estrogen action in 'vitro

THE JOURNAL OF BIOLOGICAL CREMI~TRY Vol. 247, No. 4, Issue of February 25, pp. 1299-1395, 1972

Ptinted in U.S.A.

Estrogen Action in ‘Vitro

INDUCTION OF THE SYKTHESIS OF A SPECIFIC UTERINE PROTEIN*

(Received for publication, July 12, 1971)

BENITA S. KATZENELLENBOGEN AND JACK GORSKI

From the Departments of Physiology and Biophysics, and Biochemistry, University of Illinois, Urbana, Illinois 61801

SUMMARY

Physiological concentrations of e&radio1 added in vifro to immature rat uteri in Eagle’s HeLa medium induced the near maximal (compared to in vivo) synthesis of a specific uterine protein (induced protein (IP)). The magnitude of IP induction closely corresponded to the amount of nuclear bound [3H]estradiol at different estradiol concentrations. Maximal IP induction and maximal binding of [3H]estradiol to nuclear receptor were obtained with 2 to 3 X 10e8 M

estradiol; 2 to 3 X 10es M estradiol gave a 50% response with both; and at lo-lo M, induction and binding were minimal. The greatest in vitro response at 2 X lop8 M estradiol was about 85 % of that obtained in vivo.

The coincidence of the induced proteins synthesized after either in vivo or in vitro hormone administration was shown by coelectrophoresis on polyacrylamide gels. Likewise, the over-all pattern of newly synthesized soluble proteins was the same in uteri stimulated with estrogen either in

vitro or in vivo. The time course of the rate of IP synthesis (a measure of amount of IP-synthesizing capacity) in uifro

(detectable by 15 min; 50% by 20 to 30 min) was similar to that obtained in vivo. In both situations, the increase in IP-synthesizing capacity is actinomycin D sensitive. Also, the rate of accumulation of IP-synthesizing capacity is highest initially and falls off rapidly with time (by 30 min) whether accumulation occurs in vitro or in vivo; at this time, however, the amount of estradiol is continuing to increase or remains at a high level in the nucleus.

Induction of IP synthesis shows a strict hormonal specificity. Only estrogenic compounds induced, whereas progesterone, testosterone, and insulin did not. The efficacy of induction was 1’7 &e&radio1 > diethylstilbestrol > estriol > 17 cr-estradiol. Cyclic adenosine 3’,5’-monophosphate and W, 02’-dibutyryl cyclic adenosine 3’,5’-monophosphate, at 10Va or 10-c M, did not induce IP and neither enhanced nor diminished the induction of IP due to estradiol.

These findings clearly indicate that estrogen can directly stimulate the uterus without the requirements of intact vascular or nervous systems.

* This research was supported by United States Public Health Service Grant HD-04828. National Institutes of Health Post- doctoral Fellowship F02 RD 45354, and Ford Foundation Grant 700-0333. An abstract of portions of this work has been published (1).

In the hope of better understanding the mechanism by which estrogen exerts its influence in target tissues, much attention has been directed toward analysis of the early physiological and biochemical responses of the uterus to estrogen and the correlation of these events with estrogen uptake and binding in uterine tissue. It is obvious that the development of an in vitro tissue or organ culture system capable of mimicking physiological in viva responses would greatly facilitate an accurate correlation of early biochemical changes with the various estrogen-binding phenomena that have been investigated in uterine tissue in recent years (2-5).

Previously reported responses of uteri (6-9) or placenta (10) to estrogens in vitro have required hormone concentrations of 2 X 10W5 to 1 X lo-’ M, which are 102- to 104-fold higher than the plasma concentrations of estrogen found in cycling rats or humans (11-14). In addition, the responses have generally been of a minimal magnitude compared to the in viuo responses and have not always proven to be reproducible. In view of the fact that one of the earliest responses to in vivo estrogen administration is the induction of the de novo synthesis of a specific rat uterine protein (induced protein; detectable within 40 min) (15) and of the product of an actinomycin D-sensitive step, presumably RNA, required for IP1 synthesis (detectable within 10 min) (16), it was of interest to investigate this response under in vitro conditions. In this paper, we report induction of IP under totally in vitro conditions using physiological (low9 M)

concentrations of estrogens. The many similarities between the in viva and in vitro estrogen-induced systems are discussed, and details of the correspondence between IP induction and estrogen binding are also presented.

