scholarly journals Endometrium-on-a-chip reveals the endometrial transcriptome, and protein content of secretome are altered by changes in circulating concentrations of insulin and glucose in vitro

2020 ◽  
Author(s):  
Tiago H. C. De Bem ◽  
Haidee Tinning ◽  
Elton J. R. Vasconcelos ◽  
Dapeng Wang ◽  
Niamh Forde
Keyword(s):  
Early Pregnancy ◽  
Stromal Cells ◽  
Receptor Interaction ◽  
Transcriptomic Response ◽  
Protein Coding ◽  
Endometrial Cells ◽  
Lower Chamber ◽  
Maternal Glucose

ABSTRACTThe molecular interactions between the maternal environment and developing embryo that are key for early pregnancy success are known to be influenced by factors such as the metabolic status. We are, however, limited in our understanding of the mechanism by which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success. Here we report for the first time the use of endometrium-on-a-chip microfluidics approach to produce a multi-cellular endometrium in vitro, that is exposed to glucose and insulin concentrations associated with maternal metabolic stressors. Following isolation of endometrial cells (epithelial and stromal) from the uteri of non-pregnant cows in early-luteal phase (Day 4-7 approximately) epithelial cells were seeded into the upper chamber (4-6 104 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 104 cells/mL). Three different concentration of glucose 1) 0.5 mM 2) 5.0 mM or 3) 50 mM or insulin 1) Vehicle, 2) 1 ng/mL or 3) 10 ng/mL were performed in the endometrial cells at a flow rate of 1µL/min for 72 hr to mimic the rate of secretion in vivo. Quantitative differences in the transcriptomic response of the cells and the secreted proteome of in vitro-derived uterine luminal fluid (ULF) were determined by RNA-sequencing and TMT respectively. Changes in maternal glucose altered 21 and 191 protein coding genes in epithelial and stromal cells respectively (p<0.05). While there was a dose-dependent quantitative change in protein secretome (1 and 23 proteins). Insulin resulted in limited transcriptional changes including insulin-like binding proteins that were cell specific (5, 12, and 20) but altered the quantitative secretion of 196 proteins including those involved in extracellular matrix-receptor interaction and proteoglycan signaling in cancer. Collectively, these highlight the potential mechanism by which changes to maternal glucose and insulin alter uterine function.

scholarly journals Endometrium on-a-chip reveals insulin- and glucose-induced alterations in the transcriptome and proteomic secretome

Endocrinology ◽  
2021 ◽  
Author(s):  
Tiago H C De Bem ◽  
Haidee Tinning ◽  
Elton J R Vasconcelos ◽  
Dapeng Wang ◽  
Niamh Forde
Keyword(s):  
Early Pregnancy ◽  
Stromal Cells ◽  
Protein Coding ◽  
Endometrial Cells ◽  
In Utero Environment ◽  
Lower Chamber ◽  
Transcriptional Changes ◽  
First Time ◽  
Dose Dependent

Abstract The molecular interactions between the maternal environment and the developing embryo that are key for early pregnancy success and are influenced by factors such as maternal metabolic status. Our understanding of the mechanism(s) through which these individual nutritional stressors alter endometrial function and the in utero environment for early pregnancy success is, however, limited. Here we report, for the first time, the use of an endometrium-on-a-chip microfluidics approach to produce a multi-cellular endometrium in vitro. Isolated endometrial cells (epithelial and stromal) from the uteri of non-pregnant cows in the early-luteal phase (Day 4-7), were seeded in the upper chamber of the device (epithelial cells; 4-6 10 4 cells/mL) and stromal cells seeded in the lower chamber (1.5-2 10 4 cells/mL). Exposure of cells to different concentrations of glucose (0.5, 5.0 or 50 mM) or insulin (Vehicle, 1 or 10 ng/mL) were performed at a flow rate of 1µL/min for 72 hr. Quantitative differences in the cellular transcriptome and the secreted proteome of in vitro-derived uterine luminal fluid (ULF) were determined by RNA-sequencing and Tandem Mass Tagging Mass Spectrometry (TMT-MS), respectively. High glucose concentrations altered 21 and 191 protein-coding genes in epithelial and stromal cells, respectively (p&lt;0.05), with a dose-dependent quantitative change in the protein secretome (1 and 23 proteins). Altering insulin concentrations resulted in limited transcriptional changes including transcripts for insulin-like binding proteins that were cell specific but altered the quantitative secretion of 196 proteins. These findings highlight one potential mechanism by which changes to maternal glucose and insulin alter uterine function.

scholarly journals Endometrial pyruvate kinase M2 is essential for decidualization during early pregnancy

2020 ◽  
Vol 245 (3) ◽  
pp. 357-368 ◽  
Author(s):  
Yan Su ◽  
Sujuan Guo ◽  
Chunyan Liu ◽  
Na Li ◽  
Shuang Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Pyruvate Kinase ◽  
Early Pregnancy ◽  
Stromal Cells ◽  
Embryo Implantation ◽  
Pyruvate Kinase M2 ◽  
Ishikawa Cells ◽  
Implantation Sites

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.

scholarly journals GATA2 and Progesterone Receptor Interaction in Endometrial Stromal Cells Undergoing Decidualization

Endocrinology ◽  
2020 ◽  
Vol 161 (6) ◽  
Author(s):  
Amanda Kohlmeier ◽  
Christia Angela M Sison ◽  
Bahar D Yilmaz ◽  
John S Coon V ◽  
Matthew T Dyson ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Early Pregnancy ◽  
Stromal Cells ◽  
Expression Profiles ◽  
Stem Cell Differentiation ◽  
Receptor Interaction ◽  
Rna Seq ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Binding Regions

Abstract The transcription factor GATA2 is important for endometrial stromal cell decidualization in early pregnancy. Progesterone receptor (PGR) is also critical during decidualization but its interaction with GATA2 in regulating genes and pathways necessary for decidualization in human endometrium are unclear. RNA-sequencing (RNA-seq) was performed to compare gene expression profiles (n = 3), and chromatin immunoprecipitation followed by sequencing (ChIP-seq) using an antibody against GATA2 (n = 2) was performed to examine binding to target genes in human endometrial stromal cells undergoing in vitro decidualization (IVD including estrogen, progestin, and 3′,5′-cyclic AMP analogue) or vehicle treatment. We identified 1232 differentially expressed genes (DEGs) in IVD vs vehicle. GATA2 cistrome in IVD-treated cells was enriched with motifs for GATA, ATF, and JUN, and gene ontology analysis of GATA2 cistrome revealed pathways that regulate cholesterol storage, p38 mitogen-activated protein kinase, and the c-Jun N-terminal kinase cascades. Integration of RNA-seq and ChIP-seq data revealed that the PGR motif is highly enriched at GATA2 binding regions surrounding upregulated genes in IVD-treated cells. The integration of a mined public PGR cistrome in IVD-treated human endometrial cells with our GATA2 cistrome showed that GATA2 binding was significantly enhanced at PGR-binding regions in IVD vs vehicle. Interrogating 2 separate ChIP-seq data sets together with RNA-seq revealed integration of GATA2 and PGR action to coregulate biologic processes during decidualization of human endometrial stromal cells, specifically via WNT activation and stem cell differentiation pathways. These findings reveal the key pathways that are coactivated by GATA2 and PGR that may be therapeutic targets for supporting implantation and early pregnancy.

scholarly journals Regulation and Function of Deiodinases During Decidualization in Female Mice

Endocrinology ◽  
2014 ◽  
Vol 155 (7) ◽  
pp. 2704-2717 ◽  
Author(s):  
Wen-Bo Deng ◽  
Xiao-Huan Liang ◽  
Ji-Long Liu ◽  
Zeng-Ming Yang
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Early Pregnancy ◽  
Stromal Cells ◽  
Mouse Uterus ◽  
Opposite Strand ◽  
And Function ◽  
Kinase A

