Calcium- and meiotic-spindle-independent activation of pig oocytes by the inhibition of staurosporine-sensitive protein kinases.

Abstract

The dependence of pig oocyte activation (both nuclear activation and cortical granule exocytosis) induced by staurosporine on intracellular Ca2+ rise and spindle assembly was studied. Nuclear activation was evaluated by pronuclear (PN) formation, cleavage and their developmental ability, and cortical granule (CG) exocytosis was assessed by electron microscopy and laser confocal microscopy of oocytes labelled with fluorescein isothiocyanate-peanut agglutinin. Exposure of pig oocytes of 0.3 and 3 microM protein kinase inhibitor staurosporine for 30 min resulted in the nuclear activation in 71.8% and 85.7% of the oocytes, respectively. The pronuclei in activated oocytes contained several compact nucleoli. When the cleaved 2-cell oocytes were further cultured in vitro, 93.5% developed beyond the 4-cell stage, and 12.9% developed to the morula stage after 4 days of culture. Of the oocytes treated with 3 microM staurosporine, 62.5% and 9.4% released their CGs partially and completely, respectively. The nuclear activation induced by staurosporine was overcome by the prior treatment of oocytes with okadaic acid, resulting in only 33.3% of the oocytes undergoing nuclear activation. However, when oocytes were exposed first to 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethanal ester), a cell permeate calcium chelator, or Colcemid, a meiotic spindle disrupter, and then to staurosporine, nuclear activation was observed in 74.2% and 82.3% of the oocytes, respectively. These data were the same as those in oocytes treated only with staurosporine (85.7%). The present study indicates that pig oocytes can be activated by the inhibition of staurosporine-sensitive protein kinase(s), and that this activation is dependent upon mitogen-activated protein kinase but independent of the intracellular Ca2+ rise and spindle integrity.