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BOR - Papers in Press, published online ahead of print August 20, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.016956
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BIOLOGY OF REPRODUCTION 69, 2015–2021 (2003)
DOI: 10.1095/biolreprod.103.016956
© 2003 by the Society for the Study of Reproduction, Inc.

Neuroendocrinology

Effect of Testosterone and Season on Proenkephalin Messenger RNA Expression in the Preoptic Area of the Hypothalamus in the Ram1

Christopher J. Scott2,3,4, Michael Mariani4, Iain J. Clarke5, and Alan J. Tilbrook4

Department of Physiology,4 Monash University, Victoria, Australia Prince Henry's Institute of Medical Research,5 Clayton, Victoria, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Enkephalin appears to exert an inhibitory action on LH secretion, but whether testosterone regulates enkephalin gene expression is unknown. This study tested the hypothesis that testosterone and/or season modulate preproenkephalin mRNA expression in specific areas of the hypothalamus. Romney Marsh rams were castrated (wethers) either during the breeding season or nonbreeding season and received intramuscular injections of either oil or testosterone propionate (five/group). Blood samples were taken for the assay of plasma LH and testosterone. Preproenkephalin mRNA expression was quantified in hypothalamic sections by in situ hybridization. Mean plasma LH concentrations were reduced and the interpulse interval for LH pulses was greater in testosterone propionate-treated wethers compared with oil-treated wethers, with no change in LH pulse amplitude. Testosterone propionate treatment reduced proenkephalin expression in the diagonal band of Broca, the caudal preoptic area, and the bed nucleus of the stria terminalis. Seasonal differences in proenkephalin expression were observed in the bed nucleus of the stria terminalis, lateral septum, periventricular nucleus, and paraventricular nucleus. No differences were observed between treatments in seven other regions examined. We conclude that testosterone and season regulate proenkephalin mRNA levels in the preoptic area/hypothalamus in the ram in a region-specific manner.

hypothalamus, male sexual function, neuroendocrinology, seasonal reproduction, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Testosterone, produced by the testes, exerts actions on most tissues throughout the body, including the brain [1]. Within the brain, testosterone plays a prominent role in the organization, programming, and activation of neuronal circuits [2]. In particular, testosterone exerts a negative feedback effect on gonadotropin releasing hormone (GnRH) secretion by the brain, which in turn affects the secretion of luteinizing hormone (LH) secretion from the pituitary gland [3, 4]. Testosterone action on the brain also affects behavior, including reproductive behavior [2]. The cellular mechanisms whereby testosterone causes central effects are poorly defined. Among the candidate neurons are those that produce endogenous opioid peptides such as ß-endorphin, dynorphin, and enkephalin. In this regard, testosterone enhanced the ability of the mixed opioid receptor antagonist, naloxone, to increase GnRH and LH secretion in male rats [5] and rams [6, 7]. This may be effected by antagonism of the actions of ß-endorphin or enkephalin or dynorphin, all of which are present within the neuroendocrine hypothalamus [8, 9]. Castration increased the mRNA levels of the ß-endorphin-precursor molecule pro-opiomelanocortin (POMC) in the hypothalamus of rats [9], while testosterone decreased hypothalamic POMC mRNA levels in rats [10] and rams [11]. Notably, POMC mRNA levels in the hypothalamus of the ram were altered following treatment with testosterone only at the time of the normal nonbreeding season and not during the breeding season [12]. An interaction between season and testosterone in the regulation of POMC levels was also reported for the hypothalamus of golden hamsters [13].

Treatment of wethers with the delta receptor agonist FK 33-824 inhibited LH secretion [14], suggesting that enkephalins may be inhibitory to GnRH secretion. Enkephalin levels in the hypothalamus have been reported to increase following mating in male rats, suggesting that enkephalins may also be involved in the regulation of reproductive behavior [15]. Whether or not enkephalins in the hypothalamus are regulated by testosterone is largely unknown. Met-enkephalin release from hypothalamic slices taken from castrated male rats increased following superfusion with testosterone in vitro [16]. Similarly, Met-enkephalin levels in the hypothalamus of male rats increased following treatment with the androgen nandrolone decanoate [17]. These studies suggest that enkephalin production is stimulated by testosterone in the rat. Enkephalin-producing neurones, however, are found in a number of different hypothalamic nuclei [18] and these may well be differentially regulated. Whether the activity of enkephalin-producing neurones differs with season, as is the case for POMC mRNA-producing neurones [12] is unknown, although there is evidence that this may be the case in the golden mantled ground squirrel [19]. The present study tested the hypothesis that testosterone and/or season modulate preproenkephalin mRNA expression in specific areas of the hypothalamus in the ram.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Procedure

This work was conducted in accordance with the Australian Prevention of Cruelty to Animals Act, 1986, and the NH&MRC/CSIRO/AAC Code of Practice for the Care and Use of Living Animals for Scientific Investigations. The work was approved by the Ethics Committees of Monash University and of the Victorian Institute of Animal Science.

