Receptor-mediated endocytosis of vitellogenin in the oocyte of the frog, xenopus laevis

Abstract

The yolk protein, vitellogenin, is internalized by the developing oocyte by the process of receptor-mediated endocytosis. This process is initiated in Xenopus laevis by gonadotropic hormones. This study w~s designed to characterize the endocytot-ic pathway of vitellogenin in· the Xenopus oocyte and the influence-of gonadotropins on this pathway. One of the molecular-mechanisms of this pathway involves the enzyme transglutaminase which catalyzes E(-y-glutamyl) lysine crosslinks. These studies have demonstrated the presence of transglutaminase activity in Xenopus ovary. By the use of specific inhibitors of transglutaminase, dansylcadaverine and methylamine, the activity of this enzyme has been correlated with vitellogenin uptake by the oocytes. An increase in the activities of· the ovarian transglutaminase was also observed subsequent to in vitro exposure of ovarian fragments to hCG. The relationship of calmodulin, the calcium regulator protein, to transglutaminase activity in the ovary and uptake of vitellogenin was investigated because of the calcium requirement for both of these processes. In this study, the uptake of vitellogenin was shown to be sensitive to the calmodulin-directed drug, Stelazine. However, Stelazine failed to inhibit the transglutaminase activity of the ovary, suggesting that the functional role for calmodulin in endocytosis of vitellogenin must be at another site. The common endocytotic pathway of nutritional protein such as vitellogenin involves clathrin-coated pits and vesicles. Studies utilizing SDS-PAGE and immunolabelling techniques have shown that the 180,000 M r protein, clathrin, is present in both hCG treated and untreated ovaries.Extracts of Xenopus ovary containing coated vesicles have additional proteins in common with pig brain coated vesicles, including the two clathrin light chains of 33,000 M and 36,000 M • r · r An RIA for measuring gonadotropin induced alterations in the levels of clathrin in the ovary was developed. However, ·the sensitivity of the assay was very low and proved inadequate to measure small differences in cilathrin levels in the ovary •