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Departments of Obstetrics and Gynecology and Molecular Genetics, University of Illinois (Z.F., S.Y., B.G., M.T., S.E.B.), Chicago, Illinois 60612; Prince Henry’s Institute of Medical Research, Monash University (E.S.), Victoria 3168, Australia; and Novartis Pharmaceuticals (D.E.), Basel, CH-4002, Switzerland
Address all correspondence and requests for reprints to: Serdar E. Bulun, M.D, Department of Obstetrics and Gynecology, University of Illinois, 820 South Wood Street, M/C808, Chicago, Illinois 60612. E-mail: . sbulun{at}uic.edu
Abstract
Aromatase P450 (P450arom) is the key enzyme for the biosynthesis of estrogen that is essential for the growth of human endometriosis, a pathology characterized by endometrium-like tissue on the peritoneal surfaces of abdominal organs manifest by pelvic pain and infertility. Surgically transplanted autologous uterine tissue to ectopic sites on the peritoneum in mice has been used as an animal model to study endometriosis. Using this mouse model, we evaluated the roles of the P450arom gene and aromatase enzyme activity in the growth of endometriosis represented by ectopic uterine tissues in mice. Endometriosis was induced surgically in the following groups of mice: 1) untreated transgenic mice with disrupted P450arom gene (ArKO); 2) ArKO mice treated with systemic estrogen; 3) untreated wild-type (WT) mice; 4) WT mice treated with estrogen; 5) WT mice treated with the aromatase inhibitor, letrozole; and 6) WT mice treated with letrozole and estrogen. Each group contained eight mice; +/+ littermates of ArKO mice were used as WT controls. Treatment with estrogen increased the size of ectopic uterine tissues in ArKO and WT mice significantly. The ectopic uterine lesions in untreated and estrogen-treated ArKO mice were strikingly smaller than those in untreated and estrogen-treated WT controls, respectively. Systemic treatment of WT mice with letrozole significantly decreased the lesion size in a dose-dependent manner. The addition of estrogen to letrozole treatment increased the ectopic lesion size, although these lesions were significantly smaller than those in mice treated with estrogen only. As tissue controls, the effects of these conditions on normally located (eutopic) uterine tissue were evaluated. The effects of disruption of the P450arom gene and treatments with letrozole and estrogen seemed to be more profound on ectopic tissues, suggesting that ectopic tissues might be more sensitive to estrogen for growth. We conclude that both an intact P450arom gene and the presence of aromatase enzyme activity are essential for the growth of ectopic uterine tissue in a mouse model of endometriosis.
ENDOMETRIOSIS IS CHARACTERIZED by the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites and is linked to pelvic pain and infertility. It is a common condition, affecting 1 in 10 women in the reproductive age group (1, 2, 3). Although the etiology and exact mechanism for the development of endometriosis are not well understood, both circumstantial and laboratory evidence are indicative of a critical role of estrogen in the establishment and maintenance of this disease (4). The usefulness of GnRH agonists, which suppress ovarian steroidogenesis in the management of endometriosis, is well recognized. There are, however, two important caveats, which are not addressed by the GnRH agonist treatment. First, large quantities of estrogen can be produced locally within the endometriotic cells; this represents an intracrine mechanism in addition to ovarian secretion, which is the classical mechanism for estrogen production (5). Local estrogen biosynthesis is not blocked by any of the currently used treatments for endometriosis. Second, estrogen produced in peripheral tissue sites (e.g. adipose tissue and skin fibroblasts) may give rise to significant circulating levels of estrogen (6). Additionally, defective inactivation of estrogen in endometriosis in contrast to eutopic endometrium may further increase local concentrations of the potent estrogen (i.e. estradiol) (7). Thus, the origin of estrogen in target tissues can arise from three likely sources: 1) ovarian secretion via an endocrine mechanism (endocrine), 2) peripheral formation that is sufficient to increase circulating estrogen (endocrine), and 3) local formation in the target tissue (intracrine or paracrine).
