scholarly journals Treatment of in vitro-Matured Bovine Oocytes With Tauroursodeoxycholic Acid Modulates the Oxidative Stress Signaling Pathway

Author(s):  
Elisa Mariano Pioltine ◽  
Camila Bortoliero Costa ◽  
Laís Barbosa Latorraca ◽  
Fernanda Fagali Franchi ◽  
Priscila Helena dos Santos ◽  
...  

In several species, oocyte and embryo competence are improved by the addition of endoplasmic reticulum (ER) stress inhibitors to in vitro maturation (IVM) medium and/or in vitro culture (IVC) medium. This study aimed to evaluate the effects of three concentrations of tauroursodeoxycholic acid (TUDCA; 50, 200, and 1,000 μM), a chemical chaperone for relieving ER stress, during IVM of bovine cumulus–oocyte complexes (COCs) for 24 h. Treated oocytes were analyzed for nuclear maturation, reactive oxygen species (ROS) production, mitochondrial activity, and abundance of target transcripts. In addition, the number of pronuclei in oocytes was evaluated after 18–20 h of insemination, and the rates of blastocyst and hatched blastocyst formation were evaluated after 7 and 8/9 days of culture, respectively. We further evaluated the transcript abundance of embryonic quality markers. Our findings showed that supplementation of IVM medium with 200 μM of TUDCA decreased ROS production and increased abundance of transcripts related to antioxidant activity in oocytes (CAT, GPX1, and HMOX1) and embryos (GPX1 and PRDX3). Interestingly, high concentration of TUDCA (1,000 μM) was toxic to oocytes, reducing the nuclear maturation rate, decreasing mitochondrial activity, and increasing the abundance of ER stress (HSPA5) and cellular apoptosis (CASP3 and CD40) related transcripts. The results of this study suggest that treatment with 200 μM of TUDCA is associated with a greater resistance to oxidative stress and indirectly with ER stress relief in bovine oocytes.

2014 ◽  
Vol 60 (2) ◽  
pp. 136-142 ◽  
Author(s):  
Keisuke KOYAMA ◽  
Sung-Sik KANG ◽  
Weiping HUANG ◽  
Yojiro YANAGAWA ◽  
Yoshiyuki TAKAHASHI ◽  
...  

2015 ◽  
Vol 36 (6Supl2) ◽  
pp. 4277 ◽  
Author(s):  
Angelo Bertani Giotto ◽  
Daniela Dos Santos Brum ◽  
Francielli Weber Santos ◽  
Antonio Carlos Galarça Guimarães ◽  
Cibele Garcia Moreira Gonçalves ◽  
...  

<p>Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20%) with different oocyte densities (1:10?l or 1:20?l) in the <em>in vitro </em>maturation (IVM) of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH) and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P&gt;0.05). In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P&lt;0.05). Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P&lt;0.05). In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P&lt;0.05). Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte density (P&lt;0.05). In conclusion, the results of this study indicate an interaction between oxygen tension and oocyte density, which increases ROS production in certain associations and subsequently influences the rates of <em>in vitro </em>fertilization of bovine oocytes. The improved rates of IVF were obtained when IVM was conducted using 20% oxygen tension and high oocyte density (1:20 ul).</p>


2015 ◽  
Vol 36 (6Supl2) ◽  
pp. 4277
Author(s):  
Angelo Bertani Giotto ◽  
Daniela Dos Santos Brum ◽  
Francielli Weber Santos ◽  
Antonio Carlos Galarça Guimarães ◽  
Cibele Garcia Moreira Gonçalves ◽  
...  

Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20%) with different oocyte densities (1:10?l or 1:20?l) in the in vitro maturation (IVM) of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH) and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P>0.05). In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P<0.05). Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P<0.05). In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P<0.05). Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte density (P<0.05). In conclusion, the results of this study indicate an interaction between oxygen tension and oocyte density, which increases ROS production in certain associations and subsequently influences the rates of in vitro fertilization of bovine oocytes. The improved rates of IVF were obtained when IVM was conducted using 20% oxygen tension and high oocyte density (1:20 ul).


2019 ◽  
Vol 31 (1) ◽  
pp. 205
Author(s):  
N. Pagano ◽  
K. Annes ◽  
C. De Canditiis ◽  
J. Ispada ◽  
B. Gasparrini ◽  
...  

