Kisspeptins and the control of gonadotropin secretion in male and female rodents

peptides 30 (2009) 57–66

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Review

Kisspeptins and the control of gonadotropin secretion in male and female rodents J. Roa a,b, J.M. Castellano a,b, V.M. Navarro a,b, D.J. Handelsman c, L. Pinilla a,b, M. Tena-Sempere a,b,* a

Department of Cell Biology, Physiology and Immunology, University of Co´rdoba, 14004 Co´rdoba, Spain CIBER Fisiopatologı´a de la Obesidad y Nutricio´n, Instituto de Salud Carlos III, 14004 Co´rdoba, Spain c ANZAC Research Institute, Concord Hospital, University of Sydney, Sydney, NSW 2139, Australia b

article info

abstract

Article history:

Kisspeptins, the products of KiSS-1 gene acting via G protein-coupled receptor 54 (GPR54),

Received 6 March 2008

have recently emerged as fundamental gatekeepers of gonadal function by virtue of their

Received in revised form

ability to stimulate gonadotropin secretion. Indeed, since the original disclosure of the

6 August 2008

reproductive facet of the KiSS-1/GPR54 system, an ever-growing number of studies have

Accepted 7 August 2008

substantiated the extraordinary potency of kisspeptins to elicit gonadotropin secretion in

Published on line 22 August 2008

different mammalian species, under different physiologic and experimental conditions, and through different routes of administration. In this context, studies conducted in laboratory

Keywords:

rodents have been enormously instrumental to characterize: (i) the primary mechanisms of

Kisspeptins

action of kisspeptins in the control of gonadotropin secretion; (ii) the pharmacological

KiSS-1

consequences of acute vs. continuous activation of GPR54; (iii) the roles of specific popula-

GPR54

tions of kisspeptin-producing neurons at the hypothalamus in mediating the feedback

GnRH

effects of sex steroids; (v) the function of kisspeptins in the generation of the pre-ovulatory

Gonadotropins

surge of gonadotropins; and (iv) the influence of sex steroids on GnRH/gonadotropin

LH

responsiveness to kisspeptins. While some of those aspects of kisspeptin function will

FSH

be covered elsewhere in this Special Issue, we summarize herein the most salient data,

Mouse

obtained in laboratory rodents, that have helped to define the physiologic roles and putative

Rat

pharmacological implications of kisspeptins in the control of male and female gonadotropic axis. # 2008 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gonadotropin responses to kisspeptins in rodents: pharmacological characterization . Mechanisms of action of kisspeptins in the control of gonadotropin secretion . . . . . . KiSS-1/kisspeptins and the feedback control of gonadotropin secretion in rodents . . .

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* Corresponding author at: Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, University of Co´rdoba, Avda. Mene´ndez Pidal s/n, 14004 Co´rdoba, Spain. Fax: +34 957 218288. E-mail address: [email protected] (M. Tena-Sempere). Abbreviations: GPR54, Gprotein-coupled receptor 54; GnRHAvda, Mene´ndez Pidal s/ngonadotropin-releasing hormone; LHAvda, Mene´ndez Pidal s/nluteinizing hormone; FSHAvda, Mene´ndez Pidal s/nfollicle-stimulating hormone. 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.08.009

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5. 6. 7.

1.

peptides 30 (2009) 57–66

Modulation of GnRH/gonadotropin responses to kisspeptins by sex steroids . . . . . . . . Kisspeptins: putative targets for pharmacological manipulation of gonadotropic axis? Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

The mammalian reproductive axis is a dynamically regulated neurohormonal system arranged onto three major tissues or levels of integration: the hypothalamus, the pituitary and the gonads. Within this system, also termed gonadotropic or hypothalamic–pituitary–gonadal (HPG) axis, pituitary gonadotropins, LH and FSH, are the main driving force for gonadal development, trophic maintenance and function [54]. Accordingly, diverse physiologic and pathological conditions, as well as pharmacological manipulations, affecting the gonads are conveyed via regulation of their secretion, and thus, elucidation of the mechanisms and signals involved in the regulatory network governing gonadotropin release has attracted considerable attention among physiologists and clinicians for decades [18–20]. In this context, during recent years, genetic analyses and functional studies have identified monogenic forms of infertility that derive from disruption of gonadotropin secretion (e.g., altered hypothalamic systems or pituitary responsiveness) or function (e.g., mutations in gonadotropin subunits or their receptors) [18–20,54]. These conditions, although globally rare, have been enormously instrumental to extend our knowledge on the actions and regulatory systems of pituitary gonadotropins. The synthesis and release of both gonadotropins is dictated by the pulsatile secretion of GnRH; a decapeptide synthesized by a sparse neuronal population of the forebrain, whose function is driven by the complex interaction of a plethora of excitatory and inhibitory signals, of central and peripheral origin. Indeed, given that GnRH acts upon pituitary gonadotrops to elicit gonadotropin synthesis and secretion, and considering the convergence of a wide array of regulatory cues onto GnRH neurons, these have been considered as major hierarchical element of the HPG axis, acting as essential integrators and major output pathway for the diversity of signals modulating the gonadotropic axis [11]. Notably, however, most of the primary regulators of gonadotropin secretion (from sex steroids to metabolic signals, such as leptin) do not appear to act directly onto GnRH neurons, but rather indirectly via trans-synaptic inputs [10,17]. The nature of such intermediary neuronal populations has remained ill defined for decades. Besides central regulators, the secretion of both gonadotropins is under the influence of a myriad of peripheral factors that include not only gonadal hormones, but also metabolic and environmental cues [10,11]. In any event, among the peripheral signals controlling gonadotropin secretion, gonadal steroids and peptides are by far the most relevant regulators, acting via negative and, eventually, positive feedback loops [16,29]. Thus, in both males and females, sex steroids secreted by the gonads in response to gonadotropins carry out a predominant inhibitory action upon pituitary LH and FSH

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secretion (negative feedback), which is mostly conducted at the hypothalamic level. However, selectively in the female, the rise in estradiol secretion by dominant follicles of the ovary, at the period preceding ovulation, is capable also to induce an increase in hypothalamic GnRH secretion (and GnRH selfpriming at the pituitary), thereby generating the pre-ovulatory surge of gonadotropins ( positive feedback), which ultimately triggers ovulation [16,29]. Of note, identification of discernible neuronal pathways, responsible for such a differential pattern of response to estrogen (positive vs. negative feedback) in such a sexually dimorphic manner, has remained elusive for decades, and has been the subject of considerable investigation and debate [16,29]. Anyhow, compelling experimental evidence from rodents had strongly suggested that efferent projections from the anteroventral periventricular (AVPV) nucleus of the hypothalamus play an indispensable role for the generation of estrogen-induced surge of gonadotropins [16].

