scholarly journals Heat Shock-derived Reactive Oxygen Species Induce Embryonic Mortality in In Vitro Early Stage Bovine Embryos

2008 ◽  
Vol 54 (6) ◽  
pp. 496-501 ◽  
Author(s):  
Miki SAKATANI ◽  
Kenichi YAMANAKA ◽  
Shuji KOBAYASHI ◽  
Masashi TAKAHASHI
Keyword(s):  
Reactive Oxygen Species ◽  
Heat Shock ◽  
Early Stage ◽  
Reactive Oxygen ◽  
Embryonic Mortality ◽  
Oxygen Species ◽  
Bovine Embryos

129 EFFECT OF SERUM SUPPLEMENTATION ON AMP-ACTIVATED PROTEIN KINASE (AMPK) ACTIVITY AND LIPID METABOLISM OF IN VITRO-CULTURED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 156
Author(s):  
S. Prastowo ◽  
F. Rings ◽  
D. S. Wondim ◽  
E. Tholen ◽  
C. Looft ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mitochondrial Biogenesis ◽  
Reactive Oxygen ◽  
Rna Isolation ◽  
Mitochondrial Activity ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Fluorescent Intensity ◽  
Ampk Activity

A major problem of embryos cultured in vitro with serum is cytoplasmic lipid accumulation resulting in lower cryotolerance compared with those derived from in vivo or in the absence of serum. AMPK is known as a master regulator of lipid, glucose, and protein metabolism in mammalian cells. Moreover, it has been reported as controller of acetyl-CoA carboxylase α (ACC), the gene responsible for lipid synthesis, and associated with mitochondrial biogenesis and activities in response to oxidative stress. In the present study we aimed to investigate the regulation of AMPK during serum supplementation in vitro. For this, bovine embryos were produced in vitro in SOF media supplemented with oestrous cow serum or fatty acid–free BSA as a system without serum. Triplicate pools (each 10 blastocysts) from each group were used for RNA isolation using Arcturus®PicoPure®RNA Isolation Kit (Life Technologies, USA). Reverse transcription was performed using a combination of Oligo(dT)23 and random primers. Quantification of AMPK catalytic α1 (AMPKA1), ACC, peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1A), and sterol regulatory element binding transcription factor 2 (SREBP2) transcripts were performed using ABI PRISM® 7000 SDS system (Applied Biosystems, Foster City, CA, USA) using GAPDH as internal control. Normalized log-transformed transcript amount data were statistically analysed using t-test. In addition, AMPK protein was detected by immunofluorescence, mitochondrial activity by MitoTracker® Red (Invitrogen, Carlsbad, CA, USA), and reactive oxygen species by H2DCFDA molecular probe (Life Technologies, USA), and fluorescent intensity signals were visualised under confocal laser scanning microscopy LSM 710 (Carl Zeiss, Germany). Results showed that the expression of AMPKA1, PGC1A, a mitochondrial biogenesis protein, and SREBP2, a regulator of lipid oxidation, were found to be lower (0.4-, 0.2-, and 0.7-fold, respectively; P < 0.05) in blastocysts derived from cultured with serum compared to without serum. By contrast, ACC was up-regulated in blastocysts cultured with serum by 1.8-fold (P < 0.05) compared to without serum. In comparison to blastocyst cultured without serum, a reduced fluorescent intensity was observed in AMPKA1 protein and mitochondrial activity in blastocyst cultured with serum. The presence of serum was also found to be involved in increasing reactive oxygen species accumulation in embryos cultured with serum. The reduced level of AMPK leads to increased ACC and subsequently enhanced conversion of fatty acids into lipid, which is associated with reduced mitochondrial biogenesis protein, elevated reactive oxygen species level, and reduced lipid oxidation by suppression of SREBP2. In conclusion, the presence of serum in in vitro culture environment affected the AMPK activity and thereby genes associated with lipid metabolism in early bovine embryos.

