During early pregnancy, the concerted actions of the steroid hormones estrogen (E) and progesterone (P) via their cognate receptors orchestrate the changes in the uterine epithelium that make it competent to attach to the blastocyst and initiate the process of implantation. It is followed by the proliferation and differentiation of the underlying stromal cells into morphologically and functionally distinct decidual cells, which control trophoblast invasion and embryonic growth. Concomitant with this cellular transformation, known as decidualization, an extensive vascular network develops in the stromal compartment that supports the development of the implanted embryo. Although P acting via the progesterone receptor (PR) is essential for decidualization, the role of E in this process is less clear. To address this issue, we used the Cre-Lox strategy to create conditional mutant mice in which estrogen receptor alpha (ERα) expression is abolished in the uterus. Strikingly, the uteri of these mutant mice failed to show any response to a decidual stimulus, revealing, for the first time, a critical role of ERα in stromal decidualization. We next evaluated the role of ERα in human decidualization using primary stromal cells obtained from endometrial biopsies. In this in vitro culture system, human endometrial stromal cells undergo differentiation in response to a hormonal cocktail containing E, P, and a cyclic AMP analog. We observed that siRNA-mediated knockdown of ERα expression severely impaired the decidualization process, indicating a central and conserved role of ERα signaling in human endometrial stromal differentiation. To identify the pathways regulated by ERα during decidualization, we performed global gene expression profiling using RNA isolated from intact and ERα-deficient mouse uteri 24 hours after administration of artificial decidualization stimulus. We identified several genes encoding known markers of stromal differentiation, whose expression is significantly down regulated in ERα deficient uteri. We also found that expression of several known E-regulated genes, including connexin 43 (Cx43), cysteine rich protein 61 (Cyr61), and receptor activity-modifying protein 3 (Ramp3), was markedly suppressed in the decidual uterus in the absence of ERα. Interestingly, our recent studies have shown that conditional deletion of the Cx43 gene in the uterine stromal cells results in severe impairment in the development of new blood vessels within the stromal compartment, indicating a critical role of ERα regulated pathways in neovascularization. Cyr61 and Ramp3 are known regulators of vascularization in other tissues. Consistent with the potential role of ERα in angiogenesis, we found that the expression of additional genes, encoding factors involved in endothelial cell proliferation and migration, was suppressed in ERα-deficient uteri. These factors include VEGF-D, semaphorin 4c, semaphorin 5a, semaphorin 7a, and endothelin 1. Collectively, our results uncovered a novel role for ERα and its downstream pathways in critically regulating uterine stromal differentiation and the development of an elaborate vascular network that is essential for embryo survival during early pregnancy. (Supported by U54 HD055787 as part of the NICHD/NIH Centers Program in Reproduction and Infertility Research)

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