Ultrastructural cytochemistry and radioautography of complex carbohydrates in secretory granules of epiphyseal chondrocytes

Abstract

Secretory granules of chondrocytes contain a variety of complex carbohydrates, which include glycosaminoglycans and glycoproteins necessary for the formation of the cartilage matrix. The present study has utilized ultrastructural, cytochemical, and radioautographic methods to determine the subcellular route of incorporation of sulfated and vicinal glycol-containing complex carbohydrates into secretory granules and their subsequent distribution in granules. High iron diamine specifically stained sulfated glycoconjugates in Golgi saccules, Golgi derived vacuoles, and mature secretory granules. Incubation of chondrocytes with 35SO4 resulted in significant radioautographic labeling of Golgi saccules, condensing vacuoles, intermediate granules containing moderately dense amorphous material, and mature granules containing fibrillar material. Periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing glycoconjugates in cisternae of rough endoplasmic reticulum and coated vesicles, some of which appeared to be budding from rough endoplasmic reticulum. Similar coated vesicles appeared to fuse with condensing vacuoles and granules of intermediate maturity which evidenced moderate PA-TCH-SP reactivity. These vesicles were very seldom seen associated with Golgi saccules which contained 35SO4 radiolabels and high iron diamine reactivity but lacked significant PA-TCH-SP staining. The PA-TCH-SP-positive vesicles were not associated with mature secretory granules which demonstrated strong PA-TCH-SP staining. Lysosomes demonstrated moderately heavy PA-TCH-SP staining. Amylase digestion of en bloc specimens removed all PA-TCH-SP reactive cytoplasmic glycogen, however, staining of cytoplasmic organelles was not decreased. These studies indicate that sulfated glycosaminoglycans and vicinal glycol-containing glycoproteins are transported to secretory granules by Golgi saccules and coated vesicles, respectively, and are subsequently distributed differently within the maturing secretory granules.