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Pituitary |
Department of Physiology,3 Monash University, Clayton, Victoria 3800, Australia
Prince Henry's Institute of Medical Research,4 Clayton, Victoria 3168, Australia
Baker Medical Research Institute,5 Prahran, Victoria 3181, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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anterior pituitary, gonadotropin-releasing hormone, luteinizing hormone, neuroendocrinology, stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Changes in the responsiveness of the pituitary gland to GnRH during stress have been suggested by several researchers. A decrease in the LH response to GnRH was seen in gonadectomized rams during isolation and restraint stress or after an injection of ACTH [4]. In ewes however, the LH response was reduced with ACTH treatment but not during isolation and restraint stress [4]. Others have found a decrease in the LH response to GnRH in rams subjected to isolation and restraint stress [8], in ovariectomized ewes treated with endotoxin during the breeding season [9], and in intact ewes under conditions of transport stress during the breeding season [10]. Transport stress can also cause a decrease in LH pulse amplitude in ovariectomized ewes during the nonbreeding season [11]. Therefore, there appear to be sex differences in the sites of action at which stress may suppress the secretion of LH that may also depend upon the nature of the stressor.
The hypothalamopituitary disconnected sheep provides a robust model for the in vivo study of pituitary function in isolation from the brain. In this model, the isolated pituitary gland can be stimulated with hypothalamic releasing peptides delivered i.v., and the removal of hypothalamic influence allows the study of changes in pituitary responsiveness [12]. In addition to the removal of endogenous GnRH input, the hypothalamic control of ACTH and cortisol is also rendered inoperative. Therefore, psychological stressors, such as isolation and restraint, will not cause activation of the hypothalamus-pituitary-adrenal axis [13]. A prolactin response to psychological stress is also not observed [14]. Therefore, any changes in pituitary responsiveness during a stress response are likely to be due to other systems that respond to stress. An increase in concentrations of epinephrine and norepinephrine was observed in Polish Mountain ewes in response to restraint stress [15], and increases in norepinephrine secretion during isolation have been observed in sheep [16] and goats [17]. Intravenous infusion of norepinephrine decreased basal LH secretion in ovariectomized ewes and the LH response to GnRH in ovariectomized and anestrous ewes [18]. Thus, the sympathoadrenal system may play a role in suppressing LH secretion during stress.
In addition to sex, season may influence the impact of stress on the response of the pituitary to GnRH. It is not known whether physiological systems activated during stress are altered by seasonal factors in mammals, but there is evidence for seasonal differences in basal and stress-induced secretion of the glucocorticoids in birds, reptiles, and amphibians [19]. Consequently, we tested the hypothesis that isolation and restraint stress decreases the responsiveness of the pituitary gland to exogenous GnRH and that these responses differ with sex and season.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult Romney Marsh rams and ewes were used in two experiments conducted at the Prince Henry's Institute of Medical Research Biological Resource Centre (Werribee, Australia; 38° latitude). Animals were fed twice daily, and water was available ad libitum throughout both experiments. The first experiment was conducted during the breeding season for this breed of sheep [20], and the second experiment was conducted during the nonbreeding season. In both experiments, the rams and ewes were gonadectomized at least 6 wk prior to the commencement of the experiment. All animals underwent hypothalamopituitary disconnection using the procedure described by Clarke and colleagues [12] between 2 wk and 9 mo prior to the commencement of the experiment. The mean (±SEM) time from hypothalamopituitary disconnection to the experiment was 16 ± 3 wk. Routine measurement of plasma LH concentrations in animals after hypothalamopituitary disconnection revealed concentrations below the sensitivity of the assay, confirming hypothalamopituitary disconnection. The animals were fitted with an indwelling jugular catheter (Dwellcath; Tuta Laboratories, Lane Cove, Australia) 8 days prior to the commencement of the experiment for the injection of GnRH and the collection of blood samples. Animals were housed in individual pens (approximately 1.5 m long, 0.5 m wide, and 1 m high) in an experimental shed for 1 wk prior to and during the experiment. The pens were in a room separate from the rest of the experimental facility, and there was minimal disturbance of the sheep throughout the experiment.
All animal procedures were conducted with prior institutional ethics approval under the requirements of the Australian Prevention of Cruelty to Animals Act 1986 and the NH&MRC/CSIRO/AAC Code of Practice for the Care and Use of Animals for Scientific Purposes.
