200 EFFECTS OF MELATONIN ON REACTIVE OXYGEN SPECIES GENERATION AND ACQUISITION OF EMBRYONIC DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 231
Author(s):  
P. C. Dall'Acqua ◽  
B. C. S. Leão ◽  
N. A. S. Rocha-Frigoni ◽  
M. Ambrogi ◽  
G. Z. Mingoti
Keyword(s):  
Reactive Oxygen Species ◽  
Embryonic Development ◽  
Signal Intensity ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Control Group ◽  
Oxygen Species ◽  
Nuclear Maturation ◽  
Intracellular Ros

Reactive oxygen species (ROS) are produced under normal culture conditions, but when production increases, it generates a harmful condition called oxidative stress (OS), leading to apoptosis and developmental blocks. Addition of antioxidants as melatonin to culture media has been used to minimize the effects of OS. Our hypothesis was that melatonin could improve oocyte in vitro maturation (IVM) and protect oocytes from ROS under a standard culture condition, thus increasing embryonic development. To test, cumulus-oocyte complexes were matured in TCM-199 with bicarbonate, 0.5 mg mL–1 of FSH, 100 IU mL–1 of hCG, and 10% FCS without supplementation (control group) or supplemented with 10–5 (MT5), 10–7 (MT7) or 10–9 (MT9) M melatonin for 22 h at 38.5°C and 5% CO2 in air. After IVM, a sample of oocytes (control, n = 59; MT5, n = 64; MT7, n = 77; MT9, n = 57) was stained with 1 µg mL–1 Hoechst 33342 to assess the nuclear maturation, and oocytes were classified as being in the stages of germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I or telophase I (A/T), or metaphase II (MII). To determine the intracellular ROS levels, other matured oocytes (control, n = 46; MT5, n = 59; MT7, n = 51; MT9, n = 61) were stained with 5 µM CellROX®Green (Molecular Probes, Eugene, OR, USA) and were evaluated immediately under an epifluorescence inverted microscope (excitation 485 nm; emission 520 nm). The images were recorded and further analysed by Q-Capture Pro image software. After subtraction of the background signal intensity from the measured fluorescent signal intensity values, 1 group was chosen as a calibrator (control group), and each treatment value was divided by the mean calibrator value to generate the relative expression level (in arbitrary fluorescence units). Finally, another sample of matured oocytes (control, n = 188; MT5, n = 173; MT7, n = 180; MT9, n = 178) was submitted to IVF (Day = 0), and the presumptive zygotes were cultured in SOF at 38.5°C and 5% CO2 in air, for up to 7 days. The cleavage rates and embryonic development were evaluated at Days 3 and 7 of IVC, respectively. Data were analysed by ANOVA followed by Tukey’s test (P < 0.05) and are presented as mean ± SEM. There was no effect (P > 0.05) of different concentrations of MT on nuclear status of matured oocytes, as we found no differences in the rates of GV (0% to 5.3% ± 3.4), GVBD (5.4% ± 3.2 to 16.3% ± 5.0), MI (1.7% ± 1.7 to 3.2% ± 3.2), AI/TI (0% to 5.4% ± 3.4), and MII (74.8% ± 2.7 to 87.5% ± 3.7). The cleavage rates did not differ (P > 0.05) among treatments (76.7% ± 4.4 to 83.8% ± 2.7), as well as the embryonic development to the blastocyst stage (31.2% ± 1.9 to 43.7% ± 5.7). The intracellular ROS levels decreased significantly (P < 0.05) in the MT9 group (0.75 ± 0.03) in comparison to Control (1.0 ± 0.06), MT5 (0.97 ± 0.05) and MT7 (0.94 ± 0.05). In conclusion, supplementation with 10–9 M melatonin during IVM reduced the intracellular ROS levels of oocytes without interfering with the nuclear maturation and the subsequent embryonic development to the blastocyst stage. Financial support was provided by FAPESP (#2013/07382–6).

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 208
Author(s):  
N. W. K. Karja ◽  
K. Kikuchi ◽  
M. Ozawa ◽  
M. Fahrudin ◽  
T. Somfai ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Culture Media ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Ros Production ◽  
Oxygen Species

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.

Reduced levels of intracellular reactive oxygen species and apoptotic status are not correlated with increases in cryotolerance of bovine embryos produced in vitro in the presence of antioxidants

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
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Intracellular Reactive Oxygen Species ◽  
Control Group ◽  
Bovine Embryo ◽  
Oxygen Species ◽  
Antioxidant Supplementation ◽  
In Vitro Production ◽  
Intracellular Ros

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.

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.

