Glutathione synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development

It was demonstrated that cysteamine supplementation during in vitro maturation (IVM) improves embryo development by increasing glutathione (GSH) synthesis in several species. An improved developmental competence of oocytes matured in the presence of cysteamine was also recorded in buffalo species. The purpose of this work was to investigate (1) if glutathione is de novo synthesized during in vitro maturation of buffalo oocytes, (2) if cysteamine improves buffalo embryo development via an increase in GSH synthesis, and (3) if the inhibition of glutathione synthesis by buthionine sulfoximide (BSO), in the 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 TCM199+10% fetal calf serum (FCS), 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17-beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 5mM BSO. Glutathione content was measured by high-performance liquid chromatography (HPLC) and fluorimetric analysis in immature oocytes and in oocytes matured in the different experimental conditions.In a second experiment, the mature oocytes were in vitro fertilized and cultured for 7 days in order to assess development to blastocysts (BLs). It was demonstrated that buffalo oocytes synthesize glutathione during in vitro maturation and that cysteamine increases glutathione synthesis. Furthermore, the promoting effects of cysteamine on embryo development and GSH synthesis were neutralized by buthionine sulfoximide. These results indicate that glutathione plays a critical role on buffalo embryo development.