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Reproductive Sciences Program (K.M.B., L.C.D., A.E.O., V.P., E.R.W., F.J.K.), Departments of Molecular and Integrative Physiology (K.M.B., L.C.D., A.E.O., V.P., E.R.W., F.J.K.) and Pediatrics (V.P.), University of Michigan, Ann Arbor, Michigan 48109; and Animal Reproduction and Biotechnology Laboratory (T.L.D., T.M.N., L.A.R.), Colorado State University, Fort Collins, Colorado 80523
Address all correspondence and requests for reprints to: Kellie M. Breen, Department of Reproductive Medicine, University of California, San Diego, Leichtag Biomedical Research Building, Room 349, La Jolla, California 92093-0674. E-mail: kbchurch{at}UCSD.edu.
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Introduction |
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Here, we present a series of experiments to investigate the neuroendocrine site and cellular mechanism of cortisol action. First, because previous evidence for mediation via the type II GR was obtained using RU486, a nonspecific antagonist, we sought to confirm the relevance of this receptor by determining whether a specific type II GR agonist mimics the effect of cortisol in decreasing responsiveness to GnRH. Next, we used an ovine pituitary cell culture system to determine whether cortisol can elicit this action directly on pituitary cells. This was deemed important in light of recent evidence that the effect of cortisol on responsiveness to GnRH is not expressed if the hypothalamus is surgically disconnected from the pituitary (9), raising the possibility that cortisol acts indirectly via a central mechanism. Finally, because chronic exposure to cortisol can inhibit expression of the GnRH receptor (10), we tested the hypothesis that the rapid inhibitory action of cortisol on responsiveness to GnRH results from reduced GnRH receptor mRNA and protein.
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Materials and Methods |
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Experiment 1: does a type II GR agonist mimic the inhibitory action of cortisol on pituitary responsiveness to GnRH?
Animal model and design.
We employed a pituitary clamp model used previously to demonstrate the inhibitory action of cortisol on pituitary responsiveness to GnRH (8, 11). Specifically, pulsatile GnRH secretion was chronically blocked in ovariectomized ewes by constant delivery of a luteal-phase level of estradiol via a 3-cm sc SILASTIC brand (Dow Corning, Midland, MI) implant during seasonal anestrus (12). Physiological GnRH pulses (5 ng/kg, iv, over 6 min; Sigma Chemical Co., St. Louis, MO) were infused hourly via a timer-regulated pump for 6 d to reactivate the gonadotrope and stabilize pituitary responsiveness to GnRH. On d 7 of pulsatile GnRH treatment, blood was sampled via jugular cannula at 12-min intervals for 12 h to assess LH pulse amplitude as an index of pituitary responsiveness to the exogenous GnRH pulses. For the first 6 h, no additional treatment was applied. During the next 6 h, cortisol (0.125 mg/kg·30 min, Solu-Cortef, hydocortisone sodium succinate; Pharmacia & Upjohn, Kalamazoo, MI) (n = 4 ewes) or dexamethasone (0.125 mg/kg·30 min, dexamethasone sodium phosphate; American Pharmaceutical Partners, Inc., Schaumburg, IL) (n = 7 ewes), suspended in 1 ml sesame oil, was injected sc every 30 min in an area of loose skin on the back. A vehicle or time control was not included due to limited resources and our repeated previous observation that neither vehicle nor time during the sampling period influenced plasma cortisol concentrations or the response to GnRH in this pituitary clamp model (8, 11) (also see results of experiment 3 of this study).
Data analysis.
Amplitudes of GnRH-induced LH pulses (peak minus preceding nadir) were averaged before and during cortisol or dexamethasone treatment and analyzed by repeated-measures ANOVA (rmANOVA) to determine whether these glucocorticoids reduce responsiveness to GnRH. Mean plasma cortisol concentrations before and during treatment were analyzed by rmANOVA. Hormone values were log transformed before statistical analysis to adjust for heterogeneity of variance. Significance level was set at P < 0.05.
Experiment 2: can cortisol act directly upon pituitary cells to inhibit responsiveness to GnRH?
Tissue culture.
