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Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510
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, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892. E-mail: catt{at}helix.nih.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T3–1 cells) express specific messenger RNA transcripts for GnRH
and exhibit positive immunostaining for the GnRH peptide. Each cell
type released GnRH during both static culture and perifusion, albeit in
lesser amounts than cultured hypothalamic cells and GT1–7 neurons. In
perifused pituitary cells, exposure to a GnRH agonist stimulated the
release of GnRH as well as LH. In contrast, treatment with a GnRH
receptor antagonist or with GnRH antiserum decreased basal LH release.
In pituitary cell cultures, a small proportion of gonadotrophs
exhibited high amplitude and low frequency baseline Ca2+
oscillations in the absence of GnRH stimulation. Such spontaneous
oscillations were comparable to those induced by picomolar
concentrations of GnRH and could be abolished by treatment with a GnRH
antagonist. These in vitro findings indicate that
locally produced GnRH causes low level activation of pituitary GnRH
receptors, induces spontaneous intracellular Ca2+
oscillations, and contributes to basal LH secretion in cultured
pituitary cells. In vivo, such autocrine or paracrine
actions of pituitary-derived GnRH could provide a mechanism for the
maintenance of optimal responsiveness of the gonadotrophs to pulses of
GnRH arising in the hypothalamus. The presence and actions of GnRH in
the anterior pituitary gland, the major site of expression of GnRH
receptors, suggest that local regulatory effects of the neuropeptide
could supplement the primary hypothalamic mechanism for the control of
episodic gonadotropin secretion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to the hypothalamic GnRH system, there is evidence for the production of GnRH at other sites in the brain (6) as well as in rat ovarian granulosa (7) and testicular cells (8), the human placenta (9), the immune system (10), and the pituitary gland (11). The presence of GnRH-producing cells in the pituitary gland and other extrahypothalamic tissues suggests that the autocrine actions of locally produced GnRH could have a physiological role in such processes as self-priming, inter- and intracellular signaling, and hormone secretion. Earlier studies have demonstrated the presence of GnRH immunostaining in long-term cultures of dispersed pituitary cells (11, 12, 13). Also, expression of GnRH messenger RNA (mRNA) in pituitary cells (14, 15) and enzymatic processing and release of GnRH from the pituitary gland (16, 17) have been reported. However, the physiological relevance of this locally produced GnRH has not been investigated, and its potential role in the pituitary gland has not been defined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary culture of hypothalamic and pituitary cells
Fetal rat hypothalamic tissue was dispersed into single cells by
controlled enzyme treatment (18), yielding about 2 x
106 cells/hypothalamus. Anterior pituitary tissue
was also subjected to enzymatic dispersion as described previously
(19). Hypothalamic cells (1.5 x 107) or
pituitary cells (2 x 107) were incubated
for 30 min in a small volume (400 µl) with preswollen Cytodex-2 beads
(Pharmacia, Piscataway, NJ) to allow attachment of cells, then
additional culture medium was added. The culture medium [DMEM/Ham’s
F-12 medium (1:1 vol/vol) with L-glutamate, high glucose
(4.5 mg/ml), and 10% heat-inactivated FBS; Life Technologies, Inc.] was changed every 2–3 days.
Immortalized GnRH neurons and pituitary gonadotrophs
GT1–7 GnRH neuronal cells (provided by Dr. Richard Weiner,
University of California, San Francisco, CA) and the T3–1 pituitary
gonadotroph cell line (provided by Dr. Pamela Mellon, University of
California, San Diego, CA) were grown in the same culture medium
employed for primary cultures. When cells reached confluence, they were
dispersed by trypsinization, washed twice in culture medium, and
cultured on Cytodex-2 beads for perifusion studies or in six-well
plates for other studies. The culture medium was changed at 48-h
intervals.
