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and ß in Rat Pituitary Gland Detected by Immunohistochemistry
Department of Nature Medicine, Atomic Bomb Disease Institute (E.N., S.Y.), and the Departments of Pharmacology I (Y.N.) and Histology and Cell Biology (T.K.), Nagasaki University School of Medicine, Nagasaki 852-8523; and the Department of Biochemistry, Saitama Medical School (S.I., H.H., M.M.), Saitama 350-0495, Japan
Address all correspondence and requests for reprints to: Eijun Nishihara, M.D., Department of Nature Medicine, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, Sakamoto 1–12-4, Nagasaki 852-8523, Japan. E-mail: eijun-ngs{at}umin.ac.jp
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Abstract |
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and ERß) in the pituitary using specific antibodies,
monoclonal antibody (1D5) for ER and polyclonal antibody generated
against ERß. First, we confirmed the detection of 66- and 55-kDa
bands for ER and ERß, respectively, in the rat pituitary extract
by Western blotting. Then immunostaining with these antibodies was
performed using fetal and adult Wistar rat tissues, combined with PRL
or LHß immunohistochemistry. Intense ERß signal was detected
throughout the pituitary from day 12 of gestation. However, staining
for ER only became detectable from day 17 of gestation. In contrast
with the fetal period, nuclei stained for ER were widely distributed
in the anterior lobe in the adult rat, whereas ERß-positive cells
were restricted in the anterior lobe. LHß, but not PRL, was
colocalized in ERß-positive cells. Our results indicated that the
major population of ER subtypes in the rat pituitary gland has changed
around the day of birth and that the expression of ERß may be
involved in the differentiation of pituitary cell function to
synthesize a specific hormone. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(original type) in the pituitary has been investigated in
detail by using immunohistochemistry (5, 6, 7), in situ
hybridization (8, 9), and RT-PCR (10). ER messenger RNA (mRNA) has
been demonstrated in most cells of the intermediate and anterior lobes
of the pituitary by in situ hybridization (8). Furthermore,
ER immunoreactivity has been identified in many anterior pituitary
cell types, including gonadotrophs and lactotrophs (6, 7). After cloning a novel ER, ERß (11), Kuiper et al. (12) reported a low expression of ERß mRNA in the adult rat pituitary by RT-PCR. Although the distribution of ERß mRNA in the adult rat pituitary has been recently examined by in situ hybridization (13, 14, 15), it remains to be determined whether the expression of ERß mRNA is restricted to the anterior lobes or to both anterior and intermediate lobes. In addition, the localization of ERß protein in the pituitary remains to be determined.
Wilson et al. (13) recently reported that the expression level of ERß mRNA in the prepubertal rat pituitary is higher than that in the adult rat. Although the fetus in placental mammals is exposed to relatively high levels of maternal and placental estrogens (16), it is not clear whether ERß is expressed in the rat fetal pituitary and whether the expression pattern changes during fetal life.
The functional roles of ERß have been recently reported by
analyzing ERß target cells in the adult rat pituitary. For example,
colocalization of ERß in gonadotrophs and lactotrophs has been
demonstrated using dual immunocytochemistry/in situ
hybridization (13, 14). However, it is still controversial that
gonadotrophs are the direct target cells for ERß. Although ERß can
form homodimers and heterodimers with ER in vitro (17),
Mitchner et al. (14) reported only a small percentage of
ERß colocalizes with ER in the pituitary.
In the present study we examined the cellular distribution of ER and
ERß using immunohistochemistry with autoclave-antigen retrieval in
sections of fetal and adult rat pituitary glands. The antibody for
ERß was raised by immunizing rabbits with a synthetic peptide of a
part of rat ERß, and the specificity of this antibody was confirmed
by Western blotting. Our results showed a dramatic change in the major
ER population from ERß to ER in the pituitary around the day of
birth. Finally, in combination with immunohistochemistry for PRL and
LHß, we demonstrated a significant role for ERß in the gonadotroph,
but not in the lactotroph.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of antibody
ER was detected with 1D5 antibody (DAKO Corp.,
Glostrup, Denmark). For the detection of ERß, a peptide
(CSSTEDSKNKESQNLQSQ) corresponding to the C-terminal amino residues of
rat ERß (11) was conjugated to keyhole limpet hemocyanin. Then, the
rabbit polyclonal antirat ERß antiserum was generated by immunizing
rabbits followed by purification as described previously (18). Specific
polyclonal antibody to PRL was obtained from Biogenesis
(Bournemouth, UK), and antibodies to GH, TSH, and LHß were gifts from
Dr. Wakabayashi (Gunma University, Maebashi, Japan).
