Alternatives to Classical Mitosis in Hemopoietic Tissues of Vertebrates

INTERNATIONAL REVIEW OF CYTOLOGY, VOL 60

Alternatives to Classical Mitosis in Hemopoietic Tissues of Vertebrates VIBEKEE. ENGELBERT The Ramsay Wright Zoological Laboratories, University of Toronto, Toronto, Canada I. Introduction

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11. Imprint, Smear, Fixation, and Staining Methods

111. IV. V. VI. VII. VIII. IX.

X. XI. XII.

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Methods Using Tritiated Thymidine . . . . . . . . . . . Fluorescence Method for DNA and RNA . . . . . . . . . Tissue Culture in Vitro of Hemopoietic Tissues . . . . . . . Behavior and Morphological Variations in Blast Cells and Their Nuclei . . . . . . . . . . . . . . . . . . . . . . The Occurrence and Significance of Two Spatially Separate Nuclear Masses in Blast Cells and in Differentiating Cells . . . . . . Fate of the Peripheral or Shell-like Nuclear Mass, and the Inner Nuclear Mass, in Differentiating Cells of Leukemic Mice (AKR Strain) . . . . . . . . . . . . . . . . . . . . . . Results following Injection of Tritiated Thymidine . . . . . . A . Reassociation of Vesicles and Nuclear Granules in Rabbit Spleens . . . . . . . . . . . . . . . . . . . . B. Grain Counts in Vesicular Nuclei of Rabbit Spleen and Bone Marrow . . . . . . . . . . . . . . . . . . . . Erythropoiesis in Blood of Vertebrates with Nucleated Erythocytes. Formation of Clone Cells from Nuclei of Young Mature Erythrocytes Summary . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

93 96 98 98 99 100 105

107 109 109 111

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117 117 I I8

I. Introduction Cell reproduction in hemopoietic tissues of vertebrates, and especially of mammals, has for many years occupied research workers in the biological sciences. When one is concerned with cell proliferation, one naturally looks for the occurrence of mitosis. This process, which is so easily seen in teaching material such as blastulas of fish or onion root tips and in human blood when it is cultured in vitro with mitosis-stimulating substances, is difficult to find in slide preparations of hemopoietic tissues from healthy humans or other animals. 93

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Many early hematologists, as well as later ones, attempted to find a “blood mother cell,” a hemocytoblast. Downey (1932, 1938) defined the myeloblast of Naegli as “the undifferentiated non-granular, lymphoid stem cell” of the red bone marrow which functions as the indifferent, polyvalent parent cell of all the “myeloid” elements. Downey stated further that “the hemocytoblast of Maximow and Danchakoff and the lymphoid hemoblast of Jordan and Latta are polyvalent large lymphocytes. A cell of myeloblastic structure is not recognized. The hemocytoblast of Ferrata is identical with Naegli’s myeloblast, but it may produce lymphocytes when in lymphoblastic function and it occurs in normal lymphatic tissue as well as the marrow. The important point here is the recognition of a stem cell that is lymphoid in character, that is polyvalent, and that occurs in normal lymphatic tissue as well as in bone marrow. Jordan (1938), after describing the comparative hematology of lower vertebrates, writes in “Terminology of Lymphoid Cells, “In the foregoing pages the terms hemocytoblast, hemoblast, lymphoid hemoblast and lymphocyte were used synonymously as indicating functional identity of morphologically variable multipotential blood stem cell. Morphologically the hemocytoblast appears very different from the smaller lymphocytes. However, both have identical capacities to develop into erythrocytes, thrombocytes, monocytes and granulocytes, the specific route of differentiation being presumably determined by the impingement of specific differential stimuli. ” In the same section Jordan writes later, “The typical hemocytoblast is a relatively large cell, with large vesicular nucleus and a moderately basophilic cytoplasm. The most distinctive features concern the arrangements of the chromatin in the form of minute granules, delicate nuclear membrane and the presence of one or several nucleoli usually achromatic. But it must be emphasized that the cytologic features of a hemocytoblast are not static; the cytology is subject to considerable variation, allowing for such variability, a concomitant of simple metabolism the large lymphocyte has an identical morphology.” He claims that mitosis occurs only in the large- and medium-sized cells in lymphoid nodules of chicken bone marrow. In numerous illustrations of blood cells he shows only three mitotic figures of hagfish “normoblast,” one toad lymphocyte, and one toad erythroblast. He labels three lymphocytes exhibiting slight size differences as “hemocytoblasts. ” It is just a little over 200 years since lymphocytes from lymph nodes were first described and illustrated by Hewson (1777) who called them “cells” some 60 years before the cell theory of Schwann (1839) was presented. Lymphocytes were first defined as a distinct cell type in blood and lymph by Jones (1846) at Charing Cross Hospital, London. Their motility was described by Ranvier (1873, Arnold (1887), and Askanazy (1905). Maximow (1909) considered the lymphocyte as the common mother cell of the different elements of the blood both in the embryo and in the postfetal life of mammals. Yoffey (1932-1933) and Jordan (1935) believed that the circulating lymphocytes lodge in the bone marrow, where they transform into erythrocytes. ”

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Kindred (1938, 1940, 1942) published quantitative studies of hemopoietic organs of young albino rats. Although he quotes percentages of cells in mitosis, his plates do not illustrate this process. Andreasen (1959) reported “mitotic figures counted in suspensions of cell nuclei prepared from the whole organ. The counting included all phases of mitosis and differential counting of the mitotic phases is possible,” and “that under certain conditions the lymphoid tissue was characterized by high mitotic rates, whereas under other conditions the same tissue was marked by just the opposite process, namely degeneration of lymphocytes combined with phagocytic activity carried out by the reticulum. Hamilton (1954) and Hill (1959) both believed that lymphocytic nucleoprotein was reutilized. Trowell (1957) claimed that a process of phagocytosis of pycnotic lymphocytes was an important link in the “re-utilization. ” Yoffey et al. (1958), after labeling lymphocytes of guinea pigs with tritiated thymidine, concluded that lymphoid tissue shows active synthesis of DNA. However, their results did not support the concept “either of massive re-utilization or large scale recirculation. ” Counts of blood cells at different time intervals were done by many workers. Yoffey er al. (1958) reported counts of the number of lymphocytes “in the cellular migration stream,” especially in the thoracic duct. In this way they dealt with “the high level of lymphocyte production. This group used tritium-labeled thymidine, as “this is believed to be rapidly and specifically incorporated into newly formed DNA, and in view of its precise localization in radioautographs seems to be suitable for study of cell production.” They reported 1.83 to 6.57% labeled cells depending on the time after injection of the tritiated thymidine that the counts were made. Trowell (1958) writes, “there is little doubt that the small lymphocyte originates by mitosis, followed by a shrinkage type maturation from the medium and large ones. ” The process was followed in cultures of thoracic duct lymph by Hall and Furth (1938). In similar experiments Gowans (1957) found that the daughter cells produced by mitosis were, initially at any rate, rather larger than small lymphocytes. “The medium and large lymphocytes are actively mitotic but the small lymphocytes never or rarely divide.” Trowell states further, “my own experience in a variety of species, has been that the small lymphocytes in the intestinal crypt epithelium are the only ones which can ever be found in mitosis. Very rarely we have seen mitosis of a small lymphocyte in rat lymph-node cultures. Dustin (1959) discussed mitotic growth in bone marrow of the rat by the stathmokinetic (colchicine) method, in which metaphases were believed to be arrested and thus could be counted to give an estimate of mitotic activity. We have prepared spleens of newborn mice with the Feulgen (Schiff) method and gentle squash technique (Engelbert, 1960, 1961). The very brilliantly colored “chromosomal” bodies which are found in very large numbers in configurations similar to metaphases do not appear to us as typical metaphases. Configurations similar to anaphases are never seen. The chromosomal ‘‘meta”

