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Centre for Reproduction, Endocrinology and Diabetes (J.E.B., J.C.M., K.T.O., X.F.L.) and Centre for Cardiovascular Biology & Medicine (S.D.B.), New Hunt’s House King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom; and Henry Wellcome Laboratory for Integrative Neuroscience & Endocrinology (S.L.L.), Dorothy Hodgkin Building, University of Bristol, Bristol BS1 3NY, United Kingdom
Address all correspondence and requests for reprints to: Dr. X. F. Li, Centre for Reproduction, Endocrinology and Diabetes, 2.36 New Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom. E-mail: xiao_feng.li{at}kcl.ac.uk.
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Abstract |
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-helical CRF9–41, 26 nmol) partly blocked the CGRP-induced suppression of LH pulses. Furthermore, CGRP8–37 (1 nmol) completely blocked hypoglycemic stress-induced suppression of LH pulses. These results suggest that the suppression of pulsatile LH secretion by central administration of CGRP may be mediated in part by CRH, and that CGRP may play a pivotal role in the normal physiological response of stress-induced suppression of the hypothalamic GnRH pulse generator, and hence the reproductive system. |
Introduction |
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It is well established that stress suppresses the activity of the hypothalamo-pituitary-gonadal (HPG) axis, especially the GnRH pulse generator, the central neural regulator of pituitary LH and FSH secretion, resulting in ovulatory dysfunction. For example, hypoglycemia is a well-characterized metabolic stressor that not only activates the HPA axis, but also has been shown to profoundly suppress GnRH/LH secretion in a variety of species ranging from rodents (9, 10, 11) to bovids (12, 13) to primates (14, 15, 16), including humans (17). The inverse relationship between the HPA and HPG axes has led to the suggestion that activation of the HPA axis may underlie stress-induced suppression of the GnRH pulse generator. There is overwhelming evidence that CRH plays a central role in stress-induced suppression of the reproductive neuroendocrine system. Central administration of CRH inhibits LH pulses (18, 19, 20), and central infusion of CRH receptor antagonists reverse the LH pulse-suppressing effects of a variety of stressful stimuli (9, 14, 21, 22). Because CGRP elicits central effects that resemble physiological responses during stress, in particular activation of the HPA axis, a novel inhibitory action of CGRP on reproductive function is postulated.
Despite the widespread distribution of CGRP neurons throughout the central nervous system, there are several key populations that may be important for the regulation of GnRH secretion. In the brainstem, the lateral parabrachial nucleus (PBN) contains a large number of CGRP neurons and has extensive reciprocal connections with brain areas involved in neuroendocrine and autonomic functions. This nucleus not only projects to the hypothalamic paraventricular nucleus (PVN) (23), the main controlling center of the HPA axis, but also projects to the GnRH-rich rostral preoptic area (24, 25, 26). Stressful stimuli, known to activate the HPA axis and suppress the GnRH pulse generator, result in neuronal activation, measured by increased FOS expression, within the PBN (27, 28), including its CGRP neurons (29). However, it remains to be established whether the direct projections from the PBN to the preoptic area contain CGRP neurons. CGRP neurons and fibers are also found in the preoptic area (30, 31, 32), and although CGRP-binding sites have been demonstrated in this region (31), there are no data for localization of CGRP receptors on GnRH neurons.
To test our hypothesis that CGRP has a crucial role in mediating stress-induced suppression of the reproductive neuroendocrine axis, we examined the effect of exogenous and endogenous CGRP on pulsatile LH secretion, a marker of the activity of the hypothalamic GnRH pulse generator, and determined whether a CGRP receptor antagonist can block the inhibitory effect of CGRP and insulin-induced hypoglycemic stress on LH pulses. In addition, immunocytochemical staining for c-Fos protein, the product of the immediate-early gene, c-fos, and marker of neuronal activation, was used to establish whether the preoptic area and hypothalamic PVN are implicated in the response to centrally administered CGRP.
