Effect of Interleukin-1β on Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Gene Expression in Castrated Male Rats

Journal of Neuroendocrinology, 2000, Vol. 12, 421–429

Effect of Interleukin-1b on Gonadotropin-Releasing Hormone (GnRH)and GnRH Receptor Gene Expression in Castrated Male Rats

S. S. Kang,* S. R. Kim,† S. Leonhardt,‡ H. Jarry,‡ W. Wuttke‡ and K. Kim**Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Seoul,Korea.†Department of Medicine, Division of Molecular Biology, Ehwa Research Center, College of Medicine, Ehwa Womans University, Seoul,Korea.‡Division of Clinical and Experimental Endocrinology, University of Goettingen, Germany.

Key words: IL-1b, gonadotropin-releasing hormone, GnRH receptor, gene expression.

Abstract

Increasing evidence suggests that interleukin-1b (IL-1b) regulates luteinizing hormone (LH) releaseprimarily through modulation of the gonadotropin-releasing hormone (GnRH) neuronal activity. Thisstudy was undertaken to elucidate the effect of IL-1b on GnRH as well as GnRH receptor (GnRHR)gene expression in the preoptic area. IL-1b (100 ng/rat) or saline was administered into the lateralventricle of castrated rats. RNA samples were isolated from micropunches of the preoptic area andmediobasal hypothalamus from individual brain slices and GnRH mRNA levels in the preoptic areaand GnRHR mRNA levels in the mediobasal hypothalamus were determined by competitive reversetranscription-polymerase chain reaction (RT-PCR) protocols. Serum LH concentrations weredecreased from 1 h to 3 h after IL-1b treatment, but rebounded at 5 h, while serum concentrationsof follicle-stimulating hormone (FSH) and prolactin were not altered. There were no significantchanges in GnRH mRNA levels from the micropunched preoptic area, while GnRHR mRNA levelsfrom the preoptic area and mediobasal hypothalamus micropunch samples, but not in the anteriorpituitary, showed a pattern similar to the serum LH profile following i.c.v. administration of IL-1b. Wethen examined the effect of IL-1b on the translational efficiency of the GnRH mRNA. After theseparation and fractionation of polyribosome-associated cytoplasmic RNA from the hypothalamicfragments containing the preoptic area-anterior hypothalamic area of control (saline-treated) and IL-1b-treated group 3 h after administration, GnRH transcript levels were examined from the eachfraction. IL-1b decreased the translational efficiency of the transcribed GnRH mRNA. These resultsclearly demonstrate that central administration of IL-1b suppresses the translational activity of GnRHmRNA. Moreover, GnRHR may play an important role in the modulation of GnRH neuronal activitythrough GnRHR-expressing neurones (or glia) in the hypothalamus.

Hypothalamic gonadotropin-releasing hormone (GnRH ) kines are localized throughout the rat brain. IL-1 receptorhas its highest density in the hypothalamus, olfactory bulb,release is influenced by a variety of central neurotransmitters

as well as by the feedback actions of steroid hormones (1, hippocampus, and dentate gyrus (6). Two types of IL-1,IL-1a and IL-1b, bind to the same receptors and elicit similar2). It is now widely acknowledged that cytokines also mediate

bidirectional communication between the immune and neuro- neuroendocrine responses, despite sharing only a limitedstructural homology (7).endocrine systems (3). Cytokines such as interleukin-1 (IL-1),

IL-2, and IL-6 are synthesized in both neuronal and non- In the rat, IL-1 has been shown to readily activate thehypothalamic-pituitary-adrenal (HPA) axis (8), and con-neuronal cells in the brain (4). IL-1, for example, is localized

in the infundibulum, median eminence, periventricular nuc- versely to inhibit the hypothalamic-pituitary-gonadal (HPG)axis (9). However, the influence of IL-1 on hypothalamic-leus, and other hypothalamic nuclei (5). Receptors for cyto-

Correspondence to: Kyungjin Kim, PhD, Department of Molecular Biology, College of Natural Sciences, Seoul National University, Seoul,Korea (e-mail: [email protected]).