Western blotting
Pituitary and ovarian tissues from 8-week-old rats were excised
and immediately frozen, followed by preparation for sampling, as
previously described (14). Thirty micrograms of each protein were
separated on SDS-PAGE (7.5% polyacrylamide gels) and then transferred
to nitrocellulose membranes by electroblotting. After blocking with
10% nonfat milk in Tris-buffered saline (TBS) buffer overnight at 4 C,
blots were incubated for 1 h with the monoclonal antibody against
ER at a 1:500 dilution or the polyclonal antibody against ERß at a
1:100 dilution in TBS/0.05% Triton X buffer. The membranes were
subsequently washed three times for 10 min each time with TBS/0.05%
Triton X buffer. Each membrane was treated with either goat antirabbit
or antimouse IgG-peroxidase-conjugated secondary antibody (MBL, Nagoya,
Japan) at a 1:200 dilution in 10% nonfat milk in TBS buffer for 1
h and was again washed six times for 15 min each time with TBS/0.05%
Triton X buffer. Finally, immunopositive bands were visualized with
H2O2,
3,3'-diaminobenzidine/4HCl (DAB; Wako Pure Chemicals, Osaka, Japan),
Co2+, and Ni+ according to
the method of Adams (19).
Immunohistochemistry
Tissues were fixed with 4% paraformaldehyde in PBS for 12
h, processed for 24 h in a tissue processor, and embedded in
paraffin. Each 5-µm thick tissue section was cut and mounted on
silane-coated slides. For ER and ERß immunohistochemistry,
sections were dewaxed, rehydrated, and autoclaved at 120 C for 10 min
in 10 mM citrate buffer (pH 6.0) (20). After washing in
PBS, endogenous peroxidase was blocked using 0.3%
H2O2 in methanol (15 min).
Slides were washed in PBS again and preincubated with 500 µg/ml goat
IgG and 5% BSA in PBS for 60 min at room temperature to reduce
nonspecific binding of antibodies. Sections were then reacted with the
primary antibody diluted at 1:200 for ER and at 1:100 for ERß
overnight at room temperature. After washing in 0.075% Brij 35
(Sigma, St. Louis, MO) in PBS, sections were incubated
with horseradish peroxidase (HRP)-goat antimouse IgG or HRP-goat
antirabbit IgG at a 1:100 dilution for 2 h at room temperature,
respectively. After washing in 0.075% Brij 35 in PBS, the sites of HRP
were visualized by DAB,
H2O2,
Co2+, and Ni+ using methyl
green as a counterstain.
For GH, TSH, LHß, and PRL immunohistochemistry, sections
were dewaxed and rehydrated, and endogenous peroxidase was blocked,
using the same method described above for ER and ERß
immunohistochemistry. After washing with PBS and blocking, sections
were reacted with each primary antibody diluted at 1:1000 for 1 h
at room temperature. After washing in 0.075% Brij 35 in PBS, sections
were incubated with HRP-goat antirabbit IgG at a 1:100 dilution for
2 h at room temperature. After washing in 0.075% Brij 35 in PBS,
sites of HRP were visualized by DAB and
H2O2 .using methyl green as
a counterstain.
Negative controls were prepared by reacting a few sections with normal mouse IgG or normal rabbit IgG at the same dilution instead of the specific antibody.
