Electron-Microscopic Observation on Nuclear Events During Binary Fission

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 chromonemata, 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 investigated 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) reported 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 stained 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 division 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 cytokinesis. 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) surrounded by a double membrane (e,) with pores and a few small masses 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 macronuclear 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 chromatin 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 contain 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. 10). The old, outer envelope has a few pores and the space between its two membranes becomes wider, while the newly formed inner envelope pos- sesses numerous pores at regular intervals. Inside the outer envelope, no spindle fibers are observed, but the matrix is filled with many vesicular remnants of spindle fibers. The oval-shaped area limited by a double membrane seen in Fig. 10 is thought to be a section of a part of cytoplasm produced by indentation of the nuclear envelope, because material similar to the cytoplasm is found inside it. At the end of this stage, when the macronucleus is fully elongated, fine fibrils fill the whole nucleus and only a few small nucleoli (less than 0.3 p in diameter) are seen in the macronucleus (Fig. 11). NUCLEAR EVENTS DURING BINARY FISSION IN BLEPHARISMA 339

Figs. 2-4. Stage 1 of macronuclear division.

Fig. 2. Portion of the compact macronucleus at the end of stage 1, showing chromatin bodies (cb) which are somewhat reduced in size and nucleoli (n), one of which contains a large vacuole (v). e, macronuclear envelope. x 28000. Fig. 3. Micronucleus in prophase. Chromatin threads (c) are entangled together throughout nucleus. e1, micronuclear envelope ; p, pore. x 35000. Fig. 4. Micronucleus in metaphase. Chromosomes (ch) are arranged in equatorial plane and a number of spindle fibers (sf) are present, some of which are attached to the chromosome. e1, micronuclear envelope ; p, pore. x 14300. 340 F. INABA AND Y. SOTOKAWA

Figs. 5-i 1. Stage 2 of macronuclear division. Fig. 5. Portion of the macronucleus at the beginning of stage 2. Chromatin bodies (cb) are much reduced in size, nucleoli (n) are in the course of degeneration and the spherical body (s) has greatly increased in size and in electron-density. x 25000. Fig. 6. Portion of the macronucleus at the same time as shown in Fig. 5, showing that the fine fibrils composed of two parallel filaments start from the part of low electron-density within the degenerating nucleolus (at arrow). cb, chromatin body ; n, nucleolus. x 32000. Fig. 7. Tangential section through the macronuclear envelope. Microtubules (t) which seemingly arise (at arrow) from the nuclear pores (p) are running along the nuclear envelope. cb, chromatn body. x 90000. NUCLEAR EVENTS DURING BINARY FISSION IN BLEPHARISMA 341

Fig. 8. Micronucleus in late anaphase, leaving a long tail containing a bundle of spindle fibers (sf). Chromatin material is seen as a mass of entangled threads (c) at the distal pole of the nucleus. e1, micronucleur envelope ; p, pore, x 12500. Fig. 9. Telophase micronucleus. Formation of the new nuclear envelope (e2) begins at the distal pole of the nucleus. The space between membranes of the old envelope (e1) becomes wider and only a few pores (p) remain in it. The new envelope is seen as a periodic structure of dense and less dense parts. c, chromatin thread. x 28000.

Fig. 10. Telophase micronucleus surrounded by two nuclear envelopes (el and e2). Numerous vesicular structures (ve) are found in the original nuclear matrix. The oval-shaped area (a) limited by a double membrane (e1) is supposed to be a part of the cytoplasm, probably produced by indentation of the nuclear envelope. c, chromatin thread. x 35000. 342 F. INABA AND Y. SOTOKAWA

Fig. 11. Portion of a fully elongated macronucleus. It is entirely filled with fine fibrils (cf ). A few small nucleoli (n) are found between the fibrils. Microtubules (t) arising from the flu- clear pores are visible outside the nuclear envelope (e). x 15000.

Figs. 12-15. Stage 3 of macronuclear division. Fig. 12. Portion of the macronucleus at the beginning of stage 3. Fine fibrils (cf) have begun to coil irregularly forming a mass. e, macronuclear envelope ; n, nucleolus. x 6160. NUCLEAR EVENTS DURING BINARY FISSION IN BLEPHARISMA 343

Fig. 13. Portion of the micronucleus at the beginning of stage 3. Microtubules (t) are no longer attached to the nuclear pore, but they converge forming a small mass of fibrous material (fm). cf, fine fibril ; e, macronuclear envelope ; n, nucleolus. x 33750. Fig. 14. Portion of the macronucleus in the middle of stage 3. As a result of coiling, fine fibrils are seen as chromatin bodies (cb). Micronuclei (mi) are still surrounded by two envelopes (e1 and e2). e, macronuclear envelope. x 17500. Fig. 15. Portion of the macronuclear node at the end of stage 3. Chromatin bodies (cb) and nucleoli (n) have the same appearance as in interphase. The micronucleus (mi) is now surrounded by only one envelope (e2). e, macronuclear envelope. x 17500. 344 F. INABA AND Y. SOTOKAWA

(4) Stage 3 At the beginning of this stage (Fig. 12), the fine fibrils in the macronucleus start to coil irregularly forming a mass of 100 mp in diameter. Microtubules begin to break and become free from the nuclear pores, but they seem to converge forming a small mass of fibrous material located near the nuclear envelope (Fig. 13). As a result of coiling, the fine fibrils in the macronucleus appear in cross section as scattered irregular-shaped chromatin bodies (100x 100-200 mp in dimensions), and the spaces between them become filled with the fibrillar matrix (Fig. 14). The nucleoli are still few in number and varying in size from 0.3 to 1.3 p in diameter. At this time micronuclei are still surrounded by two envelopes, although the outer one begins to disrupt. At the end of this stage (Fig. 15), all the nuclear components have almost the same appearance as in the interphase ; chromatin bodies distributed at the periphery begin to become attached to the nuclear envelope. The nucleoli increase in size and in number. The spherical bodies decrease in size and in electron-density. Microtubules are no longer visible in the cytoplasm. The micronuclei are now surrounded by only the new envelope and contain a network of chromatin threads, 60 mp thick. They seem to be in interphase.

DISCUSSION

1. Macronuclear Division

a. Chromonemata The chromonemata in the macronucleus in the interphase of Blepharisma wardsi has, as in other ciliates, the form of threads, 100 m p thick, composed of fine fibrils about 10 mp in thickness. In the early stage of binary fission when the macronuclear nodes fuse into a compact mass, they gradually become uncoiled. With the elongation of the macronucleus during the subsequent stage, all the chromonemata uncoil into fine fibrils. From autoradiographic and photometric studies on various species of ciliates, it is gene- rally recognized that the time of DNA synthesis varies in different species and even in different strains within a species (see Prescott and Stone 1967). The most complete studies by autoradiography, as well as by electron microscopy, were reported about a hypotrichous ciliate Euplotes and a hymenostome Tel rahvmena. In Euplotes, it was found that the chromatin bodies of the macronucleus uncoil into fine fibrils in the reorganization band, which passes through the macronucleus in early interphase (Faure- Fremiet et al. 1957, Kluss 1962), and DNA replication occurs when these are fine fibrils in this band (Gall 1959, Kimball and Prescott 1962, Prescott and Stone 1967). In Tetra- hymena pyriformis strain HSM, the time of DNA synthesis occupies one third of the cell cycle (Prescott and Stone 1967). However, it was reported by another author (Flickinger 1965) that no impressive morphological changes could be observed in the chromonemata throughout the cell cycle. In Blepharisina, no specific site of reorganization of the macronucleus such as that in Euplotes appeared in the cell cycle, but it NUCLEAR EVENTS DURING BINARY FISSION IN BLEPHARISMA 345 was observed that uncoiling of the chromonemata of the macronucleus begins at the period of contraction and continues during the elongation of the macronucleus. If DNA synthesis occurs during uncoiling of the chromonemata, it may be concluded that DNA synthesis occurs in stage 2 of macronuclear division of Blepharisma. The double structure of the fine chromatin fibrils is clearest in stage 2, which suggests that the chromonemata divide by endomitosis as a result of DNA replication. However, the exact stage of DNA synthesis should be studied further by autoradiographic and photometric methods.

b. Nucleoli The nucleoli grow larger in stage 1 of macronuclear division. One cause of growth may be vacuole formation inside them. They gradually degenerate during stage 2. During degeneration, they become more heterogeneous in electron-density. When the macronucleus is filled with fine fibrils, only a few nucleoli remain in the macronucleus. And then, with progress of recoiling of the fine fibrils, nucleoli gradually increase in size and in number. Elimination of nucleoli from the macronucleus during binary fission was reported in Paramecium bursaria (Ehret and Powers 1955) and Tetrahymena pyriformis (Elliott et at. 1962, Elliott 1963). No such phenomenon could be found at any stage of macronuclear division in Blepharisma wardsi.

