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Biology of Reproduction 67, 829-836 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Influence of Estradiol on NADPH Diaphorase/Neuronal Nitric Oxide Synthase Activity and Colocalization with Progesterone or Type II Glucocorticoid Receptors in Ovine Hypothalamus1

Laurence Dufourny2,,a,b, and Donal C. Skinnera,b

a Department of Clinical Veterinary Science, University of Bristol, Langford BS40 5DU, United Kingdom b Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming 82071


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) has been shown to play an important role in both the neuroendocrine reproductive and stress axes, which are closely linked. Because progesterone (P4) receptors (PRs) and glucocorticoid receptors (GRs) are not found in GnRH neurons and the NOergic system has been implicated in the control of GnRH secretion, this study aimed to ascertain whether steroids altered the NOergic system. Our first objective was to map the distribution of NO synthase (NOS) cells in the ovine preoptic area (POA) and hypothalamus and to determine whether NOS activity is enhanced by estradiol (E2) treatment. Using NADPH diaphorase (NADPHd) histochemistry, we found that NADPHd-positive neurons were spread throughout the ovine POA and hypothalamus, and that all NADPHd cells were immunoreactive for NOS. In response to estradiol, a significant increase in the number of NADPHd cells was noted only in the ventrolateral region of the ventromedial nucleus (VMNvl), with no significant difference in the POA or arcuate nucleus. Progesterone and glucocorticoid receptors were colocalized with NADPHd reactive neurons in the POA, arcuate nucleus, and VMNvl of ewes in both treatment groups. In ewes receiving estradiol, the number of NADPHd-positive cells containing steroid receptors in the POA (PR, 81%; GR, 79%) and arcuate nucleus (PR, 89%; GR, 84%) was similar, but in the VMNvl, fewer NADPHd-positive cells contained GR (PR, 88%, GR, 31%). These data show that estradiol up-regulates NOS activity in a site-specific manner and that the influence and possible interaction of progesterone and corticosteroids on NO producing cells may differ according to the neural location.

glucocorticoid receptor, hypothalamus, nitric oxide, progesterone receptor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neuroendocrine reproductive axis is profoundly affected by both the ovarian and adrenal steroids. In the ewe, as in several other species, progesterone (P4) is a powerful inhibitor of both tonic and surge secretion of GnRH [1, 2]. Similarly, cortisol has been shown to inhibit LH release [3] and this effect appears to be profoundly affected by the prevailing steroidal milieu [4]. The reproductive cycle is also delayed following stressful events such as transportation [5, 6] and isolation/restraint [4]. It is clear that these steroids do not act directly on GnRH neurons in the ewe because neither type II glucocorticoid receptors (GRs) [7] nor progesterone receptors (PRs) [8] are coexpressed in GnRH perikarya. It is probable, therefore, that an interneuronal system transduces the inhibitory input of progesterone and corticosteroids to the GnRH system.

Recent evidence suggests that nitric oxide (NO) may play a substantial role. NO is a gaseous intercellular transmitter in the central nervous system [9] produced by a NO synthase (NOS), which converts L-arginine to L-citrulline. The NOS neuronal form (NOS-I), which is constitutively expressed, is regulated by calcium/calmodulin and is dependent on oxygen and NADPH as cosubstrates [10].

In rats, there is evidence that NO participates in both the control of sexual behavior [11] and GnRH/LH release [12–14]. NOS is not found in GnRH neurons, although these neurons are often surrounded by NOS cells [15–17]. In this species, NOS synthesis is steroid-dependent and estrogen receptors {alpha} have been found in NO producing neurons of the preoptic area (POA) and hypothalamus [18, 19]. PR has also been described in NOS immunoreactive neurons from the guinea pig diencephalon [20, 21].