Experimental Procedure
Experiment 1: The effect of isolation and restraint stress on LH secretion in response to exogenous GnRH during the breeding season A cross-over design was used in experiment 1 with two groups of sheep: one group of gonadectomized rams (n = 6) and one group of gonadectomized ewes (n = 5). The experiment was conducted as two replicates, with three rams and three ewes in one replicate and three rams and two ewes in the other replicate. There were 3 experimental days in each replicate with 2 days between each experimental day. Each experimental day was divided into two periods. The first period on each day was a control period. Days 1 and 3 were designated control days, and no stress was imposed during the second period on these days. Isolation and restraint stress was imposed during the second period on Day 2.
For 1 wk prior to the commencement of the experiment and between experimental days, the animals were treated with an i.v. injection of GnRH (125 ng in 2.25 ml 50K units/L heparinized saline; Auspep, Melbourne, Australia). This treatment was previously characterized [7, 21]. An automated pump administered each injection over 6 min every 2 h. On each experimental day, the pump was stopped immediately prior to 0600 h. At 0600 h and every 2 h until 2000 h, the animals were given a hand-delivered i.v. injection of GnRH (125 ng/5 ml saline). Two GnRH injections were given prior to the commencement of the first period on each experimental day, and three injections were given in each of the two subsequent periods. Blood samples (5 ml) were taken from the jugular vein at -10, 5, 10, 20, 30, 40, and 60 min relative to each experimental GnRH injection, beginning at 0950 h and continuing until 2100 h. The plasma was harvested for measurement of the concentrations of LH and cortisol. The concentrations of LH were measured in all the samples collected, but the concentrations of cortisol were measured in the samples collected at -10, 30, and 60 min relative to each GnRH injection and in all the samples taken in the first hour after the commencement of isolation and restraint stress.
Beginning at 1530 h on Day 2, isolation and restraint stress was imposed for 5.5 h (until 2100 h) as described previously [4]. Sheep were moved into pens in a different part of the experimental facility that contained no sheep in adjacent pens. The sheep were restrained using a harness so that only the head could move freely and there was access to water. The pens were then covered with burlap so that the sheep were isolated, and there was no visual contact with other sheep.
Experiment 2: The effect of isolation and restraint stress on LH secretion in response to exogenous GnRH during the nonbreeding season In experiment 2, the design and procedure of experiment 1 was repeated, except that additional blood samples were taken at -10, -5, 2, 5, 10, 15, 20, and 25 min relative to the onset of the stress and at the same time on the control days. The samples were collected in nonheparinized tubes containing glutathione oxidation inhibitor (3% glutathione in 9.5% EGTA; Sigma, St. Louis, MO). The plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine (DOPA; a precursor of norepinephrine), and 3,4-dihydroxyphenylglycol (DHPG; a metabolite of norepinephrine) were measured in the additional samples. Catecholamine concentrations also were measured in samples taken 1 h before and 1 and 2 h after the onset of stress. As in experiment 1, LH was measured in samples taken at -10, 5, 10, 20, 30, 40, and 60 min relative to each injection of GnRH. Cortisol was measured in samples taken approximately every hour throughout the experiment and in samples taken every 0.5 h for 2 h after the onset of isolation and restraint stress.
LH Radioimmunoassay
Plasma LH concentrations were determined according to the protocol described by Lee and colleagues [22] and using NIH LH S18 as a standard. The mean (±SEM) sensitivity of the assay was 0.70 ± 0.05 ng/ml (n = 13). The intra-assay coefficient of variation was 7% at 15 ng/ml and 7% at 36 ng/ml. The interassay coefficient of variation was 11% at 8 ng/ml and 9% at 11 ng/ml.
Cortisol RIA
Concentrations of cortisol in plasma were measured after extraction with dichloromethane using a procedure previously described by Bocking and colleagues [23]. The standard used in the assay was hydrocortisone H-4001 (Sigma Chemical Co., St. Louis, MO). The mean (±SEM) sensitivity of the assay was 1.6 ± 0.06 ng/ml (n = 14). The interassay coefficient of variation was 9% at 8 ng/ml and 9% at 21 ng/ml. The intra-assay coefficient of variation was 6% at 19 ng/ml and 8% at 164 ng/ml.
