ARTICLES
A -Aminobutyric AcidB Agonist Reverses the Negative Feedback Effect of Testosterone on Gonadotropin-Releasing Hormone and Luteinizing Hormone Secretion in the Male Sheep1
Gary L. Jackson, Susanne G. Wood and David E. Kuehl
Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802
Address all correspondence and requests for reprints to: Dr. Gary L. Jackson, Department of Veterinary Biosciences, 2001 South Lincoln Avenue, Urbana, Illinois 61802. E-mail:
[email protected].
Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Infusion of baclofen, a GABAB agonist, into the medial basal hypothalamus (MBH) of castrated rams rapidly increases LH pulse amplitude without altering pulse frequency. The objectives of this study were to determine whether baclofen infusion increased LH in testosterone (T)-treated and intact rams, the increased LH was due to increased GnRH release, and FSH secretion also was increased. In the first experiment we tested the main effects and interaction of baclofen and T on FSH and LH pulse patterns in castrated rams (n = 7). In the second experiment we determined whether baclofen affected GnRH and LH pulses in intact males. Microdialysis guide cannulae were implanted bilaterally into the MBH. After recovery of the animal from surgery, the MBH was perfused using concentric microdialysis probes (2-mm tip) with artificial cerebrospinal fluid (aCSF) for a 3-h control period followed by either aCSF or 1 mM baclofen for 4 h. Blood samples were taken at 10-min intervals. T suppressed mean LH concentrations (10.4 ± 1.3 vs. 3.3 ± 1.3 ng/ml) such that LH pulses were undetectable in some T-treated animals during the control period. The change (control period vs. drug infusion period) in mean LH was greater in response to baclofen than in response to aCSF and was not altered by T. The baclofen x T interaction was nonsignificant. Mean FSH was decreased by T, but was not altered by baclofen. In the second experiment hypophyseal portal blood was collected coincident with microdialysis. Infusion of baclofen into the MBH of intact males (n = 7) resulted within 1 h in the onset of frequent and robust GnRH pulses (0.10/h before baclofen vs. 1.57/h after baclofen) that were followed either immediately or gradually by coincident LH pulses. One interpretation is that baclofen acts downstream of the site of action of T. GABAB receptors may regulate pulse amplitude in both the presence and absence of T and regulate pulse frequency by modulating the inhibitory effect of T.
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TESTOSTERONE (T) ACTS primarily within the hypothalamus via its metabolites, estradiol or dihydrotestosterone, to reduce the frequency of GnRH and LH pulses (1, 2, 3, 4). Although GnRH neurons may contain steroid receptors (5), most studies have found few or none (3, 6, 7). This has led to the idea that the negative feedback actions of T are mediated primarily via interneurons that secrete neurotransmitters or neuromodulators inhibitory to GnRH secretion. Although the identities of these neurochemicals remain conjectural, there is sufficient evidence to implicate -aminobutyric acid (GABA) as a candidate.
GABA is widely distributed throughout the hypothalamus. GABAergic neurons concentrate steroids (7, 8, 9, 10) and synapse with GnRH-secreting cell bodies in the preoptic area (11). Within the medial basal hypothalamus (MBH) there are high affinity uptake sites for GABA, a dense plexus of GABAergic neurons, and messenger RNAs coding for multiple GABA receptor subunits (12, 13, 14, 15, 16). Application of drugs that alter GABA metabolism and of GABA antagonists and agonists, both in vivo and in vitro, disrupt GnRH or LH secretory patterns (17, 18, 19, 20). Furthermore, that observation that T increases GABA turnover coincident with suppressed LH release (21, 22) is consistent with the hypothesis that GABA at least in part mediates the action of T.
However, the specific role of GABA in regulating GnRH remains unclear. Within the hypothalamus, GABA probably acts via one or both of the two major receptor types, GABAA and GABAB. The postsynaptic GABAA receptor, activation of which usually results in decreased neuronal responsiveness, probably mediates the inhibitory action of GABA. The GABAB receptors, originally thought to be strictly or largely presynaptic autoreceptors, have been less intensely investigated (23). However, observations by Scott et al. (24) that localized microinjection of baclofen, a GABAB agonist, into the medial preoptic area of estrogen-treated ewes during the anestrous period transiently elevated LH concentrations in some individuals suggested a possible role for this receptor type. This observation was extended by Ferreira et al. (17), who found that application of baclofen into the MBH of castrated rams by microdialysis resulted in prolonged and robust increases in LH pulse amplitude, with no observable effect on pulse frequency. This unexpected observation raised several questions. 1) Would baclofen increase LH in T-treated castrated and intact rams? 2) Was the increased LH secretion due to increased secretion of GnRH? 3) Was the increased secretion of LH paralleled by increased secretion of FSH? The experiments reported here were conducted to answer these questions.
