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, But Not ERß, Gene Is Expressed in Growth Hormone-Releasing Hormone Neurons of the Male Rat Hypothalamus1
Department of Medicine, Nippon Medical School, Tokyo 113-8603, Japan
Address all correspondence and requests for reprints to: Jun Kamegai, M.D., Department of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-Ku, Tokyo 113-8603, Japan. E-mail: jkamegai{at}nms.ac.jp
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Abstract |
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(ER) and ERß genes. In the medial basal hypothalamus of adult male
rat, the ER messenger RNA (mRNA) was located in medial preoptic area
(MPA) and arcuate nucleus (ARC), whereas ERß mRNA was detected in
MPA, supraoptic nucleus, and paraventricular nucleus. From studies
using adjacent sections, the distribution of ER mRNA-containing
cells appeared to overlap in part with those of GHRH and SS expressing
cells only in the ARC. On the other hand, the distribution of ERß
mRNA-containing cells does not appear to overlap with GHRH cells or SS
cells. The double label in situ hybridization studies
showed that in the ARC, 70% of GHRH neurons contain ER mRNA,
whereas less than 5% of SS neurons expressed the ER gene. These
results indicated that GHRH neurons are direct target cells for
estrogens, and estrogens may act directly on GHRH neurons through ER
during adult life to modify GH secretory patterns. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1) and ERß (2). ER has been shown to be
expressed in medial preoptic area (MPA), periventricular nucleus (PeV),
arcuate nucleus (ARC), and ventromedial nucleus (VMN) of the female rat
hypothalamus, where ERß has been detected in MPA, paraventricular
nucleus (PVN), supraoptic nucleus (SON), and ARC (2, 3, 4, 5, 6).
These differential expression patterns suggest that ER and ERß may
have unique functions which is supported by in vitro studies
where estrogen analogs have been shown to preferentially activate the
two ERs (7). These observations, coupled with the fact
that ERß can form homodimers and heterodimers with ER in
vitro (8, 9), suggest the activity of estrogens may
depend on whether a cell contains ER, ERß, or both.
There is a striking sex difference in the pattern of GH secretion in
rats (10). Male rats secrete GH in regular bursts at
3–4-h intervals, whereas the GH secretory pattern in female rats is
characterized by smaller and more frequent pulses and higher
intervening GH concentrations (10). These sexual dimorphic
GH secretory patterns have been shown to be responsible for differences
between male and female rats in body growth, hepatic steroid metabolism
and PRL receptor concentration (11, 12, 13, 14). Early reports
indicate that exposure to or deprivation of gonadal steroid hormones
during the neonatal period causes permanent alterations of the adult GH
secretory profile, suggesting that gonadal steroids have important
imprinting effects during the neonatal period (11, 15).
Recently, we have reported that short-term (6 h)
administration of a small dose (0.01 mg/rat) of dihydrotestosterone
(DHT), a nonaromatizable androgen, masculinized the GH secretory
pattern in ovariectomized rats. This masculinization of the GH
secretory pattern was not easily attained even after the administration
of a pharmacological dose of DHT to intact adult female rats
(16). In addition, Paison and colleagues have been
reported that short-term (12 h) administration of estradiol to
gonadectomized adult male rats feminized the male pattern of
spontaneous and GH-releasing hormone (GHRH) stimulated GH secretion
(17). These data indicate that gonadal steroids are
