Role of Corticotropin-Releasing Factor Receptor-2 in Stress-Induced Suppression of Pulsatile Luteinizing Hormone Secretion in the Rat
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内分泌学杂志 2005年第1期
Division of Reproductive Health, Endocrinology, and Development (X.F.L., J.E.B., K.T.O.), New Hunt’s House, King’s College London, London SE1 1UL, United Kingdom; and Henry Wellcome Laboratory for Integrative Neuroscience and Endocrinology (S.L.L.), University of Bristol, Bristol BS1 3NY, United Kingdom
Address all correspondence and requests for reprints to: Dr. Kevin O’Byrne, Division of Reproductive Health, Endocrinology, and Development, 2.36D New Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom. E-mail: kevin.o’byrne@kcl.ac.uk.
Abstract
Corticotropin-releasing factor (CRF) has been implicated as an important mediator of stress-induced inhibition of reproduction. The role of specific CRF receptor subtypes in this effect is unknown, and in the current study, we investigated the role of the CRF-R2 receptor in stress-mediated suppression of pulsatile LH section. Ovariectomized rats with sc 17?-estradiol capsules were implanted with intracerebroventricular (icv) and iv cannulae. Blood samples (25 μl) were collected every 5 min for 5 h for LH measurement. Central administration of urocortin II (0.24, 2.4, 24, or 240 nmol, icv), which selectively binds to CRF-R2, resulted in a dose-dependent suppression of LH pulses. Restraint stress (1 h) induced a profound suppression of pulsatile LH secretion and astressin2-B, a selective CRF-R2 antagonist (28 nmol icv, 10-min prerestraint), was effective in blocking this inhibitory response. These findings suggest that CRF-R2 mediates, at least in part, restraint stress-induced inhibition of LH pulses and 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
STRESS ACTIVATES THE hypothalamic-pituitary-adrenocortical (HPA) axis and suppresses the hypothalamic-pituitary-gonadal (HPG) axis, specifically pulsatile secretion of GnRH, through a mechanism that is not clearly understood. Restraint, a psychological/physical stress, results in a profound suppression of pulsatile LH secretion in a variety of species, including rats (1), sheep (2), and monkeys (3). It is well established that corticotropin-releasing factor (CRF), the principal factor driving the HPA axis during stress, is a potent inhibitor of the GnRH pulse generator (4, 5, 6). Central administration of CRF inhibits LH pulses (4, 5, 6), and suppression of LH secretion by a variety of stressful stimuli can be reversed by CRF antagonists (6, 7, 8). In addition to CRF, three CRF-related neuropeptides, urocortin (Ucn) (9), UcnII (10), and UcnIII (11), have been identified. Physiological effects of the CRF family are mediated by two receptor subtypes, CRF-R1 and CRF-R2 (12). Although CRF has a 15-fold higher affinity for CRF-R1 than for CRF-R2, Ucn binds with equally high affinity to both receptors (9). On the other hand, UcnII and UcnIII bind selectively to CRF-R2, with no appreciable activity on CRF-R1 and are thus considered endogenous ligands for CRF-R2 (10, 11).
The pivotal role of CRF in the HPA response to stress is mediated by CRF-R1 (13). Although CRF-R1 is also implicated in anxiogenic responses to stress (14), the role of CRF-R2 in mediating behavioral indices of stress is controversial with evidence for both anxiolytic- (15, 16) and anxiogenic-like effects (17, 18). However, the role of CRF-R2 in stress-induced suppression of the GnRH pulse generator has not been investigated. The present study was designed to investigate whether intracerebroventricular (icv) administration of the selective CRF-R2 agonist, UcnII, inhibits LH pulses and determine whether central administration of the selective CRF-R2 antagonist, astressin2-B, can block restraint stress-induced suppression of pulsatile LH secretion.
