The disappearance of nucleolus has been traced in the rat erythroid cells in relation with the cell specialization under varying conditions, i. e.
in anemia with or without treatment by bromouracil and aminopterin. To make the findings more reliable the observations have been made on tissue section as well as on the smeared samples as the nucleolus becomes
often indistinct in smeared cell. The results indicate that under anemic condition nucleolus is lost by the late basoplilic stage. Treatment with bromouracil retained the nucleoli and cytoplasmic basophilicity till later
stage of cell specialization suggesting some similar mechanism of RNA disintegration both in nucleolus and cytoplasm.</p

Shigehisa, Morio

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Studies on the relation between heme and nucleic acid syntheses in erythroid cell. 3. Nucleolus of erythroblast, changes in relation with cell specialization

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Acta Medica OkayamaVolume 23, Issue 1 1969 Article 1FEBRUARY 1969Studies on the relation between heme andnucleic acid syntheses in erythroid cell. 3.Nucleolus of erythroblast, changes in relationwith cell specializationMorio Shigehisa∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Studies on the relation between heme andnucleic acid syntheses in erythroid cell. 3.Nucleolus of erythroblast, changes in relationwith cell specialization∗Morio ShigehisaAbstractThe disappearance of nucleolus has been traced in the rat erythroid cells in relation withthe cell specialization under varying conditions, i. e. in anemia with or without treatment bybromouracil and aminopterin. To make the findings more reliable the observations have been madeon tissue section as well as on the smeared samples as the nucleolus becomes often indistinctin smeared cell. The results indicate that under anemic condition nucleolus is lost by the latebasoplilic stage. Treatment with bromouracil retained the nucleoli and cytoplasmic basophilicitytill later stage of cell specialization suggesting some similar mechanism of RNA disintegrationboth in nucleolus and cytoplasm.∗PMID: 4241733 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. O:,ayama 23, 1-6 (1969)STUDIES ON THE RELATION BETWEEN HEME AND NUCLEICACID SYNTHESES IN ERYTHROID CELLIII. NUCLEOLUS OF ERYTHROBLAST, CHANGES INRELATION WITH CELL SPECIALIZATIONMorio SHIGEHISADepartment oj Pathology, Okayama University Medical School,Okayama, Japan (Director: Prof S. Seno lReceived jor publication, NovembJr 18, 1968The function of nucleolus is not yet fully clarified, but generally itis believed that the nucleolus is essential for the formation of ribosome.BROWN and GURDON (1) showed inability to synthesize new ribosomalRNA in defective mutant of aquatic toad of Xenopus laevis which fails toform nucleolus. The data definitely indicate that nucleolus is essentialfor the synthesis of ribosomal RNA. CAMERON et al. (2) observed therelation between morphologic changes and the RNA and protein synthesisby using Tetrahymena pyriformis revealing that an increase in RNA synthesisfollowed by protein synthesis was initiated with the concomitant disag.gregation of the large fusion bodies into nucleoli, which ensued by bring.ing the animal into a fresh proteose peptone medium. They suggestedthat there should be a close correlation between unfused nucleoli andribisome formation. RITOSSA and SPIEGELMANN (3) reported that nucleolusis the site of ribosomal RNA synthesis from experiment of Drosophilamelanogaster. Apart from these, there are several papers (4-10) suggestinga close relation between nucleolus and r-RNA synthesis. Certain mamma·lian cells have also distinct nucleolus and some studies on RNA synthesishave been carried out by using liver nucleoli (11).In erythroblast distinct nucleolus can always be distinguished in earlystage of specialization, but soon it disappears with the advance of speciali-zation stages, i. e. proerythroblast have one or two nucleoli but basophilic,polychromatic and orthochromatic erythroblasts do not have any clearnucleolus: This may imply an increase in the RNA synthesis by thedisaggregation of the nucleoli, as the cells divide actively. However,SENO et al. (12) showed that RNA content of erythroblast decreased by onehalf at each cell division proportionate to the decrease in cell volume i. e.there is no actual increase in the RNA content during cell specialization.fhis phenomenon will suggest that there should not be a remarkable1Shigehisa: Studies on the relation between heme and nucleic acid synthesesProduced by The Berkeley Electronic Press, 1969M. SHIGEHISAr·RNA synthesis in the basophilic and lata specialization stages. But in theerythroblast whose cytoplasmic basophilicity is retained till the later stageof specialization the nucleolus is often found even in the cells of advancedspecialization. In this paper it is reported that erythroblast retains itsnucleolus till later stage of specialization in the animals receiving bromo-uracil injection.MATERIALS AND METHODSAdult Wister rats weighing 250 to 270 g were used. These rats were madeanemic by repeated phenylhydrazine-HCl injection subcutaneously, 0.3 ml of1.25 % solution per 100 g body weight once a day for three days successively.They were divided into three groups. The animals of first group were treatedwith aminopterin (AP) injection, 2 mg per 100 g body weight, and sacrificed24 hours after the AP injection. The animals of second group were injectedwith bromouracil (BU), 25 mg per 100 g body weight daily for 2 to 3 days andsacrificed 24 hours after the last injection of BU. Those of third group wereanemic control, which were killed by decapitation three days after the lastphenylhydrazine injection.The fresh bone marrow tissue was obtained from femur, smeared andstained with Giemsa. Besides these a small piece of bone marrow tissue of about1.5 mm was fixed by glutaraldehyde for 60 min, washed with distilled waterovernight, dehydrated and embedded in Epon. Sections of about 4 p. thickwere obtained by using glass knife, stained with methylgreen-thionin fornucleolus at 40°C for 30 min. The dye solution was prepared by mixing 1 %methylgreen (5 ml), 0.275 % thionin (3 ml), 0.1 M citrate buffer (pH 5.6, 12.5ml) and distilled water (29.5 ml). The nucleolus is stained purple by thistechnique.For the purpose to discriminate of the specialization stage of each cellin tissue section and in wet the diameters of cells of tissue section embeddedin Epon, smeared cells and fixed ones were compared on lymphoid cells frommesenterial lymph node of normal rat, and the changes in cell diameter byfixation and by smearing were observed estimating nuclear diameters of 300cells in one sample. Small stick of the lymph node tissue was taken on anobject glass added with a drop of blood serum, crushed gently with cover-slide and the free cells thus obtained were observed in wet under commonlight microscope immediately. Another piece of the same tissue was alsocrushed and smeared, dried, fixed with methanol and stained with Giemsa.And still other one piece was fixed with glutaraldehyde and embedded inEpon and sectioned.RESULTS AND DISCUSSIONSThe tissue sections of bone marrow of anemic animal stained by2Acta Medica Okayama, Vol. 23 [1969], Iss. 1, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss1/1Relation Between Heme and Nucleic Acid Syntheses in Erythroid Cell 3methylgreen thionin showed clearly the nucleolus in large erythroblats(7-8 fl. in nuclear diameter) having nucleolus, but not in the small ones (5fl.and smaller) i. e. the nucleolus disappears at a certain stage of specializa-tion in these cells (Fig. I, line a). On smear the cells appeared larger thanthose in tissue sections. The largest cells having distinct nucleoli of 7-8 fl.a c bEB100l/).2ju='Z~.~ 50.cIn;§'0~E~ 0 LB -.-J--+-.........,;.-L .. iFig. 1 This figure shows the changing rateof cell size by smearing and fixing withmethanol and fixing with glutaraldehyde anddehydrating through ethanol and embeddin6in Epon.Each point shows the mean value of about100 cells in each specialization stage and thecell size is reduced or enlarged proportionateto the original size after fixation, dehydrationand embedding or smearing, drying andfixing.(a) Erythroblasts in section(b) Erythroblasts in smear(c) Expection curve of erythroblasts inwet5 7 9 11 13 fJNuclear Diameter0/040302105 6 7 8 9 10 11fJcell DiameterFig. 2 Changes in cell size1.of living lymphocytes in serumby smearing and dryingThe smeared cell is flatten-ed and enlarged by about 25per cent.(a) Lympocytes in wet(b) Lympocytes in smearnuclear diameter on tissue section were com·parable to those of about 12-13 fl- on smear,which were the largest in size and had nuc-leoli. For getting the exact information onthe change in cell size by different treatmentsi. e. in tissue sections and smeared sample,the size of lymphocyte from one lymph nodewas observed comparing to these on tissuesection, smear and wet preparation.The mean value of cell diameter of Iym.phocytes proved to be 6.28 ± 0.25 fl. in wet,7.88 ± 0.54 /-t in smear (Fig. 2) and about 4.7l'-in tissue sections. The difference in the twomean values between the wet and the smearspecimens was about 1. 6l-t, 25 %.. Namely,spherical lymphocyte is enlarged by 1.25times in diameter being flattened by smear-ing, and shrunk to about 25 % by fixation,3Shigehisa: Studies on the relation between heme and nucleic acid synthesesProduced by The Berkeley Electronic Press, 19694 M. SHIGEHISAdehydration and enbedding. So, 8 If. nucleus of erythroblast on Eponsection is equal to 10.5 p. nucleus of cell in wet and 13 Ill. nucleus insmeared sample. These values coincide well with those obtained by actualestimation. Thus, the decreasing rates of cell diameter of erythroblastwith the advance of cell specialization are drown diagrammatically as thelines, a (fixed tissue), c (wet), b (smear), appearing in Fig. 1. This in-dicates that the largest proerythroblast is 8, 10.5, 13 Ii. in diameter, earlybasophilic erythroblast 6.2, 8.3, 1O.4,'J. and late basophilic one 4.8, 6.4,8. 1 /-~ in tissue section, wet and smeared samples, respectively.As can be understood from the cell size in each preparation, thenucleolus is present in 100 % of proerythroblast, in 60 % of early baso-philic erythroblast and in less than 10 96 of late basophilic erythroblast.That is, the nucleolus disappears completely by the end stage of late baso-philic erythroblast. Therefore, in the erythroblast of anemic rat theproduction of ribosome should terminate at the late basophilic stage,assuming that the nucleolus is the site of ribosome formation or thesynthesis of r-RNA. The data are consistent with those reported by SENDand collaborators (12) in that the synthesis of RNA markedly drops atpolychromatic stage as observed by the incorporation of uridine into invitro. As reported in the previous paper (13) the RNA synthesis of theerythroblast of anemic rat observed by the uridine incorporation was notaltered through the treatment with aminopterin and bromouracil. But thetreatment with bromouracil impressed the delay in the disappearance ofnucleoli. As the bromouracil treatment resulted in the delay in the disap-pearance of basophilicity of cytoplasm, the retained nucleoli till laterstage of specialization may be correlated to the delayed disintegration ofRNA in cytoplasm. (14)From such a viewpoint the nucleoli have been observed on the cellsfrom the anemic animals treated with BU. Observations were also madeon the cells from AP-treated animal in which the basophilicity decreased atrather earliar stage of specialization comparing to the anemic control. InFig. 3 the number of percentage of the nucleolus having cells at eachspecialization stage has been presented. That is, all the proerythroblastshad nucleolus, but in early basophilic erythroblast the number of nucleolushaving cells was much reduced in both anemic control and AP treatedanimals, while in those from BU treated animals nearly one half of thecells had nucleoli. And even in late basophilic erythroblast fairly a largenumber of cells had nucleoli (Fig. 3).The data indicate that the retardation of the reduction of basophili-city of RNA degradation (cytoplasmic basophilicity disappears completely4Acta Medica Okayama, Vol. 23 [1969], Iss. 1, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss1/1Relation Between Heme and Nucleic Acid Syntheses in Erythroid Cell 5100i{,..... n.oP- t:~:~ .?'0 50jjE~zProE EB LB PSpecialization StageoFig. 