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NEUROENDOCRINOLOGY |
Imperial College School of Medicine Endocrine Unit, Hammersmith Hospital, London W12 ONN, United Kingdom
Address all correspondence and requests for reprints to: Prof. S. R. Bloom, Endocrine Unit, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: s.bloom{at}ic.ac.uk
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Introduction |
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Previous studies have shown that injection of orexin A into the lateral cerebral ventricle stimulates plasma LH in ovariectomized (OVEX) steroid-replaced rats (7). By contrast, orexin A inhibits plasma LH when injected into the lateral or third ventricle in OVEX unreplaced rats (7, 8). However, the hypothalamic mechanism by which orexin A alters the HPG axis remains to be determined. These studies suggest that orexin A may be important in the regulation of the HPG axis and that the endocrine milieu may influence orexin A actions in the HPG. This ovarian steroid-dependent bimodal LH response to orexin A is similar to that of other orexigenic neuropeptides, such as NPY (9, 10). We have previously shown that orexin A stimulates NPY release from hypothalamic explants in vitro (11). We hypothesized that the hypothalamic action of orexin A on the HPG axis might be mediated through NPY, possibly via the NPY Y1 receptor.
We explored the effects of orexin A on LHRH release in hypothalamic explants from male and female rats at different stages on the estrus cycle and on LH release from dispersed pituitaries. We examined the effect of estrous cycle stage on LHRH release from hypothalamic explants in vitro and on orexin A content in hypothalami, pituitaries, and a number of other brain regions in vivo. We investigated the effects of chronic endocrine manipulations on hypothalamic orexin A and LHRH contents and on hypothalamic prepro orexin mRNA in female rats. We also measured orexin A content in pituitary and other brain regions in these female rats. We aimed to demonstrate steroid-dependent effects on orexin A release from hypothalamic explants in vitro. Finally, we examined whether NPY (via the NPY Y1 receptor) may be involved in mediating the hypothalamic stimulation of orexin A on LHRH release, using the selective NPY Y1 receptor antagonist BIBP3226 on hypothalamic explants in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and tissues
Male and female Wistar rats (Central Biological Services,
Imperial College, London, UK) (males, 150–250 g; females, 200–250 g)
were housed in cages of five with ad libitum access to food
and water under controlled temperature (21–23 C) and light (12 h of
light and 12 h of dark). Intact/normal, sham-operated and OVEX
female Wistar rats (Charles River Laboratories, Inc.,
Wilmington, MA) weighing 220–250 g were housed as above. The
stage of the estrous cycle and the effects of endocrine manipulation
were determined by examination of vaginal cytology, as previously
described (12, 13). Animals were killed by decapitation
for hypothalamic explant experiments and for harvesting of brain
regions and trunk blood in endocrine-manipulated and normally cycling
females. Animals were killed by CO2 asphyxiation
for pituitary dispersion experiments. Animal procedures undertaken were
all approved by the British Home Office Animals Scientific Procedures
Act 1986 (Project License no. PPL 90/1077 and PPL 90/1066).
Static incubation of hypothalamic explants
The static incubation system was used as previously described
(14). Rats were killed by decapitation, whole brain was
removed immediately, and a 1.7-mm hypothalamic slice was taken and
placed into tubes containing artificial cerebrospinal fluid (aCSF), as
previously described (11). The tubes were maintained at 37
C in a water bath and equilibrated with 95% O2
and 5% CO2. Before being challenged with the
agent of interest, hypothalami in all experiments were allowed an
initial 2-h equilibration period, during which 1 ml of fresh aCSF was
replaced every 60 min.
Effect of orexin A on LHRH release in hypothalamic explants harvested from male rats and from female rats at different stages of the estrous cycle. Hypothalami from female rats at different stages of the cycle were harvested during the afternoon after establishing cycle stage by vaginal smears to encourage a proportion to be captured at the late proestrous stage. Hypothalamic explant experiments using male rats were performed as usual in the morning. After the initial 2 h equilibration period, all hypothalami were incubated for 45 min with 600 µl aCSF (basal period) before being challenged with 100 nM orexin A in 600 µl aCSF for 45 min (test period). The viability of the tissue was confirmed by a 45-min exposure to 600 µl 56 mM KCl solution, with isotonicity maintained by substituting K+ for Na+ (i.e. 56 mM KCl aCSF). At the end of each period, the aCSF was removed and frozen at -20 C until measurement of LHRH.
Effect of estrous cycle stage on basal and potassium-stimulated orexin A release. To explore orexin A release from hypothalamic explants on the morning of proestrus relative to estrus and metestrus, hypothalami from female rats at different stages of the cycle were harvested, and experiments were performed in the morning. After the initial 2-h equilibration period, the hypothalami were incubated for 45 min with 600 µl aCSF (basal period) before being challenged with 600 µl KCl aCSF (56 mM) for 45 min (test period). At the end of each period, the aCSF was removed and frozen at -20 C until measurement of orexin A.
Effect of estrogen on orexin A release in hypothalamic explants harvested from males. Hypothalami from male rats were used in preference to females to simplify for cycle stage. After the initial 2-h equilibration period, hypothalami were incubated for 1 h with 600 µl aCSF (basal period) before being challenged with 100 nM 17ß-E2 in 600 µl aCSF for 1 h (test period). The viability of the hypothalamic explants was confirmed by a 1-h exposure to 600 µl 56 mM KCl aCSF. At the end of each period, the aCSF was removed and frozen at -20 C until measurement of orexin A.
Effect of a selective NPY Y1 receptor antagonist (BIBP3226) on LHRH release with orexin A. After the initial 2-h equilibration period, hypothalami harvested from male rats were incubated for 45 min with 600 µl aCSF (basal period) before being challenged with 100 nM orexin A in 600 µl aCSF with or without 1000 nM BIBP3226 or 1000 nM BIBP3226 in 600 µl aCSF alone (test periods). The viability of the hypothalamic explants was confirmed by a 45-min exposure to 600 µl 56 mM KCl aCSF. At the end of each period, the aCSF was removed and frozen at -20 C until measurement of LHRH.
Each hypothalamic explant experiment was performed either two or three times (n = 30 per experiment). Hypothalami releasing less than 100% of basal peptide after exposure to 56 mM KCl aCSF solution were assumed not to be viable and were not included in the final data.
Anterior pituitary dispersions
The anterior pituitaries that were harvested from male or
proestrous female rats (200–250 g) were dispersed, and secretion
experiments were performed using a method adapted from Childs et
al. (15) and as previously described (12, 16).
Effects of orexin A on basal and LHRH-stimulated LH release. The effects of 0.01–100 nM orexin A on basal LH release and LH release after submaximal stimulation by 10 nM LHRH were assessed in dispersed pituitaries from male rats. The effects of 0.01–100 nM orexin A on basal LH release and 0.01–1000 nM orexin A on LH release after submaximal stimulation by 1 nM LHRH were assessed in dispersed pituitaries harvested from proestrous female rats. Medium from all experimental wells was collected and frozen at -20 C until LH was measured by RIA. Three independent pituitary dispersion and secretion experiments were performed (n = 3).
Monitoring of intact estrous cycles
Rats were smeared daily for the duration of two cycles, and
cycling rats were killed at different stages of the estrous cycle
(n = 7–10 per estrous cycle stage). Orexin A peptide content was
measured in 17 brain regions harvested as described below.
Chronic endocrine manipulations in female rats and measurement of
prepro orexin mRNA by Northern blot
Female Wistar rats weighing 200–250 g were OVEX or
sham-operated in house and allowed 4 d postoperative recovery
before the 22-d study. Endocrine manipulations used and doses of
estrogen and progesterone chosen were based on a previously published
study (17). Uninjected OVEX control rats (n = 10)
were maintained for 26 d and killed during diestrus. OVEX and
physiological estrogen-replaced (E2R) rats were given daily
physiological E2 replacement by sc injection (5 µg 17ß-E2
3-benzoate per rat in 100 µl safflower oil) for 22 d and
killed, with vaginal smear appearance similar to metestrus or
estrus. Intact hyperestrogenized (HyperE2) rats (n = 10)
were given sc depot injections of 2 mg 17ß-E2 3-benzoate in 100 µl
safflower oil every 7 d and killed, with vaginal smear appearance
similar to estrus. Sham-operated control rats (n = 10) were given
daily sc injections of vehicle alone (100 µl safflower oil) and
killed in diestrus. Cycle stage was assessed by vaginal smears
performed daily for the last 8 d of the study. Animals were killed
by decapitation. Plasma was collected and snap-frozen for subsequent
measurement of LH. Hypothalami were harvested and snap-frozen for
subsequent quantification of prepro orexin mRNA by Northern blot, using
materials and methods as previously described (18).
Chronic endocrine manipulations in female rats for measurement of
orexin A and LHRH peptide contents
OVEX, sham-operated, and intact female rats were purchased from
Charles River Laboratories, Inc. (as above) and allowed to
acclimatize for 4 d before the 14-d study. Endocrine manipulations
that were used and doses of estrogen and progesterone chosen were based
on a previously published study (17). Sham-operated
control rats (n = 8) were administered daily sc injections of
vehicle alone (100 µl safflower oil). Half were killed in estrus or
metestrus and half in late proestrus. Data from these groups (data not
shown) verified results obtained for orexin A measurements in extracts
from tissues harvested from cycling females as above. OVEX control rats
(n = 8) were maintained for 14 d and killed during diestrus.
OVEX and physiological E2R rats (n = 8) were given daily
physiological E2R by sc injection (5 µg 17ß-E2 3-benzoate per rat
in 100 µl safflower oil) from days 0 to 14 of the study and killed,
with vaginal smear appearance similar to metestrus or estrus. OVEX and
physiological progesterone-replaced (ProgR) rats (n = 8) were
given daily physiological progesterone sc injection (0.5 mg
progesterone per rat in 100 µl safflower oil) from days 0 to 14 of
the study and killed, with vaginal smear appearance similar to estrus
or proestrus. Intact HyperE2 rats (n = 8) were given sc injections
of 2 mg 17ß-E2 3-benzoate per rat in 100 µl safflower oil on days
0, 4, 8, and 12 of the study and killed, with vaginal smear appearance
similar to estrus. For all rats, daily vaginal smears were performed
during the last 8 d of the study. Animals were decapitated, and
pituitaries, brain regions, and plasma were harvested as below.
Harvesting and extraction of tissues for measurement of peptide
contents
Brains from animals with intact estrous cycles after chronic
hormone manipulations were rapidly dissected into multiple anatomical
regions (19) including the hypothalamus, thalamus,
olfactory tubercles, cortex (temporal cortex sampled), septum,
striatum, midbrain, pons, medulla, cerebellum, and pituitary. Tissues
were immediately snap-frozen and stored at -70 C until extracted as
previously described (20).
Hormone measurements (RIAs)
LHRH in aCSF and tissue extracts and LH in plasma and culture
medium from pituitary dispersions were measured by RIA, as previously
described (16). Results were calculated in terms of NIDDK
standard preparation and expressed either in femtomoles per explant,
nanograms per milliliter, or percentage of basal hormone release.
Orexin A was assayed in aCSF and in tissue extracts as previously
described (20). Intra-assay and interassay coefficients of
variation were 8 and 12%, respectively, for all assays.
Statistics
All data were presented as mean ± SEM. Data
from static incubation of hypothalamic explants were expressed as
femtomoles per explant and analyzed by paired t test between
basal and test period and between basal and 56 mM
KCl aCSF period. Data from the anterior pituitary dispersions were
compared by ANOVA, with post hoc Tukey’s test (Systat,
Evanston, IL) between control and experimental groups. For peptide
measurements in extracted tissues and plasma samples, statistical
difference between control and experimental groups was determined by
ANOVA, followed by post hoc least significant
difference (Systat 8.0, Evanston, IL). In all analyses,
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and
from female rats at different stages of the estrous cycle (Fig. 1B)). Orexin A (100 nM) significantly stimulated
LHRH release in hypothalamic explants harvested in the afternoon from
female rats at late proestrus, but not estrus or metestrus (Fig. 1B).
KCl aCSF (56 mM) significantly stimulated LHRH
release, verifying the viability of the explants at all stages of the
estrus cycle (Fig. 1B).
