Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and their potential use for male fertility regulation.

COHEN, DJ, DA ROS, VG, BUSSO, D, ELLERMAN, D, MALDERA, J, GOLDWEIC, N, CUASNICU, PS

Asian Journal of Andrology

Blackwell Publishing

Año: 2007 vol. 9 p. 528 - 532

1008-682X

Protein DE is a cysteine-rich secretory protein synthetized by the proximal regions of the epididymis in response to androgens. Since it was the first identified member of the CRISP family it is also known as CRISP-1. Results from our laboratory indicated that DE is localized on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites. The finding that immunization of rats with DE produces a significant inhibition of fertility and a specific interference with the sperm fusion ability, suggests that DE represents a good epididymal contraceptive target. 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 resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family named Signature 2 (S2). To further study the involvement of this region in gamete fusion, the ability of other CRISP proteins to bind to the rat egg was analyzed in correlation with the amino acid sequence of their S2 regions. Results revealed that while testicular protein Tpx-1 (also known as CRISP-2) was capable of binding to the rat egg, human epididymal ARP and helothermine (from lizard saliva), were unable to recognize the rodent egg. Interestingly, the S2 region presented only two substitutions in Tpx-1 and four in both human ARP and helothermine when compared to S2 in DE, suggesting that the amino acid sequence of this region would be relevant for the ability of these proteins to interact with the egg. The binding of Tpx-1 to the egg surface opened the possibility for a role of this protein in gamete fusion. Subsequent experiments using both Tpx-1 and anti-Tpx-1 in in vitro fertilization studies, confirmed the participation of Tpx-1 in this step of fertilization through its binding to complementary sites in the egg. Competition studies showed that incubation of zona-free eggs with a fixed concentration of Tpx-1 and increasing amounts of DE, gradually reduced the binding of Tpx-1 to the egg, indicating that both CRISPs share the egg complementary sites. The observation that anti-DE and anti-Tpx-1 significantly inhibit sperm-egg fusion while being able to only recognize the corresponding proteins, support the idea of a functional cooperation between these two homologue CRISP molecules as a mechanism to ensure the success of fertilization. We believe these results will contribute to a better understanding of the molecular mechanisms involved in gamete fusion as well as to the development of new and safer fertility regulating methods.