PART
Iv. ACTIONSON THE FEMALEGENITAL TRACT REGULATION OF ESTROGEN RECEPTOR REPLENISHMENT BY PROGESTERONE* James H. Clark, A. J. W. Hsueh,t and Ernest J. Peck, Jr. Department of Cell Biology Baylor College of Medicine Houston, Texas 77030
INTRODUCTION The cyclic changes in female reproductive tissues have been studied extensively by reproductive biologists. The periodic variations in blood levels of estrogen and progesterone are believed to be responsible for the changes observed in uterine tissue throughout the estrous and menstrual cycles. With the discovery of steroid hormone receptors, it became apparent that variations in tissue levels of hormone receptors might also act as a regulatory step in the control of reproductive cyclicity. It has been suggested that progesterone might antagonize the action of estrogen by reducing the amount of cytoplasmic estrogen receptor, Rc.i-3 Recent work from our laboratory indicates that this is one of the mechanisms by which progesterone and nonsteroidal antiestrogens exert their antagonistic eff e c t ~ . Another ~-~ possibility is that progesterone might interfere with the nuclear binding of the receptor-estrogen complex and thus reduce or modify its effects on nucleus-mediated events. In this paper, we have examined the influence of progesterone on estrogen receptor replenishment and nuclear binding in immature and adult rats.
MATERIALSA N D METHODS Preparation of Animals and Materials
Immmature (22 days old) female Sprague-Dawley rats were purchased from Texas Inbred Mice Co. and housed in a controlled environment with 13 hr of artificial light between 7 A M and 8 PM. All rats received two daily subcutaneous injections of 17p-estradiol(2.5 pg in 0.5 ml of 0.9% saline solution that contained I % ethanol). Pretreatment with estradiol was performed to increase the sensitivity of the uterus to progesterone, presumably via estrogen's effect of increasing the synthesis of the progesterone On the third day, rats were randomly divided into three groups. Animals in one group were injected with 2.5 pg of estradiol (E) and 0.2 ml of sesame oil. Animals in a second group were injected with 2.5 pg of estradiol plus 2.5 mg of progesterone ( E + P) dissolved in 0.2 ml of sesame oil. Animals in a third group were injected with 2.5 pg of estradiol plus I mg of testosterone propionate (E T) dissolved in 0.2 ml of sesame oil. In all groups,
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*Supported by National Institutes of Health Grants HD-04985 and HD-08389 and by American Cancer Society Grant BC-92. t h e s e n t address: National Institute of Child Health and Human Development, Bethesda, Md. 20014.
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the sesame oil or steroids dissolved in oil were injected at a subcutaneous site separate from that of estradiol injection. Animals were killed at various intervals after injection to assess the effect of progesterone and testosterone propionate on the level of uterine estrogen receptors. In experiments designed to test the uterine responsiveness of the various groups to subsequent estrogen treatment, animals were further injected with 2.5 pg of estradiol in 0.5 ml of saline on Day 4 and killed 24 hr later. All animals were killed by cervical dislocation; the uteri were stripped of adhering fat and mesentery and weighed (wet weight). Some uteri were used for the determination of nuclear and cytoplasmic receptor concentrations by [3H]estradiol e x ~ h a n g e . ~Some . ' ~ uteri were saved for proteinll and DNA determinationsI2 or dried overnight in an 80°C oven for dry weight determination. Ovariectomized adult (body weight 200-250 g) female Holtzman-strain rats were purchased from Hormone Assay Co. (Chicago, Ill.) and used 3-5 weeks after the operation. All rats received one subcutaneous injection of 10 pg of 17pestradiol in 1 ml of saline solution that contained I % ethanol. On the second day, rats were randomly divided into three groups. Animals in group A were injected with 10 pg of estradiol in 1 ml of saline plus 0.5 rnl of sesame oil (E). Animals in group B were injected with 10 p g of estradiol in 1 ml of saline plus 6.5 mg of progesterone dissolved in 0.5 ml of sesame oil ( E + p). Animals in group C were injected with 10 p g of estradiol plus 5 mg of hydrocortisone 21-acetate dissolved in 0.5 ml of sesame oil ( E HA). In all groups, the sesame oil or steroids in oil were injected at separate subcutaneous sites. Twenty-four hours later (Day 3), the animals were sacrificed; the uteri were stripped of adhering fat and mesentery and weighed with luminal fluid (whole uterine weight). The uteri were then slit to release luminal fluid, pressed against filter paper, and reweighed (empty uterine weight) to enable the amount of fluid to be calculated by difference (luminal fluid weight). The uteri were then analyzed for cytoplasmic and nuclear receptor levels by the [3H]estradiol exchange assay, as described above. To determine the uterine sensitivity to further estradiol treatment, some animals were injected with 1 ml of saline or 10 pg of estradiol in I ml of saline in Day 3 and analyzed for whole or empty uterine weight, dry weight, and DNA content 24 hr later. In experiments designed to study the cytoplasmic receptor concentration in rats during the estrous cycle, Purdue Wistar virgin female rats (4-6 months old) were used. Vaginal smears were taken daily, and only adult rats that had completed at least two 4-day cycles were used. All animals were killed between 9:30 and 10:30 AM by decapitation. Their uteri were removed, and cytoplasmic receptor concentration was determined by the [) Hlestradiol exchange a ~ s a y . ~ * ' O
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Sepciration of Myometrial and Endometrial Components
To measure receptor concentration in different components of the uterine tissue, gentle scraping was used to separate rnyometrium from the rest of the uterine tissue. Uterine tissues stimulated with hormones were taken from castrate rats on Day 3 of hormone treatment and dissected carefully under a dissecting microscope. Endometrial tissues were gently scraped from the remaining myornetrial tissues by a pair of fine forceps, and these two fractions were used for the [3H]estradiol exchange assay. Histologic examination revealed that the myometrial fraction was completely devoid of endometrial components, whereas the endometrial fraction was composed of epithelial and stromal elements, in addition
