Endocrine Basis for Disruptive Effects of Cortisol on Preovulatory Events
http://www.100md.com
内分泌学杂志 2005年第4期
Reproductive Sciences Program and Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109-0404
Address all correspondence and requests for reprints to: Kellie M. Breen, Reproductive Sciences Program, University of Michigan, 300 North Ingalls Building, Room 1101 SW, Ann Arbor, Michigan 48109-0404. E-mail: breenk@umich.edu.
Abstract
Stress activates the hypothalamo-pituitary-adrenal axis leading to enhanced glucocorticoid secretion and concurrently inhibits gonadotropin secretion and disrupts ovarian cyclicity. Here we tested the hypothesis that stress-like concentrations of cortisol interfere with follicular phase endocrine events of the ewe by suppressing pulsatile LH secretion, which is essential for subsequent steps in the preovulatory sequence. Cortisol was infused during the early to midfollicular phase, elevating plasma cortisol concentrations to one third, one half, or the maximal value induced by isolation, a commonly used model of psychosocial stress. All cortisol treatments compromised at least some aspect of reproductive hormone secretion in follicular phase ewes. First, cortisol significantly suppressed LH pulse frequency by as much as 35%, thus attenuating the high frequency LH pulses typical of the preovulatory period. Second, cortisol interfered with timely generation of the follicular phase estradiol rise, either preventing it or delaying the estradiol peak by as much as 20 h. Third, cortisol delayed or blocked the preovulatory LH and FSH surges. Collectively, our findings support the hypothesis that stress-like increments in plasma cortisol interfere with the follicular phase by suppressing the development of high frequency LH pulses, which compromises timely expression of the preovulatory estradiol rise and LH and FSH surges. Moreover, the suppression of LH pulse frequency provides indirect evidence that cortisol acts centrally to suppress pulsatile GnRH secretion in follicular-phase ewes.
Introduction
A VARIETY OF stressors inhibit gonadotropin secretion and disrupt ovarian cyclicity. For example, acute restraint and/or isolation reduce(s) mean plasma LH concentrations and pulsatile LH secretion in the rat, sheep, and monkey (1, 2, 3, 4, 5, 6). Transportation inhibits pulsatile LH secretion and delays and attenuates the LH surge in sheep and cows (7, 8). Furthermore, immune/inflammatory stress suppresses pulsatile LH secretion during the preovulatory period in sheep (9) and delays or blocks the LH surge in follicular-phase ewes, cows, and monkeys (9, 10, 11). Despite evidence that stress interferes with gonadotropin secretion and ovarian cyclicity, the underlying mechanisms are not well understood. In this regard, adaptations to stress comprise an initial autonomic activation and release of catecholamines from the sympathetic nervous system, followed by a more delayed and prolonged activation of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced secretion of CRH, arginine vasopressin, ACTH, and the glucocorticoids (12, 13, 14). Each of these factors potentially mediates reproductive suppression. In actuality, the integrated actions of all of them are likely involved, with the relative importance of each depending on the nature of the stressor (12).
Our laboratory has become interested in the coincident, stress-induced increase in glucocorticoid secretion and suppression of reproductive neuroendocrine function and ovarian cyclicity in sheep. In this regard, a recent study in sheep revealed that a continuous elevation of plasma cortisol to levels observed during isolation stress (75 ng/ml) suppressed the preovulatory estradiol rise and prevented the LH surge in nonstressed, follicular-phase ewes (15). That study, however, did not address how cortisol elicits these effects. Our recent findings that cortisol acutely inhibits pulsatile LH secretion in ovariectomized ewes (16) provides a plausible hypothesis that was tested in the present study: cortisol disrupts the follicular phase by suppressing pulsatile LH secretion, which is an essential stimulus for the preovulatory estradiol rise that induces the LH surge.
Materials and Methods
Animal model
Two experiments were conducted during successive breeding seasons (experiment 1, November to December 2001; experiment 2, November to December 2002) on adult, ovary-intact Suffolk ewes maintained under standard husbandry conditions at the Sheep Research Facility (near Ann Arbor, MI; 42 degrees 18' N). Before the experiment, the ewes were moved from their natural environment to an enclosed, unheated barn in which photoperiod was regulated to simulate that of the outdoors. All procedures were approved by the Committee for the Use and Care of Animals at the University of Michigan.
The estrous cycle of sheep typically lasts 16–17 d and consists of a luteal phase of approximately 13 d and a follicular phase of approximately 3 d (17). To enable simultaneous study of multiple ewes, the follicular phase was synchronized among randomly cycling ewes using intravaginal progesterone-releasing devices [controlled internal drug release (CIDR); InterAg, Hamilton, New Zealand]. These devices maintain plasma progesterone concentrations (2–4 ng/ml) equivalent to those of the late luteal phase (18). After 16 d (adequate time for corpus luteum regression), the CIDRs were removed, leading to a rapid decrease in plasma progesterone concentrations and synchronization of follicular-phase endocrine events: increased pulsatile LH secretion, estradiol rise, and LH/FSH surges (18).
Cortisol
One day before CIDR removal, ewes were penned in groups and equipped with two indwelling jugular catheters, one for collecting blood and one for infusing cortisol. Cortisol was infused via battery-operated, programable pumps (model 6 MP; Autosyringe, Hooksett, NH) secured to the back of each ewe. The pumps allowed delivery of cortisol or vehicle without restraint and with no apparent disturbance to the sheep. In experiment 1, cortisol (Solu-Cortef, hydrocortisone sodium succinate, aqueous solution, 125 mg/ml; Pharmacia & Upjohn, Kalamazoo, MI) was dissolved in saline vehicle and continuously infused iv at a dose of 0.05 mg/kg·h. This treatment elevated plasma cortisol to approximately 30 ng/ml, less than half the maximal value of 75–80 ng/ml that we observe during isolation stress (Wagenmaker, E., A. Pytiak, and F. Karsch, unpublished observations). Therefore, in experiment 2, the infusion rate was increased; two doses were administered, 0.075 and 0.15 mg/kg·h.
Experiment 1: effect of continuous infusion of cortisol on the follicular phase
This experiment tested whether continuous infusion of cortisol would suppress pulsatile LH secretion and delay or block the preovulatory LH surge. Beginning 2 h before progesterone withdrawal (i.e. CIDR removal) and continuing for 96 h, ewes received a constant infusion of either cortisol or vehicle (n = 8/group; Fig. 1A). Jugular blood was sampled at 5-min intervals from 12 to 16 h after progesterone withdrawal to assess LH pulsatility and every 1–2 h through 96 h to evaluate the LH surge, which typically occurs approximately 48 h after CIDR removal (18).
FIG. 1. Designs for experiment 1 (A) and experiment 2 (B). Time is depicted as hours relative to progesterone withdrawal (–CIDR, arrow). Dashed lines designate the expected profile of serum progesterone (Prog) concentration. Hatched horizontal bars indicate the period of vehicle or cortisol infusion. The gray boxes and solid gray lines identify the periods of sampling for hormone analysis. Solid bars indicate the expected period of LH surges in control ewes receiving vehicle.
Experiment 2: effect of different patterns and plasma concentrations of cortisol
Experiment 1 revealed that a low constant elevation of plasma cortisol suppresses LH pulse frequency and delays the LH surge. Experiment 2 expanded on these findings by determining: 1) the effect of different patterns and plasma concentrations of cortisol, 2) the effect of cortisol on pulsatile LH secretion at different times during the follicular phase, 3) the influence of cortisol on estradiol and FSH secretion, and 4) the influence of cortisol on subsequent corpus luteum function. The design is illustrated in Fig. 1B. From 2 h before progesterone withdrawal and continuing for 96 h, ewes received one of four infusion treatments (n = 6/group): 1) vehicle, 2) maximal cortisol, continuous 75–80 ng/ml increase in plasma cortisol to approximate the peak concentration we observe during isolation, 3) half-maximal cortisol, continuous 40 ng/ml increase in plasma cortisol to approximately half the peak value observed during isolation, and 4) repeated maximal cortisol, intermittent 75–80 ng/ml increases in plasma cortisol for 4 h at 12-h intervals. The repeated maximal cortisol group was intended to reflect cortisol responses to repeated exposure to an acute stressor. During isolation, enhanced cortisol secretion is evident for 4–6 h, and repeated isolation every 12 h leads to repeated increases in cortisol secretion (Karsch, F., unpublished data).
LH pulses were monitored in blood sampled at 5-min intervals during three 4-h windows of time: 12–16, 24–28, and 36–40 h after progesterone withdrawal (early-, mid-, and late-follicular phase). In the repeated maximal group, each sampling window coincided with the 4-h elevation in cortisol. Blood was also collected less frequently from 12 h before to 96 h after progesterone withdrawal for assay of LH (2-h intervals), FSH, and estradiol (4-h intervals). Thereafter blood was sampled daily for 21 d to monitor progesterone as an index of corpus luteum function. Because plasma estradiol concentrations in sheep are extremely low, samples for estradiol analysis were collected into glass tubes cleaned in an electrolytic oven in an attempt to reduce residues that may introduce a low blank activity in the estradiol assay.