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phase” separates out into three, four, or more chromosomal groups, which remain connected; ring- or band-shaped nuclei are also found. These nuclear stages, we believe, are young stages of polymorphonuclear leukocytes. J. H. Morrison and G. B. Wilson (private communication, 1958) reported that they had treated spleens of 3-day-old rats with the Feulgen squash method. They too found no anaphases and believed that metaphase chromosomes passed directly into the telophasic state shortly to form band-shaped nuclei of young neutrophilic leukocytes. Studies in our laboratory using in vitro preparations without mitotic stimulants and a medium of calf serum and synthetic medium 1066 (Connaught Laboratories), or the animals’ own blood, were made on lymphoid tissue of normal mice with phase-contrast and cinematographic recording. Metaphase figures entering anaphase were not seen as most of the chromosomes fused forming a ringshaped nucleus with cytoplasm. In contrast, when we cultured lymphoid tissue from leukemic mice (AKR) in vifro the metaphases entered anaphase in most cases (Engelbert, 1968). Wintrobe (1967) quotes studies of bone marrow from nine healthy males in which 8.86 mitoses per 1000 cells were found, i.e., a mitotic index of 0.9%. Diggs et al. (1957) claimed that if hemopoietic tissues showed mitotic figures greater than 1% it is indicative of abnormal cell production. Classical mitosis thus presents a considerable puzzle to workers interested in the reproduction of blood cells. Experiments carried out in our laboratory with injections of tritiated thymidine showed that out of 10,000 labeled cells counted in spleens and bone marrow, only one well-labeled anaphase figure was present (Engelbert, 1967). Westermann (1974) identified and followed the developmental series of thrombocytes in four species of turtle. She states “while other (cell) types were sometimes found in mitosis no division stages were ever observed in thrombocytes. ” Wedlock (1974) reported that spleen imprints of chicks at about 16 days of incubation showed all stages of mitosis in erythrocytes. The same author injected chicks with bacteriophage OX174 at the time of hatching and at 4, 8, and 16 days after hatching. After 4 days of exposure to the phage the spleens showed young plasma cells in all stages of mitosis. In bone marrow of newly hatched chicks exposed to the phage, young plasma cells were found in groups and mitotic figures were sometimes seen in these cells. Wedlock writes that, apart from the examples mentioned above, mitosis was exceedingly rare.

11. Imprint, Smear, Fixation, and Staining Methods The imprint method, used by Downey (1938) and since by many others, was used by us as a gentle touch method (Engelbert, 1961). The cut surfaces of spleen, lymph nodes, thymus, and bone marrow were touched gently to the

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surfaces of thoroughly cleaned slides. If the bone marrow was too soft, a sterile camel-hair brush (Mills, 1964; Mills et al., 1969) was used to make smears of the marrow. Imprints of bone marrow were also made by touching a small sterile gelfoam sponge to the marrow surface, then touching the sponge to a slide, thus making an imprint (Engelbert, 1956). The dry imprint method (Shelley, 1961; (Mills, 1964; Mills et a l . , 1969) has been used extensively; afterward the dry slides are stained with the May-Griinwald-Giemsa technique, developed by Pappenheim. This stain has been our routine stain for many years. Blood smears were made as camel-hair brush smears (Mills, 1964). The brush smears avoided smudges, which occurred easily with the slide smear method, as young soft cells were damaged. In several cases we took biopsy specimens from spleens of rabbits, 900-2000 gm in weight. The rabbits were anesthetized with 1.5 to 2 cc of 1% Ibatal (sodium pentobarbital U.S.P. XIV, Ingram and Bell, Toronto) injected intravenously, using a marginal ear vein. The biopsy method produced excellent material both for dry imprints and for tissue culture specimens. The splenic incisions were packed with sterile gelfoam sponges. All but one of the rabbits recovered. Fixation in methyl alcohol was followed by MGG stain. We used this stain according to the technique of Jacobson and Webb (1952). It should be emphasized that fast fixation is imperative. Slow fixation such as one finds with formalin causes the nuclei and cells to contract thus causing artifacts. Fast fixation with acetic alcohol 1:3 for 5 to 8 seconds catches the cells and nuclei in activities that other methods may miss. Acetic alcohol is followed by two rinses in absolute ethyl alcohol and air drying. The slides are stained either with the Schiff or Feulgen method for DNA (Feulgen and Rossenbeck, 1924) or with toluidine blue. The latter method was carried out according to Momson (1958), who was the first to adopt Bonhag’s (1955) techniques for use on imprints of hemopoietic tissues. Imprints were stained in toluidine blue 0 (N.S.) at 37°C for 30-40 minutes in a 0.05% staining solution in McIlvaine ’s citric acid-disodium phosphate buffer, 1/10 strength, pH 4.0 (see Pearse, 1960). The various behavioral stages of blast cell nuclei to be described later were well stained (Engelbert, 1961, Figs. 1 and 4). We also used the Feulgen (Schiff) nuclear stain on Millipore imprints fixed in Zenker’s acetic solution. After the rinse in distilled water following fixation, the filters are put into NHCL at 60°C for 10 minutes, then rinsed in distilled water and put into Schiff’s reagent for 1 hour. This is followed by three 3-minute rinses in fresh S02-water; a 5-minute wash in running tap water; a quick rinse in 95% ethyl alcohol; 2 minutes in absolute ethyl alcohol and xylol (1 :1); finally three changes in pure xylol of 5 minutes each, followed by mounting with malinol. The Millipore filters remain transparent for a long time and allow observation with high-power objectives as well as photomicrography (Engelbert, 1961, Fig. 4). Imprints on Millipore filters have the advantage of consisting of more than

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one cell layer, forming, as it were, a thin section, without the disadvantages of having cells cut by the microtome knife.

111. Methods Using Tritiated Thymidine Mammalian tissue was prepared as dry imprints. Tissues and blood with nucleated erythrocytes had to be fixed briefly in methanol and air-dried before radioautography, as it had been found (Smith, 1969) that the large erythrocytes were damaged by the warm photographic emulsion, in which the slides had to be dipped. Three-week-old rabbits weighing 500-600 gm were injected intravenously with tritiated thymidine (Engelbert, 1967), at a dosage of 0.5 pCilgm body weight (specific activity 6.7 Cilmole). The dry imprint method was used to prepare the hemopoietic tissues for radioautography. The radioautographic technique of Car0 (1964) and Kopriwa and Leblond (1962) were used with Ilford K5 emulsion. The slides were developed after 10 days of storage at 4°C in the dark. Avian tissue was stored for 14 days. The slides were developed in D-19 Developer at 20°C for 2 minutes, then transferred to a stop bath of 1% acetic acid for 10 seconds, fixed in Kodak Rapid Fixer with hardener for 2 minutes, and washed in running water for 5 to 10 minutes. When the slides were dry they were stained with MGG stain using increased staining times.

IV. Fluorescence Method for DNA and RNA Dry imprints were used also for the acridine orange stain (Edward Gurr, Ltd., London, England) prepared as a 0.1% solution by measuring 10.0 ml of a 1% aqueous stock acridine orange solution and adding phosphate buffer to 100 ml. The slides, one at a time, were immersed in rapid succession in a series of solutions: dipped in 1% acetic acid for 30 seconds, stained in 0.1% acridine orange stain (10 seconds for mammalian tissue, 20 seconds for avian tissue), rinsed in phosphate buffer for 3 seconds, transferred to 1 M calcium chloride (to allow for differentiation of the nucleic acids) for 3 to 10 seconds, and finally rinsed in phosphate buffer for 3 seconds. The slides were then mounted with a few drops of the buffer and covered with a zero-thickness cover glass sealed on with hot paraffin. A Leitz Ortholux microscope equipped for fluorescence microscopy, with a mercury vapor lamp and the necessary activating filters as well as protective orange shielding and protective filters in the oculars, were used. It was found that an interval of 30 minutes between staining and observation of the cells produced

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the best results with avian tissue (Smith, 1969). This agrees with Bushong et al. (1968). With ultrablue light the stained tissues displayed the colors of the nucleic acids. DNA fluoresced green to yellow while RNA fluoresced orange to brick red. Hemoglobin appeared black as this substance blocks the fluorescent light, because it absorbs monochromatic light (Nairn, 1962).

V. Tissue Culture in Vitro of Hemopoietic Tissues These techniques were carried out in a special sterile room and sterile techniques were maintained throughout. In the early work we were interested in the effect of foreign proteins. Rabbits of farm stock were injected with 10 ml of sterile normal neutral horse serum per 1800 gm body weight (McMillan, 1958; McMillan and Engelbert, 1963). The injection was given by way of marginal ear veins with a sterile 26-gauge hypodermic needle. Cultures in v i m were made from a small explant of spleen or lymph nodes or thymus, approximately 1 mm3, placed on a sterile glass slide with a drop of medium. The medium was either sterile normal neutral horse serum and Earl's modification of Tyrodes solution 1:1, or serum from blood of the donor animal. Later, when it became available, we used horse serum ultrafiltrate and also Connaught Laboratories synthetic medium 1066 (courtesy of Dr. R. C. Parker and Mr. Healey). The 1066 was used sometimes with calf serum 1:l. A 22 X 40-mm sterile cover glass of zero thickness was placed carefully over a drop of medium containing the cells. Sometimes the cover glass was lifted slightly by placing sterile pieces of zero coverslips under it. The cover glass was always sealed on with hot paraffin. The culture preparations were placed in an incubator at 37.5"C, or examined immediately on a warm stage registering 33" or 35°C mounted on a Reichert Zetopan microscope equipped with positive' and negative phase contrast. An Arriflex 16-mm motion picture camera loaded with 100 ft of Eastman Tri-X-Reversal safety film, Type F278, was mounted above. A 1OX Leitz Periplan ocular was mounted in a Micro Ibso attachment especially fitted for the Arriflex camera. The normal incubator temperature of 37°C would have made it necessary to use high speeds and high light intensities in order to follow the cell movements with cinematography. As we wished to avoid cell damage that might occur with high light intensities we lowered the temperature as explained. Another culture method (McMillan, 1958; McMillan and Engelbert, 1963) consisted of taking small whole fragments of mesenteric lymph nodes and culturing them in a Maximow slide, the depression filled with horse serum and Earl's modification of Tyrodes solution 1:1. A large sterile cover glass was placed over the culture chamber; the slide was then placed in a sterile petri dish with strips of

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blotting paper soaked in sterile distilled water to minimize evaporation of the culture medium. The culture chambers were kept in an incubator at 37°C. Imprints were made five to one slide on the day the experiment was started. Six cultures were set up for each of five rabbits. Each day for 11 days imprints were made on sterile slides with the cultured tissue under sterile conditions as usual. In this way 20 or 30 sets of imprints were made from the same piece of tissue over a period of 11 days.

VI. Behavior and Morphological Variations in Blast Cells and Their Nuclei In 1938 Jordan wrote, “It must be emphasized that the cytologic features of a hemocytoblast are not static; the cytology is subject to considerable variation. ” Unfortunately, however, Jordan did not describe or illustrate the considerable variations. We believe that we have seen “the considerable variation” in practically every slide prepared, regardless of the method (we used all methods) or the type of mammal employed [we used rabbits, rats (Wistar strain), mice from Connaught Laboratories or inbred C57 mice from Jackson Laboratories, field mice, hamsters, guinea pigs, artic lemmings, a Canadian racoon, an American opossum and also leukemic mice (AKR) before the disease appeared]. One of the extreme variations consisted of a lengthening or stretching of individual nuclei; sometimes the stretching made the nucleus appear thin and threadlike. Besides the above variation we found the usual rounded nuclei. These often appeared with very little cytoplasm. The stretched nuclei always appeared with very little cytoplasm. Cultures in vitro and viewing of single cells or nuclei with high-power phase contrast optics plus cinematography allowed us to follow and photograph the changes and see the actual stretching of the nucleus. The movements exhibited by the nucleus were often in a spiraling fashion. Lewis (1931) showed a spiraling movement of myeloblasts which he cultured in v i m (see also Engelbert, 1956, 1958, 1960, 1961, 1967, 1971; McMillan, 1958; McMillan and Engelbert, 1960, 1963; Shelley, 1961; Engelbert and McMillan, 1962; Shelley et al., 1969; Westermann et al., 1970). While the nucleus in our cultures was extended or stretched, intranuclear divisions of small nuclear bodies constantly took place. Eventually nuclear granules, the products of the divisions, were released to the medium (Engelbert and McMillan, 1962; Engelbert, 1967). After being extended for a considerable time, the nucleus rounded up and a rim of cytoplasm appeared around it. The nucleus remained in this state for 10 minutes or more displaying as it were the textbook morphology. The rounding up stage appeared as a resting stage with no special activity visible until the nucleus again assumed the stretched out stage. These alternative changes went on for

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many hours and could go on for 1 day and 1 night or longer. In imprints the stretched nuclei could be seen radiating from the associated tissue, as pins from a pincushion. Figure 1 (McMillan and Engelbert, 1960) shows an elongated nucleus extended to a length of 120 pm. The twist one sees on this extended nucleus often looks as if the nucleus made a half-turn during the stretching period, part of the spiraling motion. In their study “The Development History of the Plasma Cells in the Lymph Node of the Rabbit,” McMillan and Engelbert (1963) present tables in which numbers of elongated and contracted blast cells are included in all the cell types found in normal rabbit lymph nodes as well as after injections of horse serum. They also present several graphical text figures in which the relative frequencies of elongated and contracted blast cells are shown in relation to the relative frequencies of other cells in rabbit lymph nodes. In their Fig. 333 Lucas and Jamroz (1961) show, in the thymus of a 35-day-old chick, elongated threadlike nuclei in two areas of the illustration. Both are labeled “smudged nuclei,” although there is no evidence of damage. In their Fig. 332 of a chick embryo thymus, a long threadlike nucleus passes over a red cell and over several lymphocytes from the upper middle of the figure toward the

FIG. 1 . Imprints from germinal centers in the white pulp of spleen of a rabbit injected 85 minutes previously with [3H]thymidine. Arrows indicate fusing vesicles. A very elongated well-labeled nucleus as well as a round well-labeled nucleus can be seen. (From Engelbert, 1967.)

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left corner. None of these cells are described. Another stretched nucleus, not threadlike but fairly broad, is partly hidden by an unlabeled cell with a magenta nucleus and deep blue cytoplasm. Figure 331 of the spleen from a 35-day-old chick shows a threadlike nucleus reaching from the right lower part of the figure to the upper middle. Figure 329 of an embryo spleen shows an elongated nucleus in the upper left-hand portion .of the figure. A second elongated nucleus, partially damaged, is found in the upper middle of this figure. In experiments carried out in our laboratory by Dr. Jean E. Mills (Westermann), 12 young rabbits, weighing 500-600 gm, were injected intravenously with tritiated thymidine; 12 exposure times were maintained (see Table I). From each of the 12 exposure times, 25 labeled cells from randomly chosen areas in each of six imprints of spleen were counted and classified on the basis of morphological shape into three variants: round, oblong or irregular, and elongated. When these figures were analyzed statistically for correlation (rank difference method) (Davenport and Ekas, 1936), the results suggested that an inverse relation existed between the numbers of labeled rounded or elongated cells ( r = -0.3), i.e., where numbers of rounded cells or nuclei are high, those of the elongated nuclei are low, and vice versa. From these results we must assume that the round and elongated or extended nuclei are members of the same cell population, but each shape expresses a different activity stage, such as described earlier. From the evidence presented so far we believe we must acknowledge that nuclei of lymphoid cells normally change their shape during their life history and these changes, when they are “caught” with fixatives or with cinematography of TABLE I OCCURRENCE I N RABBIT SPLEENOF CELLSWITH VARYING MORPHOLOGY CARRYING LABEL AFTER DIFFERENT TIMESOF EXPOSURE TO TRITIATED THYMIDINE

Rabbit no.

Time exposed to tritiated thymidine

Cell variant Round

1 2 3 4 5 6 7 8 9 10 11 12

5 minutes 40 minutes 85 minutes 2 hours 4 hours 8 hours 12 hours 24 hours 2 days 4 days 8 days 12 days

80 86 53 37 61 59 41 61 45 64 72 37

Oblong or irregular 36 43 40 78 48 52 52

40 31 55 45 63

Elongate

Total no. of cells

34 21 57 35 41 39 57 49 74 31 33 50

150 150 150 150 150 150 150 150 150 150 150 150

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cultures in vitro, cannot be “shrugged off” as degenerative stages or artifacts (Engelbert, 1967). During the stretching and twisting period the nucleus also releases vesicles that appear colorless with the MGG stain. Shelley (1961) demonstrated intranuclear vesicles in blast cells in rabbit lymph nodes with different fixatives and staining techniques. The study was limited to “seemingly naked nuclei” of round to ovoid form, with the long axis no greater than three times the short axis, since they make up about 77% of blast cell nuclei in a mesentric rabbit lymph node. Shelley defined an intranuclear vesicle as a pale-staining or nonstaining area, 1 p m or greater in diameter, which is enclosed by the nucleus for more than half its circumference. She found that with the May-GriinwaldGiemsa staining method, iodine vapor and methanol were equally valuable as fixatives for demonstrating intranuclear vesicles. Neutral formalin fixation and MGG staining of imprints showed statistically fewer vesicles in the nuclei. Neutral formalin kills the nucleus very slowly; the nucleus contracts squeezing out the vesicles. It is therefore necessary to fix the nuclei very quickly to maintain their morphology and contents (Engelbert, 1967). Pearse (1960) considers that formalin is the best protein fixative, but we find it is a very poor fixative for nuclei. When staining with hematoxylin following fixation with either iodine or formalin vapor, Shelley found no differences in the proportions of nuclei containing and not containing vesicles. Hematoxylin will stain vesicles containing protein alone. From the statistical analysis of intranuclear vesicles in lymph nodes of seven normal rabbits. Shelley et al. (1969) concluded that: (i) The rabbits used were homogeneous with regard to the number of blast cell nuclei displaying intranuclear vesicles. (ii) The size of the intranuclear vesicle was independent of the nuclear size. (iii) Nuclei with a larger nuclear index (width X length) tended to display a greater number of vesicles. When vesicles are viewed with high-power and negative phase-contrast optics they display a grayish color (Engelbert, 1960, Fig. 9). With the mercury bromphenol blue method (Mazia et al., 1953) the contents of vesicles stain blue indicating that the contents are protein (Pearse, 1968). During the process of elongation and stretching the nuclei release much of their nucleoplasm (Engelbert, 1958). The nucleoplasm when released has the typical appearance of ‘‘vacuolated cytoplasm or ‘‘degenerative cytoplasm (Dacie and White, 1949, Pl.IV, Figs. 2 and 4). The “vacuoles” these authors studied in erythropoiesis in human bone marrow we believe are “our vesicles,” which we shall eventually show play an important role in the formation of new cells and new nuclei in hemopoietic tissues. Downey and Weidenreich (1912) showed that lymphocytes released pieces of their cytoplasm and they believed it was a normal property of these cells. Weill (1913) and Williamson (1950) demonstrated similar phenomena. Nuclear fragments of lymphocytes were shown in culture preparations by Popoff (1927), Tschassownikow (1927), and Emmart (1936). ”

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In our laboratory Blair (1955) observed 5590 normal lymphocytes from thymi of mammals and chick embryos. She counted 4591 cells that showed one or more tubes radiating from each cell; 551 nuclear bodies were found inside tubes; Engelbert (1953) found tubes extending from the thymic cells cultured in vitro. White (1947-1948) and Frank and Dougherty (1953) reported “cytoplasmic budding” from lymphocytes. In a paper published in 1882 by Watney on the thymus, one illustration clearly shows two cells with fairly long tubes. Watney did neither label nor describe the two cells, but the details of his drawing were carefully camed out. Westermann et al. (1970) described protoplasmic fragments in hematopoietic tissues and an analysis of intranuclear vesicles in lymph node blast cells of the rabbit. They demonstrated that vesicles and cytoplasmic fragments are found in greatest number in the lymph node and spleen and are least common in the thymus and bone marrow in the rabbit. Vesicles appear to originate by the extrusion of intranuclear and intracytoplasmic vesicles mostly from cells of the lymphoid series. Vesicles and cytoplasmic fragments are absent from blood smears and extremely difficult to recognize in sections or in areas of imprints where the cells are closely applied one to another. These authors also suggest that chromatin from stretched nuclei and smaller free chromatin masses may become transferred to free vesicles and this process may function in new cell formation. Wedlock (1974) carried out an extensive investigation of differentiation of hemopoietic cells in the thymus, bursa of Fabricius, spleen, and bone marrow of chick embryos and hatched chicks (Callus domesticus) from 14 days of incubation to 16 days after hatching. Further, the cytology of normally developing organs was compared to that of organs stimulated by the bacteriophage 0x174, and numbers of all the different cell types were counted in imprints. She also used fluorescence microscopy as well as radioautography after injections with tritiated thymidine. Five chicks were used as controls for each stage. Five were used for experiments with phage for each age. Each treatment or staining method was studied in chick embryos at 14, 16, and 18 days of incubation and immediately before hatching and in young chicks at 4, 8, and 16 days after hatching. She described nuclei of three morphological types: long extended nuclei containing vesicles, spherical nuclei with webbed chromatin, and spherical nuclei with homogeneous chromatin and two nucleoli. Her illustrations show the typical long extended nuclei we have described. She reports that in the thymus, 76%of the cells counted at 14 days of incubation have very little cytoplasm. This decrease to 20% at hatching and rises to 36% at 16 days after hatching. During the entire period the lymphocytes increase steadily from 8 to 55%.In the bursa of Fabricius, 57% of the cells counted at 14 days of incubation have very little cytoplasm. This decreases to 36% at hatching but increases to 53% at 16 days. Lympnocytes increase from 8% at 14 days of incubation to 38% at 16 days after hatching. In the spleen 54% of the cells counted at 14 days of incubation were

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nuclei with little cytoplasm. Their number decreases to 13% at 18 days of incubation, but at hatching they constitute 22% and at 16 days after hatching, 34%. In bone marrow at 14 days of incubation nuclei with little cytoplasm form 31%, at hatching 15%, and at 16 days after hatching 6%. Lymphocytes in the same period increase from 2.6 to 24% at 16 days after hatching. Wedlock tested the cell counts in normal and in phage-stimulated chicks with analysis of variance. Calculations were performed by a C.G.E. time-sharing computer service. The two-factor analysis used the counts of embryonic cells and the three-factor analysis of variance used the counts of cells of hatched chicks. Probability value of F ratio: (i) indicates a significant difference in cell populations at the 5 % probability level; (ii) indicates a significant difference in cell population at the 1% probability level; and (iii) indicates a significant difference in cell populations at the 0.1% probability level.

