GPR54-Dependent Stimulation of Luteinizing Hormone Secretion by Neurokinin B in Prepubertal Rats
et al. (2012) GPR54-Dependent Stimulation of Luteinizing Hormone Secretion by Neurokinin B in
Prepubertal Rats. PLoS ONE 7(9): e44344. doi:10.1371/journal.pone.0044344
GPR54-Dependent Stimulation of Luteinizing Hormone Secretion by Neurokinin B in Prepubertal Rats
Xiao Feng Li
Yuan Shao Lin
Ming Han Hu
Stewart J. Paterson
Robert P. Millar
Stafford L. Lightman
Kevin T. O'Byrne kevin.o'
David A. Slattery, University of Regensburg, Germany
Kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn) are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus (ARC) to GnRH neurons and numerous other hypothalamic targets. Each of the KNDy neuropeptides has been implicated in regulating pulsatile GnRH/LH secretion. In isolation, kisspeptin is generally known to stimulate, and Dyn to inhibit LH secretion. However, the NKB analog, senktide, has variously been reported to inhibit, stimulate or have no effect on LH secretion. In prepubertal mice, rats and monkeys, senktide stimulates LH secretion. Furthermore, in the monkey this effect is dependent on kisspeptin signaling through its receptor, GPR54. The present study tested the hypotheses that the stimulatory effects of NKB on LH secretion in intact rats are mediated by kisspeptin/GPR54 signaling and are independent of a Dyn tone. To test this, ovarian-intact prepubertal rats were subjected to frequent automated blood sampling before and after intracerebroventricular injections of KNDy neuropeptide analogs. Senktide robustly induced single LH pulses, while neither the GPR54 antagonist, Kp-234, nor the Dyn agonist and antagonist (U50488 and nor-BNI, respectively) had an effect on basal LH levels. However, Kp-234 potently blocked the senktide-induced LH pulses. Modulation of the Dyn tone by U50488 or nor-BNI did not affect the senktide-induced LH pulses. These data demonstrate that the stimulatory effect of NKB on LH secretion in intact female rats is dependent upon kisspeptin/GPR54 signaling, but not on Dyn signaling.
The secretion of GnRH is pulsatile and is controlled by the
GnRH pulse generator [1,2]. Pubertal onset in the female is
characterized by increasingly frequent gonadotropin pulses, rising
estradiol (E2) levels and reduced sensitivity to the negative
feedback effects of E2 . Novel components essential for the
regulation of GnRH secretion, and thus physiologic pubertal
development and fertility, were discovered through mutations in
genes encoding GPR54 [4,5], the putative receptor for kisspeptin,
Neurokinin B (NKB) and its receptor (NK3R) . Kisspeptin,
NKB and NK3R are coexpressed within hypothalamic arcuate
nucleus (ARC) neurons, which might comprise the GnRH pulse
generator, along with kappa-opioid receptor (KOR) and its ligand,
dynorphin A (Dyn) . Expression of the genes encoding
kisspeptin and its receptor (Kiss1 and Kiss1R, respectively) increases
at puberty [12,13], as does NKB- and Dyn-immunoreactivity .
Moreover, exogenous kisspeptin stimulates precocious LH
secretion and puberty in rats . Current research endeavors to
unravel the complex roles of kisspeptin/GPR54, NKB/NK3R
and Dyn/KOR (collectively, KNDy) signaling systems, and the
interaction between these, in determining the activity of the
GnRH pulse generator, have employed a range of animal models.
It is widely accepted that kisspeptin mediates the stimulation of
GnRH and gonadotropin secretion [16,17], while dynorphin is
involved in the suppression of gonadotropin release [18,19].
Conversely, recent findings concerning the role and mode of
action of NKB are apparently contradictory (in part due to
differences in the species, sex, gonadal status and methodology
used) . We have recently shown that central
(intracerebroventricular or intra-ARC) administration of the NKB analog,
senktide, dose-dependently suppresses pulsatile LH secretion in
ovariectomized (OVX) rats replaced with low levels of E2 [21,22].
