Epidermal growth factor binding and receptor distribution in the mouse reproductive tract during development

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142, ‘75-85

(1990)

Epidermal Growth Factor Binding and Receptor Distribution in the Mouse Reproductive Tract during Development NANCY

L. BOSSERT, KAREN G. NELSON, KIMBERLY

A. Ross, TSUNEO TAKAHASHI,

AND JOHN A. MCLACHLAN

Laboratory of Reproductive and De-velopmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, North Carolina 27709 Accepted July 9, 1990 The ontogeny of the epidermal growth factor (EGF) receptor in the different cell types in the neonatal and immature mouse uterus and vagina was examined. Jmmunohistochemical examination of prenatal and neonatal reproductive tracts with a polyclonal antibody to the EGF receptor shows immunoreactive EGF receptors as early as Day 13 of gestation. Autoradiographic analysis of tissue sections at 3 to 17 days of age (the day of birth is Day 1) demonstrates that both uterine and vaginal epithelial and stromal cells are capable of binding ‘ZI-labeled EGF. Both the ‘ZI-labeled EGF autoradiography and immunohistochemistry in whole tissue show higher EGF receptor levels in the uterine epithelium than the uterine stroma. The presence of EGF receptors was also confirmed by affinity labeling and Scatchard analysis of isolated uterine cell types at 7 and/or 17 days of age. However, in contrast to the autoradiography and immunohistochemistry data of intact tissue, the affinity labeling and Scatchard data of isolated cells indicate that the uterine stroma contains higher levels of EGF receptor than that of the uterine epithelium. The reason for this discrepancy between the different techniques is, as yet, unknown. Regardless of the differences in the actual numbers of EGF receptors obtained, our data demonstrate that the developing mouse reproductive tract contains immunoreactive EGF receptors that are capable of binding ‘?-labeled EGF. o 1990 Academic press, ~nc.

molecules may play a role in fetal growth and development of specialized function (Adamson et al., 1981; Partanen and Thesleff, 1987). The biological effects of EGF are mediated by its binding to a specific cell surface receptor, which is a 170,000 MW transmembrane glycoprotein possessing intrinsic protein tyrosine kinase activity (Ullrich et al., 1984; Schlessinger, 1986). Functional EGF receptors have been documented in fetal and adult tissues (reviewed by Adamson and Rees, 1981). It has also been demonstrated that the number of EGF receptors in some fetal tissues increases during gestation (Adamson and Meek, 1984). Given that control of EGF receptor expression may play a role in the determination of cellular responsiveness to EGF (Clark et al., 1985), local variations in receptor content may play a regulatory role separate from any variations in local EGF concentration that may occur (Nexa et ah, 1980; Gardner et al, 1989). In the adult animal, EGF has been hypothesized to act in an autocrine and/or paracrine fashion to mediate estrogen stimulation of cell growth in the uterus and other target tissue(s) (Tomooka et ah, 1986; DiAugustine et ah, 1988). Moreover, EGF receptors have been identified in the adult uterus (Mukku and Stancel, 1985; Chakraborty et ab, 1988) and have been localized to all major uterine cell types (Chegini et ab, 1986; Lin et al, 1988). However, the presence of EGF receptors or EGF-like molecules earlier in the development of the female reproductive tract has not been documented. Since it has been shown

INTRODUCTION

The quest for the isolation, characterization, and mechanism of action of factors involved in cellular growth and differentiation has focused on an expanding number of polypeptide growth factors. One such factor is epidermal growth factor (EGF),l a single-chain polypeptide (MW 6045) originally isolated from the mouse submaxillary gland that was shown to accelerate incisor eruption and eyelid opening in neonatal mice (Cohen, 1962). EGF has subsequently been shown to induce a multitude of effects, including the enhancement of DNA synthesis in mammalian cells, both in vitro and in viva (reviewed by Carpenter and Cohen, 19’79; Carpenter and Zendegui, 1986). Although the exact physiological role of EGF is unclear, the presence of EGF and/or EGF-like molecules has been documented in embryonic and fetal tissues (Rappolee et ah, 1988; Twardzik, 1985; Freemark and Comer, 1987). Studies have shown that EGF and its receptor are present in mouse embryos as early as Day 12 of gestation (Nexti et al., 1980). These studies and others led to the suggestion that EGF and/or EGF-like 1 Abbreviations used: EGF, epidermal growth factor; ?-labeled growth factor; PBS, phosphate-buffered saEGF, ‘?odo-epidermal line; DMEM, Dulbecco’s modified Eagle medium; [3H]TdR, tritiated thymidine; HBSS, Hanks’ balanced salt solution; Hepes, N’-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; DES, diethylstilbestrol. 75

0012-1606/90 $3.00 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form rrserved.