© 2000 Blackwell Science Ltd

422 Effect of IL-1b on GnRH and its receptor gene expression

was centrifuged for 2 min at full speed to dry the membrane, total RNA waspituitary function depends on the route of administration.eluted with 30 ml of water.Systemic administration of IL-1 stimulates adrenocortico-

tropic hormone release, but does not alter luteinizing hor- Competitive RT-PCR for GnRH and GnRHR mRNAsmone (LH) release in castrated rats. In contrast, central

Cloning of mutant GnRH and GnRHR cDNAs was previously describedinjection of IL-1 interferes with GnRH release from the (17, 18). Briefly, the rat GnRH cDNA (575 bp) inserted into the pGEM4hypothalamus and LH release from the pituitary (9, 10). By vector (a gift from Dr Kelly Mayo, Northwestern University, Evanston, IL,

USA) was cleaved by ScaI (74 bp) and AsuII (310 bp) restriction enzymes,comparing the inhibitory effect of IL-1a and IL-1b on LHand ligated by T4 DNA ligase, producing a truncated mutant GnRH cDNA.secretion, it has become evident that IL-1b is relatively moreGnRH primers were synthesized based on the sequence of rat GnRH cDNAeffective than IL-1a (11). Microinfusion of IL-1b into the (19). The upstream primer is 5∞-CACTATGGTCACCAGCGGGG-3∞ and the

lateral ventricle of proestrus rats reduces in vivo GnRH downstream primer is 5∞-AGAGCTCCTCGCAGATCCCTAAGA-3∞. Thepredicted sizes of PCR products are 375 bp for native GnRH and 139 bp forrelease from the median eminence (12). There is, however,mutant GnRH. GnRHR cDNA (441 bp) obtained from the rat pituitarylittle evidence that IL-1b affects GnRH mRNA expressions.RNA using the RT-PCR procedure was cloned to the pGEM4Z vector. TheRecent evidence indicates that GnRH receptor (GnRHR) isupstream primer, 5∞-CTTGAAGCCCGTCCTTGGAGAAAT-3∞, and down-

expressed in the hypothalamus, in particular, in the medio- stream primer, 5∞-GCGATCCAGGCTAATCGCCGCCAT-3∞, were used forbasal hypothalamus (13). Moreover, we found that temporal PCR. The plasmid was cut at the PstI site and subsequently digested with

Bal31 nuclease and Mung-Bean nuclease. This DNA was ligated with T4changes in mediobasal hypothalamus GnRHR mRNADNA ligase, generating the 280 bp deletion mutant of GnRHR cDNA.expressions had an inverse relationship with changes in

Native and mutant cRNA templates were coreverse transcribed with 200preoptic area GnRH mRNA expressions after treatment with unit of RNaseH-moloney murine leukaemia virus reverse transcriptasesteroid hormones in ovariectomized rats indicating that there (GIBCO-BRL, Gaithersburg, MD, USA) in 20 ml of the RT reaction mixture.

The RT reaction was carried out at 37 °C for 30 min followed by 5-minis a reciprocal interaction between GnRH neurones andperiod at 95 °C. Subsequently, 80 ml of the PCR reaction mixture was added.GnRHR-expressing neurones (or glia) (14). Therefore, thePCR amplification was carried out with a two-step procedure to reducepresent study was designed to evaluate the effect of IL-1b onhybrids (first step: denaturation at 94 °C for 1 min, primer annealing at 60 °C

GnRH and GnRHR gene expression in the hypothalamus. for 1 min, and primer extension at 72 °C for 2 min; second step: denaturationat 85 °C for 1 min, primer annealing at 60 °C for 1 min, and primer extensionat 72 °C for 2 min). To detect GnRH mRNA levels in the preoptic area, 35

