed with estradiol has been largely translocated to the nucleus (Padykula et al., 1982). During the 72 hour steady exposure to estradiol, the concentration of nuclear estrogen receptor reaches maximal level within 24 hours and stays high for the remaining 48 hours. This hyperestrogenization produces tall columnar cells with highly euchromatic nuclei and large nucleoli. The cytoplasm acquires the ultrastructural features associated with protein synthesis for secretion in that the rough endoplasmic reticulum is voluminous (Figs. 2 and 4) and the supranuclear Golgi complex is large. Free polysomes are abundant. The overall appearance correlates well with the condition of sustained high concentration of nuclear estrogen receptor, which promotes a functional state of cellular growth and differentiation through heightened transcription and translation. UTERINE SIMPLE AND COMPLEX NUCLEAR BODIES 123 s1 s2 s3 NB1 NB2 NB3 Fig. 3a, b. ( ~ 2 7 , 5 0 0 Serial ) section analysis of the four nuclear bodies shown in Figure 2. Note the designation of the section number along the top and the nuclear body number along the left margins of Figure 3, a and b. Start structural analysis with Section 4, and refer to Figure 2 for low power orientation. NB1 and NB2 occur in five consecutive sections and, hence, have a diameter of approximately 400 nm; also, they appear to be similar in shape. NB1 is shown in its entirety, whereas only part of NB2 is present. NB1 and NB2 are small complex bodies with electron lucent cores. NB3 occurs in five sections and differs distinctly form NB1 and 2 in shape, size, and absence of a distinct core. Thus, NB3 is a simple body comprised almost entirely of filamentous material. NB4 is a large complex nuclear body with electron opaque components in its core. It is only partially included in the six sections illustrated here. In section 3, the plane-of-section passes through the capsule of this complex body; this image might be misconstrued as a simple body. 126 H.A. PADYKULA AND S.M. POCKWINSE UTERINE SIMPLE AND COMPLEX NUCLEAR BODIES 127 128 H.A. PADYKULA AND S.M. POCKWINSE UTERINE SIMPLE AND COMPLEX NUCLEAR BODIES rial, as shown for NB4 in Section 3 and somewhat in Section 4. The profile of NB4 in Section 3 might be misidentified as a simple nuclear body except for its large size. In Figure 6, nearly the full extent of a complex body is illustrated in 11 serial sections which span a nuclear area that is 880 nm thick. Visual reconstruction of this complex body suggests that it is somewhat irregularly spherical in shape. It largest diameter (S7 and 58) is 600 nm. However, as estimated from section thickness, it would be somewhat larger than 880 nm. Note that S11 and the section preceding S1 seen in isolation would be identified as simple bodies. Thus, the magnitude of error in counts of the frequency of occurrence of simple nuclear bodies would vary with both the shape, number, and size of the complex bodies. In the least, two erroneous profiles per complex nuclear body would be introduced in estimations of the frequency of occurrence of simple nuclear bodies. DISCUSSION This new evidence demonstrates, for the first time, that simple nuclear bodies exist as entities which are structurally distinct from complex nuclear bodies in rat uterine luminal epithelial cells. Since Bouteille et al. (1974) stated that simple bodies have been reported “in most tissues in which they have been carefully sought,” it seems possible that they may be regularly present in nuclei. In our experience they are more easily identifiable in highly euchromatic nuclei, rather than in heterochromatic nuclei. The structural similarity between the filamentous substance of simple nuclear bodies and the capsule of the complex nuclear bodies suggests interrelationship, especially since the center of a simple body is sometimes more lucent. In our recent investigation of the effect of 72 hours of steady estrogenic stimulation in the immature rat luminal epithelial cells, we observed a linear increase in frequency of observation of complex nuclear bodies, while the simple bodies Fig. 6. ( ~ 2 5 , 0 0 0The ) extent of complex body is partially illustrated in 12 consecutive 80 nm serial sections. Although its diameter in this plane is approximately 600 nm, it would be, in another plane, at least 800 nm since it occurs in 10 sections. Note the presence of small electron opaque granules in the cores of profiles S5,6,7, and 8. 129 remained a t approximately the base-line level (Padykula et al., 1982). Th‘is serves as a n example of differing functional response by the two types of nuclear bodies to the same hormonal stimulus. Whether or not a functional relationship exists between simple and complex nuclear bodies cannot be analyzed until their rate of turnover has been determined. The possibility that simple bodies may be precursors of complex bodies has been brought forth from review of the literature (Bouteille et al., 1974). The formation of increasing numbers of complex nuclear bodies in uterine target cells can be achieved by steady maintenance of high nuclear concentrations of estrogen receptor by steady estrogenic stimulation (Padykula et al., 1981; Padykula and Clark, 1981). Once the estrogenic stimulation is removed, the nuclear estrogen receptor concentration decreases rapidly and is accompanied by a steady decrease in the frequency of observation of complex nuclear bodies (Padykula et al., 1982). The formation and disappearance of complex bodies is without evident change in the frequency of simple bodies. Since so little is known about the origin and function of complex nuclear bodies, speculation is appropriate. They are essentially protein structures, as demonstrated by trypsin digestion (Krishnan et al., 1967). Our experimental analyses indicate that complex nuclear bodies increase during progressive cellular hypertrophy brought about by heightened transcriptional activity, and conversely decrease in number during cellular atrophy. Whether or not these complex bodies arise from simple bodies remains to be determined experimentally. The filamentous capsule contains protein, as demonstrated by protease digestion (Krishnan et al., 1967; Dupuy-Coin et al., 1972). The filamentous components of both simple and complex nuclear bodies may reflect linkage to the “fibrogranular” acidic polypeptide network known as the nuclear matrix (Berezney, 19791, which possesses specific binding sites for estrogens and androgens (Barrack and Coffey, 1980). The remarkable rapid formation and disappearance of complex nuclear bodies in direct relation to increasing and decreasing concentration of nuclear estrogen receptor suggests that they may be transient differentiations of a pre-existing protein framework. They disappear from the nuclear scene without signs of progressive degradation associated 130 H.A. PADYKULA AND S.M. POCKWINSE with the accumulation of visible debris, as it occurs during cytoplasmic autophagy. ACKNOWLEDGMENTS The assistance of Christopher D. Hebert in the photographic work is gratefully acknowledged. This research was supported by NIH research grant HD13941. LITERATURE CITED Barrack, E.R., and D.S. Coffey 119801The specific binding of estrogens and androgens to the nuclear matrix of sex hormone responsive tissues. J. Biol. Chem. 255:7265-7275. Berezney, R. (1979) Dynamic properties of the nuclear matrix. In: The Cell Nucleus, Vol. VII. H. Busch, ed. Academic Press, New York, pp. 413-456. Bouteille, M., S.R. Kalifat, and J. Delarue (1967) Ultrastructural variations of nuclear bodies in human diseases. J. Ultrastruct. Res.,19:474-486. Bouteille, M., M. Laval, and A.M. Dupuy-Coin (1974) Localization of nuclear functions as revealed by ultrastructural autoradiography and cytochemistry. In: The Cell Nucleus, Vol. I. H. Busch, ed. Academic Press, New York, pp. 1-74. Clark. J.H.. J.W. Hardin, H.A. Padykula, and C.A. Cardasis (1978)Role of estrogen receptor binding and transcriDtiona1 activitv in the stimulation of hvperestrogenism and nuclear bodies. Proc. Nat. Acid: Sci., 752781-2784. Dupuy-Coin, A.M., S.R. Kalifat, and M. Bouteille (1972) Nuclear bodies as proteinaceous structures containing ribonucleoproteins. J. Ultrastruct. Res., 38:174-187. Fitzgerald, M., and H.A. Padykula (1983) Differing Functional responses of simple and complex nuclear bodies in uterine luminal epithelial cells following estrogenic stimuli. Anat. Rec. 205:131-141. Kierszenbaum, A.L. (1969)Relationship between nucleolus and nuclear bodies in human mixed salivary tumors. J. Ultrastruct. Res., 29:459-469. Krishnan, A., B.G. Uzman, and E.T. Hedley-Whyte (1967) Nuclear bodies: A component of cell nuclei in hamster tissues and human tumors. J. Ultrastruct. Res., 19563572. LeGoascogne, C., and E.E. Baulieu (1977). Hormonally controlled “nuclear bodies” during the development of prepubertal rat uterus. Biol. Cell., 30:195-206. Monneron, A,, and W. Bernhard (1966) Fine structural organization of the interphase nucleus in some mammalian cells. J. Ultrastruct. Res., 27266-288. Padykula, H.A., L.G. Coles, C. Weston, W.C. Okulicz, W. Robidoux, and W.W. Leavitt (1982) Correlated responses of nuclear bodies and nuclear estrogen receptor to estradiol exposure and withdrawal in uterine epithelial cells. Abstract #1076, Program of the 64th Annual Meeting of the Endocrine Society, p. 348. Padykula, H.A., and J.H. Clark (1981)Nuclear bodies as functional indicators in the target cells of sex steroid hormones. In: Cell Nucleus, Vol. IX. H. Busch, ed. Academic Press, New York, pp. 309-339. Padykula, H.A., M. Fitzgerald, J.H. Clark, and J.W. Hardin (1981) Nuclear bodies as structural indicators of estrogenic stimulation in uterine luminal epithelial cells. Anat. Rec., 201:679-696.