CIIMAR - Interdisciplinary Center of Marine and Environmental Research, CIMAR Associated Laboratory, Laboratory of Cellular, Molecular and Analytical Studies, University of Porto , Porto , Portugal .
Toxicol Mech Methods. 2014 Jan;24(1):42-9. doi: 10.3109/15376516.2013.846952. Epub 2013 Oct 22.
To achieve more information about growth and development of oocytes in teleost fish or concerning toxicity testing, it is necessary to develop adequate in vitro oocyte culture conditions. Herein, initial stages of zebrafish oocytes (I, primary; II, cortical; III, vitellogenic) were analyzed under serum-free medium conditions as gonadal fragments or as separated oocyte-follicle complexes. Two vital dye staining methods (MTT, trypan blue) were applied to assess mitochondrial activity and membrane integrity of the oocytes during 4 days, and compared to morphological alterations studied by transmission electron microscopy. Vital dye staining indicated reduced viability at day 4 for all stages in both in vitro culture methods. Additionally, the viability decreased significantly in gonadal fragments at day 2 for stages III (MTT, TB) and II (TB only). Signs of degradation at the ultrastructural level (vacuoles, disintegration of endoplasmic reticulum and detachment of follicular cell layers) appeared in gonadal fragments at day 4 for stages II and III, and in separated oocyte-follicle complexes both at day 4 for stages I-III, and at day 2 for stage III. In conclusion, zebrafish oocytes at stages I and II seemed viable for 2 days as separated oocyte-follicle complexes considering their mitochondrial activity, membrane integrity and ultrastructural morphology. Cultured as gonadal fragments, the majority of analyses indicated similar results for stages I and II oocytes. In contrast, stage III oocytes seemed viable for not longer than 24 h. Results should be taken into consideration for the experimental design of in vitro assays using teleost fish oocytes.
为了获得更多关于鱼类卵母细胞生长和发育的信息或有关毒性测试的信息，有必要开发适当的体外卵母细胞培养条件。在此，在无血清培养基条件下，分析了斑马鱼卵母细胞的初始阶段（I，初级；II，皮质；III，卵黄生成），作为性腺片段或分离的卵母细胞-卵泡复合物。应用两种活细胞染料染色方法（MTT、台盼蓝）评估卵母细胞在 4 天内的线粒体活性和膜完整性，并与透射电子显微镜研究的形态变化进行比较。活细胞染料染色表明，在两种体外培养方法中，所有阶段的卵母细胞在第 4 天的活力都降低了。此外，在第 2 天，III 期（MTT、TB）和 II 期（仅 TB）的性腺片段中的活力显著降低。在第 4 天，II 期和 III 期的性腺片段以及第 4 天和第 2 天的 I-III 期的分离卵母细胞-卵泡复合物中出现了超微结构水平的降解迹象（空泡、内质网解体和卵泡细胞层分离）。综上所述，考虑到线粒体活性、膜完整性和超微结构形态，在第 2 天作为分离的卵母细胞-卵泡复合物，I 期和 II 期的斑马鱼卵母细胞似乎具有活力。作为性腺片段培养时，大多数分析结果表明 I 期和 II 期卵母细胞的结果相似。相比之下，III 期卵母细胞的活力不超过 24 小时。在使用鱼类卵母细胞进行体外试验的实验设计中，应考虑这些结果。
