Cumulus cells were isolated from bovine COCs collected from antral follicles and analyzed by immunofluorescence and confocal microscopy for (A) OGT, (B) OGA and (C) O-GlcNAc. The gray scale images (left) show the localization of each specific marker, and the merged images (right) show DNA in blue, F-actin in red, and the marker of interest (OGT, OGA, O-GlcNAc) in green. Single optical sections are shown. The arrowhead in (C) highlights O-GlcNAcylated proteins that localize to the nuclear envelope. This experiment was repeated three times with pools of cumulus cells and representative images are shown. The scale bars are 25 μm.