The Role of Kisspeptin Signaling in Reproduction Role of Kisspeptin Signaling in Reproduction ... 73...

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Xavier d'Anglemont de Tassigny and William Henry ColledgeThe Role of Kisspeptin Signaling in Reproduction

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The Role of Kisspeptin Signalingin Reproduction

Kisspeptins are a group of peptides that stimulate GnRH release and are

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.

Xavier d’Anglemont de Tassigny andWilliam Henry Colledge

Department of Physiology, Development and Neuroscience,Reproductive Physiology Group, University of Cambridge,

Cambridge, United [email protected]

Acquisition of reproductive competency is essen-tial for continuation of all species. Fertility inmammals is initiated at puberty by the pulsatilesecretion of gonadotrophin releasing hormone(GnRH) from a small number of neurons in thehypothalamus (FIGURE 1). The GnRH is releasedinto the hypophyseal portal blood system fromnerve terminals in the palisade layer of the medianeminence of the hypothalamus. The GnRH acts onthe anterior pituitary to stimulate the release of thegonadotrophic hormones, luteinizing hormone(LH), and follicle stimulating hormone (FSH). Thegonadotrophic hormones act on the gonads tostimulate sexual maturation and gametogenesis(spermatogenesis in males and oogenesis in fe-males). The gonads produce sex steroids (testos-terone in males and estrogen and progesterone infemales), which are required for gametogenesis, formaturation of accessory sex organs, and to providehormonal feedback loops that regulate GnRH andgonadotrophic hormone release under differentphysiological conditions (FIGURE 1). AlthoughGnRH neurons are a critical component of thereproductive axis, kisspeptin (Kp) peptides havebeen identified recently as vital upstream regula-tors that integrate central and peripheral signalswith GnRH release, thereby playing a pivotal rolein the control of reproduction (FIGURE 1).

Kisspeptin Expression Profile

Kisspeptins are an overlapping set of amidatedpeptides encoded by the Kiss1 gene, which is lo-cated in close proximity to the Golt1a (golgi trans-port 1 homolog A) gene in most species. In humansand mice, the Kiss1 gene consists of two codingexons downstream from at least one noncodingexon (60) (FIGURE 2). The human promoter hasbeen mapped immediately upstream of the non-coding exon and contains a TATA box and severalpotential SP1 transcription factor binding sites.These SP1 binding sites facilitate binding of AP-1�/SP1 and DRIP130/SP1 transcriptional activatorcomplexes and contribute to basal promoter activ-ity (71, 72). Two SP1 binding sites closest to the

transcriptional start site function together to facil-itate transcriptional activation by estrogen (63). Inmice, an analogous region also containing a TATAbox is located just upstream of the noncoding exonbut this has not been assessed for promoter activ-ity. Several alternatively spliced Kiss1 transcriptshave been identified in mice that contain se-quences from the first exon of the Golt1a gene(NCBI transcript accession numbers: AY707856AY707857 AY707859). Thus Kiss1 transcripts in ro-dents may be generated from both a Kiss1 pro-moter (P1) and the Golt1a promoter (P2). Thephysiological significance of this is not known butmay allow an additional level of gene regulation inmice.

Kisspeptins were originally isolated from humanplacenta (55, 78) and are derived from a largerprecursor protein (FIGURE 2). The longest kisspep-tin is 54 amino acids in length (Kp54), but shorterkisspeptins (Kp13, Kp14) have also been isolatedcorresponding to the carboxy terminus of Kp54.These shorter kisspeptins may represent degrada-tion products, but they still retain full biologicalactivity, as does a synthetic peptide of only 10amino acids (Kp10). Kp14 is highly conserved be-tween species, with the final 10 amino acids show-ing very little variation, particularly in mammals(Table 1). A series of alanine substitutions in Kp10have shown that amino acids 6 (F) and 10 (F or Y,depending on the species) are the most critical forreceptor binding (18, 36, 79). NMR structural mod-eling indicates that the COOH-terminal 7 aminoacids form a helicoid structure that is disrupted bythese alanine substitutions (36, 79).

The distribution of kisspeptin neurons in thehypothalamus varies between species (16). In therodent, in situ hybridization and immunohisto-chemistry have been used to map kisspeptin neu-rons to two discrete regions of the hypothalamus, thearcuate (ARC) nucleus and in a periventricular con-tinuum of cells within the rostral part of the thirdventricle, including the anteroventral periventricularnucleus (AVPV) (12, 13, 15, 31) (FIGURE 1). Humans(95), rhesus monkeys (102), and sheep (26, 110) haveproportionally more kisspeptin neurons in the ARC

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than in the AVPV region. Kisspeptin expression in-creases in the ARC and AVPV regions during pubertaldevelopment in rodents (15, 74, 117) and monkeys(102), consistent with kisspeptin signaling initiatingpuberty. Kisspeptin fibers have been found in thepreoptic area (POA) of adult female rats (54) andmice (15) in close association with GnRH neuroncell bodies. These associations are probably fromAVPV kisspeptin neurons and are proposed tomodulate the preovulatory GnRH/LH surge in fe-males (34). Kisspeptin immunoreactive fibers orig-inating from cell bodies in the ARC make closeapposition to GnRH axons in the median eminenceof the monkey (89) and are proposed to modulatethe pulsatile release of GnRH (FIGURE 1).

In some species, anatomical differences in thehypothalamus are found between the sexes withsome nuclei showing a neuronal density difference(105). In rodents, the AVPV region is sexually di-morphic (106, 107) with a greater number ofkisspeptin neurons in females compared with

males (15, 50). This sexual dimorphism is causedby neonatal exposure to testosterone, which is ar-omatized into estrogen and causes defeminizationof the AVPV region. Neonatal female rats treatedwith androgens (50) or estradiol (44) develop veryfew kisspeptin neurons in the AVPV, whereas cas-tration of neonatal male rats increases the numberof kisspeptin neurons in the AVPV (44). The rolethat estrogens play in this process has been exam-ined by using transgenic mice lacking alpha-feto-protein (Afp�/�), which protects the fetal brainfrom the defeminizing action of circulating estro-gens. As expected, female Afp�/� mice developedmale-like numbers of kisspeptin neurons in theAVPV (29). Gpr54�/� male mice have a feminizedAVPV region with a greater number of kisspeptinneurons than wild-type males and similar to that offemales (51). These data suggest that kisspeptinsignaling during the neonatal period is required fortestosterone production in males to defeminize theAVPV. The AVPV region also contains a sexually

FIGURE 1. The mammalian hypothalamic pituitary gonadal axisAt puberty, pulsatile secretion of gonadotrophic releasing hormone (GnRH) stimulates the anterior pituitary to re-lease the gonadotrophic hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). These act onthe gonads to promote gamete formation and the production of gonadal steroid hormones, which form feedbackloops to regulate GnRH, LH, and FSH release. Kisspeptin (Kiss1) neurons act as a principal relay for steroid feedbackon GnRH secretion. In females, high levels of estrogens and progesterone stimulate kisspeptin neurons of the AVPVto induce the preovulatory surge of GnRH/LH, whereas they inhibit KISS1 expression in the arcuate nucleus (ARC). Inthe male, GnRH and gonadotrophic hormone release are negatively regulated by circulating testosterone, partlythrough the activity of kisspeptin neurons of the ARC. POA, preoptic area; AVPV, anteroventral periventricular nu-cleus; ME, median eminence.

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dimorphic population of tyrosine hydroxylase (TH)neurons (105–107), but there is little overlap be-tween these and the kisspeptin neurons (50). Incontrast, the ARC does not display sexually dimor-phic differences in kisspeptin neuron density ordistribution in rodents (44, 50). In sheep, however,the ARC is sexually dimorphic with half the num-ber of kisspeptin neurons in rams compared withewes (10). These sexually dimorphic kisspeptinneurons co-express dynorphin (DYN) and neuro-kinin B (NKB). Prenatal testosterone treatment offemales, however, reduces DYN and NKB expres-sion but not kisspeptin expression, suggesting dif-ferent critical periods for sexual differentiation ofthese genes (10).

Kisspeptins and theReproductive Axis

The critical role that kisspeptins play in regulatingthe reproductive axis is illustrated by the conse-quences of mutations in the kisspeptin signalingpathway in mice and humans. Mice with disrup-tion of the kisspeptin receptor gene (Gpr54/Kiss1r)(24, 27, 51, 60, 100) or Kiss1 (20, 27) do not undergopubertal development, and both sexes are infertile,with poor gonadal growth and impaired gameto-genesis. Male mice have delayed spermatogenesisand produce low numbers of spermatozoa. Femalemice do not show a normal estrus cycle, fail toovulate, and do not have corpora lutea in theirovaries. This phenotype is caused by low levels ofgonadotrophic hormones (LH, FSH) and gonadalsex steroids in the bloodstream (20, 24, 51, 60, 100).Similarly, loss-of-function mutations in GPR54/KISS1R in humans cause hypogonadotrophic hy-pogonadism (21, 59, 100, 101), whereas gain-of-function mutations cause precocious puberty(118). Moreover, a missense mutation in thekisspeptin precursor protein has been found in aBrazilian boy with central precocious puberty(104). The mutation reduces serum protease deg-radation of the protein and may therefore be asso-ciated with increased kisspeptin signaling.

These reproductive defects can be directly attrib-uted to the action of kisspeptins in the hypothala-mus. Central or peripheral injection of kisspeptinstimulates gonadotrophin secretion in most spe-cies, 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 gonado-trophin secretion is mediated by kisspeptin-stim-ulated GnRH release from hypothalamic neurons.Consistent with this, the majority of GnRH neuronsexpress the kisspeptin receptor (GPR54/KISS1R),as shown by in situ hybridization or tagged expres-sion of a LacZ reporter gene (37, 40, 46, 69, 81).Kisspeptin-mediated GnRH release has been

demonstrated directly in ewes (69) and femalerhesus monkeys (52) and by inhibition of kisspeptinresponses in rodents by administration of GnRH an-tagonists (46). In addition, mice with a disruptedGpr54/Kiss1r gene cannot secrete GnRH from hypo-thalamic fragments after kisspeptin stimulation (19).

Sex steroids provide feedback loops that allowthe gonads to communicate with the hypothala-mus to regulate GnRH release (FIGURE 1). Sex ste-roids achieve this indirectly, however, since GnRHneurons do not express androgen or estrogen (ER�

receptors) (41, 45). It is now thought that kisspep-tin neurons mediate the actions of sex steroids onGnRH neurons. The majority of kisspeptin neuronsexpress estrogen receptor alpha (ER� of �90%) (26,111, 112), the androgen receptor (�65%) (112), andthe progesterone receptor (�86%) (110), consistentwith their role as mediators of sex steroid feedbackon the reproductive axis. In rodents, sex steroids

FIGURE 2. Generation of kisspeptins from the Kiss1 geneThe kisspeptin coding region (pale blue) is located within two exons ofthe Kiss1 gene. At least one upstream noncoding exon (white) hasbeen identified and the promoter (P1) in humans had been mappedimmediately upstream of this exon. The exact location of the mouseKiss1 promoter remains to be determined since Kiss1 transcripts fusedto Golt1a sequences have been found, suggesting some expressionfrom a second promoter (P2) in this species. The primary translationproduct is a 138- to 145-amino acid protein (Kp145), depending on thespecies, which contains a secretory signal sequence (dark green). Pro-teolytic cleavage generates a 54-amino acid amidated peptide (Kp54also known as metastin). In the placenta, shorter kisspeptins have beenisolated (Kp14 and Kp13), which may be degradation products fromKp54. The shorter kisspeptins all contain the same 10 amino acids (yel-low). A synthetic peptide containing only these 10 amino acids (Kp10)retains biological activity in vivo.

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differentially regulate kisspeptin expression to de-crease expression in the AVPV region (50, 111) andincrease expression in the ARC (111). Thesechanges are reversed by either testosterone or es-tradiol replacement (111, 112). Studies in otherspecies have corroborated the effects of sex ste-roids on kisspeptin expression (46, 74, 95, 103,110). In the sheep, the ARC kisspeptin neuronsmediate both the negative feedback action of pro-gesterone on GnRH release during the luteal phase(11) and the positive feedback action of estradiolexpression at the time of ovulation (25, 113). Themechanism by which sex steroids differentiallyregulate kisspeptin expression in the AVPV andARC regions had not been resolved, but estradiolmediates its feedback effects through both estro-gen response element (ERE)-dependent and ERE-independent signaling in rodents. This was shownby examining the effects of estradiol on Kiss1 ex-pression in mutant mice with ER� molecules thatcannot bind to ERE sequences (32). Stimulation ofKiss1 expression by estradiol in the AVPV requiredan ERE-dependent pathway. In contrast, inhibitionof Kiss1 expression by estradiol in the ARC wasERE-independent (32). In addition, insulin-likegrowth factor-1 (IGF-1) increases Kiss1 expressionin the AVPV but not the ARC of prepubertal femalerats in the presence of estradiol (42, 43). Theseexpression changes are consistent with kisspeptinneurons in the ARC regulating the negative feed-back effect of sex steroids on GnRH release andthose in the AVPV/PeN regions responsible for thepreovulatory GnRH/LH surge (39) (see FIGURE 4).

Kisspeptin action onGnRH neurons

Kisspeptin signaling through GPR54/KISS1R cou-ples to Gq/11 to activate phospholipase C and

increases inositol triphosphate (IP3) and diacylglyc-erol (DAG) levels in the cell (55, 73, 116) (FIGURE 3).The subsequent increase in intracellular Ca2� andDAG activates protein kinase C and initiates a ki-nase phosphorylation cascade resulting in phos-phorylation of ERK1/2. DAG also stimulates GnRHdepolarization by activation of a nonselective cat-ion channel (TRPC) and inhibition of an inwardlyrectifying potassium channel (Kir) (66, 84). Thuskisspeptins act directly on GnRH neurons to in-duce a sustained depolarization event and increasethe rate of action potential firing (37, 88). The num-ber of GnRH neurons that can respond to kisspeptinsincreases during puberty even though the expressionlevels only change slightly, suggesting posttransla-tional maturation of GPR54/KISS1R function withpuberty (37). In adult mice, �-amino-butyric acid(GABA) has a predominantly hyperpolarizing effect onGnRH neurons to inhibit GnRH release (14). Kisspep-tins have been reported to suppress the inhibitoryeffects of GABA(B) receptor signaling in GnRH neu-rons (123). Thus, under conditions of estrogen posi-tive feedback when kisspeptin levels increase in theAVPV, kisspeptins will antagonize any inhibitory ac-tion of GABA to stimulate GnRH release and LHsurge/ovulation.

After initial stimulation, continuous administra-tion of kisspeptin suppresses the reproductive axis.Continuous delivery of human Kp10 inhibited fur-ther LH release in agonadal monkeys within 24 h(90, 99). Similarly, sustained kisspeptin activity inwomen failed to elicit LH or FSH release by day 14(49). In contrast, pulsatile delivery of kisspeptin inagonadal juvenile monkeys did not suppress gona-dotropic hormone release (87). The suppressiveeffects are probably caused by desensitization ofGPR54/KISS1R rather than the GnRH receptorsince all individuals remained responsive to GnRH.Desensitization of GPR54/KISS1R signaling is me-diated by G-protein-coupled receptor serine/threonine kinase 2 (GRK2) and �-arrestin in a cellculture model (80). GPR54/KISS1R also displaysconstitutive basal activity at �5% of the maximumactivity after kisspeptin stimulation (80). This basalactivity 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 neu-rons in the hypothalamus that express GPR54/KISS1R (40, 61, 73). To distinguish between directand indirect action of kisspeptins on GnRH neu-rons, electrophysiological responses are usuallymeasured in the presence of inhibitors of trans-synaptic input from other neurons. Under theseconditions, part of the GnRH response to kisspep-tins is mediated by trans-synaptic input fromGABAergic and glutamatergic neurons, which was

