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Original Studies |
and ß in the Human Fetus1
Department of Pathology, Tohoku University School of Medicine (J.T., T.S., C.K. H.N., H.S.), Sendai, Miyagi 980-8575, Japan; Department of Geriatric Medicine, Tokyo University (S.I.), Bunkyo-ku, Tokyo 113-0033, Japan; and Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University (N.H.), Toyoake, Aichi 470-1192, Japan
Address all correspondence and requests for reprints to: Dr. Junji Takeyama, Department of Pathology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. E-mail: j-takeyama{at}patholo2.med.tohoku.ac.jp
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Abstract |
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(ER) and ß (ERß). In this study we examined the
expression and cellular localization of ER and ERß in various
human fetal tissues by semiquantitative RT-PCR (13 and 20 gestational
weeks) and immunohistochemistry (13, 20, and 38 gestational weeks),
respectively, to study the possible effects of estrogens on human fetal
tissues during development. Relatively high levels of ERß expression
were detected in various human fetal tissues, whereas those tissues
expressing ERß had markedly lower levels of ER expression. ERß
messenger ribonucleic acid expression was especially high in the
adrenal gland. ERß-immunoreactive protein was localized to the
definitive zone, but not in the fetal zone, of the adrenal cortex.
Although low levels of ERß messenger ribonucleic acid were present in
the brain, heart, lung, and kidney, ERß immunoreactivity was not
detected in these tissues. These results suggest that the effects of
estrogens in these tissues are predominantly mediated through ERß.
ERß immunoreactivity was detected in Sertoli cells and spermatogonia
in the male reproductive tract and in germ cells in the fetal testis
and epididymis. In the female reproductive tract, both ER and ERß
were immunopositive in epithelium of the oviduct. The results of the
present study have demonstrated the possible sites for estrogenic
action in the human fetus and suggest that the effects of estrogen via
ERß may play important roles in human fetal development, especially
in the definitive zone of the adrenal cortex, and in the reproductive
tissues of the developing fetus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), cloned in 1986 (2, 3), was considered
the only form of nuclear receptor that was able to bind to estrogen and
mediate its hormonal effects in the normal physiological processes of
the mammal. However, a second estrogen receptor, ERß, has been cloned
in the rat (4), mouse (5), and human
(6). ERß has been demonstrated to have highly conserved
DNA- and ligand-binding domains compared with ER (7).
Estrogen binds ERß with an affinity and specificity similar to those
of ER (7). ERß is also capable of stimulating
transcription of the target genes in a manner similar to ER,
although the degree of activation is lower than that of ER
(6). ERß has also been demonstrated to have a wide
distribution of expression in various human tissues (8, 9), including the human fetus (10), distinct from
that of ER. The differential expression of ER and ERß in these
tissues suggests more complex and diverse control mechanisms in
estrogenic actions than was originally envisioned
(11).
The production and secretion of estrogens in normal pregnancy are known
to rise continuously throughout pregnancy (12, 13). In
addition to estrogens entering the fetal circulation through the
umbilical vein (14), large quantities of estrogens are
present in the amniotic fluid and may enter the fetal circulation by
passive diffusion through the fetal skin or by swallowing and
absorption from the gastrointestinal tract (15). The role
of estrogens in the human fetus remains unclear, but their actions
through ER may play pivotal roles in the maturation of fetal tissues,
as the human fetus is exposed to large amounts of estrogens. In the
human fetus, Brandenberger et al. reported the distribution
of ER and ERß messenger ribonucleic acid (mRNA) using RT-PCR
analysis (10), but the in situ or precise
cellular localization of these receptors, which provides pivotal
information about estrogenic actions in fetal development, has not been
identified. Therefore, in this study we examined the expression of
ER and ERß to determine the potential sites of estrogenic actions
in the human fetus at 13 and 20 gestational weeks by semiquantitative
RT-PCR using fluorescent dye-labeled primers. In addition, we studied
the cellular localization of ER and ERß using
immunohistochemistry.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human fetal tissues were obtained from fetuses aged 13 weeks
(female; n = 1) and 20 weeks (male; n = 1) gestation after
elective termination in normal pregnant women at Tohoku University
Hospital and Nagaike Maternal Clinic (Sendai, Japan). Tissues from a
fetus aged 38 gestational weeks (female stillborn; n = 1) were
obtained at the time of autopsy at Tohoku University Hospital. The
cause of death was not apparent from pathological examinations of this
neonate. For positive control of the quantitation of ER mRNA, we
used the tissue from breast carcinoma associated with high expression
of ER. This research protocol was approved by the committee on the
ethics of Tohoku University School of Medicine. The ages of the fetuses
were estimated by the last menstrual date, body weight, or crown-rump
length. The specimens for RNA isolation were snap-frozen and stored at
-80 C until use, and those for immunohistochemistry were fixed in 10%
neutral formalin for 18 h at 4 C and embedded in paraffin. Total
RNA was extracted from the whole tissues by homogenizing tissue
specimens in guanidinium thiocyanate, followed by ultracentrifugation
in cesium chloride (16).
