Download The Role of Kisspeptin Signaling in Reproduction

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PHYSIOLOGY 25: 207–217, 2010; doi:10.1152/physiol.00009.2010
The Role of Kisspeptin Signaling
in Reproduction
Kisspeptins are a group of peptides that stimulate GnRH release and are
Xavier d’Anglemont de Tassigny and
William Henry Colledge
Department of Physiology, Development and Neuroscience,
Reproductive Physiology Group, University of Cambridge,
Cambridge, United Kingdom
[email protected]
required for puberty and maintenance of normal reproductive function. This
review focuses on our understanding of the way in which kisspeptin signaling
regulates mammalian fertility and how they act as central integrators of
different hormonal and physiological signals.
Kisspeptin Expression Profile
Kisspeptins are an overlapping set of amidated
peptides encoded by the Kiss1 gene, which is located in close proximity to the Golt1a (golgi transport 1 homolog A) gene in most species. In humans
and mice, the Kiss1 gene consists of two coding
exons downstream from at least one noncoding
exon (60) (FIGURE 2). The human promoter has
been mapped immediately upstream of the noncoding exon and contains a TATA box and several
potential SP1 transcription factor binding sites.
These SP1 binding sites facilitate binding of AP-1␣/
SP1 and DRIP130/SP1 transcriptional activator
complexes and contribute to basal promoter activity (71, 72). Two SP1 binding sites closest to the
1548-9213/10 ©2010 Int. Union Physiol. Sci./Am. Physiol. Soc.
transcriptional start site function together to facilitate transcriptional activation by estrogen (63). In
mice, an analogous region also containing a TATA
box is located just upstream of the noncoding exon
but this has not been assessed for promoter activity. Several alternatively spliced Kiss1 transcripts
have been identified in mice that contain sequences from the first exon of the Golt1a gene
(NCBI transcript accession numbers: AY707856
AY707857 AY707859). Thus Kiss1 transcripts in rodents may be generated from both a Kiss1 promoter (P1) and the Golt1a promoter (P2). The
physiological significance of this is not known but
may allow an additional level of gene regulation in
mice.
Kisspeptins were originally isolated from human
placenta (55, 78) and are derived from a larger
precursor protein (FIGURE 2). The longest kisspeptin is 54 amino acids in length (Kp54), but shorter
kisspeptins (Kp13, Kp14) have also been isolated
corresponding to the carboxy terminus of Kp54.
These shorter kisspeptins may represent degradation products, but they still retain full biological
activity, as does a synthetic peptide of only 10
amino acids (Kp10). Kp14 is highly conserved between species, with the final 10 amino acids showing very little variation, particularly in mammals
(Table 1). A series of alanine substitutions in Kp10
have shown that amino acids 6 (F) and 10 (F or Y,
depending on the species) are the most critical for
receptor binding (18, 36, 79). NMR structural modeling indicates that the COOH-terminal 7 amino
acids form a helicoid structure that is disrupted by
these alanine substitutions (36, 79).
The distribution of kisspeptin neurons in the
hypothalamus varies between species (16). In the
rodent, in situ hybridization and immunohistochemistry have been used to map kisspeptin neurons to two discrete regions of the hypothalamus, the
arcuate (ARC) nucleus and in a periventricular continuum of cells within the rostral part of the third
ventricle, including the anteroventral periventricular
nucleus (AVPV) (12, 13, 15, 31) (FIGURE 1). Humans
(95), rhesus monkeys (102), and sheep (26, 110) have
proportionally more kisspeptin neurons in the ARC
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Acquisition of reproductive competency is essential for continuation of all species. Fertility in
mammals is initiated at puberty by the pulsatile
secretion of gonadotrophin releasing hormone
(GnRH) from a small number of neurons in the
hypothalamus (FIGURE 1). The GnRH is released
into the hypophyseal portal blood system from
nerve terminals in the palisade layer of the median
eminence of the hypothalamus. The GnRH acts on
the anterior pituitary to stimulate the release of the
gonadotrophic hormones, luteinizing hormone
(LH), and follicle stimulating hormone (FSH). The
gonadotrophic hormones act on the gonads to
stimulate sexual maturation and gametogenesis
(spermatogenesis in males and oogenesis in females). The gonads produce sex steroids (testosterone in males and estrogen and progesterone in
females), which are required for gametogenesis, for
maturation of accessory sex organs, and to provide
hormonal feedback loops that regulate GnRH and
gonadotrophic hormone release under different
physiological conditions (FIGURE 1). Although
GnRH neurons are a critical component of the
reproductive axis, kisspeptin (Kp) peptides have
been identified recently as vital upstream regulators that integrate central and peripheral signals
with GnRH release, thereby playing a pivotal role
in the control of reproduction (FIGURE 1).