2. Gonadotropin responses to kisspeptins in rodents: pharmacological characterization As described in other chapters of this Special Issue, the emergence of kisspeptins has revolutionized our understanding of the neuroendocrine mechanisms responsible of the control of key facets of reproductive maturation and function, from brain sexual differentiation and puberty onset to the metabolic regulation of fertility [39]. Undoubtedly, however, a significant part of the research efforts in the field were initially (and are still presently) devoted to the characterization of the pharmacological effects of kisspeptins in terms of regulation of gonadotropin secretion in different species [39]. The importance of these studies is twofold: (i) to define the biological effects and functional relevance of kisspeptin in the control of the gonadotropic axis; and (ii) to provide the scientific basis for the design of protocols of pharmacological intervention of the reproductive system based in the use of kisspeptin analogs, of either agonistic or antagonistic activity. Notably, as defined elsewhere in this Special Issue, kisspeptins exist in different molecular forms (kisspeptin-54, -14, -13, and -10). Yet, although they all have the capacity to activate GPR54, potential differences in terms of biosynthesis at different sites and in vivo biopotency have not been thoroughly analyzed to date. As clear evidence of the interest drawn by the role of this system in the control of gonadotropin secretion, roughly within 1 year since the initial reports on hypogonadotropic hypogonadism in humans and mice with inactivating mutations of GPR54, a number of groups worldwide reported the ability of kisspeptins (mostly, kisspeptin-10 and kisspeptin-54) to stimulate LH secretion in a number of mammalian species, including mouse, rat, sheep and macaque [13,27,28,30,32,47,56]. Likewise,

peptides 30 (2009) 57–66

the stimulatory effects of kisspeptins on FSH secretion were initially described in the rat and later in the sheep [1,31]. More recently, kisspeptin-54 has been proven to elicit LH and, to a lesser extent, FSH secretion in humans, thus proving the conserved role of kisspeptins as potent stimulators of gonadotropin release in mammals [8,9]. Overall, the striking similarities of the effects of kisspeptins on gonadotropin secretion among different mammalian species reinforced the usefulness of rodent studies for covering the physiologic and pharmacological goals defined above. In this scenario, genetic and pharmacological studies in rodents, conducted over the last 4 years, have paved the way for the characterization of the effects and mechanisms of action of kisspeptins in the control of gonadotropin secretion. In fact, the demonstration of the state of hypogonadotropism in mice engineered to lack a functional GPR54 gene initially evidenced the involvement of the system in the control of gonadotropin secretion [12,46]; a phenomenon that has been more recently confirmed in KiSS-1 null mice [7]. Moreover, the fact that the potent stimulatory effects of kisspeptin-10 were completely blocked in GPR54 knockout mice, despite preserved pituitary responsiveness to GnRH, demonstrated that the gonadotropic effects of kisspeptin are solely mediated via GPR54 [28]. Pharmacological tests conducted in rats and mice were the first to document the extraordinarily potent LH releasing effects of kisspeptin-10 and kisspeptin-54 (metastin). Thus, threshold doses for LH stimulation were defined (depending on the study considered) between 100 fmol and 1 pmol, for protocols of intracerebroventricular (i.c.v.) or intrahypothalamic administration [13,32,34]. Moreover, such stimulatory effects were also detected after systemic injection, over a variety of routes (intravenous, subcutaneous and intraperitoneal) and a range of doses [32,59]. Based on detailed doseresponse studies conducted in vivo, the median effective dose (ED50) for LH was calculated at 2–4 pmol, for i.c.v. administration [4,32]. Concerning systemic delivery, doses as low as 0.1 mg/rat (0.3 nmol/kg BW) i.v. were sufficient to induce robust LH peaks in freely moving rats [59]. Overall, comparative analysis of the published data on the LH releasing activity of kisspeptins and other neuropeptides and neurotransmitters, such as glutamate and galanin-like peptide (GALP), demonstrate that kisspeptins are likely the most potent elicitors of the GnRH/LH axis known so far [53]. For instance, the direct comparison of the LH releasing effects of GALP and kisspeptin-10 in male rats demonstrated that, despite similarly maximal responses are achieved after stimulation with high doses of both peptides, i.e., in the nmol range, the ED50 for kisspeptin-10 was approximately 150-fold lower than for GALP [4]. As was the case for LH, rodent studies have also documented the ability of kisspeptins to stimulate FSH secretion [31]. The data available, however, evidences that the threshold doses for FSH stimulation are clearly higher than for LH, with a predicted ED50 of 400 pmol, for i.c.v. administration (i.e., 200-fold less sensitive) [31]. The mechanisms behind such divergence are likely diverse and will be discussed in following sections. In addition, the time-course for the stimulatory effects of kisspeptin on FSH release in male rats appears to be somewhat slower than for LH secretion [31].

