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Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Kevin J. Catt, M.D., Ph.D., Endocrinology and Reproduction Research Branch, Building 49, Room 6A-36, NICHD, NIH, Bethesda, Maryland 20892. E-mail: catt{at}helix.nih.gov
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Introduction |
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More recently, in vitro studies employing hypothalamic slices, cultured hypothalamic cells, and immortalized GnRH neurons (GT1 cells) have been applied to the analysis of GnRH secretion and its regulation (6, 7, 8, 9, 10, 11). Electrophysiological studies on GnRH-producing cell lines (12, 13, 14) and GnRH-containing embryonic neurons (15) have demonstrated the expression of a wide variety of voltage and ligand-gated channels. Tetrodotoxin-sensitive sodium current, as well as Ca2+ current with transient and sustained dihydropyridine-sensitive components, have been observed (13, 14). GnRH-producing neuronal cell lines also exhibit episodes of depolarizing electrical activity and fluctuations in [Ca2+]i (16, 17). The presence of connexin 26 proteins in GT1–7 cells (18) suggests that gap junction coupling between GnRH-producing neurons could serve to coordinate their pulsatile secretion. In rhesus monkeys, in vivo measurements of multiunit electrical activities (MUA) from the medialbasal hypothalamus have shown invariant synchrony between abrupt increases in frequency of MUA volleys and the initiation of LH pulses (19). Although it is not clear whether this electrical activity originates from GnRH cells or other neuronal elements, it is probable that GnRH neurons with intrinsic electrical activity participate in the formation of MUA.
The ability of GT1 cells to exhibit episodic GnRH release in the absence of other cell types indicates that intrinsic factors, such as autocrine regulation of neurosecretion, could be important determinants of pulsatile GnRH release. The finding that GT1–7 cells express GnRH receptors, agonist activation of which influences the pattern of pulsatile GnRH release by changing pulse frequency and amplitude (20), is consistent with this proposal. The present studies were performed to determine whether such autocrine regulation through endogenous GnRH receptors is also operative in normal GnRH neurons. For this purpose, cultured fetal hypothalamic cells were employed to analyze the expression of GnRH receptors, and the influence of GnRH agonist and antagonist analogs on the dynamics of GnRH release from native GnRH neurons.
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Materials and Methods |
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Perifusion procedure
Dispersed hypothalamic cells were incubated in 50-ml tubes
containing 1.5 x 107 cells, 0.3 ml preswollen
Cytodex-2 beads (Pharmacia, Piscataway, NJ), and 30 ml of
culture medium for 24 h in 5% CO2/air. The suspension
was then transferred into 60-mm dishes and culture was continued for
14–60 days, with replenishment of culture medium every second day.
Before perifusion, the cell-bead mixture was collected by sedimentation
and resuspended in Krebs-Ringer buffer continuing 1 mg/ml BSA, 1 mg/ml
glucose, 20 µM bacitracin, pH 7.4. After gassing for
1 h with 95% O2/5% CO2, the beads were
loaded into a temperature-controlled 0.5 ml chamber (Endotronics, Inc.; Minneapolis, MN). Perifusion was performed at a flow rate
of 10 ml/h at 37 C for at least 1 h to establish a stable baseline
before addition of agents made up in the same medium. Fractions were
collected at either 1- or 5-min intervals and stored at -20 C before
RIA using 125I-GnRH (Amersham, Arlington
Heights, IL), GnRH standards (Peninsula, Belmont, CA), and primary
antibody donated by Dr. V. D. Ramirez, University of Illinois
(Urbana, IL) (21). The intraassay and interassay coefficients of
variation at 80% binding in standard samples (15 pg/ml) were 12–14%,
respectively.
Static culture of hypothalamic cells and GT1–7 cells
Cells were cultured in 12-well plates [2 x
106 cells per well for hypothalamic cells and 1 x
106 cells per well for GT1–7 cells, provided by Dr.