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males (15, 50). This sexual dimorphism is caused
by neonatal exposure to testosterone, which is aromatized into estrogen and causes defeminization
of the AVPV region. Neonatal female rats treated
with androgens (50) or estradiol (44) develop very
few kisspeptin neurons in the AVPV, whereas castration of neonatal male rats increases the number
of kisspeptin neurons in the AVPV (44). The role
that estrogens play in this process has been examined by using transgenic mice lacking alpha-fetoprotein (Afp⫺/⫺), which protects the fetal brain
from the defeminizing action of circulating estrogens. As expected, female Afp⫺/⫺ mice developed
male-like numbers of kisspeptin neurons in the
AVPV (29). Gpr54⫺/⫺ male mice have a feminized
AVPV region with a greater number of kisspeptin
neurons than wild-type males and similar to that of
females (51). These data suggest that kisspeptin
signaling during the neonatal period is required for
testosterone production in males to defeminize the
AVPV. The AVPV region also contains a sexually
FIGURE 1. The mammalian hypothalamic pituitary gonadal axis
At puberty, pulsatile secretion of gonadotrophic releasing hormone (GnRH) stimulates the anterior pituitary to release the gonadotrophic hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). These act on
the gonads to promote gamete formation and the production of gonadal steroid hormones, which form feedback
loops to regulate GnRH, LH, and FSH release. Kisspeptin (Kiss1) neurons act as a principal relay for steroid feedback
on GnRH secretion. In females, high levels of estrogens and progesterone stimulate kisspeptin neurons of the AVPV
to induce the preovulatory surge of GnRH/LH, whereas they inhibit KISS1 expression in the arcuate nucleus (ARC). In
the male, GnRH and gonadotrophic hormone release are negatively regulated by circulating testosterone, partly
through the activity of kisspeptin neurons of the ARC. POA, preoptic area; AVPV, anteroventral periventricular nucleus; ME, median eminence.
208
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than in the AVPV region. Kisspeptin expression increases in the ARC and AVPV regions during pubertal
development in rodents (15, 74, 117) and monkeys
(102), consistent with kisspeptin signaling initiating
puberty. Kisspeptin fibers have been found in the
preoptic area (POA) of adult female rats (54) and
mice (15) in close association with GnRH neuron
cell bodies. These associations are probably from
AVPV kisspeptin neurons and are proposed to
modulate the preovulatory GnRH/LH surge in females (34). Kisspeptin immunoreactive fibers originating from cell bodies in the ARC make close
apposition to GnRH axons in the median eminence
of the monkey (89) and are proposed to modulate
the pulsatile release of GnRH (FIGURE 1).
In some species, anatomical differences in the
hypothalamus are found between the sexes with
some nuclei showing a neuronal density difference
(105). In rodents, the AVPV region is sexually dimorphic (106, 107) with a greater number of
kisspeptin neurons in females compared with
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Kisspeptins and the
Reproductive Axis
The critical role that kisspeptins play in regulating
the reproductive axis is illustrated by the consequences of mutations in the kisspeptin signaling
pathway in mice and humans. Mice with disruption of the kisspeptin receptor gene (Gpr54/Kiss1r)
(24, 27, 51, 60, 100) or Kiss1 (20, 27) do not undergo
pubertal development, and both sexes are infertile,
with poor gonadal growth and impaired gametogenesis. Male mice have delayed spermatogenesis
and produce low numbers of spermatozoa. Female
mice do not show a normal estrus cycle, fail to
ovulate, and do not have corpora lutea in their
ovaries. This phenotype is caused by low levels of
gonadotrophic hormones (LH, FSH) and gonadal
sex steroids in the bloodstream (20, 24, 51, 60, 100).
Similarly, loss-of-function mutations in GPR54/
KISS1R in humans cause hypogonadotrophic hypogonadism (21, 59, 100, 101), whereas gain-offunction mutations cause precocious puberty
(118). Moreover, a missense mutation in the
kisspeptin precursor protein has been found in a
Brazilian boy with central precocious puberty
(104). The mutation reduces serum protease degradation of the protein and may therefore be associated with increased kisspeptin signaling.
These reproductive defects can be directly attributed to the action of kisspeptins in the hypothalamus. Central or peripheral injection of kisspeptin
stimulates gonadotrophin secretion in most species, including humans (22, 23), monkeys (87, 99),
sheep (4, 69), pigs (62), goats (38), rats (74 –76, 119,
120), mice (31, 69), and goldfish (64). This gonadotrophin secretion is mediated by kisspeptin-stimulated GnRH release from hypothalamic neurons.
Consistent with this, the majority of GnRH neurons
express the kisspeptin receptor (GPR54/KISS1R),
as shown by in situ hybridization or tagged expression of a LacZ reporter gene (37, 40, 46, 69, 81).
Kisspeptin-mediated GnRH release has been
demonstrated directly in ewes (69) and female
rhesus monkeys (52) and by inhibition of kisspeptin
responses in rodents by administration of GnRH antagonists (46). In addition, mice with a disrupted
Gpr54/Kiss1r gene cannot secrete GnRH from hypothalamic fragments after kisspeptin stimulation (19).
Sex steroids provide feedback loops that allow
the gonads to communicate with the hypothalamus to regulate GnRH release (FIGURE 1). Sex steroids achieve this indirectly, however, since GnRH
neurons do not express androgen or estrogen (ER␣
receptors) (41, 45). It is now thought that kisspeptin neurons mediate the actions of sex steroids on
GnRH neurons. The majority of kisspeptin neurons
express estrogen receptor alpha (ER␣ of ⬃90%) (26,
111, 112), the androgen receptor (⬃65%) (112), and
the progesterone receptor (⬃86%) (110), consistent
with their role as mediators of sex steroid feedback
on the reproductive axis. In rodents, sex steroids
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dimorphic population of tyrosine hydroxylase (TH)
neurons (105–107), but there is little overlap between these and the kisspeptin neurons (50). In
contrast, the ARC does not display sexually dimorphic differences in kisspeptin neuron density or
distribution in rodents (44, 50). In sheep, however,
the ARC is sexually dimorphic with half the number of kisspeptin neurons in rams compared with
ewes (10). These sexually dimorphic kisspeptin
neurons co-express dynorphin (DYN) and neurokinin B (NKB). Prenatal testosterone treatment of
females, however, reduces DYN and NKB expression but not kisspeptin expression, suggesting different critical periods for sexual differentiation of
these genes (10).
FIGURE 2. Generation of kisspeptins from the Kiss1 gene
The kisspeptin coding region (pale blue) is located within two exons of
the Kiss1 gene. At least one upstream noncoding exon (white) has
been identified and the promoter (P1) in humans had been mapped
immediately upstream of this exon. The exact location of the mouse
Kiss1 promoter remains to be determined since Kiss1 transcripts fused
to Golt1a sequences have been found, suggesting some expression
from a second promoter (P2) in this species. The primary translation
product is a 138- to 145-amino acid protein (Kp145), depending on the
species, which contains a secretory signal sequence (dark green). Proteolytic cleavage generates a 54-amino acid amidated peptide (Kp54
also known as metastin). In the placenta, shorter kisspeptins have been
isolated (Kp14 and Kp13), which may be degradation products from
Kp54. The shorter kisspeptins all contain the same 10 amino acids (yellow). A synthetic peptide containing only these 10 amino acids (Kp10)
retains biological activity in vivo.
