REPRODUCTION-DEVELOPMENT
Estradiol Down-Regulates RF-Amide-Related Peptide
(RFRP) Expression in the Mouse Hypothalamus
C. S. Molnár, I. Kalló, Z. Liposits, and E. Hrabovszky
Laboratory of Endocrine Neurobiology (C.S.M., I.K., Z.L., E.H.), Institute of Experimental Medicine,
Hungarian Academy of Sciences, Budapest, and Department of Neuroscience (I.K., Z.L.), Faculty of
Information Technology, Pázmány Péter Catholic University, Budapest, 1083 Hungary
In most mammals, RF-amide-related peptides are synthesized in the dorsomedial hypothalamic
nucleus and regulate reproduction via inhibiting GnRH neurons and, possibly, adenohypophyseal
gonadotrophs. In the present study, we investigated the possibility that RFRP-synthesizing neurons
are involved in estrogen feedback signaling to the reproductive axis in mice. First, we used quantitative in situ hybridization and compared the expression of prepro-RFRP mRNA of ovariectomized
mice, with and without 17␤-estradiol (E2) replacement. Subcutaneous administration of E2 via
silastic capsules for 4 d significantly down-regulated prepro-RFRP mRNA expression. The underlying receptor mechanism was investigated with immunohistochemistry. In ovariectomized mice,
low levels of nuclear estrogen receptor (ER)-␣ immunoreactivity were detectable in 18.7 ⫾ 3.8%
of RFRP neurons. The majority of RFRP neurons showed no ER-␣ signal, and RFRP neurons did not
exhibit ER-␤ immunoreactivity. Results of these studies indicate that RFRP is a negatively estradiolregulated neurotransmitter/neuromodulator in mice. The estrogenic down-regulation of RFRP
expression may contribute to estrogen feedback to the reproductive axis. The issue of whether E2
regulates RFRP neurons directly or indirectly remains open given that ER-␣ immunoreactivity is
present only at low levels in a subset of these cells. (Endocrinology 152: 1684 –1690, 2011)
he decapeptide GnRH represents the primary hypothalamic neurohormone that stimulates gonadotropin
secretion from the adenohypophysis (1, 2). An inhibitory
neuropeptide named gonadotropin-inhibiting hormone
(GnIH) has also been identified in the quail hypothalamus;
GnIH inhibits gonadotropin release from the pituitary in
a dose-dependent manner (3). In addition to acting as a
release-inhibiting hormone on gonadotrophs, GnIH also
regulates fertility via influencing the neurosecretory output of hypophysiotropic GnRH neurons. Accordingly,
GnIH-immunoreactive neuronal contacts (4) and GnIH
receptors (5) are present on avian GnRH neurons. Putative
GnIH homologs, RF-amide-related peptides (RFRP-1,
RFRP-2, and RFRP-3), have also been identified in mammals (6). With some species differences, the majority of
neurons that synthesize prepro-RFRP mRNA and RFRP
peptides have been localized to the dorsomedial nucleus of
the hypothalamus in hamsters, rats, mice, and sheep (6 –
T
16) and to the intermediate periventricular nucleus in
monkeys (17). Unlike in birds where GnIH-immunoreactive axons innervate the external zone of the median eminence and, thus, have access to the hypophyseal portal
vasculature (3, 18), RFRP neurons in rodents do not project to the external zone of the median eminence (10, 12)
and do not accumulate Fluoro-Gold from the systemic
circulation (12), suggesting that RFRP regulate fertility
primarily via central mechanisms. These mechanisms include direct inhibitory actions exerted upon GnRH neurons, as indicated by RFRP-immunoreactive neuronal
contacts on GnRH neurons (10, 12, 13, 19, 20) and by the
RFRP-3-induced hyperpolarization (19) and reduced electrical activity of a large subset of GnRH neurons (21) in
slice preparations of GnRH-GFP transgenic mice.
The GnRH neuronal system, which represents the
final common pathway in the neuroendocrine control of
reproduction, responds to feedback actions of circulat-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/en.2010-1418 Received December 9, 2010. Accepted January 21, 2011.
First Published Online February 15, 2011
Abbreviations: E2, 17␤-Estradiol; ER, Estrogen receptor; GnIH, gonadotropin-inhibiting
hormone; OVX, ovariectomized; RFRP, RF-amide related peptide.
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Endocrinology, April 2011, 152(4):1684 –1690
Endocrinology, April 2011, 152(4):1684 –1690
ing 17␤-estradiol (E2). Although direct estrogen actions
upon GnRH neurons can be exerted via estrogen receptor (ER)-␤ (22–26), interneurons expressing the classical ER-␣ play a critically important role in sensing and
conveying information on circulating estrogens to the
GnRH neuronal system (27). Evidence that the RFRP
neuronal system may be involved in estrogen feedback
signaling to GnRH neurons has emerged from studies of
hamsters. RFRP neurons in this rodent species contain
ER-␣ (10) and respond with c-Fos expression to an acute
administration of E2 (10).
In the present study, we investigated further the estrogen responsiveness of the RFRP neuronal system by addressing the estrogenic regulation of prepro-RFRP gene
expression in mice. First, quantitative in situ hybridization
was used to compare the expression levels of prepro-RFRP
mRNA in ovariectomized (OVX) mice with and without
estradiol replacement. Second, to identify the receptor
mechanism underlying this regulation, the presence of nuclear ER (ER-␣ and ER-␤) in RFRP neurons was studied
with dual-label immunohistochemistry.
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1685
with a Leica SM 2000R freezing microtome (Leica Microsystems, Nussloch Gmbh, Germany). The sections were stored at
⫺20 C in 24-well tissue culture plates containing antifreeze solution [30% ethylene glycol, 25% glycerol, 0.05 M phosphate
buffer (pH 7.4)].
Quantitative analysis of prepro-RFRP mRNA levels
in OVX and OVXⴙE2 mice with in situ
hybridization
Probe preparation
Materials and Methods
To prepare a cRNA hybridization probe to prepro-RFRP
mRNA, a 424-bp cDNA fragment was amplified with PCR from
rat hypothalamic cDNA. The amplicon (corresponding to bases
136 –559 of the rat prepro-RFRP mRNA; AB040288) was inserted into plasmid vector using the pGEM-T Easy Vector System from Promega (Madison, WI). The plasmid was propagated
in DH5␣ cells (Invitrogen, Carlsbad, CA), isolated with the QIAGEN (Valencia, CA) Plasmid Maxi kit, linearized with SalI and
purified with phenol/chloroform/isoamyl alcohol, followed by
chloroform/isoamyl alcohol extractions and then precipitation
with NaCl and ethanol. The linearized transcription template
was transcribed (28) with T7 RNA polymerase in the presence of
[35S]UTP (NEN Life Science Products, Boston, MA) to yield
antisense transcripts. In pilot in situ hybridization experiments,
we established that this probe efficiently recognized the mouse,
in addition to the rat, prepro-RFRP mRNA.
Animals
Hybridization
Sixty-day-old CD1 mice (n ⫽ 15) were purchased from a local
colony bred at the Medical Gene Technology Unit of the Institute
of Experimental Medicine. They were housed in a light-controlled (12-h light, 12-h dark cycle, lights on at 0700 h) and
temperature-controlled (22 ⫾ 2 C) environment, with free access
to standard food and tap water. The studies were carried out with
permission from the Animal Welfare Committee of the Institute
of Experimental Medicine (No. A5769-01) and in accordance
with legal requirements of the European Community (Decree
86/609/EEC).
Surgeries
Nine mice were deeply anesthetized with a cocktail of ketamine (25 mg/kg), xylavet (5 mg/kg), and pipolphen (2.5 mg/kg)
in saline and OVX bilaterally. On postovariectomy d 9, they
were re-anesthetized and implanted sc with a single silastic capsule (Sanitech, Havant, UK; length ⫽ 10 mm; inner diameter ⫽
1.57 mm; outer diameter ⫽ 3.08 mm) containing either sunflower oil (OVX group; n ⫽ 5) or 100 ␮g/ml E2 (Sigma Chemical
Co., St Louis, MO) in sunflower oil (OVX⫹E2 group; n ⫽ 4).
Four days later, the mice were anesthetized and killed by transcardiac perfusion with 40 ml 4% paraformaldehyde in PBS. The
brains were removed, postfixed for 1 h, infiltrated with 20%
sucrose overnight, and then snap-frozen on dry ice. Another six
mice were OVX and treated similarly to generate OVX (n ⫽ 3)
and OVX⫹E2 (n ⫽ 3) groups and perfused with a mixture of 2%
paraformaldehyde and 4% acrolein. These mice were used in
colocalization studies of ER-␤ and RFRP-1 immunoreactivities.
Section preparation
Serial 20-␮m sections were cut in the coronal plane (determined using a mouse dissection mold) from the hypothalami
Every sixth section from each paraformaldehyde-fixed mouse
hypothalamus was mounted on silanized microscope slides from
sterile Tris-buffered saline (50 mM; pH 7.8) with a sterile paint
brush and air dried. Then the sections were processed for the
radioisotopic in situ hybridization detection of prepro-RFRP
mRNA with a modified procedure detailed elsewhere (29). After
posthybridization treatments including the ribonuclease A digestion (20 ␮g/ml, 60 min at 37 C) of probe excess and a 30-min
stringent treatment in 0.1⫻ standard saline citrate solution (1⫻
standard saline citrate solution ⫽ 0.15 M NaCl/0.015 M sodium
citrate, pH 7.0) at 60 C, the sections were rinsed briefly in 70%
ethanol and air dried.