Cell perifusion procedure
Before each perifusion, the cell-bead mixture was collected by
sedimentation, resuspended in DMEM/F12 (1:1) containing 0.1% BSA and
20 µM bacitracin (Life Technologies, Inc.),
and gassed with 95% O2-5%
CO2. After placement in multiple microchamber
modules (Cellex Biosciences, Inc., Minneapolis, MN) with an internal
volume of 0.5 ml, the bead-attached cells were perifused with medium at
a flow rate of 0.15 ml/min at 37 C. Fractions were collected at 5-min
intervals and stored at -20 C before RIA. GnRH was measured using
[125I]GnRH (Amersham Pharmacia Biotech, Chicago, IL), GnRH standards (Peninsula Laboratories, Inc., Belmont, CA), and primary antibody (donated
by Dr. V. D. Ramirez, University of Illinois, Urbana, IL). The
intra- and interassay coefficients of variation at 80% binding in
standard samples (15 pg/ml) were 12% and 14%, respectively. The
sensitivity of the assay, defined as twice the SD at the
zero dose, was 0.2 pg/tube. There was no detectable cross-reactivity of
the GnRH antibody with the GnRH agonist and antagonist analogs employed
in this study (18, 19). LH concentrations in perifusion medium were
measured by double antibody RIA using kits obtained from the NIDDK, and
results were expressed as nanograms per ml rat LH RP-3 standard. The
sensitivity of the assay was 2.5 ng/ml, and the intra- and interassay
coefficients of variation were 5% and 8%, respectively (n = 6)
(20).
Static cell culture
Hypothalamic or pituitary cells (2 x
106) were cultured in six-well plates and
maintained for 3–4 days for measurements of hormone release.
Immortalized GT1–7 or T3–1 cells (106) were
cultured in six-well plates, and GnRH and LH release was measured after
48 h of incubation. Hormone release was measured after 1-h
incubation at 37 C in DMEM/F12 medium containing 1% BSA and 20
µM bacitracin, pH 7.4. During long term culture of
pituitary cells (2 x 106/well) for 20 days,
sampling was performed by removing 1 ml medium and replacing an equal
volume of fresh medium in a total volume of 2 ml. Hormone levels were
measured at 24-h intervals, and at each time point the released hormone
was expressed as the cumulative production.
Intracellular Ca2+([Ca2+]i) measurements
For analysis of
[Ca2+]i, pituitary cells
were plated on 25-mm coverslips coated with poly-L-lysine
and cultured for 48 h. The coverslips were then washed twice,
loaded with 2 µM indo-1/AM (Molecular Probes, Inc., Eugene, OR) for 60 min at 37 C, and mounted on the stage
of an inverted Diaphot microscope attached to a dual emission
photometry system (Nikon, Garden City, NY). Cells were
excited with a 360-nm light beam, and the emission was measured at 405
and 485 nm. After the background subtraction, the ratio of these two
intensities was further converted to
[Ca2+]i using the
standard curve constructed by the addition of known concentrations of
Ca2+ to 10 µM indo-1. Data
collection, aided by the FASTINCA program (University of Cincinnati
Medical Center, Cincinnati, OH), was performed in 360-msec
intervals.
In addition to photometry, Ca2+ imaging was also performed where indicated. Coverslips with cells where loaded with 2 µM fura-2/AM (Molecular Probes, Inc.) for 60 min at 37 C, washed, and mounted on a stage of Axiovert 135 microscope (Carl Zeiss, Oberkochen, Germany) attached to the Attofluor Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). Cells were illuminated alternatively by 340- and 380-nm beams, and the emission was observed at 505 nm. Changes in [Ca2+]i are presented as the ratio of these two fluorescence intensities (F340/F380), and the estimate of the actual concentration was obtained using the Grynkiewicz equation (21). Data were obtained from two to seven gonadotrophs simultaneously at a sampling rate of one point per sec.
