Jpn. J. Protozool. Vol. 40, No. 2. (2007)

123

Original

Detection of haploid nuclear death in living Paramecium caudatum
Xianyu YANG1*, Xin GAO1 and Xinlu SHI2
1

School of Forestry and Biotechnology, Zhejiang Forestry University, Hangzhou 311300,
China, 2 Hangzhou Key Laboratory for Animal Science and Technology, Hangzhou Normal University, Hangzhou 310036, China.
Key Words: Paramecium; conjugation; apoptosis; acridine orange; Hoechst 33342

SUMMARY
During conjugation of Paramecium caudatum,
four haploid nuclei are formed after meiosis, only
one of those that enters the paroral region survives
and divides once more to form two gametic nuclei,
and the remaining three degenerate. Here, we applied the method of co-staining the living cells withacridine orange and Hoechst 33342, which had
been found effective to identify the apoptotic nuclei
in Tetrahymena, to clarify whether the meiotic nuclear degeneration in P. caudatum is an apoptotic
process. We found that three haploid nuclei outside
the paroral region were stained green-yellow, while
the survivor in the paroral region was stained blue.
When the conjugating pairs separated and the synkaryon divided twice, three degenerating haploid
nuclei were still observed and stained yellow. The*Corresponding author
Tel: +86-571-63744008 Fax: +86-571-63740809
E-mail: xianyu_yang@hotmail.com
Received: 7 June 2007; Accepted: 11 September 2007

old fragmented macronuclei, which are known to
be still alive in exconjugants, were stained blue, in
sharp contrast with the case in Tetrahymena.
These results strongly suggest that the haploid
nuclei degenerate in an apoptotic way in P. caudatum.

INTRODUCTION
Ciliates are a group of unicellular organisms
having two kinds of nuclei, small diploid germinal
micronuclei and large polygenic somatic macronuclei (Nanney, 1980), and nuclear events follow a
programmed schedule during their sexual process,i.e., conjugation. Paramecium caudatum is one of
the well-known ciliates with one micronucleus andone macronucleus, and during conjugation nuclear
events include meiosis, gametic nuclear formationand exchange, formation of synkaryon (fertilized
nucleus), three successive postzygotic nuclear divisions (synkaryon divisions), new macronuclear
differentiation and development, and old macronu-

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Nuclear death in Paramecium caudatumclear fragmentation and degeneration, meanwhile
old oral apparatus is disintegrated and replaced bya new one (Wichterman, 1986). In fact, during
conjugation of P. caudatum, there are three stages
in which the micronuclei encounter fate determination. 1). After meiosis, only one meiotic productsurvives, while the other three degenerate (Calkins
and Cull, 1907). 2). Soon after the 3rd synkaryon
division, four posterior nuclei are differentiatedinto macronuclear anlagen; four anterior nuclei
remain as presumptive micronuclei (Mikami,1980). 3). In exconjugants, only one presumptive
micronucleus is selected as a germinal nucleus forthe next generation, and the remaining three degenerate (Calkins and Cull, 1907). In the case ofold macronuclei, they break into approximately
fifty pieces, and finally, the old macronuclear fragments degenerate and are replaced by the newmacronuclei. Therefore, nuclear degeneration happens three times during the sexual process of P.
caudatum: degeneration of haploid meiotic nuclei,
extra-presumptive micronuclei elimination, andold macronuclear degeneration. However, the oldmacronuclear fragments in exconjugants remainfunctional and keep their ability to undergo DNA
synthesis and regenerate into the macronucleus(Mikami, 1988). Recently, it was reported that theold macronuclear fragments finally undergo an
apoptotic process in P. caudatum (Kimura et al.,2004). Concerning the degradation of meiotic
haploid nuclei and the extra-presumptive micronuclear elimination, it is still not clear whether they
undergo an apoptotic process.
Apoptosis is characterized by oligonucleosomesized DNA fragmentation, which can be detectedby agarose gel electrophoresis (Compton, 1992),
and it also can be detected by a TUNEL technique(Gavrieli et al., 1992). In Drosophila, acridine
orange (AO), a vital fluorescent dye, has been used
to identify apoptotic cells (Abrams et al. 1993;
White et al., 1994). In Tetrahymena, AO was combined with another vital fluorescent dye, Hoechst

33342 (HO), and, by use of these two fluorescentdyes, the normal nuclei were stained blue, and thedegenerating meiotic nuclei and old macronuclei
were stained yellow-orange or yellow-green(Mpoke and Wolfe, 1997; Santos et al., 2000).
The various advantages of AO/HO staining over
the TUNEL technique to detect apoptosis werediscussed, and AO/HO double staining was proposed as a useful method for specifically identifying apoptotic cells (Mpoke and Wolfe, 1997).
Therefore, in this experiment, we focused on the
study of meiotic haploid nuclear degradation in P.
caudatum to clarify whether this process is apoptotic by AO/HO staining. Here, we will reportthese results and discuss some related experimentresults.

MATERIALS and METHODS
Chemicals and Stock Solutions
AO was purchased from Shanghai Chemical Reagent Company (China), and Hoechst 33342 from
Beyotime Institute of Biotechnology (Haimen,
Jiangsu, China). Other chemicals were from
Hangzhou Dafang Chemical Reagent Inc (China).
Stock solutions (50x) of both AO (1 µg/ml) andHoechst 33342 (10 µg/ml) were prepared with
autoclaved water and kept in 4°C refrigerator.
Cell Culture and Conjugation Induction
Two complementary mating types of P. caudatum
were cultivated in 2.5% fresh lettuce juice diluted
with modified Dryl’s solution (Dryl, 1959), prepared by substituting KH2PO4 for NaH2PO4 (KDS). Klebsiella pneumoniae was inoculated one
day before use (Hiwatashi, 1968). The conjugation was induced by mixing highly reactive cells ofcomplementary mating types (Hiwatashi, 1968).
To obtain more concentrated conjugating pairs, weused iron-dextran particles (Vosskühler and Tiedtke, 1993) to separate conjugating pairs of P. cau-Jpn. J. Protozool. Vol. 40, No. 2. (2007)

125

datum (Yang and Takahashi, 1999). All the experiments were performed at room temperature (～23°C).

they were observed and photographed under a
Nikon 50i fluorescence microscope.

Staining, Observation, and Photographing
Ten minutes or more before the observation, 10µl each of AO and Hoechst 33342 stock solutions
was added to a 480 µl cell suspension (final concentration: 0.02 µg/ml of AO; 0.2 µg/ml of
Hoechst 33342). To make the preparations, thecells were transferred to a slide glass with as little
solution as possible, and the movement of cells
was slowed down by placing a cover glass on fourtiny drops of soft flour paste around the cells andpressing the four corners of the cover glass, asreported by Shi et al. (1985). When the cells became flattened and started to move very slowly,

RESULTS
Micronuclear Behavior at Vegetative Stage andduring Meiosis
After cells of two complementary mating
types are mixed to induce conjugation, the conjugating pairs are formed; the micronuclei then migrate out of the concavity of the macronucleus(Fig. 1a) and undergo meiosis through a long prophase including a crescent stage (Fig. 1b) to form
4 haploid nuclei (Fig. 1c, 1d). Figure 2 shows avegetative cell in which the micronucleus (arrow)is located in the concavity of macronucleus

