Ochsner Scott A, Day Anthony J, Rugg Marilyn S, Breyer Richard M, Gomer Richard H, Richards Joanne S
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Endocrinology. 2003 Oct;144(10):4376-84. doi: 10.1210/en.2003-0487. Epub 2003 Jul 17.
During ovulation, the oocyte and surrounding somatic cumulus cells contained within a specialized, mucoid matrix are released from the ovary. One matrix component, TNF-alpha-stimulated gene 6 (TSG-6), is a hyaluronan binding protein induced in cumulus cells of preovulatory follicles by the LH surge and is decreased in cumulus cells of COX-2 and prostaglandin E2 (PGE2) receptor subtype EP2 null mice that exhibit impaired ovulation and cumulus expansion. To determine if TSG-6 was hormonally induced in cumulus cells in vitro and was functional during the formation of the expanded matrix, we established a cumulus cell-oocyte complex (COC) culture system. This system was used to analyze the effects of FSH, PGE2, EP2 receptor, and selected protein kinase inhibitors on TSG-6 production as well as specific antibodies to the TSG-6 link module on TSG-6 function. We document that TSG-6 message and protein are induced by cAMP/protein kinase A/MAPK signaling pathways and that blocking these cascades prevents expansion and the production of TSG-6. FSH but not PGE2 rescued expansion and production of TSG-6 in the EP2 null COCs, indicating that generation of a cAMP signal is essential. Furthermore, disruption of the functional interactions between TSG-6, inter-alpha trypsin inhibitor, and hyaluronan with specific antibodies severely altered matrix formation and cumulus expansion, as recorded by time-lapse imaging. Collectively, these results indicate that TSG-6 mRNA is induced in cumulus cells in culture by cAMP and that the secreted TSG-6 protein is a key structural component of the mouse COC matrix.
在排卵过程中，包含在一种特殊的黏液样基质中的卵母细胞和周围的体细胞卵丘细胞从卵巢中释放出来。一种基质成分，即肿瘤坏死因子-α刺激基因6（TSG-6），是一种透明质酸结合蛋白，在排卵前卵泡的卵丘细胞中由促黄体生成素高峰诱导产生，而在环氧化酶-2（COX-2）和前列腺素E2（PGE2）受体亚型EP2基因敲除小鼠的卵丘细胞中减少，这些小鼠表现出排卵和卵丘扩展受损。为了确定TSG-6是否在体外被激素诱导产生于卵丘细胞中，以及在扩展基质形成过程中是否具有功能，我们建立了卵丘细胞-卵母细胞复合体（COC）培养系统。该系统用于分析促卵泡激素（FSH）、PGE2、EP2受体和选定的蛋白激酶抑制剂对TSG-6产生的影响，以及针对TSG-6连接模块的特异性抗体对TSG-6功能的影响。我们证明TSG-6的信使核糖核酸（mRNA）和蛋白质由环磷酸腺苷（cAMP）/蛋白激酶A/丝裂原活化蛋白激酶（MAPK）信号通路诱导产生，并且阻断这些级联反应会阻止扩展和TSG-6的产生。FSH而非PGE2挽救了EP2基因敲除COC中的扩展和TSG-6的产生，表明cAMP信号的产生至关重要。此外，用特异性抗体破坏TSG-6、α-间胰蛋白酶抑制剂和透明质酸之间的功能相互作用，会严重改变基质形成和卵丘扩展，延时成像记录了这一现象。总体而言，这些结果表明，cAMP在培养的卵丘细胞中诱导TSG-6 mRNA产生，并且分泌的TSG-6蛋白是小鼠COC基质的关键结构成分。
