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Facilitation of receptive behavior in estrogen-primed female rats by the anti-progestin, RU 486
HORMONES
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BEHAVIOR
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Facilitation of Receptive Behavior in Estrogen-Primed Female Rats by the Anti-progestin, RU 486 ERIC T. PLEIM,’ PAMELA J. CAILLIAU, MARC A. WEINSTEIN, ANNE M. ETGEN,* AND RONALD J. BARFIELD Department New
of Biological Sciences, Rutgers College, Rutgers University, Piscataway, Jersey 08855-1059; and *Departments of Neuroscience and Psychiatry, Albert Einstein College of Medicine, Bronx, New York
The progestin receptor antagonist RU 38486 (henceforth referred to as RU 486) was tested for facilitative effects on female receptive behavior in ovariectomized Long-Evans rats primed with 2 pg estradiol benzoate (EB). RU 486 (0,0.5, 1.6, or 5.0 mg) was administered 48 hr after estrogen priming. The lordosis quotient (LQ) and lordosis score (LS) were assessed 4 hr after RU 486 administration in a standardized test consisting of a lo-mount test by a stimulus mate. A significant dose effect was found by both LQ and LS, with those subjects receiving 5 mg of RU 486 being significantly more receptive than vehicle control animals. Thus RU 486 acted as a weak progestin agonist under testing conditions typical for assessment of progestin facilitation of female sexual behavior in rats. Low levels of proceptive behavior (hops and darts) were seen in a minority of the tests, and did not vary systematically as a function of the dose of RU 486 administered. We also examined the effects of RU 486 given before progesterone (P) on receptivity in a blocking paradigm and confirmed previous reports that the antagonist significantly attenuates facilitation of sexual behavior when given in combination with P. A progestin receptor assay of the cytosols of the hypothalamus-preoptic area in estrogen-primed female rats treated with 5 mg RU 486 revealed a significantly greater depletion of available cytosolic P receptors than when rats were treated with a similarly facilitating dose of P (100 pg). The results suggest a possible dual mode of action for RU 486-a weak, receptormediated agonistic effect on sexual behavior when given alone to estrogen-primed rats, and a competitive blocking effect on receptivity when administered with P. 0 1990 Academic Press. Inc.
The synthetic steroid RU 38486 (RU 486) acts as an anti-progestin in both central nervous system (CNS) and peripheral tissue due to its competitive, high affinity binding to progestin receptors and apparent lack of agonistic properties (Brown and Blaustein, 1984; Philibert, 1984). In behavioral experiments, systemic RU 486 administered prior to proges’ To whom correspondence should be addressed at Department of Biological Sciences, Rutgers University, Busch Campus, Piscataway, NJ 08855-1059. 301 0018-506X/90 $1.50 Copyright 6 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.
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terone (P) blocked the facilitative effect of P on female sexual behavior in guinea pigs and rats (Brown and Blaustein, 1984; Brown, Moore, and Blaustein, 1987; Vathy, Etgen, and Barfleld, 1987). The inhibitory effect of RU 486 apparently occurs in the same brain areas reported sensitive to the facilitating effects of P. Intracerebral implantation of RU 486 in the ventromedial nucleus of the hypothalamus (VMN) and habenula has been shown to reduce behavioral responsiveness to subsequent P treatment (Etgen and Barfield, 1986). In addition to the above effects, RU 486 has also been shown to abbreviate the period of behavioral receptivity induced by estrogen plus P treatment when administered after P in guinea pigs and rats (Blaustein, Finkbohner, and Delville, 1987; Brown and Blaustein, 1986). This effect appeared to be specific to facilitation brought about by the interaction of progestins and their receptors. An excess of RU 486 caused early termination of behavioral estrus in rats made receptive by a sequence of estradiol benzoate (EB) and P, but did not diminish responding in animals brought into a comparable level of heat by EB and a large dose of estradiol (E) in place of the P treatment (Blaustein et al., 1986). In that experiment, animals receiving RU 486 after EB and a high dose of E had significantly higher lordosis scores than their vehicle controls when tested 8 hr following the injection of E. This result suggests that RU 486 might be a mixed agonist-antagonist with respect to facilitation of female sexual behavior. In fact, RU 486 has been reported to possess dual progestin agonist-antagonist actions in experiments with human breast cancer cells (Horowitz, 1985). In viva studies with postmenopausal women have shown that RU 486 given alone displayed progestomimetic activity in the endometrium, but when used together with P, RU 486 acted as a pure antagonist (Gravanis, Schaison, George, DeBrux, Satyaswaroop, Baulieu, and Robel, 1985). However, RU 486 administered by itself has not previously been reported to possess such agonistic properties in guinea pigs or rats (Brown and Blaustein, 1984; Vathy et al., 1987). Our aim was to investigate RU 486 as a possible facilitator of female sexual behavior in estrogen-primed female rats. In a preliminary study, we found that RU 486 is able to facilitate female sexual behavior in rats primed with a substantial priming dose of estradiol benzoate (10 pg EB/animal). The first experiment reported here is a dose-response study of the facilitating effects of RU 486 given systemically to ovariectomized female rats injected with an amount of EB (2 pg/animal) which we previously had found adequate for the priming of female sexual behavior without resulting in lordosis, in the absence of a facilitating injection of P (Pleim and Barfield, 1988). We next examined the effects of RU 486 given before a facilitating dose of P, vehicle, or no injection, since previous behavioral studies of RU 486 in rats and
