Journal of Neuroendocrinology, 1999, Vol. 11, 195–201
Negative Regulation of Gonadotropin-Releasing Hormone and
Gonadotropin-Releasing Hormone Receptor Gene Expression by a
Gonadotropin-Releasing Hormone Agonist in the Rat Hypothalamus
Y.-G. Han, S. S. Kang, J. Y. Seong, D. Geum, Y.-H. Suh* and K. Kim
Department of Molecular Biology and Research Centre for Cell Differentiation, College of Natural Sciences, and *Department of
Pharmacology, College of Medicine and Department of Molecular Biology, Neuroscience Research Centre, Seoul National University, Seoul,
Korea.
Key words: gonadotropin-releasing hormone, gonadotropin-releasing hormone receptor, ultrashort loop feedback, homologous
down-regulation, gene expression.
Abstract
There exists evidence for the presence of ultrashort loop feedback circuits of gonadotropinreleasing hormone (GnRH) secretion in the hypothalamus. It is, however, uncertain whether a similar
mechanism is involved in the regulation of GnRH gene expression in vivo. Furthermore, little is
known about the regulation of GnRH receptor (GnRHR) expression in the brain. In the present study,
we examined the regulation of GnRH and its receptor gene expression by GnRH in vivo. A GnRH
agonist, [D-Ala6, des-Gly10]GnRH-ethylamide (des-Gly GnRH), was administered by
intracerebroventricular (i.c.v.) injection via the lateral ventricle of ovariectomized and estradiol
(OVX+E)-treated rats. The amounts of GnRH and GnRHR mRNA were measured in the preoptic
area (POA) and posterior mediobasal hypothalamus (pMBH) micropunch samples from individual rat
brain slices by respective competitive reverse transcription-polymerase chain reactions. The i.c.v.
administration of des-Gly GnRH significantly decreased GnRH and GnRHR mRNA expression in a
dose-and time-related manner: des-Gly GnRH (6 ng) suppressed GnRH and GnRHR mRNA
expression within 2 h, and the suppression was maintained without significant variation until 8 h
after treatment. Treatment with Antide, [N-Ac-d-Nal(2)1, pCl-d-Phe2, d-Pal(3)3, Lys(Nic)5, d-Lys(Nic)6,
Lys(iPR)8, d-Ala10]GnRH (10 ng), a potent GnRH antagonist, did not alter GnRH mRNA expression,
but prevented des-Gly GnRH-induced suppression of GnRH mRNA expression. Antide alone
decreased GnRHR mRNA expression, but failed to alter agonist-induced suppression of GnRHR
mRNA expression. These results demonstrate the existence of an ultrashort loop feedback
mechanism for GnRH gene expression in the POA, along with homologous down-regulation of
GnRHR mRNA expression in the pMBH.
Gonadotropin-releasing hormone (GnRH ) is the major regulator of reproduction in mammals. It is synthesized by
neurosecretory cells which are dispersed in the preoptic area
(POA) and adjacent sites in the rostral portion of hypothalamus, and released into the hypophyseal portal vessels. The
activity of GnRH neurones is regulated by steroids and
several neurotransmitters (1, 2) and, in addition, several
studies have suggested that there is an autoregulation of
GnRH by an ultrashort loop feedback mechanism. For
instance, the central or peripheral administration of GnRH
or its analogues altered the secretion and tissue contents of
GnRH, and serum luteinizing hormone (LH ) concentrations
(3–7). While it is evident that an ultrashort loop feedback
mechanism of GnRH operates at the level of GnRH and LH
release, there is little evidence that such a mechanism influences the level of gene expression.
The mediobasal hypothalamus (MBH ) appears to be the
principal structure involved in the ultrashort loop feedback
action of GnRH, since treatment with a GnRH agonist
suppressed basal and K+-stimulated GnRH release from
MBH fragments, but not from median eminence explants
(3). The MBH region, including the arcuate nucleus and the
Correspondence to: Dr Kyungjin Kin, Department of Molecular Biology, College of Natural Sciences, Seoul National University, Seoul
151-742, Korea.
