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目的探讨一种改良的玻璃化液冻存乳鼠卵巢,复苏后进行异体原位移植,评价其对卵母细胞成熟、受精及胚胎发育潜能的影响。方法乳鼠卵巢进行ED20和改良的EG5.5/30液玻璃化冻存,复苏后的乳鼠卵巢一部分酶解消化,通过荧光活力检测卵子的存活率;激光共聚焦显微镜观察α-tubulin的免疫荧光检测卵子内细胞骨架的变化。一部分乳鼠卵巢进行成年去势雌鼠的原位移植。标本随机分为新鲜组和新鲜移植组;ED20液冷冻组和ED20液冻融移植组;EG5.5/30液冷冻组和EG5.5/30冻融移植组。术后15 d,受体鼠同雄鼠合笼,统计移植6个月内的产仔情况;术后6个月,检测恢复动情周期的受体鼠雌二醇(E2)水平。结果玻璃化冻融乳鼠卵巢后,ED20液冷冻组和EG5.5/30液冷冻组卵子存活率(87.0%,84.6%)低于新鲜组(92.6%)(P<0.05);冻融后卵子内微丝和微管结构受损,荧光强度均低于新鲜组(P<0.05);接受冻融卵巢原位移植的受体鼠可以实现自然产仔,但两冷冻液冻融移植组首窝产仔时间、平均每窝产仔数、产仔总数均较新鲜移植组差异有统计学意义(P<0.05)。结论改良的EG5.5/30液冻存幼鼠卵巢复苏后异体原位移植同ED20液效果相似,受体鼠可诞生正常子代,但是生殖储备和生育力明显下降。
OBJECTIVE: To investigate the effects of vitrification on the development of oocyte maturation, fertilization and embryo development in ovariectomized rats. Methods The ovaries of neonatal rats were subjected to ED20 and modified EG5.5 / 30 liquid vitrification. The ovariectomized zygotes were resorbed and digested after resuscitation, and the viability of ovum was detected by fluorescence viability. The immunofluorescence of α-tubulin was observed by laser scanning confocal microscopy Detection of changes in the ovum cytoskeleton. Part of the neonatal ovariectomy adult female castration orthotopic transplantation. The specimens were randomly divided into fresh group and fresh transplantation group; ED20 liquid freezing group and ED20 liquid freezing and thawing group; EG5.5 / 30 liquid freezing group and EG5.5 / 30 freezing and thawing group. At 15 days after operation, the recipient rats and the male rats were caged to calculate the litter size within 6 months after transplantation. The estradiol (E2) levels of the recipient rats recovering from the estrous cycle were measured 6 months after the operation. Results After ovariectomized and frozen-thawed, the ovum survival rates of ED20 and EG5.5 / 30 liquid freezing groups were 87.0% and 84.6%, respectively, lower than that of fresh group (92.6%) (P <0.05) Ovarian microfilament and microtubule structure were impaired, fluorescence intensity was lower than that of fresh group (P <0.05); recipient mice receiving orthotopic transplantation of frozen-thawed ovary could achieve natural litter size, however, Litter litter time, the average number of litter size per litter, litter size compared with the fresh transplantation group, the difference was statistically significant (P <0.05). Conclusion The modified EG5.5 / 30 liquid cryopreservation of ovary resuscitation allogeneic orthotopic transplantation with ED20 liquid similar effect, recipient mice can be born normal offspring, but the reproductive reserve and fertility decreased significantly.