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.

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 Human immune interferon: induction in lymphoid cells by a calcium ionophore

1980 ◽  
Vol 29 (2) ◽  
pp. 561-563
Author(s):  
F Dianzani ◽  
T M Monahan ◽  
J Georgiades ◽  
J B Alperin
Keyword(s):  
Calcium Ionophore ◽  
Maximum Level ◽  
Calcium Ionophore A23187 ◽  
Lymphoid Cells ◽  
Ionophore A23187 ◽  
Immune Interferon ◽  
Slow Kinetics ◽  
Lymphocyte Cultures ◽  
Human Immune ◽  
Kinetics Of

Human lymphocyte cultures produced interferon after treatment with the calcium ionophore A23187, but not after treatment with potassium ionophore nonactin. Interferon production was detectable between 3 and 6 h after ionophore treatment and reached the maximum level between 12 and 24 h. Ionophore-induced interferon appeared to be immune interferon on the basis of acid lability, lack of neutralization by antibody to leukocyte interferon, slow kinetics of activation of the antiviral state, and molecular characterization by isoelectrofocusing.

245 ANALYSIS OF CORTICAL GRANULE EXUDATE OBTAINED BY CHEMICAL OOCYTE ACTIVATION IN PIGS

2011 ◽  
Vol 23 (1) ◽  
pp. 221
Author(s):  
R. Romar ◽  
M. J. Izquierdo-Rico ◽  
H. Funahashi
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Cortical Granule ◽  
Ionophore A23187 ◽  
Oocyte Activation ◽  
Cortical Granules ◽  
Isolation And Identification

Cortical granules (CG) are clue organelles in the mammalian oocyte because once released, their content modifies the zona pellucida (ZP) and oolema, thus preventing polyspermy. However, research on putative CG proteins has progressed slowly because of the picogram amount of proteins contained in CG. Isolation and identification of CG contents in porcine oocytes would help to elucidate the molecular mechanism involved in blocking polyspermic fertilization. Our objective was to study the contents of CG from in vitro-matured (IVM) porcine oocytes, and to achieve this objective, CG exudate was collected after its release from chemically activated oocytes. Oocytes were subjected to IVM in porcine oocyte medium supplemented with 50 μM β-mercaptoethanol for 44 h. After the IVM period, the ZP was removed by protease treatment (0.5% pronase in PBS), and the ZP-free oocytes were activated with calcium ionophore A23187 (6.5 μM, 2 min) in a medium consisting of 114.06 mM NaCl, 3.20 mM KCl, 0.50 mM MgCl2·6H2O, 10.00 mM sodium lactate, 0.35 mM NaH2PO4, 5.00 mM glucose, 25.07 mM NaHCO3, and 8.00 mM calcium lactate·5H2O. After activation, oocytes were transferred to fresh medium without calcium ionophore and kept for 30 min to allow release of the CG content. After this time, medium containing the CG exudate was collected, as well as the activated oocytes, and both samples were stored at –80°C until analysis. Samples were thawed and the CG proteins were concentrated by centrifugation in 10-kDa centrifugal devices (Microcon, Millipore, Billerica, MA) following the manufacturer’s instructions. The CG exudates from activated oocytes (n = 300) and activated oocytes (n = 125) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. In brief, 4% stacking and 12% separating gel was used and run using 25 mM Tris–0.2 M glycine buffer, pH 8.6, containing 0.1% SDS for 1.5 h at 150 V and room temperature. After electrophoresis, the gel was silver stained. Thirteen strong bands were identified in the CG exudate lane, with an approximate molecular mass from approximately 45 to 105 kDa. However, the lane for activated oocytes showed faint protein bands. The presence of well-defined bands in the CG exudate lane might correspond to different CG-derived proteins. These preliminary results show a new approach for studying CG content. Further proteomic analysis of the bands will help to describe specific proteins contained in these organelles, shedding light on the role of the cortical reaction in pigs. Supported by MEC and FEDER (AGL2009-12512-C02-01) and Okayama Universit R. R. was granted funding by JSPS (Ref. S-09210).

