Van der Elst J C, Nerinckx S S, Van Steirteghem A C
Centre for Reproductive Medicine, University Hospital, Brussels Free University, Belgium.
J Assist Reprod Genet. 1993 Apr;10(3):202-12. doi: 10.1007/BF01239222.
The cryopreservation of mature metaphase II-stage mouse oocytes is associated with decreased fertilizability, spindle damage, and increased polyploidy. Therefore, we investigated the outcome of cryopreservation of immature germinal vesicle-stage mouse oocytes.
Oocytes were punctured from Graafian follicles in primed F1 hybrid mice and were then released into maturation medium containing the meiotic inhibitor dibutyryl cyclic AMP. Both slow and ultrarapid freezing protocols with dimethyl sulfoxide, 1,2-proponediol, or a mixture of both agents were tested. We recorded morphological survival rates, in vitro maturation rates, and two-cell and blastocyst formation rates. Each group of frozen oocytes was compared with both unfrozen germinal vesicle-stage oocytes and metaphase II-stage oocytes.
An optimal cryosurvival rate of 78% was reached after ultrarapid freezing with 3 M dimethyl sulfoxide followed by one-step dilution, but a decreased rate of two-cell formation was observed. Freezing with a combination of dimethyl sulfoxide and 1,2-propanediol did not improve this fertilization-decreasing effect. Very low cryosurvival rates after freezing with 1,2-propanediol indicated its inappropriateness for ultrarapid freezing of immature oocytes. The rates of in vitro maturation were equivalent for frozen-thawed and freshly collected germinal vesicle-stage oocytes, independent of the freezing protocol used. We report, nevertheless, as a general characteristic for both slow and ultrarapid freezing of fully grown germinal vesicle-stage oocytes, that the in vitro development up to the blastocyst stage is inhibited despite full nuclear maturation.
We report that cryopreservation of immature germinal vesicle-stage oocytes is invariably associated with a low developmental capacity after fertilization. The rate of in vitro nuclear maturation did not equate with developmental competence. This in turn suggests the importance of cytoplasmic maturation for embryonic development.
成熟的中期II期小鼠卵母细胞的冷冻保存与受精能力下降、纺锤体损伤及多倍体增加有关。因此，我们研究了未成熟生发泡期小鼠卵母细胞冷冻保存的结果。
从经预处理的F1杂交小鼠的格拉夫卵泡中穿刺取出卵母细胞，然后将其放入含有减数分裂抑制剂二丁酰环磷腺苷的成熟培养基中。测试了使用二甲基亚砜、1,2 - 丙二醇或两者混合物的慢速和超快速冷冻方案。我们记录了形态学存活率、体外成熟率以及二细胞和囊胚形成率。将每组冷冻的卵母细胞与未冷冻的生发泡期卵母细胞和中期II期卵母细胞进行比较。
用3M二甲基亚砜进行超快速冷冻并一步稀释后，最佳冷冻存活率达到78%，但观察到二细胞形成率下降。使用二甲基亚砜和1,2 - 丙二醇的组合冷冻并没有改善这种受精能力下降的效果。用1,2 - 丙二醇冷冻后的冷冻存活率非常低，表明其不适用于未成熟卵母细胞的超快速冷冻。冷冻解冻后的生发泡期卵母细胞与新鲜收集的卵母细胞体外成熟率相当，与所使用的冷冻方案无关。然而，我们报告，作为完全成熟的生发泡期卵母细胞慢速和超快速冷冻的一般特征，尽管核完全成熟，但体外发育至囊胚阶段受到抑制。
我们报告未成熟生发泡期卵母细胞的冷冻保存总是与受精后发育能力低相关。体外核成熟率与发育能力不相等。这反过来表明细胞质成熟对胚胎发育的重要性。
