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Endocrinology Vol. 148, No. 12 5984-5990
Copyright © 2007 by The Endocrine Society

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

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

Address all correspondence and requests for reprints to: Dr. Kevin O’Byrne, Division of Reproduction and Endocrinology, 2.36D New Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, United Kingdom. E-mail: kevin.o'byrne{at}kcl.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Early life exposure to immunological challenge has programming effects on the adult hypothalamo-pituitary-adrenocortical axis stress responsivity, and stress is known to suppress GnRH pulse generator activity, especially LH pulses. We investigated the effects of neonatal exposure to endotoxin on stress-induced suppression of pulsatile LH secretion and the involvement of corticotropin-releasing factor (CRF) receptor mechanisms in adult rats. Pups at 3 and 5 d of age were administered lipopolysaccharide (LPS, 50 µg/kg, ip). At 12 wk of age, they were ovariectomized and implanted with sc 17ß-estradiol capsules and iv cannulas. Blood samples (25 µl) were collected every 5 min for 5 h for LH measurement. After 2 h of sampling, rats were given LPS (25 µg/kg, iv). CRF and CRF-R1 and CRF-R2 receptor mRNA was determined by RT-PCR in medial preoptic area (mPOA) micropunches collected at 3 h after LPS administration. There was no difference in basal LH pulse frequency between neonatal LPS- and neonatal saline-treated controls. However, neonatal endotoxin-treated rats exhibited a significantly greater LPS stress-induced suppression of LH pulse frequency. Basal mPOA CRF-R1 expression was unchanged in neonatal LPS- and neonatal saline-treated rats. However, CRF-R1 expression was significantly increased in response to LPS stress in neonatal LPS-treated animals but not in neonatal saline-treated controls. CRF and CRF-R2 expression was unchanged in all treatment groups. These data demonstrate that exposure to bacterial endotoxin in early neonatal life programs long-term sensitization of the GnRH pulse generator to the inhibitory influence of stress in adulthood, an effect that might involve up-regulation of CRF-R1 expression in the mPOA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS GROWING evidence that adverse early environments can have a profound and lifelong influence on responsivity to stress through epigenetic programming. Early life is a period of heightened susceptibility to common stressors that might permanently modify major regulatory systems such as the hypothalamo-pituitary-adrenocortical (HPA) axis (1). Indeed, exposure of neonatal rats to an immunological challenge, e.g. lipopolysaccharide (LPS), programs long-term changes in HPA activity, with increases in hypothalamic paraventricular nuclear (PVN) corticotropin-releasing factor (CRF) gene expression and basal corticosterone pulse frequency and amplitude as well as marked increases in stress-induced corticosterone release in adulthood (1, 2). In contrast to the wealth of information on the effects of early life events on the HPA axis, that on the hypothalamo-pituitary-gonadal (HPG) axis is limited and confounding. Some studies have shown that prenatal stress disrupts estrous cycles and reduces fertility (3, 4); others have shown normal cyclicity and fecundity in rats (5). Excess exposure to glucocorticoids delays puberty without affecting estrous cycles (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 reproduction. It is well established that stress suppresses the activity of the HPG axis, specially the GnRH pulse generator. CRF, a principal component in the stress response, is core to stress-induced suppression of the reproductive system (9, 10, 11, 12).

Immunological stressful stimuli (e.g. LPS) result in a profound suppression of pulsatile LH secretion in a variety of species, including rats (13, 14), sheep (15), and monkeys (16, 17). We have recently shown a differential role of CRF-R1 and CRF-R2 receptors in stress-induced suppression of LH pulses, with the metabolic (insulin-induced hypoglycemia) or immunological (LPS) stressors involving activation of CRF-R2 only, whereas the psychological stressor, restraint, involves both receptor subtypes (13, 18). The mechanisms by which stress influences reproduction are likely to involve complex interactions among a number of central pathways. The role of PVN CRF in control of LH secretion is controversial. Although there is a rise in CRF mRNA expression in the PVN in response to a variety of stressors (19) that suppress LH pulses, lesions of the PVN per se fail to interfere with the inhibitory effect of stress on LH release in rats (20). These data suggest that dysfunction of the GnRH pulse generator might involve CRF populations in addition to those of the PVN-HPA system. Additional CRF systems implicated in the control of the GnRH pulse generator include those of the medial preoptic area (mPOA). The mPOA contains a population of CRF neurons (21). Synaptic connections are found between CRF and GnRH neurons in the mPOA (22, 23). 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 secretion in the rat (27). These combined data raise the interesting possibility that adverse early life events might program increases in CRF and/or CRF receptor activity in the mPOA that down-regulate GnRH signaling across postnatal development resulting in sensitization of the HPG axis to the inhibitory effects of stress.

