RESUMEN
The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.
Asunto(s)
Acrosina/metabolismo , Proteínas del Huevo/metabolismo , Precursores Enzimáticos/metabolismo , Fertilización/fisiología , Glicoproteínas de Membrana/metabolismo , Espermatozoides/efectos de los fármacos , Suramina/farmacología , Zona Pelúcida/metabolismo , Acrosina/genética , Animales , Antineoplásicos/farmacología , Precursores Enzimáticos/genética , Femenino , Radioisótopos de Yodo/metabolismo , Masculino , Ratones , Ratones Transgénicos , Estructura Molecular , Óvulo/metabolismo , Unión Proteica , Receptores de Superficie Celular/metabolismo , Espermatozoides/metabolismo , Sulfatos/metabolismo , Zona Pelúcida/química , Glicoproteínas de la Zona PelúcidaRESUMEN
The cMG1 gene was originally identified as a mitogen-stimulated primary response gene. However, in contrast to genes such as c-fos and TIS11, cMG1 is also expressed at significant levels before and after the transient elevation induced by agonists. We have sequenced a 1.3 kb rat genomic cMG1 clone, which includes 931 bp upstream of the transcription start site identified by primer-extension analysis. A 1033 bp fragment, including this 5'-flanking sequence, directed the expression of the reporter gene chloramphenicol acetyltransferase (CAT) in transfected NIH-3T3 cells. Progressive 5'-to-3' deletion indicated that an element located between -138 and -114 was responsible for most of this basal CAT expression. DNA mobility-shift assays showed that the sequence between -143 and -105 contained binding sites for cellular proteins, the principal complexes involving nucleotides between -119 and -105. We conclude that these complexes may represent the transcription factor-DNA element interactions that determine basal cMG1 expression.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Inmediatas-Precoces , Regiones Promotoras Genéticas , Proteínas/genética , Células 3T3 , Animales , Secuencia de Bases , Factor 1 de Respuesta al Butirato , Cloranfenicol O-Acetiltransferasa/genética , ADN Complementario/química , Eliminación de Gen , Expresión Génica , Genes Reporteros , Ratones , Datos de Secuencia Molecular , Células PC12 , ARN Mensajero/metabolismo , Ratas , Análisis de Secuencia de ADN , Transfección , TristetraprolinaRESUMEN
A homologous radioimmunoassay for the measurement of epidermal growth factor (EGF) levels in pig tissues and body fluids has been developed using an antiserum to recombinant porcine EGF. The assay is highly specific, showing no cross-reactivity with a variety of other polypeptides including the structurally related protein, transforming growth factor-alpha. Furthermore, < 1% cross-reactivity was observed with mouse EGF emphasizing the necessity for homologous assays for EGF measurement. Immunoreactive EGF was present in extracts of pig kidney and pancreas (3.44 +/- 0.43 and 0.76 +/- 0.13 (S.E.M.) pmol/g wet weight respectively), but was not detected in extracts of submaxillary gland or liver. Although immunoreactive EGF was not detectable in uterine, allantoic or ovarian follicular fluids, colostrum contained EGF at biologically active concentrations (0.84 +/- 0.15 nmol/l). Immunoreactive EGF was also present in pig urine, with similar concentrations in samples from male or female animals. In addition, pig urine inhibited the binding of 125I-labelled EGF to 3T3 fibroblasts and stimulated DNA synthesis in quiescent monolayers of these cells, indicating that the immunoreactive material in urine is biologically active. Quantitative comparisons of the data presented here with that published previously indicate considerable species variation in the EGF levels of various tissues and body fluids.