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Biology of Reproduction 62, 821-830 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Paracrine Regulation of Epithelial Progesterone Receptor by Estradiol in the Mouse Female Reproductive Tract1

Takeshi Kuritab, Ki-jun Leec, Paul S. Cookec, Julia A. Taylorc, Dennis B. Lubahnc, and Gerald R. Cunha2,,a

a Department of Anatomy, University of California, San Francisco, California 94143 b Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802 c Departments of Biochemistry and Child Health, University of Missouri, Columbia, Missouri 65211


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of progesterone receptor (PR) by estradiol-17ß (E2) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E2 induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor {alpha} (ER{alpha}) knockout (ERKO) mice showed that ER{alpha} is essential for E2-induced PR expression in both vaginal epithelium and stroma, and for E2-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E2 was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E2 only in wild-type epithelium and/or stroma. Hence, in vagina, E2 induces PR directly via ER{alpha} within the tissue. Conversely, E2 down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E2 requires stromal, but not epithelial, ER{alpha}. In vitro, isolated uterine epithelial cells retained a high PR level with or without E2, which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E2 down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ER{alpha}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen and progesterone are essential for growth and function of the female reproductive tract. Estrogenic and progestational actions on target cells are mediated through estrogen receptors (ER) and progesterone receptors (PR), respectively. Both PR and ER are members of the steroid-retinoid receptor superfamily, and function as steroid-modulated transcription factors [1]. Two different ER genes, ER{alpha} and ERß, have been described, and their amino acid sequences are highly homologous in the DNA binding and ligand binding domains [2]. Although ER{alpha} and ERß show significant differences in affinity to certain estrogenic compounds [2, 3], both can bind estrogens and transactivate genes that are regulated through estrogen response elements [4]. ER{alpha} and ß proteins have been detected in many of the cell types of rodent uteri [5, 6], but studies on ER{alpha} knockout (ERKO) mice demonstrated that ER{alpha} is essential for estrogen-induced proliferation and secretory protein production in the uterus [7].

In most species, the PR is composed of two ligand-binding forms (long B form and N-terminal truncated A form) derived from one gene [8]. Recent studies with PR knockout (PRKO) mice showed that PR is essential for secondary sexual development and function of progesterone target organs. For example, decidualization, a progestational action in uterus, does not occur in uteri of PRKO mice [9].

The ER and PR levels are thought to be critical in determining cell responsiveness to steroids, and thus receptor regulation has been studied extensively. PR is one of most well-documented estrogen-regulated genes. In many target tissues, both normal and neoplastic, PR is induced by estrogen and is widely recognized as a marker for estrogen action [10]. In vitro studies have shown that human, rat and rabbit PR are induced through binding of the occupied ER to multiple estrogen-responsive regions in the 5'-region of PR gene [11–13]. In many species, estrogen up-regulates PR in almost all uterine cell types including the epithelium [14–16]. These reports are consistent with a model of estrogen regulation of PR in which occupied ER binds to the PR promoter and activates transcription of the PR gene. In contrast, estrogen administration reduces radiolabeled progesterone binding [17, 18] and PR immunoreactivity [19–21] in rat and mouse uterine epithelium. Thus, the mechanism of estrogen action on rodent uterine epithelium appears to differ from that of many other species and remains unexplained.

Many genes have been identified which are regulated by estrogen in tissues, and it has been tacitly assumed that the hormonal effects on a cell are mediated through receptors in the responding cell. Nonetheless, we have shown that the mitogenic effects of androgen on prostatic epithelium [22], and of estradiol-17ß (E2) on uterine [23], vaginal [24], and mammary epithelia [25], and the inhibitory effect of progesterone on uterine epithelial proliferation [26] are all mediated via stromal hormone receptors. Hence, in male and female reproductive tracts, proliferation of epithelial cells is regulated by steroids through a paracrine mechanism mediated via stromal steroid receptors. In all cases, epithelium also expresses functional steroid receptors, but epithelial steroid receptors are not required for the regulation of the epithelial proliferation. This fact suggests that tissue responses to a steroid may not be direct even if the responsive tissue expresses the appropriate hormone receptor.

The objective of the study reported here was to determine whether epithelial and/or stromal ER{alpha} is involved in regulation of epithelial PR by E2. Roles of ER{alpha} in regulation of PR by E2 were studied in mouse uterus and vagina by comparing PR expression in uterus and vagina between wild-type (wt) and ERKO mice. We also studied the respective roles of epithelial versus stromal ER{alpha} in the regulation of epithelial PR by E2 in mouse uterus and vagina, using tissue recombinants composed of wt or ERKO epithelium and stroma.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

All animals were maintained in accordance with the NIH Guide for Care and Use of Laboratory Animals, and all procedures described here were approved by the University of California-San Francisco and University of Illinois animal care and usage committees. ERKO mice were produced as described previously [27]. Adult female athymic nude mice and female Balb/c mice were purchased from Charles River (Wilmington, MA).

