Liu Ji-Long, Sung Li-Ying, Tian X Cindy, Yang Xiangzhong
Department of Animal Science, University of Connecticut, Storrs 06269, USA.
Biol Reprod. 2002 Dec;67(6):1853-63. doi: 10.1095/biolreprod.102.005694.
A previous study showed that with hypertonic sucrose treatment, a projection is formed in mouse metaphase II (MII) oocytes in proximity to the spindle and chromosomes, where a polarized cortical domain is located. However, little is known about the mechanisms involved in this process. Here, we designed a series of experiments to test the hypothesis that hypertonicity is the induction factor for the formation of projections in mouse MII oocytes. Our hypothesis was supported by the following evidence: 1) different concentrations of sucrose affected the formation and shape of projections, whereas serum or basic media had little effect; 2) other hypertonic sugar solutions could also induce projection formation; and 3) projections could also be induced by hypertonic NaCl solution. We then tested the hypothesis that the cytoskeleton was involved in the formation of hypertonicity-induced projections. This was investigated by culturing MII- and germinal vesicle-stage mouse oocytes in the presence or absence of cytoskeletal inhibitors, including cytochalasin B (disruption of actin filaments), nocodazole (disruption of microtubules), and taxol (polymerization of tubulin molecules). We found that none of the cytoskeletal inhibitors alone could prevent hypertonicity-induced projection formation, whereas the combination of cytochalasin B with nocodazole or with taxol blocked the formation of these projections in most matured oocytes. When immature oocytes were incubated in cytochalasin B, but not in nocodazole or taxol, the formation of an actin-rich domain and the peripheral positioning of the spindle were blocked during maturation; hence, no projections were formed, even after hypertonic sucrose treatment. Based on these observations, we propose that three components are necessary for projection formation: 1) a polarized cortical patch (e.g., an actin-rich domain), 2) rigid submembrane structures (e.g., a spindle and/or chromosomes), and 3) solid connections between the above. Any disturbance of one of these factors will affect the hypertonicity-induced projection formation. Hypertonicity-induced projection in mouse oocytes thus provides an experimental model for studies regarding cell polarity and the interaction between membrane and submembrane components.
先前的一项研究表明，用高渗蔗糖处理时，在小鼠中期II（MII）卵母细胞中靠近纺锤体和染色体的位置会形成一个突起，此处是极化皮质区域所在之处。然而，对于这一过程涉及的机制知之甚少。在此，我们设计了一系列实验来检验高渗性是小鼠MII卵母细胞中突起形成的诱导因子这一假说。我们的假说得到了以下证据的支持：1）不同浓度的蔗糖会影响突起的形成和形状，而血清或基础培养基影响很小；2）其他高渗糖溶液也能诱导突起形成；3）高渗氯化钠溶液也能诱导突起形成。然后，我们检验了细胞骨架参与高渗诱导突起形成的假说。通过在有或没有细胞骨架抑制剂的情况下培养MII期和生发泡期小鼠卵母细胞来对此进行研究，这些抑制剂包括细胞松弛素B（破坏肌动蛋白丝）、诺考达唑（破坏微管）和紫杉醇（微管蛋白分子聚合）。我们发现，单独一种细胞骨架抑制剂都不能阻止高渗诱导的突起形成，而细胞松弛素B与诺考达唑或与紫杉醇联合使用则能在大多数成熟卵母细胞中阻止这些突起的形成。当未成熟卵母细胞在细胞松弛素B中孵育时，但在诺考达唑或紫杉醇中未孵育时，在成熟过程中富含肌动蛋白区域的形成和纺锤体的外周定位会被阻断；因此，即使经过高渗蔗糖处理也不会形成突起。基于这些观察结果，我们提出突起形成需要三个成分：1）一个极化皮质斑（例如，富含肌动蛋白的区域），2）刚性的膜下结构（例如，纺锤体和/或染色体），以及3）上述两者之间的牢固连接。这些因素中的任何一个受到干扰都会影响高渗诱导的突起形成。因此，小鼠卵母细胞中的高渗诱导突起为研究细胞极性以及膜与膜下成分之间的相互作用提供了一个实验模型。