Materials and Methods
Animals and surgical procedures
Adult female Wistar rats, weighing 220–250 g, obtained from Bantin and Kingman, (Hull, UK), were housed under controlled conditions (14-h light, 10-h dark cycle, with lights on at 0700 h; temperature 22 ± 2 C) and provided with food and water ad libitum. All animal procedures were undertaken in accordance with the United Kingdom Home Office Regulations (Project License PPL: 70/4993). All surgical procedures were carried out under ketamine (100 mg/kg ip; Pharmacia and Upjohn Ltd., Crawley, UK) and Rompun (10 mg/kg ip; Bayer, Leverkusen, Germany) anesthesia. Because it is well established that 17?-estradiol (E2) replacement in ovariectomized animals enhances stress- and CRF-induced suppression of LH pulses (6, 19), rats were bilaterally ovariectomized and implanted with a SILASTIC (Dow Corning Corp., Midland, MI) capsule (inner diameter, 1.57 mm; outer diameter, 3.18 mm; supplied by Sani-tech, Havant, UK), filled to a length of 25 mm with E2 (Sigma Chemicals Ltd., Poole, UK) dissolved at a concentration of 20 μg/ml peanut oil (Sigma). The E2-containing capsules should produce circulating concentrations of E2 within the range observed during the diestrous phase of the estrous cycle (38.8 ± 1.2 pg/ml) as previously described by Maeda and colleagues (19). At the time of ovariectomy, all rats were also fitted with an icv guide cannula (22-gauge; Plastics One, Inc., Roanoke, VA) positioned into the left lateral cerebral ventricle; the coordinates for implantation were 1.5 mm lateral, 0.6 mm posterior to bregma, and 4.5 mm below the surface of the dura (20). The guide cannula was secured using dental cement (Dental Filling Ltd., Swindon, UK) and fitted with a dummy cannula (Plastics One) to maintain patency. After a 10-d recovery period, the rats were fitted with two indwelling cardiac catheters via the jugular veins (21). The catheters were exteriorized at the back of the head and secured to a cranial attachment; the rats were fitted with a 30-cm-long metal spring tether (Instec Laboratories Inc., Boulder, CO). The distal end of the tether was attached to a fluid swivel (Instec Laboratories), which allowed the rat freedom to move around the enclosure. Experimentation commenced 3 d later.
Effect of UcnII on LH pulses
The effect of mouse UcnII (Sigma) on pulsatile LH secretion was examined using the icv route 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 (21). Once connected, the animals were left undisturbed for 1 h before blood sampling commenced. Experimentation commenced between 0900 and 1000 h when blood samples were taken every 5 min for 5 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, UcnII was infused into the lateral ventricles over 4 min. Each rat received a single dose of 0.24, 2.4, 24, or 240 nmol UcnII in 4 μl of artificial cerebrospinal fluid (aCSF) (n = 7–9 per treatment group). Control rats received 4 μl of aCSF icv (n = 5).
Restraint stress and astressin2-B
To test whether the restraint stress-induced suppression on LH pulses was mediated via CRF-R2, we examined the effect of a CRF-R2 selective receptor antagonist, astressin2-B, on this response. Rats were connected to the automated blood sampling system. Blood sampling commenced between 0900 and 1100 h and continued for 5 h, as above, and blood samples assayed for LH. After 1 h 50 min of blood sampling, astressin2-B (28 nmol in 4 μl of aCSF) was administered over 4 min via icv injection (n = 9). Ten minutes later, the rats were placed in a restraint device for 60 min (1). Automated blood sampling continued uninterrupted during restraint and the 2-h postrestraint period. Control rats received aCSF in place of the CRF antagonist (n = 7). Additional controls were left to roam freely around their enclosure after infusion of aCSF (n = 5) or astressin2-B (n = 3) into the lateral cerebral ventricle. Blood samples (50 μl) were also collected manually for corticosterone measurement via the second cardiac catheter 15 min before and 30, 60, and 120 min after the onset of restraint stress.
RIA for LH and corticosterone
A double-antibody RIA supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 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 (10 μl) by RIA using a rat corticosterone kit (MP Biomedicals Inc., Costa Mesa, CA). 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 (22). 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. The effect of restraint stress on pulsatile LH secretion was calculated by comparing the mean LH interpulse interval before, during, and after restraint. For the 2-h prerestraint, the 1-h restraint, and 2-h postrestraint periods, the LH interpulse interval was calculated by dividing the appropriate period duration by the number of pulses. When there were no LH pulses during the 1-h restraint period, the LH interpulse interval assigned to the restraint period was taken as the interval from the onset of restraint to the first LH pulse in the 2-h postrestraint period. The LH interpulse interval assigned to the subsequent postrestraint period in those particular examples was calculated by dividing the 2-h postrestraint period by the number of pulses in that period. Mean levels of corticosterone after drug administration were compared with pretreatment values. Statistical significance was tested using one-way ANOVA and Dunnett’s test. P < 0.05 was considered statistically significant.
Results
Effect of UcnII on LH pulses
Administration of UcnII into the lateral cerebral ventricle resulted in a dose-dependent suppression of pulsatile LH secretion (Figs. 1, B–D, and 2). The increase in LH pulse interval in response to 0.24, 2.4, 24, or 240 nmol UcnII was evident for the duration of the 3-h posttreatment period. Control infusion of aCSF or the lowest dose of UcnII (0.24 nmol) had no effect on LH pulse frequency (Figs. 1A and 2).
