Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation.
Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation.
复制标题
L-脯氨酸在哺乳动物卵母细胞冷冻保存中的冷冻生物学特性
DOI：
10.4103/0366-6999.187846
复制
发表时间：
2016-08-20
影响因子：
6.1
通讯作者：
Qiao J
中科院分区：
文献类型：
--
作者：
Zhang L;Xue X;Yan J;Yan LY;Jin XH;Zhu XH;He ZZ;Liu J;Li R;Qiao J
背景：L-脯氨酸是一种天然、无毒的冷冻保护剂，可帮助各种动植物的细胞和组织耐受冷冻。L-脯氨酸在哺乳动物卵母细胞冷冻保存中的应用尚不多见。本研究探讨了L-脯氨酸的低温生物学特性及其在小鼠卵母细胞冷冻保存中的保护作用。方法：采用拉曼光谱法和渗透压计法研究L-脯氨酸的冷冻特性。将8周龄B6 D2 F1小鼠的成熟卵母细胞置于含有不同浓度L-脯氨酸的玻璃化冷冻液中进行玻璃化冷冻，并与对照组（15%DMSO和15%EG，不含L-脯氨酸）进行比较。采用免疫荧光法和体外受精法检测胚胎存活率、5-甲基胞嘧啶（5-mC）表达、体外受精率、2细胞率和囊胚率。资料采用卡方检验。结果：L-脯氨酸可在1 min内穿透卵母细胞膜，2.00 mol/L L-脯氨酸混合液的渗透压与对照组相近。在2.00 mol/L L-脯氨酸+7.5%DMSO +10%EG中解冻后卵母细胞的存活率显著高于对照组。L-脯氨酸联合组与对照组之间5-mC表达无差异。用2.00mol/L L-脯氨酸联合7.5%DMSO和10%EG玻璃化冷冻卵母细胞，其体外受精率、两细胞率和囊胚率与对照组相似。结论：适宜浓度的L-脯氨酸可提高小鼠卵母细胞在低浓度DMSO和EG条件下的冷冻效果，适用于人卵母细胞的玻璃化冷冻。
Background:L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation. Methods:The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG), comparing with the control group (15% DMSO and 15% EG without L-proline). The survival rate, 5-methylcytosine (5-mC) expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test. Results:L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control. Conclusions:It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification.