Use of carboxyfluorescein diacetate to study formation of permeable channels between mouse blastomeres

Abstract

The transmission of signals between cells of the early preimplantation embryo may be important in the recognition of relative cell position, thereby influencing the direction of cell differentiation^1–5. Gap junctional (water-permeable) channels may provide one route for such signal transmission⁶. A previous report⁷, in which intact, zona-free mouse embryos were impaled by microelectrodes, demonstrated that gap junction-mediated dye transfer and electrical continuity were absent between blastomeres of two- and four-cell embryos but appeared during the eight-cell stage. It is at the eight-cell stage that compaction occurs, during which cells polarize³, flatten on each other^3,8 and show the first morphological evidence of intercellular junctions^9,10. We report here the use of a simple, non-iontophoretic method to detect the passage of a low-molecular-weight dye between aggregated blastomeres of the early preimplantation mouse embryo. Cells are incubated in the nonfluorescent, non-polar reagent, 6-carboxyfluorescein diacetate (CFDA)^11,12, which enters the cell freely, where it becomes entrapped following enzymatic conversion¹³ to the hydrophilic fluorophore 6-carboxyfluorescein (CF; molecular weight 370). The labelled cells may then be aggregated with unlabelled cells and the formation of communicating channels between cells is revealed by the passage of dye from labelled to unlabelled blastomeres. CFDA provides better results than previously used fluorescein esters^14,15, which hydrolyse to a much less hydrophilic fluorophore that leaks from the cell relatively quickly^11,15. Using CFDA we have confirmed that the capacity to form channels is lacking at all stages from the mature oocyte to the four-cell embryo but appears during the first 6 h of the eight-cell stage, preceding the fully compacted state.