JAPAN. J. GENETICS Vol. 43, No. 5: 335348 (1968) ELECTRON-MICROSCOPICOBSERVATION ON NUCLEAR EVENTS DURING BINARY FISSION !N BLEPHARISMAWARDS! (CILIATA,HETEROTRICHIDA) FUMIE INABA AND YUMIKO SOTOKAWA Department of Zoology, Faculty of Science, Nara Women's University, Nara 630 Received June 6, 1968 Recent electron microscopic studies on the nuclear apparatus in ciliate protozoa re- vealed that the macronucleus in the interphase contains entangled thread-like chromo- nemata, about 100 mp thick, which appear in cross section as numerous irregular-shaped bodies composed of fine fibrils, about 10 mp thick, and that the micronucleus in the interphase contains thinner thread-like chromonemata, about 50 m p thick, which appear as a compact network in cross section. The reorganization of the chromatin bodies or threads in the macronucleus was studied by electron microscopy in certain species of hypotrich (Faure-Fremiet et al. 1957, Roth 1957, Kluss 1962, Inaba and Suganuma 1966, Chakraborty 1967). It was clearly shown that the chromatin bodies uncoil into their component fine fibrils in the reorganization band which passes through the macronucleus during the interphase in these ciliates, and then they are reorganized into chromatin bodies. Several reports of electron microscopic studies on macronuclear events during binary fission have been published. Some of them, however, were on changes of the nucleoli rather than of the chromonemata (Ehret and Powers 1955, Roth and Minick 1961, Elliott et al. 1962, Elliott 1963). Flickinger (1965) studied the morphology of the chromonemata throughout the cell cycle of Tetrahymena pyriformis, but he did not find any correlation between the morphological changes in the chromonemata and DNA synthesis. Suga- numa and Inaba (1967) studied the morphology of the macronucleus during binary fission in a hypotrichous ciliate, Urostyla grandis, and reported changes of the chromonemata following the reorganization. This paper is on electron microscopic observations on nuclear events during binary fission in a heterotrich, Blepharisma wardsi with special reference to the behavior of the chromonemata during macronuclear division. Micronuclear division was also in- vestigated to assess the stages of the macronuclear events. MATERIALS AND METHODS Blepharisma wardsi was used in this study and was obtained through the courtesy of Dr. H. I. Hirshfield of New York University, New York. Hirshfield et al. (1965) re- ported on the morphology of this species. Stock cultures were maintained at 25°C in 4 % lettuce juice (pH 6.8), inoculated with Aerobactor aerogenes, in which the cells divide once a day. About 3 days after transfer into fresh culture medium cells were found 336 F. INABA AND Y. SOTOKAWA in various stages of division. They were collected at random and fixed with 1 % os- mium tetroxide buffered to pH 7.9 with Veronal acetate buffer. After fixation for 30 minutes, they were dehydrated by passage through a series of ethanols and propylene oxide, and then embedded singly in epoxy resin by Luft's method (1961). The division stage of the cells was examined under a light microscope and material was trimmed for sectioning in the desired plane. Sections were cut with a Porter Blum ultratome, stained with uranyl acetate and lead citrate according to Reynolds' method (1963), and examined in a JEM-7 electron microscope. Parts of the samples were fixed in Schau- dinn's fixative for light microscopy, embedded in methacrylate. Sections were stained with Feulgen's reagent, methyl green-pyronin or mercuric-bromphenol-blue, and were also tested with periodic-acid-Schiff reagent. Some of the cells were fixed with 5 % aqueous silver nitrate solution for light microscopic observation and the whole mounts were stain- ed with Feulgen's reagent. RESULTS The macronucleus in the interphase of Blepharisma wardsi is in the form of 4-6 round nodes of 10-20 p diameter, connected by fine strands. The micronuclei are very small, so that it is difficult to count them under a light microscope. Macronuclear di- vision can be broadly divided into 3 stages on the basis of gross morphological changes : Stage 1. The period from aggregation of the macronuclear nodes to their fusion into a compact mass. When the first sign of fission appears as a slight expansion of the posterior half of the cell, the connective strand between the macronuclear nodes disappears and the nodes gather together in the central part of the body. Then they coalesce into a large compact mass, about 301 in diameter, at about the time when the newly developed adoral anlage becomes visible on the ventral side of the body. Stage 2. Period occupied by elongation of the macronucleus. When the constric- tion furrow appears on the middle of the body, the compact macronucleus begins elon- gating and finally becomes cylindrical. Stage 3. Period of nodulation of the macronucleus