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previously that the uterus of the prenatal/neonatal mouse is deficient in estrogen receptors (Taguchi et al., 1988; Yamashita et ah, 1989), this raises the possibility that EGF receptors may be an important component of the cellular physiology of the differentiating uterus. In this study, we have examined the cellular localization and biochemical characteristics of EGF receptors in the different cell types in the prenatal reproductive tract and the neonatal and immature mouse uterus and vagina, using immunological and autoradiographic techniques as well as affinity labeling and Scatchard analysis. MATERIALS

AND

METHODS

Tissues. Pregnant outbred CD-l [Crl:CD-l(ICR)BR] mice were sacrificed at Day 13 through 18 of gestation (Day 0 is the day of vaginal plug observation), and fetuses were collected. Fetal reproductive tracts (ovary, Wolffian and Miillerian ducts) were removed, fixed in Bouin’s solution, dehydrated through graded alcohols to xylenes, embedded in paraffin, and sectioned (4-pm sections) by standard methods. Neonatal and immature CD-1 mice were sacrificed at 3 to 17 days of age, and uteri and vaginas were obtained, incubated with ‘%I-labeled EGF, fixed in formalin, and processed for autoradiography or immunohistochemistry as described in later sections. Uterine epithelial and stromal cells were isolated from 7- and 17-day-old CD-l mice and used for EGF affinity labeling or Scatchard analysis. Materials. Receptor grade mouse EGF and HPLCpurified ol-EGF were obtained from Collaborative Research Inc. (Bedford, MA) and Biomedical Technologies, Inc. (Stoughton, MA), respectively. Diagnostic Systems Laboratories (Webster, TX) iodinated the HPLC-purified a-EGF by the chloramine-T method (sp act 150 &i/pg). A polyclonal antibody raised in rabbit to a synthetic dodecapeptide derived from the extracellular domain of the human EGF receptor was obtained from Cambridge Research Biochemicals, Inc. (Valley Stream, NY). A rabbit polyclonal antibody to EGF receptors purified from mouse liver was generously provided by Dr. Eileen Adamson at the La Jolla Cancer Research Foundation (La Jolla, CA). Vectastain ABC-AP and alkaline phosphatase substrate (black) kits were obtained from Vector Laboratories (Burlingame, CA). BCA protein assay reagents were obtained from Pierce Chemical Co. (Rockford, IL). Electrophoresis reagents were obtained from Bio-Rad Laboratories (Rockville Centre, NY). Type IV collagen was obtained from Collaborative Research Inc. All other reagents were the highest grade commercially available. Cell isolation. Uteri were removed from 7- and 17-dayold CD-l mice, stripped of mesentery, and each uterine horn was bisected. The uterine pieces were incubated in

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0.1% bovine pancreatic trypsin in Ca- and Mg-free phosphate-buffered saline (PBS), pH 7.0, at 37°C for 1 hr. Cells and small tissue fragments were collected by centrifugation at 1000~ for 5 min. The pellet was resuspended in a 1:l mixture of DMEM:Ham’s F-12 media containing penicillin (31 pg/ml), streptomycin (50 pg/ ml), 0.1% Fungizone, 0.1% bovine serum albumin (BSA), and 0.05 mMCaCl,, and epithelial cells were separated from stromal cells by centrifugation in a Percoll gradient (Tomooka et ah, 1986). This isolation method yielded approximately 2.6 X lo5 epithelial and 3.6 X lo5 stromal cells from a 7-day-old mouse uterus and 4.0 X lo5 epithelial and 5.3 X lo5 stromal cells from a 17day-old mouse uterus. lz51-EGF binding assay. EGF binding assays were performed using a modification of the method of Cruise and Michalopoulos (1986) on isolated uterine epithelial and stromal cells that were cultured for 5 days. Stromal cells were seeded in 12-well plates (Costar 3512) at a density of 3 X lo5 cells/well. Epithelial cells were seeded in collagen (Type IV)-coated 12-well plates at a density of 3 X lo5 cells/well. The basal (serum-free) culture medium consisted of a 1:l mixture of DMEM:Ham’s F-12 media containing penicillin (31 pg/ml), streptomycin (50 wg/ ml), 0.1% Fungizone, 0.05 mM CaCl,, and 0.1% BSA. Both the epithelial and stromal cells were cultured for 5 days in basal medium supplemented with transferrin (10 pg/ml), insulin (10 pg/ml), and EGF (10 rig/ml). After 5 days, the supplemented basal medium was removed, and the cultures were washed twice with 5 ml basal medium before incubation in 2 ml basal medium at 37°C for 4 hr. The cultures were washed twice as before and then incubated for 3 hr on ice in 6.33 ng ?-labeled EGF/ml with or without unlabeled EGF (0.1-5000 ng/ ml) in Hepes buffer with 0.2% BSA. Following incubation, cultures were washed three times with Hepes/BSA buffer to remove free (unbound) labeled EGF. Receptorbound EGF was solubilized by incubation of the cultures for 30 min at room temperature in 1 ml of 1 N NaOH, which was collected and counted in a Beckman y counter (70-80% efficiency). The apparent equilibrium dissociation constant (&) and the apparent binding capacity (B,,,) were calculated by linear regression analysis. DNA autoradiography. Neonatal and immature mice were injected subcutaneously with 1.5 &i rH]TdR per gram body weight. Uteri and vaginas were removed after 1 hr, fixed in 10% neutral buffered formalin, dehydrated through graded alcohols to xylenes, embedded in paraffin, and sectioned (4-pm sections) by standard methods. Deparaffinized slides were dipped in Kodak NTB2 emulsion, exposed for 3 weeks at 4”C, developed, and stained with hematoxylin and eosin. Three to six tissue sections were examined for each time point, and a minimum of 1200 epithelial cells and 2500 stromal cells were counted.

