RESUMO
OBJECTIVE: To determine the secretion of Grp78 by human oviduct epithelial cells, its association to spermatozoa, and its involvement in gamete interaction. DESIGN: Prospective study. SETTING: Basic research laboratory. SUBJECT(S): Semen samples obtained from normozoospermic volunteers. Tubal tissue provided by patients undergoing hysterectomies. Oocytes collected from women undergoing IVF-ET. INTERVENTION(S): Analysis of Grp78 expression and secretion by oviductal tissue. Gamete incubation with recombinant Grp78 (rec-Grp78). MAIN OUTCOME MEASURE(S): Assessment of protein expression and secretion by immunohistochemistry and Western immunoblotting, respectively. Evaluation of rec-Grp78 binding to human spermatozoa by immunocytochemistry, and analysis of its effect upon gamete interaction using the hemizona assay. RESULT(S): Grp78 was found in the surface of oviduct epithelial cells. Soluble Grp78 was detected in oviductal fluids from women in the periovulatory period and in oviductal tissue conditioned medium. Rec-Grp78 was able to bind to the sperm acrosomal cap, and its presence during gamete interaction led to a decrease in the number of spermatozoa bound to the zona pellucida (ZP). When calcium ions from the incubation medium were replaced by strontium, rec-Grp78 enhanced sperm-ZP interaction. CONCLUSION(S): Grp78 is expressed and secreted by oviduct epithelial cells. The protein would bind to the gametes and may modulate their interaction in a calcium-dependent manner.
Assuntos
Células Epiteliais/metabolismo , Tubas Uterinas/metabolismo , Proteínas de Choque Térmico/metabolismo , Interações Espermatozoide-Óvulo , Espermatozoides/metabolismo , Zona Pelúcida/metabolismo , Adulto , Western Blotting , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Imuno-Histoquímica , Masculino , Ciclo Menstrual , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Recombinantes/metabolismo , Técnicas de Cultura de TecidosRESUMO
Recombinant human zona pellucida protein C expressed in Chinese hamster ovary cells associates to the acrosomal region of human spermatozoa and inhibits sperm-zona pellucida interaction in the hemizona assay. Recombinant human zona pellucida protein C may be a useful tool toward the development of diagnostic methods for male factor infertility and the elucidation of the molecular basis of fertilization.
