Activation of embryonic development at fertilization is known to be triggered by long-lasting Ca²⁺ oscillations in mammalian eggs. These sperm-induced Ca²⁺ transients start as propagated waves originating in the egg’s cortex. Involvement of the inositol trisphosphate (InsP₃) receptors in this process has been demonstrated, suggesting that fertilization activates phosphoinositide metabolism (Carroll, 2001). Furthermore, a novel sperm-specific phospholipase C isoform – PLCzeta – has recently been demonstrated to mimick sperm-induced egg activation when microinjected into mammalian eggs (Saunders et al. 2002). Fertilization is thus considered to activate primarily phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis to generate InsP₃ and diacylglycerol (DAG). However, the extent and spatiotemporal pattern of PIP₂ hydrolysis are unknown.

Using molecular tools and confocal microscopy, we investigated the activation of the phosphoinositide pathway at fertilization in mouse eggs. To monitor PIP₂ levels, we microinjected the eggs with an mRNA encoding a GFP-tagged pleckstrin homology domain that selectively binds PIP₂ (PH-GFP). As another indicator of phosphoinositide signalling, we expressed a GFP-tagged conventional protein kinase C (PKCλ-GFP; Oancea & Meyer, 1998). Intracellular Ca²⁺ level was monitored simultaneously using fura-red.

PH-GFP exhibited a strong plasma membrane labelling in mouse eggs, with accumulation of the fusion protein in the microvilli of the vegetal pole. The specificity toward PIP₂ labelling was assessed by expressing a mutant version of PH-GFP that does not recognise PIP₂. At fertilization, we did not detect any significant loss of plasma membrane staining that could indicate PIP₂ hydrolysis. Rather, a net increase in PIP₂ labelling was observed. This increase in plasma membrane PIP₂ was transient, activated by the rise in cytoplasmic Ca²⁺ and could be mimicked by photoreleasing caged InsP₃. In contrast, when using ionomycin to activate PIP₂ hydrolysis, a dramatic loss of PIP₂ labelling was observed. Two toxins that inhibit cortical granule release at fertilization, jasplakinolide and Botulinum neurotoxin A, had inhibitory effects on the rise in PIP₂, suggesting that this increase in plasma membrane PIP₂ may play a role in exocytosis of the cortical granules (Halet et al. 2002).

PKCλ-GFP was found to rapidly and reversibly translocate to the plasma membrane in a manner that mirrored Ca²⁺ release at fertilization. To investigate the role of Ca²⁺ and DAG in PKC translocation, we expressed GFP fusion constructs of the isolated C1 or C2 domains of PKCλ which bind DAG or Ca²⁺, respectively. Unexpectedly, no significant translocation of C1-GFP was observed at fertilization, despite efficient translocation after stimulation with a phorbol ester or an analogue of DAG. Also, ionomycin-induced PIP₂ hydrolysis generated DAG that recruited C1-GFP to the plasma membrane. In contrast, the dynamics of C2-GFP at fertilization closely matched the dynamics of the full-length PKCλ-GFP.

These data suggest that the physiological Ca²⁺ signalling pathway at fertilization does not lead to significant PIP₂ hydrolysis or DAG accumulation in the plasma membrane. In addition, PKC translocation seems to be mediated solely via Ca²⁺ binding to the C2 domain.

This work was supported by the MRC.