Materials and methods cycles of the first step, and five cycles of the second step were used. To detectGnRHR mRNA levels in the preoptic area, mediobasal hypothalamus andAnimalspituitary, 30 (for pituitary RNA) or 40 (for preoptic area and mediobasal

Adult male rats (weight 220–250 g) were maintained under standardized hypothalamus mRNA) cycles of the first step were used. Ten ml aliquots ofconditions ( light on from 07.00 h to 1900 h, room temperature 23 °C, water PCR products were electrophoresed on an 1.5% agarose gel in TAE buffer,and food available ad lib.). One week after castration, under pentobarbital stained with ethidium bromide and photographed under UV illuminationanaesthesia, a guide cannula (o.d. 0.96 mm, stainless steel tube) with inner with Polaroid 665 type negative and positive films (Polaroid, Cambridge,stilet 24 gauge was stereotaxically implanted into the lateral ventricle according MA, USA). Negative film was used for densitometric scanning of native andto the atlas of Paxinos and Watson (15) and fixed in place with anchor screws mutant signals.and dental cement. After a recovery period of one week, the inner stilet wasremoved and then IL-1b (100 ng/rat, recombinant human interleukin-1b, Polyribosome-associated RNA separation from the hypothalamic fragmentsPBH, Hanover, Germany) was injected i.c.v. using a Hamilton syringe.

The hypothalamic fragments including the preoptic area-anterior hypothal-amic area were isolated, snap-frozen, and kept in −70 °C until use. ThePoly(A)-rich RNA isolation from micropunch samples of the preoptic area andboundaries of the preoptic area-anterior hypothalamic area was caudally themediobasal hypothalamusposterior border of the optic chiasm, frontally the apex of the isoscelesBrains were coronally sliced in a 600-mm thickness according to the atlas oftriangle-shaped region, laterally the lateral hypothalamic sulci, and trans-Paxinos and Watson (15) (for the preoptic area slice: bregma (B) 0.20 mmversely at a depth of 2–3 mm (20). Polyribosome-associated RNA wasto B–0.40 mm; for the mediobasal hypothalamus slice: B–2.60 mm to B–3.20fractionated as described elsewhere (21). Briefly, each tissue was subjected to(mm). The preoptic area and mediobasal hypothalamus were micropunchedhomogenization with lysis buffer (0.25% sodium deoxycholate, 10 mM Tris-from the frozen rat brain slices with a 1-mm-diameter stainless needle by theHCl (pH. 7.4), 1.5 mM MgCl2, 0.3 M sucrose, 0.5% NP-40). The homogenatesmethod of Palkovits and Brownstein (16). Messenger RNA was isolated fromwere centrifuged at 800×g for 20 min at 4 °C. The cytoplasmic fraction wasthe micropunched samples using a Dynabeads mRNA isolation kit (Dynalfurther centrifuged at 12,000 r.p.m. for 30 min at 4 °C. This post mitochondrialA.S., Oslo, Norway) as previously described (17, 18). Briefly, two micro-supernatant was layered over a continuous 10–40% sucrose gradient in apunches of the preoptic area and mediobasal hypothalamus from each rat17-ml polyallomer ultracentrifuge tube. In case of control, EDTA waswere sonicated in 100 ml of lysis buffer (100 mM Tris-HCl, pH 8.9, 500 mMpremixed to the gradient at a 100-mM final concentration. The gradientsLiCl, 10 mM EDTA, pH 8.0, 1% SDS, and 5 mM dithiothreitol ), placed onwere centrifuged at 100 000×g for 5 h at 4 °C in a AH-627 rotor (Sorvallice for 1 min, and briefly centrifuged. Dynabeads-oligo (dT) (5 mg/ml, 30ml )ODT-65). After the centrifugation, RNA was fractionated into 14 tubes andwere directly added to lysis-buffered samples, resuspended and then placedprecipitated with isopropanol. Pellets from the isopropanol pricipitation offor 10 min at room temperature. Using the dynal magnetic particle concen-each fraction were sonicated with lysis buffer and total RNA was extractedtrator, supernatant was removed and the mRNA captured by the Dynabeadsusing RNeasy kit as described above. Equal aliquots of purified RNA werewas washed twice with washing buffer (10 mM Tris-HCl, pH 8.5, 0.15 Mused for RT-PCR. PCR products were electrophoresed on 1.5% agarose gelLiCl, and 1 mM EDTA). The tube was then incubated at 65 °C for 3 minand stained by ethidium bromide. Gels were photographed and GnRH signalswith 30 ml of elution buffer (2 mM EDTA, pH 7.5). Eluted mRNA wason negative film were measured with a densitometric scanner.directly used as a RNA source for the reverse transcription (RT) reaction as

