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Early effect of estrogen on chromatin ultrastructure in endometrial nuclei
Molecular and Cellular Endocrinology, 19 (1980) 79-92 0 Elsevier/North-Holland Scientific Publishers, Ltd.
79
EARLY EFFECT OF ESTROGEN ON CHROMATIN ENDOMETRIAL
ULTRASTRUCTURE
IN
NUCLEI
Patrice VIC a, Marcel GARCIA b, Claude HUMEAU a and Henri ROCHEFORT b a Laboratoire d ‘Histologic, Fact&P de Mhdecine, 2, rue Ecole de Mkdecine, 34000 Montpellier, and b Unit& d’Endocrinologie Cellulaire et Mol&ulaire 60, rue de Navacelles, 34100 Montpellier (France)
(U 148) I.N.S.E.R.M.,
Received 2 January 1980; accepted 25 March 1980
The effect of estradiol on chromatin ultrastructure in interphase nuclei was studied in immature rat and lamb endometrium. Physiological doses of estradiol within the first hour transformed the condensed chromatin into dispersed chromatin both in vivo and in vitro. These ultrastructural modifications were specifically induced by hormones translocating the estrogen receptor to the nucleus of estrogen-responsive tissues. Conversely, the antiestrogen tamoxifen gave a hypercondensation of chromatin. The addition of actinomycin D, cordycepin or o-amanitin, but not of cycloheximide, prevented the effect of estradiol both on the ultrastructural change and on [ 3H]uridine incorporation, suggesting that chromatin decondensation was closely related to transcriptional activity. These results indicate that in endometrium, estrogen rapidly provokes a large and extended modification of chromatin ultrastructure, which suggest a general effect on chromatin function rather than a selective activation of a limited number of genes. Keywords:
estradiol; chromatin; endometrium;
ultrastructure.
It is generally admitted that estrogen acts in target cell nuclei by stimulating the expression of specific genes through their interaction with specific receptor proteins (RF) (Jensen et al., 1968). This has been mainly established in non-mammalian tissues for the regulation of egg white (O’Malley and Means, 1974) and yolk proteins (Green and Tata, 1976). The mechanism of action of estrogens is less.comprehensive in tissues such as uterus and mammary tumors where estrogen provokes general hypertrophia. Whether the hormone specifically stimulates the expression of a limited or of a large number of genes is unknown. Since there are more than 50000 nuclear estrogen receptors in one given target cell (Gorski and Gannon, 1976) and that no saturation is observed for nuclear translocation of the receptor (Williams and Gorski, 1972), the question of the number of different sites responding to the hormone-receptor complexes is still unanswered. The mechanism by which gene expression is regulated is also unknown. It could be due to specific interactions with DNA of regulatory proteins such as the receptor. However, the alternative would be that the hormone-receptor complex induce a more extended conforma-
80
Patrice Vie, Marcel Garcia, Claude Humeau, HenriPochefort
tional change of chromatin. We have therefore decided to look to the ultrastructure of chromatin in interphase nuclei of estrogen target cells before and after estrogen stimulation. In the absence of estrogen, the cells of endometrium and hormonedependent mammary tumor grow slowly and are blocked in an inactive G, (or G,) phase containing mostly condensed chromatin. The present paper describes a rapid dispersion of chromatin in the endometrial cells after in vivo and in vitro addition of physiological doses of estrogens *. This ultrastructural change is correlated to biochemical responses to hormones, is induced specifIcally by RE ligands and appears to be related to the transcriptional activity of chromatin.
MATERIALS
AND METHODS
(I) Material. The following steroids were kindly given by Roussel Uclaf, Romainville (France): 17@stradiol, 17arestradiol,3-deoxyestradiol, progesterone, dihydrotestosterone. The antiestrogens tamoxifen (frans-1 -(p-dimethylaminoethoxyphenyl-1,2-diphenylbut-I ene) were kindly given by ICI Laboratories (England) (Dr. Patterson) and Nafoxidine (Ur r leeA; l-(2-@-3,4-dihydro-6-methoxy-2-phenylnaphth-I -yl)-phenoxy)-ethyl)-pyrrolidine) by Upjohn Laboratories (U.S.A.) (Dr. Kadruska) resp. Antibiotics and ethidium bromide were purchased from Sigma. (2) Hormone treatments. For in vitro experiments, tissue samples of ~1 mm3 from lamb endometrium and thymus were rapidly collected after slaughtering and incubated in Eagle’s medium (95% 02/5% COZ) at 37°C under stirring for 30-90 min with or withont different hormones or drugs. For in vivo experiments, 21-dayold female rats from Wistar strain were given a S.C. injection of hormone solubilized in oil and were sacrified 1-3 h later. (3) Electron microscopy. The tissues were fmed successively with glutaraldehyde (2.5%) and osmium tetroxide (1%) in phosphate buffer at pH 7.3. They were then embedded in araldite. The sections were stained with uranyl acetate and lead citrate according to Reynolds (1963). In some cases, the deoxyribonucleoproteins were ruled out by an EDTA treatment (Bernhard, 1969). In this case, the fmation was with glutaraldehyde only and embedding in Epon. The sections were then stained (1 min) with uranyl acetate, layered on a 0.2 M EDTA solution for 20 min and finally post-stained (1 min) with lead citrate. Quantitative morphometric studies, using randomisation, were used with 5 successive degrees according to Weibel and Elias (1967). For in vitro lamb uterus incubation,
* Preliminary reports have been published: P. Vie, M. Garcia, J. And& C. Humeau and H. Rochefort (1978) Endocrine Society Meeting, Miami Beach (Florida, U.S.A.), June 14-16, Abstract No. 582; P. Vie, M. Garcia, J. And&, C. Humeau and H. Rochefort (1978) C.R. Acad. Sci. (Paris) 287,141-144.
Early effect
of estrogen on chromutin ultrastructure
81
each experimental point was performed on 10 samples of 1 mm3 tissue from 3 or 4 uteri. The samples were mixed, embedded and sectioned at 3 levels. In each experiment, 1000-2000 nuclei of epithelial cells were counted at random. The percentage of the nuclear area occupied by condensed chromatin was measured on micrographs using square paper, excluding tangential sections of nuclei. 25-50 randomised micrographs of nuclei were analysed for each case. 2 series of nuclei were then defined. The condensed chromatin nuclei contained more than 25% condensed chromatin area per total nuclear area and the dispersed chromatin nuclei contained less than 10% of condensed chromatin area per total nuclear area. Nuclei containing between 10 and 25% condensed chromatin represented only 10% of the total nuclei . (4) In vitro /‘H]leucine incorporation. Groups of 5-7 immature rats were used for each hormonal treatment. 16 h after the injection, rats were sacrificed and their uteri were incubated for 2 h in 2 ml of Eagle’s medium containing 5 &i/ml of [3H]L-leucine (spec. act., 1 Ci/mmole) in Oa/COz (9515) atmosphere. The cytosoluble proteins were then separated and the radioactivity was counted in the acidinsoluble fraction as described (Garcia and Rochefort, 1977). (S)Zn vitro /3H]un*dine incorporah*on in lamb endomeqium. After a 30-min incubation, with or without hormones, 2-mm3 samples of endometrium (z-100 mg tissue) were incubated with 5 &i/ml of [‘Hluridine (spec. act., 24 Ci/mmole) for 90 min and 37’C in OJCOz atmosphere. All procedures were then carried out at 2°C. The tissue was homogenized in Tris-HCl 10 mM, EDTA 1.5 mM buffer pH 7.4, and then precipitated by 0.5 N PCA. RNA was then hydrolysed in 0.3 N NaOH at 37°C for 1 h, the reaction was stopped by adding PCA (0.5 N final concentration). A second alkaline hydrolysis was performed on the remaining acid-insoluble material in the same conditions. [‘H]Uridine of the hydrolysates was counted in triplicate. RNA concentration was obtained by Az6e absorbance assuming that 1 mg RNA hydrolysed in 1 ml gives an Az6c of 23.
