Hochi S, Ito K, Hirabayashi M, Ueda M, Kimura K, Hanada A
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan.
Theriogenology. 1998 Mar;49(4):787-96. doi: 10.1016/S0093-691X(98)00028-4.
The effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h in TCM199 supplemented with 5% fetal bovine serum (FBS) in 3% CO2 in air. The oocytes were first exposed to 20% ethylene glycol solution and were subjected to vitrification in a solution containing 40% ethylene glycol, 18% Ficoll-70 and 0.3 M sucrose. After warming in 20 degrees C water, oocytes which had been vitrified at less than 24-h of IVM were again cultured to complete the 24-h of IVM period. Oocytes were then incubated with frozen-thawed spermatozoa in Brackett and Oliphant (BO) medium containing 60 micrograms/ml heparin and 0.25% BSA for 20 h. In vitro fertilization rates of oocytes vitrified-warmed at 0, 6, 12 and 24-h IVM were 75.2, 68.0, 82.0 and 72.4%, respectively, comparable to the rates for unvitrified control oocytes (80.6%). A higher incidence of polyspermic fertilization was observed in oocytes vitrified at 24-h IVM (44.9 vs 22.6% in the control group, P < 0.05). Vitrification of oocytes at 12-h IVM seemed to be better than that of other IVM groups, since the normal fertilization rate of all treated oocytes was the highest (36.0%) among the vitrification groups. Developmental competence of the oocytes following vitrification and in vitro fertilization (12-h IVM group) was examined by cell-free culture of presumptive zygotes up to 9 d in modified synthetic oviduct fluid (mSOF) in 5% CO2, 5% O2 and 90% N2. The cleavage rate of zygotes from vitrified oocytes 48 h after insemination was 29.8%, which was lower than that of the control group (57.0%, P < 0.05). Development to blastocysts from the vitrified oocytes (4.8%) was much lower than that of the control group (27.0%, P < 0.05). These results indicate that cryopreservation of bovine oocytes by vitrification may be affected by their maturation stage in vitro, and that developmental competence to blastocysts of cleaved oocytes following vitrification may be impaired compared with unvitrified control oocytes.
研究了体外成熟（IVM）过程中核阶段对玻璃化冷冻-解冻牛卵母细胞存活的影响。将带有紧密卵丘细胞的卵母细胞在补充有5%胎牛血清（FBS）的TCM199中于含3%二氧化碳的空气中培养0、6、12和24小时。卵母细胞首先暴露于20%乙二醇溶液中，然后在含有40%乙二醇、18%聚蔗糖-70和0.3M蔗糖的溶液中进行玻璃化。在20℃水中解冻后，在IVM少于24小时时进行玻璃化的卵母细胞再次培养以完成24小时的IVM期。然后将卵母细胞与冷冻-解冻的精子在含有60微克/毫升肝素和0.25%牛血清白蛋白的Brackett和Oliphant（BO）培养基中孵育20小时。在IVM 0、6、12和24小时时进行玻璃化冷冻-解冻的卵母细胞的体外受精率分别为75.2%、68.0%、82.0%和72.4%，与未玻璃化的对照卵母细胞的受精率（80.6%）相当。在IVM 24小时时进行玻璃化的卵母细胞中观察到多精受精的发生率较高（对照组为22.6%，IVM 24小时玻璃化组为44.9%，P<0.05）。在IVM 12小时时进行卵母细胞玻璃化似乎比其他IVM组更好，因为在玻璃化组中所有处理的卵母细胞的正常受精率最高（36.0%）。通过在改良的合成输卵管液（mSOF）中于5%二氧化碳、5%氧气和90%氮气中对假定合子进行无细胞培养直至9天，检查玻璃化和体外受精后（12小时IVM组）卵母细胞的发育能力。受精后48小时，来自玻璃化卵母细胞的合子的分裂率为29.8%，低于对照组（57.0%，P<0.05）。来自玻璃化卵母细胞的囊胚发育率（4.8%）远低于对照组（27.0%，P<0.05）。这些结果表明，牛卵母细胞的玻璃化冷冻保存可能受其体外成熟阶段的影响，并且与未玻璃化的对照卵母细胞相比，玻璃化后分裂卵母细胞发育至囊胚的能力可能受损。
