42272e38e0e7fc5caa66bd62add51575 intjoumolsci_a2020v21n20p7547.pdf 3923723d986060bd23349403773d6bb7db829363 intjoumolsci_a2020v21n20p7547.pdf 45d26e57b47cf852c82ebd000b2a2d17453dcf4e7ac508150d76b62aa9e5805e intjoumolsci_a2020v21n20p7547.pdf Title: Glutathione Ethyl Ester Protects In Vitro-Maturing Bovine Oocytes against Oxidative Stress Induced by Subsequent Vitrification/Warming Subject: 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-) 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. Keywords: cow; reactive oxygen species; cryopreservation; cryotop; spindle configuration; mitochondria; embryo development; gene expression Author: Tania García-Martínez, Meritxell Vendrell-Flotats, Iris Martínez-Rodero, Erika Alina Ordóñez-León, Manuel Álvarez-Rodríguez, Manel López-Béjar, Marc Yeste and Teresa Mogas Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Thu Oct 15 04:15:15 2020 CEST ModDate: Thu Oct 15 04:21:07 2020 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2878495 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- QZVAGO+VnURWPalladioL Type 1 Custom yes yes no 10 0 AMQXJC+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 EKIUHZ+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 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PDFMetadata: Objects: 640 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Meiotic Spindle Status of Vitrified/Warmed Bovine Oocytes after Maturation in IVM Medium Supplemented with GSH-OEt Destination: subsection.2.1 Item: Title: ROS Production Detected in Vitrified/Warmed Bovine Oocytes after Their Maturation in IVM with or without GSH-OEt Destination: subsection.2.2 Item: Title: Mitochondrial Activity and Distribution in Vitrified/Warmed Bovine Oocytes after Their Maturation in IVM Medium Supplemented with GSH-OEt Destination: subsection.2.3 Item: Title: Gene Expression in Vitrified/Warmed Bovine Oocytes after Their Maturation in IVM Medium Supplemented with GSH-OEt Destination: subsection.2.4 Item: Title: Developmental Competence and Gene Expression Determined in Embryos Derived from Vitrified/Warmed Bovine Oocytes In Vitro Matured in IVM Medium with or without GSH-OEt Destination: subsection.2.5 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Chemicals and Suppliers Destination: subsection.4.1 Item: Title: Oocyte Collection and In Vitro Maturation Destination: subsection.4.2 Item: Title: Oocyte Vitrification and Warming Destination: subsection.4.3 Children: Item: Title: Vitrification Protocol Destination: subsubsection.4.3.1 Item: Title: Warming Protocol Destination: subsubsection.4.3.2 Item: Title: In Vitro Fertilization and Embryo Culture Destination: subsection.4.4 Item: Title: Chromosome and Spindle Organization Destination: subsection.4.5 Item: Title: Reactive Oxygen Species Destination: subsection.4.6 Item: Title: Mitochondrial Activity and Distribution Destination: subsection.4.7 Item: Title: RNA Extraction, Reverse Transcription and Quantitative Real-Time PCR Analysis Destination: subsection.4.8 Item: Title: Experimental Design Destination: subsection.4.9 Children: Item: Title: Meiotic Spindle Status, ROS Production, Mitochondrial Activity and Distribution and Gene Expression Examined in Vitrified/Warmed Bovine Oocytes after Maturation in IVM Medium with or without GSH-OEt Destination: subsubsection.4.9.1 Item: Title: Developmental Competence and Gene Expression Determined in Embryos Derived from Vitrified/Warmed Bovine Oocytes In Vitro Matured in IVM Medium with or without GSH-OEt Destination: subsubsection.4.9.2 Item: Title: Statistical Analyses Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Glutathione Ethyl Ester Protects In Vitro-Maturing Bovine Oocytes against Oxidative Stress Induced by Subsequent Vitrification/Warming Author: Tania García-Martínez, Meritxell Vendrell-Flotats, Iris Martínez-Rodero, Erika Alina Ordóñez-León, Manuel Álvarez-Rodríguez, Manel López-Béjar, Marc Yeste and Teresa Mogas Subject: 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-) 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. 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