END LABELING STUDIES OF FRAGMENTED DNA IN THE AVIAN GROWTH-PLATE - EVIDENCE OF APOPTOSIS IN TERMINALLY DIFFERENTIATED CHONDROCYTES

The chondro-osseous junction has been the subject of considerable scru tiny, especially in terms of the fate and role of the terminally diffe rentiated chondrocyte. Although it has been proposed that these cells change their phenotype and survive in the epiphysis, possibly as osteo blasts, evidence from a number of other studies suggests that chondroc ytes may undergo apoptosis or programmed cell death. A useful test for programmed cell death is to end label DNA in cryosections using the c ommercial reagent ApopTagTM and detect antibody binding to fragmented DNA by epifluorescence; more direct assessments include examination of the nucleus for condensation of chromatin, evaluating fragmentation t hrough alkaline and pulsed field agarose gel electrophoresis of DNA, a nd measuring apoptosis by flow cytometry. We found that we could label cells in the proliferative and the hypertrophic region of the proxima l tibial growth plate of the chick with ApopTag. Most of the chondrocy tes in the hypertrophic region were labeled by the reagent; in contras t, few proliferative chondrocytes were stained by the end-labeling pro cedure. Both agarose and pulsed field electrophoresis were used to con firm that there was fragmentation of chondrocyte DNA. Alkaline gel ele ctrophoresis indicated that there was more fragmentation of DNA from h ypertrophic cells than from proliferative chondrocytes. Further eviden ce in support of apoptosis was provided by electron microscopic observ ation of cells in the hypertrophic region of the growth plate. We note d that many of the cells in this region of the growth plate appeared t o be undergoing programmed cell death since their nuclei contained con densed chromatin. Finally, we used flow cytometry to analyze chondrocy tes isolated from the proliferating and hypertrophic regions of the gr owth plate for apoptosis. Dual parameteric now cytometric contour plot s of Hoechst and 7-amino-actinomycin D fluorescence showed that about 8% of cells in the plate were apoptotic. Most of these cells were in h ypertrophic cartilage. In summary, the results of this investigation i ndicate that chondrocytes terminate their life history by apoptosis. W hile it is possible that the terminal labeling studies may overestimat e the number of cells undergoing this event, the data lend credence to the view that cells are removed from the epiphysis through apoptosis. If this is the case, then chondrocytes probably enter the terminal ph ase of their life as fully functioning cells and genomic, and/or local environmental conditions provide termination signals that initiate ev ents that lead to programmed cell death.