RESUMO
Neural crest cells (NCC) segregate from neural folds, swim through extracellular matrix, and differentiate in several cell types. During in vivo and in vitro dispersion, NCC display a substantial proliferative rate. Here, we have evaluated this biological behavior under the influence of different substrates. NCC exhibit the highest proliferative activity on fibronectin substrate, with shortening of the G1 period. This fact is reverted by specific antibody pre-treatment of the substrate. Taking into account that NCC migrate along narrow and restricted pathways rich in essential fibronectin, with high proliferative rates, these results indicate that the fibronectin pathway contributes to increase the cell number, leading to a high population pressure that could participate in the early dispersion of NCC
Assuntos
Animais , Embrião de Galinha , Células Cultivadas , Fibronectinas , Fase G1 , Fase G2 , Crista Neural , Fase SRESUMO
Neural crest cells (NCC) segregate from neural folds, swim through extracellular matrix, and differentiate in several cell types. During in vivo and in vitro dispersion, NCC display a substantial proliferative rate. Here, we have evaluated this biological behavior under the influence of different substrates. NCC exhibit the highest proliferative activity on fibronectin substrate, with shortening of the G1 period. This fact is reverted by specific antibody pre-treatment of the substrate. Taking into account that NCC migrate along narrow and restricted pathways rich in essential fibronectin, with high proliferative rates, these results indicate that the fibronectin pathway contributes to increase the cell number, leading to a high population pressure that could participate in the early dispersion of NCC
Assuntos
Animais , Embrião de Galinha , Células Cultivadas , Fibronectinas/farmacologia , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Crista Neural/citologia , Fase S/efeitos dos fármacosRESUMO
The dynamic parameters of mouse sperm cells exposed to follicular and oviductal fluids were assessed. Spermatozoa were tracked on a chemotactic Zigmond chamber and recorded using a videomicroscopy system. The results were evaluated with computer-supported image analysis. Follicular fluid at a dilution of 10(-4) markedly increased the proportion of spermatozoa with high velocity, and stimulated chemotactic behaviour. The highest velocities were observed in sperm cells exposed to oviductal fluid, and a greater proportion of these cells had high velocity compared with those exposed to follicular fluid. Chemotaxis was induced in spermatozoa exposed to oviductal fluid at dilutions of 10(-3) and 10(-5). These results suggest the presence of temporal subpopulations of responsive spermatozoa, considering the distance travelled towards both follicular and oviductal fluids and the proportion of sperm cells migrating towards the gradient in the highest distance ranges. This is the first report on the effect of isolated follicular and oviductal fluids on dynamic parameters and chemotaxis of mouse spermatozoa. The findings support previous work showing that the motility and directionality of mouse sperm cells is increased by factors in the microenvironment of the egg. Although the significance of these factors in vivo is unknown, it is possible that there is a relay mechanism involving sequential activity of both oviductal and follicular fluids to direct the male gametes towards the egg.
Assuntos
Quimiotaxia/fisiologia , Tubas Uterinas/metabolismo , Líquido Folicular/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Animais , Líquidos Corporais/fisiologia , Feminino , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de VídeoRESUMO
Neural crest cells (NCC) segregate from neural folds, swim through extracellular matrix, and differentiate in several cell types. During in vivo and in vitro dispersion, NCC display a substantial proliferative rate. Here, we have evaluated this biological behavior under the influence of different substrates. NCC exhibit the highest proliferative activity on fibronectin substrate, with shortening of the G1 period. This fact is reverted by specific antibody pre-treatment of the substrate. Taking into account that NCC migrate along narrow and restricted pathways rich in essential fibronectin, with high proliferative rates, these results indicate that the fibronectin pathway contributes to increase the cell number, leading to a high population pressure that could participate in the early dispersion of NCC.
Assuntos
Fibronectinas/farmacologia , Fase G1/efeitos dos fármacos , Crista Neural/citologia , Animais , Células Cultivadas , Embrião de Galinha , Fase G2/efeitos dos fármacos , Fase S/efeitos dos fármacosRESUMO
Neural crest cells (NCC) segregate from neural folds, swim through extracellular matrix, and differentiate in several cell types. During in vivo and in vitro dispersion, NCC display a substantial proliferative rate. Here, we have evaluated this biological behavior under the influence of different substrates. NCC exhibit the highest proliferative activity on fibronectin substrate, with shortening of the G1 period. This fact is reverted by specific antibody pre-treatment of the substrate. Taking into account that NCC migrate along narrow and restricted pathways rich in essential fibronectin, with high proliferative rates, these results indicate that the fibronectin pathway contributes to increase the cell number, leading to a high population pressure that could participate in the early dispersion of NCC.
RESUMO
The aim of this work was to evaluate the period of time required for the induction of changes in motility of mouse spermatozoa in response to factors from the microenvironment of oocytes. To determine the effects of the latency time, the period of preincubation before contact with the oocyte product(s), sperm samples were incubated for 15 or 90 min and then exposed to either Dulbecco's Modified Eagle's Medium (DMEM) or to a crude extract of superovulated oocytes (CE). The assays were performed in a Zigmond chamber by filling one compartment with either DMEM or CE, and the other compartment with the sperm suspension. A videomicroscopy system was used for tracking the spermatozoa. Sperm motility analysis was assessed using a semiautomatic objective method, and the following parameters were determined; (1) dynamic parameters: curvilinear velocity, linear velocity and linearity; (2) progressive motility: percentage of spermatozoa showing either circular or linear patterns of movement; and (3) directional motility: percentage of spermatozoa that moved towards either the DMEM or the CE. The results of this work showed that when the spermatozoa contacted soluble factors in CE after only 15 min of previous incubation, there was a significant increase in their dynamic parameters, change in their progressive motility, and induction of directional movement of the spermatozoa towards the CE components, while a longer period of preincubation did not significantly modify these effects. On the other hand, in the presence of culture medium (with or without addition of bovine serum albumin), the spermatozoa needed a more extended period of incubation to significantly increase their dynamic parameters and to modify their progressive motility, while maintaining a random direction of movement.
