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.

De novo synthesis of sphingolipids is essential for decidualization in mice.

Sphingolipids play multiple roles in membrane structure, signal transduction, stress responses, neural development and immune reaction. The rate of de novo synthesis pathway of sphingolipids is regulated by two key enzymes, serine palmitoyltransferase (SPT), and ketoreductase (Kds). Here, we find that the mRNA levels of three subunits of the SPT holoenzyme (Sptlc1, Sptlc2, and Ssspta) are significantly up-regulated in mouse uterine stromal cells during decidualization. The expression of Kds, which reduces 3-keto-dihydrosphingosine to dihydrosphingosine, is co-localized with Sptlc1 in mouse uteri during early pregnancy. Moreover, l-Cycloserine, a specific inhibitor of SPT, can significantly decrease the weight and number of implantation sites, and impede the decidualization process in mouse uterine stromal cells, suggesting that blockage of de novo sphingolipid synthesis may cause defective decidualization and early pregnancy loss in mice. In addition, this study also shows progesterone (P4) can stimulate the expression of both Sptlc2 and Ssspta in mouse uterus. Therefore, our study shows that de novo synthesis of sphingolipids is necessary in implantation and plays a key role in decidualization of mouse.

The Impact of Multiparity on Uterine Gene Expression and Decidualization in Mice.

It has been well established that a previous pregnancy exhibits a beneficial effect on the subsequent pregnancy. However, the underlying mechanisms have not been defined. We hypothesized that multiparity may affect decidualization process during early pregnancy. To test this hypothesis, we analyzed global gene changes associated with multiparity in the mouse uterus using RNA-sequencing (RNA-seq). We identified a total of 131 differentially expressed genes (fold change > 2 and false discovery rate < 0.05), of which 58 were downregulated and 73 genes were upregulated in the second pregnancy (SP) compared to the first pregnancy. Functional clustering analysis showed that genes involved in stress response were significantly enriched. Most importantly, a significant portion of differentially expressed genes, 14 genes or 10.7%, overlapped with the gene list associated with decidualization. Quantitative reverse transcription (RT) polymerase chain reaction (qRT-PCR) analysis confirmed a decreased expression of 4 genes (Klk1, kallikrein 1; H2-Eb1, histocompatibility 2 class II antigen E beta; Mmp7, matrix metallopeptidase 7; Pdpn, podoplanin) and an increase in expression of 2 genes (Thy1, thymus cell antigen 1; Ptgs2, prostaglandin-endoperoxide synthase 2) in SP. Beyond protein-coding genes, we also identified a differentially expressed long noncoding RNA AI506816. Our data provide new insights into the molecular mechanisms underlying the beneficial effect of multiparity.

Involvement of atypical transcription factor E2F8 in the polyploidization during mouse and human decidualization.

Polyploid decidual cells are specifically differentiated cells during mouse uterine decidualization. However, little is known about the regulatory mechanism and physiological significance of polyploidization in pregnancy. Here we report a novel role of E2F8 in the polyploidization of decidual cells in mice. E2F8 is highly expressed in decidual cells and regulated by progesterone through HB-EGF/EGFR/ERK/STAT3 signaling pathway. E2F8 transcriptionally suppresses CDK1, thus triggering the polyploidization of decidual cells. E2F8-mediated polyploidization is a response to stresses which are accompanied by decidualization. Interestingly, polyploidization is not detected during human decidualization with the down-regulation of E2F8, indicating differential expression of E2F8 may lead to the difference of decidual cell polyploidization between mice and humans.

Dysregulated LIF-STAT3 pathway is responsible for impaired embryo implantation in a Streptozotocin-induced diabetic mouse model.

The prevalence of diabetes is increasing worldwide with the trend of patients being young and creating a significant burden on health systems, including reproductive problems, but the effects of diabetes on embryo implantation are still poorly understood. Our study was to examine effects of diabetes on mouse embryo implantation, providing experimental basis for treating diabetes and its complications. Streptozotocin (STZ) was applied to induce type 1 diabetes from day 2 of pregnancy or pseudopregnancy in mice. Embryo transfer was used to analyze effects of uterine environment on embryo implantation. Our results revealed that the implantation rate is significantly reduced in diabetic mice compared to controls, and the change of uterine environment is the main reason leading to the decreased implantation rate. Compared to control, the levels of LIF and p-STAT3 are significantly decreased in diabetic mice on day 4 of pregnancy, and serum estrogen level is significantly higher. Estrogen stimulates LIF expression under physiological level, but the excessive estrogen inhibits LIF expression. LIF, progesterone or insulin supplement can rescue embryo implantation in diabetic mice. Our data indicated that the dysregulated LIF-STAT3 pathway caused by the high level of estrogen results in the impaired implantation in diabetic mice, which can be rescued by LIF, progesterone or insulin supplement.

Progesterone regulates secretin expression in mouse uterus during early pregnancy.

