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Participation in the Mouse Uterine Epithelial Secretory Response1
Department of Veterinary Biosciences, University of Illinois (D.L.B., T.S., P.S.C.), Urbana, Illinois 61802; the Department of Anatomy, University of California (T.K., G.R.C.), San Francisco, California 94143; and the Departments of Biochemistry and Child Health, University of Missouri (J.A.T., D.B.L.), Columbia, Missouri 65211
Address all correspondence and requests for reprints to: Dr. Paul Cooke, Department of Veterinary Biosciences, University of Illinois, 2001 South Lincoln Avenue, Urbana, Illinois 61802. E-mail: p-cooke{at}uiuc.edu
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Abstract |
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-positive stroma; epithelial ER is
neither necessary nor sufficient for E2-induced uterine
epithelial mitogenesis. In the present study, we addressed the question
of whether production of uterine epithelial secretory proteins and
their messenger RNAs (mRNAs) requires ER in stroma, epithelium, or
both by analyzing tissue recombinations composed of uterine tissue from
adult ER knockout (ko) and neonatal BALB/c (wt) mice. Stroma (S) and
epithelium (E) were separated by trypsinization, and four types of
uterine tissue recombinants were prepared: wt-S + wt-E, wt-S + ko-E,
ko-S + wt-E, and ko-S + ko-E. These tissue recombinants were grown as
subrenal capsule grafts in intact female nude mice for 4 weeks, at
which time the hosts were ovariectomized. To assess the production of
secretory proteins and their mRNAs, 1 week after ovariectomy the hosts
were given three daily injections of oil or E2 (100 ng),
and then 24 h later the grafts were recovered and used for either
ER or lactoferrin (LF) immunohistochemistry. To assess steady state
mRNA levels by Northern blotting, hosts received one injection of oil
or E2 24 h before harvest. ER immunohistochemistry was
used to monitor the completeness of tissue separation. In wt-S + wt-E
tissue recombinants from E2-treated hosts, the epithelium
stained intensely for LF (an abundant E2-dependent uterine
secretory protein), whereas similar tissue recombinants from
oil-treated hosts showed minimal immunostaining. Conversely, LF
immunostaining was minimal in wt-S + ko-E grafts from both oil- and
E2-treated hosts. LF staining was also minimal in ko-S +
ko-E and ko-S + wt-E tissue recombinants regardless of hormone
treatment. For Northern analyses, the epithelial content of the tissue
recombinants was monitored using the reference epithelial transcript,
E-cadherin. While all tissue recombinant groups expressed E-cadherin
mRNA, wt-S + wt-E tissue recombinants from E2-treated hosts
produced a strong, single 2.6-kb band of LF mRNA. LF transcripts were
minimal or absent in all other tissue recombinant types. Northern
blotting results identical to those seen for LF were also observed for
the uterine secretory protein complement component C3. Our data
demonstrate that both stromal and epithelial ER are required for the
production of LF protein and of LF or C3 mRNAs in response to
E2. Thus, in contrast to E2-induced epithelial
mitogenesis, which requires only stromal ER, both epithelial and
stromal ER are necessary for the production of
E2-dependent epithelial secretory proteins. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
as E2 does not stimulate UtE mitogenesis in the ER
knockout (ERKO) mouse (3). ER is expressed in both the stroma and
epithelium of the adult murine uterus, and we have recently shown that
stimulation of UtE mitogenesis by E2 is mediated indirectly
through stromal ER (1). These results indicated that epithelial
ER are neither necessary nor sufficient to mediate the epithelial
mitogenic response to E2 and raise obvious questions
regarding the role of epithelial ER in the normal uterine response
to E2.
Estradiol is also essential for the production of many UtE secretory
proteins. A number of these proteins have been identified and include
lactoferrin (LF) (4), the most abundant UtE secretory protein, the
adhesion molecule CAM 105, keratan sulfate proteoglycan (reviewed in
Refs. 3, 5), and complement component C3 (6). The inability of ERKO
mice to produce LF in response to E2 treatment (3)
indicates that ER is essential for mediating the effects of
E2 on secretory protein production by UtE cells.
