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The Journal of Clinical Endocrinology & Metabolism 90(6):3752–3756
Copyright © 2005 by The Endocrine Society
doi: 10.1210/jc.2004-1818
Estrogen Receptors and Estrogen-Metabolizing Enzymes
in Human Ovaries during Fetal Development
Tommi E. Vaskivuo, Minna Mäentausta, Svea Törn, Olayiwola Oduwole, Annika Lönnberg, Riitta Herva,
Veli Isomaa, and Juha S. Tapanainen
Departments of Obstetrics and Gynecology (T.E.V., M.M., A.L., J.S.T.) and Pathology (T.E.V., M.M., A.L., R.H., J.S.T.) and
Research Center for Molecular Endocrinology, World Health Organization Collaborating Centre for Research on
Reproductive Health, and Biocenter Oulu (S.T., O.O., V.I.), University of Oulu, FI-90014 Oulu, Finland
Estrogen action plays a crucial role in many processes throughout the
human life span, including development. Estrogens are pivotal in the
regulation of female reproduction, but little is known about their role
during ovarian development. To better understand estrogen action
during ovarian development, the expression of estrogen receptors
(ERs)-␣ and -␤ and key enzymes regulating estradiol production,
17␤-hydroxysteroid dehydrogenases (17HSDs) types 1, 2, and 7, were
analyzed in human fetal ovaries. The expression of ERs was related
to the development of ovarian follicles. Before the 26th week of fetal
life ER␣ was only occasionally detected, but from then onward, its
expression was detected in ovarian follicles. Consistent expression of
ER␤ was seen from the 20th week until term. Both ER␣ and ER␤ were
localized to the granulosa cells and oocytes. Expression of 17HSD1
E
STROGENS HAVE AN essential influence on development of the female reproductive system and the maintenance of fertility (1). Estrogen action is mediated by two
specific estrogen receptors (ERs), ER␣ and ER␤ (2, 3). They
have closely related DNA binding regions but different ligand-binding affinities (3). Their expression patterns also
differ. In the ovaries ER␣ is expressed in the theca and interstitial cells of growing follicles, whereas ER␤ is mainly
located in the granulosa cells (4). In the human fetus, the
expression of ERs is not well known. Expression of ER␣ has
been observed in early ovarian development (5), and ER␤
mRNA has been detected in midgestational human fetal
ovaries (6, 7). However, their spatial and temporal expression
patterns and roles in human ovarian development are
unclear.
Local estrogen concentrations at the tissue and cellular
level are controlled by estrogen-metabolizing enzymes. We
have previously shown that aromatase, which is the key
enzyme converting androgens to estrogens, is expressed in
human fetal ovary (8). In addition to aromatase, the 17␤hydroxysteroid dehydrogenases (17HSDs) modulate the biological activity of sex steroid hormones by catalyzing key
steps in the formation and primary metabolism of active
estrogens, androgens, and progesterone (9). Hence, 17HSDs
regulate the concentrations of active sex steroids able to bind
and 17HSD7 enzymes, catalyzing the conversion of estrone to more
active estradiol, was detected as early as at the 17th week of fetal life.
The expression of 17HSD1 displayed a pattern similar to that of ERs
and increased toward term, whereas that of 17HSD7 decreased and
was negative by the 36th week. 17HSD1 was localized to the granulosa cells, whereas 17HSD7 expression was more diffuse and was
found in both granulosa and stromal cells. 17HSD2, converting estradiol to less potent estrone, was negative in all samples studied. The
simultaneous appearance of estrogen-converting enzymes and ERs at
the time of follicle formation indicates that the machinery for estrogen
action exists in fetal ovaries and suggests a possible role for estrogens
in the developing ovary. (J Clin Endocrinol Metab 90: 3752–3756,
2005)
to their respective steroid receptors. At present, nine 17HSD
isoforms have been characterized in humans (9, 10). 17HSD1
and 17HSD7 catalyze the conversion of estrone (E1) to more
active estradiol (E2) (11, 12), whereas 17HSD2 catalyzes the
opposite reaction and inactivates sex hormones (13, 14). In
the adult human ovary 17HSD1 is expressed in granulosa
cells of growing follicles and in the corpus luteum (8, 15).
Localization of 17HSD7 in the human ovary is unknown, but
in rodents it is expressed mainly in the corpus luteum (16).
The role of estrogen action in ovarian development and
reproductive function has been extensively studied using ER
and aromatase knockout animal models (17–20). However,
the role of estrogen in human ovarian development is much
less well known, and the developmental impact of an inactivating aromatase mutation on ovarian development is difficult to assess, owing to a possible compensatory role of
maternal estrogens (21, 22). Moreover, recently discovered
putative ERs that can also exist as membrane-bound forms
further complicate the picture (23).
