51 The Gonadotropin-Releasing Hormone and Its Receptor L Jennes, University of Kentucky, Lexington, KY, USA A Ulloa-Aguirre, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico; Oregon National Primate Research Center, Beaverton, OR, USA J A Janovick, Oregon National Primate Research Center, Beaverton, OR, USA V V Adjan, University of Kentucky, Lexington, KY, USA P M Conn, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico; Oregon National Primate Research Center, Beaverton, OR, USA; Oregon Health and Science University, Portland, OR, USA ß 2009 Elsevier Inc. All rights reserved.Chapter Outline51.1 Introduction 164651.2 GnRH Neuronal Systems 164651.2.1 Embryonic Development of the GnRH System 164651.2.2 Postnatal and Adult GnRH Systems 164722.214.171.124 GnRH-containing cell bodies 1647126.96.36.199 GnRH-containing projections 1647188.8.131.52 Associations with the cerebrospinal fluid 1648184.108.40.206 Cytology of GnRH hormone neurons 164851.2.3 Regulation of GnRH Neurons 1648220.127.116.11 Catecholamines 164918.104.22.168 Glutamate 165051.2.3.3 Gamma-aminobutyric acid 16522.214.171.124 Neuropeptides 165251.3 GnRH Receptors in the Central Nervous System 165451.3.1 Localization of GnRH Receptors in the Brain 165451.3.2 Characterization of GnRH Receptors in the Brain 165451.3.3 Regulation of GnRH Receptor Expression in the Brain 165551.3.4 Functional Aspects of GnRH Receptors in the Brain 165651.4 Molecular and Cellular Mechanism of GnRH Action in the Anterior Pituitary 165651.4.1 GnRH Receptor 165651.4.2 Effector Coupling 165751.4.3 Receptor–Receptor Interactions 165751.4.4 Receptor Trafficking 1657126.96.36.199 Endoplasmic reticulum quality control system and the role of endogenous chaperone proteins 1657188.8.131.52 Mutant GnRHRs isolated from patients with hypogonadotropic hypogonadism are actually misfolded and misrouted proteins that can be rescued and restored to function 1658184.108.40.206 The ability to rescue mutant proteins using pharmacoperones has therapeutic potential 1659220.127.116.11 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 1659References 1660Further Reading 1668 1645
1646 The Gonadotropin-Releasing Hormone and Its Receptor51.1 Introduction most GnRH cells have reached their final destina- tion (Schwanzel-Fukuda and Pfaff, 1989; WrayGonadotropin-releasing hormone (GnRH) is a dec- et al., 1989a,b). However, many GnRH neuronsapeptide that is synthesized by relatively few neurons remain associated throughout life with the ganglionin the ventral forebrain. These neurons project axons terminale and the associate branches of the termi-to the median eminence of the mediobasal hypothal- nal nerve (Schwanzel-Fukuda and Silverman, 1980;amus where the hormone is released into the peri- Schwanzel-Fukuda and Pfaff, 1990, 1991; Jennes andvascular space of fenestrated capillaries and carried Schwanzel-Fukuda, 1992; Schwanzel-Fukuda et al.,through the blood to the anterior pituitary. GnRH 1992; Schwanzel-Fukuda, 1999; Jennes and Stumpf,binds to and activates specific membrane receptors 1980a). The time course of the migration of GnRHon the gonadotropes to stimulate the synthesis and neurons and initiation of GnRH peptide synthesisrelease of follicle-stimulating hormone (FSH) and has been confirmed with in situ hybridization studiesluteinizing hormone (LH). These pituitary hor- that showed that the neurons begin to synthesizemones, in turn, cause follicular growth and ovulation GnRH mRNA at E10 and that by E11 translationin the ovary, as well as steroid hormone synthesis. into GnRH peptide reaches detectable levels (WrayThe ovarian hormones, estrogen and progesterone, et al., 1989a). Additional experiments using thymi-feed back to the brain and pituitary in an inhibitory dine incorporation into newly formed DNA con-fashion throughout the reproductive cycle, except for firmed that GnRH neurons indeed originate froma very short period during proestrus when a change the olfactory placode and not from the ventricularto a positive feedback stimulates GnRH, LH, and lining (Schwanzel-Fukuda and Pfaff, 1989). ThisFSH release to induce ovulation. In the rat, the view is further supported by studies of the small-appropriate pulsatile release of GnRH is critical for eye mouse, which fails to develop eyes and olfactorythe maintenance of an estrous cycle and for repro- placodes due to a mutation in the pax-6 gene. Homo-duction in general. zygote mice do not contain GnRH neurons in the nasal region or in the forebrain, whereas hetero- zygotes contain a normal GnRH neuronal system51.2 GnRH Neuronal Systems (Dellovade et al., 1998). Several other regions in the mouse brain were described to contain neurons that51.2.1 Embryonic Development of the express GnRH transiently during the pre- and earlyGnRH System postnatal period, and it was shown that these neuronsAlthough GnRH neurons reside in the rostroventral in the lateral septum, bed nucleus of the stria term-forebrain in the adult mammal, these neurons origi- inals, and tectum are not derived from the olfactorynate in the olfactory placode. Using immunohisto- placode (Wu et al., 1995; Skynner et al., 1999). Thesechemistry, GnRH-synthesizing neurons are first cells synthesize the mammalian form of the GnRHidentified at embryonic day 11 (E11) in the epithe- decapeptide, as is suggested by their immunoreactiv-lium of the medial olfactory placode (Schwanzel- ity to a variety of well-characterized antibodies. TheFukuda and Pfaff, 1989; Wray et al., 1989a). Here, biological significance of this subset of GnRH neu-the GnRH neurons appear oval or fusiform and have rons is not understood; however, the lack of access ofnot yet developed identifiable axons (Zheng et al., these neurons to fenestrated capillaries precludes an1992). GnRH neurons begin to leave the epithelium endocrine function.at E12 as cell clusters that migrate through the crib- The exact cell lineage of GnRH neurons is notriform plate to the ganglion terminale. During this clear. It was widely accepted that GnRH neuronsearly stage, approximately 50% of the GnRH neu- were closely related to olfactory sensory neuronsrons also express galanin and this percentage declines (Wray et al., 1989b), but later studies (Hilal et al.,as the migration continues (Key and Wray, 2000). 1996) showed that in chickens and in mice carrying aOver the subsequent few days, most GnRH cells mutation in the transcription factor activator proteinenter the ventromedial forebrain as part of the roots 2a (Kramer et al., 2000) GnRH neurons are present inof the nervous terminalis, and fewer cells reach the the respiratory epithelium. Moreover, the removal ofaccessory olfactory bulb along the vomeronasal the embryonic region of the nose that would differ-nerve. By E14, GnRH cells begin to reach the septum entiate into the respiratory epithelium eliminatesand preoptic region. The migration of the GnRH GnRH neurons, whereas the removal of the olfactoryneurons continues through birth and at that time epithelium region has no impact on GnRH
The Gonadotropin-Releasing Hormone and Its Receptor 1647neuronal development (el Amraoui and Dubois, number of immunoreactive neurons rapidly declines1993). Together, these data indicate that GnRH pro- from rostral to caudal in the medial preoptic area andgenitor cells may actually be more closely related to anterior hypothalamic regions. Single GnRH neuronsrespiratory cells than to olfactory cells and more are consistently seen in a small region right above thestudies are needed to clarify the exact origin of supraoptic nuclei. In the rat, the mediobasal hypothal-GnRH neurons. amus does not contain immunoreactive GnRH Similarly, very little is known about the mechan- perikarya.isms that control the migration of GnRH neuronsfrom the nose into the forebrain. It appears that 18.104.22.168 GnRH-containing projectionsstrands of GnRH neurons travel along a track that The most extensive axonal projection reaches thecontains the neural cell adhesion molecule (NCAM), median eminence of the ventral hypothalamus throughbut not laminin, cytotactin, fibronection, or cytactin diffuse projections that include periventricular, supra-(Schwanzel-Fukuda et al., 1992, 1994). These initial chiasmatic, and subchiasmatic pathways. Some of thesefindings stimulated a series of experiments in which projections traverse many hypothalamic nuclei, suchantibodies to NCAM were administered to 10-day- as the periventricular gray, paraventricular nucleus,old embryos, and it was shown that this treatment ventromedial nucleus, and arcuate nucleus. In thedisrupted the migration of embryonic GnRH neu- median eminence, GnRH-containing axons sproutrons (Schwanzel-Fukuda et al., 1994). However, extensively and most terminals are located in thebecause GnRH neurons reach their final position at external layer next to fenestrated capillaries, with thethe appropriate time in mice that do not express highest concentrations in the regions that sur-NCAM (Schwanzel-Fukuda et al., 1995), it appears round the infundibular sulcus (Silverman et al., 1979;that NCAM is not required for an appropriate migra- Merchenthaler et al., 1980, 1984; Jennes and Stumpf,tion (for review, see Schwanzel-Fukuda (1999)). 1980 a,b; Hoffman and Gibbs, 1982). This is the siteMigrating GnRH neurons follow a track of axons where GnRH is released into the portal blood to con-in vivo and in vitro that contain the intermediate trol the activity of the anterior pituitary gonadotropes.filament peripherin (Fueshko and Wray, 1994; Wray It has been shown in the rat that a single microin-et al., 1994). Peripherin is typically expressed in jection of retrograde tracer into the median eminenceolfactory receptor neurons (Gorham et al., 1991), labels approximately 65% of all GnRH neurons,and the close spatial relation between migrating which suggests that most GnRH neurons project toGnRH neurons and olfactory axons could indicate this neurohemal contact zone (Silverman et al., 1987).interactions of the two cell types in the guidance pro- Because such an injection covers only a small portioncess or common underlying mechanisms of guidance. of the median eminence, the percentage of GnRH neurons projecting to the median eminence is proba- bly substantially higher. An additional major GnRH-51.2.2 Postnatal and Adult GnRH Systems containing projection terminates in the OVLT, which like the median eminence is a circumventricularMany reviews have been published on the anat- organ that contains fenestrated capillaries. Althoughomy of the GnRH neuronal systems in the rodent it is clear that GnRH released in the median emi-(Barry et al., 1985; Jennes and Conn, 1994; Silverman nence controls LH and FSH release, the physiologi-et al., 1994); it is beyond the scope of this chapter cal significance of GnRH released at the OVLT is notto provide a detailed description of the entire known because only a venous vascular link exists withGnRH system. the pituitary portal system. The remaining projections of GnRH-containing22.214.171.124 GnRH-containing cell bodies axons are rather limited and involve only a few axonsIn the postnatal rat, most GnRH neurons are located per site. Areas in the brain that contain GnRH axonsaround the organum vasculosum of the lamina termi- include olfactory structures, such as the main andnalis (OVLT) in a dispersed distribution pattern that accessory olfactory bulb, the islands of Calleja, andis reminiscent of an inverted Y. GnRH neurons the pyramidal region of the piriform cortex. In addi-extend from the medial portions of the horizontal tion, a few axons reach the medial and cortical nucleilimb of the diagonal band through the vertical limb of the amgdala via the ventral amygdalal–fugal path-into the medial septum. Slightly fewer neurons are way and the stria teminalis. Single axons are also seenpresent in the rostral periventricular area, and the in the ventral hippocampus and the subiculum.
