COVALENT LINKAGE BETWEEN PROTEINS OF THE INTER-ALPHA-INHIBITOR FAMILYAND HYALURONIC-ACID IS MEDIATED BY A FACTOR PRODUCED BY GRANULOSA-CELLS

The direct interaction of hyaluronic acid (HA) and proteins of the int er-alpha-inhibitor family plays a critical role in organization and st abilization of the expanding cumulus extracellular matrix (cECM) follo wing an ovulatory stimulus, Despite similarities in the morphology of cumulus oocyte complexes (COCs) expanding in vivo and in vitro, we fin d that the cECM of COCs which expand within intact follicles are more elastic and resistant to shear stress than the cECM of those stabilize d in vitro, Western blot analysis shows that only the heavy chains of inter-alpha-inhibitor are incorporated into the cECM and appears to be covalently linked to HA after stabilization in vivo while intact inte r-alpha-inhibitor is bound to the HA-enriched cECM by a non-covalent m echanism in in vitro stabilized COCs, However, purified pre-alpha-inhi bitor and HA can form covalent linkage in the presence of granulosa ce lls or with granulosa cell-conditioned medium, In addition, COCs resis tance to shear stress is also enhanced by coincubation with granulosa cells, Upon formation of the apparent covalent linkage between heavy c hains and HA in culture medium, the light chain (bikunin) is concomita ntly released into the medium as a complex with chondroitin sulfate mo ieties of inter-alpha-inhibitor supporting the possibility that HA may replace the chondroitin sulfate linkage to the heavy chains, We specu late that a factor(s) secreted by granulosa cells within the follicle may catalyze a transesterification reaction resulting in an exchange o f chondroitin sulfate with HA at the heavy chain/chondroitin sulfate j unction followed by release of chondroitin sulfate-bikunin into the fo llicular fluid. It is also possible that the consequent further stabil ization of the cECM through the covalent interaction of HA and heavy c hains of inter-alpha-inhibitor may play an important role in the proce ss of ovulation.