203 SUPPLEMENTATION WITH LINOLENIC ACID, L-CARNITINE, ALONE OR ASSOCIATED, DURING IVM RESULTED IN DECREASE OF ROS LEVELS AND APOPTOTIC INDEX OF BOVINE IN VITRO PRODUCED EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 232
Author(s):  
B. C. S. Leão ◽  
N. A. S. R. Frigoni ◽  
P. C. Dall'Acqua ◽  
M. Ambrogi ◽  
G. Z. Mingoti
Keyword(s):  
Reactive Oxygen Species ◽  
Embryonic Development ◽  
Linolenic Acid ◽  
Lipid Content ◽  
Reactive Oxygen ◽  
Antioxidant Effect ◽  
Apoptotic Index ◽  
Control Group ◽  
Oxygen Species

Supplementation of in vitro maturation (IVM) medium with linolenic acid (ALA) has been used in order to reduce oocyte lipid content and have beneficial effects on maturation and acquisition of competence for embryonic development. Besides the effect of reducing cellular lipid content, l-carnitine (l-car) has an antioxidant effect by reducing the levels of reactive oxygen species (ROS) and protecting cells from apoptosis. However, the association of ALA and l-car has never been tested. This study was conducted to evaluate the effects of supplementation of IVM medium of bovine oocytes with ALA, l-car or the association of both (ALA+l-car) on embryonic development and blastocysts reactive oxygen species (ROS) levels and occurrence of apoptosis. Cumulus-oocyte complexes (n = 2241, in 11 replicates) were matured during 22 h at 38.5°C and 5% CO2 in air, in TCM-199 medium with bicarbonate, hormones and 10% FCS (control group), also supplemented with 100 μM ALA group; or 5 mM l-car (l-car group); or 100 μM ALA associated with 5 mM l-car (ALA+l-car group). After fertilisation (Day 0), zygotes were cultured 7 days in SOF that was supplemented with 0.5% BSA and 2.5% FCS, in 5% CO2 in air at 38.5°C. The cleavage and blastocysts rates were evaluated, respectively, at Days 3 and 7. Blastocysts were stained with 5 mM of H2DCFDA (Molecular Probes, Invitrogen, Carlsbad, CA, USA) and TUNEL (In Situ Cell Death Detection Kit, Roche Applied Science, Boston, MA, USA), to evaluate the ROS levels and the blastomers apoptotic index, respectively. The ROS (n = 115) and TUNEL (n = 102) stained blastocysts were evaluated under an epifluorescence microscope (excitation 495 nm/510–550 nm and emission 404 nm/590 nm), and the ROS levels (expressed as arbitrary fluorescence units) were measured by Q-Capture Pro image software (Q Imaging, Surrey, BC, Canada). The fluorescence intensity values were subtracted from mean values of background in the images. The variables were analysed by ANOVA followed by Tukey’s test (P < 0.05) and data are presented as mean ± s.e.m. There was no effect (P > 0.05) of the supplements during IVM on cleavage and blastocysts rates (%), respectively, for control (81.1 ± 1.8 and 29.0 ± 3.1), ALA (80.5 ± 2.1 and 29.7 ± 2.3), l-car (79.5 ± 2.8 and 29.2 ± 2.3), and ALA+l-car (82.2 ± 1.1 and 30.5 ± 2.0) groups. The oocytes supplementation resulted in a decrease (P < 0.05) in ROS levels for ALA (0.84 ± 0.04), l-car (0.85 ± 0.03) and ALA+l-car (0.82 ± 0.02) groups, compared to the Control (1.00 ± 0.05). Consequently, the percentage of apoptotic blastomeres decreased (P < 0.05) after ALA (6.9 ± 1.0%), l-car (7.5 ± 1.2%) and ALA+l-car (4.6 ± 0.7%) supplementations, unlike to the Control group (12.0 ± 1.2%). In conclusion, the supplementation with ALA, l-car or ALA+l-car during IVM did not affect the blastocyst development, but led to a reduction in ROS levels and in the apoptotic index of such blastocysts. These findings may be due to some antioxidant effect of these supplements in the oocytes and/or the produced embryos. Financial support was through FAPESP (#2012/10084–4 and #2013/07382–6).

283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

2006 ◽  
Vol 18 (2) ◽  
pp. 249 ◽  
Author(s):  
N. Maedomari ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
A. Takizawa ◽  
J. Noguchi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Control Group ◽  
Nuclear Maturation ◽  
First Polar Body

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P < 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P < 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P < 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P < 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P < 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P < 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

scholarly journals Qizhi Jiangtang Jiaonang Improves Insulin Signaling and Reduces Inflammatory Cytokine Secretion and Reactive Oxygen Species Formation in Insulin Resistant HepG2 Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-8
Author(s):  
Xiao-Tian Zhang ◽  
Chun-Jiang Yu ◽  
Jian-Wei Liu ◽  
Yan-Ping Zhang ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Palmitic Acid ◽  
Hepg2 Cells ◽  
Reactive Oxygen ◽  
Glucose Consumption ◽  
Control Group ◽  
High Dose ◽  
Oxygen Species ◽  
Insulin Resistant

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.

