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Endocrinology Vol. 139, No. 3 913-921
Copyright © 1998 by The Endocrine Society

ARTICLES

Uterine and Vaginal Organ Growth Requires Epidermal Growth Factor Receptor Signaling from Stroma1

Yun Kit Hom, Peter Young, Jane F. Wiesen, Päivi J. Miettinen, Rik Derynck, Zena Werb and Gerald R. Cunha

Department of Anatomy (Y.K.H., P.Y., J.F.W., Z.W., G.R.C.) and Department of Growth and Development (P.J.M., R.D.), University of California, San Francisco, California 94143

Address all correspondence and requests for reprints to: Dr. Gerald R. Cunha, Department of Anatomy, Mail Stop 0452, University of California, San Francisco, California 94143. E-mail: grcunha{at}itsa.ucsf.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estrogens are crucial for growth and function of the female genital tract. Recently, we showed that induction of uterine epithelial proliferation by estradiol is a paracrine event requiring an estrogen receptor-positive stroma. Growth factors [such as EGF (epidermal growth factor) ligands] are likely paracrine mediators, which may directly or indirectly regulate epithelial proliferation in estrogen target organs via cell-cell interactions. In this report, we used mice with a null mutation in their EGF receptor (EGFR) to examine the role of EGFR signaling in growth of the uterus and vagina and in estrogen-induced uterine and vaginal epithelial proliferation. When WT and EGFR-knockout (EGFR-KO) uteri and vaginae were grown as renal capsule grafts in nude mice, growth of uterine and vaginal grafts of EGFR-KO mice was reduced, compared with their WT counterparts. Grafts of both EGFR-KO uteri and vaginae were about one third smaller (wet weight) than their corresponding WT organs, even though differentiation of both epithelium and mesenchyme were normal in both cases. Both wild-type and EGFR-KO vaginal grafts contained within their lumina alternating layers of cornified and mucified epithelial cell layers, indicating cyclic alteration of epithelial differentiation. In response to estradiol treatment, stromal cell labeling index (LI), as assessed by incorporation of 3H-thymidine, was severely depressed in EGFR-KO uterine and vaginal grafts vs. stromal cell LI in WT uterine and vaginal grafts. Unexpectedly, epithelium of both EGFR-KO and wild-type grafts responded comparably to estradiol with a marked elevation (~7-fold overall) of epithelial LI in response to estradiol in uterine and vaginal epithelia. These data supported the hypothesis that overall uterine and vaginal organ growth, in response to estrogen, required EGFR signaling for DNA synthesis in the fibromuscular stroma, whereas EGFR signaling was not essential for estrogen-induced epithelial growth in the uterus and vagina.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EPIDERMAL growth factor (EGF) family of ligands, which includes EGF, TGF{alpha}, heparin-binding EGF (HB-EGF), amphiregulin, heregulin, epiregulin, and several other molecules (1, 2), is implicated in uterine and vaginal development, particularly as mediators of estrogen action. The possible role of the EGF family in estrogenic effects in the female genital tract is supported by many studies. Estrogens have effects upon both EGF and EGF receptors in the uterus (3, 4) and vagina (5) in vivo. Estradiol injected into immature female rats elicited a 3-fold increase in specific, high-affinity, saturable binding of 125I-EGF to uterine membranes (6, 7). After estrogen injection, uterine EGFR binding increased between 6–12 h, remained elevated at 18 h, and declined thereafter. This increase in EGFR-binding was blocked by both cycloheximide and actinomycin D and was specifically induced by estrogens but not by nonestrogenic hormones (6). A corresponding study, using ovariectomized rats, showed a 2- to 3-fold elevation of uterine immunodetectable EGFR 18 h after injection of estradiol (8). Uterine EGFR transcripts were elevated in ovariectomized rats (9) within 3 h of estradiol injection, remained elevated at 6 h after estradiol injection, and then declined thereafter. Estrogenic effects were blocked by actinomycin D but not by puromycin. Nonestrogenic hormones did not mimic the estrogen-mediated increase in EGFR messenger RNA (mRNA) levels. However, for all of the above studies, it was unclear whether the effect of estradiol on uterine EGFR reflected changes in the epithelial, stromal, or myometrial compartments. Nonetheless, these studies suggested that estrogen-dependent growth of the uterus and vagina were mediated via EGF ligands acting through the EGFR.

