Control animals received 0.2 ml 1,2-propanediol at the same time periods. Tissue and cell fractionation, and determinations of tissue radioactivity, nuclear.
EARLY ESTROGEN ACTION: CONCOMITANT STIMULATIONS WITHIN TWO MINUTES OF NUCLEAR RNA SYNTHESIS AND UPTAKE OF RNA PRECURSOR BY THE UTERUS* BY ANTHONY R. MEANS AND TERRELL H. HAMILTON DEPARTMENT OF ZOOLOGY, THE UNIVERSITY OF TEXAS, AUSTIN
Communicated by Frederick L. Hisaw, September 20, 1966
A central problem in elucidating mechanisms of hormone action concerns whether increased synthesis of macromolecules is a cause or consequence of increased uptake and transport of their precursors by the cellular membrane. Hamilton, Widnell, and Tatal demonstrated a five- to tenfold increase in rate of uterine nuclear RNA synthesis within 20 min following administration of a single dose of estradiol-17f3 to the ovariectomized rat. Means and Hamilton2 found that this stimulation of nuclear RNA synthesis in the uterus was accompanied by an increase in the rate of uptake of uridine-H3. The immediacy and extent of the two effects-one at the level of nuclear transcription and one at the level of the cell membrane-have prompted us to investigate nuclear RNA synthesis and uterine uptake of uridineH3 at earlier periods of time in the hope of ascertaining which occurs first. Materials and Methods.-Adult Sprague-Dawley rats weighing 150-180 gm and ovariectomized at least 3 weeks were obtained from Hormone Assay Co. (Chicago, Ill.), and used in all experiments. All animals received intraperitoneally 100 MC uridine-H3 (gl 8.0 C/mmole; New England Nuclear Corp., Boston, Mass.) in 0.2 ml buffered physiological saline (Abbott Laboratories, North Chicago, Ill.) 10 min prior to killing. Estradiol-17f3 obtained from Sigma Chemical Co. (St. Louis, Mo.) was dissolved in 1,2 propanediol and 10 ug in 0.2 ml solution was administered intraperitoneally into experimental animals 2, 5, 10, 20, or 30 min prior to killing by cervical dislocation. Control animals received 0.2 ml 1,2-propanediol at the same time periods. Tissue and cell fractionation, and determinations of tissue radioactivity, nuclear RNA specific activity, RNA, and DNA, were performed as previously described.2 Results.-Administration of a single dose of estradiol-17f3 to the ovariectomized rat results within 2 min in a 40 per cent increase over control of incorporation of uridine-H3 into uterine nuclear RNA (Fig. 1). In this experiment, experimental animals received uridine-H3 10 min, and estradiol-17,B 2 min, prior to killing and removal of uteri to the ice-cold homogenizing medium;2 control animals received similarly uridine-H3 and 1,2-propanediol without hormone. Nuclear RNA specific activity continued to increase at 5 and 10 min of estrogen action, reaching a maximum of 420 per cent over control at 20 min. Thirty minlutes following administration of the hormone, nuclear RNA synthesis had declined to 330 per cent over control (Fig. 1). The decrease in specific activity of nuclear RNA after the initial, dramatic increase has previously been shown to continue to 2 hr, when it levels off at about 50 per cent over control." 2 This rate of incorporation of uridine-H3 into nuclear RNA is continued to the fourth hour of estrogen action. 2: 3 Accompanying the stimulation of incorporation of uridine-H3 into uterine nuclear RNA by estradiol-17f3 is an increase in uptake of the tritiated precursor by the organ. Figure 1 shows that the uptake is stimulated by 13 per cent at 2 min of 1I94
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600
EJ 500
Uptake of Uridine-H3
B Acid-insoluble Radioactivity U Specific Activity of Nuclear RNA
T
-400 0
0
0
300
200
0
2
5
10
20
30
Time after Estrogen (min.) FIG. 1.-Effect of estrogen on uptake of uridine-H3 by the uterus and its incorporation into acid-insoluble material and nuclear RNA. The data are expressed as per cent of control value. Brackets show the ranges of triplicate experiments. Five uteri were pooled for each experimental or control group. The ranges of variation for the acid-insoluble data are given in Table 1. The control values are: 6,920 cpm/mg homogenate DNA; 11.0% acid-insoluble radioactivity; and 1,204 cpm/mg nuclear RNA (see Table 1). For each period of time tested for estrogen action, separate control experiments were performed (see Materials and Methods). Thus the control values given here at time zero for tissue radioactivity and nuclear RNA specific activity, and for acid-insoluble radioactivity given in Table 1, represent respectively the mean for 15 control groups (i.e., three for each time period of estrogen action tested).
action by the hormone. Continued increases in uptake are noted 5, 10, 20, and 30 min following estrogen administration, with the greatest increase in rate of uptake occurring between 5 and 10 min of hormone action. This is also the time interval of greatest increase in rate of nuclear RNA synthesis. Although both the incorporation of uridine-H3 into nuclear RNA and the uptake of the precursor by the uterus increase, no significant increase in uterine wet weight was observed during the first 30 min of estrogen action (Table 1).
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TABLE 1 EARLY EFFECTS OF ESTROGEN ON THE UPTAKE OF URIDINE-H3 BY THE UTERUS AND ITS INCORPORATION INTO ACID-INSOLUBLE MATERIAL AND NUCLEAR RNA Time after estrogen (min)
Total tissue radioactivity (cpm/mg DNA)
0* 2t
6,920 8,069
5t
9,759
lot
20t 30t
14,153 15,027 17,073
Acid-insoluble fraction (%)
11.0+0.5 12.6 i 0.2 18.4+0.3 21.8+0.6 21.9+0.6
20.5 + 0.4
Specific activity nuclear RNA (cpm/mg RNA)
Uterine wet weight (mg)
1,204 1,650
57+7
2,462
54±t6 57+7 57+t5
5,526
6,364 5,136
55 4+ 3
58 + 2
Mean value for 15 separate control groups (see Materials and Methods and Fig. 1). t Mean values for triplicate experiments (see Materials and Methods). For ranges of absolute values, see Fig. 1. *
The proportion of total tissue radioactivity which is acid-insoluble is shown in Figure 1 as a function of time after administration of estradiol-17,3. Table 1 gives the absolute values for the acid-insoluble data expressed as per cent of control in Figure 1. A linear rate of increase occurred from 0 to 10 min of estrogen action, with a maximum of 22 per cent for acid-insoluble radioactivity being reached at the latter time period. The value at 20 min of hormone action is the same as at 10 min, indicating maximum efficiency of precursor utilization by the RNA-synthesizing apparatus of the uterine cell. By 30 min of estrogen action, however, the acidinsoluble radioactivity is 20.5 per cent of the total-representing a decrease from the values observed at 10 and 20 min. This trend in variation for proportion of uterine radioactivity which is acid-insoluble is paralleled by the decrease in rate of incorporation of uridine-H3 into nuclear RNA during the same time interval (Fig. 1). Discussion.-The finding that nuclear RNA synthesis is stimulated within 2 min of estrogen's acting on the uterus of the ovariectomized rat leaves little doubt that one of the earliest effects of this hormone occurs at the transcriptional level of the uterine cell. This rapid stimulation of nuclear RNA synthesis occurs considerably sooner than recently reported effects of estrogen at the level of translation or amine mobilization. These reports include binding of estradiol-17j3 to some uterine receptor molecule,4 the estrogen-stimulated synthesis of a specific cytoplasmic protein,5 and the liberation of uterine histamine.6 In addition, recent work in this laboratory with characterized polysomes isolated from the cytoplasmic fraction of the uterus demonstrates that the initial rise in polysome level occurs at 2 hr of estrogen action, with a concomitant increase at that time in polysome activity assayed in a cell-free, protein-synthesizing system.7 Gorski and others have suggested that estrogenic stimulation of RNA synthesis in the uterus depends upon a preceding synthesis of protein.5' 8, 9 Our present findings for estrogen action at earlier time periods, however, establish an effect on nuclear RNA synthesis prior to enhancement of either nuclear or cytoplasmic protein synthesis (see also refs. 1-3, 12). Szego6 has emphasized that upon reaching the uterus, estrogen may first increase the availability of substrates, ions, and possible regulatory substances prior to stimulation of RNA and protein synthesis. The worker cited, however, underestimates the rapidity of estrogen-stimulated RNA synthesis. Our findings indicate that increased uptake of precursor by the uterine cell membrane constitutes an early, if not primary, action of estrogen, but the fact that both transcription of DNA and cellular uptake of uridine-H3 are stimulated by 2 min of estrogen action
