Institute of Experimental Endocrinology and Oncology 'G. Salvatore', Second Unit, National Research Council, 80131Naples, Italy.
Department of Research Infrastructures for Marine Biological Resources, Stazione Zoologica Anton Dohrn, 80121Naples, Italy.
Zygote. 2024 Feb;32(1):38-48. doi: 10.1017/S0967199423000552. Epub 2023 Dec 5.
The actin filaments on the surface of echinoderm oocytes and eggs readily undergo massive reorganization during meiotic maturation and fertilization. In sea urchin eggs, the actin cytoskeletal response to the fertilizing sperm is fast enough to accompany Ca signals and to guide sperm's entry into the egg. Although recent work using live cell imaging technology confirmed changes in the actin polymerization status in fertilized eggs, as was previously shown using light and electron microscopy, it failed to provide experimental evidence of F-actin depolymerization a few seconds after insemination, which is concurrent with the sperm-induced Ca release. In the present study, we applied Raman microspectroscopy to tackle this issue by examining the spectral profiles of the egg's subplasmalemmal regions before and after treating the eggs with actin drugs or fertilizing sperm. At both early (15 s) and late (15 min) time points after fertilization, specific peak shifts in the Raman spectra revealed change in the actin structure, and Raman imaging detected the cytoskeletal changes corresponding to the F-actin reorganization visualized with LifeAct-GFP in confocal microscopy. Our observation suggests that the application of Raman spectroscopy, which does not require microinjection of fluorescent probes and exogenous gene expression, may serve as an alternative or even advantageous method in disclosing rapid subtle changes in the subplasmalemmal actin cytoskeleton that are difficult to resolve.
棘皮动物卵母细胞和卵子表面的肌动蛋白丝在减数分裂成熟和受精过程中容易发生大规模重组。在海胆卵中，肌动蛋白细胞骨架对受精精子的反应速度足够快，可以伴随 Ca 信号并指导精子进入卵子。尽管最近使用活细胞成像技术的工作证实了受精卵中肌动蛋白聚合状态的变化，如先前使用光和电子显微镜所示，但它未能提供在授精后几秒钟内 F-肌动蛋白解聚的实验证据，这与精子诱导的 Ca 释放同时发生。在本研究中，我们应用拉曼微光谱技术通过检查卵的亚质膜区域的光谱轮廓来解决这个问题，这些区域在用肌动蛋白药物处理卵或受精精子前后进行了处理。在受精后 15 秒（早期）和 15 分钟（晚期）的时间点，拉曼光谱中的特定峰位移揭示了肌动蛋白结构的变化，拉曼成像检测到与用 LifeAct-GFP 在共聚焦显微镜中可视化的 F-肌动蛋白重排相对应的细胞骨架变化。我们的观察表明，拉曼光谱的应用不需要荧光探针的微注射和外源基因表达，它可能是揭示亚质膜肌动蛋白细胞骨架中难以解决的快速微妙变化的替代方法，甚至是有利的方法。