FIG. 1. Representative examples showing the effects of icv injection () of 4 μl aCSF (A) and 2.4 (B), 24 (C), and 240 nmol (D) of the selective CRF-R2 agonist UcnII on pulsatile LH secretion in ovariectomized E2-replaced rats. Note the increase in LH pulse interval in response to UcnII was evident for the duration of the 3-h posttreatment period and was dose dependent. Treatment with aCSF alone had no effect on LH pulse interval. *, LH pulse.
FIG. 2. Summary showing the dose-dependent inhibitory effect of icv administration of UcnII on pulsatile LH secretion in ovariectomized E2-replaced rats. Treatment with the lowest dose of UcnII (0.24 nmol) or aCSF alone had no effect on LH interpulse intervals. *, P < 0.05 vs. 0.24 and 2.4 nmol UcnII (n = 7–9 per group) and control (aCSF) group (n = 5) at the same time point. #, P < 0.05 vs. 0.24 nmol UcnII and control (aCSF) groups at the same time point and 2 h pretreatment period.
Effect of astressin2-B on restraint stress-induced suppression of LH pulses
Restraint stress suppressed pulsatile LH secretion as previously demonstrated (1). LH pulses were profoundly suppressed immediately after the rats were replaced in restraint devices, and the LH pulse interval did not return to normal within the 2 h posttreatment period in most animals (Figs. 3B and 4). The LH pulses were completely abolished in three of eight animals during the 1-h restraint and subsequent 2-h postrestraint periods. Intracerebroventricular administration of the CRF-R2-specific antagonist (astressin2-B, 28 nmol) 10 min before the onset of restraint stress blocked the inhibition of pulsatile LH release in response to this stress (Figs. 3C and 4). Control rats treated with aCSF (Figs. 3A and 4) or astressin2-B (Figs. 3D and 4) alone without restraint did not show any change in LH pulse frequency. There was no significant difference in baseline (prerestraint) plasma concentrations of corticosterone between the astressin2-B treated and aCSF control animals (56.1 ± 13.4 ng/ml; 48.1 ± 12.8 ng/ml, respectively; mean ± SEM). Restraint stress rapidly increased circulating levels of corticosterone, which reached peak concentrations at 60 min after the onset of restraint. There were no differences in these stress-associated peak plasma levels of corticosterone between the astressin2-B treated (575.4 ± 65.8 ng/ml) and aCSF control animals (589.2 ± 49.4 ng/ml). The area under the curve for astressin2-B and aCSF-treated animal during restraint stress were 18,322 ± 1191 ng/ml·h and 18,157 ± 2081 ng/ml·h, mean ± SEM; P > 0.05, respectively. The interval for area under the curve measures was 1 min, and the reference was zero.
FIG. 3. The effects of icv injection () of the selective type 2 CRF receptor antagonist, astressin2-B, on restraint () stress-induced suppression of LH pulsatile secretion in ovariectomized E2-replaced rats. A, Representative example showing regular LH pulses in a rat administered (icv) 4 μl aCSF after 1 h 50 min of a 5-h blood sampling experiment. B, After a 2-h control blood sampling period, restraint (1 h) profoundly suppressed LH pulses. C, Administration of astressin2-B (28 nmol, icv) 10 min before the onset of restraint (1 h) completely blocked stress-induced suppression of LH pulses. D, Astressin2-B (28 nmol, icv) alone had no effect on LH pulse interval. *, LH pulse.
FIG. 4. Effect of astressin2-B on restraint stress-induced suppression of pulsatile LH secretion in ovariectomized E2-replaced rats. The mean LH interpulse interval during the 1-h restraint and 2-h postrestraint stress periods was significantly prolonged, compared with the 2-h prerestraint control period (n = 7). The inhibitory effect on LH pulses induced by restraint was reversed by administration of the selective CRF-R2 antagonist, astressin2-B (28 nmol, icv) 10 min before the onset of restraint stress (n = 9). Treatment with aCSF (n = 5) or astressin2-B (n = 3) in the absence of restraint stress had no effect on LH interpulse interval. *, P < 0.05 vs. baseline control period, restraint plus astressin2-B group 1 h restraint and 2 h postrestraint periods, and posttreatment aCSF or astressin2-B without restraint.
Discussion
Our data show for the first time that CRF-R2 plays a pivotal role in stress-induced suppression of pulsatile LH secretion. Central (icv) administration of the selective CRF-R2 antagonist, astressin2-B, blocked restraint stress-induced suppression of LH pulses in female ovariectomized, E2-replaced Wistar rats. In addition, we show for the first time that icv administration of the selective CRF-R2 agonist, UcnII, resulted in a dose-dependent suppression of LH pulse frequency. These data extend the results of previous studies showing that CRF can suppress LH pulses in a variety of species (4, 5, 6) and that nonselective CRF antagonists can block the suppression of LH pulses by various stressors, including insulin-induced hypoglycemia (6, 7) and foot shock (4).