Autoradiography
First, the sections were exposed to Kodak BioMax MR autoradiography films for 3 d and signals developed with standard
procedures. Then the slides were dipped into Kodak NTB nuclear track emulsion (Kodak, Rochester, NY) and exposed for 1
wk. The autoradiographs were developed with Kodak processing chemicals. The sections were dehydrated with 95%, followed
by 100% ethanol (5 min each), cleared with xylene (twice for 5
min), and coverslipped with DPX mounting medium (Fluka Chemie, Buchs, Switzerland).
Image analysis and statistics
The x-ray film images were scanned using a HP ScanJet 4600
flatbed scanner equipped with a transparent material adapter.
For consistency, the autoradiographic images of the most heavily
labeled four sections in each animal were selected for the quantitative analysis of prepro-RFRP mRNA expression. The digital
image files were saved with TIF extension and opened for
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E2 Regulation of RFRP Neurons
analysis with the Image J software (public domain at http://
rsb.info.nih.gov/ij/download/src/). During measurements, a threshold was set and held constant across all sections to highlight the
entire positive hybridization signal area but not background. The
autoradiographic signal in each animal was characterized with
the mean of the four bilateral integrated density measurements (sum
of pixel density values in the highlighted signal area; mean gray
value ⫻ area). The OVX and the OVX⫹E2 groups were compared
using one-way ANOVA.
The integrated density of x-ray film autoradiographs depends
both on the number of labeled neurons in the signal area and the
single-cell levels of prepro-RFRP mRNA expression in individual RFRP neurons. These two parameters were analyzed further
using the computerized image analysis of emulsion autoradiographs from the same sections that had been selected for film
analysis. Each animal was characterized with eight digital photomicrographs from the dorsomedial nuclei of these four sections. We used a consistent sampling method to reach the densest
concentration of RFRP neurons in each microscope field. The
eight microscopic images of each animal were scanned with an
AxioCam MRc 5 digital camera mounted on a Zeiss AxioImager
M1 microscope, using a ⫻20 objective lens and the AxioVision
4.6 software (Carl Zeiss, Göttingen, Germany). The TIF files
were analyzed by an investigator blind to treatments. The files
were opened with Image J, and the threshold was set to only
highlight the silver grains in the sections. Then all neurons found
bilaterally in the eight image files of each animal were identified
and selected individually, using the lasso tool of the Image J
software. The integrated density of highlighted pixels covered by
silver grains (mean gray value ⫻ area) was determined for each
neuron. Each animal was finally characterized with the mean
integrated density over individual RFRP neurons, as determined
from all cells found in the eight photomicrographs. The number
of silver grain clusters identified as RFRP neurons in the OVX
and the OVX⫹E2 groups as well as the mean integrated density
of RFRP neurons in the two treatment groups were compared
with one-way ANOVA.
Dual-label immunohistochemical experiments to
colocalize ER-␣ and RFRP-1 immunoreactivities
Paraformaldehyde-fixed sections were pretreated with a mixture of 0.5% H2O2 and Triton X-100 (30 min) and then incubated in a 1:20,000 dilution (in 2% normal horse serum) of the
C1355 ER-␣ antiserum (C1355; Millipore, Temecula, CA;
1:10,000) raised in rabbit, followed by biotinylated secondary
antibodies (Jackson ImmunoResearch Europe Ltd., Soham,
Cambridgeshire, UK; 1:500) and the ABC Elite reagent for 60
min each. The signal was visualized with nickel-intensified diaminobenzidine and then post-intensified with silver-gold (30).
Subsequently, RFRP-1 immunoreactivity was detected with
mouse monoclonal antibodies (IF3; Takeda Pharmaceutical Co.
Ltd., Japan; 1:20,000) against the C terminus of rat RFRP-1 (7),
using the biotinylated secondary antibody-ABC technique and
nonintensified diaminobenzidine as the chromogen. The duallabeled sections were mounted on microscope slides and coverslipped with DPX.
Dual-label immunohistochemical experiments to
colocalize ER-␤ and RFRP-1 immunoreactivities
The same approach as above was chosen to attempt colocalization of ER-␤ and RFRP-1 immunoreactivities, except for us-
Endocrinology, April 2011, 152(4):1684 –1690
ing acrolein/paraformaldehyde-fixed tissue sections in which the
remaining aldehydes were neutralized with sodium borohydride
(24, 25). The ER-␤ rabbit antiserum used in these studies (Z8P,
lot 01162852, 150 ng/ml; Zymed Laboratories, San Francisco,
CA) has been characterized in our previous work (24, 25).
Specificity controls
Labeling specificity was verified using various control approaches. Neurons immunoreactive for ER-␣ or ER-␤ showed a
dominantly nuclear labeling that disappeared after omission of
the primary or secondary antibodies (31) or when using hypothalamic sections from ER-␣ (32) or ER-␤ (33) knockout mice.
