Takahashi Toshifumi, Takahashi Eiji, Igarashi Hideki, Tezuka Naohiro, Kurachi Hirohisa
Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata, Japan.
Mol Reprod Dev. 2003 Oct;66(2):143-52. doi: 10.1002/mrd.10341.
In vivo post-ovulatory aging of oocytes significantly affects the development of oocytes and embryos. Also, oocyte aging alters the regulation of the intracellular calcium concentration, thus affecting Ca(2+) oscillations in fertilized oocytes. Because reactive oxygen species (ROS) are known to significantly perturb Ca(2+) homeostasis mainly through direct effects on the machinery involved in intracellular Ca(2+) storage, we hypothesized that the poor development of aged oocytes that may have been exposed to oxidative stress for a prolonged time might arise from impaired Ca(2+)-oscillation-dependent signaling. The fertilization rates of aged oocytes and of fresh oocytes treated with 100 microM hydrogen peroxide (H(2)O(2)) for 10 min were significantly lower than that of fresh oocytes. Comparing within the fertilized oocytes, blastocyst formation was decreased while embryo fragmentation was increased similarly in the aged and H(2)O(2)-treated fresh oocytes. The frequency of Ca(2+) oscillations was significantly increased whereas the amplitude of individual Ca(2+) transients was lowered in the aged and H(2)O(2)-treated fresh oocytes. The rates of rise and decline in individual Ca(2+) transients were decreased in these oocytes, indicating impaired Ca(2+) handling. When lipid peroxidation was assessed using 4,4-difluoro-5-(4-phenyl-1,3-buttadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY) in unfertilized oocytes placed in a 5% CO(2) in air atmosphere, the green fluorescence (indicating lipid peroxidation) increased faster in the aged oocytes than in the fresh oocytes. Furthermore, the green fluorescence in the aged oocytes was already approximately 20 times higher than that in the fresh oocytes at the beginning of the measurements. These findings support the idea that Ca(2+) oscillations play a key role in the development of fertilized aged oocytes.
卵母细胞在体内排卵后的老化会显著影响卵母细胞和胚胎的发育。此外，卵母细胞老化会改变细胞内钙浓度的调节，从而影响受精后卵母细胞中的钙离子振荡。由于已知活性氧（ROS）主要通过直接影响参与细胞内钙储存的机制来显著扰乱钙稳态，我们推测，长时间暴露于氧化应激的老化卵母细胞发育不良可能源于钙离子振荡依赖信号的受损。老化卵母细胞以及用100微摩尔过氧化氢（H₂O₂）处理10分钟的新鲜卵母细胞的受精率显著低于新鲜卵母细胞。在受精后的卵母细胞中进行比较，老化卵母细胞和经H₂O₂处理的新鲜卵母细胞中囊胚形成减少，而胚胎碎片化增加情况相似。老化卵母细胞和经H₂O₂处理的新鲜卵母细胞中钙离子振荡频率显著增加，而单个钙离子瞬变的幅度降低。这些卵母细胞中单个钙离子瞬变的上升和下降速率降低，表明钙离子处理受损。当在置于含5%二氧化碳的空气环境中的未受精卵母细胞中使用4,4 - 二氟 - 5 -（4 - 苯基 - 1,3 - 丁二烯基）- 4 - 硼 - 3a，4a - 二氮杂 - s - 茚满 - 3 - 十一烷酸（C11 - BODIPY）评估脂质过氧化时，老化卵母细胞中的绿色荧光（表明脂质过氧化）比新鲜卵母细胞增加得更快。此外，在测量开始时，老化卵母细胞中的绿色荧光已经比新鲜卵母细胞高约20倍。这些发现支持了钙离子振荡在老化受精卵母细胞发育中起关键作用的观点。