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guinea pigs have not reported agonistic effects of this compound when given in a blocking paradigm. Previous work has suggested that the ability of a progestin treatment to facilitate receptive behavior in estrogen-primed female rodents is directly related to the depletion of cytosolic progestin receptors (PRc) and translocation of P-occupied PRc to the nuclear compartment (Blaustein, 1982). The presumed mechanism of action of RU 486 as a blockade of P action is that RU 486 occupies PRc, preventing accumulation of Poccupied receptors in the nucleus. Supporting this interpretation, RU 486 injection after a facilitating P injection decreases the nuclear concentration of P-occupied progestin receptors and terminates behavioral estrus in guinea pigs (Brown and Blaustein, 1986). Since the receptor dynamics of RU 486-induced facilitation are unclear, we tested whether, under the conditions of our experiment, a dose of RU 486 administered alone having similar agonistic properties to a small P dose (100 pg/animal SC) would deplete the concentration of available cytosolic progestin receptors to a similar degree as the small P dose. If a large dose of RU 486 depleted PRc to a similar degree, then the nuclear, protein synthetic mechanisms of action of facilitation may be similar, but RU 486 may be less efficient in translocating receptors. On the other hand, if relatively severe PRc depletion by RU 486 is accompanied by modest behavioral effects, then facilitation by RU 486 may involve less efficient nuclear binding, protein synthesis, differences in the conformation of the steroid receptor complex (Etgen and Rob&h, 1988), or even a totally distinct nongenomic mechanism. METHOD
Animals Adult female Long-Evans rats (Blue Spruce Farms, Inc., Altamont, NY) weighing between 220-280 g were ovariectomized under Metofane anesthesia (Pitman-Moore Inc., Washington Crossing, NJ). They were housed singly in 25 x 18 x 18 cm stainless steel cages with lights off from 9:30-21:30 hr. Rat chow and water were available ad libitum. Behavioral Testing Behavioral testing commenced at least 2 and no more than 4 weeks following ovariectomy. Experimental subjects were primed with 2 pg EB 52 hr before testing. Four hours prior to testing, each animal was screened for lordosis responsiveness due to the effects of EB alone (pretest). The pretest procedure was the same as the actual behavioral tests described below except that experienced males were allowed only five mounts with pelvic thrusting. Immediately following the pretest,
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each subject received either vehicle or one of three doses of RU 486. Four hours later, experienced males were placed in 50 x 26 x 30 cm rectangular glass aquaria and allowed 10 min to adapt. Females were placed in the aquaria and allowed 10 mounts with pelvic thrusting from the male. Lordosis quotient (LQ, percentage of mounts with pelvic thrusting accompanied by lordosis by the female subject) and lordosis score (LS, average quality of lordosis in response to 10 mounts based on a 3point scale: 1 point for slight dorsoflexion to 3 points for exaggerated dorsoflexion with markedly arched back) were derived as described previously (Hardy and DeBold, 1972). The presence of proceptive behavior (hopping and darting) was noted when observed. Behavioral observers had no knowledge of the assigned treatment while scoring lordosis. Each animal was tested twice, 2 weeks apart, to eliminate possible effects of repeated administration of estradiol and RU 486 on receptivity. Eleven subjects received two of the four possible treatments in a counterbalanced order, and each test was treated as an independent event in the statistical analysis. In the second experiment three separate groups of 13 ovariectomized, estrogen-primed (2 pg of EB) rats were injected with either 500 pg of P in 0.1 ml sesame oil or 0.1 ml oil alone, 1 hr after being injected with either 5.0 mg RU 486 or vehicle control (pretreatment immediately following a five-mount pretest), or were given no facilitating injection. Behavioral testing commenced 4 hr later. Subjects ,were tested twice, once with each pretreatment (RU 486 or vehicle) 2 weeks apart counterbalanced for order of injection. Hormones
RU 486 (17p-hydroxy- 1 lp-[4-dimethylaminophenyll17 a-[ l-propynyllestra-4,9-dien-3-one; Roussel UCLAF, Romaineville, France) was dissolved in a vehicle containing 80% sesame oil, 15% benzyl benzoate, and 5% ethyl alcohol (OBE vehicle). RU 486 was administered subcutaneously in 0.4 ml vehicle at a dose of 0, 0.5, 1.6, or 5.0 mg/animal. P was dissolved in sesame oil in a concentration of 500 pg per 0.1 ml. Progestin Receptor Assay
Twenty-two adult, ovariectomized Long-Evans rats were given one of three treatments. All subjects were primed 2 days before the assay with 2 pg of EB SC in 0.1 ml sesame oil, then on the morning of the assay were given 0.4 ml of OBE vehicle, 100 pg of P in 0.4 ml OBE vehicle, or 5 mg of RU 486 in 0.4 ml of OBE vehicle. Four to five hours later, all subjects were given a receptivity test consisting of 10 mounts from a stimulus male. No vehicle-treated animals showed any receptivity, most P-treated rats were receptive, and about half the RU 486-treated subjects showed receptivity (average LQ about 60 for those responders),
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confirming the pattern seen in the behavioral experiments. Immediately following behavioral testing, unanesthetized rats were decapitated and their brains were removed for the cytosol progestin binding assay. All of the following procedures were carried out at 0-4°C. The hypothalamus-preoptic area (HPOA) of each rat was dissected out and floated in TEG buffer (10 mM Tris, 1.5 mM Na,EDTA, 10% (v/v) glycerol, pH 7.4). The tissue samples were homogenized (two HPOAs from the same treatment group constituted each sample) in glass tissue grinders with Teflon pestles in 0.84 ml TEMG buffer (TEG with 12 mM monothioglycerol). Homogenates were spun at SSOg for 10 min. The supernatants were transferred to ultracentrifuge tubes topped off with TEMG and spun at 105,OOOg for 1 hr. The supernatants, or cytosols, were incubated in duplicate overnight with [3H]R 5020 (0.5 nM final concentration) with or without IOO-fold excess of cold R 5020. Bound [3H]R 5020 was separated from free by gel filtration on Sephadex LH-20 (Pharmacia, Piscataway, NJ) columns. Incubates (200 ~1) were loaded onto the columns and washed with 200 ,ul of TEMG. Thirty minutes after loading, samples were eluted into 5-ml scintillation minivials with 700 ~1 of TEMG. Five milliliters Betafluor (National Diagnostics, Highland Park, NJ) was added to each vial, and samples were counted for 45 min on a Beckman Model 7800 scintillation counter at 55% efficiency. Protein content of the cytosols was estimated with the Bradford (1976) dye-binding method using lysozome as the protein standard. Binding measured in the presence of lOO-fold excess of cold R 5020 (nonspecific) was subtracted from that measured in the absence of cold R 5020 to yield specific binding for each sample. A total of four data points (tissue from two rats each) were obtained for the RU 486 and P treatment conditions, and three for the vehicle condition. In each of two runs of the assay RU 486 subjects were pooled according to whether or not they had responded to RU 486 with receptivity in the behavioral test. However, since no difference in PRc concentration was seen, the PRc results from responders and nonresponders were pooled for the purpose of statistical analysis. RESULTS
Experiment 1 The results of the dose-response study of RU 486 on female receptive behavior are depicted in Fig. 1 and Table 1. One-way ANOVA revealed a significant dose effect on both lordosis quotient, (F(3, 84) = 3.126, P = 0.030) and lordosis score, (F(3, 84) = 3.105, P = 0.031). There were no differences in the pretest LQ or LS among the groups. Post hoc analysis of differences among means showed that the 5.0-mg treatment significantly differed from vehicle treatment with respect to both LQ and LS (Newman-Keuls, P < 0.05; Myers, 1979). Behavioral signs
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*
0
.5 DOSE
1.6
5
RU 466 @/subject)
FIG. 1. Effect of RU 486 on female receptive behavior. Ovariectomized rats were treated with 2 pg EB in 0.1 ml sesame oil SC 48 hr prior to the facilitatory injection (SC) of 0, 0.5, 1.6, or 5.0 mg RU 486 dissolved in 0.4 ml vehicle (see text for composition). RU 486 was given immediately after a five-mount pretest with stud male rats. Animals were tested 4 hr after RU 486 treatment. *Significantly greater than vehicle controls (Newman-Keuls, P < 0.05).
of proceptivity (hopping and darting) were observed in less than a third of the experimental tests, and no significant effect of dose was found on the proportion of animals at each dose level showing proceptive behavior (Chi-square test, P > 0.05; Siegel, 1956). Experiment
2
The effects of an injection of P, oil, or no injection 1 hr after an injection of 5 mg of RU 486 or vehicle are depicted in Fig. 2. RU 486 pretreatment significantly decreased LQ in subjects facilitated with 500 pg of P compared with those facilitated with vehicle pretreatment (paired t test, t,* = 5.278, P = 0.0002). When rats were pretreated with RU 486, they showed higher lordosis quotients than when they were given vehicle whether they were injected with oil 1 hr later or given no second injection (paired t tests, t,2 = 2.88, P = 0.0138; t,* = 3.415, P = 0.0051, respectively). Experiment 3 One-way ANOVA showed a significant effect of treatment on specifically bound receptors (F(2, 8) = 13.638, P < 0.003; Fig. 3). PRc under the RU 486 condition were significantly depleted compared to those under
FACILITATION
TABLE 1 of Lordosis by RU 486
Facilitation Dose RU 486 (mg) 0
0.5 1.6 5.0
Pre-LQ k SEM 21.0 11.8 25.5 20.9
k ‘2 +
6.8 4.5 8.4 1.5
LQ? SEM 21.1 36.4 43.2 64.6
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k f k -r-
Pre-LS 2 SEM
8.3 8.8 10.0 8.3
3.4 1.5 4.3 3.9
-c + k k
1.1 0.6 1.5 1.4
LS f SEM 4.7 6.8 7.8 11.7
k l!z + k
1.5 1.7 1.9 1.6
Test with proceptive behavior 2 7 5 6
a Ovariectomized rats were primed with 2 pg EB 48 hr before SC injection of RU 486 in 0.4 ml vehicle. LQ and LS values represent group means (number of tests in each treatment group = 22).
the vehicle and P conditions, (Scheffe F test, P < 0.05).
which did not differ from each other
DISCUSSION
The results reported here indicate that RU 486 can facilitate sexual receptivity in female rats in a dose-dependent manner at physiological levels of estrogen priming. Since the highest dose used in this study is commonly given as a competitive anti-progestin, it may seem odd that the status of this synthetic hormone as a partial agonist for the facilitation of sexual behavior has apparently been overlooked in recent work using female rats (Brown ef al., 1987; Vathy et al., 1987). Since many drug and hormone antagonists are also partial agonists, it should not be surprising to find that under certain conditions RU 486 is able to mimic the behavioral actions of P to some extent. However, RU 486 did not stimulate proceptivity nearly as well as P, perhaps indicating a different mechanism of action between the two steroids. It may be significant that the agonistic action of RU 486 is usually found only when P is absent from the system. In biochemical studies, RU 486 has been found to possess dual progestin agonist-antagonist actions on estrogen-independent progestin receptors of T47D,, human breast cancer cells, and to induce P-like secretory changes in human endometrial tissue, both in the absence of P stimulation (Gravanis et al., 1985; Horowitz, 1985). In the present study, facilitation by RU 486 followed by an oil injection (as in previous blocking paradigms) was less effective than RU 486 alone, so it is easy to see how a facilitative effect might have been missed in earlier work. We can only speculate from these data that the injection of P, and to a lesser extent the injection of sesame oil, somehow disrupts the facilitating effect of RU 486. Perhaps P or other adrenal steroids
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n VEHELE **
PROGESTERONE
OIL
COIVTROL
TREA’IMENT
ta
RU486
NO INJECTION
AT 1 HOUR
FIG. 2. Effects on sexual receptivity of RU 486 given 1 hr prior to P, sesame oil, or no further injections. Animals were primed with 2 pg EB 48 hr before an injection of either 5.0 mg RU 486 or vehicle immediately followed by a five-mount pretest. Four hours following the second injection subjects were tested for sexual receptivity. *, **, and *** indicate significant dependent f tests at a = 0.05, 0.01, and 0.001, respectively.
