Kisspeptin, Neurokinin B, and Dynorphin Act in the Arcuate Nucleus to Control Activity of the GnRH Pulse Generator in Ewes
The Endocrine Society
Kisspeptin, Neurokinin B, and Dynorphin Act in the Arcuate Nucleus to Control Activity of the GnRH Pulse Generator in Ewes
Robert L. Goodman 0
Stanley M. Hileman 0
Casey C Nestor 0
Katrina L. Porter 0
John M. Connors 0
Steve L. Hardy 0
Robert P. Millar 0
Maria Cernea 0
Lique M. Coolen 0
Michael N. Lehman 0
0 Departments of Physiology and Pharmacology (R.L.G. , S.M.H., C.C.N., K.L.P., J.M.C., S.L.H.) , West Virginia University , Morgantown , West Virginia 26506; Mammal Research Institute (R.P.M.), University of Pretoria, Pretoria 0002, South Africa; University of Capetown/Medical Research Council Receptor Biology Unit (R.P.M.), University of Cape Town , 7701 Cape Town , South Africa ; Centre for Integrative Physiology (R.P.M.), University of Edinburgh , Edinburgh EH16 4SB, Scotland , United Kingdom; and Departments of Neurobiology and Anatomical Sciences (M.C., M.N.L.) and Physiology (L.M.C.), The University of Mississippi Medical Center , Jackson, Mississippi 39216
Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus play a key role in GnRH pulse generation, with kisspeptin driving GnRH release and neurokinin B (NKB) and dynorphin acting as start and stop signals, respectively. In this study, we tested this hypothesis by determining the actions, if any, of four neurotransmitters found in KNDy neurons (kisspeptin, NKB, dynorphin, and glutamate) on episodic LH secretion using local administration of agonists and antagonists to receptors for these transmitters in ovariectomized ewes. We also obtained evidence that GnRH-containing afferents contact KNDy neurons, so we tested the role of two components of these afferents: GnRH and orphanin-FQ. Microimplants of a Kiss1r antagonist briefly inhibited LH pulses and microinjections of 2 nmol of this antagonist produced a modest transitory decrease in LH pulse frequency. An antagonist to the NKB receptor also decreased LH pulse frequency, whereas NKB and an antagonist to the receptor for dynorphin both increased pulse frequency. In contrast, antagonists to GnRH receptors, orphanin-FQ receptors, and the N-methyl-D-aspartate glutamate receptor had no effect on episodic LH secretion. We thus conclude that the KNDy neuropeptides act in the arcuate nucleus to control episodic GnRH secretion in the ewe, but afferent input from GnRH neurons to this area does not. These data support the proposed roles for NKB and dynorphin within the KNDy neural network and raise the possibility that kisspeptin contributes to the control of GnRH pulse frequency in addition to its established role as an output signal from KNDy neurons that drives GnRH pulses. (Endocrinology 154: 4259 - 4269, 2013)
Gis the final common pathway for the neural control of
nRH secretion into the hypophysial portal circulation
LH. Under most endocrine conditions, GnRH secretion
occurs episodically (
), a pattern that is essential for
normal reproductive function as exposure of gonadotropes to
continuous GnRH inhibits LH secretion (
). Clearly the
GnRH neurons responsible for this episodic pattern must
release GnRH in synchrony, but the mechanisms
responsible for synchronizing their activity remain largely
unknown. There is evidence from immortalized GnRH cells
) and primary cultures of immature GnRH neurons
(5) that GnRH neurons have the inherent capacity to
produce episodic release, but the applicability of these
observations to normal adults in which GnRH neurons are
anAbbreviations: AMPL, LH pulse amplitude; ARC, arcuate nucleus; BNI, nor-binaltorphimine;
icv, intracerebroventricular; IPI, interpulse interval; KNDy, kisspeptin/neurokinin
B/dynorphin; MUA, multiunit electrical activity; NKB, neurokinin B; NMDA, N-methyl-D-aspartate;
atomically scattered is unclear. Moreover, because
kisspeptin is essential for GnRH secretion in humans (
), pulsatile GnRH secretion is normally dependent on
some afferent input.
