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date: 08 December 2019

Membrane-Initiated Estradiol Signaling in the Central Nervous System

Summary and Keywords

In the last two decades of the 20th century, key findings in the field of estrogen signaling completely changed our understanding of hormones: first, steroidogenesis was demonstrated in the CNS; second, a vast majority of cells in the nervous system were shown to have estrogen receptors; third, a second nuclear estrogen receptor (ERß) was cloned; and finally, “nuclear” receptors were shown to be present and functional in the cell membrane. Shortly thereafter, even more membrane estrogen receptors were discovered. Steroids (estrogens, in particular) began to be considered as neurotransmitters and their receptors were tethered to G protein-coupled receptor signaling cascades. In some parts of the brain, levels of steroids appeared to be independent of those found in the circulation and yet, circulating steroids had profound actions on the brain physiology. In this review, we discuss the interaction of peripheral and central estrogen action in the context of female reproduction—one of the best-studied aspects of steroid action. In addition to reviewing the evidence for steroidogenesis in the hypothalamus, we review membrane-localized nuclear receptors coupling to G protein-signaling cascades and the downstream physiological consequences for reproduction. We will also introduce newer work that demonstrates cell signaling for a common splice variant of estrogen receptor-α (ERα), and membrane action of neuroprogesterone in regulating estrogen positive feedback.

Keywords: estrogen, progesterone, membrane receptors, ERα, kisspeptin, ovulation, luteinizing hormone, gonadotropin-releasing hormone, sexual receptivity

Introduction

The model of steroids acting exclusively as ligand-gated transcription factors has been amended as our ability to interrogate cellular processes has become more sophisticated. We now understand that steroids (here we focus on the sex steroids estradiol and progesterone) can and do act on such a rapid timescale that they can be considered neurotransmitters, in addition to carrying out their classic nuclear actions. As might be expected by this shift in our understanding, we have also had to rethink synthesis of hormones. We now understand that sex hormones are synthesized not only in the gonads and associated glands, but also on demand in other locations, including the central nervous system (Reviewd in Fester & Rune 2015, Figure 1).

Membrane-Initiated Estradiol Signaling in the Central Nervous SystemClick to view larger

Figure 1. Steroidogenesis in the brain. Neurons and astrocytes express all enzymes needed for estrogen synthesis. Steroidogenesis requires the transport of cholesterol to the mitochondrial matrix where the synthesis of pregnenolone from cholesterol is catalyzed by the cytochrome P450 side-chain cleavage enzyme (P450scc, CYP11A1). Astrocytes appear to be extremely active in this process. Pregnenolone is then converted to progesterone by 3β‎-hydroxysteroid dehydrogenase (3β‎-HSD), which is converted to androgens via cytochrome P450 17a-hydroxylase (P45017a) and 17 β‎-hydroxysteroid dehydrogenase (17 β‎ -HSD). The conversion of E2 is catalyzed by aromatase (P450Arom, CYP19A1). Other cell types in the brain do not appear to have the capacity to synthesize estrogens from cholesterol. Reviewed in Fester and Rune (2015).

In this manner, steroids such as estradiol can affect central control of reproductive physiology (sexual behavior, ovulation, etc.) as well as a variety of seemingly non-reproductive physiological functions including learning and memory, energy balance, and thermoregulation. Steroid levels shift over multiple different timelines: over an animal’s lifetime (early postnatal surges, puberty, and reproductive senescence), over the course of days (along the estrous or menstrual cycle), and daily (circadian changes); and now we know that along with these changes steroid actions can occur over seconds to minutes (rapid steroid synthesis, and neurotransmitter-like actions). The effects of sex steroids observed within minutes have led researchers to focus their studies on the mechanisms of these “rapid” steroid actions. The results of these have unveiled an array of steroid receptors located on the membrane of cells, as opposed to the classic view of receptors isolated in the nucleus. Beginning at the membrane, steroids can elicit intracellular signaling pathways that result in changes such as calcium influx and release of intracellular stores of calcium, kinase activation, and protein translation—all without the need for transcriptional changes. However, these membrane-initiated signaling cascades can eventually result in gene transcription through activation of cyclic AMP response-element binding (CREB), thus all steroid signaling can ultimately affect DNA transcription, mRNA translation, and protein synthesis.

Mechanisms of Estrogen Membrane Signaling (EMS)

Membrane-Initiated Estradiol Signaling in the Central Nervous SystemClick to view larger

Figure 2. Proposed schema of estradiol (E2) signaling in neurons: (A) Classic E2 activation of ERα‎ in the nucleus to activate gene transcription via estrogen-response elements (EREs). More recent results reveal that E2 activates membrane-associated ERs (mERs). (B) E2 binds to ERα‎ localized at the neuronal (plasma) membrane, transactivating group 1 metabotropic glutamate receptor (mGluR1a) signaling. Activation of mGluR1a causes Gα‎q activation of phospholipase C (PLC) that catalyzes the hydrolysis of membrane-bound phosphatidylinositol 4,5-biphosphate (PIP2) to inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG). IP3 mediates the release of intracellular (endoplasmic reticulum) Ca2+ stores and DAG activates protein kinase C (PKC) and downstream MAPK-induced phosphorylation of CREB. (C) The Gq-mER appears to directly activate PLC, which also generates IP3 and DAG to activate PLC. IP3 releases intracellular Ca2+ stores, and DAG activates protein kinase C theta (PKCθ‎), which activates adenylyl cyclase (AC). The generation of cAMP activates PKA, which can rapidly modulate the coupling of various receptors (e.g., GABAB and mu-opioid (μ‎) receptors). Both mER pathways can ultimately signal to the nucleus, activating cAMP-responsive element binding protein (CREB)—regulating transcription. (D) Another ER is GPER, which is found either on the cell membrane or the endoplasmic reticulum. Activation of the GPER induces the release of intracellular Ca2+. Finally, (E) a splice variant of ERα‎, ERαΔ4‎, transactivates the inhibitory type II mGluRs, mGluR2/3. Activation of this signaling complex inhibits AC, lowering cAMP levels, which can lead to inhibition of L-type voltage-gated Ca2+ channels (modified and updated from P. E. Micevych & Kelly, 2012; Qiu et al., 2003).