The identical distribution patterns of the prepro-RFRP mRNA
and RFRP-1 peptide signals served as a positive control for the
specificity of the in situ hybridization and immunohistochemical
methods. The IF3 monoclonal RFRP-1 antibody revealed cell
bodies in the dorsomedial nucleus, an area ventral to it and in the
periventricular nucleus of the caudal hypothalamus, in accordance with the published distribution pattern of RFRP neurons
(10). The lack of antibody cross-reactivity with related RF-amide
neuropeptides (neuropeptides FF, AF, and SF) was also indicated
by the absence of labeled cell bodies elsewhere in the brain, including the nucleus of the solitary tract where the common precursor peptide of these neuropeptides is highly expressed (34).
Results
Prepro-RFRP mRNA levels of OVX mice decrease in
response to E2 treatment
Radioisotopic in situ hybridization studies revealed a
restricted regional distribution of prepro-RFRP mRNAsynthesizing neurons in the mouse hypothalamus that was
in accordance with results of earlier studies (10). The majority of labeled neurons were observed in the dorsomedial
nucleus, an area ventral to it, and in the periventricular
nucleus of the caudal hypothalamus. The distribution patterns were identical in the OVX and OVX⫹E2 groups, but
the signal was weaker in the latter (photographic insets in
Fig. 1). Quantitative analysis established that a 4-d E2
treatment of OVX mice significantly decreased the integrated density of x-ray film images [F(1,7) ⫽ 11.12; P ⫽
0.012; Fig. 1].
Silver grain clusters in the emulsion autoradiographs
were analyzed to determine whether E2 treatment decreased the number of detectable RFRP neurons, the single-cell levels of prepro-RFRP mRNA, or both. The integrated density analysis of silver grains over individual
neurons (Fig. 2) revealed lower single-cell levels of preproRFRP mRNA expression in the OVX⫹E2 vs. the OVX
group [F(1,7) ⫽ 8.85; P ⫽ 0.021]. In retrospect, this unbiased analysis identified significantly fewer [F(1,7) ⫽ 7.89;
P ⫽ 0.026] silver grain clusters (RFRP neurons) in
OVX⫹E2 mice (26.4 ⫾ 2.6 neurons per animal, mean ⫾
SEM), compared with OVX controls (39.0 ⫾ 3.6 neurons
per animal).
Endocrinology, April 2011, 152(4):1684 –1690
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Z8P antiserum, many ER-␤-immunoreactive cell nuclei
were detectable in the paraventricular nucleus (Fig. 3I),
but only scattered cell nuclei were labeled for ER-␤ in the
dorsomedial nucleus (Fig. 3J). ER-␤-positive RFRP neurons were not revealed (Fig. 3J).
Discussion
FIG. 1. E2 treatment reduces prepro-RFRP mRNA expression in the
dorsomedial nucleus. In situ hybridization detection of prepro-RFRP
mRNA expression in OVX mice treated sc with oil vehicle (OVX group;
n ⫽ 5) or E2 (OVX⫹E2 group; n ⫽ 4) for 4 d reveals identical signal
distribution in x-ray film autoradiographs, with reduced expression
levels in the latter treatment group (photographic insets). Results of
quantitative image analysis, shown in the diagram, confirm that the
prepro-RFRP mRNA signal (integrated density; mean gray value ⫻
signal area, expressed as percentage of the OVX group) in the
dorsomedial nucleus is lower in OVX⫹E2 vs. OVX mice. *, P ⬍ 0.05.
Nuclear ER-␤ is absent, whereas ER-␣ occurs in a
small subset of RFRP-synthesizing neurons
The use of silver-gold-intensified nickel-diaminobenzidine (30) enabled the sensitive visualization of ER-␣ (Fig.
3, A–G). A heavy ER-␣ immunolabeling was present in the
arcuate and ventromedial nuclei (Fig. 3A) and in scattered
cell nuclei within the dorsomedial and periventricular nuclei where most RFRP-1 immunoreactive cells occurred
(Fig. 3, A and B). A pale nuclear ER-␣ signal was detected
in 18.7 ⫾ 3.8% of RFRP-1-immunoreactive cells (Fig. 3,
C–E and H), whereas the majority of RFRP neurons did
not contain ER-␣ signal (Fig. 3, C, F, and G). Using the
FIG. 2. Prepro-RFRP mRNA expression by individual RFRP neurons is
reduced in response to E2 treatment. Results of the single-cell image
analysis, shown in the diagram, reveal reduced single-cell levels of
prepro-RFRP mRNA expression (integrated density; mean gray value ⫻
area of silver grains above individual neurons) in OVX mice treated sc
with E2 for 4 d (OVX⫹E2 group; n ⫽ 4) vs. their oil-treated controls
(OVX group; n ⫽ 5). Integrated density values are expressed as
percentage of the OVX group. *, P ⬍ 0.05. Individual RFRP neurons
are indicated by arrows in representative emulsion autoradiographs.