Recently four groups have proposed an important role
for a specific set of neurons in the arcuate nucleus (ARC)
in synchronizing GnRH release (
). These neurons
coexpress kisspeptin, neurokinin B (NKB), and dynorphin
(12). They are thus called kisspeptin/neurokinin
B/dynorphin (KNDy) neurons (
) and are found in sheep (
), mice (10), and goats (
). There is also evidence
that KNDy neurons are important for reproductive
function in women (
), although their role in men has been
). Although this population of neurons was
first identified based on the colocalization of these three
neuropeptides, most also contain glutamate in mice (
and sheep (
), whereas galanin and a marker for
-aminobutyric acid have been observed in a smaller percentage
of murine KNDy neurons (
Four lines of indirect evidence led to the hypothesis that
KNDy neurons were important for episodic GnRH
secretion: 1) both kisspeptin (
) and NKB (20) are critical for
normal GnRH secretion in humans; 2) KNDy neurons
form an interconnected network (
13, 21, 22
) that includes
connections between the ARC on both sides of the third
), 3) KNDy neurons contain NK3R, the
receptor for NKB (
10, 13, 24
), and 4) bursts of multiunit
electrical activity (MUA) that correlate with LH pulses are
recorded from the vicinity of KNDy neurons (25) and are
synchronized between each ARC (
). These four groups
all proposed that kisspeptin is the output to GnRH
neurons, whereas NKB acts within the KNDy network to
initiate each GnRH pulse, and dynorphin acts within this
network to inhibit KNDy neural activity and thus
terminate each pulse. There is strong evidence that kisspeptin is
critical for episodic GnRH release (
) and Kiss1r
antagonists block LH pulses in ovariectomized (OVX) ewes
). The proposed actions of NKB are supported by
reports that the stimulatory actions of an NK3R agonist,
senktide, on GnRH secretion are mediated by kisspeptin
release from KNDy neurons in several species (
that intracerebroventricular (icv) administration of NKB
accelerated the frequency of MUA in the ARC of OVX
goats (9). There is less evidence for the proposed role of
dynorphin, but iv administration of a nonspecific
endogenous opioid receptor antagonist, naloxone, prolonged
each GnRH pulse in OVX ewes (
), and icv
administration of the -opioid receptor antagonist,
nor-binaltorphimine (BNI), increased the frequency of MUA in OVX
Although there is thus significant indirect evidence for
the hypothesis that KNDy neurons are important to
GnRH pulse generation, there is no direct evidence that
kisspeptin, NKB, and/or dynorphin act in the ovine ARC
to affect endogenous GnRH secretion. Thus the primary
goal of this work was to determine what, if any, actions the
four established transmitters in KNDy neurons
(kisspeptin, NKB, dynorphin, and glutamate) have within the
ARC. Specifically, we tested the hypotheses that
kisspeptin, NKB, or glutamate act in the ARC to stimulate LH
pulse frequency, but dynorphin acts there to inhibit this
system. Our approach was to monitor episodic LH
secretion, as an index of GnRH release, before and after
) or microinjection (
receptor agonists and antagonists into the ARC of OVX
ewes. In light of evidence that a GnRH receptor antagonist
can increase GnRH pulse frequency in ewes (
GnRH directly inhibits neurons in the rodent ARC (
we also explored a possible role for GnRH neural input to
KNDy neurons. We first determined that
GnRH-containing varicosities contacted KNDy cell bodies, and then
tested the hypotheses that GnRH or orphanin-FQ, an
inhibitory endogenous opioid peptide found in GnRH
), act in the ARC to inhibit LH pulse frequency.
Materials and Methods
Adult mixed-breed blackface ewes were maintained in an
open barn and moved indoors 3?7 days prior to surgeries. Ewes
were fed a maintenance pelleted ration once per day and had free
access to water and mineral blocks. Lights were adjusted
bimonthly to mimic the duration of natural lighting. All
experiments were carried out in ewes that had been OVX for at least 2
weeks, but not more than 10 weeks, prior to any treatments.
Surgeries were carried out as previously described (
sterile conditions using 2%-4% isofluorane as anesthesia.
Ovariectomies were performed via midventral lapaoratomy and
chronic bilateral guide tubes were placed just above the ARC
(35). Animals were treated with dexamethasone, analgesic, and
penicillin, from 1 day before to 5 days after surgery (
samples (3? 4 mL) were collected by jugular venipuncture into
heparinized tubes and plasma stored at 20?C. All procedures
were approved by the West Virginia University Institutional
Animal Care and Use Committee and followed National Institutes
of Health guidelines for use of animals in research.
