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Program in Molecular Biology (M.E.W., R.J.H.) and the Department of Cell Biology, Neurobiology, and Anatomy (R.H.P., R.J.H.), Loyola University, Stritch School of Medicine, Maywood, Illinois 60153
Address all correspondence and requests for reprints to: Robert J. Handa, Ph.D., Department of Anatomy and Neurobiology, College of Veterinary Medicine, Colorado State University, Fort Collins, Colorado 80523. E-mail: rhanda{at}cvmbs.colostate.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA in adult females. In contrast, in prepubertal female
pituitaries, ERß mRNA levels are much greater than those of ER
mRNA. Film densitometric analysis of whole pituitaries, similarly
showed that ERß mRNA is greater in prepubertal pituitaries than in
adult pituitaries. However, after emulsion autoradiography, cell counts
confirmed that prepubertal and adult pituitaries differ, not in the
level of ERß mRNA expression, but in the number of cells expressing
ERß mRNA. In postnatal day 15 pituitaries, there were twice as many
cells per mm2 as in adults. A comparison between
prepubertal males and females showed that females exhibited a 2-fold
greater level of ERß mRNA expression. To determine which cell types
express ERß mRNA, we performed in situ hybridization
for ERß mRNA coupled with immunohistochemistry for FSH or PRL. In
prepubertal pituitaries, 84.5 ± 2.3% of FSH-immunoreactive cells
also express ERß. Nearly all of the PRL-immunoreactive cells lacked
ERß mRNA. These data demonstrate sex- and age-related differences in
ERß mRNA expression in the anterior pituitary. Furthermore, these
data suggest that ERß is not the specific mediator of estrogen action
in lactotrophs, whereas ERß may be the prime mediator of estrogen
action in FSH-containing gonadotrophs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß (1, 2). Both ER and ERß messenger RNA (mRNA) have
been shown by RT-PCR to be expressed in many estrogen-responsive
reproductive tissues, including the pituitary gland (3). Within the adult anterior pituitary gland, estrogen binding has been demonstrated by in vitro autoradiography to be localized within gonadotrophs, lactotrophs, somatotrophs, and thyrotrophs, with the highest amounts in gonadotrophs (4). Postnatal development of estrogen binding in the anterior pituitary shows a dramatic increase in binding around postnatal days (PND) 10–15 before leveling off to adult levels by PND 25 (5). During this period of development, there is a greater percentage of gonadotrophs among the population of cells than that in the adult pituitary (6).
The functional significance of pituitary ER has been demonstrated by the fact that estrogen can directly regulate pituitary hormones in addition to its indirect effects through modulation of hypothalamic releasing hormones. Estrogen affects the expression of both the gonadotropins, LH and FSH, in pituitary fragments (7). In addition, estrogen can directly stimulate PRL secretion both in vitro as well as in vivo (8, 9).
ER mRNA has previously been demonstrated in the intermediate and
anterior lobes of the pituitary by in situ hybridization
(10), and ER immunoreactivity has been identified in many anterior
pituitary cell types, including gonadotrophs and lactotrophs (11).
Consistent with this, Kuiper and colleagues (3) have demonstrated that
ER mRNA is the predominant species in the adult rat pituitary. As
estrogen binding has been shown to exhibit transient developmental
changes (5), we examined the level of ERß mRNA expression in
prepubertal and adult rat pituitary glands. Furthermore, we determined
the cellular distribution of ERß mRNA within the anterior
pituitary.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR
Total RNA was isolated from frozen pituitaries using guanidium
isothiocyanate by a method previously described (12). One microgram of
total RNA was reverse transcribed using an
oligo(deoxythymidine)12–15 primer and Superscript RT
(Life Technologies, Gaithersburg, MD) in a final reaction
volume of 20 µl. Ten microliters of the complementary DNA were
amplified by PCR in the presence of 2 µCi
[32P]deoxy-CTP using primers specific for the ER,
ERß, histone 3.3 genes (3, 12, 13). Histone 3.3 was run as an
internal control as previously described (12). The cycle parameters for
ER and ERß were the same: 92 C for 1 min, 57 C for 30 sec, and 72
C for 1 min for 30 cycles, as previously described (3). The expected
products of 290 bp (ER) and 262 bp (ERß) were obtained. Reaction
products were separated by nondenaturing PAGE, dried, and visualized by
film autoradiography. The ER and ERß reaction products from
separate tubes were combined and run concurrently on a single gel.
