GnRH mediates estrous behavior induced by ring A reduced progestins and vaginocervical stimulation

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Behavioural Brain Research 187 (2008) 1–8

Research report

GnRH mediates estrous behavior induced by ring A reduced progestins and vaginocervical stimulation Porfirio G´omora-Arrati a , Carlos Beyer a , Francisco Javier Lima-Hern´andez a , Maria Elena Gracia a , Anne M. Etgen b , Oscar Gonz´alez-Flores a,∗ a

Centro de Investigaci´on en Reproducci´on Animal, CINVESTAV Universidad Aut´onoma de Tlaxcala. Apdo. 62, Tlaxcala, c.p. 90000, Mexico b Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA Received 1 March 2007; received in revised form 9 June 2007; accepted 20 August 2007 Available online 22 August 2007

Abstract The present study was designed to assess the participation of gonadotropin-releasing hormone (GnRH) in the display of estrous behavior induced by application of vaginal–cervical stimulation (VCS) and by the intracerebroventricular (icv) administration of progesterone and its ring A-reduced metabolites to ovariectomized (ovx), estradiol benzoate (E2 B) primed rats. Icv injection of Antide, a GnRH-1 receptor antagonist, significantly depressed lordosis behavior in ovx, E2 B-primed rats treated with icv GnRH. Application of VCS to ovx, E2 B-primed rats facilitated both lordosis and proceptivity. These behavioral responses were significantly depressed by the icv administration of Antide. Similarly, icv Antide blocked the stimulatory effect on both lordosis and proceptive behaviors elicited by progesterone and its ring A-reduced metabolites: 5␣-pregnandione (5␣DHP), 5␣-pregnan-3␣-ol-20-one (5␣,3␣-Pgl) and 5␤-pregnan-3␤-hydroxy-20-one (5␤,3␤-Pgl) in ovx, E2 B-primed rats. By contrast, icv injection of Antide failed to interfere with the facilitatory effect of the synthetic progestin megestrol acetate on lordosis and proceptive behaviors. This progestin is not reduced in ring A. The results suggest that GnRH release is an important process in the chain of events leading to the display of estrous behavior in response to progesterone, its ring A-reduced metabolites, and VCS in female rats. © 2007 Elsevier B.V. All rights reserved. Keywords: GnRH; Progesterone; Megestrol acetate; 5␣-Pregnan-3␣-ol-20-one; 5␤-Pregnan-3␤-hydroxy-20-one; Antide; Lordosis; Proceptivity

1. Introduction A well-known action of ring A-reduced progesterone (P) metabolites is the facilitation of lordosis and proceptive behaviors in estradiol (E2 )-primed rodents [7,8,33–36,38,39,41,43]. The cellular mechanism(s) by which these progestins enhance female sexual behavior is (are) unclear. The fact that concurrent administration of the classical progestin receptor (PR) antagonist RU486 abolished the estrous behavior induced by several ring A-reduced progestins [8,10,43] implicated the participation of PRs in this response. However, progestins such as 5␣-pregnan3␣-ol-20-one (5␣,3␣-Pgl), which do not bind to the intracellular PR [70,79], induce lordosis behavior more potently than P when administered to estrogen-primed rats either intravenously or

∗

Corresponding author. Tel.: +52 246 46 21727; fax: +52 246 46 21727. E-mail address: [email protected] (O. Gonz´alez-Flores).