Although epithelial ER does not appear to be involved in the
induction of epithelial proliferation by E2, some work has
indicated that epithelial ER may be obligatory for the production of
E2-regulated secretory products by UtE. Yamashita
et al. (7) observed estrogen stimulation of LF in the
UtE of neonatal CD-1 mice. The UtE of neonatal CD-1 mice contain both
ER-positive and ER-negative cells, but LF production was only
detected in ER-positive UtE cells. These results suggest that
epithelial ER expression could be a prerequisite for secretory
protein production in UtE and that the effects of E2 on UtE
secretory proteins may be elicited directly via epithelial ER. Work
indicating that epithelial androgen receptors (AR) are obligatory for
secretory protein production in prostate and seminal vesicle (8, 9) is
also consistent with this hypothesis. However, it has not been
established whether ER in the stroma are also required or whether
epithelial ER alone are sufficient for mediating the E2
induction of UtE secretory protein production.
We have recently developed a new experimental system that uses tissues
from the ERKO mouse to study the mechanism of E2 action on
female genital tract epithelia (1, 10). The crucial feature of this
system involves enzymatically separating and recombining ERKO uterine
epithelium and stroma with that of the wild-type ER-positive BALB/c
mouse (1). This tissue separation/recombination technique (reviewed in
Ref. 10) provides a unique method for experimentally controlling the
presence of ER in either stroma or epithelium. By analyzing the
effects of a lack of stromal and/or epithelial ER on E2
responses such as epithelial secretory protein production, the role of
ER in each tissue compartment can be definitively determined.
The aim of the present study was to use the tissue recombinant
methodology to examine the relative roles of stromal and epithelial
ER in E2 regulation of the UtE secretory proteins, LF
and C3. Our results show that both stromal and epithelial ER are
required for the production of these E2-dependent
epithelial secretory proteins, in contrast to E2-induced
UtE mitogenesis, which requires only stromal ER.
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Materials and Methods |
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Tissue recombination
Uteri were removed from adult (90–120 day) ERKO and neonatal
(0–3 day) BALB/c mice that had been killed by CO2
asphyxiation or decapitation, respectively. Uterine gland formation in
BALB/c mice begins at approximately 1 week of age (12) and continues
throughout the neonatal period. Once glandular invasion of the
underlying stroma has become extensive, it is not possible to isolate
stromal tissue free of contaminating glandular epithelium, and thus
neonatal BALB/c mice were used for all studies described here. The ERKO
uterine epithelium also forms glands (13), but these glands are
rudimentary and do not preclude effective separation of the stromal and
epithelial components of the ERKO uterus. Therefore, adult ERKO females
were used for all experiments to maximize the amount of uterine tissue
that could be obtained from these animals. We have previously shown
that tissue recombinations of uterus prepared with combinations of
neonatal and adult tissue grow well and manifest normal physiological
responses (14, 15).
The homozygous ERKO genotype, initially established by multiplex PCR, was verified by confirming the presence of hypoplastic uteri and hyperemic ovaries (11). The tissue separation and recombination procedure for uterine epithelium and stroma has been described previously (14, 15, 16). Briefly, uteri from BALB/c and ERKO mice were trimmed, opened lengthwise, and incubated with 1% trypsin (Life Technologies, Grand Island, NY) in calcium- and magnesium-free HBSS for 90 min at 4 C. Uterine stroma and epithelium were then separated using gentle mechanical manipulation, as described previously (1). Stroma and epithelium obtained by this method are devoid of contamination with the other tissue (16). Uterine tissue recombinations were prepared and incubated overnight on 1% agar medium (Difco, Detroit, MI) as described previously (1). The following four tissue recombinants were prepared with ERKO (ko) or wild-type BALB/c (wt) uterine stroma (S) and epithelium (E): wt-S + wt-E, wt-S + ko-E, ko-S + wt-E, and ko-S + ko-E. Tissue recombinants were transplanted under the renal capsule of intact adult female athymic (nude) mice (Harlan Sprague-Dawley). Grafts were allowed to grow for 4 weeks, at which time all hosts were ovariectomized.
Hormone treatments
One week after ovariectomy, hosts were given oil or
E2. To determine the mechanism by which E2
elicits epithelial secretory mRNA or protein production in uterine
tissue recombinants, host animals received one or three daily
injections, respectively, of E2 (100 ng; Sigma Chemical Co., St. Louis, MO) in 0.05 ml corn oil, or vehicle
alone. Tissue recombinants were harvested 24 h after the final
injection. For Northern blotting, tissue recombinants were immediately
flash-frozen in liquid nitrogen upon harvesting and stored at -80 C
for subsequent RNA isolation. For immunohistochemical analysis,
harvested tissue recombinants were fixed in 10% neutral buffered
formalin (Sigma Chemical Co.) for 12 h at 4 C,
paraffin embedded, and sectioned at 6 µm.
RNA isolation and Northern blot analysis for lactoferrin,
complement component C3, and E-cadherin
Total RNA was extracted from uterine tissue recombinants and
whole BALB/c and ERKO uteri using the Qiagen RNeasy total RNA kit
(Qiagen, Inc., Chatsworth, CA). After quantification, RNA
(9 µg) was denatured at 55 C in the presence of deionized formamide
and 37% formaldehyde. RNA was resolved on a 1.5% agarose-formaldehyde
gel and blotted on synthetic, uncharged nylon membranes (Duralon-UV,
Stratagene, La Jolla, CA). RNA was transferred in 20
x SSC (3.0 M NaCl and 0.3 M sodium citrate, pH 7.0). The RNA in gels
and filters was visualized with ethidium bromide by UV
transillumination to confirm appropriate transfer, ensure the integrity
of RNA, and check the loading of equivalent amounts of total RNA.
Blots were prehybridized for 1.5 h at 65 C in QuikHyb hybridization solution (Stratagene). E-Cadherin complementary DNA (cDNA) probe was premixed with previously sheared and denatured salmon sperm DNA (0.1 mg/ml; Stratagene) and added directly to the QuikHyb for hybridization at 65 C for 2.5 h. The mouse cDNA probe for E-cadherin was a 1800-bp PvuII fragment of a 4300-bp cDNA obtained from Dr. Masatoshi Takeichi (Kyoto University, Kyoto, Japan). Blots were then stripped and rehybridized with LF and C3 cDNAs. To assess RNA load levels between lanes, the expression of 28S ribosomal RNA was assessed. The mouse cDNA probe for LF was a 1930-bp EcoRI fragment obtained from Dr. Christina Teng (NIEHS, Research Triangle Park, NC); the rat cDNA probe for C3 was a 840-bp EcoRI/PstI fragment obtained from Dr. Barry Komm (Wyeth-Ayerst Laboratories, Inc., Radnor, PA). The cDNA probes were labeled with [32P]deoxy-CTP by random oligonucleotide priming (Multiprime DNA labeling systems, Amersham, Arlington Heights, IL). Probes were used at 2 x 108 cpm/µg. Membranes were washed twice for 15 min each time in 2 x SSC and 0.1% SDS at room temperature, then once for 3–5 min in 0.1 x SSC and 0.1% SDS at 65 C and exposed on Kodak X-Omat/AR film (Eastman Kodak Co., Rochester, NY).
ER and LF immunohistochemistry
To immunohistochemically detect ER in uterine tissue
recombinants, antigen retrieval followed by an avidin-biotin complex
method were used as described previously (1). Nonspecific binding was
blocked using Super Block (Pierce, Rockford, IL). Slides
were incubated with either the primary monoclonal antibody (NCL-ER-LH2,
Novocastra, Burlingame, CA) or a control nonspecific IgG (Dako Corp., Carpinteria, CA) and washed, and then the secondary
biotinylated antimouse antibody (LSAB2 kit, Dako Corp.)
was applied. Diaminobenzidine (Dako Corp.) was
precipitated by streptavidin-conjugated horseradish peroxidase to
visualize the immunoreaction.
To immunohistochemically detect LF in uterine tissue recombinants as well as whole BALB/c and ERKO uteri, slides were deparaffinized, rehydrated, and washed in Tris-buffered saline with Tween-20 for 5 min. Slides were then submerged in 3% H2O2 for 10 min to inactivate endogenous peroxidase activity and washed in Tris-buffered saline with Tween-20 (5 min). Nonspecific binding was blocked using BSA. Slides were incubated with either LF antiserum containing the primary antibody or normal rabbit serum as a control at 24 C for 2 h. The LF antiserum was obtained from Dr. Christina Teng at NIEHS and has been used extensively for LF immunodetection (7, 17). The slides were washed, and a secondary donkey antirabbit IgG horseradish peroxidase-linked F(ab')2 fragment was applied. The slides were again washed, and diaminobenzidine in imidazole-containing buffer was used as the chromogen. Finally, slides were dehydrated through graded ethanol washes followed by a xylene dip and then coverslipped. Tissue recombinants from at least three separate experiments were examined using the ER and LF immunostaining procedures.
Image and data analysis
All images were captured using an Olympus Corp.
Vanox Photomicroscope with planapochromatic lenses and a Sony Digital
Photo Camera DKC-5000 (Sony, Tokyo, Japan) interfaced to a Macintosh
computer using Adobe Photoshop software (Adobe Systems Inc., San Jose,
CA). Northern blotting results were replicated at least four
times for each tissue recombinant type, derived from three or four
separate experiments. Relative levels of epithelial mRNA from the
various tissue recombinant groups were monitored between lanes in the
membrane by hybridizing for the reference epithelial transcript,
E-cadherin.
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Results |
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The E2 treatment regimen used in the present study has been
shown to produce optimal induction of UtE LF protein (7) and mRNA (3)
in vivo. LF protein expression was strong in epithelium of
wt-S + wt-E tissue recombinants from E2-injected host
animals, but immunohistochemical staining was faint in the same type of
grafts from oil-injected hosts. Furthermore, LF protein expression was
minimal in wt-S + ko-E tissue recombinants from both oil- and
E2-treated hosts (Fig. 1
). In
ko-S + wt-E and ko-S + ko-E tissue recombinants from both
E2-and oil-treated hosts, LF protein expression was also
minimal or absent. Consistent with our immunohistochemical results,
tissue recombinants composed of wt-S + wt-E from E2-treated
hosts showed strong LF mRNA expression, comparable to that observed in
uteri from intact BALB/c mice (Fig. 2).
As expected, LF mRNA expression was low in these same tissue
recombinants from oil-treated hosts. Furthermore, LF mRNA was minimal
or undetectable in wt-S + ko-E, ko-S + wt-E, and ko-S + ko-E tissue
recombinants from either E2- or oil-treated hosts.
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).
Similar RNA loading was observed under UV illumination (data not
shown); however, more critical than the amount of total RNA loaded was
verification that epithelial RNA was present in all tissue
recombinants. The impaired proliferation of ERKO or BALB/c epithelium
when associated with ERKO stroma (1) suggested that the decreased LF or
C3 mRNA signal in tissue recombinants prepared with ko-S could be due
to a lack of epithelium in these tissue recombinants rather than to a
deficiency in LF or C3 mRNA production in these epithelia caused by the
association with ko-S. To address this possibility, we examined the
expression of an epithelial-specific marker, E-cadherin mRNA (18).
Although E-cadherin levels varied between the different tissue
recombinant types, a 5.1-kb band of E-cadherin was observed in each of
the tissue recombinant types (Fig. 2). Therefore, the lack of LF or C3
mRNA in the wt-S + ko-E tissue recombinants from E2-treated
hosts and the lack of LF or C3 mRNA expression in all tissue
recombinants prepared with ko-S (ko-S + wt-E and ko-S + ko-E) are
clearly not due to an absence of epithelium in these tissue
recombinants, a finding consistent with our immunohistochemical
results.
According to the manufacturer, the LH2 antibody is specific to several
epitopes along the entire length of the ER molecule, some of which
have high homology to ERß. However, the lack of ER staining in ERKO
uterine tissue with this antibody (Fig. 3) despite the reported expression of
ERß mRNA in ERKO uterus (2) suggests that the LH2 antibody staining
seen in the present experiments is due to ER. Therefore, ER
immunohistochemistry confirmed the expected ER expression in
epithelium and stroma of the various tissue recombinants (Fig. 3) and
demonstrated the efficacy of the tissue separation and recombination
technique. Tissue recombinants composed of wt-S + wt-E showed high
levels of dark nuclear staining in both epithelial and stromal cells,
indicating the presence of ER. In wt-S + ko-E, ER was detected in
stroma but not in epithelium. Conversely, ER was detected by
immunohistochemistry in epithelium only but not stroma in ko-S+ wt-E
tissue recombinants and in neither epithelium or stroma in ko-S + ko-E
tissue recombinants (Fig. 3).
