The Gonadotropin-Releasing Hormone and Its Receptor

3.17

The Gonadotropin-Releasing Hormone and Its Receptor

Lothar Jennes, Anatomy and Neurobiology, College of Medicine, University of Kentucky, Lexington, KY, USA Alfredo Ulloa-Aguirre, Universidad Nacional Autónoma de México (UNAM)-Mexican National Institutes of Health, Mexico City, Mexico Jo Ann Janovick and P Michael Conn, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA Ó 2017 Elsevier Inc. All rights reserved. This chapter is a revision of the previous edition chapter by L. Jennes, A. Ulloa-Aguirre, J.A. Janovick, V.V. Adjan, P.M. Conn, volume 3, pp. 1645–1669, Ó 2009, Elsevier Inc.

Introduction 363 Gonadotropin-Releasing-Hormone Neuronal Systems 364 Embryonic Development of the Gonadotropin-Releasing-Hormone System 364 Postnatal and Adult Gonadotropin-Releasing-Hormone Systems 364 Gonadotropin-Releasing-Hormone-Containing Cell Bodies 364 Gonadotropin-Releasing-Hormone-Containing Projections 365 Associations with the Cerebrospinal Fluid 365 Cytology of Gonadotropin-Releasing-Hormone Neurons 365 Regulation of Gonadotropin-Releasing-Hormone Neurons 366 Catecholamines 367 Glutamate 367 g-Aminobutyric Acid 367 Neuropeptides 369 Gonadotropin-Releasing-Hormone Receptors in the Central Nervous System 370 Localization of Gonadotropin-Releasing-Hormone Receptors in the Brain 370 Characterization of Gonadotropin-Releasing-Hormone Receptors in the Brain 371 Regulation of Gonadotropin-Releasing-Hormone Receptor Expression in the Brain 371 Functional Aspects of Gonadotropin-Releasing-Hormone Receptors in the Brain 372 Molecular and Cellular Mechanism of Gonadotropin-Releasing-Hormone Action in the Anterior Pituitary 372 GnRH Receptor 372 Effector Coupling 372 Receptor–Receptor Interactions 373 Receptor Trafficking 373 Endoplasmic Reticulum Quality Control System and the Role of Endogenous Chaperone Proteins 373 Mutant GnRHRs Isolated from Patients with Hypogonadotropic Hypogonadism (HH) Are Actually Misfolded and Misrouted Proteins That Can Be Rescued and Restored to Function 373 3.17.4.4.3 The Ability to Rescue Mutant Proteins Using Pharmacoperones Has Therapeutic Potential 374 3.17.4.4.4 The Rescue Approach Appears Generally Applicable to Other Mutant GPCRs, Non-GPCR Receptors, Ion Channels, and Enzymes Associated with Disease: This Supports the Importance of Understanding the Mechanism of This Event in a Well-Defined System 374 Acknowledgments 375 References 375 3.17.1 3.17.2 3.17.2.1 3.17.2.2 3.17.2.2.1 3.17.2.2.2 3.17.2.2.3 3.17.2.2.4 3.17.2.3 3.17.2.3.1 3.17.2.3.2 3.17.2.3.3 3.17.2.3.4 3.17.3 3.17.3.1 3.17.3.2 3.17.3.3 3.17.3.4 3.17.4 3.17.4.1 3.17.4.2 3.17.4.3 3.17.4.4 3.17.4.4.1 3.17.4.4.2

3.17.1

Introduction

Gonadotropin-releasing hormone (GnRH) is a decapeptide that is synthesized by relatively few neurons in the ventral forebrain. These neurons project axons to the median eminence of the mediobasal hypothalamus where the hormone is released into the perivascular space of fenestrated capillaries and carried through the blood to the anterior pituitary. GnRH binds to and activates specific membrane receptors on the gonadotropes to stimulate the synthesis and release of luteinizing hormone

Hormones, Brain, and Behavior, 3rd edition, Volume 3

(LH) and follicle-stimulating hormone (FSH). These pituitary hormones, in turn, cause follicular growth and ovulation in the ovary, as well as steroid hormone synthesis. The ovarian hormones estrogen and progesterone feed back to the brain and pituitary in an inhibitory fashion throughout the reproductive cycle, except for a very short period during proestrus when a change to a positive feedback stimulates GnRH, LH, and FSH release to induce ovulation. In the rat, the appropriate pulsatile release of GnRH is critical for the maintenance of an estrous cycle and for reproduction in general.

http://dx.doi.org/10.1016/B978-0-12-803592-4.00060-2

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3.17.2 Gonadotropin-Releasing-Hormone Neuronal Systems 3.17.2.1 Embryonic Development of the GonadotropinReleasing-Hormone System Although GnRH neurons reside in the rostroventral forebrain in the adult mammal, these neurons originate in the olfactory placode. Using immunohistochemistry, GnRH-synthesizing neurons are first identified at embryonic day 11 (E11) in the epithelium of the medial olfactory placode (Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989a). Here, the GnRH neurons appear oval or fusiform and have not yet developed identifiable axons (Zheng et al., 1992). GnRH neurons begin to leave the epithelium at E12 as cell clusters that migrate through the cribriform plate to the ganglion terminale. During this early stage, approximately 50% of the GnRH neurons also express galanin and this percentage declines as the migration continues (Key and Wray, 2000). Over the subsequent few days, most GnRH cells enter the ventromedial forebrain as part of the roots of the nervus terminalis, and fewer cells reach the accessory olfactory bulb along the vomeronasal nerve. By E14, GnRH cells begin to reach the septum and preoptic region. The migration of the GnRH neurons continues through birth and at that time most GnRH cells have reached their final destination (Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989a,b). However, many GnRH neurons remain associated throughout life with the ganglion terminale and the associate branches of the terminal nerve (Schwanzel-Fukuda and Silverman, 1980; Schwanzel-Fukuda and Pfaff, 1990, 1991; Jennes and Schwanzel-Fukuda, 1992; SchwanzelFukuda et al., 1992; Schwanzel-Fukuda, 1999; Jennes and Stumpf, 1980a). The time course of the migration of GnRH neurons and initiation of GnRH peptide synthesis has been confirmed with in situ hybridization studies that showed that the neurons begin to synthesize GnRH mRNA at E10 and that by E11 translation into GnRH peptide reaches detectable levels (Wray et al., 1989a). Additional experiments using thymidine incorporation into newly formed DNA confirmed that GnRH neurons indeed originate from the olfactory placode and not from the ventricular lining (Schwanzel-Fukuda and Pfaff, 1989). This view is further supported by studies of the small-eye mouse, which fails to develop eyes and olfactory placodes due to a mutation in the pax-6 gene. Homozygote mice do not contain GnRH neurons in the nasal region or in the forebrain, whereas heterozygotes contain a normal GnRH neuronal system (Dellovade et al., 1998). Several other regions in the mouse brain were described to contain neurons that express GnRH transiently during the pre- and early postnatal period, and it was shown that these neurons in the lateral septum, bed nucleus of the stria terminals, and tectum are not derived from the olfactory placode (Wu et al., 1995; Skynner et al., 1999). These cells synthesize the mammalian form of the GnRH decapeptide, as is suggested by their immunoreactivity to a variety of well-characterized antibodies. The biological significance of this subset of GnRH neurons is not understood; however, the lack of access of these neurons to fenestrated capillaries precludes an endocrine function.

The exact cell lineage of GnRH neurons is not clear. It was widely accepted that GnRH neurons were closely related to olfactory sensory neurons (Wray et al., 1989b), but later studies (Hilal et al., 1996) showed that in chickens and in mice carrying a mutation in the transcription factor activator protein 2a (Kramer et al., 2000), GnRH neurons are present in the respiratory epithelium. Moreover, the removal of the embryonic region of the nose that would differentiate into the respiratory epithelium eliminates GnRH neurons, whereas the removal of the olfactory epithelium region has no impact on GnRH neuronal development (el Amraoui and Dubois, 1993). Together, these data indicate that GnRH progenitor cells may actually be more closely related to respiratory cells than to olfactory cells and more studies are needed to clarify the exact origin of GnRH neurons. Similarly, very little is known about the mechanisms that control the migration of GnRH neurons from the nose into the forebrain. It appears that strands of GnRH neurons travel along a track that contains the neural cell adhesion molecule (NCAM), but not laminin, cytotactin, fibronection, or cytactin (Schwanzel-Fukuda et al., 1992, 1994). These initial findings stimulated a series of experiments in which antibodies to NCAM were administered to 10-day-old embryos and it was shown that this treatment disrupted the migration of embryonic GnRH neurons (Schwanzel-Fukuda et al., 1994). However, because GnRH neurons reach their final position at the appropriate time in mice that do not express NCAM (Schwanzel-Fukuda et al., 1995), it appears that NCAM is not required for an appropriate migration (for review, see Schwanzel-Fukuda, 1999). Migrating GnRH neurons follow a track of axons in vivo and in vitro that contain the intermediate filament peripherin (Fueshko and Wray, 1994; Wray et al., 1994). Peripherin is typically expressed in olfactory receptor neurons (Gorham et al., 1991), and the close spatial relation between migrating GnRH neurons and olfactory axons could indicate interactions of the two cell types in the guidance process or common underlying mechanisms of guidance.

