Laboratory of Cellular and Developmental Biology, Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy.
PLoS One. 2012;7(6):e39231. doi: 10.1371/journal.pone.0039231. Epub 2012 Jun 18.
Ionomycin is a Ca(2+)-selective ionophore that is widely used to increase intracellular Ca(2+) levels in cell biology laboratories. It is also occasionally used to activate eggs in the clinics practicing in vitro fertilization. However, neither the precise molecular action of ionomycin nor its secondary effects on the eggs' structure and function is well known. In this communication we have studied the effects of ionomycin on starfish oocytes and zygotes. By use of confocal microscopy, calcium imaging, as well as light and transmission electron microscopy, we have demonstrated that immature oocytes exposed to ionomycin instantly increase intracellular Ca(2+) levels and undergo structural changes in the cortex. Surprisingly, when microinjected into the cells, ionomycin produced no Ca(2+) increase. The ionomycin-induced Ca(2+) rise was followed by fast alteration of the actin cytoskeleton displaying conspicuous depolymerization at the oocyte surface and in microvilli with concomitant polymerization in the cytoplasm. In addition, cortical granules were disrupted or fused with white vesicles few minutes after the addition of ionomycin. These structural changes prevented cortical maturation of the eggs despite the normal progression of nuclear envelope breakdown. At fertilization, the ionomycin-pretreated eggs displayed reduced Ca(2+) response, no elevation of the fertilization envelope, and the lack of orderly centripetal translocation of actin fibers. These alterations led to difficulties in cell cleavage in the monospermic zygotes and eventually to a higher rate of abnormal development. In conclusion, ionomycin has various deleterious impacts on egg activation and the subsequent embryonic development in starfish. Although direct comparison is difficult to make between our findings and the use of the ionophore in the in vitro fertilization clinics, our results call for more defining investigations on the issue of a potential risk in artificial egg activation.
离子霉素是一种 Ca(2+)-选择性载体，广泛用于增加细胞生物学实验室中的细胞内 Ca(2+)水平。它偶尔也用于在进行体外受精的临床中激活卵子。然而，离子霉素的确切分子作用及其对卵子结构和功能的次要影响并不为人所知。在本通讯中，我们研究了离子霉素对海星卵母细胞和受精卵的影响。通过使用共聚焦显微镜、钙成像以及光和透射电子显微镜，我们证明了暴露于离子霉素的未成熟卵母细胞会立即增加细胞内 Ca(2+)水平，并在皮质中发生结构变化。令人惊讶的是，当将其微注射到细胞中时，离子霉素不会产生 Ca(2+)增加。离子霉素诱导的 Ca(2+)上升后，肌动蛋白细胞骨架迅速发生变化，在卵母细胞表面和微绒毛中出现明显的解聚，并在细胞质中聚合。此外，皮质颗粒在加入离子霉素几分钟后被破坏或与白色小泡融合。这些结构变化尽管核膜破裂正常进行，但阻止了卵子的皮质成熟。在受精时，经离子霉素预处理的卵子显示 Ca(2+)反应降低、受精包被物没有升高，以及肌动蛋白纤维的有序向心移位缺失。这些改变导致单核受精卵的细胞分裂困难，并最终导致更高的异常发育率。总之，离子霉素对海星卵子的激活和随后的胚胎发育有各种有害影响。尽管我们的发现与离子载体在体外受精临床中的应用之间的直接比较具有挑战性，但我们的结果呼吁对人工卵子激活的潜在风险问题进行更明确的研究。