RNA isolation and RT-PCR
Total RNA was isolated from T3 cells, cultured pituitary
cells, GT1–7 cells, and fetal hypothalamic cells by the method of
Chomczynski and Sacchi (22) and checked for purity. RNA samples were
subjected to RT-PCR to determine their content of GnRH mRNA. Four to 6
µg total RNA from each sample were reverse transcribed into
complementary DNA in a 20-µl reaction mixture containing an
oligo(deoxythymidine) primer using SuperScript II RNase
H- reverse transcriptase (Life Technologies, Inc.). Aliquots (2.5 µl) of the RT reaction
mixture were subjected to PCR amplification for 35 cycles using the
ELONGase Amplification System (Life Technologies, Inc.) in
a final volume of 50 µl containing 1.5 mM
MgCl2, 0.2 µM of each primer, and
0.2 mM of each deoxy-NTP. The PCR conditions for each cycle
were denaturation at 94 C for 30 sec, annealing at 60 C for 25 sec, and
extension at 72 C for 30 sec. Specific primers, based on the mouse
pro-GnRH sequence (23), corresponded to amino acids -9 to -3 for the
sense primer (5'-ACTGTGTGTTTGGAAGGCTGC-3') and amino acids 57–51 for
the antisense primer (5'-TTCCAGAGCTCCTCGCAGATC-3'). The negative amino
acid numbers are within the sequence of the signal peptide (23).
The primer sequences were between the signal peptide and the GnRH-associated peptide, and spanned an intron composed of about 1300 bp. Samples without reverse transcriptase were used as controls for each cell type to exclude the possibility of contamination by genomic DNA, which would yield a 1500-bp segment together with the 198-bp fragment derived from GnRH mRNA transcripts. However, no 1500-bp product was observed under these conditions. Also, a RNA sample extracted from the optic nerve gave no 198-bp DNA product after amplification, and thus served as a negative control. The PCR products were analyzed by agarose gel electrophoresis (4% NuSieve 3:1, FMC BioProducts, Rockland, ME), and the DNA fragments were gel-purified using a QIAEX II Gel Extraction Kit (QIAGEN, Valencia, CA). DNA sequencing was performed using the Thermo Sequence radiolabeled terminator cycle sequencing kit from Amersham Pharmacia Biotech (Arlington Heights, IL).
Immunocytochemistry of cultured pituitary cells and hypothalamic
neurons
Immunostaining of LH and GnRH was performed by
avidin-biotin-peroxidase and alkaline phosphatase methods. The cultures
were washed with 0.01 M PBS, fixed in Bouin’s fluid for 30
min, washed, dehydrated, and kept dry at -70 C. For single
immunostaining the fixed cultures were rehydrated, treated with 3%
H2O2, rinsed, blocked by
incubation in 10% normal goat serum in PBS, washed, and incubated
overnight at 4 C with rabbit anti-LH serum (1:50,000; NIDDK) or rabbit
anti-GnRH serum (1:1,000; provided by Dr. V. D. Ramirez,
University of Illinois, Urbana, IL). On day 2, the slides were rinsed
and incubated with goat antirabbit IgG-biotin conjugate, followed by
avidin-biotin-peroxidase or alkaline phosphatase complex. Specific
staining for LH (blue) was visualized with a Vector blue
alkaline phosphatase substrate kit III, and GnRH (brown) was
visualized with a diaminobenzidine substrate kit for peroxidase
(Vector Laboratories, Inc., Burlingame, CA). Antibody
specificity was determined by treating cells with LH or GnRH antiserum
preadsorbed with homologous peptides.
For double immunostaining, identified pituitary gonadotrophs were repeatedly washed in PBS, incubated with rabbit IgG for 5 h at room temperature, blocked in 10% normal goat serum, washed, and processed for second immunostaining. There was no formation of positive immunoproducts when the second primary antibodies were preadsorbed with related hormones or omitted, confirming the specificity of the alkaline phosphatase reaction in the double immunostaining.
Data analysis
GnRH, LH, and LH pulses were identified, and their parameters
were determined by a computer-based algorithm cluster analysis (24).
Individual point SDs 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 upstroke and
downstroke were used to maintain false positive and false negative
error rates below 10%. The statistical significance of the pulse
parameters and hormone levels was tested by one-way ANOVA. Duncan’s
multiple range test with critical ranges level of 0.01 was used as a
post-hoc test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, a single product corresponding to the
size predicted by the GnRH primers was observed in both T3–1
gonadotrophs (lane 2) and cultured anterior pituitary cells (lane 3),
similar to that observed in positive control cells (GT1–7 neurons,
lane 4; cultured hypothalamic cells, lane 5). No such products were
obtained in the absence of reverse transcribed mRNA (lane 6),
indicating that the RNA preparation was free of genomic DNA
contamination. The DNA sequence analysis of the gel-purified 198-bp
fragments from each cell type was identical with the known nucleotide
sequence of the GnRH gene (23), thus confirming the authenticity of the
amplified DNA fragments. Consistent with the presence of its
transcripts, immunoreactive GnRH peptide was released from both
cultured pituitary cells and T3–1 gonadotrophs. The highest amount
of GnRH released per 1 x 106 cells during
1-h incubation in DMEM/F12 medium containing 1% BSA and 20 µg
bacitracin at 37 C was in GT1–7 cells, followed by hypothalamic cells,
T3–1 cells, and anterior pituitary cells (by ANOVA,
P < 0.01; Fig. 1B).
