271INFLUENCE OF ARGININE-GLYCINE-ASPARTIC ACID (RGD) IN BOVINE SPERM-EGG BINDING, AND FERTILIZATION IN VITRO

Osteopontin (OPN), a secretory RGD-containing phosphoprotein, has been identified in cow oviductal ephitelium and fluid, but its role in fertilization is unclear. RGD peptide is capable of blocking fertilization, inducing intracellular Ca2+ transients, and initiating parthenogenetic development when present during bovine fertilization in vitro.This study was conducted to determine whether in vitro sperm binding to the zona pellucida (ZP) and fertilization of bovine oocytes were affected by treating the sperm or oocytes with RGD (arginine–glycine–aspartic acid, a sequence recognized by integrins) or non-RGD-containing peptides. In vitro matured oocytes were incubated (39°C, 5% CO2 in air) for 2 hours in fertilization medium with: (1) no peptides;; (2) 50μgmL−1 RGD (Calbiochem®, San Diego, CA, USA); (3) 1000μgmL−1; (4) 50μgmL−1 non-RGD (Calbiochem®); (5) 1000μgmL−1 non-RGD. The bovine sperm from two differents bulle was collected by artificial vagina, pooled, washed twice with MTM at 500g for 10min and incubated (39°C, 5% CO2 in air) for two h at 5×107 concentration in MTM with: (6) no peptides;; (7) 50μgmL−1 RGD;; (8) 1000μgmL−1; (9) 50μgmL−1 non-RGD; (10) 1000μgmL−1 non-RGD. Following incubation, treated and untreated oocytes were washed and inseminated with 1×105 treated or untreated fresh spermatozoa per 10 oocytes;; after the sperm were recovered from a Percoll gradient (45%/90%). After 18–20h, oocytes were removed from co-culture, and washed in TL-HEPES. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per ZP counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetato-orcein and observed for the presence of pronuclei. For the five replicates, 100–120 oocytes were used for fertilization and 150–170 oocytes were used for sperm-egg binding assays. Data were analized by SAS. Treatment of sperm or oocytes with the RGD peptide significantly decreased (P<0.05) fertilization compared to the non-treated controls or those treated with non-RGD peptides: (1) 80%±3.0; (2) 42%±3.0; (3) 30.2%±3.0; (4) 78.5%±3.0; (5) 79.1%±3.0; (6) 78.9%±3.0; (7) 41.3%±3.0; (8) 29.1%±3.0; (9) 79.2%±3.0; (10) 80.2%±3.0. More sperm bound to the ZP of untreated or non-RGD-treated oocytes or sperm than those incubated with the RGD peptide: (1) 71.2±4.1; (2) 33.2±4.2; (3) 24.2±4.1; (4) 69.5±4.1; (5) 70.2±4.2; (6) 71.9±4.2; (7) 29.8±4.2; (8) 19.8±4.2; (9) 68.9±4.2; (10) 70.6±4.2. These studies demonstrated that incubation of bovine oocytes or spermatozoa with a RGD peptide inhibits sperm-egg binding and fertilization in vitro. These findings support the notion that the role of osteopontin in bovine fertilization may involve inteaction with integrins via its RGD sequence.