Brito D C C, Domingues S F S, Rodrigues A P R, Maside C, Lunardi F O, Wu X, Figueiredo J R, Pieczarka J C, Santos R R
Laboratory of Cytogenetics, Centre for Advanced Studies on Biodiversity (CEABIO), Biological Sciences Institute, Federal University of Pará, Brazil; Laboratory of Wild Animal Biology and Medicine, Federal University of Pará, Brazil.
Laboratory of Wild Animal Biology and Medicine, Federal University of Pará, Brazil.
Theriogenology. 2018 Apr 15;111:69-77. doi: 10.1016/j.theriogenology.2018.01.015. Epub 2018 Jan 31.
We aimed to evaluate the effect of two vitrification methods on the morphology and functionality of vitrified feline preantral follicles. Feline ovarian tissue was vitrified with EG + trehalose combined or not with dimethyl sulphoxide (DMSO), using two different techniques (open or closed systems). Morphology, developmental capacity and mRNA expression of markers for follicle survival and quality were assessed before and after in vitro culture (IVC). Both vitrification and culture media were serum-free. Vitrification of feline ovarian tissue from five adult domestic cats was performed with EG + trehalose combined or not with DMSO. Two systems were used: the open system solid-surface vitrification (SSV) and the closed system ovarian tissue cryosystem (OTC). Histological analysis of follicle integrity showed that the percentages of normal follicles in previously vitrified ovarian fragments decreased after 7 days of in vitro culture (IVC), independently of the protocol used. Although follicular activation was observed by Ki-67 labelling, this was accompanied by extensive follicular degeneration as detected by a 3-4-fold decrease in follicular density. Remarkable follicle activation was observed in the ovarian tissue vitrified using OTC and subjected to IVC, probably due to a higher rate of degeneration of developing follicles. Even with such follicular loss, the results are promising for the combination of EG + DMSO + trehalose in a serum-free medium when applying the SSV method, with this approach resulting in the highest rates of normal developing follicles (19%) after 7 days IVC, together with granulosa cells proliferating at the same rate observed in fresh tissue.
我们旨在评估两种玻璃化方法对玻璃化猫科动物窦前卵泡形态和功能的影响。使用两种不同技术（开放或封闭系统），将猫卵巢组织用乙二醇（EG）+海藻糖联合或不联合二甲基亚砜（DMSO）进行玻璃化处理。在体外培养（IVC）前后，评估卵泡存活和质量标志物的形态、发育能力和mRNA表达。玻璃化和培养基均无血清。对五只成年家猫的猫卵巢组织进行玻璃化处理，使用EG+海藻糖联合或不联合DMSO。使用了两种系统：开放系统固体表面玻璃化（SSV）和封闭系统卵巢组织冷冻系统（OTC）。卵泡完整性的组织学分析表明，无论使用何种方案，先前玻璃化的卵巢碎片中正常卵泡的百分比在体外培养7天后均下降。尽管通过Ki-67标记观察到卵泡激活，但同时伴随着卵泡密度降低3-4倍所检测到的广泛卵泡退化。在使用OTC玻璃化并进行IVC的卵巢组织中观察到显著的卵泡激活，这可能是由于发育中卵泡的退化率较高。即使存在这种卵泡损失，当应用SSV方法时，在无血清培养基中EG+DMSO+海藻糖的组合结果仍很有前景，这种方法在IVC 7天后产生的正常发育卵泡率最高（19%），同时颗粒细胞的增殖速率与新鲜组织中观察到的相同。