Pyruvate is a key energy substrate for the oocyte during maturation and acquisition of developmental competence. Mitochondrial activity is also essential for oocyte competence. Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase that indirectly stimulates pyruvate dehydrogenase (PDH), increasing pyruvate oxidation. PDH converts pyruvate into acetyl coenzyme A (acetyl-CoA) and thereby modulates the entry of glucose-derived carbons into the tricarboxylic acid (TCA) cycle, the main ATP production pathway within the oocyte. It was reported that DCA addition to embryo culture media improves embryo development in aged mice, by enhancing mitochondrial membrane potential (MMP) and decreasing oxidative stress (McPherson et al. 2014 Fertil. Steril. 101, 1458-1466). We hypothesised that increased pyruvate metabolism through the oxidative pathway, by stimulating PDH activity with DCA, could influence in vitro oocyte maturation. The aim of this work was to evaluate the effect of different concentrations of DCA during in vitro maturation (IVM) of bovine oocytes on maturation rate and mitochondrial activity, by assessing MMP and levels of flavin adenine dinucleotide (FADH2), nicotinamide adenine dinucleotide hydride (NADH), and reactive oxygen species (ROS). Abattoir-derived bovine cumulus-oocytes complexes (COC; n=360, over 4 replicates) were in vitro-matured with 0 (Control; n=120), 0.5mM (n=120) and 5mM (n=120) of DCA. After maturation, all matured COC were denuded by mechanical pipetting and meiotic progression was assessed by Hoechst 33342 staining and MMP by MitoTracker Red CMXRos test (Thermo Fisher Scientific, Waltham, MA, USA). Moreover, FADH2 and NADH levels were evaluated by autofluorescence (Dumollard et al. Development 134, 455-465) and ROS levels by CellRox® Green test (Thermo Fisher Scientific). Data were analysed by ANOVA, and the Tukey post hoc test was used to evaluate the difference among groups. The α-level was set at 0.05. Treatment with both concentrations of DCA decreased maturation rate (86.1, 67.8, and 67.6% in 0, 0.5, and 5mM groups, respectively; P&lt;0.05). The MMP increased in oocytes matured with the highest concentration of DCA (3.42±0.28, 4.44±0.51, and 6.32±0.89 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P&lt;0.05). In line with this, higher levels of FADH2 (3.16±0.15, 3.96±0.24, and 3.83±0.20 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P&lt;0.05) and NADH (3.86±0.14, 4.80±0.16, and 4.95±0.17 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P&lt;0.05) were found in both DCA-treated groups compared with the control. Unexpectedly, ROS levels increased in the presence of DCA (0.9±0.07, 1.30±0.12, and 1.54±0.16 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P&lt;0.05) compared with the control. These results suggest that DCA was effective in stimulating mitochondrial activity of bovine oocytes, but also resulting in increased oxidative stress that likely accounts for the decreased maturation rate. Therefore, alternative strategies should be identified for the manipulation of the oocyte metabolic profile to improve oocyte developmental competence.


2013 ◽  
Vol 25 (1) ◽  
pp. 274
Author(s):  
A. B. Giotto ◽  
A. C. G. Guimarães ◽  
C. G. M. Gonçalves ◽  
N. P. Folchini ◽  
C. I. I. U. F. Machado ◽  
...  

The reactive oxygen species (ROS) produced by animal cells and at physiological levels are responsible for several cellular functions. However, when there is an imbalance between ROS production and the antioxidant system in the cell, oxidative stress occurs and causes severe cell damage. In oocytes, ROS can affect the dynamics of maturation and early embryo development processes. Oxygen tension and the density of oocytes by medium volume during in vitro maturation (IVM) can influence ROS production. The aim of this study was to evaluate the influence of the association between oxygen tension (5 or 20%) and different oocyte densities during IVM (1 : 10 or 1 : 20 oocytes µL–1 of medium) on the ROS levels in oocytes and medium. Bovine oocytes (n = 420) were obtained from slaughterhouse ovaries by aspiration of 2- to 8-mm follicles. Quality I and II oocytes (De Loss et al. 1989 Gamete Res. 24, 197–204) were homogeneously distributed into groups of 15 oocytes per treatment: Treatment (T) 1 = 1 : 10 in 5% of O2; T2 = 1 : 10 in 20% of O2; T3 = 1 : 20 in 5% of O2; and T4 = 1 : 20 in 20% of O2. The oocytes were matured in TCM-199 supplemented with 10% oestrous mare serum, 100 µg mL–1 of epidermal growth factor, 50 µg mL–1 of LH, 5 µg mL–1 of FSH, and 22 µg mL–1 of pyruvate for 22 to 24 h at 39°C, in 5% CO2 and saturated humidity. To assay ROS production, denuded oocytes and 60-µL samples of IVM medium were evaluated by the spectrofluorometric method with 2′7′-dichlorofluorescein-diacetate, in which the fluorescence intensity emission was considered an indicator of ROS production and measured by a spectrofluorophotometer. The ROS production in oocytes and in IVM medium was expressed as units of fluorescence (UF); data were analysed by ANOVA and Duncan’s test with a 5% level of significance. Seven replications were performed. In treatment groups T1 and T3, the ROS production in oocytes was higher (P < 0.05) than in oocytes of treatment groups T2 and T4 (13.53 and 18.78 UF v. 7.92 and 6.15 UF, respectively). The ROS production in IVM medium was higher in the T1 (23.86 UF) and T2 (24.12 UF) treatment groups than in the T3 (18.78 UF) and T4 (18.57 UF) treatment groups. These results suggest an increase in ROS production in IVM oocytes under a 5% O2 atmosphere in relation to a 20% O2 atmosphere, irrespective of the oocyte density by volume of IVM medium. On the other hand, the accumulation of ROS in IVM medium seemed higher when the oocyte density was 1 oocyte to 10 µL of IVM medium, independent of the oxygen tension used. A higher level of ROS in 5% O2 tension may be caused by competition for O2 between oocyte and cumulus cells, causing a reduction in O2 levels and changing the availability of O2 to energy generation in oocytes and consequently increasing ROS generation. In this respect, 5% O2 during IVM may contribute to the onset of oxidative stress in oocytes, which may compromise fertilization and early embryo development. Further research is necessary to clarify esterase activity in oocytes and the addition of exogenous peroxidase to validate the assay. Financial support: FAPERGS (1011575) and CNPq (501763/2009).


