Corneal development: IV. Interruption of collagen excretion into the primary stroma of the cornea with l-azetidine-2-carboxylic acid

Abstract

The primary stroma of the cornea of the chick embryo contains a cell-free orthogonal ply of collagen fibrils which is delineated clearly by Gomori's silver stain for reticulin and has, in miniature, the same fibrous architecture as the mature stroma. The collagen of this matrix is synthesized by the basal cells of the corneal epithelium and deposited beneath them a layer at a time. l-Azetidine-2-carboxylic acid (LACA; 0.5 mg in 0.005 ml of water) was injected into the chorioallantoic vein of chick embryos at 5 days of incubation, when the primary stroma is cell free. LACA is an imino acid analog of l-proline, competes with proline for incorporation into collagen and prevents the excretion of the altered collagen. Fifteen minutes after the injection of LACA the Golgi apparatuses located in the basal cytoplasm of the basal epithelial cells (which are normally silver-positive until 10 days of incubation) became silver-negative. A silver-free, collagen-sparse zone appeared beneath the corneal epithelium 1 hr after the injection of LACA. At 16 hr after LACA injection the Golgi apparatuses of the basal epithelial cells once again became silver-positive. Between 18 and 24 hr after the injection, new collagen layers began to appear once more beneath the epithelium. During the following 10 days the collagen-sparse zone became more deeply buried in the stroma and became filled with newly deposited collagen, presumably of fibrocytic origin. The corneas of control animals injected with d-azetidine-2-carboxylic acid, or simultaneously with LACA and l-proline, developed normally. The results suggest that the Golgi apparatuses of the basal cells of the corneal epithelium process the collagen which is deposited in the primary stroma, confirm previous findings that the lamellae of the primary stroma are formed in sequence just beneath the corneal epithelium and open the way for an analysis of the role of the noncollagenous stromal matrix in determining the pattern of polymerization of stromal collagen.