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Table 1. Kp14 species comparison
Species
Sequence
Human
Chimp
Rhesus
Orangutan
Cat
Horse
Cow
Sheep
Mouse
Rat
Dolphin
Zebrafish
DLPNYNWNSFGLRF-NH2
DLPNYNWNSFGLRF-NH2
DLPNYNWNSFGLRF-NH2
DLPNYNWNSFGLRF-NH2
DLSAYNWNSFGLRY-NH2
LLPAYRWNSFGLRY-NH2
DVSAYNWNSFGLRY-NH2
DVSAYNWNSFGLRY-NH2
DLSTYNWNSFGLRY-NH2
DMSAYNWNSFGLRY-NH2
DLSAYNWNSFGLRY-NH2
NVAYYNLNSFGLRY-NH2
differentially regulate kisspeptin expression to decrease expression in the AVPV region (50, 111) and
increase expression in the ARC (111). These
changes are reversed by either testosterone or estradiol replacement (111, 112). Studies in other
species have corroborated the effects of sex steroids on kisspeptin expression (46, 74, 95, 103,
110). In the sheep, the ARC kisspeptin neurons
mediate both the negative feedback action of progesterone on GnRH release during the luteal phase
(11) and the positive feedback action of estradiol
expression at the time of ovulation (25, 113). The
mechanism by which sex steroids differentially
regulate kisspeptin expression in the AVPV and
ARC regions had not been resolved, but estradiol
mediates its feedback effects through both estrogen response element (ERE)-dependent and EREindependent signaling in rodents. This was shown
by examining the effects of estradiol on Kiss1 expression in mutant mice with ER␣ molecules that
cannot bind to ERE sequences (32). Stimulation of
Kiss1 expression by estradiol in the AVPV required
an ERE-dependent pathway. In contrast, inhibition
of Kiss1 expression by estradiol in the ARC was
ERE-independent (32). In addition, insulin-like
growth factor-1 (IGF-1) increases Kiss1 expression
in the AVPV but not the ARC of prepubertal female
rats in the presence of estradiol (42, 43). These
expression changes are consistent with kisspeptin
neurons in the ARC regulating the negative feedback effect of sex steroids on GnRH release and
those in the AVPV/PeN regions responsible for the
preovulatory GnRH/LH surge (39) (see FIGURE 4).
Kisspeptin action on
GnRH neurons
Kisspeptin signaling through GPR54/KISS1R couples to Gq/11 to activate phospholipase C and
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Kp14 is highly conserved between many species. The
leucine2 (L) to valine (V) or methionine (M) are conserved amino-acid substitutions (58). Differences from
the human sequence are shown in italicized bold.
increases inositol triphosphate (IP3) and diacylglycerol (DAG) levels in the cell (55, 73, 116) (FIGURE 3).
The subsequent increase in intracellular Ca2⫹ and
DAG activates protein kinase C and initiates a kinase phosphorylation cascade resulting in phosphorylation of ERK1/2. DAG also stimulates GnRH
depolarization by activation of a nonselective cation channel (TRPC) and inhibition of an inwardly
rectifying potassium channel (Kir) (66, 84). Thus
kisspeptins act directly on GnRH neurons to induce a sustained depolarization event and increase
the rate of action potential firing (37, 88). The number of GnRH neurons that can respond to kisspeptins
increases during puberty even though the expression
levels only change slightly, suggesting posttranslational maturation of GPR54/KISS1R function with
puberty (37). In adult mice, ␥-amino-butyric acid
(GABA) has a predominantly hyperpolarizing effect on
GnRH neurons to inhibit GnRH release (14). Kisspeptins have been reported to suppress the inhibitory
effects of GABA(B) receptor signaling in GnRH neurons (123). Thus, under conditions of estrogen positive feedback when kisspeptin levels increase in the
AVPV, kisspeptins will antagonize any inhibitory action of GABA to stimulate GnRH release and LH
surge/ovulation.
