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Endocrinology Vol. 148, No. 10 4993-5001
Copyright © 2007 by The Endocrine Society

Orphanin FQ: Evidence for a Role in the Control of the Reproductive Neuroendocrine System

Chad D. Foradori, Marcel Amstalden, Lique M. Coolen, Sushma R. Singh, Christine J. McManus, Robert J. Handa, Robert L. Goodman and Michael N. Lehman

Department of Biomedical Sciences (C.D.F., R.J.H.), Neuroscience Division, Colorado State University, Fort Collins, Colorado 80523; Department of Cell Biology, Neurobiology, and Anatomy (M.A., L.M.C., M.N.L.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521; and Department of Physiology and Pharmacology (S.R.S., C.J.M., R.L.G.), West Virginia University Health Sciences Center, Morgantown, West Virginia 26506-9229

Address all correspondence and requests for reprints to: Michael N. Lehman, Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario, Canada N6A 5C1. E-mail: michael.lehman{at}schulich.uwo.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Orphanin FQ (OFQ), also known as nociceptin, is a member of the endogenous opioid peptide family that has been functionally implicated in the control of pain, anxiety, circadian rhythms, and neuroendocrine function. In the reproductive system, endogenous opioid peptides are involved in the steroid feedback control of GnRH pulses and the induction of the GnRH surge. The distribution of OFQ in the preoptic area and hypothalamus overlaps with GnRH, and in vitro evidence suggests that OFQ can inhibit GnRH secretion from hypothalamic fragments. Using the sheep as a model, we examined the potential anatomical colocalization between OFQ and GnRH using dual-label immunocytochemistry. Confocal microscopy revealed that approximately 93% of GnRH neurons, evenly distributed across brain regions, were also immunoreactive for OFQ. In addition, almost all GnRH fibers and terminals in the external zone of the median eminence, the site of neurosecretory release of GnRH, also colocalized OFQ. This high degree of colocalization suggested that OFQ might be functionally important in controlling reproductive endocrine events. We tested this possibility by examining the effects of intracerebroventricular administration of [Arg14, Lys15] OFQ, an agonist to the OFQ receptor, on pulsatile LH secretion. The agonist inhibited LH pulse frequency in both luteal phase and ovariectomized ewes and suppressed pulse amplitude in the latter. The results provide in vivo evidence supporting a role for OFQ in the control of GnRH secretion and raise the possibility that it acts as part of an ultrashort, autocrine feedback loop controlling GnRH pulses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ORPHANIN FQ (OFQ), also known as nociceptin, is a member of the family of endogenous opioid peptides, which also includes ß-endorphin, enkephalins, and dynorphin (1). Whereas sharing some homology with dynorphin, OFQ does not exhibit appreciable binding affinity for classical opioid receptors (1, 2); rather it functions as an endogenous ligand for the opioid receptor like (ORL)-1 receptor (1). OFQ cells are present in the preoptic area, anterior hypothalamus, and arcuate nucleus of the hypothalamus of the rat (3) and human (4). Functionally, OFQ has been implicated in a variety of systems including pain (5), anxiety (6), cardiovascular function (7), food intake (8), circadian rhythms (9), and cognition (10). There is also evidence that OFQ may play a role in neuroendocrine function: intracerebroventricular delivery of OFQ in the rat stimulates GH and prolactin (11, 12) and has been reported to both stimulate (13) and inhibit (14) corticosterone secretion. Sinchak et al. (15) have shown that OFQ delivered into the ventromedial nucleus facilitates lordosis in female rats in a dose-dependent manner. However, there has been relatively little attention to the possible role of OFQ in reproductive neuroendocrine function, specifically in the control of GnRH secretion.

GnRH neurons, and their projections to the median eminence, control the secretion of pituitary LH and thus comprise the final common pathway for the neuroendocrine control of reproduction (16). Endogenous opioid peptides are important regulators of the GnRH system, controlling both the preovulatory surge (17) and pulsatile secretion (18) of GnRH. OFQ has been shown to inhibit forskolin-induced GnRH secretion in a dose-dependent manner from rat hypothalamic fragments (19). OFQ cells in the preoptic area and anterior hypothalamus overlap the site of a majority of GnRH perikarya (20, 21, 22). In addition, OFQ fibers and ORL-1 receptors are present in high abundance in the median eminence (4, 23, 24) in which GnRH is released in portal blood. Thus, the distribution of OFQ and its receptor overlap that of GnRH neurons and fibers, making OFQ a potential candidate for controlling GnRH and LH release.

