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Introduction
Nociceptin/orphanin FQ (OFQ) is a heptadecapeptide that
exhibits high amino acid sequence homology to the
endogenous opioid peptides, especially dynorphin
A[1]. OFQ is the putative endogenous ligand for the opioid receptor-like
1 receptor (ORL1 receptor)[2]. The ORL1 receptor has
structural and functional homology with the δ, κ, and
m classic opioid receptors. Despite its close similarity to opioid
receptors, the ORL1 receptor does not selectively bind
opioids and opioid antagonists[2]. A number of physiological
effects of OFQ have been reported, including nociceptive
modulation, and cardiovascular and renal physiological
functions[3].
The accumulated investigations suggest OFQ, like the
opiate, may be involved in the regulation of anterior
pituitary hormone secretion. It has been demonstrated that OFQ
activates the G-protein coupled, inwardly-rectifying
K+ channel and hyperpolarizes many hypothalamic neurosecretion
cells, especially in the arcuate nucleus
(ARC)[4]. OFQ administration stimulates prolactin and growth hormone release
in a dose- and time-related manner[5], and OFQ plays a
physiologically significant role in the regulation of prolactin
secretion[6]. A recent study has revealed that OFQ potently
and dose-dependently inhibits forskolin-induced,
gonadotropin-releasing hormone (GnRH) release from rat
hypothalamic fragments[7]. Our previous work demonstrates that the
central administration of OFQ might inhibit the release of
hypothalamic GnRH and decrease the level of plasma
luteinizing hormone (LH) via the ORL1 receptor in ovariectomized
(OVX) rats[8]. Such an OFQ-induced alteration of anterior
pituitary hormone secretion would implicate OFQ as an
important modulator of reproductive function.
GnRH, as an important hypothalamic decapeptide, plays
a key role in the functions of the
hypothalamic-pituitary-ovary axis (HPOA) by modulating the secretion of LH and
the follicle-stimulating hormone (FSH) from the anterior
pituitary over the estrus cycle[9]. The most marked of these
modulations is the LH surge at mid cycle, a critical event
responsible for initiating
ovulation[10]. Evidence in published studies has demonstrated that the neurotransmission of
afferent neuronal systems that are sensitive to steroids is
necessary to stimulate GnRH neurons to induce the mid-cycle
LH surge[10,11]. There is general agreement that endogenous
opioid peptides (EOP) mediate the negative feedback action
of steroid hormones on GnRH pulse frequency during the
estrus cycle[12]. The precise mechanisms underlying the
opioid neural system mediating the steroid regulation of
GnRH secretion remains to be determined.
OFQ and the ORL1 receptor are densely localized in the
pre-optic area (POA), ventromedial hypothalamus (VMH) and
ARC, all of which have been identified as the chief
hypothalamic nuclei regulating GnRH
release[9,13]. As far as the inhibitory effect of OFQ on GnRH and LH release are concerned,
we postulate that hypothalamus OFQ might be involved in
the neuroendocrinological regulation of the LH surge during
the estrus cycle. The estrogen and progesterone primed
(EBP), ovariectomized (OVX) rats have always been adopted
for studying the neuroendocrine mechanisms underlying the
LH surge during the estrus cycle[14,15]. To test the hypothesis,
we also observed the effects of the central administration of
OFQ on the LH surge of EBP-primed, OVX rats. Moreover,
the changes in the mRNA and protein levels of OFQ coupled
with the ORL1 receptor in the related hypothalamic area are
also investigated at different stages during the estrus cycle
of female rats.
Materials and methods
Animals and drugs Female Sprague-Dawley rats
weighing 160_200 g were obtained from the Animal Center of
Shanghai, Fudan University (Shanghai, China). They were
kept in an air-conditioned room with controlled lighting (12 h
light/12 h dark) and given free access to laboratory chow
and tap water. Nociceptin/orphanin OFQ
(MW 1810) and
[Nphe1]NC(1_13)NH2 (NC13;
MW=1382) were purchased from Phoenix Pharmaceutical Company (St Joseph, MD, USA).
The compounds were dissolved in artificial cerebrospinal
fluid (ACSF, 128 mmol/L NaCl, 2.5 mmol/L KCl, 1.4 mmol/L
CaCl2, 1.0 mmol/L MgCl2, and 1.2 mmol/L
Na2HPO4; pH 7.4). All other reagents and solvents were of analytical grade.
