Gorbsky G J, Simerly C, Schatten G, Borisy G G
Integrated Microscopy Resource for Biomedical Research, University of Wisconsin, Madison 53706.
Proc Natl Acad Sci U S A. 1990 Aug;87(16):6049-53. doi: 10.1073/pnas.87.16.6049.
After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5- (and -6)-carboxy-x-rhodamine], was microinjected into living oocytes. Biotinylated tubulin incorporated into the meiotic spindle to apparent equilibrium within 15 min. To assess quantitatively the rates of disassembly and assembly of the microtubules, small domains within the spindles of oocytes injected with x-rhodamine-tubulin were photobleached and their recovery was analyzed by digital imaging microscopy. Fluorescence recovery in the spindles was rapid and extensive, plateauing to an average of 83% at 4 min. The calculated half-time for turnover of the spindle microtubules was 77 sec. In contrast, fluorescence recovery of the spindle midbodies in telophase oocytes was much more limited, averaging approximately 22% at 4 min. These data indicate that most microtubules within the arrested metaphase spindle of the mouse oocyte undergo rapid cycles of assembly and disassembly. Microtubules of the telophase midbody are more stable.
排卵后，哺乳动物卵母细胞停滞于第二次减数分裂中期。我们研究了构成小鼠卵母细胞减数分裂纺锤体的微管是稳定的，还是经历着快速的组装和解聚循环。将用生物素或x-罗丹明[5-（及-6）-羧基-x-罗丹明]衍生化的猪脑微管蛋白显微注射到活的卵母细胞中。生物素化的微管蛋白在15分钟内融入减数分裂纺锤体并达到明显的平衡。为了定量评估微管的解聚和组装速率，对注射了x-罗丹明-微管蛋白的卵母细胞纺锤体内的小区域进行光漂白，并通过数字成像显微镜分析其荧光恢复情况。纺锤体中的荧光恢复迅速且广泛，在4分钟时稳定在平均83%。计算得出纺锤体微管周转的半衰期为77秒。相比之下，末期卵母细胞纺锤体中体的荧光恢复则非常有限，在4分钟时平均约为22%。这些数据表明，小鼠卵母细胞停滞中期纺锤体内的大多数微管经历着快速的组装和解聚循环。末期中体的微管更稳定。
