Artificial Decidualization Research Articles

Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS

Polycystic ovary syndrome (PCOS) is a complex common endocrine disorder affecting women of reproductive age. Ovulatory dysfunction is recognized as a primary infertile factor, however, even when ovulation is medically induced and restored, PCOS patients continue to experience reduced cumulative pregnancy rates and a higher spontaneous miscarriage rate. Hyperandrogenism, a hallmark feature of PCOS, affects ovarian folliculogenesis, endometrial receptivity, and the establishment and maintenance of pregnancy. Decidualization denotes the transformation that the stromal compart of the endometrium must undergo to accommodate pregnancy, driven by the rising progesterone levels and local cAMP production. However, studies on the impact of hyperandrogenism on decidualization are limited. In this study, we observed that primary endometrial stromal cells from women with PCOS exhibit abnormal responses to progesterone during in vitro decidualization. A high concentration of testosterone inhibits human endometrial stromal cells (HESCs) decidualization. RNA-Seq analysis demonstrated that pyruvate dehydrogenase kinase 4 (PDK4) expression was significantly lower in the endometrium of PCOS patients with hyperandrogenism compared to those without hyperandrogenism. We also characterized that the expression of PDK4 is elevated in the endometrium stroma at the mid-secretory phase. Artificial decidualization could enhance PDK4 expression, while downregulation of PDK4 leads to abnormal decidualization both in vivo and in vitro. Mechanistically, testosterone excess inhibits IGFBP1 and PRL expression, followed by phosphorylating of AMPK that stimulates PDK4 expression. Based on co-immunoprecipitation analysis, we observed an interaction between SIRT1 and PDK4, promoting glycolysis to facilitate decidualization. Restrain of AR activation resumes the AMPK/SIRT1/PDK4 pathway suppressed by testosterone excess, indicating that testosterone primarily acts on decidualization through AR stimulation. Androgen excess in the endometrium inhibits decidualization by disrupting the AMPK/SIRT1/PDK4 signaling pathway. These data demonstrate the critical roles of endometrial PDK4 in regulating decidualization and provide valuable information for understanding the underlying mechanism during decidualization.

The potential role of the prolyl isomerase Pin1 during blastocyst implantation and uterine decidualization in mice.

During early pregnancy in mice, the establishment of uterine receptivity and endometrial decidualization require the extensive proliferation and differentiation of endometrial epithelial cells or stromal cells. Pin1 has been suggested to act as a molecular 'timer' of the cell cycle and is involved in the regulation of cellular proliferation and differentiation by binding many cell-cycle regulatory proteins. However, its physiological role during early pregnancy is still not fully understood. Here, we employed immunohistochemistry to determine the spatiotemporal pattern of Pin1 expression during early pregnancy. We found that Pin1 was mainly localized in subluminal stromal cells on day 4, in the decidual zone on days 5 to 8 of pregnancy and in artificial decidualization. Using a uterine stromal cell culture system, we found that progesterone, but not estrogen, induced the expression of Pin1 in a progesterone receptor-dependent manner. Inhibition of Pin1 in the uterus leads to impaired embryo implantation and decidualization in mice. Notably, a decrease in Pin1 activation affected the functional execution of several implantation- or decidualization-related factors. These findings provide new evidence for a previously unknown function of Pin1 in mediating embryo implantation and decidualization during successful pregnancy establishment and maintenance.

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5' untranslated region (5'-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization invitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

Circadian gene Rev-erbα influenced by sleep conduces to pregnancy by promoting endometrial decidualization via IL-6-PR-C/EBPβ axis