Materials and Methods
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Adult castrated or gonad-intact male sheep, predominantly of the Suffolk breed, were maintained outdoors at the Veterinary Research Farm (Urbana, IL; latitude 40°N) until a few days before undergoing surgery for bilateral placement of guide cannulae into the brain. Thereafter they were housed indoors, exposed to artificial lighting from 0400–2000 h (16 h of light, 8 h of darkness) supplemented by natural lighting, fed a balanced pelleted diet produced by the University of Illinois feedmill, and given free access to water. Exp 1 was conducted during the late winter (January through March) of 1999, and Exp 2 was performed during the period from November 1999 through January 2000. The experimental protocol was approved by the institutional committee on laboratory animal care and was conducted in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.
Surgery
Guide cannula placement. Surgery for bilateral placement of the guide cannulae was carried out under aseptic conditions, using modifications of procedures previously described (17). Anesthesia was induced with sodium thiopental and was maintained with 3–4% halothane. The animal’s head was secured firmly in a stereotaxic instrument (Kopf Instruments, Tujunga, CA). After an incision, a circular piece of skull (2.5 cm in diameter) was removed, and the sagittal sinus was doubly ligated. The sinus was then retracted, and 0.15 cc of radiopaque dye (Renografin-76, E.R. Squibb and Sons, Princeton, NJ) was injected into the third ventricle. Lateral radiographs that outline the ventricle were used to aid in the placement of guide cannulae. The final placements were made using an x, y, z manipulator and additional radiographs. Twenty-gauge stainless steel guide cannulae 62 mm long with stylets extending an additional 1 mm (Exp 1) or 3 mm (Exp 2) were then placed bilaterally into the MBH. For Exp 1, the tips of the stylets were placed 2.8–3.0 mm above the floor of the ventricle, 1.0 mm anterior to the most anterior portion of the posterior wall of the infundibular recess, and 2.25 mm lateral to midline. Given that the microdialysis probes extended 3 mm beyond the guide tubes, the dorsoventral target of the probe tip was 0.8–1.0 mm above the floor of the ventricle. For Exp 2, the tips of the stylets were placed at this location. The cannulae and a protective cap were anchored to the skull with dental acrylic and screws, and the incision was then closed.
In all studies, butorphanol (Torbugesic, Fort Dodge Laboratories, Fort Dodge, IA) was provided as analgesic for 2 days postsurgery, and Liquamycin, LA-200 (0.1 cc/kg; Pfizer, Inc., New York, NY) antibiotic was provided for 10 days.
Portal cannula placement. For Exp 2, approximately 3 weeks after guide cannula placement the animals were reanesthetized, and a portal cannula device was installed and maintained as previously described (25, 26, 27). The only change was that the cannula device was flushed daily with a saline-heparin solution (125 U/ml) instead of saline solution.
The animals were then placed in adjacent individual pens. Approximately 7 days later the dual drug perfusion-portal blood sampling procedure was conducted.
Experimental design and protocol
Exp 1: effects of T and baclofen. The objective of this experiment was to determine whether T affected the ability of baclofen to increase the level of circulating LH. The experiment was a factorial with two factors: steroid (T vs. no T) and drug [baclofen vs. artificial cerebrospinal fluid (aCSF)]. A cross-over design was used so that within steroid groups each animal was treated with both aCSF-aCSF and aCSF-baclofen in random order. There were seven animals each within the T and no T groups. All animals had been castrated at least 60 days before receiving bilateral guide cannulas. T treatment was achieved by placing SILASTIC implants (Dow Corning, Inc., Midland, MI; 3.32 mm i.d. x 4.61 mm o.d. x 10 cm) containing packed crystalline T sc over the dorsal rib area at a dose of 1.70–2.0 cm/kg BW (25). The implants were left in place for the duration of the study.
