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Division of Endocrinology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115 (K.-H.J., L.J., J.A.M.); and Department of Biology, Boston University, Boston, Massachusetts 02215 (E.P.W.)
Address all correspondence and requests for reprints to: Joseph A. Majzoub, Division of Endocrinology, Children’s Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail: majzoub{at}a1.tch.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CRH has been proposed to negatively regulate GnRH secretion. An imbalance of central CRH has been implicated in the suppression of reproductive function in humans under stressful conditions. Patients with major depression or anorexia nervosa show suppressed reproductive function (9) and also have a high concentration of CRH in the cerebro-ventricular system (10). In rodents, intracerebroventricular (icv) administration of CRH attenuates GnRH level in the hypophysial portal circulation of female rats (11). Central, but not iv, injection of CRH also inhibits LH secretion over 5 h in a dose-dependent manner in ovariectomized female rats (12). These studies suggest a central inhibitory effect of endogenous CRH on the reproductive system, possibly acting upon GnRH neurons. Furthermore, 5 h of restraint, which activates the hypothalamic-pituitary-adrenocortical (HPA) axis and CRH gene expression (13), completely inhibits the proestrus LH surge and ovulation in intact cycling rats (14). Other stressors (undernutrition, hypoglycemia, or electric foot-shock) also inhibit LH secretion and pulsatility (15, 16, 17) as well as GnRH gene expression in the mPOA (18), and these effects are reversed following administration of a CRH antagonist in gonadectomized rats (17, 19) and monkeys (20). These results further suggest that CRH inhibits GnRH activity through CRH receptors.
However, it is unclear whether endogenous CRH is a relevant inhibitor of central reproductive function during a stress response under all circumstances. For example, arginine vasopressin (AVP) has also been found to inhibit LH secretion in monkeys (21), and CRH has been shown to stimulate LH secretion in gonadal steroid-treated sheeps (22). It is also possible that the CRH antagonists administered in many of the above studies might block the action of CRH-related molecules such as urocortin (23), rather than that of CRH.
We hypothesized that the stressor-induced inhibition of the hypothalamic-pituitary-gonadal axis activity in the mouse is mediated by CRH. To test this hypothesis, plasma levels of LH and reproductive steroid in CRH-deficient (knockout, KO) mice compared with wild-type (WT) mice were measured during restraint or food withdrawal. In some experiments, a CRH antagonist was administered to mice before stress, to determine whether stressor-induced inhibition of LH in CRH KO mice was due to a CRH-like molecule interacting with CRH receptors. Surprisingly, we found that genetic or pharmacologic CRH deficiency does not prevent stressor-mediated suppression of the murine reproductive axis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stressors
Restraint. Singly housed adult (3–5 months old) WT and CRH
KO male and female mice were subjected to restraint in ventilated 50-ml
tubes for 4 or 5 h. Tubes were designed to be small enough to
restrain a mouse so that it was able to breathe but unable to move
freely. Control mice were left unhandled in their cages. The duration
of restraint for female mice was timed to start just before the
beginning of the proestrus LH surge and to finish at the time of the
peak of the surge, 2 h before lights off.
Food withdrawal
Singly housed adult (3–4 months old) WT and CRH KO male and
female mice were subjected to food withdrawal for 36 h. All mice
were placed in new cages without food at the beginning of the food
withdrawal period until blood sample collection. Drinking water was
available throughout the experiment.
Estrous cycle monitoring
WT and CRH KO adult (3–5 months old) female mice were singly
housed on a 14-h, 10-h light-dark cycle, and estrous cycles were
monitored by daily vaginal cytology (25). Only mice regularly cycling
with an interval of 4–6 days were included in experiments.
Lateral ventricle cannulation
Singly housed adult (3–5 months old) WT and CRH KO female mice
received an icv cannula (C315; Plastics One, Roanoke, VA) into the
lateral ventricle under stereotaxic guidance (Model 900; DavidKopf
Instruments, Tujunga, CA) using 2.5% avertin anesthesia. A 26-gauge
guide cannula was inserted through the skull at coordinates 0.5-mm
posterior and 1.4-mm lateral to the bregma, and to a depth of 2.5 mm,
and a stylet was placed into the guide cannula to protect the lumen.
