Santos R R, Tharasanit T, Van Haeften T, Figueiredo J R, Silva J R V, Van den Hurk R
Departmant of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Cell Tissue Res. 2007 Jan;327(1):167-76. doi: 10.1007/s00441-006-0240-2. Epub 2006 Aug 26.
Caprine preantral follicles within ovarian fragments were exposed to or vitrified in the presence of sucrose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), or various combinations thereof. The fragments were cryopreserved by using either a conventional (CV) or a solid-surface vitrification (SSV) protocol, and the cryoprotectants were removed by equilibrating vitrified ovarian fragments in "warming solution" consisting of minimum essential medium and heat-inactivated fetal calf serum (MEM(+)) followed by washes in MEM(+) with or without sucrose. Histological analysis of follicle integrity showed that the percentages of normal follicles in ovarian fragments vitrified in sucrose mixed with EG and/or DMSO (CV method) or mixed with EG or DMSO (SSV method) followed by washes in MEM(+) plus sucrose were similar to those of controls (ovarian fragments fixed without previous vitrification). Unlike for MEM(+) (supplemented or unsupplemented by sucrose) and DMSO followed by washes in the absence of sucrose, the percentages of normal follicles found after exposure to cryoprotectant did not significantly differ from that found after vitrification, indicating that follicular degeneration was attributable to a toxic effect of cryoprotectants and not to the vitrification procedure. The viability of preantral follicles after the CV and SSV procedures was investigated by using calcein-AM and the ethidium-homodimer as "live" and "dead" markers, respectively. In both tested vitrification procedures, the highest percentages of viable follicles were observed when a mixture of sucrose and EG (70.3% for CV and 72.4% for SSV) was used. Preantral follicles were also vitrified (either by CV or SSV) in sucrose and EG and then cultured for 24 h, after which their viability was compared with that of cultured fresh and uncultured vitrified follicles. The viability of these follicles was maintained after SSV, but not after CV. Thus, the viability of caprine preantral follicles can be best preserved after SSV in a mixture of sucrose and EG, followed by washes in medium containing sucrose.
将卵巢组织块中的山羊窦前卵泡置于蔗糖、二甲基亚砜（DMSO）、乙二醇（EG）或它们的各种组合存在的条件下进行暴露或玻璃化处理。采用传统（CV）或固体表面玻璃化（SSV）方案对组织块进行冷冻保存，通过在由最低限度基本培养基和热灭活胎牛血清组成的“复温溶液”（MEM(+)）中平衡玻璃化的卵巢组织块，然后在含或不含蔗糖的MEM(+)中洗涤来去除冷冻保护剂。卵泡完整性的组织学分析表明，在蔗糖与EG和/或DMSO混合（CV法）或与EG或DMSO混合（SSV法）后进行玻璃化处理，然后在含蔗糖的MEM(+)中洗涤的卵巢组织块中，正常卵泡的百分比与对照组（未经玻璃化处理直接固定的卵巢组织块）相似。与在含或不含蔗糖的MEM(+)以及DMSO中处理后再在无蔗糖条件下洗涤不同，暴露于冷冻保护剂后发现的正常卵泡百分比与玻璃化处理后发现的正常卵泡百分比无显著差异，这表明卵泡变性归因于冷冻保护剂的毒性作用而非玻璃化过程。分别使用钙黄绿素-AM和碘化丙啶二聚体作为“活”和“死”标记物，研究CV和SSV处理后窦前卵泡的活力。在两种测试的玻璃化处理程序中，当使用蔗糖和EG的混合物时，观察到的存活卵泡百分比最高（CV法为70.3%，SSV法为72.4%）。窦前卵泡也在蔗糖和EG中进行玻璃化处理（通过CV或SSV），然后培养24小时，之后将其活力与培养的新鲜卵泡和未培养的玻璃化卵泡的活力进行比较。这些卵泡在SSV处理后活力得以维持，但在CV处理后则不然。因此，山羊窦前卵泡的活力在蔗糖和EG的混合物中采用SSV处理，然后在含蔗糖的培养基中洗涤后能得到最佳保存。