Table 1. Kp14 species comparison

Species Sequence

Human DLPNYNWNSFGLRF-NH2

Chimp DLPNYNWNSFGLRF-NH2

Rhesus DLPNYNWNSFGLRF-NH2

Orangutan DLPNYNWNSFGLRF-NH2

Cat DLSAYNWNSFGLRY-NH2

Horse LLPAYRWNSFGLRY-NH2

Cow DVSAYNWNSFGLRY-NH2

Sheep DVSAYNWNSFGLRY-NH2

Mouse DLSTYNWNSFGLRY-NH2

Rat DMSAYNWNSFGLRY-NH2

Dolphin DLSAYNWNSFGLRY-NH2

Zebrafish NVAYYNLNSFGLRY-NH2

Kp14 is highly conserved between many species. Theleucine2 (L) to valine (V) or methionine (M) are con-served amino-acid substitutions (58). Differences fromthe human sequence are shown in italicized bold.

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enhanced by estradiol (84, 85). Moreover, kisspep-tin increased inhibitory GABAergic input to GnRHneurons during the estradiol-mediated negativefeedback part of the estrous cycle (84, 85).

Physiological Regulation ofKisspeptin Signaling

Mammalian ovulation requires an LH surgebrought about by the positive feedback action ofestradiol on GnRH release. Kisspeptin expressionincreases in the AVPV region just before ovulationand during a steroid-induced LH surge in ovariec-tomized rats (114). At the time of ovulation,kisspeptin neurons in the AVPV are activated asindicated by induction of c-fos (13). Inhibition ofkisspeptin action in the POA by local injection of amonoclonal antibody abolishes the proestrous LHsurge and inhibits estrous cyclicity in rats (1, 54). Inthe absence of either GPR54/KISS1R or kisspeptinexpression in transgenic female mice, the sex ste-roid-induced GnRH/LH surge does not occur com-pared with wild-type siblings (13). In rodents, tissueablation studies have shown that the LH surge is alsogated by a circadian oscillator in the suprachiasmaticnucleus (SCN) to ensure that the surge occurs near theonset of darkness. Kisspeptin expression and c-fosneuronal activation in the AVPV region are alsosubject to circadian regulation. This was demon-strated in ovariectomized mice that were main-tained in constant darkness and still showed anestradiol-induced LH surge (94). Thus kisspeptinsignaling, regulated by both estradiol and circa-dian signals, is essential for the preovulatoryGnRH/LH surge.

Kisspeptin signaling may also play a role in mod-ulating the pulsatile release of GnRH. The mecha-nism by which this occurs is not fully understood,but recent information about the gene expressionprofile of kisspeptin neurons has provided impor-tant clues. In sheep and mice, the majority ofkisspeptin neurons in the ARC co-express dynor-phin A (DYN) and neurokinin B (NKB) (30, 77)(FIGURE 4). Of mouse ARC kisspeptin neurons,96% also express the neurokinin B receptor geneNk3r, but only 20% of kisspeptin neurons expressthe dynorphin receptor gene Kor. This may be anunderestimate, however, since others have shownby immunohistochemistry that practically all NKB-positive neurons in the rat ARC co-express DYN(3). In contrast, kisspeptin neurons in the AVPVregions of the female mouse hypothalamus showlimited expression of Dyn (33%) and NkB (10%).Co-expression of DYN and NKB in kisspeptin neu-rons may be physiologically relevant since both ofthese neuropeptides can inhibit LH secretion inrats (53, 77, 97, 98), and there is an intimate asso-ciation between ARC kisspeptin/NKB neurons and

