J. Cell Sci. 82, 223-234 (1986)
223
Printed in Great Britain © The Company of Biologists Limited 1986
HOMOTYPIC PAIR FORMATION DURING
CONJUGATION IN TETRAHYMENA
THERMOPHILA
AKIO KITAMURA»t, TOSHIRO SUGAI AND YASUKO KITAMURA*
Biological Institute, Tohoku University, Aoba-yama, Sendai 980 and Department of
Biology, Ibaraki University, Mito 310, Japan
SUMMARY
In the ciliate Tetrahymena thermopkila, conjugation has been believed to occur only between
cells of different mating types. We found the formation of homotypic pairs during normal conjugation by using micronuclear morphological markers. Homotypic pairs formed preferentially
during the first lOmin following the first pair formation and comprised about half of the pairs.
These results suggest the involvement of mating-type non-specific adhesion of cells in the initial
step of conjugation. Homotypic pairs apparently persist for at least 30min and then separate into
single cells. Homotypic pairs are also formed when conjugant pairs re-form after mechanical
separation of heterotypic pairs. Five kinds of glycosidases, three kinds of proteases and phospholipase C showed no effect on either the formation of homotypic pairs or their separation. The
relation between the mating-type substances and the molecules responsible for mating-type nonspecific adhesion of cells is discussed.
INTRODUCTION
Since the discovery of conjugation and mating types in the ciliated protozoon
Tetrahymena pyriformis (Elliott & Nanney, 1952; Elliott & Hayes, 1953) considerable effort has been expended in the analysis of the early stages of conjugation in this
species. Conjugation of T. thermophila (formerly syngen 1 of T. pyriformis; Nanney
& McCoy, 1976) is known to involve three distinct developmental events leading to
cell pairing: initiation, co-stimulation and tip transformation of the cell. Initiation
occurs when sexually mature cells are removed from growth medium to a starvation
medium of low salinity (Bruns & Brussard, 1974). The presence of cells of complementary mating types is unnecessary for this step. However, the second step,
co-stimulation, requires a transient contact between cells of different mating types
(McCoy, 1972; Bruns & Palestine, 1975; Finley & Bruns, 1980). Co-stimulation,
which is a sexual cell recognition step, induces tip transformation, a morphogenetic
change in the anterior tip at which paired cells are joined (Wolfe & Grimes, 1979),
and leads to pair formation, followed by meiosis and genetic exchange between
conjugant cells. Conjugating pairs are believed to be all heterotypic (Nanney, 1977),
although Ron (1973, 1974) reported that homotypic pairs occur at a very low
• Present address: Zoologisches Institut der Universitat Munster, SchloBplatz 5, D-4400
Munster, FRG.
f Author for correspondence.
Key words: Tetrahymena thermophila, mating, homotypic pair formation, cell surface interaction,
self-incompatibility.
224
A. Kitamura, T. Sugai and Y. Kitamura
percentage (less than 0-1 %) during normal conjugation. In this report we describe
the formation of homotypic pairs in high proportions in the earliest stage of
conjugation and the involvement of mating-type non-specific adhesion in conjugation
of Tetrahymena. We also analyse the chemical nature of this adhesion. Part of this
work has been reported in abstract form (Kitamura et al. 1984).
MATERIALS AND METHODS
Stocks and culture methods
All stocks used in this study were derived from inbred strain B of T. thermophila. Stock nA
of mating type III has a haploid micronucleus and stock 4 of mating type II has a triploid
micronucleus. They were established from progeny of asymmetrical triplet conjugation according
to the method described by Preparata & Nanney (1977). Spontaneous selfing was never observed in
any stock throughout the present study. Cells were cultured axenically in a medium containing 2 %
(w/v) proteose peptone (Difco Laboratories), 1 % yeast extract (Difco) and 0-6% glucose (Waco
Pure Chem. Co.), without shaking, at 25°C.
Conjugation experiments
Conjugation was induced as described previously (Sugai & Hiwatashi, 1974; Sugai, 1984). Cells
in late log phase were concentrated by centrifugation in a hand-operated centrifuge and washed
three times with washing solution (WS), which contained 34-2mM-NaCl, 1-lmM-KCl, 1-1 mMCaCl2, and suspended separately in the same solution to a final concentration of about 5X10 5
cells ml" 1 . The suspended cells were incubated overnight at 25 °C for initiation. Equal numbers of
cells of the two mating types were then mixed to induce conjugation at 25°C. For determination of
the extent of conjugation, samples of the cells were removed from the medium and fixed with an
equal volume of saturated picric acid. Between 300 and 500 cells were counted at various times after
mixing the mating types and the percentage of conjugation was calculated as follows:
^
_
no. of conjueants X 2
Con ugat on (%) =
:—'-%— T .
