scholarly journals Glutathione Ethyl Ester Protects In Vitro-Maturing Bovine Oocytes against Oxidative Stress Induced by Subsequent Vitrification/Warming

2020 ◽  
Vol 21 (20) ◽  
pp. 7547
Author(s):  
Tania García-Martínez ◽  
Meritxell Vendrell-Flotats ◽  
Iris Martínez-Rodero ◽  
Erika Alina Ordóñez-León ◽  
Manuel Álvarez-Rodríguez ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Ethyl Ester ◽  
Mitochondrial Activity ◽  
Cell Stage ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Mitochondrial Distribution ◽  
Targeted Gene Expression ◽  
Bovine Oocytes

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.

Developmental potential of bovine oocytes following IVM in the presence of glutathione ethyl ester

2010 ◽  
Vol 22 (4) ◽  
pp. 597 ◽  
Author(s):  
E. C. Curnow ◽  
J. P. Ryan ◽  
D. M. Saunders ◽  
E. S. Hayes
Keyword(s):  
Oxidative Stress ◽  
Ethyl Ester ◽  
Embryo Development ◽  
Buthionine Sulfoximine ◽  
Cumulus Cells ◽  
Cell Number ◽  
Developmental Potential ◽  
Novel Approach ◽  
Bovine Oocytes

Glutathione (GSH) is synthesised during oocyte maturation and represents the oocyte’s main non-enzymatic defence against oxidative stress. Inadequate defence against oxidative stress may be related to poor embryo quality and viability. In the present study, bovine oocytes were matured in vitro in the presence of GSH ethyl ester (GSH-OEt), a cell permeable GSH donor, and its effects on subsequent fertilisation and embryo development were assessed. GSH-OEt significantly increased the GSH content of IVM oocytes without affecting fertilisation or Day 3 cleavage rates. Maturation in the presence of GSH-OEt did not significantly increase the blastocyst rate compared with control oocytes. However, 5 mM GSH-OEt treatment resulted in significantly higher blastocyst total cell number. The GSH level of IVM oocytes was significantly decreased in the absence of cumulus cells and when cumulus–oocyte complexes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine oocytes and restored the rate of normal fertilisation, but not embryo development, to levels seen in control oocytes. Thus, GSH-OEt represents a novel approach for effective in vitro elevation of bovine oocyte GSH and improvement in blastocyst cell number.

161 Dichloroacetate influences the mitochondrial activity of bovine oocytes impairing meiotic progression

2019 ◽  
Vol 31 (1) ◽  
pp. 205
Author(s):  
N. Pagano ◽  
K. Annes ◽  
C. De Canditiis ◽  
J. Ispada ◽  
B. Gasparrini ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pyruvate Dehydrogenase ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Meiotic Progression ◽  
Thermo Fisher Scientific ◽  
Maturation Rate ◽  
Bovine Oocytes ◽  
Fisher Scientific

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<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<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<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<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<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.

scholarly journals Aging-related Changes in In Vitro-matured Bovine Oocytes: Oxidative Stress, Mitochondrial Activity and ATP Content After Nuclear Maturation

2014 ◽  
Vol 60 (2) ◽  
pp. 136-142 ◽  
Author(s):  
Keisuke KOYAMA ◽  
Sung-Sik KANG ◽  
Weiping HUANG ◽  
Yojiro YANAGAWA ◽  
Yoshiyuki TAKAHASHI ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Activity ◽  
Atp Content ◽  
Nuclear Maturation ◽  
Bovine Oocytes

170 EXPRESSION PATTERN OF THE Sox2 GENE IN BOVINE OOCYTES AND IN VITRO-DERIVED EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 165 ◽  
Author(s):  
T. A. L. Brevini ◽  
S. Antonini ◽  
F. Cillo ◽  
G. Pennarossa ◽  
S. Colleoni ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Inner Cell Mass ◽  
False Negative ◽  
Cell Mass ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Developmental Potential ◽  
Sox2 Expression ◽  
Bovine Oocytes