Aromatase is the key enzyme for estrogen formation in human tissues. The conversion of C19 steroids to estrogens by aromatase P450 (P450arom) takes place in a number of human tissues, e.g. ovary, placenta, adipose tissue, skin, and brain (8). We previously demonstrated significant levels of P450arom mRNA and activity in the stromal cell component of endometriotic tissues, whereas aromatase expression is either only barely detectable or most commonly absent in the eutopic endometrium (9, 10). Moreover, aromatase activity and P450arom mRNA levels in cultured endometriotic stromal cells could be induced strikingly by cAMP analogs or PGE2 to extremely high levels comparable to those found in ovarian granulosa cells or the placental syncytiotrophoblast (10). The clinical significance of local aromatase activity in endometriotic tissue was exemplified recently by the successful use of an aromatase inhibitor to treat an unusually aggressive case of recurrent postmenopausal endometriosis that was resistant to progestin (11). Collectively, these data are suggestive that aromatase expression in one or more human tissues is essential for the growth and persistence of ectopic endometrium-like lesions, i.e. endometriosis, as this pathological tissue is dependent on biologically active estradiol.
Studies in rodents in which endometriosis was surgically induced have demonstrated a critical effect of surgically transplanted uterine tissues on fertility (12, 13, 14). Estrogen gave rise to growth, whereas pregnancy and lactation caused atrophy of these ectopic uterine lesions (14). These findings have demonstrated that the rodent model of endometriosis is useful for the study of responsiveness of ectopic lesions to steroid hormones (12, 13, 14).
The aromatase knockout (ArKO) mouse has been developed as a mouse model of estrogen insufficiency by targeted disruption of the P450arom (Cyp19) gene (15). We have used ArKO mice by surgically transplanted uterine tissue to peritoneal surfaces to directly determine the role of aromatase in the development and growth of ectopic uterine lesions. We demonstrated for the first time that in mice genetically deprived of estrogen production, ectopic uterine tissues remain atrophic. Adding back systemic estrogen causes growth of this ectopic tissue in ArKO mice. By analogy, treatment of wild-type mice systemically with an aromatase inhibitor significantly reduces the size of ectopic uterine implants. Using this in vivo model, we studied the growth properties of aberrantly located (i.e. ectopic) vs. normally located (i.e. eutopic) endometrial tissue in response to genetically and pharmacologically induced estrogen deficiency and thus the role of aromatase in endometriosis.
Materials and Methods
Animals
All mice had C57BL6 backgrounds. All mice were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Disrupting the Cyp19 gene as described previously (15) generated ArKO mice. Mice were genotyped by PCR as described previously (16). We maintain an ArKO mouse colony in this laboratory and genotype them routinely. Wild-type (WT) littermates were used as controls. Animals were maintained under specific pathogen-free conditions and had unlimited access to drinking water.
Surgery
At the time of surgery, all mice were 6 months of age. We induce endometriosis-like lesions through transplantation of one of the uterine horns to the bowel mesentery as described previously (12). Briefly, the animal was placed under general anesthesia, and a midventral incision was made to expose the uterus and intestines. The left uterine horn was ligated at both ends and removed to warm medium (Ham’s F-12). The uterine horn was split longitudinally and then cut into three square pieces measuring approximately 2 mm on each dimension. Three equal pieces of left uterine tissues were sutured with serosal layer in direct apposition to the peritoneum close to a vessel, as is the endometrial layer of the uterine square facing the abdominal cavity. A single suture (4-0 VP557) was used for each piece of tissue. The abdomen was then closed using the same suture material. The mice were killed within 8 wk to assess the size, histology, and proliferating cell nuclear antigen (PCNA) content of the endometriotic lesion.
Experimental design
Initially female mice were divided into six groups. WT littermates of ArKO mice were used to assess the effects of the aromatase inhibitor letrozole. WT littermates also served as control groups for ArKO mice. Endometriosis was induced surgically in all of the following groups of mice: 1) ArKO mice, 2) untreated WT mice, 3) ArKO mice treated with conjugated estrogens (CE), 4) WT mice treated with CE, 5) WT mice treated with letrozole, and 6) WT mice treated with letrozole added to the CE regimen. The transplanted (ectopic) and eutopic uterine tissues were evaluated within 8 wk after surgery. Mice were killed with CO2 immediately before the tissues were harvested.
Estrogen supplementation
CE were purchased from Ayerst Laboratories, Inc. (Philadelphia, PA). Ten micrograms of CE were dissolved in 100 µl saline and administrated sc. Various quantities of the aromatase inhibitor letrozole [4,4'-(1H-1,2,4-triazol-1-ylmethylene) dibenzonitrile; Novartis, Basel, Switzerland ] were first dissolved in ethanol and then diluted with saline and administrated sc every day in a volume of 100 µl.
