Ball G D, Bellin M E, Ax R L, First N L
Mol Cell Endocrinol. 1982 Sep;28(1):113-122. doi: 10.1016/0303-7207(82)90045-4.
Bovine cumulus-oocyte complexes from small (1-5 mm) follicles were cultured for 24 h in 0.25 ml minimum essential medium supplemented with 10% fetal bovine serum and 20 microCi [3H]glucosamine. Treatment groups consisted of supplementing the culture medium with no hormone (control), 0.5 IU/ml follicle-stimulating hormone (FSH) or 10 mM 8-Br-adenosine cyclic monophosphate (cAMP). After culture, the complexes were fixed for light and scanning electron microscopy. Electron photomicrographs revealed that complexes induced to expand with FSH or cAMP contained a copious glycosaminoglycan (GAG) matrix extending between and around the cumulus cells. Control complexes did not exhibit expansion or an extracellular matrix. The radiolabeled GAG material was isolated for chemical identification. Chemical analyses included: (1) electrophoresis of GAG material, (2) electrophoresis of GAG material after enzyme or nitrous acid treatment, (3) thin-layer chromatography of GAG hydrolysates. The results from electrophoresis showed that the radiolabeled GAG co- migrated with hyaluronic acid. The GAG material was resistant to chondroitinase ABC and nitrous acid degradation but was digested by hyaluronidase. Complexes treated with FSH and cAMP incorporated higher (P less than 0.1 and P less than 0.025 respectively) amounts of [3H]glucosamine into hyaluronic acid than control cultures. Thin-layer chromatography identified the primary amino sugar of the GAG to be glucosamine. These data collectively showed that the radioactive GAG produced by bovine cumulus-oocyte complexes was hyaluronic acid.
从小卵泡（1 - 5毫米）采集的牛卵丘-卵母细胞复合体在含有10%胎牛血清和20微居里[3H]葡糖胺的0.25毫升最低限度基本培养基中培养24小时。处理组包括在培养基中不添加激素（对照组）、添加0.5国际单位/毫升促卵泡激素（FSH）或10毫摩尔8-溴腺苷环磷酸（cAMP）。培养后，复合体进行固定以用于光学显微镜和扫描电子显微镜观察。电子显微照片显示，用FSH或cAMP诱导扩张的复合体含有丰富的糖胺聚糖（GAG）基质，该基质在卵丘细胞之间和周围延伸。对照复合体未表现出扩张或细胞外基质。分离出放射性标记的GAG物质进行化学鉴定。化学分析包括：（1）GAG物质的电泳，（2）酶或亚硝酸处理后GAG物质的电泳，（3）GAG水解产物的薄层色谱分析。电泳结果表明，放射性标记的GAG与透明质酸共迁移。GAG物质对软骨素酶ABC和亚硝酸降解具有抗性，但可被透明质酸酶消化。用FSH和cAMP处理的复合体比对照培养物将更多（分别为P < 0.1和P < 0.025）的[3H]葡糖胺掺入透明质酸中。薄层色谱分析确定GAG的主要氨基糖为葡糖胺。这些数据共同表明，牛卵丘-卵母细胞复合体产生的放射性GAG是透明质酸。
