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Animal Science Journal (2013) 84, 369–381 doi: 10.1111/asj.12056

REV IEW AR TICLE

Kisspeptin as a master player in the central control of
reproduction in mammals: An overview of kisspeptin
research in domestic animals
Hiroaki OKAMURA, Takashi YAMAMURA and Yoshihiro WAKABAYASHI
Animal Physiology Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan

ABSTRACT
The hypothalamo-pituitary-gonadal (HPG) axis is the regulatory system for reproduction in mammals. Because secretion of
gonadotropin-releasing hormone (GnRH) into the portal vessels is the final step at which the brain controls gonadal
activities, the GnRH neuronal system had been thought to be central to the HPG axis. A newly discovered neural peptide,
kisspeptin, has opened a new era in reproductive neuroendocrinology. As shown in a variety of mammals, kisspeptin is a
potent endogenous secretagogue of GnRH, and the kisspeptin neuronal system governs both the pulsatile GnRH secretion
that drives folliculogenesis, spermatogenesis and steroidogenesis, and the GnRH surge that triggers ovulation in females.
The kisspeptin neuronal system is therefore considered a master player in the central control of mammalian reproduction,
and kisspeptin and related substances could therefore be valuable for the development of novel strategies for the
management of fertility in farm animals. To this end, the present review aimed to summarize the current research on
kisspeptin signaling with a focus on domestic animals such as sheep, goats, cattle, pigs and horses.

Key words: domestic animals, GnRH, kisspeptin, neurokinin B, reproduction.

INTRODUCTION resolved. For example, how are the two modes of

A subset of neurons in the hypothalamus synthesizes
gonadotropin-releasing hormone (GnRH) and releases
it into the portal vessels, which is the final mechanism
by which the brain controls gonadal activities. GnRH
acts on the anterior pituitary gland to stimulate the
secretion of the gonadotropins, luteinizing hormone
(LH) and follicle-stimulating hormone (FSH), which
mediate the control of the gonads by the brain. In
response to gonadotropins, the gonad releases steroid
hormones, such as estrogen and progesterone in
females and androgens in males, which in turn feed
back to the hypothalamus to alter GnRH secretion. The
reproductive system is regulated by this circuit, the
so-called hypothalamo-pituitary-gonadal (HPG) axis
(Karsch 1984). There are two modes of GnRH secre-
tion: the pulsatile mode, tonic gonadotropin secretion
that drives folliculogenesis, spermatogenesis and ster-
oidogenesis, and is controlled by negative feedback via
gonadal steroids, and the surge mode, the preovula-
tory gonadotropin discharge that induces ovulation in
females in response to positive feedback via estrogen.
Although the above-described scenario had been
accepted as the central dogma of the control of repro-
duction, several critical issues had remained to be
GnRH secretion generated, and how are steroid
hormone signals transmitted to GnRH neurons that
lack estrogen receptor alpha (ERa) mediating the feed-
back action of estradiol (E2) on GnRH secretion? A
newly discovered peptide, kisspeptin, provides essen-
tial clues to answering these long-lasting questions
and has opened a new era in reproductive endocrinol-
ogy. Here, we describe the history, anatomy and physi-
ology of kisspeptin and the kisspeptin neuronal system
and then offer a brief prospective for the potential
applications of kisspeptin and related substances to
domestic animal production. As a number of excellent
general reviews of kisspeptin have appeared elsewhere
(Maeda et al. 2007; Oakley et al. 2009; Ohkura et al.
2009a; Clarke 2011; Goodman & Lehman 2012; Smith
2012; Caraty et al. 2012a), this review aimed mainly to
summarize the knowledge accumulated in domestic
Correspondence: Hiroaki Okamura, Animal Physiology
Research Unit, National Institute of Agrobiological Sciences,
2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan. (Email:
hokamu@affrc.go.jp)
Received 20 November 2012; accepted for publication 16
January 2013.