BackgroundSleep disturbance can cause adverse pregnancy outcomes by changing circadian gene expression. The potential mechanisms remain unclear. Decidualization is critical for the establishment and maintenance of normal pregnancy, which can be regulated by circadian genes. Whether Rev-erbα, a critical circadian gene, affects early pregnancy outcome by regulating decidualization needs to be explored.MethodsQPCR, western blot and artificial decidualization mouse model were used to confirm the effect of sleep disturbance on Rev-erbα expression and decidualization. The regulatory mechanism of Rev-erbα on decidualization was assessed using QPCR, western blot, RNA-Seq, and Chip-PCR. Finally, sleep disturbance mouse model was used to investigate the effect of therapeutic methods targeting Rev-erbα and interleukin 6 (IL-6) on improving adverse pregnancy outcomes induced by sleep disturbance.ResultsDysregulation of circadian rhythm due to sleep disturbance displayed abnormal expression profile of circadian genes in uterine including decreased level of Rev-erbα, accompanied by defective decidualization. Rev-erbα could regulate decidualization by directly repressing IL-6, which reduced the expression of CCAAT/enhancer-binding protein β (C/EBPβ) and its target insulin-like growth factor binding protein 1 (IGFBP1), the marker of decidualization, by inhibiting progesterone receptors (PR) expression. Moreover, deficient decidualization, higher abortion rate and lower implantation number were exhibited in the mouse models with sleep disturbance compared with those in normal mouse. Pharmacological activation of Rev-erbα or neutralization of IL-6 alleviated the adverse effect of sleep disturbance on pregnancy outcomes.ConclusionsTaken together, Rev-erbα regulated decidualization via IL-6-PR-C/EBPβ axis and might be a connector between sleep and pregnancy outcome. Therapies targeting Rev-erbα and IL-6 might help improving adverse pregnancy outcomes induced by sleep disturbance.

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research.

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Endometrial glycogen metabolism during early pregnancy in mice.

Glucose is critical during early pregnancy. The uterus can store glucose as glycogen but uterine glycogen metabolism is poorly understood. This study analyzed glycogen storage and localization of glycogen metabolizing enzymes from proestrus until implantation in the murine uterus. Quantification of diastase-labile periodic acid-Schiff (PAS) staining showed glycogen in the glandular epithelium decreased 71.4% at 1.5 days postcoitum (DPC) and 62.13% at DPC 3.5 compared to proestrus. In the luminal epithelium, glycogen was the highest at proestrus, decreased 46.2% at DPC 1.5and 63.2% at DPC 3.5. Immunostaining showed that before implantation, glycogen metabolizing enzymes were primarily localized to the glandular and luminal epithelium. Stromal glycogen was low from proestrus to DPC 3.5. However, at the DPC 5.5 implantation sites, stromal glycogen levels increased sevenfold. Similarly, artificial decidualization resulted in a fivefold increase in glycogen levels. In both models, decidualization increased expression of glycogen synthase as determine by immunohistochemistry and western blot. In conclusion, glycogen levels decreased in the uterine epithelium before implantation, indicating that it could be used to support preimplantation embryos. Decidualization resulted in a dramatic increase in stromal glycogen levels, suggesting it may have an important, but yet undefined, role in pregnancy.

Acbp is essential for decidualization during early pregnancy in mice.

Decidualization of uterine stromal cells plays an important role in the establishment of normal pregnancy. Previous studies have demonstrated that Acyl-CoA binding protein (Acbp) is critical to cellular proliferation, differentiation, mitochondrial functions, and autophagy. The characterization and physiological function of Acbp during decidualization remain largely unknown. In the present study, we conducted the expression profile of Acbp in the endometrium of early pregnant mice. With the occurrence of decidualization, the expression of Acbp gradually increased. Similarly, Acbp expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. We applied the mice pseudopregnancy model to reveal that the expression of Acbp in the endometrium of early pregnant mice was not induced by embryonic signaling. Moreover, P4 significantly upregulated the expression of Acbp, whereas E2 appeared to have no regulating effect on Acbp expression in uterine stromal cells. Concurrently, we found that interfering with Acbp attenuated decidualization, and that might due to mitochondrial dysfunctions and the inhibition of fatty acid oxidation. The level of autophagy was increased after knocking down Acbp. During induced decidualization, the expression of ACBP was decreased with the treatment of rapamycin (an autophagy inducer), while increased with the addition of Chloroquine (an autophagy inhibitor). Our work suggests that Acbp plays an essential role in the proliferation and differentiation of stromal cells during decidualization through regulating mitochondrial functions, fatty acid oxidation, and autophagy.

BHPF exposure impairs mouse and human decidualization

Although BHPF has been widely used in plastic manufacturing as a substitute for BPA, current evidence suggests that BHPF also causes harmful effects on reproduction. However, effects of BHPF on mammalian early pregnancy are still poorly defined. This study aimed to explore the effects of BHPF on early pregnancy, especially decidualization and embryonic development in mice and human beings. The results showed that 50 and 100 mg/kg BHPF exposure reduced birth weight, and implantation site weight on the day 8 of pregnancy in mice. Because BHPF inhibits both embryo development and artificial decidualization in mice, suggesting that the detrimental effects of BHPF should be from its effects on embryo development and decidualization. Under in vitro decidualization, 10 μM BHPF inhibits decidualization and leads to disordered expression of Lamin B1 and collagen in mice. In addition, 10 μM BHPF also inhibits decidualization, and causes disordered expression of both collagen III and Lamin B1 under human in vitro decidualization. However, collagen III supplementation can rescue BHPF inhibition on decidualization. Further, our study demonstrates that BHPF impairs human decidualization through the HB-EGF/EGFR/STAT3/Collagen III pathway. Taken together these data suggest that exposure to BHPF impairs mouse and human decidualization during early pregnancy.

Loss of basigin expression in uterine cells leads to subfertility in female mice†.

Basigin (BSG) is a transmembrane glycoprotein involved in cell proliferation, angiogenesis, and tissue remodeling. BSG has been shown to be essential for male and female reproduction although little is known about its role in normal uterine function. To study the potential function of BSG in the female reproductive tract, we generated mice with conditional knockout of Bsg in uterine cells using progesterone receptor-Cre and hypothesized that BSG is required for normal pregnancy in mice. Fertility study data showed that the conditional knockout mice had significantly reduced fertility compared to controls. Ovarian function of the conditional knockout mice appeared normal with no difference in the number of superovulated oocytes collected or in serum progesterone levels between the conditional knockout and the control mice. Uterine tissues collected at various times of gestation showed increased abnormalities in implantation, decidualization, placentation, and parturition in the conditional knockout mice. Uterine cross sections on Day 5 of pregnancy showed implantation failure and abnormal uterine epithelial differentiation in a large proportion of the conditional knockout mice. There was a compromised decidual response to artificial decidualization stimuli and decreased mRNA and protein levels for decidualization genes in the uteri of the conditional knockout mice. We also observed altered protein expression of monocarboxylate transporter 1 (MCT1), as well as impaired angiogenesis in the conditional knockout uteri compared to the controls. These results support that BSG is required for successful pregnancy through its functions in implantation and decidualization.

Usp22 is expressed in mouse uterus during early pregnancy and involved in endometrial stromal cell decidualization.

While decidualization is essential for embryo implantation in the context of a normal pregnancy, the molecular basis for this process remains poorly understood. Ubiquitin-specific protease 22 (Usp22), one of the deubiquitinating enzymes, is an important regulator of tumor progression and knocking out this gene in mice results in placental vascular dysplasia and embryonic lethality. In this study, we first demonstrated that Usp22 is spatiotemporally expressed in the mouse peri-implantation uterus. Under artificial decidualization, Usp22 upregulation was detected in both in vivo and in vitro. Progesterone treatment could stimulate Usp22 expression in mouse endometrial stromal cells through progesterone/progesterone receptor (PR) pathway, which is inhibited by PR antagonist. The downregulation of Usp22 within mouse endometrial stomal cells by shRNA impaired their ability to proliferate and undergo decidualization. Taken together, these results suggest that Usp22 is involved in uterine stromal decidualization in mice.

