Presence of antioxidant in vitro maturation medium and its effects on glutathione level, spindle area and rate of in vitro fertilization

ABSTRACT

Effect of different doses of cysteamine on rate of in vitro maturation [IVM], in vitro fertilization [IVF] and glutathione [GSH] level was studied. Metaphase II [MII] spindle area was analyzed for quantification of shape and size of oocytes. Female mice were primed with 5 IU of pregnant mare's stimulating gonadotrophin. Germinal vesicle [GV] oocytes were retrieved 48 hrs later. IVM medium was supplemented with 0, 50, 100, 200 and 500 mM of cysteamine. For IVM and IVF assessment in each group, 150 GV oocytes were used. Experiments also included a group of ovulated oocytes [matured in vivo] after priming with pregnant mare's stimulating gonadotrophin and human chorionic gonadotropin. GSH level was measured by 5, 5-Dithio-bis [2nitrobenzoic acid] DTNB-GR recycling protocol in GV and MII oocytes. For IVF, MII oocytes were inseminated with mature mouse sperm and rate of two-cell embryo was measured. For immunocytochemistry of microtubule and chromosomes, MII oocytes were fixed by methanol and immunostained with alpha-and beta-microtubule antibody and Hoechst. The spindle area was then analyzed. A dose-dependent improvement was observed in IVM and IVF rate. MII development and two-cell embryo formation were increased significantly in group which received 200 micro m cysteamine compare to the control group. GSH level was increased in presence of cysteamine in group which received 200 micro m cysteamine. Spindle area was increased in all groups in vitro except for the group which received 500 micro m cysteamine. The difference between spindle area in 200 micro m cysteamine and in vivo group was not significant [P>0.05]. Administration of cysteamine improves IVM and IVF rate in a dose-dependant manner. Also cysteamine induces glutathione synthesis in MII oocyte and improves microtubule when administered at a dose of 200 micro m. Therefore, addition of cysteamine as an antioxidant can improve IVM and IVF rate by increasing of oocytes quality