After initial stimulation, continuous administration of kisspeptin suppresses the reproductive axis.
Continuous delivery of human Kp10 inhibited further LH release in agonadal monkeys within 24 h
(90, 99). Similarly, sustained kisspeptin activity in
women failed to elicit LH or FSH release by day 14
(49). In contrast, pulsatile delivery of kisspeptin in
agonadal juvenile monkeys did not suppress gonadotropic hormone release (87). The suppressive
effects are probably caused by desensitization of
GPR54/KISS1R rather than the GnRH receptor
since all individuals remained responsive to GnRH.
Desensitization of GPR54/KISS1R signaling is mediated by G-protein-coupled receptor serine/
threonine kinase 2 (GRK2) and ␤-arrestin in a cell
culture model (80). GPR54/KISS1R also displays
constitutive basal activity at ⬃5% of the maximum
activity after kisspeptin stimulation (80). This basal
activity may explain why the phenotype of Kiss1⫺/⫺
mutant mice is less severe than that of Gpr54⫺/⫺
mutant mice (9, 60).
Kisspeptins may also have indirect effects on
GnRH neurons via synaptic input from other neurons in the hypothalamus that express GPR54/
KISS1R (40, 61, 73). To distinguish between direct
and indirect action of kisspeptins on GnRH neurons, electrophysiological responses are usually
measured in the presence of inhibitors of transsynaptic input from other neurons. Under these
conditions, part of the GnRH response to kisspeptins is mediated by trans-synaptic input from
GABAergic and glutamatergic neurons, which was
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enhanced by estradiol (84, 85). Moreover, kisspeptin increased inhibitory GABAergic input to GnRH
neurons during the estradiol-mediated negative
feedback part of the estrous cycle (84, 85).
Physiological Regulation of
Kisspeptin Signaling
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Mammalian ovulation requires an LH surge
brought about by the positive feedback action of
estradiol on GnRH release. Kisspeptin expression
increases in the AVPV region just before ovulation
and during a steroid-induced LH surge in ovariectomized rats (114). At the time of ovulation,
kisspeptin neurons in the AVPV are activated as
indicated by induction of c-fos (13). Inhibition of
kisspeptin action in the POA by local injection of a
monoclonal antibody abolishes the proestrous LH
surge and inhibits estrous cyclicity in rats (1, 54). In
the absence of either GPR54/KISS1R or kisspeptin
expression in transgenic female mice, the sex steroid-induced GnRH/LH surge does not occur compared with wild-type siblings (13). In rodents, tissue
ablation studies have shown that the LH surge is also
gated by a circadian oscillator in the suprachiasmatic
nucleus (SCN) to ensure that the surge occurs near the
onset of darkness. Kisspeptin expression and c-fos
neuronal activation in the AVPV region are also
subject to circadian regulation. This was demonstrated in ovariectomized mice that were maintained in constant darkness and still showed an
estradiol-induced LH surge (94). Thus kisspeptin
signaling, regulated by both estradiol and circadian signals, is essential for the preovulatory
GnRH/LH surge.
Kisspeptin signaling may also play a role in modulating the pulsatile release of GnRH. The mechanism by which this occurs is not fully understood,
but recent information about the gene expression
profile of kisspeptin neurons has provided important clues. In sheep and mice, the majority of
kisspeptin neurons in the ARC co-express dynorphin A (DYN) and neurokinin B (NKB) (30, 77)
(FIGURE 4). Of mouse ARC kisspeptin neurons,
96% also express the neurokinin B receptor gene
Nk3r, but only 20% of kisspeptin neurons express
the dynorphin receptor gene Kor. This may be an
underestimate, however, since others have shown
by immunohistochemistry that practically all NKBpositive neurons in the rat ARC co-express DYN
(3). In contrast, kisspeptin neurons in the AVPV
regions of the female mouse hypothalamus show
limited expression of Dyn (33%) and NkB (10%).
