Evidence that a cyclic RGD-containing peptide can stimulate protrein kinase C in mouse eggs

Introduction: Spermatozoon and egg plasma membrane interactions that lead to egg activation are complex and may involve ligand–receptor interactions and signal transduction, as well as membrane fusion. In mouse egg, fertilization is followed by a series of calcium oscillations and stimulation of protein kinase C (PKC), both of which are involved in egg activation. Integrins, which are heterodimeric cell-surface glycoproteins and serve as receptors for a variety of ligands, participate in spermatozoon–egg interaction and may be involved in mediating a signal transduction cascade, resulting in the hydrolysis of phosphatidyl inositol to release inositol triphosphate and diacylglycerol, the physiological activator of PKC. In this study we have tested the ability of a cyclic peptide containing the Arg-Gly-Asp (RGD) sequence, a recognition signal for several integrins, to interfere with mouse spermatozoon–egg interaction and to stimulate PKC activity. Materials and methods: Ovulated eggs from CD1 mice were deprived of zona pellucida and incubated for 30 min in the presence of increasing amounts of cyclo(Arg-Gly-Asp-D-Phe-Val) or cyclo(Arg-Ala-Asp-D-Phe- Val), a cyclic control peptide. In a first trial, these eggs were inseminated for 30 min in the presence of the peptides and analysed for determination of sperm fusion by using a DNA-staining dye transfer technique. In a second trial, peptide-treated eggs were not inseminated but processed for the biochemical analysis of PKC activity by using MARKS as a specific PKC substrate. Results: Our experiments show that spermatozoon–egg fusion was inhibited in a dose-dependent manner when eggs were treated with the cyclic RGD peptide ranging from 25 to 500 μmol/l. In contrast, the control peptide had no effect on this event at any concentration tested. Moreover, we found that egg treatment with 500 μmol/l cyclic RGD peptide results in a significant increase in PKC activity when compared with untreated eggs. This stimulation was not observed in eggs exposed to the control peptide. Conclusions: We demonstrated that the cyclic RGD peptide we have selected for t his study interferes with spermatozoon–egg interaction byreducing egg ability to fuse with spermatozoa. The specificity of the receptor was confirmed by the fact that a cyclic peptide, not containing the RGD sequence, did not impair spermatozoon–egg fusion. Therefore, we have considered this peptide a useful tool to investigate whether integrins can stimulate transduction mechanisms in mouse eggs. When eggs were challenged with the cyclic RGD peptide, a significant increase in PKC activity was observed. The finding that this effect was not induced by the control peptide indicates that PKC can be stimulated by a signalling system activated by specific receptors for RGD-containing ligands on the oolemma. Based on present results, we suggest that at fertilization PKC may be stimulated in mouse eggs by an integrin-mediated signalling activated by spermatozoa.