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Freshly dissected neonatal and immature uteri and vaginas were cut lengthwise into 2-mm segments, which were then cut open along one side in order to expose the luminal epithelium. These tissue fragments were then incubated for 1 hr at 4°C with 120 rig/ml lz51-labeled EGF in Hanks’ balanced salt solution (HBSS) containing 20 mM Hepes, pH 7.5, and 0.2% BSA. The specific binding of ‘%I-labeled EGF was shown by incubating control tissues from mice of all ages with a loo-fold excess of unlabeled EGF (receptor grade) added to the ‘251-labeled EGF-containing medium. After incubation, the tissues were washed (2 X 5 min in binding medium followed by 1 x 5 min in binding medium without BSA) at 4°C. The tissues were then fixed in 10% neutral buffered formalin and processed for autoradiography or immunohistochemistry. Y-labeled EGF binding to unoccupied receptors did not prevent binding of the polyclonal anti-EGF receptor antibody used to localize immunoreactive EGF receptors. Slides for autoradiography were deparaffinized, dipped in Kodak NTB2 emulsion, exposed for 3-4 weeks at -7O”C, developed, and stained with hematoxylin and eosin. Immunohistochemistry. Slides of Bouin’s-fixed prenatal reproductive tracts or formalin-fixed neonatal and immature uteri and vaginas were deparaffinized and rinsed in 20% glacial acetic acid at 4°C for 15 set to inhibit endogenous alkaline phosphatase. All subsequent incubations and washes were performed at room temperature. Sections were washed in phosphate-buffered saline (PBS) for 20 min, covered with a 1.5% solution of normal goat serum in PBS for 20 min to reduce nonspecific binding, and incubated with a 1:lOO dilution of the polyclonal anti-human EGF receptor antibody (Cambridge Research Biochemicals) for 30 min. Other sections of neonatal and immature mouse uterus were washed in PBS for 20 min, covered with a 5% solution of normal goat serum in PBS for 30 min to reduce nonspecific binding, and incubated with a 1:250 dilution of the polyclonal anti-mouse EGF receptor antibody (Dr. E. Adamson). Two controls were performed on tissue sections from mice of all ages examined to ascertain whether the alkaline phosphatase color reaction product represents the localization of immunoreactive EGF receptors. To determine the endogenous, nonspecific reaction product, the EGF receptor antibody was replaced with normal rabbit serum or preimmune serum. To document the specificity of the anti-human EGF receptor antibody, the antibody was incubated overnight at 4°C with the cognate peptide to which it was raised, and the EGF receptor antibody was then replaced with the antibody-peptide mixture. The Vectastain ABC-AP and alkaline phosphatase substrate (black) kits were then used to localize the EGF receptor. Sections were subsequently dehydrated through alcohols to xylenes and

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ls51-EGF autoradiography.

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FIG. 1. Nuclear r3H]TdR incorporation in the developing mouse uterus (A) and vagina (B). Female mice were injected with [3H]TdR on various days after birth, and uteri and vaginas were removed 1 hr later and processed for autoradiography. Labeled nuclei were counted in both epithelial(0) and stromal cells (0). Each point represents the mean k SEM of 3 to 6 mice.

mounted. It should be noted that the specific immunohistochemical staining obtained is dependent upon the type of histological fixative used to preserve the tissue specimens. A$inity labeling. Freshly dissected uteri and vaginas were incubated and washed as previously described for ‘Y-labeled EGF autoradiography. To crosslink ‘%I-labeled EGF to the EGF receptor, the tissue was incubated after the final wash with 400 ~1 of 1.0 mMbis-(sulfosuccinimidyl)-suberate in 20 mM Hepes buffer for 20 min at room temperature, then terminated by the addition of excess glycine (28 mM). Tissues were mixed with 200 ~1 solubilization buffer (1% Triton X-100, 10 mM Hepes, pH 7.4, 10 pg/ml leupeptin, and 100 KU aprotinin), homogenized with a Brinkmann Polytron (15 set on ice), and spun at 1OOO.oat 4°C for 15 min to reduce

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FIG 2. Autoradiographs of ia?-labeled EGF binding in the mouse uterus at different stages of development. The uteri were incubated with ?-labeled EGF, washed, and processed for autoradiography as described in the Materials and Methods section. Labeled tissue sections were photographed before (A, C, E) and after (B, D, F, G) counterstaining with hematoxylin and eosin. (A, B) In the uterus of the 3-day-old mouse, the luminal epithelium (e) is heavily labeled, while the stroma (s) has fewer grains. At 9 days (C, D) and 15 days (E, F), the uterine luminal epithelium is still heavily labeled, particularly at the basal and lateral surfaces. (G) The uterine glands of the 7- to Ill-day old mouse exhibit

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bubbles. The supernatant was collected and spun at 40,000 rpm at 4°C for 30 min in a Beckman 42.2 Ti rotor. Protein content of the supernatant was assayed using the BCA protein assay kit. Tissue homogenates were mixed with sodium dodecyl sulfate (SDS) sample buffer, heated at 100°C for 5 min, and separated by electrophoresis in 8% SDS-polyacrylamide gels (1.5 mm thickness) with a 3% stacking gel. Molecular weight markers were run in a parallel lane. After electrophoresis (850 Vhr), gels were simultaneously fixed and stained (10% acetic acid, 40% methanol, 0.25% Coomassie brilliant blue R-250) and destained in 10% acetic acid plus 10% methanol. After drying, autoradiography was performed at -70°C with Kodak XAR-5 film. Freshly isolated uterine epithelial and stromal cells were also affinity labeled by incubation of aliquots of cells for 1 hr at 4°C with 450 rig/ml 1251-labeled EGF in HBSS with 20 mM Hepes and 0.2% BSA. The cells were washed and crosslinked as previously described for whole tissue except the cells were pelleted in a microfuge at 12,000 rpm for 1 min at 4°C between steps. Following the last wash, the cells were mixed with 200 ~1 solubilization buffer, lysed by freeze-thawing in liquid nitrogen, and spun at 40,000 rpm at 4°C for 30 min in a Beckman 42.2 Ti rotor. Preparation for electrophoresis and SDS-PAGE were performed as previously described for whole tissue extracts. RESULTS

The neonatal and immature mouse reproductive tract is in the process of profound growth and differentiation. Previous studies have demonstrated significant levels of DNA synthesis in the neonatal mouse uterus (Bigsby and Cunha, 1985; Eide, 1975). Given that strain differences in DNA synthesis may exist, nuclear [3H]thymidine incorporation was quantitated autoradiographitally in the uterus and vagina of the 3-day-old to 17-dayold CD-l mice used in our study. The results demonstrate that [3H]TdR incorporation is greater in uterine epithelium than stroma, and that there are gradual decreases in labeling in both cellular compartments as the animal matures (Fig. 1A). Although uterine stroma1 r3H]TdR incorporation exhibits an overall gradual decline, the stromal labeling index, unlike the epithelial labeling index, reaches an apparent plateau from 9 days to 15 days. By 17 days, the labeling index is approximately the same in uterine epithelium and stroma. The level of DNA synthesis is lower in the neonatal vagina