© 2013 Japanese Society of Animal Science

370 H. OKAMURA et al.

animals such as sheep, goats, cattle, pigs and horses.
Chickens are not included because it is generally
accepted that kisspeptin signaling is absent in birds.
DISCOVERY OF KISSPEPTIN
The Kiss1 gene was initially isolated as a tumor metas-
tasis gene by a group in Hershey, PA, USA, (Lee et al.
1996), the home of the famous Hershey’s Kiss choco-
lates. Thereafter, the Kiss1 gene product, kisspeptin,
was discovered to be an endogenous ligand for the
G-protein-coupled receptor, GPR54 (Kotani et al.
2001; Ohtaki et al. 2001). In 2003, two groups inde-
pendently found that inactivating mutations of the
kisspeptin receptor gene (GPR54) were associated
with idiopathic hypogonadotropic hypogonadism in
humans (de Roux et al. 2003; Seminara et al. 2003).
This human genetic finding was substantiated experi-
mentally by the generation of GPR54 null mice, which
exhibit reduced gonadal size and low levels of gona-
dotropins and steroid hormones, and fail to undergo
puberty (Funes et al. 2003; Seminara et al. 2003;
Messager et al. 2005). These studies established
kisspeptin/GPR54 signaling as a new player in the HPG
axis and thus led to the intensive and extensive
research on kisspeptin performed over the past decade.
AMINO ACID SEQUENCE
OF KISSPEPTIN
The Kiss1 gene encodes a precursor peptide that is
cleaved to produce the biologically active form of
kisspeptin, which is 52–54 amino acids long in most
mammals (Oakley et al. 2009). As shown in Table 1
(Ohkura et al. 2009b; Tomikawa et al. 2010), the
deduced amino acid sequence of kisspeptin is well
conserved across mammals. For example, the
sequence of goat kisspeptin shows 98%, 91% and
77% sequence identity to ovine, bovine, and porcine
kisspeptin, respectively. In particular, the C-terminal
10 residues, which are the minimal core sequence for
maximal receptor activation (Kotani et al. 2001;
Ohtaki et al. 2001), are identical among the species
listed with the exception that the C-terminal tyrosine
(Y) is replaced by phenylalanine (F) in primates.
The C-terminal decapeptide of kisspeptin, Kp-10, is
commercially available and is routinely used to inves-
tigate the physiological actions of kisspeptin in verte-
brates, including fishes (Kanda et al. 2008). However,
it should be noted that full-length kisspeptin, either rat
type (52 residues; Pheng et al. 2009) or human type
(54 residues; Kinoshita et al. 2005), seems to have
more potent biological activity than Kp-10 in rats.
BIOLOGICAL ACTIONS OF KISSPEPTIN
Since the initial observations in rodents (Gottsch et al.
2004; Matsui et al. 2004), the effects of centrally or
peripherally administered kisspeptin on gonadotropin
secretion have been investigated in a variety of
mammals, including domestic animals. Kisspeptin
consistently stimulates gonadotropin, namely LH,
secretion in all mammalian species examined so far,
although its efficacy seems to depend on the species,
age, sex, steroidal milieu, season and route of admin-
istration involved. Several studies have indicated that
kisspeptin has less effect on FSH secretion than on LH
secretion (Caraty et al. 2007; Lents et al. 2008; Smith
et al. 2009a).
Stimulatory effects of kisspeptin on
gonadotropin secretion
Table 2 summarizes effective doses of kisspeptin on
gonadotropin secretion in domestic animals, and a
representative profile of LH secretion induced by
kisspeptin administration in a steer is shown in
Figure 1. LH levels increased robustly after bolus intra-
venous (i.v.) administration of Kp-10 and peaked
30 min after the injection. The stimulatory effect of
Kp-10 on LH secretion lasted for approximately 1 h.
Ezzat et al. (2009) reported that i.v. injection of as low
as 3.9 nmol of Kp-10 per kg of body weight signifi-
cantly increased plasma LH and FSH concentrations in
prepubertal calves of both sexes and that the same
dose of Kp-10 was also effective, although slightly less
potent, when administered intramuscularly to male
calves. Whitlock et al. (2008, 2010) investigated the
effect of Kp-10 administration on LH secretion in adult
ovariectomized (OVX) cows. Bolus i.v. injection of
Kp-10 stimulated LH secretion, and a similar effect was
observed in OVX cows treated with estradiol cypionate
and/or progesterone (Whitlock et al. 2008). Intrave-
nous infusion of Kp-10 over 5 h in OVX cows
increased plasma LH concentrations for as long as
140 min following the start of infusion, but the stimu-
latory action of the peptide was not observed thereaf-
ter (Whitlock et al. 2010).

Table 1 Comparison of amino acid sequence organization of kisspeptin among different species
Goat 1 GAALCPS–ESSAGPRQPGPCAPRSRLIPAPRGAVLVQREKDVSAYNWNSFGLRY 53
Sheep 1 GAALCPS–ESSAGPRQPGPCAPRSRLIPAPRGAALVQREKDVSAYNWNSFGLRY 53
Cattle 1 GAALCPP–ESSAGPQRLGPCAPRSRLIPSPRGAVLVQREKDVSAYNWNSFGLRY 53
Pig 1 GTSSCQPPESSSGPQRPGLCTPRSRLIPAPRGAVLVQREKDLSAYNWNSFGLRY 54
Rat 1 –TSPCPPVENPTGHQRP–PCATRSRLIPAPRGSVLVQREKDMSAYNWNSFGLRY 52
Human 1 GTSLSPPPESSGSPQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF 54
—Kp–10—

© 2013 Japanese Society of Animal Science Animal Science Journal (2013) 84, 369–381
 KISSPEPTIN IN DOMESTIC ANIMALS 371

Smith et al. (2009a, 2011)

Hashizume et al. (2010)

Whitlock et al. (2008)
Whitlock et al. (2008)

Ohkura et al. (2009b)

Smith et al. (2009a)

Magee et al. (2009)

Caraty et al. (2007)

Lents et al. (2008)

Ezzat et al. (2009)

Saito et al. (2012)

Reference

15.6 or 39 nmol/head
Effective doses of bolus i.v. administration on Kp-10 on stimulating luteinizing hormone (LH) release in various domestic animals
Dose of kisspeptin

0.77 nmol/kg BW
3.9 nmol/kg BW
0.1 nmol/kg BW
0.1 nmol/kg BW

3.9 nmol/kg BW
15.6 nmol/head

390 nmol/head

780 nmol/head
390 nmol/head
6 nmol/head

Figure 1 A representative luteinizing hormone (LH) profile

after bolus i.v. injection of the kisspeptin decapeptide Kp-10
in a steer.
Gonadal status and steroid milieu