Paracrine models of estrogen action in the mouse uterus and vagina were considered for many years because estrogen receptors were expressed in the epithelium, stroma, and myometrial cells (10, 11, 12). Thus, it was possible that estradiol elicited epithelial effects by acting directly upon the epithelium via epithelial estrogen receptors or via estrogen receptors in stromal cells, which in turn, stimulated epithelial proliferation in a paracrine fashion. It was also unknown whether stromal proliferation was regulated by direct or paracrine action of estradiol. It was initially assumed that the myriad effects of estradiol on epithelium were mediated directly through epithelial estrogen receptors. However, analysis of estrogen receptor expression and estradiol responsiveness in the neonatal mouse uterus indicated that this was not correct. Using neonatal Balb/c mice, Cunha et al. (13) demonstrated, with steroid autoradiography, that estrogen receptors were undetectable in uterine epithelium (UtE) but were present in uterine mesenchyme (UtM). Despite the apparent lack of uterine epithelial estrogen receptors in the neonatal mouse, injection of diethylstilbestrol (DES) caused a doubling in the rate of UtE proliferation (14). One explanation for these results could be that DES induced the expression of epithelial estrogen receptors that, in turn, mediated the mitogenic effects of DES on the epithelium. However, Bigsby and Cunha (14) showed that estrogen receptors remained undetectable in the UtE, even after DES stimulation. These results suggested that the mitogenic effects of DES on neonatal UtE could be elicited via paracrine influences from estrogen receptor-positive mesenchymal cells. More recent immunohistochemical studies were consistent with this interpretation (15, 16, 17, 18).

To determine roles of epithelial vs. stromal estrogen receptors in uterine epithelial proliferation, a transgenic estrogen receptor knockout (ERKO) mouse (19) was used to produce uterine tissue recombinants in which epithelium (UtE), stroma (UtS), or both were devoid of functional estrogen receptors. In tissue recombinants prepared with wild-type (WT) uterine stroma (WT-UtS + WT-UtE and WT-UtS + ERKO-UtE), epithelial labeling index (LI) was increased severalfold by estradiol over oil-treated controls (20). In contrast, in tissue recombinants prepared with ERKO uterine stroma (ERKO-UtS + ERKO-UtE and ERKO-UtS + WT-UtE), epithelial LI was low and similar in estradiol- vs. oil-treated specimens. These data clearly demonstrated that estradiol induction of uterine epithelial proliferation was a paracrine event requiring an estrogen receptor-positive stroma. Moreover, epithelial estrogen receptors were neither necessary nor sufficient for estradiol-induced epithelial proliferation. These findings suggested the existence of paracrine mediators of stromal origin, which directly or indirectly regulated epithelial proliferation in estrogen target organs. Growth factors (such as the EGF family of ligands) are likely candidates of such putative paracrine mediators.

To examine the role of EGFR signaling in estrogen-dependent growth of the uterus and vagina, we used a transgenic mouse deficient in EGFR signaling (21), which would be predicted to exhibit impaired uterine and vaginal growth and impaired estrogenic response. These EGFR-KO mice showed growth retardation and epithelial dysfunction, which resulted in gastrointestinal and lung abnormalities resembling human diseases associated with premature birth. EGFR-KO homozygotes displayed epithelial immaturity and multiorgan failure, whereas the heterozygotes developed normally. Some homozygous EGFR-KO embryos died prenatally, but many survived into the early neonatal period before succumbing. Nevertheless, organ rudiments could be rescued from EGFR-KO neonates by grafting them into athymic nude mouse hosts so that estrogenic response could be examined. Using these methods, we investigated the complex interplay between estrogen action, paracrine stromal-epithelial interactions, and EGFR signaling in growth of the uterus and vagina.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals. EGFR-KO mice (21) were bred at the University of California, San Francisco. EGFR-KO mice were typed at birth by their so-called open eye phenotype. Normal female Balb/c mice were obtained from the Cancer Research Laboratory, University of California, Berkeley, CA. Intact female athymic nude mice were purchased from Harlan (Indianapolis, IN). For this report, 50 female EGFR-KO newborns, 48 female WT littermates, and 100 nude mice were used. 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, animal care and usage committees. Mice were maintained under controlled temperature and lighting conditions during the experiment and were given food and water ad libitum.

Microdissection and tissue recombinations. Female EGFR-KO mice, normal littermates, and normal female Balb/c mice were killed at 0–3 days postnatal, and entire genital tracts were removed by dissection. For whole-organ grafts, uteri and vaginae were trimmed as indicated (Fig. 1Go) and grafted under the renal capsule of female athymic nude mice.



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  		Figure 1. Female EGFR-KO and Balb/c mice were killed at 0–3 days postnatal, and the entire genital tract was removed by dissection. Whole-organ pieces, for grafting, were trimmed as indicated by the lines. The ovaries, uterine horns, and vagina are indicated. The bar equals 4 mm.

 
Grafting, ovariectomy, and graft harvest. Whole-organ rudiments from EGFR-KO and WT animals were transplanted beneath renal capsules of female athymic nude mouse hosts. After 1 month of growth beneath the renal capsule, the nude mouse hosts were ovariectomized; 7 days later, the nude mouse hosts were given one injection (ip) of either 125 ng estradiol (Steroids, Wilton, NH) or corn oil (control). Eighteen hours later, the hosts were injected ip with 1.5 µCi/g BW of 3H-thymidine (specific activity = 84 Ci/mmol) (Amersham, Arlington Heights, IL) and killed 2 h later. The grafts were harvested and fixed in 4% formalin. Harvested grafts were imaged using a color Lumina camera (Leaf System, South Brough, MA) to document overall size and gross morphology. Wet weights were determined for all grafts.

Histology. The grafts were harvested, fixed in 4% buffered formaldehyde, embedded in paraffin, and sectioned at 6 µm. For histological analysis, specimens were stained with hematoxylin and eosin.

Autoradiography. For analysis of epithelial and stromal LI, paraffin sections of the specimens were mounted on glass slides, dipped in NTB-II photographic emulsion (Kodak, Rochester, NY), and processed autoradiographically via standard protocols (22).

LI. Epithelial LI and stromal LI with 3H-thymidine was determined as the percentage of labeled epithelial or stromal cells in the total population of epithelial or stromal cells, as described previously (22). Individual histological sections to be scored were selected randomly, and for a given section, only regions of appropriate section orientation were scored in which the plane of section was roughly perpendicular to the plane of the epithelial basement membrane. Areas of poor section quality, tangential, or oblique orientation were excluded. For each type of graft, a minimum of 300 cells were scored per individual specimen for 3–6 replicate specimens.

Statistics. Values were expressed as the mean ± SEM of at least six different experiments. Differences among means were estimated using a Student’s unpaired t test and ANOVA. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Growth, but not differentiation, was impaired in grafted uteri and vaginae from EGFR-KO mice. In general, grafts of EGFR-KO uteri and vaginae grown for 1 month in intact female hosts were smaller than their WT littermates, as judged by wet weight at harvest and overall size of the grafts. Grafted WT uteri from estradiol-treated hosts (0.299 ± 0.114 g, n = 3) were 1.6 times larger than uteri derived from EGFR-KO mice (0.187 ± 0.065 g, n = 3). Each set of individual experiments was performed with grafts from EGFR-KO mice of slightly different ages (0–3 days postnatal). The age of the graft at transplantation affected the graft’s weight gain. Thus, overall size and weight of grafts varied from experiment to experiment because of differences in specimen age at the time of grafting. However within individual experiments, grafts of WT uteri always attained a larger size and weight than grafts of EGFR-KO uteri. Grafts of undifferentiated neonatal uterine rudiments from WT donors developed normally. Both luminal epithelium and uterine glands developed from the undifferentiated Müllerian duct epithelium of the neonatal uterus. The undifferentiated UtM of the grafted neonatal uterus differentiated into endometrial stroma and myometrium (Fig. 2aGo). Although grafts of EGFR-KO uteri were somewhat smaller than WT grafts after 1 month of growth, development of both epithelium and mesenchyme was normal and was equivalent to that of grafts of WT uteri (Fig. 2Go, a and b).