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Materials and Methods |
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Effect of CGRP on LH pulses
The effect of human CGRP (Phoenix Europe GMBH, Karlsruhe, Germany) on pulsatile LH secretion was examined using both central and peripheral routes of administration. On the morning of experimentation, an icv injection cannula (Plastics One) with extension tubing, preloaded with drug or vehicle, was inserted into the guide cannula. The distal end of the tubing was extended outside of the animal cage to allow remote infusion without disturbing the rat during the experiment. Rats were then attached via one of the two cardiac catheters to a computer-controlled automated blood sampling system, which allows for the intermittent withdrawal of small blood samples (25 µl) without disturbing the rats (35). Once connected, the animals were left undisturbed for 1 h before blood sampling commenced. Experimentation commenced between 0900 and 1100 h when blood samples were taken every 5 min for 6 h. After removal of each 25-µl blood sample, an equal volume of heparinized saline (10 U/ml normal saline; CP Pharmaceuticals Ltd., Wrexham, UK) was automatically infused into the animal to maintain patency of the catheter and blood volume. Blood samples were frozen at -20 C for later assay to determine LH concentrations. After 2 h of blood sampling, CGRP was infused into the lateral ventricles over 4 min. Each rat received a single dose of 75 pmol, 400 pmol, or 1.2 nmol CGRP in 4 µl of artificial cerebrospinal fluid (aCSF) (n = 6–11 per treatment group). Control rats received 4 µl aCSF icv (n = 7). To test the specificity of the CGRP, six rats were coadministered CGRP (400 pmol) and CGRP antagonist (1 nmol human CGRP8–37, Phoenix Europe GMBH) by icv injection. Controls received CGRP8–37 (1 nmol) alone (n = 7). Blood sampling for later LH measurement was carried out as described above.
To determine whether peripheral CGRP affects LH pulses, 400 pmol CGRP was administered by bolus iv injection after a 2-h control blood sampling period and then sampling continued for an additional 4 h as described above (n = 9). Samples were assayed for LH.
To test whether the effect of CGRP on LH pulses was mediated via CRH, animals were coadministered CGRP (400 pmol) and a CRH receptor antagonist (-helical CRF9–41, 26 nmol) by icv injection (n = 10). Blood sampling for later LH measurement was carried out as described above.
CGRP and hypoglycemic stress
To test the hypothesis that endogenous CGRP mediates hypoglycemic stress-induced suppression of pulsatile LH secretion, we examined the effect of a CGRP receptor antagonist on this response. After an overnight fast, rats were connected to the automated blood sampling system. Blood sampling commenced between 0900 and 1100 h and continued for 6 h, as above, and blood samples assayed for LH. Every 60 min, blood samples (0.10 ml) were drawn manually from the second catheter to determine blood glucose concentrations, which were measured using a Reflolux S blood glucose monitor (Boehringer Mannheim, Lewes, UK). After 1 h and 50 min of automated blood sampling, the CGRP antagonist (5 nmol CGRP8–37) was administered over 4 min via icv injection (n = 8). Ten minutes later, a single iv injection of 0.5 U/kg insulin (Nordisk Wellcome Human Insulin, Crawley, UK) in 0.2 ml saline was given. For the 45-min period after insulin administration, blood glucose concentrations were monitored every 5 min. Eight rats were treated with this regimen. In addition to the measurement of blood glucose, some of the blood samples collected manually for this purpose were split and retained for later measurement of plasma corticosterone levels. Corticosterone was measured at the following time points: -60, -0, +15, +30, +45, +60, +90, +120, and +240 min relative to the injection of insulin. Control rats (n = 7) received 4 µl aCSF icv instead of CGRP antagonist 10 min before insulin administration.
FOS immunocytochemistry and microscopic analysis
To determine the effect of CGRP on FOS expression in the brain, ovariectomized rats were administered CGRP (1.2 nmol, n = 7) or aCSF (4 µl, n = 7) by icv injection. Ninety minutes later, the rats were deeply anesthetized and transcardically perfused with heparinized saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and immediately placed in a postfix solution of 15% sucrose in 4% paraformaldehyde at 4 C until they sank. They were then transferred into a 30% solution at 4 C for overnight. Brains were stored at -80 C for later immunocytochemical detection of FOS protein. The hypothalami were coronally sectioned (30 µm), and every fourth section was used for FOS immunoreactivity. Free-floating sections were permeablized in 0.3% Triton X-100 (Sigma Chemicals Ltd.) before being incubated in 0.02% H2O2 and blocked in 2% normal goat serum in PBS. Sections were incubated in 1:100,000 polyclonal rabbit anti-c-fos primary antibody (AB-5 OSI, Oncogene Science, San Diego, CA) containing 1.25% normal goat serum at 4 C for 36 h. Sections were then rinsed before incubation in 1:1000 biotinylated goat antirabbit secondary antibody (BA-1000; Vector Laboratories, Burlingame, CA) for 2 h at room temperature followed by 1:1500 conjugated peroxidase streptavidin for an additional 2 h at room temperature. Sections were then rinsed, and Vector’s diaminobenzidine substrate kit was used to visualize FOS staining, intensified with nickel ammonium chloride (Vector Laboratories) for 5 min. Sections were then mounted on slides, dehydrated, and coverslipped.