In the present study, the role of estrogen action was investigated in human ovarian development by studying the
expression of ERs and the estrogen-metabolizing enzymes
17HSD1, 17HSD2, and 17HSD7 in fetal ovaries.
Materials and Methods
Fetal ovaries
First Published Online March 22, 2005
Abbreviations: E1, Estrone; E2, estradiol; ER, estrogen receptor; HSD,
hydroxysteroid dehydrogenase.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.
Ovaries from 10 fetuses (fetal age 13–24 and 33 wk) with normal
karyotype were obtained after spontaneous abortions and therapeutic
abortions carried out as a result of maternal disease. In addition, eight
neonates (fetal age 26 – 40 wk) who died because of perinatal asphyxia
or infection within 48 h of birth were studied. Samples with visible
autolysis or abnormal karyotype were excluded from the study. All
3752
Vaskivuo et al. • Fetal Ovary Estrogen Receptors and Metabolic Enzymes
samples were fixed in 4% buffered formaldehyde for 24 h, rehydrated,
and embedded in paraffin. Histological sections (4 ␮m) were cut and
processed for immunohistochemistry and in situ hybridization. The
study was approved by the Ethics Committee of Oulu University
Hospital.
Immunohistochemistry
Paraffin sections were deparaffinized in xylene and hydrated gradually through a series of ethanol dilutions and then washed in water.
Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol. Nonspecific binding was prevented by using fetal calf
serum at a 1:5 dilution in PBS. Primary antibody was applied to the
sample and it was incubated overnight. ER␣ was detected using mouse
monoclonal antibody (NCL-ER-6F11) raised against the N-terminal
(A/B) region of human ER␣ (working concentration 1:50 in PBS; Novocastra, Newcastle-upon-Tyne, UK). An antigen-absorbed antibody
control was not used because the antibody for ER␣ is well characterized.
ER␤ was detected using two different antibodies: rabbit polyclonal antibody (PAI-310) raised against a synthetic peptide corresponding to
C-terminal amino acid residues 467– 485 of rat ER␤ (working concentration 1:50 in PBS; Affinity BioReagents Inc., Golden, CO) and a rabbit
monoclonal antibody raised against human ER␤ (working concentration
1:300 in PBS; Santa Cruz Biotechnology, Santa Cruz, CA). For negative
controls, PBS was used instead of primary antibody. The sections were
washed in PBS before addition of the secondary antibody and incubation
for 30 min. As the secondary antibodies, biotinylated rabbit antimouse
and goat antirabbit immunoglobulins (Vectastain Elite ABC kit, Vector
Laboratories, Burlingame, CA) were used against the respective primary
antibodies. After washing in PBS, the sections were incubated for 30 min
with avidin-conjugated alkaline phosphatase (Vectastain Elite ABC Kit).
The sections were washed in PBS followed by treatment with liquid
diaminobenzidine and a wash in water. Counterstaining was performed
with hematoxylin. The sections were dehydrated through a series of
ethanol dilutions and cleared in xylene.
Staining with monoclonal ER␤ antibodies was controlled by using
antigen-absorbed antibodies, as previously described (8). Control peptides were purchased from Santa Cruz Biotechnology and corresponding antibodies were incubated with a 5-fold excess (by weight) of the
blocking peptide overnight at 4 C. The neutralized antibodies were used
according to normal immunostaining procedures.
In situ hybridization
Probes for in situ hybridization were prepared from a 376-bp fragment (nucleotides 1–376) of human 17HSD1 cDNA (24), a 380-bp fragment (nucleotides 191–570) of human 17HSD2 cDNA (13), and an 832-bp
fragment (nucleotides 39 – 870) of the human 17HSD7 cDNA (12) cloned
in pGEM-4Z plasmids (Promega, Madison, WI) and used as templates
for in vitro transcription. Sense and antisense [35S]CTP-labeled RNA
probes were transcribed with T7 or SP6 RNA polymerases using linearized plasmids as templates. Specific activities of the synthesized RNA
probes were approximately 6 ⫻ 106 cpm/␮l.
