Regulation of estradiol and progesterone receptor concentrations in cat uteri following chronic progesterone administratioN

J.sreroidBiochem. Vol. 24,No. 2.pp.587-590,1986

0022-4731/86 $3.00f0.00

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REGULATION OF ESTRADIOL AND PROGESTERONE RECEPTOR CONCENTRATIONS IN CAT UTERI FOLLOWING CHRONIC PROGESTERONE ADMINISTRATION HAROLD G.

VERHAGE*

and

RANDAL C. JAFFE~

Departments of *Obstetrics and Gynecology and tPhysiology and Biophysics, University of Illinois at Chicago, Health Sciences Center, P.O. Box 6998, Chicago, IL 60680, U.S.A. (Received 2 July 1985) Summary-The purpose of this study was to determine the early effect of progesterone (P) on the estradiol (E2) and P receptor systems in cat uteri. Ovariectomized animals were treated with & for 7 days. Animals were then treated for up to 48 h with E, and P, treated with P while being E, withdrawn, or just E, withdrawn. P treatment resulted in a significant decrease in P cytosol receptor (PcR) and a significant increase in P nuclear receptor (PnR) at all times included in this study when compared to levels measured in the E,-treated animal. E, cytosol receptor (EcR) and E, nuclear receptor (EnR) levels were significantly lower after 12 h of P treatment and remained so for the duration of this study. When EcR and EnR levels were compared after 48 h of P treatment in the presence or absence of E,, or after 48 h of I!$ withdrawal, the loss of EnR following P treatment appeared to be independent of any changes in F.cR levels or serum E, levels, and only dependent on the presence of P. These results clearly illustrate that the chronic administration of P decreases the uterine concentration of its own receptor, and suggests that P decreases the E, receptor system by a selective action within the nucleus which diminishes their ability to retain EnR.

EXPERIMENTAL

INTRODUCTION

Numerous studies suggest that if a steroid is to alter cell morphology and function the steroid-receptor complex must bind to specific nuclear binding (acceptor) sites [I]. In the hamster [2,3], rat [4] and mouse [5] the administration of P causes an immediate and significant loss of EnR. Okulicz et a1.[4] suggested that this effect of P may be a general mechanism for P regulation of E2 action in target tissue. However, a similar effect on the EnR following the administration of P was not observed in the ewe [5]. Stone et a/.[51 suggested that the difference in the effect of P on the EnR in rodents and the ewe was a result of the different effects P has on their uteri. In the ewe, P maintains tissue growth, RNA:DNA ratios and protein synthesis, whereas in rodents P is anti-uterotropic. The cat is another species where P maintains and induces tissue growth, maintains organelles associated with secretion and elevated metabolic activity, and induces mitotic activity [6,7]. The long-term (14 days) effect of P in the cat is to significantly decrease both the total E2 [8] and P [6] receptor systems. However, the early effects of P on the steroid receptor systems in this species, particularly the effect of P on the retention of the EnR, are not known. In this study we report the early effect of P on both receptor systems. P was administered in both the presence and absence of E, and these results were compared with E, withdrawn animals. Data is presented which suggests that in the cat, a species where P maintains tissue growth, P causes a loss of EnR similar to previous reports in rodents.

Steroids

[2,4,6,7-3H]E, (98.1 Ci/mmol), [1,2,6,7-3H]P (96.5 Ci/mmol), [ 17a-methyl-3H]promegestone (R5020) (86.0 Ci/mmol) and unlabeled R5020 were obtained from the New England Nuclear Corp. (Boston, Mass., U.S.A.). Unlabeled E,, DES, testosterone, P and cortisol were obtained from Sigma Chemical Co. (St Louis, MO., U.S.A.). Animals

