Neonatal Lipopolysaccharide Exposure Exacerbates Stress-Induced Suppression of Luteinizing Hormone Pulse Frequency in Adulthood
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0013-7227/07/$15.00/0 Endocrinology 148(12):5984 –5990
Printed in U.S.A. Copyright © 2007 by The Endocrine Society
doi: 10.1210/en.2007-0710
Neonatal Lipopolysaccharide Exposure Exacerbates
Stress-Induced Suppression of Luteinizing Hormone
Pulse Frequency in Adulthood
X. F. Li, J. S. Kinsey-Jones, A. M. I. Knox, X. Q. Wu, D. Tahsinsoy, S. D. Brain, S. L. Lightman, and
K. T. O’Byrne
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Division of Reproduction and Endocrinology (X.F.L., J.S.K.-J., A.M.I.K., X.Q.W., D.T., K.T.O.), Cardiovascular Division
(S.D.B.), King’s College London, Guy’s Campus, 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
Early life exposure to immunological challenge has program- after LPS administration. There was no difference in basal LH
ming effects on the adult hypothalamo-pituitary-adrenocor- pulse frequency between neonatal LPS- and neonatal saline-
tical axis stress responsivity, and stress is known to suppress treated controls. However, neonatal endotoxin-treated rats
GnRH pulse generator activity, especially LH pulses. We in- exhibited a significantly greater LPS stress-induced suppres-
vestigated the effects of neonatal exposure to endotoxin on sion of LH pulse frequency. Basal mPOA CRF-R1 expression
stress-induced suppression of pulsatile LH secretion and the was unchanged in neonatal LPS- and neonatal saline-treated
involvement of corticotropin-releasing factor (CRF) receptor rats. However, CRF-R1 expression was significantly increased
mechanisms in adult rats. Pups at 3 and 5 d of age were ad- in response to LPS stress in neonatal LPS-treated animals but
ministered lipopolysaccharide (LPS, 50 g/kg, ip). At 12 wk of not in neonatal saline-treated controls. CRF and CRF-R2 ex-
age, they were ovariectomized and implanted with sc 17- pression was unchanged in all treatment groups. These data
estradiol capsules and iv cannulas. Blood samples (25 l) were demonstrate that exposure to bacterial endotoxin in early
collected every 5 min for 5 h for LH measurement. After 2 h of neonatal life programs long-term sensitization of the GnRH
sampling, rats were given LPS (25 g/kg, iv). CRF and CRF-R1 pulse generator to the inhibitory influence of stress in adult-
and CRF-R2 receptor mRNA was determined by RT-PCR in hood, an effect that might involve up-regulation of CRF-R1
medial preoptic area (mPOA) micropunches collected at 3 h expression in the mPOA. (Endocrinology 148: 5984 –5990, 2007)
T HERE IS GROWING evidence that adverse early envi-
ronments can have a profound and lifelong influence
on responsivity to stress through epigenetic programming.
cles (6) but decreases (7) or has no effect on basal LH (6, 8).
There are no reports on the effect of early life stress on the
activity of the GnRH pulse generator, the central regulator of
Early life is a period of heightened susceptibility to common reproduction. It is well established that stress suppresses the
stressors that might permanently modify major regulatory activity of the HPG axis, specially the GnRH pulse generator.
systems such as the hypothalamo-pituitary-adrenocortical CRF, a principal component in the stress response, is core to
(HPA) axis (1). Indeed, exposure of neonatal rats to an im- stress-induced suppression of the reproductive system
munological challenge, e.g. lipopolysaccharide (LPS), pro- (9 –12).
