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Frontiers in Neuroendocrinology xxx (2011) xxx–xxx Contents lists available at ScienceDirect Frontiers in Neuroendocrinology journal homepage: www.elsevier.com/locate/yfrne Review Control of GnRH secretion: One step back Iain J. Clarke ⇑ Department of Physiology, Monash University, P.O. Box 13F, Clayton, Victoria 3800, Australia a r t i c l e i n f o Article history: Available online xxxx Keywords: Gonadotropin releasing hormone RF-amide peptide Reproduction Food intake Hypothalamus a b s t r a c t The reproductive system is controlled by gonadotropin releasing hormone (GnRH) secretion from the brain, which is finely modulated by a number of factors including gonadal sex steroids. GnRH cells do not express estrogen receptor a, but feedback is transmitted by neurons that are at least ‘one step back’ from the GnRH cells. Modulation by season, stress and nutrition are effected by neuronal pathways that converge on the GnRH cells. Kisspeptin and gonadotropin inhibitory hormone (GnIH) neurons are regulators of GnRH secretion, the former being a major conduit for transmission of sex steroid feedback. GnIH cells project to GnRH cells and may play a role in the seasonal changes in reproductive activity in sheep. GnIH also modulates the action of GnRH at the level of the pituitary gonadotrope. This review focuses on the role that kisspeptin and GnIH neurons play, as modulators that are ‘one step back’ from GnRH neurons. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction The original finding of Harris [53], that electrical stimulation of the brain caused ovulation in the rabbit, instigated his lifelong mission to delineate the means by which pituitary gonadotropin secretion is stimulated. This culminated in the development of the neurohumoral theory [54] which relies upon a unique anatomical arrangement in the median eminence and a highly efficient conduit of multiple signals from various types of hypothalamic cells to their respective target cells in the anterior pituitary. The early work of Harris and others, which led to the formulation of the neurohumoral theory was well reviewed by Donovan [42] in an earlier Harris Memorial Lecture. Whereas it became apparent that neurohumoral factors were produced by the hypothalamus and reached the anterior pituitary gland by way of the hypophysial portal blood system, it was not until the chemical identification of such factors that proof of such ‘messengers’ was obtained. The purification of thyrotropin releasing hormone (TRH) [14,16] from hypothalamic extracts was closely followed by discovery of gonadotropin releasing hormone (GnRH) [2,86,107]. Proof of secretion of GnRH into the portal system was provided by the technically challenging studies of Fink and colleagues [106]. Original work in the non-human primate provided evidence that the pattern of secretion of luteinizing hormone (LH) was pulsatile [40]. Ultimately, the exact relationship between the secretion of GnRH and that of LH from the pituitary was revealed with the creation of a model in sheep that allowed concomitant serial sampling of hypophysial portal blood and jugular ⇑ Fax: +61 3 9905 2547. E-mail address: [email protected] venous blood in conscious, sentient sheep [20,29]. This allowed the full description of the hypothalamo-pituitary–gonadal (HPG) axis, outlining how GnRH action is modulated at the level of the pituitary gonadotrope and how feedback effects of gonadal hormones modulate the secretion of GnRH and the gonadotrophins [23]. The measurement of GnRH secretion also allowed definition of physiological and environmental factors that regulate reproduction at the level of the GnRH system. This included revelation of the effects of gonadectomy [20], photoperiod/season [72], stress [92], stress levels of cortisol [89], immune response [69] and gonadal steroid feedback [25,71] on GnRH secretion. One of the most fundamental aspects of the operation of the HPG axis is the means by which sex steroids act to modulate GnRH secretion and this took some time to resolve. Since GnRH cells do not possess the relevant sex steroid receptors [57], significant efforts were made in various laboratories and species over decades (1970s to 2003) to identify steroid-receptive elements in the brain that relayed feedback information to the GnRH cells. Various cell types were found to express estrogen, progesterone and androgen receptors, but evidence of a major conduit remained elusive [127]. A significant advance was the discovery that the kisspeptin receptor (and by implication, its ligand, kisspeptin) was essential for normal reproduction [39,108]. Intense investigation over the past 7 years strongly suggests that sex steroid feedback regulation of GnRH cells is predominantly exerted via kisspeptin cells, although many other cell types in the brain also play a role. These cells are at least ‘one step back’ from the GnRH cells, allowing for integration of information of steroid feedback, season, stress, immune status, nutritional status, etc. to be synthesized by the GnRH cells and converted to a singular output of the brain that drives the reproductive system. Thus, serial and neuronal systems converge on 0091-3022/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yfrne.2011.01.001 Please cite this article in press as: I.J. Clarke, Control of GnRH secretion: One step back, Front. Neuroendocrinol. (2011), doi:10.1016/j.yfrne.2011.01.001 2 I.J. Clarke / Frontiers in Neuroendocrinology xxx (2011) xxx–xxx