3.17.2.2 Postnatal and Adult Gonadotropin-ReleasingHormone Systems Many reviews have been published on the anatomy of the GnRH neuronal systems in the rodent (Barry et al., 1985; Jennes and Conn, 1994; Silverman et al., 1994); it is beyond the scope of this review to provide a detailed description of the entire GnRH system.

3.17.2.2.1 Bodies

Gonadotropin-Releasing-Hormone-Containing Cell

In the postnatal rat, most GnRH neurons are located around the organum vasculosum of the lamina terminalis (OVLT) in a dispersed distribution pattern that is reminiscent of an inverted Y. GnRH neurons extend from the medial portions of the horizontal limb of the diagonal band through the vertical limb into the medial septum. Slightly fewer neurons are present in the rostral periventricular area, and the number of immunoreactive neurons rapidly declines from rostral to caudal in the medial preoptic area and anterior hypothalamic regions. Single GnRH neurons are consistently seen in a small region right above the supraoptic nuclei. In the rat, the

The Gonadotropin-Releasing Hormone and Its Receptor

mediobasal hypothalamus does not contain immunoreactive GnRH perikarya.

3.17.2.2.2 Gonadotropin-Releasing-Hormone-Containing Projections The most extensive axonal projection reaches the median eminence of the ventral hypothalamus through diffuse projections that include periventricular, suprachiasmatic, and subchiasmatic pathways. Some of these projections traverse many hypothalamic nuclei, such as the periventricular gray, paraventricular nucleus, ventromedial nucleus, and arcuate nucleus. In the median eminence, GnRH-containing axons sprout extensively and most terminals are located in the external layer next to fenestrated capillaries, with the highest concentrations in the regions that surround the infundibular sulcus (Jennes and Stumpf, 1980a,b; Silverman et al., 1979; Merchenthaler et al., 1980, 1984; Hoffman and Gibbs, 1982). This is the site where GnRH is released into the portal blood to control the activity of the anterior pituitary gonadotropes. It has been shown in the rat that a single microinjection of retrograde tracer into the median eminence labels approximately 65% of all GnRH neurons, which suggests that most GnRH neurons project to this neurohemal contact zone (Silverman et al., 1987). Because such an injection covers only a small portion of the median eminence, the percentage of GnRH neurons projecting to the median eminence is probably substantially higher. An additional major GnRHcontaining projection terminates in the OVLT, which like the median eminence is a circumventricular organ that contains fenestrated capillaries. Although it is clear that GnRH released in the median eminence controls LH and FSH release, the physiological significance of GnRH released at the OVLT is not known because only a venous vascular link exists with the pituitary portal system. The remaining projections of GnRH-containing axons are rather limited and involve only a few axons per site. Areas in the brain that contain GnRH axons include olfactory structures, such as the main and accessory olfactory bulb, the islands of Calleja, and the pyramidal region of the piriform cortex. In addition, a few axons reach the medial and cortical nuclei of the amygdala via the ventral amygdal-fugal pathway and the stria terminalis. Single axons are also seen in the ventral hippocampus and the subiculum. Caudal projections pass from the medial septum-diagonal band through the stria medullaris, medial habenula, and the fasciculus retroflexus to the interpeduncular nucleus from which single fibers turn dorsally into the raphe nuclei and the central gray.

3.17.2.2.3

Associations with the Cerebrospinal Fluid

A unique feature of the GnRH neuronal system that may have important functional implications is the close anatomical association of GnRH axons to the ventricular and subarachnoid cerebrospinal fluid (Jennes and Stumpf, 1980b; Burchanowski et al., 1979). Thus, GnRH fibers are present consistently in or next to the ependyma of the ventromedial portion of the septal lateral ventricles, the ventral portions of the hypothalamic third ventricle, the subfornical organ, and the aqueduct. It appears that, at least in some of these regions, GnRH axons penetrate the ependyma and thus have direct contact with the internal cerebrospinal fluid. Similar contacts occur on the outer surface

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of the brain, especially at the olfactory bulb where the ganglion terminale and the nervus terminalis reside in the subarachnoid space, the prechiasmatic cistern, and the interpeduncular cistern. Based on these anatomical findings and studies that measured GnRH levels in the cerebrospinal fluid (Van Vugt et al., 1985; Skinner et al., 1995), it is suggested that release of the hormone into the portal blood may be coupled or synchronized with the release of GnRH into the cerebrospinal fluid. This view is supported by the finding that increased levels of GnRH and LH in the blood are paralleled by increases in the GnRH concentrations in the cerebrospinal fluid. GnRH in the cerebrospinal fluid could reach distant sites of action and have prolonged effects because endo- or exopeptidases are present only in very low levels in these fluid-filled compartments (Mendez et al., 1990). The scenario of a coordinated release of GnRH could represent an efficient way by which the endocrine effects of GnRH on the pituitary gonadotropes could be coupled with the intracerebral effects of GnRH, for example the facilitation of reproductive behaviors. An additional mechanism by which GnRH release at two anatomically distinct sites could be coordinated has been suggested based on multiple retrograde labeling experiments that show that one GnRH neuron can terminate at the perivascular space of fenestrated capillaries and, through axon collaterals, at specific nuclei in the brain, such as the interpeduncular nucleus (Jennes, 1991).

3.17.2.2.4 Neurons

Cytology of Gonadotropin-Releasing-Hormone

GnRH neurons appear as unipolar or bipolar cells that can have either smooth contours or have a spiny appearance; however, the significance of these morphological differences has not been elucidated (Krisch, 1980; Liposits et al., 1984; Jennes et al., 1985; Wray and Hoffman, 1986). Ultrastructural studies have shown that GnRH neurons are rather simple neurons that have all the standard organelles required for appropriate functioning (Jennes et al., 1985; Mazzuca, 1977; Witkin and Demasio, 1990). While earlier light and electron microscopic immunohistochemical studies have failed to identify dendrites of GnRH neurons (Jennes et al., 1985; Witkin and Silverman, 1985), more recent studies using microinjections of biocytin into green fluorescent protein (GFP) expressing GnRH neurons clearly show that GnRH neurons have long, mostly unbranched dendrites which contain many spinelike extensions (Campbell et al., 2005). The GnRH dendrites often associate with dendrites of other GnRH neurons and can even share synapses which suggests that GnRH dendrites may be much more important for integrations of different signals than previously thought (Campbell et al., 2009; Campbell and Suter, 2010). GnRH axons form presynaptic specializations on the dendrites or perikarya of other hypothalamic neurons, which indicates that the GnRH peptide is released at these sites and probably acts as a neurotransmitter (Jennes et al., 1985; Witkin and Silverman, 1985). Interestingly, some of the postsynaptic neurons also contain GnRH, and it has been suggested that this kind of innervation could represent a mechanism by which the activity of GnRH neuronal system is synchronized (Witkin and Silverman, 1985; Leranth et al., 1985a). However, such contacts between GnRH neurons are fairly rare and may not

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be sufficiently frequent to represent a relevant mechanism of activity coupling among the GnRH neurons.