1648 The Gonadotropin-Releasing Hormone and Its ReceptorCaudal projections pass from the medial septum– 126.96.36.199 Cytology of GnRH hormone neuronsdiagonal band through the stria medullaris, medial GnRH neurons appear as unipolar or bipolar cellshabenula, and the fasciculus retroflexus to the inter- that can have either smooth contours or have a spinypeduncular nucleus from which single fibers turn appearance; however, the significance of these mor-dorsally into the raphe nuclei and the central gray. phological differences has not been elucidated (Krisch, 1980; Liposits et al., 1984; Jennes et al.,188.8.131.52 Associations with the 1985; Wray and Hoffman, 1986). Ultrastructural stud-cerebrospinal fluid ies have shown that GnRH neurons are rather simpleA unique feature of the GnRH neuronal system that neurons that have all the standard organelles requiredmay have important functional implications is the for appropriate functioning (Mazzuca, 1977; Jennesclose anatomical association of GnRH axons to et al., 1985; Witkin and Demasio, 1990). While earlierthe ventricular and subarachnoid cerebrospinal fluid light and electron microscopic immunohistochemical(Burchanowski et al., 1979; Jennes and Stumpf, studies have failed to identify dendrites of GnRH1980b). Thus, GnRH fibers are present consistently neurons ( Jennes et al., 1985; Witkin and Silverman,in or next to the ependyma of the ventromedial 1985), more recent studies using microinjections ofportion of the septal lateral ventricles, the ventral biocytin into green fluorescent protein expressingportions of the hypothalamic third ventricle, the sub- GnRH neurons clearly show that GnRH neuronsfornical organ, and the aqueduct. It appears that, at have long, mostly unbranched dendrites which con-least in some of these regions, GnRH axons penetrate tain many spine-like extensions (Campbell et al.,the ependyma and thus have direct contact with the 2005). GnRH axons form presynaptic specializationsinternal cerebrospinal fluid. Similar contacts occur on the dendrites or perikarya of other hypothalamicon the outer surface of the brain, especially at the neurons, which indicates that the GnRH peptideolfactory bulb where the ganglion terminale and the is released at these sites and probably acts as anervus terminalis reside in the subarachnoid space, neurotransmitter ( Jennes et al., 1985; Witkin andthe prechiasmatic cistern, and the interpeduncular Silverman, 1985). Interestingly, some of the postsyn-cistern. Based on these anatomical findings and stud- aptic neurons also contain GnRH, and it has beenies that measured GnRH levels in the cerebrospinal suggested that this kind of innervation could representfluid (Van Vugt et al., 1985; Skinner et al., 1995), it a mechanism by which the activity of GnRH neuronalis suggested that release of the hormone into the system is synchronized (Leranth et al., 1985b; Witkinportal blood may be coupled or synchronized with and Silverman, 1985). However, such contacts betweenthe release of GnRH into the cerebrospinal fluid. GnRH neurons are fairly rare and may not be suffi-This view is supported by the finding that increased ciently frequent to represent a relevant mechanism oflevels of GnRH and LH in the blood are paralleled activity coupling among the GnRH neurons.by increases in the GnRH concentrations in thecerebrospinal fluid. GnRH in the cerebrospinal 51.2.3 Regulation of GnRH Neuronsfluid could reach distant sites of action and haveprolonged effects because endo- or exopeptidases The protein synthesis and release activity of GnRHare present only in very low levels in these fluid- neurons is regulated by complex feedback loops thatfilled compartments (Mendez et al., 1990). The sce- include the gonadal steroid hormones estradiol andnario of a coordinated release of GnRH could repre- progesterone, as well as a large number of neuro-sent an efficient way by which the endocrine effects transmitter systems (for review, see Kalra (1993),of GnRH on the pituitary gonadotropes could be Kordon et al. (1994), and Levine (1997)). Estradiolcoupled with the intracerebral effects of GnRH, for exhibits an inhibitory effect on GnRH neuronal sys-example, the facilitation of reproductive behaviors. tems during all phases of the reproductive cycle,An additional mechanism by which GnRH release at except for the proestrous stage when rising estradioltwo anatomically distinct sites could be coordinated levels cause a switch to a positive feedback mode thathas been suggested based on multiple retrograde stimulates GnRH release, which in turn causes a mas-labeling experiments that show that one GnRH neu- sive surge in pituitary LH secretion that is necessaryron can terminate at the perivascular space of fene- for ovulation. The mechanisms underlying this tempo-strated capillaries and, through axon collaterals, at rary positive feedback are not known but it appearsspecific nuclei in the brain, such as the interpedun- that direct effects of estradiol on GnRH neurons arecular nucleus ( Jennes, 1991). probably not the trigger for the LH surge. GnRH
The Gonadotropin-Releasing Hormone and Its Receptor 1649neurons express only low levels of estrogen receptor- b (Hoffman, 1985). Although the limitations of the(ER-b) which is not necessary for the induction of resolution of light microscopy do not permit a distinc-ovulation (Hrabovszky et al., 2000). However, the man- tion between synaptic contacts and en passant axons,datory ER-a is not present in GnRH neurons. It is a close proximity of axons to GnRH neurons pro-therefore thought that input from steroid-sensitive vides an anatomical rationale for further in-depthinterneurons to the GnRH neurons is required for studies. Additional electron microscopic studies havethe generation of a GnRH-mediated LH surge. determined that some of these close appositions are Many studies have focused on the identification indeed synaptic in nature and that tyrosine hydroxy-of neurotransmitter systems that regulate GnRH lase (Leranth et al., 1988a; Chen et al., 1989b), gamma-neuronal activity by administering agonists or antag- aminobutyric acid (GABA) (Leranth et al., 1988b),onists during the preovulatory or estrogen- and glutamate (Goldsmith et al., 1994), or b-endorphinprogesterone-induced LH surge. Data from such (Chen et al., 1989a) are contained in these terminals.studies are summarized in several excellent reviews(Kalra, 1993; Kordon et al., 1994; Levine, 1997). Col- 184.108.40.206 Catecholamineslectively, these studies show that most manipulations Noradrenergic and adrenergic axons are in closeof a neurotransmitter system have an impact on proximity to GnRH neurons (Moore et al., 1999).GnRH-mediated LH release, either stimulating LH Some of these fibers form synaptic complexessecretion to various degrees or reducing circulating with the GnRH neurons (Leranth et al., 1988b;LH levels. Most treatments that result in a stimulation Chen et al., 1989b), and GnRH neurons express theof GnRH neurons produce a two- to fivefold increase a1B adrenergic receptor mRNA (Petersen et al.,in LH levels, which is substantially smaller than LH 1999) and protein (Hosny and Jennes, 1998). Thislevels during the surge. So far, only a sequential treat- suggests that the catecholamines participate directlyment with estrogen and progesterone has been able in the control of GnRH neuronal activity. Oneto induce proestrus-like LH levels. On the other requirement for an involvement in the regulation ofhand, most antagonists of stimulatory neurotrans- GnRH neurons is the capability of the catecholamin-mitters can prevent the surge. These findings suggest ergic neurons to convey the steroidal signal. Severalthat it is probably not a single neurotransmitter sys- studies have provided strong evidence that this istem that induces GnRH neurons to release preovula- indeed the case. Thus, noradrenergic neurons accu-tory amounts of the peptide but instead several mulate 3H-estradiol in their nuclei (Heritage et al.,systems that participate either in parallel or sequen- 1977, 1980), ER-a, and probably ER-b; mRNA istially in the induction of the surge. Interference with present in the appropriate regions A1 and A2 of theonly one of these stimulators abolishes the surge. brainstem (Shughrue et al., 1997); and many of the One approach to identifying the neurotransmitters noradrenergic and adrenergic neurons contain ER-aand neuropeptides that participate in the control of protein (Simonian and Herbison, 1997; Lee et al.,GnRH neuronal activity is to use multiple immuno- 2000a). Moreover, tyrosine hydroxylase (Liaw et al.,histochemical stainings for GnRH and select neuro- 1992) and dopamine-b-hydroxylase gene expressiontransmitter, to determine which transmitter is present (Serova et al., 2000) are stimulated by estradiol, andin axons that are next to GnRH cell bodies. These the noradrenergic and adrenergic neurons in thestudies have demonstrated that catecholaminergic brainstem respond to estradiol by transient synthesis(Ajika, 1979; Jennes et al., 1982, 1983; Hoffman et al., of the transcription factor fos (Lee et al., 2000a). The1982; Leranth et al., 1988a; Chen et al., 1989b), sero- noradrenergic and adrenergic input to the GnRHtoninergic ( Jennes et al., 1982; Kiss and Halasz, 1985), system is critical for the maintenance of pulsatilegamma-aminobutyric acidergic (GABAergic; Jennes GnRH release and the induction of a preovulatoryet al., 1983; Leranth et al., 1985a; Witkin, 1992), and and estrogen- and progesterone-induced LH surge,glutamatergic axons (Goldsmith et al., 1994; Eyigor as has been shown repeatedly with physiologicaland Jennes, 1997) were juxtaposed to GnRH neurons approaches (for review, see Kordon et al. (1994) andas were axons that contained the neuropeptides, Herbison (1997a)). Thus, the inhibition of eitherb-endorphin (Leranth et al., 1988a; Chen et al., norepinephrine or epinephrine synthesis results1989a,b), galanin (Merchenthaler et al., 1991), neuro- in a markedly reduced LH secretion in ovariecto-peptide Y (Li et al., 1999), vasoactive intestinal peptide mized rats, suggesting that both neurotransmitters(VIP; van der Beek et al., 1993, 1994; Smith et al., regulate basal pulsatile GnRH–LH release (Drouva2000), substance P (Hoffman, 1985), or neurotensin and Gallo, 1976). Similar treatments with specific
1650 The Gonadotropin-Releasing Hormone and Its Receptorsynthesis inhibitors also prevent the steroid-induced 220.127.116.11 GlutamateLH surge, indicating an important role in the genera- Immunohistochemical studies have shown that glu-tion of the preovulatory surge (Kalra et al., 1972; tamate or neuron-specific vesicular glutamate trans-Kalra and McCann, 1974; Crowley and Terry, 1981; porters (VGLUTs) are present in many axonCrowley et al., 1982; Coen and Coombs, 1983; for terminals that innervate GnRH neurons (Goldsmithreview, see Herbison (1997a)). On the other hand, et al., 1994; Lin et al., 2003), which suggests an impor-the administration of norepinephrine, epinephrine, tant stimulatory role of this excitatory neurotransmit-or a-adrenergic agonists causes a significant elevation ter in the control of GnRH neurons (Figure 1). Theof GnRH-stimulated LH levels (Rubinstein and findings that glutamatergic neurons in the septalSawyer, 1970; Gallo and Drouva, 1979; Kalra and complex and the hypothalamic arcuate and ventro-Gallo, 1983; Barraclough et al., 1984; Levine et al., medial nuclei as well as in the preoptic region express1991). These pharmacological studies are of physio- ER-a (Thind and Goldsmith, 1997; Eyigor et al.,logical relevance because measurements of norepi- 2004) and that short-term treatment with estradiolnephrine and epinephrine release (Mohankumar causes an increase in glutamate content (Luine et al.,et al., 1994) and turnover during select phases of the 1997) suggest that the glutamate neurons are affectedestrous cycle or the steroid-induced LH surge show by gonadal steroids and could convey the steroidalthat catecholamine turnover in the median eminence signals to the GnRH neurons. Glutamate release inis increased just before and during the LH surge the preoptic region is increased just prior to and(Wise et al., 1981; Wise, 1982; Adler et al., 1983; during the steroid-induced LH surge, as measuredSheaves et al., 1984). Together, based on anatomical with push–pull perfusion or in vivo dialysis ( Jarryand physiological evidence, the catecholamines can et al., 1992, 1995; Ping et al., 1994; Demling et al.,be viewed as important participants in the regulation 1985). Many studies have shown that glutamate andof GnRH release. its agonists AMPA, N-methyl-D-aspartate (NMDA), (a) (d) VGLUT2 (b) Synaptophysin (c) GnRH Overlay 20 µmFigure 1 Example of immunohistochemical triple labeling for (a) VGLUT2, (b) synaptophysin, and (c) GnRH as well as (d)an overlay of the three images, showing that many glutamatergic terminals are juxtaposed to the GnRH neuron, and someof these terminals contain synaptophysin (arrowheads). Reproduced from Lin W, McKinney K, Liu L, Lakhlani S, and Jennes L(2003) Distribution of vesicular glutamate transporter-2 messenger ribonucleic acid and protein in the septum-hypothalamusof the rat. Endocrinology 144(2): 662–670, Copyright 2003, The Endocrine Society.
The Gonadotropin-Releasing Hormone and Its Receptor 1651 GnRH GluR1 Fos OverlayFigure 2 Example of triple-label immunohistochemistry for GnRH (green), AMPA receptor subunit GluR1 (red) and fosprotein (blue), as well as an overlay of the three images. Scale bar ¼ 30 mm. Reproduced from Bailey JD, Centers A, andJennes L (2006) Expression of AMPA receptor subunits (GluR1-GluR4) in gonadotrophin releasing hormone neurons of youngand middle-aged persistently oestrous rats during the steroid-induced luteinising hormone surge. Journal ofNeuroendocrinology 18(1): 1–12, with permission from Blackwell Publishing.and kainate (KA) stimulate GnRH–LH release in vivo indicates a preferential activation of KA2-containingand in vitro in a dose-dependent manner and, con- GnRH neurons (Eyigor and Jennes, 2000). Similarly,versely, that the blockade of either glutamate receptor the expression of the AMPA subunits GluR1 andsubtype with specific antagonists prevents the surge GluR3 increases significantly in GnRH neurons at(for review, see Brann and Mahesh (1997)). the time just before and during the LH surge, again In order to identify some of the mechanisms by preferentially in GnRH neurons that are activated aswhich glutamate regulates GnRH neurons, GnRH determined by the presence of fos (Bailey et al., 2006).neurons were screened for the presence of various However, glutamate or glutamate agonist administra-glutamate receptor subunit mRNAs and proteins. tion alone produces only a modest increase inThese studies showed that GnRH neurons express GnRH–LH release, and the coordinated release ofmost, if not all, ionotropic glutamate receptor sub- other transmitters is needed to induce a full surge.units (Eyigor and Jennes, 1997; Gore et al., 1996;Ottem and Petersen, 2000; Miller and Gore, 2002; 18.104.22.168 Gamma-aminobutyric acidBailey et al., 2006). Examples are shown in Figure 2. GABA is the major inhibitory neurotransmitter in theSeveral lines of evidence suggest that these glutamate brain and strong evidence exists for an important inhib-receptors are indeed activated during the steroid- itory role in the control of GnRH neuronal activity.induced LH surge. For instance, approximately Thus, intracerebral microinjections of GABA into the50–60% of the GnRH neurons that express transiently preoptic area inhibit the proestrous surge (Herbisonthe transcription factor fos during the LH surge also and Dyer, 1991), and the administration of GABAA andexpress KA2 and GluR5 receptor subunits, which GABAB receptor antagonists has only modest effects
1652 The Gonadotropin-Releasing Hormone and Its Receptoron basal LH release; however, they greatly potentiate membrane; however, a definitive characterization ofthe stimulatory effects of norepinephrine (Hartman such receptors has not been accomplished.et al., 1990). Extracellular GABA concentrations, asmeasured with push–pull perfusion approaches, 22.214.171.124 Neuropeptidesdecline dramatically prior to and during the estrogen- There are many neuropeptides that have been showninduced LH surge, which suggests that the release of a to participate in the regulation of GnRH releasetonic inhibition of the rostral hypothalamic neurons by (for review, see Kordon et al. (1994)); it is beyondGABA parallels the secretory activity of GnRH neu- the scope of this chapter to discuss the effects of allrons ( Jarry et al., 1995). Such a release of a continuous these peptides. We focus here on neuropeptide Yinhibition may also be an important mechanism that (NPY), vasoactive intestinal polypeptide (VIP),participates in the induction of puberty. Thus, GABA b-endorphin, and kisspeptins, because we knowlevels are very high in the mediobasal hypothalamus of most about their actions and receptor expression.the prepubertal monkey and decrease during early andmidpuberty, which is the exact opposite of GnRH 51.2.3(i) Neuropeptide Ylevels (Terasawa et al., 1999). GABAergic neurons con- NPY is probably the best-understood neuropeptidecentrate 3H-estradiol in their nuclei (Flugge et al., ¨ that is part of an excitatory circuit enhancing GnRH1986); contain ER-a protein in their nuclei (Herbison release. Several studies have shown that the adminis-et al., 1993); have extracellular GABA concentrations tration of NPY stimulates GnRH release (Crowleymodulated by estrogen (for review, see Herbison et al., 1987; Crowley and Kalra, 1987; Sabatino et al.,(1997b)); and are, from an anatomical point of view, 1989), whereas intraventricular injection of specificlocated in the appropriate hypothalamic areas to inner- anti-NPY antibodies (Wehrenberg et al., 1989) orvate GnRH neurons. That GABA can control GnRH antisense oligonucleotides (Kalra et al., 1995) blocksneurons directly at the perikaryon or axon terminal is the steroid-induced LH surge. These data suggestsuggested by several studies. Thus, glutamic acid that endogenous NPY is required for the occurrencedecarboxylase (GAD), the rate-limiting enzyme in of a surge. The observations that NPY mRNA con-GABA synthesis, is present in many axons that sur- tent in the arcuate nucleus increases prior to theround GnRH neurons ( Jennes et al., 1983), and Ler- onset of an LH surge (Bauer-Dantoin et al., 1992;anth et al. (1985a) have shown with electron Sahu et al., 1994), that NPY peptide levels in themicroscopy that GAD immunoreactive axons form mediobasal hypothalamus parallel LH levels (Sahupresynaptic terminals on GnRH perikarya. Evidence et al., 1989), and NPY release is enhanced during thethat these synapses are functioning is indicated by the LH surge (Watanobe and Takebe, 1992) supportobservation that approximately 75% of the GnRH the view that the NPY neurons participate in theneurons express the b-subunit of GABAA receptors, stimulation of GnRH release.whereas the a1- or b2-subunit mRNAwas not detected The NPY neurons that are involved in the control(Petersen et al., 1993). Later studies found that approx- of GnRH neurons are located in the brainstem,imately 25% of the GnRH neurons express the a1- or where NPY is expressed in catecholaminergic neu-a2-subunit mRNA, whereas only a small percentage rons (Everitt et al., 1984) and in the arcuate nucleusof the GnRH neurons contain detectable levels of (Chronwall et al., 1985; McShane et al., 1994). Both ofthe a1- and a2-subunit protein ( Jung et al., 1998). these neuronal cell groups express receptors forThe same study detected b3-subunit mRNA in only estrogen (Sar et al., 1990), and could therefore trans-approximately 25% of the GnRH neurons; g1 mRNA mit the steroid signal to the GnRH neurons. Thewas not detected. These receptor subunits can form NPY neurons project to the median eminence,functioning receptors as determined by electrophysio- among others, where NPY is released into the peri-logical measurements of membrane patches isolated vascular space for transport to the anterior pituitaryfrom identified GnRH neurons. These studies showed or for interactions with the axon terminals of otherthat all GnRH neuronal membrane patches responded neurons. In addition, NPY-containing axons reachto GABA and that the responses were inhibited by the the medial sepum–diagonal band–rostral preopticGABAA antagonist bicuculline (Spergel et al., 1999). area where the GnRH neurons are located. EvidenceTogether, these studies show that certain GABAA for direct actions on the GnRH neurons was providedreceptor subunits are present in GnRH neurons and, by electron microscopic studies that identified NPYbased on the subunit composition, that they could in presynaptic specialization on GnRH perikaryaform functioning GABA receptors in the plasma (Tillet et al., 1989; Tsuruo et al., 1990). Because