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). Single immunostaining with a
specific GnRH antiserum revealed that GnRH was present in 11 ±
0.6% of 4141 pituitary cells counted in three experiments (Fig. 2B).
On double immunostaining with both LH and GnRH antisera, 360 of 378
LH-containing cells (95%) also exhibited brown staining for GnRH (Fig. 2, C and D). Also, some GnRH-positive cells were not positively stained
for LH, as indicated by the arrow in Fig. 2D. No
immunostaining for GnRH (brown) was detectable when the
cells were incubated in normal goat serum or with GnRH-preadsorbed
primary antibody for GnRH and subsequently treated with the Vector blue
alkaline phosphatase.
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). In
dynamic studies, GnRH was also released from pituitary cells (Fig. 3B)
and T3–1 gonadotrophs (Fig. 3C) perifused with DMEM/F12
supplemented with 1% BSA and 20 µM bacitracin,
pH 7.4, at 37 C, using flow rate of 0.15 ml/min. These results indicate
that the synthesis and release of GnRH are characteristic features not
only of hypothalamic neurons, but also of anterior pituitary cells. The
production of GnRH by mouse T3–1 gonadotrophs raises the
possibility that GnRH is cosecreted with gonadotropins in primary
cultures of pituitary cells.
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). Perifused pituitary cells also
responded to successive applications of 10 nM
[D-Ala6]Ag with a
significant (7.5 ± 0.5 vs. 2.4 ± 0.2 pg/ml;
P < 0.001; n = 4) increase in GnRH release (Fig. 3B
). Also, pituitary cells responded to successive application of 10
nM
[D-Ala6]Ag with a
significant increase in LH release (15.3 ± 2.8 vs.
7.2 ± 1.8 ng/ml; P < 0.001; n = 3).
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T3–1 gonadotrophs also responded to agonist stimulation with
a significant (30.6 ± 2.0 vs. 10.7 ± 0.2 pg/ml;
P < 0.006; n = 3) increase in GnRH release (Fig. 3C) in a manner comparable to that observed in hypothalamic cells (19, 25, 26). This was an unexpected result, as these cells do not exhibit
regulated secretion of LH -subunit in response to GnRH stimulation
(27). As shown in Fig. 3C, LH release was almost completely
unaffected by [D-Ala6]Ag
treatment, in contrast to the severalfold increase in GnRH release.
Taken together, these results suggest that a fraction of native
gonadotrophs express both gonadotropins and GnRH, and release them in
response to activation of GnRH receptors. The results further indicate
that the exocytotic pathway in T3–1 gonadotrophs is functional, but
that only GnRH is secreted in a regulated manner, whereas LH
-subunit is secreted in a constitutive manner.
Effects of GnRH antagonist analogs and anti-GnRH serum on LH
release
To study the physiological relevance of locally produced GnRH on
gonadotroph function, the effect of a potent GnRH receptor antagonist
([Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]GnRH
(SB-75; provided by Dr. A. V. Schally, V.A. Hospital, New Orleans,
LA) was studied during sustained agonist stimulation. As shown in Fig. 4A, this antagonist not only abolished the
[D-Ala6]Ag-induced plateau LH
response, but also caused a prompt decrease in LH release to below
the basal level (9.0 ± 0.2 vs. 7.1 ± 0.1;
P < 0.01; n = 4) The GnRH receptor antagonist
also significantly decreased LH release in unstimulated pituitary
cells, an effect that was reversed during the subsequent washing period
(Fig. 4B). The effect of GnRH antagonist treatment on basal LH
secretion was clearly observed when cells were perifused at flow rate
of 0.15 ml/min. These results suggest that locally produced GnRH is
sufficient to activate its receptors in pituitary gonadotrophs and
contributes to the basal rate of gonadotropin secretion.
