0013-7227/04/$15.

00/0 Endocrinology 145(10):4565– 4574

Printed in U.S.A. Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2004-0413

Developmental and Hormonally Regulated Messenger

Ribonucleic Acid Expression of KiSS-1 and Its Putative
Receptor, GPR54, in Rat Hypothalamus and
Potent Luteinizing Hormone-Releasing Activity
of KiSS-1 Peptide
V. M. NAVARRO, J. M. CASTELLANO, R. FERNÁNDEZ-FERNÁNDEZ, M. L. BARREIRO, J. ROA,
J. E. SANCHEZ-CRIADO, E. AGUILAR, C. DIEGUEZ, L. PINILLA, AND M. TENA-SEMPERE
Department of Cell Biology, Physiology and Immunology (V.M.N., J.M.C., R.F.-F., M.L.B., J.R., J.E.S.-C., E.A., L.P.,
M.T.-S.), University of Cordoba, 14004 Cordoba, Spain; and Department of Physiology (C.D.), University of Santiago
de Compostela, 15705 Santiago de Compostela, Spain

The gonadotropic axis is centrally controlled by a complex throughout the estrous cycle and was significantly increased
regulatory network of excitatory and inhibitory signals that after gonadectomy, a rise that was prevented by sex steroid
is activated at puberty. Recently, loss of function mutations of replacement both in males and females. Moreover, hypotha-
the gene encoding G protein-coupled receptor 54 (GPR54), the lamic expression of the KiSS-1 gene was sensitive to neonatal
putative receptor for the KiSS-1-derived peptide metastin, imprinting by estrogen. From a functional standpoint, intra-
have been associated with lack of puberty onset and hypogo- cerebroventricular administration of KiSS-1 peptide induced
nadotropic hypogonadism. Yet the pattern of expression and a dramatic increase in serum LH levels in prepubertal male
functional role of the KiSS-1/GPR54 system in the rat hypo- and female rats as well as in adult animals. In conclusion, we
thalamus remain unexplored to date. In the present work, provide novel evidence of the developmental and hormonally
expression analyses of KiSS-1 and GPR54 genes were con- regulated expression of KiSS-1 and GPR54 mRNAs in rat hy-
ducted in different physiological and experimental settings, pothalamus and the ability of KiSS-1 peptide to potently stim-
and the effects of central administration of KiSS-1 peptide on ulate LH secretion in vivo. Our current data support the con-
LH release were assessed in vivo. Persistent expression of tention that the hypothalamic KiSS-1/GPR54 system is a
KiSS-1 and GPR54 mRNAs was detected in rat hypothalamus pivotal factor in central regulation of the gonadotropic axis at
throughout postnatal development, with maximum expres- puberty and in adulthood. (Endocrinology 145: 4565– 4574,
sion levels at puberty in both male and female rats. Hypo- 2004)
thalamic expression of KiSS-1 and GPR54 genes changed

T HE PITUITARY GONADOTROPINS, LH and FSH, are

glycoprotein hormones primarily involved in the con-
trol of key gonadal functions such as ovulation, spermato-
nals participate in fine regulation of the gonadotropic axis,
acting at hypothalamic and/or pituitary levels (2– 4). Attain-
ment of reproductive capacity at adulthood is the end point
genesis, and sex hormone production (1). In mammals, LH of a cascade of sex developmental events that leads to pu-
and FSH secretion is controlled by a complex neuroendocrine berty. It is assumed that awakening of the neuroendocrine
network integrating central and peripheral signals. The piv- system controlling gonadotropin secretion at puberty is the
otal hierarchical factor in the central control of gonadotropin result of the concerted enhancement of excitatory signals and
secretion is the hypothalamic decapeptide LH-releasing hor- the lowering of inhibitory inputs on LHRH neurons. How-
mone (LHRH), also termed GnRH, which dictates the pul- ever, despite recent developments in the field (for review, see
satile release of LH and FSH from pituitary gonadotropes (2, Refs. 5–7), the primary mechanisms for such a maturational
3). In turn, episodic release of LHRH is exquisitely governed switch remain largely unknown.
by the interplay of a plethora of excitatory and inhibitory Recently, loss of function mutations of the gene encoding
signals at the hypothalamus (2, 3). In addition, gonadal hor- G protein-coupled receptor 54 (GPR54) were shown to be
mones (sex steroids and peptides) and other peripheral sig- unexpectedly associated with lack of puberty onset and hy-
pogonadotropic hypogonadism in both humans and rodents
Abbreviations: AUC, Area under the curve; CT, cycle threshold; DPN, (8, 9). GPR54 was initially cloned in the rat as an orphan
diarylpropionitrile; EB, estradiol benzoate; ER, estrogen receptor; GPCR, receptor with 45% sequence similarity to galanin receptors
G protein-coupled receptor; icv, intracerebroventricular; LHRH, LH- (10). Thereafter, the human ortholog of GPR54, termed
releasing hormone; ORX, orchidectomized, orchidectomy; OVX, ovari- AXOR12 or hOT7T175, was identified (11–13). A search for
ectomized, ovariectomy; PPT, propylpyrazoletriol; PRL, prolactin; T,
testosterone. the natural ligand(s) of this receptor identified a 54-amino
Endocrinology is published monthly by The Endocrine Society (http://
acid secreted peptide, derived from the proteolytic process-
www.endo-society.org), the foremost professional society serving the ing of the product of the metastasis suppressor gene KiSS-1,
endocrine community. with high affinity binding for GPR54 (11–13). This novel