VII. The Occurrence and Significance of Two Spatially Separate Nuclear Masses in Blast Cells and in Differentiating Cells

During early research work (Engelbert, 1956) it was often observed in in vitro cultures of normal rabbit spleens that some of the elongated nuclei released nuclear and cytoplasmic masses, through a tubelike opening at one end. In fixed imprints the same behavior was also observed. Sometimes the elongated nuclei were slightly broken and part of the nuclear wall lifted away. In such cases one could see small nuclear bodies lying inside the elongated nuclei. Rounded nuclei could at certain stages also be sufficiently “nonplastic” to crack open, even with a gentle imprint technique. In these cases one saw clearly that the damaged part of the nucleus constituted part of a shell-like or peripheral layer, inside which two well-developed nuclear masses were hidden (Engelbert, 1956, 1970). The shell-like or peripheral layer had small nuclear granules on both its inner and outer surfaces. These granules appeared to be produced by the “shell.” Various stages of such nuclei seen often over the years clearly demonstrated that all of the shell-like or peripheral nuclear portion finally became individual free granules and that the inner nuclear masses formed the nuclei of granulocytes. When the granules began to appear on the peripheral nuclear mass, its future existence as a cohesive mass seemed only of short duration. Often one sees only small pieces of the peripheral or shell-like nuclear mass. The granules lying in the cytoplasm are typical of those in granulocytes. In textbook illustrations of neutrophilic and eosinophilic “myelocytes” (Bloom and Fawcett,

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1968), one sees mainly the granular mass; only in later stages can the nuclei be seen, as the granules become spread farther apart. In view of this evidence we decided to run experiments with tritiated thymidine in order to find out if the two separated nuclear masses would label and thus indicate DNA synthesis. We had observed that the blood of birds (Galfus domesticus) often carried a good number of immature granulocytes, which displayed the shell-like or peripheral nuclear layer. We therefore used birds of Leghorn stock for the experiments. Chick embryos, newly hatched chicks, and pullets weighing 1500-1800 gm were used. Three pullets were injected intravenously using a brachial vein. Tritiated thymidine was injected into the coelom of 16 newly hatched chicks and into the vascular bed behind the eye of 13 chick embryos. Ten embroys, eleven newly hatched chicks, and one pullet served as controls. Imprints were made of spleens and brush smears were made of the blood. After the injections of tritiated thymidine label was found on both elongated and rounded nuclei similar to that reported for mammals (Section VI) (Engelbert, 1967). Eighteen-day chick embryos carried heavy label on the shell-like or peripheral nuclear layer of differentiating cells. If large or small pieces of this mass remained the cells were called “immature. ” If granules were fully formed and no shell-like nuclear mass remained the cells were called “mature.” For each animal used 500 granulocytes or all of the granulocytes found in 6 to 10 samples were counted. After 30 minutes of exposure to tritiated thymidine imprints from 18-day embryo spleen had 100 granulocytes; 16 were mature with label on the granules, and 37 were immature cells with label on the granules and on the remains of the shell-like nuclear layer. From one pullet blood samples were taken 3, 6, and 24 hours after injection of the isotope. At 3 hours, 275 granulocytes were counted: 173 mature cells with label on the granules, 9 mature cells with no label, 75 immature cells with label, and 18 immature cells with no label. At 6 hours, 500 granulocytes were counted: 493 mature cells with label on the granules, and 7 mature cells without label. No immature stages were found. At 24 hours, 10 samples were scanned; some cells had label but most had none. A second pullet killed 1 hour after injection had so few granulocytes in 10 samples that no counts were made. Intense erythropoiesis was present however (Smith and Engelbert, 1969) (see later). A pullet killed 2 hours after the injection had 500 granulocytes: 88 mature with label, 74 mature without label, 166 immature with label, 172 immature without label. A pullet killed as a control without an injection of tritiated thymidine had 41 eosinophils, 29 of which were mature and 12 immature, and 160 heterophils, 132 of which were mature and 28 immature. There were so few basophils in the samples that they were not reported. The eosinophils have rounded granules; the heterophils have rice-grain-shaped granules. In control animals one could easily see the difference between heterophilic and eosinophilic granulocytes, but in slides prepared with radioautography it was

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often difficult to determine the shape of the granules. However, in a good number of cases we could see the difference and both rounded and rice-grainshaped granules carried label. The label on the granules consisted of a single small “cap” on each. The label on the shell-like nuclear mass appeared as paired grains or as several joined grains which indicates DNA synthesis while granules were being formed (Engelbert, 1970, Fig. 4). On the nuclei of the granulocytes, the inner nuclear mass did not carry any label. With the Feulgen or Schiff method of staining a small area on each granule is “Feulgen or Schiff positive”; this presumably is the DNA which labels with tritiated thymidine. The mature granulocytes eventually releases granules either individually or in a small mass. This can be seen clearly in in vitro cultures and also in imprints. Finally, the grain is detached from the granule and the small mass of labeled DNA becomes free in the blood. We have seen many granulocytes in mammals with label on the granules, but never with label on the nuclei. We believe that the peripheral or shell-like nuclear mass, which produces granules, is a morphological example of what Roels (1966), in his extensive review of “the variability of DNA,” calls “metabolic DNA.” Roels wrote, “one may explain these variations by accepting two types of DNA: a staple one with genetic function and a labile one with metabolic function. ” In a paper entitled, “Turnover of DNA and Function,” Pelc (1968) wrote, “the metabolic DNA of a given type of differentiated cell consists of extra copies of the genes which are active in the cell; the metabolic DNA is the working DNA, which regulates and performs the transcription of RNA and possibly other functions of DNA, while active molecules of metabolic DNA are subject to wear and tear and are periodically renewed. DNA can thus be labeled during three periods: premitotic synthesis, formation of metabolic DNA and renewal or repair. The inner nuclear mass which becomes the nuclei of granulocytes we regard as genetic DNA and in normal tissues we have not seen these nuclei with label.

