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BOR - Papers in Press, published online ahead of print June 22, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042275
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BIOLOGY OF REPRODUCTION 73, 781–789 (2005)
DOI: 10.1095/biolreprod.105.042275
© 2005 by the Society for the Study of Reproduction, Inc.

Inhibition of Luteinizing Hormone Secretion by Localized Administration of Estrogen, but not Dihydrotestosterone, Is Enhanced in the Ventromedial Hypothalamus During Feed Restriction in the Young Wether1

Christina J. McManus 2 , Robert L. Goodman , Nancy V. Llanza , Miroslav Valent , Adam B. Dobbins , John M. Connors , and Stanley M. Hileman 

Department of Physiology and Pharmacology, West Virginia University, Morgantown, West Virginia 26506-9229


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of steroids to inhibit LH secretion is enhanced during undernutrition. To identify potential hypothalamic sites at which this enhancement may occur, we examined LH secretion in feed-restricted or fed young wethers treated with locally administered metabolites of testosterone. In experiment 1, microimplants containing crystalline estradiol-17ß (E) or cholesterol were administered via chronic guide tubes directed to the preoptic area (POA) or ventromedial hypothalamus (VMH) in fed or feed-restricted wethers. E treatment in the VMH decreased LH pulse frequency, pulse amplitude, and mean LH concentration in feed-restricted, but not fed, wethers. E may act in the POA to suppress LH under feed restriction, but definite conclusions cannot be drawn because of steroid-independent effects of feed restriction on LH pulse frequency. In experiment 2, the effect of dihydrotestosterone (DHT) in the VMH was determined. DHT administration to the VMH did not alter LH secretion in either feed-restricted or fed wethers. Thus the VMH is one site wherein E negative feedback is enhanced during feed restriction in the wether. In contrast, we found no evidence for enhanced responsiveness to androgen negative feedback within the VMH of feed-restricted wethers. We suggest that increased sensitivity within the VMH to E, but not to DHT, is important for suppressing LH secretion in undernourished male sheep.

androgen receptor, estradiol receptor, hypothalamus, luteinizing hormone, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inadequate energy availability because of insufficient nutrient intake or excessive caloric expenditure results in a reversible suppression of the hypothalamic-pituitary-ovarian axis at all levels [1]. However, inhibition of GnRH release is thought to be the primary mechanism [2–5] because the administration of exogenous GnRH in a pulsatile pattern to undernourished individuals increases plasma concentrations of LH, FSH, and gonadal steroid hormones [6– 7], restores pulsatile LH secretion in feed-deprived females [8–12], and restores fertility when administered chronically [6, 13–14]. One of the ways by which reproductive function is compromised during undernutrition is through an enhanced negative feedback potency of gonadal steroids. For example, estradiol is extremely effective in suppressing LH secretion in ovariectomized, undernourished ewes [10] or feed-deprived wethers (Hileman et al., unpublished results). Similarly, feed restriction in castrated male sheep causes only a small suppression of LH secretion, a response that is magnified by estrogen infusion [15]. Sensitivity to exogenous androgens is also greater in underfed animals as compared to ad libitum fed animals [16]. The mechanism responsible for enhanced responsiveness to steroid negative feedback during decreased energy availability is currently unknown.

The locus for the enhanced responsiveness to steroid negative feedback in conditions of negative energy balance is probably not the GnRH neurons themselves, because very few GnRH neurons contain estrogen receptors of the {alpha}-subtype (ESR1) [17–18], or androgen receptors (AR) [19]. Although recently estrogen receptors of the ß-subtype (ESR2) and estrogen-related receptors have been found to be expressed in some GnRH neurons [20–23], there is no evidence implicating these receptors in the control of GnRH release. Thus, steroid negative feedback probably is conveyed to GnRH neurons via afferents [24]. Two hypothalamic areas in which changes in responsiveness to steroid negative feedback may occur are the preoptic area (POA) and the ventromedial hypothalamus (VMH). These regions have been identified previously as areas in which significant changes in the expression of estrogen receptors occur in response to feed restriction in the ovariectomized ewe [25], the Syrian hamster [26], the mouse [27], and the rat [28]. In addition, the POA contains the majority of GnRH cell bodies [29] and local interneurons may mediate the change in responsiveness to steroid negative feedback, such as the suppression of the reproductive axis during seasonal anestrus [30]. The VMH has a number of functions, including possible involvement in the regulation of feed intake, control of tonic LH release [31], and control of some reproductive behaviors [32]. Thus, the POA and VMH may play an important role in the enhancement of steroid negative feedback during negative energy balance.

In males, both androgenic and estrogenic steroids are involved in the negative feedback regulation of the reproductive axis [33]. For example, testosterone may act directly on AR, but it may also be metabolized to the more potent androgen dihydrotestosterone (DHT) via 5{alpha}-reductase or to estradiol via aromatase [34]. Although the role of DHT as a physiological inhibitor of LH is less characterized than the role of estradiol, DHT reduces pulsatile LH secretion [35] and the conversion of testosterone to DHT by 5{alpha}-reductase is a physiologically important step in the inhibition of LH secretion by testosterone in male sheep [36]. The role of DHT during feed restriction is not well characterized.

In order to determine whether the POA and/or VMH are sites at which enhancement of the responsiveness to steroid negative feedback occurs during feed restriction, we administered estradiol-17ß (E) or DHT locally through microimplantation via chronic guide tubes directed to the POA or VMH. In another model of increased responsiveness to steroid negative feedback, the anestrous ewe, E acts in the ventromedial POA to stimulate a system involving dopamine neurons that inhibit GnRH pulsatility [24, 30]. Recent data suggest that the suppression of the reproductive axis in the growth-restricted, ovariectomized ewe occurs through the central inhibition of GnRH neurons [37] and that, in the rat, feed-restriction increases dopamine receptor function [38]. Therefore, in order to examine the role of dopamine in the response to localized steroid administration to the POA and VMH, we administered the dopamine-D2 receptor antagonist sulpiride to see if the suppression of LH in steroid-treated, feed-restricted wethers involved dopaminergic input.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General

All procedures were approved by the West Virginia University Animal Care and Use Committee and follow NIH guidelines for use of animals in research. Long-term (at least 4 mo prior to neurosurgery) castrated male sheep (West Virginia University–Davis College of Agriculture, Forestry, and Consumer Sciences, Morgantown, WV) of predominantly Suffolk breeding were used. Wethers were approximately 20 wk of age at the start of neurosurgeries. They were maintained in an indoor facility with lighting adjusted to approximate natural day length, temperature maintained between 15 and 23°C, and access to water and a daily alfalfa pellet and corn ration. Experiments were conducted during the breeding season (September–November) to ensure that any change in LH secretion was caused by changes in nutritional balance and steroid treatment and not caused by suppressive effects of an inhibitory photoperiod.

Surgical Procedures

Neurosurgeries were performed as previously described [30, 39–40] using sterile techniques with wethers under halothane (approximately 2%) anesthesia in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). After the skull was exposed, a 20 mm wide x 30 mm long hole, centered about 10 mm rostral to the bregma, was drilled and punched in the skull and the superior sagittal sinus was ligated. A lateral ventricle was temporarily cannulated by lowering a sharpened 18-G stainless steel tube just rostral to bregma and 4 mm lateral to midline, and radio-opaque dye (iohexol, Omnipaque 350; Winthrop, New York, NY) was injected (2 ml over a period of 1 min) to visualize the ventricles. Bilateral 18-gauge sharpened stainless steel guide tubes (length: 53 mm for implants directed to the POA; 55 or 57 mm for implants directed to the VMH) were lowered to a position 2 mm dorsal to the target sites for microimplants (target site for POA: 1.5 mm lateral to midline, 3 mm dorsal to supraoptic recess of the third ventricle, at the rostral point of this recess in the AP plane; target site for VMH: 2 mm lateral to midline, 4 mm dorsal to floor of the third ventricle, 1–2 mm anterior to the most anterior portion of the infundibular recess in the AP plane). After positioning, guide tubes were blocked with 22-gauge wire stylets, the lateral cannula was removed, and the exposed brain was covered with gelfoam and a fine nylon mesh. Dental acrylic was applied over the fine mesh and around cranial screws for anchorage. The upper portion of a 20-cc plastic vial with a screwable lid was cemented in place with the dental acrylic to protect the protruding guide tubes and the skin was sutured around this apparatus [36]. Dexamethasone was administered i.m. in decreasing daily doses, beginning with 20 mg on the day prior to surgery and ending 3 days afterwards with 2 mg. Penicillin (6 ml) was also injected daily during this time period and atropine (15 mg) was given immediately prior to surgery. A postsurgical analgesic (flunixin meglumine; 100 mg) was administered while the animals were recovering from anesthesia. Animals were treated with an antibiotic following all changes in implants or stylets.

