CHANGES IN YOLK PLATELET PH DURING XENOPUS-LAEVIS DEVELOPMENT CORRELATE WITH YOLK UTILIZATION - A QUANTITATIVE CONFOCAL MICROSCOPY STUDY

The variations of the pH in Xenopus yolk platelets have been estimated by fluorescence confocal microscopy and computer image processing, Fo r pH measurements in vitellogenic oocytes, the pH-sensitive fluorescen t dye, DM-NERF, was coupled to vitellogenin, and the DM-NERF-vitelloge nin was taken up by oocytes via receptor-mediated endocytosis. Dual em ission ratio measurements of internalized DM-NERF-vitellogenin indicat ed that the mature yolk platelets are mildly acidic (pH 5.6), Their pr ecursors, the primordial yolk platelelets, have a similar pH, This pH is probably sufficiently low for the partial cleavage of vitellogenin to yolk proteins, but not for yolk degradation, The yolk platelet pH a t various developmental stages was estimated by measuring the accumula tion of Acridine Orange, both in isolated yolk platelets and in disagg regated embryonic cells, During oogenesis, the yolk platelets accumula ted a constant amount of Acridine Orange, correspending to a pH of aro und 5.7, During embryogenesis, however, yolk platelets became progress ively much more acidic (pH <5). Acidification correlated with yolk deg radation in the various tissues examined, and yolk utilization was blo cked when acidification was inhibited with bafilomycin, an inhibitor o f vacuolar H+-ATPase, Bafilomycin also inhibited differentiation of ce lls isolated from stage 13-15 embryos, These data show that the yolk p latelet pH is developmentally regulated and is involved in triggering yolk degradation, Also, yolk acidification and degradation appeared to be associated with cell differentiation and with the formation of the endosomal/lysosomal compartment, typical of adult cells, but absent i n early embryos.