Evaluation of ectopic and eutopic uterine tissues
Eight weeks after peritoneal transplantation of uterine tissues in treated or untreated mice, animals were killed with CO2. Mice were immediately fixed in the supine position on a platform, the abdomen was opened, and all three dimensions of ectopic and eutopic uterine tissues were measured using a caliper. The volume of each ectopic uterine tissue was calculated by following formula: Vectopic = 0.52 x A x B x C, where A, B, and C denote width, length, and height, respectively. The volume of the cylinder-shaped eutopic uterine horn was calculated by the following formula: Veutopic = 
r2h, where r = (D1 + D2)/4, D1 and D2 are diameters at each end of the uterine horn, and h is length of the uterine horn. Tissues were also photographed using a digital camera. All images of transplanted tissues and remaining eutopic uterine horns were taken using the DP10 microscope digital camera system (Olympus Corp., Melville, NY) at x2.5 magnification. We used the remaining eutopic uterine horn as a control tissue in each mouse. These normally located eutopic uterine tissues were evaluated with respect to size, weight, and proliferation. For histological assessment, sections of ectopic tissues together with adherent organs (bowel and its mesentery) and eutopic uterine tissues were fixed in 10% neutral buffered formalin. After appropriate fixing for 2 d before dehydration, tissues were trimmed, dehydrated through ascending grades of ethyl alcohol, cleared in xylene, and infiltrated with paraffin wax. Tissues were embedded in paraffin and 5-µm sections were prepared. The sections were heat-dried and stained with hematoxylin and eosin.
Western blot
Western blotting was used to determine the levels of PCNA in ectopic uterine tissues. Briefly, tissue were homogenized in lysis buffer and transferred to nylon membrane. Nonspecific binding was blocked by incubation overnight at 4 C with Tris-buffered saline containing 0.05% Triton X-100 and 5% nonfat dry milk. Filters were exposed to antibody raised against PCNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature, and antimouse IgG-horseradish peroxidase for 40 min at room temperature. Chemiluminescent Super Signal West Femto Stable Peroxide Buffer (Santa Cruz Biotechnology, Inc.) was used to detect the signals.
Statistics
We initially used two-tailed ANOVA to determine the statistical significance of the differences between multiple groups. This was followed by Newman-Keuls multiple comparisons test to calculate differences between individual groups. The P value for the difference between the means of two groups was calculated, where 
was equal to 0.05 and power (1 - ß) was 0.80.
Results
Effects of the disruption of the P450arom (Cyp19) gene on the growth of ectopic uterine tissue
To assess the in vivo role of the P450arom gene in the growth of ectopic uterine tissue, uterine tissues were surgically transplanted to the peritoneum of the bowel mesentery in adult female ArKO and WT mice. We preserved ovaries in all groups of mice to mimic the intact function of ovary as in premenopausal women with endometriosis. First, we compared the size of ectopic lesions in untreated ArKO and WT mice (Fig. 1
, A and C). Eight weeks from the time of transplantation of uterine tissues to the peritoneum, transplants remained as small scars in untreated ArKO mice at the end of the 8-wk period (0.83 ± 0.08 mm3), whereas transplanted uterine tissues in untreated WT mice grew as round lesions (36.06 ± 1.73 mm3; Fig. 1, A and C, and Table 1). When exogenous estrogen was administered to these mice (10 µg CE, sc, every day) for 10 wk (starting from 2 wk before surgery), the size of ectopic uterine tissues increased significantly in both groups (Fig. 1, E and G, and Table 1). These gross anatomical findings were accompanied by similar change in histological sections (Fig. 1, B, D, F, and H). These findings are suggestive that a functional P450arom gene is essential for the growth of ectopic uterine implants. Exogenous estrogen partially substitutes for the effects of an intact P450arom gene, but does not fully restore the WT phenotype with respect to lesion size (see statistically significant difference between groups 3 and 4 in Table 1).
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Thus, we concluded that the growth of ectopic uterine implants is dependent on the product of the P450arom gene or estrogen. To demonstrate that aromatase enzyme activity was essential for the growth of these implants, WT animals were treated with letrozole, a potent medication that inhibits the function of the P450arom protein. Letrozole was administered sc for 4 wk with or without treatment of animals with CE before and during letrozole treatment. Letrozole treatment (10 µg/d, sc) significantly decreased the implant size (Fig. 1I
and groups 2 and 5 in Table 1), whereas adding CE (10 µg/d, sc) back to letrozole treatment partially restored the lesion size (Fig. 1K and Table 1). Add-back estrogen to letrozole-treated mice, however, did not fully restore the ectopic lesion size to that observed in controls treated with estrogen only (see statistically significant difference between groups 4 and 6 in Table 1).
