Luciano Alberto M, Goudet Ghylène, Perazzoli Federica, Lahuec Cecile, Gérard Nadine
Institute of Anatomy of Domestic Animals, Histology and Embryology, Faculty of Veterinary Medicine, University of Milan, Milan, Italy.
Mol Reprod Dev. 2006 May;73(5):658-66. doi: 10.1002/mrd.20469.
The in vitro developmental competence of equine oocytes is still low in comparison with other domestic mammals. A major factor affecting the viability of cells during in vitro culture is the increased oxidative stress. Oxidative modifications could be responsible for oocyte incompetence for in vitro maturation (IVM). Cysteamine, a glutathione (GSH) synthesis enhancer, has been shown to increase intracellular GSH content and to improve embryo development when added during IVM of bovine, porcine, and ovine oocytes. The aim of the present study was (1) to determine whether equine cumulus-oocyte complexes (COCs) benefit from the addition of cysteamine during IVM, (2) to compare the GSH content of oocytes after in vivo maturation and IVM, (3) to assess whether cysteamine administration during IVM of equine oocyte enhances early embryonic developmental capability following ICSI, (4) to study the glutathione peroxidase (GPX) mRNA level in COCs. In vivo matured COCs were collected by aspiration from preovulatory follicles, and analyzed at collection. Immature COCs were collected in vivo or from slaughterhouse ovaries and matured in culture media supplemented or not with 100 microM cysteamine. After nuclear stage assessment, oocytes were analyzed for GSH concentration and both oocytes and cumulus cells were analyzed for GPX and GAPDH mRNA. Our data showed that the maturation capability was similar in both in vivo aspirated oocytes and in those isolated from slaughterhouse ovaries. Moreover, the addition of cysteamine during IVM affected neither GSH content nor maturation rate. At the time of collection, intra-oocyte GSH content was not influenced by the chromatin status. GSH concentration was similar in in vivo and in vitro matured metaphase II (MII) stage oocytes, and was significantly higher in MII than immature germinal vesicle stage oocytes. Moreover, the presence of serum inhibited whereas its absence stimulated the accumulation of GSH within oocytes during IVM. After ICSI, a similar proportion of zygotes in each group developed beyond the two-cell stage after 72 hr of culture. Cumulus cells expressed GPX mRNA, while GPX transcript was absent in both immature and mature oocytes. Cumulus expression of GPX mRNA was significantly higher when analyzed at collection than after IVM. Taken together, our results demonstrate that in equine oocytes, GSH increases during IVM but the relative intra-oocyte content of this thiol does not affect maturation and early development efficiency after fertilization. We hypothesize that factor(s) other than GSH/GPX are responsible for the limited in vitro early developmental capability of equine oocytes.
与其他家养哺乳动物相比，马卵母细胞的体外发育能力仍然较低。体外培养过程中影响细胞活力的一个主要因素是氧化应激增加。氧化修饰可能是卵母细胞体外成熟（IVM）能力不足的原因。半胱胺是一种谷胱甘肽（GSH）合成增强剂，已被证明在牛、猪和羊卵母细胞的IVM过程中添加时，可增加细胞内GSH含量并改善胚胎发育。本研究的目的是：（1）确定马卵丘-卵母细胞复合体（COCs）在IVM过程中添加半胱胺是否有益；（2）比较体内成熟和IVM后卵母细胞的GSH含量；（3）评估马卵母细胞IVM过程中给予半胱胺是否能增强ICSI后的早期胚胎发育能力；（4）研究COCs中谷胱甘肽过氧化物酶（GPX）mRNA水平。通过抽吸从排卵前卵泡中收集体内成熟的COCs，并在收集时进行分析。未成熟的COCs在体内或从屠宰场卵巢中收集，并在补充或不补充100 microM半胱胺的培养基中成熟。在进行核阶段评估后，分析卵母细胞的GSH浓度，并分析卵母细胞和卵丘细胞的GPX和GAPDH mRNA。我们的数据表明，体内抽吸的卵母细胞和从屠宰场卵巢分离的卵母细胞的成熟能力相似。此外，IVM过程中添加半胱胺既不影响GSH含量也不影响成熟率。在收集时，卵母细胞内的GSH含量不受染色质状态的影响。体内和体外成熟的中期II（MII）期卵母细胞中的GSH浓度相似，且MII期卵母细胞中的GSH浓度显著高于未成熟的生发泡期卵母细胞。此外，血清的存在会抑制而血清的缺失会刺激IVM过程中卵母细胞内GSH的积累。ICSI后，每组中相似比例的合子在培养72小时后发育到二细胞期以上。卵丘细胞表达GPX mRNA，而未成熟和成熟卵母细胞中均未检测到GPX转录本。收集时分析的卵丘细胞中GPX mRNA的表达明显高于IVM后。综上所述，我们的结果表明，在马卵母细胞中，IVM过程中GSH增加，但这种硫醇在卵母细胞内的相对含量并不影响受精后的成熟和早期发育效率。我们推测，除GSH/GPX之外的其他因素是马卵母细胞体外早期发育能力有限的原因。