282 DIFFERENT CONCENTRATIONS OF FORSKOLIN FOR MEIOSIS BLOCK AND TO IMPROVE IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 230
Author(s):  
D. Paschoal ◽  
R. Maziero ◽  
M. Sudano ◽  
M. Guastali ◽  
L. Crocomo ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Least Squares ◽  
Control Group ◽  
In Vitro Production ◽  
Intact Cells ◽  
Significance Level ◽  
Nuclear Maturation ◽  
Oocyte Meiosis ◽  
Group Control

The inhibition of nuclear maturation allows time for the oocyte to accumulate molecules that are important for embryonic development. It was suggested that the inhibition of spontaneous nuclear IVM might allow for more time to accumulate the molecules important for embryonic development. The objective of this work was to evaluate blocking oocyte meiosis with the addition of forskolin. Slaughterhouse-derived bovine Zebu ovaries were collected and carried to the laboratory. Oocytes (n = 584) with at least 3 intact layers of cumulus cells and homogeneous cytoplasm were selected for IVM. The oocytes were transferred to drops of TCM 199 plus 10% FCS and hormones. The oocytes remained in IVM medium in 3 different concentrations of forskolin (6886), 0.1, 0.05, 0.025 mM, and a control group (withouth forskolin), for 6 h. Then they were maturated for an additional 18 h in forskolin-free medium. The first period above was an attempt to block (Block) and the second to resume (Res) the oocyte meiosis. The oocytes were incubated in a humidified atmosphere with 5% CO2 at 38.5°C in an air incubator. The oocytes were assessed for the stage of nuclear maturation, to see if they were in M II. Then oocytes were in vitro fertilized (IVF) with frozen Nelore bull semen (Bos taurus indicus). Presumptive zygotes (20–30/group) were cultured in SOFaa (synthetic oviducal fluid) supplemented with 5 mg mL–1 of BSA; the embryos were kept in an incubator with 5% CO2, 5% O2, and 90% N2 at 38.5°C and absolute humidity. On Day 7 (Day 0 = IVF) the blastocyst, the number of viable cells, and apoptosis rate (terminal deoxynucleotide transferase uridine nick-end labelling) were observed. Data were analysed with ANOVA using SAS PROC GLM (SAS Inst. Inc., Cary, NC, USA). Sources of variation in the model, including treatment and replication, were respectively considered fixed and random effects. If ANOVA was significant, the contrasts of means were performed using the least-squares difference. Data are presented as the mean and the standard error of least-squares. For all analyses, we used a significance level of 5%. No differences were observed for the stage of nuclear maturation of the oocyte (N = 336; control: 67.7 ± 8.3; F 0.025 mM, Block/Res: 67.7 ± 8.9; F 0.05 mM, Block/Res: 65.9 ± 9.8; F 0.1 mM, Block/Res: 50.2 ± 8.9), the blastocyst rate (N = 584; Control: 36.7 ± 3.7; F0.025 mM, Block/Res: 32.6 ± 3.7; F0.05 mM, Block/Res: 29.2 ± 3.7; F0.1 mM, Block/Res: 25.1 ± 3.7), and total number of intact cells (N = 10–15 embryos/group; Control:140.1 ± 13.0; F0.025 mM, Block/Res: 129.9 ± 13.0; F0.05 mM, Block/Res: 139.0 ± 13.0; F0.1 mM, Block/Res: 104.4 ± 13.0; P > 0.05). However, a higher rate of apoptosis was observed in the blastocysts produced from oocytes blocked for 6 h with the higher concentration of forskolin (N = 10–15 embryos/group): Control: 12.1 ± 2.5a; F 0.025 mM, Block/Res: 12.9 ± 2.5a; F0.05 mM, Block/Res: 13.5 ± 2.5a; F 0.1 mM, Block/Res: 30.2 ± 2.5b (P < 0.05). We conclude that all the experimental groups reached the stage of M II after the addition of forskolin and the highest concentration of forskolin caused cellular degeneration without harming embryo production on the seventh day.