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(Fig. 1B) than uterus, but by 17 days the levels of DNA synthesis are similar in both tissue types. The distribution of accessible, unoccupied EGF receptors was examined in the uterus and vagina of 3-day-old to 17-day-old mice by incubation of viable tissue fragments with ‘251-labeled EGF followed by autoradiography. Light microscopic autoradiographs clearly demonstrate the presence of ‘251-labeled EGF binding sites in both the developing CD-l mouse uterus (Fig. 2) and vagina (data not shown). Binding sites for 1251-labeled EGF in the developing uterus are present in all of the major cell types-luminal and glandular epithelium, stroma, and muscle (Fig. 2A-F). At the developmental stages examined, ‘251-labeled EGF binding is more intense in the uterine epithelium than in the stroma and muscle. The majority of silver grains within the luminal epithelial cells appear to be basally located. ‘Y-labeled EGF binding in developing uterine glands is concentrated at the epithelial-stromal interface, making precise cellular localization difficult (Fig. 2G). The specific binding of ‘%I-labeled EGF in these tissues is demonstrated by a significant reduction in labeling (approximately 90%) when excess unlabeled EGF is included in the original incubation (Fig. 2H). This reduction in ‘%Ilabeled EGF labeling is seen in tissues from mice of all ages. Vaginal epithelial and stromal cells also demonstrate ‘251-labeled EGF binding, where binding is more intense in the epithelium than the stroma at the developmental stages examined (data not shown). ‘251-labeled EGF binding sites within the stratified vaginal epithelium are predominantly localized to the basal cell population. Thus, 1251-labeled EGF binding and autoradiography demonstrates the presence of accessible, unoccupied EGF receptors in all of the major cell types of the 3-dayold to 17-day-old mouse uterus and vagina. The distribution of immunoreactive EGF receptors was examined in the reproductive tracts of prenatal, neonatal, and immature mice. To ascertain whether the alkaline phosphatase color reaction product represents the localization of immunoreactive EGF receptors, two controls were performed on tissues from mice of all ages examined. When the polyclonal anti-EGF receptor antibody is replaced with normal rabbit serum or preimmune serum to determine the endogenous, nonspecific reaction product, a very faint background is seen (Fig. 3A). To document the specificity of the anti-human EGF receptor antibody, the antibody was incubated overnight at 4°C with the cognate peptide to which it was

heavy labeling at the epithelial-stromal interface, making precise cellular localization difficult, as seen in the uterus at 13 days (arrows). (H) Autoradiograph of mouse uterus at 3 days incubated with ‘l-labeled EGF and a loo-fold excess of unlabeled EGF in the incubation medium. The labeling by ‘%I-labeled EGF is diminished significantly compared to the mouse uterus at 3 days incubated with ‘%I-labeled EGF alone (B), thereby confirming the specific binding of ‘?-labeled EGF in the mouse uterus. This control was performed for tissue from mice of all ages with similar results. Magnifications X400.

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FIG. 3. Micrographs of mouse uteri demonstrating specificity of immunohistochemical staining for EGF receptor. (A) Control section of uterus at 11 days stained with second antibody alone, demonstrating the faint stain of the endogenous, nonspecific reaction product. (B, C) Serial sections of uterus at 17 days stained with anti-EGF receptor antibody alone (B) or anti-EGF receptor antibody plus peptide antigen (dodecapeptide derived from the extracellular domain of the human EGF receptor) (C). The reduction in staining when the peptide antigen is included in the incubation mixture documents the specificity of the anti-EGF receptor antibody. These controls were performed for tissues from animals of all ages with similar results. Magnifications X380.

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raised. When the anti-human EGF receptor antibody (Fig. 3B) is replaced with the antibody-peptide mixture (Fig. 3C), there is a marked reduction in reaction product, thereby documenting the specificity of staining. Although the reproductive tracts of prenatal mice were not subjected to l%I-labeled EGF binding and autoradiography, they were examined for the distribution of immunoreactive EGF receptors. Prenatal mice exhibit immunoreactive EGF receptors in the major cell types of the developing ovary and Mtillerian and Wolffian ducts (Fig. 4). The immunoreactive EGF receptors in the Miillerian and Wolffian ducts are less abundant in stroma1 cells than in epithelial cells, where apical staining is more pronounced than basal staining (Figs. 4A-4D). As development proceeds from Day 13 to Day 18 of gestation, and the Wolffian duct regresses in female fetuses, there is no apparent alteration in the immunoreactive EGF receptor levels in either the Mtillerian or Wolffian ducts (Figs. 4E, 4F). Neonatal and immature mice exhibit homogeneously distributed immunoreactive EGF receptors in the major uterine and vaginal cell types. Similar to the ‘%I-labeled EGF binding studies, immunoreactive EGF receptors appear to be more abundant in uterine epithelium than stroma at the developmental stages examined (Fig. 5). After 13 days, discrete granule-like regions of immunoreactive EGF receptor are seen in the basal portion of uterine epithelial cells (Figs. 5C, 5D). The anti-mouse EGF receptor antibody yielded the same pattern of immunoreactivity in the neonatal and immature mouse uterus as the anti-human EGF receptor antibody, including the granule-like regions of immunoreactive EGF receptor in the basal portion of the uterine epithelial cells. Bouin’s-fixed tissue sections from 7- and 17day-old mice demonstrated a pattern of immunoreactivity similar to that seen in the prenatal reproductive tract in that epithelial staining was both apically and basally located. Immunoreactive EGF receptors are also more abundant in vaginal epithelium than stroma (data not shown). In other species, the specific high-affinity binding of EGF is associated with a uterine membrane receptor with mol wt of 150,000-170,000 (Mukku and Stancel, 1985; Hoffmann et ak, 1984). Given that our immunohistochemical and autoradiographic data document the presence of EGF receptors in mouse uterus and vagina, it was of interest to determine using affinity labeling techniques whether CD-1 mice contain a similar size EGF receptor. For these studies, viable uterine and vaginal tissue fragments were incubated with ‘%I-labeled EGF, crosslinked with disuccinimidyl suberate, extracted with detergent, and analyzed by SDS-PAGE. Autoradiography demonstrates ‘%I-labeled EGF in the uterus and vagina of 7- and 17-day old mice binds to a major receptor species of 170,000 MW and a minor spe-