X 100.
v
' 8
'
total no. of cells
For counting the numbers of homotypic and heterotypic pairs, the nuclei of pairs were fixed with
3 % formalin followed by post-fixation with Carnoy's fixative (acetic acid/ethanol, 1:3, v/v) for
lOmin, hydrolysed in 5M-HC1 for 30min at room temperature (a=20°C) and then stained with
acetic orcein. To distinguish between homotypic and heterotypic pairs we used clones of complementary mating types that differed in micronuclear size (see Results). The ratio of homotypic
pairs to total pairs induced was determined by counting about 250 pairs for each time point. For
calculation of the percentage of cells in homotypic pairs we used the following formula:
„, .
.
.
. ,_.
no. of homotypic pairs
.
.
._.
X 0
Conjugating homotypic pairs (%) =
c ™ 11 ^ 11011 ( % ) n 0 . o f total pairs
in which the percentage of conjugation was obtained by counting cells from picric acid-fixed
preparations instead of cells from acetic orcein-stained preparations, because some difficulties in
distinguishing single and paired cells were found in light-microscopic observation of the latter
preparations at lower magnifications.
Treatment of cells vrith enzymes
For glycosidase digestion, initiated cells were washed twice with WS, which contains 5 mM-2-(Nmorpholino)ethanesulphonic acid (MES) (the pH adjusted to 5-8 with NaOH), and suspended in
the same solution. Conjugating pairs usually appear 30—35 min after mixing cells of two different
mating types. A relatively small volume of concentrated enzyme solution was added to the cell
suspension immediately after the first pair was observed. Glycosidases tested were cr-mannosidase
Homotypic pairs in Tetrahymena
225
(Boehringer-Mannheim), /9-galactosidase (Sigma, grade VI), hyaluronidase (Wako Pure Chem.
Co.), /}-glucosidase (Research Division Miles Lab.) and neuraminidase (Sigma, type V). Tests of
trypsin (Sigma, type III) and ar-chymotrypsin (Sigma, type II) digestion were made by the same
procedures with WS containing S mM-Af-2-hydroxyethylpiperazine-W-2-ethanesulphonic acid
(HEPES) buffer, pH 7-8, adjusted with NaOH. The medium for treatment of cells with Pronase P
(Kaken Chem. Co.) or phoapholipase C (Sigma, type XII) was SmM-HEPES in WS, p H 7 - l .
RESULTS
Establishment of marker strains
To examine whether conjugating pairs are homotypic or heterotypic, we established clones with a micronuclear morphological marker according to the method of
Preparata & Nanney (1977). Three types of exconjugant clones differing in micronuclear size were obtained from asymmetrical triplet conjugation; one clone has
smaller than normal, presumably haploid, micronuclei (Fig. 1B,D), the second has
apparently normal-sized micronuclei and the third has larger than normal, probably
triploid, micronuclei (Fig. 1A,C). Although we do not know exactly the ploidies of
the micronuclei in these strains, our preliminary cytogenetic analysis indicates that
the micronucleus of strain nA is aneuploid.
When frequency distributions of micronuclear size were examined by measuring
the diameters of the micronuclei on photomicrographs of the stained cells, all cells of
stock nA examined were found to have micronuclei with a diameter between 1-0 and
2-0/tfn, and all cells of stock 4 were found to have micronuclei between 3-0 and
4'0/un (Fig. 2), while the average normal diploid nucleus was about 2-5[im in
diameter (not shown). As shown in Fig. 2, the diameters of micronuclei of stock nA
were never larger than 2-0fim and those of stock 4 never smaller than 3-0^m,
showing no overlapping of size between them. No significant change in nuclear size
occurred in either stock during the course of this study. These results provided us
with good morphological markers and led us to use these stocks for the determination
of homo- and heterotypic pairs.
Detection of homotypic pairs during conjugation
When inititated cells of stocks nA and 4 were brought together, the first pairs were
usually observed 25—30min after mixing. Then the percentage of cells in pairs
increased exponentially to reach the maximum (80—90%) in about 1 h (Fig. 3).
To determine whether conjugating pairs are homotypic or heterotypic, samples
were removed from the cell suspension at various intervals after mixing cells of the
complementary mating types, and then examined by staining. As shown in Fig. 4,
about 45% of the pairs were homotypic during the first lOmin following the
formation of the first pair. Both types of homotypic pairs, those between cells of
mating type II (Fig. 5B) and those between cells of type III (Fig. 5C), appeared in
almost the same numbers, being 20% and 24% of all induced pairs, respectively.