Thyroid dysfunction during human pregnancy is closely related to serious pregnancy outcome. However, the regulation and function of thyroid hormones during early pregnancy are largely unknown. We found that type II deiodinase, an enzyme converting T4 to activated T3, is highly expressed in the mouse uterus on days 3 and 4 of pregnancy. Once the embryo implants into the receptive uterus, type III deiodinase (Dio3), a mainly paternally imprinted gene for inactivating T3, is significantly induced in the stromal cells and accompanied by DNA hypermethylation of intergenic differentially CpG methylation regions in the δ-like 1 homolog-Dio3 imprinting cluster. The concentration of uterine free T3 is actually decreased after embryo implantation. T3 induces Dio3 expression both in vivo and in vitro, suggesting a positive feedback loop. T3 addition or Dio3 knockdown compromises decidualization. These results indicate that the Dio3-mediated local T3 decrease is critical for decidualization of stromal cells during early pregnancy. Furthermore, we found that progesterone regulates Dio3 expression through its cognate receptor both in vivo and in vitro. Additionally, cAMP regulates Dio3 transcription through the protein kinase A-cAMP response element-binding protein pathway. The inhibition of the protein kinase A pathway results in decreased Dio3 expression and impaired decidualization. Dio3 opposite strand (Dio3os) expressed in a similar pattern to Dio3, is transcribed from the opposite strand of Dio3 and fine-tunes Dio3 expression during decidualization. Our data indicate that Dio3 is strongly expressed and tightly controlled during decidualization.

Immunocytochemical localization of prostaglandin synthase in the ovine uterus during the oestrous cycle and in early pregnancy

1990 ◽  
Vol 2 (4) ◽  
pp. 311 ◽  
Author(s):  
LA Salamonsen ◽  
JK Findlay
Keyword(s):  
Epithelial Cells ◽  
Early Pregnancy ◽  
Stromal Cells ◽  
Cellular Localization ◽  
In Vitro Release ◽  
Oestrous Cycle ◽  
Intense Staining ◽  
Endometrial Cells ◽  
Ovine Uterus

Prostaglandin (PG) synthase has been localized by immunocytochemistry within the ovine uterus throughout the oestrous cycle and in early pregnancy. On Day 4 of the cycle, PG synthase was located primarily in the stromal cells in caruncular and intercaruncular tissue with little staining in the epithelium. On Days 14 through to 16, the most intense staining was in the luminal epithelial cells (caruncular and intercaruncular) and in epithelial cells of glands close to the uterine lumen. PG synthase was also located in the intercaruncular stromal cells, particularly close to the myometrium. Staining for the enzyme on Day 10 was intermediate between that of Day 4 and Day 14. On Day 15 of pregnancy, the pattern of staining was identical to that on Day 15 of the cycle, with no detectable difference in intensity. When endometrial cells (cycle, Day 14) were cultured with and without ovine trophoblast protein-1 (3 ng mL-1) in vitro, release of PGE and PGF2 alpha was attenuated (54% and 47% of control respectively) but no differences were observed in the intensity of staining for PG synthase in the cells. These results demonstrate marked cyclical changes in the endometrial cell types producing PGs, suggesting differential regulation of PG synthase. In addition, it appears that conceptus-induced changes in PGF2 alpha release do not occur via changes in the concentration or cellular localization of PG synthase, but rather that the activity of the enzyme is modified.

scholarly journals Interleukins Affect Equine Endometrial Cell Function: Modulatory Action of Ovarian Steroids

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Anna Z. Szóstek ◽  
Antonio M. Galvão ◽  
Takuo Hojo ◽  
Kiyoshi Okuda ◽  
Dariusz J. Skarzynski
Keyword(s):  
Cell Proliferation ◽  
Early Pregnancy ◽  
Stromal Cells ◽  
Real Time Pcr ◽  
Cell Function ◽  
Positive Control ◽  
Endometrial Cell ◽  
Ovarian Steroids ◽  
Endometrial Cells

The aim of the present study was to investigate the interaction between ovarian steroids, interleukins and prostaglandins (PG) in equine epithelial and stromal cells in vitro. In Experiment 1, cells were exposed to IL-1α(10 ng/mL), IL-1β(10 ng/mL) or IL-6 (10 ng/mL) for 24 h and cell proliferation was determined using MTT. In Experiment 2, cells were exposed to progesterone (P4; 10−7 M); 17-βestradiol (E2; 10−9 M) or P4+E2for 24 h and later medium was replaced with a fresh one treated with IL-1α, IL-1βor IL-6 (10 ng/mL, each) for 24 h. The oxytocin (OT; 10−7 M) was used as a positive control. In Experiment 3, cells were exposed to P4(10−7 M), E2(10−9 M) or P4+E2for 24 h and theIL receptormRNAs transcription was determined using Real-time PCR. Prostaglandins concentration was determined using the direct enzyme immunoassay (EIA) method. Our findings reveal a functional linking between ovarian steroids and IL-stimulated PG secretion by equine endometrial cells. This interaction could be one of the mechanisms responsible for endometrial local orchestrating events during the estrous cycle and early pregnancy.

