Identification of link protein during follicle development and cumulus cell cultures in rats

Cumulus oocyte complex (COC) expansion is induced through hyaluronic acid p roduction and accumulation of proteins of the inter-alpha-trypsin inhibitor family in the gonadotropin-stimulated cumulus cells. Link protein, a glyco protein found in cartilage, interacts specifically with hyaluronic acid and stabilizes the binding of proteoglycan monomers to hyaluronic acid to form aggregates. The aim of this study was to investigate the expression of imm unoreactive link protein during follicle development in rats and in cumulus cells in culture by immunohistochemistry and Western blot as well as by sp ecific enzyme-linked immunosorbent assay. Immunohistochemical analysis reve aled that the extracellular matrix of cumulus cells that were morphological ly at a stage of COC expansion were markedly stained for link protein, wher eas granulosa cells from immature follicles were not stained. Cumulus cells deposited link protein into the extracellular matrix in an in vitro cultur e system. The staining intensity was negated by the treatment with hyaluron idase, suggesting that the link protein is bound to hyaluronic acid. We hav e identified a 42-kDa immunoreactive link protein in rat ovary during the p reovulatory period and in COC extracts. Addition of FSH to the medium of cu mulus cells in culture supplemented with 10% FBS and oocyte-conditioned med ium resulted in an increased rate of link protein synthesis. This work sugg ests that the cumulus cells synthesize the link protein that may stabilize the binding of inter-alpha-trypsin inhibitor or dermatan sulfate proteoglyc an to hyaluronic acid to make up hyaluronic acid-rich matrix aggregate.