Insight into the neuroendocrine site and cellular mechanism by which cortisol suppresses pituitary responsiveness to gonadotropin-releasing hormone

Abstract

Stress-like elevations in plasma glucocorticoids rapidly inhibit pulsatile LH secretion in ovariectomized sheep by reducing pituitary responsiveness to GnRH. This effect can be blocked by a nonspecific antagonist of the type II glucocorticoid receptor (GR) RU486. A series of experiments was conducted to strengthen the evidence for a mediatory role of the type II GR and to investigate the neuroendocrine site and cellular mechanism underlying this inhibitory effect of cortisol. First, we demonstrated that a specific agonist of the type II GR, dexamethasone, mimics the suppressive action of cortisol on pituitary responsiveness to GnRH pulses in ovariectomized ewes. This effect, which became evident within 30 min, documents mediation via the type II GR. We next determined that exposure of cultured ovine pituitary cells to cortisol reduced the LH response to pulse-like delivery of GnRH by 50% within 30 min, indicating a pituitary site of action. Finally, we tested the hypothesis that suppression of pituitary responsiveness to GnRH in ovariectomized ewes is due to reduced tissue concentrations of GnRH receptor. Although cortisol blunted the amplitude of GnRH-induced LH pulses within 1-2 h, the amount of GnRH receptor mRNA or protein was not affected over this time frame. Collectively, these observations provide evidence that cortisol acts via the type II GR within the pituitary gland to elicit a rapid decrease in responsiveness to GnRH, independent of changes in expression of the GnRH receptor.

Figure 1

Design for primary ovine pituitary cell culture experiment. Time is depicted as day of culture. Open horizontal bar indicates treatment with medium alone. Shaded horizontal bar depicts initiation of treatment with cortisol (150 ng/ml) before testing responsiveness to GnRH on d 5. Arrows designate timing of repeated, hourly boluses of GnRH (4 ng/ml, 10 min). A, GnRH boluses were administered before (pre) and immediately after treatment onset with cortisol or medium alone (acute); B and C, GnRH boluses were administered either 24 or 48 h, respectively, after chronic treatment with cortisol or medium alone.

Figure 2

LH (•) 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.

Figure 3

LH pulse amplitude in response to hourly GnRH (5 ng/kg, iv) boluses during exposure to cortisol (○) or dexamethasone (•) in experiment 1. Amplitude is expressed as a ratio of each hourly LH response during cortisol or dexamethasone treatment to the pretreatment mean (±sem). Horizontal bar depicts the 6-h period of twice hourly sc injection of cortisol or dexamethasone.

Figure 4

LH release from ovine pituitary cells in response to three hourly boluses of GnRH (4 ng/ml, 10 min) in experiment 2. A, Cells were challenged with GnRH before (pre) and after (acute) exposure to medium alone or medium containing cortisol (150 ng/ml) on d 5 of culture; B, responsiveness to GnRH was tested on d 5 of culture after exposure to medium alone or 24 or 48 h chronic cortisol. Results represent the mean ± sem of four independent experiments each performed in sextuplicate. *, P < 0.05: A, pre vs. acute; B, medium alone vs. 24- or 48-h chronic cortisol.

Figure 5

A, Mean ± sem. LH pulse amplitude in ewes before (white bars) and during (striped bars) exposure to cortisol or vehicle in experiment 3, part 1; B and C, mean ± sem GnRH receptor and pituitary LH (B and C, respectively) in ewes exposed to cortisol (black bars) or vehicle (white bars). Cortisol and vehicle were administered by twice-hourly sc injection for 3 h. *, P < 0.01, before vs. during cortisol.

Figure 6

Mean ± sem plasma LH (•) and cortisol (○) concentration in ewes receiving a continuous 2-h infusion of vehicle (A) or cortisol (B) in experiment 3, part 2. Tick marks indicate time of administration of hourly GnRH (5 ng/kg, iv) boluses to induce each LH response. Results are mean ± sem of GnRH receptor protein (C) and GnRH receptor mRNA (D) in ewes exposed to cortisol (black bars) or vehicle (white bars). No sem indicates value is smaller than data point.