186 Protective effect of resveratrol on invitro fertilization of ovine oocytes matured under cadmium exposure

2020 ◽  
Vol 32 (2) ◽  
pp. 221
Author(s):  
A. Piras ◽  
F. Ariu ◽  
N. Martino ◽  
A. Maltana ◽  
I. Menéndez-Blanco ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Laser Scanning ◽  
Peripheral Region ◽  
Cadmium Exposure ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cortical Granules ◽  
Female Reproduction ◽  
Major Mechanism ◽  
Normal Fertilization

Cadmium (Cd) is one of the most toxic environmental and industrial heavy metals, with adverse effect on female reproduction. Earlier invitro studies have demonstrated that exposure to Cd during IVM impairs oocyte fertilization through the induction of oxidative stress (Martino et al. 2017 Reprod. Toxicol. 69, 132-145). The aim of this study was to evaluate the effect of supplementing IVM medium with resveratrol (Res), a phytoalexin with antioxidant activity, on IVF of ovine oocytes matured under Cd exposure and to evaluate whether this effect could be mediated by alterations of cortical granules (CGs) distribution and/or intracellular levels of reactive oxygen species (ROS). Cumulus-oocyte complexes collected from slaughterhouse-derived ovaries of prepubertal ewes were invitro matured with 0μM CdCl2 (control), 2μM CdCl2 (Cd group), or 2μM CdCl2 + 1μM Res (Cd-Res). Groups of MII oocytes were invitro fertilized with ram semen (Bogliolo et al. 2011 Reprod. Fertil. Dev. 23, 809-817). After 16h, oocytes were fixed with absolute ethanol, stained with Hoechst 33342, and classified as follows: normally fertilized (two pronuclei), polyspermic (more than two pronuclei), and abnormally fertilized (asynchronous pronucleus formation). Levels of ROS and distribution of CG in MII oocytes (n=25 each group) were evaluated by confocal laser scanning microscopy after staining with 2’,7’dichlorodihydrofluorescein diacetate and Alexa Fluor 488-conjugated lectin peanut agglutinin, respectively (Jiao et al. 2013 PLoS One 8, 58018). Data of fertilization and CGs distribution were analysed by Chi-squared test and ROS levels (mean±standard error) by Kruskal-Wallis test with STATA\IC 11.0 (Stata Corp.). Oocytes matured in presence of Res had higher normal fertilization and lower polyspermy rates than those matured without Res (Table 1). Levels of ROS in oocytes were lower in the Cd-Res group than in the Cd group (74.4±1.3 vs. 122.4±1.5 pixel/oocyte; P<0.05), and similar to those of the control (71.1±2.3 pixel/oocyte). The ratio of oocytes of Cd-Res group with CGs normally distributed in the peripheral region was higher than that of Cd group (91.3% vs. 65.2%; P<0.05) and comparable to that of control oocytes (95.8%). Collectively, our results showed that resveratrol protects oocytes from Cd-induced alteration of normal fertilization by preventing oxidative stress and improving the major mechanism responsible for blockage of polyspermic fertilization. Table 1.Effect of resveratrol on IVF rate of oocytes matured under cadmium exposure Group No. of total oocytes No. of MII oocytes No. of fertilized oocytes (%) Normal Polyspermy Abnormal Control 136 105 (77.2) 59 (56.2)a 23 (21.9)a 6 (5.7) Cd 146 106 (72.6) 39 (36.8)b 38 (35.8)b 7 (6.6) Cd-Res 114 88 (77.2) 47 (53.4)a 16 (18.2)a 7 (7.9) a,bValues in the same column with different superscripts differ significantly (P<0.05). This project was supported by FDS 2016 (CUP J86C18000780005).

scholarly journals Potential role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress

2001 ◽  
Vol 114 (9) ◽  
pp. 1643-1653 ◽  
Author(s):  
Z. Dastoor ◽  
J.L. Dreyer
Keyword(s):  
Oxidative Stress ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Apoptotic Cells ◽  
Transfected Cells ◽  
And Oxidative Stress

Recent studies indicating a role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in apoptosis or oxidative stress has been reported. Using confocal laser-scanning microscopy, we have investigated the cellular distribution of GAPDH in central nervous system (CNS)-derived cells (neuroblastoma mNB41A3), in non-CNS derived cells (R6 fibroblast) and in an apoptosis-resistant Bcl2 overexpressing cell line (R6-Bcl2). Induction of apoptosis by staurosporine or MG132 and oxidative stress by H(2)O(2) or FeCN enhanced the nuclear translocation of endogenous GAPDH in all cell types, as detected by immunocytochemistry. In apoptotic cells, GAPDH expression is three times higher than in non-apoptotic cells. Consistent with a role for GAPDH in apoptosis, overexpression of a GAPDH-green fluorescent protein (GAPDH-GFP) hybrid increased nuclear import of GAPDH-GFP into transfected cells and the number of apoptotic cells, and made them more sensitive to agents that induce apoptosis. Bcl2 overexpression prevents nuclear translocation of GAPDH and apoptosis in untransfected cells, but not in transfected cells that overexpress GAPDH-GFP. Our observations indicate that nuclear translocation of GAPDH may play a role in apoptosis and oxidative stress, probably related to the activity of GAPDH as a DNA repair enzyme or as a nuclear carrier for pro-apoptotic molecules.

Dynamics of cortical granule exocytosis at fertilization in living mouse eggs

1996 ◽  
Vol 270 (5) ◽  
pp. C1354-C1361 ◽  
Author(s):  
M. Tahara ◽  
K. Tasaka ◽  
N. Masumoto ◽  
A. Mammoto ◽  
Y. Ikebuchi ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cortical Granule ◽  
Free Calcium ◽  
Cortical Granules ◽  
Acetoxymethyl Ester ◽  
Living Mouse ◽  
Cortical Granule Exocytosis ◽  
Mouse Eggs

Sperm-egg fusion induces an intracellular free calcium concentration ([Ca2+]i) increase and exocytosis of cortical granules (CGs). Recently we used an impermeable fluorescent membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), to develop a method to evaluate the kinetics of exocytosis in single living cells. In this study we used digital imaging and confocal laser scanning microscopy to evaluate CG exocytosis in living mouse eggs with TMA-DPH. Time-related changes of CG exocytosis were estimated as the percent increase of TMA-DPH fluorescence. The increase of fluorescence in the egg started after sperm attachment, continued at an almost uniform rate, and ceased at 45-60 min. Whereas the [Ca2+]i increase at fertilization was transient or oscillatory, exocytosis was not always induced concomitantly with each [Ca2+]i peak. Next we used this method to determine some intracellular mediators of exocytosis in the egg. An intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and a microfilament inhibitor, cytochalasin B, blocked sperm-induced exocytosis. A guanosine 5'-triphosphate-binding protein activator, AlF4-, induced exocytosis. These results suggest that [Ca2+]i, microfilament, and guanosine 5'-triphosphate-binding proteins may be involved in CG exocytosis. In conclusion, this method has significant advantages for studying exocytosis in living eggs.

scholarly journals Curcumin Downregulates Phosphate Carrier and Protects against Doxorubicin Induced Cardiomyocyte Apoptosis

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Lu Junkun ◽  
Chu Erfu ◽  
Hasahya Tony ◽  
Li Xin ◽  
K. C. Sudeep ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Laser Scanning ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
H9c2 Cell ◽  
Malondialdehyde Content ◽  
Phosphate Carrier

Aim. To explore the effects of curcumin on phosphate carrier (PiC) and its role in protection against doxorubicin induced myocyte toxicity.Methods. Using H9c2 cell line, the cardiotoxic effect of doxorubicin and its mitigation by curcumin were studied. H9c2 cells were cultured with doxorubicin and/or curcumin at various concentrations. Analysis for apoptosis of H9c2 was done using flow cytometry. Confocal laser scanning microscopy was used to record the fluorescence intensity ratios and to determine the mitochondrial permeability transition pore (MPTP) opening state. Oxidative stress was measured using glutathione level, superoxide dismutase activities, and malondialdehyde content. The effect of doxorubicin and curcumin on PiC gene expression was measured by real-time PCR.Results. Curcumin decreased mRNA of PiC and was partly protective against oxidative stress, loss of mitochondrial transmembrane potential, and apoptosis induced by doxorubicin. Interestingly, the effect was not clearly dose dependent and the concentration of 12 mg/L was more efficient than 15 and 10 mg/L.Conclusion. Curcumin downregulates PiC and partly protects against doxorubicin induced oxidative stress and myocyte apoptosis.

scholarly journals Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore

1999 ◽  
Vol 343 (2) ◽  
pp. 311-317 ◽  
Author(s):  
Jörg HüSER ◽  
Lothar A. BLATTER
Keyword(s):  
Oxidative Stress ◽  
Living Cell ◽  
Structural Damage ◽  
Laser Scanning ◽  
Mitochondrial Permeability Transition ◽  
Electrical Potential ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Laser Scanning Microscopy ◽  
Confocal Laser

Confocal laser scanning microscopy and the potentiometric fluorescence probe tetramethylrhodamine ethyl ester were used to measure changes in membrane electrical potential (δΨm) in individual mitochondria after isolation or in the living cell. Recordings averaged over small mitochondrial populations revealed a gradual decline in δΨmcaused by the light-induced generation of free radicals. Depolarization was attenuated by dithiothreitol or acidification. In contrast, individual organelles displayed rapid spontaneous depolarizations caused by openings of the mitochondrial permeability transition pore (MTP). Repetitive openings and closings of the pore gave rise to marked fluctuations in δΨm between the fully charged and completely depolarized state. Rapid spontaneous fluctuations in δΨm were observed in mitochondria isolated from rat heart and in mitochondria in living endothelial cells. The loss of δΨm of mitochondria in the living cell coincided with swelling of the organelle and the breakdown of long mitochondrial filaments. In the individual mitochondrion, oxidative stress initially triggered pore openings of shorter duration, before prolonged openings caused the complete dissipation of δΨm and a measurable efflux of larger solutes. Generalizing this scheme, we suggest that under conditions of prolonged oxidative stress and/or cellular Ca2+ overload, short openings of MTP might serve as an emergency mechanism allowing the partial dissipation of δΨm, the fast release of accumulated Ca2+ ions and the decreased generation of endogenous oxygen radicals. In contrast, loss of matrix metabolites, swelling and other structural damage of the organelle render prolonged openings of the transition pore deleterious to mitochondria and to the cell.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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