Tissue Collection and Analysis

Histological verification of the implantation sites was performed as described previously [30]. Specifically, sodium heparin (20 000 IU) was injected i.v. 10 min before, and again immediately prior to, the administration of a lethal dose of sodium pentobarbital (about 2000 mg i.v). The head was quickly removed and perfused via both internal carotid arteries with 4 L of 4% paraformaldehyde in 0.1 M PO4 buffer containing 1.0 IU sodium heparin/ml and 0.1% NaNO3 (a vasodilator). The brains were removed and tissue blocks containing the diencephalon dissected out and stored at 4°C in this fixative overnight, and then in 0.1 M phosphate buffer containing 20% sucrose until they sank. Frozen coronal sections (50 µm thick) were cut on a microtome and every fifth section was mounted on microscope slides and stained with cresyl violet for determination of implant location [30].

Steroid Administration

Steroids (Sigma, St. Louis, MO) were administered to the POA or VMH via microimplants consisting of sterile 22 gauge blunt-ended stainless steel tubes that extended 1 mm beyond the guide tubes and into which had been tamped crystalline E (experiment 1), DHT (experiment 2), or cholesterol (C), which was used as a control for both experiments. Microimplants were tamped in steroid 50 times and their exterior was wiped clean with sterile gauze [30].

Blood Collection

On all days of blood sample collection, peripheral blood samples (4 ml) were collected via jugular venipuncture at 12-min intervals for either 4 or 6 h as described below; this frequency of blood collection was based on previous experience that allows for easy identification of LH pulses [41–42]. Blood samples were stored at 4°C after clotting. Serum then was harvested and stored at –20°C until analysis of hormone concentrations by radioimmunoassay.

Experiment 1: E Microimplants Into the POA or VMH During Feed Restriction

In September 2002, chronic guide tubes were surgically placed into the targeted hypothalamic areas (POA, n = 13; VMH, n = 13). After at least 12 days of recovery from surgery, during which time animals were fed according to NRC requirements for maintenance [43], wethers were assigned within hypothalamic area to one of two feeding levels, restricted (R) or fed (F), with the mean weight of all groups approximately equal. Thus, a total of four treatment groups were used: POA-R (n = 7), POA-F (n = 6), VMH-R (n = 7), and VMH-F (n = 6). Restricted animals were fed to lose approximately 15% of initial body weight over 8 wk. Animals in the F group were fed at maintenance levels to minimize growth of the skull during the experimental period so that guide tube location remained the same throughout the experiment. Animals were weighed weekly and diets adjusted accordingly.

Treatment with microimplants began on Day 42 of feed restriction. Based on previous work in our laboratory (McManus et al., unpublished results), this corresponds to a time when wethers should exhibit enhanced sensitivity to steroid negative feedback, but before animals lost enough weight to cause steroid-independent suppression of the reproductive axis. Blood samples were collected for 4 h on Day 42 as a pretreatment control period, immediately after which animals received microimplants containing crystalline E or C. The implants were left in neural tissue for 3 days with blood samples collected on the last day (Day 45; 6 h), and then implants were removed and replaced with sterilized 22-gauge wire stylets. Blood samples were collected after 3 days of no treatment (Day 48; 4 h). Animals then received E or C using a crossover design for 3 days until sample collection on Day 51 (6 h) was completed. Implants were then removed and replaced with wire stylets, and samples were collected for 4 h on Day 54. On Day 55, animals were killed for histological verification of implantation sites.