2020 ◽  
Vol 21 (20) ◽  
pp. 7547
Author(s):  
Tania García-Martínez ◽  
Meritxell Vendrell-Flotats ◽  
Iris Martínez-Rodero ◽  
Erika Alina Ordóñez-León ◽  
Manuel Álvarez-Rodríguez ◽  
...  

This study aimed to examine whether the addition of glutathione ethyl ester (GSH-OEt) to the in vitro maturation (IVM) medium would improve the resilience of bovine oocytes to withstand vitrification. The effects of GSH-OEt on spindle morphology, levels of reactive oxygen species (ROS), mitochondrial activity and distribution, and embryo developmental potential were assessed together with the expression of genes with a role in apoptosis (BAX, BCL2), oxidative-stress pathways (GPX1, SOD1), water channels (AQP3), implantation (IFN-τ) and gap junctions (CX43) in oocytes and their derived blastocysts. Vitrification gave rise to abnormal spindle microtubule configurations and elevated ROS levels. Supplementation of IVM medium with GSH-OEt before vitrification preserved mitochondrial distribution pattern and diminished both cytoplasmic and mitochondrial ROS contents and percentages of embryos developing beyond the 8-cell stage were similar to those recorded in fresh non-vitrified oocytes. Although not significantly different from control vitrified oocytes, vitrified oocytes after GSH-OEt treatment gave rise to similar day 8-blastocyst and hatching rates to fresh non-vitrified oocytes. No effects of GSH-OEt supplementation were noted on the targeted gene expression of oocytes and derived blastocysts, with the exception of GPX1, AQP3 and CX43 in derived blastocysts. The addition of GSH-OEt to the IVM medium before vitrification may be beneficial for embryo development presumably as the consequence of additional anti-oxidant protection during IVM.


2016 ◽  
Vol 28 (3) ◽  
pp. 349 ◽  
Author(s):  
Weiping Huang ◽  
Sung-Sik Kang ◽  
Katsuhisa Nagai ◽  
Yojiro Yanagawa ◽  
Yoshiyuki Takahashi ◽  
...  

The objective of this study was to investigate the dynamics of mitochondrial activity in in vitro-grown (IVG) bovine oocytes during pre-maturational culture (pre-IVM) and its relationship to their developmental competence upon being subjected to different pre-IVM durations. After 12-day IVG culture, oocytes were cultured for 0, 10 or 20 h with 3-isobutyl-1-methylxanthine (IBMX) as pre-IVM. Mitochondrial activity in IVG oocytes after 10 h pre-IVM was the highest among all the pre-IVM durations (P < 0.05). In addition, cleavage (79.4%) and blastocyst rates (38.9%) of embryos derived from IVG oocytes with 10 h pre-IVM were higher than those with 20 h pre-IVM (63.0 and 25.8%, respectively; P < 0.05) and similar to those of in vivo-grown oocytes (82.7 and 36.7%, respectively). To confirm the developmental ability of IVG oocytes with 10 h pre-IVM beyond the blastocyst stage in vivo, embryo transfer was attempted. Transferred embryos developed to the elongated embryonic stage (63.6%, 7/11) in the recipient uterus at Day 16 of oestrus, and a male calf was delivered (50%, 1/2). In conclusion, it was indicated that the mitochondrial activity of bovine IVG oocytes peaked at 10 h pre-IVM and was closely correlated with the nuclear maturation and developmental competences of IVG oocytes.