After cannulation, mice were allowed to recover for 1–2 estrous cycles
before experimentation. All infusions were made through a 33-gauge
internal cannula with a 10 µl Hamilton syringe under the rate control
of a syringe pump (Model 341; Orion Research, Cambridge, MA). Each
cannulation site was verified by India ink infusion through the guide
cannula into the lateral ventricle at the end of the experiment. The
infusion volume and rate for India ink were the same as those used for
the antagonist, 3 µl and 1.2 µl/min, respectively. Mice with
improperly positioned cannulation tracts were omitted from subsequent
data analysis.
Central administration of CRH antagonist
The CRH antagonist, 
-helical CRH9–41 (hCRH,
code 246–202-15), was kindly supplied by Dr. Jean Rivier (Salk
Institute, La Jolla, CA). Reconstitution of hCRH was performed as
previously reported (26). It was dissolved in 0.45%
saline-H2O with 0.1% BSA (vehicle), with the pH adjusted
under NaOH vapor. The final pH of the solution was 7.4. On a proestrus
day, each mouse received 0, 1, or 10 µg of hCRH via the icv route
(1.2 µl/min) in 3 µl of vehicle, 30 min before 4 h of
restraint, which ended 2 h before lights-off. As a control, some
animals received antagonist or vehicle via the icv route without
subsequent restraint.
Plasma sample collection
Blood sampling was performed by retro-orbital sinus phlebotomy
(
300 µl) into heparinized capillary tubes for both sexes. The
blood was collected on ice, and plasma was separated within 20 min from
cells by centrifugation at 4 C, and stored at -80 C in aliquots until
assayed. In the restraint experiments in females, blood was withdrawn
from each animal 2 h before lights-off on a day of proestrus at a
frequency of every 1–3 estrous cycles (every 4–12 days). To prevent
hypovolemia, 1 ml of sterile lactated Ringer’s and dextrose solution
(0.5% dextrose, 0.6% NaCl, 0.31% sodium lactate, 0.03% KCl, and
0.02% CaCl2, pH 5.0, 525 mOsmol/liter) was injected ip
following each phlebotomy.
In vitro ACTH secretion analysis
To verify the efficacy of the CRH antagonist, its effect on ACTH
secretion in vitro was tested. ACTH peptide was assayed in
media using a modification of previously reported methods (27) using
the mouse pituitary corticotroph cell line, AtT-20.
Hormone analysis
The LH RIA was performed by a double-antibody method using
reagents for the rat from NIDDK, NIH (rLH-I-9 for iodination,
AFP10250C; rLH-RP-3 for standard calibration (888 IU/mg), AFP7187B;
anti-rLH-RIA-11 for anti-LH antibody, AFPC697071P). LH RIA protocols
were kindly provided by Drs. Marie Gibson (Mount Sinai Medical Center,
New York, NY), Greg Miller (MGH, Boston, MA), and Jon E. Levine
(Northwestern University, Evanston, IL). Iodination of rat LH was
performed by the chloramine-T method (28). The specific activity for
rat LH iodination ranged between 0.13–0.17 mCi/µg, and the
incorporation ratio was 67–86%. The linear range of the LH standard
curve was between 0.3–5 ng/ml, and assay sensitivity was about 0.4
ng/ml, calculated as 95% of maximum binding. The precision of the LH
RIA was determined by calculating intra and interassay coefficients of
variance, which were 5.8% and 4.5%, respectively. Immunoreactive
corticosterone (ICN Pharmaceuticals, Inc.,
Orangeburg, NY), testosterone (ICN Pharmaceuticals, Inc.),
and ACTH (INCSTAR Corp., Stillwater, MN) were measured by
commercial RIA kits according to the manufacturers’ instructions or
using previously described modifications (29).
Data analysis
Results were analyzed by two-tailed Student’s t
tests. The unpaired t test was performed for the comparison
of differences between two groups, and the paired t test was
performed for the comparison of differences between two different
treatments within the same animals. Two-way ANOVA was performed for the
comparisons between multiple groups followed by multiple comparisons
using t tests with Bonferroni-Dunn posthoc correction. A
P value less than 0.05 was considered statistically
significant. All data are presented as the mean ±
SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As expected, after restraint, plasma corticosterone level rose over
4-fold in WT female mice. The plasma corticosterone level in CRH KO
female mice after restraint also increased significantly compared with
its basal level as shown in Fig. 1A
. As
shown in Fig. 1B, the proestrus LH surge in both WT and CRH KO female
mice was totally abolished following 5 h of restraint, indicating
that CRH is not required for restraint-induced suppression of the LH
surge in female CRH KO mice. A subsequent proestrus phase without
restraint (control) displayed a normal LH surge in both WT and CRH KO
female mice (Fig. 1B).
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hCRH, into a lateral ventricle of the
brains of WT and CRH KO female mice on a proestrus afternoon 30 min
before 4 h of restraint.
As shown in Fig. 2A, suppression of the
LH surge was similar after restraint in both genotypes, regardless of
whether mice were infused with vehicle or hCRH (hatched
vs. solid bars), suggesting that CRH or another
CRH-related molecule might not be involved in this suppression in
either genotype. A 10-fold higher dose (10 µg) of hCRH was also
not effective in the inhibition of restraint-induced LH surge
suppression (data not shown). Administration of hCRH (gray
bars) or vehicle (open bars) in the absence of
restraint did not disrupt the normal LH surge in either genotype,
indicating hCRH was not acting as a partial agonist. However, CRH KO
female mice showed a smaller LH surge than WT female mice in this
setting, although this was not statistically significant.
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hCRH infusion did not block the plasma corticosterone response to
restraint, as shown in Fig. 2B
(compare hatched and
solid bars). However, CRH-induced secretion of ACTH from
AtT-20 cells in vitro was attenuated by 100 or 1,000
nM of hCRH treatment (Table 1), confirming that the hCRH
preparation in the present study was bioactive.
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) were only about 50% those in
WT female mice (Fig. 1A), showing a sexually dimorphic adrenal response
to restraint. As in CRH KO female mice, CRH KO male mice showed a
significant, but attenuated increase in plasma corticosterone following
restraint despite the lack of CRH, consistent with our previous
findings (24). As shown in Fig. 3B, the plasma LH level in WT male mice
was suppressed following 5 h of restraint. CRH KO male mice showed
a similar trend (P = 0.06), suggesting a normal
suppressive response of the hypothalamic-pituitary-gonadal axis
following restraint. A normal stress response of gonadal testosterone
secretion following restraint was also present, as shown in Fig. 3C.
Basal plasma testosterone levels in WT and CRH KO male mice were
similar, and were both completely suppressed after 5 h of
restraint, consistent with the fall in LH in both genotypes.
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(females) and
5A (males). Both WT female and male mice
increased their plasma corticosterone levels markedly following
starvation. However, CRH KO female and male mice did not show any
change in plasma corticosterone levels following 36 h of food
withdrawal, suggesting an absolute requirement of CRH for HPA axis
response following this stressor, in contrast to that seen following
restraint (compare Fig. 4A, 5A with 1A,
3A). As shown in Fig. 4B, plasma LH levels in both CRH KO and WT female
mice, compared with controls, were blunted after 36 h of food
withdrawal, indicating that neither CRH nor a rise in corticosterone is
required for starvation-induced suppression of plasma LH. Male mice of
both genotypes also showed suppression of plasma LH levels, as shown in
Fig. 5B. In addition, plasma testosterone levels in male mice of both
genotypes were completely suppressed following 36 h of food
withdrawal, as shown in Fig. 5C.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hCRH, via an icv (17, 19) but not peripheral route (12, 17). An in vitro study also demonstrates a direct inhibitory
effect of CRH on GnRH release from the rat hypothalamus (35). An
anatomical study using CRH and GnRH double-labeling suggests a direct
neural connection between CRH axons and GnRH perikarya in the mPOA of
the rats (36). These results clearly suggest that the site of CRH’s
inhibitory activity is in the brain, and that CRH may act as an
important and necessary factor for the inhibition of LH secretion. Other studies concur with ours in suggesting the involvement of substances other than CRH in the negative regulation of the reproductive axis during stress (21, 34, 37). The suppression of reproductive function following activation of the HPA axis also appears to be species-specific. There is no correlation between fasting-induced suppression of LH secretion and changes in plasma cortisol levels in monkeys (38, 39), and hypoglycemia-induced activation of the HPA axis does not suppress LH secretion in women (40). Our results in mice differ from those reported in rats (12, 17). However, whether restraint is perceived to be equally stressful in mice and rats, or equally dependent upon CRH is unclear. Restraint in mice could be a greater stressor than in rats, and the suppressive effects of other stressors, such as mild electric foot-shock or immune stress, on the hypothalamic-pituitary-gonadal axis might be more readily antagonized by a CRH antagonist as previously reported (17).