the GnRH cells to determine the output of these cells in terms of secretion. A major focus of this review is the role of kisspeptin in the regulation of the GnRH system, especially in relation to work done in the ovine model. The reader is also referred to other recent reviews on the function of kisspeptin [19,90,112]. Another recently recognized modulator of reproductive function is gonadotropin inhibitory hormone (GnIH). Whereas the prevailing view was that the secretion of GnRH was the singular means by which the brain controls the reproductive system, the existence of an inhibitory system was also entertained. Gonadal factors such as luteinizing hormone release-inhibiting factor (LHRIF) [62] were proposed. Another inhibitory factor, gonadotropin surge inhibiting factor (GnSIF) [37], suppresses LH secretion with or without any effect on FSH. This latter is a protein produced by ovarian follicles, not the brain [83], but there was no convincing evidence of hypothalamic factor that negatively regulated the reproductive axis until 2000. The discovery that GnIH was present in the hypothalamus of the quail [131] and that it acted as a negative regulator of reproduction was, therefore, an important advance in our knowledge. Whilst evidence that GnIH is an important facet of the HPG axis in mammals has been tardy, work over the last few years has escalated its importance. Like kisspeptin, GnIH is an RF-amide peptide and it exerts negative effects on GnRH cells [43] and, at least in some species, the gonadotropes [33]. Recent and extensive reviews on the role of GnIH in mammals are also available [32,77,111,129,130] and this review will focus mainly on work in relation to GnIH function that has been carried out in sheep. The following review will consider the evidence that GnIH cells are also ‘one step back’ from GnRH cells and play a major role in the regulation of GnRH secretion as well as acting on the pituitary gonadotropes, in sheep at least, to negatively regulate gonadotropin synthesis and secretion. 2. Inherent properties of GnRH cells Andrew and Dudek [5] showed that magnocellular neuroendocrine cells possess inherent phasic firing patterns [5] and this was extended to a demonstration of the same in GnRH cells when the ‘labeling’ of cells by incorporation of green fluorescent protein genes through transgenics provided a means of recording from single units in vitro [79]. Others showed that multi-unit activity of the hypothalamus correlated with pulsatile LH secretion in rats [74], sheep [126] and non-human primates [138]. The work of Dudek and colleagues suggested that all neuroendocrine may cells possess an inherent phasic rhythm [4,5]. With the advent of transgenic mice in which green fluorescent protein was genetically targeted to GnRH neurons, it became possible to target individual cells, revealing that single units displayed bursting consistent with phasic secretory activity [122]. This is translated into the secretory mode as demonstrated by the phasic release of GnRH from cells of the fetal nasal placode of rhesus monkeys studied in culture [124]. One might envisage a model, based on modulation of such inherent phasic activity of the hypophysiotropic cells. On the other hand, based on the multi-unit activity generated by unknown elements in the basal hypothalamus, the concept of a ‘pulse generator’ continues to be embraced. For the reasons given above, pulse generation by neurons that are ‘upstream’ of neuroendocrine cells may not be necessary if the neuroendocrine cells themselves have inherent phasic rhythms. To achieve co-ordinate phasic rhythm of a number of neurons, such that a pulsatile discharge of the hypophysiotropic hormone occurs at regular intervals, some means of communication between the neurons would seem necessary. In the case of GnRH neurons, this appears to be achieved by dendro-dendritic connections [17]. One could infer that neuronal elements that modulate GnRH neurons are the ‘pulse generators’ and a wide range of neuronal inputs to the neurons are known; this is discussed in the next section. Irrespective of either model, it is clear that GnRH neurons function in a phasic mode, leading to the ultradian pulse pattern of GnRH secretion. In the free-running situation, unrestrained by gonadal steroid feedback, the ultradian pulse rhythm of GnRH cells (reflected in LH pulses) is roughly one hour, as well typified in nonhuman primates [40] and sheep [29,73]. It should be noted that, in contrast to the pulse pattern of LH secretion, that of FSH secretion does not rigidly reflect GnRH secretion [24]. This is because FSH secretion does not depend directly on the secretagogue properties of GnRH [28,30]. 