grams long-term changes in HPA activity, with increases in Immunological stressful stimuli (e.g. LPS) result in a pro-
hypothalamic paraventricular nuclear (PVN) corticotropin- found suppression of pulsatile LH secretion in a variety of
releasing factor (CRF) gene expression and basal corticoste- species, including rats (13, 14), sheep (15), and monkeys (16,
rone pulse frequency and amplitude as well as marked in- 17). We have recently shown a differential role of CRF-R1 and
creases in stress-induced corticosterone release in adulthood CRF-R2 receptors in stress-induced suppression of LH
(1, 2). In contrast to the wealth of information on the effects pulses, with the metabolic (insulin-induced hypoglycemia)
of early life events on the HPA axis, that on the hypothalamo- or immunological (LPS) stressors involving activation of
pituitary-gonadal (HPG) axis is limited and confounding. CRF-R2 only, whereas the psychological stressor, restraint,
Some studies have shown that prenatal stress disrupts es- involves both receptor subtypes (13, 18). The mechanisms by
trous cycles and reduces fertility (3, 4); others have shown which stress influences reproduction are likely to involve
normal cyclicity and fecundity in rats (5). Excess exposure to complex interactions among a number of central pathways.
glucocorticoids delays puberty without affecting estrous cy- The role of PVN CRF in control of LH secretion is contro-
versial. Although there is a rise in CRF mRNA expression in
First Published Online September 13, 2007 the PVN in response to a variety of stressors (19) that sup-
Abbreviations: CRF, Corticotropin-releasing factor; E2, 17-estradiol; press LH pulses, lesions of the PVN per se fail to interfere with
HPA, hypothalamo-pituitary-adrenocortical; HPG, hypothalamo-pitu- the inhibitory effect of stress on LH release in rats (20). These
itary-gonadal; LPS, lipopolysaccharide; mPOA, medial preoptic area;
PVN, paraventricular nuclear. data suggest that dysfunction of the GnRH pulse generator
Endocrinology is published monthly by The Endocrine Society (http://
might involve CRF populations in addition to those of the
www.endo-society.org), the foremost professional society serving the PVN-HPA system. Additional CRF systems implicated in
endocrine community. the control of the GnRH pulse generator include those of the
5984Li et al. • LPS Exposure and Stress-Induced LH Suppression Endocrinology, December 2007, 148(12):5984 –5990 5985
medial preoptic area (mPOA). The mPOA contains a pop- LPS and the other received saline as controls. For the immunological
ulation of CRF neurons (21). Synaptic connections are found stress experiments, LPS (25 g/kg) dissolved in 0.3 ml saline or 0.3 ml
saline alone for controls was injected iv after 2 h basal blood sampling
between CRF and GnRH neurons in the mPOA (22, 23).
for LH measurement.
GnRH neurons express CRF-R1 in the mouse (24). CRF-R1
and CRF-R2 are present in the mPOA (25, 26), and intra-
mPOA administration of CRF profoundly suppresses LH Tissue collections and quantitative RT-PCR
secretion in the rat (27). These combined data raise the in- Expression of CRF, CRF-R1, and CRF-R2 mRNA was determined by
teresting possibility that adverse early life events might pro- real-time quantitative RT-PCR in the mPOA from ovariectomized E2-
gram increases in CRF and/or CRF receptor activity in the treated rats. The animals were killed by decapitation 3 h after LPS
mPOA that down-regulate GnRH signaling across postnatal administration and blood sampling as described above, and whole
development resulting in sensitization of the HPG axis to the brains were carefully removed, frozen on dry ice, and then stored at ⫺80
C. For real-time RT-PCR study, sections (300 m) were cut on a cryostat,
inhibitory effects of stress. and bilateral punches (1 mm diameter) of the mPOA were taken from
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The aims of the present study were to test the hypothesis Bregma ⫹0.2 to ⫺0.4 mm according to the rat brain atlas of Paxinos and
that exposure to bacterial endotoxin in early neonatal life Watson (29) and following the micropunch method of Palkovits (30).
programs long-term sensitization of the GnRH pulse gener- Total RNA was extracted from the punched mPOA tissues for each rat
ator to the inhibitory influence of LPS-stress in adulthood using TRI reagent (Sigma-Aldrich) following the manufacturer’s pro-
tocol. RT was then carried out using the reverse transcriptase Suprescript
and to determine mPOA CRF and CRF receptor involvement. II (Invitrogen, Carlsbad, CA) and random primer following the manu-
facturer’s instruction.
