scholarly journals Unusual characteristics of opaque Ishikawa endometrial cells include the envelopment of chromosomes with material containing endogenous biotin in the latter stages of cytokinesis

2014 ◽  
Author(s):  
Honoree Fleming
Keyword(s):  
Confluent Monolayer ◽  
Monolayer Cell ◽  
Endometrial Cells ◽  
Daughter Cells ◽  
Spatial Extension ◽  
Polyploid Cells ◽  
Complete Genomes ◽  
Ishikawa Cells ◽  
Dynamic Population ◽  
Endogenous Biotin

We have identified a small dynamic population of opaque cells in Ishikawa endometrial cultures whose unusual characteristics include the fact that chromosomes become enveloped during the final stages of cytokinesis by material staining for endogenous biotin. Endogenous biotin, ultimately shown to be due to mitochondrial carboxylases, was detected in a membrane that wraps around aggregated nuclei in syncytia that develop as part of the differentiation of domes in Ishikawa cells. (Fleming H et al. 1998). The “wrapped chromosomes” in individual opaque Ishikawa cells stain similarly suggesting a similar origin. We were able to show that opaque cells form from transparent monolayer cells, can be polyploid, and often appear to be detaching from the colony and from the underlying substrate. We were also able to show an opaque cell fissioning asymmetrically, to give rise to a monolayer cell whose nucleus appeared to be wrapped. We believe that the cycle of differentiation of monolayer cells into opaque, polyploid cells and depolyploidization back into monolayer cells is involved in the spatial extension of cells as they develop from discrete colonies into a confluent monolayer. Wrapping of chromosomes may ensure that complete genomes are inherited by daughter cells during depolyploidization.

scholarly journals Unusual characteristics of opaque Ishikawa endometrial cells include the envelopment of chromosomes with material containing endogenous biotin in the latter stages of cytokinesis

2014 ◽  
Author(s):  
Honoree Fleming
Keyword(s):  
Confluent Monolayer ◽  
Monolayer Cell ◽  
Endometrial Cells ◽  
Daughter Cells ◽  
Spatial Extension ◽  
Polyploid Cells ◽  
Complete Genomes ◽  
Ishikawa Cells ◽  
Dynamic Population ◽  
Endogenous Biotin

We have identified a small dynamic population of opaque cells in Ishikawa endometrial cultures whose unusual characteristics include the fact that chromosomes become enveloped during the final stages of cytokinesis by material staining for endogenous biotin. Endogenous biotin, ultimately shown to be due to mitochondrial carboxylases, was detected in a membrane that wraps around aggregated nuclei in syncytia that develop as part of the differentiation of domes in Ishikawa cells. (Fleming H et al. 1998). The “wrapped chromosomes” in individual opaque Ishikawa cells stain similarly suggesting a similar origin. We were able to show that opaque cells form from transparent monolayer cells, can be polyploid, and often appear to be detaching from the colony and from the underlying substrate. We were also able to show an opaque cell fissioning asymmetrically, to give rise to a monolayer cell whose nucleus appeared to be wrapped. We believe that the cycle of differentiation of monolayer cells into opaque, polyploid cells and depolyploidization back into monolayer cells is involved in the spatial extension of cells as they develop from discrete colonies into a confluent monolayer. Wrapping of chromosomes may ensure that complete genomes are inherited by daughter cells during depolyploidization.

Fine Structure of Cytochalasin Induced Polyploid Cells

1968 ◽  
Vol 26 ◽  
pp. 122-123
Author(s):  
Awtar Krishan ◽  
Nestor Bohonos
Keyword(s):  
Fine Structure ◽  
Cytochalasin B ◽  
Present Report ◽  
Cell Monolayer ◽  
Polyploid Cell ◽  
Specific Cell ◽  
Nuclear Surface ◽  
Unsuccessful Attempt ◽  
Daughter Cells ◽  
Polyploid Cells

Cytochalasin B, a mould metabolite from Helminthosporium dermatioideum has been shown to interfere with specific cell activities such as cytoplasmic cleavage and cell movement. Cells undergoing nuclear division in the presence of cytochalasin B are unable to complete the separation of the resulting daughter cells. In time-lapse studies, the daughter cells coalesce after an initial unsuccessful attempt at separation and form large multinucleate polyploid cells. The present report describes the fine structure of the large polyploid cells induced in Earle's L-cell monolayer cultures by exposure to cytochalasin B (lγ/ml) for 92 hours.In the present material we have seen as many as 7 nuclei in these polyploid cells. Treatment with cytochalasin B for longer periods of time (6 to 7 days, with one medium change on the 3rd day) did not increase the number of nuclei beyond the 7 nuclei stage. Figure 1 shows a large polyploid cell with four nuclei. These nuclei are indistinguishable in their fine structure from those of the cells from control cultures but often show unusually large numbers of cytoplasmic invaginations and extensions of the nuclear surface (Figure 2).

scholarly journals Mitonucleons formed during differentiation of Ishikawa endometrial cells generate vacuoles that elevate monolayer syncytia: Differentiation of Ishikawa domes, Part 1

2016 ◽  
Author(s):  
Honoree Fleming
Keyword(s):  
Cell Death ◽  
Structural Changes ◽  
Apical Membrane ◽  
Signet Ring Cells ◽  
Double Membrane ◽  
Endometrial Cells ◽  
Endometrial Epithelial Cell ◽  
Signet Ring ◽  
Endogenous Biotin ◽  
Cell Profile

In 1998, we published a paper (Fleming et.al, 1998) describing some aspects of Ishikawa endometrial epithelial cell differentiation from monolayer cells into cells forming fluid-filled hemispheres called domes. The process begins with the dissolution of membranes within discrete regions of the monolayer. Nuclei from fused cells aggregate and endogenous biotin in particulate structures assumed to be mitochondria increase throughout the resulting syncytium. Endogenous biotin is also the distinguishing feature of a membrane that surrounds aggregates of multiple nuclei in a structure called a mitonucleon. The current paper includes additional observations on structural changes accompanying Ishikawa differentiation. Vacuoles form in the heterochromatin of the mitonucleon and within the biotin-containing double membrane surrounding heterochromatin. With the formation of vacuoles, the mitonucleon can be seen to rise along with the apical membrane of the syncytium in which it formed. The small vacuoles that form within the heterochromatin result in structures similar to “cells with optically clear nuclei” found in some cancers. The second larger vacuole that forms within the membrane surrounding the heterochromatin transforms the cell profile to one that resembles “signet ring” cells also observed in some cancers. Eventually the membrane surrounding the massed heterochromatin, generated three to four hours earlier, is breached and previously aggregated nuclei disaggregate. During this process heterochromatin in the mitonucleons undergoes changes usually ascribed to cells undergoing programmed cell death such as pyknosis and DNA fragmentation (Fleming, 2016b). The cells do not die, instead chromatin filaments appear to coalesce into a chromatin mass that gives rise to dome-filling nuclei by amitosis during the final three to four hours of the 20 hour differentiation (Fleming, 2016c).

scholarly journals LEFTYA Activates the Epithelial Na+ Channel (ENaC) in Endometrial Cells via Serum and Glucocorticoid Inducible Kinase SGK1

2016 ◽  
Vol 39 (4) ◽  
pp. 1295-1306 ◽  
Author(s):  
Madhuri S. Salker ◽  
Zohreh Hosseinzadeh ◽  
Nour Alowayed ◽  
Ni Zeng ◽  
Anja T. Umbach ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ussing Chamber ◽  
Endometrial Adenocarcinoma ◽  
Negative Regulator ◽  
Na Channel ◽  
Uterine Receptivity ◽  
Endometrial Cells ◽  
Ishikawa Cells ◽  
Epithelial Na Channel ◽  
Endometrial Epithelial Cells

Background: Serum & glucocorticoid inducible kinase (SGK1) regulates several ion channels, including amiloride sensitive epithelial Na+ channel (ENaC). SGK1 and ENaC in the luminal endometrium epithelium, are critically involved in embryo implantation, although little is known about their regulation. The present study explored whether SGK1 and ENaC are modulated by LEFTYA, a negative regulator of uterine receptivity. Methods: Expression levels were determined by qRT-PCR and Western blotting, ENaC channel activity by whole cell patch clamp and transepithelial current by Ussing chamber experiments. Results: Treatment of Ishikawa cells, an endometrial adenocarcinoma model cell line of endometrial epithelial cells, with LEFTYA rapidly up-regulated SGK1 and ENaC transcript and protein levels. Induction of ENaC in response to LEFTYA was blunted upon co-treatment with the SGK1 inhibitor EMD638683. ENaC levels also significantly upregulated upon expression of a constitutively active, but not a kinase dead, SGK1 mutant in Ishikawa cells. LEFTYA increased amiloride sensitive Na+-currents in Ishikawa cells and amiloride sensitive transepithelial current across the murine endometrium. Furthermore, LEFTYA induced the expression of ENaC in the endometrium of wild-type but not of Sgk1-deficient mice. Conclusions: LEFTYA regulates the expression and activity of ENaC in endometrial epithelial cells via SGK1. Aberrant regulation of SGK1 and ENaC by LEFTYA could contribute to the pathogenesis of unexplained infertility.

scholarly journals Heat Stress-Induced Multiple Multipolar Divisions of Human Cancer Cells

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 888 ◽  
Author(s):  
Chen ◽  
Liu ◽  
Huang ◽  
Li ◽  
Zhao ◽  
...  
Keyword(s):  
Heat Stress ◽  
Cancer Cells ◽  
Cell Fusion ◽  
Human Cancer ◽  
Human Cancer Cells ◽  
Daughter Cells ◽  
Mitotic Slippage ◽  
Polyploid Cells ◽  
Mcf 7

Multipolar divisions of heated cells has long been thought to stem from centrosome aberrations of cells directly caused by heat stress. In this paper, through long-term live-cell imaging, we provide direct cellular evidences to demonstrate that heat stress can promote multiple multipolar divisions of MGC-803 and MCF-7 cells. Our results show that, besides facilitating centrosome aberration, polyploidy induced by heat stress is another mechanism that causes multipolar cell divisions, in which polyploid cancer cells engendered by mitotic slippage, cytokinesis failure, and cell fusion. Furthermore, we also find that the fates of theses polyploid cells depend on their origins, in the sense that the polyploid cells generated by mitotic slippage experience bipolar divisions with a higher rate than multipolar divisions, while those polyploid cells induced by both cytokinesis failure and cell fusion have a higher frequency of multipolar divisions compared with bipolar divisions. This work indicates that heat stress-induced multiple multipolar divisions of cancer cells usually produce aneuploid daughter cells, and might lead to genetically unstable cancer cells and facilitate tumor heterogeneity.

scholarly journals Stable inhibition of interleukin 1 receptor type II in Ishikawa cells augments secretion of matrix metalloproteinases: possible role in endometriosis pathophysiology

Reproduction ◽  
2007 ◽  
Vol 134 (3) ◽  
pp. 525-534 ◽  
Author(s):  
S Guay ◽  
A Akoum
Keyword(s):  
Interleukin 1 ◽  
Receptor Type ◽  
Type Ii ◽  
Endometrial Cells ◽  
Functional Changes ◽  
Empty Vector ◽  
Ishikawa Cells ◽  
Empty Expression Vector