To assess the possible functional role of OFQ in the control of GnRH secretion, we chose the sheep as a model because of the ability to easily monitor LH pulses in this species as well as evidence implicating EOPs in the control of GnRH and LH pulses (18, 25). First, we examined possible anatomical overlap between OFQ and GnRH using dual-label immunocytochemistry and confocal microscopy. Second, we determined the effects of intracerebroventricular administration of OFQ or an ORL-1 agonist on LH secretion in ovary-intact and ovariectomized (OVX) ewes. The results reveal extensive cellular colocalization between OFQ and GnRH and provide in vivo evidence for a role of OFQ in central regulation of the reproductive neuroendocrine system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult blackface ewes, showing regular estrous cycles, were maintained in an open barn with free access to water and fed once daily with a maintenance regimen of silage. They were moved to indoor facilities 2–3 d before experimentation. Once indoors, the animals were kept two per pen under a photoperiod similar to that occurring outdoors and fed a maintenance diet of alfalfa pellets and corn. Blood samples were taken by jugular venipuncture, collected into heparinized tubes, and plasma harvested the next day and stored at –20 C until assayed. The routine handling and experimental procedures involving sheep were approved by the West Virginia University Animal Care and Use Committee.

Experiment 1: immunocytochemical examination of the overlap between OFQ and GnRH in the preoptic area and hypothalamus
This study was performed during the breeding season (October through January) in four ewes in the midluteal phase (d 6–9). Animals were heparinized (two iv injections of 25,000 U heparin given 10 min apart), then deeply anesthetized with sodium pentobarbital (~4000 mg, iv), and rapidly decapitated. The heads were perfused via both internal carotids with 6 liters of 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.3) containing 0.1% sodium nitrite and 10 U/ml heparin. Serum and ovaries were also harvested to verify hormone levels and the presence of corpora lutea consistent with midluteal sheep. After perfusion of the head, the brain was removed and a tissue block containing the septal region, preoptic area, and hypothalamus was dissected out. The tissue was stored in 4% paraformaldehyde at 4 C overnight and then placed in 30% sucrose at 4 C until infiltration was complete. Thick (50 µm) frozen coronal sections were cut and stored at –20 C in a cryopreservative solution (26) until being processed immunohistochemically for OFQ and GnRH.

Experiment 2: the effects of OFQ or an agonist for the ORL-1 receptor on LH secretion in luteal phase ewes
We stereotaxically implanted an 18-gauge stainless steel guide tube (n = 10, experiment 2) into the third ventricle using sterile techniques and gas anesthesia (4 parts oxygen: 1 part nitrous oxide, supplemented with halothane at 1–4%) as previously described (27). Briefly, a 2-cm diameter hole was drilled in the skull, the sagittal sinus ligated, and radioopaque dye injected into one lateral ventricle. Lateral and frontal x-rays were then used to place the tip of the tubing into the upper portion of the third ventricle approximately 2 mm posterior to its rostral edge. Placement into the ventricle was confirmed by back flow, or withdrawal of, cerebral spinal fluid (CSF). After protecting the surface of the brain with gel foam and a wire mesh, the tubing was plugged to prevent CSF flow, cemented in place with cranial screws and dental acrylic, and protected with a plastic cap. Animals were allowed to recover from all surgeries for at least 3 wk before experimental procedures were performed.

OFQ and the potent OFQ agonist, [Arg14, Lys15] OFQ were purchased from Tocris Bioscience (Ellisville, MO), diluted to a concentration of 1 nm per 200 µl in sterile saline, and stored at –20 C in separate aliquots. Animals were allowed to recover from neurosurgery and their estrous cycles were synchronized with prostaglandin F2{alpha} (25). All treatments were given on d 7–8 of the luteal phase and spread over three sequential ovarian cycles in December and January. For each treatment, blood samples were collected every 12 min for 36 min before and 4 h after injection of vehicle (200 µl sterile saline, n = 6), 1 nm OFQ (n = 5), or 1 nm OFQ agonist (n = 5). Injections were done over approximately 15 sec using a sterile 1-cc syringe with a 22-gauge needle cut to extend just beyond the guide tube into the third ventricle. Gentamicin and prostaglandin F2{alpha} were given through im injections at the end of each sampling period. Treatments were randomized so that no ewe received the same treatment twice, no animal received more than two treatments, and approximately the same number of treatments was given each day.

