226 TYROSINE PHOSPHORYLATION PATTERN IN PORCINE EJACULATED AND EPIDIDYMAL CAPACITATED BOAR SPERM

After ejaculation, mammalian sperm must undergo a preparation period know as capacitation to be competent to fertilize. Capacitation normally occurs in the female genital tract; however, it can also be achieved in vitro by removing seminal plasma and incubating sperm in capacitation media. In previous studies, we have demonstrated that ejaculated spermatozoa washed through a Percoll® gradient can penetrate the oocyte faster than epididymal sperm. It has been shown that capacitation is associated with tyrosine phosphorylation of sperm proteins, and many studies utilize protein phosphorylation as an indicator of sperm capacitation (Tardif S et al. 2001 Biol. Reprod 65, 784–792). The main objective of this experiment was to determine the protein tyrosine phosphorylation patterns of ejaculated and epididymal boar spermatozoa washed through a Percoll® gradient and incubated in a capacitating medium (TALP) without albumin. Ejaculated and epididymal spermatozoa were each divided into 3 groups: (1) spermatozoa without any treatment (control = C), (2) spermatozoa washed through Percoll® gradient (P), and (3) spermatozoa washed through Percoll® and incubated 3 h in TALP medium (P-3hr). The sperm plasma membranes were isolated (as described in Bravo M et al. 2005 Mol. Reprod. Dev. 71, 88–96), and sperm soluble proteins were separated by SDS-PAGE 12% acrylamide. Afterward, the proteins were transferred onto a nitrocellulose membrane and blocked with TBST-albumin 5% and were then incubated with an anti-phosphotyrosine antibody (4G10 Upstate Biotechnology, Lake Placid, NY, USA; 1:20000), then with an anti-mouse IgG (1:20000; Biorad, Hercules, CA, USA) and revealed with ECL + Plus (Amersham, Sao Paulo, Brazil). Five replicates were conducted for this experience. The results showed a band of tyrosine phosphoprotein of 19 kDa in all experimental groups except the ejaculated control group. A band with a range of 40–43 kDa appears strongly phosphorylated in the ejaculated spermatozoa, while in the epididymal spermatozoa was poorly labeled. In conclusion, the ejaculated and epididymal spermatozoa have different pattern of tyrosine phosphorylation with the same treatment. It is probably that the seminal plasma proteins could be regulated by the phosphorylation of the 19 kDa of protein. However, further studies are necessary to elicit what kind of proteins in seminal plasma change this stage of phosphorylation and their role in an IVF system. Supported by MEC (AGL2006-03495).