The percentage of the total homotypic pairs then decreased to 11 % in 30 min and to
less than 2% in 70 min (Fig. 4). Similar results were obtained when another
combination of different strains were tested (stock M-3 of mating type IV with
A. Kitamura, T. Sugai and Y. Kitamura
ma
1A
B
^^M-—ma
Fig. 1. Difference in micronuclear size between stock 4 (A,C) and stock nA (B,D). After
fixation with Carnoy's fixative, preparations were stained with acetic orcein. Outlines of
the cells are obscure in A and B. Both macro- (ma) and micronuclei (mi) are easily seen in
A, while the micronuclei can hardly be seen in B. Bar, 10/fln.
Homotypic pairs in Tetrahymena
400
n - 550
x=
S.D. = 0-26 fan
n = 550
x = 3'4/rni
S.D . = 0-28
200
I
z
0
—•— — 1 — 1 \—
10
1—
20
3-0
Size of micronuclei (,um)
—i—
4-0
i
Fig. 2. Histogram of micronuclear sizes of stock nA (open column) and of stock 4
(stippled column). Number of micronuclei («), average diameter (x), standard deviation
(S.D.).
triploid micronuclei and stock IB-1 of VI with haploid micronuclei). No external
differences between homotypic and heterotypic pairs in the mode of cell contact was
found by light-microscopic observation.
As seen in Fig. 3, the percentage of the total number of cells forming homotypic
pairs was about 7% in the first lOmin after the first pair formation. This value
continued for the succeeding 30 min and dropped to 1 % 70 min after the first pair
formation, indicating that homotypic pairs eventually separated into single cells.
In order to know how long the homotypic pairs persist in mating, we checked their
micronuclear changes at each time point by staining cells. Sugai & Hiwatashi (1974)
reported that micronuclear changes at the late stage I of meiotic prophase occur
about 60 min after the onset of conjugation. Micronuclear changes typical for this
stage was observed in most homotypic pairs 60 min after the first pair formation. On
the contrary, these changes were not seen in any homotypic pairs when tested within
30 min after the first pair formation. From these results and those shown in Fig. 3, it
is suggested that homotypic pairs can unite for at least 30 min and separate in about
70 min. For the determination of the exact duration of conjugation between
homotypic pairs, a more precise measurement is necessary using, for instance,
another morphological marker such as double monster cells.
Formation of homotypic pairs in weakly mating reactive cells
In order to know whether homotypic pair formation depends on the extent of
mating reactivity of the cells, cells with weak mating reactivity were mixed to induce
conjugation. For the preparation of these cells, initiation was carried out at a cell
density five times as high as that normally used for initiation. In Fig. 6, the kinetics
A. Kitamura, T. Sugai and Y. Kitamura
228
of pairing in the weakly reactive cells show a great delay in the appearance of the first
pair (55 min after mixing) and a slow rate (maximum pairing in about 3 h), compared
with the kinetics of pairing in highly reactive cells (Fig. 3). When acetic orcein-
20
40
60
80
Time after mixing (min)
20
40
60
Time after pairing (min)
100
80
Fig. 3. Kinetics of cell pairing and percentage of cells in homotypic pairs versus total
cells. Abscissa: time after mixing cells (upper scale) and time after the first pair formation
(lower scale). Ordinate: percentage of cells in pairs. Initiated cells of stocks nA and 4 were
mixed at 25°C to mate at time 0 in the upper scale. (O
O) Homotypic pairs;
(•
• ) total (homotypic plus heterotypic) cell pairing. Percentage of cells in pairs was
determined from counts of 300-500 cells at each point. The percentage of cells in
homotypic pairs was calculated by the formula presented in Materials and Methods. The
arrow indicates the time of appearance of the first pairs.
0-5
I
1-0
10
15
VWA
!? 20
~25
I
40
70
Fig. 4. Proportions between homotypic and heterotypic pairs during the course of
conjugation. The first pairs appeared at time 0 and about 250 pairs were examined at each
time indicated. (D), (•) and ( • ) , pairs of mating types II—III (heterotypic), II—II
(homotypic) and III—III (homotypic), respectively.
Homotypic pain in Tetrahymena
229
4
5A
B
Fig. 5. Photomicrographs of three types of conjugating pairs. A. Heterotypic pair
between mating type II and III cell; B, homotypic pair between type II cells (triploid);
C, homotypic pair between type III cells (haploid). Bar, 20/fin.
stained preparations of cells were examined for scoring the ratio of homotypic to total
pairs, 39% (108/276) of the pairs proved to be homotypic during the first 10 min
after the onset of pair formation. The value decreased to 19 % (48/259) in 30 min, to
2-8% (7/250) in 60 min, and to 0-4% (1/250) in 90 min. The kinetics of homotypic
pairing in Fig. 6 show that homotypic pairs were rarely seen 70 min after the first pair
formation. These results are reproducible and show striking similarities in the
proportions between homo- and heterotypic pairs to those presented in Fig. 4.
