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Colchicine and differentiation of macronuclear anlagen in Paramecium caudatum
Arch. Protistenk. 121 (1979): 213-222
:Miyagi College of Education, D ep artmen t of B iology, Sen da i , Japan
Colchicine and Differentiation of Macronu clear Anlagen in Paramecium caudatum By KAZUYUKI ll1IKAl\U With 4 Figures
Summary I n exc on juga n t s of Paramecium caudatu m , 4 macronuclea r a nlagen out of 8 postzygotic divi si on products with the sa me a ppearance wer e differentiated. When the postzygotic nuclear d ivi sion was inhibited by colchici ne , n ormal macronuclear anl agen wer e d ev el oped but number of t he an lagen wa s few er than n orm a l one. Th e r esult suggests a possi bi lity t hat the role of p ostzygotic nuclear di vi si on in ma cr onuclea r differen ti a ti on is t o produce r egular number of anlagen b ut n ot to develop t he m. ;\loreover, m icr onuclear differen ti a t ion seeme d to occ ur r egardless of t he inh ibi t ion of post zygot ic nuclear division . The result suggesting in de pe n de nce of macronuclear d iffer en t ia t ion a n d fra gmen ta ti on of old m acr onucleu s wa s a lso obtained .
Introduction Ciliates are unic ellular animals cont aining two kinds of nu clei, ma cronucleus and micronucleus. Pa ramecium caudatum, one of t he ciliates, cont ains each one of the tw o kinds of nuclei. Th e macronucleus controls the veget ative activity, while the micronucleus is concerned with the reprodu ctive activity, that is, meiosis and formation of gametic nu clei in sexual pr ocess. At ever y fission , mi cronu clei produce only mi cronuclei, macronuclei produce only macronuclei. Macronucl ei are developed from pr oduct s of micronuclei but micr onuclei are never produced from macronuclei. The nu clear differentiation is irreversible. Morphological changes during sexual processes were investigated long time ago (MAUPAS 1889; HERTWIG 1889 ; CALKINS and CULL 1907). During con jugat ion , gametic nu clei which are produced from micronucleus through meiosis exchange reciprocally and fertilize. The fertilized nu cleus (synkaryon) divid es 3 times and gives rise to 8 nu clei. Of t he 8 nucl ei 4 become macro nu clear anlagen , one becomes a micronucleus, a nd t he remaining 3 degenerate (Fig. 1). Th e number of the a nlage n differentiated is constant and abnormal nu mb er is very rare. It is a n in t eresting problem what controls the mechanism of macronu clear differ entiat ion. In t his report, postz ygotic nu clear divisions are ar reste d at various st ages by colchicine and differentiation of macro- and micronucleus was observed t o know the mechanism of ma cronu clear differenti ation. 15 Arch.
Protistenk. Bd. 121
214
K . l\fIKAMI
Materi als and Methods Stocks dm- I and d m-3 which belong t o mating type s V and VI resp ectively were used. These stocks wer e grown in fr esh let tu ce m ediu m. The method s of cult ure and in duct ion of con jugation we r e a s describe d b y H IWATASHI (1968). Conj uga n ts a n d exco n j uga n t s were ke pt in Dryl's solu tion (D S) (D RYL 1959). Treatment with colchicine was m a d e by trans ferring t h e cells into DS contain ing 15 m~I colchici ne (Merk) at 25 C. After a certain period of the treatme nt, t he cells wer e t rasfe rre d into fr esh D S. Cells t r eate d with colch icine and r in sed with fr esh D S were pla ced on a lb um ini ze d cover glas ses . T h e cov er glasses were a ir-dried and f ix ed in Schaudinn's solut ion (KIRB Y 1950) for 15 m in at 25 C shortl y befo re t he cover gla sses were dried com pletely . Th is m ethod p reserved t he ellipsoid form of t he cells, pro tect in g t he ce lls fr om flatte n ing whe n a ir-dried , and m a de observation of prec ise n umbe r of fr agment ed macro n ucle i , macronuclear anla gen an d m icronuelei p ossib le. SUbsequen t ly, m er curie ehloride co n ta ine d in Scha u din n's sol. was r emoved b y placing the pr epa rati on s in iodine solution (0.5 % W IV io dine , 0.5 % W IV pot a ssium i od ite a n d 30 % V IV al cohol ) for a few minutes a nd then r insed in I % (W/V) thiosul fa t e solution . Preparations wer e stain ed by F eulgen reaction a n d counterstaine d with fast-gre en F CF (0.5 %). Test for macronuclear r egen er a t ion follow ed the method of the prev io us paper (MIKAMI a nd HIWATASHI 1975). Cells with a n ew macron ue leu s developed fr om a divis io n product of the synkaryon ha ve no mating r ea ct iv it y d ur ing 50- 60 fis sions after eon juga t ion eve n under a pp ro p riate eon dit ion . H ow ever, cells with macro n uc le i r egen erat ed fr om old m a eronuclear fra gmen t s la ck t h e im matu r ity per iod and show ma t ing r ea et ivi t y soo n aft er conjuga t ion . Ma t in g rea ct ivi t y wa s t est ed at 10-1 5 fi ssi on s after conj ugation to kn o w the occurren ce of m a cronuelea r regen eration.
Results In P . caudaium, the fertili zation nu cleus (synkar yon) which is form ed by fusion of male a nd femal e nu clei norm ally divides three times and gives rise t o 8 nuclei. The conjuga nts usually sepa rate shortly aft er the fir st divi sion of t he synkaryo n. Th e 8 nuclei have t he same appe.arance whe n they were produ ced bu t a bout 2 or 3 h after the t hird division , morphological cha nges are observe d in 4 nuclei among t he 8. Th e 4 gro w t hrough a series of deve lopmental stages (anl agen stages) into the post zygotic macronuclei. Th e fate of t he remaining 4 is eit her to become a micronu cleus or t o degen erate. Nu clei were recognized as ma cronu elear anlagen when het erochromatic aggregations appear in t hem about 4 h after the pair separation (Fig. 2.1). Th e number of the aggr egations is about ten as already reported by SAITO and SATO (1961). Subsequently, the het erochromatic aggregates disintegrate gradually (Fig. 2.2). and disappeared about 7 h after t he pair separation. About at this time, volume- of the anlagen incrases and F eulgen st aina bilit y markedly redu ced. This achromatic stage of t he a nlagen is followed by a stage when t he anlage gra dua lly becomes more baso philic and F eulgenp ositivc. Th e degenerati on of 3 nuclei out of the remaining 4 nuclei seems to occur after t he differenti ation of macronu clear anl agen. It is difficult t o distinguish morphologically t he micronucl eus a nd the 3 ot her nu clei doomed to degenerate and to det ermin e crit ical t ime of t he micro nuclear differenti ati on. Appro ximately when t he t hird post zygotic nu clear division occurs, old ma cronucleus is t ra nsformed into a loosely wound skein (skeinformation) which subse quent ly break s int o a number of more or less spherical pieces referr ed to as macronuclear fra gment s.
215
Colchi cine a nd Differen ti ation of Macronucloar Anlag en Table 1. Effect of colch icine on macronuelear differen tiation Concen tr a t ion of colchi cine
T otal cells t reated
Number of macronu elear anlag en
10mM 12.5 m}l 15mM
19 16 15
2 6 12
0 5
9 2
2
3
4
12 1 1
0 0 0
0 0 0
Cell wore transferre d to eac h concen tra t ion of colchic ine within 10 min after pair separation.
Th ese frag ments are distri buted t o daughter cells during fissions following fertilizat ion . Differentiation of ma cro- and micronucleus is complete before the first fission after conjugation . Wh en conjugating pain, or cxconjugant s were transferr ed into 8-15 mM colchicine, no macronuclear anlagen were produced. Although P . caudatum is very resist ant t o colchicine, 8 -15 mM colchicine was enough t o arrest postzygotic nu clear divi sion. Th e time of colchicine treatment necessar y for t he inhibition of postzygoti c nu clear di vision depends up on concentration of th e agent (Table 1). Cells were su bject ed to 15 m~I colchicine 30 min before separat ion of conjuga ting pairs (Fig. 1). After sepa ration of t he pairs, t he exconjuga nt s were transferr ed t o fresh 15 mM colchicine and kept in it up to about 36 h afte r t he pair separation . All the t reated cells had no macronuclear a nlage (Fig. 2 - 6, 7), while control cells had 4 well-developed a nlage n which were faintl y stained by F eulgen reacti on a nd deeply by fast-green F C:F (Fig. 2-3). In t he next exper iment, t he length of the treatment was limited as shown in Tabl e 2. Treatmen t for more t han 5 h after pa ir sepa ration gave a lmost t he same inh ibit ory effect as t he 36 h treatment on the developmen t of macronuc1ear a nlagen. Treatmen t for 3 h after t he pair separati on did not inhibit the a nlage n development perfectl y . Cells were exposed t o 15 mM colchicine from various stages of conjugat ion and post conju gation (Fig. 1) t o 20 - 26 h aft er pair separation. Wh en conjugants were treated with the drug 30 min t o 1 h before pair separa t ion (Fig. I schedule of the treatment I) ,all exconjugant.s lack ma eronuclear anlagen (Fig. 3-1I). When the treatment star te d 0.5-1 h after pair separat ion , one or two mac ro nu clear anlagen developed (Fig. 3- III). The later t he stages of the treatment are shift ed, the mor e the number of the anlagen in crease and the closer it becomes to normal number of 4 (Fig. 3- III, I V). Wh en t he treatment st ar te d 2.5-3 h afte r pair sepa rat ion, norm al nu mber of 4 macronuclear anlagen developed in almost all excon juga nts (Fig. 3- V). Colchicine inhibits differentiation of macronucl ear anlagen but did not inhibit growth of t hem. Th e less was the number of macronu clear anlagen, the larger was the volume of each anlage. Under the condit ion of colchicine t rea tme nt gro wth of t he anlagen was slight ly slower t han that wit hout colchicine. The cells without macronuclear a nlage n undergo mac ro nucl ear regeneration if t hey are capa ble of gro wth. Cells trea te d according to t he schedule as in Fig. 1- II 16·
216
K.
(a)
Time table -1
Pair separat ion
+
9
!
3,
2
4
0- -0-1- - ' !
Macronucleu
MIK A:lU
!
!
o
Micronucleus
Synga~o-----
J
-0.......0
5 hours !
----- - --0 ---- --- @ - - - - - - - E)
O~O
0---8
0 0 ____ - -0 O~o
------ - 0]) ------- (3)
o
p,ir sep arat ion
(b)
Q Schedule of the treatment
(1
~---~;
•
_
1
m·------ - -- - - - - - - - - - - - - - IV .------ - - - - - - - ------< V .--- - - - - -- - - -----<
Fig. 1. (a) N orma l course of n uclea r events d u r in g la t e and post conjuga ti on . F ormation of syn kar y on occu rr ed a b out 0. 5 - 1 h b efore pai r se pa ra t ion and the first p ost zygo t ic n u clear divi sion was accomplish ed a t pair se paration . (b) Sch edule o f t he treatm ent wi th co lc h icine . Cells a t the stages ind ica t ed by dotted lin es were t ransfe rred into co lc hicine sol. a nd k ept in t h e so l. about fo r 24 h a fte r pair se pa ra t ion .