MATERIALS

The steroids used were 17@-estradiol and estriol (Mann Re- search) ; diethyIstilbestro1, testosterone, and 17a-estradiol (Sigma) ; and progesterone (Calbiochem). Stock steroid solutions were made in absolute ethanol and were diluted by addition to either Eagle’s HeLa medium (for in vitro incubation) or warm 0.15 M saline (for in vivo injection). Actinomycin D, generously supplied by Merck Sharp and Dohme was made as a stock solution (25 mg/4 ml) in deionized water. Cyclic adenosine 3’, 5’-monophosphate and N6,02’-dibutyryl cyclic adenosine 3’) 5’-monophosphate (Calbiochem) solutions were freshly made in Eagle’s HeLa medium. Porcine insulin (re-

1 The abbreviation used is: IP, induced protein.

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crystallized, 24.4 USP units per mg) was obtained from Mann Research; a stock solution (200 milliunits per ml) was made in glass-distilled water, pH 2.3, containing 2% ovalbumin. Eagle’s HeLa medium (liquid, Difco) was used without modification. L-[4,5-3HlLeucine (2.0 Ci per mmole) and Q4C]leucine (316 mCi per mmole) were from Schwarz BioResearch.

METHODS

Handling of Animals ad IIormone Administration in Vivo or irz liitro-Immature female rats, 22 to 25 days old (Holtzman), n-ere used throughout this study. When estrogen was admin- ist,ered in viva, each experimental animal was injected intraperitoneally with 5 pg of 17/?-estradiol iu 0.5 ml of 0.15 M saline. Control animals received an equal volume of saline alone. At designated time interval? after injection, the animals were decapitated one at a time, and their uteri were quickly excised, stripped of all surrounding fatty tissue, and processed as indicated. When uteri from previously untreated animals were exposed to estrogen (or ethanol in the case of controls) in vitro,

the animals were decapitated one at a time, and their uteri were quickly excised, stripped of all surrounding fatty tissue, and immediately transferred to unmodified Eagle’s HeLa medium previously warmed at 37”, containing designated concentrations of 17@-estradiol or other indicated steroid or an equal volume of ethanol. Incubations contained two to three uteri per ml of medium and were conducted in stoppered lo- or 25.ml capacity Erlenmeyer flasks maintained in a shaking water bath at 37” under an atmosphere of 95% 02 and 5% COZ. After indicated intervals of incubation with steroid or vehicle control, either (a) uteri were thoroughly rinsed with fresh 37” HeLa medium and transferred to flasks containing fresh HeLa medium (again at two to three uteri per ml) to which radioactive amino acid \yith or without actinomycin D (see below) was then added, or (b) additions of radioactive amino acid with or without actinomycin D were made directly to the original flask.

Double Isotope Labeling of Uterine Proteins and Preparation of Uterine Soluble Proteins--Uterine proteins were labeled with 20 @i per ml of L-[4, 5-3H]leucine (2.0 Ci per mmole in 10e2 M HCI) or 5 PCi per ml of L-[14C]leucine (316 mCi per mmole in 1OW M

HCl) for 2 hours at 37”, with or without 28 or 31 pg per ml of actinomycin D, in Eagle’s HeLa medium under an atmosphere of 95% 02 and 5’$J0 Con. Thi: concentration of actinomycin D has been shown to nearly completely block uterine RNA synthesis in vitro (17). Because the 14C incubations were 10% in 10W2 M I-ICI, 3H incubations were also made 10% in 10e2 M HCl in order to equalize volumes and conditions. This amount of HCI did not detectably change the pH of the HeLa medium and, whether included or omitted, had no effect on IP induction. Estrogen-treated uteri were incubated with one isotope and the controls with the second, or uteri receiving estrogen in vitro were incubated with one isotope and uteri receiving estrogen in vivo were incubated with the second. At the end of the incorporation period, the uteri of both groups \yere rinsed thoroughly three times with ice-cold 0.05Tc Na&DT-1 and homogenized sepa- rately or together in this EDTX solution (ca. six to eight uteri per ml) by means of a motor-driven Dual1 glass homogenizer. Homogenates were centrifuged for 50 min at 27,000 X g at 4” in a Servall centrifuge, and the resulting supernatant fraction was frozen until use.

Polyacrylamide Gel E2ecfrophoresis-Uterine supernatant frac- t)ions (a00 to 300 ~1; equivalent of one to two uteri per gelj were

separated on 6% polyacrylamide gels (9 x 0.7 cm) that were prepared and run in buffer (0.066 NI Tris-0.02 M boric acid-O.003 M NazEDTA), pH 8.6, as previously described (16). At the termination of a run, gels to be stained Jvere treated with 1% Amidoschwarz in 7% acetic acid for several hours and were then electrophoretically destained; gels to be counted for radioactivity determination were placed for several hours in tubes containing 7.5% acetic acid and \yere then further freed of radioactive amino acids by undergoing electrophoresis with 7.5 % acetic acid for 45 min in an electrophoretic destainer. Gels were then frozen over Dry Ice and sectioned into 1.66.mm discs with a multiple razor blade slicer. The proteins in the gel slices were eluted by incubating the discs in scintillation vials containing 0.8 ml of 1% sodium dodecyl sulfate and agitating overnight on a rotary shaker at room temperature (18). After thorough mixing with 15 ml of toluene-based scintillation fluid (0.03% dimethyl 1,4-bis-[2-(4.methyl-5.phenyloxazolyl)]-benzene and 0.5% 2,5-diphenyloxazole, New England Nuclear) that Tyas 10% in Biosolv-BBS3 (Beckman), samples were counted in a Packard Tri-Carb scintillation spectrophotometer. For double isotope gels, the counting efficiency was 27% for 3H and 43% for 14C using reduced range counts. Radioactivity determinations on duplicate samples with gels processed using the hydrogen peroxide and Nuclear-Chicago solubilizer method described previously (16, 19) gave similar radioactivity profiles and count recovery. However, as the peroxide method was considered to be more laborious and more liable to variability, the dodecyl sulfate-count elution method was used for all results reported here.

Quantitation of IP Sylzthesis-The relative rate of II’ synthesis was determined from analysis of double labeled uterine soluble experimental and control proteins that underwent coelectrophoresis on acrylamide gels. Either one of two methods was used. In the first method, which was the one used routinely, the 3H :14C ratio (or estrogen to control ratio) was determined for each gel slice, and the increase in area under the ratio curve in the IP region was determined. Thus, control area plus estrogen-increased area (total estrogen - control area) divided by control area, (A, + A,)/A,, was used as an estimate of relative rate of IP synthesis. In the second method, the ratio of counts per min in the IP band (three peak slices) to the counts per min in several gel slices above and several slices below the 11’ band was calculated for both 3H and 1%. The ratio for 3H (if estrogen) was then divided by the ratio for 14C (if control) within a gel, and the value obtained was used as an estimate of relative rate of IP synthesis.

Data calculated by both methods gave similar relative rate values within a series of gels. Such analytical procedures are necessary, as we have no way at present of determining the absolute amount of IP.

RESULTS

In Vitro IP Synthesis after Brief Exposure to Estradiol in Vivo- Previous work in this laboratory has demonstrated the estrogen- induced synthesis of IP in vivo and the dependence of this induction upon prior synthesis of the product of an actinomycin D-sensitive step (16). Fig. 1 illustrates that synthesis of IP is induced and can continue in vitro following very short (1, 5, or 10 min) pulses of estrogens in the animal. The rate of IP synthesis increases with little or no lag period after estrogen and eventually appears to reach a plateau. These kinetics are similar to those


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,DIO min seen when IP synthesis is induced in vivo (see Fig. 4). However, /’

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key ‘5 /’ after hormone injection, and there is a more marked decrease by

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In Vitro Induction of IP Synthesis after Exposure to Estradiol

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1’ .’ “0’5 mm vitro following a brief estrogen pulse in vivo, we investigated whether the induced synthesis of this protein could occur after

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administration of physiological concentrations of estradiol under ww I ‘cf / / completely in vitro conditions. Polyacrylamide gel electro-

