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Colocalization of Progesterone Receptors in Parvicellular Dynorphin Neurons of the Ovine Preoptic Area and Hypothalamus

Department of Cell Biology, Neurobiology, and Anatomy (C.D.F., L.M.C., M.E.F., M.N.L.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521; Department of Zoology and Physiology (D.C.S.), University of Wyoming, Laramie, Wyoming 82071-3166; and Department of Physiology and Pharmacology (R.L.G.), West Virginia University Health Sciences Center, Morgantown, West Virginia 26506-9229

Address all correspondence and requests for reprints to: Dr. Michael N. Lehman, Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521. E-mail: michael.lehman{at}uc.edu.

	Abstract

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Recent evidence suggests that the dynorphin- receptor opioid system acts to mediate the inhibitory effect of progesterone (P) on GnRH pulse frequency during the luteal phase of the ovine estrous cycle. It is known that progesterone receptors (PRs) are required for the actions of P on GnRH secretion. Therefore, if P acts directly on dynorphin (DYN) neurons, then these neurons should contain PRs. To test this hypothesis, we used a dual-label immunoperoxidase procedure to visualize PRs and DYN in the preoptic area (POA) and hypothalamus of ovary-intact ewes killed during the luteal phase of the estrous cycle. The PR was colocalized in more than 90% of parvicellular DYN neurons in the POA, anterior hypothalamus (AHA), and arcuate nucleus (ARC). By contrast, none of magnocellular DYN cells of the paraventricular and supraoptic nuclei coexpressed immunoreactive PRs. The high percentage of colocalization of PRs in parvicellular DYN cells of the POA, AHA, and ARC suggests that these cells are prime targets of P. In addition, DYN cells in the ARC, but not the POA or AHA, were found to receive synaptic inputs from DYN-positive axon terminals. This observation raises the possibility that an ultrashort feedback loop controls the release of DYN from ARC neurons.

	Introduction

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THE SECRETION OF GnRH into the pituitary portal blood supply is the final common pathway responsible for neuroendocrine control of reproduction. GnRH neurons play a major role in the control of reproductive function, regulating events leading to ovulation, and the shutting down of ovarian function before puberty and during other periods of infertility (1, 2, 3). The ovarian steroids, estradiol and progesterone (P), exert positive and negative feedback actions that produce changes in the pattern of pulsatile GnRH and corresponding LH release throughout the estrous or menstrual cycle (4, 5) and control the timing of the preovulatory GnRH/LH surge (1). P, in particular, is a major inhibitory brake on the GnRH system, suppressing the frequency of GnRH pulses and blocking the positive feedback actions of estrogen during the luteal phase of the ovarian cycle (1). Despite the evidence that a small number of guinea pig GnRH cells possess nuclear P receptors (PRs) (6), GnRH cells in sheep (7), hens (8), and mink (9) have been found to lack PRs. In addition, in sheep, nonclassical PRs are unlikely to play a role because administration of RU486 blocks the inhibitory influence of P on GnRH secretion (10). Therefore, this influence is likely to occur via neural afferents onto GnRH cells.

A growing body of evidence has implicated the endogenous opioid peptides (EOPs) in mediating the negative feedback influence of P on GnRH secretion during the luteal phase of the estrous cycle in sheep (1), rats (11), and primates (12). The stimulatory effects of EOP antagonists on LH release have demonstrated that EOPs inhibit LH pulse frequency during the luteal phase of the ovarian cycle of sheep (13, 14), monkeys (15, 16), and humans (17, 18). Because EOP antagonists have little, or no, effect in ovariectomized animals (13, 14, 19) and postmenopausal women (20, 21), it has been proposed that EOPs mediate the negative feedback actions of P on GnRH pulse frequency. Recent work has shown that antagonists specific to the opioid receptor, the opioid receptor subtype that has the highest affinity for the EOP dynorphin A (DYN), can preferentially block the influence of P during the luteal phase, compared with the actions of µ- and -specific antagonists (22). In addition, almost all GnRH neurons in the medial basal hypothalamus (MBH) of the sheep brain receive synaptic inputs from DYN-containing terminals (23). These observations led us to hypothesize that during the luteal phase, P acts via DYN neurons to inhibit the activity of MBH GnRH neurons and the pulsatile secretion of GnRH into the portal blood supply. One prediction from this working hypothesis is that DYN cells would contain PRs because, as noted above, RU486 blocks the inhibitory effects of P (10).

Simerly et al. (24) reported that a high percentage of DYN cells (85%) in the rat anteroventral periventricular nucleus contained the mRNA encoding PRs. To our knowledge, no one has examined DYN cells in other areas of the mammalian hypothalamus for the presence of PRs. The preoptic and hypothalamic areas containing immunoreactive PRs in the sheep (7, 25) overlap regions known in other species to contain DYN cells (26, 27, 28). Therefore, the aims of the present study were to determine the distribution of DYN immunoreactive cells in the ovine preoptic area (POA) and hypothalamus and to determine which of these DYN cells coexpress PRs. In the course of this study, we also observed frequent instances of close contacts between DYN-positive boutons and DYN-positive cell bodies in the arcuate nucleus (ARC). Because DYN-DYN contacts observed at a light microscopic level were suggestive of synaptic connections, we also used electron microscopic immunocytochemistry to determine whether these contacts were indeed synapses.

	Materials and Methods

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Animals
Adult Suffolk ewes 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 in individual pens under a photoperiod similar to that occurring outdoors. The routine handling and experimental procedures involving animals were approved by the West Virginia University Animal Care and Use Committee.

Experimental protocols
This study was performed during the breeding season (October through January) in six ewes that had demonstrated at least two normal 16- to 17-d estrous cycles (determined by monitoring either P levels or behavior with a vasectomized ram). Ewes in the midluteal phase (d 6–9) were perfused. Serum and ovaries were harvested to verify hormone levels and corpus luteum consistent with midluteal sheep. On the basis of examination of the ovaries and P serum levels, all ewes were in the luteal phase of the estrous cycle.

Tissue preparation
For light microscopic studies, six animals were heparinized (two iv injections of 25,000 U heparin given 10 min apart), then deeply anesthetized with sodium pentobarbital (2,000 mg, iv), and rapidly decapitated. The heads were perfused via both internal carotids with 6 liters 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.3) containing 0.1% sodium nitrite and 10 U/ml heparin. Following perfusion, the brain was removed and a tissue block containing the septal region, POA, 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 (29) until being processed immunohistochemically for PRs and DYN.

For electron microscopic (EM) analyses, one ewe was anesthetized with sodium pentobarbital and perfused bilaterally via the carotid arteries in situ with 6 liters of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M PB (pH 7.3) with 0.1% sodium nitrite. The first 300–400 ml fixative perfused through the carotids also contained a higher concentrated heparin solution (1000 U/ml); heparin was added to the rest of the fixative in a lesser concentration (100 U/ml). Following perfusion, the brain and attached pituitary were carefully removed from the cranium. The POA and hypothalamus were dissected out and placed in the same fixative for an additional 24 h at 4 C and then transferred to PB and stored at 4 C until sectioned. Coronal sections (50 µm) through the MBH were cut on a vibratome and stored at -20 C in a cryopreservative solution (29) until processed for EM immunocytochemistry.

RIA
P was measured by duplicate aliquots of serum sample collected from each ewe, just before it was killed, using a commercially available kit (Diagnostic Systems Laboratories, Inc., Webster, TX) (14). Sensitivity of the assay averaged 0.06 ng/ml and intra- and interassay coefficients of variation were 3.4% and 10.1%, respectively. All animals had circulating P concentrations in the luteal phase range of 1.0–2.2 ng/ml.

Light microscopic immunohistochemistry
Dynorphin A and PR were detected using a dual immunoperoxidase procedure (30, 31). In this procedure, a nuclear PR was first detected using a modified avidin-biotin-immunoperoxidase protocol (25) with nickel-enhanced diaminobenzidine as chromogen (blue-black reaction product) and the DYN was identified using the same procedure without nickel enhancement (brown reaction product). Immunohistochemistry procedures were carried out on free-floating sections at room temperature, except for incubation with primary antibodies against PR and DYN, which were performed at 4 C. Sections were washed in 0.1 M Tris solution with 0.9% saline at pH 7.6 for several hours to remove cryoprotectant. After washing, the sections were placed in 1% sodium borohydride (Sigma, St. Louis, MO) for 10 min to remove excess aldehydes. Sections were then placed into a 1% hydrogen peroxide (Sigma) solution for 10 min to eliminate endogenous peroxidase activity. Sections were incubated for 1 h in 0.1 M Tris solution with 0.9% saline at pH 7.6 with 10% normal donkey serum (NDS) (Jackson Laboratories, Inc., West Grove, PA) and 0.2% Triton X-100 (Sigma). Sections were incubated with monoclonal antibody against human PR (1:10 of prediluted stock provided from company, PR10 A9 clone, Immunotech, Marseille, France) for 60–65 h. The antibody was raised against the hormone-binding domain (922AGMVKPLLFHKK933) of the human PR; therefore, both isoforms (A and B) should have been detected. This antibody has been previously validated in sheep (25).

Following incubation, sections were washed and then placed in a solution of 0.2% Triton X-100 with biotinylated donkey antimouse IgG (1:400, Jackson Laboratories, Inc.) for 1 h. The sections were washed and incubated for 1 h in avidin-biotin-horseradish peroxidase complex (1:400, Vector Laboratories, Burlingame, CA). PR was visualized using 3,3'-diaminobenzidine with 0.02% nickel sulfate and 0.003% hydrogen peroxide as substrate. The second antigen, DYN, was then demonstrated using an avidin-biotin-immunoperoxidase procedure using diaminobenzidine without enhancement. After repeated washing the tissue was placed in 1% hydrogen peroxide in 0.1 M PB with 0.9% saline (PBS) to remove any unreacted peroxidase. The sections were then washed and incubated in PBS containing 4% NDS and 0.4% Triton X-100 for 1 h. Sections were incubated in rabbit polyclonal antibody against dynorphin A 1–17 (1:20,000 dilution, IHC 8730, Peninsula Laboratories, Inc., San Carlos, CA) for 48 h in 0.4% Triton X-100. The dynorphin A 1–17 antibody shows cross-reactivity with dynorphin A 1–13 and none with other prodynorphin derivatives such as dynorphin A 1–8, -neo-endorphin, leu-enkephalin, and dynorphin B. After incubation in primary antibody, sections were washed and incubated in biotinylated donkey antirabbit IgG (1:400), followed by avidin-biotin-horseradish peroxidase complex (1:400), and reacted using unenhanced 3,3'- diaminobenzidine as the chromogen. Immunohistochemical controls included omission of one or both of the primary antibodies from the immunostaining protocol, the absence of which completely eliminated staining for the corresponding antigen.

In addition to the protocol described above to visualize PR, antigen unmasking was performed on two of the six animals perfused for light microscopic immunohistochemistry. High-temperature antigen retrieval on 4% paraformaldehyde-fixed tissue has been previously shown to increase the visualization of many antigens not detectable without treatment, including PR (7, 32). The antigen unmasking used a similar protocol as described above. Subsequent to washing after removal from cryoprotectant the sections were washed in antigen unmasking buffer (Vector Laboratories, H-3300, 0.01 M sodium citrate pH 7.8). Sections were placed into boiling antigen unmasking buffer for 5 min and allowed to cool. In this protocol, sections were processed for PR using a mouse antiprogesterone receptor antibody (1:650 dilution; Neomarker, PgR Ab-8 MS-298-PO, Fremont, CA) using a modified protocol described in Skinner et al. (7). PgR Ab-8 is a mixture of two well-characterized clones, which recognizes both isoforms of the human PR (7, 33). The same DYN antibody was used at a concentration of 1:60,000.

Electron microscopic immunocytochemistry
Tissue perfused for EM immunocytochemistry was processed for DYN alone in a similar manner as described above. Sections were thoroughly washed in PB (pH 7.3) to remove cryoprotective solution. Sections were incubated in 0.5% hydrogen peroxide solution for 10 min to eliminate endogenous peroxidase activity and washed in PB. Sections were then placed in 0.1 M glycine made up in PB for 10 min and then washed in PB alone. Sections were incubated for 1 h at room temperature in PB with 4% NDS and 0.02% saponin (Sigma), followed by rabbit polyclonal antibody against DYN (1:20,000 dilution, IHC 8730, Peninsula Laboratories, Inc.) overnight at room temperature in 0.02% saponin and 4% NDS in PB. The remainder of the protocol for EM immunocytochemistry for DYN was the same as that described above, except that all steps were done in PB without detergent.