Our previous studies showed a marked deficiency in interleukin 1 receptor type II (IL1R2) in the endometrial tissue of women with endometriosis, particularly in epithelial cells. We believe that such a deficiency in IL1R2, a potent and specific IL1 inhibitor, makes endometrial cells more sensitive to IL1 and less capable of buffering the cytokine’s effects, which may lead to functional changes that favor endometriosis development. The main objective of our study was to stably inhibit IL1R2 expression in endometrial cells in order to evaluate the role of IL1R2 deficiency in endometriosis pathophysiology. Stable clones of Ishikawa adenocarcinoma endometrial cells transfected with IL1R2 antisense and showing downregulation of IL1R2 protein expression, or with the empty expression vector alone and showing no noticeable difference in IL1R2 expression, were selected. The downregulation of IL1R2 expression in IL1R2 antisense transfectants when compared with control cells was confirmed by ELISA, Western blot and immunofluorescence. In these cells, IL1R2 expression was markedly reduced, compared with non-transfected cells or cells transfected with the empty vector, and there was a significant increase in the basal and the IL1-β (IL1B)-induced levels of matrix metalloproteinase (MMP)-2 and MMP-9 secretion. Furthermore, a significant decrease in IL1B-induced secretion of tissue inhibitor of MMPs-1, a known MMP-9 inhibitor, was observed. These in vitro data make plausible a role for IL1R2 deficiency in the capability of endometrial cells to invade the host tissue and develop in ectopic locations.

scholarly journals Estrogen Up-Regulates Mismatch Repair Activity in Normal and Malignant Endometrial Glandular Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (10) ◽  
pp. 4863-4870 ◽  
Author(s):  
Tsutomu Miyamoto ◽  
Tanri Shiozawa ◽  
Hiroyasu Kashima ◽  
Yu-Zhen Feng ◽  
Akihisa Suzuki ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Mismatch Repair ◽  
Proliferating Cells ◽  
Endometrial Cells ◽  
Estrogen Exposure ◽  
Estrogen Receptor Positive ◽  
Ishikawa Cells ◽  
Glandular Cells ◽  
Repair Activity

Impaired mismatch repair (MMR) is reportedly crucial in the early stages of endometrial carcinogenesis. Although estrogen exposure is considered an important risk factor for endometrial carcinoma, the relationship between estrogen and MMR activity remains undetermined. The present study was undertaken to elucidate the effect of estrogen on MMR activity in normal and malignant endometrial cells. The expression of MMR proteins, hMLH1 and hMSH2, and its correlation with estrogen was examined using immunohistochemical and immunofluorescent techniques. The effect of estradiol (E2) on the expression of hMLH1/hMSH2 protein/mRNA and in vitro MMR activity using two types of heteroduplex (G/T mismatches, 2-base insertion-deletion loops) was examined in cultured normal endometrial glandular cells and estrogen receptor-positive endometrial carcinoma Ishikawa cells. Immunohistochemical expression of hMLH1 and hMSH2 in normal endometrial glands was positively correlated with the serum E2 levels. The expression of hMLH1/hMSH2 protein and mRNA was increased in normal endometrial glandular and Ishikawa cells by E2 treatment. In vitro MMR activity was up-regulated by E2 in both types of cell and heteroduplex. Immunofluorescent analysis demonstrated that E2 enhanced proliferation and hMLH1/hMSH2 expression in both cells; however, proliferating cells without hMLH1/hMSH2 expressions implying high-risk cells were more frequently observed under low E2 concentrations. Collectively, the E2-induced up-regulation of MMR activity in endometrial cells suggests that high estrogen levels act as an intrinsic defense against endometrial carcinogenesis, whereas the imbalance between cell growth and MMR under low E2 environment as seen at postmenopause is vulnerable to carcinogenesis.

scholarly journals Changes in the Expression of TBP-2 in Response to Histone Deacetylase Inhibitor Treatment in Human Endometrial Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1427
Author(s):  
Hye In Kim ◽  
Seok Kyo Seo ◽  
Seung Joo Chon ◽  
Ga Hee Kim ◽  
Inha Lee ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Expression ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Redox Status ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Ros Accumulation ◽  
Ishikawa Cells ◽  
Mrna And Protein Expression