In all experiments, mice were ovariectomized, and hormone treatment was started 2 wk later. Daily dosages of 125 ng E2 in 0.1 ml peanut oil (Sigma, St. Louis, MO) were injected i.p. Ovariectomized female wt and ERKO mice (approximately 90 days old) and host athymic mice for ERKO/wt uterine or vaginal tissue recombinants received daily injection of E2 or oil for 3 days. Twenty-four hours after the last hormone injection, animals were killed to harvest tissue samples.

Tissue Separation, Recombination, and Grafting

Details of uterine [23] or vaginal [24] tissue recombination with ERKO mice have also been described. Briefly, uteri or vaginae from wt and ERKO mice were dissected free of adherent connective tissue and cut into small pieces (4–6 segments/uterine horn and 2–4 pieces/vagina). The pieces were enzymatically dissociated by 1% trypsin (1/250; Difco Laboratories, Detroit, MI), and then stroma and epithelium were physically separated using fine surgical instruments. Stroma and epithelium were recombined on agar plates and allowed to adhere during overnight culture. After overnight culture, the tissue recombinants were grafted under the renal capsules of female athymic nude mice. Approximately 1 mo after grafting, all hosts were ovariectomized and then received hormone treatments described above.

Primary Culture of Uterine Epithelial Cells

Mature Balb/c mice (approximately 90 days old) were ovariectomized, and 2 wk after ovariectomy uterine epithelial cells were isolated as described above. Cells were embedded in a 1:1 mixture of growth factor-reduced Matrigel (Becton Dickinson Labware, Bedford, MA) and rat tail collagen, and cultured in a 1:1 mixture of Dulbecco's Modified Eagle's and Ham's F-12 media (Gibco, Gaithersburg, NY) with transferrin (5 µg/ml; Sigma) and insulin (10 µg/ml; Gibco). After 3 days of cultivation, E2 (final concentration 10-10 to 10-5 M) or vehicle alone (absolute ethanol) was added. The cells were cultured 3 more days and then fixed within the extracellular matrix as described below.

Immunohistochemistry

Anti-human PR rabbit polyclonal IgG and anti human-ER{alpha} mouse monoclonal IgG 1D5 were purchased from DAKO (Carpenteria, CA). Tissues were fixed with 4% paraformaldehyde for 3 h on ice, processed into paraffin, and then sectioned at 6 µm. Immunohistochemical detection of ER{alpha} [23] and PR [26] in mouse tissues has been described. Immunoreactivity was developed for ER{alpha} and PR by Vectastain Elite ABC kit (Vector Laboratories Inc., Burlingame, CA). The specificity of immunostaining for ER{alpha} and PR was confirmed by staining on uterus and vagina form ERKO and PR knockout mice, respectively. An additional negative control, nonimmune mouse IgG (DAKO) and rabbit IgG (Vector) also confirmed the specificity of the immunostaining (data not shown). The anti-human PR antibody recognized both PR-A (approximately 100 kDa) and -B (approximately 90 kDa) forms in Western blots of the mouse uterine proteins (data not shown).

Image Analysis

To quantitate epithelial PR, images of PR immunohistochemistry were captured with a DC330 camera (Dage-MTI Inc., Michigan City, IN) interfaced with a PowerBase 200 computer (Power Computing, Round Rock, TX) and analyzed with Scion Image 1.62a (Scion Inc., Frederick, MD). An area of epithelial nuclei was manually selected by adjusting the threshold for optical density (OD), and then OD in the yellow channel of the CMYK (cyan, magenta, yellow, black) mode was measured in the selected area. The mean of OD of controls with nonimmune IgG was used as the baseline (PR level = 0). In each group, an area equivalent to 1500–7000 nuclei was analyzed from 6 to 14 tissue recombinants. Statistical analysis was performed on a statistical analysis package StatView (Abacus Concepts Inc., Berkeley, CA). To compare OD between different tissue types and hormone treatments, two-factorial ANOVA was used for overall analysis, and Fisher's protected least-significant-difference (PLSD) test was used to determine statistical differences between groups (P < 0.01).

Preparation of Total RNA

Adult female ERKO and wt mice were ovariectomized. Two weeks later, mice were given 3 daily treatments with oil or E2 as described above. All mice were killed 24 h after the last injection, and uteri and vaginae were removed. Epithelium was isolated as described above, then flash-frozen in liquid nitrogen. Uteri and vaginae from at least 2 mice were used for each treatment group in each experiment, and the entire experiment was repeated 3 times. Total RNA was prepared from frozen uterine and vaginal epithelial tissue by using the RNeasy Mini Kit (Qiagen, Chatsworth, CA). Purity and concentration of the RNA was determined by UV (260/280) absorbance in a spectrophotometer.

Northern Blotting

For all Northern blots used, equal amounts of RNA from control (oil-treated) and E2-treated mice were electrophoresed on a 1.5% agarose formaldehyde gel. The gel was blotted to nylon membranes, and the RNA was fixed to the membrane by UV cross-linking. Murine PR cDNA [28] was a gift from Dr. G. Shyamala, Lawrence Berkeley Laboratory, Berkeley, CA. Human 28S rRNA [29] was used for a loading control. PR cDNA probe was labeled with 32P-dCTP using the multiprime DNA labeling system (Amersham, Arlington Heights, IL) and hybridized at 68°C. The hybridized membrane was washed and exposed to Kodak X-Omat x-ray film (Eastman Kodak, Rochester, NY) with intensifying screens. After hybridization with the PR cDNA probe, membranes were stripped of probe and reprobed with the 28S rRNA cDNA probe, as described previously [30]. For PR mRNA expression, the strongest band (the 6.9-kilobase [kb] band) was quantitated using a computer-linked scanning laser densitometer and RFLPrint software (Pdi, Huntington Station, NY). The relative intensity of the 6.9-kb band to the 28S band (6.9 kb/28S) was calculated in each lane. The ratio was highest in the oil-treated group. Therefore, this group was nominally considered to have a value of 100% and was used as the benchmark against which other lanes were measured. The data were analyzed by ANOVA and Fisher's PLSD tests on the InStat statistical package (GraphPad Software Inc., San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Estradiol-Regulated PR Expression in Female Reproductive Tract

In wt ovariectomized mice, ER{alpha} was detected in both epithelium and stroma of vagina (Fig. 1, a and b) and uterus (Fig. 1, g and h) in both oil and E2 treatment groups. In ERKO mice, uteri and vaginae were negative for ER{alpha} (data not shown).



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  		FIG. 1. ER{alpha} and PR localization in wt and ERKO vagina and uterus. Mature (approximately 90-day-old) wt and ERKO female mice were ovariectomized. Two weeks after ovariectomy, daily doses of 125 ng E2 in 0.1 ml peanut oil or oil only were given from Days 1 to 3. a–f) Vagina: ER{alpha} (a and b), and PR (c–f) immunostaining. Wt (a–d) and ERKO (e and f) mice were treated with oil (a, c, and e) or E2 (b, d, and f). Basal epithelium is indicated by arrowheads (b and d). ep, Epithelium; st, stroma. g–l) Uterus: ER{alpha} (g and h), and PR (i–l) immunostaining. Wt (g–j) and ERKO (k and l) mice were treated with oil (g, i, and k) or E2 (h, j, and l). ep, Epithelium; st, endometrial stroma. Quantitative results are shown in Figure 2

PR was very low to undetectable in vaginal epithelium and stroma in ovariectomized oil-treated wt and ERKO mice (Fig. 1, c and e). E2 treatment strongly induced PR in both vaginal epithelium and stroma of wt mice (Fig. 1, c and d), but not in vaginae of ERKO mice (Fig. 1, e and f). Thus, ER{alpha} is essential for the E2 induction of PR in vaginal epithelium and stroma. In wt mice, vaginal epithelial cells differentiated (cornified) and showed heterogeneity in ER{alpha} and PR expression with E2 treatment. While basal vaginal epithelium retained a high level of ER{alpha} (Fig. 1b, indicated by arrowheads), ER{alpha} signal was diminished or absent in suprabasal layers. Therefore, overall ER{alpha} level in vaginal epithelium decreased. In contrast, PR expression was strongest in apical vaginal epithelial cells and was reduced in the basal layer (Fig. 1d, indicated by arrowheads).

In the uterus of both wt and ERKO mice, both intensity of PR signal and number of PR-positive cells were low in the endometrial stroma of ovariectomized oil-treated mice, even though PR was strongly expressed in luminal and glandular uterine epithelia (Fig. 1, i and k). In wt mice, E2 treatment dramatically reduced PR in uterine epithelium, while PR was strongly induced in the endometrial stroma (Fig. 1j). In contrast, a high PR level was maintained in uterine epithelium of ovariectomized ERKO mice treated with E2 (Fig. 1l). Thus, ER{alpha} is essential for E2-induced down-regulation of PR in uterine epithelium (compare Fig. 1, j and l). Unexpectedly, PR was induced by E2 in endometrial stroma of ERKO mice (Fig. 1l). Thus, PR may be induced by E2 in endometrial stroma through an ER{alpha}-independent pathway.

The relative PR expression level in vaginal and uterine epithelia was determined by measuring OD of epithelial nuclei in PR-immunostained sections. In wt mice, morphometric analysis of PR expression clearly showed E2-induced up-regulation of PR in vaginal epithelium, and E2-induced down-regulation of PR in uterine epithelium (Fig. 2). PR expression was significantly higher in vaginal epithelium of E2-treated wt mice (Fig. 2 with *) than in vaginal epithelium of oil-treated wt mice. PR expression was significantly lower in uterine epithelium of E2-treated wt mice (Fig. 2 with **) than in uterine epithelium of oil-treated wt, or oil- or E2-treated ERKO mice (P < 0.01; Fig. 2). In contrast, E2 was unable to change the PR level in vaginal and uterine epithelia of ERKO mice, and thus, there was no significant difference in PR levels of uterine and vaginal epithelia between oil and E2 groups in ERKO mice (Fig. 2). In ERKO mice, vaginal epithelial PR levels were low in both oil- and E2-treated groups and significantly lower than PR levels in E2-treated wt samples (P < 0.01; Fig. 2).