Tab le 2. Time of t he colchi cino t re a t men t and the n umber of macron uclea r an lagen differen t ia t ed P eriod of t reatment*
No. of exconjugants ob serv ed
-
62 50 39 20
0.5""' 3 0.5 ""' 5 0. 5 ""' 7 0.5 ,,", 9
N um ber of m a cr onuclear a nlagen 0
42
4
49 37 20
0 0 0
2
3
4
8 1 2 0
0 0 0 0
8 0 0 0
* - 0.5""' 3 indicates trea t m ent with col chi cine fro m 0.5 h before pair separation to 3 h after pai r sepa ra t ion (see Fig. 1).
Colchicine and Differentiation of Macronuclear Anlagen
217
1
2
3
4
•
6
7
Fig. 2. 1. Chromatic aggreagations in a macronuclear anlage which is seen in normal course of macronuclear differentiation. 2. Macronuclear anlage is deeply stained by fast-green and chromatic aggregation in the center of the nucleus disappears shortly after this stage in normal course of macronuclear differentiation. 3. Normal exconjugant cell with 4 macronuclear anlagen (achromatic stage). 4. A colchicine-treated cell with one macronuelear anlage, one micronucleus ami a few fragments of old macronucleus, 5. A colchioine-t.reatcd cell with two maeronuclear anlagen and non-fragmented old macronucleus. 6. A colchicine-trcated cell without macronuclear anlagen. 7. Abnormal fragmentation of old macronucleus produced by treatment with colchicine.
218
K.
IV
MIKAMI
T
rJ
v
o
1 2 3 4 Number of macronuclear anlagen
F ig. 3. E ff ect of colch icine on macro nuclear differentiation. Cells wer e transferred to 15 mM colc hici ne a ccording to the schedule of the treatment a s sho wn in F ig . l ·(b) and ob served the number of macro nuclear anlagen d ifferen t ia te d . I ,...., V indicate sche d ules of the treatment shown in F ig. l o(b).
were transferred to DS and kept in it for a few hours and then, transferred to lettuce medium and grown. There were many cells, fissions of which were irreversely arrested by colchicine . Only 10-50 % of t he cells treated as ab ove could divide and grow. Th e exconjuga nt clones which were abl e to grow were examined to know whether macronuclear regeneration occurred or not. In all of these clones, ma cronuclear regeneration occurred (Tabl e 4) , while in cont rol clones (period of treatment 2.5,,-,24) frequency of macronucl ear rege neration was 26 %. Many cells, fissions of which were impeded by the colchicine treatment , become thiner and thiner and event ually dis. appeare d, probably becau se of the inhibition of gullet formation. In cells with a sma ller number of the anlage n, t he anlagen appar ently fail t o divide at some cell
219
Colchi cine an d Differentiation of Ma cr on u clea r Anl ag en
2
1
Fig. 4. Abnorm al postzygotic nuclea r divis ion by col chicine treatm ent. B oth pictures showl 2 tripolar n uc lear divisio ns . 1. A ce ll not fla t tened when prepared 2. A cell fl a t t ened by ai r d ryi ng wh en prepared.
Ta ble 3. E ffect of colchicine on the fragmen tation of old ma cronucleu s Period of
treatment"
Total excon jugants
No . of cells with no fragmented m acronucleu s
-0.5,....,3 -0.5 ,....,5 -0.5 ,....,7 - 0.5,...., 9
62 50 39 20
4 10
*
1.5
10
Mea n number of fragmen t s in fragmente d cells
in to ta l cells
29 .0 23.3 29.9 20 .0
25 .9 17. 5
18.7 10.5
same as Ta ble 2
divi sion aft er conjugation, yielding a daughter cell without macronucleus. In these cells, old macronuclear fra gment s proceed to regeneration (M I KAMI and HIWATASHI 1975). I n some exco njugant cells as shown in F ig. 2 -4, 5, fragmentation of old macronu cleus was perfect ly or par tialy inhibited but never t heless a new macronucleus (anlage) was di fferentiated. This evidence sugges ts t hat differentiation of macronu clear a nlagen is independent of fra gmentation of old macronucleus. No fusion of t he fra gments was observed in t he exconjugant s treated wit h colchicin e. I n t he colch icine treat ment, some a bnormalities in postzygotic nucl ear division was observe d . Some of t he treated cells showed mu lt ipolar post zygot ic nuclear division (Fig. 4 ). Since inhibiti on of fragment at ion of old ma cronucle us was observed in som e experime nt as
220
K.
MIKAMI
Table 4. Colchicine treatment and occurrences of macronuclear regeneration (M R) and amicronucleate clones Period of treatment
Total exconjugant clones treated
NR clones*
-0.5,....- 24 2.5,....- 24
36 31
23
o
MR clones
amicronucleate clones
36 8
o
2
* Normal macronuclear reorganization
already mentioned, effects of colchicine on skeinformation and fragmentation of old macronucleus were investigated. When cells were exposed to colchicine 30 min before pair separation or earlier, the fragmentation was inhibited. When treatment time were prolonged as shown in the first column of Table 3, inhibition of the fragmentation increased depending on the length of exposure to colchicine. It is difficult to investigate action of colchicine on the differentiation of micronucleus mainly because products of postzygotic nuclear division and differentiated micronucleus cannot be distinguished when they were in the same cytoplasm. The treatment with the schedule II produced only 2 amicronucleate clones as shown in Table 4 and other clones had one micronucleus respectively, that is, a new micronucleus was produced in spite of the inhibition of the postzygotic nuclear division. It was not clear how the 2 amicronucleate clones were produced. In P. tetraurelia, however, amicronucleate strains were frequently produced by this method and about 50 amicro nucleate strains were obtained (MIKAMI, unpublished).
Discussion What is the mechanism controlling macro- and micro nuclear differentiation in ciliates? In Tetrahymena, MAUPAS suggested that differentiation of macro- and micronucleus was correlated with brief localization of the nuclei at the opposite ends of the cell; the 2 at the posterior end becoming macronuclei (MAUPAS 1889). In P. caudatum, 4 macronuclear anlagen and a micronucleus are always formed. Does the micronucleus regularly arise in one end of the cell and the 4 macronuclei in the other? It is difficult to know whether the macronucleus regularly arises at one end of the cell in P. caudatum, because the postzygotic nuclei seem to move arround immediatelly after postzygotic division and moreover macronuclear differentiation is morphologically recognized 2 or 3 h after the third postzygotic nuclear division. NANNEY (1953) demonstrated that cytoplasm plays a crucial role in directing the activities of the nucleus in Tetrahymena. However, no locally specified cytoplasm such as germ plasm found in multicellular animals has been found in ciliates. Nevertheless, it seems to be in full agreement that nuclear differentiation can be affected by cytoplasm (MAUPAS 1889; NANNEY 1953; SONNEBORN 1954). Does postzygotic division have any contribution to the differentiation of macroand micronucleus? In P. caudatum, the third division is very interesting because the
Colchicine and Differentiation of Macronuclear Anlagen
221
spindle of the third nuclear division lies parallel to the longitudinal axis of the cell and elongates until their poles reach opposite ends of the cell as mentioned by CALKINS and CULL (1907). When the third postzygotic division was arrested by cochicine treatment, normal differentiation of macronuclear anlagen was disturbed and 2 insted of 4 anlagen were produced. In P. bursaria, EOELHAAF demonstrated that abnormities occurred in the differentiation of macronuclear anlagen when the third postzygotic division (the last postzygotic division) was subjected to colchicine (1955). The result in P. caudatum suggests a possibility that the third postzygotic nuclear division plays an important role for producing the regular number of macronuclear anlagen. Next problem is whether synkaryon (fertilization nucleus) and products of first or second postzygotic division can also develop to macro- or micronucleus. Results from the inhibition of postzygotic nuclear division by colchicine suggest that nuclei after first or seconr] postzygotic division can develop to macro- and micronucleus, though the number of macronuclear anlagen was fewer than normal one. Colchicine inhibits postzygote nuclear division but does not inhibit growth of macronuclear anlagen. When colchicine treatment inhibited the first postzygotic division, that is, the treatment stage I in Fig. 1, no macronuclear anlagen was observed in the exconjugant cells (Fig. 3-- I). However, it is very difficult to know when synkaryon has an ability to develop to macronuclear anlagen or micronucleus and whether the first postzygotic division is really necessary for macro- and micro nuclear differentiation, because the stage of synkaryon seems to be short and a possibility that colchicine arrests the process of synkaryon formation is not ruled out. Fragmentation of macronucleus was partly or perfectly inhibited by colchicine. This suggests the possibility that microtubles play an important role in skeinformation and fragmentation of old macronucleus. The fragmentation of P. caudatum continues until about 24 h after mixing of the mating types and 30-60 fragments are produced. Macronuclear anlagen can differentiate without fragmentation of old maconucleus. Therefore, macronuclear differentiation and fragmentation of old macronucleus are independent phenomena.
Acknowledgements The author thanks Dr. K. HIWATASHI for his discussion during the course of this investigation and for help in preparation of the manuscript. I would also like to thank Dr. S. KorZUMI and Dr. M. SAITO for many helpful suggestions and encouragements.
Literature CALKINS, G. N., and CULL, S. W.: The conjugation of Paramecium aurelia (caudatum). Arch. Protistcnk. 10 (1907): 375-415. DRYL, S.: Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool. 6 (1959): 25. EGELHAAF, A.: Cytologisch-entwicklungsphysiologische Untersuchungen zur Konjugation von Paramecium bursaria FOCKE. Arch. Protistenk. 100 (1955): 447-514.
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K. MIKAMI, Colchicine and Differentiation of Macronuclear Anlagen
HERTWIG, R.: Uber die Konjugation der Infusorien. Abh. bayer. Akad. Wiss. 17 (1889): 151. HIWATASHI, K.: Determination and inheritance of mating type in Paramecium caudatum. Genetics. 58 (1968): 373-386. KIRBY, H.: Materials and methods in the study of protozoa. Berkley and Los Angeles 1950. MAUPAS, E.: Le rajeunissement karyogamique chez les cilies. Arch. Zool. expo gen. Ser. 2, 7 (1889): 149-517. MIKAMI, K., and HIWATASHI, K.: Macronuclear regeneration and cell division in Paramecium caudatum. J. Protozool. 22 (1975): 536-540. NANNEY, D. L.: Nucleo-cytoplasmic interaction during conjugation in Tetrahymena. BioI. Bull. 105 (1953): 133 -148. SAITO, M., and SATO, H.: Morphological studies on the macronuclear structure of Paramecium caudatum. III. On the development of the macronucleus in the exeonjugant. Zool. Mag (Jap.) 70 (1961): 81-88. SONNEBORN, T. M.: Patterns of nucleocytoplasmic integration in Paramecium. Caryologia. 1 (1954): 307-325. Author's address. Dr. KAZUYUKI MIKAMI, Department of Biology, Mlyagi College of Education, Aoba-yama, Sendai, Japan.
