Mecanismo de participación de la proteína testicular TPX-1 en el proceso de fusión de gametas

BUSSO D, GOLDWEIC N, VALCARCEL A, COHEN DJ Y CUASNICU PS

Buenos Aires, Argetina

Jornada; VIII Jornadas Multidisciplinarias de la Sociedad Argentina de Biología.; 2006

Sociedad Argentina de Biologia

Epididymal protein DE/CRISP-1 and testicular protein Tpx-1/CRISP-2 are two highly homologous members of the CRISP (Cysteine-Rich Secretory Protein) family. DE participates in gamete fusion through complementary sites on the egg surface. Evidence suggests that Tpx-1 would also participate in fusion in human and rodent. To study the mechanisms involved in the participation of Tpx-1 in fusion, we first analyzed the presence of Tpx-1 binding sites on human oocytes. The eggs were incubated with recombinant Tpx-1 and subjected to indirect immunofluorescence (IIF). Tpx-1 bound over the entire egg surface as described for the human DE homologue. In rodents, eggs incubated with Tpx-1 presented a bright fluorescent labelling similar to that found on DE-incubated eggs. Recent results revealed that the ability of DE to bind to the egg surface resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family named Signature 2 (S2). Interestingly, Tpx-1 exhibits only two substitutions in S2 when compared to DE. In vitro competition studies were carried out to investigate whether these proteins were interacting with the same egg-complementary sites. For this purpose, zona-free eggs were incubated in medium containing a fixed concentration of recTpx-1 and increasing concentrations of DE, and the presence of Tpx-1 analyzed by IIF. Results showed that the increasing concentrations of DE resulted in a gradual decrease in fluorescent staining for Tpx-1, indicating that both proteins share binding sites on the egg surface. Together, these results support the involvement of DE and Tpx-1 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilization.