We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

We tested the hypothesis that there are sex differences in the inhibitory effect of cortisol on pulsatile LH secretion and pituitary responsiveness to GnRH in gonadectomized sheep. In experiment 1, pulsatile LH secretion was examined in gonadectomized ewes and rams infused with either saline, a low (250 microg/kg.h) or a high (500 microg/kg.h) dose of cortisol for 30 h. In experiment 2, direct pituitary actions of cortisol were assessed by monitoring LH pulse amplitude in response to exogenous GnRH in hypothalamo-pituitary disconnected ewes and rams infused with the low dose of cortisol. In experiment 1, the mean (+/-sem) plasma LH concentration was (P<0.05) reduced significantly during cortisol infusion in both sexes, but the effect was greater in rams. In ewes, LH pulse amplitude and frequency were reduced (P<0.05) at the high, but not the low, cortisol dose, whereas total LH output (LH pulse amplitude multiplied by frequency) was reduced (P<0.05) at both doses. In rams, LH pulse frequency and amplitude and total LH output were (P<0.05) reduced significantly at both cortisol doses. In experiment 2, plasma LH concentration and pulse amplitude in response to exogenous GnRH were not affected by infusion of cortisol in either sex. We conclude that gonadectomized rams are more sensitive than gonadectomized ewes to the effects of cortisol to inhibit LH secretion and that sex differences exist in the specific actions of cortisol on LH pulses. The results of experiment 2 suggest that intact hypothalamic input to the pituitary is necessary for cortisol to inhibit pituitary responsiveness to GnRH.

We have used the hypothalamo-pituitary disconnected (HPD) sheep model to investigate direct pituitary actions of cortisol to suppress LH secretion in response to exogenous GnRH. We previously observed that, during the non-breeding season, treatment with cortisol did not suppress the LH response to GnRH in HPD gonadectomised rams or ewes.1 In the present experiment, we tested the effect of cortisol on the LH response to exogenous GnRH in gonadectomised HPD sheep during the breeding season. Using a cross-over design, HPD gonadectomised Romney Marsh rams (n = 6) and ewes (n = 5) received a saline or cortisol (250 μg/kg/h) infusion for 30 h on each of two days, one week apart. All animals were treated with 125 ng i.v. injections of GnRH every 2 h during a 6 h control period preceding the infusion and during the infusion. Jugular blood samples were taken during the control period and the first 6 h and last 6 h of the infusion (over 3 LH pulses). Mean plasma concentrations of LH and LH pulse amplitudes, driven by programmed GnRH injections, were similar in gonadectomised rams and ewes and there were no significant effects of saline infusion between the control periods or the saline infusion in either sex. The amplitude of LH pulses was significantly (P

Stress-like elevations in plasma cortisol suppress LH pulse amplitude in ovariectomized ewes by inhibiting pituitary responsiveness to GnRH. Here we sought to identify the receptor mediating this effect. In a preliminary experiment GnRH and LH pulses were monitored in ovariectomized ewes treated with cortisol plus spironolactone, which antagonizes the type I mineralocorticoid receptor (MR), or with cortisol plus RU486, which antagonizes both the type II glucocorticoid receptor (GR) and the progesterone receptor (PR). Cortisol alone reduced LH pulse amplitude, but not pulsatile GnRH secretion, indicating that it reduced pituitary responsiveness to endogenous GnRH. RU486, but not spironolactone, reversed this suppression. We next tested whether RU486 reverses the inhibitory effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, frequency, and duration. Hourly GnRH pulses were delivered to ovariectomized ewes in which endogenous GnRH pulses were blocked by estradiol during seasonal anestrus. Cortisol alone reduced the amplitude of LH pulses driven by the exogenous GnRH pulses. RU486, but not an antagonist of PR (Organon 31710), prevented this suppression. Thus, the efficacy of RU486 in blocking the suppressive effect of cortisol is attributed to antagonism of GR, not PR. Together, these observations imply that the type II GR mediates cortisol-induced suppression of pituitary responsiveness to GnRH.

Stress responses are thought to act within the hypothalamopituitary unit to impair the reproductive system, and the sites of action may differ between sexes. The effect of isolation and restraint stress on pituitary responsiveness to GnRH in sheep was investigated, with emphasis on possible sex differences. Experiments were conducted during the breeding season and the nonbreeding season. In both experiments, 125 ng of GnRH was injected i.v. every 2 h into hypothalamopituitary disconnected, gonadectomized rams and ewes on 3 experimental days, with each day divided into two periods. During the second period on Day 2, isolation and restraint stress was imposed for 5.5 h. Plasma concentrations of LH and cortisol were measured in samples of blood collected from the jugular vein. In the second experiment (nonbreeding season), plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were also measured. In both experiments, there was no effect of isolation and restraint stress on plasma concentrations of cortisol in either sex. During the breeding season, there was no effect of isolation and restraint stress on plasma concentrations of LH in either sex. During the nonbreeding season, the amplitude of the first LH pulse after the commencement of stress was significantly reduced (P < 0.05) in rams and ewes. In the second experiment, during stress there was a significant increase (P < 0.05) in plasma concentrations of epinephrine in rams and ewes and significantly higher (P < 0.05) basal concentrations of norepinephrine in ewes than in rams. These results suggest that in sheep stress reduces responsiveness of the pituitary gland to exogenous GnRH during the nonbreeding season but not during the breeding season, possibly because of mediators of the stress response other than those of the hypothalamus-pituitary-adrenal gland axis.