© 1999 Blackwell Science Ltd
196 GnRH and GnRH receptor gene expression by GnRH
ventromedial nucleus, receives extensive GnRH-containing
axonal projections (8) and is the central site of reproductive
behaviour (9). It is notable that GnRH receptor (GnRHR)
transcripts were readily detectable in the MBH as demonstrated by in-situ hybridization (8) and competitive reverse
transcription-polymerase chain reaction (RT-PCR) analyses
(10). However, in contrast to the extensive studies on the
regulation of GnRHR gene expression in the pituitary, little
information is available about the regulation of GnRHR
gene expression in the brain. A recent study has reported
fluctuations in GnRHR mRNA expression in the MBH
during the oestrous cycle (11), and the fluctuations, to some
extent, seem to be attributable to gonadal steroids–i.e. the
increase of GnRHR mRNA expression during early proestrus
coincides with high circulating concentrations of estradiol.
We also found that administration of estradiol to ovariectomized (OVX ) rats caused a marked increase in GnRHR mRNA
expression in the pMBH (12). However, it is uncertain
whether GnRHR gene expression in the brain is under the
control of homologous regulation, a characteristic feature of
peptide hormone action. Alterations in target cell sensitivity
or responsiveness to subsequent hormonal stimulation is
often governed by homologous regulation (13). In fact, native
GnRH or GnRH agonists regulate pituitary GnRHR mRNA
expression in vivo and in vitro (14–18).
To address the question of whether GnRH itself affects
GnRH and GnRHR gene expression, the present study
determined GnRH mRNA expression in the POA and
GnRHR mRNA expression in the pMBH following intracerebroventricular (i.c.v.) injection of GnRH agonist into OVX
and estradiol (OVX+E)-treated rats.
Results
It has previously been shown that competitive RT-PCR
procedure is useful in quantifying GnRH and GnRHR
mRNA expression in POA and pMBH micropunch samples
derived from an individual rat (10, 19). In this study, POA
GnRH RNA samples and pMBH GnRHR RNA samples
were used to compete with 0.2 pg of mutant GnRH cRNA
and 5 fg of mutant GnRHR cRNA, respectively. Figure 1
depicts the standard curves of competitive RT-PCR for
GnRH and GnRHR mRNA determination. A constant
amount of mutant cRNAs (0.2 pg GnRH and 5 fg GnRHR)
were coamplified with serially diluted native GnRH and
GnRHR cRNAs (native to mutant: 0.1, 0.2, 0.5, 1, 2, 5, and
10). The plots of log ratio of native to mutant signals against
log concentration of native cRNAs revealed a linear relationship. The regression coefficients (r) of GnRH and GnRHR
were 0.996 and 0.979, respectively (Fig. 1).
Dose-and time-dependent reductions in the expression of
POA GnRH and pMBH GnRHR mRNA were observed
after i.c.v. injection of des-Gly GnRH. A significant reduction
in POA GnRH mRNA expression was observed following
6 ng (0.45±0.04 pg/micropunch, n=6), but not following
0.6 ng (1.40±0.31 pg/micropunch, n=8) of des-Gly GnRH
compared to control levels (1.47±0.36 pg/micropunch, n=
8) (Fig. 2). Similarly, 6 ng of des-Gly GnRH decreased
GnRHR mRNA expression (8.6±3.3 fg/micropunch, n=6)
compared to control levels (34±6.5 fg/micropunch, n=8)
(Fig. 2). Serum LH concentrations were significantly
(P<0.05) increased following 6 ng, but not following 0.6 ng
of GnRH agonist (Table 1). The injection of a relatively
large dose of GnRH into the third ventricle has previously
been reported to increase plasma LH concentrations (20).
