Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats

Hormones and Behavior 98 (2018) 77–87

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Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, progestin and estrogen receptors in estrogen-primed rats

T

Domínguez-Ordóñez R.a, Garcia-Juárez M.a, Lima-Hernández F.J.a, Gómora-Arrati P.a, ⁎ Domínguez-Salazar E.b, Blaustein J.D.c, Etgen A.M.d, González-Flores O.a,b, a

Centro de Investigación en Reproducción Animal, Universidad Autónoma de Tlaxcala-CINVESTAV, México Area de Neurosciencias, Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana, México c Department of Psychological and Brain Sciences, University of Massachusetts, Amherst, MA 01003, USA d Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA b

A R T I C L E I N F O

A B S T R A C T

Keywords: Lordosis Estradiol GnRH G1 G-protein coupled estrogen receptor 1 G15 Antide RU486 Tamoxifen

The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by intracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17β-estradiol (free E2). In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1 induced lordosis behavior at 120 and 240 min. In Experiment 2, icv injection of the GPER-1 antagonist G15 significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv injection of Antide blocked the stimulatory effect of E2 on lordosis behavior. In Experiment 3, systemic injection of either tamoxifen or RU486 significantly reduced lordosis behavior induced by icv administration of G1 in OVX, EB-primed rats. The results suggest that GnRH release activates both estrogen and progestin receptors and that this activation is important in the chain of events leading to the display of lordosis behavior in response to activation of GPER-1 in estrogen-primed rats.

1. Introduction Although lordosis in ovariectomized (OVX) rats typically requires sequential treatment with 17β-estradiol (E2) and progesterone (P), it can also be induced by high or repeated doses of E2 alone (Beyer et al., 1971; Boling and Blandau, 1939; Davidson et al., 1968; Zemlan and Adler, 1977). We and others recently also showed that unesterified 17βestradiol (free E2) administered subcutaneously (sc; DomínguezOrdóñez et al., 2015) or intracerebroventricularly (icv; DomínguezOrdóñez et al., 2015; Long et al., 2014) facilitates lordosis behavior in OVX-E2 benzoate (EB)-primed rats in the absence of P. The cellular mechanism by which E2 enhances this behavior is unclear. However, lordosis behavior induced 39.5 h after EB priming by acute sc and intracerebral E2 administration is reduced by tamoxifen (TMX), a selective estrogen receptor modulator that has both antagonist and agonist actions on estrogen receptor α and β (ERα/β) and agonist effects on G-protein coupled estrogen receptor 1 (GPER-1; Filardo et al., 2000; Filardo et al., 2002; MacGregor and Jordan, 1998; Revankar

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et al., 2005; Vivacqua et al., 2006) and by 5 mg of RU486, a classical progestin receptor (PR) antagonist, when administered sc 1 h before E2 (Domínguez-Ordóñez et al., 2015). This implicates the participation of both steroid receptors in the short latency behavioral response to E2. It is important to note that TMX has brain region-specific effects on lordosis (Howard et al., 1984). For example, TMX reduced lordosis behavior induced by P when injected into the ventromedial hypothalamus, but not when administered into the preoptic area or interpeduncular region (Howard et al., 1984). Moreover, Long et al. (2017) showed that TMX and ICI 182,780 (ICI) infused into the arcuate nucleus facilitate lordosis within 30 min in EB-primed animals. These effects are blocked by pretreatment with the GPER-1 antagonist G15, indicating that they are mediated by GPER-1. Furthermore, RU486 administered sc at 48 h after EB-priming facilitated female sexual behavior in Long-Evans rats (Pleim et al., 1990). However, when administered 1 h before P, the PR antagonist reduced P facilitation of lordosis, suggesting that RU486 has a dual effect; it may act as an antagonist in the presence of P and as an agonist in its absence.

Corresponding author at: Centro de Investigación en Reproducción Animal, Universidad Autónoma de Tlaxcala-CINVESTAV, México. E-mail address: oglezfl[email protected] (O. González-Flores).

https://doi.org/10.1016/j.yhbeh.2017.12.005 Received 28 June 2017; Received in revised form 8 November 2017; Accepted 15 December 2017 0018-506X/ © 2018 Elsevier Inc. All rights reserved.

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in our colony at Centro de Investigación en Reproducción AnimalPanotla. Animals were housed in a reversed light–dark cycle (14 h light, 10 h dark, lights on at 2300 h) and a controlled temperature (23 ± 2 °C) environment. They were fed Purina Rodent Laboratory Chow 5001 and water ad libitum. Several males were used during sexual behavior testing (see below).

Most of the biological effects of estrogens are mediated by the classical ERs, ERα and ERβ, which up- or down- regulate the expression of their target genes by binding to site-specific DNA sequences (estrogen response elements) and/or specific co-regulatory proteins, including co-activators and co-repressors. The role of each ER subtype in the expression of lordosis behavior has been explored using ER knockout female mice, antisense oligonucleotides, and ER subtypespecific agonists and antagonists (Dewing et al., 2007; Kudwa and Rissman, 2003; Mazzucco et al., 2008; Ogawa et al., 1999; Ogawa et al., 1998; Rissman et al., 1999). We recently reported that ERα and ERβ agonists each induce lordosis behavior in estrogen-primed rats, and both ER subtype-specific antagonists reduce lordosis behavior induced by free E2 (Domínguez-Ordóñez et al., 2016). More recently, E2 has been found to act on non-classical receptors localized within cell membranes or the endoplasmic reticulum (Domínguez and Micevych, 2010; Filardo and Thomas, 2012; Gaudet et al., 2015; Revankar et al., 2005). One example of this is GPER-1, also known as GPR30, which plays an important role in mediating the rapid effects of E2 (Filardo et al., 2000, 2002; Filardo and Thomas, 2012; Gaudet et al., 2015). For example, this receptor regulates the release of gonadotropin releasing hormone (GnRH) from hypothalamic neurons (Noel et al., 2009; Qiu et al., 2008; Rudolf and Kadokawa, 2013). Stimulation of GnRH neurons with G1, a selective agonist (Bologa et al., 2006), stimulates GnRH release, and treatment with G15 (GPER-1 antagonist), blocks E2-induced GnRH release (Kenealy and Terasawa, 2012). Furthermore, icv administration of either E2 or G1 significantly increases lordosis 30 min after administration to EB-primed rats, while pretreatment with G15 blocks E2 and G1 facilitation of sexual receptivity (Long et al., 2014). Thus, GPER-1 participates in the acute E2 facilitation of lordosis behavior estrogen-primed rats. GPER-1 is highly expressed in areas involved in the expression of lordosis, such as the arcuate nucleus, medial preoptic nucleus and ventromedial hypothalamus (Brailoiu et al., 2007; Hazell et al., 2009; Long et al., 2014; Qiu et al., 2008; Rudolf and Kadokawa, 2013). GnRH also facilitates lordosis behavior in E2-primed rodents that have been either OVX or OVX and adrenalectomized (Moss and McCann, 1973; Pfaff, 1973). This effect of GnRH on female sexual behavior is mediated by the GnRH-1 receptor, because Antide, a GnRH-1 receptor antagonist, blocks lordosis behavior in OVX, E2-primed rats induced by several agents including GnRH, ring A-reduced progestins, and vaginocervical stimulation (Gómora-Arrati et al., 2008). PRs also participate in lordosis behavior induced by GnRH, because RU486 inhibits GnRH-induced lordosis and proceptive behaviors (Beyer et al., 1997). The present study assessed the participation of GPER-1 and GnRH-1 receptors in the display of lordosis induced by icv administration of G1, a GPER-1 agonist, and by free E2 in OVX, estrogen-primed rats. Experiment 1 was designed to confirm prior reports that GPER-1 facilitates lordosis in OVX, estrogen-primed rats through the administration of G1 and by the icv infusion of the antagonist, G15. Because the cellular mechanism by which GPER-1 induces lordosis behavior has not been well clarified, and because GPER-1 promotes GnRH release (Terasawa and Kenealy, 2012), Experiment 2 tested the ability of the GnRH antagonist, Antide, to interfere with G1 and E2 facilitation of lordosis. Because G1 may induce lordosis behavior through GnRH release and subsequent activation of PRs and perhaps ERs, in Experiment 3, we tested the ability of the selective ER modulator, TMX, and the PR antagonist, RU486, to block lordosis facilitated by G1 in OVX, estrogenprimed rats.

2.2. Surgical procedures Female rats were bilaterally OVX under anesthesia with xylazine (4 mg/kg) and ketamine (80 mg/kg) and group housed (4/cage). One week later, they were anesthetized with xylazine (4 mg/kg) and ketamine (80 mg/kg) and placed in a Kopf stereotaxic instrument (Tujunga, CA) for implantation of a stainless steel cannula (22 gauge, 17-mm length) into the right lateral ventricle following the Paxinos and Watson (2006) atlas coordinates: anteroposterior + 0.80 mm, mediolateral − 1.5 mm, dorsoventral −3.5 mm with respect to bregma. A stainless steel screw was fixed to the skull, and both the cannula and screw were attached to the bone with dental cement. A dummy cannula (30 gauge) provided with a cap was introduced into the guide cannula to prevent clogging and contamination. Immediately after each surgical procedure, rats were injected with penicillin (165,000 IU/kg of procaine benzyl penicillin and 55,000 IU/kg of crystalline benzyl penicillin), and this continued for 3 days after surgery. After surgery, rats were housed individually in plastic cages with food and water available ad libitum for recovery until the test day. During this time, animals did not show any apparent discomfort due to isolation. All of the experiments were performed under the guidelines of the Mexican Law of Animal Protection (NOM-062-ZOO-1999) under the approval and supervision of the Institutional Committee for the Use and Care of Laboratory Animals of Centro de Investigación y de Estudios Avanzados. 2.3. Behavioral testing Tests for sexual behavior were conducted by placing females in a circular Plexiglas arena (53 cm diameter) with a male after drug administration as discussed below. The lordosis quotient [LQ = (number of lordosis / 10 mounts) × 100] and lordosis score (LS) were used to assess receptive behavior in response to the first 10 mounts. LS refers to the intensity of lordosis, which is quantified according to Hardy and DeBold (1971). This scale ranges from 0 to 3 for each individual response and consequently, from 0 to 30 for each female that received 10 mounts. In all experiments, the rats were tested at 30, 120, and 240 min after E2 or G1 infusion by an experimenter blind to treatment groups. 2.4. Chemicals E2 benzoate (EB), unesterified 17β-E2 (E2) and the GnRH-1 antagonist, Antide (acetyl-D-Ala(2-naphthyl)-D-Phe(4-Cl)-D-Ala(3-pyridyl)Ser-Lys(Nε-nicotinoyl)-D-Lys(Nε-nicotinoyl)-Leu-Lys(Nε-isopr-opyl)Pro-D-Ala-NH2) were purchased from Sigma Chemicals (St. Louis, MO). The GPER-1 agonist G1 ( ± )-1-[(3aR*,4S*,9bS*)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]ethanone and the GPER-1 antagonist G15 (3aS*,4R*,9bR*)-4-(6-Bromo1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline were purchased from Tocris Cookson (St. Louis, MO). PR antagonist, RU486, and the ER antagonist, TMX, were also purchased from Sigma Chemicals (St. Louis, MO). EB, E2 and TMX were dissolved in sesame oil vehicle. G1, G15 and Antide were dissolved in 10% DMSO, and RU486 was dissolved in sesame oil:benzyl benzoate:benzylic alcohol (80:15:5). All drugs and vehicles infused icv were administered in a volume of 1 μl. To avoid unnecessary duplication and thus minimize the numbers of animals killed, important animal welfare concerns, the same groups of vehicle (DMSO) control animals were used throughout Experiments 1 and 2.

2. Methods 2.1. Animals A total of 126 female rats were used in this study. Animals were sexually inexperienced, female Sprague Dawley rats (240–280 g) bred 78

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halothane, and 1% methylene blue was administered through the cannula. The brain was removed and sectioned in the transverse plane to confirm the cannula position in the right lateral ventricle. Animals whose cannula was not in the ventricle (n = 7) were discarded from the data analysis.

2.5. Experiment 1 2.5.1. Involvement of GPER-1 in lordosis behavior in OVX, EB-primed rats The objective of this experiment was to characterize the effect of the GPER-1 agonist, G1, on lordosis behavior and to establish a dose response curve of that compound in OVX, estrogen-primed rats. One week after cannula implantation, rats were primed with a sc injection of 5 μg (13.28 nmol) of EB in 0.1 ml of oil followed 40 h later by icv infusion of G1 or 10% DMSO vehicle. G1 was infused at the following doses in independent groups: DMSO (control, n = 11); G1, 3.5 (8.5 nmol, n = 8), 7.5 (18.2 nmol, n = 8), 15 (36.4 nmol, n = 9) or 30 μg (72.8 nmol, n = 9). Those doses were chosen based on the dose of 30 μg used by Lebesgue et al. (2009), where it was effective in inducing prolactin secretion.

2.9. Statistical analysis To assess the effects of icv administration of G1 or E2, we first used the Kruskall–Wallis test (significance level p < 0.05) for each of the three times tested. Because LQ and LS values in some groups were not normally distributed, the results of two independent groups at 30, 120, and 240 min were compared using the Wilcoxon Mann–Whitney U test. This is an acceptable alternative to the t-test with a power efficiency of 95.5% of the parametric test (Siegel and Castellan, 1995). The effects of G15 and Antide on the behavioral action of G1 and E2 and the effects of TMX and RU486 on the behavioral action of G1 were assessed by comparing the LQ obtained with G1 or E2 alone with those obtained when different antagonists were added. The Wilcoxon Mann–Whitney U test was used in this case as well. We also calculated effect sizes (Berben et al., 2012) for differences in mean LQ scores between females treated with G1 or E2 and those who received those agonists plus different inhibitors. We report Cohen's d for interpreting effect sizes for the standardized mean differences.

2.6. Experiment 2 Effect of icv administration of GPER-1 and GnRH-1 receptor antagonists on G1 induced lordosis behavior in OVX, EB-primed rats. To test the hypothesis that lordosis behavior induced by G1 involves GPER-1 or GnRH-1 receptors, one week after stereotaxic surgery, the animals were injected sc with 5 μg of EB and 39.5 h later received an icv injection of either G15 or Antide followed by G1 at 40 h as follows: 25 μg (67.5 nmol) of G15 plus 15 μg of G1; 1 μg (2.4 nmol) of Antide plus 15 μg of G1 (n = 12). G15 and Antide were administered 30 min before G1. The dose of G1 was selected based on the results of Experiment 1. The dose of G15 was selected from Long et al. (2014) while the dose of Antide was selected based on previous work from our laboratory (Gomora-Arrati, et al. 2008).

3. Results 3.1. Experiment 1 3.1.1. Icv injections of G1 facilitate lordosis behavior in OVX, estrogenprimed rats Table 1 indicates probability and Mann Whitney U values and effect sizes of different doses of G1. The control animals infused with DMSO expressed low levels of LQ at all times tested. Low LQs were observed with most doses of G1 at 30 min post-infusion (Fig. 1; see Table 1 for statistical values); only animals injected with 3.5 μg were significantly different from the control group receiving DMSO (p < 0.05). Compared to DMSO controls, G1 at 15 (p < 0.01) and 30 (p < 0.01) μg induced statistically significant increases in lordosis at 120 min whereas 3.5 (p = 0.08) and 7.5 μg (p = 0.1) were without effect. The highest level of receptivity (LQ and LS) was observed at 240 min with all four doses (Fig. 1, Table 1). Similar results were obtained for LS (Fig. 1); the doses of 3.5 and 15 μg (both p < 0.05 vs DMSO) induced increases at 30 min, but 7.5 and 30 μg had no effect at this time. At 120 min the

2.6.1. Effect of icv administration of GPER-1 and GnRH-1 receptor antagonists on E2 induced lordosis behavior in OVX, EB-primed rats One week after stereotaxic surgery, the animals were injected sc with 5 μg of EB and 40 h later received an icv injection of 2 ng of E2 (n = 11). To test the hypothesis that lordosis behavior induced by E2 involves GPER-1 or GnRH-1 receptors, we used either G15 or Antide combined with the E2 as follows: 25 μg of G15 plus E2 (n = 9); 1 μg of Antide plus E2 (n = 10). G15 and Antide were administered 30 min before E2. The dose of E2 was selected based on previous work from our laboratory (Domínguez-Ordóñez et al., 2015). 2.7. Experiment 3 2.7.1. Participation of ER and PR in lordosis behavior induced by G1 in OVX, EB-primed rats This experiment was designed to determine whether lordosis behavior induced by G1 is mediated through PRs and/or ERs. A total of 30 estrogen-primed (5 μg of EB) rats were used. At 39 h after EB injection, 9 females were injected sc with 5 mg of the progestin antagonist, RU486, and at 39.5 h, 10 females received a sc injection of 5 mg of TMX. Forty hours after EB, all rats received an icv injection of 15 μg of G1. At 39 h after EB injection, 6 control females received a sc injection of RU486 vehicle and at 39.5 h, 5 additional control animals received TMX vehicle, followed by an icv injection of 15 μg of G1. The doses of RU486 and TMX were based on previous studies in our laboratory (Beyer et al., 1997; Domínguez-Ordóñez et al., 2015). Because we have shown in multiple experiments that oil and oil:benzyl benzoate: benzylic alcohol, vehicles of TMX and RU486, respectively, do not affect the expression of lordosis behavior in estrogen-primed rats (Beyer et al., 1995, 1997; González-Mariscal et al., 1989), these groups were not included.

Table 1 Statistical values associated with lordosis quotients in rats given icv infusion of G1 when compared to control DMSO-treated rats (Experiment 1). Treatment

DMSO G1 3.5 μg (n = 8) G1 7.5 μg (n = 8) G1 15 μg (n = 9) G1 30 μg (n = 9)

2.8. Histological examination of cannula placement

Statistical value at each test time

d p< U d p< U d p< U d p< U

30 min

120 min

240 min

– 1.6 0.05 16 0.005 NS 43.5 0.2 NS 27 0.4 NS 48

– 1.6 NS 23 1.4 NS 26.5 2.7 0.01 9 1.8 0.01 15

– 1.6 0.05 14.5 1 0.05 20 1 0.05 20.5 1.6 0.01 13

Time (min) is relative to infusion of G1 or vehicle. DMSO, vehicle; G1, GPER-1 agonist; Cohen's d, effect size value; p < , probability; U, Mann Whitney U test value; NS, not significant.

One day after completion of the experiments, rats implanted with icv cannula were euthanized with an overdose of the anesthetic 79

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Fig. 1. Intracerebral infusion of 3.5 (n = 8), 7.5 (n = 8), 15 (n = 9) and 30 μg (n = 9) of G1 to OVX, EB-primed rats on LQ (A) and LS (B). Only the 3.5 μg dose of G1 induced significant increases in lordosis behavior at 30 min post injection. At 120 and 240 min after injection of G1 all doses used significantly elevated LQ and LS compared with animal infused only with vehicle (DMSO, n = 11; black bar). *p < 0.01, + p < 0.05 vs DMSO.

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Fig. 2. The facilitation of LQ (A) and LS (B) with intracerebral infusion of 15 μg of G1 (n = 9) in OVX, EB-primed rats was significantly antagonized by icv infusion of 25 μg of the GPER-1, antagonist G15 (67.5 mM; n = 9) or by icv infusion of 1 μg (628 μM) of Antide, a GnRH-1 antagonist (n = 12) at 30 and 120 min after G1 injection. LQ and LS induced with G1 at 240 min was significantly reduced by Antide and not by G15. + p < 0.05; *p < 0.01; vs G1. Data for 15 μg of G1 alone are from Fig. 1.

doses of 15 and 30 μg (both p < 0.01) increased LS, but no significant effect was found with 3.5and 7.5 μg. However, at 240 min all doses except 7.5 μg increased LS (p < 0.05 vs DMSO).

reduced at 240 min. Table 2 indicates Mann Whitney values, p values and effect sizes for G15 and Antide combined with G1. Effect sizes (Table 2) confirm that G15 was most effective at inhibiting G1 facilitation of lordosis at 30 (d = 0.6) and 120 (d = 0.8) min compared to 240 min (d = 0.42). Administration of Antide also significantly prevented the stimulatory effect of G1 on LQ (Fig. 2A) in estrogen-primed rats at 30, 120 (both p < 0.01) and 240 min (p < 0.05). Moreover, Antide significantly reduced the LS (Fig. 2B) at 30, 120 (both p < 0.01) and 240 min (p < 0.05). Antide had similar effect sizes as those obtained with G15 at 30, 120 and 240 min (see Table 2 for d values).

3.2. Experiment 2 3.2.1. Effect of G15 and Antide on lordosis behavior induced by G1 Injection of G15 significantly decreased LQ induced by G1 at 30 and 120 min (both p < 0.01; Fig. 2A). G15 also significantly decreased the LS at 30 and 120 min (both p < 0.01) (Fig. 2B). The inhibitory effect of G15 was transitory, because LQ and LS were not significantly 81

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These demonstrate the large magnitude of the inhibitory effect of those agents on lordosis behavior (Lipsey and Wilson, 2000). In Experiment 1, G1 induced lordosis with a relatively long latency. At 2 h after infusion, only the highest doses (15 and 30 μg) induced a clear response, whereas at 4 h post-injection all doses induced intense and similar levels of lordosis. In a previous report, the administration of G1 into the third ventricle induced lordosis with a very short latency of 30 min (Long et al., 2014). That study used OVX Long-Evans rats primed with 2 μg EB sc 4 times at four day intervals. It is possible that the use of different strains of rats (Sprague-Dawley vs Long-Evans) or the use of repeated doses of EB by Long et al. (2014) is responsible for the different latencies of G1 action. It should also be noted that compounds administered by an icv route diffuse into many brain areas. The compounds move from the lateral ventricle, via bulk flow to the third ventricle, then to the fourth ventricle, then over the entire surface of the brain. Thus, after icv administration of small molecules, their concentration decreases logarithmically in the brain with each millimeter of distance (Pardridge, 2012). In order to compensate for this possible dilution limitation, G1 (Experiment 1) was administered at different doses, in all cases obtaining the greatest responses at 120 and 240 min post-injection. The cellular mechanism through which G1 facilitates lordosis behavior is not clear. However, in Experiment 2, G15 reduced the facilitating effects of G1 and E2 in OVX, EB-primed rats, suggesting the participation of GPER-1. This is in agreement with previous results in rats as well as mice (Anchan et al., 2014; Long et al., 2014) because pretreatment with G15 inhibited G1 and E2 facilitation of lordosis. This corroborates the idea that E2 facilitates lordosis in part through the activation of GPER-1. GPER-1 has been found at a variety of intracellular locations in neurons as well as in the plasma membrane in brain and anterior pituitary (Brailoiu et al., 2007; Filardo et al., 2007; Funakoshi et al., 2006). GPER-1 immunoreactivity is detected in the endoplasmic reticulum and plasma membrane of neuronal perikarya and dendritic shafts as well as in spines and in clusters of vesicles in axon terminals of the hippocampus (Waters et al., 2015). GPER-1 immunostaining has also been reported in the Golgi apparatus in magnocellular oxytocin neurons (Sakamoto et al., 2007). Activation of GPER-1can induce non-genomic signaling, such as activation of the α subunit of the G protein, which in turn activates adenylyl cyclase. Because Antide inhibited lordosis induced by G1 and E2, we speculate that lordosis behavior was induced by the release of GnRH and the activation of its GnRH-1 receptor. Previous findings support this interpretation. a) In primates, GPER-1 is located in hypothalamic GnRH neurons where it mediates the rapid actions of E2 on GnRH release (Jacobi et al., 2007; Noel et al., 2009). Both E2 and G1 increase intracellular Ca+ and stimulate GnRH release, and these effects are blocked by G15 (Noel et al., 2009; Kenealy and Terasawa, 2012; Terasawa and Kenealy, 2012). b) Lordosis behavior induced by GnRH, P and some of its ring A-reduced metabolites, leptin or vaginocervical stimulation is reduced by icv administration of Antide (García-Juárez et al., 2011; Gómora-Arrati et al., 2008), supporting the idea that GnRH release is an obligatory step in the facilitation of lordosis by each of these. Interestingly, Experiment 3 demonstrated the involvement of ER and PR in the regulation of lordosis facilitation by GPER-1, because both TMX and RU486, respectively, blocked lordosis behavior induced by G1. In previous studies, TMX injected before estrogen priming in OVX rats, either into the ventromedial hypothalamus or systemically, inhibited estrogen priming of lordosis behavior (Etgen, 1979; Etgen and Shamamian, 1986; Howard et al., 1984). In addition, concurrent icv infusion of TMX with GnRH, leptin, progestins, or PGE2 significantly reduced lordosis induced by these compounds in estrogen-primed rats (Lima-Hernández et al., 2014), suggesting that ERs also participate in activation of lordosis behavior many h after the priming administration of E2. The agonist and antagonist effects of TMX may vary according to

Table 2 Statistical values associated with lordosis quotients in rats given icv infusion of the agonists G1 or E2 when compared to agonist plus the antagonists G15 or Antide (Experiment 2). Treatment

DMSO G15 + G1 (n = 9) Antide + G1 (n = 12) G15 + E2 (n = 9) Antide + E2 (n = 10)

Statistical value at each time

d p< U d p< U d p< U d p< U

30 min

120 min

240 min

– 0.6 0.01 14 0.58 0.01 20 0.02 NS 44 0.4 NS 33

– 0.8 0.01 5.5 0.76 0.01 9 0.76 0.01 9.5 0.77 0.01 6.5

– 0.42 NS 19.5 0.49 0.05 25.5 0.73 0.01 10 0.62 0.05 12

Time (min) is relative to infusion of G1, E2 or vehicle. DMSO, vehicle; G1, GPER-1 agonist; G15, GPER-1 antagonist; Antide, GnRH-1 receptor antagonist; E2 estradiol; Cohen's d, effect size value; p < , probability; U, Mann Whitney U test value; NS, not significant.

3.2.2. Effect of G15 and Antide on lordosis behavior induced by free E2 E2 infused icv induced intense lordosis behavior in EB-primed rats at 120 and 240 min (both p < 0.01). The elevation of LQ and LS by E2 was significantly reduced by G15 at 120 and 240 min (both p < 0.01) (Fig. 3); however, there was no effect on LQ or LS at 30 min. Table 2 indicates Mann Whitney U values, p values and effect sizes for G15 and Antide combined with E2. G15 had a substantial effect size (Table 2) on the inhibition of lordosis (LQ) induced by E2 at 120 (d = 0.76) and 240 (d = 0.73) min, but a small effect size at 30 min (d = 0.02). Antide significantly prevented the stimulatory effect of E2 on LQ in estrogenprimed rats at 120 (p < 0.01) and 240 min (p < 0.05), but not at 30 min. Moreover, Antide significantly reduced the elevation of LS induced by E2 (Fig. 3B) only at 120 min (p < 0.01) post-injection. The greatest effect sizes for Antide inhibition of LS induced by E2 were found at 120 and 240 min (see Table 2 for d values). 3.3. Experiment 3 3.3.1. Effect of TMX and RU486 on lordosis behavior induced by G1 Systemic administration of TMX reduced LQ (Fig. 4A) and LS (Fig. 4B) induced by G1 at 120 (p < 0.001) and 240 min (p < 0.01) but not at 30 min. Table 3 indicates Mann Whitney U values, p values and effect sizes for TMX and RU486 combined with G1. Effect sizes (Table 3) for TMX were large at 120 (d = 0.8) and 240 (d = 0.72) min but small at 30 min (d = 0.21). LQ and LS induced by G1 at 120 and 240 min were almost completely suppressed by the sc injection of RU486 (both p < 0.001 vs G1 alone). However, there was no effect at 30 min for LQ or LS. Effect sizes confirm that RU486 was highly effective at inhibiting G1-induced lordosis at 120 and 240 min (see Table 3 for d values). 4. Discussion The present study tested the hypothesis that lordosis behavior induced by GPER-1 activation in EB-primed rats involves GnRH-1 receptors, PRs and ERs. The results suggest that lordosis behavior induced through the activation of GPER-1 by E2 or G1 may depend on the release of GnRH, because icv administration of Antide blocked the facilitatory actions of both compounds. TMX and RU486 also inhibited the facilitation of lordosis by G1, suggesting the participation of both ER and PR in this process. We believe that the roles of GnRH-1 receptor, ER and PR are biologically meaningful because of the effect size estimates of the impact of G15, TMX and RU486 treatment (Tables 2 and 3). 82

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Fig. 3. The facilitation of LQ (A) and LS (B) with intracerebral infusion of 2 ng of E2 (n = 11) in OVX, EBprimed rats was antagonized significantly both by icv infusion of 25 μg of the GPER-1 antagonist G15 (n = 9) or by icv infusion of 1 μg of Antide (GnRH-1 antagonist, n = 10) at 30, 120 and 240 min after E2 infusion. *p < 0.01; + p < 0.05 vs E2.

E2 in the arcuate nucleus, effects associated with the blockade of the preovulatory-like LH surge and with suppression of the E2-induced prolactin surge (Aquino et al., 2016). On the other hand, nonsteroidal antiestrogens, like TMX, ICI or nafoxidine, mimic the effects of E2 on body weight and food intake (Wade and Blaustein, 1978; Wade and Gray, 1979; Wade and Heller, 1993; Wade et al., 1993). Another clear agonist effect of TMX is to induce GnRH self-priming (defined as an enhanced LH secretory response to the second of two pulses of identical concentration of GnRH), probably through binding ERα (Sánchez-

the behavior measured and the cellular process; for example, TMX blocks E2 induction of PR in the pituitary, but in the absence of E2, it induces PR synthesis (Roy et al., 1979; Sánchez-Criado et al., 2002). Thus, like RU486 and its action on the PR (Pleim et al., 1990), TMX may act as an antagonist in the presence of E2 or other agonists and as an agonist in the absence of E2. TMX appears to have primarily antagonist effects on selected E2-dependent reproductive processes, because TMX prevents the E2-induced increase in cFos expression in the anteroventral periventricular nucleus and also prevents PR induction by 83

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Fig. 4. LQ (A) and LS (B) were induced in OVX, EBprimed rats by icv administration of 15 μg of G1 (n = 11). The lordosis behavior induced at 30 min (LQ and LS) was not significantly reduced by sc administration of 5 mg of TMX (ER antagonist, n = 10) or 5 mg of antiprogestin RU486 (RU; n = 9). Lordosis behavior induced by G1 at 120 and 240 min post-injection was significantly inhibited by sc administration of TMX or RU486. &p < 0.001; *p < 0.01 vs G1.

that study and the present one that could account for these differing results. First, the rat strains used differed between studies (we used Sprague Dawley, whereas the Long study used the Long Evans strain). Secondly, the manner in which lordosis was induced differed importantly between the two studies. In the Long study, lordosis was induced in EB-primed rats by the administration of TMX or ICI alone; in the present study, lordosis was induced in EB-primed rats by E2 or G1, and in this case, TMX inhibited G1-dependent lordosis induction. We did not test for potential agonist effects of TMX alone. Moreover, the strongest, most rapid facilitatory effect of TMX on lordosis behavior was observed when TMX was infused directly into the arcuate nucleus in the Long study. In the present study, TMX was administered icv only, and relatively rapid inhibition of G1-induced lordosis was observed. As

Criado et al., 2002, 2005, 2006). Recent work by Long et al. (2017) explored time-dependent effects of TMX and ICI on lordosis expression in EB-primed rats when the drugs were infused via different routes. They showed that systemic administration of TMX or icv infusion of ICI facilitated lordosis 4 h later in OVX, EB-primed rats. However, when either ER antagonist was directly infused in the arcuate nucleus, they induced more intense lordosis behavior and with a shorter latency (30 min). This effect was mediated through the activation of GPER-1, because G15 reduced the lordosis induced by those compounds. In contrast to the study by Long et al. (2017), we observed that TMX, administered icv, inhibited G1 facilitation of lordosis behavior in EB-primed rats. There are some methodological differences between 84

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Ordóñez et al., 2015; García-Juárez et al., 2011; Lima-Hernández et al., 2014; Mani and Blaustein, 2012; Mani and Portillo, 2010). The fact that RU486 reduced lordosis behavior induced by G1 in the present study suggests that G1 activates PRs, perhaps indirectly via GnRH release. This idea is supported by the ability of RU486 to block GnRH selfpriming, which requires cross-communication between GnRH receptoractivated protein kinase A and the PR (Turgeon and Waring, 1994; Waring and Turgeon, 1992). Overall, the results suggest that GPER-1 is involved in the stimulation of lordosis behavior in estrogen-primed female rats. Thus, we propose that E2 and G1 bind directly to GPER-1 (upper panel, Fig. 5), which is present in GnRH neurons in the preoptic area and which stimulates an increase in intracellular calcium, inducing the release of GnRH (Naor et al., 2000; Weiner and Charles, 2001: Kraus et al., 2003: Navratil et al., 2003; Higa-Nakamine et al., 2015). GnRH neurons project to other regions of the hypothalamus (e.g., paraventricular nucleus) containing cells that express the GnRH-1 receptor (Silverman et al., 1987; Wierman et al., 2011; Schauer et al., 2015). The released GnRH may then activate its membrane receptor (GnRH-1) localized in postsynaptic neurons, which contain PRs and ERs. The GnRH1 receptor-dependent increase in second messenger production and kinase activation may then modulate lordosis behavior by activating both ERs and PRs in a ligand-independent manner (Beyer et al., 2003; Demay et al., 2001; Mani and Blaustein, 2012; Mani and Portillo, 2010). An alternative intracellular mechanism by which GnRH-1 receptors could induce lordosis behavior is by activating the mitogen-activated protein kinase (MAPK) pathway through the Src/ER/PR/MAPK complex in hypothalamic neurons (lower panel, Fig. 5; Higa-Nakamine et al., 2015; Kraus et al., 2003; Navratil et al., 2003; Naor et al., 2000). PRs bind the SH3 domain of Src through its proline-rich domain (residues 421 and 428 located in the N-terminus), while ERs interact directly with the SH2 domains through phosphotyrosine 537 located also at the N-terminus (Ballaré et al., 2003; Boonyaratanakornkit et al., 2001; Faivre and Lange, 2007; Migliaccio et al., 1998). Therefore, PR and ER could heterodimerize through the ERIDI and II (ER-interaction domain I and II) in the N-terminal domain of the PR and the ligandbinding domain of ER. Indeed others (Migliaccio et al., 1998; Boonyaratanakornkit et al., 2001) showed that activation of Src/MAPK through the progestin agonist, R5020, was blocked by administration of