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  		FIG. 2. Epithelial PR levels in uterus and vagina of wt and ERKO mice corresponding to the immunohistochemical images in Figure 1. Epithelial nuclei were isolated electronically by adjusting the threshold from immunostained slides. The OD (range from 0 to 256) was measured in the yellow channel of the CMYK mode in the epithelial nucleus. The average OD of controls with nonimmune IgG was used as the baseline (OD = 0). In each group, 1500–7000 nuclei were analyzed from 6 to 14 samples. Data are presented as mean ± SEM. All data were statistically analyzed by the factorial ANOVA test. In vaginal epithelium, PR level was significantly higher in E2-treated wt mice (*) than in oil-treated wt and oil- or E2-treated ERKO mice (P < 0.01). In uterine epithelium, PR level was significantly lower in E2-treated wt mice (**) than in oil-treated wt and oil- or E2-treated ERKO mice (P < 0.01)

PR mRNA levels were measured by Northern blot in isolated uterine and vaginal epithelia, and the results confirmed immunohistochemical findings (Fig. 3A). Northern blotting for PR mRNA in vaginal and uterine epithelia from wt and ERKO mice revealed the presence of a series of transcripts ranging from 11.5 to 4.4 kb (data not shown). A representative Northern blot and normalized densitometric data for the most abundant 6.9-kb PR transcript are shown in Figure 3, A and B, respectively. E2 treatments produced clear changes in the overall level of PR transcripts in the different tissues. Differential effects on the various transcripts were not observed, in that the relative intensities of the different transcript sizes were similar in the various treatment groups despite the marked changes in overall band intensity across the different treatment groups. In wt vaginal epithelium, overall PR mRNA expression was low in the oil-treated control group, and 6.9-kb PR mRNA expression was increased by approximately 100% by E2 treatment in vaginal epithelium (Fig. 3, A and B). In contrast, in ERKO mice, PR mRNA level was so low in vaginal epithelium following oil or E2 treatment (data not shown) that it was impossible to perform densitometric analysis.



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  		FIG. 3. Northern blot analysis of PR mRNA. A) Steady-state PR mRNA expression in wt uterine and vaginal epithelia (wt-UtE and wt-VgE, respectively) and ERKO uterine epithelium (ERKO-UtE). E2 increased PR mRNA expression in wt-VgE, but produced the opposite effect in wt-UtE. E2 did not affect PR mRNA expression in ERKO-UtE. Loading, as determined by the intensity of a 28S rRNA hybridization signal, was similar in all lanes. B) Normalized densitometric data of 6.9-kb PR mRNA expression in the various treatment groups. Signals were normalized against a 28S rRNA cDNA probe to compensate for interlane differences in RNA loading. Values of UtE are shown as a percentage of the values for the oil-treated group. Values in vaginal epithelium are shown as a percentage of the values for the E2-treated group. The data are presented as mean ± SEM of values from 3 separate experiments. All values for the oil- and E2-treated groups in the wt-VgE and wt-UtE experiments were significantly different (P < 0.01). PR mRNA expressions in the ERKO UtE oil- and E2-treated groups were not significantly different (P > 0.01)

Regulation of PR mRNA expression in wt uterine epithelium by E2 was the exact opposite of that seen in vaginal epithelium of wt mice (Fig. 3, A and B). In wt uterine epithelium, PR mRNA expression was high in the oil-treated group, and decreased by about 75% in the E2-treated group (Fig. 3B). In contrast to the clear changes in PR mRNA expression in wt uterine epithelium seen in response to E2, expression of PR mRNA remained high and unchanged in uterine epithelium of E2-treated and oil-treated ERKO mice (Fig. 3B). These results indicate that the E2 effects seen in the wt tissue require ER{alpha}, and that the mRNA results presented here strongly corroborate our immunohistochemical results on the role of E2 in modulating PR expression in reproductive tract epithelia.