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FIG. 4. Micrographs of immunoreactive EGF receptor distribution in prenatal mouse reproductive tract demonstrating intense staining in the ovary (ov) and ducts (d), and less and transverse (E) sections of Day 14 reproductive tracts showing intense staining in the immunoreactivity is still observed at Day 15 (F). Note the level of staining in the Wolffian Day 15. Magnifications: (A) X100; (B, C) X200; (D, E, F) X400.

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reproductive tracts. (A, B) Oblique section of Day 13 intense staining in the stroma (s). Longitudinal (C, D) Wolfiian (Wd) and Mtillerian (Md) ducts. The strong duct is not altered with the regression of the duct at

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ties of 150,000 MW (Fig. 6). Studies in other systems suggest that the 150,000 MW species is a proteolytic degradation product of the 170,000 MW species (Cohen et aL, 1982; Cassel and Glaser, 1982). Densitometric analysis of the autoradiographs reveals that the uterus binds two- to threefold more ?-labeled EGF per milligram protein than the vagina at 7 days. In addition, the uterus binds more ‘?-labeled EGF per milligram protein at 7 days than it does at 17 days. In all cases, the presence of excess unlabeled EGF in the original incubation abolishes the binding of ‘Y-labeled EGF to both the 170,000 MW and 150,000 MW species, suggesting that the observed bands represent binding specific for EGF receptor sites. Furthermore, analysis of EGF binding in separated uterine epithelial and stromal cells reveals the same major and minor receptor species in both uterine cell types of 7- and 17-day-old mice (data not shown). In other species, Scatchard (1949) analysis demonstrates that EGF binds to isolated uterine membranes with high affinity (Kd = 0.1-0.7 nM; Mukku and Stance& 1985; Hofmann et ah, 1984). Thus, it was of interest to determine whether purified uterine epithelial and stroma1 cells also demonstrate high affinity EGF binding. Initial Scatchard analysis of EGF binding in freshly isolated uterine epithelial and stromal cells demonstrated high affinity binding in both cell types (Kd = 0.52 and 0.73 nM, respectively; data not shown). However, the number of binding sites per cell was low, suggesting an effect of the enzymatic cell isolation procedure. Given the difficulty obtaining sufficient freshly isolated cells and the low number of EGF binding sites per freshly isolated cell, Scatchard analysis was also used to assess binding affinity of uterine epithelial and stromal cells that were cultured for 5 days in order to allow the cells to regenerate receptors lost due to the use of trypsin during cell isolation. The Scatchard plot of the binding data for both the cultured epithelial and stromal cells appeared linear (Fig. 7; correlation coefficient = 0.96 and 0.96 for stromal and epithelial cells, respectively), again suggesting a single class of noninteracting binding sites. The Kd and B,,, values are 0.11 nM and 9.44 fmole EGF bound per lo5 cells for epithelial cells and 0.15 nMand 20.65 fmole EGF bound per lo5 cells for stromal cells. These Kd values are also indicative of high-affinity binding sites. The number of binding sites per cell was calculated to be approximately 57,000 for epithelial cells and 124,000 for stromal cells. Thus, our data indicate that in vitro cell culture provides favor-

FIG. 5. Micrographs of EGF receptor immunolocalization in 1;he of neonatal and immature mice. (A) In the uterus at 3 da YST innmunoreactive EGF receptors are more abundant in the epithelil lrn (e!) than the stroma (s). (B) Immunostaining of the uterine epithelh .im is still more intense than the stroma at 7 days. At 15 days (C) and 17 ul terus

days (D), the uteri exhibit epithelial EGF receptor staining that is still slightly more intense than stromal staining. In addition, starting at 13 days, discrete granule-like regions of immunoreactive EGF receptors are now seen in the basal portion of the uterine epithelial cells (arrows). Magnifications X400.

BOSSERT ET AL. 7 DAYS

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FIG. 6. Affinity labeling of EGF receptors in vaginal tissue fragments at 7 days and 17 days. bated in the absence (-) or the presence (+) of unlabeled EGF and crosslinking was performed Materials and Methods. Extracts were analyzed lowed by autoradiography. Affinity labeling with veals that all of the tissues examined contain the ceptor species (170,000 MW) along with a minor (150,000 MW).