Hydrophobic steroids can easily penetrate lipid-soluble cell membranes and bind to receptors in the cell nucleus (Figure 2). To date, two classic “nuclear” estrogen receptors have been discovered, ERα‎ and ERβ‎. Once bound with an estrogen (estrone, estradiol, or estriol), the estrogen receptor dimerizes and associates with estrogen response element (EREs), AP-1 (activating protein-1), or SP-1 (stimulating protein-1) sites in the regulatory regions of responsive genes. This type of signaling is called “classical,” or “nuclear” and elicits unique, sometimes overlapping, patterns of gene transcriptional changes (Gaub, Bellard, Scheuer, Chambon, & Sassone-Corsi, 1990; Paech et al., 1997; Philips, Chalbos, & Rochefort, 1993; Umayahara et al., 1994; Webb et al., 1999). This was the consensus model of estrogen action, whose time course was hours to days. This timeframe was congruent with experiments in which estrogenic actions required a long time course, such as the induction of sexual receptivity. But estrogens also have actions that are more rapid, and these effects are evident within minutes. Because of their time course, these were designated “rapid actions” and do not require changes in gene expression, hence the term “non-genomic” is also used (Hayden-Hixson & Ferris, 1991; Kelly, Moss, & Dudley, 1976; McEwen & Alves, 1999; Szego & Davis, 1967). A somewhat surprising discovery was that the very same estrogen receptors, ERα‎ and ERβ‎, also participate in estrogen membrane signaling (EMS) (Figure 3). Overexpression experiments showed that approximately 3% of ERα‎ and 2% of ERβ‎ is trafficked to the cell membrane (Abraham, Todman, Korach, & Herbison, 2004; Razandi, Pedram, Greene, & Levin, 1999). The process of trafficking depends on two important factors: palmitoylation and caveolin binding.

Membrane-Initiated Estradiol Signaling in the Central Nervous SystemClick to view larger

Figure 3. Estrogen-induced ERα‎ trafficking to the membrane. Astrocytes and neurons derived from hypothalamic tissue were harvested and treated in vitro with vehicle (0 min) or estradiol (E2) for 5, 30, 60, or 120 minutes. Cells were processed using biotinylation to label membrane proteins and then processed for western blot. A. Example western blot using membrane proteins from hypothalamic astrocytes, showing ERα‎-immunoreactive bands at 66 kDa and 52 kDa (corresponding to ERα‎ and ERαΔ4‎, respectively). ERαΔ4‎ is the dominant ER, but both ERα‎ and ERαΔ4‎ are enriched in the membrane following estradiol exposure. B. Optical densities were corrected for corresponding values of ß-actin. Levels shown represent quantification of ERαΔ4‎ bands only, derived from hypothalamic astrocytes (black bars) and neurons (gray bars). Exposure to estradiol-induced trafficking of ERα‎ to the cell surface, with a maximum level observed at 30 minutes. Significant (p < 0.05) changes from baseline are indicated with * (astrocytes) or + (neurons). Images modified, with permission, from Bondar et al. (2009) and Dominguez and Micevych (2010).

Palmitoylation

In order for ERα‎ and ERβ‎, which are nuclear receptors, to be trafficked to the cell surface they must undergo palmitoylation, a modification that favors an association with the membrane. Palmitoylation is one of the most common post-translational modifications and involves the attachment of fatty acids (e.g., palmitic acid) to a protein to enhance its hydrophobicity (reviewed in Guan & Fierke, 2011; Smotrys & Linder, 2004). This alteration is often found in other membrane-localized proteins, such as postsynaptic density protein-95 kDa (PSD-95), which is commonly used as an identification marker for neural synapses. Palmitoylation is likely to be involved in intracellular trafficking of a number of nuclear receptors, androgen receptors (ARs), the progesterone receptor (PGR), and the glucocorticoid receptor (GR), in addition to ERα‎, ERβ‎. (Pedram et al., 2007). Indeed, a critical, highly conserved nine-amino acid palmitoylation motif in the ligand binding (E) domain has been identified in these receptors (Adlanmerini et al., 2014; Meitzen et al., 2013; Pedram et al., 2007; Pedram, Razandi, Lewis, Hammes, & Levin, 2014). Experimental mutation of cysteine 452 in the palmitoylation site eliminates receptor functioning on the cell membrane, while nuclear signaling remains intact, supporting the critical nature of palmitoylation to membrane association. Palmitoylation is mediated by DHHC enzymes (so named for a specific, conserved sequence motif). In the cases of ERα‎ and ERβ‎, palmitoylation occurs through the function of DHHC7 and DHHC21 in neurons (Meitzen et al., 2013; Pedram et al., 2012; Tonn Eiseniger, Woolfrey, Swanson, Schnell, Meitzen, Dell’Acqua & Mermelstein, 2018).

Caveolins

The second component of trafficking ERs to the membrane is the set of scaffolding proteins called caveolins (Cavs). Cavs are proteins that mediate binding of other proteins and receptors to form functional associations (Francesconi, Kumari, & Zukin, 2009; Patel, Murray, & Insel, 2008; Takayasu et al., 2010). There are three Cavs (named Cav1-3) that are expressed in neurons (Boulware, Kordasiewicz, & Mermelstein, 2007). Cav1 and Cav3 can associate with ERα‎ and ERβ‎ (Christensen & Micevych, 2012; Meitzen, Britson, Tuomela, & Mermelstein, 2019); reviewed in P. E. Micevych et al., 2017). Knockdown of Cav1 in the arcuate nucleus of the hypothalamus (ARH) prevents trafficking of ERα‎ to the cell membrane and abrogates estrogen actions on the induction of sexual receptivity. The type of Cav that associates with the ER also determines the downstream signaling initiated by EMS. It is worth noting that this association is not unique to estrogen receptors. Androgen receptors (ARs) also require Cav1 for membrane localization (Deng et al., 2017).

