Time-lapse confocal imaging-induced calcium ion discharge from the cumulus–oocyte complex at the time of cattle oocyte activation

2020 ◽  
Vol 32 (14) ◽  
pp. 1223
Author(s):  
Hanna J. McLennan ◽  
Melanie L. Sutton-McDowall ◽  
Sabrina Heng ◽  
Andrew D. Abell ◽  
Jeremy G. Thompson
Keyword(s):  
Cumulus Cell ◽  
Membrane Integrity ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Direct Result ◽  
Oocyte Activation ◽  
Active Efflux ◽  
Cell Membrane Integrity ◽  
Morula Stage

Oocyte activation, the dynamic transformation of an oocyte into an embryo, is largely driven by Ca2+ oscillations that vary in duration and amplitude across species. Previous studies have analysed intraoocyte Ca2+ oscillations in the absence of the oocyte’s supporting cumulus cells. Therefore, it is unknown whether cumulus cells also produce an ionic signal that reflects fertilisation success. Time-lapse confocal microscopy and image analysis on abattoir-derived cattle cumulus–oocyte complexes coincubated with spermatozoa revealed a distinct discharge of fluorescence from the cumulus vestment. This study demonstrated that this Ca2+ fluorescence discharge was an artefact induced by the imaging procedure independently of oocyte activation success. The fluorescence discharge was a direct result of cumulus cell membrane integrity loss, and future studies should consider the long-term effect of fluorescent labels on cells in time-lapse imaging. However, this study also demonstrated that the distinctive pattern of a coordinated fluorescence discharge was associated with both the presence of spermatozoa and subsequent embryo development to the morula stage, which was affected by Ca2+ chelation and a reduction in the active efflux of the fluorophore. This indicates that the cumulus vestment may have a relationship with oocyte activation at and beyond fertilisation that requires further investigation.

scholarly journals Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide

Zygote ◽  
2011 ◽  
Vol 20 (4) ◽  
pp. 333-337 ◽  
Author(s):  
Kenzo Uchikura ◽  
Masashi Nagano ◽  
Mitsugu Hishinuma
Keyword(s):  
Propidium Iodide ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Non Invasive ◽  
Maturation Rate ◽  
Cell Layers ◽  
The Relationship

SummaryWe examined the relationship between integrity of cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturational competence of feline oocytes. Feline cumulus–oocyte complexes (COCs) were collected from either small (400–800 μm) or large (≥800 μm) follicles. Immediately after collection, cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI), which penetrates only non-viable cells. Cumulus cells without PI staining were judged as having good membrane integrity. After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89.8%) was higher (p < 0.05) than that from small follicles (60.8%). There was no difference in the maturation rate of oocytes from follicles with the same size regardless of cumulus morphology. In contrast, oocytes that had cumulus cells with good membrane integrity showed a higher maturation rate (93.8%) than oocytes with poor cumulus integrity (76.9%) in large follicles (p < 0.05). We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide staining can be used to predict the maturational competence of oocytes.

153 Fertilisation of Cattle Oocytes is Linked to Novel Waves of a Ca2+ Fluorophore in Cumulus Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 216
Author(s):  
H. J. McLennan ◽  
M. L. Sutton-McDowall ◽  
S. Heng ◽  
J. G. Thompson
Keyword(s):  
Xenopus Oocytes ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Molecular Probes ◽  
Vitelline Membrane ◽  
Oocyte Activation ◽  
Sperm Binding ◽  
Significant Difference ◽  
The Media