described below.Radioimmunoassay of serum LH, FSH and prolactin

Total RNA extraction from the pituitary Serum LH concentrations were assayed using a double antibody radio-immunoassay reagent kindly provided by the National Pituitary Agency. TheTotal cytoplasmic RNA from the pituitary was extracted by RNeasy RNA

purification kit (Qiagen, Hilden, Germany). Briefly, each pituitary was tracers NIADDK-rLH-I9 were iodinated by the chloramine-T method. Theantiserum was NIADDK-rLH-S-10 and the reference preparation wassonicated in 350 ml of lysis buffer. One volume of 70% ethanol was added to

the lysate, briefly vortexed and applied onto RNeasy spin column. Spin NIADDK-rLH-RP-2. LH levels were expressed as NIADDK RP2 units.Intra- and interassay coefficients of variation were 7.2 and 11.2%, respectively.column was centrifuged for 15 s at 8,000×g, washed with 700 ml of first wash

buffer, and washed with 500 ml of second wash buffer twice. Spin column Characteristics of the prolactin and FSH assay were described elsewhere (22).

© 2000 Blackwell Science Ltd, Journal of Neuroendocrinology, 12, 421–429

Effect of IL-1b on GnRH and its receptor gene expression 423

Intra- and interassay coefficients of variation of prolactin were 7.8 and 13.1%,respectively. Those of FSH assay were 6.9 and 13.4%, respectively.

Statistical analysis

The amounts of GnRH and GnRHR mRNA were calculated from the ratioof native-to-mutant using each standard curve. Data were statistically evalu-ated using one- or two-way anova followed by Fisher’s least significantdifference test for a post-hoc comparison. Statistical significance was setat P<0.05.

Results

Effect of IL-1b on serum LH, FSH, and prolactin levels andGnRH mRNA levels in the preoptic area

A heparinized catheter was implanted into a jugular vein ofindividual rat 1 day before experiment. Sera were collectedat 10 min intervals 1 h before and 2 h after i.c.v. injection ofIL-1b (100 ng/rat) or saline. As shown in Fig. 1, i.c.v. admin-istration of IL-1b gradually reduced serum LH concentra-tions, but failed to alter FSH and prolactin concentrationsat any time point 2 h following IL-1b administration. Theseresults are in agreement with previous findings (11, 23),indicating the specific action of central IL-1b on the LHrelease. Based on the finding that IL-1b reduces hypothalamicGnRH release (12), we sought to find a correlative changein GnRH gene expression at the level of the preoptic area.Two hours after treatment of IL-1b, rats were killed andGnRH mRNA levels were determined in preoptic area micro-punch samples from individual rats by competitive RT-PCR.Unexpectedly, no change in GnRH mRNA level wasobserved (Fig. 2).

