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and ß Messenger Ribonucleic Acids in Adult Gonadotropin-Releasing Hormone Neurons1
Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom
Address all correspondence and requests for reprints to: Allan E. Herbison Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge CB2 4AT, United Kingdom.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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messenger RNA. Approximately 10% of GnRH neurons
contained ERß transcripts that were always coexistent with ER.
Single cell RT-PCR analysis of nonGnRH cells located in the medial
preoptic area revealed a similar coexpression pattern of ER and
ERß transcripts. In contrast, single striatal cells were not found to
contain ERß despite ER being present in approximately 25% of
cells. The analysis of single GnRH neurons in cycling female mice
revealed that the detection of ER and ERß transcripts was lowest
on proestrus (ER, 18% of all GnRH neurons; ERß, 0%) compared
with diestrus (44% and 6%) and estrus (75% and 19%, respectively).
Using a novel approach that enables single cell RT-PCR analysis of GnRH
neurons, we present here evidence for the cyclic expression of ER
and ERß messenger RNAs within prepubertal and adult female GnRH
neurons. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite its importance, the precise nature of estrogen’s influence on the GnRH neuron remains unresolved (2). Immunocytochemical studies have consistently failed to detect estrogen receptor (ER) protein in the GnRH neurons of many species (see Ref. 2 for review) and equally, little evidence exists for the accumulation of radioactive estradiol by these cells in vivo (3). On the other hand, there are reports of low levels of ER messenger RNA (mRNA) and estradiol binding in two different immortalized GnRH cell lines (4, 5, 6, 7) and the GnRH promotor has been shown to contain functional estrogen response elements (4, 8). Furthermore, a small population of GnRH neurons in the guinea pig have been reported to express immunoreactivity to the progesterone receptor (9), the other major gonadal steroid receptor in the female. Thus, whether or not native GnRH neurons express ERs in vivo has remained somewhat controversial. This issue is of substantial importance within the field of GnRH neurobiology as the absence of ERs in these neurons invokes the need for indirect mechanisms of estrogen action (2).
We discovered recently that individual GnRH neurons could be identified
on a morphological basis in an acute hypothalamic slice preparation.
This ability, combined with the application of single cell RT-PCR
techniques, has enabled us to examine for the presence of specific
mRNAs in individual GnRH neurons. Here we report these methods and
show, for the first time, the presence of both ER and ERß
transcripts in native GnRH neurons isolated from prepubertal and
cycling female mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Slices were placed in a recording chamber and then mounted onto the
stage of an upright microscope (Axioskop FS, Carl Zeiss, Oberkochen, Germany) fitted with differential
interference contrast optics. Slices were held submerged with a nylon
mesh and continuously superfused with oxygenated standard Krebs (10).
Three different types of neurons were sampled from the same brain; 1)
putative GnRH neurons exhibiting a bipolar-type morphology with a
vertical orientation (Fig. 1A
) were
identified within the medial septum (MS) and ventral diagonal band of
Broca/rostral preoptic area (rPOA); 2) medial preoptic area (mPOA)
neurons of variable morphology were taken from the medial preoptic
nucleus at the anteroposterior level of bregma (11); and 3) striatal
neurons, also of multiple morphology, were taken from the
caudate-putamen at the level of bregma. The mPOA neurons were selected
for analysis based on the likelihood that many would express both ER
transcripts while, conversely, the striatum is not reported to express
either ER in the rat (12).
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when filled with 8 µl of
autoclaved internal solution containing: 140 mM KCl, 10
mM EGTA, 1 mM MgCl2, 1
mM CaCl2, 10 mM HEPES (pH titrated
to 7.3 with KOH). Identified neurons were located with a x40 immersion
objective (Carl Zeiss) and approached with a patch pipette
under positive pressure. Following contact with the cell somata, steady
negative pressure was applied to the electrode to form a high
resistance seal and then the cytoplasm, but not the nucleus, of the
neuron was harvested under visual control. In some experiments, the
neuronal nature of the cell to be harvested was first ascertained by
observing their firing characteristics under current clamp conditions.
All solutions used for cell content harvesting were prepared from
autoclaved water and all glassware heated for 250 C for 4 h
(13).
Single cell multiplex RT-PCR for GnRH, ER, and ERß
transcripts
Immediately following harvesting, the contents of the individual
cell were expelled from the patch electrode into 8.5 µl of RT mixture
containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl,
3 mM MgCl2, 20 mM DTT, 0.5
mM dNTPs, 100 ng random hexamer primers, 200 ng oligo
(dT)12–15, and 20 U RNase inhibitor (RNAsin; Promega Corp., Madison, WI). Superscript II reverse transcriptase
(1 µl containing 200U Moloney murine leukemia virus; Life Technologies, Inc., Gaithersburg, MD) was added and
complementary DNA (cDNA) synthesis performed sequentially at room
temperature (5 min) and then at 42 C (1 h). Following cDNA synthesis,
reactions were snap frozen on dry ice and stored at -70 C for up to 1
week before undergoing PCR.
Multiplex PCR was undertaken on a basis similar to that described
previously (14, 15). First round PCR was performed on 5 µl of RT
product from each cell using pooled oligonucleotide pairs in a 100 µl
reaction containing 50 mM KCl, 10 mM Tris-HCl
(pH 9.0), 1.5 mM MgCl2, 0.2 mM
dNTPs, 10 pmol of each of the following oligonucleotides (GnRH F1 and
R1, ER F1 and R1, ERß F1 and R1; see Table 1), and Taq DNA Polymerase
(2.5 U; Amersham Pharmacia Biotech, Piscataway,
NJ). In addition, 1 ng aliquots of hypothalamic cDNA prepared from
total RNA extracted from C57/BL6xCBA mice were run as positive controls
and water blanks as a negative control in each PCR. Thirty-six cycles
of first round amplification were performed using a "Robocycler"
(Stratagene, Torrey Pines, CA) in thin walled 0.2
ml PCR tubes according to the following protocol: first cycle of 95 C
(3 min), 60 C (2 min), and 72 C (3 min) followed by 35 cycles of 95 C
(40 sec), 60 C (1 min) and 72 C (1 min). A final 5 min incubation at 72
C was used to "polish" the DNA termini. Second round nested PCR was
performed using 1.25 µl aliquots of the first round amplicon pool.
PCR for GnRH, ER, and ERß was then performed in separate 25 µl
reactions containing 50 mM KCl, 10 mM Tris-HCl
(pH 9.0), 1.5 mM MgCl2, 0.2 mM
dNTPs, nested oligonucleotide pairs (GnRH F2 and R2, or ER F2 and
R2, or ERß F2 and R2; 1 µM each), and 0.6 Units
Taq DNA Polymerase (Amersham Pharmacia Biotech). For some cells, glial fibrillary acidic protein (GFAP)
primers (Table 1) were also included in the PCR protocol. A further 36
cycles of amplification were undertaken using the same protocol
described for the first round PCR. Resulting amplicons (GnRH 213bp,
ER 423bp, ERß 333bp) were resolved on ethidium bromide-stained
1.5% TBE-agarose gels and photographed using a gel documentation
system (Bio-Rad Laboratories, Inc.). The identity of PCR
amplicons was confirmed by cloning, using a T/A overhang PCR cloning
strategy (pT-Easy; Promega Corp.), followed by fluorescent
dideoxy-Sanger sequencing using an ABI fluorescent sequencer at The
Babraham Institute microchemical facility.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
were identified over individual cells that were scattered throughout
the medial septum passing down to form an inverted "Y" above the
anterior recess of the third ventricle in the diagonal band of Broca
and rostral preoptic area (Fig. 1A). Hybridized cells were also
detected in the lateral preoptic area and anterior hypothalamus above
the optic chiasm and supraoptic nucleus but not elsewhere in the
brain.
Morphologically identified GnRH neurons
In total, 65 GnRH neurons harvested from 6 prepubertal female mice
and 22 adult female mice (7 diestrus, 7 proestrus, 8 estrus) were
examined for the presence of ER transcripts. The identification of GnRH
neurons on the basis of their location, orientation and bipolar
morphology (Fig. 1, B–D) was most successful in prepubertal mice,
where approximately 90% of all harvested putative GnRH neurons were
shown by RT-PCR to contain GnRH transcripts. In comparison, 68% of all
similarly identified cells within the MS and rPOA of adult female were
found to contain GnRH transcripts (range of 30–100% on an individual
animal basis). The amplicon size following GnRH PCR was approximately
213 bp (Fig. 1E), as predicted by the GnRH primers used, and sequencing
of the amplicon showed it to be identical to murine GnRH (16). Cells
harvested from brain regions outside of the known distribution of GnRH
neurons, such as the striatum, were never found to contain GnRH
amplicons (Fig. 1E).
In prepubertal mice, 11 of 18 (61%) neurons containing GnRH mRNA were
also found to contain ER transcripts and 3 (17%) of these also
expressed ERß transcripts (Fig. 2). The
presence of ER transcripts was independent of the location of GnRH
neurons as 5 of 7 MS and 6 of 11 rPOA GnRH neurons contained ER
transcripts, whereas 1 of 7 MS and 2 of 11 rPOA GnRH neurons expressed
ERß mRNA. In adult female mice, a total of 47 GnRH mRNA-containing
neurons were examined and 21 (45%) found to contain ER transcripts
(Figs. 1E and 2). ERß transcripts were identified in 5 (11%) of the
adult GnRH neurons (Fig. 1E) and, as with the prepubertal females, were
always found to coexist with ER transcripts. The analysis of adult
GnRH neurons on a topographical basis also revealed no differences in
the presence of ER transcripts between cells located in the MS (42%
with ER; 14% with ERß) compared with those in the rPOA (45% with
ER; 10% with ERß). The identity of ER and ERß amplicons
derived from GnRH neurons was confirmed by sequencing which showed them
to be identical to the murine ER (18) and ERß (19).
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). The percentage of GnRH neurons
expressing ER transcripts altered from 44% (7/16) in diestrus to
18% (2/11) in proestrus and 75% (12/16) in estrus (Fig. 3A). Although
less frequently detected, a similar trend was observed for ERß
transcripts that were found in 6%, 0% and 19% of GnRH neurons over
the same days of the cycle, respectively (Fig. 3A). To provide a
statistical analysis of ER transcript presence in GnRH neurons, the
percentage of GnRH neurons positive for ER was determined for each
animal (diestrus, 65 ± 17%; proestrus 19 ± 14%; estrus
67 ± 16%; n = 7 each group) and values subjected to
nonparametric ANOVA (P = 0.03) with posthoc Dunn’s
multiple comparison test that showed estrus animals to have
significantly higher numbers of ER-positive GnRH neurons compared
with proestrus (P < 0.05). No differences were
detected across the cycle for the relatively few GnRH neurons with
ERß transcripts.
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transcripts. Four cells (12%) were
also positive for ERß (Figs. 1E and 2) with three expressing both
ER and ERß, while the other contained only ERß transcripts.
Similar to the profile observed for GnRH neurons, the detectability of
ER transcripts in mPOA cells was dependent upon the estrous cycle stage
of the mouse with 7/13 (54%) diestrus, 2/9 (22%) proestrus, and 3/12
(25%) estrus cells found to contain ER transcripts (Fig. 3B).
Equivalent values for ERß were 12%, 0% and 17% (Fig. 3B). None of
these morphologically diverse mPOA cells were found to contain GnRH
amplicons.
We also found a number of striatal cells that were positive for ER
in adult female mice. Of 44 cells harvested, 12 (27%) were found to
contain ER transcripts. In contrast, we found no evidence for ERß
expression in any of the 44 striatal cells (Fig. 2). As noted for the
GnRH and mPOA neurons, cells from the striatum expressed ER
transcripts at different levels through the estrous cycle; diestrus
5/15 (33%), proestrus 2/13 (15%), and estrus 5/16 (31%; Fig. 3C). A
retrospective analysis of ER-positive mPOA and striatal cells revealed
no common morphology or distinguishing characteristics compared with
nonER expressing cells.
Controls
Where sequence data made it possible, all of the primers were
designed to cross intron-exon boundaries and then checked against
genomic DNA to ensure that the amplicons derived from single cell
harvests originated from RNA. The identity of GnRH, ER, and ERß
amplicons was confirmed by sequencing. In addition, a number of
procedural and other controls were always undertaken: 1) internal
pipette buffer expelled from the patch electrodes after they had been
introduced into the slice but no cell harvested (mock harvests),
underwent RT-PCR in parallel with cell contents but were consistently
PCR amplicon negative; 2) cells from the three areas under
investigation and control harvests were subjected to PCR by an
investigator blind to their identity; 3) five neurons subsequently
shown to be GnRH and ER-positive, underwent a brief period of
electrical recording before harvesting and displayed neuronal firing
characteristics (data not shown); and 4) to assess potential glial cell
"contamination," the cell contents from 12 PCR-positive GnRH
neurons (four of which were also found to be ER-positive) were
subjected to PCR for GFAP and, despite a positive band in the
hypothalamic cDNA control, were all shown to be negative.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The GnRH neurons of most mammalian species exhibit a bipolar, fusiform-type morphology with a distinct vertical orientation (20). Using 150 µm thick coronal brain slices from adult and prepubertal mice, we show here by single cell RT-PCR that the many of the MS and rPOA cells displaying these morphological characteristics contain GnRH mRNA and are, therefore, GnRH neurons. The specificity of this procedure is indicated by the correct sequencing of the GnRH amplicon, the complete absence of GnRH amplicons in cells harvested from brain regions outside the "inverted Y" GnRH distribution, and our unpublished observation that GnRH transcripts can also be PCRed from fluorescent GnRH neurons targeted through GnRH-promotor-driven transgenics. However, it is important to recognize that false negatives may occur from failure of the harvesting and/or RT-PCR procedure. Similarly, we are aware that GnRH neurons that do not display a clear bipolar morphology will not be sampled. Despite these caveats, the present procedure provides the first approach through which individual, unmodified living GnRH neurons can be investigated in a routine manner within the brain.
We provide evidence that the majority of sampled GnRH neurons contain
ER mRNA, most prominently ER, and that the frequency with which we
detect these ER transcripts by single cell RT-PCR varies significantly
across the estrous cycle. In collecting these data we have been careful
to include a series of systematic controls. These include the use of
"mock harvests," negative and positive PCR controls and the
sequencing of amplicons from GnRH neurons. Combined with the
region-specific profiles of ER transcripts and their temporal variation
across the estrous cycle, these control measures demonstrate that the
amplicons are authentic and that they do not arise from contamination
or other external factors originating from the slice or harvesting
apparatus. With respect to the GnRH neurons, we were concerned that the
ER mRNAs may have been derived from glial cells ensheathing GnRH
neurons (20). This was shown not to be the case as electrical
recordings from GnRH- and ER-positive cells demonstrated typical
neuronal firing characteristics, whereas others were shown by PCR not
to express the glial cell marker GFAP. Thus, we are confident that the
present results provide well controlled evidence for the presence of
both ER and ERß mRNAs in native GnRH neurons.
Quite strikingly, we observed that our ability to detect both ER and
ERß in adult GnRH neurons was dependent on the stage of the estrous
cycle; fewer GnRH neurons were found to express ER transcripts in
proestrous mice (18%) compared with diestrus (44%) and estrus (75%).
This fluctuating pattern of ER mRNA expression is very similar to that
reported previously in the rat preoptic area using quantitative
in situ hybridization techniques and is thought to depend
upon circulating gonadal steroid concentrations (24, 25). Although we
have not examined this hypothesis directly in our mice, the relatively
high frequency of detection of ER mRNAs in prepubertal GnRH
neurons, when ovarian steroid levels are low, might suggest a similar
steroid-dependent expression or stability of ER mRNAs in murine GnRH
neurons.
We found that approximately 50% of mPOA cells contained ER
transcripts following single cell RT-PCR, whereas only approximately
10% expressed ERß. A previous immunocytochemical experiment had
shown the presence of ER-expressing neurons in the mouse preoptic
area (26) and recent work has shown that ERß mRNA is also expressed
in this area (27). The present study provides the first data on the
coexpression of ER and ERß within neurons of the mouse brain and,
very similar to a recent report in the rat (28), we estimate that
around 25% of the mPOA cells containing ER transcripts also express
ERß mRNA. Interestingly, ERß transcripts were rarely found in
isolation and almost always coexisted with ER. We also note that the
detection of both ER transcripts in mPOA cells fluctuated across the
estrous cycle. Although not the primary objective of this study, these
observations do raise interesting issues regarding potential
similarities in the regulation of ER and ERß mRNAs within single
mPOA cells, and the possibility that ER/ERß heterodimers (29) may
be involved in regulating estrogen-responsive genes within these
neurons.
In contrast to GnRH and mPOA cells, we found no evidence for the
expression of ERß mRNA within cells taken from the striatum of female
mice. This is in good agreement with in situ hybridization
data in the rat (12). However, we did observe that approximately one
quarter of striatal cells contained ER transcripts and that the
number of positive cells detected depended upon the stage of the
estrous cycle. Immunocytochemistry (26) has not revealed ER in the
striatum of the mouse and we can find no reports describing ER mRNA
distribution within the murine brain. Interestingly, ER mRNA has
been detected in the striatum of neonatal rats (30). Thus, the level of
ER protein translated from ER transcripts in some striatal cells may
be insufficient to enable detection with immunocytochemistry in this
species. Nevertheless, it remains interesting that ER transcripts
are present in a few cells of the murine striatum as work in the rat
has provided good evidence for direct effects of estrogen in this area
(see Ref. 31).
Although we demonstrate the presence of ER mRNAs in native GnRH
neurons, it remains somewhat paradoxical that very little evidence
exists for ER protein in GnRH neurons in vivo (see
Introduction). On one hand, it may be argued that GnRH
neurons synthesize relatively low levels of ER protein that are below
the detection threshold using immunocytochemistry and receptor
autoradiography. On the other hand, it is possible that low levels of
ER transcription occur in GnRH neurons but that this is not translated.
As such, it may be entirely redundant or, as shown for other mRNAs
(32), have a functional role without being translated. In this
scenario, it is interesting to note the parallel situation in bone
where, despite good evidence for direct actions of estrogen upon bone
cells, it has been notoriously difficult to provide in vivo
evidence for ER expression in mammalian osteocytes (33). While early
immunocytochemical and binding studies failed to detect ER protein in
bone cells, the presence of ER mRNA was eventually shown by molecular
approaches including RT-PCR (34, 35, 36), and it is now thought that
osteoblasts and osteoclasts express functionally relevant ERs, but do
so at barely detectable levels (33). It is also interesting to note
that, similar to the GnRH phenotype (4, 5, 6, 7), the first evidence for ERs
within osteocytes was obtained from cultured cells in vitro
(33). Thus, the present evidence derived from PCR raises the
possibility that GnRH neurons may express low levels of ER protein. A
recent study in the acrolein-fixed, colchicine-treated rat has reported
that 17% of GnRH neurons are immunoreactive using an ER antisera
(37). We share the reservations of the authors and others (38)
regarding those data and believe that further experimentation is
clearly required to address the issue of functional ER protein in the
GnRH phenotype.
In conclusion, we report here a procedure that enables living GnRH
neurons to be identified in the acute brain slice preparation thus
overcoming one of the major technical obstacles to our understanding of
the cellular characteristics of the GnRH phenotype. Using this
procedure, we provide evidence of ER and ERß mRNA expression by
adult and prepubertal GnRH neurons using single cell RT-PCR. Similar to
mPOA cells, the presence of ER transcripts was predominant, with
ERß detected almost exclusively in coexistence with ER. The
detection of ER transcripts in GnRH neurons was dependent upon the
estrous cycle with the lowest numbers of ER-expressing GnRH neurons
found on proestrus and greatest on estrus. Together, these observations
provide the first demonstration of ER mRNAs within native GnRH neurons
and raise interesting questions regarding their function within this
important neuroendocrine phenotype.
