Dalvit G, Llanes S P, Descalzo A, Insani M, Beconi M, Cetica P
Area of Biochemistry, School of Veterinary Sciences, University of Buenos Aires, Buenos Aires, Argentina.
Reprod Domest Anim. 2005 Apr;40(2):93-7. doi: 10.1111/j.1439-0531.2004.00522.x.
In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.
体外培养导致氧浓度高于体内环境，从而使活性氧（ROS）水平升高，进而引起细胞膜脂质过氧化。α-生育酚（维生素E的活性形式）是一种抗氧化剂，可保护哺乳动物细胞免受脂质过氧化，它可由抗坏血酸再生。本研究的目的是确定在成熟培养基中添加α-生育酚和/或抗坏血酸对牛卵母细胞体外成熟（IVM）以及随后的体外受精（IVF）和胚胎发育的影响。卵丘-卵母细胞复合体（COCs）在199培养基（对照）中成熟，并添加α-生育酚和/或抗坏血酸。通过高效液相色谱（HPLC）测定COCs中α-生育酚的浓度。IVF和体外培养（IVC）在改良的合成输卵管液（mSOF）中进行。在IVM期间，COCs中天然存在的α-生育酚量减少了一半（p<0.05），不过在有抗坏血酸存在的情况下它保持不变。在添加这种抗氧化剂的培养基中成熟的COCs中检测到更多的α-生育酚（p<0.05），但添加α-生育酚加抗坏血酸可维持更高水平的α-生育酚（p<0.05）。不同处理之间在核成熟和受精百分比方面未观察到显著差异。成熟培养基中α-生育酚或抗坏血酸的存在未能改变获得的囊胚百分比，不过当同时添加两种抗氧化剂时观察到显著下降（p<0.05）。抗坏血酸在IVM期间维持了掺入COC膜中的α-生育酚的抗氧化能力。成熟过程中维生素E的活性形式损害了卵母细胞发育能力的获得。
