Keefe David, Liu Lin, Wang Wei, Silva Celso
Department of Obstetrics and Gynecology, Division of Reproductive Medicine and Infertility, Women and Infants Hospital, 101 Dudley Street, Providence, RI 02905, USA.
Reprod Biomed Online. 2003 Jul-Aug;7(1):24-9. doi: 10.1016/s1472-6483(10)61724-5.
Meiotic spindles tether the chromosomes of oocytes and have been found to be structurally abnormal in older women. Conventional methods to image the meiotic spindle, such as immunostaining or transmission electron microscopy, require prior fixation, so they cannot be used clinically, and their utility in developmental studies is limited. Spindles can also be imaged non-invasively based on their birefringence, an inherent optical property of highly ordered molecules, such as microtubules, as they are illuminated with polarized light. Polarized light microscopy has been gainfully applied to embryology for decades, but recently a digital, orientation-independent polarized light microscope, the polscope, has demonstrated the exquisite sensitivity needed to image the low levels of birefringence exhibited by mammalian spindles. Its use of nearly circularly polarized light also produces orientation-independent measures of spindle birefringence, thus providing a method to quantify spindle architecture in living oocytes. The safety and utility of polscope imaging has been demonstrated in mammalian oocytes, including those from women undergoing ICSI. Spindle imaging with the polscope provides structural information closely related to the more invasive immunostaining method, and also enables study of the dynamic architecture of spindles. Profound effects of cooling on meiotic spindles have also been shown, and polscope imaging has been used to optimize thermodynamic stability of oocytes during ICSI. It has been shown that embryos derived from oocytes with normal, intact meiotic spindles exhibit superior development after fertilization and in-vitro culture. The mechanisms underlying age-related disruption of meiotic spindles in women remain unclear, but may relate to factors residing within the chromosomes themselves, since mice engineered to shorten their telomeres exhibit structurally abnormal spindles in their oocytes, and their embryos undergo cell cycle arrest and apoptosis, a phenotype remarkably similar to that observed in oocytes and embryos from older women. A time-lapse video of a mouse oocyte imaged by polscope may be purchased for viewing on the internet at www.rbmonline.com/Article/824 (free to web subscribers).
减数分裂纺锤体束缚着卵母细胞的染色体，并且已发现在年长女性中其结构异常。成像减数分裂纺锤体的传统方法，如免疫染色或透射电子显微镜，需要事先固定，因此无法用于临床，且它们在发育研究中的效用有限。纺锤体也可基于其双折射进行无创成像，双折射是高度有序分子（如微管）的一种固有光学特性，当它们被偏振光照射时就会表现出来。偏振光显微镜已在胚胎学中得到了数十年的有效应用，但最近一种数字式、与方向无关的偏振光显微镜——偏振光镜，已展现出对哺乳动物纺锤体所呈现的低水平双折射进行成像所需的极高灵敏度。它使用近圆偏振光还能产生与纺锤体双折射无关方向的测量结果，从而提供了一种量化活卵母细胞中纺锤体结构的方法。偏振光镜成像的安全性和效用已在哺乳动物卵母细胞中得到证实，包括来自接受卵胞浆内单精子注射（ICSI）的女性的卵母细胞。用偏振光镜进行纺锤体成像可提供与侵入性更强的免疫染色方法密切相关的结构信息，还能对纺锤体的动态结构进行研究。降温对减数分裂纺锤体的深远影响也已得到证实，偏振光镜成像已被用于优化ICSI过程中卵母细胞的热力学稳定性。已表明，源自具有正常、完整减数分裂纺锤体的卵母细胞的胚胎在受精和体外培养后表现出更好的发育情况。女性减数分裂纺锤体与年龄相关的破坏背后的机制仍不清楚，但可能与染色体本身所含的因素有关，因为经过基因工程改造以缩短其端粒的小鼠在其卵母细胞中表现出结构异常的纺锤体，并且它们的胚胎会经历细胞周期停滞和凋亡，这一表型与在年长女性的卵母细胞和胚胎中观察到的非常相似。一个通过偏振光镜成像的小鼠卵母细胞的延时视频可在互联网上www.rbmonline.com/Article/824购买观看（网络订阅用户免费）。
