Abstract
BackgroundMammalian 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.MethodsAn 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.ResultsThe 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.ConclusionThe 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.
Highlights
Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands on the sperm
Integrin recognition sequences known to play a role in fertilization are the recognize the tripeptide sequence Arg-GlyAsp (RGD) sequence and other tripeptide sequences such as TDE, QDE and FEE included in the active site of fertilin beta, a component of the first molecule identified as a sperm surface protein required for sperm-oocyte fusion [14,15,16,17,18]
Effect of a cyclic RGD containing peptide on sperm-oocyte interaction As in the mouse RGD containing peptides are known to interfere with sperm-oocyte interaction [16,17], we tested the conditions under which a cyclic RGD peptide interacts effectively with the oocyte by monitoring its ability to inhibit fertilization
Summary
Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. At fertilisation the oocyte undergoes a series of rapid changes responsible for the onset of the embryonic development and the blockage of polyspermy These changes, collectively known as "oocyte activation", are under the regulation of cytoplasmic signalling events activated in the oocyte following a multi-step interaction with the fertilising sperm [1,2,3]. Integrin recognition sequences known to play a role in fertilization are the RGD sequence and other tripeptide sequences such as TDE, QDE and FEE included in the active site of fertilin beta, a component of the first molecule identified as a sperm surface protein required for sperm-oocyte fusion [14,15,16,17,18]. It has been suggested the hypothesis that sperm-oocyte binding and fusion involve combined interactions between RGD-sensitive integrins such as αv β1 and RGD-insensitive integrins such as α6 β1 integrins on the oocyte [19]
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