Co-expression of DYN and NKB in kisspeptin neurons may be physiologically relevant since both of
these neuropeptides can inhibit LH secretion in
rats (53, 77, 97, 98), and there is an intimate association between ARC kisspeptin/NKB neurons and
GnRH fibers at the median eminence (56, 89). Kisspeptin/NKB/DYN neurons also form connections
with each other (57), and this has led to the suggestion that signaling between these neuropeptides may coordinate the release of kisspeptin at
GnRH nerve terminals to generate pulsatile release
of GnRH (57, 77) (FIGURE 4). In this scheme, NKB
is proposed to synchronize and stimulate DYN
production in all kisspeptin neurons. DYN would
then reduce NKB secretion by negative feedback,
which would consequently reduce DYN levels to
generate regular pulses of NKB and DYN. Pulsatile
kisspeptin release would be driven by these pulsatile changes in NKB and DYN, which signal
through different G-proteins and could thereby act
to differentially regulate kisspeptin release. Interestingly, Kiss1, NkB, and Dyn all show similar negative regulation in expression by estradiol (77, 91,
111). In support of this hypothesis is the observation that infusion of a selective kisspeptin antagonist into the ARC nucleus can reduce LH pulse
frequency but not pulse amplitude in female rats
(65). Whatever function NKB signaling has in coordinating pulsatile kisspeptin and GnRH release,
it seems to be less physiologically important in
mice since Nk3r knockout mice are not reported to
show any reproductive defects (108). This is in
contrast to loss-of-function mutations in NKB or
NK3R in humans, which are associated with reproductive failure (28, 35, 121).
The way in which NKB acts on GnRH neurons is
not clear. In rats, GnRH neurons express the
FIGURE 3. Cellular action of kisspeptins on GnRH neurons
Kisspeptin binding to its receptor, GPR54/KISS1R, activates the G-protein Gq/11 and phospholipase C to cleave 4,5-bisphosphate (PIP2) into
inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 causes intracellular Ca2⫹ release from the endoplasmic reticulum, which activates protein kinase C and a kinase phosphorylation cascade (RAF, MEK1/2,
ERK1/2). GnRH depolarization is caused by activation of a nonselective
cation channel (TRPC) and inhibition of an inwardly rectifying potassium
channel (Kir) by DAG.
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on both GnRH and kisspeptin neurons in rodents
but only an indirect action via kisspeptin neurons
in sheep. NK3R and GPR54/KISS1R both signal via
Gq/11, so they may be expected to show synergistic
activities. Indeed, central administration of NKB
and Kp10 produced greater LH secretion in male
mice than with Kp10 administration alone (17).
Kisspeptin signaling is also involved in regulating the reproductive cycle of species that show
seasonal breeding. Several species have seasonal
breeding patterns that coordinate time of birth
with optimal environmental conditions. A reduction in kisspeptin expression during the nonbreeding (anoestrous) season has been found in sheep
(110) and hamsters (33, 92). Administration of
FIGURE 4. Interaction of kisspeptin neurons with GnRH neurons in rodents
Kisspeptin neurons are found mainly in the anteroventral periventricular (AVPV) and arcuate (ARC) regions of the rodent hypothalamus. Gonadotrophin releasing hormone (GnRH) neurons are located in the preoptic area (POA) and send fibers to the median eminence (ME). Kisspeptin neurons in
the AVPV are sexually dimorphic with greater numbers in females and are thought to regulate the preovulatory GnRH/LH surge. Kisspeptin neurons
in the ARC co-express neurokinin B (NKB) and dynorphin (DYN). The kisspeptin receptor (GPR54/KISS1R) and the neurokinin B receptor (NK3R) are
both expressed at GnRH nerve terminals. The kisspeptin neurons from the ARC form close appositions with GnRH terminals in the inner zone of the
median eminence and also project to each other. NK3R and GPR54/KISS1R are coupled to Gq/11 whereas the DYN receptor (KOR) is coupled to a
Gi/o. Inset: dynorphin and neurokinin B signaling between kisspeptin neurons may be involved in synchronizing kisspeptin release to generate GnRH
pulsatility. ⫹, Stimulatory activities; ⫺, inhibitory activities. NKB and DYN receptors signal through different G-proteins, which may allow differential
regulation of kisspeptin release.