The increase in serum LH concentrations following 6 ng of
des-Gly GnRH may be due to the diffusion of des-Gly GnRH
to the vicinity of hypophyseal portal vessel system and hence
to the pituitary gland. This increase also indicates the accurate
delivery of des-Gly GnRH into the ventricular system. Since
6 ng of des-Gly GnRH decreased GnRH and GnRHR
mRNA expression within 2 h, the time-course of this effect
was examined. As shown in Fig. 3, compared to control
mRNA levels of GnRH(1.38±0.31 pg/micropunch, n=6)
and GnRHR(33±6.4 fg/micropunch, n=6), significant
reductions in mRNA levels of GnRH (0.52±
0.09 pg/micropunch, n=5) and GnRHR (4.7±0.07 fg/
micropunch, n=5) were seen 2 h after des-Gly GnRH injection. GnRH and GnRHR mRNA levels remained low until
8 h after treatment and did not vary significantly, although
serum LH concentrations returned to basal levels by this time
point (Table 1).
To examine the specificity of des-Gly GnRH action on
GnRH and GnRHR mRNA expression, Antide (10 ng), a
potent GnRH antagonist, was administered i.c.v. 1 h before
administration of des-Gly GnRH (6 ng i.c.v.). Antide alone
failed to alter GnRH mRNA expression, but restored the
agonist-induced suppression of GnRH mRNA expression
(Fig. 4). By contrast, GnRHR mRNA expression was
decreased significantly following Antide alone and was not
recovered from agonist-induced low levels by pretreatment
of Antide (Fig. 4). Des-Gly GnRH significantly (P<0.05)
increased serum LH concentrations, but Antide failed to alter
serum LH concentrations induced by des-Gly GnRH
(Table 1).
Discussion
The present study provides for the first time in-vivo evidence
for negative regulation, by GnRH, both on its own and on
T 1. Serum Luteinizing Hormone Concentrations.
Experiment
Group
Dose-response
effects of desGly-GnRH
Control
Des-Gly-GnRH
Des-Gly-GnRH
Control
Des-Gly-GnRH
Des-Gly-GnRH
Des-Gly-GnRH
Time-course
Effects of des−
Gly-GnRH
(6 ng)
Effects of desGly-GnRH and
Antide
(0.6 ng)
(6 ng)
(0.5 h†)
(2 h)
(4 h)
Des-Gly-GnRH (8 h)
Control
Antide (10 ng)
Des-Gly-GnRH (6 ng)
Des-Gly-GnRH (6 ng)
Antide (10 ng)
n
LH (ng/nl )
8
8
6
6
6
5
5
1.83±0.25
1.47±0.18
5.98±0.62*
1.96±0.70
10.17±2.69*
13.82±2.29*
5.17±1.22
5
5
5
5
1.95±0.70
2.76±0.24
2.24±0.42
6.11±1.07*
5
8.53±2.66*
Values are mean±SEM. *P<0.05 (vs Control ). †Time after i.c.v.
injection. n=No. individual animals.
© 1999 Blackwell Science Ltd, Journal of Neuroendocrinology, 11, 195–201
GnRH and GnRH receptor gene expression by GnRH 197
(A)
(B)
Native
Mutant
Native
Mutant
10
r = 0.996
1.0
0.1
0.02
Native
Mutant
Ratio (Native/Mutant)
Ratio (Native/Mutant)
Native
Mutant
0.2
10
r = 0.979
1.0
0.1
2
0.02
Native GnRH cRNA (pg)
0.2
2
Native GnRHR cRNA (fg)
F. 1. Standard curves of competitive reverse transcription-polymerase chain reaction (RT-PCR) for gonadotropin-releasing hormone (GnRH ) and
GnRHR mRNA. A constant amount of mutant GnRH or GnRHR cRNA was coamplified with various concentration of native GnRH () or
GnRHR () cRNA, respectively. Each polymerase chain reaction (PCR) product was separated on 2% agarose gel. The plot of the log ratio of native
to mutant signals vs log concentration of native cRNAs revealed a linear relationship. () 0.2 pg of mutant GnRH cRNA was coamplified with 0.02,
0.04, 0.1, 0.2, 0.4, 1, and 2 pg of native GnRH cRNA. () 5 fg of mutant GnRHR cRNA was coamplified with 0.5, 1, 2.5, 5, 10, 25, and 50 fg of
native GnRHR cRNA.
