Novel Use of Bovine Zeta-Crystallin as a Conformational DNA Probe to Characterize a Phase Transition Zone and Terminally Differentiating Fiber Cells in the Adult Canine Ocular Lens

Abstract

Using a novel immunocytochemical staining method, we aimed to characterize the phase transition zone (PTZ) (∼100 μm) in adult ocular lenses and the process of terminal differentiation (denucleation) within normal fiber cells. The binding to DNA of zeta(ζ)-crystallin (Z-DNA-binding protein) and anti-double-stranded (ds-)-B-DNA antibody probes was found to decline gradually throughout denucleating fibers, with a precipitous decrease occurring at about 100 μm (PTZ). Nuclei of superficial fiber cells (in front of the PTZ) showed the highest DNA probe-binding values, followed by middle fibers (MF) and deep fibers (DF). With the use of ζ-crystallin, anti-ds-B-DNA antibody, and anti-single stranded (ss-) DNA antibody probes, it was possible to reveal a loss of reactivity of fiber cell ds-DNA. Ss-DNA antibody binding was seen initially in the MF and reached its highest intensity level in the DF. The pattern of ζ-crystallin probe-DNA reactivity correlates with the loss of anti-B-DNA antibody staining and decreased eosin-protein staining. These data suggest that a reorganization of DNA and intracellular protein supramolecular order in normal adult lenses occurs at a depth of about 100 μm (PTZ).