The suppressive effect of letrozole on the growth of ectopic uterine tissue was dose dependent (Fig. 2). We found that the minimum dose of letrozole that gave rise to a significant decrease in the lesion size was 5 µg/d, sc continued for 4 wk. Increases in the dose up to 20 µg/d did not result in further reduction in the ectopic lesion size (Fig. 2). Three mice were included in each group for this dose-response study (total n = 21 in Fig. 2).
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Effects of disruption of the P450arom gene or the function of its product on cell proliferation
We determined the effects of an intact P450arom gene or its product on cell proliferation in ectopic uterine lesions. We determined the protein levels of PCNA for this purpose. PCNA levels in ectopic uterine tissues were strikingly decreased in ArKO mice (Fig. 3, lane 1) and letrozole-treated WT mice (Fig. 3, lane 4) compared with untreated WT mice (Fig. 3, lane 3). The addition of CE to ArKO or letrozole-treated WT mice (Fig. 3, lanes 2 and 6) restored cell proliferation (Fig. 3). This experiment was performed three times with reproducible results (Fig. 3).
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The volume and weight of the remaining left uterine horn after surgery and treatment were determined, and eutopic uterine tissue was evaluated as an additional control (Fig. 4, A and C, and Tables 1 and 2). Significant changes in the size and weight of eutopic uterine tissue accompanied disruption of the P450arom gene and inhibition of aromatase activity with letrozole (Tables 1 and 2). These changes were similar to those seen in ectopic uterine tissues and were reversible in part by treatment with CE. Histological examination confirmed the changes in volume and weight (Fig. 4).
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and 2 and Figs. 4 and 5). These decreases, however, were not as pronounced as the corresponding conditions for ectopic uterine tissues (Table 1). In ArKO mice, the volume of the ectopic uterine tissues was 2% that in WT mice, whereas the volume of mean eutopic uterine tissues was 15% that in WT mice (Table 1). By the same token, ectopic uterine tissue volume decreased by 48% in response to letrozole, whereas letrozole-induced volume reduction was 26% for eutopic uterine tissue (Table 1). These observations suggest that ectopic uterine tissue is more dependent for growth on a functional P450arom gene and protein compared with eutopic uterine tissue.
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In this study we used ArKO mice as an in vivo model to determine the role of the P450arom gene in the growth of ectopic uterine tissue. We also employed the aromatase inhibitor letrozole to determine the role of aromatase enzyme activity in the growth of ectopic uterine tissue in WT mice. This model clearly indicated that both P450arom gene and P450arom protein with an intact enzyme activity are essential for the growth of ectopic and eutopic uterine tissues.
The antiproliferative effects of aromatase inhibitors on the eutopic endometrium have been reported previously (17). Our findings were consistent with this report. Our results were also suggestive that ectopic tissue is more sensitive to the proliferative effects of the P450arom gene and aromatase enzyme. Exogenous estrogen could be used as a surrogate treatment to induce significant growth in these tissues of mice with genetically disrupted P450arom gene or abolished aromatase enzyme activity. Thus, we demonstrated clearly that estrogen is essential for the growth of ectopic uterine tissue.
We have not analyzed the role of local aromatase expression in endometriosis, because we did not use ovariectomy in this particular study for two reasons: 1) to mimic endometriosis in premenopausal women, and 2) to study the role of ovarian aromatase expression in this model. In mice, aromatase is expressed primarily in the brain and ovaries, and estradiol is secreted only from the ovary (18, 19, 20). In our model we do not know yet whether aromatase expression is present in ectopic uterine tissues of mice. Thus, negative effects of disrupted P450arom gene or enzyme activity in this model may be a result of the absence of ovarian aromatase activity, which would preclude ovarian estradiol secretion. In fact, it was reported that sc letrozole at a dose of 5 µg/d abolished ovarian function in mice (21).
Studies are underway to determine the role of tissue specific P450arom expression in the mouse endometriosis model. Nonetheless, regardless of site-specific P450arom expression, this model allowed us to demonstrate the essential roles of the P450arom gene and enzyme in the growth of endometriosis.
Footnotes
This work was supported by NIH Grant HD-38691 (to S.E.B.).
Abbreviations: ArKO, Aromatase knockout; CE, conjugated estrogens; E, estrogen; P450arom, aromatase P450; PCNA, proliferating cell nuclear antigen; WT, wild type.
Received December 19, 2001.
Accepted April 4, 2002.
References
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