Ar ch. Protist onkd. 136 (19 88): 45-50
VEE Gust av Fischer Verlag .Ie na
Zo ol ogic al Institute, H iro shima Univer s ity, Japan
The Role of Micronuclei on Macronuclear Function in Euplotes patella
By KATSUYUKI SATO
With 2 Figures
Key words: Eup lot es patella syng en 2; lVla cr onuclear d eco mposition; Amicro nuc lea tes
Summary
'I'o investigate the role of micronuclei in E . patella, the n uclei wore remov ed from vegetative
cells and exconjugants by m icro-suet.ion . In veg etative cells, the macronuclei became visibly a be r rant. All of the cells died in a shor t time. In e xco n juga nt s, t he macronuclear a nla gen develo ped
normally a nd elong a t ed into rod-like a nlage n . Aft er elo ngatio n, however, t hey a lso degen er a t ed ,
a nd all of the ex con j uga n t s di ed out. These result.s s uggest t hat mi cronuclei a re necessary for the
conti n ue d functi ons of m a cronuclei a nd m a cr onuclear anlagen.
Introduction
Ciliat es have two kinds of nuclei ; macronuclei and mi cronuclei. Micronuclei genera lly a ct as germ nu clei in the sexual cycle, as suggest ed by SONNEBORN (1954).
In the T etrahymena pyrijormi8 complex, many naturally amicronucleate species were
reported (NANNEY and McCoy 1976). Amicronucleate stocks whi ch appeared spontaneously in cult ure and crossbreeding have been known also in Paramecium caudatum
(OSSIPOV and SKOBLO 1968), in P. multimicronucleatum, P. trichium and Oxytricha
jallax (DILLER 1965) , and E u-p lotes patella (KATASHIMA 1971). However, the importance of the micronucleu s in vegetative growth has been suggested by observations
on artificially produced amicronucleate lines of some ciliates, in whi ch loss of vigor
and mortality of cells have been reported (TAYLOR and FARBER 1924 ; SCHWARTZ 1946;
:MIYAKE 1956 ; WICHTER~IAN 1959; WELLS 1961; BORCHSENIUS and OSSIPOV 1978 ;
FOKIN and OSSIPOV 1981).
Parti cul arly, som e invest iga t ors have reported abnormalities of the oral apparatus in veget ative amicronu cleates, and also the abnormal reorganization of the oral
apparatus in amicronucleate exconjugants (DILLER 1965; MIKAMl 1979; FUJISHIMA
and WATANABE 1981; No and MIKAMl 1981). According to these reports, the abnormality of the oral apparatus seems to aff ect the viability of amicronucleates in Paramecium and Oxytricha. Few detailed studies, but, deal with changes in the macronuclei of amicronucleates (AMMERMANN 1970; GOLIKOVA 1978).
This pap er de scribes morphological macronuclear cha nges in vegetative amicronucleates and exconjugant amicronucleates which were obtained by removing the
micronu clei in E. patella.
Materials and l\'lethods
The used stocks were LX5b of mating type I, DX9a of t ype II, DXI0 and HY31 of type IV,
DY25 of type V and Al of u nknown type in Euplotes patella syngen 2. Stock Al was 10 fiss ions
46
K.
SATO
old, an offspring from the cross between stocks LX5b and DXI0. The other stocks were provided
through the kindness of Prof. R. KATASHIMA. The culture medium was 1 % wheat grain infusion
with Ghilomonas paramecium and Enterobacter aeroqenes, All cultures were kept at 24 ± 1°C.
The removal and restoration of micronuclei were carried out with a Chambers-type micromanipulator with a single needle as conceived by KOIZUMI (1974). The tip of the glass needle used
for these operations was about 3 ""m in inside diameter. The immobilization solution was 1 %
methyl cellulose. After the operations, the cells were placed in exhausted solution for one day,
and then transferred into fresh culture medium. To obtain amicronucleates, vegetative cells with
GI or early S phase macronuclei and exconjugants at 6 h after pair separation were used.
