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Neuroendocrinology |
Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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anterior pituitary, luteinizing hormone, neuroendocrinology, neurotransmitters
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the effect of GABA on LH secretion has been investigated in various hypothalamic areas, the majority of studies have dealt with the preoptic area (POA). A series of studies provide evidence that GABAergic neurons in the POA may mediate the negative feedback effect of testosterone (T) in the male rat [3, 4] and the effect of estrogen (E) on the preovulatory LH surge in the female [5]. Scott and Clarke [6] reported that injection of minute quantities of GABAA agonists and antagonists into the POA of ovariectomized (OVX) ewes during the breeding season suppressed circulating LH, while injection of GABAB analogues had no effect. However, injection of the GABAB agonist baclofen into the POA of E-treated OVX ewes during the nonbreeding season transiently increased LH in some animals [7]. From these data the authors concluded that in the ewe POA activation of GABAB receptors affects LH secretion, although there appears to be a seasonal shift in their importance.
Relatively few studies have been made of the function of GABAB receptors in the medial basal hypothalamus (MBH) of any species, but available data suggest that they also may modulate basal LH secretion. The MBH contains high-affinity uptake sites for GABA, a dense plexus of GABAergic neurons, and messenger RNA coding for multiple GABA receptor subunits [2] (for review, see [8–10]). Tetracycline-dependent release of GABA from grafted astrocytes in the median eminence of the rat disrupts estrous cycles in the female rat [11]. Infusion of the GABAB agonist baclofen into the MBH of castrated or castrated, T-treated male sheep rapidly and robustly increased the amplitude of GnRH and LH pulses with little effect on pulse frequency [12, 13]. Another agonist, SKF 97541, had similar effects in castrated rams, and that effect was blocked by coinfusion of the GABAB antagonist CGP 52432 [14]. Collectively, results of these studies suggest that GABAB receptors within the MBH may have a role in modulating basal LH secretion, at least in the male sheep. However, the mechanisms controlling LH are sexually differentiated in many species including the sheep; that is, males lack the capacity for expressing an LH surge [15], and similar infusion studies have not been reported for the female. The MBH contains the GnRH neuron terminals [16], appears to be a site at which E acts in the ewe to induce the LH surge [17], and appears capable of supporting the LH surge [18]. Thus, additional studies of the role of the GABAergic system in this region are needed in order to gain a better understanding of the control of LH secretion. In view of the fact that our previous studies on MBH GABAB neurons were confined to the male and that previous studies of the female were confined to the POA, it was deemed important to investigate the role of MBH GABAB receptors in the female. The reported experiments had two objectives: to determine 1) if infusion of GABAB agonists into the MBH of female sheep would increase basal LH secretion and 2) if infusions of a potent antagonist would alter expression of E-induced LH surges.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Surgery
Surgery for bilateral placement of the guide cannulae was carried out under aseptic conditions using procedures previously described [12]. 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 ml of a radiopaque dye (Conray 400; Mallinkrodt Inc., St. Louis, MO) 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 were then placed bilaterally into the MBH. The tips of the stylets were placed 2.8–3.0 mm above the floor of the ventricle, 1.3 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 3.0 mm beyond the guide tubes, the dorsoventral target of the probe tip was 0.8–1.0 mm above the floor of the ventricle. The cannulae and a protective cap were anchored to the skull with dental acrylic and screws, and the incision was then closed. Liquamycin, LA 200 (0.1 cc/kg; Pfizer, Inc., New York, NY), was provided for 10-days postsurgery.
Dialysis Probe and Dialysis Buffer
The microdialysis probe had a nitrocellulose hollow fiber dialysis membrane with a molecular mass cutoff of 6 kDa (Spectra/Por; Spectrum, Gardena, CA). The probe was of the concentric design adapted for use in sheep with modifications previously described [8]. It was constructed in our laboratory from 24-gauge stainless-steel tubing through which a 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 was sealed with epoxy (Devcon Corp., Riviera Beach, FL). The final length of dialysis membrane in direct contact with brain tissue was 2.0 mm. It should be noted that the maximal dorsoventral dimension of the ventromedial nucleus is approximately 3.5 mm in sheep. Drugs were dissolved in an artificial cerebrospinal fluid (CSF), which consisted of 127.6 mM NaCl, 2.4 mM KCL, 0.69 mM CaCl2, 1 mM MgSO4, 2.3 mM NaH2PO4, and 9.7 mM Na2HPO4 (pH 7.4).