Sexually mature domestic cats were housed in individual cages under full spectrum lighting. Food and water were available ad libitum. An i.m. injection of ketamine hydrochloride (25 mg/kg body wt) supplemented with acepromazine maleate (0.6 mg/kg body wt) was used to anesthetize the animals prior to surgery. Animals, bilaterally ovariectomized for at least 4 weeks, were treated with E, for 7 days by means of a tubular Silastic” implant (1 .Ocm, Dow Corning No. 601-331) placed S.C. in the midscapular region. Previous studies have shown that this results in a systemic level of E, of approx. 30 pg/ml[9, lo]. On the 7th day of E, treatment the animals were unilaterally hysterectomized. In Experiment I, animals were treated with P for either 6, 12, 24 or 48 h by means of a tubular SilasticK implant (3 x 6.0 cm, Dow Corning No. 601-331) which results in a systemic level of P of approx. 30 ng/ml[6]. The contralateral uterine horn was then removed. In Experiment

588

HAROLD G. VERHAGE and RANDAL C. JAFFE

II, the animals were either treated with Ez and P (E, + P), or the E, implant was removed at the time the P implants were inserted (-E? + P), or the E? implant was removed (- Ez). The remaining uterine horn was removed 48 h after the onset of the various treatments in Experiment II. All animals were then killed with a lethal dose of sodium pentobarbital (iv.). Preparation of tissue

The uterine horns were placed in Hank’s buffered saline solution at 0°C immediately after removal and quickly trimmed of excess fat and mesentery. The tissue was then frozen in liquid nitrogen. On the day following the removal of the second uterine horn from each animal the quantity of steroid receptors in both uterine horns of each animal were determined in the same assay. Receptor assays

Tissue was either homogenized in 4 vol Buffer A (E, receptors, 10 mM Tri-HCl, 1.5 mM EDTA, pH 7.4) or 4 vol Buffer B (P receptors, 0.5 M sucrose, 10 mM Tri-HCl, 2 mM MgCl, pH 7.2) and centrifuged at 2000 g for 10 min at 4°C [9]. All subsequent steps were carried out at @4”C except where otherwise noted. The supernatant (68 ml) was incubated for IOmin with a pellet prepared from 5 ml of dextran-coated charcoal (0.5% Norit A and 0.05% Dextran T-70 in 1.5 mM EDTA and 10mM Tris-HCl, pH 7.4 [DCC]) in order to remove endogenous steroids, then centrifuged at 2500 g for 10 min and the resulting supernatant was centrifuged at 105,OOOg for 1 h. The supernatant was used for cytosol receptor determinations. The crude nuclear pellets from the first centrifugation were washed twice by gentle rehomogenization in either Buffer A when EnR was to be measured, or Buffer C (Buffer B + 30% glycerol) when PnR was to be measured, poured through organza (fine-mesh fabric) and centrifuged at 12,000g after each wash, and resuspended in the appropriate buffer. Aliquots (300 ~1) of the cytosol or nuclear fractions were added to two parallel series of tubes, one containing the labeled steroid and the. other containing the same concentration of the labeled steroid plus a 200-fold excess of unlabeled steroid. The ligands used were [3H]E, for E, receptors, [‘HIP for the PcR and [‘H]R5020 for PnR determinations. The unlabeled steroids were DES, P and R5020, respectively. In addition, all cytosol tubes contained a 200-fold higher concentration of testosterone (E2) or cortisol (P) than the respective labeled ligand. For the EJ receptor determinations cytosols were incubated at 0°C for 2 h and nuclei at 30°C for 1 h [8] and for the P receptor determinations cytosols were incubated at 0°C for 2 h and nuclei at 22°C for 4 h (91. Bound and free steroid in the cytosol was separated by adding 300~1 DCC to each tube. Following incubation for 5 min at 0°C the tubes were centri-

fuged at 2000 g for 10 min and the radioactivity in the supernatant was determined. Nuclear incubations were terminated by centrifugation and the pellets were washed once with 1 ml of Buffer D (Buffer A + 0.2% Triton X-100). The pellets were then solubilized in 0.1 N NaOH at 90-C and counted in scintillation fluid composed of toluene, Triton X- 100 and PPO-POPOP (RPI scintillator) in a ratio of 2000 : 1000 : 126. The counting efficiency was approx. 34%. Other analytical methods