The Gonadotropin-Releasing Hormone and Its Receptor 1653NPY can bind to and activate several different the VIP-2 receptor and that VIP immunoreactivemembrane receptor subtypes, it was important to axons were present next to most of the GnRH anddetermine which subtype is expressed in GnRH neu- VIP-2 receptor positive neurons (Smith et al., 2000).rons. Previous experiments have suggested that the Together, these data suggest that VIP neurons from theactivation of the NPY-Y1 receptor subtype is suprachiasmatic nucleus participate directly in therequired for the occurrence of a steroid-induced control of GnRH neurons through VIP-2 receptors.LH surge (Kalra et al., 1992) and a preovulatory LHsurge (Leupen et al., 1997), and it was shown that 126.96.36.199(iii) Endogenous opiatesNPY-Y1 receptor expression in the hypothalamus is The endogenous opiates include three families ofgreatly elevated just prior to and during the proes- neuropeptides (the endorphins, dinorphins, and enke-trous LH surge (Xu et al., 2000). Immunohistochemi- phalins), all of which have inhibitory effects on bothcal studies have confirmed that the NPY-Y1 receptor the frequency and amplitude of GnRH-mediatedprotein is present in many GnRH nerve terminals in LH release pulses (Bruni et al., 1977; Gilbeau et al.,the median eminence; however, the protein was 1985; for review, see Kalra (1993) and Kordon et al.not detected in GnRH perikarya (Li et al., 1999). (1994)). Conversely, the administration of the generalThese findings suggest that although the NPY-Y1 opiate-receptor blocker naloxone (Bruni et al., 1977;receptor subtype probably mediates the stimulatory Van Vugt et al., 1982) or the immunoneutralization ofeffects of NPY on GnRH release in the median b-endorphin (Weesner and Malven, 1990) causes aneminence, the effects of NPY on GnRH perikarya increase in circulating LH levels, which suggests thatmay involve another NPY receptor subtype that has endogenous endorphin is an important neurotrans-not been identified. mitter that tonically inhibits the GnRH neurons. This view is supported by studies that measured decreasing51.2.3(ii) Vasoactive intestinal polypeptide b-endorphin levels in the portal vessels on the after-It is clear now that the preovulatory LH surge is noon of proestrus just prior to the onset of the LHgenerated by a coupling of the steroidal signals with surge (Sarkar and Yen, 1985), indicating that thecircadian signals that originate from the suprachias- release of the GnRH neuronal system from the tonicmatic nucleus. In this nucleus, VIP is synthesized in inhibition is a physiologically relevant event and not aa large number of neurons that are located in the pharmacological artifact.ventrolateral aspects of the nucleus. Axons from these Endorphin-containing perikarya are restricted toVIP neurons project to GnRH neurons, among the hypothalamic arcuate nucleus and a considerableothers, where close appositions have been described. number of the endorphin-positive neurons alsoInterestingly, such close contacts occur with the sub- express estrogen receptors ( Jirikowski et al., 1986),population of GnRH neurons that express fos during suggesting that this neuronal system is a strong can-the steroid-induced LH surge, which suggests that didate to propagate the estrogen signal to other neu-VIP-containing axons exert a stimulatory effect rons. From the perikarya in the arcuate nucleus,on the GnRH neurons (van der Beek et al., 1994). extensive projections reach the median eminence,Although the physiological effects of VIP on GnRH preoptic area, and medial septum–diagonal bandrelease have not been fully elucidated and are, in part, (Khatchaturian et al., 1985), among others, whichcontroversial, several lines of evidence support are the areas in which a direct innervation of thethe view that VIP stimulates GnRH release. Thus, GnRH neuronal system could occur. Indeed, presyn-the central administration of VIP antiserum or aptic b-endorphin-containing specializations onadministration of VIP antisense oligonucleotides to GnRH perikarya have been described (Leranth et al.,the suprachiasmatic nucleus delays and reduces the 1988a; Chen et al., 1989a). However, several studiesmagnitude of the steroid-induced GnRH–LH surge were unable to detect the mRNA for any of the opiate(van der Beek et al., 1999; Harney et al., 1996) and receptor subtypes (Mitchell et al., 1997; Sannella andlesions of the suprachiasmatic nucleus abolish the Petersen, 1997), and it remains to be determined ifproestrous surge (Gray et al., 1978). In order to deter- these negative data are due to limited sensitivities ofmine whether VIP acts directly on GnRH neurons, dual in situ hybridization procedures or if, indeed,immunohistochemical triple-labeling experiments GnRH neurons do not synthesize opioid receptors.were conducted using antibodies to GnRH, VIP, and A large body of evidence suggests that the opioidthe VIP-2 receptor. The results of these studies show system interacts with other neurotransmitter systems,that approximately 40% of the GnRH neurons express which in turn could influence GnRH neuronal activity
1654 The Gonadotropin-Releasing Hormone and Its Receptor(for review, see Kalra (1993)); the interactions with 51.3 GnRH Receptors in the Centralthe catecholaminergic and NPY-containing neurons Nervous Systemespecially warrant further detailed investigations. 51.3.1 Localization of GnRH Receptors in51.2.3(iv) Kisspeptins the BrainRecently, a novel peptide has been identified to be The finding that GnRH is present in presynaptica very potent stimulator of GnRH release. This terminals and in axons that project to regions in thepeptide which is derived from the Kiss1 gene is brain that are not related to the direct regulation ofsynthesized as a 145-amino-acid long peptide that is the anterior pituitary gonadotrope function and stud-further processed to a 54-, 14-, 13-, and 10-amino- ies that showed a facilitatory effect of exogenousacid long peptide (Ohtaki et al., 2001). Kisspeptin is GnRH on reproductive behaviors prompted thesynthesized in neurons of the arcuate and the search for intracerebral receptors for GnRH. Initially,anteroventral periventricular nuclei, among others, in vitro autoradiography was used to localize thewhich are critical sites in the brain involved in the GnRH binding sites in the brain, followed by bio-control of GnRH neuronal activity (Gottsch et al., chemical assays that determined the binding charac-2004; Brailoiu et al., 2005). The findings that teristics of GnRH (Badr and Pelletier, 1987; Reubikisspeptin-producing neurons in the hypothalamus et al., 1987; Jennes et al., 1988; Leblanc et al., 1988).express estrogen receptors and kisspeptin mRNA These studies showed that specific binding sites forlevels are regulated by gonadal steroids support the GnRH exist in many regions of the central nervousview that this peptide is important for the control of system that had been implicated either in the inte-GnRH neurons (Smith et al., 2005a,b). While a direct, gration of olfactory and vomeronasal cues or in sitessynaptic innervation of GnRH neurons by kisspeptin- that are known to be involved in the generation ofcontaining axon terminals has not yet been clearly emotional and reproductive behaviors. Thus, bindingestablished, many data support the view that they sites for GnRH are present in the laminae glomer-do exist. Thus, kisspeptin-54 and kisspeptin-10 are ulosa and plexiformis of the olfactory bulb, theextraordinarily potent in causing GnRH release, and nucleus olfactorius anterior, the frontal cortex at theintracerebroventricular administration of only 1-fmol sulcus rhinalis, and the piriform cortex. In the sep-(Gottsch et al., 2004) or 10-pmol peptide (Navarro tum, the dorsal and lateral portions of the lateralet al., 2005) causes a significant rise in circulating LH septal nucleus contain moderate amounts of GnRHlevels. Almost all GnRH neurons express the receptor binding sites, whereas the medial septum and thefor kisspeptin, GPR54 (Messager et al., 2005; Navarro diagonal band are not labeled. In the hypothalamus,et al., 2005), and administration of kisspeptin causes a only the arcuate nucleus is labeled and, further cau-rapid and transient expression of the transcription dally, the interpeduncular nucleus and the central grayfactor fos (Irwig et al., 2004). This finding is remark- contain measurable amounts of GnRH binding sites.able because fos expression in GnRH neurons is lim- Specific binding is also seen in the cortical nucleus ofited to a few hours during the LH surge (Lee et al., the amygdala and more prominently in the hippocam-1990, 1992; Hoffman et al., 1993), and up to now only a pus, where most label is associated with the strata oriensstrict regimen of sequential estradiol and progester- and radiatum. The highest number of GnRH bindingone administration to ovariectomized rodents was sites is measured in the parasubiculum, which is labeledable to induce fos expression in GnRH neurons mim- at the medial tip of the caudal cerebral cortex ( Jennesicking the events that occur during the preovulatory et al., 1988).surge. Together, these data suggest that kisspeptin is apotent stimulator of GnRH release and that kisspep-tin plays an important role in the regulation of GnRH 51.3.2 Characterization of GnRHneurons under physiological conditions. However, Receptors in the Brainjust like the other neuropeptides or amino acid trans- Biochemical analyses of the specificity and affinity ofmitters, kisspeptin is not absolutely required since the GnRH binding to hippocampal membrane pre-GPR54-deficient mice, although infertile, respond parations show that they are very similar when com-to estradiol followed by progesterone administration pared to the anterior pituitary GnRH receptors inwith elevated LH levels and fos expression in the that all agonists that bind to hippocampal membranesGnRH neurons (Dungan et al., 2007). also bind to the pituitary GnRH receptors with
The Gonadotropin-Releasing Hormone and Its Receptor 1655similar inhibiting concentration (IC50). In addition, dentate gyrus of the hippocampus in both male andGnRH fragments or different forms of GnRH from female rats ( Jennes et al., 1995). These data are inlower vertebrates do not bind to the hippocampal good agreement with studies that show a transientGnRH binding sites, nor do they interact with the elevation in the number of GnRH-binding sites inpituitary GnRH receptor. The only GnRH analog the hippocampus of castrated male rats (Badr et al.,that we found to bind to the pituitary GnRH receptor 1988; Ban et al., 1990). In the mediobasal hypothala-but not to hippocampal preparations is the antagonist mus, GnRH receptor mRNA levels were elevated in[D-p-Glul, D-Phe2, D-Trp3,6]GnRH ( Jennes et al., the morning of the estrogen- and progesterone-1990). The reason for this discrepancy is not known, induced LH surge, whereas the levels declined dur-but it is possible that the difference in the lipid ing the surge ( Jennes et al., 1997), which indicatesmicroenvironment may prevent the binding of this that the cells increase the synthesis of GnRH recep-analog to hippocampal binding sites. That the brain tor mRNA and probably protein in preparation forGnRH binding sites are very similar to the GnRH the LH surge and not as a consequence of the surge.receptor in the anterior pituitary was shown by in situ These data are similar to the results of Seong et al.hybridization that applied cRNA probes encoding (1998), who used competitive PCR to measurethe pituitary GnRH receptor. The results of these GnRH receptor mRNA in tissue punches of thestudies show that the mRNA is present in most areas mediobasal hypothalamus. These studies show thatof the brain where GnRH binding sites were mea- the administration of progesterone after estradiolsured, except for the interpeduncular nucleus and priming causes a dose-dependent decrease in GnRHthe central gray where no hybridization signal was receptor mRNA levels, whereas estradiol alone sti-detected. However, a hybridization signal was de- mulated GnRH receptor expression in the medioba-tected in the ventromedial nucleus and the medial sal hypothalamus. In addition, luciferase activityhabenula, and it was hypothesized that the GnRH under the control of the GnRH receptor promotorreceptor protein is synthesized in the neurons of the in transgenic mice is greatly enhanced in whole brainventromedial nucleus and transported anterogradely of ovariectomized animals that received estradiolto the central gray along a well-characterized exten- when compared to untreated ovariectomized micesive projection (Conrad and Pfaff, 1976). Similarly, (Duval et al., 2000). However, the data from thesethe protein could be synthesized in the neurons of studies with brain tissues are different from the datathe medial habenula and transported anterogradely obtained with pituitary tissue in which GnRH recep-through the fasciculus retroflexus to the interpedun- tor mRNA levels are low during the morning of thecular nucleus ( Jennes and Woolums, 1994). Addi- LH surge before they rise at noon and remain hightional studies are needed to determine if these until after the surge (Bauer-Dantoin et al., 1993). Thehypotheses are correct. The anatomical data describ- reasons for this discrepancy are not clear; it is plausi-ing the presence of GnRH receptor mRNA or protein ble that newly synthesized GnRH receptor protein inin the brain have been confirmed with polymerase the mediobasal hypothalamus is transported to distantchain reaction (PCR) for the mediobasal hypothala- sites, thus requiring more time before it reaches itsmus (Seong et al., 1998) and in transgenic mice that final location. That appropriate synthesis of thesynthesize luciferase under the control of the GnRH GnRH receptor in the brain may be important forreceptor promoter (Duval et al., 2000). Together, the occurrence of an estrogen- and progesterone-these studies clearly strengthen the view that specific induced LH surge is indicated by experiments thatreceptors for GnRH exist in select regions of the use the intraventricular administration of antisensecentral nervous system. oligonucleotides to reduce receptor synthesis. The administration of antisense oligonucleotides but not of sense oligonucleotides significantly decreases51.3.3 Regulation of GnRH Receptor GnRH receptor mRNA in the mediobasal hypothala-Expression in the Brain mus in estrogen- and progesterone-treated animalsVery little is known about the regulation of brain and causes a significant reduction in circulating LHGnRH receptor expression. We could show with (Seong et al., 1998). However, this treatment within situ hybridization that the removal of the gonads antisense oligonucleotides also reduced GnRHleads to a significant elevation of GnRH receptor receptor synthesis in the anterior pituitary; thus, it ismRNA levels in areas CAI and CA3 and in the not clear if the primary surge-blocking effect was
1656 The Gonadotropin-Releasing Hormone and Its Receptorexerted at the level of the central nervous system or activity of the target neurons. A change in the activitythe anterior pituitary. Together, these data suggest of the GnRH target neurons, many of which projectthat GnRH receptor mRNA and protein expression to the median eminence, could alter the release ofis at least in part regulated by gonadal steroids. Based GnRH, which controls pituitary gonadotrope func-on the anatomical distribution of estrogen receptors tion. Thus, GnRH neurons could participate in theand GnRH receptors, it is possible that the effects of regulation or synchronization of other GnRH neu-the gonadal steroids are exerted directly in the GnRH rons indirectly by using neurons in the arcuatereceptor synthesizing cells because ER-a is promi- nucleus as a relay station. However, many more stud-nently expressed in the arcuate nucleus, whereas ies are needed to establish the precise role thatER-b is the dominant form in the hippocampus GnRH plays inside the central nervous system.(Shughrue et al., 1997).51.3.4 Functional Aspects of GnRH 51.4 Molecular and CellularReceptors in the Brain Mechanism of GnRH Action in the Anterior PituitaryWe can only speculate about the function of GnRHreceptors in the brain. It has been known for many Much of the information regarding the molecularyears that peripheral or central administration of mechanism of GnRH has come from studies assessingGnRH can facilitate reproductive behaviors (Moss the coupling of the pituitary-derived receptor (Sealfonand McCann, 1973; Pfaff, 1973), especially when the et al., 1997; Byrne et al., 1999; Stojilkovick et al., 1994)peptide is administered into the central gray (for to effector systems. Pituitaries themselves have alsoreview, see Pfaff et al. (1994)). It is thought that the been a useful model, as have reconstituted systems incentral gray receives extensive afferent projections which receptors (including native, chimera, andfrom the ventromedial nucleus (Conrad and Pfaff, mutant receptor moieties) have been stably or tran-1976), which may transport the GnRH receptor siently transfected into systems that do not ordinarilyprotein. An enhanced GnRH receptor expression express receptors (Kaiser et al., 1997).in the neurons of the ventromedial nucleus duringthe morning of the LH surge could indicate thathigh levels of the receptor protein can reach the central 51.4.1 GnRH Receptorgray before estrus, which is the time of the cycle whenreproductive behaviors are most pronounced. The pituitary GnRH receptor has been cloned from The arcuate nucleus is one of the hypothalamic a substantial number of mammalian and premammalianregions that are essential for the maintenance species (Sealfon et al., 1997; Tsutsumi et al., 1992). Thisof regular estrous cyclicity (Bogdanove, 1964), and molecule is a member of the seven-transmembranethis nucleus contains a large number of different receptors superfamily. Because of the modest length ofneuropeptide- or monoamine-expressing neurons both the (extracellular) amino and (intracellular) car-(Chronwall, 1985), many of which also contain recep- boxyl terminal, the mammalian GnRH receptor type Itors for estradiol ( Jirikowski et al., 1986; Sar et al., (hereafter referred to only as GnRHR) is among the1990). Thus, the arcuate nucleus is generally thought smallest members of this superfamily (Ulloa-Aguirreof as a center in the brain in which the estrogen and Conn, 2000; Millar, 2003) and may approach thesignals are converted into neuronal signals. Among smallest size G-protein-coupled receptor (GPCR) thatthe estradiol target cells in the arcuate nucleus are can bind ligand and transduce an intracellular signal. Inneurons that contain NPY (Chronwall et al., 1985), birds, fish, and reptiles, the carboxyl tail is significantlyb-endorphin (Khatchaturian et al., 1985), or GABA longer, due to an extended carboxyl terminal, and this(Mugnaini and Oertel, 1985), all of which are known may reflect the altered regulation in these species,to affect GnRH release from the median eminence compared to mammals (Blomenrohr et al., 1999; Hed- ¨into the capillary plexus (for review, see Kordon et al. ing et al., 1998; Lin et al., 1998a; Millar, 2003). In the(1994)). If these neurons express GnRH receptors, as primate GnRHR, the expression levels are relativelyis suggested by the anatomical overlap of GnRH- low, an effect which appears attributable to the presencereceptor mRNA-containing neurons and the pepti- of an Lys191 not present in rat or mouse sequencesdergic and aminergic neurons, then GnRH released (Arora et al., 1999; Stanislaus et al., 1997). The removalin the arcuate nucleus could affect the secretory of this amino acid dramatically increases expression
The Gonadotropin-Releasing Hormone and Its Receptor 1657levels, and there appears to be functional interac- et al., 1999; Thomas et al., 2007) and for heterogeneoustion between modifications at this and other sites receptor regulation (Rocheville et al., 2000a,b).(Stanislaus et al., 1997). 51.4.4 Receptor Trafficking51.4.2 Effector Coupling 188.8.131.52 Endoplasmic reticulum qualityLike some other members of the GPCR’s superfamily, control system and the role of endogenousthe GnRHR appears functionally coupled to multiple chaperone proteinsG-proteins and appears significantly promiscuous, Molecular chaperones serve as a control mechanismdepending on the availability of G-proteins. Evidence for recognizing, retaining, and targeting misfolded pro-for coupling to multiple G-proteins comes from over- teins for their eventual degradation. These proteins areexpression and palmitoylation studies (Stanislaus key components of the endoplasmic reticulum (ER)et al., 1997, 1998), identification of separate sites of quality control system (QCS), a complex sorting sys-interaction (Ulloa-Aguirre et al., 1998; Arora et al., tem that identifies and separates newly synthesized1999), and other experimental approaches. This proteins according to their maturation status (Ulloa-observation has been an attractive consideration in Aguirre et al., 2004a). Although the steric character ofan explanation of how a single class of ligand inter- the protein backbone restricts the spectrum of proteinacting with a single class of receptor can regulate shapes that are recognized by the stringent qualitymultiple end points in coordinated, yet independent control mechanisms, some features displayed by pro-functions (release and biosynthesis of LH, FSH, and teins (including exposure of hydrophobic shapes,secretogranin; up- and downregulation of receptors; unpaired cysteines, immature glycans, and particularand sensitization and desensitization). Several reports sequence motifs) have been identified as important for(Kaiser et al., 1995; Pinter et al., 1999) in which differ- chaperone–protein association (Ellgaard and Helenius,ent techniques were used to provide variable levels of 2001; Dong et al., 2007). In fact, molecular chaperonesreceptor per cell suggest that differential regulation possess the ability to recognize misfolded proteins bymay be related to receptor number. In this way, the the exposure of hidden hydrophobic domains or spe-stimulus (GnRH) and the status of the target cell both cific sequences (Dong et al., 2007; Tan et al., 2004).participate in defining the responses elicited. Through this association, chaperones may stabilize unstable conformers of nascent polypeptides to pre- vent aggregation and facilitate correct folding, or51.4.3 Receptor–Receptor Interactions assembly, of the substrate via binding and release cyclesA series of observations dating back to the 1980s suggest (Hartl and Hayer-Hartl, 2002).the following: (1) Techniques that cause GnRHR to Several GPCR interacting proteins that supportassociate provoke all known actions of the releasing trafficking to the cell surface have been identified.hormone, even when the receptor is occupied by an Neither inactivation nor afterpotential A (Nina A), aantagonist (Conn et al., 1982b). (2) The occupancy of photoreceptor-specific integral membrane glycopro-the receptor by agonists is sufficient to cause receptors tein, is a molecular chaperone that facilitates cell- ˚to associate to within a distance of 50–100 A (Conn surface membrane expression of the sensory GPCRet al., 1982a,b; Cornea et al., 2001). (3) This process rhodopsin 1 in Drosophila melanogaster ; its absence(termed microaggregation, oligomerization, or, poten- leads to rhodopsin 1 ER accumulation and degrada-tially inaccurately, dimerization) is distinct from (and tion (Shieh et al., 1989; Schneuwly et al., 1989; Colleytemporally a precursor to) the process of patching, et al., 1991). Its mammalian homolog RanBP2 specif-capping, and internalization (macroaggregation) that is ically binds red/green opsin molecules and acts as aassociated with the extinction of the response system. chaperone aiding proper folding, transport, and local- These data are consonant with a role of microag- ization of the mature receptors to the cell membranegregation in GnRHR signaling. Evidence for receptor– (Ferreira et al., 1996). ODR4 is a molecular chaper-receptor interactions have now been observed for a one that assists in folding, ER exit, and/or targetingnumber of GPCRs (Conn et al., 1985; Pace et al., of olfactory GPCRs (e.g., ODR10 in the nematode1999; Harmatz et al., 1985; Hebert et al., 1996; Blakely Caenorhabditis elegans) to olfactory cilia (Dwyer et al.,et al., 2000; Heldin, 1995; Gether, 2000; Lee et al., 1998; Gimelbrant et al., 2001). Calnexin and calreti-2000b) and are suggested to be important for the expla- culin are molecular chaperones that bind a broadnation of independent mediation of responses (Pinter range of glycoproteins, including several GPCRs
1658 The Gonadotropin-Releasing Hormone and Its Receptor(e.g., the GnRHR, vasopressin-2 receptor, and the charge (12 instances); the remainder involved a gainglycoprotein hormones receptors) (Vassilakos et al., or loss of Cys (four instances) or a loss of a Pro (one1998; Schrag et al., 2003; Rozell et al., 1998; Morello instance). Each of these types of changes might beet al., 2001; Helenius et al., 1997; Brothers et al., expected to have a substantial impact on the overall2006). The action of these chaperones predominantly shape of the receptor, unlike a conservative substitu-centers on substrate N-glycans present on the newly tion (i.e., Gly for Ala) for example. As misshapenedsynthesized proteins, adding hydrophobicity to the (misfolded) proteins, the single amino acid mutantsfolding protein (Schrag et al., 2003; Rozell et al., 1998; characteristically failed the cellular QCS and mis-Morello et al., 2001; Helenius et al., 1997). When routing occurred (Ulloa-Aguirre et al., 2004a); in theN-linked glycosylation or early glycan processing cases examined, misrouting was due to retention infails, glycoproteins misfold, aggregate, and fail the the ER ( Janovick et al., 2003a; Leanos-Miranda et al., ˜QCS (Morello, et al., 2001). Receptor activity modify- 2003, 2005; Ulloa-Aguirre et al., 2004b).ing proteins (RAMPs) are proteins that interact with The corollary of the observation that these areseveral GPCRs (e.g., the calcitonin receptor-like misrouted proteins, is that, if restored to the plasmareceptor, the vasoactive intestinal polypeptide/pitui- membrane, these mutants can become functionaltary adenylate cyclase-activating peptide receptor, the ( Janovick et al., 2003a; Leanos-Miranda et al., 2003, ˜glucagon receptor, and the parathyroid hormone 2005). This happens because they have retained (orreceptor) fostering the transport of the associated regain) both the ability to bind agonist and the abilityreceptor to, and regulating its signaling function at, to transduce a signal. This differs from a prior viewthe PM (Christopoulos et al., 2003), whereas gC1q-R that most mutants are defective because they have(receptor for globular heads of C1q) interacts with the (permanently) lost the ability to bind ligand or cou-carboxyl terminus of the alpha1B-adrenergic receptor ple to effector proteins. The ability to rescue andand regulates the maturation and expression of the restore proteins to function opens a new avenue inreceptor (Xu et al., 1999). Another molecular chaper- therapeutics which appears broadly applicable toone is BiP/Grp 78, which is involved in the protective disease-causing mutants of the GnRHR.unfolded protein response; a cell stress program acti- Restoration of binding and coupling activity ofvated when misfolded proteins accumulate and/or mutants was initially shown by adding plasma mem-aggregate in the ER (Yang et al., 1998; Schroder and brane targeting sequences to defective humanKaufman, 2005). Finally, DriP78 is an ER-membrane- GnRHR mutants (i.e., making chimeras of humanassociated protein that binds to the F(x)3F(x)3F motif mutants with the C-terminal sequence found in cat-of the dopamine receptor (and presumably other fish GnRHR that increases plasma membrane target-GPCRs bearing this motif), thereby facilitating its ing of the GnRHR (Lin et al., 1998b)), or by deletingmaturation and export to the PM (Bermak et al., an amino acid (K191) found in the human (and other2001). Identification of these particular molecular primates) wild-type (WT) receptor that, when pres-chaperones is important since they represent a poten- ent, decreases routing to the plasma membrane. Suchtial target to manipulate ER retention and/or export modifications led to rescue of the mutant sequencesmechanisms, and hence a means for influencing pro- as assessed by both radioligand binding and by thetein trafficking and secretion (Aridor and Balch, 2000; ability of agonist to activate Gaq/11-protein-mediatedRivera et al., 2000). inositol phosphates (IP) production (Lin et al., 1998b; Janovick et al., 2003b).184.108.40.206 Mutant GnRHRs isolated from More recently ( Janovick et al., 2003a, 2007; Leanos- ˜patients with hypogonadotropic Miranda et al., 2003, 2005), we learned that it is possi-hypogonadism are actually misfolded and ble to use a pharmacological approach and show thatmisrouted proteins that can be rescued and small, nonpeptide molecules (pharmacologic chaper-restored to function ones or pharmacoperones) which bind the GnRHRResults over the last 7 years have led to the conclu- mutants can stabilize most of the mutant proteinssion that all of the first 17 reported single amino acid (15 of 17) in a conformation that passes the QCS;mutations of the human GnRHR that lead to hypo- these rescued mutants are not retained in the ER, butgonadotropic hypogonadism (HH) are actually mis- route correctly to the plasma membrane, where theyfolded proteins ( Janovick et al., 2002). All 17 genetic bind ligand (shown with radioligand) and successfullymutations resulted from single amino acid changes in transduce signaling (shown by IP response). Such phar-the GnRHR. Most frequently, this was a change in macoperones are, of course, lead drug candidates.
The Gonadotropin-Releasing Hormone and Its Receptor 1659220.127.116.11 The ability to rescue mutant Castro-Fernandez et al., 2005). The ability to rescueproteins using pharmacoperones has misfolded proteins with drugs presents a new thera-therapeutic potential peutic approach for a remarkably diverse palette ofPharmacoperones for the GnRHR mutants have now diseases caused by other misfolded molecules: Alz-been identified from three different chemical classes heimer’s disease, cataracts, cystic fibrosis, familial(indoles, quinolones, and erythromycin macrolides; hypercholesterolemia, HH, nephrogenic diabetesJanovick et al., 2003a) using combinational chemical insipidus, and retinitis pigmentosa, among otherslibraries. Within each chemical class, there is an (Ulloa-Aguirre et al., 2003, 2004a,b; Conn and Jano-effect of mutant rescue that is proportional to the vick, 2005; Maegawa et al., 2007; Lumb et al., 2003; Vijaffinity of binding to the receptor ( Janovick et al., et al., 2006; Biswas et al., 2004; Biswas and Das, 2004;2003a). In retrospect, it is not surprising that concen- Kim et al., 2006; Lashuel and Hirling, 2006; Cashmantrations needed for rescue differ between chemical and Caughey, 2004; Mallucci and Collinge, 2005). Inclasses of pharmacoperones since it is likely that the the case of the human GnRHR, this receptor alsospecific chemical interaction between the drugs and appears to provide opportunities for regulation bythe mutants would stabilize the mutants by distinct intentionally misfolding a portion of the WT receptor,means (i.e., by interacting with different residues on resulting in a circumstance in which a proportionthe receptor). Only two of the mutants in the group of the synthesized molecule is retained in the ERdiscussed (the first 17 GnRHR mutants reported, ( Janovick et al., 2006; Conn et al., 2006a,b). Otherexcluding deletion and truncation mutants) cannot GPCRs ( Janovick et al., 2006) appear to share thebe at least partially rescued by this (pharmacoperone) balance between the ER and the plasma membraneapproach ( Janovick et al., 2006). In these two cases, and therefore these wild-type receptors may be candi-this occurs because the mutation leads to a change dates for this rescue approach – when the therapeuticthat is so distorting to the protein structure that rescue goal is to increase the plasma membrane expression ofdoes not occur. In both of these cases (amino acids 168 these moieties.and 217), the mutation is identical, Ser ! Arg and both Accordingly, it is useful to consider potential usesmutations occur in transmembrane segments (trans- (Conn et al., 2007) of pharmacoperones in four groups:membrane segments 4 and 5, respectively), resulting (1) to prevent misfolding of molecules that do soin a highly unfavorable thermodynamic change that and lead to disease (e.g., cataracts and neurodegenera-causes a specific ( Janovick et al., 2006) effect on the tive diseases); (2) to rescue mutants (e.g., mutantreceptor configuration (Knollman et al., 2005). GnRHR in HH, cystic fibrosis transmembrane con- Because the pharmacoperones are successful in res- ductance regulator in cystic fibrosis, a-galactosidase Acuing the remaining 15 mutants – even though the loci in Fabry’s disease); and (3) to increase (or decrease) theof the mutations are widely distributed over the recep- percentage of WT molecules that route to the mem-tor – this argues for the generality of this approach for brane, when only a fraction of the total synthesizedmutants of the human GnRHR. Moreover, the drugs material is expressed at the plasma membrane, as isused as pharmacoperones were initially developed as the case for the WT hGnRHR (Conn et al. 2006a,b).oral antagonists of GnRH for use in humans; accord- There is a fourth condition in which perturbation ofingly, there is extensive safety and pharmacokinetic and the ER homeostasis results in disorders because of thepharmacodynamic data available in human and animal promotion in the synthesis of deleterious productsmodels which will make it easier to move the rescue potentially treatable with pharmacoperones; a goodtechnique to in vivo rescue situations. example of this is prion replication (Hetz et al., 2007).18.104.22.168 The rescue approach appearsgenerally applicable to other mutant GPCRs, Acknowledgmentsnon-GPCR receptors, ion channels, andenzymes associated with disease: This research was supported by NIH AG025647,This supports the importance of AG17164 and RR15592(L.J.) NIH HD-19899, TW /understanding the mechanism of this event HD-00668, RR-00163, and HD-18185 (P. Michaelin a well-defined system Conn), and grant 45991 from CONACyT, MexicoMutants of other GPCRs, non-GPCR receptors, as (Alfredo Ulloa-Aguirre). Alfredo Ulloa-Aguirre is awell as ion channels and enzymes can be rescued recipient of a Research Career Development Awardby this approach and restored to activity (e.g., ´ ´ from the Fundacion IMSS, Mexico.