Y 5 1 Id// phoretic analysis of uterine soluble proteins synthesized in vitro

G% / following a l-hour in vitro incubation with 3.7 X lo-* M 17fi-

&k estradiol (Fig. 2) demonstrates that synthesis of IP can be in- - 10

0 ;5 30 60 90 120 duced when uteri are exposed to estradiol in vitro. It is seen that TIME (MINUTES) estrogen increased the rate of incorporation of labeled amino acid

FIG. 1. Time course of the relative rate of IP synthesis after into only one protein band, IP, relative to control; reversing the

brief exposure to 17p-estradiol in vivo. Immature rats (three per isotopes used for labeling experimental or control gave the same

group) wereinjected with estradiol (5 pg) or saline aloneanddecap- results and indicated that there was not preferential uptake of one itated at 1, 5, or 10 min after injection. The uteri were excised, isotope by the induced protein. extensively rinsed with Eagle’s HeLa medium, and then incubated in 0.9 ml of Eagle’s HeLa medium at 37” under an atmosphere of

It is also of interest that, although IP synthesis is dependent

95% 02 and 5y0 CO2 for time intervals up to 120 min. Following upon accumulation of the product of an actinomycin D-sensitive

incubation, actinomycin D (31 pg per ml) and labeled leucine (*H step, the induction phenomenon cannot be ascribed to the pres- for estrogen-treated animals and 1% for controls) were added for ence of actinomycin D and, likewise, there is no superinduction a a-hour amino acid incorporation period. Control and estrogen- (20, 21) by actinomycin D (compare Fig. 2, A and B). treated uteri were homogenized together. Following centrifu- gation, the supernatant fraction was separated by polyacrylamide

Comparison of Induced Proteins Synthesized by Uteri Stimu-

gel electrophoresis, and the relative rate of IP synthesis (experi- lated with Estrogen in Vitro or in Viva-The polyacrylamide gel

mental/control) was determined by gel analysis as described under electrophoretic distribution of labeled uterine soluble proteins, “Methods.” Each point represents a single determination. after in vitro or in vivo estrogen stimulation, is seen in Fig. 3. In

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FIG. 2. Electrophoretic distribution on polyacrylamide gels of uteri) into protein for 2 hours at 37” in the presence of 28 pg per ml uterine soluble proteins synthesized in vitro following a l-hour in of actinomycin D (A) or in the absence of actinomycin D (B). vitro incubation with 3.7 X lo-,* M 17p-estradiol. Uteri (three per Control and estradiol-treated uteri in each set were homogenized group) excised from untreated animals were first incubated with together and the supernatant fraction of centrifuged homogenates either 3.7 X 10-* M estradiol or ethanol only (1o/o) in 2.0 ml of was separated by polyacrylamide gel electrophoresis. The radio- Eagle’s HeLa medium for 60 min at 37” and then allowed to incor- activity and 3H:W ratio in each gel slice were determined. porate labeled leucine (3H for estradiol-treated and 1% for control


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FIG.!% Co-electrophoresis on polyacrylamide gel of uterine soluble proteins synthesized in vitro after a l-hour in vitro or in uivo estrogen stimulation. Uteri (five per group) from animals injected intraperitoneally 60 min prior to sacrifice with 5 pg of 17& estradiol were rinsed with Eagle’s HeLa medium and then incubated with [sH]leucine and actinomycin D for 2 hours in 1.8 ml of Eagle’s HeLa medium. Uteri (five per group) excised from untreated animals were incubated with 2.0 X 10-S M 17&estradiol for 60 min in 1.8 ml of Eagle’s HeLa medium, rinsed, and transferred to 1.8 ml of fresh Eagle’s HeLa medium containing [YJ]leucine and actinomycin D for a further 2-hour incubation. Uterine soluble proteins were prepared and subjected to electrophoresis as described under “Methods.” The radioactivity and 3H:W ratio in each slice were determined.

this experiment, uteri from animals receiving 5 pg of estradiol intraperitoneally 60 min prior to sacrifice were incubated with [3H]leucine in vitro; uteri from untreated animals were excised and first, incubated with 2.0 x lo-* M e&radio1 for 60 min in vitro

and were then incubated with [W]leucine in vitro. Co-electrophoresis of equal volumes of uterine supernatant fractions after either in vitro or in vivo estrogen stimulation (Fig. 3) demonstrates the coincidence of the in vitro ([W]leucine) and in vivo ([3H]leu- tine) induced proteins. Furthermore, the magnitude of the in vitro response was approximately 85a/, of that obtained in vivo. It, should be noted that the over-all pattern of newly synthesized soluble proteins, as evidenced by correspondence of the 3H and 14C radioactivity throughout the gel, is similar after in vitro or in oivo e&radio1 administration. (The elevated 3H:14C ratio at slices 51 and 52 is in a region of low absolute counts, so that this increased ratio is probably of no significance.)

Time Course of Rate of IP Synthesis (IP-synthesizing Ca- pucity)-Fig. 4 shows the relative rate of IP synthesis as a function of duration of incubation with estrogen in vitro. After incubation with either 4.1 X lo-* M or 4.1 x 10mg M estradiol for time intervals of up to 150 min, the uteri were allowed to incorporate labeled amino acid into protein in the presence of actinomycin D. The rate at which IP is synthesized, relative to control, can be considered an index of IP-synthesizing capacity.

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g 1.0 L-- 1. m--ILI 0 15 30 60 90 120 150

TIME (MINUTES)

FIG. 4. Time course of the relative rate of IP synthesis at two different 17p-estradiol concentrations in vitro. Uteri (three per group) excised from untreated animals were incubated in 0.9 ml of Eagle’s HeLa medium containing either 4.1 X 10-S M or 4.1 X lo+ M estradiol (experimentals) or ethanol (controls) at 37’ for time intervals up to 150 min. At the end of each time period, experimental uteri were allowed to incorporate [aH]leucine and control uteri were allowed to incorporate [l%]leucine into protein in the presence of actinomycin D for 2 hours at 37”. Control and estrogen-treated uteri were homogenized together, and the supernatant fraction of centrifuged homogenates was separated by acrylamide gel electrophoresis. From analysis of such gels, the relative rate of IP synthesis (experimental/control) was determined as described under”Methods.” The solid line indicates the time course of the relative rate of IP synthesis following different periods of in vivo estradiol (5 fig per rat) and is from data reported by De- Angelo and Gorski (16).

This capacity accumulates in the period following estrogen administration and prior to the addition of actinomycin D; it therefore is an estimate of the amount, of one or more components required for IP synthesis that are produced in an actinomycin D-sensitive step. The rate of IP synthesis, or accumulation of IP-synthesizing capacity, is readily detectable by 15 min and reaches about 50% of maximal level by 20 min at the higher hormone concentration and by 30 min at the lower hormone concentration. It is of interest that both e&radio1 concentrations evoke the same final maximal response although it is reached faster at the higher hormone concentration. The early phase (0 to 60 min) of the rate curve of IP synthesis in vitro is similar to that obtained in vivo. At later times, however, the curves diverge as the apparent rate of IP synthesis falls off sharply in vivo.

The rate of change of IP-synthesizing capacity after various periods of exposure to e&radio1 is presented in Fig. 5. Whether IP-synthesizing capacity is accumulated in viva or in vitro in uteri of immature or mature ovariectomized rats, the same basic rate profile is observed; that is, the rate of accumulation of synthesizing capacity is highest, initially and falls off rapidly with time (by 30 min). Thus, the rate of accumulation of synthesizing capacity is decreasing at the same time estradiol concentration is continuing to increase (or remains at a high level) in the nucleus (see “Discussion”). However, significant amounts of estradiol have already accumulated in the nucleus at the time at which IP-synthesizing capacity is increasing at maximal rate (by 15 min) (Fig. 5).

