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).

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).

183 CALRETICULIN, A 60-kDa PROTEIN, IS EXOCYTOSED AFTER CHEMICAL ACTIVATION OF ZONA PELLUCIDA-FREE PIG OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 203
Author(s):  
R. Romar ◽  
M. D. Saavedra ◽  
H. González-Márquez ◽  
Y. Ducolomb ◽  
R. Fierro ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Chemical Activation ◽  
Calcium Ionophore ◽  
Control Group ◽  
Cortical Granule ◽  
Cortical Region ◽  
Oocyte Activation ◽  
Cortical Granules ◽  
Cortical Reaction ◽  
Rabbit Igg

Following gamete membrane fusion or artificial oocyte activation, cortical granules undergo exocytosis and the released content modifies the zona pellucida (ZP), preventing polyspermy. The specific cortical granule-derived proteins responsible for these post-fertilization events are not fully characterized. Calreticulin, a highly conserved ubiquitous protein of 60 kDa, was exocytosed from activated hamster eggs (Muñoz-Gotera et al. 2001 Mol. Reprod. Dev. 60, 405–413). Preliminary results from our laboratory have shown that calreticulin is located in the cortical area of pig oocytes (data not shown). This study was designed to test whether calreticulin is exocytosed after oocyte activation with calcium ionophore. 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 0.5% pronase in Ca2+-free PBS. After washing, the ZP-free oocytes were incubated with calcium ionophore A23187 (6.5 μM) for 2min, transferred to a 100-μL droplet of exudate medium (Romar et al. 2011 Reprod. Fertil. Dev. 23, 221 abst) and incubated at 38.5°C, 5% CO2 and saturated humidity for 30 min. After incubation, the medium containing the oocyte exudate (n = 1000) was carefully aspirated and run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The gel was then electro transferred onto a polyvinylidene fluoride (PVDF) membrane, incubated with an anti-calreticulin rabbit polyclonal antibody (1:1000) and finally conjugated to horseradish peroxidase (1:20 000) for 1 h with a monoclonal anti-rabbit IgG. Membrane visualization was accomplished using the ECL plus method and Typhoon 9410. A control group was performed with exudate collected from non-activated ZP-free oocytes. To verify cortical reaction and calreticulin exocytosis, an aliquot of activated ZP-free oocytes (n = 18) were fixed (3.7% paraformaldehyde for 30 min), permeabilized (0.1% Triton X-100 for 10 min), incubated with anti-calreticulin antibody (1:10 for 1 h) and conjugated to tetramethyl rhodamine isothiocyanate (1:400 for 1 h) with an anti-rabbit IgG. Finally, samples were incubated with peanut agglutinin conjugated to fluorescein isothiocyanate (10 μg mL–1 for 30 min), mounted and examined under a confocal microscope. No statistical analysis was made because the observations were purely qualitative. A Western blot analysis showed an immunoreactive band of ∼60 kDa, consistent with the expected size of calreticulin, in the lane containing the exudate from activated oocytes. No band was observed in the lane with the exudate collected from non-activated oocytes. Observation under confocal microscopy showed no PNA or anti-calreticulin fluorescence in the cortical region, indicating that the activated pig oocytes displayed full cortical reaction and calreticulin exocytosis during incubation time. These results show that calreticulin protein is exocytosed after the chemical activation of ZP-free pig oocytes as well as the disappearance of the cortical granule monolayer. The possible role of calreticulin on preventing polyspermy should be further investigated. Supported by MEC and FEDER (AGL2009-12512-C02-01) and CONACYT (0105961/I0110/194/09).

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.

