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Previous in vitro studies have suggested that calreticulin (CALR), which is responsible for the folding and quality control of glycoproteins, may be associated with decidualization. However, its precise role in regulating decidualization has not been explored in vivo. Here, we used pregnant rat models to examine endometrial CALR expression during the peri-implantation period. We also examined whether polypectomy, a procedure that could ameliorate infertility, alters the endometrial expression levels of CALR and several implantation factors in women diagnosed as infertile. In rats, uterine CALR was expressed at a high level at the implantation site, and a marked increase in CALR expression was observed in decidual cells of normal pregnancy. In addition, endometrial CALR expression was enhanced by either administration of estradiol-17β in the delayed implantation rat model or the artificial induction of decidualization in the pseudopregnant rat. In cultured stromal cells, siRNA-mediated silencing of CALR inhibited the decidual stimulus-induced expression of prolactin, decidual/trophoblast prolactin-related protein, and connexin 43. In humans, the endometrial expression levels of the mRNAs encoding CALR and the implantation-related factor insulin-like growth factor binding protein (IGFBP)-7 tended to increase after polypectomy. The strongest positive correlation between expression levels before polypectomy was observed for IGFBP-7 and CALR, and the strength of this correlation increased after the surgery. Thus, endometrial CALR may play a role in the formation of decidua, and the polypectomy of infertile patients may result in the co-operative expression of endometrial factors, including CALR, that could enhance endometrial receptivity.

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.

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., (© 2014 Society for Reproduction and Fertility.)

Successful implantation and placentation require that trophoblasts adhere to the uterine epithelium and penetrate the decidualized endometrium. However, the biochemical mechanisms of the establishment of pregnancy including these phenomena have not yet to be definitively elucidated. We have found that stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics, and insulin-like growth factor-binding protein (IGFBP)-related protein 1 (IGFBP-rP1, now called IGF-binding protein 7) were highly expressed in the endometrium around the time of implantation and decidualization. In this article, we review our recent findings of the research regarding the functions of these implantation-associated proteins in endocrine physiology and pharmacology. Analysis of the expression of both factors in rodent and human uterus has revealed that both factors are crucial for the process of endometrial stromal cell differentiation.

We used the library subtraction technique to identify genes specifically expressed in the rat uterus during early pregnancy. One such gene was that for stathmin, a factor that is associated with tubulin binding and the destabilization of microtubules. Stathmin was expressed at higher levels in implantation sites than in interimplantation sites on d 6 and 7 of pregnancy; the levels on d 6 and 7 were higher in implantation sites than in the entire uterus on d 3-5 of pregnancy or in nonpregnant uteri. Intense expression of stathmin mRNA was primarily limited to the subluminal stromal cells at the implantation site. Expression was also detected in the decidual zones and was accentuated during the period of decidualization (d 7-12). In the delayed implantation pregnant rat model, uterine stathmin expression was low, but increased after implantation induced by administration of 17beta-estradiol to the progesterone-primed animal. Further, decidualization in the pseudopregnant rat, induced by intrauterine infusion of oil, enhanced stathmin expression. Stathmin expression clearly increases in the uterus when stimulated by embryo implantation and decidualization and may play a role in the early stages of pregnancy.

