Two Distinct Populations of Tumor Necrosis Factor-Stimulated Gene-6 Protein in the Extracellular Matrix of Expanded Mouse Cumulus Cell–Oocyte Complexes

Abstract

After the luteinizing hormone surge, the cumulus cell-oocyte complexes (COCs) in the preovulatory follicles produce a viscoelastic extracellular matrix, a process that requires the synthesis of hyaluronan as well as the incorporation of some components of the inter-α-trypsin inhibitor (IαI) family. In this study we report, that a hyaluronan-binding protein, the translated product of tumor necrosis factor-stimulated gene-6 (TSG-6), is also specifically accumulated in this matrix. TSG-6 mRNA expression is quickly upregulated and peaks at ∼1500 copies/cell 4 h after the ovulatory stimuli as assessed by quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry reveals the colocalization of the TSG-6 protein and hyaluronan around the cumulus and granulosa cells. The TSG-6 protein exists in two distinct populations in the COC matrix as demonstrated by Western-blot analysis. One population is a monomer that is anchored to the matrix by a noncovalent interaction. The second population is a covalent complex with either of the heavy chains of IαI and is bound to hyaluronan through a strong interaction that is resistant to denaturing conditions. The specific incorporation of the TSG-6 protein into the COC matrix suggests a structural role for this molecule.