Time course effect of IL-1b on serum LH levels and GnRHmRNA levels in the preoptic area

To scrutinize whether the effect of IL-1b is time-related, aCollection interval (1=10 min)

–4 2 8

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F. 1. Effects of IL-1b on serum luteinizing hormone (LH) (), folliclesecond series of experiments was designed. Rats were killedstimulating hormone (FSH) (), and prolactin () concentrations. Aat 0, 3, and 5 h after i.c.v. injection of IL-1b. Serum LHheparinized catheter was implanted into a jugular vein of individual rat

concentrations were low at 3 h, but rebounded, reaching to 1 day before the experiment. Sera were collected at 10 min intervals. IL-two-thirds of the control value at 5 h (Fig. 3). There was 1b (100 ng/rat) or saline was injected into lateral ventricle at time 0

point (arrow). Serum LH, FSH and prolactin concentrations wereno change in serum FSH or prolactin concentrations at 3 hdetermined by respective radioimmunoassays. Since sera were insufficientand 5 h time points (Fig. 3, ). No temporal change into measure all three hormones at each time point, sera of consecutiveGnRH mRNA levels in the preoptic area was observed two time points were mixed and assayed for prolactin. Error bars indicate

(Fig. 3). the mean±SEM.

Effect of IL-1b on translational efficiency of the GnRH mRNAEffect of IL-1 receptor antagonist

To ensure the specificity of IL-1b action, an IL-1 receptor Thus far, we failed to observe any correlation in changes inserum LH concentrations and GnRH mRNA levels inantagonist was tested. IL-1 receptor antagonist (5 mg/rat,

human interleukin-1 receptor antagonist, PBH, Hanover, response to IL-1b, and we sought to investigate whether IL-1b may affect the translational efficiency of GnRH mRNA.Germany) or saline was injected into the lateral ventricle

10 min prior to IL-1b treatment. Since serum LH concentra- The translational efficiency can be determined by amount ofpolyribosome bound to the mRNA. From the hypothalamictions were lowest at the 3 h time point, rats were killed 3 h

after IL-1b treatment. Treatment with IL-1 receptor antagon- fragments obtained 3 h after icv administration of IL-1b orsaline, polyribosome-associated cytoplasmic RNA was isol-ist effectively blocked the IL-1b-induced decrease in serum

LH concentrations (Fig. 3). There was no observed change ated and layered on continuous (10–40%) sucrose gradientultracentrifugation (21). Fractions were analysed for thein GnRH mRNA levels in preoptic area micropunch samples,

or in serum FSH and prolactin concentrations (Fig. 3– ). distribution of ribosome-associated GnRH mRNA through



Log(pg GnRH cRNA))

–1 0 1

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F. 2. Effect of IL-1b on GnRH mRNA expression in micropunches of the preoptic area. Rats were decapitated 2 h after treatment with IL-1b.Both sides of the preoptic area were punched from the frozen rat brain slices with a 1-mm-diameter stainless needle according to the method ofPalkovits and Brownstein (16). () Standard curve of competitive RT-PCR for the quantification of GnRH mRNA levels. One pg of mutant GnRHcRNA was coamplified with various amounts of native GnRH cRNA (0.1, 0.2, 0.5 1, 2, 5, and 10 pg). () PCR products from two experimentalgroups were separated on the 1.5% agarose gel and stained with ethidium bromide. The ratio of native/mutant of each product was converted withstandard curve. Error bars indicate the mean±SEM.

the gradient by RT-PCR procedure. RT-PCR was performed (Fig. 5– ). It is interesting that IL-1b concomitantly affectsLH release and the expression of GnRHR. Notice that thewith the same volume of RNA eluent from each fraction.

Representative photographs of GnRH RT-PCR products temporal profile of GnRHR mRNA in the preoptic area andmediobasal hypothalamus (Fig. 5,) were similar to that offrom three experimental groups are shown in Fig. 4().