4565
4566 Endocrinology, October 2004, 145(10):4565– 4574 Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion

protein, named metastin, contains a C-terminal Arg-Phe- ceuticals Ltd. (Belmont, CA). Estradiol benzoate (EB) and testosterone
NH2 sequence distinctive for the RFamide peptide family. To (T) were purchased from Sigma-Aldrich Corp. (St. Louis, MO). The
selective agonist of estrogen receptor ␣ (ER␣), propylpyrazoletriol
date, a number of KiSS-1-derived peptides have been iden- (PPT), and the potency-selective ER␤ ligand, diarylpropionitrile (DPN)
tified (13). These have been globally termed kisspeptins, and were obtained from Tocris Cookson Ltd. (Avonmouth, UK).
include metastin (kisspeptin-54), kisspeptin-14, and kisspep-
tin-13. They all share a common C-terminal RFamide motif Experimental designs
and exhibit equal biopotency at rat and human GPR54 (13).
In addition, other C-terminal fragments of KiSS-1, such as In the first series of experiments, analysis of hypothalamic expression
of KiSS-1 and GPR54 mRNAs was conducted at different stages of
kisspeptin-10 or KiSS-1112–121, KiSS-1114 –121, and KiSS-194 –121, postnatal development. Thus, in experiment 1, hypothalamic samples
display high affinity binding for GPR54 and are provided were obtained from male rats at 1 d (n ⫽ 10), 5 d (n ⫽ 10), 10 d (n ⫽ 10),
with biological activities in different cell lines and/or in vivo 15 d (n ⫽ 5), 20 d (n ⫽ 5), 30 d (n ⫽ 5), 45 d (n ⫽ 5), 75 d (n ⫽ 5), and
systems (12, 13). 18 months (540 d; n ⫽ 5/group) postpartum, corresponding to the
neonatal (1 and 5 d), infantile (10 and 15 d), prepubertal (20 and 30 d),
Metastin was initially purified from human placenta (11). pubertal (45 d), adult (75 d), and aged stages of postnatal maturation (5).
Thereafter, significant expression of the KiSS-1 gene was also Similarly, in experiment 2, hypothalamic tissue samples were obtained
demonstrated in the brain, especially at the hypothalamus from female rats at 1 d (n ⫽ 10), 5 d (n ⫽ 10), 10 d (n ⫽ 10), 15 d (n ⫽
and basal ganglia (12, 14). Similarly, GPR54 mRNA has been 5), 20 d (n ⫽ 5), 30 d (n ⫽ 5), and 60 d (n ⫽ 5/group) postpartum,
found in placenta, several areas of the central nervous sys- corresponding to the neonatal (1 and 5 d), infantile (10 and 15 d),
prepubertal (20 d), pubertal (30 d), and adult (60 d) stages of postnatal
tem, pituitary, spinal cord, and pancreas (12, 14). Low cir- development (5). On the latter, estrous cyclicity was monitored by daily
culating levels of metastin have been detected in human vaginal cytology in adult females, and only rats with at least two con-
plasma, which increase dramatically during pregnancy (15). secutive 4-d estrous cycles were used. Representative hypothalamic
From a functional standpoint, metastin is provided with samples were obtained from adult cyclic females at proestrous (at 1000
and 1800 h), estrous (at 1000 h), diestrous d 1 (at 1000 h), and diestrous
potent antimetastasis activity on some tumors, such as mel- d 2 (at 1000 h) phases of the ovarian cycle.
anoma and papillary thyroid carcinoma (11, 16). In addition, In the next set of experiments, regulation of hypothalamic expression
it was recently proposed that KiSS-1 peptides might play a of KiSS-1 and GPR54 genes by gonadal factors was monitored in male
role in the regulation of trophoblast invasion (15) and the and female rats. Thus, in experiment 3, adult (75 d old) males were
control of some endocrine systems (13). However, the actual bilaterally orchidectomized (ORX) under light ether anesthesia, and
hypothalamic samples (n ⫽ 5/group) were obtained 2 wk after surgery.
physiological functions of KiSS-1-derived peptides remain An additional group of ORX males (n ⫽ 5) was implanted with SILAS-
largely unexplored. In this context, the demonstration that TIC brand silicon tubing (Dow Corning, Midland, MI) elastomers (40
inactivating mutations of their putative receptor led to re- mm in length; inner diameter, 0.062 cm; exterior diameter, 0.125 cm)
productive failure due to hypogonadotropic hypogonadism containing T, and hypothalami were sampled 2 wk after ORX. Similarly,
in experiment 4, bilateral ovariectomy (OVX) was performed under
highlighted a previously unsuspected role for the KiSS-1/ ether anesthesia in adult (60 d old) cycling females at random stages of
GPR54 system in the control of the gonadotropic axis (8, 9). the estrous cycle. Two weeks after OVX, rats (n ⫽ 5/group) were injected
Yet the biological effects, regulatory mechanisms, site(s) of sc daily for 3 d with 0.2 ml olive oil (used as vehicle), 25 ␮g synthetic
action, and developmental pattern of expression of this sys- EB, 1.5 mg of the selective ER␣ agonist PPT, 1.5 mg of the potency-
tem within the reproductive axis have not been explored to selective ER␤ ligand, DPN, or PPT plus DPN. The experimental protocol
and doses for the different ER ligands were selected based on previous
date. To initiate such an analysis, we report herein the ex- references, including data from our group (18, 19). In addition, in ex-
pression profile of KiSS-1 and GPR54 genes in the rat hy- periment 5, the effects of neonatal exposure to estrogen on the hypo-
pothalamus at different developmental stages and experimen- thalamic expression levels of KiSS-1 and GPR54 mRNAs were evaluated.
tal settings. In addition, the effects of central administration of In the rat, estrogenization during critical periods of sexual differentia-
tion of the hypothalamus (i.e. the perinatal period) has been reported to
a biologically active KiSS-1 peptide on LH release are studied permanently impair functioning of the reproductive axis and puberty
in vivo. onset (20). In this setting, 1-d-old male rats were injected sc with a single
dose of EB (500 ␮g/rat; dissolved in 100 ␮l olive oil), a regimen that has
been reported to induce complete estrogenization in the male rat without
Materials and Methods major systemic toxicity (20). Vehicle (oil)-injected animals served as
Animals and drugs controls. The animals (n ⫽ 6/group) were killed on d 60 postpartum.
Finally, in experiment 6, the ability of KiSS-1 peptide to centrally
Wistar rats bred in the vivarium of University of Cordoba were used. modulate LH secretion was assessed in vivo. To this end, mouse KiSS-
The day the litters were born was considered d 1 of age. The animals 1110 –119-NH2 peptide was used. This peptide is the rodent homologue of
were maintained under constant conditions of light (14 h of light, from human KiSS-1112–121-NH2 or kisspeptin 10, which was previously shown
0700 h) and temperature (22 C) and were weaned at d 21 of age in groups to maximally bind and activate GPR54 in transfected Chinese hamster
of five rats per cage with free access to pelleted food and tap water. ovary cells (12, 13). Central intracerebroventricular (icv) administration
Experimental procedures were approved by the Cordoba University of KiSS-1 peptide was conducted in prepubertal male (30 d old) and
ethical committee for animal experimentation and were conducted in female (25 d old) rats as well as in adult (75 d old) males (n ⫽ 10 –12
accordance with the European Union normative for care and use of rats/group) as previously described (21, 22). A dose of 1 nmol KiSS-1 in
experimental animals. In all experiments the animals were killed by 10 ␮l was injected per rat on the basis of previous references testing the
decapitation, and for experiments involving mRNA analysis (experi- neuroendocrine actions of different centrally administered peptides (22,
ments 1–5), the hypothalamus was dissected out, as described in detail 23). Groups of animals (n ⫽ 10 –12) were sequentially killed 15 and 60
previously (17), by a horizontal cut about 2 mm in depth with the min after icv injection. Animals injected with vehicle (physiological
following limits: 1 mm anteriorly from the optic chiasm, the posterior saline, 0.9% NaCl) served as controls. Upon decapitation, trunk blood
border of mamillary bodies, and the hypothalamic fissures. Hypotha- was collected, and serum samples were separated by centrifugation at
lamic samples were immediately removed upon decapitation, frozen in 1600 ⫻ g for 20 min and stored at ⫺20 C until use for hormone deter-
liquid nitrogen, and stored at ⫺80 C until processing for RNA analysis. minations (see below). As our initial data evidenced a potent LH-
Mouse KiSS-1110 –119-NH2, the rodent analog of the C-terminal KiSS-1 releasing effect of centrally administered KiSS-1 peptide, a detailed
decapeptide KiSS-1112–121-NH2, was obtained from Phoenix Pharma- time-course analysis of such a response was conducted in experiment 7.
Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion Endocrinology, October 2004, 145(10):4565– 4574 4567