ER, mGluR, and Cav Associations

Several questions arise about membrane estrogen receptors (ERs). First, how do ERs, which are nuclear transcription factors, signal when on the cell membrane? Second, if the number of ERs is so low on the membrane, are these ERs physiologically relevant? The answer to both questions relies on caveolin (Cav) proteins. Cavs not only target ERα‎ and ERβ‎ to the membrane, they also organize downstream signaling by associating steroid receptors with G protein-coupled receptors, which amplifies estrogen membrane signaling (EMS). While many candidates have been proposed as ER co-receptors, the most conclusive evidence is that membrane ERα‎ and ERβ‎ signal through distinct groups of metabotropic glutamate receptors (mGluRs; Boulware et al., 2005). In cells of the nervous system, ERs linked with Cav1 associate with type I mGluR (i.e., mGuR1a), initiating excitatory signaling (Boulware et al., 2007). Conversely, association with Cav3 leads to activation of type II mGluRs—producing inhibition. Thus, various Cavs allow the same ER to excite or inhibit neurons and astrocytes via associations with different mGluRs. For example, we recently determined that a common alternatively spliced ERα‎ mRNA in the brain (ERαΔ‎4, missing exon 4 [Pfeffer, Fecarotta, Castagnetta, & Vidali, 1993; Skipper, Young, Bergeron, Tetzlaff, Osborn, & Crews, 1993]) is trafficked to the cell membrane in association with Cav3. ERαΔ‎4 associates with and transduces mGluR2 leading to an inhibition of adenylyl cyclase (Wong, Scott, Johnson, Mohr, Mittelman-Smith & Micevych, in revision). While it has not been formally determined, several inhibitory actions that involve ERα‎ and mGluR2/3 may be mediated by ERαΔ‎4 (e.g., V. Chaban, Li, McDonald, Rapkin, & Micevych, 2011; Mermelstein, Becker, & Surmeister, 1996).

ERαΔ‎4 has been observed by the present authors and others during biotinylation experiments (Figure 3) that demonstrate ERα‎ on the cell surface in hypothalamic neurons and astrocytes (Bondar, Kuo, Hamid, & Micevych, 2009; Dominguez & Micevych, 2010; Dominguez, Dewing, Kuo, & Micevych, 2013; Gorosito, Lorenzo, & Cambiasso, 2008). This ER is missing exon 4 and is enriched in cell membranes. Our experiments indicate that ERαΔ‎4 is trafficked to the membrane with Cav3 and transactivates mGluR2, leading to inhibition of cAMP. We therefore hypothesize that ERαΔ‎4 may be responsible for the observed estrogen inhibition of L-type voltage-gated calcium channels (VGCC) in hippocampal neurons and dorsal root ganglion neurons (V. V. Chaban, Mayer, Ennes, & Micevych, 2003; Mermelstein et al., 1996). ERαΔ‎4 is expressed in the ARH, but does not appear to be responsible for the well-known estrogenic inhibition of kisspeptin expression (Wong et al., in revision). As we have identified both full-length ERα‎ and ERαΔ‎4 on membranes of the same cells, an interesting question is how cells determine which signaling cascade will predominate. Further studies must be done to answer this question and determine a physiological role for ERαΔ‎4.

Membrane ER Cell Biology

Resting (baseline) levels of estrogen receptors (ERs) on the membrane are very low (<5% of total cellular ER) but estrogen itself affects levels of membrane receptors. In fact, one of the main factors influencing the trafficking of ERα‎ to the membrane appears to be estradiol. Initially, exposure to estradiol induces trafficking to the membrane (Dominguez & Micevych, 2010; Figure 3). With time, however, receptor internalization occurs, causing a subsequent drop in membrane ERα‎ levels (Bondar et al., 2009). Internalization is a universal phenomenon displayed by membrane receptors to limit stimulation by removing the receptor from the membrane and to remove the ligand so that the receptor can be restimulated. The process of internalization is triggered by a ligand binding to its cognate receptor, leading to phosphorylation of the receptor by G protein-coupled receptor kinase 2 (GRK2) and the binding of β‎-arrestin-1, which leads to clathrin-mediated internalization into early endosomes. This pathway has been described for ERα‎ and mGluR1a in neurons and astrocytes (Bondar et al., 2009; Dominguez, Hu, Zhou, & Baudry, 2009; Dominguez & Micevych, 2010). In the early stages of ERα‎ internalization, receptors are recycled back to the membrane where they can be stimulated anew. Indeed, as with estradiol-induced ERα‎ trafficking, androgens increase levels of androgen receptors (ARs) on the membrane through activation of protein kinase C (PKC) (Deng et al., 2017; Dominguez & Micevych, 2010; Wong, Abrams, & Micevych, 2015). Eventually, the ERα‎-mGluR1a complex is degraded in lysosomes. This down-regulation of the receptor limits the duration of receptor stimulation. For membrane ERα‎, the down-regulation is an important way to limit signaling in the temporal domain (i.e., EMS; reviewed in P. Micevych & Sinchak, 2013). This process has also been described for androgen receptors and may be a general phenomenon of steroid receptor signaling from the membrane.

Other Membrane Estrogen Receptors

As with most signaling systems, several distinct receptors have been identified for estrogens in addition to ERα‎ and ERβ‎ (Rudolph et al., 2016). These include: GPER—an orphan G protein-coupled receptor that binds estrogen (aka GPR30; Revankar, Cimino, Sklar, Arterburn, & Prossnitz, 2005); reviewed in Filardo & Thomas, 2005, and an STX-activated Gq-mER (Figure 2; Qiu et al., 2003; reviewed in P. E. Micevych & Kelly, 2012). While there is substantial evidence for the presence and physiological action associated with GPER activation, some questions remain about its localization (Vrtacnik, Ostanek, Mencej-Bedrač, & Marc, 2014). Some investigators find GPER on the cell membrane while others suggest that it may be localized to the smooth endoplasmic reticulum, which is congruent with the release of intracellular calcium seen with GPER activation. Over the years, questions have been raised about GPER as a membrane estrogen receptor (ER) (Ahola, Alkio, Manninen, & Ylikomi, 2002; Cheng, Quinn, Graeber, & Filardo, 2011; Cheng, Graeber, Quinn, & Filardo, 2011; Pedram, Razandi, & Levin, 2006; Prossnitz & Barton, 2011). There have been suggestions that GPER may not act on its own, but rather interacts with another receptor to induce signaling (Gao, Ma, Ostmann, & Das, 2011). A candidate for such a modifier is another splice variant of ERα‎, ERα‎36 (Kang et al., 2010; Pelekanou et al., 2016) that only expresses exons 2–6 of ERα‎ DNA, indicating that ERα‎36 lacks transcriptional activation domains AF-1 and AF-2 (Z. Y. Wang & Yin, 2015). Although ERα‎36 is present in the cytoplasm and membrane, it inhibits transcriptional activity of the full-length ERα‎ (Z. Wang et al., 2006). ERα‎36 and GPER appear to interact with each other: overexpression of GPER induces ERα‎36; the selective GPER agonist, G-1 activates ERα‎36-mediated transcriptional events; and blocking ERα‎-36 prevents GPER signaling induced by its specific ligand, G-1 (Kang et al., 2010).

Another putative membrane ER is Gq-mER, which is blocked by ICI 182,780, the so-called universal ER antagonist, and is selectively activated by a tamoxifen derivative, STX (Qiu et al., 2003; Figure 2). STX has been shown to affect, in an estrogen-mimetic way, a number of physiologic functions including energy balance, lordosis behavior, and estrogen-positive feedback underlying the luteinizing hormone surge [reviewed in (Christensen & Micevych, 2013; A. W. Smith, Ronnekleiv, & Kelly, 2014; Vail & Roepke, 2018)]. In our own experiments, STX rapidly increases intracellular calcium levels, leading to the induction of progesterone synthesis in female hypothalamic astrocytes — mimicking estrogen actions (Kuo, Hamid, Bondar, Prossnitz, & Micevych, 2010). While pharmacological evidence in support of a physiological role for Gq-mER is mounting, definitive questions cannot be addressed until the receptor has been identified and cloned.

EMS Intracellular Signaling Pathways

Through both in vitro and in vivo studies, a great deal has been learned about the intracellular signaling pathways associated with estrogen membrane signaling (EMS) [recently reviewed in (P. E. Micevych et al., 2017), see Figure 2]. Since estrogen receptors (ERs) transactivate mGluRs, it is not surprising that their signaling cascades are those activated by glutamatergic signaling. Like the effects of estrogen, these actions are pleiotropic, ranging from regulating calcium to ultimately modifying transcription (Mani, Mermelstein, Tetel, & Anesetti, 2012; P. E. Micevych et al., 2017). These actions vary according to which cells are examined. In the brain, we have studied EMS in hypothalamic neurons and astrocytes. In both cell types, positive actions of estrogen were mediated through full-length ERα‎ transactivating mGluR1a. In astrocytes, estrogen rapidly induces progesterone synthesis (Figure 4) through a signaling cascade that involves the release of intracellular calcium stores via a PLC-IP3mediated mechanism, leading to activation of a calcium-dependent adenylyl cyclase, phosphorylation of protein kinase A (PKA), and activation of cholesterol transport proteins, TSPO and StAR (Chen, Kuo, Wong, & Micevych, 2014). The centrality of the increase of intracellular calcium was established by using thapsigargin to release IP3-sensitve stores, which mimicked stimulation with estrogen (P. E. Micevych et al., 2007). In neurons, rather than activating PKA, EMS initiates protein kinase C (PKC) signaling (Dewing, Christensen, Bondar, & Micevych, 2008), which is related to an estrogen-induced opioid inhibition of sexual receptivity (Dewing et al., 2007; reviewed in P. E. Micevych & Mermelstein, 2008).

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Figure 4. Anterior hypothalamic kisspeptin (kiss) signaling underlies ovulation. Estradiol (E2) signaling in kisspeptin neurons occurs via activation of membrane receptors and classical nuclear receptors. Activation of membrane ERα‎ leads to increased kiss mRNA and protein. Activation of nuclear ERα‎ causes up-regulation of progesterone (P4) receptors (PGR), which translocate to the cell membrane. Rapid PGR activation leads to activation of Src, a non-receptor tyrosine kinase, which activates Erk 1/2, leading to higher levels of kiss mRNA. Therefore, presence of P4 augments the already stimulatory effects of estradiol. The source of this P4 appears to be neuroprogesterone (neuroP), synthesized locally by hypothalamic astrocytes following stimulation of membrane-localized ERα‎. Once estradiol and P4 integrate signaling in kiss neurons, kisspeptin is released, leading to GnRH and LH surges, culminating in ovulation.

EMS-Modulated Circuits

While research in the field of steroid membrane signaling is still young and its mechanisms less well understood than the nuclear pathway, it is becoming apparent that nearly all physiological processes that are affected by estrogens contain a membrane signaling component. Estrogen membrane signaling (EMS), for example, is not limited to reproduction, but has been found to be an integral part of the formation of synaptic spines in the hippocampus, nucleus accumbens, and hypothalamus.

The hippocampus has been almost as well studied as the hypothalamus in terms of steroidogenesis (Hojo et al., 2004; MacLusky, Walters, Clark, & Toran-Allerand, 1994; Prange-Kiel, Wehrenberg, Jarry, & Rune, 2003; Roselli, Horton, & Resko, 1985; Tabatadze, Huang, May, Jain, & Woolley, 2015) and estrogenic actions, in particular on synaptic potentiation. Estradiol appears to act via pre- and postsynaptic mechanisms in both the male and female rat hippocampus to induce synaptic potentiation (Kramar et al., 2009; Oberlander & Woolley, 2016). Interestingly, a number of estrogen receptors (ERα‎, ERβ‎ and GPER) mediate these actions, but they are sexually dimorphic (Oberlander & Woolley, 2016). Presynaptically, estradiol increases glutamate release via ERα‎ in males but, in females, this is mediated by ERβ‎. In males, estradiol postsynaptic actions require ERβ‎ to increase miniature excitatory postsynaptic currents (mEPSC), sensitivity to glutamate, and glutamate-evoked calcium transients. In females, these effects are dependent on GPER. One of the important lessons of these studies is that estradiol responsiveness varies from synapse to synapse — even among synapses on the same dendrite, indicating an extraordinary specificity of estradiol modulation of neuronal activity.