3. Regulation of GnRH cells by neuronal systems within the brain Given that GnRH cells operate in a phasic manner, the modulation of ultradian secretion becomes highly relevant, since this dictates reproductive function. Such a system allows for alterations in frequency and amplitude with exquisite precision; variation in these two parameters allows very fine tuning of GnRH output. The question then becomes one of how such modulation is effected. Afferents neurons that provide either direct or indirect input to GnRH cell bodies are important in this regard, so a large number of studies employing a variety of techniques have cataloged these. Evidence exists for direct input to GnRH cells from brain stem noradrenergic cells [95,100] and the serotoninergic elements of the raphe nucleus [76], as well as various cell types of the hypothalamus and the forebrain [63,93,95,96]. In order to exert fine control over GnRH secretion, however, it is not necessary that upstream neuronal systems form a direct pathway to the GnRH cells. Thus, it is possible that pathways from regions such as the A1 noradrenergic field of the brainstem may involve inter-neuronal relays in the bed nucleus of the stria terminalis (BNST) or preoptic regions in close vicinity to the GnRH cells [91,93]. Anterograde and retrograde neuronal tracing between the arcuate nucleus of the hypothalamus (ARC) and the preoptic area of the ovine brain indicate that there is very limited direct input to GnRH cells from the former [9,97]. Thus, even though a case is made below for a predominant role of kisspeptin cells in the ARC in the regulation of GnRH cells, there is little evidence of direct neuronal projections that subserve this [9]. One caveat on this observation is that kisspeptin cells may project to dendrites of GnRH cells that are not readily seen without special techniques. On the other hand, kisspeptin cells in the lateral POA of the ovine brain do appear to provide direct neuronal input to GnRH cells [9]. The existence of multi-synaptic pathways to GnRH neurons from various regions of the brain presumably allows even further means of fine control of the reproductive system, incorporating information in relation to metabolic status, season, stress state, immune status, olfactory stimuli, etc. By 2002, various neuronal systems of the brain were known to provide input to GnRH cells and the neuroanatomical framework was reviewed [127]. Other reviews provided a description of sex steroid responsive systems that might subserve feedback regulation of GnRH cells [57]. It is notable, however, that no single neuronal system could be ascribed a predominant role in the modulation of GnRH cells by sex steroids. Noradrenergic cells of the brain stem appear important for normal function, but it is also likely that glutamatergic input plays a major role as a positive regulator of GnRH cells [35,79]. Certainly, GnRH cells in the sheep brain receive a very high level of glutamatergic input, based on confocal microscopic studies [95]. The other most common neurotransmitter in the hypothalamus is gamma amino butyric acid (GABA) and GABAergic cells appear to be major negative regulators Please cite this article in press as: I.J. Clarke, Control of GnRH secretion: One step back, Front. Neuroendocrinol. (2011), doi:10.1016/j.yfrne.2011.01.001 I.J. Clarke / Frontiers in Neuroendocrinology xxx (2011) xxx–xxx of GnRH cells [56]. Notwithstanding this simplistic model, glutamatergic and GABAergic systems are known to have both positive and negative effects on the firing patterns of GnRH cells, so much is yet to be learned as to how neuronal function is translated into pulsatile GnRH secretion. In 2003, our understanding of the neuronal circuits that are ‘one step back’ from GnRH cells received a major fillip with the revelation that kisspeptin cells are essential for reproductive function. 4. Kisspeptin As indicated above, the revelation that kisspeptins and the kisspeptin receptor GPR54 are essential for reproductive function was a major advance in reproductive neuroendocrinology. Hereafter in this review (unless stated otherwise), ‘kisspeptin’ will refer to the 10-amino acid form, although it is recognized that larger forms of the post-translational product of the Kiss1 gene are equally effective [65,75]. Mass spectrometry analysis of the arcuate nucleus of the sheep brain indicates the presence of kisspeptin-10, but not larger forms of kisspeptin (Zeng, A.I. Smith and I.J. Clarke, unpublished data). There are two major groupings of kisspeptin cells in the mammalian brain, one being in the ARC and the other being in the rostral hypothalamus/POA. In rodents, the latter group of cells is located in the anteroventral periventricular nucleus (AVPV) and preoptic periventricular nucleus (PeV) [34,51,115,116]; this region has also been called the rostral periventricular area of the third ventricle (RP3V) [58]. In the female sheep, the two synonymous populations of kisspeptin cells are in the ARC and in the lateral POA [48]. How these two groups of kisspeptin cells regulate GnRH cells has been the subject of intense study over the past 7 years. 4.1. Kisspeptin cells are major regulators of GnRH cells Kisspeptin administration causes GnRH secretion, at least on the basis of measurement of GnRH in cerebrospinal fluid following intracerebroventricular infusion of kisspeptin-10 [87]. It remains to be shown, however, that kisspeptin stimulates secretion into the hypophysial portal blood. Supporting the notion that kisspeptin exerts direct action on the GnRH cells, it has been shown that virtually all of these cells express the cognate receptor (GPR54) [52,64]. There is no good indication that kisspeptin has a direct effect on the pituitary gonadotropes to cause LH release in sheep [119]. This is in spite of the fact that at least some GPR54 expression can be detected in the pituitary gland by PCR [119]. Consistent with this, it appears that kisspeptin does not exert hypophysiotropic function because it is not secreted into the hypophysial portal blood in significant amounts [119]. Finally, in vivo studies on hypothalamopituitary disconnected, GnRH replaced sheep, kisspeptin infusion did not affect the amplitude of LH pulses [119]. Interestingly, kisspeptin cells project to the median eminence, where varicose fibers come into close apposition to GnRH fibers [99] and it is possible that there is of axo-axonic regulation of GnRH secretion at this level. In this regard, studies in the non-human primate, using push–pull perfusion of the stalk-median eminence (SME) indicated concordance between patterns of secretion of kisspeptin and GnRH [75]. In addition, levels of both peptides in push–pull samples obtained from the SME indicated a rise in associated with puberty. Furthermore, the administration of kisspeptin to the SME stimulated LH secretion. Regarding the latter finding however, it is equivocal as to whether kisspeptin acted on GnRH cell bodies in the mediobasal hypothalamus or whether it acted on secretory terminals. What is the exact link between kisspeptin and GnRH function? In other words, is up-regulation or down-regulation of kisspeptin function directly translated into a change in GnRH secretion? 3 One way of addressing this question is to prevent kisspeptin action and measure GnRH/LH secretion. The development of a kisspeptin antagonist was instructive in this regard. This peptide antagonist blocks pulsatile LH secretion, with supporting electrophysiological data as well as in vivo measurement of GnRH secretion from the primate SME to show that the effect is exerted at the level of the GnRH neuron [103]. In the ovine brain, kisspeptin cells of the lateral POA provide direct input to GnRH neurons, whereas kisspeptin cells of the ARC may regulate GnRH neurons through a poly-synaptic pathway involving cells of another type [9]. The observation that kisspeptin stimulates GnRH release from the mouse median eminence [36] also supports the concept of a possible site of action at the level of GnRH neuroterminals. 4.2. Kisspeptin cells transmit the feedback effect of sex steroids to GnRH cells One major reason for intense focus on the role of kisspeptin in the control of reproductive function is that the kisspeptin cells express steroid receptors, whereas GnRH cells do not. GnRH cells do not express estrogen receptor-a (ERa), or androgen receptors [59,61] yet it is clear that the function of secretion of GnRH is regulated by sex steroids [23]. With respect to estrogen feedback, more than 60% of kisspeptin cells in the AVPV of the female mouse brain express estrogen receptor-a (ERa), as demonstrated by in situ hybridization [118] and immunohistochemistry [34,118]. In the AVPV/PeV of the female rat brain, approximately 90% of kisspeptin cells were shown express ERa, as assessed by immunohistochemical staining [1]. In the ovine brain, virtually all of the kisspeptin cells in the ARC express ERa, whereas only 50% of the population of cells in the lateral POA do so [48]. The question arises as to what function is performed by the kisspeptin cells that do not express steroid receptors; this may be to control metabolic homeostasis as discussed below. 4.3. Kisspeptin cells produce the positive feedback effect of estrogen Based on a range of criteria, including gene expression for Kiss1, Fos activation by estrogen, immunohistochemically measured levels of kisspeptin and various studies with gene knockout and replacement models, it is clear that the AVPV/PeV population of kisspeptin cells is fundamentally involved in the positive feedback effect of estrogen and the generation of the pre-ovulatory surge in rats and mice [113,139]. It has been consistently shown across different laboratories and in both rats and mice that kisspeptin cells are upregulated in the AVPV/PeV in the rat and mouse brain in the pre-ovulatory (pro-estrous) period, whereas the arcuate kisspeptin cells are down-regulated at this time (reviewed in [110]. Thus, the AVPV/PeV kisspeptin cells are regarded as fundamental to the propagation of the positive feedback effect that elicits the pre-ovulatory surge in GnRH/LH in these species. Whereas kisspeptin cells in the ovine brain exist as two major groupings, as in rodents, the mode of estrogen positive feedback appears to be quite different. Thus, in the ewe, this involves action of estrogen within the mediobasal hypothalamus, based on studies where micro-implants of estrogen were placed in this region [13,18]. Concordant with this is a range of observations that strongly suggest the fundamental role of arcuate nucleus kisspeptin cells in the positive feedback phenomenon. Evidence for this is as follows: 1. Kisspeptin treatment causes ovulation in anestrous ewes [21]. The important principle that is demonstrated in this study is that administration of this single neuropeptide leads to a surge in LH secretion (and, by inference, a GnRH surge). This may, however, be elicited by stimulation of basal pulsatile GnRH/ Please cite this article in press as: I.J. Clarke, Control of GnRH secretion: One step back, Front. Neuroendocrinol. (2011), doi:10.1016/j.yfrne.2011.01.001 4 2. 3. 4. 5. 6. 7. I.J. Clarke / Frontiers in Neuroendocrinology xxx (2011) xxx–xxx LH secretion, leading to a rise in estrogen levels that then elicits a positive feedback effect. The positive feedback effect of estrogen may then be regarded as a secondary event, also at the level of the mediobasal hypothalamus (most likely the arcuate nucleus) in this species. More recent data also invoke a role for preoptic kisspeptin neurons in the positive feedback event in sheep [17] and non-human primates [121]. ARC and POA Kiss1 expression is upregulated in the follicular phase of the estrous cycle [44,117]. The upregulation of Kiss1 in the caudal ARC is consistent with this region of the brain being the site at which estrogen acts to cause positive feedback in the sheep (vide supra). An effect of estrogen at this level would necessarily be transmitted to GnRH cells by at least one inter-neuronal link, which could be either the kisspeptin cells in the latPOA or some other type of cell. The former would explain the upregulation of the latPOA cells, although estrogen could act directly on at least 50% of the latPOA kisspeptin cells that express ERa. The latPOA kisspeptin cells project to GnRH cells [9], so that this could be the direct conduit to GnRH cells that subserves the positive feedback response. Kisspeptin peptide levels are increased in cells of the caudal ARC in the late follicular phase [117]. This substantiates the observations on Kiss1 expression and also consolidates the notion that this region of the brain is where estrogen acts to cause positive feedback in the ewe. Kisspeptin cells are acutely activated by estrogen as assessed by Fos immunohistochemistry [117]. This particular study compared responses in ovariectomised ewes within 1 h after a challenge with estrogen, which is considered to be a transient signal to activate the positive feedback mechanism [31]. This signal activates a chain of events, leading to a time-delayed surge in GnRH secretion [26]. This study contrasted the activational state of kisspeptin neurons in the ARC in ovariectomised ewes that were receiving chronic estrogen treatment as opposed to a transient challenge. As such, it distinguished the two types of feedback, with reduced Fos labeling in cells from animals receiving chronic estrogen treatment and increased Fos labeling in those receiving an acute challenge. Whether the same neurons are involved in both types of feedback or not is unknown, but more detail on the negative feedback mechanism is given below. In the ewe brain, 90% of GnRH cells express GPR54, but there is also a number of non-GnRH cells in the POA that express the receptor and could act as relay cells for the effects of kisspeptin to regulate GnRH secretion [117]. Further studies on the regulation of the receptor will prove informative in relation to the positive feedback mechanism. Response to kisspeptin in terms of LH secretion, is increased in the follicular phase of the estrous cycle [120]. Presumably this is preparatory to GnRH surge induction. In the non-human primate, as in the ewe, it also appears that the positive feedback phenomenon is associated with upregulation of kisspeptin in both the ARC and the POA, based on in situ hybridization measures of Kiss1 [121]. This contrasts with the repeated observation that AVPV/PeV kisspeptin cells are upregulated in the rodent brain by estrogen treatment or during the pro-estrous period, with contemporaneous down-regulation of the kisspeptin cells in the ARC (vide supra). This indicates a clear cut difference between rodents on one hand and sheep/nonhuman primates on the other hand. 4.4. Kisspeptin cells mediate the negative feedback effect of ovarian steroids Kisspeptin cells appear to mediate negative feedback, as initially shown by upregulation of Kisspeptin cells following ovariec- tomy, as assessed by immunohistochemistry [94]. Furthermore, chronic treatment with estrogen alone reduces Fos in kisspeptin cells in the ARC of ovariectomised ewes [117]. Virtually all kisspeptin cells in the ARC co-express dynorphin (DYN) and neurokinin B (NKB) [50]. This has led to the naming of these cells as K (kisspeptin) N (neurokinin B) Dy (dynorphin) (KNDy) cells [22]. The KNDy cells also express both ERa and progesterone receptor at a high level [46,48], providing the necessary machinery for these KNDy cells to mediate both negative and positive feedback effects of sex steroids. There is good evidence that dynorphin plays a role in mediating the negative feedback effect of progesterone [47,49], in addition to the evidence that chronic estrogen treatment downregulates the KNDy cells in OVX ewes [117]. Further support for the notion that these cells participate in transmitting the negative feedback effect to GnRH cells is the observation that kisspeptin expression in the ARC is reduced in the luteal phase of the estrous cycle [117]. This does not mean that other cells, such as those that produce enkephalin, may not also participate in the negative feedback regulation of GnRH secretion [136], but a major role for the KNDy cells is most likely. As for the involvement of these cells in the positive feedback mechanism, any transmission from the ARC to the GnRH cells of the POA is likely to involve a poly-synaptic pathway. KNDy cells may regulate a subset of GnRH cells in the mediobasal hypothalamus, to exert the negative feedback effect [49]. 4.5. Kisspeptin cells and non-reproductive functions In spite of overwhelming evidence that kisspeptin cells exert major control over GnRH cells, there is also a range of other regulatory neuronal systems. Kisspeptin cells in both the ARC and the POA express the signaling form of the leptin receptor, ObRb, and the former group are networked with appetite regulating cells [9], allowing transmission of signals relating to metabolic state. There are two reasons why this network is of significance. Firstly, the expression of ObRb allows kisspeptin cells to respond to the relative level of body fat and this could be transmitted to GnRH cells. Reproductive function is shut down in low body condition, but this can be counteracted by leptin administration [55], which could be effected via kisspeptin cells. Second, kisspeptin cells in the ARC are reciprocally connected with neuropeptide Y and proopiomelanocortin cells in the same nucleus [9]. The high level of expression of sex steroid receptors in the kisspeptin cells may allow sex steroid regulation of appetite and energy expenditure via the other two cell types, in which expression of ERa is much lower [82,109]. Since estrogen is well known to influence metabolic function [6], this pathway could be important in this regard. Notably, the reciprocal pathway is stronger for POMC/kisspeptin than for NPY/kisspeptin [9] and in light of evidence that melanocortins stimulate the reproductive axis [7], this could form part of a network that allows stimulatory effects via more than one neuronal system, acting in concert. 5. Gonadotropin inhibitory hormone (GnIH) GnIH was identified as an hypothalamic factor that inhibits the HPG axis in the quail [131] and a large body of work has established this is as a bone fide regulatory peptide in avian species [128]. Original work by Hinuma et al. [60] identified the GnIH gene in mammals at the same time, but invoked a role in the control of prolactin secretion. The presence of orthologues of GnIH in the brains of various species as now been reported [32,111]. These have been named RF-amide related peptides (RFRP), being members of the RF-amide family, but there seems no good reason why the original nomenclature (GnIH) should not extend to all Please cite this article in press as: I.J. Clarke, Control of GnRH secretion: One step back, Front. Neuroendocrinol. (2011), doi:10.1016/j.yfrne.2011.01.001 I.J. Clarke / Frontiers in Neuroendocrinology xxx (2011) xxx–xxx species [32,111]. The GnIH gene encodes 3 peptides in birds [130] and two peptides in mammals [77]. The issue of multiple forms of nomenclature are clarified by Tsutsui et al. [130] and I have adopted the same. Thus, in birds, the gene encodes GnIH, GnIHRP1 and GnIH-RP2, whereas the gene in mammals encodes GnIH1 (RFRP-1) and GnIH-3 (RFRP-3) [130]. Early work with a polyclonal antibody against avian GnIH [131], identified immunoreactive cells in the diencephalon, pons and medulla of the mouse brain [134]. Within the hypothalamus, GnIHexpressing cells are concentrated in the dorsomedial hypothalamic nucleus, with some also seen in the posterior hypothalamus between the ventromedial nucleus and the dorsomedial hypothalamic nucleus. In the rat brain, cells were visualized in the dorsomedial nucleus and in regions surrounding the ventromedial nucleus and in the tuberomammillary nucleus [67]. With a different antiserum against the rat GnIH precursor peptide immunoreactive cells were seen to be confined to the dorsomedial nucleus of the rat brain [101]. Thus, it is important to consider these immunohistochemical findings against the possible variation in specificity of the antibodies used and this is discussed in a recent review [32]. GnIH immunoreactive cells have also been localized to the dorsomedial nucleus of the hypothalamus in hamsters, mice and rats using an antibody against white crowned sparrow GnIH [78] and this distribution was also observed with in situ hybridization [78]. More recent in situ hybridization analysis reveals GnIH mRNA expressing cells in the dorsomedial nucleus and dorsomedial parts of the ventromedial nucleus, with cells extending rostral to the anterior hypothalamus and the ventral perifornical area of the rat brain [80]. In the sheep brain, in situ hybridization identified GnIH-expressing cells in the ventral region of the paraventricular nucleus and the dorsomedial nucleus [33,38,114], with a similar distribution detected using immunohistochemistry, using either the white crowned sparrow GnIH antiserum [78,114] or an antiserum raised in guinea pigs against human GnIH-3 [98]. The location of GnIH cells in the dorsomedial nucleus in a range of species may lend clues regarding the functional significance of the peptide. In particular, dorsomedial nucleus plays a role in the regulation of energy balance (vide supra). In the male rhesus macaque, GnIH mRNA and immunoreactive cell bodies are seen in the intermediate periventricular nucleus [133]. GnIH neurons have widespread projections within the hypothalamus, the amygdala, the bed nucleus of the stria terminalis and the paraventricular nucleus of the thalamus [67]. In the rodent and primate brain, GnIH terminal beds are also found in the preoptic area, septum and diagonal band of Broca [67,133], where GnRH cells are located. Between 40% and 80% of GnRH cells show GnIHimmunoreactive varicose fibers in close proximity in the primate, rat, sheep, hamster and mouse brain [67,78,98,114,133,140] as in birds [11,135]. This provides a substrate by which GnRH cells may be regulated either directly or indirectly by GnIH. GnIHimmunoreactive terminals have also been observed in the neurosecretory zone of the median eminence in hamsters, sheep, and primates [33,38,78,133], but similar projections have not been seen in the rat [67,101]. On this basis, a role for GnIH in the regulation of the pituitary gonadotrope is proposed for the sheep, as well as the rat [88] with functional data supporting this (vide infra). The GnIH receptor (GnIH-R) was originally identified by ligand activation of an orphan G-protein coupled receptor as OT7T022 [60]. It is and is expressed at a high level in the hypothalamus, with moderate levels of expression in other brain tissues, the eye and the testis. The receptor is one of two previously named neuropeptide FF (NPFF) receptors. NPFF1 is the receptor for