Semiquantitative analysis of immunostaining for FOS was carried out on a Zeiss (Oberkochen, Germany) AxioVision microscope image system. All analyses were performed on coded slides by an investigator who did not have knowledge of the experimental treatment conditions. The boundaries of regions counted were determined by comparing the Paxinos and Watson’s rat brain atlas (34) with neuroanatomical and cytoarchitectural landmarks. Sections used for preoptic area analysis were taken from the region corresponding to bregma +0.20 to -0.40 mm, and for the hypothalamic PVN they corresponded to bregma -1.80 to -2.12 mm. The number of immunostaining cells for FOS in each brain region was determined bilaterally in four sections from each rat. Neurons expressing FOS immunoreactivity were counted with bright-field microscopy at x100 magnification. Fine focusing was performed to ensure counting of all immunostaining cells throughout the thickness of the sections.
RIA for LH and corticosterone
A double antibody RIA supplied by the National Institute of Diabetes and Digestive and Kidney Diseases was used to determine LH concentration in the 25-µl whole blood sample. The sensitivity of the assay was 0.093 ng/ml. The intraassay variation was 5.8%, and the interassay variation was 5.0%. Total corticosterone was determined in plasma (5 µl) by RIA using a rat corticosterone kit (ICN Diagnostics, Orangeburg, NY). The sensitivity of the assay was 7.5 ng/ml. The intra- and interassay variations were 11.8 and 15.4%, respectively.
Statistical analysis
Detection of LH pulses was established by use of the algorithm ULTRA (37). Two intraassay coefficients of variation of the assay were used as the reference threshold for the pulse detection. The effect of treatment on pulsatile LH secretion was calculated by comparing the mean LH pulse interval before and after administration of drug and expressed as "prolongation of LH pulse interval" as a percentage of the pretreatment control value. The decrease in blood glucose concentrations in response to insulin was determined by comparing the mean glucose level before insulin injection with the mean blood glucose concentration during the 45-min period after insulin injection. The inhibitory effect of hypoglycemic stress on LH pulses was calculated by comparing the mean LH pulse interval before insulin with the first interval after administration and expressed as prolongation of LH pulse interval as the percentage by which the mean first interval exceeds the pretreatment control value. Statistical significance was tested using one-way ANOVA and Dunnett’s test. P < 0.05 was considered statistically significant.
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Results |
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, B–D and H). The inhibitory response was immediate in onset (Fig. 1, C and D) and in the case of the highest dose (1.2 nmol CGRP) resulted in a complete suppression of LH pulses for the duration of the 4-h posttreatment period in six of nine rats, and, therefore, for the purpose of analysis, these were assigned a value of 4 h for the posttreatment LH pulse interval. In the remaining animals, one to three pulses were observed in the posttreatment period. To investigate the receptor specificity of the action of CGRP on GnRH pulse generator activity, a CGRP antagonist (1 nmol CGRP8–37) was coadministered with CGRP (400 nmol) and found to completely block the inhibitory response (Fig. 1, F and H). Intracerebroventricular administration of CGRP antagonist (1 nmol CGRP8–37) alone did not affect LH pulses (Fig. 1, E and H), nor did the administration of the vehicle, aCSF (Fig. 1, A and H). To further demonstrate that the inhibitory effect of CGRP on pulsatile LH secretion involves a central site of action, the effect of peripherally administered CGRP was examined. Intravenous injection of CGRP (400 pmol) did not affect LH interpulse interval (27.5 ± 2.2 min before vs. 26.3 ± 1.3 min after CGRP injection; mean ± SEM; P > 0.05; Fig. 1G).
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-helical CRF9–41, 26 nmol). It was found that the CRH antagonist was effective in blocking the immediate CGRP-induced suppression of LH pulses, but only for approximately the first 2 h after treatment (Fig. 2, B and C). During the second 2-h period, examined LH pulses were suppressed to a similar extent as in animals injected with CGRP (400 pmol) alone (Fig. 2, A and C).
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, A and C; P < 0.05), as previously demonstrated (14). Despite exhibiting a comparable decrease in blood glucose, administration of 5 nmol CGRP8–37 icv 10 min before the injection of insulin completely prevented the interruption of pulsatile LH release in response to this stress (Fig. 3, B and C; P < 0.05). There were no differences in stress-associated increases in plasma corticosterone between the CGRP antagonist-treated and aCSF control animals during hypoglycemic stress (17,700 ± 1,380 ng/ml·h and 17,787 ± 2,667 ng/ml·h, mean ± SEM; P > 0.05, area under the curve for CGRP antagonist- and aCSF-treated animals, respectively). The interval for area under the curve measures was 1 min, and the reference was zero.
