RESUMO
The acrosome reaction (i.e. the exocytosis of the sperm vesicle) is a prerequisite for fertilization, but its molecular mechanism is largely unknown. We have identified a cDNA clone for a gene named haprin, which encodes a haploid germ cell-specific RING finger protein. This protein is a novel member of the RBCC (RING finger, B-box type zinc finger, and coiled-coil domain) motif family that has roles in several cellular processes, such as exocytosis. It is transcribed exclusively in testicular germ cells after meiotic division. Western blot and immunohistochemical analyses showed the molecular weight of Haprin protein to be Mr approximately 82,000. It was localized in the acrosomal region of elongated spermatids and mature sperm and was not present in acrosome-reacted sperm. The specific antibody against the RING finger domain of Haprin inhibited the acrosome reaction in permeabilized sperm. These results indicated that the novel RBCC protein Haprin plays a key role in the acrosome reaction and fertilization.
Assuntos
Reação Acrossômica/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Plasma Seminal/fisiologia , Acrossomo/química , Sequência de Aminoácidos , Animais , Western Blotting , Proteínas de Transporte/química , Proteínas de Transporte/genética , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/química , DNA Complementar/genética , Fertilização , Imunofluorescência , Expressão Gênica , Biblioteca Gênica , Imuno-Histoquímica , Masculino , Meiose , Camundongos , Dados de Sequência Molecular , Peso Molecular , RNA Mensageiro/análise , Proteínas de Plasma Seminal/química , Proteínas de Plasma Seminal/genética , Análise de Sequência de DNA , Espermátides/química , Espermátides/ultraestrutura , Espermatozoides/química , Espermatozoides/ultraestrutura , Testículo/químicaRESUMO
Rat sperm epididymal glycoprotein DE belongs to the cysteine-rich secretory protein (CRISP) family and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. To investigate the molecular mechanisms underlying the role of DE in gamete fusion, in the present work we expressed DE in a prokaryotic system, and examined the relevance of carbohydrates and disulfide bonds for the biological activity of the protein. Immunofluorescence and sperm-egg fusion assays carried out in the presence of recombinant DE (recDE) revealed that this protein exhibits the ability to bind to the DE-egg binding sites and to inhibit gamete fusion, as does native DE (nDE). Comparison of the proteins indicated, however, that the inhibitory ability of recDE was significantly lower than that of nDE. This difference would not be due to the lack of carbohydrates in the bacterially expressed protein because enzymatically deglycosylated nDE was as able as the untreated protein to inhibit gamete fusion. To examine whether disulfide bridges are involved in DE activity, the presence of sulfhydryls in nDE and recDE was evaluated by the biotin-maleimide technique. Results indicated that, unlike nDE, in which all cysteines are involved in disulfide bonds, recDE contains free thiol groups. Subsequent experiments showed that reduction of nDE with dithiothreitol significantly decreased the ability of the protein to inhibit gamete fusion. Together, these results indicate that whereas carbohydrates do not have a role in DE-mediated gamete fusion, disulfide bridges are required for full biological activity of the protein. To our knowledge, this is the first study reporting the relevance of structural components for the function of a CRISP member.