Khidir M A, Stachecki J J, Krawetz S A, Armant D R
C. S. Mott Center for Human Growth and Development, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Exp Cell Res. 1995 Aug;219(2):619-23. doi: 10.1006/excr.1995.1272.
Lysolecithin was used to permeabilize embryonic cells to impermeant compounds without compromising embryo viability. Within 2 min of exposure to 0.05% lysolecithin, mouse morulae became permeable to inositol 1,4,5-trisphosphate, leading to the release of calcium from intracellular stores. Although these morulae remained permeable for at least 2 h, they developed normally to the blastocyst stage during subsequent culture in vitro. Lysolecithin permeabilization was then used to potentiate the internalization of alpha-amanitin. Pretreatment with lysolecithin for 2 min markedly accelerated the onset of transcriptional inhibition from 3 h to 10 min after alpha-amanitin addition. The rapid inhibition by alpha-amanitin of mRNA synthesis in lysolecithin-treated embryos provided a precise method for delineating developmentally important transcriptional periods. Using this protocol, we found that mRNAs required for embryonic cavitation were synthesized between 87 and 91 h post-hCG, shortly before blastocoel formation commenced.
溶血卵磷脂被用于使胚胎细胞对非渗透性化合物具有通透性，同时又不损害胚胎的活力。在暴露于0.05%溶血卵磷脂的2分钟内，小鼠桑椹胚对肌醇1,4,5-三磷酸变得具有通透性，导致细胞内储存的钙释放。尽管这些桑椹胚至少在2小时内保持通透性，但在随后的体外培养中它们正常发育到囊胚阶段。然后利用溶血卵磷脂通透化作用来增强α-鹅膏蕈碱的内化。用溶血卵磷脂预处理2分钟显著加速了转录抑制的起始，从添加α-鹅膏蕈碱后的3小时缩短至10分钟。α-鹅膏蕈碱对经溶血卵磷脂处理的胚胎中mRNA合成的快速抑制提供了一种精确的方法来描绘发育过程中重要的转录时期。使用该方案，我们发现胚胎空化所需的mRNA在hCG后87至91小时之间合成，就在囊胚腔形成开始前不久。