EBP-primed, OVX rats and the induction of the LH surge
The female rats were subjected to the surgical removal of
both ovaries (OVX) under aseptic conditions. The
experiments were carried out 4_6 weeks after the ovariectomy. The
rats were given 7.5 mg estradiol benzoate (EB)
subcutaneously in corn oil at 10.00 hours. Progesterone (5 mg) was
administered subcutaneously in corn oil 48 h after the EB
treatment. The studies were performed from 12.00 to 20.00
hours, when the steroid-induced LH surge release took place
between 13.30 and 14.30 hours[14,15].
Intracerebroventricular injection The implantation of
the cannula was performed stereotaxically under anesthesia
with sodium pentobarbital (40 mg/kg, ip). Stereotaxic
surgical procedures were used to implant 1 22 gauge, stainless
steel guide cannula with a removable 28 gauge, inner stylette
to the left lateral ventricle (bregma AP: 1.0 mm; length: 1.5
mm; height: 3.0 mm)[16]. Experiments with
intracerebroven-tricular (icv) injection were performed at least 7 d after the
operation. The ACSF was added with the protease inhibitor
(1 g/L) for preventing proteolysis after the injection of OFQ
and NC13. OFQ dissolved in 10 µL ACSF was infused through
a 28 gauge cannula, extending 0.5 mm beyond the guide
cannula. The needle was connected to a 10 µL syringe by a
polyethylene tube and the drug solutions were delivered by
an infusion pump at a flow rate of 5 µL/min. The injection
needle was maintained in place for an extensive period of 10
min after the injection. Without any restraint, the EBP-primed,
OVX rats were icv injected with ACSF (10 µL, as the control),
OFQ (20 or 200 nmol OFQ in 10 µL ACSF), NC13+OFQ (20
nmol NC13 in 2 µL ACSF, followed by 20 nmol OFQ in 8 µL
ACSF) or NC13 (20 nmol NC13 in 10 µL ACSF) at 14.00 hours.
Sequential blood samples, each 200 µL, were taken through
the indwelling cannula from the tail veins of EBP-primed,
OVX rats at 2 h intervals between 12.00 to 20.00 hours. The
same volume of physiological saline was replaced at each
bleeding. Blood plasma (50 µL) was separated from each
blood sample by centrifugation and stored at -20 °C until the
LH radioimmunoassay (RIA).
LH RIA Plasma LH was measured in duplicate samples
(n=6) by double antibody RIA using an LH RIA kit (Shanghai
Institute of Biological Products, Shanghai, China) according
to the manufacturer's instructions. Cross-reactivities to the
related compound were less than 3.0%, and the sensitivity
of the assay was 0.4 ng/mL. The intra-assay and the
interassay coefficient of variation was 3.5% and 4.8%,
respectively, over the range of 2.0_20 ng/mL.
RT-PCR analysis Daily vaginal smears were obtained
and only the rats having 2 consecutive 4 d estrus cycles
were used for the experiments. The stage of the estrus cycle
was monitored by the collection of vaginal smears at 6 h
intervals and each of the 6 animals was decapitated on
diestrus, pro-estrus, and estrus, respectively. The total
cytoplasmic RNA was isolated from the POA of the
hypothalamus as described by Lamar et
al[17] using Trizol reagent (Life Technologies, Rockville, MD, USA). The total RNA (4 µg)
was digested with DNase RNase-free enzyme to eliminate
genomic DNA, and then converted to cDNA using 200 U
Moloney murine leukemia virus reverse transcriptase
(Promega, Madison, WI, USA) in 20 µL buffer containing
0.4 mmol/L deoxynucleotide triphosphates, 20 U RNase
inhibitor, and 0.8 µg oligo
(deoxythymidine)15 (Sino-American Biotechnology Company, Shanghai, China). The
specific primers for the ORL1 receptor were
5'-CAGGC-TGTTAATGTGGCCATATG-3' and
5'-GAGCCTGAAAGCA-GACGGACACC-3' and the primers for amplifying the OFQ
were 5'-GTGACTCTGAGCAGCTCAGC-3' and 5'-TTCTGG-TTGGCCAACTTCCG-3' (synthesized at the Shanghai Sangon
Biological Engineering Technology and Service Company,
Shanghai, China)[18]. The housekeeping gene
β-actin expression was used as an internal control since it is widely
utilized for PCR reaction and did not change in the rat
hypothalamus during the estrus cycle (data not shown). The PCR
conditions were carried out as follows: denaturation at 94 °C
for 45 s, annealing at 60 °C for 45 s, and elongation at 72 °C
for 60 s with Thermus aquaticus DNA polymerase (Promega,
USA; 28 cycles for the ORL1 receptor and prepronociceptin,
and 18 cycles for β-actin). The RT-PCR products (5 µL) were
electrophoresed in a 1.6% agarose ethidium bromide gel and
visualized using the SYNGENE imaging system (GeneSnape
Software, London, UK).
