Effects of cysteamine on glutathione synthesis of buffalo (Bubalus bubalis) oocytes during in vitro maturation.

An improved developmental competence of oocytes matured in presence of cysteamine was recorded in buffalo species. The purpose of this work was to investigate (1) if cysteamine supplementation during in vitro maturation (IVM) of buffalo oocytes increases GSH synthesis, and (2) if the inhibition of glutathione synthesis during IVM by buthionine sulfoximide (BSO), in presence or absence of cysteamine, affects subsequent embryo development and GSH synthesis. Cumulus-oocytes complexes (COCs) recovered from slaughtered animals, were matured in vitro in TCM 199 + 10 % FCS, 0.5 mcg/mL FSH, 5 mcg/mL LH and 1 mcg/mL 17 beta estradiol in absence or presence of cysteamine (50 mcM), with or without 5 mM BSO at 38.5°C under 5% CO2 in humidified air for 24 hours. In experiment 1 a total number of 149 oocytes, over 8 replicates, was used for GSH measurements. Glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in oocytes immediately after follicle aspiration (before maturation) or at the end of in vitro maturation (IVM) for the remaining oocytes, which were evenly distributed in the four IVM groups. Experiment 2 was carried out on a total number of 405 COCs, over 3 replicates. COCs were randomly assigned to the four different experimental groups of maturation, in vitro fertilized and cultured for 7 days in order to assess development to blastocysts. In vitro fertilization was carried out at 38.5°C under 5% CO2 in humidified air in TALP medium supplemented with 0.2 mM penicillamine, 0.1mM hypotaurine and 0.01 mM heparin. Frozen-thawed sperm from a tested bull was treated by swim-up procedure and used at a final concentration of 20 6/ml. After 20-22h presumptive zygotes were cultured in SOF medium , supplemented with essential and non essential aminoacids and 8 mg/ml BSA, in a gas atmosphere of 5% CO2, 7% O2, and 88% N2. Differences among treatments in each experiment were analyzed by ANOVA. It was demonstrated that buffalo oocytes synthesize glutathione during IVM and that cysteamine increases glutathione synthesis. Furthermore buthionine sulfoximide neutralized the promoting effects of cysteamine on GSH synthesis and embryo development. These results indicate that cysteamine supplementation during in vitro maturation of buffalo oocytes improves embryo development by increasing GSH synthesis.