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  		Figure 2. Hematoxylin and eosin-stained sections of WT and EGFR-KO uterine and vaginal grafts. Panel a, WT uterus; panel b, EGFR-KO uterus; panel c, WT vagina; panel d, EGFR-KO vagina. For the uteri (a and b), the dotted lines delineate the stroma from the myometrium (Myo). In panel a, a uterine gland is indicated. For the vaginae (c and d), alternating cornified (C) and mucified (M) layers are indicated in the lumen. The bar equals 100 µm.

 
Grafted WT vaginae from estradiol-treated hosts (0.226 ± 0.030 g, n = 3) were 1.5 times larger than corresponding organs derived from EGFR-KO mice (0.149 ± 0.029 g, n = 3). As mentioned above, each individual experiment was performed with grafts from EGFR-KO mice of slightly different ages, which led to some variability in overall graft size and weight from experiment to experiment. However, within a given experiment, vaginal growth was consistently elevated in WT vs. EGFR-KO grafts. As was the case for uteri, grafts of neonatal vaginae of WT and KO mice developed normally and formed a highly differentiated vaginal epithelium whose differentiation varied with the stage of the estrous cycle of the host at the time of death. The lumina of the vaginal grafts were filled with alternating layers of cornified and mucified epithelial cells in both WT and EGFR-KO vaginal grafts, indicating cyclical alteration in epithelial differentiation (Fig. 2Go, c and d).

Stromal cells in grafted uteri and vaginae from EGFR-KO mice had an impaired proliferative response to estradiol. We found that overall growth (wet weight) was reduced in grafts of EGFR-KO vs. WT uteri and vaginae. To explain this difference in size of EGFR-KO uterine and vaginal grafts, we determined cell proliferation by analyzing incorporation of 3H-thymidine and stromal cell LI’s in grafts of intact WT and EGFR-KO uteri. LI for uterine stromal cells of EGFR-KO uteri in response to estradiol was indistinguishable from that of oil-treated controls (Fig. 3AGo). This contrasts with an increase in stromal cell LI of WT uterine grafts treated with estradiol, which was 4.6 times higher than that of WT uteri treated with oil and 2.3 times higher than that of its EGFR-KO counterpart. Similarly, in estradiol-treated EGFR-KO vaginal grafts, stromal cell LI was indistinguishable from that of its oil-treated counterpart (Fig. 3BGo). In contrast, in estradiol-treated WT vaginal grafts, stromal cell LI was 19.5 times higher than that of its estradiol-treated EGFR-KO counterpart and 6.4 times higher than its WT oil-treated counterpart. These data indicated that stromal response to estradiol was markedly impaired in EGFR-KO mice.



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  		Figure 3. Stromal cell LIs in WT (WT) and EGFR-KO (KO) uterine and vaginal grafts. Stromal cell LIs with 3H-thymidine were determined, as the percentage of labeled stromal cells per total stromal cells counted. A, WT and EGFR-KO uterine stroma; B, WT and EGFR-KO vaginal stroma. Each bar represents the mean of at least six different experiments ± SEM. *, Statistical significance at P < 0.05; Ut, uterus; Vg, vagina; E2, estradiol.