Protein was measured by the method of Lowry et al.[ll] and DNA was assayed by the method of Burton[ 121. The dissociation constants and number of binding sites were determined by the method of Scatchard[ 131. Data analysis

Values are expressed as the mean f SE. The results were compared by paired t-test [14]. Values were considered to be significantly different if P < 0.05. RESULTS

The early effects of P on the E, and P receptor systems in the E,-primed ovariectomized animal are shown in Figs 1 and 2. After E, treatment for 7 days one uterine horn was removed and P treatment begun. The remaining uterine horn was removed at one of the indicated times. The receptor content from each animal before and after P treatment was determined in the same assay and compared. PcR was significantly reduced at each time included in this study (Fig. 1). After 48 h of P treatment approx. 30% of the PcR binding remained compared to the binding measured in tissue removed from animals treated only with E, (Fig. 2). PnR, on the

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0.2

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2.0

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0.2

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12

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Fig. I. Time course of the effect of P on the Ez and P receptor systems. Animals were treated for 7 days with Ez. One uterine horn was then removed (0) and P treatment begun. The contralateral uterine horn was removed at the indicated time (M). Receptors in the E,-treated uterine horn and (E, + P)-treated uterine horn from each animal were measured in the same assay and compared by paired f-test. Values arc expressed as mean + SE. *P < 0.05(E2 vs Ez + P treatment).

Chronic P: effect on steroid receptor levels

6

12

24

589

48

Hours

Fig 2. Time course of the effect of P on the E, and P receptor system. Data presented in Fig. I expressed as a percentage of the receptor binding measured after P treatment compared to that measured in El-primed tissue from the same animal. other hand, was significantly elevated at each of the time points (Fig. 1). Both EcR and EnR were unaffected by P treatment

for 6 h but were significantly reduced after I2 h of P treatment and continued to remain so for the duration of the study (Fig. 1). When the binding after the onset of P treatment was expressed as a percentage of the binding measured before P treatment, EcR plateaued at approx. 60% of the pretreatment value whereas EnR continued a gradual decline until less than 40% of the binding remained after 48 h of P treatment (Fig. 2). In another experiment, animals received I& implants for 7 days. The animals were then divided into three groups: both E, and P treatment, E, withdrawal f-E,) during P treatment or E, withdrawal. Ez and P receptor levels were compared in tissue removed before and 48 h after treatment (Figs 3 and 4). In the E, + P treatment group, PcR, EcR and EnR were all significantly lower at 48 h, whereas PnR was significantly elevated (Fig. 3). In the P + E, withdrawal group, PcR and EnR were significantly lowered, PnR was slightly elevated (not significant) and there was no change in E, cytosol binding (Fig. 3). In the E, withdrawal group the only significant change

Fig. 4. Data presented in Fig. 3 expressed as a percentage of the receptor binding measured after 48 h of treatment compared to that measured in E,-primed tissue from the same animal.

was an increase in EcR. PcR, PnR and EnR showed a tendency to decrease (Fig. 3). When the level of steroid binding sites was expressed as a percentage of that in the contralateral horn which was removed before E, withdrawal and/or P treatment, it appeared that EnR was lowered to the same extent (30%) following 48 h of P treatment regardless of the presence or absence of E, (Fig. 4). This decrease could not be attributed to E& withdrawal since there was no significant change in the E, withdrawal group (Fig. 3). EcR was significantly increased in the E, withdrawal group but not in the P + E, withdrawal group. This suggests that within 48 h P prevented the accumulation of EcR. DISCUSSION

