小鼠卵慢速和快速降温及玻璃化冻存的比较研究

CRYOPRESERVATION OF OOCYTES USING SLOW COOLING,ULTRAPID COOLING AND NITRIFICATION PROTOCOLS

本文用４种冷冻模式：慢速降温（ＳＬ），超快速降温（Ｑ１）和防冻剂组成不同的玻璃化法（Ｒ－ＦＶＭ和ＭＶＭ）对昆明小鼠卵进行冷冻保存的比较研究，并用体外受精技术检测冷冻复苏卵的受精能力。ＳＬ和Ｒ－ＦＶＭ的存活率分别为５５．１±１．２和６５．９±７．９，显著高于Ｑ１（２４．２±７．３）和ＭＶＭ（４．５±２．４）其受精率分别为７２．８±１．８和７３．９±０．４显著高于Ｑ１（５８．６±１１．２）和ＭＶＭ（４１．５±８．５），而与对照组（７７．５±３．９）相似。结果表明：１）慢速降温程序（ＳＬ）和同时含有渗透性和非渗透性防冻剂的玻璃化法（Ｆ—ＲＶＭ）较适宜昆明小鼠卵的冻存；２）同时考虑冻存过程中的冰晶损伤和渗透压损伤是获得较好冻存结果的关键。

Unfertilized Kunming mouse oocytes obtained 14 h after hCG were cryopreserved employing four freezing protocols: 1) SL, the oocytes were equilibrated in 0.5, 1.0,1.5 mol/ L 1, 2-propanediol for 5, 5 and 10 min at room temperature respectively; slow cooling at a rate of-0.3 ℃ / min to-30℃. The frozen eggs were thawed in 37℃ water and moved into fresh medium after equilibration in 1.0 mol / L 1, 2-propanediol-0.2 mol / Lsucrose / PB1, 0.5 mol / L 1, 2-propane-diol / PBl, 0.2 mol / L sucrose / PBl for 5 min,respectively, 2) Ql, using a freezing medium of 3.5 mol/ L DMSO in Hepes-HTF, and cooling ultrarapidly by simply plunging into liquid nitrogen. The frozen eggs were thawed in 37℃ water and moved into fresh medium after equilibration in 0.5 mol/ L sucrose,0.25 mol / L sucrose Hepes-HTF for 5 min respectively; 3) R-FVM, vitrifying oocytes in 90% VSl and thawed in 4t bath and moved into fresh medium after equilibration in 50%VSl and 25% VSl for 10 min, respectively; 4) MVM, vitrifying oocytes in Massip's vitrification solution and thawing in 20℃ bath. The frozen eggs equilibration in 1 mol/ L sucrose for 10 min and washed 3 times by PBS. The results showed that the survival rate and fertilization rate of SL and R-FVM were similar to those of the control, but significant higher than those of Ql and MVM (P <0.05>. Our results suggested that: 1) Both slow cooling and vitrification can preseve Kunming mouse oocytes well; 2) The vitrification solution containing both permeating and nonpermeating cryoprotectants (such as VSl) was suitablefor Kunming mouse oocyte vitrification.