scholarly journals Boar sperm tyrosine phosphorylation patterns in the presence of oviductal epithelial cells: in vitro, ex vivo, and in vivo models

Reproduction ◽  
2013 ◽  
Vol 146 (4) ◽  
pp. 315-324 ◽  
Author(s):  
Victoria Luño ◽  
Rebeca López-Úbeda ◽  
Francisco Alberto García-Vázquez ◽  
Lydia Gil ◽  
Carmen Matás
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Ex Vivo ◽  
Cell Populations ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Experimental Conditions ◽  
In Vivo Models

Spermatozoa transport through the oviduct is a controlled process that regulates sperm capacitation. A crucial event involved in capacitation is protein tyrosine phosphorylation (TP). This study was undertaken to determine whether similarities exist in protein TP distribution between spermatozoa bound or unbound to oviductal epithelial cells (OEC) in three different conditions: i)in vitro, spermatozoa coincubated with OEC cultures; ii)ex vivo, spermatozoa deposited in porcine oviductal explants from slaughtered animals; iii)in vivo, in which sows were inseminated and the oviduct was recovered. The localization of phosphotyrosine protein was determined using indirect immunofluorescence. The distribution of protein TP was significantly (P<0.05) different between bound and unbound cell populations in all experiments. In sows inseminated close to ovulation, spermatozoa were found mainly in the utero–tubal junction, where spermatozoa exhibited higher proportion of flagellum phosphorylation. Spermatozoa not bound to OEC exhibited high levels of protein phosphorylation (phosphorylated equatorial subsegment and acrosome and/or phosphorylated flagellum) in theex vivoandin vivoexperiments (P<0.05). However, unbound spermatozoa coincubated with OEC inin vitroconditions tended to show intermediate levels of TP (equatorial subsegment with or without phosphorylated flagellum). In spermatozoa bound to OEC, protein TP was located in the equatorial subsegment or presented no phosphorylation (P<0.05). Although sperm capacitation conditionsin vivowere not reproduciblein vitroin our experimental conditions, sperm and OEC binding seemed to be a mechanism for selecting spermatozoa with a low level of TP inin vivo,ex vivo, andin vitroexperiments.

scholarly journals Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽  
2006 ◽  
Vol 132 (5) ◽  
pp. 721-732 ◽  
Author(s):  
Patricia Grasa ◽  
José Álvaro Cebrián-Pérez ◽  
Teresa Muiño-Blanco
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Calcium Entry Blocker ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine ◽  
Entry Blocker ◽  
Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

scholarly journals Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

2018 ◽  
Vol 315 (5) ◽  
pp. C653-C663 ◽  
Author(s):  
Kasin Yadunandam Anandam ◽  
Omar A. Alwan ◽  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Uptake Inhibition ◽  
Tnf Α ◽  
Vitro Model ◽  
Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation

Development ◽  
1995 ◽  
Vol 121 (4) ◽  
pp. 1129-1137 ◽  
Author(s):  
P.E. Visconti ◽  
J.L. Bailey ◽  
G.D. Moore ◽  
D. Pan ◽  
P. Olds-Clarke ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Tyrosine Phosphorylation ◽  
Bovine Serum ◽  
Female Reproductive Tract ◽  
Dependent Manner ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine

The molecular basis of mammalian sperm capacitation, defined functionally as those processes that confer on the sperm the acquisition of fertilization-competence either in vivo in the female reproductive tract or in vitro, is poorly understood. We demonstrate here that capacitation of caudal epididymal mouse sperm in vitro is accompanied by a time-dependent increase in the protein tyrosine phosphorylation of a subset of proteins of M(r) 40,000-120,000. Incubation of sperm in media devoid of bovine serum albumin, CaCl2 or NaHCO3, components which individually are required for capacitation, prevent the sperm from undergoing capacitation as assessed by the ability of the cells to acquire the pattern B chlortetracycline fluorescence, to undergo the zona pellucida-induced acrosome reaction and, in some cases, to fertilize metaphase II-arrested eggs in vitro. In each of these cases the protein tyrosine phosphorylation of the subset of capacitation-associated proteins does not occur. Protein tyrosine phosphorylation of these particular proteins, as well as sperm capacitation, can be recovered in media devoid of each of these three constituents (bovine serum albumin, CaCl2 or NaHCO3) by adding back the appropriate component in a concentration-dependent manner. The requirement of NaHCO3 for these phosphorylations is not due to an alkalinization of intracellular sperm pH or to an increase in media pH. Caput epididymal sperm, which lack the ability to undergo capacitation in vitro, do not display this capacitation-dependent subset of tyrosine phosphorylated proteins in complete media even after extended incubation periods, and do not fertilize metaphase II-arrested eggs in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

