RELAYIVE VOLUME CHANGES OF THE NUCLEOLUS EN RELAnou TO CELL AND mews m mum $A'E‘NUM AND TRADEECANFlg. PALQ?0$A Thesis for ”10 Degree of M. S. MICHIGAN STATE UNIVERSZTY Lee Virn Leak .1959 THE-3953 ('- LIBRAR . I‘llic‘hignn State University F37 TIVE VOLde CHANGES OF THE NUCLEOLUS IN RELATION TO CELL AND NUCLEUS IN PISUM SATIVUM AND EMSCAN‘I' IA PALUCDOSA By Lee Virn Leak AN ABSTRACT Submitted to the College of Science and Arts of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1959 / ',/ Approved By % 0% Lee Virn Leak l A knowledge of the volume changes of the nucleolus in relation to that of the cell and nucleus is of prime importance to the cytologist in considering the mitotic cycle. The phases or points of volume ins crease and decrease of the cell components must also be considered. Meristematic cells of gigum,root tips and microspore cells of Tradescantia were used as merterials for obtaining measurements of nuc- leolus, ucleus and cell. The nucleolus, nucleus and cell are clearly differentiated after specific fixing and staining procedures. The nucleolus shows a definite volume increase at the inception of active mitosis (interphase to early prophase) irregardless of nucleo- lar fusion. Thereafter volume increases cease and during late prophase the nucleolar volume is again the some or less than that in interphase. Along with nucleolar volume increase there is also a volume increase in cell and nucleus. The number of nucleoli vary from one to three in giggm,sativum and from one to four in W W- Fusion of nucleoli was observed from interphase to late prephase in.gi§gm, however, there was no indication of fusion in stages 1, 2, 3 and h in the microspore cells of W. RELATIVE VOLUME CHANGES OF THE NUCLEOLUS IN RELATION TO CELL AND NUCLEUS IN EISUM SATIVUM AND IEAQESCANTIA PALUDO§§ BY Lee Virn Leak A THESIS Submitted to the College of Science and.Arts of Michigan State University of Agriculture and.Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and.Plant.Pathology 1959 4,1,]- ‘37 I; {/03 ACKNOWLEDGEMENTS The author wishes to express his deepest gratitude to Dr. G.B._Wilson, first for the inspiration from his lectures in Cytogenetics to undertake this study, and for his most generous and continued assistance in direct- ing and carrying out this study. Also sincere appreciation for the helpful ideas obtained from Dr. L. Mericle, Dr. E. Cantino and Dr. J.H. Morrison. To Mr. P-G. Coleman for his excellent photographic services, Miss Gabriele Mdhling for her wonderful services in typing the manuscript, and other members of the cytology group and botany department, many thanks are given. In addition deepest appreciation is exPressed to the.Agricultural Experimental Station for their financial aid in this project. INTRODUCTION LITERATURE REVIEW MATERIAL AND PROCEDURE CYTOLOGICAL.EXAMINATION OBSERVATIONS DISCUSSION SUMMARY B IBLIOGRAPHY TABLE OF CONTENT PAGE ‘3 11 1.6 28 35 37 TABLES 10. 11. LIST OF TABLES Index of cell volume from squash preparations in Pisum. Index of nuclear volume from squash preparations in Pisum. Index of nucleolar volume from squash preparations in Pisum. Index of cell volume from longitudinal sections in Pisum. Index of nuclear volume from longitudinal sections in Pisum. ' Index of nucleolar volume from longitudinal sections in Pisum. Index of cell volume in microspore cells of Tradescantia. Index of nuclear volume in microspore cells of Tradescantig. Index of nucleolar volume in microspore cells of Tradescantia. Ratios of cell/nucleus. cell/nucleolus and nucleus/ nucleolus in Pisum. Ratios of cell/nucleus, cell/nucleolus and nucleus/ nucelolus in W. PA GE 13 13 14 14 In 15 15 30 30 LIST OF TEXT FIGURES TEXT FIGURES 1. (‘0 10. PLATES II. Mean volume of nucleolus from squash.preparations in gisum. Percentage volume increase of cell, nucleus and nucleolus from squash preparations in.Pisum. Frequency of cells containing one, two, three and four nucleoli from squash preparations in.Pisum. Mean volume of one, two, three and four nucleoli per stage in meristematic cells of Pisum. Percentage volume increase of cell, nucleus and nucleolus from longitudinal sections of Pisum. Percentage volume increase of cell, nucleus and nucleolus in microspore cells of Trageggantig, Mean volume of nucleolus in microspore cells of mm- Mean volume of one. two, three and four nucleoli per stage in microspore cells of Imageggantig, Frequency of cells containing one, two, three and four nucleoli in microspore cells of Trageggantia, Mean volume of nucleolus from longitudinal sections in P1 39' LIST OF PLATES A series of normal divisions showing interphase, early prephase and late prophase from.squash.preparations and longitudinal sections in.21§ygp A series of figures showing stages measured in micro- spore cells of W- PAGE 17 17 20 22 22 25 25 27 27 PAGE 18 23 INTRODUCTION The nucleolus is a spherical or oval-shaped intranuclear body, which exhibits variable degrees of affinity fer acid and basic dyes. Hucleolar formation is generally regarded as taking place during telo— phase in association with a special region of one or more chromosomes of the haploid couplement (Gates, 19h2; Kaufman, 19148: Hankansson and Levan, 1952). Battenbury and Serra, (1952) observed that the number of nucleoli per cell varies with different organisms. It may be observed as a conspicuous component of the nucleus during interphase, early'pro- phase, late prophase and telophase. The nucleolus has been the subject of extensive cytological study since its discovery by rontana in 1781. Little, however, is known at the present time of its exact function