Hormone assays
LH was assayed in duplicate aliquots (5–200 μl) of plasma using a modification (19) of a previously described RIA (20, 21) and is expressed in terms of National Institutes of Health-LH-S12. FSH was assayed in duplicate 100-μl aliquots of plasma (18) using reagents procured from the National Hormone and Pituitary Program and is expressed in terms of National Institute of Diabetes and Digestive and Kidney Diseases ovine FSH-1 (AFP 5679C). Estradiol was assayed in duplicate diethyl ether extracts of 200 μl plasma using a modification (22) of the diagnostics estradiol MAIA assay (Adaltis Inc., Bologna, Italy). Assay validity was reconfirmed by verifying accuracy of recovery of estradiol standard added to plasma from ovariectomized ewes (99.5 ± 3.8%); parallelism between serial dilutions of standard and ovine plasma samples; and negligible values in ovariectomized ewes (0.56 ± 0.1 pg/ml). Progesterone was assayed in duplicate 100-μl aliquots of unextracted plasma using the Coat-A-Count progesterone assay kit (Diagnostic Products Corp., Los Angeles, CA), validated for use in sheep (23). Total plasma cortisol was assayed in duplicate 50-μl aliquots of unextracted plasma using the Coat-A-Count cortisol assay kit (Diagnostic Products Corp.), validated for use in sheep (24). Assay sensitivity averaged 0.7 ng/ml (32 assays), 0.6 ng/ml (four assays), 0.34 pg/ml (six assays), 0.4 ng/ml (eight assays), and 0.8 ng/ml (12 assays) for the LH, FSH, estradiol, progesterone, and cortisol assays, respectively. Mean intra- and interassay coefficients of variation were 5.8 and 5.2% for LH, 6.5 and 9.8% for FSH, 10.0 and 8.6% for estradiol, 6.2 and 8.5% for progesterone, and 4.2 and 6.5% for cortisol, respectively.
Data analysis
LH pulses were identified using the Cluster pulse-detection algorithm (25). As in previous studies (24, 26), cluster sizes for both peaks and nadirs were set at two samples, and the t statistic used to identify a significant (P < 0.05) increase or decrease was 2.6. LH pulse frequency was expressed as interpulse interval and calculated as the mean interval between pulse peaks observed during the sampling window. When only one pulse occurred, the interval from the peak to the end of the sampling window was considered a conservative estimate of the pulse interval.
Estradiol concentrations were compared among groups by repeated-measures ANOVA. Estradiol rises were considered to have occurred if hormone concentrations increased 2-fold above the baseline mean and remained elevated for at least 12 h. The baseline mean was defined as the average estradiol concentration from 12 to 0 h before progesterone withdrawal. LH and FSH surges were considered to have occurred if plasma concentrations increased 3-fold (LH) or 2-fold (FSH) above the baseline mean (average LH or FSH concentration from 12–20 h after progesterone withdrawal) and remained so for at least 8 h. Latent period to the estradiol and LH/FSH surge peak was defined as the time from progesterone withdrawal (CIDR removal) to the highest value.
To normalize the distribution of values before statistical analysis, plasma hormone concentrations were log transformed and the inverse of the square root transformation was applied to each interpulse interval. Differences in single values between control and experimental groups (e.g. time to LH surge peak) were determined by one-way ANOVA. Two-way repeated-measures ANOVA was used to compare hormonal profiles over time between groups. When a significant treatment x time interaction was observed in experiment 2, which comprised four groups, post hoc analysis was conducted to identify specific treatment effects. This consisted of successively excluding data from two groups and repeating the ANOVA on the remaining two. Fisher’s exact probability test was used to identify group differences in the proportion of control and experimental ewes expressing the estradiol rise, LH, or FSH surge. Level of significance was P < 0.05.
Results
Plasma progesterone and cortisol concentrations
Mean progesterone before CIDR removal (2.4 ± 0.3 and 2.6 ± 0.4 ng/ml for experiments 1 and 2, respectively) was at a late luteal phase level and did not differ among control and cortisol-infused ewes. Progesterone decreased to undetectable values within 12 h of CIDR removal and remained undetectable during the infusion. Mean plasma cortisol before CIDR removal was low; values remained so throughout vehicle infusion (8.5 ± 1.7 and 8.3 ± 1.3 ng/ml for experiments 1 and 2, respectively). Continuous cortisol infusion elevated plasma cortisol to 27.3 ± 2.5 ng/ml in experiment 1 and either 40.8 ± 2.9 or 77.4 ± 1.5 ng/ml in experiment 2 (half-maximal and maximal groups, respectively; Fig. 2). These cortisol concentrations represent approximately one third, one half, and the maximal values we observe during isolation stress (see Fig. 2, solid vertical bar). In the repeated maximal group, plasma cortisol was elevated to 79.7 ± 2.4 ng/ml during every 4-h infusion period (data not illustrated).
FIG. 2. Mean ± SEM plasma cortisol concentration in ewes treated in experiment 1 with cortisol (Exp 1 cortisol, closed circles) and vehicle (open circles), maximal cortisol (closed circles), or half-maximal cortisol (gray circles) in experiment 2. Treatments were administered by iv infusion (hatched horizontal bar). The thick solid vertical bar indicates maximal plasma cortisol concentrations (mean ± SEM) observed in this laboratory in response to isolation (Wagenmaker, E., A. Pytiak, and F. Karsch, unpublished observations).
Experiment 1: effect of continuous infusion of cortisol on the follicular phase
All control ewes exhibited high-frequency LH pulses from 12 to 16 h after progesterone withdrawal with a mean interpulse interval of 63 ± 5 min (Fig. 3, A–D, illustrates pulse profiles in four of eight ewes). Cortisol lowered LH pulse frequency to one pulse every 93 ± 7 min (P < 0.05; Fig. 3, E–H, pulse profiles in four of eight ewes) and reduced mean LH concentrations by approximately 50% (1.6 ± 0.4 vs. 3.4 ± 0.7 ng/ml, cortisol vs. vehicle, P < 0.05). LH pulse amplitude, however, was not significantly reduced by cortisol (1.9 ± 0.5 vs. 2.9 ± 0.7 ng/ml, cortisol vs. vehicle, P > 0.05). All ewes of both groups expressed the LH surge, and there was no group difference in the magnitude of the LH surge peak (152 ± 38 vs. 183 ± 26 ng/ml, cortisol vs. vehicle, P > 0.05). However, cortisol increased the latent period from progesterone withdrawal to LH peak (70 ± 5 vs. 56 ± 4 h, cortisol vs. vehicle, P < 0.05).
FIG. 3. LH pulse patterns are shown for four ewes treated with vehicle (A–D, open circles) or cortisol (E–H, closed circles) from 12–16 h after progesterone withdrawal in experiment 1. Asterisks indicate peaks of LH pulses.
Experiment 2: effect of different patterns and plasma concentrations of cortisol
Vehicle-infused controls.
During the three windows of 5-min sampling, control ewes expressed high-frequency LH pulses typical of the follicular phase (Fig. 4). Between 12 and 40 h after progesterone withdrawal, mean frequency increased progressively from 1 pulse every 65 min to 1 pulse every 48 min, and mean LH pulse amplitude declined from 2.3 to 1.0 ng/ml (P < 0.05 in both instances; Fig. 4 and Table 1, A and B). All control ewes displayed an estradiol rise reaching a peak of 5.9 ± 0.5 pg/ml (Table 2A) and culminating in LH and FSH surges 48 ± 4 and 46 ± 3 h (LH and FSH, respectively) after progesterone withdrawal (Table 2, B and C).
FIG. 4. Plasma LH and estradiol profiles (A) and LH pulse patterns (B) in two ewes treated with vehicle in experiment 2. Follicular-phase onset was synchronized by withdrawing intravaginal progesterone devices. Horizontal open bars indicate the time of vehicle infusion. Mean ± SEM time to LH surge peak in controls is depicted by the horizontal black bar at the top. LH pulses were monitored by 5-min jugular blood sampling (B) during the early, mid-, and late follicular phase; asterisks in LH pulse windows indicate peaks of LH pulses. For clarity of presentation, plasma FSH values were omitted.
TABLE 1. Effect of cortisol treatments on LH pulse parameters in experiment 2
TABLE 2. Effect of cortisol treatments on estradiol rise and LH and FSH surge parameters in experiment 2
Maximal cortisol.
Three major disruptive effects were observed in ewes receiving maximal cortisol. First, cortisol reduced LH pulse frequency and the mean plasma LH concentration. Interpulse interval was more than twice that of controls during the last two sampling windows (Fig. 5 and Table 1A). The progressive decline in LH pulse amplitude, as seen in control ewes across the sampling windows, was not observed (Table 1B). Second, maximal cortisol interfered with the follicular phase estradiol rise. Three ewes did not exhibit the estradiol rise; values generally remained less than 2 pg/ml (Fig. 5A). The other three ewes did express sustained estradiol rises, but the peak was delayed by an average of 20 h (Fig. 5B and Table 2A, P < 0.05 vs. controls). Third, LH and FSH surges were blocked or delayed. The three ewes that failed to express estradiol rises did not display LH/FSH surges (Fig. 5A and Table 2, B and C; surge incidence P < 0.05 vs. controls). In the other three ewes, the LH/FSH surges were delayed by approximately 20 h, compared with controls (Fig. 5B and Table 2, B and C). Amplitudes of the estradiol and LH/FSH peaks, when they occurred, were not suppressed by cortisol (Table 2).
FIG. 5. Plasma LH and estradiol profiles (A) and LH pulse patterns (B) in two ewes treated with maximal cortisol in experiment 2. Black horizontal bars indicate the time of cortisol infusion. Mean ± SEM time to LH surge peak in controls is depicted by the horizontal black bar at the top. See Fig. 4 for further details.
Half-maximal cortisol.
Continuous infusion of the lower dose of cortisol had similar disruptive effects. LH pulse frequency was reduced, compared with controls; values did not differ from the maximal cortisol group (Fig. 6 and Table 1A, P < 0.05). Again, mean LH concentrations were reduced and LH pulse amplitude did not fall progressively (Table 1, B and C). Furthermore, this cortisol treatment either blocked the estradiol rise and LH/FSH surge (one ewe; Fig. 6A) or delayed these events relative to the controls (five ewes; Fig. 6B and Table 2). Again, the estradiol and LH/FSH peaks, when they occurred, were not reduced, compared with controls (Table 2).
FIG. 6. Plasma LH and estradiol profiles (A) and LH pulse patterns (B) in two ewes treated with half-maximal cortisol in experiment 2. Gray horizontal bars indicate the time of cortisol infusion. See Fig. 4 for further details.
Repeated maximal cortisol.
