RESUMO
The decapacitating fraction of human seminal plasma, which strongly interacts with concanavalin A, is constituted by high mannose-type N-linked glycoproteins, most of them of less than 44 kDa. Each component with apparent molecular mass of 30, 18, and 17 kDa respectively, as judged by SDS-PAGE, was submitted to "in gel" digestion with trypsin followed by HPLC separation of the peptides and sequencing. They were characterized at microscale as gp17, an aspartyl protease that possibly contributes to liquefaction of the seminal plasma coagulum, two fragments of human acid phosphatase (17 and 30 kDa, respectively), and a 17-kDa fragment of carboxypeptidase E. Neither the fragments of prostatic acid phosphatase nor that of carboxypeptidase E had been described before in the human seminal fluid. Very weak bands, of apparent molecular masses 44 and 52 kDa, are consistent with presence of small amounts of parent compounds, prostatic acid phosphatase and carboxypeptidase E.
Assuntos
Carboxipeptidase H/isolamento & purificação , Concanavalina A/metabolismo , Glicoproteínas/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Tirosina Fosfatases/isolamento & purificação , Sêmen/química , Fosfatase Ácida , Sequência de Aminoácidos , Carboxipeptidase H/metabolismo , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Sêmen/enzimologiaRESUMO
These studies showed that the fractionation of bovine seminal plasma based on lectin agarose affinity chromatography, employing lectins specific to asparagine linked oligosaccharides, and a lectin specific for fucosylated glycans, lead to products with an inhibitory effect on the acrosine-like protease activity. This effect decreases when glycocompounds containing fucosylated Lewis(x) structures are removed, suggesting that these compounds might have some role in the modulation of this activity in the bull. In the fraction devoid of high mannose, hybrid and non-bisecting lactosaminic oligosaccharide-containing glycocompounds, PDC-109 and aSFP proteins were detected and characterized at microscale.
Assuntos
Cromatografia de Afinidade/métodos , Endopeptidases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas/análise , Proteínas/química , Sêmen/química , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Endopeptidases/química , Lectinas/química , Masculino , Inibidores de Proteases/química , Proteínas de Plasma SeminalRESUMO
Protamine crosslinking by disulphide (-SS-) bonds in the main factor responsible for the stability of chromatin structure in mammalian spermatozoa. Sperm chromatin containing arginine/cysteine-rich protamines shows a deeply modified cytochemical reactivity (e.g. basophilia) when compared with somatic chromatic. After methanol or ethanol-acetic acid fixation and toluidine blue (TB) staining, most sperm heads in semen smear from human fertile donors exhibited a pale blue (orthochromatic) colour, while a few sperm heads exhibited violet-blue or violet (metachromatic) staining. Smears from infertile patients showed an increased amount of metachromatic sperm nuclei. After reduction of -SS- bonds by dithiothreitol, sperm heads from all smears were metachromatic, suggesting that DNA phosphates then become available for TB stacking. Micro- and macro-spectrophotometric studies confirmed the microscopic colour reaction of sperm nuclei. The ortho-/metachromatic ration seems a useful parameter for evaluation of altered chromatin structure in spermatozoa cells. Taking into account the current interest in complementary staining tests for evaluation of semen quality, the metachromatic TB reaction represents a simple cytochemical approach for detecting sperm chromatin abnormalities based on differences in -SS- crosslinking.
Assuntos
Cromatina/patologia , Infertilidade Masculina/fisiopatologia , Técnicas Reprodutivas , Espermatozoides/patologia , Cromatina/ultraestrutura , Corantes , Dissulfetos , Fertilidade , Humanos , Infertilidade Masculina/patologia , Masculino , Oligospermia/patologia , Oligospermia/fisiopatologia , Valor Preditivo dos Testes , Prognóstico , Valores de Referência , Espectrofotometria , Contagem de Espermatozoides , Cabeça do Espermatozoide/patologia , Cabeça do Espermatozoide/ultraestrutura , Motilidade dos Espermatozoides , Espermatozoides/anormalidades , Espermatozoides/fisiologia , Cloreto de TolônioRESUMO
The object of the present work was to investigate: a) The cytotoxic activity of cytophilic antibody in sera from guinea pigs sensitized with the testicular antigen; b) The presence of immune complexes. In vitro cytotoxicity induced by hyperimmune sera or by the cytophilic antibodies eluted from the corresponding macrophages, was demonstrated after incubation with either germinal cells or spermatozoa. Marked cytotoxicity was also observed in vivo when sera or antibodies were injected into the tests, as shown by sequential histologic studies. Circulating immune complexes, as tested by the 125I-C1q binding assay, were not detected in these sera. On the contrary, high levels of circulating immune complexes were found when the polyethyleneglycol precipitation test was used. It can be concluded that the cytotoxic effect induced by cytophilic antibodies of IgG2 nature, may be associated with polyethyleneglycol precipitating immune complexes.