During fertilization, multiple intracellular calcium (Ca2+) oscillations are initiated after sperm binding to the oocyte vitelline membrane. This Ca2+ signalling has been extensively studied in denuded mouse and Xenopus oocytes but minimally studied in larger mammals. Cows in particular are unusual, as the few studies on oocyte activation have observed fewer Ca2+ oscillations during fertilisation compared with mice. Furthermore, cattle intracytoplasmic sperm injection (ICSI) is inefficient, despite parthenogenetic activation occurring readily. We hypothesise that cumulus cells are important for cattle oocyte activation at fertilisation. Here, we assessed the behaviour of Ca2+oscillations in fertilising intact cattle cumulus–oocyte complexes (COC). Abattoir-derived cattle COC were matured and fertilised in vitro using Bovine Vitro Media Suite (IVF Vet Solutions). The COC were stained 3.5 h after insemination with the Ca2+ fluorescent probe Fluo4AM (5 μM, Molecular Probes Inc., Eugene, OR, USA) for 30 min, washed, and imaged every 5 min for 6 h in a Fluoview FV10i incubating time-lapse confocal microscope (Olympus) before being returned to culture. Embryo development was assessed at Day 8 to confirm fertilisation. Fluo4AM fluorescence intensity was assessed using FIJI ImageJ. Mean relative intensity over time was graphed for specific regions of interest and the area under graphs was calculated to quantify differences for comparison using a Mann-Whitney Test (mean ± SEM). Experiment 1 (4 reps of 10 COC) compared confirmed fertilised v. uninseminated; experiment 2 (2 reps of 10 COC) compared inseminated COC ± 10 μM BAPTA-AM (Ca2+ chelator, Sigma-Aldrich, St. Louis, MO, USA). There were distinct coordinated waves of differing Fluo4AM intensity in both the oocyte and the cumulus cells surrounding the confirmed fertilised oocytes. This contrasted to the random uncoordinated flashes of Fluo4AM fluorescence in the cumulus cells of the uninseminated oocytes. The fluorescence pattern in +BAPTA-AM COC matched the random flashes observed in the uninseminated group of experiment 1. The fluorescence in the media surrounding the COC immediately following the Fluo4AM waves spiked and then plateaued at a higher level of fluorescence. This was quantified by assessing the area under the graph for 1 h of the plateau following the fluorescence spike. There were no differences between confirmed fertilised (346.4 ± 41.62) and uninseminated groups (239.8 ± 32.08; P > 0.05), but this was affected by differences in cumulus dispersal due to the presence or absence of sperm. Experiment 2 used BAPTA-AM to block oocyte activation with sperm present in both groups and showed a significant difference between the fluorescence increase in the media of the 2 groups (–BAPTA-AM: 311.2 ± 31.57, +BAPTA-AM: 201.4 ± 26.59; P < 0.03). Although the physiological significance has yet to be determined, we have observed a novel Ca2+ wave in the cumulus cells that could be linked to oocyte activation in cattle. There was a significant increase in Fluo4AM fluorescence in the media surrounding the COC, which may indicate cumulus cells are releasing Ca2+ at the time of oocyte activation.

scholarly journals Non-invasive assessment of porcine oocyte quality by supravital staining of cumulus–oocyte complexes with lissamine green B

Zygote ◽  
2015 ◽  
Vol 24 (3) ◽  
pp. 418-427 ◽  
Author(s):  
Rahul Dutta ◽  
Shun Li ◽  
Konrad Fischer ◽  
Alexander Kind ◽  
Tatiana Flisikowska ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Developmental Competence ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Low Grade ◽  
Cell Membrane Integrity ◽  
Non Invasive ◽  
Lissamine Green

SummaryWe evaluated the usefulness of lissamine green B (LB) staining of cumulus–oocyte complexes (COC) as a non-invasive method of predicting maturational and developmental competence of slaughterhouse-derived porcine oocytes cultured in vitro. Cumulus cells of freshly aspirated COCs were evaluated either morphologically on the basis of thickness of cumulus cell layers, or stained with LB, which penetrates only non-viable cells. The extent of cumulus cell staining was taken as an inverse indicator of membrane integrity. The two methods of COC grading were then examined as predictors of nuclear maturation and development after parthenogenetic activation. In both cases LB staining proved a more reliable indicator than morphological assessment (P < 0.05). The relationship between LB staining and cumulus cell apoptosis was also examined. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for DNA fragmentation revealed that oocytes within COCs graded as low quality by either LB staining or visual morphology showed significantly greater DNA fragmentation (P < 0.05) than higher grades, and that LB and visual grading were of similar predictive value. Expression of the stress response gene TP53 showed significantly higher expression in COCs graded as low quality by LB staining. However expression of the apoptosis-associated genes BAK and CASP3 was not significantly different between high or low grade COCs, suggesting that mRNA expression of BAK and CASP3 is not a reliable method of detecting apoptosis in porcine COCs. Evaluation of cumulus cell membrane integrity by lissamine green B staining thus provides a useful new tool to gain information about the maturational and developmental competence of porcine oocytes.

OPS vitrification of mouse immature oocytes before or after meiosis: the effect on cumulus cells maintenance and subsequent development

Zygote ◽  
2009 ◽  
Vol 17 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Lun Suo ◽  
Guang-Bin Zhou ◽  
Qing-Gang Meng ◽  
Chang-Liang Yan ◽  
Zhi-Qiang Fan ◽  
...  
Keyword(s):  
Survival Rate ◽  
Cell Damage ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Germinal Vesicle ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Germinal Vesicle Breakdown ◽  
Immature Oocytes

SummaryCryopreservation can cause cumulus cell damage around the immature oocytes, which may result in poor subsequent development. To evaluate the effect of the meiosis stage on the cumulus cell cryoinjury and determine the suitable stage for cryopreservation in immature oocytes, mouse oocytes at germinal vesicle (GV) and germinal vesicle breakdown (GVBD) stages were vitrified using open pulled straw (OPS) method. Cumulus cells damage was scored immediately after thawing by double-fluorescent staining. The survival rate of the oocytes was evaluated and the subsequent development of oocytes was assessed through in vitro culture (IVC) and in vitro fertilization (IVF) separately. After vitrification, a higher proportion of cumulus cells of GV oocytes were damaged than those of GVBD and untreated control groups. The survival rate of vitrified GVBD oocytes (94.1%) was significantly higher (p < 0.05) than that of GV oocytes (85.4%). Oocytes vitrified at GVBD stage (55.7%) showed similar cleavage rate compared to those at GV stage (49.2%), but significantly higher (p < 0.05) blastocyst rate (40.9% vs. 27.4%). These results demonstrate that oocytes at GVBD stage remain better cumulus membrane integrity and developmental ability during vitrification than those at GV stage, indicating they are more suitable for immature oocytes cryopreservation in mice.

Quercetin in attenuation of ischemic/reperfusion injury: A review

2020 ◽  
Vol 13 ◽  
Author(s):  
Milad Ashrafizadeh ◽  
Saeed Samarghandian ◽  
Kiavash Hushmandi ◽  
Amirhossein Zabolian ◽  
Md Shahinozzaman ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Membrane ◽  
Blood Supply ◽  
Apoptotic Cell ◽  
Ischemia Reperfusion ◽  
Membrane Integrity ◽  
Therapeutic Effects ◽  
Molecular Pathways ◽  
Cell Membrane Integrity ◽  
Cell Membrane Disruption

Background: Ischemia/reperfusion (I/R) injury is a serious pathologic event that occurs due to restriction in blood supply to an organ, followed by hypoxia. This condition leads to enhanced levels of pro-inflammatory cytokines such as IL-6 and TNF-, and stimulation of oxidative stress via enhancing reactive oxygen species (ROS) levels. Upon reperfusion, blood supply increases, but it deteriorates condition, and leads to generation of ROS, cell membrane disruption and finally, cell death. Plant derived-natural compounds are well-known due to their excellent antioxidant and anti-inflammatory activities. Quercetin is a flavonoid exclusively found in different vegetables, herbs, and fruits. This naturally occurring compound possesses different pharmacological activities making it appropriate option in disease therapy. Quercetin can also demonstrate therapeutic effects via affecting molecular pathways such as NF-B, PI3K/Akt and so on. Methods: In the present review, we demonstrate that quercetin administration is beneficial in ameliorating I/R injury via reducing ROS levels, inhibition of inflammation, and affecting molecular pathways such as TLR4/NF-B, MAPK and so on. Results and conclusion: Quercetin can improve cell membrane integrity via decreasing lipid peroxidation. Apoptotic cell death is inhibited by quercetin via down-regulation of Bax, and caspases, and upregulation of Bcl-2. Quercetin is able to modulate autophagy (inhibition/induction) in decreasing I/R injury. Nanoparticles have been applied for delivery of quercetin, enhancing its bioavailability and efficacy in alleviation of I/R injury. Noteworthy, clinical trials have also confirmed the capability of quercetin in reducing I/R injury.

scholarly journals Integrating flexible electronics for pulsed electric field delivery in a vascularized 3D glioblastoma model

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Marie C. Lefevre ◽  
Gerwin Dijk ◽  
Attila Kaszas ◽  
Martin Baca ◽  
David Moreau ◽  
...  
Keyword(s):  
Electric Field ◽  
Electric Fields ◽  
Flexible Electronics ◽  
Soft Tissues ◽  
Multiphoton Microscopy ◽  
Membrane Integrity ◽  
Tumor Model ◽  
Pulsed Electric Fields ◽  
In Ovo ◽  
Cell Membrane Integrity

