Hormones and Behavior 98 (2018) 77–87

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Hormones and Behavior

journal homepage: www.elsevier.com/locate/yhbeh

Lordosis facilitated by GPER-1 receptor activation involves GnRH-1, T

progestin and estrogen receptors in estrogen-primed rats
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
b
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

A R T I C L E I N F O A B S T R A C T

Keywords: The present study assessed the participation of membrane G-protein coupled estrogen receptor 1 (GPER-1) and
Lordosis gonadotropin releasing hormone 1 (GnRH-1) receptor in the display of lordosis induced by in-
Estradiol tracerebroventricular (icv) administration of G1, a GPER-1 agonist, and by unesterified 17β-estradiol (free E2).
GnRH In addition, we assessed the participation of both estrogen and progestin receptors in the lordosis behavior
G1
induced by G1 in ovariectomized (OVX), E2-benzoate (EB)-primed rats. In Experiment 1, icv injection of G1
G-protein coupled estrogen receptor 1
G15
induced lordosis behavior at 120 and 240 min. In Experiment 2, icv injection of the GPER-1 antagonist G15
Antide significantly reduced lordosis behavior induced by either G1 or free E2. In addition, Antide, a GnRH-1 receptor
RU486 antagonist, significantly depressed G1 facilitation of lordosis behavior in OVX, EB-primed rats. Similarly, icv
Tamoxifen 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 et al., 2005; Vivacqua et al., 2006) and by 5 mg of RU486, a classical

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 17β-
estradiol (free E2) administered subcutaneously (sc; Domínguez-
Ordóñez et al., 2015) or intracerebroventricularly (icv; Domínguez-
Ordóñez 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 un-
clear. 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
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 lor-
dosis (Howard et al., 1984). For example, TMX reduced lordosis be-
havior induced by P when injected into the ventromedial hypotha-
lamus, 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 be-
havior in Long-Evans rats (Pleim et al., 1990). However, when ad-
ministered 1 h before P, the PR antagonist reduced P facilitation of
lordosis, suggesting that RU486 has a dual effect; it may act as an an-
tagonist 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: oglezflo@gmail.com (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.
R. Domínguez-Ordóñez et al.
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 (es-
trogen response elements) and/or specific co-regulatory proteins, in-
cluding 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 subtype-
specific 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). Sti-
mulation of GnRH neurons with G1, a selective agonist (Bologa et al.,
2006), stimulates GnRH release, and treatment with G15 (GPER-1 an-
tagonist), 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 re-
ceptivity (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 hypotha-
lamus (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 be-
havior 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 in-
hibits 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 fa-
cilitates lordosis in OVX, estrogen-primed rats through the adminis-
tration 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 re-
lease 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, estrogen-
primed rats.
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
Hormones and Behavior 98 (2018) 77–87
in our colony at Centro de Investigación en Reproducción Animal-
Panotla. 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).
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 keta-
mine (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 proce-
dure, 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 ad-
ministration 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 re-
sponse 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 an-
tagonist, 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-ben-
zodioxol-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-Bromo-
1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline were pur-
chased 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 se-
same oil:benzyl benzoate:benzylic alcohol (80:15:5). All drugs and ve-
hicles infused icv were administered in a volume of 1 μl. To avoid un-
necessary 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.
R. Domínguez-Ordóñez et al. Hormones and Behavior 98 (2018) 77–87