The role of pituitary-derived GnRH in the control of basal LH release
was also assessed in perifusion studies using anti-GnRH serum. The
ED50 of the anti-GnRH serum in RIA at a dilution
of 1:50,000 was 11.0 pg/ml. A 1:100 dilution of the anti-GnRH serum was
used to neutralize GnRH released from cultured pituitary cells. In
control perifusions the mean LH level was 6.2 ± 0.2 ng/ml and did
not change when the cells were exposed to heat-treated (100 C) medium
(5.8 ± 0.13 ng/ml; Fig. 5A).
Heat-inactivated anti-GnRH serum caused a small (4.9 ± 0.16
vs. 4.5 ± 0.18 ng/ml), but insignificant, rise in LH
secretion (Fig. 5B). Perifusion of pituitary cells with untreated
anti-GnRH serum caused a significant decrease in LH release (7.4
± 0.2 vs. 5.6 ± 0.15 ng/ml; P <
0.04; n = 4), as shown by the individual traces in Fig. 5, C and
D, and E, and F. The similar inhibitory effects of GnRH antagonist and
GnRH antiserum treatment suggest that locally produced GnRH
participates in the maintenance of basal LH release in perifused
pituitary cells.
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In further experiments, we compared spontaneous high amplitude
[Ca2+]i oscillations with
those induced by GnRH. Some cells (26 of 77) exhibited relatively
regular baseline [Ca2+]i
oscillations (Fig. 6A, left
panel), typical of those observed after stimulation of quiescent
gonadotrophs with 10–100 pM GnRH (right
panel). The average frequency of these oscillations was 6 ±
0.9 spikes/min, comparable to that observed in quiescent gonadotrophs
stimulated with 100 pM GnRH (7 ± 0.8
spikes/min; n = 34). In residual cells, oscillations were
irregular (Fig. 6B, left panel) and resembled the
subthreshold type of
[Ca2+]i signaling
observed in GnRH-stimulated gonadotrophs (right panel).
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, left panel. The
increase in spike frequency was also observed in two additional cells
stimulated with 1 nM GnRH. In parallel to this,
the frequency of [Ca2+]i
oscillations increased from 7 ± 0.9 to 11 ± 0.6 spikes/min
in 34 gonadotrophs initially stimulated with 100
pM and subsequently with 1
nM GnRH (Fig. 7A, right panel).
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; n = 3), again comparable to that
observed in gonadotrophs stimulated with low and subsequently with high
GnRH concentrations (Fig. 7B, right panel; n = 25).
Finally, both spontaneous (Fig. 7C, left panel;
n = 2) and GnRH-induced [Ca2+]i
oscillations (right panel; n = 27) were abolished by
the addition of thapsigargin, a blocker of endoplasmic reticulum
Ca2+-adenosine triphosphatase. These results show
that spontaneous [Ca2+]i
oscillations are very similar to those induced by GnRH and suggest that
they are initiated by the locally produced neuropeptide.
