Endocrinology Vol. 142, No. 5 1915-1922
Copyright © 2001 by The Endocrine Society
Endotoxin Inhibits Pituitary Responsiveness to Gonadotropin-Releasing Hormone1
Candace Y. Williams,
Thomas G. Harris,
Deborah F. Battaglia,
Catherine Viguié2 and
Fred J. Karsch
Reproductive Sciences Program and Department of Physiology,
University of Michigan, Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Dr. F. J. Karsch, Reproductive Science Program, University of Michigan, 300 North Ingalls Building, Room 1101 SW, Ann Arbor, Michigan 48109-0404. E-mail:
fjkarsch{at}umich.edu
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Abstract
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Immune/inflammatory challenges powerfully suppress reproductive
neuroendocrine activity. This inhibition is generally considered to be
centrally mediated via mechanisms that regulate GnRH secretion. The
present study provides two lines of evidence that bacterial endotoxin,
a commonly used model of immune/inflammatory challenge, also acts to
inhibit pituitary responsiveness to GnRH. In the first experiment,
pulsatile secretion of GnRH into pituitary portal blood and LH into
peripheral blood were monitored in ovariectomized ewes treated with a
low dose of endotoxin. Although this treatment only marginally
suppressed GnRH pulsatile secretion, it markedly disrupted LH
pulsatility. In extreme cases, the low dose of endotoxin blocked LH
pulses without inhibiting endogenous GnRH pulses, thereby uncoupling
GnRH and LH pulsatile suppression. In the second experiment, we tested
the hypothesis that endotoxin inhibits pituitary responsiveness to
exogenous GnRH pulses. Hourly pulses of GnRH were delivered to
ovariectomized ewes in which endogenous GnRH secretion was blocked.
Endotoxin suppressed the amplitude of GnRH-induced LH pulses. Together,
these observations support the conclusion that endotoxin inhibits
pituitary responsiveness to GnRH.
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Introduction
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AN IMMUNE/INFLAMMATORY challenge, such as
that associated with bacterial infection, impairs reproductive
neuroendocrine function and disrupts ovarian cyclicity
(1, 2, 3, 4, 5, 6). Endotoxin or lipopolysaccharide (LPS) is a
commonly used model of immune/inflammatory challenge that consists of
the toxic component of Gram-negative bacteria. When administered
peripherally, endotoxin activates a cascade of cytokines and
prostaglandins (PGs), as well as other mediators that induce a host of
pathophysiological responses. Such responses include the induction of
fever, activation of the neuroendocrine stress axis, and inhibition of
the neuroendocrine reproductive axis (1, 2, 3, 4, 5, 6, 7, 8, 9).
An immune/inflammatory challenge could suppress reproductive
neuroendocrine activity either by inhibiting GnRH release from the
hypothalamus and/or by inhibiting pituitary responsiveness to the GnRH
stimulus. Most prior work has focused on the hypothalamus as the site
of reproductive neuroendocrine inhibition. Observations in a wide
variety of species indicate that endotoxin or various intermediary
cytokines such as interleukin-1 (IL-1) or tumor necrosis factor-
(TNF
), can inhibit both the pulsatile and surge modes of GnRH
secretion (2, 3, 6, 10, 11, 12, 13, 14, 15). For example, systemic
endotoxin suppresses pulsatile GnRH secretion into pituitary portal
blood of ovariectomized ewes (2), and central delivery of
IL-1 inhibits GnRH release and Fos expression in GnRH neurons at the
time of the preovulatory LH surge of rats (15).
Surprisingly little research has focused on an effect of
immune/inflammatory challenge at the level of the pituitary despite
knowledge that various inhibitors of reproductive neuroendocrine
function can act at this level (gonadal steroids, stress hormones,
etc.). An initial study in the rat suggests that central
administration of IL-1 may not impair pituitary responsiveness to GnRH
(16). More recent work, however, has shown that systemic
endotoxin does inhibit the expression of GnRH receptor messenger RNA in
the anterior pituitary gland of the rat (17). The latter
observation encourages further work to assess the impact of
immune/inflammatory challenge on pituitary responsiveness to GnRH.