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neurokinin B receptor (NK3R) on axons in the median eminence but not at the GnRH cell bodies
(56). In contrast, NK3R expression has not been
found in GnRH neurons of ewes. Although GnRH
fibers in the median eminence of rats express the
NK3R, the physiological action of NKB on GnRH
neurons is not clear. Some groups have reported
inhibition of LH secretion after central administration of the neurokinin B agonist senktide in the rat
(77, 97), whereas others have observed little effect
in mice (17). In ewes, senktide stimulates LH release during the follicular phase but not the luteal
phase of the oestrus cycle (68). These disparities
may reflect the difference in NK3R expression between species, with senktide having a dual action
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Kisspeptin Agonists
and Antagonists
Kisspeptin analogs with agonistic or antagonistic
activities could be useful for the treatment of
clinical disorders such as infertility (48), premature
or delayed puberty, and prostatic or metastatic
cancers. Analogs need to be tested in vivo since
receptor binding affinities in cell culture do not
always predict biological potency in a living animal. For example, all kisspeptin family members
bind to GPR54/KISS1R with similar affinities (55),
but Kp54 is more potent than Kp10 in rodents after
peripheral injection (70, 83, 119), probably due to
differences in biostability. Thus development of
Kp10 analogs with greater in vivo stability than
Kp10 may help in the development of therapeutic
products. A Kp10 analog in which the aminoterminal tyrosine (Y) is replaced with an enantiomeric tyrosine (dYNWNSFGLRF-NH2, [dY]1Kp10)
has similar receptor binding and signaling activity
to Kp10 but shows more potent effects in vivo (18).
Peripheral administration of [dY]1Kp10 increased
plasma LH and testosterone levels in male mice
more potently than Kp10 (18), possibly due to reduced degradation of the analog.
The development of kisspeptin antagonists
should also allow an assessment of the action of
kisspeptin on specific parts of the hypothalamus or
at defined times during the estrous cycle or pregnancy. These temporo-spatial studies are not possible in the Gpr54/Kiss1r or Kiss1 knockout mice,
which have congenital absence of kisspeptin signaling in all tissues. Roseweir and colleagues (96)
generated a Kp10 antagonist (dANWNGFGdWRF,
peptide 234) that has two D-amino acid substitutions and a Ser to Gly change at position 5. In a
comprehensive analysis, peptide 234 was shown to
potently inhibit Kp10 signaling in CHO cells expressing GPR54/KISS1R and decrease Kp10stimulated GnRH neuron firing in brain slice
preparations. In vivo, central delivery of peptide
234 inhibited pulsatile GnRH release in pubertal
female rhesus monkeys and pulsatile LH secretion
in ovariectomized ewes. In rodents, peptide 234
reduced the postcastration rise in LH and attenuated Kp10-stimulated LH secretion (96). Continuous central infusion of peptide 234 delayed
puberty in female rats and prevented the preovulatory LH surge in sexual mature rats (86). Peptide
234 is probably acting as a competitive inhibitor
with better biostability than Kp10. Inhibition effects are typically observed when peptide 234 is
used at a ⫻10 –1,000 molar excess over Kp10.
For maximum utility as a therapeutic agent,
kisspeptin analogs should be capable of crossing
the blood-brain barrier after peripheral administration. Whether peptide 234 can cross the bloodbrain barrier has not been completely established.
