Visualization of chromosome assembly during the S and G2 stages of the cycle and chromosome disassembly during the G1 stage in semithin sections of mouse duodenal crypt cells and other cells

Previous examination of dividing cells in the isthmus of the mouse pyloric antrum by using semithin (0.5-micron-thick) Epon sections revealed that the prophasic condensation of chromosomes began early in the DNA-synthesizing (S) stage. In order to examine whether the same observation could be made in other proliferating cell types, the crypt base columnar cells in mouse duodenum and the hepatocytes of the rat 48 hr after partial hepatectomy were investigated by morphologic and radioautographic techniques. When crypt base columnar cells were studied in semithin Epon sections, the four phases of mitosis showed the characteristic features described by classical cytologists. Moreover, the proportion of cells in prophase and telophase was high. To relate the mitotic phases to the stages of the cell cycle, the "frequency of labeled mitoses method" provided the duration of the cell cycle, 12.3 hr, and of the S stage, 7.3 hr. From the frequency of the occurrence of mitotic phases, it was estimated that metaphase lasted 0.3 hr and anaphase 0.11 hr, in line with previous estimates. However, the durations of prophase and telophase were long, 5.9 and 1.9 hr, respectively. The whole mitotic process took over 8 hr. From the duration of prophase and cycle stages, it was calculated that 67% of the S stage was occupied by prophasic cells. In fair agreement with this estimate, 68% of the labeled cells 10 min after a 3H-thymidine injection were found to be in prophase. In regenerating hepatocytes, the morphological features and frequency of prophase and telophase cells were similar to those observed in duodenal crypt cells. While the cycle time was not measured and, therefore, the duration of cycle stages and mitotic phases could not be estimated, it is likely that their duration would be of the same order of magnitude. In conclusion, the mitotic process in duodenal crypt cells takes over 8 hr. Moreover, the crypt cells, like antral isthmal cells, show features of early prophase soon after they enter the S stage of the cycle.     

Ultrastructure of the DNA-synthesizing cells of the sinoatrial node in mouse embryos according to electron microscopic autoradiographic data

By means of electron microscopic autoradiography with 3H-thymidine a study was made of the differentiation degree of DNA synthesizing muscle cells in the sinoatrial node (SAN) of the heart conductive system of the 18 day old mouse embryos. Clear myocytes (CM), predominating in the SAN at this stage, are irregular in shape, with interdigitating protrusions. Nuclei are clear, spherical or ellipsoidal. One hour following 3H-thymidine injection, about 6% of CM display labeled nuclei; this index is considerably lower than in working ventricular myocardium. Like unlabeled myocytes, CM being in phase S contain sparse, randomly located thin myofibrilles. In some areas of the sarcoplasm, only myofilament bundles and Z-disk material can be seen. The number of CM myofibrilles is always considerably less than in the working ventricular myocytes. Accumulations of intermediate (8--11 nm) filaments are present. Mitochondria with a few cristae are not numerous. The sarcoplasmic reticulum and Golgi apparatus being relatively well developed, multivesicular bodies, centrioles, and occasional cilia are often seen. Near the centrioles (basal bodies), striated filamentous bundles are found sometimes showing periodic dense lines separated by 50--70 nm. Specialized contacts between CM are rare, being presented only by desmosomes and primitive intercalated discs. Besides CM, sparse small dark cells occur filled with myofibrilles and mitochondria. In the peripheral regions of the node "transitional" cells are seen. The SAN of the 18 day old embryo mouse heart grown due to proliferation of CM with a poorly developed myofibrillar apparatus.     

An electron microscopic study on the interstitial cells of the gizzard in the love-bird (Uroloncha domestica)

Summary A type of cells morphologically resembling fibroblasts or Schwann cells and identical with the interstitial cells as firstly described byCajal was studied electronmicroscopically.Examinations of serial sections reveal that cell membranes of these cells make close appositions (nexus) to those of all surrounding smooth muscle cells. The surfaces of these cells are also provided with nerve endings of certain axons derived from plexus myentericus.On the basis of these findings, the possible nature and function of interstitial cells are discussed.This work was supported in part by NIH Grant NB-06052.     