Image analysis
Photographs of immunostained sections around sinusoid vessels in
the anterior lobes of the pituitary were selected from separate
sections from three adult male rats. After selecting the area of tissue
section stained for ERß, the corresponding area in mirror sections
stained for ER, PRL, and LHß was photographed. One negative
control section for each paired section was also photographed. The
percentages of ER, PRL, and LHß with ERß colocalized in the
pituitary were calculated among 100 cells, which were not stained in
the negative control, for each animal.
Statistical analysis
All data were expressed as the mean ± SD.
Differences between groups were examined for statistical significance
using Student’s t test. P < 0.05 denoted
the presence of a statistically significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß and
ERß, Western blot analysis of extracts from the rat ovaries, which
are known to express both ER and ERß, was performed. As shown in
Fig. 1
, 66- and 55-kDa bands were
detected with anti-ER antibody (1D5) and anti-ERß antibody,
respectively. When the extract of the adult rat pituitary was analyzed
by Western blotting in the same way, we also found 66- and 55-kDa bands
for ER and ERß, respectively. When the first antibody was omitted,
no bands were observed (data not shown).
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and ERß in positive tissues, uterus and prostate, respectively.
Staining for ER was detected in the nuclei of glandular epithelium
and stromal cells in the endometrium of the uterus (data not shown),
whereas ERß immunoreactivity was detected in nuclei of secretary
epithelial cells of the prostate (Fig. 2A).
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and ERß. ER immunoreactivity was not
detected in Rathke’s pouch from E12 to E14 (data not shown), but
appeared in a small population of anterior lobe cells on E17 (Fig. 3, A and B). In the adult male rat
(8-week-old), nuclear staining for ER was detected in a large
population of anterior lobe cells, but was not detected in the
intermediate lobe or posterior lobe (Fig. 4). In contrast, the signal for ERß was
detected as intensely stained dots inside Rathke’s pouch on E12 (Fig. 3C). Furthermore, immunopositive cell regions changed during
development from the internal side of Rathke’s pouch to the anterior
and posterior lobes (Fig. 3, C and D–F). In other regions of the
brain, the choroid plexus and olfactory epithelium were also positive
for ERß from E17 onward. On P1, staining of the choroid plexus did
not change, but olfactory and nasal epithelia showed more abundant
signals, and the taste buds also became positive for ERß (data not
shown).
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), with fewer such cells in the intermediate
lobe. In the posterior lobe, ERß-positive cells were also fewer than
in the anterior lobe, and most staining shown in Fig. 4 represented
nonspecific signals in nerve fibers. Interestingly, the signal for
ERß, unlike that for ER, was distributed in the nucleus,
perinuclear region, and cytoplasm in positive cells in the fetal
pituitary (Fig. 2, B and C). However, in the adult pituitary, it was
detected mostly in the nuclei (Fig. 2D). For both ERs, no
differences in the expression pattern were detected between male and
female rats. Hormonal expression, including GH, PRL, TSH, and LHß, in the pituitary was detected from P1 (data not shown). The largest population among pituitary cells was GH cells, and the smallest one was LHß cells. However, these hormonal expressions could not be detected until E17, in which all sections were just adjacent to the sections examined for ERß expression.
To determine the cell types that coexpressed ERß in the pituitary of
male adult rats, we performed immunohistochemistry for ERß, ER,
LHß, and PRL proteins in mirror sections (Fig. 5). Measurement of the percentage of
cells that colocalized ERß among LHß- or PRL-positive cells in the
anterior lobes showed that 67.2 ± 5.3% (mean ±
SD; n = 3) of LHß- and 11.2 ± 0.2% (n =
3) of PRL-positive cells coexpressed ERß protein, and the difference
was statistically significant (P < 0.001). In
addition, the percentage of ER-positive cells in the anterior lobes
that colocalized ERß protein were 20.6 ± 2.1% (n =
3).