NO has also been implicated in stress regulation. NOS mRNA is increased in the paraventricular nucleus [22, 23] and anterior pituitary [22] following restraint in rats, and it has been suggested that NO acts at the hypothalamic level to modulate the physiological response to stressors. Indeed, intracerebroventricular injection of L-NAME, a NOS inhibitor, diminished ACTH response following a stress [24], and L-NAME, given subcutaneously, delayed the return of plasma corticosterone to baseline levels [25].

The distribution of NOS correlates well with that of NADPH diaphorase (NADPHd) in the rodent brain [20, 26]. Therefore, our first objective was to characterize the relationship between NADPHd and NOS-immunoreactive cells and to map the NADPHd cell distribution in the ovine brain. Our second objective was to determine whether progesterone or corticosteroids could modulate physiological functions directly through NO-producing neurons. To achieve this, PRs or GRs were detected in brains from steroid-treated ovariectomized ewes. As estradiol priming is commonly known to enhance levels of PR expression [27] and estradiol priming is critical for the ability of progesterone to inhibit GnRH secretion [28]. Our final objective was to determine whether this treatment modulates NADPHd activity in the ovine brain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Eight sexually mature Dorset Horn ewes were ovariectomized at least 4 mo before the start of the study. Animals were maintained in outdoor barns on natural photoperiod; fed daily with hay, straw, and concentrate; and were given free access to water. At the time of the experiment (early breeding season), animals were divided into two experimental groups; one received estradiol and progesterone (E2 + P4; n = 4), and the other received progesterone only (P4 only; n = 4). Animals in the E2 + P4 group received 17ß-estradiol s.c. through a 1 cm Silastic capsule for 2 wk, starting 3 wk before tissue collection. Two progesterone-releasing implants (CIDR; InterAg, Hamilton, New Zealand) were inserted intravaginally into both groups for 10 days. Twelve hours after progesterone implant removal, progesterone was readministered through the same CIDRs for 1 h, just before the animals were killed. Following these protocols, GnRH secretion is either suppressed (i.e., E2 + P4 treatment) or not modified (P4 only treatment) when progesterone is readministered [28].

Tissue Fixation

Ewes were injected i.v. with 25 000 IU heparin and immediately killed with an overdose of sodium pentobarbitone. Animals were decapitated and the brains were fixed by perfusion through both carotid arteries with 1 L of 1% sodium nitrite in 0.9% NaCl, followed by 3 L of 4% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer pH 7.4, and finally, 1 L of 20% sucrose. Brains were dissected out and left overnight in sucrose (4°C). A block containing the POA and diencephalon was embedded in Tissue-Tek (Miles Inc., Elkhart, IN) and frozen by immersion in nitrogen-cooled isopentane. Coronal sections (20 µm) were cut on a cryostat, collected on silane-coated slides [29], and frozen to -30°C until further processing.

NOS Immunolabeling

Sections were washed three times for 10 min each in 0.01 M PBS pH 7.4, then incubated for 72 h at 4°C with a mixture of PBS 0.3% Triton X-100 containing 5% normal goat serum and a rabbit polyclonal antiserum raised against NOS-I (AB1552; Chemicon, Temecula, CA) diluted to 1:500. Sections were washed, incubated for 90 min at 4°C in PBS containing goat biotinylated anti-rabbit immunoglobulin G (IgG; 1:200; Vector Laboratories, Burlingame, CA), washed, transferred into fluorescein isothiocyanate (FITC)-streptavidin (1:300; Vector Laboratories) for 1 h at 4°C, washed, and finally covered with Vectashield (Vector Laboratories). Control sections were incubated with normal goat serum or with PBS instead of the anti-NOS antibody, which resulted in the absence of any specific staining. The specificity of the immunostaining was established by the supplier because immunostaining was fully abolished by preabsorption of the antibody with residues 1414–1429 of the rat NOS-I protein. In addition, cryostat sections of rat hypothalamus fixed as described above were used as the tissue control for the presence of immunoreactivity. The distribution in the rat hypothalamus corresponded well with that described previously [30].

