Grant D. Orvis

Uterine epithelial estrogen receptor α is dispensable for proliferation but essential for complete biological and biochemical responses.

Female fertility requires estrogen to specifically stimulate estrogen receptor α (ERα)-dependent growth of the uterine epithelium in adult mice, while immature females show proliferation in both stroma and epithelium. To address the relative roles of ERα in mediating estrogen action in uterine epithelium versus stroma, a uterine epithelial-specific ERα knockout (UtEpiαERKO) mouse line was generated by crossing Esr mice with Wnt7a-Cre mice. Expression of Wnt7a directed Cre activity generated selective deletion of ERα in uterine epithelium, and female UtEpiαERKO are infertile. Herein, we demonstrate that 17β-estradiol (E2)-induced uterine epithelial proliferation was independent of uterine epithelial ERα because DNA synthesis and up-regulation of mitogenic mediators were sustained in UtEpiαERKO uteri after E2 treatment. IGF-1 treatment resulted in ligand-independent ER activation in both wild-type (WT) and UtEpiαERKO and mimicked the E2 stimulatory effect on DNA synthesis in uterine epithelium. Uterine epithelial ERα was necessary to induce lactoferrin, an E2-regulated secretory protein selectively synthesized in the uterine epithelium. However, loss of uterine epithelial ERα did not alter the E2-dependent progesterone receptor (PR) down-regulation in epithelium. Strikingly, the uterine epithelium of UtEpiαERKO had robust evidence of apoptosis after 3 d of E2 treatment. Therefore, we surmise that estrogen induced uterine hyperplasia involves a dispensable role for uterine epithelial ERα in the proliferative response, but ERα is required subsequent to proliferation to prevent uterine epithelial apoptosis assuring the full uterine epithelial response, illustrating the differential cellular roles for ERα in uterine tissue and its contribution during pregnancy.

A mesenchymal perspective of müllerian duct differentiation and regression in Amhr2-lacZ mice

The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding β‐galactosidase (β‐gal), into the AMHR‐II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2‐lacZ heterozygotes express β‐gal activity in an Amhr2‐specific pattern. In the gonads, β‐gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. β‐gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, β‐gal activity was restricted to one side of the ductal mesoepithelium, whereas in males β‐gal expression encircled the duct. β‐gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal β‐gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2‐expressing mesenchyme. Mol. Reprod. Dev. 75: 1154–1162, 2008.

Functional Redundancy of TGF-beta Family Type I Receptors and Receptor-Smads in Mediating Anti-Müllerian Hormone-Induced Müllerian Duct Regression in the Mouse

Amniotes, regardless of genetic sex, develop two sets of genital ducts: the Wolffian and Müllerian ducts. For normal sexual development to occur, one duct must differentiate into its corresponding organs, and the other must regress. In mammals, the Wolffian duct differentiates into the male reproductive tract, mainly the vasa deferentia, epididymides, and seminal vesicles, whereas the Müllerian duct develops into the four components of the female reproductive tract, the oviducts, uterus, cervix, and upper third of the vagina. In males, the fetal Leydig cells produce testosterone, which stimulates the differentiation of the Wolffian duct, whereas the Sertoli cells of the fetal testes express anti-Müllerian hormone, which activates the regression of the Müllerian duct. Anti-Müllerian hormone is a member of the transforming growth factor-beta (TGF-beta) family of secreted signaling molecules and has been shown to signal through the BMP pathway. It binds to its type II receptor, anti-Müllerian hormone receptor 2 (AMHR2), in the Müllerian duct mesenchyme and through an unknown mechanism(s); the mesenchyme induces the regression of the Müllerian duct mesoepithelium. Using tissue-specific gene inactivation with an Amhr2-Cre allele, we have determined that two TGF-beta type I receptors (Acvr1 and Bmpr1a) and all three BMP receptor-Smads (Smad1, Smad5, and Smad8) function redundantly in transducing the anti-Müllerian hormone signal required for Müllerian duct regression. Loss of these genes in the Müllerian duct mesenchyme results in male infertility due to retention of Müllerian duct derivatives in an otherwise virilized male.

Stromal-to-epithelial transition during postpartum endometrial regeneration.

Endometrium is the inner lining of the uterus which is composed of epithelial and stromal tissue compartments enclosed by the two smooth muscle layers of the myometrium. In women, much of the endometrium is shed and regenerated each month during the menstrual cycle. Endometrial regeneration also occurs after parturition. The cellular mechanisms that regulate endometrial regeneration are still poorly understood. Using genetic fate-mapping in the mouse, we found that the epithelial compartment of the endometrium maintains its epithelial identity during the estrous cycle and postpartum regeneration. However, whereas the stromal compartment maintains its identity during homeostatic cycling, after parturition a subset of stromal cells differentiates into epithelium that is subsequently maintained. These findings identify potential progenitor cells within the endometrial stromal compartment that produce long-term epithelial tissue during postpartum endometrial regeneration.