released as a consequence of a second injection are able to compete with the RU-PRc binding sufficiently to diminish the weak facilitatory effect. The focus of the current study is primarily behavioral, but we have included an assay to examine the concentration of cytoplasmic P receptors remaining in hypothalamus after RU 486 versus an approximately behaviorally equipotent dose of P. In this way, we have provided evidence that RU 486 does deplete the cytosolic receptor pool at least as efficiently as an equipotent dose of P. Though not conclusive, this result is consistent with the interpretation that this weak facilitation caused by RU 486 is receptor-mediated, but for some reason less effective than the receptor-mediated facilitation induced by P. Further studies using nuclear receptor assays and double-labeling techniques are needed to shed additional light on this question. As it stands, whether the weaker effects/pg of RU 486 on behavior relative to P are due to differences in nuclear binding, steroid-receptor conformation, protein synthesis, or some other factor, remains to be determined. Considering that several other compounds unrelated to P may also cause facilitation of sexual behavior in estrogen-primed rodents, it is even possible that RU 486 facilitation is acting through an entirely distinct mechanism.
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TREATMENT
FIG. (HPOA) 100 pg relative
3. Cytosolic progestin receptors assayed of female Long-Evans rats primed with 2 progesterone, or 5 mg RU 486. Receptors to vehicle or the small progesterone dose.
from the hypothalamus-preoptic area wg EB and treated with 0.4 ml vehicle, were significantly depleted by RU 486 (*P < 0.05, Sheffe F Test).
ACKNOWLEDGMENTS This work was supported by USPHS Grant HD04484 to RJB. We thank Dr. D. Philibert (Roussel-UCLAF, Romaineville, France) for the generous gift of RU 486. Some of this work was presented at the Society for Neuroscience Annual Meeting, 1988, in Toronto, Canada.
REFERENCES Blaustein, J. D. (1982). Alteration of sensitivity to progesterone facilitation of lordosis in guinea pigs by modulation of hypothalamic progestin receptors. Brain Res. 243, 287300. Blaustein, J. D., Finkbohner, R., and Delville, Y. (1987). Estrogen-induced and estrogenfacilitated female rat sexual behavior is not mediated by progestin receptors. Neuroendocrinology 45, 152-159. Bradford, M. M. (1976). A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Brown, T. J., and Blaustein, J. D. (1984). Inhibition of sexual behavior in female guinea pigs by a progesterone receptor antagonist. Brain Res. 301, 343-349. Brown, T. J., and Blaustein, J. D. (1986). Abbreviation of the period of sexual behavior in female guinea pigs by the progesterone antagonist RU 486. Brain Res. 373, 103113. Brown, T. J., Moore, M. J., and Blaustein, J. D. (1987). Maintenance of progesterone-
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facilitated sexual behavior in female rats requires continued hypothalmic protein synthesis and nuclear progestin receptor occupation. Endocrinology 121, 298-304. Etgen, A. M., and Bartield, R. J. (1986). Antagonism of female sexual behavior with intracerebral implants of antiprogestin RU 486: Correlation with binding to neural progestin receptor. Endocrinology 119, 1610-1617. Etgen, A. M., and Robisch, D. M. (1988). Differential extraction of estradiol- and tamoxifenreceptor complexes from hypothalamic cell nuclei. Brain Res. 452, l-10. Gravanis, A., Schaison, G., George, M., DeBrux, J., Satyaswaroop, P. G., Baulieu, E. E., and Robel, P. (1985). Endometrial and pituitary responses to the steroidal antiprogestin RU 486 in postmenopausal women. J. Clin. Endocrinol. Metab. 60, 15’6-163. Hardy, D. F., and DeBold, J. F. (1972). Effects of coital stimulation upon behavior of the female rat. .I. Comp. Physiol. Psych. 78, 400-408. Horowitz, K. B. (1985). The antiprogestin RU 38486: Receptor mediated progestin versus antiprogestin actions screened in estrogen-sensitive T47D,, human breast cancer cells. Endocrinology
116, 2236-2245.
Mullick, A., and Katzenellenbogen, B. S. (1986). Antiprogestin-receptor complexes: Differences in the interaction of the antiprogestin RU 38,486 and the progestin R5020 with the progesterone receptor of human breast cancer cells. Biochetn. Biophys. Res. Commun.
135, 90-97.
Myers, J. L. (1979). Fundamentals of Experimental Design. Allyn & Bacon, Boston. Okulicz, W. C. (1987). Effect of the antiprogestin RU-486 on progesterone inhibition of occupied nuclear estrogen receptor in the uterus. J. Steroid Biochem. 28, 117-122. Philibert, D. (1984). RU-38486: An original multi-faceted anti-hormone in vivo. In M. K. Agarwal and W. D. Gruyter (Eds.), Adrenal Steroid Antagonism, pp. 77-101. de Gruyter, New York. Pleim, E. T., and Barheld, R. J. (1988). Progesterone versus estrogen facilitation of female sexual behavior by intracranial administration to female rats. Horm. Behav. 22, 150159. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Vathy, I. U., Etgen, A. M., and Barfield, R. J. (1987). Actions of progestins on estrous behavior in female rats. Physiol. Behav. 40, 591-595.
