RESUMO
Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.
Assuntos
Metaloproteínas/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Hormônios Testiculares/fisiologia , Animais , Sítios de Ligação , Proteínas Secretadas pelo Epidídimo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Metaloproteínas/análise , Metaloproteínas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Oócitos/química , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Capacitação Espermática , Espermatozoides/química , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Hormônios Testiculares/análise , Hormônios Testiculares/farmacologiaRESUMO
A basic 54-kDa protein (pI approximately 8.8) that cross-reacts with anti-caltrin antisera has been detected and isolated by gel filtration and cation exchange chromatography from seminal vesicle content of the rat. The soluble protein spontaneously precipitated in NaHCO3-buffered solution at pH 7.8, but it was kept soluble in imidazole buffer containing EDTA and dithiothreitol at pH 7.0. In addition to the main band of 54 kDa, two faint immunoreactive fractions with molecular weights around 45,000 and 14,000 were also revealed by Western blotting. The presence of the rat caltrin sequence within the primary structure of the 54-kDa molecule has been investigated by sequencing the peptides generated by trypsin digestion. The sequence of the first 46 amino acid residues of rat caltrin has been found in one of the fragments produced by enzymatic cleavage. However, the exact location of the caltrin sequence in the whole 54-kDa protein has not been determined. The purified 54-kDa protein did not inhibit Ca2+ uptake by epididymal spermatozoa. Results indicated that this molecule represents an inactive precursor of caltrin and is enzymatically processed in the lumen of the seminal vesicle to the small and active calcium transport inhibitor protein. The immunoreactive proteins with intermediate molecular weights (45,000 and 14,000) could represent partially degraded products of the precursor. The lack of inhibitory activity of the precursor may be related to the molecule's having a size and conformation that would make it unable to interact with caltrin receptors on the sperm surface.