During the course of examining the suppressive effects of endotoxin on
GnRH secretion into pituitary portal blood of the ewe, we observed an
unexpected uncoupling between GnRH and LH pulses after endotoxin
treatment. In extreme cases, LH pulses were eliminated, whereas GnRH
pulses showed either slight or no inhibition. This provided
circumstantial evidence that endotoxin may inhibit pituitary
responsiveness to GnRH. In this report we first describe this
uncoupling of GnRH and LH pulse suppression. Next, we present follow-up
experiments performed to test the hypothesis that endotoxin inhibits
pituitary responsiveness to GnRH.
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Materials and Methods
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Experiments were conducted on adult Suffolk ewes maintained
outdoors at the Sheep Research Facility near Ann Arbor, MI. Ewes were
fed hay and grass pellets and had free access to water and mineral
licks. During the experiment, animals were housed indoors in a calm
environment. To ensure that the observed alterations in GnRH and/or LH
pulse patterns did not result from changes in ovarian steroid
secretion, all ewes were ovariectomized. Endotoxin (Escherichia
coli LPS 05:B55, Sigma, St. Louis, MO) was
administered by iv bolus. Peripheral blood was obtained via an
indwelling jugular catheter installed 1 day before sampling. Surgeries
were performed aseptically under general anesthesia. All procedures
were approved by the Committee for the Use and Care of Animals at the
University of Michigan.
Exp 1: effect of endotoxin on endogenous GnRH and LH
pulsatility
This experiment was conducted during the mid to late breeding
season (November 1997 to January 1998) on 16 ewes ovariectomized 57
months previously. Ewes were surgically fitted with an apparatus for
hypophyseal portal blood collection 24 weeks before sampling. The
surgical preparation and procedure for remote automated sampling of
hypophyseal portal blood have been described by Caraty
(18). This procedure enables sampling of portal blood from
conscious, physiologically noncompromised ewes. Portal blood for assay
of GnRH was drawn continuously from 4 h before to 10 h after
endotoxin and dispensed as 10-min fractions into 0.5 ml ice-cold
bacitracin (3 x 10-3
M). Samples of jugular blood were obtained continuously and
separated into 5- or 10-min fractions for assay of LH and cortisol.
Endotoxin was injected at a dose of 40 ng/kg. This dose is 10-fold less
than that which we use routinely to demonstrate profound suppression of
GnRH and LH pulsatility (2, 19); it is close to the
threshold for suppressing pulsatile LH secretion in ovariectomized ewes
(Armstrong, S., and F. J. Karsch, unpublished observations). For
injection, endotoxin was dissolved as 10 µg/ml in sterile saline on
the day of the experiment, vortexed vigorously, and diluted 1:10 to
provide the 40 ng/kg dose. After sample collection, ewes were killed
with a barbiturate overdose (Beuthanasia, Schering Plough Animal Health Corp., Kenilworth, NJ), and the pituitary was inspected
to confirm appropriate placement of the cut for sampling portal
blood.
Exp 2: effect of endotoxin on pituitary responsiveness to exogenous
GnRH
In this experiment we used a model in which endogenous GnRH
pulses were blocked in ovariectomized ewes. Blockade of GnRH pulses was
achieved by delivery of ovarian steroids to produce a midluteal phase
serum level of either progesterone (Run 1) or estradiol (Run 2) during
the anestrous season. These treatments were previously found to
eliminate endogenous GnRH pulsatility (20, 21, 22). For the
purpose of this study, this steroid-block model was preferable to one
involving surgical ablation of GnRH secretion (e.g.
hypothalamo-pituitary disconnection or hypothalamic lesion). The latter
approach would compromise other hypothalamic functions, such as stress
axis activation, which might directly or indirectly influence
gonadotropin secretion after endotoxin treatment. The experiment was
conducted in two runs during the anestrous season (MayJuly of 1998
and 1999). Both runs were conducted as a cross-over design in which
each ewe acted as her own control. The design of both runs is
illustrated in Fig. 1
.

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Figure 1. Design of Exp 2, Run 1 (top) and
Run 2 (bottom). Small arrowheads depict
exogenous GnRH pulses delivered hourly by iv injection (Run 1) or
infusion (Run 2). Large arrowheads designate treatment
with endotoxin or saline and, in Run 2, with flurbiprofen (Fb) or its
vehicle (V). Shaded boxes in bottom panel
indicate when jugular blood samples were taken to measure plasma GnRH
concentration. See Materials and Methods
for further details.
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Run 1 (Fig. 1
, top). Seven anestrous ewes were
ovariectomized and immediately treated sc with a 5-mm SILASTIC brand
(Dow Corning Corp., Midland, MI) estradiol implant
(22, 23) to maintain responsiveness to progesterone
(24, 25). After 9 days, the estradiol implant was removed,
and each ewe was treated with progesterone intravaginally (Controlled
Internal Drug Release Device, InterAg, Hamilton, NZ; two devices per
ewe). This treatment restored a midluteal phase concentration of serum
progesterone (46 ng/ml) (26). After 4 days of
progesterone, hourly boluses of GnRH (5 ng/kg, iv) were initiated. For
delivery, a 250 ng/ml solution of GnRH (Sigma) was
prepared by diluting a stock solution (100 µg/ml) with sterile saline
containing 0.1% BSA. Prior studies indicate the 5 ng/kg GnRH dose
creates artificial GnRH pulses that have amplitudes within the range of
endogenous GnRH pulses in ovariectomized ewes (21). After
a 12-h priming period to stabilize pituitary response to the hourly
GnRH pulses, jugular blood was sampled at 10-min intervals for 15
h to assess LH release (Fig. 1
, open bar). After a 5-h
control period, endotoxin (400 ng/kg dissolved as 10 µg/ml in sterile
saline) or vehicle was injected iv. This is the dose of endotoxin we
routinely use for reliable suppression of both GnRH and LH pulses
in ovariectomized ewes (2, 19). It causes
pathophysiological symptoms such as fever, lethargy, occasional
diarrhea, and shivering. The efficacy of the endotoxin challenge was
confirmed by serum cortisol and rectal temperature responses monitored
at 30- and 60-min intervals, respectively. After completion of
sampling, the progesterone-releasing devices were removed, and the
estradiol implant was reinserted sc for 9 days to maintain
responsiveness to progesterone negative feedback. Two weeks later, the
second part of this cross-over experiment was conducted with the
endotoxin and saline treatments reversed, such that all seven ewes
received both endotoxin and vehicle.
Run 2 (Fig. 1
, bottom). Run 1 suggested that
endotoxin inhibits pituitary responsiveness to GnRH. The results,
however, were not definitive because the progesterone treatment failed
to produce the expected complete blockade of endogenous GnRH/LH
pulsatility in some ewes. Further, in some ewes receiving saline
instead of endotoxin, responses to the hourly GnRH injections tended to
increase gradually during the 15-h observation period. This suggested
the 12-h priming period might have been insufficient to stabilize
pituitary responsiveness to the exogenous GnRH pulses. The second run
of the experiment was thus modified in two ways: the priming period was
extended to 4 days, and estradiol, rather than progesterone, was used
to block endogenous GnRH pulses. Estradiol is an extremely potent
inhibitor of pulsatile GnRH secretion in the anestrous ewe
(22). In addition, the goal of Run 2 was expanded to
determine whether endotoxin inhibits pituitary responsiveness to GnRH
via a PG-dependent mechanism. This was prompted by recent evidence that
PGs mediate the suppressive effects of endotoxin on pulsatile GnRH and
LH secretion in the ewe (19).
Run 2 was conducted on eight ewes ovariectomized 12 months previously.
Before the study, each ewe received a 3-cm estradiol implant that
maintains a luteal phase concentration of circulating estradiol (
2
pg/ml) (25). A cannula was inserted into each jugular
vein, one for GnRH delivery and one for blood collection. GnRH (5
ng/kg, 3-ml injection volume, fresh solution prepared daily) was
administered hourly as 6-min pulses using an infusion pump (MiniPuls 2,
Gilson, Middletown, WI) controlled by an electronic timer (model
6227, Fisher Scientific, Pittsburgh, PA). After a 4-day
priming period, endotoxin or saline vehicle was delivered as an iv
bolus (400 ng/kg, 0 h in Fig. 1
). In addition, each ewe received
flurbiprofen (2 mg/kg, iv;
1.0-ml injection volume;
Sigma) or its vehicle (95% ethanol) 30 min before
endotoxin (or saline) and again 5 h later. Flurbiprofen inhibits
the cyclooxygenase-1 and -2 enzymes required for PG synthesis
(27). The dose selected was previously found to reverse
the inhibition of pulsatile GnRH and LH secretion in ovariectomized
ewes and to block endotoxin-induced fever (19), which is a
PG-dependent response (28, 29).
Run 2 was conducted as a cross-over experiment in which each ewe
received three treatments: 1) vehicle for both endotoxin and
flurbiprofen (control), 2) endotoxin plus vehicle for flurbiprofen, 3)
endotoxin plus flurbiprofen. [Note, flurbiprofen alone was not given
as this treatment does not affect LH pulses in ovariectomized ewes
(19).] Blood was sampled at 10-min intervals from 6
h before endotoxin or vehicle until 12.5 h after endotoxin to
monitor pituitary responsiveness to GnRH. To quantify the
experimentally produced GnRH concentrations, aliquots of the jugular
samples surrounding six GnRH pulses (24 h before and 46 h after
endotoxin/vehicle, shaded boxes in Fig. 1
) during one run of
the cross-over experiment were dispensed into bacitracin and frozen for
subsequent GnRH assay. Rectal temperature was taken hourly to assess
efficacy of the endotoxin challenge and to confirm that flurbiprofen
blocked fever.
Assays
LH was measured in duplicate aliquots of plasma (10200 µl)
using a modification (30) of a previously described RIA
(31, 32). Mean intra- and interassay coefficients of
variation (33 assays) were 6.5% and 6.9%, respectively, and assay
sensitivity for 200 µl averaged 0.8 ng/ml NIH LH-12. For GnRH assay,
samples were centrifuged within 1 h of collection to remove cells,
and plasma was stored at -80 C until assay. A 750-µl aliquot of
sample (
600 µl plasma and 150 µl bacitracin) was extracted in 2
ml methanol. Duplicate aliquots of extract (
240 µl portal plasma)
were assayed for GnRH using a previously described RIA (33, 34). In Exp 1, all GnRH values for a given ewe were determined
in a single assay; in Exp 2, all GnRH values were determined in one
assay. Intraassay variation, assessed as median variance ratio of assay
replicates (35), averaged 6.8%, and assay sensitivity was
0.2 pg/tube. Plasma cortisol concentrations were determined in
duplicate 50-µl aliquots of unextracted plasma using the Coat-A-Count
cortisol assay kit (Diagnostic Products, Los Angeles, CA),
validated for use in sheep (2). Modifications of the kit
protocol included adding a low point on the standard curve and
increasing the incubation time with radioactive tracer to 1 h at
37 C. Mean intra- and interassay coefficients of variation (nine
assays) were 5.9% and 6.4%, respectively. Sensitivity averaged 0.8
ng/ml.
Data analysis
In Exp 1, GnRH in pituitary portal blood was assessed as a
collection rate (picograms per min) rather than a concentration. This
minimizes error due to contamination of portal samples with peripheral
blood or cerebrospinal fluid or due to changes in pituitary blood flow.
Formal pulse analysis and statistical comparisons were not performed
because this was an observational study conducted for another purpose,
and controls treated with vehicle instead of endotoxin were not
included.
In Exp 2, all exogenous GnRH pulses induced an increase in circulating
LH. In Run 1, amplitudes of these LH responses (peak minus preceding
nadir) were averaged across three 5-h periods: one preendotoxin/vehicle
(-5 to 0 h) and two postendotoxin/vehicle (05 and 510 h). The
percent change in mean amplitude of the LH response between pre- and
posttreatment periods was calculated in each ewe for both the endotoxin
and vehicle runs of the cross-over study. [The 05 h period was
removed from the analysis due to a technical problem with the animal
model (see Results).] To determine if endotoxin or vehicle
altered pituitary responsiveness to GnRH pulses, the percent change in
LH pulse amplitude within each ewe (pretreatment value vs.
value 510 h after endotoxin/vehicle) was compared by paired
t test. In Run 2, LH pulse amplitudes during the 18-h
experiment were averaged across six 3-h periods: two periods during
preendotoxin/vehicle treatment and four periods posttreatment. Next,
the percent change across the observational period was calculated by
determining the ratio of the mean LH pulse amplitude in the first
pretreatment period to that in each subsequent period. These ratios
were log transformed and analyzed by repeated measures ANOVA in which
both treatment and time were repeated measures. To identify where
significant interactions existed among treatments, each treatment was
deleted in turn, and the analysis was repeated in a pairwise manner.