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Discussion |
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, again consistent with the results
of earlier studies (3). The large differences in LF mRNA and protein
production with and without E2 in conjunction with the
tissue separation/recombination technique allow unequivocal conclusions
to be drawn about the dependence of LF production on the binding of
E2 to ER of uterine stroma vs. uterine
epithelium.
Our histochemical results for LF clearly indicate that both stromal and
epithelial ER must be present to obtain normal LF protein expression
in response to E2 treatment. In the present study,
expression of neither stromal nor epithelial ER alone resulted in
significant LF expression; thus, the absence of either stromal or
epithelial ER essentially precludes LF protein production by the
epithelium. Our immunostaining results were corroborated by Northern
blot for LF mRNA production in the various types of tissue recombinants
and again demonstrated that both stromal and epithelial ER are
necessary for the production of LF mRNA by the epithelium.
It has been postulated that LF is directly regulated by estradiol
actions on uterine epithelium in the rodent (22, 23) based partially on
the existence of an estrogen response element in the LF gene (22). The
present results show that although epithelial ER is necessary for LF
production, the presence of both epithelial ER and an estrogen
response element in the LF gene are not by themselves sufficient to
allow LF production in response to E2 in the absence of
stromal ER, emphasizing the critical role of stromal ER in normal
uterine E2 responsiveness.
The results showing that epithelial ER are necessary for epithelial
secretory production are consistent with reports of an estrogen
response element in the LF gene (22) discussed above and the
observation that LF expression was detected in ER-positive, but not
ER-negative, uterine epithelium of early postnatal CD-1 mice (7). More
recently, epithelial LF expression in fetal uteri of CD-1 mice after
diethylstilbestrol (DES) exposure has been reported (20). Although
fetal and neonatal uterine epithelia appear to be ER negative (24), DES
exposure rapidly increases the expression of UtE ER mRNA and protein
(7, 25) in neonatal mice, suggesting that uterine epithelium expressing
LF in the DES-treated fetuses might have also been expressing ER,
although this was not evaluated.
To determine whether the findings related to the relative roles of
stromal and epithelial ER in LF production are generally applicable
to other secretory proteins, we also examined the role of stromal and
epithelial ER in the production of another E2-dependent
epithelial secretory protein, complement component C3. E2
injection produces a 25-fold increase in C3 mRNA in ovariectomized rats
(6). Analysis of the production of C3 mRNA in various tissue
recombinants suggests a common regulatory pathway for the production of
uterine secretory proteins. As shown for LF, ER-positive uterine
epithelia cannot make C3 mRNA in vivo in response to
E2 when associated with an ER-negative uterine stroma.
Likewise, epithelium in tissue recombinants consisting of
ER-positive uterine stroma and ER-negative uterine epithelia does
not make C3 mRNA in response to E2. C3 mRNA production is
stimulated by E2 only in the presence of both stromal and
epithelial ER.
Northern blot comparisons of mRNA for LF or C3 in the various tissue
recombinants are only semiquantitative because tissue recombinants can
contain varying amounts of epithelium. As mentioned above, pronounced
increases in uterine LF and C3 expression in response to E2
have been observed by others (4, 6); therefore, even substantial
variations in the epithelial content of the various tissue recombinants
used in the present study (e.g. 2- to 3-fold) cannot account
for the decreased LF and C3 expression observed in tissue recombinants
that lack stromal or epithelial ER and do not affect the overall
interpretation of our results. However, we also probed our Northern
blots for E-cadherin mRNA to monitor the epithelial presence in the
various tissue recombinants and to ensure that the lack of a LF mRNA
signal in tissue recombinants, such as wt-S + ko-E and ko-S + wt-E,
from E2-treated hosts was not due to an absence of
epithelium. E-Cadherin is universally and specifically expressed in
epithelial cells and has not been reported to vary with hormonal status
of the animal (26, 27). Despite the presence of epithelium in all
tissue recombinant groups, as indicated by E-cadherin mRNA expression,
only wt-S + wt-E tissue recombinants produced LF mRNA.