or the morphological changes which.it undergoes in either the dividing or nondividing cell. Biochemical evidence indicates that the nucleolus contains ribo- nucleo-protein, and is an.activs site of incorporation of labelled nucleic acid and.protein precursors in certain cell types (Brachet.l957). It is generally assumed that there is a.progressive diminution in nucleolar volume from.interphass to its dissolution before the onset of metaphase (Swanson, 1957). Prelimdnary experiments in this laboratory have indicated, however, that the inception of mitosis is accompanied by'a corresponding nucleolar volume increase (Wilson and Morrison, 1958). In view of the little known concerning the function of the nucleo- 2 lus during cell division, it is essential that more precise cytological information be available in order to understand more thoroughly the role played by this cellular organelle in the mitotic process. The present investigtion, therefore, was carried out to determine: (1) the changes in nucleolar volume from interphase through late prophase in meristematic cells of m m, (2) changes in the nucleolus rela— tive to cell and nuclear volume change, (3) nucleolar fusion in meri- stematic cells of 21m, and ('4) changes in the nucleolus from inten- phase to prophase in microspore cells of W W- LITERATURE REVIEW Since the discovery of the cell by Robert Book in 1665 and the formulation of the cell-theory by Schleiden, (1938) and Schwann, (1839), many studies have been made on the cell as a unit as well as on its structural components. There remain however, many questions as to the function of may cellular components. W: The nucleolus is an organ of the cell which is almst universally present. It is absent or at least not detectable by present techniques in lower Cyanophyceae (Oscillatoria) as shown by Spearing, (1937). According to Montgomery, (1898) Iontana in 1781 was the first to observe the nucleolus in the epithelial cells of the eel, and described it as " a rounded or oval body with a spot in the middle”. Valentin describes the nucleolus in conjunctiva as a rounded corpuscle which appeared to form “a kind of second nucleus“ within the nucleus (Gates, 19142). The nucleolus was first discovered in plants by Schleiden (Wilson, 1898). Origin Of The nucleolus: It is generally recognized by cytolo- gists that the nucleolus is formed during teIOphase of reproductive and somatic cells in both plants and animals. Some mintain that the origin of nucleolar substance is the chromatin (Kc01intock, 1931}; Gas- persson, 1950), while others claim it to be a derivative of the peri- plasm (see Dermen, 1933: Tischler, 1992). According to Heitz (1931) and mtsuura (1938) every chromsome of the complement can be regarded as a "nucleolar chromosome" in the sense that they can all produce nucleoli under certain specified conditions. Battenbury and Serra (1952) consider that the two types of nucleolar formation are possible at telophase, namely: (1) the production of layers of pronucleolar substance over the chromosome surface and in the lacunae between them (layer type), and (2) the formation of pronucleolar droplets (droplet type). During the mitotic process the nucleolus gradually diminishes, and is usually dissolved or dissipated by metaphase. In some organ? isms, however, the nucleoli may persist throughout the mitotic period, dividing at anaphase or passing undivided to one or the other of the poles (Swanson, 1957). According to Battenbury and Serra (1952) the number of nucleoli formed at telophase varies considerably. The number of nucleolar droplets formed may exceed the number of nucleolar zones present, the former ranging from one to one or two hundred in some species. These droplets fuse, thus bringing about a decrease in number. A mutation has been.produced by Elsdale, Frischberg and Smith (1958) that reduces the nucleolar number in Xenopus lggzig, An.expected Mendelian ratio (1:2:1) is obtained among the offspring from.a cross of two heterozyb gotes, i.e., one quarter with cells containing two nucleoli, one half having but a single nucleolus in each cell, and one quarter having no nucleoli at all. Composition Of The Nucleolus: By using a preparation of ribo- nuclease, Brachet (19h1) was able to show that the basophillia of the nucleolus was due to its content of ribonucleic acid (RNA). With the 5 ultra-violet absorption spectrum Caspersson and Schultz (l9h0) observ- ed that although the nucleolus had.a nucleic acid absorption spectrum, it did not give a positive Feulgen reaction. They concluded, on this basis that the nucleolus contained ribonucleic acid. That protein is present in considerable quantities has been reported by Pollister and His (l9h8): Caspersson (1950): Furnberger et a1 (1952): Vincent and Huxley (195“); and Vincent (1955). The presence of Peulgen.positive material in nucleoli has also been reported many times in the litera- ture (Duryee and Doherty, l95h). Brashet (1957) suggested this could result from penetration of heterochromatic segments of chromosomes into portions of the nucleolus. The nucleoli of certain cells have been re— ported to contain phosphatase (Danielli, 1933; Bradfield, 1951), the OPE-synthesizing enzyme (Baltus, 195a), and minerals (Vincent, 1952: Immers, l95h). The presence of lipoidal materials has been suggested from the action of certain lipid solvents on nucleolar structure (Gates, l9h2). Observations on fusion in living and fixed material (Montgomery 1898; Strangeway, 1923: Gates, 19h2: and Hughes, 1952) suggest the nuc— leolus to be a more or less fluid body. That it exists as a coacerh