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Notwithstanding, the stimulatory effects of high doses (1 nmol/i.c.v.) of kisspeptin-10 on LH and FSH secretion were fully preserved after antagonization of ionotropic glutamate (NMDA and KA/AMPA) receptors, as well as after blockade of the endogenous nitric oxide (NO) tone [31,32]; well-known physiologic modulators of gonadotropin secretion. The functional implications of these observations are discussed below. Interestingly enough, rodent studies have also documented that the robust LH and FSH releasing effects of kisspeptins are equally detected in both male and female animals, at different stages of postnatal maturation [2]. For instance, in rats, the ability of kisspeptin to potently elicit LH secretion has been demonstrated in males and females at the neonatal, infantile, juvenile and pubertal stages of sexual maturation [2], as well as in adulthood [32,42]. On the latter, kisspeptin-10 was able to evoke significant LH responses not only in adult males, but also in cyclic female rats at different stages of the estrous cycle, as well as during pregnancy and, even, lactation [42]. Of note, however, the sensitivity to kisspeptin in terms of LH secretion appears to be significantly depressed in lactating dams; a phenomenon that might contribute to the state of hypogonadotropism linked to this condition [42]. In addition, the stimulatory effects of kisspeptins on FSH secretion have been documented in peripubertal and adult male and female rats [31,42]. Overall, such a consistency for the stimulatory effects of kisspeptins on gonadotropin secretion across sexual development and sexes further documents not only the physiologic role but also the potential pharmacological interest of the KiSS-1/GPR54 system in the control of the gonadotropic axis in mammals.

3. Mechanisms of action of kisspeptins in the control of gonadotropin secretion The initial disclosure of the extraordinarily potent releasing effects of kisspeptins on gonadotropin secretion boosted an enormous interest for the identification of the potential mechanisms involved. Several lines of evidence, accumulated over the last years, have substantiated that the primary site of action of kisspeptins in the control of the gonadotropic axis is located at hypothalamic GnRH neurons. Such experimental evidence can be summarized into the following points: (i) the potent LH and FSH releasing effects of kisspeptin-10 are completely abrogated after pre-treatment with GnRH antagonists in male and female rodents [13,27,31,32]; (ii) GnRH neurons in the rat forebrain do express GPR54 gene [21]; (iii) kisspeptin activates GnRH neurons in rodents, as evidenced by induction of c-fos expression [21], as well as potent and longlasting depolarization responses [15]; (iv) kisspeptin induces, in a dose-dependent manner, the secretion of GnRH by hypothalamic explants ex vivo [3,56]; and (v) murine cell lines parentally related to GnRH neurons, such as GT1-7 cells, express GPR54 mRNA and are able, under some conditions, to respond to kisspeptin stimulation [22,37]. In the same line, we have observed that GnRH-deficient hpg mice are unable to respond to kisspeptin stimulation in terms of LH secretion (see Fig. 1). Altogether, the above data demonstrate that hypothalamic GnRH is an obligate mediator for the gonadotropinreleasing effects of kisspeptins.

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Fig. 1 – Lack of LH responses in hpg mice i.p. injected with an effective dose of mouse kisspeptin-10 (10 mg). The hpg mouse harbors an inactivating mutation in the GnRH gene that alters its processing and renders the animal hypogonadotropic. As shown in the figure, while wildtype mice respond to systemic administration of kisspeptin-10 with a robust LH secretory peak at 15-min after injection, hpg mice failed to respond to a similar stimulus. This observation further confirms that GnRH is an indispensable mediator for the stimulatory effects of kisspeptin on gonadotropin secretion. Groups with different superscript letters are statistically (P < 0.05) different.

The physiologic relevance of such a kisspeptin-GnRH pathway for the generation of gonadotropin responses to kisspeptins is further documented by the demonstration of a close association between GnRH responses detected ex vivo and LH responses observed in vivo, at different developmental stages, in male and female rats [2]. Moreover, it has been recently demonstrated that populations of KiSS-1 neurons (such as those at the AVPV-nucleus) physically interact with GnRH neurons in the mouse forebrain [5]. In sum, the above data point out that kisspeptin input (steaming from specific KiSS-1 neurons located at discrete hypothalamic areas) drives the activation of GnRH neurons, which do express the canonical KiSS-1 receptor. Interestingly, the stimulation of GnRH neurons by kisspeptin at critical developmental stages, such as puberty, appears to be under the precise regulation of a combination of factors, including not only the enhancement of kisspeptin tone, but also plastic changes involving an elevation of the number of projections to GnRH neurons, as well as an increase in the sensitivity to kisspeptin and GPR54 signaling efficiency [39]. Of note, based on data from expression analyses in vivo, the effects of kisspeptin on GnRH neurons do not apparently involve, at least in the short-term, the transcriptional activation of GnRH gene but rather stimulation of secretion of the releasable pool of GnRH [32]. This feature, together with their capacity to act directly at nerve terminal to evoke GnRH release [6], explain the ability of kisspeptins to elicit the acute increases in circulating levels of LH described in previous sections. Until recently, the signaling pathways responsible for the stimulatory effects of kisspeptins on GnRH neurons had only been evaluated using hypothalamic explants and protocols of

pharmacological blockade of key intracellular signals/factors following in vitro stimulation with kisspeptin [2]. Using this approach, it has been suggested that the stimulatory effects of kisspeptin on GnRH secretion require the activation of phospholipase-C (PLC), mobilization of intracellular Ca2+ stores and recruitment of ERK1/2 and p38 kinases [2]. In contrast, kisspeptin-induced GnRH release was preserved in spite of the blockade of adenylate cyclase (i.e., it was not dependent on cAMP signaling) and did not apparently require the influx of extracellular Ca2+, at least in this ex vivo setting (see Fig. 2). These features are remarkably similar to those reported for GPR54 signaling using heterologous cell systems, as described in detail elsewhere in this Special Issue. In the last few months, two different papers have refined the above observations by using electrophysiological recordings and calcium imaging in GnRH neurons. These reports have documented that kisspeptin excitation of GnRH neurons is conveyed through a PLC/calcium-dependent pathway regulating multiple ion channels, including potassium and transient receptor potential (TRP) channels [25,61]. As indicated in Section 2, the profiles of LH and FSH secretion after kisspeptin stimulation appear partially different, with faster and more sensitive LH responses in male rats. One possible explanation for such a phenomenon is that stimulation of GnRH neurons with kisspeptin, at the low dose range, elicits a pattern of GnRH release that favors preferential secretion of LH. In this sense, profiles of high frequency pulses are prone to elicit LH secretion were as low frequencies favor FSH synthesis [26]. Of note, however, acute injection of kisspeptin is apparently unable to significantly alter the frequency of pulsatile release of GnRH, as indirectly evidenced by recording of hypothalamic multiunit electrical activity volleys, at least in gonadectomized female rats [24]. On the other hand, the fact that blockade of major regulatory pathways of GnRH secretion, such as glutamate and NO, did not prevent the releasing effects of kisspeptin strongly suggest that the KiSS-1/GPR54 system is independent, or eventually distal, to those central regulators in the control of GnRH neurons [31,32]. Admittedly, however, the evidence published to date on the above interactions is restricted to the testing of high doses of kisspeptin-10, which hampers the assessment of potential, subtle changes in its effects at the low dose range. Moreover, it has been recently described in mice that blockade of glutamate receptors reduced the stimulatory effects of kisspeptin on GnRH neuronal activity, which suggests that at least part of the releasing effects of kisspeptin might be mediated by activation of glutamate pathways [35]; a possibility that warrants further investigation. In addition, due to the lack of effective antagonists of GPR54, evaluation of the consequences of blockade of kisspeptin signaling on GnRH/gonadotropin responses to a diversity of central excitatory signals (including glutamate) has not been yet conducted. Finally, while it is well defined that the primary site of action of the KiSS-1/GPR54 system in the control of the reproductive axis is located at the hypothalamus, some controversy persists on the possibility of additional effects of kisspeptins directly at the pituitary level. In this sense, original reports documented either no effects or modest stimulatory actions of kisspeptin on LH secretion by rat

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Fig. 2 – Tentative model for the signaling pathways recruited following GPR54 activation by kisspeptin at the hypothalamus. Using protocols of pharmacological blockade and kisspeptin stimulation of hypothalamic explants ex vivo, it was demonstrated that the GnRH releasing effect of kisspeptin is blunted by: (i) inactivation of PLC (by means of U-73122); (ii) depletion of intracellular Ca2+ stores (by thapsigargin); and (iii) blockade of ERK1/2 and p38 kinase (by PD-98059 and SB203580, respectively). In contrast, GnRH responses were preserved after antagonization of adenylate cyclase (by MDL12,330A; not shown), inhibition of extracellular Ca+2 influx (by cadmium), or blockade of Jun N-terminal kinase (by SP600125). Likewise, GnRH responses to kisspeptin were also detected after inhibition of prostaglandin synthesis by indomethacin (not shown). Composed from data of Ref. [2].

pituitaries in vitro [39]. More recently, the ability of kisspeptin to elicit LH release acting directly at the pituitary has been further documented in rodent (Ca+2 responses have been identified in response to direct kisspeptin stimulation in rat gonadotropes), bovine and ovine species, even at the nM range [14,51,52]. Moreover, rat studies have suggested the expression and hormonal regulation of KiSS-1 gene at the pituitary [38], while in the sheep, kisspeptin has been detected in hypophysial portal blood [51]. On the latter, however, the lack of significant fluctuations in kisspeptin concentrations at key physiological states, such as the pre-ovulatory surge, has been interpreted as evidence for the lack of physiologic relevance of such direct pituitary effects [51]. Overall, while the dominant hypothalamic actions of kisspeptins on GnRH neurons is undisputed, the possibility of direct pituitary effects remains as a contentious issue that warrants further investigation.

4. KiSS-1/kisspeptins and the feedback control of gonadotropin secretion in rodents Further proof for the physiologic relevance of kisspeptin signaling in the control of gonadotropin secretion came from rodent studies addressing the potential involvement of this system in mediating the feedback effects of sex steroids. These

studies have included: (i) the characterization of the effects of changes in the sex steroid milieu on the expression patterns of KiSS-1 (and GPR54) gene at the hypothalamus; and (ii) the identification of canonical sex steroid receptors in putative KiSS-1 neurons [39]. In addition, (iii) the activation of KiSS-1 neurons by sex steroids has also been evaluated in rodents [39]. The first evidence for the potential regulation of KiSS-1 gene expression at the hypothalamus by androgen and estrogen was obtained in rat studies using male and female models of gonadectomy (GNX), with or without sex steroid replacement. Thus, GNX induced a significant rise in KiSS-1 mRNA levels at the hypothalamus that coincided with the expected rise in circulating levels of gonadotropin. In addition, sex steroid replacement of GNX rats was sufficient to prevent both hormonal (LH) and gene expression (KiSS-1) responses [30]. By the use of in situ hybridization analyses in rats and mice, the above changes (detected by semi-Q RT-PCR in whole hypothalamic fragments) were located to the arcuate nucleus (ARC) [21,49,50], a key hypothalamic center for the integration of a wide array of peripheral regulators of the gonadotropic axis. Interestingly enough, later studies in sheep and primates (including humans) have confirmed the putative role of KiSS-1 neurons at the hypothalamic infundibular/arcuate nucleus in conveying the negative feedback effects of sex steroids also in other mammalian species [44,48].

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Notwithstanding, in situ hybridization analyses in rodents disclosed also that a discrete neuronal population expressing KiSS-1 that is located at the AVPV responds to sex steroids in a diametrically opposite manner: KiSS-1 mRNA expression at this site decreases after GNX and increases following estradiol supplementation [49,50]. These observations immediately raised the possibility that KiSS-1 neurons at the AVPV might be mechanistically involved in the generation of the preovulatory surge, induced by the preceding rise of circulating estradiol. Indeed, during the last 2 years, compelling evidence has been gathered, in different physiologic rodent models, supporting that contention; findings that are exhaustively revised by Tsukamura and Maeda in this Special Issue. Overall, the data available evidence that estrogen is capable to activate the transcription of KiSS-1 gene, thereby inducing an increase in KiSS-1/kisspeptin expression that enhances the secretory activity of GnRH neurons, and thus triggers the pre-ovulatory surge of gonadotropins [39]. As revised by Kaufmann elsewhere in this Special Issue, development of KiSS-1 neurons at the AVPV is exceedingly higher in adult female rodents, thus providing the potential basis for the sexual dimorphism in the ability of estrogen to induce positive feed-back, which is selectively detected in the female [23]. The molecular mechanisms whereby the same regulator (estrogen) is able to increase KiSS-1 mRNA in AVPV neurons while it decreases its expression at the ARC remain unsolved, although it is apparent that this is not related with a differential pattern of expression of a and b forms of estrogen receptors (ER) between these two hypothalamic sites. Moreover, pharmacological and functional genomic studies in rats and mice have demonstrated that the regulatory actions of estrogen on the hypothalamic expression of KiSS-1 gene are mediated via ERa [30,41,60].

