scholarly journals Developing a reproducible protocol for culturing functional confluent monolayers of differentiated equine oviduct epithelial cells

2021 ◽  
Author(s):  
Bart Leemans ◽  
Elizabeth G Bromfield ◽  
Tom A E Stout ◽  
Mabel Vos ◽  
Hanna Van Der Ham ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Luteal Phase ◽  
Receptor Expression ◽  
Microporous Membrane ◽  
Cytokeratin 19 ◽  
Apical Surface ◽  
Microfluidic Chips ◽  
Differentiation Protocol ◽  
Oviduct Epithelial Cells

Abstract We describe the development of two methods for obtaining confluent monolayers of polarized, differentiated equine oviduct epithelial cells (EOEC) in Transwell inserts and microfluidic chips. EOECs from the ampulla were isolated post-mortem and seeded either (1) directly onto a microporous membrane as differentiated EOECs (direct seeding protocol) or (2) first cultured to a confluent de-differentiated monolayer in conventional wells, then trypsinized and seeded onto a microporous membrane (re-differentiation protocol). Maintenance or induction of EOEC differentiation in these systems was achieved by air-liquid interface introduction. Monolayers cultured via both protocols were characterized by columnar, cytokeratin 19-positive EOECs in Transwell inserts. However, only the re-differentiation protocol could be transferred successfully to the microfluidic chips. Integrity of the monolayers was confirmed by transepithelial resistance measurements, tracer flux and the demonstration of an intimate network of tight junctions. Using the direct protocol, 28% of EOECs showed secondary cilia at the apical surface in a diffuse pattern. In contrast, re-differentiated polarized EOECs rarely showed secondary cilia in either culture system (>90% of the monolayers showed <1% ciliated EOECs). Occasionally (5–10%), re-differentiated monolayers with 11–27% EOECs with secondary cilia in a diffuse pattern were obtained. Additionally, nuclear progesterone receptor expression was found to be inhibited by simulated luteal phase hormone concentrations, and sperm binding to cilia was higher for re-differentiated EOEC monolayers exposed to estrogen-progesterone concentrations mimicking the follicular rather than luteal phase. Overall, a functional equine oviduct model was established with close morphological resemblance to in vivo oviduct epithelium.

CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo

1999 ◽  
Vol 112 (6) ◽  
pp. 887-894
Author(s):  
N.A. Ameen ◽  
B. Martensson ◽  
L. Bourguinon ◽  
C. Marino ◽  
J. Isenberg ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Surface Expression ◽  
Apical Plasma Membrane ◽  
Intracellular Camp ◽  
Apical Surface ◽  
C Terminus ◽  
Confocal Immunofluorescence ◽  
Small Intestinal Villus

cAMP activated insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) channels from endosomes to the apical plasma membrane has been hypothesized to regulate surface expression and CFTR function although the physiologic relevance of this remains unclear. We previously identified a subpopulation of small intestinal villus epithelial cells or CFTR high expressor (CHE) cells possessing very high levels of apical membrane CFTR in association with a prominent subapical vesicular pool of CFTR. We have examined the subcellular redistribution of CFTR in duodenal CHE cells in vivo in response to the cAMP activated secretagogue vasoactive intestinal peptide (VIP). Using anti-CFTR antibodies against the C terminus of rodent CFTR and indirect immunofluorescence, we show by quantitative confocal microscopy that CFTR rapidly redistributes from the cytoplasm to the apical surface upon cAMP stimulation by VIP and returns to the cytoplasm upon removal of VIP stimulation of intracellular cAMP levels. Using ultrastructural and confocal immunofluorescence examination in the presence or absence of cycloheximide, we also show that redistribution was not dependent on new protein synthesis, changes in endocytosis, or rearrangement of the apical cytoskeleton. These observations suggest that physiologic cAMP activated apical membrane insertion and recycling of CFTR channels in normal CFTR expressing epithelia contributes to the in vivo regulation of CFTR mediated anion transport.

Human duodenal mucosal brush border Na+/H+ exchangers NHE2 and NHE3 alter net bicarbonate movement

2001 ◽  
Vol 281 (1) ◽  
pp. G159-G163 ◽  
Author(s):  
Maltin Repishti ◽  
Daniel L. Hogan ◽  
Vijaya Pratha ◽  
Laura Davydova ◽  
Mark Donowitz ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Brush Border ◽  
Polyclonal Antibodies ◽  
Duodenal Mucosa ◽  
Transport Processes ◽  
Apical Surface ◽  
Luminal Perfusion ◽  
Significant Step

