兔初级卵母细胞生发泡移植方法的研究

Transfer technique for germinal vesicle of primary oocytes in rabbit

目的 :以家兔为实验动物模型 ,探索卵母细胞生发泡 (GV)互换的可行性。方法 :在注射第一针卵泡刺激素 (FSH) 72 h后 ,取大耳白兔卵巢内 GV期卵母细胞 ,用显微操作和电融合的方法 ,对兔初级卵母细胞进行异卵间 GV互换 ,将重构卵培养于 TCM-1 99加 1 0 % FCS中 ,对重构卵进行成熟培养 ,最后对成熟重构卵进行卵胞浆内精子注射(ICSI)和胚胎培养。结果 :对 987枚 GV期卵母细胞进行了异卵间 GV互换 ,成功互换 2 72枚 (2 7.6 % ) ,电融合后存活 2 30枚 (84.6 % ) ,在进行成熟培养后 ,2 30枚卵有 1 78枚(77.4% )发育到 MII期 (排出第一极体 ) ,将成熟卵进行 ICSI后 ,有 1 2 6枚 (70 .8% )受精。结论 :对兔初级卵母细胞进行 GV互换是可行的 ,若将此法应用于提高高龄妇女卵母细胞的质量 ,将有助于解决她们的不育问题。

Objective: Using rabbit as a model to evaluate the feasibility of the germinal vesicle (GV)transfer. Methods: Collecting the GV stage oocytes from the ovaries of white Japan big ear rabbits at 72 h after the first follicle stimulating hormone injection. The GV-karyoplast was exchanged between the primary oocytes, the pairs of GV karyoplast-cytoplast were fused by electrical pulses, and then the reconstructed oocytes were maturated in TCM-199 with 10% FCS. Finally, the matured reconstructed oocytes were fertilized by intracytoplasmic sperm injection (ICSI) with the same species' sperms. Results: A total of 987 GV stage oocytes were manipulated. 272 (27.6%) GV-karyoplasts were removed and replaced successfully without lysis. Karyoplast fusion was accomplished in 230 for a final success rate of 84.6% (230/272). After in vitro maturation 178 of 230 (77.4%) reached metaphase II (emitted first polar body). The fertilization rate of matured reconstructed oocytes by ICSI was 70.8% (126/178). Conclusion: The result indicated that GV transfer was feasible, which could potentially become a new approach in treatment of women infertility due to advanced maternal age.