This determined which treatments differed across time.
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Results
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Exp 1: dissociation of GnRH and LH pulsatile secretion
In 10 of 16 ewes treated with the 40 ng/kg dose of endotoxin,
pulsatile GnRH and LH secretion became dissociated to varying degrees
after treatment (representative examples in Fig. 2
). Specifically, before endotoxin, GnRH
pulses corresponded to LH pulses. After endotoxin, this one to one
correspondence was much less evident. In these instances, the LH pulse
pattern was profoundly disrupted, whereas GnRH pulses were not
suppressed (Fig. 2A
), were minimally suppressed (Fig. 2B
), or were only
transiently disrupted compared with LH pulses (Fig. 2C
). Thus, this
dose of endotoxin appeared to be near or below threshold for GnRH
inhibition, but it markedly disrupted pulsatile LH secretion. In all
ewes endotoxin stimulated a large increase in circulating cortisol
within 3060 min and induced fever (data not shown).

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Figure 2. Profiles of GnRH in pituitary portal blood
(top panels) and LH in jugular blood (bottom
panels) in three representative ovariectomized ewes treated
with the low dose of endotoxin (40 ng/kg; arrows).
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Exp 2: endotoxin decreases pituitary responsiveness to exogenous
GnRH
Run 1. The time courses of LH in two representative ewes after
vehicle and the 400 ng/kg dose of endotoxin are illustrated in Fig. 3
, A and B. In five of seven ewes,
endotoxin clearly suppressed LH pulse amplitude in response to the
exogenous GnRH pulses; this effect was not seen with vehicle (Fig. 3A
).
Analysis of the results, however, was complicated, as two of the ewes
expressed LH pulses that did not coincide with the exogenous GnRH
pulses (Fig. 3B
, asterisks; top, 0.5 h after
vehicle; bottom, 3 h after endotoxin). These extra LH
pulses were presumed to reflect episodes of endogenous GnRH secretion
escaping progesterone blockade. These endogenous GnRH pulses precluded
definitive assessment of the response to exogenous GnRH pulses because
they occurred shortly before the next exogenous GnRH pulse and thus
could have altered pituitary responsiveness. Further, the transients in
circulating LH produced by the extra LH pulses obscured the response to
the exogenous GnRH pulse (see Fig. 3
). This complication was dealt with
in two ways. First, the experiment was repeated under conditions in
which extra LH pulses did not occur (Run 2, below). Second, because the
extra LH pulses occurred during the first 5 h after vehicle or
endotoxin, this period was excluded from the analysis and LH responses
05 h before endotoxin or vehicle were compared with those 510 h
after treatment. This analysis revealed no suppression in LH pulse
amplitude after vehicle, but significant suppression after endotoxin to
52 ± 3% of the pretreatment value (P
0.05;
Fig. 3C
). As expected, endotoxin stimulated cortisol secretion and
induced fever (Fig. 4
).

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Figure 4. Mean ± SEM (seven ewes) plasma
cortisol (top) and rectal temperature
(bottom) values in responses to endotoxin (open
symbols) or vehicle (solid symbols) injected at
0 h (arrows).
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Run 2. Representative time courses of LH in response to
vehicle, endotoxin, and endotoxin plus flurbiprofen are illustrated in
Fig. 5
. Fig. 6
depicts composite results comparing
percent suppression of LH pulse amplitude in all ewes over time for
each of the three treatments. In this run, LH pulses occurred only
immediately after exogenous GnRH pulses, suggesting that endogenous
GnRH pulses were totally blocked. As in Run 1, LH pulse amplitude
decreased after endotoxin (to 44 ± 4% of pretreatment value) but
not vehicle (Figs. 5
and 6
). Statistical analysis revealed a highly
significant treatment by time interaction (P <
0.0001). Further analysis to identify the source of the significant
interaction revealed that responses to both endotoxin and endotoxin
plus flurbiprofen differed from the response to vehicle
(P < 0.0001 in each case). However, the responses to
endotoxin vs. endotoxin plus flurbiprofen did not differ
from each other in terms of either treatment by time interaction
(P > 0.15) or overall mean value (P >
0.6). Thus, endotoxin suppressed the pituitary response to GnRH, and
flurbiprofen did not reverse this effect.