Materials and Methods For the quantitative PCR, the following primers were used: CRF
Animals and neonatal endotoxin exposure sense, 5⬘-CTCTCTGGATCTCACCTTCCAC-3⬘, and antisense, 5⬘-CTA-
AATGCAGAATCGTTTTGGC-3⬘; CRF-R1 sense, 5⬘-TCCACTACA-
Pregnant Sprague Dawley rats (Charles River, Manston, UK) were TCTGAGACCATTCAGTACA-3⬘, and antisense, 5⬘-TCCTGCCACC-
housed under controlled conditions (14-h light, 10-h dark cycle, with GACGCCACCTCTTCCGGA-3⬘; CRF-R2, sense, 5⬘-CTGCTGCAACT-
lights on at 0700 h; temperature, 22 ⫾ 2 C) and provided with food and CATCGACCACGAAGTGGA-3⬘, and antisense, 5⬘-CCTGGTAGATG-
water ad libitum. On the day of birth (postnatal d 0), litters were culled TAGTCCACTAAGTCACCAG-3⬘; and 28S rRNA sense, 5⬘-TTGAA-
to 10 –12 pups and randomly distributed among the dams. On d 3 and AATCCGGG-GGAGAG-3⬘, and antisense, 5⬘-ACATTGTTCCAACAT-
5 after birth, pups were injected ip with 50 g/kg endotoxin in 0.05 ml GCCAG-3⬘. The primer pairs selected for CRF, CRF-R1, and CRF-R2
sterile saline (LPS, serotype Escherichia coli 055:B5; Sigma-Aldrich, Poole, detection were designed to amplify across at least one intron, ruling out
UK), a dose known to provoke permanent HPA axis activation (1, 2). the possibility of identical size bands resulting from genomic DNA
Control pups received saline (0.05 ml). All litters were weaned at 21 d, amplification. Based on the rat CRF genomic sequence (accession no.
and female offspring were housed four to six per cage until they reached NM031019.1), the primers for CRF will amplify a fragment of 149 bp
11–12 wk of age. All animal procedures were undertaken in accordance corresponding to nucleotides 625–774 of the GenBank sequence. The
with the United Kingdom Home Office Regulations. CRF-R1 and CRF-R2 sense primer corresponds to nucleotides 901–928
and 717–743 of the GenBank sequence (NM030999.3 and NM022714.1),
Surgical procedures with cDNA products of 248 and 307 bp, respectively. The LightCycler
(Roche Biochemicals, Lewes, UK) was used for real-time quantitative
Surgical procedures were carried out under ketamine (100 mg/kg analysis of CRF, CRF-R1, and CRF-R2 mRNA expressions. The sample
ip; Pharmacia and Upjohn Ltd., Crawley, UK) and Rompun (10 cDNA prepared as above was used as a template for the PCR. During
mg/kg ip; Bayer, Leverkusen, Germany) anesthesia. Rats were bi- PCR, the amplified cDNA products were detected after each annealing
laterally ovariectomized and implanted with a SILASTIC brand cap- phase in real time using the Faststart DNA Master SYBR Green I kit
sule (inner diameter, 1.57 mm; outer diameter, 3.18 mm; Sanitech, (Roche). Each reaction included 2 l sample cDNA (optimized so that
Havant, UK), filled to a length of 25 mm with 17-estradiol (E2) sample values of the PCR product were within the standard curve), 0.5
(Sigma-Aldrich) dissolved at a concentration of 20 g/ml arachis oil l 25 m antisense and sense primers, 2 l 15 mm MgCl2, 1 l Faststart
(Sigma-Aldrich). The E2-containing capsules produced circulating
DNA master SYBR Green mix, and 4 l water to give a total reaction
concentrations of E2 within the range observed during the diestrous
volume of 10 l. The CRF reaction conditions were 10 min at 95 C for
phase of the estrous cycle (⬃38.8 ⫾ 1.2 pg/ml) as previously de-
one cycle and then 10 sec at 95 C, 10 sec at 56 C, and 10 sec at 72 C for
scribed by Maeda and colleagues (28). After a 10-d recovery period,
32 cycles. The CRF-R1 reaction conditions were 15 min at 94 C for one
the rats were fitted with two indwelling cardiac catheters via the
cycle and then 15 sec at 95 C, 30 sec at 63 C, and 16 sec at 72 C for 34
jugular veins (19). The catheters were exteriorized at the back of the
head and secured to a cranial attachment, and the rats were fitted with cycles. The CRF-R2 reaction conditions were 10 min at 95 C for one cycle
a 30-cm-long metal spring tether (Instec Laboratories Inc., Boulder, and then 10 sec at 95 C, 10 sec at 60 C, and 13 sec at 72 C for 36 cycles.