Experimental Design
Series 1: Effects of GABA agonists The objective of these four experiments was to determine if infusion of GABAB agonists into the MBH of OVX ewes would increase circulating LH. The four experiments varied with respect to specific animal model and drug regimen. In experiments 1 and 2, all animals were treated with estradiol implants, whereas in experiments 3 and 4, they were not.
Experiment 1 The objective was to determine if the GABAB agonist SKF 97541 (SKF) [19] (Tocris—Cookson, Ellisville, MO) would elevate LH in E-treated OVX ewes. There were two treatments applied to nine animals: CSF and SKF dissolved in CSF. A crossover design was used so that each ewe received each treatment in a balanced order.
Adult ewes were OVX in early August, and at the same time a subcutaneous estradiol-filled silicone implant (3.32 mm o.d.) was inserted over the ribs at a dose of 0.065 cm/kg. The implants ranged in size from 1.6 to 2.3 cm long and produced physiological circulating estradiol concentrations [20]. The implants were retained for the duration of the study. Microdialysis guide tubes were placed into the MBH in mid-September, and perfusions were done in October. Animals were exposed to natural photoperiod until OVX and then placed in a windowed barn in which natural lighting was supplemented by fluorescent lighting (10L:14D) from 0700 h until 1700 h.
Approximately 3 wk after implantation of the guide tubes and one day before drug dialysis, groups of three animals each had a catheter placed into the jugular vein to facilitate blood collection. Each animal was placed into an individual pen and allowed 12–15 h to acclimate.
On the day of treatment the stylets were removed from the guide cannulae and replaced with microdialysis probes and dialysis proceeded as described previously [12]. Each animal then received a 3-h period of control dialysis of CSF followed by a 4-h period of infusion with either CSF or SKF (10 µM). The SKF concentration was based on results of previous studies that found that 5 µM SKF elevated basal LH in castrated male sheep [10]. The microdialysis probes were not changed during infusion. During the entire 7-h dialysis period, blood samples were collected from the jugular vein at 9-min intervals. Blood was collected into glass tubes containing 100 µl heparin (125 U/ml) and then was centrifuged within 1 h of collection. Plasma was stored at -20°C until assayed for LH.
After each dialysis session, the probes were removed and replaced with sterile stylets. The animals were injected with LA-200 and returned to group pens. Subsequent dialysis sessions were conducted at 1-wk intervals.
Experiment 2 Based on the results of experiment 1, experiment 2 was conducted to determine if a higher dose of SKF would elevate circulating LH in E-treated ovariectomized ewes. There were three treatments: CSF, SKF (10 µM), and SKF (40 µM). Treatments were applied to eight animals according to a crossover design such that each animal received each treatment in partially balanced order. The protocol was similar to that of experiment 1 except that the experiment was conducted during the nonbreeding (anestrous) season in April. Ewes that had been OVX for over 6 mo were moved into a windowed barn in April. Natural light was supplemented with artificial light (16L:8D) from 0400 h to 2000 h.
Experiment 3 The objective of this experiment was to determine if the GABA agonist baclofen (Sigma, St. Louis, MO) would alter basal LH pulse parameters in OVX ewes during the breeding season. Ewes that had been OVX approximately 6 mo previously had bilateral guide cannulae implanted in October. They then were subjected to two microdialysis sessions as described in the Experiment 6 section. After a 3- to 4-wk recovery period, they were placed on this experiment, which had two treatments: CSF and baclofen (1 mM) applied to nine animals. A crossover design was used such that each animal received each treatment in a balanced order. The microdialysis and sample collection procedures were as described for experiment 1.