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Facilitation of Receptive Behavior in Estrogen-Primed Female Rats by the Anti-progestin, RU 486 ERIC T. PLEIM,’ PAMELA J. CAILLIAU, MARC A. WEINSTEIN, ANNE M. ETGEN,* AND RONALD J. BARFIELD Department New
of Biological Sciences, Rutgers College, Rutgers University, Piscataway, Jersey 08855-1059; and *Departments of Neuroscience and Psychiatry, Albert Einstein College of Medicine, Bronx, New York
The progestin receptor antagonist RU 38486 (henceforth referred to as RU 486) was tested for facilitative effects on female receptive behavior in ovariectomized Long-Evans rats primed with 2 pg estradiol benzoate (EB). RU 486 (0,0.5, 1.6, or 5.0 mg) was administered 48 hr after estrogen priming. The lordosis quotient (LQ) and lordosis score (LS) were assessed 4 hr after RU 486 administration in a standardized test consisting of a lo-mount test by a stimulus mate. A significant dose effect was found by both LQ and LS, with those subjects receiving 5 mg of RU 486 being significantly more receptive than vehicle control animals. Thus RU 486 acted as a weak progestin agonist under testing conditions typical for assessment of progestin facilitation of female sexual behavior in rats. Low levels of proceptive behavior (hops and darts) were seen in a minority of the tests, and did not vary systematically as a function of the dose of RU 486 administered. We also examined the effects of RU 486 given before progesterone (P) on receptivity in a blocking paradigm and confirmed previous reports that the antagonist significantly attenuates facilitation of sexual behavior when given in combination with P. A progestin receptor assay of the cytosols of the hypothalamus-preoptic area in estrogen-primed female rats treated with 5 mg RU 486 revealed a significantly greater depletion of available cytosolic P receptors than when rats were treated with a similarly facilitating dose of P (100 pg). The results suggest a possible dual mode of action for RU 486-a weak, receptormediated agonistic effect on sexual behavior when given alone to estrogen-primed rats, and a competitive blocking effect on receptivity when administered with P. 0 1990 Academic Press. Inc.
The synthetic steroid RU 38486 (RU 486) acts as an anti-progestin in both central nervous system (CNS) and peripheral tissue due to its competitive, high affinity binding to progestin receptors and apparent lack of agonistic properties (Brown and Blaustein, 1984; Philibert, 1984). In behavioral experiments, systemic RU 486 administered prior to proges’ To whom correspondence should be addressed at Department of Biological Sciences, Rutgers University, Busch Campus, Piscataway, NJ 08855-1059. 301 0018-506X/90 $1.50 Copyright 6 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.
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terone (P) blocked the facilitative effect of P on female sexual behavior in guinea pigs and rats (Brown and Blaustein, 1984; Brown, Moore, and Blaustein, 1987; Vathy, Etgen, and Barfleld, 1987). The inhibitory effect of RU 486 apparently occurs in the same brain areas reported sensitive to the facilitating effects of P. Intracerebral implantation of RU 486 in the ventromedial nucleus of the hypothalamus (VMN) and habenula has been shown to reduce behavioral responsiveness to subsequent P treatment (Etgen and Barfield, 1986). In addition to the above effects, RU 486 has also been shown to abbreviate the period of behavioral receptivity induced by estrogen plus P treatment when administered after P in guinea pigs and rats (Blaustein, Finkbohner, and Delville, 1987; Brown and Blaustein, 1986). This effect appeared to be specific to facilitation brought about by the interaction of progestins and their receptors. An excess of RU 486 caused early termination of behavioral estrus in rats made receptive by a sequence of estradiol benzoate (EB) and P, but did not diminish responding in animals brought into a comparable level of heat by EB and a large dose of estradiol (E) in place of the P treatment (Blaustein et al., 1986). In that experiment, animals receiving RU 486 after EB and a high dose of E had significantly higher lordosis scores than their vehicle controls when tested 8 hr following the injection of E. This result suggests that RU 486 might be a mixed agonist-antagonist with respect to facilitation of female sexual behavior. In fact, RU 486 has been reported to possess dual progestin agonist-antagonist actions in experiments with human breast cancer cells (Horowitz, 1985). In viva studies with postmenopausal women have shown that RU 486 given alone displayed progestomimetic activity in the endometrium, but when used together with P, RU 486 acted as a pure antagonist (Gravanis, Schaison, George, DeBrux, Satyaswaroop, Baulieu, and Robel, 1985). However, RU 486 administered by itself has not previously been reported to possess such agonistic properties in guinea pigs or rats (Brown and Blaustein, 1984; Vathy et al., 1987). Our aim was to investigate RU 486 as a possible facilitator of female sexual behavior in estrogen-primed female rats. In a preliminary study, we found that RU 486 is able to facilitate female sexual behavior in rats primed with a substantial priming dose of estradiol benzoate (10 pg EB/animal). The first experiment reported here is a dose-response study of the facilitating effects of RU 486 given systemically to ovariectomized female rats injected with an amount of EB (2 pg/animal) which we previously had found adequate for the priming of female sexual behavior without resulting in lordosis, in the absence of a facilitating injection of P (Pleim and Barfield, 1988). We next examined the effects of RU 486 given before a facilitating dose of P, vehicle, or no injection, since previous behavioral studies of RU 486 in rats and
