Maintenance of Gonadotropin Secretion by Glucocorticoids under Stress Conditions through the Inhibition of Prostaglandin Synthesis
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《内分泌学杂志》
Department of Veterinary Physiology, Veterinary Medical Science, University of Tokyo, Tokyo 113-8657, Japan
Abstract
We have previously reported that glucocorticoids counteract the suppressive effects of tumor necrosis factor- on both pulsatile and surge secretion of LH. This suggests that glucocorticoids have a protective effect on reproductive function under infectious stress. In the present study, we examined whether glucocorticoids maintain pulsatile LH secretion under various conditions of acute stress and the possible involvement of prostaglandins (PGs) in glucocorticoid actions. Three different types of stressors, namely infectious (lipopolysaccharide, 0.5 μg/kg), hypoglycemic (2-deoxy-D-glucose, 100 mg/kg), and restraint stress (1 h) were applied to ovariectomized rats. In ovariectomized rats, LH pulses were partially suppressed by restraint, but not by lipopolysaccharide or 2-deoxy-D-glucose. On the other hand, adrenalectomy (ADX) significantly enhanced the suppressive effects of all the stressors applied on LH pulses. Treatment with both corticosterone (25 mg/kg) and indomethacin (10 mg/kg) in ADX rats significantly attenuated the suppressive effects of these stressors on LH pulses. In addition, the immunoreactivity of cyclooxygenase-2, a PG-synthesizing enzyme, in the brain under stress conditions was much enhanced by ADX, and this was counteracted by corticosterone treatment. Similarly, an increase in body temperature under restraint stress was enhanced by ADX and suppressed by corticosterone. These results suggest that suppression of LH pulsatility by stress is mediated by PGs in the brain, and that increased release of endogenous glucocorticoids in response to stress counteracts this suppression by inhibiting PG synthesis, and thereby maintains reproductive function regardless of the nature of the stressor.
Introduction
IT IS WELL RECOGNIZED that reproductive function is suppressed under stress conditions. Suppression of the electrical activity of the GnRH pulse generator (1, 2), as well as the pulsatile and surge patterns of LH secretion (3, 4, 5, 6) under many kinds of stress conditions have been demonstrated. It has been widely accepted that this suppression of the hypothalamic-pituitary-gonadal axis is at least partially due to glucocorticoids (7, 8, 9, 10) through the activation of the hypothalamic-pituitary-adrenal axis. However, we have recently reported that glucocorticoids counteract the suppressive effects of TNF- on both pulsatile and surge secretion of LH (11, 12). This suggested that an increase in glucocorticoid secretion is involved in maintaining the function of the hypothalamic-pituitary-gonadal axis under infectious stress condition.
TNF- is one of the proinflammatory cytokines, and is present at high levels in the peripheral circulation during the acute phase of infection and after lipopolysaccharide (LPS) injection (13, 14). LPS injection mimics the events occurring during the acute phase of infection. During infectious stress, the central actions of blood-borne cytokines including TNF- are largely mediated by prostaglandins (PGs) produced in the brain (15). We have shown that indomethacin, a cyclooxygenase (COX) inhibitor, blocks TNF--induced suppression of the electrical activity of the GnRH pulse generator (16), supporting the notion that PGs mediate the suppressive effects of TNF- on pulse generator activity. Because glucocorticoids are known to suppress PG synthesis (17, 18), maintenance of LH secretion by glucocorticoids under infectious stress condition might be achieved at least partially through the suppression of PG synthesis in the brain.
As well as infectious stress, hypoglycemia and restraint stress are known for their inhibitory effects on reproductive function (13, 14). Hypoglycemic stress induced by the injection of insulin or 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, or restricted feeding was reported to suppress LH pulses in rats (3, 4), ewes (19), and monkeys (1). It has been also demonstrated that restraint stress suppresses LH surge as well as LH pulses in rats (20, 21, 22). The present study was undertaken to elucidate whether glucocorticoids might have protective effects on LH pulsatility not only under infectious stress, but also under various acute stress conditions in common. To this end, we investigated the ability of glucocorticoids to counteract the suppressive effects of hypoglycemic (2-DG) and restraint stresses, as well as infectious (LPS) stress, on LH pulses. Possible involvement of PGs in this glucocorticoid action was also examined by immunohistochemical detection of COX-2, an inducible PG-synthesizing enzyme (15), in the brain.
Materials and Methods
Animals and surgery
Adult female rats of the Wistar-Imamichi strain were obtained from the Imamichi Institute for Animal Reproduction (Tsuchiura, Japan). The animals were maintained under controlled light (lights on, 0500–1900 h), and given free access to food and water. All rats were ovariectomized (OVX) under ether anesthesia at the age of 8 wk (body weight, 200–250 g). A number of animals were also adrenalectomized (ADX). For ADX animals, four SILASTIC-brand silicon tubing implants (Dow Corning Corp., Midland, MI; 1.5 mm inner diameter; 3.0 mm outer diameter; 70 mm in length) containing corticosterone (Wako, Osaka, Japan) were implanted into each rat immediately after ADX to maintain basal serum corticosterone levels, and 0.85% saline was given as drinking water. The rats were subjected to the following experiments after at least 1 wk of recovery period. The experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals, Graduate School of Agriculture and Life Sciences, University of Tokyo.
Experimental procedure
On the day before each experiment, a SILASTIC-brand silicon tubing cannula was inserted into the jugular vein under ether anesthesia. The distal end of the cannula was tunneled sc to the back of the neck. On the day of the experiment, the animals were moved to the experimental room and allowed an adaptation period of at least 2 h. A blood sample (100–150 μl) was withdrawn through the indwelling jugular cannula from freely moving animals without anesthesia at 5-min intervals from 1200 h for the determination of LH pulses. After the withdrawal of each blood sample, an equal volume of heparinized saline (10 U/ml), in which erythrocytes were suspended, was replaced. During the blood sampling, the animals were subjected to the three types of stressors: iv injection of LPS (055: B5; Sigma, St. Louis, MO; 0.5 μg/kg body weight) or 2-DG (Sigma; 100 mg/kg) at 1300 h, or restraint stress. As restraint stress, the rats were strapped by their limbs onto a wooden restraint board from 1300–1400 h. In addition, corticosterone (Wako; 25 mg/kg) or indomethacin (Wako; 10 mg/kg) was injected sc or iv, respectively, into some of the ADX rats at 1300 h. Blood sampling was continued until 1500 h. In the restraint stress experiment, an additional group of animals, whose blood samples were obtained between 1600 and 1800 h, but not between 1300 and 1600 h, was included. The collected blood samples were allowed to clot for 1–2 h at room temperature and centrifuged at 5000 rpm for 10 min. The separated serum was stored at –80 C until assayed for LH.
Measurement of body temperature
Because PGs are known to be a potent pyrogen (23), body temperature was measured to assess PG synthesis in the brain under restraint stress. To determine body temperature, a battery-operated transmitter (E-Mitter-4000; Mini Mitter, Bend, OR) was sc implanted in the back under ether anesthesia more than 3 d before the experiment. Animals were restrained from 1300–1400 h, and a number of the ADX animals were sc injected with corticosterone (25 mg/kg) at the start of restraint stress. The body temperature was monitored every hour between 1230 and 1830 h.
Hormone assay
The serum concentrations of LH were measured by the double antibody RIA, using materials supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The reference standard for the LH assay was NIDDK-rLH-rp-3. The intra and interassay coefficients of variation for LH assays, which were calculated from five to seven replicated determinations for the pool of rat serum containing 3.0 ng/ml of LH, were 9.8 and 12.4%, respectively. Serum corticosterone concentrations were determined by RIA with a specific antibody generated in our laboratory by using corticosterone conjugated to BSA as an antigen. All the samples were measured in a single assay. The intraassay coefficient of variation was 12.6% for the pooled serum concentration of 324.9 ng/ml.
Immunohistochemistry
One hour after the injection of LPS (0.5 μg/kg) or 2-DG (100 mg/kg), or after release from the restraint, animals were anesthetized with pentobarbital sodium (30 mg/kg) and perfused via the left ventricle of the heart with saline followed by 4% paraformaldehyde in PBS (0.02 M, pH 7.2). Brains were dissected out and further fixed in the paraformaldehyde solution overnight and then soaked in a solution of 30% sucrose in PBS for 3 d. Brain sections of 30-μm thickness were made in a cryostat, for immunostaining for COX-2. Free-floating sections were rinsed with PBS for 10 min three times. Sections were then incubated with 0.3% H2O2 in methanol for 30 min at room temperature and rinsed with PBS three times. Thereafter, sections were incubated in PBS containing 4% normal rabbit serum for 2 h. Tissue sections were incubated in anti-COX-2 primary antibody (sc-1747; Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000 with 0.3% Triton X-100 in PBS) at 4 C for 72 h, washed three times with PBS, and then processed using a Vectastain ABC kit ("Elite" ABC reagent; Vector Labs, Burlingame, CA). The sections were treated for approximately 15 min in 0.5 mg/ml of diaminobenzidine tetrahydrochloride (Sigma; dissolved in 0.02 M PBS, with 0.01% hydrogen peroxide and 0.25% nickel), and observed under a light microscope (EX51; Olympus, Tokyo, Japan).
Data analyses
LH pulses were identified by means of the PULSAR computer program (24). The cut-off criteria for pulse determination, G1, G2, G3, G4, and G5 were 3.98, 2.40, 1.68, 1.24, and 0.93, respectively. The pulse amplitude denoted the difference between peak and baseline, and the pulse frequency denoted the number of pulses during each 1-h period. Overall mean LH concentrations during each 1-h period were also calculated. The data were statistically analyzed by ANOVA followed by Tukey-Kramer’s test or Dunnet’s test. Differences were considered significant at P < 0.05.