The in situ hybridization reactions were performed as previously
described (25). Hybridization signals were detected after 15-d (17HSD7
after 21 d) exposure to autoradiographic NTB2-emulsion (Eastman
Kodak, Rochester, NY) at ⫺20 C. The slides were developed using D-19
developer and Unifix solution (Eastman Kodak), following the instructions of the manufacturer. Nuclei were further stained with Hoechst
33258 (Sigma-Aldrich Finland, Helsinki, Finland), after which the slides
were mounted with glycergel (Dako A/S, Glostrup, Denmark), except
for the 17HSD7 slides, which were stained with hematoxylin and eosin
and mounted with Pertex (Histolab, Gothenburg, Sweden).
Evaluation of samples
All samples were analyzed by two independent observers (immunohistochemistry: T.E.V. and M.M.; 17HSD1 and 17HSD2 in situ hybridizations: T.E.V. and O.O.; 17HSD7 in situ hybridizations: T.E.V. and S.T.).
Immunohistochemical sections were analyzed using light microscopy,
and in situ hybridizations were analyzed using dark-field and light
microscopy. The results were evaluated by using a standard histograde
J Clin Endocrinol Metab, June 2005, 90(6):3752–3756
3753
system: ⫺, negative; ⫾, moderately positive; ⫹, positive; ⫹⫹, strongly
positive; and ⫹⫹⫹, very strongly positive.
Results
ER␣ immunohistochemistry
In the two youngest samples (13 and 14 wk) studied, no
ER␣ immunoreactivity was detected. Between wk 14 and 24,
only occasional weak staining was observed in areas in
which follicles had started to develop (Table 1). In other areas
in which follicular formation had not yet been completed,
ER␣ staining was negligible and seemed to be confined to
granulosa/pregranulosa cells and oocytes (Fig. 1A). From
the 26th week onward, nearly all oocytes were individually
enveloped by a granulosa cell layer. At this stage immunostaining of ER␣ was moderate and localized to both the
follicular granulosa cells and oocytes. However, near term
there was more individual variation, and in two samples
aged 37 and 39 wk, the staining was negligible. Immunoreactivity of ER␣ was evident only in the follicular granulosa
cells and oocytes; no staining was observed in the stroma
(Fig. 1A). Furthermore, the immunostaining was clearly localized to the cytoplasm and only occasional nuclear staining
was detected.
ER␤ immunohistochemistry
Immunoreactivity of ER␤ was negative until wk 20, except
in one sample from a 14-wk-old fetus showing weak staining.
From the 20th week onward, immunostaining was seen in
most of the follicles (Fig. 1B). Similarly to ER␣, ER␤ staining
was primarily localized to follicular granulosa cells, but oocytes also showed positive staining. Immunostaining increased gradually and reached its peak between wk 27 and
40. During this period strong staining was seen in almost all
follicles. Thereafter the staining weakened, and just before
birth there was only weak immunoreactivity in some follicles. Similarly to ER␣, ER␤ immunostaining was observed
only in follicular cells, whereas stromal cells were unstained.
17HSDs
The expression pattern of 17HSD1 mRNA in human fetal
ovaries was somewhat similar to that of estrogen receptors
(Table 1). No expression was detected before follicular deTABLE 1. Evaluation of ER-␣ and -␤, and the estrogenconverting enzymes 17HSD1, 17HSD2, and 17HSD7 in the human
ovary during fetal development
Fetal age
(wk)
13–15
16 –19
20 –23
24 –27
28 –33
34 – 41
Estrogen
receptors
Estrogen-converting enzymes
ER␣
ER␤
17HSD1
17HSD2
17HSD7
⫺
⫺
⫾
⫹⫹
⫹
⫾
⫾
⫺
⫹
⫾
⫹⫹⫹
⫹⫹
⫺
⫾
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫺
⫺
⫺
⫺
⫺
⫺
na
⫹
⫹
⫹
⫾
⫺
na, No specimen available. Grades: ⫺ negative, ⫾ moderately positive, ⫹ positive, ⫹⫹ strongly positive, ⫹⫹⫹ very strongly positive.
(Immunohistochemical analysis was performed by T.E.V. and M.M.;
17HSD1 and 17HSD2 in situ hybridizations, T.E.V. and O.O.; and
17SHD7 in situ hybridizations, T.E.V. and ST.)
3754
J Clin Endocrinol Metab, June 2005, 90(6):3752–3756
FIG. 1. Immunohistochemical analysis of ER␣ and ER␤ in fetal human ovaries. At the age of 14 wk, immunostaining of ER␣ was negative and no identifiable follicular structures could be detected (A1).