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to small fragments of myometrium. Therefore, the latter fraction was called the enriched endometrial fraction. Determination of Cytoplmmic Binding Sites by the ['HI Estradiol Exchange Assay The uterine tissues were homogenized in all-glass Kontes homogenizers under ice and then centrifuged at 800g for 10 min. The pellet fractions were used for the determination of nuclear binding sites by the [3H]estradiol exchange assay.9 The supernatant fraction was treated with an equal volume of activated charcoal
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FIGURE I . Effect of progesterone on the estrogen-induced depletion and replenishment of the cytoplasmic estrogen receptor. Immature rats received 2.5 pg of estradiol for 2 days and on the third day were injected with either estradiol ( 0 ) or estradiol plus progesterone (A). The quantity of cytoplasmic estrogen receptor was determined on Days 0-2 and at various times after estrogen or estrogen plus progesterone treatment on Day 3. All values are expressed as the percentage of control based on the receptor concentration on Day 3. Numbers in parentheses represent the numbers of experiments with four animals per experiment.
suspension (0.5% charcoal and 0.05% dextran) at 4°C for IS min to remove free steroids. The tubes were then centrifuged for 15 min at 8OOg. The supernatant fractions were dispensed into two series of tubes that contained either 13 nM [3H]estradiolfor determination of total binding or 13 nM [3H]estradiol plus a 100fold excess of diethylstilbestrol (DES) for determination of nonspecific binding. The tubes were incubated at 23°C for 18-20 hr. At the end of incubation, the tubes were cooled to 4°C for 10 min, and the charcoal suspension was added. Incubation was continued for IS min at 4"C, and the tubes were centrifuged at 800g for 15 min. Aliquots of the supernatants were added to 3 ml of ethanol and 10 ml of scintillation fluid ( 5 . 5 g of Permablend@I. Packard Instruments Co., Chicago, I l l . , in I
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medE 0 on d P 24
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FIGURE2. The effect of delayed progesterone treatment on the quantity of cytoplasmic estrogen receptor. Immature rats received 2.5 pg of estradiol for 2 days. On Day 3, progesterone was administered at various times after an injection of estradiol, and cytoplasmic receptor levels were determined 24 hr after estradiol treatment.
liter of toluene). Radioactivity was determined with a Packard liquid scintillation spectrometer at 25% efficiency. RESULTS Eflects of Progesterone on Cytoplasmic Levels of Estrogen Receptor
We have previously demonstrated that progesterone treatment causes a significant reduction in the amount of cytoplasmic estrogen receptor, R,, 24 hr The reduced R, also correafter injection in estrogen-primed immature lated with a reduced sensitivity to estrogen injection on Day 4. To gain further information on the mechanism of action of progesterone on the reduction of estrogen receptor concentration, the quantities of R, and RiE were determined by [3H]estradiol exchange at various stages of hormonal treatment. As shown in FIGURE 1, pretreatment with estradiol on Days 1 and 2 increases the concentration of R, in uterine tissue. On Day 3, animals were divided into two groups, and estradiol (E) or estradiol plus progesterone (E + P) were injected. The quantity of R, fell to low levels within 1 hr of injection and began to increase gradually at 4-8 hr. During the time of R, depletion, there was a concomitant increase in the amount of R,E in the nucleus in both treatment groups (see FIGURE 5). However, between 8 and 24 hr after injection, the R, level continued to increase in the E treatment
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group but remained constant in the E + P treatment group. This observation suggested that two separate processes were involved in the replenishment of R, and that progesterone might inhibit the replenishment process between 8 and 24 hr after estradiol injection. No significant effect of progesterone on uterine DNA or protein content was observed on Day 4.6 To further study the time sequence of progestational inhibition of R, replenishment, progesterone was injected at various intervals after estradiol treatment on Day 3, and the level of R, was measured 24 hr after estradiol injection. As can be seen in FIGURE 2, progesterone injection at 4-8 hr after estrogen injection decreased the quantity of R, to the level observed when progesterone was injected simultaneously with estradiol. However, when progesterone was injected between 8 and 24 hr after estrogen, the level of R, 24 hr after E treatment increased gradually, thus suggesting an escape from the antagonistic effect of progesterone. These data suggest the existence of two separate processes for receptor replenishment. The ability of progesterone to cause a reduction in R, appears to be specific for estrogen antagonists. Testosterone propionate ( I mg Day 3) caused a 25% reduction in R, and a slight decrease in estrogen-stimulated uterine weight (data not shown). Other steroids, such as hydrocortisone 21-acetate, have no effect on R, replenishment (to appear elsewhere). Progesterone has no effect on R, in the nonestrogen-primed uterus. Presumably, this finding reflects an insensitivity of the uterus to mogesterone that results from low quantities of progesterone recep-
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FIGURE3. Stereospecificity of the cytoplasmic estrogen receptor after estrogen plus progesterone treatment. The ability of various compounds to compete for estradiol-binding sites was measured by competitive binding analysis of uterine cytosol obtained on Day 4 from estrogen plus progesterone-treated rats. Compounds were added to receptor assays at 100 times the concentration of [)H]estradiol. Abbreviations: diethylstilbestrol, DES; progesterone, P; corticosterone, B; cortisol, F;dexamethasone, D; and testosterone, T.