112 The effect of biological extenders on invitro induction of the acrosome reaction in bovine spermatozoa

2020 ◽  
Vol 32 (2) ◽  
pp. 183
Author(s):  
L. Gatenby ◽  
K. R. Bondioli
Keyword(s):  
Acrosome Reaction ◽  
Fluorescent Microscopy ◽  
Calcium Ionophore ◽  
Fluorescein Isothiocyanate ◽  
Egg Yolk ◽  
Calcium Ionophore A23187 ◽  
Induction Treatment ◽  
Ionophore A23187 ◽  
Frozen Semen ◽  
The Mean

Biological extenders in semen are used to protect the integrity of the sperm cells during cryopreservation. The two most common extenders are egg yolk and milk, and initial experiments describing heparin capacitation for IVF were conducted with semen frozen in egg yolk extender. However, semen of many bulls used for IVF is frozen in milk-based extender, and it is not clear whether this affects the response to heparin capacitation. This study aims to assess whether the use of milk- or egg-based extenders in frozen-thawed bovine semen affects sperm's ability to undergo the acrosome reaction, utilising either the endogenous progesterone pathway or calcium ionophore A23187 to induce the acrosome reaction following heparin capacitation. Frozen semen from 12 bulls (6 using egg yolk citrate-based extender containing 20% egg yolk, and 6 using milk-based extender) was used for no less than 3 replicates each. All semen was commercially processed and cryopreserved using the same methodology. Semen was thawed and separated using a Percoll gradient and centrifugation at 400×g for 10min, then incubated for 4h in a capacitating medium containing heparin (10μgmL−1) at a concentration of 1×106 spermmL−1. Capacitated sperm were then introduced to progesterone (10μM) or A23187 (10 μM) for acrosome induction and stained with fluorescein isothiocyanate-conjugated peanut agglutinin (5μgmL−1). After treatment and staining, the sperm were analysed using flow cytometry to determine the percentage of populations with fluorescent acrosomes, indicating an exposed acrosome and an ongoing acrosome reaction. Fluorescent microscopy was also used to confirm fluorescence in acrosome-reacting samples. Results were analysed as a 2×3 factorial design by ANOVA with extender and acrosome induction treatment as factors. For egg yolk-extended semen, the mean percent of acrosome-reacted sperm was 83.7 (±6.8), 81.8 (±7), and 15.0 (±7.5) for sperm treated with progesterone, A23187, and heparin only, respectively. Compared to milk-extended semen, for which the mean percent acrosome-reacted was 15.0 (±8.2), 14.8 (±7.4), and 12.0 (±6.8) for progesterone, A23187, and heparin only, respectively. Significant differences were observed between extenders, with egg yolk-extended semen having a significantly greater response to acrosome reaction-inducing agents than semen extended with milk-based extenders (P&lt;0.05). Also, it was observed that progesterone induced a similar percentage of acrosome reactions as A23187. Variation between bulls in the response to heparin capacitation has been observed and cannot be eliminated in this study. The difference observed between extenders warrants further study with a split ejaculate design. The possibility that the semen extender used during cryopreservation affects the response to heparin capacitation suggests that this should be accounted for in IVF protocols to increase the efficiency of this technology.