Treatment with IL-1b significantly suppressed the transla- serum LH concentration (see Fig. 3). A prior treatment withIL-1 receptor antagonist marginally blocked the IL-1b-tional efficiency of the GnRH mRNA from the hypothalamus

(Fig. 4). As an internal control, we also examined the induced decrease in GnRHR mRNA levels in the preopticarea and mediobasal hypothalamus (Fig. 6,).distribution of L7 RNA encoding ribosomal protein through

the gradients from three experimental groups, which showedno significant difference between saline- and IL-1b-treatedgroups (data not shown). Figure 4 depicts the representative Discussiondistribution of total RNA through sucrose gradients fromthree experimental groups. There was no difference in total It has become increasingly evident that activation of the

immune system in response to a variety of stressors coincidesRNA distribution between saline- and IL-1b-treated groups,but in EDTA-treated group, there was a shift in peak to the with impaired reproduction (24). Administration of various

cytokines into the lateral ventricle of the brain significantlylight fractions caused by the dissociation of GnRH mRNAand translational complex of ribosomal subunits. lowered serum LH concentrations in gonadectomized rats.

Intracerebroventricular injection of IL-1b inhibited the pro-estrous LH surge and subsequent ovulation in intact femaleEffect of IL-1b on GnRHR mRNA levels from the preopticrats (9, 11). In contrast, peripheral administration of thearea, mediobasal hypothalamus, and pituitarycytokines did not significantly alter serum LH concentrationsor normal oestrous cyclicity (9), although cytokines appearThe GnRHR mRNA levels in the several brain regions

change during the oestrous cycle (13), suggesting that to exert a direct effect on the gonad and thereby to interferewith sex steroid secretion (25).GnRHR in the brain may have important roles in the

regulation of reproduction. In the present study, we simultan- In the present study, acute administration of IL-1b intothe lateral ventricle of castrated rats selectively depressedeously examined the effect of IL-1b on GnRHR mRNA

levels in the preoptic area and mediobasal hypothalamus as serum LH concentrations within 2 h. No changes in serumFSH concentrations were detected at the time pointswell as in the pituitary, which are important sites for the

regulation GnRH neuronal activity. GnRHR mRNA in the observed. These results indicate a specific action of centralIL-1b on serum LH release. The inability of IL-1b to inhibitpreoptic area and mediobasal hypothalamus, but not in the

pituitary was reduced only at 3 h after IL-1b treatment FSH release may indicate the existence of FSH-releasing



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F. 3. Time course of the effect of IL-1b on serum luteinizing hormone (LH) concentrations (), GnRH mRNA (), follicle stimulating hormone(FSH ) concentrations (), and prolactin concentrations (). Rats were killed at 0, 3, and 5 h after icv injection of IL-1b (100 ng/rat) and trunkblood was collected for the determination of hormone concentrations by radioimmunoassay. IL-1ra (5 mg/rat) or saline was injected into the lateralventricle of the 3 h group rats 10 min before IL-1b treatment. GnRH mRNA levels in the preoptic area micropunch were calculated on the basis ofthe standard plot shown in Fig. 2. Each bar indicates the mean±SEM. **P<0.01 (vs control group). ++P<0.01 (vs IL-1b-treated 3 h group).

component(s) or a more complicate mechanism for the ones (30, 31) while IL-1b stimulates CRF neurones (29, 32).Thus, CRF may be a mediator of IL-1-induced inhibition ofregulation of FSH secretion (26, 27).

The ability of IL-1b to inhibit LH secretion is believed to the GnRH-LH axis. In ovariectomized Rhesus monkey, anIL-1-induced decrease in LH secretion was reversed by CRFbe mediated through the inhibition of GnRH secretion (12,

23, 28, 29). Indeed, both types of IL-1 significantly suppressed antagonist delivered to the ventricle (33). However, in rats,central administration of different types and doses of CRFGnRH release from preoptic area-mediobasal hypothalamus

fragments, but failed to alter GnRH release in vitro from the antagonists did not alter the IL-1-induced suppression ofGnRH release and LH release (11, 12), indicating that themicrodissected median eminence (28). Microinfusion of IL-

1b into the lateral ventricle of proestrous rats reduced GnRH increase in CRF neuronal activity is not directly related tothe effects of IL-1b on GnRH neuronal activity. Indeed, thererelease in vivo (12). These results indicate that IL-1b inhibits