Calpain7 Negatively Regulates Human Endometrial Stromal Cell Decidualization in EMS by Promoting Foxo1 Nuclear Exclusion by Increasing the Phosphorylation of AKT1

Background: Endometrial receptivity damage caused by impaired decidualization may be one of the mechanisms of infertility in endometriosis (EMs). Our previous study demonstrated that CAPN7 is abnormally overexpressed in EMs. Whether CAPN7 affects the regulation of decidualization remains to be determined. Methods: The expression of CAPN7 during decidualization in endometria was examined by IHC and western blotting. The transduction of a CAPN7 recombinant adenovirus into human endometrial stromal cells was performed to investigate relevant decidualization marker genes. An artificial decidualization experiment was performed to study the role of CAPN7 in decidualization in vivo. The potential molecular mechanisms were further confirmed through immunofluorescence and coimmunoprecipitation analyses. IHC analysis was performed to evaluate differentially expressed proteins, including CAPN7, p-AKT1 and p-FoxO1, between the fertile control and EMs groups. Findings: CAPN7 expression decreased during human endometrial stromal cell (HESC) decidualization in vitro. CAPN7 negatively regulated decidualization in vitro and in vivo. In addition, CAPN7 notably promoted the phosphorylation of FoxO1 at Ser319, Ser256 and Thr24, leading to increased FoxO1 exclusion from nuclei in decidualized HESCs. Furthermore, the overexpression of CAPN7 obviously promoted the phosphorylation of AKT1, which is upstream of FoxO1, at Ser473. We also identified one conserved potential PEST sequence in the AKT1 protein and found that CAPN7 was able to hydrolyse AKT1. Interpretation: CAPN7 might serve as a promising therapeutic target for improving endometrial receptivity in women with EMs. Funding: This work was supported by the National Key Research and Development Program of China (2018YFC1004400), the National Natural Science Foundation of China (No. 81070492, No. 81871165 and No. 31872846), a grant from Jiangsu Provincial Key Research and Development (BE2018602), and a special grant for principal investigators from the Health Department of Jiangsu Province (ZDRCA2016070). Declaration of Interest: We declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article. Ethical Approval: All samples were collected with the informed consent of the patients, and approval from the ethics committee was obtained for this study.

Norepinephrine exposure restrains endometrial decidualization during early pregnancy.

Previous studies have focused on the role of norepinephrine on arrhythmias, generalized anxiety disorder, and cancer. This study aimed to investigate the effect of norepinephrine on endometrial decidualization. Artificial decidualization and norepinephrine-treated mice were established in vivo. In vitro, human endometrial stromal cells were treated with MPA and cAMP to induce decidualization. Decidual markers and important signaling molecules during decidualization were detected using quantitative real-time PCR and Western blot. RNA sequencing was performed to determine related signaling pathways. Exposure to excess norepinephrine significantly restricted the induced expression of decidualized markers Dtprp, BMP2, WNT4, and Hand2 in mice. In vitro, 10 µM norepinephrine markedly downregulated the expressions of prolactin, IGFBP1, and PLZF, which are the specifical markers of decidual stromal cells during decidualization. The gene set enrichment analysis showed a significant enrichment in neuroactive ligand-receptor interactions of norepinephrine treatment group. The α1b-adrenergic receptor expression was upregulated by norepinephrine. Interestingly, norepinephrine did not inhibit the expression of IGFBP1 in endometrial stromal cells after silencing α1b-adrenergic receptor, while significantly suppressed the induced decidualization with overexpression of α1b-adrenergic receptor. When α1b-adrenergic receptor was activated, endometrial p-PKC was significantly increased under post-treatment with norepinephrine in vivo and in vitro. In addition, norepinephrine treatment inhibited embryo and fetal development using a normal pregnancy model. Therefore, norepinephrine exposure inhibited endometrial decidualization through the activation of the PKC signaling pathway by upregulating α1b-adrenergic receptor. Our study could explain some female reproductive problems due to stress and provide some novel strategies for this disorder.

Implantation failure and embryo loss contribute to subfertility in female mice mutant for chromatin remodeler Cecr2†.

Defects in the maternal reproductive system that result in early pregnancy loss are important causes of human female infertility. A wide variety of biological processes are involved in implantation and establishment of a successful pregnancy. Although chromatin remodelers have been shown to play an important role in many biological processes, our understanding of the role of chromatin remodelers in female reproduction remains limited. Here, we demonstrate that female mice mutant for chromatin remodeler Cecr2 are subfertile, with defects detected at the peri-implantation stage or early pregnancy. Using both a less severe hypomorphic mutation (Cecr2GT) and a more severe presumptive null mutation (Cecr2Del), we demonstrate a clear difference in the severity of the phenotype depending on the mutation. Although neither strain shows detectable defects in folliculogenesis, both Cecr2GT/GT and Cecr2GT/Del dams show defects in pregnancy. Cecr2GT/GT females have a normal number of implantation sites at embryonic day 5.5 (E5.5), but significant embryo loss by E10.5 accompanied by the presence of vaginal blood. Cecr2GT/Del females show a more severe phenotype, with significantly fewer detectable implantation sites than wild type at E5.5. Some Cecr2GT/Del females also show premature loss of decidual tissue after artificial decidualization. Together, these results suggest a role for Cecr2 in the establishment of a successful pregnancy.

Notch1 is crucial for decidualization and maintaining the first pregnancy in the mouse†.

The endometrium undergoes a pregnancy-delivery-repair cycle multiple times during the reproductive lifespan in females. Decidualization is one of the critical events for the success of this essential process. We have previously reported that Notch1 is essential for artificial decidualization in mice. However, in a natural pregnancy, the deletion of Notch1 (PgrCre/+Notch1f/f, or Notch1d/d) only affects female fertility in the first 30days of a 6-month fertility test, but not the later stages. In the present study, we undertook a closer evaluation at the first pregnancy of these mice to attempt to understand this puzzling phenomenon. We observed a large number of pregnancy losses in Notch1d/d mice in their first pregnancy, which led to the subfertility observed in the first 30days of the fertility test. We then demonstrated that the initial pregnancy loss is a consequence of impaired decidualization. Furthermore, we identified a group of genes that contribute to Notch1 regulated decidualization in a natural pregnancy. Gene ontogeny analysis showed that these differentially expressed genes in the natural pregnancy are involved in cell-cell and cell-matrix interactions, different from genes that have been previously identified from the artificial decidualization model, which contribute to cell proliferation and apoptosis. In summary, we determined that Notch1 is essential for normal decidualization in the mouse uterus only in the first pregnancy but not in subsequent ones.

Increased FOXL2 expression alters uterine structures and functions†.

The transcription factor forkhead box L2 (FOXL2) regulates sex differentiation and reproductive function. Elevated levels of this transcription factor have been observed in the diseases of the uterus, such as endometriosis. However, the impact of elevated FOXL2 expression on uterine physiology remains unknown. In order to determine the consequences of altered FOXL2 in the female reproductive axis, we generated mice with over-expression of FOXL2 (FOXL2OE) by crossing Foxl2LsL/+ with the Progesterone receptor Pgrcre model. FOXL2OE uterus showed severe morphological abnormality including abnormal epithelial stratification, blunted adenogenesis, increased endometrial fibrosis, and disrupted myometrial morphology. In contrast, increasing FOXL2 levels specifically in uterine epithelium by crossing the Foxl2LsL/+ with the lactoferrin Ltficre mice resulted in the eFOXL2OE mice with uterine epithelial stratification but without defects in endometrial fibrosis and adenogenesis, demonstrating a role of the endometrial stroma in the uterine abnormalities of the FOXL2OE mice. Transcriptomic analysis of 12weeks old Pgrcre and FOXL2OE uterus at diestrus stage showed multiple signaling pathways related with cellular matrix, wnt/β-catenin, and altered cell cycle. Furthermore, we found FOXL2OE mice were sterile. The infertility was caused in part by a disruption of the hypophyseal ovarian axis resulting in an anovulatory phenotype. The FOXL2OE mice failed to show decidual responses during artificial decidualization in ovariectomized mice demonstrating the uterine contribution to the infertility phenotype. These data support that aberrantly increased FOXL2 expressions in the female reproductive tract can disrupt ovarian and uterine functions.

Comparative Analysis of Mouse Decidualization Models at the Molecular Level.