Results
Serum corticosterone levels
Serum corticosterone concentrations before (1300 h) and 1 h after (1400 h) the application of stressors in OVX, adrenal-intact rats are shown in Fig. 1. Serum corticosterone levels just before (1300 h) and 1 h after (1400 h) corticosterone injection in OVX/ADX rats, which received SILASTIC-brand silicon tubing implants containing corticosterone to maintain basal corticosterone levels, are shown in Fig. 1 as well. There was no significant difference in basal serum corticosterone levels among experimental groups. There was also no significant difference in corticosterone levels in response to different stressors. Serum corticosterone levels 1 h after the injection of corticosterone in OVX/ADX rats were not different from those 1 h after the application of stressors in OVX, adrenal-intact rats.
Serum LH profiles
Representative serum LH profiles in LPS and 2-DG experiments are shown in Fig. 2, and the mean LH concentration, pulse frequency, and pulse amplitude are summarized in Fig. 3. There were no significant differences in the parameters before the injection of LPS or 2-DG among experimental groups. The injection of neither LPS nor 2-DG had any significant effect on pulsatile LH secretion in OVX animals. On the other hand, both of them severely suppressed pulsatile LH secretion in OVX/ADX animals. The mean LH concentration after the injection of LPS was significantly lower than that before the injection, and also lower than the values in OVX rats, during the entire 2-h experimental period. Although the pulse frequency was not significantly changed after the injection of LPS, the pulse amplitude was significantly suppressed after the treatment compared with the pretreatment value, and it was also significantly smaller in the second 1-h period than were the amplitude values of OVX animals in the same period. The injection of 2-DG also significantly decreased the mean LH concentration and pulse amplitude in OVX/ADX rats compared with their values before the injection and the values from the OVX rats after the injection. The frequency of LH pulses in the second 1-h period after the injection of 2-DG was significantly lower than it was before the injection. Treatment with corticosterone at the time of LPS or 2-DG injection almost completely blocked their suppressive effects on pulsatile LH secretion. Injection of indomethacin also preserved LH pulsatility after the injection of LPS or 2-DG in OVX/ADX animals. In both corticosterone- and indomethacin-treated animals, the mean LH concentration, pulse frequency, and pulse amplitude were not affected by either LPS or 2-DG.
Representative serum LH profiles during the restraint stress are shown in Fig. 4, and the mean LH concentration, pulse frequency, and pulse amplitude are summarized in Fig. 5. As shown in Figs. 4A and 5A, LH pulse frequency and amplitude in OVX rats were significantly suppressed during restraint, but immediately recovered after release. On the other hand, in OVX/ADX rats, the suppression of LH pulses was prolonged, and mean LH concentration and pulse amplitude did not recover for at least 2 h after the release. Pulse frequency in OVX/ADX rats during restraint and after release was significantly lower than that before restraint and the corresponding value in OVX rats, respectively. Corticosterone treatment did not restore LH pulsatility at all after the release. Therefore, we stopped bleeding at 1300 h, restrained animals between 1300 and 1400 h, and took blood samples between 1600 and 1800 h in the next experiment (Figs. 4B and 5B). In this additional experiment, there was still no recovery of LH pulsatility in OVX/ADX animals, even 2–4 h after release. Mean LH concentration and pulse amplitude were significantly smaller for the entire 2 h experimental period than before restraint, and pulse frequency was also significantly lower in the first 1-h period. Both corticosterone and indomethacin almost completely restored LH pulsatility in OVX/ADX rats 2–4 h after release; the mean LH concentration, pulse frequency, and pulse amplitude in animals that received corticosterone or indomethacin were not different from those before injection.
COX-2 immunoreactivity
Although COX-2-immunoreactive (COX-2-ir) cells were hardly discernible in OVX rats 1 h after injection of LPS or 2-DG, many COX-2-ir cells were detected mainly in and around the blood vessels in several brain regions including the cortex, allocortical structures, hypothalamus, and meninges in OVX/ADX animals. Figure 6 illustrates the representative patterns of COX-2 immunoreactivity observed in the primary olfactory cortex and neighboring meninges. One hour after release from the restraint, a small number of COX-2-ir cells were detected even in OVX rats, and the number was much increased in OVX/ADX rats. However, corticosterone treatment almost completely eliminated COX-2-ir cells after stress treatment, regardless of the type of stressor.
Body temperature
Figure 7 shows changes in body temperature in the restraint stress experiment. The body temperature of OVX rats was slightly but significantly increased after release from restraint. The body temperature of OVX/ADX rats was significantly higher than that of OVX rats between 1430 and 1630 h. On the other hand, the body temperature of OVX/ADX rats treated with corticosterone after release was not different from that before restraint.
