Succu S, Leoni G G, Berlinguer F, Madeddu M, Bebbere D, Mossa F, Bogliolo L, Ledda S, Naitana S
Department of Animal Biology, University of Sassari, via Vienna 2, 07100 Sassari, Italy.
Theriogenology. 2007 Jul 1;68(1):107-14. doi: 10.1016/j.theriogenology.2007.04.035. Epub 2007 May 29.
The vitrification procedure effects on molecular and cytoskeletal components and on developmental ability of in vitro matured prepubertal ovine oocytes were evaluated. MII oocytes were divided into three groups: (1) vitrified in cryoloops (VTR); (2) exposed to vitrification solutions and rehydrated without being plunged into liquid nitrogen (EXP); (3) without further treatment as a control (CTR). Two hours after treatment, membrane integrity, assessed by propidium iodide/Hoechst staining, was lower in VTR and EXP than in CTR (70.6%, 88.5% and 95.2%, respectively). Cleavage rate after fertilization was statistically different among all groups (21.4%, 45.4% and 82.8% for VTR, EXP and CTR groups respectively; P<0.01). Blastocyst rate in VTR (0.0%) and EXP (2.8%) groups was lower (P<0.01) than in CTR (22.8%). Maturation promoting factor activity was lower (P<0.01) in VTR and EXP groups compared with CTR at both 0 h (82.2%, 83.6% and 100%, respectively) and 2 h (60% and 53.9% and 100%, respectively) after warming. Immediately after warming VTR and EXP oocytes showed a lower rate of normal spindle and chromosome configuration compared to CTR (59.1%, 48.0% and 83.3%, respectively; P<0.01). After 2 h of culture in standard conditions the percentage of oocytes with normal spindle and chromosome organization decreased in both VTR and EXP groups compared to CTR (36.4%, 42.8% versus 87.5%, respectively). In conclusion the exposition to the tested cryoprotectant solution and the vitrification in cryoloops modified cytoskeletal components and alter biochemical pathways that compromise the developmental capacity of prepubertal in vitro matured ovine oocytes.
评估了玻璃化程序对体外成熟的青春期前绵羊卵母细胞的分子和细胞骨架成分以及发育能力的影响。将MII期卵母细胞分为三组:(1) 在冷冻环中进行玻璃化处理(VTR);(2) 暴露于玻璃化溶液中并复水但未投入液氮(EXP);(3) 不做进一步处理作为对照(CTR)。处理后两小时,通过碘化丙啶/ Hoechst染色评估的膜完整性,VTR组和EXP组低于CTR组(分别为70.6%、88.5%和95.2%)。受精后的卵裂率在所有组之间存在统计学差异(VTR组、EXP组和CTR组分别为21.4%、45.4%和82.8%;P<0.01)。VTR组(0.0%)和EXP组(2.8%)的囊胚率低于CTR组(22.8%)(P<0.01)。在复苏后0小时(分别为82.2%、83.6%和100%)和2小时(分别为60%、53.9%和100%),VTR组和EXP组的成熟促进因子活性低于CTR组(P<0.01)。复苏后立即观察发现,与CTR组相比,VTR组和EXP组卵母细胞正常纺锤体和染色体构型的比例较低(分别为59.1%、48.0%和83.3%;P<0.01)。在标准条件下培养2小时后,与CTR组相比,VTR组和EXP组中具有正常纺锤体和染色体组织的卵母细胞百分比均下降(分别为36.4%、42.8% 对87.5%)。总之,暴露于测试的冷冻保护剂溶液以及在冷冻环中进行玻璃化处理会改变细胞骨架成分,并改变生物化学途径,从而损害青春期前体外成熟绵羊卵母细胞的发育能力。