The present results indicating that both stromal and epithelial ER
are required for the production of E2-dependent UtE
secretory proteins, such as LF and C3, parallel our recent data
obtained with vaginal tissue recombinants, where both stromal and
epithelial ER are required for E2-induced vaginal
epithelial cornification, normal stratification, and mucification (28).
Terminal morphological and functional epithelial differentiation
therefore appear to require epithelial ER. However, these findings
with epithelial differentiation are in contrast to our results showing
that uterine, vaginal, and mammary gland epithelial proliferations in
response to E2 are mediated entirely through ER in the
stroma and that epithelial ER is neither necessary nor sufficient
for this process (1, 28, 29). Thus, ER mediation of
E2-induced epithelial proliferation and secretory protein
production differ in a very fundamental manner. Furthermore, the
present results showing that both stromal and epithelial ER are
necessary for the production of UtE secretory proteins along with our
previous demonstration that vaginal epithelial cornification and normal
stratification are also dependent on both stromal and epithelial ER
represent the first known in vivo epithelial responses to
E2 shown to require ER in each of these tissue
compartments (28). Although estrogen treatment has been shown to
increase epithelial progesterone receptor expression in
isolated UtE cells in vitro (30), the present results
showing the essential role of epithelial ER in
E2-induced secretory protein production represent the first
function described in vivo for UtE ER and are consistent
with our recent report in vagina describing the necessity of epithelial
ER for vaginal epithelial differentiation (28).
The role of ERß in LF production, if any, is unclear. The lack of LF
mRNA in ERKO mice (3), which express uterine ERß mRNA (2), suggests
that ERß by itself is not sufficient and that ER is necessary to
mediate E2-induced LF production. However, it is not
possible to determine from this previous work or our present data
whether ERß may be necessary for LF production, and whether the
absence of ERß would impair or eliminate LF production in a uterus
that expressed ER. The recent development of the ERß knockout
mouse should provide an unequivocal answer to this question.
Immunohistochemical staining for ER in uterine tissue recombinants
provided an important control for verifying the completeness of tissue
separation and allowed identification of tissue origin in heterotypic
tissue recombinants. ER immunostaining also showed that epithelial and
stromal cells maintain the expression of ER independent of the
presence or absence of ER in the adjoining tissue.
The precise role of stromal ER in allowing uterine epithelium to
produce E2-dependent secretory proteins is not clear.
Occupancy of stromal ER by E2 could stimulate the
stromal cells to produce a growth factor(s), which then acts on the
epithelium (10, 31), presumably in concert with changes induced by
occupancy of epithelial ER by E2, to result in secretory
protein production. Conversely, occupancy of stromal ER by
E2 could reduce or abolish the production of some
inhibitory stromal factor(s) that acts tonically under normal
circumstances to prevent secretory protein production; the constitutive
expression of secretory proteins that are normally induced in response
to E2 by cultured UtE cells is consistent with this
hypothesis (5).
The effects of E2 on stromal cells could also involve changes in the stromal extracellular matrix (ECM) and/or basement membrane, which then secondarily influences epithelial cell secretory activity. The structural composition of the ECM and basement membrane is influenced by stromal regulatory factors and is important for normal epithelial growth and differentiation (32, 33, 34, 35). E2 could directly act on stromal cells to elicit changes in the ECM or the epithelial basement membrane, with secondary effects on the secretory activity of the adjacent epithelial cells. Similarly, E2 has been shown to produce changes in the production of stromal growth factors, which could then have paracrine effects on surrounding stromal cells that are essential to allow secretory protein production by the overlying epithelial cells. For example, E2 up-regulates stromal transforming growth factor-ß expression, and transforming growth factor-ß-stimulated collagen production by fibroblasts contributes to the functional stromal organization (36), which is required to support the integrity of the epithelial basement membrane and, therefore, normal epithelial growth and differentiation.
