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

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 [1‐3]. It is well established that upon fusion sperm releases into the oocyte a sperm-specific phospholipase C-zeta (PLCζ) which induces a rise in intracellular Ca²⁺ capable of releasing metaphase arrest and driving the zygote through the embryonic cell cycle [4]. Although the Ca²⁺-mediated signal transduction pathway at fertilization is not fully resolved, it seems to involve specific kinases such as protein kinase C (PKC) known to be activated in many cell types through enzyme- or G-protein-coupled receptors localised on plasma membrane [5, 6]. However, due to the effectiveness of intracytoplasmic sperm injection in most mammalian species [7], the hypothesis that receptor-mediated pathways may participate in the oocyte activation process has been poorly investigated.

It is well established that binding of sperm ligands to specific oolemma receptors is a prerequisite step in sperm-oocyte interaction leading to fertilisation [8, 9]. Candidate molecules involved in gamete interactions include integrins, transmembrane glycoproteins with heterodimeric structure (alpha-chain and beta-chain) that act as co-receptors in many cell-cell interaction [10]. Individual integrins can bind to more than one ligand and about half of them recognize the tripeptide sequence Arg-Gly-Asp (RGD) present in the extracellular matrix proteins such as fibronectin and vitronectin [11]. Integrins expressed on the surface of mouse oocytes can be divided into two groups: β1 integrins (α2 β1, α3β1, α5 β1, α6 β1 and α9β1) and αv integrins (αv β1, αvβ3, αv β5; [12, 13]). 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 [14‐18]. Recently 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].

In order to clarify the role of integrins at fertilization, it is important to consider that these molecules can serve not only as structural receptors that participate in cell-cell and cell-matrix interaction, but also as signalling receptors that regulate intracellular pH [20], intracellular free Ca²⁺ [21], inositol lipid turnover [22] and protein phosphorylation [23]. Recent research has indicated the ability of peptides containing a RGD sequence to induce intracellular Ca²⁺ transients and to initiate parthenogenetic development in amphibian and bovine oocytes [24‐26], indicating that RGD-binding receptors may function as signalling receptors in oocytes as it occurs in other cell types. Multiple intracellular signalling molecules are stimulated following integrin-dependent adhesion. These include members of mitogen-activated protein kinase (MAPK) signalling pathways, Rho family GTPases, non-receptor tyrosine kinases such as focal adhesion kinase (FAK) and Src, and members of the lipid signalling pathways such as phosphatidyl-inositol 3-kinase (PI 3-K), and protein kinase C [27‐29]. PKC signalling is considered a major regulator of oocyte activation acting both dependently and independently from the fertilization calcium signal [30, 31]. Although its role is not clearly established, it has been proposed that this kinase provides integrated signals aimed to modulate the kinetics and the extent of activation events such as Ca²⁺ spiking and cortical granule exocytosis [32].

Based on the above observations, we put forward the hypothesis that integrins may participate in the activation-associated signalling in mouse oocytes. To this end, in the present study we investigated the ability of a cyclic RGD peptide to activate a pathway leading to the stimulation of protein kinase C and cortical granule exocytosis.

In this study, we provide evidence that exposure of mouse oocytes to a cyclic RGD peptide can inhibit fertilization and induce an activation-like response which includes the activation of PKC signalling and exocytosis of cortical granules. To our knowledge, this is the first report identifying a potential role of integrins and their ligands in the signalling events underlying mouse oocyte activation.

It is well known that in the mouse RGD-containing peptides do not have a substantial inhibitory effect on sperm-oocyte interaction as it occurs in other species, but cause a partial inhibition of fertilization, an observation taken as an evidence that sperm-oocyte fusion would utilize multiple molecules and/or multiple sites on molecules [16, 17]. Although in our IVF assay a complete inhibition of sperm fusion was not achieved, the cyclic RGD peptide we employed reveals a high biological activity being about 50% inhibition observed at 250 μM, a concentration ten-fold lower than that required with linear RGD peptides[17]. Nevertheless, we are not able to establish whether higher concentrations of the peptide would have been more effective since in this study the lyophilized compound was dissolved at the maximal concentration allowed according to manufacturer's instructions. However, given that the RGD peptide inhibited sperm interaction whereas the nonRGD did not, we have taken these results as an indirect evidence of peptide binding to integrin receptors.

