Participation of CRISP proteins in the fertilization process

COHEN DJ; MALDERA JA; VASEN G; ERNESTO JI; WEIGEL MUÑOZ M; BATTISTONE MA; CUASNICÚ PS

San Pablo

Congreso; Fifth International Conference on Epididymis; 2010

Maria Christina W. Avellar y Patricia S. Cuasnicu

Mammalian fertilization is a complex multi-step process mediated by different molecules present on both gametes. Rat protein CRISP1, originally identified in our laboratory, is a member of the evolutionary conserved Cysteine-RIch Secretory Protein (CRISP) family. CRISP1 is expressed in by the proximal regions of the epididymis in response to androgens and associates with the sperm surface during epididymal transit. Evidence indicates the existence of two populations of CRISP1 on sperm: a major one, loosely bound, released during capacitation and proposed to act as a decapacitating factor, and a minor one, strongly associated, that remains on sperm after capacitation and is postulated to participate in fertilization. Recent results support the participation of a molecular complex between CRISP1 and zinc in the in vivo association of the loosely-bound population of CRISP1 with sperm, and the involvement of membranous vesicles (epididymosomes) in the mechanism by which CRISP1 strongly associates with sperm. CRISP1 localizes on the dorsal region of capacitated sperm and on the equatorial segment of acrosome-reacted cells. Consistent with these localizations, in vitro experiments using anti- CRISP1 antibodies as well as purified or recombinant protein during gamete co-incubation, support the involved of CRISP1 in both sperm-ZP binding and gamete fusion through its interaction with egg-complementary sites. The potential roles of the protein in capacitation and fertilization were further supported by the finding that capacitated sperm from CRISP1 “knock out” animals exhibited low levels of cAMP and protein tyrosine phosphorylation and presented an impaired ability to fertilize in vitro zona-intact and zona-free eggs. In spite of this, CRISP1- deficient mice were fertile, suggesting that testicular CRISP2, also involved in gamete fusion, and/or other CRISP homologues present on sperm, would compensate for the lack of CRISP1 in the mutant mice. Altogether, the presented results support both the participation of CRISP proteins in different stages of fertilization and the existence of a functional cooperation between homologue CRISPs as a mechanism to ensure the success of this process. We believe these results will contribute to a better understanding of the involvement of epididymal proteins in gamete interaction as well as to the development of new and safer fertility regulating methods. In agreement with our observations in rodents, results indicate the participation of human epididymal CRISP1 (hCRISP1) in both human sperm-ZP binding and gamete fusion through its interaction with binding sites in the human egg. In view of these results and the finding that immunization of rats with CRISP1 raises specific antibodies which inhibit animal fertility, immunization studies were carried out in male and female non-human primates. Results showed that hCRISP1 is immunogenic and that the produced antibodies enter the male reproductive tract and recognize the sperm protein without affecting sperm production or maturation. Altogether, the results obtained increase our understanding on the molecular mechanisms of gamete interaction and support the potential use of CRISP family members for the development of new and safer fertility regulating methods.