Role of Stromal-Epithelial Interactions in Regulating PR in Uterine and Vaginal Epithelia

ER{alpha} is essential for E2 regulation of PR in vaginal and uterine epithelia, as shown by the observation of E2-induced changes in vaginal and uterine epithelial PR in wt mice but not in ERKO mice. Since ER{alpha} is expressed in both epithelial and stromal cells of uterus and vagina of normal mice, it was unclear whether estrogen action on uterine and vaginal epithelial PR requires epithelial ER{alpha}, stromal ER{alpha}, or both. To study the possible role of epithelial-stromal interactions in epithelial PR regulation by E2, four types of tissue recombinants were made with vaginal or uterine epithelium (E) and stroma (S) from wt and/or ERKO mice (wt-S+wt-E, wt-S+ERKO-E, ERKO-S+wt-E, and ERKO-S+ERKO-E). Tissue recombinants were grown as subrenal capsule grafts in intact female nude mice for 4 wk, at which time all hosts were ovariectomized. All hosts received three daily injections of 125 ng E2 or oil. The genotype of the epithelium and stroma in all tissue recombinants was confirmed with ER{alpha} immunohistochemistry (see Figs. 4 and 6).



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  		FIG. 4. ER{alpha} and PR immunohistochemistry in ERKO/wt vaginal tissue recombinants. ER{alpha} (a and c) and PR (b and d). Tissue recombinants were made with vaginal epithelium and stroma from wt and/or ERKO (ko) mice, wt-S +wt-E (I), wt-S + ko-E (II), ko-S + wt-E (III), and ko-S + ko-E (IV). Tissue recombinants were grafted under the renal capsule of female nude mice and grown for approximately a month. All hosts were then ovariectomized. Two weeks after ovariectomy, all hosts received daily injection of E2 (c and d) or oil (a and b) for 3 days. Nuclei of ER{alpha} (a and c) and PR (b and d) positive cells stain brown, while negative nuclei are blue because of the counterstain

In all four types of vaginal tissue recombinants treated with oil only, PR were undetectable or very low in both vaginal epithelium and stroma (Fig. 4, I–IVb). With E2 treatment, PR was strongly expressed in both epithelium and stroma of wt-S+wt-S vaginal tissue recombinants (Fig. 4, Id) but was undetectable in ERKO-S+ERKO-E vaginal tissue recombinants (Fig. 4, IVd). In wt-S+ERKO-E and ERKO-S+wt-E tissue recombinants (Fig. 4, II and III), E2 treatment induced a high level of PR only in the wt-S or wt-E, respectively. Therefore, PR expression was directly regulated by estrogen in vivo in vaginal stroma and epithelium through ER{alpha} in the responding tissue. Morphometric analysis of epithelial PR levels in vaginal tissue recombinants showed clear differences among groups (Fig. 5A). Epithelial PR levels were low and virtually identical in all four types of vaginal tissue recombinants treated with oil. Epithelial PR levels in the E2-treated vaginal tissue recombinants prepared with wt-E (wt-S+wt-E and ERKO-S+wt-E; Fig. 5A with *) were significantly higher than those of oil-treated vaginal tissue recombinants (P < 0.01). In contrast, there was no significant difference in epithelial PR levels between the oil-treated group and E2-treated groups in uterine tissue recombinants prepared with ERKO-E (wt-S+ERKO-E and ERKO-S+ERKO-E). These results clearly demonstrate that ER{alpha} in vaginal epithelium is essential for up-regulation of vaginal epithelial PR induced by E2 treatment.



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  		FIG. 5. Epithelial PR levels in ERKO/wt vaginal and uterine tissue recombinants. A) Vaginal tissue recombinants corresponding to the immunohistochemical images in Figure 4. All data were statistically analyzed by two-factorial ANOVA and Fisher's PLSD test. The columns with * are significantly higher than others (P < 0.01). B) Uterine tissue recombinants corresponding to the immunohistochemical images in Figure 6. Data are presented as mean ± SEM. All data were statistically analyzed by two-factorial ANOVA and Fisher's PLSD tests. The columns with * are significantly lower than others (P < 0.01)

All four types of uterine tissue recombinants strongly expressed PR in epithelium of the oil-treated group (Figs. 5B and 6). In contrast, epithelial PR was down-regulated by E2 in uterine tissue recombinants prepared with wt-S (wt-S+wt-E and wt-S+ERKO-E; Fig. 6, I and II). Even though uterine wt-S+ERKO-E tissue recombinants did not express functional ER{alpha} in epithelium, E2 dramatically reduced PR immunoreactivity in the epithelium (Fig. 6II). Conversely, E2 did not down-regulate epithelial PR in uterine tissue recombinants prepared with ERKO-S (ERKO-S+wt-E and ERKO-S+ERKO-E; Fig. 6III, b and d, and IV, b and d) despite the expression of epithelial ER{alpha} in the ERKO-S+wt-E tissue recombinants. Morphometric analysis of epithelial PR levels in uterine tissue recombinants showed clear differences among groups. Epithelial PR levels were high and virtually identical in all four types of uterine tissue recombinants treated with oil (P < 0.01). Epithelial PR levels in the E2-treated uterine tissue recombinants prepared with wt-S (wt-S+wt-E and wt-S+ERKO-E; Fig. 5B with *) were significantly lower than those in oil-treated uterine tissue recombinants. In contrast, there was no significant difference in the high epithelial PR levels between the oil-treated group and E2-treated groups in uterine tissue recombinants prepared with ERKO-S (ERKO-S+wt-E and ERKO-S+ERKO-E). These results clearly demonstrate that ER{alpha} in uterine stroma is essential for down-regulation of uterine epithelial PR induced by E2 treatment, and that ER{alpha} in uterine epithelium is neither necessary nor sufficient to mediate the E2 effect on uterine epithelial PR expression. Thus, uterine epithelial PR expression is regulated by E2 through an unknown, novel paracrine mechanism that is mediated via stromal ER{alpha}.