230
A. Kitamura, T. Sugai and Y. Kitamura
Homotypic pair formation in rejoining cells
As shown in Fig. 3, the majority of conjugating pairs at the plateau of pairing
are in the form of a heterotypic union, which will last until the completion of
conjugation.
To determine whether the cells in heterotypic pairs are no longer able to form
homotypic pairs, we determined the percentages of homotypic pairs after mechanical
separation. After being washed twice with WS, the pairs were separated mechanically to single cells by placing the tip of the pipette flush against the surface of the
glass Petri dish and forcefully ejecting the cell suspension at least 10 times. Neither
breakdown of the cells nor marked change in swimming behaviour in separated cells
were seen after the completion of pair-separation.
As seen in Fig. 7, the cells began to re-pair within a few minutes and about 80 % of
cells united in 30 min. The percentage of cells in homotypic pairs was scored to be
about 16% 10 min after the appearance of the first re-formed pair and dropped to
3-5 % in 30min at a much faster rate than in normal conjugation. During the first
5 min following the first re-formation of pairs, about 32 % of the re-formed pairs were
homotypic. These results show that the mating-type non-specific attachment occurs
when pairs re-form.
|
0
80
120
Time after mixing (min)
|
,
30
60
Time after pairing (min)
160
,
90
,_
120
Fig. 6. Kinetics of cell pairing and percentage of cells in homotypic pairs in weak matingreactive cells. Abscissa: upper scale shows time after mixing them, and lower, time after
the first pair formation. Ordinate: percentage of cells in pairs indicated as in Fig. 3.
( • ) Total (heterotypic plus homotypic) pairs; (O) homotypic pairs.
Homotypic pairs in Tetrahymena
90
Time (min)
231
210
Fig. 7. Time course of homotypic pair formation after mechanical separation of pairs.
Abscissa: time after mixing cells of stocks nA and 4. Ordinate: percentage of cells in pairs
indicated as in Fig. 3. Initiated cells of stocks nA and 4 were mixed at time 0 to allow
pairing ( • ) and conjugating pairs were separated mechanically at the time indicated by
arrow. One half of the cell suspension was left for further steps of conjugation under
unchanged conditions. After the separation of pairs, samples were examined for rejoining
(A) at intervals. The percentage of cells in homotypic pairs to total cells (O) in the course
of rejoining was obtained as in Fig. 3.
Effects of various types of hydrolases on the formation of homotypic pairs
The fact that homotypic pairs appear at the initial step of conjugation suggests the
involvement of mating-type non-specific adhesion of cells in the early stage of cell
pairing.
In order to know the molecular basis of mating-type non-specific adhesion of cells
and the mechanism of pair separation from this adhesion, we tested the effects of
three kinds of proteases, five kinds of glycosidases and phospholipase C on the
formation and separation of homotypic pairs.
As shown in Figs 3 and 6, homotypic pairs are detected at the maximum ratio
10 min after the first pair formation and disappear in 80 min, irrespective of the
degree of mating reactivity of cells. We treated cells with each enzyme immediately
after sighting the first pair and examined the percentages of cells in homotypic pairs
10, 30 and 80 min after the first pair formation. No significant difference in the
percentage of cells in homotypic pairs was seen after any enzyme treatment when
compared with control cells. Concentrations of enzymes tested were 5^gml"' for
ar-mannosidase and /3-glucosidase, 100/igmP 1 for Pronase P and trypsin, and
10/igmP 1 for the rest.
232
A. Kitamura, T. Sugai and Y. Kitamura
Failure of artificial induction of homotypic pairs by chemical agents
Chemical induction of conjugation in ciliates was first reported in Paramedum
caudatum by Miyake (1958). Homotypic pairs are induced among cells of a single
mating type by treatment with high concentrations of potassium or other cations
under Ca-poor conditions. In order to know whether homotypic pairs are induced
without the presence of cells of complementary type, we attempted chemical
induction of conjugation in Tetrahymena.
After being washed twice with 1 mM-phosphate-buffered saline, pH7-2, initiated
cells were treated at a cell density of about SxlO'mP 1 with various concentrations
(6-25 mM) of KC1 plus acetamide (25 mM) and acriflavine (50/igml" 1 ), and with
ethyleneglycol-bis(/S-aminoethyl ether)-iV,./V'-tetraacetic acid (EGTA, 0-1-3 mM).
