Comizzoli P, Wildt D E, Pukazhenthi B S
Department of Reproductive Sciences, Center for Species Survival, Smithsonian's National Zoological Park, Washington, DC, USA.
Reprod Domest Anim. 2009 Jul;44 Suppl 2:269-74. doi: 10.1111/j.1439-0531.2009.01372.x.
The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro. Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8-16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes.
未成熟猫卵母细胞含有一个大的生发泡（GV），其染色质解聚，对冷冻损伤高度敏感。本研究的目的是通过研究使用白藜芦醇（Res）（一种组蛋白脱乙酰酶增强剂）使GV染色质浓缩对玻璃化冷冻过程中卵母细胞存活的影响，探索一种替代传统冷冻保存的方法。在实验1中，将裸卵暴露于0、0.5、1.0或1.5 mmol/l的Res中1.5小时，然后评估染色质结构或进行培养以评估卵母细胞的减数分裂和体外发育能力。暴露于1.0或1.5 mmol/l的Res会导致GV染色质完全去乙酰化和最显著的浓缩。与其他处理相比，1.5 mmol/l的Res浓度损害了卵母细胞达到中期II（MII）或形成囊胚的能力。在实验2中，将裸卵如实验1那样暴露于Res中，并直接（新鲜）或在玻璃化冷冻后进行体外培养。然后评估这两种类型的卵母细胞的减数分裂能力、受精能力和形成胚胎的能力。玻璃化冷冻对卵母细胞的减数分裂和发育能力产生了总体负面影响。然而，与所有相应处理相比，先前暴露于1.0 mmol/l Res的玻璃化冷冻卵母细胞在达到MII、实现早期第一次卵裂以及发育到晚期胚胎阶段（8 - 16细胞）的能力方面有所提高。总之，结果表明，与Res诱导的GV染色质浓缩相关的瞬时表观遗传修饰是完全可逆的，并且有利于玻璃化冷冻过程中卵母细胞的存活。这种方法使得首次从玻璃化冷冻的未成熟卵母细胞中产生了猫胚胎。