The aims of the present study were to test the hypothesis that exposure to bacterial endotoxin in early neonatal life programs long-term sensitization of the GnRH pulse generator to the inhibitory influence of LPS-stress in adulthood and to determine mPOA CRF and CRF receptor involvement.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and neonatal endotoxin exposure
Pregnant Sprague Dawley rats (Charles River, Manston, UK) were housed under controlled conditions (14-h light, 10-h dark cycle, with lights on at 0700 h; temperature, 22 ± 2 C) and provided with food and water ad libitum. On the day of birth (postnatal d 0), litters were culled to 10–12 pups and randomly distributed among the dams. On d 3 and 5 after birth, pups were injected ip with 50 µg/kg endotoxin in 0.05 ml sterile saline (LPS, serotype Escherichia coli 055:B5; Sigma-Aldrich, Poole, UK), a dose known to provoke permanent HPA axis activation (1, 2). Control pups received saline (0.05 ml). All litters were weaned at 21 d, and female offspring were housed four to six per cage until they reached 11–12 wk of age. All animal procedures were undertaken in accordance with the United Kingdom Home Office Regulations.

Surgical procedures
Surgical procedures were carried out under ketamine (100 mg/kg ip; Pharmacia and Upjohn Ltd., Crawley, UK) and Rompun (10 mg/kg ip; Bayer, Leverkusen, Germany) anesthesia. Rats were bilaterally ovariectomized and implanted with a SILASTIC brand capsule (inner diameter, 1.57 mm; outer diameter, 3.18 mm; Sanitech, Havant, UK), filled to a length of 25 mm with 17ß-estradiol (E2) (Sigma-Aldrich) dissolved at a concentration of 20 µg/ml arachis oil (Sigma-Aldrich). The E2-containing capsules produced circulating concentrations of E2 within the range observed during the diestrous phase of the estrous cycle (~38.8 ± 1.2 pg/ml) as previously described by Maeda and colleagues (28). After a 10-d recovery period, the rats were fitted with two indwelling cardiac catheters via the 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 a 30-cm-long metal spring tether (Instec Laboratories Inc., Boulder, CO). The distal end of the tether was attached to a fluid swivel (Instec), which allowed the rat freedom to move around the enclosure. Experimentation commenced 3 d later.

Stress procedures
On the day of experimentation, rats were attached via one of the two cardiac catheters to a computer-controlled automated blood sampling system, which allows for the intermittent withdrawal of small blood samples (25 µl) without disturbing the animals (19). Blood sampling commenced between 0900 and 1000 h when samples were collected every 5 min for 5 h for LH measurement. After removal of each 25-µl blood sample, an equal volume of heparinized saline (5 U/ml normal saline; CP Pharmaceuticals Ltd., Wrexham, UK) was automatically infused into the animal to maintain patency of the catheter and blood volume. Blood samples were frozen at –20 C for later assay to determine LH concentrations. Both neonatal LPS- and saline-treated rats were further divided into two subgroups in adulthood; one was treated with LPS and the other received saline as controls. For the immunological stress experiments, LPS (25 µg/kg) dissolved in 0.3 ml saline or 0.3 ml saline alone for controls was injected iv after 2 h basal blood sampling for LH measurement.

Tissue collections and quantitative RT-PCR
Expression of CRF, CRF-R1, and CRF-R2 mRNA was determined by real-time quantitative RT-PCR in the mPOA from ovariectomized E2-treated rats. The animals were killed by decapitation 3 h after LPS administration and blood sampling as described above, and whole brains were carefully removed, frozen on dry ice, and then stored at –80 C. For real-time RT-PCR study, sections (300 µm) were cut on a cryostat, and bilateral punches (1 mm diameter) of the mPOA were taken from Bregma +0.2 to –0.4 mm according to the rat brain atlas of Paxinos and Watson (29) and following the micropunch method of Palkovits (30). Total RNA was extracted from the punched mPOA tissues for each rat using TRI reagent (Sigma-Aldrich) following the manufacturer’s protocol. RT was then carried out using the reverse transcriptase Suprescript II (Invitrogen, Carlsbad, CA) and random primer following the manufacturer’s instruction.

For the quantitative PCR, the following primers were used: CRF sense, 5'-CTCTCTGGATCTCACCTTCCAC-3', and antisense, 5'-CTAAATGCAGAATCGTTTTGGC-3'; CRF-R1 sense, 5'-TCCACTACATCTGAGACCATTCAGTACA-3', and antisense, 5'-TCCTGCCACCGACGCCACCTCTTCCGGA-3'; CRF-R2, sense, 5'-CTGCTGCAACTCATCGACCACGAAGTGGA-3', and antisense, 5'-CCTGGTAGATG-TAGTCCACTAAGTCACCAG-3'; and 28S rRNA sense, 5'-TTGAAAATCCGGG-GGAGAG-3', and antisense, 5'-ACATTGTTCCAACATGCCAG-3'. The primer pairs selected for CRF, CRF-R1, and CRF-R2 detection were designed to amplify across at least one intron, ruling out the possibility of identical size bands resulting from genomic DNA amplification. Based on the rat CRF genomic sequence (accession no. NM031019.1), the primers for CRF will amplify a fragment of 149 bp corresponding to nucleotides 625–774 of the GenBank sequence. The CRF-R1 and CRF-R2 sense primer corresponds to nucleotides 901–928 and 717–743 of the GenBank sequence (NM030999.3 and NM022714.1), with cDNA products of 248 and 307 bp, respectively. The LightCycler (Roche Biochemicals, Lewes, UK) was used for real-time quantitative analysis of CRF, CRF-R1, and CRF-R2 mRNA expressions. The sample cDNA prepared as above was used as a template for the PCR. During PCR, the amplified cDNA products were detected after each annealing phase in real time using the Faststart DNA Master SYBR Green I kit (Roche). Each reaction included 2 µl sample cDNA (optimized so that sample values of the PCR product were within the standard curve), 0.5 µl 25 µM antisense and sense primers, 2 µl 15 mM MgCl2, 1 µl Faststart DNA master SYBR Green mix, and 4 µl water to give a total reaction volume of 10 µl. The CRF reaction conditions were 10 min at 95 C for one cycle and then 10 sec at 95 C, 10 sec at 56 C, and 10 sec at 72 C for 32 cycles. The CRF-R1 reaction conditions were 15 min at 94 C for one cycle and then 15 sec at 95 C, 30 sec at 63 C, and 16 sec at 72 C for 34 cycles. The CRF-R2 reaction conditions were 10 min at 95 C for one cycle and then 10 sec at 95 C, 10 sec at 60 C, and 13 sec at 72 C for 36 cycles. The 28S rRNA reaction conditions were 10 min at 95 C for one cycle and then 15 sec at 95 C, 10 sec at 54 C, and 5 sec at 72 C for 28 cycles. Reaction 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 done to optimize the Mg2+ concentration, to confirm PCR specificity by agarose gel electrophoresis and melting curve analysis, and to prepare the PCR products used to generate standard curves in real-time PCR. CRF, CRF-R1, and CRF-R2 were quantified against a standard curve of samples containing known CRF, CRF-R1, CRF-R2, and 28S PCR product concentrations, using the LightCycler program. 28S rRNA was quantified as a reference gene against a separate standard curve of samples containing known concentrations of 28S rRNA product. The melting curves for CRF, CRF-R1, and CRF-R2 mRNAs and 28S rRNA generated by the LightCycler program demonstrated that single products were amplified. PCR product for CRF, CRF-R1, and CRF-R2 mRNAs was sequenced and analyzed using an ABI PRISM 310 (Applied Biosystems, Foster City, CA).

RIA for LH measurement
A double-antibody RIA supplied by the National Institute of Diabetes and Digestive and Kidney Diseases was used to determine LH concentration in the 25-µl whole blood sample. The sensitivity of the assay was 0.093 ng/ml. The intraassay variation was 6.3%, and the interassay variation was 5.8%.

Statistical analysis
Detection of LH pulses was established by use of the algorithm ULTRA (31). Two intraassay coefficients of variation of the assay were used as the reference threshold for the pulse detection. The inhibitory effect of LPS stress on pulsatile LH secretion was calculated by comparing the mean LH pulse interval before LPS with the first prolonged interval after its administration. Subsequent LH interpulse intervals were defined as the recovery period for analysis. Values 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 expressed as a ratio of mRNAs for CRF or CRF receptors and 28S rRNA. Comparisons between groups were made by subjecting data to two-way ANOVA and Dunnett’s test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neonatal LPS exposure sensitized the GnRH pulse generator
Regular pulsatile LH secretion representing normal GnRH pulse generator activity was observed in both neonatal LPS and neonatal saline-treated animals during the 2-h baseline blood sampling period in adulthood, with no significant difference in LH interpulse interval between experimental groups (Fig. 1Go, A–E). Pulsatile LH secretion was suppressed within the first hour after iv injection of LPS (25 µg/kg) and generally returned to normal during the second-hour period after LPS administration in ovariectomized E2-replaced adult rats treated neonatally with saline (Fig. 1Go, B and E). Neonatal LPS-treated rats showed a much more profound suppression of LH pulses compared with the neonatal saline-treated control group in response to this immunological challenge in adulthood (Fig. 1Go, B, D, and E). LH pulse amplitude was not affected by neonatal LPS exposure (data not shown).