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  		FIG. 6. ER{alpha} and PR immunohistochemistry in ERKO/wt uterine tissue recombinants. ER{alpha} (a and c) and PR (b and d). Tissue recombinants were made with uterine epithelium and stroma from wt and/or ERKO (ko) mice, wt-S +wt-E (I), wt-S + ko-E (II), ko-S + wt-E (III), and ko-S + ko-E (IV). Tissue recombinants were grafted under the renal capsule of female nude mice and grown for approximately a month. All hosts were then ovariectomized. Two weeks after ovariectomy, all hosts received daily injection of E2 (c and d) or oil (a and b) for 3 days. Nuclei of ER{alpha} (a and c) and PR (b and d) positive cells stain brown, while negative nuclei are blue because of the counterstain

Stroma-Independent PR Expression in Cultured Uterine Epithelium

To determine whether uterine epithelial PR expression is constitutive or requires stromal factors, uterine epithelium was isolated from ovariectomized Balb/c mice (approximately 90 days old) and cultured in extracellular matrix. Six days after isolation, cultured uterine epithelium still expressed high levels of PR with and without E2 (Fig. 7). Thus, the high PR levels in isolated uterine epithelium appeared to be constitutive, and clearly E2 at a final concentration of up to 10 µM cannot down-regulate PR in isolated uterine epithelium in vitro.



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  		FIG. 7. PR immunohistochemistry in uterine epithelial primary culture. Uterine epithelia were isolated from adult Balb/c mice (approximately 90 days old) ovariectomized at least 2 wk before culture. Cells were embedded in a 1:1 mixture of growth factor-reduced Matrigel and rat tail collagen. Three days after culture, cells were treated with vehicle (a) or 10-6 M E2 (b) for 3 more days


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our understanding of hormonal regulation of PR is based on numerous in vitro and in vivo studies. Since E2 increases total PR content in many target organs including uterus, it has been assumed that E2-induced up-regulation of PR is a general phenomenon in all target cells [10]. Although mouse and rat uteri are the most widely used in vivo models for estrogen and progesterone action, until recently regulation of PR and ER have been studied mostly at the organ and not at the cellular level in these species. In this study, we have focussed on E2 regulation of epithelial PR expression in mouse uterus and vagina, and have elucidated a complex paracrine circuitry involved in estrogen-regulation of PR through the analysis of wt/ERKO tissue recombinants. Interpretation of results of tissue recombination experiments is dependent upon the purity of the isolated stroma and epithelium, since cross-contamination of the tissues used to make tissue recombinants (particularly the stroma) can negate conclusions. In the current study, the stroma and epithelium were derived from ERKO and wt mice, and through use of parallel ER{alpha} and PR immunostaining of adjacent tissue sections, the purity of the tissues composing the tissue recombinants was verified and validated in all cases.

This report is the first to show that murine vaginal PR is regulated directly by estradiol in vivo through ER{alpha} in the responding epithelial and stromal cells. PR up-regulation occurred only in vaginae of ER{alpha}-positive wt (but not ER{alpha}-negative ERKO) mice treated with estradiol. ERß, if present, cannot substitute for the absence of ER{alpha}. Moreover, in wt-S+ERKO-E and ERKO-S+wt-E vaginal tissue recombinants, only the wt-S or the wt-E showed induction of PR in response to E2, indicating that PR is regulated directly via ER{alpha} in the responding cell itself. In E2-treated wt-S+wt-E tissue recombinants, apical vaginal epithelial cells were strongly positive for PR but almost negative for ER{alpha}. This heterogeneity in ER{alpha} and PR expression appears to be a manifestation of the program of vaginal epithelial differentiation elicited by E2. Estrogen-induced vaginal epithelial cornification is a complex process involving epithelial proliferation, which results in thickening of the vaginal epithelium, followed by epithelial keratinization. The overall process requires both epithelial and stromal ER{alpha} [24]. E2-induced vaginal epithelial proliferation is regulated by a paracrine mechanism requiring ER{alpha} in the stroma cells [24]. Expression of E2-induced differentiation markers such as cytokeratin 10 in apical cells requires ER{alpha} in the vaginal epithelial cells themselves [24]. Therefore, in the vagina, E2 acts through paracrine mechanisms to elicit proliferation of basal epithelial cells and acts directly on basal and/or suprabasal cells to elicit differentiation. One aspect of vaginal epithelial differentiation is the expression of PR in cells of suprabasal layers in which ER{alpha} is exceedingly low or undetectable. This suggests that expression of epithelial PR is an event mediated by epithelial ER{alpha}. Our study is the first to emphasize that the commitment of vaginal epithelium to express PR occurs in the basal epithelial compartment, which is ER{alpha}-positive. While cells are preprogrammed to express PR, this commitment is deferred until the differentiating vaginal epithelial cells enter suprabasal or apical layers.