Ovine pituitary glands, obtained from a local abattoir (Wolverine Packing Co., Detroit, MI; December to March), were enzymatically dissociated using a two-step collagenase-viokase digestion procedure (Sigma) (13). Dispersed cells were plated (5.0 x 105 cells per well) in DMEM, supplemented with L-glutamine (1 ml/liter), gentamycin (400 µl/liter), fungisone (1 ml/liter), 10% horse serum, and 2.5% fetal calf serum (all reagents obtained from Invitrogen Life Technologies, Carlsbad, CA). Cultures were maintained at 37 C under 5% CO2. On d 5, medium was changed to serum-free DMEM, and LH responses to repeated hourly boluses of GnRH were determined in the presence or absence of cortisol. To simulate GnRH pulses in vitro, cultures were exposed to medium devoid of GnRH for 50 min followed by medium containing GnRH (4 ng/ml; Sigma) for 10 min. After each 10-min GnRH bolus, medium was removed and stored at –20 C for LH assay. This GnRH exposure was designed to approximate the duration and concentration of a natural GnRH pulse in pituitary portal blood of ovariectomized ewes (14). A preliminary study confirmed the efficacy of 10 min exposure to increasing concentrations of GnRH (0.25, 1.0, and 4.0 ng/ml) in enhancing LH release into the medium (3.0 ± 1.6, 5.6 ± 1.0, 8.8 ± 0.9 ng/ml increase over basal values, respectively; n = 2 independent experiments each performed in sextuplicate).
Experimental design.
To determine the effect of acute cortisol exposure on responsiveness to GnRH in vitro, cultures were challenged with six hourly boluses of GnRH (4.0 ng/ml, 10 min) on d 5 of culture. For the first three boluses, no additional treatment was applied (Fig. 1A
, pre period). Beginning 30 min after the third GnRH bolus and continuing for the next three hourly GnRH boluses (acute period), cells were cultured in medium containing 150 ng/ml cortisol (Pharmacia & Upjohn) or medium alone. To investigate effects of chronic cortisol, additional cells were treated with medium containing cortisol or medium alone during the final 24 or 48 h of culture before testing responsiveness to three hourly GnRH boluses (Fig. 1, B and C, 24- or 48-h chronic period). The cortisol concentration in medium mimicked maximal plasma cortisol levels in ewes during an acute immune/inflammatory stress (15). Basal LH concentrations were determined at corresponding times in medium of untreated cultures and subtracted from GnRH-stimulated LH responses before data analysis.
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Experiment 3: does cortisol reduce pituitary concentrations of GnRH receptor?
Part 1 (endogenous GnRH pulse model).
During the nonbreeding season, blood was collected from 14 ovariectomized ewes via jugular cannula for 6 h at 10-min intervals for assessment of LH pulse amplitude. For the first 3 h, no treatment was applied. During the final 3 h of sampling, vehicle (sesame oil) or cortisol (0.125 mg/kg in vehicle; Pharmacia & Upjohn) was administered by sc injection every 30 min (n = 7 per group). Immediately after sampling, ewes were injected with a barbiturate overdose (Fatal Plus; Vortech Pharmaceuticals, Dearborn, MI), and the pituitary gland was removed, divided midsagittally, frozen on dry ice, and stored at –80 C for measurement of GnRH receptor protein and pituitary LH.
Part 2 (pituitary clamp model).
Part 1 demonstrated that cortisol lowered LH pulse amplitude but not tissue concentrations of the GnRH receptor. Part 2 was conducted to confirm this finding using a more powerful animal model that allowed precise control of the GnRH stimulus and to analyze acute cortisol effects on GnRH receptor gene expression as well as protein content.
The pituitary clamp model described in experiment 2 was employed here. Eighteen ovariectomized ewes received hourly GnRH boluses for 6 d to stabilize gonadotrope responsiveness. Beginning 3 h before blood sampling on d 7, the ewes were disconnected from the GnRH pump, and the hourly boluses of GnRH were delivered manually (over 20 sec) via jugular cannula. This allowed sampling at more precise times relative to delivery of a GnRH pulse. While continuing to receive pulsatile delivery of GnRH, blood was sampled via jugular catheter at 10-min intervals for 5 h. For the first 3 h, all ewes (n = 18) received continuous infusion of vehicle (saline). During the latter 2 h, half the ewes received continuous infusion of cortisol (0.40 mg/kg·h; Pharmacia & Upjohn) (n = 9 per group). Cortisol was administered by continuous iv infusion (as opposed to sc injection used in part 1) to decrease the latency in achieving a target plasma cortisol level of approximately 125 ng/ml. Thirty minutes after the final GnRH injection, ewes were euthanized, and the pituitary was collected for measurement of GnRH receptor protein and mRNA as described for part 1.
Isolation and quantification of mRNA.
Total cellular RNA was obtained from one half of each pituitary gland using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Integrity of the RNA was determined by OD absorption ratio OD260nm:OD280nm between 1.7 and 2.0. First-strand cDNA was synthesized from 2 µg total RNA with iScript cDNA Synthesis Kit per the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA).
