Disrupted Function of Tumor Necrosis Factor-α-Stimulated Gene 6 Blocks Cumulus Cell-Oocyte Complex Expansion
Scott A. Ochsner, Anthony J. Day, Marilyn S. Rugg, Richard M. Breyer, Richard H. Gomer, Joanne S. Richards
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
... 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.