Starting approximately 4 weeks after implantation of the guide cannulas and 1 day before drug dialysis, groups of three animals had a catheter inserted into the jugular vein for the purpose of blood collection. Each animal was placed into an individual metabolism pen and allowed 12–16 h to acclimate. On the day of the experiment, the stylets were removed from the guide cannulas and replaced by microdialysis probes. The probes were anchored in place by paper tape. The probes were connected to an infusion pump (Sage Instruments, Boston, MA) by means of microline tubing (Cole Parmer, Chicago, IL). Each animal then received a 4-h period of control dialysis of aCSF at a flow rate of 2 µl/min. During the subsequent 4 h, either dialysis of aCSF was continued or the solution was switched to one containing baclofen (1 mM; Sigma, St. Louis, MO). The probes were not changed. Due to dead space that was filled with aCSF within the probe assembly, it required approximately 30 min for the baclofen to move from the inlet to the probe tip and into the surrounding tissue. During the entire 8-h dialysis period, jugular blood samples were collected at 10-min intervals. Blood samples were collected in glass tubes containing 100 µl heparin (125 U/ml) and were centrifuged within 1 h of collection. Plasma was stored at -20 C until assayed for LH. After each dialysis session, the microdialysis probes were replaced by sterilized stylets, and the animals were returned to group pens after receiving Liquamycin LA-200. Subsequent dialysis sessions were conducted at 1-week intervals.
Exp 2: effects on GnRH secretion. The objectives of this experiment were to determine whether 1) baclofen would elevate LH in intact males; and 2) the baclofen-induced elevation in LH was accompanied by coincident elevation of GnRH secretion.
Adult males, approximately 1–2 yr old were subjected to placement of bilateral guide tubes as described for Exp 1. Three weeks later each animal was fitted with a portal-cannulation device. Then 7–10 days later they were subjected to simultaneous bilateral microdialysis and collection of portal blood using procedures described previously (25, 26, 27).
Approximately 12–14 h before this procedure three catheter assemblies were inserted into the jugular veins. Two were used to administer heparin and one to collect blood. Pairs of animals were placed into adjacent individual pens and allowed to acclimate. On the morning of the procedure, the stylets were removed, dialysis probes were inserted, and the dialysis of aCSF was started. Approximately 10–15 min later heparinization of the animal was started, and portal sampling was conducted as described previously (25, 26, 27). To maintain stable blood flow heparin was infused at a rate of 200 IU/kg·h and was supplemented with periodic injection as necessary.
Microdialysis of aCSF was continued until baseline portal and jugular blood samples had been collected for 3 h. Then the microdialysate was changed to a 1-mM baclofen solution, and microdialysis was continued for 4 h. Given that in previous experiments we repeatedly had found no effect of aCSF during the second half of perfusion, that treatment was not included in this experiment. During the entire procedure jugular blood samples were collected continuously into 9-min fractions, whereas portal samples were collected into 4.5 or 3 min (animal 9915) fractions. The animals were killed after the dialysis-portal collection procedure.
Dialysis probe and dialysis buffer
The microdialysis probe had a nitrocellulose hollow fiber dialysis membrane with a molecular mass cut-off of 6 kDa (Spectra/Por, Spectrum, Gardena, CA). The probe was of a concentric design adapted for use in sheep with modifications previously described (17). It was constructed in our laboratory from 24-gauge stainless steel tubing through which fused silica tubing passed (Polymicro Technologies, Phoenix, AZ) and exited from the microline inlet. The silica tubing extended 1.75 mm from the stainless guide around which the dialysis membrane (length, 2.5 mm) was fitted. The distal end of the dialysis membrane was sealed with epoxy (Devcon Corp., Danvers, MA). The final length of dialysis membrane in direct contact with brain tissue was 2 mm. It should be noted that the maximal dorsoventral dimension of the ventromedial nucleus is approximately 3.5 mm in sheep (28). The aCSF consisted of 127.6 mM NaCl, 2.5 mM KCl, 0.69 mM CaCl2, 1 mM MgSO4, 2.3 mM NaH2PO4, and 9.7 mM Na2HPO4 (pH 7.4).
