Regular articleEstrogen receptors regulate the estrous behavior induced by progestins, peptides, and prostaglandin E2
Introduction
Progestins and some non-steroidal agents, such as gonadotropin-releasing hormone (GnRH; Beyer et al., 1982, Beyer et al., 1995, Foreman and Moss, 1977, González-Flores et al., 2006, González-Mariscal and Beyer, 1988, Ramírez-Orduña et al., 2007, Riskind and Moss, 1983, Sakuma and Pfaff, 1980, Wu et al., 2006) and prostaglandin E2 (PGE2; Beyer et al., 1997, Dudley and Moss, 1976, Hall and Luttge, 1977, Rodriguez-Sierra and Komisaruk, 1977) facilitate lordosis behavior in ovariectomized (ovx) rats primed with estradiol (E2). The time course of lordosis facilitation is similar to that induced by progesterone (P; see Moralí and Beyer, 1979). None of these agents have affinity for either classical estrogen receptors (ERs) or progestin receptors (PRs).
Several researchers (Beyer et al., 2003, Blaustein, 2003, Mani et al., 1997) proposed that the PR acts as a common molecular mediator for the various agents that activate lordosis behavior. This idea is supported by the finding that administration of the PR antagonist, RU486, blocks estrous behavior normally induced by P, several ring A-reduced progestins (Beyer et al., 1995), GnRH, cyclic nucleotides, PGE2 (Beyer et al., 1997), leptin (García-Juárez et al., 2011), dopamine (Mani et al., 1996), and by vagino-cervical stimulation (VCS; Auger et al., 1997, González-Flores et al., 2008) in ovx rats primed with E2. This role of PR as a common molecular mediator in lordosis behavior is unlikely to be due to its classic transcriptional action at the genome, but rather by rapidly activating signaling systems such a protein kinases involved in the expression of female sexual behavior in rats (Beyer et al., 2003, Mani and Portillo, 2010, Mani et al., 1996, Mani et al., 1997).
Recent findings show that the activation of the Src–PR–mitogen-activated protein kinase (MAPK) complex is essential for the facilitation of lordosis behavior by P and other agents (González-Flores et al., 2010, Lima-Hernández et al., 2012). The mechanism by which P stimulates the Src–MAPK pathway has been well studied in some cells possessing both ER and PR (for review see, Boonyaratanakornkit et al., 2008, Edwards, 2005, Lange, 2004), and many laboratories now suggest that functional c-Src is required for intracellular signaling initiated by both growth factors and steroid hormones. For example, PR through its N-terminus binds directly to SH3 domains in c-Src and associates via the N-terminal with ER, whereas ERs interact with the SH2-domain of c-Src (Ballare et al., 2003, Boonyaratanakornkit et al., 2001, Migliaccio et al., 2007). These interactions are required for Src–MAPK activation by progestins and estrogens (Boonyaratanakornkit et al., 2001, Faivre et al., 2005, Migliaccio et al., 1998) in that the antiprogestin RU486 and antiestrogens, such as tamoxifen and ICI182780, inhibit steroid stimulation of the Src–MAPK complex.
In the present study we hypothesized that the classical ER participates in the facilitation of lordosis not only by P, as was reported by several workers in rodents (Etgen, 1979, Kow and Pfaff, 1975, Micevych and Sinchak, 2014), but also by ring A-reduced progestins, by non-steroidal agents such as GnRH, PGE2 and leptin, and by VCS. For this purpose, an infusion of tamoxifen, an agent that has a mixed agonist/antagonist action at ERs, or the pure ER antagonist ICI182780 (ICI) was administered intracerebroventricularly (icv) shortly before application of VCS or infusion of agents that facilitate lordosis.