In order to determine whether dopamine was involved in the steroid-induced suppression of LH during feed restriction, the dopamine-D2 receptor antagonist sulpiride was administered (1.2 mg/kg, i.m.) to all wethers after 4 h of frequent blood sample collection on Days 45 and 51 of feed restriction [30]. Frequent sample collection continued for an additional 2 h after the administration of sulpiride (for a total of 6 h of blood collection). Because sulpiride induces an immediate increase in episodic LH secretion when LH is suppressed by dopaminergic inhibition [30], 2 h of frequent blood sample collection was considered sufficient to assess the response to sulpiride.

Experiment 2: DHT Microimplants Into the VMH During Feed Restriction

Previous studies in which DHT or testosterone were administered locally to the hypothalamus of wethers for 5 to 7 days failed to alter LH secretion, possibly because of a reduction in androgen receptor expression because of the loss of testosterone in long-term castrated animals [31]. Therefore, we increased the duration of steroid treatment in order to test the effect of DHT in the hypothalamus on LH secretion. This precluded a crossover design, so R or F wethers were treated with microimplants containing either DHT or C. Thus, a total of four treatment groups were used: R with DHT (R-DHT; n = 7), R with C (R-C; n = 6), F with DHT (F-DHT; n = 6), and F with C (F-C; n = 6).

In September 2003, bilateral chronic guide tubes were surgically placed into the VMH of 25 wethers. After at least 7 days of recovery from surgery, during which time animals were fed according to National Research Council requirements for maintenance [43], wethers were assigned to one of two feeding levels, R (n = 13) or F (n = 12), so that mean body weight of the groups was approximately equal. Restricted animals were fed to lose approximately 15% of initial body weight over 8 wk and animals in the fed group were fed at maintenance levels. After 4 wk of feed restriction, animals were assigned to steroid treatments (DHT or C) so that the mean weight of the wethers in the two steroid treatments was approximately equal within feeding regimens.

Beginning on Day 28 of feed restriction, animals were treated with microimplants containing crystalline DHT or C. Implants were replaced every 7 days so that animals were continuously exposed to DHT or cholesterol for 28 days. The lumen of the used implants was examined upon replacement; all implants had some steroid remaining at the time of replacement. Frequent blood samples were collected for 4 h at 12-min intervals on Days –1, 28, 35, 42, 49, and 55 relative to the start of feed restriction. Implants were inserted or replaced following sample collection on Days 28, 35, 42, and 49. On Day 56, animals were killed for histological verification of implantation sites.

Radioimmunoassay Analysis

Concentration of LH was determined in 50-, 100- or 200-µl aliquots by radioimmunoassay, using a modification of a previously described method [30, 39]. Values are expressed in terms of the ovine standard, NIH S24. Radioiodinated ovine LH (AFP-8614B, courtesy of A.F. Parlow, NIDDK) was used as tracer and primary antiserum was AFP-192279 (courtesy of A.F. Parlow, NIDDK; dilution 1:2 000 000). The sensitivity (95% confidence interval at 0 ng/ml) averaged 0.08 ng/tube. Intra-assay coefficients of variation (CV) averaged 10.9% and 15.4% respectively, for serum pools displacing radiolabeled LH to approximately 37% and 61% of the total bound, and interassay CVs were 12.9% and 19.9% for the same serum pools.

Data Analysis

A pulse of LH was defined as previously described: any increase in concentration in which 1) concentrations were elevated relative to prenadirs and postnadirs for at least two consecutive samples, 2) the pulse peaked within two sampling intervals, 3) the increment between peak and nadir concentrations exceeded the prenadir and postnadir values by at least two standard deviations of the peak value, and 4) the amplitude exceeded the sensitivity of the assay [30, 41]. If a statistically significant increase in concentration was detected at the end of sampling so that no decrement in concentration could be determined, this increase was considered a pulse. Significant effects of hormone treatment and feeding regimen on LH parameters and body weight were identified using two-way ANOVA for repeated measures and paired Student t-tests (one-tailed). In experiment 1, no differences were found in response to treatment with empty implants when comparisons were made within groups, indicating that no carryover effect occurred because of estrogen treatment. Therefore, for clarity, the data are presented as comparisons with respective pretreatment periods. Analysis of the effects of sulpiride (experiment 1) was conducted via two-way ANOVA for repeated measures for the 2-h periods prior to and after administration of sulpiride. Results are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: E Microimplants Into the POA or VMH During Feed Restriction

Guide tube placements are shown in Figure 1. Histological analysis indicated that five of the six POA-F wethers and seven of the seven POA-R wethers had correct placements of the guide tubes. Histological preparations from the remaining animal were not available. Chronic guide tubes were correctly positioned in five of the six VMH-F wethers and five of the seven VMH-R wethers. One VMH-R wether had an incorrect placement (too anterior plus one guide tube in the third ventricle). Histological preparations from the two remaining animals were not available. Data from animals with incorrect placements or for which histological preparations were not available were not included in the analyses.