RESULTS (I) Effect of estradiol (EJ on chromatin ultrastructure in the interphase nuclei of endometn’um When lamb endometrium was incubated in vitro with 20 nM estradiol (E,) for 60 or 90 min, the condensed chromatin of the epithelial cell nuclei was transformed into dispersed chromatin. In control experiment, when endometrium was incubated without Ez, clumps of condensed chromatin were located near the nuclear envelope (Fig. 1). After Ez treatment, chromatin was dispersed in the whole of the nucleoplasm. It was thin, dusty and homogeneously spread out. In some cases, a border of condensed chromatin remained visible in close contact with the inner nuclear mem-
82
Patrice Vie, Marcel Garcia, Claude Humeau, Hem-i Rochefort
Fig. 1. In vitro effect of Ea in lamb endometrium. Lamb endometrium was incubated 90 min as indicated in the Materials and Methods in the absence (a and c) or presence (b and d) of 20 nM E2. The nuclei were then stained either classically (X7000, a and b) or with EDTA technique (X3000 c and d) as described in Methods. Insert (c and d) represents a higher magnification (X30 000) of EDTA staining. The arrows exhibit the perichromatic fib&. cc, condensed chromatin; nu, nucleolus; dc, dispersed chromatin.
brane. The nucleolus size was also generally increased after E2 treatment. The cytoplasmic organelles appeared normal at this stage. This effect was significant when the proportion of nuclei containing dispersed chromatin was counted for lOOO3000 nuclei as described in the Methods (Table 1). The area of condensed chromatin per nucleus was found to decrease from 27 to <5%. The EDTA staining of nuclei showed an increase of the number of perichromatic fibrils after E, incubation,
83
Early effect of estrogen on chromatin ultrastructure
Table 1 Early in vitro effects of Ea in lamb endometrium Control % Nuclei with dispersed chromatin
14k
5=
% Condensed chromatin area/per nucleus
275
3
Uridine incorporation (cem/ccg RNA)
p value
E2
480 * 30
87k
7
<5 702 f 16
Lamb endometrium was incubated with 20 nM E2 for 60 or 90 min as in the Methods. Chromatin ultrastructure and uridine incorporation into RNA were determined in parallel as indicated in Methods. a Mean *SD of 3-9 separate expts.
whilst the concentration of perichromatic and interchromatic granules remained unmodified. This effect was specific of estrogen target tissues as it was not found in non-target cells. The thymocytes nuclei displayed the same condensed chromatin after incubation with or without E2 (Fig. 2). In the fibroblast (not shown) and lymphocyte (Fig. 5) located in the same endometrium, the degree of dispersion of chromatin was not markedly modified by E2 treatment. Since the chromatin ultrastructure is highly dependent on ionic strength (Laval and Bouteille, 1973) we have
Fig. 2. Absence of in vitro effect of Ea on lamb thymocytes out (a) or with (b) 20 nM Ea (X7000).
incubated in vitro for 90 min with-
84
Patrice Vie, Marcel Garcia, Claude Humeau, Henri Rochefort
controlled that the condensation of chromatin was identical in nuclei which had been incubated for 1 h at 37’ in culture medium and in nuclei observed directly without incubation. A similar dispersion of chromatin was observed after 30-min incubation but to a lesser degree. The same modifications of endometrial chromatin were observed 1 or 3 h after in vivo administration of a physiological dose (2.5 /.tg) of Ea to immature rat. The content of the nucleus was transformed into homogeneous chromatin and a thin layer of condensed chromatin remained at the periphery of the nucleus (Fig. 3).
Fig. 3. In vivo effect of steroids on rat endometrial nuclei (X6000). 3 h after S.C. injection of oil (a) 2.5 pg, Ez (b), 10 mg progesterone (c) or 10 mg DHT (d), the nuclei of immature rat endometrium were treated as described in Methods.
Early effect of estrogen on chromtin
85
ultrastructure
The volume of nucleolus were markedly increased by Ez and DHT treatment. The nuclei from tibroblast and myometrial cells did not appear to be modified by Ez treatment at this time. Conversely, the liver of male Xenopus laevi specifically stimulated by estrogens for vitellogenin induction and not for cell growth (Green and Tata, 1976) did not show any significant modification of chromatin 8.5 h after an in vivo injection of 1 mg Ez via lymph vessel. At this early time, the vitellogenin mRNA begins to increase (Ryffel, 1978). (2) Homone specificity The effect of other steroids and RE ligands was then tested both in vitro on lamb uteri and in vivo on immature rat uteri. After in vitro incubation of lamb uteri with 0.1 PM 3deoxyestradiol or 17a-estradio1, the tine structure of chromatin was not modified. High concentrations of progesterone administered either in vitro or in vivo were also inefficient (Table 2). On the other hand, pharmacological doses of DHT provoked in vitro and in vivo (Table 2, Fig. 3d) the transformation of the majority of the condensed chromatin into dispersed chromatin. These results are in agreement with the binding affinities of the androgens for the RE and with their efficiency to stimulate the rat uterus estrogen induced protein (Ruh and Ruh, 1975; Garcia and Rochefort, 1977). Moreover, in the rat uterus, the in vivo effect of hormones on chromatin dispersion was correlated with the stimulation of [3H]leucine incorporation into protein and the RE nuclear translocation (Table 2). Estradiol was the most active hormone on these 3 parameters, high doses of DHT were also active, but progesterone (10 mglrat) which does not bind to RE was
Table 2 Early in vitro effects of Ez, DHT and progesterone Oil % Nuclei with dispersed alb chromatm (1- 3 h)
10+5c
% 13H] Leu into proteins b (6-14 h)
100
% Nuclear Ea receptor b (l-3 h)
13i 3
in immature rat uterus E2
Prog.
DHT
80 * 10
15 f 10
275 f 70
222 + 39
90
55*
2
lO*
5
112 f 22 12*
Hormones were administered either in vitro a (estradiol 20 nM, DHT and progesterone 10 mg/rat).
3
1 PM) or
in vivo b (E2 2.5 pg, DHT and progesterone
The percentages of nuclear receptors compared with the total extractable E2 receptors after injection of DHT and progesterone are from Rochefort and Garcia (1976) and after E2 mjection from Anderson et al. (1973). c Mean *SD of 2-7 separate expts.
86
Patrice Vie, Marcel Garcia, Claude Humeau, Henri Rochefort
totally inactive. These results suggested that the chromatin dispersion was only seen with the hormones able to interact with the RE. We then looked for the effect of the estrogen antagonists Tamoxifen and Nafoxidine which are known to prevent estrogen action via a RE-mediated process. After 90 min in vitro incubation of lamb endometrium with Tamoxifen or Nafoxidine (1 PM), the tine structure of chromatin in epithelial cells was never dispersed as in the case of estradiol. In some cases (3 Expts.) the ultrastructure of chromatin was not apparently modified as compared to control nuclei. In other cases (4 Expts.), the nucleus exhibited a hypercondensation of chromatin with important clumps of condensed chromatin (Fig. 4). In this case, the percentage of the condensed chromatin area per nucleus increased from 27% in control to 50% in treated cells. This last aspect was mostly seen in glandular epithelial cells but less frequently in the surface epithelial cells of lamb endometrium. Neither nuclear bodies nor nucleolar channel systems were observed. The cytoplasmic organelles were not altered strongly suggesting the absence of any cytotoxicity. It was controlled in these conditions that Tamoxifen binds to RE and translocates it to the nucleus (unpublished). When Tamoxifen (1 PM) and Ez (20 nM) were added together in the incubating medium, the chromatin was dispersed as with Ez alone. In these conditions, one knows that Ez is more efficient than Tamoxifen to bind to RE and activate it, since no competition was observed when the 2 ligands were added at the same time to the incubated tissue. This was probably due to the slower rate of entry into the
Fig. 4. In vitro effect of Tamoxifen endometrium for 90 min (X5000),
1 pM alone (a) and with Ez 10 nM (b), incubated SC, supercondensation of chromatin.
with lamb
Early effect of estrogen an chromatin ultrastructure
87
cells of the antiestrogen (Rochefort and Capony, 1973). Thus, this last experiment showed that Tamoxifen had no effect when its intracellular binding to RE was prevented by Es. These results suggested that the mechanism of the interaction and action of RE in chromatin was probably different when it was occupied by E2 or by an antiestrogen. (3) Relationship with transcription The ultrastructural modification of chromatin appeared to be approximately contemporary of the estrogen stimulation of transcription. This was shown by measuring uridine ~co~oration into RNA. As already shown in an other system (Go&i, 1973), RNA synthesis was found to be significantly increased from 90 to 120 min following Es addition to lamb endometrium incubated in vitro (Table 1). Since the effects of Es on chromatin dispersion and on transcription were both detected at early stages, we raised the question of the cause-relation~ip between the 2 Ea stimulated events. We have tried to answer this question by adding in vitro to lamb endometrium, specific inhibitors of RNA transcription (Fig. 5). (a) The efficiency of actinomycin D differed according to its sequence of administration. When 1 j.&i actinomycin D was added to endometrium 15 min before Ez treatment, the chromatin remained partially condensed as in’ control nuclei. While some nuclear microsegregation due to actinomycin D was observed as already described (Bernhard, 1971). When actinomycin D was administered 15 min after E2 treatment, the dispersion of chromatin was not prevented (Fig. 5). (b) The effect of ~-amanitin,another transcription inhibitor acting via a different mechanism by preventing RNA polymerase B activities was then tested. When 1 PM o-amanitin was incubated alone or 15 min before Es addition no change in the amount of condensed chromatin was observed, suggesting that this drug also prevented chromatin transformation. Conversely, the dispersion of chromatin was obtained when the drug was ad~nistered 15 mm after Ez. In all cases, the drug provoked the usual modifications of nuclear structure such as splitting of the nucleolus and the appearance of many perichromatic granules (Fig. 5) (Fiume and Laschi, 1965 ; Barsotti and Marinozzi, 1979). (c) Ethidium bromide, another intercalating drug is known to inhibit most of the tr~sc~ption (Waring, 1964) and a part of the RE-nuclear translocation (Andre et al., 1977). After in vitro treatment by ethidium bromide (2 mM), the chromatin was no longer dispersed by Ea. It showed the typical modifications induced by this drug such as bleaching out of dispersed chromatin, and unsticking of condensed chromatin from the nuclear envelope (Andre et al., 1977). (d) Cordycepin (I 00 a) which blocks specifically polyadenylation and the premRNA processing also prevented the Es-induced dispersion of chromatin. Conversely, cycloheximide, a protein-synthesis inhibitor, was inefficient to block Ez effect when administrated in vitro at 50-500 ,uM concentration 30 min before Ez treatment of lamb uteri. We therefore concluded that the antibiotics blocking transcription also blocked
Patrice Vie, Marcel Garcia, Claude Humeau, Henri Rochefort
Fig. 5. In vitro effects of antibiotics (X6000). Lamb endometrium was successively incubated in vitro either with antibiotics alone (15 min) then with antibiotics + Ea (45 min), or with Es alone (15 min) then with E2 + antibiotics (45 min). (a) actinomycin D (1 &M) before Er (0.1 HM), (b) E2 (0.1 PM) before actinomycin D (1 PM), (c) cu-amanitin (1 PM) before E2 (20 nM), (d) Es (20 nM) before a-amanitin (1 PM). cc, condensed chromatin; nu, nucleolus; L, lymphocyte; arrow, segregation of the nucleolus.
Early effect of estrogen on chromatin ultrastructure
a9
the ultrastructural modification of chromatin induced by E2 and that RNA synthesis was required for inducing this chromatin dispersion while protein synthesis was not.
DISCUSSION A transformation of the in situ chromatin from a condensed to a dispersed stair has already been shown after several days of treatment with E2, both in the endometrium of castrated women (Sirtori and Bosisio-Bestetti, 1967) and in primary culture of rabbit endometrium (Berliner and Gerschenson, 1976). However, due to the delay between the time of hormone addition and that of electron-microscopical examination, 1:he relevance of this transformation to the mechanism of action of estrogen was not obvious. In this paper, we now report that the dispersion of chromatin in interphase nuclei of lamb and rat endometrium is induced rapidly, within 1 h, and specifically by RE ligands both in vitro and in vivo. Among the different steroids tested, only those able to interact efficiently with RE were active in dispersing chromatin. In addition to physiological doses of estrogens, pharmacological doses of DHT which act via the RE to stimulate uterine cell growth (Rochefort and Garcia, 1976; Garcia and Rochefort, 1977) were also able to disperse chromatin while progesterone was ineffective at any concentration. However, antiestrogen which also binds RE did not induce chromatin dispersion in endometrial nuclei. Conversely, in some cases, a further clumping of chromatin was observed after in vitro addition of Tamoxifen, but this was prevented when the RE were occupied by estrogens, strongly suggesting that this additional effect of antiestrogen was RE-mediated. In the early stages after hormonal treatment, we saw neither nucleolar channel (Armstrong and More, 1974), nor nuclear bodies (Clark et al., 1978) nor the chromatin dispersion described by Tseng et al. (1979) in hepatocytes of Xenopus laevi, since these modifications were observed several days after hormone treatment. A similar clumping of chromatin has been observed with glucocorticoid in lymphocytes (Nicholson et al., 1979). These observations suggest that the RE might induce in chromatin both positive and negative types of responses depending on the nature of the ligand bound to it. However, the effect of antiestrogen should be obtained in vivo before concluding that antiestrogen provokes a different transformation of chromatin than that induced by estrogen. We have then tried to specify the relationship between the effect of estrogen on chromatin dispersion and on the stimulation of transcription in the same uterine cells. Both effects occur rapidly after hormone addition. RNA accumulation in uterus is known to be stimulated very rapidly within 5 min (Hamilton et al., 1968). While this study was in progress, chromatin dispersion was shown to occur within 15 and 30 min after E2 treatment in endometrium of ovariectomized rats (VasquezNin et al., 1978) suggesting that chromatin dispersion could be a direct effect of Ez.
90
Patrice Vie, Marcel Garcia, Claude Humeau, Hem-i Rochefort
We have confirmed this, 30 min after in vitro incubation of lamb uterus with E?. Here we present ultrastructural and biochemical evidence that transcription is stimulated when chromatin has been decondensed by estrogens. In fact, the EDTA staining of nuclei showed a large increase of perichromatic fibrils corresponding to the pre-mRNA species, 60-90 min after Ez treatment while [3H]uridine incorporation into RNA was stimulated by Ez. In order to specify whether one phenomenon was responsible for the other, we used antibiotics blocking transcription by different mechanisms. All antibiotics appeared to block the Ez-induced dispersion of chromatin, even though the effect of the drugs alone might in some cases mask the Ez effect. This suggested that the stimulation of transcription by EZ was required to observe chromatin dispersion and that this dispersion could be a consequence of transcription stimulation. However when chromatin had been dispersed by Ez, a further inhibition of transcription could not reverse the dispersed chromatin into a condensed state within 60-90 min. Actinomycin D has also been shown to prevent the estrogen-induced processing of its nuclear receptor in MCFT cells (Horwitz and McGuire, 1978). However, this inhibition was specific for a few intercalating drugs while chromatin dispersion appeared to be inhibited by any antibiotics blocking transcription. It is obvious that chromatin dispersion can only be observed when chromatin is condensed in the absence of estrogen. This is the case for tissues which grow slowly without estrogens, such as endometrium or estrogen-dependent mammary tumors (Vie et al., 1978; and unpublished results). In fact, this effect was not observed in the hormone-independent CaH mouse mammary tumors (Vie et al., in preparation) and in the liver of male Xenopus Zaevi 8.5 h after Ez treatment when the concentration of vitellogenin mRNA began to increase, while the growth was not stimulated (Green and:Tata, 1976; Ryffel, 1978). However, a similar chromatin dispersion has been described in Xenopus laevi liver 12 h after Ez injection, when general protein synthesis but not cell division was increased by Ez (Bergink et al., 1977). The chromatin dispersion appears thus to be a general phenomenon observed when dormant cells in a Ge or inactive Gr stage of the cell cycle are recruted by hormones into an active Gr stage (Epifanova, 1966; Ringertz, 1969; Gerschenson et al., 1977) and restricted to tissues whose growth and/or general protein synthesis is stimulated by hormone. The same modification is observed by other mitogens such as phytohemagglutinin in lymphocytes (Tokuyasu et al., 1968) or in regenerating liver (Derenzini et al., 1977). We do not know whether this extended and early modification of chromatin involves the stimulation by hormones of a large or limited number of genes. However, this suggests that the specific triggering of a few genes by regulatory proteins as observed in procaryotes is not the only mechanism possible for steroid-hormone action. The extent of modification of chromatin structure by hormones has also been suggested independently by Johnson et al. (1979) and O’Malley and Means (1974) who showed a large increase of E. coli RNA polymerase binding sites to DNA in great excess as compared to the number of hormone-receptor sites. The
Early effect of estrogen on chromatin ultrastructure
91
mechanism of this large modification of chromatin structure, either reversible and due to direct interaction of chromatin with receptor protein or ions, or irreversible and due to covalent modifications of proteins, is unknown. To conclude, the early dispersion of chromatin by estrogens suggest another mechanism of action than the discrete stimulation of a limited number of specific genes. This effect appears mostly visible in cells which are quiescent without hormones as in uterus and some estrogen-dependent mammary tumors. Preliminary experiments with human-breast cancer tissue incubated in vitro with or without Ea also suggest that this morphological test might be of interest in hormone-dependent cancer to predict the type of hormone which will stimulate tumor growth.
ACKNOWLEDGEMENTS We thank Prof. P. Sentein for providing the electron microscopic facilities and Dr. Andre who performed some experiments. The help of Ariane Legrand and Micheline Cros for electron microscopy and of E. Barrie and M. Paolucci for preparing the manuscript was greatly appreciated. This work has been supported by grants from the Delegation a la Recherche Scientifique et Technique and the Institut National de la Sante et de la Recherche Medicale.
REFERENCES Anderson, J.N., Peck Jr., E.J., and Clark, J.H. (1973) Endocrinology 92, 14881495. Andre, J., Vie, P., Humeau, C., and Rochefort, H. (1977) Mol. Cell. Endocrinol. 8, 225-241. Armstrong, E.M., and More, LA. (1974) Cytobios 11, 13-16. Barsotti, P., and Marinozzi, V. (1979) J. Submicrosc. Cytol. 11.1, 13-73. Baxter, J.D., Johnson, L.K., and Lan, N. (1978) 5th Int. Congr. Hormonal Steroids, New Delhi (India) 30 Oct.-4 Nov., J. Steroid Biochem. 9, abstract 838. Bergink, E.W., Tseng, M.T., and Wittliff, J.L. (1977) Cytobiologie, Z. Exp. Zellforsch. 14, 362377. Berliner, J.A., and Gerschenson, L.E. (1976) J. Steroid Biochem. 7, 153-158. Bernhard, W. (1969) J. Ultrastruct. Res. 27, 250-265. Bernhard, W. (1971) Advances in Cytopharmacology, 1st Int. Symp. Cell Biol. Cytopharmacol., Venice (Italy), Raven, New York. Clark, J.H., Anderson, J.N., and Peck, E.J. (1973) Adv. Exp. Med. Biol. 36, 15-59. Clark, J.H., Hardin, J.W., Padykula, H.A., and Cardasis, C.A. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 75,2781-2784. Derenzini, M., Lorenzoni, E., Marinozzi, V., and Barsotti, P. (1977) J. Ultrastruct. Res. 59, 250-262. Epifanova, 0.1. (1966) Exp. Cell. Res. 42,562-577. Fiume, L., and Laschi, R. (1965) Sperimentale 111,288-297. Garcia, M., and Rochefort, H. (1977) Steroids 29, 111-126. Gerschenson, L.E., Conner, E., and Mura, J.T. (1977) Endocrinology 100, 1468-1471.
