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  • Title: Study of protein kinase C antagonists on cortical granule exocytosis and cell-cycle resumption in fertilized mouse eggs.
    Author: Ducibella T, LeFevre L.
    Journal: Mol Reprod Dev; 1997 Feb; 46(2):216-26. PubMed ID: 9021753.
    Abstract:
    Although pharmacological agonists of protein kinase C (PKC) stimulate some events of mammalian egg activation, including cortical granule (CG) exocytosis, it is not known if these events are dependent on PKC activation during the normal process of fertilization. In order to examine the potential role of PKC in CG exocytosis, this study investigated whether PKC agonists faithfully mimic CG release and whether PKC antagonists block fertilization-induced CG release in mature mouse eggs. Phorbol ester (TPA, 2.5 ng/ml) treatment resulted in an atypical pattern of CG release in which there was a greater net loss of CGs in the equatorial region of the egg than in the region opposite the spindle. This pattern also was in contrast to that during fertilization, in which CG release occurred randomly throughout the cortex. Fertilization experiments utilized two different PKC inhibitors, bisindolylmaleimide (5 microM) and chelerytherine (0.8 microM), targeted to both the "conserved" substrate and ATP binding domains of PKC. Simultaneous use of both inhibitors at maximal concentrations (compatible with fertilization and above their IC50S) resulted in no detectable inhibition of CG release in treated fertilized eggs compared to controls. In addition no inhibition of anaphase onset was observed in treated fertilized eggs. Activity of the inhibitors was verified by demonstrating that they blocked the induction of CG loss by TPA. Moreover, 1 microM staurosporine, a potent but less specific antagonist of PKC, also did not block CG loss whereas the metaphase-anaphase transition was temporarily inhibited. The results indicate that TPA does not faithfully mimic CG release in fertilized eggs, that a role for PKC in CG release at fertilization remains to be established, and that other calcium-dependent effectors may be involved in CG exocytosis.
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