Experiment 4 The objective of this experiment was to determine if the GABA agonists baclofen and SKF would alter basal LH pulse parameters in ovariectomized ewes during the nonbreeding season. Long-term OVX ewes were removed from outdoors in mid-February and placed in a windowed barn. Natural lighting was supplemented with artificial lighting (16L:8D) from 0400 h to 2000 h. Guide tubes were implanted in late February, and microdialysis procedures were initiated starting 2–3 wk postsurgery. There were three treatments: CSF, baclofen (1 mM), and SKF (40 µM) applied to nine ewes. Treatment order was assigned according to three 3 x 3 partially balanced Latin squares such that each animal received each treatment in a balanced order. Perfusion and blood collection procedures were as described for experiment 1.
Series 2: Effects of GABA B antagonists The objective of these experiments was to determine if infusion of the GABAB antagonist CGP 52432 (CGP)[21] (Tocris-Cookson, Ellisville, MO) into the MBH would alter the LH surge induced by injection of estradiol benzoate (EB) into OVX ewes. We reasoned that if the agonists stimulated LH, then antagonists should either inhibit LH or have no effect. The two experiments differed with respect to specific treatment and sampling regimens.
Experiment 5 All ewes were ovariectomized in August, and each was given an estradiol implant and subjected to surgery as described for experiment 1. After being subjected to the two microdialysis sessions of experiment 1, the animals were allowed to recover for 2 wk and then utilized for this experiment.
This experiment was conducted as a 2 x 2 factorial with steroid (EB vs. oil) and drug (CGP 250 µM vs. CSF) as the two factors. A Latin square design was used so that each of the eight ewes received each of the four treatments in a balanced order.
One day before drug treatment, groups of four ewes each had a catheter inserted into the jugular vein for purposes of blood collection and then were placed into individual pens. At 1000 h (10 h before start of drug treatment), each was injected with either 50 µg of EB in 0.5 ml corn oil or corn oil only. At approximately 0700 h the next morning, blood sampling was started and then continued at 1-h intervals until 1600. Stylets were removed from the guide cannulae and replaced with microdialysis probes previously filled with either CSF or CGP solution. Microdialysis was immediately started and continued for 8 h. After the dialysis session, the probes were replaced with sterile stylets. The animals were injected with Liquamycin and returned to group pens. The interval between subsequent dialysis sessions was 1 wk with a 2-wk interval between EB injections.
Experiment 6 In view of the lack of effect of CGP noted in the Experiment 5 section, the protocol for experiment 6 was modified. This experiment differed from the previous one in that long-term OVX animals were not given estradiol implants, CGP was used at a higher dose (500 µM), and CGP infusions and blood collection were started before EB injection. Infusions and blood collection were maintained for 24 h.
There were two treatments: EB plus CGP and EB plus CSF. A crossover design was used so that each of nine ewes received each treatment in a partially balanced order. Ewes which had been OVX approximately 6 mo previously had bilateral guide cannulae implanted in October. At that time they were moved into a windowed barn. Natural lighting was supplemented with artificial lighting (10L:14D); 2–3 wk later, the jugular veins were cannulated, and the ewes placed in individual pens and allowed to acclimate for 16 h.
At approximately 0800 h the next morning, the stylets were removed and replaced with filled microdialysis probes. Infusion continued for 24 h. Each animal was injected with 50 µg of EB as soon as infusion was started (approximately 0830). Blood sampling was started immediately before insertion of the probes and was continued at 1-h intervals for 24 h. After the microdialysis session, animals were treated with Liquamycin and then returned to group pens for a 3-wk recovery period before the second EB injection and microdialysis session.
Hormone Assay
Plasma samples were assayed in duplicate for LH using a previously described radioimmunoassay validated for use in our laboratory [22]. The sensitivity was 1 ng/ml NIH LH S-20 at 90% binding. The intraassay coefficient of variation was 3.7%, and the interassay coefficients of variation were 8.4%, 4.2%, and 6.3% for low, medium, and high internal standards, respectively. Values for LH interpulse interval (IPI) and LH pulse amplitude were determined using the Pulsar algorithm [23].