The neuroendocrine and behavioral response to stress is important for survival and is clearly complex, involving many neural circuits, transmitters, and receptors. The CRF system is clearly critical to many of these responses, and there is increasing evidence for specificity of the CRF-R1 and CRF-R2 systems. It is well established that CRF-R1 is the primary receptor subtype that mediates the HPA response to stress and CRF-R1 knockout mice show a markedly blunted ACTH response to restraint stress (23). Interestingly, restraint stress also induces UcnII expression in approximately 50% of the parvocellular cells of the paraventricular nucleus (PVN) (24), although withdrawal of corticosteroid feedback, another classic activator of CRF, has no effect on UcnII mRNA, suggesting that although these cells are activated by stress, they are not part of the classic HPA neuroendocrine control system. It has been suggested that changes in HPA responses to stress may involve functional antagonism between CRF-R1 and CRF-R2 (10). CRF-R2 knockout mice exposed to restraint stress show rapid elevations of ACTH levels, and the increased corticosterone levels are sustained much longer, compared with the wild-type animals (25). Central administration of CRF-R2 selective antagonists, both sauvagine and astressin2-B, did not alter stress-induced ACTH secretion (26, 27). Furthermore, although astressin2-B effectively blocked restraint stress-induced suppression of pulsatile LH secretion, it did not affect the stress-induced rise of corticosterone in the present study.
The role of PVN CRF in the control of pulsatile LH secretion is controversial. Although there is a rise in CRF mRNA expression in the PVN in response to a variety of stressors that suppress LH pulses, including hypoglycemic stress, the degree of activation of the HPA axis did not always parallel the degree of the suppression of LH pulses (21). Moreover, we have recently shown that complete habituation of LH pulse suppression in response to repeated restraint stress was accompanied by maintenance of positive HPA axis activation, indicated by markedly increased corticosterone levels (1). This suggests differential control mechanisms underlying the response of the HPA and HPG axes to stress. Furthermore, CRF receptor antagonists can block the inhibitory effect of hypoglycemic stress on the GnRH pulse generator without attenuating the stress-induced rise in glucocorticoids (7). It is also reported that bilateral electrolytic lesions of the PVN fail to prevent the inhibition of LH secretion in response to various stressors (28). These observations suggest that differential control mechanisms underlie the response of the HPA and HPG axes to restraint stress, and the interaction between reproductive function and stress may involve the CRF outside the PVN.
In addition to its presence in the PVN, UcnII mRNA is also expressed in other stress-related cell groups including the arcuate nucleus of the hypothalamus and the locus ceruleus (10). The locus ceruleus is of particular interest because this brain stem nucleus is strongly implicated in stress-induced suppression of the hypothalamic GnRH pulse generator via a CRF-mediated mechanism (29). Central administration of UcnII induces Fos expression, a mark of neuronal activation, in the central nucleus of the amygdala, the bed nucleus of the stria terminalis, and the PVN (10); areas that have been implicated in physiological and behavioral responses to stress. In keeping with the differential effects of CRF and UcnII, the receptors CRF-R1 and CRF-R2 exhibit distinct distribution patterns in the brain. Studies in rats have revealed an abundance of CRF-R1 in pituitary, cerebral cortex, sensory relay nuclei, and cerebellum, whereas CRF-R2 is generally localized to specific subcortical structures, including the lateral septum and various hypothalamic nuclei, in particular the ventromedial nucleus (30). CRF-R1 and CRF-R2 receptors are also both present in the bed nucleus of the stria terminalis and the amygdala complex, although differential anatomical location is evident in both structures (30). At the level of the GnRH neurons themselves, there is no clear evidence of a close relationship with CRF-R2 terminals, but in view of the evidence for CRF-R2 regulation of pulsatile LH secretion demonstrated in our studies, there is clearly a need for further experimentation to elucidate the neuroanatomical substrate underlying this effect.
In summary, the present study provides evidence that CRF-R2 mediates, at least in part, restraint stress-induced suppression of pulsatile LH secretion. This was demonstrated by blockade of stress-induced suppression of pulsatile LH secretion by central administration of the selective CRF-R2 antagonist, astressin2-B. Furthermore, central administration of UcnII resulted in a profound suppression of LH pulses, which is consistent with a role for CRF-R2 in regulation of pulsatile LH secretion. Whereas the current study did not address a potential pituitary site of action for UcnII on LH secretion, this cannot be excluded. Whether UcnII itself or another CRF-R2 ligand is involved in the CRF-R2 mediated inhibition of LH pulses in response to stress remains to be investigated. Establishing the UcnII neuronal populations involved in regulation of the reproductive axis and its detailed mechanism at the cellular and molecular levels will be of interest in a better understanding of reproductive dysfunction caused by depression and stress-related disorders.