CO). The distal end of the tether was attached to a fluid swivel The 28S rRNA reaction conditions were 10 min at 95 C for one cycle and
(Instec), which allowed the rat freedom to move around the enclosure. then 15 sec at 95 C, 10 sec at 54 C, and 5 sec at 72 C for 28 cycles. Reaction
Experimentation commenced 3 d later. conditions for CRF, CRF-R1, and CRF-R2 mRNA and 28S rRNA were
optimized separately to give the best results for each primer and for the
different quantities of target in samples. Preliminary experiments were
Stress procedures done to optimize the Mg2⫹ concentration, to confirm PCR specificity by
On the day of experimentation, rats were attached via one of the two agarose gel electrophoresis and melting curve analysis, and to prepare
cardiac catheters to a computer-controlled automated blood sampling the PCR products used to generate standard curves in real-time PCR.
system, which allows for the intermittent withdrawal of small blood CRF, CRF-R1, and CRF-R2 were quantified against a standard curve of
samples (25 l) without disturbing the animals (19). Blood sampling samples containing known CRF, CRF-R1, CRF-R2, and 28S PCR product
commenced between 0900 and 1000 h when samples were collected concentrations, using the LightCycler program. 28S rRNA was quanti-
every 5 min for 5 h for LH measurement. After removal of each 25-l fied as a reference gene against a separate standard curve of samples
blood sample, an equal volume of heparinized saline (5 U/ml normal containing known concentrations of 28S rRNA product. The melting
saline; CP Pharmaceuticals Ltd., Wrexham, UK) was automatically in- curves for CRF, CRF-R1, and CRF-R2 mRNAs and 28S rRNA generated
fused into the animal to maintain patency of the catheter and blood by the LightCycler program demonstrated that single products were
volume. Blood samples were frozen at ⫺20 C for later assay to determine amplified. PCR product for CRF, CRF-R1, and CRF-R2 mRNAs was
LH concentrations. Both neonatal LPS- and saline-treated rats were sequenced and analyzed using an ABI PRISM 310 (Applied Biosystems,
further divided into two subgroups in adulthood; one was treated with Foster City, CA).5986 Endocrinology, December 2007, 148(12):5984 –5990 Li et al. • LPS Exposure and Stress-Induced LH Suppression
RIA for LH measurement CRF-R2 mRNA expression in mPOA was low because sig-
A double-antibody RIA supplied by the National Institute of Diabetes nificant fluorescence often did not appear until later than 33
and Digestive and Kidney Diseases was used to determine LH concen- cycles of PCR. We have regarded the CRF-R2 still detectable
tration in the 25-l whole blood sample. The sensitivity of the assay was in this region using the present techniques, because there was
0.093 ng/ml. The intraassay variation was 6.3%, and the interassay an absence of nonspecific amplification in the negative con-
variation was 5.8%.