NADPHd Histochemistry

NADPHd histochemistry was performed as previously described [31]. Briefly, after three washes in PBS, sections were incubated in PBS 0.3% Triton X-100 containing 1.1 mM ß-NADPH (Boehringer, Bracknell, U.K.) and 0.1 mM nitroblue tetrazolium (Sigma, St. Louis, MO) for 1 h at 38°C. After incubation, sections were rinsed in PBS, dehydrated in alcohol, and mounted in dibutyl phthalate xylene (BDH Laboratory Supplies, Poole, U.K.) or processed for PR or GR immunocytochemistry.

Double Labeling for NADPHd and PR or GR

NADPHd histochemistry was first performed on sections from all animals. Sections were washed in PBS, boiled three times for 3 min in 10 mM citrate buffer pH 6.0, cooled at room temperature for 30 min, and placed for 10 min in a solution of 40% methanol, 1% H2O2, and PBS. Sections from ewes treated with E2 + P4 were then washed and incubated for 72 h at 4°C in PBS 0.3% Triton-X100 containing 5% normal goat serum and a mouse monoclonal anti-human PR antibody (PgRAb8; Neomarkers, Fremont, CA) diluted to 1.5 µg/ml. Sections from ewes treated with P4 only were incubated for 72 h at 4°C in PBS 0.3% Triton X-100 containing 5% normal goat serum and a rabbit polyclonal anti-GR antibody (PA1-511; Affinity Bioreagents, Inc., Golden, CO) diluted 1:800. Sections were washed and incubated for 90 min at 4°C in biotinylated goat secondary IgG (1:200; Vector Laboratories), washed, and incubated for 90 min at room temperature with the Vector Elite kit (1:50). Immunoreactivity was visualized using 3,3'-diaminobenzidine [32].

The specificity of the anti-PR antibody in ewes was previously checked [8], and characteristics of the anti-GR antibody have been published previously [33]. Preadsorption of this antibody with a saturating amount of synthetic peptide corresponding to the target sequence of GR (2 µg/ml; Affinity Bioreagents) resulted in loss of immunoreactivity in ewes.

Analysis of Results

Randomly selected sections of the hypothalamus were examined under a Leica DMRB microscope equipped with a L4 filter (450–490 nm) to visualize FITC-labeled NOS neurons. Sections were examined and some pictures of NOS-immunoreactive neurons were stored using an image acquisition system. The same sections were examined again after NADPHd histochemistry, and further images were taken of the previously stored microscopic fields. Determination of the coexistence of NOS with NADPHd activity was performed with a computer by comparing the two images.

NADPHd-positive cells and double-labeled neurons (NADPHd + PR on sections of ewes treated with E2 + P4 or NADPHd + GR on sections of ewes with P4 only) were observed every 200 µm. A semiquantitative analysis was performed to determine the influence of estrogen on NADPHd activity. NADPHd cells were counted on three hemisections taken at the same levels in the POA, arcuate nucleus, and ventrolateral aspect of the ventromedial nucleus (VMNvl) for each animal. The mean number of NADPHd cells per three hemisections was determined in each group in each neural area, and data were analyzed statistically using the Student t-test.

Double-labeled neurons were counted in the whole POA (5–6 hemisections per animal), arcuate nucleus (10 hemisections per animal), and VMNvl (7 hemisections per animal) on hemisections taken every 400 µm. The percentage of NADPHd-positive neurons expressing steroid receptors was then calculated in each area for each animal, and the mean (± SEM) percentage was then determined for each group.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Relationship Between NADPHd-Positive and NOS-Immunoreactive Cells

NOS immunoreactive neurons in the ewe brain displayed a weak cytoplasmic yellow-green fluorescence (Fig. 1, A–C). Comparison of the same section of NOS-immunolabeling with the distribution of NADPHd cells stained dark violet after histochemistry (Fig. 1, D–F) revealed that all NADPHd neurons possess NOS immunoreactivity in each region examined (i.e., arcuate nucleus; Fig. 1, A and D, and VMNvl; Fig. 1, B and E, C and F).



