Warburg-like Glycolysis and Lactate Shuttle in Mouse Decidua during Early Pregnancy

Decidualization is an essential process of maternal endometrial stromal cells to support pregnancy. Although it is known that enhanced glucose influx is critical for decidualization, the underlying mechanism in regulating glucose metabolism in decidua remains insufficiently understood. Here, we demonstrate that aerobic glycolysis-related genes and factors are all substantially induced during decidualization, indicating the existence of Warburg-like glycolysis in decidua. In vitro, progesterone activates hypoxia-inducible factor 1α (Hif1α) and c-Myc through Pi3k-Akt signaling pathway to maintain aerobic glycolysis in decidualizing cells. Knocking down of pyruvate kinase M2 (Pkm2) attenuates the induction of decidual marker gene. Decidual formation in vivo is also impaired by glycolysis inhibitor 3-bromopyruvate. Besides, lactate exporter monocarboxylate transporter 4 (Mct4) is induced in newly formed decidual cells, whereas lactate importer Mct1 and proliferation marker Ki-67 are complementarily located in the surrounding undifferentiated cells, which are supposed to consume lactate for proliferation. Hif1α activation is required for lactate-dependent proliferation of the undifferentiated cells. Inhibition of lactate flux leads to compromised decidualization and decelerated lactate-dependent proliferation. In summary, we reveal that Warburg-like glycolysis and local lactate shuttle are activated in decidua and play important roles for supporting early pregnancy.     

Post-implantation mouse conceptuses produce paracrine signals that regulate the uterine endometrium undergoing decidualization

The uterus undergoes a series of dramatic changes in response to an implanting conceptus that, in some mammalian species, includes differentiation of the endometrial stroma into decidual tissue. This process, called decidualization, can be induced artificially in rodents indicating that the conceptus may not be essential for a proper maternal response in early pregnancy. In order to test this hypothesis, we determined if and how the conceptus affects uterine gene expression. We identified 5 genes (Angpt1, Angpt2, Dtprp, G1p2 and Prlpa) whose steady-state levels in the uterus undergoing decidualization depends on the presence of a conceptus. In situ hybridization revealed region-specific effects which suggested that various components of the conceptus and more than one signal from the conceptus are likely responsible for altering decidual cell function. Using cell culture models we found that trophoblast giant cells secrete a type I interferon-like molecule which can induce G1p2 expression in endometrial stromal cells. Finally, decidual Prlpa expression was reduced in the uterus adjacent to Hand1- and Ets2-deficient embryos, suggesting that normal trophoblast giant cells in the placenta are required for the conceptus-dependent effects on Prlpa expression in the mesometrial decidua. Overall, these results provide support for the hypothesis that molecular signals from the mouse conceptus have local effects on uterine gene expression during decidualization.     

Ovarian steroid receptors and activated MAPK in the regional decidualization in rats

Though the decidua serves a critical function in implantation, the hormonal regulated pathway in decidualization is still elusive. Here we describe in detail the regional distribution and the effects of progesterone receptors (PGR), estrogen receptors (ESR), and MAPK activation on decidualization. We showed an increase in PGR A, PGR B, ESR1, and phosphorylated MAPK3-1 proteins (p-MAPK3-1), but not in ESR2, in the decidual tissue up to Day 8 of pregnancy. PGR was predominantly found in the nuclei of mesometrial decidual cells and of undifferentiated stromal cells where it colocalizes with ESR2 and ESR1. In the antimesometrial decidua, all the receptors showed cytoplasmic localization. MAPK was activated exclusively in undifferentiated stromal cells of the junctional zone between the antimesometrial and mesometrial decidua and at the border of the antimesometrial decidua. Treatment with the progesterone antagonist onapristone and/or the estrogen antagonist faslodex reduced the extent of decidual tissue and downregulated the levels of PGR and ESR1. The expression level of ESR2 was affected only by the progesterone receptor antagonist, while neither the antiprogestin nor the antiestrogen significantly modified the p-MAPK3-1 level. The inhibition of MAPK3-1 phosphorylation by PD98059 impaired the extent of decidualization and the closure reaction of the implantation chamber, and significantly downregulated ESR1. These results confirm a role of both steroid receptors in the growth and differentiation of the different decidual regions and suggest a new function for p-MAPK3-1 in regulating expression levels of ESR1, thereby maintaining the proliferation capacity of stromal cells and limiting the differentiation process in specified regions of decidual tissues.     

Corticotropin releasing factor decidualizes human endometrial stromal cells in vitro. Interaction with progestin

Decidualization of endometrial cells is a hormone-dependent process of differentiation which occurs during the menstrual cycle and pregnancy. Recent in vitro studies have revealed that cAMP and its generators induce decidualization of stromal cells isolated from proliferative endometrium and that progestins enhance the effect of cAMP. Since corticotropin releasing factor (CRF) generates cAMP and prostaglandins in other organs, in the present study the effect of CRF, a hypothalamic factor also produced by decidua and fetal membranes, on in vitro decidualization of endometrial stromal cells was evaluated. The addition of CRF to a culture medium of stromal cells induced in vitro decidualization, as indicated by morphologic changes from elongated fibroblast-like cells into larger and round cells and by the release of prolactin in the medium. The effect of CRF on stromal cells and on prolactin release was significantly augmented by the coincubation in the presence of medroxyprogesterone acetate. This observation indicates CRF as a novel factor of decidualization and confirms that progestins act as enhancers of the expression of decidual products.     

Decreased NDRG1 expression is associated with pregnancy loss in mice and attenuates the in vitro decidualization of endometrial stromal cells

Embryo implantation is an essential step for a successful pregnancy, and any defect in this process can lead to a range of pregnancy pathologies. The objective of this study was to explore the role of N‐myc downregulated gene 1 (NDRG1) in embryo implantation. It was found that uterine NDRG1 expression has a dynamic pattern during the estrous cycle in nonpregnant mice and that uterine NDRG1 expression was elevated during the implantation process in pregnant mice. The distinct accumulation of NDRG1 protein signals was observed in the primary decidual zone adjacent to the implanting embryo during early pregnancy. Furthermore, uterine NDRG1 expression could be induced by activated implantation or artificial decidualization in mice. Decreased uterine NDRG1 expression was associated with pregnancy loss in mice and was associated with recurrent miscarriages in humans. The in vitro decidualization of both mouse and human endometrial stromal cells (ESCs) was accompanied by increased NDRG1 expression and downregulated NDRG1 expression in ESCs effectively inhibited decidualization. Collectively, these data suggest that NDRG1 plays an important role in decidualization during the implantation process, and the abnormal expression of NDRG1 may be involved in pregnancy loss.     

PGF2alpha induced differential expression of genes involved in turnover of extracellular matrix in rat decidual cells

In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2α) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2α in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2α sixty genes were significantly down-regulated whereas only six genes were up-regulated (from a total of 1176 genes studied). Interestingly, the majority of the genes inhibited by PGF2α are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2α. The results strongly suggest that the abortificient role of elevated levels of PGF2α after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation.     

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.     

Gamma‐amino butyric acid and the A‐type receptor suppress decidualization of mouse uterine stromal cells by down‐regulating cyclin D3

Uterine decidualization, characterized by stromal cell proliferation and differentiation into polyploid decidual cells, is critical to the establishment of pregnancy in mice, although the mechanism underlying this process remains poorly understood. This study is the first to investigate the expression of gamma‐amino butyric acid (GABA) and the GABA A‐type receptor π subunit (GABPR) in the early‐pregnancy mouse uterus and their roles in decidualization. The expression of GABRP was detected from Day 4 to 8 of pregnancy. The effects of GABA and GABA A‐type receptor on cell proliferation and apoptosis were investigated using the Cell Titer 96® AQueous One Solution Cell Proliferation Assay and flow cytometry. The levels of cyclin D3 protein were measured in cultured stromal cells artificially induced to undergo decidualization, and treated with GABA and a GABA A‐type receptor agonist or antagonist, respectively, at the same time. mRNA expression of gabrp in implantation sites was lower than that in inter‐implanted sites. GABA and GABRP protein were localized in the luminal and glandular epithelium, stromal cells, and decidual cells. In vitro, GABPR protein level was decreased in cultured stromal cells during the decidualization process. The addition of GABA and the GABA A‐type receptor agonist Muscimol inhibited stromal cell proliferation, promoted apoptosis, and arrested cells in S‐phase, followed by decreased expression of cyclin D3. These results show that in mice, GABA was actively involved in inhibiting stromal cell proliferation and suppresses decidualization progress through GABA A‐type receptors by down‐regulating cyclin D3 level. Mol. Reprod. Dev. 80: 59–69, 2013. © 2012 Wiley Periodicals, Inc.     