337 MITOCHONDRIA AND REACTIVE OXYGEN SPECIES IN PREBUPERTAL LAMB OOCYTES BEFORE AND AFTER IN VITRO MATURATION

2010 ◽  
Vol 22 (1) ◽  
pp. 325
Author(s):  
M. E. Dell'Aquila ◽  
B. Ambruosi ◽  
R. Guastamacchia ◽  
F. Binetti ◽  
E. Ciani ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Female Reproductive Tract ◽  
Nuclear Chromatin ◽  
Oxygen Species ◽  
Intracellular Ros ◽  
Before And After ◽  
Group A ◽  
Group B

Juvenile in vitro embryo transfer (JIVET) reduces the generation interval and increases the rate of genetic gain. The developmental competence of in vitro-produced embryos is strictly related to oocyte quality. Oxidative stress in the oocyte is an emerging problem in reproductive in vitro technologies, due to the gas atmosphere used to incubate oocytes and the lack of physiological defense mechanisms available in the female reproductive tract. The major source of reactive oxygen species (ROS) is represented by mitochondria where ROS are produced during oxidative phosphorylation. The aim of the present study was to analyze mitochondria and ROS in ovine prepubertal oocytes before and after IVM in order to clarify their suitability in JIVET programs. Cumulus-oocyte complexes from the ovaries of 38 slaughtered prepubertal (less than 8 months of age) lambs of the Comisana breed were analyzed at retrieval (group A) or after IVM (group B; Ambruosi et al. 2009 Theriogenology 71, 1093-1104). After cumulus cell removal, all oocytes underwent nuclear chromatin, mitochondria and ROS evaluation by confocal analysis of fluorescence distribution and intensity. Hoechst 33258 and Mitotracker Orange CMTM Ros (Molecular Probes Inc., Eugene, OR) were used to label nuclear chromatin and mitochondria (Ambruosi et al. 2009) and 2′,7′-dichloro-dihydro-fluorescein diacetate was used for ROS labelling (Hashimoto et al. 2000 Mol. Reprod. Dev. 57, 353-360). Out of 65 oocytes from group A, 38 oocytes with regular size (>130 μm in diameter), morphology and nuclear chromatin at the GV stage were selected for analysis. One-hundred-thirty-eight oocytes underwent IVM (group B). Nuclear maturation rate (metaphase II with 1st polar body extruded) was 54%, 75/138. All MII oocytes were used for analysis. Significantly higher rate of oocytes from group B showed heterogeneous (large aggregates, clusters, pericortical, perinuclear) mitochondrial (mt) distribution pattern than oocytes from group A (55%, 41/75 v. 29%, 11/38, respectively; P < 0.05) which showed uniform distribution of small mt aggregates. Fluorescent intensity of mt labeling did not differ between groups (43.05 ± 16.15 v. 45.89 ± 10.36, for group A and B respectively; NS). In most of the oocytes from both groups, intracellular ROS were distributed in small or large aggregates (35/38, 92% and 62/75, 83%). No statistical difference was observed for intracellular ROS levels between oocytes from group A (66.36 ± 13.2) and group B (72.84 ± 20.63; NS). The culture conditions used in this study provided normal mt distribution and intracellular ROS levels. Qualitative and quantitative evaluation of mitochondria and intracellular ROS could be useful to improve in vitro culture methods in ovine prepubertal oocytes.

Supplementation of kaempferol to in vitro maturation medium regulates oxidative stress and enhances subsequent embryonic development in vitro

Zygote ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 59-64
Author(s):  
Yuhan Zhao ◽  
Yongnan Xu ◽  
Yinghua Li ◽  
Qingguo Jin ◽  
Jingyu Sun ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Caspase 3 ◽  
Treated Group ◽  
Control Group ◽  
Oxygen Species

SummaryKaempferol (KAE) is one of the most common dietary flavonols possessing biological activities such as anticancer, anti-inflammatory and antioxidant effects. Although previous studies have reported the biological activity of KAE on a variety of cells, it is not clear whether KAE plays a similar role in oocyte and embryo in vitro culture systems. This study investigated the effect of KAE addition to in vitro maturation on the antioxidant capacity of embryos in porcine oocytes after parthenogenetic activation. The effects of kaempferol on oocyte quality in porcine oocytes were studied based on the expression of related genes, reactive oxygen species, glutathione and mitochondrial membrane potential as criteria. The rate of blastocyst formation was significantly higher in oocytes treated with 0.1 µm KAE than in control oocytes. The mRNA level of the apoptosis-related gene Caspase-3 was significantly lower in the blastocysts derived from KAE-treated oocytes than in the control group and the mRNA expression of the embryo development-related genes COX2 and SOX2 was significantly increased in the KAE-treated group compared with that in the control group. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased after KAE treatment. Mitochondrial membrane potential (ΔΨm) was increased and the activity of Caspase-3 was significantly decreased in the KAE-treated group compared with that in the control group. Taken together, these results suggested that KAE is beneficial for the improvement of embryo development by inhibiting oxidative stress in porcine oocytes.

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