Sexual Receptivity

In many species, sexually receptive females assume a posture in which the spine is in a lordotic position (arched back), which allows a male to intromit leading to fertilization of an ovulated egg. Thus, lordosis is operationally defined (Beach 1976) and occurs in rodents on the afternoon of proestrous. In the 20th century, a number of investigators demonstrated that lordosis behavior requires appropriate steroid concentrations that would stimulate specific brain circuits (e.g., Blaustein, Finkbohner, & Delville, 1987; Fadem, Barfield, & Whalen, 1979; Feder, Blaustein, & Nock, 1979; Johnston & Davidson, 1979; Lisk & Barfield, 1975; reviewed in Pfaff, Sakuma, Ko, Lee, & Easton, 2005). Thus, lordosis is the characteristic sexual behavior observed in female rodents that are appropriately primed with hormones (reviewed in P. Micevych & Christensen, 2012b). Sexual receptivity is typically expressed 48 hours after estradiol treatment in ovariectomized rodents and has been shown to be dependent on estradiol-induced transcription at nuclear ERs augmented by membrane-initiated signaling (Vasudevan, Kow, & Pfaff, 2005). However, immediately following estradiol treatment, female rodents are not sexually receptive. This phenomenon has been observed for decades, but studied only relatively recently. This refractory period is undoubtedly due in part to the time it takes for novel protein transcription to occur (over the course of hours to days; see Parsons, Rainbow, Pfaff, & McEwen [1982]). It is now known that this period also reflects the early inhibitory actions of estradiol. An active inhibition of lordosis is observed immediately (within minutes) of estradiol administration. Multiple studies have provided an understanding of a comprehensive pathway in which activation of cells in the ARH leads to lordosis behavior. Counterintuitively, the effect of estrogen on lordosis behavior is biphasic: first, there is an initial inhibition of behavior; then lordosis is activated approximately 20 hours after estrogen stimulation. Neurons expressing neuropeptide Y (NPY) respond to estradiol and release NPY locally within the ARH. This stimulates nearby proopiomelanocortin/β‎-endorphin (POMC/β‎-END) neurons, which express NPY-Y1 and GABAB receptors (Figure 5; Mills, Sohn, & Micevych, 2004; Sinchak et al., 2013). In turn, β‎-END is released from ARH neurons, whose terminals are located in the medial preoptic nucleus (MPN), stimulating the internalization of mu opioid receptors (MOR). MOR neurons innervate neurons in the ventromedial nucleus (VMN), which in turn control hypothalamic output governing lordosis behavior. Internalization of MOR causes active inhibition of lordosis behavior, lasting for approximately 20 hours. At that point, progesterone relieves this inhibition and allows for expression of sexual receptivity (Sinchak & Micevych, 2001). Therefore, the inhibitory cascade initiated by estradiol is rapid in onset and relatively short-lived.

Cellular Mechanisms Governing Sexual Receptivity

Within NPY neurons, a rapid signaling cascade is initiated by estradiol binding to membrane-associated ERα‎ (Figure 5). ERα‎ transactivates mGluR1a, leading to a rise in free intracellular calcium (Dewing et al., 2008; Dominguez et al., 2013; P. E. Micevych, Rissman, Gustafsson, & Sinchak, 2003). The functional association of mGluR1a is necessary and highlighted by experiments in which direct activation of mGluR1a or PKC in the absence of estradiol leads to quantifiable µ-opioid receptor (MOR) internalization, a reliable marker for β‎-endorphin release in the medial preoptic nucleus necessary for the display of lordosis (Dewing et al., 2007). Internalization is a proxy for activation of receptors, as mentioned for ERα‎ and ERαΔ‎4.

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Figure 5. EMS signaling in NPY neurons leads to lordosis behavior. Neurons in the arcuate nucleus of the hypothalamus (ARH) expressing neuropeptide Y (NPY) express membrane ERα‎. Activation of this receptor leads to transactivation of metabotropic glutamate receptor 1a (mGluR1a) and activation of a protein kinase C (PKC) pathway, culminating in local NPY release. ARH proopiomelanocortin/ß-endorphin (POMC/ß-END) neurons expressing the Y1 receptor are activated by NPY, leading to release of ß-END in the medial preoptic nucleus. Ultimately, this will lead to lordosis behavior.

Estrogenic Regulation of Morphological Plasticity

Much of the morphological plasticity of the central nervous system (CNS) is due to dendritic changes, specifically the shape and number of dendritic spines. These spines are small protrusions from the dendritic shaft that receive synaptic input, usually excitatory. Dendritic spines appear in many neurons and have multiple morphologies classified as filopodial, thin, mushroom-shaped, and stubby, which indicate their functional state. The spine’s morphology can change rapidly through both activity-dependent and -independent processes (reviewed in Rochefort & Konnerth, 2012). Several factors influence spine morphology including the arrangement of the cytoskeleton (Honkura, Matsuzaki, Noguchi, Ellis-Davies, & Kasai, 2008). Due to their importance as sites of synaptic interactions and their labile nature, investigators have studied dendritic spines to observe how steroids alter brain function.

Since the classic observations of estrogenic alterations of synapses in the ARH (Matsumoto & Arai, 1979, 1981), the spinal cord nucleus of the bulbocavernosus (SNB; Forger & Breedlove, 1987; Kurz, Senglaub, & Arnold, 1986), and hippocampus (Woolley & McEwen, 1992, 1993), it has been become clear that sex-steroid hormones affect brain structure. Experiments demonstrate that many steroid actions target dendrite length, spine density, morphology, and gap junctions (Christensen, Dewing, & Micevych, 2011; Frankfurt, Gould, Woolley, & McEwen, 1990; Gould Woolley, Frankfurt, & McEwen, 1990; Griffin & Flanagan-Cato, 2008; Griffin, Ferri-Kolwicz, Reyes, Van Bockstaele, & Flanagan-Cato, 2010; Gross et al., 2016; Leranth, Shanabrough, & Horvath, 2000; Matsumoto, Arnold, Zampighi, & Micevych, 1988; Matsumoto, Micevych, & Arnold, 1988; Staffend, Loftus, & Meisel, 2011; Staffend, Hedges, Chemel, Watts, & Meisel, 2014). Although the most dramatic effects occur during development, significant steroid regulation of dendritic structure also occurs in adulthood. In the CA1 region of the hippocampus, the density of spines and synapses fluctuates during the estrus cycle due to the actions of estradiol and progesterone (Woolley & McEwen, 1993). Recent work focusing on estradiol actions on hippocampal spines in relation to cognitive function has been reviewed (Luine & Frankfurt, 2012). However, sex steroids have been shown to modulate dendritic structure throughout the CNS (reviewed in Cooke & Woolley, 2005).

The first concerted efforts concentrated on spines in the hypothalamus. In particular, the ventromedial nucleus (VMH) was targeted. Here, estradiol increased spine density and dendritic branching mimicking changes during the estrous cycle (Calizo & Flanagan-Cato, 2000, 2002; Frankfurt et al., 1990; Madeira, Ferreira-Silva, & Paula-Barbosa, 2001; reviewed in Griffin & Flanagan-Cato, 2011). Our own work has focused on the ARH, a hypothalamic site important for estradiol actions on reproduction and energy balance, among other physiological processes. Estradiol treatment induces dendritic spines in ovariectomized rats (Christensen et al., 2011). Four hours of estradiol treatment is sufficient to increase ARH spine density, which remains stable for 48 hours. The newly formed spines were filopodial, a morphology considered immature and unstable (see Kasai, Matsuzaki, Noguchi, Yasumatsu, & Nakahara, 2003). With time, their morphology evolved and matured. In the ARH of our ovariectomized rats, mushroom-shaped spines appear approximately 20 hours after estradiol treatment. These mushroom-shaped spines are considered mature, stable, and functional. The spines contain the postsynaptic density composed of receptors and anchoring proteins that allow for efficient synaptic transmission (Holtmaat, Wilbrecht, Knott, Welker, & Svoboda, 2006; Knott, Holtmaat, Wilbrecht, Welker, & Svoboda, 2006; Matsuzaki et al., 2001; Yoshihara, De Roo, & Muller, 2009). This 20-hour window corresponds with the time course after estradiol treatment when lordosis can be activated by P4 treatment in OVX rats, suggesting that both spinogenesis and spine maturation are critical for regulating sexual receptivity. Indeed, preventing spine formation abrogates the ability of estradiol to induce sexual receptivity (lordosis; reviewed in Micevych & Christensen, 2012a).

EMS Mechanisms Regulating Spinogenesis

Spines have a core cytoskeleton composed of β‎-actin. The formation of spines requires the rearrangement of this core to deform the membrane. Because much of the actin in the brain is concentrated in spines, spinogenesis correlates with an observed increase in β‎-actin immunoreactivity. In the ARH, we inhibited β‎-actin polymerization with cytochalasin D, which prevented spinogenesis and sexual receptivity (Christensen et al., 2011). Actin remodeling is highly regulated by cofilin whose activity in turn is regulated by membrane-initiated signaling. Cofilin, an actin depolymerizing factor, is inactivated by phosphorylation. Estradiol increases cofilin phosphorylation (inactivation) within an hour of treatment. Moreover, antagonism of mGluR1a prevents this, indicating that EMS regulates cofilin activity. The deactivation of cofilin allows the establishment of new spines (Bamburg, McGough, & Ono, 1999; Meng et al., 2002). The stabilization and maturation of spines appears to require that estradiol increases the expression of genes involved in spine maturation. While we have examined the expression of various pre- and postsynaptic markers, the results have not allowed us to conclude whether estradiol is acting directly in the nucleus or through membrane-to-nuclear EMS (Rudolph et al., 2016). The two-step wiring hypothesis proposes that the newly formed filopodial spines are labile until stabilized by another stimulus (Srivastava et al., 2008; reviewed in Srivastava et al., 2011). We have not yet identified this critical stabilizing input in the hypothalamus. It is tempting to think that salient reproductive input (olfactory and/or tactile) may be the needed stimuli, but the time course does not align with maturation, which occurs before a mating encounter. Thus, the site/mechanism of estradiol action regulating spine maturation has not been resolved in the ARH.

The nucleus accumbens is another region that has been studied to understand estradiol regulation of spines. This nucleus is involved in processing motivation, reward, and positive reinforcement, and has been implicated in motivation for sexual behavior (Been, Hedges, Vialou, Nestler, & Meisel, 2013; Moore, Himmler, Teplitzky, Johnson, & Meisel, 2017; reviewed in P. E. Micevych & Meisel, 2017). The nucleus accumbens is divided into a shell and a core region, sometimes referred to as limbic and motor parts of the reward circuitry, respectively (reviewed in Pierce & Kumaresan, 2006). In the core, estradiol decreases spine density on medium spiny neurons, but in the shell estradiol increases spine density (Peterson, Mermelstein, & Meisel, 2015; Staffend et al., 2011; Staffend et al., 2014). These results point to an intriguing distinction that is a consequence of activating mGluR1a versus mGluR5 (Gross et al., 2016). Although both receptors are considered members of the group I mGluRs, they have distinct actions on dendritic spines. Thus, mGluR1a receptors coupled with mERα‎ increase spines in the accumbens shell, as they do in the hypothalamus (Christensen, Dewing, & Micevych (2011), while ERα‎-mGluR5 interactions reduce spines in the accumbens core and hippocampus (Huang & Woolley, 2012). These interesting findings point out that our understanding of the interactions of mERα‎ with various mGluR is far from complete and awaits further investigation.