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Gorski, J. (1973) in: Handbook of Physiology, Vol. 2, Geiger, S.R. (Ed.), Am. Phys. Sot., pp. 525-536. Gorski, J., and Gannon, F. (1976) Annu. Rev. Physiol. 38425-450. Green, C.D., and Tata, J.R. (1976) Cell 7, 131-139. Hamilton, T.H., Widnell, C.C., and Tata, J.R. (1968) J. Biol. Chem. 243,408-417. Horwitz, K.B., and McGuire, W.L. (1978) J. Biol. Chem. 253,6319-6322. Jensen, E.V., Suzuki, T., Kawashima, T., Stumpf, W.E., Jungblut, P.W., and DeSombre, E.R. (1968) Proc. Natl. Acad. Sci. (U.S.A.) 59,632-638. Johnson, L.K., Lan, N.C., and Baxter, J.D. (1979) J. Biol. Chem. 254, 7785-7794. Laval, L., and Bouteille, M. (1973) Exp. Cell. Res. 76, 337-348. Means, A.R., and Hamilton, T.H. (1966) Proc. Natl. Acad. Sci. (U.S.A.) 56,686-694. O’Malley, B.W., and Means, A.R. (1974) Science 183,610-620. Nicholson, M.L., Voris, B.P., and Young, D.A. (1979) Cancer treatment reports, 1st Int. Congr. Hormones and Cancer, 63, 1196, abstract 269. Reynolds, F. (1963) J. Cell. Biol. 17, 208-211. Ringertz, N. (1969) in: Handbook of Molecular Cytology (North-Holland, Amsterdam). Rochefort, H., and Capony, F. (1973) C.R. Acad. Sci. (Paris) 276,2321-2324. Rochefort, H., and Garcia, M. (1976) Steroids 28,549-560. Ruh, T.S., and Ruh, M.F. (1975) Endocrinology 97, 1144-1150. Ryffel, G.U. (1978) Mol. Cell. Endocrinol. 12, 237-246. Sirtori, C., and Bosisio-Bestetti, M. (1967) Cancer Res. 27, 367-376. Tokuyasu, K., Madden, S.C., and Zeldis, L.J. (1968) J. Cell. Biol. 39,630-660. Tsai, S.Y., Harris, S.E., Tsai, M.J., and O’Malley, B. (1976) J. Biol. Chem. 251,4713-4721. Tseng, M.T., Bergink, E.W., and Wittliff, J.L. (1979) Cytobiologie, Z. Exp. Zellforsch. 20, 143149. Vasquez-Nin, G.H., Echeverria, O.M., Molina, E., and Fragoso, J. (1978) Acta Anat. 102, 308318. Waring, M.J. (1964) Biochim. Biophys. Acta 87, 358-361. Weibel, E.R., and Elias, H. (1967) in: Quantitative Methods in Morphology, Springer, Berlin. Williams, D., and Gorski, J. (1972) Proc. Natl. Acad. Sci. (U.S.A.) 69, 3464-3468.
79
EARLY EFFECT OF ESTROGEN ON CHROMATIN ENDOMETRIAL
ULTRASTRUCTURE
IN
NUCLEI
Patrice VIC a, Marcel GARCIA b, Claude HUMEAU a and Henri ROCHEFORT b a Laboratoire d ‘Histologic, Fact&P de Mhdecine, 2, rue Ecole de Mkdecine, 34000 Montpellier, and b Unit& d’Endocrinologie Cellulaire et Mol&ulaire 60, rue de Navacelles, 34100 Montpellier (France)
(U 148) I.N.S.E.R.M.,
Received 2 January 1980; accepted 25 March 1980
The effect of estradiol on chromatin ultrastructure in interphase nuclei was studied in immature rat and lamb endometrium. Physiological doses of estradiol within the first hour transformed the condensed chromatin into dispersed chromatin both in vivo and in vitro. These ultrastructural modifications were specifically induced by hormones translocating the estrogen receptor to the nucleus of estrogen-responsive tissues. Conversely, the antiestrogen tamoxifen gave a hypercondensation of chromatin. The addition of actinomycin D, cordycepin or o-amanitin, but not of cycloheximide, prevented the effect of estradiol both on the ultrastructural change and on [ 3H]uridine incorporation, suggesting that chromatin decondensation was closely related to transcriptional activity. These results indicate that in endometrium, estrogen rapidly provokes a large and extended modification of chromatin ultrastructure, which suggest a general effect on chromatin function rather than a selective activation of a limited number of genes. Keywords:
estradiol; chromatin; endometrium;
ultrastructure.
It is generally admitted that estrogen acts in target cell nuclei by stimulating the expression of specific genes through their interaction with specific receptor proteins (RF) (Jensen et al., 1968). This has been mainly established in non-mammalian tissues for the regulation of egg white (O’Malley and Means, 1974) and yolk proteins (Green and Tata, 1976). The mechanism of action of estrogens is less.comprehensive in tissues such as uterus and mammary tumors where estrogen provokes general hypertrophia. Whether the hormone specifically stimulates the expression of a limited or of a large number of genes is unknown. Since there are more than 50000 nuclear estrogen receptors in one given target cell (Gorski and Gannon, 1976) and that no saturation is observed for nuclear translocation of the receptor (Williams and Gorski, 1972), the question of the number of different sites responding to the hormone-receptor complexes is still unanswered. The mechanism by which gene expression is regulated is also unknown. It could be due to specific interactions with DNA of regulatory proteins such as the receptor. However, the alternative would be that the hormone-receptor complex induce a more extended conforma-
80
Patrice Vie, Marcel Garcia, Claude Humeau, HenriPochefort
tional change of chromatin. We have therefore decided to look to the ultrastructure of chromatin in interphase nuclei of estrogen target cells before and after estrogen stimulation. In the absence of estrogen, the cells of endometrium and hormonedependent mammary tumor grow slowly and are blocked in an inactive G, (or G,) phase containing mostly condensed chromatin. The present paper describes a rapid dispersion of chromatin in the endometrial cells after in vivo and in vitro addition of physiological doses of estrogens *. This ultrastructural change is correlated to biochemical responses to hormones, is induced specifIcally by RE ligands and appears to be related to the transcriptional activity of chromatin.
MATERIALS
AND METHODS
(I) Material. The following steroids were kindly given by Roussel Uclaf, Romainville (France): 17@stradiol, 17arestradiol,3-deoxyestradiol, progesterone, dihydrotestosterone. The antiestrogens tamoxifen (frans-1 -(p-dimethylaminoethoxyphenyl-1,2-diphenylbut-I ene) were kindly given by ICI Laboratories (England) (Dr. Patterson) and Nafoxidine (Ur r leeA; l-(2-@-3,4-dihydro-6-methoxy-2-phenylnaphth-I -yl)-phenoxy)-ethyl)-pyrrolidine) by Upjohn Laboratories (U.S.A.) (Dr. Kadruska) resp. Antibiotics and ethidium bromide were purchased from Sigma. (2) Hormone treatments. For in vitro experiments, tissue samples of ~1 mm3 from lamb endometrium and thymus were rapidly collected after slaughtering and incubated in Eagle’s medium (95% 02/5% COZ) at 37°C under stirring for 30-90 min with or withont different hormones or drugs. For in vivo experiments, 21-dayold female rats from Wistar strain were given a S.C. injection of hormone solubilized in oil and were sacrified 1-3 h later. (3) Electron microscopy. The tissues were fmed successively with glutaraldehyde (2.5%) and osmium tetroxide (1%) in phosphate buffer at pH 7.3. They were then embedded in araldite. The sections were stained with uranyl acetate and lead citrate according to Reynolds (1963). In some cases, the deoxyribonucleoproteins were ruled out by an EDTA treatment (Bernhard, 1969). In this case, the fmation was with glutaraldehyde only and embedding in Epon. The sections were then stained (1 min) with uranyl acetate, layered on a 0.2 M EDTA solution for 20 min and finally post-stained (1 min) with lead citrate. Quantitative morphometric studies, using randomisation, were used with 5 successive degrees according to Weibel and Elias (1967). For in vitro lamb uterus incubation,
* Preliminary reports have been published: P. Vie, M. Garcia, J. And& C. Humeau and H. Rochefort (1978) Endocrine Society Meeting, Miami Beach (Florida, U.S.A.), June 14-16, Abstract No. 582; P. Vie, M. Garcia, J. And&, C. Humeau and H. Rochefort (1978) C.R. Acad. Sci. (Paris) 287,141-144.
Early effect
of estrogen on chromutin ultrastructure
81
each experimental point was performed on 10 samples of 1 mm3 tissue from 3 or 4 uteri. The samples were mixed, embedded and sectioned at 3 levels. In each experiment, 1000-2000 nuclei of epithelial cells were counted at random. The percentage of the nuclear area occupied by condensed chromatin was measured on micrographs using square paper, excluding tangential sections of nuclei. 25-50 randomised micrographs of nuclei were analysed for each case. 2 series of nuclei were then defined. The condensed chromatin nuclei contained more than 25% condensed chromatin area per total nuclear area and the dispersed chromatin nuclei contained less than 10% of condensed chromatin area per total nuclear area. Nuclei containing between 10 and 25% condensed chromatin represented only 10% of the total nuclei . (4) In vitro /‘H]leucine incorporation. Groups of 5-7 immature rats were used for each hormonal treatment. 16 h after the injection, rats were sacrificed and their uteri were incubated for 2 h in 2 ml of Eagle’s medium containing 5 &i/ml of [3H]L-leucine (spec. act., 1 Ci/mmole) in Oa/COz (9515) atmosphere. The cytosoluble proteins were then separated and the radioactivity was counted in the acidinsoluble fraction as described (Garcia and Rochefort, 1977). (S)Zn vitro /3H]un*dine incorporah*on in lamb endomeqium. After a 30-min incubation, with or without hormones, 2-mm3 samples of endometrium (z-100 mg tissue) were incubated with 5 &i/ml of [‘Hluridine (spec. act., 24 Ci/mmole) for 90 min and 37’C in OJCOz atmosphere. All procedures were then carried out at 2°C. The tissue was homogenized in Tris-HCl 10 mM, EDTA 1.5 mM buffer pH 7.4, and then precipitated by 0.5 N PCA. RNA was then hydrolysed in 0.3 N NaOH at 37°C for 1 h, the reaction was stopped by adding PCA (0.5 N final concentration). A second alkaline hydrolysis was performed on the remaining acid-insoluble material in the same conditions. [‘H]Uridine of the hydrolysates was counted in triplicate. RNA concentration was obtained by Az6e absorbance assuming that 1 mg RNA hydrolysed in 1 ml gives an Az6c of 23.
RESULTS (I) Effect of estradiol (EJ on chromatin ultrastructure in the interphase nuclei of endometn’um When lamb endometrium was incubated in vitro with 20 nM estradiol (E,) for 60 or 90 min, the condensed chromatin of the epithelial cell nuclei was transformed into dispersed chromatin. In control experiment, when endometrium was incubated without Ez, clumps of condensed chromatin were located near the nuclear envelope (Fig. 1). After Ez treatment, chromatin was dispersed in the whole of the nucleoplasm. It was thin, dusty and homogeneously spread out. In some cases, a border of condensed chromatin remained visible in close contact with the inner nuclear mem-
82
Patrice Vie, Marcel Garcia, Claude Humeau, Hem-i Rochefort
Fig. 1. In vitro effect of Ea in lamb endometrium. Lamb endometrium was incubated 90 min as indicated in the Materials and Methods in the absence (a and c) or presence (b and d) of 20 nM E2. The nuclei were then stained either classically (X7000, a and b) or with EDTA technique (X3000 c and d) as described in Methods. Insert (c and d) represents a higher magnification (X30 000) of EDTA staining. The arrows exhibit the perichromatic fib&. cc, condensed chromatin; nu, nucleolus; dc, dispersed chromatin.
brane. The nucleolus size was also generally increased after E2 treatment. The cytoplasmic organelles appeared normal at this stage. This effect was significant when the proportion of nuclei containing dispersed chromatin was counted for lOOO3000 nuclei as described in the Methods (Table 1). The area of condensed chromatin per nucleus was found to decrease from 27 to <5%. The EDTA staining of nuclei showed an increase of the number of perichromatic fibrils after E, incubation,
83
Early effect of estrogen on chromatin ultrastructure
Table 1 Early in vitro effects of Ea in lamb endometrium Control % Nuclei with dispersed chromatin
14k
5=
% Condensed chromatin area/per nucleus
275
3
Uridine incorporation (cem/ccg RNA)
p value
E2
480 * 30
87k
7
<5 702 f 16
Lamb endometrium was incubated with 20 nM E2 for 60 or 90 min as in the Methods. Chromatin ultrastructure and uridine incorporation into RNA were determined in parallel as indicated in Methods. a Mean *SD of 3-9 separate expts.
whilst the concentration of perichromatic and interchromatic granules remained unmodified. This effect was specific of estrogen target tissues as it was not found in non-target cells. The thymocytes nuclei displayed the same condensed chromatin after incubation with or without E2 (Fig. 2). In the fibroblast (not shown) and lymphocyte (Fig. 5) located in the same endometrium, the degree of dispersion of chromatin was not markedly modified by E2 treatment. Since the chromatin ultrastructure is highly dependent on ionic strength (Laval and Bouteille, 1973) we have
Fig. 2. Absence of in vitro effect of Ea on lamb thymocytes out (a) or with (b) 20 nM Ea (X7000).
incubated in vitro for 90 min with-
84
Patrice Vie, Marcel Garcia, Claude Humeau, Henri Rochefort
controlled that the condensation of chromatin was identical in nuclei which had been incubated for 1 h at 37’ in culture medium and in nuclei observed directly without incubation. A similar dispersion of chromatin was observed after 30-min incubation but to a lesser degree. The same modifications of endometrial chromatin were observed 1 or 3 h after in vivo administration of a physiological dose (2.5 /.tg) of Ea to immature rat. The content of the nucleus was transformed into homogeneous chromatin and a thin layer of condensed chromatin remained at the periphery of the nucleus (Fig. 3).
Fig. 3. In vivo effect of steroids on rat endometrial nuclei (X6000). 3 h after S.C. injection of oil (a) 2.5 pg, Ez (b), 10 mg progesterone (c) or 10 mg DHT (d), the nuclei of immature rat endometrium were treated as described in Methods.
Early effect of estrogen on chromtin
85
ultrastructure
The volume of nucleolus were markedly increased by Ez and DHT treatment. The nuclei from tibroblast and myometrial cells did not appear to be modified by Ez treatment at this time. Conversely, the liver of male Xenopus laevi specifically stimulated by estrogens for vitellogenin induction and not for cell growth (Green and Tata, 1976) did not show any significant modification of chromatin 8.5 h after an in vivo injection of 1 mg Ez via lymph vessel. At this early time, the vitellogenin mRNA begins to increase (Ryffel, 1978). (2) Homone specificity The effect of other steroids and RE ligands was then tested both in vitro on lamb uteri and in vivo on immature rat uteri. After in vitro incubation of lamb uteri with 0.1 PM 3deoxyestradiol or 17a-estradio1, the tine structure of chromatin was not modified. High concentrations of progesterone administered either in vitro or in vivo were also inefficient (Table 2). On the other hand, pharmacological doses of DHT provoked in vitro and in vivo (Table 2, Fig. 3d) the transformation of the majority of the condensed chromatin into dispersed chromatin. These results are in agreement with the binding affinities of the androgens for the RE and with their efficiency to stimulate the rat uterus estrogen induced protein (Ruh and Ruh, 1975; Garcia and Rochefort, 1977). Moreover, in the rat uterus, the in vivo effect of hormones on chromatin dispersion was correlated with the stimulation of [3H]leucine incorporation into protein and the RE nuclear translocation (Table 2). Estradiol was the most active hormone on these 3 parameters, high doses of DHT were also active, but progesterone (10 mglrat) which does not bind to RE was
Table 2 Early in vitro effects of Ez, DHT and progesterone Oil % Nuclei with dispersed alb chromatm (1- 3 h)
10+5c
% 13H] Leu into proteins b (6-14 h)
100
% Nuclear Ea receptor b (l-3 h)
13i 3
in immature rat uterus E2
Prog.
DHT
80 * 10
15 f 10
275 f 70
222 + 39
90
55*
2
lO*
5
112 f 22 12*
Hormones were administered either in vitro a (estradiol 20 nM, DHT and progesterone 10 mg/rat).
3
1 PM) or
in vivo b (E2 2.5 pg, DHT and progesterone
The percentages of nuclear receptors compared with the total extractable E2 receptors after injection of DHT and progesterone are from Rochefort and Garcia (1976) and after E2 mjection from Anderson et al. (1973). c Mean *SD of 2-7 separate expts.
86
Patrice Vie, Marcel Garcia, Claude Humeau, Henri Rochefort
totally inactive. These results suggested that the chromatin dispersion was only seen with the hormones able to interact with the RE. We then looked for the effect of the estrogen antagonists Tamoxifen and Nafoxidine which are known to prevent estrogen action via a RE-mediated process. After 90 min in vitro incubation of lamb endometrium with Tamoxifen or Nafoxidine (1 PM), the tine structure of chromatin in epithelial cells was never dispersed as in the case of estradiol. In some cases (3 Expts.) the ultrastructure of chromatin was not apparently modified as compared to control nuclei. In other cases (4 Expts.), the nucleus exhibited a hypercondensation of chromatin with important clumps of condensed chromatin (Fig. 4). In this case, the percentage of the condensed chromatin area per nucleus increased from 27% in control to 50% in treated cells. This last aspect was mostly seen in glandular epithelial cells but less frequently in the surface epithelial cells of lamb endometrium. Neither nuclear bodies nor nucleolar channel systems were observed. The cytoplasmic organelles were not altered strongly suggesting the absence of any cytotoxicity. It was controlled in these conditions that Tamoxifen binds to RE and translocates it to the nucleus (unpublished). When Tamoxifen (1 PM) and Ez (20 nM) were added together in the incubating medium, the chromatin was dispersed as with Ez alone. In these conditions, one knows that Ez is more efficient than Tamoxifen to bind to RE and activate it, since no competition was observed when the 2 ligands were added at the same time to the incubated tissue. This was probably due to the slower rate of entry into the
Fig. 4. In vitro effect of Tamoxifen endometrium for 90 min (X5000),
1 pM alone (a) and with Ez 10 nM (b), incubated SC, supercondensation of chromatin.
with lamb
Early effect of estrogen an chromatin ultrastructure
87
cells of the antiestrogen (Rochefort and Capony, 1973). Thus, this last experiment showed that Tamoxifen had no effect when its intracellular binding to RE was prevented by Es. These results suggested that the mechanism of the interaction and action of RE in chromatin was probably different when it was occupied by E2 or by an antiestrogen. (3) Relationship with transcription The ultrastructural modification of chromatin appeared to be approximately contemporary of the estrogen stimulation of transcription. This was shown by measuring uridine ~co~oration into RNA. As already shown in an other system (Go&i, 1973), RNA synthesis was found to be significantly increased from 90 to 120 min following Es addition to lamb endometrium incubated in vitro (Table 1). Since the effects of Es on chromatin dispersion and on transcription were both detected at early stages, we raised the question of the cause-relation~ip between the 2 Ea stimulated events. We have tried to answer this question by adding in vitro to lamb endometrium, specific inhibitors of RNA transcription (Fig. 5). (a) The efficiency of actinomycin D differed according to its sequence of administration. When 1 j.&i actinomycin D was added to endometrium 15 min before Ez treatment, the chromatin remained partially condensed as in’ control nuclei. While some nuclear microsegregation due to actinomycin D was observed as already described (Bernhard, 1971). When actinomycin D was administered 15 min after E2 treatment, the dispersion of chromatin was not prevented (Fig. 5). (b) The effect of ~-amanitin,another transcription inhibitor acting via a different mechanism by preventing RNA polymerase B activities was then tested. When 1 PM o-amanitin was incubated alone or 15 min before Es addition no change in the amount of condensed chromatin was observed, suggesting that this drug also prevented chromatin transformation. Conversely, the dispersion of chromatin was obtained when the drug was ad~nistered 15 mm after Ez. In all cases, the drug provoked the usual modifications of nuclear structure such as splitting of the nucleolus and the appearance of many perichromatic granules (Fig. 5) (Fiume and Laschi, 1965 ; Barsotti and Marinozzi, 1979). (c) Ethidium bromide, another intercalating drug is known to inhibit most of the tr~sc~ption (Waring, 1964) and a part of the RE-nuclear translocation (Andre et al., 1977). After in vitro treatment by ethidium bromide (2 mM), the chromatin was no longer dispersed by Ea. It showed the typical modifications induced by this drug such as bleaching out of dispersed chromatin, and unsticking of condensed chromatin from the nuclear envelope (Andre et al., 1977). (d) Cordycepin (I 00 a) which blocks specifically polyadenylation and the premRNA processing also prevented the Es-induced dispersion of chromatin. Conversely, cycloheximide, a protein-synthesis inhibitor, was inefficient to block Ez effect when administrated in vitro at 50-500 ,uM concentration 30 min before Ez treatment of lamb uteri. We therefore concluded that the antibiotics blocking transcription also blocked
Patrice Vie, Marcel Garcia, Claude Humeau, Henri Rochefort
Fig. 5. In vitro effects of antibiotics (X6000). Lamb endometrium was successively incubated in vitro either with antibiotics alone (15 min) then with antibiotics + Ea (45 min), or with Es alone (15 min) then with E2 + antibiotics (45 min). (a) actinomycin D (1 &M) before Er (0.1 HM), (b) E2 (0.1 PM) before actinomycin D (1 PM), (c) cu-amanitin (1 PM) before E2 (20 nM), (d) Es (20 nM) before a-amanitin (1 PM). cc, condensed chromatin; nu, nucleolus; L, lymphocyte; arrow, segregation of the nucleolus.