Immunohistochemistry The stage of the estrus cycle
was monitored by the collection of vaginal smears at 6 h
intervals and each of the 6 animals were deeply anesthetized
with 75 mg/kg sodium pentobarbital ip on diestrus, pro-estrus,
and estrus, respectively. They were transcardially perfused
with 200 mL of 0.9% NaCl, followed by 300 mL fixative
containing 4% paraformaldehyde (PF) in 0.1 mol/L phosphate
buffer (PB; pH 7.4). The brains were removed and postfixed
for 48 h in 4% PF with 10% sucrose and sectioned coronally
on cryostat (Leica CM1800, Nussloch, Germany) at 30 µm
and stored in cryoprotectant (sodium PB, pH 7.4, with
0.9% saline, 30% sucrose, and 30% ethylene glycol) at -20 °C
until use. The floating rat brain sections were washed in 0.01
mol/L phosphate-buffered saline (PBS) to remove the
cryoprotectant. The sections were incubated for 60 min in
bovine serum albumin (BSA) diluent (0.01 mol/L PBS with
0.9% NaCl, 5% BSA, and 0.3% Triton X-100), then
transferred to a solution containing rabbit anti-rat OFQ antibody
(1:1000, Phoenix Pharmaceutical Company, St Joseph, MO,
USA) or a rabbit anti-rat GnRH antibody (1:2000, Chemicon
Interna-tional, Temecula, California, USA) in BSA diluent,
and incubated at 24 °C for 48 h. After the primary antibody
incubation, the sections were washed in 0.01 mol/L PBS and
incubated with biotinylated goat anti-rabbit
immunoglobulin G (IgG; 1:1000) for 60 min at room temperature, followed
by an avidin biotin complex coupled to horseradish
peroxidase (1:1000, Vector Elite Kit, Vector
Laboratories, Burlingame, CA, USA) for 60 min, also at room temperature.
Immuno-staining was visualized with a 0.04% solution of
3,38-diaminobenzidine tetrahydrochloride (DAB) containing
2.5% nickel chloride and 0.01%
H2O2 dissolved in 0.1 mol/L
sodium acetate. After 5 min in DAB, the reaction was
terminated by 2 consecutive washes in 0.9% NaCl. The sections
were mounted from 0.9% NaCl onto polylysine-subbed
microscope slides, dehydrated with graded alcohol, followed
by xylene, and coverslipped with Permount (Fisher
Scientific Company, Pittsburgh, PA, USA). Immunohistochemical
bright field staining was viewed with a microscope (Nikon,
Hasunuma, Tokyo, Japan) with camera attachments, and the
number and the mean optical density of the immunoactive
neurons and fibers were observed and analyzed under the
support of Image Master VDS (Amersham Pharmacia Biotech,
Piscataway, NJ, USA).
Western blotting The dissection of the medial basal
hypothalamus (MBH) fragments and protein isolation of the
above samples were performed as described by Lamar
et al[17]. The protein samples (100 µg/sample) were obtained with the
assistance of a BCA-100 protein quantitative analysis kit
(Shenergy Biocolor BioScience Technology Company, Shanghai, China) and were separated by 10% SDS-PAGE.
