Original Paper Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
Received: September 19, 2013 Accepted after revision: June 26, 2014 Published online: July 7, 2014
Activation of the GPR30 Receptor Promotes Lordosis in Female Mice Divya Anchan a Amber Gafur b Kazuhiro Sano c Sonoko Ogawa c Nandini Vasudevan a, b a Neuroscience Program, Tulane University and b Cell and Molecular Biology Department, Tulane University, New Orleans, La., USA; c Laboratory of Behavioral Neuroendocrinology, University of Tsukuba, Tsukuba, Japan
Abstract Background/Aims: Estrogens are important effectors of reproduction and are critical for upregulating female reproductive behavior or lordosis in females. In addition to the importance of transcriptional regulation of genes by 17β-estradiol-bound estrogen receptors (ER), extranuclear signal transduction cascades such as protein kinase A (PKA) are also important in regulating female sexual receptivity. GPR30 (G-protein coupled receptor 30), also known as GPER1, a putative membrane ER (mER), is a G protein-coupled receptor that binds 17β-estradiol with an affinity that is similar to that possessed by the classical nuclear ER and activates both PKA and extracellular-regulated kinase signaling pathways. The high expression of GPR30 in the ventromedial hypothalamus, a region important for lordosis behavior as well as kinase cascades activated by this receptor, led us to hypothesize that GPR30 may regulate lordosis behavior in female rodents. Method: In this study, we investigated the ability of G-1, a selective agonist of GPR30, to regulate lordosis in the female mouse by administering this agent prior to progesterone in an estradiol-progesterone priming paradigm prior to testing with stud males. Results: As expected, 17β-estradiol benzoate (EB), but not sesame oil, increased lordosis behavior in female mice. G-1 also in-
© 2014 S. Karger AG, Basel 0028–3835/14/1001–0071$39.50/0 E-Mail [email protected] www.karger.com/nen
creased lordosis behavior in female mice and decreased the number of rejective responses towards male mice, similar to the effect of EB. The selective GPR30 antagonist G-15 blocked these effects. Conclusion: This study demonstrates that activation of the mER GPR30 stimulates social behavior in a rodent model in a manner similar to EB. © 2014 S. Karger AG, Basel
Introduction
Estrogen is a critical determinant of reproductive behavior in female rodents, exemplified by the sexually dimorphic lordosis posture [1]. The lordosis posture is controlled by neurons in the periaqueductal grey, which receive inputs from neurons in the ventromedial hypothalamus (VMH). Lesions in the VMH abolished lordosis [2] while administration of 17β-estradiol into the VMH restored lordosis behavior that was lost in ovariectomized (OVX) rats [3]. These studies suggest that 17β-estradiol signaling in the VMH is sufficient to induce lordosis in rats. Estrogens can signal by using both nongenomic and genomic modes of signaling in cells [4, 5]. The genomic mode of signaling involves the binding of the cognate ligand, 17β-estradiol, to either the α or β isoform of the estrogen receptor (ER), transforming these
Divya Anchan and Amber Gafur contributed equally to this article.
Nandini Vasudevan Cell and Molecular Biology Department, Tulane University 2000 Percival Stern Hall New Orleans, LA 70118 (USA) E-Mail nandini @ tulane.edu
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Key Words Female mice · G-1 · GPR30 · Lordosis · Membrane estrogen receptor
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compound with a Ki of 11 nM for GPR30 [26], an affinity similar to that exhibited by 17β-estradiol (5.7 nM) for GPR30 [26]. In addition, G-1 is specific for GPR30 and does not bind ERα or ERβ, even at micromolar concentrations [26]. GPR30 activation can be blocked by the synthetic antagonist G-15, which has a binding affinity of 20 nM [27] for this receptor. GPR30 can rapidly activate PKA signal transduction pathways in SKBR3 cells, which lack ERα and ERβ [22], and can mobilize calcium in hypothalamic astrocytes [28]. Furthermore, the GPR30-mediated activation of epidermal growth factor (EGF) signaling and consequent ERK signaling [29] was implicated in neuroprotection afforded by 17β-estradiol in the CA1 region of the hippocampus exposed to ischemia [30]. Though administration of G-1 into the cerebral ventricles of female ovarihysterectomized rats could not mimic the negative feedback of 17β-estradiol on luteinizing hormone (LH) secretion, it increased short-latency prolactin secretion, similar to 17β-estradiol [31]. These studies demonstrate that some of the actions of 17β-estradiol in the central nervous system could be due to non-genomic signaling mediated by activation of GPR30. Indeed, similar to the dense distribution of ERα in the VMH, the high expression of GPR30 [32] in the ventrolateral VMH, as well as its rapid activation of the PKA pathway, suggest that this receptor may contribute to a rapid effect of 17β-estradiol on lordosis behavior. The main hypothesis tested in this study was that GPR30 activation by a selective agonist, G-1, can increase lordosis behavior in OVX female mice. A second hypothesis was that male interactions with females treated with G-1 would be similar to those with 17β-estradiol-treated females. We found that the administration of G-1, combined with progesterone, caused an increase in lordosis in female OVX mice and reduced rejective responses, which were similar to the effects of 17β-estradiol. This increase in lordosis by G-1 treatment could be blocked by the GPR30 antagonist, G-15. This suggests that activation of the putative mER GPR30 is sufficient for the display of lordosis behavior in female mice. Methods Animals Two groups of adult C57BL/6J female mice (16–22 weeks of age; Charles River, Wilmington, Mass., USA, and Clea Inc., Tokyo, Japan) were test subjects in replicate experiments. Females were individually housed and adult male C57BL/6J mice (14–20 weeks of age; Charles River and Clea Inc.) were housed in groups under a 12-hour light:12-hour dark cycle (lights off at noon) with food and water provided ad libitum. Female mice were OVX under isoflurane anesthesia and allowed to rest for 10 days after surgery to
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proteins into ligand-dependent transcription factors that regulate transcription at target genes in an hourly time frame [6, 7]. On the other hand, non-genomic signaling by 17β-estradiol includes the far more rapid activation of kinases and calcium flux [8, 9], presumably initiated by an ER at the plasma membrane (mER), whose identity remains unclear [10, 11]. Lordosis behavior is often studied by treating OVX rodents with 17β-estradiol followed by progesterone [12, 13]. Silencing of the ERα in the VMH abolished the transcriptional upregulation of the progesterone receptor (PR) by estrogen and prevented proceptive and lordosis behaviors in female mice [14], demonstrating the importance of ERα in lordosis. Apart from the accepted importance of the transcriptional actions of 17β-estradiol in inducing lordosis [15], a membrane-limited estradiol conjugate, such as E2-BSA (17β-estradiol conjugated to bovine serum albumin), induced the PR [16] when used alone and activated lordosis behavior [17] when it was coupled with a second administration of 17β-estradiol into the VMH. This suggests that non-genomic actions of estrogens contribute to lordosis behavior. Furthermore, knocking down caveolin, a protein that anchors ERα to the plasma membrane, in