In contrast to the usual pattern of estrogenic regulation of PR, we have shown for the first time in the mouse uterine epithelium that an exceptionally high level of PR is constitutively expressed in the absence of either estrogen or ER{alpha}. In wt mice, uterine epithelial PR is down-regulated by E2 via an ER{alpha}-dependent mechanism since uterine epithelial PR is not down-regulated in ERKO mice. This is the first study to show that the low level of ERß present in the uterus in ERKO mice [31] is not capable of substituting for the absence of ER{alpha} in the regulation of uterine epithelial PR. On the basis of tissue recombinant studies, E2-induced down-regulation of uterine epithelial PR is mediated through novel paracrine mechanisms mediated by stromal ER{alpha}.

The spectrum of steroid receptors and the underlying mechanisms regulating receptor content are specific aspects of epithelial differentiation in hormone target organs. For example, induction of prostatic differentiation by urogenital sinus mesenchyme in the androgen-receptor (AR)-negative bladder epithelium involves AR expression in the induced epithelium, which is an appropriate differentiation marker for prostatic epithelium [32]. Likewise, ER{alpha} and PR are appropriate differentiation markers for vaginal and uterine epithelial cells. These ER{alpha} and PR differentiation markers confer upon uterine and vaginal epithelial cells the possibility of direct hormonal regulation of hormone-dependent proteins. In light of the common embryological origin of uterine and upper vaginal epithelia from the Müllerian duct [33, 34], it is interesting that on the basis of analysis of ERKO mice, ER{alpha} is required for PR expression in vaginal epithelium but not in uterine epithelium. In the mouse and rat, simple columnar uterine epithelium and stratified cervical/vaginal epithelium can be distinguished by their morphology and other markers such as cytokeratins [35]. Tissue recombination experiments have shown that neonatal uterine and vaginal epithelia can be induced by mesenchyme to change their differentiation pattern provided the epithelia are derived from mice during the first week after birth. Thus, neonatal Müllerian vaginal epithelium recombined with uterine mesenchyme differentiates as a simple columnar uterine epithelium. Likewise, uterine epithelium recombined with vaginal mesenchyme differentiates into an epithelium that expresses morphological and biochemical features of vaginal epithelium [36, 37]. Our preliminary studies demonstrate that regulation of PR expression in vaginal and uterine epithelia is determined by the final epithelial phenotype expressed. For example, when neonatal Müllerian vaginal epithelium is recombined with uterine mesenchyme, the vaginal epithelium undergoes uterine differentiation and the induced uterine epithelium maintains a high level of PR in the absence of E2, a unique feature of uterine epithelial differentiation. However, vaginal epithelium from adult mice combined with uterine mesenchyme mostly retains the stratified squamous vaginal phenotype and requires E2 to maintain its PR, which is an indication of vaginal epithelial differentiation (unpublished data). In this experiment, the adult vaginal epithelium is determined (presumably as a result of prior mesenchymal induction) and maintains vaginal differentiation and the vaginal pattern of PR regulation even though it has been recombined with uterine mesenchyme. These data suggest that the differential regulation of PR expression in uterine and vaginal epithelia is an integral component of tissue-specific cyto-differentiation induced by stroma during development. In turn, PR expression results in the differential expression of other genes regulated by progesterone, thus determining specific hormonal responses of vaginal and uterine epithelia.

Uterine epithelial PR expression appears to be constitutive and independent of stromal factors, because PR is maintained at a high level in the absence of estrogen in isolated uterine epithelial cells cultured on or in extracellular matrix. Therefore, E2 does not down-regulate uterine epithelial PR by antagonizing stromal factors that stimulate epithelial PR. Instead, uterine epithelial PR appears to be down-regulated by stromal paracrine signals that antagonize the constitutive expression of epithelial PR. The nature of the paracrine stromal signal is currently unknown, but certain gene knockout mice may be useful in implicating or excluding certain candidate molecules. A mouse that is deficient for the stromal signaling molecule should be impaired in down-regulation of uterine epithelial PR by E2.