Des-Gly-GnRH (ng)
GnRH mRNA (pg/micropunch)
CTL
0.6
(B)
Des-Gly-GnRH (ng)
6
1.5
1.0
0.5
8
8
6
CTL
0.6
6
Des-Gly-GnRH (ng)
GnRHR mRNA (fg/micropunch)
(A)
CTL
0.6
6
8
8
6
CTL
0.6
6
40
30
20
10
Des-Gly-GnRH (ng)
F. 2. Dose effects of des-Gly GnRH on GnRH mRNA levels in the preoptic area (POA) () and gonadotropin-releasing hormone receptor
(GnRHR) mRNA levels in the posterior mediobasal hypothalamus (pMBH ) (). Des-Gly GnRH (0.6 or 6 ng) was i.c.v. injected at 15.00 h. Control
(CTL) rats received 5 ml of saline. Rats were killed at 17.00 h. Each mRNA from the preoptic area (POA) and the pMBH micropunch was coamplified
with 0.2 pg of mutant GnRH cRNA and 5 fg of mutant GnRHR cRNA, respectively. Three representative PCR samples per experimental group are
shown in upper panels. Bars in lower panel represent the mean (± SEM ) amount of GnRH and GnRHR mRNA levels, respectively. Numbers on
the bars indicate individual animals. *P<0.05 (vs Control ).
© 1999 Blackwell Science Ltd, Journal of Neuroendocrinology, 11, 195–201
198 GnRH and GnRH receptor gene expression by GnRH
(A)
Time after injection (h)
CTL
0.5
2
4
(B)
Time after injection (h)
8
CTL
0.5
2
4
8
40
1.5
1.0
0.5
CTL
0.5
2
4
n = 5–6
GnRHR mRNA (fg/micropunch)
GnRH mRNA (pg/micropunch)
n = 5–6
30
20
10
8
CTL
Time after injection (h)
2
0.5
4
8
Time after injection (h)
F. 3. Temporal changes in gonadotropin-releasing hormone (GnRH ) mRNA levels in the POA () and GnRHR mRNA levels in the posterior
mediobasal hypothalamus (pMBH ) () after des-Gly GnRH administration. Des-Gly GnRH (6 ng) was i.c.v. injected into each group of rats at
09.00 h, 13.00 h, 15.00 h, or 16.30 h. Control (CTL) rats received 5 ml of saline at 15.00 h. Rats were killed at 17.00 h. Numbers indicate individual
animals. *P<0.05 (vs Control ).
GnRH mRNA (pg/micropunch)
CTL
Des-GlyDes-GlyGnRH
GnRH Antide +Antide
1.5
1.0
0.5
5
5
5
5
Des-Gly-GnRH
–
+
–
+
Antide
–
–
+
+
(B)
CTL
GnRHR mRNA (fg/micropunch)
(A)
Des-GlyDes-GlyGnRH
GnRH Antide +Antide
40
30
20
10
5
5
5
5
Des-Gly-GnRH
–
+
–
+
Antide
–
–
+
+
F. 4. Effects of Antide pretreatment before des-Gly GnRH administration on gonadotropin-releasing hormone (GnRH ) mRNA levels in the POA
() and GnRHR mRNA levels in the posterior mediobasal hypothalamus (pMBH ) (). Antide (10 ng) was i.c.v. injected at 14.00 h, and 1 h later
des-Gly GnRH (6 ng) was injected. Control (CTL) rats received 5 ml of saline at 14.00 h and 15.00 h. All rats were then killed at 17.00 h. The numbers
on the bars indicate individual animals. *P<0.05 (vs Control ).