2.5. Experiment 1 halothane, and 1% methylene blue was administered through the
cannula. The brain was removed and sectioned in the transverse plane
2.5.1. Involvement of GPER-1 in lordosis behavior in OVX, EB-primed rats to confirm the cannula position in the right lateral ventricle. Animals
The objective of this experiment was to characterize the effect of the whose cannula was not in the ventricle (n = 7) were discarded from the
GPER-1 agonist, G1, on lordosis behavior and to establish a dose re- data analysis.
sponse curve of that compound in OVX, estrogen-primed rats. One week
after cannula implantation, rats were primed with a sc injection of 5 μg 2.9. Statistical analysis
(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 To assess the effects of icv administration of G1 or E2, we first used
independent groups: DMSO (control, n = 11); G1, 3.5 (8.5 nmol, the Kruskall–Wallis test (significance level p < 0.05) for each of the
n = 8), 7.5 (18.2 nmol, n = 8), 15 (36.4 nmol, n = 9) or 30 μg three times tested. Because LQ and LS values in some groups were not
(72.8 nmol, n = 9). Those doses were chosen based on the dose of normally distributed, the results of two independent groups at 30, 120,
30 μg used by Lebesgue et al. (2009), where it was effective in inducing and 240 min were compared using the Wilcoxon Mann–Whitney U test.
prolactin secretion. This is an acceptable alternative to the t-test with a power efficiency of
95.5% of the parametric test (Siegel and Castellan, 1995).
2.6. Experiment 2 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
Effect of icv administration of GPER-1 and GnRH-1 receptor an- assessed by comparing the LQ obtained with G1 or E2 alone with those
tagonists on G1 induced lordosis behavior in OVX, EB-primed rats. obtained when different antagonists were added. The Wilcoxon
To test the hypothesis that lordosis behavior induced by G1 involves Mann–Whitney U test was used in this case as well. We also calculated
GPER-1 or GnRH-1 receptors, one week after stereotaxic surgery, the effect sizes (Berben et al., 2012) for differences in mean LQ scores be-
animals were injected sc with 5 μg of EB and 39.5 h later received an icv tween females treated with G1 or E2 and those who received those
injection of either G15 or Antide followed by G1 at 40 h as follows: agonists plus different inhibitors. We report Cohen's d for interpreting
25 μg (67.5 nmol) of G15 plus 15 μg of G1; 1 μg (2.4 nmol) of Antide effect sizes for the standardized mean differences.
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 3. Results
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 3.1. Experiment 1
laboratory (Gomora-Arrati, et al. 2008).
3.1.1. Icv injections of G1 facilitate lordosis behavior in OVX, estrogen-
2.6.1. Effect of icv administration of GPER-1 and GnRH-1 receptor primed rats
antagonists on E2 induced lordosis behavior in OVX, EB-primed rats Table 1 indicates probability and Mann Whitney U values and effect
One week after stereotaxic surgery, the animals were injected sc sizes of different doses of G1. The control animals infused with DMSO
with 5 μg of EB and 40 h later received an icv injection of 2 ng of E2 expressed low levels of LQ at all times tested. Low LQs were observed
(n = 11). To test the hypothesis that lordosis behavior induced by E2 with most doses of G1 at 30 min post-infusion (Fig. 1; see Table 1 for
involves GPER-1 or GnRH-1 receptors, we used either G15 or Antide statistical values); only animals injected with 3.5 μg were significantly
combined with the E2 as follows: 25 μg of G15 plus E2 (n = 9); 1 μg of different from the control group receiving DMSO (p < 0.05). Com-
Antide plus E2 (n = 10). G15 and Antide were administered 30 min pared to DMSO controls, G1 at 15 (p < 0.01) and 30 (p < 0.01) μg
before E2. The dose of E2 was selected based on previous work from our induced statistically significant increases in lordosis at 120 min whereas
laboratory (Domínguez-Ordóñez et al., 2015). 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
2.7. Experiment 3 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
2.7.1. Participation of ER and PR in lordosis behavior induced by G1 in 30 min, but 7.5 and 30 μg had no effect at this time. At 120 min the
OVX, EB-primed rats
This experiment was designed to determine whether lordosis be- Table 1
havior induced by G1 is mediated through PRs and/or ERs. A total of 30 Statistical values associated with lordosis quotients in rats given icv infusion of G1 when
estrogen-primed (5 μg of EB) rats were used. At 39 h after EB injection, compared to control DMSO-treated rats (Experiment 1).
9 females were injected sc with 5 mg of the progestin antagonist,
Treatment Statistical value at each test time
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 30 min 120 min 240 min
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 DMSO – – –
G1 3.5 μg d 1.6 1.6 1.6
TMX vehicle, followed by an icv injection of 15 μg of G1. The doses of (n = 8) p< 0.05 NS 0.05
RU486 and TMX were based on previous studies in our laboratory U 16 23 14.5
(Beyer et al., 1997; Domínguez-Ordóñez et al., 2015). Because we have G1 7.5 μg d 0.005 1.4 1
shown in multiple experiments that oil and oil:benzyl benzoate: (n = 8) p< NS NS 0.05
U 43.5 26.5 20
benzylic alcohol, vehicles of TMX and RU486, respectively, do not af-
G1 15 μg d 0.2 2.7 1
fect the expression of lordosis behavior in estrogen-primed rats (Beyer (n = 9) p< NS 0.01 0.05
et al., 1995, 1997; González-Mariscal et al., 1989), these groups were U 27 9 20.5
not included. G1 30 μg d 0.4 1.8 1.6
(n = 9) p< NS 0.01 0.01
U 48 15 13
2.8. Histological examination of cannula placement
Time (min) is relative to infusion of G1 or vehicle. DMSO, vehicle; G1, GPER-1 agonist;
One day after completion of the experiments, rats implanted with Cohen's d, effect size value; p < , probability; U, Mann Whitney U test value; NS, not
icv cannula were euthanized with an overdose of the anesthetic significant.