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

182 CHEMICAL ACTIVATION OF ZONA PELLUCIDA-FREE OOCYTES PROVOKES FULL CORTICAL REACTION: AN APPROACH TO STUDY CORTICAL GRANULE-DERIVED PROTEINS IN PIGS

2012 ◽  
Vol 24 (1) ◽  
pp. 203
Author(s):  
M. D. Saavedra ◽  
R. Romar ◽  
H. González-Márquez ◽  
Y. Ducolomb ◽  
R. Fierro ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Chemical Activation ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Cortical Granule ◽  
Ionophore A23187 ◽  
Peanut Agglutinin ◽  
Cortical Granules ◽  
Cortical Reaction ◽  
The Mean

The cortical reaction is a mechanism that prevents polyspermy by cortical granule content being released into the periviteline space, modifying the zona pellucida (ZP). Knowledge about specific cortical granule-derived proteins has progressed slowly because these organelles contain only picogram quantities of proteins. An efficient method for collecting cortical granule content would help in its study; chemical activation of ZP-free oocytes has been successfully used in the murine model (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Calcium ionophore A23187 is an effective chemical stimulator for provoking the cortical reaction in ZP-intact pig oocytes. However, the commonly used protocol (50 μM for 5min) cannot be employed with ZP-free oocytes because the oolemma is damaged, oocyte lysed and medium contaminated with ooplasm content, which is necessary to reduce the time and ionophore concentration (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst). The objective of this study was to evaluate the efficiency of this activation protocol for provoking the cortical reaction in ZP-free oocytes by assessment with confocal and electron microscopy. Immature cumulus–oocyte complexes from Landrace × Large White gilts were in vitro matured for 44 h in an NCSU-37 medium. After maturation, the oocytes were stripped of cumulus cells and their ZP were removed with pronase. Then, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM for 2min), transferred to an exudate medium and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. Control ZP-free oocytes were incubated without being activated. After incubation, ionophore-treated (n = 10) and control oocytes (n = 18) were used to assess the presence of a cortical granule monolayer. An aliquot was fixed, permeabilized (0.1% Triton), incubated with peanut agglutinin lectin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min) and examined under a confocal microscope. Presence or absence of a cortical granule monolayer at the equator level was recorded. Another aliquot was fixed and processed for electron microscopy observation. The cortical granules in the whole oocytes were counted and results are presented as the mean ± standard error of the mean. No cell lysis was observed in control or activated ZP-free oocytes after treatment and incubation time. The confocal study showed that the activation protocol provokes a full cortical reaction in 100% of A23187-treated oocytes, given that no peanut agglutinin labeling was observed in the cortical area. Presence of a cortical granule monolayer under the oolemma was observed in 100% of control oocytes. Cortical granule release was confirmed by electron microscopy. Control oocytes had 5.90 ± 1.78 cortical granules per 5 μm of oolemma, whereas activated oocytes exhibited a significant reduction (P < 0.05) of up to 0.71 ± 0.20. In conclusion, the presented activation protocol by using ZP-free oocytes is a valid method for provoking a complete cortical reaction and could be employed in the future as an efficient method to collect cortical granule-derived proteins in pig oocytes. Supported by CONACYT (0105961/I0110/194/09), MEC and FEDER (AGL2009-12512-C02-01).

Platelets from Bleeding Simmental Cattle Have a Long Delay in both ADP-activated Expression of GpllB-IIIA Receptors and Fibrinogen-dependent Platelet Aggregation

1996 ◽  
Vol 76 (06) ◽  
pp. 1047-1052 ◽  
Author(s):  
Mony M Frojmovic ◽  
Truman Wong ◽  
Gene P Searcy
Keyword(s):  
Platelet Aggregation ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Receptor Expression ◽  
Ionophore A23187 ◽  
Activated Platelets ◽  
Simmental Cattle ◽  
Adhesive Proteins ◽  
Kinetics Of