Fig. 1. Schematic representation of major nuclear stages during the conjugation of Paramecium caudatum. a.
Soon after the formation of a conjugating pair. Micronuclei have left the concavity of the macronuclei. b. Crescentstage of 1st meiotic prophase. c. 1st meiotic division. d. 2nd meiotic division. One meiotic product enters the paroral region (dot-lined areas). e. 3rd prezygotic nuclear division. The nucleus enters the paroral region and divides
mitotically to form gametic nuclei, and the remaining three degenerate (crosses). f. Synkaryon formation. g. 1st
synkaryon division. h. 2nd synkaryon division. i. 3rd synkaryon division. At this stage, cells become rounder andshorter. Four nuclei locate in the posterior region, and the other four locate in the anterior region of the cell. The
old macronuclei show a thick skein form. j. Nuclear differentiation. Arrowhead: macronuclear anlagen having
aggregated chromatin; arrow: spherical presumptive micronuclei. There are many old macronuclear fragments. k.
Development of macronuclear anlagen and germinal nuclear selection. Arrowhead: more developed macronuclear
anlagen; arrow: one selected micronucleus; crosses: presumptive micronuclei destined to be degenerated.

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Nuclear death in Paramecium caudatum

Fig. 2. An AO/HO co-stained vegetative Paramecium
caudatum. Arrow: a micronucleus in the concavity ofthe macronucleus. Triangle: a macronucleus. Many
yellow-orange or yellow food vacuoles were observed.

(triangle); both of them are stained blue by costaining of AO and Hoechst 33342, while the foodvacuoles are stained yellow or yellow-orange.
Figure 3A shows a conjugating pair soon after pairformation approximately 2 h after conjugation, andthe micronuclei migrated out of the concavity ofthe macronucleus. Figure 3B shows a conjugating
pair soon after 1st meiotic division, and Fig 3C
shows one that entered 2nd meiotic division approximately 12 h after the induction of conjugation. Figure 3D shows a conjugating pair after 2nd
meiotic division. Except in Fig. 3C, both micronuclei (arrows) and macronuclei (triangle) are stained
blue (Fig. 3A, 3B, 3D), while both are stained
greenish-blue in Fig. 3C. Many trapped AO particles show brilliant orange-red color in Fig. 3C.
The Process of Meiotic Nuclear Degeneration
Soon after meiosis, one of the haploid nucleithat enters the paroral region survives (Fig. 1d) andcompletes 3rd prezygoic nuclear division to form
two gametic nuclei: a migratory pronucleus and astationary pronucleus (Fig. 1e). In contrast, theremaining three nuclei outside the paroral regions
degenerate (Fig. 1e, 1f). Figure 3E shows a conjugating pair at the stage of 3rd prezygotic division,and Fig. 3F shows one soon after 3rd prezygotic
division, approximately 14 h after conjugation;
here, both the surviving meiotic nuclei or the gametic nuclei (solid arrows) and macronuclei(triangles) are stained blue, while two or threegreen-yellow degenerating nuclei (hollow arrows)
are observed in each conjugant. In this experiment, 52 cells around the third prezygotic divisionwere observed; and two green-yellow degenerating
nuclei were observed in 24 cells, and three wereobserved in 28 cells.
A Long Process of Haploid Degeneration
To see how long the degenerating haploid
nuclei remain in the cells, the cells at later stages
were also observed. After reciprocal exchange of
migratory pronuclei, synkaryons are formed anddivide three times successively to produce eight
nuclei (Fig. 1g-1i). Usually conjugating pairs
separate after 1st synkaryon division and become
exconjugant cells (exconjugants) (Wichterman,
1986). Approximately 16 h after conjugation,
most exconjugants finished 2nd synkaryon divisionand entered 3rd division. Here, 13 such cells wereobserved, and, in each cell 4 synkaryon division
products and 1 macronucleus were stained blue,
while 3 round structures were stained yellow, asshown in Fig. 4A, which were considered to be thedegenerating meiotic nuclei. Around 18 h after the
induction of conjugation, most cells were at theanaphase or early telophase of 3rd synkaryon division, and one or two yellow-stained structures
were still observed in 3 out of 11 cells (Fig. 4B),
while no such structures were observed in the remaining cells.
Old Macronuclear Behavior in Exconjugants
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127

