During pregnancy, progesterone inhibits the growth-promoting actions of estrogen in the uterus. However, the mechanism for this is not clear. The attenuation of estrogen-mediated proliferation of the uterine epithelium by progesterone is a prerequisite for successful implantation. Our study reveals that progesterone-induced expression of the basic helix-loop-helix transcription factor Hand2 in the uterine stroma suppresses the production of several fibroblast growth factors (FGFs), which act as paracrine mediators of mitogenic effects of estrogen on the epithelium. In mouse uteri lacking Hand2, continued induction of these FGFs in the stroma maintains epithelial proliferation and stimulates estrogen-induced pathways, resulting in impaired implantation. Thus, Hand2 is a critical regulator of the uterine stromal-epithelial communication that directs proper steroid regulation conducive for the establishment of pregnancy.

A critical role of progesterone (P) during early pregnancy is to induce differentiation of the endometrial stromal cells into specialized decidual cells that support the development of the implanting embryo. The P-induced signaling pathways that participate in the formation and function of the decidual cells remain poorly understood. We report here that the expression of the bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGF␤ superfamily, is induced downstream of P action in the mouse uterine stroma during decidualization. To determine the function of BMP2 during this differentiation process, we employed a primary culture system in which undifferentiated stromal cells isolated from pregnant mouse uterus undergo decidualization. When recombinant BMP2 was added to these stromal cultures, it markedly advanced the differentiation program. We also found that siRNA-mediated silencing of BMP2 expression in these cells efficiently blocked the differentiation process. Gene expression profiling experiments identified Wnt4 as a downstream target of BMP2 regulation in stromal cells undergoing decidualization. Attenuation of Wnt4 expression by siRNAs greatly reduced stromal differentiation in vitro, indicating that it is a key mediator of BMP2-induced decidualization. We also observed a remarkable induction in the expression of BMP2 in human endometrial stromal cells during decidualization in vitro in response to steroids and cAMP. Addition of BMP2 to these cultures led to a robust enhancement of Wnt4 expression and stimulated the differentiation process. Collectively, our studies uncovered a unique conserved pathway involving BMP2 and Wnt4 that mediates P-induced stromal decidualization in the mouse and the human.The steroid hormones progesterone (P) 2 and estrogen (E) act in concert to control uterine competency for embryo implantation (1). A primary role of P is to induce differentiation of the endometrial stromal cells into morphologically distinct decidual cells (2, 3). In mice, this differentiation process, termed decidualization, is a prerequisite for successful implantation and is initiated at the time of blastocyst attachment to the uterine epithelium on day 4.5 of pregnancy. The attachment reaction is followed by the proliferation and differentiation of the stromal cells surrounding the implanting blastocyst to form the decidual bed (2, 3). The decidual cells are thought to produce hormones and cytokines that are critical for embryo development, secrete factors that control trophoblast invasion and serve an immunoregulatory function during pregnancy (4, 5). However, P-induced signaling molecules that participate in the formation and function of this unique tissue remain poorly understood.It is well established that the majority of the physiological effects of P are mediated via the intracellular progesterone receptors (PRs) (6). Strong evidence in favor of an essential role of PR in P-mediated responses in the uterus came from a mouse model carrying a null mutation of this gene (7). Ablation of P...

