Kobayashi H, Sun G W, Hirashima Y, Terao T
Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka, Japan.
Endocrinology. 1999 Aug;140(8):3835-42. doi: 10.1210/endo.140.8.6917.
Cumulus oocyte complex (COC) expansion is induced through hyaluronic acid production and accumulation of proteins of the inter-alpha-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-alpha-trypsin inhibitor or dermatan sulfate proteoglycan to hyaluronic acid to make up hyaluronic acid-rich matrix aggregate.
卵丘卵母细胞复合体（COC）的扩张是通过促性腺激素刺激的卵丘细胞中透明质酸的产生以及α-胰蛋白酶抑制剂家族蛋白质的积累来诱导的。连接蛋白是一种存在于软骨中的糖蛋白，它与透明质酸特异性相互作用，并稳定蛋白聚糖单体与透明质酸的结合以形成聚集体。本研究的目的是通过免疫组织化学、蛋白质印迹以及特异性酶联免疫吸附测定法，研究大鼠卵泡发育过程中以及培养的卵丘细胞中免疫反应性连接蛋白的表达。免疫组织化学分析显示，形态上处于COC扩张阶段的卵丘细胞的细胞外基质中连接蛋白染色明显，而未成熟卵泡的颗粒细胞则未染色。卵丘细胞在体外培养系统中将连接蛋白沉积到细胞外基质中。用透明质酸酶处理可使染色强度消失，这表明连接蛋白与透明质酸结合。我们在大鼠卵巢排卵前期以及COC提取物中鉴定出一种42 kDa的免疫反应性连接蛋白。在补充有10%胎牛血清和卵母细胞条件培养基的培养卵丘细胞培养基中添加促卵泡激素（FSH），可导致连接蛋白合成速率增加。这项工作表明，卵丘细胞合成连接蛋白，该蛋白可能稳定α-胰蛋白酶抑制剂或硫酸皮肤素蛋白聚糖与透明质酸的结合，以形成富含透明质酸的基质聚集体。
