For the purpose to clarify the control mechanism of erythroid cell differentiation, the author observed morphologic changes in bone-marrow cells and circulating
red cells in phenylhydrazine anemia of rabbits by introducing a mass of red cells into vein at one time and reached the following conclusions. 1. After red cell transfusion in a mass to animal showing a marked hematopoietic activity, anisocytosis or macrocytosis becomes distinct with the appearance of big reticulocytes and red cells as large as four times the normal in
volume. This suggests, judging from their volume, the accelerated denucleation of erythroblast as early as at the late basophilic stage. 2. Observations on bone marrow at this stage revealed the reduction in the number of erythroblasts of undifferentiated type with the increase of rather differentiated ones. In erythroid islet, undifferentiated cells are found surrounding
a reticulum cell located in the center, while well differentiated ones in the outskirt area are situated near the sinusoid. Such a cell arrangement suggests that the erythroid cell requires a high oxygen tension for its differentiation. 3. From these observations and other results obtained from the studies on reticulocyte maturation and RNA synthesis of erythroblast, the author stresses that erythroid cells can differentiate as long as it is provided with a certain level of oxygen, even though it may develop m-RNA for differentiation. In other words, there should be two steps in the differentiation of erythroblast, the first is m-RNA synthesis induced by the information and the second is the somatic
protein synthesis with oxygen supply. This seems to be directly connected to the control mechanism of hematopoiesis by oxygen.</p

Matsuoka, Kenichi

English

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 19, Issue 5 1965 Article 1OCTOBER 1965Studies on reticuloendothelial system andhemato-poiesis, III. Relationship betweendifferentiation of erythroblast and oxygentensionKenichi Matsuoka∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on reticuloendothelial system andhemato-poiesis, III. Relationship betweendifferentiation of erythroblast and oxygentension∗Kenichi MatsuokaAbstractFor the purpose to clarify the control mechanism of erythroid cell differentiation, the authorobserved morphologic changes in bone-marrow cells and circulating red cells in phenylhydrazineanemia of rabbits by introducing a mass of red cells into vein at one time and reached the followingconclusions. 1. After red cell transfusion in a mass to animal showing a marked hematopoietic ac-tivity, anisocytosis or macrocytosis becomes distinct with the appearance of big reticulocytes andred cells as large as four times the normal in volume. This suggests, judging from their volume,the accelerated denucleation of erythroblast as early as at the late basophilic stage. 2. Observationson bone marrow at this stage revealed the reduction in the number of erythroblasts of undifferen-tiated type with the increase of rather differentiated ones. In erythroid islet, undifferentiated cellsare found surrounding a reticulum cell located in the center, while well differentiated ones in theoutskirt area are situated near the sinusoid. Such a cell arrangement suggests that the erythroidcell requires a high oxygen tension for its differentiation. 3. From these observations and otherresults obtained from the studies on reticulocyte maturation and RNA synthesis of erythroblast,the author stresses that erythroid cells can differentiate as long as it is provided with a certain levelof oxygen, even though it may develop m-RNA for differentiation. In other words, there shouldbe two steps in the differentiation of erythroblast, the first is m-RNA synthesis induced by theinformation and the second is the somatic protein synthesis with oxygen supply. This seems to bedirectly connected to the control mechanism of hematopoiesis by oxygen.