Drug and hormone administration
Agonists to NK3R (NKB and senktide) and antagonists to
NK3R (SB222200), -opioid receptors (BNI),
N-methyl-D-aspartate (NMDA) receptors (MK-801), and orphanin-FQ
receptors (UFP-101) were purchased from Tocris Bioscience. The
antagonist to Kiss1r, p271, which inhibits LH secretion when given
icv to OVX ewes (
) was prepared by Dr R. Millar, and the
GnRH receptor antagonist, acyline, was a gift from the Eunice
Kennedy Shriver National Institute of Child Health and Human
Development. Crystalline drugs were stored at either 20?C or
room temperature based on the manufacturer?s
recommendations. Microimplants were cut to either extend 1 mm beyond the
end of the guide tube (experiment 1) or to the tip of the guide tube
(other experiments). The lumen of microimplants was filled by
tamping sterile 22-gauge tubing in crystalline drug at least 60
) and then cleaning the outside with sterile gauze. For
microinjections of Kiss1r antagonist (experiments 3 and 4), stock
solution (26.4 mg/mL or 0.011 mmol/mL, stored at 20?C) was
diluted on the day of the experiment in sterile saline and 150 nL
rapidly injected (over 30 sec) into each hemisphere using sterile
1 L Hamilton syringes with fixed needles that extended to the
tip of the guide tube.
Hypothalamic tissue was collected for immunocytochemistry
and histological determination of treatment sites as previously
). Briefly, ewes were heparinized and killed with an
iv overdose of sodium pentobarbital (8 ?12 mL Euthasol;
Webster Veterinary). When breathing stopped, the head was removed
and perfused via internal carotids with 6 L of 4%
paraformaldehyde in 0.1 M phosphate buffer containing 10 U/mL heparin
and 0.1% NaNO3. Tissue blocks were removed and stored at
4?C in fixative overnight and then in 30% sucrose. After sucrose
infiltration, 45- m-thick frozen coronal sections were cut using
a freezing microtome. For most experiments, every fifth section
through the ARC was stained with cresyl violet and examined to
determine site of treatment. For immunocytochemistry, 12
parallel series of sections (540 m apart) were stored at 20?C in
Tissues for analysis of GnRH inputs to KNDy neurons were
collected during an artificial follicular phase (
). Briefly, OVX
ewes (n 3) were treated with one 0.5-cm-long estradiol implant
and two controlled internal drug release progesterone implants
for 10 ?11 days. One day after controlled internal drug release
progesterone implant removal, four 3-cm-long estradiol
implants were inserted sc to simulate the preovulatory estradiol rise
and tissue collected 18 hours later as described above.
Experiments examining the effects of receptor agonists and
antagonists were performed in OVX ewes over a 3-year period,
and although the general protocol remained the same, minor
details evolved. During the first year (experiments 1?3), blood
samples were collected every 12 minutes for 2 hours before to 4
hours after insertion of microimplants or microinjections.
Thereafter (experiments 4 and 5), blood samples were collected every
10 minutes for 3 hours before to 4 hours after insertion of
microimplants or microinjections. At the end of blood sampling,
microimplants were removed and animals were given an im
injection of 4 mL Gentamicin (Webster Veterinary Supplies)
prophylactically. For each experiment, all ewes received all
treatments (including controls) with treatment order randomized
among the animals in that experiment. There was no effect of
order of treatment in any experiment. In most cases, experiments
were performed in the breeding season (mid-September through
the end of December), but two experiments were done in early
anestrus (March through April). Clear LH pulses are evident in
OVX ewes throughout the year, but LH pulse frequencies are
slightly higher in December than July (
). Because the
experimental design used each animal as its own control, this seasonal
variation did not affect the analyses.
Experiment 1: does endogenous kisspeptin act in the ARC to control LH pulses?
Ewes from a previous study that had guide tubes targeting the
ARC to test the effects of RU486 in OVX ewes treated with
estradiol and progesterone (
) were used in this experiment.
After the last RU486 treatment in February, the peripheral
estradiol and progesterone implants were removed, and the
animals allowed to recover for 3 weeks. In early March, the effects
of empty (control) or Kiss1r antagonist (p271)-filled
microimplants that extended 1 mm beyond the end of the guide tubes
were determined using a crossover design (n 4) with 4 days
Experiment 2: does endogenous NKB act in the
ARC to control LH pulses?
Another group of ewes from the RU486 study (
) was used
in this experiment. After the last RU486 treatment in September,
the peripheral implants were removed, and the animals were
allowed to recover for 6 weeks. In November, the effects of
empty (control) or SB222200-filled microimplants that extended
just to the end of the guide tubes were determined using a
crossover design (n 7) with 7 days between replicates.
Experiment 3: what is the effect of a low dose of
Kiss1r antagonist in the ARC?
In mid-December, the animals (n 7) used in experiment 2
received either bilateral microinjections of 500 pmol of p271 per
side or 150 nL saline (as controls), and LH pulses were monitored
for 2 hours before and 4 hours after injection (this volume was
chosen based on preliminary data indicating that control 200 nL
injections into the ARC inhibited LH pulses). Treatments were
then repeated using a crossover design 3 days later.
Experiment 4: what is the effect of a higher dose of Kiss1r antagonist in the ARC?
Experiment 3 was replicated the next breeding season
(October) in a new group of OVX ewes (n 6) with 2 nmol of p271
or vehicle per side and samples collected every 10 minutes for 3
hours before and 4 hours after microinjection using a crossover
design with 6 days between treatments.
Experiment 5: what are the actions of KNDy neurotransmitters, GnRH, and orphanin-FQ in the ARC?
This experiment tested the local effects of NKB, BNI (
-opioid receptor antagonist), acyline (GnRH receptor antagonist),
UFP-101 (orphanin-FQ receptor antagonist), or MK-801
(NMDA receptor antagonist) using microimplants into the
ARC. Three sets of OVX ewes were used in this experiment,
which took place over the period of a year. The first set (n 6)
received NKB, BNI, and control treatments in September, but
only four of these had correct placements. Therefore, two more
replicates were done; the next set (n 7) received BNI, acyline,
MK-801, and control treatments in April, whereas the last set
(n 6) received senktide (as part of a different experiment
presented elsewhere), NKB, UFP-101, and control treatments in
September. There were 4 ?7 days between replicates in each set.
Immunocytochemistry for kisspeptin, GnRH, and synaptophysin
Triple-label immunofluorescence was conducted on tissue
sections from the middle ARC for kisspeptin, GnRH, and
synaptophysin (a marker for presynaptic terminals), as previously
). Briefly, to visualize kisspeptin and GnRH, tissue
sections were coincubated in monoclonal mouse anti-GnRH
serum (1:8000; Covance) and polyclonal rabbit antikisspeptin-10
serum (1:200 000; gift from A. Caraty, Universit? Tours,
Nouzilly France) for 17 hours. Kisspeptin was visualized with Alexa
555 goat antirabbit (1:100; 30 min; Invitrogen). Next, GnRH
was visualized using biotinylated goat antimouse (1:250: 1 h;
Jackson ImmunoResearch Laboratories), ABC (1:500; Vector
Laboratories), biotinylated tyramide (TSA; 1:250; diluted in PBS
with 1 L of 3% H2O2 per milliliter of total volume; 10 min;
PerkinElmer Life Sciences; catalog number NEL700A), and
Alexa 488-conjugated streptavidin (1:100; 30 min; Jackson
ImmunoResearch Laboratories). Next, sections were incubated
with monoclonal mouse antisynaptophysin (1:200; Sigma;
incubated for 17 h) and Cy5-conjugated goat antimouse (1:100; 30
min; Jackson ImmunoResearch Laboratories). Sections were
mounted on gelatinized slides, dried, and coverslipped with
gelvatol. Specificity and validation of these antibodies in sheep
tissues has been previously described (
controls included omission of one of the primary antibodies from the
protocol; this resulted in complete elimination of labeling for the
corresponding antigen without any effect on the others.
Using a Zeiss LSM-510 laser-scanning confocal microscope
system, images of the ARC of the hypothalamus were captured
in Z-stacks of 1 m optical sections. Alexa 555 fluorescence
(kisspeptin) was imaged with a HeNe1 laser and a 543-nm
emission filter, Alexa 488 fluorescence (GnRH) with an Argon laser
and a 488-nm emission filter, and Cy5 fluorescence
(synaptophysin) with a HeNe2 laser and a 633-nm emission filter. KNDy
cells (30 ?54/ewe) in hemisections (4 ?5/ewe) from the middle
ARC were analyzed for contacts containing GnRH and
synaptophysin. Images were pseudocolored showing kisspeptin in
blue, GnRH in green, and synaptophysin in red to optimally
illustrate GnRH close appositions onto kisspeptin neurons.
