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Regular Article |
a Instituto de Biología y Medicina Experimental, (1428) Buenos Aires, Argentina
b Instituto de Ginecología y Fertilidad, (1122) Buenos Aires, Argentina
c Hokkaido University School of Medicine, Sapporo 060, Japan
ABSTRACT
Human epididymal sperm protein ARP, a member of the cysteine-rich secretory proteins (CRISP) family, exhibits significant homology with rat epididymal protein DE, a candidate molecule for mediating sperm-egg fusion in rodents. The aim of this study was to investigate the involvement of ARP in human gamete fusion. Sequential extraction of proteins from ejaculated human sperm revealed the existence of a population of ARP that is tightly associated with the sperm surface and thus, potentially capable of participating in gamete interaction. Exposure of capacitated human sperm to a polyclonal antibody against recombinant ARP (anti-ARP) produced a significant and concentration-dependent inhibition in the ability of human sperm to penetrate zona-free hamster eggs. This inhibition was not due to a deleterious effect on the gametes because anti-ARP affected neither sperm viability or motility, nor egg penetrability. The antibody did not inhibit the occurrence of spontaneous or Ca2+ ionophore-induced acrosome reaction, nor did it inhibit the ability of sperm to bind to the oolema, supporting a specific inhibition of the antibody at the sperm-egg fusion level. As a relevant evidence for a role of ARP in gamete fusion, the existence of complementary sites for this protein on the surface of human eggs was investigated. Experiments in which zona-free human oocytes discarded from in vitro fertilization programs were exposed to ARP, fixed, and subjected to indirect immunofluorescence revealed the presence of specific ARP-binding sites on the entire surface of the human egg, in agreement with the fusogenic properties of the human oolema. Together, these results strongly support the participation of ARP in the sperm-egg fusion process, suggesting that this protein would be the functional homologue of DE in humans.
First decision: 27 February 2001.
1 Supported by World Health Organization grant H9/181/R429, CONRAD grant MFG-97-29, and National Ministry of Health grant to P.S.C., and PLACIRH Re-entry grant PRE-014/97 to D.A.E. D.J.C., D.A.E., and D.B. are Research Fellowship recipients from the National Research Council of Argentina (CONICET). P.S.C. is a Research Career Award recipient from CONICET.
2 Correspondence: Patricia S. Cuasnicu, Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, (1428) Buenos Aires, Argentina. FAX: 54 11 4786 2564; cuasnicu{at}dna.uba.ar
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