”

VIII. Fate of the Peripheral or Shell-like Nuclear Mass, and the Inner Nuclear Mass, in Differentiating Cells of Leukemic Mice (AKR Strain) Imprints of spleen, thymus, and lymph nodes from AKR mice, with detectable enlargements of the inguinal or axiliary lymph nodes, had many immature plasma cells with a broad border of basophilc cytoplasm and the pale central area characteristic of the developmental stages of this cell. It is known to appear in response to antigens. The Gooch virus is considered the causative agent in murine leukemia (Metcalf, 1966). Fully developed plasma cells were rare or missing entirely. The immature plasma cells shed their cytoplasm to a large extent. The blast cell nuclei of

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elongated shape were split lengthwise into individual nuclear strands in many cases. Intact blast cell nuclei extruded basophilic granular substance and also nucleoli. The vesicles produced were not the normal type which do not stain with MGG and thus appear colorless. Instead the blast cell nuclei produced a great many hemoglobin-containing vesicles. In cats with leukemia the blast cells also produced great numbers of hemoglobin-carrying vesicles (V. E. Engelbert, unpublished). In leukemic mice the peripheral nuclear mass labels with tritiated thymidine, but the label appears to consist of single grains, and not of several joined grains, as in normal animals. The normal relationship between the shelllike or peripheral nuclear mass and the inner nuclear mass is not maintained, even in these relatively early stages of murine leukemia. The peripheral nuclear mass “expels” as it were the inner nuclear mass. The latter has a ring-shaped or lobed nucleus surrounded by pale nongranular cytoplasm. The expelled mass is “the leukocyte” seen commonly in leukemias. The shell-like nuclear mass, now an empty shell, gradually breaks up and small labeled pieces or fragments can be found scattered over the imprint. In the later stages of murine leukemia extreme enlargements of thymus, spleen, and lymph nodes take place. At this time most of the cells in these organs undergo classical mitosis. The cells undergoing mitosis appear to be enlarged plasma cell nuclei; these cells have no cytoplasm. Elongated naked blast cell nuclei are still often found and may transform into plasma cells (McMillan and Engelbert, 1960, 1963). Other cell types are not found at this stage. The drug cytosine arabinoside, produced by the Upjohn Co. and tested by them on animals with leukemia, was used by us in order to see if any cytological effect could be found. Dr. E. L. Masson of the Upjohn Co. of Canada gave us a sample as well as a copy of the company’s unpublished records of its use and effect. After four daily intraperitoneal injections of 0.5 ml tripledistilled water in which 20 mg of cytosine arabinoside was dissolved, nine mice were examined as to weight of spleen, thymus, and lymph nodes. Imprints were made as usual from the three organs. Weights of the three organs were mostly normal or nearly normal. Imprints of the treated mice showed a much more scanty cell population; they were less dense than imprints of nontreated mice, as if a large part of the cell population had been expelled from the three organs. Plasma cells were reduced in number, but some were still found even in mice treated early (as soon as enlargement of lymph nodules could be detected). The plasma cell persisted especially in the thymus. The shedding of their cytoplasm also persisted. The splitting of the elongated blast nuclei was much decreased. The hemoglobin vesicles were often entirely absent and the normal colorless (with MGG) vesicles were back in large numbers. Classical mitosis was not seen in the treated “apparently healthy looking” mice. However, the most significant change was that the shell-like nuclear mass and the inner nuclear mass appeared to maintain their close relationship. Differentiation was thus not totally interrupted as in the nontreated mice, in which the two nuclear masses, peripheral and

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inner, became separated and the first one was destroyed. The label on cells in the spleen was in paired or small clumps of grains, which was a sign that differentiating cells “were working” on DNA synthesis. Normal granulocytes were found in bone marrow. The work with leukemic mice is quoted here because it sheds additional light on the importance of the two associated nuclear masses, the peripheral and the inner mass, and their importance in differentiation (Engelbert, 1971).

IX. Results following Injection of Tritiated Thymidine A. REASSOCIATION OF VESICLES A N D NUCLEAR GRANULES I N RABBITSPLEENS In Section VI, production of vesicles by blast cell nuclei was reported fully and the work of Shelley, Westermann, McMillan, and Engelbert described. The elongated nuclei release their vesicular contents into a central mass or “nest” of vesicles. The nuclei radiate out from the edge of this central mass. Soon however the nuclei move into the mass of vesicles and come to lie close to vesicular membranes. Figure 1 shows an elongated well-labeled nucleus “caught” as it moves between vesicles; one end of this nucleus adheres closely to a large partially stained vesicle on the left. In the same figure small vesicles can be seen fusing (arrows) and forming larger vesicles. In Fig. 2 the black masses surrounding the colorless large vesicles (Ve) are the labeled densely packed elongated nuclei. In a vesicle at the upper center (Ve) labeled nuclear granules can be seen entering the colorless vesicle. The individual grains cannot be seen on the surrounding nuclei. When labeled nuclei are condensed or contracted, they appear completely black as in Fig. 2. In Plate 3 of Engelbert (1967) one can see both condensed black areas and stretched areas of the same nucleus. In the stretched part one can count the individual grains. Figure 2 in the present paper presents a large vesicle at the right side with a good label (Lb). One can see the grains are two to four times the size of individual grains, indicating that further DNA synthesis is taking place. A vesicle in the lower right-hand comer appears to be entered by labeled nuclear granules around its periphery. It may be reasonable to assume that the nuclear granules enter the vesicles in vesicular blebs formed from the vesicular membrane. In Fig. 3 in the upper center a vesicle with a good label and stainable content (MGG) has along its lower periphery the remains (Nb) of one of the nuclei or “the” nucleus which contributed its contents of labeled DNA. We now call such vesicles “vesicular nuclei. Other almost colorless vesicles are surrounded by black rims of labeled nuclei. In Fig. 4 a well-labeled vesicular nucleus in DNA synthesis shows the remains (Nb) of a contributing nucleus on its upper left periphery. Stained but not labeled vesicles are also present. ”

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FIG.2. Vesicles (Ve) with a tight rim of labeled nuclei (mows) around them. Gradual entrance of nuclear material into vesicles can be detected by grains above lightly labeled vesicles and staining of the vesicular substance-LLheavy label on a former vesicle that now has acquired labeled DNA. (From Engelbert, 1967.)

It should be mentioned at this time that we have seen the morphology and behavior of nuclear granules in our work with live preparations of in vitro cultures. Individual granules divide first forming clumps because they lie close together. Shortly after they part. We believe that the label on the vesicular nuclei means that these cells will differentiate, forming either granulocytes, erythrocytes, or lymphocytes. Thomas (1959) and Yoffey (1960) suggested that this label meant division of the whole cell. Andreasen (1959) reported ‘‘degeneration of lymphocytes” which we believe was due to his observation of the large nonstaining vesicles shown above. He also thought that the degeneration was “combined with phagocytic activity of the reticulum.” We have not seen cells of the reticulum often in imprints, but we have sometimes seen their nuclei in imprints on Millipore filters. The author wrote in 1960 that “reticular cells and other mesenchymal elements, which form connective tissue in the animal body, constitute a group of cells, where normal mitosis is

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FIG. 3. Nuclear rim (arrows) around vesicles (Ve). Nb, Remains of nuclear border or nuclear rim around a well-labeled vesicular cell. (From Engelbert, 1967.)

relatively easy to demonstrate. However, we still need to know how blood cells are formed. ” Yoffey said “the term reticular has been fraught with such difficulties that I avoid it. But you start off with a primitive cell, which goes through a number of divisions” (Yoffey et al., 1959, p. 58). In the same discussion Yoffey denies that “extensive pycnosis” is found in “germinal centers.” He says further, “Frankly we don’t believe that you find very many of these cells dying in healthy animals. ” We do not believe in the death of lymphocytes either. The idea of “pycnosis” may have originated from the fact that, in contracted form, the blast cell nuclei stain very dark and appear totally black when labeled. We believe that the stages of vesicles and “vesicular nuclei” are the cells Yoffey (1973) calls “transitional cells” and lists as “pale transitionals, “basophilic transitionals, ” and finally “blast cells. ”

”

B. GRAINCOUNTS IN VESICULAR NUCLEI OF RABBITSPLEEN AND BONE MARROW Figure 5 presents grain counts in individual cells from spleens of four rabbits killed 5 minutes, 85 minutes, 2 hours, and 1 day after injections with tritiated