GnRH fibers at the median eminence (56, 89). Kis-speptin/NKB/DYN neurons also form connectionswith each other (57), and this has led to the sug-gestion that signaling between these neuropep-tides may coordinate the release of kisspeptin atGnRH nerve terminals to generate pulsatile releaseof GnRH (57, 77) (FIGURE 4). In this scheme, NKBis proposed to synchronize and stimulate DYNproduction in all kisspeptin neurons. DYN wouldthen reduce NKB secretion by negative feedback,which would consequently reduce DYN levels togenerate regular pulses of NKB and DYN. Pulsatilekisspeptin release would be driven by these pulsa-tile changes in NKB and DYN, which signalthrough different G-proteins and could thereby actto differentially regulate kisspeptin release. Inter-estingly, Kiss1, NkB, and Dyn all show similar neg-ative regulation in expression by estradiol (77, 91,111). In support of this hypothesis is the observa-tion that infusion of a selective kisspeptin antago-nist into the ARC nucleus can reduce LH pulsefrequency but not pulse amplitude in female rats(65). Whatever function NKB signaling has in co-ordinating pulsatile kisspeptin and GnRH release,it seems to be less physiologically important inmice since Nk3r knockout mice are not reported toshow any reproductive defects (108). This is incontrast to loss-of-function mutations in NKB orNK3R in humans, which are associated with repro-ductive failure (28, 35, 121).

The way in which NKB acts on GnRH neurons isnot clear. In rats, GnRH neurons express the

FIGURE 3. Cellular action of kisspeptins on GnRH neuronsKisspeptin binding to its receptor, GPR54/KISS1R, activates the G-pro-tein Gq/11 and phospholipase C to cleave 4,5-bisphosphate (PIP2) intoinositol triphosphate (IP3) and diacylglycerol (DAG). IP3 causes intracel-lular Ca2� release from the endoplasmic reticulum, which activates pro-tein kinase C and a kinase phosphorylation cascade (RAF, MEK1/2,ERK1/2). GnRH depolarization is caused by activation of a nonselectivecation channel (TRPC) and inhibition of an inwardly rectifying potassiumchannel (Kir) by DAG.

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neurokinin B receptor (NK3R) on axons in the me-dian eminence but not at the GnRH cell bodies(56). In contrast, NK3R expression has not beenfound in GnRH neurons of ewes. Although GnRHfibers in the median eminence of rats express theNK3R, the physiological action of NKB on GnRHneurons is not clear. Some groups have reportedinhibition of LH secretion after central administra-tion of the neurokinin B agonist senktide in the rat(77, 97), whereas others have observed little effectin mice (17). In ewes, senktide stimulates LH re-lease during the follicular phase but not the lutealphase of the oestrus cycle (68). These disparitiesmay reflect the difference in NK3R expression be-tween species, with senktide having a dual action

on both GnRH and kisspeptin neurons in rodentsbut only an indirect action via kisspeptin neuronsin sheep. NK3R and GPR54/KISS1R both signal viaGq/11, so they may be expected to show synergisticactivities. Indeed, central administration of NKBand Kp10 produced greater LH secretion in malemice than with Kp10 administration alone (17).

Kisspeptin signaling is also involved in regulat-ing the reproductive cycle of species that showseasonal breeding. Several species have seasonalbreeding patterns that coordinate time of birthwith optimal environmental conditions. A reduc-tion in kisspeptin expression during the nonbreed-ing (anoestrous) season has been found in sheep(110) and hamsters (33, 92). Administration of

FIGURE 4. Interaction of kisspeptin neurons with GnRH neurons in rodentsKisspeptin neurons are found mainly in the anteroventral periventricular (AVPV) and arcuate (ARC) regions of the rodent hypothalamus. Gonadotro-phin releasing hormone (GnRH) neurons are located in the preoptic area (POA) and send fibers to the median eminence (ME). Kisspeptin neurons inthe AVPV are sexually dimorphic with greater numbers in females and are thought to regulate the preovulatory GnRH/LH surge. Kisspeptin neuronsin the ARC co-express neurokinin B (NKB) and dynorphin (DYN). The kisspeptin receptor (GPR54/KISS1R) and the neurokinin B receptor (NK3R) areboth expressed at GnRH nerve terminals. The kisspeptin neurons from the ARC form close appositions with GnRH terminals in the inner zone of themedian eminence and also project to each other. NK3R and GPR54/KISS1R are coupled to Gq/11 whereas the DYN receptor (KOR) is coupled to aGi/o. Inset: dynorphin and neurokinin B signaling between kisspeptin neurons may be involved in synchronizing kisspeptin release to generate GnRHpulsatility. �, Stimulatory activities; �, inhibitory activities. NKB and DYN receptors signal through different G-proteins, which may allow differentialregulation of kisspeptin release.