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Acknowledgments |
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Footnotes |
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Received May 13, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and -ß mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
knockout
mouse. Endocrinology 138:5649–5652 immunoreactivity in neurons of the rat forebrain.
Endocrinology 139:5267–5270. Mol Endocrinol 11:1486–1496-immunoreactivity in gonadotrophin-releasing
hormone-expressing neurones. J Neuroendocrinol 11:331–335[CrossRef][Medline]
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K. Wada, L. Hu, N. Mores, C. E. Navarro, H. Fuda, L. Z. Krsmanovic, and K. J. Catt Serotonin (5-HT) Receptor Subtypes Mediate Specific Modes of 5-HT-Induced Signaling and Regulation of Neurosecretion in Gonadotropin-Releasing Hormone Neurons Mol. Endocrinol., January 1, 2006; 20(1): 125 - 135. [Abstract] [Full Text] [PDF]
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C. Bodo, A. E. Kudwa, and E. F. Rissman Both Estrogen Receptor-{alpha} and -{beta} Are Required for Sexual Differentiation of the Anteroventral Periventricular Area in Mice Endocrinology, January 1, 2006; 147(1): 415 - 420. [Abstract] [Full Text] [PDF]
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 D. A. Cunha, E. M. Carneiro, M. d. C. Alves, A. G. Jorge, S. M. de Sousa, A. C. Boschero, M. J. A. Saad, L. A. Velloso, and E. M. Rocha Insulin secretion by rat lachrymal glands: effects of systemic and local variables Am J Physiol Endocrinol Metab, November 1, 2005; 289(5): E768 - E775. [Abstract] [Full Text] [PDF]
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C. A. Christian, J. L. Mobley, and S. M. Moenter Diurnal and estradiol-dependent changes in gonadotropin-releasing hormone neuron firing activity PNAS, October 25, 2005; 102(43): 15682 - 15687. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, A. Bae, R. S. Swerdloff, A. Iranmanesh, and C. Wang Experimentally Induced Androgen Depletion Accentuates Ethnicity-Related Contrasts in Luteinizing Hormone Secretion in Asian and Caucasian Men J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1632 - 1638. [Abstract] [Full Text] [PDF]
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 A. Cariboni, F. Pimpinelli, S. Colamarino, R. Zaninetti, M. Piccolella, C. Rumio, F. Piva, E. I. Rugarli, and R. Maggi The product of X-linked Kallmann's syndrome gene (KAL1) affects the migratory activity of gonadotropin-releasing hormone (GnRH)-producing neurons Hum. Mol. Genet., November 15, 2004; 13(22): 2781 - 2791. [Abstract] [Full Text] [PDF]
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J. S. Jorgensen, C. C. Quirk, and J. H. Nilson Multiple and Overlapping Combinatorial Codes Orchestrate Hormonal Responsiveness and Dictate Cell-Specific Expression of the Genes Encoding Luteinizing Hormone Endocr. Rev., August 1, 2004; 25(4): 521 - 542. [Abstract] [Full Text] [PDF]
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J. L. Temple, E. Laing, A. Sunder, and S. Wray Direct Action of Estradiol on Gonadotropin-Releasing Hormone-1 Neuronal Activity via a Transcription-Dependent Mechanism J. Neurosci., July 14, 2004; 24(28): 6326 - 6333. [Abstract] [Full Text] [PDF]
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S. L. Petersen, E. N. Ottem, and C. D. Carpenter Direct and Indirect Regulation of Gonadotropin-Releasing Hormone Neurons by Estradiol Biol Reprod, December 1, 2003; 69(6): 1771 - 1778. [Abstract] [Full Text] [PDF]