GnIH, whereas NPFF2 is the receptor for NPFF, a RF-amide peptide family initially proposed to modulate the actions of morphine [84,141]. GnIH-R was also identified by Dockray [41] as GPR147 (named RFR-2). GnIH-R has now been identified in range of species including the 5 rat [15], human [15] and sheep [38]. In the sheep brain, it is expressed in the suprachiasmatic, supraoptic nucleus and periventricular nuclei as well as the pars tuberalis. GnIH-R is expressed by GnRH cells in the avian brain [12,132], but this has not yet been shown in the brain of a mammal. It will also be important to identify other types of cell types that express GnIH-R, such as those regulating appetite (vide infra). In spite of the earliest indications that GnIH was present in the brains of mammals and that it negatively regulates reproductive function [60], recognition that it is a significant regulatory peptide in mammals has been unforthcoming until recently. The recent description of the presence of GnIH orthologues and GnIH-R in the brains of a range of mammalian species and the projections of GnIH neurons to relevant neuroendocrine cells strongly suggest a role in the regulation of reproduction and food intake. Functional data now substantiate its significance. 5.1. GnIH input to GnRH cells and putative regulation of GnRH secretion Varicose fibers immunoreactive for GnIH are seen in close apposition to GnRH cells in the sheep [114] and the non-human primate [121]. With this anatomical framework, functional evidence of direct action is offered by the demonstration that GnIH-3 administered by intracerebroventricular injection reduced plasma LH secretion in ovariectomized (OVX) hamsters [78] and gonad-intact rats [66,67]. Others, however, showed that icv administration of GnIH-3 was not effective in reducing plasma LH levels in OVX, estrogen treated rats [3] and OVX rats [88]. ICV administration of GnIH an antisense oligonucleotide increased plasma LH levels to pre-pubertal rats [66], but this did not cause advancement of puberty. Any regulation of GnRH cells by GnIH may be direct, predicated on the neuronal input to GnRH cells, but it could also be indirect. In support of the latter, GnIH was shown to reduce neuronal activation in the anteroventral periventricular (AVPV) region as well as in GnRH neurons (based on Fos labeling) with a surgeinduction protocol in rats [3]. In this study, however, the LH surge was not significantly reduced in magnitude. These functional data are consistent with observe projections of GnIH cells to GnRH cells [98], as is other evidence of function, demonstrated by inhibition of the firing rate of GnRH neurons in brain slices from male and female mice [43,140]. Whereas 43% of GnRH cells showed reduced firing with GnIH treatment, 9% showed an increased firing rate [43]. Further studies, including localization and quantification of GnIH-R will elucidate the full significance of the peptide in the control of reproduction. 5.2. GnIH action on the pituitary gonadotropes Intravenously (iv) administered GnIH-3 had no effect on basal LH secretion in OVX rats and only minimal (albeit statistically significant) effects on GnRH-stimulated secretion [101]; was interpreted to mean that GnIH has no major effect on gonadotropes. Others [88] showed a reduction in plasma LH levels in OVX rats 2 h after iv administration, with lack of effect of icv administration. In OVX ewes, systemically administered GnIH-3 consistently reduces pulsatile LH secretion [33], without any effect on the plasma levels of other pituitary hormones, such as growth hormone or prolactin; similar results have also been obtained in the bovine [68]. The dose-dependent reduction in GnRH-stimulated LH secretion that is seen in cultures of rat, sheep and bovine pituitary cells, supports the notion of a direct effect of GnIH on gonadotropes [33,68,88], although some contrary data have also been reported in studies of effects on rat pituitary cells in vitro [3]. Such dissimilar results may be due to variable culture conditions or may relate to Please cite this article in press as: I.J. Clarke, Control of GnRH secretion: One step back, Front. Neuroendocrinol. (2011), doi:10.1016/j.yfrne.2011.01.001 6 I.J. Clarke / Frontiers in Neuroendocrinology xxx (2011) xxx–xxx possible species differences in peripheral GnIH activity. Other lines of evidence also suggest direct pituitary action of GnIH, in sheep, at least. Firstly, GnIH-3 eliminates the GnRH-stimulated mobilization of intracellular calcium in gonadotropes, which is considered mandatory for gonadotropin release [33]. Second, GnRH-stimulated upregulation of LHb mRNA levels is also negated by GnIH, which may be due to reduced phosphorylation of extracellular signalregulated kinase (ERK) [105]. These data indicate a direct action of GnIH on the pituitary gonadotrope to reduce both synthesis and secretion of LH. In vitro treatment of ovine pituitary cell cultures also show an effect of GnIH-3 to reduce FSH secretion in response to GnRH [33] as well as reducing FSHb mRNA levels [105]. Thus, GnIH may inhibit the production/secretion of both gonadotropins. 