Gasparrini Bianca, Sayoud Hichem, Neglia Gianluca, Matos Daniel Gustavo de, Donnay Isabelle, Zicarelli Luigi
DISCIZIA, Faculty of Veterinary Medicine, Federico II University, 80137 Naples, Italy.
Theriogenology. 2003 Sep 15;60(5):943-52. doi: 10.1016/s0093-691x(03)00098-0.
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.
已证明，在体外成熟（IVM）过程中补充半胱胺可通过增加几种物种中谷胱甘肽（GSH）的合成来改善胚胎发育。在水牛物种中也记录到，在半胱胺存在下成熟的卵母细胞具有更高的发育能力。本研究的目的是调查：（1）水牛卵母细胞在体外成熟过程中是否重新合成谷胱甘肽；（2）半胱胺是否通过增加GSH合成来改善水牛胚胎发育；（3）在有或没有半胱胺的情况下，丁硫氨酸亚砜胺（BSO）对谷胱甘肽合成的抑制是否会影响随后的胚胎发育和GSH合成。从屠宰动物中回收的卵丘-卵母细胞复合体（COCs），在不存在或存在半胱胺（50μM）、有或没有5mM BSO的情况下，于含有10%胎牛血清（FCS）、0.5μg/ml促卵泡素（FSH）、5μg/ml促黄体素（LH）和1μg/ml 17-β-雌二醇的TCM199中进行体外成熟。通过高效液相色谱（HPLC）和荧光分析测定未成熟卵母细胞以及在不同实验条件下成熟的卵母细胞中的谷胱甘肽含量。在第二个实验中，将成熟的卵母细胞进行体外受精并培养7天，以评估其发育至囊胚（BLs）的情况。结果表明，水牛卵母细胞在体外成熟过程中合成谷胱甘肽，且半胱胺可增加谷胱甘肽的合成。此外，丁硫氨酸亚砜胺抵消了半胱胺对胚胎发育和GSH合成的促进作用。这些结果表明，谷胱甘肽在水牛胚胎发育中起关键作用。
