Canine decidualization in vitro: extracellular matrix modification, progesterone mediated effects and selective blocking of prostaglandin E2 receptors.
ABSTRACT: Recently, we established an in vitro model with immortalized dog uterine stromal (DUS) cells for investigations into canine-specific decidualization. Their capability to decidualize was assessed with cAMP and prostaglandin (PG) E2. Here, we show that the effects of PGE2 are mediated through both of the cAMP-mediating PGE2 receptors (PTGER2/4). Their functional inhibition suppressed gene expression of PRLR and PGR in DUS cells. We also assessed the effects of cAMP and PGE2 on selected extracellular matrix components and CX43, and showed that cAMP, but not PGE2, increases COL4, extracellular matrix protein 1 (ECM1) and CX43 protein levels during in vitro decidualization, indicating a mesenchymal-epithelial decidual transformation in these cells. Thus, although PGE2 is involved in decidualization, it does not appear to regulate extracellular matrix. Further, the role of progesterone (P4) during in vitro decidualization was addressed. P4 upregulated PRLR and PGR in DUS cells, but these effects were not influenced by PGE2; both P4 and PGE2 hormones appeared to act independently. P4 did not affect IGF1 expression, which was upregulated by PGE2, however, it suppressed expression of IGF2, also in the presence of PGE2. Similarly, P4 did not affect PGE2 synthase (PTGES), but in the presence of PGE2 it increased PTGER2 levels and, regardless of the presence of PGE2, suppressed expression of PTGER4. Our results indicate a reciprocal regulatory loop between PGE2 and P4 during canine in vitro decidualization: whereas P4 may be involved in regulating PGE2-mediated decidualization by regulating the availability of its receptors, PGE2 regulates PGR levels in a manner dependent on PTGER2 and -4.
Project description:Oviductal glycoprotein 1 (OVGP1), an oviductin, is involved in the maintenance of sperm viability and motility and contributes to sperm capacitation in the oviduct. In this study, the regulatory effects exerted by prostaglandin E2 (PGE2) and F2? (PGF2?) on OVGP1 expression via their corresponding receptors in bovine oviductal epithelial cells (BOECs) were investigated. BOECs were cultured in vitro, and their expression of receptors of PGE2 (PTGER1, PTGER2, PTGER3, and PTGER4) and PGF2? (PTGFR) was measured using RT-qPCR. Ca2+ concentration was determined with a fluorescence-based method and cAMP was quantified by enzyme-linked immunosorbent assays to verify activation of PTGER2 and PTGFR by their corresponding agonists in these cells. OVGP1 mRNA and protein expression was measured using RT-qPCR and western blotting, respectively, following PTGER2 and PTGFR agonist-induced activation. PTGER1, PTGER2, PTGER4, and PTGFR were found to be present in BOECs; however, PTGER3 expression was not detected. OVGP1 expression was significantly promoted by 10-6 M butaprost (a PTGER2 agonist) and decreased by 10-6 M fluprostenol (a PTGFR agonist). In addition, 3 ?M H-89 (a PKA inhibitor) and 3 ?M U0126 (an ERK inhibitor) effectively inhibited PGE2-induced upregulation of OVGP1, and 5 ?M chelerythrine chloride (a PKC inhibitor) and 3 ?M U0126 negated OVGP1 downregulation by PGF2?. In conclusion, this study demonstrates that OVGP1 expression in BOECs is enhanced by PGE2 via PTGER2-cAMP-PKA signaling, and reduced by PGF2? through the PTGFR-Ca2+-PKC pathway.
Project description:Although GPR64 has an important role for male fertility, its physiological roles in the female reproductive system are still unknown. In the present study, immunohistochemical analysis reveals a spatiotemporal expression of GPR64 in the uterus during early pregnancy. Observation of remarkable induction of GPR64 expression in uterine decidual cells points to its potential physiological significance on decidualization. The decidualization of uterine stromal cells is a key event in implantation. Progesterone (P4) signaling is crucial for the decidualization of the endometrial stromal cells for successful pregnancy. Therefore, we examined ovarian steroid hormone regulation of GPR64 expression in the murine uterus. P4 induced GPR64 expression in the epithelial and stromal cells of the uterus in ovariectomized wild-type mice, but not in PRKO mice. ChIP analysis confirmed that PGR proteins were recruited on progesterone response element of Gpr64 gene in the uteri of wild-type mice treated with P4. Furthermore, the expression of GPR64 was increased in human endometrial stromal cells (hESCs) during in vitro decidualization. Interestingly, small interfering RNA (siRNA)-mediated knockdown of GPR64 in hESCs remarkably reduced decidualization. These results suggest that Gpr64 has a crucial role in the decidualization of endometrial stromal cells.