Hormone assay
Plasma samples were assayed in duplicate for LH using a previously described RIA validated for use in our laboratory (25). The sensitivity was 0.5 ng/ml NIH LH S2O at 90% binding. The intraassay coefficient of variation was 3.4%, and the interassay coefficient of variation averaged 10.0% for three pooled sera. Values for mean LH, LH interpulse interval, and LH pulse amplitude were determined using the Pulsar algorithm (29). The G values were set as G(1) = 2.2 and G(2, 3, 4, 5) = 2.0 for LH and G(1) = 3.4, G(2) = 3.3, and G(3, 4) = 3.2 for GnRH. FSH was assayed using the assay kit provided by the NIDDK as previously validated in our laboratory (30). FSH RP-2 was used as the standard. The assay sensitivity was 0.5 ng/ml. Intra- and interassay coefficients of variation for four pooled sera averaged 3.1% and 12.4%.
T also was assayed using a previously described RIA validated for use in our laboratory (25). The sensitivity was approximately 0.4 ng/ml, and the intra- and interassay coefficients of variation were 6.3% and 7.6%, respectively.
GnRH was assayed using antibody BDS-037 in a previously validated procedure (31). The sensitivity was 2.0 pg/ml; intra- and interassay coefficients of variation were 8.7% and 15.4%, respectively. With this assay there was no detectable background for GnRH in peripheral blood.
Histology
At the end of the experiment, the animals were killed. The brains were removed after perfusion with saline via carotid artery, followed by 4% formalin fixative, after which the hypothalami were isolated and immersed in fixative. The sections collected were histologically processed and stained with Luxol fast blue to localize probe placement. Evaluation of probe placement was made with the aid of diagrams from Lehman et al. (32).
Analysis of data
For Exp 1, data across treatments were analyzed by two-way ANOVA with repeated measures for one factor (drug). As pulses of LH were not detectable in some T-treated animals, the across-treatment analysis was confined to changes in mean LH and FSH concentrations. This analysis was based on the , i.e. change in values during the control or first half of each infusion vs. those during the experimental or last 3 h of the second half of infusion. Thus, (drug period minus control period) in response to aCSF-aCSF was compared with that in response to aCSF-baclofen across both T and no T groups. Analysis of the effects of baclofen on pulse parameters within the castrated (no T) group was performed using one-way ANOVA with repeated measures. For Exp 2, analysis of interpulse interval could not be performed due to absence of pulses during aCSF infusion. Thus, analysis of the effect of baclofen on GnRH pulse frequency was performed using the nonparametric Wilcoxon sign-rank test. Analysis of effects of baclofen on mean GnRH and LH was performed using one-way ANOVA for repeated measures after transformation of the data following tests for homogeneity of variance.
Results
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
Histology
All probes were confined to the MBH (Fig. 1) either in or near the ventromedial nuclei.
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Figure 1. Diagram showing aggregate location of bilateral dialysis probe placement in 14 male sheep in which the target was the MBH. The anterior-most section is to the left. , Location in castrated animals; •, location in T-treated animals. arc, Arcuate nucleus; cp, cerebral peduncle; dm, dorsomedial nucleus; III, third ventricle; fx, fornix; me, median eminence; mt, mammillothalamic tract; ot, optic tract; vm, ventromedial nucleus.
Changes in LH and FSH
The T implants produced a mean circulating T concentration of 3.7 ± 0.16 vs. 0.6 ± 0.03 ng/ml in controls (P < 0.01). T reduced the mean basal (prebaclofen) LH concentration (10.4 ± 1.3 vs. 3.3 ± 1.3 ng/ml; P < 0.01) to the extent that it was not feasible to analyze effects of baclofen on pulse parameters in castrated vs. T-treated animals. Thus, comparison of the effects of baclofen in these two groups was limited to analysis of changes in mean LH concentrations. Representative responses to baclofen and aCSF by T-treated animals are shown in Fig. 2. The main effect of baclofen compared with that of aCSF was highly significant (P < 0.01). The interaction between steroid and baclofen was nonsignificant, i.e. baclofen increased mean LH concentrations in both castrated and castrated T-treated animals (Fig. 3). Notably, in some cases (e.g. Fig. 2, sheep 9904 and 9906), the rise of LH in T-treated animals occurred as an apparent increase in basal concentrations unaccompanied by the appearance of distinct LH pulses.
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Figure 2. Representative secretory profiles of LH in four castrated T-treated male sheep subjected to two separate bilateral infusions by microdialysis of aCSF only (left panels) or aCSF followed by 1 mM baclofen into the MBH (right panel). Data on effects of only aCSF in sheep 9908 were not obtained due to a damaged probe.
