Evidence for the participation of human epididymal protein CRISP1 in sperm-zona pellucida interaction

MALDERA JA; BUSSO D; CHIRINOS M; RAFFO G; BLAQUIER JA; LARREA F; CUASNICU PS

Philadelphia, USA

Congreso; 34th Annual Meeting of American Society of Andrology; 2009

American Society of Andrology

Human epididymal CRISP1 (Cysteine RIch Secretory Protein 1), as its rodent counterpart, associates with sperm during maturation and participates in gamete fusion through complementary sites in the oolema. Based on recent observations showing that rodent CRISP1 is also involved in the previous stage of sperm-zona pellucida (ZP) interaction, in the present work we investigated the participation of human CRISP1 (hCRISP1) in this specific step of the fertilization process. For this purpose, human hemizona (HZ) were inseminated with human capacitated sperm in the presence of anti-hCRISP1 polyclonal antibody. As controls, the corresponding HZ were co-incubated with sperm in the presence of normal rabbit IgG or in medium alone (C). Results showed that anti-hCRISP1 produced a significant inhibition in the number of bound sperm per HZ compared to controls (anti-hCRISP1 vs. IgG p<0.005; anti-hCRISP1 vs. C p<0.01). Insemination of HZ in the presence of bacterially-expressed hCRISP1 (coupled to maltose binding protein, MBP), also produced a significant reduction in the number of sperm bound per HZ compared to HZ incubated in medium alone (p<0.005) or medium containing MBP (p<0.005). Indirect immunofluorescence experiments using human ZP-intact eggs confirmed the ability of hCRISP1 to bind to the ZP in addition to the reported binding of the protein to the oolema. Finally, with the aim of identifying the ZP ligand of hCRISP1, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 or MBP in ELISA experiments. Results revealed that hCRISP1 specifically interacted with ZP3 in a dose-dependent manner. Together, these results indicate that human CRISP1, as its rodent homologue, participates not only in sperm-egg fusion but also in the previous stage of sperm-ZP interaction.