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Glucocorticoid Maintains Pulsatile Secretion of Luteinizing Hormone under Infectious Stress Condition

Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan

Address all correspondence and requests for reprints to: Masugi Nishihara, Ph.D., D.V.M., Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan. E-mail: amnishi{at}mail.ecc.u-tokyo.ac.jp.

	Abstract

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We have previously shown that TNF-, a major proinflammatory cytokine, suppressed hypothalamic GnRH pulse generator activity and that this inhibitory effect was enhanced by -helical CRH, a CRH receptor antagonist. The present study was conducted to elucidate the involvement of glucocorticoid (GC) in modulating LH pulses under infectious stress condition. Adrenalectomy (ADX) markedly enhanced the suppressive effect of TNF- (1 µg), injected iv, on LH pulses in ovariectomized (OVX) rats. Pretreatment with a sc injection of corticosterone (10 mg) almost completely restored LH pulses after TNF- injection in OVX/ADX animals. Injection of TNF- increased the number of c-Fos-immunoreactive cells in the supraoptic nucleus (SON), the dorsomedial hypothalamic nucleus (DMH), and the parvocellular region of the paraventricular nucleus (PVN), which was more prominent in OVX/ADX than OVX animals except in the DMH. Pretreatment with corticosterone decreased the number of Fos-immunoreactive cells in the PVN and SON but not in the DMH. These results suggest that GC has a potent protective effect on LH pulsatility under conditions of infectious stress, the mechanism of which involves at least the suppression of the excitability of PVN and SON neurons. In addition, the DMH does not seem to mediate the central action of GC, though it may play an important role in inducing pathophysiological reactions to invasive stress.

	Introduction

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IT IS NOW WELL established that immune signals have a broad influence on the neuroendocrine systems (1, 2), the most prominent being the suppression of the hypothalamic-pituitary-gonadal (HPG) axis as well as the activation of the hypothalamic-pituitary-adrenal (HPA) axis. Along this line, we have previously shown that bacterial endotoxin lipopolysaccharide (LPS) suppresses the electrical activity of the hypothalamic GnRH pulse generator, which governs the pulsatile secretion of LH, and that this inhibitory effect is, at least partially, mediated by TNF- (3). TNF- has been detected in extremely large amounts in the peripheral circulation after LPS injection or during the acute phase of infection, and it is regarded as one of the major cytokines responsible for the coordination of host defense mechanisms (4, 5).

Central actions of blood-borne cytokines, including TNF-, are largely mediated by prostaglandins (PGs) produced in the brain (6, 7, 8). In addition, CRH, the release of which is stimulated by cytokines through PGs, has an inhibitory effect on GnRH pulse generator activity (9, 10). We have therefore attempted to elucidate the possible involvement of PGs and/or the PG-CRH system in mediating TNF-’s effect and have shown that indomethacin, a cyclooxygenase inhibitor, blocked the suppression of GnRH pulse generator activity by TNF-, whereas -helical CRH, a CRH receptor antagonist, enhanced the suppressive effect of TNF- on pulse generator activity (6). These observations led us to hypothesize that a resultant increase in glucocorticoid (GC) secretion, after the activation of the HPA axis, might favor the HPG axis under infectious stress, though negative effects of GC on LH secretion have been reported under various experimental conditions (11, 12, 13, 14, 15).

In the present study, to explore the role of GC in modulating the HPG axis under infectious stress condition, we investigated the effects of ADX and replacement of corticosterone on TNF--induced suppression of pulsatile LH secretion in ovariectomized (OVX) rats. In addition, immunohistochemical observations were conducted using the expression of c-Fos protein, a product of the immediate early gene c-fos (16), as a marker for neuronal excitation to identify hypothalamic substrates involved in the response to TNF- and its modulation by GC.

	Materials and Methods

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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 OVX, under ether anesthesia, at the age of 8 wk (body weight, 230–250 g). A number of animals were also adrenalectomized (ADX). For ADX animals, 0.85% saline was given as drinking water. The rats were subjected to the experiments described below, after at least 2 wk of recovery. The experiments were conducted according to the guidelines for the care and use of laboratory animals, Graduate School of Agriculture and Life Sciences, the University of Tokyo.