An electron microscopic study of the transport of peroxidases in the endothelium of mouse aorta

Summary Aortic endothelium presents a continuous barrier to diffusion of macromolecules. The cell margins overlap for long distances and there are multiple points of contact between the cell membranes at which the intercellular cleft is reduced to 30–40 Å or less, and free diffusion of lanthanum is impeded at some points of apposition. Macromolecular transport through the endothelium of mouse aorta was studied with the help of horseradish peroxidase (HRP) and bovine milk lactoperoxidase. Following injection of 0.25–0.5 mg of HRP no tracer was detected in the intercellular clefts even though it was seen in plasmalemmal vesicles and subendothelial space. However, when 5 mg of HRP was injected in either 0.05 or 0.5 ml of saline, transport of the enzyme occurred through both the intercellular clefts and via the plasmalemmal vesicles. On the other hand, lactoperoxidase of m.w. 82000 was transported through the plasmalemmal vesicles only. The findings were discussed with reference to the transport of serum lipoproteins and it was suggested that low and high density lipoproteins would be transported via the plasmalemmal vesicles.The excellent technical help of Miss R. Ben-Moshe and Mrs. A. Mandeles is gratefully acknowledged. This study was supported in part by a grant from the Myra Kurland Heart Fund, Chicago, Ill., and by a grant 06-101-1 of the National Institute of Health, United States Public Health Service.     

An electron microscopic and histochemical study of the secretory cells inHydra viridis

Summary The fresh water coelenterateHydra viridis possesses a unique distribution of mucous and serous secretory cells in the gastrodermis. The mucous cells are found only in the hypostome, a region devoid of the serous zymogen cells. On the other hand, the zymogen cells are found extending from the tentacles to the peduncle. Histochemical stains indicated that the two hypostomal mucous cells, spumous and granular, secreted an acid mucopolysaccharide, and incorporated radiosulfate. The radiosulfate label was not sensitive to hyaluronidase digestion, but was removed by acid methanolysis. In contrast, the secretory product of the zymogen cell was rich in proteins and a PAS-positive moiety (unsulfated).The ultrastructure of these cells was correlated with their histochemical staining properties. It was demonstrated that glutaraldehyde preserved the ultrastructure of the secretory granules better than osmium, and also preserved more components within the granules. The mucous cell granules contained an electrolucent and an electron dense component. The cells were both PAS-positive and alcianophilic. After osmium fixation the dense component was lost and the cells were primarily alcianophilic. Osmium also failed to preserve the electron dense component in the zymogen cells.Observations of corresponding thick and thin sections showed a cell containing granules similar to the granules seen in mouse Paneth cells. The dense core was osmiophilic and the lighter halo was alcianophilic.These results lead us to conclude that the electrolucent filamentous component is an alcianophilic acid mucopolysaccharide and the dense granular component is probably a PAS-positive material.

---

Zusammenfassung Der FrischwassercölenteratHydra viridis weist eine einzigartige Verteilung von mukösen und serösen sekretorischen Zellen in der Gasterodermis auf. Die mukösen Zellen finden sich nur im Hypostom, in welchem seröse Zymogenzellen fehlen. Die Zymogenzellen andereseits finden sich von den Tentaklen bis zum Pedunkulus. Histochemische Methoden zeigten, daß die zwei Typen hypostomaler muköser Zellen, d.h. spumöse und granuläre, ein saures Mukopolysaccharid ausscheiden und radioaktives Sulfat einbauen. Der Radiosulfat-Markierer war nicht sensitiv gegenüber Hyaluronidase, konnte aber entfernt werden mit saurem Methanol. Im Gegensatz dazu war das Produkt der Zymogenzellen reich an Proteinen und enthielt PAS-positives Material.Die Feinstruktur dieser Zellen war korreliert mit diesen histochemischen Befunden. Glutaraldehyd erhielt die Feinstruktur der Sekretgranula besser und fixierte mehr Komponenten als Osmium. Die Granula der mukösen Zellen enthielten elektronendichte und -durchsichtige Komponenten; diese Zellen färbten sich mit PAS und Alcyan. Nach Osmium-Fixierung war die elektronendichte Komponente abwesend und die Zellen waren hauptsächlich alcyanophil. Auch in den Zymogenzellen vermochte Osmium nicht, die elektronendichte Komponente zu erhalten. Beobachtungen an alternierenden dicken und dünnen Schnitten zeigten eine Zelle mit Körnern ähnlich den Granulen von Maus Paneth-Zellen. Das dichte Zentrum dieser Granula war osmiophil, der hellere Halo alcyanophil.Diese Resultate lassen uns schließen, daß die elektronen-durchsichtige filamentöse Komponente ein alcyanophiles Mukopolysaccharid ist; das dichte, zentrale Material ist wahrscheinlich PAS-positiv.

---

---

This paper was prepared from a thesis submitted in partial fulfillment for the degree of Master of Arts.

---

This work was supported by the National Institutes of Health grant no. GM-11218.

---

I wish to thank Dr.Marcus Singer for permission to use the E. M. facilities in the Dept. of Anatomy, Case Western Reserve University, and Dr.Joseph A.Grasso for instruction in the techniques of electron microscopy and the use of his facilities.     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.