RESUMO
OBJECTIVE: This study was undertaken to clone and express a recombinant human zona pellucida protein 3 and to characterize its biologic activities as a sperm ligand and an inducer of the acrosome reaction. STUDY DESIGN: Human ovarian teratocarcinoma (PA-1) cells were transfected with an expression vector containing human zona pellucida protein 3 complementary deoxyribonucleic acid with a sequence coding for a 6-histidine tail introduced into its 3' end. Purification of the secreted glycoprotein was performed by sequential affinity (lectin and nickel--nitrilotriacetic acid) and ion-exchange chromatography. RESULTS: Western blot analysis confirmed a molecular weight of approximately 65 kd for the purified product. A cell-free translation system revealed a correctly sized protein backbone of 47 kd. The recombinant human zona pellucida protein 3 demonstrated specific, potent, and dose-dependent competitive inhibition of sperm-zona pellucida binding in vitro under hemizona assay conditions. Recombinant human zona pellucida protein 3 also stimulated the acrosome reaction of live sperm. This effect was fast, dose dependent, and capacitation time dependent. Furthermore, advance incubation with pertussis toxin, an inactivator of heterotrimeric G proteins, blocked recombinant human zona pellucida protein 3--induced acrosomal exocytosis. CONCLUSION: The recombinant human zona pellucida protein 3 expressed in PA-1 cells manifested the full spectrum of expected biologic activities. It therefore represents a valuable tool for examination of human fertilization and the design of new strategies in diagnosis of male factor infertility and in contraception.
Assuntos
Proteínas do Ovo/farmacologia , Glicoproteínas de Membrana/farmacologia , Ovário/fisiologia , Receptores de Superfície Celular , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/fisiologia , Western Blotting , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Proteínas do Ovo/biossíntese , Proteínas do Ovo/genética , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Neoplasias Ovarianas , Ovário/metabolismo , Toxina Pertussis , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/fisiologia , Transfecção , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologia , Glicoproteínas da Zona PelúcidaRESUMO
The effect was studied of a low dose of gamma-ray preexposure on the frequency and molecular spectrum of radiation-induced mutations at the hprt locus in a human T-cell leukemia line. When the cells were preexposed to 0.01 Gy of gamma-rays, the yield of mutations induced by a subsequent 2-Gy challenge dose was reduced by 60%, compared with the 2 Gy of irradiation alone. The data of Southern blot analysis showed that 47% of the mutants induced by 2 Gy in the cells without low-dose preexposure were of the deletion or rearranged mutations type. In contrast, in the low-dose radioadapted cells the proportion of this type of 2-Gy-induced mutations decreased to 28%. This is close to the control level (22%) of spontaneous mutations. Our results confirm that a low dose of gamma-ray preexposure leads to a decreased susceptibility to gene deletions and rearrangements after high-dose irradiation.