until the completion of cyto- kinesis. With the progress of cytokinesis, the elongated macronucleus modulates into a beaded form. Each of the daughter cells receives a share of nodes, occasionally unequal in number. Electron Microscopic Observations (1) Interphase As shown in Fig. 1, the macron>>clear node in the interphase is surrounded by a double membrane, 23 mp thick, in which numerous pores of 100 mp diameter are found. Chromatin material is seen in cross section as irregular-shaped, Feulgen-positive, dense bodies of 100 x 100-300 mp in dimensions. They are composed of fine fibrils of about 10 mp thick. These chromatin bodies are scattered throughout the fibrillar matrix of low electron-density, but those distributed at the periphery of the nucleus are attached to the inner membrane of the nuclear envelope. The round nucleoli of 0.5-3 tc diameter NUCLEAR EVENTS DURING BINARY FISSION IN BLEPHARISMA 337 Fig. 1. Portion of a macronuclear node of Blepharisma wardsi in interphase, showing the nuclear envelope (e) with numerous pores (p), chromatin bodies (cb), nucleoli (n), and a spherical body (s) embedded in the matrix (mx). Also visible are an interphase micronucleus (mi) sur- rounded by a double membrane (e,) with pores and a few small mas- ses of fibrous material (fm). x 8280. identified by pyronin staining, are scattered between the chromatin bodies. They are also composed of fine fibrils, but they look rather heterogeneous in electron-density. Occasionally, one or two homogeneous, Feulgen-negative, spherical bodies of low electron- density are found in a node. They resemble in appearance the carbohydrate-protein complex described by Kennedy (1965) in B. undulans. However, they cannot be as- sumed to be homologous with the latter, because of their low density and negative reaction for periodic-acid-Schiff, and are tentatively considered as protein-containing bodies because they stain with mercuric-bromphenol-blue. A small number of tiny masses composed of fibrous substance (250 x 50 mp) are scattered in the cytoplasm close to the macronucleus. The micronuclei are round, about 1 i diameter and usually found near the macro- nuclear node. They are surrounded by a double, porous membrane similar to that of the macronucleus. Chromatin material appears as a network of threads, about 60 m,o thick. (2) Stage 1 At the beginning of this stage, nucleoli and spherical bodies in the aggregated mac- 338 F. INABA AND Y. SOTOKAWA ronuclear nodes begin to enlarge, while the chromatin bodies show little change. At the end of the stage when the macronuclear nodes fuse together into a mass, the chro- matin bodies appear smaller (100-200 mp in diameter) and are more sparsely distributed (Fig. 2). The nucleoli in some cases enlarge to a diameter of about 5 p and often con- tain a large vacuole (0.5-2 p in diameter), the inside of which has an appearance similar to that of the matrix. Spherical bodies increase in size and in electron-density at this time. Micronuclei at the beginning of this stage are considered to be in the prophase of mitosis. They contain entangled chromonemata of 40-60 mi thickness (Fig. 3). At the end of this stage, they proceed to the metaphase in which they are a large spindle shape (3 x 5 p), and contain several round chromosomes (0.3-0.4 p in diameter) in the equatorial plane. A number of spindle fibers (23 m~c in diameter) are observed running from one pole to the other, some attached to the chromosome (Fig. 4). (3) Stage 2 At the beginning of this stage, when the macronucleus is slightly elongated (Fig. 5), the chromatin bodies in the macronucleus decrease considerably in size and in number, and the spaces between them are filled with fine twisted fibrils composed of two parallel filaments (each 5 mp thick). The nucleoli become smaller, varying in size from 0.3 to 2 p in diameter, and are strikingly heterogeneous in electrondensity. Sometimes fine chromatin fibrils composed of two parallel filaments are seen starting from the area of lower electron-density inside the nucleolus (Fig. 6). In contrast to the nucleoli, the spherical bodies continue to increase in size to a diameter of about 4 p and become more electron-dense (Fig. 5). A number of microtubules, 23 mp in diameter, appear in the cytoplasm near the nuclear envelope, running along the longitudinal axis of the elonga- ting macronucleus. These microtubules seem to arise from the pores on the nuclear envelope (Fig. 7). Micronuclei at this time are in anaphase or telophase (Figs. 8 and 9). It is noticed that the formation of the new nuclear envelope just around the chromatin mass begins at the distal pole of the daughter micronucleus (Fig. 9). The new envelope is seen as a periodic structure composed of dense and less dense parts, representing the pores and the double membranes, respectively. Thus the micronucleus becomes surrounded by two envelopes (Fig.