Liposomes fuse with sperm cells and induce activation by delivery of impermeant agents

Sperm cell activation is a critical step in fertilization. To directly inve stigate the cell signaling events leading to sperm activation it is necessa ry 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 (Rls) and NBD phosphatidylethanolamine (NBD DH PE)/rhodamine phosphatidylethanolamine (rhod DHPE) fusion assays in bulk sa mples, (2) membrane transfer of fluorescence from liposome membranes labele d with R18 and rhodamine-tagged phosphatidylethanolamine (TRITC DHPE), and (3) lumenal transfer of impermeant calcium ions from liposomes to sperm cel ls, a process that stimulated sperm cell activation. Intermediate-sized uni lamellar liposomes (98.17 +/- 15.34 nm) were prepared by the detergent-remo val technique using sodium cholate as the detergent and a phosphatidylcholi ne/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) lipid compositio n. In the Rls fusion assays, self-quenching increased logarithmically with increasing concentrations of Rls in the liposome membranes; addition of unl abeled sperm to R18-labeled liposomes lead to a rapid release of self-quenc hing. In the NBD DHPE/rhod DHPE resonance energy transfer (RET) fusion assa y, RET was rapidly reduced under similar conditions. In addition, individua l 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 i n a time-dependent increase in sperm cell activation (7.5-fold over control s after 15 min). We conclude that liposomes can be used for direct loading of membrane-impermeant agents into sea squirt sperm cell cytoplasm, and tha t delivery occurs via fusion and content intermixing. (C) 1999 Elsevier Sci ence B.V. All rights reserved.