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Research Article |
Reproductive Sciences Program and Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109
ABSTRACT
Season can profoundly influence activity of the hypothalamic-pituitary-adrenal axis and alter reproductive neuroendocrine responsiveness to stress and gonadal steroids. Here we tested the hypothesis that the inhibitory effect of a stress-like increment in plasma concentration of the adrenal steroid cortisol on pulsatile LH secretion varies with season. LH pulse patterns were monitored prior to and during the administration of cortisol in the same seven ovariectomized ewes during three stages of the yearly breeding cycle: breeding season, transition to anestrus, and midanestrus. The elevation in cortisol mimicked the rise in plasma level of cortisol in response to an immune/inflammatory stress. During all three seasons, cortisol acutely suppressed the pulsatile release of LH. This inhibition reflected a marked reduction of LH pulse amplitude and a minimal suppression of LH pulse frequency. Of interest, the suppressive effect of this physiologic increment in cortisol did not vary across seasons. This provides initial evidence that, in ovariectomized ewes, cortisol-induced suppression of pulsatile LH secretion differs from that of gonadal steroids in that it is not profoundly influenced by season.
cortisol, gonadotropin-releasing hormone, luteinizing hormone, seasonal reproduction, stress
INTRODUCTION
It is well known that stress can suppress gonadotropin secretion and impair ovarian cyclicity [1–4]. Associated with these disruptive effects are activation of the hypothalamic-pituitary-adrenal (HPA) axis and elevation in plasma glucocorticoids. We and others have shown that stress-like increments in plasma levels of the adrenal steroid cortisol, similar to concentrations observed during an acute psychosocial or immune/inflammatory stress, potently inhibit gonadotropin secretion in both ovariectomized and follicular phase ewes [5–7]. These observations are consistent with the concept that activation of the HPA axis and increased glucocorticoid secretion contribute to stress-induced suppression of reproductive neuroendocrine function.
Like other neuroendocrine regulatory systems, activity of the HPA axis and neuroendocrine responsiveness to stress vary throughout the course of the year. For example, basal levels of glucocorticoids undergo seasonal variations in numerous mammalian species (chipmunks, squirrels, deer, sheep, baboons and humans [8–12]), and the magnitude of stress-induced increases in glucocorticoids varies with season in a wide variety of vertebrates [11]. In addition, humans express changes in responsiveness of nonvascular tissues to topical corticosteroids depending on the time of the year [13], and patients with depression experience seasonal fluctuations in responsiveness of the HPA axis to the negative feedback actions of synthetic glucocorticoids [14]. Of particular interest to the present study, recent evidence in sheep suggests there is a seasonal change in stress-induced suppression of reproductive neuroendocrine activity. Specifically, isolation/restraint inhibits gonadotropin secretion in rams and ewes during the nonbreeding season but not during the breeding season [15].
These observations, in conjunction with extensive evidence documenting a dramatic seasonal variation in responsiveness of the reproductive neuroendocrine axis to the negative feedback action of gonadal steroids [16–18], leads to the hypothesis that the inhibitory effects of the adrenal corticosteroids on gonadotropin secretion vary with season. In the present study, we tested this hypothesis by comparing the effect of a fixed increment in plasma cortisol on pulsatile LH secretion in the same ewes at three times of the year, coinciding with the breeding season, transition to anestrus, and mid-anestrus.
MATERIALS AND METHODS
Both experiments were conducted on mature Suffolk ewes maintained outdoors under standard husbandry conditions at the Sheep Research Facility near Ann Arbor, Michigan. The ewes were fed hay and alfalfa pellets and had free access to water and mineral licks. Surgery for ovariectomy was performed aseptically under general anesthesia at least 5 mo prior to use. All procedures were approved by the Committee for the Use and Care of Animals at the University of Michigan.
Preliminary Experiment to Select Cortisol Dose
In the breeding season (October), 24 ovariectomized ewes (n = 6/group) received vehicle (sesame oil) or one of three cortisol doses (0.05, 0.1, 0.25 mg/kg suspended in sesame oil; hydrocortisone sodium succinate, Solu-cortef, Pharmacia & Upjohn, Kalamazoo, MI). Blood for assay of LH and cortisol was collected at 10-min intervals by jugular catheter for 12 h. During the first 6 h, no treatment was applied. For the next 4 h, cortisol or vehicle was administered by sc injection every 30 min to an area of loose skin on the back.
Main Experiment
During months corresponding with the breeding season (October), the transition to anestrus (March), and midanestrus (June), cortisol was administered to the same seven ovariectomized ewes. On each occasion, blood was sampled every 10 min from 6 h before to 6 h after the onset of treatment. As in the preliminary experiment, for the first 6 h, no treatment was applied. Cortisol was administered for the next 6 h by sc injection every 30 min at a dose of 0.125 mg/kg suspended in sesame oil. This cortisol dose is slightly greater than the intermediate dose used in the preliminary experiment, which significantly reduced LH pulse amplitude, but not to the extent observed with the highest dose. The dose in the main experiment produces an elevation in plasma cortisol mimicking that induced by an acute immune/inflammatory stress (
110 ng/ml [6]) and has been shown to inhibit pituitary responsiveness to GnRH in ovariectomized ewes [19]. A vehicle control was not included in the main experiment, as we have repeatedly observed this treatment neither elevates plasma cortisol values nor inhibits pulsatile LH secretion in ovariectomized ewes [preliminary experiment and Refs. 6, 19, 20].
Hormone Assays
LH concentrations were determined in duplicate aliquots (25–200 µl) of plasma using a modification [21] of a previously described RIA [22, 23] and are expressed in terms of NIH-LH-S12. The mean intra- and interassay coefficients of variation were 4.9% and 5.2%, respectively, and assay sensitivity averaged 0.69 ng/ml (22 assays). Total plasma cortisol concentrations were determined in duplicate 50-µl aliquots of unextracted plasma using the Coat-A-Count cortisol assay kit (Diagnostic Products Corporation, Los Angeles, CA) validated for use in sheep [24]. Mean intra- and interassay coefficients of variation were 5.8% and 4.8%, respectively (17 assays). Assay sensitivity averaged 0.62 ng/ml.
Data Analysis
LH pulses were identified using the Cluster pulse detection algorithm [25]. As explained elsewhere [6, 19], cluster sizes for peaks and nadirs were set at 1 and 2, respectively, and the t-statistic used to identify a significant increase or decrease was 2.6. LH pulse amplitude was defined as the difference between a peak and its preceding nadir. Total pulsatile LH output was calculated as the product of pulse frequency x mean pulse amplitude. Before statistical analysis, plasma hormone concentrations were log transformed and pulse frequencies were square root transformed to normalize the distribution across a range of values. To identify significant effects of cortisol on LH secretion, values for pre- and posttreatment periods were obtained in every ewe for each parameter (e.g., LH pulse frequency, mean LH pulse amplitude, total pulsatile output, and mean plasma LH). In the preliminary experiment, two-way repeated measures ANOVA was used to identify differences in the effects of increasing doses of cortisol on LH secretion. In the main experiment, the effects of cortisol on LH secretion within each season were determined by one-way repeated measures ANOVA. Next, two-way repeated measures ANOVA was used to identify significant interactions (i.e., seasonal effects) for all LH parameters. In addition, LH pulse parameters during the 6-h period prior to treatment were compared across seasons by two-way repeated measures ANOVA to identify steroid-independent seasonal effects. Two-way repeated measures ANOVA was used to test for seasonal differences in plasma cortisol concentrations produced by the treatment. Significance level was set at P 
0.05.
RESULTS
Preliminary Experiment
Mean cortisol concentrations were less than 15 ng/ml prior to treatment and during vehicle administration. Twice-hourly injections of the graded doses of cortisol elevated plasma cortisol concentrations within 2 h to 36.6 ± 3.7, 80.5 ± 4.6, and 198.2 ± 20.7 ng/ml (hereafter designated low, intermediate, and high, respectively) and remained elevated throughout the 4-h treatment period. Vehicle did not alter any parameter of LH secretion (LH pulse frequency, LH pulse amplitude, total pulsatile output, or mean plasma LH). All three cortisol doses reduced LH pulse amplitude (P < 0.05; Table 1) and caused corresponding decreases in total pulse output and mean LH value (data not shown). The reduction in LH pulse amplitude in response to the high dose of cortisol was twice as great as that produced by the intermediate dose (P < 0.05; Table 1). None of the cortisol doses reduced LH pulse frequency (data not shown).
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Main Experiment
Prior to treatment, mean plasma cortisol concentrations were less than 10 ng/ml in all seasons. During administration of cortisol, plasma cortisol concentrations increased within 2 h to 109.2 ± 9.8, 106.6 ± 5.6, and 121.8 ± 9.7 ng/ml (breeding, transition, and anestrous seasons, respectively) and remained elevated throughout the 6-h treatment period (Fig. 1). These cortisol values did not differ with season.
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LH pulse profiles in four representative ewes receiving cortisol in all three seasons are illustrated in Figures 2 and 3. Composite results for all seven ewes comparing LH pulse frequency, LH pulse amplitude, total pulsatile output, and mean plasma LH concentration prior to and during cortisol in all seasons are presented in Table 2. As reported previously [26–28], seasonal changes were observed in the patterns of pulsatile LH secretion in ovariectomized ewes in the absence of exogenous steroid (i.e., prior to cortisol). In particular, LH pulse frequency decreased (P < 0.001) and pulse amplitude increased (P < 0.01) as ewes progressed from the breeding season to anestrus. In all seasons, cortisol unambiguously suppressed pulsatile LH secretion. Inhibition was evident within 1–2 h and was maintained throughout the 6-h treatment period, although LH pulses were readily detected during cortisol treatment (Figs. 2 and 3). The statistical analysis revealed suppressive effects of cortisol within each season. Importantly, there was not a significant interaction of these effects among seasons for any of the parameters evaluated, indicating that the inhibitory effects of this increment in cortisol did not vary seasonally. Within each season, cortisol significantly reduced LH pulse amplitude (P < 0.001), total LH pulsatile output (P < 0.001), and mean plasma LH concentration (P < 0.001). Cortisol also lowered LH pulse frequency (P < 0.05) in the breeding season and anestrus but did not significantly affect frequency during the transition to anestrus. In contrast to the marked reduction of LH pulse amplitude, however, LH pulse frequency was lowered minimally (1 pulse/6 h or less; Table 2).
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DISCUSSION
Prior evidence demonstrates seasonal changes in both basal and stress-induced increases in glucocorticoid secretion [8–12] and in responsiveness to some of the many diverse actions of corticosteroids [13, 14]. Further, inhibitory effects of stress on gonadotropin secretion can also vary over the course of the year [15]. In contrast to these reports, the present study demonstrates that a fixed, stress-like increment in plasma cortisol inhibits pulsatile LH secretion to a similar extent during times corresponding to the breeding season, transition to anestrus, and mid-anestrus. This finding provides initial evidence that the inhibitory effect of glucocorticoids on reproductive neuroendocrine activity in ovariectomized ewes does not vary with season.
In considering the foregoing conclusion, however, it is important to acknowledge that a seasonal change in responsiveness to only one dose of cortisol was formally tested. Thus, it is possible that a maximally suppressive dose was used and a seasonal effect would have been disclosed with a lower dose. We do not favor this alternative interpretation because the preliminary experiment suggests that a maximally effective dose of cortisol was not employed. The reduction of LH pulse amplitude produced by 109 ng/ml cortisol in the breeding season of the main experiment (36% decrease) was intermediate to that observed with 80 and 198 ng/ml cortisol at the same time of year in the preliminary experiment (22% and 45%, respectively). Further, none of the doses of cortisol completely inhibited LH pulsatility, leaving open the potential for greater suppression. Thus, while our conclusion must be qualified by the possibility that a seasonal effect might be disclosed with a lower dose of cortisol, the present study permits the conclusion that the inhibitory effect of a plasma cortisol concentration of 110 ng/ml (a high stress-like level) does not vary with season.
This observation is somewhat unexpected in light of extensive evidence that the negative feedback effects of gonadal steroids on pulsatile LH secretion are far greater in anestrus than during the breeding season [16–18]. To begin to understand why the suppressive effects of a corticosteroid on pulsatile LH secretion in ovariectomized ewes do not vary seasonally whereas those of a gonadal steroid such as estradiol do, it is helpful to consider how these different steroids express their inhibitory effects. The seasonal change in the negative feedback action of estradiol is due to an effect on frequency of LH pulses that reflects a change at the hypothalamic level [18, 26]. Estradiol potently inhibits GnRH (and thus LH) pulse frequency in ovariectomized ewes during the anestrous season, but this effect is not evident during the breeding season. Unlike estradiol, cortisol does not inhibit GnRH secretion in ovariectomized ewes [19], and its acute, suppressive effect on frequency of LH pulses is minimal, 10% or less as observed in this study. Rather than powerfully inhibiting frequency, cortisol reduces LH pulse amplitude by suppressing pituitary responsiveness to GnRH [19]. Thus, the absence of a seasonal change in the inhibitory effect of cortisol on pulsatile LH secretion in ovariectomized ewes may reflect the lack of a major hypothalamic effect to suppress LH pulse frequency.
The aforementioned interpretation must be tempered by three considerations. First, in contrast to the effect of cortisol in the ovariectomized ewe, this glucocorticoid markedly inhibits LH pulse frequency in ovary-intact ewes during the follicular phase of the estrous cycle [7], suggesting a hypothalamic component of cortisol action in the presence of ovarian steroids. Therefore, it would be interesting to determine if season influences the inhibitory effect of cortisol on pulsatile LH secretion in ovary-intact ewes or in ovariectomized ewes treated with ovarian steroids. This would be challenging, however, because of seasonal changes in the negative feedback action of the ovarian steroids themselves. The second qualifier to the present conclusion is that, in contrast to the acute effect of cortisol as studied here, more prolonged treatment with cortisol can reduce LH pulse frequency to a greater extent in ovariectomized ewes (unpublished observations). This raises the possibility that the duration of cortisol exposure influences the mode of reproductive neuroendocrine suppression. If this possibility were to be substantiated by a formal study, it would be interesting to test for seasonal changes in response to chronic cortisol exposure. A third qualifier is that cortisol significantly (albeit minimally) suppressed LH pulse frequently during the breeding and anestrous seasons, without affecting frequency during the transition to anestrus. Importantly, the statistical analysis revealed no significant effect of season. Therefore, despite these reservations, the present finding permits the conclusion that the acute inhibitory effect of a physiologic increment in cortisol on pulsatile LH secretion in ovariectomized ewes does not change with season.
As mentioned previously, our present findings stand in contrast to previous evidence illustrating seasonal changes in HPA activity and neuroendocrine responsiveness to stress. For example, the glucocorticoid response to acute capture and restraint changes throughout the course of the year in amphibians, reptiles, and birds [11]. Of particular relevance to the present study, isolation/restraint stress inhibits pituitary responsiveness to exogenous GnRH in gonadectomized sheep during anestrus but not during the breeding season [15]. That determination was made in sheep in which the hypothalamus was surgically disconnected from the pituitary. Because this surgery removes neurosecretory terminals controlling ACTH as well as LH, there was no stress-induced increase in cortisol secretion [15]. Thus, the seasonal change in stress-induced suppression of pituitary responsiveness to GnRH in that model cannot be attributed to a change in the secretion of or responsiveness to cortisol. The more likely explanation, which fits nicely with the present findings, is that mediators other than cortisol (i.e., catecholamines, opioids) or differences in the cognitive perception of stress account for seasonal changes in this reproductive neuroendocrine response to stress.
In summary, this study contributes novel evidence that responsiveness of the reproductive neuroendocrine axis to the adrenal steroid cortisol is not influenced by season. Specifically, we observed that a defined, stress-like increment in cortisol suppresses pulsatile LH secretion in ovariectomized ewes to a similar extent during times coincident with the breeding season, the transition to anestrus, and mid-anestrus. During all three seasons, the inhibitory effect of cortisol is attributed primarily to a reduction of LH pulse amplitude, with LH pulse frequency only minimally suppressed. Although the inhibitory effect of gonadal steroids is greatly influenced by season, the response to cortisol in ovariectomized ewes appears to differ in this regard.
ACKNOWLEDGMENTS
The authors would like to thank Doug Doop and Gary McCalla for their exceptional animal care; Emily Adams, Heather Billings, Lisa Modrick, Amy Oakley, Andrew Pytiak, Elizabeth Wagenmaker, and Emily Wessinger for their contribution to the completion of this study; and Dr. Alan Tilbrook for his careful review of this manuscript.
FOOTNOTES
1 Supported by NIH-HD-30773, T32-HD07048, and the Office of the Vice President for Research at the University of Michigan. A preliminary report was presented at the 37th annual meeting of the Society for the Study of Reproduction, Vancouver, BC, Canada, 1–4 August 2004. 
2 Correspondence: Kellie M. Breen, Reproductive Sciences Program, University of Michigan, 300 N. Ingalls Building, Room 1115 SW, Ann Arbor, MI 48109-0404. FAX: 734 936 8620; breenk{at}umich.edu
Received: 21 July 2005.
First decision: 15 August 2005.
Accepted: 7 September 2005.
REFERENCES
This article has been cited by other articles:
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  A. E. Oakley, K. M. Breen, A. J. Tilbrook, E. R. Wagenmaker, and F. J. Karsch Role of Estradiol in Cortisol-Induced Reduction of Luteinizing Hormone Pulse Frequency Endocrinology, June 1, 2009; 150(6): 2775 - 2782. [Abstract] [Full Text] [PDF]
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 E. R. Wagenmaker, K. M. Breen, A. E. Oakley, B. N. Pierce, A. J. Tilbrook, A. I. Turner, and F. J. Karsch Cortisol Interferes with the Estradiol-Induced Surge of Luteinizing Hormone in the Ewe Biol Reprod, March 1, 2009; 80(3): 458 - 463. [Abstract] [Full Text] [PDF]
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C. A. Stackpole, I. J. Clarke, K. M. Breen, A. I. Turner, F. J. Karsch, and A. J. Tilbrook Sex Difference in the Suppressive Effect of Cortisol on Pulsatile Secretion of Luteinizing Hormone in Sheep Endocrinology, December 1, 2006; 147(12): 5921 - 5931. [Abstract] [Full Text] [PDF]
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