Specificity of in Vitro IP Induction-Only estrogenic compounds (17P-estradiol, diethylstilbestrol, estriol, 17cr-estradiol) were able to elicit IP synthesis in vitro, whereas progesterone (10e6 or lo-* M) and testosterone (lo-8 M) did not. At lo-lo M


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I Y- IN VIVO E,, MATURE

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TIME (MINUTES)

FIG. 5. Rate of change of IP-synthesizing capacity after various periods of exposure to 17p-estradiol. Immature rat uteri were incubated in vitro with either 4.1 X lo-* M (0-O) or 4.1 X 10e9 M (O---O) estradiol. R t f h a e o c ange of IP-synthesizing capacity following in vivo estradiol administration to immature rats (5 pg per rat, A- - -A) or mature, ovariectomized rats (10 rg per rat, t-m) have been calculated from data reported by DeAngelo and Gorski (16). Rates are expressed in arbitrary units. Data on the amount of bound [3H]estradiol in immature rat uterine nuclear extracts as a function of time of incubation in vitro with 2 X lo-* M [sHIestradio at 37” (A-A) are presented for comparison (4).

all estrogens gave essentially no induction (see also Fig. 6), and at 10V6 M all gave the same degree of induction. At 10-S or low9 M the most efficient inducer was 17/3-estradiol (set at 100%) > diethylstilbestrol (-60 %) > estriol (~50 ‘%) > 17cr-estradiol (~30%). These values for relative effectiveness in inducing IP synthesis are very similar to values reported for the relative potency of these compounds based on vaginal mitotic count data (22).

E$ect of Cyclic Adenosine S’,6’-Monophosphate and Dibutyryl Cyclic Admosine S’,5’-Monophosphate on in Vitro Induction of IP Synthesis-Cyclic adenosine 3’) 5’-monophosphate and N6,02’-dibutyryl cyclic adenosine 3’,5’-monophosphate, at concentrations of lop3 or 10e5 M, gave no detectable induction of IP synthesis (l-hour incubation, 37”). When these compounds (lOma M) were added simultaneously with estradiol, the cyclic nucleotides neither enhanced nor diminished the induction of IP due to estradiol.

Dose-Response Curve with 17&Estradiol; Correspondence between Magnitude of IP Induction and Amount of Nuclear Bound [8H]EstradioZ-The magnitude of IP induction (after 60 min of incubation) is depicted as a function of 17&estradiol concentration in Fig. 6. For comparison, the amount of nuclear bound [3H]estradiol as a function of in vitro hormone concentration (again after 60 min of incubation) (4) is also plotted.

LOG M (17B-ESTRADIOL)

FIG. 6. Effect of in vitro 17p-estradiol concentration on the rate of IP synthesis. Uteri (five per group) excised from untreated rats were incubated in 2.0 ml of Eagle’s HeLa medium containing various concentrations of 17p-estradiol (or ethanol for controls) for 60 min at 37”. At this time, [aH]leucine plus actinomycin D were added to experimental flasks and [Wlleucine plus actinomycin D were added to control flasks, and amino acid incorporation was allowed to proceed for 2 hours at 37”. Uterine soluble proteins were subjected to electrophoresis on polyacrylamide gels, and IP synthesis was quantitated as described under “Methods.” Each point is the average of two determinations, except for values at 1 X 10-T M and 2 X lo-& M, which represent single determinations. Results are expressed relative to IP induction with 3 X 10e8 M estradiol being 100%. Values for bound 13H1178-estradiol _ .- in immature rat uterine nuclear extracts after a 60.min incubation with various concentrations of [aH]estradiol in Eagle’s HeLa medium at 37” are from Giannopoulos and Gorski (4).

Estradiol at 2 to 3 X lo-* M gave a maximal response (IP induction and nuclear binding) and at 2 to 3 x 10eg M gave a 50% response; at lo-lo M, induction and binding (the latter not shown) were minimal. At high estradiol concentrations (above 10-T M) there was a decreased response; in a single experiment, 2 X 10m6 M hormone gave 68% of the maximal level of IP induction.

Capacity of Excised Uteri to Respond to 17fi-Estradiol Xtimula- tion after Various Periods of Incubation in Estrogen-jree Medium- When uteri were incubated in Eagle’s HeLa medium at 37” for various periods of time prior to addition of estradiol and then subsequently assayed for rate of IP synthesis, it was seen that the uteri rapidly lost their ability to synthesize IP at maximal rate (Fig. 7). By 1 hour, estradiol was able to elicit only about 400/, of the initial response and there was thereafter a more gradual, yet continual, decline in ability of the uteri to respond to estradiol stimulation with the induced synthesis of IP. How- ever, even after 8 hours of incubation, the tissue still showed 18 7, of the initial response.