Discussion
In the present study, ADX itself did not affect the pulsatile pattern of LH secretion under normal conditions as reported by us (11, 25) and other groups (9). However, the injection of LPS or 2-DG significantly suppressed LH pulsatility in OVX/ADX animals, although it had no such effect in adrenal-intact animals. This indicates that the adrenal glands have a protective effect on LH secretion under hypoglycemic as well as infectious stress conditions. The injection of corticosterone into the OVX/ADX animals, by which serum corticosterone levels attained those in adrenal-intact animals 1 h after the exposure to the stressors, almost completely restored pulsatile LH secretion, indicating that the protective effect of the adrenal gland on LH pulsatility is attributable to the increase in endogenous glucocorticoids released in response to stressors. Unlike infectious and hypoglycemic stresses, restraint stress had potent negative influences on LH pulsatility even in OVX animals, which is consistent with previous reports (20, 26). Although LH pulses recovered immediately after release in OVX rats, they did not recover by 4 h after release in OVX/ADX rats. However, replacement of corticosterone in OVX/ADX rats restored LH pulsatility at least 2 h after release. Therefore, it is probable that glucocorticoids play a pivotal role in maintaining LH pulsatility in the presence of the acute stressors applied in the present study.
Although there have been many reports suggesting that glucocorticoids have an inhibitory effect on LH secretion (7, 8, 9, 10), recent studies have demonstrated that glucocorticoids are not involved in the acute suppression of LH pulses after LPS treatment in rats (27) and ewes (28). Moreover, it is reported that LPS-induced inhibition of LH secretion in rats is significantly reversed by dexamethasone, a synthetic glucocorticoid (29). We have also shown that corticosterone maintains LH pulses (11) and surge (12) in rats, even after TNF- injection. Thus, although a large amount of exogenous glucocorticoids indeed appears to suppress LH secretion (7, 10), we suggest that endogenous glucocorticoids released in response to acute stress favor gonadotropin secretion. A possibility that the effect of glucocorticoids on LH secretion in nonstressed animals is different from that in stressed animals also cannot be ruled out.
In the present study, indomethacin, an inhibitor of PG synthesis, restored LH pulsatility as effectively as corticosterone under all the stress conditions applied in OVX/ADX animals. It has been already shown that the suppressive effect of infectious stress on LH secretion is mediated by PGs (16, 30). The present results suggest that PGs mediate the effect of hypoglycemic and restraint stress, as well as infectious stress, on LH pulsatility. In support of our results, it has been shown that both hypoglycemia (31) and restraint (25) induce PG synthesis in the brain. In this context, we conducted an immunohistochemical study of COX-2 in the brain. After LPS injection, COX-2-ir cells were much increased by ADX in and around the blood vessels in several regions of the brain, including the cortex, hypothalamus, and meninges. This distribution is consistent with the previous reports (32, 33), which demonstrated COX-2-ir cells in rat brain, mainly in the endothelial cells of brain vessels, after LPS treatment. COX-2 immunoreactivity in these regions of the brain was also enhanced by hypoglycemic and restraint stress, supporting our notion that PGs may be common mediators of acute stresses in the brain. Although how different stressors could converge to cause an increase in PG synthesis remains to be resolved, it might be noteworthy to mention that many stressors could induce hypothalamic IL-1 expression, which is a major cytokine inducing PG synthesis (34). IL-1 may be a possible candidate that mediates the information of various stressors entering the hypothalamus through different routes/mechanisms and, in turn, stimulates COX-2 expression.
The synthesis of PG is known to be inhibited by glucocorticoids via suppression of the induction of COX-2 (18) as well as facilitation of the biosynthesis of lipocortin-1 (17), which inhibits the activity of phospholipase A2, an enzyme that catalyzes the release of fatty acids from the sn-2-position of membrane phospholipids (35). Our observation that the number of COX-2-ir cells in OVX/ADX rats was reduced by corticosterone treatment suggests that glucocorticoids maintain LH secretion by inhibiting the expression of COX-2 at least partially, and hence PGs, in the brain. This is further supported by the effect of corticosterone on body temperature. In this study, the body temperature of ADX/OVX rats was significantly higher than that of OVX rats after restraint, and this was reversed by corticosterone. Because PG is regarded as a potent pyrogen (23, 25), the present observation suggests that restraint stress induces PG synthesis in the brain, and that this is effectively suppressed by glucocorticoids. In addition, glucocorticoid elevation induced by insulin or 2-DG is reported to suppress the synthesis of PG in brain (36). Taken together, these observations lead us to suggest that glucocorticoids prevent the suppression of LH pulsatility via inhibition of PG synthesis in the brain under acute stress conditions.
In conclusion, the present study demonstrates that PGs mediate the suppressive effect on LH pulsatility common to infectious, hypoglycemic, and restraint stress. We also suggest that increased release of endogenous glucocorticoids in response to stress counteracts the effect of stress by inhibiting PG synthesis in the brain, and thereby maintains LH secretion regardless of the nature of the stressor. Activation of hypothalamic-pituitary-adrenal axis may be involved in the maintenance, rather than the suppression, of reproductive function under acute stress conditions.
Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research (13854007, 17208025 to M.N.) and Research Fellowships for Young Scientists (1611663 to T.M.) from the Japan Society for the Promotion of Science.
First Published Online November 17, 2005
Abbreviations: ADX, Adrenalectomized; COX, cyclooxygenase; COX-2-ir, COX-2-immunoreactive; 2-DG, 2-deoxy-D-glucose; LPS, lipopolysaccharide; OVX, ovariectomized; PG, prostaglandin.