Interferon-tau (IFNtau), produced by the trophectoderm of ruminant ungulates, binds to the type I IFN receptor (IFNAR) located at the uterine endometrium in a paracrine manner. Since IFNtau attenuates the secretory pattern of an endometrial luteolysin, prostaglandin F2alpha, IFNtau has been considered as a conceptus factor implicated in the process of maternal recognition of pregnancy. Here we report the presence of IFNAR subunit (IFNAR1) in ovine conceptuses during the period of peri-implantation development and demonstrate that 125I-human (h) IFNalpha binds to membrane preparations from ovine corpus luteum and conceptus. Using an antibody against hIFNAR1, immunohistochemical analysis revealed that IFNAR1 protein was present in day 14 and 16 conceptuses (day 0 = day of estrus) and luminal and glandular epithelia of the endometrium. Conceptus membrane proteins analyzed by western blot with the same antibody displayed immunoreactive bands at 95, 60 and 55 kDa while endometrial membrane proteins showed bands at 200, 95 and 55 kDa. Northern blot analysis revealed that IFNAR1 mRNA was present in days 15-19 conceptuses and day 18-19 allantoic membranes. Receptor binding studies indicated that 125I-hIFNalpha binding to day 16, but not earlier, conceptus membrane proteins could be displaced with hIFNalpha or ovine IFNtau. Based on Scatchard analysis, day 16 conceptus membranes contained 28 fmol IFNAR/mg protein with a dissociation constant of 300 pM. Cross-linking experiments demonstrated that 125I-hIFNalpha-receptor complex migrated at 120 kDa, indicating that the receptor component(s) was approximately 100 kDa. These data provide evidence that although the binding does not occur until day 16, ovine conceptuses possess IFNAR1 near or at the time of implantation, suggesting that IFNtau, a factor produced by the trophectoderm of ruminant ungulates, could act on the conceptus in an autocrine manner. In addition to functioning as an antiluteolytic factor, therefore, IFNtau may have a direct effect on conceptus development.

The appropriate differentiation of endometrial stromal cells (ESCs) into decidual cells is required for embryo implantation and subsequent placentation into humans. Decidualization is accompanied by the appearance of senescent-like cells. We recently reported the secretory phase-specific downregulation of endometrial progesterone receptor membrane component 1 (PGRMC1) and enhanced decidualization upon PGRMC1 knockdown and inhibition in cultured ESCs. However, it remains unknown whether PGRMC1 is involved in cellular senescence during decidualization. Here, we showed that the small interfering RNA (siRNA)-mediated knockdown of PGRMC1 and the inhibition of PGRMC1 by AG-205 increased the expression of the transcription factor forkhead box protein O1 (FOXO1) and the senescence-associated β-galactosidase activity in cAMP analog- and progesterone-treated ESCs. Furthermore, the knockdown of FOXO1 repressed the decidual senescence induced by siRNA-based PGRMC1 knockdown or AG-205 treatment. Taken together, the decreased PGRMC1 expression in ESCs may accelerate decidualization and cellular senescence via the upregulation of FOXO1 expression for appropriate endometrial remodeling and embryo implantation during the secretory phase. [ABSTRACT FROM AUTHOR]

Decidualization — the differentiation of endometrial stromal cells (ESCs) into decidual cells — is a crucial step for successful embryo implantation and placentation that is initiated in the secretory phase of the menstrual cycle. During decidualization, ESCs undergo proliferation arrest and secrete inflammatory mediators, including senescence-associated secretory phenotype (SASP). Although several senolytic agents improve age-related diseases, their effects on cellular senescence in decidualizing ESCs has not been explored. To do this, we treated decidualized ESCs with the senolytic agents Quercetin (Que), Dasatinib (Das), and BPTES. Que decreased the number of senescence-associated β-galactosidase (SA-β-Gal) positive cells and expression of senescence markers in ESCs treated with the decidual stimulus (dibutyryl-cAMP plus progesterone: DP). Concomitantly, Que markedly increased the expression of the decidualization markers IGFBP1, PRL, and FOXO1, in decidualizing ESCs. Similar to Que, Das also stimulated decidualization. Treatment with a combination of Que and Das synergistically increased the expression of decidualization markers and senescence markers compared with treatment with Que or Das alone. However, BPTES did not enhance the expression of decidualization markers. These results imply that treatment with Que and/or Das can remove senescent decidual cells and enhance the decidualization of the rest of ESCs. Thus, senolytic modulation of abnormal ESC decidualization could alleviate infertility caused by dysfunctions of endometrial receptivity and embryo implantation. • Decidualization of endometrial stromal cells induces cellular senescence. • Senolytic reagents Quercetin and Dasatinib remove decidual senescent cells. • Other senolytic reagent BPTES does not affect decidualization and cellular senescence. • Quercetin and Dasatinib synergistically promote decidualization. [ABSTRACT FROM AUTHOR]