80 REACTIVE OXYGEN SPECIES LEVEL IN PIG EMBRYOS CULTURED IN PRESENCE OF HYALURONAN

2016 ◽  
Vol 28 (2) ◽  
pp. 169
Author(s):  
B. Gajda ◽  
M. Kucia ◽  
Z. Smorag ◽  
M. Romek

It has been reported that during in vitro embryo culture reactive oxygen species (ROS) are generated and are detrimental to embryo development. A recent study (Smorag et al., Proc. 9th ICPR, 2013, 110) demonstrated that an addition of 1 mg mL–1 of hyaluronan (HA) to porcine embryo culture medium improves the development of zygote to blastocyst stage and the quality of produced embryos. Moreover, the embryos cultured with HA showed lower inner mitochondrial membrane potential (Romek et al., 2015 Proc. Symp. Progress in Cell Biology: Mitochondria and Chloroplast, Krakow, 31). Based on the beneficial effect provided by supplementation of HA during embryo culture, we investigated the ROS level in porcine embryos cultured with HA. Porcine zygotes were obtained surgically after flushing the oviducts of superovulated and inseminated gilts. In the experimental group, zygotes were cultured up to the blastocyst stage in NCSU-23 medium supplemented with 1 mg mL–1 of HA (CROMA, Pharma GmbH, Leobendorf, Austria), in an atmosphere containing 5% CO2 in air, at 39°C. In the control group, HA supplementation was omitted. To measure ROS level, embryos at the stages 2–4 and 8–16 cell, morula, and blastocyst (experimental group) and zygote, 2–4 and 8–16 cell, morula, and blastocyst (control group) were labelled with 5 μM CM-H2DCFDA dye (Molecular Probes Inc., OR, USA) for 30 min at 39°C. Labelled embryos were then examined under a Nikon Eclipse microscope with a CCD camera. The total amount of fluorescence emitted from each individual embryos and proportional to the ROS level was measured in arbitrary units. The data were analysed using one-way ANOVA and post-hoc Tukey test. ROS level (mean ± standard error of the mean) in the experimental group was 8.21 ± 2.65 (n = 25), 10.31 ± 3.13 (n = 18), 9.08 ± 2.89 (n = 21), and 20.45 ± 2.38 (n = 31) for 2–4 cell, 8–16 cell, morula, and blastocyst, respectively, whereas in the control group was 9.15 ± 3.43 (n = 15), 7.11 ± 3.13 (n = 18), 8.67 ± 3.04 (n = 19), 11.47 ± 2.46 (n = 29), and 54.74 ± 2.89 (n = 21) for zygote, 2–4 cell, 8–16 cell, morula, and blastocyst, respectively. For experimental and control groups, ROS levels remained unchanged up to morula. On the contrary, at the blastocyst stage from the experimental group ROS level decreased significantly (P ≤ 0.05) in comparison with blastocysts from the control group. In conclusion, porcine blastocysts derived from zygotes cultured with supplementation of 1 mg of HA possess a significantly lower ROS level than blastocysts cultured without HA. This suggests that HA supplementation in culture medium can reduce the ROS level in porcine cultured blastocysts. The project was funded by the National Science Center based on decision number DEC-2012/07/B/NZ9/01326.

2016 ◽  
Vol 28 (2) ◽  
pp. 169
Author(s):  
M. Romek ◽  
M. Kucia ◽  
B. Gajda ◽  
Z. Smorag

