Effect of glucose in the culture medium on development of horse oocytes matured and microfertilized in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1067 ◽  
Author(s):  
T Azuma ◽  
YH Choi ◽  
S Hochi ◽  
N Oguri
Keyword(s):  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cell Stage ◽  
Morula Stage ◽  
Fetal Bovine ◽  
Oviduct Fluid ◽  
Effect Of Glucose

The development of in-vitro matured and microfertilized horse oocytes was examined in vitro. Fertilized oocytes were produced by 20-h insemination of in-vitro matured and partially zona-removed oocytes with frozen spermatozoa that had been treated with caffeine/calcium ionophore A23187 (fertilization rate 34.2%, monospermy rate 76.9%). Embryonic development was assessed by the number of nuclei stained with Giemsa solution. In Experiment 1, a continuous 8-day culture of the microfertilized oocytes in TCM199 or modified synthetic oviduct fluid (m-SOF) supplemented with 10% fetal bovine serum or 0.1% polyvinyl alcohol (PVA) in 5% O2, 5% CO2 and 90% N2 resulted in very few embryos developing beyond the 8-cell stage. In Experiment 2, the effects of different glucose concentrations (0, 0.5, 5.5 mM) in m-SOF/PVA during Days 1-4 and Days 5-8 of culture were examined. Proportions of oocytes having more than one nucleus ranged from 17.7% to 44.7% among the combinations of glucose concentrations. Supplementation with glucose at 0.5 mM during Days 1-4 followed by 5.5 mM during Days 5-8 resulted in the best embryo development; 12/55 (21.8%) nuclei-positive oocytes developed to the 8-16-cell stage, 11 (20.0%) developed to the 17-50-cell stage, and 5 (9.1%) comprised more than 50 cells and were assumed to be at the morula stage.

scholarly journals Parthenogenetic Activation of Porcine Oocytes by Calcium Ionophore A23187

2005 ◽  
Vol 48 (1) ◽  
pp. 60-67 ◽  
Author(s):  
S. Yin ◽  
M. Hishinuma ◽  
K. Hamana ◽  
J. Sekine
Keyword(s):  
Calcium Ionophore ◽  
Developmental Rate ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cleavage Rate ◽  
North Carolina State University ◽  
Activation Rate ◽  
Morula Stage ◽  
Almost All

Abstract. This study was designated to clarify the influence of activation of porcine matured oocytes by calcium ionophore on in vitro development of the parthenotes. The follicular oocytes were matured, activated and cultured in North Carolina State University-23 (NCSU-23) medium supplemented with 10% porcine follicular fluid (pFF). The in vitro-matured oocytes were exposed to calcium ionophore at concentrations of 12.5, 25 or 50 μM for 3, 5, 7 or 9 min. The activation rate of the oocytes increased as concentration of ionophore decreased, being at 27–33 and 68–77 % for the oocytes treated with 50 and 12.5 μM ionophore, respectively. Almost all activated oocytes were haploid. The highest cleavage rate (76%) and developmental rate to morula (41%) were observed in the oocytes treated with 12.5 μM ionophore for 5 min. However, development to blastocyst was observed only in the oocytes treated with 25 μM ionophore for 3 and 5 min (3 and 4% of treated oocytes, respectively). We concluded that the activation treatment of the porcine oocytes with 12.5 μM ionophore for 5 min provided the highest develop-mental rate to morula, but this treatment is not sufficient to overcome a developmental block at the morula stage.

205 CAPACITATION OF STALLION SPERMATOZOA EVALUATED BY FERTILIZATION OF BOVINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 181
Author(s):  
M. R. Hudson ◽  
G. E. Seidel Jr ◽  
E. L. Squires ◽  
B. E. Spizzirri ◽  
D. J. Walker ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Cell Divisions ◽  
Bovine Oocytes