In OVX adult ewes treated with E2, i.v. injection of

Kp-10 during the anestrous season significantly stimu-
lated LH and FSH secretion, and the human (hKp-10)
Intact (breeding season)

OVX + E2 (anestrous)

and rat (rKp-10) types of the decapeptide exerted

Intact (luteal phase)

Intact (luteal phase)

similar effects (Caraty et al. 2007). Smith et al. (2009a,

Intact (anestrous)

2011) reported that the effect of Kp-10 administration

on gonadotropin secretion varied with the stage of the
OVX + E2

Castrated

estrous cycle and the season in ewes with intact

ovaries. The LH response to Kp-10 was greater in the
Intact

Intact

Intact
OVX

late-follicular phase compared with the luteal phase

during the breeding season (Smith et al. 2011), and the
effect of Kp-10 on LH but not FSH secretion was
greater during the anestrous season than in the luteal
Male, female

phase during the breeding season (Smith et al. 2009a).

BW, body weight; E2, estradiol; OVX, ovariectomized; P, progesterone.

In male castrated goats, plasma LH concentrations

Female
Female
Female
Female
Female

Female
Female
Female

were promptly elevated after bolus i.v. administration

Male
Male
Sex

of Kp-10, and the stimulatory effect of the peptide on

LH secretion lasted for several hours (Ohkura et al.
2009b). Similar effects of Kp-10 were observed in pre-
and post-pubertal male goats (Saito et al. 2012), and
Young (pre- and post-pubertal)

cycling female goats during the luteal phase

(Hashizume et al. 2010).
In prepubertal gilts, intracerebroventricular (i.c.v.)
Young (prepubertal)

Young (prepubertal)

administration of as low as 7.8 nmol of Kp-10 signifi-

cantly elevated serum LH and FSH concentrations. On
the other hand, a dose of 780 nmol of Kp-10 was
required to increase serum LH concentrations when it
was administered peripherally, and serum FSH con-
Adult

Adult

Adult

Adult

centrations were not affected even by i.v. injection of

Age

3900 nmol of the peptide (Lents et al. 2008).

In cycling adult mares, bolus i.v. administration of
390 nmol of Kp-10 during the luteal phase signifi-
cantly increased serum LH and FSH concentrations,
Animal
Table 2