To test this hypothesis, cultured pituitary cells were treated with the
potent GnRH receptor antagonist,
[N-Ac-D-Nal
(2)1,D-pCl-Phe2,D-Trp3,D-hArg
(Et2)6,D-Ala10]GnRH
(detirelix; 0.5 µg/ml). In quiescent cells, detirelix did not affect
basal [Ca2+]i (Fig. 8A). However, in spontaneously active
cells with basal [Ca2+]i
oscillations of 6 ± 0.6 min-1 (n =
25; from 10 recordings) the antagonist consistently abolished the
[Ca2+]i oscillations
(Fig. 8, B–D). Conversion of the nonoscillatory
[Ca2+]i responses to 100
nM GnRH (Fig. 7B) into the oscillatory pattern
(8 ± 0.8 min-1; n = 25;
P < 0.05; from 10 recordings) typically elicited by
low concentrations of GnRH was usually observed after antagonist
blockade of GnRH receptors (Fig. 8, right side of tracings
A–D). These results indicate that the apparently spontaneous
[Ca2+]i oscillations are,
in fact, elicited by locally produced GnRH. As LH secretion from
gonadotrophs is controlled by
[Ca2+]i (30), the
occurrence of such oscillations provides an explanation for the
inhibitory effects of GnRH antagonists on basal LH release (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study has addressed the potential regulatory actions of
locally formed GnRH at the pituitary level by several approaches. In
accord with earlier reports (14, 15), we confirmed the presence of
specific GnRH transcripts in pituitary cells and T3–1 gonadotrophs
as well as positive immunostaining in anterior pituitary cells. Also,
readily measurable release of the GnRH peptide was observed in short
and long term static cultures of pituitary cells as well as
immortalized gonadotrophs and perifused pituitary cells. When
perifusion studies were performed at a slow flow rate (0.15 ml/min),
GnRH release was detected under basal conditions as well as during GnRH
agonist stimulation. The latter finding demonstrates that GnRH is
cosecreted with LH and suggests that the locally produced peptide and
its receptors might be involved in an autoregulatory feedback
mechanism.
Supportive evidence for the participation of locally produced GnRH in the control of gonadotroph function was provided by the inhibitory action of the potent GnRH receptor antagonist, SB-75, on basal LH release. In earlier studies on pituitary cells perifused with medium at 1 ml/min, basal LH release was very low and was not affected by removal of extracellular Ca2+ and abolition of spontaneous electrical activity (28). However, the basal LH release observed when cells were perifused at flow rate of 0.15 ml/min was significantly higher and was consistently reduced after the addition of a GnRH receptor antagonist. When the role of ligand-independent activity of GnRH receptors in basal LH release was tested by the addition of anti-GnRH serum, a significant decrease in secretion was observed. These findings suggest that a low level of GnRH receptor activation by endogenous GnRH contributes to the basal rate of LH release in vitro.
The central role of [Ca2+]i in the control of gonadotropin secretion by GnRH has been well established (34). In pituitary gonadotrophs, low picomolar concentrations of GnRH induce irregular, low amplitude [Ca2+]i transients that are classified as subthreshold responses. At higher concentrations, GnRH promotes high amplitude baseline [Ca2+]i oscillations at a frequency (ranging from 2–25/min) that is dependent on the agonist concentration (29). Such responses to agonist stimulation and the ability of each Ca2+ spike to promote exocytosis (30) are consistent with the concept that gonadotropin secretion is frequency coded by the intracellular calcium signal. Also, only high amplitude [Ca2+]i spikes are able to trigger exocytosis (35). Thus, the partial dependence of basal LH release on locally produced GnRH implies that some gonadotrophs must exhibit baseline [Ca2+]i spiking in the absence of exogenous GnRH. In accord with this, spontaneous [Ca2+]i oscillations very similar to those elicited by subthreshold and low threshold concentrations of GnRH were observed in a small proportion of gonadotrophs. Furthermore, the ability of GnRH receptor blockade to abolish such oscillations demonstrates that GnRH receptors participate in their generation.
These findings indicate that endogenously produced GnRH activates the GnRH receptors expressed in pituitary gonadotrophs, leading to subthreshold and low frequency baseline [Ca2+]i oscillations that elicit a sufficient degree of exocytosis to contribute to the basal level of LH secretion. This local effect of pituitary GnRH could serve to maintain an optimal degree of sensitivity of the gonadotroph population to periodic stimulation by hypothalamic GnRH. This would essentially correspond to an intrinsic form of the well known priming effect of exogenous GnRH, which enhances secretory responses to subsequent GnRH stimulation (36, 37, 38).
Received September 20, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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X 174 DNA/HaeIII digest markers. Reactions without RT
(lane 6) and without RNA sample (lane 7) served as controls. GnRH
transcripts of 198 bp, as predicted by the GnRH primers used, were
detected in all samples. B, Release of GnRH by normal and immortalized
pituitary and hypothalamic cells. Data are the mean ±
SE of six replicates for each cell type.
Asterisks indicate significant differences in GnRH
release compared to the cultured pituitary cells.