 
Estradiol induces epithelial growth in grafted uteri and vaginae of EGFR-KO mice. The proliferative response to estradiol in grafts of WT and EGFR-KO uteri and vaginae was assessed by determining LI. As shown in Fig. 4AGo, UtE of both EGFR-KO and WT uterine grafts responded comparably to estradiol by incorporation of 3H-thymidine. Epithelial LI in WT uterine grafts increased approximately 4-fold in response to estradiol, relative to WT uterine grafts treated with oil. Epithelial LI in EGFR-KO uterine grafts treated with estradiol increased approximately 9 times, relative to that of EGFR-KO uterine grafts treated with oil. Epithelial LI’s for WT and EGFR-KO uterine grafts treated with oil were not significantly different from each other at P < 0.05.



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  		Figure 4. Epithelial cell LIs in grafts of WT (WT) and EGFR-KO (KO) uteri and vaginae. Epithelial cell LI with 3H-thymidine was determined, as the percentage of labeled epithelial cells per total epithelial cells counted. A, WT and EGFR-KO uterus; B, WT and EGFR-KO vagina. Each bar represents the mean of at least six different experiments ± SEM. *, Statistical significance at P < 0.05. Ut, Uterus; Vg, vagina; E2, estradiol.

 
As was the case for the uterus, both EGFR-KO and WT vaginal grafts exhibited a marked epithelial proliferative response to estradiol (Fig. 4BGo). Epithelial LI’s in WT vaginal grafts treated with estradiol were elevated approximately 3-fold, relative to epithelial LI of WT vaginal grafts treated with oil. Similarly, EGFR-KO vaginal grafts treated with estradiol exhibited an increase in epithelial LI approximately 11 times higher than that of EGFR-KO vaginal grafts treated with oil. Epithelial LI’s for WT and EGFR-KO vaginal grafts treated with oil were not significantly different from each other at P < 0.05.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Growth of organs containing an epithelial parenchyma was generally impaired in EGFR-KO mice, which led to a spectrum of deleterious lesions, principally in the lung and gastrointestinal tract, that compromised postnatal survival. Though EGFR-KO mice usually died in the neonatal period, undifferentiated uteri and vaginae could be isolated from neonatal EGFR-KO mice and grafted into athymic nude hosts. In this way, it was possible to grow these undifferentiated neonatal organs sufficiently long in an endocrinologically normal environment to achieve full maturation and thereby enable the study of the estrogenic response in EGFR-KO uterine and vaginal grafts. Using these methods, we investigated the interplay between estrogen action and EGFR signaling. As shown in Fig. 2Go, histological analysis of uterine and vaginal grafts indicated that their epithelia grew and differentiated normally when grafted into intact female nude mouse hosts. Our group routinely grafted embryonic and neonatal organs from both male and female rodents. These renal capsule grafts displayed normal growth, morphogenesis, and differentiation. Moreover, markers of adult function were expressed by grafts of embryonic or neonatal organ rudiments usually by 3–4 weeks after grafting. For example, lactotransferrin was expressed by neonatal uterine grafts or homotypic neonatal uterine tissue recombinants (UtM + UtE) grown for 4 weeks in adult female hosts (20). For this reason, it was important to recognize that our experimental model did not relate to hormonal response of neonatal organs but instead, more appropriately, to postnatal organs at on advanced stage of functional differentiation comparable, in many ways, to adult organs.

The EGFR-KO mouse was described to undergo multiorgan failure (21), which led to its demise in the early neonatal period. Given the short life span of the EGFR-KO mouse, it was not possible to determine whether congenital abnormalities were reversible with time. Moreover, in the original description of the EGFR-KO mouse (21), only a limited number of organs were examined. Given this background it was striking that uterine and vaginal development was so normal in uterine and vaginal grafts from EGFR-KO neonatal mice. Perhaps the effects of EGFR-KO were variable in different organs. Alternatively, developmental abnormalities that existed during development of EGFR-KO uteri and vaginae were not observed at the end of our experiments because, given the extended period of growth in the nude mouse hosts, such abnormalities could be repaired through compensatory mechanisms. In any case, it should be emphasized that the embryonic and early neonatal development of the female Müllerian ducts and the urogenital sinus was normal in EGFR-KO mice. For this reason, the neonatal female genital tract of the EGFR-KO mouse was slightly smaller than, but otherwise indistinguishable from, the WT.