0166-4328/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2007.08.017

directly into the brain [7,8,39]. This suggests that ring A-reduced progestins activate the PR in a ligand independent mechanism through signaling pathways triggered at the membrane level. Indeed, several protein kinase inhibitors reduce estrous behavior induced by ring A-reduced progestins [38,39,41]. This effect (PR activation) could be produced by a direct action of the progestins at the membrane level of neurons possessing PRs or indirectly by releasing neurotransmitters or neuromodulators capable of activating intracellular signaling mechanisms in these PR neurons. P and some of its ring A-reduced metabolites influence gonadotropin secretion by acting at the hypothalamic level. Thus, several workers using ovariectomized (ovx), E2 -primed rats found that 5␣-pregnanedione (5␣-DHP), 5␣,3␣-Pgl and 5␤,3␤-pregnanolone (5␤,3␤-Pgl) potently stimulate the release of gonadotropins by activating GnRH secretion from the hypothalamus [24,30,31,44,57,59,75,82,83]. Indeed, 5␤,3␤-Pgl is 1000 times more potent than P in inducing GnRH release

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both in vivo and in vitro [62,66–68]. The release of hypothalamic GnRH by ring A-reduced progestins is relevant for its stimulatory effect on lordosis behavior; as initially shown by Moss et al. [53–55], this peptide can trigger female sexual behavior in ovx or ovx-adrenalectomized rats primed with E2 [10,21,32,42,45,73,74,81]. At least two forms of GnRH, GnRH-I and GnRH-II, are present in mammals. The presence of two GnRH isoforms suggests the existence of two cognate GnRH receptor subtypes, type 1 and 2 [51,56,76]. The GnRH type 1 receptor participates in the expression of female sexual behavior, because Antide, a type 1 receptor antagonist, blocked lordosis behavior induced by GnRH in ovx rats primed with E2 benzoate (E2 B) [45,81]. The purpose of the present study was to use the GnRH1 antagonist Antide to test the hypothesis that the release of GnRH participates in the facilitation of estrous behavior by ring A-reduced progestins. Because vaginal–cervical stimulation (VCS) both enhances lordosis behavior [28,37,46,48,72] and promotes GnRH release [11,16,64,78], we also tested the capacity of Antide to interfere with VCS facilitation of lordosis. The capacity of Antide to block the stimulatory effect of megestrol acetate (MA) on estrous behavior was also explored, because this progestin cannot be ring A-reduced and does not stimulate GnRH release. 2. Material and methods 2.1. Animals and surgeries A total of 134 Sprague–Dawley female rats (240–280 g body weight), bred in our colony in Tlaxcala, were used. They were maintained under controlled temperature (23 ± 2 ◦ C) and light (14:10; L:D) conditions and fed Purina rat chow and water ad libitum. Females were bilaterally ovx under ether anesthesia and housed in groups of four. Two weeks after ovx, the females were anesthetized with xylazine (4 mg/kg) and ketamine (80 mg/kg), placed in a Kopf stereotaxic instrument (Tujunga, CA, USA), and implanted with a stainless steel cannula (22 gauge, 17 mm long; Plastics One, Roanoke, VA, USA) into the right lateral ventricle following coordinates from the atlas of Paxinos and Watson [63] (A/P + 0.80 mm, M/L −1.5 mm, D/V −3.5 mm with respect to bregma). A stainless steel screw was fixed to the skull, and both cannula and screw were attached to the bone with dental cement. An insert cannula (30 gauge) provided with a cap was introduced into the guide cannula to prevent clogging and contamination. Animal care and all the experimental procedures adhered to the Mexican Law for the Protection of Animals.

2.2. Testing procedures Tests for sexual behavior (receptivity and proceptivity) were conducted by placing females in a circular Plexiglas arena (53 cm in diameter) with a sexually active male. The lordosis quotient [LQ = (number of lordosis/10 mounts) × 100] was used to assess receptive behavior. Proceptivity was evaluated by determining the incidence of hopping, darting, and ear-wiggling across the whole receptivity test [49]. We considered an animal proceptive when showing two of these behaviors during the testing period. This criterion was used since in our Sprague–Dawley rats only a small proportion of animals will display the three proceptive behaviors together. This may be due to the fact that our Sprague–Dawley rats rarely (<10%) show darting in our testing conditions.

2.3. Chemicals The steroids used were: E2 B; P; 5␣-DHP; 5␣,3␣-Pgl; 5␤,3␤-Pgl; and MA (17-hydroxy-6-methylpregna-4,6-diene-3,20-dione acetate). Other drugs

used were: GnRH-1 and the GnRH-1 receptor antagonist Antide [Acetyl-dAla(2-naphthyl)-d-Lys(N␧-nicotinoyl)-d-Lys(N␧-nicotinoyl)-Leu-Lys(N␧isopropyl)Pro-d-Ala-NH2 ]. Steroids and Antide were purchased from Sigma (St. Louis, MO, USA). GnRH was purchased from Peninsula Laboratories (Belmont, CA, USA). E2 B was always administered sc in 0.1 ml sesame oil.

2.4. Experiment 1 2.4.1. Effect of Antide on estrous behavior induced by GnRH-1 This experiment determined whether the lordosis behavior induced by GnRH-1 is mediated by activation of the GnRH-1 receptor. One week after implantation of a cannula in the right lateral ventricle, 19 ovx rats were primed with 5 ␮g of E2 B (hour 0). Thirty-nine hours later, 1 ␮g of Antide or vehicle (saline) in a 1 ␮l volume was administered intracerebroventricularly (icv). One hour after Antide or saline (40 h post E2 B), an icv injection of 50 ng of GnRH-1 was administered. The dose of Antide was taken from the study of Kauffman and Rissman [45], while the dose of the GnRH-1 was selected from our previous experiment, in which a dose response curve for icv GnRH was established in ovx rats treated with 5 ␮g E2 B [69]. The dose of 50 ng elicited maximal responses in both lordosis and proceptive behaviors [69]. The number of animals in the control group was 8 (saline + GnRH), while the number of animals in the treatment group was 11 (Antide plus GnRH). The behavioral tests were conducted at 60, 120 and 240 min after GnRH administration. Previous studies both from our laboratory and others have found that lordosis behavior appears at 60 min following GnRH administration and reaches its maximal level at 120 min, declining thereafter (240 min; [54,60,74]).

2.5. Experiment 2 2.5.1. Effect of Antide on estrous behavior induced by VCS Ovx rats were injected with 5 ␮g of E2 B. Forty hours later, animals were divided into two treatment groups (n = 8/group). One group received manual flank stimulation plus VCS and saline, and the other one received the same stimulation plus Antide. Manual flank stimulation consisted of palpations applied with the finger and thumb to both flanks and with the palm of the hand to the perineal area of the rat. VCS consisted of 150 g of pressure into the vagina and cervix through a calibrated vaginal probe [37,46] applied together with manual flank stimulation for approximately 5 s. Antide or saline (1 ␮l) was administered icv 1 h before stimulation. Immediately (0 min), 120 and 240 min following stimulation, females were placed in a circular plexiglas arena until they received 10 mounts with pelvic thrusts from experienced males.

2.6. Experiment 3 2.6.1. Effect of Antide on estrous behavior induced by P and MA In this experiment we used two progestins: P, which, in the brain, is reduced at C5 (ring A-reduction) to yield initially 5␣-DHP and subsequently pregnanolones by a further reduction at C3, and MA, which can not be reduced in ring A due to the presence of a double bond at C6 [60,61,79]. Ovx females were primed with 2 ␮g of E2 B and 39 h later, 1 ␮g of Antide was administered icv in 1 ␮l of saline. The dose of E2 B (2 ␮g) has been found in previous experiments to effectively prime ovx rats to the action of both P and its ring A-reduced metabolites at the dosages employed in this study. One hour later either P or MA was administered icv at a dose of 130 ng dissolved in 1 ␮l of oil. This dose of P has been observed in a previous study to elicit a maximal response when icv administered to ovx, E2 B(2 ␮g) primed rats. Some animals (n = 8/group) were assigned to receive P or MA plus saline, and other rats (n = 8/group) received these progestins in combination with Antide. As a further control, eight females only received Antide plus the progestin vehicle (oil). Behavioral tests were carried out at 30, 120 and 240 min after progestin administration.

2.7. Experiment 4 2.7.1. Effect of Antide on estrous behavior induced by ring A-reduced metabolites of P Animals were primed with 2 ␮g of E2 B for 39 h as in Experiment 3. One hour after the icv administration of 1 ␮g of Antide to E2 B-primed females,

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ring A-reduced progestins dissolved in 1 ␮l of oil were administered icv, at the following doses: 13 ng for 5␣-DHP and 5␣,3␣-Pgl, and 130 ng for 5␤,3␤Pgl. These doses were found to be highly effective in eliciting estrous behavior from dose response curves previously established in our laboratory [39]. Animals (n = 8/group) were assigned to receive one of the three progestins plus saline (vehicle for Antide) or one of three progestins in combination with Antide.

2.8. Histological confirmation of cannula placement Twenty-four hours after completion of the experiments, females were deeply anesthetized with ether, and 1% methylene blue was administered through the cannula. The brain was removed and sectioned in the transverse plane to check the cannula position in the right lateral ventricle. Eight animals whose cannula was not in the ventricle were discarded from the experiment.

2.9. Statistical analysis The effect of Antide on the behavioral actions of GnRH, VCS and progestins was assessed by comparing the LQs and proceptivity obtained with each agent plus vehicle versus those obtained when Antide was administered. Because the distribution of LQ values in some groups was not normal, a Wilcoxon–Mann–Whitney U-test was used to compare the two independent groups [15]. Fischer’s exact probability test was used to compare the proportion of proceptive females among experimental groups [15,22].

3. Results 3.1. Experiment 1: Effects of Antide on estrous behavior induced by GnRH-I Fig. 1 shows the lordosis behavior induced by 50 ng of GnRH administered icv. LQ scores were high enough at 120 min after GnRH infusion (55 ± 7) to reveal that the group receiving Antide in combination with GnRH exhibited significantly less lordosis than those given GnRH alone (p < 0.05).

Fig. 2. Effect of icv injection of 1 ␮g of Antide on the stimulatory effect of vaginal–cervical stimulation (VCS) on lordosis and proceptive behaviors of ovx, E2 B-treated rats. Facilitation of lordosis and proceptivity by VCS (n = 8) at 120 min was inhibited by Antide. * p < 0.01 vs. Antide plus VCS (n = 8).

3.2. Experiment 2: Effects of Antide on estrous behavior induced by VCS Fig. 2 shows the effect of Antide on the estrous behavior (lordosis and proceptivity) induced by VCS. Although VCS started to facilitate lordosis behavior almost immediately in some animals, both LQ scores and proceptivity were highest at 120 min after VCS. The icv administration of Antide 1 h before VCS significantly reduced lordosis and suppressed proceptive behavior induced by VCS at 120 min (p < 0.01). 3.3. Experiment 3: Effects of Antide on estrous behavior induced by P and MA

Fig. 1. Effect of icv injection of 1 ␮g of Antide on the stimulatory effect of GnRH (50 ng; n = 8) on lordosis behavior of ovx, E2 B-treated rats. Facilitation of lordosis by GnRH at 120 min was inhibited by Antide administration. + p < 0.05 vs. Antide plus GnRH (n = 11).

Table 1 shows the effects on both lordosis and proceptive behavior of the icv administration of 130 ng of P or MA alone and combined with 1 ␮g of Antide. At this dose, both progestins induced significant lordosis and proceptive behaviors, with the best response obtained at 120 min. Antide significantly blocked

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Table 1 Effect of 1 ␮g of Antide on sexual behavior (lordosis and proceptivity) induced by 130 ng or P and MA in ovx rats primed with 2 ␮g of E2 B N

30 min

120 min

240 min

LQ, mean ± S.E.

%Proceptive females

LQ, mean ± S.E.

%Proceptive females

LQ, mean ± S.E.