SummaryWe have previously reported that platelets from bleeding Simmental cattle do not aggregate in vitro in response to ADP, collagen and calcium ionophore A23187, though calcium mobilization and myosin light chain phosphorylation do occur. The aggregation abnormality, measured by aggregometry, was ascribed to abnormal cytoskeletal expression, with the maximal numbers of activated GpIIb-IIIa receptors per platelet no different from that seen in normal bovine platelets activated with ADP. We have therefore sought to compare the kinetics of microaggregation with the rate of expression of GpIIb-IIIa receptors required for mediating fibrinogen (Fg)-dependent platelet aggregation, to provide a more direct molecular explanation for the aggregation abnormality. We compared aggregation kinetics of ADP-activated platelets using both aggregometry and particle counting to monitor microaggregation. Fibrinogen receptor expression was monitored with FITC-labelled human Fg and with the reporting antibody for activated GpIIb-IIIa, FITC-PAC1, using flow cytometry. The affected platelets show a marked delay in onset of microaggregation for ADP-activated platelets stirred with human Fg, paralleded by an unusual delay in activated GpIIb-IIIa receptor expression (DARE) for otherwise competent Fg binding. The on-rates for Fg binding to platelets maximally pre-activat-ed with PMA are identical for normal and affected platelets, whether comparing the binding of human or bovine Fg. The unique DARE syndrome explains the observed delay in aggregation of platelets from affected Simmental cattle and predicts the bleeding problems due to delayed binding of adhesive proteins.

scholarly journals Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils

1986 ◽  
Vol 239 (3) ◽  
pp. 723-731 ◽  
Author(s):  
P G Heyworth ◽  
A W Segal
Keyword(s):  
Respiratory Burst ◽  
Protein A ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Receptor Signal ◽  
Latex Beads ◽  
Respiratory Burst Oxidase ◽  
Kinetics Of

Phosphorylation of a 47 kDa protein in human neutrophils is induced by phorbol 12-myristate 13-acetate (PMA), opsonized latex beads, fMet-Leu-Phe, calcium ionophore A23187 and fluoride. All of these stimuli activate the specialized microbicidal respiratory burst of neutrophils, and in each case the kinetics of activation correspond with the kinetics of phosphorylation of the 47 kDa protein. Trifluoperazine (50 microM) and chlorpromazine (100 microM), inhibitors of calmodulin and protein kinase C, abolish the increase in oxygen consumption and selectively prevent phosphorylation of the 47 kDa protein after PMA stimulation. Treatment of neutrophils with pertussis toxin totally inhibits both superoxide production and phosphorylation of this protein in response to fMet-Leu-Phe, but not in response to PMA, indicating that a GTP-binding protein modulates the fMet-Leu-Phe receptor signal. Phosphorylation of the 47 kDa protein, a phenomenon absent from the neutrophils of subjects with autosomal recessive chronic granulomatous disease, which lack the respiratory burst, appears to be the common trigger for activation of the burst in normal neutrophils.

scholarly journals 5MPF AND MAP KINASES IN OVINE OOCYTES: EFFECTS OF ENUCLEATION AND CAFFEINE ON ACTIVITY AND DEVELOPMENT OF NUCLEAR TRANSFER EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 125 ◽  
Author(s):  
J-H. Lee ◽  
K.H.S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Map Kinases ◽  
Chromatin Condensation ◽  
Calcium Ionophore ◽  
Cumulus Cells ◽  
Calcium Ionophore A23187 ◽  
Meiotic Spindle ◽  
Ionophore A23187 ◽  
Chi Square ◽  
Chi Square Test