the arcuate nucleus decreased ERα localization at the membrane and attenuated lordosis receptivity [18], suggesting that membrane-initiated ERα signaling in the arcuate nucleus modulates lordosis behavior [11]. Signal transduction cascades also influence lordosis; the lordosis enhancing actions of progesterone were blocked by infusion of a protein kinase A (PKA) inhibitor in the ventral tegmental area in hamsters and rats [19]. In addition, elevation of cyclic AMP, the second messenger in the PKA pathway, in the VMH attenuated the inhibitory effect of serotonin on lordosis behavior [20]. Lastly, 17β-estradiol increased, within 1 h, phospho-cyclic AMP response element-binding protein in the ventrolateral VMH of female mice via ERα activation, since this increase was lost in the ERαknockout mouse but maintained in the ERβ-knockout mouse [21]. Hence, since the PKA pathway modulates lordosis and is inducible rapidly by 17β-estradiol via ERα in a region of the brain critical for lordosis (i.e. the ventrolateral VMH), signaling via an mER that activates this pathway could contribute to lordosis behavior. In addition to ERα at the plasma membrane, GPR30 (Gprotein coupled receptor 30), also known as GPER1, a former orphan G protein-coupled receptor, is also a candidate mER based on its high affinity binding to 17β-estradiol [22] and its ability to localize in the plasma membrane [23, 24] of MCF-7 cells. GPR30 activation is mimicked by a synthetic agonist, G-1 [25], a substituted dihydroquinoline
Hormone Regimen 17β-Estradiol benzoate (EB; Sigma Inc., St. Louis, Mo., USA), progesterone (Sigma Inc.) and G-1 and G-15 (Tocris, Bristol, UK) were dissolved in sesame oil (Sigma Inc.). The G-1 and G-15 suspensions were gently heated to 55 ° C and stirred for 24 h. We used EB to induce lordosis behaviors in OVX female mice as per Ogawa et al. [13]. In experiment I, OVX females were randomly divided into three treatment groups (12/treatment group): those that received vehicle (100 μl sesame oil), those that received 10 μg EB s.c. (in 100 μl sesame oil) and those that received 50 μg G-1 s.c. (in 100 μl sesame oil), 48 and 24 h before behavioral testing. In experiment II (n = 8 animals/treatment group), in addition to the three groups described in experiment I, a fourth group of animals received 10 μg EB and 250 μg G-15 s.c. (in 100 μl sesame oil) and a fifth group received 50 μg G-1 and 250 μg G-15 s.c., 48 and 24 h before behavioral testing. G-15 was administered to the animal 30 min before the administration of G-1 or EB. Four to 6 h before testing, every female animal, including vehicle-treated animals, received a subcutaneous injection of 500 μg progesterone in 100 μl sesame oil. Behavioral testing commenced 2 h after lights off, under red light, by the introduction of a trained stimulus male into the females’ home cage. Males were trained once a week for sexual experience with OVX females treated with EB and progesterone for a period of 4 weeks. Tests were digitally recorded using a standard resolution black and white camera (Ganz FC-62D) and the videos evaluated by an observer blinded to the hormone treatment of the female subjects. We used a single test, similar to that used by Sinchak et al. [33], in order to study the neural correlates of G-1-regulated lordosis in the future, without the confounds of multiple tests or injections.
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Behavioral Analysis Females could have different types of bouts with stimulus males, with a bout being defined as an encounter of any duration between males and females that is complete when either the male or female moves half the body length of the female away [13]. Bouts were classified as lordotic, rejective or social, as defined below [13]. Lordotic bouts were bouts where the female displayed lordosis in response to male mounts or intromissions. Lordosis responses were scored for a maximum of 15 mounts or a minimum of 10 mounts from the male; females that did not receive 10 mounts during the 30-min test were eliminated from the analysis. Hence, in experiment I, 3 vehicle-treated females were removed from the analysis. In experiment II, 1 animal from each treatment group except the EB-treated group was removed from the analysis. The quality of the lordosis responses of the females was scored using a scale from 0 to 3 similar to the scale used by Sinchak et al. [33]. A zero score implied that the female allowed a mount or intromission by the male but was not immobile; this parameter has been considered as lordosis behavior by Koonce and Frye [34]. A score of 1 was given when the female stayed still for the male to mount or intromit and a score of 2 was given when the female showed slight dorsiflexion of the spine. A score of 3 was given when the female showed complete dorsiflexion of the spine and extension of the legs in response to a male mount
GPR30 Can Regulate Lordosis in Mice
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Fig. 1. EB, but not G-1, treatment increased uterine wet weight in female mice. OVX female mice were treated with sesame oil (vehicle), 10 μg EB or 50 μg G-1 s.c. 48 and 24 h before testing for lordosis behavior. Uterine wet weight was measured at sacrifice, 2 days after the sex behavior tests. One-way ANOVA followed by Tukey’s post hoc test were used to compare uterine wet weights between the groups. Data represent means ± SEM. * p < 0.05 vs. other groups.
or intromission. Any of these scores (0–3) displayed by the female in response to a male mount or intromission categorized the bout as a lordosis bout and was used to calculate the lordosis quotient (LQ). The LQ was calculated by the percent of lordosis occurrences divided by the number of male mounts. Hence, both the LQ and the average lordosis scores (LS) denote the sexual receptivity of the female. We also analyzed rejection behaviors of the female such as kicking, standing up or running away quickly from the attempted mounts of the male. Bouts that contained any of these behaviors in response to a male mount were classified as rejective bouts and not given LS. We also separately analyzed social interactions initiated by the male to the female over the entire 30-min test by scoring genital sniffing, touching of the female’s back with forepaws and chasing of the female by the male. Bouts that contained any of these elements were classified as social bouts [13]. Two days after the sex behavior test, mice were sacrificed and uterine wet weight recorded. A one-way ANOVA followed by Tukey’s post hoc test (Graph Pad Prism, La Jolla, Calif., USA) was used to compare various parameters between treatment groups. Bartlett’s test for differences in variances amongst treatment groups showed no significant difference in variance. The D’Agostino-Pearson omnibus normality showed no deviation from normality amongst treatment groups. A value of p < 0.05 was considered statistically significant in all cases.
Results
Female mice treated with EB showed an increase in uterine wet weight compared to mice treated with vehicle or G-1 (fig. 1; F2, 21 = 11.33, p = 0.0005). The uterine Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
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heal and also to allow for reduction in circulating hormone levels. Care was taken to see that cages of females or males were not changed within 48 h prior to behavioral testing. All housing conditions and tests were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Tulane University Institutional Animal Care and Use Committee.
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OVX female mice were treated with sesame oil (vehicle; n = 9), 10 μg EB (n = 12) or 50 μg G-1 (n = 12) s.c. 48 and 24 h before testing for lordosis behavior. Progesterone (500 μg s.c.) was administered 4–6 h before lordosis behavior testing. LQ (a), LS (b), the latency to the first
lordosis bout (c) and the number of rejective bouts in response to mounts (d) were scored by an observer blinded to the experimental status. One-way ANOVA followed by Tukey’s post hoc test was used to compare the lordosis parameters among the groups. Data represent means ± SEM. * p < 0.05, ** p < 0.06, *** p < 0.07, vs. vehicle.
weights of G-1- and vehicle-treated mice were not significantly different from each other. In experiment I, mice treated with either EB or G-1 showed a significantly higher LQ than vehicle-treated mice (fig. 2a; F2, 29 = 7.475; p = 0.0024); EB- and G-1-treated animals were not significantly different from each other. However, though EB-treated mice showed a higher average LS than the vehicle-treated group (fig. 2b; F2, 30 = 3.611; p = 0.0393), G1-treated mice were not statistically different from either the vehicle- or EB-treated mice. The latency to the first lordotic bout was significantly shorter in the G-1-treated animals when compared to vehicle (fig. 2c; F2, 31 = 3.769; p = 0.0343); the latency in the EB-treated group was close to statistical significance when compared to vehicle (p = 0.06). The number of rejective bouts in response to male mounts was also significantly lower in the EB-treated group when compared to vehicle (fig. 2d; F2, 30 = 4.348; p = 0.0220); the lower number of rejective bouts in the G-
1-treated group was close to statistical significance when compared to the vehicle-treated group (p = 0.07). Social interactions initiated by the male were also measured for the entire duration of the 30-min tests for all females. As can be seen, in experiment I, the EB- and G-1-treated groups did not differ from each other or from vehicle in the number of social bouts (fig. 3a; F2, 33 = 0.4137; p = 0.6646), the latency to the first social bout (fig. 3b; F2, 33 = 1.102; p = 0.3422) or the percentage of bouts that had genital sniffs by the male (fig. 3c; F2, 33 = 0.5233; p = 0.5974). Both EB- and G-1-treated females elicited more stimulation from the males, as shown by the greater number of bouts of males touching the females’ back with their paws compared to the females treated with vehicle alone (fig. 3d; F2, 33 = 4.780; p = 0.0150). The EB and G-1 groups did not differ from each other in any social interaction. In experiment II, we administered the G-1 antagonist G-15 (250 μg) along with
Fig. 2. G-1 treatment increased lordosis behavior in female mice.
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Fig. 3. G-1 increased social interactions initiated by males. OVX
female mice were treated with sesame oil (vehicle; n = 12), 10 μg EB (n = 12) or 50 μg G-1 (n = 12) s.c. 48 and 24 h before testing for lordosis behavior. Progesterone (500 μg s.c.) was administered 4–6 h before lordosis behavior testing. The number of social bouts (a), the latency to the first social bout (b), and the
either EB or G-1 (s.c.) 48 and 24 h before behavioral testing. G-15 blocked the G-1-induced increase in LQ (fig. 4a; F4, 31 = 12.17; p < 0.0001) and largely suppressed the EB-induced increase in LQ (p = 0.06 in EB- + G15-treated group compared to the EB-treated group). Similarly, G-15 also blocked the G-1-induced increase in LS (fig. 4b; F4, 33 = 8.767; p < 0.0001) and the G-1-induced decrease (fig. 4d; F4, 33 = 13.36; p < 0.0001) in the number of rejective bouts. The decrease in LS (fig. 4b) in animals that received both EB and G-15 was close to significance (p = 0.06) when compared to animals that received EB alone. Similarly, animals that received EB and G-15 had a greater number of rejective bouts (fig. 4d), which approached statistical significance when compared to animals that received EB alone (p = 0.06). G-15 did not block the ability of G-1 to reduce the latency to the first lordotic bout compared to vehicle treatment (fig. 4c; F4, 33 = 3.0; p = 0.0324). GPR30 Can Regulate Lordosis in Mice
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percent of bouts with genital sniffs from the male (c) and with the male’s paws on the female’s back (d) were scored by an observer blinded to the experimental status. One-way ANOVA followed by Tukey’s post hoc test was used to compare the social parameters among the groups. Data represent means ± SEM. * p < 0.05 vs. vehicle.
Discussion
The dose and route of delivery of EB (10 μg/mouse s.c.) that we used have been employed previously to stimulate lordosis behavior in OVX female mice [13, 33]. In accordance with the previous literature [35–37], this dose generated a uterotrophic response, reflected by the increase in uterine wet weight at sacrifice. G-1 injections, on the other hand, did not increase uterine wet weight; this is in agreement with a previous study in which a range of G-1 doses, from 0.5 to 100 μg/mouse, did not stimulate uterine weight gain [38]. In mice, administration of propylpyrazole triol, an ERα-selective agonist, but not diarylpropionitrile, an ERβ-selective agonist, increased uterine weight, suggesting that ERα may be the principal ER by which 17β-estradiol exhibits its uterotrophic actions [39]. We chose the doses of 50 μg of G-1 and 10 μg EB administered subcutaneously because previous studies have Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
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behavior. OVX female mice were treated with sesame oil (vehicle; n = 7), 10 μg EB (n = 8), 50 μg G-1 (n = 7), EB with 250 μg G-15 (n = 7) or G-1 with 250 μg G-15 (n = 7) s.c. 48 and 24 h before testing for lordosis behavior. Progesterone (500 μg s.c.) was administered 4–6 h before lordosis behavior testing. LQ (a), LS (b), the
used a similar ratio, i.e. ∼4:1 G-1 to 17β-estradiol given subcutaneously to female OVX mice [40]. The dose of G-15 we used was based on the ratio (∼8:1) of G-15 (40 μg/day) to G-1 (5 μg/day) used by Hammond et al. [41] to test cognition in OVX female rats. This dose decreased the memory improvement seen with estrogen and G-1 in an alternating T-maze task [41]. We used a slightly lower ratio, i.e. 5:1 G-15 (250 μg) to G-1 (50 μg). Lordosis Behavior In agreement with the previous literature [13, 33, 42], EB increased LQ and LS, and decreased both the latency to the first lordotic bout and the number of rejective bouts. The increase in LS, which measures the intensity of lordosis, was also similar to that seen in other studies 76
Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
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latency to the first lordosis bout (c) and the number of rejective bouts in response to mounts (d) were scored by an observer blinded to the experimental status. One-way ANOVA followed by Tukey’s post hoc test was used to compare measures among the groups. Data represent means ± SEM. * p < 0.05 vs. vehicle, ** p < 0.05 vs. G-1-treated group, *** p < 0.06 vs. EB-treated group.
[13, 33, 42]. G-1, a selective agonist of the GPR30 receptor, also increased LQ, and decreased the latency to the first lordotic bout and the number of rejective bouts. However, in all cases, vehicle-treated animals showed a higher LQ than expected; this may in part be due to our scoring methodology, where females that were not immobile but allowed a male mount were given an LS of 0. Bouts that contained such elements were counted as lordosis bouts [34]. Since EB presumably can activate multiple receptors (i.e. GPR30, ERα and ERβ) while G-1 selectively activates GPR30, the significantly lower number of rejective bouts in the EB-treated group versus the G1-treated group, in which the number was close to statistical significance, could be due to the additional role of ligand-bound ERα in EB-treated animals that does not Anchan/Gafur/Sano/Ogawa/Vasudevan
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the G-1- or the EB-treated group (online suppl. fig. 1; for all online suppl. material, see www.karger.com/ doi/10.1159/000365574; F4, 32 = 1.833; p = 0.1467). EB significantly increased the number of bouts in which the males had their paws on the females’ backs, similar to experiment I, while the increase in the G-1-treated group compared to the vehicle-treated group approached significance (p = 0.06). The males continued to investigate the G-15-treated females despite the increased number of rejective bouts by these females. In addition, though G-15 decreased the EB effect on lordosis behavior, it was not decreased to control levels for either LQ or the number of rejective bouts. The G-15 suppression of EB-induced lordosis behavior approached significance (p = 0.06) when compared to EB treatment alone. This suggests that GPR30 activation contributes to the EB effect on lordosis, but that EB may also increase lordosis via other ER that are not inhibited by G-15. Alternatively, this could also be because different aspects of lordosis behavior require different magnitudes of activation of the GPR30 receptor, and the dose of G-15 that we employed in this study was insufficient to block GPR30 activation completely; future studies with mice that have a genetic deletion of the GPR30 gene would be useful to distinguish between these two possibilities. GPR30-knockout mice do not show impaired fertility [10], though a rigorous analysis of lordosis behavior remains to be done. This study suggests that GPR30 activation is sufficient, but not completely necessary, for lordosis behavior in female mice.
GPR30 Can Regulate Lordosis in Mice
Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
Molecular Mechanisms Underlying Lordosis The understanding of the mechanisms by which 17β-estradiol enhances lordosis is incomplete, though initiation depends on the β-endorphin activation of μ-opioid receptors in the medial preoptic nucleus, which leads to the disinhibition of VMH neurons, whose activation is required for lordosis [49]. However, the μ-opioid receptor internalization required for this is caused by 17β-estradiol [50] acting on ERα and not on GPR30 [51]. Transcriptional upregulation of PR by ERα is also a critical component of 17β-estradiol-driven lordosis behavior [52, 53]. G-1 can also activate transcription, though not all genes that are regulated by 17β-estradiol are also regulated by G-1. In ovarian cancer cells, both 17β-estradiol and G-1 upregulated the mRNA for pS2, c-fos and cyclin A and D1; however, PR was upregulated solely by 17β-estradiol via an ERα-dependent mechanism [54]. Though our time frame of G-1 administration (i.e. 48 h before lordosis behavior testing) would be expected to activate genomic signaling, the specific genes, including PR, 77
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occur in G-1-treated animals. Activation of one of the other ER by EB is supported by the data that show that the selective GPR30 antagonist G-15 blocked the G-1 action to a greater extent (i.e. to vehicle levels) than it blocked the EB action in LQ, LS and the number of rejective bouts. Removal of ERα [13], but not ERβ [43], by genetic ablation abolished lordosis behaviors and increased rejective behaviors toward stud males. In agreement with this finding, administration of the ERα-selective agonist propylpyrazole triol, but not the ERβ-selective agonist diarylpropionitrile, increased both proceptive and lordosis behavior in the female rat [44], although a recent study showed that propylpyrazole triol can activate GPR30 to increase ERK activation in endometrial cancer cells that lack ERα [45]. In addition, intracerebroventricular administration of antisense oligonucleotides to ERα, but not to ERβ, decreased lordosis in female rats, showing the critical need for ERα in lordosis behavior in female rodents [46]. Alternatively, the dose of G-1 used in our study might be insufficient to reduce the number of rejective bouts to the same extent as EB. The use of G-15 in this study also shows that the G-1-mediated increase in lordosis behavior is due to specific activation of GPR30. We also measured male-initiated social interactions. There was no difference in the amount of male investigation of any female, as evidenced by the lack of difference in the number of social bouts or the percent of bouts that involved genital sniffs, an initial approach behavior by the male, across treatment groups. Blocking progesterone synthesis using either trilostane or aminoglutethimide did not abolish lordosis behavior in rats, but greatly attenuated proceptive behavior [47]. Since all treatment groups received equal amounts of progesterone and because progesterone is important for the appetitive behaviors that precede the receptive lordosis behavior [48], differences using a combined estradiol and progesterone priming paradigm in mice may not be readily apparent. Therefore, the effect of G-1 on proceptive behaviors might be investigated in the future in the estradiol-only priming regimen in rats rather than in mice. Both G-1and EB-treated females showed a greater percentage of bouts in which males placed their paws on the females’ backs; this could be because of the lower rates of rejection amongst females of these groups compared to females of the vehicle-treated groups. Surprisingly, G-15 did not block all the aspects of G1-driven lordosis. For example, G-15 did not block the reduced latency to the first lordotic bout seen in the G1-treated group or decrease the number of bouts in which the males had their paws on the females’ backs in either
or processes activated by GPR30 that drive lordosis behavior remain unknown. Since GPR30 activation is sufficient and ERα appears to be necessary for lordosis behavior in female mice, the molecular mechanism of GPR30 activation of lordosis may require the presence of ERα. GPR30 activation could be upstream of ERα action; our previous study showed that G-1 can phosphorylate ERα at serine 118, albeit in males in the ventral hippocampus [55]. In the mouse spermatogonial cell line GC-1, transcription of the c-fos gene and ERK1/2 activation by 17β-estradiol requires both ERα and GPR30, since these were sensitive to the ER-reducing inhibitor, ICI 182780, as well as to an siRNA to GPR30 [56]. To our knowledge, no study has reported the colocalization or interaction of ERα and GPR30 in hypothalamic neurons. Apart from the GPR30 activation of PKA and ERK that might enhance lordosis behavior, GPR30 also increases calcium in a number of different cell types. In COS-7 cells transfected with GPR30, intracellular calcium rises rapidly (within 10 min of 17β-estradiol application) in an EGF receptor-dependent manner [57]. In the same cell line transfected with ERα, 17β-estradiol also increased calcium rapidly, but in an EGF receptor-independent and PI3K-dependent manner [57], suggesting that though 17β-estradiol can signal via both receptors to achieve the same endpoint, the signal transduction mechanisms may be different. In LH-releasing hormone (LHRH) neurons, both 17β-estradiol and a membranelimited estradiol conjugate, the estradiol dendrimer conjugate, rapidly increased calcium oscillations, which is a
measure of LHRH neuron excitation [58]. Blocking GPR30 action in these neurons using an siRNA reduced both the estradiol dendrimer conjugate- and 17β-estradiolinduced calcium oscillations, while the pure ER antagonist, ICI 182780, had no effect [59]. Since LHRH facilitates lordosis when infused into the medial preoptic area [60] or the VMH [61], it is possible that GPR30 activates lordosis behavior via stimulation of LHRH release.
Conclusion
Previous studies have shown an effect of chronic or acute administration of G-1 on nonsocial behaviors such as learning and memory [62] and anxiety [40, 55] in female and male rodents. Here, we show that GPR30 activation is sufficient to drive a classic 17β-estradiol-regulated reproductive social behavior, lordosis, in female mice. It remains to be seen if GPR30 activation is absolutely necessary for lordosis behavior induced by 17β-estradiol by using animals that lack GPR30.
Acknowledgments We thank Kim Scarmardo for animal maintenance and Xiao Kai for help with some of the initial stud male training. We also acknowledge the Newcomb Fellows Grant (Tulane University) that provided partial support to A.G. N.V is supported by both the NSF CAREER grant IOS-1053716 and by Tulane University startup funds.
1 Pfaff DW: Estrogens and Brain Function: Neural Analysis of a Hormone-Controlled Mammalian Reproductive Behavior. New York, Springer, 1980. 2 Pfaff DW, Sakuma Y: Deficit in the lordosis reflex of female rats caused by lesions in the ventromedial nucleus of the hypothalamus. J Physiol (Lond) 1979;288:203–210. 3 Davis PG, McEwen B, Pfaff DW: Localized behavioral effects of tritiated estradiol implants in the ventromedial hypothalamus of female rats. Endocrinology 1979; 104: 893– 903. 4 Vasudevan N, Pfaff DW: Membrane-initiated actions of estrogens in neuroendocrinology: emerging principles. Endocr Rev 2007; 28: 1– 19. 5 Micevych PE, Dewing P: Membrane-initiated estradiol signaling regulating sexual receptivity. Front Endocrinol (Lausanne) 2011; 2:26.