5. Modulation of GnRH/gonadotropin responses to kisspeptins by sex steroids In addition to the transcriptional effects described above, evidence is also mounting that sex steroids are able to modulate net GnRH/gonadotropin responsiveness to kisspeptin in the female rat; a phenomenon that might contribute also to the generation of the pre-ovulatory surge of gonadotropins [41]. In this sense, recent pharmacologic and electrophysiological studies in rats and mice have jointly pointed out that relative GnRH/LH responses to kisspeptin are decreased in GNX animals, while estrogen replacement is able to rescue the state of maximal responsiveness [35,41]. Indeed, using GNX rats, we have demonstrated that the combined administration of estradiol (or a selective agonist of ERa) and progesterone induces supra-maximal LH responses to kisspeptin [41,42]; a phenomenon that is in line with previous findings of our group in cyclic female rats that showed cycle-dependent fluctuations in the pattern of gonadotropin responses to kisspeptin, with maximal LH responsiveness during the proestrus-toestrus transition, i.e., at the time of the pre-ovulatory surge [42]. Moreover, using a selective antagonist of ERa, we have recently shown that acute blockade of ERa signaling does not only impedes the generation of the pre-ovulatory surge and subsequent ovulation, but induces also a marked decrease in net LH and FSH responses to kisspeptin in cyclic female rats at

proestrus [40,41]. These observations are in good agreement with recent data in GNX mice, where estrogen has been shown to enhance kisspeptin-stimulated GnRH neuronal activity [35]. Overall, these observations evidence that, in addition to transcriptional effects on KiSS-1 gene, estrogen is able to increase the responsiveness of GnRH neurons to kisspeptin stimulation; a phenomenon that is likely to contribute to the full expression of the pre-ovulatory surge of gonadotropins. Indeed, it has been recently demonstrated that neuronal ERa signaling is indispensable for the induction of LH surges by estrogen [60]. In this context, it is tempting to hypothesize that part of this stimulatory action is mediated via its ability to enhance GnRH responsiveness to kisspeptin. Interestingly, in contrast to ERa, antagonization of ERb in cyclic female rats failed to alter the endogenous pre-ovulatory surge of LH and to block ovulation, but significantly enhanced the magnitude of acute LH responses to kisspeptin [41]. Moreover, modest, but detectable, inhibitory effects on LH responses to kisspeptin were observed in GNX female rats supplemented with a selective ERb agonist. In striking contrast, selective blockade of ERb attenuated FSH responses to kisspeptin in cyclic female rats at proestrus [40]. Altogether, the above data illustrates the complexity of ER signaling in setting GnRH/ gonadotropin responsiveness to kisspeptin, with a dominant positive role of ERa, but a dual mode of action of ERb: subtle inhibitory effect on LH secretion [41]; moderate stimulatory effect on FSH secretion [40]. The former may operate as negative modifier of GnRH/LH responses to kisspeptin; a phenomenon that could contribute to partially restrain LH secretion at certain physiological states. In addition, the differential roles of ERb signaling on LH and FSH secretion might be mechanistically relevant for the dissociation of gonadotropin secretion at the preovulatory phase of the cycle, at least in rodents [40]. Finally, as mentioned above, administration of progesterone together with estrogen (or selective ERa ligands) to GNX female rats induced a state of maximal responsiveness to kisspeptin in terms of LH secretion [41,42]. The mechanisms for such a stimulatory action of progesterone are unclear, but might reflect its pituitary effects, rather than primary changes in GnRH responsiveness to kisspeptin, as suggested by comparative analyses on the effects of kisspeptin and GnRH itself following antagonization of progesterone receptors (PR) [41]. In this sense, it is well known that PRs at the gonadotrope are essential for the generation of GnRH self-priming and the pre-ovulatory surge. Worthy to note, despite the documented roles of progesterone in the negative and positive feedback regulation of gonadotropin secretion in the female, the effects of this sex steroid on the expression of KiSS-1 gene, at different hypothalamic nuclei, have not been reported to date in rodents. Yet, expression analyses in the sheep have documented the ability of progesterone to partially suppress KiSS-1 mRNA levels in the ARC [48].

6. Kisspeptins: putative targets for pharmacological manipulation of gonadotropic axis? The physiologic and pharmacological features of the KiSS-1/ GPR54 system, as major stimulator of gonadotropin secretion