3 Number percent of erythroblasthaving nucleolus. Rats were made anemic bythree injections of phenylhydrazne, 6.7 mgper animal per day for three days and afterthree-day interval they were treated withaminopterin and bromouracil.BU : The cells from those treated with bromo-uracil, 24 hours after 4 injections, 25 mg per100 g body weight daily for four days. AP:The cells from the animals treated with amino-pterin, 24 hours after the single injection 2 mgper 100 g body weight. C: Anemic control. ProE: Lagre sized proerythroblast, EB: Earlybasophilic Ebl. LS: Lat~ basophilic EBI.P : polychromatic Ebl. 0: orthochromatic Ebl.by the treatment with RNase) is somehow correlated to the disappearanceof nucleolus, irrespective of the RNA synthesis. The mechanism is notclear at present, but it seems to be similar to disintegration mechanism ofRNA both in cytoplasm and nucleoli.SUMMARYThe disappearance of nucleolus has been traced in the rat erythroidcells in relation with the cell specialization under varying conditions, i. e.in anemia with or without treatment by bromouracil and aminopterin.To make the findings more reliable the observations have been made ontissue section as well as on the smeared samples as the nucleolus becomesoften indistinct in smeared cell. The results indicate that under anemiccondition nucleolus is lost by the late basoplilic stage. Treatment withbromouracil retained the nucleoli and cytoplasmic basophilicity till laterstage of cell specialization suggesting some similar mechanism of RNAdisintegration both in nucleolus and cytoplasm.ACK.NOWLEDGEMENTThe author is indebted much to Frofessor Satin::aru SENO for his beneficial guidanceand encouragement throughout the course of this work. Many thanks are due to the mem-bers of tl-.e Department of Patl-.ology for invaluable suggestions, and to Miss H. HIGASHIOKAfor her technical assistance.REFERENCES1. BROWN, D. D. and GURDON, J. B.: Absence of ribosomal RNA synthesis in the anudeo-lated mutant of Xenopus laevis. Proc. Nat. Acad. Sci. 51, 139, 19645Shigehisa: Studies on the relation between heme and nucleic acid synthesesProduced by The Berkeley Electronic Press, 196962.3.4.5.6.7.8.9.10.11.12.13.14.M. SHIGEHISACAMERON, 1. L. and GUILE, E., E. JR.: Nucleolar and biochemical changes duringunbalanced growth of Tetrahymena pyriformis. ]. Cell Biol. 26, 845, 1965RITOSSA, F. M. and SPIEGELMANN, S.: Localization of DNA complementary to ribosomalRNA in the nucleolus organizer region of Drosophila melanogaster. Proc. Nat. Acad. Sci. 53,737, 1965MCCONKEY, E. H. and HOPKINS, J. W.: The relationship of the nucleolus to the synthesisof ribosomal RNA in HeLa cells. Proc. Nat. A cad. Sci. 51, 1197, 1964SIRLIN, J. L., TANNDER, C. J. and JACOB, J.: The relationship between the nucleolusorganizer and nucleolar RNA. Exptl. Cell Res. 31, 611, 1963BusH, H., MURAMATSU, M., ADAMS, H., STEELE, W. J., LIAU, M-C. and SMETANA, K.:Isolation of nucleoli, Exptl. Cell Res. Suppl. 9, 150, 1963PERRY, R. P.: The cellular sites of synthesis of ribosomal and 4S RNA. Proc. Nat.Acad. Sci. 48, 2179, 1962SIRLIN, J. L., JACOB, J. and KATO, K. -1.: The relation of messenger to nucleolar RNA.Exptl. Cell Res. 27, 355, 1962EDSTROM, J-E., GRAMPP, W. and SCOR, N.: The intracellular distribution and hetero-genity of ribonucleic acid in starfish oocytes. ]. Biophys. Biochim. Cytol. 11, 549, 1961SIRLIN, J. L.: Cell sites of RNA and protein synthesis in the salivary gland of Smittia(Chironomidae), Exptl. Cell Res. 19, 177, 1960MURAMATSU, M.: RNA synthesis in mammalian cells. Protein, Nucleic Acid and En<.yme 12,922, 1967 (in Japanese)SENO, S., MIYAHARA, M., ASAKURA, H., OCHI, 0., MATSUOKA, K. and TOYAMA, T.:.Macrocytosis resulting from early denucleation of erythroid precursors. Blood 24, 582,1964SHIGEHISA, M.: Studies on the relation between heme and nucleic acid syntheses inerythroid cell. II. Nucleic acid synthesis in erythroblast of anemic rat treated withaminopterin and bromouracil. Acta Med. Okayama 22, 319, 1968SHIGEHISA, M.: Studies on the relation between heme and nucleic acid synthesis inerythroid cell. 1. Effects of aminopterin and bromouracil on hemopoiesis and hemoglobinsynthesis in anemic rat. Acta Med. Okayama 22, 251, 19686Acta Medica Okayama, Vol. 23 [1969], Iss. 1, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol23/iss1/1

Studies on the relation between heme and nucleic acid syntheses in erythroid cell. 3. Nucleolus of erythroblast, changes in relation with cell specialization

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