Jpn. J. Protozool. Vol. 38, No. 2. (2005)

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OriginalInhibition of mating pair formation by wheat germ agglutinin and wheat
germ agglutinin-binding substances in Paramecium caudatum cells
Ariko FUKE1,2* and Katsuyuki SATO1
1

Department of Biology, Naruto University of Education, Takashima, Naruto-cho, Naruto-shi,Tokushima, 772-8502, 2Joint Graduate School in the Science of School Education, Hyogo
University of Teacher Education, Shimokume, Yashiro-cho, Kato-gun, Hyogo, 637-1494, JapanSUMMARYINTRODUCTION

In this study, we revealed that the lectin wheatgerm agglutinin (WGA) and its specific binding
sugars, N-acetylneuraminic acid (NeuAc) and Nacetylglucosamine (GlcNAc), inhibited mating pairformation in Paramecium caudatum. The concentrations that caused 50% inhibition (IC50) were 17nM, 3 mM and 30 mM, respectively. Using FITCWGA, it was shown fluorescence-cytochemicallythat WGA bound to the adhesion site between conjugated cells; this binding was inhibited by NeuAc
but not GlcNAc. Moreover, the binding of FITCWGA was maintained appreciably when its localization was changed in correspon-dence with progression of conjugation until exchange of gametic
nuclei was completed at which point the binding
between both cells disintegrated. These results
strongly suggested that NeuAc plays a primary rolein mating pair formation in P. caudatum as a specific WGA binding sugar.

Conjugation is the sexual process of ciliates
such as Paramecium caudatum. During this process, cells form a mating pair through mutual recognition of complementary mating types. Conjugation in P. caudatum proceeds through the following steps: immediately after mixing cells of complementary mating types, cells adhere with eachother through their cilia (mating reaction)(Takahashi et al., 1974; Kitamura and Hiwatashi,1976). The cilia then degenerate at the ventral sideof adherence and within about 60 minutes after
onset of the mating reaction, they adhere anteriorventrally along their surface (holdfast union).
Then, within around 4 hours after beginning themating reaction, cells adhere ventrally along their
entire surface (paroral union). During this step,
cells are united firmly as both cell membranes
become partly fused (Vivier and André, 1961;
Hiwatashi and Kitamura, 1985). Cells then exchange gametic nuclei derived from micronuclei
through the oral region where the cell membranes
have fused; this forms a fertilized nucleus. Thereafter, conjugated cells leave each other and amacro- and micronuclei are newly formed from thefertilized nucleus in each individual cell
(Fujishima, 1988).

*Corresponding author
TEL : +81-88-683-6663
E-mail: fuke02@abelia.ocn.ne.jp
Received: 22 Oct. 2004; Accepted: 2 May 2005.

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WGA-binding substances in Paramecium caudatum

In this way, conjugation is a phenomenon,
characterized by mating pair formation in which
adherence between individual cells is mediated bytheir membranes. Attempts have been made toinvestigate the involvement of specific complex
polysaccharides in the cell membrane during mating pair formation using lectins with considerable
specificities in recognizing certain sugar residues
in the complex. For example, Concanavalin A(Con A) does not have any inhibitory effect onmating pair formation of Paramecium tetraurelia(Pape et al., 1988), but it does effect other ciliates
including P. caudatum (Casci and Hunaggel, 1988;
Evelyn et al., 1995; Frisch and Loyter, 1977; Tsukii and Hiwatashi, 1978). Mating pair formationsof P. tetraurelia and P. primaurelia were inhibitedby another lectin, wheat germ agglutinin (WGA)(Pape et al., 1988; Delmonte et al., 1997). Moreover, in P. tetraurelia, Pape et al. (1988) observed
specific labeling of fluorescein isothiocyanateconjugated WGA (FITC-WGA) at the adhesion
site between conjugated cells. These results
strongly suggest that these lectins inhibit mating
pair formation by recognizing and then binding
specifically to the sugar of the complex polysaccharide at the adhesion site in these ciliates.
Since these works indicate that inhibition bylectin might be species specific to some extent, andsince no attempts have been made to investigate theinhibitory effect of WGA on mating pair formationin P. caudatum cell, we investigated the effect ofWGA. The effects of various concentrations ofWGA-specific binding sugars, N-acetylneuraminicacid (NeuAc) and N-acetylglucosamine (GlcNAc),
were examined quantitatively to identify the sugarthat plays a primary role in mating pair formation ofP. caudatum. Moreover, we followed the changes
in localization of the specific WGA binding sitewith progress of conjugation using FITC-WGA.

MATERIALS AND METHODS
Cell culture and preparation of cell suspension
KNZ2 (mating type E) and KNZ5 (matingtype O) strains of P. caudatum were provided byMikami, Environment Education Center, Miyagi
University of Education, and cultured at 25±1°C
by adding fresh medium everyday for 4 days. Themedium was prepared by growing Klebsiella pneumoniae in fresh lettuce juice medium (pH6.8) for 1
day at 25±1°C. Cells were collected by manual
centrifugation then precipitated cells were washedtwice in Dryl’s solution (Dryl, 1959) and incubated at 25±1°C for 1 hour after cell density wasadjusted to 2500 cells/ml with Dryl’s solution.
Effect of WGA and its specific sugars on mating pair formation and calculations of the rateof mating pair formation
WGA, NeuAc and GlcNAc, purchased from
Sigma (St Louis, MO, USA), were stored at –20°C
then dissolved in phosphate buffered saline (PBS)(pH 7.0) immediately before use at concentrationsof 28 µM, 25 mM and 1 M, respectively. For individual inhibition tests of the lectin and its specificsugars, upon mixing equal volumes of suspensionsof cells of complementary mating types in a depression glass slide, the WGA, NeuAc andGlcNAc-PBS solutions were added at maximum
final concentrations of 83 nM, 5 mM and 100 mM,
respectively.
Four hours after mixing, both the total number of cells and number of pairs were counted in
the cell mixture after fixing with a saturated picricacid solution (Nacalai Chemcal Co. Ltd., Kyoto,Japan). Fixation was performed in a petri dish using a drop of cell suspension mixed with saturated
picric acid. As control experiments, the lectin or
conjugated sugars were omitted from the incubation medium. Each inhibition and control experiment was run at least in triplicate. The rate of mating pair formation was calculated by the followingJpn. J. Protozool. Vol. 38, No. 2. (2005)

equation from the average of three experiments:
rate of mating pair formation (%) ={(Total numberof mating pairs) / (Total number of cells/2)}×100.
Cytochemical staining of conjugated cells withFITC-WGA
Equal volumes of cell suspension with complementary mating types were mixed in a small
Erlenmeyer flask then incubated at 25±1°C. At
each prescribed conjugation step, aliquots of cellsuspension were centrifuged manually to collectconjugated cells, which were then suspended in
Dryl’s solution. FITC-WGA (Sigma) dissolved in
PBS (pH 7.0) at 1 mg/ml was added to each suspension at a final concentration of 30 µg/ml thenincubated at 25±1°C for 15 minutes. After centrifuging at 600 ×g for 1 minute, precipitated cells
were suspended and incubated in Dryl’s solution at25±1°C for 10 minutes. After a second round ofcentrifugation at 600×g for 1 minute, cells werethen suspended and fixed in 4% paraformaldehyde(Wako Pure Chemical Indus-tries, Ltd., Osaka,Japan) dissolved in PBS (pH 7.0) at room tempera-165

ture for 15 minutes. Cells were then washed twicein PBS (pH 7.0) by incubation at room temperaturefor 5 minutes combined with manual centrifugation. To confirm the conjugating step, cell nuclei
were stained with 0.0001%(W/V) 4,6-diamino-2phenylindole (DAPI) (Wako Pure Chemical Industries, Ltd.). The localization of FITC-WGA wasobserved under a fluorescence microscope(OPTIPHOT-2, Nikon, Tokyo, Japan) under conditions of B excitation (excitation at 450-490 nm).
Inhibition of WGA binding to the adhesion site
in the presence of NeuAc or GlcNAc
Before adding to the conjugated cells (4 hours
after cell mixing), 30 µg/ml FITC-WGA was preincubated with 4 mM NeuAc or 100 mM GlcNAc
in PBS (pH 7.0) at 25°C for 20 minutes. Cells
were incubated in WGA-NeuAc or WGA-GlcNAc
mixtures at 25°C for 15 minutes then washed,
fixed and observed as described above. Attempts
were also made to determine whether cells could
maintain their integrity under the maximum concentration of GlcNAc (200 mM).