Systemic injection of peptide 234 fused with penetratin, a cationic cell-penetrating peptide, can inhibit LH and FSH release in response to central
injection of Kp10 (86). This does not prove that the
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kisspeptins during the nonbreeding season can stimulate the reproductive axis and induce testicular
growth in hamsters (92) and ovulation in ewes
(4). Signals controlling these seasonal changes in
kisspeptin expression may include photoperiod acting via melatonin (8, 92) and food restriction (82).
Kisspeptins also play a role in integrating signals
about metabolic status to the reproductive axis.
Low body weight associated with undernutrition
can suppress the reproductive axis, and food restriction has been shown to decrease kisspeptin
expression in adult rodents (5, 67). Undernutrition
during fetal development may also affect kisspeptin expression and influence reproductive function
in adulthood. Prenatal undernutrition in rats results in reduced Kiss1 expression at postnatal day
16 and delayed vaginal opening, which can be reversed by chronic central injection of kisspeptin
(47). Mice that are deficient in leptin (Ob/Ob) have
reduced kisspeptin expression compared with
wild-type, which is reversed by leptin infusion (67).
Similarly, central administration of leptin normalizes the low kisspeptin levels found in an experimentally induced diabetic rat model (6, 7). Around
40% of kisspeptin neurons in the ARC co-express
the leptin receptor (109), suggesting that kisspeptin
neurons monitor peripheral fat reserves via leptin to
modulate the reproductive axis under conditions of
negative energy balance. Modulation of kisspeptin
expression by leptin may be mediated by the mammalian target of rapamycin (mTOR) protein since
mTOR activation stimulates the reproductive axis
and inactivation reduces kisspeptin expression in the
ARC (93). Melanin-concentrating hormone (MCH)
may also provide a link between energy status and
reproduction since this hormone is upregulated during fasting and can inhibit the action of kisspeptin on
some GnRH neurons (122).
Other factors that influence the reproductive
axis may do so via kisspeptin neurons. Melanocortins, which stimulate LH release in several mammalian species, have been shown to increased Kiss1
expression in the dorsolateral POA after infusion of
an agonist into the lateral ventricle of luteal stage
ewes (2). Short-term exposure to alcohol has also
been shown to reduce Kiss1 expression in the AVPV
and ARC nuclei (115). These affects of alcohol may
be mediated by reduced insulin-like growth factor
1 (IGF-1) signaling since alcohol reduces IGF-1
levels in the bloodstream (115) and IGF-1 can stimulate Kiss1 expression in the AVPV region of prepubertal female rats (42, 43).
213
REVIEWS
fusion protein can cross the blood-brain barrier,
however, since inhibition may be at the level of the
median eminence where the centrally injected
Kp10 may still act, and it was not reported whether
the same effects were found with peptide 234 lacking penetratin.
Summary and
Unanswered Questions
No conflicts of interest, financial or otherwise, are declared by the author(s).
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Kisspeptin signaling through the G-protein-coupled receptor, GPR54/KISS1R, is crucial for initiation of puberty and maintenance of mammalian
fertility. Kisspeptin neurons act as central integrators of external and physiological signals within the
hypothalamus. They are potent stimulators of
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females. There are still unresolved questions, however, such as which molecules act upstream of
Kiss1 to regulate expression and what the mechanism is by which the two populations of kisspeptin
neurons (AVPV and ARC) are differentially regulated by sex steroids. In addition, how does NKB/
DYN signaling regulate the activity of ARC
kisspeptin neurons and what other neuropeptides
may be involved? Why do NK3R mutations cause
infertility in humans but not in mice? Also, is it true
that the AVPV neurons are solely responsible for
the GnRH/LH surge in females, whereas the ARC
neurons act at GnRH nerve terminals to regulate
tonic pulsatile GnRH/LH release? Finally, what role
does GPR54/KISS1R oligomerization or interaction
with other membrane receptors have in modulating the activity of kisspeptins on GnRH neurons?
The answers to these questions should provide us
with important knowledge about the central mechanisms controlling the mammalian reproductive
axis. 䡲
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