Proteoglycans and Proteins in the Extracellular Matrix of Mouse Cumulus Cell–Oocyte Complexes

Abstract

In the preovulatory follicle, the oocyte is surrounded by ∼1000 closely associated cumulus cells forming the compact form of the cumulus cell–oocyte complex (COC). In response to the gonadotropin surge, the COC in a follicle destined for ovulation undergoes expansion when the cumulus cells synthesize and organize an extensive extracellular matrix enriched in hyaluronan. Successful expansion of the COC appears to be essential for ovulation and ultimately for fertilization. We studied this processin vitroby isolating compact COCs from preovulatory mouse follicles and incubating them under conditions which promote COC expansion by retention of newly synthesized hyaluronan (HA) in the extracellular matrix around the cells. [3H]Leucine and [35S]sulfate were used as precursors to label macromolecules synthesized by the cells that may be necessary for organizing the HA in this matrix. After labeling, expanded COCs were washed to remove medium and any labeled molecules that were not associated with the matrix. Macromolecules selectively associated with the matrix were then solubilized by digesting the expanded COCs briefly withStreptomyceshyaluronidase, an enzyme that specifically cleaves HA. Cells were removed by centrifugation, and the digest supernate was analyzed by molecular sieve chromatography and SDS–PAGE. A dermatan sulfate proteoglycan of large hydrodynamic size (>1 million Da) and a ∼46-kDa protein were the predominant labeled species identified. The proteoglycan has properties similar to proteoglycans such as aggrecan and versican which interact specifically with HA. The ∼46-kDa protein has the same molecular size as the link protein which interacts with HA and HA-binding proteoglycans to form stable ternary complexes in a variety of extracellular matrices. We propose that the dermatan sulfate proteoglycan and the ∼46-kDa protein synthesized by the cumulus cells form similar ternary complexes that are necessary for retaining HA in the COC matrix and hence are required for successful COC expansion.