Previous work with the cat demonstrated that E, administration to long-term ovariectomized animals caused a significant increase in both the EcR and EnR when compared to the ovariectomized control [8]. The administration of P for 14 days, in the presence or absence of continuous E,, reduced the receptor levels to values equal to those observed in 1.5 Qz $ 0.6 control animals. Our current data demonstrate that 1.0 “, g 0.4 P sibilantly reduces EcR and EnR within 12 h of i 0.5 ‘p I!! 0.2 administration. EnR was further reduced at each time 2 5 .EF point over the 48 h included in this study. 6 .P 1.5 I y 0.3 The effect of P on the EnR was independent of the I presence of E, since P reduced EnR to the same ,I 0.2 1.0 * extent in both the group treated concurrently with E, 0.5 ; g 0.1 a. and the group in which the Ez implant was removed as P treatment was begun. EnR was also significantly P+E, P-E, -E, P+E, P-E2 -E, lower after 48 h in both P-treated groups than in the E2 withdrawn group (which received no P). If P were Fig. 3. E, and P receptor levels in animals treated for 7 days preventing the formation of the E,-receptor complex with E,, then for 48 h with P in the presence (P + E,) or absence (P - E,) of E2, or undergoing E2 withdrawal (-I$). one would expect EnR levels to be similar in the One uterine horn was removed after 7 days of E, treatment P + Ez withdrawal and Ez withdrawal groups. Since (a) and the contralateral uterine horn was removed 48 h we did not observe this, the decline in EnR is not due ). Receptors in both uterine horns from the same animal were measured in the same assay and compared by to P preventing the formation of the E,-receptor paired r-test. Values are expressed as mean + SE. *P -c0.05. complex. This concept is further supported by the

590

HAROLDG. VERHAGEand RANDALC. JAFFE

cytosol data since EcR levels were significantly lower after 48 h in the P + E, group but not in the P + E, withdrawal group. Therefore, an explanation for the P-related decline in EnR must include some mechanism for decreasing the retention of the Ez---receptor complex within the nucleus, similar to what has been observed in the hamster [2,3, 151, rat [4], mouse [S] and the fetal guinea-pig~l4]. However, it did take longer for the decline in EnR to become signi~cant following the onset of P treatment in the cat compared to rodents. This could be the result of species variation or possibly the manner in which P was administered. Here, P was administered by S.C. implants, whereas in rodents P was administered by S.C. injection. Administration of E, to the ovariectomized cat induces hypertrophy, hyperplasia and the synthesis of a secretory substance that is packaged in apical secretory granules in the epithelial cells of the uterine glands [lo]. This secretory substance (CUPED) is present in the uterine lumen in E,-treated cats 117, 181. When P is added to the treatment regimen, there is an additional wave of hypertrophy and hyperplasia. a cessation of the production of CUPED, and a wave of glycogen synthesis [6,7]. The quantity of CUPED within the uterine lumen is greatly reduced after 2 days of P treatment and absent after 4 days [ 171. The changes in the compartmentalization of the steroid receptors induced by P treatment correlate well with these changes in morphological and biochemical parameters in the cat endometrium. If one assumes that DNA transcription is the primary focal point of steroid action [I], then within the cat uterus E, regulates a set of inducible genes (including the gene controlling the synthesis of CUPED), and the action of P is two-fold: (I) to prevent the transcription of at least some of the Ez-regulated genes (i.e. CUPED); and (2) to initiate the transcription of its own set of inducible genes (including those responsible for the synthesis of glycogen). The initial event in the action of P would be the increase in PnR as observed here and elsewhere [19]. An immediate effect of the increase in PnR is the decreased in EnR retention which could account for the antiestrogenjc action of P. The ability of P to trigger its own set of inducible genes is not dependent on Ez priming in the cat since a similar sequence of morphological events was observed when P was administered adrenalectomized cats [6].

to ovariectomized

and

Acknowledgements-The

technical assistance of Patricia A. Mavrogianis and the secretarial assistance of Margarita Guerrero are gratefully appreciated. This work was supported by NIH Grant HD-11404. REFERENCES 1. O’Malley B. W.: Steroid hormone action in eucaryotic

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cytochemical localization of a cat uterine protein that is estrogen dependent (CUPED). Biol. Reprod. 32 (1985) 1229-1235. 19. Verhage H. G., Boomsma R. A., Murray M. K. and

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