The stromal changes induced by occupancy of stromal ER by
E2 that are essential for allowing uterine epithelium to
produce E2-dependent secretory proteins may also involve
mechanisms other than simple changes in growth factor signaling by
stromal cells. For example, E2 stimulation of stromal cells
could result in changes in enzymatic modification or changes in
bioavailability of growth factors, their receptors, binding proteins,
or enzymes that modify the activity of the molecules involved (10, 31),
thus making identification of any stromal paracrine factor(s)
particularly elusive.
The concept that epithelial ER could be involved in the production
of UtE proteins, but not mitogenesis, is supported by previous work in
the male. The use of a mutant mouse strain lacking functional AR has
provided information on the relative role of stromal and epithelial AR
in androgen-induced epithelial proliferation and secretory protein
production in organs such as the prostate and seminal vesicle. Mice
with the testicular feminization (Tfm) mutation have a frameshift
mutation in the AR gene that results in a truncated and inactive AR
(37). Affected males have testes and produce testosterone, but their
cells cannot respond to androgens. Wild-type mesenchyme from the fetal
urogenital sinus (UGS; the precursor of the prostate in the male) can
induce Tfm bladder epithelium to form prostatic epithelium that
responds mitogenically to androgen (8). However, this epithelium does
not express AR or prostatic secretory proteins (9). Thus, epithelial AR
appear necessary for the production of secretory proteins even though
epithelial growth and morphogenesis appear to be mediated by
mesenchymal/stromal AR. Although recombining tissues from Tfm and
wild-type mice has provided important information, there are certain
intrinsic limitations to this system that preclude examining the role
of stromal AR in prostatic epithelial secretory activity. In the
absence of stromal AR, the UGS of a Tfm male mouse cannot form prostate
and instead differentiates into vagina. Hence, an AR-negative UGS Tfm
mesenchyme plus wild-type bladder epithelium tissue recombinant
does not produce prostate. Therefore, the necessity of stromal AR for
prostatic development means that it is impossible to obtain an
AR-deficient prostatic stroma, so the autonomous ability of prostatic
epithelial AR to mediate the production of prostatic androgen-dependent
secretory proteins when associated with an AR-deficient prostatic
stroma cannot be evaluated. Similarly, this limitation means that the
necessity of stromal AR for mediating the production of
androgen-dependent secretory proteins also cannot be definitively
established. Our finding that both stromal and epithelial ER are
necessary for normal E2-induced UtE secretory protein
production strongly implies that a similar mechanism is involved in AR
mediation of androgen-induced secretory protein production in prostate
and that stromal AR may also be obligatory for prostatic epithelial
secretory protein production in vivo.
In summary, E2-induced UtE secretory responses, such as the
production of LF mRNA and protein and C3 mRNA, require both stromal and
epithelial ER. These are the first known functions described for UtE
ER in vivo. The mechanism of E2-induced UtE
secretory protein production is mechanistically different from that of
E2-induced uterine and vaginal epithelial proliferation,
but the requirement for both stromal and epithelial ER for
E2-stimulated production of UtE secretory proteins is
similar to the mechanism we have recently described for
E2-induced vaginal epithelial differentiation (28). Taken
together, UtE secretory protein production and vaginal epithelial
differentiation represent the first known epithelial responses to
E2 shown to require both stromal and epithelial ER. The
obligatory requirement for epithelial ER in UtE secretory protein
production parallels AR mediation of secretory protein production in
male reproductive organs and indicates that dependence of secretory
protein production on epithelial sex steroid receptors such as AR and
ER may be common to both male and female reproductive organs.