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1 3 H I ESTRADIOL (nMI
I I H3- ESTRADIOL (Llmde x lo9,
FIGURE 4. Binding parameters of the cytoplasmic estrogen receptor after estrogen plus progesterone treatment. Uterine cytosol was incubated with various concentrations of ['Hlestradiol, and the quantity of bound receptor was determined as described in the text. The data are plotted as a saturation curve (I& and a double reciprocal plot (right).
Properties of Cytoplasmic Estrogen Receptor After Progesterone Treatment
Progesterone could alter steroid binding parameters of R, rather than cytoplasmic levels of R, and thereby affect the ability of the uterus to respond to estrogen. To examine some of these properties, cytosol was prepared from estras AND diol plus progesterone-treated animals, as described under MATERIAI
METHODS. To test the steroid specificity of R, on Day 4, various steroids were used as potential competitors for [3 Hlestradiol binding by R, in the charcoal adsorption assay. Estrogen binding was inhibited by the presence of a 250-fold excess of DES but not by progesterone, corticosterone. cortisol, dexamethasone, or testosterone (FIGURE 3). The dissociation constant, Kdr was examined by incubating uterine cytosol with various concentrations of [3 Hlestradiol or [3 Hlestradiol plus a 100-fold 4). The saturation characteristics and K d (6 x M) did excess of DES (FIGURE not differ significantly from those of the estrogen receptor found in control uteri and reported by other^.^.^,^ Nuclear Accumulation and Retention of Estrogen Receptor in Estrogen- or Estrogen plus Progesterone-Treated Animals
We have suggested that retention of the receptor-estrogen complex for longer than 4-6 hr is a requirement for true uterine growth.lLI5 Therefore, any interaction that interferes with this process is likely to be antagonistic. Accumulation and retention of R,E were measured at I , 6, and 24 hr after injection of estradiol on Days 3 and 4 in immature animals (FIGURES 5 & 6). On Day 3, the quantity of R,E did not differ between either group: however, the total quantity of R,E that was retained at the 4- and 24-hr points in the E + P-treated group was less than that in the E-treated group (FIGURE 5). On Day 4 , the quantity of R,E was significantly reduced in E' + P-treated animals 1 hr after injection, as compared to estrogentreated animals. By 6 hr, the quantity of R,E in E P-treated animals had declined to levels that were not significantly different from those of controls,
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whereas the level of R,E in estradiol-treated rats was maintained above control 6). Because R, in the E P-treated group was lower on Day 4, the levels (FIGURE decrease in R,E that was observed in this group may be caused by a reduction in the quantity of R, that is available for translocation.
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Effrct of Progesterone on Quantity of Estrogen Receptor and Uterine Responsiveness in Adult Castrate Rats
As shown in FIGURE 7, progesterone treatment caused a significant decrease in cytoplasmic estrogen receptor, R,, content. A reduction is also observed in levels of nucleus-bound estrogen receptor, although these quantities in all groups were small and therefore have little influence on the total amount of receptor. The decrease in quantity of R, does not result from a general depressive action of progesterone on uterine protein synthesis, because the decrease is significant. even when the data are expressed as sites per milligram of protein (25.9 1.9 pmol/100 mg of protein for the E pretreatment; 1 I. 1 -+ 1.7 pmoV100 mg of protein for the E + P pretreatment group, p < 0.01). We have previously shown that testosterone has a weak effect on R, replenishment and that this effect correlates with its weak antiestrogenicity.6 To further examine the specificity of the depressive effect of progesterone on uterine R, content, estrogen-treated adult castrate rats were injected with hydrocortisone 21acetate, as explained under MATERIALSA N D METHODS.This treatment caused no
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FIGURE5. The effect of progesterone on the nuclear retention of estrogen receptor on Day 3. Immature rats received 2.5 pg of estradiol for 2 days and on the third day were injected with either estradiol (0) or estradiol plus progesterone (a). The quantity of R,E was determined I , 4, and 24 hr after iiection.