Experiment 3: the effects of an ORL-1 receptor agonist on LH secretion in ovariectomized ewes
Because preliminary studies showed suppression of episodic LH secretion in some OVX ewes after injections of 100–200 µl of vehicle into the third ventricle (data not shown), we next used infusions (60 µl/h) of vehicle (sterile saline) or OFQ agonist (5 nmol/h). We chose to use the OFQ agonist because it was slightly more effective in experiment 2 and to avoid administration of a protease inhibitor. A 1.5-in.-long, 18-gauge BD Precision Glide needle (Fisher Scientific, Fair Lawn, NJ) with a hub for Luer-Lok syringes was stereotaxically implanted into the third ventricle as described for experiment 2 and ewes were OVX via midventral laparotomy. Ewes were allowed to recover at least 2 wk after surgery before treatments were begun. Infusions were done using a battery-operated syringe pump (Graseby Medical, St. Paul, MN) strapped to the back of each ewe. The day before infusions, the two ewes in each pen were separated into individual stalls using a wooden panel that allowed animals free movement. The next morning a sterile 1-cc syringe was loaded with vehicle or OFQ agonist and connected to polypropylene tubing that terminated in a 22-gauge stainless steel cannula glued inside a syringe tip that fit snugly inside the hub of the chronically implanted needle. The end of the cannula was cut so it protruded less than 1 mm from the implanted needle into the third ventricle, and the entire apparatus had been sterilized the day before use. The syringe was placed in the syringe pump and the cannula inserted into the third ventricle after four blood samples had been collected; blood collection then continued every 12 min for 4 h. Treatments were given using a cross-over design with a week between replicates in March (four ewes received OFQ agonist; three saline in the first replicate). All animals were treated with gentamicin after the completion of each infusion.

Immunohistochemistry
OFQ and GnRH were detected using a dual-immunofluorescent procedure, which was carried out on free-floating sections at room temperature. A series of every fourth section through the preoptic area and hypothalamus was washed in 0.1 M PB with 0.9% saline (PBS) for several hours to remove cryoprotectant. After washing, the sections were incubated in PBS containing 4% normal donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and 0.4% Triton X-100 (Sigma, St. Louis, MO) (PBSTX) for 1 h. Sections were then incubated in PBSTX containing a rabbit polyclonal antibody against OFQ (1:3000-Neuromics Antibodies, Northfield, MN; catalog no. RA10106) for 24 h. After incubation, sections were washed and then placed in a solution of PBSTX with Cy3-conjugated affinipure donkey antirabbit IgG (1:200; Jackson ImmunoResearch Laboratories) for 1 h. After incubation, the sections were washed and then incubated in PBS containing 4% normal goat serum; (Jackson ImmunoResearch Laboratories) and 0.4% Triton X-100 (Sigma) (PBSTX) for 1 h. The sections were then incubated overnight in mouse anti-GnRH (1:100; Sternberger Monoclonals, Lutherville, MD; catalog no. SMI41). After incubation the sections were washed and incubated with Alexa 488 conjugated goat antimouse IgG (1:200; Molecular Probes, Eugene, OR). After washing in PB, each series was mounted on slides and coverslipped with gelvatol to prevent fading (28). Immunohistochemical controls included omission of each of the primary antibodies from the immunostaining protocol, the absence of which completely eliminated staining for the corresponding antigen. In addition, preabsorption for 24 h at 4 C of diluted antiserum for OFQ and GnRH with nanomolar concentrations of the corresponding peptide supplied by the companies of origin resulted in elimination of all specific staining.

Sections were viewed under a Nikon Microphot-FXA fluorescent microscope (Nikon Microscopes, Tokyo, Japan). For each animal, all single- and double-labeled GnRH neurons in the diagonal band of Broca (three sections/animal), preoptic area (five sections/animal), anterior hypothalamic area (five to six sections/animal), and mediobasal hypothalamus (defined as that area extending from rostral to caudal levels of the median eminence, including the rostral and middle portions of the arcuate nucleus; seven to eight sections/animal) were counted and the percentage of double-labeled OFQ/GnRH in each region were calculated. Results were reported as mean ± SEM. Statistical comparison among OFQ/GnRH colocalization in between brain regions was analyzed using ANOVA, followed by Sheffe post hoc comparisons. In addition, representative sections containing double-labeled neurons and fibers were examined using an LSM 510 laser scanning confocal microscope (Carl Zeiss Microscopy, Jena, Germany). Digital images were acquired and imported into Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA). Images were not altered in any way except for minor adjustments of brightness and contrast.

RIAs
Concentrations of LH were determined in duplicate 100- or 200-µl aliquots by RIA, using a modification of a previously described method (27). Values are expressed in terms of the ovine standard, NIH S24. Radioiodinated ovine LH (AFP-8614B, courtesy of Dr. A. F. Parlow, National Hormone and Peptide Program, Torrance, CA) was used as tracer and the primary antiserum was AFP-192279 (courtesy of Dr. A. F. Parlow; dilution 1:2,000,000). The sensitivity (95% confidence interval at 0 ng/ml) averaged 0.06 ng/tube. Intraassay coefficients of variation (CVs) averaged 13.9 and 10.4%, respectively, for serum pools displacing radiolabeled LH to approximately 36 and 57% of the total bound counts. Interassay CVs were 16.9 and 20.3% for the same serum pools. Progesterone was measured in selected samples (150 µl) to confirm that ewes were in the luteal phase using a commercially available kit (Diagnostic Systems Laboratories, Inc., Webster, TX); the single assay used had a sensitivity of less than 0.03 ng/ml and an intraassay CV of 7% for a pool producing 40% displacement.

Statistical analysis of LH pulses
LH pulses were identified using established criteria (29). For experiment 2, the average pulse frequency and mean LH concentration for the 4 h after injection were calculated for each ewe and effects of treatment compared with controls by the Mann-Whitney test and one-way ANOVA respectively. For experiment 3, the infusion period was divided into 2-h bins (0–2 and 2–4 h) and average pulse frequency, amplitude, and mean LH concentrations calculated for each ewe during each time period. LH pulse frequency was then analyzed using Friedman’s two-way ANOVA and differences in LH pulse amplitude and concentrations analyzed by two-way ANOVA for repeated measures, with treatment and time as the two main effects. When significant differences were observed, Dunnett’s post hoc test was used to determine which groups differed from blank controls. The level of statistical significance was set at 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1: anatomical distribution and colocalization of OFQ and GnRH immunoreactivity
Distribution of OFQ immunoreactive cells and fibers in the sheep preoptic area and hypothalamus.
Scattered OFQ immunoreactive (-ir) cells and fibers were seen in the diagonal band of Broca, the ventromedial preoptic area (vmPOA) at the level of the OVLT (Figs. 1Go and 2Go, A–C) and in the posterior division of bed nucleus of the stria terminalis. Distinct cells with a moderate level of OFQ-ir were observed dorsal and lateral to the supraoptic nucleus in the lateral hypothalamic area that did not extend into the ventrolateral portion of the anterior hypothalamic area (AHA). At the level of the AHA, a modest number of cells with low levels of OFQ-ir were present in the parvocellular part of the paraventricular nucleus (PVN) along the lateral border. There were a small number of cells in the magnocellular division of the PVN. Scattered, darkly stained fibers were identified throughout the PVN. OFQ-ir fibers were seen ventral to the PVN, coursing dorsoventrally within the periventricular zone. No immunolabeling was found in the suprachiasmatic nucleus or ventromedial nucleus. Caudally, the retrochiasmatic area contained numerous fibers. The periventricular nucleus contained a few, moderately stained neurons adjacent to the third ventricle. The arcuate nucleus contained moderately stained cells and fibers throughout its rostral-to-caudal extent. OFQ-ir cells were more numerous in its dorsomedial part, and fibers within the arcuate primarily maintain a dorsal-to-ventral orientation (Figs. 1Go and 2Go). No OFQ-ir cells were found in the medial mammillary nucleus, but there were OFQ-ir fibers present in this region. The most intense fiber labeling in the hypothalamus was seen in the median eminence (ME). The ME contained an intense immunolabeled fiber plexus (Fig. 3Go). OFQ-ir fibers were located at all rostral-caudal levels of the ME but most heavily concentrated in the external zone.


Figure 1
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  		FIG. 1. Camera lucida drawings depicting the distribution of GnRH-ir cells (open circles), GnRH cells that colocalized with OFQ (filled circles), and OFQ-ir cells lacking GnRH (triangles) in the ovine preoptic area (POA) (A), AHA (B–D), mediobasal hypothalamus (MBH) (E–G), and posterior hypothalamus (H). ac, Anterior commissure; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; CP, cerebral peduncle; DMH, dorsomedial hypothalamic nucleus; fx, fornix; GP, globus pallidus; IC, internal capsule; ir, infundibular recess; LHA, lateral hypothalamic area; mr, mammillary recess; mt, mammillothalamic tract; OC, optic chiasm; OT, optic tract; OVLT, organum vasculosum of the lamina terminalis; PE, periventricular nucleus; PMd, premammillary nucleus, dorsal division; PMv, premammillary nucleus, ventral division; PT, pars tuberalis of the anterior pituitary; RE, reunions thalamic nucleus; SCN, suprachiasmatic nucleus; SI, substantia innominata; SM, supramammillary nucleus; SON, supraoptic nucleus; TM, tuberomammillary nucleus; VMH, ventromedial hypothalamic nucleus; ZI, zona incerta; 3V, third ventricle.

 

Figure 2
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  		FIG. 2. Confocal images showing OFQ (red), GnRH (green), and their colocalization (yellow) in the ovine preoptic area and hypothalamus. A–C, Low-power view of the preoptic area at the level of the organum vasculosum of the lamina terminalis (OVLT) showing OFQ and GnRH cells and their colocalization (e.g. white arrows). Bar, 100 µm. D–F, High-power images of a double-labeled OFQ/GnRH cell; note that the immunofluorescence for OFQ has a punctate appearance in the cytoplasm as opposed to the more diffuse appearance of the GnRH signal. Bar, 20 µm. G–I, Single-labeled OFQ cells (e.g. red arrow) in the arcuate nucleus. Note that many GnRH fibers coursing through this region are also OFQ positive (e.g. white arrows). 3v, Third ventricle. Bar, 100 µm.

 

Figure 3
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  		FIG. 3. Low- (A–C) and high- (D–F) power confocal images showing colocalization (yellow) of OFQ (red) and GnRH (green) in fibers in the ME. zi, Internal zone; ze, external zone; pt, pars tuberalis of the anterior pituitary. Note in panels D–F that OFQ (e.g. red arrow) and GnRH (e.g. green arrow) are not always present in the same intraaxonal segments. Bar (C), 200 µm; (F), 20 µm.

 
Colocalization of OFQ and GnRH
Extensive colocalization of OFQ and GnRH in both cells and fibers was seen throughout the preoptic area and hypothalamus. Overall, 92.97 ± 1.11% of GnRH-ir cells were found to colocalize OFQ. Dual-labeled OFQ/GnRH cells (Fig. 1Go, filled circles) were seen in the diagonal band of Broca, vmPOA (Fig. 2Go, A–F), the anterior hypothalamic area, and mediobasal hypothalamus, with no significant regional differences in their degree of colocalization (df = 3, F = 2.503, P = 0.1089) (Table 1Go). Fibers containing both OFQ and GnRH were also located in these regions (e.g. Fig. 2Go, G–I). The greatest extent of OFQ/GnRH fiber colocalization appeared to be in the ME, particularly in its external zone (Fig. 3Go, A–C). In contrast to its colocalization in almost all GnRH cells and fibers, not all OFQ cells in these regions were immunopositive for GnRH. For example, in the arcuate nucleus, many single-labeled OFQ neurons were seen (Fig. 2GGo). Likewise, OFQ-ir cells in the PVN were not immunoreactive for GnRH. Finally, many OFQ-ir fibers in the hypothalamus, such as those seen in and around the supraoptic nucleus and PVN, lacked GnRH.


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  		TABLE 1. Mean numbers (±SEM) of GnRH cells in the diagonal band (DB), preoptic area (POA), AHA, and mediobasal hypothalamus (MBH) that either colocalized OFQ or were single labeled, and the mean percentages of colocalization (±SEM) in each region

 
The appearance of GnRH and OFQ immunolabeling in individual cells and fibers was different when viewed under high-power confocal microscopy. GnRH-ir was diffuse and homogenous throughout the cytoplasm, whereas OFQ-ir often had a more punctate appearance within the cytoplasm (Fig. 2Go, D–F). Similarly, in fibers immunoreactive for both OFQ and GnRH, the two peptides appeared to be differentially localized, with OFQ and GnRH not always being present in either the same beaded varicosity or the thin portions of fibers located between adjacent varicosities (Fig. 3Go, D–F).

Experiment 2: effects of OFQ and an OFQ agonist on LH secretion during the luteal phase
Progesterone concentrations on the day of treatment averaged 2.1 ± 0.3 ng/ml and did not differ among treatment groups. In controls, all ewes had at least one LH pulse (Fig. 4Go) and the majority had two or three pulses. In ewes treated with either OFQ or the OFQ agonist, all animals had either one or no LH pulses after injection. The agonist produced a significant decrease in LH pulse frequency (Fig. 5Go), but the effects of OFQ were not statistically significant (P < 0.06). Mean LH concentrations also tended to be lower in both treated groups (Fig. 5Go), but neither effect was statistically significant (df = 2, F = 2.67, P = 0.10). The low number of LH pulses in the treated groups precluded an analysis of LH pulse amplitudes.


Figure 4
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  		FIG. 4. LH pulse patterns from representative luteal phase ewes injected into the third ventricle (arrows) with vehicle (top panel), OFQ (middle panel), or an OFQ agonist (bottom panel). Solid circles depict peaks of LH pulses.

 

Figure 5
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  		FIG. 5. Effects of injection of saline (C), OFQ (O), or OFQ agonist (A) into the third ventricle on mean LH concentrations (left panel) and LH pulse frequency (right panel) in luteal phase ewes. *, P < 0.05 vs. vehicle injection.

 
Experiment 3: effects of the OFQ agonist on LH secretion in OVX ewes
Because the OFQ agonist was more effective than OFQ in luteal phase ewes and recently published data had suggested the use of a protease inhibitor with OFQ (40), we tested the agonist only in OVX animals. In six of the seven ewes, infusion of the agonist clearly suppressed episodic LH secretion, compared with infusion of vehicle (Fig. 6Go). The inhibitory actions of the agonist on mean LH levels were evident only during the last 2 h of the infusion (Fig. 7Go) resulting in a significant effect of time (df = 1, F = 25.4, P < 0.01) and a time x treatment interaction (df = 1, F = 5.15, P < 0.05) in the two-way ANOVA. Interestingly, a similar delayed effect was observed in LH pulse amplitude (time: df = 1, F = 7.81, P < 0.05; time x time: df = 1, F = 7.71, P < 0.05), but pulse frequency was significantly suppressed during both the first and last 2 h of the infusion (Fig. 7Go). The OFQ agonist may have produced a stronger inhibition than evident in the mean data because the one ewe that did not respond had evidence of an infection in the third ventricle postmortem; data from this ewe were included in the analysis because we could not be sure when the infection occurred.


Figure 6
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  		FIG. 6. LH pulse patterns in a representative OVX ewe infused in the third ventricle over a 4-h period (shaded bar) with sterile saline (top panel) and OFQ agonist (bottom panel). Solid circles depict peaks of LH pulses.