R. I. Weiner, University of California San Francisco (San
Francisco, CA) (22)] in 2 ml of culture medium. GnRH agonist
(des-Gly10-[D-Ala6]GnRH
N-ethylamide; [D-Ala6]Ag, 50
pM) and antagonist ([D-pGlu1,
D-Phe2, D-Trp3,6]GnRH;
[D-pGlu]Antag, 50 pM) analogs were added to
the culture wells immediately after plating the cells. In some
experiments, an additional GnRH antagonist analog
([Ac-D-Nal (2)1,
D-Phe(pCl)2, D-Pal
(3)3, D-Cit6,
D-Ala10]GnRH; SB-75, donated by Dr. A. V.
Schally, VA Hospital, New Orleans, LA) was used. Initial levels of GnRH
(time "0") in control and treated groups were measured immediately
after plating the cells and adding the peptides. GnRH production was
measured at 24-h intervals after removing 1 ml of medium and replacing
1 ml of fresh medium, and was calculated as cumulative production over
8 to 9 days of culture. The GnRH content of cultured hypothalamic cells
and GT1–7 neurons was measured by RIA after brief sonication in 1
M Na2CO3, followed by adjustment of
the pH of samples to 7.5.
Radioligand binding assays
Plasma membrane receptors for GnRH (GnRH-R) were analyzed by
binding studies with
125I-des-Gly10-[D-Ala6]GnRH
N-ethylamide (Hazleton, VA). The radioligand (150
pM) and nonradioactive peptides were added in 100-µl
aliquots to 1-week-old monolayer cultures of hypothalamic cells
maintained in 12-well Falcon plates. After incubation to equilibrium
for 60 min at room temperature, the cells were washed three times with
ice-cold PBS containing 0.1% BSA, then solubilized in 1 M
NaOH containing 0.1% SDS and analyzed for bound radioactivity in a
-spectrometer.
GnRH receptors were also analyzed in membrane fractions prepared from
cultured fetal hypothalamic cells. After 10 days in culture, cells were
washed twice with binding buffer containing 10 mM
(Tris[hydroxymethyl]aminomethane)-Trizma base, 0.1% BSA, 1
mM DL-dithiothreitol, 10 µg/ml aprotinin, 0.1
mM phenylmethylsulfonyl fluoride, and 1 mM EDTA
in PBS, pH 7.4. The cells were broken in a glass homogenizer, briefly
sonicated, and sedimented at 400 µg for 15 min. The resulting pellets
were centrifuged at 10,000 x g for 30 min and resuspended
in binding buffer. For the assay procedure, 50–100 µg receptor
protein was incubated with 150 pM 125I-labeled
[D-Ala6]Ag for 60 min on ice in the presence
of increasing concentration of the test peptides. Nonspecific binding
was assessed in the presence of 1 µM of
[D-Ala6]Ag. Incubations were performed in
12 x 75-mm borosilicate glass tubes in a total volume of 0.5 ml
and terminated by dilution with 4 ml ice-cold PBS (pH 7.4), followed by
filtration through glass fiber filters (GF/C Whatman Daigger Scientific, Wheeling, IL) in a multiple holder. The
filters were washed three times with 4 ml PBS, and retained
radioactivity was determined in a -spectrometer.
Preparation of hypothalamic RNA
Total RNA was prepared from hypothalamic tissue and cells by the
acid guanidinium thiocyanate-phenol-chloroform method as described by
Chomczynski and Sacchi (23). Hypothalami were removed from adult rats
by dissection and rapidly frozen on dry ice, and 10-day cultures of
fetal rat hypothalamic cells were washed with ice-cold PBS and frozen
on dry ice. RNA concentrations and purity were determined
spectrophotometrically at 260 and 280 nm (24).
RT-PCR
RT-PCR was carried out using the GeneAmp Thermostable
rTth reverse transcriptase RNA PCR kit (Perkin Elmer, Norwalk, CT). First strand complementary DNA (cDNA) was
synthesized using total RNA and random primers
(Invitrogen, San Diego, CA) and PCR amplification was
performed with gene-specific primers based on sequences in the
transmembrane domains of the mouse pituitary GnRH receptor (25). The
sequence of the primers used for PCR were sense (1S)
5'-GTGACCGTGACTTTCTTC-3'; and antisense (7AS) 5'-GTCGAAGCACGGGTTTAG-3'.