Many of the organs that were adversely affected in the EGFR-KO mouse were composed of epithelium and mesenchyme. In the present study, we found that the uterus and vagina of EGFR-KO mice exhibited a generalized growth deficit of 34–38%. Although EGFR signaling was absent simultaneously in both epithelium and mesenchyme in EGFR-KO mice, our data suggested that the lack of functional EGFR in uterine and vaginal stroma was the key event accounting for overall organ hypoplasia in the female genital tract and impaired overall growth of EGFR-KO vs. WT uteri and vaginae. Tritiated-thymidine LI studies supported this conclusion. It should be recognized that 3H-thymidine incorporation did not differentiate DNA replication from DNA repair. However, all controls and experimental conditions reported in this study behaved normally with respect to known proliferative (DNA synthetic) response to estradiol. LIs of the stroma of EGFR-KO uterine and vaginal grafts treated with estradiol demonstrated a complete absence of proliferative response to estradiol. The growth deficiency of the EGFR-KO uterus and vagina could also be caused by a higher rate of apoptosis, although this was not measured in this study. Estradiol is known to inhibit apoptosis in granulosa cells of the rat ovarian follicle, and the lack of EGFR signaling here could inhibit estrogen-induced apoptosis. In any case, EGFR signaling seems to be required for optimal estrogen-dependent stromal growth in the uterus and vagina. Because the epithelium forms only about 10%, whereas the fibromuscular wall forms about 90% of the uterus (23), impaired growth of the stroma more profoundly affected overall organ size than impaired growth of the epithelium, which only constituted a small fraction of the uterus. Our results were consistent with a model in which the estrogen-receptor-mediated action of estradiol in either the epithelium or stroma elicited production of EGF ligands that subsequently interacted with the EGFR on stromal cells and stimulated stromal proliferation. The impaired stromal DNA synthesis in EGFR-KO mice indicated that estradiol by itself was not a complete mitogen for uterine or vaginal stromal cells, but instead that EGF ligands produced by the stromal cells and acting in an autocrine manner, or EGF ligands produced by the epithelium and acting as a paracrine manner, were involved in estrogen-induced growth of uterine and vaginal stromal cells.

Parallel studies in estrogen receptor-deficient (ERKO)/WT tissue recombinants clearly demonstrated that estradiol induction of uterine epithelial proliferation was a paracrine event requiring estrogen receptor-positive stroma (20). The current study extended these observations using EGFR-KO uteri and vaginae. We found that estrogen-induced epithelial growth mediated by stromal estrogen receptors was normal, suggesting that estrogen-induced stroma-mediated epithelial growth did not require the EGFR signaling pathway in the epithelial cells. Furthermore, the results of this study indicated that a functional stromal EGFR pathway was not required for stromal production of estrogen-induced paracrine factors necessary for epithelial cell growth but could be more crucial for uterine and vaginal stromal growth. It was reported that the preimplantation uterus differentially expressed full-length (EGFR-fl) and a truncated (EGFR-tr) forms of the EGFR (24). The EGFR-fl is a fully functional receptor, whereas the EGFR-tr is a secreted protein, which is not thought to have any direct cell signaling capabilities. In situ hybridization studies indicated that EGFR-fl transcripts were found only in the uterine stroma and myometrium, but not in the epithelium, whereas EGFR-tr message was detected in all major uterine cell-types (24). These findings were corroborated by immunohistochemical results showing that EGFR was detected only in the uterine stroma, deciduum, and myometrium, but not in the uterine luminal or glandular epithelium of the early pregnant mouse (25). Additionally, EGF ligands were shown to bind to a variety of other erb-B receptors (26). Using in situ hybridization, erb-B2 mRNA was detected primarily in uterine epithelial cells on days 1–4 of pregnancy in the mouse with the highest level found on day 1 (27). Further analysis showed that ovariectomized mice, treated with estradiol, up-regulated erb-B2 expression in the UtE by 3.5-fold using a combination of RT-PCR and in situ hybridization (27). Thus, it was possible that in EGFR-KO mice, EGF ligands could still be important for uterine/vaginal epithelial growth by signaling through these other receptors. Given the complexity of the EGF ligand family, it was not surprising that deletion of a single growth factor gene, such as TGF{alpha}, did not compromise the health or fertility of TGF{alpha}-KO mice (28). Taken together, these data imply that UtE was not the direct target for the effects of EGF-type growth factors and that their mitogenic effects were actually mediated by paracrine mechanisms involving other uterine cell-types expressing EGFR. Arguing against this interpretation are in vitro studies that showed a direct effect of EGF upon isolated UtE (29, 30). Using a collagen gel culture system, dissociated uterine and vaginal epithelial cells responded to EGF with growth in a serum-free, defined culture medium (29, 30). Unlike the in vivo situation, however, estradiol did not stimulate growth for vaginal or uterine epithelial cells in a similar collagen gel culture system (31), so comparisons between in vitro and in vivo results were difficult to reconcile. In any case, we could not rule out the possibility that, in WT mice, EGFR signaling in the epithelium occurred and was involved in estrogen-induced uterine and vaginal epithelial proliferation in vivo. However, in EGFR-KO mice, other ligand-receptor systems could clearly compensate for the lack of EGFR in the epithelium. One such possibility was erb-B2, which is a receptor subtype, capable of binding EGF-related ligands in uterine epithelial cell proliferation (27).