The proximal duodenal mucosa secretes HCO[Formula: see text] that serves to protect the epithelium from injury. In isolated human duodenal enterocytes in vitro, multiple luminal membrane proteins are involved in acid/base transport. We postulated that one or more isoforms of the Na+/H+ exchanger (NHE) family is located on the apical surface of human duodenal mucosal epithelial cells and thereby contributes to duodenal mucosal HCO[Formula: see text] transport. Duodenal biopsies were obtained from human volunteers, and the presence of NHE2 and NHE3 was determined by using previously characterized polyclonal antibodies (Ab 597 for NHE2 and Ab 1381 for NHE3). In addition, proximal duodenal mucosal HCO[Formula: see text] transport was measured in humans in vivo in response to luminal perfusion of graded doses of amiloride; 10−5–10−4 M amiloride was used to inhibit NHE2 and 10−3 M amiloride to inhibit NHE3. Both NHE2 and NHE3 were localized principally to the brush border of duodenal villus cells. Sequential doses of amiloride resulted in significant, step-wise increases in net duodenal HCO[Formula: see text] output. Inhibition of NHE2 with 10−5 M and 10−4 M amiloride significantly increased net HCO[Formula: see text] output. Moreover, there was an additional, equivalent increase ( P < 0.05) in duodenal HCO[Formula: see text] output with 10−3 M amiloride, which inhibited NHE3. We conclude that 1) NHE2 and NHE3 are localized principally to the brush border of human duodenal villus epithelial cells; 2) sequential inhibition of NHE2 and NHE3 isoforms resulted in step-wise increases in net HCO[Formula: see text]output; 3) NHE2 and NHE3 participate in human duodenal villus cell HCO[Formula: see text] transport; and 4) the contribution of NHE-related transport events should be considered when studying duodenal HCO[Formula: see text] transport processes.

scholarly journals Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells

2014 ◽  
Vol 25 (20) ◽  
pp. 3095-3104 ◽  
Author(s):  
Keiko Saegusa ◽  
Miyuki Sato ◽  
Katsuya Sato ◽  
Junko Nakajima-Shimada ◽  
Akihiro Harada ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Intestinal Cells ◽  
Intermediate Filament Protein ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Microtubule Network

Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.

scholarly journals Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo

Reproduction ◽  
2019 ◽  
Vol 158 (1) ◽  
pp. 85-94 ◽  
Author(s):  
Meriem Hamdi ◽  
María J Sánchez-Calabuig ◽  
Beatriz Rodríguez-Alonso ◽  
Sandra Bagés Arnal ◽  
Kalliopi Roussi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Metabolic Response ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Transcriptomic Response ◽  
Early Embryos ◽  
Oviduct Epithelial Cells

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.

Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells

1993 ◽  
Vol 105 (4) ◽  
pp. 1025-1043 ◽  
Author(s):  
M. Berryman ◽  
Z. Franck ◽  
A. Bretscher
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Cytoskeletal Proteins ◽  
Specific Cell ◽  
Apical Surface ◽  
Tissue Sections ◽  
Differentiated Cells

Ezrin and moesin are two cytoskeletal proteins originally purified from human placenta that are 74% identical in overall protein sequence. They are believed to be membrane-cytoskeletal linking proteins because they share sequence homology with erythrocyte band 4.1 and colocalize with actin specifically in microvilli and membrane ruffles in cultured cells. To determine if ezrin and moesin share similar distributions in vivo, we studied their localizations with respect to F-actin in tissue sections. Surprisingly, ezrin and moesin exhibited very different cellular distributions. Ezrin was highly concentrated and colocalized with actin on the apical surface of many epithelial cell types. During enterocyte differentiation, the pattern of expression and redistribution of ezrin was consistent with it performing a role in microvillus assembly. Immunoelectron microscopy in differentiated cells revealed that ezrin was restricted mainly to the plasma membrane of microvilli and other actin-rich surface projections. Moesin was found in endothelial cells and was also enriched in the apical microvilli of a restricted set of epithelial cells. All polarized cell types with abundant microvilli contained one or both proteins, suggesting that ezrin and moesin perform related functions. However, the differential expression of ezrin and moesin indicates that they have distinct properties, which are uniquely adapted to specific cell types.

scholarly journals Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells.