Analysis of the mRNAs for ER and ERß
Semiquantitative analysis of ER, ERß, and ß-actin mRNAs
was carried out in the total RNA fractions from various human fetal
tissues by RT-PCR using a specific pair of fluorescent dye-labeled
primers and their standard RNAs, as previously described (17, 18).
Oligonucleotide primers for RT-PCR were synthesized as shown in Table 1
. Quantitative analyses of each specimen
were repeated three times, and the data were presented as the mean
value.
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The polyclonal antibody for ERß was produced by immunizing a
rabbit with synthesized peptides of the C-terminal region of ERß and
was purified on affinity columns bound with the synthetic peptide.
Immunoblot analysis of ER- or ERß-transfected cells demonstrated
that the polyclonal antibody (1:500 dilution) specifically detected
ERß protein, but did not react with ER protein (19).
The monoclonal antibody for ER was commercially obtained
(Immunotech, Marseilles, France).
Immunohistochemistry
Paraffin-embedded tissues (3.0-µm sections) were mounted onto
clean MAS-coated glass slides (Matsunami Co. Ltd., Tokyo, Japan).
Autoclave treatment for 5 min at 120 C in 0.01 mol/L citrate buffer, pH
6.0, was employed after deparaffinization for antigen retrieval
(20). The slides were then immersed in methanol containing
0.3% hydrogen peroxide for 30 min to block endogenous peroxidase
activity. Normal rabbit serum (for ER) or normal goat serum (for
ERß) was incubated for 30 min at room temperature to decrease
nonspecific staining. Sections were then incubated with the primary
antibody for ER (1:50 dilution) or ERß (1:500 dilution) for
18 h at 4 C. After washing, the specimens were incubated with
EnVision+, peroxidase, mouse (for ER) or rabbit (for ERß;
DAKO Corp., Carpinteria, CA) for 1 h at room
temperature, followed by incubation in a solution containing 0.05%
hydroxychloride (pH 7.6), 0.06 mmol/L 3,3'-diaminobenzidine (DAB), and
2 mmol/L hydrogen peroxide. Immunoreactive sections were counterstained
with hematoxylin. For negative controls, preimmune mouse serum (for
ER) or rabbit serum (for ERß) were used instead of primary
antibodies. No specific immunoreactivity was detected in these
sections. In addition, ERß immunoreactivity was completely
obliterated by absorbing the antibody with an excessive amount of
antigen. Experiments were repeated twice, and there were no differences
of the patterns of immunolocalization between the two experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß
are summarized in Table 2. The mRNA
levels of ER and ERß in the RNA fractions were determined by
RT-PCR analysis using a fluorescent primer in the presence of an
internal standard RNA. All data were also expressed after adjustment by
ß-actin mRNA levels of the same specimens. The highest level of ERß
mRNA expression was detected in the adrenal gland obtained from a fetus
at 20 gestational weeks. ERß mRNA expression was also detected in the
brain, heart, lung, and kidney. The levels of ER mRNA in all fetal
tissues examined were markedly low.
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and ERß are
summarized in Table 2
, and representative illustrations of ER and
ERß immunohistochemistry are summarized in Figs. 1-3. ERß immunoreactivity in the fetal
adrenal cortex was present only in the definitive zone, not in the
fetal zone (Fig. 1, A and B). ER immunoreactivity was not detected
in the fetal adrenal glands examined in this study. In the male
reproductive tract (20 weeks gestation), ERß immunoreactivity was
detected in Sertoli cells constituting the great majority of the
seminiferous epithelium (Fig. 2A).