Thus, 1 nmol KiSS-1 peptide was icv injected into adult male rats (n ⫽ Spain). Quantification of the intensity of RT-PCR signals was carried out
10 –12 rats/group), and systemic blood samples (300 ␮l) were obtained by densitometric scanning using an image analysis system (1-D Man-
by jugular venipuncture before (0 min) and 15, 30, 45, 60, 90, 120, and ager, TDI Ltd., Madrid, Spain), and values of the specific targets were
180 min after central administration of KiSS-1. normalized to those of internal controls to express arbitrary units of
relative expression. In all assays, liquid controls and reactions without
RNA analysis by semiquantitative RT-PCR RT resulted in negative amplification.

Total RNA was isolated from hypothalamic samples using the single- RNA analysis by real-time RT-PCR
step, acid guanidinium thiocyanate-phenol-chloroform extraction
method (24). Hypothalamic expression of KiSS-1 and GPR54 mRNAs To verify changes in gene expression observed by final-time RT-PCR,
was assessed by RT-PCR, optimized for semiquantitative detection, real-time RT-PCR was performed in selected experimental samples us-
using the primer pairs and conditions indicated in Table 1. For each ing the iCycler iQ Real-Time PCR detection system (Bio-Rad Labora-
target, RT-PCR amplification was routinely conducted using two dif- tories, Hercules, CA). In detail, KiSS-1 and GPR54 mRNA levels were
ferent sets of primers, which were generated on the basis of the pub- assayed in representative hypothalamic samples from different stages of
lished sequences of rat KiSS-1 and GPR54 genes (GenBank accession nos. postnatal development in males (5-, 15-, 30-, 45-, and 75-d-old rats) and
AY196983.1 and NM023992.1, respectively) and designed to span the females (5-, 15-, 30-, and cyclic 60-d-old rats on diestrous d 1) and in
intron sequences. In addition, hypothalamic expression of LHRH neonatally estrogenized adult male rats. The synthesized cDNAs were
mRNA was assessed in selected experimental groups using the primer further amplified (1/10th) in triplicate by PCR using SYBR Green I as
pair and conditions described in Table 1. As an internal control for RT fluorescent dye and 1⫻ iQ Supermix containing 50 mm KCl, 20 mm
and reaction efficiency, amplification of a 240-bp fragment of S11 ribo- Tris-HCl, 0.2 mm deoxy-NTPs, 3 mm MgCl2, and 2.5 U iTaq DNA
somal protein mRNA was carried out in parallel in each sample, as polymerase (Bio-Rad Laboratories) in a final volume of 25 ␮l. The PCR
indicated in Table 1. cycling conditions were as follows: initial denaturation and enzyme
For amplification of the targets, 2 ␮g total RNA were used for RT-PCR activation at 95 C for 5 min, followed by 40 cycles of denaturation at 95
in two consecutive separate steps. In addition, to enable appropriate C for 15 sec, annealing at 62.5 C (KiSS-1), 63.5 C (GPR54) or 58 C (RP-S11)
amplification in the exponential phase for each target, PCR amplifica- for 15 sec, and extension at 72 C for 1 min. Product purity was confirmed
tions of specific signals and RP-S11 transcript were carried out in sep- by dissociation curves and random agarose gel electrophoresis. No-
arate reactions with different number of cycles, but using similar template controls were included in all assays, yielding no consistent
amounts of the corresponding cDNA templates generated in single RT amplification. Calculation of the relative expression levels of the target
reactions, as previously described (25, 26). PCRs consisted of a first mRNAs was conducted based on the cycle threshold (CT) method (27).
denaturing cycle at 97 C for 5 min, followed by a variable number of The CT for each sample was calculated using iCycler iQ real-rime PCR
cycles of amplification, defined by denaturation at 96 C for 30 sec, detection system software with an automatic fluorescence threshold (Rn)
annealing for 30 sec, and extension at 72 C for 1 min. A final extension setting. Accordingly, fold expression of target mRNAs over reference
cycle of 72 C for 15 min was included. Annealing temperature was values was calculated by the equation 2⫺⌬⌬CT, where ⌬CT is determined
adjusted for each target and primer pair: 62.5 C for KiSS-1 products, 63.5 by subtracting the corresponding RP-S11 CT value (internal control)
C for GPR54 products, and 58 C for LHRH and RP-S11 transcripts. from the specific CT of the target (KiSS-1 or GPR54), and ⌬⌬CT is
Different numbers of cycles were tested to optimize amplification in the obtained by subtracting the ⌬CT of each experimental sample from that
exponential phase of PCR. In detail, analysis of intensity of PCR signals of the reference sample (taken as reference value 100). For each exper-
as a function of the number of amplification cycles revealed a strong imental group assayed, reference samples were arbitrarily taken from
linear relationship among cycles 28 –38 in the case of KiSS-1 and GPR54 5-d-old rat hypothalamus (postnatal development) and control adult
transcripts (with correlation coefficients r2 ⱖ 0.97) and among cycles hypothalamic samples (estrogenization model). No significant differ-
20 –28 in the case of RP-S11 (r2 ⫽ 0.982). On this basis, the numbers of ences in CT values were observed for RP-S11 between the treatment
PCR cycles indicated in Table 1 were chosen for additional semiquan- groups.
titative analysis of specific targets and RP-S11 internal control.
PCR-generated DNA fragments were resolved in Tris-borate-buff- Hormone measurement by specific RIA
ered 1.5% agarose gels and visualized by ethidium bromide staining.
The specificity of PCR products was confirmed by direct sequencing Serum LH, prolactin (PRL), and GH levels were measured in a vol-
using a fluorescent dye termination reaction and an automated se- ume of 10 –25 ␮l using a double-antibody method and RIA kits supplied
quencer (Central Sequencing Service, University of Cordoba, Cordoba, by the NIH (Dr. A. F. Parlow, NIDDK National Hormone and Peptide