Assuntos
Complexo Antígeno-Anticorpo/análise , Autoanticorpos/imunologia , Macrófagos/imunologia , Espermatozoides/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Citotoxicidade Imunológica , Cobaias , Soros Imunes , Masculino , Orquite/etiologia , Orquite/imunologia , Testículo/citologia , Testículo/imunologiaRESUMO
En trabajpos previos (1) demostramos la presencia de anticuerpos citofilicos antiespermáticos (ACA) en suero de cobayos con orquitis inmunológica experimental, encontrándose una estrecha relación entre lesión testicular y ACA. Dichos ACA fueron asilados mediante elución de la superficie de macrófagos, demosntrando su naturaleza de IgG2 (12). Este trabajo tiene por objeto estudiar la capacidad citotóxica tanto in vitro, como in vitro, de ACA extraídos (HE) de sueros provenientes de cobayos con alto título de ACA (HS). Asimismo, en dichos sueros se investigó la presencia de complejos inmunes circulantes (CIC). Se demostró citotoxicidad in citro sobre: a) células germinales aisladas de tésticulo normal (Tabla 1) incubadas con HS (1/32) y con el anticuerpo aislado HE (1/128); b) espermatozoides (Tabla 1) incubados con HE (1/64). Se demostro citotoxicidad in vivo (Tabla 2) mediante el estudio histológico de testículos de cobayos normales a los cuales se inyecto HS y HE en el espacio subalbugineo. Con HS se hallo intensa orquitis desde el primer día post-inyección. Con HE las lesiones testiculares se observaron a partir del día 7. La incubación in vitro o la inyección en el espacio subalbugíneo utilizando suero normal o fracción citofgílica aislada de suero normal no demostratron citotoxicidad en ningún caso. La presencia de CIC se investigó ne HS mediante: 1) la técnica de combinación 125I-C1q(19), con la cual no se obtuvieron datos significativos en relación al suero normal (Tabla 3); 2) el test de precipitación de polietilenglicol (4) el cual permitió detectar nivelles elevados de CIC en HS (Tabla 4). Estos hallazgos sugieren que los ACA poseen actividad citotóxica sobre el tejido testicular. Esta citotoxicidad podría estar relacionada con los CIC precipitables con polietilenglicol (AU)
Assuntos
Animais , Masculino , Complexo Antígeno-Anticorpo/análise , Autoanticorpos/imunologia , Macrófagos/imunologia , Espermatozoides/imunologia , Citotoxicidade Imunológica , Orquite/etiologia , Orquite/imunologia , Testículo/citologia , Testículo/imunologia , Cobaias , Complexo Antígeno-Anticorpo/imunologia , Soros ImunesRESUMO
The object of the present work was to investigate: a) The cytotoxic activity of cytophilic antibody in sera from guinea pigs sensitized with the testicular antigen; b) The presence of immune complexes. In vitro cytotoxicity induced by hyperimmune sera or by the cytophilic antibodies eluted from the corresponding macrophages, was demonstrated after incubation with either germinal cells or spermatozoa. Marked cytotoxicity was also observed in vivo when sera or antibodies were injected into the tests, as shown by sequential histologic studies. Circulating immune complexes, as tested by the 125I-C1q binding assay, were not detected in these sera. On the contrary, high levels of circulating immune complexes were found when the polyethyleneglycol precipitation test was used. It can be concluded that the cytotoxic effect induced by cytophilic antibodies of IgG2 nature, may be associated with polyethyleneglycol precipitating immune complexes.
RESUMO
En trabajpos previos (1) demostramos la presencia de anticuerpos citofilicos antiespermáticos (ACA) en suero de cobayos con orquitis inmunológica experimental, encontrándose una estrecha relación entre lesión testicular y ACA. Dichos ACA fueron asilados mediante elución de la superficie de macrófagos, demosntrando su naturaleza de IgG2 (12). Este trabajo tiene por objeto estudiar la capacidad citotóxica tanto in vitro, como in vitro, de ACA extraídos (HE) de sueros provenientes de cobayos con alto título de ACA (HS). Asimismo, en dichos sueros se investigó la presencia de complejos inmunes circulantes (CIC). Se demostró citotoxicidad in citro sobre: a) células germinales aisladas de tésticulo normal (Tabla 1) incubadas con HS (1/32) y con el anticuerpo aislado HE (1/128); b) espermatozoides (Tabla 1) incubados con HE (1/64). Se demostro citotoxicidad in vivo (Tabla 2) mediante el estudio histológico de testículos de cobayos normales a los cuales se inyecto HS y HE en el espacio subalbugineo. Con HS se hallo intensa orquitis desde el primer día post-inyección. Con HE las lesiones testiculares se observaron a partir del día 7. La incubación in vitro o la inyección en el espacio subalbugíneo utilizando suero normal o fracción citofgílica aislada de suero normal no demostratron citotoxicidad en ningún caso. La presencia de CIC se investigó ne HS mediante: 1) la técnica de combinación 125I-C1q(19), con la cual no se obtuvieron datos significativos en relación al suero normal (Tabla 3); 2) el test de precipitación de polietilenglicol (4) el cual permitió detectar nivelles elevados de CIC en HS (Tabla 4). Estos hallazgos sugieren que los ACA poseen actividad citotóxica sobre el tejido testicular. Esta citotoxicidad podría estar relacionada con los CIC precipitables con polietilenglicol