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Figure 3. Comparison of effects of infusion of aCSF-aCSF vs. aCSF-baclofen on changes in mean LH concentrations in castrated (n = 7; ) and castrated T-treated (n = 7; ) male sheep. Each bar represents the mean ± SEM. The main effect of baclofen was significantly different (P < 0.01) from that of aCSF, and there was no interaction between steroid and drug.
Within the castrated group there were significant effects of baclofen, compared with aCSF, on changes in both mean pulse amplitude (3.19 ± 1.3 vs. -1.07 ± 0.45 ng/ml; P < 0.01) and peak pulse amplitude (4.61 ± 2.01 vs. -1.72 ± 0.53 ng/ml; P < 0.01). Baclofen did not affect interpulse interval (0.6 ± 0.03 vs. 0.6 ± 0.06 h).
Although T reduced mean FSH concentrations (10.31 ± 1.39 vs. 3.99 ± 1.27 ng/ml; P < 0.01), baclofen had no significant effect on FSH in either castrated or T-treated animals (data not shown).
GnRH in intact rams
This experiment was conducted to determine whether the baclofen-induced increases in LH concentrations were accompanied by increased GnRH secretion. Representative results obtained from four of seven rams are shown in Fig. 4. In all animals no or few GnRH and LH pulses were detected before baclofen infusion. However, baclofen treatment induced robust GnRH pulses within a few minutes in six of the seven animals. Overall, mean GnRH pulse frequency changed from 0.10 ± 0.06 to 1.57 ± 0.48/h (P < 0.03) during baclofen infusion. LH pulses first appeared either at the same time as GnRH pulses (Fig. 4, sheep 9916) or only after a variable delay (Fig. 4, sheep 9915). Subsequently, GnRH and LH pulses were wholly coincident, although in cases where GnRH pulses were very frequent, the LH pulses became obscured. Baclofen treatment increased the mean GnRH concentration from 3.41 ± 0.63 to 8.50 ± 2.92 pg/ml (P < 0.05) and the mean LH concentration from 0.79 ± 0.09 to 5.00 ± 2.03 ng/ml (P < 0.01).
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Figure 4. Representative changes in patterns of LH in jugular blood and of GnRH in portal blood of four rams subjected to bilateral infusion by microdialysis of aCSF followed by infusion of 1 mM baclofen into the MBH.
Discussion
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
These results show that 1) baclofen greatly increased LH concentrations in castrated, T-treated castrated, and intact male sheep; 2) this effect was mediated by increased release of GnRH; and 3) the rapid increases in GnRH and LH were not accompanied by changes in FSH concentrations.
The noted effect of baclofen in castrated animals confirms previous observations (17) of increased pulse amplitude without an effect on pulse frequency. A similar effect was observed in some (sheep 9914), but not all, T-treated animals. Another response pattern in T-treated animals was a clear increase in mean circulating LH, unaccompanied by LH pulses. An explanation for this nonpulsatile release was not readily apparent; thus, it was important to determine the effect of baclofen on GnRH release. This was accomplished in Exp 2, albeit in intact males. Although there are limitations in making inferences from data obtained in the intact male to the other two models, the results support the conclusion that the effects of baclofen on LH are mediated entirely or wholly through increased secretion of GnRH.
These results of Exp 2 are useful for several reasons. First, they show that the effect of baclofen on GnRH secretion is rapid and robust. This observation suggests the availability of a readily releasable pool of GnRH and that baclofen was acting relatively close to the infusion site. Second, they help explain the nonpulsatile release of LH in response to baclofen shown by some T-treated animals in Exp 1. In some animals (e.g. sheep 9915), the anterior pituitary of either T-treated or intact males was not readily responsive to the initial pulse(s) of GnRH, but became so after repeated priming pulses. Whether this initial unresponsiveness was due to secondary effects of prolonged suppression of GnRH by T, direct effects of T on the pituitary, or both is not clear. However, the results do demonstrate that a full response can be achieved relatively rapidly and again illustrate the well known priming action of GnRH.