TABLE 1. Oligonucleotide primer pairs used for RT-PCR amplification of KiSS-1, GPR54 and RP-S11 transcripts in hypothalamic
samples

Target Oligo-primers Expected size (bp) PCR cycles

KiSS-1 (A) KiSS-1 sense (5⬘-TGG CAC CTG TGG TGA ACC CTG AAC-3⬘)
202 32
KiSS-1 antisense 1 (5⬘-ATC AGG CGA CTG CGG GTG GCA CAC-3⬘)

KiSS-1 (B) KiSS-1 sense (5⬘-TGG CAC CTG TGG TGA ACC CTG AAC-3⬘)
301 32
KiSS-1 antisense 2 (5⬘-GCC ACC TGC CTC CTG CCG TAG CGC-3⬘)

GPR54 (A) GPR54 sense (5⬘-TGT GCA AAT TCG TCA ACT ACA TCC-3⬘)
194 32
GPR54 antisense 1 (5⬘-AGC ACC GGG GCG GAA ACA GCT GC-3⬘)

GPR54 (B) GPR54 sense (5⬘-TGT GCA AAT TCG TCA ACT ACA TCC-3⬘)
303 32
GPR54 antisense 2 (5⬘-AGC AGG TAT AGG GCC AGC AGG TTG-3⬘)

LHRH LHRH sense (5⬘-GCA CTA TGG TCA CCA GCG GG-3⬘)
477 32
LHRH antisense (5⬘-CAT GGA TCT CAG CGT CAA TG-3⬘)

RP-S11 S11 sense (5⬘-CAT TCA GAC GGA GCG TGC TTA C-3⬘)
240 24
S11 antisense (5⬘-TGC ATC TTC ATC TTC GTC AC-3⬘)
For KiSS-1 and GPR54 targets, two different primer combinations (A and B), whose sequences are included, were routinely used for analysis.
The expected size of the generated cDNA products and the number of cycles selected for RT-PCR analysis are indicated for each signal.
4568 Endocrinology, October 2004, 145(10):4565– 4574 Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion

FIG. 1. Developmental profile of ex-

pression of KiSS-1 and GPR54 genes in
rat hypothalamus throughout postna-
tal maturation in the male. In the left
panels, representative RT-PCR assays
are presented of expression levels of
KiSS-1 and GPR54 mRNAs in hypotha-
lamic samples from 1-, 5-, 10-, 15-, 20-,
30-, 45-, and 75-d-old as well as 18-
month-old male rats. Parallel amplifi-
cation of S-11 ribosomal protein mRNA
served as internal control. In the right
panels, semiquantitative values are the
mean ⫾ SEM of at least three indepen-
dent determinations. Groups with dif-
ferent superscript letters are statisti-
cally different (P ⬍ 0.05, by ANOVA
followed by Student-Newman-Keuls
multiple range test).

Program, Bethesda, MD). Rat LH-I-9, PRL-I-6, and GH-I-7 were labeled TABLE 2. Real-time RT-PCR data from triplicate analyses of
with 125I by the chloramine-T method, and the hormone concentrations hypothalamic KiSS-1 and GPR54 mRNA levels in representative
were expressed using reference preparations LH-RP-3, PRL-RP-3, and experimental groups
GH-RP-2 as standards. Intra- and interassay coefficients of variation
were less than 8% and 10%, respectively. The sensitivities of the assays mRNA levels (% over reference)
were 20, 10, and 5 pg/tube for LH, PRL, and GH, respectively. The Groups
KiSS-1 GPR54
accuracy of hormone determination was confirmed by assessment of rat
serum samples of known hormone concentrations used as external Male (5 d) 102.0 ⫾ 6.0 105.0 ⫾ 7.0
controls. Male (15 d) 110.0 ⫾ 7.0 112.0 ⫾ 6.0
Male (30 d) 55.0 ⫾ 4.0a 47.5 ⫾ 5.0a
Presentation of data and statistics Male (45 d) 292 ⫾ 17.5a 214.0 ⫾ 12.5a
Male (75 d) 112.5 ⫾ 9.0 115.0 ⫾ 7.0
Semiquantitative RT-PCR analyses were carried out in duplicate from Female (5 d) 105.0 ⫾ 8.0 100.0 ⫾ 6.0
at least three independent RNA samples of each experimental group. For Female (15 d) 60.0 ⫾ 6.0a 61.0 ⫾ 5.0a
generation of RNA samples, two or three hypothalamic fragments were Female (30 d) 186.0 ⫾ 10.0a 172.0 ⫾ 9.0a
pooled before isolation, and the generated samples were processed Female (60 d, 115.0 ⫾ 7.0 105.0 ⫾ 7.0
independently. Real-time RT-PCR analyses were conducted in triplicate. diestrous d)
Semiquantitative RNA data are presented as the mean ⫾ sem. Serum LH Control (60 d) 112.0 ⫾ 8.0 102.0 ⫾ 5.0
determinations were conducted in duplicate, with a total number of Neoestrogenized 30.9 ⫾ 4.0a 115.0 ⫾ 4.0
10 –12 samples/determinations per group. Hormonal data are presented
Representative samples from three settings were assayed: 1) dif-
as the mean ⫾ sem. In addition, integrated LH secretory responses were
ferent stages of postnatal development in males, 2) different stages of
expressed, when appropriate, as the area under the curve (AUC), cal-
postnatal development in females, and 3) neonatally estrogenized
culated following the trapezoidal rule, over a 180-min period. Results
male rats. Relative mRNA levels in the experimental groups are
were analyzed for statistically significant differences using ANOVA,
expressed as percentage over reference values, which were arbitrarily
followed by the Student-Newman-Keuls multiple range test (SigmaStat
set at male 5-d (1), female 5-d (2), and control (3); underlined. Ref-
2.0; Jandel Corp., San Rafael, CA). P ⱕ 0.05 was considered significant.
erence values were assigned a level of 100%; the other values were
normalized accordingly. The percent expression over reference values
Results was calculated for each group by the equation 2⫺⌬⌬CT.
KiSS-1 and GPR54 mRNA levels in rat hypothalamus a
P ⱕ 0.05 vs. corresponding reference values (by ANOVA, followed
during development and the estrous cycle by Student-Newman-Keuls multiple range test).

