ABSTRACT
In a recent paper, Gomori (1941) has described a histochemical method for the localization of phosphatase in animal tissues. The method depends on the fact that the enzyme is not destroyed by moderate treatment with 80 % alcohol, in which the tissues are fixed. After fixation the tissues are sectioned; the sections are incubated with sodium glycerophosphate and calcium chloride in buffered-solution and calcium phosphate is deposited at the site of phosphatase activity. This can be rendered visible by treatment with cobalt nitrate solution followed by ammonium sulphide, when the site of the phosphatase is marked by a black precipitate. By the use of such methods he has described, among other things, the occurrence of phosphatase in capillary endothelial cells in different animals, not with absolute regularity, but in a large number of cases. In those instances in which a positive reaction was not obtained it was shown to be an idiosyncrasy of the particular tissue rather than a failure of the method. It seemed possible therefore that an attempt to separate endothelial cells from other mesenchymal cells growing in vitro might be assisted by the use of this method.
Hanging-drop tissue cultures were made from 8 or 9-day chick embryos and heart tissue was selected for its content of endothelium. The tissues were explanted into a fluid medium consisting of the exudate derived from a clot formed from equal parts of cock plasma and embryo extract. In this medium heart tissue grows very well as a single layer of cells on the surface of the coverslip. The outgrowth is undoubtedly mixed and shows regions of fibroblast-like growth and others where structures resembling endothelial membranes are formed. Myoblasts, fibroblasts, endothelial cells, histiocytes and undifferentiated mesenchyme cells are among the constituents which may take part in the formation of the outgrowth where they are in an ideal condition for histological examination.
Such cultures were grown for 24 or 48 hr. and then washed in Ringer solution to free them from the protein-containing medium. They were then fixed in 80 % alcohol for from4to 24 hr., brought down towaterand then divided into three groups. The cultures in group 1 were incubated in water for hr. at 37o C. Those in group 2 were incubated for the same time in calcium chloride and sodium barbital, while those of the third group were incubated in sodium glycerophosphate, sodium barbital and calcium chloride. The actual solutions used were:
At first sight the results on tissue cultures appeared to be disappointing. Most of the cells in group 3 cultures were browner than those in group 2, but there was no one class of cells which constantly showed any markedly positive reaction. In those cells which had well-developed centrospheres, the centrosphere was notice-ably brown in appearance, but there was no indication that any particular cell type contained any quantity of active phosphatase. However, an examination of the cells which were undergoing mitosis at the time of fixation brought out some interesting points.
In the accompanying illustrations are shown different stages of mitosis as seen in cultures incubated with calcium chloride alone (group 2) which show the distribution of existing phosphates, and also as seen in similar cultures incubated with sodium glycerophosphate as well (group 3), indicating the presence of phosphates produced during incubation and therefore probably due to the action of phosphatase. It is quite clear that the chromosomes have become heavily coated and that the cytoplasm of the dividing cells also shows a conspicuous darkening. The cell membrane appears to be separated from the cytoplasm by a clear and colourless area which does not appear in group 2 cultures and seems to be due, in part at least, to an impregnation of the cell surface which does not seem to occur in control cultures. Whether there is actually an ectoplasm and an endoplasm in these cells is not certain; it may well be an artefact brought about by the alcohol fixation and the subsequent treatment, but there can be little doubt that the phosphatase reaction shows up the chromosomes, the inner mass of cytoplasm, the centriole sometimes, and the cell membrane of the dividing cell. In most resting cells the nucleoli show some tendency to be blackened, but otherwise the cells remain uniformly negative, except as already stated for some conspicuous centrospheres.
It is tempting to connect the phosphatase activity here described with the reorganization of the nucleic acid and nucleoprotein content of the cell which takes place at mitosis, but it remains for future work to elucidate the true significance of its occurrence. Living cultures incubated with a drop of 0·2 % sodium glycerophosphate in Ringer solution added to a drop of the usual exudate medium grew well, but on subsequent fixation and treatment with cobalt nitrate and ammonium sulphide they showed no difference from the controls, so that, as might be expected, the accumulation of calcium phosphate which occurs in the fixed cells is an artificial phenomenon, and normally the final product of the phosphatase activity is not calcium phosphate, whatever else it may be.
SUMMARY
The chromosomes of dividing cells give a strongly positive reaction for the presence of phosphatase.
ACKNOWLEDGEMENTS
The experiments reported in this paper were carried out with the help of funds received from the British Empire Cancer Campaign, to whom the author wishes to record his gratitude.
REFERENCE
EXPLANATION OF PLATE 1
Plate I
On the left, Figs. 1-6a show drawings of cells as seen after incubation with calcium chloride and subsequent treatment for the demonstration of phosphates. Controls.
On the right, Figs. I-VI show drawings of cells as seen after incubation with calcium chloride and sodium glycerophosphate, followed by treatment for the demonstration of phosphates. Positive phosphatase reaction.
The numbers indicate corresponding stages of cell division. 1 and I, Resting cells, a and II, Early prophase. 3 and III, Illa, Late prophase. 4 and IV, Metaphase. 5 and V, Anaphase. 6, 6a and VI, Telophase.
On the left, Figs. 1-6a show drawings of cells as seen after incubation with calcium chloride and subsequent treatment for the demonstration of phosphates. Controls.
On the right, Figs. I-VI show drawings of cells as seen after incubation with calcium chloride and sodium glycerophosphate, followed by treatment for the demonstration of phosphates. Positive phosphatase reaction.
The numbers indicate corresponding stages of cell division. 1 and I, Resting cells, a and II, Early prophase. 3 and III, Illa, Late prophase. 4 and IV, Metaphase. 5 and V, Anaphase. 6, 6a and VI, Telophase.