STAT3 regulates uterine epithelial remodeling and epithelial-stromal crosstalk during implantation.

Embryo implantation is regulated by a variety of endometrial factors, including cytokines, growth factors, and transcription factors. Earlier studies identified the leukemia inhibitory factor (LIF), a cytokine produced by uterine glands, as an essential regulator of implantation. LIF, acting via its cell surface receptor, activates the signal transducer and activator of transcription 3 (STAT3) in the uterine epithelial cells. However, the precise mechanism via which activated STAT3 promotes uterine function during implantation remains unknown. To identify the molecular pathways regulated by STAT3, we created SW(d/d) mice in which Stat3 gene is conditionally inactivated in uterine epithelium. The SW(d/d) mice are infertile due to a lack of embryo attachment to the uterine luminal epithelium and consequent implantation failure. Gene expression profiling of uterine epithelial cells of SW(d/d) mice revealed dysregulated expression of specific components of junctional complexes, including E-cadherin, α- and β-catenin, and several claudins, which critically regulate epithelial junctional integrity and embryo attachment. In addition, uteri of SW(d/d) mice exhibited markedly reduced stromal proliferation and differentiation, indicating that epithelial STAT3 controls stromal function via a paracrine mechanism. The stromal defect arose from a drastic reduction in the production of several members of the epidermal growth factor family in luminal epithelium of SW(d/d) uteri and the resulting lack of activation of epidermal growth factor receptor signaling and mitotic activity in the stromal cells. Collectively, our results uncovered an intricate molecular network operating downstream of STAT3 that regulates uterine epithelial junctional reorganization, and stromal proliferation, and differentiation, which are critical determinants of successful implantation.

Lhx1 is required in Müllerian duct epithelium for uterine development.

The female reproductive tract organs of mammals, including the oviducts, uterus, cervix and upper vagina, are derived from the Müllerian ducts, a pair of epithelial tubes that form within the mesonephroi. The Müllerian ducts form in a rostral to caudal manner, guided by and dependent on the Wolffian ducts that have already formed. Experimental embryological studies indicate that caudal elongation of the Müllerian duct towards the urogenital sinus occurs in part by proliferation at the ductal tip. The molecular mechanisms that regulate the elongation of the Müllerian duct are currently unclear. Lhx1 encodes a LIM-homeodomain transcription factor that is essential for male and female reproductive tract development. Lhx1 is expressed in both the Wolffian and Müllerian ducts. Wolffian duct-specific knockout of Lhx1 results in degeneration of the Wolffian duct and consequently the non-cell-autonomous loss of the Müllerian duct. To determine the role of Lhx1 specifically in the Müllerian duct epithelium, we performed a Müllerian duct-specific knockout study using Wnt7a-Cre mice. Loss of Lhx1 in the Müllerian duct epithelium led to a block in Müllerian duct elongation and uterine hypoplasia characterized by loss of the entire endometrium (luminal and glandular epithelium and stroma) and inner circular but not the outer longitudinal muscle layer. Time-lapse imaging and molecular analyses indicate that Lhx1 acts cell autonomously to maintain ductal progenitor cells for Müllerian duct elongation. These studies identify LHX1 as the first transcription factor that is essential in the Müllerian duct epithelial progenitor cells for female reproductive tract development. Furthermore, these genetic studies demonstrate the requirement of epithelial-mesenchymal interactions for uterine tissue compartment differentiation.

Critical Tumor Suppressor Function Mediated by Epithelial Mig-6 in Endometrial Cancer

Endometrial cancer is preceded by endometrial hyperplasia, unopposed estrogen exposure, and genetic alterations, but the precise causes of endometrial cancer remain uncertain. Mig-6, mainly known as a negative regulator of the EGF receptor, is an important mediator of progesterone signaling in the uterus, where it mediates tumor suppression by modulating endometrial stromal-epithelial communications. In this study, we investigated the function of Mig-6 in the uterine epithelium using a tissue-specific gene knockout strategy, in which floxed Mig-6 (Mig-6(f/f)) mice were crossed to Wnt7a-Cre mice (Wnt7a(cre+)Mig-6(f/f)). Wnt7a(cre+)Mig-6(f/f) mice developed endometrial hyperplasia and estrogen-dependent endometrial cancer, exhibiting increased proliferation in epithelial cells as well as apoptosis in subepithelial stromal cells. We documented increased expression of NOTCH1 and BIRC3 in epithelial cells of Wnt7a(cre+)Mig-6(f/f) mice and decreased expression of the progesterone receptor (PR) in stromal cells. Progesterone therapy controls endometrial growth and prevents endometrial cancer, but the effectiveness of progesterone as a treatment for women with endometrial cancer is less clear. We noted that the hyperplasic phenotype of Wnt7a(cre+)Mig-6(f/f) mice was prevented by progesterone treatment, whereas this treatment had no effect in PR(cre/+)Mig-6(f/f) mice where Mig-6 was deleted in both the epithelial and stromal compartments of the uterus. In contrast, activation of progesterone signaling in the stroma regulated proliferation and apoptosis in the epithelium via suppression of ERα signaling. In summary, our results establish that epithelial Mig-6 functions as a critical tumor suppressor that mediates the ability of progesterone to prevent the development of endometrial cancer.