We have also shown that intra-ARC injection of a KOR agonist,
U50488, similarly decreases LH pulse frequency, and
demonstrated that the inhibitory influence of ARC NK3R activation on
pulsatile LH secretion is dependent on Dyn/KOR signaling .
On the contrary, the presence of relatively high levels of E2, such
as those in intact rats or in OVX rats treated with high levels of E2,
appear to reverse the inhibitory influence of NKB/NK3R
signaling, rather stimulating LH secretion . Indeed, E2 levels
have long been known to play such a modulatory role in the
regulation of numerous neuropeptides . Thus it appears that
NKB flexibly mediates the effects of E2 negative feedback on the
HPG axis, at least in the rat. While considerable advances have
recently been made in the understanding of the roles of KNDy
signaling systems in modulating pulsatile LH secretion in adults,
little is currently known about the involvement of these
neuropeptides in regulating LH secretion in the developing
Although kisspeptin/GPR54 signaling has been considered
a prerequisite for normal reproductive development and fertility,
a recent study demonstrated that reproductive maturation can
occur in transgenic female mice apparently devoid of kisspeptin
neurons through conditional ablation . However the same
study also documents disrupted cyclicity and infertility in mice in
which kisspeptin neurons were ablated postnatally . It has
recently been demonstrated that both NKB and kisspeptin analogs
stimulate LH secretion in prepubertal agonadal male macaques
. The same study reported that desensitization of GPR54
abrogated the senktide-induced increase in LH levels ,
suggesting that NKB elicits LH secretion in a kisspeptin/GPR54
dependent fashion. The kisspeptin/GPR54-dependent nature of
HPG axis stimulation by NKB has since been evidenced in male
GPR54-knockout mice . These findings are consistent with the
notion that other stimulatory cues may compensate for the lack of
kisspeptin/GPR54 signaling during pubertal development. To this
end there is no consensus on the importance of kisspeptin/GPR54
signaling in reproductive development.
There is evidence for sexual dimorphism of ARC kisspeptin/
NKB neurons in prepubertal mice  and rats , as well as for
sex differences in the regulation of LH secretion in rodent species
[28,29]. Currently little is known about the involvement of the
kisspeptin/GPR54 signaling system in mediating the effects of
NKB/NK3R signaling on LH secretion in the prepubertal female.
Therefore in the present study we investigated whether, like in the
male monkey and the male mouse, NK3R agonism stimulates
pulsatile LH secretion in the prepubertal female rat. Since
presently the kisspeptin-NKB interaction is poorly understood,
we also investigated whether central effects of senktide are
mediated by the kisspeptin/GPR54 system. Finally, we tested
the hypothesis that the inhibitory Dyn/KOR signaling system is
not involved in the modulation of pulsatile LH secretion under
Materials and Methods
All animal procedures were undertaken in accordance with the
Animals (Scientific Procedures) Act UK, 1986, and were approved
by the Kings College London Ethical Review Panel Committee.
All surgical procedures were carried out under anesthesia induced
by ketamine (Vetalar, 100 mg/kg, ip; Pfizer, Sandwich, UK) and
xylazine (Rompun, 10 mg/kg, ip; Bayer, Leverkusen, Germany),
and all efforts were made to minimize suffering.
