Intrahypothalamic injection of RU486 antagonizes the lordosis induced by ring A-reduced progestins

Phystology & Behavtor, Vol 46, pp 435--438 ©Pergamon Press plc, 1989 Pnnted m the U S A

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Intrahypothalamic Injection of RU486 Antagonizes the Lordosis Induced by Ring A-Reduced Progestins GABRIELA GONZALEZ-MARISCAL,

OSCAR GONZ,~,LEZ-FLORES AND CARLOS BEYER t

Centro de Investigaci6n en Reproducci6n Animal, CINVESTA V-Universidad Autdnoma de Tlaxcala Apdo, Postal 62, Tlaxcala 90 000, Tlax, Mexico R e c e i v e d 22 February 1989

GONT_a6~EZ-MARISCAL, G., O GONZALEZ-FLORES AND C BEYER. lntrahypothalamic injection of RU486 antagonizes the lordosis induced by ring A-reduced progestins. PHYSIOL BEHAV 46(3) 435-438, 1989.--We explored the possibility that nng A-reduced progestms facdltate lordosis in estrogen primed rats through their interaction with an mtracellular progestin receptor (PR) by ustng RU486 This drug binds with high affimty to the PR, thus preventing the action of progesterone (P). Ovanectomized estrogen-pnmed rats (2 wg estradlol benzoate 40 hr earher) were bilaterally rejected into the ventromedial hypothalanuc nucleus (VMHN) with 1 wg of P, 5ot-pregnanedlone or 313,513-pregnanolone m 1 p,l oll All three progesfins effectively facihtated lordosis, tested at four and eight hours after mtrahypothalanuc rejections The abdlty of RU486 to counteract progestm-lnduced lordosis was assessed by refusing 10 p,g of this agent into the VMHN along with any of the progesnns RU486 antagomzed the lordosis reduced not only by P (67% reduction) but also that reduced by 5et-pregnanedlone and by 313,513-pregnanolone (47% and 93% reductions, respectively). Results suggest that rang A-reduced progestms may act through the PR mechanism to faclhtate lordosis, 1 e , in a fashion similar to P Progestlns

Lordosis

Ventromedial hypothalamus

Ant~progeslans

RU486

Progestln receptor

that specific antagonists of PR would block the lordosis induced by P but not that induced by ring A-reduced progestms. In the present work we tested this prediction by investigating the effects of RU38486 (RU486), a specific progestin antagonist (18, 30, 34, 38, 46), on the lordosis induced by P, 5ct-pregnanedione and 313,513-pregnanolone.

SEVERAL progestins facilitate lordosis when implanted into the ventromedial hypothalamic nucleus (VMHN) of estrogen-primed rats (4, 9, 36, 39, 40). These progestins include both compounds with a delta-4-3-keto structure as well as those reduced in ring A. We recently reported that the intrahypothalamic infusion of oil microdepots containing 5 p,g of progesterone (P), 5et-pregnane3,20-dione (5ct-pregnanedione) or 313-hydroxy-513-pregnan-20-one (313,513-pregnanolone) reliably facilitated lordosis in ovariectomized estrogen-primed rats (4). It has been postulated that an intracellular mechanism-including the binding of the progestin to an intracellular receptor, its transport to the nucleus and the consequent induction of protein synthesis--is involved in the lordogenic action of P and of other delta-4-3-keto pregnanes (5, 25, 26, 37). Although this idea fits well with the facilitation of lordosis by P or R5020, steroids which bind with good affinity to the intracellular progestin receptor (PR), it does not appear appropriate to explain the effects of ring A-reduced pregnanes which bind poorly to PR (8, 16, 19, 28, 44). These last progestins could act through alternative mechanisms, since they exert potent effects on neuronal excitability (10, 11, 15, 20, 24, 29, 31, 35, 41) by interacting with various membrane processes (11, 13, 15, 23, 27, 32, 33) which do not involve their binding to PR. Based on the above considerations we predicted

METHOD

Animals and Surgical Procedures Wistar female rats (220-250 g), bred in our colony, were maintained under controlled temperature (23---2°C) and light (14 hr light: 10 hr dark) conditions. They were fed Purina rat chow and water ad lib. Subjects (Ss) were ovariectomized under ether anesthesia and, two weeks later, stainless steel cannulae were bilaterally implanted into the VMHN as previously described (4).