1660 The Gonadotropin-Releasing Hormone and Its ReceptorReferences cytoplasmic carboxyl-terminal tail of a nonmammalian gonadotropin-releasing hormone receptor in cell surface expression, ligand binding and receptor phosphorylationAdler B, Johnson M, Lynch C, and Crowley W (1983) and internalization. Molecular Pharmacology 56: 1229–1237. Evidence that norepinephrine and epinephrine systems Bogdanove EM (1964) The role of the brain in the regulation of mediate the stimulatory effects of ovarian hormones on pituitary gonadotropin secretion. Vitamins and Hormones luteinizing hormone and luteinizing hormone-releasing (NY) 22: 205–260. hormone. Endocrinology (Baltimore) 113: 1431–1438. Brailoiu GC, Dun SL, Ohsawa M, et al. (2005) KiSS-1 expressionAjika K (1979) Simultaneous localization of LHRH and and metastin-like immunoreactivity in the rat brain. Journal of catecholamines in rat hypothalamus. Journal of Anatomy Comparative Neurology 481(3): 314–329. 128: 331–347. Brann DW and Mahesh VB (1997) Excitatory amino acids:Aridor M and Balch WE (2000) Perspectives: Drug Evidence for a role in the control of reproduction and anterior delivery. Regulating export of ER cargo. Science 287: pituitary hormone secretion. Endocrine Reviews 18(5): 816–817. 678–700.Arora KK, Chung HO, and Catt KJ (1999) Influence of a species- Brothers SP, Janovick JA, and Conn PM (2006) Calnexin- specific extracellular amino acid on expression and function mediated retention of the human gonadotropin-releasing of the human gonadotropin-releasing hormone receptor. hormone receptor. Journal of Molecular Endocrinology 37: Molecular Endocrinology 13: 890–896. 479–488.Badr M, Marchetti B, and Pelletier G (1988) Modulation of Bruni JF, Van Vugt D, Marshall S, and Meites J (1977) Effect of hippocampal LHRH receptors by sex steroids in the rat. naloxone, morphine and methionine enkephalin of serum Peptides (NY) 9: 441–442. prolactin, luteinizing hormone, follicle stimulating hormone,Badr M and Pelletier G (1987) Characterization and thyroid stimulating hormone and growth hormone. Life autoradiographic localization of LHRH receptors in the rat Sciences 21: 461–466. brain. Synapse 1: 567–571. Burchanowski BJ, Knigge KM, and Sternberger LA (1979)Ban E, Crumeyrolle-Arias M, Latouche J, et al. (1990) GnRH Rich ependymal investment of luliberin (LHRH) fibers receptors in rat brain, pituitary and testis: Modulation revealed immunocytochemically in an image like that from following surgical and gonadotropin-releasing hormone golgi stain. Proceedings of the National Academy of agonist-induced castration. Molecular and Cellular Sciences of the United States of America 76(12): 6671–6674. Endocrinology 70: 99–107. Byrne B, McGregor A, Taylor PL, Sellar R, Rodger FE,Bailey JD, Centers A, and Jennes L (2006) Expression of Fraser JM, and Eidne KA (1999) Isolation and AMPA receptor subunits (GluR1–GluR4) in gonadotrophin- characterization of the marmoset gonadotropin releasing releasing hormone neurones of young and middle-aged hormone receptor. Ser140 of the DRS motif is substituted persistently oestrous rats during the steroid-induced by Phe. Journal of Endocrinology 163: 447–456. luteinising hormone surge. Journal of Neuroendocrinology Campbell RE, Han SK, and Herbison AE (2005) Biocytin filling of 18(1): 1–12. adult gonadotropin-releasing hormone neurons in situBarraclough CA, Wise PM, and Selmanoff MK (1984) A role for reveals extensive, spiny, dendritic processes. Endocrinology hypothalamic catecholamines in the regulation of 146(3): 1163–1169. gonadotropin secretion. Recent Progress in Hormone Cashman NR and Caughey B (2004) Prion diseases – close to Research 40: 487–529. effective therapy? Nature Reviews Drug Discovery 3:Barry J, Hoffman GE, and Wray S (1985) LHRH-containing 874–884. ¨ ¨ systems. In: Bjorklund A and Hokfelt T (eds.) Handbook of Castro-Fernandez C, Maya-Nunez G, and Conn PM (2005) Chemical Neuroanatomy, vol. 4, pp. 166–215. Amsterdam: Beyond the signal sequence: Protein routing in health and Elsevier. disease. Endocrine Reviews 26(4): 479–503.Bauer-Dantoin AC, Hollenberg AN, and Jameson JL (1993) Chen W-P, Witkin JW, and Silverman AJ (1989a) b Endorphin Dynamic regulation of gonadotropin releasing hormone and gonadotropin-releasing hormone synaptic input to receptor mRNA levels in the anterior pituitary gland during gonadotropin-releasing hormone neurosecretory cells the rat estrous cycle. Endocrinology (Baltimore) 133: in the male rat. Journal of Comparative Neurology 286(1): 1911–1914. 85–95.Bauer-Dantoin AC, Urban JH, and Levine JE (1992) Chen W-P, Witkin JW, and Silverman AJ (1989b) Gonadotropin Neuropeptide Y gene expression in the arcuate nucleus is releasing hormone (GnRH) neurons are directly innervated increased during preovulatory luteinizing hormone surges. by catecholamine terminals. Synapse 3: 288–290. Endocrinology (Baltimore) 131: 2953–2958. Christopoulos A, Christopoulos G, Morfis M, et al. (2003) NovelBermak JC, Li M, Bullock C, and Zhou Q-Y (2001) receptor partners and function of receptor activity-modifying Regulation of transport of the dopamine D1 receptor by proteins. Journal of Biological Chemistry 278: 3293–3297. a new membrane-associated ER protein. Nature Cell Chronwall BM (1985) Anatomy and physiology of the Biology 3: 492–498. neuroendocrine arcuate nucleus. Peptides (NY)Biswas A and Das KP (2004) Role of ATP on the interaction of 6(supplement 2): 1–11. a-crystallin with its substrates and its implications for the Chronwall BM, DiMaggio DA, Massari VJ, Pickel VM, molecular chaperone function. Journal of Biological Ruggero DA, and O’Donohue TL (1985) The anatomy of Chemistry 279: 42648–42657. neurupeptide-Y-containing neurons in rat brain.Biswas S, Harris F, Dennison S, Singh J, and Phoenix DA (2004) Neuroscience 15(4): 1159–1181. Calpains: Targets of cataract prevention? Trends in Coen C and Coombs M (1983) Effects of manipulating Molecular Medicine 10: 78–84. catecholamines on the incidence of the preovulatory surgeBlakely BT, Rossi FMV, Tillotson B, Palmer M, Estelles A, and of luteinizing hormone and ovulation in the rat: Evidence for a Blau HM (2000) Epidermal growth factor receptor necessary involvement of hypothalamic adrenaline in the dimerization monitored in live cells. Nature Biotechnology normal or midnight surge. Neuroscience 10: 187–206. 18: 218–222. Colley NJ, Baker EK, Stamnes MA, and Zuker CS (1991) The ¨Blomenrohr M, Heiding A, Sellar R, Leurs R, Bogerd J, cyclophilin homolog ninaA is required in the secretory Eidne KA, and Willars GB (1999) Pivotal role for the pathway. Cell 67: 255–263.
The Gonadotropin-Releasing Hormone and Its Receptor 1661Conn PM and Janovick JA (2005) A new understanding Dungan HM, Gottsch ML, Zeng H, et al. (2007) The of protein mutation unfolds. American Scientist 93: role of kisspeptin-GPR54 signaling in the tonic 314–321. regulation and surge release of gonadotropin-releasingConn PM, Janovick JA, Brothers SP, and Knollman PE (2006a) hormone/luteinizing hormone. Journal of Neuroscience ‘Effective inefficiency’: Cellular control of protein trafficking 27(44): 12088–12095. as a mechanism of posttranslational regulation. Journal of Duval DL, Farrls AR, Quirk CC, Nett TM, Hamernik DL, and Endocrinology 190(1): 13–16. Clay CM (2000) Responsiveness of the ovineConn PM, Knollman PE, Brothers SP, and Janovick JA (2006b) gonadotropin-releasing hormone receptor gene to Protein folding as post-translational regulation: Evolution of estradiol and gonadotropin-releasing hormone is not a mechanism for controlled plasma membrane expression detectable in vitro but is revealed in transgenic mice. of a G-protein coupled receptor. Molecular Endocrinology Endocrinology (Baltimore) 141(3): 1001–1010. 20(12): 3035–3041. Dwyer ND, Troemel ER, Sengupta P, and Bargmann C (1998)Conn PM, Rogers DC, and McNeil R (1982a) Potency Odorant receptor localization to olfactory cilia is mediated by enhancement of a GnRH agonist: GnRH-receptor ODR-4, a novel membrane-associated protein. Cell 93: microaggregation stimulates gonadotropin release. 455–466. Endocrinology (Baltimore) 111: 335–337. el Amraoui A and Dubois PM (1993) Experimental evidence forConn PM, Rogers DC, Seay SG, Jinnah H, Bates M, and an early commitment of gonadotropin-releasing hormone Luscher D (1985) Regulation of gonadotropin release, GnRH neurons, with special regard to their origin from the receptors, and gonadotrope responsiveness: A role for ectoderm of nasal cavity presumptive territory. GnRH receptor microaggregation. Journal of Cellular Neuroendocrinology 57(6): 991–1002. Biochemistry 7: 13–21. Ellgaard L and Helenius A (2001) ER quality control: Towards anConn PM, Rogers DC, Stewart JM, Neidel J, and Sheffield T understanding at the molecular level. Current Opinion in Cell (1982b) Conversion of a gonadotropin releasing hormone Biology 13: 431–437. antagonist to an agonist. Nature (London) 296: 653–655. Everitt BJ, Hokfelt T, Terenius L, Tatemoto K, Mutt V, andConn PM, Ulloa-Aguirre A, Ito J, and Janovick JA (2007) Goldstein M (1984) Differential co-existence of neuropeptide G-protein coupled receptor trafficking in health and disease: Y (NPY)-like immunoreactivity with catecholamines in the Lessons learned to prepare for therapeutic mutant rescue central nervous system of the rat. Neuroscience 11(2): in vivo. Pharmacological Reviews 59: 225–250. 443–462.Conrad LC and Pfaff DW (1976) Efferents from medial basal Eyigor O and Jennes L (1997) Expression of glutamate forebrain and hypothalamus in the rat. Part II: An receptor subunit mRNAs in gonadotropin-releasing hormone autoradiographic study of the anterior hypothalamus. neurons during the sexual maturation of the female rat. Pharmacological Reviews 169(2): 221–261. Neuroendocrinology 66(2): 122–129.Cornea A, Janovick JA, Maya-Nunez G, and Conn PM (2001) Eyigor O and Jennes L (2000) Kainate receptor subunit-positive Gonadotropin releasing hormone receptor gonadotropin-releasing hormone neurons express c-Fos microaggregation: Rate monitored by fluorescence during the steroid-induced luteinizing hormone surge in the resonance energy transfer. Journal of Biological Chemistry female rat. Endocrinology (Baltimore) 141(2): 779–786. 3(276): 2153–2158. Eyigor O, Lin W, and Jennes L (2004) Identification of neuronesCrowley WR, Hassid A, and Kalra SP (1987) Neuropeptide Y in the female rat hypothalamus that express oestrogen enhances the release of luteinizing hormone (LH) receptor-alpha and vesicular glutamate transporter-2. induced by LH-releasing hormone. Endocrinology Journal of Neuroendocrinology 16(1): 26–31. (Baltimore) 120: 941–945. Ferreira PA, Nakayama TA, Pak WL, and Travis GH (1996)Crowley WR and Kalra SP (1987) Neuropeptide Y stimulates the Cyclophilin-related protein RanBP2 acts as chaperone for release of luteinizing hormone-releasing hormone from medial red/green opsin. Nature 383: 637–640. basal hypothalamus in vitro. Modulation by ovarian hormones. ¨ Flugge G, Oertel WH, and Wuttke W (1986) Evidence for Neuroendocrinology 46(2): 97–103. estrogen-receptive GABAergic neurons in the preoptic/Crowley WR and Terry LC (1981) Effects of an epinephrine anterior hypothalamic area of the rat brain. synthesis inhibitor, SKF64139, on the secretion of luteinizing Neuroendocrinology 43(1): 1–5. hormone in ovariectomized female rats. Brain Research Fueshko S and Wray S (1994) LHRH cells migrate on peripherin 204(1): 231–235. fibers in embryonic olfactory explant cultures: An in vitroCrowley WR, Terry LC, and Johnson MD (1982) Evidence for model for neurophilic neuronal migration. Developmental the involvement of central epinephrine systems in the Biology 166(1): 331–348. regulation of luteinizing hormone, prolactin, and growth Gallo R and Drouva S (1979) Effects of intraventricular infusion hormone release in female rats. Endocrinology (Baltimore) of catecholamines on luteinizing hormone release in 110(4): 1102–1107. ovariecromized and ovariectomized, steroid primed rats.Dellovade TL, Pfaff DW, and Schwanzel-Fukuda M (1998) The Neuroendocrinology 29: 149–162. gonadotropin releasing hormone system does not develop in Gether U (2000) Uncovering molecular mechanisms involved in small-eye (Sey) mouse phenotype. Brain Research. activation of G protein-coupled receptors. Endocrine Developmental Brain Research 107(2): 233–240. Reviews 21(1): 90–113.Demling J, Fuchs E, Baumert M, and Wuttke W (1985) Preoptic Gilbeau PH, Almirez RG, Holaday JW, and Smith CG (1985) catecholamine, GABA, and glutamate release in Opioid effects on plasma concentration of luteinizing ovariectomized and ovariectomized estrogen primed rats hormone and prolactin in the adult rhesus monkey. utilizing pushpull cannula technique. Neuroendocrinology Journal of Clinical Endocrinology and Metabolism 60: 41: 212–218. 299–305.Dong C, Filipeanu CM, Duvernay MT, and Wu G (2007) Gimelbrant AA, Haley SL, McClintock TS (2001) Olfactory Regulation of G protein coupled receptor export trafficking. receptor trafficking involves conserved regulatory steps. Biochimica et Biophysica Acta 1768: 853–870. Journal of Biological Chemistry 276(10): 7285–7290.Drouva S and Gallo R (1976) Catecholamine involvement in Goldsmith PC, Thind KK, Perera AD, and Plant T (1994) episodic luteinzing hormone release in adult ovariectomized Glutamate-immunoreactive neurons and their rats. Endocrinology (Baltimore) 99: 651–658. gonadotropin-releasing hormone-neuronal interactions in
