Department of Biology, University of Toronto, 1265 Military Trail, Scarborough, Canada.
Kingston General Hospital, Kingston, Canada.
Plant Methods. 2014 Oct 15;10(1):33. doi: 10.1186/1746-4811-10-33. eCollection 2014.
Meiosis progression in the more recent past has been investigated using 5-bromo-2'-deoxyuridine (BrdU) uptake by S-phase meiocytes undergoing DNA replication. BrdU uptake is detected by reaction with BrdU antibody followed by epifluorescent microscopy examination of chromosome spreads and/or squashes. We here report using confocal microscopic examination of intact meiocytes in conjunction with the new thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The simplicity of the EdU detection coupled with confocal examination of anthers provides a more exact temporal description of meiotic prophase I progression in Arabidopsis and opens up the possibility of examining the coordination of microsporocyte development with the other tissues of the anther.
Using our time course protocol, we have determined the duration of wild type Arabidopsis leptotene to be 5 h, zygotene -6 h, pachytene -10 h and a diplotene duration of approximately 1 h. We estimate G2 duration to be approximately 7 h based on the timing of the initial appearance of EdU signal in early leptotene meiocytes. In addition we have found that DNA replication in meiocytes is not done synchronously with the associated tapetal layer of cells. The EdU labeling suggests that S-phase replication of meiocyte DNA precedes the duplication of tapetal cell DNA.
The increased number of meiotic staging criteria that can be assessed in our confocal analysis, as compared to chromosome spreading or squashing, makes the identification of even the early and late portions of the prophase I substages attainable. This enhanced staging coupled with the ability to easily generate large data sets at hourly time points makes it possible to more exactly determine substage duration and to detect modest temporal abnormalities involving meiocyte entrance into and/or exit from leptotene, zygotene and pachytene. Confocal analysis also makes it possible to study the relationships between different cell types within the flower bud as meiosis proceeds.
减数分裂进程在更近的过去已经通过 S 期减数分裂细胞的 DNA 复制过程中摄取 5-溴-2'-脱氧尿苷（BrdU）来研究。BrdU 摄取通过 BrdU 抗体反应检测，随后对染色体铺片和/或压片进行荧光显微镜检查。我们在此报告使用完整减数分裂细胞的共焦显微镜检查，结合新的胸苷类似物 5-乙炔基-2'-脱氧尿苷（EdU）。EdU 检测的简单性与花药中的共焦检查相结合，为拟南芥减数分裂前期 I 进程提供了更准确的时间描述，并为研究小孢子发生与花药其他组织的协调提供了可能性。
使用我们的时间进程方案，我们确定野生型拟南芥细线期的持续时间为 5 小时，合线期为 6 小时，粗线期为 10 小时，二价体期持续约 1 小时。我们根据早期细线期减数分裂细胞中 EdU 信号初始出现的时间估计 G2 期持续时间约为 7 小时。此外，我们还发现减数分裂细胞中的 DNA 复制与相关的绒毡层细胞不是同步进行的。EdU 标记表明，减数分裂细胞 DNA 的 S 期复制先于绒毡层细胞 DNA 的复制。
与染色体铺片或压片相比，我们的共焦分析中可以评估的减数分裂分期标准数量增加，使得即使是前期 I 亚期的早期和晚期部分也可以识别。这种增强的分期加上以小时为时间点轻松生成大量数据集的能力，使得更准确地确定亚期持续时间并检测涉及减数分裂进入和/或退出细线期、合线期和粗线期的适度时间异常成为可能。共焦分析还使得有可能在减数分裂进行时研究花蕾内不同细胞类型之间的关系。