Histone deacetylase inhibitors (HDACi) induce apoptosis preferentially in cancer cells by caspase pathway activation and reactive oxygen species (ROS) accumulation. Suberoylanilide hydroxamic acid (SAHA), a HDACi, increases apoptosis via altering intracellular oxidative stress through thioredoxin (TRX) and TRX binding protein-2 (TBP-2). Because ROS accumulation, as well as the redox status determined by TBP-2 and TRX, are suggested as possible mechanisms for endometriosis, we queried whether SAHA induces apoptosis of human endometrial cells via the TRX–TBP-2 system in endometriosis. Eutopic endometrium from participants without endometriosis, and ectopic endometrium from patients with endometriosis, was obtained surgically. Human endometrial stromal cells (HESCs) and Ishikawa cells were treated with SAHA and cell proliferation was assessed using the CCK-8 assay. Real-time PCR and Western blotting were used to quantify TRX and TBP-2 mRNA and protein expression. After inducing oxidative stress, SAHA was applied. Short-interfering TRX (SiTRX) transfection was performed to see the changes after TRX inhibition. The mRNA and protein expression of TBP-2 was increased with SAHA concentrations in HESCs significantly. The mRNA TBP-2 expression was decreased after oxidative stress, upregulated by adding 2.5 μM of SAHA. The TRX/TBP-2 ratio decreased, apoptosis increased significantly, and SiTRX transfection decreased with SAHA. In conclusion, SAHA induces apoptosis by modulating the TRX/TBP-2 system, suggesting its potential as a therapeutic agent for endometriosis.

scholarly journals Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Marina Segura-Benítez ◽  
María Cristina Carbajo-García ◽  
Ana Corachán ◽  
Amparo Faus ◽  
Antonio Pellicer ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Proteomic Analysis ◽  
Embryo Implantation ◽  
Endometrial Receptivity ◽  
Biological Processes ◽  
Endometrial Cells ◽  
Isolation Methods ◽  
Ishikawa Cells ◽  
Endometrial Epithelial Cells ◽  
Implantation Success

Abstract Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

scholarly journals Serum estradiol levels in controlled ovarian stimulation directly affect the endometrium

2017 ◽  
Vol 59 (2) ◽  
pp. 105-119 ◽  
Author(s):  
Kamran Ullah ◽  
Tanzil Ur Rahman ◽  
Hai-Tao Pan ◽  
Meng-Xi Guo ◽  
Xin-Yan Dong ◽  
...  
Keyword(s):  
Embryo Implantation ◽  
Controlled Ovarian Hyperstimulation ◽  
Endometrial Receptivity ◽  
Protein Profiling ◽  
Differentially Expressed ◽  
Differentially Expressed Proteins ◽  
Serum Estradiol ◽  
Endometrial Cells ◽  
Ishikawa Cells ◽  
Estradiol Levels

Previous studies have shown that increasing estradiol concentrations had a toxic effect on the embryo and were deleterious to embryo adhesion. In this study, we evaluated the physiological impact of estradiol concentrations on endometrial cells to reveal that serum estradiol levels probably targeted the endometrium in controlled ovarian hyperstimulation (COH) protocols. An attachment model of human choriocarcinoma (JAr) cell spheroids to receptive-phase endometrial epithelial cells and Ishikawa cells treated with different estradiol (10−9 M or 10−7 M) concentrations was developed. Differentially expressed protein profiling of the Ishikawa cells was performed by proteomic analysis. Estradiol at 10−7 M demonstrated a high attachment rate of JAr spheroids to the endometrial cell monolayers. Using iTRAQ coupled with LC–MS/MS, we identified 45 differentially expressed proteins containing 43 significantly upregulated and 2 downregulated proteins in Ishikawa cells treated with 10−7 M estradiol. Differential expression of C3, plasminogen and kininogen-1 by Western blot confirmed the proteomic results. C3, plasminogen and kininogen-1 localization in human receptive endometrial luminal epithelium highlighted the key proteins as possible targets for endometrial receptivity and interception. Ingenuity pathway analysis of differentially expressed proteins exhibited a variety of signaling pathways, including LXR/RXR activation pathway and acute-phase response signaling and upstream regulators (TNF, IL6, Hmgn3 and miR-140-3p) associated with endometrial receptivity. The observed estrogenic effect on differential proteome dynamics in Ishikawa cells indicates that the human endometrium is the probable target for serum estradiol levels in COH cycles. The findings are also important for future functional studies with the identified proteins that may influence embryo implantation.

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