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  		FIG. 1. Bilateral placements for chronic guide tubes directed to the mPOA (A–B, circles) and the VMH (C–D, squares) in experiment 1. Placements for F animals are indicated by closed symbols; for R animals, open symbols. Black crosses indicate an incorrect placement

Mean body weight was lower in POA-R than in POA-F by Week 5 of restriction (P = 0.041; Fig. 2A) and was lower in VMH-R than in VMH-F by Week 3 (P = 0.009; Fig. 2B). Differences in mean body weight between the F and R groups for each placement remained significant (P < 0.05) for the remainder of the experiment. Compared with initial body weights, the mean body weight of POA-F wethers was not significantly different from initial levels at any point (P > 0.05), but the mean body weight in POA-R wethers was significantly lower than initial body weight during Week 6 (P = 0.01) and Week 8 (P = 0.03). In VMH-F wethers, body weight was significantly greater than initial body weight during Week 5 (P = 0.02), Week 6 (P = 0.05), and Week 8 (P = 0.05), but in VMH-R wethers, body weight was significantly lower than initial body weight only at Week 8 of feed restriction (P = 0.03).



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  		FIG. 2. Mean ± SEM body weight (kg) for R (open symbols) versus F (closed symbols) wethers with chronic guide tubes directed to the mPOA (A, circles) or the VMH (B, squares) in experiment 1. Treatment with E or C began after blood sample collection on Day 42 and continued through Day 45 and began after blood sample collection on Days 48 and continued through Day 51, as indicated by the boxes. Only animals with correct placements of the chronic guide tubes to the respective area as indicated by histological analysis were included (*P < 0.05 versus F)

As indicated in Figure 3A, localized administration of E to the POA of R wethers was associated with a decrease in the mean number of LH pulses per 4 h as compared to the number of LH pulses per 4 h in F wethers during treatment with E-containing microimplants (P = 0.049). However, an overall difference between POA-F and POA-R groups (P = 0.031) without a group by steroid-treatment interaction (P = 0.718) indicated that LH pulsatility also was reduced by feed restriction per se. There was also no significant difference in the decrement in LH pulse frequency induced by E in F ({Delta}: 0.4 ± 0.9 pulses per 4 h) versus R ({Delta}: 0.8 ± 0.6 pulses per 4 hr) wethers. Mean concentrations of LH and amplitude of LH pulses were not different between POA-F and POA-R (P > 0.05) for any treatments (see Table 1).



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  		FIG. 3. Mean ± SEM LH pulses per 4 h for F and R wethers with chronic guide tubes directed to the mPOA (A) or the VMH (B) in experiment 1 during cholesterol (C) and estrogen (E) treatment with respective pretreatment periods (*P < 0.05 versus F; +P < 0.05 versus C; °P < 0.05 versus respective pretreatment). Representative individual pulsatile LH profiles in four wethers from experiment 1 are presented in C–F. Pulse peaks are denoted by filled circles


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  		TABLE 1. Mean concentration (ng/ml) and pulse amplitude (ng/ml) of LH in fed and food-restricted wethers in Experiment 1 during cholesterol (C) and estrogen (E) treatment with respective pretreatment periods.a

