During ovulation, the oocyte and surrounding somatic cumulus cells contained within a specialized, mucoid matrix are released from the ovary. One matrix component, TNF-␣-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 E 2 (PGE 2 ) receptor subtype EP2 null mice that exhibit impaired ovulation and cumulus expansion. To determine if TSG-6 was hormonally

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... 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, PGE 2 , 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 PGE 2 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-␣ 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. (Endocrinology 144: 4376 -4384, 2003) Abbreviations: COC, Cell-oocyte complex; COX-2, cyclooxygenase 2; EP2, PGE 2 receptor subtype; hCG, human chorionic gonadotropin; I␣I, inter-␣ trypsin inhibitor; PMSG, pregnant mare serum gonadotropin; PGE 2 , prostaglandin E 2 ; SDS, sodium dodecyl sulfate; SDS/2-ME, 2% SDS and 6% ␤-mercaptoethanol; TSG-6, TNF-␣-stimulated gene 6.