In this study, we demonstrate that E2 down-regulates
prepro-RFRP mRNA expression of OVX mice. In addition, we show that nuclear ER-␤ is absent, whereas
ER-␣ occurs in a subset (18.7 ⫾ 3.8%) of RFRP neurons
in OVX mice.
Estrogenic down-regulation of prepro-RFRP mRNA
expression may be involved in estrogen feedback
mechanisms
We found that a 4-d E2 regimen down-regulates prepro-RFRP mRNA levels in OVX mice. This observation
suggests that in the presence of high E2, RFRPs may exert
a reduced inhibition on the reproductive axis via actions
on GnRH neurons, gonadotrophs, or both. We propose
that the withdrawal of the inhibitory RFRP tone from the
reproductive axis in proestrus when E2 levels are high may
play a physiological role in positive estrogen feedback. In
contrast with the estrogenic regulation of prepro-RFRP
mRNA expression we report here in mice, a recent quantitative PCR study has found no difference between prepro-RFRP mRNA levels of OVX rats vs. rats killed at the
time of a LH surge induced by exogenous E2 and progesterone administration (35). It requires clarification to
what extent this discrepancy is due to differences between
species, animal treatments, or sensitivity of the applied
RNA quantification methods. In ewes, morphological evidence exists that the active reproductive status coincides
with a reduced RFRP inhibitory tone, as reflected by decreased numbers of RFRP-3-immunoreactive cell bodies
and fiber contacts on GnRH neurons during breeding season (13). Interestingly however, E2 treatment did not affect prepro-RFRP mRNA levels of OVX ewes in the same
study (13).
Evidence for ER-␣ in a subset of RFRP neurons
Our colocalization experiments were carried out with
dual-label immunohistochemistry in an attempt to identify which ER isoform may account for the estrogenic regulation of prepro-RFRP mRNA expression. These experiments found no evidence for ER-␤ in RFRP neurons using
the same antiserum and optimized detection method that
successfully detected ER-␤ in GnRH neurons of the rat in our
previous studies (24, 25) and ER-␤ in the paraventricular
nucleus of the mouse in the present study. On the other hand,
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FIG. 3. Use of dual-label immunohistochemistry provides evidence for the absence of ER-␤ and the presence of ER-␣ within a small subset
(18.7%) of RFRP-1-immunoreactive neurons in OVX mice. A, Use of the silver-gold-intensified Ni-diaminobenzidine chromogen reveals a high
density of darkly labeled ER-␣-immunoreactive cell nuclei (black signal) in the arcuate (ARC) and ventromedial (VMN) nuclei; B, heavily labeled cell
nuclei are also present in the dorsomedial nucleus (DMN) where the majority of RFRP-1-immunoreactive neuronal cell bodies (brown cytoplasmic
labeling) occur; C–G, as shown in the high-power images of framed areas in B, typical RFRP neurons are either devoid of the nuclear ER-␣ signal
(white arrows; C, F, and G) or exhibit weak ER-␣ labeling (black arrows; C, D, and E); H, very few RFRP neurons show medium, and none show
high labeling intensity. Arrowheads in C point to ER-␣-immunoreactive nuclei in non-RFRP neurons. I and J, The application of silver-goldintensified Ni-diaminobenzidine to detect ER-␤ immunoreactivity reveals a large number of labeled nuclei in the paraventricular nucleus (PVN; I) but
only a few labeled cells in the DMN (arrowhead in J). RFRP-1-immunoreative cells in the DMN (arrows) do not contain ER-␤ signal (arrowhead) in
the high-power inset. Scale bars, 50 ␮m (A, B, I, and J) and 10 ␮m (C–H and high-power inset in J).
we found low levels of ER-␣ signal in a relatively small subset
(18.7 ⫾ 3.8%) of RFRP neurons, raising the possibility that
E2 may act via ER-␣ to regulate prepro-RFRP mRNA expression directly. Alternatively, the estrogenic regulation of
RFRP neurons may also be indirect, considering the relatively low levels of ER-␣ in RFRP cells.
The RFRP-synthesizing neurons are found similarly in
the dorsomedial nucleus in Syrian hamsters, rats, and mice
(10). However, differences in reproductive physiology of
rodent species may also relate to the RFRP neuronal system. Although in our present study we found only low
levels of ER-␣ in 18.7 ⫾ 3.8% of RFRP neurons in mice,
40% of RFRP neurons express ER-␣ in the Syrian hamsters (10). In Syrian hamsters, melatonin-sensitive neurons
of the dorsomedial hypothalamic nucleus play a critical
role in the seasonal onset of reproductive quiescence in the
Endocrinology, April 2011, 152(4):1684 –1690
short-day photoperiod, which can be prevented by the
lesion of the dorsomedial nucleus (36, 37). Whether RFRP
neurons are specifically involved in the seasonal regulation of reproduction requires clarification.