In situ hybridization
In situ hybridization for ERß mRNA was performed by
methods previously described (14). A [35S]UTP-labeled
antisense complementary RNA probe was generated by in vitro
transcription of a specific ERß PCR fragment with an SP6 promoter on
the 3'-end. The probe corresponds to nucleotides 1183–1514 relative to
the start site (2). The 5'-end contained a T7 promoter so that sense
strand probes could also be generated. These promoters were added by
PCR with receptor-specific primers that contained an eight-nucleotide
overhang complementary to primers containing the promoters (15). The
probe specificity was determined by the lack of hybridization with a
sense control probe. The hybridization pattern observed in the brain
with this probe is identical to that observed with other ERß probes
(16).
Tissue sections were acetylated with 0.25% acetic anhydride, dehydrated in graded alcohols, and air-dried. Sections were incubated in a hybridization solution (50% formamide, 0.60 M NaCl, 0.02 M Tris, 0.01 M EDTA, 10% dextran sulfate, 2 x Denhart’s solution, 50 mM dithiothreitol, 0.2% SDS, 100 mg/ml salmon sperm DNA, 500 mg/ml total yeast RNA, and 50 mg/ml yeast transfer RNA) containing the radiolabeled probe at a concentration of 2 x 107 cpm/ml at 55 C overnight. After hybridization, the slides were rinsed in 2 x SSC (standard saline citrate), and nonhybridized RNA was digested with 30 mg/ml ribonuclease A for 30 min at 37 C. The final wash stringency was 0.1 x SSC at 60 C. For autoradiographic detection of hybridization, slides were exposed to autoradiographic film for 1–3 days or were dipped in nuclear tract emulsion (Kodak NTB-3, Eastman Kodak Co., Rochester, NY), air-dried, and exposed for 3–7 days at 4 C.
Immunohistochemistry
After in situ hybridization, but before
autoradiography, immunohistochemistry was performed using standard
procedures with sterile buffers and serum (17). Tissue for FSH or PRL
immunohistochemistry were incubated overnight with the NIDDK rabbit
antirat primary antibodies (FSH, 1:500; PRL, 1:15,000) at room
temperature in PBS [pH 7.0; containing 2% normal goat serum
(NGS)]. A biotinylated goat antirabbit secondary antibody (1:500;
Kirkegaard & Perry, Inc., Gaithersburg, MD) was then
incubated for 2 h. The complex was visualized by
streptavidin-horseradish peroxidase (Kirkegaard & Perry)
with diaminobenzidine (0.5 mg/ml) as the chromogen. After detection of
immunoreactive cells, slides were dipped in photographic emulsion
(Kodak NTB-3). A brown cytoplasmic oxidation product overlaid by
exposed silver grains was indicative of a double-labeled cell.
Image analysis
The number of grains per fixed area approximating a cell was
counted using a video camera (Sony XC-77) connected to a Zeiss
Axioplan microscope (New York, NY), an Apple Power Macintosh 7100
computer, and NIH Image (version 1.57) software. A
grain-counting macro originally written by Dr. Karl Beykirch
(University of California School of Medicine, Los Angeles, CA) and
adapted for our use by Dr. Alan Nagahara (Loyola University) was used.
The macro counted the density of silver grains, identified by darkfield
microscopy, by calculating the number of pixels present above a
threshold defined by the user. Measurements were taken from 10 cells
from 3 different sections for each animal, and the mean of the 30
measurements was averaged to give the mean for that animal. The data
were expressed as the percentage of area covered by grains (mean grain
area). For cell counts, a labeled cell was defined as one that
contained over 5 times the number of background grains.
