Introduction There is increasing research interest in the role of inositol- containing compounds in preimplantation embryo development. Inositol is essential for continued cell proliferation and growth of rabbit blastocysts (Fahy and Kane, 1992) and it also stimulates hatching of hamster blastocysts (Kane and Bavister, 1988). A major role of inositol is as part of the phosphatidylinositol (PtdIns) signal transduction system in which receptor-stimulated activation of PtdIns-dependent phospholipase C (PLC) leads to hydrolysis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2 ) to produce the two second messengers: inositol(1,4,5)trisphosphate (Ins(1,4,5)P 3 ), which acts to release Ca 2+ from internal Ca 2+ stores, and diacylglycerol, which activates protein kinase C (for a review, see Berridge, 1992). Mouse (Kane et al., 1992), rabbit (Fahy and Kane, 1993) and cattle (Hynes et al., 2000) embryos, and mouse embryonic stem (ES) cells (Duffy and Kane, 1996) incorporate inositol into the phosphoinositides and inositol phosphates of the PtdIns signal transduction system. In addition, inhibitors of PLC inhibit mouse blastocyst formation (Stachecki and Armant, 1996). Perhaps the most important evidence for an essential role for phosphoinositide compounds in preimplantation development is the demon- stration that knocking out the PLC β3 gene prevents development of mouse embryos to the blastocyst stage (Wang et al., 1998). Another role for inositol is in the formation of glycosylphosphatidylinositol (GPI) lipids, which function as anchors for the attachment of surface proteins to the plasma membrane of various cell types (for a review, see Low, 1987). The GPI anchors typically contain phosphatidylinositol inserted in the cell membrane, connected to a carbohydrate core structure consisting of glucosamine, mannose, galactose and ethanolamine, to which the protein is attached by a phosphodiester linkage (for reviews, see Englund, 1993; McConville and Menon, 2000). Another characteristic of the GPI anchors is that they may be cleaved to release the protein by endogenous PtdIns-specific phospholipases (Brewis et al., 1994). Glycosylphosphatidylinositols are also involved in the signal transduction pathways of numerous hormones and growth factors such as insulin (Saltiel and Cuatrecasas, 1986), and growth factors and cytokines, including insulin- like growth factor I (IGF-I), epidermal growth factor (EGF), and transforming growth factor β1 (TGF-β1) as well as interleukins 1 and 2 (reviewed by Jones and Varela-Nieto, 1998). Insulin and other growth factors activate PLC to hydrolyse GPI, resulting in the release of the polar head group, an inositol-containing oligosaccharide called an Nature of glycosylphosphatidylinositols produced by mouse embryonic stem cells L. R. Quinlan and M. T. Kane* Department of Physiology, National University of Ireland, Galway, University Road, Galway, Ireland Reproduction (2001) 122, 785–791 Research Incorporation of [ 3 H]inositol into mouse embryonic stem cells of the CCE cell line leads to the labelling of the three common phosphoinositides, phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and a fourth unknown lipid (lipid X). Incubation with [ 3 H]glucosamine results in the labelling of lipid X and at least one other lipid that co-migrates with phosphatidylinositol (lipid Y), indicating that both of these lipids are putative glycosylphosphatidylinositols. In this study, the incorporation of other possible glycosylphos- phatidylinositol precursors, ethanolamine, mannose and galactose, into lipids X and Y was examined. Galactose was incorporated into lipids X and Y, and ethanolamine and mannose into lipid Y only. Inhibitors of glycosylphos- phatidylinositol biosynthesis pathways, mannosamine and 2-fluoro-2-deoxyglucose, both significantly inhibited ethanolamine incorporation into lipid Y. A high glucose concentration (25 mmol l –1 ) abolished the action of both inhibitors. Phospholipase C treatment of embryonic stem cells that had been labelled in culture with [ 3 H]ethanolamine caused a large release of ethanolamine label into the incubation medium and markedly decreased the amount of ethanolamine-labelled lipid Y remaining in the cell membranes. These effects were almost totally abolished by incubation with mannosamine before ethanolamine labelling. These studies strongly indicate that lipid Y is a member of the protein anchor class of glycosylphosphatidylinositol, whereas lipid X is a member of the signal transduction inositol phosphoglycan class of glycosylphosphatidylinositol. © 2001 Society for Reproduction and Fertility 1470-1626/2001 *Correspondence Email: michael.kane@nuigalway.ie