College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai-an City, People's Republic of China.
Biol Reprod. 2012 Nov 1;87(5):105. doi: 10.1095/biolreprod.112.103127. Print 2012 Nov.
This study tested the hypothesis that oocyte aging could be prevented for a longer time by reducing the culture temperature while supplementing the culture medium with more pyruvate. Newly ovulated mouse oocytes were cultured at various temperatures for various times in HCZB medium (Kimura and Yanagimachi, Biol Reprod 1995; 52:709-720) containing various concentrations of pyruvate before examining for aging parameters and developmental potential. The increase in susceptibility to activating stimuli was efficiently prevented when oocytes were cultured in HCZB with 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 96 h, and 5°C for 48 h. Satisfactory blastocyst development of both parthenotes and fertilized zygotes was achieved after oocyte culture in HCZB containing 10.27 mM pyruvate at 37°C for 6 h, 25°C for 24 h, 15°C for 36 h, and 5°C for 24 h. Transfer of two-cell embryos or blastocysts showed no difference between newly ovulated control oocytes and oocytes cultured at 15°C for 36 h in either term pregnancy, live young per pregnant recipient, live young/transferred embryos, or birth weight of young. Oocytes with impaired developmental potential after culture at 15°C for 96 h and at 5°C for 48 h showed unrecoverable decreases in the content of glutathione, the glutathione/oxidized glutathione ratio, the BCL2 content, and in the numbers of oocytes with normal spindles and cortical granule distribution, suggesting induction of oxidative stress, which caused oocyte apoptosis and cytoskeleton alterations by downregulating BCL2. Because oocytes cultured at 15°C for 36 h were activated or fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 42 h without impairing their developmental potential.
这项研究检验了这样一个假设，即在补充培养基中添加更多丙酮酸的同时降低培养温度，可以使卵母细胞衰老的时间更长。在 Kimura 和 Yanagimachi 的研究中，新排出的小鼠卵母细胞在含有不同浓度丙酮酸的 HCZB 培养基中于不同温度下培养不同时间（Biol Reprod 1995; 52:709-720），然后检测其衰老参数和发育潜能。当卵母细胞在含有 10.27 mM 丙酮酸的 HCZB 中于 37°C 培养 6 小时、25°C 培养 24 小时、15°C 培养 96 小时和 5°C 培养 48 小时时，其对激活刺激的敏感性增加得到了有效预防。在含有 10.27 mM 丙酮酸的 HCZB 中于 37°C 培养 6 小时、25°C 培养 24 小时、15°C 培养 36 小时和 5°C 培养 24 小时后，孤雌激活胚胎和受精卵都能获得令人满意的囊胚发育。将二细胞胚胎或囊胚移植到受体中后，无论是足月妊娠、活产仔数/受孕受体数、活产仔数/移植胚胎数，还是仔鼠出生体重，新排出的卵母细胞与在 15°C 培养 36 小时的卵母细胞之间都没有差异。在 15°C 培养 96 小时和 5°C 培养 48 小时后，发育潜能受损的卵母细胞的谷胱甘肽含量、谷胱甘肽/氧化型谷胱甘肽比值、BCL2 含量以及具有正常纺锤体和皮质颗粒分布的卵母细胞数量均不可恢复性降低，提示诱导了氧化应激，这通过下调 BCL2 导致卵母细胞凋亡和细胞骨架改变。由于在 15°C 培养 36 小时的卵母细胞在 6 小时恢复培养后被激活或受精，因此成功地将排卵小鼠卵母细胞的衰老时间延长了 42 小时，而不会损害其发育潜能。
