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 alpha -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.

Proteome changes of porcine follicular fluid during follicle development

“…Numerous proteins that contribute to blood coagulation and fibrin degradation through plasminogen (PLG) or proteolysis by thrombin activation [61] were detected. For example, alpha-2-macroglobulin (A2M), a protease inhibitor that binds small or large proteinases such as the plasmin that regulates proteolytic activity [63], influences cumulus cells expansion by inhibiting zinc-dependent metalloproteases [64]. In this way, the excess of A2M lead a reduction in porcine cumulus expansion [64, 65].…”

“…For example, alpha-2-macroglobulin (A2M), a protease inhibitor that binds small or large proteinases such as the plasmin that regulates proteolytic activity [63], influences cumulus cells expansion by inhibiting zinc-dependent metalloproteases [64]. In this way, the excess of A2M lead a reduction in porcine cumulus expansion [64, 65]. Clusterin (CLU), another protease inhibitor, is an important regulator of the complement [66], preventing apoptosis of follicular cells [67].…”

Comparative Analysis of Porcine Follicular Fluid Proteomes of Small and Large Ovarian Follicles

“…In the present study, only the LNA protein dataset revealed significant enrichment, and the proteins having the highest number of interactions play important roles on cytoplasmic maturation. Amongst these proteins, plasminogen [47], AMBP [57], and alpha-2-macroglobulin [58] positively influence cumulus expansion, while alpha-2-HS-glycoprotein regulates the binding of spermatozoa on zona pellucida [59], which could influence the incidence of polyspermy in pigs [14]. In addition, the histidine-rich glycoprotein (HRG) protein may act on blood coagulation [60] inhibiting the action of vascular endothelial growth factor [61].…”