Localized administration of E to the VMH caused a decrease in the number of LH pulses per 4 h in R, but not in F, wethers (P = 0.020; Fig. 3B). A significant decrease in LH pulse frequency was also observed in R wethers during E treatment as compared to the respective untreated sampling period (P = 0.039), with the decrements ({Delta}: 0.6 ± 0.5 pulses per 4 hr for F wethers versus {Delta}: 2.2 ± 0.4 pulses per 4 hr for R wethers) also being significantly different. No significant difference in the number of LH pulses per 4 h was found between VMH-F and VMH-R in response to C treatment or during any of the control blood collections (P > 0.05); although frequency tended to be lower (P = 0.056) in R wethers prior to C, there were no main effects of feed restriction (P = 0.138). This trend suggests that steroid-independent effects in VMH-R wethers (similar to those in the POA-R animals) were beginning to occur. It is important to note that there was a significant (P = 0.05) interaction of E and level of nutrition indicating that E was more effective in VMH-R than in VMH-F wethers. Both mean LH and amplitude of LH pulses were significantly reduced in VMH-R wethers as compared to VMH-F wethers only during treatment with estrogen-containing implants (P = 0.039 and P = 0.04, respectively, see Table 1). No other differences in mean LH or amplitude of LH pulses were significant (P > 0.05).

Sulpiride treatment did not stimulate LH secretion in R, E-implanted wethers (Fig. 4). LH pulse frequency in response to sulpiride administration was similar during E treatment for F and R wethers (P > 0.05), indicating that the suppression of mean LH in response to localized E administration in R animals was not increased by the administration of the dopamine receptor antagonist. Sulpiride treatment also did not reverse the steroid-independent suppression of LH in the POA-R wethers, because mean LH concentration and pulse amplitude were not altered by sulpiride administration (data not shown; P > 0.05).



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  		FIG. 4. Mean ± SEM LH pulses per 2 h for F and R wethers in experiment 1 before and after treatment with Sulp during C and E treatment (A, POA; B, VMH). Representative individual pulsatile LH profiles in four wethers prior to and in response to sulpiride treatment (administered at time indicated by the arrow) from experiment 1 are presented in C–F. Pulse peaks are denoted by filled circles

Experiment 2: DHT Microimplants Into the VMH During Feed Restriction

Histological analysis indicated that six of the seven R-DHT, four of the six R-C, six of the six F-DHT, and six of the six F-C wethers had correct placements of the guide tubes in the rostral VMH (see Fig. 5). One R-C wether had an incorrect placement (too anterior). Histological preparations from the two remaining animals were not available. Data from animals with incorrect placements or for which histological preparations were not available were not included in the analyses.



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  		FIG. 5. Bilateral placements for chronic guide tubes directed to the VMH (B–C) in experiment 2 (R-DHT, open circles; R-C, open squares; F-DHT, closed circles; F-C, closed squares). Black crosses in A indicate an incorrect placement into the mPOA

Mean body weight was lower in R-DHT than in F-DHT wethers by Week 6 of restriction (P = 0.0.038; Fig. 6) and in R-C than in F-C by Week 7 (P = 0.019; Fig. 6). Differences in mean body weight between the F and R groups for each steroid-treatment remained significant (P < 0.05) for the remainder of the experiment. Compared to initial body weight, F-C wethers gained 19.6 ± 6.4% (a significant change in body weight; P = 0.04), F-DHT wethers gained 15.7 ± 3.7% (P > 0.05), R-DHT wethers lost 12.0 ± 3.2% (P > 0.05), and R-C wethers lost 13.3 ± 2.2% (P > 0.05).



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  		FIG. 6. Mean ± SEM body weight (kg) in food-restricted and F wethers in experiment 2 (R-DHT, open circles; R-C, open squares; F-DHT, closed circles; F-C, closed squares; *P < 0.05 R-DHT versus F-DHT; +P < 0.05 R-C versus F-C). Treatment with DHT or C began after blood sample collection at Week 4 and continued through blood sample collection at Week 8 as indicated by the box

Treatment of R wethers with DHT implants did not cause a significant reduction in LH pulse frequency (P > 0.05; Fig. 7), pulse amplitude, or mean LH (data not shown). Thus, no consistent decrease in LH secretion was observed in response to feed restriction or to localized DHT administration to the VMH.