The profiles of hypothalamic expression of KiSS-1 and

GPR54 mRNAs were first evaluated in samples from male they were moderate at birth and up to d 15 postpartum,
rats at different stages of postnatal development, from the declined at the prepubertal stage (d 20 and 30 postpartum),
neonatal period to adulthood and aging. Stages of postnatal and sharply increased thereafter, with maximum expression
maturation were defined on the basis of previous references levels at puberty (45-d-old males; Fig. 1). Such an expression
(for a review, see Ref. 5), and timing of puberty onset in male profile was confirmed by real-time RT-PCR analysis of rep-
(45 d old) and female (30 –35 d old) rats was defined on the resentative hypothalamic samples that showed maximum
basis of previously accepted criteria (5) and recording of expression levels in 45-d-old male rats (Table 2).
external signs of reproductive maturation, such as vaginal Similarly, hypothalamic expression of KiSS-1 and GPR54
opening and balanopreputial separation. Persistent expres- mRNA was persistently detected in female rats throughout
sion of KiSS-1 and GPR54 mRNAs was detected in male rat postnatal development, with moderate levels during the neo-
hypothalamus by means of semiquantitative RT-PCR at all natal period, which decreased significantly in the prepuber-
age points studied (1-, 5-, 10-, 15-, 20-, 30-, 45-, and 75-d-old tal stage and maximally increased at puberty (30-d-old fe-
rats as well as aged 18-month-old animals). However, hy- males; Fig. 2). Again, this expression profile was confirmed
pothalamic levels of KiSS-1 and GPR54 mRNAs significantly by real-time RT-PCR analysis of representative hypotha-
changed, in a coordinated manner, during the study period; lamic samples that showed maximum expression levels in
Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion Endocrinology, October 2004, 145(10):4565– 4574 4569

FIG. 2. Developmental profile of expres-

sion of KiSS-1 and GPR54 genes in rat
hypothalamus throughout postnatal mat-
uration in the female. In the left panels,
representative RT-PCR assays are pre-
sented of expression levels of KiSS-1 and
GPR54 mRNAs in hypothalamic samples
from 1-, 5-, 10-, 15-, 20-, 30-, and 60-d-old
female rats. In the latter, hypothalamic
samples in the diestrous d 1 (D1) phase of
the ovarian cycle were used. Parallel
amplification of S-11 ribosomal protein
mRNA served as an internal control. In the
right panels, semiquantitative values are
the mean ⫾ SEM of at least three indepen-
dent determinations. Groups with differ-
ent superscript letters are statistically dif-
ferent (P ⬍ 0.05, by ANOVA followed by
Student-Newman-Keuls multiple range
test).

FIG. 3. Profile of expression of KiSS-1

and GPR54 genes in rat hypothalamus at
different stages of the estrous cycle in the
rat. In the left panels, a representative
RT-PCR assay is presented of expression
levels of KiSS-1 and GPR54 mRNAs in
hypothalamic samples obtained from
adult (60 d old) cyclic females at
proestrous (P; at 1000 and 1800 h), es-
trous (E; at 1000 h), diestrous d 1 (D1; at
1000 h), and diestrous d 2 (D2; at 1000 h)
phases of the ovarian cycle. For compar-
ative purposes, expression analyses in
45- and 75-d-old male rats (m45 and m75)
are also shown. Parallel amplification of
S-11 ribosomal protein mRNA served as
an internal control. In the right panels,
semiquantitative values are the mean ⫾
SEM of at least three independent deter-
minations. Groups with different super-
script letters are statistically different
(P ⬍ 0.05, by ANOVA followed by Student-
Newman-Keuls multiple range test).