Histology
At the end of each experiment, the animals were killed. The brains were removed after perfusion via carotid artery with 0.9% saline solution, followed by 10% formalin fixative, after which the hypothalmi were isolated and immersed in fixative. Alternatively, the hypothalmi were removed, frozen on dry ice, and stored at -80°C until sectioned on a freezing microtome. The sections collected were histologically processed and stained with Luxol fast blue or O-toluidine blue to localize probe placement. Evaluation of probe placement was made with the aid of diagrams from Lehman et al. [16].
Analysis of Data
For experiments 1–4, the differences ("delta" in each LH pulse parameter [mean, amplitude, and interpulse interval]) between the first 3 h and the last 3 h of infusion were calculated. Subsequently, the mean differences were compared using analysis of variance for repeated measures followed by Newman-Keuls tests [24]. For experiments 5 and 6, two parameters were analyzed: highest LH concentrations achieved and interval from injection of EB to the time that the highest LH concentration was achieved. Comparison among or between groups was done by analysis of variance for repeated measures and by Newman-Keuls tests (experiment 5).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Data from animals with damaged guide cannulae or in which probes were outside the target area (usually one probe in the third ventricle and one far lateral) were excluded from analysis. As a result, data from six, five, eight, eight, six, and six animals were analyzed for experiments 1–6, respectively. A schematic representation of probe placement in the six animals retained for analysis of experiment 1 is shown in Figure 1. Similar probe placement was obtained in the other experiments.
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Experiment 1: Effect of SKF (10 µM) during the breeding season Mean LH concentrations were relatively low in all animals during infusion of CSF (3.7 ± 5 ng/ml) and remained thus throughout the perfusion procedure. Infusion of SKF had no significant effect (P > 0.05) on any of the LH pulse parameters (data not shown).
Experiment 2: Effect of SKF (10 and 40 µM) during the nonbreeding season Basal LH concentrations were at or below assay sensitivity in all trials (1.2 0.2 ng/ml). Consequently, the data were not subjected to analysis by Pulsar. The effects of SKF were equivocal. In two animals, LH remained at this low concentration throughout treatment. In two animals, a single robust and seemingly prolonged "pulse" occurred during treatment with both doses of SKF (Fig. 2). In one animal, a robust pulse occurred during treatment with 40 µM SKF; however, in one trial this animal also had a pulse during pretreatment with CSF. Stated in another way, during the second half of perfusion, none of five animals had a pulse in response to CSF, two of five had one pulse during treatment with 10 µM SKF, and three of five had one pulse during treatment with 40 µM SKF.
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Experiment 3: Effect of baclofen during the breeding season Although baclofen tended to elevate mean LH (6.3 ± 0.8 vs. 6.8 ± 0.8 ng/ml) and LH pulse amplitude (2.4 ± 0.7 vs. 3.0 ± 0.7 ng/ml), the effect did not reach statistical significance (P > 0.05) (Fig. 3). This reflected considerable variation in responses with only four of eight animals showing a subjective elevation of LH in response to baclofen, while two appeared to show no response, and two appeared to show a decrease.
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Experiment 4: Effect of baclofen and SKF during the nonbreeding season Both baclofen (1 mM) and SKF (40 µM) increased mean LH (P < 0.05) (8.9 ± 0.8 vs. 13.6 ± 1.8 and 9.2 ± 1.0 vs. 12.4 ± 1.5 ng/ml) and LH pulse amplitude (P < 0.05) (4.7 ± 0.9 vs. 8.8 ± 1.2 and 4.8 ± 1.6 vs. 7.6 ± 1.0 ng/ml) as compared to CSF (Fig. 4). Neither affected LH interpulse intervals. There was no difference (P > 0.05) between effects of baclofen and SKF.
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Experiment 5: Effect of CGP on LH surge The results are illustrated in Figure 5. Although the sampling duration was too short to monitor peak "surge" concentrations in some animals, EB injection clearly elevated LH (P < 0.01). CGP had no significant effect (P > 0.05) on either concentration or interval from start of blood collection to time of highest LH concentration.
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Experiment 6: Effect of CGP on LH surge E-treatment produced robust elevation of LH over time, but there was no significant effect (P > 0.05) of CGP on either peak LH concentrations or on time between injection and occurrence of peak concentrations (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The equivocal, or lack of, effect of infusing SKF into the MBH of E-treated ewes on basal LH was somewhat surprising in light of previous studies showing that concentrations as low as 5 µM significantly elevated LH in castrated rams [14] and that 1 mM baclofen rapidly and robustly elevated both GnRH and LH pulse amplitude in T-treated castrated rams [13]. Notably, these weak responses to SKF in the E-treated ewe were observed during both the breeding and the anestrous seasons. However, since we cannot equate the degree or level of negative feedback produced by E-implants versus T-implants in separate studies, it is difficult to make comparisons between studies. On the one hand, the differing responses of rams and ewes may reflect a sex difference in the role of MBH GABAB receptors, while, on the other, it may be that the E-implants were so effective as to block responses either to transmitters within the hypothalamus or to GnRH by the anterior pituitary. In this respect, we note that Scott and Clarke [7] reported that during the anestrous period, injection of baclofen into the POA elevated LH in some E-treated OVX ewes but not in OVX ewes not treated with E. In that case, E-treatment appeared to have increased the response to the agonist. The reason for the different effects of E on responses to baclofen noted in the two studies are not obvious but may be related either to the methods of treatment with either E or baclofen or to some other differences in protocol.
It was not the original intent of our studies to compare responses between E-treated and non-E-treated ewes, but in light of the very slight response shown by E-treated ewes in experiments 1 and 2, we chose to then investigate the response in the absence of E-treatment. In contrast to the slight response to 40 µM SKF shown by E-treated OVX ewes during the nonbreeding season (experiment 2), both 40 µM SKF and baclofen clearly elevated LH pulse amplitude and mean LH concentrations in OVX ewes not treated with E during the nonbreeding season (experiment 4). A similar though not statistically significant effect of baclofen was noted in non-E-treated ewes during the breeding season. Whereas these observations are not sufficient to indicate a clear effect of E on responsiveness to GABAB agonists, they are important in indicating a possible role of the MBH GABAB system in regulating basal LH in the ewes. In this respect, results from the ewe and ram are similar; however, evaluation of the data from our previous studies [12–14] suggests that the responses were more robust in castrated rams than in these ewes. This suggests a sex difference in the precise role of the GABAB system in regulating the pulse mode of LH secretion in this species. Obviously, direct comparisons between sexes and between OVX and OVX E-treated ewes will be required in order to address these issues.
Slight differences in the protocols of experiments 3 and 4 preclude direct statistical comparison of responses to baclofen by OVX ewes in the two experiments. However, the finding that baclofen elicited a clear response in the nonbreeding season while it caused only a trend during the breeding season may reflect an effect of season on this response. Scott and Clarke [7] reported a seasonal difference in responses of ewes subjected to injections of GABAergic drugs into the POA. They also found a greater response during the nonbreeding season. The data from each of the two studies, while limited, are consistent in indicating a seasonal influence on the role of GABA in modulating LH secretion in the ewe.