AbstractGlioblastoma is a highly aggressive brain tumor, very invasive and thus difficult to eradicate with standard oncology therapies. Bioelectric treatments based on pulsed electric fields have proven to be a successful method to treat cancerous tissues. However, they rely on stiff electrodes, which cause acute and chronic injuries, especially in soft tissues like the brain. Here we demonstrate the feasibility of delivering pulsed electric fields with flexible electronics using an in ovo vascularized tumor model. We show with fluorescence widefield and multiphoton microscopy that pulsed electric fields induce vasoconstriction of blood vessels and evoke calcium signals in vascularized glioblastoma spheroids stably expressing a genetically encoded fluorescence reporter. Simulations of the electric field delivery are compared with the measured influence of electric field effects on cell membrane integrity in exposed tumor cells. Our results confirm the feasibility of flexible electronics as a means of delivering intense pulsed electric fields to tumors in an intravital 3D vascularized model of human glioblastoma.

10-gingerol induces oxidative stress through HTR1A in cumulus cells: in-vitro and in-silico studies

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Kiptiyah Kiptiyah ◽  
Widodo Widodo ◽  
Gatot Ciptadi ◽  
Aulanni’am Aulanni’Am ◽  
Mohammad A. Widodo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Culture ◽  
Superoxide Dismutase ◽  
In Silico ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Sod Activity ◽  
In Silico Studies ◽  
Incubation Periods

AbstractBackgroundWe investigated whether 10-gingerol is able to induce oxidative stress in cumulus cells.MethodsFor the in-vitro research, we used a cumulus cell culture in M199, containing 10-gingerol in various concentrations (0, 12, 16, and 20 µM), and detected oxidative stress through superoxide dismutase (SOD) activity and malondialdehyde (MDA) concentrations, with incubation periods of 24, 48, 72, and 96 h. The obtained results were confirmed by in-silico studies.ResultsThe in-vitro data revealed that SOD activity and MDA concentration increased with increasing incubation periods: SOD activity at 0 µM (1.39 ± 0.24i), 12 µM (16.42 ± 0.35ab), 16 µM (17.28 ± 0.55ab), 20 µM (17.81 ± 0.12a), with a contribution of 71.1%. MDA concentration at 0 µM (17.82 ± 1.39 l), 12 µM (72.99 ± 0.31c), 16 µM (79.77 ± 4.19b), 20 µM (85.07 ± 2.57a), with a contribution of 73.1%. Based on this, the in-silico data uncovered that 10˗gingerol induces oxidative stress in cumulus cells by inhibiting HTR1A functions and inactivating GSK3B and AKT˗1.Conclusions10-gingerol induces oxidative stress in cumulus cells through enhancing SOD activity and MDA concentration by inhibiting HTR1A functions and inactivating GSK3B and AKT˗1.

scholarly journals Physiological, Morphological and Antioxidant Responses of Pediococcus pentosaceus R1 and Lactobacillus fermentum R6 Isolated from Harbin Dry Sausages to Oxidative Stress

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1203
Author(s):  
Huan Zhang ◽  
Jianhang Xu ◽  
Qian Chen ◽  
Hui Wang ◽  
Baohua Kong
Keyword(s):  
Oxidative Stress ◽  
Electron Microscopy ◽  
Antioxidant Properties ◽  
Membrane Integrity ◽  
Starter Cultures ◽  
Lactobacillus Fermentum ◽  
Fermented Foods ◽  
Pediococcus Pentosaceus ◽  
Cell Membrane Integrity ◽  
Dry Sausages

As functional starter cultures and potential probiotics, the ability of lactic acid bacteria to resist oxidative stress is essential to maintain viability and functional properties. This study investigates the effects of H2O2 at different concentrations (0, 1, 2, and 3 mM) on the physiological, morphological, and antioxidant properties of Pediococcus pentosaceus R1 and Lactobacillus fermentum R6 isolated from Harbin dry sausages. The increase in H2O2 concentration induced a significant increase in reactive oxygen species and a decrease in intracellular ATP levels (p < 0.05). Based on scanning electron microscopy, transmission electron microscopy, and electric conductivity analysis, H2O2 stress caused cell deformation, the destruction of cell membrane integrity, partial loss of the cytoplasm, and an increase in the cell conductivity of both strains. H2O2 stress with 1 mM or 2 mM concentrations could effectively improve the scavenging rates of free radicals, the activities of superoxide dismutase and glutathione peroxide, and the total antioxidant capacity of both strains (p < 0.05). In conclusion, an appropriate oxidative stress contributed to the activation of the antioxidant defense system of both strains, conferred strains a better effect in inhibiting the oxidation of fermented foods, and improved the health of the host.

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