Fig. 3. AO/HO co-stained conjugating pairs of P. caudatum. Triangles: macronuclei; solid arrows: micronuclei or
meiotic products; hollow arrows: degenerating haploid meiotic nuclei. A – D. Both micronuclei or meiotic products
and macronuclei were stained blue or greenish-blue. E and F. Both surviving meiotic products and gametic nuclei
were stained blue, while the degenerating haploid nuclei out of the paroral cone were stained green-yellow. A. A
conjugating pair soon after the pair formation approximately 2 h after conjugation and the micronuclei migrated outof the concavity of macronucleus. Both micronuclei and macronuclei were stained blue. B. A conjugating pair soonafter 1st meiotic division. There were 2 round 1st meiotic division products. C. A conjugating pair soon after 1st
meiotic division approximately 12 h after the induction of conjugation. Many chromosomes were observed in eachdivision product. Many trapped AO particles showed bright orange-red. D. A conjugating pair soon after 2nd meiotic division approximately 13 h after conjugation. E. A conjugating pair at 3rd prezygotic division approximately
14 h after conjugation. Many trapped AO particles showed bright orange-red. F. A conjugating pair soon after 3rd
prezygotic division approximately 14 h after conjugation. Scale bars: 20 µm.

Fig. 4. AO/HO co-stained exconjugants of Paramecium caudatum. White arrowheads: two contractile vacuoles. A.
An exconjugant after 2nd synkaryon division approximately 16 h after conjugation. Arrows: 4 synkaryon division
products; hollow arrows: degenerating haploid meiotic nuclei. B. An exconjugant at the earlier telophase of 3rd synkaryon division approximately 18 h after conjugation. Arrows: telophase nuclei of 3rd synkaryon division; hollow arrows: degenerating haploid nuclei; question mark: a haploid nucleus delayed to degenerate. The old macronucleus
started to break. C. An exconjugant 72 h after the induction of conjugation of P. caudatum. White arrows: macornuclear anlagen; black arrow: a presumptive micronucleus. Scale bars: 20 µm.

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Nuclear death in Paramecium caudatum

macronuclear anlagen, and the remaining four are
presumptive micronuclei (Fig. 1j). Through aprocess of complicated DNA rearrangement, themacronuclear anlagen become more developed,
and one of the presumptive micronuclei is selected, showing a spindle-like shape, while theother three eventually degenerate (Fig. 1k) (Yangand Takahashi, 2000). In all 17 exconjugants, 72 h
after conjugation, 4 macronuclear anlagen and 1 to
4 spindle-shaped presumptive micronuclei were
stained blue, and there were about 50 extra bluestained structures and some orange-red or orange
structures (Fig. 4C).

DISCUSSION
During conjugation of Paramecium caudatum, 4 haploid nuclei are formed after meiosis, andonly one of the nuclei that enters the paroral region
survives and finishes 3rd prezygotic division toform gametic nuclei, while the remaining threedegenerate (Fig. 1e) (Calkins and Cull, 1907; Yanagi, 1987). In Tetrahymena thermophila, 3 of 4
haploid meiotic products also degenerate, and boththe meiotic nuclear degeneration and the oldmacronuclear degeneration have been demonstrated to be an apoptotic process (Davis et al.,
1992; Mpoke and Wolfe, 1996, 1997; Santos et al.,2000). Therefore, it is reasonable to predict thatthe meiotic products of P. caudatum degenerate ina similar way, even though the issue has not beenthoroughly examined yet. To clarify this issue, thedegeneration of meiotic products of P. caudatum
was here studied by AO/HO co-staining method.
The results obtained show that the meiotic products outside the paroral region were stained yellow-orange or green-yellow at around 3rd prezygotic nuclear division (hollow arrows in Fig. 3E
and 3F), while the nuclei located in the paroral
region were stained blue. In contrast to this, themicronuclei or the meiotic products at the earlier

stages were always stained blue (Fig. 3B – 3D).
All these results suggested that haploid nuclei degenerate in an apoptotic way in P. caudatum as
they do in Tetrahymena (Santos et al., 2000).
Concerning when the meiotic products completely disappear from the conjugating cells, there
have been no clear descriptions. The current study
showed that degenerating meiotic nuclei existed
until 3rd synkaryon division (Fig. 4A and 4B), and,in Tetrahymena thermophila, the degenerating
haploid nuclei were observed in the conjugating
pairs after the end of 1st synkaryon division(synkaryon divides twice in Tetrahymena insteadof three times in P. caudatum) (Santos et al., 2000)
indicating a longer degenerating process of meiotic
nuclei in Paramecium than in Tetrahymena.
Regarding the old macronuclei of P. caudatum, their fragments have been demonstrated to befunctional in exconjugants that keep their ability
for DNA synthesis, and, when the new macronuclear anlagen fail to develop into functional macronuclei they are able to regenerate into the macronuclei (MR) (Mikami, 1988). In this report, there
were about fifty blue-stained structures and 16
orange-red or orange structures in the exconjugants of P. caudatum 72 h after mixing of the complementary mating types (Fig. 4C). The blue
structures were demonstrated to be the macronuclear fragments by staining the same cells withcarbol fuchsin solution (Carr and Walker, 1961)(data not shown), and this observation indicatedthat the old macronuclear fragments were functional and their degeneration was not initiated in
exconjugants of P. caudatum (Mikami, 1988). In
two other species of Paramecium, the macronuclear fragments (in P. tetraurelia) or condensed oldmacronuclei (in P. bursaria) were also stained bluein the exconjugants 48 hours or more after conjugation by AO/HO co-staining (data not shown).
Recently, it was reported that the old macronuclear
degeneration was an apoptotic process and that it
was initiated around the fifth cell cycle after theJpn. J. Protozool. Vol. 40, No. 2. (2007)

conjugation, indicating the delay of old macronuclear degeneration in P. caudatum (Kimura et al.,2004), which might be the reason that degenerating macronuclear fragments were not observed inthis study. In Tetrahymena, the degeneration ofold macronuclei has also been demonstrated to bean apoptotic process, which is initiated soon after
nuclear differentiation (Mpoke and Wolfe, 1997)
and much earlier than that in P. caudatum.
Concerning the three extra presumptive micronuclei in exconjugants, their degradation was
confirmed morphologically by carbol fuchsin
staining (Yang and Takahashi, 2000). In contrast
to this, Taka et al. (2006) demonstrated the division potency of the extra-presumptive micronuclei
even after the germinal micronuclear selection by
micronuclear elimination. In exconjugants asshown in Fig. 4C, there are about 50 old macronuclear fragments (stained blue) in different sizes and
16 orange-red or orange colored structures, whichmight be food vacuoles or the degenerating presumptive micronuclei themselves, as was mentioned above. Therefore, it is difficult to distinguish the degenerating presumptive micronuclei
from the other structures. To clarify whether the
extra-presumptive micronuclei degenerate in an
apoptotic way, special efforts might be needed toeliminate the interference of other structures suchas food vacuoles.
Finally, we have to explain the different colors of Figs. 2 – 4. Based on the colors，the pictures could be divided into a “blue group” including Figs. 2, 3A, 3B, 3D, and 3F and a “green”
group including Figs. 3C, 3E, and 4A - 4C. In thecourse of the observation of the cells co-stained by
AO and HO, both micronuclei and macronuclei
were stained blue at the beginning, except for thedegenerating meiotic nuclei (Figs. 3E and 3F) or
food vacuoles (Figs. 2 and 4C), and the cytoplasm
was stained so faintly that it was hard to recognize
the outline of the cells. However, when the observation time of cells became longer, both the nuclei

129

and the cytoplasm changed gradually to greenish.
Therefore, depending on the timing of photography, the functional surviving nuclei were sometimes blue and sometimes greenish blue.

ACKNOWLEDGEMENTS
We thank Prof. L. Q. Zhang and Dr. L. J. Ma
for their kind supports by providing us a Nikon 50i
fluorescent microscope. This work was partially
supported by the grants from National Natural
Science Foundation of China (No. 30670297,
30670222).

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