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guinea pigs have not reported agonistic effects of this compound when given in a blocking paradigm. Previous work has suggested that the ability of a progestin treatment to facilitate receptive behavior in estrogen-primed female rodents is directly related to the depletion of cytosolic progestin receptors (PRc) and translocation of P-occupied PRc to the nuclear compartment (Blaustein, 1982). The presumed mechanism of action of RU 486 as a blockade of P action is that RU 486 occupies PRc, preventing accumulation of Poccupied receptors in the nucleus. Supporting this interpretation, RU 486 injection after a facilitating P injection decreases the nuclear concentration of P-occupied progestin receptors and terminates behavioral estrus in guinea pigs (Brown and Blaustein, 1986). Since the receptor dynamics of RU 486-induced facilitation are unclear, we tested whether, under the conditions of our experiment, a dose of RU 486 administered alone having similar agonistic properties to a small P dose (100 pg/animal SC) would deplete the concentration of available cytosolic progestin receptors to a similar degree as the small P dose. If a large dose of RU 486 depleted PRc to a similar degree, then the nuclear, protein synthetic mechanisms of action of facilitation may be similar, but RU 486 may be less efficient in translocating receptors. On the other hand, if relatively severe PRc depletion by RU 486 is accompanied by modest behavioral effects, then facilitation by RU 486 may involve less efficient nuclear binding, protein synthesis, differences in the conformation of the steroid receptor complex (Etgen and Rob&h, 1988), or even a totally distinct nongenomic mechanism. METHOD
Animals Adult female Long-Evans rats (Blue Spruce Farms, Inc., Altamont, NY) weighing between 220-280 g were ovariectomized under Metofane anesthesia (Pitman-Moore Inc., Washington Crossing, NJ). They were housed singly in 25 x 18 x 18 cm stainless steel cages with lights off from 9:30-21:30 hr. Rat chow and water were available ad libitum. Behavioral Testing Behavioral testing commenced at least 2 and no more than 4 weeks following ovariectomy. Experimental subjects were primed with 2 pg EB 52 hr before testing. Four hours prior to testing, each animal was screened for lordosis responsiveness due to the effects of EB alone (pretest). The pretest procedure was the same as the actual behavioral tests described below except that experienced males were allowed only five mounts with pelvic thrusting. Immediately following the pretest,
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each subject received either vehicle or one of three doses of RU 486. Four hours later, experienced males were placed in 50 x 26 x 30 cm rectangular glass aquaria and allowed 10 min to adapt. Females were placed in the aquaria and allowed 10 mounts with pelvic thrusting from the male. Lordosis quotient (LQ, percentage of mounts with pelvic thrusting accompanied by lordosis by the female subject) and lordosis score (LS, average quality of lordosis in response to 10 mounts based on a 3point scale: 1 point for slight dorsoflexion to 3 points for exaggerated dorsoflexion with markedly arched back) were derived as described previously (Hardy and DeBold, 1972). The presence of proceptive behavior (hopping and darting) was noted when observed. Behavioral observers had no knowledge of the assigned treatment while scoring lordosis. Each animal was tested twice, 2 weeks apart, to eliminate possible effects of repeated administration of estradiol and RU 486 on receptivity. Eleven subjects received two of the four possible treatments in a counterbalanced order, and each test was treated as an independent event in the statistical analysis. In the second experiment three separate groups of 13 ovariectomized, estrogen-primed (2 pg of EB) rats were injected with either 500 pg of P in 0.1 ml sesame oil or 0.1 ml oil alone, 1 hr after being injected with either 5.0 mg RU 486 or vehicle control (pretreatment immediately following a five-mount pretest), or were given no facilitating injection. Behavioral testing commenced 4 hr later. Subjects ,were tested twice, once with each pretreatment (RU 486 or vehicle) 2 weeks apart counterbalanced for order of injection. Hormones
RU 486 (17p-hydroxy- 1 lp-[4-dimethylaminophenyll17 a-[ l-propynyllestra-4,9-dien-3-one; Roussel UCLAF, Romaineville, France) was dissolved in a vehicle containing 80% sesame oil, 15% benzyl benzoate, and 5% ethyl alcohol (OBE vehicle). RU 486 was administered subcutaneously in 0.4 ml vehicle at a dose of 0, 0.5, 1.6, or 5.0 mg/animal. P was dissolved in sesame oil in a concentration of 500 pg per 0.1 ml. Progestin Receptor Assay
Twenty-two adult, ovariectomized Long-Evans rats were given one of three treatments. All subjects were primed 2 days before the assay with 2 pg of EB SC in 0.1 ml sesame oil, then on the morning of the assay were given 0.4 ml of OBE vehicle, 100 pg of P in 0.4 ml OBE vehicle, or 5 mg of RU 486 in 0.4 ml of OBE vehicle. Four to five hours later, all subjects were given a receptivity test consisting of 10 mounts from a stimulus male. No vehicle-treated animals showed any receptivity, most P-treated rats were receptive, and about half the RU 486-treated subjects showed receptivity (average LQ about 60 for those responders),
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BY RU
486
305
confirming the pattern seen in the behavioral experiments. Immediately following behavioral testing, unanesthetized rats were decapitated and their brains were removed for the cytosol progestin binding assay. All of the following procedures were carried out at 0-4°C. The hypothalamus-preoptic area (HPOA) of each rat was dissected out and floated in TEG buffer (10 mM Tris, 1.5 mM Na,EDTA, 10% (v/v) glycerol, pH 7.4). The tissue samples were homogenized (two HPOAs from the same treatment group constituted each sample) in glass tissue grinders with Teflon pestles in 0.84 ml TEMG buffer (TEG with 12 mM monothioglycerol). Homogenates were spun at SSOg for 10 min. The supernatants were transferred to ultracentrifuge tubes topped off with TEMG and spun at 105,OOOg for 1 hr. The supernatants, or cytosols, were incubated in duplicate overnight with [3H]R 5020 (0.5 nM final concentration) with or without IOO-fold excess of cold R 5020. Bound [3H]R 5020 was separated from free by gel filtration on Sephadex LH-20 (Pharmacia, Piscataway, NJ) columns. Incubates (200 ~1) were loaded onto the columns and washed with 200 ,ul of TEMG. Thirty minutes after loading, samples were eluted into 5-ml scintillation minivials with 700 ~1 of TEMG. Five milliliters Betafluor (National Diagnostics, Highland Park, NJ) was added to each vial, and samples were counted for 45 min on a Beckman Model 7800 scintillation counter at 55% efficiency. Protein content of the cytosols was estimated with the Bradford (1976) dye-binding method using lysozome as the protein standard. Binding measured in the presence of lOO-fold excess of cold R 5020 (nonspecific) was subtracted from that measured in the absence of cold R 5020 to yield specific binding for each sample. A total of four data points (tissue from two rats each) were obtained for the RU 486 and P treatment conditions, and three for the vehicle condition. In each of two runs of the assay RU 486 subjects were pooled according to whether or not they had responded to RU 486 with receptivity in the behavioral test. However, since no difference in PRc concentration was seen, the PRc results from responders and nonresponders were pooled for the purpose of statistical analysis. RESULTS
Experiment 1 The results of the dose-response study of RU 486 on female receptive behavior are depicted in Fig. 1 and Table 1. One-way ANOVA revealed a significant dose effect on both lordosis quotient, (F(3, 84) = 3.126, P = 0.030) and lordosis score, (F(3, 84) = 3.105, P = 0.031). There were no differences in the pretest LQ or LS among the groups. Post hoc analysis of differences among means showed that the 5.0-mg treatment significantly differed from vehicle treatment with respect to both LQ and LS (Newman-Keuls, P < 0.05; Myers, 1979). Behavioral signs
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*
0
.5 DOSE
1.6
5
RU 466 @/subject)
FIG. 1. Effect of RU 486 on female receptive behavior. Ovariectomized rats were treated with 2 pg EB in 0.1 ml sesame oil SC 48 hr prior to the facilitatory injection (SC) of 0, 0.5, 1.6, or 5.0 mg RU 486 dissolved in 0.4 ml vehicle (see text for composition). RU 486 was given immediately after a five-mount pretest with stud male rats. Animals were tested 4 hr after RU 486 treatment. *Significantly greater than vehicle controls (Newman-Keuls, P < 0.05).
of proceptivity (hopping and darting) were observed in less than a third of the experimental tests, and no significant effect of dose was found on the proportion of animals at each dose level showing proceptive behavior (Chi-square test, P > 0.05; Siegel, 1956). Experiment
2
The effects of an injection of P, oil, or no injection 1 hr after an injection of 5 mg of RU 486 or vehicle are depicted in Fig. 2. RU 486 pretreatment significantly decreased LQ in subjects facilitated with 500 pg of P compared with those facilitated with vehicle pretreatment (paired t test, t,* = 5.278, P = 0.0002). When rats were pretreated with RU 486, they showed higher lordosis quotients than when they were given vehicle whether they were injected with oil 1 hr later or given no second injection (paired t tests, t,2 = 2.88, P = 0.0138; t,* = 3.415, P = 0.0051, respectively). Experiment 3 One-way ANOVA showed a significant effect of treatment on specifically bound receptors (F(2, 8) = 13.638, P < 0.003; Fig. 3). PRc under the RU 486 condition were significantly depleted compared to those under
FACILITATION
TABLE 1 of Lordosis by RU 486
Facilitation Dose RU 486 (mg) 0
0.5 1.6 5.0
Pre-LQ k SEM 21.0 11.8 25.5 20.9
k ‘2 +
6.8 4.5 8.4 1.5
LQ? SEM 21.1 36.4 43.2 64.6
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k f k -r-
Pre-LS 2 SEM
8.3 8.8 10.0 8.3
3.4 1.5 4.3 3.9
-c + k k
1.1 0.6 1.5 1.4
LS f SEM 4.7 6.8 7.8 11.7
k l!z + k
1.5 1.7 1.9 1.6
Test with proceptive behavior 2 7 5 6
a Ovariectomized rats were primed with 2 pg EB 48 hr before SC injection of RU 486 in 0.4 ml vehicle. LQ and LS values represent group means (number of tests in each treatment group = 22).
the vehicle and P conditions, (Scheffe F test, P < 0.05).
which did not differ from each other
DISCUSSION
The results reported here indicate that RU 486 can facilitate sexual receptivity in female rats in a dose-dependent manner at physiological levels of estrogen priming. Since the highest dose used in this study is commonly given as a competitive anti-progestin, it may seem odd that the status of this synthetic hormone as a partial agonist for the facilitation of sexual behavior has apparently been overlooked in recent work using female rats (Brown ef al., 1987; Vathy et al., 1987). Since many drug and hormone antagonists are also partial agonists, it should not be surprising to find that under certain conditions RU 486 is able to mimic the behavioral actions of P to some extent. However, RU 486 did not stimulate proceptivity nearly as well as P, perhaps indicating a different mechanism of action between the two steroids. It may be significant that the agonistic action of RU 486 is usually found only when P is absent from the system. In biochemical studies, RU 486 has been found to possess dual progestin agonist-antagonist actions on estrogen-independent progestin receptors of T47D,, human breast cancer cells, and to induce P-like secretory changes in human endometrial tissue, both in the absence of P stimulation (Gravanis et al., 1985; Horowitz, 1985). In the present study, facilitation by RU 486 followed by an oil injection (as in previous blocking paradigms) was less effective than RU 486 alone, so it is easy to see how a facilitative effect might have been missed in earlier work. We can only speculate from these data that the injection of P, and to a lesser extent the injection of sesame oil, somehow disrupts the facilitating effect of RU 486. Perhaps P or other adrenal steroids
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n VEHELE **
PROGESTERONE
OIL
COIVTROL
TREA’IMENT
ta
RU486
NO INJECTION
AT 1 HOUR
FIG. 2. Effects on sexual receptivity of RU 486 given 1 hr prior to P, sesame oil, or no further injections. Animals were primed with 2 pg EB 48 hr before an injection of either 5.0 mg RU 486 or vehicle immediately followed by a five-mount pretest. Four hours following the second injection subjects were tested for sexual receptivity. *, **, and *** indicate significant dependent f tests at a = 0.05, 0.01, and 0.001, respectively.