Accepted for publication November 10, 2005.
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Abstract
We have previously reported that glucocorticoids counteract the suppressive effects of tumor necrosis factor- on both pulsatile and surge secretion of LH. This suggests that glucocorticoids have a protective effect on reproductive function under infectious stress. In the present study, we examined whether glucocorticoids maintain pulsatile LH secretion under various conditions of acute stress and the possible involvement of prostaglandins (PGs) in glucocorticoid actions. Three different types of stressors, namely infectious (lipopolysaccharide, 0.5 μg/kg), hypoglycemic (2-deoxy-D-glucose, 100 mg/kg), and restraint stress (1 h) were applied to ovariectomized rats. In ovariectomized rats, LH pulses were partially suppressed by restraint, but not by lipopolysaccharide or 2-deoxy-D-glucose. On the other hand, adrenalectomy (ADX) significantly enhanced the suppressive effects of all the stressors applied on LH pulses. Treatment with both corticosterone (25 mg/kg) and indomethacin (10 mg/kg) in ADX rats significantly attenuated the suppressive effects of these stressors on LH pulses. In addition, the immunoreactivity of cyclooxygenase-2, a PG-synthesizing enzyme, in the brain under stress conditions was much enhanced by ADX, and this was counteracted by corticosterone treatment. Similarly, an increase in body temperature under restraint stress was enhanced by ADX and suppressed by corticosterone. These results suggest that suppression of LH pulsatility by stress is mediated by PGs in the brain, and that increased release of endogenous glucocorticoids in response to stress counteracts this suppression by inhibiting PG synthesis, and thereby maintains reproductive function regardless of the nature of the stressor.
Introduction
IT IS WELL RECOGNIZED that reproductive function is suppressed under stress conditions. Suppression of the electrical activity of the GnRH pulse generator (1, 2), as well as the pulsatile and surge patterns of LH secretion (3, 4, 5, 6) under many kinds of stress conditions have been demonstrated. It has been widely accepted that this suppression of the hypothalamic-pituitary-gonadal axis is at least partially due to glucocorticoids (7, 8, 9, 10) through the activation of the hypothalamic-pituitary-adrenal axis. However, we have recently reported that glucocorticoids counteract the suppressive effects of TNF- on both pulsatile and surge secretion of LH (11, 12). This suggested that an increase in glucocorticoid secretion is involved in maintaining the function of the hypothalamic-pituitary-gonadal axis under infectious stress condition.
TNF- is one of the proinflammatory cytokines, and is present at high levels in the peripheral circulation during the acute phase of infection and after lipopolysaccharide (LPS) injection (13, 14). LPS injection mimics the events occurring during the acute phase of infection. During infectious stress, the central actions of blood-borne cytokines including TNF- are largely mediated by prostaglandins (PGs) produced in the brain (15). We have shown that indomethacin, a cyclooxygenase (COX) inhibitor, blocks TNF--induced suppression of the electrical activity of the GnRH pulse generator (16), supporting the notion that PGs mediate the suppressive effects of TNF- on pulse generator activity. Because glucocorticoids are known to suppress PG synthesis (17, 18), maintenance of LH secretion by glucocorticoids under infectious stress condition might be achieved at least partially through the suppression of PG synthesis in the brain.
As well as infectious stress, hypoglycemia and restraint stress are known for their inhibitory effects on reproductive function (13, 14). Hypoglycemic stress induced by the injection of insulin or 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, or restricted feeding was reported to suppress LH pulses in rats (3, 4), ewes (19), and monkeys (1). It has been also demonstrated that restraint stress suppresses LH surge as well as LH pulses in rats (20, 21, 22). The present study was undertaken to elucidate whether glucocorticoids might have protective effects on LH pulsatility not only under infectious stress, but also under various acute stress conditions in common. To this end, we investigated the ability of glucocorticoids to counteract the suppressive effects of hypoglycemic (2-DG) and restraint stresses, as well as infectious (LPS) stress, on LH pulses. Possible involvement of PGs in this glucocorticoid action was also examined by immunohistochemical detection of COX-2, an inducible PG-synthesizing enzyme (15), in the brain.
Materials and Methods
Animals and surgery
Adult female rats of the Wistar-Imamichi strain were obtained from the Imamichi Institute for Animal Reproduction (Tsuchiura, Japan). The animals were maintained under controlled light (lights on, 0500–1900 h), and given free access to food and water. All rats were ovariectomized (OVX) under ether anesthesia at the age of 8 wk (body weight, 200–250 g). A number of animals were also adrenalectomized (ADX). For ADX animals, four SILASTIC-brand silicon tubing implants (Dow Corning Corp., Midland, MI; 1.5 mm inner diameter; 3.0 mm outer diameter; 70 mm in length) containing corticosterone (Wako, Osaka, Japan) were implanted into each rat immediately after ADX to maintain basal serum corticosterone levels, and 0.85% saline was given as drinking water. The rats were subjected to the following experiments after at least 1 wk of recovery period. The experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals, Graduate School of Agriculture and Life Sciences, University of Tokyo.