Apoptosis Might Contribute to the Thrombocytopenia in Heparin-Induced Thrombocytopenia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2545-2545
Author(s):  
Tatiana A. Nevzorova ◽  
Elmira R. Mordakhanova ◽  
Anastasia A. Ponomareva ◽  
Izabella A. Andrianova ◽  
Rustem I. Litvinov ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Immune Complexes ◽  
Secretory Granules ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Time Dependent ◽  
Ionophore A23187 ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is a prothrombotic autoimmune complication of heparin therapy. Thrombocytopenia and thrombosis in HIT patients are caused by immune complexes containing pathogenic antibodies against platelet factor 4 (PF4)/glycosaminoglycan complexes. Mechanisms of platelet activation and/or destruction in HIT are not fully understood. Phosphatidylserine expression is a marker of platelet activation that contributes to the procoagulant function. On the other hand, phosphatidylserine expression is generally an early marker of cell apoptosis, which, similarly to other cells, controls platelet life span. The aim of this study was to investigate if apoptosis might play a role in HIT. Gel-filtered normal platelets were incubated for 15 or 60 min with recombinant PF4 (10 µg/ml) and KKO antibodies (50 µg/ml) and studied by electron microscopy and flow cytometry using fluorescently labeled markers of cell activation and apoptosis, such as annexin V, antibodies to CD62P (P-selectin) and MitoTracker DeepRed FM. Platelets and platelet-derived microparticles were identified by flow cytometry using labeled antibodies to CD41 and electron microscopy. Calcium ionophore A23187 (10 µM) was used as a positive control. Incubation of platelets with PF4+KKO caused fast expression of P-selectin on platelets comparable with calcium ionophore A23187 stimulation, suggesting that platelets were fully activated by PF4+KKO within 15 min, when they also started to produce CD41 and annexin-positive microparticles. Activation of platelets with PF4+KKO for 60 minutes led to a further increase in phosphatidylserine expression on their surface, with a time-dependent reduction of mitochondrial membrane potential, which reflects a disturbance of energy metabolism and is characteristic of cell apoptosis. Scanning electron microscopy showed that platelets treated with PF4+KKO or A23187, unlike untreated cells, displayed dramatic morphological changes with a loss of discoid shape, formation of filopodia, and microvesiculation. By transmission electron microscopy, the PF4+KKO-treated platelets had an irregular shape due to formation of plasma membrane invaginations and pseudopodia. Formation of an increasing number of intracellular vacuoles and enlargement of the lumen of the open canalicular system were observed. Some vacuoles contained various inclusions, such as secretory granules, membrane components, and grainy particles. The number of secretory granules in the PF4+KKO-treated cells was dramatically reduced. In all cases, formation of microparticles of various shapes and sizes was observed. These results indicate that the PF4-containing pathogenic immune complexes induce strong and time-dependent platelet activation leading to procoagulant microparticle formation that may contribute to thrombosis. At the same time, the results strongly suggest that the HIT-like immune complexes likely induce platelet apoptosis that can be an important mechanism of thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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.

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.

Effects of glucocorticoids on prostaglandin formation by human amnion

1990 ◽  
Vol 68 (6) ◽  
pp. 671-676 ◽  
Author(s):  
William Gibb ◽  
Jean-Claude Lavoie
Keyword(s):  
Calcium Ionophore ◽  
Inhibitory Effect ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Isolated Cells ◽  
Stimulatory Action ◽  
Human Amnion ◽  
Human Labor ◽  
Epidermal Growth

The human amnion may be an important source of prostaglandins involved in the onset of human labor and therefore it is important to define the factors that regulate their formation in this tissue. In the present study we demonstrate that glucocorticoids inhibit prostaglandin production by freshly isolated amnion cells. The inhibitory action of the glucocorticoids, however, changes to a stimulatory action when the cells are maintained in primary culture for a few days. For both inhibition and stimulation, concentrations of 10−8 M dexamethasone or greater were required to give significant effects, and estradiol and progesterone had no effect on the prostaglandin output of the cells. Epidermal growth factor (EGF), which has previously been found to stimulate prostaglandin output by confluent amnion cells, did not alter prostaglandin output of cells initially placed in culture. Furthermore, the stimulatory action of EGF and dexamethasone appeared additive. The calcium ionophore A23187 stimulated prostaglandin output in freshly isolated cells and accentuated the inhibitory effect of dexamethasone. These studies indicate that prostaglandin formation by human amnion during pregnancy could be regulated by glucocorticoids. These steroids are easily available to the amnion by way of cortisone conversion to Cortisol by the maternal decidua. The results also indicate that amnion is capable of responding to glucocorticoids in both a stimulatory and inhibitory fashion and whether one or both actions are of importance in vivo is a question that is as yet unresolved.Key words: prostaglandins, amnion, fetal membranes, glucocorticoids, labor, pregnancy.

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