Project description:The steroid hormones 17?-estradiol and progesterone are critical regulators of endometrial stromal cell differentiation, known as decidualization, which is a prerequisite for successful establishment of pregnancy. The present study using primary human endometrial stromal cells (HESCs) addressed the role of estrogen receptor-? (ESR1) in decidualization. Knockdown of ESR1 transcripts by RNA interference led to a marked reduction in decidualization of HESCs. Gene expression profiling at an early stage of decidualization indicated that ESR1 negatively regulates several cell cycle regulatory factors, thereby suppressing the proliferation of HESCs as these cells enter the differentiation program. ESR1 also controls the expression of WNT4, FOXO1, and progesterone receptor (PGR), well-known mediators of decidualization. Whereas ESR1 knockdown strongly inhibited the expression of FOXO1 and WNT4 transcripts within 24 hours of the initiation of decidualization, PGR expression remained unaffected at this early time point. Our study also revealed a major role of cAMP signaling in influencing the function of ESR1 during decidualization. Using a proteomic approach, we discovered that the cAMP-dependent protein kinase A (PKA) phosphorylates Mediator 1 (MED1), a subunit of the mediator coactivator complex, during HESC differentiation. Using immunoprecipitation, we demonstrated that PKA-phosphorylated MED1 interacts with ESR1. The PKA-dependent phosphorylation of MED1 was also correlated with its enhanced recruitment to estrogen-responsive elements in the WNT4 gene. Knockdown of MED1 transcripts impaired the expression of ESR1-induced WNT4 and FOXO1 transcripts and blocked decidualization. Based on these findings, we conclude that modulation of ESR1-MED1 interactions by cAMP signaling plays a critical role in human decidualization.
Project description:The uterine response to the presence of embryos is poorly understood in the domestic dog (Canis familiaris). The intimate embryo-maternal cross-talk, which begins following the hatching of blastocysts and embryo attachment leads to strong structural and functional remodelling of the uterus. A part of this process is decidualisation, comprising morphological and biochemical changes that result in formation of maternal stroma-derived decidual cells. These are an integral part of the canine placenta materna, which together with the maternal vascular endothelium are the only cells of the canine endotheliochorial placenta able to resist trophoblast invasion. These cells are also the only ones within the canine placenta expressing the progesterone receptor (PGR). Understanding the decidualisation process thus appears essential for understanding canine reproductive physiology.Here, we investigated the capability of canine uterine stromal cells to decidualise in vitro, thereby serving as a canine model of decidualisation. A dbcAMP-mediated approach was chosen during a time course of 24 - 72 h. Tissue material from six (n?=?6) healthy, dioestric bitches was used (approximately 2 weeks after ovulation). Cells were characterized by differential staining, nearly 100 % of which were vimentin-positive. Scanning and transmission electron microscope analyses were applied, and morphological changes were recorded with a live cell imaging microscope. Expression of several decidualisation markers was investigated.The in vitro cultured stromal cells acquired characteristics of decidual cells when incubated with 0.5 mM dbcAMP for 72 h. Their shape changed from elongated to rounded, while ultrastructural analysis revealed higher numbers of mitochondria and secretory follicles, and an increased proliferation rate. Elevated expression levels of IGF1, IGF2, PRLR and ER? were observed in decidualised cells; PRL and ER? remained mostly below the detection limit, while PGR remained unaffected. The expression of smooth muscle ? actin (?SMA), another decidualisation marker, was strongly induced. Among prostaglandin system members, levels of COX2 (PTGS2) and of PGE2-synthase (PTGES) were upregulated. Expression of the PGE2 receptors, PTGER2 and PTGER4, was clearly detectable.An in vitro decidualisation model with canine uterine stromal cells was successfully established, allowing future, more detailed studies to be undertaken on the underlying molecular and endocrine mechanisms of canine decidualisation.
Project description:Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is expressed in many organs, including female reproductive organs, and is a stem cell marker in the stomach and intestinal epithelium, hair follicles, and ovarian surface epithelium. Despite ongoing studies, the definitive physiological functions of Lgr5 remain unclear. We utilized mice with conditional deletion of Lgr5 (Lgr5(d/d)) in the female reproductive organs by progesterone receptor-Cre (Pgr(Cre)) to determine Lgr5's functions during pregnancy. Only 30% of plugged Lgr5(d/d) females delivered live pups, and their litter sizes were lower. We found that pregnancy failure in Lgr5(d/d) females was due to insufficient ovarian progesterone (P4) secretion that compromised decidualization, terminating pregnancy. The drop in P4 levels was reflected in elevated levels of P4-metabolizing enzyme 20?-hydroxysteroid dehydrogenase in corpora lutea (CL) inactivated of Lgr5. Of interest, P4 supplementation rescued decidualization failure and supported pregnancy to full term in Lgr5(d/d) females. These results provide strong evidence that Lgr5 is critical to normal CL function, unveiling a new role of LGR5 in the ovary.