Acknowledgments
The authors thank Dr. A. F. Parlow (NIDDK) for providing the LH RIA kit and J. Rivier and W. W. Vale for their generous gift of astressin2-B.
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Address all correspondence and requests for reprints to: Dr. Kevin O’Byrne, Division of Reproductive Health, Endocrinology, and Development, 2.36D New Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom. E-mail: kevin.o’byrne@kcl.ac.uk.
Abstract
Corticotropin-releasing factor (CRF) has been implicated as an important mediator of stress-induced inhibition of reproduction. The role of specific CRF receptor subtypes in this effect is unknown, and in the current study, we investigated the role of the CRF-R2 receptor in stress-mediated suppression of pulsatile LH section. Ovariectomized rats with sc 17?-estradiol capsules were implanted with intracerebroventricular (icv) and iv cannulae. Blood samples (25 μl) were collected every 5 min for 5 h for LH measurement. Central administration of urocortin II (0.24, 2.4, 24, or 240 nmol, icv), which selectively binds to CRF-R2, resulted in a dose-dependent suppression of LH pulses. Restraint stress (1 h) induced a profound suppression of pulsatile LH secretion and astressin2-B, a selective CRF-R2 antagonist (28 nmol icv, 10-min prerestraint), was effective in blocking this inhibitory response. These findings suggest that CRF-R2 mediates, at least in part, restraint stress-induced inhibition of LH pulses and 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
STRESS ACTIVATES THE hypothalamic-pituitary-adrenocortical (HPA) axis and suppresses the hypothalamic-pituitary-gonadal (HPG) axis, specifically pulsatile secretion of GnRH, through a mechanism that is not clearly understood. Restraint, a psychological/physical stress, results in a profound suppression of pulsatile LH secretion in a variety of species, including rats (1), sheep (2), and monkeys (3). It is well established that corticotropin-releasing factor (CRF), the principal factor driving the HPA axis during stress, is a potent inhibitor of the GnRH pulse generator (4, 5, 6). Central administration of CRF inhibits LH pulses (4, 5, 6), and suppression of LH secretion by a variety of stressful stimuli can be reversed by CRF antagonists (6, 7, 8). In addition to CRF, three CRF-related neuropeptides, urocortin (Ucn) (9), UcnII (10), and UcnIII (11), have been identified. Physiological effects of the CRF family are mediated by two receptor subtypes, CRF-R1 and CRF-R2 (12). Although CRF has a 15-fold higher affinity for CRF-R1 than for CRF-R2, Ucn binds with equally high affinity to both receptors (9). On the other hand, UcnII and UcnIII bind selectively to CRF-R2, with no appreciable activity on CRF-R1 and are thus considered endogenous ligands for CRF-R2 (10, 11).
The pivotal role of CRF in the HPA response to stress is mediated by CRF-R1 (13). Although CRF-R1 is also implicated in anxiogenic responses to stress (14), the role of CRF-R2 in mediating behavioral indices of stress is controversial with evidence for both anxiolytic- (15, 16) and anxiogenic-like effects (17, 18). However, the role of CRF-R2 in stress-induced suppression of the GnRH pulse generator has not been investigated. The present study was designed to investigate whether intracerebroventricular (icv) administration of the selective CRF-R2 agonist, UcnII, inhibits LH pulses and determine whether central administration of the selective CRF-R2 antagonist, astressin2-B, can block restraint stress-induced suppression of pulsatile LH secretion.
Materials and Methods
Animals and surgical procedures
Adult female Wistar rats, weighing 220–250 g, obtained from Bantin and Kingman, (Hull, UK), were housed under controlled conditions (14-h light, 10-h dark cycle, with lights on at 0700 h; temperature 22 ± 2 C) and provided with food and water ad libitum. All animal procedures were undertaken in accordance with the United Kingdom Home Office Regulations (Project License PPL: 70/4993). All surgical procedures were carried out under ketamine (100 mg/kg ip; Pharmacia and Upjohn Ltd., Crawley, UK) and Rompun (10 mg/kg ip; Bayer, Leverkusen, Germany) anesthesia. Because it is well established that 17?-estradiol (E2) replacement in ovariectomized animals enhances stress- and CRF-induced suppression of LH pulses (6, 19), rats were bilaterally ovariectomized and implanted with a SILASTIC (Dow Corning Corp., Midland, MI) capsule (inner diameter, 1.57 mm; outer diameter, 3.18 mm; supplied by Sani-tech, Havant, UK), filled to a length of 25 mm with E2 (Sigma Chemicals Ltd., Poole, UK) dissolved at a concentration of 20 μg/ml peanut oil (Sigma). The E2-containing capsules should produce circulating concentrations of E2 within the range observed during the diestrous phase of the estrous cycle (38.8 ± 1.2 pg/ml) as previously described by Maeda and colleagues (19). At the time of ovariectomy, all rats were also fitted with an icv guide cannula (22-gauge; Plastics One, Inc., Roanoke, VA) positioned into the left lateral cerebral ventricle; the coordinates for implantation were 1.5 mm lateral, 0.6 mm posterior to bregma, and 4.5 mm below the surface of the dura (20). The guide cannula was secured using dental cement (Dental Filling Ltd., Swindon, UK) and fitted with a dummy cannula (Plastics One) to maintain patency. After a 10-d recovery period, the rats were fitted with two indwelling cardiac catheters via the jugular veins (21). The catheters were exteriorized at the back of the head and secured to a cranial attachment; the rats were fitted with a 30-cm-long metal spring tether (Instec Laboratories Inc., Boulder, CO). The distal end of the tether was attached to a fluid swivel (Instec Laboratories), which allowed the rat freedom to move around the enclosure. Experimentation commenced 3 d later.