5.3. Effect of GnIH on food intake GnIH cells project to appetite regulating cells within the lateral hypothalamic area, ventromedial nucleus and arcuate nucleus in the ovine brain [98], with similar projections also seen in the primate [133]. This suggests a functional role for GnIH in the regulation of food intake [67,88,123]. GnIH was originally shown to stimulate food intake in birds [123] and similar data have now been obtained in rats [66,67,88]. Thus ICV injections of GnIH increased food intake in rats [66,67,88]. This is consistent with the role of the dorsomedial nucleus in the regulation of appetite and energy balance [104], but little was known of the cell types involved in energy balance within this nucleus. In sheep projections of GnIH cells to cells that produce neuropeptide Y or pro-opiomelanocortin in the arcuate nucleus and those that produce orexin or melanin concentrating hormone in the lateral hypothalamus [98] suggests a role in the control of energy balance, but functional studies are required. The dual function of GnIH in relation to reproduction and appetite is similar to that of other peptides such as neuropeptide Y (which stimulates food intake and negatively regulates reproduction in the sheep [10,27]) and melanocortins (which reduce food intake and stimulate reproduction [7,8,45,137]). 5.4. Kisspeptin and GnIH as key regulators in seasonal breeding Sheep are seasonal breeders, being sexually active in response to short day photoperiod [70,85]. Whereas it has been known for some years that seasonality in sheep is due to alterations in the frequency of generation of GnRH/LH pulses [102], as well as an alteration in the negative feedback effect of ovarian steroids [81], there was no identification of a neural substrate that changes with season and controls GnRH secretion accordingly. The A15 dopaminergic nucleus was identified as a key center in the control of seasonality [125] but as to how this is connected to GnRH secretion is not yet clear. With the revelation of key regulatory function of kisspeptin and GnIH, it is hardly surprising that this has been investigated in relation to seasonal breeding. In the ewe, kisspeptin production (ARC) and input to GnRH cells is reduced in seasonal anestrus and increases at onset of the breeding season [114]. Also in the ewe, GnIH protein expression is higher during the nonbreeding season than in the breeding season [114]. Terminal projections from GnIH cells to GnRH neurons are increased during the non-breeding season [114]. Thus, it appears that the activity of GnIH may be a contributing factor to the inhibition of the reproductive system during the non-breeding season. Similarly, GnIH gene expression in Soay ewes on long day photoperiod was higher than in ewes on a short day protocol [38]. Nevertheless, when these ewes were held on extreme long day photoperiods (20 h or 22 h light), the effect on GnIH gene expression was lost. It was concluded that GnIH may not play a major role in seasonality [38], but the inhibitory effect may be amplified by an increase in input to GnRH cells during the non-breeding season [114]. Further work, including measurement of GnIHR expression in different seasons may be informative. Reciprocal changes in kisspeptin activity indicate that both RF-amide peptides play a role in seasonal changes in reproductive activity. Intervention studies such as reducing GnIH function in the non-breeding season would be instructive. 6. Conclusions In the last decade, the emergence of kisspeptin and GnIH as major neuroendocrine regulators of reproduction, the latter having gained acceptance only in the last few years. This has led to a significant revision of our understanding as to how reproduction is controlled by the brain, especially through the feedback effects of gonadal steroids. The accumulated data on the role of kisspeptin is far more extensive than that for GnIH and further work is required to understand the degree to which GnIH functions in normal physiology. There is a need to develop a reliable assay for GnIH and to ascertain whether the peptide is secreted into hypophysial portal blood; this would substantiate the notion that GnIH acts on the pituitary gonadotropes. A more detailed understanding of the regulation of the receptors for these two peptides would be informative. As for the growth hormone axis, that is controlled by the opposing actions of somatostatin and growth hormone releasing hormone, it now appears that the reproductive system is also under control of two peptides with opposing action. GnIH or relevant analogs could potentially be developed for the treatment of diseases such as endometriosis, prostate cancer and precocious puberty; all conditions where reproductive function needs to be down-regulated. Such agents could offer realistic alternatives to the use of superactive GnRH agonists or antagonists to suppress gonadal steroid levels. Kisspeptin may have some utility for the induction of ovulation [19]. Whereas our knowledge of the neuroendocrine control of reproduction has been expanded by the recognition of the respective roles of kisspeptin and GnIH, we should not lose sight of the fact that many neuronal systems act to control GnRH secretion. The output of GnRH neurons, as measured in the hypophysial portal blood, is the sum of multiple inputs to the GnRH cells, being the integration of neuronal systems transducing sex steroid feedback, season, stress, immune status, nutritional status and mood. Nevertheless, the kisspeptin and GnIH systems appear to be only ‘one step back’ from the GnRH neurons, providing important modulatory function. References [1] S. Adachi, S. Yamada, Y. Takatsu, H. Matsui, M. Kinoshita, K. Takase, H. Sugiura, T. Ohtaki, H. Matsumoto, Y. Uenoyama, H. Tsukamura, K. Inoue, K. Maeda, Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats, J. Reprod. 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