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Received: September 19, 2013 Accepted after revision: June 26, 2014 Published online: July 7, 2014
Activation of the GPR30 Receptor Promotes Lordosis in Female Mice Divya Anchan a Amber Gafur b Kazuhiro Sano c Sonoko Ogawa c Nandini Vasudevan a, b a Neuroscience Program, Tulane University and b Cell and Molecular Biology Department, Tulane University, New Orleans, La., USA; c Laboratory of Behavioral Neuroendocrinology, University of Tsukuba, Tsukuba, Japan
Abstract Background/Aims: Estrogens are important effectors of reproduction and are critical for upregulating female reproductive behavior or lordosis in females. In addition to the importance of transcriptional regulation of genes by 17β-estradiol-bound estrogen receptors (ER), extranuclear signal transduction cascades such as protein kinase A (PKA) are also important in regulating female sexual receptivity. GPR30 (G-protein coupled receptor 30), also known as GPER1, a putative membrane ER (mER), is a G protein-coupled receptor that binds 17β-estradiol with an affinity that is similar to that possessed by the classical nuclear ER and activates both PKA and extracellular-regulated kinase signaling pathways. The high expression of GPR30 in the ventromedial hypothalamus, a region important for lordosis behavior as well as kinase cascades activated by this receptor, led us to hypothesize that GPR30 may regulate lordosis behavior in female rodents. Method: In this study, we investigated the ability of G-1, a selective agonist of GPR30, to regulate lordosis in the female mouse by administering this agent prior to progesterone in an estradiol-progesterone priming paradigm prior to testing with stud males. Results: As expected, 17β-estradiol benzoate (EB), but not sesame oil, increased lordosis behavior in female mice. G-1 also in-
© 2014 S. Karger AG, Basel 0028–3835/14/1001–0071$39.50/0 E-Mail [email protected] www.karger.com/nen
creased lordosis behavior in female mice and decreased the number of rejective responses towards male mice, similar to the effect of EB. The selective GPR30 antagonist G-15 blocked these effects. Conclusion: This study demonstrates that activation of the mER GPR30 stimulates social behavior in a rodent model in a manner similar to EB. © 2014 S. Karger AG, Basel
Introduction
Estrogen is a critical determinant of reproductive behavior in female rodents, exemplified by the sexually dimorphic lordosis posture [1]. The lordosis posture is controlled by neurons in the periaqueductal grey, which receive inputs from neurons in the ventromedial hypothalamus (VMH). Lesions in the VMH abolished lordosis [2] while administration of 17β-estradiol into the VMH restored lordosis behavior that was lost in ovariectomized (OVX) rats [3]. These studies suggest that 17β-estradiol signaling in the VMH is sufficient to induce lordosis in rats. Estrogens can signal by using both nongenomic and genomic modes of signaling in cells [4, 5]. The genomic mode of signaling involves the binding of the cognate ligand, 17β-estradiol, to either the α or β isoform of the estrogen receptor (ER), transforming these
Divya Anchan and Amber Gafur contributed equally to this article.
Nandini Vasudevan Cell and Molecular Biology Department, Tulane University 2000 Percival Stern Hall New Orleans, LA 70118 (USA) E-Mail nandini @ tulane.edu
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Key Words Female mice · G-1 · GPR30 · Lordosis · Membrane estrogen receptor
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compound with a Ki of 11 nM for GPR30 [26], an affinity similar to that exhibited by 17β-estradiol (5.7 nM) for GPR30 [26]. In addition, G-1 is specific for GPR30 and does not bind ERα or ERβ, even at micromolar concentrations [26]. GPR30 activation can be blocked by the synthetic antagonist G-15, which has a binding affinity of 20 nM [27] for this receptor. GPR30 can rapidly activate PKA signal transduction pathways in SKBR3 cells, which lack ERα and ERβ [22], and can mobilize calcium in hypothalamic astrocytes [28]. Furthermore, the GPR30-mediated activation of epidermal growth factor (EGF) signaling and consequent ERK signaling [29] was implicated in neuroprotection afforded by 17β-estradiol in the CA1 region of the hippocampus exposed to ischemia [30]. Though administration of G-1 into the cerebral ventricles of female ovarihysterectomized rats could not mimic the negative feedback of 17β-estradiol on luteinizing hormone (LH) secretion, it increased short-latency prolactin secretion, similar to 17β-estradiol [31]. These studies demonstrate that some of the actions of 17β-estradiol in the central nervous system could be due to non-genomic signaling mediated by activation of GPR30. Indeed, similar to the dense distribution of ERα in the VMH, the high expression of GPR30 [32] in the ventrolateral VMH, as well as its rapid activation of the PKA pathway, suggest that this receptor may contribute to a rapid effect of 17β-estradiol on lordosis behavior. The main hypothesis tested in this study was that GPR30 activation by a selective agonist, G-1, can increase lordosis behavior in OVX female mice. A second hypothesis was that male interactions with females treated with G-1 would be similar to those with 17β-estradiol-treated females. We found that the administration of G-1, combined with progesterone, caused an increase in lordosis in female OVX mice and reduced rejective responses, which were similar to the effects of 17β-estradiol. This increase in lordosis by G-1 treatment could be blocked by the GPR30 antagonist, G-15. This suggests that activation of the putative mER GPR30 is sufficient for the display of lordosis behavior in female mice. Methods Animals Two groups of adult C57BL/6J female mice (16–22 weeks of age; Charles River, Wilmington, Mass., USA, and Clea Inc., Tokyo, Japan) were test subjects in replicate experiments. Females were individually housed and adult male C57BL/6J mice (14–20 weeks of age; Charles River and Clea Inc.) were housed in groups under a 12-hour light:12-hour dark cycle (lights off at noon) with food and water provided ad libitum. Female mice were OVX under isoflurane anesthesia and allowed to rest for 10 days after surgery to
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proteins into ligand-dependent transcription factors that regulate transcription at target genes in an hourly time frame [6, 7]. On the other hand, non-genomic signaling by 17β-estradiol includes the far more rapid activation of kinases and calcium flux [8, 9], presumably initiated by an ER at the plasma membrane (mER), whose identity remains unclear [10, 11]. Lordosis behavior is often studied by treating OVX rodents with 17β-estradiol followed by progesterone [12, 13]. Silencing of the ERα in the VMH abolished the transcriptional upregulation of the progesterone receptor (PR) by estrogen and prevented proceptive and lordosis behaviors in female mice [14], demonstrating the importance of ERα in lordosis. Apart from the accepted importance of the transcriptional actions of 17β-estradiol in inducing lordosis [15], a membrane-limited estradiol conjugate, such as E2-BSA (17β-estradiol conjugated to bovine serum albumin), induced the PR [16] when used alone and activated lordosis behavior [17] when it was coupled with a second administration of 17β-estradiol into the VMH. This suggests that non-genomic actions of estrogens contribute to lordosis behavior. Furthermore, knocking down caveolin, a protein that anchors ERα to the plasma