Sox2 is a member of the Sox (SRY-related HMGbox) family. It acts to maintain developmental potential and marks the pluripotent lineage of the early mouse embryo; in particular, as in the case of Oct-4 and Nanog, Sox2 is expressed specifically in the inner cell mass (ICM) and in the epiblast of this species. Moreover, it plays an important role in the transcription network that maintains stem cell pluripotency, interacting with other factors such as Oct-4 and Nanog. Little information is available on this gene in bovine; therefore aims of the present study were: a) to identify and characterize the Sox2 expression profile in bovine oocytes and preimplantation embryos; and b) to investigate its expression pattern in ICM and trophectoderm (TE). Bovine oocytes and embryos were obtained by in vitro maturation and fertilization; blastocysts at Day 7 post-insemination underwent microsurgery to separate TE from ICM. mRNA was isolated from 3 pools, each consisting of 5 MII oocytes, 2-, 4-, 8-, and 16-cell embryos, morulae, blastocysts, ICMs, and TEs. Semi-quantitative analysis of Sox2 expression was performed in the exponential phase of PCR amplification using rabbit globin as exogenous control. Data were analyzed with one-way ANOVA, followed by multiple pairwise comparisons with Tukey test (SigmaStat 2.03, SPSS, Inc., Chicago, IL, USA). Values are presented as mean � SEM and differences of P ≤ 0.05 are considered significant. In order to rule out false negative results, PCR amplifications of isolated ICMs and TEs were extended to the plateau phase. Fragment identity was confirmed by sequencing. Comparison of bovine Sox2 cDNA sequence (EMBL AM774325) with databases revealed a 98%, 93%, and 87% homology with sheep, human, and mouse, respectively. Sox2 mRNA was detectable in oocytes as well as in embryos at the different developmental stages analyzed. Semi-quantitative expression studies revealed that Sox2 was present as both maternal and embryonic transcript; in particular, a statistically significant increase from the 8-cell stage, concomitant with embryo genome activation, was observed. Differently from the mouse, Sox2 was expressed in both bovine ICM and TE, resembling the profile previously shown for Oct-4 (van Eijk et al. 1999 Biol. Reprod. 60, 1093–1103), and suggesting that Sox2 expression might be regulated by Oct-4 also in bovine, as described in mouse and human. These findings also suggest that its expression may become restricted to the ICM only at the expanded hatched stage, as previously described for Oct-4 in pig embryos (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). This work was supported by PRIN 2006, FIRST 2005, TECLA-MIUR, and EUROSTELLS-ESF.

Antioxidant supplementation during in vitro culture improves mitochondrial function and development of embryos from aged female mice

2015 ◽  
Vol 27 (6) ◽  
pp. 975 ◽  
Author(s):  
Elena Silva ◽  
Alison F. Greene ◽  
Kevin Strauss ◽  
Jason R. Herrick ◽  
William B. Schoolcraft ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Blastocyst Stage ◽  
Mitochondrial Activity ◽  
Blastocyst Development ◽  
Female Mice ◽  
Expression Of Genes ◽  
Blastocyst Quality ◽  
Maternal Aging ◽  
Young Mice

Maternal aging results in reduced oocyte and blastocyst quality, thought to be due, in part, to mitochondrial dysfunction and accumulation of reactive oxygen species. To reduce oxidative stress, the antioxidants α-lipoic acid (ALA; 10 µM), α-tocopherol (250 µM), hypotaurine (1 mM) and N-acetylcysteine (NAC; 1 mM), and sirtuin (100 ng mL–1) were added to embryo culture medium (AntiOX) and compared with a control (CON) without antioxidants to assess blastocyst development after in vitro maturation and fertilisation of oocytes from aged B6D2F1 female mice (13.5 months). Development to the blastocyst stage increased in the AntiOX compared with CON group (87.6% vs 72.7%, respectively; P < 0.01), in addition to higher mitochondrial membrane potential and ATP levels in the AntiOX group. Expression of genes associated with oxidative stress (PI3K, FOXO3A and GLRX2) was upregulated in the CON compared with AntiOX group. In addition to AntiOX, a medium containing only NAC and ALA (rAntiOX) was used to culture embryos from young CF1 females (6–8 weeks). More blastocysts developed in the rAntiOX compared with CON group (64.1% vs 43.3%, respectively; P < 0.01), although AntiOX (48.0% blastocysts) did not result in improved development in young mice. Antioxidants improved mitochondrial activity, gene expression and development in embryos of older female mice, whereas a reduced level of antioxidants during culture was beneficial to embryos from young mice.

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

2021 ◽  
Vol 9 ◽  
Author(s):  
Elisa Mariano Pioltine ◽  
Camila Bortoliero Costa ◽  
Laís Barbosa Latorraca ◽  
Fernanda Fagali Franchi ◽  
Priscila Helena dos Santos ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Er Stress ◽  
Mitochondrial Activity ◽  
Ros Production ◽  
High Concentration ◽  
Tauroursodeoxycholic Acid ◽  
Chemical Chaperone ◽  
Nuclear Maturation ◽  
Bovine Oocytes

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.

scholarly journals Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karina Cañón-Beltrán ◽  
Yulia N. Cajas ◽  
Serafín Peréz-Cerezales ◽  
Claudia L. V. Leal ◽  
Ekaitz Agirregoitia ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Akt Signaling Pathway ◽  
Cell Stage ◽  
Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

scholarly journals NRF2-mediated signaling is a master regulator of transcription factors in bovine granulosa cells under oxidative stress condition

2021 ◽  
Author(s):  
Mohamed Omar Taqi ◽  
Mohammed Saeed-Zidane ◽  
Samuel Gebremedhn ◽  
Dessie Salilew-Wondim ◽  
Ernst Tholen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Proliferation ◽  
Endoplasmic Reticulum ◽  
Transcription Factors ◽  
Endoplasmic Reticulum Stress ◽  
Granulosa Cells ◽  
Mitochondrial Activity ◽  
Small Interference Rna ◽  
Nrf2 Signaling Pathway

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.

scholarly journals Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3111
Author(s):  
Po-Yu Lin ◽  
Denny Yang ◽  
Chi-Hsuan Chuang ◽  
Hsuan Lin ◽  
Wei-Ju Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Functional Features ◽  
Early Mouse ◽  
Extraembryonic Tissues

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

Sign in / Sign up

Export Citation Format

Share Document