79
R. Domínguez-Ordóñez et al. Hormones and Behavior 98 (2018) 77–87

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.

80
R. Domínguez-Ordóñez et al.
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).
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
Hormones and Behavior 98 (2018) 77–87
Fig. 2. The facilitation of LQ (A) and LS (B) with in-
tracerebral 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.
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 facil-
itation 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 pre-
vented 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).
R. Domínguez-Ordóñez et al. Hormones and Behavior 98 (2018) 77–87

Table 2 These demonstrate the large magnitude of the inhibitory effect of those
Statistical values associated with lordosis quotients in rats given icv infusion of the agents on lordosis behavior (Lipsey and Wilson, 2000).
agonists G1 or E2 when compared to agonist plus the antagonists G15 or Antide
In Experiment 1, G1 induced lordosis with a relatively long latency.
(Experiment 2).
At 2 h after infusion, only the highest doses (15 and 30 μg) induced a
Treatment Statistical value at each time clear response, whereas at 4 h post-injection all doses induced intense
and similar levels of lordosis. In a previous report, the administration of
30 min 120 min 240 min G1 into the third ventricle induced lordosis with a very short latency of
DMSO – – – 30 min (Long et al., 2014). That study used OVX Long-Evans rats
G15 + G1 d 0.6 0.8 0.42 primed with 2 μg EB sc 4 times at four day intervals. It is possible that
(n = 9) p< 0.01 0.01 NS the use of different strains of rats (Sprague-Dawley vs Long-Evans) or
U 14 5.5 19.5 the use of repeated doses of EB by Long et al. (2014) is responsible for
Antide + G1 d 0.58 0.76 0.49
the different latencies of G1 action. It should also be noted that com-
(n = 12) p< 0.01 0.01 0.05
U 20 9 25.5 pounds administered by an icv route diffuse into many brain areas. The
G15 + E2 d 0.02 0.76 0.73 compounds move from the lateral ventricle, via bulk flow to the third
(n = 9) p< NS 0.01 0.01 ventricle, then to the fourth ventricle, then over the entire surface of the
U 44 9.5 10
brain. Thus, after icv administration of small molecules, their con-
Antide + E2 d 0.4 0.77 0.62
(n = 10) p< NS 0.01 0.05 centration decreases logarithmically in the brain with each millimeter
U 33 6.5 12 of distance (Pardridge, 2012). In order to compensate for this possible
dilution limitation, G1 (Experiment 1) was administered at different
Time (min) is relative to infusion of G1, E2 or vehicle. DMSO, vehicle; G1, GPER-1 ago- doses, in all cases obtaining the greatest responses at 120 and 240 min
nist; G15, GPER-1 antagonist; Antide, GnRH-1 receptor antagonist; E2 estradiol; Cohen's
post-injection.
d, effect size value; p < , probability; U, Mann Whitney U test value; NS, not significant.
The cellular mechanism through which G1 facilitates lordosis be-
havior is not clear. However, in Experiment 2, G15 reduced the facil-
3.2.2. Effect of G15 and Antide on lordosis behavior induced by free E2
itating effects of G1 and E2 in OVX, EB-primed rats, suggesting the
E2 infused icv induced intense lordosis behavior in EB-primed rats at
participation of GPER-1. This is in agreement with previous results in
120 and 240 min (both p < 0.01). The elevation of LQ and LS by E2
rats as well as mice (Anchan et al., 2014; Long et al., 2014) because
was significantly reduced by G15 at 120 and 240 min (both p < 0.01)
pretreatment with G15 inhibited G1 and E2 facilitation of lordosis. This
(Fig. 3); however, there was no effect on LQ or LS at 30 min. Table 2
corroborates the idea that E2 facilitates lordosis in part through the
indicates Mann Whitney U values, p values and effect sizes for G15 and
activation of GPER-1. GPER-1 has been found at a variety of in-
Antide combined with E2. G15 had a substantial effect size (Table 2) on
tracellular locations in neurons as well as in the plasma membrane in
the inhibition of lordosis (LQ) induced by E2 at 120 (d = 0.76) and 240
brain and anterior pituitary (Brailoiu et al., 2007; Filardo et al., 2007;