The mouse is widely used to study decidualization and there are three well-established mouse models of decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). However, the extent of similarity and difference between these models at the molecular level remains largely unknown. Here, we performed a comparative analysis using the RNA-seq approach. In the NPD model, which is thought to be the golden standard of mouse decidualization, we found a total of 5277 differentially expressed genes, with 3158 genes being up-regulated and 2119 genes being down-regulated. A total of 4294 differentially expressed genes were identified in the AD model: 1127 up-regulated genes and 3167 down-regulated genes. In comparison to NPD, 1977 genes were consistently expressed, whereas only 217 genes were inconsistently expressed, indicating that AD is a reliable model for mouse decidualization. In the IVD model, RNA-seq analysis revealed that 513 genes were up-regulated and 988 genes were down-regulated. Compared to NPD, 310 genes were consistently expressed, whereas 456 genes were inconsistently expressed. Moreover, although the decidualization marker Prl8a2 (prolactin family 8 subfamily a member 2) was up-regulated, the widely-used marker Alpl (alkaline phosphatase liver/bone/kidney) was down-regulated in the IVD model. Therefore, we suggest that the IVD model should be optimized to mimic NPD at the transcriptomic level. Our study contributes to an increase in the knowledge about mouse models of decidualization.

Endometrial pyruvate kinase M2 is essential for decidualization during early pregnancy.

Embryo implantation is essential for normal pregnancy. Decidualization is known to facilitate embryo implantation and maintain pregnancy. Uterine stromal cells undergo transformation into decidual cells after embryo attachment to the endometrium. Pyruvate kinase M2 (PKM2) is a rate limiting enzyme in the glycolysis process which catalyzes phosphoenolpyruvic acid into pyruvate. However, little is known regarding the role of PKM2 during endometrial decidualization. In this study, PKM2 was found to be mainly located in the uterine glandular epithelium and luminal epithelium on day 1 and day 4 of pregnancy and strongly expressed in the decidual zone after embryo implantation. PKM2 was dramatically increased with the onset of decidualization. Upon further exploration, PKM2 was found to be more highly expressed at the implantation sites than at the inter-implantation sites on days 5 to 7 of pregnancy. PKM2 expression was also significantly increased after artificial decidualization both in vivo and in vitro. After PKM2 expression was knocked down by siRNA, the number of embryo implantation sites in mice on day 7 of pregnancy was significantly reduced, and the decidualization markers BMP2 and Hoxa10 were also obviously downregulated in vivo and in vitro. Downregulated PKM2 could also compromise cell proliferation in primary endometrial stromal cells and in Ishikawa cells. The migration rate of Ishikawa cells was also obviously suppressed by si-PKM2 according to the wound healing assay. In conclusion, PKM2 might play an important role in decidualization during early pregnancy, and cell proliferation might be one pathway for PKM2 regulated decidualization.

Gene UCHL1 expresses specifically in mouse uterine decidual cells in response to estrogen.

UCHL1 is expressed specifically in the brain and gonads of almost all studied model organisms including Drosophila, zebrafish, amphibians, and mammals, suggesting a high degree of evolutionary conservation in its structure and function. Although UCHL1 has been involved in spermatogenesis in mice, its specific expression in mammal placenta remains elusive. Our previous work has revealed that UCHL1 is highly expressed in oocytes, and has been involved in mouse ovarian follicular development. Here, we further examined UCHL1 expression change in endometria during early natural pregnancy, with different stages of the estrous cycle and pseudopregnancy as control. The UCHL1 gene deletion model showed that UCHL1 protein is associated with endometrial development, and its deletion leads to infertility. Notably, we demonstrate evidence showing the distinct expression pattern of UCHL1: weak expression over the uterine endometria, strong expression in decidualized stromal cells at the implantation site with a peak at pregnancy D6, and a shift with primary decidualization to secondary decidualized zones. Using the delayed implantation, the delayed implantation activation, and the artificial decidualization models, we have demonstrated that strong expression of UCHL1 occurred in response to decidualization and estrogen stimulation. These observations suggest that during the early proliferation and differentiation of mouse uterine decidua, UCHL1 expression is up-regulated, and formed an unique intracellular distribution mode. Therefore, we proposed that UCHL1 is involved in decidualization, and possibly in response to estrogen regulation.