important determinants of the adult pattern of GH secretion during
adult life. However, the mechanisms by which gonadal steroids modulate
the GH axis of the adult rat are not clear. Therefore, in the present
study we used double label in situ hybridization in the
adult male rat brain to determine whether GHRH neurons and/or
somatostatin (SS) neurons coexpress the ER or ERß gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Probe preparation
The rat ER riboprobe plasmid was obtained from Dr. Koike S
(1). The plasmid consisted of a 1.9 kb complementary DNA
(cDNA) inserted into pBluescript II SK (+). After BamHI was
used to linearize the cDNA, the 35S-labeled
antisense riboprobe was generated through the use of T7 RNA polymerase
and an SP6/T7 transcription kit (Roche Molecular Biochemicals Yamanouchi, Tokyo, Japan). The rat ERß riboprobe
plasmid was obtained from Dr. G. Kuiper (2). The
plasmid consisted of a 2.6-kb cDNA inserted into pBluescript KS. After
NotI was used to linearize the cDNA, the
35S-labeled antisense riboprobe was generated
through the use of T3 RNA polymerase (Roche Molecular Biochemicals, Yamanouchi, Japan). Digoxigenin- or
35S-labeled GHRH and SS probes were prepared as
previously described (18). The rat GHRH cDNA (a gift from
Dr. K. E. Mayo) was constructed into the
EcoRI/PstI site of pBluescript II SK (+), and the
rat SS cDNA (a gift from Dr. M. R. Montminy) was constructed into
the HindIII/EcoRI site. To produce antisense and
sense RNA probes for GHRH messenger RNA (mRNA), the plasmid was
linearized with EcoRI and PstI, respectively. To
produce antisense and sense RNA probes for SS mRNA, the plasmid was
linearized with HindIII and EcoRI, respectively.
Radioactive and digoxigenin-labeled RNA copies were synthesized from
linearized plasmids with 35S-UTP and
digoxigenin-UTP, respectively, using an SP6/T7 transcription kit. The
synthesized RNA copies, 0.4 kb (GHRH and SS) in length, were
precipitated with ethanol and solubilized in 1 x TE.
Single label in situ hybridization
Frozen coronal sections, 30 µm thick, were cut with a
cryostat, mounted onto Silan-coated slides (Shin-Etsu Chemical Co.,
Tokyo, Japan), and air-dried. The medial basal hypothalamus from the
rostral end of the third ventricle to the caudal end of ARC was
processed for in situ hybridization. The hybridization
protocol was described previously (18). In brief, sections
were dried, digested by proteinase K (10 µg/ml), and acetylated with
0.25% acetic anhydride. Sections were rinsed briefly in 2x saline
sodium citrate (SSC), then air-dried. The probe was dissolved in
hybridization buffer (10 mM Tris, pH 8.0,
containing 50% formamide, 10% dextransulfate, 1x Denhardt’s
solution, 12 mM EDTA, 30 mM
NaCl, 0.5 mg/ml yeast transfer RNA, and 10 mM
dithiothreitol). Thereafter, 2 x 106 dpm of
a probe in 100 µl buffer were applied to each slide and hybridized at
55 C overnight. The slides were rinsed in 4 x SSC and digested
with ribonuclease-A (20 µg/ml) for 30 min, followed by two 5-min
changes of 2 x SSC, 10 min in 1 x SSC, 10 min in 0.5
x SSC at room temperature, and 30 min in 0.1 x SSC at 55 C. The
slides were dehydrated in ethanol and then air dried. These sections
were dipped in Konica NR-M2 autoradiography emulsion (Konica, Tokyo,
Japan), exposed for 10 days (ER and ERß mRNA) or 5 days (GHRH and
SS mRNA), and developed. The sections were counterstained with cresyl
violet. As a control for nonspecific labeling, sense ER, ERß,
GHRH, and SS probes generated by T3,
T7, T3, and
T3 RNA polymerases, respectively, were used on
some adjacent sections from experimental animals and no specific signal
was detected.