Functional analysis of ectodermal β-catenin during external genitalia formation

β‐catenin is a molecule belonging to the armadillo family of proteins that is a crucial core‐component of cellular adherens junctions, and a component of the canonical Wnt‐signaling pathway. We attempted to analyze the functional significance of ectodermal‐derived β‐catenin during the development of the mouse genital tubercle, a mammalian anlage of the external genitalia. For this purpose, the conditional loss of function mouse mutant Wnt7a‐Cre;β‐catf/f was utilized. Loss of ectodermal β‐catenin leads to the formation of urethral cleft during preputial uprising. Although expression of E‐cadherin was retained in the genital tubercle ectoderm of mutants, probably through plakoglobin compensatory expression, expression of other crucial adherens junction components such as α‐catenin and F‐actin in the cell‐cell border were distinctly reduced. We also showed that β‐catenin is necessary for the expression of its transcriptional downstream target Lef‐1 which was localized in the basal layer of the preputial ectoderm, excluding the midventral region at E15.5. Such specialized region was observed to possess cytoplasmic β‐catenin expression at this stage. Coincidentally, mitotically active cells were also found in the basal layer of the preputial ectoderm excluding the midventral region. In mutant genital tubercle, cell proliferation in the preputial ectoderm was decreased. Taken together, we suggest that ectodermal β‐catenin is necessary not only to maintain adherens junction integrity, but also to regulate cell proliferation possibly through Lef‐1 functions. Thus, β‐catenin is shown to perform dual functions, initially as an adhesion molecule and later on as a possible transcription factor.

Abstract 2197: Epithelial Mig-6 is critical for a tumor suppressor function in endometrial cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Normal endometrial function requires a balance of progesterone (P4) and estrogen (E2) action. A bias toward E2 action and away from P4 action underlies the tumorigenesis of endometrial adenocarcinoma which is the fourth most common cancer in women. In previous studies, we have identified mitogen-inducible gene 6 (Mig-6) as a downstream target of PR (progesterone receptor) action in the uterus. Mig-6 is expressed in both the epithelium and stroma of the uterus. Conditional ablation of Mig-6 in all compartments of the uterus (PRcre/+ Mig-6f/f) results in infertility and leads to the development of endometrial hyperplasia and estrogen-induced endometrial cancer. Our central hypothesis is that Mig-6 is a critical mediator of stromal-epithelial communication in pregnancy, steroid hormone regulation and tumor suppressor function. In order to understand the role of epithelial Mig-6 in the uterus, we generated a mouse model in which we specifically ablate epithelial endometrial Mig-6 using Wnt7a-cre mice (Wnt7acre+ Mig-6f/f). Wnt7acre+ Mig-6f/f mice are subfertile due to embryo abortion and display normal P4 attenuation of E2-mediated uterine hypertrophy. However, Wnt7acre+ Mig-6f/f mice develop endometrial hyperplasia at 5 months of age as well as estrogen-induced endometrial cancer. The expression of PR is reduced in Wnt7acre+ Mig-6f/f mice compared to control Mig-6f/f mice. Interestingly, the hyperplasia exhibited by the Wnt7acre+ Mig-6f/f mice is reversed by P4 treatment, contrary to what is observed in PRcre/+ Mig-6f/f mice. Also, the expression of ERα, as well as its target genes Muc-1, Clca3, Ltf and Birc1, is significantly decreased in the Wnt7acre+ Mig-6f/f mice as compared to the control and PRcre/+ Mig-6f/f after P4 treatment. Concurrently, stromal Mig-6 expression is significantly increased in Wnt7acre+ Mig-6f/f mice indicating that P4-induced stromal Mig-6 can reverse the hyperplasia seen in the Wnt7acre+ Mig-6f/f mice by regulating ERα expression level. These data suggest that epithelial Mig-6 plays an important tumor suppressor role during tumorigenesis in the uterus. (Supported by NIH, R01 HD057873) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2197. doi:10.1158/1538-7445.AM2011-2197