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
The complex mechanism of intracellular transport is regulated by free calcium in different manners. Calcium binding proteins regulate several aspects of the vesicle fusion mechanism mediated by NSF (N-ethylmaleimide sensitive fusion factor). At least in some regulated exocytosis, calcium-binding proteins are the trigger for fusion downstream of NSF, Still, calcium-binding proteins, such as annexins, may be part of a different fusion mechanism mediating some specific transport steps or working in parallel to the NSF-dependent fusion process. Calcium is not the only ion necessary for the function of factors involved in vesicular transport. A zinc requirement has been also proposed. One of the zinc-dependent factors is probably a protein with a cysteine-rich region that coordinates zinc and binds phorbol esters. Although protein kinase C is the more prominent family of proteins carrying this domain, the factor necessary for transport does not appear to function as a kinase.
Assuntos
Transporte Biológico , Proteínas de Ligação ao Cálcio/fisiologia , Cálcio/fisiologia , Metaloproteínas/fisiologia , Proteínas de Transporte Vesicular , Zinco/fisiologia , Animais , Proteínas de Transporte/fisiologia , Linhagem Celular , Vesículas Revestidas/fisiologia , Cães , Exocitose/fisiologia , Líquido Intracelular/metabolismo , Rim , Fusão de Membrana , Proteínas Sensíveis a N-Etilmaleimida , Ésteres de Forbol/metabolismo , Ligação Proteica , Proteína Quinase C/fisiologiaRESUMO
The complex mechanism of intracellular transport is regulated by free calcium in different manners. Calcium binding proteins regulate several aspects of the vesicle fusion mechanism mediated by NSF (N-ethylmaleimide sensitive fusion factor). At least in some regulated exocytosis, calcium-binding proteins are the trigger for fusion downstream of NSF, Still, calcium-binding proteins, such as annexins, may be part of a different fusion mechanism mediating some specific transport steps or working in parallel to the NSF-dependent fusion process. Calcium is not the only ion necessary for the function of factors involved in vesicular transport. A zinc requirement has been also proposed. One of the zinc-dependent factors is probably a protein with a cysteine-rich region that coordinates zinc and binds phorbol esters. Although protein kinase C is the more prominent family of proteins carrying this domain, the factor necessary for transport does not appear to function as a kinase.
Assuntos
Animais , Cães , Transporte Biológico , Cálcio , Proteínas de Ligação ao Cálcio , Metaloproteínas/fisiologia , Zinco , Linhagem Celular , Ésteres de Forbol/metabolismo , Exocitose , Rim , Líquido Intracelular/metabolismo , Fusão de Membrana , Ligação Proteica , Proteína Quinase C/fisiologia , Proteínas de Transporte/fisiologia , Vesículas Revestidas/fisiologiaRESUMO
The complex mechanism of intracellular transport is regulated by free calcium in different manners. Calcium binding proteins regulate several aspects of the vesicle fusion mechanism mediated by NSF (N-ethylmaleimide sensitive fusion factor). At least in some regulated exocytosis, calcium-binding proteins are the trigger for fusion downstream of NSF, Still, calcium-binding proteins, such as annexins, may be part of a different fusion mechanism mediating some specific transport steps or working in parallel to the NSF-dependent fusion process. Calcium is not the only ion necessary for the function of factors involved in vesicular transport. A zinc requirement has been also proposed. One of the zinc-dependent factors is probably a protein with a cysteine-rich region that coordinates zinc and binds phorbol esters. Although protein kinase C is the more prominent family of proteins carrying this domain, the factor necessary for transport does not appear to function as a kinase.(AU)
Assuntos
Animais , Cães , RESEARCH SUPPORT, NON-U.S. GOVT , RESEARCH SUPPORT, U.S. GOVT, NON-P.H.S. , Transporte Biológico , Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Metaloproteínas/fisiologia , Zinco/fisiologia , Proteínas de Transporte/fisiologia , Linhagem Celular , Vesículas Revestidas/fisiologia , Exocitose/fisiologia , Líquido Intracelular/metabolismo , Rim , Fusão de Membrana , Ésteres de Forbol/metabolismo , Ligação Proteica , Proteína Quinase C/fisiologiaRESUMO
Previous studies in our laboratory indicated that immunization of male and female Wistar and Lewis rats with epididymal protein DE, resulted in the development of anti-DE antibodies in over 90% of the animals, with a significant and reversible reduction of fertility. In the present study, ELISA assays performed to analyze the evolution of the immune response indicated that antibody levels in the sera of immunized animals reached a maximum at 8 weeks after the initial injection and then gradually decreased, returning to control values by the end of the sixth month. Western blot experiments demonstrated that the immune sera specifically recognized DE in epididymal sperm extracts and epididymal cytosol, while no reaction was observed with different reproductive and essential organs. The immune sera were also capable of recognizing DE on the surface of both fresh and capacitated sperm as indicated by indirect immunofluorescence experiments. Finally, the exposure of sperm to immune sera prior to uterine insemination resulted in a significant (P < 0.05) reduction in the percentage of fertilized eggs compared to controls, with no effect on sperm motility and viability, nor on their ability to undergo capacitation. Together, these results support the participation of the raised antibodies as mediators of the antifertility effect and suggest a specific interference at the sperm-egg interaction level.