Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Theriogenology. 2011 Jun;75(9):1652-60. doi: 10.1016/j.theriogenology.2010.12.028. Epub 2011 Feb 26.
The objective of this study was to investigate the potential of swamp buffalo oocytes vitrified-warmed at the metaphase of the second meiotic cell division (M-II) stage to develop to the blastocyst stage after parthenogenetic activation (PA) or intracytoplasmic sperm injection (ICSI). In Experiment 1, we examined the effects of exposure time of oocytes to cryoprotectants (CPA) on their in vitro development after PA. In vitro matured (IVM) oocytes were placed in 10% dimethylsulfoxide (DMSO) + 10% ethylene glycol (EG) for 1 min and then exposed to 20% DMSO + 20% EG + 0.5 M sucrose for 30 s, 45 s or 60 s (1 min + 30 s, 1 min + 45 s and 1 min + 60 s groups, respectively). The oocytes were then exposed to warming solution (TCM199 HEPES + 20% FBS and 0.5M sucrose) for 5 min and then washed in TCM199 HEPES + 20% FBS for 5 min. IVM oocytes without CPA treatments served as a control group. The viability assessed by fluorescein diacetate (FDA) staining was 100% in all groups. The developmental rates after PA to the blastocyst stage between 1min+30s (16%) and control (26%) groups did not differ significantly, but they were significantly higher than those in 1 min + 45 s (10%) and 1 min + 60 s (2%) groups. In Experiment 2, we examined the effect of two CPA exposure times, 1 min + 30 s and 1 min + 45 s on the in vitro development after PA of oocytes vitrified by the microdrop method. The viabilities in vitrified 1 min + 30 s, 1 min + 45 s and the control (without CPA treatments) groups were not different (97%, 95% and 100%, respectively). The development of surviving oocytes to the blastocyst stage in the vitrified 1 min + 30 s group (8%) was significantly higher than that in the vitrified 1 min + 45 s group (4%) and significantly lower than those in control group (26%). In Experiment 3, we examined the effect of two CPA exposure times, 1 min + 30 s and 1 min + 45 s on in vitro development after ICSI of vitrified oocytes. Viabilities in vitrified oocytes among 1 min + 30 s, 1 min + 45 s and control groups were not different (96%, 91% and 100%, respectively). After ICSI, vitrified-warmed oocytes were activated and oocytes with the second polar body were cultured for 7 days. The development of ICSI oocytes to the blastocyst stage in the vitrified 1 min + 30 s group (11%) was significantly higher than that in the vitrified 1 min + 45 s (7%) group and significantly lower than those in control group (23%). In conclusion, our study demonstrated that the 1 min + 30 s CPA treatment regimen could yield the highest blastocyst formation rates after PA and ICSI for oocytes vitrified by the microdrop method.
本研究旨在探讨水牛卵母细胞在第二次减数分裂中期（M-II）阶段经玻璃化冷冻-解冻后，通过孤雌激活（PA）或胞质内精子注射（ICSI）发育为囊胚的潜力。在实验 1 中，我们研究了卵母细胞暴露于冷冻保护剂（CPA）的时间对 PA 后体外发育的影响。体外成熟（IVM）的卵母细胞先在 10%二甲基亚砜（DMSO）+10%乙二醇（EG）中孵育 1 分钟，然后暴露于 20% DMSO+20% EG+0.5 M 蔗糖中 30 秒、45 秒或 60 秒（1 分钟+30 秒、1 分钟+45 秒和 1 分钟+60 秒组）。然后，将卵母细胞暴露于解冻液（TCM199 HEPES+20% FBS 和 0.5M 蔗糖）中 5 分钟，然后在 TCM199 HEPES+20% FBS 中洗涤 5 分钟。未进行 CPA 处理的 IVM 卵母细胞作为对照组。所有组的活率均通过荧光二乙酸酯（FDA）染色评估为 100%。PA 后发育至囊胚阶段的比率在 1 分钟+30 秒（16%）和对照组（26%）之间没有显著差异，但显著高于 1 分钟+45 秒（10%）和 1 分钟+60 秒（2%）组。在实验 2 中，我们研究了两种 CPA 暴露时间（1 分钟+30 秒和 1 分钟+45 秒）对微滴法玻璃化冷冻卵母细胞 PA 后体外发育的影响。玻璃化冷冻的 1 分钟+30 秒、1 分钟+45 秒和对照组（未进行 CPA 处理）的活率无差异（分别为 97%、95%和 100%）。玻璃化冷冻的 1 分钟+30 秒组中存活卵母细胞发育为囊胚的比率（8%）显著高于 1 分钟+45 秒组（4%），显著低于对照组（26%）。在实验 3 中，我们研究了两种 CPA 暴露时间（1 分钟+30 秒和 1 分钟+45 秒）对玻璃化冷冻卵母细胞 ICSI 后体外发育的影响。玻璃化冷冻卵母细胞在 1 分钟+30 秒、1 分钟+45 秒和对照组之间的活率无差异（分别为 96%、91%和 100%）。ICSI 后，将玻璃化解冻的卵母细胞激活，并用第二极体培养 7 天。玻璃化冷冻的 1 分钟+30 秒组中 ICSI 卵母细胞发育为囊胚的比率（11%）显著高于 1 分钟+45 秒组（7%），显著低于对照组（23%）。总之，我们的研究表明，对于微滴法玻璃化冷冻的卵母细胞，CPA 处理 1 分钟+30 秒可以获得最高的 PA 和 ICSI 后囊胚形成率。