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I. M. Abraham, S.-K. Han, M. G. Todman, K. S. Korach, and A. E. Herbison Estrogen Receptor {beta} Mediates Rapid Estrogen Actions on Gonadotropin-Releasing Hormone Neurons In Vivo J. Neurosci., July 2, 2003; 23(13): 5771 - 5777. [Abstract] [Full Text] [PDF]
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J. F. Couse, M. M. Yates, V. R. Walker, and K. S. Korach Characterization of the Hypothalamic-Pituitary-Gonadal Axis in Estrogen Receptor (ER) Null Mice Reveals Hypergonadism and Endocrine Sex Reversal in Females Lacking ER{alpha} But Not ER{beta} Mol. Endocrinol., June 1, 2003; 17(6): 1039 - 1053. [Abstract] [Full Text] [PDF]
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S. J. Krajewski, T. W. Abel, M. L. Voytko, and N. E. Rance Ovarian Steroids Differentially Modulate the Gene Expression of Gonadotropin-Releasing Hormone Neuronal Subtypes in the Ovariectomized Cynomolgus Monkey J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 655 - 662. [Abstract] [Full Text] [PDF]
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S. Khosravi and P. C. K. Leung Differential Regulation of Gonadotropin-Releasing Hormone (GnRH)I and GnRHII Messenger Ribonucleic Acid by Gonadal Steroids in Human Granulosa Luteal Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 663 - 672. [Abstract] [Full Text] [PDF]
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S. B. Pillai, J. M. Jones, and R. D. Koos Treatment of Rats with 17{beta}-Estradiol or Relaxin Rapidly Inhibits Uterine Estrogen Receptor {beta}1 and {beta}2 Messenger Ribonucleic Acid Levels Biol Reprod, December 1, 2002; 67(6): 1919 - 1926. [Abstract] [Full Text] [PDF]
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L. Dufourny and D. C. Skinner Progesterone Receptor, Estrogen Receptor {alpha}, and the Type II Glucocorticoid Receptor Are Coexpressed in the Same Neurons of the Ovine Preoptic Area and Arcuate Nucleus: A Triple Immunolabeling Study Biol Reprod, November 1, 2002; 67(5): 1605 - 1612. [Abstract] [Full Text] [PDF]
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K.-i. Matsuda, I. Ochiai, M. Nishi, and M. Kawata Colocalization and Ligand-Dependent Discrete Distribution of the Estrogen Receptor (ER){alpha} and ER{beta} Mol. Endocrinol., October 1, 2002; 16(10): 2215 - 2230. [Abstract] [Full Text] [PDF]
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N. Sharifi, A. E. Reuss, and S. Wray Prenatal LHRH Neurons in Nasal Explant Cultures Express Estrogen Receptor {beta} Transcript Endocrinology, July 1, 2002; 143(7): 2503 - 2507. [Abstract] [Full Text] [PDF]
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C. S. Nunemaker, R. A. DeFazio, and S. M. Moenter Estradiol-Sensitive Afferents Modulate Long-Term Episodic Firing Patterns of GnRH Neurons Endocrinology, June 1, 2002; 143(6): 2284 - 2292. [Abstract] [Full Text] [PDF]
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 M. Potier, M. Karl, F. Zheng, S. J. Elliot, G. E. Striker, and L. J. Striker Estrogen-Related Abnormalities in Glomerulosclerosis-Prone Mice : Reduced Mesangial Cell Estrogen Receptor Expression and Prosclerotic Response to Estrogens Am. J. Pathol., May 1, 2002; 160(5): 1877 - 1885. [Abstract] [Full Text] [PDF]
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S. Pompolo, C. J. Scott, and I. J. Clarke Selective Regulation of Glutamic Decarboxylase Isoform 65, But Not Isoform 67, in the Bed Nucleus of the Stria Terminalis and the Preoptic Area of the Ewe Brain Across the Estrous Cycle Endocrinology, February 1, 2002; 143(2): 544 - 550. [Abstract] [Full Text] [PDF]
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 E. J. Wagner, O. K. Ronnekleiv, and M. J. Kelly The Noradrenergic Inhibition of an Apamin-Sensitive, Small-Conductance Ca2+-Activated K+ Channel in Hypothalamic gamma -Aminobutyric Acid Neurons: Pharmacology, Estrogen Sensitivity, and Relevance to the Control of the Reproductive Axis J. Pharmacol. Exp. Ther., October 1, 2001; 299(1): 21 - 30. [Abstract] [Full Text] [PDF]
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T. R. Pak, G. R. Lynch, and P.-S. Tsai Testosterone and Estrogen Act via Different Pathways to Inhibit Puberty in the Male Siberian Hamster (Phodopus sungorus) Endocrinology, August 1, 2001; 142(8): 3309 - 3316. [Abstract] [Full Text] [PDF]
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C. McGarvey, P. S. Cates, A. N. Brooks, I. A. Swanson, S. R. Milligan, C. W. Coen, and K. T. O'Byrne Phytoestrogens and Gonadotropin-Releasing Hormone Pulse Generator Activity and Pituitary Luteinizing Hormone Release in the Rat Endocrinology, March 1, 2001; 142(3): 1202 - 1208. [Abstract] [Full Text] [PDF]
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D. C. Skinner, A. Caraty, and R. Allingham Unmasking the Progesterone Receptor in the Preoptic Area and Hypothalamus of the Ewe: No Colocalization with Gonadotropin-Releasing Neurons Endocrinology, February 1, 2001; 142(2): 573 - 579. [Abstract] [Full Text] [PDF]
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 M. POTIER, S. J. ELLIOT, I. TACK, O. LENZ, G. E. STRIKER, L. J. STRIKER, and M. KARL Expression and Regulation of Estrogen Receptors in Mesangial Cells: Influence on Matrix Metalloproteinase-9 J. Am. Soc. Nephrol., February 1, 2001; 12(2): 241 - 251. [Abstract] [Full Text]
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G. L. Jackson, S. G. Wood, and D. E. Kuehl A {gamma}-Aminobutyric AcidB Agonist Reverses the Negative Feedback Effect of Testosterone on Gonadotropin-Releasing Hormone and Luteinizing Hormone Secretion in the Male Sheep Endocrinology, November 1, 2000; 141(11): 3940 - 3945. [Abstract] [Full Text] [PDF]
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F. J. Hayes, S. B. Seminara, S. DeCruz, P. A. Boepple, and W. F. Crowley Jr. Aromatase Inhibition in the Human Male Reveals a Hypothalamic Site of Estrogen Feedback J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3027 - 3035. [Abstract] [Full Text]
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M. Xu, J. H. Urban, J. W. Hill, and J. E. Levine Regulation of Hypothalamic Neuropeptide Y Y1 Receptor Gene Expression during the Estrous Cycle: Role of Progesterone Receptors Endocrinology, September 1, 2000; 141(9): 3319 - 3327. [Abstract] [Full Text] [PDF]
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C. J. Scott, A. J. Tilbrook, D. M. Simmons, J. A. Rawson, S. Chu, P. J. Fuller, N. H. Ing, and I. J. Clarke The Distribution of Cells Containing Estrogen Receptor-{alpha} (ER{alpha}) and ER{beta} Messenger Ribonucleic Acid in the Preoptic Area and Hypothalamus of the Sheep: Comparison of Males and Females Endocrinology, August 1, 2000; 141(8): 2951 - 2962. [Abstract] [Full Text] [PDF]
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R. Maggi, F. Pimpinelli, L. Molteni, M. Milani, L. Martini, and F. Piva Immortalized Luteinizing Hormone-Releasing Hormone Neurons Show a Different Migratory Activity in Vitro Endocrinology, June 1, 2000; 141(6): 2105 - 2112. [Abstract] [Full Text] [PDF]
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P. E. Chappell, J. Lee, and J. E. Levine Stimulation of Gonadotropin-Releasing Hormone Surges by Estrogen. II. Role of Cyclic Adenosine 3',5'-Monophosphate Endocrinology, April 1, 2000; 141(4): 1486 - 1492. [Abstract] [Full Text] [PDF]
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