Figure 1
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  		FIG. 1. A–D, Representative examples illustrating the effects of iv injection ({downarrow}) of saline (0.3 ml) or LPS (25 µg/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 µg/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.

 
Effects of neonatal LPS exposure on CRF and CRF receptor mRNA expression in mPOA
CRF, CRF-R1, and CRF-R2 mRNAs were detected in the tissue micropunched from the mPOA of all experimental groups. In the mPOA, basal expression of CRF mRNA was similar in neonatal LPS-treated rats and neonatal saline-treated controls in adulthood (Fig. 2AGo). Immunological stress induced by LPS administration in adulthood did not change the ratio of CRF mRNA over 28S rRNA in either neonatal LPS-treated rats or neonatal saline-treated controls (Fig. 2AGo). The basal mean levels of CRF-R1 mRNA expression in the mPOA were not significantly different between neonatal LPS-treated animals and neonatal saline-treated controls in adulthood (Fig. 2BGo). CRF-R1 mRNA levels of expression were not changed 3 h after LPS stress in the adult neonatal saline-treated controls. However, CRF-R1 mRNA expression in the mPOA was remarkably increased at 3 h after LPS stress in the adult neonatal LPS-treated animals (P < 0.05) (Fig. 2BGo). CRF-R2 mRNA expression in mPOA was low because significant fluorescence often did not appear until later than 33 cycles of PCR. We have regarded the CRF-R2 still detectable in this region using the present techniques, because there was an absence of nonspecific amplification in the negative controls at this stage in all PCR experiments. Although more obvious individual variations in CRF-R2 mRNA expression was observed, levels of CRF-R2 mRNA in the mPOA did not change significantly in response to LPS stress in adulthood in either neonatal LPS-treated or neonatal saline-treated control groups (Fig. 2CGo). There were no significant differences detected in basal CRF-R2 mRNA levels in the mPOA between neonatal LPS-treated rats and neonatal saline-treated controls (Fig. 2CGo).


Figure 2
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  		FIG. 2. Effects of neonatal LPS (Neo-LPS, 50 µg/kg ip on postnatal d 3 and 5) exposure or neonatal saline treatment (Neo-Salin, 0.05 ml ip on postnatal d 3 and 5) on CRF, CRF-R1, and CRF-R2 mRNA expression in the mPOA in response to acute LPS stress (25 µg/kg iv in 0.3 ml saline) in adulthood. CRF, CRF-R1, and CRF-R2 mRNA levels were measured in adult brain micropunch samples from the mPOA using real-time RT-PCR. There was no significant difference in levels of CRF (A), CRF-R1 (B), and CRF-R2 (C) mRNA expression in response to control saline injections in adult rats treated neonatally with LPS or saline. However, CRF-R1 expression was significantly increased 3 h after LPS stress in neonatal LPS-treated adult rats (B). No significant difference in CRF or CRF-R2 mRNA levels was detected in the mPOA in response to LPS stress in neonatal saline or neonatal LPS treatment groups (A and C). *, P < 0.05 vs. neonatal LPS group administered saline or neonatal saline group exposed to LPS stress in adulthood; n = 5–9 per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is considerable evidence that increased activity of central CRF systems is a persisting neurobiological consequence of early life stress, which is now considered an important factor in modulating susceptibility to the development of various pathologies from metabolic to affective disorders in adulthood. It is well established that exposure of neonatal rats to LPS programs long-term changes in HPA axis activity with decreases in hippocampal and hypothalamic glucocorticoid receptors attenuating corticosterone negative feedback and hence increasing PVN CRF and basal corticosterone pulse frequency and amplitude as well as markedly increasing stress-induced corticosterone release in adulthood (1, 2). Similar mechanisms may operate for the reproductive neuroendocrine system. This study reveals the novel finding that exposure to bacterial endotoxin in early neonatal life programs long-term sensitization of the GnRH pulse generator to the inhibitory influence of stress in adulthood. Although neonatal LPS exposure failed to alter basal mPOA CRF-R1 expression in adulthood, we have shown for the first time a significant LPS stress-induced increase in CRF-R1 mRNA expression in the mPOA of adult female rats that was only seen in those animals that had been exposed neonatally to an endotoxin challenge.

