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DEVELOPMENT OF THE EMBRYO
Harborside C
7:30 AM-10:00 AM

(134) GLUTHATIONE SYNTHESIS BY BOVINE MORULAE AND BLASTOCYSTS PRODUCED IN VITRO.

Feugang, Jean¹, Sayoud, Hichem¹, Dessy, Franz¹, Donnay, Isabelle¹, ¹ Catholic University of Louvain, Louvain-la-Neuve, BE

ABSTRACT- Oxidative stress is increased in in vitro culture conditions and may contribute to the reduced efficiency of in vitro production of embryos. Moreover, the increase in embryo oxidative metabolism from compaction to blastulation could generate a rise in reactive oxygen species (ROS). Reduced glutathione (GSH) plays an important role in protecting the cell from these ROS. The aim of this study was to evaluate the variations in GSH content of IVP bovine embryos during compaction and blastulation in the presence of reducing agents or inhibitors of GSH synthesis. In vitro-derived bovine embryos were obtained from abattoir oocytes after IVM/IVF and IVC in SOF medium containing 5% fetal calf serum under 5% CO_2,, 5% O_2, and 90 % N_2,. Morulae were collected at day 5 post-insemination (pi) and cultured in the absence or in the presence of an inhibitor (0.4mM Buthionine sulfoximine - BSO) or a stimulator (0.1mM b-mercaptoethanol) of GSH synthesis. In parallel, blastocysts were produced without any selection at day 5 pi. Blastocysts of each group were collected at day 7 pi. GSH was evaluated in groups of 10 to 16 embryos (Day 5 morulae or Day 7 blastocysts) by HPLC separation (C-18 reversed-phase column) combined to fluorescence detection after derivatization with O-phthaldehyde. Experiments were repeated 5 times and each group was evaluated in duplicate. Mean GSH levels (mean±sem) were compared using ANOVA 1 followed by PLSD Fisher^'s test. GSH content of morulae (0.78±0.15 pmole/embryo) was not statistically different from in blastocysts. Selection of morulae at day 5 pi did not affect the GSH content of blastocysts evaluated at day 7 pi (0.42±0.16 pmole/embryo and 0.43±0.13 pmole/embryo for the blastocysts deriving from selected and non selected morulae, respectively). The level of GSH in blastocysts was significantly reduced by BSO (0.021±0.006 pmole/embryo) or increased by b-mercaptoethanol (1.66±0.16 pmole/embryo). In conclusion, bovine blastocysts synthesized glutathione. It is possible to stimulate this synthesis in order to reinforce endogenous protection of embryonic cells against ROS. HPLC system allows to evaluate GSH content in small numbers of embryos, compared to the currently used enzymatic methods.

KEY WORDS: Bovine embryo, In vitro culture, Gluthatione, Oxidative stress