VAGINA

viable uterine and Samples were incua loo-fold excess of as described under by SDS-PAGE fol‘=I-labeled EGF reexpected major redegradative species

able conditions for the EGF receptor to become reestablished in that there is a 16- to 36-fold increase in receptor number compared to freshly isolated cells. Furthermore, freshly isolated and cultured stromal cells consistently demonstrate more EGF receptors than the corresponding epithelial cells. This has been confirmed with data obtained from affinity labeling experiments. Regardless of the differences in the actual numbers of EGF receptors obtained, our data from immunohistochemistry, autoradiography, affinity labeling, and Scatchard analysis demonstrate that the mouse reproductive tract contains EGF receptors at the developmental stages examined. DISCUSSION

This study demonstrates that the developing mouse uterus and vagina contain an immunoreactive EGF receptor that is capable of binding lz51-labeled EGF. Similar to EGF binding observed in the uterus of the adult rat (Lin et ah, 1988) and human (Hofmann et al., 1984; Chegini et al., 1986), ‘%I-labeled EGF binds to the major cell types (epithelium, stroma, and myometrium) of the developing mouse uterus and vagina. The basal localization of silver grains seen here in the uterine and vaginal epithelium has also been documented in the oral epithelium (Cho et al., 1988) and rat intestine (Scheving et aZ., 1989). The ‘251-labeled EGF binding pattern observed in the mouse vaginal epithelium is similar to that reported for human skin, where the binding sites of ‘251-labeled EGF are primarily located in the mitotically active, basal keratinocytes and appear in diminished numbers as the degree of differentiation of these cells progresses (Nanney et ab, 1984). Immunoreactive EGF receptors have been reported in the human uterus (Damjanov et al., 1986) as well as

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other tissues such as skin (Nanney et ak, 1984; Green and Couchman, 1985) and stomach (Mori et al., 1987). Although heavy basal staining for EGF receptors has been documented in gastric parietal cells (Mori et ah, 1987), the discrete granule-like regions of immunoreactive EGF receptor seen here in the basal portion of mouse uterine epithelial cells at 13 days of age have not been documented elsewhere. The significance of these granule-like regions is, as yet, unknown, although they could represent clusters of EGF receptors. The tissue sections used for EGF receptor immunolocalization had previously been exposed to ‘251-labeled EGF, and it has been documented in A431 cells that EGF treatment results in receptor clustering that reaches a maximum within 10 min but is maintained for 1 hr, before returning to the control situation within another hour (van it has been shown by Belzen et al., 1988). Furthermore, immunofluorescent techniques that EGF receptors remain sequestered in intracellular membranous structures following internalization (Decker, 1988). Although the affinity labeling incubations in this study were performed at 4°C so that receptor internalization would not occur, the immunoreactive granule-like regions seen here might represent endogenous EGF receptors that had been previously internalized and sequestered in intracellular structures. Our 1251-labeled EGF autoradiography and immunohistochemistry data obtained from studies of intact tissue indicate that the EGF receptor is more prominent in uterine epithelium than stroma. Yet our data from 1251labeled EGF affinity labeling and Scatchard analysis of

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FIG. 7. Scatchard analysis of EGF binding to cultured uterine epithelial and stromal cells. Binding data from 17-day-old uterine epithelial (0) and stromal (0) cells that were cultured for 5 days is plotted according to Scatchard (1949) to give Kd and B,,,, values of 0.11 nM and 9.44 fmol EGF bound/lO’cells for epithelial cells and 0.15 nMand 20.65 fmol EGF bound/lO’ cells for stromal cells. The number of binding sites per cell was calculated to be approximately 57,000 for epithelial cells and 124,000 for stromal cells.