membrane, in the arcuate nucleus decreased ERα localization at the membrane and attenuated lordosis receptivity [18], suggesting that membrane-initiated ERα signaling in the arcuate nucleus modulates lordosis behavior [11]. Signal transduction cascades also influence lordosis; the lordosis enhancing actions of progesterone were blocked by infusion of a protein kinase A (PKA) inhibitor in the ventral tegmental area in hamsters and rats [19]. In addition, elevation of cyclic AMP, the second messenger in the PKA pathway, in the VMH attenuated the inhibitory effect of serotonin on lordosis behavior [20]. Lastly, 17β-estradiol increased, within 1 h, phospho-cyclic AMP response element-binding protein in the ventrolateral VMH of female mice via ERα activation, since this increase was lost in the ERαknockout mouse but maintained in the ERβ-knockout mouse [21]. Hence, since the PKA pathway modulates lordosis and is inducible rapidly by 17β-estradiol via ERα in a region of the brain critical for lordosis (i.e. the ventrolateral VMH), signaling via an mER that activates this pathway could contribute to lordosis behavior. In addition to ERα at the plasma membrane, GPR30 (Gprotein coupled receptor 30), also known as GPER1, a former orphan G protein-coupled receptor, is also a candidate mER based on its high affinity binding to 17β-estradiol [22] and its ability to localize in the plasma membrane [23, 24] of MCF-7 cells. GPR30 activation is mimicked by a synthetic agonist, G-1 [25], a substituted dihydroquinoline
Hormone Regimen 17β-Estradiol benzoate (EB; Sigma Inc., St. Louis, Mo., USA), progesterone (Sigma Inc.) and G-1 and G-15 (Tocris, Bristol, UK) were dissolved in sesame oil (Sigma Inc.). The G-1 and G-15 suspensions were gently heated to 55 ° C and stirred for 24 h. We used EB to induce lordosis behaviors in OVX female mice as per Ogawa et al. [13]. In experiment I, OVX females were randomly divided into three treatment groups (12/treatment group): those that received vehicle (100 μl sesame oil), those that received 10 μg EB s.c. (in 100 μl sesame oil) and those that received 50 μg G-1 s.c. (in 100 μl sesame oil), 48 and 24 h before behavioral testing. In experiment II (n = 8 animals/treatment group), in addition to the three groups described in experiment I, a fourth group of animals received 10 μg EB and 250 μg G-15 s.c. (in 100 μl sesame oil) and a fifth group received 50 μg G-1 and 250 μg G-15 s.c., 48 and 24 h before behavioral testing. G-15 was administered to the animal 30 min before the administration of G-1 or EB. Four to 6 h before testing, every female animal, including vehicle-treated animals, received a subcutaneous injection of 500 μg progesterone in 100 μl sesame oil. Behavioral testing commenced 2 h after lights off, under red light, by the introduction of a trained stimulus male into the females’ home cage. Males were trained once a week for sexual experience with OVX females treated with EB and progesterone for a period of 4 weeks. Tests were digitally recorded using a standard resolution black and white camera (Ganz FC-62D) and the videos evaluated by an observer blinded to the hormone treatment of the female subjects. We used a single test, similar to that used by Sinchak et al. [33], in order to study the neural correlates of G-1-regulated lordosis in the future, without the confounds of multiple tests or injections.
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Behavioral Analysis Females could have different types of bouts with stimulus males, with a bout being defined as an encounter of any duration between males and females that is complete when either the male or female moves half the body length of the female away [13]. Bouts were classified as lordotic, rejective or social, as defined below [13]. Lordotic bouts were bouts where the female displayed lordosis in response to male mounts or intromissions. Lordosis responses were scored for a maximum of 15 mounts or a minimum of 10 mounts from the male; females that did not receive 10 mounts during the 30-min test were eliminated from the analysis. Hence, in experiment I, 3 vehicle-treated females were removed from the analysis. In experiment II, 1 animal from each treatment group except the EB-treated group was removed from the analysis. The quality of the lordosis responses of the females was scored using a scale from 0 to 3 similar to the scale used by Sinchak et al. [33]. A zero score implied that the female allowed a mount or intromission by the male but was not immobile; this parameter has been considered as lordosis behavior by Koonce and Frye [34]. A score of 1 was given when the female stayed still for the male to mount or intromit and a score of 2 was given when the female showed slight dorsiflexion of the spine. A score of 3 was given when the female showed complete dorsiflexion of the spine and extension of the legs in response to a male mount
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Fig. 1. EB, but not G-1, treatment increased uterine wet weight in female mice. OVX female mice were treated with sesame oil (vehicle), 10 μg EB or 50 μg G-1 s.c. 48 and 24 h before testing for lordosis behavior. Uterine wet weight was measured at sacrifice, 2 days after the sex behavior tests. One-way ANOVA followed by Tukey’s post hoc test were used to compare uterine wet weights between the groups. Data represent means ± SEM. * p < 0.05 vs. other groups.
or intromission. Any of these scores (0–3) displayed by the female in response to a male mount or intromission categorized the bout as a lordosis bout and was used to calculate the lordosis quotient (LQ). The LQ was calculated by the percent of lordosis occurrences divided by the number of male mounts. Hence, both the LQ and the average lordosis scores (LS) denote the sexual receptivity of the female. We also analyzed rejection behaviors of the female such as kicking, standing up or running away quickly from the attempted mounts of the male. Bouts that contained any of these behaviors in response to a male mount were classified as rejective bouts and not given LS. We also separately analyzed social interactions initiated by the male to the female over the entire 30-min test by scoring genital sniffing, touching of the female’s back with forepaws and chasing of the female by the male. Bouts that contained any of these elements were classified as social bouts [13]. Two days after the sex behavior test, mice were sacrificed and uterine wet weight recorded. A one-way ANOVA followed by Tukey’s post hoc test (Graph Pad Prism, La Jolla, Calif., USA) was used to compare various parameters between treatment groups. Bartlett’s test for differences in variances amongst treatment groups showed no significant difference in variance. The D’Agostino-Pearson omnibus normality showed no deviation from normality amongst treatment groups. A value of p < 0.05 was considered statistically significant in all cases.
Results
Female mice treated with EB showed an increase in uterine wet weight compared to mice treated with vehicle or G-1 (fig. 1; F2, 21 = 11.33, p = 0.0005). The uterine Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
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heal and also to allow for reduction in circulating hormone levels. Care was taken to see that cages of females or males were not changed within 48 h prior to behavioral testing. All housing conditions and tests were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Tulane University Institutional Animal Care and Use Committee.