 

Figure 7
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  		FIG. 7. Effects of OFQ agonist infusion on mean LH concentrations, LH pulse amplitude (AMPL), and LH pulse frequency (FREQ) during the first 2 h (0–2 h) and the second 2 h (2–4 h) of infusion. *, P < 0.05 vs. vehicle infused control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The overall distribution of OFQ-ir cells and fibers we observed in the ovine preoptic area and hypothalamus was very similar to that reported previously in the rat (3, 30) and human (4). However, in addition to populations of single-labeled OFQ cells in these areas, we found an exceptionally high percentage of GnRH-ir cells that colocalized OFQ. Double-labeled OFQ/GnRH cells were not regionally restricted, and a similar high percentage of colocalization (>90%) was seen in each brain region in which GnRH cells are found (Table 1Go), even though the total number of GnRH neurons in each region varied as expected (22, 31, 32). The high percentage of colocalization suggests that OFQ is functionally important in the control of GnRH secretion. Indeed, we found that intracerebroventricular infusions of an OFQ agonist significantly reduced the frequency of LH pulses in both intact luteal phase and OVX ewes. Although these inhibitory effects were observed with an agonist, there is strong evidence that this OFQ analog interacts with the ORL-1 and not the classical opioid receptors (41). In light of this specificity and evidence that OFQ is the endogenous ligand for the ORL-1 receptor (1), these data provide in vivo evidence for a role of OFQ in regulation of the ovine reproductive neuroendocrine system.

It is of note that the observations of OFQ/GnRH colocalization reported here are based on brain sections obtained from ewes during the midluteal phase of the estrous cycle, during which pulsatile GnRH secretion is under the negative feedback influence of progesterone (29). Recent evidence in the female rat indicates that mRNA levels of both OFQ and its cognate receptor, ORL-1, in the preoptic area and hypothalamus are regulated by physiological levels of gonadal steroids (30). Specifically, in the medial preoptic nucleus of the rat, both estradiol and progesterone were required to significantly elevate OFQ mRNA levels above that seen in OVX rats. Hence, it would be worthwhile to compare OFQ expression in GnRH neurons in intact animals at different times during the estrous cycle and between OVX and steroid-replaced ewes. Nonetheless, because we found inhibitory effects of OFQ in both intact and OVX ewes, it would seem unlikely steroid-induced changes in OFQ or ORL-1 expression are critical for the ability of OFQ to inhibit GnRH pulses.

The distribution of OFQ-ir described here and that described in rats (3, 30) and human (4) suggests OFQ is expressed in areas (vmPOA, arcuate nucleus) that have been implicated in the neuroendocrine control of reproduction. These areas have also been found to express high levels of other neuropeptides, specifically other endogenous opioid peptides, and gonadal steroid receptors (estrogen and progesterone receptors) (33, 34). Indeed, the location of OFQ-ir cells in the ovine arcuate nucleus is similar to that of dynorphin (35, 36), neurokinin B (37), enkephalin (38), ß-endorphin (36), dopamine (39), and neuropeptide Y (39). However, in preliminary studies, OFQ and dynorphin were not colocalized in the arcuate nucleus (Foradori, C. D., unpublished data) which, considering the high degree of colocalization between dynorphin and neurokinin B in this area (35), suggests that OFQ also does not colocalize with the latter neuropeptide.

The in vivo inhibition of LH pulse frequency and amplitude in this study complements and extends previous findings of inhibitory effects of OFQ on GnRH release from the rodent hypothalamus. An earlier study reported that OFQ at concentrations of 100-1000 nM inhibited forskolin-induced GnRH secretion from male rat hypothalamic slices but had no effect on basal nonstimulated GnRH release (19). A report published while this work was in progress (40) did observe inhibitory effects in hypothalamic slices on nonstimulated GnRH secretion, but these required doses of 2–20 mM OFQ. They also reported that central administration of OFQ in the rat inhibited GnRH and LH levels, but relatively modest effects (generally 20–30% inhibition) were observed and these occurred only at one time point, even when OFQ was given continuously. Relatively high doses (20–200 nmol per single injection; 2–20 nmol/min push-pull perfusion) of OFQ were required, compared with the effective doses of the OFQ agonist (1 nmol per single injection; 5 nmol/h infusion) used in this study. This difference in effective doses may reflect the use of the OFQ agonist which is 5–30 times more potent than OFQ (41), but direct comparisons of doses between these two species are complicated because of the much larger volume of ventricular CSF in sheep.

