Decreased opsin mRNA and immunoreactivity in progressive rod-cone degeneration (prcd): Cytochemical studies of early disease and degeneration

Abstract

Opsin mRNA level and immunoreactivity were examined by in situ hybridization and immunocytochemistry in normal and progressive rod cone degeneration (prcd)-affected dogs. In situ hybridization used 35S- and/or 3 H-labeled bovine opsin cRNA probes; immunocytochemistry used six monoclonal mouse anti-bovine opsin antibodies (MAb1) that are specific to different regions of the N-terminal, loop v-vi and the C-terminal domains. Optimal labeling and histological resolution at the single cell level were achieved with semi-thin sections of DGD wax-embedded tissues; it was possible to correlate the cytochemical observations with the disease staging in topographically defined regions that exhibited different disease severity. In early disease (stages 0-1), opsin mRNA levels and immunoreactivity were normal. During the transition from disease to degeneration (stage 2), however, opsin mRNA was reduced sharply; it then rapidly became undetectable in late stages of degeneration (stages 3, 4). Reduction of immunoreactivity was seen with all the MAbs in stages 2 and 3, but the degree of reduction varied remarkably in different regions of the protein molecule; immunoreactivity was reduced more in the cytoplasmic regions, particularly in the phosphorylation sites and the far end of the C-terminal domain. In contrast, the epitopes of the N-terminal domain that are located in the intradiscal compartment were better preserved. It is noteworthy that, in stages 2 and 3, many rod cells still survived despite the decrease in the mRNA level and immunoreactivity. The results indicate that early disease in prcd-affected rods is not initiated by a reduction of opsin mRNA or the protein quantity. However, opsin expression disappears in early degeneration and before cell death. The differences in immunoreactivity with disease may result either from alterations in the protein structure or configuration, or from selective loss of epitopes located in the cytoplasmic domains of the molecule. © 1994 Academic Press. All rights reserved.