Project description:Connexin43 (Cx43) containing gap junctions play an important role in bone homeostasis, yet little is known about the second messengers communicated by Cx43 among bone cells. Here, we used MC3T3-E1 pre-osteoblasts and UMR106 rat osteosarcoma cells to test the hypothesis that cAMP is a second messenger communicated by bone cells through Cx43 containing gap junctions in a manner that is sufficient to impact osteoblast function. Overexpression of Cx43 markedly enhanced the activity of a cAMP-response element driven transcriptional luciferase reporter (CRE-luc) and increased phospho-CREB and phospho-ERK1/2 levels following expression of a constitutively active Gs? or by treatment with prostaglandin E2 (PGE2), 3-Isobutyl-1-methyl xanthine (IBMX) or forskolin. The Cx43-dependent potentiation of signaling in PGE2 treated cells was not accompanied by a further increase in cAMP levels, suggesting that the cAMP was shared between cells rather than Cx43 enhancing cAMP production. To support this, we developed a novel assay in which one set of cells expressing constitutively active Gs? (donor cells) were co-cultured with a second set of cells expressing a CRE-luc reporter (acceptor cells). Using this assay, activation of a CRE-luc reporter in the acceptor cells was both Cx43- and cell contact-dependent, indicating communication of cAMP among cells. Finally, we showed that Cx43 increased the cAMP-dependent mRNA expression of receptor activator of nuclear factor kappa B ligand (RANKL) and enhanced the repression of the sclerostin mRNA, implying a potential mechanism for the modulation of tissue remodeling. In total, these data demonstrate that Cx43 can communicate cAMP between cells and, more importantly, that the communicated cAMP is sufficient to impact signal transduction cascades and the expression of key bone effector molecules between interconnected cells.
Project description:Successful implantation and maintenance of pregnancy require the transformation of uterine endometrial stromal cells into distinct decidualized cells. Although estrogen and progesterone (P4) receptors are known to be essential for decidualization, the roles of steroid receptor coregulators in this process remain largely unknown. In this study, we have established a key role for the coregulator, repressor of estrogen receptor activity (REA), in the decidualization of human endometrial stromal cells (hESCs) in vitro and of the mouse uterus in vivo. Our studies revealed that the level of REA normally decreases to half as hESC decidualization proceeds and that uterine reduction of REA in transgenic heterozygous knockout mice or small interfering RNA knockdown of REA in hESC temporally accelerated and strongly enhanced the differentiation process, as indicated by changes in cell morphology and increased expression of biomarkers of decidualization, including P4 receptor. Findings in hESC cultured in vitro with estradiol, P4, and 8-bromo-cAMP over a 10-day period mirrored observations of enhanced decidualization response in transgenic mice with heterozygous deletion of REA. Importantly, gene expression and immunohistochemical analyses revealed changes in multiple components of the Janus kinase/signal transducer and activator of transcription pathway, including marked up-regulation of signal transducer and activator of transcription 3 and IL-11, master regulators of decidualization, and the down-regulation of several suppressor of cytokine signaling family members, upon reduction of REA. The findings highlight that REA physiologically restrains endometrial stromal cell decidualization, controlling the timing and magnitude of decidualization to enable proper coordination of uterine differentiation with concurrent embryo development that is essential for implantation and optimal fertility.
Project description:Human endometrial stromal cells (ESCs) differentiate into decidual cells by the action of progesterone, which is essential for implantation and maintenance of pregnancy. We previously reported that glucose uptake by human ESCs increases during decidualization and that glucose is indispensable for decidualization. Although glucose transporter 1 (GLUT1) is upregulated during decidualization, it remains unclear whether it is involved in glucose uptake. Here, we attempted to determine the role of GLUT1 during decidualization as well as the factors underlying its upregulation. ESCs were incubated with cAMP to induce decidualization. Knockdown of GLUT1 suppressed cAMP-increased glucose uptake and the expressions of specific markers of decidualization, IGF-binding protein-1 (IGFBP-1), and prolactin (PRL). To investigate the regulation of GLUT1 expression, we focused on CCAAT enhancer-binding protein β (C/EBPβ) and Wilms' tumor 1 (WT1) as the upstream transcription factors regulating GLUT1 expression. Knockdown of either C/EBPβ or WT1 suppressed cAMP-increased GLUT1 expression and glucose uptake. cAMP treatment also increased the recruitment of C/EBPβ and WT1 to the GLUT1 promoter region. Interestingly, cAMP increased the H3K27 acetylation (H3K27ac) and p300 recruitment in the GLUT1 promoter region. Knockdown of C/EBPβ or WT1 inhibited these events, indicating that both C/EBPβ and WT1 contribute to the increase of H3K27ac by recruiting p300 to the GLUT1 promoter region during decidualization. These findings indicate that GLUT1 is involved in glucose uptake in ESCs during decidualization, thus facilitating the establishment of pregnancy.
Project description: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.
Project description:Decidualization is critical for the embryonic implantation and successful pregnancy. ATRA can suppress in-vitro decidualization of human endometrial stromal cells (hESCs) induced by MPA and estrogen treatment. However, the mechanism by which RA suppressed estrogen and progesterone induced decidualization of mESCs is not clear. We used microarrays to investigate the mechanism by which all-trans RA (ATRA) regulates the decidualization of endometrial stroma cells (mESCs). mESCs were isolated at day 4 of pseudopregnancy and cultured with administration of E2 and P4 in the presence or absence of ATRA for 72h.