Our recent study (Romek et al., Proc. of 29th Scientific Meeting of A.E.T.E., 2013, 196) demonstrated that high hydrostatic pressure (HHP) decreased the potential of the inner mitochondrial membrane in porcine embryos from morula to blastocyst stage. Therefore, the aim of this study was to find out if HHP treatment of cultured porcine embryos has an effect on production of reactive oxygen species (ROS) in these cells. Gilts were superovulated and inseminated using standard methods. Then zygotes were surgically collected after flushing the oviducts of the donors gilts 22 to 24 h after insemination. Obtained zygotes were cultured in NCSU-23 (North Carolina State University-23) medium up to the blastocyst stage, in an atmosphere containing 5% CO2 in air at 39°C. In the experimental group, embryos at zygote, 2- to 4-cell, 8- to 16-cell, morula and blastocyst stages were treated with 20 MPa of hydrostatic pressure (HHP100, Cryo-Innovation Ltd., Szeged, Hungary) for 60 min at 39°C with an interval of 60 min between HHP treatment and subsequent embryo staining. For the control group of embryos at the same stage of development, the HHP treatment was omitted. An additional group of blastocysts derived after culture was analysed 4 h after the HHP treatment. ROS level was measured using 5 μM CM-H2DCFDA fluorescent dye (Molecular Probes Inc., Eugene, OR, USA). Embryos from the experimental and control groups were stained for 30 min at 39°C and then analysed under a Nikon Eclipse microscope equipped with a CCD camera. The total amount of fluorescence emitted from each individual embryo was measured. The data (in arbitrary unit) were analysed using one-way ANOVA and post-hoc Tukey test. After HHP zygote treatment, the percentage of obtained blastocysts was 67.01, whereas in control group it was 63.95 (P < 0.05). ROS level proportional to the measured amount of fluorescence (mean ± standard error of the mean) was 9.15 ± 2.70 (n = 15), 7.11 ± 2.46 (n = 18), 8.67 ± 2.4 (n = 19), 11.47 ± 1.94 (n = 29), and 54.74 ± 2.28 (n = 21) for zygote, 2- to 4-cell, 8- to 16-cell, morula, and blastocyst stage of the control group, respectively. After HHP treatment, the ROS level was 7.39 ± 2.4 (n = 19), 6.66 ± 2.28 (n = 21), 9.14 ± 2.61 (n = 16), 7.23 ± 2.28 (n = 21), 33.06 ± 2.4 (n = 19) for zygote, 2- to 4-cell, 8- to 16-cell, morula, and blastocyst stage, respectively, and 35.57 ± 2.4 (n = 10) for blastocyst 4 h after HHP treatment. In conclusion, (1) HHP treatment of porcine zygotes improve embryo developmental potential; (2) ROS level in both control and experimental groups remained unchanged up to morula stage, whereas at the blastocyst stage, after HHP treatment ROS level significantly decreased (P < 0.05) in comparison with nontreated blastocysts; (3) HHP treatment on porcine blastocysts resulted in a lowered level of ROS that remained unchanged for 4 h. These results suggest that HHP treatment could improve the quality of cultured porcine blastocysts. The project was funded by the National Science Centre based on decision number DEC-2012/07/B/NZ9/01326.


2007 ◽  
Vol 19 (1) ◽  
pp. 208
Author(s):  
N. W. K. Karja ◽  
K. Kikuchi ◽  
M. Ozawa ◽  
M. Fahrudin ◽  
T. Somfai ◽  
...  

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 �M (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 � 2.3, 39.3 � 1.7, and 42.5 � 2.7, respectively) was significantly higher (P &lt; 0.01) than those in the control (33.4 � 1.6) and DPI-0.5 (32.7 � 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P &lt; 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 �M significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.


2007 ◽  
Vol 61 (1-2) ◽  
pp. 43-52 ◽  
Author(s):  
Tsvetan Chaprazov ◽  
Ivan Borissov ◽  
Evgeni Slavov ◽  
Petco Dzhelebov

Staphylococcus aureus is the leading pathogenic cause of nosocomial infections, especially in bacteriaemia and sepsis. Phagocytosis is known to be an essential factor of innate immunity. The aim of this study was to evaluate phagocytic activity of leucocytes in experimental bacteriaemia. Bacteriaemia was induced in six adult male mongrel dogs (experimental group) by intravenous injection of Staphylococcus aureus isolate (1.2x109 cells/ml). Phagocytic activity was evaluated before infection (0 h.), and on the 2nd, 6th, 24th, 48th hour, and on the 7th, 14th and 21st day after infection. Six control animals were tested in the same dynamics. Phagocytic activity was evaluated by using the Nitrotetrazolium blue reduction (NBT) test, and the immune fluorescence method (Samnaliev et al., 1995) for detection of phagocytic index and percentage of phagocyting leucocytes (FITC marked Staphylococcus aureus were used). Percentage of phagocyting leucocytes showed an increase in experimental animals, compared to control animals, on the 24th h. Production of reactive oxygen species, evaluated by NBT, showed changes within experimental group as follows: an increase on the 24th h. compared to the 2nd h., 6th h., and the 14th day after infection; and an increase on 48th h., compared to the 2nd h. and 14th day. Comparison of reactive oxygen species production between groups, revealed an increase in the experimental group, as compared to the control group, on the 24th h. and on the 48th h. In conclusion cell elements of innate immunity are mostly activated on the first two days after inducing Staphylococcus aureus bacteriaemia in dogs.


Author(s):  
Arnab Banerjee ◽  
Debasmita Das ◽  
Rajarshi Paul ◽  
Sandipan Roy ◽  
Ankita Bhattacharjee ◽  
...  