vate is suggested.by Ehrenberg (l9u6). Others, however, maintain that it contains variable quantities of proteins with high densities and appears as a send-solid body (Pollister and His, l9h7; Vincent, 1955). Englgglazgtnngjign: Numerous hypotheses of nucleolar function have been suggested. Vincent (1955) gives the following list: (1) to shield the chromosomes from the cyt0plasm during mitosis; (2) to serve as a transfer of chromosomal influence to the cytoplasm; (3) to func— 6 tion as a storage point for the materials produced at a restricted rate by the chromosomes: (h) to operate as a site for limiting rates of reac- tion necessary for mintenance. of cytoplasmic synthesis; ( 5) to serve as a site of accumulation of chromsomal or intranuclear products; (6) to serve as a site of accumulation of unutilized and/or unusable materials of cytoplasmic origin which enter the nucleus but cannot re- turn to the cytoplasm; and (7) to function as a reservior of energy source for nuclear activity. None of these suggested functions has much experimental basis. There is very little information on the relationship of the nuc- leolus to the cell or nucleus, or on its volume change from interphase to its dissolution or to the state where it is no longer recognizable. MATERIAL AND PROCEDURE For cytological investigation of the cell, nucleus and nucleolus, a fixative and staining procedure must be used that will differentiate these cellular components with a minimum amount of’physical distortion. A method which met these criteria would allow one to make accurate meas- 'urements of the cell components in question. Several fixatives were tested in order to obtain preparations in which chromatin, nucleolus, and cytoplasm could be observed. The ”alco- hol-formal-acetic' fixative (Battenbury, 1951) and numerous combinations of methanol, chloroform and propionic acid were tested. These did not give satisfactory results for*accurate measurements of nucleolus, nuc- leus and cell volumes. By using 15 parts of.ETOH (ethyl alcohol), 1 part 40 per cent formaldehyde and 3 parts propionic acid the desired re- sults were obtained (Morrison, Leak and Wilson, 1959). The meristems of young root tips (2-3 cms. basipetally from the root cap) of zigug_sativum var; Alaska, and microspore cells of Tradegr gang pgludog were used as materials for measurements. Neither the giggg_nor Tradesgggtia was treated.with chemicals prior to fixation, so measurements were made of ”normal” tissue. The peas were soaked overnight in glass distilled water at 25°C. They were then roll- ed in paper toweling and the rolls were placed vertically in beakers one third filled with glass distilled water. The peas were allowed to garb minate in an incubator at 25°C for #8 hours. The root tips were ex- cised (one centimeter basipetally from the root cap) and fixed in 15 parts 95 per cent,ITOH, 1 part #0 per cent formaldehyde and.3 parts pro- pionic acid. While in the fixative the root tips were placed in a vase uum and evacuated from 1—2 hours, after which they were removed from the vacuum and allowed to fix for 12 hours under refrigeration. Sguash Prgparations: The tissue was hydrolyzed in 1 normal HCl for 15 minutes at 60°C, stained with Schiff's Reagent for 30 minutes, then stained in aceto-carmine from 10-15 minutes. After staining with aceto- carmine the meristmes were removed from the excised root tips and squashed in a drop of .05 per cent fast green in #5 per cent acetic acid. A cover glass was set over the squashed material and the underside of the slide was gently heated. The slide was then.p1aced.between several layers of filter*paper and pressure applied. After squashing the slides were placed in 90 per cent TBA for 2h hours, removed.and made permanent with diaphane. W: The root tips were removed from the fixa- tive, dehydrated and embedded in.paraffin (as cited by Mericle, 1957). Longitudinal sections were cut with the microtome at 16 microns, the sec- tions then mounted on slides and allowed to dry, after which the parap ffin.was removed with xylene. The tissue was hydrolyzed in 1 normal ECl for 18 minutes at 60°C, and stained with Schiff's Reagent for 30 minutes. This was fellowed by additional staining with.aceto-carmine for 3-5 min- utes, and counter staining in .05 per cent fast green in.h5 per cent acetic acid for 2 minutes. The tissue was transferred to a staining jar containing equal volumes of alcohol and xylene for 1-2 minutes, and then cleared in xylene and mounted in clarite. Procedure For Handlinngicrogpore Cells: Buds were selected in series from flowering heads of Izgdgfiggnjig_plants with stages ranging from the tetrad thrmugh the first microspore division. The fixative used for the root tips did not give the differentiation necessary for accurate examination of the cell components. After various proportions of 95 per cent ETOH, ho per cent fermaldehyde and propionic acid were tested it was found that 15 parts of 95 per cent ETOH, 5 parts of #0 per cent formaldehyde and 3 parts of prOpionic acid gave the desired results (clearly defined.boundaries) for accurate measurements of cell, nucleus and nucleolus. Microgpore Smear’Preparations: The six anthers were dissected from each.bud and.p1aced on separate slides. By gently teasing the any there with the flattened end of’a glass red, the microspore cells were extracted with very little damage to the cell walls, and with little contamination from the anther wall. The microspore cells readily ad, hered to the surface of the dry slides, which were quickly placed in a staining jar containing the fixative and allowed to fix