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, A and E) or PVN (Fig. 4, B and E) 90 min after icv administration of 4 µl aCSF. In contrast, the number of FOS-positive cells increased dramatically in the preoptic area (Fig. 4, C and E) and PVN (Fig. 4, D and E) after CGRP (1.2 nmol icv) administration.
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Discussion |
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Although the mechanisms underlying this action of CGRP on the reproductive neuroendocrine axis are not yet known, CGRP has been shown to evoke a classic stress response, namely stimulation of the HPA axis with a rise in circulating levels of corticosterone, which is mediated by CRH (2). It is well established that CRH also plays a central role in stress-induced suppression of the GnRH pulse generator. Central infusion of a CRH receptor antagonist reverses the LH pulse-suppressing effects of a variety of stressful stimuli, including insulin-induced hypoglycemia, fasting, and foot shock (9, 14, 21, 22). We found that central coadministration of a CRH antagonist with CGRP was able to block the CGRP-induced suppression of LH pulses. Interestingly, whereas the effect of CGRP on LH pulse frequency was evident for at least 4 h, the duration of the posttreatment period examined, the CRH antagonist was only effective for the first 2 h. This could be due to the fact that a single injection of CRH antagonist icv is only effective for a limited period. In this regard, we have observed, in preliminary studies, that a single administration of CRH antagonist into the brainstem nucleus, the locus coeruleus, will only block restraint stress-induced suppression of LH pulses for approximately 30 min, with repeated injections required to maintain effectiveness (Mitchell, J. C., and K. T. O’Byrne, personal observation). Despite the limited duration of the CRH antagonist effect, such a blockade for the initial 2 h postinjection clearly indicates an involvement of CRH in the LH response to central CGRP. Although an evaluation of the effect of icv administration of CRH antagonist alone on LH pulse frequency was not carried out in the present study, we have observed that such treatment does not affect pulsatile LH secretion in the rat (Cates, P. S., and K. T. O’Byrne, personal observation) or rhesus monkey (14). Thus, we propose that the inhibitory effect of CGRP on pulsatile release of LH is mediated, at least in part, by CRH. Moreover, these data raise the possibility that CGRP may be a higher-order regulator of stress-induced suppression of the GnRH pulse generator, involving a novel interaction with CRH. Nevertheless, a direct action of CGRP on the GnRH pulse generator cannot be excluded.
The CGRP neuronal populations involved in the suppression of the reproductive axis remain to be established. However, the presence of CGRP fibers and binding sites has been demonstrated in several key brain regions involved in the regulation of the stress and reproductive axes, including the preoptic area, PVN, and limbic nuclei (31, 32). Indeed, intraparaventricular nuclear administration of CGRP stimulates ACTH and corticosterone release (40), and in vitro studies have shown that CGRP stimulates the release of both ACTH secretagogues, CRH and vasopressin, from hypothalamic explants (40). However, the role of PVN CRH in the control of pulsatile LH secretion is controversial. Although there is a rise in CRH mRNA expression in the PVN in response to a variety of stressors that suppress LH pulses, including hypoglycemic stress (36), and various pharmacological experiments have provided evidence that stress-activated inputs, including noradrenergic input, to the PVN suppress LH pulses via CRH (38), bilateral electrolytic lesions of the PVN fail to prevent the inhibition of LH secretion in response to various stressors (41). Furthermore, there is a scarcity of PVN projections to the GnRH-rich regions of the preoptic area (25, 26). Although the CGRP receptor antagonist completely blocked hypoglycemic stress-induced suppression of pulsatile LH secretion, it did not affect the stress-induced rise in corticosterone. Similarly, CRH receptor antagonists can block the inhibitory effect of hypoglycemic stress on the GnRH pulse generator (9, 14, 15), without attenuating the stress-induced rise in glucocorticoids (14). Although the simultaneous release of CRH and vasopressin in hypoglycemic stress (42, 43) provides an explanation for the continuing activation of the HPA axis in the presence of a CRH receptor antagonist, the same may be true in the presence of a CGRP receptor antagonist. Nevertheless, we have previously shown that vasopressin does not mediate the hypoglycemic-induced inhibition of GnRH pulse generator frequency in the rat (35) and rhesus monkey (14). Thus, it is arguable whether there is likely to be any contribution from the central components of the HPA axis, viz. the CRH and vasopressin cells in the PVN, toward the suppression of LH release in response to hypoglycemic stress.