Effect of UcnII on LH pulses
The effect of mouse UcnII (Sigma) on pulsatile LH secretion was examined using the icv route 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 (21). Once connected, the animals were left undisturbed for 1 h before blood sampling commenced. Experimentation commenced between 0900 and 1000 h when blood samples were taken every 5 min for 5 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, UcnII was infused into the lateral ventricles over 4 min. Each rat received a single dose of 0.24, 2.4, 24, or 240 nmol UcnII in 4 μl of artificial cerebrospinal fluid (aCSF) (n = 7–9 per treatment group). Control rats received 4 μl of aCSF icv (n = 5).
Restraint stress and astressin2-B
To test whether the restraint stress-induced suppression on LH pulses was mediated via CRF-R2, we examined the effect of a CRF-R2 selective receptor antagonist, astressin2-B, on this response. Rats were connected to the automated blood sampling system. Blood sampling commenced between 0900 and 1100 h and continued for 5 h, as above, and blood samples assayed for LH. After 1 h 50 min of blood sampling, astressin2-B (28 nmol in 4 μl of aCSF) was administered over 4 min via icv injection (n = 9). Ten minutes later, the rats were placed in a restraint device for 60 min (1). Automated blood sampling continued uninterrupted during restraint and the 2-h postrestraint period. Control rats received aCSF in place of the CRF antagonist (n = 7). Additional controls were left to roam freely around their enclosure after infusion of aCSF (n = 5) or astressin2-B (n = 3) into the lateral cerebral ventricle. Blood samples (50 μl) were also collected manually for corticosterone measurement via the second cardiac catheter 15 min before and 30, 60, and 120 min after the onset of restraint stress.
RIA for LH and corticosterone
A double-antibody RIA supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 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 (10 μl) by RIA using a rat corticosterone kit (MP Biomedicals Inc., Costa Mesa, CA). 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 (22). 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. The effect of restraint stress on pulsatile LH secretion was calculated by comparing the mean LH interpulse interval before, during, and after restraint. For the 2-h prerestraint, the 1-h restraint, and 2-h postrestraint periods, the LH interpulse interval was calculated by dividing the appropriate period duration by the number of pulses. When there were no LH pulses during the 1-h restraint period, the LH interpulse interval assigned to the restraint period was taken as the interval from the onset of restraint to the first LH pulse in the 2-h postrestraint period. The LH interpulse interval assigned to the subsequent postrestraint period in those particular examples was calculated by dividing the 2-h postrestraint period by the number of pulses in that period. Mean levels of corticosterone after drug administration were compared with pretreatment values. Statistical significance was tested using one-way ANOVA and Dunnett’s test. P < 0.05 was considered statistically significant.
Results
Effect of UcnII on LH pulses
Administration of UcnII into the lateral cerebral ventricle resulted in a dose-dependent suppression of pulsatile LH secretion (Figs. 1, B–D, and 2). The increase in LH pulse interval in response to 0.24, 2.4, 24, or 240 nmol UcnII was evident for the duration of the 3-h posttreatment period. Control infusion of aCSF or the lowest dose of UcnII (0.24 nmol) had no effect on LH pulse frequency (Figs. 1A and 2).
FIG. 1. Representative examples showing the effects of icv injection () of 4 μl aCSF (A) and 2.4 (B), 24 (C), and 240 nmol (D) of the selective CRF-R2 agonist UcnII on pulsatile LH secretion in ovariectomized E2-replaced rats. Note the increase in LH pulse interval in response to UcnII was evident for the duration of the 3-h posttreatment period and was dose dependent. Treatment with aCSF alone had no effect on LH pulse interval. *, LH pulse.