Spermatogenic germ cells and interstitial cells also demonstrated ERß
immunoreactivity (Fig. 2A). ERß immunoreactivity was also present in
epithelial cells of the epididymis and interstitial cells around the
ducts (Fig. 2B). ER immunoreactivity was not detected in the male
reproductive organs. In the female reproductive tract (38 weeks
gestation), immunoreactivity for both ER and ERß was detected in
epithelium of the oviduct (Fig. 3, A and
B). ER and ERß immunoreactivity was below the level of detection
of immunohistochemistry in fetal brain, heart, lung, liver, kidney,
gastrointestinal tract, ovary, and uterus.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA was undetectable or was
present at very low levels in human fetal tissues examined at both 13
and 20 weeks gestation, whereas ERß mRNA was detected in various
human fetal tissues at these gestational periods. These results are
consistent with the previous RT-PCR study reported by Brandenberger
et al. (10). These results also suggest that
the actions of estrogens in human fetal tissues are predominantly
mediated through ERß, rather than ER. Among the various human fetal tissues examined in this study, the highest level of ERß expression was detected in the adrenal gland. The human fetal adrenal cortex is composed of two morphologically distinct zones, the definitive zone and the fetal zone. In vitro studies have demonstrated that the definitive zone secretes predominantly cortisol, whereas the fetal zone secretes large quantities of dehydroepiandrosterone sulfate and little cortisol (21). In this study, ERß immunoreactivity was present exclusively in the definitive zone of the fetal adrenal gland. Previous studies have shown that steroid production in human fetal adrenal cortical cells in vitro is modulated by estrogen (22, 23). Pepe et al. reported that estrogens are also involved in possible interactions between the placenta and fetus, such as the induction of maturation of the fetal hypothalamic pituitary adrenocortical axis (24, 25). These results and the findings reported in this study suggest that the estrogenic actions via ERß in the definitive zone of the fetal adrenal gland may play crucial roles in steroidogenesis as well as in the maturation of the fetal adrenal cortex. Further investigations are required, however, to clarify the precise role of ERß expression in the fetal adrenal gland’s definitive zone.
We examined immunoreactivity for both ER and ERß in the
reproductive tracts of the male (20 gestational weeks) and female (38
gestational weeks) fetus, but could not quantitate the mRNA transcript
by RT-PCR due to the unavailability of specimens. There were no
apparent macro- or microscopic abnormalities in the stillborn 38-week
gestation fetus, but we could not completely exclude an unknown
medical/physiological abnormalities that might have affected the
results of the study. In the male reproductive tract, the seminiferous
epithelium, composed mostly of Sertoli cells and a few germ cells,
demonstrated marked ERß immunoreactivity. This finding is also
consistent with prior RT-PCR data from Brandenberger et al.,
who reported high levels of ERß mRNA in testis (10). In
addition, marked ERß immunoreactivity was detected in the epididymis.
ER immunoreactivity, however, was not detected in this tissue. These
results suggest that the effects of estrogens, predominantly via ERß,
may play important roles in the development of male reproductive
organs. In the female reproductive tract, immunoreactive ER and
ERß were both detected in epithelium of the oviduct, which suggests
that estrogenic actions, through both ER and ERß, may modulate the
development of the oviduct. We could not detect immunoreactivity for ER
subtypes in the uterus and ovary in the 38-week gestational fetus,
although previous studies have demonstrated that ERs were involved in
the development of the fetal uterus (26) and oocytes
(27). The protein levels of ERs present in these tissues
may be too low to allow detection of their expression using
immunohistochemistry. The difference between previous reports and the
current data may be explained by that in gestational ages. The
number of cases examined in this study was limited, and further
investigations are required to clarify these discrepancies.
In summary, we have examined the expression and cellular localization
of ER and ERß in the human fetus. The results of our present study
suggest that the effects of estrogens are mediated predominantly
through ERß and may play important roles in the development of the
adrenal gland and male reproductive organs. Moreover, our findings
appear to suggest that the estrogenic actions in female
reproductive organs are likely to be mediated via both ER and
ERß.
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Acknowledgments |
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Footnotes |
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Received September 9, 2000.
Revised January 9, 2001.
Accepted January 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß. Endocrinology. 138:863–870. (ER-) and ß
(ER-ß) mRNA in the midgestational human fetus. J Clin Endocrinol
Metab. 82:3509–3512.This article has been cited by other articles:
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E. D. Albrecht, R. B. Billiar, G. W. Aberdeen, J. S. Babischkin, and G. J. Pepe Expression of Estrogen Receptors {alpha} and {beta} in the Fetal Baboon Testisand Epididymis Biol Reprod, April 1, 2004; 70(4): 1106 - 1113. [Abstract] [Full Text] [PDF]
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