RESUMEN
AIMS/HYPOTHESIS: The aim of this study was to investigate whether gestational diabetes mellitus, which occurs in the microvascular remodelling phase of placental development, causes alterations in surface expression of tight and adherens junctional molecules involved in endothelial barrier function and angiogenesis. METHODS: Term placenta, delivered by elective Caesarian section, from normal pregnancy (n = 5) and those complicated by gestational diabetes (n = 5) were perfusion-fixed and analysed by indirect immunofluorescence and confocal scanning microscopy. Using systematic random sampling, the surface expression of endothelial junctional proteins and the relative incidences of immunostained vessels were compared between the two study groups. Total vessel lengths were measured by stereological techniques. RESULTS: The adherens junctional molecules, vascular-endothelial cadherin and beta-catenin, and the tight junctional molecules, occludin and zonula occludens-1 were localised to paracellular clefts in both study groups. The diabetic placentae showed pronounced reductions in the intensity of immunofluorescence and in the number of immuno-positive vessels. A corresponding statistically significant increase (from 19% to 56%) in the percentage of vessels showing junctional anti-phosphotyrosine immunoreactivity was found. The differences observed represented real changes in the absolute lengths of immunostained regions along the vessels. The stereological measurements failed to detect any statistically significant change in the combined length of fetal vessels in gestational diabetic placenta. CONCLUSION/INTERPRETATION: Our results suggest that even short duration diabetic insult, alters the surface expression of placental junctional proteins. This alteration could be mediated by the tyrosine-phosphorylation pathway. The changes suggest impaired barrier function rather than accelerated vascular growth.
Asunto(s)
Cadherinas/análisis , Proteínas del Citoesqueleto/análisis , Diabetes Gestacional/fisiopatología , Endotelio Vascular/fisiopatología , Placenta/anatomía & histología , Placenta/irrigación sanguínea , Embarazo/fisiología , Transactivadores , Antígenos CD , Peso al Nacer , Cesárea , Diabetes Gestacional/tratamiento farmacológico , Endotelio Vascular/fisiología , Femenino , Edad Gestacional , Hemoglobina Glucada/análisis , Humanos , Recién Nacido , Insulina/uso terapéutico , Proteínas de la Membrana/análisis , Ocludina , Tamaño de los Órganos , Fosfoproteínas/análisis , Valores de Referencia , Proteína de la Zonula Occludens-1 , beta CateninaRESUMEN
Tight and adherens junctions are major determinants of endothelial integrity. Molecules present therein have been implicated in vascular permeability, stability of junctions, angiogenesis and intracellular signalling. Using immunofluorescence and confocal scanning microscopy, the adherens junctions (AJs) in human placental vessels were found to contain the entire cadherin-catenin complex predicted from in vitro studies. Vascular endothelial cadherin (VE-cadherin) clusters were co-localized with beta-catenin, an important signal transduction ligand, and with alpha-catenin, which is thought to link the complex to the peri-junctional actin. Antibodies to plakoglobin, a molecule shown to be a component of stable adherens junctions, revealed immunoreactivity in clefts of stromal villous vessels, but weak or negative immunoreactivity in intermediate and terminal villi. Tight junctional molecules demonstrated a differential surface expression. Within the same villous tree, arteries, veins and arterioles contained occludin but the exchange vessels in terminal villi were immunonegative. ZO-1, however, was present throughout. Ultrastructurally, there were no differences in frequency, position or dimension of tight junctions in these vessels. They showed a consistent 4 nm separation between outer membrane leaflets regardless of their location in the vascular tree. Occludin is not necessary for formation of tight junctions in the placenta; it may have an accessory role providing stability or added adhesiveness to tight junctions of large vessels. Its absence in terminal villous microvessels, along with the weak plakoglobin immunoreactivity in AJs, suggest that the junctions here are less stable. This may allow the increased plasticity necessary in terminal villi for continual growth, proliferation and solute exchange.
Asunto(s)
Cadherinas/análisis , Vellosidades Coriónicas/irrigación sanguínea , Proteínas del Citoesqueleto/análisis , Endotelio Vascular/química , Uniones Estrechas/química , Transactivadores , Adulto , Antígenos CD , Adhesión Celular , Vellosidades Coriónicas/ultraestructura , Desmoplaquinas , Endotelio Vascular/citología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Microscopía Confocal , Embarazo , Tercer Trimestre del Embarazo , Uniones Estrechas/ultraestructura , alfa Catenina , beta Catenina , gamma CateninaRESUMEN
Ultrastructural, immunochemical, fluorescence and stereological studies were undertaken on human villous trophoblast from 13 weeks of gestation to term. The aim was to describe and quantify morphological changes during proliferation, differentiation and apoptosis in cytotrophoblast and syncytial regions of non-aggregated and aggregated nuclei. Numbers of trophoblast nuclei increased continuously from 13 weeks. In term placentae, intrasyncytial differentiation was characterized ultrastructurally by gradual decreases in nuclear size and packing density accompanied by nucleolar regression, and increasing heterochromatinization, envelope convolution and packing density of nuclear pore complexes. In densely packed areas, nuclear profiles resembled interlocking jigsaw pieces. Occasionally, these 'pre-apoptotic' nuclei were associated with annulate lamellae. Rarely, nuclear changes terminated in apoptosis with a characteristic pattern of condensed peripheral chromatin, a central island of euchromatin, no nucleoli and no discernible nuclear pores. Apoptotic nuclei were seen singly and within dense nuclear aggregations. Similar spatial patterns of nuclei and chromatin were seen in propidium iodide-stained sections at 13-41 weeks. Whilst the relative incidence of intensely fluorescent nuclei remained constant, absolute numbers increased linearly during gestation and correlated positively with the volume of syncytial knots. Nuclei labelled for DNA fragmentation occurred very infrequently and were also found in nuclear clusters as well as singly. We suggest that nuclear differentiation in syncytium has two phases: on entering syncytium, nuclei become committed to a long programmed pre-apoptotic phase which leads to a short apoptotic execution phase. We propose further that clustered nuclei (pre-apoptotic and apoptotic) in syncytial knots probably represent the extrusion component of normal continuous epithelial turnover.