Table 1 The rate of mating pair formation of P. caudatum cells under various concentrationsof WGA, NeuAc and GlcNAc.

WGA
(nM)
0

Rate of mating pair formation (%)
68

NeuAc
(mM)
0

Rate of mating pair formation (%)
58

GlcNAc.
(mM)
0Rate of mating pair formation (%)
59

1439

154203728

192424020

421.4328606.2562.241280

1.268n.d.*

50.9

100

1.0*: Not determined

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WGA-binding substances in Paramecium caudatum

pairs in the absence of WGA, the rate in the presence of WGA largely decreased with increasing
concentration. A less than 5% rate was observedwith more than 42 nM of WGA. In both the inhibition tests in the absence of NeuAc and GlcNAc,around 60% of cells engaged in mating pair formation. But, as in the case of WGA, rates of mating
pair formation decreased in the presence of eachsugars at increasing concentrations. By dividing by
the control rate, all values were normalized andplotted against the concentration of lectin and its
specific sugars (Fig. 1). From these relationships,
the value representing 50% inhibition (IC50) of
mating pair formation was estimated empirically ataround 17 nM, 3 mM and 30 mM for WGA,
NeuAc and GlcNAc, respectively.

Fig. 1. The relationship between the concentrationsof WGA, NeuAc and GlcNAc, and normalized rates
of mating pair formation. These normalized late averaged of three experiments.

RESULTS
Inhibition of mating pair formation by WGA
and conjugated sugars
The mating pair formation of P. caudatum
was investigated at various concentrations ofWGA, NeuAc and GlcNAc, as summarized inTable 1. Although 68% of cells formed mating

Cytochemical staining of conjugated cells with
FITC-WGA
Using FITC-WGA, changes in the localization of WGA binding sites were indicated clearlywith progression of conjugation, as shown in thefluorescent photomicrographs in Fig. 2. Before
mixing, somatic P. caudatum cells were not labeled by FITC-WGA except for in the paroral region (Fig. 2A). Two hours after cells of complementary mating types were mixed, fluorescence ofFITC-WGA could be detected only in the anteriorventral part where cells adhered (Fig. 2B). With
the progress of conjugation, marked fluorescence
was observed clearly in the ventral part where cells
were united firmly (Fig. 2C). The fluorescence wasmaintained while the cells were united (Figs. 2CG); however, when the bond between the cells
disintegrated, fluorescence was detected only in asmall fraction of the ventral part where the cells
had previously been united, and in the paroral region (Figs. 2H and I).
Inhibition of WGA binding to the adhesion site
by NeuAc and GlcNAc
To define the specific WGA binding sugar at

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167

Fig. 2. The fluorescence micrograph of cells stainedwith FITC-WGA. Pictures were taken every 2 h during0-16 h after cells mixing. A: 0 h after the cell mixing
B: 2 h after the cell mixing. The anterior-ventral (arrowhead) and oral region (o) of conjugated cell werestained with FITC-WGA. C: 4 h, D: 6 h, E: 8 h, F: 10 h,and G: 12 h after cell mixing. The adhesion sites of theconjugated pairs were stained. H:14 h and I:16 h after
the cell mixing. The oral regions of the cells werestained. The arrows indicate specific staining site. Bars
represent 50 µm in length.

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WGA-binding substances in Paramecium caudatum

the adhesion site of conjugated cells, cells wereincubated in FITC-WGA-NeuAc or FITC-WGAGlcNAc. The final concentrations of FITC-WGA,
NeuAc and GlcNAc were 30 µg/ml, 4 mM and
100 mM, respectively. NeuAc but not GlcNAc(Fig. 3B) inhibited cytochemical staining of FITCWGA in the adhesion site between cells (Fig. 3C).
Even in the presence of a high concentration ofGlcNAc (200 mM), the inhibition of staining was
not observed. Sometimes the surfaces of the cellbodies as well as the cilia of the conjugating cells
were stained by FITC-WGA. Since, however, thisstaining was shown to be independent of the presence or absence of sugars, it appears to be nonspecific.

DISCUSSION

Fig. 3. Inhibition of FITC-WGA staining by NeuAc
and GlcNAc. A: Conjugated cells (4 h after cell mixing) treated with 30 µg/ml FITC-WGA. B:Conjugatedcells treated with 30 µg/ml FITC-WGA saturated with
100 mM GlcNAc. The adhesion sites of the conjugatedcells were stained. The arrows indicate specific stainingsite. C: Conjugated cells treated with 30 µg/ml FITCWGA saturated with 4 mM NeuAc. Staining of FITCWGA in the adhesion site was not detectable. Bars represent 50 µm in length.

It was previously reported that WGA inhibits
mating pair formation in P. tetraurelia and P. primaurelia (Pape et al., 1988, Delmonte et al.,1997). In this study, we indicated quantitatively
that WGA also inhibits mating pair formation in P.
caudatum cells at an IC50 of 17 nM (Table 1 andFig. 1). We also showed fluorescence cytochemically that WGA bound specifically to theadhesion site between conjugated cells (Figs. 2CG). Moreover, FITC-WGA staining in the adhesion site could not be detected after the bond between the conjugated cells disintegrated (Figs. 2Hand I). WGA localization changed in correspondence with progression of conjugation (Fig. 2).
These results suggest that mating pair formation inP. caudatum cells is inhibited by WGA throughspecific binding to sugar in the complex polysaccharide that is crucial to mating pair formation.
Tsukii and Hiwatashi (1978) reported theinhibition of mating pair formation in P. caudatum
cells in the presence of higher effective concentrations of Con A (480 nM) than required for complete inhibition by WGA (40 nM). This might sug-

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gest that either Con A and WGA specific bindingsugars are responsible for mating pair formationin P. caudatum or that Con A non-specificallybinds to WGA-specific binding sugar. Since they
did not show the IC50 of Con A, we could notcompare our data with theirs. It appears necessary to conduct the inhibition test under various
concentrations of Con A and through cytochemical staining of FITC-Con A as shown in thisstudy.
Both WGA specific binding sugars, NeuAc
and GlcNAc, also showed considerable inhibitory
effects on mating pair formation in P. caudatumcells (Table 1). NeuAc showed a lower order ofmagnitude of IC50 than GlcNAc (Fig. 1). Moreover, NeuAc but not GlcNAc blocked WGA
binding to the adhesion site between conjugatedcells (Figs. 3B and C). These results suggest that
NeuAc bound specifically and then masked thesugar recognition site of WGA on the cell membrane that was crucial for cell adherence duringmating pair formation. Inhibition of mating pairformation seems to be caused not by toxicity ofWGA, NeuAc and GlcNAc, because P. caudatum cells remained alive for 24 hours with alltested concentrations of lectin and sugars. It istherefore necessary to elucidate the details of themolecular mechanism of mating pair formation,in particular, the involvement of the complex
polysaccharide that contains NeuAc as a residue.
In P. tetraurelia, FITC-WGA staining in theadhesion site was found at 4 and 7 h after initiation of conjugation, as shown here for P. caudatum. Lüthe and Plattner (1986) reported, however, that WGA binding substances were alreadylocalized in the expected adhesion site before
starting conjugation in P. tetraurelia cells. Moreover, Pape et al. (1988) described that staining of
the adhesion site between P. tetraurelia cells by
FITC-WGA was not blocked by 200 mM NeuAc
or neuraminidase treatment of the cell, but by
200 mM GlcNAc. These are not coincidental

169

with the results of this study; WGA binding sites
could not be visualized by FITC-WGA cytochemically until adhesion of the cells started and NeuAc
was primarily the specific binding sugar to WGA.
The crucial complex poly-saccharide and its behavior in the conjugation steps therefore appear todiffer among species of Paramecium.
It could be concluded from the results of thisstudy that a specific WGA binding complex polysaccharide is involved in cell adhesion during mating pair formation in P. caudatum cells. Furthermore, WGA, NeuAc, and GlcNAc inhibited theprogression of conjugation; nuclear events werestopped at an early stage (data not shown). WGA
binding substances therefore seem to be necessary
for progress of conjugation as well as cell adhesion.

ACKNOWLEDGEMENT
We thank Dr. Kazuyuki Mikami of Miyagi
University of Education for providing P. caudatum(KNW 2 and KNZ 5). We also thank Dr. Kensuke
Chikamori and Dr. Koji Shimizu for providing
helpful advice.

REFERENCES
Casci, R. J. and Hufnagel, L. A. (1988) Cell pairing during mating in Tetrahymena:Ⅰ. Does
phagocytosis or a cell surface receptor participate in Con A block? J. Protozool., 25, 424430.
Delmonte Corrado , M. U., Locatelli, D., Paleari,L. and Bottipoli, G. (1997) Lectin-bindindsites involved in Paramecium primaurelia
mating pair formation. J. Euk. Microbiol., 44,603-608.
Dryl, S. (1959) Antigenic transformation in Paramecium Aurelia after homologous antiserum
treatment during autogamy and conjugation. J.

170

WGA-binding substances in Paramecium caudatum

Protozool .Suppl., 6, 25.
Plümper, E., Freiburg, M. and Heckmann, K.
(1995) Inhibition of pair formation by Concanavalin A and Concanavalin A-binding
glycoconjugates in Euplotes octocarinatus.
J. Euk. Microbiol., 42, 166-173.
Frisch, A. and Loyter, A. (1977) Inhibition ofconjugation in Tetrahymena pryiformis by
Con A. Localization of Con A-binding sites.
Exp.Cell Res., 110, 337-346.
Fujishima, M. (1988) Conjugation. In: Paramecium. Görtz, H. D.(ed.). Springer-Verlag,Berlin and Heidelberg, pp. 70-84.
Hiwatashi, K. and Kitamura, A. (1985) Fertilization in Paramecium. In: Biology of Fertilization. Metz, C. B. (ed.), Academic Press, New
York, vol.1, pp.57-85.
Kitamura, A. and Hiwatashi, K. (1976) Mating
reactive membrane vesicles from cilia ofParamecium caudatum. J. Cell Biol., 69,736-740.
Lüthe,N. and Plattner, H. (1986) Lectin bindingsites in Paraecium tetraurelia cells. II.
Labeling analysis predominantly of nonsecretory components. Histochemistry, 85,377-388.
Ofer,L., Levkovitz,H. and Loyter, A. (1976) Conjugation in Tetrahymena pryiformis. The effect of polylysin, concanavalin A, and bivalent
metals on the conjugation prosess. J.CellBiol., 70, 287-293.
Pape, R., Haack-Bell, B., Lüthe, N. and Plattner,H. (1988) Conjugation of Paramecium
tetraurelia cells: selective wheat germ agglutinin binding, reversible local trichocyst detachment and secretory function repair. J.Cell Sci.,
90, 37-49.
Takahashi, M., Takeuchi,N. and Hiwatashi ,K.
(1974) Mating agglutination of cilia detachedfrom complementary mating types of Paramecium. Exp. Cell Res., 87, 415-417.
Tsukii ,Y. and Hiwatashi,K. (1978) Inhibition ofearly event of sexual process in Paramecium
by Concanavalin A. J. Exp. Zool., 205, 439446.
Vivier, E. and André, J. (1961) Données structurales et ultrastructurales nonvelles sur la
conjugaison de Paramecium caudatum. J.
Protozool., 8, 416-426.