LH concentrations were measured as previously described in
duplicate with a RIA using 100 L of plasma and reagents
provided by the National Hormone and Peptide Program. LH assay
sensitivity averaged 0.07 ng/tube (NIH S24) with intra- and
interassay coefficients of variation of 5.5% and 11.2%,
Pulses were identified using established criteria (
average LH concentration, LH pulse amplitude (AMPL), and
interpulse interval (IPI) determined for the following periods:
pretreatment (2 or 3 h, depending on experiment), 0 ?2 hours,
and 2? 4 hours after insertion of microimplants or
microinjections. These values for each experiment were analyzed for
differences between agonist/antagonist and control treatments
using two-way ANOVA with repeated measures (main effects of
time and treatment) with Bonferroni?s t test used to determine
differences between individual values. If a drug increased IPI
beyond 2 hours, this analysis would underestimate drug effects.
Therefore, in these cases, the maximum duration between two
pulses before treatment was compared with this variable during
treatment using two-way ANOVA with repeated measures. P
.05 was statistically significant.
Analysis of GnRH input to KNDy neurons
In the three animals examined, a total of 133 kisspeptin
cells were analyzed and 16.7% 4.5% of these were
contacted by at least one GnRH/synaptophysin-positive
terminal. GnRH terminals were observed in contact with
both kisspeptin cell bodies (Figure 3) and dendrites.
Synaptophysin was colocalized in all GnRH varicosities in
close apposition to KNDy cells, confirming their identity
as presynaptic terminals. Kisspeptin cells bearing GnRH
inputs were not located in any specific region within the
middle ARC but instead were evenly distributed
throughout the cell group at this level. To facilitate a concise
description, the results of the rest of the experiments have
been grouped based on the neurotransmitter being tested,
rather than chronologically.
Sites of microimplantation and microinjection
Based on previous evidence on the spread of drugs from
similar treatments (
), the microimplant or
microinjection sites on both sides of the hypothalamus had to be within
1 mm of the ARC to be considered a correct placement. Using
this criterion 28 of 36 ewes were accepted for analysis of LH
data, with sites concentrated just before or above the start of
the infundibular recess in the anterior-posterior axis
(Supplemental Figure 1, published on The Endocrine Society?s
Journals Online web site at http://endo.endojournals.org).
Missed injections were either dorsal (n 3), through the base
of the hypothalamus (n 2), in the third ventricle (n 2), or
posterior to the ARC in the mammillary body (n 1).
Effects of Kiss1r antagonist
In experiment 1, microimplants containing p271 that
extended beyond the guide tubes into the ARC inhibited
LH pulses after a delay of 1?2 hours in three of four ewes
and significantly increased IPI (Figure 1) and the
maximum time between pulses (before: 51 11 min; after:
117 15 min). However, there was also an inhibition of
LH concentrations with control treatments (Table 1). This
was primarily due to a decrease in LH pulse amplitude
were 141% 13% longer than the
preinjection average (compared with
96% 10% with control
microinjection). Neither dose significantly
altered LH pulse amplitude (Figure 1) or
mean LH concentrations (Table 1).
Effects of NK3R antagonist
Treatment of OVX ewes with the
NK3R antagonist, SB222200,
disrupted episodic LH secretion in all
five ewes and significantly increased
IPI, whereas there was no effect of
empty tubing (Figure 2). Because this
experiment was done in mid-late
December, control IPIs were lower than
in the previous work. Statistical
analysis indicated significant main
effects of time, treatment, and an
interaction of the two. Two ewes had a
complete inhibition during the
posttreatment period; in the other three
ewes, the hiatus in LH pulses was
shorter, ranging from 84 to 108
minutes (Figure 2). Thus, the longest
hiatus between pulses was significantly
greater postimplantation (148 27
min) than before (62.4 7.0 min),
whereas there was no significant
differences in these variables with
control treatments. Mean LH
concentrations were significantly inhibited
by SB222200 (Table 1), but LH pulse
amplitude was not analyzed in this
group because three of five ewes had
no pulses during one time interval. There was no
significant effect of control treatments on mean LH (Table 1) or
LH pulse amplitude (Figure 2). Interestingly, in the two
ewes with misplaced microimplants (one posterior and
one dorsal), SB222200 appeared to increase episodic LH
secretion (Supplemental Figure 2). These data were not
statistically analyzed because of the low animal number.