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Figure 5. LH responses to hourly exogenous GnRH pulses in
Exp 2, Run 2. LH pulse profiles are shown for two representative ewes
treated with vehicle (top panels), endotoxin
(middle panels), and endotoxin plus flurbiprofen
(bottom panels). This was a cross-over experiment in
which each ewe received all three treatments. Fb, Flurbiprofen; V, its
vehicle. Solid symbols indicate times of exogenous GnRH
pulses. Arrows depict times of treatment with endotoxin,
flurbiprofen, or vehicle.
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Figure 6. Summary of LH responses in Exp 2, Run 2, for ewes
treated with vehicle, endotoxin, or endotoxin plus flurbiprofen. Values
are expressed as the mean ± SEM (eight ewes) LH pulse
amplitude (percentage of basal value) during five 3-h time periods
(indicated along x-axis) relative to that of the first
pretreatment period (-6 to -3 h). See text (Materials and
Methods) for further details.
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Unexpectedly, LH pulse amplitude in some vehicle-treated ewes appeared
to increase 912 h posttreatment (Figs. 5B
and 6
). Of interest, the
increment in plasma GnRH produced by the exogenous GnRH pulses also
increased later in the sampling period, possibly accounting for this
late increase in LH pulse amplitude (P
0.001, by
paired t test on mean peak values -5 to -2 h
vs. 4 to 6 h; Fig. 7
).
The reason for this late increase in GnRH pulse amplitude is not known.
The overall mean amplitude of the GnRH pulses produced in jugular
plasma by the infusion, which should be comparable to that in portal
plasma during GnRH infusion (36), was 81.4 ± 11.2
pg/ml. This was approximately 2-fold larger than the natural GnRH
pulses amplitude we previously observed in ovariectomized ewes during
the anestrous season, the time of year this study was performed
(41.8 ± 5.5 pg/ml; recalculated from Ref. 22).
Further, the exogenous GnRH pulses produced a more prolonged increment
in plasma GnRH than that seen during a natural pulse (compare patterns
in Figs. 2
and 7
). Endotoxin or flurbiprofen did not alter the time
course of circulating GnRH produced by exogenous GnRH pulses (not
shown).

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Figure 7. Time course of GnRH concentration in peripheral
plasma produced by delivery of GnRH pulses to ovariectomized ewes in
which endogenous GnRH pulses were blocked in Exp 2, Run 2. Values are
the mean ± SEM (8 ewes) for three GnRH pulses before
and three GnRH pulses after treatment with endotoxin or vehicle.
Hormonal profiles are discontinuous because not all samples were
assayed for GnRH.
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As in Run 1, endotoxin induced fever (Fig. 8
). Flurbiprofen prevented this fever
response, suggesting effective blockade of PG synthesis.

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Figure 8. Mean ± SEM (eight ewes) rectal
temperature values in response to vehicle, endotoxin, and endotoxin
plus flurbiprofen in Exp 2, Run 2. Note that flurbiprofen blocked
endotoxin-induced fever.
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Discussion
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This study provides two lines of evidence that endotoxin can
impair reproductive neuroendocrine activity by suppressing the
pituitary response to GnRH. Exp 1 suggests endotoxin inhibits
responsiveness to endogenous GnRH pulses; Exp 2 indicates the same
holds true for exogenous GnRH pulses. In Exp 1, GnRH and LH pulse
suppression were uncoupled in ovariectomized ewes treated with a low
dose of endotoxin (40 ng/kg). This treatment markedly disrupted LH
pulsatility in all ewes tested. In contrast, this dose of endotoxin
only marginally suppressed pulsatile GnRH secretion, and in some ewes,
it appeared to be essentially ineffective in this regard. The
uncoupling of GnRH and LH pulse suppression after the low dose of
endotoxin complements the earlier finding (2) that
recovery from suppression of GnRH pulsatility can precede the recovery
of pulsatile LH secretion in ovariectomized ewes treated with a higher
dose of endotoxin (400 ng/kg). Taken together, these observations
provided circumstantial evidence that endotoxin inhibits the pituitary
response to GnRH, and they prompted a more definitive test of this
possibility.