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significant decrease in either cytoplasmic or nuclear estrogen receptor concentration (FIGURE 7). These data suggest that the depressive effect of progesterone on estrogen receptor level is hormone specific. To demonstrate that the reduced level of estrogen receptor does reflect a decrease in uterine responsiveness in progesterone-treated animals, animals in E and E + P pretreatment groups were injected with E or saline on Day 3, and the uterine weights were measured 24 hr later. The uterine weights (whole, empty, and dry) and luminal fluid weights at various stages of treatment were measured, and the results are shown in FIGURE 8. Progesterone depressed the whole uterine weight dramatically within 24 hr after treatment (Day 3). A significant decrease in
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FIGURE6. Nuclear accumulation and retention of receptor-estrogen complex in estradiol or estradiol plus progesterone-treated animals on Day 4. Immature female rats were treated with estradiol(0) or estradiol plus progesterone (El) as described in the text. On Day 4, they were injected with 2.5 pg of estradiol, and the nuclear level of the complex was measured 0, I , and 6 hr after treatment.
uterine luminal fluid weight was also observed, which appears to be responsible for the reduction in whole uterine weight. Slight reductions in empty uterine and dry weights were observed on Day 3. Estradiol injection on Day 3 further increased the difference in whole uterine and luminal fluid weights between the two groups on Day 4. Estradiol treatment on Day 3 did not increase the uterine weight responses in the E + P pretreatment group but increased significantly that of the E pretreatment group, thus indicating a decreased responsiveness of the E + P pretreatment group to further estrogen treatment. No appreciable difference among treatment groups was observed when empty uterine weights were compared to those of saline-injected controls (TABLE1).
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E tP
E
. -
FIGURE 7. The effect of Droeesterone and hvdrocortisone acetate on the auantitv of estrogen receptor in the uteri of adult castrate rats. Three groups of castrate rats were given 10 pg of estradiol. Twenty-four hours later, the animals were injected with 10 pg of estradiol (E), 10 pg of estradiol plus 6.5 mg of progesterone (E + P), or 10 pg of estradiol plus 5 mg of hydrocortisone acetate ( E + HA). Twenty-four hours later, the animals were sacrificed, and and total (M) estrogen receptor their uteri were analyzed for cytoplasmic ( 0 ) .nuclear (I), contents by the ['Hlestradiol exchange assay. The data represent the mean SE from six to eight experiments with three or four animals per experiment.
*
TABLEI PROGESTERONE ANTAGONISM OF ESTROGEN-INDUCED UTERINEGROWTH* Percent Percent Percent PreWhole of Empty. of Dry of treatment Treatment Uterine Saline Utenne Saline Weight Saline (Day 2) (Day 3) Weight (mg) Control Weight (mg) Control (mg) Control E(1O)t 528.02 51.0 187 237.026.4 157 47.1 2 1.8 144 E S(6) 282.0251.8 100 151.02 9.6 100 32.6+ 3.2 100 E(9) 176.02 3.7 114 163.02 3.6 116 3 2 . 9 2 1 . 1 109 E+P S(6) 155.5 2 11.8 100 141.8 2 8.8 100 30.3 t 1.6 100 *All animals were injected with 10 pg of 17p-estradiol on Day 1. Twenty-four hours later (Day 2), they,were divided into two groups. Animals in the E pretreatment group were injected with 10 p g of estradiol plus 0.5 ml of sesame oil. Animals in the E + P pretreatment group were injectetl with 10 pg of estradiol plus 6.5 mg of progesterone dissolved in 0.5 ml of sesame oil. On Day 3, animals in both groups were injected with either 1 ml of saline (S) or 10 pg of estradiol dissolved in I ml of saline (E). All animals were sacrificed 24 hr after the last injection, and uterine weights were determined. tNumbers in parentheses represent the numbers of animals used for each determination.
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However, estradiol treatment on Day 3 increased the uterine weight responses in both E and E + P pretreatment groups, as compared to those of saline-injected controls. However, the ability of estradiol to increase uterine weight was significantly depressed (p < 0.01) in the E + P pretreatment group, as compared with its effect on the estrogen pretreatment group. Estradiol treatment after estrogen pretreatment caused dramatic uterine growth over the saline control
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FIGURE 8. The effect of progesterone on uterine weight response in adult castrate rats. All animals were injected with 10 pg of 17fl-estradiol on Day 1 . Twenty-four hours later (Day 2), they were divided into two groups. The animals were injected with either 10 p g of estradiol plus 0.5 ml of sesame oil ( 0 ) or with 10 pg of estradiol plus 6.5 mg of progesterone dissolved in 0.5 ml sesame oil (0).On Day 3, the animals in both groups were injected with 10 p g of estradiol. Animals were sacrificed at various days of the experiment, and their uterine weights (whole, empty, and dry) and luminal fluid weights were determined, a s described in MATERIALS AND METHODS.The data represent the mean -+ SE from at least 10 animals in each group. group, whereas estradiol treatment in the E + P pretreatment group caused only very slight stimulation. These data indicate that the antagonistic effect of progesterone on uterine growth response correlates with its depressive effect on the quantity of estrogen receptor (FIGURE 7). The possible influence of progesterone on uterine DNA content was examined because it has been shown that progesterone antagonizes estrogen-induced cell di-
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vision in the chick On Day 3 , uterine DNA content was not significantly different between the two pretreatment groups (1.39 ? 0.10 mghterus in the E pretreatment group; 1.33 ? 0.12 in the E + P pretreatment group). Twenty-four hours after injection of estradiol on Day 3 , uterine DNA content increased in both the E + P pretreatment group (1.67 2 0.15 mghterus) and the E pretreatment group (1.51 2 0.11 mghterus). Thus, no antagonistic effect of progesterone on uterine DNA synthesis was demonstrated. The effect of hydrocortisone 21-acetate (HA) on uterine responsiveness was also tested. Animals in the E + HA pretreatment group were injected with estrogen on Day 3 , and the uterine weights were measured 24 hr later. Hydrocortisone 21-acetate did not cause any appreciable change in any of the uterine growth parameters when compared to those in the estrogen pretreatment group 7). (FIGURE Measurement of Estrogen Receptor Content in Myometrial and Enriched Endometrial Fractions of E- and E + P-Treated Animals
To establish that receptor replenishment is similar both in endometrium and in myometrium, the endometrium and myometrium from adult castrate rats were carefully separated, as described under MATERIALS A N D METHODS.On Day 3 of treatment. estrogen receptor content was measured in nuclear and cytoplasmic 9). A significant inhibition of fractions of these two uterine components (FIGURE cytoplasmic receptor content in E + P-treated animals was observed in both myometrium and enriched endometrium. In addition, a similar decrease was also demonstrated in nuclear fractions of both tissue types. Cyclic Flirctrration of Estrogen Receptor Content During the Estrous Cycle
The concentration of estrogen receptor in the cytoplasmic fraction of the uterus was measured during different stages of the estrous cycle. As shown in FIGURE10, no significant difference in R, content could be found during diestrus, proestrus, and estrus, whereas a significant decrease was observed at metestrus. To rule out the possibility that the observed reduction in R, was due to a decreased affinity of R, for estrogen, the dissociation constant of the receptorestrogen complex was determined for each stage of the cycle: metestrus, 0.87 0. I 1 nM; diestrus. 0.97 2 0.11 nM; proestrus, 0.97 0.15 nM; and estrus, 1.21 2 0.20 nM. None of these values is significantly different (p < 0.01). As we have shown in previous work, the nuclear level of estrogen receptor varies with the blood level of estrogeni6 and constitutes a significant amount of total uterine receptor. These nuclear values have been included in FIGURE10 to show the variation in total receptor content during the estrous cycle. Total uterine receptor content or receptor content per milligram of DNA reached high levels during diestrus and proestrus and decreased to the lowest level at metestrus (FIGURE 10).
*
*
DISCUSSION The results of this study demonstrate that progesterone interferes with the replenishment of the cytoplasmic estrogen receptor, R,, in the estrogenized immature rat uterus. In addition, this reduction is correlated with a reduced sensitivity
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6ot
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FIGURE9. Concentration of cytoplasmic and nuclear estrogen receptor in the endometrium ( 0 )and myometrium (El) of the adult rat uterus. Ovariectomized adult rats were pretreated with estrogen or estrogen plus progesterone as described in text, and the quantity of estrogen receptor was measured in the endometrium and myometrium on Day 3. of the uterus to the action of e ~ t r o g e n . ~Cytoplasmic ,'~ receptor'replenishment appears to involve two phases. The first phase, which occurs 4-8 hr after injection, may represent a recycling of R, that is not blocked by progesterone (FIGURES 1& 2). The quantity of R, that is replenished equals that which was depleted, and therefore recycling through some deactivation-reactivation mechanism could exist to account for this phase of replenishment. The second phase, which occurs 8-24 hr after injection, may involve synthesis of R, that is blocked by progesterone treatment. The quantity of R, that is replenished during phase 2 is 1& approximately twofold greater than that which was initially present (FIGURES 2). The increase in R, was inhibited by progesterone, even when the injection of 2). This finding implies that progesterone progesterone was delayed 8 hr (FIGURE may act on some late event in the sequence that leads to new R, synthesis. These data suggest that R, replenishment in estrogen-primed animals may be accomplished by two mechanisms: a recycling of existing R, after nuclear accumulation, which is not blocked by the presence of progesterone, and a synthesis of new R,, which is blocked by progesterone. The replenishment of cytoplasmic receptors for glucocorticoids by a process that does not depend on protein and RNA syntheses has been proposed by Ishii et ul.1s*19 and Rousseau et It is possible that the first phase of replenishment that was observed in this report is of this type. The second phase of R, replenishment may result from cellular hypertrophy induced by estrogen. The involvement of protein synthesis in the replenishment process has been suggested by Gorski ef