PCR primers to detect the ovine GnRH receptor were designed from the known ovine sequence (GenBank accession nos. L43841, L43842, and L42937) reported by Campion et al. (16). Forward and reverse primers for the ovine GnRH receptor were 5'-ACCAGGCCTCTAGCAGTGAA-3' and 5'-CTTTTTCACCTTCAGCTGCC-3', respectively. Forward and reverse primers for actin (housekeeping gene) were 5'-TCTGGCACCACACCTTCTAC-3' and 5'-GGTCATCTTCTCACGGTTGG-3', respectively. Each primer pair was validated to ensure amplification of the proper size DNA fragment and that no amplification of genomic DNA occurred. PCR products were subcloned into pGemT Easy vector (Promega, Madison, WI), and the plasmids were sequenced to confirm the identity of both ovine GnRH receptor and actin DNA fragments.
Real-time PCR was performed using the Bio-Rad iCycler under the following conditions: 95 C for 3 min, followed by 36 cycles of 95 C for 30 sec, 59 C for 30 sec, and 72 C for 60 sec. Separate reactions were made to amplify GnRH receptor and actin and consisted of 10% of the cDNA synthesis reaction added to 300 nM of each primer in iQ SYBR Green Supermix (Bio-Rad). A standard curve for each primer set consisted of four serial dilutions (1:10) of sequenced PCR product. All samples were tested in duplicate within a single run. Values for GnRH receptor gene were standardized to actin and expressed as relative units. A melting curve analysis was performed to confirm that a single amplicon was generated in each reaction, and its size (226 and 105 bp, GnRH receptor and actin, respectively) was verified by gel electrophoresis.
GnRH receptor protein analysis.
One half of each pituitary gland was used to prepare individual partially purified membrane fractions from which GnRH receptor concentration was quantified according to the standard curve technique of Nett et al. (17). Briefly, a standard curve was generated by incubating increasing quantities of a bovine pituitary membrane pool with 0.2 nM [125I]D-Ala6-GnRH-Pro9-ethyl-amide ([125I]D-Ala6). The amount of specifically bound [125I]D-Ala6 in each pituitary membrane preparation was compared with the standard curve, and the concentration of GnRH receptor was calculated. All samples within part 1 or 2 were run in duplicate in a single assay. Results are expressed as total receptor concentration (free plus bound) per milligram pituitary.
Data analysis.
Pituitary concentrations of LH, GnRH receptor protein, and GnRH receptor mRNA were compared by one-way ANOVA to assess an effect of cortisol. LH pulse amplitude, frequency, and mean LH concentration before and during treatment in each ewe were compared by rmANOVA. In part 1, endogenous LH pulses were identified using the Cluster pulse-detection algorithm (18). Cluster sizes for peaks and nadirs were set at 1 and the t statistic used to identify a significant increase and decrease was 2.6. Hormone values were log transformed, and square root transformation of pulse frequencies was preformed before statistical analysis.
Hormone analyses
LH concentrations were determined in duplicate aliquots (25–100 µl) of plasma, cell culture medium, or pituitary homogenate using a modification (19) of a previously described RIA (20, 21). Values for plasma and cell culture medium are expressed in terms of NIH-oLH-S12. Mean intra- and interassay coefficients of variation were 4.0 and 6.0%, respectively; assay sensitivity averaged 0.60 ng/ml (32 assays). Values for pituitary homogenates were determined in a single assay and are expressed in terms of NIH-oLH-S24; intraassay coefficient of variation was 5.0%, and assay sensitivity was 0.61 ng/ml. Total plasma cortisol concentrations were determined in duplicate 50-µl aliquots of unextracted plasma using the Coat-A-Count cortisol assay kit (Diagnostic Products Corp., Los Angeles, CA), validated for use in sheep (6). Mean intra- and interassay coefficients of variation were 7.1 and 9.8%, respectively (14 assays); assay sensitivity averaged 0.51 ng/ml.
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Results |
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illustrates plasma profiles of cortisol and LH in one representative ewe that received cortisol and two representative ewes that received dexamethasone. Before treatment, cortisol was low and stable (8.1 ± 2.7 vs. 4.3 ± 0.8 ng/ml for cortisol and dexamethasone groups, respectively). Injection of cortisol every 30 min elevated plasma cortisol concentrations within 2 h to 86.6 ± 15.2 ng/ml. Injection of dexamethasone every 30 min reduced plasma cortisol to 1.4 ± 0.7 ng/ml (P < 0.001) during the 6-h treatment period, confirming dexamethasone evoked negative feedback within the hypothalamic-pituitary-adrenal axis via the type II GR (22, 23).
