Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes.
BACKGROUND
Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes.


METHODS
An In-Vitro-Fertilization assay (IVF) was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val), was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val), were evaluated.


RESULTS
The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P < 0.05, Student-Newman-Keuls Method). When the peptide was applied to the oocytes at these concentrations, a dose-dependent increase of PKC activity was observed, in association with a loss of cortical granules ranging from 38+/-2.5 % to 52+/-5.4 %. Evaluation of meiotic status revealed that cyclic RGD peptide was ineffective in inducing meiosis resumption under conditions used in the present study.


CONCLUSIONS
The presents results provide evidence that a cyclic RGD peptide highly effective in inhibiting sperm-oocyte interaction stimulates in mouse oocytes the activation of PKC and the exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for a full oocyte activation response. This study may contribute to the understanding of possible negative effects of skipping gamete interaction in IVF techniques.
Tatone
Carla
C
Department of Biomedical Sciences and Technologies, University of L'Aquila, Via Vetoio, 67100 L'Aquila, Italy. ctatone@univaq.it
Carbone
Maria Cristina
MC
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro; D066298:In Vitro Techniques; D016023:Integrins / Q000187:drug effects / Q000502:physiology*; D008297:Male; D008540:Meiosis / Q000187:drug effects; D008561:Membrane Fusion / Q000187:drug effects; D051379:Mice; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013084:Sperm-Ovum Interactions / Q000187:drug effects*
D016023:Integrins; D010456:Peptides, Cyclic; C092113:cyclo(arginyl-glycyl-aspartyl-phenylalanyl-valyl); D011493:Protein Kinase C