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acting primarily on GnRH neurons, have led to the proposal that kisspeptins may constitute a suitable target for therapeutic intervention of the gonadotropic axis [39]. Indeed, manipulation of the KiSS-1 system (with either activation or antagonization) might be theoretically beneficial in a diversity of pathological conditions, including puberty disorders, endocrine-related tumors, endometriosis and ovarian insufficiency. Admittedly, some of those conditions are currently treated by using GnRH analogs. Yet, it must be recognized also that activation of GPR54 signaling by kisspeptins, as a mean to stimulate gonadotropin secretion, might hold optimal physiologic characteristics vs. stimulation of the gonadotropic axis by pharmacological boluses of GnRH, as the former is likely to induce the secretion of the endogenous releasable pool of GnRH. In the above context, different experimental studies in rodents, published to date, clearly illustrate on the potential usefulness of kisspeptin analogs in the manipulation of the gonadotropic axis. Thus, in addition to the potent gonadotropin-releasing effects of a single bolus described in previous sections, protocols of repeated administration of kisspeptin-10 in rats (four boluses of 30 nmol/kg BW every 75 min) were able to induce a pattern of repeated LH pulses, without decrement in terms of amplitude, duration or secretory mass, thus providing the basis for the design of procedures for robust, short-term activation of the gonadotropic axis [59]. Interestingly, these observations are in line with reports in juvenile monkeys and female sheep, where intermittent injections of short-term infusions of kisspeptins have been shown to elicit sustained LH secretory responses [1,36]. The therapeutic interest of the above findings is reinforced by the fact that such responses were obtained after systemic administration of kisspeptins, which further stresses the feasibility of the design of amenable protocols of pharmacological intervention based on the use of GPR54 agonists. At the other extreme of the spectrum of gonadotropin responses to kisspeptins, protocols of chronic subcutaneous administration of kisspeptin to male rats have been reported to down-regulate the gonadotropic axis, with extinguished LH responses within 48 h and testicular atrophy in the long-term (13-days of infusion) [55]. Likewise, protocols of continuous infusion of kisspeptin in monkeys have evidenced that LH secretory responses to kisspeptin may desensitize also in primates [45]; a finding of potential therapeutic interest given the lack of antagonists of GPR54. The mechanisms of such desensitization remain to be fully solved, although the possibility of down-regulation of GPR54 has been suggested. Anyhow, we have recently obtained evidence that gonadotropin responses to continuous administration of kisspeptin do vary depending on the hormone (LH vs. FSH), the stage of sexual maturation (puberty vs. adulthood) and the functional state of the gonadotropic axis (fed ad libitum vs. undernutrition) [43]. Thus, in our experiments, the loss of LH stimulation after continuous kisspeptin exposure was accompanied by persistent elevation of FSH levels all through the infusion period in adult female rats. These observations strongly suggest that potential desensitization of gonadotropin responses to kisspeptin does not solely involve downregulation at the receptor level, but may include also changes

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on the patterns of GnRH secretion; a possibility of pharmacological interest that merits further investigation. Additional efforts in this pharmacological front include the identification/development and biological testing of analogs of endogenous kisspeptins, with either agonistic or antagonistic activity. Given the original recognition of their anti-metastatic properties, peptidergic analogs of kisspeptins, of low-molecular weight, have been designed [33,57,58]. Yet, biological testing of those compounds has been restricted to heterologous cell reporter systems and, to our knowledge, analyses of promising candidates (such as compounds FM059a and C34) in terms of induction of gonadotropin secretion in rodent models have not been reported to date, and are currently in progress in our laboratory. Similarly, natural products with ability to activate or inactivate GPR54 in vitro might be optimal candidates for in vivo testing. Finally, generation of full antagonists of GPR54 is eagerly awaited, as these may provide: (i) an optimal tool for physiologic studies on the roles of kisspeptins in the control of the gonadotropic axis, as well as on related and non-related systems; and (ii) a therapeutic option for a diversity of pathological conditions where GnRH analogs are currently in use.

7.

Conclusions

In this review, we have summarized the state-of-the-art of a particular aspect of KiSS-1 physiology that has drawn considerable attention in the last years; namely, the roles of kisspeptins as essential regulators of gonadotropin secretion and, hence, putative pharmacological targets for therapeutic intervention of the reproductive axis. In this context, molecular and pharmacological studies in rodents, as revised herein, have paved the way for the characterization of the indispensable function of kisspeptins, and their receptor GPR54, in the regulation of gonadotropin secretion in mammals, in both sexes, at different stages of sexual development and under different functional states. Indeed, some of the observations originally made in laboratory rodents have been replicated and confirmed in other mammalian species, including humans. For instance, as it was originally described in mice and rats, KiSS-1 neurons at the infundibular/arcuate nucleus seems to be involved in the negative feedback regulation of gonadotropin secretion in human and non-human primates [44]. Likewise, as it is the case in cyclic female rats, maximal gonadotropin responses to kisspeptin have been detected at the pre-ovulatory phase of the menstrual cycle in women [9]. Overall, such commonalities in the physiology of kisspeptins across mammals reinforce the potential for clinical translation of data arising from experimental rodent studies, which are likely to continue and expand in the years to come.

Acknowledgments The authors wish to thank the continuous support and efforts of Enrique Aguilar and other members of the research team at the Physiology Section of the University of Cordoba, as well as of Carlos Dieguez, from the Department of Physiology of the

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University of Santiago de Compostela, Spain, in different studies on neuroendocrine aspects of kisspeptin physiology, which have been partially revised in this work. The experimental work from the authors’ laboratory summarized in this review has been supported by grants BFI 2002-00176 and BFU 2005-07446 from Ministerio de Educacio´n y Ciencia, Spain, funds from Instituto de Salud Carlos III (Project PI042082 and CIBER Fisiopatologı´a de la Obesidad y Nutricio´n), and EU research contract EDEN QLK4-CT-2002-00603. CIBER is an initiative of Instituto de Salud Carlos III (Ministerio de Sanidad, Spain).