Catecholamine Detection
Epinephrine, norepinephrine, DOPA, and DHPG were extracted from 500 µl of plasma using alumina adsorption. The amount of each compound was measured using electrochemical detection (Model 5100A coulometric detector; Environmental Sciences Associates, San Francisco, CA) after separation using HPLC (25 cm Altex Ultrasphere column, ODS 4.6 mm x 25 cm, 5-µm particle size; Beckman Instruments, Porterville, CA) according to a previously described procedure [24]. The sensitivity of this procedure was 15 pg/ml.
Statistical Analyses
The parameters measured were LH pulse amplitude, LH nadir, and mean plasma concentrations of LH and cortisol. LH pulse amplitude was defined as the peak LH concentration recorded after each GnRH injection minus the LH nadir preceding the injection. LH nadir was defined as the plasma concentration of LH at 10 min before each GnRH injection. The mean LH and cortisol concentrations were calculated by taking the mean of all data within a period for each hormone. All data were analyzed using a repeated-measures ANOVA with sex as the between-subjects variable. LH pulse amplitude and the LH nadir were analyzed with day and pulse as within-subject variables. The mean concentrations of LH and cortisol were analyzed with day and period as within-subject variables. The concentrations of norepinephrine, epinephrine, DOPA, and DHPG were analyzed with day and time as within-subject variables. For each set of data, the homogeneity of variance was assessed. Square-root or log transformation was applied where appropriate. When necessary, one unit was added to each data point before log transformation. Post hoc comparisons between groups were made using least significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the breeding season (experiment 1), there were no significant changes in the LH pulse amplitude, mean LH concentrations, or LH nadir preceding each GnRH injection when isolation and restraint stress was imposed (Fig. 1). During the nonbreeding season (experiment 2), there was a significant effect (P < 0.05) of isolation and restraint stress on LH pulse amplitude. On Day 2, the amplitude of the fourth LH pulse (the first pulse during isolation and restraint stress) was significantly less (P < 0.05) than that of the third pulse (the pulse immediately preceding the onset of stress). There were no effects of isolation and restraint stress on mean plasma concentrations of LH or on the LH nadir preceding each GnRH injection (Fig. 2). There were no significant differences between the sexes, so the data for rams and ewes were combined for Figures 1 and 2.
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Plasma Concentrations of Cortisol
In experiments 1 and 2, there were no significant changes in plasma concentrations of cortisol when isolation and restraint stress was imposed. In experiment 1, the mean (±SEM) concentrations of cortisol were 5.9 ± 1.3 ng/ml on Day 1, 5.6 ± 0.6 ng/ml on Day 2, and 4.0 ± 0.5 ng/ml on Day 3. In experiment 2, the mean plasma concentrations of cortisol were 7.2 ± 1.0 ng/ml, 7.9 ± 1.3 ng/ml, and 7.1 ± 1.1 ng/ml on Days 1, 2, and 3, respectively. There were no significant differences between the sexes, so the data for males and females were combined.
Plasma Concentrations of Catecholamines
Epinephrine In rams, plasma concentrations of epinephrine were significantly greater (P < 0.05) than prestress concentrations from 2 to 180 min after the onset of isolation and restraint stress (Fig. 3). Plasma concentrations of epinephrine in the samples taken 60, 10, and 5 min before the onset of stress on Day 2 were not significantly different from each other. There were no consistent significant changes in the plasma concentrations of epinephrine on Day 1 or Day 3 in rams.
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In ewes, plasma concentrations of epinephrine were significantly greater (P < 0.05) than prestress concentrations from 2 to 25 min after the commencement of the isolation and restraint stress (Fig. 3). At 60 and 180 min after the onset of stress, the plasma concentrations of epinephrine were similar to those in the prestress period. There were no significant changes in the plasma concentrations of epinephrine in the period before isolation and restraint stress, and there were no consistent significant differences in the plasma concentrations of epinephrine on Day 1 or Day 3 in ewes. Plasma concentrations of epinephrine did not differ significantly in a consistent manner between rams and ewes.