In Exp 2 we directly tested the hypothesis that endotoxin inhibits
pituitary responsiveness to exogenous GnRH pulses, using ovariectomized
sheep in which endogenous GnRH secretion was blocked by ovarian
steroids. Note, in this experiment we used a higher dose of endotoxin
(400 ng/kg), as it is the dose we have typically used to inhibit both
GnRH and LH pulsatility in ovariectomized ewes (2, 19).
Additionally, prior results describing the time course of GnRH and LH
suppression suggested that this dose may inhibit pituitary
responsiveness to GnRH (2).] The results of Run 1
supported the hypothesis that endotoxin reduced the amplitude of the LH
response to exogenous GnRH pulses, although technical difficulties
precluded definitive conclusion. These difficulties were overcome in
Run 2, and the results demonstrated conclusively that endotoxin
inhibited pituitary responsiveness to GnRH.
It is important to consider our findings in the context of the prior
conclusion that the pituitary response to GnRH in the rat is not
suppressed by central delivery of IL-1, one component of the cytokine
cascade that mediates responses to immune/inflammatory challenge
(16). Although seemingly at odds with the present
conclusion, the design of the two studies differed substantially:
vastly different GnRH doses, single vs. multiple GnRH
injections, assessment of response to GnRH at one vs.
multiple time points, etc. Most notably, the
immune/inflammatory challenges themselves differed: a specific cytokine
delivered centrally in the prior study and peripheral endotoxin used
here. In essence, the prior study in rats is not comparable to that
reported here, and it does not weaken our present conclusion that
endotoxin inhibits pituitary responsiveness to GnRH.
Our study indicates the effect of endotoxin on responsiveness to GnRH
is evident regardless of whether the pituitary is stimulated by
endogenous or exogenous GnRH pulses (Exp 1 and 2, respectively).
Nevertheless, from the data presented it might seem as if endotoxin had
a more potent inhibitory effect when endogenous GnRH pulses stimulated
the pituitary (compare Figs. 2
and 5
). It is important to point out,
however, that the two experiments were not strictly comparable, because
different doses of endotoxin were employed, and the exogenous GnRH
pulses in Exp 2 did not fully replicate those produced endogenously in
Exp 1. In addition, the animal models differed in the two experiments,
ovariectomized ewes in Exp 1 and steroid-treated ovariectomized ewes in
Exp 2. As discussed below, ovarian steroids modify responses to immune
challenge. Thus, a quantitative comparison between responses in the two
experiments is not appropriate. The main point is that endotoxin
compromises pituitary responsiveness regardless of whether endogenous
or exogenous GnRH pulses stimulate the pituitary.
The present demonstration that endotoxin inhibits pituitary
responsiveness to GnRH raises questions related to the mechanisms that
mediate this effect. Recent work in rodents has shown that systemic
endotoxin decreases GnRH receptor gene expression in the pituitary
(17). This effect was seen within 3 h, a time frame
consistent with the endotoxin-induced suppression of pituitary
responsiveness that we observed in the ewe. We can hypothesize,
therefore, that endotoxin reduces GnRH receptor expression in sheep,
thereby suppressing the responsiveness of the gonadotrope to GnRH.
Further work is required to test this hypothesis and to assess other
mechanisms within the pituitary that may mediate the response to this
immune challenge.
It is also important to assess whether endotoxin acts directly upon the
pituitary to exert these effects and whether it acts via the same
intermediates that generate other pathophysiological responses to
immune/inflammatory stimuli. At the level of GnRH secretion, endotoxin
stimulates a cascade of peripheral and central cytokines such as IL-1
and TNF
that, together with enhanced synthesis of PGs and perhaps
hormones of the neuroendocrine stress axis, inhibit GnRH release
(7, 8, 9, 10, 11, 19, 37, 38). The question arises of whether the
same mediators suppress LH secretion at the level of the pituitary.
Recent evidence indicates that endotoxin enhances pituitary expression
of IL-1, TNF
, and both their receptor messenger RNAs
(39), but whether these cytokines act locally within the
pituitary to suppress responsiveness to GnRH remains to be tested. With
regard to PGs, we recently demonstrated that administration of a PG
synthesis inhibitor, flurbiprofen, abolished endotoxin-induced
suppression of pulsatile GnRH and LH secretion in the ovariectomized
ewe (19). Here, we tested whether flurbiprofen would also
reverse endotoxin-induced suppression of pituitary responsiveness to
GnRH. To our surprise, flurbiprofen was ineffective in this regard,
although it did abolish the generation of fever, suggesting that PG
synthesis was effectively blocked.