Experimental procedure
On the day before each experiment, a SILASTIC-brand silicon tubing cannula was inserted into the jugular vein under ether anesthesia. The distal end of the cannula was tunneled sc to the back of the neck. On the day of the experiment, the animals were moved to the experimental room and allowed an adaptation period of at least 2 h. A blood sample (100–150 μl) was withdrawn through the indwelling jugular cannula from freely moving animals without anesthesia at 5-min intervals from 1200 h for the determination of LH pulses. After the withdrawal of each blood sample, an equal volume of heparinized saline (10 U/ml), in which erythrocytes were suspended, was replaced. During the blood sampling, the animals were subjected to the three types of stressors: iv injection of LPS (055: B5; Sigma, St. Louis, MO; 0.5 μg/kg body weight) or 2-DG (Sigma; 100 mg/kg) at 1300 h, or restraint stress. As restraint stress, the rats were strapped by their limbs onto a wooden restraint board from 1300–1400 h. In addition, corticosterone (Wako; 25 mg/kg) or indomethacin (Wako; 10 mg/kg) was injected sc or iv, respectively, into some of the ADX rats at 1300 h. Blood sampling was continued until 1500 h. In the restraint stress experiment, an additional group of animals, whose blood samples were obtained between 1600 and 1800 h, but not between 1300 and 1600 h, was included. The collected blood samples were allowed to clot for 1–2 h at room temperature and centrifuged at 5000 rpm for 10 min. The separated serum was stored at –80 C until assayed for LH.
Measurement of body temperature
Because PGs are known to be a potent pyrogen (23), body temperature was measured to assess PG synthesis in the brain under restraint stress. To determine body temperature, a battery-operated transmitter (E-Mitter-4000; Mini Mitter, Bend, OR) was sc implanted in the back under ether anesthesia more than 3 d before the experiment. Animals were restrained from 1300–1400 h, and a number of the ADX animals were sc injected with corticosterone (25 mg/kg) at the start of restraint stress. The body temperature was monitored every hour between 1230 and 1830 h.
Hormone assay
The serum concentrations of LH were measured by the double antibody RIA, using materials supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The reference standard for the LH assay was NIDDK-rLH-rp-3. The intra and interassay coefficients of variation for LH assays, which were calculated from five to seven replicated determinations for the pool of rat serum containing 3.0 ng/ml of LH, were 9.8 and 12.4%, respectively. Serum corticosterone concentrations were determined by RIA with a specific antibody generated in our laboratory by using corticosterone conjugated to BSA as an antigen. All the samples were measured in a single assay. The intraassay coefficient of variation was 12.6% for the pooled serum concentration of 324.9 ng/ml.
Immunohistochemistry
One hour after the injection of LPS (0.5 μg/kg) or 2-DG (100 mg/kg), or after release from the restraint, animals were anesthetized with pentobarbital sodium (30 mg/kg) and perfused via the left ventricle of the heart with saline followed by 4% paraformaldehyde in PBS (0.02 M, pH 7.2). Brains were dissected out and further fixed in the paraformaldehyde solution overnight and then soaked in a solution of 30% sucrose in PBS for 3 d. Brain sections of 30-μm thickness were made in a cryostat, for immunostaining for COX-2. Free-floating sections were rinsed with PBS for 10 min three times. Sections were then incubated with 0.3% H2O2 in methanol for 30 min at room temperature and rinsed with PBS three times. Thereafter, sections were incubated in PBS containing 4% normal rabbit serum for 2 h. Tissue sections were incubated in anti-COX-2 primary antibody (sc-1747; Santa Cruz Biotechnology, Santa Cruz, CA; 1:1000 with 0.3% Triton X-100 in PBS) at 4 C for 72 h, washed three times with PBS, and then processed using a Vectastain ABC kit ("Elite" ABC reagent; Vector Labs, Burlingame, CA). The sections were treated for approximately 15 min in 0.5 mg/ml of diaminobenzidine tetrahydrochloride (Sigma; dissolved in 0.02 M PBS, with 0.01% hydrogen peroxide and 0.25% nickel), and observed under a light microscope (EX51; Olympus, Tokyo, Japan).
Data analyses
LH pulses were identified by means of the PULSAR computer program (24). The cut-off criteria for pulse determination, G1, G2, G3, G4, and G5 were 3.98, 2.40, 1.68, 1.24, and 0.93, respectively. The pulse amplitude denoted the difference between peak and baseline, and the pulse frequency denoted the number of pulses during each 1-h period. Overall mean LH concentrations during each 1-h period were also calculated. The data were statistically analyzed by ANOVA followed by Tukey-Kramer’s test or Dunnet’s test. Differences were considered significant at P < 0.05.