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FIG.4. Nb, Remains of nucleus that have contributed DNA to the well-labeled vesicular cell. (From Engelbert, 1967.)

thymidine (Engelbert, 1967). Total labeled cells counted are shown at the upper left in each square. The lower grain counts shown as short columns represent vesicles which have begun to accumulate nuclear granules from the blast cells surrounding them (see Fig. 2). The tall columns with grain counts above 10 grains present vesicular nuclei that have not only accumulated nuclear granules from blast cells surrounding them, but in which the nuclear granules have begun “mini-mitoses,’’ and thus have started the second growth phase of vesicular nuclei. The grain counts 1 day after injection of tritiated thymidine show that 60% of the vesicular nuclei examined have entered the second growth phase. If one examines Figs. 1-4, it is clear that vesicles with a high grain count are all in the second growth phase, increasing DNA content through mini-mitoses. Clumps of two, four, or more granules originate through such divisions. From the clumps of granules individual granules will move away and possibly divide again later (see basic nuclear units, Engelbert, 1956). The decrease of grain counts in Fig. 5 must therefore not be regarded as a “dilution effect,” where the

2or 113

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0

-

100

6RAIN COUNT: INTERVALS OF 20

FIG.5 . Grain counts in individual cells from spleens of four rabbits killed 5 minutes, 85 minutes, 2 hours, and I day after injections of tritiated thymidine. (From Engelbert, 1967.)

cells with lower grains counts are division products of cells with higher grain counts. The differences in grain numbers counted in the splenic cells from the four rabbits must be regarded and evaluated as cells in growth, in which the increase and accumulation of DNA is the main function taking place. This increase of DNA content eventually leads to various stages of differentiation whereupon different blood cells will be produced by the spleen. In tissue cultures I have seen vesicles gradually accumulate small granular bodies (V. E. Engelbert, unpublished). The most frequent grain counts in spleen and bone marrow cells of the four rabbits were compared in Fig. 15 of Engelbert (1967). At 1.5 hours after injection of tritiated thymidine, the cells of the spleen had the highest grain count, 110, the bone marrow cells, 25. At 3 hours and continuing to the second day the most frequent splenic grain count was 15 grains per cell. The bone marrow counts varied: 30 grains at 3 hours, 10 grains at 7 . 5 hours, 25 grains at 12 hours, and 15 grains at 1 day after injection of the isotope. The grain counts in both organs then declined steadily giving 5 grains or less on the 9th day. To obtain the best measure of DNA accumulation and consequential growth in hemopoietic cells, grain counts after one injection of tritiated thymidine should be taken early during the first day and not later than 24 hours after the injection.

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Thomas (1959) and Yoffey (1960) interpreted cells with lower grain counts as products of direct mitosis of the cells with higher grain counts. Such an interpretation does not agree with the work presented here.

X. Erythropoiesis in Blood of Vertebrates with Nucleated Erythrocytes. Formation of Clone Cells from Nuclei of Young Mature Erythrocytes In 1960, in studies of blood semars of nucleated erythrocytes of birds, amphibians, and fishes, I suggested that the nucleus of the red blood cell in its younger stages behaved as a mother cell. Pouchlike extensions of the nuclear membrane with nuclear contents pinched off outside the mother cell. Recently, a paper by Komocki (1929) was brought to my attention. This author wrote “Uber die Abstammung der Erythrocyten der niederen Wirbeltiere von den sogenannt nackten Kernen. He had published several earlier papers on his work with turtles and, later, worked with salamanders. Morashita (1 957) believed that leukocytic cells were produced by extrusion of cytoplasm from the nucleated erythrocyte of the toad. He did not mention any involvement of the nucleus in this process. We have observed intranuclear divisions in living erythrocytes. The small nuclear granules divide one at the time; then one part moves away toward the nuclear membrane leaving a “whitish track” in the nucleoplasm. In imprint preparations it is very easy to see when these intranuclear divisions are taking place because the whitish tracks show up very well. Lucas and Jamroz (1961, Fig. 228) show in detail nuclei of erythrocytes from blood of a chick embryo heart on the 10th day of incubation. The small nuclear granules and also the whitish tracks which we regard as a sign that “mini-divisions” are taking place within the nuclear membrane are very clear in this illustration. These authors, however, use terms from mammalian hematology to describe the various erythrocytes. Smith (1969) described and illustrated the formation of small nuclear buds or blebs from erythrocytes in peripheral blood of chick embryos and hatched chicks. She used camel-hair brush smears and four staining methods, May-Griinwald-Giemsa, fluroescence with acridine orange stain, mercury bromphenol blue stain, and the Schiff (or Feulgen) method, as well as labeling with tritiated thymidine. She showed that more than one bleb could originate on a nucleus at the same time although one at a time seemed usual. The bleb finally became a pouch at its outer end. While the May-Griinwald-Giemsa method showed development and finally the free new cell very well, the fluorescence method with acridine orange stain presented the best detail. In the latter preparations the mother cell nucleus fluoresced yellow and the hemoglobin black as it absorbed monochromatic light (Nairn, 1962). The blebs, while still attached to the mother nucleus or finally free, also stained yellow. The free new cells, the ”

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“clone cells,” soon showed that the yellow material had concentrated and stained brighter yellow in the center in which the new nucleus was forming. The surrounding “cytoplasm” while still yellowish began to show portions of brick red color indicating the formation of RNA. Soon all the cytoplasm was brick red and the nucleus, dense and yellow, the same color and shape as in other cells. The cytoplasm gradually became black as hemoglobin was formed. The mercury bromphenol blue stained the mother cell, but left the blebs completely colorless. The Feulgen method stained the nuclei of erythrocytes well, but the early stages of clone cells although magenta in color were pale. However, the fact that they stained as DNA instead of protein (bromphenol blue) we believe is significant. Smith counted over 86,000 cells and only one red cell showed any resemblance to pro- or metaphases, but no ana- or telophases were found. The frequency of clone cells ranged from 3.7 to 6 and 8%. Lucas and Jamroz (1961) reported 0.2% cells in mitosis in chicken bone marrow. The same authors also show two clone cells (Fig. 226, Nos. 15, 16) still attached to the mother erythrocyte; both are called “smudged primary erythrocytes.” In Fig. 2 a clone cell between two erythrocytes (No. 6) is called a squashed erythrocyte nucleus. In our photographs the early stages of clone cells in Fig. 12 of Smith and Engelbert (1969) form as basket-like structures. This, however, is a temporary stage and soon the strands form a bleb. During differentiation of the clone cells into erythrocytes in chicks Smith found that approximately 50% of the clone cells remained adherent to the mother cell. Both erythrocytes and clone cells were labeled in peripheral blood of chicken exposed to tritiated thymidine for 24 hours. Deutsch (1970) investigated clone formation in the peripheral blood of the white sucker, Cutostomus commersoni, after labeling with tritiated thymidine for various exposure intervals. He found that 10% of the cells of peripheral blood were clone cells. Both clone cells and erythocytes labeled after exposure to the isotope and 13 to 18% of the saclike extensions of clone cells (called Stage 11) had mean grain counts of 235 to 301 grains per cell. The greatest uptake of tritiated thymidine was observed about the forty-eighth hour and the fifth day of exposure. Counts of clone cells in peripheral blood of seven species of New Zealand birds and one species of Canadian birds were made by Engelbert and Young (1970b). In blood from one kiwi, 18% of the 1500 cells counted were clone cells. In kiwi No. 2 (8 years old), of 2000 cells 41% were clone cells; in weka No. 1, of 1500 cells 23% were clone cells; in weka No. 2, of 2400 cells, 10% were clone cells; in weka No. 3, of 1500 cells, 14% were clone cells. In yellow-eyed penguin, of 2000 cells, 5% were clone cells. Blue or fairy penguin (on nest with two eggs) had, out of 2000 cells, 12% clone cells. Takahe (Notorismantelli) had, out of 2000 cells, 15%clone cells. h k e h o No. 1 (broken leg) had, out of 1500 cells, 5% clone cells. h k e h o No. 2 (healthy) had, out of