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OVX female mice were treated with sesame oil (vehicle; n = 9), 10 μg EB (n = 12) or 50 μg G-1 (n = 12) s.c. 48 and 24 h before testing for lordosis behavior. Progesterone (500 μg s.c.) was administered 4–6 h before lordosis behavior testing. LQ (a), LS (b), the latency to the first
lordosis bout (c) and the number of rejective bouts in response to mounts (d) were scored by an observer blinded to the experimental status. One-way ANOVA followed by Tukey’s post hoc test was used to compare the lordosis parameters among the groups. Data represent means ± SEM. * p < 0.05, ** p < 0.06, *** p < 0.07, vs. vehicle.
weights of G-1- and vehicle-treated mice were not significantly different from each other. In experiment I, mice treated with either EB or G-1 showed a significantly higher LQ than vehicle-treated mice (fig. 2a; F2, 29 = 7.475; p = 0.0024); EB- and G-1-treated animals were not significantly different from each other. However, though EB-treated mice showed a higher average LS than the vehicle-treated group (fig. 2b; F2, 30 = 3.611; p = 0.0393), G1-treated mice were not statistically different from either the vehicle- or EB-treated mice. The latency to the first lordotic bout was significantly shorter in the G-1-treated animals when compared to vehicle (fig. 2c; F2, 31 = 3.769; p = 0.0343); the latency in the EB-treated group was close to statistical significance when compared to vehicle (p = 0.06). The number of rejective bouts in response to male mounts was also significantly lower in the EB-treated group when compared to vehicle (fig. 2d; F2, 30 = 4.348; p = 0.0220); the lower number of rejective bouts in the G-
1-treated group was close to statistical significance when compared to the vehicle-treated group (p = 0.07). Social interactions initiated by the male were also measured for the entire duration of the 30-min tests for all females. As can be seen, in experiment I, the EB- and G-1-treated groups did not differ from each other or from vehicle in the number of social bouts (fig. 3a; F2, 33 = 0.4137; p = 0.6646), the latency to the first social bout (fig. 3b; F2, 33 = 1.102; p = 0.3422) or the percentage of bouts that had genital sniffs by the male (fig. 3c; F2, 33 = 0.5233; p = 0.5974). Both EB- and G-1-treated females elicited more stimulation from the males, as shown by the greater number of bouts of males touching the females’ back with their paws compared to the females treated with vehicle alone (fig. 3d; F2, 33 = 4.780; p = 0.0150). The EB and G-1 groups did not differ from each other in any social interaction. In experiment II, we administered the G-1 antagonist G-15 (250 μg) along with
Fig. 2. G-1 treatment increased lordosis behavior in female mice.
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Fig. 3. G-1 increased social interactions initiated by males. OVX
female mice were treated with sesame oil (vehicle; n = 12), 10 μg EB (n = 12) or 50 μg G-1 (n = 12) s.c. 48 and 24 h before testing for lordosis behavior. Progesterone (500 μg s.c.) was administered 4–6 h before lordosis behavior testing. The number of social bouts (a), the latency to the first social bout (b), and the
either EB or G-1 (s.c.) 48 and 24 h before behavioral testing. G-15 blocked the G-1-induced increase in LQ (fig. 4a; F4, 31 = 12.17; p < 0.0001) and largely suppressed the EB-induced increase in LQ (p = 0.06 in EB- + G15-treated group compared to the EB-treated group). Similarly, G-15 also blocked the G-1-induced increase in LS (fig. 4b; F4, 33 = 8.767; p < 0.0001) and the G-1-induced decrease (fig. 4d; F4, 33 = 13.36; p < 0.0001) in the number of rejective bouts. The decrease in LS (fig. 4b) in animals that received both EB and G-15 was close to significance (p = 0.06) when compared to animals that received EB alone. Similarly, animals that received EB and G-15 had a greater number of rejective bouts (fig. 4d), which approached statistical significance when compared to animals that received EB alone (p = 0.06). G-15 did not block the ability of G-1 to reduce the latency to the first lordotic bout compared to vehicle treatment (fig. 4c; F4, 33 = 3.0; p = 0.0324). GPR30 Can Regulate Lordosis in Mice
20 10 0
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percent of bouts with genital sniffs from the male (c) and with the male’s paws on the female’s back (d) were scored by an observer blinded to the experimental status. One-way ANOVA followed by Tukey’s post hoc test was used to compare the social parameters among the groups. Data represent means ± SEM. * p < 0.05 vs. vehicle.
Discussion
The dose and route of delivery of EB (10 μg/mouse s.c.) that we used have been employed previously to stimulate lordosis behavior in OVX female mice [13, 33]. In accordance with the previous literature [35–37], this dose generated a uterotrophic response, reflected by the increase in uterine wet weight at sacrifice. G-1 injections, on the other hand, did not increase uterine wet weight; this is in agreement with a previous study in which a range of G-1 doses, from 0.5 to 100 μg/mouse, did not stimulate uterine weight gain [38]. In mice, administration of propylpyrazole triol, an ERα-selective agonist, but not diarylpropionitrile, an ERβ-selective agonist, increased uterine weight, suggesting that ERα may be the principal ER by which 17β-estradiol exhibits its uterotrophic actions [39]. We chose the doses of 50 μg of G-1 and 10 μg EB administered subcutaneously because previous studies have Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
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Fig. 4. Blockade of GPR30 reduces EB- and G-1-driven lordosis
behavior. OVX female mice were treated with sesame oil (vehicle; n = 7), 10 μg EB (n = 8), 50 μg G-1 (n = 7), EB with 250 μg G-15 (n = 7) or G-1 with 250 μg G-15 (n = 7) s.c. 48 and 24 h before testing for lordosis behavior. Progesterone (500 μg s.c.) was administered 4–6 h before lordosis behavior testing. LQ (a), LS (b), the
used a similar ratio, i.e. ∼4:1 G-1 to 17β-estradiol given subcutaneously to female OVX mice [40]. The dose of G-15 we used was based on the ratio (∼8:1) of G-15 (40 μg/day) to G-1 (5 μg/day) used by Hammond et al. [41] to test cognition in OVX female rats. This dose decreased the memory improvement seen with estrogen and G-1 in an alternating T-maze task [41]. We used a slightly lower ratio, i.e. 5:1 G-15 (250 μg) to G-1 (50 μg). Lordosis Behavior In agreement with the previous literature [13, 33, 42], EB increased LQ and LS, and decreased both the latency to the first lordotic bout and the number of rejective bouts. The increase in LS, which measures the intensity of lordosis, was also similar to that seen in other studies 76
Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
10
5
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d
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*
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* G-1 + G-15
latency to the first lordosis bout (c) and the number of rejective bouts in response to mounts (d) were scored by an observer blinded to the experimental status. One-way ANOVA followed by Tukey’s post hoc test was used to compare measures among the groups. Data represent means ± SEM. * p < 0.05 vs. vehicle, ** p < 0.05 vs. G-1-treated group, *** p < 0.06 vs. EB-treated group.