Several EGF ligands are produced in the uterus and vagina. Uterine and vaginal epithelial cells were stimulated by estradiol to produce EGF and/or TGF{alpha} (5, 32). However, in the uterus, EGF seemed to be secreted apically into the uterine lumen (32) and, thus, could not be available for interaction with EGFR in either stromal or epithelial cells. Similarly, vaginal epithelium (stimulated in vivo by estradiol) expressed TGF{alpha} transcripts in suprabasal cell layers (33), which again raised the possibility that EGF ligands produced by vaginal epithelium could be unavailable for interaction with stromal or epithelial EGFR. TGF{alpha} expression also was found in the mouse uterus during the periimplantation period (day 1–4 of pregnancy) in a cell-type specific manner (34, 35). By in situ hybridization and immunoblot analysis, TGF{alpha} (34) and proTGF{alpha} (35), respectively, were localized in the luminal and glandular epithelia on days 1–4 of pregnancy, and many of the stromal cells expressed TGF{alpha} on days 3–4 of pregnancy. In the uterus of ovariectomized adult rats, the production of HB-EGF was stimulated in uterine stromal cells by progesterone (P) or P followed by estradiol. Such hormonal treatments repressed HB-EGF expression in the UtE, whereas estradiol alone increased HB-EGF expression in the epithelium. For ovariectomized adult mice, coinjection of P plus estradiol stimulated HB-EGF expression in uterine stromal cells, as detected by in situ hybridization, whereas estradiol alone increased expression of HB-EGF only in the epithelium (36, 37). Amphiregulin was induced by P in the uterine luminal epithelium of ovariectomized mice (38), whereas TGF{alpha} expression was stimulated by DES in the uterine epithelial cells, with only a modest increase in TGF{alpha} expression in uterine stromal cells (32). Thus, EGF ligands were produced by both epithelial and stromal cells, and therefore, proliferation of uterine and vaginal stromal cells could be elicited via either autocrine or paracrine mechanisms.

Thus, our results (using an EGFR null mutant mouse) showed that EGFR signaling was required for estrogen-induced proliferation of uterine and vaginal stromal cells but challenged the generally accepted notion that the EGFR receptor signaling system was crucial for estrogen-induced proliferation of uterine and vaginal epithelial cells.

If EGFR were not needed for estrogen-induced epithelial growth, then some other signaling pathway(s) in the uterus and vagina was used to elicit epithelial mitogenesis mediated via estrogen receptors in the stroma. Of the many possible compensatory growth factor pathways which could play key roles in estradiol-induced epithelial growth, keratinocyte growth factor (KGF), HGF, and insulin-like growth factor 1 (IGF-1) were worthy of consideration.

