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Identification of Link Protein during Follicle Development and Cumulus Cell Cultures in Rats

Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-3192, Japan

Address all correspondence and requests for reprints to: Dr. Hiroshi Kobayashi, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-3192, Japan.

Cumulus oocyte complex (COC) expansion is induced through hyaluronic acid production and accumulation of proteins of the inter--trypsin inhibitor family in the gonadotropin-stimulated cumulus cells. Link protein, a glycoprotein 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 immunoreactive link protein during follicle development in rats and in cumulus cells in culture by immunohistochemistry and Western blot as well as by specific enzyme-linked immunosorbent assay. Immunohistochemical analysis revealed that the extracellular matrix of cumulus cells that were morphologically at a stage of COC expansion were markedly stained for link protein, whereas granulosa cells from immature follicles were not stained. Cumulus cells deposited link protein into the extracellular matrix in an in vitro culture system. The staining intensity was negated by the treatment with hyaluronidase, suggesting that the link protein is bound to hyaluronic acid. We have identified a 42-kDa immunoreactive link protein in rat ovary during the preovulatory period and in COC extracts. Addition of FSH to the medium of cumulus cells in culture supplemented with 10% FBS and oocyte-conditioned medium resulted in an increased rate of link protein synthesis. This work suggests that the cumulus cells synthesize the link protein that may stabilize the binding of inter--trypsin inhibitor or dermatan sulfate proteoglycan to hyaluronic acid to make up hyaluronic acid-rich matrix aggregate.

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