The experiment used adult Romney Marsh rams and was conducted at Prince Henry's Institute of Medical Research, Werribee, Australia (38°S). During the experiment, the animals were kept under natural photoperiod and had free access to feed and water. Rams were surgically castrated under halothane anaesthesia 1 wk prior to the commencement of experimental procedures. Groups of 10 animals were used during the breeding season (April–May) and nonbreeding season (November–December) for this breed of sheep [20]. Within seasons, rams were randomly allocated to one of two groups (n = 5/group) of similar mean bodyweight. One group received i.m. injections of testosterone propionate (Sigma Chemical Company, St. Louis, MO) for 7 days commencing a week after surgery (8 mg every 12 h). This dose of testosterone propionate was chosen because it significantly reduces plasma LH levels in both seasons [21]. The second group was given i.m. injections of peanut oil vehicle (1 ml every 12 h) for 7 days. On the seventh day of treatment, blood samples were taken every 10 min for 12 h via indwelling jugular venous catheters (Dwellcath, Tuta Laboratories, Lane Cove, Australia) inserted the previous day.

Tissue Collection

On the day following blood sampling, the sheep were killed in pairs consisting of an oil-treated and a testosterone propionate-treated wether, by an overdose of sodium pentobarbital (Lethabarb; Virbac, Peakhurst, NSW, Australia). These pairings were maintained for the in situ hybridization studies. The final testosterone propionate and oil injections were given on the morning of tissue collection, approximately 1 h prior to the commencement of the tissue collection from the first pair of animals. The heads were removed and perfused through both carotid arteries with 2 L normal saline containing 25 000 U heparin, followed by 1.5 L 4% paraformaldehyde in 0.1 M phosphate buffer, the final 0.5 L containing 20% sucrose. The brain was then removed, the hypothalamus dissected out and postfixed at 4°C in fixative containing 30% sucrose for 14 days. Cryostat sections were cut in the coronal plane at a thickness of 20 µm, collected into cryoprotectant with 2% paraformaldehyde and stored at -20°C.

Radioimmunoassays

Plasma concentrations of LH were measured by radioimmunoassay as described by Lee et al. [22], using NIH-oLH-S18 as standard. Seven assays were conducted with an average sensitivity of 0.5 ng/ml, an intraassay coefficient of variation less than 13% over the range 3.3–18.9 ng/ml and an interassay coefficient of variation less than 14%. Plasma concentrations of testosterone were measured using a commercial kit (Coat-A-Count, Diagnostic Products Corporation, Los Angeles, CA), using one assay for all samples measured in each season.

In Situ Hybridization

In situ hybridization was conducted using 35S-dUTP-labeled riboprobe according to the protocol of Simmons et al. [23] as described in detail previously [24]. We used a 693-base pair (bp) rat proenkephalin cDNA inserted in pGem3 [25]. The amplification, purification, and linearization of plasmid DNA were performed using standard techniques [26]. The proenkephalin gene encodes for met-enkephalin and leu-enkephalin. Matched sections from oil- and testosterone propionate-treated wether from the same season were mounted on the same Super Frost Plus slide (Menzel Glasser, Braunscheig, Germany) and air dried overnight. Matched sections from animals from the other season were also mounted on another slide. Thus, anatomically matched sections from one animal of each treatment group were hybridized in the same run. There were five hybridization runs, each run containing all the tissue analysed from one animal (16 sections) from each treatment group. The slides were coded such that the subsequent image analysis was conducted with the operator blind to the treatments.

For hybridization, 200 µl of hybridization solution (5 x 106 cpm/ml) was applied to each slide, covered with a glass coverslip, and the slides placed in a humidified plastic container and incubated at 53°C for 16 h. Following posthybridization washes, the slides were placed in apposition to a phosphoimager plate overnight to verify the success of the hybridization and then dipped in Ilford K5 photographic emulsion (Ilford Australia, Mt. Waverley, Victoria, Australia) and exposed for 3 days. The dipped slides were then developed using Ilford Phenisol X-ray developer, fixed, and lightly counterstained with 1% cresyl violet.

