3种玻璃化液对新生鼠卵巢的渗透反应及冻存效果的比较

目的:探索适宜新生鼠卵巢保存的玻璃化液和冷冻方案。方法:观察新生SD大鼠卵巢在预平衡液及3种玻璃化液中不同时间段的表面积变化,冻融后进行组织学观察和成年SD大鼠肾被膜下异体移植后在体活力分析。结果:新生鼠卵巢在预平衡液中出现渗透性脱水变化,移入EFS40(A组)、EG5.5(B组)、EG5.5/30(C组)3种玻璃化液中,再次剧烈皱缩。3min后,卵巢表面积分别为等渗液对照组表面积的63.2%、82.4%、70.8%。此脱水状态的卵巢玻璃化冻融后形态完整的卵泡百分率均与新鲜移植组(D组)无显著性差异(P>0.05);异体移植后,动情周期出现率和动情周期出现时间均与D组无显著性差异(P>0.05)。各冷冻移植组存活移植物均可见不同发育阶段的各级卵泡,但卵泡数目少于D组,移植20d时A组与D组间有显著性差异(P<0.05);移植60d时B组卵泡数少于D组,组间有差异(P<0.05);C组在各时间点上取材的卵泡数与D组均无差异(P>0.05)。结论:在预平衡液中15min、改良的EG5.5/30液中3min的二步渗透平衡法适宜新生鼠卵巢的玻璃化冷冻。

Comparison of Osmotic Respond and Cryoprotective Effect of Three Vitrification Solutions on Newborn Rat Ovaries

Objective：To study the suitable vitrification solution and to find a efficient protocol to preserve newborn rat ovaries.Methods：The osmotic superficial area changes of newborn rat ovaries were observed in pre-equilibrated solution and 3 vitrification solutions at different time.The cryoprotective effect was estimated by observing the morphologic structure and by allotransplanting post-thawing newborn rat ovaries under the capsule of sex-natural rats＇ kidneys,observing the follicular viability.Results：The shrankage of newborn rat ovaries was observed in the pre-equilibrated solution.When newborn rat ovaries were exposed to EFS40（group A）,EG5.5（group B）,EG5.5/30（group C）solutions,the newborn ovaries were rapidly shrunk again,the superficial area of the newborn rat ovaries was 63.2%,82.4%,70.8%,separately in 3 min.In this dehydrational condition,newborn rat ovaries were performed by vitrification.There was no significant difference in rate of normal morpho-logical follicles between vitrification group and fresh control group（group D）（P0.05）.Then allotransplanting under kidney capsule,as for the initiation rate and days of estrus after allotransplantation,there was no significant difference between vitrification and fresh transplanted groups（P0.05）.Survival ovarian trans-plants in each vitrification groups could witness the developing follicles at different stages.The number of the follicles was less than that in group D.After 20 d of transplanting,the number of the follicles in group A was significantly lower than that in group D（P0.05）.And after 60 d of transplanting,the number of the follicles in group B was significantly lower than that in group D（P0.05）.But at the different time point there was no significant difference between group C and group D（P0.05）.Conclusion：These results indicated that the vitrification protocol of pre-equilibrated solution for 15 min and modified EG5.5/30 solution for 3 min suits for cryopreservation of newborn rat ovaries.