Cory A. Rubel

WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse

WNT4, a member of the Wnt family of ligands, is critical for the development of the female reproductive tract. Analysis of Wnt4 expression in the adult uterus during pregnancy indicates that it may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo. To investigate the role of Wnt4 in adult uterine physiology, conditional ablation of Wnt4 using the PRC” mouse model was accomplished. Ablation of Wnt4 rendered female mice subfertile due to a defect in embryo implantation and subsequent defects in endometrial stromal cell survival, differentiation, and responsiveness to progesterone signaling. In addition to altered stromal cell function, the uteri of PITe/+ Wnt4f/f (Wnt4d/d) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands and the emergence of a p63‐positive basal cell layer beneath the columnar luminal epithelial cells. The altered epithelial cell phenotype was further escalated by chronic estrogen treatment, which caused squamous cell metaplasia of the uterine epithelium in the Wnt4d/d mice. Thus, WNT4 is a critical regulator not only of proper postnatal uterine development, but also embryo implantation and decidualization.—Franco, H. L., Dai, D., Lee, K. Y., Rubel, C. S., Roop, D., Boerboom, D., Jeong, J.‐W., Lydon, J.‐P., Bagchi, I. C., Bagchi, M. K., DeMayo, F. J. WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse. FASEB J. 25, 1176–1187 (2011). www.fasebj.org

Research Resource: Genome-Wide Profiling of Progesterone Receptor Binding in the Mouse Uterus

Progesterone (P4) signaling through its nuclear transcription factor, the progesterone receptor (PR), is essential for normal uterine function. Although deregulation of PR-mediated signaling is known to underscore uterine dysfunction and a number of endometrial pathologies, the early molecular mechanisms of this deregulation are unclear. To address this issue, we have defined the genome-wide PR cistrome in the murine uterus using chromatin immunoprecipitation (ChIP) followed by massively parallel sequencing (ChIP-seq). In uteri of ovariectomized mice, we identified 6367 PR-binding sites in the absence of P4 ligand; however, this number increased at nearly 3-fold (18,432) after acute P4 exposure. Sequence analysis revealed that approximately 73% of these binding sites contain a progesterone response element or a half-site motif recognized by the PR. Many previously identified P4 target genes known to regulate uterine function were found to contain PR-binding sites, confirming the validity of our methodology. Interestingly, when the ChIP-seq data were coupled with our microarray expression data, we identified a novel regulatory role for uterine P4 in circadian rhythm gene expression, thereby uncovering a hitherto unexpected new circadian biology for P4 in this tissue. Further mining of the ChIP-seq data revealed Sox17 as a direct transcriptional PR target gene in the uterus. As a member of the Sox transcription factor family, Sox17 represents a potentially novel mediator of PR action in the murine uterus. Collectively, our first line of analysis of the uterine PR cistrome provides the first insights into the early molecular mechanisms that underpin normal uterine responsiveness to acute P4 exposure. Future analysis promises to reveal the PR interactome and, in turn, potential therapeutic targets for the diagnosis and/or treatment of endometrial dysfunction.

Constitutive activation of smoothened leads to female infertility and altered uterine differentiation in the mouse.

Previous work has identified Indian hedgehog (Ihh) as a major mediator of progesterone signaling during embryo implantation. Ihh acts through its downstream effector smoothened (Smo) to activate the GLI family of transcription factors. In order to gain a better understanding of Ihh action during embryo implantation, we expressed a Cre-recombinase-dependent constitutively activated SMO in the murine uterus using the Pgrtm2(cre)Lyd (PRcre) mouse model [Pgrtm2(cre)Lyd+Gt(ROSA)26Sortm1(Smo/EYFP)Amc+ (PRcre/+SmoM2+)]. Female PRcre/+SmoM2+ mice were infertile. They exhibited normal serum progesterone levels and normal ovulation, but their ova failed to be fertilized in vivo and their uterus failed to undergo the artificially induced decidual response. Examination of the PRcre/+SmoM2+ uteri revealed numerous features such as uterine hypertrophy, the presence of a stratified luminal epithelial cell layer, a reduced number of uterine glands, and an endometrial stroma that had lost its normal morphologic characteristics. Microarray analysis of 3-mo-old PRcre/+SmoM2+ uteri demonstrated a chondrocytic signature and confirmed that constitutive activation of PRcre/+SmoM2+ increased extracellular matrix production. Thus, constitutive activation of Smo in the mouse uterus alters postnatal uterine differentiation which interferes with early pregnancy. These results provide new insight into the role of Hedgehog signaling during embryo implantation.

Epithelial-Stromal Interaction and Progesterone Receptors in the Mouse Uterus

Healthy uterine function depends on the balanced interaction of the ovarian steroids estrogen and progesterone (P4) signaling through their respective receptors. The expression of each receptor is regulated by the other through crucial cross talk between the epithelial and stromal compartments. Ablation of the progesterone receptor (PR) results in complete infertility in mice, and evidence increasingly demonstrates that the PR is a major mediator of epithelial-stromal cross talk and events leading to the disruption of this communication can lead to P4 resistance in the uterus. This resistance, through impaired P4 signaling, can be at the level of the PR itself, coregulators, and downstream effectors. The mechanisms underlying P4 resistance is of critical importance in womens health because this defect is seen in a wide variety of diseases including infertility, endometriosis, endometrial carcinoma, polycystic ovarian syndrome, and leiomyomas. By using mouse models of PR signaling, many of these mechanisms are beginning to be elucidated and aid in the development of effective therapies for treatment of uterine diseases.

GATA2 is expressed at critical times in the mouse uterus during pregnancy.

In mammals, such as mouse and human, timely production of the progesterone receptor (PR) in the proper uterine compartments is critical for preparing the uterus for the initiation and maintenance of pregnancy. Developmentally, the expression of GATA2, a member of the six member zinc-finger family of transcription factors, has been shown to be necessary for multiple non-related tissues, such as the hematopoietic system, adipose maturation and the urogential system. We recently identified Gata2 as a potential progesterone target gene in the mouse uterus; however, the expression of the GATA genes in the mouse uterus during pregnancy has not been demonstrated. In the present study, we examined the expression of GATA2 protein during the phases of pregnancy, including early pregnancy where progesterone (P4) signaling is critical in order to facilitate the window of receptivity for embryo implantation and during the decidualization of the uterine stroma, a process of cellular proliferation and differentiation which is necessary for maintenance of the invading embryo until placentation occurs. Here, we report that GATA2 protein is expressed in the uterine luminal and glandular epithelium pre-implantation, spatio-temporally co-localizing with that of the PR. Additionally, GATA2 continues to be expressed in the decidualized stroma throughout early pregnancy indicating a role in the maintenance of decidual cells. Based on these findings, we conclude that GATA2 is expressed during critical phases of early pregnancy, similar to that of the PR, and that it may play a major role in mediating P4 signaling in the mouse uterus.