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  		FIG. 7. Mean ± SEM LH pulses per 4 h for F and R wethers in experiment 2 (R-DHT, open circles; R-C, open squares; F-DHT, closed circles; F-C, closed squares). Treatment with DHT or C began after blood sample collection at Week 4 and continued through blood sample collection at Week 8. Representative individual pulsatile LH profiles in four wethers from experiment 2 are presented in B–E. Pulse peaks are denoted by filled circles


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the first experiment indicate that the VMH is a site in which the responsiveness to E-negative feedback is enhanced during feed restriction in young castrate male sheep. LH pulse frequency was clearly decreased in response to localized administration of E to the VMH in R, but not in F, wethers. This enhanced responsiveness was specific to E because no change in LH pulse frequency occurred in either VMH-R or VMH-F wethers in response to localized administration of C. The mechanism by which this enhancement of responsiveness to E-negative feedback occurs is currently unknown. One potential mechanism is that the number of cells expressing ESR1 may be altered in response to changes in energy balance. In the ewe, a significant decrease in the number of cells expressing ESR1 in the VMH was found in response to 50 days of restricted feed intake [25]. Similar results have been found in other species [26–28]. It is possible that E could diffuse from the VMH to the arcuate nucleus (ARC), where small but consistent increases in ESR1 have been reported to occur in feed-restricted ewes [25] and hamsters [26]. We are currently investigating the possibility that changes in the expression of ESR1 also occur within the VMH and ARC of the feed-restricted wether.

We observed a significant decrease in LH pulse frequency in POA-R wethers compared to POA-F wethers in response to localized administration of E. However, because an overall decrease in the LH pulse frequency was detected in POA-R as compared to POA-F wethers during control treatment periods, it is difficult to separate steroid-dependent effects from steroid-independent suppression of the reproductive axis. It is unclear why a steroid-independent suppression of LH occurred in the POA-R wethers because the amount and percentage of body weight lost during the experiment were similar to those for the other R wethers in this study. Clearly, steroid-independent suppression of the reproductive axis occurs in response to long-term alterations in feed availability in the sheep. Both estradiol-treated and untreated ovariectomized, growth-restricted lambs show low levels of serum LH [10], and inhibition of GnRH pulsatility by undernutrition is evident in the absence of ovarian steroids [44]. This has made the assessment of the steroid-dependent effects of nutrient restriction more difficult [15]. Whether steroid-dependent and steroid-independent suppression occur via similar, separate, or overlapping mechanisms remains to be determined.

In contrast to the results from the first experiment, localized administration of a nonaromatizable metabolite of testosterone, DHT, to the VMH of R wethers was not associated with any changes in LH secretion. These results were somewhat surprising because an enhanced sensitivity to testosterone is exhibited in rats in response to short-term feed deprivation [45]. In addition, an enhancement of sensitivity to the blockade of AR has been shown to occur during feed restriction in the male rat, and undernutrition enhances androgen-mediated feedback suppression of GnRH that leads to an inhibition of LH secretion [16]. However, testosterone-negative feedback on LH and FSH is diminished in the long-term-castrated ram [46], and the time interval after castration affects responsiveness to short-term starvation in adult male rats [47]. Long-term castration has been shown to result in a decrease in the expression of pituitary AR, but not estrogen receptors, in rams [48]. The effect of long-term castration on the expression of AR within the hypothalamus of male sheep is not known. We attempted to circumvent the issue by prolonging the period of exposure to DHT, but cannot rule out the possibility that the failure of intrahypothalamic DHT to alter LH secretion was because of decreased expression of AR in long-term castrates. Nonetheless, the lack of response to DHT observed in the present study is similar to that seen in adult long-term castrated rams administered DHT to the arcuate-ventromedial hypothalamus during an inhibitory photoperiod [31]. Those results lead to the suggestion that control of LH (and GnRH) secretion by testosterone may require aromatization [31]. However, blockade of the reduction of testosterone to DHT reduces the ability of testosterone to suppress LH secretion in long-term-castrated sheep, indicating that the conversion of testosterone to DHT is a physiologically important step in the negative feedback control of LH release by testosterone [36]. In that regard, it is important to note that our study examined the responsiveness of only one hypothalamic site, the VMH, to localized administration of DHT. In the intact ram, AR-expressing cells are also found within the medial POA and the infundibular and premammillary nuclei, as well as the ventromedial nucleus [19, 49]. Thus, it is possible that enhancement of responsiveness to DHT may occur within an area other than the VMH or may require action in multiple areas. Alternatively, the absence of effect of DHT implants may be due to a very limited diffusion of DHT; the diffusion of DHT in brain tissue is not known, although it is likely to be similar to that of E [32] and testosterone [31], which is about 1 mm from similar microimplants.