30-d-old female rats (Table 2). In adult cyclic females, relative pothalamic KiSS-1 mRNA levels 2 wk after surgery, which
levels of KiSS-1 and GPR54 mRNAs throughout the estrous was associated with a moderate rise in GPR54 mRNA
cycle were persistently higher than those in age-matched expression (Fig. 4). In this setting, serum LH levels sig-
males. The expression levels of these transcripts significantly nificantly increased 2 wk after ORX, whereas hypotha-
varied along the cycle, with peak levels on diestrous d 1 for lamic steady state levels of LHRH mRNA remained un-
KiSS-1 and GPR54 and low levels at estrus for KiSS-1 (Fig. 3). affected (Fig. 4). T replacement (40-mm SILASTIC brand
elastomers containing T) blunted the rise in circulating LH
Hypothalamic KiSS-1 and GPR54 mRNA levels after levels and totally prevented the increase in hypothalamic
gonadectomy and estrogenization KiSS-1 mRNA levels 2 wk after ORX. Similarly, T supple-
Regulation of hypothalamic expression of KiSS-1 and mentation blocked the moderate increase in GPR54 mRNA
GPR54 mRNAs by gonadal factors was evaluated using after ORX and partially suppressed hypothalamic LHRH
models of male and female gonadectomy. Because these mRNA levels (Fig. 4).
experimental manipulations are known to induce dra- In the female rat, hypothalamic levels of KiSS-1 and, to a
matic changes in the functioning of the gonadotropic axis, lesser extent, GPR54 mRNAs significantly increased 2 wk
serum LH levels and hypothalamic LHRH mRNA expres- after OVX. Estradiol replacement to OVX rats totally pre-
sion were also monitored. Orchidectomy (ORX) of adult vented such a response; a phenomenon that was mimicked
animals resulted in a significant, clear-cut increase in hy- by the administration of the selective agonist of ER␣, PPT,
4570 Endocrinology, October 2004, 145(10):4565– 4574 Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion

FIG. 4. Profile of expression of KiSS-1, GPR54, and LHRH genes in rat hypothalamus in adult (75 d old) rats, 2 wk after ORX with or without
T replacement. In the left panels, a representative RT-PCR assay is presented of expression levels of the targets in hypothalamic samples from
control males (Co), ORX rats, and ORX animals implanted with SILASTIC brand capsules containing T (ORX⫹T). Two independent samples
per group are presented. Parallel amplification of S-11 ribosomal protein mRNA served as an internal control. In the right panels, semiquan-
titative values of gene expression levels are the mean ⫾ SEM of at least three independent determinations. In addition, serum LH levels in the
different experimental groups are presented. Groups with different superscript letters are statistically different (P ⬍ 0.05, by ANOVA followed
by Student-Newman-Keuls multiple range test).

but not of the potency-selective ligand of ER␤, DPN. The Effects of icv administration of KiSS-1 peptide on
responses to the combined administration of PPT and DPN LH secretion
were not significantly different from those of administration To evaluate its role in the central control of the gonado-
of PPT alone (Fig. 5). As was the case in males, serum LH tropic axis, the effects of icv administration of the active
levels in gonadectomized females changed in parallel to hy-
fragment of KiSS-1 peptide, mouse KiSS-1110 –119-NH2, on LH
pothalamic KiSS-1 transcript levels, with a significant in-
secretion were tested in prepubertal and adult rats. In pre-
crease in LH concentrations in OVX females that was pre-
pubertal animals, central injection of 1 nmol/rat KiSS-1 pep-
vented by administration of estradiol and PPT, but not DPN.
tide elicited a clear-cut increase in serum LH levels in both
In contrast, 2-wk OVX resulted in a significant decrease in
males (30 d old) and females (25 d old), with maximal stim-
hypothalamic LHRH mRNA levels that were stimulated by
ulation (⬃10- to 12-fold increase over controls) 15 min after
estradiol replacement as well as by PPT and DPN adminis-
tration. For the latter, hypothalamic LHRH mRNA levels injection. In contrast, serum PRL levels were moderately
were maximally increased by administration of the potency- inhibited by central administration of KiSS-1 peptide to pre-
selective ER␤ ligand DPN (Fig. 5). pubertal animals. Likewise, in adult male rats, icv injection
In addition, the effects of neonatal estrogen exposure on of 1 nmol KiSS-1 peptide induced a significant increase in
the expression of KiSS-1, GPR54, and LHRH mRNAs in the serum LH levels 15 and 60 min after administration. In this
hypothalamus and on serum LH levels were monitored in setting, serum PRL levels remained unaffected after central
adult male rats. Inappropriate exposure to estrogenic com- administration of KiSS-1 peptide (Fig. 7). Serum GH levels
pounds during critical periods of sex differentiation of the were not modified by icv injection of KiSS-1 (1 nmol/rat) in
hypothalamus in the rat has been reported to induce a pleth- either prepubertal males (1.1 ⫾ 0.26 ng/ml at 15 min after
ora of reproductive defects through mechanisms that remain KiSS-1 vs. 1.85 ⫾ 0.6 ng/ml in controls) or prepubertal fe-
partially unexplored (20). Neonatal administration of EB (500 males (0.84 ⫾ 0.14 ng/ml at 15 min after KiSS-1 vs. 0.81 ⫾ 0.16
␮g/rat, on d 1 postpartum) induced a significant decrease in ng/ml in controls). Because our initial data evidenced a very
serum LH concentrations and hypothalamic KiSS-1 mRNA potent LH releasing activity of KiSS-1, a detailed time-course
levels in 60-d-old animals. In contrast, hypothalamic expres- analysis of the LH response to icv injection of 1 nmol/rat
sion of GPR54 and LHRH mRNAs remained unaffected in KiSS-1 was conducted in adult animals. Central injection of
neonatally estrogenized adult males (Fig. 6). The profile of KiSS-1 resulted in a dramatic increase in serum LH levels
expression of KiSS-1 and GPR54 mRNAs after neonatal es- over basal (0 min) values that peaked 15 min after peptide
trogenization was confirmed by real-time RT-PCR analysis of injection. Moreover, serum LH concentrations in KiSS-1-
representative hypothalamic samples (Table 2). injected animals were higher than those in paired vehicle-
Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion Endocrinology, October 2004, 145(10):4565– 4574 4571

FIG. 5. Profile of expression of KiSS-1, GPR54, and LHRH genes in rat hypothalamus in adult (60 d old) rats, 2 wk after OVX with or without
estrogen replacement, or administration of the selective ER␣ agonist PPT, the potency-selective ER␤ ligand DPN, or PPT plus DPN to OVX
rats. Parallel amplification of S-11 ribosomal protein mRNA served as an internal control. In the right panels, semiquantitative values of gene
expression levels are the mean ⫾ SEM of at least three independent determinations. In addition, serum LH levels in the different experimental
groups are presented. Groups with different superscript letters are statistically different (P ⬍ 0.05, by ANOVA followed by Student-Newman-
Keuls multiple range test).

