Molecular mechanisms involved in gamete interaction: Evidence for the participation of cysteine rich secretory proteins (CRISP) in sperm-egg fusion.

DA ROS V, BUSSO D, COHEN DJ Y CUASNICU, PS.

Madrid, España

Simposio; 10th International Symposium on Spermatology.; 2006

Epididymal protein DE and testicular protein Tpx-1 are two cysteine rich secretory proteins also known as CRISP-1 and CRISP-2, respectively.  DE/CRISP-1 is localized on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites   Recent results using bacterially-expressed recombinant fragments of DE as well as synthetic peptides, revealed that the ability of DE to bind to the egg surface and inhibit gamete fusion resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family (Signature 2). Given the high degree of homology between DE/CRISP-1 and Tpx-1/CRISP-2, we also explored the potential participation of the testicular intra-acrosomal protein in gamete fusion. Results showing the ability of recombinant Tpx-1 to bind to the egg surface (evaluated by indirect immunofluorescence (IIF)), and to significantly inhibit zona-free egg penetration, supported the involvement of this protein in gamete fusion through its interaction with egg-binding sites. Interestingly, Tpx-1 exhibits only two substitutions in Signature 2 when compared to this region in DE. Considering that both DE and Tpx-1 were capable of binding to the egg surface and inhibit gamete fusion, IIF competition studies were carried out to investigate whether these proteins were interacting with the same egg-complementary sites. For this purpose, eggs were first incubated with a fixed concentration of Tpx-1, exposed to increasing concentrations of DE, and finally analyzed for the presence of Tpx-1 by IIF (and viceversa). Results revealing the ability of each protein to displace the other from the egg surface indicated that both proteins would be sharing a common egg-binding site. Together, these results support the involvement of both epididymal DE/CRISP-1 and testicular Tpx-1/CRISP-2 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilization.