Hyaluronic acid-binding proteins (HABPs) are necessary for expansion of the cumulus-oocyte complex (COC) during oocyte maturation. In this study, to obtain the detailed information of HABPs during cumulus expansion, we examined the expression of HABPs in porcine COCs during in vitro maturation (IVM). After maturation culture, proteins were extracted from porcine COCs and separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After transfer, the membranes were subjected to ligand blotting with biotinylated hyaluronic acid (bHA) or fluorescein isothiocyanate-labeled hyaluronic acid (FITC-HA). Furthermore, the extracted proteins were subjected to immunoprecipitation, Western blotting, and immunofluorescence analysis to dissect the HABPs. Ligand blotting with FITC-HA could detect HABPs. Using this ligand-blotting method, 13 and 14 bands of HABPs were detected in porcine COCs after 0 and 48 h in culture, respectively. Of these, the level of expression of 85-kDa HABP increased with cumulus expansion during IVM and was newly detected after culture. Immunoprecipitation, Western blotting, and immunofluorescent analysis confirmed that the 85-kDa HABP corresponded to CD44 and that it existed on/in the membrane of cumulus cells. The present results indicated that HABP expressed in porcine COCs during IVM, particularly CD44, may form a network of the matrices in the extracellular space of the oocyte with cumulus expansion during IVM.
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