Terasaki M, Jaffe L A
Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
J Cell Biol. 1991 Sep;114(5):929-40. doi: 10.1083/jcb.114.5.929.
The ER of eggs of the sea urchin Lytechinus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiIC18(3) (DiI) in soybean oil; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles. Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex. Since diffusion of DiI is confined to continuous bilayers, the spread of the dye supports the concept that the ER is a cell-wide, interconnected compartment. In time lapse observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was stationary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave apparently coincident with the calcium wave and becoming most marked by 2-3 min. By 5-8 min the ER returned to an organization similar to that of the unfertilized egg. The cortical network also changed at fertilization; it became disrupted and eventually recovered. DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis. DiI-stained membranes accumulated in the region of the microtubule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the egg; this accumulation persisted near the centrosomes and zygote nucleus throughout pronuclear fusion and the first two mitotic cycles. We have used a new method to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization.
通过向海胆（Lytechinus pictus）卵的内质网（ER）显微注射二羰花青DiIC18(3)（DiI）在大豆油中的饱和溶液来进行染色；染料从油滴扩散到整个卵的内质网膜中，但没有扩散到其他细胞器中。共聚焦显微镜显示，大的潴泡贯穿卵的内部，皮质处有管状膜网络。由于DiI的扩散局限于连续的双层膜，染料的扩散支持了内质网是一个细胞范围内相互连接的区室的概念。在延时观察中，内部潴泡处于持续运动中，而皮质内质网是静止的。受精后，内部内质网似乎变得更精细地划分，开始时是一个明显与钙波同时出现的波，并在2 - 3分钟时最为明显。到5 - 8分钟时，内质网恢复到类似于未受精卵的组织状态。皮质网络在受精时也发生了变化；它变得紊乱，最终恢复。DiI标记使得在原核迁移和有丝分裂期间能够对内质网进行连续观察。当精子核和中心粒（精子星体）迁移到卵的中心时，DiI染色的膜聚集在围绕它们的微管阵列区域；在整个原核融合和前两个有丝分裂周期中，这种聚集一直持续在中心体和受精卵核附近。我们使用了一种新方法来观察活细胞中内质网的空间和时间组织，并且我们证明了受精时内质网有显著的重组。