The primers used for nested PCR were sense (3S)
5'-CTCAGCTATCTGAAGCTCTTC-3'; and antisense (6AS)
5'-GACGACAAAGGAGGTAGCG-3'. The numbers in parentheses refer
to the transmembrane domains. Nested PCR was performed in combination
with primers 1S and 6AS, or 3S and 6AS. The reaction conditions for
amplification were 94 C for 1 min, 45 C for 1 min, and 72 C for 1 min
for 35 cycles, and were carried out in a Perkin Elmer
Cetus DNA thermal cycler. Twenty-microliter aliquots of the 100 µl
PCR reaction mixture were then electrophoresed on a 1.0% agarose gel
and visualized by staining with ethidium bromide (24). A control
without the reverse transcriptase step was performed to exclude the
possibility of contamination with genomic DNA. The authenticity of the
PCR-amplified products was confirmed by Southern blot analysis using
a32P-labeled GnRH receptor cDNA probe (25), and a final
stringent wash with 0.5x saline-sodium-citrate (SSC) and 0.1% SDS at
55 C.
Immunocytochemical determination of GnRH and GnRH receptor in
cultured hypothalamic neurons
For immunocytochemical localization of GnRH and GnRH-R in
cultured fetal hypothalamic cells, the enzymatically dispersed cells
were plated on glass chamber slides in standard culture medium at a
density of 105 cells/well. After 3 days, the medium was
supplemented with 5-fluoro-2-deoxyuridine (80 µM) and
culture was continued for 3 days. Before immunostaining the culture
medium was removed and the slides were washed with 0.01 M
PBS, fixed with Bouin’s fluid for 30 min, washed, dehydrated, and kept
dry at -70 C. Immunostaining for GnRH and GnRH-R was performed by the
avidin-biotin peroxidase and alkaline phosphatase methods,
respectively. After hydration the cells were treated with 3%
H2O2, rinsed, blocked by incubation in 10%
normal goat serum in PBS, washed, and incubated overnight at 4 C with
an anti-GnRH polyclonal antibody (1:1000; generously provided by Dr.
V. D. Ramirez). On day 2, the slides were rinsed and incubated in
goat antirabbit IgG-biotin conjugate (1:500) followed by avidin-biotin
peroxidase complex (1:350). GnRH staining was visualized with a
diaminobenzidine substrate kit for peroxidase (Vector, Burlingame, CA).
After GnRH staining, cells were rinsed, blocked by incubation in 10%
normal goat serum, washed, and incubated overnight with a polyclonal
antiserum to the GnRH-R (1:500) at 4 C. The GnRH-R antiserum was raised
in a rabbit immunized with bovine thyroglobulin conjugated with a
synthetic peptide corresponding to the third extracellular loop of the
mouse GnRH-R (residues 291–306). The cells were then incubated in goat
antirabbit IgG-biotin conjugate (1:500, Vector,
Burlingame, CA) and an avidin-biotin alkaline phosphatase complex. The
GnRH-R antigenic sites were visualized using a Vector Blue
Alkaline phosphatase substrate kit III. Antibody specificity was
determined by treating cells with GnRH antibody preadsorbed with
synthetic GnRH or without primary antibody substituted with normal goat
serum for GnRH-R. In double immunostaining, no blue reaction product
was formed when the GnRH-R antibody was omitted after GnRH
immunostaining was completed, confirming the specificity of the
alkaline phosphatase reaction for the GnRH-R.
Data analysis
GnRH pulses were identified, and their parameters were
determined by computerized cluster analysis (26). Individual point
standard deviations were calculated using a power function variance
model from the experimental duplicates. A 2 x 2 cluster
configuration and a t statistic of 2 for the upstroke and
downstroke were used to maintain false-positive and false-negative
error rates below 10%. The statistical significance of the pulse
parameters was tested by using one-way ANOVA.
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), and the lack of displacement by
unrelated peptides (100 nM) including angiotensin II, TRH,
oxytocin, and arginine vasopressin (not shown). The
concentration-dependent inhibition of
125I-[D-Ala6]Ag binding by
unlabeled GnRH, and GnRH agonist and antagonist analogs, is illustrated
in Fig. 1A. The estimated IC50 values for each competition
curve were 23 nM for GnRH, 1.6 nM for the
[D-Ala6]Ag, and 0.2 nM for the
[D-pGlu]Antag. Scatchard analysis of the data for GnRH
and its agonist and antagonist analogs revealed the presence of both
high and low affinity binding sites. The Kd values of the
high affinity sites were 1.4 nM for GnRH, 0.9
nM for [D-Ala6]Ag, and 0.3
nM for [D-pGlu]Antag. Those of the low
affinity sites were 638 nM for GnRH, 478 nM for
[D-Ala6]Ag, and 91 nM for
[D-pGlu]Antag (Fig. 1A).
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). Southern hybridization using
GnRH receptor cDNA probes further confirmed the authenticity of the PCR
amplified products. No such products were obtained in the absence of
reverse transcribed mRNA, indicating that the RNA preparation was free
of genomic DNA contamination. These results demonstrate the expression
of the GnRH receptor gene in the hypothalamus, consistent with the
presence of GnRH receptor sites in these cells (Fig. 1A).
Immunostaining with a specific GnRH antiserum revealed that about 2%
of the cultured hypothalamic cells were GnRH-containing neurons with
typical bipolar morphology. The brown reaction product
characteristic of the GnRH immunocytochemical precipitate complex was
distributed throughout the cytoplasm and primary processes and was
absent from the nucleus. On double immunostaining with both GnRH and
GnRH receptor antisera, a majority of the cells that were positive for
GnRH also exhibited blue granular staining for GnRH-R. This
was predominantly distributed at the plasma membrane of bipolar neurons
(Fig. 1C) and in some monopolar neurons (Fig. 1D). No immunostaining
for GnRH-R (blue) was detectable when GnRH positive cells
(brown) were incubated in normal goat serum without primary
antibody for GnRH-R, and subsequently treated with the Vector
blue alkaline phosphatase reagent (Fig. 1E).
Effects of GnRH receptor agonist and antagonist analogs in static
cultures
Static cultures of hypothalamic cells contained 1074 ± 83 pg
per 106 cells, and released 27.4 ± 1.2 pg/ml into the
incubation medium (n = 6). Depolarization with 50 mM
KCl caused a significant increase in GnRH release (121 ± 8 pg/ml;
P < 0.01; n = 6) with a concomitant decrease in
GnRH content to 671 ± 72 pg/106 cells
(P < 0.05; n = 6; Fig. 2). Static cultures of immortalized GnRH
neurons contained 1442 ± 105 pg GnRH per 106 cells,
and released 49 ± 1.4 pg/ml into the incubation medium (n =
6). Depolarization with 50 mM KCl increased GnRH release to
163 ± 24 pg/ml (P < 0.01; n = 6), with a
concomitant decrease in GnRH content to 769 ± 98 pg/ml
(P < 0.05; n = 6; Fig. 2).
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) and GT1–7 cells (Fig. 3B) was
measured under basal conditions and during treatment with low
concentrations of the two analogs. At the zero time point, no
immunoreactive GnRH was detectable in medium of controls and treated
cells. The basal release of GnRH increased from 3.9 ± 1.4 pg/ml
on day 1 of culture to 17.2 ± 1.8 pg/ml (P <
0.01) on day 3 in culture in GT1–7 cells, and from 7.4 ± 0.8
pg/ml on day 8 of culture to 14.5 ± 0.6 (P <
0.01) day 5 in culture in control hypothalamic cells. Daily addition of
50 pM [D-Ala6]Ag to the culture
medium did not significantly change GnRH release from either cell type,
and the cumulative GnRH profile was similar to that observed in
controls. However, sustained antagonist treatment with 50
pM [D-pGlu]Antag caused a prominent increase
in GnRH release during the first three days of culture, followed by a
monotonic decrease to a level comparable to that observed in untreated
cells.
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). Sample collections at 1-min
intervals provided four to five data points per peak, and revealed
clearly defined episodes of GnRH release. Under basal conditions, the
mean amplitude of each episode of GnRH release, calculated from the
five highest points for each peak, was 7.6 ± 1.3 pg/ml. Transient
depolarization by exposure to 35 mM KCl significantly
increased the mean peak amplitude to 12.3 ± 2.4 pg/ml
(P < 0.05). Over 2.5 h of perifusion, 160 data
points were collected and 9 peaks were detected by cluster analysis. In
this experiment, the mean interval between peaks was 15.3 ± 3.0
min and the mean peak width was 14 ± 3.6 min.
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). The number of peaks
detected was the same (9/2.5 h), the mean interval between peaks was
unchanged at 16.8 ± 4.5 min, and there was a slight decrease in
mean peak width (11.6 ± 4.3 min) compared with the original data
set. There was also a close correlation between the curve for GnRH
release obtained by calculating the cumulative value during 5-min
collections and that derived from the original data set (Fig. 4C).
After this transformation, nine peaks were detected by cluster
analysis, the mean interval between peaks was 16.2 ± 2.3 min, and
the mean peak width was 11.6 ± 3.5 min. Because analysis of 5-min
fractions obtained at a flow rate of 10 ml/h was adequate to monitor
episodic GnRH release, these conditions were used in subsequent
perifusion experiments.
The biological activity of GnRH released by hypothalamic cells was
evaluated by directing the perifusion medium from the hypothalamic
cells into a downstream chamber containing anterior pituitary cells.
Basal LH release was low before connection with a chamber containing
hypothalamic cells (Fig. 4D, open circles) and increased
significantly after connection was established (closed
circles).
Cultured hypothalamic neurons released GnRH in a pulsatile manner for
up to 2 months. As the age of the cultured cells increased from 15–60
days, there was an increase in peak frequency and a decrease in peak
amplitude. The interval between GnRH pulses decreased with duration of
culture from 35 ± 6.1 min to 26.4 ± 15.7 min, 13.7 ±
4.1 min, and 15.4 ± 4.5 min at 15 days, 30 days, 45 days, and 60
days, respectively. The mean peak amplitude increased from 14.4 ±
2.3 pg/ml on day 15 to 38.0 ± 2.4 pg/ml on day 30, and decreased
thereafter to 10.2 ± 4.4 pg/ml and 3 ± 1.4 pg/ml at day 60
(Fig. 5).
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). First, sustained agonist activation
of hypothalamic GnRH receptors by continuous exposure to 1
nM [D-Ala6]Ag extended the
interpulse period from 25.0 ± 4.9 min to 33.3 ± 6.3 min,
and increased the average peak height from 17.1 ± 2.1 pg/ml to
25.5 ± 2.3 pg/ml (P < 0.01, Fig. 6A). More
prominent reduction of spikes frequency and increases in spike
amplitude were observed during sustained exposure of hypothalamic cells
to 10 nM [D-Ala6]Ag. The
interpeak interval increased from 22.5 ± 10.0 min to 50.0 ±
8.5 min during 3 h of treatment, and the mean peak height
increased from 4.9 ± 1.7 pg/ml to 13.8 ± 2.1 pg/ml.
Exposure of perifused hypothalamic cells to 100 nM
[D-Ala6]Ag caused a further decrease in pulse
frequency, and only a single large peak with mean height of 35.5
± 3.2 pg/ml was observed (Fig. 6B). Thus, increases in agonist
concentration caused an increase in the interpeak interval and
amplitude of GnRH pulses, leading to less frequent but more prominent
episodes of GnRH release. As shown in Fig. 6C, the GnRH agonist analog
did not cross-react in the GnRH RIA over a wide range of
concentrations.
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) rose from the basal value of 8.1
± 0.9 pg/ml to 14.8 ± 2.0 pg/ml during the 3-h treatment period.
The progressive increase in GnRH release during prolonged antagonist
treatment was followed by a prominent peak during washout of SB-75 and
returned to near-control levels. Similar increases in basal GnRH
release were observed in six such experiments during treatment with the
potent GnRH antagonist (not shown). In perifused GT1–7 neurons,
treatment with the [D-pGlu]Antag also increased GnRH
release and was followed by a transient peak response (Fig. 7B). As
shown in Fig. 7C, neither of the GnRH antagonist analogs cross-reacted
in the GnRH RIA over a wide range of concentrations.