For cytological observation, cells were air-dried, fixed in MFG solution (methyl alcohol: formalin: acetic acid = 17: 2 : 1), and hydrolyzed for 1 h in 4 N HCI at room temperature. Then the
cells were stained by the Feulgen reaction and counterstained with 0.5 % Fast green FCF. Especially for the measurement of the width of macronuclei, 15-17 cells were stained by acetic carmine. The maximal width of the macronuclei (without the replication band) at Gl or early S
dhase was measured as the width of each macronucleus.
Results
The viability of vegetative amicronucleates was greatly limited (Table 1). In
stocks LX5b and DX9a, about 80 % of the cells could divide into two daughter cells
within 2 days after removing micronuclei. These daughter cells, however, died out
before the next division, even though they lived for about a week. Meanwhile, about
20 % of the cells died out within about a week, without division. A similar result was
obtained when micronuclei were removed from cells in a stock of recent origin. There-
Table 1. Viability of vegetative amicronucleates and exconjugants amicronucleate after micronuclear removal
Used stocks
No. of examined Alive
cells
Dead
No division
1 div.
~
7
7
11
25
24
21
o
o
o
o
1
o
10
16
5
31
33
o
o
o
Vegetative cells
Micronuclei removed
LX5b
DX9a
Al
32
31
32
o
o
o
Cytoplasm removed
LX5b
30
29
55
24
Micronuclei restored
LX5b
Exconjugants
Micronuclei removed
LX5bX DXlO
HY3IxDY25
31
34
o
o
1
Micronuclei restored
LX5bxDXIO
45
24
19
2
o
50
45
5
o
o
Untreated
LX5bxDXIO
2 div.
47
Amicronucleates in Euplotee
1
2
3
Fig. 1. Macronuclear changes in vegetative amicronucleates after micro nuclear removal. Bar
presents 20 [tm. 1. Condensation of a macronucleus in a cell 1 d after removal. 2. A partial breakdown (arrow) of macronucleus in a cell 3 d after removal. 3. A resorbed macronucleus in a cell
5 d after removal.
fore, the death of amicronucleates is probably not related to the aging of the stocks.
In control experiments, the cytoplasm (about 200 pm) close to micronuclei was removed. 29 of the 30 sham operated cells gave rise clones. In other experiments, micronuclei were drawn into a needle and immediately put back to their original position.
24 of the 55 cells whose micronuclei were restored grew into clones. But the others died
out. In a sample of cells in which micronuclear restoration was attempted, only 5 of
the 15 cells had micronuclei. Presumably all or most of the amicronucleates that
developed clones had been provided with effective micronuclear replacements. The
micronucleus is apparently necessary for the growth of vegetative cells.
Morphological changes of macronuclei were investigated after removing micronuclei. The shape of the macronuclei was normal in all cells at one day after removal.
At cellular division, the normal condensation of the macronuclei occurred (Fig. 1--1).
In the daughter cells, however, the macronuclei could not enter the next S phase,
then decomposed. The macronuclei did not retain their usual C-shape, but became
broad in their width. The mean width of the macronucleus in cells at 4 or 5 h after
removal was 5.9 ± 0.4 pm (95 % confidence limit), corresponding to that of ordinary
cells. Two and 3 d later, the width of the macronuclei in the daughter cells reached
10.0 ± 0.8 pm and 9.5 ± 0.9 pm, respectively. In addition, the macronuclei began
to break down 2 d after the removal (Fig. I-II). Their chromatin began to be resorbed, so that they became smaller in the following days (Fig. I-III). The same decomposition of macronuclei was observed in the amicronucleates that did not divide.
Do macronuclear anlagen also decompose in exconjugant amicronucleates? If so,
when does it start? To answer the questions, micronuclei were removed from 6 h
old exconjugants. In amicronucleates from both crosses, LX5b X DXlO and HY3I X
DY25, the cells usually survived for about a week after the removal. Most operated
cells died out without the first postconjugant division. On the other hand, when micronuclei were removed, and then put back in cells in the same way, 53 % of the restored cells could divide into clones. Most of the remaining cells remained alive for
about a week without division, and then died out (Table 1).