The results of CGP infusions were highly uniform in not altering characteristics of LH surges. Interpretation of "negative" results is difficult, but two interpretations are either that the dose of CGP was not high enough or that activation of GABAB receptors in the MBH are not critical for induction of the surge. We do not favor the first in that the higher dose of CGP used was found previously to block effects of a coinfused agonist on basal LH in the ram and tended to lower basal LH [14]. Also, it is noted that CGP 52432 is among the most potent and freely soluble of currently available GABAB antagonists [21] with an IC50 of 85 nM. The second interpretation is consistent with the data, but little is known about the relationship between GABA in the MBH and control of the LH surge. To our knowledge, this is the only study that has investigated that relationship. The results are somewhat puzzling in that in the ewe the MBH appears to be a critical site for the action of E in inducing the surge [17]. Also, studies on the effect of hypothalamic deafferentation in the ewe [18] indicate the MBH is sufficient to support the LH surge, although input from the POA seems to be needed for a quantitatively normal surge. In comparison, there is evidence that GABAergic neurons terminating in the POA play a significant role in regulating both the basal and the surge modes of LH secretion [25–27]. Particularly relevant are studies in the ewe by Robinson and colleagues, who found that POA GABA concentrations fell coincident with the LH surge in the ewe [27] and that progesterone treatment prevented both the LH surge and the temporal decline in GABA [28]. The anatomical source of the GABA measured in the POA in these studies is not at all clear, nor is the degree of interaction of GABAergic neurons located in the MBH and POA.
Full understanding of the role of GABA in regulating LH remains elusive. This is due in part to variable results obtained by multiple approaches ranging from in vivo studies such as those described here to recent investigations of effects of applying GABA directly to individual GnRH neurons [29]. In the latter case, rapid direct stimulation of GABAA receptors caused a transitory increase in excitability of GnRH neurons, whereas prolonged exposure reduced excitability. Apparently, direct activation of GABAB receptors inhibited GnRH neuron excitability. These results clearly contrast with results of the present and many other studies in which GABAergic drugs were applied to adjacent tissues. The complexity of the system is illustrated by observations that E can decrease the hyperpolarization response of GABAergic neurons to baclofen by apparently modulating GABAB autoreceptor function and that the effects of E on some GABAergic neurons changes over time [30]. Thus, it appears that GABA may act at multiple sites to affect GnRH and LH secretion and that the sum effect depends on multiple background factors, such as photoperiod and steroid levels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gary L. Jackson, Department of Veterinary Biosciences, 2001 South Lincoln Avenue, Urbana, IL 61802. FAX: 217 244 1652; g-jackson{at}uiuc.edu
Received: 16 July 2003.
First decision: 8 August 2003.
Accepted: 12 September 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-aminobutyric acid turnover: effect of castration on -aminobutyric acid turnover in microdissected brain regions of the male rat. J Neurochem 1993 60:2254-2264[CrossRef][Medline]-aminobutyric acid (GABA) is mediated by GABAA receptors but not by GABAB receptors. Endocrinology 1993 132:1789-1796[Abstract]-aminobutyric acid may be involved in seasonal changes in the negative feedback effects of estrogen on gonadotropin-releasing hormone secretion levels in the ewe. Endocrinology 1993 133:2904-2912[Abstract]1 subunit is expressed in distinct neuronal populations. Molecular Brain Research 1999 15:121-132-aminobutyric acid production near luteinizing hormone-releasing hormone nerve terminals disrupts estrous cyclicity in the rat. Endocrinology 2001 142:2102-2114-aminobutyric acid subtypes in the arcuate-ventromedial region of the castrated ram. Endocrinology 1996 137:3453-3460[Abstract]-aminobutyric acid agonist reverses the negative feedback effect of testosterone on gonadotropin-releasing hormone and luteinizing hormone secretion in the male sheep. Endocrinology 2000 141:3940-3945-aminobutryic receptors excites gonadotropin-releasing hormone neurons. Mol Endocrinol 2002 16:2872-2891This article has been cited by other articles:
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  A. L. Bogusz, S. L. Hardy, M. N. Lehman, J. M. Connors, S. M. Hileman, J. H. Sliwowska, H. J. Billings, C. J. McManus, M. Valent, S. R. Singh, et al. Evidence that {gamma}-Aminobutyric Acid Is Part of the Neural Circuit Mediating Estradiol Negative Feedback in Anestrous Ewes Endocrinology, June 1, 2008; 149(6): 2762 - 2772. [Abstract] [Full Text] [PDF]
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