TB filled in by Laureen Please see later in the article for the Editors' Summary

During early pregnancy, steroid hormones estrogen (E) and progesterone (P) regulate a complex series of interactions between the implanting embryo and the uterus by controlling the proliferation and differentiation of uterine epithelium and stroma in a timely manner. To identify the steroid-regulated genes that control these functions, we performed messenger RNA profiling of mouse uterine tissues at the time of implantation. Our analysis revealed that the expression of the transcription factor CCAAT͞enhancer-binding protein ␤ (C͞EBP␤) is rapidly induced in the pregnant uterus at the time of blastocyst attachment. The expression of C͞EBP␤ increased further during the decidualization phase of pregnancy and was localized in the proliferating as well as the decidualized stromal cells surrounding the implanted embryo. Administration of E or P to ovariectomized females induced C͞EBP␤ expression in both uterine epithelium and stroma, showing a dual regulation of this gene by these hormones. The female C͞EBP␤-null mice are infertile. We, therefore, assessed steroid-hormone-dependent responses in the uteri of these mice. We observed that E-induced proliferation of uterine epithelial cells is markedly compromised in the absence of C͞EBP␤. Most strikingly, there was a complete lack of response of the C͞EBP␤-deficient uteri to an artificial deciduogenic stimulus, indicating a critical role of this transcription factor in regulating the decidualization program. Further analysis revealed defects in steroid-induced stromal cell proliferation and differentiation in C͞EBP␤-null uteri. Collectively, our studies established that C͞EBP␤ is a key mediator of steroid responsiveness of the epithelium and stroma in the mouse uterus.estrogen ͉ progesterone ͉ implantation ͉ decidualization T he physiological functions of the mammalian uterus are profoundly influenced by the concerted actions of steroid hormones estrogen (E) and progesterone (P). Waves of steroidhormone-induced cell proliferation and differentiation dictate the cyclical changes that occur in the uterine epithelium during the reproductive cycle (1-4). During pregnancy, these hormones orchestrate the changes in the uterine epithelium that make it competent to attach to the blastocyst to initiate the process of implantation (1-4). Subsequently, E and P regulate a series of complex interactions between the developing embryo and the cells in the stromal compartment leading to the formation of the decidua, which nourishes the embryo and maintains early pregnancy. Although the details of these events vary in different species, the central roles played by E and P in controlling various phases of early pregnancy are common to many mammals (5).The mouse has served as an important animal model to study the regulation of uterine functions by E and P (6-8). The development of mutant mouse models lacking the estrogen receptor (ER) ␣ and progesterone receptor (PR) has firmly established the requirement of these hormones and their downstream signaling pathways for successful establishmen...

The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture.

Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies in the mouse have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is essential for this differentiation process. BMP2 is markedly induced in human primary endometrial stromal cells (HESCs) as they undergo differentiation in response to steroid hormones and cAMP. The present study was undertaken to identify the BMP2-mediated molecular pathways in primary cultures of HESCs during decidualization. Using gene expression profiling, we identified wingless-related murine mammary tumor virus integration site 4 (WNT4) as a target of BMP2 regulation during decidualization. Attenuation of WNT4 expression in HESCs by small interfering RNA administration greatly reduced BMP2-induced stromal differentiation. Additionally, adenovirus-mediated overexpression of WNT4 in HESCs markedly advanced the differentiation program, indicating that it is a key regulator of decidualization. The stimulatory effect of WNT4 was accompanied by the accumulation of active β-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical WNT signaling pathway. Functional inhibition of WNT4/β-catenin pathway by Dickkopf-1, an inhibitor of the canonical WNT signaling, or small interfering RNA-mediated silencing of β-catenin expression, greatly reduced the BMP2- and WNT4-induced decidualization. Gene expression profiling revealed that Forkhead box protein O1, a forkhead family transcription factor and previously reported regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. Taken together, these studies uncovered a linear pathway involving BMP2, WNT4/β-catenin, and Forkhead box protein O1 that operates in human endometrium to critically control decidualization.

Uterine RNA was isolated 72 h after the initiation of decidualization and subjected to quantitative PCR analysis using gene-specific primers for ER␣, alkaline phosphatase (Alkp), BMP2, connexin 43 (Cx43), and PRP. P and PϩAI represent uterine RNA from ovariectomized mice treated with P and P plus letrozole, respectively. www.pnas.org/cgi

Environmental and occupational exposure to bisphenol A (BPA), a chemical widely used in polycarbonate plastics and epoxy resins, has received much attention in female reproductive health due to its widespread toxic effects. Although BPA has been linked to infertility and recurrent miscarriage in women, the impact of its exposure on uterine function during early pregnancy remains unclear. In this study, we addressed the effect of prolonged exposure to an environmental relevant dose of BPA on embryo implantation and establishment of pregnancy. Our studies revealed that treatment of mice with BPA led to improper endometrial epithelial and stromal functions thus affecting embryo implantation and establishment of pregnancy. Upon further analyses, we found that the expression of progesterone receptor (PGR) and its downstream target gene, HAND2 (heart and neural crest derivatives expressed 2), was markedly suppressed in BPA-exposed uterine tissues. Previous studies have shown that HAND2 controls embryo implantation by repressing fibroblast growth factor and the MAPK signaling pathways and inhibiting epithelial proliferation. Interestingly, we observed that down-regulation of PGR and HAND2 expression in uterine stroma upon BPA exposure was associated with enhanced activation of fibroblast growth factor and MAPK signaling in the epithelium, thus contributing to aberrant proliferation and lack of uterine receptivity. Further, the differentiation of endometrial stromal cells to decidual cells, an event critical for the establishment and maintenance of pregnancy, was severely compromised in response to BPA. In summary, our studies revealed that chronic exposure to BPA impairs PGR-HAND2 pathway and adversely affects implantation and the establishment of pregnancy.