48
K.
S AT O
MF
1
2
3
Fig. 2. Ch anges of m acro n uclear anlagen in ex con juga nt amicronucleates . B ar presents 20 11m.
1. A nor m al ova l anlage. 2. An elonga t ing a nlage with a partial breakdown (a rrow). 3. An a b no r mal elongating a nla ge (MaA) a nd a pycnotic p reconjugant macro n ucle ar fr agm ent (MF).
In exco njugant amicronucleates, all t hese nu clear cha nges wer e t he same a s t hose
in normal cells up to the elongat ion of macronuclear anlage (Fi g. 2-1). However,
during or afte r its elongat ing, the anlage broke down partially in some ca ses (Fig .
2-II). In other cases, a n elongat ing anlag e was faintly st ained , suggest ing no occurrence of DNA synthesis in the ma cronuclear a nlage (Fi g. 2-III). Therefore, onl y
elongating rnacronuclear anlag en decomposed.
Discussion
Loss of vit ality and vigor in amicronucleates of E. patella was in agreeme nt with
some of t he previous observations (TAYLOR and FARBER 1924 ; KIMBALL 1941; KLOETZEL 1980 ; MIKAl\1l et al. 1985). Furthermore, as resul t s, the decomposition of vegetative ma cronuclei and elongat ing ma cronuclear anlagen occurred. In E . octocarinai u s, a similar decomposition was observed in spontaneous amicronucleates (K UHLMANN, person al communica t ion ) and a micronucleates in which t he mi cronuclei were
removed (MIKMU et al. 1985). Also in vegetative amicronucleates of the other ciliat es
(AMMERMANN 1970; GOLIKOVA 1978), morphological and functional changes of macronuclei have been reported, alo ng with loss of ability to divide normally. I n amieronucleate Paramecium bursaria (GOLIKOVA 1978), the macronuclei were fragment ed
occasionally. In Stylonychia mytilus (AMMERMANN 1970), pseudomicronuclei were
formed from the macronu clei. These observations suggest an intera ction between
t he macronucleus and t he mi cronucleu s. Interestingly, if mi cronuclei were removed
from E . patella ex conjugants at an early stage of ma cronuclear development, the oval
macronuclear anlagen at the st age dev elop ed normally into elon gating anlagen. But
the elongating anlagen also decomposed. Micronuclei might playa similar rol e in
both vegetative cells and excon jugants, but not be of major consequen ce in macronuclear development. The st ruct ure of the elongat ing anlagen resembles that of vegetative ma cronuclei. Both kinds of nuclei are C-shaped and ha ve a number of small
dense chromatin masses (TURNER 1930 ; KLOETZEL 1970) . In E . woodrujji (RAO 1968),
Amicronucleates in Euplotes
49
the elongating anlagen undergo active RNA synthesis. Therefore, functional macronuclei might decompose after micronuclear removal. As to other roles of the micronucleus in Euplotes, MIKAMI et al. (1985) provide evidence that the micronuclei perform functions also for the initiation of macronuclear DNA replication. The present
observations suggest that micronuclei play important roles not only in DNA replication but also in macronuclear function. When micronuclei are eliminated, macronuclei
seem to lose all their functions, that is, the abilities to divide, to replicate DNA, and
to synthesize RNA.
Acknowledgements
The author is grateful to Prof. Dr. R. KATASHIMA for his invaluable advice and encouragement. Thanks are also extended to Drs. D. L. NANNEY, K. HECK1\IANN and H.- 'V. KUHLMANN
for valuable comments and criticism of the manuscript.
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Author's address: KATSUYUKI SATO, Department of Natural Science, Naruto University of
Teacher Education, Takashima, Naruto 772, Japan.