trols at this stage in all PCR experiments. Although more
obvious individual variations in CRF-R2 mRNA expression
Statistical analysis was observed, levels of CRF-R2 mRNA in the mPOA did not
Detection of LH pulses was established by use of the algorithm change significantly in response to LPS stress in adulthood
ULTRA (31). Two intraassay coefficients of variation of the assay were in either neonatal LPS-treated or neonatal saline-treated con-
used as the reference threshold for the pulse detection. The inhibitory trol groups (Fig. 2C). There were no significant differences
effect of LPS stress on pulsatile LH secretion was calculated by
comparing the mean LH pulse interval before LPS with the first detected in basal CRF-R2 mRNA levels in the mPOA be-
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prolonged interval after its administration. Subsequent LH interpulse tween neonatal LPS-treated rats and neonatal saline-treated
intervals were defined as the recovery period for analysis. Values controls (Fig. 2C).
given in the text and figures represent means ⫾ sem. Quantification
of mRNAs for CRF, CRF-R1, and CRF-R2 and 28S rRNA were carried
out on all micropunched mPOA tissue samples and the values ex- Discussion
pressed as a ratio of mRNAs for CRF or CRF receptors and 28S rRNA.
Comparisons between groups were made by subjecting data to two- There is considerable evidence that increased activity of
way ANOVA and Dunnett’s test. P ⬍ 0.05 was considered statistically central CRF systems is a persisting neurobiological con-
significant. sequence of early life stress, which is now considered an
important factor in modulating susceptibility to the de-
Results velopment of various pathologies from metabolic to af-
Neonatal LPS exposure sensitized the GnRH fective disorders in adulthood. It is well established that
pulse generator exposure of neonatal rats to LPS programs long-term
Regular pulsatile LH secretion representing normal changes in HPA axis activity with decreases in hippocam-
GnRH pulse generator activity was observed in both neo- pal and hypothalamic glucocorticoid receptors attenuat-
natal LPS and neonatal saline-treated animals during the ing corticosterone negative feedback and hence increasing
2-h baseline blood sampling period in adulthood, with no PVN CRF and basal corticosterone pulse frequency and
significant difference in LH interpulse interval between amplitude as well as markedly increasing stress-induced
experimental groups (Fig. 1, A–E). Pulsatile LH secretion corticosterone release in adulthood (1, 2). Similar mech-
was suppressed within the first hour after iv injection of anisms may operate for the reproductive neuroendocrine
LPS (25 g/kg) and generally returned to normal during system. This study reveals the novel finding that exposure
the second-hour period after LPS administration in ovari- to bacterial endotoxin in early neonatal life programs long-
ectomized E2-replaced adult rats treated neonatally with term sensitization of the GnRH pulse generator to the
saline (Fig. 1, B and E). Neonatal LPS-treated rats showed inhibitory influence of stress in adulthood. Although neo-
a much more profound suppression of LH pulses com- natal LPS exposure failed to alter basal mPOA CRF-R1
pared with the neonatal saline-treated control group in expression in adulthood, we have shown for the first time
response to this immunological challenge in adulthood a significant LPS stress-induced increase in CRF-R1
(Fig. 1, B, D, and E). LH pulse amplitude was not affected mRNA expression in the mPOA of adult female rats that
by neonatal LPS exposure (data not shown). was only seen in those animals that had been exposed
neonatally to an endotoxin challenge.
Because the neonatal LPS paradigm does not affect pul-
Effects of neonatal LPS exposure on CRF and CRF receptor satile LH secretion under basal nonstress conditions, in this
mRNA expression in mPOA unstressed state in adulthood, the hypothalamic GnRH pulse
CRF, CRF-R1, and CRF-R2 mRNAs were detected in the generator is functioning normally in the ovariectomized E2-
tissue micropunched from the mPOA of all experimental replaced animal. However, these animals are markedly sen-
groups. In the mPOA, basal expression of CRF mRNA was sitized to stress-induced perturbation of the GnRH pulse
similar in neonatal LPS-treated rats and neonatal saline- generator, although a possible caveat is the acute nature of
treated controls in adulthood (Fig. 2A). Immunological stress the stressful stimulus used. Additional studies using chronic
induced by LPS administration in adulthood did not change or repeated stress paradigms might be necessary to deter-
the ratio of CRF mRNA over 28S rRNA in either neonatal mine in more detail the long-term consequence to reproduc-
LPS-treated rats or neonatal saline-treated controls (Fig. 2A). tive function. Nevertheless, the finding of early life program-
The basal mean levels of CRF-R1 mRNA expression in the ming in the HPG axis by stress exposure may help to explain
mPOA were not significantly different between neonatal the individual variations or large population differences in
LPS-treated animals and neonatal saline-treated controls in various reproductive dysfunctions under stress conditions.