Disruptive effects of the intermittent cortisol treatment were less severe than those caused by continuous infusion. This treatment reduced LH pulse frequency and mean LH concentrations relative to values in controls (Fig. 7 and Table 1, A and C). Although the suppression in frequency across time did not differ significantly among the three cortisol treatments, the mean interval between pulses in the intermittent cortisol group was 15–30 min less than that in the maximal cortisol group during the last two sampling windows. In contrast to the other two cortisol groups, LH pulse amplitude did decrease with time (P < 0.05) and did not differ from values in control ewes. Sustained estradiol rises and LH/FSH surges were expressed by all ewes receiving intermittent cortisol, and neither timing nor amplitude of the peak hormone values differed significantly with respect to values in the controls (Fig. 7 and Table 2). Although not significantly delayed, peak values for all three hormones occurred an average of 6–9 h later than in the control ewes.
FIG. 7. Plasma LH and estradiol profiles (A) and LH pulse patterns (B) in two ewes treated with repeated maximal cortisol in experiment 2. Black horizontal boxes indicate the time of repeated cortisol infusions. See Fig. 4 for further details.
Plasma FSH concentrations.
FSH was assayed at 30-min intervals during the three windows of frequent sampling to assess FSH secretion before the surge. Mean values did not differ among control and cortisol-infused ewes (Table 3).
TABLE 3. Effect of cortisol treatments on mean FSH (ng/ml) concentrations before the surge in experiment 2
Effects on the subsequent luteal phase.
After treatment, all control and cortisol-infused ewes expressed a rise in progesterone indicative of a luteal phase. Onset of this luteal phase (day progesterone exceeded 0.5 ng/ml) was delayed by 1 d in the maximal cortisol group (control vs. maximal cortisol: 5.3 ± 0.2 vs. 6.2 ± 0.4 d after CIDR removal; P < 0.05). Neither the duration of the luteal phase (days progesterone > 0.5 ng/ml) nor the integrated amount of progesterone secreted during the luteal phase (sum of all values) differed among control and cortisol-infused ewes (data not shown).
Discussion
In sheep, as in other species, increased pulsatile LH secretion is an essential stimulus for generating the follicular phase rise in estradiol secretion and subsequent expression of the LH and FSH surges (17, 27, 28). The present study provided an initial test of the hypothesis that cortisol interferes with expression of the preovulatory sequence of endocrine events by suppressing pulsatile LH secretion. Our results support this hypothesis by demonstrating that elevated plasma cortisol concentrations reduce LH pulse frequency in follicular-phase ewes and delay or block the resultant estradiol rise and gonadotropin surge. Although the estradiol rise and LH surge were sometimes not observed during cortisol infusion, it is assumed they occurred soon after the infusion ended because these ewes subsequently expressed plasma progesterone profiles indicative of functional corpora lutea. Overall, our findings reinforce the recent report that a stress-like increment in plasma cortisol disrupts the preovulatory estradiol rise and LH surge (15). Beyond this, the present study provides novel insight into the underlying mechanism by linking this effect to suppression of pulsatile LH secretion and, in particular, LH pulse frequency.
Although consistent with the hypothesis that reduced pulsatile LH secretion underlies the effect of cortisol on follicular phase events, our study does not exclude the possibility that other actions of cortisol also interfere with timely expression of the estradiol rise and LH surge. For example, cortisol might suppress estradiol synthesis by reducing ovarian responsiveness to gonadotropic hormones. At the level of the ovary, LH enhances estradiol secretion by stimulating thecal cell biosynthesis of androgens, which are then converted to estradiol by granulosa cells under the influence of FSH (29). Glucocorticoid receptors have been identified in granulosa cells of the rat (30), and glucocorticoids reduce responsiveness of human and rat granulosa cells to gonadotropic hormones (31, 32). Either impaired ovarian responsiveness to FSH or reduced FSH secretion would attenuate LH-stimulated estradiol synthesis and compromise the preovulatory estradiol rise. Although cortisol did not suppress basal FSH secretion before the surge, our study did not assess follicular responsiveness to FSH. Further work is warranted to determine whether the disruptive influence of cortisol on the follicular phase is expressed at the ovarian as well as the neuroendocrine level.
Another way that cortisol could interfere with the follicular phase is by suppressing those processes required for induction of the LH surge. Specifically, cortisol could either prevent generation of the positive feedback signal (i.e. enhanced estradiol secretion) or reduce neuroendocrine responsiveness to that signal. The absence or delay of the estradiol rise associated with lowered LH pulse frequency in most of our ewes is clearly consistent with the former possibility. However, two lines of evidence suggest cortisol also interferes with responsiveness to the positive feedback signal. First, the LH surge was delayed by approximately 24 h in some of the cortisol-treated ewes that expressed preovulatory-like estradiol rises at the usual time (e.g. Figs. 5B and 7B). Second, other studies provide direct evidence that a stress-like increment in plasma cortisol delays the LH surge induced by a fixed exogenous estradiol stimulus in an artificial follicular-phase model (33). Thus, cortisol likely interferes with timely expression of the LH surge by inhibiting both generation of, and responsiveness to, the positive feedback signal.
Two unexpected observations from the present study are noteworthy. The first pertains to the apparent difference in LH suppression in follicular phase and ovariectomized ewes and the possible dependence on ovarian steroid milieu. The major effect of cortisol on LH pulses in follicular phase ewes was a reduction of frequency. This implies primarily a hypothalamic effect and suppression of GnRH pulse frequency, a possibility we are presently testing. This contrasts with the effect we observed in ovariectomized ewes not replaced with gonadal steroids, in which cortisol acts acutely to reduce LH pulse amplitude by suppressing pituitary responsiveness to GnRH rather than by inhibiting pulsatile GnRH secretion (16). We cannot discount a pituitary effect of cortisol in the present study because responsiveness to GnRH is regulated by an interaction of multiple factors including the plasma estradiol level and GnRH pulse frequency, both of which appear to be influenced by cortisol in follicular phase ewes. Nevertheless, the more marked suppression of LH pulse frequency in follicular-phase ewes, compared with ovariectomized ewes, is highly interesting because it suggests ovarian steroids enhance the interaction of cortisol with central mechanisms controlling pulsatile GnRH secretion. Although this remains to be tested formally, two recent findings are consistent with this possibility: the type II glucocorticoid receptor is coexpressed with estradiol receptor- and the progesterone receptor in hypothalamic neurons (34), and gonadal steroids profoundly influence stress-induced suppression of pulsatile LH secretion (35).
The second unexpected observation was an apparent uncoupling of the suppressive effects of cortisol on LH pulses and the estradiol rise/LH surge in ewes receiving intermittent cortisol infusions. Specifically, cortisol suppressed LH pulse frequency but did not significantly affect either the preovulatory estradiol rise or LH surge. It should be noted, however, that LH pulses were monitored only during the intermittent 4-h cortisol infusions; LH secretion may have recovered during the intervening 8 h when cortisol was not infused. Of interest, intermittent cortisol tended to increase the latent period to the peaks of the preovulatory estradiol rise and gonadotropin surges, although this trend was not significant. Furthermore, the suppressive effect of intermittent cortisol on LH pulses also exhibited a trend to be less than that of continuous cortisol infusion. Thus, we suspect that LH pulses were not as severely suppressed, nor were the estradiol rise and gonadotropin surges as severely compromised, by the intermittent compared with the continuous cortisol infusion.
A final point pertains to the physiologic and pathophysiologic relevance of the various cortisol treatments employed. With regard to the continuous 96-h infusion, several clinical disorders as well as intense exercise are associated with prolonged elevations in glucocorticoids (36, 37). For example, in Cushing’s syndrome, plasma cortisol is chronically elevated and plasma gonadotropin concentrations and pituitary responsiveness to GnRH are reduced (38, 39). Furthermore, hypercortisolemia and reduced LH pulsatility are evident in patients suffering from major depression, anorexia nervosa, and hypothalamic amenorrhea (40, 41, 42, 43, 44, 45). Although peak glucocorticoid responses are not sustained during chronic stress due to habituation and negative feedback within the HPA axis, it is noteworthy that suppression of LH pulses was achieved with sustained plasma cortisol level far less than the maximal values we observe during psychosocial stress (experiment 1). With regard to the repeated cortisol treatment, this paradigm simulated cortisol elevations during repeated stress (46, 47) (Wagenmaker, E., and F. Karsch, unpublished observations) and may be likened to the diurnal hypercortisolemia observed in women with functional hypothalamic amenorrhea (43, 44). Thus, the cortisol treatments in the present study are likely to be relevant for understanding reproductive dysfunction associated with certain stressors and various clinical disorders.
Collectively, the present observations support the hypothesis that stress-like increments in plasma cortisol interfere with the follicular phase by suppressing the development of high frequency LH pulses, which compromises timely expression of the preovulatory estradiol rise and LH and FSH surges. Furthermore, our finding that cortisol reduces LH pulse frequency provides indirect evidence that cortisol acts centrally to suppress GnRH pulsatile secretion in follicular phase ewes. Although these observations support an important role of glucocorticoids in the suppression of reproductive function in response to stress, they do not discount a mediatory role of other hormones activated in response to stress (e.g. catecholamines CRH, ACTH). Finally, it is important to emphasize that successful reproduction requires follicular maturation and estradiol biosynthesis, induction of the LH surge, ovulation, and expression of sexual behavior to be coordinated within a tight time frame. The present finding that physiologically relevant elevations in plasma cortisol disrupt timely expression of preovulatory endocrine events warrants further work to assess how cortisol interferes with these events, the impact of these disturbances on fertility, and the importance of heightened cortisol secretion to reproductive dysfunction in response to certain stressors and pathophysiological states.
Acknowledgments
The authors sincerely thank Doug Doop and Gary McCalla for their expertise in animal care. We also thank Emily Adams, Kari Dugger, and Lisa Modrick for their technical assistance in collecting blood samples and conducting hormone assays; Amy Oakley and Andrew Pytiak for their contribution to the interpretation of the results; and Dr. Morton Brown for his help with data analysis. Finally, we are grateful to Drs. Gordon D. Niswender, Vasantha Padmanabhan, and Leo E. Reichert, Jr. for supplying RIA reagents.
References
Rasmussen DD, Malven PV 1983 Effects of confinement stress on episodic secretion of LH in ovariectomized sheep. Neuroendocrinology 36:392–396
Matteri RL, Watson JG, Moberg GP 1984 Stress or acute adrenocorticotrophin treatment suppresses LHRH-induced LH release in the ram. J Reprod Fertil 72:385–393
Norman RL, Smith CJ 1992 Restraint inhibits luteinizing hormone and testosterone secretion in intact male rhesus macaques: effects of concurrent naloxone administration. Neuroendocrinology 55:405–415
Norman RL, McGlone J, Smith CJ 1994 Restraint inhibits luteinizing hormone secretion in the follicular phase of the menstrual cycle in rhesus macaques. Biol Reprod 50:16–26
Akema T, Chiba A, Shinozaki R, Oshida M, Kimura F, Toyoda J 1996 Acute immobilization stress and intraventricular injection of CRF suppress naloxone-induced LH release in ovariectomized estrogen-primed rats. J Neuroendocrinol 8:647–652
Tilbrook AJ, Canny BJ, Serapiglia MD, Ambrose TJ, Clarke IJ 1999 Suppression of the secretion of luteinizing hormone due to isolation/restraint stress in gonadectomized ewes and rams is influenced by sex steroids. J Endocrinol 160:469–481
Nanda AS, Dobson H, Ward WR 1990 Relationship between an increase in plasma cortisol during transport-induced stress and failure of oestradiol to induce a luteinising hormone surge in dairy cows. Res Vet Sci 49:25–28
Dobson H, Trebble JE, Phogat JB, Smith RF 1999 Effect of transport on pulsatile and surge secretion of LH in ewes in the breeding season. J Reprod Fertil 116:1–8
Battaglia DF, Krasa HB, Padmanabhan V, Viguié C, Karsch FJ 2000 Endocrine alterations that underlie endotoxin-induced disruption of the follicular phase in ewes. Biol Reprod 62:45–53
Peter AT, Bosu WT, DeDecker RJ 1989 Suppression of preovulatory luteinizing hormone surges in heifers after intrauterine infusions of Escherichia coli endotoxin. Am J Vet Res 50:368–373
Xiao E, Xia-Zhang L, Barth A, Zhu J, Ferin M 1998 Stress and the menstrual cycle: relevance of quality in the short- and long-term response to a 5-day endotoxin challenge during the follicular phase in the rhesus monkey. J Clin Endocrinol Metab 83:2454–2460
Sapolsky RM, Romero M, Munck AU 2000 How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55–89
Carrasco GA, Van de Kar LD 2003 Neuroendocrine pharmacology of stress. Eur J Pharmacol 463:235–272
Minton JE 1994 Function of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system in models of acute stress in domestic farm animals. J Anim Sci 72:1891–1898
Macfarlane MS, Breen KM, Sakurai H, Adams BM, Adams TE 2000 Effect of duration of infusion of stress-like concentrations of cortisol on follicular development and the preovulatory surge of LH in sheep. Anim Reprod Sci 63:167–175
Breen KM, Karsch FJ 2004 Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level? Endocrinology 145:692–698
Goodman RL 1994 Neuroendocrine control of the ovine estrous cycle. In: Knobil E, Neill JD, eds. The physiology of reproduction. Vol 2. New York: Raven Press; 659–709
Van Cleeff J, Karsch FJ, Padmanabhan V 1998 Characterization of endocrine events during the periestrous period in sheep after estrous synchronization with controlled internal drug release (CIDR) device. Domest Anim Endocrinol 15:23–34
Hauger RL, Karsch FJ, Foster DL 1977 A new concept for control of the estrous cycle of the ewe based on the temporal relationships between luteinizing hormone, estradiol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology 101:807–817
Niswender GD, Midgley Jr AR, Reichert Jr LE 1968 Radioimmunologic studies with murine, ovine and porcine luteinizing hormone. In: Rosenborg E, ed. Gonadotropins. Los Altos, CA: GERON-X; 299–306
Niswender GD, Reichert Jr LE, Midgley Jr AR, Nalbandov AV 1969 Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166–1173
Evans NP, Dahl GE, Glover BH, Karsch FJ 1994 Central regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by estradiol during the period leading up to the preovulatory GnRH surge in the ewe. Endocrinology 134:1806–1811
Padmanabhan V, Evans NP, Dahl GE, McFadden KL, Mauger DT, Karsch FJ 1995 Evidence for short or ultrashort loop negative feedback of gonadotropin-releasing hormone secretion. Neuroendocrinology 62:248–258
Battaglia DF, Bowen JM, Krasa HB, Thrun LA, Viguié C, Karsch FJ 1997 Endotoxin inhibits the reproductive neuroendocrine axis while stimulating adrenal steroids: a simultaneous view from hypophyseal portal and peripheral blood. Endocrinology 138:4273–4281
Veldhuis JD, Johnson ML 1986 Cluster analysis: a simple, versatile and robust algorithm for endocrine pulse detection. Am J Physiol 250:E486–E493
Debus N, Breen KM, Barrell GK, Billings HJ, Brown M, Young EA, Karsch FJ 2002 Does cortisol mediate endotoxin-induced inhibition of pulsatile luteinizing hormone and gonadotropin-releasing hormone secretion? Endocrinology 143:3748–3758
Baird DT, McNeilly AS 1981 Gonadotrophic control of follicular development and function during the oestrous cycle of the ewe. J Reprod Fertil Suppl 30:119–133
Campbell BK, Baird DT, McNeilly AS, Scaramuzzi RJ 1990 Ovarian secretion rates and peripheral concentrations of inhibin in normal and androstenedione-immune ewes with an autotransplanted ovary. J Endocrinol 127:285–296
Gore-Langton RE, Armstrong DT 1994 Follicular steroidogenesis and its control. In: Knobil E, Neill JD, ed. The physiology of reproduction. Vol 1. New York: Raven Press; 571–627
Schreiber JR, Nakamura K, Erickson GF 1982 Rat ovary glucocorticoid receptor: identification and characterization. Steroids 39:569–584
Hsueh AJ, Erickson GF 1978 Glucocorticoid inhibition of FSH-induced estrogen production in cultured rat granulosa cells. Steroids 32:639–648
Michael AE, Pester LA, Curtis P, Shaw RW, Edwards CR, Cooke BA 1993 Direct inhibition of ovarian steroidogenesis by cortisol and the modulatory role of 11?-hydroxysteroid dehydrogenase. Clin Endocrinol 38:641–644
Wagenmaker ER, Breen KM, Tilbrook AJ, Turner AI, Clarke IJ, Karsch FJ 2004 Does cortisol disrupt the positive feedback effects of estradiol on the preovulatory LH surge? Biol Reprod 70(Suppl 1):130 (Abstract)
Dufourny L, Skinner DC 2002 Progesterone receptor, estrogen receptor , and the type II glucocorticoid receptor are coexpressed in the same neurons of the ovine preoptic area and arcuate nucleus: a triple immunolabeling study. Biol Reprod 67:1605–1612
Tilbrook AJ, Turner AI, Clark IJ 2000 Effects of stress on reproduction in non-rodent mammals: the role of glucocorticoids and sex differences. Rev Reprod 5:105–113
Villanueva AL, Schlosser C, Hopper B, Liu JH, Hoffman DI, Rebar RW 1986 Increased cortisol production in women runners. J Clin Endocrinol Metab 63:133–136
Loucks AB, Mortola JF, Girton L, Yen SS 1989 Alterations in the hypothalamic-pituitary-ovarian and the hypothalamic-pituitary-adrenal axes in athletic women. J Clin Endocrinol Metab 68:402–411
Boccuzzi G, Angeli A, Bisbocci D, Fonzo D, Giadano GP, Ceresa F 1975 Effect of synthetic luteinizing hormone-releasing hormone (LH-RH) on the release of gonadotropins in Cushing’s disease. J Clin Endocrinol Metab 40:892–895
Luton J-P, Thieblot P, Valcke J-C, Mahoudeau JA, Bricaire H 1977 Reversible gonadotropin deficiency in male Cushing’s disease. J Clin Endocrinol Metab 45:488–495
Halbreich U, Asnis GM, Shindledecker R, Zumoff B, Nathan RS 1985 Cortisol secretion in endogenous depression. II. Time-related functions. Arch Gen Psychiatry 42:909–914
Pfohl B, Sherman B, Schlechte J, Stone R 1985 Pituitary-adrenal axis rhythm disturbances in psychiatric depression. Arch Gen Psychiatry 42:897–903
Gold PW, Gwirtsman H, Avgerinos PC, Nieman LK, Gallucci WT, Kaye W, Jimerson D, Ebert M, Rittmaster R, Loriaux DL, Chrousos GP 1986 Abnormal hypothalamic-pituitary-adrenal function in anorexia nervosa. Pathophysiologic mechanisms in underweight and weight-corrected patients. N Engl J Med 314:1335–1342
Suh BY, Liu JH, Berga SL, Quigley ME, Laughlin GA, Yen SS 1988 Hypercortisolism in patients with functional hypothalamic amenorrhea. J Clin Endocrinol Metab 66:733–739
Berga SL, Mortola JF, Girton L, Suh B, Laughlin G, Pham P, Yen SS 1989 Neuroendocrine aberrations in women with functional hypothalamic amenorrhea. J Clin Endocrinol Metab 68:301–308
Meller WH, Grambsch PL, Bingham C, Tagatz GE 2001 Hypothalamic pituitary gonadal axis dysregulation in depressed women. Psychoneuroendocrinology 26:253–259
Smith RF, Dobson H 2002 Hormonal interactions within the hypothalamus and pituitary with respect to stress and reproduction in sheep. Domest Anim Endocrinol 23:75–85
Bhatnagar S, Vining C 2003 Facilitation of hypothalamic-pituitary-adrenal responses to novel stress after repeated social stress using the resident/intruder paradigm. Horm Behav 43:158–165(Kellie M. Breen, Heather )
Address all correspondence and requests for reprints to: Kellie M. Breen, Reproductive Sciences Program, University of Michigan, 300 North Ingalls Building, Room 1101 SW, Ann Arbor, Michigan 48109-0404. E-mail: breenk@umich.edu.