A third aspect of baclofen’s action in the intact male is that in some animals there was a clear increase in both the frequency and the amplitude of measurable GnRH pulses. Although the increase in pulse frequency may simply have reflected an increase in amplitude, it seems unlikely for at least two reasons. The first is that T decreases GnRH pulse frequency, with little effect on amplitude (25). The second is that GnRH pulse frequency in some intact males during baclofen treatment approached that in castrated males (1.6 vs. 1.7 pulses/h). Accordingly, the data can be interpreted as suggesting that GABAB receptors also regulate the pulse generator. In castrated animals that effect may not be apparent because the pulses already are occurring at a near-maximal rate. In contrast, in the intact or T-treated animal this effect of baclofen is detectable because T suppresses the pulse rate, and baclofen reverses this action of T. These observations also indicate that baclofen probably acts downstream from the site of action of T.
The specific sites and mechanisms by which baclofen exerts these effects is unknown. A priori there are at least three possible general modes of action: direct stimulation of the GnRH neuron, direct disinhibition of stimulatory interneurons, and inhibition of inhibitory interneurons. The first two seem relatively unlikely. Although baclofen alters the firing rate of GT-1 cells in vitro, the effect is inhibitory (33). There are several potential stimulatory systems; however, the net effect of baclofen is to reduce neuronal activity (23) by either direct inhibition or disinhibition of GABA-secreting neurons. Thus, it appears improbable that stimulatory systems are directly activated by baclofen, although there is evidence that activation of GAGAB receptors facilitates activation of adenyl cyclase by neurotransmitters such as norepinephrine (34). That action potentially could increase the effect of a stimulatory neurotransmitter.
On the other hand, the wide distribution of GABAB receptors and the presence of several putative inhibitory neuronal systems that affect GnRH secretion lend credence to the third possibility. The anatomical distribution of GABAB receptors within the brain has not been described in the sheep, but in the rat their wide distribution includes several hypothalamic nuclei (35, 36, 37, 38, 39). GABAB receptors are located on cell bodies, axons, and dendrites (40); thus, they have the potential for both pre- and postsynaptic effects. Although two morphological studies found a relative paucity of GABAB receptor protein on GABA-synthesizing neurons (37, 38), many physiological data indicate that presynaptic GABAB receptors act as autoreceptors to suppress GABA release (41, 42). Of equal or greater interest is the clear presence of heterologous GABAB receptors on non-GABAergic neurons, e.g. glutaminergic or dopaminergic (40, 41). Thus, GABAB receptors may have an important role in regulating the secretion of several neurotransmitters or neuromodulators. Although critical double labeling studies have not been reported, there is support for the concept that activation of GABAB receptors affects the release or action of a variety of neurotransmitter systems, including the glutaminergic and dopaminergic systems (43, 44, 45, 46, 47, 48). For example, baclofen reduced the release of dopamine from striatal slice preparations and other experimental systems (45, 46) and inhibited the effect of heroin on self-administrative behavior (49).
Many data implicate the GABAergic, dopaminergic, and opiatergic systems as exerting an inhibitory influence on GnRH secretion in sheep and other species (17, 18, 19, 50, 51, 52); thus, each is a potential target for the action of baclofen. Inhibition of any of these systems, either directly or indirectly, has the potential to increase GnRH and LH release.
The failure of baclofen treatment, and subsequently increased GnRH secretion, to effect equally rapid increases in FSH and LH concentrations is consistent with previous observations made in the ram, namely, that postcastration LH concentrations rise within a few hours, whereas the rise of FSH starts several days later (53) and that administration of exogenous GnRH increases LH much more rapidly than it does FSH (54). Our results simply underscore, and clearly demonstrate, the relative unresponsiveness of the FSH secretory system to GnRH.
Finally, it is not yet obvious how these observations relate to the normal regulation of GnRH pulse frequency and amplitude. The most parsimonious interpretation would be that the GABAergic system normally constrains the GnRH secretory system by acting at distinct sites to regulate both frequency and amplitude and that localized activation of GABAB receptors is an integral part of a multineuronal control system. However, the available data are neither sufficiently consistent nor complete to provide a clear understanding of these relationships.
Acknowledgments
We thank Mrs. J. Thompson for carrying out the histological procedures, the National Hormone and Pituitary Agency (Baltimore, MD) for the ovine LH and FSH assay kit, Dr. Bruce Schanbacher for the anti-GnRH antibody, and Dr. Jan Roser (University of California, Davis, CA) for the LH antibody.
Footnotes
1 This work was supported by NIH Grant HD-27453 and USDA Grant AG99–35203–7737.
Received April 24, 2000.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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