The present study clearly shows that CRH deficiency does not prevent or even attenuate stressor-induced LH suppression in mice. Plasma LH levels were decreased following restraint or food withdrawal in both WT and CRH KO mice, suggesting that CRH is not a required inhibitory regulator of LH secretion during the stress response judged by our CRH KO mice model. Therefore, it is possible that other central factors known to restrain LH secretion, such as endogenous opioids (3, 33, 34, 41, 42), cytokines (4), or AVP (21, 43), may instead act together to inhibit LH secretion (1) in CRH deficiency to compensate the lack of CRH. For example, endogenous opioids have been shown to be involved in the inhibition of LH secretion in rats (33, 34, 41), in ewes (3), and in monkeys (42). Furthermore, nonCRH pathways controlling GnRH down-regulation during stress have been suggested (30, 34). In this regard, a more careful investigation for the potential roles of opioids and AVP in the absence of CRH would be beneficial.
It is also possible that another hypothalamic neuropeptide or CRH receptor ligand, such as urocortin, has increased input to GnRH neurons during pre- or postnatal development of the brain of CRH KO mice as a compensatory response to CRH deficiency. Compensatory or counteractive overexpression of related molecules has been reported in other gene knockout models (44, 45). However, CRH or urocortin are unlikely to mediate this response because the CRH antagonist used in the present study, which binds to both CRH type 1 and type 2 receptors (46, 47), was not able to block the restraint-induced suppression of the proestrus LH surge, even in WT mice.
Central hCRH infusion did not attenuate plasma corticosterone
secretion following restraint, even in WT female mice. Some (48, 49),
but not all (50, 51) studies have found that central infusion of the
CRH antagonist, hCRH, can block stressor-induced corticosterone
secretion in rats. In addition, hCRH has been shown to have variable
inhibitory effects on CRH action in different biological systems at
different doses (52), and to have poor CRH antagonist activity compared
with other CRH antagonist compounds (53). Although the same lot of
hCRH used in this study effectively prevented the secretion of ACTH
from mouse pituitary tumor cell line AtT-20 cells at 100 nM
concentration, the in vitro and in vivo
conditions are quite different. Although at high doses, hCRH may
have a partial agonist effect, this seems unlikely because this
activity was not seen at any dose of hCRH tested. However, it is
still possible that in our study, due to technical difficulties,
hCRH did not reach the correct sites within the brain to block CRH
receptors.
In conclusion, genetic or pharmacologic CRH deficiency does not prevent
suppression of plasma LH levels in mice following restraint or food
withdrawal. Furthermore, the involvement of other CRH-like molecules
acting on CRH receptors is unlikely, because central infusion of
hCRH does not prevent restraint-induced suppression of the LH surge
in either WT or CRH KO mice.
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Acknowledgments |
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hCRH, Marie Gibson, Greg
Miller, and Jon E. Levine for LH RIA protocols, John A. M.
Mattheij and Catherine Rivier for critical discussions, Stacie C.
Weninger for help with brain ventricle cannulation, and Chris Lage and
Allison Carrigan for animal maintenance. |
Footnotes |
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2 Present address: Division of Genetics, Brigham and Women’s
Hospital, Harvard Medical School, 20 Shattuck Street, Boston,
Massachusetts 02115.
Received August 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and 
-receptor
agonists on plasma luteinizing hormone (LH), prolactin, and the
estrogen-induced LH surge in ovariectomized ewes. Endocrinology 137:3640–3648[Abstract]
-helical corticotropin-releasing
factor9–41 in three bioassay systems. Endocrinology 129:1312–1316[Abstract]
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