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ul.2 and Cidlowski and Muldoon.22 Recently, Mester and B a ~ l i e usuggested ~~ that the replenishment of R, in uteri of immature rats involves two separate processes. The first process, which occurs 0-6 hr after injection, could not be blocked by cycloheximide, whereas the second process, which occurs 6- 1 1 hr after injection, was dependent on protein synthesis. Thus, in cell types that grow in size or number in response to a steroid hormone, R, replenishment may involve both recycling and synthesis of new receptor, whereas in cells that d o not grow in response to their target steroid, R, replenishment may involve only recycling. The reduction in estrogen binding by R, that occurs after progesterone treatment could have resulted from changes in the steroid binding specificity and/or the 3 & 4). However, these parameters appear to be binding affinity of R, (FIGURES unchanged by progesterone treatment. The effect of progesterone on decreased R, does not appear to be a pharmacologic one, because the half-maximal dose of 0.5 mg of progesterone is well within the generally accepted "physiologic" ranges for the hormone. 1 7 . 2 8 If reduced R, on Day 4 is related to the reduced uterine growth that is observed 24 hr the quantity of R, that is translocatable to the nucleus and/or the reA
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FIGURE10. Concentration of estrogen receptor in cytoplasmic and nuclear fractions of rat uterus during the estrous cycle. Uterine tissues were taken from rats on the days of proestrus (P),estrus (E),metestrus (M), or diestrus (D).Cytoplasmic (El) and nuclear ( 0 ) fractions were prepared, and the [)H]estradiol exchange assay was used to measure the estrogen receptor concentrations in both fractions. Tissue fractions were incubated with various concentrations of either [3H]estradiol alone or [3H]estradiol plus diethylstilbestrol (DES). Specific binding was obtained by subtracting the amount of [)H]estradiol bound in the presence of DES from total binding in the absence of DES. The quantity of estradiol receptor at various stages of the cycle was obtained from double-reciprocal plots. The K J values were also determined for the various stages: metestrus, 0.98 nM; diestrus, 0.97 nM; proestrus. 0.97 nM: and estrus, 1.21 nM.
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tention time for nucleus-bound acceptor, R,, should be reduced. As shown in FIGURE 6, the level of R, I and 6 hr after injection of estradiol is significantly lower in progesterone-pretreated animals. We have shown that long-term nuclear retention of R,E correlates with the stimulation of true uterine growth.lSls Therefore, any circumstance that results in diminished nuclear retention time or number of R,E is likely to cause reduced uterine growth. Progesterone also causes a significant decrease in the number of cytoplasmic estrogen receptors, R,, in the adult rat uterus (FIGURE 7). This decrease is correlated with a reduced uterine sensitivity to estrogen (FIGURE 8). As discussed earlier, cytoplasmic receptor replenishment may involve both recycling and de novo synthesis of R,, and progesterone may have a preferential inhibitory effect on synthesis alone. The possibility that these processes occur in an independent manner in different uterine cell types cannot be ruled out; however, the demonstration that receptor replenishment occurs both in endometrium and in myometrium makes this hypothesis unlikely (FIGURE 1). The depressive effect of progesterone on levels of R, appears to be specific for estrogen antagonists. We have shown that nonsteroidal estrogen antagonists, such as nafoxidine hydrochloride (Upjohn I I ,000-A) and testosterone propionate, antagonize receptor replenishment and estrogen r e s p o n s i v e n e ~ s . ~In* ~contrast, .~~ hydrocortisone 21-acetate had no effect on either receptor level or on uterine responsiveness to estrogen (FIGURE 7). Progesterone did not antagonize estrogen-induced elevations in uterine DNA. Therefore, it is not likely that progesterone exerts its antagonism by inhibiting estrogen-induced cell division, as has been reported for the chick oviduct.24Because the adult rat uterus is a fully differentiated organ, whereas the chick oviduct is not, these differences in progesterone action may reflect basic differences in the state of differentiation of these organs. The antagonistic effect of progesterone may function during the estrous cycle in the rat (FIGURE 10 & Reference 23). The total quantity of receptor is low at metestrus and elevated at other times during the cycle (FIGURE 10). Blood levels of progesterone are high in late proestrus and early and may be the stimulus for the reduced levels of estrogen receptor observed during metestrus. Cyclic changes in reproductive tracts are the most characteristic feature of female reproduction. The demonstration of progesterone antagonism of estrogen receptor levels during the rat estrous cycle provides a very interesting model for the study of these cyclic changes. As described above, estrogen acts to promote uterine growth and to increase the tissue content of progesterone whereas progesterone antagonizes the action of estrogen by decreasing tissue levels of estrogen receptor. The interaction of these two ovarian hormones at the receptor level provides a basis for the cyclic changes observed in uterine tissues during the estrous cycle of the rat. This mechanism probably functions in primates, because cytoplasmic fractions of human uterus and monkey oviduct bind less estrogen during the luteal phase than during the follicular phase of the menstrual The ability of progesterone to antagonize and/or modify the action of estrogen may form a basis for interhormonal control mechanisms. This interplay between estrogen and progesterone may be considered in the following way (FIGURE 1 I). Estrogen, E, binds to the cytoplasmic estrogen receptor, RE, to f o r m a complex,R:p E that translocates to the nucleus, RE * E. This complex is responsible for the stimulation of events that lead to an increase in both R: and the cytoplasmic progesterone receptor, R;. Estrogen also stimulates uterine hypertrophy and
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hyperplasia (FIGURE 4). The elevated levels of RE augment the ability of the uterus to respond to progesterone. Progesterone binds to its cytoplasmic receptor to form RE P, which undergoes translocation to the nucleus to form R: P, and elicits the characteristic progestational responses that prepare the uterus for implantation. Progesterone also reduces Re, and the nuclear retention time for R;* E, thereby decreasing the ability of the uterus to respond in a totally estrogendirected fashion. Thus, progesterone reduces and/or redirects the ability of the uterine cells to respond to estrogen in such a way as to produce an appropriate uterine environment for implantation and pregnancy. This scheme does not rule out other possible interactional and control points, such as membrane effects, events not mediated by receptors, and steroid effects on pituitary hormone secretion. It is presented as a model that may provide a framework for testing ideas.