An Innovative Ex Vivo Vascular Bioreactor as Comprehensive Tool to Study the Behavior of Native Blood Vessels Under Physiologically Relevant Conditions

2019 ◽  
Vol 2 (4) ◽  
Author(s):  
Noemi Vanerio ◽  
Marco Stijnen ◽  
Bas A. J. M. de Mol ◽  
Linda M. Kock
Keyword(s):  
Shear Stress ◽  
Blood Vessels ◽  
Ultrasound Imaging ◽  
Ex Vivo ◽  
Biological Response ◽  
Vessel Diameter ◽  
Tissue Growth ◽  
In Vivo Models

Abstract Ex vivo systems represent important models to study vascular biology and to test medical devices, combining the advantages of in vitro and in vivo models such as controllability of parameters and the presence of biological response, respectively. The aim of this study was to develop a comprehensive ex vivo vascular bioreactor to long-term culture and study the behavior of native blood vessels under physiologically relevant conditions. The system was designed to allow for physiological mechanical loading in terms of pulsatile hemodynamics, shear stress, and longitudinal prestretch and ultrasound imaging for vessel diameter and morphology evaluation. In this first experience, porcine carotid arteries (n = 4) from slaughterhouse animals were cultured in the platform for 10 days at physiological temperature, CO2 and humidity using medium with blood-mimicking viscosity, components, and stability of composition. As expected, a significant increase in vessel diameter was observed during culture. Flow rate was adjusted according to diameter values to reproduce and maintain physiological shear stress, while pressure was kept physiological. Ultrasound imaging showed that the morphology and structure of cultured arteries were comparable to in vivo. Histological analyses showed preserved endothelium and extracellular matrix and neointimal tissue growth over 10 days of culture. In conclusion, we have developed a comprehensive pulsatile system in which a native blood vessel can be cultured under physiological conditions. The present model represents a significant step toward ex vivo testing of vascular therapies, devices, drug interaction, and as basis for further model developments.

scholarly journals How Can Biomolecules Improve Mucoadhesion of Oral Insulin? A Comprehensive Insight using Ex-Vivo, In Silico, and In Vivo Models

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 675 ◽  
Author(s):  
Mariana Amaral ◽  
Ana Sofia Martins ◽  
José Catarino ◽  
Pedro Faísca ◽  
Pradeep Kumar ◽  
...  
Keyword(s):  
In Silico ◽  
Ex Vivo ◽  
Polymeric Nanoparticles ◽  
Spherical Shape ◽  
Diabetic Rats ◽  
Laser Scattering ◽  
In Vivo Models ◽  
Mean Size

Currently, insulin can only be administered through the subcutaneous route. Due to the flaws associated with this route, it is of interest to orally deliver this drug. However, insulin delivered orally has several barriers to overcome as it is degraded by the stomach’s low pH, enzymatic content, and poor absorption in the gastrointestinal tract. Polymers with marine source like chitosan are commonly used in nanotechnology and drug delivery due to their biocompatibility and special features. This work focuses on the preparation and characterization of mucoadhesive insulin-loaded polymeric nanoparticles. Results showed a suitable mean size for oral administration (<600 nm by dynamic laser scattering), spherical shape, encapsulation efficiency (59.8%), and high recovery yield (80.6%). Circular dichroism spectroscopy demonstrated that protein retained its secondary structure after encapsulation. Moreover, the mucoadhesive potential of the nanoparticles was assessed in silico and the results, corroborated with ex-vivo experiments, showed that using chitosan strongly increases mucoadhesion. Besides, in vitro and in vivo safety assessment of the final formulation were performed, showing no toxicity. Lastly, the insulin-loaded nanoparticles were effective in reducing diabetic rats’ glycemia. Overall, the coating of insulin-loaded nanoparticles with chitosan represents a potentially safe and promising approach to protect insulin and enhance peroral delivery.

scholarly journals Bcl-2 overexpression in type II epithelial cells does not prevent hyperoxia-induced acute lung injury in mice

2010 ◽  
Vol 299 (3) ◽  
pp. L312-L322 ◽  
Author(s):  
Isabelle Métrailler-Ruchonnet ◽  
Alessandra Pagano ◽  
Stéphanie Carnesecchi ◽  
Karim Khatib ◽  
Pedro Herrera ◽  
...  
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Lung Injury ◽  
Ex Vivo ◽  
Lung Damage ◽  
Type Ii Cells ◽  
Type Ii ◽  
Lung Weight

