Garrett F E, Goel S, Yasul J, Koch R A
Sperm Cell Biology and Gamete Ultrastructure Laboratory, Department of Biological Science, California State University, Fullerton, P.O. Box 6850, Fullerton, CA 92834-6850, USA.
Biochim Biophys Acta. 1999 Feb 4;1417(1):77-88. doi: 10.1016/s0005-2736(98)00258-2.
Sperm cell activation is a critical step in fertilization. To directly investigate the cell signaling events leading to sperm activation it is necessary to deliver membrane impermeant agents into the cytoplasm. In this study, the use of liposomes as possible agent-loading vectors was examined using (1) the octadecylrhodamine B (R18) and NBD phosphatidylethanolamine (NBD DHPE)/rhodamine phosphatidylethanolamine (rhod DHPE) fusion assays in bulk samples, (2) membrane transfer of fluorescence from liposome membranes labeled with R18 and rhodamine-tagged phosphatidylethanolamine (TRITC DHPE), and (3) lumenal transfer of impermeant calcium ions from liposomes to sperm cells, a process that stimulated sperm cell activation. Intermediate-sized unilamellar liposomes (98.17+/-15.34 nm) were prepared by the detergent-removal technique using sodium cholate as the detergent and a phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) lipid composition. In the R18 fusion assays, self-quenching increased logarithmically with increasing concentrations of R18 in the liposome membranes; addition of unlabeled sperm to R18-labeled liposomes lead to a rapid release of self-quenching. In the NBD DHPE/rhod DHPE resonance energy transfer (RET) fusion assay, RET was rapidly reduced under similar conditions. In addition, individual sperm became fluorescent when TRITC DHPE-labeled liposomes were incubated with unlabeled sperm cells. Incubation of sperm cells with empty liposomes did not significantly affect sperm cell activation and did not alter cell morphology. However, incubation with Ca (10 mM)-loaded liposomes resulted in a time-dependent increase in sperm cell activation (7.5-fold over controls after 15 min). We conclude that liposomes can be used for direct loading of membrane-impermeant agents into sea squirt sperm cell cytoplasm, and that delivery occurs via fusion and content intermixing.
精子细胞激活是受精过程中的关键步骤。为了直接研究导致精子激活的细胞信号事件，有必要将膜不透性试剂导入细胞质。在本研究中，使用（1）在大量样品中进行的十八烷基罗丹明B（R18）和NBD磷脂酰乙醇胺（NBD DHPE）/罗丹明磷脂酰乙醇胺（rhod DHPE）融合测定，（2）来自用R18和罗丹明标记的磷脂酰乙醇胺（TRITC DHPE）标记的脂质体膜的荧光膜转移，以及（3）从脂质体到精子细胞的不透性钙离子的内腔转移，该过程刺激精子细胞激活，来研究脂质体作为可能的试剂装载载体的用途。使用胆酸钠作为去污剂，通过去污剂去除技术制备中等大小的单层脂质体（98.17±15.34nm），脂质组成是磷脂酰胆碱/磷脂酰乙醇胺/胆固醇（2:1:1摩尔比）。在R18融合测定中，随着脂质体膜中R18浓度的增加，自猝灭呈对数增加；将未标记的精子添加到R18标记的脂质体中导致自猝灭迅速释放。在NBD DHPE/rhod DHPE共振能量转移（RET）融合测定中，在类似条件下RET迅速降低。此外，当将TRITC DHPE标记的脂质体与未标记的精子细胞一起孵育时，单个精子会发出荧光。将精子细胞与空脂质体孵育不会显著影响精子细胞激活，也不会改变细胞形态。然而，与负载Ca（10 mM）的脂质体孵育导致精子细胞激活呈时间依赖性增加（15分钟后比对照高7.5倍）。我们得出结论，脂质体可用于将膜不透性试剂直接装载到海鞘精子细胞细胞质中，并且递送是通过融合和内容物混合发生的。
