In vitro Uptake of Exogenous α-Fetoprotein by Chicken Dorsal Root Ganglia

Abstract

Exogenous chicken α-Fetoprotein (AFP) was added to embryonic chick dorsal root ganglia plated on gelatin-coated tissue culture dishes at different stages during the differentiation process and its intracellular uptake demonstrated by immunocytochemical methods. Other embryonic serum proteins were added as a control. Morphologically well-differentiated neurons (ganglion cells) appeared positively labeled for AFP, contrasting with weakly stained other cell types: these included spindle-shaped cells identified as Schwann cells and larger, often closely packed cells, which could be less mature neurons. Fibroblasts were found negative or faintly stained. No AFP was noticeable in cultures grown in the absence of the protein. These results suggest that the presence of AFP in the developing CNS is for the most part, if not entirely, due to protein uptake as opposed to in situ synthesis.

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“…in fetal stages of the developing central nervous system [55] or in mesenchy mal cells of the developing kidney [23], In addition, it may be added that the expression of AFP-and SAmRNAs by some fetal tissues, other than the liver, is not a common phenomenon [22], Incubation of S and F cells with exogenous AFP showed no differences in the uptake of the protein as detected by immunostaining (a similar observation in S cells and fibroblasts of cultured chicken root ganglia was reported earlier [14]). Nevertheless, when the intensities of staining (measured by the end points of staining) of AFP-incubated cells were separately compared with their non-incubated counterparts, some differences were ob served.…”

“…With regard to endogenous AFP and exoge nous AFP uptake used as differential markers, more complex mixed cultures containing S, F and additional neuronal cells can be analyzed, e.g., organized peripheral nerve primary cell culture or, as described earlier, chicken spinal ganglia cultures [14], Similarly, mouse neuroblastoma cells [15], fetal mouse or chicken primary neuronal cells [13,14] can also be studied using this approach. It is necessary to add that, in contrast to neu ronal cultured cells, the glial cells do not seem to internal ize AFP in vitro [13,14,29]; the cultured glial-like cells accompanying neuronal cells internalize AFP, although relatively weakly [13,14]. In this work, accessory cul tured glial cells, primary astrocytes and a glioma cell line, studied by incubating with FITC-AFP, sometimes pre sented a very weak and irregular labelling (both types of culture were GFA protein-positive).…”

“…After a 48-hour incubation, the medium was removed and the plates incubated for 2-3 h in serum-free medium to deplete cells of endogenous bovine AFP. Fresh medium containing 100 pg/ml of the following purified native proteins or their FITC conjugates [16] were then added (1 ml/plate): mouse or rat AFP [13]; mouse or rat SA as positive control [14,15], or ovalbumin and lysine as negative controls [15,16]. Moreover, FITC conjugates of bovine AFP and SA were tested.…”

“…In both cases, the cultures treated with native AFP or with FITC-AFP, we used as a positive control of AFP uptake C-1300 mouse neuroblastoma cells [14,45] and B-104 rat neuroblastoma cells (gift of L. Culp, CWRU, Cleveland, Ohio). These poorly differentiated cells do not show the presence of endogenous AFP, using anti-AFP antibodies, but internalize AFP in vitro [15].…”

“…The technique of AFP uptake, in which the cultures are maintained without serum for only about 4 h, was described earlier using different cell culture systems [13][14][15][16][17] also containing F and S cells [14]. In the present work, both S and F cells were able to take up the native AFP protein; on the contrary, after incubation of the cultures with FITC-AFP, only F cells and not S cells gave The results of microfluorescence quantitation are expressed as the average ± SD of the fluorescence intensity and of dispersion index (300 fields of 10 pm2 of each S and F culture were consid ered).…”