3.17.2.3 Regulation of Gonadotropin-Releasing-Hormone Neurons The protein synthesis and release activity of GnRH neurons is regulated by complex feedback loops that include the gonadal steroid hormones estradiol and progesterone, as well as a large number of neurotransmitter systems (for review, see Kalra, 1993; Herbison, 2006; Levine, 1997). Estradiol exhibits an inhibitory effect on GnRH neuronal systems during all phases of the reproductive cycle, except for the proestrous stage when rising estradiol levels cause a switch to a positive feedback mode that stimulates GnRH release, which in turn causes a massive surge in pituitary LH secretion that is necessary for ovulation The mechanisms underlying this temporary positive feedback are not known, but it appears that direct effects of estradiol on GnRH neurons are probably not the trigger for the LH surge. GnRH neurons express only low levels of estrogen receptor–b which is not necessary for the induction of ovulation (Hrabovszky et al., 2000). However, the mandatory estrogen receptor-a is not present in GnRH neurons. It is therefore thought that input from steroid-sensitive interneurons to the GnRH neurons is required for the generation of a GnRHmediated LH surge. A large number of studies have begun to identify neurons in the brain that regulate GnRH neuronal activity. Thus, anteroand retrograde transport studies of tracers in combination with immunohistochemistry have identified several regions in the brain that are involved in the control of GnRH neurons, such as the arcuate and ventromedial or the anteroventral periventricular nuclei (Wright and Jennes, 1993; Van der Beek et al., 1997; Simonian et al., 1999). More recently, modified pseudorabies virus that can replicate only in the presence of Cre recombinase was microinjected into the medial septumdiagonal band of transgenic mice whose GnRH neurons express Cre recombinase (Campbell, 2007; Yoon et al., 2005; Campbell and Herbison, 2007). Thus, the virus could replicate only in GnRH neurons and was transported retrogradely to the cells that innervate GnRH neurons. This approach provided valuable results and identified extensive input to the GnRH neurons from many hypothalamic, limbic, and brainstem structures. However, it is not entirely clear which of the identified cells are true first-order afferent neurons or second- or third-order cells since the virus is passed on at synaptic junctions and the length of the survival time of the animals after receiving the virus determines how many orders of neurons are infected. Based on the approach developed by Horowitz et al. (1999), Boehm et al. (2005) generated a transgenic mouse model in which GnRH neurons synthesize GFP and barley lectin. While GFP remains in the GnRH neurons, barley lectin is transported anterogradely and retrogradely and passed on either to the cells that are innervated by GnRH neurons or to the neurons that provide synaptic input to GnRH neurons. For the first time, these studies revealed extensive neuronal networks involving about 50 000 neurons that are directly involved in the regulation of the GnRH neuronal system. Besides regions that were known to participate in the regulation

of GnRH neuronal activity, such as the arcuate or ventromedial nuclei, many novel areas were identified that were previously not associated with the control of GnRH neurons. Thus, many cells in the anterior cortical amygdaloid nucleus, the lateral hypothalamus, nucleus reuniens, and paraventricular thalamic nuclei contained lectin indicating that these cells either received synaptic input from GnRH neurons or they innervated GnRH neurons. The absence of GnRH fibers in some of these regions is a clear indicator that the lectinpositive cells did not receive input by GnRH axons but instead had taken up the lectin at presynaptic terminals that innervated GnRH neurons. These studies show that relatively few GnRH neurons in a rodent brain integrate inputs from a large number of different brain regions that are involved in the processing and generation of olfactory, pheromonal, behavioral, or endocrine information. Many studies have focused on the identification of neurotransmitter systems that regulate GnRH neuronal activity by administering agonists or antagonists during the preovulatory or estrogen- and progesterone-induced LH surge. Data from such studies are summarized in several excellent reviews (Kalra, 1993; Herbison, 2006; Levine, 1997). Collectively, these studies show that most manipulations of a neurotransmitter system have an impact on GnRH-mediated LH release, either stimulating LH secretion to various degrees or reducing circulating LH levels. Most treatments that result in a stimulation of GnRH neurons produce a two- to fivefold increase in LH levels, which is substantially smaller than LH levels during the surge. So far, only a sequential treatment with estrogen and progesterone has been able to induce proestrus-like LH levels. On the other hand, most antagonists of stimulatory neurotransmitters can prevent the surge. These findings suggest that it is probably not a single neurotransmitter system that induces GnRH neurons to release preovulatory amounts of the peptide but instead several systems that participate either in parallel or sequentially in the induction of the surge. Interference with only one of these stimulators abolishes the surge. One approach to identifying the neurotransmitters and neuropeptides that participate in the control of GnRH neuronal activity is to use multiple immunohistochemical stainings for GnRH and select neurotransmitter, to determine which transmitter is present in axons that are next to GnRH cell bodies. These studies have demonstrated that catecholaminergic (Ajika, 1979; Jennes et al., 1982, 1983; Hoffman et al., 1982; Leranth et al., 1988a; Chen et al., 1989a), serotoninergic (Jennes et al., 1982; Kiss and Halasz, 1985), g-aminobutyric acidergic (GABAergic; Jennes et al., 1983; Leranth et al., 1985b; Witkin, 1992), and glutamatergic axons (Goldsmith et al., 1994; Lin et al., 2003) were juxtaposed to GnRH neurons as were axons that contained the neuropeptides, b-endorphin (Leranth et al., 1988a; Chen et al., 1989a,b), galanin (Merchenthaler et al., 1991), neuropeptide Y (Li et al., 1999), vasoactive intestinal peptide (van der Beek et al., 1993, 1994; Smith et al., 2000), substance P (Hoffman, 1985), or neurotensin (Hoffman, 1985). Although the limitations of the resolution of light microscopy do not permit a distinction between synaptic contacts and en passant axons, a close proximity of axons to GnRH neurons provides an anatomical rationale for further in-depth studies. Additional electron microscopic

The Gonadotropin-Releasing Hormone and Its Receptor

studies have determined that some of these close appositions are indeed synaptic in nature and that tyrosine hydroxylase (Leranth et al., 1988a; Chen et al., 1989a), GABA (Leranth et al., 1988b), glutamate (Goldsmith et al., 1994), or b-endorphin (Chen et al., 1989b) is contained in these terminals.

3.17.2.3.1

Catecholamines

Noradrenergic and adrenergic axons are in close proximity to GnRH neurons (Moore et al., 1999). Some of these fibers form synaptic complexes with the GnRH neurons (Chen et al., 1989a; Leranth et al., 1988b), and GnRH neurons express the a1B adrenergic receptor mRNA (Petersen et al., 1999) and protein (Hosny and Jennes, 1998). This suggests that the catecholamines participate directly in the control of GnRH neuronal activity. One requirement for an involvement in the regulation of GnRH neurons is the capability of the catecholaminergic neurons to convey the steroidal signal. Several studies have provided strong evidence that this is indeed the case. Thus, noradrenergic neurons accumulate 3H-estradiol in their nuclei (Heritage et al., 1977, 1980), estrogen receptor-a, and probably estrogen receptor-b, mRNA is present in the appropriate regions A1 and A2 of the brain stem (Shughrue et al., 1997), and many of the noradrenergic and adrenergic neurons contain estrogen receptor-a protein (Simonian and Herbison, 1997; Lee et al., 2000). Moreover, tyrosine hydroxylase (Liaw et al., 1992) and dopamine-b-hydroxylase gene expression (Serova et al., 2000) are stimulated by estradiol, and the noradrenergic and adrenergic neurons in the brain stem respond to estradiol by transient synthesis of the transcription factor fos (Lee et al., 2000). The noradrenergic and adrenergic input to the GnRH system is critical for the maintenance of pulsatile GnRH release and the induction of a preovulatory and estrogen- and progesteroneinduced LH surge, as has been shown repeatedly with physiological approaches (for review, see Herbison, 1997a, 2006). Thus, the inhibition of either norepinephrine or epinephrine synthesis results in a markedly reduced LH secretion in ovariectomized rats, suggesting that both neurotransmitters regulate basal pulsatile GnRH–LH release (Drouva and Gallo, 1976). Similar treatments with specific synthesis inhibitors also prevent the steroid-induced LH surge, indicating an important role in the generation of the preovulatory surge (Kalra et al., 1972; Kalra and McCann, 1974; Crowley and Terry, 1981; Crowley et al., 1982; Coen and Coombs, 1983); for review, see Herbison (1997a, 2006). On the other hand, the administration of norepinephrine, epinephrine, or a-adrenergic agonists causes a significant elevation of GnRH-stimulated LH levels (Rubinstein and Sawyer, 1970; Gallo and Drouva, 1979; Kalra and Gallo, 1983; Barraclough et al., 1984; Levine et al., 1991). These pharmacological studies are of physiological relevance because measurements of norepinephrine and epinephrine release (Mohankumar et al., 1994) and turnover during select phases of the estrous cycle or the steroid-induced LH surge show that catecholamine turnover in the median eminence is increased just before and during the LH surge (Wise et al., 1981; Wise, 1982; Adler et al., 1983; Sheaves et al., 1984). Together, based on anatomical and physiological evidence, the catecholamines can be viewed as important participants in the regulation of GnRH release.