Molecular weight standards were loaded onto each gel to
verify the stained proteins. The proteins were then
electro-blotted onto polyvinylidene difluoride membranes (BioRad,
Hercules, CA, USA). The polyclonal antibodies against the
ORL1 receptor (1:500, Santa Cruz Bio-technology, Santa Cruz,
CA, USA) and β-actin (1:500, Boster Biotechnology
Com-pany, Wuhan, China) were used to detect changes of the
ORL1 receptor protein and β-actin (as the internal control)
according to the manufacturer's protocols. The secondary
goat anti-rabbit IgG (1:200, Sino-American Biotechnology
Company, China) was conjugated to horseradish peroxidase
in conjunction with the enhanced chemiluminescence
system (ECI, Amersham Biosciences, Little Chalfont, UK) which
allowed for the visualization of proteins. Protein
determination was made using the SYNGENE imaging system
(Gene-Snape Software, UK).
Statistical analysis All results were expressed as mean±
SD and were analyzed by SPSS statistical software (SPSS
Inc, Chicago, Illinois, USA). The data in Figure 1 were
analyzed by using a 2-way repeated measures ANOVA with time
as the repeated measure. The other results were analyzed by
1-way ANOVA, and the significance of difference was
determined by the Newman-Keuls test. When only 2 treatment
groups were compared, Student's t-test was used. A
probability of P<0.05 was considered to be statistically significant.
Results
Effect of OFQ and NC13 icv injection on EBP-primed
OVX rats As reported in previous
studies[14,15], it was apparent that the LH level in the control group began to
increase at 14.00 hours (6.72±0.85 ng/mL), but with no
statistical significance, as compared with the base LH level at 12.00
hours (3.68±0.54 ng/mL). The significant steroid-induced
plasma LH surge in EBP-primed, OVX rats took place at 16.00
hours (9.79±1.18 ng/mL) and reached its peak at about 18.00
hours (12.38±1.32 ng/mL). In the animals receiving icv
injections of 20 and 200 nmol OFQ at 14.00 hours, the
steroid-induced LH surges also occurred in the same period, but
with lower amplitudes. The LH levels of the 20 and 200 nmol
OFQ groups at 16.00 and 18.00 hours decreased significantly
in comparison with the control group at the same time
(P<0.05). OFQ showed a dose-related inhibitory effect on the
LH surge of EBP-primed, OVX rats (Figure 1). The
significantly decreased LH surge at 18.00 hours in the 20 nmol OFQ
group (9.27±1.33 ng/mL) was abolished by pretreatment with
20 nmol NC13, a competitive antagonist of the ORL1
receptor (11.98±1.57 ng/mL). No change in the LH surge at 18.00
hours was observed with a single icv injection of 20 nmol
NC13 (Figure 2).
Change of OFQ and ORL1 mRNA levels in the POA
during the estrus cycle The relative mRNA level of OFQ in
the POA on pro-estrus (0.36±0.09) was observed to decrease
significantly than those on diestrus and estrus (0.59±0.11
and 0.51±0.12; P<0.05), and the discrepancy between diestrus
and estrus did not reach statistical significance (Figure 3
and 4). The relative levels of the ORL1 receptor mRNA
expression in the POA were not significantly different among
the 3 stages of the estrus cycle (Figure 4).
Change of OFQ-immunoreactive neurons and GnRH-IR
fibers in related areas of the hypothalamus during the
estrus cycle Throughout the rostral half of the hypothalamus,
some anterior and lateral hypothalamic areas such as the
diagonal band of Broca (DBB), paraventricular nucleus,
supraoptic nuclei, and the medial POA (mPOA) contained
sparse, moderately stained OFQ-immunoreactive (OFQ-IR)
neurons immediately adjacent to the third ventricle, with
moderate fiber and terminal labeling. The ARC also
contained moderately stained cells and fibers, with numerous
puncta throughout its rostral to caudal extent. Lateral to the
ARC, lightly stained neurons were present in the VMH
nucleus VMH (Figure 5). The number of OFQ-IR neurons in
the mPOA (19.6±3.6) and VMH (21.8±4.8) in the
hypothalamus on pro-estrus decreased significantly than those on
diestrus (25.8±4.2 and 29.2±4.6) and estrus (24.8±4.4 and
30.4±4.2; P<0.05). The site-specific downregulation of
OFQ-IR neurons on pro-estrus was also observed in ARC, but not
in DBB (Figure 6). There were no statistical significances
detected between diestrus and estrus in those hypothalamic
nucleus mentioned earlier.
The median eminence (ME) contained an intense GnRH
immunoreactive (GnRH-IR) fiber plexus and numerous puncta.