Hormone Treatments One week after implantation, Ss received 2 )xg estradiol benzoate (EB; SC in 0.1 ml carthamus oil). Forty hours later they were lightly anesthetized with ether and one of the following treatments was delivered as an oil solution into the VMHN:

~Requests for reprints should be addressed to Carlos Beyer.

435

GONZALEZ-MARISCAL ET AL.

436

TABLE 1 ANTAGONISM OF PROGESTIN-INDUCED LORDOSIS BY SIMULTANEOUS INFUSION OF RU486 INTO VMHN

Response at 4hr Group

Treatment

1

oil

2 3 4

10p.gRU486 1 ixg progesterone 1 i~g progesterone + 10 ~g RU486 1 p.g 5et-pregnanedmne 1 p~g 5et-pregnanedlone + 10 ~g RU486 1 i~g 3[3,513-pregnanolone 1 p.g 313,513-pregnanolone + 10 ~g RU486

5 6 7 8

N 6

LQ 0 +-

8hr Responsive Ss

LS 0

0

-+

0

0%

LQ 0 -+

0

LS

Responsive Ss

0 _+ 0

0%

6 15 20

0 -+ 0 49 _+ 43* 16 -+ 32:~

0 10 3

_+ 0 -+ 10* -+ 7:~

0% 60% 30%

0 _+ 0 51 +- 46* 27 +_ 38

0 +_ 0 l0 -+ 10* 6 -4- 9

0% 60% 55%

13 19

60 _+ 43* 32 -+ 37:~

14 7

-+ l l t +- 9§

84% 58%

61 _+ 40* 37 -+ 39

15 _+ 9[ 9 -+ 10

77% 58%

11 ll

45 + 45* 3 -+ 9:~

12 _+ 11" 0 6 -+ 2¶

54% 9%

50 _+ 41" 19 _+ 31

12 -+ 10" 7 _+ 9

63% 36%

Ovanectomlzed rats received, 40 hr after estrogen pnnung (2 ~g EB), bilateral infusions of the above-shown treatments in 1 Ixl od into the VMHN The lordosis quotient (LQ) and lordosis score (LS) were determined four and eight hours later Data show means --- standard devlatmn. *p-<0.05, tp-<0 01 versus Group 1, :~p-<0.05, §p<0 025, ¶p-<0 01 versus the corresponding group receiving only progestin

Group Group Group Group Group Group Group Group

1: 2: 3: 4: 5" 6" 7: 8.

oll vehicle 10 p,g RU486 1 p,g progesterone 1 p.g progesterone + 10 p.g RU486 1 p.g 5et-pregnanedione 1 p,g 5a-pregnanedione + 10 txg RU486 1 Ixg 313,513-pregnanolone 1 Ixg 313,513-pregnanolone + 10 Ixg RU486

(n = (n = (n = (n = (n = (n = (n = (n =

6) 6) 15) 20) 13) 19) 11) 11)

All solutions were admlmstered bilaterally m 1 p.1 along approximately 15 sec. This volume was required for the adequate solution of both RU486 and the progestms. RU486 was a generous girl of ROUSSEL-UCLAF (Romalnville, France). The other steroids were obtained from Sigma (St. Lores, MO). The vehicle used for dissolving progestins and RU486 was carthamus oll containing 5% benzyl alcohol and 15% benzyl benzoate. The dose used for intrahypothalamic injections was selected from a study exploring dose-response relationships of various progestins for facilitating lordosis behavior (Beyer, Gonzfdez-Flores and Gonz~lezMariscal, in progress).

Behavioral Testing Immediately prior to intrahypothalamic injections a pretest for lordosis was conducted to determine receptivity levels induced by EB only. Ss showing lordosis behavior in response to estrogen were discarded from the experiment. Testing was done four and eight hours after intrahypothalamic injections in circular Plexiglas arenas containing a sexually vigorous male. The lordosis quotient (LQ) and the lordosis score (LS) were evaluated for each female as follows: LQ = mean number of lordosis in 10 mounts × 100 LS = mean lordosis intensity in 10 mounts × 10 Lordosis intensity was rated in a scale of 0 to 3 as described by Hardy and De Bold (12). The occurrence of proceptive behaviors (hopping, darting, ear-wiggling) was also recorded.