Rac1 Regulates Endometrial Secretory Function to Control Placental Development

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.     

The rat as an animal model for fetoplacental development: a reappraisal of the post-implantation period

Following implantation in rodents, the uterine stromal fibroblasts differentiate into densely packed decidual cells. This process, called decidualization, is well-orchestrated and progresses both antimesometrially and mesometrially, creating two regions with distinctive cellular morphologies. In addition, subsequent placental development is dependent on the invasion of the trophoblast, the process intimately linked to the endometrial tissue remodelling and depending largely on the environment created by the decidua; this phenomenon is crucial for the establishment and maintenance of pregnancy. The key mechanisms underlying the maternal tissue remodelling and trophoblast invasion remain poorly understood. The rat, just like human beings, exhibits a highly invasive type of placental development, the haemochorial placentation. For obvious ethical reasons, the studies of endometrial tissue remodelling throughout pregnancy in humans are greatly limited. Although the rat differs somewhat from humans with regards to the implantation process, it is an appropriate model for studying the mechanisms of decidualization as well as subsequent remodelling of the uterine tissues and fetoplacental development. As decidual remodelling is very closely linked to placentation and the maternal-fetal interactions in the rat show several important similarities to human placentation, the morphological alterations occurring during the post-implantation period in the rat have been addressed in the present review.     

子宫内膜蜕膜化的特征及其调节因素

子宫内膜向蜕膜的转化是正常着床和妊娠的一个重要特征,对于胚泡着床是必不可少的。在蜕膜化过程中,子宫内膜基质细胞在形态和生理等方面都发生了很大的变化。蜕膜化过程受多种因素的调节,包括cAMP、胰岛素样生长因子结合蛋白-1(IGFBP-1)、自然杀伤细胞、同源盒基因-10(HOXA10)、激活素等。但对蜕膜化的机制及调节等仍不清楚。     

Differential expression and anti-oxidant function of glutathione peroxidase 3 in mouse uterus during decidualization

Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H₂O₂). Based on our data, GPX3 may play a major role in reducing H₂O₂ during decidualization.     

Dysfunction of Liver Receptor Homolog-1 in Decidua: Possible Relevance to the Pathogenesis of Preeclampsia

Preeclampsia (PE) is a multisystem disorder unique to Homo sapiens that is known to cause maternal and perinatal mortality and morbidity. Between 5–7% of all pregnancies are affected by PE and it is responsible for approximately 50,000 maternal deaths annually. The pathogenesis of PE remains poorly understood. However, the results of this study indicated that insufficient decidualization plays a significant role. NR5A1 and NR5A2 are orphan members of the Ftz-F1 subfamily of nuclear receptors and are involved in mammal follicular development, female reproduction, steroidogenesis, and decidualization. The expression of NR5A1 and NR5A2 in the human decidua and their functions during decidualization were investigated using in vitro cultured cells by real-time PCR, immunohistochemistry, western blotting, and siRNA techniques. The results demonstrated that the levels of NR5A2 mRNA and protein in the decidual tissues of women with PE were lower than those of normal pregnant women. However, the levels of NR5A1 mRNA and protein did not significantly differ between groups. The expression of NR5A2 was upregulated after in vitro decidualization, but the expression of NR5A1 remained low and showed no difference compared with that of the control cells. Knocking down of NR5A2 in human endometrial stromal cells (hESC) resulted in a significant reduction in their expression of decidualization markers (IGFBP1 and PRL) and signaling pathway molecules (WNT4 and BMP2) (P < 0.05). From these data, we concluded that NR5A2 is pivotal for the decidualization of decidual tissues and cultured human endometrial stromal cells. Disorders of the endometrium in decidual tissues may be associated with the abnormal decidualization thought to cause PE.