2021 ◽  
Vol 85 (3) ◽  
pp. 493-501
Author(s):  
Masashi Mikami ◽  
Ohba Takuya ◽  
Yuta Yoshino ◽  
Shinsuke Nakamura ◽  
Kenichi Ito ◽  
...  

ABSTRACT The Asian traditional medicinal plant Acorus calamus and its component α-asarone exhibited various biological activities, such as antiinflammation and antioxidant effects. In the present study, we investigated the in vitro effects of A. calamus extract and α-asarone on oxidative stress- and endoplasmic reticulum (ER) stress–induced cell death in hippocampal HT22 cells. A. calamus extract and α-asarone both significantly suppressed cell death induced by the oxidative stress inducer l-glutamate and ER stress inducer tunicamycin. A. calamus extract and α-asarone also significantly reduced reactive oxygen species (ROS) production induced by l-glutamate. Moreover, A. calamus extract and α-asarone suppressed the phosphorylation of protein kinase RNA-like ER kinase (PERK) induced by tunicamycin. These results suggest that A. calamus extract and α-asarone protect hippocampal cells from oxidative stress and ER stress by decreasing ROS production and suppressing PERK signaling, respectively. α-Asarone has potential as a potent therapeutic candidate for neurodegenerative diseases, including Alzheimer's disease.


Author(s):  
Mohamed Omar Taqi ◽  
Mohammed Saeed-Zidane ◽  
Samuel Gebremedhn ◽  
Dessie Salilew-Wondim ◽  
Ernst Tholen ◽  
...  

AbstractTranscription factors (TFs) are known to be involved in regulating the expression of several classes of genes during folliculogenesis. However, the regulatory role of TFs during oxidative stress (OS) is not fully understood. The current study was aimed to investigate the regulation of the TFs in bovine granulosa cells (bGCs) during exposure to OS induced by H2O2 in vitro. For this, bGCs derived from ovarian follicles were cultured in vitro till their confluency and then treated with H2O2 for 40 min. Twenty-four hours later, cells were subjected to various phenotypic and gene expression analyses for genes related to TFs, endoplasmic reticulum stress, apoptosis, cell proliferation, and differentiation markers. The bGCs exhibited higher reactive oxygen species accumulation, DNA fragmentation, and endoplasmic reticulum stress accompanied by reduction of mitochondrial activity after exposure to OS. In addition, higher lipid accumulation and lower cell proliferation were noticed in H2O2-challenged cells. The mRNA level of TFs including NRF2, E2F1, KLF6, KLF9, FOS, SREBF1, SREBF2, and NOTCH1 was increased in H2O2-treated cells compared with non-treated controls. However, the expression level of KLF4 and its downstream gene, CCNB1, were downregulated in the H2O2-challenged group. Moreover, targeted inhibition of NRF2 using small interference RNA resulted in reduced expression of KLF9, FOS, SREBF2, and NOTCH1 genes, while the expression of KLF4 was upregulated. Taken together, bovine granulosa cells exposed to OS exhibited differential expression of various transcription factors, which are mediated by the NRF2 signaling pathway.


2017 ◽  
Vol 44 (4) ◽  
pp. 288-293 ◽  
Author(s):  
Shiho Yamadera ◽  
Yuya Nakamura ◽  
Masahiro Inagaki ◽  
Isao Ohsawa ◽  
Hiromichi Gotoh ◽  
...  

Aim: To examine the effects of vitamin E-coated dialyzer on oxidative stress in vitro. Methods: A dialyzer with a synthetic polymer membrane (APS-11SA) and vitamin E-coated dialyzer (VPS-11SA) were connected to a blood tubing line, and U937 cells were circulated in the device. The circulating fluid was collected at 1, 2, 5, 10, 25, and 50 cycles, which are estimated numbers of passes through the dialyzer. Intracellular reactive oxygen species (ROS) production, malondialdehyde (MDA), and Cu/Zn-superoxide dismutase (SOD) were quantified. Results: Intracellular ROS production was increased in the first cycle by APS-11SA and was decreased throughout the experiment by VPS-11SA. Intracellular ROS production in the VPS-11SA device was lower, and MDA levels were decreased. MDA levels were lower during VPS-11SA processing than during APS-11SA processing. Cu/Zn-SOD levels remained unchanged. Conclusion: Our results highlight anti-oxidative-stress effects of a vitamin E-coated dialyzer.


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