(d = 0.73) min, but a small effect size at 30 min (d = 0.02). Antide
Funakoshi et al., 2006). GPER-1 immunoreactivity is detected in the
significantly prevented the stimulatory effect of E2 on LQ in estrogen-
endoplasmic reticulum and plasma membrane of neuronal perikarya
primed rats at 120 (p < 0.01) and 240 min (p < 0.05), but not at
and dendritic shafts as well as in spines and in clusters of vesicles in
30 min. Moreover, Antide significantly reduced the elevation of LS in-
axon terminals of the hippocampus (Waters et al., 2015). GPER-1 im-
duced by E2 (Fig. 3B) only at 120 min (p < 0.01) post-injection. The
munostaining has also been reported in the Golgi apparatus in mag-
greatest effect sizes for Antide inhibition of LS induced by E2 were
nocellular oxytocin neurons (Sakamoto et al., 2007). Activation of
found at 120 and 240 min (see Table 2 for d values).
GPER-1can induce non-genomic signaling, such as activation of the α
subunit of the G protein, which in turn activates adenylyl cyclase.
3.3. Experiment 3 Because Antide inhibited lordosis induced by G1 and E2, we spec-
ulate that lordosis behavior was induced by the release of GnRH and the
3.3.1. Effect of TMX and RU486 on lordosis behavior induced by G1 activation of its GnRH-1 receptor. Previous findings support this in-
Systemic administration of TMX reduced LQ (Fig. 4A) and LS terpretation. a) In primates, GPER-1 is located in hypothalamic GnRH
(Fig. 4B) induced by G1 at 120 (p < 0.001) and 240 min (p < 0.01) neurons where it mediates the rapid actions of E2 on GnRH release
but not at 30 min. Table 3 indicates Mann Whitney U values, p values (Jacobi et al., 2007; Noel et al., 2009). Both E2 and G1 increase in-
and effect sizes for TMX and RU486 combined with G1. Effect sizes tracellular Ca+ and stimulate GnRH release, and these effects are
(Table 3) for TMX were large at 120 (d = 0.8) and 240 (d = 0.72) min blocked by G15 (Noel et al., 2009; Kenealy and Terasawa, 2012;
but small at 30 min (d = 0.21). LQ and LS induced by G1 at 120 and Terasawa and Kenealy, 2012). b) Lordosis behavior induced by GnRH, P
240 min were almost completely suppressed by the sc injection of and some of its ring A-reduced metabolites, leptin or vaginocervical
RU486 (both p < 0.001 vs G1 alone). However, there was no effect at stimulation is reduced by icv administration of Antide (García-Juárez
30 min for LQ or LS. Effect sizes confirm that RU486 was highly ef- et al., 2011; Gómora-Arrati et al., 2008), supporting the idea that GnRH
fective at inhibiting G1-induced lordosis at 120 and 240 min (see release is an obligatory step in the facilitation of lordosis by each of
Table 3 for d values). these.
Interestingly, Experiment 3 demonstrated the involvement of ER
4. Discussion and PR in the regulation of lordosis facilitation by GPER-1, because
both TMX and RU486, respectively, blocked lordosis behavior induced
The present study tested the hypothesis that lordosis behavior in- by G1. In previous studies, TMX injected before estrogen priming in
duced by GPER-1 activation in EB-primed rats involves GnRH-1 re- OVX rats, either into the ventromedial hypothalamus or systemically,
ceptors, PRs and ERs. The results suggest that lordosis behavior induced inhibited estrogen priming of lordosis behavior (Etgen, 1979; Etgen and
through the activation of GPER-1 by E2 or G1 may depend on the re- Shamamian, 1986; Howard et al., 1984). In addition, concurrent icv
lease of GnRH, because icv administration of Antide blocked the facil- infusion of TMX with GnRH, leptin, progestins, or PGE2 significantly
itatory actions of both compounds. TMX and RU486 also inhibited the reduced lordosis induced by these compounds in estrogen-primed rats
facilitation of lordosis by G1, suggesting the participation of both ER (Lima-Hernández et al., 2014), suggesting that ERs also participate in
and PR in this process. We believe that the roles of GnRH-1 receptor, ER activation of lordosis behavior many h after the priming administration
and PR are biologically meaningful because of the effect size estimates of E2.
of the impact of G15, TMX and RU486 treatment (Tables 2 and 3). The agonist and antagonist effects of TMX may vary according to