Statistical Analysts The facflitaUon of lordosis by lntrahypothalamic infusion of progestins was determined by comparing LQ and LS values of groups 3, 5 and 7 agmnst those seen in Group 1, that received only the oll vehicle. The ability of RU486 to counteract the lordosis reduced by the different progestms was assessed by companng LQ and LS values of groups receiving RU486 + progestin against the corresponding groups recewing only progestin. Mann-Whitney's U-test was used for all comparisons (42).

Histologwal Analysis Upon completion of experiments, Ss were sacrificed with an overdose of ether and they were then perfused through the heart with 10% formalin. The brains were removed, fixed, embedded in paraffin, cut in the transverse plane and stained with hematoxilineosin to verify cannula placement. Only animals showing the tip of the cannula within 0.5 mm from the limit of the VMHN were included in the analysis. RESULTS

Table 1 shows that the intrahypothalamic infusion of 1 pLg of all the progestins tested significantly stimulated lordosis. Maximal LQ and LS values were usually obtained already at four hours after mtrahypothalamlc injections. Lordosis parameters remained essentially unchanged at eight hours. The magmtude of the response induced by 5et-pregnanedione and by 313,513-pregnanolone thd not significantly differ from that induced by P. Proceptive behaviors were not reduced by any progestin at any test. The infusion of RU486 along with P significantly reduced both LQ and LS values by 67%. Surprisingly, RU486 antagonized also the action of 5et-pregnanedione and of 3 [3,513-pregnanolone. Thus, it reduced the LQ and LS of the former by 47% and 50%, respecUvely (p<0.05; p<0.025) and it practically abohshed the effect of the latter (93% and 95% reductions m LQ and LS, respectively; p < 0 . 0 5 and p<0.01). The effect of RU486 was transitory, since at eight hours after mtrahypothalamic injections

PROGESTINS, RU486 AND LORDOSIS

437

groups 4, 6 and 8 showed higher LQ and LS values than those displayed at four hours. Moreover, at eight hours no significant statistical difference was obtained between groups receiving RU486 and those receiving only the progestins (i.e., groups 3, 5 and 7, respectively). The intrahypothalamic mfusion of RU486 alone did not stimulate lordosis nor did it induce any overt alterations in motor activity, thereby ruling out the possibility that RU486 antagonized progestin-induced lordosis through nonspecific effects.

DISCUSSION The present results confirm and extend previous findings showing stimulation of lordosis by the administration of P (4, 9, 36, 39, 40) and some ring A-reduced progestins (4, 9, 39) into the VMHN of estrogen-primed rats. The ~nhibitory effect of intrahypothalamic RU486 upon the action of intrahypothalamic P also agrees with previous findings showing that this agent reduces (7) or abolishes (5) the lordosis reduced by systemic P. The molecular mechanism involved in the antiprogestational action of RU486 has been well characterized (2, 6, 14, 18, 22). Thus, RU486 inhibits the genomic actions of P by binding to PR. This binding stabilizes the 8S form of PR, thus preventing its interaction with DNA [for review, see (2)]. The fact that the lordogenic actmn of P was depressed by RU486 supports the idea that this effect is mediated by the binding of the steroid to its intracellular PR. This conclusion is also supported by studies showing a good correlation between mtrahypothalamic PR levels and the capacity of P to stimulate lordosis in rats (1, 5, 7, 25, 26, 37). Moreover, P binds with good affinity to the intracellular PR (8, 16, 19, 28, 44). In contrast to P, ring A-reduced progestins, which are natural metabohtes of P (3, 6, 17), bind poorly to PR--particularly those reduced in the 513-position. Although the absolute values of binding affinities reported for the nng A-reduced progestins vary widely (8, 16, 19, 28, 44), it is clear that they bind to PR with 10-

to 1,000-fold less affinity than P. Moreover, ring A-reduced progestins exert powerful effects on neuronal excitability through their interaction with membrane proteins or phospholipids (10, 11, 15, 20, 24, 29, 31, 35, 41). Therefore, on the basis of their pharmacology and their poor binding to PR it has been suggested that ring A-reduced progestins stimulate lordosis through other mechanisms than their binding to PR (4,9). If this were the case, RU486, which acts intracellularly through PR, would not interfere with the lordosis induced by ring A-reduced progestins. The present results, however, do not support the participation of such an alternative mechanism, but, rather, suggest that ring A-reduced progestins use, in a similar manner to P, their interaction with PR to facilitate lordosis. It must be recalled that drugs showing a low affinity for a given receptor--as is the case of ring A-reduced progestins towards PR--are not inactive but, rather, less potent than those with high affinity (47,48). This implies that quantitatwely similar responses can be obtained with both low and high affinity drugs if a sufficiently large dose is administered, a condition that probably occurred in our experiment. An alternative explanatmn for the present results would be that RU486 competes not only for PR but also for membrane-bound receptors mediating other effects of ring A-reduced progestins, such as: enhancement of GABAergic activity (13, 23, 27, 33, 43), membrane stabilization (10, 11, 15, 31, 41) and alterations in the release of neurotransmitters or neuromodulators (21, 32, 45). These membrane effects could theoretically be involved in the facilitation of lordosis. However, the chemical structure of RU486 (a delta-4-3-keto-19-nor-progestin) is different from that assocmted to the membrane actions exerted by progestins. Moreover, we have recently shown that RU486 failed to prevent or diminish the anesthetic action of 3et-hydroxy-513-pregnan-20-one in rats, a response involving an action at the membrane level. Therefore, though it is not possible to rule out an action of RU486 on one of the various membrane effects of ring A-reduced progestins, our present data favour the idea that all progestins--P and its ring A-reduced metabolites--facilitate lordosis by using a common cellular mechanism, i.e., their interaction with PR.

REFERENCES 1. Ahdleh, H ; Brown, T. J , Wade, G. N., Blaustem, J D. Hypothalamic nuclear progestin receptors and the duration of sexual receptivity m ovanectomized and ovanectonuzed-hysterectonuzed rats. Physlol Behav. 36-211-215, 1986 2 Banheu, E. E Antihormone-sterold hormonal activity, heat shock protein hsp90 and receptors. Horm. Res. 28 181-195; 1987. 3 Bertacs, S. J., Bertacs, P. J., Clarke, J L . ; Karavolas, H. J Distribution and ovarian control of progestm-metabolizmg enzymes in various rat hypothalarmc regions J. Steroid Biochem. 26 321-328; 1987. 4 Beyer, C., Gonz~dez-Manscal, G.; Equ~ar, J. R.; G6mora, P. Lordosis faclhtatton In estrogen-primed rats by mtrabrain rajectlon of pregnaoes. Pharmacol. Blochem. Behav. 28:919-926; 1988 5. Brown, T. J , Moore, M J , Blaustem, J. D. Mmntenance of progesterone-facilitated sexual behavmr m female rats requires conunued hypothalanuc protein synthesis and nuclear progestin receptor occupation. Endocrinology 121:298-304, 1987. 6. Celotta, F.; Melcangi, R. C., Negd-Cesl, P.; Ballaino, M ; Martini, L. Differential dlstnbutmn of the 5a reductase m the central nervous system of the rat and mouse: are white matter structures of the brain target tissue for testosterone acnon? J. Steroid Blochem. 26:125-129; 1987 7. Etgen, A M., Barfield, R J. Antagomsm of female sexual behavior with mtracerebral implants of antiprogestin RU38486: correlatmn with bin&ng to neural progestin rec eptors. Endocnnology 119"1610-1617, 1986. 8. Ghoshal, A , Feuer, G. Structural requtrements for progesterone binding to the hepatic endoplasmic reticulum in the female rat.

