Mechanisms involved in the participation of testicular protein TPX-1 in the gamete fusion process

BUSSO, D; GOLDWEIC, NM; VALCARCEL, A; COHEN, DJ; CUASNICÚ, PS

Buenos Aires, Argentina

Congreso; VIII Jornadas Multidisciplinarias de la Sociedad Argentina de Biología (SAB); 2006

Sociedad Argentina de Biología (SAB)

Epididymal protein DE and testicular protein Tpx-1 are two homologous members of the CRISP (Cysteine-Rich Secretory Protein) family. Evidence from our group indicates that both proteins participate in gamete fusion in rodents and human. While DE mediates gamete fusion through its binding to egg complementary sites, the mechanisms underlying the functional role of Tpx-1 remain unknown. To investigate the involvement of egg binding sites for Tpx-1, zona-free human and mouse eggs were incubated with bacterially-expressed Tpx-1 coupled to MBP (Maltose Binding Protein) (recTpx-1), and then subjected to indirect immunofluorescence (IIF) using anti-MBP. While fluorescent labeling was observed over the entire human egg surface, coincident with the fusion ability of the human oolema, mouse eggs exhibited labeling over the egg surface and a negative area as described for DE in mouse eggs. In view of recent results showing that the egg binding ability of DE  resides in a region of 12 amino acids in which Tpx-1 exhibits only two substitutions when compared to DE, we investigated whether these two proteins were interacting with the same egg-complementary sites. Zona-free mouse eggs were incubated in medium containing a fixed concentration of recTpx-1 and increasing amounts of purified DE, and the binding of recTpx-1 to the egg analyzed by IIF. Results showed that exposure to DE resulted in a gradual decrease in the fluorescent staining for Tpx-1, indicating that both proteins share binding sites on the egg. These results support the participation of both DE and Tpx-1 in gamete fusion through their interaction with egg binding sites, suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilization.