Proteoglycan synthesis by corneal explants from developing embryonic chicken.

Abstract

Proteoglycan biosynthesis in developing chicken corneas was studied. Corneas free of scleral rims were prepared from 5- to 18-day-old chick embryos and labeled in vitro with [3H]glucosamine and [35S]sulfate. Then, the labeled medium and corneal proteoglycans were analyzed. Day 5 corneas synthesized mainly chondroitin sulfate/dermatan sulfate proteoglycan and a sudden increase in keratan sulfate proteoglycan synthesis was found on Day 7. Keratan sulfate proteoglycan in Day 7 cornea appeared to be synthesized by fibroblasts which invaded the stroma between Day 5 and Day 7. The proportion of keratan sulfate proteoglycan synthesis increased with advancing embryonic age until Day 14 and declined a little on Day 18. The relative proportion of chondroitin sulfate/dermatan sulfate proteoglycan synthesis changed in a reverse curve during Day 7 to Day 18. Because embryonic cornea begins to become transparent on Day 14, this change in proteoglycan synthesis may be correlated with the onset of transparency. The labeled glycosaminoglycans were isolated from cultured embryonic corneas by pronase digestion, and then digested with chondroitinase ABC or keratanase II. Disaccharides in the enzymatic digests were analyzed by HPLC, and the extents and positions of sulfation of proteoglycans were determined. Sulfation of keratan sulfate proteoglycan continued to increase with advancing age until Day 18. Moreover, it has been shown by HPLC of unsaturated disaccharides of chondroitinase ABC digests that, whereas a comparable amount of non-sulfated "chondroitin sulfate/dermatan sulfate" was synthesized during Day 7 to Day 9, its amount declined during late development (Day 12 to Day 18). Such increases in sulfation of proteoglycans with advancing age may be correlated with progress of corneal transparency, which begins on Day 14 and continues until hatching. Analysis of proteoglycans of the culture medium has shown that most of the medium proteoglycans were keratan sulfate proteoglycan and free keratan sulfate chain (or keratan sulfate chain with a short peptide) during all ages after Day 7, although the major proteoglycan of Day 5 medium was chondroitin sulfate/dermatan sulfate proteoglycan. Sulfation of medium keratan sulfate also increased with advancing age. In addition, the proportion of free keratan sulfate chain in the medium increased during late corneal development, suggesting that catabolism of keratan sulfate proteoglycan might be preferentially accelerated during those times.