[13]

[14]

[15]

references [16]

[1] Caraty A, Smith JT, Lomet D, Ben Said S, Morrissey A, Cognie J, et al. Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes. Endocrinology 2007;148:5258–67. [2] Castellano JM, Navarro VM, Fernandez-Fernandez R, Castano JP, Malagon MM, Aguilar E, et al. Ontogeny and mechanisms of action for the stimulatory effect of kisspeptin on gonadotropin-releasing hormone system of the rat. Mol Cell Endocrinol 2006;257–258:75–83. [3] Castellano JM, Navarro VM, Fernandez-Fernandez R, Nogueiras R, Tovar S, Roa J, et al. Changes in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition. Endocrinology 2005;146:3917–25. [4] Castellano JM, Navarro VM, Fernandez-Fernandez R, Roa J, Vigo E, Pineda R, et al. Effects of galanin-like peptide on luteinizing hormone secretion in the rat: sexually dimorphic responses and enhanced sensitivity at male puberty. Am J Physiol Endocrinol Metab 2006;291: E1281–9. [5] Clarkson J, Herbison AE. Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons. Endocrinology 2006;147:5817–25. [6] d’Anglemont de Tassigny X, Fagg LA, Carlton MB, Colledge WH. Kisspeptin can stimulate gonadotropin-releasing hormone (GnRH) release by a direct action at GnRH nerve terminals. Endocrinology 2008;149:3926–32. [7] d’Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG, et al. Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci USA 2007;104:10714–9. [8] Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, et al. Kisspeptin-54 stimulates the hypothalamic–pituitary–gonadal axis in human males. J Clin Endocrinol Metab 2005;90:6609–15. [9] Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson M, Ramachandran R, et al. Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. J Clin Endocrinol Metab 2007;92:3958–66. [10] Fernandez-Fernandez R, Martini AC, Navarro VM, Castellano JM, Dieguez C, Aguilar E, et al. Novel signals for the integration of energy balance and reproduction. Mol Cell Endocrinol 2006;254–255:127–32. [11] Fink G. Neuroendocrine regulation of pituitary function: general principles. In: Conn PM, Freeman ME, editors. Neuroendocrinology in physiology and medicine. Totowa, NJ: Humana Press; 2000. pp. 107–134. [12] Funes S, Hedrick JA, Vassileva G, Markowitz L, Abbondanzo S, Golovko A, et al. The KiSS-1 receptor GPR54 is essential

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

for the development of the murine reproductive system. Biochem Biophys Res Commun 2003;312:1357–63. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, et al. A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 2004;145:4073–7. Gutierrez-Pascual E, Martinez-Fuentes AJ, Pinilla L, TenaSempere M, Malagon MM, Castano JP. Direct pituitary effects of kisspeptin activation of gonadotrophs somatotrophs stimulation of luteinising hormone growth hormone secretion. J Neuroendocrinol 2007;19:521–30. Han SK, Gottsch ML, Lee KJ, Popa SM, Smith JT, Jakawich SK. Activation of gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty. J Neurosci 2005;25:11349–56. Herbison AE. Estrogen positive feedback to gonadotropinreleasing hormone (GnRH) neurons in the rodent: the case for the rostral periventricular area of the third ventricle (RP3V). Brain Res Rev 2008;57:277–87. Herbison AE, Pape JR. New evidence for estrogen receptors in gonadotropin-releasing hormone neurons. Front Neuroendocrinol 2001;22:292–308. Huhtaniemi I, Ahtiainen P, Pakarainen T, Rulli SB, Zhang FP, Poutanen M. Genetically modified mouse models in studies of luteinising hormone action. Mol Cell Endocrinol 2006;252:126–35. Huhtaniemi IT. The role of mutations affecting gonadotrophin secretion and action in disorders of pubertal development. Best Pract Res Clin Endocrinol Metab 2002;16:123–38. Huhtaniemi IT, Themmen AP. Mutations in human gonadotropin and gonadotropin-receptor genes. Endocrine 2005;26:207–17. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, et al. Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 2004;80:264–72. Jacobi JS, Martin C, Nava G, Jeziorski MC, Clapp C, Martinez de la Escalera G. 17-b-Estradiol directly regulates the expression of adrenergic receptors and kisspeptin/GPR54 system in GT1-7 GnRH neurons. Neuroendocrinology 2007;86:260–9. Kauffman AS, Gottsch ML, Roa J, Byquist AC, Crown A, Clifton DK, et al. Sexual differentiation of Kiss1 gene expression in the brain of the rat. Endocrinology 2007;148:1774–83. Kinsey-Jones JS, Li XF, Luckman SM, O’Byrne KT. Effects of kisspeptin-10 on the electrophysiological manifestation of gonadotropin-releasing hormone pulse generator activity in the female rat. Endocrinology 2008;149:1004–8. Liu X, Lee K, Herbison AE. Kisspeptin excites gonadotropinreleasing hormone (GnRH) neurons through a phospholipase C/calcium-dependent pathway regulating multiple ion channels. Endocrinology 2008;149:4605–14. Marshall JC, Dalkin AC, Haisenleder DJ, Paul SJ, Ortolano GA, Kelch RP. Gonadotropin-releasing hormone pulses: regulators of gonadotropin synthesis and ovulatory cycles. Recent Prog Horm Res 1991;47:155–87 [discussion 88–9]. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T. Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 2004;320:383–8. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, et al. Kisspeptin directly stimulates gonadotropinreleasing hormone release via G protein-coupled receptor 54. Proc Natl Acad Sci USA 2005;102:1761–6. Naftolin F, Garcia-Segura LM, Horvath TL, Zsarnovszky A, Demir N, Fadiel A, et al. Estrogen-induced hypothalamic

