RESUMO
Testicular protein Tpx-1, also known as CRISP-2, is a cysteine-rich secretory protein specifically expressed in the male reproductive tract. Since the information available on the human protein is limited to the identification and expression of its gene, in this work we have studied the presence and localization of human Tpx-1 (TPX1) in sperm, its fate after capacitation and acrosome reaction (AR), and its possible involvement in gamete interaction. Indirect immunofluorescence studies revealed the absence of significant staining in live or fixed non-permeabilized sperm, in contrast to a clear labelling in the acrosomal region of permeabilized sperm. These results, together with complementary evidence from protein extraction procedures strongly support that TPX1 would be mainly an intra-acrosomal protein in fresh sperm. After in vitro capacitation and ionophore-induced AR, TPX1 remained associated with the equatorial segment of the acrosome. The lack of differences in the electrophoretic mobility of TPX1 before and after capacitation and AR indicates that the protein would not undergo proteolytical modifications during these processes. The possible involvement of TPX1 in gamete interaction was evaluated by the hamster oocyte penetration test. The presence of anti-TPX1 during gamete co-incubation produced a significant and dose-dependent inhibition in the percentage of penetrated zona-free hamster oocytes without affecting sperm motility, the AR or sperm binding to the oolema. Together, these results indicate that human TPX1 would be a component of the sperm acrosome that remains associated with sperm after capacitation and AR, and is relevant for sperm-oocyte interaction.
Assuntos
Glicoproteínas/análise , Glicoproteínas/metabolismo , Capacitação Espermática/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/química , Anticorpos/farmacologia , Moléculas de Adesão Celular , Glicoproteínas/antagonistas & inibidores , Humanos , Masculino , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismoRESUMO
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
We describe 2 patients with adult-onset type II citrullinemia who developed transient hypoproteinemia and jaundice in early infancy. Liver histology showed a marked fatty change and fibrosis. After the patients had lived without symptoms to the ages of 5 and 16 years, respectively, the diagnosis was made by genetic analysis.