172 ADMINISTRATION OF GLUTATHIONE OR THIOREDOXIN TO MEDIUM REDUCES INTRACELLULAR REDOX STATUS AND IMPROVES EMBRYONIC DEVELOPMENT TO THE BLASTOCYST STAGE OF PORCINE IVM/IVF OOCYTES

Abstract

Successful in vitro production of blastocysts from immature oocytes can be carried out using in vitro oocyte maturation (IVM), fertilization (IVF), and embryo culture (IVC) at a high level of repeatability in the porcine. However, the rates of in vitro development of IVM/IVF oocytes to the blastocyst stage remained around 20%. The environment in vitro is so simple and materially limited that there exist several stressors in vitro that disturb normal embryo development. Oxidative stress, which is caused by excess production of reactive oxygen species, is a major disturbing factor for the development of pre-implantation embryos in vitro. The series of present experiments were conducted using culture conditions with enhanced reducing capacity by the addition of glutathione (GSH) or thioredoxin to the culture medium to monitor developmental competence of porcine embryos and to verify their intracellular redox status. Cumulus-oocyte complexes were obtained from ovaries recovered from prepubertal gilts. Putative zygotes were produced by IVM of oocytes, followed by IVF (designated as Day 0). They were then cultured in modified NCSU-37 media containing GSH or thioredoxin as an antioxidant, or without any antioxidant (control), and blastocyst development rates on Day 6 were monitored. In addition, intracellular GSH content as a reducing parameter and intracellular H₂O₂ level as an oxidative parameter were measured; the intracellular redox status in the embryo was verified by the ratio of the GSH to the H₂O₂. Measurements in each group were replicated six times. Percentages of the embryos that developed to the blastocyst stage were significantly increased when 0.5 or 1.0 µM GSH (29.6 ± 2.7% or 30.4 ± 3.5%, and P < 0.05 or 0.01, respectively) or 1.0 mg/mL thioredoxin (30.6 ± 2.4%, P < 0.01) was added to the medium compared to the percentage in the control group (20.1 ± 2.2%). Intracellular redox status in embryos at the 8- to 12-cell stage or blastocysts was drastically reduced in GSH- or thioredoxin-added groups compared to that in the control group (P < 0.05 to 0.001). Furthermore, GSH or thioredoxin addition to the medium increased total cell numbers (48.3 ± 2.1 to 49.2 ± 2.1) and lowered ratios of apoptotic cells (6.2 ± 0.6% to 7.0 ± 0.7%) in blastocyst compared to those values in the control group (P < 0.05; cell number = 39.3 ± 2.0, apoptosis rate = 11.1 ± 1.1%) (37 to 53 embryos in each group were used for the TUNEL assay). These results suggest that the administration of GSH or thioredoxin to the culture medium improves in vitro embryonic development after IVM/IVF of oocytes, and that these beneficial effects are associated with maintenance of the intracellular redox status in a reduced state in porcine embryos.