AbstractBackgroundIn the present era, obesity is increasing rapidly, and high dietary intake of lipid could be a noteworthy risk factor for the occasion of obesity, as well as nonalcoholic fatty liver disease, which is the independent risk factor for type 2 diabetes and cardiovascular disease. For a long time, high-lipid diet (HLD) in “fast food” is turning into part of our everyday life. So, we were interested in fulfilling the paucity of studies by means of preliminary evaluation of these three alternative doses of HLD on a rat model and elucidating the possible mechanism of these effects and divulging the most alarming dose.MethodsThirty-two rats were taken, and of these, 24 were fed with HLD in three distinctive compositions of edible coconut oil and vanaspati ghee in a ratio of 2:3, 3:2 and 1:1 (n = 8), orally through gavage at a dose of 10 mL/kg body weight for a period of 28 days, whereas the other eight were selected to comprise the control group.ResultsAfter completion of the experiment, followed by analysis of data it was revealed that hyperlipidemia with increased liver and cardiac marker enzymes, are associated with hepatocellular injury and cardiac damage. The data also supported increased proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α). As oxidative stress parameter increased in both liver and heart, there is also an increased in TNF-α due to an increased expression of inducible nitric oxide (NO) synthase, which led to a high production of NO. Moreover, HLD treatment explicitly weakens reasonability of hepatocytes and cardiomyocytes conceivably through G0/G1 or S stage capture or perhaps by means of enlistment of sub-G0/G1 DNA fragmentation and a sign of apoptosis.ConclusionsBased on the outcomes, it tends to be inferred that consequences of the present examination uncovered HLD in combination of 2:3 applies most encouraging systemic damage by reactive oxygen species generation and hyperlipidemia and necroapoptosis of the liver and heart. Hence, outcome of this study may help to formulate health care strategy and warns about the food habit in universal population regarding the use of hydrogenated and saturated fats (vanaspati ghee) in diet.


Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 994-999
Author(s):  
Y Niwa ◽  
T Sakane ◽  
Y Miyachi ◽  
T Kanoh ◽  
K Somiya

We assessed the generation of reactive oxygen species (ROS: O2-, H2O2, OH . , chemiluminescence) by neutrophils and monocytes from six patients with infectious mononucleosis, ten patients with other viral diseases, and ten normal controls. Neutrophils from infectious mononucleosis patients showed markedly decreased generation of all reactive oxygen species, compared with the two control groups; this abnormality persisted for four to eight weeks after disease onset. Monocytes from these patients generated normal levels of ROS. Normal neutrophils incubated with T lymphocytes from infectious mononucleosis patients generated significantly less of each ROS than did those incubated with T cells from either control group. T cell-mediated suppression of ROS generation required both OKT4+ cells from infectious mononucleosis patients and OKT8+ cells from either patients or normals. We conclude that the generation of reaction oxygen species in neutrophils is suppressed in patients with infectious mononucleosis, at least in part, by interacting subsets of T lymphocytes.


Author(s):  
Pei Zhang ◽  
Jing Liao ◽  
Xiaoju Wang ◽  
Zhengping Feng

IntroductionDiabetes and osteoporosis are common metabolic diseases. Abnormal high glucose can lead to the apoptosis of osteoblasts. Autophagy is a highly conserved cellular process that degrades proteins or organelles. In the present study, we comparatively analyzed the effects of high glucose and glucose fluctuation on apoptosis and autophagy of MC3T3-E1 osteoblasts.Material and methodsMC3T3-E1 cells were respectively treated with different concentrations of D-glucose: 5.5 mM for the control group, 25 mM for the high glucose group and 5.5/25 mM for the glucose fluctuation group.ResultsHigh glucose and glucose fluctuation decreased MC3T3-E1 proliferation and activated autophagy. Also, high glucose and glucose fluctuation might induce the production of reactive oxygen species, decline the mitochondrial membrane potential and trigger apoptosis. The differences in the glucose fluctuation treatment group were more significant. Moreover, N-acetylcysteine, an antioxidant reagent, dramatically eliminated the intracellular reactive oxygen species induced by high glucose and glucose fluctuation, and significantly inhibited the autophagy and apoptosis in MC3T3-E1 osteoblasts. Furthermore, treatment with chloroquine, an inhibitor of autophagy, significantly increased the apoptosis of MC3T3-E1 osteoblasts.ConclusionsHigh glucose, especially high glucose fluctuation, inhibits proliferation and promotes apoptosis and autophagy of MC3T3-E1 osteoblasts. This may occur through inducing oxidative stress and mitochondrial damage in the osteoblasts.


