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<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).

Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells

2000 ◽  
Vol 12 (2) ◽  
pp. 15 ◽  
Author(s):  
H. T. Cheong ◽  
K. Ikeda ◽  
M. A. Martinez Diaz ◽  
S. Katagiri ◽  
Y. Takahashi
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Calcium Ionophore ◽  
Developmental Rate ◽  
Cumulus Cells ◽  
Calcium Ionophore A23187 ◽  
Serum Starvation ◽  
Ionophore A23187 ◽  
Oocyte Collection

This study tested the effects of oocyte collection method, activation protocol and maturational age of recipient oocytes on the in vitro development of nuclear transfer embryos reconstructed with cultured cumulus cells. Cumulus cells synchronized in G0/G1 phase by serum-starvation culture were transferred into enucleated oocytes that were collected by aspiration or dissection method and cultured for 33 or 44 h. Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and cycloheximide (CHXM), and cultured for 6 days. Oocyte collection methods, activation treatment in the presence of cytochalasin B and activation protocols did not affect the developmental rate of embryos reconstituted with 44-h-matured recipients. However, the development of embryos reconstituted with 33-h-matured recipients was significantly improved (P<0.05) by activation with the combination of electric pulse and CHXM. The present study shows that reconstituted porcine embryos derived from cultured cumulus cells can develop to the blastocyst stage, and that their development can be improved by reconstruction with young oocyte cytoplasts following activation with a combination of electric pulse and CHXM.

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.

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.

Optimisation of porcine oocyte activation following nuclear transfer

Zygote ◽  
2000 ◽  
Vol 8 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Tao Tao ◽  
Zoltán Macháty ◽  
Lalantha R. Abeydeera ◽  
Billy N. Day ◽  
Randall S. Prather
Keyword(s):  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Calcium Ionophore ◽  
Subsequent Development ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Oocyte Activation ◽  
Free Medium ◽  
Activation Rate

Experiments were conducted to examine the effects of (a) different activation methods, (b) incubation time in calcium-free medium and (c) bisbenzimide staining on the activation and subsequent development of pig oocytes. Oocytes were matured in vitro and activated by one of the following methods: combined thimerosal/dithiothreitol (DTT) treatment, calcium ionophore A23187 treatment followed by incubation in the presence of 6-dimethylaminopurine (6-DMAP), electroporation, and electroporation followed by incubation with cytochalasin B. There were no significant differences in the activation rate (ranging from 70.0% to 88.3%) and the percentage of cleaved embryos after activation (ranging between 48.8% and 58.8%) among the four treatment groups (p < 0.05). The rate of development to the blastocyst stage in oocytes activated by thimerosal/DTT (10.0%) or electroporation followed by cytochalasin B treatment (12.3%) was significantly higher (p < 0.05) than in the group activated with A23187/6-DMAP (2.5%). Both the activation rate and the rate of blastocyst formation in oocytes that were incubated in Ca2+-free medium for 8 h before thimerosal/DTT activation were significantly lower (p < 0.05) than in those incubated for 0, 1 or 4 h. Intracellular Ca2+ measurements revealed that the Ca2+ homeostasis in these oocytes were severely altered. Staining of oocytes with 5 μg/ml bisbenzimide for 2 h decreased the quality of blastocysts and increased the rate of degenerated embryos at day 6. Two activation protocols (thimerosal/DTT and electroproation) were used for activation after nuclear transfer; the rate of nuclear formation did not differ in the oocytes activated by the two different methods.

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

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.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

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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