adulthood (Fig. 2B). CRF-R1 mRNA levels of expression Furthermore, we observed a significant disruption in estrous
were not changed 3 h after LPS stress in the adult neonatal cyclicity, examined by vaginal cytology, in the neonatal LPS-
saline-treated controls. However, CRF-R1 mRNA expression treated animals in adulthood (Wu, X. Q., and X. F. Li, per-
in the mPOA was remarkably increased at 3 h after LPS stress sonal observation), which might also suggest a long-term
in the adult neonatal LPS-treated animals (P ⬍ 0.05) (Fig. 2B). increased vulnerability of the reproductive system.Li et al. • LPS Exposure and Stress-Induced LH Suppression Endocrinology, December 2007, 148(12):5984 –5990 5987
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FIG. 1. A–D, Representative examples illustrating the effects of iv injection (2) of saline (0.3 ml) or LPS (25 g/kg) on pulsatile LH secretion
in ovariectomized E2-replaced adult rats, which were neonatally treated (on postnatal d 3 and 5) with vehicle saline (0.05 ml ip, A and B) or
LPS (50 g/kg ip, C and D), respectively. E, Summary showing the inhibitory effect of LPS stress on pulsatile LH secretion, calculated by
comparing the mean LH pulse interval before LPS with the first prolonged interval after its administration. Neo-Sal⫹Sal, Neonatal saline and
iv saline; Neo-LPS⫹Sal, neonatal LPS and iv saline; Neo-Sal⫹LPS, neonatal saline and iv LPS; Neo-LPS⫹LPS, neonatal LPS and iv LPS.
Subsequent LH pulse intervals were defined as the recovery period for analysis. The inhibitory effect of LPS stress on pulsatile LH secretion
was enhanced in adult rats exposed neonatally to endotoxin. #, P ⬍ 0.05 vs. baseline LH pulse interval in the same treatment group; *, P ⬍
0.05 vs. neonatal saline control group at same experimental time point.
The possible mechanism of sensitization of the HPG axis suppression of the GnRH pulse generator; e.g. functional
by neonatal LPS exposure might involve neonatal program- hypothalamic amenorrhea is associated with hypercorti-
ming of the HPA axis, because HPA hyperactivity is char- solism and disruption of pulsatile LH secretion (32). It is well
acteristic of this model (1), and the inverse relationship be- established that CRF plays a pivotal role in stress-induced
tween the HPA and HPG axes had led to the hypothesis that suppression of the GnRH pulse generator. CRF inhibits LH
activation of the HPA system during stress may drive the pulses (12), and CRF antagonists reverse the LH pulse-sup-5988 Endocrinology, December 2007, 148(12):5984 –5990 Li et al. • LPS Exposure and Stress-Induced LH Suppression
pression bear no relationship to the degree of LH pulse
suppression (19). Most importantly, lesions of the PVN per se
fail to interfere with the inhibitory effect of stress on LH
release in rats (20). Furthermore, there is an absence of PVN
CRF neurons projecting to the GnRH-rich region of the
mPOA (33). These data suggest that the increased vulnera-
bility of the GnRH pulse generator to stress might involve
neonatal programming of CRF systems located at sites other
than the PVN.