%Proceptive females

saline + P Antide + P

9 9

45 ± 7 4 ± 3*

11 0

63 ± 12 17 ± 7*

55 11+

33 ± 9 29 ± 8

11 12

saline + MA Antide + MA

8 8

49 ± 9 59 ± 11

37 37

76 ± 6 66 ± 9

62 40

54 ± 10 51 ± 7

50 25

Estrous behavior in E2 B-primed animals was tested 30, 120 and 240 min after application of progestins. Antide was administered into the right lateral ventricle 60 min before progestins. * p < 0.01; + p < 0.05 vs. corresponding group receiving Antide plus progestins.

the lordosis induced by P at 30 and 120 min (p < 0.01) and also inhibited the proceptivity induced at 120 min (p < 0.05). On the other hand, Antide did not modify the facilitation of lordosis or proceptive behaviors by MA. Indeed, LQ values were slightly higher in the group receiving MA plus Antide than in the group injected with MA plus vehicle at 30 min after icv injection. Values obtained with Antide alone (data not shown) were not different from those obtained with the combination of Antide and P. 3.4. Experiment 4: Effect of Antide on estrous behavior induced by ring A-reduced metabolites of P Fig. 3 shows that icv infusion of ring A-reduced progestins significantly stimulated lordosis behavior at 120 min

post-infusion, when maximum LQ values were apparent. Lower values were seen on the 30 and 240 min tests (panel A). All progestins induced some proceptive behavior, and maximal proceptivity was observed at 120 min (panel B). Two progestins, 5␣,3␣-Pgl and 5␤,3␤-Pgl, induced the same proportion of proceptive females (75%) while 5␣-DHP induced proceptivity in 62.5% of females. Administration of Antide significantly decreased LQ scores in animals infused with 5␣-DHP and 5␣,3␣-Pgl at all the times tested, with the greatest inhibition at 120 min (see Fig. 3, panel A for p-values). In females given 5␤,3␤-Pgl, Antide blocked lordosis significantly at 30 min (p < 0.01) and almost completely suppressed lordosis behavior at 120 min (p < 0.001). By 240 min after progestin injection, no significant differences between Antide-treated females and those receiving 5␤,3␤-Pgl were noted. At the 120 min test, Antide

Fig. 3. Effect of icv injection of 1 ␮g of Antide on the stimulatory effect of 5␣-DHP (13 ng; n = 8), 5␣,3␣-Pgl (13 ng; n = 8) and 5␤,3␤-Pgl (130 ng; n = 8) on lordosis (panel A) and proceptivity (panel B) of ovx, E2 B-treated rats. Facilitation of lordosis by the ring A-reduced progestins at 30 and 120 min after steroid infusion were inhibited by Antide. Facilitation of proceptive behavior by the ring A-reduced progestins at 120 min was inhibited by Antide. + p < 0.05; * p < 0.01; ** p < 0.001 vs. corresponding group receiving Antide plus progestin.