FIG. 6. Hypothalamic profile of expres-

sion of KiSS-1, GPR54, and LHRH genes
in neonatally estrogenized adult (60 d
old) male rats. In the left panels, repre-
sentative RT-PCR assays are shown of
expression levels of KiSS-1, GPR54, and
LHRH mRNAs in hypothalamic samples
obtained from control males (C1–C4)
and neonatally estrogenized male rats
(EB1–EB4). Four independent samples
per group are presented. Parallel ampli-
fication of S-11 ribosomal protein mRNA
served as an internal control. In the
right panels, semiquantitative values of
gene expression levels are the mean ⫾
SEM of at least four independent deter-
minations. In addition, serum LH levels
in the different experimental groups are
presented. **, P ⬍ 0.05 vs. correspond-
ing controls (by ANOVA followed by
Student-Newman-Keuls multiple range
test).

injected animals during the 180-min study period following Discussion

drug administration. Accordingly, integrated LH secretion
during this time frame (180 min), as estimated by the AUC, Recently, a role for the KiSS-1/GPR54 system in control
was significantly increased by central icv injection of 1 nmol of the gonadotropic axis and puberty onset has emerged
KiSS-1 (Fig. 8). (8, 9). Yet the biological effects, regulatory mechanisms,
4572 Endocrinology, October 2004, 145(10):4565– 4574 Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion

FIG. 7. Serum LH and PRL levels in

prepubertal 30-d-old males (A), prepu-
bertal 25-d-old females (B), and adult
75-d-old males (C) at 15 and 60 min
after central icv administration of ve-
hicle or 1 nmol/rat KiSS-1 in 10 ␮l. **,
P ⬍ 0.01 vs. corresponding vehicle-
injected controls (by ANOVA followed
by Student-Newman-Keuls multiple
range test).

FIG. 8. Serum LH levels in adult (75 d old) male

rats before (0 min) and 15, 30, 45, 60, 90, 120, and
180 min after central icv administration of vehi-
cle or 1 nmol/rat KiSS-1 in 10 ␮l. In addition to the
profiles of mean LH levels in the experimental
groups, the integrated secretory responses, as the
AUC over the study period (180 min), are shown.
**, P ⬍ 0.01 vs. corresponding vehicle-injected
controls; a, P ⬍ 0.01 vs. KiSS-1 injected group
at 15 min (by ANOVA followed by Student-
Newman-Keuls multiple range test).

site(s) of action, and developmental pattern of expression in both male and female rats. Despite low circulating levels
of this system within the reproductive axis have not been of pituitary gonadotropins before puberty, prepubertal
explored to date. To our knowledge, this is the first study LHRH neurons are intrinsically able to increase their
to report the developmental and hormonally regulated secretory activity after certain experimental manipula-
pattern of expression of the genes encoding KiSS-1 and its tions, such as electrical stimulation or administration of
putative receptor, GPR54, in rat hypothalamus. Our ex- N-methyl-d-aspartate, an agonist of ionotropic excitatory
pression analyses during postnatal development showed amino acid receptors (5). Thus, it is evident that puberty
that the lowest hypothalamic mRNA levels of KiSS-1 and onset is mainly due to activation of central excitatory
GPR54 occur during the prepubertal stage, whereas max- inputs during the juvenile-pubertal transition period.
imum expression of these genes was observed at puberty Among others, neuronal systems using the excitatory
Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion Endocrinology, October 2004, 145(10):4565– 4574 4573