1982 ◽  
Vol 95 (1) ◽  
pp. 333-339 ◽  
Author(s):  
G Greenburg ◽  
E D Hay
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Mesenchymal Cells ◽  
Apical Surface ◽  
Collagen Gels ◽  
Mesenchymal Transformation

This study of epithelial-mesenchymal transformation and epithelial cell polarity in vitro reveals that environmental conditions can have a profound effect on the epithelial phenotype, cell shape, and polarity as expressed by the presence of apical and basal surfaces. A number of different adult and embryonic epithelia were suspended within native collagen gels. Under these conditions, cells elongate, detach from the explants, and migrate as individual cells within the three-dimensional lattice, a previously unknown property of well-differentiated epithelia. Epithelial cells from adult and embryonic anterior lens were studied in detail. Elongated cells derived from the apical surface develop pseudopodia and filopodia characteristic of migratory cells and acquire a morphology and ultrastructure virtually indistinguishable from that of mesenchymal cells in vivo. It is concluded from these experiments that the three-dimensional collagen gel can promote dissociation, migration, and acquisition of secretory organelles by differentiated epithelial cells, and can abolish the apical-basal cell polarity characteristic of the original epithelium.

scholarly journals Interactions of pathogenic and commensal strains of Mannheimia haemolytica with differentiated bovine airway epithelial cells grown at an air-liquid interface

2017 ◽  
Author(s):  
Daniel Cozens ◽  
Erin Sutherland ◽  
Miquel Lauder ◽  
Geraldine Taylor ◽  
Catherine C. Berry ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Airway Epithelial Cells ◽  
Respiratory Epithelium ◽  
Mannheimia Haemolytica ◽  
Sudden Increase ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Upper Respiratory Tract

AbstractMannheimia haemolyticaserotype A2 is a common commensal species present in the nasopharynx of healthy cattle. However, prior to the onset of bovine pneumonic pasteurellosis, there is sudden increase inM. haemolyticaserotype A1 within the upper respiratory tract. The events during this selective proliferation of serotype A1 strains are poorly characterised. In this investigation, a differentiated bovine airway epithelial cell culture was used to study the interactions of A1 and A2 bovine isolates with the respiratory epithelium. This model reproduced the key defences of the airway epithelium, including tight junctions and mucociliary clearance. Although initial adherence of the serotype A1 strains was low, by 12 hours post-infection the bacteria was able to traverse the tight junctions to form foci of infection below the apical surface. The size, density and number of these foci increased with time, as did the cytopathic effects observed in the bovine bronchial epithelial cells. Penetration ofM. haemolyticaA1 into the sub-apical epithelium was shown to be through transcytosis but not paracytosis. Commensal A2 bovine isolates however were not capable of colonising the model to a high degree, and did not penetrate the epithelium following initial adherence at the apical surface. This difference in their ability to colonise the respiratory epithelium may account for the sudden proliferation of serotype A1 in the onset of pneumonia pasteurellosis. The pathogenesis observed was replicated by virulent A2 ovine isolates; however colonisation was 10-fold lower in comparison to bovine A1 strains. This investigation provides new insight into the interactions ofM. haemolyticawith bovine airway epithelial cells which are occurringin vivoduring pneumonia pasteurellosis.

scholarly journals Effect of Cell Polarization on Hepatitis C Virus Entry

2007 ◽  
Vol 82 (1) ◽  
pp. 461-470 ◽  
Author(s):  
Christopher J. Mee ◽  
Joe Grove ◽  
Helen J. Harris ◽  
Ke Hu ◽  
Peter Balfe ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Scavenger Receptor ◽  
Cell Polarization ◽  
Receptor Expression ◽  
Cell Junction ◽  
Apical Surface ◽  
Viral Receptor ◽  
Lateral Cell

ABSTRACT The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.

Adhesion of calcium oxalate monohydrate crystals to anionic sites on the surface of renal epithelial cells

1996 ◽  
Vol 270 (1) ◽  
pp. F192-F199 ◽  
Author(s):  
J. C. Lieske ◽  
R. Leonard ◽  
H. Swift ◽  
F. G. Toback
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Calcium Oxalate ◽  
Kidney Stones ◽  
Membrane Surface ◽  
Calcium Oxalate Monohydrate ◽  
Apical Surface ◽  
Renal Epithelial Cells ◽  
Renal Tubular Cells

Adhesion of microcrystals to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones. The role of membrane surface charge as a determinant of the interaction between renal epithelial cells (BSC-1 line) and the most common crystal in kidney stones, calcium oxalate monohydrate (COM), was studied in a tissue culture model system. Adhesion of COM crystals to cells was blocked by cationized ferritin. Other cations that bind to cells including cetylpyridinium chloride and polylysine, as well as cationic dyes such as Alcian blue, also inhibited adhesion of COM crystals, but not all polycations shared this effect. Specific lectins including Triticum vulgaris (wheat germ agglutinin) blocked crystal binding to the cells. Furthermore, treatment of cells with neuraminidase inhibited binding of crystals. Therefore, anionic cell surface sialic acid residues appear to function as COM crystal receptors that can be blocked by specific cations or lectins. In vivo, alterations in the structure, function, quantity, or availability of these anionic cell surface molecules could lead to crystal retention and formation of renal calculi.

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