90 INTRACELLULAR REACTIVE OXYGEN SPECIES IN BOVINE EMBRYOS CULTURED IN VITRO WITH CATALASE UNDER VARIOUS OXYGEN TENSIONS

2012 ◽  
Vol 24 (1) ◽  
pp. 157 ◽  
Author(s):  
N. A. S. Rocha ◽  
B. C. S. Leão ◽  
M. F. Accorsi ◽  
G. Z. Mingoti
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Embryonic Development ◽  
Embryo Development ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Intracellular Ros ◽  
Oxygen Tensions

The production of reactive oxygen species (ROS), such as superoxide anion (O2–), hydroxyl radical (OH–) hydrogen peroxide (H2O2) and organic peroxides, is a normal process that occurs in the cellular mitochondrial respiratory chain. The high oxygen tension in in vitro culture (IVC) conditions is believed to induce oxidative stress, as a result of increase in ROS intracellular production, that can be correlated with embryonic developmental failure. Supplementation with antioxidants during IVC appears to increase the resistance of bovine embryos to the oxidative stress and consequently improve embryo development. The aim of this study was to evaluate the effects of antioxidant (catalase) and oxygen tensions during IVC on the embryonic development and quantification of intracellular ROS. Cumulus–oocyte complexes (COC; n = 337) were in vitro matured (IVM) in TCM-199 supplemented with 0.2 mM pyruvate, 25 mM sodium bicarbonate, 75 μg mL–1 gentamicin, 10% FCS and hormones for 24 h at 38.5°C and 5% CO2 in air. Then they were fertilized and the presumptive zygotes were cultured in SOFaa medium without (control) or with 100 UI catalase (CAT) for 7 days at 38.5°C in one of 2 types of humified atmosphere: 5% CO2 in air (≈20% O2) or in gaseous mixture (7% O2, 5% CO2 and 88% N2). The cleavage rate was evaluated at 72 hours post-insemination (hpi) and the embryonic development at 168 hpi. At this time, the level of intracellular ROS was measured using the fluorescent probe 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Invitrogen, Oakville, Canada), at 5 μM (Bain et al. 2011 Reprod. Fertil. Dev. 23, 561–575). Stained embryos were imaged immediately using an inverted microscope and analysed by Q-Capture Pro image software (QImaging, Surrey, BC, Canada). The signal intensity values of embryos were subtracted by the average of backgrounds in the images. Embryo development was analysed by chi-squared test and means of the intensity of fluorescence were compared by ANOVA followed by Tukey's test (P < 0.05). The cleavage rates were 84.04%a (control 20% O2), 77.55%a (CAT 20% O2), 77.03%a (control 7% O2) and 71.83%a (CAT 7% O2). The embryonic development rates were 40.43%a (control 20% O2), 33.67%a (CAT 20% O2), 20.27%b (control 7% O2) and 16.90%b (CAT 7% O2). The fluorescent intensity were 3.9 ± 0.4a (control 20% O2), 1.8 ± 0.2b (CAT 20% O2), 2.7 ± 0.2ab (control 7% O2) and 2.8 ± 0.2ab (CAT 7% O2). Although catalase did not significantly affect blastocyst frequencies (P > 0.05), embryo development was adversely affected by reduced O2 tension (P < 0.05). H2DCFDA staining indicated a significant (P < 0.05) reduction in the levels of intracellular ROS within embryos cultured with catalase under 20% O2 compared with the control group in the same O2 tension. Additionally, a consistent but insignificant reduction in intracellular ROS within embryos cultured under 7% O2 was found. We can conclude that supplementation with catalase to IVC medium at 20% O2 is suitable for lowering intracellular ROS levels in IVP bovine embryos, without lowering the rates of blastocysts production. This finding corroborates with theory that antioxidants are beneficial to embryo quality. Alta Genetics Brazil, Deoxi Biotecnologia.