FIG. 2. Summary showing the dose-dependent inhibitory effect of icv administration of UcnII on pulsatile LH secretion in ovariectomized E2-replaced rats. Treatment with the lowest dose of UcnII (0.24 nmol) or aCSF alone had no effect on LH interpulse intervals. *, P < 0.05 vs. 0.24 and 2.4 nmol UcnII (n = 7–9 per group) and control (aCSF) group (n = 5) at the same time point. #, P < 0.05 vs. 0.24 nmol UcnII and control (aCSF) groups at the same time point and 2 h pretreatment period.
Effect of astressin2-B on restraint stress-induced suppression of LH pulses
Restraint stress suppressed pulsatile LH secretion as previously demonstrated (1). LH pulses were profoundly suppressed immediately after the rats were replaced in restraint devices, and the LH pulse interval did not return to normal within the 2 h posttreatment period in most animals (Figs. 3B and 4). The LH pulses were completely abolished in three of eight animals during the 1-h restraint and subsequent 2-h postrestraint periods. Intracerebroventricular administration of the CRF-R2-specific antagonist (astressin2-B, 28 nmol) 10 min before the onset of restraint stress blocked the inhibition of pulsatile LH release in response to this stress (Figs. 3C and 4). Control rats treated with aCSF (Figs. 3A and 4) or astressin2-B (Figs. 3D and 4) alone without restraint did not show any change in LH pulse frequency. There was no significant difference in baseline (prerestraint) plasma concentrations of corticosterone between the astressin2-B treated and aCSF control animals (56.1 ± 13.4 ng/ml; 48.1 ± 12.8 ng/ml, respectively; mean ± SEM). Restraint stress rapidly increased circulating levels of corticosterone, which reached peak concentrations at 60 min after the onset of restraint. There were no differences in these stress-associated peak plasma levels of corticosterone between the astressin2-B treated (575.4 ± 65.8 ng/ml) and aCSF control animals (589.2 ± 49.4 ng/ml). The area under the curve for astressin2-B and aCSF-treated animal during restraint stress were 18,322 ± 1191 ng/ml·h and 18,157 ± 2081 ng/ml·h, mean ± SEM; P > 0.05, respectively. The interval for area under the curve measures was 1 min, and the reference was zero.
FIG. 3. The effects of icv injection () of the selective type 2 CRF receptor antagonist, astressin2-B, on restraint () stress-induced suppression of LH pulsatile secretion in ovariectomized E2-replaced rats. A, Representative example showing regular LH pulses in a rat administered (icv) 4 μl aCSF after 1 h 50 min of a 5-h blood sampling experiment. B, After a 2-h control blood sampling period, restraint (1 h) profoundly suppressed LH pulses. C, Administration of astressin2-B (28 nmol, icv) 10 min before the onset of restraint (1 h) completely blocked stress-induced suppression of LH pulses. D, Astressin2-B (28 nmol, icv) alone had no effect on LH pulse interval. *, LH pulse.
FIG. 4. Effect of astressin2-B on restraint stress-induced suppression of pulsatile LH secretion in ovariectomized E2-replaced rats. The mean LH interpulse interval during the 1-h restraint and 2-h postrestraint stress periods was significantly prolonged, compared with the 2-h prerestraint control period (n = 7). The inhibitory effect on LH pulses induced by restraint was reversed by administration of the selective CRF-R2 antagonist, astressin2-B (28 nmol, icv) 10 min before the onset of restraint stress (n = 9). Treatment with aCSF (n = 5) or astressin2-B (n = 3) in the absence of restraint stress had no effect on LH interpulse interval. *, P < 0.05 vs. baseline control period, restraint plus astressin2-B group 1 h restraint and 2 h postrestraint periods, and posttreatment aCSF or astressin2-B without restraint.
Discussion
Our data show for the first time that CRF-R2 plays a pivotal role in stress-induced suppression of pulsatile LH secretion. Central (icv) administration of the selective CRF-R2 antagonist, astressin2-B, blocked restraint stress-induced suppression of LH pulses in female ovariectomized, E2-replaced Wistar rats. In addition, we show for the first time that icv administration of the selective CRF-R2 agonist, UcnII, resulted in a dose-dependent suppression of LH pulse frequency. These data extend the results of previous studies showing that CRF can suppress LH pulses in a variety of species (4, 5, 6) and that nonselective CRF antagonists can block the suppression of LH pulses by various stressors, including insulin-induced hypoglycemia (6, 7) and foot shock (4).
The neuroendocrine and behavioral response to stress is important for survival and is clearly complex, involving many neural circuits, transmitters, and receptors. The CRF system is clearly critical to many of these responses, and there is increasing evidence for specificity of the CRF-R1 and CRF-R2 systems. It is well established that CRF-R1 is the primary receptor subtype that mediates the HPA response to stress and CRF-R1 knockout mice show a markedly blunted ACTH response to restraint stress (23). Interestingly, restraint stress also induces UcnII expression in approximately 50% of the parvocellular cells of the paraventricular nucleus (PVN) (24), although withdrawal of corticosteroid feedback, another classic activator of CRF, has no effect on UcnII mRNA, suggesting that although these cells are activated by stress, they are not part of the classic HPA neuroendocrine control system. It has been suggested that changes in HPA responses to stress may involve functional antagonism between CRF-R1 and CRF-R2 (10). CRF-R2 knockout mice exposed to restraint stress show rapid elevations of ACTH levels, and the increased corticosterone levels are sustained much longer, compared with the wild-type animals (25). Central administration of CRF-R2 selective antagonists, both sauvagine and astressin2-B, did not alter stress-induced ACTH secretion (26, 27). Furthermore, although astressin2-B effectively blocked restraint stress-induced suppression of pulsatile LH secretion, it did not affect the stress-induced rise of corticosterone in the present study.
The role of PVN CRF in the control of pulsatile LH secretion is controversial. Although there is a rise in CRF mRNA expression in the PVN in response to a variety of stressors that suppress LH pulses, including hypoglycemic stress, the degree of activation of the HPA axis did not always parallel the degree of the suppression of LH pulses (21). Moreover, we have recently shown that complete habituation of LH pulse suppression in response to repeated restraint stress was accompanied by maintenance of positive HPA axis activation, indicated by markedly increased corticosterone levels (1). This suggests differential control mechanisms underlying the response of the HPA and HPG axes to stress. Furthermore, CRF receptor antagonists can block the inhibitory effect of hypoglycemic stress on the GnRH pulse generator without attenuating the stress-induced rise in glucocorticoids (7). It is also reported that bilateral electrolytic lesions of the PVN fail to prevent the inhibition of LH secretion in response to various stressors (28). These observations suggest that differential control mechanisms underlie the response of the HPA and HPG axes to restraint stress, and the interaction between reproductive function and stress may involve the CRF outside the PVN.
In addition to its presence in the PVN, UcnII mRNA is also expressed in other stress-related cell groups including the arcuate nucleus of the hypothalamus and the locus ceruleus (10). The locus ceruleus is of particular interest because this brain stem nucleus is strongly implicated in stress-induced suppression of the hypothalamic GnRH pulse generator via a CRF-mediated mechanism (29). Central administration of UcnII induces Fos expression, a mark of neuronal activation, in the central nucleus of the amygdala, the bed nucleus of the stria terminalis, and the PVN (10); areas that have been implicated in physiological and behavioral responses to stress. In keeping with the differential effects of CRF and UcnII, the receptors CRF-R1 and CRF-R2 exhibit distinct distribution patterns in the brain. Studies in rats have revealed an abundance of CRF-R1 in pituitary, cerebral cortex, sensory relay nuclei, and cerebellum, whereas CRF-R2 is generally localized to specific subcortical structures, including the lateral septum and various hypothalamic nuclei, in particular the ventromedial nucleus (30). CRF-R1 and CRF-R2 receptors are also both present in the bed nucleus of the stria terminalis and the amygdala complex, although differential anatomical location is evident in both structures (30). At the level of the GnRH neurons themselves, there is no clear evidence of a close relationship with CRF-R2 terminals, but in view of the evidence for CRF-R2 regulation of pulsatile LH secretion demonstrated in our studies, there is clearly a need for further experimentation to elucidate the neuroanatomical substrate underlying this effect.
In summary, the present study provides evidence that CRF-R2 mediates, at least in part, restraint stress-induced suppression of pulsatile LH secretion. This was demonstrated by blockade of stress-induced suppression of pulsatile LH secretion by central administration of the selective CRF-R2 antagonist, astressin2-B. Furthermore, central administration of UcnII resulted in a profound suppression of LH pulses, which is consistent with a role for CRF-R2 in regulation of pulsatile LH secretion. Whereas the current study did not address a potential pituitary site of action for UcnII on LH secretion, this cannot be excluded. Whether UcnII itself or another CRF-R2 ligand is involved in the CRF-R2 mediated inhibition of LH pulses in response to stress remains to be investigated. Establishing the UcnII neuronal populations involved in regulation of the reproductive axis and its detailed mechanism at the cellular and molecular levels will be of interest in a better understanding of reproductive dysfunction caused by depression and stress-related disorders.
Acknowledgments
The authors thank Dr. A. F. Parlow (NIDDK) for providing the LH RIA kit and J. Rivier and W. W. Vale for their generous gift of astressin2-B.
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