CALCIUM-INDEPENDENT AND MEIOTIC-SPINDLE-INDEPENDENT ACTIVATION OF PIGOOCYTES BY THE INHIBITION OF STAUROSPORINE-SENSITIVE PROTEIN-KINASES

The dependence of pig oocyte activation (both nuclear activation and c ortical granule exocytosis) induced by staurosporine on intracellular Ca2+ rise and spindle assembly was studied. Nuclear activation was eva luated 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 flu orescein isothiocyanate-peanut agglutinin. Exposure of pig oocytes of 0.3 and 3 mu M protein kinase inhibitor staurosporine for 30 min resul ted in the nuclear activation in 71.8% and 85.7% of the oocytes, respe ctively. The pronuclei in activated oocytes contained several compact nucleoli. When the cleaved 2-cell oocytes were further cultured in vit ro, 93.5% developed beyond the 4-cell stage, and 12.9% developed to th e morula stage after 4 days of culture. Of the oocytes treated with 3 mu M staurosporine, 62.5% and 9.4% released their CGs partially and co mpletely; respectively The nuclear activation induced by staurosporine was overcome by the prior treatment of oocytes with okadaic acid, res ulting in only 33.3% of the oocytes undergoing nuclear activation. How ever, when oocytes were exposed first to ,2-bis(O-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (acetoxymethanal ester), a cell permeate ca lcium chelator, or Colcemid, a meiotic spindle disrupter, and then to staurosporine, nuclear activation was observed in 74.2% and 82.3% of t he oocytes, respectively These data were the same as those in oocytes treated only with staurosporine (85.7%). The present study indicates t hat pig oocytes can be activated by the inhibition of staurosporine-se nsitive protein kinase(s), and that this activation is dependent upon mitogen-activated protein kinase but independent of the intracellular Ca2+ rise and spindle integrity.