Because the neonatal LPS paradigm does not affect pulsatile LH secretion under basal nonstress conditions, in this unstressed state in adulthood, the hypothalamic GnRH pulse generator is functioning normally in the ovariectomized E2-replaced animal. However, these animals are markedly sensitized to stress-induced perturbation of the GnRH pulse generator, although a possible caveat is the acute nature of the stressful stimulus used. Additional studies using chronic or repeated stress paradigms might be necessary to determine in more detail the long-term consequence to reproductive function. Nevertheless, the finding of early life programming in the HPG axis by stress exposure may help to explain the individual variations or large population differences in various reproductive dysfunctions under stress conditions. Furthermore, we observed a significant disruption in estrous cyclicity, examined by vaginal cytology, in the neonatal LPS-treated animals in adulthood (Wu, X. Q., and X. F. Li, personal observation), which might also suggest a long-term increased vulnerability of the reproductive system.

The possible mechanism of sensitization of the HPG axis by neonatal LPS exposure might involve neonatal programming of the HPA axis, because HPA hyperactivity is characteristic of this model (1), and the inverse relationship between the HPA and HPG axes had led to the hypothesis that activation of the HPA system during stress may drive the suppression of the GnRH pulse generator; e.g. functional hypothalamic amenorrhea is associated with hypercortisolism and disruption of pulsatile LH secretion (32). It is well established that CRF plays a pivotal role in stress-induced suppression of the GnRH pulse generator. CRF inhibits LH pulses (12), and CRF antagonists reverse the LH pulse-suppressing effects of a variety of stressful stimuli (9, 13, 18). Nevertheless, the role of PVN CRF in control of LH secretion is controversial. Stress-induced increases in PVN CRF expression 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 vulnerability 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 region (25, 26), and the marked suppression of pulsatile LH secretion by intra-mPOA administration of CRF (our unpublished 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 increased 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 reproductive 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 hypoglycemic stress that suppresses LH pulses (19). Furthermore, 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 expression in the mPOA of adult female rats treated neonatally with saline suggests that the mPOA CRF receptor system is not involved in acute LPS stress-induced suppression 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 that would most probably represent a submaximal response and thus more clearly reveal differential responsivity. Nevertheless, additional studies at different time points might be necessary to exclude the involvement of mPOA CRF receptor systems in the inhibitory effects of LPS stress on pulsatile LH secretion. However, in sharp contrast to the neonatal saline-treated animals, acute LPS stress evoked a marked increase in CRF-R1 in the mPOA of adult rats neonatally exposed to endotoxin. CRF-R2 expression was unaffected in these animals. These data raise the interesting possibility that the sensitizing effect of neonatal LPS exposure on stress-induced suppression of the GnRH pulse generator might involve a CRF-R1 mechanism in the mPOA that is susceptible to programming by early life stressors and thus underlie, at least in part, vulnerability to stress-related reproductive dysfunction later in life. Whether this change in stress responsivity is mediated by epigenetic programming, including changes in the DNA methylation status of the promoter region of the CRF-R1 gene, remains to be determined. It must be recalled, however, that we have previously shown that the inhibitory effect of LPS and hypoglycemic stress on LH secretion was attenuated by administration of a CRF-R2- but not a CRF-R1-selective antagonist (13). The significance of this apparent dichotomy remains to be determined. It may be argued, however, that other critical CRF and CRF receptor neuronal populations underlie the acute inhibitory response to LPS stress, including the prominent CRF-R2 expression sites of the ventral lateral septum, the medial nucleus of the amygdala, and the posteromedial aspect of the bed nucleus of the stria terminalis that have extensive projections to the GnRH-rich regions of the mPOA (25, 26, 33). Furthermore, given the increasing evidence for the presence of functional CRF receptors in glia in the central nervous system (37) and CRF-mediated cytokine production in microglia (38), we cannot exclude the possibility of CRF-R1 in microglia contributing important mechanisms to the interaction between immunological challenges and the reproductive neuroendocrine system in stress-related disruption of the GnRH pulse generator. Nevertheless, it appears that the mPOA, especially its CRF-R1 constituent, is part of the circuitry responsible for the long-term sensitization of the GnRH pulse generator to the inhibitory influence of stress as a result of adverse early life events.


   Footnotes
 
This work was supported by the Wellcome Trust.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 13, 2007

Abbreviations: CRF, Corticotropin-releasing factor; E2, 17ß-estradiol; HPA, hypothalamo-pituitary-adrenocortical; HPG, hypothalamo-pituitary-gonadal; LPS, lipopolysaccharide; mPOA, medial preoptic area; PVN, paraventricular nuclear.

Received May 25, 2007.

Accepted for publication September 4, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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