Animals and Surgical Procedures
Adult Sprague-Dawley rats (Charles River, Margate, UK) were
allocated into breeding pairs. Litters were assessed daily, and the
day of birth was considered postnatal day one (PND 1). Excess
male offspring were culled to reduce litters to 1012 pups on PND
3. Pups were weaned onto standard rat chow (RM1; SDS Diets,
Witham, UK) on PND 21 and were housed 45 per cage under
controlled conditions (12:12 h light/dark cycle, lights on 0700 h,
temperature, 2262uC) and provided with food and water ad
On PND 26 female rats were fitted with a unilateral guide
cannula (22 gauge; Plastics One, Roanoke, VA) directed towards
the left lateral ventricle, the coordinates for implantation being
1.2 mm lateral, 1 mm posterior to Bregma, and 4.5 mm below the
surface of the dura, according to the rat brain atlas of Paxinos and
Watson . The guide cannula was secured using dental cement
(Dental Filling, Swindon, UK), and fitted with a dummy cannula
(Plastics One) to maintain patency . Correct cannula
placement was confirmed by the observation of gravitational
meniscus movement upon insertion of an internal injection
cannula (Plastics One) with extension tubing preloaded with
artificial cerebrospinal fluid (aCSF). At the time of icv cannulation
rats were also fitted with a single indwelling cardiac catheter via
the right jugular vein . The catheter was exteriorized at the
back of the head and enclosed within a 30-cm long lightweight
metal spring tether (Instec Laboratories, Boulder, CO) secured to
a cranial attachment. The distal end of the tether was attached to
a fluid swivel (Instec Laboratories), allowing the rat to move freely.
After surgery, animals were housed singly and allowed 2 d
recovery before experimentation.
On the morning of experimentation, 1 ml normal saline
(Animalcare, Dunnington, UK) was intravenously (iv)
administered over 10 min and the rats were then attached via the cardiac
catheters to a computer-controlled automated blood sampling
system, which enables intermittent withdrawal of 30-ml blood
samples without disturbing the rats . Once connected, the
animals were left undisturbed for 1 h before blood sampling
commenced (between 1000 and 1200 h) then blood samples were
taken every 5 min for 3 h. After removal of each blood sample, an
equal volume of heparinized saline (50 U heparin sodium/ml
normal saline; Wockhardt, Wrexham, UK) was infused. Blood
samples were incubated on ice during the experiment and then
frozen at 220uC.
After a period of control blood sampling an icv injection
cannula with extension tubing, preloaded with drug or vehicle
(aCSF), was inserted into the guide cannula. The distal end of the
tubing was extended outside of the cage to allow remote infusion
without disturbing the rat. In order to reduce the effects of stress
caused by cannula insertion, rats were habituated to handling
twice daily for 2 days. Each injection was given in a volume of 4 ml
vehicle, with 2 ml air separating the treatments. Each treatment
regimen was preloaded within a single injection
cannula-tubingsyringe assembly. After the bleeding procedure, rats were replaced
with 1.5 ml whole blood, i.v. over 15 min. A further 1 ml whole
blood was administered i.v. over 10 min the following day to
preserve the hematocrit and maintain blood volume. Animals were
monitored each morning for vaginal opening as a marker of
Both experiments were repeated 24 times with a 48 h interval
between repetitions (age 2834 d), and only rats that had not
undergone vaginal opening were included. Treatments were
randomized in each repetition of both experiments following
a crossover design, whereby each repetition featured at least one
rat subjected to each of the treatment groups. If any rat was
assigned to the same treatment group more than once, the mean of
the replicates was analysed.
Effect of NK3R Agonist on LH Secretion Following
PreTreatment with GPR54 Antagonist
Infusion regimen consisted of three consecutive injections of
vehicle or 2 nmol selective GPR54 antagonist, Kp-234 (Tocris,
Bristol, UK), each administered over 5 min, 20 min apart, with
a further injection of vehicle or 300 pmol selective NK3R agonist,
senktide (Tocris), administered 15 min following the first injection.
The treatment groups were as follows: Kp-23463+ vehicle
(negative control; n = 3), vehicle 63+ senktide (positive control;
n = 3), Kp-234 63+ senktide (n = 9).