1662 The Gonadotropin-Releasing Hormone and Its Receptor the monkey hypothalamus. Endocrinology (Baltimore) formaldehyde-induced fluorescence. Journal of 134(2): 858–868. Comparative Neurology 176: 607–630.Gore AC, Wu TJ, Rosenberg JJ, and Roberts JL (1996) Heritage AS, Stumpf WE, Sar M, and Grant LD (1980) Brainstem Gonadotropin-releasing hormone and NMDA receptor catecholamine neurons are target sites for sex steroid gene expression and colocalization change during hormones. Science 207: 1377–1379. puberty in female rats. Journal of Neuroscience 16(17): Hetz C, Castilla J, and Soto C (2007) Perturbation of 5281–5289. endoplasmic reticulum homeostasis facilitates prionGorham JD, Baker H, Kegler D, and Ziff EB (1991) The replication. Journal of Biological Chemistry 282: expression of the neuronal intermediate filament protein 12725–12733. peripherin in the rat embryo. Brain Research. Developmental Hilal EM, Chen JH, and Silverman AJ (1996) Joint migration Brain Research 57(2): 235–248. of gonadotropin-releasing hormone (GnRH) andGottsch ML, Cunningham MJ, Smith JT, et al. (2004) A role for neuropepride Y (NPY) neurons from olfactory placode to kisspeptins in the regulation of gonadotropin secretion in the central nervous system. Journal of Neurobiology 31(4): mouse. Endocrinology 145(9): 4073–4077. 487–502.Gray GD, Soderstein P, Tallentire D, and Davidson JM (1978) Hoffman GE (1985) Organization of LHRH cells: Differential Effects of lesions in various structures of the apposition of neurotensin, substance P and catecholamine suprachiasmatic-preoptic region on LH regulation axons. Peptides (NY) 6(3): 439–461. and sexual behavior in female rats. Neuroendocrinology Hoffman GE and Gibbs FP (1982) LHRH pathways in rat 25(3): 174–191. brain: ‘Deafferentation’ spares a sub-chiasmaticHarmatz D, Ji TH, and Middaugh CR (1985) Aggregation state of LHRH projection to the median eminence. Neuroscience the gonadotropin receptor. Biochemical and Biophysical 7(8): 1979–1993. Research Communications 127(2): 687–692. Hoffman GE, Wray S, and Goldstein M (1982) Relationship ofHarney JP, Scarbrough K, Rosewell KL, and Wise PM (1996) catecholamines and LHRH: Light microscopic study. Brain In vivo antisense antagonism of vasoactive intestinal peptide Research Bulletin 9(1–6): 417–430. in the suprachiasmatic nuclei causes aging-like changes in Hoffman GE, Smith MS, and Verbalis JG (1993) c-Fos and the estradiol-induced luteinizing hormone and prolactin related immediate early gene products as markers of activity surges. Endocrinology (Baltimore) 137(9): 3696–3701. in neuroendocrine systems. Frontiers in NeuroendocrinologyHartl FE and Hayer-Hartl M (2002) Molecular chaperones in 14(3): 173–213. the cytosol: From nascent chain to folded protein. Science Hosny S and Jennes L (1998) Identification of alpha1B 295: 1852–1858. adrenergic receptor protein in gonadotropin releasingHartman RD, He JR, and Barraclough CA (1990) Gamma- hormone neurones of the female rat. Journal of aminobutyric acid-A and -B receptor antagonists increase Neuroendocrinology 10(9): 687–692. luteinizing hormone-releasing hormone neuronal Hrabovszky E, Shughrue PJ, Merchenthaler I, Hajszan T, responsiveness to intracerebroventricular norepinephrine in Carpenter CD, Liposits Z, and Petersen SL (2000) Detection ovariectomized estrogen-treated rats. Endocrinology of estrogen receptor-beta messenger ribonucleic acid and (Baltimore) 127(3): 1336–1345. 125I-estrogen binding sites in luteinizing hormone-releasingHebert TE, Moffett S, Morello J-P, Loisel TP, Bichet DG, hormone neurons of the rat brain. Endocrinology (Baltimore) Barret C, and Bouvier M (1996) A peptide derived from 141(9): 3506–3509. b2-adrenergic receptor transmembrane domain inhibits both Irwig MS, Fraley GS, Smith JT, et al. (2004) Kisspeptin activation receptor dimerization and activation. Journal of Biological of gonadotropin releasing hormone neurons and regulation Chemistry 271(27): 16384–16392. of KiSS-1 mRNA in the male rat. Neuroendocrinology 80(4):Heding A, Vrecl M, Bogerd J, McGregor A, Sellar R, Taylor PL, 264–272. and Eidne KA (1998) Gonadotropin-releasing hormone Janovick JA, Brothers SP, Cornea A, et al. (2007) Refolding of receptors with intracellular carboxyl-terminal tails undergo misfolded/misrouted mutant G-protein coupled receptor: acute desensitization of total inositol phosphate production Mechanism of pharmacoperone function. Molecular and and exhibit accelerated internalization kinetics. Journal of Cellular Endocrinology 272(1): 77–85. Biological Chemistry 273(9): 11472–11477. Janovick JA, Goulet M, Bush E, Greer J, Wettlauffer DG, andHeldin CH (1995) Dimerization of cell surface receptors in signal Conn PM (2003a) Structure–activity relations of successful transduction. Cell (Cambridge, MA) 80: 213–233. pharmacologic chaperones for rescue of naturally occurringHelenius S, Trombetta ES, Hebert DN, and Simons JF (1997) and manufactured mutants of the GnRHR. Journal of Calnexin, calreticulin and the folding proteins. Trends in Cell Pharmacology and Experimental Therapeutics 305(2): Biology 7: 193–200. 608–614.Herbison AE (1997a) Noradrenergic regulation of cyclic GnRH Janovick JA, Knollman PE, Brothers SP, Ayala-Yanez R, secretion. Reviews of Reproduction 2: 1–6. Aziz AS, and Conn PM (2006) Regulation of G-proteinHerbison AE (1997b) Estrogen regulation of GABA coupled receptor trafficking by inefficient plasma transmission in rat preoptic area. Brain Research Bulletin membrane expression: Molecular basis of an evolved 44(4): 321–326. strategy. Journal of Biological Chemistry 281(13):Herbison AE and Dyer RG (1991) Effect on luteinizing hormone 8417–8425. secretion of GABA receptor modulation in the medial ˜ Janovick JA, Maya-Nunez G, and Conn PM (2002) Rescue of preoptic area at the time of proestrous luteinizing hormone hypogonadotropic hypogonadism-causing and surge. Neuroendocrinology 53(3): 317–320. manufactured GnRH receptor mutants by a specific protein-Herbison AE, Robinson JE, and Skinner DC (1993) Distribution folding template: Misrouted proteins as a novel diseased of estrogen receptor-immunoreactive cells in the preoptic etiology and therapeutic target. Journal of Clinical area of the ewe. Co-localization with glutamic acid Endocrinology and Metabolism 87(7): 3255–3262. decarboxylase but not luteinizing hormone-releasing Janovick JA, Ulloa-Aguirre A, and Conn PM (2003b) Evolved hormone. Neuroendocrinology 57(4): 751–759. regulation of gonadotropin-releasing hormone receptor cellHeritage AS, Grant LD, and Stumpf WE (1977) 3H-estradiol surface expression. Endocrine 22(3): 317–327. in catecholamine neurons of rat brain stem: Jarry H, Hirsch B, Leonhardt S, and Wuttke W (1992) Amino Combined localization by autoradiography and acid neurotransmitter release in the preoptic area of rats
The Gonadotropin-Releasing Hormone and Its Receptor 1663 during the positive feedback actions of estradiol on LH Kaiser UB, Conn PM, and Chin WW (1997) Studies of release. Neuroendocrinology 56(2): 133–140. gonadolropin-releasing hormone (GnRH) action using GnRHJarry H, Leonhardt S, Schwarze T, and Wuttke W (1995) receptor-expressing pituitary cell lines. Endocrine Reviews Preoptic rather than mediobasal hypothalamic amino acid 18: 46–70. neurotransmitter release regulates GnRH secretion during Kaiser UB, Sabbagh E, Katzenellenbogen RA, Conn PM, and the estrogen-induced LH surge in the ovariectomized rat. Chln WW (1995) A mechanism for the differential regulation Neuroendocrinology 62(5): 479–486. of gonadotropin subunit gene expression by gonadotropin-Jennes L (1991) Dual projections of gonadotropin releasing releasing hormone. Proceedings of the National Academy of hormone containing neurons to the interpeduncular nucleus Sciences of the United States of America 92: 12280–12284. and to the vasculature in the female rat. Brain Research Kalra SP (1993) Mandatory neuropeptide-steroid signaling for 545(1–2): 329–333. the preovulatory luteinizing hormone-releasing hormoneJennes L, Beckman WC, Stumpf WE, and Grzanna R (1982) discharge. Endocrine Reviews 14: 507–537. Anatomical relationships of serotoninergic and Kalra PS, Bonavera JJ, and Kalra SP (1995) Central noradrenalinergic projections with the GnRH system in administration of antisense oligodeoxynucleotides to septum and hypothalamus. Experimental Brain Research neuropeptide Y (NPY) mRNA reveals the critical role of newly 46(3): 331–338. synthesized NPY in regulation of LHRH release. RegulatoryJennes L, Brame B, Centers A, Janovick JA, and Conn PM Peptides 59(2): 215–220. (1995) Regulation of hippocampal gonadotropin Kalra SF, Fuentes M, Fournier A, Parker SL, and Crowley WR releasing hormone (GnRH) receptor mRNA and (1992) Involvement of the Y-l receptor subtype in the GnRH-stimulated inositol phosphate production by gonadal regulation of lutelnizing hormone secretion by neuropeptide steroid hormones. Brain Research. Molecular Brain Y in rats. Endorinology (Baltimore) 130(6): 3323–3330. Research 33(1): 104–110. Kalra SP and Gallo R (1983) Effects of intraventricularJennes L and Conn PM (1994) Gonadotropin-releasing administration of catecholamines on luteinizing hormone hormone and its receptors in rat brain. Frontiers in release in morphine-treated rats. Endocrinology (Baltimore) Neuroendocrinology 15: 51–77. 113(1): 23–28.Jennes L, Dalati B, and Conn PM (1988) Distribution of Kalra PS, Kalra S, Krulich L, Fawcett C, and McCann SM (1972) gonadrotropin releasing hormone agonist binding sites in the Involvement of norepinephrine in transmission of the rat central nervous system. Brain Research 452(1–2): stimulatory influence of progesterone on gonadotropin 156–164. release. Endocrinology (Baltimore) 90: 1168–1176.Jennes L, Eyigor O, Janovick JA, and Conn PM (1997) Kalra SP and McCann SM (1974) Effects of drugs modifying Brain gonadotropin releasing hormone receptors: catecholamine synthesis on plasma LH and ovulation in the Localization and regulation. Recent Progress in Hormone rat. Neuroendocrinology 15: 79–91. Research 52: 475–491. Key S and Wray S (2000) Two olfactory placode derived galaninJennes L, Janovick JA, Braden T, and Conn PM (1990) subpopulations: Luteinizing hormone-releasing hormone Gonadotropin releasing hormone binding sites in rat neurones and vomeronasal cells. Journal of hippocampus: Different structure/binding relationships Neuroendocrinology 12(6): 535–545. compared to the anterior pituitary. Molecular and Cellular Khatchaturian H, Lewis ME, Tsou K, and Watson SJ (1985) Neuroscience 1: 121–127. b-Endorphin, a-MSH. ACTH, and related peptides. In:Jennes L and Schwanzel-Fukuda M (1992) Ontogeny of ¨ ¨ Bjorklund A and Hokfelt T (eds.) Handbook of Chemical gonadotropin-realeasing hormone-containing neuronal Neuroanatomy, vol. 4, pp. 216–272. Amsterdam: Elsevier. ¨ ¨ systems in mammals. In: Bjorklund A and Hokfelt T (eds.) Kim W, Kim Y, Min J, Kim DJ, Chang YT, and Hecht MH (2006) Handbook of Chemical Neuroanatomy, vol. 10, pp. 573–597. A high-throughput screen for compounds that inhibit Amsterdam: Elsevier. aggregation of the Alzheimer’s peptide. ACS ChemicalJennes L and Stumpf WE (1980a) LHRH-systems in the brain of Biology 1: 359–369. the golden hamster. Cell and Tissue Research 209(2): Kiss J and Halasz B (1985) Demonstration of serotoninergic 239–256. axons terminating on luteinizing hormone-releasing hormoneJennes L and Stumpf WE (1980b) LHRH-Neuronal projection to neurons in the preoptic area of the rat using a combination of the inner and outer surface of the brain. Neuroendocrinology immunocytochemistry and high resolution autoradiography. Letters 2: 241–246. Neuroscience 14(1): 69–78.Jennes L, Stumpf WE, and Sheedy ME (1985) Ultrastructural Knollman PE, Janovick JA, Brothers SP, and Conn PM (2005) characterization of gonadotropin-releasing hormone Parallel regulation of membrane trafficking and dominant- (GnRH)-producing neurons. Journal of Comparative negative effects by misrouted GnRH receptor mutants. Neurology 232: 534–547. Journal of Biological Chemistry 280(26): 24506–24514.Jennes L, Stumpf WE, and Tappaz ML (1983) Anatomical Kordon C, Drouva SV, Martinez de la Escalera G, Weiner RI, relationships of dopaminergic and GABAergic systems with Knobil E, and Neill JD (1994) Role of classic and peptide the GnRH-systems in the septo-hypothalamic area. neurotransmitters in the neuroendocrine regulation of Immunohistochemical studies. Experimental Brain Research luteinizing hormone and prolactin. In: Knobil E and Neill J 50(1): 91–99. (eds.) The Physiology of Reproduction, vol. 1,Jennes L and Woolums S (1994) Localization of pp. 1621–1681. New York: Raven Press. gonadotropin realizing hormone receptor mRNA in rat brain. Kramer PR, Guerrero G, Krishnamurthy R, Mitchell PJ, and Endocrine 2: 521–528. Way S (2000) Ectopic expression of luteinizingJirikowski GF, Merchenthaler I, Reiger GE, and Stumpf WE hormone-releasing hormone and peripherin in the (1986) Estradiol target sites immunoreactive for respiratory epithelium of mice lacking transcription factor beta-endorphin in the arcuate nucleus of rat and mouse AP-2alpha. Mechanisms of Development 94(1–2): 79–94. hypothalamus. Neuroscience Letters 65(2): 121–126. Krisch B (1980) Two types of luliberin-immunoreactiveJung H, Shannon EM, Fritschy JM, and Ojeda SR (1998) perikarya in the preoptic area of the rat. Cell and Tissue Several GABAA receptor subunits are expressed in LHRH Research 212(3): 443–455. neurons of juvenile female rats. Brain Research 780(2): Lashuel HA and Hirling H (2006) Rescuing defective vesicular 218–229. trafficking protects against a-synuclein toxicity in cellular
1664 The Gonadotropin-Releasing Hormone and Its Receptor and animal models of Parkinson’s disease. ACS Chemical Li C, Chen P, and Smith MS (1999) Morphological evidence for Biology 1: 420–424. direct interaction between arcuate nucleus neuropeptide Y ˜Leanos-Miranda A, Ulloa-Aguirre A, Janovick JA, and (NPY) neurons and gonadotropin-releasing hormone Conn PM (2005) In vitro coexpression and pharmacological neurons and the possible involvement of NPYYl receptors. rescue of mutant GnRH receptors causing Endocrinology (Baltimore) 140(11): 5382–5390. hypogonadotropic hypogonadism in humans expressing Liaw JJ, He JR, Hartman RD, and Barraclough CA (1992) compound heterozygous alleles. Journal of Clinical Changes in tyrosine hydroxylase mRNA levels in medullary Endocrinology and Metabolism 90(5): 3001–3008. A1 and A2 neurons and locus coeruleus following castration ˜Leanos-Miranda A, Ulloa-Aguirre A, Ji TH, Janovick JA, and and estrogen replacement in rats. Brain Research. Molecular Conn PM (2003) Dominant-negative action of disease- Brain Research 13(3): 231–238. causing GnRHR-mutants: A trait that potentially coevolved Lin X, Cornea A, Janovick JA, and Conn PM (1998a) with decreased plasma membrane expression of GnRHR in Visualization of unoccupied and occupied gonadotropin- humans. Journal of Clinical Endocrinology and Metabolism releasing hormone receptor in living cells. Molecular and 88(7): 3360–3367. Cellular Endocrinology 146: 27–37.Leblanc P, Crumeyrolle M, Latouche J, et al. (1988) Lin X, Janovick JA, Brothers S, Blomenrohr J, Bogerd J, and Characterization and distribution of receptors for Conn PM (1998b) Addition of catfish gonadotropin-releasing gonadotropin-releasing hormone in the rat hippocampus. hormone (GnRH) receptor intracellular carboxyl-terminal tail Neuroendocrinology 48(5): 482–488. to rat GnRH receptor alters expression and regulation.Lee E, Moore CT, Hosny S, Centers A, and Jennes L (2000a) Molecular Endocrinology 12: 161–171. Expression of estrogen receptor-alpha and c-Fos in Lin W, McKinney K, Liu L, Lakhlani S, and Jennes L (2003) adrenergic neurons of the female rat during the steroid- Distribution of vesicular glutamate transporter-2 messenger induced LH surge. Brain Research 875(1–2): 56–65. ribonucleic acid and protein in the septum-hypothalamus ofLee SP, O’Dowd BF, Ng GY, et al. (2000b) Inhibition of cell the rat. Endocrinology 144(2): 662–670. surface expression by mutant receptors demonstrates that Liposits Z, Setalo G, and Flerko B (1984) Application of the D2 dopamine receptors exist as oligomers in the cell. silver–gold intensified 3.30 -diaminobenzidine chromogen to Molecular Pharmacology 58: 120–128. the light and electron microscopic detection of the luteinizingLee WS, Smith MS, and Hoffman GE (1990) Luteinizing hormone-releasing hormone system of the rat brain. hormone-releasing hormone neurons express Fos protein Neuroscience 13(2): 513–525. during the proestrous surge of luteinizing hormone. Luine VN, Grattan DR, and Selmanoff M (1997) Gonadal Proceedings of the National Academy of Sciences of the hormones alter hypothalmic GABA and glutamate levels. United States of America 87: 5163–5167. Brain Research 747: 165–168.Lee WS, Smith MS, and Hoffman GE (1992) cFos activity Lumb MJ, Birdsey GM, and Danpure CJ (2003) Correction of an identifies recruitment of luteinizing hormone-releasing enzyme trafficking defect in hereditary kidney stone hormone neurons during the ascending phase of the disease in vitro. Biochemical Journal 374: 79–87. proestrous luteinizing hormone surge. Journal of Maegawa GHB, Tropak M, Buttner J, Stockley T, Kok F, Neuroendocrinology 4: 161–166. Clarke JTR, and Mahuran DJ (2007) Pyrimethamine as aLeranth C, MacLusky NJ, Sakamoto H, Shanabrough M, and potential pharmacological chaperone for late-onset Naftolin F (1985a) Glutamic acid decarboxylase-containing forms of GM2 gangliosidosis. Journal of Biological axons synapse on LHRH neurons in the rat medial preoptic Chemistry 282: 9150–9161. area. Neuroendocrinology 40(6): 536–539. Mallucci G and Collinge J (2005) Rational targeting for prionLeranth C, MacLusky NJ, Shanabrough M, and Naftolin F (1988) therapeutics. Nature Reviews Neuroscience 6: 23–34. Catecholaminergic innervation of luteinizing hormone- Mazzuca M (1977) Light and electron microscopic aspects of releasing hormone and glutamic acid decarboxyase the neuro-secretory cell. In: Vincent JD and Kordon C (eds.) immunopositive neurons in the rat medial preoptic area. Cell Biology of Hypothalamic Neurosecretion, vol. 280, Electron-microscopic double label and degeneration study. pp. 273–288. Paris: CNRS. Neuroedocrinology 48: 591–602. McShane TM, Wise PM, and Jennes L (1994) Neuropeptide-YLeranth C, MacLusky NJ, Shanabrough M, and Naftolin F neurons projectioning to the medial septum–diagonal band (1988b) ImmunohistochemicaI evidence for synaptic do not have access to fenestrated capillaries in the rat brain. connections between pro-opiomelanocorim- Molecular and Cellular Neuroscience 5(5): 459–465. immunoreactive axons and LH-RH neurons in the preoptic Mendez M, Cruz C, Joseph-Bravo P, Wilk S, and Charli JL area of the rat. Brain Research 449: 167–176. (1990) Evaluation of the role of prolyl endopeptidase andLeranth C, Segura LMG, Palkovits M, MacLusky NJ, pyroglutamyl peptidase 1 in the metabolism of LHRH and Shanabrough M, and Naftolin F (1985b) The LH-RH TRH in brain. Neuropeptides 17(2): 55–62. containing neuronal network in the preoptic area of the rat: Merchenthaler I, Gorcs T, Setalo G, Petrusz P, and Flerko B Demonstration of LH-RH containing nerve terminals in (1984) Gonadotropin-releasing hormone (GnRH) neurons synaptic contact with LH-RH neurons. Brain Research 345: and pathways in the rat brain. Cell Tissue Research 237(1): 332–336. 15–29.Leupen SM, Besecke LM, and Levine JE (1997) Neuropeptide Y Merchenthaler I, Kovacs G, Lavasz G, and Setalo G (1980) Y1-receptor stimulation is required for physiological The preoptico-infundibular LH-RH tract of the rat. amplification of preovulatory luteinizing hormone surges. Brain Research 198(1): 63–74. Endocrinology (Baltimore) 138(7): 2735–2739. Merchenthaler I, Lopez FJ, Lennard DE, and Negro-Vilar ALevine JE (1997) New concepts of the neuroendocrine (1991) Sexual differences in the distribution of regulation of gonadotropin surges in rats. Biology of neurons coexpressing galanin and luteinizing hormone- Reproduction 56(2): 293–302. releasing hormone in rhe rat brain. Endocrinology (Baltimore)Levine JE, Bauer-Dantoin AC, Besecke LM, et al. (1991) 129(4): 1977–1986. Neuroendocrine regulation of the luteinizing hormone- Messager S, Chatzidaki EE, Ma D, et al. (2005) Kisspeptin releasing hormone pulse generator in the rat. Recent directly stimulates gonadotropin-releasing hormone release Progress in Hormone Research 47: 97–153. via G protein-coupled receptor 54. Proceedings of the
The Gonadotropin-Releasing Hormone and Its Receptor 1665 National Academy of Sciences of the United States of Ping L, Mahesh VB, and Brann DW (1994) Release of glutamate America 102(5): 1761–1766. and aspartate from the preoptic area during theMillar RP (2003) GnRH I and type II GnRH receptors. Trends in progesterone-induced LH surge: In vivo microdialysis Endocrinology and Metabolism 14(1): 35–43. studies. Neuroendocrinology 59: 318–324.Miller BH and Gore AC (2002) N-methyl-D-aspartate receptor Pinter JH, Janovick JA, and Conn PM (1999) subunit expression in GnRH neurons changes during Gonadotropin-releasing hormone receptor concentration reproductive senescence in the female rat. Endocrinology differentially regulates intracellular signaling pathways in 143(9): 3568–3574. GGH3 cells. Pituitary 2: 181–190.Mitchell V, Prevot V, Jennes L, Aubert JP, Croix D, and Reubi JC, Palacios JM, and Maurer R (1987) Specific Beauvillain JC (1997) Presence of mu and kappa luteinizing-hormone-releasing hormone receptor binding opioid receptor mRNAs in galanin but not in sites in hippocampus and pituitary: An autoradiographical GnRH neurons in the female rat. NeuroReport 8(14): study. Neuroscience 21(3): 847–856. 3167–3172. Rivera VM, Wang X, Wardwell S, et al. (2000) Regulation ofMohankumar PS, Thyagarajan S, and Quadri SK (1994) protein secretion through controlled aggregation in the Correlations or catecholamine release in the medial endoplasmic reticulum. Science 287: 826–830. preoptic area with proestrous surges of luteinizing hormone Rocheville M, Lange DC, Kumer U, Patel SC, Patel RC, and and prolactin: Effects of aging. Endocrinology (Baltimore) Patel YC (2000a) Receptors for dopamine and somatostatin: 135(1): 119–126. Formation of hetero-oligomers with enhanced functionalMoore CT, Lee E, Tuggle B, Eyigor O, and Jennes L (1999) activity. Science 288: 154–157. Clutamategic and adrenergic innervation of Rocheville M, Lange DC, Kumer U, Patel SC, Patel RC, and Gonadotropin releasing hormone neurons in rat brain. Patel YC (2000b) Subtypes of the somatostatin receptor Advances in Reproduction 3: 293–302. assemble as functional homo- and heterodimers. Journal of ¨ ¨Morello JP, Salahpour A, Petaja-Repo UE, et al. (2001) Biological Chemistry 275(11): 7862–7869. Association of calnexin with wild type and mutant AVPR2 Rozell TG, Davis DP, Chai Y, and Segaloff DL (1998) that causes nephrogenic diabetes insipidus. Biochemistry Association of gonadotropin receptor precursors with the 23: 6766–6775. protein folding chaperone calnexin. Endocrinology 139:Moss RL and McCann SM (1973) Induction of mating 1588–1593. behavior in rats by luteinizing hormone-releasing factor. Rubinstein L and Sawyer CH (1970) Role of catecholamines in Science 181(95): 177–179. stimulating the release of pituitary ovulating hormone(s) inMugnaini E and Oertel WP (1985) An atlas of the rats. Endocrinology (Baltimore) 86(5): 988–995. distribution of GABAergic neurons and terminals in the Sabatino FD, Collins P, and McDonald JK (1989) Neuropeptide- rat CNS as revealed by GAD immunohistochemistry. In: Y stimulation of luteinizing hormone-releasing hormone ¨ ¨ Bjorklund A and Hokfelt T (eds.) Handbook of secretion from the median eminence in vitro by estrogen- Chemical Neuroanatomy, vol. 4, pp. 436–608. Amsterdam: dependent and extracellular Ca2+-independent Elsevier. mechanisms. Endocrinology (Baltimore) 124(5): 2089–2098.Navarro VM, Castellano JM, Fernandez-Fernandez R, et al. Sahu A, Crowley WR, and Kalra SP (1994) Hypothalamic (2005) Characterization of the potent luteinizing hormone- neuropeptide-Y gene expression increases before the releasing activity of KiSS-1 peptide, the natural ligand of onset of the ovarian steroid-induced luteinizing GPR54. Endocrinology 146(1): 156–163. hormone surge. Endocrinology (Baltimore) 134(3):Ohtaki T, Shintani Y, Honda S, et al. (2001) Metastasis 1018–1022. suppressor gene KiSS-1 encodes peptide ligand of a Sahu A, Jacobson W, Crowley WR, and Kalra SP (1989) G-protein-coupled receptor. Nature 411(6837): 613–617. Dynamic changes in neuropeptide Y concentrationsOttem EN and Petersen SL (2000) The majority of LHRH in the median eminence in association with neurons in the MEPO/OVLT express NMDARI. Society for preovulatory luteinizing hormone release in the rat. Neurosciences, 30th Annual Meeting. (Abstract 540.4). Neuroendocrinology 1: 83–87. New Orleans, LA. Sannella MI and Petersen SL (1997) Dual label in situPace AJ, Gama L, and Breitwieser GE (1999) Dimerization of the hybridization studies provide evidence that luteinizing calcium-sensing receptor occurs within the extracellular hormone-releasing hormone neurons do not domain and is eliminated by Cys 6 Ser mutations at Cys101 synthesize messenger ribonucleic acid for mu, kappa, or and Cys236. Journal of Biological Chemistry 274(17): delta opiate receptors. Endocrinology (Baltimore) 138(4): 11629–11634. 1667–1672.Petersen SL, Curran MA, Hrabovszky EL, and Ottem EN (1999) Sar M, Sahn A, Crowley WR, and Kalra SP (1990) Localization Neurotransmitter regulation of GnRH synthesis and release. of neuropeptide-Y immunoreactivity in estradiol- 81st Annual Meeting of the Endocrine Society. (Abstract concentrating cells in the hypothalamus. Endocrinology S19–2). San Diego, CA. (Baltimore) 127(6): 2752–2756.Petersen SL, McCrone S, Adelman JP, and Mahan LC (1993) Sarkar DK and Yen SS (1985) Changes in beta-endorphin-like GABAA, receptor subunit mRNA in cells of the preoptic area: immunoreactivity in pituitary portal blood during the estrous Colocalization with LHRH mRNA using dual-label in situ cycle and after ovariectomy in rats. Endocrinology hybridization histochemistry. Endocrine 1: 29–34. (Baltimore) 116(5): 2075–2079.Pfaff DW (1973) Luteinizing hormone-releasing Schneuwly S, Shortridge RD, Larrivee DC, Ono T, Ozaki M, and factor potentiates lordosis behavior in Pak WL (1989) Drosophila ninaA gene encodes an typophysectomized ovariectomized female rats. Science eye-specific cyclophilin (cyclosporine A binding protein). 182(117): 1148–1149. Proceedings of the National Academy of Sciences of thePfaff DW, Schwartz-Giblin S, McCarthy MM, and Kow L-M United States of America 86: 5390–5394. (1994) Cellular and molecular mechanisms of female Schrag JD, Procopio DO, Cygler M, Thomas DY, and reproductive behavior. In: Knobil E and Neill JD (eds.) The Bergeron JJM (2003) Lectin control of protein folding and Physiology of Reproduction, pp. 107–220. New York: Raven sorting in the secretory pathway. Trends in Biochemical Press. Sciences 28: 49–57.
1666 The Gonadotropin-Releasing Hormone and Its ReceptorSchroder M and Kaufman RJ (2005) The mammalian Silverman AJ, Jhamandas J, and Renaud LP (1987) Localization unfolded protein response. Annual Review of Biochemistry of luteinizing hormone-releasing hormone (LHRH) neurons 74: 739–789. that project to the median eminence. Journal ofSchwanzel-Fukuda M (1999) Origin and migration of Neuroscience 7(8): 2312–2319. luteinizing hormone-releasing hormone neurons Silverman AJ, Krey LC, and Zimmerman EA (1979) A in mammals. Microscopy Research and Technique 44(1): comparative study of the luteinizing hormone releasing 2–10. hormone (LHRH) neuronal networks in mammals. Biology ofSchwanzel-Fukuda M, Abraham S, Crossin KL, Edelman GM, Reproduction 20(1): 98–110. and Pfaff DW (1992) Immunocytochemical demonstration Silverman AJ, Livne I, and Witkin JW (1994) The gonadotropin- of neural cell adhesion molecule (NCAM) along the releasing hormone, neuronal systems: migration route of luteinizing hormone-releasing hormone Immunocytochemistry and in situ hybridization. (LHRH) neurons in mice. Journal of Comparative Neurology In: Knobil E and Neill JD (eds.) The Physiology of 321(1): 1–18. Reproduction, pp. 1683–1709. New York: Raven Press.Schwanzel-Fukuda M and Pfaff DW (1989) Origin of luteinlzing Simonian SX and Herbison AE (1997) Differential hormone-releasing hormone neurons. Nature (London) 338: expression of estrogen receptor and neuropeptide Y by 161–164. brainstem A1 and A2 noradrenaline neurons. NeuroscienceSchwanzel-Fukuda M and Pfaff DW (1990) The migration of 76(2): 517–529. luteinizing hormone-releasing hormone (LHRH) neurons Skinner DC, Malpaux B, Velaleu B, and Caraty A (1995) from the medial olfactory placode into the medial basal Luteinizing hormone (LH)-releasing hormone in third forebrain. Experientia 46(9): 956–962. ventricular cerebrospinal fluid of the ewe: Correlation with LHSchwanzel-Fukuda M and Pfaff DW (1991) Migration of LHRH- pulses and the LH surge. Endocrinology (Baltimore) 136(8): immunoreactive neurons from the olfactory placode 3230–3237. rationalizes olfacto-hormonal relationships. Journal of Skynner MJ, Slater R, Sim JA, Allen ND, and Herbison AE (1999) Steroid Biochemistry and Molecular Biology 39(4B): Promoter transgenics reveal multiple gonadotropin- 565–572. releasing hormone-l-expressing cell populations of differentSchwanzel-Fukuda M, Pfaff DW, Crossin KL, Cremer H, embryological origin in mouse brain. Journal of Hardelin J-P, and Petit C (1995) Luteinizing-releasing Neuroscience 19(14): 5955–5966. hormone (LHRH) neurons migrate normallin neural Smith JT, Cunningham MJ, Rissman EF, Clifton DK, and cell adhesion molecule (N-CAM) deficient mice. Steiner RA (2005a) Regulation of Kiss1 gene expression in Chemical Senses. Association for Chemoreception the brain of the female mouse. Endocrinology 146(9): Sciences Meeting Abstracts. Association for 3686–3692. Chemoreception Sciences. Smith JT, Dungan HM, Stoll EA, et al. (2005b)Schwanzel-Fukuda M, Reinhard GR, Abraham S, Crossin KL, Differential regulation of KiSS-1 mRNA expression by sex Edelman GM, and Pfaff DW (1994) Antibody to neural steroids in the brain of the male mouse. Endocrinology 146 cell adhesion molecule can disrupt the migration of (7): 2976–2984. luteinizing hormone-releasing hormone neurons into the Smith MJ, Jennes L, and Wise EM (2000) mouse brain. Journal of Comparative Neurology 342(2): Localization of the VIP2 receptor protein on GnRH 174–185. neurons in the female rat. Endocrinology (Baltimore)Schwanzel-Fukuda M and Silverman AJ (1980) The nervus 141(11): 4317–4320. terminalis of the guinea pig: A new luteinizing hormone- ¨ Spergel DJ, Kruth U, Hanley VF, Sprengel R, and Seeburg PH releasing hormone (LHRH) neuronal system. Journal of (1999) GABA- and glutamate-activated channels in green Comparative Neurology 191(2): 213–225. fluorescent protein-tagged gonadotropin-releasing hormoneSealfon SC, Weinstein H, and Miller RP (1997) Molecular neurons in transgenic mice. Journal of Neuroscience 19(6): mechanisms of ligand interaction with the 2037–2050. gonadotropin-releasing hormone receptor. Endocrine Stanislaus D, Janovick JA, Brothers S, and Conn PM (1997) Reviews 18: 180–205. Regulation of Gq/11a by the GnRH receptor. MolecularSeong JY, Kang SS, Kam K, Han YG, Kwon HB, Ryu K, and Endocrinology 11: 738–746. Kim K (1998) Differential regulation of gonadotropin- Stanislaus D, Ponder S, Ji TH, and Conn PM (1998) releasing hormone (GnRH) receptor expression in the Gonadotropin-releasing hormone couples to multiple G posterior mediobasal hypothalamus by steroid hormones: proteins in rat gonadotropes and in GGH3 cells: Evidence Implication of GnRH neuronal activity Brain Research. from palmitoylation and overexpression of G proteins. Molecular Brain Research 53(1–2): 226–235. Biology of Reproduction 59: 579–586.Serova L, Rivkin M, and Sabban EL (2000) Estrodiol can Stojilkovick SS, Reinhart J, and Catt KJ (1994) regulate dopamine B-hydroxylase transcription. Society Gonadotropin-releasing hormone receptors: Structure for Neurosciences, 30th Annual Meeting. (Abstract 19.4). and signal transduction pathways. Endocrine Reviews 15: New Orleans, LA. 462–499.Sheaves R, Warburton E, Laynes R, and Mackinnon P (1984) Tan CM, Brady AE, Nickols HH, Wang Q, and Limbird LE (2004) Adrenaline concentration and turnover in the arcuate Membrane trafficking of G protein-coupled receptors. nucleus and median eminence during the critical period in Annual Review of Pharmacology and Toxicology 44: the rat. Brain Research 323(2): 326–329. 559–609.Shieh BH, Stamnes MA, Seavello S, Harris GL, and Zuker CS Terasawa E, Luchansky LL, Kasuya E, and Nyberg CL (1999) (1989) The ninaA gene required for visual transduction in An increase in glutamate release follows a decrease in Drosophila encodes a homologue of cyclosporin A-binding gamma aminobutyric acid and the pubertal increase in protein. Nature 338: 67–70. luteinizing hormone releasing hormone release in theShughrue PJ, Lane MV, and Merchenthaler I (1997) female rhesus monkeys. Journal of Neuroendocrinology Comparative distribution of estrogen receptor and mRNA in 11(4): 275–282. the rat central nervous system. Journal of Comparative Thind KK and Goldsmith PC (1997) Expression of estrogen and Neurology 388(4): 507–525. progesterone receptors in glutamate and GABA neurons of
The Gonadotropin-Releasing Hormone and Its Receptor 1667 the pubertal female monkey hypothalamus. cerebrospinal fluid of ovariectomized rhesus monkeys: Neuroendocrinology 65(5): 314–324. Correlation with luteinizing hormone pulses. EndocrinologyThomas RM, Nechamen CA, Mazurkiewicz JE, Muda M, (Baltimore) 117(4): 1550–1558. Palmer S, and Dias JA (2007) Follicle-stimulating hormone Van Vugt GT, Sylvester PW, Aylsworth CF, and Meites J (1982) receptor forms oligomers and shows evidence of Countreraction of gonadal steroid inhibition of LH by carboxyl-terminal proteolytic processing. Endocrinology naloxone. Neuroendocrinology 34: 274. 148(5): 1987–1995. Vassilakos A, Michalak M, Lehrman MA, and Williams DB (1998)Tillet Y, Caldani M, and Batailler M (1989) Anatomical Oligosaccharide binding characteristics of the molecular relationships of monoaminergic and neuropeptide chaperones calnexin and calreticulin. Biochemistry 37: Y-containing fibres with luteinizing hormone-releasing 3480–3490. hormone systems in the preoptic area of the sheep brain: Vij N, Fang S, and Zeitlin PL (2006) Selective inhibition of Immunohistochemical studies. Journal of Chemical endoplasmic reticulum-associated degradation rescues Neuroanatomy 2(6): 319–326. DF508-cystic fibrosis transmembrane regulator andTsuruo Y, Kawano H, Kagotani Y, et al. (1990) suppresses interleukin-8 levels. Journal of Biological Morphological evidence for neuronal regulation of luteinizing Chemistry 281: 17369–17378. hormone- releasing hormone-containing neurons by Watanobe H and Takebe K (1992) Evidence that neuropeptide Y neuropeptide Y in the rat septo-preoptic area. Neuroscience secretion in the median eminence increases prior to the Letters 110(3): 261–266. luteinizing hormone surge in ovariectomized steroid-primedTsutsumi M, Zhou W, Millar PP, et al. (1992) Cloning and rats: Estimation by push–pull perfusion. Neuroscience functional expression of a mouse gonadotropin-releasing Letters 146(1): 57–59. hormone receptor. Molecular Endocrinology 6(7): Weesner GD and Malven PV (1990) Intracerebral 1163–1169. immunoneutralization of beta-endorphin and met-Ulloa-Aguirre A and Conn PM (2000) G protein-coupled enkephalin disinhibits release of pituitary luteinizing hormone receptors and G proteins. In: Conn PM and Means AR (eds.) in sheep. Neuroendocrinology 52: 382–388. Principles of Molecular Regulation, pp. 3–25. Totowa, NJ: Wehrenberg WB, Corder R, and Gaillard RC (1989) A Humana Press. physiological role for neuropeptide Y in regulating theUlloa-Aguirre A, Janovick JA, Brothers SP, and Conn PM estrogen/progesterone induced luteinizing hormone surge (2004a) Pharmacological rescue of conformationally- in ovariectomized rats. Neuroendocrinology 49: 680–682. defective proteins: Implications for the treatment of human Wise PM (1982) Norepinephrine and dopamine activity in disease. Traffic 5(11): 821–837. microdissected brain areas of the middle-aged and ˜Ulloa-Aguirre A, Janovick JA, Leanos-Miranda A, and Conn PM young rat on proestrus. Biology of Reproduction 27(3): (2003) Misrouted cell surface receptors as a novel 562–574. disease etiology and potential therapeutic target: The case Wise P, Rance N, and Barraclough C (1981) Effects of estradiol of hypogonadotropic hypogonadism due to GnRH and progesterone on catecholamine turnover rates in resistance. Expert Opinion on Therapeutic Targets 7(2): discrete hypothalamic regions in ovariectomized rats. 175–185. Endocrinology (Baltimore) 108: 2186–2193. ˜Ulloa-Aguirre A, Janovick JA, Leanos-Miranda A, and Conn PM Witkin JW (1992) Increased synaptic input to (2004b) Misrouted cell surface GnRH receptors as a disease gonadotropin-releasing hormone neurons in aged, etiology for congenital isolated hypogonadotropic virgin, male Sprague-Dawley rats. Neurobiology of Aging hypogonadism. Human Reproduction Update 10(2): 13(6): 681–686. 177–192. Witkin JW and Demasio K (1990) Ultrastructural differences ¨¨ ¨Ulloa-Aguirre A, Stanislaus D, Arora V, Vaananen J, Brothers S, between smooth and thorny gonadotropin-releasing Janovick JA, and Conn PM (1998) The third intracellular loop hormone neurons. Neuroscience 34(3): 777–783. of the rat gonadotropin-releasing hormone (GnRH) receptor Witkin JW and Silverman AJ (1985) Synaptology of LHRH couples the receptor to Gs- and Gq/11-mediated signal neurons in rat preoptic area. Peptides (NY) 6: 263–271. transduction pathways: Evidence from loop fragment Wray S, Grant P, and Gainer H (1989a) Evidence that cells transfection in GGH3 cells. Endocrinology (Baltimore) 139: expressing luteinizing hormone-releasing hormone mRNA 2472–2478. in the mouse are derived from progenitor cells in thevan der Beek EM, Swarts HJ, and Wiegant VM (1999) Central olfactory placode. Proceedings of the National Academy administration of antiserum to vasoactive intestinal of Sciences of the United States of America 86(20): peptide delays and reduces luteinizing hormone and 8132–8136. prolactin surges in ovariectomized, estrogen-treated rats. Wray S and Hoffman G (1986) Postnatal morphological changes Neuroendocrinology 69(4): 227–237. in rat LHRH neurons correlated with sexual maturation.van der Beek EM, van Oudheusden HJ, Buijs RM, van der Neuroendocrinology 43: 93–97. Donk HA, and van den Hurk R (1994) Preferential induction Wray S, Key S, Qualls R, and Fueshko SM (1994) A subset of of c-fos immunoreactivity in vasoactive intestinal peripherin positive olfactory axons delineates the polypeptide-innervated gonadotropin-releasing hormone luteinizing hormone releasing hormone neuronal migratory neurons during a steroid-induced luteinizing hormone surge pathway in developing mouse. Developmental Biology in the female rat. Endocrinology (Baltimore) 134(6): 166(1): 349–354. 2636–2644. Wray S, Nieburgs A, and Elkabes S (1989b) Spatiotemporal cellvan der Beek EM, Wiegant VM, van der Donk HA, and van den expression of luteinizing hormone-releasing hormone in the Hurk R (1993) Lesions of the suprachiasmatic nucleus prenatal mouse: Evidence for an embryonic origin in the indicate the presence of a direct vasoactive intestinal olfactory placode. Brain Research. Developmental Brain polypeptide-containing projection to gonadotrophin- Research 46(2): 309–318. releasing hormone neurons in the female rat. Journal of Wu TJ, Gibson MJ, and Silverman AJ (1995) Gonadotropin- Neuroendocrinology 5(2): 137–144. releasing hormone (GnRH) neurons of the developlngVan Vugt DA, Diefenbach WD, Akton E, and Ferin M (1985) tectum of the mouse. Journal of Neuroendocrinology 7(12): Gonadotropin-releasing hormone pulses in third ventricular 899–902.
1668 The Gonadotropin-Releasing Hormone and Its ReceptorXu Z, Hirasawa A, Shinoura H, and Tsujimoto G (1999) hormone-immunoreactive neurons in the medial olfactory Interaction of the alpha(1B)-adrenergic receptor with gClq-R, placode and basal forebrain of embryonic mice. a multifunctional protein. Journal of Biological Chemistry Neuroscience 46(2): 407–418. 274: 21149–21154.Xu M, Urban JH, Hill JW, and Levine JE (2000) Regulation of hypothalamic neuropeptide Y Yl receptor gene expression during the estrous cycle: Role of progesterone receptors. Further Reading Endocrinology (Baltimore) 141(9): 3319–3327.Yang Y, Turner RS, and Gaut JR (1998) The chaperone BiP/ Ulloa-Aguirre A, Janovick JA, Miranda AL, Conn PM (2006) GRP78 binds to amyloid precursor protein and decreases G protein-coupled receptor trafficking: Understanding the Ab40 and Ab42 secretion. Journal of Biological Chemistry chemical basis of health and disease. ACS Chemical Biology 273: 25552–25555.Zheng LM, Pfaff DW, and Schwanzel-Fukuda M (1992) Electron 1(10): 631–638. microscopic identification of luteinizing hormone-releastng
Biographical SketchLothar Jennes is professor in the Department of Anatomy and Neurobiology at the University of Kentucky. He received hisgraduate training at the University of Salzburg, Austria, and postdoctoral training at INSERM in Lille, France, theUniversity of North Carolina in Chapel Hill, and Duke University in Durham. His laboratory focuses on theneuroendocrine control of reproduction with particular emphasis on the factors and mechanisms that controlgonadotropin-releasing hormone neurons in young and senescent animals. He is the author or co-author of over 100research publications.Alfredo Ulloa-Aguirre was born in 1949 in Mexico City. He obtained his MD, DSc, and specialty degrees in Internal Medicineand Reproductive Endocrinology at the National University of Mexico. In 1980 he was awarded a Rockefeller FoundationFellowship to attend the Division of Reproductive Biology of the University of Pennsylvania in Philadelphia, Pennsylvania,USA, as a posdoctoral fellow in reproductive biology and endocrinology. In 1996 and 2003, he was a visiting professor at theOregon National Primate Research Center, Oregon Health Sciences University in Beaverton, Oregon, USA where he iscurrently a collaborating scientist. He leads the Research Unit in Reproductive Medicine at the Mexican Institute of SocialSecurity and has published prolifically and received many awards and honors for his scientific achievements. He has been amember of many editorial boards including Archives of Medical Research, Human Reproduction, Endocrine, Reproductive BiomedicineOnline and Reproductive Biology and Endocrinology, and Recent Patents on Endocrine, Metabolic & Immune Drug Discovery. The researchfocus of his group includes the study of the structure–function relationship of gonadotropins and gonadotropin receptors, theGnRH receptor, and the neuroendocrine regulation of gonadotropin secretion.Jo Ann Janovick is a senior research associate and registered pharmacist and has been in the Conn laboratory for 23 years,contributing to research, technique development, and training. She is a collector of Pacific Northwest Indian art and isdevoted to her two dogs.Valeriya V. Adjan, MD, is a scientist II in the Department of Anatomy and Neurobiology at the University of Kentucky.Before joining the University of Kentucky in 2002, she was an internal medicine physician in Rostov, Russia. At UK, shebecame interested in the field of reproductive biology and joined the Jennes lab to study the role of neurotransmitters thatcontrol GnRH release.P. Michael Conn is associate director and senior scientist of the Oregon National Primate Research Center and professor ofphysiology and pharmacology, and cell and developmental biology at OHSU. He has written extensively on the molecularmechanism of hormone action and on topics related to neuroscience, endocrinology, physiology, pharmacology, andmolecular biology. He is an outspoken supporter of the humane use of animals in research and the importance of publiceducation about the importance of science.