Large, densely stained fibers filled the more medial aspect of
ME in its rostral part (Figure 7). As expected, a significant
increase in the mean optic density of GnRH-IR fibers in ME
was observed on pro-estrus (27.6±4.4), as compared with
those on diestrus and estrus (19.4±2.7 and 20.4±3.1; Figure 8).
Western blot analysis There were no statistically
significant differences detected in the relative ORL1 receptor
protein levels in the MBH among the diestrus (0.38±0.08),
pro-estrus (0.32±0.07), and estrus (0.43±0.12; Figure 9). The
Western blot finding was consistent with the change of the
ORL1 receptor mRNA expression in the POA.
Discussion
GnRH is the primary brain signal responsible for the
release of LH and FSH from the anterior pituitary gland. Many
classical neurotransmitters and neuropeptides alter GnRH
neuronal activity through direct and sometimes indirect
actions[19]. EOP constitutes an important inhibitory
component of the neural circuitry that regulates GnRH secretion,
and a significant decrease in the inhibitory opioid tone is
critical for the generation of a LH surge during the estrus
cycle[11]. Immunohistochemical staining and an
in situ
hybridization study have indicated that OFQ and the ORL1
receptor are abundant in the hypothalamus, especially in the
mPOA, VMH, supraoptic nuclei, ARC, DBB, and ME, many
of which are closely relevant for the regulation of GnRH
release[20]. The homology between the opiate peptides and
OFQ, as well as between their receptors, coupled with the
localization of OFQ and its receptor in the hypothalamus,
suggest a neuroendocrine role for OFQ in
reproduction[13, 21].
Previous studies have shown that OFQ can potently
inhibit GnRH release from MBH fragments in
vitro and from the POA of OVX rats in
vivo[7,8]. These studies offer
important clues about a possible participation of OFQ in the
regulation of the HPOA. Our initial experiments confirmed that
OFQ icv injections of 20 or 200 nmol can significantly
decrease plasma LH levels of OVX rats, and the strongest
effects occurred 2 h after
administration[8]. Based on the results,
we hypothesize that OFQ is most likely a predominant
regulator of GnRH and LH secretion, at least in rats. In addition,
the present study reveals that the central administration of
20 or 200 nmol OFQ at 14:00 h results in significant decreases
of pre-ovulatory LH surges induced in the EBP-primed, OVX
rats. Commonly accepted as the ideal animal model adopted
for reproduction research, the EBP-primed, OVX rats may
help us to elucidate the neuroendocrine mechanisms
underlying the LH surge during the estrus
cycle[14,15]. Due to the limits of observing periods and sampling intervals in our
experiments, we can not exclude the possibility that OFQ is
not only able to decrease, but can also delay pre-ovulatory
LH surges in the EBP-primed, OVX rats.
As the first identified selective ORL1 receptor antagonist,
[Nphe1]NC(1_13)NH2, that is, NC13, has allowed us to
characterize biological effects that are clearly mediated through
the ORL1 receptor in vitro and in
vivo[3]. The inhibitory effect of 20 nmol OFQ on the LH surge in the EBP-primed,
OVX rats can be reversed by pretreatment with 20 nmol
selective ORL1 receptor antagonist NC13. In our
preliminary experiments, it was found that an icv injection of 20
nmol NC13 alone has no marked influence on plasma LH
levels in OVX rats. These results further indicate that the
decrease of pre-ovulatory LH secretion induced by OFQ
administration is mediated by the ORL1 receptor in the brain.
There has been no evidence of OFQ receptors on the
anterior pituitary gland until now, implying that OFQ might have
no direct effect on pituitary hormone secretion. Taken
together, it is strongly suggested that by interacting with its
own receptor, the ORL1 receptor, OFQ elicits its inhibitory
effect on hypothalamus GnRH release, thus decreasing the
plasma LH surge in the estrus cycle.
