title: Addition of glutathione or thioredoxin to culture medium reduces intracellular redox status of porcine IVM/IVF embryos, resulting in improved development to the blastocyst stage.
abstract: The present series of experiments investigated the effect of a reducing environment created by addition of reduced glutathione (GSH) or thioredoxin (TRX) to in vitro culture medium on the developmental competence of in vitro produced porcine embryos, and their intracellular redox status. Porcine cumulus-oocyte complexes were collected from ovaries matured and fertilized in vitro. The putative zygotes were then cultured for 6 days in modified NCSU-37 medium with or without (control) GSH or TRX, and their developmental competence was evaluated. In addition, the intracellular redox status of the cultured embryos was compared quantitatively using an index based on the ratio of the intracellular GSH content relative to the intracellular H(2)O(2) level. The proportion of embryos that developed to the blastocyst stage was significantly increased when 0.5 or 1.0 microM GSH (29.6% or 30.4%, P < 0.05 or 0.01, respectively) or 1.0 mg/ml TRX (30.6%, P < 0.01) was added to the medium compared to that without any supplementation (control; 20.1%). The intracellular redox status of embryos at the 8- to 12-cell stage or the blastocyst stage in the group cultured in the presence of GSH or TRX was significantly reduced in comparison with the control (P < 0.05 to 0.001). Furthermore, administration of GSH or TRX enhanced the total cell number (from 48.3 to 49.2) and lowered the proportion of apoptotic cells (from 6.2% to 7.0%) in blastocysts compared with the control (cell number 39.3; apoptosis rate 11.1%, P < 0.05). These results suggest that GSH or TRX can improve the in vitro development of porcine embryos, while maintaining an intracellular reductive status.
authors.affiliation: Genetic Diversity Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Ozawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagai
authors.forename: Mokhamad
authors.initials: M
authors.lastname: Fahrudin
authors.forename: Ni Wayan Kurniani
authors.initials: NW
authors.lastname: Karja
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kaneko
authors.forename: Junko
authors.initials: J
authors.lastname: Noguchi
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Ohnuma
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D000818:Animals; D017209:Apoptosis; D001755:Blastocyst / Q000502:physiology*; D003470:Culture Media / Q000737:chemistry*; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro; D005978:Glutathione / Q000008:administration & dosage / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D013552:Swine; D013879:Thioredoxins / Q000008:administration & dosage / Q000378:metabolism*
chemical_list: D003470:Culture Media; D016877:Oxidants; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D005978:Glutathione