82
R. Domínguez-Ordóñez et al.
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 an-
tagonist effects on selected E2-dependent reproductive processes, be-
cause TMX prevents the E2-induced increase in cFos expression in the
anteroventral periventricular nucleus and also prevents PR induction by
83
Hormones and Behavior 98 (2018) 77–87
Fig. 3. The facilitation of LQ (A) and LS (B) with in-
tracerebral infusion of 2 ng of E2 (n = 11) in OVX, EB-
primed 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-
R. Domínguez-Ordóñez et al.
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 adminis-
tration of TMX or icv infusion of ICI facilitated lordosis 4 h later in OVX,
EB-primed rats. However, when either ER antagonist was directly in-
fused in the arcuate nucleus, they induced more intense lordosis be-
havior 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
Hormones and Behavior 98 (2018) 77–87
Fig. 4. LQ (A) and LS (B) were induced in OVX, EB-
primed 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 adminis-
tration 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-in-
jection 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 im-
portantly between the two studies. In the Long study, lordosis was in-
duced 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
R. Domínguez-Ordóñez et al. Hormones and Behavior 98 (2018) 77–87

Table 3 Ordóñez et al., 2015; García-Juárez et al., 2011; Lima-Hernández et al.,

Statistical values associated with lordosis quotients in rats given icv infusion of G1 2014; Mani and Blaustein, 2012; Mani and Portillo, 2010). The fact that
compared to the antagonists TMX and RU486 (Experiment 3).
RU486 reduced lordosis behavior induced by G1 in the present study
Treatment Statistical value at each time suggests that G1 activates PRs, perhaps indirectly via GnRH release.
This idea is supported by the ability of RU486 to block GnRH self-
30 min 120 min 240 min priming, which requires cross-communication between GnRH receptor-
activated protein kinase A and the PR (Turgeon and Waring, 1994;
DMSO – – –
TMX + G1 d 0.21 0.8 0.72 Waring and Turgeon, 1992). Overall, the results suggest that GPER-1 is
(n = 10) p< NS 0.001 0.01 involved in the stimulation of lordosis behavior in estrogen-primed
U 24 2 7 female rats. Thus, we propose that E2 and G1 bind directly to GPER-1
RU486 + G1 d 0.15 0.84 0.88 (upper panel, Fig. 5), which is present in GnRH neurons in the preoptic
(n = 9) p< NS 0.001 0.001
area and which stimulates an increase in intracellular calcium, inducing
U 28 1.5 0.5
the release of GnRH (Naor et al., 2000; Weiner and Charles, 2001: Kraus
Time (min) is relative to infusion of G1 or vehicle. G1, GPER-1 agonist; TMX, tamoxifen, et al., 2003: Navratil et al., 2003; Higa-Nakamine et al., 2015). GnRH
selective ER modulator; RU486, PR antagonist; Cohen's d, effect size value; p < , prob- neurons project to other regions of the hypothalamus (e.g., para-
ability; U, Mann Whitney U test value; NS, not significant. ventricular nucleus) containing cells that express the GnRH-1 receptor
(Silverman et al., 1987; Wierman et al., 2011; Schauer et al., 2015). The
noted previously, this suggests that TMX may act as an antagonist in the released GnRH may then activate its membrane receptor (GnRH-1) lo-
presence of E2 or other agonists and as an agonist in the absence of E2. calized in postsynaptic neurons, which contain PRs and ERs. The GnRH-
At the level of neural mechanisms, the different effects of TMX on 1 receptor-dependent increase in second messenger production and