Assuntos
Oócitos/fisiologia , Motilidade dos Espermatozoides/fisiologia , Interações Espermatozoide-Óvulo , Espermatozoides/fisiologia , Animais , Quimiotaxia/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: In vertebrate embryos, migration of trunk neural crest cells (NCC) proceeds mainly in two streams: a dorsoventral path between the neural tube and somites, and a dorsolateral one between somites and ectoderm. This last pathway is taken by melanocyte precursor cells (MPC) homing the skin, while pigment cells seeding internal organs and the peritoneal wall follow the dorsoventral pathway. Early routes taken by subpopulations of NCC have been well documented using the quail-chick chimaera system and monoclonal antibodies to NCC. However, very little is known about the advanced migratory behavior of MPC, which determines their late distribution patterns at different embryonic axial levels. METHODS: Histological sections of neck, thorax, and abdomen of 6.5 to 9 day quail embryos submitted to DOPA reaction (tyrosinase activity) were used. In four concentric areas--dorsal and ventrally subdivided--the relative density of MPC was determined by morphometric methods. RESULTS: The relative regional density of MPC from their individualization as DOPA-positive putative pigment cells until their definitive seeding in the epidermis showed a progressively higher cell density from deeper to peripheral zones in all three levels studied, with peaks of cell density suggesting a centrifugal pattern occurring in at least two waves of migratory cells. CONCLUSIONS: The spatial distribution of the MPC varies according to both the axial level and the developmental stage of the embryo. Furthermore, the general pattern of centrifugal distribution observed might be attributed to a different timing of cell differentiation closely related to their migratory behavior.
Assuntos
Coturnix/embriologia , Melanócitos/citologia , Crista Neural/citologia , Células-Tronco/citologia , Animais , Contagem de Células , Diferenciação Celular , Movimento Celular/fisiologia , Di-Hidroxifenilalanina/metabolismo , Embrião não Mamífero , Melanócitos/fisiologia , Monofenol Mono-Oxigenase/metabolismo , Crista Neural/fisiologia , Células-Tronco/fisiologiaRESUMO
A method is presented for softening the hard tissues of stem and strobile of Equisetum giganteum allowing the preparation of representative histological slides suitable for teaching and research. Segments of aerial portions of the Equisetum giganteum shoot system were fixed with formaldehyde:ethanol:acetic acid (5:10:5) for 24 hr at room temperature, washed in distilled water and immersed in a mixture of 5% hydrofluoric acid and 0.5% sulfuric acid for 1 hr at room temperature. Hydrofluoric acid has a higher affinity for silica components, and the sulfuric acid acts as a catalyst favoring the separation of calcium silcates. This simple, inexpensive and rapid method allows paraffin sections to be prepared while preserving the topographic microanatomy by decreasing technical artifacts produced by conventional softeners, and preserving PAS-positive polysaccharides.
Assuntos
Inclusão em Parafina/métodos , Plantas/ultraestrutura , Manejo de Espécimes , TensoativosRESUMO
We have previously demonstrated that directional migration of neural crest cells (NCC) is associated with a high cell density, resulting from an active cell proliferation. It is also known that treatment with retinoic acid (RA) causes a dose-dependent inhibition of proliferation of some cell types, and that administration of RA during the early stages of embryonic development, induces cranio-facial abnormal patterns corresponding to NCC derivatives. In view of these findings, it was of interest to determine if exogenous RA is a potential modulator of the mitotic rate of NCC, and to explore the hypothesis of an inhibitory effect exerted by RA on the proliferative behaviour of NCC in vivo and in vitro. Homogenates of RA-treated chick embryos showed a low [3H]dT incorporation, indicating a generalized diminution of DNA synthesis. The labelling index (LI = number of labelled cells/total number of cells) revealed that NCC from RA-treated and control embryos had higher values of [3H]dT incorporation than neural tube cells (P < 0.0001). Autoradiographs of RA-treated chick embryos showed a significantly lower [3H]dT incorporation in NCC at the prosencephalic and mesencephalic levels, as well as in the neural tube cells at the prosencephalic, mesencephalic and rhombencephalic levels, than in control chick embryos (P < 0.0001). NCC cultures treated with 1 or 10 microM RA had a significantly lower LI than in cultures treated with 0.1 microM RA or control cultures (P < 0.04). In chick embryos, the mitotic index of NCC was 0.026 for RA-treated and 0.033 for controls, while the duration of the cell cycle was significantly longer in the NCC of RA-treated embryos (approximately 40 h) than in controls (approximately 25 h). The length of the cell cycle phases of NCC was similar in both experimental conditions, except for G1 phase, which was significantly longer in the RA-treated group than in controls. These results show that RA blocks DNA synthesis and lengthens the proliferative behaviour of NCC both in early chick embryos and in vitro, effects that could modify the morphogenetic patterns of NCC distribution through a decreased cell population.
Assuntos
Divisão Celular/efeitos dos fármacos , Crista Neural/citologia , Tretinoína/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , DNA/biossíntese , Inibidores do Crescimento/farmacologiaRESUMO
A method is presented for in situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions "in ovo." The chick embryo is placed in a "shell-less" culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo+membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.