In studies of integrin functions in gametes and somatic cells, synthetic peptides containing the RGD (Arg-Gly-Asp) motif have been extensively used as the inhibitors of integrin-ligand interactions. Although the inhibitory activity of disintegrins depends mainly from their primary structure, structural and functional studies suggest that the receptor binding ability of these proteins lies in subtle positional requirements of the tripeptide RGD that is harboured in a defined hairpin loop (the disintegrin loop) projecting from the disintegrin core. This has led to the study of small, chemically synthesised, cyclic-RGD peptides, which exert more potency than linear RGD in integrin binding assay [40]. Thus it is likely that the cyclic RGD peptide used in the present study mimics the physiological action of RGD-containing proteins, supporting the view that, along with proteins with other tripeptide sequence such as fertilin [14], RGD-containing polypeptides located on sperm membrane, such as vitronectin [41, 42], play an important role in gamete interactions leading to fertilization.

The analysis of PKC activity in oocytes exposed to the cyclic RGD peptide at concentrations effective in inhibiting sperm fusion revealed a significant increase in the activity of this enzyme. This finding supports the hypothesis that under this condition an oocyte-integrin signalling cascade is activated to switch on an oocyte phospholipase C leading to increased production of inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG) [43]. DAG activates PKC and IP₃ triggers Ca²⁺ release from intracellular stores. The observation that oocytes exposed to the RGD peptide undergo a significant loss of cortical granules further suggests that under, our experimental conditions, a RGD-sensitive receptor on the oolemma has activated signalling pathways similar to those triggered by sperm at fertilization. Moreover, the occurrence of cortical granule exocytosis in association with an increased PKC activity supports the role for this kinase in the regulation of this event. On the other hand, being the loss of cortical granules a Ca²⁺-dependent event [44], present results might be an indirect evidence that oocytes exposed to the RGD peptide had undergone an increase of intracellular Ca²⁺ as suggested by a previous study [45]. There is still the possibility that PKC activity in response to the RGD peptide represents that observed at fertilization in the absence of a Ca²⁺ signal and probably supported by Ca²⁺-independent PKC isotypes [31]. In this respect, previous results based on the use of PKC agonists and antagonists, suggested that exocytosis can be triggered independently either by Ca²⁺ rise and PKC [46, 47]. Oocyte exposure to a RGD peptide seems to be responsible for a reorganization of actin network similar to that induced by sperm [48]. Moreover, as discussed by Tsaadon et al. [49], PKC may regulate the cytoskeletal dynamic underlying exocytosis enabling the process of vesicle fusion with plasma membrane.

A further observation associated with present results is that RGD-associated signalling leads to PKC activation and cortical granule exocytosis but is not able to stimulate meiosis resumption. This supports the hypothesis that, in contrast to PKC activation achieved by pharmacological agonists, the activation of a PKC signalling through a receptor-mediated mechanism, is not the sufficient trigger for the activation of the anaphase-promoting complex/cyclosome (APC/C) pathway leading to meiosis resumption [32, 50]. In contrast to our results in the mouse, in bovine oocytes a release of meiotic arrest is observed after exposure to RGD peptides [25], it is likely that RGD-sensitive receptors might be capable of activating additional pathways.

Although further investigation will be needed, our results suggest that, at fertilization, a sperm membrane protein containing a RGD sequence may interact with a RGD-sensitive receptor on the oolemma activating a cascade of signalling pathways involved in oocyte activation. Given that in the mouse and bovine sperm injection can induce an abnormal Ca²⁺ response with developmental consequences [51, 52], it can be speculated that a possible role of integrin-mediated pathways may be that to cooperate with those activated in the cytosol by other sperm molecules [4] in order to correctly orchestrate oocyte activation events. Although studies on animal models must be interpreted with caution, this hypothesis raises the need to better investigate the consequences of skipping gamete interaction at surface level in a number of assisted reproductive technologies.