Environmental procedure
The day before experiments, a SILASTIC brand (Dow Corning, Midland, MI) 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 to spend at least 2 h of adaptation period. A blood sample (100–150 µl) was withdrawn through the indwelling jugular cannula from freely moving animals, without anesthesia, at 5-min intervals for 4 h starting at 1200 h. After the withdrawal of each blood sample, an equal volume of heparinized saline (10 U/ml), in which erythrocytes were suspended, was replaced. At 1300 h, i.e. 1 h after the start of blood sampling, OVX and OVX/ADX animals were administered either saline (50 µl) or TNF- (R & D Systems, Minneapolis, MN; 1 µg/50 µl saline) through the indwelling jugular cannula. Corticosterone (Wako, Osaka, Japan; 10 mg/0.3 ml sesame oil) was sc injected into some of the OVX/ADX animals treated with TNF- at 1200 h. According to our preliminary study, this corticosterone treatment resulted in serum corticosterone levels of between 100–200 ng/ml within 1 h, which was maintained for at least 6 h thereafter. The collected blood samples were allowed to clot for 1–2 h at room temperature and were centrifuged at 5000 rpm for 15 min. The separated serum was stored at -80 C until assayed.

Hormone assay
The serum concentrations of LH were measured with a rat LH RIA kit (Amersham-Pharmacia Biotech, Piscataway, NJ). The intra- and interassay coefficients of variation (CVs) for LH assays, which were calculated from five replicated determinations for the pool of rat serum containing 12.9 ng/ml of LH, were 4.1% and 5.8%, respectively. Pulse detection was done based on the methods reported previously (17, 18, 19). Briefly, an LH pulse was defined when the CVs of LH values composing both the ascending and descending phases of each LH peak exceeded two times the corresponding intraassay CV. The pulse amplitude denoted the difference between the peak and the preceding nadir, 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.

Immunocytochemistry
At 1400 h, i.e. 1 h after the injection of TNF- or saline, four animals from all the experimental groups were anesthetized with pentobarbital sodium (30 mg/kg, ip) and perfused, via the left ventricle of the heart, with saline followed by 4% paraformaldehyde in 0.02 M PBS (pH 7.2). Brains were dissected out and further fixed in the paraformaldehyde solution overnight and then in a solution of 30% sucrose in 0.02 M PBS for 3 d. Brain sections of 30-µm thickness were made in a cryostat, and one out of every four slices was used for immunostaining for c-Fos protein. 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 blocking solution (Block Ace, Snow Brand Milk Products Co., Sapporo, Japan) for 2 h. Tissue sections were incubated in anti-Fos primary antibody (Oncogene Research Products, Cambridge, MA; 1:7000 with 0.3% Triton X-100) at 4 C for 72 h, washed three times with 0.02 M PBS 0.03% Triton X-100 (PBST), and then processed using a Vectastain ABC kit (Vector Labs, Elite ABC reagent, Burlingame, CA). The sections were treated for approximately 15 min in 0.5 mg/ml diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO; dissolved in 0.02 M PBS, with 0.01% hydrogen peroxide and 0.25% nickel chloride). Cells were counted with the image analyzing application IPLab (Scanalytics, Fairfax, VA).

Statistical analysis
The data were analyzed by ANOVA followed by Tukey-Kramer’s test or paired t test. Differences were considered significant at P < 0.05.

	Results

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Serum LH profiles
Representative serum LH profiles in each group are shown in Fig. 1. The mean LH concentration, pulse frequency, and pulse amplitude before the injection of saline or TNF- are listed in Table 1, and relative changes in these parameters, after the injection, are summarized in Fig. 2. As shown in Table 1, there were no significant differences in the parameters before the injection among experimental groups. Saline injection affected the pulsatile pattern of LH secretion in neither the OVX nor the OVX/ADX group. The injection of TNF- only partially affected pulsatile LH secretion in OVX animals; pulse frequency in the third 1-h period after the injection was significantly decreased, as compared with that before the injection, whereas mean LH concentration and pulse amplitude were not changed by the treatment. On the other hand, TNF- severely suppressed pulsatile LH secretion in OVX/ADX animals. The mean LH concentration after the injection was significantly lower than that before the injection, and also the values of other groups, during the entire experimental period of 3 h. The injection of TNF- into OVX/ADX animals decreased the pulse frequency, though the difference was not significant in the second 1-h period after the treatment. Pulse amplitude remained suppressed after the second 1-h period, compared with the pretreatment value, and it was also significantly smaller than the values of other groups in the third 1-h period. Pretreatment with corticosterone, 1 h before the TNF- injection, completely blocked the suppressive effect of TNF- on pulsatile LH secretion. In corticosterone-pretreated animals, the mean LH concentration, pulse frequency, and pulse amplitude were not affected by TNF- injection.

View larger version (40K): [in this window] [in a new window]   FIG. 1. Representative profiles of serum LH. The profiles of two individual animals in each experimental group are shown. Saline or TNF- (1 µg) was iv injected into OVX and OVX/ADX rats, 1 h after the start of blood sampling, which is denoted by vertical dotted lines. Corticosterone (CORT, 10 mg) was sc injected into OVX/ADX rats, 1 h before TNF- injection. Asterisks, Statistically defined LH pulses.

View this table: [in this window] [in a new window]   TABLE 1. Mean serum LH concentration, LH pulse frequency, and pulse amplitude before the injection of TNF- or saline

View larger version (51K): [in this window] [in a new window]   FIG. 2. Relative changes in the mean LH concentration, and frequency and amplitude of LH pulses during each 1-h period after TNF- (1 µg) or saline injection. CORT (10 mg) was sc injected 1 h before TNF-. Mean values for 1 h before TNF- or saline injection, which are listed in Table 1, are defined as 100%. Each column and vertical bar represents the mean ± SEM. Values with different letters are significantly different (P < 0.05, ANOVA followed by Tukey-Kramer test). *, Significantly different from the values before TNF- or saline injection (P < 0.05, paired t test).

View larger version (107K): [in this window] [in a new window]   FIG. 3. Immunostaining for c-Fos protein in the PVN, SON, and DMH, 1 h after TNF- (1 µg) or saline injection. CORT (10 mg) was sc injected 1 h before TNF-. Scale bar, 100 µm for PVN and DMH, and 50 µm for SON.

View larger version (28K): [in this window] [in a new window]   FIG. 4. The number of c-Fos-immunoreactive cells in the PVN, SON, DMH, and VMH. The sections were cut 30-µm thick, cells were counted every four sections throughout each nucleus, and the counts were summed. Each column and vertical bar represents the mean ± SEM. Values with different letters are significantly different (P < 0.05, ANOVA followed by the Tukey-Kramer test).

	Discussion

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It has been well established that central actions of TNF-, as well as other cytokines, are largely mediated by PGs produced in the hypothalamus (7, 8). We have also shown that an inhibitory effect of TNF- on the electrical activity of the GnRH pulse generator is, at least partially, mediated by PGs (6). On the other hand, GC is known to inhibit PG synthesis via facilitation of the biosynthesis of lipocortin-1 (21), which inhibits the activity of phospholipase A2, an enzyme catalyzing the release of fatty acids from the sn-2-position of membrane phospholipids (22). It is therefore suggested that the mechanism by which corticosterone restored pulsatile LH secretion after TNF- injection involves the suppression of PG synthesis in the hypothalamus.

Contrary to the present observation, there are many reports demonstrating that GC has an inhibitory effect on LH secretion (11, 12, 13, 14, 15). Under unstressful conditions, a large amount of exogenous GC seems to suppress LH secretion in both intact rats (12) and humans (13). Because the susceptibility of LH pulsatility to stress is enhanced by estrogen (23), excess GC may tend to suppress LH secretion in intact subjects, in which ovarian steroids are present. In addition, there is a report that GC is inhibitory of LH secretion in rats under stressful conditions (14). In this report, the authors demonstrated that foot shock-stress suppressed LH secretion in OVX rats, but not OVX/ADX rats, and suggested that GC played a primary role in the stress-induced suppression of LH secretion. The difference between their results and ours may depend on the difference between the stressors. Glucocorticoid would effectively prevent the suppression of LH pulsatility by the stress, the effect of which is mediated by PGs in the hypothalamus, as in the case of infectious stress. Different effects on pituitary LH release might also reflect differences in serum corticosterone levels between the two experimental conditions, because there are two different receptor systems for corticosteroids, i.e. the mineralocorticoid and GC receptor systems; and high levels of corticosterone activate the GC system, whereas lower levels activate primarily the mineralocorticoid system (24, 25).

