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
b
CIBER Fisiopatologa 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
Contents
1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2. Gonadotropin responses to kisspeptins in rodents: pharmacological characterization. . . . . . . . . . . . . . . . . . . . . . . . . 58
3. Mechanisms of action of kisspeptins in the control of gonadotropin secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4. KiSS-1/kisspeptins and the feedback control of gonadotropin secretion in rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
p e p t i d e s 3 0 ( 2 0 0 9 ) 5 7 6 6
a r t i c l e i n f o
Article history:
Received 6 March 2008
Received in revised form
6 August 2008
Accepted 7 August 2008
Published on line 22 August 2008
Keywords:
Kisspeptins
KiSS-1
GPR54
GnRH
Gonadotropins
LH
FSH
Mouse
Rat
a b s t r a c t
Kisspeptins, the products of KiSS-1 gene acting via G protein-coupled receptor 54 (GPR54),
have recently emerged as fundamental gatekeepers of gonadal function by virtue of their
ability to stimulate gonadotropin secretion. Indeed, since the original disclosure of the
reproductive facet of the KiSS-1/GPR54 system, an ever-growing number of studies have
substantiated the extraordinary potency of kisspeptins to elicit gonadotropin secretion in
different mammalianspecies, under different physiologic and experimental conditions, and
through different routes of administration. In this context, studies conducted in laboratory
rodents have been enormously instrumental to characterize: (i) the primary mechanisms of
action of kisspeptins in the control of gonadotropin secretion; (ii) the pharmacological
consequences of acute vs. continuous activation of GPR54; (iii) the roles of specic popula-
tions of kisspeptin-producing neurons at the hypothalamus in mediating the feedback
effects of sex steroids; (v) the function of kisspeptins in the generation of the pre-ovulatory
surge of gonadotropins; and (iv) the inuence of sex steroids on GnRH/gonadotropin
responsiveness to kisspeptins. While some of those aspects of kisspeptin function will
be covered elsewhere in this Special Issue, we summarize herein the most salient data,
obtained in laboratory rodents, that have helped to dene the physiologic roles and putative
pharmacological implications of kisspeptins in the control of male and female gonadotropic
axis.
# 2008 Elsevier Inc. All rights reserved.
* 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: 1tesem@uco.es (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.
avai l abl e at www. sci encedi r ect . com
j our nal homepage: www. el sevi er . com/ l ocat e/ pept i des
0196-9781/$ see front matter # 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2008.08.009
5. Modulation of GnRH/gonadotropin responses to kisspeptins by sex steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
6. Kisspeptins: putative targets for pharmacological manipulation of gonadotropic axis? . . . . . . . . . . . . . . . . . . . . . . . . 62
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
1. 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
hypothalamicpituitarygonadal (HPG) axis, pituitary gonado-
tropins, LH and FSH, are the main driving force for gonadal
development, trophic maintenance and function [54]. Accord-
ingly, diverse physiologic and pathological conditions, as well
as pharmacological manipulations, affecting the gonads are
conveyed via regulation of their secretion, and thus, elucida-
tion of the mechanisms and signals involved in the regulatory
network governing gonadotropin release has attracted con-
siderable attention among physiologists and clinicians for
decades [1820]. In this context, during recent years, genetic
analyses and functional studies have identied monogenic
forms of infertility that derive fromdisruptionof gonadotropin
secretion (e.g., altered hypothalamic systems or pituitary
responsiveness) or function (e.g., mutations in gonadotropin
subunits or their receptors) [1820,54]. 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 bothgonadotropins 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 gonado-
trops 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
dened for decades.