Effect of NK3R Agonist on LH Secretion Following
PreTreatment with KOR Agonist or Antagonist
Senktide (300 pmol) or vehicle were administered 15 min
following icv pre-treatment with 400 nmol KOR agonist
(U50488; Tocris), or 6.8 nmol KOR antagonist (nor-BNI; Tocris),
or vehicle. The treatment groups were as follows: U50488+ vehicle
(negative control; n = 4), nor-BNI + vehicle (negative control;
n = 4), vehicle + senktide (positive control; n = 5), U50488+
senktide (n = 9), nor-BNI + senktide (n = 10).
RIA for LH Measurement
A double-antibody RIA (NIDDK, Bethesda, MD) was used to
determine LH concentrations in the blood samples .
Referenced preparation was rLH-RP-3. The sensitivity of the
assay was 0.093 ng/ml. The intra-assay variation was 7.3% and
the inter-assay variation was 8.7%.
Verification of LH pulses was established using the algorithm
ULTRA . Two intra-assay coefficients of variation of the LH
RIA were used as the reference threshold for pulse detection. The
effect of treatments on LH secretion was calculated by comparing
the area under the LH profile (AUC) in the 30-min period
immediately following the first injection with that in the 30-min
baseline (pre-treatment) period immediately before the time of
injection, using SigmaPlot v.11 (Systat Software, San Jose, CA).
Where a spontaneous LH pulse, defined as a pulse that did not
coincide with the timing of treatments, was detected within the
30min period immediately before the time of the first injection, the
baseline period used in analysis was designated as any 30-min
period within the control (pre-injection) bleeding period devoid of
LH pulses. Values given in the text and figures represent mean 6
SEM. Statistical significance was tested using one-way ANOVA
and Duncans New Multiple Range post-hoc test. P,0.05 was
Effect of NK3R Agonist on LH Secretion Following
PreTreatment with GPR54 Antagonist
In order to investigate the effect of NK3R agonism on the HPG
axis and explore the interaction between the NKB/NK3R and
kisspeptin/GPR54 signaling systems in the prepubertal female rat,
we analysed patterns of LH secretion following icv injections of
senktide in the presence and absence of Kp-234 (Fig. 1).
Coadministration of Kp-234 and vehicle (Fig. 1 A, B) had no effect on
LH secretion (AUC pre-treatment versus post-treatment,
10.361.4 ng/ml.min versus 11.162.3 ng/ml.min; P.0.05).
Senktide administered with vehicle induced a single LH pulse (AUC
pre-treatment versus post-treatment, 9.261.8 ng/ml.min versus
21.963.3 ng/ml.min; P,0.05), which coincided invariably with
the timing of the senktide injection (Fig. 1 C). Spontaneous LH
pulses (Fig. 1 B, E) occurred randomly. The senktide-induced LH
pulse was blocked by the co-administration of Kp-234 (Fig. 1 D, E;
AUC pre-treatment versus post-treatment, 11.560.8 ng/ml.min
versus 13.060.8 ng/ml.min; P.0.05).
Effect of NK3R Agonist on LH Secretion Following
PreTreatment with KOR Agonist or Antagonist
To establish whether Dyn/KOR signaling is involved in the
control of pulsatile LH secretion in the prepubertal female rat, and
whether the Dyn/KOR and NKB/NK3R signaling systems
interact in this animal model, we monitored LH secretion profiles
following icv administration of senktide in rats pretreated with
U50488 or nor-BNI (Fig. 2). Administered with vehicle, neither
U50488 (Fig. 2 A), nor nor-BNI (Fig. 2 B), affected baseline LH
levels (AUC pre-treatment versus post-treatment, 10.360.5 ng/
ml.min versus 11.061.4 ng/ml.min and 9.260.4 ng/ml.min
versus 10.360.4 ng/ml.min, respectively; P.0.05). Single LH
pulses induced by senktide administration (AUC pre-treatment
versus post-treatment, 10.361.4 ng/ml.min versus 24.864.4 ng/
ml.min; P,0.05) were unaffected by pre-treatment with U50488
(Fig. 2 C; AUC pre-treatment versus post-treatment, 9.460.9 ng/
ml.min versus 26.666.0 ng/ml.min; P,0.05) or nor-BNI (Fig 2
D; AUC pre-treatment versus post-treatment, 8.760.4 ng/ml.min
versus 25.664.2 ng/ml.min; P,0.05).