Abstract
Stress activates the hypothalamo-pituitary-adrenal axis leading to enhanced glucocorticoid secretion and concurrently inhibits gonadotropin secretion and disrupts ovarian cyclicity. Here we tested the hypothesis that stress-like concentrations of cortisol interfere with follicular phase endocrine events of the ewe by suppressing pulsatile LH secretion, which is essential for subsequent steps in the preovulatory sequence. Cortisol was infused during the early to midfollicular phase, elevating plasma cortisol concentrations to one third, one half, or the maximal value induced by isolation, a commonly used model of psychosocial stress. All cortisol treatments compromised at least some aspect of reproductive hormone secretion in follicular phase ewes. First, cortisol significantly suppressed LH pulse frequency by as much as 35%, thus attenuating the high frequency LH pulses typical of the preovulatory period. Second, cortisol interfered with timely generation of the follicular phase estradiol rise, either preventing it or delaying the estradiol peak by as much as 20 h. Third, cortisol delayed or blocked the preovulatory LH and FSH surges. Collectively, our findings support the hypothesis that stress-like increments in plasma cortisol interfere with the follicular phase by suppressing the development of high frequency LH pulses, which compromises timely expression of the preovulatory estradiol rise and LH and FSH surges. Moreover, the suppression of LH pulse frequency provides indirect evidence that cortisol acts centrally to suppress pulsatile GnRH secretion in follicular-phase ewes.
Introduction
A VARIETY OF stressors inhibit gonadotropin secretion and disrupt ovarian cyclicity. For example, acute restraint and/or isolation reduce(s) mean plasma LH concentrations and pulsatile LH secretion in the rat, sheep, and monkey (1, 2, 3, 4, 5, 6). Transportation inhibits pulsatile LH secretion and delays and attenuates the LH surge in sheep and cows (7, 8). Furthermore, immune/inflammatory stress suppresses pulsatile LH secretion during the preovulatory period in sheep (9) and delays or blocks the LH surge in follicular-phase ewes, cows, and monkeys (9, 10, 11). Despite evidence that stress interferes with gonadotropin secretion and ovarian cyclicity, the underlying mechanisms are not well understood. In this regard, adaptations to stress comprise an initial autonomic activation and release of catecholamines from the sympathetic nervous system, followed by a more delayed and prolonged activation of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced secretion of CRH, arginine vasopressin, ACTH, and the glucocorticoids (12, 13, 14). Each of these factors potentially mediates reproductive suppression. In actuality, the integrated actions of all of them are likely involved, with the relative importance of each depending on the nature of the stressor (12).
Our laboratory has become interested in the coincident, stress-induced increase in glucocorticoid secretion and suppression of reproductive neuroendocrine function and ovarian cyclicity in sheep. In this regard, a recent study in sheep revealed that a continuous elevation of plasma cortisol to levels observed during isolation stress (75 ng/ml) suppressed the preovulatory estradiol rise and prevented the LH surge in nonstressed, follicular-phase ewes (15). That study, however, did not address how cortisol elicits these effects. Our recent findings that cortisol acutely inhibits pulsatile LH secretion in ovariectomized ewes (16) provides a plausible hypothesis that was tested in the present study: cortisol disrupts the follicular phase by suppressing pulsatile LH secretion, which is an essential stimulus for the preovulatory estradiol rise that induces the LH surge.
Materials and Methods
Animal model
Two experiments were conducted during successive breeding seasons (experiment 1, November to December 2001; experiment 2, November to December 2002) on adult, ovary-intact Suffolk ewes maintained under standard husbandry conditions at the Sheep Research Facility (near Ann Arbor, MI; 42 degrees 18' N). Before the experiment, the ewes were moved from their natural environment to an enclosed, unheated barn in which photoperiod was regulated to simulate that of the outdoors. All procedures were approved by the Committee for the Use and Care of Animals at the University of Michigan.
The estrous cycle of sheep typically lasts 16–17 d and consists of a luteal phase of approximately 13 d and a follicular phase of approximately 3 d (17). To enable simultaneous study of multiple ewes, the follicular phase was synchronized among randomly cycling ewes using intravaginal progesterone-releasing devices [controlled internal drug release (CIDR); InterAg, Hamilton, New Zealand]. These devices maintain plasma progesterone concentrations (2–4 ng/ml) equivalent to those of the late luteal phase (18). After 16 d (adequate time for corpus luteum regression), the CIDRs were removed, leading to a rapid decrease in plasma progesterone concentrations and synchronization of follicular-phase endocrine events: increased pulsatile LH secretion, estradiol rise, and LH/FSH surges (18).
Cortisol
One day before CIDR removal, ewes were penned in groups and equipped with two indwelling jugular catheters, one for collecting blood and one for infusing cortisol. Cortisol was infused via battery-operated, programable pumps (model 6 MP; Autosyringe, Hooksett, NH) secured to the back of each ewe. The pumps allowed delivery of cortisol or vehicle without restraint and with no apparent disturbance to the sheep. In experiment 1, cortisol (Solu-Cortef, hydrocortisone sodium succinate, aqueous solution, 125 mg/ml; Pharmacia & Upjohn, Kalamazoo, MI) was dissolved in saline vehicle and continuously infused iv at a dose of 0.05 mg/kg·h. This treatment elevated plasma cortisol to approximately 30 ng/ml, less than half the maximal value of 75–80 ng/ml that we observe during isolation stress (Wagenmaker, E., A. Pytiak, and F. Karsch, unpublished observations). Therefore, in experiment 2, the infusion rate was increased; two doses were administered, 0.075 and 0.15 mg/kg·h.
Experiment 1: effect of continuous infusion of cortisol on the follicular phase
This experiment tested whether continuous infusion of cortisol would suppress pulsatile LH secretion and delay or block the preovulatory LH surge. Beginning 2 h before progesterone withdrawal (i.e. CIDR removal) and continuing for 96 h, ewes received a constant infusion of either cortisol or vehicle (n = 8/group; Fig. 1A). Jugular blood was sampled at 5-min intervals from 12 to 16 h after progesterone withdrawal to assess LH pulsatility and every 1–2 h through 96 h to evaluate the LH surge, which typically occurs approximately 48 h after CIDR removal (18).
FIG. 1. Designs for experiment 1 (A) and experiment 2 (B). Time is depicted as hours relative to progesterone withdrawal (–CIDR, arrow). Dashed lines designate the expected profile of serum progesterone (Prog) concentration. Hatched horizontal bars indicate the period of vehicle or cortisol infusion. The gray boxes and solid gray lines identify the periods of sampling for hormone analysis. Solid bars indicate the expected period of LH surges in control ewes receiving vehicle.
Experiment 2: effect of different patterns and plasma concentrations of cortisol
Experiment 1 revealed that a low constant elevation of plasma cortisol suppresses LH pulse frequency and delays the LH surge. Experiment 2 expanded on these findings by determining: 1) the effect of different patterns and plasma concentrations of cortisol, 2) the effect of cortisol on pulsatile LH secretion at different times during the follicular phase, 3) the influence of cortisol on estradiol and FSH secretion, and 4) the influence of cortisol on subsequent corpus luteum function. The design is illustrated in Fig. 1B. From 2 h before progesterone withdrawal and continuing for 96 h, ewes received one of four infusion treatments (n = 6/group): 1) vehicle, 2) maximal cortisol, continuous 75–80 ng/ml increase in plasma cortisol to approximate the peak concentration we observe during isolation, 3) half-maximal cortisol, continuous 40 ng/ml increase in plasma cortisol to approximately half the peak value observed during isolation, and 4) repeated maximal cortisol, intermittent 75–80 ng/ml increases in plasma cortisol for 4 h at 12-h intervals. The repeated maximal cortisol group was intended to reflect cortisol responses to repeated exposure to an acute stressor. During isolation, enhanced cortisol secretion is evident for 4–6 h, and repeated isolation every 12 h leads to repeated increases in cortisol secretion (Karsch, F., unpublished data).
LH pulses were monitored in blood sampled at 5-min intervals during three 4-h windows of time: 12–16, 24–28, and 36–40 h after progesterone withdrawal (early-, mid-, and late-follicular phase). In the repeated maximal group, each sampling window coincided with the 4-h elevation in cortisol. Blood was also collected less frequently from 12 h before to 96 h after progesterone withdrawal for assay of LH (2-h intervals), FSH, and estradiol (4-h intervals). Thereafter blood was sampled daily for 21 d to monitor progesterone as an index of corpus luteum function. Because plasma estradiol concentrations in sheep are extremely low, samples for estradiol analysis were collected into glass tubes cleaned in an electrolytic oven in an attempt to reduce residues that may introduce a low blank activity in the estradiol assay.
Hormone assays
LH was assayed in duplicate aliquots (5–200 μl) of plasma using a modification (19) of a previously described RIA (20, 21) and is expressed in terms of National Institutes of Health-LH-S12. FSH was assayed in duplicate 100-μl aliquots of plasma (18) using reagents procured from the National Hormone and Pituitary Program and is expressed in terms of National Institute of Diabetes and Digestive and Kidney Diseases ovine FSH-1 (AFP 5679C). Estradiol was assayed in duplicate diethyl ether extracts of 200 μl plasma using a modification (22) of the diagnostics estradiol MAIA assay (Adaltis Inc., Bologna, Italy). Assay validity was reconfirmed by verifying accuracy of recovery of estradiol standard added to plasma from ovariectomized ewes (99.5 ± 3.8%); parallelism between serial dilutions of standard and ovine plasma samples; and negligible values in ovariectomized ewes (0.56 ± 0.1 pg/ml). Progesterone was assayed in duplicate 100-μl aliquots of unextracted plasma using the Coat-A-Count progesterone assay kit (Diagnostic Products Corp., Los Angeles, CA), validated for use in sheep (23). Total plasma cortisol was assayed in duplicate 50-μl aliquots of unextracted plasma using the Coat-A-Count cortisol assay kit (Diagnostic Products Corp.), validated for use in sheep (24). Assay sensitivity averaged 0.7 ng/ml (32 assays), 0.6 ng/ml (four assays), 0.34 pg/ml (six assays), 0.4 ng/ml (eight assays), and 0.8 ng/ml (12 assays) for the LH, FSH, estradiol, progesterone, and cortisol assays, respectively. Mean intra- and interassay coefficients of variation were 5.8 and 5.2% for LH, 6.5 and 9.8% for FSH, 10.0 and 8.6% for estradiol, 6.2 and 8.5% for progesterone, and 4.2 and 6.5% for cortisol, respectively.