Besides central regulators, the secretion of both gonado-
tropins is under the inuence 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 gonadotropinsecretion, 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
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 self-
priming at the pituitary), thereby generating the pre-ovulatory
surge of gonadotropins ( positive feedback), which ultimately
triggers ovulation [16,29]. Of note, identication of discernible
neuronal pathways, responsible for such a differential pattern
of response to estrogen(positive vs. negative feedback) insuch
a sexually dimorphic manner, has remained elusive for
decades, and has been the subject of considerable investiga-
tion 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 understand-
ing 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
signicant part of the research efforts in the eld 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 impor-
tance of these studies is twofold: (i) to dene the biological
effects and functional relevance of kisspeptin in the control of
the gonadotropic axis; and (ii) to provide the scientic 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 dened
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
systeminthe control of gonadotropinsecretion, roughly within
1 year since the initial reports on hypogonadotropic hypogo-
nadism 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 stimu-
late LHsecretion in a number of mammalian species, including
mouse, rat, sheepandmacaque [13,27,28,30,32,47,56]. Likewise,
p e p t i d e s 3 0 ( 2 0 0 9 ) 5 7 6 6 58
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 gonado-
tropin release in mammals [8,9]. Overall, the striking simila-
rities of the effects of kisspeptins on gonadotropin secretion
among different mammalian species reinforced the usefulness
of rodent studies for covering the physiologic and pharmaco-
logical goals dened 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 conrmed 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 pre-
served 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
rst to document the extraordinarily potent LH releasing
effects of kisspeptin-10 and kisspeptin-54 (metastin). Thus,
threshold doses for LH stimulation were dened (depending
on the study considered) between 100 fmol and 1 pmol, for
protocols of intracerebroventricular (i.c.v.) or intrahypotha-
lamic administration [13,32,34]. Moreover, such stimulatory
effects were also detected after systemic injection, over a
variety of routes (intravenous, subcutaneous and intraper-
itoneal) and a range of doses [32,59]. Based on detailed dose-
response studies conducted in vivo, the median effective dose
(ED50) for LH was calculated at 24 pmol, for i.c.v. adminis-
tration [4,32]. Concerning systemic delivery, doses as low as
0.1 mg/rat (0.3 nmol/kg BW) i.v. were sufcient to induce
robust LH peaks in freely moving rats [59]. Overall, compara-
tive analysis of the published data on the LH releasing activity
of kisspeptins and other neuropeptides and neurotransmit-
ters, 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 simi-
larly 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 thresholddoses for FSHstimulationare clearly higher than
for LH, with a predicted ED50 of 400 pmol, for i.c.v. admin-
istration (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 kisspeptinonFSHrelease inmale
rats appears to be somewhat slower than for LH secretion [31].
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 func-
tional 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 signicant 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 signicantly 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 identication 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 antago-
nists in male and female rodents [13,27,31,32]; (ii) GnRH
neurons in the rat forebrain do express GPR54 gene [21]; (iii)
kisspeptinactivates GnRHneurons inrodents, as evidenced by
induction of c-fos expression [21], as well as potent and long-
lasting 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-decient hpg mice are unable to
respond to kisspeptinstimulationinterms of LHsecretion (see
Fig. 1). Altogether, the above data demonstrate that hypotha-
lamic GnRH is an obligate mediator for the gonadotropin-
releasing effects of kisspeptins.
p e p t i de s 3 0 ( 2 0 0 9 ) 5 7 6 6 59
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 specic
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,
suchas puberty, appears to be under the precise regulationof 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 efciency [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 GnRHrelease [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 Ca
2+
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 inux of extracellular Ca
2+
, 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 rened 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 regu-
lating multiple ion channels, including potassium and
transient receptor potential (TRP) channels [25,61].
As indicated in Section 2, the proles of LH and FSH
secretion after kisspeptin stimulation appear partially differ-
ent, 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 GnRHrelease that favors preferential
secretion of LH. In this sense, proles 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 signicantly 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 kisspeptinstrongly 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, whichhampers 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 dened 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
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 wild-
type 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.
p e p t i d e s 3 0 ( 2 0 0 9 ) 5 7 6 6 60
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
identied in response to direct kisspeptin stimulation in rat
gonadotropes), bovine and ovine species, evenat the nMrange
[14,51,52]. Moreover, rat studies have suggestedthe expression
and hormonal regulation of KiSS-1 gene at the pituitary [38],
while in the sheep, kisspeptin has been detected in hypophy-
sial portal blood [51]. On the latter, however, the lack of
signicant uctuations 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
systemin 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
identication 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 beenevaluated inrodents [39].
The rst 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 signicant 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 sufcient 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-QRT-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 conrmed 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].
