Membrane-initiated estradiol signaling regulating sexual receptivity

Abstract

Estradiol has profound actions on the structure and function of the nervous system. In addition to nuclear actions that directly modulate gene expression, the idea that estradiol can rapidly activate cell signaling by binding to membrane estrogen receptors (mERs) has emerged. Even the regulation of sexual receptivity, an action previously thought to be completely regulated by nuclear ERs, has been shown to have a membrane-initiated estradiol signaling (MIES) component. This highlighted the question of the nature of mERs. Several candidates have been proposed, ERα, ERβ, ER-X, GPR30 (G protein coupled estrogen receptor), and a receptor activated by a diphenylacrylamide compound, STX. Although each of these receptors has been shown to be active in specific assays, we present evidence for and against their participation in sexual receptivity by acting in the lordosis-regulating circuit. The initial MIES that activates the circuit is in the arcuate nucleus of the hypothalamus (ARH). Using both activation of μ-opioid receptors (MOR) in the medial preoptic nucleus and lordosis behavior, we document that both ERα and the STX-receptor participate in the required MIES. ERα and the STX-receptor activation of cell signaling are dependent on the transactivation of type 1 metabotropic glutamate receptors (mGluR1a) that augment progesterone synthesis in astrocytes and protein kinase C (PKC) in ARH neurons. While estradiol-induced sexual receptivity does not depend on neuroprogesterone, proceptive behaviors do. Moreover, the ERα and the STX-receptor activation of medial preoptic MORs and augmentation of lordosis were sensitive to mGluR1a blockade. These observations suggest a common mechanism through which mERs are coupled to intracellular signaling cascades, not just in regulating reproduction, but in actions throughout the neuraxis including the cortex, hippocampus, striatum, and dorsal root ganglias.

Keywords: STX; cell signaling; estrogen receptor; lordosis behavior; neuroprogesterone; neurosteroids.

Figure 1

Effects of blocking the synthesis of progesterone and progesterone receptors in the CNS on sexual behavior. Blocking either progesterone synthesis (A,B) with aminoglutethimide (AGT) or trilostane (TRI) or activation of progesterone receptors (C) with RU486 reduces expression of proceptive behaviors, but has no effect on expression of lordosis in OVX/ADX rats treated every 4 days with 10 μg 17β-estradiol benzoate (EB) and then 1 h before the test with EB and drug. Animals were tested 53–56 h after the initial EB injection for sexual receptivity, as measured by lordosis quotient (LQ; Beach, ; Beach and LeBoeuf, 1967) and expression of proceptive behaviors (proceptivity scale; Tennent et al., 1980). Treatments to block progesterone synthesis or progesterone receptors were delivered subcutaneously and started just before the EB treatment and on the following mornings before testing. AGT (10 mg per treatment) blocks P450 side chain cleavage that converts cholesterol to pregnenolone (A); TRI blocks the enzyme 3β-hydroxysteroid dehydrogenase (16.5 mg per treatment; 3β-HSD) which converts pregnenolone to progesterone (B). RU486 (5 mg per treatment) is a progesterone receptor antagonist (C). Data are means ± SEM of 12 animals. * Represents significantly less than control treatment within behavior group as determined by Mann–Whitney, where p > 0.05 (from Micevych et al., 2008).

Figure 2

Comparison of estradiol with STX-induced μ-opioid receptor (MOR) in the medial preoptic nucleus (MPN). Estradiol, STX, LY 367,385, and control (aCSF) were injected into the arcuate nucleus of the hypothalamus of OVX rats and then transcardially perfused 30 min later with chilled 0.9% saline followed by 4% paraformaldehyde in Sorenson’s buffer. Sections from the arcuate nucleus (ARH) were processed for MOR internalization using rabbit primary antibodies directed against MOR (1:24,000; Neuromics). Histogram illustrates the ability of STX to induce MOR internalization similar to EB, as measured by immunofluorescence staining intensity in the MPN. Blocking mGluR1a with the antagonist LY 367,385 attenuates STX-induced MOR internalization. * Represents p < 0.05 compared to control as determined by one-way ANOVA.

Figure 3

Schematic representation of estradiol’s regulation of mER trafficking. Upon activation by a ligand (E2), membrane ER is internalized and the agonist-receptor complex is phosphorylated by G protein coupled receptor kinases (GIRKs). β-arrestins are attached to the receptor along with adaptor/scaffolding proteins (e.g., caveolin) and rapidly internalized into early endosomes. At this point, the receptor is either dissociated from its agonist and recycled back to the plasma membrane or degraded by fusing with lysosomes. Abbreviations: E2, estradiol; ER, estrogen receptor; CAV, caveolin; mGluR1, metabotropic glutamate receptor 1; PKCθ, protein kinase Cθ.

Figure 4

Estradiol-induced μ-opioid receptor (MOR) internalization in the medial preoptic nucleus (MPN) is dependent on protein kinase C θ (PKCθ) activation in the arcuate nucleus of the hypothalamus (ARH). This graph represents two experiments: animals infused with a PKC inhibitor, bisindolylmaleimide (BIS; 50 nmol, gray bars), and animals infused with a PKC activator, phorbol 12,13-dibutyrate (PDBu; 25 nmol, black bars). Animals sacrificed 30 min after EB injections displayed a significant increase in MOR internalization compared to oil injected animals. Antagonizing with BIS attenuated this EB-induced MOR internalization in the MPN. PDBu did not increase the level of internalization above that seen in EB-only treated animals. However the PKC activator did induced MOR internalization in the absence of EB treatment suggesting that estradiol and PDBu act through the same signaling pathway. The mGluR1 antagonist (LY367385) and agonist (DHPG) were also infused in combination with BIS or PDBu to determine whether PKC activation was downstream of ER/mGluR1 signaling. These data correlate with behavioral data that show that rats treated with BIS before EB were less receptive than aCSF-treated, EB-primed rats, and that PDBu did not induce lordosis behavior in animals that were not treated with a sub-behavioral dose of EB (2 μg; Dewing et al., ; Yildirim et al., 2008). * = statistical significance at the p < 0.05 level compared with aCSF + oil group as determined by two-way ANOVA and post hoc analysis (from Dewing et al., 2008).