c. Spherical bodies The spherical bodies in Blepharisma wardsi are quite unlike the nucleoli in shape and in general appearance, but are similar in some ways to the carbohydrate-protein complex found by Kennedy (1965) in the interphase macronucleus of Blepharisma undulans. They are, however, different from the latter in electron-density and in carbohydrate content. The spherical bodies are considered to be protein-containing bodies, because they stain with mercuric-bromphenol-blue. The behavior of these is unlike that of the nucleoli during the division cycle ; they reach their maximum size and highest electron-density when the chromonemata are wholly uncoiled and thereafter they pro- gressively decrease in size and in electron-density. Although their metabolic role is unknown, they may be related to replication of the chromonemata.

d. Microtubules Microtubules were observed in the stage of the macronuclear division in several species of ciliate (Roth and Minick 1961, Roth and Shigenaka 1964, Raikov 1965, Carasso and Favard 1965, Suganuma and Inaba 1967). A number of microtubules can be found along the longitudinal axis of the macronucleus of B. wardsi during its elongation. It is supposed that the microtubules arise from the nuclear pores and terminate in a small mass of fibrous material near the nuclear envelope, though the exact nature of the mass is unknown. In most of the ciliates investigated by electron microscopy such as Tetra- hymena (Roth and Minick 1961), Nassula and Nassulopsis (Raikov 1965), Campanella (Carasso and Favard 1965) and Urostyla (Suganuma and Inaba 1967), microtubules were found between the chromonemata inside the dividing macronucleus. In Diplodium (Roth and Shigenaka 1964) they were found on the inside as well as on the outside of the 346 F. INABA AND Y. SOTOKAWA

macronucleus during division. In the present species, they are found only in the cytoplasm outside the nuclear envelope. It is clear that these extranuclear microtubules participate in the elongation of the macronucleus by pulling the nuclear envelope, but independently of the chromonemata. During the nodulation of the macronucleus, they become free from the nuclear envelope, and after complete nodulation, they are no longer detectable in the cytoplasm.

2. Micronuclear Division Division of the micronucleus seems to progress more rapidly than that of the macronucleus. When the macronucleus becomes a compact mass, the micronuclei enter the prophase of mitosis. Metaphase occurs at the beginning of elongation of the macronucleus and telophase is found before the uncoiling of the chromonemata of the macronucleus is complete. Recently, an excellent electron microscopic study on micronuclear division was made by Jenkins (1967) in Blepharisma americanum. He failed to find any pores in the micronuclear envelope in interphase. In the present species, however, we observe numerous pores on the micronuclear envelope both in the interphase and in the division stages. Our observation on the formation of the new micronuclear envelope in late telophase is in accord with that of Jenkins. In Tetrahymena pyriformis, Elliott (1963) reported seeing an intranuclear body during micronuclear division, which he supposed to be a centriole. No such body could be found during micronuclear division of Blepharisma. We agree with Jenkins' opinion that micronuclear division in Blephari- sma is closed-acentric mitosis.

SUMMARY

The nuclear changes during binary fission in a heterotrichous ciliate, Blepharisma wardsi were studied by electron microscopy. The macronucleus in the interphase is in the form of 4-6 nodes. At the beginning of binary fission, the macronuclear nodes aggregate and coalesce into a mass (stage 1), then the macronucleus elongates (stage 2) and finally becomes nodulatod (stage 3). The chromatin bodies in the macronucleus gradually uncoil into their component fine fibrils at the end of stage 1, and the macronucleus becomes entirely filled with these fine fibrils in stage 2, then the fine fibrils gradually recoil to form chromatin bodies in stage 3. Nucleoli decrease both in size and in number in stage 2 and increase again in size and number during stage 3. The spherical bodies containing protein, on the other hand, have maximum size and electron-density in stage 2. The macronuclear envelope per- sists throughout the division cycle. The extra-nuclear microtubules seem to arise from the nuclear pores. These microtubules become most prominent in stage 2, and probably participate in the elongation of the macronucleus. Micronuclear division is initiated in stage 1, proceeds rapidly and ends in stage 2 of macronuclear division. NUCLEAR EVENTS DURING BINARY FISSION IN BLEPHARISMA 347

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