scholarly journals NADPH Oxidase 5 and Melatonin: Involvement in Ram Sperm Capacitation

2021 ◽  
Vol 9 ◽  
Author(s):  
Sara Miguel-Jiménez ◽  
Blanca Pina-Beltrán ◽  
Silvia Gimeno-Martos ◽  
Melissa Carvajal-Serna ◽  
Adriana Casao ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Sperm Capacitation ◽  
Mammalian Sperm ◽  
Ram Sperm ◽  
Mammalian Spermatozoa ◽  
Nadph Oxidase 5

Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O2⋅–), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O2⋅– and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets

2007 ◽  
Vol 97 (03) ◽  
pp. 425-434 ◽  
Author(s):  
Dmitry Kireev ◽  
Nadezhda Popenko ◽  
Aleksei Pichugin ◽  
Mikhail Panteleev ◽  
Olga Krymskaya ◽  
...  
Keyword(s):  
Blood Platelets ◽  
Calcium Ionophore ◽  
Coagulation Factors ◽  
In Vitro Models ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Factor X ◽  
Activated Platelets

SummaryPlatelet microparticles (PMPs) are small vesicles released from blood platelets upon activation. The procoagulant activity of PMPs has been previously mainly characterized by theirability to bind coagulation factors VIII and Va in reconstructed systems. It can be supposed that PMPs can contribute to the development of thrombotic complications in the pathologic states associated with the increase of their blood concentration. In this study we compared procoagulant properties of calcium ionophore A23187-activated platelets and PMPs using several in-vitro models of hemostasis. Surface densities of phosphatidylserine, CD61, CD62P and factor X bound per surface area unit were determined by flow cytometry. They were 2.7-, 8.4-, 4.3-, and 13-fold higher for PMPs than for activated platelets, respectively. Spatial clot growth rate (Vclot) in the reaction-diffus ion experimental model and endogenous thrombin potential (ETP) were determined in plasma, which was depleted of phospholipid cell surfaces by ultra-centrifugation and supplemented with activated platelets or PMPs at different concentrations. Both Vcllot and ETP rapidly increased with the increase of PMP or platelet concentration until saturation was reached. The plateau values of Vclot and ETP for activated platelets and PMPs were similar. In both assays, the procoagulant activity of one PMP was almost equal to that of one activated platelet despite at least two-orders-of-magnitude difference in their surface areas. This suggests that the PMP surface is approximately 50- to 100-fold more procoagulant than the surface of activated platelets.

scholarly journals MiR-302e attenuates allergic inflammation in vitro model by targeting RelA

2018 ◽  
Vol 38 (3) ◽  
Author(s):  
Lifeng Xiao ◽  
Li Jiang ◽  
Qi Hu ◽  
Yuru Li
Keyword(s):  
Inflammatory Cytokines ◽  
Luciferase Activity ◽  
Allergic Inflammation ◽  
Calcium Ionophore ◽  
Allergic Diseases ◽  
Calcium Ionophore A23187 ◽  
Human Mast Cell ◽  
Ionophore A23187 ◽  
Down Regulation

Allergic inflammation is the foundation of allergic rhinitis and asthma. Although microRNAs are implicated in the pathogenesis of various diseases, information regarding the functional role of microRNAs in allergic diseases is limited. Herein, we reported that microRNA-302e (miR-302e) serves as an important regulator of allergic inflammation in human mast cell line, HMC-1 cells. Our results showed that miR-302e is the dominant member of miR-302 family expressed in HMC-1 cells. Moreover, the expression of miR-302e was significantly decreased in response to phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 or ovalbumin (OVA) stimulation. Overexpression of miR-302e blocked PMA/A23187 or OVA induced the increase in inflammatory cytokines levels, such as IL-1β, IL-6, tumor necrosis factor (TNF)-α and thymic stromal lymphopoietin, while miR-302 inhibition further promoted the release of these cytokines. Mechanistically, we found that miR-302e is a novel miRNA that targets RelA, a gene known to be involved in regulating inflammation, through binding to the 3′-UTR of RelA mRNA. Ectopic miR-302e remarkably suppressed the luciferase activity and expression of RelA, whereas down-regulation of miR-302e increased RelA luciferase activity and expression. Pharmacological inhibition of NF-κB reversed the augmented effect of miR-302e down-regulation on inflammatory cytokines level. Taken together, the present study demonstrates miR-302e limits allergic inflammation through inhibition of NF-κB activation, suggesting miR-302e may play an anti-inflammatory role in allergic diseases and function as a novel therapeutic target for the treatment of these diseases.

scholarly journals Prevention of calpain-dependent degradation of STK38 by MEKK2-mediated phosphorylation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Atsushi Enomoto ◽  
Takemichi Fukasawa ◽  
Hiroki Tsumoto ◽  
Masataka Karube ◽  
Keiichi Nakagawa ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Terminal Region ◽  
Calpain Inhibitor ◽  
Ionophore A23187 ◽  
Serine Threonine Kinase ◽  
Defective Mutant ◽  
Cleavage Assay

Abstract Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-family implicated in the regulation of cell division and morphogenesis. However, the molecular mechanisms underlying STK38 stability remain largely unknown. Here, we show that treatment of cells with either heat or the calcium ionophore A23187 induced STK38 degradation. The calpain inhibitor calpeptin suppressed hyperthermia-induced degradation or the appearance of A23187-induced cleaved form of STK38. An in vitro cleavage assay was then used to demonstrate that calpain I directly cleaves STK38 at the proximal N-terminal region. Deletion of the N-terminal region of STK38 increased its stability against hyperthermia. We further demonstrated that the MAPKK kinase (MAP3K) MEKK2 prevented both heat- and calpain-induced cleavage of STK38. MEKK2 knockdown enhanced hyperthermia-induced degradation of STK38. We performed an in vitro MEKK2 assay and identified the key regulatory site in STK38 phosphorylated by MEKK2. Experiments with a phosphorylation-defective mutant demonstrated that phosphorylation of Ser 91 is important for STK38 stability, as the enzyme is susceptible to degradation by the calpain pathway unless this residue is phosphorylated. In summary, we demonstrated that STK38 is a calpain substrate and revealed a novel role of MEKK2 in the process of STK38 degradation by calpain.

scholarly journals Tulathromycin Exerts Proresolving Effects in Bovine Neutrophils by Inhibiting Phospholipases and Altering Leukotriene B4, Prostaglandin E2, and Lipoxin A4Production

2014 ◽  
Vol 58 (8) ◽  
pp. 4298-4307 ◽  
Author(s):  
Carrie D. Fischer ◽  
Stephanie C. Duquette ◽  
Bernard S. Renaux ◽  
Troy D. Feener ◽  
Douglas W. Morck ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Lipid Signaling ◽  
Prostaglandin E ◽  
Ionophore A23187 ◽  
Proinflammatory Mediators ◽  
Bovine Neutrophils

ABSTRACTThe accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4(LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4[LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytesin vitroand inMannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammationin vivoand characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4and prostaglandin E2(PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2(PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

In vitro induction of the acrosome reaction in ovine spermatozoa by calcium ionophore A23187

2003 ◽  
Vol 51 (1) ◽  
pp. 103-109 ◽  
Author(s):  
Ö. Uçar ◽  
T. J. Parkinson
Keyword(s):  
Phase Contrast ◽  
Acrosome Reaction ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Contrast Microscopy ◽  
The Relationship ◽  
In Vitro Induction ◽  
Artificial Vagina

The relationship between concentration of calcium ionophore A23187 and incubation time upon the proportion of spermatozoa undergoing acrosome reaction (AR) in vitro was investigated in rams from a commercial artificial insemination (AI) program. Two ejaculates were collected by artificial vagina from each of nine rams of three breeds (Finn Dorset, Charolais and Suffolk) aged 8-36months. Each ejaculate was diluted in a skimmed milk extender. Spermatozoa were thereafter incubated for 45 or 60min in modified Tyrode's medium (TALP) which contained either zero, 0.1 or 1.0µM/l A23187. After fixing in 10% formaldehyde, the number of spermatozoa that had undergone AR was determined by phase contrast microscopy. In pre-incubation samples, 21.3± 3.3% of spermatozoa had undergone AR. Percentages of acrosome reacted spermatozoa were significantly (P<0.001) increased after incubation with A23187. After incubation with 0.1µM/l A23187 for 45 and 60min there were 22.4±3.0% and 31.7±4.3% acrosome reacted spermatozoa, respectively. After incubation with 1.0µM/l A23187 for 45 and 60min there were 46.2±6.5% and 53.8±5.9% acrosome reacted spermatozoa, whilst corresponding numbers in control samples were 17.0±2.7% and 22.3±4.2%. There was also a significant (P<0.001) effect of individual animals upon the responses to different concentrations of A23187. These findings indicate that (i) A23187 can be used to assess the AR of ovine spermatozoa in vitro and (ii) there are effects of individual animals upon the proportion of spermatozoa undergoing AR.