Sheep
Cattle

Horse

whereas 780 nmol of Kp-10 administration in the late

Goat

Pig

follicular phase was insufficient to induce ovulation

Animal Science Journal (2013) 84, 369–381 © 2013 Japanese Society of Animal Science
372 H. OKAMURA et al.
(Magee et al. 2009). The effect of Kp-10 on LH secre-
tion in pony mares was less potent during the late
follicular phase than during other follicular stages
(Decourt et al. 2010).
Sites of kisspeptin action on
gonadotropin secretion
Several lines of evidence indicate that the action of
kisspeptin on gonadotropin secretion is mediated cen-
trally by GnRH. First, the majority of GnRH neurons in
mice (Irwig et al. 2004) and sheep (Smith et al. 2009b)
express kisspeptin receptor gene, GPR54. Second,
peripheral administration of kisspeptin activates GnRH
neurons, as reflected by Fos (a marker of neuronal
activation) expression, in rats (Matsui et al. 2004).
Third, kisspeptin stimulates GnRH release in vitro from
hypothalamic tissues from mice (d’Anglemont de
Tassigny et al. 2008), rats (Uenoyama et al. 2011) and
sheep (Smith et al. 2011). Fourth, i.v. injection of
kisspeptin increases the concentration of GnRH in the
portal vessels, with a parallel rise in peripheral LH
concentrations, in sheep (Smith et al. 2011; Li et al.
2012) and goats (Tanaka et al. 2012). Finally, the effect
of kisspeptin on gonadotropin secretion is blocked by
pretreatment with a GnRH receptor antagonist in
rodents (Gottsch et al. 2004; Irwig et al. 2004; Matsui
et al. 2004) and monkeys (Plant et al. 2006) and by
hypothalamo-pituitary disconnection, which prevents
the delivery of GnRH to the pituitary (Clarke et al.
1983), in sheep (Smith et al. 2008a).
The expression of GPR54 in the anterior pituitary in
some animals (Richard et al. 2009), including sheep
(Smith et al. 2008a), suggests that kisspeptin may also
act at the pituitary level. Indeed, in vitro studies in
which Kp-10 was added to pituitary cell cultures
derived from sheep (1 nmol/L; Smith et al. 2008a),
pigs (10 nmol/L; Suzuki et al. 2008) and cattle
(1000 nmol/L; Suzuki et al. 2008; Ezzat et al. 2010)
showed that Kp-10 increased the concentration of LH
in the culture medium. Therefore, it is possible that
kisspeptin acts at dual sites, both the hypothalamus
and the pituitary. However, the biological significance
of the latter is completely unclear at present.
Other actions of kisspeptin
Peripheral administration of Kp-10 increased growth
hormone (GH) secretion in OVX cows treated with
estradiol cypionate and/or progesterone (Whitlock
et al. 2008) but had no effect without the concomitant
steroid treatment (Whitlock et al. 2008, 2010).
Kadokawa et al. (2008) reported that i.v. injection of
Kp-10 elevated plasma GH concentrations in prepu-
bertal heifers. However, another study failed to
observe any effect of Kp-10 on GH secretion in prepu-
bertal calves of either sex (Ezzat et al. 2009). In sheep,
i.v. injection of doses of Kp-10 that potently stimulated
© 2013 Japanese Society of Animal Science
LH secretion had no effect on plasma GH, prolactin
and cortisol concentrations regardless of the season or
the stage of the estrous cycle (Smith et al. 2009a;
Whitlock et al. 2010), whereas i.c.v. administration of
Kp-10 significantly increased GH secretion in OVX
ewes (Whitlock et al. 2010). Neither i.v. nor i.c.v. injec-
tion of Kp-10 affected GH secretion in prepubertal gilts
(Lents et al. 2008) or GH or prolactin secretion in
luteal-phase female goats (Hashizume et al. 2010).
Although it seems that the stimulation of GH secretion
is the sole endocrine action of kisspeptin outside of the
HPG axis observed to date, there is controversy as to
the conservation of this effect between species or even
within the same species, and the biological significance
of this action of kisspeptin remains far from clear.
A previous report in mice indicated that kisspeptin/
GPR54 signaling is not required for male or female
copulatory behavior (Kauffman et al. 2007). As far as
we know, there is no study published to date examin-
ing the effects of kisspeptin on sexual behavior in
domestic animals.
ANATOMY OF KISSPEPTIN NEURONS
Distribution
The distribution of kisspeptin-producing neurons has
been examined by in situ hybridization and/or immu-
nohistochemistry in sheep (Estrada et al. 2006;
Franceschini et al. 2006; Smith et al. 2007; Merkley
et al. 2012), goats (Takase et al. 2007; Ohkura et al.
2009b), pigs (Tomikawa et al. 2010) and horses
(Decourt et al. 2008; Magee et al. 2009). Kisspeptin
neurons are found almost exclusively in two discrete
areas within the hypothalamus, rostrally in the preop-
tic area (POA) and caudally in the arcuate nucleus
(ARC). Alternatively, the rostral population is found in
the periventricular nucleus rather than the POA in
pigs (Tomikawa et al. 2010). This distribution pattern is
similar to that in rodents, in which the rostral popu-
lation of kisspeptin neurons is concentrated in the
anteroventral periventricular nucleus (AVPV) and
associated areas (Clarkson & Herbison 2006; Adachi
et al. 2007). Although kisspeptin-immunoreactive
neurons were also observed in several other areas of
the equine hypothalamus (Magee et al. 2009), this
study may need to be interpreted with caution, as the
antibody used seems to cross-react with other antigens
in addition to kisspeptin (Pompolo et al. 2006;
Goodman et al. 2007; Smith et al. 2008b).
Although the projections of kisspeptin neurons have
not been systematically examined in domestic
animals, the initial study in sheep revealed that fibers
containing kisspeptin are present throughout the
hypothalamus, being most abundant in the POA, ARC
and median eminence (ME) (Franceschini et al. 2006).
A more detailed analysis in mice demonstrated that
kisspeptin fibers project not only to most of the
Animal Science Journal (2013) 84, 369–381

