LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, Bedfordshire, UK.
Theriogenology. 2012 Jan 1;77(1):28-38. doi: 10.1016/j.theriogenology.2011.07.009.
Successful cryopreservation is usually measured in terms of cell survival. However, there may also be more subtle effects within cells that survive. Previous studies on zebrafish have produced evidence of mitochondrial DNA (mtDNA) damage in cryopreserved embryonic blastomeres and, after exposure to cryoprotectants, alterations in mtDNA replication in embryos and decreased mitochondrial membrane potential, mtDNA and ATP production in ovarian follicles. This study shows that the decreased ATP levels previously observed in stage III zebrafish ovarian follicles exposed to ≥3 M methanol persisted in those follicles that subsequently developed to stage IV. However, the decreased mtDNA levels were restored in those follicles. In order to determine whether mitochondrial distribution and/or their transport network was affected by the methanol exposure, immunocytochemistry analysis of tubulin and mitochondrial cytochrome c oxidase I (COX-I) was performed, along with phalloidin staining of polymerized actin. Neat arrangements of all proteins were observed in control follicles, with COX-I and tubulin being colocalized near granulosa cell nuclei, while actin formed hexagonal and/or polygonal structures nearer granulosa cell membranes and projected into the oocyte surface. Exposure to methanol (2 to 4 M) disrupted the COX-I and tubulin arrangements and the hexagonal and/or polygonal actin distribution and actin projections into the oocyte. These effects were still observed in those follicles that developed to stage IV, although the severity was reduced. In summary, the disruption to function and distribution of mitochondria in ovarian follicles exposed to >2 M methanol may be mediated via disruption of the mitochondrial transport system. Some recovery of this disruption may take place after methanol removal and subsequent follicle maturation.
成功的冷冻保存通常以细胞存活率来衡量。然而，在存活的细胞内可能还有更微妙的影响。以前对斑马鱼的研究表明，冷冻保存的胚胎卵裂球中存在线粒体 DNA（mtDNA）损伤，并且在暴露于冷冻保护剂后，胚胎中的 mtDNA 复制发生改变，线粒体膜电位降低，卵母细胞中的 mtDNA 和 ATP 产生减少。本研究表明，以前在暴露于≥3M 甲醇的 III 期斑马鱼卵母细胞中观察到的 ATP 水平降低在随后发育到 IV 期的卵泡中仍然存在。然而，mtDNA 水平在这些卵泡中得到了恢复。为了确定线粒体的分布和/或其运输网络是否受到甲醇暴露的影响，进行了微管蛋白和线粒体细胞色素 c 氧化酶 I（COX-I）的免疫细胞化学分析，并进行了聚合肌动蛋白的鬼笔环肽染色。在对照卵泡中观察到所有蛋白质的整齐排列，COX-I 和微管蛋白在颗粒细胞核附近共定位，而肌动蛋白形成靠近颗粒细胞膜的六边形和/或多边形结构，并投射到卵母细胞表面。暴露于甲醇（2 至 4M）破坏了 COX-I 和微管蛋白的排列以及六边形和/或多边形肌动蛋白的分布和肌动蛋白向卵母细胞的投射。在发育到 IV 期的卵泡中仍然观察到这些效应，尽管严重程度降低。总之，暴露于>2M 甲醇的卵巢卵泡中线粒体的功能和分布的破坏可能是通过破坏线粒体运输系统介导的。甲醇去除和随后的卵泡成熟后，这种破坏可能会有一些恢复。
