Purpose: To determine the morphologic and biochemical events preceding the breakdown of fiber cell nuclei in the primate lens.
Methods: Monkey lens slices were labeled with fluorescent probes and optically sectioned using a confocal microscope. The distribution of nuclear histones was visualized by immunofluorescence. DNA and cellular membranes were imaged simultaneously by staining with SYTO 17 and 3,3'-dihexyloxacarbocyanine iodide, respectively. The condition of fiber cell DNA during differentiation was determined by an in situ DNA fragmentation assay. The assay was adapted to allow the detection of DNA fragments with 3'-OH or 3'-PO4 termini.
Results: Monkey lens fiber nuclei passed through distinct stages before disintegrating. In the outer cell layers, the nuclei were large, smooth, and oval-shaped with prominent nucleoli. Deeper in the lens, they had a flattened profile with whorls of membranous material and nucleic acid accumulated at one end. At this point, histone immunofluorescence was reduced and the nucleoli had a characteristic, spoked appearance. At the border of the organelle-free zone, the intracellular membranes (including the nuclear envelope) disappeared, and particulate material was released from the nuclei into the cytoplasm. This material was stained by SYTO-17 and the DNA fragmentation assay, indicating that it contained fragmented DNA with 3'-OH termini.
Conclusions: The denucleation process in the primate lens differs from that described recently in the embryonic chicken lens. In particular, the extrusion of nuclear material and persistence of DNA-rich particles in the fiber cytoplasm are novel features. One similarity between the denucleation process in these species is the appearance of 3'-OH ends in the DNA after the loss of the nuclear membrane.