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In the present studies, all neuronal pathways and interconnections within the hypothalamus were disrupted by dispersion and culture of the hypothalamic cell population. Nevertheless, pulsatile GnRH secretion from such cultured cells was reestablished in vitro and resembled the profile of GnRH release from intact hypothalamic explants (8) and that observed in pituitary portal blood vessels (37). Dispersed hypothalamic cells attached to cytodex beads retain the ability to form interconnections and continue to generate a neuropeptide secretory pattern similar to that observed in vivo (38). The persistence of such a pattern, with changes in pulse frequency and amplitude during 2 months in culture, indicates that neuropeptide release from a reconstituted neuronal network can occur in the absence of inputs from extrahypothalamic neurons and peripheral endocrine glands. Several studies have shown that immortalized GnRH-producing neuronal cell lines (GT1–1 and GT1–7) also exhibit an oscillatory pattern of GnRH release (31, 32, 39). These observations, and the present findings in cultured hypothalamic cells, suggest that rhythmic activity is an intrinsic property of the GnRH neurons.
We have previously reported that GnRH receptors are expressed in GT1–7
cells and that their activation by GnRH agonist analogs influences the
pattern of episodic GnRH release from perifused cultures. Other studies
have shown that GnRH receptors are widely distributed throughout the
brain, and that their density and mRNA levels fluctuate during the
estrous cycle (40, 41). Considerable overlapping of the brain areas
that contain GnRH-producing cells, and those that exhibit expression of
GnRH receptor mRNA, has been reported (42). These findings are
consistent with the possibility that an ultrashort-autocrine feedback
mechanism may contribute to the control of GnRH secretion from the
hypothalamus (20). This hypothesis has been supported by our present
finding that immunocytochemically identified GnRH neurons in cultured
hypothalamic cells also express GnRH-R that are demonstrable by
immunostaining with a receptor antiserum. Also, GnRH-R transcripts were
present in cultured hypothalamic cells as well as in hypothalamic
tissue from adult animals, and their molecular characteristics were
similar to those found in the pituitary gland and 
T3 cells (43).
These receptors bind native GnRH and its potent analogs in a similar
manner to the GnRH receptors that are present in GT1 cells and T3
gonadotrophs (20). The expression of GnRH receptors in hypothalamic
GnRH neurons provides a physiological basis for a number of earlier
published observations on feedback actions of GnRH on its secretion
in vivo. Thus, indirect evidence for the operation of an
ultrashort feedback mechanism in the control of GnRH secretion has been
obtained by the analyzing the effects of intraventricular injection of
GnRH on LH release (44, 45).
More direct evidence for an autocrine action of GnRH was obtained from studies with GT1–7 cells, in which rapid increases in [Ca2+]i were elicited by treatment with a potent GnRH agonist (20). In addition, activation of GnRH receptors caused transient hyperpolarization that was followed by recovery of electrical activity with increased spike frequency (46). In perifused GT1–7 cells, treatment with GnRH agonists also increased GnRH release, consistent with the rapid elevation in [Ca2+]i, and caused a dose-dependent change in pulse amplitude and frequency. Increasing agonist concentrations caused an increase in peak amplitude, with prolongation of the interpeak interval reduced basal GnRH oscillations (20). In the present study, the receptor-mediated actions of GnRH agonists on the neuronal network of cultured hypothalamic cells likewise caused a decrease in frequency and an increase in amplitude of the GnRH pulses. Thus, autocrine feedback leads to switching of the basal pulsatile pattern of release to one characterized by less frequent but more prominent episodes of GnRH secretion. This observation demonstrates that the modulatory actions of GnRH receptor activation on neurosecretion from immortalized GnRH neurons are also evident in the neural network formed in cultured hypothalamic cells. It is of interest that increased release of GnRH from the stalk-median eminence region of ovariectomized rhesus monkeys has been observed during prolonged agonist infusion (47). However, in ovariectomized rats the GnRH concentration in hypophyseal portal plasma was reduced by GnRH agonist treatment (45). These contradictory results could reflect differences in the experimental models and time of sampling, since both stimulatory and inhibitory effects were observed during long-term sample collection.