In nuclear transfer (NT) embryos, exposure of the donor chromatin to the MII cytoplasm results in premature chromatin condensation (PCC) which may be beneficial for nuclear reprogramming (Campbell KHS and Alberio R 2003 Reprod. Suppl. 61, 477–494). Following enucleation, maturation promoting factor (MPF) activity in murine oocytes is primarily associated with the meiotic spindle. This reduced MPF activity in the cytoplast may result in decreased PCC and reprogramming. Conversely, increasing cytoplast MPF activity may increase reprogramming. The aims of this study were to perform quantitative analysis of MPF and MAPK activities in ovine oocytes: 1. at anaphase/telophase I (A/TI) or MII; 2. following enucleation; 3. following treatment with caffeine (an inhibitor of Myt1/Wee1 activity). The development of ovine NT embryos reconstructed using caffeine-treated oocytes as cytoplast recipients was then determined. Oocytes were matured in TCM 199, 10% FBS, 5μgmL−1 FSH, 5μgmL−1 LH,1μgmL−1 estradiol, 0.3mM sodium pyruvate and 100μM cysteamine. 15h post-onset of maturation (hpm) oocytes were stripped of cumulus cells and enucleated in HSOF containing 5μgmL−1 Hoechst 33342 and 7.5μgmL−1 cytochalasin B (CB). Control oocytes were sham-enucleated by removing an equal volume of cytoplasm. Oocytes were cultured in SOF±10mM caffeine. Groups of 10 oocytes were sampled and analyzed for MPF and MAPK activities as previously described (Ye JP et al., 2003 Reproduction 125, 645–656). For NT, primary foetal fibroblasts were quiesced in DMEM containing 0.1% FBS for 2–3 days. Cell fusion was induced with two DC pulses of 25VcM-1 for 80μs. 3 methods of NT were compared: A. fusion 20hpm, activation 21hpm; B. fusion 24hpm, activation 25hpm; C. 10mM caffeine 18–24hpm, fusion 24hpm, activation 25hpm. All oocytes were activated in HSOF containing 5μgmL−1 calcium ionophore (A23187), cultured in SOF with 10μgmL−1 of cycloheximide and 7.5μgmL−1 CB for 5h, and then transferred to mSOFaaBSA medium, all at 5% CO2, 5% O2 and 90% N2 at 39°C. On Day 2 cleavage was assessed and 10% FBS added to the medium. Development to blastocyst was assessed on Day 7. All data were analyzed by chi-square test. Both MPF and MAP kinase activities were increased at MII compared to A/TI (P&lt;0.05). There were no differences in activities of both kinases between intact and enucleated oocytes. Following enucleation, both kinase activities were identical in all groups, reaching maximum activities 24hpm followed by a slow decline. Caffeine increased the activity of both kinases (MPF in particular) in all groups. Following 5 replicates (total oocytes 145, 143, 144 for NT methods A,B,C, respectively), no significant differences were observed between fusion (82.1%, 67.8%, 67.4%), cleavage (90.8%, 88.7%, 89.7%) or development to blastocyst (20.2%, 18.6%, 25.8%). Analysis of total cell numbers on limited numbers of blastocysts (7, 6, 7) were NS(70.9±38.5, 69.3±25.4, 93.3±17.8).

Growth factors and the primary cilium in BALB/c 3T3 cells

1987 ◽  
Vol 45 ◽  
pp. 830-831
Author(s):  
R. W. Tucker ◽  
N. S. More ◽  
S. Jayaraman
Keyword(s):  
Growth Factors ◽  
Primary Cilium ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Calcium Ionophore ◽  
Growth Stimulation ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
3T3 Cells ◽  
Microtubule Depolymerization

The mechanisms by which polypeptide growth factors Induce DNA synthesis in cultured cells is not understood, but morphological changes Induced by growth factors have been used as clues to Intracellular messengers responsible for growth stimulation. One such morphological change has been the transient disappearance of the primary cilium, a “9 + 0” cilium formed by the perinuclear centriole in interphase cells. Since calcium ionophore A23187 also produced both mitogenesis and ciliary changes, microtubule depolymerization might explain ciliary disappearance monitored by indirect immunofluorescence with anti-tubulin antibody. However, complete resorption and subsequent reformation of the primary cilium occurs at mitosis, and might also account for ciliary disappearance induced by growth factors. To settle this issue, we investigated the ultrastructure of the primary cilium using serial thin-section electron microscopy of quiescent BALB/c 3T3 cells before and after stimulation with serum.

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