GnRH neuronal activity by acting at the level of GnRH is evidence that endogenous opioid peptides (9, 11, 28, 31),prostaglandins (23, 34), excitatory amino acids (35), andperikarya rather than at the level of the GnRH nerve ter-

minals. It also appears that the preoptic area may represent catecholamines (34) all participate in the regulation of GnRHneurones through central IL-1, and to date, there is noa primary target for central IL-1b in modulating the activity

of GnRH neurones that project to the median eminence. evidence that IL-1 can directly act on GnRH neurones.However, by RT-PCR based cloning, we have recently foundThere have been several reports that corticotropin-releasing

factor (CRF) exerts an inhibitory influence on GnRH neur- expression of IL-1 receptor type-I and type-II mRNAs in the



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F. 4. Effects of IL-1b on the translational activity of GnRH mRNA. () Representative photographs of GnRH RT-PCR products through sucrosegradient from three experimental groups. Polyribosome profile analysis of GnRH mRNA 3 h after IL-1b (100 ng/rat, i.c.v.) or saline injection.Polyribosome-associated cytoplasmic RNA was isolated from hypothalamus, sucrose-fractionated and aliquoted in 14 fractions. RT-PCR wasperformed to determine the amount of GnRH mRNA in each fraction. () The maximal intensity of PCR-amplified GnRH mRNA signals throughthe fraction of three treatment groups was represented as mean±SEM (n=8). Control gradients had a maximal intensity of approximately fraction3.24, whereas IL-1b shifted this to fraction 6.41. *P<0.05 (vs control group). **P<0.01 (vs control group). () Distribution of total RNA fromgradients of three experimental groups.

GnRH producing GT1-1 cell line (data not shown). Although 1b effectively reduced the expression of c-fos in GnRHneurones (12) as well as GnRH mRNA levels (10) whenin vivo evidence is needed to demonstrate a direct action of

IL-1 on GnRH neurones, it appears that IL-1 may also affect administered 6 h or 9.5 h before the LH surge. However, inthe present study, no significant change in GnRH mRNAGnRH neurones directly. Thus, the influence of IL-1 on the

GnRH-LH axis seems to involve a complex cascade levels in the preoptic area was observed (Figs 2, 3). Infact, there is no close correlation between serum LH concen-mechanism.

There is a close correlation between GnRH release and trations and GnRH mRNA level. We examined the transla-tional efficiency of the GnRH mRNA in the preoptic areaserum LH concentrations (12, 21, 28, 29). Hypothalamic

GnRH neurones express immediate early genes such as c-fos 3 h after IL-1b treatment by separation of polyribosome-associated cytoplasmic RNA followed by RT-PCR deter-during the proestrous LH surge (36). Central injection of IL-



CTL 3h 5h

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Y=0.736X–0.043r=0.982

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10pg0.1pg5fg0.05fg2fg0.02fg

nativeGnRHR

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F. 5. Standard curves of competitive RT-PCR for the quantification of GnRHR mRNA expression in the preoptic area (), mediobasalhypothalamus () and pituitary (). Titration experiments were performed with different amounts of mutant GnRHR cRNA. Effects of IL-1b onGnRHR mRNA levels in micropunches of the preoptic area ( ) and mediobasal hypothalamus (), and from the pituitary ( ). Rats were decapitatedat 0, 3, and 5 h after i.c.v. injection of IL-1b (100 ng/rat). Each bar indicates the mean±SEM. *P<0.05 (vs control group).

mination of translatable GnRH mRNA. Treatment with IL- be involved in the regulation of GnRH release (37, 38). Themediobasal hypothalamus GnRHR mRNA levels fluctuated1b significantly shifted the distribution of ribosome-associated