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  		FIG. 1. Microphotographs of 20 µm frontal sections of ewes treated with E2 + P4 showing neuronal NOS-containing neurons detected with immunofluorescence in the (A) arcuate and (B and C) VMNvl. After photography, coverslips were carefully removed and histochemistry for NADPHd was performed on the same sections (D–F). Comparison of the two images revealed that NOS-immunoreactive cells were NADPHd-positive (arrows). Scale bar = 25 µm

Distribution of NADPHd-Positive Neurons in the Ovine Hypothalamus

The distribution of NADPHd-positive neurons is the same in ewes treated with E2 + P4 and P4 only. At the most rostral level of the POA (Fig. 2A), NADPHd-stained neurons were found predominantly in the diagonal band of Broca, where they were more concentrated in the vertical portion (Fig. 3A) and around the fornix. Some scattered neurons were also found around the anterior commissure and along the lateral ventricle (Fig. 2A). Many moderately stained NADPHd cells were found throughout the medial POA (Fig. 2, B and C, and Fig. 3B). In contrast, very few faintly stained neurons were evident in the supraoptic nucleus (Fig. 2, B and C). Some densely stained cells surround the fornix (Fig. 2, B–D) and many strongly colored NADPHd cells were observed in the magnocellular part of the paraventricular nucleus; most labeled neurons in the parvocellular part were weakly stained (Fig. 2, D and E, and Fig. 3C). Some moderately stained neurons were spread in the anterior hypothalamic area and in the periventricular area (Fig. 2D). Few cells were detected in the border of the ventromedial nucleus with the neighboring third ventricle or in the central ventromedial nucleus (Fig. 2, E–G, and Fig. 3D). In contrast, in the VMNvl, where neurons were larger than in the other parts of the nucleus, many darkly stained, densely clustered NADPHd neurons were found (Fig. 2, F and G), and they extended dorsally toward the perifornical area (Fig. 2F). The dorsomedial nucleus (Fig. 2, F and G) contained some small moderately stained neurons. Scattered intensely colored neurons were also observed throughout the rostrocaudal extension of the lateral hypothalamic area and along the optic tract (Fig. 2, F and G). Small moderately stained NADPHd-positive neurons were detected throughout the rostrocaudal extension of the arcuate nucleus (Fig. 2, F–H), but they were more numerous in the caudal part (Fig. 3E). The ventral premamillary (Fig. 3F) and the paramamillary areas that appear well demarcated on the basis of NADPHd histochemistry, showed large strongly stained neurons that were densely packed (Fig. 2H).



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  		FIG. 2. Representative drawings of 20-µm coronal sections (A–H) through the POA and hypothalamus of ewes treated with P4 only showing the localization of NADPHd-stained neurons (dots). Each dot represents two NADPHd-positive cells. ac, Anterior commissure; AHA, anterior hypothalamic area; AR, arcuate nucleus; DBB, diagonal band of Broca; DM, dorsomedial nucleus; f, fornix; LHA, lateral hypothalamic area; lPOA, lateral preoptic area; LS, lateral septum; lv, lateral ventricle; ME, median eminence; mPOA, medial preoptic area; mt, mamillothalamic tract; oc, optic chiasm; ot, optic tract; Pa, paramamillary area; pt, anterior pituitary, pars tuberalis; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; st, stria terminalis; VMN, ventromedial nucleus; VP, ventral part of the premamillary area



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  		FIG. 3. Microphotographs of 20-µm coronal sections throughout the vertical subdivision of the diagonal bands of Broca (A), the medial POA (B), the paraventricular nucleus (C), the dorsolateral and central ventromedial nucleus (D), the periventricular hypothalamic area (D), the caudal arcuate nucleus (E) and the ventral premamillary area (F) of a ewe treated with P4 only after NADPHd histochemistry. AR, Arcuate nucleus; DBBv, vertical portion of the diagonal band of Broca; f, fornix; Pe, periventricular area; mPOA, medial preoptic area; PVN, paraventricular nucleus; VP, ventral part of the premamillary area. Bar = 45 µm in A–C and 105 µm in D–F

Effect of Estradiol Treatment on NADPHd Cell Number

To assess the effect of estradiol on NADPHd-positive cells, the number of stained neurons was compared in the POA, the arcuate nucleus, and the VMNvl between ewes treated with E2 + P4 and P4 only (Fig. 4). Quantitative analysis revealed that ewes treated with E2 had significantly (P < 0.01) more NADPHd-stained neurons in the VMNvl, where the number of cells in animals treated with E2 + P4 was nearly double that found in ewes treated with P4 only (Fig. 4). In contrast, the number of NADPHd-stained cells in the POA or arcuate nucleus was not significantly modified (P > 0.6; Fig. 4).



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  		FIG. 4. Influence of estradiol on the number of NADPHd stained cells in the POA, arcuate nucleus (AR), and VMNvl in ewes treated with E2 + P4 (n = 4; black bars) and P4 only (n = 4; white bars). ***P < 0.01; Student t-test E2 + P4 vs. P4 only

Colocalization of NADPHd with PR

The distribution of PR-immunopositive cells colored in brown from DAB obtained in ewes treated with E2 + P4 concurs with previous findings [8]. The distribution of PR-stained and NADPHd-stained cells overlapped at the level of the medial POA (Fig. 5A), VMNvl (Fig. 5B), and arcuate nucleus (Fig. 5, C and D). Double-labeled neurons were noted by the dark blue coloration associated with NADPHd in their cytoplasm and the brown DAB product linked to PR in their nucleus (Fig. 5, A–D). They were found throughout the rostrocaudal extension of these three areas, but the largest concentration occurred in the ventromedial nucleus (Fig. 5B). These double-stained neurons represented 81.3% ± 3.2% of the NADPHd cells in the medial POA (260–380 NADPHd cells/ewe), 88.9% ± 0.8% in the VMNvl (850–1150 NADPHd cells/ewe) and 90.0% ± 1.5% in the arcuate nucleus (400–490 NADPHd cells/ewe).



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  		FIG. 5. Microphotographs of 20-µm brain sections from ewes treated with E2 + P4 (A–D) or with P4 only (E–H) after NADPHd histochemistry combined with PR (A–D) or GR (E–H) immunocytochemistry at the level of the POA (A and E), VMNvl (B and F) and arcuate nucleus (C, D, G, and H). Double-labeled neurons show both cytoplasmic dark violet staining from NADPHd histochemistry and the brown nuclear precipitate from DAB associated to immunocytochemistry of steroidal receptors. Note the difference in intensity of NADPHd staining at the level of the VMNvl between ewes treated with E2 + P4 (B) and ewes treated with P4 (F) only. Bar = 45 µm in A–C and E–G, and 10 µm in D and H

Colocalization of NADPHd with GR

GR immunoreactive neurons in the ewe hypothalamus were spread widely as described previously [7]. The highest concentrations of GR cells occur in the medial POA, arcuate nucleus, and ventromedial nucleus. GR was also expressed to a lesser extent in the other diencephalic areas. GR distribution overlapped that of NADPHd neurons mainly in the medial POA (Fig. 5E), VMNvl (Fig. 5F), arcuate nucleus (Fig. 5, G and H), and to a smaller degree in the paraventricular nucleus, dorsomedial nucleus, and ventral premamillary area. Double-labeled neurons showed well-defined cellular compartments that were distinguishable by their respective color as described for PR (Fig. 5, E–H). They were observed in the regions where distributions of GR and NADPHd overlap except in the paraventricular nucleus. The largest subpopulation of GR immunoreactive cells showing NADPHd activity was observed in the arcuate nucleus (Fig. 5, G and H). Quantitative analysis revealed that 84.3% ± 1.6% of NADPHd cells contain GR in the arcuate nucleus (450–650 NADPHd cells/ewe), 79.0% ± 1.3% in the medial POA (250–340 NADPHd cells/ewe) but only 31.8% ± 6.1% in the VMNvl (350–800 NADPHd cells/ewe).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study describes the neuronal distribution of NADPHd-cells, which are NOS-immunoreactive, in the POA and hypothalamus of the ewe. Moreover, we have shown that the number of NADPHd cells varies in a site-specific manner in response to estradiol treatment, and that both progesterone and the corticosteroids target these cells.

Our demonstration that ovine NADPHd-positive cells contain neuronal NOS immunoreactivity is consistent with previous studies in the rat [26, 34, 35], guinea pig [20], and primates [26, 36].

The distribution of NADPHd cells in ovine brain is globally consistent with previous studies in rats [31, 37], guinea pigs [20], primates [38], and humans [39]. The POA and VMNvl contain NADPHd/NOS-positive neurons in all species examined to date [18, 20, 38–40]. A few species-dependent differences were also noted, mainly at the level of supraoptic, paraventricular, and arcuate nuclei. The supraoptic nucleus of the ewe contains very few faintly stained NADPHd-positive neurons, which concurs with findings in the guinea pig [20], but this region is strongly labeled in rats [31, 37]. In contrast, NADPHd cells were undetectable in the supraoptic nucleus of monkeys [38] or humans [39]. More stained neurons were noted in the magnocellular part of the paraventricular nucleus than in the parvocellular area, which is consistent with reports in rodents [20, 31, 37] and cats [40], whereas few NADPHd/NOS neurons were apparent in the paraventricular nucleus of monkeys [38] or humans [39]. In agreement with a study on the macaque monkey [38], NADPHd-stained neurons were observed throughout the rostro-caudal extension of the arcuate nucleus of the ewe, with the greatest density of cells at the caudal level. This distribution contrasts with that of rats, in which no [31] or very few [30] cells were detected, and in guinea pigs, in which NOS cells were found only in the caudal part of the arcuate nucleus [20].

Ewes treated with E2 + P4 showed an higher number of NADPHd cells than in ewes treated with P4 only, suggesting that estradiol increases the number of NADPHd cells. This result agrees with previous studies in rats showing a direct influence of female gonadal steroids on NOS synthesis, or activity in the central and peripheral system, or both [41–43]. It is interesting that this regulation appears exclusive to the VMNvl of ewes, as previously reported in guinea pigs [20] and in several studies on rats [18, 42, 43]. No significant modulation of NOS activity appears in the ovine POA following estrogen treatment; this differs from a previous study on ovariectomized rats, in which an increase of NADPHd activity was detected in the medial POA following estradiol treatment for 2 days [19]. The duration of the steroid treatment and species-specific response to estradiol priming may explain this discrepancy. Alternatively, our results may reflect an increase in PR by estradiol, which allows a greater induction of NADPHd by progesterone.

We have demonstrated the coexistence of PR with NADPHd activity in ovine brain, suggesting a direct effect of progesterone on neurons that produce NO. This result concurs with previous data obtained in guinea pigs, in which approximately 10% of NOS cells in the POA and 55% in the ventrolateral nucleus, which is homologous to the ovine VMNvl, show PR immunoreactivity [20]. It is interesting that the percentage of NADPHd cells containing PR was higher in ewes than in guinea pigs. Although this may suggest a more important role for NO in physiological processes in the brain of ewes, we believe this disparity probably results from the use of an antigen retrieval technique in our study, which has been shown to significantly increase the number of PRs detected in the brains of guinea pigs [29]. In the ovine brain, we are unable to detect PR if no unmasking has been carried out [8].

We recently demonstrated that all PR-immunoreactive neurons contain estrogen receptor {alpha} in the ewe POA and hypothalamus [44]. Thus, data from the present study can be related to previous studies performed in rats [19], in which 80% of NADPHd neurons were estimated to contain estrogen receptors in the POA and VMNvl.

