Simerly C, Nowak G, de Lanerolle P, Schatten G
Division of Reproductive Sciences, Oregon Regional Primate Research Center, Departments of Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97006, USA.
Mol Biol Cell. 1998 Sep;9(9):2509-25. doi: 10.1091/mbc.9.9.2509.
To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome-cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.
为了探究非肌肉肌球蛋白II亚型在小鼠配子发生、受精及早期发育过程中的作用，我们使用针对肌球蛋白IIA和IIB亚型的单特异性抗体进行了定位和显微注射研究。每种肌球蛋白II抗体均可识别卵母细胞中的一种205 kDa蛋白，但不能识别成熟精子中的该蛋白。肌球蛋白IIA和IIB在减数分裂成熟过程及受精后表现出差异表达：只有IIA亚型可检测到中期纺锤体或在有丝分裂的分裂沟中积累。在未受精的卵母细胞中，两种肌球蛋白亚型均在覆盖中期停滞的第二次减数分裂纺锤体的皮质中呈极化分布。用秋水仙酰胺破坏纺锤体后，皮质极化发生改变：分散的减数分裂染色体引发肌球蛋白IIA和微丝在每个染色体团块附近组装。在精子融合过程中，两种肌球蛋白II亚型均集中在第二极体分裂沟和精子融合锥中。在功能实验中，显微注射肌球蛋白IIA抗体可破坏减数分裂成熟过程，使细胞停滞在中期II，这可能是由于纺锤体相关的肌球蛋白IIA蛋白耗竭以及抗体与染色体表面结合所致。相反，显微注射肌球蛋白IIB抗体可阻断秋水仙酰胺处理的成熟卵母细胞中微丝介导的染色体分散，提示其在介导染色体-皮质肌动球蛋白相互作用中发挥作用。单独或共同注射的两种肌球蛋白II抗体均不会阻断第二极体形成、体外受精或胞质分裂。最后，显微注射一种不可磷酸化的20 kDa调节性肌球蛋白轻链可特异性阻断精子融合锥解体并阻碍细胞周期进程，提示干扰肌球蛋白II磷酸化会影响受精。因此，传统肌球蛋白通过参与偏心减数分裂纺锤体定位（译者注：此处原文为eccentric meiotic spindle positioning，直译为偏心减数分裂纺锤体定位，可能是指纺锤体在卵母细胞中的不对称分布等情况，具体含义需结合专业知识进一步理解）、精子融合锥动态变化及胞质分裂来打破卵母细胞中的皮质对称性。尽管小鼠精子不表达肌球蛋白II，但不同的肌球蛋白II亚型在早期胚胎发育过程中可能具有不同的作用。