Sites of action of RFRP on the reproductive axis
Although it is generally agreed that GnIH/RFRP neurons innervate GnRH cells in birds, rodents, sheep, and
primates (4, 10, 12, 13, 19, 20), an unresolved paradox
relates to the putative adenohypophyseal site of action of
RFRP peptides in various species. GnIH/RFRP-immunoreactive terminals do occur in the neurosecretory zone in
birds (3, 18), sheep (14), and monkeys (17) but not in
rodents (10, 12). Furthermore, RFRP-synthesizing neurons in rats do not accumulate Fluoro-Gold from the systemic circulation, indicating that they have no access to the
hypophyseal portal circulation to act on the adenohypophysis (12). Indeed, the RFRP antagonist RF9 is not
capable of eliciting LH secretion from static pituitary cultures (38). Conversely, in support of the direct adenohypophyseal effect of RFRP-3, a dose-dependent reduction
in GnRH-stimulated LH secretion has been reported in
adenohypophyseal cultures of various species, including
rodents (14, 39, 40).
In summary, in this study, we demonstrate that E2
down-regulates prepro-RFRP mRNA expression of OVX
mice, which may play a role in estrogen feedback to the
reproductive axis. The observation of the weak ER-␣ signal in a small subset (18.7%) of RFRP neurons may indicate that E2 directly regulates prepro-RFRP gene expression, although the possibility that the action is indirect
also needs to be considered.
Acknowledgments
We thank Takeda Pharmaceutical Co. Ltd. for kindly providing
the RFRP-1 antibody for this study and Hajni Bekó for expert
technical assistance.
Address all correspondence and requests for reprints to: Erik
Hrabovszky, M.D., Ph.D., Laboratory of Endocrine Neurobiology,
Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, 1083 Hungary. E-mail: hrabovszky@koki.hu.
This study was supported by the National Science Foundation
of Hungary (OTKA K69127, K83710, and T73002) and the
Hungarian Health Research Council Fund (ETT 122/2009). The
research leading to these results has received funding from the
European Community’s Seventh Framework Program (FP7/
2007–2013) under Grant Agreement 245009.
Disclosure Summary: All of the authors have nothing to
declare.
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References
1. Matsuo H, Baba Y, Nair RM, Arimura A, Schally AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:
1334 –1339
2. Burgus R, Butcher M, Amoss M, Ling N, Monahan M, Rivier J,
Fellows R, Blackwell R, Vale W, Guillemin R 1972 Primary structure of the ovine hypothalamic luteinizing hormone-releasing factor
(LRF) (LH-hypothalamus-LRF-gas chromatography-mass spectrometry-decapeptide-Edman degradation). Proc Natl Acad Sci USA
69:278 –282
3. Tsutsui K, Saigoh E, Ukena K, Teranishi H, Fujisawa Y, Kikuchi
M, Ishii S, Sharp PJ 2000 A novel avian hypothalamic peptide
inhibiting gonadotropin release. Biochem Biophys Res Commun
275:661– 667
4. Bentley GE, Perfito N, Ukena K, Tsutsui K, Wingfield JC 2003
Gonadotropin-inhibitory peptide in song sparrows (Melospiza
melodia) in different reproductive conditions, and in house sparrows
(Passer domesticus) relative to chicken-gonadotropin-releasing hormone. J Neuroendocrinol 15:794 – 802
5. Ubuka T, Kim S, Huang YC, Reid J, Jiang J, Osugi T, Chowdhury
VS, Tsutsui K, Bentley GE 2008 Gonadotropin-inhibitory hormone
neurons interact directly with gonadotropin-releasing hormone-I
and -II neurons in European starling brain. Endocrinology 149:268 –
278
6. Hinuma S, Shintani Y, Fukusumi S, Iijima N, Matsumoto Y, Hosoya
M, Fujii R, Watanabe T, Kikuchi K, Terao Y, Yano T, Yamamoto
T, Kawamata Y, Habata Y, Asada M, Kitada C, Kurokawa T, Onda
H, Nishimura O, Tanaka M, Ibata Y, Fujino M 2000 New neuropeptides containing carboxy-terminal RFamide and their receptor in
mammals. Nat Cell Biol 2:703–708
7. Fukusumi S, Habata Y, Yoshida H, Iijima N, Kawamata Y, Hosoya
M, Fujii R, Hinuma S, Kitada C, Shintani Y, Suenaga M, Onda H,
Nishimura O, Tanaka M, Ibata Y, Fujino M 2001 Characteristics
and distribution of endogenous RFamide-related peptide-1.
Biochim Biophys Acta 1540:221–232
8. Yano T, Iijima N, Kakihara K, Hinuma S, Tanaka M, Ibata Y 2003
Localization and neuronal response of RFamide related peptides in
the rat central nervous system. Brain Res 982:156 –167
9. Yano T, Iijima N, Hinuma S, Tanaka M, Ibata Y 2004 Developmental expression of RFamide-related peptides in the rat central
nervous system. Brain Res Dev Brain Res 152:109 –120
10. Kriegsfeld LJ, Mei DF, Bentley GE, Ubuka T, Mason AO, Inoue K,
Ukena K, Tsutsui K, Silver R 2006 Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals.
Proc Natl Acad Sci USA 103:2410 –2415
11. Bentley GE, Kriegsfeld LJ, Osugi T, Ukena K, O’Brien S, Perfito N,
Moore IT, Tsutsui K, Wingfield JC 2006 Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory
hormone (GnIH) in birds and mammals. J Exp Zool A Comp Exp
Biol 305:807– 814
12. Rizwan MZ, Porteous R, Herbison AE, Anderson GM 2009 Cells
expressing RFamide-related peptide-1/3, the mammalian gonadotropin-inhibitory hormone orthologs, are not hypophysiotropic
neuroendocrine neurons in the rat. Endocrinology 150:1413–1420
13. Smith JT, Coolen LM, Kriegsfeld LJ, Sari IP, Jaafarzadehshirazi
MR, Maltby M, Bateman K, Goodman RL, Tilbrook AJ, Ubuka T,
Bentley GE, Clarke IJ, Lehman MN 2008 Variation in kisspeptin
and RFamide-related peptide (RFRP) expression and terminal connections to gonadotropin-releasing hormone neurons in the brain: a
novel medium for seasonal breeding in the sheep. Endocrinology
149:5770 –5782
14. Clarke IJ, Sari IP, Qi Y, Smith JT, Parkington HC, Ubuka T, Iqbal
J, Li Q, Tilbrook A, Morgan K, Pawson AJ, Tsutsui K, Millar RP,
Bentley GE 2008 Potent action of RFamide-related peptide-3 on
pituitary gonadotropes indicative of a hypophysiotropic role in the
1690
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
Molnár et al.
E2 Regulation of RFRP Neurons
negative regulation of gonadotropin secretion. Endocrinology 149:
5811–5821
Dardente H, Birnie M, Lincoln GA, Hazlerigg DG 2008 RFamiderelated peptide and its cognate receptor in the sheep: cDNA cloning,
mRNA distribution in the hypothalamus and the effect of photoperiod. J Neuroendocrinol 20:1252–1259
Legagneux K, Bernard-Franchi G, Poncet F, La Roche A, Colard C,
Fellmann D, Pralong F, Risold PY 2009 Distribution and genesis of
the RFRP-producing neurons in the rat brain: comparison with melanin-concentrating hormone- and hypocretin-containing neurons.
Neuropeptides 43:13–19
Ubuka T, Lai H, Kitani M, Suzuuchi A, Pham V, Cadigan PA, Wang
A, Chowdhury VS, Tsutsui K, Bentley GE 2009 Gonadotropininhibitory hormone identification, cDNA cloning, and distribution
in rhesus macaque brain. J Comp Neurol 517:841– 855
Ukena K, Tsutsui K 2001 Distribution of novel RFamide-related
peptide-like immunoreactivity in the mouse central nervous system.
Neurosci Lett 300:153–156
Wu M, Dumalska I, Morozova E, van den Pol AN, Alreja M 2009
Gonadotropin inhibitory hormone inhibits basal forebrain vGluT2gonadotropin-releasing hormone neurons via a direct postsynaptic
mechanism. J Physiol 587:1401–1411
Johnson MA, Tsutsui K, Fraley GS 2007 Rat RFamide-related peptide-3 stimulates GH secretion, inhibits LH secretion, and has variable effects on sex behavior in the adult male rat. Horm Behav
51:171–180
Ducret E, Anderson GM, Herbison AE 2009 RFamide-related peptide-3, a mammalian gonadotropin-inhibitory hormone ortholog,
regulates gonadotropin-releasing hormone neuron firing in the
mouse. Endocrinology 150:2799 –2804
Herbison AE, Pape JR 2001 New evidence for estrogen receptors in
gonadotropin-releasing hormone neurons. Front Neuroendocrinol
22:292–308
Hrabovszky E, Shughrue PJ, Merchenthaler I, Hajszán T, Carpenter
CD, Liposits Z, Petersen SL 2000 Detection of estrogen receptorbeta messenger ribonucleic acid and 125I-estrogen binding sites in
luteinizing hormone-releasing hormone neurons of the rat brain.