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Acknowledgments |
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Footnotes |
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Received April 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621
knockout mouse. Biol Reprod 59:470–475This article has been cited by other articles:
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 H. Cardenas and W. F. Pope Attenuation of Estrogenic Effects by Dihydrotestosterone in the Pig Uterus Is Associated with Downregulation of the Estrogen Receptors Biol Reprod, February 1, 2004; 70(2): 297 - 302. [Abstract] [Full Text] [PDF]
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K. S. Grant and C. R. Wira Effect of Mouse Uterine Stromal Cells on Epithelial Cell Transepithelial Resistance (TER) and TNF{alpha} and TGF{beta} Release in Culture Biol Reprod, September 1, 2003; 69(3): 1091 - 1098. [Abstract] [Full Text] [PDF]
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 P. M. Ismail, J. Li, F. J. DeMayo, B. W. O'Malley, and J. P. Lydon A Novel LacZ Reporter Mouse Reveals Complex Regulation of the Progesterone Receptor Promoter During Mammary Gland Development Mol. Endocrinol., November 1, 2002; 16(11): 2475 - 2489. [Abstract] [Full Text] [PDF]
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C. T. Teng, C. Beard, and W. Gladwell Differential Expression and Estrogen Response of Lactoferrin Gene in the Female Reproductive Tract of Mouse, Rat, and Hamster Biol Reprod, November 1, 2002; 67(5): 1439 - 1449. [Abstract] [Full Text] [PDF]
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T. Sato, G. Wang, M. P. Hardy, T. Kurita, G. R. Cunha, and P. S. Cooke Role of Systemic and Local IGF-I in the Effects of Estrogen on Growth and Epithelial Proliferation of Mouse Uterus Endocrinology, July 1, 2002; 143(7): 2673 - 2679. [Abstract] [Full Text] [PDF]
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H. Engelhardt, B. A. Croy, and G. J. King Conceptus Influences the Distribution of Uterine Leukocytes During Early Porcine Pregnancy Biol Reprod, June 1, 2002; 66(6): 1875 - 1880. [Abstract] [Full Text] [PDF]
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 D. L. Buchanan, S. Ohsako, C. Tohyama, P. S. Cooke, and T. Iguchi Dioxin Inhibition of Estrogen-Induced Mouse Uterine Epithelial Mitogenesis Involves Changes in Cyclin and Transforming Growth Factor-{beta} Expression Toxicol. Sci., March 1, 2002; 66(1): 62 - 68. [Abstract] [Full Text] [PDF]
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A. D. Papaconstantinou, B. R. Fisher, T. H. Umbreit, P. L. Goering, N. T. Lappas, and K. M. Brown Effects of {beta}-Estradiol and Bisphenol A on Heat Shock Protein Levels and Localization in the Mouse Uterus Are Antagonized by the Antiestrogen ICI 182,780 Toxicol. Sci., October 1, 2001; 63(2): 173 - 180. [Abstract] [Full Text] [PDF]
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H. Wang, H. Eriksson, and L. Sahlin Estrogen Receptors {alpha} and {beta} in the Female Reproductive Tract of the Rat During the Estrous Cycle Biol Reprod, November 1, 2000; 63(5): 1331 - 1340. [Abstract] [Full Text]
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D. L. Buchanan, T. Sato, R. E. Peterson, and P. S. Cooke Antiestrogenic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Mouse Uterus: Critical Role of the Aryl Hydrocarbon Receptor in Stromal Tissue Toxicol. Sci., October 1, 2000; 57(2): 302 - 311. [Abstract] [Full Text] [PDF]
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A. D. Papaconstantinou, T. H. Umbreit, B. R. Fisher, P. L. Goering, N. T. Lappas, and K. M. Brown Bisphenol A-Induced Increase in Uterine Weight and Alterations in Uterine Morphology in Ovariectomized B6C3F1 Mice: Role of the Estrogen Receptor Toxicol. Sci., August 1, 2000; 56(2): 332 - 339. [Abstract] [Full Text] [PDF]
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T. Kurita, K.-j. Lee, P. S. Cooke, J. P. Lydon, and G. R. Cunha Paracrine Regulation of Epithelial Progesterone Receptor and Lactoferrin by Progesterone in the Mouse Uterus Biol Reprod, April 1, 2000; 62(4): 831 - 838. [Abstract] [Full Text]
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 J. F. Couse and K. S. Korach Estrogen Receptor Null Mice: What Have We Learned and Where Will They Lead Us? Endocr. Rev., June 1, 1999; 20(3): 358 - 417. [Abstract] [Full Text]
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D. Chen, P. Ganapathy, L.-J. Zhu, X. Xu, Q. Li, I. C. Bagchi, and M. K. Bagchi Potential Regulation of Membrane Trafficking by Estrogen Receptor {alpha} via Induction of Rab11 in Uterine Glands during Implantation Mol. Endocrinol., June 1, 1999; 13(6): 993 - 1004. [Abstract] [Full Text]
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