EMS Modulation of Male Sexual Behavior

It has been known for decades that in males as in females, many of the actions of testosterone (aggression, nociception, learning and memory) are due to the aromatization to estradiol (reviewed in Cornil, Ball, & Balthazart, 2012; Laredo, Villalon Landeros, & Trainor, 2014). As in females, rapid actions of estradiol were demonstrated in male rats and Japanese quail (Cornil, Dalla, Papadopoulou-Daifoti, Baillien, & Balthazart, 2006; Cross & Roselli, 1999; Taziaux, Keller, Bakker, & Balthazart, 2007). These experiments demonstrated that rapid estradiol action affected the motivation for behavior rather than copulation (reviewed in Cornil, Ball, & Balthazart, 2015). In male birds, estradiol action on motivational behavior is mediated by ERβ‎ transactivating mGluR1a (Seredynski, Balthazart, Ball, & Cornil, 2015). The source of the estradiol, in male birds, is likely to be centrally produced estradiol, which stimulates membrane ERβ‎ and modulates male sexual motivation. As mentioned, the brain is capable of de novo synthesis of estradiol (Figure 1). Interestingly, such rapid estradiol actions on male motivation for sexual behavior have also been observed in rodents (Kaufman, Kelly, & Roselli, 2013). Currently, it is not clear whether the same ER-mGluR1a mechanism is involved in male rodents as in male birds and female rodents. However, GPER and Gq-mER do not appear to be involved in membrane-initiated estradiol signaling underlying rodent copulatory behaviors (Seredynski et al., 2015). In female rats, blocking steroidogenesis in the brain prevents proceptive but not receptive behaviors and these behaviors require neuroP and the classic, nuclear progesterone receptor (Micevych, Soma, & Sinchak, 2008; Micevych & Dewing, 2011).

Progesterone (P4)

Estrogen membrane signaling (EMS) acts in hypothalamic astrocytes to dramatically increase progesterone (P4) synthesis (Figure 4). This response is sexually differentiated and developmentally regulated. Only postpubertal female astrocytes respond to estradiol: neither prepubertal nor male astrocytes up-regulate P4 synthesis in response to estradiol stimulation (Kuo, Hamid, Bondar, Dewing, Clarkson, & Micevych, 2010). Blocking steroidogenesis in the hypothalamus of gonadally intact, cycling rats prevents the LH surge, indicating that local neuroprogesterone is a key component of the estrogen-positive feedback surge mechanism (Micevych & Sinchak, 2011). This P4 may act on neurons that express the neuropeptide kisspeptin in the hypothalamus (Figure 4). Kisspeptin neurons stimulate GnRH release from the hypothalamus but likely also integrate progesterone feedback to initiate the LH surge. Strong evidence for this was observed in an elegant study in which the progesterone receptor (PGR) was knocked out in kisspeptin neurons, preventing estrogen-positive feedback of the LH surge and ovulation (Stephens et al., 2015). This finding suggests that P4 activation of PGR in kisspeptin neurons mediates estrogen action in the LH surge. Initially, it was unknown how P4 acted in kisspeptin neurons but this response appears to be via P4 membrane-initiated signaling (PMS). While there is even less information about membrane P4 signaling compared to that of estradiol, we do know that progestins can also elicit rapid cellular responses. For example, in the CNS, progesterone has been found to rapidly affect neural responsiveness, reproduction, and neuroprotection (Bashour & Wray, 2012; Parsons, MacLusky, Krey, Pfaff, & McEwen, 1980; Petitti & Etgen, 1989; Schumacher, Colrini, Pfaff, & Mcewen, 1990; Sleiter et al., 2009; S. S. Smith, Waterhouse, Chapin, & Woodward, 1987; S. S. Smith, Waterhouse, & Woodward, 1987). With respect to hypothalamic control of reproduction, there are indications that PMS affects GnRH release (Ke & Ramirez, 1987; Sleiter et al., 2009). Indeed, superfusion of P4-3-BSA (a membrane-impermeable progesterone construct) stimulates GnRH release, giving a strong indication that membrane-associated receptors mediate P4 action in the GnRH pathway.

Mechanisms of Membrane P4 Signaling

As with estradiol, several progestin receptors and binding proteins have been found on cell membranes. However, given the critical nature of PGR in fertility and sexual behavior (e.g., Chappell & Levine, 2000; Stephens et al., 2015), and given the large role classical estrogen receptors (ERs) play in EMS, it appears likely that classical PGR on the membrane mediates at least some of the rapid effects observed with P4 stimulation. As with classical ERs, PGR contains a nine-amino acid motif, critical for palmitoylation, allowing trafficking to the cell membrane (Pedram et al., 2007). The palmitoylation sequence also allows binding with caveolin proteins which determine the association of ERs with mGluRs. Presently, however, there are no experimental results that support a PGR-mGluR signaling complex, as has been described for ERα‎ and ERβ‎. Future experiments will likely reveal whether such an mGluR association exists with PGR as well.

Recent evidence suggests a different mechanism for PGR. Several in vitro studies have pointed to a distinct role for membrane-localized PGR through association with Src proteins—a family of non-receptor tyrosine kinases (Boonyaratanakornkit et al., 2001; Boonyaratanakornkit et al., 2007; Migliaccio et al., 1998; Migliaccio et al., 2002; Mittelman-Smith, Wong, & Micevych, 2018). The physical association occurs at a proline-rich domain present in PGR that binds to the SH3 domain of Src kinase (Boonyaratanakornkit et al., 2001). Once activated, Src kinase phosphorylates downstream effectors such as MAPK/ERK.

The integration of estrogen and progestin signaling occurring in kisspeptin neurons is still being dissected. Results from our laboratory indicate that centrally produced P4 may augment the stimulatory effects of peripheral estradiol, leading to increased production and release of kisspeptin, discussed further in the next section “Kisspeptin System—Overall Picture” (Mittelman-Smith et al., 2018).

Kisspeptin System—Overall Picture

Ovulation occurs as a culmination of a series of events often described as a positive feedback loop. In this loop, estradiol released by growing ovarian follicles stimulates the hypothalamus, causing release of GnRH into the pituitary portal blood, resulting in an LH surge and ovulation. GnRH neurons themselves do not express ERα‎ (e.g., Herbison, Pape, Simonian, Skynner, & Sim, 2001; Hrabovszky et al., 2000; Shivers et al., 1983), though knockout studies in the mouse demonstrated ERα‎ to be the critical receptor mediating this positive feedback (Dubois et al., 2015; Wintermantel et al., 2006). Therefore, attention was focused on an “upstream population” of neurons that might mediate estradiol’s stimulatory effects on the GnRH surge. Neurons in the area of the hypothalamus called the RP3V, expressing the neuropeptide kisspeptin, have now been accepted as the primary site of estrogen action in the positive feedback mechanism governing the luteinizing hormone (LH) surge. The rationale for this involves the key findings listed in Table 1.

Table 1. Principal Findings Pointing to Kisspeptin Neurons as Mediators of Estradiol Positive Feedback on the LH Surge. For In-Depth Information about the Kisspeptin System. See Roa et al. (2009), Roseweir and Millar (2009), J. T. Smith, Popa, Clifton, Hoffman, and Steiner (2006).

Finding

Source

RP3V kisspeptin mRNA and protein are induced by estradiol stimulation, in vivo and in vitro.

(Mittelman-Smith et al., 2015; J. T. Smith, Cunningham, Rissman, & Steiner, 2005; J. T. Smith, 2013)

RP3V kisspeptin neurons express ERα‎, the necessary receptor for estrogen-positive feedback.

(J. T. Smith et al., 2005)

Kisspeptin neurons directly project to GnRH neurons, which express kisspeptin receptor (Kiss1R or GPR54).

(Wintermantel et al., 2006)

RP3V kisspeptin neurons express estradiol-inducible PGR in vivo and in vitro.

(Mittelman-Smith et al., 2015; Zhang et al., 2014)

PGR expression is critical to the LH surge.

(Chappell & Levine, 2000)

The neuropeptide kisspeptin is the most potent stimulator of GnRH neurons.

(e.g., Han et al., 2005)

EMS in the Kisspeptin System

While it is well established that estradiol induces kisspeptin mRNA and protein in the RP3V, intracellular signaling mechanisms governing this up-regulation are still being characterized. Our research supports EMS in this process: E-6-BSA (membrane-impermeable estradiol construct) stimulates kisspeptin mRNA in vitro to the same degree observed with free estradiol (Mittelman-Smith, Wong, Kathiresan, & Micevych, 2015). However, others have found this up-regulation to be estrogen-response element (ERE)-dependent in a knockout mouse model (Gottsch et al., 2009). A synthesis of the in vitro and in vivo data suggests that, in whole animal studies, EMS upregulates kisspeptin expression, which is augmented by neuroP acting through membrane PGR. The ERE-dependent part of this pathway is transcription of PGR but not of kisspeptin directly. Further results will shed light on the extent to which EMS vs. nuclear action affects kisspeptin production and release (Figure 4).

The neural P4 signaling that augments estradiol in RP3V kisspeptin neurons depends on PGR for the LH surge (Stephens et al., 2015). In vitro studies in our laboratory support a role for membrane-localized PGR in this pathway: R5020 (a PGR selective activator) stimulates phosphorylation of ERK1/2 in kisspeptin cells within 5 minutes. This is mimicked by a Src family activator, suggesting that membrane PGR activation involves co-activation of Src (Mittelman-Smith et al., 2018). Indeed, classical PGR has been linked to Src activation in other model systems. While estradiol alone is capable of inducing kisspeptin expression and release, it is dramatically enhanced in the presence of progesterone derived from astrocytes (i.e., neuroprogesterone; Mittelman-Smith et al. [2018]). These results came from in vitro co-culture experiments in which P4 augments the effects of estradiol in kisspeptin neurons. In vivo, estradiol is insufficient to induce an LH surge when hypothalamic steroid synthesis is blocked. This indicates that estradiol from the ovary and neural-derived P4 are involved in triggering the LH surge. Moreover, if kisspeptin expression is prevented in vivo, estradiol or P4 are incapable of triggering an LH surge (Delhousey, Chuon, Mittelman-Smith, Micevych, & Sinchak in revision). Moreover, when P4 synthesis is blocked, estradiol will not elicit an LH surge but microinfusion of kisspeptin into the OVLT (a region containing GnRH cell bodies) will. This suggests that estradiol primes kisspeptin neurons by upregulating PGR, which allows for further progesterone up-regulation of kisspeptin to induce the GnRH/LH surge.

Conclusion

The discovery of a constellation of estrogen and progesterone receptors suggests that steroid signaling is highly nuanced in the nervous system. Our studies of these actions are in their infancy. As methodology becomes more sophisticated, we will better understand how different receptors in the same neuron influence function (Connor & Wang, 2018). One such example is the estradiol induction of sexual receptivity. Three types of signaling are involved: EMS through the ERα‎-mGluR1a complex, direct nuclear activation of transcription through ERα‎, and EMS through GPER/GPR30 (Long et al., 2017; reviewed in . Micevych & Sinchak, 2013). Each of these actions have a temporal sequence related to the final output—lordosis. The initial EMS induces a refractory period during which the female is unresponsive to the male. During this time, estradiol acting at nuclear receptors induces receptors (e.g., PGR) and neuropeptides. Finally, estradiol can act on GPER/GPR30 to activate lordosis behavior or, as in the gonadally intact female rodent, progesterone from the luteinized follicle stimulates lordosis. Our understanding of the cellular consequences of multiple ERs and progesterone receptors is limited. For example, we have shown that both ERα‎ and ERαΔ‎4 are found together in a number of neurons and astrocytes. Full-length ERα‎ couples with mGluR1a and activates a positive signaling cascade, while ERαΔ‎4 activates an inhibitory cascade leading to a decrease of cAMP levels and inhibition of L-type VGCC (Boulware et al., 2005; V. V. Chaban et al., 2003; V. Chaban et al., 2011; Wong, et al., in revision. It may be that the inhibitory ERαΔ‎4 provides a mechanism for constraining estradiol-induced activation. In other words, estradiol stimulation may control both facilitation of postsynaptic activity and its cessation. The first decades of this millennium have been extremely productive in terms of understanding the entire scope of steroid-signaling strategies. Steroids, especially estrogens, are now in the same position as most other intercellular signaling molecules, with numerous receptors that allow for a host of neuronal and glial responses leading to control of movement, complex behaviors, and cognition. Our challenge is to continue to unpack these actions at a molecular level.

Acknowledgments

Research from our laboratory was supported by DA013185, HD042635, HD007228 and the David Geffen School of Medicine at UCLA.

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