from 3-5 min~ utes. After fixation the tissue was washed in distilled water for 3 minutes, hydrolyzed in 1 normal 301 for 3 minutes at room temperature and stained with Schiff's Reagent for 3-5 minutes. The slides were rinsed with 1 normal HCl, stained in aceto-carmine for 2 minutes, counr ter stained with .05 per cent fast green in #5 per cent acetic acid for l ndnute. Cover glasses were placed on the slides and the measurements were made from temporary mounts. The mounts were placed.in.a glass chamber that was moistened with R5 per cent acetic acid and stored under 10 refrigeration to prevent drying. In root tips of 21m and the microspore cell of W the nucleolus is stained a bluish green. The aceto-carmine is used to in- tensify the staining of the chromosomes. With the above technique the boundaries of the nucleus and nucleolus are clearly defined in both the meristemtic cells of 21m and the microspore cells of W. 11 CYTOLOGICAL EXAMINATION The preparations were examined.with a 97X oil objective and 25X oc- ulars. A 25X ocular containing a micrometer scale was used for measur- ing the width and length of cell, nucleus and nucleolus. The index of volume of cell, nucleus and nucleolus from squash.preparations and lone gitudinal sections of 21m, and microspore cells of mm were calculated by using the formulae (LL93. (Wilson, 19148) and 913:3. As- suming the microspore cells of Ingflgiggntig_to be oblate sphergids the formula flggggb.was also used to calculate the index of cellular volume, (Tables 1-6). Velume indices of the meristematic cells in root tips of isum were calculated from diameter measurements of a randomly chosen sample of 50 cells per slide in the interphase, early prophase and late prOphase stages. Four slides for a total of 200 cells for each of the three stages were examined from.the sectioned material. From the squash.pre— parations a total of 100 cells were measured for each of the three stages. Measurements and statistical analysis of data obtained for both squash preparations and longitudinal sections were made and compared in order to detect any changes ascribable to difference in methodology. One hundred cells were used for measurements of nucleoli, nuclei and cells for each of the four stages from the microspore cells of Inflamm- 12 Hucleoli of three hundred cells were chosen at random from prepa- rations of meristematic and microspore cells to determine whether fu- sion occurs, and if so, to what degree. The data obtained were ana- lyzed statistically to test the significance of observed differences between stages. 13 Table 1. Index of cell volume in squash preparations in interphase, early prephase and late prophase in Pisum. No. INDEX or vomm 31101: memo inn-3 11.1.1113 3 2 1111131223153 100 1+.o7 1.009 7.76 1.009 O E. rnormsr 100 5.75" 1.009 10.91» 1.009 L. racemes 100 5.90. 1.009 11.24. 1.009 t Significant volume increase from interphase. Table 2. Index of nuclear volume in squash preparations in interphase, early prophase and late prophase in Pisum. No. mm or vomm sacs mm 9151-2 (13113 3 2 INTERPHASE 100 . . 69 1. 003 1. 30 1. 003 . I s. racemes 100 1.10 1.005 2.10 1.005 L. norms;- 100 " 0" :.Significant volume increase from interphase. Nuclear membrane has disappeared. Table 3. Index of nucleolar volume in squash preparations in interphase, early prophase and late proplnse in ELEM- No. 01,011.15 mm or VOLUME sues memo 111.113 3 2 mmnrmsr 100 .013 1.001 .021» 1.001 . n. PROPHASE 100 .029' 1.002 .055 1.002 . L. racemes 100 .021" 1.002 .039 1. 002 ' Significant volume increase from inter-phase. 11+ Table lb. Index of cell volume in longitudinal sections in interphase, early prephase and late prophase in Rim. No. mm or VOLUME STAGE masons]: 5121.2 1.1wa 3 2 Inmmsr 200 1.32 1.005 2.06 1.005 E. paormsr 200 1.77" 1.005 3.31' 1.005 . L. racemes 200 2.08' 1.006 3.87 1.006 1' Significant volume increase from interphase. Table 5. Index of nuclear volume in longitudinal sections in interphase, early prophase and late prophase in w. No. INDEX or vomnm snot mum 31:12 (1:113 3 2 1111113911181: 200 .25 1.002 .146 1.002 r. mormsr 200 .Iu' 1.002 .78' 1.002 I. i. L. paormsr 200 o 0 Significant volume increase from interphase. s ' Nuclear membrane has disappeared. Table 6. Index of nucleolar volume in longitudinal sections in interphase, early prephase and late prophase in 21mm. No. or arms mm or ‘70me sum means]: Mg m)? 3 2 1111112111151: 200 .013 1.002 .020 1.002 ’ r. PROPHASE 200 .025' 1.002 .039 1.002 ‘ L. Paormsr. 200 .021’ 1.001 .030 1.001 . Significant volume increase from interphase. Table 7. Index of cell volume in stage 1, 2, 3 and 1+ 15 in microspore cells of W. No. INDEX or VOLUME STAGE masons i321: W JHLLLB 2 1 100 5.59 2.000 10.65 2.000 5.75 2.000 2 100 5.59 2.000 10.65 2.000 6.09 2.000 3 100 9.23" 2.000 15.63' 2.000 9.53‘ 2.000 0 100 11.53" 2.000 19.68m 2.000 12.00‘ 2.000 t Significant volume increase from stage 1. Table 8. Index of nuclear volume in stage 1, 2, 3 and l; in microspore cells of W. No. mm or VOLUME sum 1122503311 $12.23. 1.1.2.213 1 100 i .53 3.009 1.002 2.009 2 100 .53 2.009 1.00 2.002 3 100 1.00' 2.003 2.70. 2.003 0 100 3.06" 2.003 5.83‘ 2.003 . Significant volume increase from stage 1. Table 9. Index of nucleolar volume in stages 1, 2, 3 and 0 in microspore cells of W No. 01‘. cans INDEX or VOLUME sues msmn 3mg (1:!)3 3 2 ' 1 100 .025 2.002 .007 2.002 2 100 .050' .2. 003 .091' 2. 003 O I 3 100 .210 2.003 .389 2.003 $ . 0 100 .290 2.005 .551 2.005 ' Significant volume increase from stage 1. 