Single cell proteomics profiling reveals that embryo-secreted TNF-α plays a critical role during embryo implantation to the endometrium

Abstract Background It has been long-known that endometrium-secreted cytokines play a critical role during embryo implantation. However, whether cytokines secreted from the embryo are relevant to the process of embryo implantation remains unclear. Methods The concentration of cytokines in embryo culture medium was tested using a newly developed, high-sensitivity single-cell proteomic platform and evaluated in comparison to embryo quality and clinical outcome. The effect of TNF-α on embryo and endometrium Ishikawa cells was investigated using immunofluorescence staining, CCK- 8 assay, TUNEL staining, and RT-qPCR. Results Of the 10 cytokines measured, only TNF-α concentration was significantly higher in the group with embryo implantation failure. Immunofluorescence staining showed that the expression of TNF-α was unevenly distributed in blastocysts, and the expression level was significantly correlated with the blastocyst inner cell mass (ICM) quality score. Gene profiling showed that addition of TNF-α led to increased expression of tumor necrosis factor receptor 1 (TNFR1) and apoptosis-related genes and that this could be inhibited by the TNF-α receptor inhibitor entanecept (ETA). In addition, an increased expression of water and ion channels, including AQP3, CFTR, ENaCA and CRISP2 was also observed which could also be inhibited by ETA. Conclusions Our results show that higher embryo-secreted TNF-α levels are associated with implantation failure through activation of TNF-α receptor, and TNF-α may be an independent predictor for pre-transfer assessment of the embryo development potential in IVF patients.

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Single Cell Proteomics Profiling Revealing that Embryo Secreted TNF-α play Critical Role during Embryo Implantation to Endometrium

10.21203/rs.3.rs-122459/v1 ◽

2020 ◽

Author(s):

Lv Jiao ◽

Shan Xudong ◽

Yang Haoxuan ◽

Wen Yuting ◽

Zhang Xueguang ◽

...

Keyword(s):

Single Cell ◽

Embryo Quality ◽

Embryo Implantation ◽

Critical Role ◽

Cell Mass ◽

Gene Profiling ◽

Immunofluorescence Staining ◽

Tunel Staining ◽

Implantation Failure ◽

Tnf Α

Abstract Problem: Although it has long been known that endometrium secreted cytokines play critical role during embryo implantation, whether cytokines secreted from embryo is relevant to embryo quality and is actively involved in embryo attachment remain unclear.Method of study: The concentration of cytokines in embryo culture medium were tested by a new developed high-sensitive single cell proteomic platform, compared with embryo quality and clinical outcome. The effect of TNF-α on embryo and endometrium Ishikawa cell was investigated using immunofluorescence staining, CCK- 8 assay, TUNEL staining, and RT-qPCR reaction.Results: Of the 10 cytokines measured, only TNF-α concentration is significantly higher in group of embryo implantation failure. Immunofluorescence staining showed that the expression of TNF-α was unevenly distributed in blastocysts, and the expression level was significantly correlated with blastocysts inner cell mass (ICM) quality score. Adding TNF-α caused significant increase of apoptotic cells, which could be inhibited by TNF-α receptor blocker entanecept (ETA). Gene profiling showed that adding TNF-α lead to increased expression of TNFR1 and apoptosis related genes, as well as ion channel genes, including CFTR, ENaCA, AQP3 and CRISP2, and the increase can be inhibited by ETA. Conclusion: In conclusion, our result showed that higher TNF-α level is associated with implantation failure through activation of TNF-α receptor, and TNF-α maybe an independent predictor for pre-transfer assessment of the embryo development potential in IVF patients.

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The Mechanism of Rhein Ameliorating the Survival Rate of Transplanted Mesenchymal Stem Cells by Improving Myocardial Microenvironment in Acute Myocardial Infarction

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2021.2570 ◽

2021 ◽

Vol 11 (4) ◽

pp. 684-689

Author(s):

Xuejun Deng ◽

Xiaojun He ◽

Gang Huang ◽

Dongmei Yu ◽

Xiaozhen Lin

Keyword(s):