This report provides evidence to indicate that selective
activation of NK3R in gonadal-intact prepubertal female rats
robustly elicits single LH pulses. We also demonstrate herewith
that senktide-induced LH pulses are potently blocked by Kp-234,
thus indicating that the stimulatory effect of NK3R agonism on
LH secretion in this animal model is GPR54-dependent. These
findings concur with previous data from the prepubertal male
monkey  and adult GPR54-knockout male mouse  that
suggest that a hierarchical functional relationship links the NKB/
NK3R and kisspeptin/GPR54 signaling systems. Furthermore,
our data indicate that an endogenous Dyn tone is not apparent in
this animal model, that augmentation or inhibition of Dyn/KOR
signaling does not alter baseline LH secretion in prepubertal
female rats, and that the senktide-induced LH pulses are
unaffected by neuropharmacological modulation of Dyn/KOR
signaling. This is the first investigation of dynorphinergic
mechanisms in the control of pulsatile LH secretion in the
developing female rat, though it is in agreement with previous
investigations interrogating prepubertal sensitivity to non-specific
opioid blockade .
In the final stages of manuscript preparation another article was
published documenting the stimulatory effect of icv administration
of senktide on LH secretion in intact prepubertal female rats .
Despite some redundancy in the observations made by this and the
present work, our data not only provides better resolution to the
dynamics of the temporal changes in LH levels following senktide
administration, but also furthers the notion of NKB-kisspeptin
interaction in the generation of pulsatile gonadotropin secretion.
Our results show that the senktide-induced increase in LH levels,
as previously documented in this animal model , in fact
resembles a single LH pulse that bears similarity to LH pulses
arising spontaneously, which are perhaps driven by endogenous
NKB/kisspeptin tones. Furthermore, a complete absence of LH
pulses, senktide-induced and spontaneous, was observed when
Kp234 was administered. Additionally, there was no effect of Kp-234
on basal LH levels (as defined by the absence of spontaneous or
senktide-induced pulses). We also observed that the administration
of Kp-234 immediately following a spontaneous LH pulse had
a tendency to decrease LH levels to baseline, thereby reducing the
duration and/or the amplitude of the LH pulse, though we did not
specifically investigate the effect of GPR54 antagonism on
spontaneous LH pulses.
nor-BNI (B, D). Central infusion of U50488 or nor-BNI had no effect on basal LH levels (A, B). Likewise, neither U50488, nor nor-BNI, affected the
senktide-induced LH pulses (C, D). The 30-min post-treatment period commenced at the time of the senktide/vehicle injection (long arrow). Area
under the curve (AUC) values in the 30-min pre-treatment (baseline) period (AUC1) and the 30-min post-treatment period (AUC2) for the five
treatment groups are compared in the experiment summary (E). *P,0.05 versus 30-min pre-treatment (baseline) period within the same treatment
group, as well as versus the same 30-min period within the group treated with U50488 and vehicle, and with nor-BNI and vehicle; n = 4210 per
It has recently been suggested from studies in transgenic mice
lacking kisspeptin neurons that kisspeptin/GPR54 signaling is not
essential for pubertal development and fertility, though acute
ablation of GPR54 neurons in adults disrupted physiologic
cyclicity and resulted in infertility . Likewise, despite severe
hypogonadotropic hypogonadism, a proportion of male and
female Kiss1- and Kiss1r-knockout mice exhibited spermatogenesis
and vaginal estrus, respectively , and Kiss1/Kiss1r
doubleheterozygotes are fertile with only mild effects on reproductive
function . In the recent years more and more similarities
between the effects of GPR54 and NK3R activation on the HPG
axis in prepubertal rodents have been documented. First, LH