16 OBSERVATIONS Measurements of nucleoli in meristematic cells of ELM m indicate quite clearly that there is a significant increase in volume from interphase to early prophase (Test figure 1). This increase is then followed by a decrease from early pr0phase with dissolution nor- mally occuring by metaphase. W: The interphase cells in meri- stematic tissue of m m are mostly isodiametric with a spheri- cal nucleolus centrally located. The cell volume increases from inten- phase to early prophase by #1 per cent and then remains constant or in- creases very slightly from early prophase to late prophase (Text figure 2). The interphase nucleus is characterized by the presence of a chro- matin reticulum and one to four nucleoli (Plate I - fig. 1). There is a 60 per cent volume increase in the nucleus from interphase to early prOphase (Text fig. 2). By late prophase the nuclear membrane has dis- solved leaving the chromosomes and nucleolus or nucleoli centrally orientated in the cell. There is a 123 per cent nucleolar volume increase from interphase to early prophase, but thereafter a decrease to late prophase, when the Volume is again about the same as or slightly less than that in inter- Phase (Text fig. 2). There were one to four nucleoli observed in the interphage stage, and one to three in early and late prophase stages. Text fig. 1 Mean volume of nucleolus during interphase, early prophase and late prophase in meristematic cells of 21m from squash preparations. Text fig. 2 Percentage volume increase of cell. nucleus and nucleolus based on per cent increase from interphase to early pro- phase and interphase to late prophase in squash preparations from 21am root tips. 17 Ten figural n u p h p - p p b p 0 5 0 5 0 5 0 5 o 5 5 4. 4 3 3 2 2 1 1 0 0 0 0 0 0 0 0 0 0 0 m2246> x¢JOmAUDz no xmazm L.PROPHASE E. PROPHASE INTERPHASE STAGES Text l'igure 2 Dean. a 1100st wasmus 150 100 mm i t n ’." 50 L. PROPHASE E. PROPHASE INTERPHASE STAGES 0\ DESCRIPTION OF PIJRTE PLAT?!) I Interphase cell with one nucleolus centrally located from squash preparations of 23511311 root tips. Early prophase cell with nucleus containing two nucleoli from squash preparations of 21m root tips. Late prophase cell containing one nucleolus with nuclear mem- brane showing signs of dissolution in squash preparations of ELEM root tips. Interphase cell with nucleus containing multinucleoli from longitudinal section of 213m root tip. Early prophase cell showing nucleus with nucleolus centrally located in longitudinal section of am root tip. Late prophase cell with one nucleolus in longitudinal section of 21m root tip. ' ”a ,._n--——.h ——___-9_————u—-—“-fifl "'-—*— " 19 In nuclei which contained a single nucleolus the latter is more or less centrally located with a pronounced vacuole. When more than one nucleo- lus is present they may be of equal or unequal volumes. Out of 300 cells counted in the interphase stage, #2 per cent con, teined a single nucleolus, 03 per cent two nucleoli, in per cent three and l per cent four (Text fig. 3). In early prophase 78 per cent of the cells contained one nucleolus, 21 per cent two nucleoli and l per cent had three nucleoli, with no cells containing feur (Text fig. 3). The number of nucleoli per cell in late prephase was not significantly dif— ferent from the number in early prophase, there being 80.? per cent COD? taining one nucleolus, 19 per cent with two nucleoli and .3 per cent containing three, with no cells being observed with feur (Text fig. 3). The increase in the number of cells with one nucleolus from interphase to early prophase indicates fusion in early prephase. The volume of the compound (fused) nucleolus is greater than the sum of the nucleolar vol- umes when there are two or more nucleoli in interphase, early prephase and late prephase (Text fig. h). Cells, nuclei and nucleoli all show a significant increase in vol- ume from interphase to early prophase. The increase in cell volume from early prophase to late prophase is not as great as its volume increase from interphase to early prophase (Table l). The nuclear membrane has disappeared by late prophase (Table 2) and the nucleolus has decreased in volume in transition from early to late prophase, indicating that dissolution has begun (Table 3). Text fig. 3 Frequency of cells containing one, two, three and four nucleoli in interphase, early prophase and late prophase from squash preparations in BM. Text fig. l+ Mean volume of one, two, three and four nucleoli per stage in meristematic cells of mm. 20 D on: NUCLsows [53 W0 NUCLECLI mas nucuzcm - roux NUCLEOLI L . PROPHASE Text figure 3 T p h b p r - 0 0 0 0 O 0 l zoza 33%; ». STAGES E . PROPHASE INTERN-[ASE Text figure it a on NUCLBOLUS m NUCLECIJ . ms: woman I ma 1mm: “Annex, . 050 .030“F' .040 83.5.» to mez— . 020 .010 L . PROPHASE E . PROPHASB I NTERPHASE 21 In order to check possible distortion produced by the squash tech- nique, similar measurements were made of sectioned material. Upon exam- ination of data from the sectioned material it was observed that the cell, nucleus and nucleolus showed a volume increase from interphase to early prophase and late prophase as was observed'in the squash prepare- tions (Text fig. 5). Both techniques showed similar trends in the index of volume increase for cell, nucleus and nucleolus (Tables 1—6. Text figs. 2 and 3). Microgpore Cells Of Tradescantia: Measurements of cells, nuclei and nucleoli were made from post meiotic interphases to prephase of the first microspore division. During this period four stages can be dis- tinguished on morphological grounds, and have been designated as stages 1. 