3.17.2.3.2

367

Glutamate

Immunohistochemical studies have shown that glutamate or neuron-specific vesicular glutamate transporters (VGLUTs) are present in many axon terminals that innervate GnRH neurons (Goldsmith et al., 1994; Lin et al., 2003) which suggests an important stimulatory role of this excitatory neurotransmitter in the control of GnRH neurons (Figure 1). The findings that glutamatergic neurons in the septal complex and the hypothalamic arcuate and ventromedial nuclei as well as in the preoptic region express estrogen receptor-a (Thind and Goldsmith, 1997; Eyigor et al., 2004) and that short-term treatment with estradiol causes an increase in glutamate content (Luine et al., 1997) suggest that the glutamate neurons are affected by gonadal steroids and could convey the steroidal signals to the GnRH neurons. Glutamate release in the preoptic region is increased just prior to and during the steroid-induced LH surge, as measured with push–pull perfusion or in vivo dialysis (Jarry et al., 1992, 1995; Ping et al., 1994; Demling et al., 1985). Many studies have shown that glutamate and its agonists AMPA, N-methyl-D-aspartate (NMDA), and kainate (KA) stimulate GnRH–LH release in vivo and in vitro in a dose-dependent manner and, conversely, that the blockade of either glutamate receptor subtype with specific antagonists prevents the surge (for review, see Brann and Mahesh, 1997). In order to identify some of the mechanisms by which glutamate regulates GnRH neurons, GnRH neurons were screened for the presence of various glutamate receptor subunit mRNAs and proteins. These studies showed that GnRH neurons express most, if not all ionotropic glutamate receptor subunits (Gore et al., 1996; Ottem and Petersen, 2000; Miller and Gore, 2002; Bailey et al., 2006; Eyigor and Jennes, 1997). Examples are shown in Figure 2. Several lines of evidence suggest that these glutamate receptors are indeed activated during the steroid-induced LH surge. For instance, approximately 50–60% of the GnRH neurons that express transiently the transcription factor fos during the LH surge also express KA2 and GluR5 receptor subunits, which indicates a preferential activation of KA2-containing GnRH neurons (Eyigor and Jennes, 2000). Similarly, the expression of the AMPA subunits GluR1 and GluR3 increases significantly in GnRH neurons at the time just before and during the LH surge, again preferentially in GnRH neurons that are activated as determined by the presence of fos (Bailey et al., 2006). However, glutamate or glutamate agonist administration alone produces only a modest increase in GnRH–LH release and the coordinated release of other transmitters is needed to induce a full surge.

3.17.2.3.3

g-Aminobutyric Acid

GABA is the major inhibitory neurotransmitter in the brain and strong evidence exists for an important inhibitory role in the control of GnRH neuronal activity. Thus, intracerebral microinjections of GABA into the preoptic area inhibit the proestrous surge (Herbison and Dyer, 1991), and the administration of GABAA and GABAB receptor antagonists have only modest effects on basal LH release; however, they greatly potentiate the stimulatory effects of norepinephrine (Hartman et al., 1990). Extracellular GABA concentrations, as measured with push–pull perfusion approaches, decline dramatically prior to and during the estrogen-induced LH surge, which suggests that the release of a tonic inhibition of the rostral hypothalamic

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The Gonadotropin-Releasing Hormone and Its Receptor

(a)

(d)

VGLUT2 (b)

Synaptophysin (c)

GnRH

Overlay

20 μm

Figure 1 Example of immunohistochemical triple labeling for VGLUT2 (a), synaptophysin (b), and GnRH (c) as well as an overlay of the three images (d), showing that many glutamatergic terminals are juxtaposed to the GnRH neuron, and some of these terminals contain synaptophysin (arrowheads).

GnRH

GluR1

Fos

Overlay

Figure 2 Example of triple-label immunohistochemistry for GnRH (green), AMPA receptor subunit GluR1 (red), and fos protein (blue) as well as an overlay of the three images. Scale bar ¼ 30 mm.

The Gonadotropin-Releasing Hormone and Its Receptor

neurons by GABA parallels the secretory activity of GnRH neurons (Jarry et al., 1995). Such a release of a continuous inhibition may also be an important mechanism that participates in the induction of puberty. Thus, GABA levels are very high in the mediobasal hypothalamus of the prepubertal monkey and decrease during early and mid-puberty, which is the exact opposite of GnRH levels (Terasawa et al., 1999). GABAergic neurons concentrate 3H-estradiol in their nuclei (Flügge et al., 1986); contain estrogen receptor-a protein in their nuclei (Herbison et al., 1993); have extracellular GABA concentrations modulated by estrogen (for review, see Herbison, 1997b); and are, from an anatomical point of view, located in the appropriate hypothalamic areas to innervate GnRH neurons. That GABA can control GnRH neurons directly at the perikaryon or axon terminal is suggested by several studies. Thus, glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis, is present in many axons that surround GnRH neurons (Jennes et al., 1983), and Leranth et al., 1985b have shown with electron microscopy that GAD immunoreactive axons form presynaptic terminals on GnRH perikarya. Evidence that these synapses are functioning is indicated by the observation that approximately 75% of the GnRH neurons express the b-subunit of GABAA receptors, whereas the a1- or b2-subunit mRNA was not detected (Petersen et al., 1993). Later studies found that approximately 25% of the GnRH neurons express the a1- or a2-subunit mRNA, whereas only a small percentage of the GnRH neurons contain detectable levels of the a1- and a2-subunit protein (Jung et al., 1998). The same study detected b3-subunit mRNA in only approximately 25% of the GnRH neurons; g1 mRNA was not detected. These receptor subunits can form functioning receptors as determined by electrophysiological measurements of membrane patches isolated from identified GnRH neurons. These studies showed that all GnRH neuronal membrane patches responded to GABA and that the responses were inhibited by the GABAA antagonist bicuculline (Spergel et al., 1999). Together, these studies show that certain GABAA receptor subunits are present in GnRH neurons and, based on the subunit composition, that they could form functioning GABA receptors in the plasma membrane; however, a definitive characterization of such receptors has not been accomplished.

3.17.2.3.4

Neuropeptides

There are many neuropeptides that have been shown to participate in the regulation of GnRH release (for review, see Leranth et al., 1985a); it is beyond the scope of this review to discuss the effects of all these peptides. We focus here on neuropeptide Y (NPY), vasoactive intestinal peptide, b-endorphin, and kisspeptins because we know most about their actions and receptor expression. l

Neuropeptide Y: NPY is probably the best understood neuropeptide that is part of an excitatory circuit enhancing GnRH release. Several studies have shown that the administration of NPY stimulates GnRH release (Crowley et al., 1987; Crowley and Kalra, 1987; Sabatino et al., 1989), whereas intraventricular injection of specific anti-NPY antibodies (Wehrenberg et al., 1989) or antisense oligonucleotides (Kalra et al., 1995) block the steroid-induced LH surge. These data suggest that endogenous NPY is required for the