© 1999 Blackwell Science Ltd, Journal of Neuroendocrinology, 11, 195–201
GnRH and GnRH receptor gene expression by GnRH 199
its receptor gene expression in the hypothalamus. The i.c.v.
administration of a GnRH agonist, des-Gly GnRH into
OVX+E-treated rats significantly reduced POA GnRH
mRNA expression within 2 h. The half-life of GnRH mRNA
was estimated to be about 22 h in hypothalamic GT1 neuronal
cells (21). Our recent in-vitro study with GT1 cells have
shown that suppression of GnRH transcription following
another GnRH agonist, Buserelin, required more than 6 h
(22). In postnatal hypothalamic slice explant cultures, however, the turnover rate of GnRH mRNA was extremely rapid,
with a half-life of less than 15 min and followed by much
slower decay (23). It seems likely that GnRH agonists may
cause both the inhibition of transcription and/or the increase
of GnRH mRNA turnover. Agonist-induced suppression of
GnRH mRNA expression was prevented by pretreatment
with a GnRH antagonist, Antide, while Antide alone did not
affect GnRH mRNA expression (Fig. 4). Li and Pelletier
have shown that administration of Antide induced an increase
in GnRH mRNA expression, as determined by in-situ hybridization (24). They used hypophysectomized male rats, where
the GnRH ultrashort loop feedback might be hyper-active,
in the absence of short and/or long loop feedback. In support
of this view, they observed no decrease in GnRH mRNA
expression following injection of either GnRH or GnRH
agonist. Thus, the antagonist-induced increase in GnRH
mRNA expression may be due to blocking GnRHR which
mediates the negative feedback action of GnRH. In the
present study, we used OVX+E-treated rats where the negative feedback action of E on the GnRH activity can occur.
In this situation, blockade of GnRHR by Antide has little
effect on GnRH mRNA expression, whereas activation of
GnRHR by des-Gly GnRH is able to decrease GnRH mRNA
expression. The route through which the ultrashort loop
feedback mechanism operates remains uncertain. Although
GnRH neurones make synaptic contacts with each other in
the POA (25), it is unclear whether GnRH neurones express
GnRHR. It is possible that des-Gly GnRH reduces GnRH
mRNA expression in the POA through other neuronal connections. In addition, the possibility of LH short loop feedback cannot be excluded, as treatment with hCG decreased
GnRH mRNA expression in hypothalamic GT1-7 cells (26).
The reduction in pMBH GnRHR mRNA expression by
des-Gly GnRH was also rapid, and the dose-related and
time-course responses were similar to those of the decline in
POA GnRH mRNA expression. A significant reduction
in pMBH GnRHR mRNA expression was seen 2 h after i.c.v.
administration of des-Gly GnRH(6 ng), suggesting that
GnRHR mRNA has a relatively short half-life. This possibility is supported by the studies showing that GnRHR mRNA
expression was decreased by GnRH agonists within 2 h both
in the pituitary (16) and aT3–1 gonadotroph cell line (17).
The rapid changes of GnRHR mRNA expression both in the
MBH (11) and the pituitary (27) during the oestrous cycle
also suggest this possibility. These rapid changes in GnRHR
mRNA expression reveal the importance of transcriptional
regulation in the surface GnRHR fluctuation. Actually, it
has been shown that GnRHR mRNA expression altered in
parallel with GnRHR numbers (17, 27).
By contrast, the agonist-induced decrease in GnRHR
mRNA expression in the pMBH was not reversed by pretreat© 1999 Blackwell Science Ltd, Journal of Neuroendocrinology, 11, 195–201
ment with Antide (Fig. 4). Recently, it has been shown that
another potent GnRH antagonist, cetrorelix, decreased
GnRHR content as well as GnRHR mRNA expression in
the pituitary (28, 29). Similarly, in our study, Antide alone
decreased GnRHR mRNA expression in the pMBH. Thus,
Antide may have an intrinsic property to decrease GnRHR
mRNA expression, although it can block the agonist-induced
decrease in GnRH mRNA expression. In this case, GnRHR
mRNA expression in rats that received both des-Gly GnRH
and Antide might be expected to show additive or synergistic
changes. However, no such changes were observed. One
plausible interpretation is that 6 ng of des-Gly GnRH and/or
10 ng of Antide caused a maximal decrease in GnRHR
mRNA expression. As shown in Fig. 3(,) maximal decrease
in GnRHR mRNA expression occurred within 2 h of i.c.v.
injection, and no further decrease was observed until 8 h. At
present, the discrepancy between the effects of Antide on
GnRH mRNA and GnRHR mRNA expression cannot be
fully explained. It is also possible to consider the difference
in signal transduction cascades triggered by the two GnRH
analogues. For instance, in rat luteal cells, GnRH agonists
activated phospholipases A2 and C, but some GnRH antagonists activated only phospholipase A2 (30). The blockade
of a stimulatory effect of agonists on phospholipase C by
GnRH antagonists may result in the failure to activate protein
kinase C (PKC ), thus blocking the signal cascade necessary
for decreasing GnRH mRNA expression. In the case of
GnRHR expression, PKC may not be a mediator of homologous down-regulation. In fact, McArdle et al. (31) have shown
that depletion of PKC does not alter the ability of GnRH to
down-regulate its own receptors.
In the present study, we did not observe up-regulation of
GnRHR mRNA expression in the pMBH. This was not
unexpected, since the experimental model was chosen to
address the issue of homologous down-regulation, rather
than up-regulation of GnRHR mRNA expression. There are
high levels of GnRHR mRNA in OVX+E-treated rats, and
these up-regulated levels decreased dramatically at the time
of progesterone-induced LH (GnRH ) surge in OVX+Etreated rats (12). Administration of either des-Gly GnRH or
Antide in to the OVX+E-treated rats suppressed GnRHR
mRNA expression in the MBH. It appears that GnRH may
exert tonic stimulatory influence on GnRHR mRNA expression in the pMBH. Studies on GnRHR in the pituitary have
demonstrated both up-and down-regulation of GnRHR
mRNA expression subsequent to exposure to GnRH or
GnRH agonists. Intermittent stimulation of GnRHR which
mimics the pulsatile release of GnRH induced up-regulation
of GnRHR mRNA expression (14, 15, 18); whereas continuous exposure to GnRH or GnRH agonists, specifically at
high doses, caused a reduction in GnRHR mRNA expression
in the pituitary (16, 17).
In summary, the present study showing that GnRH mRNA
expression in the POA is down-regulated by a GnRH agonist,
suggests the presence of ultrashort loop feedback circuit for
GnRH neuronal activity at the level of gene expression.
Furthermore, the finding of homologous down-regulation of
GnRHR mRNA expression in the pMBH suggests a complex,
yet coordinated interaction in the GnRH neuronal apparatus.
200 GnRH and GnRH receptor gene expression by GnRH
Materials and methods
Animals and experimental design
Female Sprague-Dawley rats (10 weeks of age, Seoul National University
Animal Breeding Centre, Korea) were housed in temperature-controlled
conditions under 14 h light and 10 h dark photocycle ( light on at 06.00 h)
with food and water supplied ad libitum. All rats were ovariectomized under
ether anaesthesia. At the same day, under pentobarbital (7.5 mg/kg body
weight (b.w.)) and ketamine hydrochloride (25 mg/kg b.w.) anaesthesia, a
polyethylene tube (o.d. 1.05 mm, i.d. 0.35 mm) with inner stylet (27 gauge)
was stereotaxically implanted into a lateral ventricle (L 2 mm, P 2 mm, V
4 mm from the bregma point) (32) and fixed in place with anchor screws and
dental cement. After a recovery period of 1 week, Silastic capsules (30 mm
in length, o.d. 3.175 mm, i.d. 1.575 mm, Dow Corning, Silastic MedicalGrade Tubing, Midland, MI, USA) containing 17b-estradiol (180 mg/ml in
sesame oil ) were implanted at 10.00 h. Two days after 17b-estradiol implantation, the inner stylet was removed under light ether anaesthesia and GnRH
analogueue dissolved in 5 ml of saline was i.c.v. injected at designated times.
