RESUMO
Fusion between gametes is a key event in the fertilization process involving the interaction of specific domains of the sperm and egg plasma membranes. During recent years, efforts have been made toward the identification of the specific molecular components involved in this event. The present work will focus on the best characterized candidates for mediating gamete membrane fusion in mammals. These molecules include members of the ADAM (a disintegrin and a metalloprotease domain) family, i.e., testicular proteins fertilin alpha, fertilin beta, and cyritestin, which are thought to interact with integrins in the egg plasma membrane through their disintegrin domains, and a member of the cysteine-rich secretory proteins (CRISP) family, i.e., epididymal protein DE, which participates in an event subsequent to sperm-egg binding and leading to fusion through specific complementary sites localized on the fusogenic area of the egg surface. The identification and characterization of these molecules will contribute not only to a better understanding of the molecular mechanisms underlying mammalian sperm-egg fusion but also to the development of new methods for both fertility regulation and diagnosis and treatment of human infertility.
Assuntos
Interações Espermatozoide-Óvulo/fisiologia , Proteínas ADAM , Animais , Cricetinae , Proteínas do Ovo/fisiologia , Feminino , Fertilinas , Glicoproteínas/fisiologia , Humanos , Masculino , Mamíferos/genética , Mamíferos/fisiologia , Fusão de Membrana , Glicoproteínas de Membrana/fisiologia , Mesocricetus , Metaloendopeptidases/fisiologia , Camundongos , Ratos , Proteínas e Peptídeos Salivares/fisiologia , Proteínas de Plasma Seminal/fisiologia , Especificidade da Espécie , Vacinas Anticoncepcionais , Zona Pelúcida/fisiologiaRESUMO
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 Ca(2+) 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.
Assuntos
Glicoproteínas/fisiologia , Glicoproteínas de Membrana , Oócitos/química , Proteínas e Peptídeos Salivares/fisiologia , Proteínas de Plasma Seminal/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Animais , Sítios de Ligação , Western Blotting , Cricetinae , Feminino , Fertilização in vitro , Técnica Indireta de Fluorescência para Anticorpo , Glicoproteínas/metabolismo , Humanos , Masculino , Oócitos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Proteínas de Plasma Seminal/metabolismo , Espermatozoides/metabolismoRESUMO
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
Rat epididymal glycoprotein DE associates with the dorsal region of the sperm head during sperm maturation, migrates to the equatorial segment (ES) with the acrosome reaction (AR), and is involved in gamete membrane fusion. In the present study we examined the association of DE with the sperm surface and the relationship of this interaction with the behavior and function of the protein. Cloning and sequencing of DE revealed a lack of hydrophobic domains and the presence of 16 cysteine residues in the molecule. Experiments in which cauda epididymal sperm were subjected to different extraction procedures indicated that while most of the protein is removable from sperm by mild ionic strength, a low amount of DE, resistant to even 2 M NaCl, can be completely extracted by agents that remove integral proteins. However, the lack of hydrophobic domains in the molecule and the failure of DE to interact with liposomes, does not support a direct insertion of the protein into the lipid bilayer. These results, and the complete extraction of the tightly bound protein by dithiothreitol, suggest that this population would correspond to a peripheral protein bound to a membrane component by strong noncovalent interactions that involve disulfide bonds. While ELISA experiments showed that no protein could be extracted by NaCl from capacitated sperm, indirect immunofluorescence studies revealed the ability of the NaCl-resistant protein to migrate to the ES. Together, these results support the existence of two populations of DE: a major, loosely bound population that is released during capacitation, and a minor strongly bound population that remains after capacitation, migrates to the ES with the AR, and thus would correspond to the one with a role in gamete fusion.
Assuntos
Metaloproteínas/metabolismo , Espermatozoides/metabolismo , Hormônios Testiculares/metabolismo , Animais , Membrana Celular/metabolismo , Clonagem Molecular , Proteínas Secretadas pelo Epidídimo , Masculino , Metaloproteínas/genética , Ratos , Ratos Sprague-Dawley , Capacitação Espermática/fisiologia , Hormônios Testiculares/genéticaRESUMO
Rat epididymal protein DE associates with the sperm surface during maturation and participates in sperm-egg fusion. Immunization of male rats with DE raised specific antibodies and produced a significant reduction in the animals' fertility. The present study focused on determining the in vivo mechanism involved in fertility inhibition. Wistar males were injected with DE, and antibody levels and animal fertility were evaluated. Results revealed an association between the two parameters, since animals with absorbance values lower than 0.5 in ELISA presented high fertility rates (66%, 100%) while those with absorbance values higher than 0.5 exhibited the lowest fertility rates (0%, 33%). Histological studies showed no evidence of orchitis, epididymitis, or vasitis in DE-immunized animals. ELISA results revealed the presence of anti-DE antibodies in epididymal and vas deferential fluids. Indirect immunofluorescence and ELISA experiments indicated that these antibodies would not interfere with the synthesis or secretion of DE or with its association with the sperm surface. Finally, while epididymal sperm recovered from DE-immunized animals presented no changes in motility, viability, or ability to undergo capacitation and acrosome reaction, they exhibited a significant decrease in their ability to fuse with zona-free eggs, with no effect on their ability to bind to the oolemma. Together these results indicate that immunization of male rats with epididymal protein DE specifically interferes with the sperm fertilizing ability, supporting the use of epididymal proteins for contraceptive vaccine development.