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occurrence of a surge. The observations that NPY mRNA content in the arcuate nucleus increases prior to the onset of an LH surge (Bauer-Dantoin et al., 1992; Sahu et al., 1994), that NPY peptide levels in the mediobasal hypothalamus parallel LH levels (Sahu et al., 1989), and that NPY release is enhanced during the LH surge (Watanobe and Takebe, 1992) support the view that the NPY neurons participate in the stimulation of GnRH release. The NPY neurons that are involved in the control of GnRH neurons are located in the brain stem, where NPY is expressed in catecholaminergic neurons (Everitt et al., 1984) and in the arcuate nucleus (Chronwall et al., 1985; McShane et al., 1994). Both of these neuronal cell groups express receptors for estrogen (Sar et al., 1990) and could therefore transmit the steroid signal to the GnRH neurons. The NPY neurons project to the median eminence, among others, where NPY is released into the perivascular space for transport to the anterior pituitary or for interactions with the axon terminals of other neurons. In addition, NPY-containing axons reach the medial septum-diagonal band–rostral preoptic area where the GnRH neurons are located. Evidence for direct actions on the GnRH neurons was provided by electron microscopic studies that identified NPY in presynaptic specialization on GnRH perikarya (Tillet et al., 1989; Tsuruo et al., 1990). Because NPY can bind to and activate several different membrane receptor subtypes, it was important to determine which subtype is expressed in GnRH neurons. Previous experiments have suggested that the activation of the NPY-Y1 receptor subtype is required for the occurrence of a steroid-induced LH surge (Kalra et al., 1992) and a preovulatory LH surge (Leupen et al., 1997), and it was shown that NPY-Y1 receptor expression in the hypothalamus is greatly elevated just prior to and during the proestrous LH surge (Xu et al., 2000). Immunohistochemical studies have confirmed that the NPY-Y1 receptor protein is present in many GnRH nerve terminals in the median eminence; however, the protein was not detected in GnRH perikarya (Li et al., 1999). These findings suggest that although the NPY-Y1 receptor subtype probably mediates the stimulatory effects of NPY on GnRH release in the median eminence, the effects of NPY on GnRH perikarya may involve another NPY receptor subtype that has not been identified. l Vasoactive Intestinal Peptide: It is clear now that the preovulatory LH surge is generated by a coupling of the steroidal signals with circadian signals that originate from the suprachiasmatic nucleus. In this nucleus, vasoactive intestinal peptide is synthesized in a large number of neurons that are located in the ventrolateral aspects of the nucleus. Axons from these vasoactive intestinal peptide neurons project to GnRH neurons, among others, where close appositions have been described. Interestingly, such close contacts occur with the subpopulation of GnRH neurons that express fos during the steroid-induced LH surge, which suggests that vasoactiveintestinal-peptide-containing axons exert a stimulatory effect on the GnRH neurons (van der Beek et al., 1994). Although the physiological effects of vasoactive intestinal peptide on GnRH release have not been fully elucidated and are, in part, controversial, several lines of evidence support the view that vasoactive intestinal peptide stimulates GnRH release. Thus, the central administration of vasoactive intestinal

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peptide antiserum or administration of vasoactive intestinal peptide–antisense oligonucleotides to the suprachiasmatic nucleus delays and reduces the magnitude of the steroidinduced GnRH–LH surge (van der Beek et al., 1999; Harney et al., 1996) and lesions of the suprachiasmatic nucleus abolish the proestrous surge (Gray et al., 1978). In order to determine whether vasoactive intestinal peptide acts directly on GnRH neurons, immunohistochemical triple-labeling experiments were conducted using antibodies to GnRH, vasoactive intestinal peptide, and the vasoactive intestinal peptide-2 receptor. The results of these studies show that approximately 40% of the GnRH neurons express the vasoactive intestinal peptide-2 receptor and that vasoactive intestinal peptide-immunoreactive axons were present next to most of the GnRH and vasoactive intestinal peptide-2 receptor-positive neurons (Smith et al., 2000). Together, these data suggest that vasoactive intestinal peptide neurons from the suprachiasmatic nucleus participate directly in the control of GnRH neurons through vasoactive intestinal peptide-2 receptors. l Endogenous Opiates: The endogenous opiates include three families of neuropeptides, the endorphins, dinorphins, and enkephalins, all of which have inhibitory effects on both the frequency and amplitude of GnRH-mediated LH release pulses (Bruni et al., 1977; Gilbeau et al., 1985); for review, see Kalra (1993) and Herbison (2006). Conversely, the administration of the general opiatereceptor blocker naloxone (Bruni et al., 1977; Van Vugt et al., 1982) or the immunoneutralization of b-endorphin (Weesner and Malven, 1990) causes an increase in circulating LH levels, which suggests that endogenous endorphin is an important neurotransmitter that tonically inhibits the GnRH neurons. This view is supported by studies that measured decreasing b-endorphin levels in the portal vessels on the afternoon of proestrus just prior to the onset of the LH surge (Sarkar and Yen, 1985), indicating that the release of the GnRH neuronal system from the tonic inhibition is a physiologically relevant event and not a pharmacological artifact. Endorphin-containing perikarya are restricted to the hypothalamic arcuate nucleus and a considerable number of the endorphin-positive neurons also express estrogen receptors (Jirikowski et al., 1986), suggesting that this neuronal system is a strong candidate to propagate the estrogen signal to other neurons. From the perikarya in the arcuate nucleus, extensive projections reach the median eminence, preoptic area, and medial septum-diagonal band (Khatchaturian et al., 1985), among others, which are the areas in which a direct innervation of the GnRH neuronal system could occur. Indeed, presynaptic b-endorphin-containing specializations on GnRH perikarya have been described (Leranth et al., 1988a; Chen et al., 1989b). However, several studies were unable to detect the mRNA for any of the opiate receptor subtypes (Mitchell et al., 1997; Sannella and Petersen, 1997), and it remains to be determined if these negative data are due to limited sensitivities of dual in situ hybridization procedures or if, indeed, GnRH neurons do not synthesize opioid receptors. A large body of evidence suggests that the opioid system interacts with other neurotransmitter systems, which in turn could influence GnRH

neuronal activity (for review, see Kalra, 1993); the interactions with the catecholaminergic and NPY-containing neurons especially warrant further detailed investigations. l Kisspeptins: A novel peptide has been identified to be a very potent stimulator of GnRH release. This peptide which is derived from the Kiss1 gene is synthesized as a 145 amino acid long peptide that is further processed to a 54, 14, 13, and 10 amino acid long peptide (Ohtaki et al., 2001). Kisspeptin is synthesized in neurons of the arcuate and the anteroventral periventricular nuclei, among others, which are critical sites in the brain involved in the control of GnRH neuronal activity (Gottsch et al., 2004; Brailoiu et al., 2005). The findings that kisspeptin producing neurons in the hypothalamus express estrogen receptors and kisspeptin mRNA levels are regulated by gonadal steroids support the view that this peptide is important for the control of GnRH neurons (Smith et al., 2005a,b). While a direct, synaptic innervation of GnRH neurons by kisspeptin-containing axon terminals has not yet been clearly established, many data support the view that they do exist. Thus, kisspeptin-54 and kisspeptin-10 are extraordinarily potent in causing GnRH release and intracerebroventricular administration of only 1 fmol (Bruni et al., 1977) or 10 pmol peptide (Navarro et al., 2005) causes a significant rise in circulating LH levels. Almost all GnRH neurons express the receptor for kisspeptin, GPR54 (Messager et al., 2005; Irwig et al., 2004) and administration of kisspeptin causes a rapid and transient expression of the transcription factor fos (Irwig et al., 2004). This finding is remarkable because fos expression in GnRH neurons is limited to a few hours during the LH surge (Lee et al., 1990, 1992; Hoffman et al., 1993) and up to now only a strict regimen of sequential estradiol and progesterone administration to ovariectomized rodents was able to induce fos expression in GnRH neurons mimicking the events that occur during the preovulatory surge. Together, these data suggest that kisspeptin is a potent stimulator of GnRH release and that kisspeptin plays an important role in the regulation of GnRH neurons under physiological conditions. However, just like the other neuropeptides or amino acid transmitters, kisspeptin is not absolutely required since GPR54-deficient mice, although infertile, respond to estradiol followed by progesterone administration with elevated LH levels and fos expression in the GnRH neurons (Dungan et al., 2007).

3.17.3 Gonadotropin-Releasing-Hormone Receptors in the Central Nervous System 3.17.3.1 Localization of Gonadotropin-Releasing-Hormone Receptors in the Brain The finding that GnRH is present in presynaptic terminals and in axons that project to regions in the brain that are not related to the direct regulation of the anterior pituitary gonadotrope function and studies that showed a facilitatory effect of exogenous GnRH on reproductive behaviors prompted the search for intracerebral receptors for GnRH. Initially, in vitro autoradiography was used to localize the GnRH binding sites in the brain, followed by biochemical assays that determined the binding characteristics of GnRH (Badr and Pelletier, 1987; Reubi et al., 1987; Jennes et al., 1988; Leblanc et al., 1988). These