Effect of pterostilbene on development, equatorial lipid accumulation and reactive oxygen species production of in vitro‐produced bovine embryos

2020 ◽  
Vol 55 (11) ◽  
pp. 1490-1500
Author(s):  
Froylan Sosa ◽  
Salvador Romo ◽  
Michael E. Kjelland ◽  
Horacio Álvarez‐Gallardo ◽  
Sandra Pérez‐Reynozo ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Lipid Accumulation ◽  
Reactive Oxygen Species Production ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Species Production

Synergistic Effect Of Nifurtimox and Inhibition Of Hsp70/Hsp90 In Treatment Of Neuroblastoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5567-5567
Author(s):  
Karin Melanie Rohrer ◽  
Gernot Bruchelt ◽  
Rupert Handgretinger ◽  
Ursula Holzer
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Heat Shock ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Neuroblastoma Cell Lines

Abstract Neuroblastoma is the most common solid cancer in childhood with high relapse and mortality rates. Furthermore, high risk neuroblastoma is often accompanied by an infaust prognosis. The 5-nitrofuran nifurtimox, usually used in the treatment of Chagas disease, showed cytotoxic effects against neuroblastoma in vitro and in experimental therapy, which is presumably due to the formation of oxidative stress. Inducing oxidative stress is a well investigated and suitable strategy in the treatment of malignant diseases in vitro but often encounters difficulties in clinical administration. Thus, nifurtimox as a well-established drug represents a promising new approach in treating neuroblastoma. Combining the induction of reactive oxygen species by application of nifurtimox with a blockade of the cells’ own stress response might even increase the cytotoxic effects. The chaperones heat shock protein 70 and 90 (Hsp70/Hsp90) are responsible for refolding or degrading damaged proteins, especially after stress situations such as heat or oxidative stress. Therefore, the roles of Hsp70 and Hsp90 were investigated in more detail. The commercially available human neuroblastoma cell lines IMR-32, LA-N-1 and the cell line LS, which has been established in the children’s hospital Tuebingen, were exposed to increasing doses of nifurtimox (0.070 mM to 0.348 mM) and incubated for 1, 2 or 3 days. It could be observed that cell viability of all cell lines was significantly and dose-dependently reduced (p<0.01) after nifurtimox treatment. An average reduction of cell viability by 50% was achieved after 24h incubation with 0.348 mM nifurtimox (LS and IMR-32). The assumption that nifurtimox induces the formation of reactive oxygen species could be confirmed. The amount of intracellular reactive oxygen species was significantly increased (p<0.05) in a dose-dependent manner in all cell lines after 24h. Furthermore, expression levels of heat shock proteins Hsp70 and Hsp90 were investigated. Western blot analysis revealed increased intracellular expression levels for both heat shock proteins after 24h nifurtimox treatment. Concluding that Hsp70 and Hsp90 have important roles in tumor cell survival, it was decided to specifically inhibit Hsp90. For this purpose, the neuroblastoma cell lines were treated with the geldanamycin analog 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). After inhibition of Hsp90 cells were additionally incubated with the previously used dosages of nifurtimox. A significant higher reduction of the cell viability (p<0.001) could be observed for all neuroblastoma cell lines compared to the application of nifurtimox or 17-DMAG alone. In conclusion, nifurtimox increases oxidative stress in neuroblastoma cell lines leading to significantly decreased cell viability. The specific inhibition of Hsp90 additionally intensifies this effect. The findings suggest that the combined administration of nifurtimox and the specific Hsp90 inhibitor 17-DMAG leads to a synergistic and favorable effect in the treatment of neuroblastoma. More importantly, being an approved medication and well investigated in a wide variety of clinical trials, nifurtimox and 17-DMAG are easy accessible and create a promising new approach not only in neuroblastoma treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reactive Oxygen Species in Host Plant Are Required for an Early Defense Response against Attack of Stagonospora nodorum Berk. Necrotrophic Effectors SnTox