Double label in situ hybridization
Serial frozen coronal sections, 6 µm thick at 30-µm
intervals, were cut with a cryostat, mounted onto Silan-coated slides,
and air-dried. The medial basal hypothalamus from the rostral end of
the third ventricle to the caudal end of the ARC was processed for
in situ hybridization. The hybridization protocol was
described previously (18). The
35S-labeled ER complementary RNA (cRNA)
probe was mixed with either the digoxigenin-labeled GHRH or SS cRNA
probe in hybridization buffer (2 x 106 dpm
35S-labeled probe and 50 ng digoxigenin-labeled
probe in 100 µl buffer). The sections were then coverslipped and
hybridized at 55 C overnight. The coverslips were removed, and the
slides were rinsed in three 5-min changes of 4 x SSC and digested
with ribonuclease-A (20 µg/ml) for 30 min, followed by two 5-min
changes of 2 x SSC, 10 min in 1 x SSC, 10 min in 0.5
x SSC at room temperature, and 30 min in 0.1 x SSC at 55C. The
slides were then washed briefly in buffer A (100
mM Tris-HCl, pH 7.5, and 150
mM NaCl) and incubated for 30 min at room
temperature in blocking reagent (Roche Molecular Biochemicals). They were then washed briefly with buffer A and
incubated for 3 h at 37 C in antidigoxigenin alkaline
phosphatase-conjugated antibody (Roche Molecular Biochemicals), diluted 1: 1000 in buffer A. They were rinsed in
two 15-min changes of buffer A and for 2 min in buffer B (100
mM Tris-HCl (pH 9.5), 100
mM NaCl, and 50 mM
MgCl2) at room temperature. The slides were then
incubated for 3 h at 37 C in buffer B with 0.34 mg/ml nitroblue
tetrazolium salt (Roche Molecular Biochemicals), 0.18
mg/ml 5-bromo-4-chloro-3-indolyl phosphate toluidinium salt
(Roche Molecular Biochemicals), and 240 µg/ml
levamisole. The chromogen reaction was halted by rinsing the slides in
10 mM Tris-HCl, pH 8.0, and 1.0
mM EDTA. The slides were placed in 70% ethanol
for 15 sec and then air dried. The slides were dipped in 60% Collodion
(Wako Chemical Co., Tokyo, Japan) dissolved in isoamyl acetate, air
dried, and dipped in autoradiography emulsion. After 10 days of
exposure, the slides were developed and mounted in an aqueous mounting
medium. To assess the nonspecific labeling with digoxigenin-labeled
probes, sense RNA probes were used on some adjacent sections from
experimental animals and no specific signal was detected.
Data analysis
To estimate the percentage of GHRH cells or SS cells
coexpressing the ER gene, the purple-stained digoxigenin-labeled
GHRH or SS cells were first isolated under brightfield illumination. To
identify cells that had been labeled by the digoxigenin color reaction,
we followed some of the criteria described by Marks et al.
(19). As 6-µm thick sections were used in the present
study, whole cells plus partial cells that contained the nucleus were
separated from those containing only a small fraction of the cell body.
ER mRNA levels were quantified by counting the autographic grain
number on a single cell using emulsion autography of in situ
hybridization. A 20-µm square grid was fixed on grains overlying the
cell, and the grain number within the grid was counted under
brightfield illumination for a high power field (x1,000). Counting the
grain numbers was performed by an operator unaware of the treatment’s
group assignment (i.e. GHRH/ER or SS/ER), as
previously described (20). The background was assessed by
counting grain numbers in a grid placed over perikarya-free regions of
the ARC. Cells were considered specifically labeled if the grain
density was at least three times the background level. Using the rat
brain atlas of Paxinos and Watson (21) as an anatomical
guide, we divided the ARC into three regions of approximately equal
length in the rostral-caudal plane, as previously described
(20). The rostral part started where the beginning of ARC,
and the middle with the rostral border of the dorsomedial nucleus. The
end of the caudal part was the end of ARC. The middle and caudal parts
were formed by dividing the area from the start of the middle part to
the end of the caudal part into two equal parts. For each rat, seven
sections from each part of the ARC (a total of 21 sections/animal) were
used for the analysis. Four rats were used for the analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß mRNA distribution mRNA was located in distinct cell populations in the
medial basal hypothalamus of adult male rat. Intense hybridization
signal of ER mRNA was observed in MPA and ARC (Fig. 1A
). However, PeV and VMN, area with
abundant ER mRNA in the ovariectomized female hypothalamus
(6), contain only few signals of ER mRNA (Fig. 2A). To estimate the localization of
neurons expressing the ER gene, adjacent sections from the same rat
were processed for in situ hybridization for GHRH and SS
mRNA (Fig. 1, A, C, and D). In the ARC, ER mRNA (Fig. 1A) was seen
in the ventrolateral region of the ARC, where GHRH mRNA signals (Fig. 1C) were observed, and the dorsomedial region of the ARC, where SS mRNA
signals (Fig. 1D) were observed. In these areas, the distribution of
these cells appeared to overlap in part with that of cells containing
ER mRNA. On the other hand, in other areas of the medial basal
hypothalamus than the ARC, the distribution of ER mRNA-containing
cells does not overlap with that of GHRH cells or SS cells.
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). However,
few positive cells for ERß mRNA were observed in the ARC (Fig. 1B),
where ERß mRNA is observed in ovariectomized female rat hypothalamus
(6). The distribution of ERß mRNA-containing cells in
these areas do not appear to overlap with GHRH cells or SS expressing
cells (Figs. 1, B–D, and 2, B and C).
Colocalization of ER mRNA and GHRH or SS mRNA
To determine if GHRH mRNA or SS mRNA is coexpressed with ER
mRNA in ARC neuron, tissue section were simultaneously hybridized with
35S-UTP labeled-ER cRNA probe and
digoxigenin-labeled GHRH cRNA or SS cRNA probe. Figure 3 shows a representative coronal section
of the ARC processed through double label, in situ
hybridization for ER mRNA and GHRH (Fig. 3, A and B) or SS (Fig. 3, C and D) mRNA, respectively. Few SS neurons in the ARC coexpressed
ER mRNA (Fig. 3, C and D). When assessed, less than 7% of SS
neurons that hybridized as positive as for SS mRNA were also positive
for ER mRNA in the ARC. In contrast, 70% of GHRH gene-expressing
cells in the ARC expressed the ER gene, and there was no significant
regional variation through the rostral-caudal parts of the ARC (Table 1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA, whereas GHRH neurons do
not contain ERß mRNA. We also found that few SS cells in PeV and ARC
of the male rat hypothalamus express ERs. These results indicated that
estrogen may act directly on GHRH neurons through ER during adult
life to modify GH secretory pattern.
In adult male rat, our study showed that the expression of ERs was
observed in restricted regions of the medial basal hypothalamus. These
observations are in agreement with earlier studies demonstrating
region-specific expression of ERs by in situ hybridization
using female rat hypothalamus (5, 6). However, the
specific pattern of hybridization signal for ERs appear to be different
between genders. For example, in the PeV, an area with abundant ER
mRNA in the ovariectomized female hypothalamus (6), only
few neurons contains signals for ER mRNA in male. In addition, few
positive cells for ERß mRNA was observed in the ARC of male rat
hypothalamus, where ERß mRNA is observed in ovariectomized female
(6). These data are consistent with previous reports that
ER levels are higher in females than males using in vitro
binding assays (22). Thus, our morphological data suggest
that there may be a sexual dimorphism in ER expression as well as in
ERß expression. We also demonstrated that, in ARC and PeV, where GHRH
and SS neurons are located, the distribution of ER mRNA-containing
cells do not overlap with that of ERß mRNA-containing cells,
suggesting ER and ERß are not typically colocalized within a
single neuron of the ARC and PeV. Although the ability of the estrogen
receptor subtypes to heterodimerize is known (8, 9), these
findings suggest that the majority of GHRH neurons expressing ER in
the ARC may act specifically with its homodimer form.
It has been reported that the existence of numerous estrogen targets
cells in rat hypothalamus detected by in vivo
autoradiography using radiolabeled ligand (23). Our
results showed that 70% of GHRH gene-expressing cells in the ARC
expressed the ER gene, and there was no significant regional
variation through the rostral-caudal parts of the ARC. These findings
are in agreement with a previous report that a subpopulation of
immunoreactive GHRH-containing neurons takes up
[3H]estradiol in the female rat
(24). However, in the same report, 20–30% of GHRH
containing neurons showed nuclear concentration of
[3H]estradiol in the central portion of ARC,
whereas 10–15% of GHRH neurons showed it in the anterior portion of
ARC (24). It remains to be determined if this discrepancy
is due to differences in 1) experimental animal model (intact adult
male rat vs. ovariectomized female rats) or 2) parameters
studied (ER mRNA; in situ hybridization vs.
estrogen binding site; in vivo autoradiography). We also
demonstrated that few SS cells in PeV and ARC of the male rat
hypothalamus contains ERs. These observations are in agreement with
earlier reports that SS-immunopositive neurons including those in the
PeV do not express ER (25, 26). These results taken
together with the fact that no estrogen-receptor binding consensus
sequence exists in the SS gene promoter (27) suggest that
the direct target cells for estrogens in the hypothalamus are not SS
but GHRH neurons. Specifically, estrogen may be involved in the
regulation of GH secretion in the male rat by acting on GHRH neurons
through ER. Because GHRH neurons are located near aromatase
expressing cells (28), it should be noted that the
possibility exist that androgens locally aromatized to estrogens could
also modulate GHRH neuronal function.
The mechanisms by which estrogen exerts its effects on GH secretion through GHRH neuron are largely unknown. There are several reports that suggest that gonadal steroids modulate GHRH mRNA, whereas others suggest the opposite. Estrogen administration reduced hypothalamic GHRH mRNA content in male rats (29). In contrast, hypothalamic GHRH mRNA is not altered by gonadectomy or pharmacological sex steroid replacement therapy in adult male and female rats (30, 31). Furthermore, it is also reported that orchidectomy plus estrogen replacement significantly increases GHRH concentration of the hypothalamus (32), whereas there is no difference in GHRH peptide concentrations between two sexes (33, 34). Thus, at present it is not clear whether GHRH concentrations and synthesis are altered by the gonadal steroids. Alternatively, the possibility of direct estrogen’s effect on GHRH release cannot be excluded. Indeed, although no direct evidence is available that demonstrates differences in GHRH release between male and female rats, indirect evidence based on passively immunized rats suggests that baseline GHRH secretion is slightly elevated with low amplitude GHRH pulses in females, and that baseline GHRH values are lower with higher amplitude GHRH pulses in male (35, 36, 37). In addition, it is also reported that estrogen has a membrane effect other than genomic effect (38, 39). 17ß-estradiol has immediate actions on median eminence endothelial cells via nongenomic signaling pathways to leading to nitric oxide-stimulated GnRH release (40). In this regard, the acute effect of estrogen on GHRH neuron may be through nongenomic mechanisms of estrogen actions.
In conclusion, we demonstrate that the majority of GHRH neurons in ARC
have ER, but not ERß, and few SS cells in PeV and ARC have ER
or ERß in adult male rat, suggesting that GHRH neurons are direct
target cells for estrogens. Based on these findings, we suggest that
GHRH may play a crucial role in determining GH secretory pattern in
adult rats. Although functional studies are necessary for full
understanding of ERs function to modulate GH secretory pattern, gonadal
environment can have a major impact on GH secretion during adult life.
In addition, because there is a discrepancy in the PeV localization of
ER mRNA containing cells between in male and in female brains, it
may give us further information to perform the colocalization study in
female rat.
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Acknowledgments |
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cDNA and Dr. G. Kuiper for
providing ERß cDNA. |
Footnotes |
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Received July 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß in the rat pituitary gland detected by
immunohistochemistry. Endocrinology 141:615–620 and-ß mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
and ERß at AP1 sites. Science 227:1508–1510
and ß form heterodimers on DNA. J Biol
Chem 272:19858–19862. Mol Endocrinol 11:1486–1496This article has been cited by other articles:
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