Myocardial Infarction ◽

Stem Cells ◽

Survival Rate ◽

Heart Function ◽

Immunofluorescence Staining ◽

Inflammatory Factors ◽

Tunel Staining ◽

Myocardial Tissue ◽

Tnf Α ◽

Myocardial Fibers

Background: The paper investigated the mechanism of Rhein improving the ischemic myocardial microenvironment, promoting the survival rate of transplanted BMSCs and functional recovery of damaged myocardium by alleviating myocardial ERS-mediated hyperinflammation and apoptosis after AMI. Material and Methods: A model of myocardial infarction was established. BLI was used to detect the survival rate of transplanted stem cells at 1, 7, 14, 21 and 28 days after surgery. TUNEL staining was used to assess apoptosis. ERS-related protein CHOP immunofluorescence staining was used to assess ERS level. The expressions of ERS-related biomarkers ATF4, CHOP, GRP78 and GRP94 were detected by Western Blot. The inflammatory factors IL-6, TNF-α and IL- 10 of myocardial tissue were detected by ELISA. CD31 immunization was performed 28 days after surgery. Fluorescence staining was used to assess tissue angiogenesis. Results: Rhein combined with BMSCs could improve cardiac function, decrease apoptosis and myocardial CHOP expression. WB showed that the expressions of ATF4, CHOP, GRP78 and GRP94 in myocardial tissue of MI rats were decreased. ELISA showed that Rhein can inhibit the expressions of pro-inflammatory factors IL-6 and TNF-α, and promote anti-inflammatory factors IL-10 expression. CD31 immunofluorescence staining showed that Rhein can promote the formation of neovascularization in infarcted myocardium. Conclusion: In AMI, myocardial ERS is activated. Rhein inhibits ERS and the mediated inflammation and oxidative stress after AMI, inhibits apoptosis, improves the survival rate of transplanted BMSCs, enhances BMSCs to promote neovascularization, inhibits myocardial fibers, and improves heart function.

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Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling

10.1101/2020.01.28.922856 ◽

2020 ◽

Author(s):

Giuliano Giuseppe Stirparo ◽

Agata Kurowski ◽

Stanley Eugene Strawbridge ◽

Hannah Stuart ◽

Thorsten Edwin Boroviak ◽

...

Keyword(s):

Transcription Factors ◽

Single Cell ◽

Inner Cell Mass ◽

Cell Mass ◽

Ectopic Expression ◽

Specific Gene ◽

List Type ◽

Early Mouse

AbstractOCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism.Highlights and noveltyMajor pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markersJAK/STAT signalling is defective in OCT4 null embryosOCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identityOCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency

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Progesterone-regulated Hsd11b2 as a barrier to balance mouse uterine corticosterone

Journal of Endocrinology ◽

10.1530/joe-19-0349 ◽

2020 ◽

Vol 244 (1) ◽

pp. 177-187 ◽

Cited By ~ 1

Author(s):

Hong-Tao Zheng ◽

Tao Fu ◽

Hai-Yi Zhang ◽

Zhen-Shan Yang ◽

Zhan-Hong Zheng ◽

...

Keyword(s):

Early Pregnancy ◽

Mouse Embryo ◽

Stromal Cells ◽

Inner Cell Mass ◽

Local Level ◽

Embryo Implantation ◽

Cell Mass ◽

Type I ◽

Mouse Uterus ◽

Ovariectomized Mice

Glucocorticoids (GCs) are essential for mouse embryo implantation and decidualization. Excess GCs are harmful for mouse embryo implantation and decidualization. 11β-Hydroxysteroid dehydrogenases type I and II (Hsd11b1/Hsd11b2) are main enzymes for regulating local level of GCs. Hsd11b2 acts as the placental glucocorticoid barrier to protect the fetus from excessive exposure. Although effects of GCs on the fetus and placenta in late pregnancy have been extensively studied, the effects of these adrenal corticosteroids in early pregnancy are far less well defined. Therefore, we examined the expression, regulation and function of Hsd11b1/Hsd11b2 in mouse uterus during early pregnancy. We found that Hsd11b2 is highly expressed in endometrial stromal cells on days 3 and 4 of pregnancy and mainly upregulated by progesterone (P4). In both ovariectomized mice and cultured stromal cells, P4 significantly stimulates Hsd11b2 expression. P4 stimulation of Hsd11b2 is mainly mediated by the Ihh pathway. The uterine level of corticosterone (Cort) is regulated by Hsd11b2 during preimplantation. Embryo development and the number of inner cell mass cells are suppressed by Cort treatment. These results indicate that P4 should provide a low Cort environment for the development of preimplantation mouse embryos by promoting the expression of uterine Hsd11b2.

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Implantation Failure in Female Kiss1−/− Mice Is Independent of Their Hypogonadic State and Can Be Partially Rescued by Leukemia Inhibitory Factor

Endocrinology ◽

10.1210/en.2013-1916 ◽

2014 ◽

Vol 155 (8) ◽

pp. 3065-3078 ◽

Cited By ~ 32

Author(s):

Michele Calder ◽

Yee-Ming Chan ◽

Renju Raj ◽

Macarena Pampillo ◽

Adrienne Elbert ◽

...