scholarly journals Experimental Type 2 Diabetes Differently Impacts on the Select Functions of Bone Marrow-Derived Multipotent Stromal Cells

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 268
Author(s):  
Jonathan Ribot ◽  
Cyprien Denoeud ◽  
Guilhem Frescaline ◽  
Rebecca Landon ◽  
Hervé Petite ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Adipogenic Differentiation ◽  
Therapeutic Modality ◽  
Multipotent Stromal Cells ◽  
Cell Therapies

Bone marrow-derived multipotent stromal cells (BMMSCs) represent an attractive therapeutic modality for cell therapy in type 2 diabetes mellitus (T2DM)-associated complications. T2DM changes the bone marrow environment; however, its effects on BMMSC properties remain unclear. The present study aimed at investigating select functions and differentiation of BMMSCs harvested from the T2DM microenvironment as potential candidates for regenerative medicine. BMMSCs were obtained from Zucker diabetic fatty (ZDF; an obese-T2DM model) rats and their lean littermates (ZL; controls), and cultured under normoglycemic conditions. The BMMSCs derived from ZDF animals were fewer in number, with limited clonogenicity (by 2-fold), adhesion (by 2.9-fold), proliferation (by 50%), migration capability (by 25%), and increased apoptosis rate (by 2.5-fold) compared to their ZL counterparts. Compared to the cultured ZL-BMMSCs, the ZDF-BMMSCs exhibited (i) enhanced adipogenic differentiation (increased number of lipid droplets by 2-fold; upregulation of the Pparg, AdipoQ, and Fabp genes), possibly due to having been primed to undergo such differentiation in vivo prior to cell isolation, and (ii) different angiogenesis-related gene expression in vitro and decreased proangiogenic potential after transplantation in nude mice. These results provided evidence that the T2DM environment impairs BMMSC expansion and select functions pertinent to their efficacy when used in autologous cell therapies.

scholarly journals Reovirus-induced cell-mediated immunity for the treatment of multiple myeloma within the resistant bone marrow niche

2021 ◽  
Vol 9 (3) ◽  
pp. e001803
Author(s):  
Louise M E Müller ◽  
Gemma Migneco ◽  
Gina B Scott ◽  
Jenny Down ◽  
Sancha King ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Immune Responses ◽  
Stromal Cells ◽  
Tumor Burden ◽  
Effector Memory ◽  
In Vivo Model ◽  
Bm Niche

BackgroundMultiple myeloma (MM) remains an incurable disease and oncolytic viruses offer a well-tolerated addition to the therapeutic arsenal. Oncolytic reovirus has progressed to phase I clinical trials and its direct lytic potential has been extensively studied. However, to date, the role for reovirus-induced immunotherapy against MM, and the impact of the bone marrow (BM) niche, have not been reported.MethodsThis study used human peripheral blood mononuclear cells from healthy donors and in vitro co-culture of MM cells and BM stromal cells to recapitulate the resistant BM niche. Additionally, the 5TGM1-Kalw/RijHSD immunocompetent in vivo model was used to examine reovirus efficacy and characterize reovirus-induced immune responses in the BM and spleen following intravenous administration. Collectively, these in vitro and in vivo models were used to characterize the development of innate and adaptive antimyeloma immunity following reovirus treatment.ResultsUsing the 5TGM1-Kalw/RijHSD immunocompetent in vivo model we have demonstrated that reovirus reduces both MM tumor burden and myeloma-induced bone disease. Furthermore, detailed immune characterization revealed that reovirus: (i) increased natural killer (NK) cell and CD8+ T cell numbers; (ii) activated NK cells and CD8+ T cells and (iii) upregulated effector-memory CD8+ T cells. Moreover, increased effector-memory CD8+ T cells correlated with decreased tumor burden. Next, we explored the potential for reovirus-induced immunotherapy using human co-culture models to mimic the myeloma-supportive BM niche. MM cells co-cultured with BM stromal cells displayed resistance to reovirus-induced oncolysis and bystander cytokine-killing but remained susceptible to killing by reovirus-activated NK cells and MM-specific cytotoxic T lymphocytes.ConclusionThese data highlight the importance of reovirus-induced immunotherapy for targeting MM cells within the BM niche and suggest that combination with agents which boost antitumor immune responses should be a priority.

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