Regions through the ARC containing close associations between DYN-immunoreactive (ir) fibers and DYN-ir cells were dissected out. These tissue pieces were postfixed in 2% osmium tetroxide containing 1.5% potassium ferricyanide (34), dehydrated, and flat embedded in Epon 812. Semithin (1 µm) sections were cut and examined for the presence of ir cells. Ultrathin (70 nm) sections were cut from the remaining tissue block, mounted on formvar-coated grids, and stained with uranyl acetate and lead citrate. Sections were examined using a JEOL 1230 electron microscope, and images of cells and terminals captured at a magnification of x4,000 and x10,000.

Analysis
The locations of single- and double-labeled DYN cells were examined in a series of every sixth section through the POA and hypothalamus of each ewe. Cells were considered doubled labeled if a ring of DYN-ir cytoplasm completely surrounded a PR-ir positive nucleus through multiple planes of focus. For quantitative assessments of the percentage of double-labeled cells, we analyzed three sections from each of the following areas that contained both PR-ir and DYN-ir cells: the POA, anterior hypothalamic area (AHA), and ARC (rostral, middle, caudal levels). For each animal, the total number of single-labeled DYN, singled-labeled PR, and double-labeled PR/DYN cells were counted in each area, and the percentage of DYN-ir cells that contained PR-ir nuclei was calculated. Images of double-labeled material were captured using an optronic digital camera attached to a microscope (Leica Corp., Deerfield, IL). The mean diameter of DYN-ir cell bodies was calculated from a sample of 10–20 cells in each region.

	Results

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PR-ir cells
PR-ir cells were identified by the presence of dense nuclear reaction product; in addition, cytoplasmic PR-ir was seen in the ventromedial hypothalamic nucleus (VMH). Cells immunoreactive for PR were located in the POA and several well-defined regions in the MBH. The greatest number of PR-ir cells in the MBH were found in the AHA, VMH, and ARC. PR-ir cells in the POA were heaviest in its ventromedial portion (Fig. 1A) at the level of the organum vasculosum of the lamina terminalis. These cells extended caudally as a continuum into the AHA region adjacent to the third ventricle (Fig. 1B). PR-ir cells in the VMH were located in a cluster that extended from the fornix toward the ventral surface of the MBH (Fig. 1C). PR-ir cells were distributed throughout the rostral-caudal extent of the ARC, with sparse cells in the rostral portion of the ARC (Fig. 1C) and greater numbers of cells in the middle (Fig. 1D) and caudal (Fig. 1E) portions of the nucleus. In addition to these regions, a few PR-ir cells were seen in the ventrolateral septum and the bed nucleus of the stria terminalis. A few cells containing PR were also seen in the rostral extent of the paraventricular nucleus (PVN) and supraoptic nucleus (SON).

View larger version (33K): [in this window] [in a new window]   Figure 1. Camera lucida drawings of ewe coronal sections with insets depicting the boxed areas at higher magnification, showing the distribution of single-labeled PR-ir cells (gray dots), single-labeled DYN-ir cells (open triangles), and double-labeled DYN-ir cells with PR-ir nuclei (filled triangles) in the POA (A), AHA (B), and rostral (C), middle (D), and caudal (E) levels of the ARC. OVLT, Organum vasculosum of the lamina terminalis. Scale bars, 2 mm and 1 mm.

View larger version (18K): [in this window] [in a new window]   Figure 2. Histogram depicting (A) the mean (± SEM) number of DYN-ir cells and (B) mean (± SEM) percentages of DYN-ir cells with PR-ir nuclei in the sheep POA; AHA; and rostral, middle, and caudal levels of the ARC.

View larger version (126K): [in this window] [in a new window]   Figure 3. Photomicrographs of single- and dual-immunoperoxidase labeled PR-ir and DYN-ir cells in the POA (A), PVN (B), and ARC (C–F) of the sheep brain. Examples of double-labeled DYN/PR cells (brown cytoplasm with blue-black nuclei) as well as single-labeled PR cells (blue-black nuclei) are seen in the POA (A) and ARC (C–F). In contrast, DYN-ir cells of the rostral PVN (B) and SON only rarely contained PR-ir nuclei. E shows an example of close contacts observed between DYN-positive boutons (arrows) and a double-labeled DYN/PR cell in the ARC. F shows double-labeled DYN/PR cells in the ARC from a section pretreated for antigen unmasking (see text). Scale bars: A ,15 µm; B, 10 µm; C, 25 µm; D, 25 µm; E, 20 µm; F,20 µm.

View larger version (112K): [in this window] [in a new window]   Figure 4. Electron micrographs of DYN-ir neurons and terminals in the ovine ARC. A, Low-power electron photomicrograph depicting a DYN-ir terminal (asterisk) contacting a DYN-ir soma (nc, nucleus of this cell). An adjacent nonimmunoreactive ARC neuronal soma is seen (nc', nucleus). B, High-power electron photomicrograph showing a DYN-ir axon terminal (at) in synaptic contact with a portion of a DYN-ir soma. Note the presence of DYN-ir dense core vesicles (arrows) in both the presynaptic terminal and postsynaptic soma. Scale bars indicated in figure.

	Discussion

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To our knowledge, the only other hypothalamic neuropeptide that shows as high a percentage of coexpression with gonadal steroid receptors is neurokinin B (NKB) (48). Specifically, 97% of NKB-ir cells in the sheep ARC were shown to coexpress ERs (48). The similar percentage of neuropeptide/steroid receptor colocalization, and the overlap in their distribution in the ARC, raises the possibility that DYN and NKB neurons represent the same population of cells. Regardless of whether DYN colocalize with NKB or other neuropeptides, based on their high degree of PR colocalization, DYN cells appear to be a major target for P’s actions in the ovine POA and hypothalamus.

The distribution of PR-ir cells we observed is very similar to that previously described in the sheep (7, 25) and includes PR-ir cells in the POA, AHA, bed nucleus of the stria terminalis, VMH, and ARC. The areas we observed in the sheep hypothalamus immunoreactive for DYN also were very similar to that previously documented in the diencephalon of the rat (27, 49, 50), hamster (28), monkey (26), and human (51). DYN perikarya were identified in the PVN, SON, POA, AHA, and ARC. However, a single previous study in the ewe described more limited and different distribution of DYN cells (52). This study reported DYN cells in the suprachiasmatic nucleus in which we did not observe them; conversely, this previous report did not note the presence of dynorphin cells in the PVN, ARC, POA, or AHA. The discrepancies between our results could be due to the specificity of the antibody used in the previous study. Preliminary observations suggest that the distribution of preprodynorphin mRNA containing cells in the sheep brain closely parallels the DYN cell distribution reported here (53).

Although the percentage of parvicellular DYN cells that contained PR was high in all hypothalamic areas analyzed, a possible caveat to this observation is that we may have underestimated the total number of DYN cells in these areas because of the lack of colchicine pretreatment of the animals. We believe this is unlikely because tissue sections processed after antigen unmasking showed similar numbers of total DYN cells and percentages of PR/DYN colocalization. Likewise, the number and distribution of immunoreactive DYN cells corresponds to those seen with in situ hybridization for preprodynorphin mRNA in the ovine ARC (53). In addition, we may not have detected all PR-ir nuclei because of the high concentrations of endogenous P present in the luteal phase animals. However, in contrast to some antibodies against ER (54), there is no evidence that the PR antibodies we used preferentially recognize ligand-unbound receptor. In addition, Dufourny and Skinner (55) have recently reported evidence that suggests that the number of PR-ir cells in the ovine hypothalamus does not change during different stages of the ovarian cycle. However, Scott et al. (56) have shown that the number of cells expressing PR mRNA is reduced in some areas of the ovine hypothalamus (ventromedial nucleus and ARC) during the luteal phase of the estrous cycle. Likewise, studies in the rodent have shown an increase in the number of cells PR-ir during or preceding times of higher estrogen levels (57, 58). Therefore, the possibility remains that we may have underestimated the number of PR-positive cells. Nonetheless, considering the high extent of colocalization of PR and DYN, it is unlikely that this would have affected the observations reported here.

The presence of PR in a high percentage of DYN cells supports the hypothesis that these cells convey the negative feedback influence of P on pulsatile GnRH secretion during the luteal phase of the estrous cycle. Consistent with this, we have observed synapses between DYN terminals and GnRH neurons in the ovine POA and MBH (23). In addition, a receptor specific antagonist has been shown to block P negative feedback, matching the effect of naloxone, a general opioid receptor antagonist (22).

In the middle and caudal regions of the ARC, we noted that many PR/DYN double-labeled cells appeared to be surrounded by DYN-ir fibers and varicosities. These associations between DYN cells and fibers were not observed among single- or double-labeled cells in the POA and AHA or among DYN cells in the SON or PVN. Our EM observations confirmed that these contacts were axosomatic and axodendritic synapses. If synaptically released DYN is to affect DYN cell bodies in the ARC, then these neurons should contain the opioid receptor (KOR). DYN and KOR are coexpressed in the magnocellular vasopressin cells of the PVN and SON in rats as well as in the neurosecretory terminals of these cells in the posterior pituitary (59). Because receptor signaling reduces intracellular calcium and inhibits transmitter release, the coexpression of DYN and KOR are thought to represent components of an ultrashort inhibitory feedback loop in this neuroendocrine system (59). DYN-DYN connections in the ARC may comprise a similar inhibitory feedback loop that regulates the activity of DYN cells and their response to P. However, it should be noted that we do not know whether the DYN-DYN contacts in the ARC are between DYN cells of the same nuclei (ARC-ARC) or may reflect communications between DYN cells in different regions (e.g. POA-ARC). Finally, because EOPs have been implicated in regulating the frequency and shape of GnRH pulses (60), it is tempting to speculate that DYN-DYN synapses could comprise a functional component of the GnRH pulse generator. Regardless of the specific function of these DYN-DYN connections, the observation that they are only observed in the ARC suggests a distinct role of this subpopulation of parvicellular DYN neurons.

Ultimately, the action of P on DYN neurons is conveyed to the GnRH system. It has been proposed that this influence is mediated at the level of the MBH via inputs onto either GnRH cell bodies or GnRH terminals in the median eminence (61). One possibility is that local DYN neurons in the arcuate nucleus, which coexpress PR, provide this input; however, the present results do not exclude the possibility that parvicellular DYN cells located more rostrally in the POA or AHA may contribute to this input. It should also be pointed out that DYN contacts are observed on GnRH perikarya in the POA (23) and that local administration of EOP antagonists in this area increases LH secretion (62). Thus, tract tracing and/or lesion experiments are necessary to determine precisely which DYN cells are afferent to GnRH neurons. In addition, although the high degree of PR/DYN colocalization suggest a role for these neurons in the feedback influence of P on GnRH neuroendocrine function, it is possible that one or more of these DYN populations may also mediate other functions of P, such as the priming of estrous behavior (1). Because PR cells are thought to be a subset of ER cells, these DYN populations may also play a role in the actions of estrogen on the GnRH system. The functional role of specific DYN cell populations could be addressed by using indices of activation such as Fos or changes in cellular DYN mRNA levels. Additionally, local injections of antisense oligomers or RU486 could address possible differences among dynorphin cells in the POA, AHA, and ARC in their behavioral or neuroendocrine functions.

In summary, we have found that a large majority of parvicellular DYN cells in the ovine POA and hypothalamus coexpress nuclear PR. In contrast, magnocellar DYN cells of the PVN and SON did not contain PR. Thus, parvicellular DYN neurons in the ovine POA and hypothalamus constitute a prime target for the influence of P in the sheep brain. Finally, we observed many instances of double-labeled PR/DYN cells in the ARC that received synaptic inputs from DYN-ir terminals. This latter observation raises the possibility that a network of DYN neurons in the ovine hypothalamus functions as a single unit to convey the influence of P upon the GnRH system, and perhaps other systems as well.

	Acknowledgments

 

	Footnotes

 
This work was supported by NIH Grant R01-HD-39916-01 and USDA Grant 2000-02132 (to M.N.L.) and NIH Grant T32-DK-59803-01 (to C.D.F.).

Abbreviations: AHA, Anterior hypothalamus; ARC, arcuate nucleus; DYN, dynorphin; EM, electron microscopic; ER, estrogen receptor; ir, immunoreactive; KOR, opioid receptor; MBH, medial basal hypothalamus; NDS, normal donkey serum; NKB, neurokinin B; P, progesterone; PB, phosphate buffer; POA, preoptic area; PR, progesterone receptor; PVN, paraventricular nucleus; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus.

Received June 5, 2002.

Accepted for publication July 16, 2002.

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