I. Effects d Estrogen E+RE-;~REE C
C
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Estrogen directed lresponses
I ncreased responsiveness to estrogen Increased responsiveness to progesterone
-Cellular hypertrophy and hyperplasi a
II. Effects d Progesterone on Estrogenized Uterus P
P+R,=R,
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\ Progesterone directed f responses
Decreased respon si veiiess to estrogen Decreased estrogen dlrected responses Progestational (Secretory) uterus
FIGURE 1 1 . Interaction of estrogen and progesterone in the control of receptor level and uterine growth. See DISCUSSION for details of this Figure. Abbreviations: E, estrogen; R:, cytoplasmic estrogen receptor; R: E. nuclear receptor-estrogencomplex; P, progesterone; R:. cytoplasmic progesterone receptor: RE P, cytoplasmic receptor-progesteronecomplex; RK P, nuclear receptor-progesterone complex.
We conclude that progesterone antagonizes and redirects the ability of uterine cells to respond to estradiol by decreasing the quantity of R,. This reduction of R, decreases the number of receptor-estrogen complexes that are translocated and retained in uterine nuclei. Thus, the ability of estrogen to stimulate uterine growth is altered and greatly reduced. These interactions may occur during the estrous cycle of the rat and probably form a basic control mechanism by which the reproductive tract oscillates each cycle. These studies indicate that the traditional measurement of hormone levels in the blood offers an incomplete picture for understanding the action of steroid hormones under various physiologic states. Responsiveness of target organs may vary as the result of changes in receptor concentration, an important variable in the control of reproductive function that can be evaluated only by measurement of tissue levels of hormone receptors. ,.
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Annals New York Academy of Sciences REFERENCES
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LEUNG,B. S. & G. H. SASAKI. 1973. Biochem. Biophys. Res. Commun. 55: 11801185. BRENNER, R. M., J. A. RESKO& N. B. WEST. 1974. Endocrinology 95: 1094-1099. MESTER,J., D. MARTEL,A. PSYCHOYOS & E.-E. BAULIEU.1974. Nature (London) 250 766,767. CLARK,J. H., J. N. ANDERSON & E. J. PECK,JR. 1973. Steroids 22: 707-718. CLARK,J. H., J. N. ANDERSON& E. J. PECK,JR. 1974. Nature (London) 251: 446,447. HSUEH,A. J. W., E. J. PECK& J. H. CLARK.1975. Nature (London) 254: 337,338. MILGROM,E., L. THI, M. ATGER& E.-E. BAULIEU.1973. J. Biol. Chem. 248: 63666371. LEAVITT,W., D. 0. TOFT,C. A. STROTT & B. W. O'MALLEY.1974. Endocrinology 94: 1041-1047. ANDERSON, J., J. H. CLARK& E. J. PECK,JR. 1972. Biochem. J. 126: 561 -567. KATZENELLENBOGEN, J. A., H. J. JOHNSON & K. E. CARLSON.1973. Biochemistry 12: 4091 -40%. LOWRY,0. H., N. J. ROSEBROUGH, A. L. FARR& R. J. RANDALL.1951. J. Biol. Chem. 193: 265-269. BURTON,K. 1966. Biochem. J. 6 2 315-318. ANDERSON, J. N., J. H. CLARK& E. J. PECK,JR. 1972. Biochem. Biophys. Res. Commun. 48: 1460- 1468. ANDERSON, J. N., E. J. PECK,JR. & J. H. CLARK.1975. Endocrinology 96:160- 167. CLARK,J. H., J. N. ANDERSON & E. J. PECK,JR. 1973. Advan. Exp. Med. Biol. 36: 15. CLARK,J. H., J. ANDERSON & E . J. PECK,JR. 1973. Science 176: 528,529. HSUEH,A. J. W., E. J. PECK,JR. & J. H. CLARK.1976. Endocrinology 98: 438. ISHII,D. N. & L. J. ARONOW.1974. Steroid Biochem. 4: 593-599. MIDDLEBROOK, J. L., M. D. WONG,D. N. ISHII& L. ARONOW.1975. Biochemistry 14: 180-185. ROUSSEAU,G. G., J. D. BAXTER,S. J. HIGGINS& G. M. TOMKINS. 1973. J. Mol. Biol. 7 9 593 -60 I . GORSKI,J., M. SARFF& J. CLARK.1971. In Advances in the Biosciences. G. Raspe, Ed. Vol. 7: 6. Pergamon Press, Vieweg. CIDLOWSKI, J. A. & T . G. MULDOON.1974. Endocrinology95 1621-1627. MESTER,J. & E.-E. BAULIEU.1975. Biochem. J. 146: 617-623. OKA,T. & R. T. SCHIMKE. 