Data analysis
LH pulses were identified using the Cluster pulse-detection algorithm (25). As in previous studies (24, 26), cluster sizes for both peaks and nadirs were set at two samples, and the t statistic used to identify a significant (P < 0.05) increase or decrease was 2.6. LH pulse frequency was expressed as interpulse interval and calculated as the mean interval between pulse peaks observed during the sampling window. When only one pulse occurred, the interval from the peak to the end of the sampling window was considered a conservative estimate of the pulse interval.
Estradiol concentrations were compared among groups by repeated-measures ANOVA. Estradiol rises were considered to have occurred if hormone concentrations increased 2-fold above the baseline mean and remained elevated for at least 12 h. The baseline mean was defined as the average estradiol concentration from 12 to 0 h before progesterone withdrawal. LH and FSH surges were considered to have occurred if plasma concentrations increased 3-fold (LH) or 2-fold (FSH) above the baseline mean (average LH or FSH concentration from 12–20 h after progesterone withdrawal) and remained so for at least 8 h. Latent period to the estradiol and LH/FSH surge peak was defined as the time from progesterone withdrawal (CIDR removal) to the highest value.
To normalize the distribution of values before statistical analysis, plasma hormone concentrations were log transformed and the inverse of the square root transformation was applied to each interpulse interval. Differences in single values between control and experimental groups (e.g. time to LH surge peak) were determined by one-way ANOVA. Two-way repeated-measures ANOVA was used to compare hormonal profiles over time between groups. When a significant treatment x time interaction was observed in experiment 2, which comprised four groups, post hoc analysis was conducted to identify specific treatment effects. This consisted of successively excluding data from two groups and repeating the ANOVA on the remaining two. Fisher’s exact probability test was used to identify group differences in the proportion of control and experimental ewes expressing the estradiol rise, LH, or FSH surge. Level of significance was P < 0.05.
Results
Plasma progesterone and cortisol concentrations
Mean progesterone before CIDR removal (2.4 ± 0.3 and 2.6 ± 0.4 ng/ml for experiments 1 and 2, respectively) was at a late luteal phase level and did not differ among control and cortisol-infused ewes. Progesterone decreased to undetectable values within 12 h of CIDR removal and remained undetectable during the infusion. Mean plasma cortisol before CIDR removal was low; values remained so throughout vehicle infusion (8.5 ± 1.7 and 8.3 ± 1.3 ng/ml for experiments 1 and 2, respectively). Continuous cortisol infusion elevated plasma cortisol to 27.3 ± 2.5 ng/ml in experiment 1 and either 40.8 ± 2.9 or 77.4 ± 1.5 ng/ml in experiment 2 (half-maximal and maximal groups, respectively; Fig. 2). These cortisol concentrations represent approximately one third, one half, and the maximal values we observe during isolation stress (see Fig. 2, solid vertical bar). In the repeated maximal group, plasma cortisol was elevated to 79.7 ± 2.4 ng/ml during every 4-h infusion period (data not illustrated).
FIG. 2. Mean ± SEM plasma cortisol concentration in ewes treated in experiment 1 with cortisol (Exp 1 cortisol, closed circles) and vehicle (open circles), maximal cortisol (closed circles), or half-maximal cortisol (gray circles) in experiment 2. Treatments were administered by iv infusion (hatched horizontal bar). The thick solid vertical bar indicates maximal plasma cortisol concentrations (mean ± SEM) observed in this laboratory in response to isolation (Wagenmaker, E., A. Pytiak, and F. Karsch, unpublished observations).
Experiment 1: effect of continuous infusion of cortisol on the follicular phase
All control ewes exhibited high-frequency LH pulses from 12 to 16 h after progesterone withdrawal with a mean interpulse interval of 63 ± 5 min (Fig. 3, A–D, illustrates pulse profiles in four of eight ewes). Cortisol lowered LH pulse frequency to one pulse every 93 ± 7 min (P < 0.05; Fig. 3, E–H, pulse profiles in four of eight ewes) and reduced mean LH concentrations by approximately 50% (1.6 ± 0.4 vs. 3.4 ± 0.7 ng/ml, cortisol vs. vehicle, P < 0.05). LH pulse amplitude, however, was not significantly reduced by cortisol (1.9 ± 0.5 vs. 2.9 ± 0.7 ng/ml, cortisol vs. vehicle, P > 0.05). All ewes of both groups expressed the LH surge, and there was no group difference in the magnitude of the LH surge peak (152 ± 38 vs. 183 ± 26 ng/ml, cortisol vs. vehicle, P > 0.05). However, cortisol increased the latent period from progesterone withdrawal to LH peak (70 ± 5 vs. 56 ± 4 h, cortisol vs. vehicle, P < 0.05).
FIG. 3. LH pulse patterns are shown for four ewes treated with vehicle (A–D, open circles) or cortisol (E–H, closed circles) from 12–16 h after progesterone withdrawal in experiment 1. Asterisks indicate peaks of LH pulses.
Experiment 2: effect of different patterns and plasma concentrations of cortisol
Vehicle-infused controls.
During the three windows of 5-min sampling, control ewes expressed high-frequency LH pulses typical of the follicular phase (Fig. 4). Between 12 and 40 h after progesterone withdrawal, mean frequency increased progressively from 1 pulse every 65 min to 1 pulse every 48 min, and mean LH pulse amplitude declined from 2.3 to 1.0 ng/ml (P < 0.05 in both instances; Fig. 4 and Table 1, A and B). All control ewes displayed an estradiol rise reaching a peak of 5.9 ± 0.5 pg/ml (Table 2A) and culminating in LH and FSH surges 48 ± 4 and 46 ± 3 h (LH and FSH, respectively) after progesterone withdrawal (Table 2, B and C).
FIG. 4. Plasma LH and estradiol profiles (A) and LH pulse patterns (B) in two ewes treated with vehicle in experiment 2. Follicular-phase onset was synchronized by withdrawing intravaginal progesterone devices. Horizontal open bars indicate the time of vehicle infusion. Mean ± SEM time to LH surge peak in controls is depicted by the horizontal black bar at the top. LH pulses were monitored by 5-min jugular blood sampling (B) during the early, mid-, and late follicular phase; asterisks in LH pulse windows indicate peaks of LH pulses. For clarity of presentation, plasma FSH values were omitted.
TABLE 1. Effect of cortisol treatments on LH pulse parameters in experiment 2
TABLE 2. Effect of cortisol treatments on estradiol rise and LH and FSH surge parameters in experiment 2
Maximal cortisol.
Three major disruptive effects were observed in ewes receiving maximal cortisol. First, cortisol reduced LH pulse frequency and the mean plasma LH concentration. Interpulse interval was more than twice that of controls during the last two sampling windows (Fig. 5 and Table 1A). The progressive decline in LH pulse amplitude, as seen in control ewes across the sampling windows, was not observed (Table 1B). Second, maximal cortisol interfered with the follicular phase estradiol rise. Three ewes did not exhibit the estradiol rise; values generally remained less than 2 pg/ml (Fig. 5A). The other three ewes did express sustained estradiol rises, but the peak was delayed by an average of 20 h (Fig. 5B and Table 2A, P < 0.05 vs. controls). Third, LH and FSH surges were blocked or delayed. The three ewes that failed to express estradiol rises did not display LH/FSH surges (Fig. 5A and Table 2, B and C; surge incidence P < 0.05 vs. controls). In the other three ewes, the LH/FSH surges were delayed by approximately 20 h, compared with controls (Fig. 5B and Table 2, B and C). Amplitudes of the estradiol and LH/FSH peaks, when they occurred, were not suppressed by cortisol (Table 2).
FIG. 5. Plasma LH and estradiol profiles (A) and LH pulse patterns (B) in two ewes treated with maximal cortisol in experiment 2. Black horizontal bars indicate the time of cortisol infusion. Mean ± SEM time to LH surge peak in controls is depicted by the horizontal black bar at the top. See Fig. 4 for further details.
Half-maximal cortisol.
Continuous infusion of the lower dose of cortisol had similar disruptive effects. LH pulse frequency was reduced, compared with controls; values did not differ from the maximal cortisol group (Fig. 6 and Table 1A, P < 0.05). Again, mean LH concentrations were reduced and LH pulse amplitude did not fall progressively (Table 1, B and C). Furthermore, this cortisol treatment either blocked the estradiol rise and LH/FSH surge (one ewe; Fig. 6A) or delayed these events relative to the controls (five ewes; Fig. 6B and Table 2). Again, the estradiol and LH/FSH peaks, when they occurred, were not reduced, compared with controls (Table 2).
FIG. 6. Plasma LH and estradiol profiles (A) and LH pulse patterns (B) in two ewes treated with half-maximal cortisol in experiment 2. Gray horizontal bars indicate the time of cortisol infusion. See Fig. 4 for further details.
Repeated maximal cortisol.
Disruptive effects of the intermittent cortisol treatment were less severe than those caused by continuous infusion. This treatment reduced LH pulse frequency and mean LH concentrations relative to values in controls (Fig. 7 and Table 1, A and C). Although the suppression in frequency across time did not differ significantly among the three cortisol treatments, the mean interval between pulses in the intermittent cortisol group was 15–30 min less than that in the maximal cortisol group during the last two sampling windows. In contrast to the other two cortisol groups, LH pulse amplitude did decrease with time (P < 0.05) and did not differ from values in control ewes. Sustained estradiol rises and LH/FSH surges were expressed by all ewes receiving intermittent cortisol, and neither timing nor amplitude of the peak hormone values differed significantly with respect to values in the controls (Fig. 7 and Table 2). Although not significantly delayed, peak values for all three hormones occurred an average of 6–9 h later than in the control ewes.