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suppressed the intense proceptive behavior induced by all three progestins (data not shown; p < 0.05 for 5␣-DHP; p < 0.01 for 5␣,3␣-Pgl and 5␤,3␤-Pgl). 4. Discussion In agreement with previous studies [45,81], our results show that Antide blocks lordosis behavior induced by GnRH-I in ovx, E2 B-primed rats, indicating that the peptide acts through the GnRH 1 receptor. Antide also prevented the facilitation of estrous behavior produced by VCS, suggesting the participation of GnRH release induced by VCS in this response. The idea that GnRH mediates the facilitation of lordosis behavior induced in ovx rats by VCS was proposed by Crowley et al. [23], who found that VCS was more effective in eliciting lordosis in hypophysectomized than in ovx rats. They speculated that the absence of both short (pituitary-hypothalamus) and long (ovary-hypothalamus) feedback loops in the hypophysectomized rats disinhibited GnRH secretion. Moreover, they found that administration of dihydrotestosterone, which inhibits GnRH release, markedly attenuated the effect of VCS on lordosis behavior of hypophysectomized rats. Our present result strongly supports the idea that VCS acts through GnRH release. Moreover, this interpretation is consistent with reports showing that VCS releases GnRH [11,16,21,78] and that VCS induces c-Fos mRNA and Fos-like immunoreactivity in GnRH neurons in the septum and anterior preoptic area [64,65]. Because Antide also decreased estrous behavior induced by P and its ring A-reduced metabolites, we can speculate that these behaviors, i.e., lordosis and proceptivity, are also stimulated by GnRH release from the hypothalamus. This explanation is supported by reports that ring A-reduced progestins induce GnRH release [24,30,31,44,57,59,75,82,83]. Moreover, 5␣,3␣Pgl, which may be synthesized by both neurons and glial cells [71], can directly and rapidly stimulate hypothalamic neurons to secrete this peptide, presumably by interacting with GABAA receptors [24]. In contrast to the above mentioned findings, the stimulatory effect of MA on estrous behavior was not altered by Antide. Because MA cannot be reduced to ring A metabolites due to structural impediments, it is likely that -4-3-keto progestins such as MA stimulate estrous behavior by binding with high affinity to a number of PRs sufficient to trigger estrous behavior [12–14,25,40,53]. Therefore, one might question why Antide blocked the behavioral actions of P, which also binds the PR with high affinity [13,15,25,53]. A possible explanation for this paradoxical finding could be that P mainly acts through its metabolism to 5␣-reduced progestins. This interpretation is supported by reports of rapid ring A-reduction of P in the hypothalamus of rats [17,18,44] and that finasteride, an inhibitor of 5␣-reductase, significantly depresses the stimulatory action of P on reproductive behaviors in both hamsters and rats [35,36]. As shown in Fig. 4, our present data suggest that lordosis behavior can be elicited by progestins through two distinct pathways: (a) the classical direct pathway in which progestins with high affinity for the PR, such as MA, freely enter the neurons possessing PRs and bind to these molecules to trigger a sequence of

Fig. 4. Proposed cellular mechanisms involved in the facilitation of estrous behavior by the delta-4,3-keto progestins and MA (pathway A) and by ring A-reduced progestins (pathway B). The following discrete events are indicated in the figure: Pathway A, direct ligand dependent pathway: step (1), passage of MA through the membrane and binding to PR; step (2), the PR complex is translocated to the nucleus; step (3), the PR-hormone-coativator complex interacts with DNA and, step (4), synthesis of proteins and estrous behavior. Pathway B, indirect ligand-independent pathway: step (1), GnRH release by 5␣- and 5␤-progestins from GnRH neurons; step (2), GnRH interaction at membrane with GnRH-1 receptor; step (3), activation through Gs of adenylyl cyclase-cAMP-PKA signaling system; step (4), phosphorylation and activation of the PR-coactivator complex; step (5) interaction of PR-coactivator complex with DNA and step (6) synthesis of proteins leading tofacilitation of estrous behavior. Abbreviations: MA, Megestrol acetate; PR, progestin receptor; GnRH, gonadotropin releasing hormone; PKA, protein kinase A.

events, leading to gene transcription; and (b) an indirect, ligandindependent pathway in which progestins which bind weakly the PR, like ring A-reduced pregnanolones, release GnRH. GnRH acts at a membrane receptor, the GnRH-1 subtype, to trigger a second messenger signaling cascade that will facilitate estrous behavior by activating PRs. Several second messenger-kinase cascades can facilitate lordosis behavior in E2 primed, ovx rats. Thus, the cAMP-kinase A [3,5,6,9,10,39,50,69], cGMPkinase G [4,19,20,29,38,39,41], protein kinase C [39,47,52], and mitogen-activated protein kinase systems [1,26,41] have all been implicated in the expression of estrous behavior in ovx E2 primed rats [10,12,27,50]. In the case of GnRH, pharmacological inhibition of the cAMP-kinase A system prevents the stimulatory effect of the peptide on lordosis behavior in ovx, E2 primed rats. Moreover, the stimulatory effect of GnRH on lordosis behavior is potentiated and prolonged by the concurrent administration of phosphodiesterase inhibitors [5], which prevent cAMP degradation. Furthermore, the GnRH-1 recep-