Asunto(s)
Apoptosis/fisiología , Vellosidades Coriónicas/ultraestructura , Citoplasma/ultraestructura , Membrana Nuclear/ultraestructura , Trofoblastos/ultraestructura , Diferenciación Celular/fisiología , División Celular/fisiología , Citoplasma/metabolismo , Fragmentación del ADN , Desarrollo Embrionario y Fetal/fisiología , Células Epiteliales/fisiología , Colorantes Fluorescentes , Edad Gestacional , Células Gigantes/citología , Humanos , Inmunohistoquímica , Microscopía Confocal , Trofoblastos/metabolismoRESUMEN
Congenital anomalies occur up to four times more frequently in diabetic pregnancy than in the nondiabetic population. Although past work has shown that maternal hyperglycemia and hyperketonemia may increase embryonic abnormalities, recent experimental evidence suggests that low insulin levels may also contribute to diabetic embryopathy. This study investigated the effects of guinea pig serum (whose insulin is inactive in rat systems) on rat embryonic growth and development in culture. Supplementation of guinea pig serum with pork insulin at low (1 ng/ml) and high (5 ng/ml) physiological concentrations and insulinlike growth factors (IGF) I and II were also studied. Culture of rat embryos from the early headfold stage in guinea pig serum resulted in poor embryonic growth and development with a 92% rate of anomalies. Supplementation of guinea pig serum with zinc-binding pork insulin significantly improved rat embryonic growth and development (46% anomaly rate) especially between the first 5 and 21 h of the period of organogenesis. This evidence supports our most recent findings that low insulin levels, as encountered in untreated diabetic pregnancy, may contribute to the increased risk of congenital abnormality. Insulin at low physiological concentrations improved growth, whereas higher physiological concentrations were required to increase growth and development. IGF-I or IGF-II supplementation improved rat embryonic growth and development but failed to match that of the controls, indicating that other growth factors including insulin may also be required.
Asunto(s)
Anomalías Inducidas por Medicamentos , Embrión de Mamíferos/fisiología , Desarrollo Embrionario y Fetal , Factor II del Crecimiento Similar a la Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Animales , Medios de Cultivo , Embrión de Mamíferos/efectos de los fármacos , Femenino , Cobayas , Técnicas de Cultivo de Órganos , Embarazo , Ratas , Ratas Endogámicas , Factores de Riesgo , Especificidad de la Especie , PorcinosRESUMEN
The effect of supplementing grass silage with fishmeal on growth, muscle composition and the rate of muscle protein synthesis was investigated in young Friesian steers with and without oestradiol implants. The effect of the beta-adrenergic agonist cimaterol was simultaneously investigated in animals fed on silage alone. Treatments lasted for 9 or 10 weeks. Fishmeal supplementation significantly increased animal growth rates (P less than 0.001) and the weights of three dissected muscles (P less than 0.001) compared with the silage-fed controls. These effects were further enhanced in animals also implanted with oestradiol. Muscle weights expressed as a proportion of body-weight were increased by fishmeal, suggesting that protein deposition had been enhanced. No further increase in the proportional muscle weights was obtained with oestradiol. Muscle dry matter content tended to be increased in both implanted and non-implanted animals receiving fishmeal compared with controls, but the proportions of protein, fat and ash were relatively constant. The intramuscular lipid composition was slightly altered by fishmeal. Muscle protein fractional synthetic rates (FSR), measured by continuous infusion of [3H]tyrosine, were increased by fishmeal in all three muscles of both implanted and non-implanted animals. There were no differences, however, due to oestradiol, over non-implanted fishmeal animals. This suggests that oestradiol may increase muscle accretion by reducing protein degradation rate. Cimaterol significantly increased longissimus dorsi (P less than 0.05) and vastus lateralis (P less than 0.01) muscle weights but had no effect on semitendinosus muscle weight or live-weight gain. The proportion of protein was increased (P less than 0.001) and the fat content reduced (P less than 0.05) in all three muscles but intramuscular lipid composition was not markedly affected. Whilst methylhistidine: creatinine excretion was reduced by cimaterol, FSR were increased in the l. dorsi and v. lateralis muscles suggesting beta-agonists have effects on both protein synthesis and protein degradation.