However, diffuse and weak ER␤ staining could be observed (A2). By
the 27th week, granulosa cells had enveloped the oocytes, and immunostaining of both receptors could be detected in the follicular cells
(A3, A4). At the fetal age of 33 wk, staining of both ␣- and ␤-receptor
was observed in the primordial follicles (A5, A6). Higher magnification of the ovaries at 33 wk shows that the staining of both ␣- and
␤-receptors were mostly located to cytoplasm, and only occasional
nuclear staining was observed (B1, B2). Endometrium was used as a
positive control for ER␣ (B3). Section in which the specific antibody
was omitted was used as negative control (B5). To ensure the specificity of the ER␤ immunostaining, antigen-absorbed antibodies were
used. Antibodies normally revealed strong staining in the follicular
cells (B4), but after antigen absorption the staining was negative or
negligible, reflecting the specificity of the analysis (B6). Scale bar, 5
␮m.
velopment had started and follicular structures were observed. Weak expression was seen as early as at 17 wk, but
after the 22nd week, strong expression was observed in all
samples. The expression was clearly localized to the granulosa cells, whereas oocytes were negative. In the few growing
follicles that were seen during the last trimester, 17HSD1
mRNA expression was clearly up-regulated (Fig. 2). No
17HSD2 mRNA expression was detected in any of the fetal
ovarian samples.
Moderate expression of 17HSD7 mRNA was observed in
fetal ovary at 17 wk, which was the earliest time point at
which it was analyzed, owing to the limited amount of tissue
available. The enzyme was mainly localized in the stromal
and pregranulosa cells, but expression in the oocyte cannot
be excluded (Fig. 2). Thereafter the expression of 17HSD7
decreased toward term and was negative by the 36th week.
Vaskivuo et al. • Fetal Ovary Estrogen Receptors and Metabolic Enzymes
FIG. 2. In situ hybridization of 17HSD1 and 17HSD7. At the gestational age of 14 wk, no detectable 17HSD1 signal was observed (A1).
However, the highest expression of 17HSD7 was already seen at wk
17 (A2). At the 22nd week, increased 17HSD1 expression could be
localized to the granulosa cells of early follicles (A3), whereas the level
of 17HSD7 signals at the 27th week was lower than in early gestation
(A4). 17HSD1 expression reached its maximum before term. Additionally, occasional follicles that had developed past the primordial
stage also showed high expression (A5). In contrast, 17HSD7 expression decreased toward term and was negative at 38 wk (A6). Higher
magnification of 17HSD1 expression shows that the gene is expressed
in the granulosa cells but not in the oocytes (B1). 17HSD7 expression
was more diffuse and signals were detected both in granulosa cells
and oocytes (B2). Placental tissue was used as a positive control for
17HSD1 analysis (B3) and adrenal for 17HSD7 (B4). Sense control
shows weak background staining in both mRNA assays (B5, B6).
Scale bar, 5 ␮m.
Discussion
ERs and estradiol-producing 17HSD1 and 17HSD7 enzymes that enhance estrogen bioactivity are present in the
human ovary during fetal development. Clear expression of
ER␣ and ER␤ was seen from the 20th week onward. Both ERs
were localized in the granulosa cells and also in the oocytes.
The highest expression of both ER␣ and ER␤ was observed
at the beginning of the last trimester. The expression patterns
of the ERs were similar, but the immunostaining of ER␤ was
more pronounced than that of ER␣. Similarly, ER␤ is the
dominant ER in the adult ovary, in which it is expressed
mainly in the granulosa cells of growing follicles, whereas
ER␣ is localized to theca and interstitial cells (4). Weak expression of 17HSD1 was detected at the age of 17 wk, whereafter it increased toward term. However, the highest expression of 17HSD7 was observed as early as at wk 17, this being
the earliest time point at which it was studied. After 17 wk
17HSD7 expression decreased, and it was negative at the 36th
Vaskivuo et al. • Fetal Ovary Estrogen Receptors and Metabolic Enzymes
week. Expression of 17HSD2 was negative in all samples
studied.
Neither of the ERs was clearly present in the ovary before
the 20th fetal week. Therefore, it seems likely that estrogen
action is not essential for early ovarian development or follicle formation. This is supported by knowledge gained from
studies on ER knockout animals (17–20). The ovaries of
␣ERKO, ␤ERKO, and ␣␤ERKO mice develop normally (18 –
20). Although ␣ERKO female mice are infertile, they show
normal follicular development until preovulatory stages, in
which follicular growth is arrested (20). ␤ERKO female mice
seem to suffer only from mild functional ovarian disorders
because they are capable of ovulating and bearing offspring
(18). On the other hand, as expected, ␣␤ERKO mice have the
most severely disturbed phenotype: ovarian development
seems to follow a normal path at least to the stage at which
primordial follicles are developed. The finding that estrogen
receptors are predominantly expressed in the human ovary
only after the follicles have developed indicates that early
ovarian development in humans is similarly independent of
estrogen action. However, after the development of ovarian
follicles the ovaries of ␣␤ERKO mice start to express various
abnormal features. In postnatal life their ovaries undergo
redifferentiation from follicles into structures that resemble
seminiferous tubules of the testis (19).