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Animals and surgeries
We used a total of 194 Sprague–Dawley female rats (240–280 g body weight), bred in our colony in Tlaxcala, Mexico. They were maintained under controlled temperature (23 ± 2 °C) and light conditions (14:10 L:D) with Purina rat chow and water provided ad libitum. Rats were bilaterally ovx under anesthesia with xylazine (4 mg/kg) and ketamine (80 mg/kg) and housed in acrylic cages in groups of four. Two weeks after ovariectomy, the females were anesthetized with xylazine and ketamine, placed in a Kopf
Effect of tamoxifen on estrous behavior induced by P and 5α- and 5β-reduced progestins
This experiment was performed to assess the participation of ERs in estrous behavior induced by progestins with a variety of structures. One week after cannula placement, all females were injected s.c. with 5 μg of E2B. At 39.5 h after E2B either tamoxifen or oil vehicle was infused, and 40 h after E2B progestins were infused. All compounds were infused icv in a volume of 1 μl delivered through a plastic catheter (Clay Adams; PE 10 No. 7401) fitted to a Hamilton syringe (10 μl) inserted into the
Effect of tamoxifen on estrous behavior induced by GnRH and leptin
This experiment was performed to assess the participation of ER in estrous behavior induced by GnRH and leptin. One week after cannula placement, all females were injected s.c. with 5 μg of E2B and 39.5 h later received an icv infusion of vehicle (oil) followed at 40 h by icv GnRH or leptin as described in Experiment 1. GnRH was dissolved in distilled water and injected at a concentration of 50 ng/1 μl (n = 9). Leptin (1 mg) was initially dissolved in 111 μl of Tris (10 mM, pH = 8). From this original
Effect of tamoxifen on estrous behavior induced by PGE2 and VCS
This experiment was performed to assess the participation of ERs in estrous behavior induced by PGE2 and VCS. One week after cannula placement, all females in the PGE2 groups were injected s.c. with 5 μg of E2B, and 39.5 h later, some rats received vehicle (oil, n = 8) or tamoxifen (n = 9). At 40 h after E2B, rats received icv infusions of PGE2 as described in Experiment 1. PGE2 was injected at a concentration of 1 μg/1 μl of saline. Forty hours after E2B injection and 30 min after infusion of vehicle
Effect of ICI, a pure ER antagonist, on estrous behavior induced by P and GnRH
This experiment was conducted to confirm that estrous behavior induced by P and GnRH, which have similar capabilities to induce lordosis in estrogen-primed female rats, is through the activation of ERs. We used the pure ER antagonist, ICI, because it has little or no agonist activity and competitively inhibits estradiol binding to the ER with a binding affinity that is 89% that of estradiol (Wakeling and Bowler, 1987). One week after cannula placement, all females were injected s.c. with 5 μg of
Testing procedures
Tests for sexual behaviors (receptivity and proceptivity) were conducted by placing females in a circular Plexiglas arena (53 cm diameter) with a sexually active male. Tests were conducted during the dark phase of the light–dark cycle. The lordosis quotient [LQ = (number of lordosis / 10 mounts) × 100] was used to assess receptive behavior. Proceptivity was evaluated by determining the incidence of hopping, darting and ear-wiggling during the entire receptivity test. We considered an animal proceptive
Verification of injection sites
One day after the behavioral test, rats were deeply anesthetized with ether, and 1% methylene blue was administered through the cannula. The brain was removed and sectioned in the transverse plane to check the cannula position in the lateral ventricle. Any rat whose cannula was not in the ventricle was discarded from the study.
Statistical analysis
The effect of tamoxifen on the behavioral action of hormones and VCS was assessed by comparing the LQs obtained with these agents alone versus those obtained when tamoxifen was also administered. To compare the effects of tamoxifen as a function of the time after hormone and VCS administration, we used a Friedman two-way (F) test. Because the distribution of LQ values in some groups was not normal, a Wilcoxon–Mann–Whitney U test was used to compare two independent groups at the different times
Tamoxifen reduces lordosis and proceptivity induced by P and most of its 5α- and 5β-reduced metabolites
The Friedman test shows statistically significant differences among groups that received progestin alone or progestin plus tamoxifen at the three times tested (P, F = 21, df = 5, p < 0.001; 5α-DHP, F = 31, df = 5, p < 0.001; 5β-DHP, F = 12.7, df = 5, p < 0.02). In females administered P, tamoxifen inhibited lordosis significantly at 120 (p < 0.01) and 240 min (p < 0.01) after progestin injection. Tamoxifen significantly decreased LQ scores in animals infused with 5α-DHP at 60 (p < 0.05), 120 (p < 0.01) and 240 min (p <
Discussion
Estrous behavior in ovx rats, as in nearly all mammalian species, can be stimulated by repeated administration of low doses of estrogens alone (E2, estrone, estriol; Beyer et al., 1982, Larsson et al., 1976) for several days (e.g., 5 to 7). This process operates in vivo in conditions of persistent estrus induced by age or by continuous illumination in rats (Takeo, 1984). However, in gonadally intact rats undergoing natural estrous cycles, estrous behavior is regulated in its level and duration
Acknowledgments
The authors gratefully acknowledge the technical assistance of Guadalupe Domínguez-López. This work was supported by CONACYT/134291 and by CACyPI UAT-2014.
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