Structural Changes during Development in Bovine Fetal Epiphyseal Cartilage

Abstract

Sedimentation coefficients of approximately 150 S show that proteoglycan aggregates from bovine fetal epiphyseal cartilage are exceptionally large. To determine the structural basis for the unusually large size of these proteoglycan aggregates, identify changes in proteoglycan structure with changing developmental age, and provide a basis for demonstrating the structural modifications which may occur in growth plate proteoglycan aggregates during endochondral ossification, we examined the molecular architecture and dimensions of fetal epiphyseal proteoglycans by electron microscopy. The eight bovine epiphyseal cartilages studied ranged in fetal age from 168 to 241 days. Proteoglycans were extracted in 4 M guanidium hydrochloride containing protease inhibitors and isolated by equilibrium density gradient centrifugation under associative and dissociative conditions. Electron micrographs were made from monolayer preparations of proteoglycan-cytochrome c mixtures on nitrocellulose support films. The overall molecular architecture of the proteoglycan aggregates from fetal epiphyseal cartilages was similar to that of aggregates from other cartilages and showed a single, unbranched central hyaluronic acid filament to which many proteoglycan monomers were attached. However, the dimensions of the fetal proteoglycans differed strikingly from those of proteoglycans from mature cow nasal or immature calf nasal cartilage. Specifically, proteoglycan aggegates from bovine fetal epipyseal cartilage showed: (a) longer hyaluronic acid central filaments; (b) greater numbers of proteoglycan monomers per aggregate; (c) closer spacing of proteoglycan monomers along the hyaluronic acid central filament; and (d) longer proteoglycan monomer core proteins. Proteoglycan monomers bound to hyaluronate consisted of two segments: (1) a peripheral thick segment, composed of the chondroitin sulfate chains condensed along the peripheral portion of the protein core, which corresponds to the chondroitin sulfate-rich region; and, (2) a central thin segment, devoid of visible glycosaminoglycan chains, which attaches directly to the hyaluronic acid central filament and contains the hyaluronic acid binding region and a portion of the keratan sulfate-rich region. The contribution of the thin segment to total monomer length decreased as total monomer length increased.Thus, in longer monomers the thick segment contributed more to total monomer length and the thin segment contributed less. Both the thin and thick segments of monomers from fetal epiphyseal cartilage were longer than the corresponding segments of calf nasal cartilage and mature bovine nasal cartilage monomers. The shorter thin segments in older cartilage may indicate the presence of more keratan sulfate while the shorter thick segments may indicate differences in monomer biosynthesis or degredation. In addition, monomer lengths in aggegrates from fetal epiphyseal cartilage were less variable than the monomer lengths in aggregates from the older cartilages which may also reflect differences in biosynthesis or in proteolytic degredation. Aggregate hyaluronate filament length, number of monomers per aggregate, and spacing between monomers were not related to fetal age, but aggregate monomer length decreased with increasing fetal age. This description of the molecular architecture and dimensions of fetal epiphyseal cartilage proteoglycans provides an essential quantitative baseline for elucidating the structural changes which may occur in growth plate proteoglycans during endochondral ossification.