:Miyagi College of Education, D ep artmen t of B iology, Sen da i , Japan
Colchicine and Differentiation of Macronu clear Anlagen in Paramecium caudatum By KAZUYUKI ll1IKAl\U With 4 Figures
Summary I n exc on juga n t s of Paramecium caudatu m , 4 macronuclea r a nlagen out of 8 postzygotic divi si on products with the sa me a ppearance wer e differentiated. When the postzygotic nuclear d ivi sion was inhibited by colchici ne , n ormal macronuclear anl agen wer e d ev el oped but number of t he an lagen wa s few er than n orm a l one. Th e r esult suggests a possi bi lity t hat the role of p ostzygotic nuclear di vi si on in ma cr onuclea r differen ti a ti on is t o produce r egular number of anlagen b ut n ot to develop t he m. ;\loreover, m icr onuclear differen ti a t ion seeme d to occ ur r egardless of t he inh ibi t ion of post zygot ic nuclear division . The result suggesting in de pe n de nce of macronuclear d iffer en t ia t ion a n d fra gmen ta ti on of old m acr onucleu s wa s a lso obtained .
Introduction Ciliates are unic ellular animals cont aining two kinds of nu clei, ma cronucleus and micronucleus. Pa ramecium caudatum, one of t he ciliates, cont ains each one of the tw o kinds of nuclei. Th e macronucleus controls the veget ative activity, while the micronucleus is concerned with the reprodu ctive activity, that is, meiosis and formation of gametic nu clei in sexual pr ocess. At ever y fission , mi cronu clei produce only mi cronuclei, macronuclei produce only macronuclei. Macronucl ei are developed from pr oduct s of micronuclei but micr onuclei are never produced from macronuclei. The nu clear differentiation is irreversible. Morphological changes during sexual processes were investigated long time ago (MAUPAS 1889; HERTWIG 1889 ; CALKINS and CULL 1907). During con jugat ion , gametic nu clei which are produced from micronucleus through meiosis exchange reciprocally and fertilize. The fertilized nu cleus (synkaryon) divid es 3 times and gives rise to 8 nu clei. Of t he 8 nucl ei 4 become macro nu clear anlagen , one becomes a micronucleus, a nd t he remaining 3 degenerate (Fig. 1). Th e number of the a nlage n differentiated is constant and abnormal nu mb er is very rare. It is a n in t eresting problem what controls the mechanism of macronu clear differ entiat ion. In t his report, postz ygotic nu clear divisions are ar reste d at various st ages by colchicine and differentiation of macro- and micronucleus was observed t o know the mechanism of ma cronu clear differenti ation. 15 Arch.
Protistenk. Bd. 121
214
K . l\fIKAMI
Materi als and Methods Stocks dm- I and d m-3 which belong t o mating type s V and VI resp ectively were used. These stocks wer e grown in fr esh let tu ce m ediu m. The method s of cult ure and in duct ion of con jugation we r e a s describe d b y H IWATASHI (1968). Conj uga n ts a n d exco n j uga n t s were ke pt in Dryl's solu tion (D S) (D RYL 1959). Treatment with colchicine was m a d e by trans ferring t h e cells into DS contain ing 15 m~I colchici ne (Merk) at 25 C. After a certain period of the treatme nt, t he cells wer e t rasfe rre d into fr esh D S. Cells t r eate d with colch icine and r in sed with fr esh D S were pla ced on a lb um ini ze d cover glas ses . T h e cov er glasses were a ir-dried and f ix ed in Schaudinn's solut ion (KIRB Y 1950) for 15 m in at 25 C shortl y befo re t he cover gla sses were dried com pletely . Th is m ethod p reserved t he ellipsoid form of t he cells, pro tect in g t he ce lls fr om flatte n ing whe n a ir-dried , and m a de observation of prec ise n umbe r of fr agment ed macro n ucle i , macronuclear anla gen an d m icronuelei p ossib le. SUbsequen t ly, m er curie ehloride co n ta ine d in Scha u din n's sol. was r emoved b y placing the pr epa rati on s in iodine solution (0.5 % W IV io dine , 0.5 % W IV pot a ssium i od ite a n d 30 % V IV al cohol ) for a few minutes a nd then r insed in I % (W/V) thiosul fa t e solution . Preparations wer e stain ed by F eulgen reaction a n d counterstaine d with fast-gre en F CF (0.5 %). Test for macronuclear r egen er a t ion follow ed the method of the prev io us paper (MIKAMI a nd HIWATASHI 1975). Cells with a n ew macron ue leu s developed fr om a divis io n product of the synkaryon ha ve no mating r ea ct iv it y d ur ing 50- 60 fis sions after eon juga t ion eve n under a pp ro p riate eon dit ion . H ow ever, cells with macro n uc le i r egen erat ed fr om old m a eronuclear fra gmen t s la ck t h e im matu r ity per iod and show ma t ing r ea et ivi t y soo n aft er conjuga t ion . Ma t in g rea ct ivi t y wa s t est ed at 10-1 5 fi ssi on s after conj ugation to kn o w the occurren ce of m a cronuelea r regen eration.
Results In P . caudaium, the fertili zation nu cleus (synkar yon) which is form ed by fusion of male a nd femal e nu clei norm ally divides three times and gives rise t o 8 nuclei. The conjuga nts usually sepa rate shortly aft er the fir st divi sion of t he synkaryo n. Th e 8 nuclei have t he same appe.arance whe n they were produ ced bu t a bout 2 or 3 h after the t hird division , morphological cha nges are observe d in 4 nuclei among t he 8. Th e 4 gro w t hrough a series of deve lopmental stages (anl agen stages) into the post zygotic macronuclei. Th e fate of t he remaining 4 is eit her to become a micronu cleus or t o degen erate. Nu clei were recognized as ma cronu elear anlagen when het erochromatic aggregations appear in t hem about 4 h after the pair separation (Fig. 2.1). Th e number of the aggr egations is about ten as already reported by SAITO and SATO (1961). Subsequently, the het erochromatic aggregates disintegrate gradually (Fig. 2.2). and disappeared about 7 h after t he pair separation. About at this time, volume- of the anlagen incrases and F eulgen st aina bilit y markedly redu ced. This achromatic stage of t he a nlagen is followed by a stage when t he anlage gra dua lly becomes more baso philic and F eulgenp ositivc. Th e degenerati on of 3 nuclei out of the remaining 4 nuclei seems to occur after t he differenti ation of macronu clear anl agen. It is difficult t o distinguish morphologically t he micronucl eus a nd the 3 ot her nu clei doomed to degenerate and to det ermin e crit ical t ime of t he micro nuclear differenti ati on. Appro ximately when t he t hird post zygotic nu clear division occurs, old ma cronucleus is t ra nsformed into a loosely wound skein (skeinformation) which subse quent ly break s int o a number of more or less spherical pieces referr ed to as macronuclear fra gment s.
215
Colchi cine a nd Differen ti ation of Macronucloar Anlag en Table 1. Effect of colch icine on macronuelear differen tiation Concen tr a t ion of colchi cine
T otal cells t reated
Number of macronu elear anlag en
10mM 12.5 m}l 15mM
19 16 15
2 6 12
0 5
9 2
2
3
4
12 1 1
0 0 0
0 0 0
Cell wore transferre d to eac h concen tra t ion of colchic ine within 10 min after pair separation.
Th ese frag ments are distri buted t o daughter cells during fissions following fertilizat ion . Differentiation of ma cro- and micronucleus is complete before the first fission after conjugation . Wh en conjugating pain, or cxconjugant s were transferr ed into 8-15 mM colchicine, no macronuclear anlagen were produced. Although P . caudatum is very resist ant t o colchicine, 8 -15 mM colchicine was enough t o arrest postzygotic nu clear divi sion. Th e time of colchicine treatment necessar y for t he inhibition of postzygoti c nu clear di vision depends up on concentration of th e agent (Table 1). Cells were su bject ed to 15 m~I colchicine 30 min before separat ion of conjuga ting pairs (Fig. 1). After sepa ration of t he pairs, t he exconjuga nt s were transferr ed t o fresh 15 mM colchicine and kept in it up to about 36 h afte r t he pair separation . All the t reated cells had no macronuclear a nlage (Fig. 2 - 6, 7), while control cells had 4 well-developed a nlage n which were faintl y stained by F eulgen reacti on a nd deeply by fast-green F C:F (Fig. 2-3). In t he next exper iment, t he length of the treatment was limited as shown in Tabl e 2. Treatmen t for more t han 5 h after pa ir sepa ration gave a lmost t he same inh ibit ory effect as t he 36 h treatment on the developmen t of macronuc1ear a nlagen. Treatmen t for 3 h after t he pair separati on did not inhibit the a nlage n development perfectl y . Cells were exposed t o 15 mM colchicine from various stages of conjugat ion and post conju gation (Fig. 1) t o 20 - 26 h aft er pair separation. Wh en conjugants were treated with the drug 30 min t o 1 h before pair separa t ion (Fig. I schedule of the treatment I) ,all exconjugant.s lack ma eronuclear anlagen (Fig. 3-1I). When the treatment star te d 0.5-1 h after pair separat ion , one or two mac ro nu clear anlagen developed (Fig. 3- III). The later t he stages of the treatment are shift ed, the mor e the number of the anlagen in crease and the closer it becomes to normal number of 4 (Fig. 3- III, I V). Wh en t he treatment st ar te d 2.5-3 h afte r pair sepa rat ion, norm al nu mber of 4 macronuclear anlagen developed in almost all excon juga nts (Fig. 3- V). Colchicine inhibits differentiation of macronucl ear anlagen but did not inhibit growth of t hem. Th e less was the number of macronu clear anlagen, the larger was the volume of each anlage. Under the condit ion of colchicine t rea tme nt gro wth of t he anlagen was slight ly slower t han that wit hout colchicine. The cells without macronuclear a nlage n undergo mac ro nucl ear regeneration if t hey are capa ble of gro wth. Cells trea te d according to t he schedule as in Fig. 1- II 16·
216
K.
(a)
Time table -1
Pair separat ion
+
9
!
3,
2
4
0- -0-1- - ' !
Macronucleu
MIK A:lU
!
!
o
Micronucleus
Synga~o-----
J
-0.......0
5 hours !
----- - --0 ---- --- @ - - - - - - - E)
O~O
0---8
0 0 ____ - -0 O~o
------ - 0]) ------- (3)
o
p,ir sep arat ion
(b)
Q Schedule of the treatment
(1
~---~;
•
_
1
m·------ - -- - - - - - - - - - - - - - IV .------ - - - - - - - ------< V .--- - - - - -- - - -----<
Fig. 1. (a) N orma l course of n uclea r events d u r in g la t e and post conjuga ti on . F ormation of syn kar y on occu rr ed a b out 0. 5 - 1 h b efore pai r se pa ra t ion and the first p ost zygo t ic n u clear divi sion was accomplish ed a t pair se paration . (b) Sch edule o f t he treatm ent wi th co lc h icine . Cells a t the stages ind ica t ed by dotted lin es were t ransfe rred into co lc hicine sol. a nd k ept in t h e so l. about fo r 24 h a fte r pair se pa ra t ion .
Tab le 2. Time of t he colchi cino t re a t men t and the n umber of macron uclea r an lagen differen t ia t ed P eriod of t reatment*
No. of exconjugants ob serv ed
-
62 50 39 20
0.5""' 3 0.5 ""' 5 0. 5 ""' 7 0.5 ,,", 9
N um ber of m a cr onuclear a nlagen 0
42
4
49 37 20
0 0 0
2
3
4
8 1 2 0
0 0 0 0
8 0 0 0
* - 0.5""' 3 indicates trea t m ent with col chi cine fro m 0.5 h before pair separation to 3 h after pai r sepa ra t ion (see Fig. 1).
Colchicine and Differentiation of Macronuclear Anlagen
217
1
2
3
4
•
6
7
Fig. 2. 1. Chromatic aggreagations in a macronuclear anlage which is seen in normal course of macronuclear differentiation. 2. Macronuclear anlage is deeply stained by fast-green and chromatic aggregation in the center of the nucleus disappears shortly after this stage in normal course of macronuclear differentiation. 3. Normal exconjugant cell with 4 macronuclear anlagen (achromatic stage). 4. A colchicine-treated cell with one macronuelear anlage, one micronucleus ami a few fragments of old macronucleus, 5. A colchioine-t.reatcd cell with two maeronuclear anlagen and non-fragmented old macronucleus. 6. A colchicine-trcated cell without macronuclear anlagen. 7. Abnormal fragmentation of old macronucleus produced by treatment with colchicine.
218
K.
IV
MIKAMI
T
rJ
v
o
1 2 3 4 Number of macronuclear anlagen
F ig. 3. E ff ect of colch icine on macro nuclear differentiation. Cells wer e transferred to 15 mM colc hici ne a ccording to the schedule of the treatment a s sho wn in F ig . l ·(b) and ob served the number of macro nuclear anlagen d ifferen t ia te d . I ,...., V indicate sche d ules of the treatment shown in F ig. l o(b).
were transferred to DS and kept in it for a few hours and then, transferred to lettuce medium and grown. There were many cells, fissions of which were irreversely arrested by colchicine . Only 10-50 % of t he cells treated as ab ove could divide and grow. Th e exconjuga nt clones which were abl e to grow were examined to know whether macronuclear regeneration occurred or not. In all of these clones, ma cronuclear regeneration occurred (Tabl e 4) , while in cont rol clones (period of treatment 2.5,,-,24) frequency of macronucl ear rege neration was 26 %. Many cells, fissions of which were impeded by the colchicine treatment , become thiner and thiner and event ually dis. appeare d, probably becau se of the inhibition of gullet formation. In cells with a sma ller number of the anlage n, t he anlagen appar ently fail t o divide at some cell
219
Colchi cine an d Differentiation of Ma cr on u clea r Anl ag en
2
1
Fig. 4. Abnorm al postzygotic nuclea r divis ion by col chicine treatm ent. B oth pictures showl 2 tripolar n uc lear divisio ns . 1. A ce ll not fla t tened when prepared 2. A cell fl a t t ened by ai r d ryi ng wh en prepared.
Ta ble 3. E ffect of colchicine on the fragmen tation of old ma cronucleu s Period of
treatment"
Total excon jugants
No . of cells with no fragmented m acronucleu s
-0.5,....,3 -0.5 ,....,5 -0.5 ,....,7 - 0.5,...., 9
62 50 39 20
4 10
*
1.5
10
Mea n number of fragmen t s in fragmente d cells
in to ta l cells
29 .0 23.3 29.9 20 .0
25 .9 17. 5
18.7 10.5
same as Ta ble 2
divi sion aft er conjugation, yielding a daughter cell without macronucleus. In these cells, old macronuclear fra gment s proceed to regeneration (M I KAMI and HIWATASHI 1975). I n some exco njugant cells as shown in F ig. 2 -4, 5, fragmentation of old macronu cleus was perfect ly or par tialy inhibited but never t heless a new macronucleus (anlage) was di fferentiated. This evidence sugges ts t hat differentiation of macronu clear a nlagen is independent of fra gmentation of old macronucleus. No fusion of t he fra gments was observed in t he exconjugant s treated wit h colchicin e. I n t he colch icine treat ment, some a bnormalities in postzygotic nucl ear division was observe d . Some of t he treated cells showed mu lt ipolar post zygot ic nuclear division (Fig. 4 ). Since inhibiti on of fragment at ion of old ma cronucle us was observed in som e experime nt as
220
K.
MIKAMI
Table 4. Colchicine treatment and occurrences of macronuclear regeneration (M R) and amicronucleate clones Period of treatment
Total exconjugant clones treated
NR clones*
-0.5,....- 24 2.5,....- 24
36 31
23
o
MR clones
amicronucleate clones
36 8
o
2
* Normal macronuclear reorganization
already mentioned, effects of colchicine on skeinformation and fragmentation of old macronucleus were investigated. When cells were exposed to colchicine 30 min before pair separation or earlier, the fragmentation was inhibited. When treatment time were prolonged as shown in the first column of Table 3, inhibition of the fragmentation increased depending on the length of exposure to colchicine. It is difficult to investigate action of colchicine on the differentiation of micronucleus mainly because products of postzygotic nuclear division and differentiated micronucleus cannot be distinguished when they were in the same cytoplasm. The treatment with the schedule II produced only 2 amicronucleate clones as shown in Table 4 and other clones had one micronucleus respectively, that is, a new micronucleus was produced in spite of the inhibition of the postzygotic nuclear division. It was not clear how the 2 amicronucleate clones were produced. In P. tetraurelia, however, amicronucleate strains were frequently produced by this method and about 50 amicro nucleate strains were obtained (MIKAMI, unpublished).
Discussion What is the mechanism controlling macro- and micro nuclear differentiation in ciliates? In Tetrahymena, MAUPAS suggested that differentiation of macro- and micronucleus was correlated with brief localization of the nuclei at the opposite ends of the cell; the 2 at the posterior end becoming macronuclei (MAUPAS 1889). In P. caudatum, 4 macronuclear anlagen and a micronucleus are always formed. Does the micronucleus regularly arise in one end of the cell and the 4 macronuclei in the other? It is difficult to know whether the macronucleus regularly arises at one end of the cell in P. caudatum, because the postzygotic nuclei seem to move arround immediatelly after postzygotic division and moreover macronuclear differentiation is morphologically recognized 2 or 3 h after the third postzygotic nuclear division. NANNEY (1953) demonstrated that cytoplasm plays a crucial role in directing the activities of the nucleus in Tetrahymena. However, no locally specified cytoplasm such as germ plasm found in multicellular animals has been found in ciliates. Nevertheless, it seems to be in full agreement that nuclear differentiation can be affected by cytoplasm (MAUPAS 1889; NANNEY 1953; SONNEBORN 1954). Does postzygotic division have any contribution to the differentiation of macroand micronucleus? In P. caudatum, the third division is very interesting because the
Colchicine and Differentiation of Macronuclear Anlagen
221
spindle of the third nuclear division lies parallel to the longitudinal axis of the cell and elongates until their poles reach opposite ends of the cell as mentioned by CALKINS and CULL (1907). When the third postzygotic division was arrested by cochicine treatment, normal differentiation of macronuclear anlagen was disturbed and 2 insted of 4 anlagen were produced. In P. bursaria, EOELHAAF demonstrated that abnormities occurred in the differentiation of macronuclear anlagen when the third postzygotic division (the last postzygotic division) was subjected to colchicine (1955). The result in P. caudatum suggests a possibility that the third postzygotic nuclear division plays an important role for producing the regular number of macronuclear anlagen. Next problem is whether synkaryon (fertilization nucleus) and products of first or second postzygotic division can also develop to macro- or micronucleus. Results from the inhibition of postzygotic nuclear division by colchicine suggest that nuclei after first or seconr] postzygotic division can develop to macro- and micronucleus, though the number of macronuclear anlagen was fewer than normal one. Colchicine inhibits postzygote nuclear division but does not inhibit growth of macronuclear anlagen. When colchicine treatment inhibited the first postzygotic division, that is, the treatment stage I in Fig. 1, no macronuclear anlagen was observed in the exconjugant cells (Fig. 3-- I). However, it is very difficult to know when synkaryon has an ability to develop to macronuclear anlagen or micronucleus and whether the first postzygotic division is really necessary for macro- and micro nuclear differentiation, because the stage of synkaryon seems to be short and a possibility that colchicine arrests the process of synkaryon formation is not ruled out. Fragmentation of macronucleus was partly or perfectly inhibited by colchicine. This suggests the possibility that microtubles play an important role in skeinformation and fragmentation of old macronucleus. The fragmentation of P. caudatum continues until about 24 h after mixing of the mating types and 30-60 fragments are produced. Macronuclear anlagen can differentiate without fragmentation of old maconucleus. Therefore, macronuclear differentiation and fragmentation of old macronucleus are independent phenomena.
Acknowledgements The author thanks Dr. K. HIWATASHI for his discussion during the course of this investigation and for help in preparation of the manuscript. I would also like to thank Dr. S. KorZUMI and Dr. M. SAITO for many helpful suggestions and encouragements.
Literature CALKINS, G. N., and CULL, S. W.: The conjugation of Paramecium aurelia (caudatum). Arch. Protistcnk. 10 (1907): 375-415. DRYL, S.: Antigenic transformation in Paramecium aurelia after homologous antiserum treatment during autogamy and conjugation. J. Protozool. 6 (1959): 25. EGELHAAF, A.: Cytologisch-entwicklungsphysiologische Untersuchungen zur Konjugation von Paramecium bursaria FOCKE. Arch. Protistenk. 100 (1955): 447-514.
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K. MIKAMI, Colchicine and Differentiation of Macronuclear Anlagen
HERTWIG, R.: Uber die Konjugation der Infusorien. Abh. bayer. Akad. Wiss. 17 (1889): 151. HIWATASHI, K.: Determination and inheritance of mating type in Paramecium caudatum. Genetics. 58 (1968): 373-386. KIRBY, H.: Materials and methods in the study of protozoa. Berkley and Los Angeles 1950. MAUPAS, E.: Le rajeunissement karyogamique chez les cilies. Arch. Zool. expo gen. Ser. 2, 7 (1889): 149-517. MIKAMI, K., and HIWATASHI, K.: Macronuclear regeneration and cell division in Paramecium caudatum. J. Protozool. 22 (1975): 536-540. NANNEY, D. L.: Nucleo-cytoplasmic interaction during conjugation in Tetrahymena. BioI. Bull. 105 (1953): 133 -148. SAITO, M., and SATO, H.: Morphological studies on the macronuclear structure of Paramecium caudatum. III. On the development of the macronucleus in the exeonjugant. Zool. Mag (Jap.) 70 (1961): 81-88. SONNEBORN, T. M.: Patterns of nucleocytoplasmic integration in Paramecium. Caryologia. 1 (1954): 307-325. Author's address. Dr. KAZUYUKI MIKAMI, Department of Biology, Mlyagi College of Education, Aoba-yama, Sendai, Japan.