The integrin-binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (Amphibia) oocytes

Abstract

Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits a5, aV and b1 in oocytes. In sperm, we could detect only the aV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-b1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosinecontaining proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. b1 subunit) as adhesion proteins.

Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs

“…Subsequently, tyrosine phosphorylation pattern was analyzed in cytosolic protein samples obtained from oocytes and embryos at 1, 5 and 10 mpf. Genistein was found to abolish some of the typical phosphorylation changes that were previously associated with fertilization in Rhinella arenarum (Mouguelar et al, 2011), like dephosphorylation of proteins of 33 and 42 kDa, and phosphorylation of 70 kDa proteins (Fig. 1A).We also evaluated the effect of genistein on the physiological dephosphorylation due to fertilization of the p42-MAPK protein using a specific anti p-ERK 1/2 antibody (see above).…”

“…These assays were performed essentially as described in Mouguelar et al, (2011). Briefly, groups of 6-8 jelly-intact oocytes were placed in wells filled with 100 ml of R406 (100 mM, 200 mM or vehicle control) solution in 50% SB.…”

“…Egg water was supplemented to eliminate any dilution of the factors that are necessary for successful fertilization (Arranz and Cabada, 2000). Fertilization was carried out adding 5 ml of sperm stock solution to reach the desired final sperm concentration (3.3 10 5 cells/ml, see Mouguelar et al, (2011) for details on the sperm concentration). After 25 min, the eggs were washed three times with 100 ml of 50% SB each.…”

“…In fact, it has been described that tyrosine phosphorylation increases in the first minutes following fertilization of Xenopus oocytes (Sato et al, 1998), Rhinella arenarum oocytes (Mouguelar et al, 2011), rat eggs (Ben-Yosef et al, 1998) and ascidian eggs (Ueki and Yokosawa, 1997), and as early as 1 min after fertilization in sea urchin eggs (Peaucellier et al, 1988). In all these species, this increase can be detected during the latent period, i.e., before the fertilization calcium signal (Ciapa and Epel, 1991).…”

“…Nevertheless, the identity of most of the key members involved in these signaling pathways has not yet been elucidated. Recently, we have described the phosphorylation at tyrosine residues of at least two proteins of 60 kDa and 70 kDa in R. arenarum egg cytosolic fractions ten minutes post-fertilization (mpf) (Mouguelar et al, 2011).…”