Early effect of estrogen on chromatin ultrastructure
a9
the ultrastructural modification of chromatin induced by E2 and that RNA synthesis was required for inducing this chromatin dispersion while protein synthesis was not.
DISCUSSION A transformation of the in situ chromatin from a condensed to a dispersed stair has already been shown after several days of treatment with E2, both in the endometrium of castrated women (Sirtori and Bosisio-Bestetti, 1967) and in primary culture of rabbit endometrium (Berliner and Gerschenson, 1976). However, due to the delay between the time of hormone addition and that of electron-microscopical examination, 1:he relevance of this transformation to the mechanism of action of estrogen was not obvious. In this paper, we now report that the dispersion of chromatin in interphase nuclei of lamb and rat endometrium is induced rapidly, within 1 h, and specifically by RE ligands both in vitro and in vivo. Among the different steroids tested, only those able to interact efficiently with RE were active in dispersing chromatin. In addition to physiological doses of estrogens, pharmacological doses of DHT which act via the RE to stimulate uterine cell growth (Rochefort and Garcia, 1976; Garcia and Rochefort, 1977) were also able to disperse chromatin while progesterone was ineffective at any concentration. However, antiestrogen which also binds RE did not induce chromatin dispersion in endometrial nuclei. Conversely, in some cases, a further clumping of chromatin was observed after in vitro addition of Tamoxifen, but this was prevented when the RE were occupied by estrogens, strongly suggesting that this additional effect of antiestrogen was RE-mediated. In the early stages after hormonal treatment, we saw neither nucleolar channel (Armstrong and More, 1974), nor nuclear bodies (Clark et al., 1978) nor the chromatin dispersion described by Tseng et al. (1979) in hepatocytes of Xenopus laevi, since these modifications were observed several days after hormone treatment. A similar clumping of chromatin has been observed with glucocorticoid in lymphocytes (Nicholson et al., 1979). These observations suggest that the RE might induce in chromatin both positive and negative types of responses depending on the nature of the ligand bound to it. However, the effect of antiestrogen should be obtained in vivo before concluding that antiestrogen provokes a different transformation of chromatin than that induced by estrogen. We have then tried to specify the relationship between the effect of estrogen on chromatin dispersion and on the stimulation of transcription in the same uterine cells. Both effects occur rapidly after hormone addition. RNA accumulation in uterus is known to be stimulated very rapidly within 5 min (Hamilton et al., 1968). While this study was in progress, chromatin dispersion was shown to occur within 15 and 30 min after E2 treatment in endometrium of ovariectomized rats (VasquezNin et al., 1978) suggesting that chromatin dispersion could be a direct effect of Ez.
90
Patrice Vie, Marcel Garcia, Claude Humeau, Hem-i Rochefort
We have confirmed this, 30 min after in vitro incubation of lamb uterus with E?. Here we present ultrastructural and biochemical evidence that transcription is stimulated when chromatin has been decondensed by estrogens. In fact, the EDTA staining of nuclei showed a large increase of perichromatic fibrils corresponding to the pre-mRNA species, 60-90 min after Ez treatment while [3H]uridine incorporation into RNA was stimulated by Ez. In order to specify whether one phenomenon was responsible for the other, we used antibiotics blocking transcription by different mechanisms. All antibiotics appeared to block the Ez-induced dispersion of chromatin, even though the effect of the drugs alone might in some cases mask the Ez effect. This suggested that the stimulation of transcription by EZ was required to observe chromatin dispersion and that this dispersion could be a consequence of transcription stimulation. However when chromatin had been dispersed by Ez, a further inhibition of transcription could not reverse the dispersed chromatin into a condensed state within 60-90 min. Actinomycin D has also been shown to prevent the estrogen-induced processing of its nuclear receptor in MCFT cells (Horwitz and McGuire, 1978). However, this inhibition was specific for a few intercalating drugs while chromatin dispersion appeared to be inhibited by any antibiotics blocking transcription. It is obvious that chromatin dispersion can only be observed when chromatin is condensed in the absence of estrogen. This is the case for tissues which grow slowly without estrogens, such as endometrium or estrogen-dependent mammary tumors (Vie et al., 1978; and unpublished results). In fact, this effect was not observed in the hormone-independent CaH mouse mammary tumors (Vie et al., in preparation) and in the liver of male Xenopus Zaevi 8.5 h after Ez treatment when the concentration of vitellogenin mRNA began to increase, while the growth was not stimulated (Green and:Tata, 1976; Ryffel, 1978). However, a similar chromatin dispersion has been described in Xenopus laevi liver 12 h after Ez injection, when general protein synthesis but not cell division was increased by Ez (Bergink et al., 1977). The chromatin dispersion appears thus to be a general phenomenon observed when dormant cells in a Ge or inactive Gr stage of the cell cycle are recruted by hormones into an active Gr stage (Epifanova, 1966; Ringertz, 1969; Gerschenson et al., 1977) and restricted to tissues whose growth and/or general protein synthesis is stimulated by hormone. The same modification is observed by other mitogens such as phytohemagglutinin in lymphocytes (Tokuyasu et al., 1968) or in regenerating liver (Derenzini et al., 1977). We do not know whether this extended and early modification of chromatin involves the stimulation by hormones of a large or limited number of genes. However, this suggests that the specific triggering of a few genes by regulatory proteins as observed in procaryotes is not the only mechanism possible for steroid-hormone action. The extent of modification of chromatin structure by hormones has also been suggested independently by Johnson et al. (1979) and O’Malley and Means (1974) who showed a large increase of E. coli RNA polymerase binding sites to DNA in great excess as compared to the number of hormone-receptor sites. The
Early effect of estrogen on chromatin ultrastructure
91
mechanism of this large modification of chromatin structure, either reversible and due to direct interaction of chromatin with receptor protein or ions, or irreversible and due to covalent modifications of proteins, is unknown. To conclude, the early dispersion of chromatin by estrogens suggest another mechanism of action than the discrete stimulation of a limited number of specific genes. This effect appears mostly visible in cells which are quiescent without hormones as in uterus and some estrogen-dependent mammary tumors. Preliminary experiments with human-breast cancer tissue incubated in vitro with or without Ea also suggest that this morphological test might be of interest in hormone-dependent cancer to predict the type of hormone which will stimulate tumor growth.
ACKNOWLEDGEMENTS We thank Prof. P. Sentein for providing the electron microscopic facilities and Dr. Andre who performed some experiments. The help of Ariane Legrand and Micheline Cros for electron microscopy and of E. Barrie and M. Paolucci for preparing the manuscript was greatly appreciated. This work has been supported by grants from the Delegation a la Recherche Scientifique et Technique and the Institut National de la Sante et de la Recherche Medicale.
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