Effects of NKB and antagonists to the -opioid receptors and NMDA receptors
We next examined the effects of NKB and receptor
antagonists for two other neurotransmitters in KNDy
neurons. There was again no effect of empty microimplants on
LH pulse frequency or amplitude (Figures 4 and 5).
NKBcontaining microimplants clearly decreased IPI in seven of
nine ewes and produced a more modest decrease in the
other two ewes. This decrease in IPI was sustained in most
(Figure 1), although this was not statistically significant
(P .075); amplitude was not analyzed with the
antagonist treatment because three animals had no LH pulses in
the final 2 hours of treatment. Because of the effects of
control treatment in this experiment, we next tested the
effects of placement of empty microimplants to the tip the
guide tube (instead of beyond it into tissue). This treatment
had no effect on episodic LH secretion (data not shown,
but evident in subsequent control treatments), so in all
subsequent experiments, microimplants and needles for
microinjections were lowered to the tip of the guide tubes.
The next two experiments tested the effects of two
doses of this Kiss1r antagonist administered by
microinjection. The lower dose (500 pmol/side) was ineffective
(Supplemental Table 1), but the higher dose (2 nmol/side)
produced a modest increase in IPI just after injection
(Figure 1). This effect was consistently seen when all ewes were
treated with antagonist, so that IPI after the microinjection
Data presented are mean ( SEM) for time periods before and 0 ?2 and 2? 4 hours after start of treatments. All treatments were via microimplants,
except for 2 nmol of p271, which were administered by microinjections.
a P .05 vs control (before treatment) values by two-way ANOVA.
ewes, so IPI was significantly less during both treatment
periods than before treatment (Figures 4 and 5). There was no
effect of NKB on LH pulse amplitude (Figure 5) or mean LH
concentrations (Table 1), and NKB had no effect in the ewes
with missed microimplantation sites (data not shown).
We used BNI and MK-801 to test for the roles of
dynorphin and glutamate, respectively, in the ARC. Because there
was no previous work demonstrating the effectiveness of
MK-801 in ewes, we first tested this antagonist in another
area (the retrochiasmatic area of anestrous ewes) in which
NMDA receptors have been implicated in inhibition of LH
pulse frequency (
). Insertion of microimplants containing
MK-801 into this area (n 7) significantly increased LH
pulse frequency (2.6 0.6 pulses per 4 h) compared with
controls (1.0 0.3 pulses per 4 h). The effects of BNI were
initially tested in the same six ewes that received NKB but
successfully in only three ewes (two had incorrect placements
and one animal that had received NKB had an obstructed
guide tube). Therefore, we examined the effects of BNI, and
MK-801, in an additional seven ewes, five of which had
Administration of BNI markedly decreased IPI in the
2-hour postimplantation in six of seven ewes, and this
decreased IPI was maintained during the last 2 hours in
four of them so that IPI was significantly lower in both
periods (Figures 4 and 5). There was no significantly effect
of BNI on LH pulse amplitude (Figure 5) or mean LH
concentrations (Table 1). MK-801 had no significant
effect on IPI or LH pulse amplitude (Figure 5) or mean LH
concentrations (Table 1). It should be noted that the
increased LH pulse frequency in these ewes when treated
with BNI serves as a useful positive control.
Possible effects of GnRH input to the ARC
afferents onto KNDy neurons were
evident, we examined the possible role
of two constituents of these neurons,
GnRH and orphanin-FQ, using
antagonists to their receptors: acyline
and UFP-101, respectively.
Although acyline increased IPI in one
ewe, neither antagonist had a
statistically significant effect on LH pulse
amplitude, IPI (Figure 6), or mean
LH (Table 1) in these OVX ewes.
frequency, whereas dynorphin acts in this region to hold
pulse frequency in check. In contrast, these data do not
support an important role for glutamate, acting via
NMDA receptors, or orphanin-FQ in the control of LH
pulse frequency in OVX ewes. Similarly, GnRH does not
appear to be important for control of
episodic secretion, even though
GnRH neurons send afferent
projections to a subset of the KNDy cell
population. The decrease in LH
secretion seen after empty guide tubes
were inserted into tissue but not
when they extended just to the tip of
the guide tubes (Figures 1? 4),
although unexpected, supports the
hypothesis that this area plays an
important role in driving episodic
The present study provides the
first direct evidence in sheep that
KNDy neurons receive
monosynaptic input from GnRH neurons and is
consistent with confocal analyses
GnRH-immunoreactive close contacts onto kisspeptin
neurons in monkeys (
GnRH/synaptophysin-containing inputs were identified only at the
light microscopic level, we have previously demonstrated
that synaptophysin-positive close contacts seen with
confocal analysis reliably indicate bona fide synapses when
the same material is examined under the electron
). Because KNDy neurons also project to GnRH
neurons in both the preoptic area
and medial basal hypothalamus
), this observation raises the
possibility of reciprocal connections
between these sets of neurons.