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kisspeptins during the nonbreeding season can stim-ulate the reproductive axis and induce testiculargrowth in hamsters (92) and ovulation in ewes(4). Signals controlling these seasonal changes inkisspeptin expression may include photoperiod act-ing via melatonin (8, 92) and food restriction (82).

Kisspeptins also play a role in integrating signalsabout metabolic status to the reproductive axis.Low body weight associated with undernutritioncan suppress the reproductive axis, and food re-striction has been shown to decrease kisspeptinexpression in adult rodents (5, 67). Undernutritionduring fetal development may also affect kisspep-tin expression and influence reproductive functionin adulthood. Prenatal undernutrition in rats re-sults in reduced Kiss1 expression at postnatal day16 and delayed vaginal opening, which can be re-versed by chronic central injection of kisspeptin(47). Mice that are deficient in leptin (Ob/Ob) havereduced kisspeptin expression compared withwild-type, which is reversed by leptin infusion (67).Similarly, central administration of leptin normal-izes the low kisspeptin levels found in an experi-mentally induced diabetic rat model (6, 7). Around40% of kisspeptin neurons in the ARC co-expressthe leptin receptor (109), suggesting that kisspeptinneurons monitor peripheral fat reserves via leptin tomodulate the reproductive axis under conditions ofnegative energy balance. Modulation of kisspeptinexpression by leptin may be mediated by the mam-malian target of rapamycin (mTOR) protein sincemTOR activation stimulates the reproductive axisand inactivation reduces kisspeptin expression in theARC (93). Melanin-concentrating hormone (MCH)may also provide a link between energy status andreproduction since this hormone is upregulated dur-ing fasting and can inhibit the action of kisspeptin onsome GnRH neurons (122).

Other factors that influence the reproductiveaxis may do so via kisspeptin neurons. Melanocort-ins, which stimulate LH release in several mamma-lian species, have been shown to increased Kiss1expression in the dorsolateral POA after infusion ofan agonist into the lateral ventricle of luteal stageewes (2). Short-term exposure to alcohol has alsobeen shown to reduce Kiss1 expression in the AVPVand ARC nuclei (115). These affects of alcohol maybe mediated by reduced insulin-like growth factor1 (IGF-1) signaling since alcohol reduces IGF-1levels in the bloodstream (115) and IGF-1 can stim-ulate Kiss1 expression in the AVPV region of pre-pubertal female rats (42, 43).

Kisspeptin Agonistsand Antagonists

Kisspeptin analogs with agonistic or antagonisticactivities could be useful for the treatment of

clinical disorders such as infertility (48), prematureor delayed puberty, and prostatic or metastaticcancers. Analogs need to be tested in vivo sincereceptor binding affinities in cell culture do notalways predict biological potency in a living ani-mal. For example, all kisspeptin family membersbind to GPR54/KISS1R with similar affinities (55),but Kp54 is more potent than Kp10 in rodents afterperipheral injection (70, 83, 119), probably due todifferences in biostability. Thus development ofKp10 analogs with greater in vivo stability thanKp10 may help in the development of therapeuticproducts. A Kp10 analog in which the amino-terminal tyrosine (Y) is replaced with an enantio-meric tyrosine (dYNWNSFGLRF-NH2, [dY]1Kp10)has similar receptor binding and signaling activityto Kp10 but shows more potent effects in vivo (18).Peripheral administration of [dY]1Kp10 increasedplasma LH and testosterone levels in male micemore potently than Kp10 (18), possibly due to re-duced degradation of the analog.

