The optimal decidualization of endometrial stromal cells (ESCs) following embryo implantation is one of the critical steps to establish pregnancy in rodents and humans. This step is intricately regulated by ovarian hormones. Using in vitro human ESCs model, we previously showed that activation of a cAMP mediator, exchange protein directly activated by cAMP (EPAC), promotes ovarian steroid- or cAMP analog-induced decidualization. However, expressions and functions of EPAC and RAP1 in the uterus during pregnancy have not yet been examined. In this study, we found that the expression of EPAC2 and RAP1 was markedly upregulated in the decidual cells at the implantation sites on days 7 and 9 of pregnancy in rats. Furthermore, both delayed-implantation and artificial decidualization models showed that EPAC2 and RAP1 expression was enhanced in decidual cells. Significant activation of cAMP-responsive element-binding protein (CREB), a central transcriptional factor of cAMP signaling, was observed in decidual cells. These spatiotemporal expressions of protein related EPAC pathway are overlapped by sites with activated cAMP signaling, indicating the association of EPAC signaling with decidualization. Strikingly, further studies in in vitro rat decidualization model showed that the cAMP analog and medroxyprogesterone stimulated the expression of decidual markers, while knockdown of EPAC1/2 and RAP1 attenuated the expressions of these markers. Together, these findings suggest that EPAC and RAP1 are the crucial factors for endometrial decidualization in rat pregnancy.

BACKGROUND The mononuclear villous cytotrophoblast (CTB) differentiates and fuses to the multinucleated syncytiotrophoblast (STB), which produces hCG and progesterone. cAMP-mediated intracellular pathways are involved in the process of endocrine differentiation and fusion (syncytialization). The exchange protein directly activated by cAMP (Epac) is a mediator of cAMP signaling. We examined the differential roles of Epac and protein kinase A (PKA) signaling in the cell fusion and differentiation of trophoblast-derived BeWo cells. METHODS Epac1 and Epac2 were localized in human placental tissue (n = 9) by immunohistochemistry. The PKA-selective cAMP analog (N⁶-phenyl-cAMP, Phe) or Epac-selective cAMP analog (CPT) was tested for effects on hCG and progesterone production, and syncytialization in BeWo cells. The effect of knockdown of Epac or its downstream target molecule (Rap1) on syncytialization was evaluated. RESULTS Epac1 and Epac2 proteins were expressed in villous CTB, STB, stroma, blood vessels and extravillous CTB of the placenta. Phe increased the expression of hCGα/β mRNA and secretion of hCG protein in BeWo cells (P < 0.01 versus control). CPT-stimulated production of hCG (P < 0.05), albeit to a lesser extent than Phe. Progesterone production was also enhanced by Phe or CPT (P < 0.01 and P < 0.05, respectively). CPT or a stable cAMP analog (dibutyryl-cAMP: Db) increased the number of syncytialized BeWo cells (P < 0.01), whereas Phe did not stimulate fusion. CPT- or Db-induced syncytialization was observed, even in the presence of a PKA inhibitor. Knockdown of Epac1 or Rap1 repressed the Db-, CPT- or forskolin-induced cell fusion. CONCLUSIONS The Epac signaling pathway may be associated with the cAMP-mediated functional differentiation and syncytialization of human trophoblasts.

Decidualization of human endometrial stroma and gland development is mediated through cyclic AMP (cAMP), but the role of intracellular calcium ion (Ca²⁺) on cAMP mediated-signaling in human endometrial stroma and glandular epithelia has not been well-characterized. The present study was designed to investigate the role of intracellular Ca²⁺ on cAMP mediated-decidualization and gland maturation events, which can be identified by the up-regulation of prolactin and IGF-binding protein (IGFBP)1 in human endometrial stromal cells (ESCs), and cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) and glandular epithelial EM-1 cells. Increases in decidual prolactin and IGFBP-1 transcript levels, induced by cAMP-elevating agents forskolin or dibutyryl cyclic AMP, were inhibited by Ca²⁺ influx into ESCs with Ca²⁺ ionophores (alamethicin, ionomycin) in a dose-dependent manner. Conversely, inhibitors of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channel (VDCC), nifedipine and verapamil, enhanced the decidual gene expression. Furthermore, dantrolene, an inhibitor of Ca²⁺ release from the intracellular Ca²⁺ store, up-regulated prolactin and IGFBP-1 expression. Ca²⁺ ionophores decreased intracellular cAMP concentrations, whereas nifedipine, verapamil or dantrolene increased cAMP concentrations in ESCs. In glandular epithelial cells, similar responses in COX2 expression and PGE2 production were found when intracellular cAMP levels were up-regulated by decreases in Ca²⁺ concentrations. Thus, a marked decrease in cytosolic Ca²⁺ levels caused the elevation of cAMP concentrations, resulting in enhanced expression of implantation-related factors including decidual markers. These findings suggest that fluctuation in cytosolic Ca²⁺ concentrations alters intracellular cAMP levels, which then regulate differentiation of endometrial stromal and glandular epithelial cells.

We investigated the relationship between prostaglandin (PG) F(2)(alpha)-induced functional luteal regression and apoptosis in the rodent ovary. Administration of PGF(2)(alpha) significantly decreased the serum levels of progesterone within 12 h after treatment in pseudopregnant mice. Apparent signals were detected in luteal tissues at 24 to 72 h by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick endo labeling (TUNEL) assay. PGF(2)(alpha) significantly increased the levels of the cleavage nuclear poly (ADP-ribose) polymerase fragment and transforming growth factor-beta (TGF-beta) mRNA within 48 h. PGF(2)(alpha) (10 microM) decreased progesterone secretion 6 to 72 h after its addition in luteinized ovarian cells, whereas C2-ceramide (25 microM) decreased progesterone levels by 44% 72 h after its addition. DNA fragmentation was not observed in the cultured cells treated with PGF(2)(alpha) for 24 h, although cells incubated with C2-ceramide showed fragmentation. Treatment with PGF(2)(alpha) for 1 h caused a distinct decrease in luteinizing hormone (LH)-induced cyclic AMP production. Thus, the inhibitory effect of PGF(2)(alpha) on progesterone secretion may be caused by both the blockage of the functional LH receptor and the activation of apoptotic signaling.