Table 3 Statistical values associated with lordosis quotients in rats given icv infusion of G1 compared to the antagonists TMX and RU486 (Experiment 3). Treatment

DMSO TMX + G1 (n = 10) RU486 + G1 (n = 9)

Statistical value at each time

d p< U d p< U

30 min

120 min

240 min

– 0.21 NS 24 0.15 NS 28

– 0.8 0.001 2 0.84 0.001 1.5

– 0.72 0.01 7 0.88 0.001 0.5

Time (min) is relative to infusion of G1 or vehicle. G1, GPER-1 agonist; TMX, tamoxifen, selective ER modulator; RU486, PR antagonist; Cohen's d, effect size value; p < , probability; U, Mann Whitney U test value; NS, not significant.

noted previously, this suggests that TMX may act as an antagonist in the presence of E2 or other agonists and as an agonist in the absence of E2. At the level of neural mechanisms, the different effects of TMX on lordosis behavior between these two studies could also be due to the fact that TMX, in some experimental conditions, inhibits GnRH release. For example, TMX effectively prevented the positive, but not negative, feedback effects of E2 (Donath and Nishino, 1998; Gao et al., 2002; Aquino et al., 2016). The inhibitory effect of RU486 on the actions of G1 also agrees with previous reports showing that this PR antagonist reduces lordosis behavior induced by a variety of compounds (Beyer et al., 1995; Beyer et al., 1997; Domínguez-Ordóñez et al., 2015; Etgen and Shamamian, 1986; García-Juárez et al., 2011; González-Mariscal et al., 1989; Mani and Portillo, 2010; Mani and Blaustein, 2012; Vathy et al., 1987). On the other hand, in certain experimental conditions, RU486 exerts agonist effects on lordosis behavior (Pleim et al., 1990). However, an agonist effect of the antiprogestin was only observed in the absence of P. Several hormone antagonists, like RU486 and TMX, induce partial agonist effects in the absence of natural hormone by a mechanism that is not well understood. Some studies suggest that PRs are common molecular effectors for compounds with diverse chemical structures that induce lordosis behavior in OVX, estrogen-primed rats (Beyer et al., 2003; Domínguez-

Fig. 5. Proposed cellular mechanisms involved in the facilitation of lordosis behavior by GPER-1 activation. Upper panel: E2 and G1 bind to GPER-1 receptor located in the membrane of GnRH neurons in the preoptic area, activating a G protein (GP) to stimulate the adenylyl cyclase-cAMP-protein kinase A (AC-cAMPPKA) system. This increases intracellular calcium (Ca++) concentration, which induces GnRH release. Lower panel: GnRH interaction with the membrane GnRH-1 receptor, located in postsynaptic hypothalamic neurons. GnRH-1 receptors interact with a GP to activate the Src-ER-PR-MAPK-complex. PR binds to the SH3 domain, whereas the ER binds to the Sh2 domain of Src (see Discussion for references). These interactions activate MAPK. In turn, MAPK phosphorylates the PR, leading to the facilitation of lordosis behavior. TMX and RU486 bind to ER and PR, respectively, where they destabilize the Src complex, preventing the activation of MAPK and the expression of lordosis. Abbreviations: GPER-1, G-protein coupled estrogen receptor 1; G1, GPER-1 agonist; E2 17β-estradiol; PR, progestin receptor; ER, estrogen receptor; GnRH, gonadotropin releasing hormone; MAPK, mitogen-activated protein kinase.

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TMX, suggesting the participation of both the ER and PR. We propose that the ER and PR may be participating as anchor proteins forming the Src/ER/PR complex and subsequently activating MAPK. In turn, MAPK would be the component that activates the ER (Jiang et al., 2007; Kato et al., 1995; Law et al., 2009) and PR (Hagan et al., 2012; Lange, 2004; Shen et al., 2001). This cellular mechanism could participate in the regulation of lordosis behavior induced by several compounds in OVX, estrogen-primed rats (González-Flores et al., 2010). In support of this proposal, we showed that icv administration of both PD98059 (inhibitor of MAPK) or PP2 (inhibitor of Src) reduced lordosis behavior induced by icv administration of GnRH (González-Flores et al., 2009; Lima-Hernández et al., 2012). Moreover, in vitro studies showed that GnRH activation of its GnRH-1 receptor activates the Src–MAPK pathway (Higa-Nakamine et al., 2015; Kraus et al., 2003). Based on these data, the fact that lordosis behavior induced by E2 or G1 was blocked with either RU486 or TMX supports the participation of Src/PR/ER/MAPK complex in the regulation of lordosis. In T47D cells in vitro, the progestin R5020 activates this pathway. Moreover, both RU486 and TMX inhibit progestin stimulation of c-Src and the MAPK, Erk-2 (Migliaccio et al., 1998). In addition, because Antide also reduced lordosis behavior induced by E2 and G1, we suggest that these compounds may facilitate lordosis by promoting GnRH release from the hypothalamus, which acts on the GnRH-1 receptor to activate the Src-MAPK pathway. Nonetheless, this is only a hypothetical model meant to stimulate further experimentation.

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