Despite efforts to control for circadian influences on the stress response by conducting the experiments at the same time of day, it must be recognized that experiments commenced between 0900 and 1100 h, which represents a circadian period in which responsiveness of the HPA system to perturbation is probably at a minimum (44). Thus, we cannot rule out the possibility that the absence of a differential effect of the CGRP antagonist vs. vehicle on hypoglycemic stress-induced corticosterone release may be attributable to these circadian influences. Similarly, sleep-related modulation of the HPA axis response must be considered (45), because experimentation commenced at the beginning of the rest phase in the rat, and thus may have also attributed to an absence of CGRP modulation of the corticosterone response to hypoglycemia.
In addition to the PVN, however, a strategic anatomical overlap between CGRP and CRH neurons is also evident in the limbic nuclei, the central nucleus of the amygdala (46), and the bed nucleus of the stria terminalis (47), which are also strongly implicated in stress-induced activation of the HPA axis (48). Furthermore, intraamygdala injections of CGRP induce stress-related behaviors (7, 49), and the amygdala has also been shown to have an inhibitory influence on LH secretion (50). Although CGRP-binding sites, neurons, and fibers are found in the preoptic area (31, 32), functional interactions with GnRH and/or CRH neurons in this region remain to be examined. Nevertheless, we have shown that icv administration of CGRP induced marked neuronal activation, measured by FOS expression, in the preoptic area. However, the neurochemical phenotype of the FOS-activated neurons remains to be examined.
Although we have shown that CGRP plays a pivotal role in hypoglycemic stress-induced suppression of the reproductive neuroendocrine axis, the heterogeneity in the neurochemical mediators of different stressors and stressor-specific neural circuitry in the central nervous system (48) necessitates study of the role of CGRP in other stress paradigms that impact on the reproductive axis. In addition, the CGRP-related peptide, adrenomedullin, has been shown to evoke stress responses, including activation of the HPA axis (51), and may therefore also affect the activity of the GnRH pulse generator. This is currently under investigation.
It is interesting to note that CGRP is also implicated in the regulation of anorectic behavior (5). Fasting is a potent stimulus for suppression of the GnRH pulse generator in both animals and humans (33, 52). There is also increasing evidence that caloric deficits underlie exercise-induced disturbances in menstrual cyclicity (53). Thus, study of a potential role for CGRP in coordinating behavioral and neuroendocrine sequelae of metabolic perturbations is timely. A newly developed nonpeptide CGRP receptor antagonist that is currently undergoing clinical trials for the treatment of neurovascular disorders (54) represents a new class of compounds that can target CGRP receptors. Establishing the role of CGRP in stress responses may be of clinical interest in providing a new and more effective treatment strategy for ovulatory dysfunction caused by depression and stress-related disorders. This is the first report showing that CGRP can influence the reproductive system. CGRP acts centrally to profoundly suppress the activity of the hypothalamic GnRH pulse generator and is critically involved in hypoglycemic stress-induced suppression of the reproductive system.
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Acknowledgments |
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Footnotes |
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X.F.L. and J.E.B. are joint first authors.
Abbreviations: aCSF, Artificial cerebrospinal fluid; CGRP, calcitonin gene-related peptide; E2, 17ß-estradiol; HPA, hypothalamo-pituitary-adrenocortical (axis); HPG, hypothalamo-pituitary-gonadal (axis); icv, intracerebroventricular; PBN, parabrachial nucleus; PVN, paraventricular nucleus.
Received November 26, 2003.
Accepted for publication January 7, 2004.
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) of (A) 4 µl aCSF, (B) 75 pmol CGRP, (C) 400 pmol CGRP (D) 1.2 nmol CGRP, (E) 1 nmol CGRP receptor antagonist (CGRP8–37), and (F) 400 pmol CGRP and 1 nmol CGRP8–37 on pulsatile LH secretion in ovariectomized rats. G, The effect of a bolus iv injection (
) of insulin (0.5 U/kg). Injection of icv aCSF did not prevent the interruption of LH pulses caused by hypoglycemic stress. CGRP8–37 completely blocked the hypoglycemic stress-induced interruption of LH pulses. Blood glucose and corticosterone levels were not significant difference between the CGRP8–37 and aCSF-treated animals. C, Summary showing the blockade of the inhibitory response to insulin-induced hypoglycemia by CGRP8–37. #, P < 0.05 vs. aCSF control; n = 7–8 per group. *, LH pulse.