Secretin, a classical gastrointestinal and neuroendocrine peptide, plays an important role in maintaining the body fluid balance. However, the expression and regulation of secretin in the reproductive system are still unknown. In our study, secretin is specifically expressed in the decidua on days 5 to 8 of pregnancy. Secretin expression is not detected under delayed implantation but is stimulated after estrogen activation and under artificial decidualization. Progesterone induces secretin expression in ovariectomized mice and cultured stromal cells, which is abrogated by specific LY294002. Because secretin is mainly localized in the decidua and also strongly expressed during in vitro decidualization, secretin may play a role during mouse decidualization through regulating cyclic adenosine monophosphate level.

Cyclic adenosine monophosphate-induced argininosuccinate synthase 1 expression is essential during mouse decidualization.

L-Arginine (L-Arg), a conditional essential amino acid in adults, has been shown to enhance pregnancy outcome. Argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl) are the key enzyme for L-Arginine (L-Arg) biosynthesis. Based our microarray analysis, Ass1 expression is upregulated significantly at implantation site on day 5 of pregnancy compared to that at inter-implantation site. However, the expression, regulation and function of Ass1 during early pregnancy remain unknown. Here we found that Ass1 is highly expressed in mouse decidua and uterine stromal cells undergoing decidualization, and Asl is weakly expressed in mouse decidua and uterine stromal cells undergoing decidualization. α-Methyl-DL-aspartic acid (MDLA), a specific inhibitor for Ass1, can significantly increase the rate of embryonic reabsorption. Under in vitro induced decidualization, MDLA clearly inhibits the expression of decidual/trophoblast prolactin-related protein (Dtprp), a marker for decidualization in mice. Only Ass1 expression is induced by cAMP through PKA/p-Creb signaling pathway. Results from our cell culture models further indicates that the high level of L-Arg enhances stromal proliferation, while enzymatic activity or Ass1 expression level is essential to determine the magnitude of both mouse and human decidualization. Interestingly, L-Arg at high concentration down-regulates Ass1 and Asl expression by negative feedback to maintain L-Arg homeostasis. These findings highlight that cAMP-induced Ass1 expression is important in controlling the magnitude of decidualization through regulating L-Arg level.

Crystallin αB acts as a molecular guard in mouse decidualization: regulation and function during early pregnancy.

Although decidualization is crucial for the establishment of successful pregnancy, the molecular mechanism underlying decidualization remains poorly understood. Crystallin αB (CryAB), a small heat shock protein (sHSP), is up-regulated and phosphorylated in mouse decidua. In mouse primary endometrial stromal cells, CryAB is induced upon progesterone treatment via HIF1α. In addition, CryAB is strongly phosphorylated through the p38-MAPK pathway under stress or during in vitro decidualization. Knockdown of CryAB results in the increase of apoptosis of stromal cells and inhibits decidualization under oxidative or inflammatory stress. Our data indicate that CryAB protects decidualization against stress conditions.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) induction on Snail expression during mouse decidualization.

Embryo implantation requires a precise synchronism between the receptive uterus and activated blastocyst and is regulated by complicated molecular networks. Although many implantation-related genes have been identified, the crosstalk among them is still unknown. Snail, a transcription repressor, plays a central role during epithelial-mesenchymal transition. Our previous study showed that Snail is highly expressed at implantation site in mouse uterus. This study was to examine how Snail is related with other implantation-related genes in mice. Uterine stromal cells were isolated from mouse uteri on day 4 of pregnancy and treated with HB-EGF. Snail was induced significantly by HB-EGF. By using specific inhibitors and siRNA, we demonstrated that HB-EGF induction on Snail expression is dependent on the EGFR-ERK-Stat3 pathway. Cox-2 was regulated by Snail. The current findings demonstrate that Snail can relate with HB-EGF, Stat3 and Cox-2 and may play a role during mouse embryo implantation and decidualization.

Temporal and spatial expression of AIF in the mouse uterus during early pregnancy.

Apoptosis-inducing factor (AIF) is a phylogenetically old, bifunctional protein with a pro-apoptotic function and redox activity. AIF regulates apoptosis and also plays a role in the defense against stress depending on its subcellular localization. Embryo implantation is a complicated process, in which an activated blastocyst interacts with a receptive uterus. The expression and regulation of AIF were investigated in this study in the mouse uterus during early pregnancy, pseudopregnancy, delayed implantation, artificial decidualization and under hormonal treatment using in situ hybridization, immunohistochemistry and real-time PCR. During early pregnancy, temporally and spatially regulated patterns of AIF expression were found in the mouse uterus; AIF expression in the luminal epithelium and glandular epithelium is regulated by steroid hormones; AIF mRNA expression in the stroma is influenced by the active blastocyst; and AIF protein was found to be located in the cytoplasm rather than the nucleus through confocal microscope. Our data suggest that AIF might play an important role during mouse embryo implantation and that the role of AIF might be implemented through its physiological activity rather than through its pro-apoptotic function in the mouse uterus during this period.