ABSTRACT
Para avaliar o efeito de sistemas de cultivo in vitro na ultra-estrutura celular, foram cultivados ovócitos com o cumulus compacto distribuídos em três tratamentos: I - meio de cultivo constituído de meio de cultura de tecido (TCM) acrescido de FSH (20µg/ml) e soro inativado de vaca em estro (10 por cento); II - meio de cultivo acrescido de células da granulosa em suspensäo (2 x 10 elevado a sexta potência/ml); III - monocamada de células da granulosa em meio de cultivo. As amostras foram obtidas em zero, 12, 18, 24 e 30 horas de cultivo e submetidas aos procedimentos para a análise ultra-estrutural. As características verificadas nos ovócitos foram caracterizadas em cinco configuraçöes (C1 a C5), levando em consideraçäo vários estádios de maturaçäo. Para esta classificaçäo foram consideradas as modificaçöes ultra-estruturais no interior do ovócito, sendo C1 um ovócito sem alteraçäo indicativa de reativaçäo da meiose, C2, C3 e C4 estádios intermediários e C5 um ovócito maduro caracterizado principalmente pela configuraçäo cromossômica em metáfase II e pela distribuiçäo dos grânulos corticais em posiçöes solitárias nas proximidades da membrana citoplasmática. Foram verificados os seguintes resultados, para os tratamentos I, II e III, respectivamente: zero hora (C1 e C2, C1 e C2, C1 e C2), 12 horas (C4, C3 e C3), 18 horas (C5, C4 e C4), 24 horas (C5, C5 e C5) e 30 horas (C5, C5 e C5). No cultivo sem células da granulosa, as características que configuram um ovócito maduro sob aspecto ultra-estrutural já ocorrem com 18 horas de cultivo
Subject(s)
Cattle , Microscopy, Electron , OocytesABSTRACT
Gut absorption is one of the first requirements for the study of the mechanism of a possible anti-inflammatory action of proteases, such as orally administered trypsin. Porcine trypsin absorption was studied in isolated jejunal loops of rats (female Holtzman and male Wistar) and guinea pig (males) by open-loop perfusion. Trypsin was dissolved in Tyrode solution and the solution perfused at a rate of 0.5 ml/min, at 37§C. Trypsin activity, total protein, and sodium and potassium concentrations were assayed in the jejunal effluent; the values were unchanged throughout the experiments, which lasted 45 to 120 min. Using a high sensitivity ELISA (i.e. pg/ml), trypsin absorption could be demonstrated by determination of the enzyme in the mesenteric venous blood (samples of 0.5 ml); the enzyme concentration increased with time of perfusion. The linear range-specificity for intact trypsin varied from 1 to 500 ng/well. In this assay polyclonal antibodies prepared against trypsin-TLCK were utilized. Whereas trypsin concentration in the perfused lumen was practically constant at 0.12 mg/ml, the concentration of absorbed trypsin in mesenteric vein blood increased from about 100 ng/ml at time zero to 1.8 µg/ml, after 45 min of perfusion. Histological and ultrastructural examination of the jejunal mucosa before and after perfusion revealed that the brush-border, basal membrane, and junctional complexes were fully preserved, thus eliminating the possibility that trypsin might have destroyed the structures, thereby reaching the blood circulation. The present data indicate that µg quantities of trypsin were absorbed by the isolated jejunal loop of the rat