Experiment 1
This tested the dose–response effect of a GnRH agonist ([-Ala6, desGly10]GnRH-ethylamide, des-Gly GnRH, Sigma, St Louis, MO, USA) on
GnRH mRNA expression in the POA and GnRHR mRNA expression in
the pMBH. Des-Gly GnRH(0.6 or 6 ng) was i.c.v. injected at 15.00 h. The
control rats received 5 ml of saline. Rats were killed at 17.00 h.
Experiment 2
Experiment 2 examined the time-course effect of des-Gly GnRH on GnRH
and GnRHR mRNA expressions for 30 min, 2 h, 4 h, and 8 h after treatment.
Des-Gly GnRH (6 ng) was i.c.v. injected at 09.00 h, 13.00 h, 15.00 h, or
16.30 h. Control rats received 5 ml of saline at 15.00 h. Rats were killed
at 17.00 h.
Experiment 3
This experiment examined whether des-Gly GnRH exerts its effect through
the GnRHR. Ten ng of a GnRH antagonist, Antide ([N-Ac--Nal(2)1, pCl-Phe2, -Pal(3)3, Lys(Nic)5, -Lys(Nic)6, Lys(iPR)8, -Ala10]GnRH,
Sigma) was i.c.v. injected at 14.00 h 1 h prior to des-Gly GnRH injection.
Control rats received 5 ml of saline twice at 14.00 h and 15.00 h. Rats were
killed at 17.00 h.
Blood samples were collected to determine serum LH concentrations.
Brains were immediately frozen on dry ice and stored at −70 °C until use.
Brains were coronally sliced into 600-mm thick slices according to the atlas
of Paxinos and Watson (32) (for the POA slice: bregma (B) 0.20 mm to B
−0.40 mm; for the pMBH slice: B −2.60 mm to B −3.20 (mm). POA
and pMBH were punched from the frozen rat brain slices with a 1-mm
diameter stainless needle according to the method of Palkovits and
Brownstein (33).
mRNA isolation
mRNA was isolated from the micropunched samples using a Dynabeads
mRNA isolation kit (Dynal A.S., Oslo, Norway) as previously described (10,
19). Briefly, two micropunches of the POA and pMBH from individual rat
were sonicated in 100 ml of lysis buffer (100 mM Tris-HCl, pH 8.9, 500 mM
LiCl, 10 mM EDTA, pH 8.0, 1% SDS, and 5 mM dithiothreitol ), placed on
ice for 1 min, and briefly centrifuged. Dynabeads-oligo (dT ) (5 mg/ml, 30 ml )
were directly added to lysis-buffered samples, resuspended and then placed
for 10 min at room temperature. Using the Dynal Magnetic Particle
Concentrator, supernatant was removed and the mRNA captured by the
Dynabeads was washed twice with washing buffer (10 mM Tris-HCl, pH 8.5,
0.15 M LiCl, and 1 mM EDTA). The tube was then incubated at 65 °C for
3 min with 30 ml of elution buffer (2 mM EDTA, pH 7.5). Eluted mRNA
(3 ml ) was directly used as a RNA source for the RT reaction.
Oligonucleotides
GnRH primers were synthesized based on the sequences of the rat GnRH
cDNA (34). The upstream primer, 5∞-CACTATGGTCACCAGCGGGG3∞(20mer) is located at 5∞ end of the first exon and downstream primer,
5∞-AGAGCTCCTCGCAGATCCCTAAGA-3∞(24mer) is near the 5∞ end of
the third exon. Primers were designed such that the predicted sizes of PCR
products were 375 bp for native GnRH and 139 bp for mutant GnRH (19).