2, 3 and 0 (Plate II, figs. 1—0). Stage 1: This is the stage which follows the formation of the te- trad. Stage 1 is characterized by a spheriodal cell with an oval shaped nucleus which is centrally located. In the nucleus there are l to 1+ nu- cleoli of variable sizes randomly distributed. Stag 2: The second stage is recognizable by an elongation of the cell, and movement of the nucleus to one end. While there is no signif- icant change in volume of the cell and nucleus over that of stage 1, there is a significant change in nucleolar volume (Tables 7, 8 and 9). The number of nucleoli per nucleus remains the same as in stage 1. Stage 3: This stage is characterized by a significant volume in- crease in both cell and nucleus, as well as in the nucleolus (Tables 7, 8 and 9). The number of nucleoli remain constant as in the two preced- ing stages. Text fig. 5 Percentage volume increase of cell, nucleus and nucleolus based on per cent increase from interphase to early pro- phase and from interphase to late pr0phase in longitudinal sections of 21103. Text fig. 6 Percentage volume increase of cell, nucleus and nucleolus based on per cent volume increase from stage 1 for stages 2, 3 and b. in microspore cells of Tradesgntia m. Text 119110 5 \\\\\\\\\\\\m Text Figure 6 \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ CCCCCCCCCCCC CCCCCCCCCCCCC .............. ....... u U n 0 0000000 n m .n U n, u U 0000 STAGES Fig. h DESCRIPTION OF PLATE PLATE II Stage one with nucleus containing three nucleoli from smear pre- parations of mdcrospore cells of Tradescantia paludosa. Stage two showing movement of nucleus to one end of cell with the nucleus containing two nucleoli in microspore cells of magmatic.- Stage three showing nucleus containing one nucleolus, which con- tains a vacuole. The cell, nucleus and nucleolus showing a marked increase in volume from the preceding stage in microspore cells of W. Stage four showing movement of nucleus back to the center of cell, with the nucleus containing two nucleoli in microspore cells of W.- Plate II 20 $1252.33 The volume of cell, nucleus and nucleoli continue to show a significant increase over that of the previous stage. This stage is also characterized by the movement of the nucleus back to the center of the cell, where it is embedded in a mass of cytoplasm which extends the width of the cell (Plate II, fig. 1+). The average number of nucleo- li per nucleus has not changed from that found‘in the preceding stages. The cell volume remains relatively constant from stages l to 2, but by stage 3 it has increased by 65 per cent (Text fig. 6) of its original size (stage 1). This increase continues through stage '4, resulting in a 108 per cent increase in volume over that of stage 1 (Text fig. 6).. There is no significant volume increase in the nucleus from stage 1 to 2. By stage 3 the nucleus has increased by 171 per cent from stage 1, and by stage 1+ to 1177 Per cent of its original volume (Text fig. 6). Nucleoli of microspore cells are visible as deep bluish-green spherical bodies which are more or less centrally located when there is only a single nucleolus per cell and randomly distributed in nuclei with more than one nucleolus. The nucleolus shows a continuous volume increase from stages 1 through 1+ in microspore cells of W (Text fig. 7). The per cent increase from stage 1 to stages 2, .3 and h is 100 per cent, 756 per cent and 1060 per cent respectively (Text fig. 6). In stages 1 through 1+ the volume of the single nucleolus is greater than the sum of the nucleolar volumes when there are two or more nucleo- 11 per nucleus (Text fig. 8). Text fig. 7 Mean volume of nucleolus during stages 1, 2, 3 and h in microspore cells of We... Text fig. 8 Mean volume of one, two, three and four nucleoli per stage in microspore cells of W. INDEX OF VOLUME INDEX 0? VOLUME .300 .275 .250 .225 .200 .175 - .150 .125 .100 .075 .050 .025 .100 .090 .0b0 .070 .060 .050 .040 .030 .020 Text figure 7 25 ST1615 Text l‘igure 8 D on: .\'1'(‘l.I-T()l.l'S B Two mum” THRH. 9.10”an - FOL’R Nl'CLEmJ ST1G£S a w \\\ 7//////// 26 In microspore cells of Izafiggggptig, neither cell nor nucleus show a significant volume increase from stage 1 to stage 2, but both show an increase in volume by stages 3 and h. The nucleolus, however, shows a continuous volume increase from stages 1 through 0. Three hundred cells were counted from the feur stages to determine whether or not there is significant fusion of multiple nucleoli. Exam- ination of the histogram as shown in Text figure 9 indicates that the number remains relatively constant throughout stages one to four. There is, therefore, no evidence that nucleolar fusion occurs during interb phase and prophase in ndcrospore cells of Tradescantia. Text fig. 9 Frequency of cells containing one, two, three and four nucleoli in microspore cells of W Text fig. 10 Mean volume of nucleolus during interphase, early prophase and late prophase in meristematic cells of film W from longitudinal sections. Text flan-09 [3 ONE NUCLEOLUS a Two NUCLEOLI runes NUCLEOLI I POUR NUCLEOLI \x\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ \\ .. s\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ca \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ 50 0 0 . 20 10 Zomba—Ema m. STAGES Textl‘igure 10 INTERWASE 84:40.» ”EACNAUDZ mo xme: - p p p - b p 5 0 5 0 5 0 5 0 5 4. M 3 3 2 2 1 l 0 0 0 0 0 0 0 0 0 L . PROPEASE E . PROPHA SE STAGES 28 DISCUSSION The onset of cell division in both meristematic cells of'2133m_and microspore cells of Iggdgggantia, is characterized by an overall in- crease in volume of cell, nucleus and nucleoli. This volume increase suggests several questions with regard to the function and relationship of cell components, e.g., (1) Is cell volume increase necessary for cells to divide? (2) Is the volume increase restricted to the cell or must there also be an increase in nucleus and nucleolus? (3) Do vol- ume increases of components coincide? (0) What is the relationship of nucleolar volume to that of the cell and nucleus? One approach to the elucidation of the above, is an examination of cell, nuclear and nucleo- lar volumes at the stage prior to division (interphase) and the first stages of division (early prophase and late prophase). The stage at which no morphological activity may be observed.was termed the "Resting Stage" by the early cytologists. It is now recognized that the so-call- ed interphase cell is in a metabolically active state. It seems to be well established, for example, that DNA-synthesis occurs during interh phase (Howard and Pelc, 1951; Vincent, 1952 and.Brachet, 1957). WWW There is a significant in- crease in cell volume from interphase through late prophase. It is apparent that the major proportion of this increase is occuring during the onset of division; i.e. from interphase to early prophase, with rel- atively little increase from early to late prephase. This volume in- 29 crease may be due to either an increase in membrane permeability of cell and nucleus, which allows an increase in uptake of water and solutes, or a synthetic process with the incorporation of proteinaceous materials into the nucleus, or a combination of the two (Sirlin and Waddington, 1956 and Brachet, 1957). Some workers maintain that the relationship between nucleus and cell is always constant, while others hold that no such constancy exists, but rather, that there is a wide variability in the relative size increase of cells and nuclei (see Trombetta, 1939). In the meri- stematic cells of ELM there appears to be a reasonably constant rela- tionship, between relative rates of volume change in cell and nucleus, with the ratio being 6:1 in interphase and 5:1 in early prOphase (Table 10). Observations in the present study indicate a significant increase in nucleolar volume in meristematic cells of Rim from interphase to early prophase. The increase ceases during early prophase, and the vol- ume begins to decrease. By metaphase the nucleolus has dissolved. An examination of the sectioned material also showed a nucleolar volume increase from interphase to early prephase (Text fig. 10). These find- ings are not in agreement with those of Hankansson and Levan (1902), who found that the size of the nucleolus in w is the same during resting (interphase) stage and the early pr0phase stages. Wm: The fusion of nucleoli has been fre- quently reported to occur in a variety of cell types (Gates, 19U2). The fusion of nucleoli commences in late telophase (Gates, 19102; Hankansson and Levan, 1902: Duryee and Doherty, 1951+). As shown by Heitz (1930), 30 Table 10. Showing ratios of cell/nucleus, cell/nucleolus and nucleus / nucleolus in mm. CELL CELL STAGE NUCIEUS NUCLEOLUS INTERPRASE 6 313 E. PROPHASE 5 198 L. PROPHASE - 281 NUCLEUS NUCLEOLUS 53 3? Table 11. Showing ratios of cell/nucleus, cell/nucleolus and nucleus / nucleolus in W. CELL CELL STAGE NUCLEUS NUCLEOLUS 1 11 223 2 11 111 6 u; h h 39 NUCLEUS NUCLEOLUS 21 10 10 31 when the nucleolus organizing portions of two chromosomes lie close to- gether in the telophase nucleus, the chance of fusion between the two developing nucleoli is greatly enhanced. Woods (1937) suggested, on the basis of volumetric studies of root tip nucleoli in m, that the relative amount of plastin generated in telephase may play a part in the fusion of nucleoli, with large amounts having a tendency to fa- vor fusion, thus giving a reduction in nucleolar number. However, in the meristemtic cells of 21m fusion continues through interphase and early prephase. The degree of nucleolar fusion was determined by count- ing the number of nucleoli in three hundred cells of each stage (Text fig. 3). There was no indication of budding or fragmentation of nucleo- li in the meristemtic cells of m m. Liircrospore Cells 0f Tradescantia paludosa: A volume increase in cell components preceded microspore division as was found in meristem— atic cells of 21m. The relative volumes of cell, nucleus and nucleo- lus of microspore cells were examined from four stages (1, 2, 3 and 1+) as explained earlier. There is no significant increase in cell volume from stage 1 to stage 2. However, by the end of stage 3 the cell volume has increased by almost half its original value (Stage 1), and this increase contin- ues through stage it (Text fig. 6). There is also no volume increase in the nucleus from stages 1 to 2. It is apparent that the cell and nucleus are synchronized with re— gard to time of volume increase, in that neither increases in stages 1 and 2, while both increase through stages 3 and h. The magnitude of nuclear volume increase, however, is much greater than that of the cell 32 (Table 11). In stage 3 and h the nuclear volume is increasing at a greater rate than that of the cell. This greater increase of nuclear volume gives a decreasing cell/nucleus ratio in stages 3 and u respec- tively (Table 11). The cell/nucleus ratio of the microspore cells of W differ from the ratio in meristematic cells of m, in that the latter it remains relatively constant (Table 10). Bryan (1951) observed the microspore cells of I;gfigggan&1a,that: (l) the DNA content of deve10ping microspores increases during the long interphase period (Stages 1-3); and (2) this increase is rather slow during early interphase (Stage 