lordosis behavior between these two studies could also be due to the kinase activation may then modulate lordosis behavior by activating
fact that TMX, in some experimental conditions, inhibits GnRH release. both ERs and PRs in a ligand-independent manner (Beyer et al., 2003;
For example, TMX effectively prevented the positive, but not negative, Demay et al., 2001; Mani and Blaustein, 2012; Mani and Portillo,
feedback effects of E2 (Donath and Nishino, 1998; Gao et al., 2002; 2010).
Aquino et al., 2016). An alternative intracellular mechanism by which GnRH-1 receptors
The inhibitory effect of RU486 on the actions of G1 also agrees with could induce lordosis behavior is by activating the mitogen-activated
previous reports showing that this PR antagonist reduces lordosis be- protein kinase (MAPK) pathway through the Src/ER/PR/MAPK com-
havior induced by a variety of compounds (Beyer et al., 1995; Beyer plex in hypothalamic neurons (lower panel, Fig. 5; Higa-Nakamine
et al., 1997; Domínguez-Ordóñez et al., 2015; Etgen and Shamamian, et al., 2015; Kraus et al., 2003; Navratil et al., 2003; Naor et al., 2000).
1986; García-Juárez et al., 2011; González-Mariscal et al., 1989; Mani PRs bind the SH3 domain of Src through its proline-rich domain (re-
and Portillo, 2010; Mani and Blaustein, 2012; Vathy et al., 1987). On sidues 421 and 428 located in the N-terminus), while ERs interact di-
the other hand, in certain experimental conditions, RU486 exerts ago- rectly with the SH2 domains through phosphotyrosine 537 located also
nist effects on lordosis behavior (Pleim et al., 1990). However, an at the N-terminus (Ballaré et al., 2003; Boonyaratanakornkit et al.,
agonist effect of the antiprogestin was only observed in the absence of 2001; Faivre and Lange, 2007; Migliaccio et al., 1998). Therefore, PR
P. Several hormone antagonists, like RU486 and TMX, induce partial and ER could heterodimerize through the ERIDI and II (ER-interaction
agonist effects in the absence of natural hormone by a mechanism that domain I and II) in the N-terminal domain of the PR and the ligand-
is not well understood. binding domain of ER. Indeed others (Migliaccio et al., 1998;
Some studies suggest that PRs are common molecular effectors for Boonyaratanakornkit et al., 2001) showed that activation of Src/MAPK
compounds with diverse chemical structures that induce lordosis be- through the progestin agonist, R5020, was blocked by administration of
havior 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 sti-
mulate the adenylyl cyclase-cAMP-protein kinase A (AC-cAMP-
PKA) system. This increases intracellular calcium (Ca++) con-
centration, 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, pre-
venting 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.

85
R. Domínguez-Ordóñez et al.
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 adminis-
tration 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 in-
duced by E2 or G1 was blocked with either RU486 or TMX supports the
participation of Src/PR/ER/MAPK complex in the regulation of lor-
dosis. 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 re-
ceptor to activate the Src-MAPK pathway. Nonetheless, this is only a
hypothetical model meant to stimulate further experimentation.
Acknowledgement
This work was supported by Consejo Nacional de Ciencia y
Tecnología (CONACYT) Grant Number 134291 and by PRODEP Grant
Number DSA/103.5/16/12094.
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