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tor can be linked with a Gs protein that activates the adenylyl cyclase-cAMP kinase A cascade [2,80]. Because GnRH can also activate other signaling systems, i.e., protein kinase C or mitogen-activated protein kinase [27,56,58,77,84], the participation of other signaling mechanisms in this process cannot be excluded and should be tested. An interesting possibility, indicated in Fig. 4, is that both ligand-dependent (direct) and ligand-independent (indirect) pathways converge on a common “final effector”, most likely the complex constituted by the PR and associated proteins (co-activators). This idea is supported by the finding that the antiprogestin RU486 blocks the stimulatory effect of both progestins and GnRH on estrous behavior in ovx, estrogen-primed rats [8,9,43]. Acknowledgments The authors gratefully acknowledge the excellent technical assistance of Guadalupe Dom´ınguez-L´opez. This work was supported by PROMEP/103.5/04/1409 program and by DHHS grant R37 MH41414. References [1] Acosta-Martinez M, Gonz´alez-Flores O, Etgen AM. The role of progestin receptors and the mitogen-activated protein kinase pathway in delta opioid receptor facilitation of female reproductive behaviors. Horm Behav 2006;49:458–62. [2] Arora KK, Krsmanovic LZ, Mores N, O’Farrell H, Catt KJ. Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropinreleasing hormone receptor. J Biol Chem 1998;273:25581–6. [3] Beyer C, Canchola E, Larsson K. Facilitation of lordosis behavior in the ovariectomized estrogen primed rat by dibutyryl cAMP. Physiol Behav 1981;26:249–51. [4] Beyer C, Fern´andez-Guasti A, Rodr´ıguez-Manzo G. Induction of female sexual behavior by GTP in ovariectomized estrogen primed rats. Physiol Behav 1982;28:1073–6. [5] Beyer C, G´omora P, Canchola E, Sandoval Y. Pharmacological evidence that LH-RH action on lordosis behavior is mediated through a rise in cAMP. Horm Behav 1982;161:107–12. [6] Beyer C, Gonz´alez-Mariscal G. Elevation in hypothalamic cyclic AMP as a common factor in the facilitation of lordosis in rodents: a working hypothesis. Ann NY Acad Sci 1986;474:270–81. [7] Beyer C, Gonz´alez-Mariscal G, Eguibar JR, G´omora P. Lordosis facilitation in estrogen primed rats by intrabrain injection of pregnanes. Pharmacol Biochem Behav 1988;31:919–26. [8] Beyer C, Gonz´alez-Flores O, Gonz´alez-Mariscal G. Ring A reduced progestins potently stimulate estrous behavior in rats: paradoxical effect through the progesterone receptor. Physiol Behav 1995;58:985–93. [9] Beyer C, Gonz´alez-Flores O, Gonz´alez-Mariscal G. Progesterone receptor participates in the stimulatory effect of LHRH, prostaglandin E2 and cyclic AMP on lordosis and proceptive behavior in rats. J Neuroendocrinol 1997;9:609–14. [10] Beyer C, Gonz´alez-Flores O, Garcia-Ju´arez M, Gonz´alez-Mariscal G. Non-ligand activation of estrous behavior in rodents: cross-talk at the progesterone receptor. Scand J Psychol 2003;44:221–9. [11] Bibeau CE, Tobet SA, Anthony ELP, Carroll RS, Baum MJ, King JC. Vaginocervical stimulation of ferrets induces release of luteinizing hormone-releasing hormone. J Neuroendocrinol 1991;3:29–36. [12] Blaustein JD. Progestin receptors: neuronal integrators of hormonal and environmental stimulation. Ann N Y Acad Sci 2003;1007:238–50. [13] Blaustein JD, Feder HH. Nuclear progestin receptors in guinea-pig brain measured by an in vitro exchange assay after hormonal treatment that affects lordosis. Endocrinology 1980;106:1061–9.

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