Although insulin has been reported to increase protein synthesis in mouse uteri (23) and in chick oviduct (24) in culture, and to augment the effects of estrogen on uterine growth (25, 26), inclusion of insulin (20 milliunits per ml) in 37” incubations

had little effect on the rate of loss of IP inducibility. Loss of tissue responsiveness is markedly temperature de-

pendent. When uteri were incubated in Eagle’s HeLa medium at O-1” prior to addition of estradiol, the tissue showed a very slight (under 5%) decrease in responsiveness over time periods of at least 4 hours (Fig. 7).


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OL._~ L.-.. LIL----L -.-i. I --- .I

0 1 2 3 4 5 6 7 8 HOURS IN CULTURE BEFORE ESTRADIOL

FIG. 7. Capacity of excised uteri to respond to 17p-estradiol stimulation after various periods of incubation in estrogen-free medium. Experimental and control uteri (three per group) were incubated in 0.9 ml of Eagle’s HeLa medium at O-l”, 37”, or 37” in medium containing 0.82 rg per ml (20 USP milliunits per ml) of insulin for time intervals up to 8 hours. After addition of 4.1 X 10-S M estradiol (in ethanol) or ethanol alone (l.loj, for controls), flasks were incubated at 37” for 60 min. Flasks then received 31 pg per ml of actinomycin D (except insulin-containing incubations, which received 28 pg per ml of actinomycin D) ; experimental flasks received in addition either 20 pCi of [aH]leucine or 5 pCi of [‘“Cl- leucine, and corresponding control flasks received the oppositely labeled leucine. Amino acid incorporation was for 2 hours at 37”; then control and estrogen-treated uteri were rinsed with and sep- arately homogenized in 0.05y0 Na,EDTA. Homogenates were centrifuged and the resultingsupernatant fractions were subjected to co-electrophoresis on polyacrylamide gels. The relative rate of IP synthesis was estimated by gel analysis as described under “Methods” and the data were expressed as percemage of initial rate of IP synthesis.

DISCUSSION

The foregoing data demonstrate the estrogenic induction of IP synthesis under in vitro conditions. By several criteria, the in viva and in vitro estrogen-induced systems are very similar. The electrophoretic patterns on polyacrylamide gels of uterine- soluble proteins synthesized in vitro after administration of estrogen in vivo or in vitro are essentially the same. Likewise, the induced proteins synthesized after either in vivo or in vitro hormonal administration migrate to the same positions on coelectrophoresis (Fig. 3). In both the in vivo (27) and in vitro situations, induction of IP synthesis shows a strict hormonal specificity. Only estrogenic compounds induce, whereas progesterone, testosterone, and insulin do not. Among several estrogenic compounds, the effectiveness of induction followed the sequence, 17&estradiol > diethylstilbestrol > estriol > 17a-estradiol, thus showing a close correlation between IP induction, relative

affinity for the uterine binding proteins, and estrogenic potency of these compounds (5, 22, 28-31).

Although several workers have reported responses of whole uteri (7, 9), endometrial tissue slices (6, 8), and chick oviduct (32) to estrogen in vitro, such responses have required consid- erably higher concentrations of estrogen (i.e. 2 x 10m5 to 1 X lo-’ M estradiol) than we have employed and have usually been of a minimal magnitude compared with the in vivo response. For example, Mayo1 and Thayer (9) have reported approsi- mately 20% of the normal in vivo induction of the synthesis of a uterine protein(s) in vitro but only by administration of lOafold higher (2.5 x 10e6 M) estrogen concentrations than physiological. It is not clear, however, from comparisons of their electrophoretic gel patterns with ours, whether the protein they report is the same as our induced protein. Estradiol concentration in the peripheral plasma of rats following infusion with estradiol to half-maximal saturation of uterine receptor sites (14) or in the plasma of normal cycling rats (33) is 1 to 2 X lo-lo M. Similar values reported for the human indicate that the peripheral plasma estradiol concentration varies between 1 x lo-lo and 2 x 10Vg 31 during the female menstrual cycle (11-13).