Interestingly, the expression of ERs in fetal mouse ovary
seems to differ from that seen in humans. ER␣ seems to be
the dominant ER in mouse ovary during fetal life, whereas
ER␤ expression is negligible or negative (26). The present
data suggest that this might not be the case in the human fetal
ovary. These species-specific differences in ER expression
may reflect differences in the role of ERs during ovarian
development.
Women with aromatase deficiency display disturbed
ovarian function, with morphological findings similar to
those in polycystic ovary syndrome and in ␣ERKO mice (27,
28). It seems that early ovarian development and formation
of follicles occurs properly, and the observed defects are the
result of a lack of estrogen action in postnatal life. Although
the present findings and our previous observation that aromatase is expressed in the fetal ovary (8) suggest that the fetal
ovary is capable of producing estrogens, the actual role of
estrogens in ovarian development is difficult to assess, owing
to the compensatory role of maternal estrogens during fetal
life (28).
The enzyme 17HSD1 is the key regulator of estrogen metabolism in the adult human ovary, converting E1 into more
potent E2 (11, 29). Whereas 17HSD7 performs a similar function to that of 17HSD1 in catalyzing E1 to E2, it is less efficient
(12). In addition to the conversion of E1 to E2, human 17HSD7
has other substrates in steroid hormone metabolism (12) and
it participates in cholesterol biosynthesis (30). The expression
of 17HSD1 had a pattern clearly similar to those of the ERs.
The enzyme was first observed at the 17th fetal week, and
clear expression of both ER␣ and ER␤ was detected shortly
thereafter. The expression of 17HSD1 increased toward term,
but that of 17HSD7 had a different pattern: it was seen at the
17th fetal week, and it decreased toward term. Its expression
was negligible or negative after the 28th fetal week when the
expression patterns of 17HSD1 and ER␤ reached their max-
J Clin Endocrinol Metab, June 2005, 90(6):3752–3756
3755
ima. The localization of 17HSD1 was clearly restricted to the
granulosa cells, whereas no expression was found in the
oocytes. The localization of 17HSD7 was not as clear as that
of 17HSD1, but it was mainly found in the stromal and
pregranulosa cells. The fact that 17HSD7 has an important
role in the rodent ovary but a less clear function in humans
underlines species-specific differences in the reproductive
system. Its expression in early fetal ovaries, however, emphasizes its possible function in estrogen metabolism during
human ovarian development (12, 16).
The concurrence of follicle formation with the expression
of estrogen-converting enzymes and ERs could indicate that
estrogens play a physiological role in late fetal life (31). We
have previously discovered that aromatase is expressed in
the human fetal ovary, indicating that the capacity to produce estrogens from precursor steroids exists already before
birth (8). Together with the present finding, that 17HSD1 is
expressed from midgestation onward, the presence of aromatase further augments the hypothesis that during the last
trimester fetal ovary is capable of producing estrogens and
E2. To further underline the importance of fetal ovary as a
functioning estrogen-producing organ, 17HSD2, which inactivates E2 to less potent E1, was completely absent in all
analyzed stages of ovarian development.
The present work demonstrates that ERs and estrogenconverting enzymes are expressed in the human fetal ovary.
The high expression of ER␣, ER␤, and 17HSD1 during the
last trimester of fetal life and the highest expression of
17HSD7 during midgestation suggest different roles of
17HSD1 and 17HSD7 in development. Furthermore, the lack
of 17HSD2 and increasing activity of 17HSD1 during gestation reflect increased estradiol production toward term. The
simultaneous appearance of ERs and 17HSD1 and 17HSD7
further indicates that the machinery for estrogen action already exists during fetal life and suggests a possible role of
estrogens in the developing ovary after follicle formation.
Acknowledgments
Received September 14, 2004. Accepted March 3, 2005.
Address all correspondence and requests for reprints to: Juha S.
Tapanainen, M.D., Ph.D., Department of Obstetrics and Gynecology,
FI-90014 University of Oulu, Finland. E-mail: [email protected].
This work was supported by grants from the Academy of Finland,
Sigrid Juselius Foundation, and Oulu University Hospital.
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