FIG. 7. Plasma LH and estradiol profiles (A) and LH pulse patterns (B) in two ewes treated with repeated maximal cortisol in experiment 2. Black horizontal boxes indicate the time of repeated cortisol infusions. See Fig. 4 for further details.
Plasma FSH concentrations.
FSH was assayed at 30-min intervals during the three windows of frequent sampling to assess FSH secretion before the surge. Mean values did not differ among control and cortisol-infused ewes (Table 3).
TABLE 3. Effect of cortisol treatments on mean FSH (ng/ml) concentrations before the surge in experiment 2
Effects on the subsequent luteal phase.
After treatment, all control and cortisol-infused ewes expressed a rise in progesterone indicative of a luteal phase. Onset of this luteal phase (day progesterone exceeded 0.5 ng/ml) was delayed by 1 d in the maximal cortisol group (control vs. maximal cortisol: 5.3 ± 0.2 vs. 6.2 ± 0.4 d after CIDR removal; P < 0.05). Neither the duration of the luteal phase (days progesterone > 0.5 ng/ml) nor the integrated amount of progesterone secreted during the luteal phase (sum of all values) differed among control and cortisol-infused ewes (data not shown).
Discussion
In sheep, as in other species, increased pulsatile LH secretion is an essential stimulus for generating the follicular phase rise in estradiol secretion and subsequent expression of the LH and FSH surges (17, 27, 28). The present study provided an initial test of the hypothesis that cortisol interferes with expression of the preovulatory sequence of endocrine events by suppressing pulsatile LH secretion. Our results support this hypothesis by demonstrating that elevated plasma cortisol concentrations reduce LH pulse frequency in follicular-phase ewes and delay or block the resultant estradiol rise and gonadotropin surge. Although the estradiol rise and LH surge were sometimes not observed during cortisol infusion, it is assumed they occurred soon after the infusion ended because these ewes subsequently expressed plasma progesterone profiles indicative of functional corpora lutea. Overall, our findings reinforce the recent report that a stress-like increment in plasma cortisol disrupts the preovulatory estradiol rise and LH surge (15). Beyond this, the present study provides novel insight into the underlying mechanism by linking this effect to suppression of pulsatile LH secretion and, in particular, LH pulse frequency.
Although consistent with the hypothesis that reduced pulsatile LH secretion underlies the effect of cortisol on follicular phase events, our study does not exclude the possibility that other actions of cortisol also interfere with timely expression of the estradiol rise and LH surge. For example, cortisol might suppress estradiol synthesis by reducing ovarian responsiveness to gonadotropic hormones. At the level of the ovary, LH enhances estradiol secretion by stimulating thecal cell biosynthesis of androgens, which are then converted to estradiol by granulosa cells under the influence of FSH (29). Glucocorticoid receptors have been identified in granulosa cells of the rat (30), and glucocorticoids reduce responsiveness of human and rat granulosa cells to gonadotropic hormones (31, 32). Either impaired ovarian responsiveness to FSH or reduced FSH secretion would attenuate LH-stimulated estradiol synthesis and compromise the preovulatory estradiol rise. Although cortisol did not suppress basal FSH secretion before the surge, our study did not assess follicular responsiveness to FSH. Further work is warranted to determine whether the disruptive influence of cortisol on the follicular phase is expressed at the ovarian as well as the neuroendocrine level.
Another way that cortisol could interfere with the follicular phase is by suppressing those processes required for induction of the LH surge. Specifically, cortisol could either prevent generation of the positive feedback signal (i.e. enhanced estradiol secretion) or reduce neuroendocrine responsiveness to that signal. The absence or delay of the estradiol rise associated with lowered LH pulse frequency in most of our ewes is clearly consistent with the former possibility. However, two lines of evidence suggest cortisol also interferes with responsiveness to the positive feedback signal. First, the LH surge was delayed by approximately 24 h in some of the cortisol-treated ewes that expressed preovulatory-like estradiol rises at the usual time (e.g. Figs. 5B and 7B). Second, other studies provide direct evidence that a stress-like increment in plasma cortisol delays the LH surge induced by a fixed exogenous estradiol stimulus in an artificial follicular-phase model (33). Thus, cortisol likely interferes with timely expression of the LH surge by inhibiting both generation of, and responsiveness to, the positive feedback signal.
Two unexpected observations from the present study are noteworthy. The first pertains to the apparent difference in LH suppression in follicular phase and ovariectomized ewes and the possible dependence on ovarian steroid milieu. The major effect of cortisol on LH pulses in follicular phase ewes was a reduction of frequency. This implies primarily a hypothalamic effect and suppression of GnRH pulse frequency, a possibility we are presently testing. This contrasts with the effect we observed in ovariectomized ewes not replaced with gonadal steroids, in which cortisol acts acutely to reduce LH pulse amplitude by suppressing pituitary responsiveness to GnRH rather than by inhibiting pulsatile GnRH secretion (16). We cannot discount a pituitary effect of cortisol in the present study because responsiveness to GnRH is regulated by an interaction of multiple factors including the plasma estradiol level and GnRH pulse frequency, both of which appear to be influenced by cortisol in follicular phase ewes. Nevertheless, the more marked suppression of LH pulse frequency in follicular-phase ewes, compared with ovariectomized ewes, is highly interesting because it suggests ovarian steroids enhance the interaction of cortisol with central mechanisms controlling pulsatile GnRH secretion. Although this remains to be tested formally, two recent findings are consistent with this possibility: the type II glucocorticoid receptor is coexpressed with estradiol receptor- and the progesterone receptor in hypothalamic neurons (34), and gonadal steroids profoundly influence stress-induced suppression of pulsatile LH secretion (35).
The second unexpected observation was an apparent uncoupling of the suppressive effects of cortisol on LH pulses and the estradiol rise/LH surge in ewes receiving intermittent cortisol infusions. Specifically, cortisol suppressed LH pulse frequency but did not significantly affect either the preovulatory estradiol rise or LH surge. It should be noted, however, that LH pulses were monitored only during the intermittent 4-h cortisol infusions; LH secretion may have recovered during the intervening 8 h when cortisol was not infused. Of interest, intermittent cortisol tended to increase the latent period to the peaks of the preovulatory estradiol rise and gonadotropin surges, although this trend was not significant. Furthermore, the suppressive effect of intermittent cortisol on LH pulses also exhibited a trend to be less than that of continuous cortisol infusion. Thus, we suspect that LH pulses were not as severely suppressed, nor were the estradiol rise and gonadotropin surges as severely compromised, by the intermittent compared with the continuous cortisol infusion.
A final point pertains to the physiologic and pathophysiologic relevance of the various cortisol treatments employed. With regard to the continuous 96-h infusion, several clinical disorders as well as intense exercise are associated with prolonged elevations in glucocorticoids (36, 37). For example, in Cushing’s syndrome, plasma cortisol is chronically elevated and plasma gonadotropin concentrations and pituitary responsiveness to GnRH are reduced (38, 39). Furthermore, hypercortisolemia and reduced LH pulsatility are evident in patients suffering from major depression, anorexia nervosa, and hypothalamic amenorrhea (40, 41, 42, 43, 44, 45). Although peak glucocorticoid responses are not sustained during chronic stress due to habituation and negative feedback within the HPA axis, it is noteworthy that suppression of LH pulses was achieved with sustained plasma cortisol level far less than the maximal values we observe during psychosocial stress (experiment 1). With regard to the repeated cortisol treatment, this paradigm simulated cortisol elevations during repeated stress (46, 47) (Wagenmaker, E., and F. Karsch, unpublished observations) and may be likened to the diurnal hypercortisolemia observed in women with functional hypothalamic amenorrhea (43, 44). Thus, the cortisol treatments in the present study are likely to be relevant for understanding reproductive dysfunction associated with certain stressors and various clinical disorders.
Collectively, the present observations support the hypothesis that stress-like increments in plasma cortisol interfere with the follicular phase by suppressing the development of high frequency LH pulses, which compromises timely expression of the preovulatory estradiol rise and LH and FSH surges. Furthermore, our finding that cortisol reduces LH pulse frequency provides indirect evidence that cortisol acts centrally to suppress GnRH pulsatile secretion in follicular phase ewes. Although these observations support an important role of glucocorticoids in the suppression of reproductive function in response to stress, they do not discount a mediatory role of other hormones activated in response to stress (e.g. catecholamines CRH, ACTH). Finally, it is important to emphasize that successful reproduction requires follicular maturation and estradiol biosynthesis, induction of the LH surge, ovulation, and expression of sexual behavior to be coordinated within a tight time frame. The present finding that physiologically relevant elevations in plasma cortisol disrupt timely expression of preovulatory endocrine events warrants further work to assess how cortisol interferes with these events, the impact of these disturbances on fertility, and the importance of heightened cortisol secretion to reproductive dysfunction in response to certain stressors and pathophysiological states.
Acknowledgments
The authors sincerely thank Doug Doop and Gary McCalla for their expertise in animal care. We also thank Emily Adams, Kari Dugger, and Lisa Modrick for their technical assistance in collecting blood samples and conducting hormone assays; Amy Oakley and Andrew Pytiak for their contribution to the interpretation of the results; and Dr. Morton Brown for his help with data analysis. Finally, we are grateful to Drs. Gordon D. Niswender, Vasantha Padmanabhan, and Leo E. Reichert, Jr. for supplying RIA reagents.
References
Rasmussen DD, Malven PV 1983 Effects of confinement stress on episodic secretion of LH in ovariectomized sheep. Neuroendocrinology 36:392–396
Matteri RL, Watson JG, Moberg GP 1984 Stress or acute adrenocorticotrophin treatment suppresses LHRH-induced LH release in the ram. J Reprod Fertil 72:385–393
Norman RL, Smith CJ 1992 Restraint inhibits luteinizing hormone and testosterone secretion in intact male rhesus macaques: effects of concurrent naloxone administration. Neuroendocrinology 55:405–415
Norman RL, McGlone J, Smith CJ 1994 Restraint inhibits luteinizing hormone secretion in the follicular phase of the menstrual cycle in rhesus macaques. Biol Reprod 50:16–26
Akema T, Chiba A, Shinozaki R, Oshida M, Kimura F, Toyoda J 1996 Acute immobilization stress and intraventricular injection of CRF suppress naloxone-induced LH release in ovariectomized estrogen-primed rats. J Neuroendocrinol 8:647–652
Tilbrook AJ, Canny BJ, Serapiglia MD, Ambrose TJ, Clarke IJ 1999 Suppression of the secretion of luteinizing hormone due to isolation/restraint stress in gonadectomized ewes and rams is influenced by sex steroids. J Endocrinol 160:469–481
Nanda AS, Dobson H, Ward WR 1990 Relationship between an increase in plasma cortisol during transport-induced stress and failure of oestradiol to induce a luteinising hormone surge in dairy cows. Res Vet Sci 49:25–28
Dobson H, Trebble JE, Phogat JB, Smith RF 1999 Effect of transport on pulsatile and surge secretion of LH in ewes in the breeding season. J Reprod Fertil 116:1–8
Battaglia DF, Krasa HB, Padmanabhan V, Viguié C, Karsch FJ 2000 Endocrine alterations that underlie endotoxin-induced disruption of the follicular phase in ewes. Biol Reprod 62:45–53
Peter AT, Bosu WT, DeDecker RJ 1989 Suppression of preovulatory luteinizing hormone surges in heifers after intrauterine infusions of Escherichia coli endotoxin. Am J Vet Res 50:368–373
Xiao E, Xia-Zhang L, Barth A, Zhu J, Ferin M 1998 Stress and the menstrual cycle: relevance of quality in the short- and long-term response to a 5-day endotoxin challenge during the follicular phase in the rhesus monkey. J Clin Endocrinol Metab 83:2454–2460
Sapolsky RM, Romero M, Munck AU 2000 How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55–89
Carrasco GA, Van de Kar LD 2003 Neuroendocrine pharmacology of stress. Eur J Pharmacol 463:235–272
Minton JE 1994 Function of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system in models of acute stress in domestic farm animals. J Anim Sci 72:1891–1898
Macfarlane MS, Breen KM, Sakurai H, Adams BM, Adams TE 2000 Effect of duration of infusion of stress-like concentrations of cortisol on follicular development and the preovulatory surge of LH in sheep. Anim Reprod Sci 63:167–175
Breen KM, Karsch FJ 2004 Does cortisol inhibit pulsatile luteinizing hormone secretion at the hypothalamic or pituitary level? Endocrinology 145:692–698
Goodman RL 1994 Neuroendocrine control of the ovine estrous cycle. In: Knobil E, Neill JD, eds. The physiology of reproduction. Vol 2. New York: Raven Press; 659–709
Van Cleeff J, Karsch FJ, Padmanabhan V 1998 Characterization of endocrine events during the periestrous period in sheep after estrous synchronization with controlled internal drug release (CIDR) device. Domest Anim Endocrinol 15:23–34
Hauger RL, Karsch FJ, Foster DL 1977 A new concept for control of the estrous cycle of the ewe based on the temporal relationships between luteinizing hormone, estradiol and progesterone in peripheral serum and evidence that progesterone inhibits tonic LH secretion. Endocrinology 101:807–817
Niswender GD, Midgley Jr AR, Reichert Jr LE 1968 Radioimmunologic studies with murine, ovine and porcine luteinizing hormone. In: Rosenborg E, ed. Gonadotropins. Los Altos, CA: GERON-X; 299–306
Niswender GD, Reichert Jr LE, Midgley Jr AR, Nalbandov AV 1969 Radioimmunoassay for bovine and ovine luteinizing hormone. Endocrinology 84:1166–1173
Evans NP, Dahl GE, Glover BH, Karsch FJ 1994 Central regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by estradiol during the period leading up to the preovulatory GnRH surge in the ewe. Endocrinology 134:1806–1811
Padmanabhan V, Evans NP, Dahl GE, McFadden KL, Mauger DT, Karsch FJ 1995 Evidence for short or ultrashort loop negative feedback of gonadotropin-releasing hormone secretion. Neuroendocrinology 62:248–258
Battaglia DF, Bowen JM, Krasa HB, Thrun LA, Viguié C, Karsch FJ 1997 Endotoxin inhibits the reproductive neuroendocrine axis while stimulating adrenal steroids: a simultaneous view from hypophyseal portal and peripheral blood. Endocrinology 138:4273–4281
Veldhuis JD, Johnson ML 1986 Cluster analysis: a simple, versatile and robust algorithm for endocrine pulse detection. Am J Physiol 250:E486–E493
Debus N, Breen KM, Barrell GK, Billings HJ, Brown M, Young EA, Karsch FJ 2002 Does cortisol mediate endotoxin-induced inhibition of pulsatile luteinizing hormone and gonadotropin-releasing hormone secretion? Endocrinology 143:3748–3758
Baird DT, McNeilly AS 1981 Gonadotrophic control of follicular development and function during the oestrous cycle of the ewe. J Reprod Fertil Suppl 30:119–133
Campbell BK, Baird DT, McNeilly AS, Scaramuzzi RJ 1990 Ovarian secretion rates and peripheral concentrations of inhibin in normal and androstenedione-immune ewes with an autotransplanted ovary. J Endocrinol 127:285–296
Gore-Langton RE, Armstrong DT 1994 Follicular steroidogenesis and its control. In: Knobil E, Neill JD, ed. The physiology of reproduction. Vol 1. New York: Raven Press; 571–627
Schreiber JR, Nakamura K, Erickson GF 1982 Rat ovary glucocorticoid receptor: identification and characterization. Steroids 39:569–584
Hsueh AJ, Erickson GF 1978 Glucocorticoid inhibition of FSH-induced estrogen production in cultured rat granulosa cells. Steroids 32:639–648
Michael AE, Pester LA, Curtis P, Shaw RW, Edwards CR, Cooke BA 1993 Direct inhibition of ovarian steroidogenesis by cortisol and the modulatory role of 11?-hydroxysteroid dehydrogenase. Clin Endocrinol 38:641–644
Wagenmaker ER, Breen KM, Tilbrook AJ, Turner AI, Clarke IJ, Karsch FJ 2004 Does cortisol disrupt the positive feedback effects of estradiol on the preovulatory LH surge? Biol Reprod 70(Suppl 1):130 (Abstract)
Dufourny L, Skinner DC 2002 Progesterone receptor, estrogen receptor , and the type II glucocorticoid receptor are coexpressed in the same neurons of the ovine preoptic area and arcuate nucleus: a triple immunolabeling study. Biol Reprod 67:1605–1612
Tilbrook AJ, Turner AI, Clark IJ 2000 Effects of stress on reproduction in non-rodent mammals: the role of glucocorticoids and sex differences. Rev Reprod 5:105–113
Villanueva AL, Schlosser C, Hopper B, Liu JH, Hoffman DI, Rebar RW 1986 Increased cortisol production in women runners. J Clin Endocrinol Metab 63:133–136
Loucks AB, Mortola JF, Girton L, Yen SS 1989 Alterations in the hypothalamic-pituitary-ovarian and the hypothalamic-pituitary-adrenal axes in athletic women. J Clin Endocrinol Metab 68:402–411
Boccuzzi G, Angeli A, Bisbocci D, Fonzo D, Giadano GP, Ceresa F 1975 Effect of synthetic luteinizing hormone-releasing hormone (LH-RH) on the release of gonadotropins in Cushing’s disease. J Clin Endocrinol Metab 40:892–895
Luton J-P, Thieblot P, Valcke J-C, Mahoudeau JA, Bricaire H 1977 Reversible gonadotropin deficiency in male Cushing’s disease. J Clin Endocrinol Metab 45:488–495
Halbreich U, Asnis GM, Shindledecker R, Zumoff B, Nathan RS 1985 Cortisol secretion in endogenous depression. II. Time-related functions. Arch Gen Psychiatry 42:909–914
Pfohl B, Sherman B, Schlechte J, Stone R 1985 Pituitary-adrenal axis rhythm disturbances in psychiatric depression. Arch Gen Psychiatry 42:897–903
Gold PW, Gwirtsman H, Avgerinos PC, Nieman LK, Gallucci WT, Kaye W, Jimerson D, Ebert M, Rittmaster R, Loriaux DL, Chrousos GP 1986 Abnormal hypothalamic-pituitary-adrenal function in anorexia nervosa. Pathophysiologic mechanisms in underweight and weight-corrected patients. N Engl J Med 314:1335–1342
Suh BY, Liu JH, Berga SL, Quigley ME, Laughlin GA, Yen SS 1988 Hypercortisolism in patients with functional hypothalamic amenorrhea. J Clin Endocrinol Metab 66:733–739
Berga SL, Mortola JF, Girton L, Suh B, Laughlin G, Pham P, Yen SS 1989 Neuroendocrine aberrations in women with functional hypothalamic amenorrhea. J Clin Endocrinol Metab 68:301–308
Meller WH, Grambsch PL, Bingham C, Tagatz GE 2001 Hypothalamic pituitary gonadal axis dysregulation in depressed women. Psychoneuroendocrinology 26:253–259
Smith RF, Dobson H 2002 Hormonal interactions within the hypothalamus and pituitary with respect to stress and reproduction in sheep. Domest Anim Endocrinol 23:75–85
Bhatnagar S, Vining C 2003 Facilitation of hypothalamic-pituitary-adrenal responses to novel stress after repeated social stress using the resident/intruder paradigm. Horm Behav 43:158–165(Kellie M. Breen, Heather )