[13, 33, 42]. G-1, a selective agonist of the GPR30 receptor, also increased LQ, and decreased the latency to the first lordotic bout and the number of rejective bouts. However, in all cases, vehicle-treated animals showed a higher LQ than expected; this may in part be due to our scoring methodology, where females that were not immobile but allowed a male mount were given an LS of 0. Bouts that contained such elements were counted as lordosis bouts [34]. Since EB presumably can activate multiple receptors (i.e. GPR30, ERα and ERβ) while G-1 selectively activates GPR30, the significantly lower number of rejective bouts in the EB-treated group versus the G1-treated group, in which the number was close to statistical significance, could be due to the additional role of ligand-bound ERα in EB-treated animals that does not Anchan/Gafur/Sano/Ogawa/Vasudevan
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the G-1- or the EB-treated group (online suppl. fig. 1; for all online suppl. material, see www.karger.com/ doi/10.1159/000365574; F4, 32 = 1.833; p = 0.1467). EB significantly increased the number of bouts in which the males had their paws on the females’ backs, similar to experiment I, while the increase in the G-1-treated group compared to the vehicle-treated group approached significance (p = 0.06). The males continued to investigate the G-15-treated females despite the increased number of rejective bouts by these females. In addition, though G-15 decreased the EB effect on lordosis behavior, it was not decreased to control levels for either LQ or the number of rejective bouts. The G-15 suppression of EB-induced lordosis behavior approached significance (p = 0.06) when compared to EB treatment alone. This suggests that GPR30 activation contributes to the EB effect on lordosis, but that EB may also increase lordosis via other ER that are not inhibited by G-15. Alternatively, this could also be because different aspects of lordosis behavior require different magnitudes of activation of the GPR30 receptor, and the dose of G-15 that we employed in this study was insufficient to block GPR30 activation completely; future studies with mice that have a genetic deletion of the GPR30 gene would be useful to distinguish between these two possibilities. GPR30-knockout mice do not show impaired fertility [10], though a rigorous analysis of lordosis behavior remains to be done. This study suggests that GPR30 activation is sufficient, but not completely necessary, for lordosis behavior in female mice.
GPR30 Can Regulate Lordosis in Mice
Neuroendocrinology 2014;100:71–80 DOI: 10.1159/000365574
Molecular Mechanisms Underlying Lordosis The understanding of the mechanisms by which 17β-estradiol enhances lordosis is incomplete, though initiation depends on the β-endorphin activation of μ-opioid receptors in the medial preoptic nucleus, which leads to the disinhibition of VMH neurons, whose activation is required for lordosis [49]. However, the μ-opioid receptor internalization required for this is caused by 17β-estradiol [50] acting on ERα and not on GPR30 [51]. Transcriptional upregulation of PR by ERα is also a critical component of 17β-estradiol-driven lordosis behavior [52, 53]. G-1 can also activate transcription, though not all genes that are regulated by 17β-estradiol are also regulated by G-1. In ovarian cancer cells, both 17β-estradiol and G-1 upregulated the mRNA for pS2, c-fos and cyclin A and D1; however, PR was upregulated solely by 17β-estradiol via an ERα-dependent mechanism [54]. Though our time frame of G-1 administration (i.e. 48 h before lordosis behavior testing) would be expected to activate genomic signaling, the specific genes, including PR, 77
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occur in G-1-treated animals. Activation of one of the other ER by EB is supported by the data that show that the selective GPR30 antagonist G-15 blocked the G-1 action to a greater extent (i.e. to vehicle levels) than it blocked the EB action in LQ, LS and the number of rejective bouts. Removal of ERα [13], but not ERβ [43], by genetic ablation abolished lordosis behaviors and increased rejective behaviors toward stud males. In agreement with this finding, administration of the ERα-selective agonist propylpyrazole triol, but not the ERβ-selective agonist diarylpropionitrile, increased both proceptive and lordosis behavior in the female rat [44], although a recent study showed that propylpyrazole triol can activate GPR30 to increase ERK activation in endometrial cancer cells that lack ERα [45]. In addition, intracerebroventricular administration of antisense oligonucleotides to ERα, but not to ERβ, decreased lordosis in female rats, showing the critical need for ERα in lordosis behavior in female rodents [46]. Alternatively, the dose of G-1 used in our study might be insufficient to reduce the number of rejective bouts to the same extent as EB. The use of G-15 in this study also shows that the G-1-mediated increase in lordosis behavior is due to specific activation of GPR30. We also measured male-initiated social interactions. There was no difference in the amount of male investigation of any female, as evidenced by the lack of difference in the number of social bouts or the percent of bouts that involved genital sniffs, an initial approach behavior by the male, across treatment groups. Blocking progesterone synthesis using either trilostane or aminoglutethimide did not abolish lordosis behavior in rats, but greatly attenuated proceptive behavior [47]. Since all treatment groups received equal amounts of progesterone and because progesterone is important for the appetitive behaviors that precede the receptive lordosis behavior [48], differences using a combined estradiol and progesterone priming paradigm in mice may not be readily apparent. Therefore, the effect of G-1 on proceptive behaviors might be investigated in the future in the estradiol-only priming regimen in rats rather than in mice. Both G-1and EB-treated females showed a greater percentage of bouts in which males placed their paws on the females’ backs; this could be because of the lower rates of rejection amongst females of these groups compared to females of the vehicle-treated groups. Surprisingly, G-15 did not block all the aspects of G1-driven lordosis. For example, G-15 did not block the reduced latency to the first lordotic bout seen in the G1-treated group or decrease the number of bouts in which the males had their paws on the females’ backs in either
or processes activated by GPR30 that drive lordosis behavior remain unknown. Since GPR30 activation is sufficient and ERα appears to be necessary for lordosis behavior in female mice, the molecular mechanism of GPR30 activation of lordosis may require the presence of ERα. GPR30 activation could be upstream of ERα action; our previous study showed that G-1 can phosphorylate ERα at serine 118, albeit in males in the ventral hippocampus [55]. In the mouse spermatogonial cell line GC-1, transcription of the c-fos gene and ERK1/2 activation by 17β-estradiol requires both ERα and GPR30, since these were sensitive to the ER-reducing inhibitor, ICI 182780, as well as to an siRNA to GPR30 [56]. To our knowledge, no study has reported the colocalization or interaction of ERα and GPR30 in hypothalamic neurons. Apart from the GPR30 activation of PKA and ERK that might enhance lordosis behavior, GPR30 also increases calcium in a number of different cell types. In COS-7 cells transfected with GPR30, intracellular calcium rises rapidly (within 10 min of 17β-estradiol application) in an EGF receptor-dependent manner [57]. In the same cell line transfected with ERα, 17β-estradiol also increased calcium rapidly, but in an EGF receptor-independent and PI3K-dependent manner [57], suggesting that though 17β-estradiol can signal via both receptors to achieve the same endpoint, the signal transduction mechanisms may be different. In LH-releasing hormone (LHRH) neurons, both 17β-estradiol and a membranelimited estradiol conjugate, the estradiol dendrimer conjugate, rapidly increased calcium oscillations, which is a
measure of LHRH neuron excitation [58]. Blocking GPR30 action in these neurons using an siRNA reduced both the estradiol dendrimer conjugate- and 17β-estradiolinduced calcium oscillations, while the pure ER antagonist, ICI 182780, had no effect [59]. Since LHRH facilitates lordosis when infused into the medial preoptic area [60] or the VMH [61], it is possible that GPR30 activates lordosis behavior via stimulation of LHRH release.
Conclusion
Previous studies have shown an effect of chronic or acute administration of G-1 on nonsocial behaviors such as learning and memory [62] and anxiety [40, 55] in female and male rodents. Here, we show that GPR30 activation is sufficient to drive a classic 17β-estradiol-regulated reproductive social behavior, lordosis, in female mice. It remains to be seen if GPR30 activation is absolutely necessary for lordosis behavior induced by 17β-estradiol by using animals that lack GPR30.
Acknowledgments We thank Kim Scarmardo for animal maintenance and Xiao Kai for help with some of the initial stud male training. We also acknowledge the Newcomb Fellows Grant (Tulane University) that provided partial support to A.G. N.V is supported by both the NSF CAREER grant IOS-1053716 and by Tulane University startup funds.
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