released as a consequence of a second injection are able to compete with the RU-PRc binding sufficiently to diminish the weak facilitatory effect. The focus of the current study is primarily behavioral, but we have included an assay to examine the concentration of cytoplasmic P receptors remaining in hypothalamus after RU 486 versus an approximately behaviorally equipotent dose of P. In this way, we have provided evidence that RU 486 does deplete the cytosolic receptor pool at least as efficiently as an equipotent dose of P. Though not conclusive, this result is consistent with the interpretation that this weak facilitation caused by RU 486 is receptor-mediated, but for some reason less effective than the receptor-mediated facilitation induced by P. Further studies using nuclear receptor assays and double-labeling techniques are needed to shed additional light on this question. As it stands, whether the weaker effects/pg of RU 486 on behavior relative to P are due to differences in nuclear binding, steroid-receptor conformation, protein synthesis, or some other factor, remains to be determined. Considering that several other compounds unrelated to P may also cause facilitation of sexual behavior in estrogen-primed rodents, it is even possible that RU 486 facilitation is acting through an entirely distinct mechanism.
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309
TREATMENT
FIG. (HPOA) 100 pg relative
3. Cytosolic progestin receptors assayed of female Long-Evans rats primed with 2 progesterone, or 5 mg RU 486. Receptors to vehicle or the small progesterone dose.
from the hypothalamus-preoptic area wg EB and treated with 0.4 ml vehicle, were significantly depleted by RU 486 (*P < 0.05, Sheffe F Test).
ACKNOWLEDGMENTS This work was supported by USPHS Grant HD04484 to RJB. We thank Dr. D. Philibert (Roussel-UCLAF, Romaineville, France) for the generous gift of RU 486. Some of this work was presented at the Society for Neuroscience Annual Meeting, 1988, in Toronto, Canada.
REFERENCES Blaustein, J. D. (1982). Alteration of sensitivity to progesterone facilitation of lordosis in guinea pigs by modulation of hypothalamic progestin receptors. Brain Res. 243, 287300. Blaustein, J. D., Finkbohner, R., and Delville, Y. (1987). Estrogen-induced and estrogenfacilitated female rat sexual behavior is not mediated by progestin receptors. Neuroendocrinology 45, 152-159. Bradford, M. M. (1976). A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Brown, T. J., and Blaustein, J. D. (1984). Inhibition of sexual behavior in female guinea pigs by a progesterone receptor antagonist. Brain Res. 301, 343-349. Brown, T. J., and Blaustein, J. D. (1986). Abbreviation of the period of sexual behavior in female guinea pigs by the progesterone antagonist RU 486. Brain Res. 373, 103113. Brown, T. J., Moore, M. J., and Blaustein, J. D. (1987). Maintenance of progesterone-
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facilitated sexual behavior in female rats requires continued hypothalmic protein synthesis and nuclear progestin receptor occupation. Endocrinology 121, 298-304. Etgen, A. M., and Bartield, R. J. (1986). Antagonism of female sexual behavior with intracerebral implants of antiprogestin RU 486: Correlation with binding to neural progestin receptor. Endocrinology 119, 1610-1617. Etgen, A. M., and Robisch, D. M. (1988). Differential extraction of estradiol- and tamoxifenreceptor complexes from hypothalamic cell nuclei. Brain Res. 452, l-10. Gravanis, A., Schaison, G., George, M., DeBrux, J., Satyaswaroop, P. G., Baulieu, E. E., and Robel, P. (1985). Endometrial and pituitary responses to the steroidal antiprogestin RU 486 in postmenopausal women. J. Clin. Endocrinol. Metab. 60, 15’6-163. Hardy, D. F., and DeBold, J. F. (1972). Effects of coital stimulation upon behavior of the female rat. .I. Comp. Physiol. Psych. 78, 400-408. Horowitz, K. B. (1985). The antiprogestin RU 38486: Receptor mediated progestin versus antiprogestin actions screened in estrogen-sensitive T47D,, human breast cancer cells. Endocrinology
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Mullick, A., and Katzenellenbogen, B. S. (1986). Antiprogestin-receptor complexes: Differences in the interaction of the antiprogestin RU 38,486 and the progestin R5020 with the progesterone receptor of human breast cancer cells. Biochetn. Biophys. Res. Commun.
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Myers, J. L. (1979). Fundamentals of Experimental Design. Allyn & Bacon, Boston. Okulicz, W. C. (1987). Effect of the antiprogestin RU-486 on progesterone inhibition of occupied nuclear estrogen receptor in the uterus. J. Steroid Biochem. 28, 117-122. Philibert, D. (1984). RU-38486: An original multi-faceted anti-hormone in vivo. In M. K. Agarwal and W. D. Gruyter (Eds.), Adrenal Steroid Antagonism, pp. 77-101. de Gruyter, New York. Pleim, E. T., and Barheld, R. J. (1988). Progesterone versus estrogen facilitation of female sexual behavior by intracranial administration to female rats. Horm. Behav. 22, 150159. Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Vathy, I. U., Etgen, A. M., and Barfield, R. J. (1987). Actions of progestins on estrous behavior in female rats. Physiol. Behav. 40, 591-595.