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).
Orexin A did not influence basal LH release in dispersed pituitaries
from males [basal 100 ± 10.62% vs. orexin A (100
nM) 98 ± 9.8% and orexin A (0.1
nM) 96.2 ± 8.2%, P = NS]
or proestrous female rats [basal 100 ± 10.3% vs.
orexin A (100 nM) 83.9 ± 14.8% and orexin
A (0.1 nM) 100.9.2 ± 7.9%,
P = NS]. Orexin A did not influence LHRH-stimulated LH
release in dispersed pituitaries from males [LHRH (10
nM) 194.3 ± 10.8% vs. orexin A
(100 nM) + LHRH (10 nM)
190 ± 7.9% and orexin A (0.1 nM) + LHRH
(10 nM) 194.9 ± 7.5%, P =
NS], but dose responsively inhibited LHRH-stimulated LH release in
dispersed pituitaries from proestrous female rats (Fig. 2).
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)). Midbrain orexin A
peptide content was higher at late proestrus relative to early
proestrus, estrus, metestrus, and diestrus (Fig. 3B). Medulla orexin A
peptide content was significantly higher at late proestrus than at
early proestrus and metestrus (Fig. 3C). Thalamus orexin A was higher
at late proestrus relative to all other stages of the estrous cycle
(Fig. 3D). No differences in orexin A peptide content were found
between different stages of the estrous cycle in any other brain region
examined (data not shown).
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, A–F) and on hypothalamic LHRH
content (Fig. 4G) and plasma LH
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), midbrain (Fig. 4B), medulla (Fig. 4C), thalamus
(Fig. 4D), olfactory tubercle (Fig. 4E), and cortex (Fig. 4F) relative
to OVEX controls. After chronic E2R, orexin A peptide content was
reduced in these same brain regions, although this only reached
significance in the thalamus (OVEX 25.9 ± 2.3 vs. E2R
18.8 ± 1.3 pmol/g of tissue; P < 0.05) (Fig. 4D). Chronic ProgR did not alter orexin A peptide content in any brain
region examined (Fig. 4, A–F). Hence, orexin A content in the HyperE2
group was also significantly lower than in the ProgR group for
hypothalamus (Fig. 4A), midbrain (Fig. 4B), thalamus (Fig. 4D),
olfactory tubercles (Fig. 4E), and cortex (Fig. 4F). No change in
orexin A peptide content was noted after any of the hormonal
manipulations in any other brain region examined (data not shown).
In contrast to orexin A peptide, which was lower after chronic HyperE2,
LHRH content was significantly higher in hypothalamus relative to OVEX
controls (Fig. 4G). No orexin A peptide was detected in pituitaries
harvested from any of the treatment groups (data not shown).
Effect of gonadal steroid hormone manipulation on prepro orexin
mRNA in hypothalami harvested from female rat (Fig. 5, panels A and B) and plasma LH
Plasma LH was significantly lower in the OVEX E2R and intact
HyperE2 groups relative to the OVEX group (OVEX 102.7 ± 6.9
vs. E2R 5.6 ± 0.5 and HyperE2 3.6 ± 0.3 ng/ml;
P < 0.000), verifying that the endocrine
manipulations were successful. There were no observable differences in
hypothalamic prepro orexin mRNA after any of the hormonal manipulations
relative to sham-operated control animals (Fig. 5, panels A and B).
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)). There was also a
slightly increased basal release of orexin A from hypothalamic explants
harvested at late proestrus (on the morning of late proestrus) relative
to estrus and metestrus, although this did not reach significance (Fig. 6). KCl (56 mM)-stimulated orexin A release was
significantly greater than basal orexin A release for explants
harvested at all stages of the estrous cycle (Fig. 6).
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Effect of a selective NPY Y1 receptor antagonist (BIBP 3226) on
LHRH release with orexin A: a possible mechanism of orexin A effect on
LHRH release (Fig. 7)
Orexin A did not stimulate LHRH release in the presence of the NPY
Y1 selective receptor antagonist, BIBP3226, and BIBP3226 alone did not
alter LHRH release in hypothalamic explants harvested from male rats
(Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Orexin A inhibited LHRH-stimulated LH release in dispersed anterior pituitaries from proestrous female rats, but not male rats, again suggesting the importance of gonadal milieu. Similarly, NPY has been shown to have effects on LH release in vitro that are dependent on gonadal steroid milieu (23, 24). The opposite effects of orexin A on the HPG at the pituitary and hypothalamus are not unusual and have been reported for other peptides (25). We were unable to detect orexin A peptide in whole pituitaries using our RIA, which would be expected if orexin A reached the pituitary via the hypophyseal-portal circulation. Recently, studies by Date et al. (26), using pooled pituitaries in their RIAs, immunohistochemistry, and in situ hybridization, have demonstrated the presence of both orexin peptide and receptors (types 1 and 2) in anterior, posterior, and intermediate pituitary lobe tissues and of orexin fibers in the median eminence (26). This suggests that orexin neurons may project to the hypothalamo-pituitary portal system in the median eminence, thus influencing anterior pituitary function.
Orexin A is synthesized almost exclusively in the posterolateral hypothalamus, and fibers project to many intra- and extrahypothalamic sites. We found a lower orexin A peptide content in hypothalami harvested from female rats at late proestrus relative to all other stages of the cycle. We also found a higher orexin A peptide content in midbrain, medulla, and thalamus harvested at late proestrus relative to all other stages of the cycle. These observations might reflect greatest release of orexin A from hypothalamic neuronal cell bodies into nerve endings at proestrus, resulting in lower hypothalamic orexin A levels at late proestrus. The higher orexin A levels in midbrain, medulla, and thalamus at late proestrus relative to other stages of the cycle are interesting and may reflect important reproductive roles for orexin A at these extrahypothalamic sites. These areas have been implicated in sex cycle-specific behaviors, such as lordosis and sexual receptivity in female rats (27, 28), suggesting a role for orexin A in these behaviors. There was no change in orexin A peptide content in temporo-frontal cortex, olfactory tubercles, inferior and superior colliculi, septum, pons, or amygdala with any stage of the cycle.
Hypothalamic LHRH content was significantly higher after chronic HyperE2 relative to OVEX rats. This is consistent with published reports (29) and suggests that peptide content is highest when release is at its lowest, for example due to feedback inhibition by chronic exogenous E2R or HyperE2. Our measurements of hypothalamic LHRH confirm that our hormone manipulation was successful.
After chronic HyperE2, orexin A content was significantly lower in hypothalamus, midbrain, medulla, thalamus, olfactory tubercle, and cortex relative to OVEX rats. Although chronic E2R tended to reduce orexin A peptide content in the same regions, this was only significant in the thalamus. These changes suggest that estrogen may cause orexin A to be released from specific sites. Hypothalamic synthesis of orexin A may not keep pace with its release, resulting in a reduction of orexin A in these sites.
It is interesting that chronic gonadal hormone manipulation changed orexin A peptide content in regions where orexin A content altered through the cycle (i.e. midbrain, medulla, and thalamus). In addition, chronic HyperE2 altered orexin A content in the cortex and olfactory tubercles, regions that are known to be sexually dimorphic (30, 31, 32). During pregnancy, when gonadal steroid concentrations are very elevated, there are alterations in arousal that may be influenced by orexin A.
No changes in orexin A peptide content were noted in any brain region after chronic ProgR in OVEX rats, relative to OVEX controls. Progesterone action requires an estrogen background for the synthesis of progesterone receptors (33). The absence of estrogen in these OVEX ProgR rats might compromise any potential progestogenic effect on orexin A release. Similarly, the changes in orexin A content observed after chronic E2R in OVEX rats were not significant. The absence of progesterone in these OVEX E2R rats may have attenuated the estrogenic effects on orexin A release.
Chronic endocrine manipulations for measurement of hypothalamic prepro orexin mRNA were carried out for 26 d, compared with 14 d for orexin A peptide measurements. This was to ensure adequate time for message changes to occur. No change in prepro orexin mRNA was found in hypothalami after chronic endocrine manipulations. The differences in hypothalamic orexin A peptide content in cycling and endocrine-manipulated animals were of a small order of magnitude. This may explain why no changes in prepro orexin mRNA were observed. In addition, the methods used may have been insufficiently sensitive to detect relatively small changes in message expression.
Changes in message expression after chronic endocrine manipulations, if they exist, are therefore likely to very small. We explored whether the changes we observed in orexin A peptide content could be due to changes in orexin A release rather than altered expression. We found that in hypothalamic explants harvested from female rats in vitro, orexin A release with 56 mM KCl aCSF was greatest on the morning of proestrus relative to estrus and metestrus. There was also a trend for increased basal orexin A release on the morning of proestrus relative to estrus or metestrus. This increased release on the morning of proestrus is consistent with the reduced hypothalamic orexin A peptide content we find at late proestrus.
E2 secreted by growing ovarian follicles has been considered classically to be the neural trigger for the preovulatory LHRH surge (33). In fact, at proestrus, plasma E2 peaks earlier than the reproductive hormones progesterone, LH, FSH, and PRL (34). We investigated whether 17ß-E2 3-benzoate altered orexin A release in vitro. To simplify for cycle stage, and because orexin A stimulated LHRH release in hypothalami harvested from male rats, we investigated the effect of 17ß-E2 3-benzoate on orexin A release using hypothalamic explants from male rats. There is increasing evidence that estrogens exert both genomic and nongenomic (acute) effects (35). Our experimental incubations were extended to 1 h to allow the minimum time for any potential genomic effect (36). Published studies also report peptide release after gonadal steroid incubations of only 1 h in vitro (37). The stimulation of orexin A release by 17ß-E2 3-benzoate in vitro may be mediated by genomic, nongenomic, or an indirect effect.
Hypothalamic prepro-NPY gene expression was found to increase after administration of ovarian steroids in OVEX rats, and the increase occurred before the onset of the LH surge induced (38). Furthermore, prepro-NPY mRNA levels in the medial basal hypothalamus were higher on the afternoon of proestrus and during the day of estrus and correlated with levels of LHRH mRNA throughout the estrous cycle (39). Hypothalamic NPY peptide content was also shown to rise during the proestrous phase of the estrous cycle (40). These changes in NPY gene expression and peptide appear to occur at a stage later than the changes in orexin A release observed in our experiments, but still before the LH surge. We have previously shown that orexin A stimulates NPY release from hypothalamic explants in vitro (11). It could be that NPY mediates the stimulatory effect of orexin A on LHRH release at the hypothalamus and that this effect occurs via the NPY Y1 receptor, which is known to mediate the LHRH surge. Consistent with this hypothesis, we have shown that a selective NPY Y1 receptor antagonist, BIBP3226, abolishes the stimulatory effect of orexin A on LHRH release from hypothalamic explants in vitro. We were unable to explore this further in vivo because intracerebroventricular administration of BIBP3226 causes a specific behavioral side effect known as barrel-rolling (41, 42). Despite being a specific high-affinity Y1 receptor antagonist, the use of BIBP 3226 in vivo is therefore restricted.
Our results suggest orexin A has important interactions in the HPG axis. However, it would be necessary to demonstrate a stimulatory effect of orexin A on LHRH release in vivo to confirm the physiological relevance. It could be that orexin A release at proestrus plays a role in stimulating the LHRH surge. The effect of orexin A on LHRH release may be mediated through NPY, possibly via the NPY Y1 receptor. Furthermore, the hypothalamic release of orexin A may be under the regulatory influence of gonadal steroid hormones.
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Acknowledgments |
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Footnotes |
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Received June 19, 2001.
Accepted for publication August 31, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R. E. Campbell, K. L. Grove, and M. S. Smith Gonadotropin-Releasing Hormone Neurons Coexpress Orexin 1 Receptor Immunoreactivity and Receive Direct Contacts by Orexin Fibers Endocrinology, April 1, 2003; 144(4): 1542 - 1548. [Abstract] [Full Text] [PDF]
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, P < 0.05 basal LHRH release at estrus
vs. basal LHRH release at proestrus.
25–35 per cycle stage). Rats were
smeared, and experiments were performed in the morning. Late proestrous
smears reflected orexin A release on the morning of proestrus. *,
P < 0.05 vs. estrus; **,
P < 0.004 vs. metestrus.