However, it is not clear whether true
reciprocal connections exist because
only 45%-60% of GnRH neurons
receive KNDy inputs,
GnRH-containing contacts on KNDy neurons
were seen in only 17% of cells, and
the specific source of these GnRH
inputs remains to be determined. The
presence of GnRH contacts onto
KNDy neurons is consistent with the
hypothesis that GnRH plays a role in
controlling the activity of these
neurons and could provide a simple
explanation for termination of each
GnRH pulse. This hypothesis is
supported by the report that the
GnRHreceptor antagonist, Nal-Glu,
stimulated GnRH pulse frequency in
luteal phase ewes (
), but we
observed no effect of acyline microimplants in the ARC on
LH pulse frequency or amplitude. It thus seems unlikely
that GnRH input to KNDy neurons plays an important
role in episodic GnRH secretion, although we cannot rule
out the possibility that an insufficient number of GnRH
receptors were blocked with the microimplants of acyline.
Nevertheless, this conclusion is consistent with other data
in sheep (
) and the inability of either a GnRH receptor
agonist or antagonist to alter bursts of MUA in OVX
). In contrast, GnRH increased bursting of MUA
in the rat (
) and stimulated the GnRH release from the
murine GT1?7 cells (
Because acyline was ineffective, we also tested whether
another product of ovine GnRH neurons, orphanin-FQ, is
important for episodic LH secretion. Orphanin-FQ is
found in all GnRH neurons and exogenous orphanin-FQ
inhibits LH pulse frequency in OVX ewes (
microimplants of UFP-101 into the ARC had no effect on
episodic LH secretion, we conclude that orphanin-FQ
released from these inputs is probably not important for
steroid-independent control of GnRH pulses. This
conclusion is consistent with the recent observation that icv
administration of this antagonist increased episodic LH
secretion only in the presence of progesterone (
Although the failures of acyline and UFP-101 to affect LH
secretion do not rule out roles for other components of
GnRH neurons in control of GnRH pulses, they raise the
possibility that the GnRH input to KNDy neurons plays
another role. One possibility is that this input may be
important for modifying KNDy cell function during the
preovulatory GnRH surge because MUA activity
decreases, or is completely eliminated, during the LH surge
in monkeys (
), goats (
), and rats (
Although these results do not support a role for
inhibitory GnRH neural input, they are consistent with the
hypothesis that dynorphin release inhibits KNDy cell activity
to terminate each pulse because BNI microimplants
consistently increased LH pulse frequency. These data
confirm and extend previous data demonstrating that icv
administration of BNI accelerates bursts of MUA in OVX
). In contrast, BNI had no effect on LH pulse
frequency in OVX rats when given icv (
) or into the ARC
), so there may be significant species differences for the
role of dynorphin in pulse generation. In this regard,
recent evidence supports a role for dynorphin in
estrogennegative feedback in rats (
), but endogenous opioid
peptides do not appear to be involved in this action in ewes
). In contrast, there is strong evidence that dynorphin
participates in progesterone-negative feedback in both
pregnant rats (
) and luteal-phase ewes (
). Thus, it
appears that dynorphin inhibits episodic LH secretion in
rats, goats, and sheep, but this inhibitory activity is evident
only in OVX animals in the latter two species.
Of the three possible stimulatory transmitters in KNDy
neurons, the data reported here support a role for NKB
and kisspeptin, but not glutamate, in maintaining episodic
GnRH secretion. The lack of effect of the NMDA receptor
antagonist is somewhat surprising because KNDy neurons
receive significant glutamatergic input (
NMDA stimulates bursting activity of KNDy neurons in
slice preparations (
) and induces pulsatile LH secretion
in sheep (
) and other species (
) including primates
). However, the lack of effect of MK-801 is consistent
with an earlier report that icv administration of a different
NMDA receptor antagonist had no effect on LH pulse
patterns in OVX lambs (
). Perhaps glutamate acts via
both NMDA and
2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid receptors to stimulate KNDy neurons
so that the blockade of both receptor types is needed to
produce an effect.