The Gonadotropin-Releasing Hormone and Its Receptor

studies showed that specific binding sites for GnRH exist in many regions of the central nervous system that had been implicated either in the integration of olfactory and vomeronasal cues or in sites that are known to be involved in the generation of emotional and reproductive behaviors. Thus, binding sites for GnRH are present in the laminae glomerulosa and plexiformis of the olfactory bulb, the nucleus olfactorius anterior, the frontal cortex at the sulcus rhinalis, and the piriform cortex. In the septum, the dorsal and lateral portions of the lateral septal nucleus contain moderate amounts of GnRH binding sites, whereas the medial septum and the diagonal band are not labeled. In the hypothalamus, only the arcuate nucleus is labeled and, further caudally, the interpeduncular nucleus and the central gray contain measurable amounts of GnRH binding sites. Specific binding is also seen in the cortical nucleus of the amygdala and more prominently in the hippocampus, where most label is associated with the strata oriens and radiatum. The highest number of GnRH binding sites is measured in the parasubiculum, which is labeled at the medial tip of the caudal cerebral cortex (Jennes et al., 1988). The results of the above localization of GnRH receptor by binding studies were confirmed in transgenic mice which carried an alkaline phosphatase reporter gene under the control of the GnRH receptor promotor. These studies show that brain GnRH receptor expression begins early postnatally in the hippocampus, lateral septum, and amygdala as well as in the median preoptic region (Schang et al., 2011). Using an animal model that expresses yellow fluorescent protein under the control of the GnRH promotor, additional cells were identified to express the GnRH receptor. These sites include various regions of the amygdala, septum, hippocampus, and thalamus, as well as mesencephalon and pontine reticular nucleus (Wen et al., 2011). These data suggest that GnRH can affect numerous systems in the brain that are associated with sexual behavior, odor/pheromone processing, food/energy balance, and many other processes.

3.17.3.2 Characterization of Gonadotropin-ReleasingHormone Receptors in the Brain Biochemical analyses of the specificity and affinity of the GnRH binding to hippocampal membrane preparations show that they are very similar when compared to the anterior pituitary GnRH receptors in that all agonists that bind to hippocampal membranes also bind to the pituitary GnRH receptors with similar inhibiting concentration (IC50). In addition, GnRH fragments or different forms of GnRH from lower vertebrates do not bind to the hippocampal GnRH binding sites nor do they interact with the pituitary GnRH receptor. The only GnRH analog that we found to bind to the pituitary GnRH receptor but not to hippocampal preparations is the antagonist [D-pGlul, D-phe2, D-trp3,6]GnRH (Jennes et al., 1990). The reason for this discrepancy is not known, but it is possible that the difference in the lipid microenvironment may prevent the binding of this analog to hippocampal binding sites. That the brain GnRH binding sites are very similar to the GnRH receptor in the anterior pituitary was shown by in situ hybridization that applied cRNA probes encoding the pituitary GnRH receptor. The results of these studies show that the mRNA is present in most areas of the brain where GnRH binding sites were measured,

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except for the interpeduncular nucleus and the central gray where no hybridization signal was detected. However, a hybridization signal was detected in the ventromedial nucleus and the medial habenula, and it was hypothesized that the GnRH receptor protein is synthesized in the neurons of the ventromedial nucleus and transported anterogradely to the central gray along a well-characterized extensive projection (Conrad and Pfaff, 1976). Similarly, the protein could be synthesized in the neurons of the medial habenula and transported anterogradely through the fasciculus retroflexus to the interpeduncular nucleus (Jennes and Woolums, 1994). Additional studies are needed to determine if these hypotheses are correct. The anatomical data describing the presence of GnRH receptor mRNA or protein in the brain has been confirmed with polymerase chain reaction (PCR) for the mediobasal hypothalamus (Seong et al., 1998) and in transgenic mice that synthesize luciferase under the control of the GnRH receptor promoter (Duval et al., 2000). Together, these studies clearly strengthen the view that specific receptors for GnRH exist in select regions of the central nervous system.

3.17.3.3 Regulation of Gonadotropin-Releasing-Hormone Receptor Expression in the Brain Very little is known about the regulation of brain GnRH receptor expression. We could show with in situ hybridization that the removal of the gonads leads to a significant elevation of GnRH receptor mRNA levels in areas CAI and CA3 and in the dentate gyrus of the hippocampus in both male and female rats (Jennes et al., 1995). These data are in good agreement with studies that show a transient elevation in the number of GnRH binding sites in the hippocampus of castrated male rats (Badr et al., 1988; Ban et al., 1990). In the mediobasal hypothalamus, GnRH receptor mRNA levels were elevated in the morning of the estrogen- and progesterone-induced LH surge, whereas the levels declined during the surge (Jennes et al., 1997), which indicates that the cells increase the synthesis of GnRH receptor mRNA and probably protein in preparation for the LH surge and not as a consequence of the surge. These data are similar to the results of Seong et al. (1998), who used competitive PCR to measure GnRH receptor mRNA in tissue punches of the mediobasal hypothalamus. These studies show that the administration of progesterone after estradiol priming causes a dose-dependent decrease in GnRH receptor mRNA levels, whereas estradiol alone stimulated GnRH receptor expression in the mediobasal hypothalamus. In addition, luciferase activity under the control of the GnRH receptor promotor in transgenic mice is greatly enhanced in whole brain of ovariectomized animals that received estradiol when compared to untreated ovariectomized mice (Duval et al., 2000). However, the data from these studies with brain tissues are different from the data obtained with pituitary tissue in which GnRH receptor mRNA levels are low during the morning of the LH surge before they rise at noon and remain high until after the surge (Bauer-Dantoin et al., 1993). The reasons for this discrepancy are not clear; it is plausible that newly synthesized GnRH receptor protein in the mediobasal hypothalamus is transported to distant sites, thus requiring more time before it reaches its final location. That appropriate synthesis of the GnRH receptor in the brain

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may be important for the occurrence of an estrogen- and progesterone-induced LH surge is indicated by experiments that use the intraventricular administration of antisense oligonucleotides to reduce receptor synthesis. The administration of antisense oligonucleotides but not of sense oligonucleotides significantly decreases GnRH receptor mRNA in the mediobasal hypothalamus in estrogen- and progesterone-treated animals and causes a significant reduction in circulating LH (Seong et al., 1998). However, this treatment with antisense oligonucleotides also reduced GnRH receptor synthesis in the anterior pituitary; thus, it is not clear if the primary surgeblocking effect was exerted at the level of the central nervous system or the anterior pituitary. Together, these data suggest that GnRH receptor mRNA and protein expression is at least in part regulated by gonadal steroids. Based on the anatomical distribution of estrogen receptors and GnRH receptors, it is possible that the effects of the gonadal steroids are exerted directly in the GnRH receptor synthesizing cells because ERa is prominently expressed in the arcuate nucleus, whereas estrogen receptor-b is the dominant form in the hippocampus (Shughrue et al., 1997).

3.17.3.4 Functional Aspects of Gonadotropin-ReleasingHormone Receptors in the Brain We can only speculate about the function of GnRH receptors in the brain. It has been known for many years that peripheral or central administration of GnRH can facilitate reproductive behaviors (Moss and McCann, 1973; Pfaff, 1973) especially when the peptide is administered into the central gray (for review, see Pfaff et al., 1994). It is thought that the central gray receives extensive afferent projections from the ventromedial nucleus (Conrad and Pfaff, 1976), which may transport the GnRH receptor protein. An enhanced GnRH receptor expression in the neurons of the ventromedial nucleus during the morning of the LH surge could indicate that high levels of the receptor protein can reach the central gray before estrus, which is the time of the cycle when reproductive behaviors are most pronounced. The arcuate nucleus is one of the hypothalamic regions that are essential for the maintenance of regular estrous cyclicity (Bogdanove, 1964), and this nucleus contains a large number of different neuropeptide- or monoamine-expressing neurons (Chronwall, 1985), many of which also contain receptors for estradiol (Sar et al., 1990; Jirikowski et al., 1986). Thus, the arcuate nucleus is generally thought of as a center in the brain in which the estrogen signals are converted into neuronal signals. Among the estradiol target cells in the arcuate nucleus are neurons that contain NPY (Chronwall et al., 1985), b-endorphin (Khatchaturian et al., 1985), or GABA (Mugnaini and Oertel, 1985), all of which are known to affect GnRH release from the median eminence into the capillary plexus (for review, see Herbison, 2006). If these neurons express GnRH receptors, as is suggested by the anatomical overlap of GnRH receptor mRNA-containing neurons and the peptidergic and aminergic neurons, then GnRH released in the arcuate nucleus could affect the secretory activity of the target neurons. A change in the activity of the GnRH target neurons, many of which project to the median eminence, could alter the release of GnRH, which controls pituitary gonadotrope function. Thus, GnRH neurons could participate in the regulation or

synchronization of other GnRH neurons indirectly by using neurons in the arcuate nucleus as a relay station. However, many more studies are needed to establish the precise role that GnRH plays inside the central nervous system.