Generally, PR is up-regulated by estrogens and down-regulated by progesterone (see references in the review [10]). Immunohistochemical studies have shown that estrogens up-regulate PR expression in uterine epithelium of many species: human [38], primates [14], cat [15], dog [39], and ewe [16, 40]. In most cases, PR disappears as a result of estrogen withdrawal, and thus uterine epithelial PR expression is generally thought to be estrogen- and ER{alpha}-dependent. Rat and mouse uterine epithelial PR is regulated by estrogen, but in a manner exactly opposite that of most other species. The physiological significance of the rodent pattern of regulation of uterine epithelial PR is not presently clear. A fundamental difference between these laboratory rodents and those animals whose uterine epithelial PR is up-regulated by estrogen is length of reproductive cycle. The length of the estrous cycle is 4–5 days in mice and rats, while in humans and many other primates the menstrual cycle is about 1 mo in duration. Though the cycle is short, rodent uteri go through proliferative, differentiative, and apoptotic responses to E2 and progesterone during their cycle similar to those of other mammalian species. We speculate that the rodent specific pattern of uterine epithelial PR regulation may be required to accommodate the highly compressed length of the estrous cycle and to establish pregnancy in the short reproductive cycle. In general, PR is down-regulated in endometrial epithelium at the time of implantation [15, 16], and the down-regulation of epithelial PR is achieved by a gradually elevated progesterone level. Since rats and mice must go through this process in a very short time, the specific regulation of uterine epithelial PR may be essential to ready uterus for implantation and decidualization.

Important clues to transcriptional regulation can frequently be found within the promoter of a gene. The structure of the promoter region and putative transcription factors interacting with the PR promoter have been identified for many species [11, 41–44]. The sequence of the 5' promoter-untranslated region of the mouse PR gene has high homology (85%) with the corresponding sequence for the rat and moderately high homology with the human (59%) and rabbit (57%) sequences [41]. In vitro studies have shown that estrogen-occupied ER can activate the PR promoter sequence of rat, rabbit, and human PR genes [11–13]. Given the dramatic difference in PR regulation in uterine versus vaginal epithelia in the mouse, or between mouse uterine PR versus uterine PR of other species, promoter activity may be radically different in rodent uterine epithelium versus that in other tissues in which PR is up-regulated in response to E2. The differential PR regulation in mouse uterine and vaginal epithelia must be due to usage of different promoter sites and/or differential expression of transcription factors interacting with the PR promoter. We therefore propose that uterine stroma induces the uterine epithelial phenotype, and that one aspect of this phenotype is constitutive expression of uterine epithelial PR as described above. Estrogen stimulates stroma via its ER{alpha} to produce a paracrine factor(s), which down-regulates constitutive PR in uterine epithelium. The 5' region of mouse PR gene contains putative regulatory binding sites for several transcription factors such as activator protein-1 (AP-1) sites [41]. AP-1 may be involved in regulation of uterine epithelial PR because components of AP-1, c-fos, jun-B, and jun-D, are induced by E2 in uterine epithelium of the mouse and rat [45–47].

The power of the tissue recombination approach for analysis of hormonal response has been discussed previously [26, 48]. Many genes have been identified which are regulated by estrogen and progestin in the uterus and vagina. Our group has emphasized the importance of paracrine regulation of epithelial function by steroids [22, 23,26, 48]. Understanding the cellular mechanism of steroid action on target tissues will require determination of whether a given effect of hormone is mediated by stromal or epithelial hormone receptors. If steroid receptors are simultaneously present in both epithelial and stromal cells, epithelial response to a tropic hormone may be mediated via either direct or paracrine mechanisms. Tissue recombinant studies are ideally suited to determining the respective roles of epithelial versus stromal hormone receptors. Knowledge of the respective roles of epithelial and stromal ER{alpha} is essential for the design and interpretation of hormonal effects in vivo.

Finally, we have shown an unexpected E2 induction of PR in endometrial stromal cells of ERKO mice. This result contradicts the original report made by Couse et al. [7] that E2 had no effect on whole-uterine PR levels in ERKO mice. However, others [21] and our studies have clearly demonstrated that PR is differentially regulated by E2 in epithelium versus stroma and myometrium. Levels of RNA, protein, or steroid binding in the uterine epithelium (which contributes only 5–10% of the uterus) can be masked in whole-organ homogenates by changes in the stroma/myometrium [49]. Proper analysis of uterine epithelial PR expression requires analysis of tissue sections by immunohistochemistry or in situ hybridization, or through direct analysis of isolated uterine epithelium. Our study is the first to examine epithelial PR regulation in vivo using freshly isolated uterine epithelium. Such an approach eliminates the uninterpretable results obtained from whole uterine homogenates. The mechanism of this ER{alpha}-independent PR regulation in endometrial stroma is currently under investigation [50].


   FOOTNOTES
 
First decision: 21 September 1999.

1 Supported by NIH Grants AG-13784 and DK47517 (to G.R.C), AG-15500 (to P.S.C.), and ES-08272 (to D.B.L.). Back

2 Correspondence: Gerald R. Cunha, P.O. Box 0452, Department of Anatomy, University of California San Francisco, San Francisco, CA 94143. FAX: 415 502 2270; grcunha{at}itsa.ucsf.edu Back

Accepted: November 5, 1999.

Received: July 30, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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