In view of the presence of CRF neurons in the mPOA
(21), the presence of CRF-R1 and CRF-R2 within this re-
gion (25, 26), and the marked suppression of pulsatile LH
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secretion by intra-mPOA administration of CRF (our un-
published observation), the CRF system in the mPOA
might play an important role in mediating this stress-
induced inhibitory response. Furthermore, the presence of
synaptic connections between CRF and GnRH neurons (22,
23) and the expression of CRF-R1 in GnRH neurons (24)
raise the possibility of direct actions of CRF on the GnRH
system. There is marked activation, measured by in-
creased c-FOS expression, of the mPOA in response to LPS
stress (34). However, there is very little known about the
mPOA CRF system in relation to stress-induced repro-
ductive dysfunction, and although CRF-R1, and to a lesser
extent CRF-R2, signals are present in this brain area, they
are generally diffuse (25, 26). In the present study, we
show that LPS stress did not alter the level of CRF mRNA
expression in the mPOA, which is in agreement with our
previous finding of no change in CRF mRNA expression
in this brain region in response to insulin-induced hypo-
glycemic stress that suppresses LH pulses (19). Further-
more, a decrease or no change in CRF mRNA levels was
observed in the mPOA in response to restraint stress (35).
The failure of LPS stress to alter CRF-R1 or CRF-R2 ex-
pression in the mPOA of adult female rats treated neo-
natally with saline suggests that the mPOA CRF receptor
system is not involved in acute LPS stress-induced sup-
pression of the GnRH pulse generator. CRF receptor
mRNA is up-regulated in the PVN at 3 h, reaching a
maximum at 6 h, and then declining by 9 h after LPS
administration (36). The rationale for detection of CRF
receptor gene expression at 3 h after treatment in the
present study was based on the selection of a time point
FIG. 2. Effects of neonatal LPS (Neo-LPS, 50 g/kg ip on postnatal d that would most probably represent a submaximal re-
3 and 5) exposure or neonatal saline treatment (Neo-Salin, 0.05 ml ip sponse and thus more clearly reveal differential respon-
on postnatal d 3 and 5) on CRF, CRF-R1, and CRF-R2 mRNA ex-
pression in the mPOA in response to acute LPS stress (25 g/kg iv in
sivity. Nevertheless, additional studies at different time
0.3 ml saline) in adulthood. CRF, CRF-R1, and CRF-R2 mRNA levels points might be necessary to exclude the involvement of
were measured in adult brain micropunch samples from the mPOA mPOA CRF receptor systems in the inhibitory effects of
using real-time RT-PCR. There was no significant difference in levels LPS stress on pulsatile LH secretion. However, in sharp
of CRF (A), CRF-R1 (B), and CRF-R2 (C) mRNA expression in re- contrast to the neonatal saline-treated animals, acute LPS
sponse to control saline injections in adult rats treated neonatally
with LPS or saline. However, CRF-R1 expression was significantly stress evoked a marked increase in CRF-R1 in the mPOA
increased 3 h after LPS stress in neonatal LPS-treated adult rats (B). of adult rats neonatally exposed to endotoxin. CRF-R2
No significant difference in CRF or CRF-R2 mRNA levels was de- expression was unaffected in these animals. These data
tected in the mPOA in response to LPS stress in neonatal saline or raise the interesting possibility that the sensitizing effect
neonatal LPS treatment groups (A and C). *, P ⬍ 0.05 vs. neonatal
LPS group administered saline or neonatal saline group exposed to
of neonatal LPS exposure on stress-induced suppression of
LPS stress in adulthood; n ⫽ 5–9 per group. the GnRH pulse generator might involve a CRF-R1 mech-
anism in the mPOA that is susceptible to programming by
pressing effects of a variety of stressful stimuli (9, 13, 18). early life stressors and thus underlie, at least in part,
Nevertheless, the role of PVN CRF in control of LH secretion vulnerability to stress-related reproductive dysfunction
is controversial. Stress-induced increases in PVN CRF ex- later in life. Whether this change in stress responsivity isLi et al. • LPS Exposure and Stress-Induced LH Suppression Endocrinology, December 2007, 148(12):5984 –5990 5989