Calcium-independent stimulation of glycogenolysis by arginine vasotocin and catecholamines in liver of the axolotl (Ambystoma mexicanum) in vitro

1986 ◽  
Vol 109 (1) ◽  
pp. 75-84 ◽  
Author(s):  
P. A. Janssens ◽  
J. Kleineke ◽  
A. G. Caine
Keyword(s):  
Organ Culture ◽  
Calcium Ionophore ◽  
Phosphodiesterase Inhibitors ◽  
Calcium Ionophore A23187 ◽  
Ambystoma Mexicanum ◽  
Arginine Vasotocin ◽  
Maximal Response ◽  
Ionophore A23187 ◽  
Glucose Release

ABSTRACT Arginine vasotocin (AVT) caused a concentrationdependent increase of glycogen phosphorylase a activity, breakdown of glycogen and release of glucose, when added to pieces of axolotl liver in organ culture. The concentration causing half-maximal response (EC50) was about 1 nmol/l. These actions of AVT were unaffected by the adrenergic antagonists propranolol, yohimbine and prazosin, but were blocked by equimolar amounts of d(CH2)5Tyr(Me)AVT, a synthetic antagonist of vasopressin. Arginine vasotocin similarly caused glycogenolysis in isolated perfused axolotl liver where the EC50 was about 0·1 nmol/l. The glycogenolytic action of AVT (10 nmol/l) was sustained for at least 3 h in Ca2+ -free perfusion and longer in organ culture. No increase in Ca2+ concentration in the effluent perfusion medium was apparent during AVT-induced glucose release. Omission of Ca2+ from the medium, together with addition of EGTA (2·5 mmol/l) to the organ culture, had only a slight inhibitory effect upon the rate of glycogenolysis brought about by AVT and did not inhibit the glycogenolytic action of catecholamines. Addition of the calcium ionophore A23187 (5 μmol/l) neither caused glucose release nor abolished the glycogenolytic action of AVT added subsequently. Nevertheless, A23187 caused increased loss of 45Ca from Ca2+-loaded liver pieces whereas AVT was without effect. There was a slight accumulation of cyclic AMP (cAMP), but not cGMP, in axolotl liver pieces cultured in the presence of 0·1 μmol AVT/l and this was accentuated in the presence of phosphodiesterase inhibitors. We conclude that, in contrast to the position in mammals, Ca2+ is not involved in the glycogenolytic actions of AVT or catecholamines in axolotl liver. Preliminary experiments suggest that the same is true in the carp and we suggest that the involvement of Ca2+ in regulation of hepatic glucose release may not have evolved until after the amphibians separated from the ancestors of the mammals. J. Endocr. (1986) 109, 75–84

Production of transgenic porcine blastocysts by hand-made cloning

2004 ◽  
Vol 16 (3) ◽  
pp. 315 ◽  
Author(s):  
P. M. Kragh ◽  
G. Vajta ◽  
T. J. Corydon ◽  
S. Purup ◽  
L. Bolund ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Transfer ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Reconstructed Embryos ◽  
Green Fluorescent

Recently, a zona-free technique for bovine somatic cell nuclear transfer (NT) with no requirement for micromanipulation (i.e. hand-made cloning (HMC)) has been described. The present study demonstrates the application of the HMC technique in the production of transgenic porcine blastocysts. In vitro-matured zona-free porcine oocytes were bisected manually using a microblade and halves containing no chromatin (i.e. the cytoplasts) were selected. Two cytoplasts were electrofused with one transgenic fibroblast expressing enhanced green fluorescent protein and reconstructed embryos were activated in calcium ionophore (A23187) followed by 6-dimethylaminopurine. Subsequently, embryos were cultured in NCSU-23 medium supplemented with 4 mg mL–1 bovine serum albumin for 7 days. In five replicates, 93.0 ± 7.0% (mean ± s.e.m.) of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, 37/37 and 28/28). On Day 7 after activation, the respective blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), 7% (1/15), 7% (2/28), 3% (1/37) and 7% (2/28), resulting in an average of 6.0 ± 0.8%. Enhanced green fluorescent protein was expressed in all cells of all eight developing blastocysts. Efforts are now directed towards the production of offspring from such transgenic NT blastocysts.

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