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2000 cells, 12% clone cells. Kea had, out of 2000 cells, 7% clone cells. The young white-throated Canadian sparrow had 18% clones in 2000 cells; the mature white-throated Canadian sparrow had 12% clones in 2000 cells. In studies of erythropoiesis in peripheral blood of tuatara (Sphenodon punctatus) and turtle (Muluclemys terrapin), Engelbert and Young (1970a) found the following: Sphenodon punctatus

Clone cells Immature clone cells with hemoglobin forming Mature clone cells Lymphocytes Granulocytes

No. 1

No. 2

No. 3

No. 4

29 1

165

67

160

90 1653 13 9

I789 23 7

I880 37 6

1813 8 11

Malaclemys terrapin

Clone cells Immature clone cells with hemoglobin forming Mature clone cells Lymphocytes Granulocytes Thrombocytes were observed in both species but not counted

No. I

No. 2

No. 3

149

150

I83

235 I101 10

126 1213 Very few, not counted 10

100 1241 5

Recently I examined slides of blood from a New Zealand tree frog; the clone cells were so numerous that over 60% of erythrocytes on the slides had clone cells attached. Examination of numbers of clone cells in fish and birds might be important in estimating the condition of health of these animals; the same can be applied to reptiles and amphibians where ecology and the balance of nature are of interest. One can say with conviction that the clone cell has been present for a very long time as Sphenodon has a history of over 200 million years. It is an important feature of blood cell formation and should not be ignored as it has to date by biologists and hematologists; it could be of great help to them and to the species involved.

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XI. Summary 1 . The search for a blood mother cell, a hemocytoblast, as well as for evidence of mitosis by many workers. 2. Methods used in tracing of behavior and morphological variations in blood cells and their nuclei including in vitro methods and cinematography. 3. The production of vesicles with protein content by nuclei in hemopoietic organs. 4. Two spatially separate nuclear masses, one peripheral the other centrally located. Only the peripheral mass labels with tritiated thymidine in normal animals. 5 . The state of the two nuclear masses in mice with leukemia (AKR strain). 6 . Accumulation by free nuclear vesicles of small nuclear granules in spleen of rabbits. The granules when inside the vesicles undergo mini-mitoses and thus synthesis of DNA takes place. 7. Grain counts in cells of spleen of rabbits after injection of tritiated thymidine show lower and higher grain counts. The lower grain counts indicate the accumulation of nuclear granules by vesicles, thus the first growth stage. The second growth stage is the increase in DNA by mini-mitoses of the nuclear granules. Thus two phases of growth create large nuclear contents. Soon these cells differentiate to form various new blood cells. 8. Erythropoiesis in peripheral blood of vertebrates with nucleated erythrocytes takes place by formation of nuclear buds from young mature erythrocytes. The new cells are clone cells. They can be found in fish, amphibians, reptiles, chick embryos, and adult birds. Sphenodon punctatus and seven species of New Zealand birds were included in this study.

XII. Conclusion Alternatives to mitosis are: (a) metaphases changing directly to nuclei in mammals; (b) development of vesicular nuclei through two growth phases, accumulation of nuclear granules and rapid increase in DNA by mini-mitoses of these granules; and (c) development of clone cells from nuclear buds of nucleated erythrocytes.

ACKNOWLEDGMENTS

I am indebted to Dr. Jean E. M. Westermann, McMaster University, Canada, for valuable criticism, to Professor Donald B . McMillan, University of Western Ontario, Canada, Mrs. Jessica

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Shelley, Mrs. Natasha Bikadoroff Smith, Mr. M. Deutsch, and Dr. Diana Wedlock for the use of their material. Table I and Figs. 1-5 are published with the kind permission of the editor of Haematologica Larim, Milano. I am sincerely grateful to principal secretary, Miss A. M. Sorensen, Museum of Natural History, Aarhus, for the secretarial work of Miss Karen Berg.

REFERENCES Andreasen, E. (1959). In ”The Kinetics of Cellular Proliferation” (F. Stohlman, Jr., ed.), p. 19. Grune & Stratton, New York. Andreasen, E., and Christensen, S. (1949). Anar. Rec. 103, 401. Arnold, J. (1887). Arch. Mikrosk. Anat. 30, 205. Askanazy, M. (1905). Zentralol. Allg. Parhol. Parhol. Anar. 16, 897. Blair, M. H. (1955). M.A. Thesis, University of Toronto, Toronto. Bloom, W., and Fawcett, D. W. (1968). “A Textbook of Histology,” 8th ed. Saunders, Philadelphia, Pennsylvania. Bonhag, P. F. (1955). J. Morphol. 96, 381. Bushong, S. C., Watson, J. A., and Atchison, R. W. (1968). Slain Technol. 43, 273. Caro, L. (1964). In “High Resolution Autoradiography” (D. M. Prescott, ed.), Vol. 1, p. 327. Academic Press, New York. Christensen, S. (1950). Acra Anar. 10, 233. Dacie, J. V., and White, J. C. (1949). J. Clin. Pathol. 2, 1. Davenport, C. B., and Ekas, M. P. (1936). “Statistical Methods in Biology, Medicine and Psychology.” Wiley, New York. Deutsch, M. (1970). M.Sc. Thesis, University of Toronto, Toronto. Deutsch, M., and Engelbert, V. E. (1970). Can. J . Zool. 48, 1241. Diggs, L. W., Sturm, D., and Bell, A. (1957). “The Morphology of Human Blood Cells.” Saunders, Philadelphia, Pennsylvania. Downey, H. (1932). In “Special Cytology. The Form and Functions of the Cell in Health and Disease” (E. V. Cowdry, ed.), Vol. 2, p. 653. Harper (Hoeber), New York. Downey, H. (1938). In “Handbook of Hematology” (H. Downey, ed.), Vol. 111, p. 1963. Hafner, New York. Downey, H., and Weidenreich, F. (1912). Arch. Mikrosk. Anat. 80, 360. Dustin, P., Jr. (1959). In “The Kinetics of Cellular Proliferation” (F. Stohlman, Jr., ed.), p. 50. Grune & Stratton, New York. Emmart, E. W. (1936). Anar. Rec. 66, 59. Engelbert, V. E. (1953). Can. J. 2001.31, 106. Engelbert, V. E. (1956). Can. J. Zool. 34, 707. Engelbert, V. E. (1958). Can. J. 2001.36, 131. Engelbert, V. E. (1960). Can. J . Zool. 38, 189. Engelbert, V. E. (1961). Can. J. 2001.39, 367. Engelbert, V. E. (1967). Haemarol. Lar. 10, 65. Engelbert, V. E. (1968). Haemarol. Lar. 11, 349. Engelbert, V. E. (1970). Haemarol. Lar. 13, 1. Engelbert, V. E. (1971). Haernarol. Lar. 14, 1. Engelbert, V. E., and McMillan, D. B. (1962). Can. J . 2001.40, 83. Engelbert, V. E., and Young, A. D. (1970a). Can. J. Zool. 48, 209.

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