hypothalamic nuclei (except the suprachiasmatic and
ventromedial nuclei) but also to extra-hypothalamic
areas such as the bed nucleus of the stria terminalis,
medial amygdala, periaqueductal gray matter, and
locus coeruleus (Clarkson & Herbison 2009), implicat-
ing kisspeptin in a variety of physiological functions
beyond reproduction.
Sensitivity of kisspeptin neurons to
gonadal steroids
Although ERa plays pivotal roles in both the negative
(Dorling et al. 2003) and positive (Wintermantel et al.
2006) feedback effects of E2 on GnRH secretion, GnRH
neurons themselves do not express ERa (Lehman et al.
1993). Therefore, some other population of neurons
must mediate the effect of E2 action on GnRH
neurons. Among several subsets of neurons, kisspeptin
neurons are conspicuous in that virtually all of them
express ERa in the ARC and approximately 50% of
them do so in the POA in ewes (Franceschini et al.
2006). Moreover, approximately 90% of kisspeptin
neurons also contain progesterone receptor in the
ovine ARC (Smith et al. 2007).
In rodents, E2 is well established as up-regulating
the expression of kisspeptin in the AVPV and down-
regulating its expression in the ARC (Smith et al. 2005;
Adachi et al. 2007). In domestic animals, there is cur-
rently only fragmentary evidence for the regulation of
kisspeptin expression by steroid hormones.
The rostral population of kisspeptin neurons express
Kiss1 messenger RNA (mRNA) at higher levels during
the late-follicular phase than during the luteal phase
in ewes (Smith et al. 2009b) and up-regulate Kiss1
expression in response to high levels of E2 in pigs
(Tomikawa et al. 2010). In goats, a subset of neurons in
the POA of mature males expresses Kiss1 and kisspep-
tin peptide (Matsuyama et al. 2011), but this expres-
sion is completely eliminated by castration (Ohkura
et al. 2009b). These observations are similar to the
findings in rodents. On the other hand, the action of
E2 in ewes remains debatable, because a similar E2
treatment increased the expression of Kiss1 in the
ovine POA in one report (Smith et al. 2008b) but not in
another (Smith et al. 2007).
In gonadectomized goats, a large number of kisspep-
tin neurons can be observed in the ARC, being much
abundant at its caudal portion in both females
(Fig. 2A) and males (Ohkura et al. 2009b), whereas
the distribution of kisspeptin neurons is extremely
sparse in intact animals (Matsuyama et al. 2011), sug-
gesting a strong inhibition of kisspeptin expression by
androgen throughout the ARC. In OVX pigs, the
expression of Kiss1 in the ARC is decreased by E2, but
only in the most caudal portion of the nucleus
(Tomikawa et al. 2010). In OVX ewes, the number of
kisspeptin-expressing neurons in the ARC is signifi-
cantly higher in vehicle-control animals than in those
Animal Science Journal (2013) 84, 369–381
KISSPEPTIN IN DOMESTIC ANIMALS 373
treated with a low dose of E2 (Smith et al. 2007,
2008b, 2009b), indicating the down-regulation of
kisspeptin expression by E2 as in other species. On the
other hand, the number of kisspeptin-expressing
neurons in the middle and/or caudal portions of the
ARC is higher during the late-follicular phase than
during the luteal phase in cycling ewes (Estrada et al.
2006; Smith et al. 2009b). Interestingly, the middle
and caudal portions of the ovine ARC contain greater
numbers of kisspeptin neurons in mid-luteal-phase
ewes than in intact rams (Cheng et al. 2010). There-
fore, the effect of gonadal steroids on kisspeptin
expression in the ARC seems to vary among species,
sexes and the portions of the nucleus, and must there-
fore be mediated by a complex mechanism, especially
in sheep.
Interaction of kisspeptin neurons with
GnRH neurons
Approximately 40% to 50% of the GnRH neurons in
the POA and medial basal hypothalamus (MBH)
receive direct apposition of kisspeptin fibers on their
cell bodies in ewes, and during the breeding season this
percentage increases to virtually 100% in the MBH,
although not in the POA (Smith et al. 2008b). Such
apposition occurs only in a small percentage of GnRH
neurons in the POA in male goats (Matsuyama et al.
2011), male mice (Clarkson & Herbison 2006) and
castrated male monkeys (Ramaswamy et al. 2008). On
the other hand, kisspeptin fibers associate extensively
with GnRH axons in the ME in both females (ewes:
Smith et al. 2011; mares: Decourt et al. 2008; rats:
Uenoyama et al. 2011) and males (goats: Ohkura et al.
2009b; Matsuyama et al. 2011; monkeys: Ramaswamy
et al. 2008) as shown (Fig. 2B). Electron microscopy has
revealed that the axon terminals of kisspeptin neurons
do in fact closely contact GnRH axon terminals in goats
(Matsuyama et al. 2011) and rats (Uenoyama et al.
2011). Therefore, although kisspeptin neurons may
interact with either cell bodies or the axon terminals of
GnRH neurons, the former is unlikely to be the major
site of kisspeptin action in males. These differences in
the anatomy of the kisspeptin-GnRH interaction may
reflect different effects of kisspeptin action on GnRH
secretion: the control of the pulsatile and surge modes
of GnRH secretion.
CONTROL OF GnRH SECRETION BY
KISSPEPTIN NEURONS
Central infusion of a GPR54 antagonist (Roseweir et al.
2009) blocked or attenuated both pulsatile LH secre-
tion (Roseweir et al. 2009; Smith et al. 2011; Goodman
et al. 2012) and the E2-induced LH surge (Smith et al.
2011) in ewes. Functional inactivation of GPR54 by
chronic administration of potent GPR54 agonists