The decreases in episodic LH secretion observed in this study, could reflect actions of the OFQ agonist at either the pituitary or hypothalamus. Although OFQ was administered centrally, it could have been transported from the third ventricle to the pituitary via the portal circulation and inhibited LH pulse amplitude. The 2-h delay in effects of the OFQ agonist on LH pulse amplitude is consistent with this possibility. Because OFQ is found in virtually all GnRH neurons (this study) and terminals in the external zone of the median eminence in both sheep and rats (3), it is likely that OFQ is normally released into the hypophyseal portal circulation. In addition, the discrete intracellular localization of GnRH and OFQ in the same fibers (Fig. 2Go) indicates these neuropeptides are packaged in separate secretory vesicles and raises the possibility of differential release of GnRH and OFQ into the portal circulation.

Although some of the data reported here could be accounted for by actions of the OFQ agonist at the pituitary, the inhibition of LH pulse frequency most likely reflects a central action of this peptide because it occurred rapidly after intracerebroventricular administration, and measurements of GnRH in portal blood of sheep have demonstrated that changes in LH pulse frequency reflect antecedent changes in GnRH pulse frequency (42). OFQ could inhibit GnRH pulse frequency either directly or indirectly by inhibiting stimulatory interneurons. Confirmation of a direct action of OFQ awaits the demonstration that GnRH cells in vivo express ORL-1. However, this possibility is supported by reports of high expression of ORL-1 in the ME and that OFQ inhibits the electrical activity of GnRH neurons in vitro by activating a subset of inwardly rectifying K+ channels (43).

Physiologically, OFQ may mediate the negative feedback actions of ovarian steroids, a possibility that could be tested by determining whether OFQ neurons contain estrogen or progesterone receptors. Alternatively, the high level of coexpression of OFQ with GnRH suggests that it acts as part of an ultrashort feedback loop in the regulation of GnRH secretion. Ultrashort feedback loops have been proposed to form part of the mechanism regulating the intrinsic pulsatility of GnRH neurons. The pulsatile release of GnRH from immortalized GnRH cell lines (44) and explants of olfactory placode (45) have led to the hypothesis that GnRH itself is responsible for synchronization of these neurons, but the discovery that GnRH neurons in vivo also produce OFQ creates a potential alternative explanation. Although OFQ colocalization within GnRH neurons is suggestive of an autocrine mechanism of action, our results do not exclude the possibility that the inhibitory effects of OFQ on GnRH secretion may also be mediated by release from other OFQ neurons, in the preoptic area/hypothalamus or elsewhere, that are presynaptic to GnRH neurons. In this regard, GnRH release from immortalized GnRH cell lines is generally not as tightly coupled as episodic GnRH secretion into the hypophyseal portal vessels, so other neural elements may be important for normal pulse generation in vivo (46).

In summary, we found extensive colocalization of the endogenous opioid peptide, OFQ, within GnRH cells in the ewe, as well as within GnRH terminals of the ME, the site of neurosecretion controlling the release of LH by the anterior pituitary. In addition, intracerebroventricular infusion of an OFQ agonist inhibited LH pulse frequency in both luteal phase and OVX ewes and suppressed pulse amplitude in OVX ewes. Taken together, these observation raise the intriguing possibility that OFQ may be acting as ultrashort feedback loop to synchronize GnRH pulses.


   Footnotes
 
This work was supported by National Institutes of Health Grants R01 HD39916 (to M.N.L.) and 5F32NS049892-02 and Lalor Foundation (to C.D.F.).

Current address for L.M.C. and M.N.L.: Department of Anatomy and Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1.

Disclosure Statement: C.D.F., M.A., L.M.C., S.R.S., C.J.M., R.J.H., R.L.G., and M.N.L. have nothing to declare.

First Published Online July 5, 2007

Abbreviations: AHA, Anterior hypothalamic area; CSF, cerebral spinal fluid; CV, coefficient of variation; ir, immunoreactive; ME, median eminence; OFQ, Orphanin FQ; ORL, opioid receptor like; OVX, ovariectomized; PB, phosphate buffer; PBSTX, PBS containing normal donkey serum and Triton X-100; PVN, paraventricular nucleus; vmPOA, ventromedial preoptic area.

Received January 4, 2007.

Accepted for publication June 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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