mediated by epigenetic programming, including changes ectomized rhesus monkey: reversal by a corticotropin-releasing factor antag-
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in the central nervous system (37) and CRF-mediated cy- 16. Xiao E, Zhang XL, Ferin M 2000 Inhibitory effects of endotoxin on LH secretion
in the ovariectomized monkey are prevented by naloxone but not by an
tokine production in microglia (38), we cannot exclude the interleukin-1 receptor antagonist. Neuroimmunomodulation 7:6 –15
possibility of CRF-R1 in microglia contributing important 17. Xiao E, Xia-Zhang L, Vulliemoz N, Rivier J, Ferin M 2007 Astressin B, a
mechanisms to the interaction between immunological corticotropin-releasing hormone receptor antagonist, accelerates the return to
normal luteal function after an inflammatory-like stress challenge in the rhesus
challenges and the reproductive neuroendocrine system in monkey. Endocrinology 148:841– 848
stress-related disruption of the GnRH pulse generator. 18. Li XF, Bowe JE, Lightman SL, O’Byrne KT 2005 Role of corticotropin-releasing
Nevertheless, it appears that the mPOA, especially its factor receptor-2 in stress-induced suppression of pulsatile luteinizing hor-
mone secretion in the rat. Endocrinology 146:318 –322
CRF-R1 constituent, is part of the circuitry responsible for 19. Li XF, Mitchell JC, Wood S, Coen CW, Lightman SL, O’Byrne KT 2003
the long-term sensitization of the GnRH pulse generator to Oestradiol and progesterone on hypoglycaemic stress-induced suppression of
the inhibitory influence of stress as a result of adverse pulsatile luteinizing hormone release and on corticotropin-releasing hormone
mRNA expression in the rat. J Neuroendocrinol 15:468 – 476
early life events. 20. Rivest S, Rivier C 1991 Influence of the paraventricular nucleus of the hy-
pothalamus in the alteration of neuroendocrine functions induced by inter-
mittent footshock or interleukin. Endocrinology 129:2049 –2057
21. Swanson LW, Sawchenko PE, Rivier J, Vale WW 1983 Organization of ovine
Acknowledgments corticotropin-releasing factor immunoreactive cells and fibers in the rat brain:
an immunohistochemical study. Neuroendocrinology 36:165–186
Received May 25, 2007. Accepted September 4, 2007. 22. MacLusky NJ, Naftolin F, Leranth C 1988 Immunocytochemical evidence for
Address all correspondence and requests for reprints to: Dr. Kevin direct synaptic connections between corticotrophin-releasing factor (CRF) and
gonadotrophin-releasing hormone (GnRH)-containing neurons in the preoptic
O’Byrne, Division of Reproduction and Endocrinology, 2.36D New area of the rat. Brain Res 439:391–395
Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, 23. Dondi D, Piccolella M, Messi E, Demissie M, Cariboni A, Selleri S, Piva F,
United Kingdom. E-mail: kevin.o’byrne@kcl.ac.uk. Samara A, Consalez GG, Maggi R 2005 Expression and differential effects of
This work was supported by the Wellcome Trust. the activation of glucocorticoid receptors in mouse gonadotropin-releasing
Disclosure Statement: The authors have nothing to disclose. hormone neurons. Neuroendocrinology 82:151–163.
24. Jasoni CL, Todman MG, Han SK, Herbison AE 2005 Expression of mRNAs
encoding receptors that mediate stress signals in gonadotropin-releasing hor-
mone neurons of the mouse. Neuroendocrinology 82:320 –328
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9th International Symposium on Avian Endocrinology
Leuven, Belgium
11–15 July 2008
The Symposium on Avian Endocrinology will cover a wide variety of endocrinological research in birds related
to growth and development, food intake and energy metabolism, reproduction and gametogenesis, maternal
investment in eggs, brain plasticity, biorhythms, behavior and stress, immune function, functional genomics,
ecology and evolution, ecotoxicology, and endocrine disruption.
Organizers: Veerle Darras and Eddy Decuypere
Location: Universiteitshallen, Naamsestraat 22, B-3000 Leuven, Belgium
Website: www.isae2008.be
Contact: Email: veerle.darras@bio.kuleuven.be
Fax: 32-16-324262You can also read