Bcl-2 is an anti-apoptotic molecule preventing oxidative stress damage and cell death. We have previously shown that Bcl-2 is able to prevent hyperoxia-induced cell death when overexpressed in a murine fibrosarcoma cell line L929. We hypothesized that its specific overexpression in pulmonary epithelial type II cells could prevent hyperoxia-induced lung injury by protecting the epithelial side of the alveolo-capillary barrier. In the present work, we first showed that in vitro Bcl-2 can rescue murine pulmonary epithelial cells (MLE12) from oxygen-induced cell apoptosis, as shown by analysis of LDH release, annexin V/propidium staining, and caspase-3 activity. We then generated transgenic mice overexpressing specifically Bcl-2 in lung epithelial type II cells under surfactant protein C (SP-C) promoter (Tg-Bcl-2) and exposed them to hyperoxia. Bcl-2 did not hinder hyperoxia-induced mitochondria and DNA oxidative damage of type II cell in vivo. Accordingly, lung damage was identical in both Tg-Bcl-2 and littermate mice strains, as measured by lung weight, bronchoalveolar lavage, and protein content. Nevertheless, we observed a significant lower number of TUNEL-positive cells in type II cells isolated from Tg-Bcl-2 mice exposed to hyperoxia compared with cells isolated from littermate mice. In summary, these results show that although Bcl-2 overexpression is able to prevent hyperoxia-induced cell death at single cell level in vitro and ex vivo, it is not sufficient to prevent cell death of parenchymal cells and to protect the lung from acute damage in mice.

scholarly journals Cross-talk between LAM cells and fibroblasts may influence alveolar epithelial cell behavior in Lymphangioleiomyomatosis

2021 ◽  
Author(s):  
Debbie Clements ◽  
Suzanne Miller ◽  
Roya Babaei-Jadidi ◽  
Mike Adam ◽  
S. Steven Potter ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cross Talk ◽  
Ex Vivo ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Dependent Manner ◽  
Epithelial Migration ◽  
Alveolar Epithelial

Lymphangioleiomyomatosis (LAM) is a female specific cystic lung disease in which TSC2 deficient LAM cells, LAM-Associated Fibroblasts (LAFs) and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial cells (AT2 cells). We hypothesised that the behaviour of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared to parenchymal AT2 cells, demonstrated by increased Ki67 expression. Further, nodular AT2 cells express proteins associated with epithelial activation in other disease states including Matrix Metalloproteinase 7, and Fibroblast Growth Factor 7 (FGF7). In vitro, LAF conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, which is a potential mediator of fibroblast-epithelial crosstalk, in an mTOR dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and that fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behaviour. Fibroblast-derived FGF7 may contribute to the cross-talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

scholarly journals Impact of Bicarbonate-β-Lactam Exposures on Methicillin-Resistant Staphylococcus aureus (MRSA) Gene Expression in Bicarbonate-β-Lactam-Responsive vs. Non-Responsive Strains

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1650
Author(s):  
Selvi C. Ersoy ◽  
Blake M. Hanson ◽  
Richard A. Proctor ◽  
Cesar A. Arias ◽  
Truc T. Tran ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Ex Vivo ◽  
In Vivo Models ◽  
Methicillin Resistant ◽  
Genetic Perturbations ◽  
Functional Relevance

Methicillin-resistant Staphylococcus aureus (MRSA) infections represent a difficult clinical treatment issue. Recently, a novel phenotype was discovered amongst selected MRSA which exhibited enhanced β-lactam susceptibility in vitro in the presence of NaHCO3 (termed ‘NaHCO3-responsiveness’). This increased β-lactam susceptibility phenotype has been verified in both ex vivo and in vivo models. Mechanistic studies to-date have implicated NaHCO3-mediated repression of genes involved in the production, as well as maturation, of the alternative penicillin-binding protein (PBP) 2a, a necessary component of MRSA β-lactam resistance. Herein, we utilized RNA-sequencing (RNA-seq) to identify genes that were differentially expressed in NaHCO3-responsive (MRSA 11/11) vs. non-responsive (COL) strains, in the presence vs. absence of NaHCO3-β-lactam co-exposures. These investigations revealed that NaHCO3 selectively repressed the expression of a cadre of genes in strain 11/11 known to be a part of the sigB-sarA-agr regulon, as well as a number of genes involved in the anchoring of cell wall proteins in MRSA. Moreover, several genes related to autolysis, cell division, and cell wall biosynthesis/remodeling, were also selectively impacted by NaHCO3-OXA exposure in the NaHCO3-responsive strain MRSA 11/11. These outcomes provide an important framework for further studies to mechanistically verify the functional relevance of these genetic perturbations to the NaHCO3-responsiveness phenotype in MRSA.

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