Norepinephrine, DOPA, and DHPG There were no significant changes in norepinephrine concentrations when isolation and restraint stress was imposed; however, at all time points the plasma concentrations of norepinephrine in ewes were significantly higher (P < 0.05) than those in rams (Fig. 4). Neither isolation and restraint stress nor sex significantly influenced plasma concentrations of DOPA (Day 1: 1.5 ± 0.8 ng/ml; Day 2: 1.6 ± 0.7 ng/ml; Day 3: 1.4 ± 0.7 ng/ml) or DHPG (Day 1: 1.0 ± 0.2 ng/ml; Day 2: 1.2 ± 0.3 ng/ml; Day 3: 1.1 ± 0.3 ng/ml) in any consistent manner.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanisms by which stress decreased the LH response of the pituitary to GnRH during the nonbreeding season are unknown. Nevertheless, it appears that ACTH and cortisol were not involved, because the hypothalamus-pituitary-adrenal axis of these animals was rendered inoperable because of the disconnection of the hypothalamic inputs to the pituitary gland. This disconnection was confirmed in our animals by the lack of significant increase in cortisol during isolation and restraint stress. The decrease in LH responsiveness to GnRH was only observed after the first GnRH injection, given 0.5 h after the commencement of the stress, and was not observed after the second and third injections, given 2.5 and 4.5 h after commencement of the stress, respectively. These results suggest that there was an immediate, short-term effect of isolation and restraint stress on pituitary responsiveness in these animals. The mediators of this rapid effect have not been identified, but epinephrine may be involved, at least during the nonbreeding season; there was a contemporaneous increase in epinephrine secretion in both rams and ewes during isolation and restraint stress at this time. This hypothesis has not been tested. An effect of norepinephrine is not supported by these results because there was no significant increase in peripheral plasma concentrations of norepinephrine during isolation and restraint stress, in contrast to the findings of others [15–17]. The highly variable nature of plasma concentrations of norepinephrine in sheep may have accounted for the lack of a significant rise in norepinephrine during stress in the current study. Neural modulation of secretory cells of the pituitary may also be a mechanism by which short-term changes in pituitary responsiveness can be regulated; synapses between nerve fibres and secretory cells of the anterior pituitary have been identified on gonadotropes in the rat [27].
The results of these studies suggest that mechanisms by which stress decreases LH secretion may be dependent on seasonal factors. Previous studies revealed no seasonal change in the release of LH from the pituitary in response to GnRH [7], although there are seasonal differences in the negative feedback effects of inhibin and testosterone on the secretion of the gonadotropins [21]. Seasonal factors may be able to alter the susceptibility of the reproductive axis to stress. The mechanisms by which this alteration may occur are unknown. Melatonin secretion increases in sheep during periods of shortening day length [28], and melatonin receptors have been identified in the pars tuberalis of the ovine pituitary [29], a site rich in gonadotropes. There are also seasonal changes in metabolic activity [30], and the hypothalamus-pituitary-adrenal axis response to stress differs between fat and thin sheep [31]. Consequently, metabolic activity may also be important in regulating the susceptibility of the pituitary LH response to GnRH during stress.
In this study, there were no sex differences in the maximal concentrations of circulating epinephrine during isolation and restraint stress, although the duration of the response was longer in rams than in ewes. There was a sex difference in the basal plasma concentrations of norepinephrine; the plasma concentrations of norepinephrine were consistently higher in ewes than in rams. In contrast, in a previous study in sheep [32] no sex differences in basal or stress-induced concentrations of central norepinephrine were found. There are sex differences in the response of the hypothalamus-pituitary-adrenal axis to stress, and these responses vary with the type of stressor and are influenced by the presence of the sex steroids [33, 34]. Evidence also exists for sex differences in the activation of the sympathoadrenal system in response to stress. For example, the sympathetic nervous system is less reactive in women than in men [35]. Weinstock and colleagues [36], however, found that in rats the female sympathoadrenal system was more sensitive to footshock and a novel environment than was that of males. Despite the differences between males and females in activation of various stress systems, our results indicate that there is no difference between gonadectomized male and female hypothalamopituitary disconnected sheep in the impact of isolation and restraint stress on release of the LH in response to GnRH.
These results of these experiments indicate that changes in pituitary responsiveness to GnRH occur during isolation and restraint stress in hypothalamopituitary disconnected rams and ewes during the nonbreeding season but not during the breeding season. This effect probably is due to mechanisms independent of increases in the secretion of ACTH and cortisol. Components of the sympathoadrenal system may be involved in decreasing the pituitary responsiveness to GnRH, but this possibility has not been explored. Further work is needed to investigate the mechanisms by which stress decreases pituitary responsiveness and the effect of seasonal factors on these mechanisms.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Alan Tilbrook, Department of Physiology, Building 13F, Monash University, Clayton, Victoria 3800, Australia. FAX: 61 3 9905 2547; alan.tilbrook{at}med.monash.edu.au
Received: 16 February 2003.
First decision: 3 March 2003.
Accepted: 21 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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