∗Copyright c©OKAYAMA UNIVERSITY MEDICAL SCHOOLActa MeJ. Okayama 19, 217-225 (1965)STUDIES ON RETICULOENDOTHELIAL SYSTEM ANDHEMATOPOIESISIll. RELATIONSHIP BETWEEN DIFFERENTIATION OFERYTHROBLAST AND OXYGEN TENSIONKenichi MATSUOKADepartment of Pathology, Okayama University Medical School, Okayama, Japan(Director: Prof. S. Seno)Received for publicatiD/l, September 10, 1965In the previous papers1.2, it was described that reticuloendothelial cells orreticulum cells found in the center of erythroid islets play an important role forthe differentiation of cells. The author stressed that reticuloendothelial cellsshould give the information to the stem cells necessary for their differentiation.According to the concept obtained from the studies on bacteria, the developmentof inducer should be connected directly to the release of messenger RNA (m-RNA) for the synthesis of somatic proteins. However, it is obvious from thecytological observations that proerythroblasts, whose nuclei are very active inRNA synthesis\ possibly m-RNA synthesis, do not synthesize hemoglobin5 whichappears only at polychromatic stage, as revealed by the routine Giemsa staining.This discrepancy found between the stage m-RNA synthesis and somatic proteinsynthesis might be due to some factor that acts to inhibit hemoglobin synthesis.SENO and associates4 have clearly demonstrated that the maturation of reticulo-cytes, or hemoglobin synthesis accompanied by RNA degradation, requires ahigh level of energy which is produced in the process of oxidative phosphory-lation. By the inhibition of respiration by KeN or the ATP formation due tothe uncoupling agent for oxidative phosphorylation, the maturation of reticulo-cytes is depressed or greatly retarded6•These basic experiments have led the author to seek if the differentiationof young erythroblasts could be accelerated under highly oxygenated environment.As is well known, erythropoiesis is extremely suppressed under high oxygentension, but those that received information for differentiation may maturerapidly by synthesizing hemoglobin in their early developmental stage. In viewof this, observations were carried on the bone-marrow cells and the circulatingdenucleated cells of rabbit previously made anemic by phenylhydrazine injection,and the animal was transfused with a mass of red cells from normal animal inone dose. In this paper, it is reported that the differentiation of erythroblasts isaccelerated on early denucleation of erythroblast or on appearance of a striking2171Matsuoka: Studies on reticuloendothelial system and hemato-poiesis, III.Produced by The Berkeley Electronic Press, 1965218 K. MATsUOKAmacrocytosis, with a decrease of undifferentiated erythroblasts and an increaseof differentiated ones in number in the bone marrow.MATERIALS A..ND METHODSAdult male rabbits weighing 2.5-3.0 kg were used. Red cell count, hema-tocrit, hemoglobin content and red cell diameter were observed at certain in-tervals. The animals showing normal hematologic picture received subcutaneousinjection of phenylhydrazine hydrochloride, 1.5 cc of 2.5 % solution in distilledwater, daily three to four times by the method reported in the previous paper2•Two to three days after the last injection when anemia reached its critical pointwith an activated hematopoiesis or a marked reticulocytosis, five animals weresacrificed for the observation of bone-marrow tissue. Other five animals receivedthe intravenous introduction of red cells from a normal rabbit in a one massdose. The red cell suspension was prepared as follows: blood 80-100 cc wastaken by cardiac puncture from one non-anemic animal by using sodium citrate1/10 volume of 3.8 % solution as anticoagulant. The blood was centrifuged andthe precipitated red cells were isolated by removing blood plasma and buffy coat.Then the cells were washed with physiological saline solution three times byrepeated centrifugations and finally suspended in 50cc of saline solution. Imme-diately after preparation the cell suspension, the total red cells obtained fromone healthy animal, was introduced into ear vein of each anemic animal.Twenty-four hours after the introduction of cell suspension, the rabbitswere sacrificed for the observation of erythroid islet. The tissues of bone mar-row and spleen were sectioned and stained by the routine method for observa-tion with light microscope. Silver staining7 was resorted to in the case of ob-servation of reticular fibers.Price-Jones curves wece drawn on each animal at three stages, i. e. beforethe injection of phenylhydrazine and the red cell transfusion, and 24 hours afterthe red cell introduction. The curves were drawn on red cells in wet prepara-tion; they were photographed at the magnification of 100 X 10 and red cells inprint were picked up at random for the measurement of their diameters, taking200 to 300 cells for one curve.OBSERVATIONS AND RESULTSBy repeated injections of phenylhydrazine, the red cell count became lowerand at the critical stage where it reached 2 to 3 days after the last injection,the count was less than 2 million per cu mm, and the red cell diameter wasincreased markedly and Price-Jones curve showed a right shift, a macrocytosis,which is shown by the solid thin-line in Fig. 1.2Acta Medica Okayama, Vol. 19 [1965], Iss. 5, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol19/iss5/1Reticuloendothelial System and Hematopoiesis 219Fig. 1 Price-Jones curves of ablood-transfused rabbit afterphenylhydrazine Injections.Broken line; before the in-jection, solid thin-line: afterthe injection, solid thick-line:2! hours after blood trans-fusion.220 ~: ~.'3Xl,I, :,180 ,,E 160 J ,Eil,,~ 140 I,Q ,,)( 120 I! !E 100i!0;'-' 80 J,60 ,,40.,20 ,,)10 11 12 13 14Cell dlometer ()')Fig. 2 Demonstration of mac-rocytes in the circulatingblood of the same animalas in Fig. 1 and Table 1.Xl,5003Matsuoka: Studies on reticuloendothelial system and hemato-poiesis, III.Produced by The Berkeley Electronic Press, 1965220 K. MATSUOKATable 1 Change of circulating-blood picture of the same animal as in Fig. 1. RBC:red cell count (x 104/ cu mm), RC: percentage of reticulocytes in the circulatingerythroid cells (%), Ht: hematocrit (%), Hb: hemoglobin content (g/dl), MCV:mean corpuscular volume of circulating erythroid cells (cu p.).Hb I MCV I3.33J~ _9.60 I 8913.74 I 622040Ht391.251.46.5RC-'------------- Hemogram- I RBCTreatment -----__before injection of phenylhydrazine I 630after 4 injections of phE:nylhydazine I 175~~_~~~~r~~~~l~_b!~~ transfusion I 506-----'-----'-------'-------'----------Histologic observations of bone marrow of the animal sacrificed at this stagerevealed a large number of erythroblastic islets occupying almost the whole areaof the bone marrow. As reported previously2, in the center of each islet one ortwo reticulum cells were situated, around which were erythroblasts in variousdifferentiation stages, arranged radially. In the center of the islet there werefound many large erythroblasts with big nucleoli and basophilic cytoplasm, andthese were thought to be the stern cells or proerythroblasts. Surrounding thesecell groups there were more mature cells or smaller cells, probably basophilicerythroblasts, and on the margin of the islet just adjacent to the sinusoidal wall,there were very small erythroblasts with dark nuclei and weakly basophiliccytoplasm, probably polychromatic or orthochromatic erythroblasts. Some ofthe mature nucleated cells were in the cavity of sinusoid surrounding the islet,which was filled with denucleated cells, reticulocytes and mature erythrocytes(Figs. 3, 4). In the sections containing stained reticular fibers, the sinusoidalstructures were distinct (Fig. 5). In the extramedullary erythropoietic foci ofspleen, a similar cellular configuration as observable in the bone marrow wass~en, but in spleen the erythroid islet was smaller than the one in the bonemarrow, and the reticulum cell in the center was directly surrounded by a ma·ture nucleated cell layer (Fig. 6).At this stage, the other animals received intravenous injection of red cellsuspension, by which red cell counts and hemoglobin contents reached nearlynormal level. Twenty·four hours after this treatment a marked anisocytosis wasFigs. 3, 4 Section of bone marrow of a phenylhydrazine injected rabbit, showing erythroblasticislets. Reticulum cells are found in the center of the islets and large undifferentiatederythrobla!ots are situated in the central part, while small differentiated ones in the outerlayer. In sinusoidal cavities more mature erythroblasts are in company with mature erythro-cytes (H-E). ><1,000Fig. 5 The same section with staining for reticular fibers by Pap's methQ(j7. X I, 000Fig. 6 Section of spleen of the same animal as in Fig. 3, showing the islets in the sinusoidalcavities. Note that these islets are smaller than in bone marrow and erythroblasts sur·rounding reticulum cells are of more mature type. X 5304Acta Medica Okayama, Vol. 19 [1965], Iss. 5, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol19/iss5/1Reticuloendothelial System and Hematopoiesis 2215Matsuoka: Studies on reticuloendothelial system and hemato-poiesis, III.Produced by The Berkeley Electronic Press, 1965222· K. MATSUOKAFigs. 