One interpretation of this finding is that PGs do not mediate
endotoxin-induced suppression of pituitary responsiveness. There is,
however, another explanation, one related to the presence or absence of
estradiol. Our prior observation that flurbiprofen prevented
endotoxin-induced LH suppression was obtained in ovariectomized ewes
not replaced with ovarian steroids (19). Of considerable
interest, we recently obtained preliminary evidence that flurbiprofen
failed to reverse endotoxin-induced LH suppression in ovariectomized
ewes treated with estradiol (Breen, K. M., and F. J. Karsch,
unpublished data). Importantly, in the present study the response to
flurbiprofen was tested in ovariectomized ewes treated with estradiol.
Substantial evidence demonstrates that estradiol influences responses
to immune/inflammatory stimuli. For example, IL-1 inhibits LH secretion
in ovariectomized monkeys, but estradiol protects against this
inhibitory effect (11, 40). Estradiol regulates production
of the proinflammatory cytokines IL-1, IL-6, and TNF
(41) and influences the extent to which endotoxin
stimulates cytokine synthesis (42). Collectively, these
findings not only suggest that estradiol regulates the response to
immune challenge, but also that it does so by modulating underlying
intermediary pathways and possibly dependence upon PGs. Thus, we
suggest that in the presence of estradiol, a pathway not involving PG
synthesis mediates endotoxin-induced suppression of pituitary
responsiveness to GnRH.
Although this putative intermediary pathway remains to be identified,
we are intrigued by the possibility that it may involve cortisol.
Recent work indicates that an elevation of cortisol to levels that we
observed during endotoxin challenge inhibits expression of the GnRH
receptor and suppresses pulsatile LH secretion in gonadectomized sheep,
provided that they are treated with estradiol (43, 44). In
the absence of estradiol, cortisol is ineffective. Although the acute
stimulation of cortisol does not appear to be necessary for
endotoxin-induced inhibition of LH pulsatility in ovariectomized ewes
(45), a mediatory role for cortisol in the presence of
estradiol has not yet been tested. Further experiments are thus
warranted to determine whether, in the presence of estradiol, endotoxin
inhibits pituitary responsiveness to GnRH via a PG-independent
mechanism, possibly one involving cortisol.
In summary, this study provides exciting new evidence that the
pituitary is an important site for reproductive inhibition in response
to immune/inflammatory challenge. Specifically, we observed that
systemic endotoxin suppresses pituitary responsiveness to GnRH. It is
now important to ascertain whether this inhibition at the level of the
pituitary is influenced by the ovarian steroid milieu, if this
inhibitory effect changes during the course of the ovulatory cycle, and
what mediators are involved. Such studies would help clarify mechanisms
for the well documented disruptive effects of immune/inflammatory
challenge on ovarian cyclicity.
 |
Acknowledgments
|
|---|
The authors are sincerely indebted to Doug Doop and Gary McCalla
for maintaining the sheep and for expert assistance with the animal
experimentation; to Martha Brown for conducting and analyzing GnRH
RIAs; to Dr. Vasantha Padmanabhan, Dr. Heather Billings, Nathalie
Briard, and Ms. Aphrodite Nikolovski for their help with conducting and
interpreting experiments; to Dr. Morton Brown and Lei Liu for help with
data analysis; and to Drs. Gordon D. Niswender, Leo E. Riechert, Jr.,
and Alain Caraty for supplying RIA reagents.
 |
Footnotes
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|---|
1 This work was supported by NIH Grant HD-30773, the Center for the
Study of Reproduction (NIH Grant P30-HD-18258), Standards and Reagents,
Data Analysis and Sheep Research Core Facilities, and the Office of the
Vice President for Research at the University of Michigan. A
preliminary report of this work has appeared (46 ). 
2 Present address: INSERM, U-501, IFR Jean Roche,
Faculté Nord, boulevard Pierre Dramard, 13016 Marseilles Cedex
16, France. 
Received September 19, 2000.
 |
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