secretion is stimulated by both senktide [42,45] and kisspeptin
. Second, we present data that implicate the kisspeptin/
GPR54 signaling system as a prerequisite of the stimulatory effects
of NK3R activation on pulsatile LH secretion, since transient
deactivation of kisspeptin/GPR54 signaling blocks
senktide-induced LH secretion. Third, kisspeptin infusion advances pubertal
onset , while antagonism of NK3R  or GPR54  results
in pubertal delay. Finally, the expression of both kisspeptin and
NKB increases through peripubertal maturation . These
observations are consistent with the notion that kisspeptin/GPR54
signaling is not essential for pubertal development [27,43,44],
since apparently the NKB/NK3R system might be able to
compensate for its absence. Because not all ARC NKB/Dyn
neurons express kisspeptin , conditional ablation of neurons
expressing Kiss1 should preserve a population of NKB neurons
potentially capable of compensating for the lack of kisspeptin/
GPR54 signaling in driving pubertal initiation and onset. Indeed,
anatomical evidence from the rat shows that NKB neurons project
to and form numerous close appositions with GnRH neurons [47
50]. However, recent evidence indicates that isolated mouse
GnRH neurons are insensitive to NKB, while senktide robustly
elicits firing of kisspeptin neurons . Indeed, in the absence of
kisspeptin neurons, other neuronal pathways may relay the
stimulatory signals induced by NK3R activation to GnRH
neurons . Further research is necessary to establish whether
kisspeptin/GPR54 signaling is indispensible for reproductive
development and fertility.
In many mammalian species puberty onset is preceded by
a period of insensitivity to the negative feedback effects of gonadal
steroids and opioid peptides. It has therefore been postulated that
endogenous opioids mediate the restraint of the HPG axis during
sexual maturation. We report herewith that indeed neither
augmentation, nor blockade, of Dyn/KOR signaling alters the
pattern of LH secretion in ovary-intact prepubertal female rats. It
has been shown that in prepubertal male monkeys ,
prepubertal gilts , female rabbits in the late prepubertal stage
, as well as in prepubertal female rats , but not in
prepubertal ewes [52,53], that sensitivity to the non-selective
opioid antagonist, naloxone, which has stimulatory effects on LH
secretion in younger juveniles and adult animals, diminishes upon
approach of pubertal onset. This is also apparent in prepubescent
boys [37,38] and girls , although there is evidence that
mopioid receptor signaling is responsible for this phenomenon .
Our data support these findings.
Our results indicate that LH pulses evoked by central NK3R
activation are not affected by the modulation of Dyn/KOR
signaling. This observation contrasts with the notion that in adult
OVX rats senktide-induced suppression of pulsatile LH secretion
is Dyn/KOR-dependent [21,22]. However, since the effects of
KOR activation on LH secretion are invariably inhibitory, it is of
little surprise that such a signaling system is not involved in
senktide-induced LH pulse generation. It is highly plausible that
diminished sensitivity to U50488, probably as a result of decreased
KOR expression and density, underlies the apparent
ineffectiveness of pharmacological modulators of Dyn/KOR signaling in
interacting with the LH response to senktide administration.
Address all correspondence and requests for reprints to: Prof. Kevin T.
OByrne, Division of Womens Health, 2.92W Hodgkin Building, Kings
College London, Guys Campus, London, SE1 1UL, United Kingdom.
The authors wish to thank Dr Parlow of the National Institute of
Diabetes, Digestive and Kidney Diseases for providing the LH RIA kit.
Conceived and designed the experiments: PG XFL KTO. Performed the
experiments: PG YSL MHH LE. Analyzed the data: PG XFL LE KTO.
Contributed reagents/materials/analysis tools: SJP RPM SLL. Wrote the
paper: PG KTO. Mentoring and manuscript review: SJP RPM SLL KTO.
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