Steroids 43:621-630, 1984. 9 Glaser, J. H , Etgen, A. M , Barfield, R. J. Intrahypothalamlc effects of progestras on estrous behavior and progestm receptor binding. Physiol. Behav. 34.871-877; 1985. 10 Gyermek, L., Genther, G , Flermng, N. Some effects of progesterone and related steroids on the central nervous system. Int. J. Neuropharmacol. 6 191-198; 1967 11 Gyermek, L., Soyka, L. F Steroid anesthetics Anesthesiology 43'331-344, 1975. 12. Hardy, D. F , DeBold, J. F. The relationship between levels of exogenous hormones and the display of lordosis by the female rat Horm. Behav. 2:287-297; 1971. 13 Hamson, N. L.; Majewska, M. D.; Harnngton, J W . ; Barker, J. L. Structure-activity relanonships for steroid interacUon with the G A B A A receptor complex. J. Pharmacol Exp. Ther 241:346-353; 1987 14. Heikinhe~mo, O., Kontula, K., Crozatto, H ; Spitz, I., Luukainen, T.; Lahtaeenmliki, P. Plasma concentrations and receptor binding of RU486 and its metabohtes m humans. J. Steroid Biochem. 26: 279-284; 1987 15. Holzbauer, M. Physiological aspects of steroids with anesthenc properties. Med. Biol. 54:227-242, 1976. 16. Iswan, S.; Colas, A. E.; Karavolas, H. J. Binding of 5ct-dihydroprogesterone and other progestlns to female rat anterior p~tmtary extracts. Steroids 47:189-203; 1986. 17 Karavolas, H. J., Bertics, P. J.; Hodges, D.; Rudle, N Progesterone processing neuroendocrine structures. In: Celom, F , Martmi, L., eds. Metabolism of hormonal steroids in the neuroendocnne structures New York: Raven Press, 1984:149-170.

G O N Z i d . ~ E Z - M A R I S C A L ET AL.

438

18 Kawai, S., Nieman, L. E.; Brandon, D. D., Udelman, R , Lonaux, B L , Chorusos, G P Pharmacokanetic properties of the antiglucocorticold and antaprogesterone steroid RU486 in man. J Pharmacol Exp Ther 241 401-406, 1987. 19 Kontula, K , Janne, O R , de Jager, E., de Visser, J., Zeelen, F Progesterone binding proteins, in vitro binding and biological activity of different steroidal hgands. Acta Endocnnol. 78 574-592; 1975 20. Kubh-Garfias, C., Cervantes, M , Beyer, C Changes in multiunit actiwty and EEG induced by the administration of natural progestins to flaxedil immobilized cats. Brain Res 114 71-81, 1976 21 Kubh-Garfias, C.; Azpema, E., Villanueva-Tello, T., PonceMonter, H. Inhibition of noradrenallne release by 513-progestlns in cerebral cortex slices. Proc West. Pharmacol. Soc 26 135-138, 1983 22 Lahtennmaka, P., Heikmheimo, O , Croxatto, H , Spitz, I , Shoupe, C.; Birgerson, L., Luukkainen, T Pharmacoklnetics and metabolism of RU486 J Steroid Biochem 27 4-6, 1987 23. Lambert, J. J., Peters, J. A., Cottrell, G. A. Actions of synthetic and endogenous steroids on the GABA A receptor Trends Pharmacol Sci 8 224-227, 1987 24 Landgren, S , Aasly, J., Backstrom, T , Dubrovsky, B , Danielsson, E The effect of progesterone and its metabohtes on the intenctal epileptiform discharge in the cat's cerebral cortex Acta Physiol Scand. 131.33-42, 1987 25 McEwen, B. S. Neural gonadal steroid action Science 211 13031311, 1981 26. McGinnis, M. Y., Parsons, B., Rainbow, T C , Krey, L C , McEwen, B S Temporal relationship between cell nuclear progestin receptor levels and sexual receptivity following intravenous progesterone administration. Brain Res 218:365-371, 1981 27 Majewska, M D , Harrison, N L , Schwartz, R. D , Barker, J. L , Paul, S M Steroid hormone metabohtes are barbiturate-like modulators of the GABA receptor Science 232:1004-1007; 1986. 28. Moguflewsky, M.; Raynaud, J P Progestin binding sites in the rat hypothalamus, pituitary and uterus. Steroids 30'99-109, 1977 29. Norberg, L., Wahlstrom, G., Backstrom, T The anaesthetic potency of 3o~-hydroxy-5a-pregnan-20-one and 3cx-hydroxy-513-pregnan-20one deternuned with an intravenous EEG-threshold method in male rats. Pharmacol. Toxlcol 61"42-47, 1987 30 Okuhcz, W Effect of the antiprogestm RU486 on progesterone inhibition of occupied nuclear estrogen receptor in the uterus J Steroid Biochem 28 117-122, 1987 31 P'an, S Y , Laubach, G D. Steroid central depressants Met Horm Res 3 415-475, 1964 32 Park, O. P , Ramfrez, V D. Pregnanolone, a metabohte of progesterone, stimulates LHRH release in vitro and in VlVO studies Brain Res. 437'245-252, 1987. 33 Peters, J. A , Kirkness, E W Callachan, H., Lambert, J J , Turner,