Of significance is the fact that there is a close correspondence between the magnitude of 11 induction and the amount of nuclear bound [3H]estradiol (4) at different estradiol concentrations (Fig. 6). Maximal IP induction and maximal binding of [3H]estradiol to nuclear receptor are obtained with 2 to 3 x lo-* M estradiol, and 2 to 3 x lop9 M estradiol gives a 50% response with both. Thus, the in vitro induction of IP clearly occurs in the physiological range and appears to parallel the dose-response of estrogen binding in the uterus (see Fig. 6).

The observation in this report that actinomycin D in vitro blocks the uterine response to in vitro estrogen strengthens the hypothesis advanced earlier (16), namely, that estrogen is inducing the synthesis of a specific RNA for IP; this in vitro system eliminates at least some of the obvious toxic effects caused by in vivo administration of actinomycin D. However, while it is tempting to speculate that the actinomycin D sensitivity of IP production implies de novo messenger RNA synthesis and that the rate of IP synthesis is proportional to the amount of accumulated IP-RNA, the complexities of transcription and translation processes in the tissues of higher animals, as discussed elsewhere (24)) indicate that this view may be a simplistic one. Therefore, the term IP-synthesizing capacity is used to indicate encompass- ment of all factors that may be involved in determining the rate of IP synthesis, including possible message synthesis, as well as factors involved in its transport, stability, and translation.

If there is direct mediation of IP-synthesizing capacity by receptor-bound estradiol, one might expect the rate of synthesis of this capacity to be proportional to the amount of nuclear bound estradiol. The accumulation of nuclear bound e&radio1 is a rapid process, reaching approximately 80% of maximal in 15 min. At these early times (prior to 15 min), there may well be proportionality between rate of synthesis of this capacity and amount of nuclear bound estradiol, but because of experimental limitations in our measurement of small amounts of IP, we cannot at present accurately monitor accumulation of this capacity before 15 min. However, from 15 min on, when nuclear estradiol increases from about 80% of maximal to maximal, the rate of accumulation of IP-synthesizing capacity is already rapidly falling off (Fig. 5).


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Issue of February 25. 1972 B. 8. Katxenellenboym and .J. Gorski 1305

It is obvious that a more complex model is necessary to explain the data on estrogen binding and IP synthesis. A simple feed- back mechanism by which newly synthesized IP inhibits further production of synthesizing capacity is unlikely since synthesis of IP does not start until 45 to 60 min after estrogen, well after the onset of the decline in rate of accumulation of IP-synthesizing capacity. I f RNA synthesis is the critical factor in IP-synthesizing capacity, then some secondary process may shut off RNA synthesis or affect its translation. The decline in tissue responsiveness observed after incubation in estrogen-free medium (Fig. 7) may account for some of this fall-off in rate of accumulation of IP-synthesizing capacity with time. However, the fact that this decline in rate of accumulation also occurs in viva (Fig. 5) suggests that a secondary process is probably involved.

It is also possible that the nuclear binding of estrogen as meas- ured in the total tissue is not critically related to the phenomenon reported here and that we must be careful in using binding data as our sole evidence for localizing the site of action of a hormone.

hlthough cyclic adenosine 3’) 5’.monophosphate or its dibutyryl analog have been implicated in the action of several protein hormones (34)) considerable controversy exists regarding their possible participation in the action of steroidal hormones (3.538). Neither of these compounds alone, at 10e3 or lop6 M,

was able to elicit the induction of IP; and when added simultaneously with estradiol, they neither enhanced nor diminished the induction of IP due to estradiol. Thus, at least in this estrogen- induced response, cyclic AMP does not appear to participate.

It is apparent from the in, vitro inducibility of this response that intact nervous and vascular systems are not required; therefore, these systems are unlikely candidates for the primary action of this hormone.

The hormone-responsive mammalian system described here, with rapid induction of uterine IP synthesis in vi&o, offers a unique opportunity for studying the regulation of a specific protein’s synthesis by estradiol. This system should prove useful in allowing a clear correlation of early biochemical changes with the various estrogen-binding phenomena that have been investigated recently in uterine tissue (2, 4, 5, 39).

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Benita S. Katzenellenbogen and Jack GorskiUTERINE PROTEIN

: INDUCTION OF THE SYNTHESIS OF A SPECIFICin VitroEstrogen Action

1972, 247:1299-1305.J. Biol. Chem.

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