The present study demonstrated that the number of Fos-ir cells in the SON and the parvocellular region of the PVN was increased 1 h after the injection of TNF-. This is consistent with the previous report investigating the effect of LPS on c-Fos expression in the brain (26). The expression of c-Fos in these brain regions was much enhanced by ADX, which was suppressed by the pretreatment with corticosterone. This difference in c-Fos expression levels probably accounts for the difference in LH responses; LH pulses in only OVX/ADX rats were suppressed 1 h after TNF- injection. In the parvocellular region of the PVN, there is a population of neurosecretory neurons containing CRH or both CRH and AVP; and, in the magnocellular regions of the PVN and SON, AVP neurons are located (27). It is therefore suggested that neurons containing CRH and/or AVP, both of which are known to participate in the activation of the HPA axis (27), were activated by TNF-. It has been demonstrated that neurons in both the PVN and the SON contain GC receptors (28) and that the AVP gene promoter has a consensus GC response element that represses AVP gene expression (29). Glucocorticoid may directly act on the neurosecretory neurons in the PVN and SON and suppress their excitability, as well as through the inhibition of PG synthesis mentioned above, thereby maintaining pulsatile LH secretion.

In the present study, the number of Fos-ir cells in the DMH, but not in the VMH, was increased after the injection of TNF- in OVX animals. It has been shown that Fos-ir cells in the DMH are increased by several forms of stress, such as the blowing of air on the face (30). Further, chemical stimulation by means of agents inducing the excitation or disinhibition of neurons in the DMH causes the pattern of physiological changes that are seen under stress, e.g. increases in heart rate, arterial pressure, and body temperature (31), and increases plasma ACTH levels (32, 33). In addition, neurons in the DMH send efferents directly to the parvocellular PVN (33, 34). The present observation provides additional evidence that the DMH is also involved in inducing biological responses to infectious stress. Interestingly, however, neither ADX nor pretreatment with corticosterone affected the number of Fos-ir cells in the DMH after TNF- injection. This suggests that the DMH transmits stress signals to the PVN but does not mediate the protective effect of GC against stress.

It has been suggested that CRH from the PVN is the primary substance causing pathophysiological reactions to stress (35, 36, 37). Inhibitory effects of CRH on reproductive function, such as the suppression of LH secretion by CRH (9, 10, 38, 39, 40) and recovery of LH secretion by -helical CRH under stressful conditions (41), have been also reported. We have previously shown, however, that intracerebroventricular injection of -helical CRH enhanced TNF--induced suppression of the GnRH pulse generator (6). Taking the present results into account, it is suggested that, at least under conditions of infectious stress, CRH may be involved in the maintenance of GnRH pulse generator activity through the activation of the HPA axis.

In conclusion, the present study suggests that GC has a potent protective effect on LH pulsatility by suppressing the excitability of neurons in the SON and PVN under infectious stress condition. In addition, it is also suggested the DMH is involved in inducing responses to infectious stress but not in mediating the protective effect of GC on LH pulsatility.

	Footnotes

 
This work was supported by a Grant-in-Aid for Scientific Research, the Japan Society for the Promotion of Science (13854007, to M.N.).

T.M. and E.W. contributed equally to this work.

Abbreviations: ADX, Adrenalectomy (adrenolectomized); CV, coefficient of variation; DMH, dorsomedial hypothalamic nucleus; Fos-ir, Fos-immunoreactive; GC, glucocorticoid; HPA, hypothalamic-pituitary-adrenal; HPG, hypothalamic-pituitary-gonadal; LPS lipopolysaccharide; OVX, ovariectomized; PG, prostaglandin; PVN, paraventricular nucleus; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus.

Received October 25, 2002.

Accepted for publication April 7, 2003.

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