KGF fits many of the criteria considered essential for a mesenchymal mediator of epithelial development. Uterine tissue from cycling and ovariectomized monkeys, treated with combinations of estradiol and P, expressed KGF mRNA, which was increased in animals in the luteal phase or in animals treated with P (39). Thus, KGF was suggested to be a P-induced, stromally-derived, progestomedin. We found that KGF, injected directly into newborn female mice, stimulated uterine epithelial growth (unpublished results). However, KGF was highly induced after incisional wounding of the skin (40, 41, 42) and the bladder (43). Thus, it was perhaps worth considering whether the apparent induction of KGF in the uterus by P was secondary to apoptotic damage associated with reduced estrogen levels in the luteal phase. In any case, KGF could be an important paracrine mediator for uterine epithelial growth.

Hepatocyte growth factor (HGF, scatter factor) is mitogenic for epithelial cells of a number of estrogen-sensitive organs, including the mammary gland (44, 45, 46) and uterus (45). HGF transcripts were detected by RT-PCR in the adult mouse uterus, and c-met mRNA expressed in the UtE by in situ hybridization (45). HGF expressed in mammary stromal cells, stimulated ductal branching, and inhibited production of secretory proteins in organ culture (45). Proliferation of primary mouse mammary epithelial cells was stimulated by coculture with primary mouse mammary fibroblasts that produced HGF (44). Thus, HGF is another potential mediator of stromal effects on epithelial growth in the female genital tract.

IGF-1 also has been suggested to be a mediator of estrogen-stimulated proliferation in the uterus. IGF-1 and IGF-1 receptor expression were up-regulated in response to estrogen (47, 48, 49). Immature rats, implanted sc with pellets containing estradiol, exhibited an elevation in uterine IGF-1 and IGF-1 receptor mRNA after 72 h of treatment (50). Transgenic mice (homozygous for a null mutation of the IGF-1 gene) had thin, flaccid uteri with a wet weight only 13% that of WT mice (51). Another transgenic mouse, overexpressing IGF-binding protein-1 showed a significant reduction in both estradiol- and IGF-1-induced uterine DNA synthesis, compared with WT mice (52). IGF-1 expression was found in the uterine glandular and luminal epithelial cells on days 1–2 of pregnancy, whereas stromal cells, on days 3–4 of pregnancy and decidual cells on days 5–6 of pregnancy seemed to be the predominant sites of IGF-1 production (53). Treatment of ovariectomized mice with P and/or estradiol induced IGF-1 expression. Estradiol specifically induced IGF-1 in uterine epithelial cells, whereas P induced IGF-1 in stromal cells (53). The combination of both estradiol and P further stimulated IGF-1 expression in the uterine stroma. Taken together, these studies suggested that KGF, HGF, and IGF-1 were important for estradiol-induced epithelial growth and that they could compensate for KO of the EGFR signaling pathway.

We conclude that EGFR signaling was required for estrogen-induced uterine and vaginal stromal growth but not for estrogen-induced growth of uterine or vaginal epithelium. The resulting impaired stromal growth in EGFR-KO mice led to overall organ hypoplasia. Given the more extensive contribution of mesenchyme to the overall mass of the uterus, and the fact that uterine and vaginal mesenchyme played a central role as an inducer of uterine and vaginal development (54), the absence of EGFR signaling in the mesenchyme of EGFR-KO mice seemed to account, in large part, for the generalized hypoplasia of estrogen-sensitive female genital tract organs. Although EGFR signaling could be used during estrogen-induced uterine and vaginal epithelial proliferation in vivo in wild-type mice, other, more important growth factor systems could act in conjunction with EGF to elicit epithelial growth. Clearly, other ligand-receptor systems did compensate for the lack of EGFR function in estrogen-induced proliferation of uterine and vaginal epithelia.


   Footnotes
 
1 This research was supported by Grants AG-13784 (to G.R.C.), HD-26732 (to Z.W.), and CA-54826 (to R.D.), and by a postdoctoral fellowship from the American Cancer Society (PF-4184, to J.F.W.) Back

Received August 13, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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