The chemical identity of the estrogen-responsive neurons that change responsiveness to steroid negative feedback in response to altered energy balance has yet to be identified. The decrease in GnRH (and LH) secretion associated with undernutrition occurs primarily through central inhibition of GnRH neurons because treatment of growth-restricted hypogonadal ewes with the nonspecific neural inhibitor sodium pentobarbital increases pulsatile LH secretion [37]. The anestrous ewe also exhibits an increased response to E-negative feedback, which is caused by activation of a system involving dopamine neurons that inhibit GnRH pulsatility [30]. In order to determine whether undernutrition works via similar neural systems, we also tested whether the suppressive effect of E in R wethers could be overcome by an injection of the dopamine-D2 receptor antagonist sulpiride. In the current experiment, no increase in LH pulse frequency was observed in response to sulpiride administration, indicating that the system involved in the enhanced responsiveness to E-negative feedback during negative energy balance does not involve the D2 receptor. Double-labeling immunocytochemistry has shown that substantial numbers of ESR1-immunoreactive cells within the POA of ewes contain the inhibitory neurotransmitter gamma amino-butyric acid, whereas the ESR1-immunoreactive cells in the ventromedial nucleus of the ewe and the AR-immunoreactive cells in the ventromedial nucleus of the ram contain the neuropeptide somatostatin [50], which has been shown to be inhibitory to LH in the ewe [51]. Because E also may have diffused to the ARC in experiment 1, it is possible that metabolically responsive neurons such as neuropeptide Y neurons in this area could be involved in this effect. The role of these neurochemicals, along with others, in the suppression of the reproductive axis in conditions of negative energy balance has yet to be fully elucidated.

The interpretation of data from these microimplants is based on the assumption that the amount of E (and DHT) released from the microimplants produces local effects and does not reach other hypothalamic areas via diffusion. This assumption is supported by two lines of evidence: 1) that independent estimates of the diffusion of radioactive estradiol [32] and testosterone [31] from this type of implant concur that the distance is probably limited to 1 mm, and 2) that the ability of microimplants to inhibit LH are site specific [31–32, 52–53] even though the sites are within a few mm of each other. Thus, we are confident that the effects observed are caused by actions on steroid-responsive cells within or near the sites of implantation.

In conclusion, E, but not DHT, acts locally within the ventromedial hypothalamus to cause a suppression of LH (and presumably GnRH) during feed restriction but not under conditions of adequate feed intake in the wether. E may act locally in the preoptic area to cause a suppression of LH under conditions of feed restriction, but definite conclusions cannot be drawn because differences in LH pulse frequency were observed that might represent steroid-independent effects of feed restriction. Further studies are required to determine whether enhanced responsiveness to steroid negative feedback also occurs via AR in other locations and to identify the neurochemical nature of the inhibitory signal(s) that respond to estradiol and that suppress GnRH secretion during negative energy balance.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. A.F. Parlow and the National Hormone and Peptide Program for LH RIA reagents. Special thanks to Paul Harton II and Robert McTaggart for assistance in histological preparations, Dr. Robert Pitts, Karie Hardy, Heather Clemmer, and Sarah Beamer for animal care at the WVU Food Animal Research Facility, and Dr. Steven L. Hardy for input and feedback during the experiment.


   FOOTNOTES
 
1 Supported by National Research Initiative Competitive Grant 2002-35203-11259 to C.J.M. from the USDA Cooperative State Research, Education and Extension Service, NIH HD 17864 to R.L.G., and USDA 2001-35203-10835 to S.M.H. Abstracts containing some of these data were presented at the 33rd annual meeting of the Society for Neuroscience, New Orleans, Louisiana, 8–12 November 2003, and at the 37th annual meeting of the Society for the Study of Reproduction, Vancouver, British Columbia, Canada, 1–4 August 2004. Back

2 Correspondence: Christina J. McManus, Department of Physiology and Pharmacology, West Virginia University, P.O. Box 9229, Morgantown, WV 26506-9229. FAX: 304 293 3850; cmcmanus{at}hsc.wvu.edu Back

Received: 4 April 2005.

First decision: 27 April 2005.

Accepted: 20 June 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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