“Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion”.

BUSSO D, COHEN DJ, GOLDWEIC N, MALDERA J, CUASNICÚ PS.

Tampa, Florida, USA.

Congreso; 32th Annual Meeting of American Society of Andrology; 2007

Epididymal protein DE and testicular protein Tpx-1 are two cysteine-rich secretory proteins known as CRISP1 and CRISP2, respectively. Rat CRISP1 (rCRISP1) 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 fragments of rCRISP1 as well as synthetic peptides revealed that the ability of rCRISP1 to bind to the egg surface resides in a region of 12 amino acids corresponding to an evolutionarily conserved motif of the CRISP family named Signature 2. Interestingly, mouse CRISP2 (mCRISP2) exhibits only two substitutions in Signature 2 when compared to rCRISP1, and it is also capable of binding to the rat egg, opening the possibility for a role of mCRISP2 in gamete fusion. In view of this, in the present work we have used the mouse model to examine both the sub-cellular localization of CRISP2 and its involvement in fertilization. Results from indirect immunofluorescence and protein extraction experiments support that mCRISP2 is an intra-acrosomal component that remains associated with sperm after capacitation and acrosome reaction. In vitro fertilization assays using zona pellucida-intact mouse eggs show that a polyclonal antibody against the protein significantly decreases the percentage of penetrated eggs with a concomitant accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility and/or acrosome reaction supporting a specific participation of mCRISP2 at the sperm-egg fusion level. In agreement with this, recombinant mCRISP2 (recCRISP2) specifically binds to the fusogenic area of mouse eggs as previously reported for rCRISP1. The similar localization of CRISP1- and CRISP2-binding sites on the egg, and the high homology of the corresponding Signature 2 regions, suggest that both proteins might interact with the same egg binding sites. In vitro competition studies show that incubation of zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rCRISP1, reduce the binding of recCRISP2 to the egg suggesting that the two proteins interact with common egg complementary sites. These results provide evidence for the involvement of both epididymal CRISP1 and testicular CRISP2 in gamete fusion, supporting the idea of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilization.