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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.

Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure–toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure–toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.

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