title: Hampered cumulus expansion of porcine cumulus-oocyte complexes by excessive presence of alpha2-macroglobulin is likely mediated via inhibition of zinc-dependent metalloproteases.
abstract: In vitro maturation (IVM) in serum causes hampered expansion of porcine cumulus-oocyte complexes (COCs) due to excessive alpha2-macroglobulin (A2M). This study investigated two hypotheses that could explain the effect of A2M: (i) binding of epidermal growth factor (EGF) to A2M, followed by its decreased availability; and (ii) inhibition of zinc-dependent metalloproteases. Cumulus expansion was evaluated based on the diameter of the COCs, the proportion of COCs participating in a floating cloud and the proportion of COCs with loss of cumulus cells. The first hypothesis of decreased EGF availability was tested by increasing the EGF concentration (20 and 50 ng/mL vs. 10 ng/mL), but was not confirmed because cumulus expansion did not improve. To verify the second hypothesis of inhibited zinc-dependent metalloproteases, the effect of tissue inhibitor of metalloproteases-3 (TIMP-3) on cumulus expansion during IVM with and without A2M was investigated. To immuno-neutralize A2M, serum was pre-incubated with A2M antibodies. Impaired cumulus expansion because of TIMP-3 could only be observed during IVM in 10% of serum with A2M antibodies. No effect of TIMP-3 was observed in medium without A2M antibodies. These results indicate that A2M and TIMP-3 share a common target, a zinc-dependent metalloprotease. Future research is directed toward the identification of the protease involved.
authors.affiliation: Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Ibaraki, Japan.
authors.forename: Ruth
authors.initials: R
authors.lastname: Appeltant
authors.affiliation: MSD Animal Health Belgium, Brussels, Belgium.
authors.forename: Josine
authors.initials: J
authors.lastname: Beek
authors.affiliation: Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Dominiek
authors.initials: D
authors.lastname: Maes
authors.affiliation: Social Science Unit, Institute for Agricultural and Fisheries Research, Merelbeke, Belgium.
authors.forename: Jo
authors.initials: J
authors.lastname: Bijttebier
authors.affiliation: Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
authors.forename: Katleen
authors.initials: K
authors.lastname: Van Steendam
authors.affiliation: Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Hans
authors.initials: H
authors.lastname: Nauwynck
authors.affiliation: Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Soom
mesh_terms: D000818:Animals; D005260:Female; D059471:In Vitro Oocyte Maturation Techniques; D045726:Metalloproteases / Q000037:antagonists & inhibitors*; D009865:Oocytes / Q000502:physiology*; D013552:Swine; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000502:physiology; D015032:Zinc; D000511:alpha-Macroglobulins / Q000502:physiology*
chemical_list: D019717:Tissue Inhibitor of Metalloproteinase-3; D000511:alpha-Macroglobulins; D045726:Metalloproteases; D015032:Zinc
keywords: alpha2-macroglobulin; cumulus expansion; in vitro maturation; porcine; serum