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1586
Author(s):  
Svetlana Veselova ◽  
Tatyana Nuzhnaya ◽  
Guzel Burkhanova ◽  
Sergey Rumyantsev ◽  
Igor Maksimov
Keyword(s):  
Reactive Oxygen Species ◽  
Early Stage ◽  
Reactive Oxygen ◽  
Triticum Aestivum L ◽  
Redox Status ◽  
Stagonospora Nodorum ◽  
Ros Generation ◽  
Ros Production ◽  
Oxygen Species ◽  
Necrotrophic Effectors

Reactive oxygen species (ROS) play a central role in plant immune responses. The most important virulence factors of the Stagonospora nodorum Berk. are multiple fungal necrotrophic effectors (NEs) (SnTox) that affect the redox-status and cause necrosis and/or chlorosis in wheat lines possessing dominant susceptibility genes (Snn). However, the effect of NEs on ROS generation at the early stages of infection has not been studied. We studied the early stage of infection of various wheat genotypes with S nodorum isolates -Sn4VD, SnB, and Sn9MN, carrying a different set of NE genes. Our results indicate that all three NEs of SnToxA, SnTox1, SnTox3 significantly contributed to cause disease, and the virulence of the isolates depended on their differential expression in plants (Triticum aestivum L.). The Tsn1–SnToxA, Snn1–SnTox1and Snn3–SnTox3 interactions played an important role in inhibition ROS production at the initial stage of infection. The Snn3–SnTox3 inhibited ROS production in wheat by affecting NADPH-oxidases, peroxidases, superoxide dismutase and catalase. The Tsn1–SnToxA inhibited ROS production in wheat by affecting peroxidases and catalase. The Snn1–SnTox1 inhibited the production of ROS in wheat by mainly affecting a peroxidase. Collectively, these results show that the inverse gene-for gene interactions between effector of pathogen and product of host sensitivity gene suppress the host’s own PAMP-triggered immunity pathway, resulting in NE-triggered susceptibility (NETS). These results are fundamentally changing our understanding of the development of this economical important wheat disease.

scholarly journals The Effects of Bioactive Compounds from Blueberry and Blackcurrant Powder on Oat Bran Pastes: Enhancing In Vitro Antioxidant Activity and Reducing Reactive Oxygen Species in Lipopolysaccharide-Stimulated Raw264.7 Macrophages

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 388
Author(s):  
Xiao Dan Hui ◽  
Gang Wu ◽  
Duo Han ◽  
Xi Gong ◽  
Xi Yang Wu ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Antioxidant Activity ◽  
Phenolic Compounds ◽  
Antioxidant Capacity ◽  
Bioactive Compounds ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Raw264.7 Macrophages ◽  
Oat Bran

In this study, blueberry and blackcurrant powder were chosen as the phenolic-rich enrichments for oat bran. A Rapid Visco Analyser was used to form blueberry and blackcurrant enriched oat pastes. An in vitro digestion process evaluated the changes of phenolic compounds and the in vitro antioxidant potential of extracts of pastes. The anthocyanidin profiles in the extracts were characterised by the pH differential method. The results showed that blueberry and blackcurrant powder significantly increased the content of phenolic compounds and the in vitro antioxidant capacity of pastes, while the total flavonoid content decreased after digestion compared to the undigested samples. Strong correlations between these bioactive compounds and antioxidant values were observed. Lipopolysaccharide-stimulated RAW264.7 macrophages were used to investigate the intracellular antioxidant activity of the extracts from the digested oat bran paste with 25% enrichment of blueberry or blackcurrant powder. The results indicated that the extracts of digested pastes prevented the macrophages from experiencing lipopolysaccharide (LPS)-stimulated intracellular reactive oxygen species accumulation, mainly by the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signalling pathway. These findings suggest that the bioactive ingredients from blueberry and blackcurrant powder enhanced the in vitro and intracellular antioxidant capacity of oat bran pastes, and these enriched pastes have the potential to be utilised in the development of the functional foods.

Sign in / Sign up

Export Citation Format

Share Document