家兔卵巢组织玻璃化冷冻保存的实验研究

Research on vitrification cryopreservation of rabbit ovarian tissue

目的采用直接覆盖玻璃化冷冻(DCV法)和半麦管法玻璃化冷冻(HS法)处理家兔卵巢组织,探讨两种冷冻方法对卵泡组织形态学及超微结构的影响。方法将10只健康雌性新西兰家兔的卵巢组织块随机分配组成新鲜对照组、DCV法和HS法玻璃化冷冻组3个实验组,观察和比较各组间卵巢内卵泡的组织形态学和超微结构变化。结果 (1)与对照组卵巢组织中的原始卵泡及初级卵泡的形态正常率(分别为88.11%和72.86%)相比,冷冻复苏后的原始卵泡及初级卵泡形态正常率在DCV法(分别为72.66%和55.08%)和HS法(分别为71.73%和56.18%)均显著降低(P<0.05),且初级卵泡的形态正常率均显著低于原始卵泡(P<0.05),但两个冷冻组间无显著差异(P>0.05);(2)观察卵泡超微结构的改变:两种玻璃化冷冻法主要损伤的是细胞浆中的线粒体,细胞核也会出现核固缩现象。结论 DCV法和HS法玻璃化冷冻均能保存家兔卵巢组织内的卵泡,但冷冻所导致的原始卵泡和初级卵泡损伤仍不可避免,其中初级卵泡损伤更明显。这两种冷冻法处理后的卵泡组织形态学无显著性变化,但在一定程度上会损伤卵泡中各组成部分的细胞超微结构。

Objective： To study the effect of cryopreservation with direct cover vitrification（DCV）or hemi straw vitrification（ HS） on the morphology and ultrastructure of the ovarian follicles in rabbit. Methods： The ovarian tissues of 10 healthy female rabbits were randomly assigned to 3 groups： control group,DCV and HS vitrification group. The histomorphology and ultrastructure of ovarian follicles were observed and compared. Results： Compared with primordial follicles and primary follicles in control group（88.11 % and 72.86%） ,the morphological normal rate after frozen-thawed was significantly decreased in DCV vitrification group（72. 66% and 55.08%）and HS vitrification group（71.73% and 56. 18%）（P〈0.05）, and the normal morphology of primary follicles was significantly lower than that of primordial follicles （P〈0.05）. However, there was no significant difference between the two vitrification groups（P〈0.05）. These two kinds of vitrification methods mainly damaged the mitochondria in the cytoplasm,and the nuclei also showed condensation. Conclusions： The follicles in the rabbit ovarian tissue can be preserved by DCV and HS vitrification, but the damage of primary follicle and primary follicle caused by cryopreservation is still inevitable, and the primary follicle damage is more obvious. There is no significant change in the histomorphology of the follicles after cryopreservation,but the ultrastructure of the follicle ceils is damaged to a certain extent.