(Matsui et al. 2012) completely eliminated LH pulses in
© 2013 Japanese Society of Animal Science
374 H. OKAMURA et al.
Figure 2 The distribution of kisspeptin neurons in the goat
medial basal hypothalamus (MBH). (A) Immunofluorescence
staining for kisspeptin (green) in the arcuate nucleus (ARC)
of an ovariectomized goat. Kisspeptin neurons are densely
packed in the caudal portion of the ARC. (B) Dual
immunofluorescence for kisspeptin (red) and
gonadotropin-releasing hormone (GnRH; green) at the
lateral aspect of the median eminence in a castrated male
goat. Kisspeptin fibers make intimate associations with
GnRH fibers at the external layer of the median eminence
(ME) at the level of the tuberoinfundibular sulcus. ARC,
arcuate nucleus; MEe, external layer of the ME; MEi,
internal layer of the ME; pt, pars tuberalis; 3V, third
ventricle. Scale bars, 100 mm.
male (Ohkura et al. 2011) and female (Wakabayashi
et al. 2011) goats. Furthermore, immunoneutraliza-
tion of endogenous kisspeptin activity disrupted the
estrous cycle in rats (Kinoshita et al. 2005). Together
with earlier human genetic findings (de Roux et al.
2003; Seminara et al. 2003) and subsequent studies in
transgenic mice (Funes et al. 2003; Seminara et al.
2003; Dungan et al. 2007), these results clearly show
that kisspeptin plays pivotal roles in the control of both
the pulsatile and surge modes of GnRH secretion.
Control of the pulsatile GnRH secretion
by kisspeptin neurons
The pulsatile discharge of GnRH into the portal vessels
is governed by neural substrates, the so-called GnRH
pulse generator, that periodically fire a volley of action
potentials (Knobil 1981; Kawakami et al. 1982; Mori
et al. 1991; Nishihara et al. 1994). Recent electrophysi-
ological studies in goats have demonstrated that activ-
ity of the GnRH pulse generator is monitored at close
vicinity of the ARC kisspeptin neurons in both males
(Ohkura et al. 2009b) and females (Wakabayashi et al.
2010). Moreover, kisspeptin has been shown in
monkeys to be secreted episodically into the ME, and
these kisspeptin pulses are temporally associated with
pulsatile GnRH secretion (Keen et al. 2008). These
results, together with other findings, have led to the
compelling hypothesis that the population of kisspep-
tin neurons in the ARC is the intrinsic source of the
GnRH pulse generator (Navarro et al. 2009; Lehman
et al. 2010; Maeda et al. 2010; Rance et al. 2010).
Virtually all kisspeptin neurons in the ARC
co-express neurokinin B (NKB) and dynorphin A
© 2013 Japanese Society of Animal Science
(Dyn) in both sheep (Goodman et al. 2007; Cheng et al.
2010) and goats (Wakabayashi et al. 2010; Matsuyama
et al. 2011), and these neurons have therefore
been termed KNDy (kisspeptin/NKB/Dyn) neurons
(Lehman et al. 2010). KNDy neurons are surrounded
by their own dense network of varicose fibers in sheep
(Foradori et al. 2002, 2006) and goats (Wakabayashi
et al. 2010) and are bilaterally interconnected by NKB
axons in rats (Krajewski et al. 2010) and goats
(Wakabayashi et al. 2013). Therefore, KNDy neurons
likely form a neuronal network interconnected by
axon collaterals and/or dendrites. This hypothesis is
supported by the physiological finding that the activity
of the GnRH pulse generator is synchronous between
the ARCs on both sides of the brain (Wakabayashi et al.
2013). Such a neuronal network would serve as a
framework for synchronizing the bursting activities of
the KNDy neurons and thereby generate discrete
neural signals to induce pulsatile GnRH secretion.
Like kisspeptin, NKB has also recently attracted con-
siderable attention. First, Topaloglu et al. (2009) discov-
ered that inactivating mutations of Tac3 and Tacr3,
which encode NKB and its receptor (NK3R), respec-
tively, produce gonadotropin deficiency and pubertal
failure in humans, suggesting the involvement of NKB/
NK3R signaling in the control of pulsatile GnRH secre-
tion. This suggestion is supported by experimental
observations in goats that bolus i.c.v. administration of
NKB immediately activates the GnRH pulse generator
(Wakabayashi et al. 2010) and further, that continuous
i.v. infusion of senktide, a selective NK3R agonist, pro-
duces intermittent bursts of the GnRH pulse generator
with corresponding LH pulses (Wakabayashi et al.
2012). One possible site of this action of NKB might be
a subset of KNDy neurons, as KNDy neurons express
NK3R (Amstalden et al. 2010; Wakabayashi et al. 2012)
and i.c.v. administration of senktide-activated KNDy
neurons in rats (Navarro et al. 2011a) and sheep
(Sakamoto et al. 2012). Moreover, NKB has been
shown to elicit trains of action potentials in mouse brain
slices containing KNDy neurons (Navarro et al. 2011b).
Collectively, it is plausible that NKB is involved in the
generation of the intermittent bursts of the GnRH pulse
generator via an auto-feedback loop of the KNDy neu-
ronal network and that kisspeptin transmits this activ-
ity to the GnRH neurons (Fig. 3) (Maeda et al. 2010;
Wakabayashi et al. 2010). Although Dyn has been sug-
gested to terminate the burst activity (Wakabayashi
et al. 2010), the precise role and action sites of Dyn in
the KNDy network remain unclear.
Control of the GnRH surge by
kisspeptin neurons
The rostral population of kisspeptin neurons (in the
AVPV) is well documented as playing a critical role in
triggering the preovulatory GnRH/LH surge in rodents
Animal Science Journal (2013) 84, 369–381
 KISSPEPTIN IN DOMESTIC ANIMALS 375