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raises the question of causality for the two responses. It could be argued that a 40 per cent increase in nuclear RNA synthesis and a 13 per cent increase in uptake of RNA precursor at 2 min of estrogen action indicates that synthesis precedes uptake. It could also be argued that a small increase in tissue radioactivity represents a much greater number of precursor molecules than the number of molecules needed to account for the increase in synthesis. There is also the problem of single versus dual or multiple primary sites of action for estrogen. Three alternate hypotheses for the primary action or actions of estrogen can be envisioned: (i) estrogen first directly or indirectly stimulates RNA synthesis in the nucleus, which necessitates changes in cellular membrane permability or function allowing increases in uptake of precursors needed for such synthesis; (ii) estrogen exerts its primary action on the outer membrane of the uterine cell, resulting in an influx of precursor which permits increased synthesis of nuclear RNA; and (iii) a dual or multiple effect of estrogen-one or several at the level of the cellular and/or nuclear membrane and one or several at the level of transcription of DNA. Any one or combination of these hypotheses is of course compatible with the current concept of estrogen control of cytoplasmic protein synthesis via direct or indirect effects on the genome., 2, 10-12 Furthermore, any of these suggestions could be consistent with the idea that estrogen in its initial action first binds to some uterine receptor molecule4 or-and this we now suggest-binds or in some other way interacts with a receptor site in or on the membrane of the uterine cell. The findings presented here do not permit the selection of any single hypothesis for the primary mechanism(s) of estrogen action. It is clear, however, that the primary effect(s) of the hormone on the uterus of the ovariectomized adult rat is extremely rapid and that the biosynthetic processes involved are intimately related to membrane phenomena. This statement has precedence in previous findings from this laboratory for in vivo effects of estrogen on protein synthesis in the subcellular fractions of the uterus.12 Thirty minutes after administration of estradiol-1713 to the ovariectomized rat, rates of subcellular protein synthesis are depressed. This depression of protein synthesis is accompanied by a decrease in the uptake by the uterus of various labeled amino acids. As the rate of protein synthesis recovers and subsequently increases over the control at later periods of estrogen action, the uptake of precursor by the organ follows a parallel course. Finally, the elucidation of the primary mechanism(s) or site(s) of action for estrogen awaits the development of new techniques or the perfection of old ones which allow a higher resolution of two variables: (a) the time required for estrogen to reach the uterus and to initiate in sequential or simultaneous order its membrane and biosynthetic effects; (b) the near-instantaneous assay of these responses at the levels of transcription of DNA and function of cellular and nuclear membranes. Summary.-Estrogen stimulates both the synthesis of nuclear RNA and the uptake of RNA precursor by the uterus within 2 min following administration of the hormone to the ovariectomized adult rat. The immediacy of these effects clearly illustrates that estrogen in some unknown way stimulates membrane activity and transcription of DNA. This results in a very rapid synthesis of a few specific molecules of RNA which presumably initiate and support the subsequent stimulation of uterine protein synthesis.
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* This research was supported by a USPHS research grant HD-00726-05 to T. H. Hamilton and by an NIH predoctoral fellowship (1-F1-GM-30, 939-01) to A. R. Means. 1 Hamilton, T. H., C. C. Widnell, and J. R. Tata, Biochim. Biophys. Acta, 108, 168 (1965). 2 Means, A. R., and T. H. Hamilton, these PROCEEDINGS, 56, 686 (1966). 3 Hamilton, T. H., C. C. Widnell, and J. R. Tata, in preparation. 4 Toft, D., and J. Gorski, these PROCEEDINGS, 55, 1574 (1966). 5 Notides, A., and J. Gorski, these PROCEEDINGS, 56, 230 (1966). 6 Szego, Clara M., Federation Proc., 24, 1343 (1965). 7 Teng, Ching-sung, and T. H. Hamilton, in preparation. 8 Mueller, G. C., J. Gorski, and Y. Aizawa, these PROCEEDINGS, 47, 164 (1961). Gorski, J., W. D. Noteboom, and J. A. Nicoletti, J. Cellular Comp. Physiol., 66 (Suppl. 1), 91 (1965) 10 Mueller, G. C., in Mechanisms of Hormone Action, ed. P. Karlson (Stuttgart: George Thieme, Verlag, 1965), p. 228. Segal, S. J., 0. W. Davidson, and K. Wada, these PROCEEDINGS, 54, 782 (1965). 12 Means, A. R., and T. H. Hamilton, Biochim. Biophys. Acta (1966), in press.