GnRH mRNA. Since the distribution of control L7 RNA during the oestrous cycle (13). We recently found thattreatment of oestrogen evoked a marked increase in GnRHRwas not significantly changed by IL-1b treatment, we believe

that this effect of IL-1b is specific to the GnRH mRNA mRNA levels in the mediobasal hypothalamus of ovariectom-ized rats (14). These results indicate that GnRHR in thespecies. The shift of GnRH mRNA distribution from heavy

to light fraction implies that the number of ribosome associ- mediobasal hypothalamus may be involved in the regulationof GnRH neuronal activity. It is of importance to note thatated with GnRH mRNA was decreased. As described by

Gore and his colleague (21), the reduced attachment of GnRHR mRNA levels from the preoptic area and mediobasalhypothalamus altered with a pattern similar to that of serumribosome to GnRH mRNA can cause a decrease in the rate

of translational initiation. Although we are unable to measure LH concentrations (Fig. 5,). These data suggest thatGnRHR in the preoptic area and mediobasal hypothalamusthe transcription rate and mRNA stability of GnRH mainly

because of the scattered distribution and relative low number may mediate the effect of IL-1b on GnRH–LH axis. However,pretreatment with an IL-1 receptor antagonist marginallyof GnRH neurones in vivo, these results clearly indicate that

central IL-1b may have a fast effect on GnRH gene expression blocks the IL-1b-induced decrease in GnRHR mRNA expres-sion in the preoptic area and mediobasal hypothalamus, whilethrough post-transcriptional modulation. It is then likely that

decreased GnRH secretion accompanied by changing transla- clearly blocking the suppressive effect of IL-1b on LH release.This result is somewhat controversial and remains to betional efficiency. Only a small fraction of GnRH is released

such that the stored pool of GnRH is essentially unaltered. resolved.In summary, the present study provides for the first timeIt appears that newly synthesized GnRH is readily secreted

without incorporation into the stored pool, as shown in other in vivo evidence that central IL-1b may affect GnRH neuronalactivity through the inhibition of the translational efficiencyneuroendocrine systems (reviews: (1, 2)).

There is increasing evidence that GnRHR in the brain may of the GnRH mRNA, and suggest the possible involvement



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17 Kim K, Jarry H, Knoke I, Seong JY, Leonhardt S, Wuttke W.

CTL IL-1b IL-1ra+IL-1b

Gn

RH

rec

epto

r m

RN

A(f

g/M

BH

mic

rop

un

ch)

(B)

2.0

1.5

1.0

0.5

0.0

n=11

CTL IL-1b IL-1ra+IL-1b

Gn

RH

rec

epto

r m

RN

A(f

g/P

OA

mic

rop

un

ch)

(A)

0.4

0.3

0.2

0.1

0.0

n=110.5

Competitive PCR for quantitation of GnRH mRNA level in a singleF. 6. Effect of IL-1 receptor antagonist (IL-1ra) on preoptic area ()micropunch of the rat preoptic area. Mol Cell Endocrinol 1993; 97:and mediobasal hypothalamus () GnRHR mRNA expressions. IL-1ra153–158.(5 mg/rat) or saline was injected into lateral ventricle 10 min prior to IL-

18 Seong JY, Jarry H, Khunemuth S, Leonhardt S, Wuttke W, Kim K.1b treatment. Rats were killed 3 h after IL-1b treatment. Each barEffect of GABAergic compounds on gonadotropin-releasing hormoneindicates the mean±SEM, and mRNA values were calculated on thereceptor gene expression in the rat. Endocrinology 1995; 136: 2587–2593.basis of standard plots shown in Fig. 5. *P<0.05 (vs control group).

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23 Rivest S, Rivier C. Centrally injected interleukin-1 beta inhibits thehypothalamic LHRH secretion and circulating LH levels via prosta-Accepted 2 November 1999glandins in rats. J Neuroendocrinol 1993; 5: 445–450.

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Effect of Interleukin-1β on Gonadotropin-Releasing Hormone (GnRH) and GnRH Receptor Gene Expression in Castrated Male Rats