Results
Serum corticosterone levels
Serum corticosterone concentrations before (1300 h) and 1 h after (1400 h) the application of stressors in OVX, adrenal-intact rats are shown in Fig. 1. Serum corticosterone levels just before (1300 h) and 1 h after (1400 h) corticosterone injection in OVX/ADX rats, which received SILASTIC-brand silicon tubing implants containing corticosterone to maintain basal corticosterone levels, are shown in Fig. 1 as well. There was no significant difference in basal serum corticosterone levels among experimental groups. There was also no significant difference in corticosterone levels in response to different stressors. Serum corticosterone levels 1 h after the injection of corticosterone in OVX/ADX rats were not different from those 1 h after the application of stressors in OVX, adrenal-intact rats.
Serum LH profiles
Representative serum LH profiles in LPS and 2-DG experiments are shown in Fig. 2, and the mean LH concentration, pulse frequency, and pulse amplitude are summarized in Fig. 3. There were no significant differences in the parameters before the injection of LPS or 2-DG among experimental groups. The injection of neither LPS nor 2-DG had any significant effect on pulsatile LH secretion in OVX animals. On the other hand, both of them severely suppressed pulsatile LH secretion in OVX/ADX animals. The mean LH concentration after the injection of LPS was significantly lower than that before the injection, and also lower than the values in OVX rats, during the entire 2-h experimental period. Although the pulse frequency was not significantly changed after the injection of LPS, the pulse amplitude was significantly suppressed after the treatment compared with the pretreatment value, and it was also significantly smaller in the second 1-h period than were the amplitude values of OVX animals in the same period. The injection of 2-DG also significantly decreased the mean LH concentration and pulse amplitude in OVX/ADX rats compared with their values before the injection and the values from the OVX rats after the injection. The frequency of LH pulses in the second 1-h period after the injection of 2-DG was significantly lower than it was before the injection. Treatment with corticosterone at the time of LPS or 2-DG injection almost completely blocked their suppressive effects on pulsatile LH secretion. Injection of indomethacin also preserved LH pulsatility after the injection of LPS or 2-DG in OVX/ADX animals. In both corticosterone- and indomethacin-treated animals, the mean LH concentration, pulse frequency, and pulse amplitude were not affected by either LPS or 2-DG.
Representative serum LH profiles during the restraint stress are shown in Fig. 4, and the mean LH concentration, pulse frequency, and pulse amplitude are summarized in Fig. 5. As shown in Figs. 4A and 5A, LH pulse frequency and amplitude in OVX rats were significantly suppressed during restraint, but immediately recovered after release. On the other hand, in OVX/ADX rats, the suppression of LH pulses was prolonged, and mean LH concentration and pulse amplitude did not recover for at least 2 h after the release. Pulse frequency in OVX/ADX rats during restraint and after release was significantly lower than that before restraint and the corresponding value in OVX rats, respectively. Corticosterone treatment did not restore LH pulsatility at all after the release. Therefore, we stopped bleeding at 1300 h, restrained animals between 1300 and 1400 h, and took blood samples between 1600 and 1800 h in the next experiment (Figs. 4B and 5B). In this additional experiment, there was still no recovery of LH pulsatility in OVX/ADX animals, even 2–4 h after release. Mean LH concentration and pulse amplitude were significantly smaller for the entire 2 h experimental period than before restraint, and pulse frequency was also significantly lower in the first 1-h period. Both corticosterone and indomethacin almost completely restored LH pulsatility in OVX/ADX rats 2–4 h after release; the mean LH concentration, pulse frequency, and pulse amplitude in animals that received corticosterone or indomethacin were not different from those before injection.
COX-2 immunoreactivity
Although COX-2-immunoreactive (COX-2-ir) cells were hardly discernible in OVX rats 1 h after injection of LPS or 2-DG, many COX-2-ir cells were detected mainly in and around the blood vessels in several brain regions including the cortex, allocortical structures, hypothalamus, and meninges in OVX/ADX animals. Figure 6 illustrates the representative patterns of COX-2 immunoreactivity observed in the primary olfactory cortex and neighboring meninges. One hour after release from the restraint, a small number of COX-2-ir cells were detected even in OVX rats, and the number was much increased in OVX/ADX rats. However, corticosterone treatment almost completely eliminated COX-2-ir cells after stress treatment, regardless of the type of stressor.
Body temperature
Figure 7 shows changes in body temperature in the restraint stress experiment. The body temperature of OVX rats was slightly but significantly increased after release from restraint. The body temperature of OVX/ADX rats was significantly higher than that of OVX rats between 1430 and 1630 h. On the other hand, the body temperature of OVX/ADX rats treated with corticosterone after release was not different from that before restraint.
Discussion
In the present study, ADX itself did not affect the pulsatile pattern of LH secretion under normal conditions as reported by us (11, 25) and other groups (9). However, the injection of LPS or 2-DG significantly suppressed LH pulsatility in OVX/ADX animals, although it had no such effect in adrenal-intact animals. This indicates that the adrenal glands have a protective effect on LH secretion under hypoglycemic as well as infectious stress conditions. The injection of corticosterone into the OVX/ADX animals, by which serum corticosterone levels attained those in adrenal-intact animals 1 h after the exposure to the stressors, almost completely restored pulsatile LH secretion, indicating that the protective effect of the adrenal gland on LH pulsatility is attributable to the increase in endogenous glucocorticoids released in response to stressors. Unlike infectious and hypoglycemic stresses, restraint stress had potent negative influences on LH pulsatility even in OVX animals, which is consistent with previous reports (20, 26). Although LH pulses recovered immediately after release in OVX rats, they did not recover by 4 h after release in OVX/ADX rats. However, replacement of corticosterone in OVX/ADX rats restored LH pulsatility at least 2 h after release. Therefore, it is probable that glucocorticoids play a pivotal role in maintaining LH pulsatility in the presence of the acute stressors applied in the present study.
Although there have been many reports suggesting that glucocorticoids have an inhibitory effect on LH secretion (7, 8, 9, 10), recent studies have demonstrated that glucocorticoids are not involved in the acute suppression of LH pulses after LPS treatment in rats (27) and ewes (28). Moreover, it is reported that LPS-induced inhibition of LH secretion in rats is significantly reversed by dexamethasone, a synthetic glucocorticoid (29). We have also shown that corticosterone maintains LH pulses (11) and surge (12) in rats, even after TNF- injection. Thus, although a large amount of exogenous glucocorticoids indeed appears to suppress LH secretion (7, 10), we suggest that endogenous glucocorticoids released in response to acute stress favor gonadotropin secretion. A possibility that the effect of glucocorticoids on LH secretion in nonstressed animals is different from that in stressed animals also cannot be ruled out.
In the present study, indomethacin, an inhibitor of PG synthesis, restored LH pulsatility as effectively as corticosterone under all the stress conditions applied in OVX/ADX animals. It has been already shown that the suppressive effect of infectious stress on LH secretion is mediated by PGs (16, 30). The present results suggest that PGs mediate the effect of hypoglycemic and restraint stress, as well as infectious stress, on LH pulsatility. In support of our results, it has been shown that both hypoglycemia (31) and restraint (25) induce PG synthesis in the brain. In this context, we conducted an immunohistochemical study of COX-2 in the brain. After LPS injection, COX-2-ir cells were much increased by ADX in and around the blood vessels in several regions of the brain, including the cortex, hypothalamus, and meninges. This distribution is consistent with the previous reports (32, 33), which demonstrated COX-2-ir cells in rat brain, mainly in the endothelial cells of brain vessels, after LPS treatment. COX-2 immunoreactivity in these regions of the brain was also enhanced by hypoglycemic and restraint stress, supporting our notion that PGs may be common mediators of acute stresses in the brain. Although how different stressors could converge to cause an increase in PG synthesis remains to be resolved, it might be noteworthy to mention that many stressors could induce hypothalamic IL-1 expression, which is a major cytokine inducing PG synthesis (34). IL-1 may be a possible candidate that mediates the information of various stressors entering the hypothalamus through different routes/mechanisms and, in turn, stimulates COX-2 expression.
The synthesis of PG is known to be inhibited by glucocorticoids via suppression of the induction of COX-2 (18) as well as facilitation of the biosynthesis of lipocortin-1 (17), which inhibits the activity of phospholipase A2, an enzyme that catalyzes the release of fatty acids from the sn-2-position of membrane phospholipids (35). Our observation that the number of COX-2-ir cells in OVX/ADX rats was reduced by corticosterone treatment suggests that glucocorticoids maintain LH secretion by inhibiting the expression of COX-2 at least partially, and hence PGs, in the brain. This is further supported by the effect of corticosterone on body temperature. In this study, the body temperature of ADX/OVX rats was significantly higher than that of OVX rats after restraint, and this was reversed by corticosterone. Because PG is regarded as a potent pyrogen (23, 25), the present observation suggests that restraint stress induces PG synthesis in the brain, and that this is effectively suppressed by glucocorticoids. In addition, glucocorticoid elevation induced by insulin or 2-DG is reported to suppress the synthesis of PG in brain (36). Taken together, these observations lead us to suggest that glucocorticoids prevent the suppression of LH pulsatility via inhibition of PG synthesis in the brain under acute stress conditions.
In conclusion, the present study demonstrates that PGs mediate the suppressive effect on LH pulsatility common to infectious, hypoglycemic, and restraint stress. We also suggest that increased release of endogenous glucocorticoids in response to stress counteracts the effect of stress by inhibiting PG synthesis in the brain, and thereby maintains LH secretion regardless of the nature of the stressor. Activation of hypothalamic-pituitary-adrenal axis may be involved in the maintenance, rather than the suppression, of reproductive function under acute stress conditions.
Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research (13854007, 17208025 to M.N.) and Research Fellowships for Young Scientists (1611663 to T.M.) from the Japan Society for the Promotion of Science.
First Published Online November 17, 2005
Abbreviations: ADX, Adrenalectomized; COX, cyclooxygenase; COX-2-ir, COX-2-immunoreactive; 2-DG, 2-deoxy-D-glucose; LPS, lipopolysaccharide; OVX, ovariectomized; PG, prostaglandin.
Accepted for publication November 10, 2005.
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