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 Ca
2+
stores (by thapsigargin); and (iii) blockade of ERK1/2 and p38 kinase (by PD-98059 and SB-
203580, respectively). In contrast, GnRH responses were preserved after antagonization of adenylate cyclase (by MDL-
12,330A; not shown), inhibition of extracellular Ca
+2
influx (by cadmium), or blockade of Jun N-terminal kinase (by SP-
600125). Likewise, GnRH responses to kisspeptin were also detected after inhibition of prostaglandin synthesis by
indomethacin (not shown). Composed from data of Ref. [2].
p e p t i de s 3 0 ( 2 0 0 9 ) 5 7 6 6 61
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
diametricallyoppositemanner: KiSS-1mRNAexpressionat 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 pre-
ovulatory 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; ndings that are exhaustively
revised by Tsukamura and Maeda in this Special Issue. Overall,
the data available evidence that estrogen is capable to activate
thetranscriptionof KiSS-1gene, therebyinducinganincreasein
KiSS-1/kisspeptin expression that enhances the secretory
activity of GnRH neurons, and thus triggers the pre-ovulatory
surge of gonadotropins [39]. As revisedby Kaufmannelsewhere
inthis 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-1mRNAinAVPVneurons whileit 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 hypotha-
lamic sites. Moreover, pharmacological andfunctional genomic
studies inrats 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 kisspep-
tininthe female rat; a phenomenonthat might contribute also
to the generation of the pre-ovulatory surge of gonadotropins
[41]. In this sense, recent pharmacologic and electrophysio-
logical studies in rats and mice have jointly pointed out that
relative GnRH/LH responses to kisspeptin are decreased in
GNXanimals, 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
phenomenonthat is inline withprevious ndings of our group
in cyclic female rats that showed cycle-dependent uctua-
tions in the pattern of gonadotropin responses to kisspeptin,
with maximal LH responsiveness during the proestrus-to-
estrus 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 LHand FSHresponses to kisspeptin in cyclic female rats at
proestrus [40,41]. These observations are in good agreement
with recent data inGNXmice, where estrogenhas beenshown
to enhance kisspeptin-stimulated GnRHneuronal 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 signicantly 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
supplementedwitha selective ERb agonist. Instriking contrast,
selective blockade of ERb attenuated FSHresponses to kisspep-
tin 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 onFSHsecretion[40]. Theformer mayoperateasnegative
modier of GnRH/LH responses to kisspeptin; a phenomenon
that couldcontributetopartiallyrestrainLHsecretionat 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 progester-
one 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 reect 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 steroidonthe expressionof KiSS-1 gene, at different
hypothalamic nuclei, have not been reported to date in
rodents. Yet, expression analyses in the sheep have docu-
mented 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
p e p t i d e s 3 0 ( 2 0 0 9 ) 5 7 6 6 62
acting primarily on GnRH neurons, have led to the proposal
that kisspeptins may constitute a suitable target for ther-
apeutic intervention of the gonadotropic axis [39]. Indeed,
manipulation of the KiSS-1 system (with either activation or
antagonization) might be theoretically benecial in a diversity
of pathological conditions, including puberty disorders,
endocrine-related tumors, endometriosis and ovarian insuf-
ciency. Admittedly, some of those conditions are currently
treated by using GnRHanalogs. Yet, it must be recognized also
that activation of GPR54 signaling by kisspeptins, as a mean to
stimulate gonadotropin secretion, might hold optimal phy-
siologic 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 gonado-
tropin-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 gonado-
tropic axis [59]. Interestingly, these observations are in line
with reports in juvenile monkeys and female sheep, where
intermittent injections of short-term infusions of kisspep-
tins have been shown to elicit sustained LH secretory
responses [1,36]. The therapeutic interest of the above
ndings 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 nding 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 gonado-
tropin 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. under-
nutrition) [43]. Thus, in our experiments, the loss of LH
stimulation after continuous kisspeptin exposure was accom-
panied by persistent elevation of FSH levels all through the
infusion period in adult female rats. These observations
strongly suggest that potential desensitization of gonadotro-
pin responses to kisspeptin does not solely involve down-
regulation at the receptor level, but may include also changes
on the patterns of GnRH secretion; a possibility of pharma-
cological interest that merits further investigation.
Additional efforts in this pharmacological front include the
identication/development andbiological 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-mole-
cular weight, have been designed [33,57,58]. Yet, biological
testing of those compounds has been restricted to hetero-
logous cell reporter systems and, to our knowledge, analyses
of promising candidates (such as compounds FM059a and C-
34) 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 conrmed in other mam-
malian 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 commonal-
ities in the physiology of kisspeptins across mammals
reinforce the potential for clinical translation of data arising
fromexperimental rodent studies, whichare 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
p e p t i de s 3 0 ( 2 0 0 9 ) 5 7 6 6 63
University of Santiago de Compostela, Spain, in different
studies on neuroendocrine aspects of kisspeptin physiology,
which have been partially revised in this work. The experi-
mental 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 Fisiopatologa 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).
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