Statistics
Data were analyzed across age by one-way ANOVA. The
Student-Newman-Keuls test was used to make post-hoc
comparisons. For the adult vs. prepubertal animal study,
three animals were analyzed. In the prepubertal male
vs. female study, five animals were analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß mRNA in prepubertal and adult female rat
pituitaries and ERß mRNA (Fig. 1
). Histone 3.3 served as an internal
loading control and did not differ (data not shown). The level of ER
expression was greater in the adult pituitary compared with that in the
PND 15 female. ERß expression was greater in the PND 15 than in the
adult female. Although it is difficult to make a direct comparison
between two different genes by RT-PCR because of different primer
efficiencies and product sizes and, thus, different specific activities
of products, it appears that in the adult animal, ER mRNA was
expressed at a much greater level than ERß mRNA. In the infantile
female, the opposite appeared to be true; ERß mRNA expression was
much greater than that of ER mRNA.
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), with a
region of high expression immediately adjacent to the posterior
pituitary in the PND 15 animals (Fig. 2). The PND 15 female contained a
considerably higher level of expression compared with both the adult
and PND 15 male. Sense strand probes served as a negative control and
demonstrated no hybridization (Fig. 2).
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). Single cell analysis showed that the
number of grains per cell was not different between the adult and PND
15 female (Fig. 4, A–C); however, the
number of labeled cells per unit area differed significantly
(P < 0.001; Fig. 4D). There were more than twice as
many ERß mRNA-expressing cells in the prepubertal female as in the
adult female.
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).
After emulsion autoradiography, the mean grain area was twice as great
in females as in males.
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). Of the PRL-ir
cells, on the other hand, only 14.8 ± 4.5% (n = 3)
coexpressed ERß (Fig. 6B). Approximately half of the ERß
mRNA-expressing cells did not possess either FSH-ir or PRL-ir.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and that it is
restricted to the anterior lobe of the pituitary. In the prepubertal
animal, the level of expression is much greater in females than in
males. Furthermore, we determined that in the prepubertal female, ERß
mRNA is expressed in nearly all FSH-containing gonadotrophs, whereas it
is virtually absent in lactotrophs.
These data replicate a previous study using RT-PCR, which showed that
ER is expressed at a greater level than ERß in the adult female
pituitary (3). In the prepubertal pituitary, however, the opposite is
true. ERß mRNA is expressed at a much greater level than ER mRNA.
It remains to be seen whether the mRNA levels reflect protein levels in
these animals.
Using in situ hybridization we determined that the
anatomical localization of ERß mRNA expression is restricted to
anterior lobe of the pituitary. After film autoradiography, it appears
that the intermediate lobe was labeled as well; however, microscopic
examination after emulsion autoradiography reveals that expression is,
in fact, limited to the anterior lobe, with a dense population of
labeled cells adjacent to the intermediate lobe. This area has been
previously described as a sex zone that has been shown to contain
primarily gonadotrophs (19). These results suggest that ERß could
mediate a specific action of estrogen in the anterior lobe of the
pituitary gland, as both the intermediate lobe and the anterior lobe
contain ER mRNA (20, 21). In addition, this observation suggests
that ER is the sole mediator of estrogen’s actions in the
intermediate lobe.
ERß mRNA levels are dramatically elevated in the prepubertal female compared with the male. This sex difference in ERß mRNA may play a role in the differential gonadotropin secretion seen in these animals (17, 22). During the prepubertal period, gonadotropin secretion is very high in the female compared with that in the male. We have recently shown that estrogen may play a role in the elevated secretion of specifically FSH in the prepubertal female (23). It is possible that ERß could potentially be mediating this sex difference.
When the expression of ERß mRNA is examined at the cellular level, the level of expression per cell remained constant in the female pituitary across puberty. In contrast, it appears that the population of cells not expressing ERß changes. This is consistent with our observation that ERß mRNA is not expressed in the lactotrophs, but is expressed in a significant population of gonadotrophs. In the prepubertal animal, gonadotrophs make up a greater percentage of anterior pituitary cells than in the adult (6, 18, 24). The number of immunoreactive lactotrophs increases dramatically after the third to fourth week of postnatal life (25), and thus, it is possible that increased lactotroph proliferation dilutes the population of cells expressing ERß. This observation coupled with previous data using RT-PCR demonstrate a potential pitfall in the use of RT-PCR for examining the expression of a gene from total RNA obtained from a heterogeneous population of cells.
In our hands, the double label technique we have employed here is effective in identifying both immunoreactivity and silver grains. There is, however, an inherent degree of background false positives and false negatives. As the cells of the pituitary are densely packed, there is an unavoidable overlap of cells. Thus, a nonimmunoreactive cell lying underneath an immunoreactive cell may contribute to the silver grains seen, resulting in a false positive. Because the percentage of ERß-negative FSH-ir cells is approximately the same as the percentage of positive PRL-ir cells, we believe that the limits of precision for this technique are approximately 10–15%. As we observed that 85% of FSH-ir cells also expressed ERß mRNA, it appears that most FSH-containing gonadotrophs are ERß mRNA positive, and nearly all lactotrophs are ERß mRNA negative.
Whereas most FSH-ir gonadotrophs express ERß, all ERß-expressing cells did not contain FSH-ir. The identity of these other cells could be LH monohormonal gonadotrophs. The number of FSH-negative, ERß-positive cells is greater than that which can be accounted for simply by the number LH monohormonal gonadotrophs that should be present in the PND 15 female rat (18). Thus, an alternate possibility is that some are somatotrophs or thyrotrophs. As somatotrophs have a common origin with the lactotrophs, it may be that thyrotrophs are the other major cell type expressing ERß mRNA. This remains to be determined.
The finding that lactotrophs do not express ERß mRNA provides a
potential mechanism for pituitary proliferation. Estrogen has been
shown to dramatically increase pituitary size as a result of lactotroph
proliferation (26). PRL-secreting adenomas are the most common
pituitary tumor found, and recent studies have used the estrogen
induction of pituitary hyperplasia in the rat as a model of human
pituitary adenoma (27). Given that lactotrophs do not express ERß,
but do express ER (11), lactotroph-specific cell proliferation is
presumably mediated through genes that are activated by ER
homodimers. As other cell types, in particular gonadotrophs, appear to
have both forms of the estrogen receptor, regulation of their hormone
secretion could be mediated through ß-homodimers or - and ß-
heterodimers. Differential transcriptional activation of promoter
elements has been demonstrated with homodimers and heterodimers (28).
Ultimately, the advent of specific antagonists to ER could allow the
targeted reduction of lactotroph proliferation potentially without
altering the role of estrogen in gonadotrophs mediated through
ERß.
Interestingly, a sex difference in the pituitary response to estrogen
has been demonstrated (29). Estrogen administration increases pituitary
androgen receptor levels and pituitary growth more rapidly in females
than in males. This increased sensitivity to estrogen may in part be
due to the presence of differential levels of ERß or ER between
the sexes, although it remains to be seen whether these sex differences
are present in the adult.
The data presented in this study demonstrate that ERß is expressed in the anterior pituitary and may be a mediator of estrogen action in the gonadotroph. Furthermore, sex differences, developmental changes, and cell-specific expression of ERß indicate that it may be an important mediator of estrogen action in the pituitary. The identities of such ERß-mediated cellular processes in the pituitary warrant further investigation.
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Footnotes |
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2 Present address: Department of Physiology, University of Kentucky,
Lexington, Kentucky 40536.
Received February 20, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Endocrinology 138:863–870 and -ß mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
and ß form heterodimers on DNA. J Biol
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