amino acids, noradrenaline and neuropeptide Y, have been produced estrogen after aromatization of testis-derived T
involved in such phenomena (4, 5). Our current data suggest promotes hypothalamic masculinization during a develop-
that, in addition, enhanced expression of both components of mental frame that spans from embryonic d 17.5 to d 10
the KiSS-1/GPR54 system at the hypothalamus might con- postpartum (20). Indeed, key events in hypothalamic func-
tribute to activation of the gonadotropic axis at puberty. This tion, such as the expression of ER␣ and ER␤ genes (28), are
contention is supported by our functional in vivo data, be- imprinted by neonatal estrogen. Our current data point out
cause central administration of KiSS-1 peptide to prepubertal that the neonatal endocrine (estrogen) milieu can also im-
male and female rats was able to significantly increase serum print the pattern of expression of the KiSS-1 gene in male rat
LH levels from low prepubertal values to levels similar to hypothalamus. In fact, relative mRNA levels of KiSS-1 at the
those in adult animals. Nevertheless, although the above hypothalamus were persistently suppressed in adults by
data might indicate a role for the hypothalamic KiSS-1 sys- neonatal estrogenization. In contrast, hypothalamic expres-
tem as a trigger of puberty onset, a permissive action of this sion of neither GPR54 nor LHRH genes was altered in adult-
novel factor in puberty development cannot be excluded on hood by neonatal exposure to high doses of estrogen. Again,
the basis of our current results. Experiments involving changes in LH levels closely paralleled those in hypotha-
chronic central administration of KiSS-1 peptide to immature lamic KiSS-1 mRNA levels, because neonatal exposure to
animals are presently in progress in our laboratory to address estrogen also induced a persistent decrease in serum LH
this issue. concentrations in adult male rats. Although this association
Additional evidence for the involvement of the hypotha- may not be causative, the contribution of persistently de-
lamic KiSS-1/GPR54 system in the regulation of the gona- creased KiSS-1 expression at the hypothalamus to the pleth-
dotropic axis is indirectly provided by its modulation by ora of developmental and functional defects in the gonado-
gonadal factors. Expression levels of KiSS-1 and GPR54 mR- tropic axis after neonatal exposure to supraphysiological
NAs significantly varied during the estrous cycle, with peak doses of estrogen merits further investigation.
levels on diestrous d 1 for KiSS-1 and GPR54 and low levels Central administration of KiSS-1 peptide elicited a very
at estrus for KiSS-1. This observation strongly suggests that potent secretory response in terms of LH secretion in pre-
circulating gonadal hormones might participate in regula- pubertal and adult animals. In the latter, a strikingly long-
tion of the hypothalamic expression of KiSS-1 and GPR54 lasting response to central icv administration of 1 nmol
genes. Accordingly, gonadectomy, in both males and fe- KiSS-1 was observed in time-course analysis over a 180-min
males, resulted in a significant increase in KiSS-1 and, to a period. Such an LH-releasing effect was not unspecific, be-
lesser extent GPR54, mRNA expression at the hypothalamus. cause serum GH levels remained unaffected, and serum PRL
These responses can be attributed to the removal of sex levels were moderately inhibited by icv injection of KiSS-1.
steroid inhibitory inputs, because they were prevented by The fact that central injection of KiSS-1 peptide was able to
replacement of ORX and OVX animals with T and EB, re- potently stimulate LH secretion before puberty and in adult
spectively. Moreover, activation of ER␣ by the selective li- males also supports the contention that this system may play
gand PPT, but not ER␤, by the potency-selective agonist DPN a relevant role in the regulation of the gonadotropic axis in
was able to mimic the effects of estrogen supplementation in adulthood. This phenomenon has been previously demon-
OVX rats, thus suggesting the involvement of ER␣ pathways strated for other hypothalamic systems, such as erbB-2/
in this phenomenon. The above responses in terms of hy- erbB-4, which has been involved not only in puberty onset,
pothalamic gene expression in the different experimental but also in regulation of the reproductive axis in the adult
groups closely paralleled the changes in serum LH levels cyclic female rat (29). As indicated above, a moderate de-
after gonadectomy and hormonal replacement. In contrast, crease in serum PRL levels was detected after icv injection of
transcriptional regulation of the LHRH gene at the hypo- KiSS-1 to prepubertal rats. Although a rise in serum PRL
thalamus did not clearly follow changes in circulating LH levels is detected during puberty in the rat (5), high PRL
values. Taken together, our data strongly suggest that the levels are frequently associated with low gonadotropin se-
hypothalamic KiSS-1/GPR54 system may play a role not cretion. Thus, it tempting to speculate that the combined
only in activation of the gonadotropic axis at puberty, but stimulatory and inhibitory effects of hypothalamic KiSS-1 on
also in its regulation in adulthood. Moreover, our present LH and PRL secretion, respectively, might be relevant for
results suggest that the effects of KiSS-1 on hypothalamic maximal activation of the gonadotropic axis at puberty onset.
LHRH, if any, are apparently not conducted at the transcrip- The hypothalamic targets and molecular mechanisms by
tional level. Alternatively, LHRH-independent actions of which central administration of KiSS-1 peptide modulates
KiSS-1 in the control of LH secretion (e.g. directly at the LH (and eventually PRL) secretion are presently under in-
pituitary level, acting as a hypophysiotropic neuropeptide) vestigation. In this context, detailed characterization of the
cannot be excluded. These phenomena are currently under pattern of expression of both components (ligand and re-
investigation at our laboratory. ceptor) of the KiSS-1/GPR54 system within the hypothala-
In addition to acute regulation by gonadal hormones, hy- mus will help to characterize the mode of action of KiSS-1 in
pothalamic expression of KiSS-1 mRNA appears to also be neuroendocrine control of the reproductive axis.
sensitive to the organizing effects of neonatal estrogen. Act- In summary, our current data point out that the genes
ing at critical periods of sex development, estrogen has been encoding KiSS-1 peptide and its putative receptor, GPR54,
involved in the functional organization of the hypothalamic- are expressed in the rat hypothalamus in a developmental
pituitary unit responsible for the control of gonadotropin and hormonally regulated manner (by gonadally derived
secretion throughout the life span (20). In the male rat, locally factors), and that central administration of KiSS-1 peptide is
4574 Endocrinology, October 2004, 145(10):4565– 4574 Navarro et al. • Hypothalamic KiSS-1/GPR54 System and LH Secretion

able to selectively and potently elicit LH secretion in pre- Tan CP, Tang-Nguyen AT, George SR, O’Dowd BF 1999 Discovery of a
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Acknowledgments 15. Horikoshi Y, Matsumoto H, Takatsu Y, Ohtaki T, Kitada C, Usuki S, Fujino M
2003 Dramatic elevation of plasma metastin concentrations in human pregnancy:
RIA kits for hormone determinations were kindly supplied by Dr. metastin as a novel placental-derived hormone in humans. J Clin Endocrinol
A. F. Parlow, NIDDK National Hormone and Peptide Program (Be- Metab 88:914 –919
thesda, MD). We are indebted to C. Bellido and A. Mayen for their 16. Ringel MD, Hardy E, Bernet VJ, Burch HB, Schuppert F, Burman KD, Saji M
outstanding assistance with some of the experimental studies. 2002 Metastin receptor is overexpressed in papillary thyroid cancer and ac-
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Received March 30, 2004. Accepted June 29, 2004. 2402
Address all correspondence and requests for reprints to: Dr. Manuel 17. Burcelin R, Thorens B, Glausner M, Gaillard RC, Pralong FP 2003 Gonado-
tropin-releasing hormone secretion from hypothalamic neurons: stimulation
Tena-Sempere, Physiology Section, Department of Cell Biology, Phys-
by insulin and potentiation by leptin. Endocrinology 144:4484 – 4491
iology, and Immunology, Faculty of Medicine, University of Cordoba, 18. Harris HA, Katzenellenbogen JA, Katzenellenbogen BS 2002 Characteriza-
Avda. Menéndez Pidal s/n, 14004 Cordoba, Spain. E-mail: fi1tesem tion of the biological roles of estrogen receptors, ER␣ and ER␤, in estrogen
@uco.es. target tissues in vivo through the use of an ER␣-selective ligand. Endocrinology
V.M.N. and J.M.C. contributed equally to this work and should be 143:4172– 4177
considered as joint first authors. 19. Tena-Sempere M, Navarro VM, Mayen A, Bellido C, Sanchez-Criado JE 2004
This work was supported by Grants BFI 2000-0419-CO3-03 and BFI Regulation of estrogen receptor (ER) isoform messenger RNA expression by
200-00176 from Ministerio de Ciencia y Tecnologı́a, Spain, and European different ER ligands in female rat pituitary. Biol Reprod 70:671– 678
Union Research Contract EDEN QLK4-CT-2002-00603. 20. Tena-Sempere M, Pinilla L, Gonzalez LC, Aguilar E 2000 Reproductive
disruption by exposure to exogenous estrogenic compounds during sex dif-
ferentiation: lessons from the neonatally estrogenized male rat. Curr Top
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