Image Analysis

Semiquantitative image analysis was conducted on dipped autoradiograms as described previously [24]. Grain counting was conducted under brightfield conditions at 400x magnification using a Fuji HC-2000 high-resolution digital camera and Analytical Imaging Station 4.0 software (Imaging Research Inc., St Catharine's, ON, Canada). Cells were regarded as labeled if grain counts were more than 5 times background (which was typically less than 5 grains per equivalent cellular area). From each treatment group, 1 section per animal was selected from the central (mid) region of each nucleus and, from each section, 10 labeled cells were selected at random from throughout the whole nucleus. For the calculation of cell density, two sections (from approximately 1 mm apart) per animal were selected for each nucleus and cells were counted manually under darkfield conditions at 100x magnification within an eyepiece grid placed in the center of the nucleus. When used at this magnification, the grid covers 0.81 mm2. All densities were converted to cell number per mm2.

Statistical Analysis

The data for plasma concentrations of LH were subjected to pulse analysis. Pulses of LH were defined according to Karsch et al. [27] as abrupt increases that were greater than the assay sensitivity, that exceeded the previous value by at least three times the standard deviation of the previous value, and that were followed by a progressive decline at a rate consistent with the reported half-life for LH of 29 min [28]. The interpulse interval was calculated as the time (in minutes) between successive peaks for defined pulses. In the event that no pulses occurred in the sampling interval, the interpulse interval was the total time of sampling (i.e., 720 min) and for one pulse it was half the time of sampling. The amplitude of LH pulses was calculated as the difference between the peak and the preceding nadir. All statistical analysis of the hormone and image analysis data was by two-factor ANOVA. Post hoc analysis was by least significant differences. Homogeneity of variance was tested using Levene test of equality of error variances and, when necessary, square-root transformations were performed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Plasma Hormone Concentrations

Plasma testosterone levels were undetectable in oil-treated wethers but were significantly (P < 0.001) higher in wethers treated with testosterone propionate (Fig. 1A). Plasma testosterone concentrations were significantly (P < 0.001) higher in the wethers treated with testosterone propionate in the nonbreeding season than those similarly treated during the breeding season (Fig. 1A).



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  		FIG. 1. A) Mean plasma testosterone concentrations (ng/ml), (B) mean plasma LH concentrations (ng/ml), (C) LH interpulse interval (min), and (D) LH pulse amplitude (ng/ml) in wethers treated with oil or testosterone propionate during the breeding season or nonbreeding season. *P < 0.05; **P < 0.01; ***P < 0.001. Note that no pulses of LH were detected in plasma from wethers treated with testosterone propionate during the nonbreeding season and hence no pulse amplitude could be calculated

Mean plasma concentrations of LH were significantly (P < 0.001) lower in testosterone propionate-treated wethers than in oil-treated wethers (Fig. 1B). There was no significant effect of season or season x hormone treatment interaction. There was no pulsatile LH secretion in all wethers treated with testosterone propionate during the nonbreeding season and in 2/5 wethers in the breeding season. As a result, the interpulse interval for LH pulses was significantly (P < 0.01) higher in testosterone propionate-treated wethers compared with oil-treated wethers (Fig. 1C). The interpulse interval was also significantly (P < 0.01) higher in wethers sampled during the nonbreeding season compared with the breeding season and there was a season x hormone treatment interaction (P < 0.01). Post hoc analysis revealed that the interpulse interval was significantly (P < 0.05) higher in testosterone propionate-treated wethers than in oil-treated wethers in both seasons. In addition, the interpulse interval was significantly (P < 0.05) higher in testosterone propionate-treated wethers in the nonbreeding season compared with similarly treated wethers in the breeding season. By contrast, there was no seasonal difference in the interpulse interval in oil-treated wethers. The amplitude of LH pulses was not significantly different between groups (P > 0.05, Fig. 1D).