To our knowledge, this is the first study to demonstrate a high degree of coexistence between NADPHd/NOS activity and GR in a mammalian brain. This suggests a direct control of NO production by corticosteroids released following a stress within many hypothalamic areas, including regions that are highly involved in the control of reproductive function such as the POA, arcuate nucleus, and ventromedial nucleus. Coexistence of NADPHd activity with PR or GR is close to 80%–90% in the arcuate nucleus and POA. It is apparent therefore that many NO-producing neurons from the arcuate nucleus and POA contain both PRs and GRs, and we are currently investigating this hypothesis. In contrast, it seems likely that only a small subset of NADPHd-positive neurons contains both PRs and GRs in the VMNvl, in which only 30% of NO-producing neurons contain GRs, whereas this percentage reaches 90% for PRs. Taken together, these data may mean that progesterone and corticosteroids act through convergent pathways on reproduction and stress regulation at the level of the POA and arcuate nucleus, but mainly through divergent pathways within the VMNvl, where the NOergic system may be more sensitive to gonadal steroids.

The physiological importance of PRs and GRs in NOS-containing neurons of the POA, arcuate nucleus, and ventromedial nucleus remains to be elucidated. However, because NO is known to increase secretion of many neuropeptides, including GnRH [12, 14] and ß-endorphin [45], many hypotheses may be postulated. Studies of glucocorticoids have shown that both corticosterone [46] and dexamethasone [47] decrease NOS mRNA in the rat hippocampus and in neuroblastoma cells. Dexamethasone treatment also diminished NADPHd staining in the spinal cord of rats [48]. Our data suggest that glucocorticoids, which can delay ovulation [4], may inhibit GnRH secretion partly through having a direct influence on NO production. The influence of progesterone on neuronal NOS mRNA is not known, although the potent inhibitory effect of progesterone on GnRH secretion [28] suggests that PRs located in NO-producing neurons may inhibit NOS synthesis, activity, or both in order to slow down NO production and thereby modulate GnRH synthesis, secretion, or both. The inhibitory effect of progesterone on GnRH secretion in the ewe appears mediated by neurons within the ventromedial/arcuate nucleus region [49, 50]. Thus, NO that is synthesized and released locally could act via autocrine pathways, paracrine pathways, or both to modulate the activity of neighboring neurons and, thereby, transduce the effect of progesterone. In this context, neuronal NOS knockout mice exhibited a lower number of ß-endorphin neurons, which is a potent inhibitor of GnRH secretion [51] in the arcuate nucleus [45]. Alternatively, because NOS is also present in axonal terminals [52] and neuronal subpopulations from the arcuate nucleus and ventromedial nucleus project toward areas potentially involved in GnRH release [53–55], then NO produced in these nuclei under progesterone or glucocorticoid control could potentially affect GnRH release.

In conclusion, these data show for the first time that the NOergic system of the ewe is extremely receptive to estradiol treatment and specifically within the VMNvl region of the hypothalamus. Furthermore, there is substantial colocalization of both PRs and GRs with NO neurons in the POA and arcuate nucleus. Moreover, although PRs were significantly colocalized with NO cells in the VMNvl, coexistence of NO with GRs is more sporadic in this area. These data demonstrate that NOS activity in these regions is highly dependent on the steroidal status of the animal and suggest that NO may play a fundamental role in the intercellular communications that control GnRH secretion and regulation of the stress axis.


   FOOTNOTES
 
First decision: 8 March 2002.

1 L.D. was supported by Wellcome Trust Travelling Fellowship 061765/Z/00/Z. Back

2 Correspondence: Laurence Dufourny, University of Wyoming, Department of Zoology and Physiology, Biological Science Building, Room 428, PO Box 3166, Laramie, WY 82071-3166. FAX: 307 766 5625; dufourny{at}uwyo.edu Back

Accepted: April 4, 2002.

Received: February 16, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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