Abstract
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 microM), Wortmannin (1 microM), and a combination of Wortmannin (1 microM), KT5720 (75 nM), and Iso-H7 (50 microM)--resulted in significantly higher pronuclear development (74.0%, 18.0%, and 35.0%, respectively) than in the negative control, H7 (10 microM; 2.0-12.4% depending upon the replication). Treatment with HA100 (250 microM) 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 microM; n = 17), KT5720 (75 nM; n = 16), and Iso-H7 (50 microM; 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 microM; 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 microM)-treated oocytes. Treatment with HA100 (250 microM) 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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