The development of kisspeptin antagonistsshould also allow an assessment of the action ofkisspeptin on specific parts of the hypothalamus orat defined times during the estrous cycle or preg-nancy. These temporo-spatial studies are not pos-sible in the Gpr54/Kiss1r or Kiss1 knockout mice,which have congenital absence of kisspeptin sig-naling in all tissues. Roseweir and colleagues (96)generated a Kp10 antagonist (dANWNGFGdWRF,peptide 234) that has two D-amino acid substitu-tions and a Ser to Gly change at position 5. In acomprehensive analysis, peptide 234 was shown topotently inhibit Kp10 signaling in CHO cells ex-pressing GPR54/KISS1R and decrease Kp10-stimulated GnRH neuron firing in brain slicepreparations. In vivo, central delivery of peptide234 inhibited pulsatile GnRH release in pubertalfemale rhesus monkeys and pulsatile LH secretionin ovariectomized ewes. In rodents, peptide 234reduced the postcastration rise in LH and attenu-ated Kp10-stimulated LH secretion (96). Continu-ous central infusion of peptide 234 delayedpuberty in female rats and prevented the preovu-latory LH surge in sexual mature rats (86). Peptide234 is probably acting as a competitive inhibitorwith better biostability than Kp10. Inhibition ef-fects are typically observed when peptide 234 isused at a �10 –1,000 molar excess over Kp10.

For maximum utility as a therapeutic agent,kisspeptin analogs should be capable of crossingthe blood-brain barrier after peripheral adminis-tration. Whether peptide 234 can cross the blood-brain barrier has not been completely established.Systemic injection of peptide 234 fused with pen-etratin, a cationic cell-penetrating peptide, can in-hibit LH and FSH release in response to centralinjection of Kp10 (86). This does not prove that the

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fusion protein can cross the blood-brain barrier,however, since inhibition may be at the level of themedian eminence where the centrally injectedKp10 may still act, and it was not reported whetherthe same effects were found with peptide 234 lack-ing penetratin.

Summary andUnanswered Questions

Kisspeptin signaling through the G-protein-cou-pled receptor, GPR54/KISS1R, is crucial for initia-tion of puberty and maintenance of mammalianfertility. Kisspeptin neurons act as central integra-tors of external and physiological signals within thehypothalamus. They are potent stimulators ofGnRH release and mediate sex steroid feedback onthe reproductive axis. Kisspeptin neurons in thearcuate region of the hypothalamus regulate thetonic pulsatile release of GnRH, whereas those inthe AVPV generate the preovulatory LH surge infemales. There are still unresolved questions, how-ever, such as which molecules act upstream ofKiss1 to regulate expression and what the mecha-nism is by which the two populations of kisspeptinneurons (AVPV and ARC) are differentially regu-lated by sex steroids. In addition, how does NKB/DYN signaling regulate the activity of ARCkisspeptin neurons and what other neuropeptidesmay be involved? Why do NK3R mutations causeinfertility in humans but not in mice? Also, is it truethat the AVPV neurons are solely responsible forthe GnRH/LH surge in females, whereas the ARCneurons act at GnRH nerve terminals to regulatetonic pulsatile GnRH/LH release? Finally, what roledoes GPR54/KISS1R oligomerization or interactionwith other membrane receptors have in modulat-ing the activity of kisspeptins on GnRH neurons?The answers to these questions should provide uswith important knowledge about the central mech-anisms controlling the mammalian reproductiveaxis. �

No conflicts of interest, financial or otherwise, are de-clared by the author(s).

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Vol. 25, August 2010

d’Anglemont de Tassigny, Colledge WH. The role of kisspeptin signaling in reproduction. Physiology 25: 207–217, 2010; doi:10.1152/physiol.00009.2010.–The last sentence on page 209 and continuing onto page 210 should read, “In rodents, sex steroids differentiallyregulate kisspeptin expression to increase expression in the AVPV region (50, 111) and decrease expression in the ARC (111).”

CORRIGENDUMPHYSIOLOGY 25: 378, 2010; doi:10.1152/physiol.00110.2010

1548-9213/10 ©2010 Int. Union Physiol. Sci./Am. Physiol. Soc.378



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