Distribution of Cells Expressing Proenkephalin mRNA

The distribution of cells in the preoptic area/hypothalamus that contained proenkephalin mRNA is shown in a series of autoradiograms from a representative ram (Fig. 2). Proenkephalin mRNA-containing cells were detected in a number of hypothalamic nuclei. High levels of expression were found in the rostral and caudal regions of the medial preoptic area, medial septum, bed nucleus of the stria terminalis (Fig. 3), periventricular nucleus, paraventricular nucleus, and the ventromedial nucleus. Strong expression was also found in the diagonal band of Broca, lateral septum, lateral preoptic area, anterior hypothalamic area, ventral premamillary nucleus, and posterior hypothalamus. Scattered cells (up to eight per section) were found lateral to the paraventricular nucleus, in the dorsomedial nucleus and in the arcuate nucleus. These latter three populations of cells were weakly labeled and were only evident after slides had been placed under emulsion for over a week and were not subjected to image analysis. There were no labeled cells detected in the suprachiasmatic nucleus, supraoptic nucleus, or median eminence. No signal was detected when sections were hybridized with the sense strand or when sections hybridized with the antisense probe were pretreated with RNAse A (not shown).



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  		FIG. 2. Film autoradiograms showing the distribution of proenkephalin mRNA-containing cells in the preoptic area/hypothalamus of a representative ram. Scale bar = 5 mm. A–H) These figures show 20-µm sections covering the main enkephalin mRNA-containing regions (rostral to caudal). 3V, Third ventricle; AHA, anterior hypothalamic area; BNST, bed nucleus of the stria terminalis; CPOA, caudal preoptic area; DBB, diagonal band of Broca; DMH, dorsomedial nucleus of the hypothalamus; LPOA, lateral preoptic area; LS, lateral septum; MS, medial septum; OC, optic chiasm; PeVN, periventricular nucleus; PH, posterior hypothalamus; PVN, paraventricular nucleus; RPOA, rostral preoptic area; VMH, ventromedial nucleus of the hypothalamus; VPMN, ventral premamillary nucleus.



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  		FIG. 3. Low power dark-field photomicrographs of the bed nucleus of the stria terminalis hybridized with antisense mRNA for proenkephalin from a wether treated with oil (A, C) or testosterone propionate (B, D) during the breeding season (A, B) or nonbreeding season (C, D). Scale bar = 200 µm. AC, anterior commissure

The Effect of Testosterone Propionate Treatment or Season on the Expression of Proenkephalin mRNA

The number of silver grains/labeled cell in the 13 regions studied is shown in Figure 4. The number of silver grains/cell was significantly greater in both the diagonal band of Broca (P < 0.01) and bed nucleus of the stria terminalis (P < 0.001) of controls (oil-treated wethers) vs. wethers treated with testosterone propionate. In the bed nucleus of the stria terminalis, there was also a significantly (P < 0.001) higher number of silver grains/cell in animals killed during the breeding season compared with the nonbreeding season and a significant (P < 0.001) season x hormone interaction. Post hoc analysis revealed that the number of silver grains/cell in the bed nucleus of the stria terminalis was higher in oil-treated wethers than in wethers treated with testosterone propionate in both seasons (P < 0.05 in both cases). In addition, the number of silver grains/cell in the bed nucleus of the stria terminalis of oil-treated wethers was higher (P < 0.001) in the breeding season than in the nonbreeding season. The number of silver grains/cell in the bed nucleus of the stria terminalis of testosterone-treated wethers did not differ between seasons. There was a significantly (P < 0.05) greater number of silver grains/cell in the periventricular nucleus of wethers killed during the breeding season compared with the nonbreeding season. The number of silver grains/cell in the paraventricular nucleus was significantly (P < 0.01) higher in the nonbreeding season compared with the breeding season. No differences (P > 0.05) were observed in the number of silver grains/cell in any of the other groups.



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  		FIG. 4. Histogram showing the mean (± SEM) number of silver grains/cell for proenkephalin mRNA in various regions of the preoptic area/hypothalamus in wethers treated with oil or testosterone propionate during the breeding season or nonbreeding season. *P < 0.05; **P < 0.01; ***P < 0.001. For abbreviations, see Figure 2

The density of proenkephalin mRNA-containing cells in the 13 regions studied is shown graphically in Figure 5. There was a significantly lower density of proenkephalin mRNA-containing cells in the caudal preoptic area (P < 0.05) of testosterone-treated wethers compared with oil-treated wethers. The density of proenkephalin mRNA-containing cells was higher in the breeding season compared with the nonbreeding season in the lateral septum (P < 0.01) and periventricular nucleus (P < 0.01). In the paraventricular nucleus, however, the density of proenkephalin mRNA-containing cells was higher (P < 0.01) in the nonbreeding season compared with the breeding season. In all other nuclei, the density of proenkephalin mRNA-producing cells was the same in all groups.