34.

35

36 37

38

39

40

41 42 43

44

45

46

47 48.

A J. Modulation of the GABA A receptor by depressant barbiturates and pregnane steroids. Br. J Pharmacol 94'1257-1269, 1988 Phihbert, D., Moguilewsky, M , Mary, I , Lecaque, D , Toumemine, C , Secchl, J , Deraedt, R Pharmacological profile of RU486 in animals In Bauheu, E G , Segal, S J., eds. The antlprogestln steroid RU486 and human fertility control. New York Plenum Press, 1985 49-62 Phillis, J W. Potentiation of the depression by adenosine of rat cerebral cortical neurones by progestational agents Br J Pharmacol 89 693-702; 1986 Powers, J B Facilitation of lordosis in ovanectomized rats by lntracerebral progesterone implants Brain Res. 48 311-325, 1972 Rainbow, T C , McGlnnls, M. Y , Davis, P G , McEwen, B S Apphcatlon of anisomycln to the lateral ventromedial nucleus of the hypothalamus inhibits the activation of sexual behavior by estradiol and progesterone Brain Res 233 417-423, 1982. Ranch, M , Loosefelt, H , Phllibert, D , Milgrom, E. Mechanism of action of an antlprogesterone, RU486, in the rabbit endometnum Effects of RU486 on the progesterone receptor and on the expression of the uteroglobin gene Eur. J. Pharmacol 148 213-218, 1985 Rodrfguez-Manzo, G , Cruz, M L , Beyer, C Facilitation of lordosis behavior in ovanectormzed estrogen primed rats by medial preoptic implantation of 513,313-pregnanolone a nng A-reduced progesterone metabohte. Physiol. Behav 36 277-281, 1986 Rubm, G S , Barfield, R J Progesterone in the ventromedial hypothalamus facilitates estrous behavior in ovarlectomized estrogen primed rats. Endocnnology 113 797-804, 1983 Selye, H Anesthetic effect of steroid hormones Proc Soc Exp Biol Med 46 ll6-121, 1941 Siegel, S Non-parametric statistics for the behavioral sciences New York McGraw Hill Book Co , 1956 Simmonds, M A , Turner, J P Potentiators of responses to activation of GABA A receptors Neuropharmacology 26 923-930, 1987 Smith, H. E.; Smith; R G , Toft, D O , Neergaard, J R , Burrows, E P , O'Malley, B W Binding of steroids to progesterone receptor proteins in chick oviduct and human uterus. J Biol Chem 249 5924-5932, 1974 Su, T P , London, E D , Jaffe, J H Steroid binding at cr receptor suggests link between endocnne, nervous and immune systems Science 240:219-221, 1988 Takeda, A Progesterone and antiprogesterone (RU38486) modulation of estrogen-inducible glycoprotein (USP-1) synthesis and secretion in rat uterine epithelial cells Endocnnology 122 1559-1564, 1988 Tallarida, R J , Jacob, L S The dose-response relation in pharmacology New York' Sprlnger-Verlag, 1979 Yamamura, H I Neurotransmitter receptor binding New York Raven Press, 1978