2021 ◽  
Vol 8 (32) ◽  
pp. 3023-3027
Author(s):  
Namrata Shrivastava ◽  
Vaibhav Shrivastava ◽  
Manish Pandey

BACKGROUND Infertility is defined as the inability to conceive after at 1 year of regular unprotected intercourse. Male contributes to almost half of infertility cases and in almost 30 % of cases, no definite aetiology is identified, and hence, male infertility is labelled idiopathic in these cases. Oxidative energy production mechanisms are almost always accompanied by reactive oxygen species (ROS), generation whose too much concentrations can lead to extensive protein damage and cytoskeletal modifications and inhibit cellular mechanisms. A number of laboratory techniques have been developed to evaluate oxidative stress by measuring ROS level in the semen. In recent times antioxidant supplements have been proposed as useful agents to increase the scavenging capacity of seminal plasma, controversy still surrounds their actual clinical utility. METHODS 34 male patients were included in this study. Reactive oxygen species detection was done by Flowcytometry using dichloroflurosecindiacetate (DCFH-DA). RESULTS The ROS in the patient group was found to be significantly higher 29.821 (5.6300 than the control group 22.162 (1.6331 having p value < 0.001). The ROS (29.821 ± 5.6300) was found to be significantly reduced after 3 months of antioxidant therapy which got reduced to 19.893 ± 4.2299 respectively. CONCLUSIONS Our study demonstrates that infertile men have significantly higher level of ROS (as measured by flowcytometry) & lower sperm count (oligospermia), decreased progressive & total motility and increased immotile sperms as compared to healthy fertile men. This study further proves that antioxidant therapy based on a combination of carnitine, zinc, coq10, lycopene and vitamin C & E for 3 months is associated with a decrease of ROS as measured by flowcytometry & a variable degree of improvement in above mentioned semen parameters. KEYWORDS Reactive Oxygen Species, Male Infertility


2015 ◽  
Vol 2015 ◽  
pp. 1-8
Author(s):  
Xiao-Tian Zhang ◽  
Chun-Jiang Yu ◽  
Jian-Wei Liu ◽  
Yan-Ping Zhang ◽  
Chao Zhang ◽  
...  

We analyzed the effects of a traditional Chinese medicine, Qizhi Jiangtang Jiaonang (QJJ), on insulin resistance (IR) in vitro. After an in vitro model of IR was established by treating human liver cancer cells (HepG2 cells) with palmitic acid, the cells were then treated with various concentrations of QJJ. Treatment with 400 µM palmitic acid for 24 h induced IR in HepG2 cells. The survival rate for HepG2 cells in the IR group was significantly lower than that of the untreated control group (P< 0.001); however, QJJ restored HepG2 cell survival (P< 0.001). As compared with HepG2 cells in the IR group, QJJ at all doses analyzed significantly increased glucose consumption (allP< 0.05). Moreover, treatment with all the QJJ doses significantly reduced the mean intracellular reactive oxygen species levels as compared with the IR group (allP< 0.05). Furthermore, high-dose QJJ reduced both TNF-αand IL-6 levels as compared to the IR group (allP< 0.05). QJJ ameliorated the altered PI3K, GLUT4, and RAGE expression observed with IR. In conclusion, QJJ can improve IR in HepG2 cells, which may be mediated through the IRS-1/PI3K/GLUT4 signaling pathway as well as regulation of NF-κB-mediated inflammation and oxidative stress.


2014 ◽  
Vol 26 (6) ◽  
pp. 797 ◽  
Author(s):  
Nathália A. S. Rocha-Frigoni ◽  
Beatriz C. S. Leão ◽  
Ériklis Nogueira ◽  
Mônica F. Accorsi ◽  
Gisele Z. Mingoti

The effects of intracellular (cysteine and β-mercaptoethanol) and extracellular (catalase) antioxidant supplementation at different times during in vitro production (IVM and/or in vitro culture (IVC)) on bovine embryo development, intracellular reactive oxygen species (ROS) levels, apoptosis and re-expansion rates after a vitrification–thawing process were examined. Blastocyst frequencies were not affected by either antioxidant supplementation (40.5%–56.4%) or the timing of supplementation (41.7%–55.4%) compared with control (48.7%; P > 0.05). Similarly, antioxidants and the moment of supplementation did not affect (P > 0.05) the total number of blastomeres (86.2–90.5 and 84.4–90.5, respectively) compared with control (85.7). However, the percentage of apoptotic cells was reduced (P < 0.05) in groups supplemented during IVM (1.7%), IVC (2.0%) or both (1.8%) compared with control (4.3%). Intracellular ROS levels measured in Day 7 blastocysts were reduced (P < 0.05) in all groups (0.60–0.78), with the exception of the group supplemented with β-mercaptoethanol during IVC (0.88), which did not differ (P > 0.05) from that in the control group (1.00). Re-expansion rates were not affected (P > 0.05) by the treatments (50.0%–93.0%). In conclusion, antioxidant supplementation during IVM and/or IVC reduces intracellular ROS and the rate of apoptosis; however, supplementation does not increase embryonic development and survival after vitrification.


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