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Study of protein kinase C antagonists on cortical granule exocytosis and cell-cycle resumption in fertilized mouse
1Department of Obstetrics and Gynecology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
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.
Insights
Protein kinase C (PKC) agonists do not fully replicate fertilization-induced cortical granule (CG) release in mouse eggs. PKC inhibitors also failed to block CG release during fertilization, suggesting other mechanisms are involved.
Area of Science:
- Reproductive Biology
- Cellular Signaling
- Developmental Biology
Background:
- Protein kinase C (PKC) activation is known to influence mammalian egg activation events, including cortical granule (CG) exocytosis.
- However, the specific role and necessity of PKC activation in CG exocytosis during natural fertilization remain unclear.
Purpose of the Study:
- To investigate if pharmacological agonists of PKC accurately mimic CG release during fertilization.
- To determine if PKC antagonists inhibit CG release induced by fertilization in mature mouse eggs.
Main Methods:
- Mature mouse eggs were treated with phorbol ester (TPA), a PKC agonist, to observe CG release patterns.
- Fertilization experiments were conducted using two PKC inhibitors, bisindolylmaleimide and chelerytherine, to assess their effect on CG release.
- The activity of inhibitors was confirmed by their ability to block TPA-induced CG loss.
Main Results:
- TPA treatment induced an atypical CG release pattern, concentrated in the egg's equatorial region, unlike the random distribution during fertilization.
- Simultaneous application of maximal concentrations of two PKC inhibitors did not inhibit fertilization-induced CG release.
- A less specific PKC antagonist, staurosporine, also failed to block CG release, though it temporarily inhibited anaphase onset.
Conclusions:
- The PKC agonist TPA does not faithfully reproduce the CG release observed during natural fertilization.
- The involvement of PKC in CG exocytosis during fertilization requires further investigation.
- Other calcium-dependent signaling pathways likely play a significant role in mediating CG exocytosis.
