Cortvrindt R, Smitz J, Van Steirteghem A C
Centre for Reproductive Medicine, University Hospital and Medical School, Dutch-speaking Brussels Free University, Belgium.
Hum Reprod. 1996 Dec;11(12):2648-55. doi: 10.1093/oxfordjournals.humrep.a019187.
Mechanically isolated intact early preantral follicles (100-130 microns diameter) from 14 day old mice were cryopreserved by a slow freezing protocol with dimethyl sulphoxide and then matured in vitro for 12 days after rapid thawing. Minor freeze damage observed after 1 day of in-vitro culture included ablation of the theca cell layer and granulosa cell dehydration, resulting in disruption of intercellular contacts with the oocyte and between granulosa cells. Of the follicles, 24% were irreversibly damaged and had a collapsed oocyte. The remaining majority of the follicles had an intact oocyte as evaluated by ultrastructural analysis. Follicles with an intact oocyte were cultured in vitro and, after an initial retarded development, the final number of fully grown oocytes ovulated in vitro was not different from that of unfrozen controls. Cryopreserved early preantral follicles matured in vitro responded to stimulus with human chorionic gonadotrophin in a similar way to controls, with mucification of the oocyte-cumulus complex, germinal vesicle breakdown and extrusion of the first polar body of the oocyte. These cryopreserved, in-vitro matured oocytes had the potential to fertilize and develop to hatched blastocysts.
从14日龄小鼠中机械分离出完整的早期窦前卵泡（直径100 - 130微米），采用含二甲基亚砜的慢速冷冻方案进行冷冻保存，然后在快速解冻后进行12天的体外成熟培养。体外培养1天后观察到的轻微冷冻损伤包括卵泡膜细胞层消融和颗粒细胞脱水，导致与卵母细胞以及颗粒细胞之间的细胞间接触中断。在这些卵泡中，24%受到不可逆损伤，卵母细胞塌陷。通过超微结构分析评估，其余大多数卵泡的卵母细胞完整。将卵母细胞完整的卵泡进行体外培养，在最初发育迟缓后，体外排卵的完全成熟卵母细胞的最终数量与未冷冻对照组无差异。冷冻保存的早期窦前卵泡在体外成熟后，对人绒毛膜促性腺激素刺激的反应与对照组相似，表现为卵母细胞 - 卵丘复合体的粘液化、生发泡破裂以及卵母细胞第一极体的排出。这些冷冻保存、体外成熟的卵母细胞具有受精并发育至孵化囊胚的潜力。