Keyword(s):

Leukemia Inhibitory Factor ◽

Hypogonadotropic Hypogonadism ◽

Embryo Implantation ◽

Critical Role ◽

Inhibitory Factor ◽

Implantation Failure ◽

Mouse Uterus ◽

Female Mice ◽

Wild Type Female ◽

Neuroendocrine Axis

The hypothalamic kisspeptin signaling system is a major positive regulator of the reproductive neuroendocrine axis, and loss of Kiss1 in the mouse results in infertility, a condition generally attributed to its hypogonadotropic hypogonadism. We demonstrate that in Kiss1−/− female mice, acute replacement of gonadotropins and estradiol restores ovulation, mating, and fertilization; however, these mice are still unable to achieve pregnancy because embryos fail to implant. Progesterone treatment did not overcome this defect. Kiss1+/− embryos transferred to a wild-type female mouse can successfully implant, demonstrating the defect is due to maternal factors. Kisspeptin and its receptor are expressed in the mouse uterus, and we suggest that it is the absence of uterine kisspeptin signaling that underlies the implantation failure. This absence, however, does not prevent the closure of the uterine implantation chamber, proper alignment of the embryo, and the ability of the uterus to undergo decidualization. Instead, the loss of Kiss1 expression specifically disrupts embryo attachment to the uterus. We observed that on the day of implantation, leukemia inhibitory factor (Lif), a cytokine that is absolutely required for implantation in mice, is weakly expressed in Kiss1−/− uterine glands and that the administration of exogenous Lif to hormone-primed Kiss1−/− female mice is sufficient to partially rescue implantation. Taken together, our study reveals that uterine kisspeptin signaling regulates glandular Lif levels, thereby identifying a novel and critical role for kisspeptin in regulating embryo implantation in the mouse. This study provides compelling reasons to explore this role in other species, particularly livestock and humans.

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mTOR Is Essential for Growth and Proliferation in Early Mouse Embryos and Embryonic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.24.15.6710-6718.2004 ◽

2004 ◽

Vol 24 (15) ◽

pp. 6710-6718 ◽

Cited By ~ 381

Author(s):

Mirei Murakami ◽

Tomoko Ichisaka ◽

Mitsuyo Maeda ◽

Noriko Oshiro ◽

Kenta Hara ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Size ◽

Inner Cell Mass ◽

Critical Role ◽

Cell Mass ◽

Embryonic Stem ◽

Mouse Embryos ◽

Early Mouse

ABSTRACT TOR is a serine-threonine kinase that was originally identified as a target of rapamycin in Saccharomyces cerevisiae and then found to be highly conserved among eukaryotes. In Drosophila melanogaster, inactivation of TOR or its substrate, S6 kinase, results in reduced cell size and embryonic lethality, indicating a critical role for the TOR pathway in cell growth control. However, the in vivo functions of mammalian TOR (mTOR) remain unclear. In this study, we disrupted the kinase domain of mouse mTOR by homologous recombination. While heterozygous mutant mice were normal and fertile, homozygous mutant embryos died shortly after implantation due to impaired cell proliferation in both embryonic and extraembryonic compartments. Homozygous blastocysts looked normal, but their inner cell mass and trophoblast failed to proliferate in vitro. Deletion of the C-terminal six amino acids of mTOR, which are essential for kinase activity, resulted in reduced cell size and proliferation arrest in embryonic stem cells. These data show that mTOR controls both cell size and proliferation in early mouse embryos and embryonic stem cells.

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A multiscale model via single-cell transcriptomics reveals robust patterning mechanisms during early mammalian embryo development

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008571 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1008571

Author(s):

Zixuan Cang ◽

Yangyang Wang ◽

Qixuan Wang ◽

Ken W. Y. Cho ◽

William Holmes ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Embryo Development ◽