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) and by composite presentation expressing the amplitude of each LH pulse after treatment onset as percentage of pretreatment mean (Fig. 3). Maximal suppression to about 30% of pretreatment values occurred within 90 min. Dexamethasone also significantly suppressed the amplitude of GnRH-induced LH pulses (Figs. 2, B and C, and 3; P < 0.05). As for cortisol, maximal reduction during dexamethasone was to about 30% of pretreatment values at 90 min. Although the duration of suppression appeared to be greater in ewes receiving dexamethasone, this was not documented statistically by a significant treatment x time interaction in the rmANOVA (P > 0.1).
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illustrates average LH release (over basal values) from pituitary cell cultures in response to three hourly boluses of GnRH administered during exposure to medium containing cortisol or medium alone. In cells cultured in medium alone, the magnitude of LH release did not differ between the first three and final three GnRH boluses (Fig. 4A). In contrast, exposure to cortisol during the 3-h acute period blunted the response to GnRH to approximately 30% of pretreatment values (pre vs. acute cortisol, P < 0.05; Fig. 4A). This suppression was evident by the first GnRH bolus administered 30 min after cortisol onset, illustrating the rapidity of the response to cortisol in vitro (pre vs. 0.5-h acute LH response: 13.3 ± 4.8 vs. 5.9 ± 1.5 ng/ml; P < 0.05). Chronic exposure to cortisol for either 24 or 48 h produced a similar reduction in responsiveness to the three hourly GnRH boluses (medium alone vs. 24- or 48-h chronic cortisol, P < 0.05; Fig. 4B).
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As observed previously in ovariectomized ewes (8, 11) and in contrast to vehicle, cortisol reduced LH pulse amplitude by about 50% (P< 0.01; Fig. 5A). Statistical analysis also revealed inhibition of mean LH concentration but no change in pulse frequency (before vs. during cortisol, mean LH and frequency, respectively: 31.5 ± 3.9 vs. 26.7 ± 3.0 ng/ml, P < 0.01; 4.4 ± 0.3 vs. 4.4 ± 0.2 pulses/3 h, P > 0.1). In contrast to the reduction in LH pulse amplitude, concentrations of GnRH receptor protein or pituitary LH content were not significantly affected by cortisol (P > 0.1; Fig. 5, B and C, respectively).
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displays average plasma LH values during delivery of five pulses of GnRH and mean plasma cortisol concentrations in vehicle- or cortisol-treated ewes. Plasma cortisol remained less than 10 ng/ml in ewes that received vehicle and in ewes before cortisol administration (Fig. 6A); continuous infusion of cortisol elevated plasma concentrations to approximately 110 ng/ml during the final 2 h of sampling (Fig. 6B).
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). In contrast, cortisol significantly blunted responsiveness to GnRH boluses administered 30 and 90 min after cortisol onset (P < 0.05, mean suppression reached 38% at 90 min; Fig. 6B). As in part 1, this acute suppression in LH was not associated with reduced pituitary concentrations of GnRH receptor protein (Fig. 6C). Similarly, cortisol did not lower GnRH receptor mRNA (Fig. 6D). |
Discussion |
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Although previous studies revealed direct inhibitory effects of glucocorticoids on response to GnRH in primary bovine and porcine pituitary cell culture (25, 26), the present study extends those observations by showing the direct action of cortisol can be elicited within a rapid time frame and that it reduces the response to pulse-like stimulation by GnRH. We observed this rapid response both in vitro and in vivo. Specifically, the inhibitory effect of cortisol in dispersed ovine pituitary cells exposed to pulse-like GnRH stimulation occurred within 30 min, and possibly earlier because this was the first time point monitored. Consistent with this response in vitro, cortisol blunted responsiveness to physiological GnRH boluses within 30 min (12% reduction) in ovariectomized sheep in which endogenous GnRH pulses were blocked. Of interest, this inhibition was enhanced (38%) during administration of the subsequent GnRH bolus that occurred 90 min after cortisol onset. This increase in suppression may reflect greater efficacy of the higher plasma cortisol concentration (
80 ng/ml at 30 min vs. 110 ng/ml at 90 min) and/or the latency for maximal suppression of GnRH-induced LH release. Taken together, these results identify a rapid and direct effect of cortisol at the pituitary level and warrant further work to identify the cellular mechanism responsible for this effect.
With regard to cellular mechanisms, increasing evidence suggests the mode of cortisol action is dependent on the duration of cortisol exposure. For example, glucocorticoids reduced gonadotropin gene expression in LβT2 cells (27) and blocked the estrogen-dependent increase in GnRH receptor expression in vivo(10), but both of these effects were observed after 24–48 h. Taking into account that the suppression in responsiveness to GnRH observed here was evident within 30 min and was independent of reduced pituitary LH content and GnRH receptor number or gene expression, it seems reasonable to consider a nongenomic glucocorticoid effect. In this regard, studies in rat and pig pituitary cell cultures suggest glucocorticoids can inhibit signaling mechanisms downstream of the GnRH receptor, including protein kinase C and cAMP (28, 29). Furthermore, recent evidence in a human pituitary cell line suggests glucocorticoids activate the MAPK pathway within 30 min, via a nongenomic mechanism involving the type II GR (30). The possibility of such a mechanism is particularly interesting in light of recent evidence that another steroid hormone, estradiol, can inhibit GnRH-induced LH release in ovariectomized ewes via a pathway independent of genomic action (31, 32). Therefore, a fruitful area of future research will be to determine whether cortisol acts rapidly by a nongenomic mechanism to inhibit responsiveness to GnRH via the type II GR.
Finally, our finding that cortisol can act directly on anterior pituitary cells to cause rapid inhibition of responsiveness to GnRH needs to be reconciled with recent observations that this glucocorticoid failed to inhibit the response to GnRH if the pituitary is surgically disconnected from the hypothalamus of gonadectomized sheep. In the hypothalamo-pituitary-disconnect (HPD) sheep model, cortisol did not reduce the amplitude of LH pulses driven by repeated pulse-like boluses of exogenous GnRH (9). One interpretation of that finding is cortisol acts indirectly via the hypothalamus to elicit a mediator that acts on the pituitary to inhibit its response to GnRH, a mediator akin to the gonadotropin-inhibiting factor recently suggested to exist in birds and mammals (33, 34, 35). A second explanation is that undisturbed communication with the hypothalamus is required to maintain cells in the anterior pituitary responsive to regulatory molecules, such as cortisol. In HPD sheep treated with GnRH, activity of the gonadotrope is reinstated, but other pituitary cell types may remain compromised and unable to respond to cortisol due to the prolonged absence of other hypophysiotropic stimuli. In this regard, our finding that cortisol evokes a rapid reduction in responsiveness to GnRH in cultured ovine pituitary cells indicates cortisol can act directly at the level of the pituitary, but it does not address whether the gonadotrope is the target for this action of cortisol. The present conclusion that cortisol can act acutely at the pituitary level to inhibit responsiveness to GnRH is substantiated by recent in vivo evidence that cortisol does, in fact, inhibit GnRH-induced LH release in HPD sheep if they are also treated with estradiol (36). Collectively, these observations encourage future work to investigate pituitary cell types and mechanisms by which cortisol acts to inhibit responsiveness to GnRH.
In summary, the present studies indicate a stress-like increment in plasma cortisol can act directly upon the pituitary gland via the type II GR to elicit a rapid decrease in responsiveness to GnRH independent of changes in GnRH receptor expression. We recently determined that this action of cortisol is physiologically relevant in that it is necessary for the suppression of pituitary responsiveness to GnRH induced by psychosocial stress. We emphasize, however, that stress also activates central pathways that inhibit pulsatile GnRH secretion (3, 4, 37, 38), and it is likely that factors other than cortisol mediate this inhibition because cortisol itself does not inhibit GnRH pulse frequency or amplitude in the ovariectomized ewe in the absence of gonadal steroids. Overall, these observations provide support for a mechanism whereby glucocorticoids contribute to the acute suppression of reproductive function during stress.
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Acknowledgments |
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Footnotes |
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Preliminary reports have appeared in the 38th Annual Meeting of the Society for the Study of Reproduction, 2005 [Biol Reprod 72(Suppl 1):137 and 648] and the 39th Annual Meeting of the Society for the Study of Reproduction, 2006 [Biol Reprod 74(Suppl 1):423].
Disclosure Statement: The authors have nothing to disclose.
First Published Online October 25, 2007
Abbreviations: GR, Glucocorticoid receptor; HPD, hypothalamo-pituitary-disconnect; rmANOVA, repeated-measures ANOVA.
Received June 12, 2007.
Accepted for publication October 16, 2007.
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) and cortisol (
) responses in one representative ewe that received cortisol (A) and two representative ewes that received dexamethasone (B and C) in experiment 1. Horizontal bar depicts the 6-h period of twice-hourly sc injection of cortisol or dexamethasone. Tick marks indicate time of administration of hourly GnRH (5 ng/kg, iv) boluses to induce each LH pulse. Note, cortisol scale for ewes receiving dexamethasone is expanded to visualize the decrease in plasma cortisol concentration during treatment.
