Parthenogenetic activation of pig eggs by exposure to protein kinase inhibitors

Abstract

The objective of the present study was to assess the effect of low concentrations of protein kinase inhibitors on activation. Pig eggs were electrostimulated or cultured with the following: 10 µM 1-[5-isoquinolinylsulfonyl]-2-methylpiperazine, HCl H7 for 24 h; 100 µM H7 for 24 h; 10 nM staurosporine for 24 h; or with 20 µM staurosporine for 20 min followed by Whitten’s medium for 24 h. Rates of pronuclear formation in eggs (n = 1240) subjected to these treatments were: untreated, 6·2%; electrostimulated, 77·1%; 10 M H7, 10·0%; 100 µM H7, 65%; 10 nM staurosporine, 24·2%; and 20 µM staurosporine, 67·3% (significance at P £ 0· 05: 10 µM H7 vs untreated, not significant; 20 µM staurosporine vs 100 µM H7, not significant). Percentages of eggs (n = 125) expressing a 22-kDa band after treatment were: untreated, 37·5%; electrostimulated, 100%; 10 µM H7, 72%; 100 µM H7, 66·7%; 10 nM staurosporine, 40 ·0%; and 20 µM staurosporine, 77· 3% (significance at P ≤ 0·10: 100 µM H7, 10 nM staurosporine and 20µM staurosporine vs 10 µM H7, not significant; 100 µM H7 and 10 nM staurosporine vs untreated, not significant). Transmission electron microscopy of ultrathin sections of treated eggs revealed that cortical granules were present in over half the untreated eggs, as well as over half of the eggs treated with 100 µM H7 or 10 nM staurosporine; in contrast, all cortical granules were absent from electrically-activated eggs. The results indicate that long-term exposure of eggs to low concentrations of broad-spectrum protein kinase inhibitors induces some of the events commonly associated with fertilization.