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  		FIG. 5. Histogram showing the mean (± SEM) number of labeled cells per mm2 for proenkephalin mRNA in various regions of the preoptic area/hypothalamus in wethers treated with oil or testosterone propionate during the breeding season or nonbreeding season. *P < 0.05; **P < 0.01. For abbreviations, see Figure 2


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have mapped the distribution of proenkephalin mRNA-producing cells in the preoptic area and hypothalamus of the ram and have shown that testosterone and season regulate the level of mRNA expression in a region-specific manner. The distribution of proenkephalin mRNA-producing cells in the hypothalamus of the ram was similar to that previously found in the female sheep [18] and was also similar to the distribution of met-enkephalin that was seen with immunohistochemistry [8]. An earlier study by Matthews et al. investigating the distribution of proenkephalin mRNA-producing cells in the hypothalamus of the ram [29] found such cells only in the paraventricular nucleus and suprachiasmatic nucleus. That study used a cDNA probe, which is less sensitive than the cRNA probe used in the present study [23], and this may explain some of the differences in distribution that we observed in our study. The reason why the study of Matthews et al. [29] found proenkephalin mRNA-containing cells in the suprachiasmatic nucleus when we and others [e.g., 8, 18] find no evidence of enkephalin-producing cells in this nucleus in sheep is unclear.

The paraventricular and periventricular nuclei showed clear but opposing responses to season. The functional significance of these changes is unknown, but similar reciprocal changes in proenkephalin mRNA in the periventricular and paraventricular nuclei were observed following changes in adiposity [30]. In that study, proenkephalin expression was lower in the periventricular nucleus and higher in the paraventricular nucleus in fat sheep compared with thin sheep [30]. Enkephalins have been implicated in the control of food intake, stimulating feeding in sheep [31], and there is a seasonal variation in voluntary feed intake in sheep [32, 33]. Considering that adiposity is a regulator of feed intake, the similarity with the results from the present study suggest a role for enkephalin-producing cells in the paraventricular and periventricular nuclei in the seasonal regulation of voluntary feed intake. Further work is required to address this possibility.

The results of this study suggest that testosterone and/or its metabolites regulate expression of proenkephalin in three regions of the preoptic area/hypothalamus of the ram, viz. the diagonal band of Broca, bed nucleus of the stria terminalis, and the caudal preoptic area. In the ram, these regions contain androgen receptors [34] and estrogen receptors [24] and thus the actions of testosterone on the proenkephalin mRNA-producing cells may be a direct one. Whether these androgen or estrogen receptor-containing neurones produce enkephalins is not known in males of any species. Regardless, regulation by testosterone of enkephalins in the hypothalamus has been reported previously in the rat. Enkephalin release from hypothalamic slices was greater in slices taken from castrated male rats compared with slices taken from intact rats [16], an action that was blocked in testosterone-treated rats, suggesting an inhibitory action of testosterone on enkephalins in the hypothalamus. In another study, proenkephalin mRNA levels in the hypothalamus of male rats were reported to decrease following castration [35], an action that was blocked by estrogen treatment, suggesting a stimulatory role for testicular steroids on proenkephalin mRNA expression. Differences between studies may relate to the nuclei studied because our results indicate regional differences in the steroid regulation of proenkephalin expression. In this regard, the absence of an effect of testosterone propionate on proenkephalin mRNA expression in the ventromedial nucleus may be of particular importance. Studies in the ewe have shown changes in enkephalin mRNA expression in the ventromedial nucleus across the estrous cycle [18] and following estrogen treatment in ovariectomized ewes [36]. Estrogen implants into the ventromedial nucleus of the ewe stimulate a preovulatory-like surge in GnRH/LH secretion as well as induce estrous behavior [37]. Similar treatments in the ram do not do so [38]. This raises the possibility that the female-specific actions of the ventromedial nucleus to regulate reproduction may involve, at least in part, a role for the enkephalins. A similar result has been reported for the rat, [39], where there was no change in proenkephalin mRNA levels in the ventromedial nucleus of castrated male rats following treatment with testosterone or estrogen, despite increases in proenkephalin mRNA levels following estrogen treatment in female rats. A sex difference in the regulation of the promoter for the proenkephalin gene in the ventromedial nucleus has been reported [40], which may explain these results.