1969. Science 163: 83.84. VANBRUNT,L. A. 1972. M. S. Thesis. Purdue University. Lafayette, Ind. HORI,T., M. IDE& T. MIYAKE.1968. Endocrinol. Japon. 15: 215-219. HASHIMOTO.I., D. M. HENRICKS,L. L. ANDERSON& R. M. MELAMPY.1968. Endocrinology 82: 333-340. SHAIKH,A. A. 1971. Biol. Reprod. 5: 297-301. BUTCHER,R. L., W. E. COLLINS& N. W. FUGO.1974. Endocrinology 94: 1704-171 I. BRUSH,M. G., R. W. TAYLOR&R. J. B. KING. 1%7. J. Endocrinol. 3 9 599-605. EVANS,L. H . & P. HAHNELL.1971. J. Endocrinol. 50: 209-218. BRENNER, R. M., J. A. RESKO& N. B. WEST. 1974. J. Endocrinol. 95: 1094-1 101. TSENG.L. & E. GURPIDE.1975. J. Clin. Endocrinol. Metab. 41: 402.
DISCUSSION DR. C. A. B. CLEMETSON (Methodist Hospital of Brooklyn, Brooklyn, N. Y.): In our studies of the rat uterus, we also observed secretion of water, sodium, and potassium after treatment with estrogens for 3 days. When we give progesterone, we get an immediate and almost total reabsorption of water, sodium, and po-
Clark et al. : Receptor Replenishment Regulation
177
tassium (Clemetson, C. A. B., U. L. Verma & S. J. DeCarlo. Unpublished results). DR. CLARK:I agree with you. In the mature animal, if you give progesterone, you immediately see a loss of luminal fluid. Luminal fluid is, however, quite different from the secretory products that progesterone induces. DR. CLEMETSON: I have been studying rats with ligatures on alternate horns. INFECTED FLUIDS FROM ONE RAT NOT ANALYZED 640- NO ANALYTICAL DATA EXCLUDED
560 600
520 480
-
PROGESTERONE
EFFFCTS OF ESTRADIOL 17-8
PLUS PROGESTERONE
t
1
0
t
"
t
1
t
"
t
"
t
t
~
t
~
t
1
t
'
12 24 36 48 6 0 72 84 96 108 120 TIME IN HOURS
FIGUREDI. Effects of administration of continued doses of estradiol versus estradiol initially and estradiol plus progesterone thereafter in the rat.
One horn is ligatured top and bottom, and the other horn is not ligatured, so that any fluid that accumulates in the ligatured horn presumably is a secretion from the endometrial cells. This fluid is completely reabsorbed with progesterone; what puzzles me is why I'm getting complete abolition of the estrogen secretory effect and reabsorption by progesterone, while you are getting a partial blocking effect. Our treatment consists of estrogen alone, and we then continue estrogen in one
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Annals New York Academy of Sciences
group and add progesterone in the other group. With the continued estrogen plus progesterone, we get total reabsorption of the fluid (FIGURE DI). TABLEDI shows the total potassium and total sodium per luminal fluid washing in the same experiments. The total sodium value, in nanoequivalents per uterine horn, is 37,000 with estradiol alone in 10 injections, whereas after estradiol treatment, six injections, and treatment with estradiol and progesterone, four injections, there are dramatic decreases in sodium, potassium, and water. I submit that this action is one of the main functions of progesterone in mammals, because it holds the membranes up against the uterine wall. If you didn’t have the progesterone, the estrogen secretion would blow the membranes away from the uterine wall. However, I’m still a little puzzled as to why I’m getting complete blocking and you’re getting a partial effect. DR. CLARK:It is well known that progesterone causes the resorption of estrogen-induced luminal fluid. This effect of progesterone is only one of the many possible points of antagonism of estrogen action. Our experiments on response also show dramatic effects on luminal fluid (see FIGURE 8) and do not conflict with your data. TABLEDI TOTALPOTASSIUM A N D SODIUM VALUESPER LUMINAL WASHING I N EXPERIMENTS W I T H ESTRADIOL VERSUSESTRADIOL PLUS PROGESTERONE I N THE RAT Volume Hormonal Injection (fil/horn) Estradiol, 10 injections 327 ? 210 n 8 Estradiol, six injections, then estradiol plus progesterone, four injections 3.4 k 0.86 n 8 P