The local effects of NKB in the ARC to stimulate LH
pulse frequency seen here confirm and extend previous
data demonstrating that icv administration of NKB
accelerates MUA bursts in OVX goats (
). Interestingly, in that
study NKB inhibited LH concentrations; because that
effect was not observed in this work, one can infer that NKB
acts in the ARC to stimulate MUA and LH pulse frequency
but produces inhibitory effects on LH secretion elsewhere
in the ovine hypothalamus. We also did not see the typical
inverse relationship between pulse frequency and
amplitude, probably because of the short duration of the
treatment. These results are consistent with accumulating data
from a number of species, including sheep, that the
stimulatory effects of NK3R agonists are mediated by
kisspeptin release from KNDy neurons (
). It should be
noted, however, that senktide inhibited episodic LH
secretion and bursts of MUA in OVX rats (67); interestingly,
this action of senktide appears to be mediated by
). Although there are thus considerable data on
the actions of exogenous NK3R agonists, this is the first
report that an NK3R antagonist (SB222200) disrupts
episodic LH secretion in OVX animals. In contrast, in OVX
rats, SB222200 (
) and another specific NK3R
) had no effect on episodic LH secretion. Thus, as
was the case with dynorphin, there appear to be species
differences in the role of endogenous NKB, but the
opposite effects of NKB and the NK3R antagonist in this study
provides strong support for the proposed role of NKB in
initiating each GnRH pulse in sheep. The infertility (
and suppression of episodic LH secretion (
) seen in
humans with inactivating mutations of NKB signaling are
also consistent with this postulated role for NKB.
The ability of microimplants of a Kiss1r antagonist to
inhibit LH pulses and for microinjections of this
antagonist to slow pulse frequency suggests that endogenous
kisspeptin may act in the ARC to stimulate LH pulse frequency
in OVX ewes. This is somewhat surprising because
exogenous kisspeptin did not alter the frequency of MUA in
OVX goats (
) and rats (
), but our data are consistent
with the effects of intra-ARC injections of a similar Kiss1r
antagonist (peptide 234) in OVX rats (
). It should be
noted that both studies of MUA used iv injection of
kisspeptin-10 so this peptide may not have reached the ARC
at effective concentrations. One possible explanation for
the effects of the antagonist is that it diffused to the median
eminence to block kisspeptin effects on GnRH terminals
), but this is unlikely because it produced only a 40%
increment in IPI in our study and no effects on LH pulse
amplitude of intra-ARC administration were observed
when this antagonist was given into the ARC of sheep or
). Although endogenous kisspeptin appears to act
within the ARC to stimulate GnRH/LH pulse frequency,
the specific neurons affected remain to be determined.
There is evidence for Kiss1r mRNA in the ARC of several
species, including sheep and rats (
), but the only cellular
analysis has been done in sheep, in which Kiss1r mRNA
was found only in cells that did not express Kiss1 mRNA
In conclusion, the results indicate that input from
GnRH neurons to the ARC is likely not important to the
control of GnRH pulse frequency, but endogenous
kisspeptin may act in this area to stimulate GnRH pulse
frequency. The data also provide strong support for the
hypotheses that NKB acts in the ARC to initiate each GnRH
pulse, whereas the inhibitory actions of dynorphin are
important for pulse termination in ewes.
We thank Heather Bungard, Jennifer Lydon, and Cheri Felix
(West Virginia University Food Animal Research Facility) and
Dr Margaret Minch for the care of the animals and Paul Harton
for his technical assistance in sectioning tissue. We also thank Dr
Al Parlow and the National Hormone and Peptide Program for
reagents used to measure LH and the Contraception and
Reproductive Health Branch of the Eunice Kennedy Shriver National
Institute of Child Health and Human Development for acyline.
Address all correspondence and requests for reprints to:
Robert L. Goodman, PhD, Department of Physiology and
Pharmacology, PO Box 9229, West Virginia University, Morgantown,
West Virginia 26506. E-mail: .
This work was supported by National Institutes of Health
Grants R01-HD039916 and RO1-HD017864.
Disclosure Summary: The authors have nothing to disclose.
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