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山东大学学报（医学版） ›› 2014, Vol. 52 ›› Issue (11): 27-32.doi: 10.6040/j.issn.1671-7554.0.2014.561

• 基础医学 • 上一篇    下一篇

改良的玻璃化液冻存乳鼠卵巢异体原位移植后生殖功能的恢复

王红燕1,2, 王燕蓉1, 徐仙1,3, 赵君利1,3, 哈灵侠1,3, 贾韶彤1,3, 刘春莲1,3   

  1. 1. 宁夏医科大学 生育力保持教育部重点实验室, 宁夏 银川 750004;
    2. 宁夏医科大学 总医院人类精子库, 宁夏 银川 750004;
    3. 宁夏医科大学 总医院生殖医学中心, 宁夏 银川 750004
  • 收稿日期:2014-08-26 发布日期:2014-11-10
  • 通讯作者: 王红燕.E-mail:1099382469@qq.com E-mail:1099382469@qq.com
  • 基金资助:
    国家自然科学基金地区项目(81460243);宁夏回族自治区科技攻关项目(2012ZYS239);宁夏高等学校科学研资项目(NGY2013079)

Fertility restore of allotransplantation of vitrified-warmed junvenile ovaries treated with a modified vitrification solution

WANG Hongyan1,2, WANG Yanrong1, XU Xian1,3, ZHAO Junli1,3, HA Lingxia1,3, JIA Shaotong1,3, LIU Chunlian1,3   

  1. 1. Key Laboratory of Fertility Preservation and Maintenance, Ministry of Education, Ningxia Medical University, Yinchuan 750004 Ningxia, China;
    2. Human Sperm Bank of The Total Hospital, Ningxia Medical University, Yinchuan 750004 Ningxia, China;
    3. Center of Reproductive Medicine of Total Hospital, Ningxia Medical University, Yinchuan 750004 Ningxia, China
  • Received:2014-08-26 Published:2014-11-10

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摘要： 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.

关键词: 体外受精, 生殖潜能, 玻璃化冷冻, 幼鼠卵巢, 原位移植

Abstract: Objective To explore the development and fertility potential of oocytes retrieved from surviving prepubertal mice ovarian grafts treated with orthotopic transplantation after a modified solution cryopreservation by vitrification. Methods Prepubertal mice ovaries were cryopreserved by vitrification using ED20 and modified EG5.5/30 solutions. Part of the fresh and vitrified-warmed ovaries were treated with enzyme digestion to test the survival rate of eggs with fluorescence dynamic method. The fluorescence intensity quantitative analysis of microfilament and microtubules of GV oocytes were evaluated with laser confocal microscopy. Part of fresh and vitrified-warmed ovaries were allotransplanted under the ovarian capsule of ovariectomized adult female mice recipients. All juvenile mice were randomly divided into six groups: fresh group and fresh orthotopic transplantation group; ED20 solution vitrified-warmed group and ED20 solution vitrified-warmed orthotopic transplantation group; modified EG5.5/30 solution vitrified-warmed group and modified EG5.5/30 solution vitrified-warmed orthotopic transplantation group. To evaluate the fertility and developmental potential of the fresh and vitrified-warmed surviving ovarian grafts, 15 days after orthotopic transplantation, female micerecipients that restored estrous cycle were mated with male mice and serum levels of estradiol (E2) in female mice recipients was measured. Results Vitrified-warmed prepubertal mice ovaries showed a high survival rate of oocytes although a remarkable reduction from 92.6% (fresh group) to 87.0% (ED20 solution vitrified-warmed group) and 84.6% (EG5.5/30 solution vitrified-warmed group) was observed respectively (P<0.05). Results of quantitative analysis of microfilament and microtubules of GV oocytes decreased significantly in the two cryopreserved groups when compared with those of fresh group (P<0.05). Female mice recipients that received vitrified-warmed juvenile ovaries could successfully give birth after mating. The first litter time, litter size and total litter size of the two vitrified-warmed orthotopic transplantation groups showed marked differences compared with fresh orthotopic transplantation group (P<0.05). Conclusion Juvenile mice ovaries treated with cryopreservation by vitrification in the modified EG5.5/30 solution and allotransplantation do not affect the development of eggs and reproductive potential, similar in ED20 solution. Female mice recipients can grive normal offspring, but reproductive reserve and fertility significantly decreased.

Key words: Orthotopic transplantation, Juvenile mouse ovary, Vitrification, In vitro fertilization, Reproductive potential

中图分类号: 

  • R737.3
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