The reason for the seasonal difference in circulating testosterone levels is unclear. The difference is unlikely to be due to bodyweight because the sheep treated during the nonbreeding season, which had the higher plasma testosterone levels, were heavier than the breeding season animals. It is also unlikely that there were seasonal differences in the clearance rate of the testosterone because a study using the same breed of sheep showed no seasonal differences in circulating testosterone levels when using subcutaneous testosterone implants [41]. The possibility exists that there were seasonal differences in the rate that the propionate molecule is cleaved from testosterone and the free steroid is released into the circulation.

Given the seasonal difference in the plasma testosterone levels of the testosterone propionate-treated animals, the season x steroid interaction that we observed in the bed nucleus of the stria terminalis must be interpreted with some caution. We believe, however, that the interaction is not entirely due to the seasonal difference in circulating testosterone levels. The degree to which testosterone treatment reduced the number of silver grains/cell was greater during the breeding season (22.2% reduction in the breeding season and 8.8% reduction in the nonbreeding season). If the higher testosterone levels in the nonbreeding season were a major factor, then the reverse result might be expected. Thus, our results suggest that the ability of testosterone to reduce enkephalin mRNA levels in the bed nucleus of the stria terminalis is greater during the breeding season than in the nonbreeding season.

The functional significance of the seasonal shift in the ability of testosterone to regulate enkephalin mRNA in the bed nucleus of the stria terminalis is unclear, but a similar seasonal shift in the ability of testosterone to regulate gene expression has been reported for pro-opiomelanocortin mRNA expression in the arcuate nucleus of rams [11, 12] and golden hamsters [13]. Lesioning the bed nucleus of the stria terminalis in hamsters inhibits short photoperiod-induced testicular regression [42]. This raises the possibility that enkephalins in the bed nucleus of the stria terminalis are involved in the seasonal variation in GnRH secretion observed in species such as the hamster and sheep.

In our study, testosterone propionate clearly suppressed LH secretion in both seasons, as we have shown previously [21]. This was most likely to have been effected through inhibition of GnRH secretion [3], but the action is unlikely to be directly on GnRH neurones because these do not express androgen receptors [30] and little or no estrogen receptor {alpha} [43]. Accordingly, it is most likely that the actions are mediated via inhibitory system(s), which do express these receptors. It would seem unlikely, however, that enkephalins have a direct action on GnRH neurones in the ram to mediate the negative feedback actions of testosterone. Enkephalins are considered to be predominantly inhibitory neurotransmitters [44], so that one might expect any feedback actions of testosterone to be associated with an increased level of enkephalin mRNA expression. As observed, however, testosterone had no effect on enkephalin mRNA expression in most regions examined and caused a decrease in three regions. Studies in rats suggest that GnRH neurones do not express mRNA for the three major opioid receptor subtypes [45], but this does not preclude the possibility that enkephalinergic neurones may act as interneurones, which indirectly relay testosterone regulation of GnRH neurones. Testosterone has been reported to inhibit GnRH secretion in rams without having a measurable effect on GnRH mRNA [11], however, so it remains possible that testosterone may influence enkephalin release without altering proenkephalin mRNA levels. All of our results must be considered with this caveat.

In summary, we have examined the role of testosterone and season in the regulation of proenkephalin mRNA in 13 different regions of the preoptic area and hypothalamus in the ram. The results indicate that regulation varies in a region-specific manner. This is consistent with the heterogeneous structure and function of the hypothalamus and is indicative of a role for enkephalins in a diverse array of physiological functions.


   ACKNOWLEDGMENTS
 
We thank Dr. Anne Turner for assistance with surgery and blood sampling, Bruce Doughton, Karen Briscoe, Linda Morrish, and Adam Link for animal care and assistance with surgery and tissue collection.


   FOOTNOTES
 
1 Supported by grants to C.J.S. and A.J.T. from the National Health and Medical Research Council of Australia and Monash University. Back

2 Correspondence: Chris Scott, School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2678, Australia. FAX: 61 2 69332587; chscott{at}csu.edu.au Back

3 Current address: School of Biomedical Science, Charles Sturt University, Wagga Wagga, New South Wales, Australia Back

Received: 5 March 2003.

First decision: 18 March 2003.

Accepted: 7 August 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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