In contrast to the agonist-induced changes in the pattern of GnRH release from hypothalamic neurons and GT1 cells, treatment with specific receptor antagonists such as SB-75 (48) and [D-pGlu]Antag caused substantial increases in GnRH release from both cell types. In static cultures, this was manifested over several days in the presence of low antagonist concentrations. In perifused hypothalamic neurons and GT1 cells, blockade of GnRH receptors abolished the basal episodic release of GnRH and initiated a prolonged phase of nonpulsatile neuropeptide secretion. These findings indicate that autocrine activation of GnRH receptors in GnRH neurons is an important component of pulsatile GnRH secretion. Furthermore, the progressive rise in GnRH release during sustained antagonist blockade suggests that autocrine activation of neuronal GnRH receptors by low GnRH concentrations periodically inhibits the constitutive release of GnRH from the GnRH neuronal network. Such constitutive secretion is probably dependent on the intrinsic excitability of the GnRH neuron, as indicated by the spontaneous firing of such neurons (14, 15, 46) and the ability of tetrodotoxin to inhibit basal GnRH secretion (39). The extent to which such autocrine inhibitory actions of GnRH are related to the activation of inhibitory G proteins (49, 50), and the suppression of second messenger signaling via the calcium and/or cAMP pathways to regulate exocytosis, has yet to be determined. It is noteworthy that increases in the size and frequency of GnRH pulses have been observed in ovariectomized ewes during GnRH antagonist administration (51).
The present findings, and observations in other experimental models, indicate that normal GnRH-producing neurons coexpress GnRH and GnRH receptors and exhibit spontaneous electrical activity that controls basal GnRH release. These characteristics are appropriate for the operation of an oscillator that is controlled by both positive and negative ultrashort-loop autocrine feedback mechanisms. The existence of such autoregulatory actions of GnRH is relevant to the maintenance and control of the episodic mode of gonadotropin secretion. The operation of such an oscillator in vivo is influenced by hormonal modulation from peripheral endocrine glands, neuropeptides, and neurotransmitters, by activation of specific plasma-membrane receptors and channels. GnRH neurons are also modulated through synaptic and gap connections within the neuronal network that connects hypothalamic and extrahypothalamic regions. Within such a complex regulatory system, the intrinsic oscillatory capacity of GnRH-producing neurons provides the basal mode of pulsatile GnRH release, and permits the generation of the midcycle LH surge that triggers ovulation.
Received May 18, 1998.
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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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T. J. Wu, S. K. Mani, M. J. Glucksman, and J. L. Roberts Stimulation of Luteinizing Hormone-Releasing Hormone (LHRH) Gene Expression in GT1-7 Cells by Its Metabolite, LHRH-(1-5) Endocrinology, January 1, 2005; 146(1): 280 - 286. [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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K. Haneda and Y. Oka Selective Modulation of Voltage-Gated Calcium Channels in the Terminal Nerve Gonadotropin-Releasing Hormone Neurons of a Teleost, the Dwarf Gourami (Colisa lalia) Endocrinology, October 1, 2004; 145(10): 4489 - 4499. [Abstract] [Full Text] [PDF]
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 J. A.F. Huirne, A. C.D. van Loenen, R. Schats, J. McDonnell, P. G.A. Hompes, J. Schoemaker, R. Homburg, and C. B. Lambalk Dose-finding study of daily gonadotropin-releasing hormone (GnRH) antagonist for the prevention of premature luteinizing hormone surges in IVF/ICSI patients: antide and hormone levels Hum. Reprod., October 1, 2004; 19(10): 2206 - 2215. [Abstract] [Full Text] [PDF]
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A. J. Martinez-Fuentes, L. Hu, L. Z. Krsmanovic, and K. J. Catt Gonadotropin-Releasing Hormone (GnRH) Receptor Expression and Membrane Signaling in Ea |
