Effect of myosin light chain kinase, protein kinase A, and protein kinase C inhibition on porcine oocyte activation

Previous studies have shown that exposure to broad-spectrum protein kinase inhibitors results in parthenogenetic activation of metaphase II arrested porcine oocytes. The objective of this study was to determine the effect of inhibitors of myosin light chain kinase and other protein kinases on pronuclear development, dephosphorylation of a 25-kDa protein, and cortical granule exocytosis. Metaphase II arrested oocytes were obtained by in vitro maturation. Cumulus-free oocytes were cultured with specific inhibitors in modified Whitten's medium for 24 h. Treatment with inhibitors that should inhibit myosin light chain kinase-HA100 (250 mu M), Wortmannin (1 mu M), and a combination of Wortmannin (1 mu M), KT5720 (75 nM), and Iso-H7 (50 mu M)-resulted in significantly higher pronuclear development (74.0%, 18.0%, and 35.0%, respectively) than in the negative control, H7 (10 mu M; 2.0-12.4% depending upon the replication). Treatment with HA100 (250 mu M) resulted in the dephosphorylation of the 25-kDa protein to a 22-kDa protein in 80.0% (n = 10) of oocytes exposed. However, Wortmannin (1 mu M; n = 17), KT5720 (75 nM; n = 16), and Iso-H7 (50 mu M; n = 19) treatment individually and in combination (n = 19) did not result in significant (p < 0.05; n = 19) dephosphorylation over the negative control, H7 (10 mu M; n = 19). HA100 treatment resulted in significant cortical granule exocytosis when evaluated by laser confocal microscopy. In addition, protein kinase assays revealed lower myosin light chain kinase activity in electroactivated oocytes (p < 0.05) and protein kinase inhibitor-treated oocytes (p < 0.05) than in negative controls, nonelectroactivated oocytes, and H7 (10 mu M)-treated oocytes. Treatment with HA100 (250 mu M) resulted in pronuclear formation, dephosphorylation, of the 25-kDa protein, and some release of cortical granules. These observations suggest that inhibition of myosin light chain kinase, protein kinase A, and protein kinase C results in activation of porcine oocytes.