The secretion of LH, which is assumed to reflect the GnRH
secretion from the hypothalamus into the portal vein, shows
2 remarkably different patterns. One is pulsatile LH
secretion and the other is pre-ovulatory LH surge
secretion[16]. The ovarian steroid hormone estradiol feeds back at the
hypothalamus to regulate the patterns of release of GnRH
and the gonadotropins. Estradiol exerts an inhibitory effect
on the GnRH secretion on diestrus and estrus of the estrus
cycle of female rats, while the rising circulating estradiol
levels cause an increase in GnRH release only during pro-
estrus[12,22]. Recent evidence has demonstrated that the
neurotransmission of afferent neuronal systems that are
receptive to estradiol is necessary to drive reproductive
cyclicity[10]. Using the double in situ
histochemistry method in female rats, it has been observed that the estrogen receptor beta
mRNA co-expressed with OFQ mRNA in some nucleus of the
hypothalamus, indicating that estrogen may hold a direct
effect on OFQ neurons[23]. Quite recently, it was reported
that OFQ mRNA levels in the hypothalamus were also
regulated by steroids in EBP-primed, OVX
rats[24]. Taken together, it is strongly suggested that OFQ is likely to play a role in
mediating the negative feedback effects of estrogen on GnRH
release.
Studies in the past few decades have demonstrated that
the neuronal component responsible for inducing the GnRH
and LH surges is located in the POA and MBH of the
hypothalamus[20]. In our study, we observed OFQ mRNA
expression in the POA across the estrus cycle and found that the
levels were reduced in the POA on pro-estrus. The higher
level of OFQ mRNA expression on diestrus and estrus is
consistent with an inhibitory role for OFQ because GnRH
and LH secretion are restrained at this time by the combined
negative feedback effects of estrogen and progesterone. The
number of OFQ-IR neurons in the mPOA, VMH, and ARC
was also found to decrease more significantly on pro-estrus
than those on diestrus and estrus. This finding suggests
the pre-ovulatory decline of OFQ mRNA and the protein
levels in those chief nucleus governing GnRH surge secretion
is likely to be associated with the onset of the pre-ovulatory
LH surge in cyclic female rats. Undoubtedly, it needs more
strong evidence to confirm that a decrease in OFQ tone in
the POA is involved in the activation of GnRH neurons. Our
next study will focus on determining the OFQ release in the
POA during the estrus cycle by microdialysis.
GnRH is synthesized in neuronal cell bodies diffusely
distributed across the basal forebrain and is secreted from
neuronal terminals in ME[21]. Because of the difficulties in
studying the diffusely distributed GnRH neurons and
measuring the arbitrary volume of GnRH secretion in
hypophyseal portal blood, we examined GnRH neuronal terminals in
ME during the estrus cycle using immunohistochemistry and
computer-assisted techniques. To some degree, the number
and mean optic density of GnRH-IR fibers in ME reflect the
content of immunoassayable GnRH released into the
fenestrated capillaries in ME, which is commensurate with the
quantities of GnRH transported from hypothalamic GnRH
neurons to the pituitary portal system. Therefore, the
significant increase in the mean optic density of GnRH-IR fibers
observed in our experiments in ME on pro-estrus is parallel
with the GnRH and the LH surge that occurred at the same
time. These results also confirm the feasibility and accuracy
of ascertaining the different stages in the estrus cycle by
vaginal smear investigations. Admittedly, an obvious
question that remains to be answered is whether the down
regulation of OFQ mRNA and protein level in POA occurs just
before the pre-ovulatory LH surge on pro-estrus in cycling
rats.
We have also measured the mRNA and protein levels of
the ORL1 receptor in the POA and MBH in the 3 stages
during the estrus cycle. The high expression level of the
ORL1 receptor mRNA and protein was detected in the POA
and VMH, which is consistent with the localization of the
ORL-1 receptor in the hypothalamus of
rats[21]. The results presented in our study show that the mRNA level of the
ORL1 receptor in the POA on pro-estrus has no evident
difference with those on diestrus and estrus. Accordingly, the
Western blot analysis indicated that there are no statistical
significant changes in the protein levels of the ORL1
receptor during the estrus cycle. These data indicate that periodic
steroid hormone fluctuations in the estrus cycle have no
influence on the ORL1 receptor mRNA level, which is
inconsistent with what has been documented by other
investigators who found that sex-related differences in the ORL1
receptor gene expression appear to be determined in part by
estrogen levels[25].
Although the precise neuroendocrine mechanisms for
the LH surge in cycling rats remain to be elucidated, the
current findings support our hypothesis that the
hypothalamic endogenous OFQ might participate in the regulation of
GnRH and LH secretion. Taken together with earlier studies,
these results suggest that OFQ may be a novel
hypothalamic modulator of reproduction.
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