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, G and H–J) and in the adult period (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
ERß in the fetal and adult rat pituitary immunohistochemically. The
major findings of our study were the following. 1) The expression of
ERß protein in the fetal rat pituitary gland is higher than that of
ER. 2) The major population of ER subtypes has changed in the
pituitary around the day of birth. 3) The cellular distribution of
ERß is not clear during fetal life. 4) In the adult pituitary, the
expression of ERß protein is largely confined to gonadotroph
cells. Estrogen binding is detected in the rat brain from E21 (21) and in the mouse pituitary from E17 (22). The rat glucocorticoid receptor mRNA, such as the nuclear hormone receptor ER, is present in Rathke’s pouch from E13 (23). In the mouse fetus, various transcription factors, such as Ptx1, Prop1, Pit1, and SF1, present in Rathke’s pouch from about E12 influence the development of pituitary gland (24). Therefore, in our study we analyzed the expression of ERs in the rat pituitary from E12 to adulthood by immunohistochemistry.
First, we showed that the expression of ER in the fetal pituitary
was lower than that during the adult period. Furthermore, we showed
that the expression of ER was detected in the nuclei of the anterior
lobe cells from E17, demonstrating that this event occurs in an earlier
period of fetal life than that was previously reported (25). In
contrast, a high expression of ERß was detected in Rathke’s pouch
from E12, and the distribution of ERß spread from inside Rathke’s
pouch to the anterior and posterior lobes during the fetal period. This
finding is in agreement with that reported in the human midgestational
fetus, where high amounts of ERß mRNA were described in the pituitary
by RT-PCR (26). The distribution of ERß in the adult was mainly
restricted in clusters in the anterior lobe. These findings suggest
that the main ER subtypes in the rat pituitary change around the day of
birth. The exact role of ERß in the pituitary during the fetal period
and the functional significance of high expression remain unknown at
present. Furthermore, the mechanisms leading to the change in the major
ER population from ERß to ER are not clear.
In the adult, our study showed that the expression of ERß was
restricted to the anterior lobes. Gonadotrophs are distributed in
clusters around sinusoid vessels in the anterior lobe, including the
sex zone (27). Our results showed that the distribution of ERß was
similar to that of the gonadotroph in the adult pituitary. We also
analyzed the adult pituitary cell types that colocalize ERß and LHß
or PRL. Our results showed that 67% of LHß-positive cells were also
positive for ERß, whereas only 11% of PRL-positive cells were
positive for ERß. These findings suggest that the direct target cells
for ERß in the pituitary are more gonadotrophs than
lactotrophs. In agreement with this finding, Wilson et
al. (13) reported that although ERß mRNA was not expressed in
the lactotrophs, it was expressed in 85% of FSH-positive cells.
Although ERß/ER heterodimers are preferentially formed over ERß
homodimers (17), only 21% of ER-positive cells, which were widely
spread in the anterior lobes of adult rats, coexpressed ERß. These
findings suggest that the majority of cells expressing ERß may act
specifically with ERß homodimers in the pituitary.
In our studies, ERß immunoreactivity in the fetal pituitary was
detected in both the nucleus and cytoplasm, whereas ER
immunoreactivity was limited to the nuclei of anterior lobe cells. As
we confirmed that ERß was solely localized in the nuclei of rat ovary
and uterus using the same antibody, our findings could be considered a
specific feature of ERß distribution in pituitary cells. Moreover,
from around P1, we detected GH, PRL, TSH, and LHß expression in the
pituitary and the major population among these hormones in this period
was not LHß-positive cells, in accordance with previous reports (28, 29). In contrast, ERß expression has been detected from E12.
Considering the close connection of LHß cells with ERß expression
in the adult, ERß might play different roles in the pituitary during
the fetal and adult periods.
In summary, the specific antibodies for ERs used in our study are useful for identifying both forms of ERs in the fetal and adult rat pituitaries. Our results of ER subtype change in the major population around the day of birth may play a role in the differentiation of pituitary cells, but functional studies are necessary for full understanding of ERß function. In the adult rat, gonadotroph-specific expression of ERß indicates that estrogen may regulate the function of gonads through ERß.
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Acknowledgments |
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Received July 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Endocrinology 138:863–870 (ER-) and
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and ß form heterodimers on DNA. J Biol
Chem 272:19858–19862 (ER-) and ß
(ER-ß) mRNA in the midgestational human fetus. J Clin Endocrinol
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