In vitro fertilization in the horse does not work reliably. Several methods of capacitating sperm in other species fail in the horse. The goal of this experiment was to develop a method to capacitate equine spermatozoa using calcium ionophore A23187 or phosphatidylcholine 12 (PC12). We also studied effects of maturing bovine oocytes for 24 or 28 h on fertilizability by capacitated equine sperm, hypothesizing that longer maturation would yield oocytes more easily fertilized by equine spermatozoa. Two sets of bovine oocytes were aspirated from 3 to 8 mm follicles of abattoir ovaries 4 h apart, but fertilized at the same time. On the day of fertilization, semen from 1 of 3 stallions was collected, evaluated, and centrifuged through 33% Percoll to remove seminal plasma. The resultant pellet was extended to 5 × 107 cells mL–1 in M199 containing 0.6% BSA, 2 mm caffeine, and 5 mm CaCl2. Sperm were treated with A23187 (1 or 3 μm) or PC12 (40 or 70 μm) or both A23187 and PC12 (1 μm/40 μm) in 500- μL aliquots. Sperm were incubated at 39°C for 10 min (for A23187 and combination treatments) or 15 min (for PC12 treatments), and then diluted 1:20 for fertilization. Oocytes from each maturation time were fertilized using the same semen preparation for each treatment. Oocytes and sperm were incubated together for 18 h in FCDM in 5% CO2 at 39°C (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596). Presumptive zygotes were cultured for 30 h in CDM-1, vortexed to remove cumulus cells, and evaluated for cleavage. Oocytes were also co-incubated with killed sperm to determine the level of parthenogenesis. Cleaved embryos were stained with orcein to ensure that each cell had a nucleus. Number of cell divisions were recorded as 0 for a 1-cell, 1 for a 2-cell, 1.5 for a 3-cell, etc. More oocytes cleaved after 28 h (18%) than 24 h (14%) maturation (P < 0.01). Sperm of Stallion 1 resulted in higher overall cleavage (24%) than Stallions 2 or 3 (11 and 12%; P < 0.01). Highest cleavage was seen with 28 h maturation and 70 μm PC12 and 3 μm A23187 (27 and 24%, respectively). The most cell divisions were seen with 28 h maturation and 70 μm PC12 (0.48); 28 of the 49 cleaved in this treatment reached ≥4-cell stage. In conclusion, both A23187 and PC12 were able to capacitate equine sperm in a dose-dependent manner as determined from cleavage of bovine oocytes matured for 28 h; maturation for the conventional 24 h was an inferior model for this purpose. Table 1. Mean responses of bovine oocytes fertilized by equine sperm

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.

The appearance of glycoconjugates associated with cortical granule release during mouse fertilization

Development ◽  
1988 ◽  
Vol 102 (3) ◽  
pp. 595-604 ◽  
Author(s):  
S.H. Lee ◽  
K.K. Ahuja ◽  
D.J. Gilburt ◽  
D.G. Whittingham
Keyword(s):  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Meiotic Spindle ◽  
Cortical Granule ◽  
Ionophore A23187 ◽  
Perivitelline Space ◽  
Cell Stage ◽  
Artificial Activation ◽  
First Time ◽  
Kinetics Of

For the first time we have shown with appropriately labelled lectins that fucosyl- and sialyl-rich glycoconjugates are released into the perivitelline space of the mouse oocyte after activation by the fertilizing spermatozoon or artificial activation by the calcium ionophore A23187 or ethanol. The glycoconjugates show a punctate distribution over the oocyte surface except for the microvilli-free area overlying the second meiotic spindle from which they are absent. Their appearance in the perivitelline space is associated with the release of the cortical granule suggesting that they represent part of the cortical granule exudate. Soon after the glycoconjugates appear, they begin to aggregate. The process continues until the beginning of cytokinesis at first cleavage when a single large aggregate is found within the cleavage furrow. Most of the labelled glycoconjugates disappear by the late 2-cell stage and no evidence was found for their presence during the later preimplantation period. This technique is suitable for monitoring the kinetics of the cortical reaction in mammalian oocytes and investigating the importance of the glycoconjugates in early preimplantation period.

Mitogenesis by serum and PDGF is independent of PI degradation and PKC in VSMC

1989 ◽  
Vol 256 (4) ◽  
pp. C886-C892 ◽  
Author(s):  
M. Kihara ◽  
P. J. Robinson ◽  
S. H. Buck ◽  
R. C. Dage
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Growth Control ◽  
Calcium Ionophore ◽  
Rat Aorta ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Pi Turnover ◽  
Fetal Bovine ◽  
20 Nm

Capacities of serum, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) on phosphatidylinositol (PI) degradation and cell growth were compared in cultured vascular smooth muscle cells (VSMC) from rat aorta. The role of protein kinase C (PKC) in growth control was also evaluated using polymixin B, a selective inhibitor of PKC. Both dialyzed and nondialyzed fetal bovine serum (FBS) in concentrations from 2 to 20% stimulated [3H]thymidine incorporation into DNA and cell growth without producing corresponding increases in PI turnover. Moreover, both PDGF (40-160 ng/ml) and FGF (6.25-150 ng/ml) also stimulated mitogenesis, but PDGF was more effective although less potent. Mitogenic amounts of PDGF did not stimulate PI turnover, whereas a maximally mitogenic amount of FGF (50 ng/ml) did produce a slight increase. Polymixin B inhibited PKC activity (IC50, 32 microM) from these cells but failed to suppress DNA synthesis produced by 10% FBS or PDGF (50 ng/ml). However, it did suppress that by FGF (50 ng/ml). Angiotensin II (10(-11)-10(-7) M) and phorbol 12,13-dibutyrate (PDB, 1-20 nM) were not mitogenic in the presence or absence of insulin (10 micrograms/ml) or the calcium ionophore A23187 (0.25-4 microM), under serum-free conditions. Instead, PDB inhibited mitogenesis of cells maintained under 0.2% FBS or stimulated with insulin (10 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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