7, 8 Change of histologic picture by blood transfusion on a phenylhydrazine injectedrabbit. Fig. 7 is the picture before transfusion, predominantly showing large undifferentiatederythroblasts. Compared with this, Fig. 8 after transfusion shows an increase of smallmore differentiated erythroblasts. X 530induced, the Price-Jones curve showing three peaks as indicated by the solidthick-line in Fig. 1.The left peak with the base of 6 to 9.5 p. and with the summit at 7.5/1. cor-responds to the Price-Jones curve of normal red cells and mainly composed ofintroduced red cells; the middle one, with the base of 7.5 to 13.5/1. and withthe summit at 10/1., to that found before the red cell transfusion or that of ma-crocytes developed by phenylhydrazine injection; and the right one with thesummit at 11.5-12/1. is a new one appearing after transfusion. No peak ap-peared on the right side of the last one. Bone-marrow tissues taken 24 hoursafter the introduction of red cell suspension showed a picture still retaining activehemopoiesis. A large number of erythroblasts constituting erythroid islets wasobserved microscopically. However, in contrast to the tissue at critical stage ofanemia, erythroblasts of a relatively mature type were predominant, while large6Acta Medica Okayama, Vol. 19 [1965], Iss. 5, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol19/iss5/1Reticuloendothelial System and Hematopoiesisimmature ones were relatively less in number (Figs. 7, 8).DISCUSSION223According to the extensive studies of WEICKER8, the nuclear volume oferythroblast reduces by one half at each cell division, and that of orthochromaticerythroblast is only 1/16 of that of the tetraploid proerythroblast. SENO and as-sociates· have demonstrated that the cell volume of erythroblasts is also reducedby one half at each cell division and the cell size becomes smaller and smallerwith the advance of maturation, their cytoplasmic volume being reduced propor-tionately to the reduction rate of nuclear volume. This means that the red cell sizeindicates directly the denucleation st~:=(', as no cell division occurs after denuclea-tion9• Erythroid cell production may require a certain high level of oxygen.OHEDA10 stated that in the bone marrow of young rabbit arterial branches reachdirectly the epiphyseal area where the arterioles are densely distributed and ac-tive hematopoiesis can be seen. Under such a highly oxygenated environment,undifferentiated cells seem to be located in the area of relatively low oxygen,such as the central part of erythroid islet. It is the general concept in biologythat undifferentiated cells live in relatively anaerobic environments while the diff-erentiated ones in the aerobic environment. According to WARBURd\ the cells pro-liferating actively as tumor cells consume less oxygen than general somatic cells;the former get much energy from anaerobic glycolysis, while the latter relylargely on the energy produced by oxidative phosphorylation. This seems tosuggest that the cell cannot differentiate without getting the energy liberated byoxidative phosphorylation, even though it develops m-RNA for its differentia-tion. Erythroid cells are not an exception. The cellular configuration of erythro-blastic islet, in which undifferentiated young cells are located in the central partof the islet and differentiated ones in the outer layer, is consistent with this view,because it is thought that oxygen tension is rather low in the central area thanin the outer layer, which directly faces the sinusoidal lumen.As just mentioned, extramedullary hematopoietic foci found in spleen de-velop in the sinusoidal cavities having a sufficient blood supply, and these isletsare mainly composed of rather mature erythroblasts.In any area where the blood supply is supposed to be well maintained,there should be erythroblasts of more differentiated type and in hypoxic areaundifferentiated ones, indicating that erythroid cell proliferation and differentia-tion are controlled by oxygen tension, probably enhancing the proliferation ofundifferentiated cells with suppressed somatic protein synthesis in hypoxia andvice versa.7Matsuoka: Studies on reticuloendothelial system and hemato-poiesis, III.Produced by The Berkeley Electronic Press, 1965224 K. MATSUOKASUMMARYFor the purpose to clarify the control mechanism of erythroid cell differentia-tion, the author observed morphologic changes in bone-marrow cells and cir-culating red cells in phenylhydrazine anemia of rabbits by introducing a mass ofred cells into vein at one time and reached the following conclusions.1. After red cell transfusion in a mass to animal showing a marked hema-topoietic activity, anisocytosis or macrocytosis becomes distinct with the appear-ance of big reticulocytes and red cells as large as four times the normal involume. This suggests, judging from their volume, the accelerated denucleationof erythroblast as early as at the late basophilic stage.2. Observations on bone marrow at this stage revealed the reduction in thenumber of erythroblasts of undifferentiated type with the increase of ratherdifferentiated ones. In erythroid islet, undifferentiated cells are found surround-ing a reticulum cell located in the center, while well differentiated ones in theoutskirt area are situated near the ·sinusoid. Such a cell arrangement suggeststhat the erythroid cell requires a high oxygen tension for its differentiation.3. From these observations and other results obtained from the studies onreticulocyte maturation and RNA synthesis of erythroblast, the author stressesthat erythroid cells can differentiate as long as it is provided with a certain levelof oxygen, even though it may develop m-RNA for differentiation. In otherwords, there should be two steps in the differentiation of erythroblast, the firstis m-RNA synthesis induced by the information and the second is the somaticprotein synthesis with oxygen supply. This seems to be directly connected tothe control mechanism of hematopoiesis by oxygen.lXEEERENCES1. MATSUOKA, K.: Studies on reticuloendothelial system and hematopoiesis. 1. Studies ofextramedullary hematopoiesis. Acta. Med. Okayama 19, 107, 19652. MATSUOKA, K.: Studies on reticuloendothelial system and hematopoiesis. H. A study onthe morphology and function of erythroblastic islet. Acta Med. Okayama 19, 161. 19653. ]ACOB, E. and MONOD, ].: Genetic regulatory mechanism in the synthesis of protein. f.Mol. Bioi. 3. 318, 19614. SENO. S.: Studies on the differentiation of erythroblast by using radio isotopes. ActaHaem. fap. 27, 718, 19645. SENO, S.: Globin synthesis in erythroblast, in correlation with the syntheses of total protein,RNA, DNA and cell differentiation. Acta Haem. fap., 24, 629, 19616. MIYAHARA, M., SENO, S., HAYASHI, K. and OCHI, 0.: Accumulation of hypoxanthinein reticulocytes affected by 2,4-dinitrophenol and antimycin A. Biochim. Biophys. Acta 95,598, 19657. PAP, T.: Ein neue Methode zur Implegnation des Reticulums. Zentralbl. all. Path. u.path. Anat. 47, 116, 19308Acta Medica Okayama, Vol. 19 [1965], Iss. 5, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol19/iss5/1Reticuloendothelial System and Hematopoiesis 2258. WEICKER, H.: Das Erythron unter physiologischen und pathologischen Bedingungen. An-na/es Nestle 20, 13, 19639. LOWENSTEIN, L. M.: Studies on reticulocyte division. Exptl. Cell Res. 17, 336, 195910. OHEDA. W.: Embryological studies on blood vessels of bone marrow. Blood vessels ofbone marrow in femur in the case of newborn and young rabbits. Okayama-lgakkai-Zasshi(in Japanese) 66, 563, 195411. WARBURG, 0.: On the origin of cancer cells. Science 123. 309, 19569Matsuoka: Studies on reticuloendothelial system and hemato-poiesis, III.Produced by The Berkeley Electronic Press, 1965

Studies on reticuloendothelial system and hemato-poiesis, III. Relationship between differentiation of erythroblast and oxygen tension

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