3.17.4 Molecular and Cellular Mechanism of Gonadotropin-Releasing-Hormone Action in the Anterior Pituitary Much of the information regarding the molecular mechanism of GnRH has come from studies assessing the coupling of the pituitary-derived receptor (Sealfon et al., 1997; Byrne et al., 1999; Stojilkovick et al., 1994) to effector systems. Pituitaries themselves have also been a useful model, as have reconstituted systems in which receptors (including native, chimera, and mutant receptor moieties) have been stably or transiently transfected into systems that do not ordinarily express receptors (Kaiser et al., 1997).

3.17.4.1

GnRH Receptor

The pituitary GnRH receptor has been cloned from a substantial number of mammalian and premammalian species (Sealfon et al., 1997; Tsutsumi et al., 1992). This molecule is a member of the seven-transmembrane receptors superfamily. Because of the modest length of both the (extracellular) amino and (intracellular) carboxyl terminal, the mammalian GnRH receptor type I (hereafter referred to only as ‘GnRHR’), is among the smallest members of this superfamily (Ulloa-Aguirre and Conn, 2000; Millar, 2003) and may approach the smallest size G protein-coupled receptor (GPCR) that can bind ligand and transduce an intracellular signal. In birds, fish, and reptiles, the carboxyl tail is significantly longer, due to an extended carboxyl terminal, and this may reflect the altered regulation in these species, compared to mammals (Millar, 2003; BlomenrThr et al., 1999; Heding et al., 1998; Lin et al., 1998a) In the primate GnRHR, the expression levels are relatively low, an effect which appears attributable to the presence of a Lys (Pfaff et al., 1994) not present in rat or mouse sequences (Arora et al., 1999; Maya-Nunez et al., 1997). The removal of this amino acid dramatically increases expression levels, and there appears to be functional interaction between modifications at this and other sites (Maya-Nunez et al., 1997).

3.17.4.2

Effector Coupling

Like other members of the GPCR superfamily, the GnRHR appears functionally coupled to multiple G proteins via conformational selectivity, and appears significantly promiscuous, depending on the availability of G proteins (Naor and Huhtaniemi, 2013). Evidence for coupling to multiple G proteins (including the Gaq/11, Gas, and Gai/o proteins) as well as activation of non-G protein-mediated pathways comes from overexpression and palmitoylation studies (Stanislaus et al., 1997, 1998), identification of separate sites of interaction (Arora et al., 1999; Ulloa-Aguirre et al., 1998), and other experimental approaches in distinct cell contexts (Naor and Huhtaniemi, 2013; Fink et al., 2010).

The Gonadotropin-Releasing Hormone and Its Receptor

This observation has been an attractive consideration in an explanation of how a single class of ligand interacting with a single class of receptor can regulate multiple end points in coordinated, yet independent functions (release and biosynthesis of LH, FSH, and secretogranin; up- and downregulation of receptors; and sensitization and desensitization). Several reports (Kaiser et al., 1995; Pinter et al., 1999) in which different techniques were used to provide variable levels of receptor per cell suggest that differential regulation may be related to receptor number. In this way, the stimulus (GnRH) and the status of the target cell both participate in defining the responses elicited.

3.17.4.3

Receptor–Receptor Interactions

A series of observations dating back to the 1980s suggest (1) techniques that cause GnRHR to associate provoke all known actions of the releasing hormone, even when the receptor is occupied by an antagonist (Conn et al., 1982a), (2) the occupancy of the receptor by agonists is sufficient to cause receptors to associate to within a distance of 50–100 Ǻ (Conn et al., 1982a,b; Cornea et al., 2001), and (3) this process (termed microaggregation, oligomerization, or, potentially inaccurately, dimerization) is distinct from (and temporally a precursor to) the process of patching, capping, and internalization (macroaggregation) that is associated with the extinction of the response system. These data are consonant with a role of microaggregation in GnRHR signaling. Evidence for receptor–receptor interactions have now been observed for a number of GPCRs (Moore et al., 1999; Conn et al., 1985; Pace et al., 1999; Harmatz et al., 1985; Hebert et al., 1996; Blakely et al., 2000; Heldin, 1995; Gether, 2000) and are suggested to be important for the explanation of independent mediation of responses (Pinter et al., 1999; Thomas et al., 2007) and for heterogeneous receptor regulation (Rocheville et al., 2000a,b).

3.17.4.4

Receptor Trafficking

3.17.4.4.1 Endoplasmic Reticulum Quality Control System and the Role of Endogenous Chaperone Proteins Molecular chaperones serve as a control mechanism for recognizing, retaining, and targeting misfolded proteins for their eventual degradation. These proteins are key components of the endoplasmic reticulum (ER) quality control system (QCS), a complex sorting system that identifies and separates newly synthesized proteins according to their maturation status (Ulloa-Aguirre et al., 2004a). Although the steric character of the protein backbone restricts the spectrum of protein shapes that are recognized by the stringent quality control mechanisms, some features displayed by proteins, including exposure of hydrophobic shapes, unpaired cysteines, immature glycans, and particular sequence motifs, have been identified as important for chaperone–protein association (Ellgard and Helenius, 2001; Dong et al., 2007). In fact, molecular chaperones possess the ability to recognize misfolded proteins by the exposure of hidden hydrophobic domains or specific sequences (Dong et al., 2007; Tan et al., 2004). Through this association, chaperones may stabilize unstable conformers of nascent polypeptides to prevent aggregation and facilitate correct folding, or

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assembly, of the substrate via binding and release cycles (Hartl and Hayer-Hartl, 2002). Several GPCR interacting proteins that support trafficking to the cell surface have been identified. Nina A (neither inactivation nor afterpotential A), a photoreceptor-specific integral membrane glycoprotein, is a molecular chaperone that facilitates cell surface membrane expression of the sensory GPCR rhodopsin 1 in Drosophila melanogaster; its absence leads to rhodopsin 1 ER accumulation and degradation (Shieh et al., 1989; Schneuwly et al., 1989; Colley et al., 1991). Its mammalian homolog RanBP2 specifically binds red/green opsin molecules and acts as a chaperone aiding proper folding, transport, and localization of the mature receptors to the cell membrane (Ferreira et al., 1996). ODR4 is a molecular chaperone that assists in folding, ER exit and/or targeting of olfactory GPCRs (e.g., ODR10 in the nematode Caenorhabditis elegans) to olfactory cilia (Dwyer et al., 1998). Calnexin and calreticulin are molecular chaperones that bind a broad range of glycoproteins, including several GPCRs (e.g., the GnRHR, vasopressin-2 receptor, and the glycoprotein hormones receptors) (Vassilakos et al., 1998; Schrag et al., 2003; Rozell et al., 1998; Morello et al., 2001; Helenius et al., 1997; Brothers et al., 2006). The action of these chaperones predominantly centers on substrate N-glycans present on the newly synthesized proteins, adding hydrophobicity to the folding protein (Schrag et al., 2003; Rozell et al., 1998; Morello et al., 2001; Helenius et al., 1997). When N-linked glycosylation or early glycan processing fails, glycoproteins misfold, aggregate, and fail the QCS (Morello et al., 2001). RAMPs (receptor activity modifying proteins) are proteins that interact with several GPCRs (e.g., the calcitonin receptor-like receptor, the vasoactive intestinal peptide/pituitary adenylate cyclaseactivating peptide receptor, the glucagon receptor, and the parathyroid hormone receptor) fostering the transport of the associated receptor to, and regulating its signaling function at, the PM (Christopoulos et al., 2003), whereas gC1q-R (receptor for globular heads of C1q) interacts with the carboxyl terminus of the alpha1B-adrenergic receptor and regulates the maturation and expression of the receptor (Xu et al., 1999). Another molecular chaperone is BiP/Grp 78, which is involved in the protective unfolded protein response; a cell stress program activated when misfolded proteins accumulate and/or aggregate in the ER (Yang et al., 1998; Schroder and Kaufman, 2005). Finally, DriP78 is an ER membrane-associated protein that binds to the F(x)3F(x)3F motif of the dopamine receptor (and presumably other GPCRs bearing this motif) thereby facilitating its maturation and export to the PM (Bermak et al., 2001). Identification of these particular molecular chaperones is important since they represent a potential target to manipulate ER retention and/ or export mechanisms, and hence a means for influencing protein trafficking and secretion (Aridor and Balch, 2000; Rivera et al., 2000).