The mating reactivity of the cells was not noticeably affected by these treatments. In
spite of trying many techniques suitable for chemical induction of conjugation in
Paramedum, no pairs were observed in Tetrahymena.
DISCUSSION
In Tetrahymena, studies of the occurrence of intraclonal conjugation have been
made intensively using selnng stocks (Nanney, 1953; Allen & Nanney, 1958). The
most important fact that emerged from those investigations was that intraclonal
conjugating pairs in selnng stocks do not consist of cells of the same mating type but
of cells of different mating types. Thus, clones undergoing selfing conjugation are
mosaic for mating types. There has been no evidence of the formation of homotypic
pairs in cell populations of pure mating type except for the report by Ron (1973,
1974), who demonstrated the rare occurrence (less than 0-1 %) of homotypic pairs
during conjugation using radioactively labelled cells. However, it seems uncertain
that the pairs induced were truly homotypic, because natural selfing was observed
frequently in one of the stocks he used and homotypic pairs were detected only in this
stock (Ron, 1974). In this study we demonstrate the formation of both types of
homotypic pairs, those between cells of one mating type and those between cells of
the other type, using two kinds of established marker strains in which natural selfing
was never seen.
This study confirmed that T. thermophila has a well-established self-incompatibility system in conjugation. This self-incompatibility system has been generally
accepted through genetical cross-breeding analysis with genetic markers, but little is
known about the exact step in conjugation at which the self-incompatibility reaction
occurs. The results obtained here reveal that self-incompatibility is not ensured by
the system of mating type-specific adhesion of cells but is achieved by the mechanism
of separation of homotypic pairs. This shows that mating-type non-specific adhesion
must be involved in the early stages of Tetrahymena conjugation. Unexpectedly,
however, homotypic pairs occur only in cells united early following mixing, while the
majority of pairs occurring subsequently are heterotypic (Fig. 3). One possible
explanation for this phenomenon is that only the most reactive cells can unite
Homotypic pairs in Tetrahymena
233
precociously in a mating-type non-specific manner. This hypothesis is, however,
ruled out by our results that demonstrate that homotypic pairs also appear when cells
with weak mating reactivity are mixed for conjugation (Fig. 6). It is likely that
conjugating cells secrete substances that inhibit the further formation of homotypic
pairs. Our preliminary experiments have shown that homotypic pair formation is
significantly affected by cell-free fluid from a conjugating cell suspension.
Since no enzyme tested affected the formation of homotypic pairs, we could not
clarify the chemical nature of this non-specific adhesion or the relation between
the adhesion molecule and the mating-type substances, which are hypothetical substances responsible for the co-stimulation reaction. The most plausible explanation
for the identity of the cell adhesion molecules is that mating-type specific and nonspecific adhesion molecules would take part in the adhesion, since the pairs formed
after the early pairs have formed are believed to be almost all heterotypic (Fig. 3) and
separated conjugant cells retain a high ability for non-specific cell adhesion (Fig. 7).
For the molecular basis of this mating-type specific adhesion, two explanations are
possible. It might be mediated by complementary substances different from the
mating-type substances. Alternatively, mating-type substances themselves may be
involved in the cellular adhesion. In this case, accumulation of those molecules
during co-stimulation may be caused by a mechanism similar to the positive feedback control of mating-type substances suggested to operate in Euplotes crassus
(Heckmann & Siegel, 1964; Dini & Miyake, 1982) and Oxytricha bifaria (Esposito
et al. 1976). Although further evidence is necessary to decide which explanation
is correct, we are strongly inclined to the latter view, because our experiments
(Kitamura & Sugai, unpublished data) showed a remarkable similarity betwen the
sensitivities to various kinds of enzymes in co-stimulation and heterotypic pair
formation.
The results suggest that the homotypic pairs split into single cells after their brief
contact. As to homotypic pair separation, we could not discover its molecular
mechanism since all enzyme treatments tested had no effect. One reasonable
hypothesis for homotypic pair separation is that the interaction between cells of
complementary mating types is necessary not only for triggering cell union but also
for its maintenance. Probably, partner cells of a heterotypic pair, which are loosely
united at first, can continue to stimulate each other to form a firm union. On the
other hand, homotypic pairs will soon fall apart because they lack mating-type
interactions. The most probable candidate for such mating-type interactions is the
co-stimulation reaction, and ciliary contact between complementary mating types
would be an essential part of this interaction.
We thank Dr K. Hiwatashi for helpful comments on the manuscript and Dr K. Heckmann for
reading it.
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(Received22 October 1985 -Accepted 15 November 1985)