1 and 2) and extremely rapid Just prior to microspore prophase (Stage 3). The nuclear volume increases as ob- served in the present study correlates well with the rate of increase in DNA as observed by Bryan. These findings suggest that the nuclear DNA synthesis is associated with nuclear volume increase. This volume increase also may be associated with an increase in permeability of the nuclear membrane. Bahr and Beerman (195k) observed in giant nuclei that the rate of transport through.the membrane per unit area is higher than that in nuclei and cells of normal size. Nucleolar volume increase begins in stage 1 and continues through stage 4, while cell and nuclear volume increases commence in or Just be- fore stage 3. The nucleolar volume increase in stage 1 through stage h is not due to fusion, as the number per nucleus remains relatively constant in each of the four stages (Text fig. 9). The foregoing observation.suggests that the nucleolar volume increase is due to either synthesis or the incorporation of nuclear substances into the nucleolus, this uptake ‘1) KO being influenced by the metabolic and nutritive state of the nucleus and cell (Gates, 1942). Gates observed that nucleolar size was found to be greatly influenced by nutrition. By growing pieces of leaves in sugar solutions he fbund that the nucleoli were larger, and contained more carbohydrates, than nucleoli of leaves kept in darkness. These re- sults indicated that the nucleolus is an important storage organ in leaf cells. Bretschnider and Hirsch (1937) observed that in eggs of certain teleosts (Lamelli branch) the nucleus and nucleolus at first grow coincidently; then the latter steps while the former continues its growth. The present study indicates that similar volume relationships exists in meristematic cells of'Bignn, while this condition was not ob- served in microspore cells of Ixafigfiggntia, It is apparent that the nucleolar volume is increasing at a rela- tively faster rate than that of the cell or nucleus, the order of mag- nitude being nucleolus, nucleus and cell (Text fig. 6). In regards to nucleolar volume per nucleus it was observed that in meristematic cells of 21m and the microspore cells of W the volume of the compound nucleolus (fused nucleoli) was consistently larger than the sum of the nucleolar volumes when there were two or more nucleoli (Text fig. 8). This condition was also reported by deMole (1928) and.Bhatia (1938). Similarly Parthasarathy (1938) found in the pollen mother cells of rice at diakenesis that the volume of the single fusion nucleolus was greater than the sum of the volumes when there were two nucleoli. It was feund in a polyploid series of unsgg, that nucleolar size increases with increase in degree of ploidy (Wilson, 19148). 31+ There was no indication of fusion in the four stages examined in the microspore cells of W, in contrast to that which was found in the meristematic cells of 21m m 3.5 SUMMARY 1. The problem of nucleolar volume increase, fusion. its relation to nuclear and cell volume increase from interphase to late prophase in meristematic cells of 21m and from interphase to prophase during post meiotic interphase of W was studied. 2. Methods for fixation and staining of cells for cytological study of nucleoli, nuclei and cells were developed. 3. The relative volumes of cell, nucleus and nucleolus were calcu- lated for the interphase and. prophase stages in the meristematic cells of m m, and the microspore cells of W W. The data were statistically analyzed to ascertain the size relationship existing between cell components prior to and during the onset of divi— sion. LL. Measurements were mde using squash preparations and longitu- dinal sections from meristematic tissue of 21m to determine if there were significant differences in the relative volumes of cell components in the preparations of the two methods. There was no appreciable dif- ference in the ratio of volume increase exhibited by nucleus, nucleolus and cell in the two types of preparations. 5. The question of volume increase in cell, nucleus and nucleolus is discussed. There is a rapid increase in nuclmr, nucleolar and cell volumes from interphase to early prephase. Howard and Pelc (1951) re— ported that this phase of division is characterized by a high degree of 36 protein synthesis. 6. The ratios of cell/nucleus, cell/nucleolus and nucleus/nucleo- lus were considered. The results indicate that the nucleolar volume increases at a much faster rate than that of cell or nucleus in the meristematic cells of mm and the microspore cells of W. The magnitude of increase is in the order of nucleolus, nucleus and cell. 7. It was observed that nucleolar size (volume) increases from interphase through.early prophase. Following early prophase the volume of the nucleolus decreases in late prephase and has disapppeared by meta- phase in meristematic cells of 23522, This observation of nucleolar volume increase from interphase to early prophase is not in agreement with the findings of Hankansson and Levan (19h2). 8. Three hundred nucleoli per stage were counted to determine the degree of fusion in both aim and mm. It was found that the per cent of nuclei containing one nucleolus is significantly increased from interphase to early prophase in.£1§gm,indicating nucleolar fusion. In Iggdggggniig, the percentage of cells with l, 2, 3 and h nucleoli remained constant through the stages measured. It is apparent that fur sion does not occur in microspore cells of Ezafleggaptig, from the inter- phase through prophase. 9. 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