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enzymatically separated uterine cells consistently demonstrate more EGF receptors in the stroma than the epithelium. There are several explanations which may account for this discrepancy. Stromal EGF receptors may exist in a masked state in whole tissue such that they are not detectable by tissue autoradiography or immunohistochemistry. The process of cell separation may unmask the receptors such that they become available for biochemical analysis of ‘%I-labeled EGF binding (DiAugustine et aZ., 1988). Alternatively, differences in cell separation conditions may render epithelial EGF receptors more susceptible to enzymatic proteolysis than stromal EGF receptors, resulting in fewer epithelial receptors being detected by biochemical assays. Nevertheless, our study consistently demonstrates that uterine stromal cells, whether freshly isolated or cultured, have more biochemically detectable EGF receptors than uterine epithelial cells. This suggests that the uterine stroma may have an inherently greater capacity to produce EGF receptors than the epithelium, and that the availability of the stromal EGF receptor is modulated by the complex cell-to-cell and cell-to-matrix interactions in whole tissue in vivo. Given that external and internal factors such as phorbol esters and vasopressin can reduce EGF receptor affinity (Das, 1982) and that receptors can be rendered inactive by structural modification, it is also plausible that the discrepancy between the biochemical and tissue analyses may be due to the existence of differences in the degree of structural modification of the EGF receptors in the various uterine cell types. Developmental changes in EGF receptor expression are not unique to the uterus. Studies have demonstrated time-dependent increases in both the EGF receptor and an EGF-like molecule in different embryonic and fetal mouse tissues (Nexe et al., 1980; Adamson and Meek, 1984). It has been hypothesized that developmental receptor variations of this kind may provide a method of cellular control separate from the hormone-mediated receptor down- and up-regulation described in other systems (Nexe et ah, 1980). A corollary to this hypothesis is that developmental changes in EGF receptor levels may be a function of different roles EGF receptors may play during different stages of growth and differentiation (Adamson and Meek, 1984; Scheving et al., 1989). It has been suggested that EGF receptors may be important for regulation of proliferation in relatively undifferentiated, embryonic cells and become important for stimulation of differentiation as the tissues mature. In adult tissues, EGF receptors may play a role not only in the stimulation of tissue repair after damage, but also in the maintenance of differentiated functions, such as ion transport in the mature kidney (Vehaskari et al., 1989).

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Given that EGF receptors are present in the prenatal reproductive tract as well as all of the uterine and vaginal cell types, it is feasible that EGF and its receptor may play an important role in the regulation of morphogenesis, growth, and differentiation of the mouse reproductive tract. It is known that estrogens regulate the growth of the uterus and other target tissues, although the mechanism(s) by which they do so is unknown. One hypothesis regarding the mechanism of estrogen action is that estrogen control of growth involves polypeptide growth factors. It is of interest to note that at a point in reproductive tract development when rapid growth is occurring and when epithelial estrogen receptors are not immunohistochemically or autoradiographically detectable (i.e., up to 4 days of age in CD-1 mice) (Taguchi et aZ., 1988; Yamashita et ah, 1989), epithelial EGF receptors are. Thus, the possibility exists that the high levels of DNA synthesis at this critical stage of development are due to endogenous EGF or EGF-like molecules. This is of particular significance in light of reproductive tract abnormalities seen in diethylstilbestrol(DES)-treated animals (McLachlan et al., 1980; Newbold et al., 1983), especially in structures derived from the Miillerian duct, where we have demonstrated immunoreactive EGF receptor molecules beginning at Day 13 of gestation. Some of the DES-induced abnormalities may be due to perturbations of the EGF and EGF receptor pathway following inappropriate exposure to exogenous estrogen. Further studies are in progress to determine whether the immunoreactive EGF receptors that are detected in the Mtillerian duct and its derivatives are functional as the reproductive tract proceeds along its developmental path, whether estrogen modifies EGF receptor levels at these developmental stages, and whether estrogen causes persistent alterations in the EGF receptor of the mouse reproductive tract. The authors thank Dr. Eileen D. Adamson for generously providing the antibody to the EGF receptor. REFERENCES ADAMSON, E. D., DELLER, M. J., and WARSHAW, J. B. (1981). Functional EGF receptors are present on mouse embryo tissues. Nature (London) 291,656-659. ADAMSON, E. D., and MEEK, J. (1984). The ontogeny of epidermal growth factor receptors during mouse development. Deu. Biol. 103, 62-70.

ADAMSON, E. D., and REES, A. R. (1981). Epidermal growth factor receptors. Mol. Cell Rio&em. 34,129-152. BIGSBY, R. M., and CUNHA, G. R. (1985). Effects of progestins and glucocorticoids on deoxyribonucleic acid synthesis in the uterus of the neonatal mouse. Endocrinology 117,2520-2526. CARPENTER, G., and COHEN, S. (1979). Epidermal growth factor. Annu. Rev. B&hem

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HOFMANN, G. E., RAO, C. V., BARROWS, G. H., SCHULTZ, G. S., and SANFILIPPO, J. S. (1984). Binding sites for epidermal growth factor in human uterine tissues and leiomyomas. J Clin. Endocrinol. Metub. 58, 880-887. LIN, T.-H., MUKKU, V. R., VERNER, G., KIRKLAND, J. L., and STANCEL, G. M. (1988). Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types. BioL Reprod. 38, 403-411. MCLACHLAN, J. A., NEWBOLD, R. R., and BULLOCK, B. C. (1980). Long-

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