Primers were chosen to flank introns so that the amplified GnRH DNA was
readily distinguished from a possible contaminating genomic DNA.
GnRHR primers were synthesized based on the sequence of the rat GnRHR
cDNA (35). The 5∞ primer, 5∞-CTTGAAGCCCGTCCTTGGAGAAAT3∞(24mer) and downstream primer, 5∞-GCGATCCAGGCTAATCGCCGCCAT-3∞(24mer) were used for PCR. Primers were designed such that the
sizes of PCR products were 441 bp for native GnRHR and 280 bp for mutant
GnRHR (10).
Competitive RT-PCR
The pGEM4 vectors containing native or mutant GnRH cDNAs (19) were
linearized by digestion with Nhe 1. The pGEM4Z vectors containing native
or mutant GnRHR cDNAs (10) were cut by Pvu II. The native and deletion
mutant cRNAs were synthesized by T7 RNA polymerase as previously
described (19). The concentration of native and mutant GnRH and GnRHR
cRNAs were measured with UV spectrophotometer at A . Native and
260
mutant cRNA templates were coreverse transcribed by 100 unit of RNase
H− moloney murine leukaemia virus (MMLV )-reverse transcriptase
(Promega) in 10 ml of reaction mixture containing 25 pmol random hexamer,
8 units of RNase inhibitor, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM
MgCl , 10 mM DTT, and 1 mM dNTP. The RT reaction was carried out at
2
37 °C for 60 min followed by 5-min period at 94 °C. Subsequently, 40 ml of
PCR reaction mixture containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl,
1.5 mM MgCl , 5 pmol of upstream and downstream primers, and 2.5 units
2
of Taq DNA polymerase (Perkin Elmer Cetus Corp., Branchburg, NJ, USA)
was added. Since native and mutant cRNAs were coamplified forming hybrids
between native and mutant DNA, PCR amplification was carried out with a
two-step procedure to prevent hybrids formation (first step: denaturation at
94 °C for 50 s, primer annealing at 60 °C for 1 min, and primer extension at
72 °C for 1 min 40 s; second step: denaturation of hybrids only at 87 °C for
50 s, primer annealing at 60 °C for 1 min, primer extension at 72 °C for 1 min
40 s). To detect GnRH mRNA from the POA and GnRHR mRNA from
the pMBH, the 35 (for GnRH mRNA) or 40 (for GnRHR mRNA) cycles
of the first step and five cycles of the second step were used. After the second
step, further primer extension at 72 °C for 10 min was performed. Six ml
aliquots of PCR products were electrophoresed on 2% agarose gel in TAE
buffer (0.04 M Tris-acetate, 0.001 M EDTA), stained with ethidium bromide,
and photographed under UV illumination with Polaroid 665 type negative
and positive films. Negative film was used for densitometric scanning of
native and mutant signals.
Radioimmunoassay of serum LH
Blood LH concentrations were assayed using a double antibody radioimmunoassay reagent kindly provided by the National Pituitary Agency. The tracers
NIADDK-rLH-I9 were iodinated by the chloramine-T method. The antiserum
was NIADDK-rLH-S-10 and the reference preparation was NIADDK-rLHRP-2. LH concentrations were expressed as NIADDK RP2 units. The intraand interassay coefficients of variation were #7.6 and 10.0%, respectively.
Data analysis
GnRH and GnRHR signals on Polaroid 665 negative film (Polaroid,
Cambridge, MA, USA) were measured with a densitometric scanner (Hoefer,
San Francisco, CA, USA). The amounts of GnRH and GnRHR mRNA
levels were calculated from the ratio of native to mutant on the basis of each
standard curve and expressed mean±SEM. Statistically significant differences
between groups were determined by one-way anova followed by Fisher’s least
significant difference test for a post-hoc comparison. Statistical significance
was set at P<0.05.
Acknowledgements
This work was supported by grants from the Korea Science and Engineering
Foundation ( KOSEF ) through the Research Centre for Cell Differentiation.
Accepted 17 August 1998
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