Endocrinology 141:3506 –3509
Hrabovszky E, Steinhauser A, Barabás K, Shughrue PJ, Petersen SL,
Merchenthaler I, Liposits Z 2001 Estrogen receptor-␤ immunoreactivity in luteinizing hormone-releasing hormone neurons of the rat
brain. Endocrinology 142:3261–3264
Kalló I, Butler JA, Barkovics-Kalló M, Goubillon ML, Coen CW
2001 Oestrogen receptor ␤-immunoreactivity in gonadotropin releasing hormone-expressing neurones: regulation by oestrogen.
J Neuroendocrinol 13:741–748
Hrabovszky E, Kalló I, Szlávik N, Keller E, Merchenthaler I, Liposits
Z 2007 Gonadotropin-releasing hormone neurons express estrogen
receptor-␤. J Clin Endocrinol Metab 92:2827–2830
Wintermantel TM, Campbell RE, Porteous R, Bock D, Gröne HJ,
Todman MG, Korach KS, Greiner E, Pérez CA, Schütz G, Herbison
AE 2006 Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons
and fertility. Neuron 52:271–280
Hrabovszky E, Kalló I, Steinhauser A, Merchenthaler I, Coen
Endocrinology, April 2011, 152(4):1684 –1690
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
CW, Petersen SL, Liposits Z 2004 Estrogen receptor-␤ in oxytocin and vasopressin neurons of the rat and human hypothalamus:
immunocytochemical and in situ hybridization studies. J Comp
Neurol 473:315–333
Hrabovszky E, Petersen SL 2002 Increased concentrations of radioisotopically-labeled complementary ribonucleic acid probe, dextran
sulfate, and dithiothreitol in the hybridization buffer can improve
results of in situ hybridization histochemistry. J Histochem Cytochem 50:1389 –1400
Liposits Z, Setalo G, Flerko B 1984 Application of the silver-gold
intensified 3,3⬘-diaminobenzidine chromogen to the light and electron microscopic detection of the luteinizing hormone-releasing hormone system of the rat brain. Neuroscience 13:513–525
Sárvári M, Hrabovszky E, Kalló I, Galamb O, Solymosi N, Likó I,
Molnár B, Tihanyi K, Szombathelyi Z, Liposits Z 2010 Gene expression profiling identifies key estradiol targets in the frontal cortex
of the rat. Endocrinology 151:1161–1176
Moffatt CA, Rissman EF, Shupnik MA, Blaustein JD 1998 Induction of progestin receptors by estradiol in the forebrain of estrogen
receptor-␣ gene-disrupted mice. J Neurosci 18:9556 –9563
Shughrue PJ, Askew GR, Dellovade TL, Merchenthaler I 2002 Estrogen-binding sites and their functional capacity in estrogen receptor double knockout mouse brain. Endocrinology 143:1643–1650
Nystedt JM, Brandt A, Vilim FS, Ziff EB, Panula P 2006 Identification of transcriptional regulators of neuropeptide FF gene expression. Peptides 27:1020 –1035
Quennell JH, Rizwan MZ, Relf HL, Anderson GM 2010 Developmental and steroidogenic effects on the gene expression of RFamide
related peptide and its receptor in the rat brain and pituitary gland.
J Neuroendocrinol 22:309 –316
Lewis D, Freeman DA, Dark J, Wynne-Edwards KE, Zucker I 2002
Photoperiodic control of oestrous cycles in Syrian hamsters: mediation by the mediobasal hypothalamus. J Neuroendocrinol 14:294 –
299
Maywood ES, Bittman EL, Hastings MH 1996 Lesions of the melatonin- and androgen-responsive tissue of the dorsomedial nucleus
of the hypothalamus block the gonadal response of male Syrian
hamsters to programmed infusions of melatonin. Biol Reprod 54:
470 – 477
Pineda R, Garcia-Galiano D, Sanchez-Garrido MA, Romero M,
Ruiz-Pino F, Aguilar E, Dijcks FA, Blomenröhr M, Pinilla L, van
Noort PI, Tena-Sempere M 2010 Characterization of the potent
gonadotropin-releasing activity of RF9, a selective antagonist of
RF-amide-related peptides and neuropeptide FF receptors: physiological and pharmacological implications. Endocrinology 151:
1902–1913
Murakami M, Matsuzaki T, Iwasa T, Yasui T, Irahara M, Osugi T,
Tsutsui K 2008 Hypophysiotropic role of RFamide-related peptide-3 in the inhibition of LH secretion in female rats. J Endocrinol
199:105–112
Kadokawa H, Shibata M, Tanaka Y, Kojima T, Matsumoto K,
Oshima K, Yamamoto N 2009 Bovine C-terminal octapeptide of
RFamide-related peptide-3 suppresses luteinizing hormone (LH) secretion from the pituitary as well as pulsatile LH secretion in bovines. Domest Anim Endocrinol 36:219 –224