Inner Cell Mass ◽

Cell Mass ◽

Mammalian Embryo ◽

Early Mammalian Embryo ◽

Cell Gene Expression ◽

Cell Gene ◽

Cell Cell

During early mammalian embryo development, a small number of cells make robust fate decisions at particular spatial locations in a tight time window to form inner cell mass (ICM), and later epiblast (Epi) and primitive endoderm (PE). While recent single-cell transcriptomics data allows scrutinization of heterogeneity of individual cells, consistent spatial and temporal mechanisms the early embryo utilize to robustly form the Epi/PE layers from ICM remain elusive. Here we build a multiscale three-dimensional model for mammalian embryo to recapitulate the observed patterning process from zygote to late blastocyst. By integrating the spatiotemporal information reconstructed from multiple single-cell transcriptomic datasets, the data-informed modeling analysis suggests two major processes critical to the formation of Epi/PE layers: a selective cell-cell adhesion mechanism (via EphA4/EphrinB2) for fate-location coordination and a temporal attenuation mechanism of cell signaling (via Fgf). Spatial imaging data and distinct subsets of single-cell gene expression data are then used to validate the predictions. Together, our study provides a multiscale framework that incorporates single-cell gene expression datasets to analyze gene regulations, cell-cell communications, and physical interactions among cells in complex geometries at single-cell resolution, with direct application to late-stage development of embryogenesis.

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BMP-treated human embryonic stem cells transcriptionally resemble amnion cells in the monkey embryo

Biology Open ◽

10.1242/bio.058617 ◽

2021 ◽

Author(s):

Sapna Chhabra ◽

Aryeh Warmflash

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Single Cell ◽

Human Embryonic Stem Cells ◽

Inner Cell Mass ◽

Cell Mass ◽

Embryonic Stem ◽

Cell Types ◽

Human Embryos ◽

Early Human

Human embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their in vivo counterparts. Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to recently published single cell transcriptomic data from early human embryos (Xiang et al., 2019). Our analyses reveal several discrepancies in the published human embryo dataset, including misclassification of putative amnion, intermediate and inner cell mass cells. These misclassifications primarily resulted from similarities in pseudogene expression, highlighting the need to carefully consider gene lists when making comparisons between cell types. In the absence of a relevant human dataset, we utilized the recently published single cell transcriptome of the early post implantation monkey embryo to discern the identity of BMP-treated hESCs. Our results suggest that BMP-treated hESCs are transcriptionally more similar to amnion cells than trophectoderm cells in the monkey embryo. Together with prior studies, this result indicates that hESCs possess a unique ability to form mature trophectoderm subtypes via an amnion-like transcriptional state.

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Cell Lineage-Specific Signaling of Insulin and Insulin-Like Growth Factor I in Rabbit Blastocysts

Endocrinology ◽

10.1210/en.2007-0821 ◽

2007 ◽

Vol 149 (2) ◽

pp. 515-524 ◽

Cited By ~ 39

Author(s):

Anne Navarrete Santos ◽

Nicole Ramin ◽

Sarah Tonack ◽

Bernd Fischer

Keyword(s):

Growth Factor ◽

Phosphoenolpyruvate Carboxykinase ◽

Target Genes ◽

Inner Cell Mass ◽

Expression Patterns ◽

Critical Role ◽

Cell Mass ◽

Cell Lineage ◽

Pathway Expression ◽

Igf I

The insulin/IGF system plays a critical role in embryo growth and development. We have investigated the expression of insulin receptor (IR) and IGF-I receptor (IGF-IR) and the activation of their downstream pathways in rabbit 6-d-old blastocysts. IR was expressed in embryoblast (Em, inner cell mass) and trophoblast (Tr) cells, whereas IGF-IR was localized mainly in Em. Isoform A (IR-A) represents the main insulin isoform in blastocysts and was found in Em and Tr cells. IR-B was detectable only in Tr. IR/IGF-IR signaling pathways were analyzed after stimulation with insulin (17 nm) or IGF-I (1.3 nm) in cultured blastocysts. Insulin stimulated Erk1/2 in Em and Tr and Akt in Tr but not in Em. IGF-I activated both kinases exclusively in Em. The target genes c-fos (for MAPK kinase-1/Erk signaling) and phosphoenolpyruvate carboxykinase (PEPCK, for PI3K/Akt signaling) were also specifically regulated. Insulin down-regulated PEPCK RNA amounts in Tr by activation of the phosphatidylinositol 3-kinase/Akt pathway. Expression of c-fos by insulin and IGF-I was different with respect to time and fortitude of expression, mirroring again the specific IR and IGF-IR expression patterns in Em and Tr. Taken together, we show that IGF-I acts primarily mitogenic, an effect that is cell lineage-specifically restricted to the Em. By contrast, insulin is the growth factor of the Tr stimulating mitogenesis and down-regulating metabolic responses. As soon as blastocyst differentiation in Em and Tr has been accomplished, insulin and IGF-I signaling is different in both cell lineages, implying a different developmental impact of both growth factors.

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