peptides 30 (2009) 57–66

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

synaptic plasticity and pituitary sensitization in the control of the estrogen-induced gonadotrophin surge. Reprod Sci 2007;14:101–16. Navarro VM, Castellano JM, Fernandez-Fernandez R, Barreiro ML, Roa J, Sanchez-Criado JE, et al. Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 peptide. Endocrinology 2004;145:4565–74. Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, et al. Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. Endocrinology 2005;146:1689–97. Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, et al. Characterization of the potent luteinizing hormone-releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 2005;146: 156–63. Niida A, Wang Z, Tomita K, Oishi S, Tamamura H, Otaka A, et al. Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity. Bioorg Med Chem Lett 2006;16:134–7. Patterson M, Murphy KG, Thompson EL, Patel S, Ghatei MA, Bloom SR. Administration of kisspeptin-54 into discrete regions of the hypothalamus potently increases plasma luteinising hormone and testosterone in male adult rats. J Neuroendocrinol 2006;18:349–54. Pielecka-Fortuna J, Chu Z, Moenter SM. Kisspeptin acts directly and indirectly to increase GnRH neuron activity and its effects are modulated by estradiol. Endocrinology 2008;149:1979–86. Plant TM, Ramaswamy S, Dipietro MJ. Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropinreleasing hormone discharges. Endocrinology 2006;147:1007–13. Quaynor S, Hu L, Leung PK, Feng H, Mores N, Krsmanovic LZ, et al. Expression of a functional g protein-coupled receptor 54-kisspeptin autoregulatory system in hypothalamic gonadotropin-releasing hormone neurons. Mol Endocrinol 2007;21:3062–70. Richard N, Galmiche G, Corvaisier S, Caraty A, Kottler ML. KiSS-1 and GPR54 genes are co-expressed in rat gonadotrophs and differentially regulated in vivo by oestradiol and gonadotrophin-releasing hormone. J Neuroendocrinol 2008;20:381–93. Roa J, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M. New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function. Front Neuroendocrinol 2008;29:48–69. Roa J, Vigo E, Castellano JM, Gaytan F, Garcia-Galiano D, Navarro VM, et al. Follicle-stimulating hormone responses to kisspeptin in the female rat at the preovulatory period: Modulation by estrogen and progesterone receptors. Endocrinology 2008;149:5783–90. Roa J, Vigo E, Castellano JM, Gaytan F, Navarro VM, Aguilar E, et al. Opposite roles of estrogen receptor (ER)a and ERb in the modulation of luteinizing hormone responses to kisspeptin in the female rat: implications for the generation of the preovulatory surge. Endocrinology 2008;149:1627–37. Roa J, Vigo E, Castellano JM, Navarro VM, FernandezFernandez R, Casanueva FF, et al. Hypothalamic expression of KiSS-1 system and gonadotropin-releasing effects of kisspeptin in different reproductive states of the female rat. Endocrinology 2006;147:2864–78. Roa J, Vigo E, Garcia-Galiano D, Castellano JM, Navarro VM, Pineda R, et al. Desensitization of gonadotropin responses to kisspeptin in the female rat: analyses of LH and FSH

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53] [54]

[55]

[56]

[57]

[58]

[59]

65

secretion at different developmental and metabolic states. Am J Physiol Endocrinol Metab 2008;294: E1088–96. Rometo AM, Krajewski SJ, Voytko ML, Rance NE. Hypertrophy and increased kisspeptin gene expression in the hypothalamic infundibular nucleus of postmenopausal women and ovariectomized monkeys. J Clin Endocrinol Metab 2007;92:2744–50. Seminara SB, Dipietro MJ, Ramaswamy S, Crowley Jr WF, Plant TM. Continuous human metastin 45-54 infusion desensitizes G protein-coupled receptor 54-induced gonadotropin-releasing hormone release monitored indirectly in the juvenile male Rhesus monkey (Macaca mulatta): a finding with therapeutic implications. Endocrinology 2006;147:2122–6. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr JS, Shagoury JK, et al. The GPR54 gene as a regulator of puberty. N Engl J Med 2003;349: 1614–27. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM. Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 2005;102:2129–34. Smith JT, Clay CM, Caraty A, Clarke IJ. KiSS-1 messenger ribonucleic acid expression in the hypothalamus of the ewe is regulated by sex steroids and season. Endocrinology 2007;148:1150–7. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA. Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 2005;146:3686–92. Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, et al. Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology 2005;146:2976–84. Smith JT, Rao A, Pereira A, Caraty A, Millar RP, Clarke IJ. Kisspeptin is present in ovine hypophysial portal blood, but does not increase during the preovulatory luteinizing hormone surge: evidence that gonadotropes are not direct targets of kisspeptin in vivo. Endocrinology 2008;149:1951–9. Suzuki S, Kadokawa H, Hashizume T. Direct kisspeptin-10 stimulation on luteinizing hormone secretion from bovine and porcine anterior pituitary cells. Anim Reprod Sci 2008;103:360–5. Tena-Sempere M. GPR54 and kisspeptin in reproduction. Hum Reprod Update 2006;12:631–9. Tena-Sempere M, Huhtaniemi I. Gonadotropins and gonadotropin receptors. In: Fauser BCJ, editor. Reproductive medicine—molecular, cellular and genetic fundamentals. New York: Parthenon Publishing; 2003. pp. 225–244. Thompson EL, Murphy KG, Patterson M, Bewick GA, Stamp GW, Curtis AE, et al. Chronic subcutaneous administration of kisspeptin-54 causes testicular degeneration in adult male rats. Am J Physiol Endocrinol Metab 2006;291: E1074–82. Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, et al. Central and peripheral administration of kisspeptin-10 stimulates the hypothalamic-pituitarygonadal axis. J Neuroendocrinol 2004;16:850–8. Tomita K, Narumi T, Niida A, Oishi S, Ohno H, Fujii N. Fmoc-based solid-phase synthesis of GPR54-agonistic pentapeptide derivatives containing alkene- and fluoroalkene-dipeptide isosteres. Biopolymers 2007;88: 272–8. Tomita K, Niida A, Oishi S, Ohno H, Cluzeau J, Navenot JM, et al. Structure-activity relationship study on small peptidic GPR54 agonists. Bioorg Med Chem 2006;14:7595–603. Tovar S, Vazquez MJ, Navarro VM, Fernandez-Fernandez R, Castellano JM, Vigo E, et al. Effects of single or repeated

66

peptides 30 (2009) 57–66

intravenous administration of kisspeptin upon dynamic LH secretion in conscious male rats. Endocrinology 2006;147:2696–704. [60] Wintermantel TM, Campbell RE, Porteous R, Bock D, Grone HJ, Todman MG, et al. Definition of estrogen receptor pathway critical for estrogen positive feedback to

gonadotropin-releasing hormone neurons and fertility. Neuron 2006;52:271–80. [61] Zhang C, Roepke TA, Kelly MJ, Ronnekleiv OK. Kisspeptin depolarizes gonadotropin-releasing hormone neurons through activation of TRPC-like cationic channels. J Neurosci 2008;28:4423–34.