Figure 3 A schematic illustration of the novel hypothalamic-pituitary-gonadal (HPG) axis. The two populations of kisspeptin
neurons in the preoptic area (POA) and arcuate nucleus (ARC) are suggested to be located upstream of the
gonadotropin-releasing hormone (GnRH) neurons in the HPG axis and to control the surge and pulse modes of
GnRH/luteinizing hormone (LH) secretion, respectively. Neurokinin B (NKB) likely plays a role in the pulse-generating
mechanism of the ARC kisspeptin neurons. ME, median eminence; och, optic chiasm; Pit, pituitary; pt, pars tuberalis.

(Oakley et al. 2009; Tsukamura et al. 2010; Khan &
Kauffman 2012). First, E2 treatment dramatically
enhances the expression of kisspeptin in the AVPV
(Smith et al. 2005; Adachi et al. 2007; Tomikawa et al.
2012). Second, the expression of kisspeptin in the
AVPV is highest at the time of the LH surge (Smith
et al. 2006; Adachi et al. 2007). Third, the E2 treatment
that induces the LH surge in OVX animals also acti-
vates the majority of the kisspeptin neurons in the
AVPV (Smith et al. 2006; Adachi et al. 2007). Mating
stimulus has been shown to activate POA kisspeptin
neurons to induce ovulation in musk shrews, which
are reflex ovulators (Inoue et al. 2011). Fourth, a brief
application of Kp-10 induces prolonged activation of
GnRH neurons in mouse brain slices (Han et al. 2005;
Pielecka-Fortuna et al. 2008).
In domestic animals, on the other hand, the role of
kisspeptin in evoking the LH surge has not been fully
evaluated. A few reports in several animal models
have suggested that, as in rodents, the kisspeptin
neurons in the POA are involved in the LH
surge. OVX sows subjected to the E2 treatment suf-
ficient to induce an LH surge and estrous behavior
comparable to those observed in intact animals,
exhibited significantly increased expression of
kisspeptin in the rostral (periventricular nucleus) but
not the caudal (ARC) population of kisspeptin
neurons (Tomikawa et al. 2010). In OVX ewes, the
expression of kisspeptin in the POA was markedly
enhanced by even a low dose of E2, which produces
circulating E2 levels during the luteal phase (Smith
et al. 2008b). Hoffman et al. (2011) demonstrated
using an excellent synchronized follicular phase
model in cycling ewes that Fos was induced con-
comitantly in kisspeptin neurons and in GnRH
neurons in the POA, but not in kisspeptin neurons in
the ARC, at the time of the GnRH/LH surge. These
results suggest that the POA kisspeptin-GnRH system
is involved in the mechanism of GnRH surge genera-
tion in ewes (Fig. 3).

Animal Science Journal (2013) 84, 369–381 © 2013 Japanese Society of Animal Science
376 H. OKAMURA et al.
Interestingly, another group showed that Fos was
induced in kisspeptin neurons not only in the POA but
also in the ARC during the LH surge in cycling ewes
(Merkley et al. 2012). Moreover, the number of
kisspeptin-expressing neurons in the ARC increases
during the late-follicular phase in this species (Estrada
et al. 2006; Smith et al. 2009b). Therefore, a subset of
kisspeptin neurons in the ARC may also participate in
the induction of the GnRH/LH surge in ewes (Caraty
et al. 1998; Merkley et al. 2012). However, ARC
kisspeptin neurons seem to be homogeneous in terms
of molecular identity: virtually all of them express
kisspeptin, NKB, Dyn, NK3R, ERa and progesterone
receptor (Oakley et al. 2009; Lehman et al. 2010;
Goodman & Lehman 2012). Further studies are
required to clarify whether there is a specific subpopu-
lation of kisspeptin neurons in the ovine ARC and
whether the mechanism by which kisspeptin controls
the GnRH surge differs between rodents and sheep.
APPLICATIONS OF KISSPEPTIN AND
RELATED SUBSTANCES: PROSPECTIVE
Studies investigating potential applications of kisspep-
tin to the management of fertility in domestic animals
have just begun. Although any conclusions would be
premature at this point, a few promising results have
nevertheless been demonstrated (Caraty et al. 2010,
2012a).
Synchronization of ovulation
by kisspeptin
To investigate the effect of Kp-10 on the induction of
the LH surge, Kp-10 was infused intravenously at a
rate of 480 nmol/h for 8 h in progesterone-primed
cyclic ewes (Caraty et al. 2007). The LH surge occurred
within 2 h after the start of the infusion in the Kp-10-
treated group but later and with variable timing in
vehicle control-treated animals. Subsequent examina-
tion showed no difference in the number of corpora
lutea between the Kp-10 and vehicle-treated groups.
These results strongly suggest that kisspeptin could be
used as a new tool for synchronization of ovulation
and thereby improve the efficiency of artificial insemi-
nation and in vitro fertilization programs (Caraty et al.
2010, 2012a).
However, in pony mares a similar treatment with
Kp-10 (infusion for 3 days at a rate of 2370 nmol/h) in
the late follicular phase failed to advance or synchro-
nize the LH surge (Caraty et al. 2012a) despite the fact
that Kp-10 can stimulate gonadotropin secretion in
mares (Magee et al. 2009; Decourt et al. 2010).
Acceleration of puberty by kisspeptin
Redmond et al. (2011) investigated whether kisspeptin
activates the HPG axis in prepubertal ewe lambs.
Treatment of lambs (28 weeks of age) with hourly i.v.
© 2013 Japanese Society of Animal Science
injections of Kp-10 (15.6 nmol) for 24 h increased LH
secretion to the levels comparable to those of the surge
in cycling adult ewes; this was followed by a rise in
progesterone secretion within 5 days after the treat-
ment. These results indicate that intermittent Kp-10
administration, which might mimic increasing activity
of the kisspeptin neuronal system during the prepu-
bertal period, induces ovulation and formation of the
corpus luteum, suggesting that kisspeptin has potential
for advancing puberty in domestic animals. Because
the duration of the induced luteal activity was shorter
than that of the normal luteal phase, the method
employed in this study might require further modifi-
cation before being applied in the field.
Development of effective
kisspeptin analogues
Kp-10 is clearly a potent GnRH secretagogue. Never-
theless, relatively large amounts of kisspeptin, com-
pared with those of GnRH analogues, were necessary
to obtain the expected outcomes in the aforemen-
tioned studies in sheep (Caraty et al. 2007), pigs (Lents
et al. 2008) and horses (Magee et al. 2009; Caraty et al.
2012a). Therefore kisspeptin analogues with more
powerful GnRH/LH secretion-inducing activity would
be highly desirable for practical applications in domes-
tic animals. Several groups have recently reported the
development of novel kisspeptin analogues (Tomita
et al. 2008; Curtis et al. 2010; Matsui et al. 2010, 2012).
Future studies of the effects of these analogues on LH
secretion in domestic animals are highly anticipated.
Mode of kisspeptin administration
The action of kisspeptin on gonadotropin secretion
seems to largely depend on the mode of its adminis-
tration. Continuous i.v. infusion of Kp-10 increased E2
secretion and induced the LH surge in seasonally
anestrous ewes (Caraty et al. 2007; Sébert et al. 2010),
suggesting that kisspeptin can be utilized to stimulate
the gonadal activity. However, it should be noted that
continuous exposure of the gonadotrophs to GnRH
causes desensitization of the GnRH receptors, which
results in the abrogation of gonadotropin secretion
(Knobil 1980). Likewise, continuous administration of
kisspeptin or its analogues has been shown to suppress
the secretion of gonadotropins and/or gonadal steroid
hormones in monkeys (Seminara et al. 2006), rats
(Matsui et al. 2012), sheep (Messager et al. 2005) and
goats (Ohkura et al. 2011). Therefore, the optimal dose
of kisspeptin and duration of the treatment might be
carefully determined for a given species, when con-
tinuous administration of kisspeptin is applied to
improve reproductive efficacy in domestic animals.
Caraty et al. (2012b) have demonstrated in ewes
that intermittent Kp-10 administration leads to inter-
mittent rises in GnRH and LH secretion (Caraty et al.
Animal Science Journal (2013) 84, 369–381

2012b) mimicking the pulsatile nature of physiological
secretion of GnRH/LH in vivo (Karsch 1984). The result
clearly indicate that intermittent repeated administra-
tion of Kp-10 avoids desensitization of kisspeptin
receptors as well as GnRH receptors, and suggests that
this mode of administration would be an alternative
protocol for the kisspeptin application. Indeed, it has
been reported that hourly i.v. injection of Kp-10 main-
tained a train of LH discharges for 48 h in juvenile
monkeys (Plant et al. 2006) and induced ovulation in
prepubertal lambs (Redmond et al. 2011).
Application of NK3R agonists to
facilitate pulsatile GnRH/LH secretion
As shown in Figure 3, the studies on the neural
mechanism of the generation of the GnRH pulse
have proposed the hypothesis that NKB plays a
pivotal role in the GnRH pulse generation cascade
(Maeda et al. 2010; Rance et al. 2010; Wakabayashi
et al. 2010). This hypothesis has been experimentally
addressed in a preliminary study using OVX+E2
goats (Yamamura et al. 2012). Continuous subcutane-
ous infusion of senktide (1440 nmol/h) for 24 h
increased the frequency of the GnRH pulse generator
activity by approximately 1.5-fold over the pre-
infusion value, and the effect of senktide was main-
tained throughout the infusion period. The results
suggest, although the dose of senktide used in that
study was still very high, that high-frequency pulsa-
tile LH secretion can be produced by continuous
administration of senktide, a method more preferable
over intermittent repeated administration in the field
of animal management.
There are several situations in which an insufficient
LH pulse frequency causes reproductive disorders,
such as domestic animals with undernutrition or a
negative energy balance (Schillo 1992) and women
with anorexia nervosa (Allouche et al. 1991) or exer-
cise amenorrhea (Veldhuis et al. 1985). The LH pulse-
stimulating action of senktide implies that NK3R
agonists may hold promise as novel therapeutic drugs
to accelerate or improve gonadal activity in humans as
well as domestic animals.
Conclusion
Emerging studies over the last decade revealed that the
kisspeptin neuronal system plays an essential role as
the gatekeeper of reproduction and thereby governs
both the pulsatile and surge modes of GnRH secretion
(Fig. 3). Although translational and field studies by a
few groups have just begun, the reported biological
actions of kisspeptin and NKB strongly suggest that
analogues of kisspeptin and NKB would be useful in
the development of novel strategies for the manage-
ment of fertility in domestic animals.
Animal Science Journal (2013) 84, 369–381
KISSPEPTIN IN DOMESTIC ANIMALS 377
ACKNOWLEDGMENTS
This work was supported in part by a grant from the
Ministry of Agriculture, Forestry and Fisheries of Japan
(Research Program on Innovative Technologies for
Animal Breeding, Reproduction and Vaccine Develop-
ment, REP-2001), and by a Grant-in Aid for Young
Scientists (B), 24780275 (TY) and 23780285 (YW) from
the Japan Society for the Promotion of Science.
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