3.17.4.4.2 Mutant GnRHRs Isolated from Patients with Hypogonadotropic Hypogonadism (HH) Are Actually Misfolded and Misrouted Proteins That Can Be Rescued and Restored to Function Results over the last 7 years have led to the conclusion that from the 37 inactivating mutations (including deletions of

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two large sequences) in the GNRHR described to date as a cause of partial or complete forms of normosmic HH, at least 17 are actually misfolded proteins (Aridor and Balch, 2000). All these 17 genetic mutations resulted from single amino acid changes in the GnRHR. Most frequently this was a change in charge (12 instances); the remainder involved a gain or loss of Cys (4 instances) or a loss of a Pro (1 instance). Each of these types of changes might be expected to have a substantial impact on the overall conformation of the receptor, unlike a conservative substitution (i.e., Gly for Ala) for example. As mis-shapened (‘misfolded’) proteins, the single amino acid mutants characteristically failed the cellular QCS and misrouting occurred (Ulloa-Aguirre et al., 2004a); in the cases examined, misrouting was due to retention in the ER (Janovick et al., 2003a; Leaños-Miranda et al., 2003, 2005; Ulloa-Aguirre et al., 2004b). The detected mutations in the human GNRHR are distributed along the entire coding sequence of the receptor with few exceptions, the intracellular loop (IL) 1 and the extracellular loop (EL) 3. In fact, in a number of these GnRHR mutants the function may be partially or completely rescuable in vitro and in vivo by physical, genetic, and/or pharmacologic means, whenever the mutation does not replace critical residues involved in agonist binding or effector activation. The corollary of the observation that these are misrouted proteins is that, if restored to the plasma membrane, these mutants can become functional (Janovick et al., 2003a; Leaños-Miranda et al., 2003, 2005). This happens because they have retained (or regain) both the ability to bind agonist and the ability to transduce a signal. This differs from a prior view that most mutants are defective because they have (permanently) lost the ability to bind ligand or couple to effector proteins. The ability to rescue and restore proteins to function opens a new avenue in therapeutics which appears broadly applicable to disease-causing mutants of the GnRHR. Restoration of binding and coupling activity of mutants was initially shown by adding plasma membrane-targeting sequences to ‘defective’ human GnRHR mutants i.e., making chimeras of human mutants with the C terminal sequence found in catfish GnRHR that increases plasma membrane targeting of the GnRHR (Lin et al., 1998b) or by deleting an amino acid (K (Pfaff et al., 1994)) found in the human (and other primates) wild type (WT) receptor that, when present, decreases routing to the plasma membrane. Such modifications led to rescue of the mutant sequences as assessed by both radioligand binding and by the ability of agonist to activate Gaq/11 proteinmediated inositol phosphates (IP) production (Lin et al., 1998b; Janovick et al., 2003b). More recently (Janovick et al., 2003a, 2007; LeañosMiranda et al., 2003, 2005) we learned that it is possible to use a pharmacological approach and show that small, nonpeptide molecules (pharmacologic chaperones or ‘pharmacoperones’) which bind the GnRHR mutants can stabilize most of the mutant proteins (15 of 17) in a conformation that passes the QCS; these rescued mutants are not retained in the ER, but route correctly to the plasma membrane, where they bind ligand (shown with radioligand) and successfully transduce signaling (shown by IP response). Such pharmacoperones are, of course, lead drug candidates.

3.17.4.4.3 The Ability to Rescue Mutant Proteins Using Pharmacoperones Has Therapeutic Potential Pharmacoperones for the GnRHR mutants have now been identified from three different chemical classes (indoles, quinolones, and erythromycin macrolides (Janovick et al., 2003a) using combinational chemical libraries. Within each chemical class there is an effect of mutant rescue that is proportional to the affinity of binding to the receptor (Janovick et al., 2003a). In retrospect, it is not surprising that concentrations needed for rescue differ between chemical classes of pharmacoperones since it is likely that the specific chemical interaction between the drugs and the mutants would stabilize the mutants by distinct means (that is, by interacting with different residues on the receptor). Only two of the mutants in the group discussed above (the first 17 GnRHR mutants reported, excluding deletion and truncation mutants) cannot be at least partially rescued by this (pharmacoperone) approach (Janovick et al., 2006). In these two cases, this occurs because the mutation leads to a change that is so distorting to the protein structure that rescue does not occur. In both of these cases (amino acids 168 and 217), the mutation is identical, Ser / Arg and both mutations occur in transmembrane segments (transmembrane segments 4 and 5, respectively), resulting in a highly unfavorable thermodynamic change that causes a specific (Janovick et al., 2006) effect on the receptor configuration (Knollman et al., 2005). Because the pharmacoperones are successful in rescuing the remaining 15 mutants – even though the loci of the mutations are widely distributed over the receptor – this argues for the generality of this approach for mutants of the human GnRHR. Moreover, the drugs used as pharmacoperones were initially developed as oral antagonists of GnRH for use in humans; accordingly there is extensive safety and pharmacokinetic and pharmacodynamic data available in human and animal models which will make it easier to move the rescue technique to in vivo rescue situations. The efficacy of rescue of mutant GnRHR has been shown in vivo using a mouse model expressing a mutant that causes HH in humans and in mice (Janovick et al., 2013) and highthroughput screens have been devised to identify other pharmacoperones for GnRHR mutants (Conn and Janovick, 2011; Janovick et al., 2011; Conn et al., 2014a; Smithson et al., 2013) as well as disease-causing mutants of the vasopressin type 2 receptor and enzyme mutants that cause hyperoxaluria (Conn et al., 2014a,b, 2015; Ulloa-Aguirre et al., 2014, 2015; Madoux et al., 2015; Tao and Conn, 2014).

3.17.4.4.4 The Rescue Approach Appears Generally Applicable to Other Mutant GPCRs, Non-GPCR Receptors, Ion Channels, and Enzymes Associated with Disease: This Supports the Importance of Understanding the Mechanism of This Event in a Well-Defined System Mutants of other GPCRs, non-GPCR receptors, as well as ion channels, and enzymes can be rescued by this approach and restored to activity (for example, Castro-Fernandez et al., 2005). The ability to rescue misfolded proteins with drugs presents a new therapeutic approach for a remarkably diverse palette of diseases caused by other misfolded molecules: Alzheimer’s disease, cataracts, cystic fibrosis, familial hypercholesterolemia, HH, nephrogenic diabetes insipidus, and retinitis

The Gonadotropin-Releasing Hormone and Its Receptor

pigmentosa, among others (Ulloa-Aguirre et al., 2004a,b, 2003; Conn and Janovick, 2005; Maegawa et al., 2007; Lumb et al., 2003; Vij et al., 2006; Biswas et al., 2004; Biswas and Das, 2004; Kim et al., 2006; Lashuel and Hirling, 2006; Cashman and Caughey, 2004; Mallucci and Collinge, 2005). In the case of the human GnRHR, this receptor also appears to provide opportunities for regulation by ‘intentionally’ misfolding a portion of the WT receptor, resulting in a circumstance in which a proportion of the synthesized molecule is retained in the ER (Janovick et al., 2006; Conn et al., 2006a,b). Other GPCRs (Janovick et al., 2006) appear to share the ‘balance’ between the ER and the plasma membrane and therefore these WT receptors may be candidates for this rescue approach – when the therapeutic goal is to increase the plasma membrane expression of these moieties. Accordingly, it is useful to consider potential uses (Conn et al., 2007) of pharmacoperones in four areas: (1) to prevent misfolding of molecules that do so and lead to disease (e.g., cataracts, neurodegenerative diseases); (2) to rescue mutants (e.g., mutant GnRHR in HH, cystic fibrosis transmembrane conductance regulator in cystic fibrosis, a-galactosidase A in Fabry’s disease); and (3) to increase (or decrease) the percentage of WT molecules that route to the membrane, when only a fraction of the total synthesized material is expressed at the plasma membrane, as is the case for the WT human GnRHR (Conn et al., 2006a,b). There is a fourth condition in which perturbation of the ER homeostasis results in disorders because of the promotion in the synthesis of deleterious products potentially treatable with pharmacoperones; a good example of this is prion replication (Hetz et al., 2007).

Acknowledgments This work was supported by National Institutes of Health Grants DK085040 and DK099090, NIH AG 025047 the Hyperoxaluria Foundation, and CONACyT grant 240619.

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